Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies of tumor promoters and drug-metabolic enzymes in hamster buccal pouch mucosa Zhang, Dong M. 1993

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

Item Metadata

Download

Media
831-ubc_1993_spring_zhang_dong_ming.pdf [ 14.21MB ]
Metadata
JSON: 831-1.0086188.json
JSON-LD: 831-1.0086188-ld.json
RDF/XML (Pretty): 831-1.0086188-rdf.xml
RDF/JSON: 831-1.0086188-rdf.json
Turtle: 831-1.0086188-turtle.txt
N-Triples: 831-1.0086188-rdf-ntriples.txt
Original Record: 831-1.0086188-source.json
Full Text
831-1.0086188-fulltext.txt
Citation
831-1.0086188.ris

Full Text

STUDIES OF TUMOR PROMOTERS AND DRUG-METABOLIC ENZYMES INHAMSTER BUCCAL POUCH MUCOSAbyDONG MING ZRANGB.D.Sc., Beijing Medical College, 1983A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCESinTHE FACULTY OF GRADUATE STUDIES(OMSS, Faculty of Dentistry)We accept this thesis as conformingto the required andardTHE UNIVERSITY OF BRITISH COLUMBIAFebruary 1993© Dong Ming Zhang, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Tel.' ‘, tjr, (-/The University of British ColumbiaVancouver, CanadaDate --C) 4-/ (Signature)DE-6 (2/88)AbstractLack of tumor promoters has been the major obstacle in theuse of the hamster buccal pouch mucosa (HBPM) model. Twoexperiments were designed to investigate the effects of two newmouse skin tumor promoters, okadaic acid (OA) and methylmethanesulfonate (MMS), on HBPM.Short term effects of OA were studied. A single application of10 1.1.g of OA in 0.1 ml of acetone produced marked inflammation aswell as an increased mitotic rate (p<0.01) as compared to that of thecontrol. It, therefore, seems that OA possesses some essentialproperties of tumor promoter. Long term study is necessary to provethat it is a potent tumor promoter in HBPM.Tumor promoting effects of MMS were examined in a long termexperiment. 25 hamsters were divided into 3 groups. In group I,pouches of 10 hamsters were initiated with 7,12-dimethylbenzanthracene (DMBA), then promoted with MMS for 10weeks. In group II, pouches of 10 hamsters were initiated with MMSand promoted with MMS. In group III, pouches of 5 hamsters wereinitiated with DMBA (right pouches) or MMS (left pouches), andpromoted with acetone. The results showed that MMS had moderatetumor promoting effects but no tumor initiating effects in the model.Gamma-glutamyl transpeptidase (GGT) and placentalglutathione S-transferase (GST-P) have been found to be increasedduring HBPM carcinogenesis. Whether such increases are oncofetalremains to be answered. There are few studies on the normaldistribution of GGT and GST-P in hamster tissues. One experimentwas designed to study their tissue distributions during thedevelopment of hamster pouches and several selected organs andtissues. The results showed no GGT and GST-P activities in hamsterpouches during their development. The expression of GGT and GST-Pactivities during HBPM carcinogenesis may represent an acquiredgenetic alteration instead of oncofetal reversion. GGT was found inepithelial cells, particularly those with 'brush borders', in severalorgans and tissues, supporting the hypothesis that GGT mayparticipate in amino acid transportation. Rarely, GGT was also notedin mensenchymal cells. GST-P was observed in few organs and onlyexpressed in epithelium.TABLE OF CONTENTSAbstract^ i iTable of contents^ ivList of Tables viiLITERATURE REVIEW^ 1I. Chemical carcinogenesis^ 11. Multistage carcinogenesis: 1Initiation^ 2Promotion 2Progression^ 52. Tumor marker 53. The Hamster buccal pouch mucosa (HBPM) model^74. New tumor promoters^ 8Okadaic acid (OA) 9Methyl methanesulphonate (MMS)^ 1 0II. Gamma-glutamyl transpeptidase (GGT): 1 21. Structure^ 122. Metabolic roles 1 23. GGT and carcinogenesis^ 1 44. Tissue distribution 1 5III. Placental glutathione S-transferase (GST-P):^1 81. Structure^ 192. Metabolic roles 1 93. GST-P and carcinogenesis^ 204. Tissue distribution 23ivEXPERIMENT^ 2 7Experiment I.In vivo mitotic activity of OA on HBPM^ 271. Objective^ 272. Materials and method^ 27AnimalsOA dosage determinationAnimal treatmentMitosis assay3. Results^ 29Gross and histologyMitosis assay4. Discussion^ 30Experiment II.In vivo tumor promoting effect of MMS on HBPM^3 21. Objective^ 322. Materials and methods^ 3 2AnimalsMMS dosage determinationAnimal treatment3. Results^ 34GrossHistology4. Discussion^ 3 7Experiment III.Developmental studies on GGT and GST-P in hamsters'fetal, newborn and adult tissues and organs^3 9V1. Objective^ 3 92. Materials and methods^ 3 9AnimalsGGT staining methodGST-P staining method3. Results^ 41Intra- and para-oral organsExtra-oral organs4. Discussion^ 46GGTGST-P5. Conclusion^ 4 8Tables^ 50Figures 54Bibliography^ 7 9viList of TablesTable 1^Mitotic Activity of OA on HBPM^ 5 1Table 2.1 Initiation-promotion experimental design^5 2Table 2.2 Tumor promoting effect of MMS on HBPM 52Table 2.3 MMS tumor promoting results in HBPM^5 3Table 3^Normal distribution of GGT/GST-P in developingand adult tissues and organs of Syrian hamster^5 4viiLITERATURE REVIEWI. Chemical carcinogenesis1. Multistage carcinogenesisSir Percival Pott (1775) first noted a connection betweenhuman skin cancer and exposure to soot. Years later, Yamagiwa andIchikawa (1918) succeeded in introducing tumors in rabbit ears byrepetitive topical application of crude coal tar. Since then, severalgroups of chemical carcinogens have been identified and a number ofexperimental animal models for the study of carcinogenesis havebeen established. Among the carcinogens, polycyclic aromatichydrocarbone (PAH) carcinogens is the most common environmentalcarcinogens causing human cancers including oral cancers (Dipple,1985). Cramer and Stowell (1943) showed that a single largeapplication of carcinogen 20-methylcholanthrene, a kind of PAH,induced tumors in mouse skin. It was found that, in the mouse skinmodel, a single subthreshold dose of a carcinogen followed byrepetitive application of noncarcinogenic irritants, such as croton oiland wounding, also produced tumors (Berenblum and Shubik,1947a,b). It is now generally accepted that carcinogenesis is amultistage process in several animal systems, consisting of thedistinct and sequential stages of initiation, promotion, andprogression (Friedwald & Rous, 1944; Berenblum & Shubik, 1947a,b;Marks & Fiirstenberger, 1987).InitiationInitiation refers to a permanent DNA change in one or a fewcells that have been exposed to a carcinogen at a level that isinsufficient to cause a neoplasm (Boutwell, 1989). This permanentchange is generally considered to be caused at a genetic level.Brookes and Lawley (1964) confirmed the somatic mutation theoryby demonstrating that PAH carcinogens bind covalently to cutaneousDNA, and that the binding capacity of the carcinogen correlated withits tumorigenicity. This theory has been supported by many otherstudies (Bishop, 1982; Weinberg, 1982; Bister & Jansen, 1986; Sell etal., 1987; Yuspa & Poirier, 1988). DNA synthesis at this stage isprobably required for fixation of the mutant gene in daughter cellsand thus is responsible for the irreversibility of the initiated cells(Cayama et al., 1978).PromotionPromotion has been described as the reversible clonalexpansion of previously initiated cells that grow faster than thesurrounding normal cells so as to develop into a visible neoplasm(Boutwell, 1989; Pitot et al., 1989). In contrast to initiation, tumorpromotion appears to proceed along an epigenetic route, which couldbe evoked by either a promoter or a complete carcinogen. The latterhas both tumor initiating effects and tumor promoting effects(Quintanilla et al., 1986; Brown et al., 1986). However, there isevidence indicating that DNA damage or mutation also occurs duringthe promotion process. Contrary to initiation, promotion occurs over along period of time, is reversible at early stages (Boutwell, 1964),and shows a distinct threshold below which promotion effects willnot be observed (Diamond et al., 1980). The prolonged process, thepresence of a threshold and early reversible nature of promotionmake it the most important step in the study of carcinogenesis, asinterruption of tumor promotion is much more feasible clinically ascompared to that of the rapid, irreversible process of tumor initiation(Slaga et al., 1980).Boutwell (1964) has further divided promotion into two stages.The first of which is conversion, whereby initiated cells areconverted to the dormant tumor cells. This stage is promoter-specificbecause it can be induced only by either a complete carcinogen or anoncarcinogenic promoter. The second stage is propagation, wherebythe dormant tumor cells multiply to form a tumor mass. This stage isless specific. Not only complete carcinogens and tumor promoters butalso hyperplasiogenic or mitogenic agents may turn the dormanttumor cells into a tumor. This subdivision of promotion has beensupported by a number of subsequent studies (Slaga et al, 1980;Fiirstenberger et al., 1981).Although tumor promoters have no tumorigenicity when testedalone, they remarkably increase tumor yield when repetitively usedfollowing initiation. Tumor promoters exhibit a broad spectrum ofbiological effects (Johnson et al., 1987; Marks et al., 1988; Marks,1990). The essence of promotion is, however, believed by manyinvestigators to be the selective stimulative growth force for theinitiated cells relative to the surrounding cells (Solt & Farber, 1976;Farber, 1984). In skin, all tumor promoters induce hyperplasticchanges, supporting the hypothesis that epidermal hyperplasia isessential for promotion. However, not all skin hyperplasiogenic orhyperproliferative agents are promoters.The extensive studies of phorbol esters, particularly 12-0-tetradecanoylphorbol-13-acetate (TPA), have yielded some resultsregarding the biochemical events and mechanisms of tumorpromotion. In mouse skin, two prominent events occur almostimmediately after treatment with phorbol esters: one is the inductionof the arachidonic acid cascade, resulting in production ofprostaglandins and metabolites along the lipoxygenase pathway, andanother is the activation of protein kinase C (PKC). Prostaglandins areinvolved in the induction of epidermal hyperproliferation. Theactivation of PKC by the substitution of TPA promoter fordiacylglycerol opens the signal transduction pathway normallycontrolled via growth factors (Johnson et al., 1987) and results inepidermal hyperplasia (Marks & Fiirstenberger, 1987). Other umorpromoters that are structurally dissimilar to phorbol esters have alsobeen found to act through the PKC pathway and therefore are calledTPA-type of tumor promoters. Tumor promoters that act throughother mechanisms are called non-TPA-type tumor promoters, whichmay affect phosphoprotein phosphotase, such as okadaic acid, leadingto amplification of protein phosphorylation (Haystead et al., 1989), ormay induce a long-lasting increment of intracellular Ca2++, such asdetergent (Setälä et al., 1954), organic peroxide (Kensler & Taffee,1986) and thapsigargin (Thastrup et al., 1990), evoking the skinwound response.ProgressionThe stage of progression is believed to be characterizedprimarily by its karyotypic instability and evolution to malignancy.The development of irreversible, aneuploid malignant neoplasmsdistinguishes progression from both initiation and promotion. Thisprocess is generally considered to be the effect of the accumulativegene mutations, as it may be augmented by treating papilloma withinitiators (Hennings et al., 1983).2. Tumor markerTumor markers have been defined as the specific biochemicaland/or molecular characteristics, products, and changes produced ina host suffering neoplasia (Beer & Pitot, 1987). Neoplasia also couldbe characterized by a loss of normal cellular markers (Miller &Miller, 1974).The significance of studying tumor markers lies in threeaspects: (1) Since the recognizable morphological changes forpotential malignant transformation occur late in the multistepprocess, it is essential to develop markers such as enzymes to labelthe carcinogen-altered cells from the early stages of carcinogenesis inorder to study them. (2) Since only a small percentage of thepremalignant lesions will develop into malignancy, it is veryimportant to develop tumor markers for an evaluation of themalignant transformation potential of premalignant lesions, and forthe early diagnosis of cancer. (3) An understanding of the keyenzymatic changes may lead to a natural approach to intervene theprocess at the early stage of carcinogenesis through specificchemotherapy and thus to alters the enzymatic activity.Tumor markers have been studied most extensively in the ratliver model and multiple tumor markers have been identified. Thepreneoplastic and neoplastic cells are characterized by theirphenotypic diversity. The availability of multiple tumor markersmay lead to the detection of those phenotypes which have thegreatest propensity to progress to carcinomas and of thosetreatments which increase such lesions. The availability of multipletumor markers is also important in screening for differentcarcinogens, promoters or progressors since different classes of theseagents may promote different initiated cells which express certainmarkers. In the liver, it has been reported that altered hepatic foci(AHF) expressing multiple markers may have a faster growth rateand are more autonomous than single marked AHF.Malignant tumors are known to share many features withnormal embryonic tissue, such as possession of enzymes and proteinsthat are normally present in embryonic tissues but low or absent innormal adult tissues. These enzymes or proteins are called carcino-embryonic (oncofetal) proteins or markers. Generally, the amount ofoncofetal proteins present in neoplastic cells is higher than theirnormal embryonic counterparts.The oncofetal hypothesis of carcinogenesis is derived fromfindings that there are analogies between the differentiation ofnormal fetal tissues and the reverse, the loss of differentiation bytumors (Knox, 1972). It has been proposed that the appearance ofoncofetal protein may reflect de-differentiation of neoplasia(Novogrodsky et al., 1976). The hypothesis, therefore, suggests thatcancer may be viewed as a problem in normal biologicaldevelopment. Studies of the relationship between cancers and theirnormal embryonic tissues may lead to a better understanding of themechanisms of malignant transformation and the regulation ofembryogenesis.3. The HBPM modelSince Salley's discovery of the HBPM model in 1954, thissystem has remained the most useful one in the study of oralcarcinogenesis. The cheek pouches are bilateral evaginations of oralmucosa and provide ample mucosal tissue. Contralateral pouchs canserve as controls and the pouches are easily visualized andaccessible. The pouch epithelium is susceptible to a number ofcarcinogens, including 7,12-dimethylbenzanthracene (DMBA) whichproduces predictable stepwise changes in the mucosa and aconsistent production of carcinomas in all treated animals (Salley,1954; Morris, 1961). A number of studies have shown that there is aconsistent time frame of tumor development with triweekly paintingwith 0.5% DMBA. Dysplasia develops around 6 to 8 weeks, earlysquamous cell carcinomas (SCCs) develop around 8 to 10 weeks andinvasive SCCs develop around 10 to 12 weeks (Salley, 1954; Morris,1961; Shklar, 1972; Freedman & Shklar, 1978). It has been shownthat the sequential changes of hyperkeratosis, dysplasia andsquamous cell carcinomas in the HBPM during carcinogenesis arecomparable to human oral lesions of epithelial hyperplasia, dysplasiaand carcinoma (Santis et al., 1964; MacDonald, 1973).Another advantage of using hamsters in the study ofcarcinogenesis is that the incidence of spontaneous neoplasms inhamsters is much lower than those in mice and rats (Van Hoosier &Trentin, 1979; Pour et al., 1979), with only rare cases having beenreported in the literature (Zhang, 1989), and none of these weresquamous cell carcinomas.DMBA carcinogenesis in HBPM is also susceptible to a numberof cocarcinogenic agents, particularly to those presumptivelyassociated with human oral carcinogenesis, such as alcohol andchronic mechanical irritation. There is, however, no potent promoterfound for this model, although weak promoting agents such asbenzoyl peroxide have been identified. The prototype mouse skinpromoters, such as TPA, are ineffective in the HBPM model. An invitro study by O'Brien and Diamond (1979) showed that TPA did notaffect cell growth or DNA synthesis of hamster cells as it did inmouse epidermal cells. Additionally, hamster cells rapidlyinactivated TPA, while there was little, if any, such metabolism inmouse skin epidermis. This may explain why TPA is an ineffectivepromoter in the HBPM carcinogenesis. As discussed above, promotionis a very important step in carcinogenesis and is critical indetermining if a tumor will develop. The lack of an effectivepromoter has been the biggest obstacle in exploring manyfundamental aspects of carcinogenesis using the HBPM model.4. New tumor promotersThe phorbol ester group of tumor promoters has beenextensively investigated and the results of these studies havecontributed tremendously to our understanding of tumor promotion.In the past several years, there has also been considerable interest innon-TPA type tumor promoters in the mouse skin model. Thesestudies of new promoters have greatly improved our understandingof tumor promotion and have provided possibilities that some ofthem are also potent tumor promoters for HBPM model.Okadaic acidOkadaic acid (OA C44H68013) is a polyether fatty acid with a m.wt. of 805.02. It has been first isolated from two marine sponges(Tachibana et al., 1981) and later found in several types of marineplankton, which are the food of these marine sponges (Murakami etal., 1981; Yasumoto et al., 1984). It causes skin irritation andgastroenteritis in humans (Murata et al., 1982). Recently, it han beenshown to be an effective tumor promoter in the mouse skin model(Suganuma at al., 1988), and in some cell culture systems (Redpath &Proud, 1989).While phorbol ester group of tumor promoters such as TPA,and other TPA-type of tumor promoters are believed to functionmainly through binding with and subsequent activation of PKC, OAdoes not bind with PKC. Studies have shown that OA is a very potentinhibitor of serine/threonine-specific protein phosphatases 1 and 2Ain any cellular event (Cohen, 1989), but has no direct inhibitingeffect on the activity of any of eight known protein kinases. Sincethese two protein phosphatases are the chief enzymes that reversethe action of PKC, their inhibition causes a net increase ofphosphorylated proteins (Haystead at al., 1989).Methyl methanesulphonateMethyl methanesulphonate (MMS C2H60 3S) is an alkylatingagent (AA) with a m. wt. of 110.13 and covalenty binds to thechemical groups of biological molecules that have an excess ofelectrons (nucleophiles). This binding is known as alkylation (Kohn,1979). MMS is a direct-acting compound and does not requiremetabolic activation (Kleihues & Coopers, 1976; Garte et al., 1985). Ithas been found that MMS shows carcinogenic effect in a number ofanimal models, such as the rat nasal mucosa model. This effect isusually observed after a long term treatment (IARC, 1974;Sellakumar et al., 1987). In contrast, in some other models, MMS hasbeen shown not to exhibit any tumor initiating effects (Frei & Venitt,1975; Pegg, 1983; Fiirstenberger et al., 1989).The reactivity of AAs toward nucleophiles can be defined interms of reaction mechanisms and the dependence of reaction rateson the nucleophilic strength of the receptor atoms (Vogel &Natarajan, 1981). The reactivity of AAs has been expressed by theSwain-Scott substrate constant s, which is a measure of thesensitivity of AA to the strength n of the nucleophile with which itreacts. Studies have shown that there is a general, direct correlationbetween chromosome breaking efficiency, cytotoxicity and s value,and a general inverse correlation between s value and the ability ofAAs to induce point mutation.Studies have shown that mutagenicity of an alkylating agentcorrelates with its carcinogenicity (Newbold et al., 1980). Theinefficiency of MMS as a tumor initiating agents in a number ofmodels has been explained to be the result of its low mutagenicity10(Loveless, 1969; Newbold et al., 1980; Morris et al., 1982; Pegg, 1983;Natarajan et al., 1984;). MMS has a high s value, hence it has as samelow mutagenic but high clastogenic and cytotoxic effects as do otherAAs with high s. Low level production of 06-guanine methylation byAAs with high s value, such as MMS, contributes to the lowmutagenicity. An increased level of 06-guanine methylation has beenfound to parallel increased mutagenicity (Frei & Lawley, 1976; Suteret al., 1980; Newbold et al., 1980). Also, it has been proposed that thelow mutagenicity of AAs with high s is because (1) they inhibit SH-group and, (2) they are very cytotoxic, causing death of the mutantcells (Vogel & Natarajan, 1981).Although induction of mutations in somatic cells has beenconsidered the most likely mechanism by which AAs might initiateneoplastic growth, other mechanisms can not be ruled out (Pegg,1983). The possibilities include induction of latent viral genes byAAs, synergistic effects of AAs and viruses and alteration of hostimmunocompetence (Pegg, 1983).In the mouse skin model, MMS is a rather powerful stage I(conversion) tumor promoter, although it is not carcinogenic by itself(Fiirstenberger et al., 1989). This is not surprising since clastogeniceffects are characteristic of stage I promoters. The correlationbetween clastogenicity and conversion in tumor promotion has beenreviewed in terms of induction of prooxidant states (Cerutti, 1985),which is critical in the generation of chromosomal aberrations in skintumor promotion (Fiisternberger et al., 1989).II. Gamma-glutamyl transpeptidase (GGT)I. StructureGamma-glutamyl transpeptidase (GGT) is a plasma membranebound glycoprotein elaborated in endoplasmic reticulum andtransported to the plasma membrane via the Golgi apparatus (Ishii etal., 1986). It is composed of two subunits which are located on theextracellular side of cell membrane (Horiuchi et al., 1978; Marathe etal., 1979). The heavy subunit anchors GGT in plasma membrane(Matsuda et al., 1983), while the light subunit noncovalently binds tothe heavy subunit (Hughey & Curthoys, 1976). Both of subunits areresponsible for its catalytic function (Garde11 & Tate, 1981).2. Metabolic rolesGGT catalyzes the initial step in the utilization of glutathione (y-glutamylcysteinylglycine, GSH) in which the y-glutamyl moiety of thistripeptide is transferred to an acceptor, which may be an amino acid,dipeptide, or GSH itself (Tate & Meister, 1981). GGT is the majorenzyme in y-glutamyl cycle, a metabolic pathway that accounts forthe enzymatic synthesis and degradation of glutathione (Elce &Broxmeyer, 1976; Meister, 1976; Samuels, 1977; McIntyre &Curthoys, 1979).The biological significance of GGT is not entirely clear. Besidesits role in GSH metabolism and the maintenance of intracellular GSHlevels, participation in amino acid transport across cell membranes,detoxification of electrophiles, peptide storage, storage and transportof cysteine, and cell proliferation have been suggested (Rosalki,1975).G a mm a —glutamyl cycle as a possible amino acid transportsystem was first proposed by Orlowski and Meister in 1970. Thistheory was later supported by a large number of studies includingstudies on the location of GGT enzymes. Marked GGT activity hasbeen found in the brush border of epithelial cells lining the proximalconvoluted tubules and loops of Henle, in the surface epithelial cellsof the small intestine, especially the jejunum, and in the choroidplexus. All of the epithelial cells in these locations function in activeamino acid absorption, supporting the hypothesis that GGT may playan important role in amino acid transportation. Conflicting reportsregarding GGT's role in amino acid transportation, however, also exist(Curthoys & Hughey, 1979).A large number of studies has shown that GGT may have animportant function in the detoxification of electrophiles through itsrole in maintaining intracellular GSH level and through themercapturic acid pathway. The first step of the mercapturic acidpathway is the glutathione conjugation reaction, in which a group ofisoenzymes known as glutathione transferases (GSTs) catalyzes theconjugation of electrophilic compounds with reduced glutathione,thus protecting macromolecules such as DNA from attack bycarcinogenic agents (Degen & Neumann, 1978; Chasseaud, 1979;Moldeus & Jernstrom, 1983). The second step of this pathway iscatalyzed by GGT which removes the y-glutamyl moiety from the GSHconjugates to form cysteine derivatives, which subsequently undergoacetylation to form N-acetyl-L-cysteine derivatives (mercapturic13acids) (Curthoys & Hughey, 1979). The mercapturic acids are solubleand readily excreted through kidney and bile ducts (Boyland &Chasseaud, 1969), making this pathway to be one of the mostimportant detoxification processes in the body.3. GGT and carcinogenesisGGT as a tumor marker has been studied extensively in liverand several other experimental models. It is significantly elevated inboth preneoplastic and neoplastic hepatic lesions and in a variety ofexperimentally induced or in human premalignant and malignantepithelial lesions, such as human oral precancerous lesions and SCCs(De Young et al., 1978; Buxman et al., 1979; Fiala, 1979; Fiala et al.,1979a,b; Gerber & Thung, 1980; Uchida et al., 1981; Calderon-Solt &Solt 1985; Mock et al., 1987). However, human lymphocytic leukemiashows decreased GGT activity (Novogrodsky et al., 1976; Hultberg &Sjogren, 1980).Of the biochemical markers for recognition of earlypreneoplastic lesions, GGT is one of the best studied. Elevated GGTactivity has been noticed as early as after a single application ofsubcarcinogenic dose of hepatocarcinogens (Scherer et al., 1972;Scherer & Emmelot, 1975a,b, 1976; Hanigan & Pitot, 1985). GGT(+)cells may develope chromosome abnormality (Miyazaki et al., 1985).Moreover, the transcription of several proto-oncogenes duringhepatocarcinogenesis has been studied. The expression of H-ras andc-myc gene has been found to be elevated in GGT(+) cells (Sinha etal., 1986).The usefulness of GGT as a tumor marker has been explored inthe HBPM model (Solt, 1981; Solt & Shklar 1982; Zhang & Mock 1987,1989, 1992; Zhang, 1989). Normally the adult cheek mucosaepithelium is devoid of detectable GGT activity. However, hamsterstreated with carcinogens progressively developed discrete foci of GGTpositive cells. GGT activity disappeared with formation of overtneoplasms (Zhang & Mock, 1987; Zhang 1989).The loss of GGT activity during multistep carcinogenesis hasalso been noted in liver, although it was much less dramatic andobvious (Tatematsu et al., 1988b). The loss of GGT staining in tumorsmay indicate a further step toward malignancy.The exact role of GGT in carcinogenesis is not clear. It has beensuggested that cells with higher GGT levels may have a bettercapacity of detoxification than those with lower GGT, resulting in aselective growth advantage required for further transformation(Laishes et al., 1978).4. Tissue distributionIn most mammals, such as humans, laboratory rodents, brownbears, dogs, oxes, when adult tissues are assayed for GGT activity, thestrongest GGT activity is noted in kidney, with weaker levels inpancreas, and still weaker activity in liver and negligible in otherorgans (Glenner et al., 1961). GGT activity in the same organ varies indifferent species of animals. After the introduction of histochemicalmethods for the localization of GGT, a number of studies have beenperformed to establish the localization of GGT in a variety of tissuesand cells. The precise localization of transpeptidase is important in15view of its proposed roles in transport and detoxification processes,as well as its usefulness as an oncofetal marker (Tate & Meister,1981). As summarized by Tate and Meister (1981), histochemicalstudies have shown that, in general, high GGT activity is seen in cellswhich exhibit secretory or absorptive functions, including theepithelial cells of renal proximal tubules, jejunum, duodenum, bileduct, epididymis, prostate, testis, seminal vesicles, choroid plexus,ciliary body, retinal pigment epithelium, bronchioles, thyroidfollicles, mammary glands, hepatocytes of canalicular regions of liver,pancreatic acinar and ductile epithelial cells, post-secretoryameloblasts and odontoblasts of developing teeth, and the epitheliumof the uterine endometrium (Marathe et al., 1979; Albert et al., 1961,1964, 1966, 1970; Ruthenburg et al., 1969; Fiala et al., 1976, 1977;Adjarov et al., 1979; Dawson et al., 1979; Ahlund-Lindqvist &Lindskog, 1985). High GGT activity has also been noted in non-secretory or non-absorptive epithelial cells, such as the granular cellsof the maturing ovarian follicle and the follicular sheath of growinghair (Buxman et al., 1979).GGT activity in different organs and tissues also variesdepending upon the stage of the development of an organ and tissue.In many species studied, including human, mouse, rat, rabbit,hamster, and guinea pig, fetal tissues in general exhibit much higherGGT activity than adult tissues with the exception of kidney, which isthe main source of GGT in the adult (Albert et al., 1964, 1970).GGT activity during the development of organs and tissues hasbeen studied in detail in the rat and humans. Their fetal andneonatal brain, lung, and particularly liver, show much higher GGT16activity than that in the adult organs, whereas, there is a steadyincrease in the GGT activity in kidney during its development. Adulthuman kidney contains 11 times greater GGT activity than the fetalrenal tissue (Albert et al., 1970a,b). Interestingly, studies haveshown that hepatic and lung carcinomas developnig in rat, humanand other mammals showed a marked increase in GGT activity(Tatematsu et al., 1985; Yamamoto et al., 1988), suggesting GGTenzyme activity in these organs is oncofetal in nature. On thecontrary, renal cell carcinomas showed decreased GGT activity(Albert, 1965; Flemming et al., 1977; Tsuda et al., 1985),In other organs and tissues, increased GGT in preneoplastic andneoplastic lesions has not been found to be oncofetal in nature (DeYoung et. al., 1978; Adjarow et al., 1979; Traynor et al., 1988).Only one study has investigated GGT activity in adult hamsterorgans and tissues (Albert et al., 1964). The results showed thathamster kidneys contained the highest level of GGT and the pancreasshowed a moderate amount of GGT. Only trace amounts, or no GGTwere found biochemically in other organs and tissues, including liver,spleen, gastrointestinal tract, adrenals, ovary, uterus, epididymis,testicle, submandibular salivary gland, and lung. When examinedhistochemically, GGT activity was found mainly in the cells of theproximal convoluted tubules in kidney, in the external secretoryportion of the cell cytoplasm of the secretory follicle in pancreas, andin the cylindric cells of the mucous membranes of the small and largeintestine. Slight GGT activity was demonstrated in the cytoplasm ofthe glandular cells and of the secretory ducts. Although no GGTactivity was noted in hepatocytes, a slight positive reaction was17occasionally observed in the wall of bile canaliculi and somereticuloendothelial cells. GGT activity was not observedhistochemically in other organs, including stomach and lung. Theresults of this study have not been confirmed by other investigatesand the study did not investigated GGT activity in hamster oral ornasal mucosa and skin. Several studies on oral carcinogenesis haveshown that adult hamster pouches contain no demonstrable GGTactivity. No study has investigated GGT activity in fetal or neonatalhamster tissues and organs, including hamster pouches, salivaryglands, and odontogenic tissues.III. Placental glutathione transferase (GST-P)Glutathione S-transferases (GSTs) are a family ofmultifunctional proteins composed of dimeric subunits. They werefound initially in rat liver (Booth et al., 1961) and later isolated fromrat liver cytosolic supernatant fraction (Habig et al., 1974a),disclosing the multiple isoenzymes acting on a broad spectrum ofuniversal substrates. Of many forms of GSTs identified in variousorgans of various species, rat, mouse and human's are best studied(Mannervik, 1985; Mannervik et al., 1985, 1987; Hayes et al., 1987).GSTs had been named alphabetically in relation to isoelectricpoints and molecular weight (MW) of the subunits (Habig et al.,1974b; Bass et al., 1977). Later, they were divided into three groups:basic, neutral, and acidic, according to isoelectric- or chromato-focusings (Sugioka et al., 1985; Hayes et al., 1986a,b). Recently, basedon similar properties in structure and catalysis shared by major18subunits, GSTs were grouped into three classes: a, p., and 7C. Thisclassification is species-independent (Mannervik et al., 1985) andreflects the evolutionary relationship between species.Placental glutathione S-transferase (GST-P), a neutral form ofGSTs with a m. wt. of 23,307, was first purified from rat placenta bySato et al. in 1984. Of particular relevance to rat GST-P (neutral) arehuman placental form of GST (GST-n, acidic) and mouse GST M II(basic). All these three GSTs belong to it class of GSTs according to thespecies-independent classification (Mannervik et al., 1985), sharemany properties and are immuno-crossreactive to each other (Sato,1989). For example, the anti-rat GST-P antibody has been found tobe cross-reactive in many species such as mouse, hamster, dog, horseand human (Moore et al., 1985; Roomi et al., 1985a; Zhang & Mock,1992).1. StructureThe identified forms of GSTs are composed of homodimer orheterodimer subunits (isoforms). The dimers may be separated, inorder of their molecular weights, into monomers by means ofelectrophoresis (Bass et al., 1977; Kitahara et al., 1983a; Satoh et al.,1985) or chromatography (Ketterer et al., 1987; Ostlund-Farrants etal., 1987).2. Metabolic rolesAs mentioned above, GS Ts are intimately related to GGT intheir metabolism as both GSTs and GGT participate in mercapturicpathway metabolism and therefore in the detoxification of19electrophilic compounds such as carcinogens. In addition, certainforms of GSTs have selenium-independent GSH peroxidase activitytoward lipid peroxides via activating P-450 (Kitahara et al., 1983b;Meyer et al., 1985; Ketterer et al., 1987). Furthermore, Ligandin, abasic form of GSTs, and several other form of GSTs, has been shownto function as binding or carrier proteins for a wide spectrum ofexogenous materials such as dyes, cholic acid, steroid hormone,hematin, leukotriene and carcinogens (Smith et al., 1977; Jakoby,1978; Chasseaud, 1979; Ketterer et al., 1985; Mantle et al., 1987).3. GST-P and carcinogenesisGST-P, together with GGT, has been found to be the best tumormarkers in the rat liver model (Cameron et al., 1978; Ogawa et al.,1980; Hsu et al., 1981; Moore et al., 1987). Among hepatocarcinogenstested, with the exception of the peroxisome proliferator group ofhepatocarcinogens (Numoto et al., 1984; Rao et al., 1984, 1986a,b,1987a,b, 1988; Goel et al., 1986; Glauert et al., 1986; Greaves et al.,1986; Hendrich et al., 1987; Wirth et al., 1987), GST-P and GGT markmore altered hepatic foci than any other hepatic tumor markers (Itoet al., 1988). GST-P(+) foci appear early during hepatic carcinogenesisand have been detected immunohistochemically within 48 hfollowing a single dose of a carcinogen (Moore et al., 1986; Sato,1988). The staining persisted for at least 6 months, suggesting anirreversible property (Takahashi et al., 1987; Sato, 1988). Thenumber of the positive cells in the foci is proportional to the increasein the dosage of a carcinogen (Moore et al., 1987a).There is evidence indicating that GST-P may be a better tumormarker than GGT in hepatic carcinogenesis. Unlike GGT, GST-P is notinducible by administration of a large variety of promoters and otherdrugs (Roomi et al., 1985b; Satoh et al., 1985; Ito et al., 1988),although it is slightly inducible by some antioxidants (Tatematsu etal., 1985, 1987, 1988b) and by ethoxyquine (Thamavit et al., 1985;Manson et al., 1987) in periportal areas. Such weak activity does notinterfere with detection of GST-P foci. However, GGT can be inducedso strongly by various promoters and drugs that the enzyme-alteredneoplastic foci are no longer recognizable (Tatematsu et al., 1985;Fischer et al., 1986). GST-P staining scores more altered hepatic focithan GGT staining (Tatematsu et al., 1985). While GGT staining israpidly lost following withdraw of carcinogen treatment (Moore etal., Tatematsu et al., 1988a,b), GST-P staining is relatively stable(Sato, 1989).GST-P was also found to be elevated in preneoplastic andneoplastic lesions in a number of experimental models (Moore et al.,1985). and in human organs such as colon (Kodate et al., 1986; Peterset al., 1989), uterine cervix (Shiratori et al., 1987), esophagus andstomach (Tsutsumi et al., 1987), liver (Sato et al., 1987), kidney (Sheaet al., 1987), and lung (Nakagawa et al., 1988). Recently, GST-P hasbeen found to be a potential tumor marker in the HBPM model(Zhang & Mock, 1992).The GST-P gene is located on chromosome 1q43 (Masuda et al.,1986) and is associated with cis-acting regulatory elements (Okudaet al., 1987). It has been found that both up and downstreamenhancers of GST-P gene contain sequences which resemble that of21TPA response element. The activation of GST-P gene has beenreported to involve AP-1 production which functions in trans-activation (Okuda et al., 1988; Sakai et al., 1988). The GST-P geneexpression is shown to be related to activation of oncogenes, such asHa-ras (Power et al., 1987), metallothionein-ras (Li et al., 1988), SV40 and Jun (Imler et al., 1988). Because GST-P mRNA level is verylow in normal tissue and may be modified in the promotion stage,the expression of GST-P in neoplasia is considered to be a result ofgene altaration or regulation at transcriptional level (Muramatsu etal., 1987).While the role of GST-P in carcinogenesis is not clear, a numberof possible roles have been suggested. As discussed above, similar toGGT, it has been proposed that the higher level of GST-P inpreneoplastic and neoplastic cells may have better capacity indetoxification, resulting in a selective growth advantage required forfurther transformation. GST-P is known to possess selenium-independent glutathione peroxidase activity toward lipid peroxidesvia activation of P-450 (Kitahara et al., 1983b; Meyer et al., 1985;Ketterer et al., 1987). It has been suggested that GST-P expressionmay be related to the inhibition of lipid peroxidation which has beenconsidered to play an important role(s) during tumor promotion. Theclearance of lipid hydroperoxides involves a series of conjugationsteps initiated by GST-P-dependent GSH peroxidase. Thus a chain ofreactive factors such as reduced GSH, NADPH and G6PD is en blocincreased (Demi & Oesterle, 1980; Kitahara et al., 1983a,b; Sato et al.,1987).4. Tissue distributionA number of studies have investigated the distribution of GST-P in the tissues of adult rats, mice and humans, but few studies havedealt with fetal tissues.In rats, the protein content of GST-P is generally low or absentin normal tissues, including placenta, fetal lung and livers, adult lung,livers, regenerating livers, heart, testis, prostate, spleen, muscles, andis significantly high in kidney and pancreas (Satoh et al., 1985; Sato,1989). Using immunohistochemical methods, strong GST-P staining isfound in adult kidney tubular epithelium (Tsuda et al., 1985),pancreas ductular cells (Moore at al., 1985), small intestine columnarepithelium (Mannervik et al., 1987), skin epidermis, lung bronchiolarepithelium (Yamamoto et al., 1988) and brain astroglia cell (Tsuchidaet al., 1987). GST-P is negative in hepatocytes, but weakly positive inbile ductular cells and placenta.In the mouse, GST MIT, corresponding to class it GST, is found insignificant amounts in the livers of male adult mice but are low infemales. No GST-P can be demonstrated in other organs, includingheart, lung, kidney, intestine, gall bladder and skin. Skin papillomasshow no GST-P staining (Roomi et al., 1985a).In humans, the corresponding GST-n or human GST-P is presentnormally in the epithelial cells of a wide variety of tissues andorgans in contrast to the limited tissue distribution of GST-P in thenormal rat and its absence in almost all tissues and organs in mice,except in male mouse livers. In human fetus, GST-P is the main orthe only isoenzyme in placenta, lung, kidney, brain and intestine(Koskelo et al., 1981; Polidoro et al., 1982; Koskelo, 1983; Pacifici et23al., 1986, Shiratori et al., 1987). A high level of GST-P is found infetal liver (Mannervik 1979; Polidoro et al., 1980; Warholm et al.,1980; Koskelo et al., 1981) and stomach (Tsutsumi et al., 1987). Inadults, GST-P is the major type of GSTs in lung, brain and spleen(Polidoro et al., 1982; Pacifici et al., 1986, Koskelo et al., 1981;Koskelo 1983). GST-P is abundant in human skin epidermis(Konohana et al., 1990). It can be detected also in adult breast (Ilio etal., 1986), kidney (Ilio et al., 1987), and intestine (Peters et al.,1989), and it is absent in the liver (Van der Jagt et al., 1985; Roomiet al., 1985a; Soma et al., 1986).Using immunohistochemical methods, Tsutsumi et al., (1987)showed that GST-P strongly stained the surface mucous cells andglands located in the fundic, pyloric and cardiac areas of the stomachof a human fetus aged 18 weeks. The staining decreased as thefetuses aged and no staining was evident in the 34-week-old fetuses.The adult stomach showed only slight staining in the parietal cells offundic glands.Very few studies have dealt with the normal distribution ofGST-P in hamsters. Using rabbit anti-rat GST-P antibody, Roomi et al.(1985a) found that the liver cytosol of most animals, includinghamsters, showed no GST-P activity, while horse and mouse livercytosol reacted. In the course of studying hamster pancreatic andhepatic carcinogenesis, Moore et al., (1985) observed no GST-Pstaining in hamster liver and pancreas. GST-P activity has not beeninvestigated in other hamster organs and tissues, including pouchmucosa, salivary gland parenchyma and the odontogenic apparatus.The normal distribution of GST-P in oral mucosa, salivaryglands and odontogenic tissues has not been studied in any species.As mentioned above, GST-P has been found to be a usefultumor marker, mainly in a variety of epithelial preneoplastic andneoplastic lesions in a number of experimental animals and inhumans. The increase or decrease of GST-P in neoplasia seems to bein reverse relationship with the normal distribution of GST-P intissues and organs. As summarized by Sato (1989), in cell typesnormally expressing large amounts of GST-P, such as human and ratkidney tubular epithelium, a decrease has been noted duringcarcinogenesis (Tsuda et al., 1985; Di Ilio et al, 1987; Kurata et al.,1987; ), and often an increase is observed in organs normally notexpressing GST-P, such as acinar cell lesions of the rat and hamsterpancreas and in squamous metaplasias and squamous cell carcinomasin the rat lung. In rat colon carcinoma, GST-P is not expressed, incontrast to positive findings reported for human colonic adenomasand carcinomas. The normal human colon is negative for GST-P(Kodate et al., 1986).The increase or decrease of GST-P in neoplasia seems to beoncofetal in nature in a number of organs, such as human and ratcolon, and human kidney, liver and stomach. In some other organs,however, GST-P is not an oncofetal marker.EXPERIMENTAs discussed above, there is no potent tumor promoter foundin the HBPM model, although weak promoting agents such as benzoylperoxide have been established. Tumor promotion is an importantstep in carcinogenesis. The lack of an effective promoter has been thebiggest obstacle in exploring many fundamental aspects ofcarcinogenesis using the HBPM model.Experiments I and II were designed to explore the tumorpromotion potential of two new tumor promoters, OA and MMS, onHBPM. Since OA is extremely expensive, only a pilot study wasdesigned (Experiment I) to investigate its irritating effects on HBPM.It is well know that most, if not all, tumor promoters of skin andmucosa are irritants, although the reverse is not true (Shubik, 1950).GGT and GST-P have been found to be increased during HBPMcarcinogenesis. Whether such increases are oncofetal remains to beanswered. No study has investigated GUT and GST-P expression infetal or neonatal hamster pouches or hamster oral mucosae. There isonly one study that investigated the normal tissue distribution ofGGT in hamster tissues. There are no data available regarding thenormal tissue distribution of GST-P in hamster tissue with exceptionof adult hamster liver and pancreas. Experiment III was designed toinvestigate the normal tissue distribution of GUT and GST-P duringthe development of hamster pouches and several selected organs andtissues. The results should improve our understanding of the normaldistributions and functions of GGT and GST-P in hamster tissues andorgans, and of the enzymatic increase in neoplasia of the model.26Experiment I:^In vivo mitotic activity of okadaic acid onhamster buccal pouch mucosa1. ObjectiveTo study the effect of OA on hamster buccal pouch epitheliumthrough gross observation and mitosis assay.2. Materials and methodsAnimalsNon-inbreeding male Syrian hamsters (Charles River BreedingLaboratories, MA) aged 8-9 weeks and weighing 105-135 g wereused. Animals were fed a commercial stack diet (Puria FormulaChow) and tap water ad libitum and maintained in the standardizedconditions of temperature and humidity with a 12 h light/dark cycle(06:00-18:00 light).OA dosage determinationGenerally, the doses of test carcinogens used in the HBPMmodel are higher than those used in the mouse skin model. Since adosage of 10 jig OA in 0.1 ml acetone was used in the mouse skinmodel for tumor promotion (Suganuma et al., 1988), concentrationsof 20 lig and 10 ii.g OA in 0.1 ml acetone were chosen for the dosagetrial.Both pouches of two hamsters were treated once with 20 lig or10 ilg OA (Sigma Chemical Company, St. Louis, MO) in 0.1 ml acetoneand sacrificed the second day. All pouches showed obviousinflammation with marked edema, erythema and focal ulceration.27Because ulceration was much more severe on the pouches treatedwith the higher concentration of OA, it was determined that thelower concentration would be used for the study.Animal treatmentSix hamsters were randomized into two groups with threeanimals in each group. Animals were anesthetized by carbon dioxideinhalation. Buccal pouches were cleaned with tap water and driedwith gauzes. 10 lig OA in 0.1 ml acetone (stored at -20°C) was appliedtopically on the surface of a pouch with the aid of a micropipette. Thepouch was then inserted back into the hamster's mouth after theacetone had almost evaporated. Both pouches of the threeexperimental hamsters were treated with OA. In the control group,the right pouches of hamsters were treated similarly with 0.1 mlacetone (also stored at -20°C), and the left pouches were leftuntreated.Mitosis assayThe method used to assay mitoses was described by Scragg andJohnson (1980) and slightly modified.Nineteen and a half hours after the treatment, all animals weregiven 0.1% vinblastin (VLB, Sigma Chemical Company, St. Louis, MO)intraperitoneally at a dose of 4 mg/kg body weight (Thilagaratnamand Main, 1972). All injections were performed between 08:30 and09:30 in order to minimize the influence from diurnal variation(Scragg & Johnson, 1980). The animals were then sacrificed 4.5 hlater (24 h after OA application) by carbon dioxide inhalation. Thepouches were surgically removed and examined grossly. Alongitudinal strip of pouch mucosa approximately 10 mm wide wasdissected from the underlying muscle along the entire length of apouch wall and rolled onto a disposable needle in an anteroposteriordirection to form a compact cylinder. Two strips of pouch mucosa perpouch were collected.Specimens were fixed in 10% buffered neutral formalin for oneweek and then underwent normal paraffin processing after theneedles were removed. After the rolled edge of a mucosa cylinderwas embedded at right angles to the block face, three 5 II sectionsseparated by at least 500 [t, were taken from each roll. Hence sixsections were sampled from each pouch. The slides were stained withhaematoxylin and eosin.All 72 sections were examined with a light microscope at X400magnification. The number of arrested metaphase figures in threethousand basal cells per section was recorded. Student t test wasemployed for statistical analysis.3. ResultsGross and microscopicThe control pouches, both untreated and acetone treated, wereunremarkable (Fig. 1.1a). OA-treated pouches appeared heavilyinflamed. Redness, edema, petechiae, ulceration and shrinkage ofpouch walls were obvious (Fig. 1.1b).On microscopic examination, OA-treated pouches demonstratedacute inflammation with numerous dilated blood vessels, ulcers anda heavy infiltration of neutrophiles.29Mitosis assayThe blocked metaphase figures were ball-shaped or wreath-like with condensed clumped or annular chromatin (Fig. 1.2). Mitosisindex (MI) expressed as number of metaphase figures per 100 basalcells was considerably elevated in OA treated pouches (5.9±1.0) ascompared with those of two controls (p<0.01). There was nosignificant statistical difference in MI between the untreated(4.36±0.72) and the acetone treated (4.11±0.43) control pouches(p>0.05) (Table 1.1).4. DiscussionAlthough the phorbol ester group of tumor promoters,especially TPA, are potent tumor promoters on mouse skin (VanSuuren, 1969), phorbol esters have no reliable promoting effect onhamster buccal pouch epithelium (Silberman & Shklar, 1963),possibly due to the existence of enzymes that rapidly inactivatephorbol esters (O'Brien & Diamond, 1979). Study of the initiation-promotion mechanism of carcinogenesis in the HBPM model has beenhampered, owing to a lack of accepted tumor promoter to carry outthe promotion procedures, so far routinely performed by a completecarcinogen DMBA. As a result, the subsequent work in exploring apreneoplastic marker for the HBPM model is inevitably hindered.There are two reasons for choosing OA for the study. The firstconsideration in choosing a potential tumor promoter for thisexperiment is that it has been proved in other systems to be a tumorpromoter. The second consideration is that the potential tumorpromoter should be structurally and mechanistically different from30the TPA-type tumor promoters. OA fulfils both criteria: it is a potenttumor promoter in the mouse skin model (Suganuma et al., 1988),and it has a different chemical structure and functional pathway ascompared to the phorbol ester group of promoters, as OA does notreact with phorbol ester receptors.The results of this study showed that OA produced markedinflammatory effects after only a single treatment at a dose (10 jig)similar to that used in the mouse skin model (Suganuma et al., 1988).The mitotic activity of OA-treated pouches was significantly higherthan those of the controls, indicating that OA stimulated pouchepithelial cell proliferation. Such properties are essential features formost, if not all, skin and mucosa tumor promoters (Shubik, 1950).Long term study is needed to investigate the possible tumorpromoting effects of OA on carcinogenesis in the HBPM. In contrast,teleocidin, a TPA-type mouse skin tumor promoters, showed noirritating effect on HBPM after 8 weeks of topical treatment(unpublished data, L. Zhang) with a dosage (15 lig) 6 times thedosage used in the mouse skin model (Suganuma et al., 1988). If OAproves to be a potent tumor promoter in the HBPM model with longterm study, it would seem that HBPM is resistant to TPA and TPA-type of tumor promoters in general, but may respond to non-TPA-type promoters.Experiment II:^In vivo tumor promoting effect ofmethylmethanesulfonate (MMS) onhamster buccal pouch mucosa1. ObjectiveTo test the potential tumor promoting effects of MMS in theHBPM model.2. Materials and methodsAnimalsAs in experiment I.MMS dosage determinationSince the promotion dosage of MMS used in the mouse skinmodel is 10% (100 gmol, Fiirstenberger et al., 1989), and sincehamster pouch mucosa, in general, requires higher a dosage ofchemicals than the mouse skin model, we first tested the effects ofMMS in the HBPM medel using the same or higher concentrations.The right pouches of 3 hamsters were painted with 10%, 20%, or 30%MMS in acetone (stored at -4°C) respectively. The left pouches weretreated with acetone similarly and was used as controls. In 24 h,animals were anesthetized by carbon dioxide inhalation and poucheswere pulled out and examined with the naked eye. All MMS treatedpouches showed very strong inflammatory responses in the form ofsevere edema, petechiae and ulceration. It was decided that suchstrong irritating effects would not be tolerated by hamsters in a longterm study. Subsequently we tested the effects of lower32concentrations of MMS on HBPM. The right pouches of 6 hamsterswere painted with 1%, or 5%, or 10% of MMS. In 24 h, the hamsterswere anesthetized and the pouches were examined. Pouches treatedwith 5% and 10% of MMS showed strong inflammatory responseswhile pouches treated with 1% MMS demonstrated only mildinflammation, which might be tolerated by hamsters in a long termstudy. Therefore, the concentration of 1% MMS was chosen for alonger term test. The right pouches of 6 hamsters were painted with1% MMS triweekly. After 2 weeks of promotion, half of the animalsshowed bleeding from mouth and anus, and muscle stiffness. Oneanimal demonstrated decerebrate rigidity, and all animals lookedsick. It was finally decided that a 0.5% MMS would be used in thelong term study.Animal treatmentIn a study of the ideal tumor initiating dosage for the HBPMmodel, McGaughey et al. (1984) found that treating hamster pouchestriweekly with 0.2% DMB A for two weeks was the best initiatingprotocol as such treatment did not produce any changes withoutfurther treatment but yielded highest tumor incidence withsubsequent applications of a tumor promoter. Therefore, it wasdecided that this initiating protocol would be used in thisexperiment. Twenty-five hamsters were divided into 3 groups andsubjected to the following treatment (Table 2.1):Group I (DMBA + MMS or nothing): Both pouches of 10hamsters were painted with 0.2% DMBA (Sigma Chemical Company,St. Louis, Mo.) in acetone triweekly for 2 weeks, and left untreated33for 10 weeks. The right pouches then were promoted with topical0.5% MMS biweekly and the left pouches were left untreated.Group II (MMS + MMS or nothing): Both pouches of 10hamsters were painted with 0.5% MMS triweekly for 2 weeks, andleft untreated for 10 weeks. The right pouches then were promotedwith 0.5% MMS biweekly while the left pouches were left untreated.Group III (DMBA or MMS + acetone): The right pouches of 5hamsters were painted with 0.2% DMBA, while the left pouches werepainted with 0.5% MMS triweekly for 2 weeks. The animals thenwere left untreated for 10 weeks and subsequently promoted withacetone biweekly.During the promotion period, the animals were anesthetizedwith carbon dioxide inhalation, and the pouches were examinedperiodically for the appearance of tumors. After 10 weeks ofpromotion, four tumors appeared in the Group I animals. It wasdecided to terminate the experiment at this time and all the animalswere sacrificed by carbon dioxide inhalation.Samples of skin and hair were excised from hamsters with hairdiscoloring in Group I and from control hamsters. All pouches wereexcised and examined grossly. Both the pouch and skin specimenswere fixed in 10% formalin for 1 week, and subsequently processedand embedded in paraffin wax. Five p.m sections were cut, stainedwith hematoxylin and eosin and examined under a light microscope.3. ResultsGrossGroup I (DMBA + MMS or nothing): Muscle stiffness and achange of fur color from dark brown to light grey were noted inthree of the ten hamsters (Fig. 2.1). At the end of the experiment, allthe animals appeared ill with weight loss as compared to the othergroups. Two of the ten hamsters demonstrated a total of four tumorson the DMBA-initiated, MMS-promoted right pouches. The tumorsappeared either smooth surfaced or cauliflower-like (Fig. 2.2). Thecolor of the tumors varyed from grayish to reddish. The size of thetumors ranged from 3-6 mm in diameter, averaging 4.5 mm All theDMBA-initiated, MMS-promoted right pouches showed shrinkage andmarked thickening of the mucosa, and were erythematous withoccasional ulcerations (Fig. 2.2). The unpromoted left pouches wereunremarkable.Group II (MMS + MMS or nothing), Group III (DMBA or MMS +acetone): No obvious toxic effects of MMS, such as decreases in bodyweight and changes in hair color, were noted in MMS-treated animalsas compared to Group I animals (Fig. 2.1). All the pouches lookedunremarkable (Fig. 2.2).HistologyGroup I (DMBA + MMS or nothing): There were nomicroscopically recognizable changes, including the amount ofmelanin and number of melanocytes, in the skin and hair ofhamsters showing gross hair discoloration as compared to the controlanimals.Epithelium of the DMBA-initiated, MMS-promoted rightpouches showed generalized, marked acanthosis and hyperkeratosis35with frequent down-growth of rete ridges. In many areas, theepithelium was at least three times as thick as the the epithelium inthe control groups (Fig. 2.3). Patches of inflammatory infiltrationwere noted and the inflammatory cells were either primarily chronicinflammatory cells, such as lymphocytes and plasma cells or mixedacute and chronic inflammatory cells (Fig. 2.4c). As shown in Table2.2, all right pouches showed generalized, moderate dysplasia and 5of the 10 right pouches demonstrated areas of severe dysplasia (Fig.2.3b,c). The 4 tumors, from the two hamsters, showed a papillary orpebbly surface and were lined with moderately to severelydysplastic epithelium (Fig. 2.4a). Areas of invasion were noted (Fig.2.4b).The DMBA-initiated but unpromoted left pouches showedoccasional thickening of the lining epithelium and occasional patchesof chronic inflammation. Two of the ten left pouches showed smallareas of dysplasia: one pouch from a hamster with tumors on theoposite pouch demonstrated one small focus of moderate to severedysplastic change and the other pouch revealed two small areas withmild to moderate dysplasia.Group II (MMS + MMS or nothing): Both the MMS-initiated,MMS-promoted right pouches and the unpromoted left pouches wereunremarkable, although occasional rete ridge formations were notedin the right pouches.Group III (DMBA or MMS + acetone): Both the DMBA-initiated,acetone-promoted right pouches and the MMS-initiated, acetone-promoted left pouches were unremarkable, although occasionalpatches of chronic inflammatory cells were noted in the rightpouches.Statistically, DMBA-initiated, MMS-promoted right pouches ingroup I showed significant differences compared to the controls interms of tumor yield, tumor rate (P<0.05), and dysplasia rate (P<0.01)(Table 2.3).4. DiscussionThe results of the study showed that the alkylating agent MMSinduced dysplasias and tumors in HBPM initiated with 0.2% DMBAfor 2 weeks, and hence it is a tumor promoter in this model. Thestudy also demonstrated that MMS is not carcinogenic in the HBPMmodel, under the conditions of the study.Two animals in Group I also demonstrated one or two smallareas of dysplastic change in the left pouches that were only initiatedwith DMBA but not promoted with MMS. In contrast, similarlyDMBA-initiated pouches of Group III animals showed no dysplasia. Itis possible that the dysplastic changes noted in the left pouches ofGroup I animals were a result of cross-contamination of MMS fromthe right pouch treatment or a result of systemic effects of MMS inthese animals. Nonetheless, tumors were noted only in the MMS-promoted right pouches and the presence of tumors and dysplasticchanges in the MMS-promoted right pouches were statistically higherthan those in the left unpromoted pouches in the Group I animals(Table 2.3).In hamsters initiated with DMBA, MMS produced marked toxiceffects, such as a decrease in body weight, general sickness, muscle37stiffness and a change in hair color. Surprisingly, in those hamstersreceived no DMBA treatment, similar MMS treatment did notproduce obvious toxic effects. Therefore, it seems that the toxiceffects resulted from combined effects of DMBA and MMS.The mechanism of MMS tumor promotion remains unclear. Thepromoting effect of MMS is generally believed to be due to itsclastogenic effects (Cerutti, 1982), which include chromosomeaberrations, such as chromosome breaking and sister-chromatidexchange (Natarajan et al., 1984).In summary, this study demonstrated that MMS is an effectivetumor promoter but is not a carcinogenic agent in the HBPM model.The mechanism of tumor promotion by MMS probably results fromits clastogenic activity. The establishment of a new tumor promotershould prove to be useful in future studies of stage-wise changes,including enzymatic changes, during HBPM carcinogenesis.Experiment III: Developmental studies on GGT and GST-Pin hamsters'fetal, newborn and adult tissues and organs1. ObjectiveTo study the tissue and organ distribution of GOT and GST-Pduring hamster development. In particular, to study GGT and GST-Pdistributions during hamster pouch development in order to find outif the enzymes' induction during HBPM carcinogenesis is oncofetal innature.2. Materials and methodsAnimalsThe maintenance of animals was similar to that in experimentI. Animals were randomly bred. Two non-inbred hamsters fromdifferent mothers were sacrificed at day 9, 13, and 15 of gestationand day 1, 3, 6, and 10 after birth, respectively. The same number ofadult female animals was used. The following tissues and organswere quick frozen in liquid nitrogen: pouch epithelial cord, oralmucosa, tongue, tooth bud, salivary gland, skin, nasal and sinuscavity, kidney, liver, stomach, intestine, and lung.Serial sections (10 p.m) were cryostat cut and mounted ongelatine coated slides. They were then fixed in cold acetone for 5minutes. GOT histochemical staining and GST-P immunohistochemicalstaining were carried out.GGT staining methodThe histochemical demonstration of GGT was performedaccording to the method described by Rutenburg et al. (1969).The sections were incubated for 2 h at room temperature in amedium composed of y-glutamy1-4-methoxy-2-naphthylamide(GMNA, Polysciences Inc., Warrington, PA), glycylglycine free base(Sigma Chemical Company, St. Louis, MO) and Fast Blue BB salt 'Gurr'(BDH Inc., Toronto, Canada). They were rinsed in a sequence of 0.85%NaC1 (GIBCO Inc., Grand Island, NY), 0.1 M cupric sulphate (BDH Inc.,Toronto, Canada), and 0.85% NaC1 again. GGT cleaves y-glutamylgroups from GMNA and transfers it to glycylglycine. Theenzymatically liberated naphythylamine carrying the negativelycharged methoxy is bridged with the positively charged diazoniumsalt present in the mixture to form a copper-chelated azo dye. Sincethe dye is rapidly formed and is insoluble, it is restricted to the cellswith GGT activity so that diffusion artifact is minimal. Aftercompletely dried, sections were counterstained in hematoxylin,covered with glycerin as mounting medium, and sealed with nailpolish.Sections from the kidney of an adult normal hamster wereused as a positive control.GST-P staining methodImmunohistochemical demonstration of GST-P was performedusing the avidin-biotin-peroxidase complex (ABC) method asdescribed by Hsu et al. (1981). This technique was developed fromperoxidase-antiperoxidase (PAP) staining (Sternberger et al., 1970).40Avidin is a 68,000 glycoprotein with four binding sites for biotin.Covalently coupling of biotin to antigens such as immunoglobulin orthe peroxidase molecule makes it possible for them to crosslink withavidin. Hence, avidin and biotin serve as a link for each other bywhich a three dimensional amplification of antigen-antibody reactionis achieved via three steps: biotinylated secondary antibody, avidin,and biotinylated horseradish peroxidase.The sections were incubated for 30 min in 0.3% 11202 (BDH Inc.,Toronto, Canada) in methanol (BDH Inc., Toronto, Canada) to quenchthe endogenous peroxidase. They were washed then incubated for 30min with diluted normal goat serum to block non-specific bindingsites. After blotting the excess serum from slides, primary antibody(rabbit anti-rat GST-P) at 1:300 dilution was added to sections.Following a 2 night incubation in fridge, sections were incubated in asequence of diluted biotinylated secondary antibody (goat anti-rabitIgG), ABC reagent, and peroxidase substrate, diaminobenzidinetetrahydrochloride (DAB). The sections were counterstained withhematoxylin and permanently mounted.For positive control, carcinogen-treated rat liver rich in GST-P(+) nodules was used. For negative control, primary antibody wassubstituted for phosphate-buffered saline.3. ResultsThe results of GGT and GST-P staining are summarized in Table3.1.When the hamsters were examined at day 9 of gestation, noorgan formation was observed. Sporadic GGT positive cells were41noted, and were mainly distributed around some cavities. No GST-Pactivity was noted at this stage.Intra- and para-oral organsCheek pouch. The formation of cheek pouch was noted 3 daysbefore birth as an epithelial bud growing inward from the oralepithelial lining (Fig. 3.1a). The epithelial bud grew caudally as anepithelial column as observed on day 1 before birth (Fig. 3.1b) andcontinued to grow on day 1 and 3 after birth (Fig. 3.1c,d). At day 6after birth, there was cytodifferentiation characterized by theappearance of keratohyalin-containing cells and cornified cells in themiddle layers of the epithelial column. There was also liquefaction ofthe middle layers of the epithelium, indicating initiation of pouchcavity formation (Fig. 3.1e). At day 10 after birth, a pouch cavity wasformed through liquefaction of epithelial cells in the center of theepithelial islands (Fig. 3.1f). Neither GGT nor GST-P activity wasnoted at any stage of pouch development.Oral mucosa. The pre-natal, neo-natal and post-natal tonguemucosae showed no detectable GGT. Aggregates of strongly GGT-positive cells, however, were noted in adult dorsal tongue mucosa.Figure 3.2a demonstrates a tongue section and figure 3.2b a tonguemucosa stripping specimen. The strongly GUT positive cells areconfined to the connective tissue of fungiform papillae. They arespindle or stellate in shape (Fig. 3.2c). There was also a weak GGTstaining in the stratified squamous epithelial cells adjacent to thosestrongly GGT positive, spindle or stellate mesenchymal cells. No GST-P(+) reaction was recorded in the tongue mucosa. The remaining oral42mucosa was negative for GGT and GST-P at any stage of thedevelopment.Tooth. As the development stage of a tooth varies in differentteeth, for a given age of a hamster, the description of GGT and GST-Pstaining will be based mainly on the degree of enamel organformation and amount of dental hard tissue formation instead of onthe age of the hamsters.Before and after birth, both the enamel organ and odontoblastsstained for GGT, though the staining of the enamel organ appearedearlier and stronger than that of odontoblasts. Occasional positiveGGT staining was observed in dental lamina (Fig. 3.3a), mainly in thearea in which a bell shaped enamel organ was forming. When theenamel organ was formed the stellate reticulum and stratumintermedium cells were the first to stain with GGT prior to, and earlyin, the deposition of dental hard tissues (Fig. 3.3b,c). The GGT stainingof these structures gradually weakened and finally disappeared withthe increment of dental hard tissues. Ameloblasts and odontoblastsexhibited GGT activity in their secretion stage following theformation of dental hard tissue. Some GGT staining was also noted inthe dental hard tissues, including both enamel and dentin,particularly the predentin.No GST-P(+) reaction was recorded during tooth development.Ten day-old and adult teeth were investigated for GGT andGST-P activity because of their high calcium content.Salivary gland. Some acini lobules of minor salivary glandsdemonstrated weak GGT positive staining from day 1 after birth (Fig.3.4a) and became stronger on day 3 and day 6 (Fig. 3.4b), while some43other lobules besides those of positively stained glands showed noreactivity. The ducts of minor salivary glands showed ambiguousstaining on day 3 and day 6 after birth. Minor salivary glands werenot studied for GGT in 10 day-old and adult hamsters.Parotid glands were examined in 10 day-old and adulthamsters, when the glands could be easily identified and removed.Two large lobules separated by connective tissue were present. Onelobule demonstrated positive GGT staining in the acini in both 10-dayold and adult hamsters (Fig. 3.4c). The acini of the other lobule werenegative for GGT. The ducts in both lobules showed ambiguousstaining in 10 day-old hamsters, but were positive in adult hamsters.No GST-P(+) reaction was recorded in either major or minorsalivary glands.Nasal and sinus mucosa. Very strong GGT activity was noted inthe pseudostratified ciliated epithelial lining cells, especially on theside near the lumen and the cilia on day 1, before and after birth(Fig. 3.5a). Moderate GGT staining was present in a similar location atday 3 after birth (Fig. 3.5b). Other days were negative.No GST-P staining was noted.Ten day-old and adult nasal and sinus mucosa were notinvestigated for GGT and GST-P activity.Extra-oral organsSkin. The epidermis showed GGT(-) in the early embryonicstage. Few skin appendages were noted in the early embryonic stageand they were negative for GGT. Immediately before birth and afterbirth as well as in the adult, numerous hair follicles were formed.44The germinal matrix of both external and internal hair root sheaths,located in the deep portion of the hair follicle, became strongly GGTpositive. The epidermis remained negative during the time of thestudy (Fig. 3.1c,d,e & 3.6).No GST-P(+) reaction was recorded.Kidney. Strong GGT activity was demonstrated in renal tubularcells, especially the brush borders in all animals after organformation (Fig. 3.7). The highest staining intensity was noted in theadult kidney.No GST-P(+) reaction was recorded.Liver. Focal weak GGT staining was noted in hepatocytes beforebirth (-3 and -1 days) but negative after birth. Weak GGT stainingwas noted occasionally in the biliary duct cells before birth and atday 1 and day 3 after birth, but absent in 10 day-old and adulthamsters.GST-P was negative before birth. A diffuse staining, moderatein intensity, was noted in hepatocytes at day 1 after birth (Fig. 3.8a),but the staining became weak at day 3 after birth (Fig. 3.8b),negative at day 6 after birth (Fig. 3.8c), ambiguous at day 10 afterbirth (Fig. 3.8d). Moderate staining was noted again in adult hamster(Fig. 3.8e). The biliary duct cells showed weak staining in allhamsters sacrificed after birth, but the number of positive duct cellsdecreased with age (Fig. 3.8).Gastrointestinal tract. On both days before birth (-3 and -1days), the lining epithelial cells of the gastrointestinal tract,especially the villi on the luminal surface, were strongly GGT positive(Fig. 3.9a,b). No GST-P staining was noted before birth.45After birth, it was possible to differentiate stomach fromintestine and the staining results of the two organs were as follows:Stomach. There was no GUT activity detectable in neonatalhamsters. However, the adult stomach, in the region close toesophagus (fundus), moderate GGT staining was noted in the gastricglands (Fig. 3.9c). A weak GST-P staining was observed in the luminalsurface of the lining epithelium at days 1, 3, 6 and 10, but absent inadults (Fig. 3.9d).Intestine. Strong GGT staining was noted in the epithelial liningcells, especially in the luminal surface and the villi after birth (Fig.3.9 e-i). No GST-P activity was noted.Lung. There was no GGT staining in the embryonic respiratorysystem. After birth the lining epithelium of the airways, particularlythe cilia of the respiratory epithelium including trachea, bronchi andbronchioles, showed weak GGT staining at day 1, moderate stainingat day 3 and 6, strong staining at day 10, and moderate staining inadult hamsters (Fig. 3.10a-e). No alveoli were stained at any stage oflung development.Weak GST-P staining was noted in the bronchiolar liningepithelium, mainly on the cilia on day 1 after birth (Fig. 3.10f).4. DiscussionGGTThe expression of GGT in preneoplasia and in neoplasms inseveral organs and tissues has been shown to be oncofetal in naturewhile in other organs, it is not. Increased GGT has been observedduring hamster pouch carcinogenesis. The results of this study46showed that there was no GGT activity in hamster pouch at any stageof its development. Therefore, the expression of GGT activity duringcarcinogenesis of HBPM may represents an acquired gene alterationinstead of re-expression of a phenotype that is presented in normalembryonic development.GGT activity was demonstrated in a number of hamster tissuesand organs in this study, primarily in epithelial cells. GGT activitywas particularly prominent in cells with 'brush borders', such asepithelial cells of the intestinal mucosa, nasal or sinus mucosa,airways, renal tubules and of ameloblasts. The term 'brush border'denotes a specific plasma membrane structure with numerousfinger-like processes with a large surface area, and is intimatelyassociated with the transport of carbohydrates, irons and amino acids(Ãhlund-Lindqvist & Lindskog, 1985). The results of this study aresimilar to those of other studies in other species, and support thehypothesis that GGT may participate normally in amino acidtransportation.As mentioned above, one study investigated GGT activityhistochemically in a number of adult hamster organs and tissues(Albert et al., 1964). The results from this current study, in general,agree with those from Albert et al.: both studies showed GGT activityin renal tubules, in the lining epithelium of bowels, in the glandularcells and secretory ducts, but no GGT activity in hepatocytes in adulthamsters. While, Albert et al. (1964) found no GGT activityhistochemically in adult hamster lung and stomach, the presentstudy showed moderate GGT staining in the fundus region of gastricglands and the lining epithelium of bronchi and bronchioles.47Occasionally, GGT activity was noted also in mesenchymal cells:odontoblasts and some stellate or spindle cells in the lamina propriaof fungiform papilla of adult hamster tongue. Although it is obviousthat these stellate and spindle cells are mesenchymal cells, theirexact cell type is not clear. Their exclusive location in the connectivetissue papilla of fungiform papillae seems to rule out the possibilityof fibroblasts as fibroblasts are abundant throughout the tonguemucosa lamina propria. The nature of them is not clear.GST-PUnlike the wide distribution of GGT during the normaldevelopment of hamster tissues and organs, few cells showed GST-Pstaining. GST-P activity was notably absent in all hamster intraoralorgans and tissues, including the hamster pouch. This suggests thatthe expression of GST-P activity during carcinogenesis of HBPM mayrepresent an acquired gene alteration instead of re-expression of aphenotype that is presented in normal embryonic development.Only three organs, liver, lung and stomach, showed weak tomoderate GST-P activity and all the staining was confined toepithelial cells.5. Conclusion1) GGT/GST-P activity was not found during the developmentof hamster buccal pouch and oral mucosa. Therefore, it seems thatthe induction of the enzymes in HBPM carcinogenesis is not oncofetalin nature, but tumor-associated;2) GGT is found in a number of organs and tissues and islocated mainly in the epithelial cells, but occasionally inmesenchymal cells; whereas, GST-P is observed in few organs and isonly in epithelium.3) The predominant location of GGT activity in epithelial cellswith 'brush borders' supports the hypothesis that GGT normallyparticipates in amino acid transportation.TABLESTable 1.1^Mitotic Activity of OA on HBPMAnimalnoPouchsideRollnoMI(%)aOA Acetone none1 L 1 5.90 5.104.97 4.604.43 4.102 5.20 6.236.10 4.675.97 4.70R 1 5.67 3.473.20 3.633.70 3.802 6.37 4.105.87 3.737.86 4.132 L 1 5.43 3.976.20 3.805.17 3.672 7.27 5.076.53 4.976.83 4.13R 1 5.87 4.736.20 3.605.87 4.172 6.60 4.037.07 4.206.27 4.373 L 1 6.23 2.636.80 4.576.30 3.072 6.37 4.406.13 3.674.93 5.07R 1 6.93 3.976.43 4.034.90 4.232 5.20 4.035.97 5.276.10 4.49Pooled data X 5.91 4.36 4.11Sx(±) 1.00 0.72 0.43P <.001b >0.05ca Mitotic Index expressed as percentage of metaphase cells in 3,000 basalcells.b Comparison between OA-treated pouches and controls.C Comparison between acetone-treated and untreated pouches.10^R^0.2% DMBA0.2% DMBAII^10^R^0.5% MMS0.5% MMSIII^5^R^0.2% DMBA0.5% MMS0.5% MMSnone0.5% MMSnoneacetoneacetonenonenonenonenonenonenoneTable 2.1^Initiation-promotion experimental designGroup^Animal^Pouch^Initiation^NT^Promotionno (31w, 2wks)^(10wks)^(2/w,10wks)NT: no treatmentDMBA: dimethylbenz(a)anthraceneMMS: methylmethanesulfonate#1w: times of treatment per weekTable 2.2^Tumor promoting effect of MMS on HBPM^Group^Pouch^TumorYielda40II^ 00III 00P value^<.05(x2)TumorRateb2(20%)0(0%)0(0%)0(0%)0(0%)0(0%)<.05(u)DysplasiaRateb 10(100%)2(20%)0(0%)0(0%)0(0%)0(0%)<.01(u)a Number of tumors in each group.b Number of animals bearing nodule or dysplasia in each group.Animal 1 2 3 4 5 6 7 8 9 10GI^right(DMBA+MMS)Degree ofdysplasiaaMo & S Mo Mo Mo & S Mo Mo Mo & S Mo Mo& S Mo & SExtent ofdysplasiab-H- ++ -H-+ +-F+-H-+ -H-+ -H-+ +++ -H-+ -H-FTumor 3 - - 11' inepithelialthicknessc+++ +++ -H-+ ++ + + +-H--H-+ i-F+ i-H-GI Degree ofleft (DMBA dysplasia Mo to S Mi to Mo - _+NT)Extent ofdysplasia+ + _ -Tumor - - _1' inepithelialthickness+ - ++Gil^Dysplasia &(MMS + MMS^tumoror NT)T inepithelialthickness Gill (DMBA or Dysplasia &MMS +^tumoracetone)T inepithelialthickness--Table 2.3^MMS tumor promoting results in HBPMa Mi: mild dysplasia; Mo: moderate dysplasia; S: severe dysplasia.b +: 1 or 2 small focal area(s); -H-+: generalized.C +: slightly increased in thickness; ++: moderately increased; +++: marked increased.52+/-^+ / -+/ -^+1-Table 3.1^Normal distribution of GGT/GST-P in developing and adult tissues and organs ofSyrian hamsterOrgan Tissues or Cells Age (Day) 3^-1^1^3^6^10^AdultToothDental laminaEnamel organDental papillaSalivary glandNasal & sinus mucosaepitheliaameloblastsStratum intermediumstellate reticulumenamel matrixodontoblastsdentinaciniductslining epithelia Intra- & para-oral organsPouch mucosa^lining epitheliaOral mucosa lining epitheliaTongue mucosa^lining epitheliaconnective tissueExtra-oral organsSkinKidneyLiverLungGastrointestinal tractsStomachIntestineepidermishair folliclestubular epitheliahepatocytesbiliary ductsacinibronchial/bronchiolepitheliaepitheliaepitheliaepithelia-/+^-1++++/-^+++/--/++^+/+++++/-^+++/-+++/-^+++/--1++++/-53FIGURESFig. 1.1 a) An acetone-treated hamster pouch demonstrating noabnormalities. b) An okadaic acid treated hamster pouchdemonstrating erythema, edema, petechia, ulceration and shrinkageof the pouch wall.Fig. 1.2. A photomicrograph showing numerous ball-shaped, blockedmetaphase mitosis figures in the hamster pouch epithelium treatedwith okadaic acid (H.E., high power view).55Fig. 2.1. The DMBA-initiated, MMS-promoted hamster (black arrow)demonstrating a change of fur color from dark brown to light grey ascompared to the control animal (white arrow).Fig. 2.2. Tumors (arrows) in the DMBA-initiated and MMS-promotedpouches. The two control pouches on the right side of the pictureshowing no abnormality.57Fig. 2.3. Histological examination. a) Untreated pouch mucosa. b & c)DMBA-initiated, MMS-promoted pouch showing severe dysplasia andmarked acanthosis and hyperkeratosis with down-growth of reteridges. The thickness of epithelium is at least 3 times that of thecontrols (H-E, high power view).I •10..#4A se1/ ei4t•, 4.. 44..•-11:\t‘k.,59Fig. 2.4. a) A photomicrograph demonstrating a papillary tumor in aDMBA-initiated, MMS-promoted pouch. b) A photomicrographshowing an island of invasive squamous epithelium. c)A DMBA-initiated, MMS-promoted pouch showing a patch of inflammatoryinfiltration of lymphocytes and plasma cells (H-E, low power view).61Fig. 3.1. GGT-staining for the development of hamster cheek pouch. a)The formation of the cheek pouch as an epithelial bud (arrow) 3 daysbefore birth. b, c & d) The growth of the bud to form long epithelialcord 1 day before birth, and 1, 3, 6 and 10 days after birth. e) 6-dayold new born hamster cheek pouch showing keratohyalin-containingcells and cornified cells as well as liquefaction of these cells in themiddle layer of the cord. f) A 10-day old hamster pouch (low powerview).Figures c), d), and e) also show GUT positive staining ingerminal matrix of external and internal hair root sheaths.• •■•^•^411••1 S1:".--. 4, • -^' . i•••I 4 4:' . ( ,r. --t• —• - ' ""Vl'• °-, le 4`•^•,... -• . , 1; fg.*, iz",:ei:44.-,`...:- .'' sf.._ - • . ..^, -. . . *.: . -..k.-0.' ,...„.-vd -,......t..:.„^:,-.. ' .• , • "6IC'''. '‘ 4, ' *Tel; • - --..... • ' , f ‘Ve1„.^. •^.^,„•• ......^4..4 4 ...,8'..1Y.t.' -^' . -^• • -^-‘.. 4.„, •^.... • .....,„^•a , ,^,„^, .1,-, ....!,t,- n ,..-.• og„. ,. • 1.••^"•7,..„ . •••^4 ^. .: . . . . . , g r 5 .14.5it •/:^••4 • . .". •■•.• • _.^-, • 1 4^, • '••••' . ••e- ::?^ .."'..e.;;; 7'....k.%:se.‘L,--;:t • • • ., .......{. c _,.... rst„.........?z: .1.,, -,. ! .„oil '1 ,Ir Itc.c.•;•....at-,:- ... .0 • • .2. ,^AJ.--et...IL 4 .- ....-•• ,....NI 0;7.4 i-,41‘.4r:"1,; 1, ..#4 1' Z- :',P,••^t•k• ',. .0 '•: 4...,• ,•_ Ur. it- ' • •••••:6 • ' ;^- S 5'.:f.44:t • •Vs • 1,4: •4. t..../rjer.0^' A' ,/," • -• fee:. ■ .5. t * .^.4I :' if '11•4:^" 10 ',^J-4.• 1,—. ,- - - . ' •r• * • •I^l•^%^'^" . .1. •^." •• .1^\ .1^4," ; • :. a'...• ..;."•.".' 4 • 7 %‘•..1'.1* •• ^'.^'. ^. ..- ' ..4.1,..„....„..,:i• - -• • • '. • .•, •^•4.^6.. •.‘. P,^S*,..• • 6 le• . 4^ •- 1,- S^-4111*-0f0-.44`.3''1,• ■ I• .1..■ V "A• dr -." s. ••••.... . ,.,^. a,• ,' 4 *Z" I 4 • - 4 .11. .. -"' • r "' - 1P, Tr*"^1,,s‘^P^sr . • .4 ".• ^. ....^• ,,.. • 4 ' •(.4 ,4 /42 • . ilk."1 re 7^.. ••:". 4..1,1. ,s, Vitis,•, • ,iejt...... _.••• ...: • -' - .• ,. I . t^., . l•^41r^4. . •^'^, k•. .-• 11 .^-^...-' .^.' . ' •^.1^4 ' , ea" v* - - - - —• - r -:^-..,.^.....-..--^.:..^.^-• --, - - - ...... A63Fig. 3.2. GGT-staining of dorsal tongue mucosa of adult hamster'ssection and strip specimens. a & b) Low power view showing strongGGT(+) cells in the connective tissue papillae of fungiform papillae(low power view). c) High magnification view of the spindle orstellate shaped GGT positive cells (high power view).Fig. 3.3. GGT-staining for tooth developing-related structures. a) Aphotomicrograph showing GGT(+) staining in dental lamina, where abell-shaped enamel organ was forming. b & c) Photographs showingstrong GGT(+) staining in the enamel organs (low power view).65Fig. 3.4. GGT-staining of hamster salivary glands. a) A weak GGT(+)staining of the lobules of acini of minor salivary glands on the section1 day after birth. b) Positive GGT staining on day 6 after birth. c)GGT(+) staining of parotid gland of adult hamsters (low power view).Fig. 3.5. GGT-staining on the sections of nasal and sinus mucosa. a) Aphotomicrograph showing strong GGT staining in the pseudostratifiedciliated epithelial lining cells 1 day after birth. b) A moderate GGTstaining in similar location on day-3 section after birth (low powerview).67Fig. 3.6. GGT staining of hamster skin. Photomicrographs showingnegative GGT staining in epidermis but strong GGT(+) staining in thegerminal matrix of both external and internal hair root sheaths onsections from a) 1 day before birth, b) 1 day after birth, c) 10 dayafter birth, and d) adult hamsters (low power view).111,(7• 4* .4 44 °• 0 35 f":,grjv .00. gotrt^•a • AB• 00 ,30o0869Fig. 3.7 a-g. GGT staining of hamster kidneys. Photomicrographsshowing strong GGT staining in renal tubular cells in all sections fromthe designed days (low power view).71Fig. 3.8. GST-P antibody staining for hamster livers. a) A diffuse,moderate GST-P staining in hepatocytes 1 day after birth (low powerview). b) A weak GST-P staining in hepatocytes 3 days after birth(low power view), c) A negative GST-P staining of hepatocytes 6 daysafter birth (medium power view) and d) An ambiguous GST-Pstaining in hepatocytes 10 days after birth (medium power view). e)A moderate GST-P staining in the adult liver hepatocytes (low powerview). f) A positive GST-P staining in the control rat liver (low powerview).73Fig. 3.9. The enzyme staining of hamster gastrointestinal tracts. a &b) Photomicrographs showing GGT staining in the villi of the liningepithelial cells 1 and 3 days before birth. c) A moderate GGT stainingin the gastric glands of adult fundus. d) A weak GST-P staining in theluminal surface of adult stomach lining epithelium. e-i) A strong GGTstaining in lining epithelium, particularly the lumical surface ofintestine (low power view).75Fig. 3.10. Enzyme staining of hamster respiratory lining epithelium.a-e) Photomicrographs showing GGT staining in the respiratory liningepithelium of trachea, bronchi and bronchioles on 1-, 3-, 6-, 10-dayold and adult hamsters respectively (low power view). f) A weakGST-P staining in the similar regions on day 1 sections after birth(medium power view).77BIBLIOGRAPHYAdjarov D, Ivanov E, Petrova S, Savoy G et al (1979) Developmentalstudies on y-glutamyltransferase in rat intestinal mucosa.Enzyme, 24:358-361.Ahlund-Lindqvist C and Lindskog S (1985) Activity of y-glutamyltransferase in developing teeth of the rat. Scand J Dent Res,93:421-425.Albert Z (1965) Gamma-glutamyl transpeptidase in cancers ofdifferent human human organs. Nature, 205:407.Albert Z, Orlowski J, Orloski M and Szewczuk A (1964) Histochemicaland biochemical investigations of y-glutamyl transpeptidase inthe tissues of man and laboratory rodents. Acta Histochem,18:78-89.Albert Z, Orlowski M, Rzucidlo Z and Orlowska J (1966) Studies on y-glutamyl transpeptidase activity and its histochemical locationin the central nervous system of man and different animalspecies. Acta Histochem, 25:312-320.Albert Z, Orlowski M and Szewczuk A (1961) Histochemicaldemonstration of y-glutamyltranspeptidase. Nature, 191:767-768.Albert Z, Rzucidlo Z and Starzyk H (1970a) Biochemical andhistochemical investigations of the gamma glutamyltranspeptidase in embryonal and adult organs of man. ActaHistochem, 37:74s-79s.Albert Z, Rzucido Z and Starzyk H (1970b) Comparative biochemicaland histochemical studies on the activity of y-glutamyltranspeptidase in the organs of fetal, new born and adult rats.Acta Histochem, 37:34-39.Bass NM, Kirsch RE, Tuff SA, Marks I et al (1977) Ligandinheterogeneity: evidence that the two non-identical subunits arethe monomers of two distinct proteins. Biochim Biophys Acta,492:163-175.Beer DG and Pitot HC (1987) Biological markers characterizing thedevelopment of preneoplastic and neoplastic lesions in rodentliver. Arch Toxicol, 10(s):68-80.Berenblum I and Shubik P (1947a) The role of croton oil applicationsassociated with a single painting of carcinogen in tumorinduction of the mouse's skin. Br J Cancer, 1:379-382.Berenblum I and Shubik P (1947b) A new quantitative approach tothe study of the stages of chemical carcinogenesis in mouseskin. Br J Cancer, 1:383-391.Bishop JM (1982) Retroviruses and cancer genes. Adv Cancer Res,37:1-32.Bister K and Jansen HW (1986) Oncogenes in retroviruses and cells:biochemistry and molecular genetics. Adv Cancer Res, 47:99-1 8 8 .Booth J, Boyland E and Sims P (1961) An enzyme from rat livercatalyzing conjugations with glutathione. Biochem J, 79:516-5 2 4 .Boutwell RK (1964) Some biological effects of skin carcinogenesis.Prog Exp Tumor Res, 4:207-250.Boutwell RK (1989) Model systems for defining initiation, promotion,and progression of skin neoplasms. In: Skin Carcinogenesis:Mechanisms and Human Relevance. Alan R Liss Inc., pp3-15.Boyland E and Chasseaud LF (1969) The role of glutathione andglutathione S -transferases in mercapturic acid biosynthesis.Adv Enzymol, 32:173-219.Brookes P and Lawley PD (1964) Evidence for the binding ofpolynuclear aromatic hydrocarbons to the nucleic acids ofmouse skin. Relation between carcinogenic power ofhydrocarbons and their binding to deoxyribonucleic acid.Nature, 202:781-784.Brown K, Quintanilla M, Ramsden M, Kerr JB et al (1986) V-ras genesfrom Harvey and Balb Murine sarcoma viruses can act asinitiators of Two-stage mouse skin carcinogenesis. Cell, 46:447-457 .Buxman MM, Kocarnik MJ and Holbrook KA (1979) Differentiationmarkers in fetal epidermis: transglutaminase andtranpeptidase. J Invest Derm, 72:171-177.Calderon-Solt L and Solt DB (1985) Gamma-glutamyl transpeptidasein precancerous lesions and carcinomas of oral, pharyngeal, andlaryngeal mucosa. Cancer, 56:138-143.Cameron R, Kellen J, Kolin A et al (1978) Gamma-glutamyltransferasein putative premalignant liver cell populations duringhepatocarcinogenesis. Cancer Res, 38:823-829.Cayama E, Tsuda H, Sarma DSR and Farber E (1978) Initiation ofchemical carcinogenesis requires cell proliferation. Nature,275:60-62.Cerutti P (1982) Abnormal oxygen metabolism in bloom syndrome.In: Progress in Mutation Research. Natarajan A and Altman J(eds). Elsevier, Amsterdam, pp203-214.Cerutti P (1985) Prooxidant states and tumor promotion. Science,227:375-381.Chasseaud LF (1979) The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens andother electrophilic agents. Adv Cancer Res, 29:175-274.Cramer W and Stowell RE (1943) Skin carcinogenesis by a singleapplication of 20-methylcholanthrene. Cancer Res, 3:46-53.Curthoys NP and Hughey RP (1979) Characterization andphysiological function of rat renal y-glutamyltranspeptidase.Enzyme, 24:383-403.Dawson J, Smith GD, B oak J and Peters T (1979) Gamma-glutamyltransferase in human and mouse breast tumors. Clin Chim Acta,96:37-42.Degen DGH and Neumann HG (1978) The major metabolite ofaflatoxin B1 in the rat is a glutathione conjugate. Chem-BiolInteractions, 22:239-255.Demi E and Oesterle D (1980) Histochemical demonstration ofenhanced glutathione content in enzyme-altered islandsinduced by carcinogens in rat liver. Cancer Res, 40:490-491.De Young LM, Richards WL, Bonzelet W, Tsai LL et al (1978)Localization and significance of y-glutamyl transpeptidase innormal and neoplastic mouse skin. Cancer Res, 38:3697-3701.Diamond L, O'Brien TG and Baird WM (1980) Tumor promoters andthe mechanism of tumor promotion. Adv Cancer Res, 32:1-74.Di Ilio C, Del Boccio G, Aceto A and Federici G (1987) Alteration ofglutathione transferase isoenzyme concentrations in humanrenal carcinoma. Carcinogenesis, 8:861-864.Di Ilio C, Del Boccio G, Massoud R and Federici G (1986) Glutathionetransferase of human breast is closely related to transferase ofhuman placenta and erythrocytes. Biochem Int, 13:263-269.Dipple A (1985) Polycyclic aromatic hydrocarbons andcarcinogenesis: an introduction. In: Polycyclic Hydrocarbonsand Carcinogenesis. ACS Symposium Series 283. Harvey RG(ed), Am Chem Soc, Washington, ppl-18.Elce JS and Broxmeyer B (1976) Gamma-glutamyltransferase of ratkidney. Biochem J, 153:223-232.Emmelot P and Scherer E (1977) Multiple-hit kinetics of tumorformation, with special reference to experimental liver andhuman lung carcinogenesis and some general conclusions.Cancer Res, 37:1702-1708.Farber E (1984a) Cellular biochemistry of the stepwise developmentof cancer with chemicals: GHA Clowes memorial lecture. CancerRes, 44:5463-5474.Farber E (1984b) Pre-cancerous steps in carcinogenesis: theirphysiological adaptive nature. Biochim Biophys Acta, 738:171-180.Fiala S, Mohindru A, Kettering WG and Fiala AE et al (1976)Glutathione and y-glutamyl transpeptidase in rat liver duringchemical carcinogenesis. J Nat! Cancer Inst, 57:591-598.Fiala S, Fiala AE, Keller RW and Fiala ES (1977) Gamma-glutamyltranspeptidase in colon cancer induced by 1, 2-dimethylhydrazine. Arch Gesch-wulstforsch, 47:117-122.Fiala S (1979) Gamma-glutamyltransferase during chemicalcarcinogenesis in rat liver. In: Carcinoembryonic Proteins. Vol2. Elsevier, Amsterdam, pp693-698.Fiala S and Trout E (1979a) Histochemical detection of high y-glutamyl transferase activity in human epithelial tumors. J CellBiol, 83:abst Z2928.Fiala S, Trout E, Pragani B and Fiala E (1979b) Increased y-glutamyltransferase activity in human colon cancer. Lancet, 1145.Fischer G, Lilienblum W, Ullrich D and Bock KW (1986)Immunohistochemical differentiation of y- glut amyltranspeptidase in focal lesions and in zone I of rat liver aftertreatment with chemical carcinogens. Carcinogenesis, 7:1405-1 4 1 0 .Fleming N, Groscurth P and Kistler GS (1977) The activity anddistribution of gamma-glutamyl transpeptidase (y-GT) inhuman foetal organs. Histochem, 51:209-218.Freedman A and Shklar G (1978) Alcohol and hamster buccal pouchcarcinogenesis. Oral Surg, 46:794-805.Frei JV and Lawley PD (1976) Tissue distribution and mode of DNAmethylation in mice by methyl methanesulfonate and N-methyl-N'-nitro-nitrosoguanidine: Lack of thymic lymphomainduction and low extent of methylation of target tissue at 0-6of guanine. Chem Biol Interact, 13:215-222.Frei JV and Venitt S (1975) Chromosome damage in the bone marrowof mice treated with the methylating agents methylmethanesulfonate and N-methyl-N-nitrosourea in the presenceof caffeine, and its relationship with thymoma induction. MutatRes, 30:89-96.Friedwald WF and Rous P (1944) The initiating and promotingelements in tumor production. J Exp Med, 80:101-125.Fiirstenberger G, Berry DL, Sorg B and Marks F (1981) Skin tumorpromotion by phorbol esters is a two stage process. Proc Nat!Acad Sci USA, 78:7722-7726.Fiirstenberger G, Schurich B, Kaina B and Petrusevska RT (1989)Tumor induction in initiated mouse skin by phorbol esters andmethyl methanesulfonate: correlation between chromosomaldamage and conversion (stage I of tumor promotion) in vivo.Carcinogenesis, 10:749-752.Garde11 SJ and Tate SS (1981) Subunit interactions in y-glutamyltranspeptidase. J Biol Chem, 256:4799-4804.Garte SJ, Hood AT, Hochwalt AE, D'Eustachio et al (1985) Carcinogenspecificity in activation of transforming genes by direct-actingalkylating agents. Carcinogenesis, 6:1709-1712.Gerber MA and Thung SN (1980) Enzyme patterns in humanhepatocellular carcinoma. Am J Pathol, 98:395-400.Glauert HP, Beer D, Rao MS and Schwarz M et al (1986) Induction ofaltered hepatic foci in rats by the administration ofhypolipidemic peroxisome proliferators alone or following asingle dose of diethylnitrosamine. Cancer Res, 46:4601-4606.Glenner GG and Folk JE (1961) Glutamyl peptidases in rat and guineapig kidney slices: histochemical demonstration of y-glutamyltranspeptidase-like activity. Nature, 192:338.Goel SK, Lalwani ND and Reddy JK (1986) Peroxisome proliferationand lipid peroxidation. Cancer Res, 46:1324-1330.Guthenberg C and Mannervik B (1979) Purification of glutathione S-transferases from rat lung by affinity chromatography.Evidence for an enzyme form absent in rat liver. BiochemBiophys Res Commun, 86:1304-1310.Habig WH, Pabst MJ, Fleischner G, Gatmaitan Z et al (1974a) Theidentify of glutathione S-transferase B with ligandin, a majorbinding protein of liver. Proc Nati Acad Sci USA, 71:3879-3882.Habig WH, Pabst MJ and Jakoby EB (1974b) Glutathione S-transferases. J Biol Chem, 249:7130-7139.Hanigan MH and Pitot HC (1985) Gamma-glutamyltranspeptidase: itsrole in hepatocarcinogenesis. Carcinogenesis, 6:165-172.Hayes JD and Mantle TJ (1986a) Use of immuno-blot techniques todiscriminate between the glutathione S-transferase Yf, Yk, Ya,Yn/Yb and Yc subunits and to study their distribution inextrahepatic tissues. Biochem J, 233:779-788.Hayes JD and Mantle TJ (1986b) Anomalous electrophoretic behaviorof the glutathione S-transferase Ya and Yk subunits isolatedfrom man and rodents. Biochem J, 237:731-740.Hayes JD, McLellan LI, Stockman PK, Howie AF, Hussey AJ andBeckett GJ (1987) Human glutathione S-transferases; Apolymorphic group of detoxification enzymes. In: Glutathione S-Transferases and Carcinogenesis. Mantle TJ, Pickett CB andHayes JD (eds). Taylor and Francis, New York, pp3-17.Haystead TAJ, Sim ATR, Carling D, Honnor RC, Tsukitani Y, Cohen Pand Hardie DG (1989) Effects of the tumor promoter okadaicacid on intracellular protein phosphorylation and metabolism.Nature, 337:78-81.Hendrich S and Pitot HC (1987) Enzymes of glutathione metabolismas biochemical markers during hepatocarcinogenesis. CancerMetast Rev, 6:155-178.Hennings H, Shores M, Wenk ML, Spangler EF et al (1983) Malignantconversion of mouse skin tumors is increase by tumor initiatorsand unaffected by tumor promoters. Nature, 304:67-69.Horiuchi S, Inoue M and Morino Y (1978) Gamma-glutamyltranspeptidase: sidedness of its active site on renal brushborder membrane. Eur J Biochem, 87:429-437.Howett MK, Pegg AE and Rapp R (1979) Enhancement of biochemicaltransformation of mammalian cells by herpes simplex virusfollowing nitrosomethylurea treatment. Cancer Res, 39:1041.Hsu SM, Raine L and Fanger H (1981) Use of avidin-biotin-peroxidasecomplex (ABC) in immunoperoxidase techniques: a comparisonbetween ABC and unlabeled antibody (PAP) procedures Jhistochem Cytochem, 29:577-580.Hughey RP and Curthoys NP (1976) Comparison of the size andphysical properties of y-glutamyltranspeptidase purified fromrat kidney following solubilization with papain or with trixonX-100. J Biol Chem, 251:7863-7870.Hultberg B and Sjogren U (1980) Gamma-glutamyl transpeptidaseactivity in normal and leukemic leukocytes. Acta Haemat,63:132-135.IARC Monographs on: Methyl Methanesulphonate. In: Evaluation ofthe Carcinogenic Risk of Chemicals to Man. Vol 7, by two IARCworking groups on the evaluation of the carcinogenesis risk ofchemicals to man which met in Lyon., pp253-260.Imler JL, Schatz C, Wasylyk C, Chatton B et al (1988) A Harvey-rasresponsive transcription element is also responsive to a tumor-promoter and to serum. Nature, 332:275-278.Ishii H, Ebihara Y, Okuno F, Munakata Y et al (1986) Gamma-glutamyl transpeptidase activity in liver of alcoholics and itshistochemical localization. Alcohol Clin Exp Res, 10:81-85.Ito N, Tsuda H, Tatematsu M, Inoue T et al (1988) Enhancing effect ofvarious hepatocarcinogens on induction of preneoplasticglutathione S-transferase placental form positive foci in rats: anapproach for a new medium-term bioassay system.Carcinogenesis, 9:387-394.Jakoby WB (1978) The glutathione S-transferases: a group ofmultifunctional detoxification proteins. Adv Enzymol, 46:383-4 14 .Johnson MD, Housey GM, Kirschmeier PT and Weinstein IB (1987)Molecular cloning of gene sequences regulated by tumor85promoters and mitogens through protein kinase C. Mol Cell Biol,7:2821-2829.Kensler TW and Taffee BG (1986) Free radicals in tumor promotion.Adv Free Radical Biol Med, 2:347-387.Ketterer B, Meyer DJ, Coles B, Taylor JB et al (1985) Glutathionetransferase and carcinogenesis. In: Antimutagenesis andAnticarcinogenesis: Mechanisms. Shankel DM, Hartman PE, KadaT and Holl...nder A (eds). Plenum Publishing, London, pp103-126 .Ketterer B, Tan KH, Meyer DJ and Coles B (1987) Glutathionetransferases: a possible in the detoxication of DAN and lipidhydroperoxides. In: Glutathione S-transferases andCarcinogenesis. Mantle TJ, Pickett CB and Hayes JD (eds). Taylor& Francis, London, pp149-163.Kitahara A, Satoh K and Sato K (1983a) Properties of the increasedglutathione S-transferase A form in rat preneoplastic hepaticlesions induced by chemical carcinogens. Biochem Biophys ResCommun, 112:20-28.Kitahara A, Yamazaki T, Ishikawa T, Camba EA et al (1983b) Changesin activities of glutathione peroxidase and glutathionereductase during chemical hepatocarcinogenesis in the rat.Gann, 74:649-655.Kleinhues P and Cooper HK (1976) Repair excision of alkylated basesfrom DNA in vivo. Oncology, 33:86-88.Knox WE (1972) Enzyme Patterns in Fetal, Adult and Neoplastic RatTissues. Basel S and Karger AG.Kodate C, Fukushi A, Narita T and Kuda H (1986) Human placentalform of glutathione S-transferase (GST-p) as a newimmunohistochemical marker for human colonic carcinoma.Gann, 77:226-229.Kohn KW (1979) DNA as a target in cancer chemotherapy:Measurement of macromolecular DNA damage produced inmammalian cells by anticancer agents and carcinogens.Methods Cancer Res, 16:291-345.Konohana A, Konohana I, Schroeder WT, O'Brien WR, Amagai M, GreerJ, Shimizu N, Gammon WR, Siciliano MJ, Duvic M (1990)Placental Glutathione-S-transferase pi mRNA is abundantlyexpressed in human skin. J Invest Dermat, 95:119-126.Koskelo K (1983) Isoelectric focusing of glutathione S-transferases:Comparision of the acidic transferases from human liver,kidney, lung, spleen and placenta. Scand J Clin Lab Invest,43:133.Koskelo K, Valmet E and Tenhunen R (1981) Purification andcharacterization of an acid glutathione S-transferase fromhuman lung. Scand J Clin Lab Invest, 41:683-689.Laishes BA, Ogawa K, Roberts E and Farber E (1978) Gamma-glutamyltranspeptidase: a positive marker for cultured rat liver cellsderived from putative premalignant and malignant lesions. JNati Cancer Inst, 60:1009-1015.Li Y, Seyama T, Godwin AK, Winokur TS et al (1988) MTrasT24, ametallothionein-ras fusion gene, medulates expression incultured rat liver cells of two gene associated with in vivo livercancer. Proc Nati Acad Sci USA, 85:344-348.Loveless A (1969) Possible relevance of 0-6 alkylation ofdeoxyguanosine to the mutageniciy and carcinogenicity ofnitrosamines and nitrosamides. Nature. 223:206-207.Luebke RW, Rogers RR, Riddle MM, Rowe DG and Smialowicz RJ(1987) Alteration of immune function in mice followingcarcinogen exposure. Immunopharmacology, 13:1-9.MacDonald DG (1973) Evaluation of epithelial atypia scoring inexperimental and human oral premalignancy. J Dent Res,52:923.Mannervik B (1985) The isoenzymes of glutathione transferase. AdvEnzymol Relat Areas Mol Biol, 57:357-414.Mannervik B, Alin P, Guthenberg C, Jensson H et al (1985)Identification of three classes of cytosolic glutathionetransferase common to several mammalian species: correlationbetween structural data and enzymatic properties. Proc NatiAcad Sci USA, 82:7202-7206.Mannervik B, Alin P Danielson UH, Guthenberg C et al (1987) Threeclasses of mammalian glutathione transferases and theiroccurrence in the rat and the mouse. In: Glutathione S-transferases and Carcinogenesis. Mantle TJ, Pickett CB andHayes JD (eds). Taylor & Francis, London.Manson MM, Green JA and Driver HE (1987) Ethoxyquin aloneinduces preneoplastic changes in rat kidney whilst preventinginduction of such lesions in liver by aflatoxin Bl.Carcinogenesis, 8:723-728.Mantle TJ, Pickett CB and Hayes JD (1987) Glutathione S-transferasesand Carcinogernesis. Taylor & Francis, London, New York andPhilaselhpia.Marathe GV, Nash B, Haschemeyer RH and Tate SS (1979)Ultrastructural localization of y-glutamyl transpeptidase in ratkidney and jejunum. FEBS Lett, 107:436-440.Marks F (1990) Hyperplastic transformation, the response of the skinto irritation and injury. In: Skin Pharmacology and Toxicology.Galli CL, Marinovich DM and Hensby CN (eds). Plenum Press,NY, pp121-145.Marks F and Fiirstenberger G (1987) From the normal cell to cancer:the multistage process of experimental skin carcinogenesis. In:Concepts and Theories in Carcinogenesis. Maskens AP, EbbesenP and Burny A (eds). Elsevier, Amsterdam, pp169-148.Marks F, Fiirstenberger G, Geschwendt M, Rogers M et al (1988) Thewound response as a key element for an understanding ofmultistage carcinogenesis in skin. In: Chemical Carcinogenesis.Feo F, Pani P, Columbano A and Garcea R (eds). Plenum Press,NY, pp217-234.Matsuda Y, Tsuji A and Katunuma N (1983) Studies on the structureof y-glutamyltranspeptidase. III. Evidence that the aminoterminus of the heavy subunit is the membrane bindingsegment. J Biochem, 93:1427-1433.McGaughey C, Jensen J and Taghdiri K (1984) Hamster cheek pouchas a bioassay system in short term screening for tumorinitiating and promoting agents. Optimization of parametersand observations of effects of cytotoxicity on tumor promotionby retinyl acetate. JEPTO, 5:283-297.McIntyre TM and Curthoys NP (1979) Comparison of the hydrolyticand transfer activities of rat renal y-glutamyltranspeptidase. JBiol Chem, 254:6499-6504.Meister A (1976) Glutathione: metabolism and function via theglutamyl cycle. Life Sci, 15:177-190.Meyer DJ,J, Beale D, Hong Tan KH, Coles B et al (1985) Glutathionetransferases in primary rat hepatomas: the isolation of a formwith GSH peroxidase activity. FEBS Lett, 184:139-143.Miller EC and Miller JA (1974) Biochemical mechanisms of chemicalcarcinogenesis. In: The Molecular Biology of Cancer. Busch H(ed). Academic Press, NY, pp377-402.Milo GE, Blakeslee JR, Hart R and Yohn DS (1978) Chemical carcinogenalterations S V40 virus induced transformation of normalhuman cell populations in vitro. Chemi.-Biol Interactions,22: 185.Miyazaki M, Handa Y and Sato J (1985) Transformation of primarycultured and co-cultured adult rat liver cells by 3'-methy1-4-dimethylaminoazobenzene. Acta Med Okayama, 39:231-238.Mock D, Whitestone B and Freeman J (1987) Gamma-glutamyltranspeptidase activity in human oral squamous cell carcinoma.Oral Surg, 64:197-201.Moldeus P and Jernstrom B (1983) Interaction of glutathione withreactive intermediates. In: Functions of Glutathione:Biochemical, Physiological, and Clinical Aspects. Larsson A,Orrenivs S, Holmgren A and Mannervik B (eds). Raven Press,NY, pp99-108.Moore MA, Hacker HJ and Bannasch P (1983) Phenotypic instabilityin focal and nodular lesions induced in a short term system inthe rat liver. Carcinogenesis, 4:595-603.Moore MA, Satoh K, Kitahara A and Sato K et al (1985) A proteincross-reacting immunohistochemically with rat glutathione S-transferase are placental form as a marker for preneoplasia inSyrian hamster pancreatic and hepatocarcinogenesis. Gann,76:1-4.Moore MA, Nakamura T and Ito N (1986) Immunohistochemicallydemonstrated glucose-6-phosphate dehydrogenase, y-glutamyltranspeptidase, ornithine decarboxylase and glutathione S-transferase enzymes: absence of direct correlation with cellproliferation in rat liver putative preneoplastic lesions.Carcinogenesis, 7:1419-1424.Moore MA, Tsuda H, Hacker HJ, Sato K et al (1987a) Altered enzymeexpression in propylnitrosamine-induced Syrian hamster lunglesions. Virchows Arch(B), 53:272-278.Moore MA, Nakagawa K, Satoh K, Ishikawa T et al (1987b) SingleGST-P positive liver cells: putative initiated hepatocytes.Carcinogenesis, 8:483-486.Moore MA, Nakagawa K and Ishikawa T (1988) Selection pressureand altered hepatocellular islands after a single injection ofaflatoxin B 1. Gann, 79:187-194.Morris AL (1961) Hamster cheek pouch response to varying lengthsof carcinogen exposure. J Dent Res 40:3-15.Morris SM, Heflich RH, Beranek DT and Kodell RL (1982) Alkylation-induced sister-chromatid exchanges correlated with reducedcell survival, not mutations. Mutat Res, 105:163-168.Murakami Y, Oshima Y and Yasumoto T (1982) Identification ofokadaic acid as a toxic component of a marine dinoflagellateprorocentrum lima. Bull Jpn Soc Sci Fish, 48: 69-72.Muramatsu M, Okuda A, Kano T and Sakai M (1987) Structrue andregulation of rat glutathione S-transferase P gene. In:Glutathione S-Transferase and Carcinogenesis. Mantle TJ,90Pickett CB and Hayes JD (eds). Taylor & Francis, London, NewYork and Philadelphia, pp111-119.Murata M, Shimatani M, Sugitani H, Oshima Y and Yasumoto T(1982)Isolation and structure elucidation of the causative toxinof the diarrhetic shellfish poisoning Bull Jpn Soc Sci Fish, 48:549-552.Nakagawa K, Yokota J, Wada M, Sasaki Y et al (1988) Levels ofglutathione S-transferase p mRNA in human lung cancer celllines correlate with the resistance to cisplatin and carboplatin.Gann, 79:301-304.Natarajan AT, Simons JWIM, Vogel EW and Van Zeeland AA (1984)Relationship between cell killing, chromosomal aberrations,sister-chromatid exchanges and point mutations induced bymonofunctional alkylating agents in Chinese hamster cells.Mutat Res, 128:31-40.Newbold RF, Warren W, Medcalf ASC and Amos J (1980)Mutagenicity of carcinogenic methylating agents is associatedwith a specific DNA modification. Nature, 283:596-599.Novogrodsky A, Tate SS and Meister A (1976) Gamma-glutamyltranspeptidase, a lymphoid cell surface marker: relationship toblastogenesis, differentiation, and neoplasia. Proc Nati Acad SciUSA, 73:2414-2418.Numoto S, Furukawa K, Furuya K and Williams GM (1984) Effects ofthe hepatocarcinogenic peroxisome-proliferating hypolipidemicagents clofibrate and nafenopin on the rat liver cell membraneenzymes y-glutamyltranspeptidase and alkaline phophataseand on the early stages of liver carcinogenesis. Carcinogenesis,5:1603-1611.O'Brien TG and Diamond L (1979) Effects of tumor-promoting phorboldiester on hamster cell in culture. Carcinogenesis, 7:801-805.Ogawa K, Solt D and Farber E (1980) Phenotypic diversity as an earlyproperty of putative preneoplastic hepatocyte populations inliver carcinogenesis. Cancer Res, 40:725-733.Okuda A, Sakai M and Muramatsu M (1987) The structure of the ratglutathione S-transferase P gene and related pseudogenes. JBiol Chem, 262:3858-3863.Okuda A, Sakai M and Muramatsu M (1988) Structure and regulatoryelements of glutathione S-transferase P gene. Proc Am AssocCancer Res, 29:1818.Orlowski M and Meister A (1970) The y-glutamyl cycle: A possibletransport system for amino acids. Proc Natl Acad Sci, 67:1248-1255.Ostlund-Farrants AK, Meyer DJ, Coles B, Southan C et al (1987) Theseparation of glutathione transferase subunits by usingreverse-phase high-pressure liquid chromatography. BiochemJ, 245:423-428.Pacifici GM, Warholm M, Guthenberg C, Mannervik B et al (1986)Organ distribution of glutathione transferase isoenzymes in thehuman fetus: differences between liver and extrahepatictissues. Biochem Pharmacol, 35:1616-1619.Pegg AE (1983) Alkylation and subsequent repair of DNA afterexposure to dimethylnitrosamine and related carcinogens. RevBiochem Toxicol, 5:83-133.Peters W, Nagergast F and Wobbes T (1989) Glutathione-S-transferases in normal and cancerous human colon tissues.Carcinogenesis, 10:2371-2374.Pitot HC, Campbell HA, Maronpot R, Bawa N, Rizvi TA, Xu Y, Sargent L,Dragan Y, Pyron M (1989) Toxicologic Pathology, 17:594-612.Polidoro G, Di Ilio C, Arduini A and Federici G (1982) Glutathioneperoxidase and glutathione S-transferase activities in humanfetal tissues. Inability of acidic form of glutathione S-transferase to catalyse the reduction of organic hydroperoxide.Biochem Int, 4:637-645.Pott P (1775) Chiurgical observations. In: Cancer Scrotitis. Hawes,Clarke and Clooins(eds), London, pp734-736.Pour PY and Althoff J (1979) Comparative studies on spontaneoustumor incidence based on systematic histologic examination ofrat and hamster strains of the same colony. Prog. exp. TumorRes, 24:199-206.Power C, Sinha S, Webber C, Manson M et al (1987) Tranformationrelated expression of glutathione S-transferase P in rat livercells. Carcinogenesis, 8:797-801.Quintanilla M, Brown K, Ramsden M and Balmain A (1986)Carcinogenic specific mutation and amplification of Ha-rasduring mouse skin carcinogenesis. Nature, 322:78-80.Rao MS, Lalwani ND and Reddy JK (1984) Sequential histologic studyof rat liver during peroxisome proliferator 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-acid (Wy-14,643)-inducedcarcinogenesis. J Nati Cancer Inst, 73:983-990.Rao MS, Subbarao V and Reddy JK (1986a) Peroxisome proliferator-induced hepatocarcinogenesis: histochemical analysis ofpreneoplastic and neoplastic lesions for y- glutamyltranspeptidase activity. J Nati Cancer Inst, 77:951-956.Rao MS, Tatematsu M, Subbarao V, Ito N et al (1986b) Analysis ofperoxisome proliferator-induced preneoplastic and neoplasticlesions of rat liver for placental form of glutathione S-transferase and y-glutamyltranspeptidase. Cancer Res, 46:5287-5290.Rao MS, Kokkinakis DM, Subbarao V and Reddy JK (1987a)Peroxi some proliferator-induced hepatocarcinogenesis: levels ofactivating and detoxifying enzymes in hepatocellularcarcinomas induced by ciprofibrate. Carcinogenesis, 8:19-23.Rao MS, Usuda N, Subbarao V and Reddy JK (1987b) Absence of y-glutamyl transpeptidase activity in neoplastic lesions inducedin the liver of male F-344 rats by d-(2-ethylhexy) phthalate, aperoxisome proliferator. Carcinogenesis, 8:1347-1350.Rao MS, Nemali MR, Usuda N, Scarpelli DG et al (1988) Lack ofexpression of glutathione S-transferase P, y-glutamyltranspeptidase, and a-fetoprotein messenger RNAs in livertumors induced by peroxisome proliferators. Cancer Res,48:4919-4925.Redpath NT and Proud CG (1989) The tumor promoter okadaic acidinhibits reticulocyte-lysate protein synthesis by increasing thenet phosphorylation of elongation factor 2. Biochem Journal,262:69-75.Roomi MW, Satoh K, Sato K and Farber E (1985a) Natural distributionof placental glutathione S-transferase and its modulation bychemicals. Fed Proc (FASEB), 44:521.Roomi MW, Ho RK, Sarma DSR and Farber E (1985b) A commonbiochemical pattern in preneoplastic hepatocyte nodulesgenerated in four different models in the rat. Cancer Res,45:564-571.Rosalki SB (1975) Gamma-glutamyltranspeptidase. Adv Clinic Chem,17:53-107.Rutenburg AM, Kim H, Fishbein JW, Hanker JS et al (1969)Histochemical and ultrastructural demonstration of y-glutamyltranspeptidase activity. J Histochem Cytochem, 17:517-526.Sakai M, Okuda A and Murammatsu M (1988) Multiple regulatoryelements and phorbol 12-0-tetradecanoate 13-acetateresponsiveness of the rat placental glutathione transferasegene. Proc Natl Acad Sci USA, 85:9456-9460.Salley JJ (1954) Experimental carcinogenesis in the cheek pouch ofthe Syrian hamster. J Dent Res, 33:253-262.Samuels S (1977) Transport of the large-neutral amino acids by they-glutamyl cycle. A proposal. J Theor Biol, 64:729-738.Santis H, Shklar G and Chauncey HH (1964) Histochemistry ofexperimentally induced leukoplakia and carcinoma of thehamster buccal pouch. Oral Surg, 17:307-318.Sato K, Satoh K, Hatayama I, Tsuchida et al (1987) Placentalglutathione S-transferase as a marker for preneoplastic tissues.In: Glutathione S-transferases and Carcinogenesis. Mantle TJ,Pickett CB and Hayes JD (eds). Laylor & Francis, London,pp127-137.Sato K (1988) Glutathione S-transferases and hepatocarcinogenesis.Jpn J Cancer Res (Gann), 79:556-572.Sato K (1989) Glutathione transferases as markers of preneoplasiaand neoplasia. Advances in Cancer Res, 52: 205-255.Sato S and Sugimura T (1976) Isoenzymes of carbohydrate enzymes.Methods Cancer Res: 12:259-315.Satoh K, Kitahara A, Soma Y, Inaba Y et al (1985) Purification,induction, and distribution of placental glutathione transferase:a new marker enzyme for preneoplastic cells in the ratchemical hepatocarcinogenesis. Proc Natl Acad Sci USA,82:3964-3968.Scherer E, Hoffmann M, Emmelot P and Friedrich-Freksa H (1972)Quantitative study on foci of altered liver cells induced in therat by a single dose of diethylnitrosamine and partialhepatectomy. J Natl Cancer Inst, 49:93-106.Scherer E and Emmelot P (1975a) Kinetics of induction and growth ofprecancerous liver cell foci, and liver tumor formation bydiethylnitrosamine in the rat. Eur J Cancer, 11:689-696.Scherer E and Emmelot P (1975b) Foci of altered liver cells inducedby a single dose of diethylnitrosamine and partial hepatectomy:their contribution to hepatocarcinogenesis in the rat. Eur JCancer, 11:145-154.Scherer E and Emmelot P (1976) Kinetics of induction and growth ofenzyme-deficient islands involved in hepatocarcinogenesis.Cancer Res, 36:2544-2554.Scragg MA and Johnson NW (1980) A comparison of in vivo and invitro stathmokinetic methods for the study of cell proliferationin hamster oral epithelia. Cell Tissue Kinet, 13:359-369.Sell S, Hunt JM, Knoll BJ and Dunsford HA (1987) Cellular eventsduring hepatocarcinogenesis in rats and the question ofpremalignancy. Adv Cancer Res, 48:37-111.Sellakumar A, Snyder CA and Albert RE (1987) Inhalationcarcinogenesis of various alkylating agents. J Nati Cancer Inst,79:285-289.Setala K, Setälä H and Holsti P (1954) A new and physiochemicallywell-defined group of tumor promoting (carcinogenic) agentsfor mouse skin. Science, 120:1075-1076.Shea TC and Henner WD (1987) Glutathione transferase it in humantumors. In: Glutathione S-transferases and Carcinogenesis.Mantle TJ, Pickett CB and Hayes JD (eds). Laylor & Francis,London, pp227-230.Shiratori Y, Soma Y, Maruyama H, Dato S et al (1987)Immunohistochemical detection of the placental form ofglutathione S-transferase in dysplastic and neoplastic humanuterine cervix lesion. Cancer Res, 47:6806-6809.Shklar G (1972) Experimental oral pathology in Syrian hamster. ProgExp Tumor Res, 16:518-538.Shubik P (1950) Studies on the promoting phase in the stages ofcarcinogenesis in mice, rats, rabbits, and guinea pigs. CancerRes, 10:13-17.Silberman S and Shklar G (1963) The effect of a carcinogen (DMBA)applied to the hamster pouch in combination with croton oil.Oral Surg. 16:1344-1355.Sinha S, Marshall CJ and Neal GE (1986) Gamma-glutamyltranspeptidase and the ras-induced transformation of a ratliver cell line. Cancer Res, 46:1440-1445.Slaga TJ, Fischer SM, Nelson K and Gleason GL (1980) Studies on themechanisms of skin tumor promotion: evidence for severalstages of promotion. Proc Natl Acad Sci USA, 77:3659-3663.Smith GJ, Ohi VS and Litwack G (1977) Ligandin, the glutathione S-transferases, and chemically induced hepatocarcinogenesis: areview. Cancer Res, 38:8-14.Solt D^(1981) Localization of gamma-glutamyl transpeptidase inhamster buccal pouch epithelium treated with 7,12-dimethylbenz(a)anthracene. JNCI, 67:193-200.Solt D and Farber E (1976) New principle for the analysis of chemicalcarcinogenesis. Nature, 263:701-703.Solt DB and Shklar G (1982) Rapid induction of y-glutamyltranspeptidase-rich intraepithelial clones in 7,12-dimethylbenzanthracene-treated hamster buccal pouch. CancerRes, 42:285-291.Soma Y, Satoh K and Sato K (1986) Purification and subunit-structural and immunological characterization of fiveglutathione S-transferases in human liver, and the acidic formas a hepatic tumor marker. Biochim Biophys Acta, 869:247-258 .Sternberger LA, Hardy PH, Cuculis JJ and Meyer HG (1970) Theunlabeled antibody enzyme method of immunohistochemistry.Preparation and properties of soluble antigen-antibodycomplex (horseradish peroxidase-antihorseradish peroxidase)and its use in identification of spirochetes. J HistochemCytochem, 18:315-333.Suganuma M, Fujiki H, Suguri H, Yoshizawa S et al (1988) Okadaicacid: An additional non-phorbol-2-tetradecanoate-13-acetate-type tumor promoter. Prot Natl Acad Sci. USA, 85:1768-1771.Sugioka Y, Kano T, Okuda A, Sakai M et al (1985) Cloning and thenucleotide sequence of rat glutathione S-transferase P cDNA.Nuc Acids Res, 13:6049-6057.Suter W, Brennand J, McMillan S and Fox M (1980) Relativemutagenicity of antineoplastic drugs and other alkylatingagents in V79 Chinese hamster cells, independence of cytotoxicand mutagenic responses. Mutat Res, 73:171-181.Szent-GyOrgyi A (1978) The Living State and Cancer. Marcel DekkerInc, NY.Tachibana K, Scheuer PJ, Tsukitani Y, Kikuchi H et al (1981) Okadaicacid, a cytotoxic polyether from two marine sponges of thegenus Halichondria. J Am Chem Soc, 103:2469-2471.Takahashi S, Tsutsumi M, Nakae D, Denda A et al (1987) Persistenteffect of a low dose of pre-administered diethylnitrosamine onthe induction of enzyme-altered foci in rat liver.Carcinogenesis, 8:509-513.Tate SS and Meister A (1981). Gamma-glutamyl transpeptidase:catalytic, structural and functional aspects. Mol Cell Biochem,39:357-68.Tatematsu M, Mera Y, Ito N, Satoh K et al (1985) Relative merits ofimmunohistochemical demonstrations of placental, A, B and Cforms of glutathione S-transferase and histochemicaldemonstration of y-glutamyltransferase as markers of alteredfoci during liver carcinogenesis in rats. Carcinogenesis, 6:1621-1626 .Tatematsu M, Tsuda H, Shirai T, Masui T et al (1987) Placentalglutathione S-transferase as a new marker forhepatocarcinogenesis: in vivo short-term screening forhepatocarcinogens. Toxicol Pathol, 15:60-68.Tatematsu M, Mera Y, Inoue T, Satoh K et al (1988a) Stablephenotypic expression of glutathione S-transferase in rat liverpreneoplastic and neoplastic lesions. Carcinogenesis, 9:215-220.Tatematsu M, Aoki T, Kagawa M, Mera Y et al (1988b) Reciprocalrelationship between development of glutathione S-transferasepositive liver foci and proliferation of surrounding hepatocytesin rats. Carcinogenesis, 9:221-225.Thamavit W, Tatematsu M, Ogiso T, Mera Y et al (1985) Dose-dependent effects of butylated hydroxyanisole, butylatedhydroxytoluene and ethoxyquin in induction of foci of rat livercells containing the placental form of glutathione S-transferase.Cancer Lett, 27:295-303.Thastrup 0, Cullen PJ, Drobak MR, Hanley MR et al (1990)Thapsigargin, a tumor promoter, discharges intracellular Ca2+stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Nati Acad Sci. USA, 87:2466-2470.Traynor OJ, Lee YC, Whittacker KB, Zinsmeister AR, Moss DW andWood CB (1988) Gamma glutamyl transferase activity in ratcolon during experimental colonic carcinogenesis. J Surg Oncol,37:52-55.Tsuchida S, Izumi T, Shimizu T, Ishikawa T et al (1987) Purificationof a new acidic glutathione S-transferase, GST-YnlYn2, with ahigh leukotriene-C4 synthase activity from brain. Eur JBiochem, 170:159-164.Tsuda H, Moore MA, Asamoto M, Satoh K et al (1985) Comparison ofthe various forms of glutathione S-transferase with glucose-6-phosphate dehydrogenase and y-glutamyltranspeptidase asmarkers of preneoplastic and neoplastic lesions in rat kidneyinduced by N-ethyl-N-hydroxyethylnitrosamine. Gann, 76:919-929.Tsutsumi M, Sugisaki T, Makino T, Miyagi N et al (1987) Oncofetalexpression of glutathione-S-transferase placental form inhuman stomach carcinomas. Jpn J Cancer Res (Gann), 78:631-633.Uchida T, Miyata H and Shikata T (1981) Human hepatocellularcarcinoma and putative precancerous disorders. Arch PatholLab Med, 105:180-186.Van der Jagt DL, Hunsaker LA, Garcia KB and Royer RE (1985)Isolation and characterization of the multiple glutathione S-transferases from human liver. J Biol Chem, 260:11603-11610.Van Hoosier GL Jr, Trentin JJ (1979) Naturally occuring tumors ofthe Syrian hamsters. Prog. exp. Tumor Res, 23:1-12.Van Suuren BL (1969) Tumor-promoting agents in two-stagecarcinogenesis. In: Progress in Experimental Tumor Research.Vol 11. Homburger F (ed). Karger AG, Basel, pp31-68.Vogel E and Natarajan AT (1981) The relation between reactionkinetics and mutagenic action of mono-functional alkylatingagents in higher eukaryotic systems: interspecies comparisons.In: Chemical mutagens. Vol 7. De Serres FJ and Hollander A(eds). Plenum Press, NY, pp295-336.Warholm M, Guthenberg C, Mannervik B, von Bahr C et al (1980)Identification of a new glutathione S-transferase in hamsterliver. Acta Chem Scand Serv, B34:607-610.Weinberg RA (1982) Oncogenes of spontaneous and chemicallyinduced tumors. Adv Cancer Res, 36:149-163.Wirth PJ, Rao MS and Evarts RP (1987) Coordinate polypeptideexpression during hepatocarcinogenesis in male F-344 rats:comparison of the Solt/Farber and Reddy models. Cancer Res,47:2839-2851.Yamagiwa K and Ichikawa K (1918) Experimental study of thepathogenesis of carcinoma. J Cancer Res, 3:1-29.Yamamoto K, Yokose Y, Nakajima A, Emimoto H et al (1988)Comparative histochemical investigation of the glutathione S-transferase placental form and y-glutamyl transpeptidaseduring N-nitrosobis (2-hydroxyprophol) amine-induced lungcarcinogenesis in rats. Carcinogenesis, 9:399-404.Yasumoto T, Murato M, Oshima Y, Matsumoto GK et al (1984)Diarrhetic shellfish poisoning. In: Seafood Toxins. AmericanChemistry Society, pp207-214.Yuspa SH and Poirier MG (1988) Chemical carcinogenesis: fromanimal models to molecular models in one decade. Adv CancerRes, 50:25-70.Zhang L (1989) Gamma-glutamyl transpeptidase and related enzymeactivity in hamster buccal pouch carcinogenesis. Ph.D. thesis,University of Toronto.Zhang L and Mock D (1987) Gamma-glutamyl transpeptidase activityduring carcinogenesis of hamster buccal pouch epithelium.Carcinogenesis, 8:977-981.Zhang L. and Mock D (1992) Effect of benzoyl peroxide on two-stageoral carcinogenesis and gamma-glutamyl transpeptidase inhamster. J. Oral Pathol Med, 21(6):270-274.

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:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0086188/manifest

Comment

Related Items