UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effect of Arsenicals on cell suspension cultures of Catharanthus Roseus (Madagascan periwinkle) Hettipathirana, Deepthi Indika 1992

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1992_fall_hettipathirana_deepthi_indika.pdf [ 4.23MB ]
Metadata
JSON: 831-1.0061749.json
JSON-LD: 831-1.0061749-ld.json
RDF/XML (Pretty): 831-1.0061749-rdf.xml
RDF/JSON: 831-1.0061749-rdf.json
Turtle: 831-1.0061749-turtle.txt
N-Triples: 831-1.0061749-rdf-ntriples.txt
Original Record: 831-1.0061749-source.json
Full Text
831-1.0061749-fulltext.txt
Citation
831-1.0061749.ris

Full Text

THE EFFECT OF ARSENICALS ON CELL SUSPENSION CULTURES OFCATHARANTHUS ROSEUS (MADAGASCAN PERIWINKLE)byDEEPTHI INDIKA HETI’IPATHIRANAB. Sc., University of Colombo, Sri Lanka, 1985A THESIS SUBMITI’ED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF CHEMISTRYWe accept this thesis as conformingto the required standardTHE UNWERSITY OF BRITISH COLUMBIAJUNE 1992© Deepthi Indika Hettipathirana, 1992Signature(s) removed to protect privacyIn presenting this thesis in partial fufflhlment of the requirements for an advanced degree atThe University of British Columbia, I agree that the library shall make it freely availablefor reference and study. I further agree that permission for extensive copying of this thesisfor scholarly purposes may be granted by the Head of my Department or by his or herrepresentatives. It is understood that copying or publication of this thesis for financial gainshall not be allowed without my written permission.Department of ChemistryThe University of British Columbia2075 Wesbrook PlaceVancouver, CanadaV6T 1W5Date: 17 June 1992Signature(s) removed to protect privacyABSTRACTAlthough the biotransformation of simple arsenic compounds to a variety of complexarsenosugars inside marine plants is well documented, the role of terrestrial plants has notbeen thoroughly investigated. The effect of arsenicals on a terrestrial plant,Catharanthus roseus, is examined by using cell suspension cultures as a model system. Theminimum inhibitory concentration of arsenate is low at 5 pg mL1 and it is the most toxicspecies to cell suspension cultures of C. roseus. Arsenite and methylarsonate (MMA) have aless toxic effect. Dimethylarsinate (DMA) is the least toxic of the arsenic species studied,with normal growth rates observed with up to 50 pg mL1 of arsenic in the medium.Significant incorporation of arsenic by cell suspension cultures of C. roseus isobserved when grown in media containing arsenicals. The toxicity to each arsenical seems tobe a direct function of uptake. Speciation of arsenic in cell extracts shows that C. roseus cellsare capable of both methylation and demethylation of arsenicals. However, there is noevidence for the formation of significant levels of complex organoarsenic species.The biosynthesis of indole alkaloids by C. roseus plants as well as tissue and cellcultures has been well documented. Addition of arsenicals into the alkaloid productionmedium, APM, changes the pattern of alkaloid accumulation. The effect is dependent on thearsenic species, its concentration, as well as the time of application. Treatment with thearsenicals, arsenate, arsenite, MMA and DMA, during early growth stages, has an inhibitoryeffect on alkaloid production. Although DMA is the least toxic to growth, it has a drasticeffect on alkaloid production. Tryptamine, an early precursor of the indole alkaloids,accumulates in cells treated with DMA indicating that the initial step of condensation oftryptamine with secologanin is inhibited.11Treatment with arsenicals during the early stationary phase of culture growthenhances the accumulation of some alkaloids, although some are suppressed. For example,treatment with DMA on day 22 of growth results in increased levels of an unidentifiedalkaloid (MW 354) whereas production of catharanthine is completely suppressed.1H spin-echo NMR spectroscopy of intact cells of C. roseus facilitates monitoringchanges inside the cells on treatment with arsenicals. This in situ detection method isnoninvasive and nondestructive in comparison to other available biochemical methods.Short term uptake of the arsenicals, MMA and DMA, by C. roseus cells that had reachedstationary phase in 1-B5 medium, is followed by using the Carr-Purcell-Meiboom-Gill pulsesequence. An increase in the peak height of the methylarsenic resonance over a period of11 hours, is indicative of uptake of each arsenical. However, there is no evidence of anybiotransformation products in the 1H NMR spectra. The accumulation site of DMA isprobably the vacuole as is seen from the change in the chemical shift of DMA as it moves intoa compartment of lower pH.Biochemical changes associated with the presence of arsenicals are evident in the1H NMR spectra of C. roseus cells isolated at different stages in the growth cycle. Althoughuptake has been demonstrated by other analytical techniques, the resonances correspondingto both MMA and DMA are not observed in the 1H NMR spectra of cells growing in mediacontaining each arsenical. The association of these arsenicals with large biomolecules- in thecell may account for the absence of these resonances.‘UTABLE OF CONTENTSAbstract • iiI..istof’I’abls.ixListofFigures xiList of.Abbrev-iat.ions. . .xviAcknowledgements xviiiChapter 1General Introduction 11.1 Biological Transformation of Arsenic 11.1.1 Redox transformation between arsenate and arsenite 21.1.2 Biomethylation 21.1.3 Organoarsenic compounds in marine organisms 51.1.3.1 Proposed mechanism for the biosynthesis of arsenosugars. . . .71.1.3.2 Conversion of arsenosugars to arsenobetaine 81.2 Overview of Thesis 10Chapter 2Uptake and Biotransformation of Arsenicals by Cell Suspension Cultures ofCatharanthusroseus 112.1 Introduction 112.1.lWhyplants’ 112.1.2 Why arsenic in plants’ 112.1.3 Freshwater algae 122.1.4 Arsenic in terrestrial plants 142.1.4.1 Soil arsenic availability 142.1.4.2 Arsenic uptake by plants 142.1.5 Biochemistry of arsenic in terrestrial plants 152.1.5.1 Arsenite 162.1.5.2 Arsenate 162.1.5.3 Organoarsenicals 182.1.6 Scope of work 212.2 Experimental 222.2.1 Instrumentation 222.2.2 Chemicals and reagents 22iv2.2.3 Culture methods.232.2.3.1 Culture maintenance 232.2.3.2 Effect of arsenic compounds on the growth of C. roseuscell suspension cultures 232.2.4 Analytical procedures 242.2.4.1 Graphite Furnace Atomic Absorption Spectrometry ( GFAA). 242.2.4.2 Hydride Generation Atomic AbsorptionSpectrometry ( HGAA) 262.2.4.3 Hydride Generation - Gas Chromatography - AtomicAbsorption Spectrometry ( HG-GC-AA) 272.2.4.4 Wet digestion of freeze dried cell samples 292.2.4.5 Extraction of cells for arsenic determination 292.2.4.6 UV decomposition of cell extracts 292.3 Results and Discussion 312.3.1 Analytical methodology 312.3.1.1 Graphite Furnace Atomic Absorption Spectrometry 312.3.1.2 Hydride Generation Atomic Absorption Spectrometry 332.3.1.3 Hydride Generation - Gas Chromatography - AtomicAbsorption Spectrornetry ( HG-GC-AA) 362.3.2 The effect of arsenic compounds on the growth of cellsuspension cultures of Catharanthus roseus 372.3.2.1 General growth pattern of a C. roseus cellsuspension culture 272.3.2.2 Growth of cell suspension cultures of C. roseusin 1-B5 medium containing arsenic compounds 392.3.2.3 Effect of arsenate 402.3.2.4 Effect of arsenite 422.3.2.5 Effect of methylarsonate 432.3.2.6 Effect of dimethylarsinate 442.3.3 Uptake of arsenicals by C. roseus cell suspension cultures 452.3.3.1 Uptake of arsenate 452.3.3.2 Uptake of arsenite 472.3.3.3 Uptake of methylarsonate 482.3.3.4 Uptake of dimethylarsinate 502.3.4 Determination of total arsenic by using Neutron Activation Analysis. . 522.3.4.1 Total arsenic in cells grown in 1-B5 medium 522.3.4.2 Total arsenic in cells grown in AikaloidProduction Medium 542.3.5 Quantitation and speciation of arsenic in C. roseus cell extracts 562.3.5.1 Total arsenic in base extracts 562.3.5.2 Efficiency of extraction 592.3.5.3 tJV decomposition / HGAA 602.3.5.4 Speciation of arsenic in cell extracts 622.3.6 Summary 67VChapter 3Effect ofArsenic Compounds on Alkaloid Production by Cell SuspensionCultures of Catharanthus roseus 683.1 Introduction 683.1.1 Biosynthesis of inonoterpenoid indole alkaloids 703.1.2 Factors affecting alkaloid production by C. roseus cellsuspension cultures 733.1.2.1 Medium composition 743.1.2.2 Other environmental factors 753.1.3 Elicitation of alkaloid production 753.1.3.1 Fungal elicitors 763.1.3.2 Abscisic acid 763.1.3.3 Inorganic salts 773.1.3.4 Vanadyl sulphate 773.1.4 Analytical methods 773.1.4.1 Thin Layer Chromatography 783.1.4.2 High Performance Liquid Chromatography 793.1.4.3 Gas Chromatography - Mass Spectrometry 803.1.4.4 Supercritical Fluid Chromatography - Mass Spectrometry. . .803.1.4.5 Thermospray Liquid Chromatography-Mass spectrometry. . .813.1.5 Scope of work 833.2 Experimental 853.2.1 Instrumentation and analytical methods 853.2.1.1 NMR and Mass Spectrometry 853.2.1.2 Thermospray Liquid Chromatography-Mass Spectrometry(LC-MS) 853.2.1.3 High Performance Liquid Chromatography C HPLC) 863.2.1.4 Thin Layer Chromatography (PLC ) 873.2.2 Culture methods 873.2.2.1 Growth conditions 873.2.2.2 Effect of arsenic compounds on growth 883.2.2.3 Effect of time of application of arsenic compounds 893.2.3 Extraction of alkaloids from cells 893.2.4 Chemicals and reagents 903.3 Results and Discussion 913.3.1 Comparison of growth characteristics of C. roseus cellsuspension cultures in standard 1-B5 medium and AlkaloidProduction Medium (APM) 913.3.2 Effect of arsenic compounds on growth of C. roseus cellsuspension cultures in APM .933.3.3 HPLC analysis of indole alkaloids 96vi3.3.3.1 HPLC analysis of alkaloid composition of cells after22 days of growth 973.3.3.2 HPLC analysis of alkaloid composition of cells after29daysofgrowth 993.3.4 Application of Thermospray Liquid Chromatography- Mass Spectrometry for the analysis of indole alkaloids 1023.3.5 Cell alkaloid composition after 22 days of growth 1053.3.5.1 LC-MS analysis of C. roseus cells grown in APM for 22 days. 1053.3.5.2 LC-MS analysis of 22 day old cells grown in APMcontaining arsenate 1083.3.5.3 LC-MS analysis of 22 day old cells grown in APMcontaining methylarsonat.e 1113.3.5.4 LC-MS analysis of 22 day old cells grown in APMcontaining dimethylarsinate 1113.3.6 LC-MS analysis of alkaloid extracts from cells grown in APMcontaining arsenic compounds for 29 days 1163.3.6.1 LC-MS analysis of C. roseus cells grown in APM for29days 1163.3.6.2 LC-MS analysis of 29 day old cells grown in APMcontaining arsenate 1173.3.6.3 LC-MS analysis of 29 day old cells grown in APMcontaining methylarsonate 1223.3.6.4 LC-MS analysis of 29 day old cells grown in APMcontaining dimethylarsinate 1223.3.7 Effect of time of application of arsenic compounds 1273.3.7.1 LC-MS analysis of C. roseus cells treated with arseniccompounds on day 22 of the growth cycle 1273.3.7.2 LC-MS analysis of C. roseus cells treated with arseniccompounds on day 11 of the growth cycle 1363.3.8 Isolation and structure elucidation of tryptamine 1443.3.8.1 Isolation of tryptamine 1443.3.8.2 Conversion to N-acetyltryptamine 1453.3.9 Summary 1463.3.9.1 Alkaloid composition in 22 and 29 day old C. roseus cellsuspension cultures 1473.3.9.2 Effect of arsenate on alkaloid production 1483.3.9.3 Effect of methylarsonate on alkaloid production 1493.3.9.4 Effect of dimethylarsinate on alkaloid production 150WIChapter 4Application of Whole Cell NMR Techniques to Study the Interaction of ArsenicCompounds withCathczranthus roseus 1524.1 Introduction 1524.1.1 1H NMR spectroscopy of intact cells . 1534.1.2 3lp I’iIR spectroscopy . . . . 1544.1.3 CNMRspectroscopy 1554.1.4 The application of 1-H Spin-echo NMR spectroscopy of intact cells. . . .1564.1.5 Scope of work 1584.2 Experimental 1594.2.1 NMR parameters 1594.2.2 Culturegrowth 1604.2.3 NMR monitoring of cells growing in APM containingarsenic compounds 1604.2.4 Uptake of arsenic compounds 1604.3 Results and Discussion 1614.3.1 General features of 1H NMR spectra of C. roseus cells 1614.3.2 1H NMR studies of C. roseus cells grown in theAlkaloid Production Medium 1634.3.2.1 Changes in the control culture in APM with growth phase. . 1634.3.2.2 NMR monitoring of cells growing in APMcontaining arsenate 1664.3.2.3 NMR monitoring of cells growing in APMcontaining arsenite 1694.3.2.4 NMR monitoring of cells growing in APM containingznethylarsonate 1714.3.2.5 NMR monitoring of cells growing in APM containingdimethylarsinate 1754.3.3 1H Spin-echo NMR studies on uptake and short term effectsof arsenicals on C. roseus cells 1784.3.3.1 Changes in a control cell sample in the NMR tube 1784.3.3.2 Uptake of methylarsonate 1814.3.3.3 Uptake of dimethylarsinate 1854.3.3.4 Effect of arsenite 188434 l3 NMR spectroscopy of C. roseus cells 1914.3.5 Summary 193Chapter 5Conclusions 195Bibliography 200AppendixA 211yinLIST OF TAflLESTABLES PAGE2.1 Furnace operating parameters for the determination of arsenic in plantcellextract.s 252.2 Typical sampling parameters for Standard Additions Method forGFA.Aaiialysis. . 252.3 Operating conditions for the continuous Hydride Generation AtomicAbsorptionassembly2.4 Reaction conditions of conversion of inorganic and methylarseniccompounds to volatile arsines 352.5 Neutron Activation Analysis (NAA ) results of total arsenic inC. roseus cells grown in arsenic spiked 1-B5 media 542.6 Neutron Activation Analysis ( NAA) results of total arsenic inC. roseus cells grown in arsenic spiked APM media 552.7 Results of total arsenic analysis in base extracts of cells grownin 1-B5 medium spiked with arsenate 572.8 Results of total arsenic analysis in base extracts of cells grownin 1-B5 medium spiked with arsenite .572.9 Results of total arsenic analysis in base extracts of cells grownin 1-B5 medium spiked with methylarsonat.e 582.10 Results of total arsenic analysis in base extracts of cells grownin 1-B5 medium spiked with dimethylarsinate 582.11 Results of total arsenic analysis in cell residues after base extraction 592.12 Percentage recovery obtained for some standard compounds and cell extractsafter UV irradiation in open and sealed quartz tubes 612.13 Results of analysis of cell extracts by UV decomposition / HGAAin sealed tubes 622.14 Results of HG-GC-AA analysis of extracts of cells grown in arsenate 642.16 Results of HG-GC-AA analysis of base extracts of cells grown inMMA and DMA 66ix3.1 The Minimum Inhibitory Concentration (MIC ) values of arsenicalsfor C. roseus cell suspension cultures grown in the standard 1-B5and APM media 943.2 HPLC, retention times of Catharanthus alkaloids 973.3 LC-MS analysis of alkaloid extracts from C. roseus cells grown in APMfor22days 1063.4 LC-MS analysis of alkaloid extracts from C. roseus cells grown in APMcontaining 3 ppm of arsenate for 22 days 1093.5 LC-MS analysis of alkaloid extracts from C. roseus cells grown in APMcontaining 6 ppm of methylarsonate for 22 days 1123.6 LC-MS analysis of alkaloid extracts from C. roseus cells grown in APMcontaining 40 ppm of dimethylarsinate for 22 days 1143.7 LC-MS analysis of alkaloid extracts from C. roseus cells growninAPMfor29days 1183.8 LC-MS analysis of alkaloid extracts from C. roseus cells grown inAPM containing 3 ppm of arsenate ( Cell age 29 days) 1203.9 LC-MS analysis of alkaloid extracts from C. roseus cells grown inAPM containing 6 ppm of methylarsonate (Cell age 29 days) 1233.10 LC-MS analysis of alkaloid extracts from C. roseus cells grown inAPM containing 25 ppm of dimethylarsinate (Cell age 29 days) 1253.11 LC-MS analysis of alkaloid extracts from C. roseus cells treated with3 ppm of arsenate on day 22 of growth cycle 1293.12 LC-MS analysis of alkaloid extracts from C. roseus cells treated with6 ppm of methylarsonate on day 22 of growth cycle 1313.13 LC-MS analysis of alkaloid extracts from C. roseus cells treated with25 ppm of dirnethylarsinate on day 22 of growth cycle 1343.14 LC-MS analysis of alkaloid extracts from C. roseus cells treated with3ppmofarsenateondayllofgrowthcycle 1373.15 LC-MS analysis of alkaloid extracts from C. roseus cells treated with6 ppm of methylarsonate on day 11 of growth cycle 1403.16 LC-MS analysis of alkaloid extracts from C. roseus cells treated with 25 ppm ofdimethylarsinate on day 11 of growth cycle 142xLIST OF FIGURESFIGURES PAGE1.1 Challenger’s mechanism for the biological methylation of arsenic 41.2 Structure of S-adenosylmethionine ( SAM) 41.3 Structure of several organoarsenic compounds found in marine animals 61.4 Structures of arsenosugars isolated from marine algae 61.5 Proposed mechanism for formation of arsenosugars from dirnethylarsinic acid. . .71.6 Proposed mechanism for formation of arsenobetaine from arsenosugars 92.1 Schematic diagram of the continuous Hydride Generation AtomicAbsorption assembly 262.2 Schematic diagram of Hydride Generation - Gas Chromatography - AtomicAbsorption C HG-GC-AA ) apparatus 282.3 General growth curve of a cell suspension culture of C. rose usin 1-B5 medium 0 Dry cell weight; B Fresh cell weight 382.4 The effect of arsenate on the biomass yield of C. roseus 412.5 The effect of arsenate on the growth curve of C. roseusConcentration of arsenate in the medium 0 0 ppm; B 2 ppm; 0 4 ppm 412.6 The effect of arsenite on the biomass yield of C. roseus0 Dry cell weight; B Fresh cell weight 422.7 The effect of MMA on the biomass yield of C. roseus0 Dry cell weight; B Fresh cell weight 432.8 The effect of DMA on the biomass yield of C. roseus0 Dry cell weight; B Fresh cell weight 442.9 Variation in % uptake of arsenate with time of incubationInitial concentrations of arsenate in the media were 0 2 ppm and I 4 ppm. . . . 472.10 Variation in 0 % uptake of arsenite and I cellular arsenic content with theinitial concentration in the medium ( after 13 days of growth) 482.11 Variation in 0 % uptake of MMA and B cellular arsenic content with theinitial concentration in the medium C after 12 days of growth ) 49xi2.12 The variation in 0 % uptake of DMA and • cellular arsenic contentwith the initial concentration in the medium ( after 13 days of growth ) 513.1 Bisindole allc.aloids 693.2 Major classes of indole alkaloids and their representative alkaloids 713.3 The biosynthetic pathway of indole alkaloids 723.4 Schematic diagram of the Thermospray Liquid Chromatography-MassSpectrometry ( LC-MS ) assembly .863.5 Growth curves of C. roseus cell suspension cultures grown in 0 1-B5and IAPMmedia 923.6 The variation of dry cell weight of C. roseus cultures with theconcentration of the arsenical in the APM mediumA Arsenate; B Arsenite; C MMA; D DMA 953.7 HPLC traces of a cell extract of a control C. roseus culture(Culture age is 22 days at the time ofharvest.)UV detection at (a) 280 nm; (b) 254 nm 983.8 HPLC traces of cell extracts of C. roseus cultures C Culture age is 22 daysat the time ofharvest; UV detection at 280 nm).Culture media contained(a) arsenate (3 ppm) (b) MMA (6 ppm) (c) DMA (40 ppm) 1003.9 HPLC traces of cell extracts of C. roseus cultures ( Culture age is 29 daysat the time of harvest; UV detection at 280 nm).Culture media contained(a) control (no arsenic ) (b) arsenate (3 ppm) (c) MMA (5 ppm)(d) DMA (25 ppm) 1013.10 Thermospray LC-MS analysis of catharanthine (a) Total Ion Chromatogramwhere peak A = impurity peak B = catharanthineand the mass spectral scan of peak B (catharanthine) 1043.11 Thermospray LC-MS analysis of alkaloid extracts from cells grown inAPM for 22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks B and I 1073.12 Thermospray LC-MS analysis of alkaloid extracts from cells grown inAPM containing 3 ppm of arsenate for 22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks G and I 110xii3.13 Thermospray LC-MS analysis of alkaloid extracts from cells grown inAPM containing 6 ppm of methylarsonate for 22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks F and I 1133.14 Therrnospray LC-MS analysis of alkaloid extracts from cells grown inAPM containing 40 ppm of dimethylarsinate for 22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks C and X 1153.15 Therniospray LC-MS analysis of alkaloid extracts from cells grown in APMfor 29 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks E and J’ 1193.16 Thermospray LC-MS analysis of alkaloid extracts from cells grown in APMcontaining 3 ppm of arsenate for 29 daysTotal Ion Chromatograrn (a) and mass spectral scans of peaks E’ and J 1213.17 Thermospray LC-MS analysis of alkaloid extracts from cells grown in APMcontaining 6 ppm of methylarsonate for 29 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks F’ and X 1243.18 Thertnospray LC-MS analysis of alkaloid extracts from cells grown in APMcontaining 25 ppm of dimethylarsinate for 29 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks D and X 1263.19 Thermospray LC-MS analysis of alkaloid extracts from cells treated with3 ppm of arsenate on day 22 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks F and G 1303.20 Thermospray LC-MS analysis of alkaloid extracts from cells treated with6 ppm of methylarsonate on day 22 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks 0’ and I 1323.21 Thermospray LC-MS analysis of alkaloid extracts from cells treated with25 ppm of dimethylarsinate on day 22 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks 0 and J 1353.22 Thermospray LC-MS analysis of alkaloid extracts from cells treated with3 ppm of arsenate on day 11 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks F and F’ 1383.23 Thermospray LC-MS analysis of alkaloid extracts from cells treated with6 ppm of methylarsonate on day 11 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks F and X 1413.24 Therinospray LC-MS analysis of alkaloid extracts from cells treated with25 ppm of dimethylarsinate on day 11 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks 0 and I 143xm4.1 A schematic representation of the Carr-Purcell-Meiboom-GiII (CPMG)pulse sequence 1594.2 400 MHz 1H NMR spectra of a cell suspension of C. roseus ( Cells were grownin 1-B5 medium for 10 days and then suspended in D20. ) 1624.3 Spin-echo NMR spectra of C. roseus cell suspensions grown in AlkaloidProduction Medium (APM ), No arsenic was added to the medium.(a) 6 hours after transferring to the new medium(b) 2 days after transfer(c) 8 days after transfer(d) 23 days after transfer 1654.4 Spin-echo NMR spectra of C. roseus cell suspensions grown in AlkaloidProduction Medium (APM ) containing 3 ppm of arsenate(a) 2 days after transferring to the new medium containing arsenate(b) 5 days after transfer(c) 12 days after transfer(d) 23 days after transfer 1684.5 Spin-echo NMR spectra of C. roseus cell suspensions grown in AlkaloidProduction Medium (APM ) containing 4 ppm of arsenite(a) 2 days after transferring to the new medium containing arsenite(b) 5 days after transfer(c) 15 days after transfer(d) 28 days after transfer 1704.6 Spin-echo NMR spectra of C. roseus cell suspensions grown in AlkaloidProduction Medium (APM ) containing 4 ppm of methylarsonate (MMA)(a) 6 hours after transferring to the new medium containing MMA(b) 8 days after transfer(c) 15 days after transfer(d) 23 days after transfer 1744.7 Spin-echo NMR spectra of C. roseus cell suspensions grown in AlkaloidProduction Medium (APM ) containing 15 ppm of dimethylarsinate ( DMA)(a) 6 hours after transferring to the new medium containing DMA(b) 8 days after transfer(c) 17 days after transfer(d) 28 days after transfer 1774.8 1H spin-echo spectra of a suspension of C. roseus cells(Cells were grown for 10 days in 1-B5 medium prior to harvesting.)Each spectrum was recorded(a)lh, (b)3h, (c)5h, (d)8hafter packing into the NMR tube 180xiv4.9 1H spin-echo spectra of a suspension of C. roseus cells treated with MMA(0.3 mg/0.5 mL of packed cells)Each spectrum was recorded(a)lh, (b)3h, (c)7h, (d)lOhafter packing into the NMR tube and treatment with MMA 1834.10 The variation of the relative signal intensity of methylarsenicresonance (MIR ) with time 1844.11 The 1H spin-echo NMR spectrum of a suspension of C. roseus cells packedinaNMRtubefor5days 1854.12 1H spin-echo spectra of a suspension of C. roseus cells treated with DMA(0.15 mg/0.5 mL of packed cells)Each spectrum was recorded(a)lh, (b)3h, (c)5h, (d)8hafter packing into the NMR tube and treatment with DMA 1874.13 The variation of the relative signal intensity of dimethylarsenicresonance (DJR ) with time 1884.14 1H spin-echo spectra of a suspension of C. roseus cells treated with arsenite(3 mg/O.5 mL of packed cells)Each spectrum was recorded(a)lh, (b)3h, (c)5h, (d)8hafter packing into the NMR tube and treatment with arsenite 1904.15 13C NMR spectra of C. roseus cell suspensions in D20(a) 13C NMR spectrum of cells grown in APM for 23 days(b) 13C NMR spectrum of cells grown in 1-B5 medium for 11 days 192xvLIST OF ABBREVIATIONSAC-3 a cell line of Cathczranthus roseus derived from a leaf explantAAS atomic absorption spectrometryAPM alkaloid production medium for cell suspension cultures of C. roseus1.B5 standard growth medium for cell suspension cultures of C. roseusCAS ceric ammonium sulphateCPMG Carr-Purcell-Meiboom-GillDMA dimethylarsinate, also dimethylarsinic acidElMS electron ionization mass spectrometryGO-MS gas chromatography - mass spectrometryGFAA graphite furnace atomic absorption spectrometryHGAA hydride generation atomic absorption spectrometryHPLC high performance liquid chromatographyi.d. inner diameterLC-MS liquid chromatography - mass spectrometryMIC minimum inhibitory concentrationMMA methylarsonate, also methylarsonic acidMS mass spectrometryMW molecular weightxn/z mass to charge ratioNMR nuclear magnetic resonanceo.d. outer diameterP1 phosphateppm parts per million, also igxv’RSD relative standard deviationSAM S-adenosylmethionineSFC-MS supercritical fluid chromatography - mass spectrometryT2 spin-spin relaxation timeTLC thin layer chromatographyUV ultravioletv/v volume per volumew/v weight per volumexviiACKNOWLEDGEMENTSI would like to extend my sincere gratitude to my research supervisor,Dr. W. R. Cullen, for his guidance and encouragement throughout the course of this project.I would also like to extend my special thanks to the members of my guidancecommittee, Dr. J. P. Kutney, Dr. J. McPherson and Dr. C. Orvig for their valuablesuggestions.The cell line AC-3 was generously supplied by Dr. J. P. Kutney, in whose laboratoryit has been developed and extensively characterized. I wish to thank Mr. G. Hewitt andco-workers in the Biological Facility of Chemistry Department for their support during tissueculture experiments and also valuable discussions. Thanks also go to the technical staff ofthis department, particularly to Dr. G. Eigendorf and his staff members for their assistancewith the mass spectrometry experiments. I would also like to acknowledge Dr. 0. Chan andstaff members in the NMR facility.I thank Dr. K. Reimer and Ms. D. Reimer, Royal Roads Military College, Victoria, fortheir contribution in arsenic speciation studies, during and after their stay at UBC. Thanksare also extended to the members of my research group, both past and present, for helpfuldiscussions and friendship.Finally, I would like to thank my husband for his patience and encouragementduring the past few years.xvmCHAPTER 1GENERAL INTRODUCTIONArsenic is one of the most widely distributed elements in the earth’s crust and in thebiosphere. The arsenic content in the earth’s crust is 1.5 -2 mg kg; it ranks twentieth inabundance in relation to other elements. 1,2 Arsenic generally occurs as the sulfides, realgar(As4S), orpiinent (As2S3)and arsenopyrite (FeAsS ) and as the arsenides of copper, iron,cobalt and lead. Natural processes such as weathering, volcanic and biological activity resultin the mobilization of arsenic. Anthropogenic activity is also responsible for emission ofarsenic into the atmosphere whence it is redistributed by rain and dry fallout. The majoranthropogenic activities include smelting and burning of fossil fuels.2’3Arsenic compounds have a long history of use for their toxic and medicinalproperties. Arsenic trioxide (white arsenic) gained the reputation of a dangerous poison inthe Middle Ages and until the nineteenth century was a preferred homicidal poison.4Medicinal use dates back to the fourth century BC, when a paste of realgar was in use as atreatment for ulcers. During the early 1900s, several organoarsenicals were developed aschemotherapeutical agents against human sleeping sickness and syphilis.3’4 There is littleuse of arsenicals in medicine today. More recently, arsenic compounds, especially arsenictrioxide and arsenate have been implicated as carcinogens but no definite proof has beenreported.41.1 BIOLOGICAL TRANSFORMATION OF ARSENICMost environmental transformations of arsenicals take place in the soil, insediments, in plants and animals and in zones of biological activity in the oceans. The1simplest environmental transformations are probably redox transformation betweenarsenate and arsenite and biomethylation. The synthesis of more complex organoarsenicalshave been reported mostly in marine organisms.21.1.1 Redox transformation between arsenate and arseniteVarious biological systems are capable of the reduction of arsenate to arsenite Thebioreduction of arsenate has been reported in aerobic and anaerobic bacteria. The presenceof arsenite in oxygenated sea water was attributed to the reduction of arsenate by marinebacteria and phytoplankton.5’6The role of Chiarella pyrenoidosa, a fresh water alga, in thereduction of arsenate was established by Blasco et al.7 The reduction of arsenate to arsenitehas been described in higher plants, animals as well as man.3Oxidation of arsenite to arsenate has been described in several bacterial strainsincluding a soil bacterium, Alcahgenes.8 A Pseudomorias strain produced arsenate underaerobic conditions only.91.1.2 BiomethylationThe biomethylation of arsenic was first described in detail by Challenger after heidentified the poisonous gas produced by molds growing on wall paper astrimethylarsine (Me3As ).10 The molds growing on the wall paper colored with arseniccontaining pigments produced a gas with a characteristic garlic like odor that caused severalpoisoning incidents. Early workers like Gosio and Biginelli established the source of the odorbut they identified the gas incorrectly asEt2AsH.In 1945, Challenger11reviewed his work on the identification of the mold metabolite,Me3As and proposed a mechanism for the methylation of arsenate (Figure 1.1). Challenger2proposed that methylation involved reduction and oxidative transfer of a methyl group froma methyl donor such as betaine, methionine or a choline derivative. It was observed thatarsenate, arsenite, methylarsonate and dimethylarsinate can all act as substrates for theproduction of trimethylarsine by Scopuiariopsis brevicaulis.Further work by Challenger11’2involving the incorporation of labelled precursors,strongly suggested that an active form of methionine can transfer its methyl group toarsenic. This active form of methionine was subsequently identified asS-adenosylmethionine ( SAM )13, the structure of which is given in Figure 1.2.Recent work by Cullen et al.1446 using labelled L-methionine-methyl-d3showedthat the CD3 group is transferred intact to a variety of arsenicals by Scopulariopsis andCandida cultures thus supporting the proposal that SAM is the source of [CH3i in thescheme given in Figure 1.1.In the proposed mechanism, each inethylation step is preceded by a reduction step.The identity of the reductant species has been investigated. 17 A range of thiols and dithiolsincluding cysteine, glutathione and dithiothreitol were found to be capable of carrying outthe reduction via a two electron transfer.’7”8 Lipoic acid is another possible candidatewhich reduces several arsenicals including trimethylarsine oxide to As(III) species. 18The biological methylation of arsenicals is ubiquitous in nature. In addition tovarious microorganisms, higher organisms including some plants, mice, monkeys and manare known to inethylate inorganic arsenic compounds.193(CH3)AsFigure 1.1 Challenger’s mechanism for the biological methylation of arsenicThe arsenic (III) intermediates in braces are unknown.0 IC — CH— 2 2 SHO INHFigure 1.2 Structure of S-adenosylmethionine (SAM)2e [CH3]H3AsO4 As(OH)3 CH3AsO(OH)2CH3AsO(OH)2(CH3)2AsO(OH)2e {CH3As(O )2} (CH3)2AsO(OH)2e [CH3Jr {(CH3)As(OH)} (CH3)AsOOH41.1.3 Organoarsenic compounds in marine organismsThe biosynthesis of a variety of complex organoarsenicals by marine fauna and florahas been under investigation for the past two decades. The concentration of arsenic in seawater is in the range 1-8 ng mL and deviations are dependent on geographicaldistribution, depth, biological activity and anthropogenic input. 1,20 The uptake of arsenate,the predominant form of arsenic in sea water, by marine organisms is well recognized asthey contain substantially higher concentrations of arsenic.21’2 Evidence indicates that alarge proportion of this arsenic is present as organoarsenic compounds and isnon-toc.6’7223The first successful isolation of an organoarsenical from a marine organism wasreported by Edmonds et al.23 Arsenobetaine (2 ) was isolated from the western rock lobster,Panulirats cygnus and characterized. Since this discovery, arsenobetaine has been shown tobe the most abundant arsenical in marine animals including fish, clams, mussels andscallops.24’5Other organoarsenicals that have been identified in marine animals arearsenocholine ( 3 ), the tetramethylarsonium ion (4 ) and trimethylarsine oxide ( 5 )•25,26Further work by Edmonds and Francesconi resulted in the isolation of arseniccontaining sugar derivatives from marine algae. The water soluble arsenicals extracted frombrown algae, Ecklonia radiata27 and Hizikici fusiforme28 were the arsenosugars (6) a - e.Arsenosugars of similar structure isolated from the kidney of giant clam, Tridacna maximain western Australia were proposed to be the metabolic products of symbiotic, unicellulargreen algae living in the clam.29 Other workers have shown that arsenosugars andarsenolipids are ubiquitous in a variety of marine algae.30’1 In addition, a5trimethylarsonium analogue of 6 a C 7) (ie. 6 a with the Me2As(O) group replaced byMe3A.s ) has been reported in the marine alga, Sargassum thunbergii.3°(CH3)A&CH2COO (CH3)AsCH2CO(2) (3)(CH3)4As (CH3)AsO(4) (5)Figure 1.3 Structure of several organoarsenic compounds found in marineanimalscH3o= f — cHCH2— R’OH OHR R’6 a -OH -OSO3H6b -OH -OH6 c -OH -SO3H6 d -NH2 -SO3H6 e -OH P(OH)CH2CH(OH)CFigure 1.4 Structures of arsenosugars isolated from marine algae61.1.3.1 Proposed mechanism for the biosynthesis of arsenosugarsA biosynthetic route for the formation of arsenosugars was proposed by Edmonds andFrancesconi32and is given in Figure 1.5.The initial formation of dimethylarsinic acid from arsenate, is postulated to followthe Challenger mechanism of methylation (See Section 1.1.2). However, the final reductionand methylation to trimethylarsine does not occur. Instead it is proposed that the adenosylgroup of the methylating agent SAM is transferred to the arsenic atom. Enzymatichydrolysis of the resulting intermediate ( 8) would lead to (9) which could form thearsenosugars (6 a - e) by reaction with available algal metabolites.32 A recent report byEdmonds et al.33 on the isolation of an arsenic containing nucleoside ( 8), a keyintermediate in the scheme, from the kidney of giant clam, Tridacna maxima gives furtherevidence for this mechanism.O:AscH2 ___JN(CH3)2AsO(OH) .im.- {(C113)2As(OH)} SAM(8)(6)Figure 1.5 Proposed mechanism for formation of arsenosugars fromdimethylarsinic acid40 = As(9)71.1.3.2 Conversion of arsenosugars to arsenobetaineIt is still not known how marine animals accumulate arsenobetaine or (CH3)4As+.Experiments indicated that marine animals acquire their arsenic burden through the foodweb rather than directly from sea water.21’32 Edmonds et aL32’4 proposed that thearsenosugars found in algae are likely to be the precursors of arsenobetaine and theconversion takes place in the food chain.The facile transformation of arsenosugars ( 6) present in the sea weedEcklonia radiata into dimethyloxarsylethanol C 10) on anaerobic decomposition supports thisview, and the scheme given in Figure 1.6 was put forward.34 Parts of this process are mostlikely to occur in the marine sediments, probably microbially mediated. It is not knownwhether the route to arsenobetaine (2) from ( 10) would proceed via arsenocholine C 3) ordimethylarsinoylacetic acid ( 110 .. 0II Oxidation II(6) (CH3)2AsCHCO (CH3)2AsCHCOj(10) (11)a) 2e a) 2eb) CH3 b) CH3Oxidation(CH3)AsCH2CO (CH3)AsCH2COj(3) (2)Figure 1.6 Proposed mechanism for formation of arsenobetaine fromarsenosugars8The isolation of trimethylarsenoriboside (7) from a marine alga suggests analternative hpothesis for the origin of arsenobetaine ( 2 )3O Anaerobic degradation of ( 7)can directly produce arsenocholine which can be converted to (2) inside the marine animalor elsewhere.36 Arsenobetaine has been recently identified in the water coiumn.It has been proposed that the formation of these organoarsenic compounds in marineorganisms is a mechanism for the detoxification of arsenic. Not only are these compoundsnon-toxic in the marine organisms that produce them but they remain non-toxic wheningested by other organisms in the food chain.1’291.2 OVERVIEW OF THESISThe work described in this thesis is carried out with the aim of understandingseveral aspects of the interaction of arsenic with a terrestrial plant, Catharanthus roseus(Madagascan periwinkle). The effect of several arsenicals on cell suspension cultures ofC. roseus as well as the biotransformation of arsenicals by the plant system is examined.Chapter 2 deals with the uptake and biotransformation of arsenic in cell suspensioncultures of C. roseus. Quantitation and speciation of arsenic inside the cells and in theexternal medium is carried out. The analytical techniques utilized, in particular severalmodes of Atomic Absorption Spectrometry, are described.Chapter 3 describes a study of the effect of arsenic on the secondary metabolism ofC. roseus cell suspension cultures. The production of indole alkaloids by this plant system iswell documented. Elicitation and suppression effects on alkaloid production by arsenicalsare investigated, with a view to using this information for understanding the chemistry ofarsenic inside the plant cell.In Chapter 4, the novel application of whole cell NMR techniques to C. roseus cells isdescribed. Spin-echo ‘H NMR spectroscopy of plant cells enables monitoring the effect ofarsenicals in vivo, without disturbing the biological system.10CHAPTER 2UPTAKE AND BIOTRANSFORMATION OF ARSENICALS BY CELL SUSPENSIONCULTURES OF CATHARANTHUS ROSEUS2.1 INTRODUCTION2.1.1 Why plants?Plants are important intermediate reservoirs through which trace elements fromprimary sources move to other living systems. Of basic importance are the quantities andforms of the trace element that enter the plant reservoir, distribute within it and finally,move into other compartments of the ecosystem.Plants may be passive receptors of the trace element or they may exert control overuptake or rejection of various forms of the element. They may be capable of converting it todifferent species, probably as a means of detoxification.2.1.2 Why arsenic in plants?Arsenic levels found in terrestrial plants growing in uncontaminated soils usuallyfall between 0.01 -5 ppm (dry weight basis) but much higher concentrations have beenreported from contaminated sites. Lists of arsenic concentrations in various plants growingin contaminated and uncontaminated soils have been compiled by several workers.38’9Widespread use of arsenicals in agriculture has resulted in a number of studies onarsenic in terrestrial plants. Inorganic arsenicals, especially lead and calcium arsenate,have been used as effective insecticides in orchards and cotton fields since the turn of thecentury and after 20 - 40 years of treatment, these soils accumulated several hundred ppm ofUarsenic. Sodium arsenite has been used as a weed killer and soil sterilant. Herbicidalcontrol of grassy and broad leaf weeds is obtained by using methylarsonate anddimethylarsinate.38 Extensive compilations of arsenicals used in agriculture and theirapplication rates are available.3’401The arsenic content in plants has been used as a reliable indication of metal depositsin the surrounding area, especially of gold, in geochemical prospecting surveys.2’38. TheDouglas fir, Pseudotsuga menziesii, shows a remarkable ability to take up large amounts ofarsenic and thus can be used as an indicator plant.42 Arsenic levels as high as 10000 ppm(dry weight basis) have been recorded in the most recent growth of trees situated close to amineralization site. Another accumulator plant is Agrostis tenuis which contains higharsenic levels when grown near derelict gold mines.43’4 Foliage of the plant containedarsenic concentrations as high as 3470 ppm (dry weight).In another study, gold and arsenic levels in Festuca rubra, a grass growing on minewastes, were studied to assess the use of arsenic as a path finder in gold prospecting.45Arsenic concentrations as high as 440 ppm were detected in roots of Bermuda grass,Cynodon dactylon when grown on contaminated soils but leaves and stems contained lowerlevels.462.1.3 Freshwater algaeArsenic accumulation by various fresh water algae has been under investigation.The ability of marine algae to take up arsenate and biotransform it to organoarseniccompounds is well documented. (See Section 1.1.3.) Freshwater algae are expected to havea similar ability, which may become important in fresh water bodies contaminated witharsenic from natural as well as industrial processes.12Three algal species Chiorella, Phaeodactylum and Skeletonema accumulated arsenicfrom the medium containing 1-30 mg L1 of arsenic and concentration factors as high as240 - 2800 were reported.47 An arsenic tolerant strain of Chiorella vulgaris was found togrow optimally in media containing high arsenic concentrations, up to 2000 mgL1.48Bioaccumulation increases with arsenic concentration and is dependent on the growthmedium composition. Levels as high as 50000 ppm (dry weight) were recorded in somecultures.48 An arsenic resistant strain of blue green alga, Nostoc sp. was investigated as ameans of removing arsenic from polluted waters.49 This species can accumulate botharsenate and arsenite. The arsenic burden in several algae species including Nostoc andChiorella, grown in media containing arsenate was present as both niethylated andnon-methylated species.5° The predominant arsenic species in the cells werenon-methylated and were found to be bound to other cell components. Methylated arsenicspecies were found mainly in the lipid soluble fraction.5°The biotransformation of arsenate in fresh water green algae was reported inanother study.51 Rhizoclonium sp. was exposed to[74A.s) arsenate for one week and thenextracted with hot ethanol. The incorporated arsenic was present as lipid and water solublelipid related compounds. The chlorophytes, Chara and Nitella, similarly producedarsenolipids as well as methylarsenicals.51 The reduction of arsenate to arsenite byChiorella pyrenoidosa was reported.8A striking difference appears to exist between the way in which marine organismsand terrestrial organisms deal with arsenic. In contrast to the marine organisms, terrestrialorganisms seem to convert inorganic arsenic only to simple methylarsenic compounds.51The only exception appears to be freshwater algae which are capable of biotransformation ofarsenic to more complex organoarsenic compounds.132.1.4 Arsenic in terrestrial plants2.1.4.1 Soil arsenic availabilityThe total arsenic content in soils is not a valid indicator of possible arsenicphytotoxicity. Arsenic may be bound to other soil constituents thus limiting its uptake byplants.38’9Arsenate is the predominant arsenic species in aerobic soils and is associated withneutral hydrous oxides, mostly of iron and aluminum, on clay particles, similar tophosphate.38 Soils of high clay content fix arsenate to a higher degTee compared to sandysoils.52 The mobility of methylarsenicals is also dependent on the clay content.52Microbial activity plays a significant role in the interconversion of arsenicals in soilsas well as the release of volatile arsenic species into the atmosphere. Redox transformationsbetween arsenate and arsenite in soils is proposed to be a combination of chemical andmicrobiological processes.39 The methylation of arsenicals by soil fungi and bacteria resultsin the release of volatile arsines from soils. The microbial degradation of organoarsenicals,MMA and DMA, was also reported.522.1.4.2 Arsenic uptake by plantsArsenic must be present in solution to be available for uptake by plant root systems;thus arsenic fixed in the soil is not available to plants. Studies on arsenic uptake byHordeum (barley) seedlings showed that arsenate uptake is 3 to 4 times higher than forarsenite. The organoarsenicals, MMA and DMA are taken up to a lesser extent. Sachsand Michaels demonstrated that root absorption by bean plants increases in the orderDMA < MMA < arsenite <arsenate.14Uptake of arsenicals after foliar applications has been investigated. The toxicityeffect of arsenite is rapid and it kills foliar tissue before any translocation can occur.55 Botharsenate an1 DMA behave similarly at high concentrations and some translocation results inelevated arsenic levels throughout the plant.MMA causes the least contact toxicity and shows rapid translocation.56 After leafuptake, redistribution throughout the plant depends on its growth stage.57 MMA is usuaflytransported towards actively growing areas and storage areas.58’9The accumulation of MMA by several vascular aquatic plants including Eichornia,Alternanthera, Hydrophyla and Lemna was reported.6°Thus these plants, rooted as well asfloating, can serve as a sink for removing MMA from natural waters.2.1.5 Biochemistry of arsenic in terrestrial plantsThe herbicidal action of arsenicals has been the subject of several reviews,55’96but surprisingly little is known about the mode of action of arsenicals in terrestrial plantsystems.The problem is complicated because of the variable toxicity patterns shown by thearsenicals. Toxicity varies widely depending on the chemical form of arsenic. Thedevelopment of analytical methods for speciation of arsenicals is essential for identifyingspecific transformations that may take place in soils as well as in plants.Toxicity is also dependent on the plant species; widely different tolerancecharacteristics are observed in various plants. This feature is made use of when somearsenicals are used as selective herbicides. Also some plants do adapt to growing in soilscontaining high levels of arsenicals.3815Another factor that complicates the picture is the differences in translocation anddistribution in the plants. The toxicity response of the plant is also influenced by the relativematurity of the plant species and its rate of growth at the time of the application.2.1.51 ArseniteGenerally, arsenite is found to be the most toxic of arsenic compounds to plantsystems. High concentrations of arsenite cause rapid contact injury in roots and in leavesafter foliar application.38 This rapid effect is attributed to reaction with sulfhydryl groups ofproteins thus disrupting membrane enzyme systems.62The dramatic effect of arsenite on protein synthesis by a higher plant has beenreported. The pattern of protein synthesis changes rapidly when soybean seedlings aretreated with arsenite ( 100 ii.M ). The changes are found to be identical with heat shockwhen the synthesis of normal proteins is greatly decreased and a new set of proteins, ‘heatshock proteins’ is induced. A similar effect, but to a smaller extent, has been observed withcadmium, but other heavy metals studied did not mimic heat shock in soy bean seedlings.632.1.5.2 ArsenateAsher and Reay53 studied the mutual effect of uptake of arsenate and phosphate inbarley seedlings. Inhibition of arsenate uptake was observed at high phosphateconcentrations. They suggested that both phosphate and arsenate are transported by acommon carrier mechanism. Similar observations have been made in other biologicalsystems.64Arsenate is a competitive inhibitor of phosphate and acts as an uncoupler ofoxidative phosphorylation.65 However, if arsenate is reduced to the trivalent form inside the16plant, it can then inhibit enzyme systems by combining with suithydryl groups. Arsenite hasbeen extracted from several plants including pine seedlings and corn following the uptake of[As] arsenate via roots.51Methylation and further biotransformation of arsenate by terrestrial plants has beenreported by only a few workers. Nissen and Benson51 investigated tomato plants growing insoils deficient in nitrogen, phosphorus and both these nutrients. Nitrogen deficiency inducedno methylation while phosphorus deficiency resulted in methylation of arsenic to a certainextent, especially in the leaves. Appreciable niethylation occurred in plants deficient in boththe nutrients.51Pyles and Woolson66 analyzed several vegetable crops that were grown in soilscontaminated with arsenate. In broccoli, cabbage, green beans, lettuce and potato peel, mostof the arsenic was extracted into the methanollwater phase. Arsenate and low levels ofmethylarsonate were detected in this extract. The presence of complex organoarsenicals wasalso suggested as the total arsenic was not recovered by arsine generation prior to digestionin hot 2 N NaOH. Beets, potato flesh, Swiss chard and tomato contained arsenic extractableinto chloroform, indicating arsenicals of non-polar lipid nature.66Aquatic plants are known to be more tolerant to arsenic than terrestrial plants.Several plants growing in arsenic rich waters of the Waikato river were reported toaccumulate high levels of arsenic.67 The submerged species Ceratophyllum exhibitedarsenic levels as high as 650 ppm.67 Several fresh water plants including floating Lemnaand submerged Sagittaria were reported to transform arsenate to methylated species as wellas to arsenolipids.51 The arsenic species were not identified. The tolerance of aquatic plantsto high arsenic levels, thus may be attributed to their ability to biotransforin it to non-toxicspecies.17Recently, studies on the interaction of arsenate with other soil nutrients werereported. Increasing Se (VI) in the medium had an antagonistic effect on arsenate uptake inalfalfa ( Medkago sativa) sand cultures.68 But increasing the arsenate concentration in themedium caused a significant increase in shoot Se concentration and uptake. The resultantdrop in yield is associated with Se toxicity. In a study involving cauliflower(Brassica oleracea ), arsenate was found to have no inhibitory effect on molybdate uptake.692.1.5.3 OrganoarsenicalsLittle is known about the biochemistry of the organoarsenicals, MMA and DMA,inside the plants. Their toxicity response is usually slow.38 The mode of action ofmethylarsonate is not well understood although it is a major, selective herbicide. Activity ofmethylarsonate on Johnson grass (Sorghum halpense) and purple nutsedge(Cyperus rotundus) makes this herbicide an important part in weed control in cotton.41’6Selective control of crab grass (Digitaria sp.) and dallis grass (Paspalus sp. ) in establishedturf is also provided by methylarsonates with minimal injury to turf grass species.41The source of this selectivity of action is still not known. Evidence for thecombination of methylarsonate with a plant rnetabolite was reported on application of14C-methylarsonate.56’7Sckerl and Frans56 isolated a metabolite of MMA from Johnsongrass, which showed a positive reaction to ninhydrin. They suggested that MMA may becombined with histidine or one of its analogues. Duble et al. reported that MMA remainedintact but it may have been complexed to a plant component, both in purple nutsedge57 andin Bermuda grass.7° A similar ninhydrin positive complex was reported in bean(Pha8eolus vulgaris (L.)) plants on treatment with sodium14C-methylarsonate.5 Thiscomplex accounted for about 60% of the MMA taken up and the rest was unchanged MMA.18In general, the arsenic-carbon bond of MMA seems to remain intact inside the plants.Some [14C) CO2 evolution was detected in wheat ( Triticum aestivum) following treatmentwith [4C] MMA but was suggested to be as a result of soil degradation.71 The majorportion of MMA was extracted with methanol as the unmodified arsenical but small amountsof the arsenical remained unextracted in some bound insoluble form.7The use of ferric salts of MMA as a fungicide in the rice fields in Japan prompted astudy on arsenic metabolism in rice. The sodium salt of MMA taken up by hydroponicallygrown rice plants, remained intact, mostly in the roots.72 However, very low levels ofinorganic arsenic as well as dimethyl and trimethyl arsenic species were detected in roots aswell as shoots. Relatively large amounts of transformed products such as inorganic arsenic,dimethyl and trimethylarsenic species were detected in the nutrient medium through rootexudation. There is the possibility that these transformations result from the activity ofalgae or microorganisms associated with the plant roots or in the nutrient solution but theresults suggest that both methylated and demethylated arsenic species are produced by therice seedlings.72The mode of action of MMA in Johnson grass was investigated by Knowles andBenson.73 Foliar application of MMA resulted in the accumulation of malic acid in treatedleaves. This was attributed to inhibition of NADP-malic enzyme by a photoreductionproduct of MMA.73 MMA itself does not inhibit the function of a preparation of this enzyme.The enzyme plays a key role in CO2 transport in the photosynthesis of this C4 plant; itsinhibition deprives the plant of its source of CO2 thus resulting in the selective eliminationof Johnson grass.74Dimethylarsinic acid, a non-selective herbicide, is less widely used than MMA. It is ageneral contact toxicant and the usage is restricted to non-crop areas.6’ Very few studies19are reported on the interaction of this arsenical in plants. Sachs and Michaels54 examinedthe metabolism of DMA in black valentine bean plants. DMA was extracted unchanged intowater but about 5% of the extractable amount remained bound to the insoluble residue. Thebound arsenic was not identified. Demethylation or reduction to a trivalent species was notobserved.202.1.6 Scope of workThe objective of the present study is to investigate the uptake and biotransformationof several asenic compounds in a terrestrial plant, Catharanthus roseus. C. roseus is a fastgrowing subshrub, growing up to 30- 120 cm height and bears rose or white flowers. Thismedicinal plant is well known as the source of a large number of indole alkaloids, some ofwhich have important pharmacological activity.75Cell suspension cultures were used in the study as an alternative to using wholeplants. Cell culture techniques are rapid and efficient and the environmental andnutritional conditions can be controlled uniformly and precisely. The relative’yundifferentiated nature of the cultured cells reduces the complications of differences inmorpholor in an intact plant76In general, terrestrial plants are suggested to possess limited ability to biotransformarsenic species, unlike marine organisms. To date very little information is available on thebiochemical interaction of arsenicals with terrestrial plants.In this study, the effect of four arsenic species, arsenate, arsenite, inethylarsonateand dimethylarsinate, on C. roseus cell suspension cultures is investigated. The effect ongrowth as well as the uptake of arsenicals is examined. Biotransformation of arsenicals, inparticular methylation, is monitored, employing several analytical techniques such ashydride generation and graphite furnace atomic absorption methods. The possibility offormation of more complex organoarsenic species is also examined.212.2 EXPERIMENTAL2.2.1 InstrumentationAtomic Absorption SpectrometryA Varian Techtron Model AA 1275 single beam atomic absorption spectrometer wasused for the arsenic determination. It was fitted with a Varian Spectra AA hollow cathodelamp operating at 7 m.A. The atomic absorption signal was monitored at 193.7 nm in alldeterminations. The spectrometer was equipped with a deuterium background corrector anda HP 82905A printer.A Varian Techtron GTA-95 atomizer accessory fitted with pyrolytically coatedgraphite tubes was used to achieve graphite furnace atomization. Argon was used as thepurge gas.2.2.2 Chemicals and reagentsAll chemicals used were of analytical grade and obtained from commercial sourcesunless otherwise stated. Standard stock arsenic solution (1000 jig mL4) for GFAA wasprepared by dissolving 1.3203 g of arsenic trioxide (As203,Fisher Scientific) and 2 g ofNaOH in 20 mL of water. The solution was diluted to 200 mL, neutralized with HCI ( 12 M)and made up to 1000 rnL. Stock solutions of arsenate(NaHAsO4.7H0,Fisher Scientific),arsenite (NaAsO2,J. T. Baker Chemicals), methylarsonate (CH3AsO(ONa)2Alfa) anddimethylarsinate (CH3)2AsO(OH), Fisher Scientific) were prepared by dissolvingappropriate weights in deionized water to give solutions containing 1000 jig mL1 of arsenic.These solutions were diluted as necessary to prepare working solutions.22Deionized water C Aquanetics Aqua Media System ) was used for solution preparationfor atomic absorption methods and deionized - distilled water was used for the preparation ofculture media. All glassware and plasticware were cleaned by soaking overnight in 2% (v/v)Extran solution, rinsing with water and soaking in HCI ( 1 M) overnight. Utensils werethen rinsed with water and deionized water until the wash was neutral to litmus.22.3 Culture methods22.3.1 Culture maintenanceC. roseus cell line AC-3 was initiated from a mature leaf explant of a C. roseus plant.Cell suspension cultures were maintained in 1-B5 medium at 26 °C on gyratory shakers at150 rpm. The composition of 1-B5 medium introduced by Gamborg et al.77 supplementedwith 2,4 D. ( 1 mg L4 ), is given in Appendix 1. Subculturing was performed every 10 days.22.32 Effect of arsenic compounds on the growth of C. roseus cell suspensionculturesFor experiments described here, 100 mL of 1-B5 liquid medium in a 250 mLErlenmeyer flask was inoculated with 15 mL of a 10 day old cell suspension and incubatedon a gyratory shaker ( 150 rpm in the absence of light at 26 °C. In the control cultures, themedia did not contain any arsenic. Known concentrations of arsenic compounds, arsenate,arsenite, MMA and DMA were added to the other cultures. Each experiment was done inquadruplicate.After an appropriate incubation time, usually 10 or more days when the cultureshave reached the stationary phase, cells were collected on a pre-weighed Miracloth filter23(Calbiochem-Behring, USA) by using a water aspirator. The spent medium was removedand stored frozen (-20 °C) until further analysis. The harvested cells were washed withdistilled water (50 mL) to remove excess medium, drained under vacuum and weighed toobtain the fresh cell weight. Harvested cells were stored frozen ( -20 °C). To obtain the drycell weight, the frozen cell samples were freeze dried and then weighed.In order to study the effect of arsenate on the growth cycle, cultures containing twodifferent concentrations of arsenate, 2 and 4 ppm, and control cultures were grown asdescribed in the previous section. Two cultures of each were harvested at different times intheir growth cycle. Both fresh and dry cell weights were obtained.2.2.4 Analytical procedures2.2.4.1 Graphite Furnace Atomic Absorption Spectrometry ( GFAA)Furnace operating parameters were optimized for analysis of plant cell extracts. Thetemperature, time and gas flow for each step were varied to obtain the maximum absorbancesignal during atomization.The sample as well as the arsenic standard solutions were injected(volume 5-10 i.tL) to the graphite furnace by using the automatic delivery system of theGTA 95 accessory. It was mixed with 20 p1., of modifier (nickel nitrate ( 100 ppm) orpalladium nitrate ( 100 ppm in 2% w/v citric acid) solutions prior to injection.The optimized furnace operating parameters for the determination of arsenic in plantcell extracts are given in Table 2.1. The standard additions method was used in analyzingplant cell extracts and the parameters used are given in Table 2.2.24Table 2.1 Furnace operating parameters for the determination of arsenic inplant cell extracts.Step Temperature Time Gas Flow Comment# (°C) (sec) (L mm4)1 75 5 3.0 dry2 90 40 3.0 dry3 120 10 3.0 dry4 1250 30 3.0 ash5 1250 1.0 0 ash6 2300 1.0 0 atomize7 2300 1.0 0 atomize8 2300 2.0 3.0 cleanTable 2.2 Typical sampling parameters for Standard Additions Method forGFAA analysisSolution Standard Sample Blank ModifierVolume Volume Volume Volume(ML) (ML) (tL) (iL)Blank- 12 20Additioni 2 2 8 20Addition2 4 2 6 20Addition3 6 2 4 20Addition4 8 2 2 20Addition5 10 2 0 20Sample - 2 10 20252.2.4.2 Hydride Generation Atomic Absorption Spectrometry (HGAA)Determination of total reducible arsenic speciesFor Hydride Generation Atomic Absorption (HGAA) measurements, a continuoushydride generation assembly illustrated in Figure 2.1 was used. It consisted of aGilson Miniplus 2 four channel peristaltic pump which was used to withdraw the sample andmix it with hydrochloric acid or buffer solution. This solution was then reacted with sodiumborohydride solution (2% w/v) and introduced into the gas-liquid separator via a 20 turnmixing coil. The gases were led into an open-ended T-shaped quartz cuvette(8.5 cm x 1 cm(o.d.)) mounted in the air/acetylene flame of a standard Varian burner. Theoperating conditions previously established by Cullen and Dodd78 were utilized and areshown in Table 2.3.Figure 2.1 Schematic diagram of the continuous Hydride GenerationAtomic Absorption assemblyReaction CoilGas-LiquidSeparatorPeristaltic PumpSampleAcidNaBH4DrainLJPressureRegulator26Table 2.3 Operating conditions for the continuous Kydride Generation AtomicAbsorption assemblyUptake flow Sample 7.5 mL mm4HCI 2.0 mL mm4NaBH4 4.0 niL mimi4Carrier gas flow Nitrogen 0.6 L mm4HCI concentration 4 MNaBH4concentration 2% (w/v) in0.1% (w/v) NaOH solutionDetermination of arseniteA citrate buffer solution at pH 6.2 was prepared by adding 10% (w/v) citric acid tosodium citrate (1 M) to adjust pH. This buffer solution was pumped in place of HCI (4 M)during HGAA analysis. The operating conditions given in Table 2.3 were used with theexception of the carrier gas flow rate which was increased to 1.0 L mmn12.2.4.3 Hydride Generation - Gas Chromatography - Atomic AbsorptionSpectrometry ( HG-GU-AA)A schematic diagram of the HG-GC-AA apparatus is given in Figure 2.2. An aliquotof sample was introduced into a Teflon reaction vessel, together with either 4 M HCI or Trisbuffer ( pH 6). A 4% (w/v) solution of KBH4 was slowly injected into the solution. Thearsines produced were then swept from the reaction vessel by a helium flow through a dryice-alcohol bath to remove water and trapped in a Teflon coil immersed in liquid nitrogen.After 5 mm, the Teflon coil was removed from the liquid nitrogen and immersed in a 60°C27water bath. The released arsines were then lead to a Porapak P-S (ChromatographicSpecialties, Canada) packed Teflon column in a CC oven C Varian 3700 CC). ThePorapak P-S packing was previously silanized using Silyl-8 (Chromatographic Specialties,Canada). The temperature of the column was carefully programmed such that the arsineswere separated. The effluent from the GO was swept to an InstrumentationLaboratories 351 Atomic Absorption Spectrometer, where it was combusted in a hydrogen-airflame in a quartz cuvette. The signal was monitored at 193.7 nm and processed by using aHewlett-Packard 3390A integrator.ReactionVesselWater BathDrainReleasing ArsinesFigure 22 Schematic diagram of Hydride Generation - Gas Chromatography -Atomic Absorption (HG-GC-AA) apparatusTrapping ArsinesSampling ValveSampleLiquid N2TrapValveHe Supplydry-ice/ alcoholWater TrapGas Chromatograph282.2.4.4 Wet digestion of freeze dried cell samplesWet cligestion was carried out in a 250 mL round bottom flask fitted with a speciallydesigned stopper and an air condenser containing a diffusion funnel.79 The stopper, funneland plugs were made of Teflon. To the freeze dried cell sample (0.25 g) in the flask, nitricacid (3 mL, 69%), sulfuric acid (1 mL, 98%) and hydrogen peroxide (3 mL, 30%) wereadded. The flask was then fitted with the air condenser assembly and heated for 3 h at250 °C by using a heating mantle. After the flask was cooled to an ambient temperature, thedigestate was transferred to a 100 mL volumetric flask and made up to the mark withdeionized water. A known quantity of NBS certified orchard leaves and a reagent blankwere digested along with each group of samples analyzed. The total arsenic in the digestatewas determined by HGAA using the conditions listed in Table 2.3.2.2.4.5 Extraction of cells for arsenic determinationFreeze dried cells or frozen then thawed fresh cells were homogenized in a blenderwith the extraction medium. Both methanol and 1 M NaOH solution were used as extractionmedia. The cell suspension was then placed in a sonic bath for 1 h. The residue was filteredoff to obtain the cell extract. The procedure was repeated two more times and the resultantextracts were analyzed separately. The concentration of total arsenic in cell extracts wasdetermined by using GFAA and HGAA techniques.2.2.4.6 UV decomposition of cell extractsAliquots of the cell extracts (either methanol or base) were made up to 50 mL byadding deionized water to give working concentrations between 0.01 - 0.1 ppm arsenic. Thesamples (50 mL) were placed in quartz containers (2.5 cm o.d.) that were left open or29sealed. The tubes that were to be sealed were constructed with a narrow neck. After placingthe sample in the tube, it was deaerated by briefly pumping under vacuum and sealed at aconstriction in the neck.The tubes were irradiated for a specified time using a 1200 W medium pressure lamp(Hanovia). The tubes, maximum 24, were arranged around the lamp in a fan cooledcarousel.78 After irradiation, the solutions were allowed to cool, reconstituted to volume inthe case of open tubes, and analyzed by HGAA. In all experiments, the AA signals of thesolution were measured before and after the irradiation.302.3 RESULTS AND DISCUSSION2.3.1 Analytical methodology2.3.1.1 Graphite Furnace Atomic Absorption SpeetrometryGraphite Furnace Atomic Absorption Spectrometry (GFAA) has become one of themost widely used analytical methods for the analysis of arsenic in a variety of samples.80’1The popularity of GFAA methods over other analytical methods stems from its superiorsensitivity and requirement of very small volumes of sample. These advantages arecounteracted to some degree by matrix interferences encountered especially inenvironmental and biological samples.82’3In GFAA, the atomizer is an electrically heated graphite tube, which is aligned in thepath of light from the arsenic hollow cathode lamp. The sample is injected onto the innertube wall of the graphite furnace. The tube is then heated in several stages.84 In the first‘dry’ stage, the solvent is evaporated quickly without sputtering. The second ‘ash’ stage(350 - 1400 °C) removes matrix components by vaporization and decomposition. In thethird ‘atomize’ stage, the analyte is rapidly vaporized and atomized and the analyteatomic absorption signal is detected, the height or area of which is related to the amount ofanalyte present.Operating parameters have to be selected for different types of samples to achievethe best analytical sensitivity and eliminate matrix effects. Programming of operatingparameters such as heating temperature, time and gas flow rate is possible with the GTA-95accessory and the furnace parameters used in the analysis of Catharanthus cell extracts aregiven in Table 2.1.31Chemical modifIcation of the sample is essential in the analysis of arsenic in manybiological samples. Chemical modifiers reduce the volatility of the analyte atom and allowthe use of relatively high ashing temperatures to eliminate matrix components.81’2 Nickelnitrate is commonly used in the analysis of arsenic, where the added nickel forms a stablearsenide which atomizes at a higher temperature. Thus the matrix material can be removedwithout any resultant loss in the analyte.Palladium has emerged as a very effective chemical modifier for arsenic and manyother elements.85’6 Palladium salts have to be reduced to Pd(0) metal to act as an effectivemodifier and a reducing agent such as ascorbic acid or citric acid is added to the modifiersolution. Palladium was found to form a stable intermetallic compound with arsenic in thegraphite firnace.87 This allows the use of ashing temperatures up to 1450 °C, without anyloss of arsenic by volatilization.In the GFAA analysis of plant cell extracts, both nickel and palladium modifiers wereused. Typically 20 L of nickel nitrate (100 ppm) is added to 10 i.iL of sample beforeinjection. Aiternatively, 20 iL of a palladium nitrate (50 ppm) in citric acid (2% w/v) wasused. Careful sample preparation also serves to eliminate the interferences from a biologicalmatrix. Dilution of the cell extract several fold, where possible, aids in reducinginterferences. It is also necessary to prepare standards which are matched to the sample.84For example, in the analysis of base extracts, an enhancement in the GFAA signal isobserved in the presence of NaOH. This enhancement effect, which is associated with thepresence of an easily ionizable element, has been well documented.88 Standards wereprepared such that they contain a similar concentration of NaOH to the samples.Use of the standard additions method proved to be useful in the analysis of plant cellextracts and the sampling parameters are given in Table 2.2. In this method, the mismatch32between standards and the sample is minimized because all standards are prepared from theactual sample.Calibration, limit of detection and precision of GFAA analysisTypically, calibration plots were produced daily for the arsenic compounds byplotting absorbance against concentration of arsenic for a series of standard arsenicsolutions. The GFAA response for arsenate, arsenite, methylarsonate and dimethylarsinatewas identical. Thus one calibration curve, which is typically linear from 10 to 250 ngarsenic, can be used for all four arsenic compounds. The relative standard deviation fortwenty injections of 10 iL of 20 ng mL arsenite solution was calculated to be 7.4%.The limit of detection, which is equivalent to the analyte concentration giving asignal equal to the blank plus three standard deviations of the blank, was determined to be10 ng ml.2.3.1.2 Hydride Generation Atomic Absorption SpectrometryThe Hydride Generation Atomic Absorption Spectrometry (HGAA) has found wideapplication in the determination of a number of elements such as As, Se, Sb, Pb and Sn.89’9°The technique is based on the conversion of the analyte to its volatile hydride. The hydrideis then transported to a heated quartz cuvette, generally by a carrier gas, and atomizedthere. The concentration of analyte atoms formed is measured by atomic absorption. Thissampling technique may be used to separate and preconcentrate analyte from the samplematrices, thereby removing potential matrix interferences commonly encountered in thedirect analysis of solutions.33The HGAA technique is used to determine the total reducible arsenic species thatproduce volatile arsine8. Commonly, they are arsenate, arsenite, methylarsonate anddimethylarsinate. An aqueous sodium borohydride solution is usually used as the arsinegenerating agent.The schematic diagram of the continuous HGAA apparatus used in the present studyis given in Figure 2.1 and was previously described by Cullen and Dodd.78 The operatingconditions used in the determination of total reducible arsenic species are given in Table 2.3.A solution of 4 M HCI is used to maintain the pH of the solution at pH 1 or lower so that allfour arsenicals are quantitatively converted to their volatile arsines.The test solution (sample), HC1 and sodium borohydride solution are pumped by aperistaltic pump and swept into a mixing coil with the carrier gas N2. The generation ofarsines (Equation 2.1) and the decomposition of excess sodium borohydride (Equation 2.2)takes place simultaneously.91As(OH)3+ BH4 + H > AsH + H3B0 + H2 Eq. 2.1BH4 + 3H20 + H > H3BO + 4H2 Eq. 2.2Gaseous arsines and H2 are separated from the solution in a gas-liquid separatingcell and swept into the heated quartz atomizer. The atomizer is heated by an air-acetyleneflame and is aligned in the optical path of the spectrometer. The absorbance is recordedwhen a stable signal is obtained.The arsine generation is pH dependent which allows for some speciation of a mixtureof arsenic compounds as described in Table 2.4.90,92,93 Further speciation can be achievedby trapping (cryofocusing) the amine mixture. Because of the volatility differences, theslow warming of the trap results in the sequential release of the arsines.94 Alternatively,GO separation can be used to determine different arsenicals after cryofocusing.9534Determination of arsenite by HGAADetermination of arsenit.e in the presence of arsenate is achieved by careful control ofthe pH of the reaction mixture. The effect of pH on arsine generation is related to the plC5 ofthe individual arsenic compound.92 Table 2.4 shows the piCa data and the reductionconditions of different arsenic compounds.Table 2.4 Reaction conditions of conversion of inorganic and methylarseniccompounds to volatile arsines92Arsenic pKa1 Reduction Product B.P.Compound pHarsenite 9.23 1-6 AsH3 -55arsenate 2.25 1 AsH3 -55methylarsonate 4.58 1-3 CH3As2 2dimethylarsinate 6.19 1-4 (CH3)2As 36trirnethylarsine 1 (CH3)As 70oxideThe conversion of arsenite to AsH3 proceeds quantitatively from pH 0 to about 6whereas the conversion of arsenate drops to near zero at pH 4. Several buffered systems,including citrate92’3 (pH 5.5), phthalate94 (pH 5.5) and acetate96 (pH 6), have beensuccessfully used for the determination of arsenite in the presence of other arseniccompounds.35In the present study, a 1 M citrate buffer solution which is pumped in place of4 M HCI, was utilized to control the pH at 6.2. The selective conversion of arsenite isaccomplished with minimal interference from arsenate, methylarsonate and in particulardimethylarsinate.Calibration, limit of detection and precision of HGAA analysisThe response of three arsenic compounds arsenite, arsenate and MMA was found tobe identical. DMA, however, gives a lower response based on the arsenic content. This mayresult from incomplete reaction, condensation of dirnethylarsine in transport tubes and/orincomplete atomization. This phenomenon was previously reported by Arbab-Zavar andHoward.96Calibration graphs for standard arsenate and DMA show a linear relationship up to100 ng mL’S Based on 20 replicate analyses, the relative standard deviation of thetechnique was 2.0% for 20 ng mL4 arsenite solution and 2.4% for 20 ng mL4 DMA solution.The limit of detection was determined to be 0.5 ng mL1A typical calibration curve for arsenit.e only determination by the HGAA technique islinear up to 100 ng mL4. The detection limit is 2 ng mL1 and the relative standarddeviation for the analysis of a 10 rig mL4 arsenite solution is 5.2%.2.3.1.3 Bydride Generation - Gas Chromatography - Atomic AbsorptionSpectrometry (HG-GC-AA)The HG-GC-AA method for speciation of reducible arsenic compounds, arsenate,arsenite, MMA and DMA, is also based on the generation of volatile arsines. The arsines arecryofocused which allows for preconcentration. On raising the temperature of the trap, the36arsines are swept into a GO column where they are separated. Atomic absorption is used asthe mode of detection.97 Several versions of this technique have been publishedpreviously.95’89For the determination of total inorganic arsenic (As(V) and As(III)), MMA andDMA, the sample is acidified to pH 1 by the addition of 4 M HCI. At this pH, furtheraddition of KBH4 converts both arsenite and arsenate to arsine (AsH3), MMA tomethylarsine and DMA to dimethylarsine. These arsines are detected according to theirretention times.Arsenite can be determined by buffering the sample to pH 6 with Tris-HCI bufferbefore reaction with KBH4 and detecting any AsH3 generated. Thus the arsenateconcentration can be evaluated from the difference between total inorganic arsenic andarsenite concentrations.Detection limits were found to be as follows: arsenite, 0.12 ng; arsenate, 0.25 ng;MMA and DMA, 0.25 ng. The relative standard deviation for the analysis of 5 ng of eacharsenic species is 5%.2.3.2 The effect of arsenic compounds on the growth of cell suspension culturesof Catharanthug roseus2.32.1 General growth pattern of a C. roseus cell suspension cultureThe growth curve of a cell suspension culture of C. roseus grown in 1.B5 medium wasobtained by plotting the fresh and dry weights of C. roseus cells against the age of the cultureand is depicted in Figure 2.3. This growth pattern is typically observed in a batch cultureand was previously described for C. roseus cell suspensions.MO2 Other growth37.—4.)c.)—characteristics such as cell number, mitotic index, RNA and DNA content have also beenpreviously monitored during the growth of C. roseus cell suspension cultures.103’4The fresh weight and the dry weight of cells per culture increase after a short lagphase, usually of a day or two duration. The lag phase is followed by the growth phaseduring which the biomass increases as a result of cell division and expansion. The cultureusually approaches stationary phase when the biomass reaches a maximum and remainsconstant. This is as a result of depletion of one or more nutrients in the medium thuslimiting the growth. In 1-B5 medium, the limiting nutrient is sucrose, the carbon source,and the C. roseus cell suspension cultures reach stationary phase after 7 - 10 days ofincubation. Following stationary phase, there can be a decrease in weight of cells due to celllysis.3025201510.——505 10 15Time of Incubation ( days )Figure 2.3 General growth curve of a cell Suspension culture of C. roseus in 1-B5medium, D Dry Cell Weight; Fresh Cell Weight0 20382.3.2.2 Growth of cell suspension cultures of C. roseus in 1-B5 medium containingarsenic compoundsThe Minimum Inhibitory Concentration (MIC) of each arsenic compound forC. roseu,s was estimated by using a graph of dry weight of cells at stationary phase againstthe initial concentration of arsenic compound. MIC values, the lowest concentration testedat which growth is prevented, have been useful in expressing tolerance of microorganisms tovarious inhibitors.105 In the present study, the MIC is defined as the concentration of thearsenic species at which the biomass of the culture is 50% or less than that of the control.At concentrations below the MIC of arsenic compounds, cultures of C. roseus appearhealthy and biomass yields are comparable to those of control cultures grown in the absenceof these additives. The appearance of the whole culture, cells and the residual medium isidentical with that of the control. Cell suspensions are generally off-white in color andconsist of small cell aggregates and single cells. One exception is seen in the cultures grownin the presence of methylarsonate (MMA). In these cultures, discoloration of cells isobserved regardless of the concentration of MMA.At concentrations above the MIC of arsenicals, there is considerable aggregation anddiscoloration of cells. The cell density is markedly low and the biomass drops indicatinginhibition of growth. Cultures show darkening within 24 h after adding the arseniccompound at high concentrations, the extent of discoloration depending on the concentration.This effect has been observed in cells treated with fungal elicitors106 and also on addition ofhigh concentrations of vanadyl sulfate107 and is very likely related to stress.392.3.2.3 Effect of arsenate on the growth of C. roseusA graph of dry weight of cells against concentration of arsenate in the medium isdepicted in Figure 2.4. The growth is suppressed at concentrations above 5 ppm. The MICof arsenate is assigned to 5 ppm of arsenic as arsenate, the concentration at which the dryweight of the culture is 50% or less than that of the control (See Figure 4.2). Arsenate isfound to be the most toxic form of arsenic for growth of C. roseus cell suspensions. Thehigher toxicity of arsenate in C. roseus cell suspension cultures probably arises as a result ofmore facile uptake ( See Section 2.3.3.1).The cells grown at higher concentrations of arsenate reach a maximum biomass laterin the growth cycle as seen from the cell weight after 17 days of growth, Figure 2.3. Thispoints to a delay in growth in the presence of arsenate that is confirmed by studying thegrowth pattern of cell suspension cultures at different concentrations of arsenate. Theresults are depicted in Figure 2.5. In the presence of arsenate, a longer lag phase results in adelay in reaching the stationary phase. This effect is more pronounced at higherconcentrations as is seen from the growth curve at 4 ppm of arsenate. This phenomenoncould indicate an initial iithibition of growth until the cells adapt to the arsenic containingenvironment.40C’—SC’._ —Figure 2.5 The effect of arsenate on the growth curve of C. roseusConcentration of arsenate in the medium 0 ppm, control; . 2 ppm;a 4ppm.0 2 4 6 8 10Arsenate Concentration (ppm )Figure 2.4 The effect of arsenate on the biomass yield of C. roseusCells harvested after 0 13 days of growth and • 17 days of growth.1.21.00.80.60.40.20.00 4 8 12Time of Incubation ( days )16 20412.3.2.4 Effect of arseniteThe variation of dry and fresh cell weight with the concentration of arsenite isdepicted in Figure 2.6. At low concentrations, arsenite does not exert any adverse effect onthe cell yield. A drastic drop in cell yield is observed at concentrations above 10 ppm. TheMIC of arsenite is estimated to be 10 ppm of arsenic as arsenite; thus arsenite is less toxic toC. roseus cell suspensions than arsenate.Generally, arsenite is known to be more toxic to biological systems than arsenate.Inorganic arsenic(111) can bind to suithydryl groups on many enzymes in a living cell62 andinterfere with the normal metabolism, which accounts for its higher toxicity. However, thecell suspension cultures of C. roseus exhibit the reverse of this toxicity pattern.252015 =i-1050.0 03 6 9 12 15Arsenite Concentration ( ppm )Figure 2.6 The effect of arsenite on the biomass yield of C. roseuso Dry Cell Weight; • Fresh Cell WeightCells harvested after 13 days of growth in media containing theindicated initial concentration of arsenite as arsenic.1.21.00.80.60.40.20422.3.2.5 Effect of methylarsonateMethylarsonate (MMA), a widely used selective herbicides shows an unusualtoxicity pattern for C. roseus cell suspensions. Cells grown in media containing MMA showdiscoloration and a lower cel1 density even at concentrations below the MIC of the arsenical.The other three arsenic compounds studied do not elicit a toxicity response below their MIC.There is a gradual decrease in biomass yield with increasing concentrations of MMAas seen in Figure 2.7. The dry biomass is about 10% less than that of the control even at1 ppm of the arsenical. The biomass yield is reduced to 50% of that of the control at a MMAconcentration of 8 ppm.025‘nv15 ._.‘10 •00- —5Figure 2.7 The effect of MMA on the biomass yield of C. roseusDry Cell Weight; • Fresh Cell WeightCells harvested after 12 days of growth in media containing theindicated initial concentration of MMA as arsenic.1.21.00.80.60.40.20 5 10 15MMA Concentration (ppm)020432.3.2.6 Effect of dimethylarsinateDimethylarsinate (DMA) is found to be the least toxic of the arsenic compoundsstudied, to C. roseus. The data of Figure 2.8 show that even at 20 ppm of arsenic, the dry cellweight after 12 days of growth is little changed from that of the control. However, the freshcell weight is significantly lower. A rapid increase in fresh cell weight takes place close tothe stationary phase when cells incorporate water. Thus, the lower fresh weight mayindicate that cultures have not reached the stationary phase at the time of harvesting. Thedry weight drops close to 50% of that of the control at an arsenical concentration between 50and 80 ppm. This MIC is the highest of the arsenicals under study.‘- —3020j10-02000 50 100 150DMA Concentration (ppm)Figure 2.8 The effect of DMA on the biomass yield of C. roseusD Dry Cell Weight; • Fresh Cell WeightCells harvested after 13 days of growth in media containing theindicated initial concentration of DMA as arsenic.442.3.3 Uptake of arsenicals by C. roseus cell suspension culturesThe uptake of arsenicals by C. roseus cells was estimated from the differencebetween the initial arsenic concentration in the medium and that remaining on completion ofgrowth. The arsenic content in the spent medium as well as the washings (containing anyexcess medium) was obtained by using GFAA and HGAA. The arsenic determination wascarried out on combined spent media from four replicate cultures. Loss of arsenic from themedium resulting from adsorption to the culture flask is minimal as determined by blankruns in the absence of cells. However, any loss from adsorption onto the surface of cells isnot accounted for in these measurements.The percentage (% ) uptake is expressed as the ratio of arsenic taken up by the cellsto the initial arsenic added to the medium in Figures 2.9 - 2.12. The uptake is alsorepresented as the amount of arsenic incorporated per gram of dry cells which gives a bettermeasure of the arsenic concentration inside the cells.2.3.3.1 Uptake of arsenateThe % uptake of arsenic against the age of culture at different concentrations ofarsenate is given in Figure 2.9. In both cultures containing 2 ppm and 4 ppm of arsenic asarsenate, rapid arsenic uptake is observed. After 3 days of growth, 95% of the arsenic istaken up by the cells from the medium containing 2 ppm of arsenate whereas 76% of arsenicis taken up from the medium containing 4 ppm of arsenate. This increases up to 99% byday 12 of incubation for both cultures.Rapid uptake of arsenate can be related to the uptake of phosphate, a structuralanalog. A common transport system for phosphate and arsenate has been proposed forseveral biological systems such as bacteria108’9 and yeasts.° Rapid uptake of arsenate45was reported in Candida humkola whereas arsenite was taken up much slower. Asherand Reay pr’posed that the mutual effect of phosphate and arsenate uptake in barleyseedlings is consistent with a common carrier mechanism having a higher affinity forphosphate than arsenate.The uptake of phosphate by C. roseus cell suspensions has been previouslystudied.102 Approximately, 46% of phosphate in Murashige-Skoog ( MS ) medium was takenup during the first day of growth (the initial concentration of phosphate is 1.25 mM). Therate reached a plateau around 58% between days 3 and 5 and then increased up to 90% byday 10 of culture growth.102 In another study, more than 95% of phosphate was taken up bythe C. rosezis cells within 48 h after inoculation, from modified MS media containing initialphosphate concentrations between 0- 2 mM.In the present work, a different growth medium (1-B5) containing 1.1 mMphosphate, is used. Although a direct correlation is not possible because of the differentmedium composition and other variables such as cell density, uptake of phosphate byC. roseus cells from the 1-B5 medium can be expected to be rapid. The facile uptake ofarsenate may indicate a common transport mechanism for the two anions in C. roseus cells.The speciation of arsenic in the residual medium was carried out by using HGAA asdescribed in Section 2.3.1.2. The arsenical left in the medium is predominantly arsenite(95%). A control study on autoclaved media containing arsenate but no plant cells failed tofind any arsenite. Thus the reduction of arsenate to arsenite is attributed to the living plantcells and may be a part of a detoxification process, since arsenite seems to be less toxic toC. roseus. Several biological systems including several plants such as pine and corn areknown to carry out the reduction of arsenate.5146100908070 -_______________0 4 8 12 16Time of incubation ( days )Figure 2.9 Variation in % uptake of arsenate with time of incubationInitial concentrations of arsenate in the media were 2 ppm and• 4 ppm.2.3.3.2 Uptake of arseniteThe percentage of arsenic taken up by the cells as a function of the initialconcentration of arsenite in the medium is given in Figure 2.10. The arsenic uptake pergram of dry cells is also shown. Very high levels of uptake C> 95% ) after 12 days of growthis obtained for arsenite concentrations below 10 ppm. Above this concentration, there is adrastic drop in uptake. The cut-off level is found to be coincident with the MIC for thisarsenic species. This drop in uptake can be related to the decrease in cell density above theMIC.I . I2047The arsenic concentration in cells (jig g dry weight) shows an increase with theinitial arsenic level in the medium, Figure 2.10. The highest values ( 1425 and 1550 jigg4)are seen at initial concentrations of 10 and 12 ppm respectively which are coincident with asharp drop in dry cell weight (See Figure 2.6). It drops to 490 jig g1 at 14 ppm of arsenitein the medium.Analysis of the residual media by using the HGAA method shows that inorganicarsenic left in the medium remain unchanged as arsenite.Figure 2.10 Variation in a % uptake of arsenite and s cellular arseniccontent with the initial concentration in the medium(after 13 days of growth)2.3.3.3 Uptake of methylarsonateThe uptake curve for MMA is depicted in Figure 2.11. The % uptake shows a gradualdecrease as the initial concentration of MMA is increased. This trend closely resembles that10080604020020001600120080040000 3ArseniteI I6 9Concentration12(ppm)1548Figure 2.11604020400030000Variation in % uptake of MMA and . cellular arseniccontent with the initial concentration in the medium(after 12 days of growth)seen in the biomass curve, (Figure 2.7), indicating that uptake of MMA is related to thenumber of living cells in the culture. At the MIC of MMA (8 ppm), the % uptake ofarsenical after 12 days of growth is 67% that of the initial arsenic content. It continues todrop as the initial concentration is increased.However, the arsenic concentration in cells (pg g ) continues to increase as theinitial MMA concentration in the medium is increased. This increase which can be related tothe decrease in the dry cell weight, is more pronounced at concentrations above 8 ppm(MIC). Even though the total arsenic incorporated into cells does not show a drasticincrease, it is high relative to the weight of cells in the culture.10080.—20001000 ‘.50 5 10 15 20MMA Concentration (ppm)492.3.3.4 Uptake of dimethylarsinateThe uptake of DMA was plotted against initial DMA concentration in the medium,(Figure 2.12). The uptake of DMA is found to be much less than for the inorganicarsenicals, arsenite and arsenate, as well as for MMA. At low concentrations of DMA, %uptake is significant, 65% at 2 ppm of DMA in the medium but it drops rapidly as theconcentration of DMA is increased. When the initial DMA concentration is 10 ppm, the% uptake af.er 13 days drops to 25% but optimum cell growth is apparent from the high cellyield. Uptake is closer to 1% of initial arsenic content in the medium at concentrationshigher than 40 ppm.Similarly, the arsenic concentration incorporated into cells (p.g g4 dry cell weight)is much less than for the other arsenicals, Figure 2.12. The highest concentration,454 ug g4, is obtained at 30 ppm of arsenic as DMA in the medium. It remains below300 jig g4 at higher concentrations of DMA in the medium.Organoarsenicals such as MMA and DMA are proposed to pass through biologicalmembranes, mainly by passive diffl.ision.2 Recently, Cullen and Ne1son1 2 estimated thepermeability coefficients of MMA and DMA through a liposome bilayer, which is used as asimple model for diffusion across biological membranes. The permeability of DMA was foundto be 330 times higher (4.5 x 10-11 1) compared to that of MMA (1.4 x 10-13 cm s4)at pH 7.4. The structural difference between the permeants, the substitution of a -CH3group for an -OH group in DMA, accounts for the higher permeability of this arsenical.At pH 5.5 (initial pH of the growth media for C. roseus ), the permeability value forDMA can be expected to be even higher as the extent of ionization of this arsenical(pICa 6.19 )2 is lower at this pH. For MMA ( pKai 4.58), the change would not be aspronounced.50Figure 2.12— .—The variation in % uptake of DMA and icellular arseniccontent with the initial concentration in the medium(after 13 days of growth)The smaller values of uptake of DMA compared to MMA observed in C. roseus cellsystems indicate that forces other than passive diffusion come into play. C. roseus cellsappear to have the ability to exclude DMA at higher concentrations of DMA in the medium.This ability probably stems from either an active process which prevents DMA uptakethrough the cell membrane or an efflux mechanism where DMA is excreted out of cells.Thus, the lower toxicity of DMA attested by the high MIC, can be attributed to the lowuptake of the arsenical by C. roseus cells.1008060402000 20 40 60 80 100DMA Concentration (ppm)512.3.4 Determination of total arsenic by using Neutron Activation AnalysisThe total arsenic in C. roseu.8 cells grown in different growth media containingseveral arsenic compounds was determined by using Neutron Activation Analysis (NAA).(NAA was performed at Queen’s University.) The arsenic content in the cells is expressedas total arsenic ( jig) per dry weight ( g) of cells.2.3.4.1 Total arsenic in cells grown in 1.B5 mediumThe results of NAA analysis of cells grown in 1-B5 medium containing differentarsenic species is listed in Table 2.5. The results show the differences in the efficiency ofincorporation of the four arsenicals from the 1-B5 medium.Cells that grew in both arsenate and arsenite containing media show high levels ofarsenic accumulation. Accumulation increases, as the initial arsenical concentration in themedium increases. But at concentrations above the MIC of each arsenical, a drop in arsenicaccumulation is usually observed. An exception is arsenate. C. roseus cells grown in amedium containing 9.4 ppm arsenate, well above its MIC, exhibit an arsenic level as high as1650 jig g1 of cells. Arsenite is also taken up efficiently as seen in the culture grown in10 ppm of arsenite which exhibits an arsenic level of 1226 jig g’ of cells. Some plants areknown to accumulate very high levels of arsenic when grown in soils or media containinghigh arsenic concentrations. The Douglas fir, Pseudotsuga menziesii is reported toincorporate large amounts of arsenic. Levels as high as 1000- 10000 jig g4 arsenic on a dryweight basis has been detected in trees grown near a mineralization site.42C. roseus cells growing in 1-B5 medium containing methylated arsenicals such asMMA and DMA also show elevated arsenic levels. Cells treated with 10 ppm of MMA andDMA accumulated 697 and 154 pg g’ of cells respectively. These levels, particularly that of52DMA, are much smaller than after inorganic arsenical treatment. Cells, when treated with200 ppm DMA, accumulated 573 igg4arsenic, which amounts to 1% of the initial arsenic inthe culture medium. A similar trend is observed from the uptake curve (See Figure 2.12).These accumulation patterns again indicate that C. roseus cell cultures are capab1e ofshutting off uptake of DMA at higher concentrations.The presence ofhigh arsenic concentrations in living C. roseus cells indicates that thecells are capable of detoxification and/or sequestration of these arsenicals in cellcompartments such that they do not disable cell metabolic processes.The comparison of arsenic content in C. roseus cells obtained by the analysis ofresidual media, Section 2.3.3, to those obtained by using NAA, shows a good correlation inthe cases where cells were treated with arsenate, arsenite and DMA. An exception is seen inthe cells treated with MMA. At low concentrations of MMA, the estimates of cellular arseniccontent obtained by the two methods show agreement. But at higher concentrations,especially above 8 ppm (MIC ), arsenic estimate in the cells is lower by NAA compared to theother method (Figure 2.11). This observation may be explained by some loss of arsenicduring the freeze drying process in the cells treated with high concentrations of MMA. Anyarsenic adsorbed onto the cell surface may be lost during freeze drying. Loss of arsenicspecies from the medium as volatile arsines during the growth may also result in anoverestimation of % uptake.53Table 2.5 Neutron Activation Analysis (NAA) results of total arsenic inC. roseus cells grown in arsenic spiked 1-B5 mediaArsenic Initial Culture Dry Cell NAACompound As Level Age Weight Results( ppm) ( days) (g/culture) (jig g cells)arsenate 2 17 0.769 213arsenate 4 17 0.979 338arsenate 9.4 17 0.157 1650arsenite 2 13 0.857 177arsenite 7 13 0.886 603arsenite 10 13 0.59 1 1226arsenite 14 13 0.118 543MMA 2 12 0.809 190MMA 6 12 0.777 463MMA 10 12 0.50.4 697MMA 15 12 0.197 594DMA 2 12 1.115 122DMA 10 12 0.966 154DMA 20 12 0.997 141DMA 50 12 0.629 250DMA 200 13 0.346 5732.3.42 Total arsenic in cells grown in Alkaloid Production MediumArsenic accumulation in C. roseus cells grown in a different medium, AlkaloidProduction Medium (APM), was investigated and the NAA results are given in Table 2.6.Overall, the arsenic levels (pgg1) in these cells are lower than those observed in cellsgrown in 1-B5 medium. This result may be due to the larger dry weights of cells per cultureusually observed in this medium which has a higher concentration of sucrose (5%)compared to the 1.B5 medium (2% )•77 A high cell density as well as high accumulation of54organic components result in a larger cell weight per culture volume in APM. Thus, theweight of arsenic relative to that of the organic content of the cells is lower compared to thatin 1-B5. In spite of the lower arsenic concentration inside the cells, the toxicity limits of thearsenic compounds expressed as MIC values do not show a large variation in APM.(See Chapter 3.)The uptake of the arsenicals from APM shows a similar trend to that observed in the1-B5 medium. The two inorganic arsenicals show high levels of arsenic accumulation insidethe cells on a dry weight basis and the two organoarsenicals, MMA and DMA, again exhibitlow incorporation, even at high arsenic concentrations.Table 2.6 Neutron Activation Analysis (NAA) results of total arsenic inC. roseus cells grown in arsenic spiked APM mediaArsenic Initial Culture Dry Cell NAACompound As Level Age Weight Results( ppm) ( days) (g/culture) (gig g cells)control - 21 3.012 < 1.5arsenate 2 21 2.913 62arsenate 3 21 1.567 181arsenite 4 22 3.142 119arsenite 6 22 2.985 199MMA 6 25 0.575 66MMA 10 25 0.452 62.2DMA 10 21 1.966 69.5DMA 30 21 1.078 173552.3.5 Quantitation and speciation of arsenic In C. roseus cell extractsCell extracts were analyzed by using GFAA and HGAA. Three or more replicateswere carried out for each sample and the mean value is quoted along with the standarddeviation of the mean. The concentrations are quoted on a dry weight basis unless otherwisenoted.2.3.5.1 Total arsenic in base extractsBetween 50 - 100 mL of extraction medium, a 1 M solution of NaOH, was used foreach 1 g of dry cells. The results of arsenic determination of base extracts of C. roseus cellsgrown in arsenate, arsenite, MMA and DMA containing media by using GFAA and HGAAare given in Tables 2.7, 2.8, 2.9 and 2.10 respectively.GFAA analysis determines the total arsenic concentration in the extract, whereasHGAA determines only the total reducible arsenic species, arsenate, arsenite, MMA andDMA, (See Section 2.3.1.2). If more complex organoarsenic species are present in a sample,GFAA analysis would give a higher result in comparison to that obtained by using HGAA.The results listed in Table 2.7 show that a major portion of the arsenic content of thecells is removed in the first extract and subsequent extracts remove smaller amounts. In theanalysis of the first base extract of all cell samples, GFAA underestimates the arsenicconcentration to a certain extent. This may be a direct consequence of high organic matrix inthe cell extracts, which could lead to some inaccuracy in GFAA measurement. Theprecautions taken include dilution of the extract several hundred fold, use of a chemicalmodifier and the use of standard additions method (See Section 2.3.1.1). Otherwise, GFAAand HGAA estimates are similar, which indicates the absence of a appreciable concentrationof organoarsenic species in the extract. In a few extracts, slightly higher concentration of56arsenic was detected by GFAA, which may indicate the presence of organoarsenic species.These extracts were analyzed further by HGAA after UV decomposition.Table 2.7 Results of total arsenic analysis in base extracts of cells grown in1-B5 medium spiked with arsenateSample Initial Extract Volume GFAA HGAA Total As(Cell As Level of Ex. extractedweight) ( ppm) ( mL) (ppm) (ppm) ( p.g g)(RSD=7%) (RSD=3%) C GFAA)arsenate 2.2 1 55 2.02 2.20 121 ± 7(0.918g) 2 67 0.20 0.15 14.6±0.9dry weight 3 62.5 0.15 0.10 10.2 ± 0.6Total arsenic extracted 146 ± 9arsenate 1.6 1 78 1.15 1.26 89.7 ± 6.3(22.947 g) 2 60 0.52 0.50. 31.2 ± 2.1wet weight 3 60 0.23 0.22 13.8 ± 0.8Total arsenic extracted C ig / 22.95 g fresh weight) 135 ± 9Table 2.8 Results of total arsenic analysis in base extracts of cells grown in1.B5 medium spiked with arseniteSample Initial Extract Volume GFAA HGAA Total As(Dry cell As Level of Ex. extractedweight) ( ppm) ( mL) (ppm) (ppm) ( igg4)(RSD=7%) (RSD=3%) ( GFAA)arsenite 6 1 61 5.00 5.16 382 ± 15(0.798 g) 2 75 0.77 0.79 72.4 ± 3.23 75 0.06 0.06 5.6 ± 0.2Total arsenic extracted 460 ± 18arsenite 9 1 54 11.86 12.0 655 ± 25(0.978 g) 2 65 1.60 1.79 106 ± 4.83 90 0.20 0.20 18.4 ± 0.8Total arsenic extracted 779 ± 3057Table 2.9 Results of total arsenic analysis in base extracts of cells grown inlBS medium spiked with methylarsonateSample Initial Extract Volume GFAA HGAA Total As(Dry cell As Level of Ex. extractedweight) ( ppm) ( mL) (ppm) (ppm) ( tg(RSD=7%) (RSD=3%) C GFAA)MMA 2 1 78 1.78 1.90 170 ± 8.5(0.8 16 g) 2 61 0.267 0.275 20.2 ± 1.53 66 0.007 0.005 0.5 ± 0.3Total arsenic extracted 191 ± 10MMA 6 1 63.5 4.52 4.66 446 ±22(0.643 g) 2 83 0.13 0.13 16.8 ± 0.83 68 0.02 0.02 2.1 ± 0.1Total arsenic extracted 465 ± 23Table 2.10 Results of total arsenic analysis in base extracts of cells grown in1-B5 medium spiked with dimethylarsinateSample Initial Extract Volume GFAA HGAA Total As(Dry cell As Level of Ex. extractedweight) ( ppm) ( mL) (ppm) (ppm) ( p.g g4)(RSD=7%) (RSD=3%) C GFAA)DMA 6 1 70 1.98 1.90 145 ± 6(0.95 1 g) 2 61 0.25 0.21 16.0 ± 0.93 75 0.03 0.02 2.4 ± 0.1Total arsenic extracted 163 ± 7DMA 14 1 68 1.72 1.62 110 ± 5.6(1.059 g) 2 45 0.45 0.35 19.1 ± 1.13 65 0.06 0.06 3.7 ± 0.2Total arsenic extracted 133±7582.3.5.2 Efficiency of extractionThe analysis of arsenic in the cell residues after base extraction gives an estimate ofthe efficiency of extraction with 1 M NaOH as the extractant. The total arsenic in the cellresidues after three consecutive base extractions was analyzed by using HGAA techniquefollowing acid digestion. Some results are presented in Table 2.11.Table 2.11 Results of total arsenic analysis in cell residues after base extractionArsenical Initial Total As Total As % As left in thegrown in As Conc. in cell extracted residueResidue into base(ppm) (rig) (gig)arsenate 2.2 0.5 134 0.4arsenate 1.6 0.7 135 0.5arsenite 6 0.63 367 0.2MMA 2 1.63 156 1.0DMA 6 2.0 156 1.3DMA 14 2.25 141 1.6These results indicate that base extraction is capable of extracting out 98% or moreof the total arsenic from plant cells grown in the presence of all four arsenicals.592.3.5.3 UV decomposition I HGAAArsenicals that are not reduced to volatile arsines are not detected by HGAA. Thesearsenicals include many naturally occurring organoarsenicals such as arsenobetaine,arsenocholine and arsenosugara. Various digestion methods have been employed to convertorganoarsenic species into simpler inorganic arsenic species. Both wet digestion of samplewith various combinations of acids79’82 and dry ashing with magnesium nitrate113 havebeen used with success on dry samples. However, these methods are difficult to applydirectly if the sample is already in solution, as is the case with cell extracts.A photooxidation method developed by Cullen and Dodd78 has been shown to besuccessful in breaking down organoarsenic compounds in solution, mainly to arsenate. Thismethod was utilized in the analysis of some methanol and base extracts of C. roseus cellsgrown in the presence of different arsenicals.The essential feature of the UV - HGAA technique is the irradiation of the sample insolution for a specified time (usually between 30 - 120 mm ) with a 1200 W medium pressurelamp (See Section 2.2.4.6). All arsenic compounds present in solution are converted toarsenate allowing the subsequent quantitation of arsenic by GFAA or HGAA techniques.The results of the UV - HGAA analysis of several cell extracts (both methanol andbase) given in Table 2.12 show that UV irradiation in open quartz tubes results in loss ofarsenic. The longer the irradiation time, the larger is the loss. Irradiation of aqueoussolutions of several standard arsenic compounds in open quartz tubes also results in a loss,usually about 10%.This observation led to the use of modified quartz containers which are sealed priorto irradiation. A good recovery for the four standard arsenic compounds is obtained with thisprocedure, as shown in Table 2.12. The inorganic arsenic compounds arsenate and arsenite60as well as MMA show close to 100% recovery whereas DMA yields a > 100% signal. Thisenhanced signal for DMA on irradiation is consistent with the breakdown of DMA toarsenate, which shows greater sensitivity for HGAA. (AsH3gives a greater absorbance thanMe2AsH, Section 2.3.1.2. )Base extracts of C. roseus cells grown in the presence of different arsenicals wereanalyzed using the sealed tube (IV irradiation followed by HGAA and the results are given inTable 2.13. The extracts do not display a significant increase in HGAA signal on irradiationfor 120 mm.A 6% increase in the signal of extract of DMA grown cells can be explained by theconversion of DMA, the major intracellular arsenic species to arsenate. However, thepresence of low levels of more complex organoarsenic species cannot be ruled out.Table 2.12 Percentage recovery obtained for some standard compounds and ceflextracts after UV irradiation in open and sealed quartz tubesSample Arsenic Average % Recovery after UV irradiation for 45 mmConcentration(ppm) Open Tube Sealed Tubearsenate 0.025 88 98arsenite 0.025 86 98MMA 0.025 87 99DMA 0.025 88 105Base Extract 0.02 92 105arsenate(2 ppm)(Dilution x50)MeOH Extract 0.02 80 104arsenate(3 ppm)61Table 2.13 Results of analysis of cell extracts by UV decomposition / HGAA insealed tubesSample Initial Extract Volume As Averagearsenic Conc. % recoveryconc. (HGAA) after UV(ppm) (mL) (ppm)arsenate 2.2 base 1 55 2.20 103arsenite 6 base 1 61 5.16 99MMA 6 basel 63 4.66 102DMA 6 base 1 70 1.90 1062.3.5.4 Speciation of arsenic in cell extractsSpeciation of arsenic present in cell extracts was carried out by using the HG-GC-AAtechnique (See Section 2.3.1.3). Arsenicals that are converted to volatile arsines such asarsenate, arsenite, MMA and DMA as well as trimethylarsine oxide (TMAO) can bequantitated by this technique. The results of analysis of both methanol and base extracts ofcells grown in the presence of arsenate are given in Table 2.14.In the first two base extracts of a freeze dried cell sample, arsenate was the onlyarsenical present. Neither arsenite nor methylarsenicals are present to a detectableconcentration. In the third extract, dimethylarsenic species is detected.In the analysis of base extracts of a non-freeze dried sample of cells grown inarsenate, both monomethyl and dimethyl arsenic species are detected, even though at lowconcentrations (Table 2.14). Methylated products account for 0.4% of the total arsenicextracted.62There is one previous report of methylation of arsenate by terrestrial plants such aspine seedlings and corn.51 Methylation of arsenic was detected only when the plants weresubjected to nitrate and/or phosphate deficient conditions prior to exposure to74As[arsenatel. Under optimum conditions of growth, no methylation has been observed.51However, C. roseus cell suspension cultures growing under normal conditions do appear tomethylate arsenate, although to a small extent.The analysis of methanol extracts of cells grown in arsenate shows the presence ofboth methyl and dimethyl arsenic species in all extracts but only in trace amounts.Significant levels of arsenite, 56% of the total arsenic extracted, are present in contrast tothe absence of arsenite in the base extracts C See Table 2.14).Apparently the extraction of cells with base ( 1 M NaOH) results in the conversion ofintracellular arsenite to arsenate. In 1 M NaOH, the redox potential of arsenate to arseniteconversion is -0.08 eV.4 Thus, oxidation of any arsenite present should be feasible. Acontrol study was carried out involving the base extraction of C. roseus cells that had reachedstationary phase. The cells were spiked with arsenite (200 .tg arsenic per 1 g (dry) cells)and then extracted with 60 mL of 1 M NaOH. The cell residue was extracted again withbase after spiking with a similar dose of arsenite. Approximately 80% arsenic present inboth the extracts is arsenate. However, only 10% of arsenite is oxidized in a standardsolution of arsenite in 1 M NaOH medium. These results indicate that some cell componentsextracted into 1 M NaOH causes the oxidation of arsenite to arsenate.63Table 2.14 Results of BG-GC-AA analysis of extracts of cells grown in arsenateSample Extract Extract Arsenic Content ( .tg)(Cell Volume As(iii) As(v) Methyl Dimethylweight) (mL) As AsArsenate base 1 55 - 93.7 - -2.2 ppm base 2 67 - 14.5 - -(0.918 g) base 3 63 - 3.8 0.11Total Arsenic Extracted 112.3 (99.9%)- 0.11 (0.1%)Total Arsenic in cell sample C extract + residue ) = 112.8 igArsenate base 1 78 - 68.6 0.13 0.031.6 ppm base 2 60 8.6 0.07 0.05(Wet wt. base 3 60 - 1.8 - 0.0322.95 g)Total Arsenic Extracted - 79.0(99.6%) 0.20 (0.3%) 0.11 (0.1%)Total Arsenic in cell sample ( extract + residue ) = 80.0 pgArsenate MeOH 1 50 6.0 14.3 d d(3 ppm) MeOH 2 50 9.2 4.0 d d(0.947 g) MeOH 3 50 16.9 6.4 d dTotal Arsenic Extracted 32.1 (56%) 24.7 (44%)Total Arsenic in cell sample ( extract + residue ) = 97.0 pg64The results of HG-GC-AA analysis of base extracts of cells grown in MMA and DMAare given in Table 2.16. In the extracts of cells grown in the presence of MMA, a majorfraction (95%) of the incorporated arsenic is present as a monomethyl arsenic species. Thisis most probably unchanged MMA. Even if MMA is associated with another cell componentinside the cell, this association is cleaved during the base extraction process.Diniethyl arsenic species are present in both extracts 1 and 2; 3.6% of the totalarsenic extracted is methylat.ed. Demethylation of MM.A is also detected; inorganic arsenic ispresent in all three base extracts. Deniethylation is found in 0.7% of the total arsenicextracted. A control study involving the incubation of a standard MMA solution for a lengthof time without any added C. roseus cells did not show any conversion.C. roseus cell grown in the presence of DMA are also capable of methylation as wellas deniethylation of the arsenical as can be seen from the results given in Table 2.16.Demethylation takes place to a greater extent. Methylarsenic species ( 10.5%) are detectedin all three extracts. Low levels of inorganic arsenic species (0.3%) formed by cleavage oftwo methyl groups are also detected. The detection of a peak with a retention timecorresponding to trimethylarsine indicates that further methylation of DMA may take placein C. roseus cell cultures. Quantitation of this peak was not possible because of the lowconcentration.In a previous study on hydroponically grown rice seedlings, a dimethyl arsenicspecies as well as inorganic arsenate has been detected in the nutrient solution into whichplants have been transferred after exposure to MMA.72 This phenomenon was proposed tobe root exudation of these transformed species.65In the present study, products of both methylation and demethylation of MMA aswell as DMA is observed in plant cell extracts. Thus the formation as well as the cleavage ofthe As-C bond of MMA and DMA in C. roseus cell cultures is unequivocally established.Table 2.16 Results of HG-GC-AA analysis of base extracts of cells grown in MMAand DMASample Extract Extract Arsenic Content ( u.g)(Dry Volume As(v) Methyl Dimethyl Trimethylweight) ( mL) & As(iii) As As AsMMA 1 78 0.2 106.0 4.42 ppm 2 61 0.6 13.4 0.2(0.816g) 3 66 0.1 0.8 -Total Arsenic Extracted 0.9 (0.7%)120.2 (95.5%) 4.6 (3.6%)Total Arsenic in cell sample (extract + residue ) = 126.7 jigDMA 1 70 - 5.3 45.14 ppm 2 61 0.1 0.9 7.3 d(0.895 g) 3 75 0.1 0.1 0.8 dTotal Arsenic Extracted 0.2 (0.3%) 6.3 (10.6%)53.2 (89.1%) dTotal Arsenic in cell sample (extract + residue ) = 60.9 jig662.3.6 SummaryThe biotransformation of elements in biological systems is an important link in theenvironmental cycling of elements. Although biotransformation of arsenicals to a variety ofcomplex arsenosugars inside marine plants is well documented, the role of terrestrial plantshas not been thoroughly investigated. In the present study involving a model system of aterrestrial plant, the variable effect of different arsenic species on cell suspension cultures ofC. roseus is examined.The minimum inhibitory concentration of arsenate is low at 5 pg mL1 and thus isthe most toxic species to cell suspension cultures of C. roseus. Arsenite and MMA have alower toxic effect. DMA is the least toxic of the arsenic species studied, where optimalgrowth is observed up to 50 pg mL1 of DMA in the medium. The uptake shows a similartrend to toxicity, with arsenate uptake the highest. The low uptake of DMA probablyaccounts for its low toxicity.The speciation of arsenic in cell extracts by using HGAA and HG-GC-AA methodsshows biotransformation of arsenicals by C. roseus cell suspension cultures. Somemethylation is observed when cells are treated with arsenate, MMA and DMA.Demethylation of the arsenicals MMA and DMA is also observed in C. roseus cell suspensioncultures.The formation of more complex organoarsenic species by C. roseus cell suspensioncultures is not apparent from UV decomposition / HGAA analysis of cell extracts, Section2.3.5.3. Decomposition of organoarsenic species to arsenate during UV irradiation wouldresult in an increase in the arsenic levels detected by HGAA. Only a small increase in theabsorbance is demonstrated in C. roseus cell extracts which may also be indicative of thepresence of dhnethylarsenic species. The cell suspension cultures of C. roseus, seem to lackthe ability to convert simple arsenicals to more complex organoarsenicals even though this iscommonplace in marine plants. 67CHAPTER 3EFFECT OF ARSENIC COMPOUNDS ON ALKALOU) PRODUCTION BY CELLSUSPENSION CULTURES OF CATHARANTHUS ROSE US3.1 INTRODUCTIONCatharanthzts roseus (L.) G. Don, Madagascan periwinkle, is a well knownmedicinal plant belonging to the plant family Apocynaceae. It is a fast growing subshrubgrowing up to 30 - 120 cm and bears rose or white flowers.75 To date more than 80 indolealkaloids have been isolated from different parts of the plant. Extensive research has beendone on the isolation and conversion of these alkaloids, many of which have importantpharmacological activity.75’116 The most notable of these therapeutic alkaloids are thebisindole alkaloids, vinblastine (1) and vincristine (2), shown in Figure 3.1, which are used incancer chemotherapy.117World wide interest for the last decade has been focussed on the production ofsecondary metabolites of C. roseus by cell culture methods. It is widely recognized thatcultured plant cells represent a potential source of valuable phytochemicals.118 Culturemethods become more important as the supply of plants is limited by geographical location.Moreover, the yield of natural products is low and affected by seasonal variation. A yearround production of alkaloids under controlled environmental conditions and themanipulation of the culture conditions to increase the yields are possible advantages of cellculture methods.Only 43 monomeric indole alkaloids have been isolated from C. roseus cell cultures todate although some cell lines were found to produce alkaloids at higher levels than found inintact plants.118 These monomeric alkaloids include catharanthine and vindoline which are68of interest as biosynthetic precursors of the valuable bisindole alkaloids. But the targetcompounds, vinbiastine and vincristine have so far not been isolated from cell suspensioncultures. They have been detected only in callus and organ cultures of C. roseus.119However, C. roseus cell suspension cultures and their cell free extracts have beenutilized to couple catharanthine and vindoline to form 3’,4’-anhydrovinblastine, anintermediate in the biosynthesis of vinbiastine type alkaloids.° On incubation of 3’,4’ -anhydrovinbiastine with growing cell8 and enzyme extracts of C. roseus, both vinbiastine andvincristine have been detected.121’2 Evidence for the formation of bisindole alkaloids incell free extracts suggest that cell suspension cultures possess the enzymes needed for theseconversions but that probably one or more of the earlier steps of the biosynthesis are blockedin the cultured, undifferentiated cells.1 R=CH32 R=CHOFigure 3.1 Bisindole alkaloidsHCO2H3693.1.1 Biosynthesis of monoterpenoid indole RikaloldsAll ionoterpenoid indole alkaloids consist of two structural elements: tryptaminewith an indole nucleus, and a C9 or ClO monoterpene unit. C. roseus plant systems produceindole alkaloids belonging to the three major classes, corynanthe - strychnos, aspidospermaand iboga, possessing three different skeletal forms in the monoterpenoid unit.123 Alkaloidsthat illustrate each of the three classes are ajmalicine ( 3 ) [corynanthe],akuammicine ( 4 ) [strychnos), vindoline (5) [aspidosperma] and catharanthine ( 6) [iboga].These alkaloids and the skeletal type of each class are illustrated in Figure 3.2.Biosynthesis of indole alkaloids has been under intense investigation during the lasttwo decades utilizing whole plants and seedlings as well as tissue and cell cultures ofC. roseus. Until 1975, the pathway of indole alkaloid formation was investigated in viva byusing radio tracer rnethods.24 Lately, the availability of cell cultures and cell free systemscoupled with new analytical methods have provided an alternative approach. As a result, aconsiderable part of the biosynthetic pathway and the enzymes involved in it are now known.The biosynthetic pathway of indole alkaloids is given in Figure 3•3123 The first stepin the indole alkaloid biosynthesis is known to be the enzymatic, stereospecific condensationof tryptamine (7 ) with the tnonoterpene unit secologanin (8).Tryptamine is derived from the amino acid tryptophan whose decarboxylation iscatalyzed by the enzyme tryptophan decarboxylase. The monoterpenoid origin of secologaninwas established by incorporation studies of mevalonate which is the basic unit ofrnonoterpenes. Various incorporation studies showed that two mevalonate units,transformed after a series of steps, combine to give geraniol which gives rise to loganinbefore being converted to secologanin.470Ajmalicine (3)(Corynanthe)Vindoline (5)(Aspidosperma)Akuammicine (4)(Strychnos)Catharanthine (6)(Iboga)cH3Figure 32 Major classes of indole alkaloids and their representative alkaloidsH3C•0cH3•cH371Tryptamine (7) I Secologanin (8)CH3O2Strictosidine (9)Aspidospermae.g. VindolinineVindolineFigure 33 The biosynthetic pathway of indole alknloidsCorynanthee.g. AjmalicineYohimbineIbogae.g. CatharanthineHCH3O2 •••Aglycone/VStrychnose.g. Akuammicine72Condensation of tryptamine and secologanin gives rise to the glucoalkaloidstrictosidine C 9); the reaction is catalyzed by strictosidine synthase.125 The biologicalconversion of strictosidine into the three major classes of indole alkaloids has been observed.The first step in this sequence is the hydrolysis of strictosidine to remove the sugar moiety.Then in several steps which are not entirely clear, corynanthe type alkaloids, ajmalicine andtetrahydroalstonine are formed.126The sequential formation of alkaloids has been observed in germinating C. roseusseedlings.127 The onset of alkaloid production was detected between 25 - 40 hours and allalkaloids present were of corynanthe type. An aspidosperma type alkaloid, tabersonineappeared after 50 hours. Catharanthine, an iboga alkaloid was detected only after100- 160 hours of germination time. The aspidosperma alkaloid vindoline was accumulatedto detectable levels only after 200 hours of germination time. These observations followed byvarious labelling experiments124 led to the hypothesis that the alkaloids of the corynanthetype are converted into those of the aspidosperma type, which are themselves converted intoalkaloids of the iboga type.3.1.2 Factors affecting nlknloid production by C. roseus cell suspension culturesThe production of alkaloids by C. roseus cell suspension cultures is governed by thecharacteristics of the cell line as well as the environmental conditions of the culture. Highyielding cell lines are established by screening cultures derived from various explantsincluding seeds, anther and leaf: Even the high yielding cell lines show variation in theirbiosynthetic capacity over time with repeated subculturing.104 The development offavorable culture conditions including the medium composition and other environmentalconditions is critical.733.1.2.1 Medium compositionThe first investigations of the influence of medium composition on indole alkaloidformation in C. roseus cell suspension cultures was performed by Zenk et al.’28 and Carewand Krueger129 in 1977. Zenk and co-workers made use of a two phase culture systemwhere cells were grown in a growth medium rich in nutrients and transferred to an alkaloidproduction medium (APM ) of different nutritional composition.128 The alkaloid productionmedia developed by other groups bear considerable resemblance to that of Zenk et al. 129,130The carbon source in the medium plays a crucial role in both the growth and thealkaloid production.131 Though a variety of substrates can be used, sucrose and glucose arefound to give optimum utilization. In a sucrose rich medium, the growth as well as alkaloidaccumulation is greatly stimulated.131 Zenk et al. used 5% (w/v) of sucrose in APM asopposed to 2% (w/v) or less in other growth media.128Phosphate has been reported to inhibit alkaloid accumulation in C. roseus, eventhough it enhances growth.132 Alkaloid production occurs only when intracellularphosphate has been exhausted below inhibitory levels. Zenk et al.’28 drastically reduced theconcentration of phosphate in Alkaloid Production Medium as compared to growth media.Plant growth regulators also play a role in the induction and inhibition of secondarymetabolism. For example 2,4-dichlorophenoxyacetic acid ( 2,4-D) and naphthylaceticacid ( NAA) greatly suppress alkaloid formation.133 Zenk et al.128 report thatbenzyladenine and indole-3-acetic acid (I.AA) are promoters affording relatively high levelsof alkaloids.There are other components in the medium such as vitamins and trace elementswhich could play an important role in alkaloid production.743.1.2.2 Other environmental factorsIncreased production of the indole alkaloids serpentine34 and catharanthine135was reported in heterotrophic C. roseus cell suspension cultures when exposed to light.However, photosynthetically active cell cultures of C. roseus were found to accumulate verylow levels of indole alkaloids. Transferring these photoautotrophic cells to a sucrose richmedium results in fast accumulation of alkaloids. 136,137The effect of temperature on alkaloid production has been investigated. Even thoughoptimum growth was observed between 27 -35 °C, alkaloid production is inhibited attemperatures above 27 O(• 138 Temperatures below 20 °C were reported to stimulate138 aswell as inhibit139 alkaloid accumulation in cell suspension cultures of C. roseus. Thecontradictory results are attributed to the different experimental conditions includingmedium composition and cell line.3.1.3 Elicitation of nlknloid productionChanges in the pattern of alkaloid accumulation in cell suspension culturescompared to the intact plant suggest that some metabolic pathways are blocked in theculture mode. Attempts to “switch” these pathways back on by altering nutritional levels inthe medium met with some success ( See Section 3.1.2.1). Another method presently used toenhance product accumulation in cultured cells is through the use of elicitors. The aim ofusing elicitors on C. roseus cell suspension cultures is not only to induce the production ofvalu.able bisindole alkaloids, which to date have not been detected in cell cultures, but also toincrease the production of monomeric alkaloids including catharanthine and vindoline in ashorter period of time.75Application of various stresses to the cell culture system was reported to stimulatesecondary metabolite production in a variety of plant cultures including C. roseus.’4° Theaddition of microbiafly derived elicitors (biotic) and chemically defined (abiotic) elicitorswhich stimulate secondary metabolism are approaches now being considered for thecommercial production ofuseful phytochemicals from plant cell cultures.3.1.3.1 Fungal elicitorsSuccessful use of fungal homogenates on C. roseus cell cultures was reported byEilert et al.’41 Several cell lines responded to the addition of a fungal elicitor by theaccumulation of tryptamine within 24 h, and catharanthine, ajmalicine and other monomericalkaloids within 72 h. Vindoline or dimeric alkaloids were not detected. The type of fungalhomogenate and the concentration, and age of the cell culture at the time of application ofelicitor all influence the response from each cell line. 1413.1.3.2 Abseisic acidAbscisic acid, another elicitor investigated, is a natural plant growth regulatorysubstance.’42 Even though abscisic acid is most often associated with inhibition of growthand biosynthetic processes, it had no significant effect on growth but was a potent stimulatorof intracellular alkaloid accumulation in C. roseus cell suspension cultures. The responsedepended upon the cell line, the concentration of abscisic acid and the growth phase at whichcells were treated.763.1.3.3 Inorganic saltsPlants are known to produce abscisic acid as a response to a variety of stressesincluding chilling and water stress. The stimulatory effect of abscisic acid led to aninvestigation of the effect of osmotic or salt stress on alkaloid accumulation by cellsuspensions of C. roseus. Cultures were subjected to osmotic stress by treating them withthe inorganic salts, sodium chloride and potassium chloride on day 5 of the growth cycle. 143Increased accumulation of alkaloids was observed depending on the concentration of saltsadded. At higher concentrations of sodium chloride (>0.5 g/ 60 mL culture), intracellularcatharanthine yield was reduced but significant levels of the alkaloids were detected in thespent culture medium, suggesting that some cell lysis may have occurred.3.1.3.4 Vanadyl sulphateIncreased accumulation of indole alkaloids in C. roseus cell suspension cultures wasreported on treatment with another abiotic elicitor, vanadyl sulphate.1°7’44 Bothajmalicine and catharanthine levels showed a 50% increase over control levels on treatmentwith 25 ppm of vanadyl sulphate, but concentrations over 100 ppm resulted in a drop inalkaloid levels. Cell response to vanadyl sulphate was also found to vary with the cell age attime of application. Treatment early on in the growth cycle could even inhibit alkaloidproduction whereas treatment during early stationary phase resulted in elevated levels ofalkaloids.3.1.4 Analytical methodsSeparation and characterization of indole alkaloids in plant extracts can be animposing problem due to the large number and different amounts of components normally77present in an extract. The ideal analytical technique would not only be sensitive and highlyspecific for he metabolites under study but permit fast analysis of a large number ofsamples. Radioimmunoassay is a highly specific technique used for screening trace levels ofalkaloids in crude cell extracts.’28”45’146 Several chromatographic methods are inwidespread use for the analysis of Catharanthus alkaloids and are discussed below.8.1.4.1 Thin Layer ChromatographyThin Layer Chromatography (TLC) has been widely used in the qualitative andquantitative determination of alkaloids in C. roseus plants and cultures. Bothunidimensional and two dimensional TLC was used as a criterion of purity for identifIcationas well as monitoring column fractional separation. 147Detection was achieved by the use of spray reagents including Dragendorif reagentand ferric aminonium sulphate reagent.148 But the most widely acclaimed spray reagent forCatharanthus alkaloids is the ceric ammonium sulphate (CAS) reagent first introduced byCone et al.147 A comprehensive evaluation of the use of CAS spray reagent for theidentification of 63 Catharanthus alkaloids was published in 1963.149 Alkaloids wereclassified into 8 major classes on the basis of the first major color produced by the CASreagent. Assignment to subclasses is accomplished by characteristic changes to the originalcolor with time. After an alkaloid had been assigned to a class and a subclass, identificationwas made by the Rf value in three different solvent systems.Several alkaloids were not detected with the CAS spray reagent.149 Among them,perivine could be detected under UV light and serpentine has a native yellow color prior tospraying CAS reagent. A two dimensional TLC technique together with the CAS reagent toseparate a mixture of dimeric alkaloids was also reported.15078The color reaction with the CAS reagent is influenced by the concentration ofalkaloid and the presence of residual solvent. Excess spraying of the reagent may also alterthe color developed. TLC is also relatively insensitive and slow. In spite of these limitations,TLC continue to be used for quick screening of mixtures of alkaloids.3.1.4.2 High Performance Liquid ChromatographyHigh Performance Liquid Chromatography (HPLC) on a reversed phase column iscommonly used for alkaloid analysis.5 Prepuriflcation of plant and cell extracts is usuallyrequired prior to HPLC. Classical procedures involve the extraction of alkaloids into anorganic solvent followed by several partitionings between different solvents in order toeliminate co-occurring plant znetabolites.152 Purification can also be achieved byfractionation of crude cell extracts on cation exchange cartridges153 and silica cartridges.154Separation on C18 reversed phase columns was achieved using both gradient155 andisocratic156 elution with either UV or fluorometric detection. UV detection is widely used atwavelengths 254 nm and 280 nm. It is a rapid technique to screen a complex mixture andpeak identification is based on co-chromatography with known standards. However,co-elution of different alkaloids and the need to evaluate the purity of separated peaks arelimitations.Use of dual wavelength detector156 or two UV detectors in series157 operating attwo different wavelengths was reported to eliminate some of these limitations. A ratio plotof absorbance at 280 nm against 254 nm is plotted alongside the chromatogram which helpsto identify the peaks containing co-eluting alkaloids.156 Rapidly scanned UV spectraprovided by either a multiple wavelength UV-visible detector or a photodiode array UV79detector facilitate the identification of unknown peaks. Fluorometric detection has also beenused successfully for HPLC separation of Catharanthus alkaloids. 1583.1.4.3 Gas Chromatography - Mass SpectrometryGas Chromatography - Mass Spectrometry (GO-MS) methods for the simultaneousdetection of several Catharanthus alkaloids have been reported.159’6°The relatively lowvolatility of catharanthus alkaloids restricts the use of GO in their analysis. Analysis ofnon-volatile analytes can be achieved by prederivatization into a volatile form. Use of thinstationary phase film in the column to facilitate rapid movement and the use of helium gasas carrier gas proved to be beneficial. 159Fractions collected from HPLC were analyzed by GO-MS and showed co-elution ofseveral components in HPLC.159 Ajmalicine and vindoline could be detected by GO-MS butcatharanthine had to be derivatized into a more volatile form. GO-MS was also used toscreen minute amounts of vindoline in cell extracts from C. roseus cell suspensions. 1603.1.4.4 Supercritical Fluid Chromatography - Mass SpectrometrySupercritical Fluid Chromatography (SFC) recently emerged as an efficienttechnique for the separation of various polar compounds. It is also well-suited to interfacingwith a mass spectrometer. Successful application of SFC-MS for separation of a mixture ofcatharanthus alkaloids was reported recently.161Near baseline separation of a complex mixture of alkaloids was achieved in arelatively short time. Both electron impact ionization and thermospray filament-onionization was u.sed.16 El spectra provided significant structural information and served toidentif’ even minor components in the mixture. Thermospray spectra with minimal80fragmentation enhanced the sensitivity of detection and indicated the presence of more thanone component in a peak. About 60 monoterpene indole alkaloids were detected in a leafextract of C. roaeus using SFC-MS.1613.1.4.5 Thermospray Liquid Chromatography-Mass spectrometryOn-line coupling of HPLC separation to a mass spectrometric detector provides aversatile analytical tool. Collection of compounds as they elute from HPLC and analysis bymass spectrometry was all that was possible before the advent of LC-MS techniques. Thedifficulty of efficient trapping particularly minor components and minute amounts ofsamples analyzed on HPLC columns made this a time-consuming and tedious method.The combination of LC with MS presented a major challenge. LC employs solutionscontaining non-volatile solutes and buffers at atmospheric pressure or above at flow rates of1 mL min1 whereas a mass spectrometer requires ions in the gas phase at low backgroundpressures (10-6 torr )•162 Any LC-MS interface must accomplish nebulization andvaporization of the liquid, ionization of the sample, removal of the excess solvent vapor andextraction of the ions into the mass analyzer. Several LC-MS interfacing techniquesincluding Direct Liquid Injection, Electrospray and Thermospray have been developedduring the last fifteen years which overcome the basic incompatibility between LC andMS.62In the thermospray interface, controlled partial vaporization of the LC effluent takesplace before it enters the ion source of the mass spectrometer. This is accomplished byapplication of heat to the capillary tube connecting LC and MS.163 Liquid flow from the LCcontaining a volatile electrolyte such as ammonium acetate, is partially vaporized and81nebulized in the directly heated capillary (vaporizer) to produce a jet of vapor containing amist of fine droplets and particles. 162,163The jet of vapor and droplets is carried at a high speed into the heated ion source,and continues to vaporize due to the rapid heat input from surrounding hot vapor. Anycharged ions in the solution form charged droplets and undergo ion evaporation to producegas phase molecular ions. These ions, usually in MX+ form where X = H, NH4 are sampledinto the mass spectrometer and the excess vapor is pumped away by a vacuum pump. 162Thermospray is a soft ionization technique applicable to a broad range of flow ratesand LC conditions. The mass spectra are composed primarily of molecular adduct ions withminimal fragmentation.For optimum performance of thermospray LC-MS, operating temperatures arecritical especially that of the vaporizer. For the best sensitivity, sufficient heat must beapplied to completely vaporize the sample. At the same time, the sample must not be heatedso much as to cause pyrolysis or other changes in the sample. The vaporizer tip (probe), aswell as the ion source temperatures have to be carefully controlled.There are several limitations in thermospray LC-MS analysis.162 It usually allowsunambiguous determination of molecular weight but fragmentation is either absent orinsufficient to allow any structure elucidation of unknown compounds. Sensitivity iscompound dependent and for some compounds, thermospray ionization does not occur. Highorganic solvent levels in the mobile phase can also suppress ionization. Some of theseshortcomings can be circumvented by the use of an auxiliary filament ionization mode wherean electron beam is placed in the thermospray jet. This ionization mode can be used withorganic solvents and gives rise to more fragmentation, thus permitting structural studies.82Thermospray LC-MS is an attractive technique for analysis of indole alkaloids as itfacilitates the analysis of complex mixtures of non-volatile, structurally similar polarcompounds with minimal sample manipulation. The analysis of Catharanthus alkaloids byusing thermospray 1.0-MS was previously reported by Auriola et al. in 1989.164 Reversedphase HPLC separation was achieved with isocratic elution with a mixture of0.1 M ammonium acetate buffer and acetonitrile as the mobile phase. Ammonium acetateplayed a dual role here. Ammonium ions decrease polar interactions by masking free silanolgroups on the bonded stationary phase as well as act as the electrolyte in the thermosprayionization. The detection of protonated molecular ions of several indole alkaloids wasreported.1643.1.5 Scope of workSome arsenicals are used as selective herbicides (e.g. methylarsonate againstJohnson grass) whereas others like arsenate, arsenite and dimethylarsinate have a broaderherbicidal action.41 This variation may arise from a variety of factors including thebiotransformation pathways of arsenic in vivo, differences in uptake and differences in thesusceptibility of different cell types to the effects of arsenic.Arsenic compounds are well documented to inhibit several enzymes in biologicalsystems.4’62 The inhibition of enzymes by arsenicals is species dependent as charge andsteric effects vary. Studies on a variety of isolated enzyme systems suggest that the trivalentarsenicals inhibit an enzyme by interacting with the sulfhydryl groups of the enzyme.62,’5Other mechanisms of inhibition have also been proposed which may not involve directreaction between arsenic and the enzyme. Arsenicals could react with the substrate or anintermediate of the reaction or structurally similar organoarsenicals may competitively83inhibit binding of the enzyme to the substrate.4 Pentavalent arsenate may directly inhibitenzymes as it can substitute for phosphate in enzyme catalyzed reactions such asphosphorylation.166 Alternatively, arsenate may be reduced to the trivalent form in thebiological system and disrupt enzyme activity.4There are reports of arsenicals enhancing enzyme activity, especially at lowconcentrations. This effect has been observed for some other heavy metals as wells Catalaseactivity was stimulated by 5.5 mM dimethylarsinate and also by arsenite.62 Dihydrofolatereductase was stimulated 17% by 10 mM arsenite.’67 The mechanisms involved have notbeen clarified. Some possible mechanisms include the arsenical inactivating a natura’inhibitor and/or increasing the active form of the enzyme.62The response of soybean seedlings to arsenite exposure was identical to heat shock,when a new set of proteins known as heat shock proteins were produced.63 Arsenite wasalso reported to mimic heat shock in other biological systems including Drosophila andmammalian cells.168’9 This specific action of an arsenical on protein synthesis suggeststhe possibility that arsenicals may also exert an effect on enzymes involved in secondarymetabolism in plants.The influence of arsenicals on a complex metabolic pathway such as alkaloidbiosynthesis which involves a series of enzymes cannot be predicted. The presentinvestigation involves a study of any elicitation or inhibition effects of arsenicals on alkaloidproduction by C. roseus cell suspension cultures. Changes in the alkaloid composition ontreatment with arsenicals may provide information about where and how arsenic interactswith the enzymes involved in the secondary metabolism.843.2 EXPERIMENTAL8.2.1 Instrumentation and analytical methods3.2.1.1 NMR and Mass Spectrometry1H NMR spectra were run at 300 MHz and 400 MHz by using Varian XL 300 andBruker WH 400 spectrometers. Chemical shifts are quoted relative to tetramethylsilane asexternal standard.Low resolution electron ionization mass spectra were recorded on Kratos MS 50 andMS 80 mass spectrometers. High resolution mass spectra were n.m on a Kratos MS 50instrument. Chemical ionization mass spectra were recorded on a Delsi-Nermag RiO- bCquadrupole mass spectrometer by using NH3 as the carrier gas.32.12 Thermospray Liquid Chromatography-Mass Spectrometry (LC-MS)Chromatographic conditions were as described for HPLC. A Waters M510 pump wasused for solvent delivery at 0.9 mL mm4 and the samples were injected with the aid of aRheodyne Model 7125 injector (loop volume 20 jiL). An ammonium acetate (1 M) solutionwas added to the solvent stream after the column by using a Waters 6000A pump at a flowrate of 0.1 mL mm4as depicted in the schematic diagram in Figure 3.4.The thermospray system used was a Vestec Kratos Thermospray interfaced to aKratos MS 80 RFA double focusing mass spectrometer. The thermospray probe temperaturewas 120 DC and the ion source temperature was 220 °C. Dilute solutions ofpoly(ethylene)glycol polymers which afford MNH4ions, were used for calibration.85mobilephase0.9 mL/minammoniumacetate (1M)0.1 mL/minFigure 3.4 Schematic diagram of the Thermospray LiquidChromatography-Mass Spectrometry (LC-MS ) assembly3.2.1.3 High Performance Liquid Chromatography (HPLC)The HPLC system consisted of Waters M45 and M510 pumps coupled to a WatersAutomated Gradient Controller. The sample was introduced via a Waters U6K injector. AWaters M418 variable wavelength UV detector and associated Waters QA-1 Data Systemwas used for detection. When necessary, fractions were collected by using a GilsonMicrofractionator.Two reversed phase columns were used in alkaloid separation. In early studies, aWaters p.-Bondapak C18 (3.9 mm (i.d.) x 30 cm) steel column was used. Isocratic elutionwith water- acetonitrile (60:40) containing 0.1% (v/v) triethylamine as modifier at a flowrate 1 mL mm4was typically used.U’’ chromatogram86Later use of a Phenomenex Bondclone (3.9 mm (i.d.) x 30 cm) steel column requiredmodification to the mobile phase. Water - acetonitrile (54:46) containing 0.15% (v/v)triethylamine was used at a flow rate of 1 niL mm4. Detection was typically at 280 nm;254 mu was occasionally used where noted.3.2.14 Thin Layer Chromatography ( ThC)Analytical TLC was performed by using precoated Merck silica gel 60 P254(0.25 mm ) TLC plates. Ceric ammonium sulfate C 1% w/v) in concentrated phosphoric acidsolution was used as the spray reagent for visualizing the alkaloid spots.Preparative Layer Chromatography was performed on pre-coated Merck silicagel 60 P254 (2 mm thickness) plates developed with EtOAc : CHCI3CH3O (10:7:4) orCH2I : EtOH ( 10:1 ) solvent systems. Visualization was achieved by using a UV lamp andby spraying a narrow strip at the edge with ceric ammonium sulfate solution. The alkaloidcontaining bands were scraped off and the adsorbed alkaloids were dissolved in anappropriate solvent before carrying out further analysis by MS and NMR.3.2.2 Culture methods32.2.1 Growth conditionsCell suspensions of C. roseus used were subcultures of the cell line AC-3, derivedfrom a leaf explant of a mature plant and were maintained in 1-B5 medium77 at 26 °C ingyratory shakers at 150 RPM. On the tenth day of growth, cells were transferred to AlkaloidProduction Medium (APM ))28 containing various concentrations of arsenic compounds.The shake flask cultures were incubated at 26 °C in a gyratory shaker for an appropriate87time before harvesting. The cells were harvested by filtration through Miracloth and werestored frozen until extraction.32.2.2 Effect of arsenic compounds on growthCells were grown as described in the previous section in 250 mL Erlenmeyer flaskscontaining 100 mL of APM. The control cultures did not contain added any arsenic. Knownamounts of arsenic compounds, arsenate, arsenite, methylarsonate and dimethylarsinatewere added to the other flasks. Each experiment was done in quadruplicate.Each flask was inoculated with 15 mL of the inoculum and incubated for 21 to22 days before harvesting. The fresh weight of cells was obtained before freezing. Cells fromtwo flasks at each concentration were freeze dried and weighed. Fresh cell samples keptfrozen at -20 °C, were used for alkaloid extractionIn order to obtain large quantities of cells for alkaloid extraction, the culture growthwas scaled up. Each culture was grown in 15 1 L flasks, each containing 400 mL of APM.Media were made up to an arsenic concentration below the Minimum InhibitoryConcentration (MIC) of each arsenic compound. The arsenic concentrations used were3 ppm of arsenate, 6 ppm of arsenite, 6 ppm of methylarsonate and 25 ppm ofdimethylarsinate. No arsenic was added to the control cultures.Each flask was inoculated with 60 mL of a 10-day old cell suspension and cultureswere incubated for 22 or 29 days before harvesting. Harvested cells were kept frozen(-20 °C ) until extraction.883.2.2.3 Effect of time of application of arsenic compoundsCultures were grown in 1 L flasks containing 400 mL of APM. Each flask wasinoculated with 60 mL of a 10 day old inoculum. Arsenic solutions were filter sterilized byusing 0.22 un filter units and added to the medium at the beginning of growth and after 11and 22 days of incubation. The arsenical concentrations studied were 2.5 ppm of arsenate,5 ppm of methylarsonate and 25 ppm of dimethylarsinate. All cultures were harvested after29 days of incubation.3.2.3 Extraction of 1k1olds from cellsThe cells were thawed and homogenized in methanol by using a UltraTurraxhomogenizer. The resulting cell suspensions were sonicated for one hour before filtering offthe residue. The residue was re-extracted with methanol and extracts were combined. Themethanol extract was concentrated and HC1 ( 1 M) was added (volume ratio 7:3aqueous : methanol). This solution was washed with petroleum ether twice andconcentrated to remove all methanol. The resulting solution was washed twice with ethylacetate; it was then neutralized (NaHCO3),the pH adjusted to ca 9.5 (10 M NaOH) andextracted with ethyl acetate several times. The combined extracts were dried overanhydrous Na2SO4and the solvent was evaporated. The residue was redissolved in ethylacetate to obtain the alkaloid extract. The residue that was insoluble in ethyl acetate wassoluble in methanol and did not contain any detectable alkaloids as judged by TLC.The spent medium, the residual medium after the cells were filtered off, wasadjusted to pH 2 with 6 M HC1 and extracted with ethyl acetate. The aqueous solution wasthen adjusted to pH 9.5 with 10 M NaOH and extracted with ethyl acetate.8932.4 Chemicals and reagentsAll chemicals used were of analytical grade and obtained from commercial sources.The alkaloid catharanthine hydrochloride was obtained from Dr. J. P. Kutney (UBC) andthe alkaloids ajmalicine, vixidoline, vindolinine, epivindolinine and anhydrovinblastine wereprovided by Dr. J. Balsevich (Plant Biotechnology Institute, Saskatoon).N-Acetyltryptamine was prepared by using literature methods.All solvents used for HPLC were of HPLC grade and were filtered through 0.45 lImMillipore filters. Deionized - distilled water was used for HPLC after filtration throughMillipore 0.5 jim filters. Double distilled water was used in all culture media.903.3 RESULTS AND DISCUSSION3.8.1 Comparison of growth characteristics of C. roseus cell suspension culturesIn standard 1-B5 medium and Alkaloid Production Medium (APM)The Alkaloid Production Medium (APM) designed by Zenk et al.8 was used togrow C. roseus cell suspension cultures. The major differences in nutrient composition from1-B5 growth medium are increased sugar content (5% (w/v) as opposed to 2% (w/v)), lowerconcentrations of both nitrate and phosphate and absence of 2,4-dichiorophenoxyacetic acid(2,4-D) as the growth regulator. Indole acetic acid and 6-benzylaminopurine are usedinstead as growth regulators in APM. (The composition of 1-B5 and APM media is given inAppendix A.)Growth of C. roseus cell suspension cultures was monitored in 1-B5 and APM mediaand the biomass, both fresh as well as dry cell weight after freeze drying, were plottedagainst the age of the culture as illustrated in Figure 3.5. In the growth medium (1-B5),culture growth is fast and the culture reaches the stationary phase in 10-11 days when onenutrient becomes limiting. In APM, the growth is slower and it reaches stationary phase in20-2 1 days. The biomass at stationary phase is greater in APM which is consistent with thehigher concentration of sucrose in the medium. Storage of sugar inside the cells results in anincrease in biomass; later decrease in biomass occurs as starch reserves are utilized by thecells.Kutney et al. reported the presence of alkaloids in C. rose us cell suspension culturesgrowing in APM as early as 2 weeks after inoculation.’52 However, maximum accumulationof alkaloids was observed in the third to fifth week of culture. After six to eight weeks thealkaloid content was found to diminish, which indicates that alkaloids may be catabolized.91I.504540353025201510iI0 5 10 15 20 25 30 35Age of Culture ( days )5432100 5 10 15Age of Culture20 25( days )30 35Figure 3.5 Growth curves of C. roseus cell suspension cultures grown iii0 1-B5 and • APM media92Cells grown in 1-B5 and APM media were screened for indole alkaloids by using TLCand HPLC. TLC separation of alkaloid extracts from cells grown in APM for 22 days showeda larger number of spots with the ceric ammonium sulphate chromogenic reagent comparedto the two spots observed from cells grown in 1-B5. HPLC analysis also shows a largervariety of alkaloids in larger concentrations in APM grown cells.3.3.2 Effect of arsenic compounds on growth of C. roseus cell suspension culturesInAPMThe growth of C. roseus cell cultures in APM containing various concentrations ofarsenic compounds was monitored. The variation in dry cell weight of cultures with theconcentration of arsenicals (arsenate, arsenite, methylarsonate (MMA) anddimethylarsinate (DMA)) in the media is illustrated in Figure 3.6. The minimuminhibitory concentration (MIC) of each arsenical can be estimated with the aid of thesegraphs. The MIC is defined as the arsenical concentration at which the biomass of theculture is 50% or less than that of the control culture into which no arsenical is added (SeeSection 2.3.2).The toxicity of the four arsenicals in APM and 1-B5 media can be compared using theestimated MIC values, which are listed in Table 3.1. (The MIC values of arsenicals forC. roseus cell suspension cultures growing in 1-B5 medium were discussed in Section 2.3.2.)Concentrations above 3 ppm of arsenate inhibits growth in APM as seen from the dry cellweight after 21 days of growth, whereas in 1-B5 medium the MIC of arsenate is estimated tobe 5 ppm at the stationary phase. Similarly, a greater toxicity effect of other arsenicals isevident in C. roseus cell suspension cultures grown in APM compared to the 1-B5 medium.933.5Figure 3.6—..———The variation of dry cell weight of C. roseus cultures with theconcentration of the arsenical in the APM medium(The dry cell weights were obtained after 23 dayi of growth in theAPM medium containing different concentrations of the arsenicals.)A Arsenate; B Arsenite; C MMA; D DMA.3.02.52.01.51.00.50.00 2 4 6 8Concentration of Arsenical ( ppm )——c-)0 20 40 60 80 100Concentration of Arsenical ( ppm )94Table 3.1 The Minimum Inhibitory Concentration (MIC) values of arsenicalsfor C. roseus cell suspension cultures in the standard 1-B5 and APMmediaArsenic Minimum Inhibitory Concentration (ppm )Compound 1-B5 Alkaloid ProductionMedium Mediumarsenate 5 3arsenite 10 7methylarsonate 8 3dimethylarsinate 50-80 20* The MIC values of arsenicals in APM are estimated from data presented in Figure 3.6. The MICvalues in 1-B5 media are discussed in Section 2.3.2.The higher toxicity response in APM in comparison to 1-B5 medium may be relatedto the differences in the nutrient composition or the growth characteristics of the cultures inthe two media or a combination of both. For example, the phosphate concentration in APMis 0.5 mM whereas it is 1.1 mM in 1-B5 medium. The lower phosphate concentration in APMmay lead to an increased uptake of arsenate because phosphate is a competitive inhibitor ofarsenate uptake ( See Section 2.3.3.1). Moreover, the longer lag phase in APM may result ina lower cell density in the culture during the first stages of growth. The resultant higherarsenical concentration per cell may result in the higher toxicity.Further experiments on the effect of arsenicals on alkaloid production were allcarried out at concentrations at or below the MIC of each arsenic compound.953.3.3 BPLC analysis of indole &ksiloidsReversed phase HPLC coupled to a variable wavelength ultraviolet (UV) detectorwas used to screen indole alkaloids in cell extracts from C. roseus. Isocratic elution with amixture of aqueous and organic solvent resulted in the separation of these polar metabolites.Peak tailing was minimized by adding a modifier, triethylamine (0.1% v/v) to the mobilephase to mask any free silanol groups on the reversed phase C18 packing.152 A typicalHPLC trace showed a number of components in the alkaloid extract. Attempts were made toidentifS’ some components by using retention time data from the available alkaloidstandards. As retention times are dependent on the column and mobile phase, standardshave to be injected each working day. Some typical retention time data for alkaloidstandards are given in Table 3.2.For quantitative work, peak height or peak area ratios of the different alkaloidfractions can not be used as a measure of concentration. It is necessary to construct acalibration curve for each alkaloid at the specified wavelength.A limitation with the HPLC separation of alkaloids is the co-elution of two or morecomponents making identification and quantitation by UV detection difficult. Identificationmay be possible following collection of individual fractions from the HPLC separation andsubjecting these to mass spectrometry by using electronic or chemical ionization. This is atime consuming, tedious method. On-line coupling of HPLC to a mass spectrometerovercomes some of these limitations.96Table 3.2 Typical HPLC retention times of Catharanthus alkaloids(a) Waters p.-Bondapak C18 column, mobile phase H20:CH3CN, 60:40;0.1% (v/v) triethylamine added.(b) Phenomenex Bondclone C18 column, mobile phase H20:CH3CN, 53:47;0.15% (v/v) triethylamine added.Alkaloid Retention Time ( mm)(a) (b)Vindolinine* 6.4 9.5.... *Epwindohnrne 6.9 10.8Vindoline 8.0 14.9Catharanthine 10.4 21.5Ajrnalicine 10.7 21.8Anhydrovinblastine 17.7 39.2* A mixture of the two alkaloids was injected.3.3.3.1 UPLC analysis of 1ka1oid composition o’f cells after 22 days of growthThe HPLC traces of alkaloid extracts from 22 day old C. roseus cells are given inFigure 3.7. They illustrate the difference in sensitivity of the detector to the alkaloids at thetwo wavelengths. The peak eluting at 20 niln can be identified as ajmalicine on the basis ofits retention time. However, a catharanthine standard was found to co-elute with ajmalicineeven though the two alkaloids elute at different times when injected alone as given in97Table 3.2. Thus the possibility that this peak contains catharanthine cannot be eliminatedon the basis of retention times.Vindoline, anhydrovinbiastine, vindolinine and epivindolinine do not seem to bepresent in the cell extracts and the unavailability of standards prevented the identification ofother peaks on the basis of retention times.(a)I I I I I0 2 4 6 8 10 12 14 16 18 20 22Retention Time ( mm)Figure 3.7 HPLC traces of a cell extract of a control C. roseus oulture (Cultureage was 22 days at the time of harvest.)UV detection at (a) 280 nm (b) 254 nm.Plot height scale (Aft 2) is 7 during plotting.(b)98The alkaloid profiles of cells grown in the presence of arsenic compounds for 22 daysare given in Figure 3.8. An overall suppression of alkaloid accumulation is apparent fromthese traces that can be directly related to the decrease in biomass. In addition, a variationin relative intensity of peaks is observed.In the HPLC traces of all three cell extracts from arsenical treated cultures, the peakat 20 mm is low in intensity, indicating the suppression of ajmalicine and/or catharanthineproduction. This is most noticeable in dimethylarsinate treated cells. In rnethylarsonatetreated cells, peaks eluting at 8.2 mm and 12.6 mm are of greater relative intensity whereasin others, they are simply suppressed. In the HPLC trace of dimethylarsinate treated cellextract, an extra peak is prominent eluting around 6.8 mm (peak T) that is not detected ina significant amount in any other cell extract including the control.3.3.32 HPLC analysis of nlknloid composition of cells after 29 days of growthC. rose us cell suspension cultures grown in APM were reported to accumu’atealkaloids up to 33 days.104 The cell suspension cultures were allowed to grow up to 29 daysin the presence of arsenic compounds and their alkaloid profiles studied. HPLC traces of thecell extracts from control as well as arsenical treated cultures are given in Figure 3.9.The HPLC trace of a cell extract from a control culture grown up to 29 days does notshow any notable differences in alkaloid content from an extract of 22 day old cells(Figure 3.9 a). A broad peak at 25 mm may be assigned to ajmalicine and/or catharanthineon the basis of the co-elution of standards.Cells treated with arsenicals exhibit suppressed alkaloid production after 29 days ofgrowth as depicted in Figure 3.9 b - d. Both methylarsonate and dimethylarsinate treatedcells contain very low levels of ajmalicine and/or catharanthine eluting around 25.0 mm. The99extract of dimethylarsinate treated cells contains an extra peak eluting at 6.0 mm which isabsent in other extracts. This peak is also present in the HPLC trace of 22 day olddimethylarsinate treated cells. A detailed discussion of the alkaloid content in these extractswill be given in Section 3.3.5. The alkaloid profiles of cells treated with arsenic compoundsat different phases in the growth cycle will also be discussed in Section 3.3.7.Figure 3.8I I I I I I I I I I0 2 4 6 8 10 12 14 16 18 20Retention Time ( mm)BPLC traces of cell extracts of C. roseus cultures ( Culture age is22 days at the time of harvest IN detection at 280 nnz; Plot heightscale (Aft 2 ) is 5.)Culture media contained (a) arsenate (3 ppm) (b) MMA (6 ppm)(c) DMA (40 ppm).(a)(b)(c)T1000 2 4 6 8 10 12 14 16 18 20 22Retention Time ( mm)Figure 3.9 HPLC traces of cell extracts of C. roseus cultures I Culture age is29 days at the time of harvest UV detection at 280 nm; Values forplot height scale (Att 2) are 7 in trace (a) and 5 In (b), (c) and (d).)Culture media contained (a) control ( no arsenic) (b) arsenate (3 ppm)(c) MMA (5 ppm) (d) DMA (25 ppm).(a)(b)(c)T(d)I I I I I I I I • I • I1013.3.4 Application of Thermospray Liquid Chromatography- Mass Spectrometryfor the analysis of indole alknloidsThermospray 1.0-MS is a valuable technique to screen cell extracts for the presenceof a variety of alkaloids. Both retention time and mass spectral information aid in theidentification of the alkaloid. As therrnospray LC-MS is a soft ionization techniciue, spectraare primarily composed of molecular adduct ions with minimal fragmentation.The results of thermospray LC-MS analysis of a standard indole alkaloid,catharanthine, is depicted in Figure 3.10. The total ion chromatogram shows an impurity(peak A) eluting after 5 minutes and catharanthine (peak B) eluting around 15 minutes.The mass spectrum of peak B shows a protonated molecular ion peak, MH+ (ni/z 337 ) as thebase peak. No significant fragment ions or ammonium adduct ions are present.Similar LC-MS spectra are observed for several monomeric indole alkaloids.Ajmalicine elutes around 15 mm and the mass spectrum consists only of the peak at mlz 353assigned to the MH ion. Similarly, the LC-MS spectra of vindolinine, epivindolinine andvindoline all contain a single peak (mlz 337, 337 and 457 respectively) corresponding to theMH ion of each alkaloid.The minimal fragmentation of monomeric indole alkaloids during thermosprayLC-MS analysis limits the information available for identification of the alkaloids and it isnot possible to differentiate between two isomers with the same retention time as onlymolecular weight information is available. But this feature is an advantage in evaluatingthe purity of LC peaks. Because monomeric indole alkaloids produce only MH+ ions underthese conditions, a scan giving rise to a spectrum containing several significant peaks wouldbe indicative of the presence of several compounds of the appropriate molecular weights.Lack of fragmentation also enhances the sensitivity of detection of alkaloids.102Quantitative information from therinospray LC-MS is limited. The intensities ofpeaks observed in the thermospray mass spectrum do not necessarily reflect the proportionsof different alkaloids present because the optimum thermospray conditions such as vaporizerand ion source temperatures are not identical for all alkaloids. Quantitation is possible byestablishing a calibration curve for each alkaloid of interest relative to the intensity of aninternal standard added to each sample.103I I300m/z400Figure 3.10 Thermospray LC-MS analysis of catharanthine (a) Total IonChromatograrn where peak A = impuritypeak B - catharanthine (b) Mass spectral scan ofpeak B (catharanthine).Retention Time(I)C04-.Ca)>4-?a)Scan NumberB100----P03372601043.3.5 CeU alkaloid composition after 22 days of growth3.3.5.1 LC-MS analysis of C. roseug cells grown in APM for 22 daysC. roseus cells grown in APM for 22 days were examined for alkaloid production byusing thertnospray LC-MS. The total ion chromatogram of the alkaloid extract and massspectral scans of several peaks are depicted in Figure 3.11. The assignments aresummarized in Table 3.3.The major component in peak B, Figure 3.11, elating at 4.0 mm has m/z 339. It canbe assigned to perivine on the basis of its molecular weight. Peak D contains severaicomponents of mlz 341, 371, 353 and 387 which could be (MHY peaks of several unassignedalkaloids. Peak F eluting at 8.8 mm contains a compound of in./z 355. This can be assignedto yohimbinet, a corynanthe alkaloid, on the basis of its molecular weight. Retention timedata are not available. This tentative assignment is denoted by the (t ) sign. The otherpossibilities for this compound are sitsirikine and isositsirikine, both corynanthe alkaloids.The major component in peak 0, eluting at 12.0 mm, has a m/z 323. This could beassigned to akuamrnicine on the basis of its molecular weight. The other components ofrn/z 353 and 385 are not assigned.The mass spectrum of the peak I eluting between 14 - 15 mm shows the co-elution oftwo components of m/z 353 and 337, Figure 3.11. The former is assigned to ajmalicine andthe latter to catharanthine based on both retention time data and molecular weightinformation. Catharanthine and ajmalicine have similar retention times as discussed above,Table 3.2. Thus LC-MS provides strong evidence for the presence of a minor concentration ofcatharanthine in the 22 day old C. roseus cell suspension.105Table 3.3 LC.MS analysis of nlkRloid extracts from C. roseus cells grown inAPM for 22 daysPeak Retention Time m/z Peak AsignmentA 2.6 339,371B 4.0 339 perivinetC 4.3 192 ?D 4.6 341,371 (MHY peaks of353, 387 several alkaloidsE 7.2 371 ?F 8.8 355 yohimbinetG 12.0 323(major) akuamrnicinet353,385I 14-15.0 353(rnajor) ajmalicine337(minor) catharanthineThe assignment is based only on the molecular weight information from thermosprayLC-MS results.106Retention Time (mm)2:25 4:54 7:22 95O I2I9 1447 1716 j9:44 22:13I - I I I I II337-—I I200 300353400 500mlzFigure 311 Thermospray LC-MS analysis of 1ksi1oid extracts from cellsgrown in APM for 22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks Bandl0:00100 —• (a)50 FB4--AC - II> I LIiro- ijiH’I 50 100 150 200 250 300 350 400 450Scan NumberB100>p0>%U,Ca)4-C219371m/z45001073.3.5.2 LC.MS analysis of 22 day old cells grown in APM containing arsenateThe results of LC-MS analysis of the alkaloid fraction of cells grown in APMcontaining 3 ppm of arsenate is illustrated in Figure 3.12 and Table 3.4. An overallsuppression of alkaloid production is observed in this culture: many of the alkaloid peaksobserved in the control culture are present here at a lower intensity. These include peaks A,E, G and I and some alkaloids are markedly absent including catharanthine in peak I.Peak I contains only ajmalicine (ni/z in arsenate treated cells although in the control,both ajmalicine and catharanthine co-elute in this peak. Peak F in the control containing acompound of rn/z of 355 is also absent in arsenate treated cells.108Table 3.4 LC-MS analysis of nlknloid extracts from C. roseus cells grown inAPM containing 8 ppm of arsenate for 22 daysPeak Retention Time m/z Peak Assignment(nun)A 2.6 339 ?B 3.8 219,249 ?401C 4.3 180,219 7D 4.6 297(major) antirhine3a875.0 607 ?E 7.2 371a ?323(minor)G 12 323a(major) akuammicinet353,385I 14-15.0 353a ajmalicine(a) Present in the control (22 days old)109Retention Time (mm)5o-(I)Ca)C4)>04,-225 453 72O 949 I2I7 I446 17,14 19,43_22III I350m/zFigure 3.12 Thermospray LC-MS analysis of nlknloid extracts from cellsgrown In APM containing 3 ppm of arsenate for 22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks GandlI .d•9:00 -—--(a)DACI I I I100 200I I I I300 400Scan numberG 100 I1008060ci)Cci)4-&.4020035380>U.,Ca)4—C 401385 20250 350m/z0 I2501103.3.5.3 LC-MS analysis of 22 day old cells grown in APM containingmtthylarsonateCells grown in APM containing 6 ppm of methylarsonate for 22 days also showsuppressed alkaloid production compared to the control. But some of the characteristicalkaloid peaks found in control are detected as depicted in Figure 3.13 and Table 3.5. Peak Feluting between 7.6 - 9 mm contains a compound of ni/z 355 which is tentatively assigned toyohimbinet in the control. The broad peak I eluting between 14- 15 mm contains only onecompound of mn/z 353 indicating the presence of ajmalicine and the absence of catharanthine.3.3.5.4 LC-MS analysis of 22 day old cells grown in APM containingdimethylarsinateCells grown in APM containing 40 ppm of dimethylarsinate do not show any of thecharacteristic alkaloids found in the control culture. Even though the cultures show normalgrowth, alkaloid production is apparently suppressed as depicted in Figure 3.14 andTable 3.6.There is evidence for accumulation of high levels of tryptamine. The peak X elutingat 5.1 mm contains a compound of mlz 203. This compound is identified asN-acetyltryptamine, formed from tryptamine during extraction by ethyl acetate (SeeSection 3.3.8.2). Tryptamine (MW 160) itself would not be detected by LC-MS as the lowercalibration mass limit was 167 for all LC-MS runs.111Table 8.5 LC-MS analysis of s1kk1oid extracts from C. roseus cells grown inAPM containing 8 ppm of methylarsonate for 22 daysPeak Retention Time mlz Peak Assignment(mm)A 2.6 339 7B 4.0 perivinetC 4.3 169,309 ?D 4.6 371,341 mixture353a,387a(minor)F 8.8 355a(major) yohimbinet337,369G 12.0 297I 14-15.0 ajmalicine(a) Present in the control (22 days old)112Retention Time (mm)0:00 2:25 4:53 7:22 9:50 12:19 1447 17:16 19:41100I I I _I ID (a)-U)a)Ca) ->0BC F G-,I 50 100 150 200 250 300 350 400Scan Number100 F 355 100 I> >4- 4-U,50 50C C369169 337I Ii Li ,200 300 400 200 300 400m/z m/zFigure 3.13 Thermospray LC-MS analysis of lki1oid extracts from cellsgrown in APM containing 6 ppm of methylarsonate for 22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks Fand I113Table 3.6 LCMS analysis of s1ksa1old extracts from C. TO8U8 cells grown inAPM containing 40 ppm of dimethylarsinate for 22 daysPeak Retention Time m/z Peak Assignment(mm)A 2.6 259,282 7B 4.0 211,314C 4.3 297 antirhinetC’ 4.45 219D 4.6 180,219 7X 5.1 203 N-acetyltryptamineX’ 7.3 275,171 ?(a) Present in the control (22 days old)114>4-U,ci)4-CU), 504-ICC>4-.04,Figure 3.14 Thermospray LC-MS analysis of nlkRloid extracts from cellsgrown in APM containing 40 ppm of dimethylarsinate for22 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks CandXRetention Time (mm)I 50 100 150 200 250 300 350Scan Numbertoo. xC.297>4-ci)a)4-C0 I I 1200 300 400m/z m/z3c1153.3.6 LC-MS analysis of sikaloid extracts from cells grown in APM containingarsenic compounds for 29 daysAs a result of changes in IIPLC conditions as well as therinospray MS conditions, theretention times in this series of experiments cannot be directly compared with the LC-MSresults from 22 day old cells shown in Figures 3.11 -3.14. The changes included the use of aPhenomenex Bondclone column as well as modified chromatographic conditions. (SeeSection 3.2.1.3). The standard ajmalicine eluted around 27 mm as opposed to 15 mm in theprevious analysis.3.3.6.1 LC-MS analysis of C. roseus cells grown in APM for 29 daysThe cells grown for 29 days in APM show a greater diversity in the alkaloids detectedas depicted in Figure 3.15 and Table 3.7. Some of the characteristic peaks observed in the22 day old cells are still present after 29 days. These include peak B containing in/z 339,assigned to perivinet, peak F containing mhz 355, assigned to yohimbine and peak J’containing ajmalicine.There are some unknown metabolites eluting in peaks A, C and D. Peaks G and Heluting at 11 and 12 mm can be assigned to vindolinine and epivindolinine. A standardmixture of these two alkaloids was found to elute at these retention times and each gives acharacteristic mass of 337. These alkaloids are not detected in 22 day old cultures.The predominant species in peak I eluting between 15 - 16 mm has a miz 323 and isassigned to akuammicine. This compound is present in 22 day old cells as well. Peak I’eluting at 24 mm contains a compound of mhz 325 which can be tentatively assigned tolochnerine. The broad peak J eluting around 26 minutes shows some separation. The firstportion J contains only a mass of 337, indicating catharanthine. The second portion J’ shows116mlz 353 (ajmalicine) as the base peak and mhz 337 (catharanthine) at a smaller intensity.Mixtures of ajmalicine and catharanthine usually co-elute as a broad band. Althoughcatharanthine is a minor component in cells after 22 days of growth, it is found at a largerrelative concentration after 29 days.3.3.6.2 LC-MS analysis of 29 day old cells grown in APM containing arsenate- In the cells grown in the presence of arsenate ( 3 ppm) for 29 days, many of thealkaloid peaks are suppressed compared to the control as illustrated in Figure 3.16 andTable 3.8. Alkaloids that are suppressed include yohimbinet (mhz 355) eluting at 9 mm(peak F), vindolinine and epivindolinine eluting at 11 and 12 mm (peaks G, H) andlochnerinet (mhz 325) eluting at 24.0 mm (peak I’). Catharanthine is also completely absentand only ajmalicine is detected in the late eluting peak J.These cells exhibit an alkaloid composition similar to that of cells grown in arsenateup to 22 days. The main differences are the disappearance of the metabolite of mhz 297which eluted at 4.6 mm and the presence of an alkaloid of mhz 383 eluting at 8.8 mm in cellsgrown up to 29 days. This latter metabolite is not observed in the control and can beassigned to lochnerininet on the basis of its molecular weight.117Table 8.7 LC-MS analysis of RlkRlojd extracts from C. roeus cells grown inAPM for 29 daysPeak Retention Time m/z Peak AssignmentA 2.6 353,371 ?B 4.0 339(niajor) perivinetC 4.5 341,387 7D 5.2 371 7E 7.8(broad) 371 mitraphyllinetF 9.0 355(major) yohimbinetF’ 10.0 355 yohimbinet297(minor) antirhinetG 11.0 337 vindolinineandH 12.5 337 epivindolinineI 16.1 323(major) akuammicinet355I’ 24.0 325 lochnerinetJ 27 337 catharanthine3’ 28.5 353(major) ajmalicine337 catharanthine118->4— -U) -Cw50--I-C100>‘criCa)4-,- 50a)>4-,0a)txRetention Time (mm)100->U)Cw50--4-C0-Figure 3.15 Thermospray LC-MS analysis of alkaloid extracts from cellsgrown in APM for 29 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks Eand J’119Scan NumberE 37’ JI 3370353I I200 300 400 500m/zI I200 300 400 500m/zTable 8.8 LC-MS analysis of filkaloid extracts from C. roseus cells grown inAPM containing 3 ppm of arsenate (Cell age 29 days)Peak Retention Time m/z Peak Assignment(mm)A 2.6 219B 4.0 9a perivinetC 4.5 430D 5.5 371 ?E 6.5 371a mitraphyllinet8.8 383(major) lochnerinine371I 16.0 akuammicineJ 28-29 ajmalicine(a) Present in the control.120Figure 3.16 Thermospray LC-MS analysis of &ks1oid extracts from cellsgrown in APM containing 3 ppm of arsenate for 29 daysTotal Ion Chromatogram (a) and mass spectral scans ofpeaks E’ and JRetention Time (mm)34:33 39:29U)C4)CI500Scan NumberJ 353> -383100->4-Cl)Cci4-C0E’243I -.I I I200 400m/z200 400m/z1213.3.6.3 LC-MS analysis of 29 day old cells grown in APM containingmethylarsonateThe results of LC-MS analysis of extracts from cells grown in the presence ofmethylarsonate are given in Figure 3.17 and Table 3.9. Suppression of alkaloid production isevident. The alkaloids that have been completely suppressed include vindolinine/ epivindolinine (m/z 337) and akuammicinet (m/z 323), which are observed in the controlcell extract. Both catharanthine and ajmalicine are also absent in these cells. Ajmalicinewas observed in the cells grown in the presence of methylarsonate after 22 days as depictedin Figure 3.13. The absence of ajmalicine in cells after 29 days of growth in methylarsonatemay indicate that its concentration is too low to be detected because it is either transformedor catabolized inside the cells over time. Cell lysis could also release alkaloids into themedium resulting in a lower intracellular concentration.Peak X eluting at 10.8 mm contained antirhinet (rn/z 297) which is present in thecontrol cell extract at a low concentration. This metabolite was previous’y observed inC. rosezss cell suspension cultures.8 The peak eluting at 24 mm containsm/z 325 (lochnerine), also observed in the control. A new metabolite of m/z 369 eluting at13.5 mm may be assigned to horhammericinet on the basis of its molecular weight.3.3.6.4 LC-MS analysis of 29 day old cells grown in APM containingdimethylarsinateThe results of LC-MS analysis of cells grown in the presence ofdimethylarsinate ( 25 ppm), show suppression of alkaloid production as illustrated inFigure 3.18 and Table 3.10. Similar results were observed in the cultures harvested after22 days.122Ajmalicine was detected at a low concentration as peak J eluting around 28.5 mm; itwas not evident in the HPLC trace. Peak D eluting around 5.2 mm contains m/z 203indicating N-acetylated derivative of trypt.amine, as well as some unidentified metabolites ofmlz 214 and 371. Broad peak E contains yohimbine (mlz 355) and another metabolite ofmlz 369. Peak X eluting at 14 mm contains m/z 369, previously seen in methylarsonatetreated cells and assigned to horhammericine.Table 3.9 LC-MS analysis of Rlkiiloid extracts from C. roseus cells grown inAPM containing 6 ppm of methylarsonate (Cell age 29 days)Peak Retention Time mlz Peak Assignment(mm)A 2.6 339 7B 3.8 9a perivinetC 4.5 387 ?D 5.1 371 mixture341,401F 8.6 355a yohimbinetF’ 10.8 297a antirhineX 13.5 369 horhammericinetI’ 24.0 325a lochnerinet(a) Present in the control.123Retention Time (mm)234 9:50 1446 19:43 24:39 29:36100- I ID (a)K-.‘In -CCC ->53 100 200 300 400 500 600Scan NumberF’ 297 100 X 36950 50383—i1 I__200 400 200 400m/z m/zFigure 3.17 Thermospray LC-MS analysis of alkaloid extracts from cellsgrown in APM containing 6 ppm of methylarsonate for 29 daysTotal Ion Chroniatogram (a) and mass spectral scans ofpeaks F’ and X124Table 3.10 LC-MS analysis of nlknloid extracts from C. roseus cells grown inAPM containing 25 ppm of dimethylarsinate (Cell age 29 days)Peak Retention Time m/z Peak Assignment(mm)A 3.0 339.388B 4.0 341,405 7C 4.8 180,367 7D 5.2 214,371(major) 7203 N-acetyltryptamineE 8-9(broad) 355a(major) yohimbmnet369X 14.0 369 horhammericinet409J 28.5 3a ajmalicine(a) Present in the control.125>%U)Ca)C4)>0a)Retention Time (mm)0:00 4:51 9:48 14:45 1941 24:38 29:34 34:31 3928Figure 3.18 Thermospray LC-MS analysis of silksiloid extracts from cellsgrown in APM containing 25 ppm of dimethylarsinate for29 daysTotal Ion Chromatogram (a) and mass spectral scans of peaks DandXI tOO 200 300 400 500 600 700 800Scan Numberg IOO>4—Cl)Cci)4—214 D371273lw203Li369409x196200 400m/zLI200 400mlz1263.3.7 Effect of time of application of arsenic compoundsThe elicitor response is reported to be dependent on the growth stage of the cultureat the time of elicitor application in many p1ant systems.17° Treatment with elicitors atdifferent times during the growth cycle has resulted in a variable response in C. roseus cellsuspension cultures.141’4 For example, in cells treated early in the growth cycle withvanadyl sulphate, alkaloid accumulation is inhibited whereas in cells treated on day 12,accumulation of both ajmalicine and catharanthine increased by 50% over the controlcultures.107’44Because interaction of the arsenic compounds with the plant cells may also vary withthe growth stage of the plant cell culture, arsenicals were added to the C. roseus cellsuspension cultures growing in APM at different times in their growth cycle and the effect onalkaloid production was monitored. The changes in alkaloid production on addition ofarsenicals at the beginning of growth is discussed in Section 3.3.6. The arsenicals have beenadded to the culture medium at the time of inoculation and will be referred to as day 0 (timezero ) application.3.3.7.1 LC-MS analysis of C. roseus cells treated with arsenic compounds onday 22 of the growth cycleC. roseus cell suspension cultures growing in APM were treated with the arsenicals,arsenate, MMA and DMA on day 22 of incubation and allowed to grow up to 29 days beforeharvesting. The cultures had reached the stationary phase and had already started alkaloidaccumulation at the time of treatment. The cell density in the culture is a maximum at thistime and thus the concentration of the arsenical per cell is at a minimum. The changes inthe alkaloid production as a response to the treatment of arsenic compounds at this stagewas investigated by using thermospray LC-MS.127ArsenateFigure 3.19 and Table 3.11 present the results of LC-MS analysis of extracts fromcells treated with 3 ppm of arsenate on day 22 of culture. The alkaloid composition of thesecells is found to be similar to that of a control culture harvested after 29 days. Most of thealkaloids in the control are present in this extract. The only exception is the absence of apeak eluting at 16 mm containing rn/z 323 assigned to akuammicine, an aspidospermaalkaloid. Peaks J and J’ eluting at about 27 mm assigned to ajmalicine and catharanthineare enhanced in the arsenate treated cells. Whether enhancement is due to one particularalkaloid or both cannot be established by LC-MS results.MethylarsonateThe results of LC-MS analysis of cells treated with 6 ppm of methylarsonate onday 22 of growth are illustrated in Figure 3.20 and Table 3.12. In contrast to the treatmentwith arsenate on day 22, an overall suppression of alkaloid production is evident. Some ofthe alkaloids are completely absent whereas some are found in lower quantities. The peakeluting at 16 mm (mlz 323) assigned to akuammicinet is absent. The peak eluting around26 mm containing ajrnalicine and catharanthine is drastically reduced and a very smallpercentage of it is catharanthine. Peak B (mlz 339), peak F’ (mlz 297), peak G (mlz 337)and peak I (m/z 325 ) are observed in the control as well. Peak G’ contains horhammericinet(m/z 369), a metabolite not observed in the control. This compound is also detected in cellcultures treated with methylarsonate at the beginning of growth.Even though C. roseu cell cultures have accumulated alkaloids by day 22 when thecultures were treated with methylarsonate, a drastic reduction in alkaloids is observed after29 days of growth. This data together with the observation of turbidity in the spent mediumsuggests that cell lysis takes place on treatment with methylarsonate.128Table 3.11 LC-MS analysis of &knloid extracts from C. roseus cells treated with8 ppm of arsenate on day 22 of growth cycle (Cells harvested onday 29 of growth)Peak Retention Time m/z Peak Assignment(mm)A 3.8 219.261 ?B 4.2 perivinetD 5.2 371 ?E 7.5-8.0 369F 8.8 355a yohimbinet0 10.7 337a,29 antirhinet,vindolinine andH 12.4 epivindolinineX 22.6 760I 25.0 325a lochnerinetJ 25.5-26.5 337a(major) catharanthine353a26.5-27.5 353a(major) ajmalicine337a(a) Present in the control.129Figure 3.19 Thermospray LC-MS analysis of alknloid extracts from cellstreated with 3 ppm of arsenate on day 22 of growthTotal Ion Chroinatogram (a) and mass spectral scans of peaks FandGRetention Time (mm)15:20 20:30 25:39 30:48U)Ca)Ca)>0a)gi>U•)Cci)CScan NumberG 337 F 355gi>4—U)Cci)C29750;0-200 400m/zi. Ii L JI I I200 400mlz130Table 3.12 LC-MS analysis of nlknloid extracts from C. roseus cells treated with6 ppm of methylarsonate on day 22 of growth cycle (Cells harvestedon day 29 of growth)Peak Retention Time m/z Peak Assignment(mm)A 3.5 219.339 ?B 4.2 perivinetD 5.5 371a ?F 8-9(broad) yohimbinetF’ 10.8 297a antirhinetG 12.8 337a vindolinine andepivindolinine14.5 369 horhammericinet239 ?I 25.0 325a lochnerinetJ 26-27(broad) 3a ajmalicinecatharanthine(a) Present in the control.131Figure 3.20 Thermospray LC-MS analysis of alkaloid extracts from cellstreated with 6 ppm of methylarsonate on day 22 of growthTotal Ion Chromatogram (a) and mass spectral scans ofpeaks G’ and IRetention Time (mm)5:05 10:13 15:22 20:30 25:39 30:49I I I I I35:56O 00D (a)-(I,PoBF.1 I I I I(00 200 300 400Scan Number369>U)CG’239500 600 •700I 325>U)G)4—C1000m/zI I I200 400m/z132DimethylarsinateCells treated with 25 ppm of dimethylarsinate on day 22 show some significantchanges in their alkaloid composition as depicted in Figure 3.21 and Table 3.13. The peak Jeluting around 26 mm contains exclusively ajmalicine and catharanthine is absent.Generally, catharanthine production seems to be accelerated after about 22 days of growthas is seen on comparison of the alkaloid profile after 22 and 29 days (See Tables 3.3and 3.7). The addition of dimethylarsinate seems to have effectively halted the productionof catharanthine.The broad intense peak F eluting between 8.4 and 9.4 minutes contains a metaboliteof rnlz 355. This peak F is observed in the control and is assigned to yohimbinet. Howeveron treatment with dimethylarsinate, this metabolite has been produced to a greater extent.Peak G consisted of a mixture of components including masses 337 and 297, which could be amixture of epivindolinine / vindolinine and antirhine. The mass 203 detected around 5 mmin other dimethylarsinate treated cells indicating tryptamine accumulation is completelyabsent in this case. This was consistent with the HPLC trace where the extra peak Tassigned to tryptamine was found to be absent. Thus the treatment on day 22 withdimethylarsinate does not result in tryptamine accumulation.133Table 3.13 LC-MS analysis of silkaloid extracts from C. roseus cells treated with25 ppm of dimethylarsinate on day 22 of growth cycle (Cellsharvested on day 29 of growth)Peak Retention Time m/z Peak Assignment(mm)A 4.3 341 ?B 4.7 perivineD 5.5 371E 6.8-7.4 369,339355F 8.4-9.4 355a(major) yohimbinetG 10.8-11.5 337a29 mixture355,325 epi/vindolinineand antirhine12.2-13 353,369 ?I 18.0 3a akuammnicinetI’ 19.5-20.5 353 7J 26-28 ajmnalicine(a) Present in the control.134>%Cl)Ca)>fr,0a)Retention Time (mm)Figure 3.21 Thermospray LC-MS analysis of alkaloid extracts from cellstreated with 25 ppm of dimethylarsinate on day 22 of growthTotal Ion Chromatograrn (a) and mass spectral scans of peaks Gand JScan NumberG 297 J337 353LI—I00>U)ci)-Ic:—QiI I I200 400m/zI I I200 400mlz1353.3.72 LC-MS analysis of C. roseus cells treated with arsenic compounds onday 11 of the growth cycleArsenateThe results of LC-MS analysis of alkaloid extracts from cells treated with 3 ppm ofarsenate on day 11 are given in Figure 3.22 and Table 3.14. A drastic suppression of alkaloidproduction similar to the treatment at the beginning of growth is observed in these cellstreated with arsenate. Both ajmalicine and catharanthine are completely absent. Themetabolite assigned to antirhinet (mlz 297 ) eluting around 10.8 mm is also detected in thecontrol. Another alkaloid lochnerinet (m/z 325), also present in the control, is detected at alow concentration. A metabolite of m/z 369 eluting around 14.5 minutes is assigned tohorhammericinet and is previously detected in some arsenical treated cells even though it isabsent in the control culture.In contrast to day 22 application, the day 11 treatment of arsenate has an inhibitoryeffect on the alkaloid production. The dissimilarity could arise due to the differences in celldensity and the variable metabolic requirements of the cells depending on the growth stageat the time of application.136Table 3.14 LC-MS analysis of silknloid extracts from C. roseus cells treated with3 ppm of arsenate on day 11 of growth cycle (Cells harvested onday 29 of growth)Peak Retention Time m/z Peak Assignment(mm)A 3.5 239 7B 4.4 341 ?D 5.5 371 7E 7-8.0 339,387F 10.8 297a(major) antirhinet355F’ 14-15 357369 horhammericinetG 22-23 196,239 ?I 25.5 325a lochnerinet(a) Present in the control.137—S-U)->4-.0 -0-p p p100 200 300 400Scan Number>(I)C-I-Figure 3.22 Thermospray LC-MS analysis of alkaloid extracts from cellstreated with 3 ppm of arsenate on day 11 of growthTotal Ion Chroniatograrn (a) and mass spectral scans of peaks Fand F’000100- —Retention Time (mm)5:05 10:13 15:20 2027 25:34 30:41I I I I I35:50D (a)500 600 7001100.297355F’196357200 400mlz m/z138MethylarsonateA drastic reduction in alkaloid production is observed on treating C. roseus cells withinethylarsonate on day 11 of culture growth as illustrated in Figure 3.23 and Table 3.15.Several unknown metabolites are observed in this extract. Peak F containing mlz 355 is alsodetected in the control and is assigned to yohimbinet. Peak J contains ajmalicine (tn/z 353)but not catharanthine.DimethylarsinateFigure 3.24 shows the results of thermospray LC-MS analysis of cells treated with25 ppm of dimethylarsinate on day 11. The results are also listed in Table 3.16. Suppressionof alkaloid production is evident although not as pronounced as in the cells treated withdimethylarsinate on day 0, Figure 3.18. Several masses such as 355 (peak F ), 325 ( peak I)and 323 (peak H) indicate to the presence of some corynanthe alkaloids but all at very lowconcentrations. Ajmalicine and catharanthine are absent in this extract.Here again, the presence of trypt,arnine is revealed by the mass spectrum of peak D,containing tnlz 203 assigned to N-acetyltryptamine. The HPLC trace also shows the extrapeak assigned to tryptamine. Thus the treatment of C. roseus cultures withdimethylarsinate on both day 0 and day 11 results in tryptamine accumulation.139Table 3.15 LCMS analysis of n1ks1oid extracts from C. roseus ceUs treated with6 ppm of methylarsonate on day ii of growth cycle (Cells harvestedon day 29 of growth)Peak Retention Time rnfz Peak Assignment(mm)A 4.2 341,196 ?D 5.2 ?E 7.0 239,196 7F 9.5-10.0 355aF’ 11.5-12.5 239297a antirhinetX 15.5- 16.5 196,239369 horhammericinetG 21-23 239J 27-28 ajmalicine(a) Present in the control.1400Ca)C>4-a0a)Retention15:22Time (mm)2031I i I IFigure 3.23 Thermospray LC-MS analysis of R1ks1oid extracts from cellstreated with 6 ppm of methylarsonate on day 11 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks FandXScan NumberF239369355I I I 0 I400 200m/z m/z200 460141Table 3.16 LC-MS analysis of R1ks1oid extracts from C. roseus cells treated with25 ppm of dimethylarsinate on day 11 of growth cycle (Cellsharvested on day 29 of growth)Peak Retention Time mlz Peak Assignment(mm)A 3.5 339a 7B 4.2 341 ?D 5.2 3718 7203 N-acetyltryptamineE 7.5-8.2 369, ?F 9.0 355a yohimbineF’ 11.5-12.5 297a,355 mixture337G 14-15(broad) 369 horhammericinetH 19.5-20.5 323a akuammicinetI 28-29 325 lochnerinet(a) Present in the control.142Figure 3.24 Thermospray LC-MS analysis of alkaloid extracts from cellstreated with 25 ppm of dimethylarsinate on day 11 of growthTotal Ion Chromatogram (a) and mass spectral scans of peaks Gand IRetention Time (mm)20:31 25:40U)C4)4.’Ca,>4.’0a’Scan Number369G173239 297I I I ii325I196200 400m/z260 400m/z1433.3.8 Isolation and structure elucidation of tryptamine3.3.8.1 Isolation of tryptamlneIn order to identi1’ the extra peak T observed in the HPLC traces ofdimethylarsinate treated cells (Figures 3.8 c, and 3.9 d), alkaloid extracts from cells grownin dimethylarsinate were subjected to HPLC and the fractions comprising peak T collected.The combined fractions were found to contain more than one component when analyzed byusing TLC. Preparative TLC separation was carried out with a 10:7:4 mixture ofEtOAc : CHCI3 : CH3O as the developing solvent system. An intense band that does notmove up with the solvent (Rf = 0.05) gives a bright yellow color on spraying with CASreagent and develops an orange rim five minutes after spraying. The compound giving riseto this band was isolated by scraping the silica off the plate and extracting with ethy1acetate.In the electron ionization mass spectrum (ElMS ) of the fraction, the parent ion wasassigned to the highest mass rniz 160.1000 with a molecular formula of C10H2N2(calc. 160.0999). The base peak m/z 130 indicated that the compound was a simple indoebase. The 1H NMR spectrum in (CD3)2C0 showed only two peaks in the high field region;2.99 ppm (t,7 Hz) and 3.50 ppm (t,7 Hz) indicating the presence of two -CH2 groups. Therest of the signals are in the aromatic region and chemical shifts were characteristic of aindole nucleus. These data suggest the compound to be tryptamine. Comparison withauthentic material confirmed this metabolite to be tryptamine.1443.8.82 Conversion to N-acetyltryptamineThe identification of trypt.amine was hampered by the slow conversion of themetabolite into a different component.. Alkaloid extracts from dimethylarsinate treated cellsin ethyl acetate show peak T eluting at 8 mm (Figure 3.9 d) accounting for 40% of thealkaloid content. Within several weeks, this peak completely disappears and another peak Nshows increased peak area. On separation by preparative TLC, this new component elutesat Rf= 0.74 and turns a yellow color with an orange rim on spraying with the CAS reagent.This metabolite was extracted from silica gel by using ethyl acetate or chloroform andsubjected to NMR and MS.ElMS shows mlz 143 as the base peak and mlz 130 as 90% of the base peak. Asseveral higher masses were observed including 370, 366 and 202, the molecular weight couldnot be confirmed. But Chemical Ionization MS spectrum contains rnlz 203 as the highestmass confirming 202 as the molecular weight. This spectrum also shows some fragments ofm/z 143 and 130.The NMR spectrum indicated the presence of some impurity, but two major peaks at3.6 ppm (q) and 2.98 ppm (t) and the characteristic peaks in the aromatic region suggestedN-acetyltryptamine. Later comparison with authentic sample confirmed the identity.Base catalysed N-acetylation of tryptamine was observed when tryptamine.HCI saltwas converted to the free base. When ethyl acetate was used to extract tryptamine frombasified aqueous solution, the resultant free base contained some N-acetylated derivative(6% ) in the HPLC trace. But when dichloromethane was used as the extracting solvent, theN-acetylated derivative was not detected.N-acetyltryptamine was previously observed in cell suspension cultures of C. roseusthat have been treated with fungal homogenates.141 A bacterial culture was also reported145to produce N-acetyltryptamine as well as tryptamine.171 In dimethylarsinate treated cellsuspensions, tryptamine accumulation was confirmed but N-acetyltryptamine apparently isan artifact formed during the extraction and on standing.3.3.9 SuninnryThe effect of arsenic compounds on the secondary metabolism of C. roseus cellsuspension cultures is dependent on complex interactions between the arsenical and theplant cell. This holds true for any elicitor used to stimulate secondary plant metabolism.The expression of some secondary biochemical features of the plants can be repressed in theculture systems and may need a stimulus for expression. The added elicitor could be thestimulus needed. Optimization of an elicitor response can only be achieved empirically anddetailed predictions for a system cannot be made.The response of cultured plant cells to an elicitor is affected by a number of factors,some of which relate to the properties of the elicitor and others to the cultured cells. Theelicitor specificity and concentration as well as the time the elicitor is in contact with the cellculture may influence the response.Variation in expression of biochemical capability among cell culture lines derivedfrom the same plant species or even the same plant is well documented.104 Variableresponse of several C. roseus cell lines to a fungal elicitor has been reported.141 In thepresent study, the response of one cell line, AC-3, derived from a leaf explant of a matureC. roseus plant, was studied. Ideally, an extensive study on a number of a cell lines shouldbe carried out.Culture conditions and nutrient composition play a role in the elicitation behavior ofplant cell cultures. Alkaloid Production Medium with an optimized nutrient composition was146used throughout this study on the effect of arsenicals on alkaloid production. A differentmedium such as 1-B5 growth medium may result in a different response on application of thearsenicals.The elicitor response also proved to be dependent on the growth stage of the cellculture in several C. roseus culture systems. The effect of a fungal elicitor varied with thetime of application; a 5 day old C. roseus culture responded by producing N-acetyltryptaminewhereas a 10 day old cell culture accumulated a whole spectrum of indole alkaloids.1-4Theresponse to vanadyl sulphate107 and abscisic acid’42 were also affected by the growth stageof the culture. The effect of application of arsenic compounds at different times in growthcycle on the alkaloid production by C. roseus cell suspension cultures was investigated in thepresent study.3.3.9.1 Alkaloid composition in 22 and 29 day old C. roseus cell suspensionculturesComparison of the alkaloid accumulation in C. roseus cells grown for 22 and 29 daysshows a wider spectrum of alkaloids in 29 day old cells. In 22 day old cells, the alkaloidsidentified or tentatively assigned include perivinet (an aspidosperma alkaloid), yohitnbinetand ajmalicine (corynanthe alkaloids) and akuammicine (strychnos alkaloid). Smallamounts of catharanthine, an iboga alkaloid, are detected co-eluting with ajmalicine.After 29 days, cells still contain perivinet (aspidosperma) and ajmalicine andyohimbinet (corynanthe). Akuammicinet, the only strychnos alkaloid detected is alsopresent after 29 days. Vindolinine and epivindolinine, both aspidosperma alkaloids arepresent in 29 day old cells although they are not detected after 22 days of growth.Catharanthine (iboga) is found at a much higher concentration. Obviously, the production147of catharanthine increases during the time between 22 - 29 days whereas that of ajmalicinediminishes. This provides evidence for the sequential formation of alkaloids; corynanthe andstrychnos alkaloids are produced earlier in the growth cycle and are followed byaspidosperma alkaloids and finally the iboga alkaloids.3.3.9.2 Effect of arsenate on silksiloid productionTreatment with arsenate at the beginning of growth has a suppressing effect onalkaloid production. Overall, alkaloid content decreases after 22 and 29 days of growthcompared to the control and only a few alkaloids observed in the control are detected. Theyare perivinet, akuammicine (strychnos) and ajmalicine (corynanthe). The production ofcatharanthine is completely suppressed in these arsenate treated cells.Application of arsenate on day 11 of growth has an even greater inhibitory effect onalkaloid production compared to the day 0 application. Many of the alkaloids observed in thecontrol are absent in this culture. Only 1ochnerine (m/z 325 ) and antirhine (m/z 297 ) arepresent in the control culture and the rest cannot be assigned. By day 11, the cell culture isin its growth phase.Arsenate treatment on day 22 does not have a suppression effect on alkaloidproduction. Alkaloid production has already commenced by day 22 and arsenate may have astimulatory effect on the production of specific alkaloids. This is most obvious in theincreased peak area of later eluting peak J where ajmalicine and catharanthine coelute,Figure 3.19.1483.3.9.3 Effect of methylarsonate on alkaloid productionMethylarsonate has an inhibitory effect on cell alkaloid production when added atthe beginning of growth. Cells harvested after 22 days of growth contains a few alkaloidalmetabolites which are assigned to ajmalicine and yohimbine, both corynanthe alkaloids.After 29 days, ajmalicine is completely absent but several other alkaloidal metabolites aredetected including antirhinet (ni/z 297), which were present in the control culture at lowconcentrations. The failure to detect ajmalicine in cells after 29 days could indicate that it ispresent at very low concentrations or it is transformed or catabolized inside the cells overtime. Cell lysis can also release alkaloids into the medium resulting in a lower intracellularconcentration of alkaloids.Application of methylarsonate after 11 days of growth resulted in low alkaloidaccumulation. A few of the characteristic alkaloids detected in the control cells are found inthese cells but only at low concentrations. Cells treated on day 22 contain a variety ofalkaloids including catharanthine of iboga class, all at low concentrations. The metabolite ofm/z 297 is again present in these cells. Another alkaloid that consistently appeared inmethylarsonate treated cells is horhaxnmericinet (mlz 369). It is not present in the controlculture but has been previously reported in C. roseus cultures.1’8The overall low intracellular concentration of alkaloids in methylarsonate treatedcells can be attributed to cell lysis releasing alkaloids in to the residual medium. This issupported by the low cell yield (both fresh and dry cell weights) of cultures treated withmethylarsonate on day 11 as well as day 0. The cell yields after treatment on day 22 is notreduced more than 10% of those treated with other arsenic compounds. Cell lysis could alsobe suggested by the turbid appearance of the spent medium.1493.3.9.4 Effect of dimethylarsinate on alkaloid productionTreatment of cultures with ditnethylarsinate during the early growth stages resultsin a drastic inhibitory effect on alkaloid production. But the cell culture growth is notaffected as cell yields are bigher than those treated with other arsenic compounds. Theculture appearance also is not affected and is comparable to that of the control.The common feature in all cultures treated with dimethylarsinate on day 0 andday 11 is tryptamine accumulation. It is detected as an extra peak T on HPLCseparation C Figure 3.8, 3.9) and accounted for about 40% of the total peak area whenanalyzed soon after extraction. N-acetyltryptarnine, an artifact produced by the reaction ofethyl acetate with tryptamine, is detected by using LC-MS.Tryptamine accumulation has been detected in C. roseus cell suspension culturestreated with other elicitors. These elicitors include fungal homogenates141 and abscisicacid. 142 Treatment with vanadyl sulphate on day 5 of growth resulted in tryptamineaccumulation but not when treated on day iO.107,1Elevated tryptamine levels in cells can result from increased tryptamine productionassociated with the stimulation of the enzyme, tryptophan decarboxylase (TDC). However,the net suppression of alkaloid accumulation in these cultures suggest that the next step,condensation of tryptamine with secologanin is most likely to be inhibited. The strictosidineproduced in this step is the precursor to all three groups of alkaloids produced by C. rose usplant systems, Figure 3.3. This step could be blocked as a result of two processes:* Dimethylarsinate inhibits activity of the enzyme, strictosidine synthase.* Dimethylarsinate has an inhibitory effect on at least one of the steps involved inthe production of secologanin.150The inhibitory effect of dimethylarsinate on the activity of an enzyme can again stemfrom two different ways:* Dimethylarsinate acts as an inhibitor of the enzyme.* The enzyme is not synthesized in the presence of dimethylarsinate.Further studies need to be carried out to investigate these possibilities.When C. roseus cells are treated with dimethylarsinate on day 22, tryptamineaccumulation is not detected. This observation fits in with the notion that dimethylarsinateblocks alkaloid production early in the pathway; by day 22 of growth, tryptaminecondensation has already taken place, and thus the addition of dimethylarsinate does nothave any profound overall effect on alkaloid accumulation.Thus in this culture, several corynanthe, strychnos and aspidosperma alkaloids aredetected which are also found in the control. But a notable absence is that of catharanthine.Catharanthine is produced to a large extent only after day 22. Thus, dimethylarsinate addedon day 22 can effectively stop catharanthine production. This indicates thatdimethylarsinate interferes with the pathway leading to catharanthine. A metabolite ofrnlz 355 (tentatively assigned to yohimbine (corynanthe)) is found to accumulate in thesecells. This may be the result of a blockage of a step leading to catharanthine, an ibogaalkaloid.151CHAPTER 4APPLICATION OF WHOLE CELL NMR TECHNIQUES TO STUDY THEINTERACTION OF ARSENIC COMPOUNDS WITH CATHARANTHUS ROSEUS4.1 INTRODUCTIONNMR spectroscopy is an important technique for the study of biological fluids andintact cells. It is nondestructive and noninvasive and allows the direct study of ceflularmetabolic processes at a molecular level. The most often studied nuclei in bioogica1 systemsare 1H, 13C and 31P, other nuclei including 15N, 23Na and 19F being studied to a lesserextent.172Use of in vivo NMR spectroscopy to study the effect of an added chemical species onplant cell metabolism has unique advantages. In vivo NMR spectroscopy offers thecapability to follow metabolic changes continuously on a single sample and it is free fromuncertainties associated with biochemical methods involving analysis of cell extracts. It a’soyields information on compartmentation in plant cells.173 The site at which a chemica’species is accumulated is important in the evaluation of its effect on metabolism. Membranetransport of any molecule that gives an observable NMR signal can also be detecteddirectly.174However, a limitation in NMR spectroscopy of intact cells is its relative insensitivity.NMR spectroscopy is suitable for studying only the most abundant chemical species insidethe cells. Thus, in vivo NMR techniques have been largely restricted to the study of majorpathways of metabolism.1524.1.1 1H NMR spectroscopy of intact cells1H NMR spectroscopy in principle, has an advantage in that its sensitivity is muchbetter than those of NMR methods involving other nuclei. But the abundance of hydrogen inbiological material results in complicated 1H NMR spectra which makes their study moredifficult. Another problem associated with 1H NMR studies is the high concentration ofwater in biological fluids. The water resonance obscures a large portion of the spectrum andalso creates a dynamic range problem during data acquisition.Various approaches are employed to simplify the proton NMR spectra of biologicalsystems. To eliminate the water resonance, a variety of water suppression methods are inuse. Presaturation of the water resonance is used successfully in many biologicalsystems.172 The broad envelope of signals arising from membrane and plasma proteins canbe eliminated by using specific pulse sequences which make use of the difference inrelaxation times of large protein molecules and smaller solute molecules in the cells.172The Hahn spin-echo pulse sequence, one such approach, establishes a delay inacquisition in order to selectively eliminate signals from macromolecules. Application ofspin-echo NMR spectroscopy to intact red blood cells was first reported in 1977.175Spin-echo NMR studies of intact blood cells and plasma are of great clinical interest as theycan provide information on diseases and drug metabolism.176’7 It has also been used tostudy the binding of several heavy metal species including Hg (II) and CH3Hg inerythrocytes.178 Another application is the measurement of pH inside the humanerythrocyte.179 An indicator species, imidazole, is introduced into the cells and its chemicalshift is used to obtain a value of 7.3 for the internal pH of red blood cells. 1791H spin-echo NMR studies have been used to monitor the biochemistry of arsenicalsin human erythrocytes.180 Dramatic changes in the NMR spectra of erythrocytes on153exposure to dimethylarsinate (DMA) indicates oxidative stress. The decrease in intensity ofthe DMA signal with time is attributed to the reduction of the arsenical to aMe2As-S- species that is bound to a transmembrane protein.180 The adduct formation ofanother arsenical, phenyldichioroarsine with suiphydryl containing compounds in guinea pigred blood cells was investigated by using 1H spin-echo NMR spectroscopy.1811H Correlation NMR spectroscopy has been used to study the microorganism, E. coilwhere metabolites that diffuse through the cell membrane are observed.182 Several cellularcomponents were identified and some dynamic aspects of glucose metabolism in E. coil weremonitored by using time course studies.1H NMR spectroscopy has seldom been used for direct studies of plant cells or tissue.The few applications to date have used the shape and the multi peak pattern of the watersignal of plant leaves to obtain information on leaf structure.1834.1.2 31P NMR spectroscopyTo date, the 31P nucleus has been studied most in in vivo metabolic studies. Thisnucleus has a natural abundance of 100% and a high inherent NMR sensitivity. Becausethere are few phosphorus containing molecules at detectable levels, 31P NMR spectra ofintact cells are relatively simple and easily interpreted. For the same reason, however,information available from 31P NMR is limited.The first report of a 31P NMR study of whole tissue was that of Moon and Richards,on human erythrocytes.184 Since then, a large number of applications of 31p NMR havebeen reported including the elucidation of intracellular pH18548land the study of energyutilization 188 in living plant cells.15431P NMR studies of Catharanthus roseus (periwinkle) cells were first reported in1984 and signals from several phosphorus containing metabolites were assigned.189 Twopeaks arise from inorganic phosphate (P1), in the cytoplasm and the vacuole, because theposition of the P1 resonance is dependent on pH. From the chemical shift positions of the P1resonances, internal pH values of 7.3 and 5.7 were found for the cytoplasm and the vacuolerespectively. 190A 31P NMR study of isolated vacuoles of C. roseus was carried out.’9’ Cells wereincubated in phosphate enriched medium prior to the isolation of vacuoles and 31P NMRspectroscopy was used to follow the kinetics of H+ exchange across the tonoplast, themembrane surrounding the vacuole. 1914.1.3 13C NMR spectroscopy13C NMR spectroscopy of intact cells and tissue suffers from the inherent low NMRsensitivity and the low natural abundance of the 13C nucleus. 13C NMR spectra at naturalabundance require significantly longer accumulation times than 1H or 31P. For this reason,‘3C NMR studies of intact tissue have been limited to systems which are relatively stable asa function of time.In order to study rapid metabolic processes in intact cells by using 13C NMR,13C enriched compounds are incorporated into the cells. This has the advantage that arelatively simple spectrum is obtained that contains resonances from the enriched compoundand its metabolites superimposed on a much weaker background.192Natural abundance as well as enriched 13C NMR studies of several biologicalsystems have been reported.192’3 Time course 13C NMR studies of U- 13Ci glucose fedE. coli cells aided in monitoring glycolysis intermediates and products in different growth155environments.193’4 Prominent resonances from glucose and fructose were present in the13C NMR spectra of fruit of the plantAucubajaponica (aoki )19513C NMR spectroscopy was also used to study crassulacean acid metabolism (CAM)in intact leaves of a plant, Kalanchoe.196 Signals from C-4 malate were observed afterovernight exposure of the leaves to 13C02. The chemical shift of malate is pH dependentwhich allowed the estimation of vacuolar pH from the whole leaf spectrum. 1964.1.4 The application of ‘H Spin-echo NMR spectroscopy of intact cellsThe original Hahn spin-echo pulse sequence (90°-t-180°-t-acquisition), creates atime delay (2t) between signal generation and accumulation.’97’198 A 1800 refocusingpulse is applied midway through the spin-spin relaxation time. This pulse eliminatesmagnetic field inhomogeneity effects. 197In a spin-echo NMR spectrum, broad signals from large molecules in the cells areselectively eliminated. This technique is based on the different rates of decay of transversemagnetization of the various protons.197 Specifically, the shorter the spin-spin relaxationtime (T2), the faster the rate of decay. Large molecules have short spin-spin relaxationtimes. Thus, the signals arising from the large molecules can be eliminated from thespectrum by delaying acquisition until their transverse magnetization has decayed to asufficiently small value. Only the signals from the smaller molecules in the cytosol are thenobserved in the spin-echo spectrum.The Carr-Purcell-Meiboom-Gil] (CPMG) pulse sequence, (90°- (t180°t )-acquisition) is a modified version of Hahn spin-echo technique and makes use of multiple180° refocusing pulses during the spin-spin relaxation period. The CPMG pulse sequence incombination with a presaturation pulse to suppress the water resonance, has been used to156obtain NMR spectra of human erythrocytes and plasma.172’97 Recently, Verpoorte et al.-99used a 1H NMR technique employing the CPMG pulse sequence to analyze the cell extractsand the medium from a Tabernaemontana divaricata plant cell suspension culture. Theyhave not been successful in their attempt to apply this technique to intact plant cells. 199There are several difficulties associated with the spin-echo technique. Theintensities of the resonances depend on the spin-spin relaxation time of each solute speciesand thus do not reflect the absolute concentration.197 The coupling constants are alsoaffected. The spectra have phase modulated signals and as a result, peak integration is notpossible.20° However, the peak heights of signals do reflect the relative ratio of the solutespecies. Thus, the relative change in concentration of a solute species can be determined bythe introduction of a suitable reference compound or the identification of an invariant speciesinside the celL20°1574.1.5 Scope of workThe application of 1H spin-echo NMR spectroscopy to study intact cells of C. roseus isnovel. The growth and secondary metabolism of C. roseus cell suspension cultures have beenextensively studied by previous workers, (See Chapter 3). Much is known about the effectof medium composition and abiotic (chemical) as well as biotic elicitors on alkaloidproduction of these plant cell cultures but a biochemical basis for these changes has not beenestablished. In vivo NMR studies may facilitate the direct observation of any changes in theprimary metabolism on application of these external agents.In the present study, the interaction of arsenicals with C. roseus cells wasinvestigated by using 1H spin-echo NMR spectroscopy. Two approaches have been employed.The first involves the recording of NMR spectra at different times in the growth cycle of theC. roseus culture growing in the presence of an arsenical and facilitates the monitoring of thelong term effects of the arsenical. In the second approach, 1H spin-echo NMR spectroscopy isused to follow the rapid, biochemical changes continuously in a cell sample on treatmentwith large doses of arsenic compounds. Transport of the arsenic compounds, methylarsonateand dimethylarsinate, across the cell membrane and accumulation inside the cells are alsoinvestigated.1584.2 EXPERIMENTAL4.21 NMR parametersC. roseus cells were harvested at the stationary phase, washed three times withdeuterium oxide (D20, MSD Isotopes, Canada) to remove excess medium and then packedinto a 5-mm NMR tube (Norell 507-HP). 1H NMR spectra were recorded on aBruker WH 400 spectrometer using a standard 5-mm probe. The Carr-Purcell-Meiboom-Gill(CPMG) pulse sequence was used to obtain the spin-echo NMR spectra and the delay time(t) was typically 30 ms. A schematic representation of this pulse sequence is given inFigure 4.1. A small presaturation pulse was applied to the water resonance prior toaccumulation. An acquisition time of 0.426 a was employed, the spectra] width was 5000 Hz.Typically, 90° and 180° pulse widths were 11 and 22 s, respectively. The free inductiondecay was collected in 4 K of data points zero filled to 32 K A 0.1 Hz line broadeningfunction was applied during Fourier transformation. All samples were maintained at27±2° C and spun at 20 Hz during data collection. Generally, 200 transients were collectedfor each spectrum.900 1800AcquisitionOerDn fl ‘ I I‘SoturotionchannelFigure 4.1 A schematic representation of the Carr-Purcell-Meiboom-GIU(CPMG ) pulse sequence1594.2.2 Culture growthC. roseus cell suspension cultures were grown as described in Section 2.2.3. Culturesgrown in both 1-B5 and APM media were monitored by LH spin-echo NMR spectroscopy after10 and 21 days of growth.4.2.3 NMR monitoring of cells growing in APM containing arsenic compoundsC. roseus cell suspension cultures were grown in 250 mL flasks containing 100 mL ofAPM each. The control cultures did not contain any added arsenic. Others contained either3 ppm of arsenate, 4 ppm of arsenite, 4 ppm of methylarsonate or 15 ppm ofdimethylarsinate. Cells were removed aseptically at different times of growth and preparedfor the NMR experiment as described in section 4.2.1.4.2.4 Uptake of arsenic compoundsC. roseus cells (0.5 mL, packed cell volume) from a culture grown in 1-B5 mediumthat had reached stationary phase, were prepared for the NMR experiment as outlined inSection 4.2.1. A specific amount of the arsenic compound was dissolved in 0.5 mL of D20prior to adding to the NMR tube.1604.3 RESULTS AND DISCUSSION4.3.1 General features of 1H NMR spectra of C. roseus cellsA typical 1H NMR spectrum of C. roseus cells obtained after suppression of the waterresonance is depicted in Figure 4.2 a. The cells are from a C. roseus cell suspension culturegrown in 1-B5 medium that had reached stationary phase after 10 days of growth. Thisspectrum is characterized by a water resonance (4.7 ppm) and a broad envelope ofoverlapping signals arising primarily from the membrane and plasma proteins. To eliminatethis broad envelope of resonances, a modified version of the spin-echo pulse sequence, CarrPurcell-Meiboom-Gill (CPMG) pulse sequence (Figure 4.1 ) was used.172 This pulsesequence creates a time delay (4t = 120 ms) between signal generation and accumulation.The selective elimination of the broad, poorly resolved signals from large protein molecules isachieved on the basis of their short relaxation times (See Section 4.1.4). Only the signalsfrom the small molecules in the cytoplasm are observed in the resulting spectrum depicted inFigure 4.2 b.The CPMG NMR spectrum of C. roseus cells (Figure 4.2 b) contains several narrowresonances F, Q and R which probably arise from small, motile molecules in the cells. PeakS, T and U are substantially broader than the other resonances and may arise from slowlytumbling species in the storage vacuoles. These resonances are characteristic of spin-echoNMR spectra of C. roseus cells that have reached stationary phase in 1-B5 medium eventhough some variation in the relative intensities can be observed.1H spin-echo NMR spectroscopy allows the observation of small molecules in the cell,usually substrates and products of primary metabolism, present iii mM concentrations. 172These spin-echo NMR detectable solutes may include organic acids such as members of the161tricarboxylic acid (PCA) cycle, amino acids and sugars, both mono and disaccharides. Thesesolutes are present in the cytosol and the vacuolar sap of plant cells. 173,201 These signalswere further investigated only if they were shown to be affected in the presence of arsenicals.(a)I I I I II I I1 0.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1 .0 0.0Chemical Shift (ppm)HR4.0 3.0 2.0 1.0 0.0Chemical Shift (ppm)Figure 4.2 400 Mhz 1-H NMR spectra of a cell suspension of C. roseus(Cells were grown in 1-B5 medium for 10 days and thensuspended in 1)20.)(a) The spectrum measured with suppression of water signal bythe application of a presaturation pulse(b) The spin-echo spectrum measured using the CPMG pulsesequence, t = 30 ms, number of scans = 200(b)I I7.0 6.0 5.01624.3.2 1H NMR studies of C. roseu cells grown in the Alkaloid Production MediumC. roseus cells grown in Alkaloid Production Medium (APM) were monitored byusing 1H spin-echo NMR spectroscopy with the aim of identifying any biochemical changes inthe cells associated with the addition of arsenicals into the medium. APM was designed tooptimize alkaloid production by C. roseus cell cultures and has a different nutrientcomposition from that of the 1-B5 medium (See Appendix A). The effect of arsenicals onalkaloid production by C. roseus cell suspension cultures grown in APM was described inChapter 3.4.3.2.1 Changes in the control culture in APM with growth phase1H spin-echo NMR speetroscopy was used to monitor C. roseus cells from a controlculture growing in APM into which no arsenic has been added. A spectrum of cells grown in1-B5 medium for 10 days, then transferred to APM, is given in Figure 4.3 a. The spectrumwas obtained six hours after transferring to the new medium. The broadresonance W centered at 4.77 ppm arises from water and has been suppressed using apresaturation pulse. The intense resonances between 3.4 and 4.4 ppm and the smallresonance S’ at 5.41 ppm are assigned to sucrose on the basis of their chemical shiftpositions. Thus, the rapid uptake of sucrose into the cells from the medium, APM, whichcontains 5% sucrose is evident. The spectrum also contains resonances from othercomponents present in 1-B5 grown cells that had reached stationary phase. These peaks arepresent at 3.0, 2.76, 2.68, 2.54 and 2.45 ppm.Two days after transfer, the resonances attributed to sucrose (S and S’) are stillpresent, (Figure 4.3 b). But other resonances are absent with the exception of the peaks at2.45 ppm (Peak A) and at 2.12 ppm (Peak B), both of which have increased in intensity.163Peak A can be assigned to succinate, a tricarboxylic acid (TCA) cycle acid, on the basis ofthe chemical shift position whereas peak B which has a negative amplitude, is not assigned.After eight days in the APM medium, cells are in the growth phase and the spectrumshows some changes in its solute composition (Figure 4.3 c). High sugar levels are stillevident from peaks labelled S. Presence of both glucose and sucrose is suggested by the twopeaks arising from the hydrogen atoms on anomeric carbon atoms at 5.21 ppm (S”) and5.41 ppm ( S’ ) respectively.Sucrose in the medium is probably taken up by the cells into the cytoplasm andsubsequently converted to starch to be stored in starch granules. Sucrose can be utilized bythe cells only after conversion to glucose.201 Glucose is expected to be present in the cells atall times, but should be especially evident at times when cells have high energyrequirements, such as during growth phase. The presence of both glucose and sucrose after8 days of growth possibly indicate a lowering of sucrose levels and an increase in glucoselevels relative to 2 day old cells.Another change evident in the NMR spectrum of 8 day old cells is the decrease inintensity of both peaks A and B. This could be a result of utilization of these components bythe cells. Peak C at 1.17 ppm does not show a change in intensity.The H NMR spectra of cells 12 and 15 days after transfer, show essentially thesame trend of decreasing intensity ofpeaks A and B. A new resonance at 3.0 ppm is detectedafter 15 days. Sugar peaks CS) still dominate the area between 3.4 and 4.4 ppm, and thusmay mask any new peaks in this region.The spectrum of 23 day old cells grown in APM is given in Figure 4.3 d. Cells containlarge amounts of sugar and it is mostly glucose (peaks S and 5”). A broad multipletbetween 3.1 and 2.9 ppm is observed.164Chemical Shift (ppm)Figure 4.3 Spin-echo NMR spectra of C. roseus cell suspensions grown inAlkaloid Production Medium (APM)No arsenic was added to the medium.(a) 6 hours after transferring to the new medium(b) 2 days after transfer(c) 8 days after transfer(d) 23 days after transfer(a)HB,Z4B(d)SnI tjC6.0 5.5 5.0 4.5i,.. IllIlli iii4.0 3.5 3.0 2.5 2.0 1 .5 1 .0 0.5165C. roseus cell suspension cultures reach stationary phase around 23 days whengrown in APM.104 The high sugar concentration in APM probably accounts for therelatively high sugar content in the cells even up to 28 days of growth. The NMR spectra ofthe cells grown in APM show several differences from those grown in 1-B5 medium. Aresonance at 3.0 ppm is prominent in the NMR spectrum of 1-B5 growncells (See Figure 4.2 b ); it is present in the spectra of APM grown cells at stationary phase,but at a smaller intensity relative to the resonances attributed to the sugars. Several othercomponents present in 1-B5 grown cells are not detected in APM grown cells such as thespecies corresponding to the signals at 2.76, 2.68, 2.54 and 2.45 ppm.4.3.2.2 NMR monitoring of cells growing in APM containing arsenateFigure 4.4 shows the spin-echo NMR spectra of cells growing in the presence of3 ppm of arsenate. Six hours after transferring to the APM medium containing arsenate, thespectrum is swamped by the signals associated with sucrose indicating a high concentrationinside the cells. The NMR spectrum of arsenate treated cells after two days of growth isshown in Figure 4.4 a. Major changes from that of the control are evident. Sucrose is stillthe major component as seen from peaks S (between 3.4 - 4.4 ppm ) and peak 5’ at 5.41 ppm.Peaks at 3.2 and 3.0 ppm present here are not seen in the parallel control at two days ofgrowth. The cell metabolites corresponding to these peaks are present in the cells atstationary phase when grown in 1-B5 medium. But after transferring to the new medium,these components quickly disappear from the cytosol as seen in the control, Figure 4.3 b.Another difference in arsenate treated cells is that components that give rise to theresonances A and B in the NMR spectrum are present at much lower concentrations.166In 1H spin-echo NMR spectra of arsenate treated cells, the peaks A and B show anincrease in intensity only after 5 days as illustrated in Figure 4.4 b. Sugar peaks dominatethe spectrum and the peaks D (3.0 ppm) and E (3.2 ppm) are still present. The out-of-phase peak C at 1.17 ppm is larger in intensity compared to the control.The NMR spectrum of 12 day old arsenate treated cells shows a remarkablesimilarity to that of the 2 day old control cells as seen in the Figure 4.4 c. The only differenceis the higher intensity of peak C at 1.17 ppm and the accompanying peaks C’ and C”. It isevident that some changes in the biochemical content of the cells are delayed after theaddition of arsenate. This effect can be related to the delay in growth previously observed inC. roseus cell suspension cultures in the presence of arsenate (See Section 2.3.2.1).A decrease in the intensity of resonances A and B is observed after 15 days. By23 days, they completely disappear as seen from the NMR spectrum of cells given inFigure 4.4 d. The prominent peaks are from the sugar, sucrose, in comparison to the controlwhere glucose is present. This difference could be the direct result of a delay in growth, oran inhibition of growth resulting in a lower cell number when treated with arsenate, thatleads to an increased sucrose concentration per cell. Higher sucrose concentration probablymasks any glucose that is present.The resonance C (at 1.17 ppm) which has a greater intensity in arsenate treatedcells in comparison to the control cells, can be attributed to ethanol. The methyl resonance ofethanol falls at 1.17 ppm at physiological pH. The signal arising from the CH2 group ofethanol has a chemical shift position of 3.6 ppm and thus would be buried under the moreintense resonances from sucrose. The resonance C is even more pronounced in the spectrumof 28 day old cells and probably indicates accumulation of ethanol in cells because of cellstress in the presence of arsenate.167H6.0 5.0I I4.0 3.0I I • I I2.0 1.0 0.0Chemical Shift (ppm)Figure 4.4 Spin-echo NMR spectra of C. roseus cell suspensions grown inAlkaloid Production Medium (APM) containing 3 ppm ofarsenate(a) 2 days after transferring to the new medium containingarsenate(b) 5 days after transfer(c) 12 days after transfer(d) 23 days after transferS(a)s,(b)ACB1684.3.2.3 NMR monitoring of cells growing in APM containing arseniteThe spin-echo NMR spectra of cells growing in APM containing 4 ppm of arsenite aregiven in Figure 4.5. Cells, two days after transferring to the new medium, do not show anylag in their metabolism compared to the control. Biochemical changes seen in the controlspectrum are observed here as well. Resonances A and B are intense in the NMR spectrum(Figure 4.5 a). The presence of both sucrose and glucose is demonstrated by the peaks S’and S” at 5.41 ad 5.21 ppm respectively.Five days after transfer, cells give rise to a NMR spectrum essentially similar to thatof the control as depicted in Figure 4.5 b. Further changes in the biochemical composition ofthese cells with time show a similar trend to that of the control. After 15 days, the onlydifference from the control is the relatively higher intensity of the peak C at 1.17 ppm thatwas assigned to ethanol. The accumulation of ethanol could indicate cell stress similar toarsenate treated cells.At stationary phase, cells contain sucrose as well as glucose (S’ and S”) and asimilar trend is observed after 28 days as shown in Figure 4.5 d. A higher level of ethanol(peak C ) is also evident in these cells compared to the control.169S6.0ChemicaJ Shift (ppm)Figure 4.5 Spin-echo NMR spectra of C. roseus cell suspensions grown inAlkaloid Production Medium (APM) containing 4ppzn of arsenite(a) 2 days after transferring to the new medium containingarsenite(b) 5 days after transfer(c) 15 days after transfer(d) 28 days after transferH(a)S, S”AC‘B(b)(c)(d)5.0I I • I4.0 3.0 2.0 1 .0 0.01704.3.2.4 NMR monitoring of cells growing in APM containing methylarsonateThe cells growing in APM containing 4 ppm of methylarsonate (MMA) weremonitored by 1H spin-echo NMR spectroscopy. Six hours after transferring to the mediumcontaining MMA, the NMR spectrum of cells is given in Figure 4.6 a. It is dominated by theresonances arising from the high concentration of sucrose (peaks S and peak S’). Asignificant feature is the absence of a methylarsenic signal expected to be present at1.75 ppm. MMA is known to be taken up by the cells as seen from the analysis of extracts ofcells grown in MMA and the residual medium by using atomic absorption spectrometry (SeeChapter 2). The absence of this signal could be a result of a low concentration of MMA.Another possibility is the association of MMA species with larger molecules in the cytoplasm.The bound species would acquire a short relaxation time related to its large molecular sizeand thus would not appear in the spin-echo NMR spectrum of cells.Mter two days, peaks A and B are prominent in addition to the peaks S and S’assigned to sucrose. Biochemical contents of the cells show a close similarity to that of thecontrol up to 5 days of growth. The NMR spectrum of cells after eight days of growth in APMcontaining MMA, show some changes from that of the 8 day old control, as illustrated inFigure 4.6 b. Sugars still dominate the spectrum, both sucrose CS’) and glucose (S”) beingpresent. Peaks A and B show a decrease in intensity with time. Peaks D (3.0 ppm) andE (3.2 ppm) are present. An additional peak D’ at 3.1 ppm is present. The out-of-phasepeak C at 1.17 ppm assigned to ethanol has a high intensity similar to the spectra of otherarsenical treated cells. By 12 days, peaks A and B almost disappear, and peak C hasincreased in intensity even to a greater extent.All NMR spectra recorded after 15 days of growth in APM containing MMA, showcharacteristic changes in the appearance of the spectra. Peaks are much narrower compared171to those in the spectra of the control and the other arsenical treated cells. The signal to noiseratio is also poor. These changes probably arise from some physical changes in the cells.Cells usually acquire a fine texture when grown in MMA and cell lysis is prevalent. Whetherthis causes the change in the NMR spectra is not known.After 15 days of growth, cells still contain sugar, mostly glucose as depicted in theNMR spectrum in Figure 4.6 c. Peak 0 shows a decrease in intensity compared topeak 0’ (3.1 ppm) and peak E (3.2 ppm). The most significant is the appearance of a newresonance N at 2.21 ppm.In the NMR spectrum of cells after 17 days of growth, peaks 0, E and F disappear,however the new peak N is still evident. A similar spectrum is obtained for cells after23 days of growth as depicted in Figure 4.5 d. After 28 days of growth, most of the cells havelysed and it was difficult to obtain cells for NMR monitoring.C. rose us cell cultures growing in a medium containing MMA exhibit an unusuallylow pH. A 22 day old control culture has a pH of 5.8 whereas a MMA treated culture has apH of 3.8. This lowering of pH suggests that acidic components are accumulated in cells ontreatment with MMA. Previously, malic acid accumulation was reported in Johnson grasswhen treated with MMA where MMA acts as a specific herbicide against Johnson grass.73Malate accumulation has been observed in C. roseus cells when subjected to osmoticstress.202’3 However, the presence of malic acid is not evident in the NMR spectra ofC. rose us cells treated with MMA. At pH 3.9, malic acid is expected to give rise to tworesonances at 2.8 and 2.7 ppm which converge to give a single broad peak at 2.9 ppm at moreacidic conditions.In an attempt to establish the identity of the characteristic resonance at 2.21 ppm(peak N), 1H NMR spectra of extracts of cells grown in media containing MMA were172obtained. Both methanol and aqueous cell extracts resuspended in D20 (pH 3.75) do notcontain a metabolite which gives rise to a signal at 2.21 ppm. The NMR spectrum of asuspension of the cell residue in D20 does not show this extra resonance either. Therefore,during the extraction process, the metabolite either undergoes decomposition or itsmolecular environment is altered such that it does not gives rise to the expected NMR signal.This extra resonance N in the NMR spectrum may be assigned to MMA itself, whichis possibly sequestered in the vacuoles. The lower pH (<3.8) in combination with a weakassociation with another molecule in the vacuole may cause the shift in chemical shift. (Thechemical shift of MMA in D20 at pH 3.8 is 1.91 ppm.) The absence of a resonanceassignaNe to MMA in the cell extracts may indicate that MMA is now bound to a largermolecule with which it comes into contact during the extraction; the resultant shortrelaxation time may be responsible for its non-appearance in the spin-echo NMR spectrum.On spiking the cell extract with a relatively large dose of MMA C 5 mg / 1 mL of cell extract),the methyl resonance of MMAis observed at 1.91 ppm.173SFigure 4.6 Spin-echo NMR spectra of C. roseug cell suspensions grown inAlkaloid Production Medium (APM) containing 4ppm ofmethylarsonate (MMA)(a) 6 hours after transferring to the new medium containing MMA(b) 8daysaftertransfer(c) 15 days after transfer(d) 23 days after transfer(a)H5,(b)SA( c)BC(d)NI I IC6.0 5.0 4.0 3.0Chemical Shift (ppm)2.0 1 .0 0.01744.3.2.5 NMR monitoring of cells growing in APM containing dimethylarsinateThe transfer of cells into APM containing 15 ppm of dimethylarsinate (DMA) doesnot result in any drastic changes as illustrated in the 1H spin-echo NMR spectra given inFigure 4.7. Six hours after transfer into the new medium, the NMR spectrum of cellsresembles that of the control (Figure 4.7 a). Both sucrose and glucose are present asrevealed by the peaks S, 5’ and S”. Other metabolites found in 10 day old cells grown in 1-B5medium are also observed. Here again, the dimethylarsenic resonance is not observed whichmay indicate a relatively low concentration of DMA inside the cell. Uptake studies showthat DMA transport is not as rapid as that of MM.A, (See Section 2.3.3.4). An alternativeexplanation is that any DMA taken up is invisible in the spin-echo spectrum because of itsassociation with larger molecules in the cell.The spectra of cells treated with DMA resemble those of the control through days twoto five and no delay in growth can be detected. Resonances A and B increase in intensity byday 2 and then decrease again with time as the corresponding metabolites are utilized by thecells. A large amount of sucrose is present similar to the control.After 8 days of growth, the NMR spectrum of cells, Figure 4.7 b, exhibits a fewdifferences from that of the control. The major sugar is sucrose in DMA treated cells. Theother difference is an extra resonance X at 1.92 ppm, a negative peak. The same trend isobserved after 12 and 15 days.Figure 4.7 c shows the NMR spectrum of cells after 17 days of growth in the mediumcontaining DMA. The major sugar present is sucrose (peak S’). Peak A at 2.43 ppm hasalmost disappeared but peak B at 2.15 ppm is still present. The new peak X at 1.9 ppm ismore evident. The out-of-phase peak at 1.2 ppm may indicate the accumulation of ethanol.175At the stationary phase (after 23 days), the NMR spectrum of DMA treated cellscontain peak S’ indicating sucrose as the major sugar and also shows the new peak X. Thespectrum of cells after 28 days of growth is given in Figure 4.7 d which exhibits several newpeaks associated with stationary phase such as the broad peaks at 2.67 ppm and 2.26 ppm aswell as the peaks D (3.0 ppm) and E (3.2 ppm). The new peak X associated with DMAtreatment is present as a negative peak at 1.9 ppm. This resonance could be assigned toacetate on the basis of chemical shift; however acetate appears as a positive peak in a CPMGNMR spectrum. Another possibility is the assignment of peak X to a dimethylarsenicmoiety, but again, DMA appears as a positive peak at 1.84 ppm in spectra obtained with theCPMG pulse sequence in the physiological pH range. Association of the dimethylarsenicmoiety with another small molecule in the cell may result in the shape and the phase changebut no evidence for this can be obtained from the NMR spectrum. It is conceivable that anyresonances from the rest of this molecule are buried under the large sugar resonances.176S2.0 1 .0 0.0Spin-echo NMR spectra of C. roseus cell suspensions grown inAlkaloid Production Medium (APM) containing 15 ppm ofdimethylarsinate (DMA)(a) 6 hours after transferring to the new medium containing DMA(b) 8 days after transfer(c) 17 days after transfer(d) 28 days after transfer(a)S,(b)A(c)B(d)SL6.0Figure 4.75.0 4.0 3.0I I I I I IChemical Shift (ppm)1774.3.3 1H Spin-echo NMR studies on uptake and short term effects of arsenicals onC. roseus cellsTime course 1H NMR spectroscopy of intact cells can be successfully used to followrapid metabolic changes continuously in a single cell sample. The interaction of arsenicalswith animal cells has been previously monitored by using 1H spin-echo NMR spectroscopy.Dimethylarsinate induced changes were observed in the 1H spin-echo NMR spectra ofhuman erythrocytes.18° The interaction of another arsenical, phenyldichioroarsine, withother cellular components in guinea pig red blood cells has also been studied by using NMRspectroscopy.181In the present study, spin-echo NMR spectroscopy is used to follow the rapid changesin C. rose us cells continuously on exposure to arsenic compounds. The transport of thearsenicals, MMA and DMA, across the cell membrane into these plant cells can also bemonitored.4.3.3.1 Changes in a control cell sample in the NMR tubeMature C. rose us cells that have reached stationary phase in standard 1-B5 mediumwere used in the study. Cells that are packed into a NMR tube, usually in deuterated water,are under stress and expected to undergo changes in their biochemical content with time.Changes in a C. roseus cell sample (0.5 mL of packed cells in 0.5 mL of D20) weremonitored as a function of time and the resultant NMR spectra are given in Figure 4.8.Several signals from small molecules in the cells can be seen in the spectrumrecorded one hour after packing into the NMR tube (Figure 4.8 a). Peaks F, Q and R arenarrow peaks indicating small, motile molecules. Peaks S, T and U are substantiallybroader probably because ofT2 effects and restricted motility.178Peak R at 3.0 ppm, a prominent resonance in the spectrum, is characteristic of allcells at stationary phase when grown in 1-B5 medium. The intensity of the peak is notaffected with time in the NMR tube. Species corresponding to peaks P and Q are observed inall cells but relative concentrations are observed to vary.The spectra obtained 3, 5 and 8 hours after packing into the NMR tube are given inFigure 4.8 b, c and d. These spectra illustrate some of the changes that take place in thecells with time. The broad, phase modulated peak U centered around 2.5 ppm changes itsshape rapidly, accompanied by a decrease in intensity. After eight hours, the peak isreplaced by a positive, broad peak. Whether this peak corresponds to one species or severalis not known.Both peaks S (2.76 ppm) and T C 2.66 ppm ) decrease in intensity with time andprobably arise from the same molecule. These chemical shifts are indicative of citrate whichis known to be present in C. roseus cells at significant concentrations.202 After eight hours,a small, broad peak remains centered at 2.7 ppm. The disappearance of peaks S and T couldbe a result of either the molecule being used up in the metabolic processes in the cell orhydrogenldeuterium exchange.The new peaks 0 (3.63 ppm) and X ( 1.15 ppm), that become more intense withtime, can be assigned to ethanol, a product of anaerobic respiration by plant cells. Peak V at1.29 ppm can be assigned to lactate on the basis of its chemical shift at physiological pH.This peak increases in intensity as a function of time, indicating the accumulation of lactate.Peak Q also shows an increase in intensity with time. Another broad peak U’ centered at2.3 ppm first appears after 4 hours, and is not assigned.179H(a)7.0Chemical Shift (ppm)Figure 4.8 1H spin-echo spectra of a suspension of C. roseus cells(Cells were grown for 10 days in 1-B5 medium prior toharvesting.)Each spectrum was recorded(a) 1 h (b) 3 h (c) 5 h (d) 8 hafter packing into the NMR tube(b)(c)(d)Uvx6.0 5.0 4.0 3.0 2.0 1 .0 0.0•1804.3.3.2 Uptake of methylarsonateMethylarsonate (MMA) was introduced into C. roseus cells at two dosages. At thehigh dosage (3 mg / 0.5 mL of the cell suspension ), the methylarsenic NMR signal was foundto swamp the signals from cell components. Cells were monitored after treatment with alower dosage of MMA (0.3 mg / 0.5 mL of cell suspension), and the NMR spectra aredepicted in Figure 4.9. At the lower dosage, the intensity of the methylarsenic signal(Peak M) at 1.79 ppm increases with time. The relative signal intensity of themethylarsenic resonance is measured against the invariant signal R and its variation withtime which is consistent with cellular uptake of MMA, is given in Figure 4.10.Any NMR resonance in the spectrum of an intact cell sample may consist of a signalfrom a substrate outside the cell and a signal from a substrate inside the cell. 174,198 Theintensity of signal from inside the cell is greater when the spin-echo technique is used. Thus,an increase in peak intensity is associated with cell uptake of the substrate when a moietymoves from a NMR less sensitive (outside) to a more sensitive region (inside ).198 Theincrease in intensity of peak M is rapid at first and slows down after about 3 hours. Thisindicates that the cytosolic capacity of the cell to accumulate MMA is larger than its abilityto transform it immediately.Transport of a species across a cell membrane can be detected by the accompanyingchemical shift change if the pH of the two compartments vary, subject to the presence of apH sensitive group in the substrate. But in the case of MMA, the change in chemical shift isnot pronounced during a pH change from 7.4 (cytoplasm, outside, 1.77 ppm) to pH 5.7(vacuolar sap, 1.80 ppm).Inverting the NMR tube and centrifuging the contente, removes the cells to thecapped end and allows a simple method of analyzing the supernatant. Eleven hours after181adding MMA to the cells, very little MMA was found to be left in the supernatant, confirmingthat the bulk of the arsenical is inside the cell.Other changes in the spectra with time, Figure 4.9, have been observed in a C. roseuscell control run over a period of time without adding an arsenical, Figure 4.8. The peaks 0,P, U’, V and X increase in intensity. These could be byproducts of cell metabolism, someprocesses being accelerated due to the rigid conditions in the NMR tube.A new peak at 1.88 ppm CM’) was observed about 5 hours after adding MMA andfound to increase in intensity slowly. This peak can be assigned to acetate ordimethylarsenic signal on the basis of its chemical shift. Methylation of MMA by C. roseuscells was observed in previous speciation studies, but whether or not it takes place duringthe time frame of the experiment (a few hours) has not been confirmed. A methylatedproduct would have to be present in a high enough concentration to be detected in the NMRspectrum.A peak at 1.9 ppm was observed in the NMR spectrum of a a cell sample packed in aNMR tube for 5 days (Figure 4.11) and is assigned to acetate. These cells were underconsiderable stress and acetate was assumed to be a product of cell lysis. If acetate ispresent, M’ in Figure 4.9, it indicates that MMA exerts considerable stress on the cells,accelerating cell lysis.182HR7.0I I I I i I I I I I IChemical Shift (ppm)Figure 4.9 1H spin-echo spectra of a suspension of C. roseus cells treated withMMA (0.3 mg/0.5 mL of packed cells)Each spectrum was recorded(a)lh (b)3h (c)7h (d)lOhafter packing into the NMR. tube and treatment with MMA.(a)(b)p(c)(d)QRM X6.0 5.0 4.0 3.0 2.0 1.0 0.01830.40-..—0.30.—0.25020Time Lapsed (h)Figure 4.10 The variation of the relative signal intensity of methylarsenicresonance (M/R) with timeThe addition of M.MA to a cell sample which had been in a NMR tube for 5 daysprovided a good control. (These cells are judged to be dead in so far as no metabolism wasexpected because the cells were anaerobic and without a carbon source. ) There is no changein the intensity of the methylarsenic signal indicating that MMA is not taken up by thesecells. Incubation after adding sucrose to the sample also afforded no change.0 2 4 6 8 10 121844.0 3.5 3.0 2.5 2.0 1 .5 1 .0 0.5Chemical Shift (ppm)Figure 4.11 The 1H spin-echo NMR spectrum of a suspension of C. roseuscells packed in a NMR tube for 5 days4.3.3.3 Uptake of dimethylarsinateUptake of dimethylarsinate (DMA) by cells that had reached stationary phase wasmonitored by using spin-echo NMR spectroscopy after treating the cells withDMA ( 0.15 mg / 0.5 mL of cell suspension). A higher dose would swamp the signals fromthe cell components. The H spin-echo NMR spectra obtained at various time intervals aftertreatment are shown in Figure 4.12.The dimethylarsenic resonance labelled D is intense compared to the resonancesfrom other cell metabolites. One hour after treating the cells with DMA, dimethylarsenicresonance is at 1.75 ppm and it gradually shifts to 1.83 ppm four hours after treatment.Thereafter, the position remains constant. This lower field shift of the chemical shift of thedimethylarsenic resonance may demonstrate the movement of DMA into the vacuoles. At pH5.5±2, the vacuolar pH of C. roseus cells,190’1 the dimethylarsenic resonance appears aty1851.83 ppm, whereas at pH 7.3 and higher (pH of cytoplasm and outside medium), a chemicalshift of 1.7 is expected. Thus, the major accumulation site of DMA is demonstrated to be thevacuoles in C. roseus cells.An increase in intensity of the dirnethylarsenic resonance C peak D ) with time is seenin Figure 4.12. Figure 4.13 shows the variation of the relative intensity of signal Dmeasured against the invariant signal R, as a function of time. The increase in signalintensity with time demonstrates the transport of the substrate DMA from the NMR lesssensitive outside to the more sensitive inside. The uptake appears to be rapid for the first3 hours after treatment at this dosage and a slower increase is observed up to 11 hours.The biochemical changes in the cells with time, observed in other cell samples, areapparent in the spectra in Figure 4.12. However, there are several exceptions. Significantaccumulation of ethanol is not observed in these cells as peaks. at 1.15 (X) and3.65 ppm C 0) are absent. Lactate (peak V) does accumulate to a lesser extent. An out-of-phase peak at 1.05 ppm (peak X’) increases with time and has not been assigned.A resonance at 1.9 ppm is first observed in this cell sample after 7 hours(Figure 4.12 c) and increases in intensity with time; this peak is also present in MMAtreated cells and is assigned to acetate.Another resonance at 1.78 ppm is also detected after 7 hours of incubation and s’owlyincreases with time. This peak is not assigned. It could be the signal from a metabolicproduct of the substrate, DMA itself186(a)(c)7.0HDChemical Shift (ppm)Figure 4.12 1H spin-echo spectra of a suspension of C. roseus cells treatedwith DMA (0.15 mg/0.5 mL of packed cells)Each spectrum was recorded(a) 1 h (b) 3 h (c) 5 h (d) 8 hafter packing into the NMR tube and treatment with DMA.(b)(d)I • I6.0 5.0• I • I • I I4.0 3.0 2.0 1 .0 0.01873.5.—2.5::12Figure 4.13 The variation of the relative signal intensity of dimethylarsenicresonance (DIR) with time4.3.3.4 Effect of arseniteArsenate or arsenite uptake cannot be monitored by 1H NMR spectroscopy but theresultant changes in the spectra can be observed. The 1H spin-echo NMR spectra of a cellsample treated with arsenite (3 mg/ 0.5 mL of cell suspension) are given in Figure 4.14.The biochemical changes accompanying cell stress are much more rapid in C. roseus cellsafter adding arsenite compared with the control cell sample, Figure 4.8. The broad peak U ispositive in this spectrum. This resonance U is usually out-of-phase in spectra of other cellsamples packed in the NMR tube and is slowly altered to a positive peak over several hours.I . I . I . I . I2 4 6 8 10Time Lapsed ( h)188The spectra obtained several hours after adding arsenite (Figure 4.14 b, c) show thechanges observed in other samples such as an increase in the intensity of peaks P, Q and theappearance of a broad peak at 2.3 ppm. Ethanol (peaks 0 and X) and lactate (peak V)accumulation is apparent both of which indicate cell stress. A broad new peak around1.0 ppm is detected three hours after treatment.189(a)(c)vJChemical Shift (ppm)Figure 4.14 1H spin-echo spectra of a suspension of C. roseus cells treatedwith arsenite (3 mg/04 niL of packed cells)Each spectrum was recorded(a)lh (b)3h (c)5h (d)8hafter packing into the NMR tube and treatment with arsenite.(b)H(d)QP RUVX7.0I I I p I p p p p p I I I I p6.0 5.0 2.0 1 .04.0 3.0 0.01904.3.4 ‘Sc NMR spectroscopy of C. roseus cellsThe natural abundance ‘Sc NMR spectra of whole cells do not yield muchinformation about the cell components. 13C spectra of C. roseus cells at stationary phasegrown in 1-B5 medium and APM are shown in Figure 4.15. After growth in APM for23 days, the 13C NMR spectrum of cells show signals from glucose exclusively and isidentical to a spectrum of a standard solution of glucose. Signals from minor cell componentsare completely swamped by the very high concentration of glucose in the cells. In contrast,cells grown in 1-B5 medium for 11 days do not contain such a high sugar content and theNMR spectrum shows peaks from minor cell components.The 13C NMR spectra of cell samples that were treated with the arsenicals, MMAand DMA, were recorded. These spectra are characterized by poor signal to noise ratios.This may be a result of rapid changes in the biochemical content of cells associated witharsenical treatment within the time frame of the NMR experiment (a few hours). Hence,natural abundance 13C NMR spectroscopy does not prove to be a useful technique inobserving the interaction of arsenicals with living plant cells.However, the use of 13C enriched substrates such as MMA and DMA, which areincorporated into the cells is expected to yield valuable information about the uptake andmetabolism of the specific substrate. The spectra which can be recorded during a relativelyshort time, will consist of resonances from the enriched compound and their metabolitessuperimposed on much weaker background resonances.191(a)________________________I I I I I I I I I I80.0 70.0 60.0 50.0 40.0 30.0Chemical Shift (ppm)Chemical Shift (ppm)Figure 4.15 13C NMR spectra of C. roseus cell suspensions in D20(a) 13C NMR spectrum of cells grown in APM for 23 days(b) 1C NMR spectrum of cells grown in 1-B5 medium for11 daysI I11 0.0 1 00.0 90.0(b)I I- I I I I i i I I I I I200.0 1 60.0 1 20.0 80.0 40.0 0.01924.3.5 SummaryNMR spectroscopy of biological systems facilitates noninvasive, in-situ detection andanalysis of biochemical species. The study of intact plant cells by using 111 spin-echo NMRspectroscopy is a novel application although spin-echo NMR techniques have been previouslyutilized in studies of animal cells and biofluids. In contrast to NMR spectra of animal cells,most of which are of mammalian erythrocytes, the NMR spectra of cells from C. roseussuspension cultures are highly variable. This feature probably reflects the differencebetween a heterogeneous developing cell culture and fully differentiated subunits of asophisticated organism. The NMR spectra of C. roseus cells show variability depending onthe growth stage as well as the nutrient medium in which the cells are grown. These factorshave to be considered when 1H NMR spectroscopy is utilized in monitoring metabolicpathways in plant cell cultures.The CPMG pulse sequence creates a time delay (4 t ) between signal generation andaccumulation thus eliminating resonances from large molecules such as proteins on the basisof their short spin - spin relaxation times. The spectra thus obtained contain resonancesonly from small or highly mobile molecules. The NMR spectra of C. roseus cells arecomplicated and further work is required for the complete assignment of resonances arisingfrom various cell metabolites.The short term uptake of methylarsenicals is monitored continuously by1H spin-echo NMR spectroscopy in cells that had reached stationary phase in 1-B5 medium.Monitoring of cells in a D20 solution containing the methylarsenicals, either MMA or DMA,shows an increase in the peak height of methylarsenic resonance over a period of 11 hours,corresponding to the uptake of each. However, there is no evidence of any biotransformationproducts of arsenicals in the 1H spin-echo NMR spectra. Low concentration of any193transformation products may preclude their detection by the NMR method. Theaccumulation site of DMA is probably the vacuole as is seen from the change in the chemicalshift of DMA when it moves into a compartment of lower pH.The longer term effects of arsenicals on the metabolism of C. roseus cells during itsgrowth cycle are monitored by 1H spin-echo NMR spectroscopy. Comparison with a controlculture aids in identif’ing any changes in the cellular contents associated with the presenceof arsenicals. Although accumulation of arsenicals has been demonstrated by otheranalytical techniques such as NAA and AAS, resonances corresponding to both MMA andDMA are not usually observed in the 1H NMR spectra of cells grown in the presence of thesearsenicals in Alkaloid Production Medium. The association of these arsenicals with largebiomolecules in the cell may account for the absence of these resonances. In this event, thespin spin relaxation time of the arsenic species will shorten and the signals will not be seenin the spin-echo NMR spectrum. A resonance at 2.2 ppm is observed only in cells growing inAPM containing MMA longer than 15 days and can be assigned to the arsenical, MMA. Thechemical shift is indicative of high acidity and an altered environment around the species.194CHAPTER 5CONCLUSIONSVarious aspects of the interaction of arsenicals with a terrestrial plant have beeninvestigated by using cell suspension cultures of Catharanthus roseus as a model system.The Minimum Inhibitory Concentration (MIC) of arsenic compounds to C. roseus wasestimated on the basis of their effect on growth. The toxicity effects show a variation withthe growth medium, that may stem from changes in metabolic processes, including uptake ofarsenicals.The MIC of arsenate is the lowest in both growth media, standard 1B5 medium andAlkaloid Production Medium. The high toxicity may arise from the mode of action of thearsenical as well as a facile uptake mechanism. Arsenic concentrations as high as1650 j.ig (1 of dry cells are detected when C. roseus cell suspension cultures are grown inmedia containing 9.4 jig mL of arsenate, indicating an efficient uptake mechanism, whichmay be the same as that of phosphate. Arsenite is less toxic than arsenate for C. roseus,although the reverse is usually observed in other biological systems.The toxic limit for methylarsonate (MMA), a selective herbicide, is close to that ofarsenite and is also dependent on the growth medium. Dimethylarsinate (DMA) is the leasttoxic of the arsenicals, with normal growth rates observed up to 50 jig mL4 of arsenic in themedium. The low uptake of DMA by C. roseus probably accounts for its low toxicity.Accumulated levels of DMA in cells are low, only 250 jig g4 after 13 days of growth in amedium containing 50 jig mL4 DMA. Thus C. roseus cells exhibit an ability to exclude thisarsenical from cells, which may be attributed either to an active process which prevents the195uptake of DMA through the cell membrane, or to an efflux mechanism where DMA isexcreted out of cells.The biotransformation processes of arsenic in C. roseus were investigated. Speciationof arsenic in cell extracts shows methylation of arsenate, MMA and DMA by C. roseus.Demethylation of both MMA and DMA takes place to a greater extent than methylation andthis is the first report of demethylation of arsenicals in a terrestrial plant system. There isno evidence for the formation of significant levels of complex organoarsenic species inC. roseus cells. This terrestrial plant system seems to lack the ability to produce complexorganoarsenic compounds, although it is commonplace in both marine and fresh waterplants.The effect of various arsenic compounds on the secondary metabolism of C. roseus cellsuspension cultures was investigated with a view to understanding the mode of action ofarsemcals in plant systems. The effect is found to vary with the arsenic species and itsconcentration as well as the time of application.Arsenate, MMA and DMA, all have an overall inhibitory effect on alkaloid productionby C. roseus cell suspension cultures, when added early in the growth cycle, at both day 0and day 11. Treatment with arsenate or MMA results in the production of lowconcentrations of a number of alkaloids, that can be isolated after 22 and 29 days of growth.Catharanthine, an iboga alkaloid is noticeably absent. Iboga alkaloids are produced last inthe biosynthetic pathway and the arsenicals may have either influenced the production ofthe precursors of this alkaloid or inhibited a step in the synthesis of catharanthine itself.DMA has a drastic effect on alkaloid production although the rate of growth has beenunaffected: tryptamine, an early precursor of indole alkaloid biosynthesis, accumulates inthese cells. Stimulation of the enzyme, tryptophan decarboxylase (TDC), may give rise to196increased levels of tryptamine, which in turn should increase the overall production of indolealkaloids. Since this is not observed, the accumulation of tryptamine may be attributed tothe inhibition of tryptarnine utilization to produce strictosidine which is the precursor to allthree groups of indole alkaloids produced in C. roseus plant systems. There are at least twoways in which DMA may inhibit the condensation of tryptamine with the terpenoid,secologanin, to produce strictosidine. DMA may either inhibit the activity of the enzyme,strictosidine synthase, thereby inhibiting strictosidine production, or inhibit at least one ofthe steps involved in the production of secologanin. DMA may act as an inhibitor of theenzyme itself or it may inhibit the synthesis of the enzyme.Application of arsenicals to growing C. roseus cultures during the stationary phasehas a variable response which is dependent on the arsenic species. Unlike the otherarsenicals, the treatment with MMA on day 22 has an inhibitory effect on alkaloidproduction. This inhibitory effect of MMA can probably be attributed to cell lysis, therebyreleasing any previously produced alkaloids into the residual medium. The treatment witharsenate or DMA on day 22 has a stimulatory effect on the production of some alkaloids.When a C. roseus culture is treated with 3 ppm of arsenate on day 22 of the culture, anenhancement in overall production of alkaloids is observed. High levels of catharanthine aredetected in these cells. Treatment with 25 ppm of DMA at the stationary phase results in adistinct pattern in the alkaloids produced. Catharanthine is completely absent indicatingthat the production of the iboga group of alkaloids is inhibited by DMA. Ajmalicine, an earlyappearing corynanthe alkaloid, is detected although at a smaller concentration. Severalother alkaloidal components are detected at high concentrations, including yohimbine,vindolinine and epivindolinine.197Noninvasive, in-situ detection of biochemical species inside the plant cells wasachieved by using a 1H spin-echo NMR spectroscopic method. The CPMG pulse sequencecreates a time delay (4 t) between signal generation and accumulation thus eliminatingresonances from large molecules such as proteins on the basis of their short spin - spinrelaxation times. The spectra thus obtained contain resonances only from small or highlymobile molecules. 1H NMR spectra of C. roseus cells, obtained at different times in thegrowth cycle reveal biochemical changes associated with growth stage and the spectra showvariation depending on the nutrient composition in the growth medium. Further work isrequired for the complete assignment of resonances arising from various cell metabolites.The short term uptake of the methylarsenicals, MMA and DMA, by C. roseus cellswas monitored continuously over a period of 11 hours. The increase in the peak height of theniethylarsenic resonance corresponds to the accumulation of the arsenical in the cells.However, there is no evidence of any biotransformation products of the arsenicals in the1H spin-echo NMR spectra, probably because their concentrations are too low.The pH dependence of the chemical shift position of the methylarsenic resonanceprovides information about the chemical environment surrounding the arsenic species. Ashift in the position of methylarsenic resonance of DMA from 1.75 ppm to 1.82 ppm over aperiod of 4 hours indicates that DMA moves into a more acidic intracellular compartment,possibly the vacuole. This phenomenon of sequestering the arsenical from the rest of thecellular components as well as the low uptake may contribute to the low toxicity of thisarsenical to the growth of C. roseus cells.The longer term effects of arsenicals on the metabolism of C. roseus cells during itsgrowth cycle were monitored by using 1H spin-echo NMR spectroscopy. Comparison with acontrol culture aids in identifring any changes in the cellular contents associated with the198presence of arsenicals. In particular, the accumulation of some metabolites such as lactateand ethanol, dicates the cell stress that accompanies the treatment with arsenicals. Themethylarsenic resonances are conspicuously absent in the cells growing in alkaloidproduction medium containing either MMA or DMA. Since uptake and accumulation ofMMA and DMA by C. roseus cells are confirmed by other analytical methods, the absence ofthe methylarsenic resonances in these spectra can be attributed to the association of thesearsenicals with large biomolecules in the cells. In this event, the spin spin relaxation time ofthe arsenic species would shorten and the signals would not be seen in the spin-echo NMRspectrum. In cells growing in the presence of MMA, a new resonance is observed at achemical shift position 2.2 ppm after 15 days of growth. The shift in position of theresonance from 1.75 ppm expected at physiological pH, may indicate to an alteredenvironment around the arsenic species such as high intracellular acidity.Although 1H spin-echo NMR spectroscopy of intact plant cells retains many of theadvantages of an in-situ detection method, it has several limitations in monitoring the effectof arsenicals on plant cells. The complicated spectra preclude the complete assignment ofresonances arising from many plant metabolites. The uptake of inorganic arsenic speciescannot be monitored and any association of methylarseriicals with intracellular proteins andlarge biomolecules can only be predicted from the disappearance of the methylarsenicresonance.199BIBLIOGRAPHY1 Nationai Research Council, Arsenic; National Academy of Sciences: Washington, DC,1977,p16.2. CulIen, W. K; Reimer, K. J. Chem. Rev. 1989,89, 713.3. National Research Council of Canada The Effects ofArsenic in the CanadianEnvironment; Ottawa, 1978.4. Squibb, K. S.; Fowler, B. A. In Biological and Environmental Effects ofArsenic;Fowler, B. A., Ed.; Elsevier: Amsterdam, 1983; p 233.5. Andreae, M. 0.; Klumpp, D. W. Environ. Sd. Tech. 1979, 13, 738.6. Sanders, J. G.; Windom, H. L. Est.Coast Mar. Sci. 1980, 10, 555.7. Blasco, F.; Gaudin, C.; Jeanjean, R. C. R. Hebd. Seances Acad. Sci. Ser. D 1971,273,812.8. Osborne, F. H.; Ehlich, H. L. J. Appl. Bacteriol. 1976,41,295.9. Philips, S. E.; Taylor, M. L. Appi. Environ. Microbiol. 1976,32,392.10. Challenger, F.; Higginbottom, C.; Ellis, L. J. Chem. Soc. 1933,95.11. Challenger, F. Chem. Rev. 1945, 36, 315.12. Challenger, F.; Lisle, D. B.; Dransfield, P. B. J. Chem. Soc. 1954, 1760.13. Cantoni, G. L. J. Biol. Chem. 1953,204,403.Cantoni, G. L. J. Am. Chem. Soc. 1952, 74, 2942.14. Cullen, W. R.; Froese, C. L.; Lui, A.; McBride, B. C.; Patmore, D. J.; Reimer, M. J.Organomet. Chem. 1977, 139, 61.15. Cullen, W. R.; McBride, B. C.; Reimer, M. Bull. Environ. Contam. Toxicol. 1979,21,157.16. Cullen, W. R.; Erdman, A. E.; McBride, B. C.; Pickett, A. W. J. Microbiol. Methods1983, 1, 297.17. Cullen, W. R.; McBride, B. C.; Reglinski, J. J. Inorg. Biochem. 1984,21,45.18. Cuflen, W. R.; McBride, B. C.; Reglinski, J. J. Inorg. Biochem. 1984,21, 179.20019. Thayer, J. S. Organometallic Compounds and Living Organisms; Academic: New York,1984; p 189.20. Faust, S. D.; Aly, 0. M. Chemistry ofNatural Waters; Ann Arbor: Michigan, 1981;p 320.21. Klumpp, D. W. Mar. Biol. 1980,58,257; 265.22. Lunde, G. J. Sd. Food Agric. 1973,24, 1021.Lunde, G. Environ. Health Perspect. 1977,19,47.23. Edmonds, J. S.; Franceseoni, K. A.; Canon, J. R.; Raston, C. L.; Skelton, B. W.; White,A. H. Tetrahedron Lett. 1977,18, 1543.24. Maher, W.; Butler, E. Appl. Organomet. Chem. 1988,2, 191.25. CuIlen, W. R.; Dodd, M. Appl. Organomet. Chem. 1989,3, 79.26. Norm, H.; Ryhage, R.; Christakopoulos, A.; Sandstroem, M. Chemosphere 1983,12,299.Shiomi, K; Kakehasi, Y.; Yanianaka, H.; Kikuchi, P. AppI. Organomet. Chem. 1987,1,177.27. Edmonds, J. S.; Franceseoni, K A. Nature 1981,289,602.Edmonds, J. S.; Francesconi, K A. J. Chem. Soc. Perkin Trans. 11983,2375.28. Edmonds, J. S.; Morita, M.; Shibata, Y. J. Chem. Soc. Perkin Trans. I 1987,577.29. Edmonds, J. S.; Francesconi, K A. J. Chem. Soc. Perkin Trans. I 1982,2989.30. Shibata, Y.; Morita, M. Agric. Biol. Chem. 1988,52, 1087.31. Wrench, J. J.; Addison, R. F. Can. J. Fish Aquat. Sci. 1981,38,518.32. Edmonds, J. S.; Francesconi, K A. Experientia 1987,43,553.Edmonds, J. S.; Francesconi, K A. Appl. Organomet. Chem. 1988,2,297.33. Francesconi, K A.; Stick, R. V.; Edmonds, J. S. J. Chem. Soc. Chem. Commun. 1991,928.34. Edmonds, J. S.; Francesconi, K A.; Hansen, J. A. Experientia 1982,38,643.35. Francesconi, K. A.; Stick, R. V.; Edmonds, J. S. Experientia 1990,46,464.36. Francesconi, K. A.; Edmonds, J. S. Sci. Total Environ. 1989, 79, 59.37. Cullen, W. R.; Nelson, J., UBC, unpublished results.20138. Wauchope, R. D. In Arsenic: Industrial, Biomedical and Environmental Perspectives;Lederer, W. H.; Fensterheim, R. J., Eds.; Von Nostrand: New York, 1983; p 348.39. Kabata-Pendias, A.; Pendias, H. Trace Elements in Soils and Plants; CRC: Florida,1984.40. Woolson, E. A. In Biological and Environmental Effects ofArsenic; Fowler, B. A., Ed.;Elsevier: Amsterdam, 1983, p 51.41. National Research Council, Arsenic: Medical and Biological Effects of environmentalPollutants, National Academy of sciences, Washington, D.C., 1977, p 233.42. Warren, E. A.; Delavault, R. E.; Barakso, J. Econ. Geol. 1964,59, 1381.43. Porter, E. K.; Peterson, P. J. Sci. Total Environ. 1975,4,365.44. Girling, C. A.; Peterson, P. J.; Minski, M. J. Sci. Total Environ. 1978,10, 79.45. Otte, M. L.; Rozema, J.; Beek, M. A.; Kater, B. J.; Broekman, R. A. Sci. Total Environ.1990, 97, 839.46. Weaver, R. W.; Melton, J. R.; Wang, D.; Duble, R. L. Environ. Pollut. (Ser. A) 1984,33,133.47. Lunde, G. Acta Chem. Scand. 1973,27, 1586.48. Maeda, S.; Nakashima, S.; Takeshita, T.; Higashi, S. Sep. Sci. Technol. 1985,20, 153.49. Maeda, S.; Kumeda, K.; Maeda, M.; Higashi, S.; Takeshita, T. Appl. Organomet. Chem.1987, 1, 363.50. Maeda, S.; Wada, H.; Kumeda, K.; Onove, M.; Ohki, A.; Higashi, S.; Takeshita, T.Appl. Organomet. Chem. 1987, 1, 465.51. Nissen, P.; Benson, A. A. Physiol. Plant. 1982,54,446.52. Bibhas, R. Intern. Pest Control 1975, 17,9.53. Asher, C. J.; Reay, P. F. Aust. J. Plant Physiol. 1979,6,459.54. Sachs, R. M.; Michaels, J. L. Weed &i. 1971, 19, 558.55. Ashton, F. M.; Crafts, A. S. Mode ofAction of Herbicides; Wiley-Interscience:New York, 1973; p 147.56. Sckerl, M. M.; Frans, R. E. Weed Sci. 1969, 17, 421.57. Duble, R. L.; Holt, E. C.; McBee, G. G. Weed Sci. 1968, 16, 421.20258. Wauchope, R. D.; Richard, E. P.; Hurst, H. R. Weed Sci. 1982,30,405.59. Hiltbold, A. E. In Arsenical Pesticides; Woolson, E. A., Ed.; ACS Symposium Series 7;American Chemical Society: Washington, DC, 1975; p 53.60. Anderson, A. C.; Abdelghani, A. A.; McDonell, D. Sci. Total Environ. 1980, 16, 95.Anderson, A. C.; Abdelghani, A. A.; McDonell, D.; Craig, L. J. Plant Nutr. 1981,8, 193.61. Woolson, E. A. Herbicides - Chemistry, Degradation and Mode ofAction; MarcelDekker: New York, 1976; p 741.62. Webb, J. L. Enzyme and Metabolic Inhibitors; Academic: New York, 1966; p 505.63. Czarnecka, E.; Edelman, L.; Schoffi, F.; Key, J. L. Plant Mol. Biol. 1984,3,45.64. Cullen, W. R.; McBride, B. C.; Pickett, W. A. Appl. Organomet. Chem. 1990,4, 119.65. Benson, A. A.; Katayama, M.; Knowles, F. C. Appl. Organomet. Chem. 1988,2,349.66. Pyles, R. A.; Woolson, E. A. J. Agric. Food Chem. 1982,30,866.67. Reay, P. F. J. Appi. Ecol. 1972,9,557.68. Rhattak, R. A.; Haghnia, G. H.; Mikkelsen, R. L.; Page, A. L.; Bradford, G. R. J.Environ. Qual. 1989,18,355.69. Blatt, C. R. In Plant Nutrition and Physiology; Applications; VanBeusichem, M. L.,Ed.; Kiuwer Academic: Boston, 1990; p 303.70. Duble, R. L.; bit, E. C.; McBee, 0. 0. J. Agric. Food Chem. 1969,17, 1247.71. Domir, S. C.; Woolson, E. A.; Kearney, P. C.; Isensee, A. R. J. Agric. Food Chem. 1976,24, 1214.72. Odanaka, Y.; Tsuchiya, N.; Matano, 0.; Goto, S. J. Agric. Food Chem. 1985,33,757.73. Knowles, F. C.; Benson, A. A. Plant Physiol. 1983, 71,235.74. Benson, A. A.; Knowles, F. C. In Photosynthesis; Akoyunoglou, 0., Ed.; Proc. mt.Congr. 5th meeting, 1980; p 33.75. Farnsworth, N. R. Lloydia 1961,24,105.76. Wetter, L D.; Constabel, F. Plant Tissue Culture Methods; National Research Councilof Canada: Ottawa, 1981.77. Gamborg, 0. L.; Miller, R. H.; Ojima, K. Exp. Cell Res. 1968,50, 151.20378. Cullen, W. R.; Dodd, M. Appl. Organomet. Chem. 1988,2, 1.79. Hoenig, M.; DeBorger, R. Spectrochim. Acta 1983, 38B, 873.80. Brooks, R. R.; Ryan, D. E.; Zhang, H. Anal. Chim. Acta 1981, 131, 1.81. Chakrabarti, D.; Irgolic, K. J.; Adams, F. Intern. J. Environ. Anal. Chem. 1984, 17,241.82. Matousek, J. P. Prog. Anal. Atom. Spectros. 1981, 4, 247.83. Van Loon, J. C. Selected Methods of Trace Analysis: Biological and EnvironmentalSamples; Wiley: New York, 1985.84. Rothery, E., Ed.; Analytical Methods for Graphite Tube Atomizers; Varian Techtron,Victoria, Australia, 1982.85. Shan, X. Q.; Ni, Z. M.; Zhang, L. Anal. Chim. Acta 1983,151, 179.86. Schlemmer, G.; Welz, B. Spectrochim. Acta 1986, 41B, 1157.87. Styris, D. L.; Prell, L. J.; Redfied, D. A. Anal. Chem. 1991,63, 503.88. Sturgeon, R. E.; Berman, S. S. Anal. Chem. 1981,53,632.89. Robbins, W. B.; Caruso, J. A. AnaL Chem. 1979,51, 894.90. Braman, R. S.; Johnson, D. L.; Foreback, C. C.; Ammons, J. M.; Bricker, J. L.Anal. Chem. 1977,49, 621.91. Agterdenbos, J.; Bax, D. Fresenius Z. Anal. Chem. 1986, 323, 783.92. Anderson, R. K.; Thompson, M.; Culbard, E. Analyst 1986, 111, 1143; and 1153.93. Aggett, J.; Aspell, A. C. Analyst 1976, 101, 341.94. Howard, A. G.; Arbab-Zavar, M. H. Analyst 1981, 106, 213.95. Andreae, M. 0. Anal. Chem. 1977,49; 820.96. Arbab-Zavar, M. H.; Howard, A. G. Analyst 1980,105, 744.97. Reimer, K J. Appl. Organomet. Chem. 1989,3,475.98. Odanaka, Y.; Tsuchiya, N.; Matano, 0.; Goto, S. Anal. Chem. 1983,55,929.99. Mukai, H.; Ambe, Y. Anal. Chim. Acta 1987, 193, 219.204100. MacCarthy, J.; Ratcliffe, D.; Street, H. E. J. Exp. Bot. 1980, 31, 1315.101. Hirose, F.; Ashihara, H. Physiol. Plant. 1984,60, 532.102. Ashihara, H.; Tokoro, T. J. Plant Physiol. 1985,118,227.103. Kanamori, I.; Ashihara, H.; Komamine, A. Z. P’flanzenphysiol. Bd. 1973, 93S, 437.104. Kurz, W. G. W.; Chatson, K. B.; Constabel, F.; Kutney, J. P.; Choi, L. S. L.;Kolodziejczyk, P.; Sleigh, S. K.; Stuart, K. L.; Worth, B. R. Phytoehemistry 1980,19,2583.105. Irvine, J.; Jones, E. B. C. J. Inst. Wood Sd. 1975, 7, 20.106. Berlin, J.; Mollenschott, C.; DiCosmo, F. Z. Naturaforsch. 1987, 42C, 1101.107. Tallevi, S. G.; DiCosmo, F. Planta Medica 1988,54, 149.108. Friedberg, I. Bwchim. Biophys. Acta 1977,466,451.109. WiUski, G. R.; Malamy, M. H. J. Bacteriol. 1980, 144, 366.110. Vidal, F. V.; Vidal, V. M. V. Mar. Bid. 1980,60, 1.111. Knobloch, K. H.; Berlin, J. Plant Cell Tiss. Org. Cult. 1983,2, 333.112. Cullen, W. R.; Nelson, J. Appi. Organomet. Chem., in press.113. Lawrence, J. F.; Michalik, P.; Tam, G.; Canache, H. B. S. J. Agric. Food Chem. 1986,34, 315.114. CRC Handbook of Chemistry and Physics; Weast, R. C.; Astle, M. J., Eds.; 60th ed.,CRC: Florida, 1980.115. Manville, J.F.; McMullen, L H.; Reimer, K J. J. Econ. Entomol. 1988, 81, 1691.116. Svoboda, G. H.; Blake, D. A. In The Catharanthus Alkaloids; Taylor, W. I.;Farnsworth, N. R., Eds.; Marcel Dekker: New York, 1975; p 45.117. Blasko, G.; Cordell, G. A. Antitumor Bisindole Alkaloids from Cathararithusroseus (L.) Brossi, A.; Suffness, M., Eds.; The Alkaloids: Chemistry and PharmacologySeries 37, Academic: New York, 1990; p 1.118. Lounasmaa, M.; Galambos, J. InFortsch. Progress in Chemistry of Organic NaturalProducts Herz, W.; Grisebach, H.; Kirby, G. W.; Tamm, C. H. Eds.; 1989; 55, 89.119. Miura, Y. K.; Hirata, K; Kurano, N. Agric. Biol. Chem. 1987,51,611.205120. Endo, T. A.; Goodbody, J.; Vucovic, J.; Misawa, M. Phytochemistiy 1988,27,2147.121. Kutney, J. P. Heterocycles 1987,25,617.122. Kutney, J. P.; Botta, B.; Boulet, A.; Buschi, C. A.; Choi, L. S. L.; Golinski, J.; Guinulka,M.; Hewitt, G. M.; Lee, G.; McHugh, M.; Nakano, J.; Nikido, T.; Onodera, J.; Perez, I.;Salisbury, P.; Singh, M.; Suen, R.; Tsukamoto, H. Heterocycles 1988,27, 629.123. Groger, D. In Biochemistry ofAlkaloids; Mothes, K.; Schutte, H. R.; Luckner, M., Eds.;Deutscher Verlag Berlin, 1985; p 278.124. Attar-Ur-Rahman; Basha, A. Biosynthesis ofIndole Alkaloids; InternationalMonographs in Chemistry, 7, Clarendon: Oxford, 1983.125. Stockight, J.; Zenk, M. H. J. C. S. Chem. Comm. 1977, 646.126. Parry, K J. In The Catharanthus Alkaloids; Taylor, W. I.; Farnsworth, N. R., Eds.;Marcel Dekker: New York, 1975; p 141.127. Scott, A. I. Ace. Chem. Res. 1970,3, 151.128. Zenk, M. H.; El-Shagi, H.; Arens, H.; Stockight, J.; Weiller, E. W.; Deus, B. In PlantTissue Cultures and Its Biotechnological Applications; Barz, W. H.; Reinhardt, E.;Zenk, M. H., Eds.; Springer Verlag: Berlin, 1977; p 27.129. Carew, D. P.; Krueger, K J. Lloydia 1977, 40, 326.130. DeLuca, V., Kurz, W. G. W. In Cell Culture Somatic Cell Genetics ofPlants; Constabel,F.; Vasil, I. K., Eds.; Academic: New York, 1988; .VoL 5.131. Kurz, W. G. W.; Constabel, F. CRC Crit. Rev. Biotechnol. 1985,2, 105.132. Knobloch, K. H.; Berlin, J. Plant Cell Tiss. Org. Cult. 1983,2, 333.133. Morris, P. Planta Medica 1986,52, 122.134. Knobloch, K H.; Bast, G.; Berlin, J. Phytochemistry 1982,21,591.135. Drapeau, D.; Blanch, H. W.; Wilke, C. R. Planta Medica 1987, 53, 373.136. Huseman, W. H.; Fischer, K.; Mittelbach, I.; Hubner, S.; Richter, 0.; Barz, W. InPrimary and &cond.ary Metabolism ofPlant Cell Cultures; Kurz, W. 0. W., Ed.;Springer-Verlag Berlin, 1989; Vol. 2, p 43.137. Tyler, R. P.; Kurz, W. 0. W.; Panchuk, B. D. Plant Cell Reports 1986,5,427.138. Courtois, D.; Guern, J. Plant Sci. Lett. 1980, 17,473.206139. Morris, P. Plant Cell Reports 1986,5,427.140. DiCosmo, F.; Towers, G. H. N. In Recent Advances in Phytochemistry; Timmermann, B.N.; Steelink, C.; Loewus, F. A., Eds.; 1984; Vol. 18, p 97.141. Eilert, U.; Constabel, F.; Kurz, W. G. W. J. Plant Physwl. 1986,126, 11.142. Smith, J. I.; Smart, N. J.; Kurz, W. G. W.; Misawa, M. Planta Medwa 1987,53,470.143. Smith, J. I.; Smart, N. J.; Kurz, W. G. W.; Misawa, M. J. Exp. Botany 1987,38, 1501.144. Smith, J. I.; Smart, N. J.; Misawa, M.; Kurz, W. G. W.; Talevi, S. 0.; DiCosmo, F. PlantCell Reports 1987,6. 142.145. Kutney, J. P.; Choi, L. S. L.; Worth, B. R. Phytochemisti-y 1980,19,2083.146. Deus-Newnann, B.; Stockight, J.; Zenk, M. H. Planta Medwa 1987,53, 184.Lapinjoki, S.; Verajankorva, H.; Heiskanen, J.; Niskanen, M.; Huhtikangas, A.;Lounasmaa, M. Planta Medica 1987,53,565.147. Cone, N. J.; Miller, K; Neuss, N. J. Pharm. Sci. 1963,52,688.148. Jakovljevic, I. M.; Seay, L. D.; Shaffer, K W. J. Pharm. Sci. 1964,53, 553.149. Farnsworth, N. K; Blomster, R. N.; Damratoski, D.; Meer, W. A.; Cammarato, L. V.Lloydia 1964,27,302.150. Farnsworth, N. K; Hilinski, I. M. J. Chromatogr. 1965,18, 184.151. Verpoorte, R.; Svendson, A. B. Chromatography ofAlkaloids, Part B: Gas LiquidChromatography and High Performance Liquid Chromatography; Elsevier:Amsterdam, 1984; p 331.152. Kutney, J. P.; Choi, L. S. L.; Kolodziejczyk, P.; Sleigh, S. K; Stuart, K. L.;Worth, B. R.; Kurz, W. 0. W.; Chatson, K. B.; Constabel, F. Phytochemistry 1980,19,2589.153. Kohl, W.; Witte, B.; Hofle, 0. Planta Medica 1983,47, 177.154. Renaudin, J. P. J. Chromatogr. 1984,291, 165.155. Verzele, M.; Taeye, L. D.; Van Dyck, J.; Decker, 0. D.; DePauw, C. J. Chromatogr.1981,214,95.156. Naaranlahti, T.; Nordstrom, M.; Huhtikangas, A.; Lounasmaa, M. J. Chromatogr.1987,410, 488.207157. Van Der Heijden, R.; Lamping, P. J.; Out, P. P.; Wijnsma, R.; Verpoorte, R. J.Chromatogr. 1987,396,287.158. Renaudin, J. P. PhysioL Veg. 1985,23,381.159. Ylinen, M.; Suhonen, P.; Naaranlahti, T.; Lapinjoki, S. P.; Huhtikangas, A. J.Chromatogr. 1990,505,429.160. Naaranlahti, T.; Lapinjoki, S. P.; Huhtikangas, A.; Toivonen, L.; Kurten, U.;Kauppinen, V.; Lounasmaa, M. Planta Medica 1989,55,155.161. Balsevich, J.; Hogge, L R.; Berry, A. J.; Games, D. E.; Mylchreest, I. C. J. NaturalProd. 1988, 51, 1173.162. Yergey, A. L.; Edmonds, C. G.; Lewis, I. A. S.; Vestal, M. L. LiquidChromatography I Mass Spectrometry; Plenum: New York, 1990; p 31.163. Blakley, C. R.; Carmody, J. J.; Vestal, M. L. J. Amer. Chem. Soc. 1980, 102,5931.Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983,55,750.164. Auriola, S.; Ranta, V-P; Naaranlahti, T.; Lapinjoki, S. P. J. Chromatogr. 1989,474,181.165. Massey, V.; Hofmann, T.; Palmer, G. J. Biol. Chem. 1962,237,3820.166. Gresser, M. J. J. Biol. Chem. 1981,256,5981.167. Blakley, R. L.; McDougall, B. M. J. Biol. Chem. 1961,236, 1163.168. Ashburner, N.; Bonner, J. F. Cell 1979, 17, 241.169. Wang, C. H.; Gomer, R. H.; Lazarides, E. Proc. Natl. Acad. Sci. USA 1981,78,3531.170. Eilert, U. In Cell Culture Somatic Cell Genetics ofPlants; Vasil, I. K Ed. Academic:New York, 1988; V.4, p 174.171. Perley, J. E.; Stowe, B. B. Biochem. J. 1966, 100, 169.172. Rabenstein, D. L.; Millis, K. K.; Strauss, E. J. Anal. Chem. 1988,60, 1380A.173. Roberts, J. K M. Ann. Rev. Plant Physiol. 1984,35,375.174. Brindle, K. M.; Campbell, I. D. Quart. Rev. Biophys. 1987,19, 159.175. Brown, F. F.; Campbell, I. D.; Kuchel, P. W.; Rabenstein, D. L. FEBS Lett. 1977,82,12.176. Nicholson, J. K; Buckingham, M. J.; Sadler, P. J. Biochem. J. 1983,211, 605.208177. Reglinski, J.; Smith, W. E.; Suckling, C. J.; Al-Kabban, M.; Stewart, M. J.; Watson, I.D. Clin. Chim. Acta 1988,175,285.178. Rabenstein, D. L.; Isab, A. A.; Reid, R. S. Biochim. Biophys. Acta 1982,696, 53.179. Rabenstein, D. L; Isab, A. A. Anal. Biochem. 1982, 121, 423.180. Reglinski, J.; Smith, W. E.; Sturroek, D. Magn. Reson. Med. 1988,6,217.181. Dill, K.; O’Connor, R. J.; MeGown, E. I Inorg. Chim. Acta 1987, 138, 95.182. Ogino, T.; Arat.a, Y.; Fujiwara, S. Biochemistry 1980, 19,3684.Ogino, T.; Arata, Y.; Fujiwara, S.; Shoun, H.; Beppu, T. J. Magn. Reson. 1978,31, 523.183. McCain, D. C. In Nuclear Magnetic Resonance - Modern Methods ofPlant Analysis;Linskens, H. F.; Jackson, J. F., Eds.; Springer Verlag: Berlin, 1986; Vol. 2, p 127.184. Moon, R. B.; Richards, J. H. J. Biol. Chem. 1973,248,7276.185. Kuchel, P. W. CRC Crü. Rev. Anal. Chem. 1981, 12, 154.186. Gardian, D. G.; Radda, G. K; Richards, R. E.; Seeley, P. J. In Biochemical ApplicationsofMagnetic Resonance; Shulman, R. G., Ed.; Academic: New York, 1979; p 463.187. Roberts, J. K M.; Ray, P. M.; Wade-Jardetzki, N.; Jardetzki, 0. Nature (London) 1980,283,870.188. Foyer, C.; Walker, D.; Spencer, C.; Mann, B. Biochem. J. 1982,202,429.189. Brodelius, P.; Vogel, H. J. Annals New York Acad. Sci. 1984,434,496.190. Vogel, H. J.; Brodelius, P.; Lilja, H.; Lohmeier-Vogel, E. M. Methods Enzymol. 1987,135B, 512.191. Mathieu, Y.; Guern, J.; Kurkdjian, A.; Manigault, P.; Manigault, J.; Zielinska, P.;Gillet, B.; Beloeil, J. C.; Lallemand, J-YPlant Physiol. 1989,89, 19.192. Norton, R. S. Bull. Magn. Reson. 1980,3,29.193. Shulman, R. G.; Brown, P. R.; Ugurbil, K.; Ogawa, S.; Cohen, S. M.; DenHollander, J.A. Science 1979,205, 160.194. Ugurbil, K; Shulman, R. G.; Brown, T. II In Biological Applications ofMagneticResonance; Shulman, R. G., Ed.; Academic: New York, 1979; p 537.195. Kainosho, M. Tetrahedron Lett. 1976,4279.196. Stidham, M. A.; Moreland, D. E.; Siedow, J. N. Plant Physiol. 1983, 73, 517.209197. Rabenstein, D. L.; Nakashima, T. T. Anal. Chem. 1979,51, 1465A.198. Brindle, K M.; Brown, F. F.; Campbell, I. D.; Grathwohl, C.; Kuchel, P. W. Biochem. J.1979,180,37.Brown, F. F.; Campbell, I. D. Phil. Trans. R. Soc. Lond. 1980, B289, 395.199. Schripsema, J.; Erkelens, C.; Verpoorte, R. Plant Cell Reports 1991,9, 527.200. Reglinski, J.; Hoey, S.; Smith, W. E.; Sturrock, R. D. J. Biol. Chem. 1988,263, 12360.201. Goodwin, T. W.; Mercer, E. I. Introduction to Plant Biochemistry; Pergamon: Oxford,1982; 2nd ed., p 18.202. Renaudin, J. P.; Brown, S. C.; Barbier-Brygoo, H.; Guern, J. Physiol. Plant. 1986,68,695.203. Rudge, K.; Morris, P. In Plant Vacuoles- Their Importance in SoluteCompartmentation in Cells and Applications in Plant Biotechnology; Mann, B., Ed.;Plenum: New York, 1986; p 535.210APPENDIX AComposition of 1-B5 medium for C. roseuscell suspension culturesNutrients mg L1Sucrose 20000NaH2PO4 150KNO3 2500(NH4)2S0 134MgSO.7H0 250CaCI2.2H 150Iron 28Micronutrients ( tng L4 ): MnSO4.H20,10;H3B0,3; ZnSO4.7H20,2;KI, 0.75;Na2MoO4.2H0,0.25; CuSO4.5H0,0.025;CoCI2.6H0,0.025Vitamins (mg L4 ): nicotinic acid, 1; thiamine.HC1; 10;pyridoxine.HC1, 1; myo-inositol, 1002,4-dichiorophenoxyacetic acid ( 2,4-D) 1 mg L211Composition of Alkaloid Production Medium forC. roseus cell suspension culturesNutrients nig L1Sucrose 50000KH2PO4 68KNO3 950NH4O3 720MgSO.7H20 185CaC1.2H 220Iron 55.9Micronutrients ( mg L’ ): MnSO4.H20,7; ZnSO4.7H20,4;H3B0,2.4;glycine, 2; KI, 0.38;(NH6Mo7O24.40,0.09; CuSO4.5H20,0.01.Vitamins ( mg L ): myo-inositol, 20; nicotinic acid, 1; pyridoxine.HCI, 0.1;thiamine.HCI, 0.1.Growth Reçulators (mg L ): folic acid, 0.5; biotin, 0.05;indole-3-acetic acid, 0.17, 6-benzylaminopurine, 1.1212

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0061749/manifest

Comment

Related Items