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Clastogenic activity in urine of individuals occupationally exposed to pesticides See, Raymond Hugh 1986

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CLASTOGENIC ACTIVITY IN URINE OF INDIVIDUALS OCCUPATIONALLY EXPOSED TO PESTICIDES By RAYMOND HUGH SEE B.Sc., The U n i v e r s i t y of B r i t i s h Columbia, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department o f Pathology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September 1986 (a) wRaymond Hugh See, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of \ aZA^t<rz^^ The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 M/fi-n ABSTRACT Epidemiological evidence suggests that many human cancers may be a t t r i b u t e d to environmental factors. Since the number of chemicals introduced into the environment i s increasing at an alarming rate, measures must be taken to reduce human exposure. There i s thus a growing need for the development of relevant and s e n s i t i v e procedures for monitoring human exposure to environmental carcinogens and mutagens. The objective of t h i s thesis was to evaluate the f e a s i b i l i t y of using urine analysis to monitor i n d i v i d u a l exposure to p e s t i c i d e s . Pesticides are widely used chemicals i n a g r i c u l t u r e , and some are known to possess genotoxic properties. In t h i s study, urine samples were c o l l e c t e d from 21 orchardists ( a l l non-smokers) i n the Okanagan Val l e y when they were engaged i n the a p p l i -cation of p e s t i c i d e s during the f r u i t growing seasons i n 1984 and 1985. As c o n t r o l s , urine was c o l l e c t e d from these same individuals during the pre-spraying as well as the post-spraying seasons. In addition, 18 i n d i v i d u a l s from an a g r i c u l t u r a l research station i n the Okanagan region (including 16 non-sprayers and 2 sprayers) were recruited to provide urine samples during the same time periods as the orchardists. As controls outside the f r u i t growing region, i n d i v i d u a l s from Vancouver and Grand Forks, B.C. were r e c r u i t e d to provide one urine specimen for t h i s study. The urine samples were concentrated by reversed-phase high pressure l i q u i d chromatography and then tested for t h e i r a b i l i t y to induce chromosome aberrations ( i . e . , clastogenic a c t i v i t y ) i n cultured Chinese hamster ovary (CHO) c e l l s . Furthermore, an attempt was made to examine the e x f o l i a t e d u r o t h e l i a l c e l l s for the presence of micronuclei as a potential in vivo i n d i c a t o r of damage by genotoxic constituents i n the urine. Urine samples obtained from the orchardists 16 to 24 hours a f t e r p e s t i c i d e a p p l i c a t i o n in 1984 resulted in l e v e l s of clastogenic a c t i v i t y undistinguishable from normal control l i m i t s . The f a i l u r e to demonstrate increased clastogenic a c t i v i t y i n the urine may have been due to (1) exposure to pesticides below the detection l i m i t s of the procedure, (2) the lack of genotoxicity in the agents sprayed, and (3) rapid p e s t i c i d e metabolism and excretion i n the urine. In the follow-up study of 1985, a l l urine voids were c o l l e c t e d on the evening of the day of pesticide spraying ( i . e . , within 8 hours of exposure). Using t h i s sampling protocol, the assay r e s u l t s showed that (1) urine samples c o l l e c t e d from the orchardists and the a g r i c u l t u r a l research s t a t i o n workers during the non-spraying periods revealed no s i g n i f i c a n t d i f f e r e n c e i n clastogenic a c t i v i t y compared to the reference control group from Vancouver and Grand Forks, and (2) clastogenic a c t i v i t y of urine samples c o l l e c t e d during the spraying period in 1985 was s i g n i f i c a n t l y elevated for the o r c h a r d i s t group (P<0.001; Tukey's non-parametric multiple comparisons test) but not for the a g r i c u l t u r a l research s t a t i o n personnel. The high urinary c l a s t o g e n i c a c t i v i t y found for the orchardists was a t t r i b u t e d to heavy exposure to p e s t i c i d e s during the mixing, formulation and a p p l i c a t i o n process and the lack of compliance by the sprayers to wear f u l l p r o t e c t i v e gear i n hot weather. Cigarette smoking was another factor a f f e c t i n g urine c l a s t o g e n i c i t y together with p e s t i c i d e exposure. Cigarette smokers from Grand Forks and the Okanagan a g r i c u l t u r a l research s t a t i o n demonstrated s i g n i f i c a n t l y higher urinary clastogenic a c t i v i t y than non-smokers (P<0.001; Mann-Whitney U test) . i i i No dose-response r e l a t i o n s h i p between the number of c i g a r e t t e s smoked and u r i n a r y c l a s t o g e n i c a c t i v i t y was evident f o r the group of smokers assayed. A l l of the above e f f e c t s were obtained without metabolic a c t i v a t i o n i n v i t r o , suggesting that the c l a s t o g e n i c agents i n the urine were d i r e c t -a c t i n g . In a large proportion of the u r i n e samples t e s t e d , low but s i g n i f i c a n t ( r e l a t i v e to solvent c o n t r o l s ) l e v e l s of c l a s t o g e n i c a c t i v i t y were observed i n the urine of unexposed non-smokers, i n d i c a t i n g the r o l e of other f a c t o r s i n the appearance of ur i n e c l a s t o g e n i c i t y . U r i n a r y pH and c r e a t i n i n e d i d not d i f f e r among the study groups. No data were obtained from the a n a l y s i s of mi c r o n u c l e i i n e x f o l i a t e d u r o t h e l i a l c e l l s . The s c a r c i t y of c e l l s among the subjects made i t d i f f i c u l t to determine the frequency of micronucleated u r o t h e l i a l c e l l s . On the ba s i s of the present research, the monitoring of u r i n e samples f o r g e n o t o x i c i t y appears to be a u s e f u l t o o l f o r assessing human exposure t o environmental carcinogens and mutagens. Urine a n a l y s i s i s not only v a l u a b l e i n q u a l i t a t i v e l y demonstrating exposure to g e n e t i c a l l y hazardous agents, but i s a l s o a promising procedure f o r assessing the e f f i c a c y of preventive measures which are implemented to reduce f u r t h e r exposure. i v TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Tables x L i s t of Figures xi Acknowledgements xiv INTRODUCTION 1 1. Rationale for Monitoring Human Exposure to Environmental Carcinogens and Mutagens 1 2. Objectives of the Research 6 2.1 The Study Group 7 2.2 E x t r a c t i o n of Genotoxic Constituents from Urine 7 3. Genetic Endpoints 8 MATERIALS AND METHODS 9 1. Subjects 9 2. Working Conditions of the Sprayers 10 3. C o l l e c t i o n of Urine Samples 10 4. Urine A n a l y s i s -. 15 4.1 M i c r o n u c l e i A n a l y s i s i n E x f o l i a t e d U r o t h e l i a l C e l l s 15 4.2 Cr e a t i n i n e Determination 17 4.3 A d d i t i o n a l Measurements 18 4.4 Assay f o r Clastogenic A c t i v i t y i n Urine 18 4.4.1 Reversed-phase high pressure l i q u i d chromatography 18 4.4.2 Concentration of urine samples 19 4.4.3 C e l l l i n e 21 4.4.4 C e l l c u l t u r e s 22 4.4.5 Preparation of c e l l s f o r cytogenetic assay 22 4.4.6 A d d i t i o n of urine concentrates 23 4.4.7 Harvesting of CHO c e l l s and s l i d e preparations ... 23 4.4.8 Ana l y s i s of metaphase p l a t e s for chromatid aberrations 24 4.4.9 S t a t i s t i c a l a n a l y s i s 24 v Page RESULTS 27 1. P r e l i m i n a r y Studies 27 1.1 Controls for CHO C e l l Chromosome Aberration Assay 27 1.2 S t a b i l i t y of Urine Extracts 27 1.3 S e l e c t i o n of an E l u t i o n Scheme 30 2. E f f e c t of Varying Degrees of P e s t i c i d e Exposure on Urine C l a s t o g e n i c i t y 35 2.1 Reference Control Group (No Exposure to Pest i c i d e s ) 35 2.2 A g r i c u l t u r a l Research S t a t i o n Personnel (Low Exposure to P e s t i c i d e s ) 41 2.3 P e s t i c i d e A p p l i c a t o r s (High Exposure to Pe s t i c i d e s ) 44 2.3.1 Phase 1: Urine c o l l e c t e d 16-24 hours a f t e r p e s t i c i d e a p p l i c a t i o n 49 2.3.2 Phase 2: Urine c o l l e c t e d w i t h i n 8 hours of p e s t i c i d e usage 57 2.4 Summary of the E f f e c t s of P e s t i c i d e Exposure on Urine C l a s t o g e n i c i t y 67 3. E f f e c t of Smoking on Urine C l a s t o g e n i c i t y 71 4. An a l y s i s of Mi c r o n u c l e i i n E x f o l i a t e d Bladder C e l l s 82 5. A d d i t i o n a l Urine Parameter Measurements 82 DISCUSSION 85 1. Urine C l a s t o g e n i c i t y Associated with P e s t i c i d e Exposure 85 2. Confounding Factors A f f e c t i n g the Urine C l a s t o g e n i c i t y Assay .. 98 2.1 Smoking and Genotoxic A c t i v i t y i n the Urine 98 2.2 Urinary Clastogenic A c t i v i t y Unrelated to C i g a r e t t e Smoking 104 2.3 Urine T o x i c i t y 106 3. Monitoring Urine Genotoxicity as a Means of Detecting Exposure to Environmental Carcinogens and Mutagens: L i m i t a t i o n s and A p p l i c a t i o n s 108 4. Outlook 112 SUMMARY 114 REFERENCES 118 v i Page APPENDICES 128 1 BLANK QUESTIONNAIRES 128 IA Questionnaire D i s t r i b u t e d to Orchardists and A g r i c u l t u r a l Research S t a t i o n Workers on the Day of Urine C o l l e c t i o n 129 IB Questionnaire Used to Obtain L i f e s t y l e and Dietary Information from Grand Forks and Vancouver Residents on the Day of Urine C o l l e c t i o n 130 2 GRAND FORKS RESIDENTS 132 2A Compilation of L i f e s t y l e and Dietary Information for Grand Forks Residents on the Day of Urine C o l l e c t i o n i n September 1985 133 2B Percentage of Aberrant Metaphases Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples Co l l e c t e d from Grand Forks Residents i n September 1985 ... 134 2C Extent of Chromatid Damage per Metaphase Pl a t e Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Grand Forks Residents i n September 1985 135 3 VANCOUVER RESIDENTS 136 3A Compilation of L i f e s t y l e and Dietary Information f o r Vancouver Residents on the Day of Urine C o l l e c t i o n i n J u l y 1985 - 137 3B Percentage of Aberrant Metaphases Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Vancouver Residents i n J u l y 1985 138 3C Extent of Chromatid Damage per Metaphase P l a t e Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Vancouver Residents i n J u l y 1985 139 4 OKANAGAN VALLEY ORCHARDISTS 140 4A Compilation of L i f e s t y l e and Dietary Information f o r Orchardists from the Okanagan V a l l e y During the Days of Urine Sampling , 141 4B Percentage of Aberrant Metaphases Induced by Urine Fractions Prepared from Urine Samples C o l l e c t e d from Orchardists i n May 1984 142 v i i Page 4C Percentage of Aberrant Metaphases Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n June 1984 144 4D Extent of Chromatid Damage per Metaphase Plate Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n June 1984 145 4E Percentage of Aberrant Metaphases Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n J u l y 1984 146 4F Extent of Chromatid Damage per Metaphase Plate Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n J u l y 1984 147 4G Percentage of Aberrant Metaphases Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n October 1984 148 4H Extent of Chromatid Damage per Metaphase Plate Induced by Urine E x t r a c t s (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n October 1984 .. 149 41 Percentage of Aberrant Metaphases Induced by Urine Ex t r a c t s (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n March 1985 150 4J Extent of Chromatid Damage per Metaphase P l a t e Induced by Urine E x t r a c t s (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n March 1985 .... 151 4K Percentage of Aberrant Metaphases Induced by Urine E x t r a c t s (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n August 1985 152 4L Extent of Chromatid Damage per Metaphase P l a t e Induced by Urine E x t r a c t s (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from Orchardists i n August 1985 ... 153 4M P r o t e c t i v e C l o t h i n g and Spraying Equipment Used by Orchardists During the Spraying Periods i n 1984 and 1985 .. 154 5 AGRICULTURAL RESEARCH STATION PERSONNEL FROM OKANAGAN VALLEY .. 155 5A Compilation of L i f e s t y l e and Dietary Information f or A g r i c u l t u r a l Research S t a t i o n Personnel During the Days of Urine C o l l e c t i o n i n 1984 and 1985 156 v i i i Page 5B Percentage of Aberrant Metaphases Induced by Urine Fractions Prepared from Urine Samples C o l l e c t e d from Non-Smoking A g r i c u l t u r a l Research S t a t i o n Personnel i n May 1984 1 5 7 5C Percentage of Aberrant Metaphases Induced by Urine Ext r a c t s (50% Acetone Eluate) Prepared from Urine Samples Co l l e c t e d from A g r i c u l t u r a l Research S t a t i o n Personnel i n March 1985 • • 1 5 8 5D Extent of Chromatid Damage per Metaphase Pl a t e Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from A g r i c u l t u r a l Research S t a t i o n Personnel i n March 1985 1 5 9 5E Percentage of Aberrant Metaphases Induced by Urine Ext r a c t s (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from A g r i c u l t u r a l Research S t a t i o n Personnel i n August 1985 1 6 0 5F Extent of Chromatid Damage per Metaphase Pl a t e Induced by Urine Extracts (50% Acetone Eluate) Prepared from Urine Samples C o l l e c t e d from A g r i c u l t u r a l Research S t a t i o n Personnel i n August 1985 ^ 1 i x LIST OF TABLES Page Table 1 E f f e c t of Human Exposure to Environmental Chemicals on Genotoxicity i n Urine 4 2 Details of Urine Sampling During 1984 and 1985 11 3 Types of Chromatid Aberrations 25 4 Chromosome Aberration A c t i v i t y Induced by Negative Controls 28 5 S t a b i l i t y of a Few Urine Extracts Over a One-Month Period 29 6 Clastogenic A c t i v i t y of Urine Fractions from Orchardists and A g r i c u l t u r a l Research Station Workers During the 1984 Spraying Season 36 7 Pesticides Used by Sprayers on the Day of Urine C o l l e c t i o n i n May 1984 37 8 Pesticides Used by Sprayers on the Day of Urine C o l l e c t i o n i n June 1984 55 9 Pesticides Used by Sprayers on the Day of Urine C o l l e c t i o n i n July 1984 56 10 Pesticides Used by Sprayers on the Day of Urine C o l l e c t i o n i n August 1985 66 11 E f f e c t of Pesticide Exposure and Timing of Urine C o l l e c t i o n on Urinary Clastogenic A c t i v i t y i n Sprayers (Percent C e l l s with Chromatid Aberrations) .. 68 12 Intergroup Comparisons of Clastogenic A c t i v i t y i n Urine Extracts During the Pre-Spraying Period i n March 1985 69 13 Intergroup Comparisons of Clastogenic A c t i v i t y i n Urine Extracts During the Spraying Period i n August 1985 70 14 Relationship Between the Number of Cigarettes Smoked and Clastogenic A c t i v i t y i n Urine 81 15 Mean Urine Parameter Values Among the Study Groups ... 83 x LIST OF FIGURES Figure Page 1 Diagramatic I l l u s t r a t i o n of the Urine C o l l e c t i o n Periods f o r the Orchardists During the Spraying and Non-Spraying Seasons i n 1984 and 1985 14 2 Study P r o t o c o l 16 3 Summary of Urine Processing .. 20 4 Clastogenic A c t i v i t y of I n d i v i d u a l Urine Fractions Prepared from Urine C o l l e c t e d from Orchardists i n May 1984 32 5 Clastogenic A c t i v i t y of I n d i v i d u a l Urine Fractions Prepared from Urine C o l l e c t e d from A g r i c u l t u r a l Research S t a t i o n Personnel i n May 1984 34 6 Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Cr e a t i n i n e Equivalence f o r the Reference Control Group (Non-Smoking Residents of Grand Forks and Vancouver) 40 7A I n d i v i d u a l V a r i a t i o n s i n the Percentage of Aberrant Metaphases Induced by Urine Extracts (Fraction A; 50% Acetone Eluate) from the Reference Control Group .. 43 7B I n d i v i d u a l V a r i a t i o n s i n the Average Frequency of Chromatid Exchanges per Metaphase Induced by Urine E x t r a c t s ( F r a c t i o n A; 50% Acetone Eluate) from the Reference Control Group 43 8A Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Cre a t i n i n e Equivalence for Non-Smoking A g r i c u l t u r a l Research S t a t i o n Workers During the Pre-Spraying Period i n March 1985 46 8B Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Creatinine Equivalence f o r Non-Smoking A g r i c u l t u r a l Research S t a t i o n Workers During the Spraying Period i n August 1985 46 9A I n d i v i d u a l V a r i a t i o n s i n the Percentage of Aberrant Metaphases Induced by Urine E x t r a c t s (Fraction A; 50% Acetone Eluate) from Non-Smoking A g r i c u l t u r a l Research S t a t i o n Workers During the Pre-Spraying (March 1985) and Spraying (August 1985) Periods 48 x i Figure Page 9B I n d i v i d u a l V a r i a t i o n s i n the Average Frequency of Chromatid Exchanges per Metaphase Induced by Urine E x t r a c t s (Fraction A; 50% Acetone Eluate) from Non-Smoking A g r i c u l t u r a l Research S t a t i o n Workers During the Pre-Spraying (March 1985) and Spraying (August 1985) Periods 48 10A Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y a t 4.0 mg/ml Creatinine Equivalence for Orchardists During the Spraying Season i n June and J u l y 1984 51 10B Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Creatinine Equivalence for Orchardists During the Post-Spraying Period i n October 1984 51 11A I n d i v i d u a l V a r i a t i o n s i n the Percentage of Aberrant Metaphases Induced by Urine Extracts (Fraction A; 50% Acetone Eluate) from Orchardists During the Spraying (June/July) and Post-Spraying (October) Periods i n 1984 .. 54 11B I n d i v i d u a l V a r i a t i o n s i n the Average Frequency of Chromatid Exchanges per Metaphase Induced by Urine E x t r a c t s (Fraction A; 50% Acetone Eluate) from Orchardists During the Spraying (June/July) and Post-Spraying (October) Periods i n 1984 54 12 Dose-Response Curves of Urine Extracts Prepared from Urine C o l l e c t e d During the Pre-Spraying (March 1985) and Spraying (August 1985) Periods from 4 Representative P e s t i c i d e Sprayers 59 13A Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Creatinine Equivalence for P e s t i c i d e Sprayers During the Pre-Spraying Period i n March 1985 .... 62 13B Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Creatinine Equivalence for P e s t i c i d e Sprayers During the Spraying Period i n August 1985 62 14A I n d i v i d u a l V a r i a t i o n s i n the Percentage of Aberrant Metaphases Induced by Urine Extracts (Fraction A; 50% Acetone Eluate) from Orchardists During the Pre-Spraying (March) and Spraying (August) Periods i n 1985 ... 65 14B I n d i v i d u a l V a r i a t i o n s i n the Average Frequency of Chromatid Exchanges per Metaphase Induced by Urine E x t r a c t s (Fraction A; 50% Acetone Eluate) from Orchardists During the Pre-Spraying (March) and Spraying (August) Periods i n 1985 65 x i i Figure Page 15 Dose-Response Curves of Urine E x t r a c t s Prepared from Urine C o l l e c t e d from 4 Non-Smoking A g r i c u l t u r a l Research S t a t i o n Workers i n March and August 1985 73 16 Dose-Response Curves of Urine Ex t r a c t s Prepared from Urine C o l l e c t e d from 4 Smoking A g r i c u l t u r a l Research S t a t i o n Workers i n March and August 1985 75 17A Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Creatinine Equivalence for Non-Smokers (Orchardists, A g r i c u l t u r a l Research S t a t i o n Personnel, Grand Forks and Vancouver Residents) 78 17B Frequency D i s t r i b u t i o n of Urinary Clastogenic A c t i v i t y at 4.0 mg/ml Creatinine Equivalence for Smokers ( A g r i c u l t u r a l Research S t a t i o n Personnel and Grand Forks Residents) 78 18A I n d i v i d u a l V a r i a t i o n s i n the Percentage of Aberrant Metaphases Induced by Urine E x t r a c t s (Fraction A; 50% Acetone Eluate) from Non-Smoking and Smoking I n d i v i d u a l s 80 18B I n d i v i d u a l V a r i a t i o n s i n the Average Frequency of Chromatid Exchanges per Metaphase Induced by Urine Ext r a c t s (Fraction A; 50% Acetone Eluate) from Non-Smoking and Smoking I n d i v i d u a l s 80 x i i i ACKNOWLEDGEMENTS I would l i k e to express my gratitude to my supervisor, Dr H.F. Stich, for his guidance and support throughout the course of this research. I am also indebted to Dr R.H.C. San for his expert advice and guidance. I would l i k e to thank Drs B.P. Dunn and M.P. Rosin for their useful comments and suggestions. I would l i k e to express my thanks to Belinda Wong for her help in preparing the figures and to Dolores Fatur for her computer assistance. I am also grateful to the s t a f f and students of the Environmental Carcinogenesis Unit at the B.C. Cancer Research Centre for the i r help and support. I would also l i k e to express my appreciation to the study subjects, whose p a r t i c i p a t i o n made t h i s project possible. F i n a l l y , my sincere gratitude to Mrs Rosemary Pritchard for typing t h i s thesis. F i n a n c i a l support was provided by Health and Welfare Canada through a grant awarded to Dr H.F. Stich . xiv INTRODUCTION 1 . Rationale for Monitoring Human Exposure to Environmental Carcinogens and Mutagens Epidemiological evidence suggests that 60 to 80% of a l l human cancers are the res u l t of l i f e s t y l e and environmental factors and are therefore, in p r i n c i p l e , preventable (Higginson, 1979). These figures are derived from two li n e s of evidence: (1) the i d e n t i f i c a t i o n of cer t a i n environmental factors i n the etiology of human cancers, and (2) the variations that exist in cancer incidence among di f f e r e n t population groups. Apart from solar u l t r a v i o l e t l i g h t as a causative factor i n skin cancer, emphasis has been placed on environmental chemicals playing a role i n human cancers ( M i l l e r , 1 9 7 8 ) . The basis for this comes from the i d e n t i f i c a t i o n of s p e c i f i c chemicals carcinogenic for humans (e.g., v i n y l chloride, 8-naphthylamine, asbestos) and the strong association between human lung cancer and cigarette smoking (M i l l e r , 1 9 7 8 ) . Currently, man i s exposed to a rapidly p r o l i f e r a t i n g l i s t of synthetic chemicals i n the environment. According to an estimate i n 1 9 7 7 , approximately 5 0 , 0 0 0 synthetic chemicals were in use, with about 1 , 0 0 0 more being introduced each year (Maugh, 1 9 7 8 ) . With the aid of short-term _in v i t r o test systems (e.g., Ames Salmonella mutagenicity test) to complement animal cancer bio-assays, many of these chemicals have been shown to induce harmful e f f e c t s . The potential health hazards imposed by these chemicals have indeed raised some concerns. To minimize exposure to harmful chemicals, the impact of genotoxic chemicals ( i . e . , toxicants capable of damaging the genome) on man must be determined. Epidemiological studies on exposed human populations represent 1 one method of r i s k evaluation. However, the long latency period (10-30 years) between exposure to the genotoxic agents and the manifestation of the disease has made t h i s approach d i f f i c u l t . The latency problem was h i g h l y evident i n the detection of adverse health e f f e c t s i n c i g a r e t t e smokers. During the 1900's, c i g a r e t t e smoking became popular, but i t was not u n t i l the mid-1920"s that lung cancer m o r t a l i t y rates increased, and another 20 years before any a s s o c i a t i o n was made between the two events (Brusick et a l . , 1981) . This l a g period was also found f o r many types of cancer caused by the atomic bomb and for cancers observed i n factory workers exposed to i n d u s t r i a l chemicals (Ames, 1979). Consequently, there i s the necessity to e s t a b l i s h e a r l y monitoring techniques to i d e n t i f y chemical hazards long before c l i n i c a l symptoms become apparent. Short-term i n v i t r o assays of pure chemicals have aided i n the discovery of the genotoxic a c t i o n of many chemicals. The induction of reverse mutations i n Salmonella typhimurium, f o r example, i s a widely used t e s t . However, such t e s t s do not permit the assessment of human exposure to environmental agents. The t e s t s also do not simulate the number of complex ways i n which chemical agents may i n t e r a c t i n b i o l o g i c a l systems. At present, two methods are widely a p p l i c a b l e f o r monitoring the e f f e c t s of genotoxic chemicals on exposed populations. One method involv e s the a n a l y s i s of c e l l s and t i s s u e s of the exposed i n d i v i d u a l f o r evidence of cytogenetic damage such as micronuclei or chromosome aberrations. The second approach involves the a n a l y s i s of _in v i t r o t a r get i n d i c a t o r s such as m i c r o b i a l or mammalian c e l l s f o r genotoxic damage a f t e r exposure to body f l u i d s from exposed i n d i v i d u a l s . This t h e s i s w i l l focus on the body f l u i d most widely analyzed, namely urine. 2 The rationale behind the analysis of urine i s based on the knowledge that ingestion or metabolic transformation of endogenous and exogenous substances may result i n the excretion of metabolites into the urine. The detection of the metabolites in urine would indicate that absorption of the precursor compound has occurred and that c e l l s in various organs are at ri s k of exposure during systemic d i s t r i b u t i o n of the parent compound. The advantage of assaying urine for genotoxic chemicals i s that this procedure takes into account the metabolic and physiological conversion of chemicals in an in t a c t organism, a situation which i s d i f f i c u l t to reproduce under i n v i t r o conditions. In addition, hosts of factors (e.g., enzyme induction, d i e t , stress) which are present in organisms but not in in v i t r o systems, may a f f e c t the metabolic course of the chemicals (Legator, 1982). By following the occurrence of genotoxic a c t i v i t y i n the urine of exposed ind i v i d u a l s , one can assess the degree of uptake of the genetic toxicant and thus proceed to minimize i t s exposure. Another advantage of analyzing human urine i s the a c c e s s i b i l i t y of the samples. Urine c o l l e c t i o n procedures are non-invasive and can be conducted on a repeated basis. In addition, the analysis of urine for genotoxicity may be coupled with quantitative chemical analysis. O v e r a l l , the analysis of urine samples i s simple and rapid. Unfortunately, the concentration of genotoxic metabolites i n the urine may be r e l a t i v e l y low, requiring extraction and concentration procedures for adequate detection. This problem was solved by Yamasaki and Ames (1977), who used non-polar Amberlite XAD-2 res i n to concentrate compounds from the urine of cigarette smokers. Since then, t h i s approach has been used to examine many types of exposure. Table 1 l i s t s some representative studies by other investigators. As i s evident from Table 1, the use of urine analysis has 3 TABLE 1 EFFECT OF HUMAN EXPOSURE TO ENVIRONMENTAL CHEMICALS ON GENOTOXICITY IN URINE Genotoxicity Exposure Genotoxicity Assay i n Urine^ Reference L i f e s t y l e : Smoking Passive smoking Salmonella mutagenicity Salmonella mutagenicity Salmonella mutagenicity Chinese hamster ovary c e l l s , chromosome aberrations^ Salmonella mutagenicity Salmonella mutagenicity + + + + + Yamasaki and Ames (1977) Aeschbacher and Chappuis (1981) Caderni and Dolara (1983) Dunn and C u r t i s (1985) Bos et a l . (1983) Sorsa et a l . (1985) Diet: Coffee d r i n k i n g F r i e d pork meat Vegetarian d i e t Occupat iona1: Anesthetic gases A n t i n e o p l a s t i c drugs Chinese hamster ovary c e l l s , chromosome aberrations Salmonella mutagenicity Salmonella mutagenicity Salmonella mutagenicity Salmonella Salmonella Salmonella mutagenicity mutagenicity mutagenicity + + Dunn and C u r t i s (1985) Baker et a l . (1982) Sasson et a l . (1985) McCoy et a l . (1979) Falck et a l . (1979) Anderson et a l . (1982) Everson et a l . (1985) Carbon electrode production Salmonella mutagenicity Pasquini et a l . (1982) TABLE 1 (cont'd) Genotoxicity Exposure Genotoxicity Assay i n Urine Reference Occupational (cont'd): Chemical manufacture Salmonella mutagenicity + Dolara et a l . (1981) Salmonella mutagenicity + K r i e b e l et a l . (1983) Epichlorohydrin Salmonella mutagenicity + Legator et a l . (1978) Rubber manufacture Salmonella mutagenicity + Falck et a l . (1980) Medical: Crude c o a l t a r Salmonella mutagenicity • + Wheeler et a l . (1981) Cyclophosphamide Salmonella mutagenicity + Siebert and Simon (1973) Metronidazole Salmonella mutagenicity + Legator et a l . (1975) N i r i d a z o l e Salmonella mutagenicity + Legator et a l . (1975) N i t r o f u r a n t o i n Salmonella mutagenicity + Wang et a l . (1977) Assayed f o r reverse mutations i n s t r a i n s of Salmonella typhimurium. Assayed f o r chromosome aberrations i n Chinese hamster ovary c e l l s . +, p o s i t i v e ; -, negative. enabled the i d e n t i f i c a t i o n of many individuals at risk for exposure to genotoxic agents. Some studies have even led to the instalment of i n t e r -vention methods. For example, pharmaceutical personnel handling antineo-p l a s t i c drugs in horizontal laminar-flow hoods were found to have mutagenic urine (Anderson et a l . , 1982). With the conversion to v e r t i c a l laminar-flow hoods and better protective measures, a two-year follow-up study showed a decrease in the mutagenicity of the urine. These findings i l l u s t r a t e the usefulness of such a monitoring procedure. 2. Objectives of the Research The aims of t h i s research were to accomplish the following: (a) To conduct a f e a s i b i l i t y study on the use of urine analysis as a means of assessing human exposure to suspected genotoxic hazards. (b) To determine whether exposure to pesticides, which are commonly used environmental chemicals, w i l l r e s u l t in elevated levels of genotoxic a c t i v i t y i n the urine. (c) To examine the effects of confounding factors such as cigarette smoke on urine genotoxicity. (d) To apply analysis of micronuclei in exfoliated u r o t h e l i a l c e l l s as a means of assessing i n vivo genotoxic damage to the di v i d i n g basal c e l l s of the epithelium. The selection of the study group, the urine extraction procedures, and the genetic endpoints of t h i s study w i l l now be b r i e f l y described. 6 2.L The Study Group j The study groups i n t h i s p r o j e c t were comprised of i n d i v i d u a l s with varying degrees of p e s t i c i d e exposure. P e s t i c i d e s are one of the most widely used chemicals i n a g r i c u l t u r e and represent a large proportion of the chemicals to which man i s exposed (Yoder et a l . , 1973). In 1977, an estimated 1.4 b i l l i o n pounds of synthetic organic p e s t i c i d e s were produced i n the world (Plimmer, 1982). Because of the phenomenal use of p e s t i c i d e s , the i m p l i c a t i o n s of these chemicals to human health have received much a t t e n t i o n . The acute t o x i c e f f e c t s of p e s t i c i d e i n g e s t i o n or a c c i d e n t a l overexposure are well-known, with 100,000 non-fatal cases of human poisoning reported each year (Plimmer, 1982). Organophosphate i n s e c t i c i d e s , w e l l -known f o r t h e i r a b i l i t y to i n h i b i t a c e t y l c h o l i n e s t e r a s e i n plasma and c e l l s , represent the major cause of occupational poisoning. Although the acute t o x i c i t y of some p e s t i c i d e s i s w e l l - e s t a b l i s h e d , the genetic consequences of p e s t i c i d e s to man are l i t t l e known. Many reports have focused on the genotoxic p r o p e r t i e s of pure p e s t i c i d e s i n laboratory t e s t systems. Very few studies have been performed on i n d i v i d u a l s o c c u p a t i o n a l l y exposed to these chemicals. The i n t e n t of t h i s research i s to provide more information about the p o t e n t i a l long-term adverse e f f e c t s of p e s t i c i d e s on man. 2.2 E x t r a c t i o n of Genotoxic Constituents from Urine The m a j o r i t y of the studies l i s t e d i n Table 1 used non-polar XAD-2 r e s i n to e x t r a c t genotoxic materials from the urine of exposed i n d i v i d u a l s . Recently, preparative reversed-phase high pressure l i q u i d chromatography (RPLC) was shown to be superior i n terms of performance and recovery to XAD-2 re s i n s i n the e x t r a c t i o n of non-polar u r i n a r y genotoxic c o n s t i t u e n t s ( C u r t i s and 7 Dunn, 1985). The research described in t h i s thesis used the methodology of Curtis and Dunn (1985) i n an e f f o r t to increase the s e n s i t i v i t y of the urine genotoxicity assay. 3. Genetic Endpoints Two genetic endpoints were used in t h i s study: (a) The frequency of chromosome aberrations induced by urine extracts on Chinese hamster ovary (CHO) c e l l s . Chromosome damage in CHO c e l l s has been extensively used in studies of environmental carcinogens and mutagens. For example, Ishidate and Odashima (1977) used the chromosome aberration assay to i d e n t i f y 25 out of 34 (73.2%) known carcinogens. In t h i s project, the assay w i l l be used as a rapid screen for genotoxicity in the urine of exposed individuals. (b) The determination of the micronucleus frequency i n exfoliated urinary bladder c e l l s . Micronuclei are acentric chromosome fragments derived from aberrant chromosomes (Stich et a l . , 1983). Their presence in the cyto-plasm of e x f o l i a t e d c e l l s as DNA-containing bodies indicates the capacity of genotoxins to i n f l i c t genetic damage on the dividing basal c e l l s of the epithelium (Stich and Rosin, 1984). The micronucleus test has been successfully applied to e x f o l i a t e d urinary bladder c e l l s of smokers and coffee drinkers (Stich and Rosin, 1984) as well as of b i l h a r z i a l patients (Raafat et a l . , 1984). An increase in the micronuclei frequency of the bladder c e l l s together with a demonstration of genotoxic a c t i v i t y i n the urine would provide strong evidence of exposure to environmental contaminants. 8 MATERIALS AND METHODS 1. Subjects The study was c a r r i e d out i n 1984 and L985. Seventy-two healthy male and female subjects on normal d i e t s p a r t i c i p a t e d i n t h i s p r o j e c t . ' Questionnaires were given to a l l p a r t i c i p a n t s to c o l l e c t information on the working environ-ment, p e s t i c i d e usage, p r o t e c t i v e c l o t h i n g , medication, and l i f e s t y l e and d i e t a r y h a b i t s . Blank copies of the questionnaires are given i n Appendix I A and IB. Urine samples were c o l l e c t e d from the f o l l o w i n g groups of i n d i v i d u a l s : (a) 21 non-smoking male o r c h a r d i s t s from Naramata and Summerland, B.C. (average age, 38.2 years). These subjects sprayed p e s t i c i d e s h e a v i l y during the f r u i t growing season from May to August. L i f e s t y l e and d i e t a r y information for t h i s group i s given i n Appendix 4A. (b) 18 male personnel (5 smokers, 13 non-smokers) at an a g r i c u l t u r a l research s t a t i o n i n Summerland (average age, 48.1 years). Only 2 i n d i v i d u a l s sprayed p e s t i c i d e s i n t h i s group. The others were a n a l y t i c a l chemists, plant p a t h o l o g i s t s , and workers on experimental farms who used only small amounts of p e s t i c i d e s . However, these workers may be exposed to low l e v e l s of p e s t i c i d e s that may have d r i f t e d from the a p p l i c a t i o n s i t e s near the s t a t i o n . L i f e s t y l e and d i e t a r y information for t h i s group i s shown i n Appendix 5A. (c) Reference c o n t r o l group: 22 male subjects (12 smokers, 10 non-smokers; average age, 54.4 years) r e s i d i n g i n Grand Forks, a n o n - a g r i c u l t u r a l area. In a d d i t i o n , 11 non-smoking males and females from Vancouver were included i n t h i s group. A l l of the subjects i n t h i s group are bel i e v e d to have no exposure to p e s t i c i d e s . L i f e s t y l e and d i e t a r y information f o r t h i s group i s given i n Appendix 2A and 3A. 9 2. Working Conditions of the Sprayers In general, the p e s t i c i d e sprayers wore p r o t e c t i v e c l o t h i n g c o n s i s t i n g of c o v e r a l l s , gloves and boots i n a d d i t i o n to h a l f - f a c e r e s p i r a t o r s . Complete d e t a i l s of the p r o t e c t i v e gear worn by each sprayer are given i n Appendix 4M. I t should be noted, however, that p r o t e c t i v e equipment was not worn c o n s i s t e n t l y throughout the p e s t i c i d e a p p l i c a t i o n process. Hot weather u s u a l l y made r e s p i r a -t o r s and p r o t e c t i v e c l o t h i n g uncomfortable to wear, and they were thus frequently discarded i n the middle of spraying. Each sprayer was responsible for pouring and mixing the concentrated chemicals ( i . e . , f o r mulation), t r a n s p o r t i n g the chemicals to the a p p l i c a t i o n s i t e , applying the p e s t i c i d e s , and cleaning the a p p l i c a t i o n equipment. Both l i q u i d and s o l i d formulations were used. The spraying period v a r i e d from 3 days to one week, with 1 to 10 working hours per day. Most of the sprayers used a wide v a r i e t y of p e s t i c i d e s during the course of a working day, i n c l u d i n g organophosphates, carbamates, and organochlorine compounds. A i r b l a s t sprayers, which can d e l i v e r high and low volumes of spray, were predominantly used. 3. C o l l e c t i o n of Urine Samples I t was decided that a l o n g i t u d i n a l study would be most i d e a l for t h i s p r o j e c t since v a r i a b l e s such as metabolic and l i f e s t y l e d i f f e r e n c e s would be e s s e n t i a l l y eliminated. The research was d i v i d e d i n t o two p a r t s . Part 1 was designed to evaluate the c o n t r i b u t i o n of p e s t i c i d e usage towards urine c l a s t o g e n i c i t y . Part 2 examined confounding r i s k f a c t o r s such as c i g a r e t t e smoking which may complicate the i n t e r p r e t a t i o n of the observations from Part 1. Table 2 shows d e t a i l s of the sampling periods for the research p r o j e c t . 10 TABLE 2 DETAILS OF URINE SAMPLING DURING 1984 AND 1985 Group C o l l e c t i o n Number Date of C o l l e c t i o n Sampling Period Orchardists Orchardists Orchardists Orchardists Orchardists Orchardists Vancouver res i d e n t s Grand Forks res i d e n t s C l C2 C3 C4 C5 C6 Research s t a t i o n C7 personnel Research s t a t i o n C8 personnel Research s t a t i o n C9 personnel Phase 1 Phase 2 CIO C l l 25 May 1984 20 June 1984 17 J u l y 1984 15 October 1984 25 March 1985 10 August 1985 25 May 1984 25 March 1985 A l l urine voids 16-24 hours a f t e r p e s t i c i d e exposure A l l urine voids 16-24 hours a f t e r p e s t i c i d e exposure A l l urine voids 16-24 hours a f t e r p e s t i c i d e exposure F i r s t morning v o i d upon waking E a r l y evening voids A l l urine voids 0-8 hours a f t e r p e s t i c i d e exposure F i r s t morning void upon waking E a r l y evening voids 10 August 1985 E a r l y evening voids 10-20 J u l y 1985 Earl y evening voids 11 September 1985 E a r l y evening voids 11 Part 1 Part 1 was fur t h e r subdivided i n t o two phases: a) Phase 1. In 1984, at l e a s t one s i n g l e urine sample was c o l l e c t e d from each of the o r c h a r d i s t s during the peak spraying season (May to August). The urine specimens tested during t h i s period were e a r l y morning samples c o n s i s t i n g of a l l voids c o l l e c t e d between 16 and 24 hours a f t e r p e s t i c i d e a p p l i c a t i o n . An off-season morning v o i d sample was c o l l e c t e d from each of the o r c h a r d i s t s during October 1984 (no p e s t i c i d e usage) as a c o n t r o l . b) Phase 2. Based on the observations made during phase 1, i t was suspected that the c o l l e c t i o n of urine at 16 to 24 hours post-spraying may have passed the time at which p e s t i c i d e s are metabolized and excreted. Consequently, i n August 1985, a l l urine specimens were c o l l e c t e d from the o r c h a r d i s t s w i t h i n 8 hours of p e s t i c i d e a p p l i c a t i o n . A l a t e afternoon sample was al s o c o l l e c t e d from the same i n d i v i d u a l s i n March 1985 to serve as a c o n t r o l (pre-spraying sample). Figure 1 summarizes the urine c o l l e c t i o n schedule f o r phases 1 and 2. Part 2 Urine voids were al s o c o l l e c t e d from the 18 a g r i c u l t u r a l research s t a t i o n personnel on the dates i n d i c a t e d i n Table 2. Evening voids were g e n e r a l l y c o l l e c t e d l a t e i n the afternoon between 4 and 8 p.m. In September 1985, a s i n g l e urine specimen was provided by i n d i v i d u a l s from Grand Forks and Vancouver. . The urine voids were a l s o c o l l e c t e d l a t e i n the afternoon (4 to 8 p.m.). The c l a s t o g e n i c a c t i v i t y of urine from the smokers i n t h i s second part of the study was compared to that of non-smokers. 12 FIGURE 1 Diagramatic i l l u s t r a t i o n - of the urine c o l l e c t i o n periods f o r the o r c h a r d i s t s during the spraying and non-spraying seasons. Indicated are the urine c o l l e c t i o n numbers during phase 1 (1984) and phase 2 (1985). 13 P h a s e 1 P h a s e 2 ALL urine specimens were coLlected i n s t e r i L e 500-ml polyethylene b o t t l e s (Nalgene, Rochester, NY) without any preservatives. B o t t l e s were r e f r i g e r a t e d by the p a r t i c i p a n t s during the c o l l e c t i o n period u n t i l f u l l . The b o t t l e s were than brought to the a g r i c u l t u r a l research s t a t i o n where each was l a b e l l e d with a code number. Before f r e e z i n g , each sample was centrifuged to obtain e x f o l i a t e d urinary bladder c e l l s (see secti o n 4.1 on micronuclei a n a l y s i s of e x f o l i a t e d u r o t h e l i a l c e l l s ) . The supernatant was packed i n i c e and shipped to our laboratory where they were stored at -20°C u n t i l a n a l y s i s . In t o t a l , 186 urine samples were c o l l e c t e d from 72 i n d i v i d u a l s . 4. Urine Analysis The urine specimens were processed for an a l y s i s as o u t l i n e d i n Figure 2. 4.1 M i c r o n u c l e i a n a l y s i s j n e x f o l i a t e d u r o t h e l i a l c e l l s F r e s h l y c o l l e c t e d urine samples from each subject were c e n t r i f u g e d at 600 g f o r 10 min. The supernatant was removed and stored at -20°C f o r clastogen a n a l y s i s . The c e l l p e l l e t was resuspended and f i x e d i n f r e s h l y prepared Carnoy's s o l u t i o n of 3:1 e t h a n o l / g l a c i a l a c e t i c a c i d (v/v) f o r 20 min at room temperature. The c e l l suspension was then r e c e n t r i f u g e d f o r 10 min and resuspended i n a few drops of the same f i x a t i v e . Drops of c e l l suspension were t r a n s f e r r e d to precleaned s l i d e s and a i r - d r i e d overnight. The c e l l s were st a i n e d according to the procedures described by S t i c h et a l . (1982). B r i e f l y , smears were pretreated i n 1 N HC1 f o r 2 min at room temperature, placed for 6 min i n t o 1 N HC1 at 60°C, t r a n s f e r r e d to 1 N HC1 at room temperature for 2 rain, r i n s e d i n d i s t i l l e d water, put i n t o S c h i f f ' s reagent f o r 90 min, and r i n s e d twice with running water. The preparations were counterstained for 15 U r i n e S a m p l e C o l l e c t i o n F r o m : O r c h a r d i s t s R e t . S i n . P e r s o n n e l G r a n d F o r k s R e s i d e n t s V a n c o u v e r R e s i d e n t s P a r a m e t e r s M e a s u r e d . P H . V o l u m e U r i n e C o n c e n t r a t i o n B y R e v e r s e d - P h a a e H P L C E x f o l i a t e d C r e a t i n i n e B l a d d e r C e l l s M i c r o n u c l e i C h r o m o s o m e E n d - P o i n t s A b e r r s l l o n s Figure 2 S t u d y P r o t o c o l 16 5 sec with f a s t green (Fisher S c i e n t i f i c , Fairlawn, NJ) dissolved i n 95% ethanol. The preparations were dehydrated through immersion i n 95% ethanol, butanol, butanol/xylene, two changes of xylene, and mounted i n Permount. Two to four s l i d e s were made for each i n d i v i d u a l . A minimum of 300 i n t a c t uro-t h e l i a l c e l l s were analyzed to determine the frequency of micronucleated c e l l s . The c r i t e r i a f or scoring are w e l l - e s t a b l i s h e d (Heddle and Salamone, 1981; Heddle et a l . , 1981; S t i c h et a l . , 1982). A p a r t i c l e was scored as a micronucleus when i t contained the f o l l o w i n g p r o p e r t i e s : ( i ) less than one-t h i r d of the nuclear diameter, ( i i ) located w i t h i n the c e l l cytoplasm but not attached to the nucleus, ( i i i ) s i m i l a r s t a i n i n g i n t e n s i t y as the main nucleus, and (iv) located w i t h i n the same f o c a l plane as the nucleus. Micronuclei found i n non-nucleated c e l l s were excluded to avoid confusion with karyorhexis. A l l s l i d e s were coded p r i o r to a n a l y s i s to avoid b i a s . 4.2 C r e a t i n i n e determination C r e a t i n i n e i s a metabolic end-product eliminated i n the urine. T o t a l c r e a t i n i n e e x c r e t i o n i s almost constant from day to day for a given i n d i v i d u a l , and i t s e x c r e t i o n i s unaltered by the extent of urine d i l u t i o n (Kapur and Pandav, 1985). The c r e a t i n i n e concentration of each urine sample was measured using a m o d i f i c a t i o n of the procedure described by Iosefsohn (1982). Colour reagents were prepared by adding 1.5 ml of 10 mM p i c r i c a c i d ( A l d r i c h Chemical Co., Milwaukee, WI) and 0.5 ml of 600 mM sodium hydroxide to l a b e l l e d t e s t tubes. A small a l i q u o t taken from each urine sample was d i l u t e d f o u r f o l d with d i s t i l l e d water. F i f t y u l of the d i l u t e d u r i n e , water or c r e a t i n i n e standard (Sigma Chemical Co., St Louis, MO) were p i p e t t e d i n t o the tubes. The reaction mixtures were incubated at room temperature for 20 min. Absorbances were 17 m e a s u r e d a g a i n s t b l a n k s c o n t a i n i n g the 50 u l of w a t e r a t 520 nm i n a d u a l beam P e r k i n - E l m e r Lambda 3 s p e c t r o p h o t o m e t e r . C r e a t i n i n e l e v e l s o f the unknowns w e r e d e t e r m i n e d by p r e p a r i n g a s t a n d a r d c u r v e w i t h c r e a t i n i n e c o n c e n t r a t i o n s r a n g i n g f r o m 0.10 to 1.0 mg/ml. A l l assays were done i n d u p l i c a t e . The c r e a t i n i n e d e t e r m i n a t i o n s e r v e d to a d j u s t for v a r y i n g u r i n e c o n c e n t r a t i o n s a n d d i f f e r e n c e s i n b o d y w e i g h t b e t w e e n i n d i v i d u a l s . 4.3 A d d i t i o n a l m e a s u r e m e n t s The pH o f e a c h u r i n e s a m p l e was m e a s u r e d w i t h an O r i o n R e s e a r c h pH m e t e r . I n a d d i t i o n , t h e u r i n e v o l u m e s f o r e a c h i n d i v i d u a l w e r e d e t e r m i n e d . 4.4 A s s a y f o r c l a s t o g e n i c a c t i v i t y i n u r i n e 4.4.1 R e v e r s e d - p h a s e h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y R e v e r s e d - p h a s e h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y (RPLC) was' c a r r i e d o u t u s i n g a W a t e r s P r e p L C / S y s t e m 500A p r e p a r a t i v e l i q u i d c h r o m a t o g r a p h ( W a t e r s S c i e n t i f i c , M i l f o r d , M A), w h i c h i n c o r p o r a t e s i t s own h i g h c a p a c i t y s o l v e n t pump, a u n i v e r s a l r e f r a c t i v e i n d e x d e t e c t o r a n d c h a r t r e c o r d e r . A W a t e r s P r e p Pak 500/C18 r e v e r s e d - p h a s e c o l u m n (5 cm x 33 cm; v o i d v o l u m e a p p r o x i m a t e l y 500 m l ) , w h i c h i s s t a b l e b e t w e e n pH 2 t o 8, was u s e d . A l l s o l v e n t s e m p l o y e d w e r e HPLC g r a d e a n d w e r e h e l i u m - d e g a s s e d b e f o r e b e i n g pumped i n t o t h e c o l u m n . E a c h d a y b e f o r e u s e , t h e c o l u m n was f l u s h e d w i t h 1 l i t r e o f a c e t o n e ( F i s h e r S c i e n t i f i c , V a n c o u v e r , B.C.) f o l l o w e d b y 2 l i t r e s o f 2.5 mM p h o s p h o r i c a c i d ( A l l i e d C h e m i c a l s , C a n a d a ) . 18 4.4.2 Concentration of urine samples The concentration procedure i s schematically summarized i n Figure 3. Urine samples were thawed i n a 37°C waterbath and a c i d i f i e d to pH 3.0 with phosphoric a c i d . The a c i d i f i c a t i o n step was necessary to (i) suppress i o n i z a t i o n of weak acids (unfortunately, the pH cannot be made a l k a l i n e enough, commensurate with reversed-phase column s t a b i l i t y , to prevent i o n i z a t i o n of organic bases), and ( i i ) p r e c i p i t a t e proteins which may clog the RPLC column. Once a c i d i f i e d , the urine samples were f i l t e r e d through coarse f l u t e d paper to remove any gelatinous p r e c i p i t a t e s . The f i l t e r e d samples, ranging from 100 to 500 ml i n volume, were introduced i n t o the column by a Waters Prep 500 LC pump u n i t (Waters S c i e n t i f i c , M i l f o r d , MA) at a flow rate of 250 ml/min. The column was then flushed with 1 l i t r e of 2.5 mM phosphoric a c i d to remove unretained m a t e r i a l . This wash was subsequently, discarded since i t contains high concentrations of s a l t s and urea which makes i t unsuitable f o r t e s t i n g . The organic m a t e r i a l on the column was subsequently eluted according to one of the f o l l o w i n g schemes. (a) Sequential e l u t i o n . In t h i s procedure, 900 ml of 5% acetone/95% 2.5 mM phosphoric a c i d , 500 ml of 15% acetone/85% 2.5 mM phosphoric a c i d , and 500 ml of 50% acetone/50% 2.5 mM phosphoric a c i d were pumped i n t o the column s e q u e n t i a l l y to e l u t e the organic m a t e r i a l adsorbed to the column. As a r e s u l t , three f r a c t i o n s of i n c r e a s i n g hydrophobicity (each f r a c t i o n i s approximately 500 ml) were generated. For subsequent d i s c u s s i o n , the 5% acetone eluate w i l l be r e f e r r e d to as f r a c t i o n 1, the 15% acetone eluate as f r a c t i o n 2, and the 50% acetone eluate as f r a c t i o n 3. 19 U r l n « T h a w « d — A c i d i f i e d F i l t e r e d F t e v e r s e d - P h a s e H P (. c. S e q u e n t i a l E l u t i o n 1 ) 5 % A c e t o n e 2 ) 1 5 % A c e t o n e 3 ) 5 0 % A c e t o n e p H A d j u s t m e n t 2 . 5 m M H - j P O ^ W a s h E l u t i o n W i t h F r a c t i o n A 5 0 % A c e t o n e E l u a t e R o t a r y - E v a p o r a t e d ( R e m o v e s A c e t o n e ) E l u a t e F r e e z e - O r i e d d u e S u s p e n d e d I n W a t e r U r i n e E x t r a c t Figure 3 S u m m a r y O f U r i n e P r o c e s s i n g 20 (b) Whole e l u t i o n . A l l the organic material on the column was eluted with 500 ml of 50% acetone/50% 2.5 mM phosphoric a c i d (v/v) to generate only a s i n g l e u rine .composite. On the basis of the r e s u l t s obtained from the three urine f r a c t i o n s using sequential e l u t i o n , the majority of the urine samples were el u t e d according to the method o f "whole e l u t i o n " . This permitted the processing of a large number of u r i n e samples for comparison. For future reference, the s i n g l e urine composite w i l l be designated as f r a c t i o n A. With both the above methods, c o l l e c t i o n of the eluates was monitored with a d i f f e r e n t i a l refTactometer b u i l t i n t o the HPLC system. F r a c t i o n cut points were determined by noting s h i f t s i n the detector baseline i n response to changes i n acetone concentrations. Once c o l l e c t e d , the f r a c t i o n s were r o t a r y evaporated at 40°C to remove the acetone and n e u t r a l i z e d with 1 M sodium hydroxide. The samples were then t r a n s f e r r e d to 900-ml pyrex glass trays covered with aluminum f o i l . Each t r a y was loaded onto a V i r t i s Console 12 f r e e z e - d r i e r where the urine f r a c t i o n s were frozen down to -40°C. Upon f r e e z i n g , the samples were d r i e d under vacuum at a she l f temperature of 10°C for 48 hours. A f t e r d r y i n g , the powdered urine e x t r a c t (sodium phosphate from n e u t r a l i z e d phosphoric a c i d plus urine components) was d i s s o l v e d i n 10 ml d o u b l e - d i s t i l l e d water and d i v i d e d i n t o two a l i q u o t s : one f o r c l a s t o g e n i c i t y a n a l y s i s , and the other f o r s t a b i l i t y determination. A l l e x t r a c t s were stored at -20°C when not i n use. 4.4.3 C e l l l i n e Chinese hamster ovary (CHO) f i b r o b l a s t s were k i n d l y donated by Dr L.D. Skarsgard (Medical Biophysics U n i t , B r i t i s h Columbia Cancer Research 21 Centre). This i s a pseudodiploid line with a modal chromosome number of 21. The low chromosome number, the stable karyotype, the large morphology of the chromosomes,.and the short generation time (12-14 hours) make this c e l l line i d e a l for the study of induced chromosome aberrations. 4.4.4 C e l l cultures CHO c e l l s were grown as monolayer cultures using MEM (Eagle's Minimal Essential Medium, Grand Island B i o l o g i c a l Co., Burlington, Ont.) supplemented with 10% f e t a l c a l f serum, a n t i b i o t i c s ( p e n i c i l l i n G, 125 ug/ml; streptomycin sulfate, 29.6 ug/ml; kanamycin, 100 ug/ml; fungizone, 2.5 ug/ml) 2 and sodium bicarbonate (1 mg/ml). Stock cultures were maintained in 75 cm p l a s t i c culture flasks (Falcon Plastics) at 37°C i n a water-saturated 5% CC>2 incubator. 4.4.5 Preparation of c e l l s for cytogenetic assay The methods employed have been described previously by Stich and 2 Kuhnlein (1979). Approximately 50,000 CHO c e l l s were seeded onto 22 mm coverslips i n 35 mm p l a s t i c dishes (Falcon P l a s t i c s ) . The c e l l s were incubated with MEM (10% f e t a l c a l f serum) at 37°C for about 2 days to allow the c e l l s time to resume exponential growth. Experiments were begun before the c e l l s reached 60% confluency for the following reasons: (i) to ensure adequate exposure to the urine extracts, and ( i i ) to avoid poor spreading of metaphase chromosomes due to overcrowding of the c e l l s . 22 4.4.6 Ad d i t i o n of urine concentrates On the day of the experiment, several d i l u t i o n s of the urine e x t r a c t s were made with serum-free MEM to obtain various concentrations of c r e a t i n i n e equivalence. The term " c r e a t i n i n e equivalence" i s that used by C u r t i s and Dunn (1985): " 4.0 mg/ml c r e a t i n i n e equivalence means that per m i l l i l i t r e of f i n a l volume, the e x t r a c t s applied to the c e l l s contained the organic m a t e r i a l i s o l a t e d from a volume of urine o r i g i n a l l y containing 4 mg c r e a t i n i n e " . Culture medium was removed from the p e t r i dishes by suction and replaced with 1 ml of serum-free medium containing urine e x t r a c t at the desired concentration. The c e l l s were incubated with the urine e x t r a c t f o r 3 hours at 37°C. Following the exposure pe r i o d , the urine e x t r a c t s were removed and cu l t u r e s were washed twice with serum-free MEM. A f t e r the a d d i t i o n of 2 . 0 ml of f r e s h MEM (supplemented with 1 0 % f e t a l c a l f serum), the c u l t u r e s were incubated f o r a f u r t h e r 1 6 hours. A l l experiments were performed under yellow f l u o r e s c e n t l i g h t i n g . For a given dose of urine e x t r a c t , at l e a s t one s l i d e was made. However, experiments were repeated f or some c r i t i c a l concentrations. As negative c o n t r o l s f o r each experiment, the f o l l o w i n g were included: (i ) serum-free medium, ( i i ) reagent b l a n k - d i s t i l l e d water ( 5 0 0 ml) passed through the RPLC column which was then e l u t e d with 5 0 % acetone, and ( i i i ) solvent blank (4% acetone (v/v) i n serum-free MEM). This concentration of acetone i s f a r i n excess of any minute traces of acetone that may remain i n the urine e x t r a c t s a f t e r r o t a r y evaporation and freeze-drying. 4.4.7 Harvesting of CHO c e l l s and s l i d e preparations Four hours before h a r v e s t i n g , 0 . 1 ml of c o l c h i c i n e ( 0 . 1 % i n serum-free MEM) was added to the c u l t u r e s to accumulate the c e l l s at metaphase. At 23 the end of the incubation p e r i o d , the c e l l s were treated with a hypotonic s o l u t i o n of 1% sodium c i t r a t e (w/v) f o r 20 min, then f i x e d i n a 3:1 s o l u t i o n (v/v) of 95% e t h a n o l / g l a c i a l a c e t i c a c i d (20 min). Once a i r - d r i e d , the c e l l s were stained with 2% aceto-orcein and dehydrated. Each c o v e r s l i p was dipped b r i e f l y i n xylene and mounted onto clean glass s l i d e s . 4.4.8 Analysis of metaphase p l a t e s for chromatid aberrations S l i d e s from each of the experiments were coded p r i o r to a n a l y s i s . Whenever p o s s i b l e , a minimum of 100 well-spread metaphase p l a t e s were selected at random and examined f o r the presence of chromatid breaks and exchanges. Aberrations were scored using the c r i t e r i a of Hsu (1982). The more commonly observed aberrations are shown i n Table 3. Results were reported i n two d i f f e r e n t ways: ( i ) the percentage of metaphases with at l e a s t one chromatid a b e r r a t i o n , and ( i i ) the average number of chromatid exchanges per metaphase c e l l . 4.4.9 S t a t i s t i c a l a n a l y s i s A l l s t a t i s t i c a l comparisons were performed using non-parametric methods. Clastogenic a c t i v i t y of urine samples from the a g r i c u l t u r a l research s t a t i o n workers before and during p e s t i c i d e exposure was evaluated f o r s i g n i f i c a n c e by the Wilcoxon paired-sample t e s t . The Tukey's m u l t i p l e comparisons t e s t was used to evaluate d i f f e r e n c e s i n the c l a s t o g e n i c a c t i v i t y of urine of o r c h a r d i s t s during d i f f e r e n t p e s t i c i d e exposure periods. This t e s t was a l s o used to examine d i f f e r e n c e s i n the mean c l a s t o g e n i c a c t i v i t y among the three study groups ( o r c h a r d i s t s , a g r i c u l t u r a l research s t a t i o n workers and reference c o n t r o l s ) . The Mann-Whitney U t e s t was used to evaluate 24 TABLE 3 T y p e s O f C h r o m a t i d A b e r r a t i o n s W [1 ti w 1 C h r o m a t i d G a p C h r o m a t i d B r e a k s C h r o m a t i d E x c h a n g e s Diagram modified from Dean and Danford (1984). 25 urinary c r e a t i n i n e values and urinary pH. In a d d i t i o n , urinary clastogenic a c t i v i t y data for smokers and non-smokers were examined for di f f e r e n c e s using t h i s s t a t i s t i c a l t e s t . C o r r e l a t i o n c o e f f i c i e n t s were c a l c u l a t e d by Spearman's rank c o r r e l a t i o n procedure. A d e s c r i p t i o n of a l l these s t a t i s t i c a l t e s t s can be found i n Zar (1984). 26 RESULTS 1. P r e l i m i n a r y Studies 1.1 Controls for CHO C e l l Chromosome Aberration Assay Before evaluating the clastogenic ( i . e . , chromosome-breaking) a c t i v i t y of urine e x t r a c t s , the spontaneous breakage frequency of CHO c e l l s had f i r s t to be determined. Table 4 summarizes the breakage frequencies observed with various solvents. In general, only 0 to 1% of the metaphases showed evidence of aberrations. These aberrations consisted only of breaks and gaps. No exchanges were found i n these c o n t r o l s . The m i t o t i c index of the CHO c e l l s was estimated by determining the number of metaphases i n a thousand c e l l s . T y p i c a l l y , t h i s f i g u r e was between 9 and 12%. 1.2 S t a b i l i t y of Urine E x t r a c t s Urine e x t r a c t s were stored at -20°C u n t i l tested. To determine the s t a b i l i t y of the e x t r a c t s under these c o n d i t i o n s , o c c a s i o n a l samples were re t e s t e d to see i f l o s s of c l a s t o g e n i c a c t i v i t y had taken place. Table 5 i l l u s t r a t e s t h a t four e x t r a c t s tested one week and one month a f t e r urine c o l l e c t i o n appeared to be s t a b l e . The response of the CHO c e l l s v a r i e d somewhat during the two time periods, p a r t i c u l a r l y at high doses. However, the maximum l e v e l of c l a s t o g e n i c a c t i v i t y w i t h i n the dose range tes t e d remained c o n s i s t e n t over the duration of storage. A d e c i s i o n was therefore made to compare the maximum c l a s t o g e n i c a c t i v i t y over the defined dose range of 1.0 to 8.0 mg/ml c r e a t i n i n e equivalence. The upper l i m i t represented concentrations t o x i c t o CHO c e l l s , while the lower l i m i t represented physio-l o g i c a l c r e a t i n i n e concentrations t y p i c a l l y present i n the u r i n a r y bladder. 27 TABLE 4 CHROMOSOME ABERRATION ACTIVITY INDUCED BY NEGATIVE CONTROLS Control Percent Metaphases with Chromatid Aberrations S l i d e 1 S l i d e 2 Serum-free medium o 1 1.0 D i s t i l l e d water concentrate 2 0 4% Acetone (v/v) 2.0 0 ^No detectable aberrations i n the metaphases examined. D i s t i l l e d water was passed through the RPLC column and eluted with 500 ml 50% acetone/50% 2.5 mM phosphoric a c i d . The eluate was then r o t a r y evaporated to remove the acetone, n e u t r a l i z e d , f r e e z e - d r i e d , and f i n a l l y r e c o n s t i t u t e d i n 10 ml d i s t i l l e d water. A 70% concentrate/30% serum-free MEM (v/v) d i l u t i o n was tested. 28 TABLE 5 STABILITY OF A FEW URINE EXTRACTS OVER A ONE-MONTH PERIOD Percent CeLLs with Chromatid Aberrations Creatinine Equivalence (mg/mL) Extract Tested a t 1 0.0 2.0 4.0 6.0 8.0 1 1 week o 2 5.0 26. L T 3 T 1 month L.O 3.0 L9.0 24.0 T 2 1 week 0 0 L7.0 26.0 T 1 month L.O 3.0 L2.0 22.0 T 3 1 week 0 9.0 23.0 T T 1 month L.O 6.0 26.0 28.0 T 4 1 week 0 0 3.0 5.0 T 1 month L.O 2.0 5.0 7.0 T Ex t r a c t s were tested at times i n d i c a t e d a f t e r urine c o i L e c t i o n . i No detectable aberrations i n the metaphases examined. T = t o x i c . 29 1.3 Selection of an Elution Scheme As described i n Materials and Methods, two methods of eluting the hydrophobic organic material from the RPLC column were used in this study: (1) sequential elution with 5%, 15% and 50% acetone to generate three fractions of increasing hydrophobicity, and (2) elution with only 50% acetone to obtain a single urine composite extract. Using 50% acetone only as an eluant also elutes materials which would be present in the 5% and 15% acetone fractions. Sequential e l u t i o n offers the advantage of characterizing genotoxic materials according to t h e i r hydrophobicity. However, unlike the l a t t e r method, three fractions must be analyzed rather than a single urine composite. Preliminary studies were performed with the sequential elution method on urine obtained during the spraying period i n May 1984 from non-smoking orchardists and non-smoking a g r i c u l t u r a l research station personnel. The objective was to determine whether clastogenic a c t i v i t y would appear predominantly i n any one of the three urine fractions. The aberration frequencies found for each urine fraction at varying urine doses are depicted i n Appendices 4 and 5, respectively, for the orchardists and a g r i c u l t u r a l research station workers. Figures 4 and 5 show, on an i n d i v i d u a l basis, the maximum clastogenic a c t i v i t y observed for each of the three urine fractions. No consistent pattern was found i n the appearance of clastogenic a c t i v i t y i n any of the three fractions for both the orchardists and the a g r i c u l t u r a l research s t a t i o n personnel. In some cases, clastogenic a c t i v i t y was observed i n a l l three f r a c t i o n s , while i n other instances, the a c t i v i t y was detectable i n only one of the three fractions. 30 FIGURE 4 Clastogenic a c t i v i t y of urine of orchardists from phase 1 (May 1984) during the spraying season. Urine fractions were prepared and assayed for clastogenic a c t i v i t y as described i n Materials and Methods. Each bar represents the maximum clastogenic a c t i v i t y over the dose range tested (1.0 to 8.0 mg/ml creatinine equivalence) for 1 of the 3 fractions prepared from the urine of an in d i v i d u a l . 31 o a w 09 a < a E o c o a c a eg 2 30-20-10 -S I 3 2 S 3 S 4 S 5 S 6 S 7 S 9 S 1 0 S 1 1 S 1 2 S 1 3 S 1 4 S 1 6 S 1 6 S 1 9 S 2 0 F r a c t i o n 2 ( 1 5 % A c e t o n e ) \AYAY7\ 40 20 -8 1 S 2 S 3 3 4 S 5 3 6 S 7 S 9 S 1 0 S 1 1 3 1 2 S 1 3 S 1 4 3 1 6 3 1 • 3 1 9 S 2 0 F r a c t i o n 3 ( 5 0 % A c e t o n e ) y rt 8 1 8 2 S 3 8 4 8 6 S 6 8 7 8 9 S 1 0 8 1 1 8 1 2 8 1 3 S 1 4 8 1 6 8 1 6 8 1 9 8 2 0 S u b j e c t N u m b e r Figure 4 32 FIGURE 5 Clastogenic a c t i v i t y of urine of a g r i c u l t u r a l research s t a t i o n workers (non-sprayers) from phase 1 (May 1984) during the spraying season. Urine f r a c t i o n s were prepared and assayed for c l a s t o g e n i c a c t i v i t y as described i n Ma t e r i a l s and Methods. Each bar represents the maximum clastogenic a c t i v i t y over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence) f o r 1 of the 3 f r a c t i o n s prepared from the urine of an i n d i v i d u a l . 33 F r a c t i o n 1 ( 6 % A c e t o n e ) R 1 R 3 R 6 R 7 R 8 R 6 R 1 0 R 1 1 R 1 3 R 1 4 R 1 6 F r a c t i o n 2 ( 1 5 % A c e t o n e ) R l R 3 R 6 B 7 R 8 R 9 R 1 0 R 1 1 R 1 3 R 1 4 R 1 5 F r a c t i o n 3 ( 5 0 % A c e t o n e ) R 1 R 3 R 6 R 7 R 8 R 9 R 1 0 R 1 1 R 1 3 R 1 4 R 1 5 S u b j e c t N u m b e r Figure 5 34 Table 6 shows the mean values of c l a s t o g e n i c a c t i v i t y represented on a group b a s i s . Despite the a p p l i c a t i o n of p e s t i c i d e s ( l i s t e d i n Table 7) 16 to 24 hours p r e v i o u s l y , o r c h a r d i s t s show no s i g n i f i c a n t d i f f e r e n c e s i n urine g e n o t o x i c i t y compared to a g r i c u l t u r a l research s t a t i o n personnel. The g e n o t o x i c i t y of the corresponding urine f r a c t i o n s d i d not d i f f e r f o r the two study groups (P>0.50; Mann-Whitney U- t e s t ) . However, the p r e l i m i n a r y study does suggest that urine g e n o t o x i c i t y may be due to more than one urine c o n s t i t u e n t , as i n f e r r e d from the presence of clastogenic a c t i v i t y i n a l l three urine f r a c t i o n s i n some i n d i v i d u a l s . Based on the lack of consistency i n the appearance of c l a s t o g e n i c a c t i v i t y i n the three urine f r a c t i o n s and on the heavy amount of work involved i n the a n a l y s i s of three f r a c t i o n s f o r a large number of urine samples, a d e c i s i o n was made to e x t r a c t organic m a t e r i a l from a l l subsequent samples with 50% acetone only. 2. E f f e c t of Varying Degrees of P e s t i c i d e Exposure on Urine C l a s t o g e n i c i t y A l l of the urine concentrates from t h i s point on were generated by e l u t i n g the RPLC column with 50% acetone. The eluate w i l l be r e f e r r e d to as f r a c t i o n A f o r future reference. A l l f i g u r e s given w i l l be expressed as the mean ± standard d e v i a t i o n . 2.1 Reference Control Group (No Exposure to P e s t i c i d e s ) Non-smoking i n d i v i d u a l s (n=21) r e s i d i n g i n Grand Forks and Vancouver, B.C., were used as a reference c o n t r o l group. Smokers from these two areas were excluded from t h i s part of the study. None of the reference c o n t r o l subjects 35 TABLE 6 CLASTOGENIC ACTIVITY OF URINE FRACTIONS FROM ORCHARDISTS AND AGRICULTURAL RESEARCH STATION WORKERS DURING THE SPRAYING PERIOD IN MAY 1984 Maximum Clastogenic A c t i v i t y (Mean ± Standard D e v i a t i o n ) 1 Percent C e l l s with Chromatid Aberrations F r a c t i o n 1 Fra c t i o n 2 F r a c t i o n 3 Group (5% Acetone) (15% Acetone) (50% Acetone) 4.6 ± 3.6 6.6 ± 5.4 7.4 ± 7.5 7.8 ± 8.7 (P>0.50) (P>0.50) A g r i c u l t u r a l s t a t i o n workers (May 1984) ± ' Orchardists (May 1984) 4.6 + 2.2 (P>0.50) Maximum cl a s t o g e n i c a c t i v i t y of urine f r a c t i o n s over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). S i g n i f i c a n c e values (as determined by the Mann-Whithey U test) are r e l a t i v e to urine f r a c t i o n s from a g r i c u l t u r a l research s t a t i o n workers. 36 TABLE 7 PESTICIDES USED BY SPRAYERS ON THE DAY OF URINE COLLECTION IN MAY 1984 P e s t i c i d e s Sprayed on Day of Urine C o l l e c t i o n Orchardist Number P e s t i c i d e Use 1 . . 2 Genotoxicity Reference 1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 19 20 Azinphos-methyl I + Alam et a l . , 1974 / / / / / / / / Benomyl F + Kappas et a l . , 1976 / Carbaryl I + Ishidate et a l . , 1977 / / Daminozide PG / Diazinon I + Matsuoka et a l . , 1979 / / / D i n i t r o c r e s o l I / Dodine F - Carere et a l . , 1978 / Endosulfan I - Fahrig, 1974 / Glyphosate H - Moriya et a l . , 1983 / / Paraquat H + Parry, 1973 / / Simazine H + Tomkins & Grant, 1976 / Thiophanate-methyl F Moriya et a l . , 1983 / / T o t a l number of spraying hours 6 9 4 3 2 5 3 6 3 3 4 6 3 3 4 2 7 ^ I , i n s e c t i c i d e ; F, fungicide; PG, plant growth regulator; H, he r b i c i d e . 2 +, p o s i t i v e r e s u l t ; negative r e s u l t ; blanks i n d i c a t e no information a v a i l a b l e for the chemical reported any exposure to p e s t i c i d e s . Urine samples were c o l l e c t e d from each subject i n September 1985 and concentrated by reversed-phase high pressure l i q u i d chromatography. The urine e x t r a c t s were then assayed for the a b i l i t y to induce chromatid aberrations i n CHO c e l l s . Appendices 2 and 3 show the c l a s t o g e n i c (chromosome-breaking) a c t i v i t y of the urine e x t r a c t s at various c r e a t i n i n e equivalences. Results are expressed i n two ways: (1) percentage of metaphases with at l e a s t one chromatid break or exchange, and (2) average number of chromatid exchanges per metaphase c e l l . The l a t t e r was determined by summing the t o t a l number of exchange figures and d i v i d i n g by the number of metaphases analyzed. As mentioned p r e v i o u s l y , negative c o n t r o l s t y p i c a l l y showed between 0 and 1% of c e l l s with chromatid a b e r r a t i o n s , and only breaks and gaps were found. The data i n Appendices 2 and 3 i n d i c a t e that the urine of the reference c o n t r o l group e x h i b i t e d low but s i g n i f i c a n t l e v e l s of c l a s t o g e n i c a c t i v i t y compared to those found f o r c o n t r o l d i s t i l l e d water concentrates (P<0.001; Mann-Whitney U t e s t ) . Dose-related increases i n c l a s t o g e n i c a c t i v i t y were observed f o r many urine e x t r a c t s . Clastogenic responses were generally found at concentrations between 2.0 and 5.0 mg/ml c r e a t i n i n e equivalence. Beyond t h i s concentration range, CHO c e l l s showed evidence of m i t o t i c i n h i b i t i o n i n a d d i t i o n to t o x i c i t y , the l a t t e r i n d i c a t e d by a decrease i n c e l l number and the presence of pyknotic n u c l e i . Figure 6 describes the d i s t r i b u t i o n of urine c l a s t o g e n i c i t y among the reference c o n t r o l i n d i v i d u a l s at a c r e a t i n i n e equivalence of 4.0 mg/ml. At t h i s c oncentration, the urine e x t r a c t s of 76% of the subjects d i d not induce more than 5% metaphases with chromatid aberrations. The urine e x t r a c t s of 19% of the i n d i v i d u a l s demonstrated c l a s t o g e n i c a c t i v i t y between 6 and 10% 38 FIGURE 6 Frequency d i s t r i b u t i o n of ur i n a r y clastogenic a c t i v i t y at 4.0 mg/ml c r e a t i n i n e equivalence for the reference c o n t r o l group (non-smoking residents of Grand Forks and Vancouver). 39 o - H 1 1 \ r-0 1 6 1 0 1 5 2 0 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 6 40 metaphases with chromatid a b e r r a t i o n s , while only 5% of the c o n t r o l group subjects showed a c t i v i t y w i t h i n the range of 11 to 15% aberrant metaphases. The maximum cl a s t o g e n i c a c t i v i t y found over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence) i s shown f o r each i n d i v i d u a l i n Figures 7A and 7B. For the group, a mean percentage of 5.2 ± 2.3 metaphases contained at l e a s t one chromatid a b e r r a t i o n , with an average number of 0.09 ± 0.02 chromatid exchanges per metaphase c e l l . These f i g u r e s strongly suggest (P<0.001; Mann-Whitney U-test) that r e l a t i v e to the baseline l e v e l s of cla s t o g e n i c a c t i v i t y (0 to 1% aberrant metaphases) observed with d i s t i l l e d water concentrates, urine contains m a t e r i a l s capable of inducing genetic damage. 2.2 A g r i c u l t u r a l Research S t a t i o n Personnel (Low Exposure to P e s t i c i d e s ) Unlike the reference c o n t r o l group where p e s t i c i d e exposure was known to be small or non-existent, i n d i v i d u a l s from the a g r i c u l t u r a l research s t a t i o n were at r i s k during the spraying period due to the p o s s i b l e d r i f t of p e s t i c i d e s from the s i t e of a p p l i c a t i o n . Urine samples were obtained from 11 non-smoking a g r i c u l t u r a l research s t a t i o n personnel during the pre-spraying (March) and spraying (August) periods i n 1985. The urines were concentrated as described p r e v i o u s l y and assayed for the presence of cla s t o g e n i c m a t e r i a l s . The percentage of metaphases with chromatid aberrations and the average number of chromatid exchanges per metaphase c e l l at various urine doses are shown f o r the two time periods i n Appendix 5. For the maj o r i t y of urine e x t r a c t s , dose-related responses were observed. Clastogenic a c t i v i t y was g e n e r a l l y detectable at 3.0 mg/ml c r e a t i n i n e equivalence, with signs of t o x i c e f f e c t s appearing at about 6.0 mg/ml concentration. 41 r FIGURE 7A I n d i v i d u a l v a r i a t i o n s i n the percentage of aberrant metaphases induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from the reference c o n t r o l group. Each bar represents the maximum percentage of aberrant metaphases obtained over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). FIGURE 7B I n d i v i d u a l v a r i a t i o n s i n the average frequency of chromatid exchanges per metaphase induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from the reference c o n t r o l group. Each bar represents the maximum number of chromatid exchanges per metaphase obtained over the dose range tes t e d (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). 42 M e a n ± S.D. a 1 ' '///. i 5.2 s 1 2.3 1 2 I 1 Figure 7A M e a n • s.D. 1 0 . \ oe 1 1 o . 0 7 1 1 | 'A 7\ / / Figure 7B Figures 8 A and 8 B i l l u s t r a t e the frequency d i s t r i b u t i o n of urinary c l a s t o g e n i c a c t i v i t y for the two time frames at 4 . 0 mg/ml c r e a t i n i n e equivalence.. L i t t l e d i f f e r e n c e was apparent i n the frequencies during the pre-spraying and spraying periods. During both these periods, over 8 0 % of the i n d i v i d u a l s e x h i b i t e d urine c l a s t o g e n i c i t y w i t h i n the range of 0 to 5% metaphases with chromatid aberrations. Over both periods, no i n d i v i d u a l s showed urine c l a s t o g e n i c i t y of more than 1 0 % aberrant metaphases. I l l u s t r a t e d i n Figures 9 A and 9 B i s the observed maximum clastogenic a c t i v i t y over the dose range tested ( 1 . 0 to 8 . 0 mg/ml c r e a t i n i n e equivalence). For the pre-spraying p e r i o d , an average of 5 . 4 ± 2 . 0 aberrant metaphases ( 0 . 1 1 ± 0 . 0 6 chromatid exchanges per metaphase c e l l ) was found for the urine specimens tested. During the spraying p e r i o d , the urines demonstrated an average a c t i v i t y of 6 . 3 ± 3 . 1 % metaphases with chromatid a b e r r a t i o n s , with an average frequency of 0 . 1 3 + 0 . 0 9 chromatid exchanges per metaphase c e l l . Comparison of these mean values by the Wilcoxon paired sample t e s t (Zar, 1 9 8 4 ) showed no s i g n i f i c a n t d i f f e r e n c e ( P > 0 . 5 0 ) . I t therefore appears that i n d i r e c t exposure to p e s t i c i d e s of a g r i c u l t u r a l research s t a t i o n workers may be i n s u f f i c i e n t to induce any fur t h e r chromosome-damaging a c t i v i t y i n the urine above that of normal/baseline l e v e l s . 2 . 3 P e s t i c i d e A p p l i c a t o r s (High Exposure to Pest i c i d e s ) The e f f e c t of p e s t i c i d e exposure on urine c l a s t o g e n i c i t y was studied by analyzing the urine of 2 1 non-smoking p e s t i c i d e a p p l i c a t o r s , i n c l u d i n g two i n d i v i d u a l s who sprayed at the a g r i c u l t u r a l research s t a t i o n . The study was c a r r i e d out i n two separate phases. Phase 1 was done i n 1 9 8 4 and Phase 2 i n the f o l l o w i n g year. The two phases d i f f e r e d mainly i n the timing of the 44 FIGURE 8A Frequency d i s t r i b u t i o n of u r i n a r y c l a s t o g e n i c a c t i v i t y at r4.0 mg/ml c r e a t i n i n e equivalence for non-smoking a g r i c u l t u r a l research s t a t i o n workers during the pre-spraying period i n March 1985. FIGURE 8B Frequency d i s t r i b u t i o n of u r i n a r y c l a s t o g e n i c a c t i v i t y at 4.0 mg/ml c r e a t i n i n e equivalence for non-smoking a g r i c u l t u r a l research s t a t i o n workers during the spraying period i n August 1985. 45 r 2 0 1 0 1 5 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 8A l 0 1 6 1 0 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 8B 46 FIGURE 9A I n d i v i d u a l v a r i a t i o n s i n the percentage of aberrant metaphases induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from non-smoking a g r i c u l t u r a l research s t a t i o n workers during the pre-spraying (March 1985) and spraying (August 1985) periods. Each bar represents the maximum percentage of aberrant metaphases obtained over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). FIGURE 9B I n d i v i d u a l v a r i a t i o n s i n the average frequency of chromatid exchanges per metaphase induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from non-smoking a g r i c u l t u r a l research s t a t i o n workers during the pre-spraying (March 1985) and spraying (August 1985) periods. Each bar represents the maximum number of chromatid exchanges per metaphase obtained over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). 47 M a r c h B e f o r e S p r a y Figure 9A A u g u s t D u r i n g S p r a y M e a n t S . D . 0 . 1 1 • 0 . 0 6 0 . 1 3 t 0 . 0 9 'An M a r c h B e f o r e 8 p r a y A u g u s t O u r l n g S p r a y Figure 9B 48 urine c o l l e c t i o n s . In general, morning urine voids were c o l l e c t e d during phase 1 while evening urines were used i n phase 2. The same subjects p a r t i c i p a t e d - i n both phases with the exception of a few i n d i v i d u a l s who were absent on the day of urine c o l l e c t i o n . 2.3.1 Phase 1: Urine c o l l e c t e d 16-24 hours a f t e r p e s t i c i d e a p p l i c a t i o n In phase 1, urine samples were c o l l e c t e d from the p e s t i c i d e sprayers ( a l l non-smokers) during the spraying periods i n May, June and J u l y . Each sprayer submitted a morning urine v o i d 16 to 24 hours a f t e r p e s t i c i d e a p p l i c a t i o n . As a c o n t r o l , a follow-up sample was obtained from each sprayer during the post-spraying period i n October 1984. Specimens c o l l e c t e d i n May 1984 were used i n the study of the sequential e l u t i o n method (see sectio n 4.4.1 i n M a t e r i a l s and Methods). Consequently, only the June and J u l y samples were compared with the post-spraying October urine since these l a t t e r samples were concentrated by the whole e l u t i o n method. Urinary c l a s t o g e n i c a c t i v i t y data f or each i n d i v i d u a l f or both the spraying and post-spraying periods are d e t a i l e d i n Appendix 4. Dose-responses were g e n e r a l l y observed f o r each sample up to high urine concentrations where t o x i c i t y became evident. P e s t i c i d e exposure during t h i s phase d i d not make the u r i n e more t o x i c towards the CHO c e l l s , since the post-spraying c o n t r o l urine samples demonstrated the same extent of t o x i c i t y . Figures 10A and 10B i l l u s t r a t e the frequency d i s t r i b u t i o n of u r i n a r y c l a s t o g e n i c a c t i v i t y at 4.0 mg/ml c r e a t i n i n e equivalence. From these graphs, the frequency d i s t r i b u t i o n of u r i n e g e n o t o x i c i t y during both the spraying and post-spraying periods appears to be s i m i l a r . During these two time periods, the urines of 90 to 95% of the subjects were unable to induce more than 5% 49 FIGURE 10A Frequency d i s t r i b u t i o n of clastogenic a c t i v i t y in urine extract at 4.0 mg/ml creatinine equivalence for orchardists during the spraying season i n June and July 1984. Urine was collected 16 to 24 hours aft e r p esticide application. FIGURE 10B Frequency d i s t r i b u t i o n of clastogenic a c t i v i t y i n urine extract at 4.0 mg/ml creatinine equivalence for orchardists during the post-spraying period i n October 1984 (morning urine voids). 50 1 0 0 -I 3 •o 1 3 C r 2 0 0 1 5 1 0 1 5 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 10A 1 0 0 - 80H 3 •o T3 c 6 0 H 4 0 2 0 - 1 0 - + - r T T 1 6 T 2 0 0 1 6 1 0 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 10B 51 aberrant metaphases. Therefore p e s t i c i d e exposure during t h i s phase d i d not appear to a f f e c t the urinary c l a s t o g e n i c a c t i v i t y of the sprayers. The maximum cla s t o g e n i c a c t i v i t i e s obtained over the dose range of 1 . 0 to 8 . 0 mg/ml c r e a t i n i n e equivalence are presented i n Figures 11A and 11B on an i n d i v i d u a l b a s i s . Despite i n t r a - and i n t e r - i n d i v i d u a l v a r i a t i o n s , the group mean values of cla s t o g e n i c a c t i v i t y during both time i n t e r v a l s were not d i f f e r e n t . Urine c o l l e c t e d during the spraying season (June/July) induced aberrations i n an average of 5.6 ± 4 . 0 % of the metaphases, with an average frequency o f 0 . 1 5 ± 0 . 1 2 chromatid exchanges per metaphase c e l l . These f i g u r e s were not s i g n i f i c a n t l y d i f f e r e n t ( P > 0 . 5 0 ; Wilcoxon paired-sample t e s t ) from the a c t i v i t y found for the "post-spray" urines ( 5 . 7 ± 2 . 7 % aberrant metaphases; 0 . 1 4 ± 0 . 0 9 chromatid exchanges per metaphase c e l l ) . The presence of u r i n a r y clastogenic a c t i v i t y during the post-spraying period i n d i c a t e s that some f a c t o r s other than p e s t i c i d e exposure may be responsible f o r the g e n o t o x i c i t y . Tables 8 and 9 l i s t the d i f f e r e n t types of p e s t i c i d e s used along with the t o t a l hours of spraying time during the days of urine c o l l e c t i o n . Azinphos-methyl was the predominant p e s t i c i d e used by the sprayers on the days of ur i n e sampling. In many instances, more than one p e s t i c i d e was a p p l i e d by an i n d i v i d u a l sprayer during the course of the day. As i s evident from the t a b l e s , many of these p e s t i c i d e s possess genotoxic p r o p e r t i e s . With the exception of one or two i n d i v i d u a l s , a l l of the sprayers applied at l e a s t one genotoxic p e s t i c i d e i n June and J u l y . However, even though geno-t o x i c p e s t i c i d e s were used by many sprayers, no o v e r a l l increase i n ur i n a r y c l a s t o g e n i c a c t i v i t y beyond normal l i m i t s was observed among the sprayers i n t h i s phase. 52 FIGURE 11A I n d i v i d u a l v a r i a t i o n s i n the percentage of aberrant metaphases induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from o r c h a r d i s t s during the spraying (June/July) and post-spraying (October) periods i n 1984. Each bar represents the maximum percentage of aberrant metaphases obtained over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). FIGURE 1 IB-I n d i v i d u a l v a r i a t i o n s i n the average frequency of chromatid exchanges per metaphase induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from o r c h a r d i s t s during the spraying (June/July) and post-spraying (October) periods i n 1984. Each bar represents the maximum number of chromatid exchanges per metaphase obtained over the dose range t e s t e d (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). 53 M e a n ^ S . O . 5.6 + 4.0 5.7 • 2.7 m i J u n e J u l y D u r i n g S p r a y O c t o b e r A f t e r S p r a y Figure 1LA " M o a n t S . O . 0.15 I 0.12 0.141 0.09 9' m u J u n e J u l y D u r i n g S p r a y O c t o b e r A f t e r S p r a y Figure 11B 54 TABLE 8 PESTICIDES USED BY SPRAYERS ON THE DAY OF URINE COLLECTION IN JUNE 1984 Pesticides Sprayed on Day of Urine Co l l e c t i o n Orchardist Number Pesticide Use Genotoxicity Reference 1 2 3 4 5 6 7 8 10 11 12 13 14 15 18 19 20 Ametryne I / / / / Azinphos-methyl I + Alam et a l . , 1974 • / / / / / / Captan F + Bridges, 1975 / Carbaryl I + Ishidate et a l . , 1977 / Cyhexatin M - Moriya et a l . , 1983 / Daminozide PG / Diazinon I + Matsuoka et a l . , 1979 / • 7 D i n i t r o c r e s o l I / / Endosulfan I - Fahrig, 1974 / Ethephon PG - Moriya et a l . , 1983 / / / Glyphosate H - Moriya et a l . , 1983 / Paraquat H + Parry, 1973 / • Pentanochlor H f Simazine H + Tomkins & Grant, 1976 / / / Thiophanate-methyl F — Moriya et a l . , 1983 / / Total number of spraying hours 14 3 8 7 4 8 4 9 5 3 2 5 9 5 2 10 8 in s e c t i c i d e ; F, fungicide; M, miticide; PG, plant growth regulator; H, herbicide. +, p o s i t i v e result; -, negative result; blanks indicate no information available for the chemical. TABLE 9 PESTICIDES USED BY SPRAYERS ON THE DAY OF URINE COLLECTION IN JULY 1984 P e s t i c i d e s Sprayed on Day of Urine C o l l e c t i o n Orchardist Number P e s t i c i d e Use 1 2 Genotoxicity Reference 3 5 10 11 12 13 14 15 16 17 19 Azinphos-methyl I + Alam et a l . , 1974 / / / / / / / Benomyl F + Kappas et a l . , 1976 / Captan F + Bridges, 1975 / Chinomethionat F / / Daminozide PG Dimethoate I + Van Bao, 1974 / / Endosulfan I - Fahrig, 1974 / / Ethion I - Moriya et a l . , 1983 / Glyphosate H - Moriya et a l . , 1983 / / Paraquat H + Parry, 1973 / / Pentanochlor H Thiophanate-methyl F — Moriya et a l . , 1983 / T o t a l number of spraying hours 2 8 14 2 6 6 5 8 4 3 4 I , i n s e c t i c i d e ; F, fungicide; PG, pla n t growth regulator; H, he r b i c i d e . +, p o s i t i v e r e s u l t ; -, negative r e s u l t ; blanks i n d i c a t e no information a v a i l a b l e f o r the chemical. On the day of urine c o l l e c t i o n , the average number of spraying hours per day was 6.8 for the June samples and 5.6 for the J u l y urines. Despite s l i g h t d i f f e r e n c e s i n the spraying time during these two months, the maximum clastogenic a c t i v i t y was not s i g n i f i c a n t l y d i f f e r e n t (June: 5.2 ± 4.0% aberrant metaphases; J u l y : 6.3 ± 3.4% aberrant metaphases; P>0.50; Wilcoxon paired-sample t e s t ) . 2.3.2 Phase 2: Urine c o l l e c t e d w i t h i n 8 hours of p e s t i c i d e usage Based on the negative f i n d i n g s from phase 1, a d e c i s i o n was made to modify the timing of the urine c o l l e c t i o n i n phase 2. This d e c i s i o n was influenced by a rec e n t l y published report that changes i n urine g e n o t o x i c i t y may be time-dependent (Kobayashi and Hayatsu, 1984). Consequently, i n phase 2, urine was c o l l e c t e d w i t h i n a few hours of p e s t i c i d e usage. Two sets of urine c o l l e c t i o n s were made from the sprayers i n 1985. A sample was c o l l e c t e d from each sprayer during the pre-spraying p e r i o d i n March. These urines were c o l l e c t e d w i t h i n a 4-hour pe r i o d i n the l a t e afternoon. This specimen was followed by the c o l l e c t i o n of an "exposure" sample during the spraying p e r i o d i n August. Each sprayer was i n s t r u c t e d to give a l l u r i n e s voided w i t h i n 8 hours of p e s t i c i d e a p p l i c a t i o n . The induction of chromatid aberrations i n CHO c e l l s by the urine concentrates i s d e t a i l e d f o r each i n d i v i d u a l i n Appendix 4. Dose-response curves of urine samples from four representative p e s t i c i d e sprayers are shown i n Figure 12. Compared to the pre-spraying p e r i o d , urine specimens c o l l e c t e d during the spraying p e r i o d e x h i b i t e d potent c l a s t o g e n i c a c t i v i t y , as i n d i c a t e d by the sharply increased frequency of chromatid aberrations. The c l a s t o g e n i c a c t i v i t y was dose-dependent but not l i n e a r , and occurred over 57 FIGURE 12 Dose-response curves of urine e x t r a c t s prepared from urines c o l l e c t e d during the pre-spraying (March 1985) and spraying (August 1985) periods from 4 representative p e s t i c i d e sprayers. Urinary c l a s t o g e n i c a c t i v i t y f or March ( o ) and for August ( A ) i s shown for each subject. 58 c o a 3 0 < o E o 20 -J • icH JZ a « 2 S u b j e c t : S 7 / / • - 0 - 0 - 0 — o - o - c - o * o e.o s.o * C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) C r e a ~7—? 1 1 r 0 2.0 4.0 6.0 8.0 t i n i n e E q u i v a l e n c e ( m g / m l ) 3 0 20H 1 0 ^ OH S u b j e c t : S 1 0 / • - o - o - o T T — 1 1 f~ 2 0 4 . 0 e.o 8 . 0 — I 1 r C r e e i l n l n . E q u i v a l e n c e ( . . / . I ) C r e a t i n i n e Equ^lnV, ( m g / m , ) Figure 12 59 a narrow concentration range. In a number of "exposure" samples ( i . e . , urines c o l l e c t e d during the spraying period in August), moderate a c t i v i t y was apparent-at concentrations as low as 2.0 mg/ml creatinine equivalence. Variations were found in the concentrations at which pronounced clasto-genic a c t i v i t y appeared, as shown in Figure 12. The percentage of aberrant metaphases generally increased with urine dose to a maximum, then decreased with a further increase i n dose. In many cases, the highest a c t i v i t i e s were found at near toxic urine concentrations. T o x i c i t y was usually noted between 5.0 and 8.0 mg/ml creatinine equivalence. At these concentrations, a decrease i n clastogenic a c t i v i t y was t y p i c a l l y observed since severely aberrant c e l l s lyse rapidly and are not detected. T o x i c i t y was prominent at high creatinine doses i n urines c o l l e c t e d during both the pre-spraying and spraying periods. Figures 13A and 13B contrast the d i s t r i b u t i o n of urinary clastogenic a c t i v i t y f o r the two periods at 4.0 mg/ml creatinine equivalence. In March, no i n d i v i d u a l s demonstrated any a c t i v i t y beyond 10% c e l l s with chromatid aberrations. Eighty percent of the subjects showed genotoxicity ranging from 0 to 5% aberrant metaphases. However, a change i n d i s t r i b u t i o n was observed a f t e r exposure to pesticides. At 4.0 mg/ml creatinine concentration, the urines of a larger proportion of individuals showed the a b i l i t y to induce high frequencies of chromatid aberrations. For example, the urines of 38% of the sprayers, a f t e r pesticide usage, demonstrated more than twice the t y p i c a l baseline l e v e l s of aberrations ( i . e . , 0 to 5% metaphases with chromatid aberrations). These sprayers included two a g r i c u l t u r a l research s t a t i o n workers who sprayed pesticides as an occupation. 60 FIGURE 13A Frequency d i s t r i b u t i o n of urinary clastogenic a c t i v i t y at 4.0 mg/ml creatinine equivalence for pesticide sprayers during the pre-spraying period i n March 1985. FIGURE 13B Frequency d i s t r i b u t i o n of urinary clastogenic a c t i v i t y at 4.0 mg/ml creatinine equivalence for pesticide sprayers during the spraying period i n August 1985. 61 1 0 0 -m « 8 0 -3 •o * 6 0 -c * 4 0 -2 0 -0 1 S 1 0 1 5 2 0 2 5 3 0 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 13A 0 1 6 1 0 1 6 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 13B 62 The maximum cla s t o g e n i c a c t i v i t y over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence) f o r each sprayer i s depicted i n Figures 14A and 14B. During the pre-spraying p e r i o d , an average of 4.9 ± 3.5% of the metaphases e x h i b i t e d chromatid aberrations, with a frequency of 0.10 ± 0.09 chromatid exchanges per metaphase c e l l . With exposure to p e s t i c i d e s , the aberration frequency was s i g n i f i c a n t l y elevated to 19.9 ± 10.2% c e l l s with chromatid aberrations (P<0.001; Wilcoxon paired-sample t e s t ) . The extent of damage per metaphases was also s i g n i f i c a n t l y increased, reaching an average frequency of 0.70 ± 0.50 chromatid exchanges per metaphase c e l l (P<0.001; Wilcoxon paired-sample t e s t ) . M u l t i p l e exchanges and fragmentation of the chromosomal complement were frequently observed. Table 10 presents a l i s t of the p e s t i c i d e s used by the sprayers on the day of urine c o l l e c t i o n . The most commonly used p e s t i c i d e s were the i n s e c t i c i d e s azinphos-methyl and phosalone. On average, each subject sprayed a t o t a l of 4.2 hours per day. Spearman rank c o r r e l a t i o n t e s t i n d i c a t e d no c o r r e l a t i o n between the t o t a l number of hours of spraying and the observed maximum cla s t o g e n i c a c t i v i t y (r s=0.317; P>0.20). Since many sprayers used a combination of p e s t i c i d e s during the course of one day, a c o r r e l a t i o n of a s p e c i f i c p e s t i c i d e with the u r i n a r y clastogenic a c t i v i t y was not f e a s i b l e . However, i t i s worth p o i n t i n g out that 4 sprayers (S13, S14, S17 and S20), who used only a s i n g l e p e s t i c i d e on that day, demonstrated high u r i n a r y c l a s t o -genic a c t i v i t y when spraying e i t h e r phosalone, simazine or paraquat. Of the 17 p e s t i c i d e s sprayed on the day of sampling, 9 (53%) had been reported to e x h i b i t genotoxic a c t i v i t y _in v i t r o (Table 10). The use of such genotoxic p e s t i c i d e s may ex p l a i n the increase i n uri n a r y c l a s t o g e n i c a c t i v i t y a s s o c iated with p e s t i c i d e exposure. 63 FIGURE 14A I n d i v i d u a l v a r i a t i o n s i n the percentage of aberrant metaphases induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from o r c h a r d i s t s during the pre-spraying (March) and spraying (August) periods i n 1985. Each bar represents the maximum percentage of aberrant metaphases obtained over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). Included are two a g r i c u l t u r a l research s t a t i o n workers (1) who were engaged i n p e s t i c i d e spraying during the f r u i t growing season. FIGURE 14B I n d i v i d u a l v a r i a t i o n s i n the average frequency of chromatid exchanges per metaphase induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from o r c h a r d i s t s during the pre-spraying (March) and spraying (August) periods i n 1985. Each bar represents the maximum number of chromatid exchanges per metaphase obtained over the dose range t e s t e d (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). Included are two a g r i c u l t u r a l research s t a t i o n workers (1) who were engaged i n p e s t i c i d e spraying during the f r u i t growing season. 64 M a r c h B e f o r e S p r a y A u g u s t D u r i n g S p r a y Figure L4A M e a n 1 S.D. 0. 1 0 t 0.09 0.70 * 0.52 (1) St - H ( n } M a r c h B e f o r e S p r a y A u g u s t D u r i n g 8 p r a y Figure 14B 65 TABLE 10 PESTICIDES USED BY SPRAYERS ON THE DAY OF URINE COLLECTION IN AUGUST 1985 P e s t i c i d e s Sprayed on Day of Urine C o l l e c t i o n Orchardist Number P e s t i c i d e Use 1 Genotoxicity ^  Reference 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 3 3 20 21 Azinphos-methyl I + Alam et a l . , 1974 / / / / / / / Benomyl F + Kappas et a l . , 1976 Captan F + Bridges, 1975 / / / Carbaryl I • + Ishidate et a l . , 1977 / Cyhexatin M - Moriya et a l . , 1983 / / / / / / Diazinon I + Matsuoka et a l . , 1979 Dimethoate I + Van Bao, 1974 / / D i n i t r o c r e s o l I / / / Dodine F - Carere et a l . , 1978 / Endosulfan I - Fahrig, 1974 Ethephon PG - Moriya et a l . , 1983 / Ethion I - Moriya et a l . , 1983 / Nab am F Paraquat Phosalone H I + Parry, 1973 / / / / / / / / / / Simazine H + Tomkins & Grant, 1976 / Ziram F —• Moriya et a l . , 1983 / T o t a l number of spraying hours 5 2 4 3 5 4 4 4 2 6 2 2 2 4 5 8 2 8 7 ^ I , i n s e c t i c i d e ; F, fungicide; M, m i t i c i d e ; 1 PG, plant growth r e g u l a t o r ; H, , h e r b i c i d e . '+, p o s i t i v e r e s u l t ; -, negative r e s u l t ; blanks i n d i c a t e no information a v a i l a b l e f o r the chemical. A g r i c u l t u r a l research s t a t i o n sprayers. 2.4 Summary of the E f f e c t s of P e s t i c i d e Exposure on Urine C l a s t o g e n i c i t y Table 11 gives a s t a t i s t i c a l summary of the r e s u l t s obtained during phases 1 and 2. The cla s t o g e n i c a c t i v i t y of urine c o l l e c t e d 16 to 24 hours a f t e r p e s t i c i d e a p p l i c a t i o n was w i t h i n normal baseline l i m i t s . M o d i f i c a t i o n of the sampling procedure to w i t h i n 8 hours of p e s t i c i d e usage r e s u l t e d i n a s i g n i f i c a n t increase i n uri n a r y g e n o t o x i c i t y compared to non-exposure baseline l i m i t s . The cla s t o g e n i c a c t i v i t y of the l a t t e r samples was s i g n i f i -c a n t l y higher- than a l l other samples c o l l e c t e d over the course of the two phases. Table 11 also demonstrates that l i t t l e v a r i a t i o n e x i s t s i n the c l a s t o -g e n i c i t y of samples c o l l e c t e d during periods of no p e s t i c i d e usage. The mean u r i n a r y c l a s t o g e n i c a c t i v i t i e s f or the samples of October 1984 and March 1985 were not s i g n i f i c a n t l y d i f f e r e n t (P>0.50; Wilcoxon paired-sample t e s t ) . However, the presence of g e n o t o x i c i t y i n the urine a f t e r the d i s -continued use of p e s t i c i d e s does i n d i c a t e that other f a c t o r s such as d i e t may play some c o n t r i b u t i n g r o l e . Tables 12 and 13 summarize the r e s u l t s f or the three major groups i n the study. Comparisons between groups during the pre-spraying period i n March 1985 showed that the u r i n a r y c l a s t o g e n i c a c t i v i t y of both o r c h a r d i s t s and a g r i c u l t u r a l research s t a t i o n workers d i d not d i f f e r from that of the reference c o n t r o l group. With the advent of p e s t i c i d e a p p l i c a t i o n , only the sprayers demonstrated elevated u r i n a r y c l a s t o g e n i c a c t i v i t y . The non-spraying a g r i -c u l t u r a l research s t a t i o n personnel d i d not e x h i b i t urine c l a s t o g e n i c i t y above b a s e l i n e l e v e l s despite the p o t e n t i a l exposure to p e s t i c i d e s i n the a i r . Elevated c l a s t o g e n i c a c t i v i t y was only pronounced i n the two research s t a t i o n workers (R17 and R18) who sprayed p e s t i c i d e s . 67 TABLE 11 EFFECT OF PESTICIDE EXPOSURE AND TIMING OF URINE COLLECTION ON URINARY CLASTOGENIC ACTIVITY IN SPRAYERS (PERCENT CELLS WITH CHROMATID ABERRATIONS) Q Value S i g n i f i c a n c e A VS. B 0. 14 P>0.50 A VS . c 0. 89 P>0.50 A vs. D 3. 82 P<0.001 B vs. C 0. 74 P>0.50 B vs. D 3. 97 P<0.001 C vs. D 4. 85 P<0.001 A: urine c o l l e c t e d 16-24 hours a f t e r p e s t i c i d e a p p l i c a t i o n during June/July 1984. B: morning urine c o l l e c t e d during the post-spraying period i n October 1984. C: evening urine c o l l e c t e d during the pre-spraying period i n March 1985. D: urine c o l l e c t e d w i t h i n 8 hours of p e s t i c i d e a p p l i c a t i o n i n August 1985. Q Values were determined from non-parametric Tukey's m u l t i p l e comparisons t e s t (Zar, 1984). 1 Group 68 TABLE 12 INTERGROUP COMPARISONS OF CLASTOGENIC ACTIVITY (PERCENTAGE OF ABERRANT METAPHASES) IN URINE EXTRACTS DURING THE PRE-SPRAYING PERIOD IN MARCH 1985 Number of Subjects Q Value Significance Pesticide sprayers (March 1985) 22 0.65 P>0.50 vs. Reference control group3 11 (September 1985) Pesticide sprayers (March 1985) 22 0.54 P>0.50 vs. Research station personnel 11 (March 1985) Research station personnel 11 0.87 P>0.50 vs. Reference control group 11 (September 1985) ''"All the groups consist of non-smoking subjects only. 2 Q values were determined by st a t i s t i c a l evaluation of group means using Tukey's multiple comparisons test (Zar, 1984). 3Subjects from Vancouver and Grand Forks. Group1 69 TABLE 13 INTERGROUP COMPARISONS OF CLASTOGENIC ACTIVITY (PERCENTAGE OF ABERRANT METAPHASES) IN URINE EXTRACTS DURING THE SPRAYING PERIOD IN AUGUST 1985 Group Number of Subjects Q Value S i g n i f i c a n c e P e s t i c i d e sprayers (August 1985) 21 4.67 P<0.001 vs. Reference c o n t r o l group" (September 1985) 11 P e s t i c i d e sprayers (August 1985) 21 3.10 P<0.01 Research s t a t i o n personnel (August 1985) 11 Research s t a t i o n personnel (August 1985) 11 0.77 P>0.50 vs. Reference c o n t r o l group (September 1985) 11 A l l the groups c o n s i s t of non-smoking subjects only. 2Q values were determined by s t a t i s t i c a l e valuation of group means using Tukey's m u l t i p l e comparisons t e s t (Zar, 1984). Subjects from Vancouver and Grand Forks. 70 F i n a l l y , the reference c o n t r o l group c o n s i s t i n g of Grand Forks and Vancouver re s i d e n t s showed low l e v e l s of urine g e n o t o x i c i t y , providing f u r t h e r evidence of other endogenous or exogenous f a c t o r s that may r e s u l t i n urine c l a s t o g e n i c i t y . 3. E f f e c t of Smoking on Urine C l a s t o g e n i c i t y Since the u r i n e of c i g a r e t t e smokers has been found to contain mutagenic (not clastogenic) compounds capable of reversing Salmonella b a c t e r i a from an auxotrophic to an autotrophic s t a t e of n u t r i t i o n (Yamasaki and Ames, 1977), the e f f e c t s of smoking habits were evaluated. The groups used i n t h i s p a r t of the study were the same as those p r e v i o u s l y described i n part 1 of the p r o j e c t . Smokers from Grand Forks and the Summerland a g r i c u l t u r a l research s t a t i o n were compared to non-smokers from Grand Forks, Vancouver and the Okanagan region ( i n c l u d i n g the o r c h a r d i s t s and the a g r i c u l t u r a l research s t a t i o n workers). Figures 15 and 16 show the r e s u l t s obtained for 4 r e p r e s e n t a t i v e non-smokers and smokers, r e s p e c t i v e l y . For both groups, a dose-related increase i n c l a s t o g e n i c a c t i v i t y was observed. However, i n smokers, the increase i n a c t i v i t y was more pronounced, as i n d i c a t e d by the steeper slopes of the curves. At high urine doses, c l a s t o g e n i c a c t i v i t y for both groups dropped sharply as a r e s u l t of t o x i c i t y . The graphs also i n d i c a t e that the i n i t i a l slopes of the curves are q u i t e s i m i l a r . This e f f e c t i s only noticeable up to about 4.0 mg/ml c r e a t i n i n e equivalence. Above t h i s con-c e n t r a t i o n r e s u l t s vary, with some curves continuing to r i s e and others f a l l i n g s teeply. 71 FIGURE 15 Dose-response curves of urine e x t r a c t s prepared from urines c o l l e c t e d i n March 1985 and August 1985 from 4 representative non-smokers from the a g r i c u l t u r a l research s t a t i o n . Urinary c l a s t o g e n i c a c t i v i t y f o r March (O) and f o r August ( • ) i s shown for each subject. 72 3(H 2<H i o n OH S u b j e c t : R 6 O - O - C v A — A—A< *~i 1 1 1 r— fi • 2 . 0 4 . 0 6 . 0 8 . 0 c o a 30 < to E 2 20 «, i o n o 09 CB £. Q. eg • 0 S u b j e c t : R 8 X T" 0 —I 1 1 r— 2 . 0 4 . 0 6 . 0 8 . 0 C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) 3 0 - 1 2 0 H 1 0 H OH S u b j e c t : R 1 4 - - S S I ' o-o T 0 I I I 1— 2 . 0 4 . 0 6 . 0 8 . 0 CO c o I 3 0 < CO E o 2 0 £ o a cs 1 0 H • oH S u b j e c t : R 1 5 • — • — A — 4 \ T 0 I I I r — 2 . 0 4 . 0 6 . 0 8 . 0 C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) Figure 15 73 FIGURE 16 Dose-response curves of urine e x t r a c t s prepared from urines c o l l e c t e d i n March 1985 and August 1985 from 4 representative smokers from the a g r i c u l t u r a l research s t a t i o n . Urinary clastogenic a c t i v i t y f or March (O) and f o r August ( • ) i s shown f o r each subject. 74 3 0 H 20 H 10H 0 i S u b j e c t : R 4 2=2** T 1 I I 1 ' 0 - 2 . 0 4 . 0 6 . 0 8 . 0 " 0 2 . 0 4 . 0 6 . 0 8 . 0 C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) 3 0 H 20H 10H a) C O « I 30H < I 20-1 • 1o^ a (0 -c a 2 o H S u b j e c t : R 1 6 , 0 ft "I J i 1 r — 0 2.0 4 . 0 6 . 0 8 . 0 C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) Figure 16 75 Figures 17A and 17B present the frequency d i s t r i b u t i o n of clastogenic a c t i v i t y for urine of non-smokers and smokers at 4.0 mg/ml creatinine equivalence. Over 80% of the urine extracts from non-smokers demonstrated low clastogenic responses, with between 0 and 5% metaphases having chromatid aberrations. However, only 30% of the smokers showed a c t i v i t y within this low range. The urine of most smokers was found to induce aberrations i n between 10 and 25% of the examined metaphases. Therefore a larger proportion of smokers show higher chromatid aberration frequencies. Figures 18A and 18B i l l u s t r a t e the maximum clastogenic response for each i n d i v i d u a l . Despite i n t e r - i n d i v i d u a l v ariations, the urine of smokers e l i c i t e d more chromatid damage than that of non-smokers. An average of 14.0 ± 6.0% of examined metaphases were aberrant when exposed to the urine of smokers. In the same experiment, urine of non-smokers induced aberrations i n only an average of 5.4 ± 2.7% of the metaphases examined. Analysis of these means by the Mann-Whitney U test showed high levels of significance (P<0.001). Scores from in d i v i d u a l smokers ranged from 1 to 26% metaphases with chromatid aberrations. Only one smoker showed low clastogenic a c t i v i t y . The extent of damage per metaphase c e l l was also higher for smokers. A f i v e -f o l d increase i n the average number of chromatid exchanges per metaphase c e l l was found for the urine of smokers (0.50 ± 0.30 chromatid exchanges per metaphase c e l l ) compared to that of non-smokers (0.10 ± 0.07 chromatid exchanges per metaphase c e l l ) . Again, these values were also s i g n i f i c a n t (P<0.001), as determined by the Mann-Whitney U test. Table 14 i l l u s t r a t e s the re l a t i o n s h i p between the number of cigarettes smoked and the observed maximum clastogenic a c t i v i t y i n the urine. Individuals consuming the same number of 76 FIGURE 17A Frequency d i s t r i b u t i o n of urinary c l a s t o g e n i c a c t i v i t y at 4.0 mg/ml c r e a t i n i n e equivalence for non-smokers ( o r c h a r d i s t s , research s t a t i o n personnel. Grand Forks and Vancouver r e s i d e n t s ) . FIGURE 17B Frequency d i s t r i b u t i o n of ur i n a r y clastogenic a c t i v i t y at 4.0 mg/ml c r e a t i n i n e equivalence for smokers (research s t a t i o n personnel and Grand Forks r e s i d e n t s ) . 77 1 0 0 -<0 3 T 3 * 60H " O c 4 0 -I 2 0 -T T 5 - r — 1 0 2 0 — 1 — 2 5 0 1 5  1 6 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 17A 3 0 1 0 0 -« 8 0 - | 3 I 6 0 - 1 •o c 4 0 -2 0 -- i r r o 1 T 6 1 0 16 20 26 —r— 3 0 % M e t a p h a s e s W i t h C h r o m a t i d A b e r r a t i o n s Figure 17B 78 FIGURE 18A I n d i v i d u a l v a r i a t i o n s i n the percentage of aberrant metaphases induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from non-smoking and smoking i n d i v i d u a l s . Each bar represents the maximum percentage of aberrant metaphases obtained over the dose range tested (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). A pipe smoker (1) i s included. FIGURE 18B I n d i v i d u a l v a r i a t i o n s i n the average frequency of chromatid exchanges per metaphase induced by urine e x t r a c t s ( f r a c t i o n A; 50% acetone eluate) from non-smoking and smoking i n d i v i d u a l s . Each bar represents the maximum number of chromatid exchanges per metaphase obtained over the dose range t e s t e d (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). A pipe smoker (1) i s included. 79 M e a n 1 S . D . 5 . 4 t 2 . 7 14.oi 6 . 0 ( 1 ) M e a n t S . D . 0 . 1 0 t 0 . 0 7 0 . 5 0 1 0 . 3 0 ( 1 ) N o n - S m o k e r s S m o k e r s Figure 18B 80 TABLE 14 RELATIONSHIP BETWEEN THE NUMBER OF CIGARETTES SMOKED AND CLASTOGENIC ACTIVITY IN URINE Subject Number of Cig a r e t t e s per Day Percent Metaphases with Chromatid Aberrations^ G10 3 2 12.0 R2 8 11.0 G13 10 1.0 G20 10 6.7 G4 12 22. 5 R4 15 17.1 R16 15 14.0 G2 15 25.5 G15 15 15.6 G16 15 6.7 R5 20 15.0 R12 20 13.0 Gl 20 13.0 G5 20 11.0 G8 20 15.8 G22 25 16.7 G17 30 21.0 Maximum value over dose range t e s t e d (1.0 to 8.0 mg/ml c r e a t i n i n e equivalence). i Pipe smoking only. 81 c i g a r e t t e s showed wide v a r i a t i o n s i n urine g e n o t o x i c i t y . Analysis of the data by Spearman rank c o r r e l a t i o n i n d i c a t e d no c o r r e l a t i o n between the average number of c i g a r e t t e s smoked per day and the frequency of aberrations (r =0.400; P>0.50). s The only pipe smoker i n the study also showed comparable l e v e l s of a c t i v i t y to c i g a r e t t e smokers, with a maximum a c t i v i t y of 12.0% metaphases with chromatid aberrations. Thus the data i n t h i s study show that c i g a r e t t e smoking i s associated with an increase i n clastogenic a c t i v i t y i n the urine and, consequently, may represent a confounding f a c t o r i n any urine a n a l y s i s . 4. A n a l y s i s of M i c r o n u c l e i i n E x f o l i a t e d Bladder C e l l s An attempt was made to analyze the e x f o l i a t e d u r o t h e l i a l c e l l s of p e s t i c i d e sprayers f o r the presence of micronuclei, an i n v i v o i n d i c a t i o n of genotoxic damage. Unfortunately, e x f o l i a t e d u r o t h e l i a l c e l l s i n the u r i n e of male subjects are not abundant, and large volumes of urine are g e n e r a l l y required to obtain an adequate number of c e l l s . Because of the s c a r c i t y of c e l l s i n the s l i d e preparations, i t was not f e a s i b l e to score f o r m i c r o n u c l e i . As a r e s u l t , no data on micronuclei were a v a i l a b l e . 5. A d d i t i o n a l Urine Parameter Measurements Mean u r i n a r y pH and c r e a t i n i n e values are depicted i n Table 15 f o r the various study groups. The mean pH of the urines ranged from 5.97 to 6.22, while mean c r e a t i n i n e concentrations ranged from 1.21 to 1.65 mg/ml. For a given group, these two parameters d i d not vary s i g n i f i c a n t l y over the periods 82 TABLE 15 MEAN URINE PARAMETER VALUES AMONG THE STUDY GROUPS Urine pH Urine C r e a t i n i n e (mg/ml) Group (Mean ± S.D.) (Mean + S.D.) Reference c o n t r o l group 6.06 ± 0.47 1.36 ± 0.43 (September 1985) Research s t a t i o n personnel: Before spraying (March 1985) 6.22 ± 0.46 1.65 + 0.37 During spraying (August 6.03 ± 0.60 1.60 ± 0.77 1985) (P>0.50)1 (P>0.50) 1 Sprayers: Phase 1: During spraying 6.14 ± 0.42 1.38 ± 0.75 (June 1984) A f t e r spraying 6.10 ± 0.39 1.41+0.54 (October 1984) (P>0.50) : (P>0.50) 1 Phase 2: Before spraying 5.97 ± 0.57 1.46 ± 0.88 (March 1985) During spraying 6.15+0.42 1.60 ± 0.55 (August 1985) (P>0.50) L (P>0.50) L Non-smokers 6.09 ± 0.51 1.50+0.56 Smokers 6.21+0.59 1.21 ± 0.82 (P>0.50)2 (P>0.50) 2 ^ S i g n i f i c a n t values f o r urine samples c o l l e c t e d during spraying and non-spraying p e r i o d s , as determined by the Mann-Whitney U-test. 2 S i g n i f i c a n t values r e l a t i v e to non-smokers, as determined by the Mann-Whitney U-test. 83 of urine sampling. No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n urinary pH and c r e a t i n i n e concentration were noted for p e s t i c i d e sprayers during periods of exposure and non-exposure. S i m i l a r l y , urinary pH and c r e a t i n i n e l e v e l s d i d not d i f f e r for smokers and non-smokers. 84 DISCUSSION In t h i s study, the urines of p e s t i c i d e sprayers and smokers were assayed f o r genotoxic a c t i v i t y to gain information on the possible genetic hazards associated with exposure to these environmental contaminants. The f o l l o w i n g chapters w i l l discuss the f i n d i n g s , i m p l i c a t i o n s , p o s s i b l e confounding f a c t o r s , and an o v e r a l l e v aluation of urine a n a l y s i s as a monitoring t o o l for assessing human exposure to environmental carcinogens and mutagens. 1. Urine C l a s t o g e n i c i t y Associated with P e s t i c i d e Exposure The current i n v e s t i g a t i o n i n d i c a t e s that i n d i v i d u a l s exposed to high concentrations of p e s t i c i d e s show s u b s t a n t i a l genotoxic a c t i v i t y i n t h e i r u r i n e s . In c o n t r a s t , pre-spraying urine samples (March 1985; phase 2) c o l l e c t e d from the same subjects demonstrated only low "baseline" l e v e l s of c l a s t o g e n i c a c t i v i t y . The mean percentage of CHO c e l l s with chromatid aberrations increased f i v e f o l d f o r the group during the peak spraying season. Moreover, the mean number of chromatid exchanges per metaphase c e l l increased by a f a c t o r of seven during t h i s p e r i o d , s t r o n g l y implying the a s s o c i a t i o n of p e s t i c i d e exposure with the high u r i n a r y clastogenic a c t i v i t y . The potency of the urine e x t r a c t s i n inducing chromatid breaks and exchanges v a r i e d between i n d i v i d u a l s . Exchange f i g u r e s were the most frequent type of chromatid a b e r r a t i o n , followed by chromatid breaks. Some e x t r a c t s were capable of inducing aberrations i n 20 to 40 percent of the examined metaphases. This high l e v e l of genotoxic damage i s comparable to that observed i n c e l l s exposed to potent clastogens. For example, comparable l e v e l s of c l a s t o g e n i c a c t i v i t y have been observed i n mammalian c e l l s exposed f o r 3 hours 85 to 2 to 4 x 10 M N-methyl-N'-nitro-N-nitrosoguanidine, 2 to 4 x 10 M -5 daunomycin, or 2 to 8 x 10 M a f l a t o x i n ( S t i c h et a l . , 1980). Genotoxicity of urine from p e s t i c i d e sprayers has not been previously reported. However, evidence of the a b i l i t y of p e s t i c i d e s to induce cyto-genetic damage i n humans i s known. Yoder et a l . (1973) noted a marked increase i n the chromatid l e s i o n s of lymphocyte c u l t u r e s prepared from i n d i v i d u a l s exposed during heavy spraying periods. The aberrations were p a r t i c u l a r l y s t r i k i n g among workers exposed p r i m a r i l y to he r b i c i d e s . Crossen et a l . (1978) and Dulout et a l . (1985) both found a s i g n i f i c a n t l y elevated incidence of si s t e r - c h r o m a t i d exchanges (another endpoint for geno-t o x i c damage) i n p e r i p h e r a l lymphocyte chromosomes of subjects o c c u p a t i o n a l l y exposed to p e s t i c i d e s . However, some of these findings have not been substantiated by other i n v e s t i g a t o r s (Rabello et a l . , 1975; Hogstedt et a l . , 1980; de Cassia Stocco et a l . , 1982; Steenland et a l . , 1985). Differences i n the degree of exposure or the use of d i f f e r e n t p e s t i c i d e s may have contri b u t e d to the negative f i n d i n g s . The high urine c l a s t o g e n i c i t y associated with p e s t i c i d e exposure was only observed i n ur i n e specimens c o l l e c t e d w i t h i n 8 hours post-spraying i n August 1985 (phase 2). Urine c o l l e c t e d 16 to 24 hours post-spraying during both June and J u l y 1984 (phase 1) demonstrated no s i g n i f i c a n t increase i n cl a s t o g e n i c a c t i v i t y over c o n t r o l b a s e l i n e values. Several explanations may account f o r the low a c t i v i t y observed during phase 1. A glance at Tables 8 and 9 shows that a l l but two i n d i v i d u a l s sprayed at l e a s t one p e s t i c i d e with known genotoxic a c t i v i t y in v i t r o . A reason for the low urinary c l a s t o g e n i c a c t i v i t y observed among the , o r c h a r d i s t s i n phase 1 may be that these p e s t i c i d e s are not genotoxic _in v i v o . The metabolism of an i n t a c t organism may convert 86 these genotoxic chemicals i n t o harmless metabolites. However, i t i s worth p o i n t i n g out that the p e s t i c i d e s used during phases 1 and 2 d i d not d i f f e r s u b s t a n t i a l l y . Another explanation for the low a c t i v i t y may be that the o r c h a r d i s t s were a c t u a l l y unexposed to any of the p e s t i c i d e s sprayed, or that the l e v e l s of p e s t i c i d e s to which they were exposed were too low to be detected. This explanation must be considered a strong p o s s i b i l i t y , even though the types of p r o t e c t i v e gear worn and the average t o t a l number of spraying hours during both phases were not c o n t r a s t i n g l y d i f f e r e n t . An a l t e r n a t i v e explanation i s that excretion of p e s t i c i d e s and t h e i r metabolites i n t o the urine i s a r a p i d process, o c c u r r i n g w i t h i n a matter of hours a f t e r exposure. By 16 to 24 hours post-exposure, the l e v e l of metabolites i n the urine may be very low, p o s s i b l y beyond the s e n s i t i v i t y l i m i t s of the chromosome aberration assay. A few i n v e s t i g a t o r s have r e c e n t l y reported t i m e - r e l a t e d increases i n mutagenic (not clastogenic) a c t i v i t y c o i n c i d i n g with mutagen exposure. For example, u r i n a r y mutagenic a c t i v i t y peaked at 2 to 6 hours i n i n d i v i d u a l s consuming a meal of f r i e d beef (Sousa et a l . , 1985). S i e b e r t and Simon (1973) found peaks of g e n e t i c a l l y a c t i v e metabolites appearing a f t e r 4 to 6 hours i n the urine of p a t i e n t s t r e a t e d with the drug cyclophosphamide. According to some i n v e s t i g a t o r s , p e s t i c i d e e x c r e t i o n i n t o the urine may be as r a p i d . High concentrations of phosalone, an i n s e c t i c i d e commonly sprayed during phase 2, and i t s metabolites were found i n human ur i n e 4 to 5 hours a f t e r spraying' (Drevenkar et a l . , 1979). E x c r e t i o n of paranitrophenol and a l k y l phosphate f o l l o w i n g i n g e s t i o n of methyl or e t h y l parathion by human subjects occurred w i t h i n 4 to 8 hours (Morgan et a l . , 1977). Carpenter e t a l . (1961) demonstrated that c a r b a r y l administered o r a l l y to r a t s 87 was metabolized r a p i d l y and appeared i n the urine w i t h i n 24 hours. On the basis of these reports and on the fi n d i n g s of high clastogenic a c t i v i t y i n the u r i n e w i t h i n 8 hours of p e s t i c i d e exposure (phase 2), i t can be speculated that the maximum excretion rate of p e s t i c i d e s generally occurs w i t h i n hours of exposure. This i n d i c a t e s the importance of c o l l e c t i n g urine at a pe r i o d when the metabolite ex c r e t i o n rate i s high or optimal. I n t e r i n d i v i d u a l v a r i a t i o n s were found i n the l e v e l s of clastogenic a c t i v i t y during the spraying period i n phase 2 (August 1 9 8 5 u r i n e s ) . Some i n d i v i d u a l s had extremely high a c t i v i t y , whereas a few showed moderate to low l e v e l s of a c t i v i t y . These v a r i a t i o n s may r e f l e c t d i f f e r e n c e s i n absorption, d i s t r i b u t i o n , metabolism and excr e t i o n of p e s t i c i d e s between i n d i v i d u a l s . A l t e r n a t i v e l y , they may also be an i n d i c a t i o n of the v a r i a t i o n s i n personal working habits and hygiene. For example, occupational exposure to p e s t i c i d e s may occur i n a v a r i e t y of s i t u a t i o n s . According to Coutts ( 1 9 7 9 ) , contact with p e s t i c i d e s may occur during ( 1 ) the formulation process, (2) the tr a n s p o r t of p e s t i c i d e s to the s i t e of a p p l i c a t i o n , (3) the t r a n s f e r of p e s t i c i d e s from the storage container to the a p p l i c a t i o n equipment, (4) the a p p l i c a t i o n process, ( 5 ) the d r i f t of p e s t i c i d e s beyond the a p p l i c a t i o n s i t e , and ( 6 ) the cleaning of a p p l i c a t i o n equipment. Careless p r a c t i c e s by sprayers during one of the above processes may have r e s u l t e d i n s i g n i f i c a n t exposure to the p e s t i c i d e s . Studies have shown that exposure i s greatest during the mixing and handling of concentrated p e s t i c i d e s p r i o r to the a p p l i c a t i o n process (Coutts, 1 9 7 9 ) . The route of exposure i s also an important f a c t o r . Of the three most common routes (dermal, i n h a l a t i o n and i n g e s t i o n ) , dermal exposure represents the major one by which p e s t i c i d e s are absorbed (Wolfe et a l . , 1 9 6 7 ; Coutts, 1 9 7 9 ) . A study of p e s t i c i d e a p p l i c a t o r s showed that 8 8 r e s p i r a t o r y exposure was only 0.02 to 5.8% of the t o t a l (dermal plus r e s p i r a t o r y ) exposure (Wolfe et a l . , 1967). A major reason for low r e s p i r a t o r y exposure values i s that spray drops, ranging from 50 to 400 ym i n diameter, are u s u a l l y larger than the 10 pm diameter required to reach the lungs (Coutts, 1979). According to recommended g u i d e l i n e s , each o r c h a r d i s t i n t h i s study was required to wear standardized p r o t e c t i v e c l o t h i n g c o n s i s t i n g of h a l f - f a c e s h i e l d s , gloves, rubber s u i t s and boots, while spraying. Despite these p r o t e c t i v e measures, high urinary c l a s t o g e n i c a c t i v i t y was s t i l l very evident. Since each sprayer was responsible f o r mixing h i s own p e s t i c i d e concentrates and d i l u t i n g them with the appropriate s o l v e n t s , i t i s p o s s i b l e that these workers, i n s p i t e of p r o t e c t i v e gear, may have ingested, inhaled or absorbed p e s t i c i d e s while working i n dusty atmospheres. The i n e f f e c t i v e n e s s of p r o t e c t i v e equipment to completely impede p e s t i c i d e exposure has been reported by many i n v e s t i g a t o r s . F r a n k l i n et a l . (1981), i n a study of a p p l i c a t o r s from the Okanagan V a l l e y , found small amounts of p e s t i c i d e s on the chest, shoulders and arms of the sprayers even when o v e r a l l s were worn. In the same study, a s i g n i f i c a n t amount (25 to 47%) of p e s t i c i d e residues on the outside of the c l o t h i n g were found on the s k i n surface. Brokopp et a l . (1981) noted that u r i n a r y a l k y l phosphate metabolites from the use of organophosphorous compounds were elevated even i n i n d i v i d u a l s wearing p r o t e c t i v e c l o t h i n g . Since exposure to p e s t i c i d e s i s p r i m a r i l y dermal, f a c t o r s such as a c c i d e n t a l s p i l l a g e or the penetration of p e s t i c i d e s through c l o t h i n g may r e s u l t i n s i g n i f i c a n t exposure. The presence of abrasions or p e r s p i r a t i o n on the s k i n may f u r t h e r contribute to the absorption of p e s t i c i d e s . 89 Dermal exposure may also be increased i f there i s a lack of compliance by the sprayers to wear p r o t e c t i v e c l o t h i n g . Respiratory masks, boots and rubber s u i t s are qui t e bulky and uncomfortable. During hot weather sprayers may d i s c a r d t h e i r p r o t e c t i v e c l o t h i n g , thus experiencing a higher dermal exposure. As i n d i c a t e d i n Appendix 4M, many sprayers neglected to wear f u l l p r o t e c t i v e gear while applying p e s t i c i d e s . In a d d i t i o n , high humidity and ambient temperatures, which are common i n the Okanagan V a l l e y , have been found to increase the dermal absorption of p e s t i c i d e s (Wojeck et a l . , 1981). The importance of s u f f i c i e n t p r o t e c t i o n was i l l u s t r a t e d by Crossen et a l . (1978) who noted higher rates of sister-chromatid exchanges i n the lymphocytes of inadequately protected sprayers. Other v a r i a b l e s may fur t h e r i n f l u e n c e the degree of exposure of each i n d i v i d u a l . For instance, the use of orchard a i r b l a s t sprayers, which d i r e c t p e s t i c i d e s upward i n t o the a i r , may r e s u l t i n greater exposure than boom-type sprayers which d i r e c t the spray downwards. Wolfe et a l . (1967) found t h a t the a p p l i c a t i o n of parathion to orchards by a i r b l a s t sprayers was twice as hazardous to the sprayers as the a p p l i c a t i o n of t h i s chemical on crops with a boom duster. In our study, a i r b l a s t sprayers were more commonly used than boom dusters and, as a r e s u l t , exposure of sprayers to p e s t i c i d e s would be expected to be greater. In a d d i t i o n to the type of spray equipment used by the a p p l i c a t o r s , wind may al s o be an important f a c t o r . Windy days may be expected t o increase the d r i f t and therefore the exposure of p e s t i c i d e s from the a p p l i c a t i o n s i t e . To our knowledge, spray operations were halted during strong windy days. The multitude of fa c t o r s a f f e c t i n g exposure makes i t d i f f i c u l t to l a b e l one v a r i a b l e as responsible f o r the high urine c l a s t o g e n i c i t y . Since the 90 p r o t e c t i v e equipment worn by each i n d i v i d u a l was not standardized, the degree of exposure was expected to vary among i n d i v i d u a l s . Furthermore, because most sprayers applied two or more p e s t i c i d e s during the course of a day, i t i s d i f f i c u l t to determine with c e r t a i n t y the s p e c i f i c p e s t i c i d e s responsible for urine g e n o t o x i c i t y . Because no chemical a n a l y s i s was performed i n t h i s study, the nature of the clastogens i n the urine i s not known. To i s o l a t e or c h a r a c t e r i z e any of the metabolites would prove to be a d i f f i c u l t task as a r e s u l t of the complexity of the urine c o n s t i t u e n t s . Comutagens, antimutagens, compounds a f f e c t i n g clastogen s o l u b i l i t y , and a host of other f a c t o r s could increase, suppress, or completely mask the a c t i o n of the clastogens (Stich et a l . , 1980). Since no e x t e r n a l source of metabolic a c t i v a t i o n was used, the geno-t o x i c substances are presumed to be d i r e c t - a c t i n g . Moreover, since the genotoxic materials were able to bind to a C18 reversed-phase column, one would expect them to be r e l a t i v e l y hydrophobic. The most hydrophobic compounds would be a n t i c i p a t e d to penetrate c e l l u l a r membranes without d i f f i c u l t y . Without knowing the s p e c i f i c nature of the u r i n a r y clastogens, one can only speculate on the cause of the high g e n o t o x i c i t y observed i n the urine of sprayers. P e s t i c i d e metabolites i n the urine may themselves be d i r e c t l y responsible f o r the increase i n g e n o t o x i c i t y . Exposure to organophosphate or carbamate i n s e c t i c i d e s commonly r e s u l t s i n the r a p i d appearance of a l k y l phosphate and phenolic metabolites i n the urine (Shafik, 1980). While l i t t l e i s known about the c l a s t o g e n i c i t y of a l k y l phosphate metabolites, phenolic compounds have been demonstrated to induce clastogenic damage i n CHO c e l l s ( S t i c h et a l . , 1981). Conversely, p e s t i c i d e s may enhance clastogenic a c t i v i t y 91 in the urine by an in d i r e c t mode of action. Certain agents, although not themselves potent mutagens, have been shown to synergize the effects of mutagenic agents. For example, 2-aminoanthracene, in conjunction with the urine of smokers, exhibited a synergistic mutagenic effect (Hannan et a l . , 1981). The mutagenic a c t i v i t y of 2-acetylaminofluorene was potentiated when combined with urine from either smokers or non-smokers (Mortelmans et a l . , 1981). Pesticides and t h e i r metabolites may also act in t h i s manner since many are known enzyme inducers. Organochlorine i n s e c t i c i d e s , for instance, are inducers of the l i v e r microsome enzymes, while organophosphate i n s e c t i c i d e s i n h i b i t these same enzymes (Hodgson et a l . , 1980). The production of a reactive intermediate or an inactive compound w i l l depend on the balance of a c t i v a t i n g and detoxifying enzymes available i n the exposed tissue (Brooks, 1980), and t h i s i n turn may be influenced by the degree of exposure to pe s t i c i d e s . In addition, pesticides have been shown to modify the action of drugs and chemicals by competing for the same conjugating enzyme systems. Pesticides metabolized by glutathione conjugation may synergize the t o x i c i t y of drugs such as acetaminophen by exhausting the available supply of l i v e r glutathione (Doroiigh, 1983). Consequently, without a d e t o x i f i c a t i o n step, these drugs become toxic for the human body. As indicated i n Table 10, many of the pesticides used by the sprayers are genotoxic i n i n v i t r o tests. In f a c t , some are known to possess carcinogenic properties i n animals. Dimethoate, for example, has been shown to induce carcinomas and sarcomas i n Osborne-Mendel rats (Reuber, 1984). Captan, a fungicide, i s capable of increasing the rate of polypoid carcinoma formation i n the duodenum of mice (Cipriano, 1980). Many of the l i s t e d pesticides are also organophosphorous compounds. Organophosphorous pesticides 92 are noted for t h e i r a b i l i t y to react with DNA by t r a n s a l k y l a t i o n , which may e x p l a i n why many organophosphates possess mutagenic a c t i v i t y Ln vivo (Epstein and Legator, 1 9 7 1 ) . Of the three major classes of p e s t i c i d e s (organophosphates, organochlorines and carbamates), the majority of the c a r c i n o g e n i c i t y l i t e r a t u r e has centered on organochlorine compounds. At present, l i t t l e i s known about the carcinogenic p r o p e r t i e s of carbamates and organophosphorous chemicals. The a p p l i c a t o r s i n t h i s study were considered to be h e a v i l y exposed to p e s t i c i d e s since each i n d i v i d u a l was i n the f o c a l point of the spraying s i t e . However, the p o t e n t i a l f o r exposure may also e x i s t f o r i n d i v i d u a l s r e s i d i n g near the area of a p p l i c a t i o n . A study by P i l i n s k a y a and L'Vova ( 1 9 7 9 ) showed t h a t s t r u c t u r a l chromosome aberrations i n the lymphocytes of i n d i v i d u a l s l i v i n g i n high p e s t i c i d e usage areas were s i g n i f i c a n t l y increased compared to those of i n d i v i d u a l s r e s i d i n g i n areas where the c e n t r a l i z e d use of p e s t i c i d e s was l i m i t e d . In our study, with the exception of two i n d i v i d u a l s who sprayed p e s t i c i d e s , no d i f f e r e n c e s i n the u r i n a r y c l a s t o g e n i c a c t i v i t y were observed f o r the a g r i c u l t u r a l research s t a t i o n personnel during the pre-spraying and spraying periods i n March and August 1 9 8 5 , r e s p e c t i v e l y . During both periods, the clastogenic a c t i v i t y of the urine was w i t h i n the l i m i t s found f o r that of the reference c o n t r o l group (non-smoking re s i d e n t s of Vancouver and Grand Forks). The r e s u l t may i n d i c a t e that the exposure l e v e l s of the a g r i c u l t u r a l research s t a t i o n workers may be i n s u f f i c i e n t to e l i c i t u r i n a r y c l a s t o g e n i c a c t i v i t y . The two workers who d i d spray i n August showed s i g n i f i c a n t increases over pre-spraying periods, s t r o n g l y suggesting an a s s o c i a t i o n between high l e v e l s of p e s t i c i d e exposure and c l a s t o g e n i c a c t i v i t y i n the urine. Therefore, based on the high a c t i v i t y observed i n the 93 21 p e s t i c i d e a p p l i c a t o r s (2 a g r i c u l t u r a l research s t a t i o n sprayers plus 19 Okanagan orchardists) and the low a c t i v i t y found i n the urine of non-smoking, non-spraying a g r i c u l t u r a l research s t a t i o n personnel during the phase 2 spraying p e r i o d , i t may be surmised that the increase i n cla s t o g e n i c a c t i v i t y was r e l a t e d to p e s t i c i d e exposure rather than to exposure r e l a t e d to the general environment (e.g., d r i n k i n g water). I t should be emphasized that the t e s t e d doses of organic m a t e r i a l i n the urine e x t r a c t s are not very d i f f e r e n t from those present i n urine i n the bladder. C r e a t i n i n e concentrations i n the urine t y p i c a l l y ranged from 0.5 to 2.5 mg/ml. Many of the urine e x t r a c t s obtained from the o r c h a r d i s t s during the spraying period were a c t i v e i n v i t r o at 4.0 mg c r e a t i n i n e equivalence per m i l l i l i t r e of t i s s u e c u l t u r e medium or l e s s . Thus the concentrations of genotoxic m a t e r i a l s used i n the assay are reasonably comparable to the concentrations to which bladder mucosal c e l l s may be exposed. No r e l a t i o n s h i p was observed between ur i n a r y pH and exposure to e i t h e r p e s t i c i d e s or c i g a r e t t e smoke. The u r i n a r y pH f o r most i n d i v i d u a l s was ge n e r a l l y s l i g h t l y a c i d i c , ranging from pH 5.1 to 7.6. The r o l e of pH has, i n the past, been found to be important. Some i n v e s t i g a t o r s have observed that an a c i d i c u r i n a r y pH increases the carcinogenic potency of some bladder carcinogens (e.g., 8-naphthylamine) i n laboratory animals (Willems and de Raat, 1985). In a d d i t i o n , a c i d u r i a has also been demonstrated to promote the d e t e r i o r a t i o n of the surface mucous coat of the bladder epithelium ( B a l i s h et a l . , 1982). A breakdown of t h i s b a r r i e r would enhance the i n t e r -a c t i o n of urine and i t s genotoxic metabolites with the d i v i d i n g basal c e l l s of the epithelium. 94 The urine extracts in t h i s study were assayed without the use of deconjugating enzymes such as B-glucuronidase and aryl sulfatase. However, Legator et a l . (1978) have reported that CHO c e l l s contain s u f f i c i e n t enzymes to cleave conjugated compounds. The use of deconjugation mechanisms i s important since many reactive chemicals are detoxified by conjugation with mercapturic acids, glucuronic acids and sulfates (Legator et a l . , 1982). Without deconjugation, some clastogenic compounds may be d i f f i c u l t to detect in the urine. Nevertheless, there i s the counter-argument that the use of deconjugating enzymes would permit one to test for molecules _in v i t r o that were probably b i o l o g i c a l l y inactive ^ n vivo. In addition, the use of deconjugation enzymes may r e s u l t i n the breakdown of genotoxic materials i n the urine (Connor et a l . , 1983). Since only one sample was co l l e c t e d from each applicator during the spraying period in August 1985, i t was d i f f i c u l t to evaluate the day-to-day variations i n an in d i v i d u a l with respect to urinary excretion of clastogenic materials. Since sprayers use many d i f f e r e n t pesticides each day, one would expect considerable variations i n clastogenic a c t i v i t y from day to day due to differences i n the pattern of metabolic transformation and excretion as well as differences i n the quantity of metabolites excreted. As w i l l be discussed i n a l a t e r chapter, urine t o x i c i t y may also a f f e c t the reproduci-b i l i t y of clastogenic a c t i v i t y i n repeated urine samples from the same in d i v i d u a l . Even though clastogenic agents were demonstrated i n the urine of sprayers, two important issues must be raised. F i r s t l y , CHO c e l l s do not possess the -protective mechanisms that are present i n the intact organism, nor do they have the same metabolic c a p a b i l i t i e s as an intact organism. As a r e s u l t , the 95 question must be brought up as to whether cultured c e l l s may be more susceptible to genetic damage than dividing c e l l s present in the human body. Secondly, the re s u l t s do not reveal whether genotoxic chemicals in the urine are able to penetrate the mucosa of the urinary t r a c t to i n f l i c t genetic damage on the dividing basal c e l l s . In an e f f o r t to answer these two questions,'the micronucleus test (Stich et a l . , 1983) was attempted on e x f o l i a t e d u r o t h e l i a l c e l l s from the sprayers. Micronuclei represent acentric chromosome and chromatid fragments that arise from chromosome and chromatid damage by genotoxic substances to dividing basal c e l l s of the epithelium (Stich et a l . , 1983). Unfortunately, due to the scarcity of the u r o t h e l i a l c e l l s i n the urine, no data were obtained. However, based on previous findings of elevated micronuclei frequencies i n the urine of smokers (Stich and Rosin, 1984), one may speculate that there may be an increase of micronucleated c e l l s i n the urine of pesticide sprayers. This would indicate the a b i l i t y of clastogens i n the urine to cause genetic damage i n vivo. The discussion of genotoxic damage i n v i t r o or i n vivo would not be complete without mentioning i t s relevance to environmental carcinogenesis. Chromosome aberrations may have d i f f e r e n t consequences depending on both the c e l l type involved and the extent of damage. C e l l s which are severely damaged, as indicated by a large number of chromosome exchanges and breaks, w i l l usually die due to the loss of v i t a l genetic information (Dean and Danford, 1984). However, i f the c e l l s are able to survive the damage, a tumour (involving somatic c e l l s ) or a mutation (involving germinal c e l l s ) may be the end r e s u l t (Palmer et a l . , 1972). Currently, the link between chromosome aberrations and cancer i s strong. There i s evidence to support the 96 b e l i e f that d e l e t i o n s and t r a n s p o s i t i o n s of chromosomal ma t e r i a l may be a c r u c i a l step i n the i n i t i a t i o n of tumours. For instance, many carcinogens (e.g., i o n i z i n g r a d i a t i o n ) are potent inducers of chromosome damage (Ishidate and Odashima, 1977; H o l l s t e i n et a l . , 1979). S p e c i f i c chromosomal t r a n s l o c a t i o n s and d e l e t i o n s are also associated with p a r t i c u l a r types of cancers (Yunis, 1983; Parmiter et a l . , 1986), i m p l i c a t i n g chromosome damage as an important f a c t o r i n some types of human cancers. A c l a s s i c example are i n d i v i d u a l s with the hereditary d i s o r d e r , Bloom's syndrome. Characterized by growth and immune d e f i c i e n c i e s , these p a t i e n t s show high frequencies of chromosome aberrations and exchanges (Cairns, 1983). More s i g n i f i c a n t l y , an elevated incidence of a v a r i e t y of cancers i s observed among t h i s group (Cairns, 1983). In view of the evidence that chromosome aberrations may be an important step i n carcinogenesis, the f i n d i n g of clastogens i n the urine of p e s t i c i d e sprayers may reveal the p o t e n t i a l exposure of human c e l l s to environmental carcinogens. Thus these i n d i v i d u a l s may be at carcinogenic r i s k . There i s evidence that some p e s t i c i d e s may be harmful to man. Axelson and Sundell (1974) found an elevated incidence of s o f t - t i s s u e sarcomas i n i n d i v i d u a l s exposed to phenoxy a c i d h e r b i c i d e s . An increased r i s k of lung cancer was shown i n pesticide-exposed male a g r i c u l t u r a l workers ( B a r t h e l , 1981). C u r r e n t l y , epidemiological data show that a g r i c u l t u r a l workers from the Okanagan V a l l e y do not demonstrate any p e s t i c i d e — r e l a t e d increase i n the incidence rates of cancer. However, p e s t i c i d e use, p a r t i c u l a r l y organo-phosphates and carbamates, has g r e a t l y increased only over the l a s t two decades, and i t may be another 20 years (owing to the lag period) before any evidence of carcinogenesis becomes apparent. The f i n d i n g of g e n o t o x i c i t y i n 97 the urine of sprayers c e r t a i n l y i l l u s t r a t e s the need to reduce p e s t i c i d e exposure, h o p e f u l l y , before adverse e f f e c t s are observed. In summary, the present i n v e s t i g a t i o n showed an increase i n urine g e n o t o x i c i t y associated with p e s t i c i d e exposure. This a s s o c i a t i o n was evident only for urine c o l l e c t e d w i t h i n 8 hours of p e s t i c i d e usage, implying that p e s t i c i d e e x c r e t i o n i n the urine may be rapi d ( i . e . , w i t h i n hours). Differences i n personal work h a b i t s , hygiene and-metabolism may have contributed to the i n t e r i n d i v i d u a l v a r i a t i o n s i n clastogenic a c t i v i t y . The high urinary c l a s t o g e n i c a c t i v i t y may be i n t e r p r e t e d i n one of two ways. The r a p i d clearance of clastogens from the body may be taken as evidence of diminished r i s k . On the other hand, such f a c i l e e x c r e t i o n may mean that organs such as the bladder w i l l be exposed to high concentrations of genotoxic m a t e r i a l (Barnes and Weisburger, 1984). 2. Confounding Factors A f f e c t i n g the Urine C l a s t o g e n i c i t y Assay In part one of the research p r o j e c t , the e f f e c t s of p e s t i c i d e exposure on urine c l a s t o g e n i c i t y were explored without consideration of confounding f a c t o r s . The second pa r t of the i n v e s t i g a t i o n examined the f a c t o r s which may confound the assay f o r g e n o t o x i c i t y i n the urine. 2.1 Smoking and Genotoxic A c t i v i t y i n the Urine The urine of smokers was analyzed i n t h i s i n v e s t i g a t i o n for two reasons: (1) to demonstrate c i g a r e t t e smoking as a confounding f a c t o r to the urine g e n o t o x i c i t y assay, and (2) to use the smoker population as a p o s i t i v e c o n t r o l group for the eva l u a t i o n of p e s t i c i d e exposure on urine c l a s t o g e n i c i t y . Epidemiological data have c l e a r l y shown a strong a s s o c i a t i o n between c i g a r e t t e smoking and various human cancers, such as b r o n c h i a l carcinoma 98 (Notani and Sanghvi, 1974; Doll and Peto, 1976; Jussawalla and Javin, 1979) and cancers of the lower urinary t r a c t (Fraumeni, 1968; Cole et a l . , 1971). The cancer r i s k was found to be a function of the number and types of cigarettes smoked (Doll and Peto, 1978). Furthermore, the frequencies of s t r u c t u r a l chromosome aberrations in the blood lymphocytes of smokers were s i g n i f i c a n t l y higher than those found in non-smokers (Hopkins and Evans, 1979; Obe et a l . , 1982). The incidence of sister-chromatid exchanges (a sensitive indicator of DNA damage) was 50% greater in heavy smokers compared to non-smokers (Husgafvel-Pursiainen et a l . , 1980; Vijayalaxmi and Evans, 1982). These reports serve to i l l u s t r a t e some of the detrimental effects associated with cigarette smoking. Yamasaki and Ames (1977) f i r s t demonstrated that urine concentrates from cigarette smokers induced frameshift mutations in h i s t i d i n e - d e f i c i e n t Salmonella typhimurium strains. This r e s u l t has since been confirmed by others s p e c i f i c a l l y investigating the e f f e c t s of smoking (Aeschbacher and Chappuis, 1981; Putzrath et a l . , 1981; Caderni and Dolara, 1983). Studies of smoking e f f e c t s on urine genotoxicity have also been done on mammalian c e l l s . Guerrero et a l . (1979) observed that the urine of smokers induces a higher frequency of sister-chromatid exchanges i n human d i p l o i d f i b r o b l a s t s than the urine of non-smokers. Beek et a l . (1982) found dose-dependent increases i n the frequency of sister-chromatid exchanges in CHO c e l l s exposed to urine extracts. However, i n t h i s study, smokers did not show an o v e r a l l increase i n a c t i v i t y . Recently, in our laboratory, Dunn and Curtis (1985) demonstrated the presence of clastogenic agents i n the urine of smokers using cultured CHO c e l l s . However, i n t h e i r study, urine concentrates were frac-tionated into 3 portions, each with a d i f f e r e n t degree of hydrophobicity. 99 With the methodology used i n t h i s project, a l l the organic material i n the urine was combined into a single composite extract. Using the extraction procedure described e a r l i e r (section 4.4.2b of Materials and Methods), the urine of smokers was found to be s i g n i f i c a n t l y more clastogenic than that of non-smokers, as indicated by the high frequencies of chromosome aberrations and the high extent of damage per metaphase. Furthermore, the clastogenic a c t i v i t y in the urine appeared reasonably reproducible with repeated urine samplings, since smokers demonstrated variable but consistently high levels of a c t i v i t y compared to non-smokers. No li n e a r c o r r e l a t i o n was found between the number of cigarettes smoked per day and the urinary clastogenic a c t i v i t y for the group. Wide v a r i a b i l i t y i n a c t i v i t y was observed among individuals smoking the same number of cigarettes per day. The absence of a dose-response relationship has also been reported by others (Recio et a l . , 1982; Kado et a l . , 1985). These investigators attributed the v a r i a b i l i t y to a number of factors, including fluctuations i n the patterns of absorption of cigarette smoke, differences i n metabolism and excretion, and variations in smoking habits such as the depth and frequency of smoke inhalation. Although no dose-response was observed for the group of smokers i n these studies, a linear dose-response was detected by Recio et a l . (1982) i n an individual who smoked an increasing number of cigarettes each day. Another factor which may explain the v a r i a -b i l i t y of clastogenic a c t i v i t y may be the tar content of the cigarettes. Smokers consuming low-tar cigarettes have exhibited weaker urine mutagenicity than those who use high-tar cigarettes (Yamasaki and Ames, 1977; Recio et a l . , 1982; Kriebel et a l . , 1985). In fa c t , i t has been reported that individuals who use low-tar cigarettes have less of a cancer risk than smokers 100 of high-tar c i g a r e t t e s (Recio et a l . , 1982). The r i s k for cancer i s a l s o much lower for c i g a r and pipe smokers (Reif, 1981) than for c i g a r e t t e smokers. The only pipe smoker i n our study (subject G10) showed only moderate c l a s t o -genic a c t i v i t y compared to the other c i g a r e t t e smokers. Therefore the low a s s o c i a t i o n between the amount of t a r i n c i g a r e t t e s and cancer r i s k may be r e f l e c t e d by low genotoxic a c t i v i t y i n the urine (Recio et a l . , 1982). In our study, the t a r content of the c i g a r e t t e s was not recorded. The nature of the clastogenic m a t e r i a l s i n the urine of smokers has yet to be determined. Cigarette smoke i s a h i g h l y complex mixture of chemicals with over 3000 con s t i t u e n t s (Falck, 1982). Included are suspected carcinogens, cocarcinogens, promoters and mutagens (Falck, 1982). Cigarette smoke condensate possesses t u m o u r - i n i t i a t i n g a c t i v i t y i n rodents (Wynder and Hoffmann, 1968) and i s mutagenic i n the Ames Salmonella t e s t with metabolic a c t i v a t i o n (Hutton and Hackney, 1975). Unfortunately, the components i n c i g a r e t t e smoke c o n t r i b u t i n g to the induction of lung and other cancers remain unknown (Sato et a l . , 1977). Only a few studies have attempted to i d e n t i f y the a c t u a l chemical metabolites i n the urine of c i g a r e t t e smokers. Putzrath et a l . (1981) reported the mutagenicity of many non-polar f r a c t i o n s of urine of c i g a r e t t e smokers, suggesting the a c t i o n of a number of chemicals. Connor et a l . (1983) found trace amounts of the bladder carcinogen, 2-aminonaphthalene, and a considerable amount of i t s metabolite, 2-amino-7-naphthol, i n the urine of a smoker. Dunn and C u r t i s (1985) provided i n d i r e c t evidence that phenolic compounds present i n the urine of c i g a r e t t e smokers (and coffee drinkers) may be p a r t l y responsible for the g e n o t o x i c i t y . Phenolic compounds themselves are c l a s t o g e n i c ( S t i c h et a l . , 1981) , but they may also autooxidize to form a c t i v e oxygen species such as hydrogen peroxide and superoxide (Hanham et a l . , 101 1983; Dunn and C u r t i s , 1985). Dunn and C u r t i s (1985) proposed that the ur i n a r y c l a s t o g e n i c a c t i v i t y i n smokers may be mediated by these a c t i v e oxygen species based on two f i n d i n g s : (1) the reduction of clastogenic a c t i v i t y by the a d d i t i o n of catalase and superoxide dismutase, and (2) the a b i l i t y of the urine e x t r a c t s to produce hydrogen peroxide at a l k a l i n e pH. From our study, i t can only be gathered that the clastogens were r e l a t i v e l y hydrophobic (since they bound to the reversed-phase column) and that they were d i r e c t - a c t i n g since no metabolic a c t i v a t i o n in v i t r o was required. The f i n d i n g of d i r e c t - a c t i n g genotoxic m a t e r i a l s i n the urine of smokers i s i n contrast to the r e s u l t s obtained i n the Ames Salmonella t e s t where genotoxic responses were dependent on metabolic a c t i v a t i o n i n v i t r o (Yamasaki and Ames, 1977; K r i e b e l et a l . , 1983; Falck et a l . , 1982). This may i n d i c a t e the presence of both d i r e c t and i n d i r e c t ( i . e . , r e q u i r i n g metabolic a c t i v a t i o n ) a c t i n g genotoxic compounds, f u r t h e r i l l u s t r a t i n g the complex nature of the urine of smokers. Since the smokers i n our study used c i g a r e t t e s over the course of the day, the timing of urine sampling was not as c r i t i c a l as for that of the uri n e o f sprayers. Several i n v e s t i g a t o r s have examined the k i n e t i c s of mutagen e x c r e t i o n i n the urine of smokers. Kado et a l . (1985) discovered that peak mutagenic a c t i v i t y appeared 4 to 5 hours a f t e r the beginning of smoking, with a c t i v i t y g r adually decreasing to pre-smoking baseline l e v e l s i n approximately 12 to 18 hours a f t e r c e s s a t i o n of smoking. Kobayashi and Hayatsu (1984) suggested that the e x c r e t i o n of genotoxic materials i n t o the urine may be an acute r e a c t i o n and not a chronic one. In t h e i r i n v e s t i g a t i o n , the urine mutagenicity of ex-smokers was comparable to the l e v e l s of non-smokers. The r a p i d clearance of genotoxic substances i n the urine of smokers 102 i s s i m i l a r to that of p e s t i c i d e sprayers. I n t e r e s t i n g l y , the h a l f - l i f e f o r the e l i m i n a t i o n of phenol i n the urine has been reported to be 4 to 8 hours (Hunter, 1968), which i s close to the value for peak genotoxic a c t i v i t y reported by Kado et a l . (1985). The e f f e c t of urine of smokers on the bladder mucosal c e l l s has been analyzed by S t i c h and Rosin (1983). Using micronuclei a n a l y s i s , the percentage of e x f o l i a t e d micronucleated u r o t h e l i a l c e l l s of smokers and alcohol d r i n k e r s was t e n f o l d higher than non-smoker c o n t r o l values. This high frequency of micronucleated c e l l s strongly emphasizes the damaging p o t e n t i a l of the urine of smokers to the urinary bladder. This may perhaps e x p l a i n why bladder cancer i s second only i n incidence to lung cancer among smokers i n Western cou n t r i e s (Cole et a l . , 1971). The appearance of genotoxic m a t e r i a l s i n the urine of smokers i n d i c a t e s that c i g a r e t t e smoking i s d e f i n i t e l y a confounding f a c t o r i n any assay f o r urine g e n o t o x i c i t y . The l e v e l of a c t i v i t y found for the smokers was not s i g n i f i c a n t l y d i f f e r e n t from that of p e s t i c i d e sprayers. Urine c l a s t o -g e n i c i t y r e l a t e d to smoking has made i t d i f f i c u l t to assay urine for geno-t o x i c i t y r e l a t e d to occupational exposure. Several p u b l i c a t i o n s have suggested that c i g a r e t t e smoking may act s y n e r g i s t i c a l l y with other environ-mental compounds to enhance urine mutagenicity (Wheeler et a l . , 1981; Rueff et a l . , 1982; C r e b e l l i et a l . , 1985; Heussner et a l . , 1985). Falck et a l . (1980) found that the urine of rubber in d u s t r y workers who smoked was more mutagenic than urine of e i t h e r non-smoking workers or smoking non-workers. Hannan et a l . (1981) demonstrated the s y n e r g i s t i c a c t i o n of the urine of smokers on mutagenicity when combined with the carcinogen 2-aminoanthracene. The most l o g i c a l explanation for the synergism between smoking and occupational 103 exposure on urine mutagenicity i s the enzymatic induction that occurs with c i g a r e t t e smoking (Jusko, 1979; Conney, 1982). Camus et a l . (1984) showed that pretreatment of enzyme inducers i n carcinogen-administered mice may increase urine mutagenicity l e v e l s . The above studies a l l i l l u s t r a t e the e f f e c t s of a c t i v e smoking. However, the passive i n h a l a t i o n of c i g a r e t t e smoke may very w e l l i n t e r f e r e with urine studies since the former i s such a widespread environmental contaminant. Findings of increased urine mutagenicity r e l a t e d to passive smoking have been reported (Yamasaki and Ames, 1977; Bos et a l . , 1983; Sorsa et a l . , 1985). Bos et a l . (1983) observed that the increase i n urine mutagenicity i n passive smokers was about 4% of that found for a c t i v e smokers. Epidemiological evidence has already shown the h e a l t h hazards of passive smoking, since there i s an increased r i s k of lung cancer among non-smokers exposed to the tobacco smoke of t h e i r smoking spouses ( G a r f i n k e l , 1981; Hirayama, 1981; Trichopoulos et a l . , 1981). C e r t a i n l y , these r e s u l t s i l l u s t r a t e the importance of r e s t r i c t i n g urine studies to non-smokers when attempting to demonstrate occupational exposure to environmental chemicals other than c i g a r e t t e smoke. Smokers should only be used as a p o s i t i v e c o n t r o l population. 2.2 Urinary Clastogenic A c t i v i t y Unrelated to Cigarette Smoking Low l e v e l s of u r i n a r y c l a s t o g e n i c a c t i v i t y were detected i n many subjects despite the absence of exposure to p e s t i c i d e s or c i g a r e t t e smoke. Compared to d i s t i l l e d water concentrate c o n t r o l s , the a c t i v i t y observed i n the i n d i v i d u a l s was s i g n i f i c a n t l y increased. Questionnaires d i d not reveal any unusual exposure which may account f o r the increase. Thus f a c t o r s other than c i g a r e t t e smoking or p e s t i c i d e exposure may contribute to urine g e n o t o x i c i t y . 104 Similar findings of low levels of urine mutagenicity unrelated to occupation or smoking have been reported (Guerrero et a l . , 1979; Beek et a l . , 1982; Barale et a l . , 1985; Everson et a l . , 1985). Recently, various dietary factors a f f e c t i n g the urine mutagenicity assay have been discovered. Mutagenic a c t i v i t y has been detected in non-smoking individuals after the ingestion of a f r i e d beef meal (Sousa et a l . , 1985) or a f r i e d pork or bacon meal (Baker et a l . , 1982), but not microwaved meat (Sousa et a l . , 1985). These authors attributed the mutagenic a c t i v i t y to browning reactions between fats and amines to form heterocyclic aromatic hydrocarbons which are mutagenic i n b a c t e r i a l systems (Sugimura and Sato, 1983; Sugimura, 1985). Urine mutagenicity was also increased in another study where subjects were r e s t r i c t e d to a vegetarian diet consisting of soy products, nuts, f r u i t s and vegetables (Sasson et a l . , 1985) . Although the agents responsible for the mutagenicity were not known, Sasson et a l . (1985) suggested that quercetin, a flavonoid found in foods of plant o r i g i n , may be involved. These studies demonstrate that consumption of mutagen-containing foods may be p a r t l y responsible for the presence of urine genotoxicity. The use of medication may also be a contributing factor. Patients treated with the antitumour drug, cyclophosphamide (Siebert and Simon, 1973), and with the antischistosomal drugs, niridazole and metronidazole (Legator et a l . , 1975) , excreted mutagenic urine. P s o r i a t i c patients receiving crude coal tar therapy have also exhibited mutagenic a c t i v i t y i n the urine (Wheeler et a l . , 1981). Some drugs may synergize the mutagenic a c t i v i t y of urine i n combination with other agents. For example, Recio et a l . (1982) noted that the use of an a r t h r i t i c drug (Ascription A.D.) increased the mutagenic a c t i v i t y of the urine of a cigarette smoker f i v e f o l d above that of urine of other smokers. The few 105 individuals in our study who used medication did not show elevated c l a s t o -genic a c t i v i t y . However, the potential confounding e f f e c t s of drugs, as i l l u s t r a t e d by the above studies, do point to the need to exercise caution in the interpretation of r e s u l t s . F i n a l l y , the appearance of low levels of clastogenic a c t i v i t y may be the r e s u l t of unknown exposure to environmental contaminants such as automobile exhaust (Ohnishi et a l . , 1980). In any urine study, i t i s v i r t u a l l y impossible to control for exposure to such environmental substances. The only solution would be to analyze repeated urine samplings to establish a baseline to account for i n t e r f e r i n g exposures. Because of the various confounding factors, one must exercise caution in interpreting urine genotoxicity r e s u l t s . Urine contains a highly complex mixture of metabolites and s a l t s . Exposure to d i f f e r e n t environmental contaminants w i l l change the nature of the urinary metabolites. I t i s important to remember that the observed urinary clastogenic a c t i v i t y represents the net e f f e c t of the interactions between urine constituents. I t i s therefore d i f f i c u l t to pinpoint a single causative factor. Without proper i d e n t i f i c a t i o n of the metabolites responsible for the clastogenic a c t i v i t y , one may, at best, make only an association between two events, e.g., exposure to pesticides and the appearance of clastogens i n the urine. 2.3 Urine T o x i c i t y Urine t o x i c i t y i s another confounding factor that merits some discussion. Toxic responses were frequently observed i n the urine samples from the subjects in t h i s study. The e f f e c t did not appear to be exposure-related since the urine of control individuals also showed toxic responses. Concentrations at which toxic responses were evident were very s i m i l a r i n a l l i n d i v i d u a l s , with 106 t o x i c i t y generally appearing at 5.0 to 8.0 mg/ml c r e a t i n i n e equivalence of urine e x t r a c t . The urine of the subjects also appeared to contain antimitogenic f a c t o r s since several urine concentrates were able to induce m i t o t i c i n h i b i t i o n i n CHO c e l l s . The t o x i c materials appeared to be a normal c o n s t i t u e n t i n urine rather than an a r t i f a c t of the e x t r a c t i o n procedure. D i s t i l l e d water concentrate (generated from the e l u t i o n of d i s t i l l e d water from the RPLC column with 50% acetone) d i d not e l i c i t t o x i c i t y at the same concentrations at which urine doses were t o x i c . Other i n v e s t i g a t o r s have encountered urine t o x i c i t y using the Ames Salmonella t e s t with metabolic a c t i v a t i o n (Recio et a l . , 1984; Everson et a l . , 1985; Heussner et a l . , 1984; K r i e b e l et a l . , 1985). Beek et a l . (1982) a l s o noted t o x i c u rine e f f e c t s on CHO c e l l s . The nature of the t o x i c components i s not known. Some have a t t r i b u t e d the t o x i c i t y to non-genotoxic components of the urine ( K r i e b e l et a l . , 1985). Recio et a l . (1984) b e l i e v e d that some of these agents may have b a c t e r i o -s t a t i c p r o p e r t i e s . Aeschbacher and Chappuis (1981) found the t o x i c i t y to be associated w i t h the incubation of u r i n e with 6-glucuronidase enzyme. They i n f e r r e d t h a t deconjugation releases metabolites which are t o x i c to the i n v i t r o i n d i c a t o r c e l l system. The importance of t o x i c i t y i s suggested i n Figure 16, where repeated u r i n e samples from a g r i c u l t u r a l research s t a t i o n smokers show more v a r i a t i o n at the higher t o x i c doses compared to the lower doses. This f i n d i n g i s concordant with t h a t found f o r urine of smokers by K r i e b e l et a l . (1985). T o x i c i t y could be avoided by s e l e c t i n g l e s s t o x i c doses f o r t e s t i n g , but u n f o r t u n a t e l y , i n many cases, a pronounced cl a s t o g e n i c response i s seen only 107 at near toxic dose l e v e l s . The roost probable explanation for th i s i s that at high urine doses, CHO c e l l s may be so "sick" that they become vulnerable to chromosome damage by clastogenic agents i n the urine. Irrespective of i t s cause, because of the p o s s i b i l i t y that t o x i c i t y may mask, suppress or even i n t e r f e r e with the detection of clastogens in urine samples, one must be conscious of false negatives due to th i s confounding factor. 3. Monitoring Urine Genotoxicity as a Means of Detecting Exposure to Environmental Carcinogens and Mutagens: Limitations and Applications The present study demonstrates the value of using urine analysis as a means of assessing human exposure to environmental contaminants. I t i s necessary to emphasize here that the study i s only an attempt to make a q u a l i t a t i v e association between exposure to a potential carcinogen or mutagen and abnormalities observed i n the proposed genetic endpoints. Several advantages of the bioassay of urine are i l l u s t r a t e d i n t h i s investigation. Unlike epidemiological studies, urine analysis for genotoxicity takes into account the variations i n exposure regimes, and therefore the screening procedure can pinpoint i n d i v i d u a l r i s k (Guerrero et a l . , 1979). With the implementation of such screening techniques, exposure to high-risk environ-ments may be minimized long before the occurrence of any i r r e v e r s i b l e pathological changes. The assay of urine for genotoxicity does not measure the a c t i v i t y of just one metabolite, but the a c t i v i t y of a combination of metabolites present i n the urine as a r e s u l t of complex exposures. Within the urine are thousands of chemicals, some of which may be i n h i b i t o r s or enhancers of genotoxic a c t i v i t y . The assay of urine samples measures the net e f f e c t of the chemicals present 108 together, taking i n t o c o n s i d e r a t i o n the s y n e r g i s t i c or antagonistic i n t e r -a ctions that may be s i g n i f i c a n t . Yet another advantage of monitoring urine for g e n o t o x i c i t y i s the demonstration of b i o l o g i c a l or genetic a c t i v i t y . U n like chemical a n a l y s i s , where the presence of urinary metabolites i s determined, the assay of urine a c t u a l l y demonstrates that the metabolites are not t o t a l l y i n a c t i v e , but possess s u f f i c i e n t b i o l o g i c a l a c t i v i t y to cause harm, such as the induction of chromosome aberrations i n CHO c e l l s . Urine monitoring i s most u s e f u l i n evaluating human exposure to m u l t i p l e agents. Rarely i s an i n d i v i d u a l ever exposed to a s i n g l e agent. In the majority of cases, the extent of exposure or the route (e.g., dermal, r e s p i r a t o r y , ingestion) w i l l not be known. For example, i n our i n v e s t i g a t i o n , most sprayers were exposed to a v a r i e t y of p e s t i c i d e s and i t was unknown whether t h i s mixture would be genotoxic _in v i v o . This question can be answered by urine g e n o t o x i c i t y assays. The actual presence of genotoxic a c t i v i t y c o i n c i d i n g with the time of exposure to the suspected genotoxin serves as evidence that exposure had occurred and that c e l l s i n various organs were thus at r i s k f o r genetic damage while the chemicals were being s y s t e m a t i c a l l y d i s t r i b u t e d throughout the body. Thus the bioassay of urine i s u s e f u l f o r i d e n t i f y i n g carcinogenic and mutagenic r i s k s associated with various work environments. However, the a n a l y s i s of urine for ge n o t o x i c i t y i s not without i t s l i m i t a t i o n s . U n l i k e chemical a n a l y s i s , there i s a lack of s e n s i t i v i t y f o r s p e c i f i c chemicals (Brusick et a l . , 1981). As demonstrated i n t h i s study, although c l a s t o g e n i c a c t i v i t y was observed with p e s t i c i d e usage, the i d e n t i t y of the clastogens remains unknown. Because of the unknown nature of the compounds, the assay r e s u l t s can only be i n t e r p r e t e d i n a q u a l i t a t i v e manner 109 i n terms of exposure assessment. To gain more u s e f u l information ( q u a n t i t a t i v e assessment), chemical techniques (e.g., a n a l y t i c a l high-pressure l i q u i d chromatography) must be employed i n conjunction with the examination for urine g e n o t o x i c i t y . Moreover, our studies and those of others (Yamasaki and Ames, 1977; Kado et a l . , 1985; Sousa et a l . , 1985) show that urine a n a l y s i s may be u s e f u l only for monitoring recent exposures. Cumulative exposures cannot be detected (Vainio et a l . , 1984). Depending on the x e n o b i o t i c , metabolites may be e l i m i n a t e d i n t o the urine w i t h i n one to perhaps two days. Beyond t h i s p e r i o d , the l e v e l s of metabolites i n the urine may be too low f o r d e t e c t i o n . Thus i t i s necessary to e i t h e r determine the e x c r e t i o n k i n e t i c s of the metabolites or to obtain m u l t i p l e urine samples a f t e r exposure to an agent. Since both choices are l i k e l y to be i m p r a c t i c a l , the c o l l e c t i o n of urine a few hours a f t e r exposure or l a t e i n the evening may be a reasonable a l t e r n a t i v e . K r i e b e l et a l . (1985) found that an evening urine sample provided a good estimation of the mutagen concentrations of a 24-hour urine sample. Nevertheless, regardless of the sampling schedule used, the c o l l e c t i o n of urine samples f a r beyond the optimal metabolite e x c r e t i o n p e r i o d w i l l r e s u l t i n l e v e l s of genotoxic a c t i v i t y u n d i s t i g u i s h a b l e from normal background values. Decomposition of u r i n a r y clastogens may present a problem i n the a n a l y s i s of u r i n e . Some metabolites are s h o r t - l i v e d and may never be detected i n the urine. Only those metabolites with long h a l f - l i v e s can be assayed. Even storage at -20°C does not ensure the s t a b i l i t y of the e x t r a c t components. A few i n d i v i d u a l s have i n v e s t i g a t e d the s t a b i l i t y of urine concentrates. Putzrath et a l . (1981) observed the l o s s of genotoxic a c t i v i t y of a few urine 110 concentrates with storage. Beek et a l . (1982) also noted decreased genotoxic a c t i v i t y with re-used urine extracts. In both cases, the authors attributed the loss of a c t i v i t y to repeated freezing and thawing of the urine extracts. Genotoxins may also be l o s t through the extraction and concentration procedures. For example, with our methods, v o l a t i l e compounds (e.g., nitrosamines) may be l o s t through the rotary evaporation or freeze-drying process. Loss of a c t i v i t y may also occur through limitations i n the extraction procedures. Because of the presence of high s a l t concentrations in the urine, hydrophilic materials are d i f f i c u l t to i s o l a t e . Toxicity of the s a l t s to mammalian c e l l s also makes i t impossible to test hydrophilic components i n urine. This t o x i c i t y problem was evident when freeze-dried unconcentrated urine was assayed for genotoxic material. New procedures must be developed to extract polar organic compounds i n urine. F i n a l l y , caution must be used in the interpretation of genotoxicity data. The absence of genotoxicity in some cases does not always necessarily indicate a lack of exposure. Metabolites may be present in concentrations below the detectable l i m i t s of the assay system. In addition, compounds excreted through routes other than urine (e.g., lungs, feces, sweat) may never be detected by urine assays. Some chemicals may also be biotransformed into products that cannot be reactivated i n v i t r o (Falck, 1982). Glucuronide and su l f a t e conjugates may be re a d i l y cleaved by the inclusion of ^-glucuronidase and sulfatase enzymes i n the assay system (Yamasaki and Ames, 1977; Legator et a l . , 1982). In contrast, chemicals conjugated to glutathione cannot be e a s i l y cleaved to y i e l d the compound in i t s o r i g i n a l form. Instead, glutathione conjugates are l i k e l y to be metabolized such that the glutathione-metabolite 111 linkage i s retained (Dorough, 1983). This class of metabolites i s not detectable i n urine clastogenicity or mutagenicity tests (Ramel, 1984). Other methods such as those that assay for mercapturic acids and other thioethers must be used to detect glutathione conjugates (van Doom et a l . , 1981). Another factor to consider i s that although absorption of the compound by the body may have occurred, the suspected agent may not have yet been metabolized. This pertains, i n p a r t i c u l a r , to l i p o p h i l i c substances that p e r s i s t i n the body for a long period of time. The slow release of these compounds from tissue stores w i l l make t h e i r detection quite d i f f i c u l t . 4. Outlook The urinary excretion of genotoxic metabolites appears to be a useful index i n screening work environments and detecting exposure to p o t e n t i a l environmental carcinogens and mutagens. Already the analysis of urine for genotoxicity has demonstrated the carcinogenic or mutagenic r i s k of ind i v i d u a l s exposed to a variety of agents, including i n d u s t r i a l chemicals, therapeutic drugs and cigarette.) smoke. The a p p l i c a b i l i t y of t h i s procedure w i l l c e r t a i n l y extend to other environmental agents i n the future. The screening of urine for genotoxicity has i t s l i m i t a t i o n s . Future research i s s t i l l warranted i n the following areas: (a) The i d e n t i f i c a t i o n of the genotoxic components present i n urine. Chemical analysis w i l l have to be< employed to reach any conclusions regarding quantitative r i s k assessment. (b) The representation of urine concentrate components to those of the o r i g i n a l urine samples. Extraction and concentration procedures may a l t e r the proportion of urine components that were present i n vivo. Loss of i n h i b i t o r s 112 during the e x t r a c t i o n procedure may, f o r example, unmask the action of a u r i n a r y metabolite i n v i t r o which would otherwise have been i n a c t i v e in v i v o . (c) The development of b e t t e r e x t r a c t i o n procedures to include a wider spectrum of u r i n a r y compounds such as the polar metabolites. (d) The usefulness of urine monitoring as a p r e d i c t o r of adverse health e f f e c t s . Although the presence of genotoxins i n urine i s i n d i c a t i v e of exposure to an environmental contaminant, no conclusions can be drawn about i t s b i o l o g i c a l consequences. (e) The relevance of using u r i n e g e n o t o x i c i t y as the only measure of environmental exposure. Urine represents only one route by which ex c r e t i o n products are eliminated. Thus the coupling of the urine screening procedure with other assay systems (e.g., a n a l y s i s of mutagens i n feces) may give more rep r e s e n t a t i v e information than the use of urine a n a l y s i s alone. In c o n c l u s i o n , the search f o r genotoxins i n urine i s a valuable t o o l f o r monitoring exposure to p o t e n t i a l environmental carcinogens and mutagens. Despite i t s few l i m i t a t i o n s , the advantages of such a procedure cannot be ignored. 113 SUMMARY The objective of t h i s study was to evaluate the f e a s i b i l i t y of using urine analysis as a means of assessing human exposure to p o t e n t i a l l y hazardous environmental agents. Urine was collected from 21 orchardists ( a l l non-smokers) i n the Okanagan Valley when they were engaged i n the application of pesticides during the f r u i t growing season i n 1984 and 1985. Control urine samples were co l l e c t e d from these same individuals during the pre-spraying and post-spraying seasons. In addition, 18 a g r i c u l t u r a l research station personnel i n the Okanagan region (including 16 non-sprayers and 2 professional sprayers) provided urine samples at the same time as the orchardists. As reference controls outside the f r u i t growing region, subjects from Vancouver and Grand Forks, B.C. were recruited to provide one urine specimen for the study. The urine was concentrated by reversed-phase high pressure l i q u i d chromatography and tested for the a b i l i t y to induce chromosome aberrations (clastogenic a c t i v i t y ) i n cultured Chinese hamster ovary (CHO) c e l l s . An attempt was also made to examine the exf o l i a t e d u r o t h e l i a l c e l l s for the presence of micronuclei as a p o t e n t i a l i n vivo indicator of damage by genotoxic agents i n the urine. The following r e s u l t s were obtained: 1. The e f f e c t s of pesticide exposure on urine genotoxicity were analyzed. Urine obtained from orchardists during periods of non-pesticide usage (samples from October 1984 and March 1985) showed comparable l e v e l s of clastogenic a c t i v i t y to non-smoking control individuals from Vancouver and Grand Forks. The c o l l e c t i o n of urine samples 16 to 24 hours af t e r 114 p e s t i c i d e exposure i n 1984 (June and July) f a i l e d to demonstrate increased c l a s t o g e n i c a c t i v i t y beyond the normal c o n t r o l l i m i t s . However, a f o l l o w -up study during the spraying season i n August 1985 revealed that the c o l l e c t i o n of urine w i t h i n 8 hours of p e s t i c i d e exposure r e s u l t e d i n s i g n i f i c a n t l y elevated l e v e l s of c l a s t o g e n i c a c t i v i t y , as determined by Turkey's non-parametric m u l t i p l e comparisons t e s t . 2. The 16 a g r i c u l t u r a l research s t a t i o n personnel who were not engaged i n p e s t i c i d e spraying d i d not demonstrate any o v e r a l l increase i n u r i n a r y c l a s t o -genic a c t i v i t y during the spraying period (August 1985) compared to the pre-spraying p e r i o d (March 1985). The two i n d i v i d u a l s from the research s t a t i o n who d i d spray p e s t i c i d e s showed increased u r i n a r y c l a s t o g e n i c a c t i v i t y . 3. The s i n g l e urine specimen from non-smokers from Vancouver and Grand Forks only showed low l e v e l s of u r i n a r y c l a s t o g e n i c a c t i v i t y r e l a t i v e t o solvent c o n t r o l s . 4. C i g a r e t t e smoking was found to be a confounding f a c t o r i n the urine c l a s t o g e n i c i t y assay. The urine of c i g a r e t t e smokers from the Okanagan research s t a t i o n and from Grand Forks demonstrated s i g n i f i c a n t l y higher c l a s t o g e n i c a c t i v i t y than that of non-smokers from the same area (as determined by the Mann-Whitney U t e s t ) . The extent of the response was not c o r r e l a t e d with the number of c i g a r e t t e s smoked. 5. A high proportion of the non-smoking subjects i n t h i s study ( i n d i v i -duals from Vancouver, Grand Forks and the Okanagan region) showed low l e v e l s of u r i n a r y c l a s t o g e n i c a c t i v i t y during periods when p e s t i c i d e usage had ceased (compared to the 0 to 1% aberrant metaphases observed with d i s t i l l e d water concentrates.) This may i n d i c a t e that other f a c t o r s (e.g., d i e t ) may r e s u l t i n urine g e n o t o x i c i t y . 115 6. The clastogenic a c t i v i t y of the urine extracts occurred over a narrow concentration, ranging from 1.0 to 8.0 mg/ml creatinine equivalence. The concentrations at which peak a c t i v i t y was observed varied between the d i f f e r e n t urine extracts. Chromatid exchanges were the major aberration found i n CHO c e l l s . 7. Many of the urine extracts e l i c i t e d t o x i c i t y i n CHO c e l l s at the upper l i m i t s of the concentration range tested. Urine from individuals who sprayed pesticides as well as from those who did not spray showed similar toxic e f f e c t s within the same dose range. 8. Micronuclei analysis on ex f o l i a t e d u r o t h e l i a l c e l l s could not be performed due to the sca r c i t y of c e l l s i n the urine. Based on the above observations, the following conclusions can be made: 1. The elevated urinary clastogenic a c t i v i t y i n orchardists during the pest i c i d e spraying period compared to that during the non-spraying period indicates that the increase may be related to exposure to pesticides. 2. The excretion of pesticides and t h e i r metabolites i n the urine i s speculated to be a rapid process (within hours of exposure). This i s supported by three l i n e s of evidence: (a) the f a i l u r e to demonstrate increased clastogenic a c t i v i t y i n urine c o l l e c t e d 16 to 24 hours after p e sticide application i n 1984. (b) the demonstration of increased clastogenic a c t i v i t y i n urine c o l l e c t e d within 8 hours of exposure i n 1985. (c) published reports of pesticide excretion i n the urine of humans within hours of exposure. 116 This speculation i s based on the assumption that the type of pesticide used, the type of protective gear worn, and the degree of pesticide exposure were si m i l a r during the two years of t h i s study. 3. The toxic e f f e c t of urine extracts on CHO c e l l cultures does not appear to be related to pesticide spraying or cigarette smoking, since control urine samples showed si m i l a r toxic effects within the same dose range. 4. The clastogens i n the urine of a l l subjects were direct-acting ( i . e . , independent of metabolic activation) and r e l a t i v e l y non-polar (able to bind to a C18 reversed-phase column). 5. Since urine from cigarette smokers showed elevated clastogenic a c t i v i t y compared to that from non-smokers, t h i s l i f e s t y l e habit must be taken into consideration i n any study of occupational exposure to genotoxic agents. 6. The assay for genotoxicity in the urine i s a useful procedure for monitoring exposure to environmental carcinogens and mutagens, but i t does have i t s l i m i t a t i o n s . Notable among these limitations are the i n a b i l i t y to extract polar compounds from the urine, the potential loss of genotoxic chemicals due to decomposition i n the urine, and the i n a b i l i t y to detect metabolites excreted through other routes (e.g., feces). 7. The analysis of urine for genotoxicity has already been used to assess human exposure to cigarette smoke, therapeutic drugs and i n d u s t r i a l chemicals. The use of t h i s monitoring procedure w i l l c e r t a i n l y continue i n the future. Used either alone or i n conjunction with other techniques, urine analysis w i l l have important applications in the search for more genotoxic chemicals i n the environment. 117 REFERENCES Aeschbacher, H.U. and Chappuis, C. 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Science 221: 227-236 (1983). Zar, J.H. , i n : B i o s t a t i s t i c a l Analysis, 2nd ed., Prentice-Hall, Englewood C l i f f s , N.J. (1984). 127 APPENDIX 1 BLANK QUESTIONNAIRES 128 APPENDIX IA QUESTIONNAIRE DISTRIBUTED TO ORCHARDISTS AND AGRICULTURAL RESEARCH STATION WORKERS ON THE DAY OF URINE COLLECTION NAME/CODE LOCATION SAMPLES - INVESTIGATOR ADDRESS/ PHONE AGE TOBACCO TYPE/ AMOUNT SMOKE OR CHEW VEGETARIAN TEA/COFFEE CUPS/DAY ALCOHOL TYPE/ AMOUNT MEDICATION DATE OF SAMPLING PESTICIDES USED AMOUNT HOURS OF EXPOSURE SPRAYING EQUIPMENT PROTECTIVE CLOTHING 1 2 9 APPENDIX IB QUESTIONNAIRE USED TO OBTAIN LIFESTYLE AND DIETARY INFORMATION FROM GRAND FORKS AND VANCOUVER RESIDENTS ON THE DAY OF URINE COLLECTION CODE NUMBER DATE NAME ADDRESS • PHONE OCCUPATION AGE SEX 1. Do you c u r r e n t l y consume any type of tobacco? I f yes, what type ( i . e . , c i g a r e t t e s , p ipe, c i g a r s , etc.) How much do you consume per day ( i . e . , no. of c i g a r e t t e s , bowls, c i g a r s ) Are you constantly exposed to someone who smokes? Do you drink coffee or tea on a d a i l y b a s i s ? Number of cups of coffee per day Number of cups of tea per day 3. Do you r e g u l a r l y take vitamin supplements? I f yes, what type Have you been takin g any medication r e c e n t l y ? I f yes, when and what type Please estimate the number of times per week that you consume a food product from each of the f o l l o w i n g food groups: Red meat P o u l t r y F i s h Eggs M i l k products Vegetables Breads/cereals F r u i t s 5. Please estimate the type and amount of alc o h o l that you consume (use e i t h e r the d a i l y or weekly space): D a i l y Weekly 6. Do you use any p e s t i c i d e s ? I f yes, give the name and the amount C o l l e c t i o n Dates of Urine Sampling: Time Time Time Time Time Time 130 APPENDIX IB (cont'd) PLEASE GIVE A DESCRIPTION OF. YOUR DIET WITHIN THE LAST 24 HOURS. IF YOU HAVE SMOKED ANY CIGARETTES OR CONSUMED ANY ALCOHOL (E.G., BEER, WINE, ETC.) WITHIN THE LAST 24 HOURS, PLEASE STATE THE AMOUNT. 131 APPENDIX 2 GRAND FORKS RESIDENTS 132 A P P E N D I X 2A COMPILATION OF LIFESTYLE AND DIETARY INFORMATION FOR GRAND FORKS RESIDENTS ON THE DAY OF URINE COLLECTION IN SEPTEMBER 1985 S u b j e c t N u m b e r Sex Age Smoking ( Y e a r s ) ( C i g a r e t t e s / D a y ) C o f f e e Consumption (Cups/Day) A l c o h o l ( L i t r e s / D a y ) M e d i c a t i o n G l M 62 20 6 1.20 0 G2 M 47 15 0 0 0 G3 M 70 0 2 0 & G4 M 64 12 3 0.75 G5 M 43 20 10 0.50 0 G6 M 52 0 8 0 0 G7 M 35 0 7 0.25 0 G8 M 57 20 5 0 0 G9 M 59 o. 2 0.50 0 G10 M 70 3 2 1 0.50 0 G i l M 58 0 0 0 0 G12 M 71 0 3 0 0 G13 M 50 10 8 0 0 G14 M 40 0 4 0 0 G15 M 59 15 4 0.25 0 G16 M 41 15 3 0 0 G17 M 43 30 20 0 o. G18 M 71 0 2 0.25 MD G19 M 34 0 1 0 0 G20 M 49 10 6 0 0 G21 M 57 0 2 0.25 o. G22 M 66 25 3 0 MD MD, m e d i c a t i o n : A.S.H. f o r a r t h r i t i s . 2 P i p e smoking o n l y . ^MD, m e d i c a t i o n : h y d r o c h l o r t h y a n a t e . MD, m e d i c a t i o n : chlorphosphamide. 133 APPENDIX 2B PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM GRAND FORKS RESIDENTS IN SEPTEMBER 1985 Percent Metaphases with Chromatid Aberrations Urine Parameters Creatinine Equivalence (mg/ml) Subject Volume Creatinine Number (ml) pH (mg/ml) o 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Gl 410 5.71 0.34 1.0 0 0 11.0 13.0 , MI9.7 2 m3 T T T G2 350 6.02 1.69 0< 1.0 10.0 25.5 16.0 MI20. 0 T T T G3 420 5.73 0.43 0 0 0 0 0 1.0 1.0 T T G4 385 5.55 0.81 1.0 0 0 0 13.0 22.5 T T T G5 355 5.75 0.40 0 0 0 4.0 11.0 T T T T G6 285 5.76 1.14 0 0 0 0 0 0 2.0 T T G7 245 6.23 1.29 1.0 1.0 1.0 3.0 5.0 T T T T G8 235 6.73 3.46 1.0 0 0 0 1.0 1.0 15.8 T T G9 200 5.41 0.91 1.0 0 0 0 11.7 MI2.5 T T T G10 290 5.44 1.12 0 0 2.0 12.0 11.0 4.0 T T T G i l 270 6.50 1.00 0 0 0 4.0 5.6 3.0 MI8.3 T T G12 295 5.61 0.85 0 0 0 4.0 6.0 MIO.O T T T G13 290 7.31 1.16 1.0 0 0 0 1.0 T T T T G14 240 6.62 0.88 0 0 0 0 0 0 0 1.0 1.0 G15 185 7.07 0.65 0 1.0 5.0 4.0 15.6 T T T T G16 275 6.52 0.93 0 0 2.0 5.0 6.7 T T T T G17 280 5.69 2.07 0 0 1.0 2.1 21.0 T T T T G18 95 5.70 1.42 0 0 0 2.0 2.0 T T T T G19 135 6.06 1.34 0 0 0 0 0 3.0 T T T G20 225 5.85 2.60 1.0 0 0 1.0 3.0 6.7 MI2.4 T T G21 280 5.72 1.57 0 0 0 1.0 2.0 2.3 5.0 T T G22 290 6.64 0.58 0 2.0 13.0 14.4 16.7 T T T T '70% d i s t i l l e d water concentrate per 30% MEM (v/v). i 'MI, mitotic i n h i b i t i o n : less than 40 metaphases observed at urine dose indicated. *T, toxic. No detectable aberrations i n the metaphases examined. 134 APPENDIX 2C EXTENT OF CHROMATID DAMAGE PER METAPHASE PLATE INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM GRAND FORKS RESIDENTS IN SEPTEMBER 1985 Average Number o f Chromatid Exchanges per C e l l C r e a t i n i n e E q u i v a l e n c e (mg/ml) S u b j e c t — - • Number 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Gl 0.00 0.00 0.00 0.51 0.48 T T T G2 0.00 0.07 0.50 1.33 0.69 MI0.65 T T T G3 0.00 0.00 0.00 0.00 0.00 0.05 0.05 T T G4 0.00 0.00 0.00 0.00 0.46 0.79 T T T G5 0.00 0.00 0.00 0.06 0.32 T T T T G6 0.00 0.00 0.00 0.00 0.00 0.00 0.12 T T G7 0.00 0.00 0.01 0.07 0.15 T T T T G8 0.00 0.00 0.00 0.00 0.00 0.00 0.60 T T G9 0.00 0.00 0.00 0.00 0.23 MI0.10 T T T G10 0.00 0.00 0.00 0.27 0.29 0.14 T T T Gil 0.00 0.00 0.00 0.07 0.08 0.05 MI0.17 T T G12 0.00 0.00 0.00 0.08 0.14 MI0.00 T T T G13 0.00 0.00 0.00 0.00 0.02 T T T T G14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 G15 0.00 0.00 0.09 0.08 0.38 T T T T G16 0.00 0.00 0.01 0.10 0.67 T T T T G17 0.00 0.00 0.00 0.10 0.83 T T T T G18 0.00 0.00 0.00 0.03 0.03 T T T T G19 0.00 0.00 0.00 0.00 0.00 0.08 T T T G20 0.00 0.00 0.00 0.00 0.07 0.16 MI0.05 T T G21 0.00 0.00 0.00 0.01 0.04 0.04 0.13 T T G22 0.00 0.01 0.46 0.43 0.52 T T T T '70% d i s t i l l e d water c o n c e n t r a t e per 30% MEM ( v / v ) . T, t o x i c . 'MI, m i t o t i c i n h i b i t i o n : l e s s than 40 metaphases observed a t dose i n d i c a t e d . 135 APPENDIX 3 VANCOUVER RESIDENTS 136 APPENDIX 3A COMPILATION OF LIFESTYLE AND DIETARY INFORMATION FOR VANCOUVER RESIDENTS ON THE DAY OF URINE COLLECTION IN JULY 1985 Coffee Subject Smoking Consumption Alcohol Number Sex (Cigarettes/Day) (Cups/Day) (Litres/Day) Medication VI M 0 4 0 0 V2 M 0 2 0 0 V3 M 0 5 0 0 V4 M 0 3 0 0 V5 F 0 4 0 0 V6 M 0 5 0 0 V7 F 0 3 0 0 V8 F 0 4 0 0 V9 M 0 6 0 0 VlO M 0 5 0 0 V l l F 0 2 0 0 137 A P P E N D I X 3B P E R C E N T A G E O F A B E R R A N T M E T A P H A S E S I N D U C E D BY U R I N E E X T R A C T S (50* A C E T O N E E L U A T E ) P R E P A R E D FROM U R I N E S A M P L E S C O L L E C T E D FROM VANCOUVER R E S I D E N T S I N J U L Y 1985 Urine Parameters S u b j e c t V o l u m e Number (ml) pH Creatinine (mg/ml) P e r c e n t M e t a p h a s e s w i t h C h r o m a t i d A b e r r a t i o n s Creatinine Equivalence (mg/ml) 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 VI 150 6.82 1.25 o 2 0 0 1.0 2.0 2.0 4.0 T 3 T V2 185 5.95 1.11 0 0 0 3.0 4.0 MIO.O 4 T T T V3 220 6.21 1.59 1.0 0 0 0 1.0 3.0 T T T V4 110 6.85 1.67 0 0 0 2.0 4.0 MI9.1 MIO.O T T V 5 125 7.10 1.20 1.0 0 1.0 2.0 3.0 5.0 8.0 T T V6 160 6.93 0.98 1.0 0 1.0 2.0 4.0 3.0 T T T V7 130 6.50 1.95 1.0 0 0 1.0 1.0 6.0 T T T V8 155 6.48 1.88 1.0 0 0 1.0 3.0 T T T T V9 140 6.89 1.77 0 0 1.0 1.0 4.0 4.0 6.0 T T VlO 145 7.02 1.62 0 0 4.0 7.0 9.0 11.0 MI8.4 T T V l l 165 7.14 1.88 0 0 2.0 1.0 6.0 5.0 T T T 70% distilled water concentrate per 30* MEM (v/v). i 'No detectable aberrations in the metaphases examined. 'T, toxic. 'MI, mitotic inhibition: less than 40 metaphases observed at urine dose indicated. 138 A P P E N D I X 3C E X T E N T O F C H R O M A T I D DAMAGE PER M E T A P H A S E P L A T E I N D U C E D BY U R I N E E X T R A C T S ( S 0 \ A C E T O N E E L U A T E ) P R E P A R E D FROM U R I N E S A M P L E S C O L L E C T E D FROM VANCOUVER R E S I D E N T S I N J U L Y 1 9 8 5 A v e r a g e N u m b e r o f C h r o m a t i d E x c h a n g e s p e r C e l l C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) S u b j e c t N u m b e r o1 1 . 0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0 V I 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 2 T2 T V2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 2 M I 0 . 0 0 3 T T T V 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 T T T V4 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 M I 0 . 1 6 M I 0 . 0 0 T T V 5 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0.02 0 . 0 6 T T V 6 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 2 T T T V 7 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 T T T V 8 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 4 T T T T V 9 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 5 0 . 0 7 0 . 0 8 T T V I O 0 . 0 0 0 . 0 0 0 . 0 5 0 . 0 9 0 . 1 4 0 . 1 8 M I 0 . 2 4 T T V l l 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 5 0 . 0 7 T T T 1 7 0 % d i s t i l l e d w a t e r c o n c e n t r a t e p e r 30% MEM ( v / v ) . T , t o x i c . 'MI, m i t o t i c I n h i b i t i o n : l e s s t h a n 4 0 m e t a p h a s e s o b s e r v e d a t d o s e I n d i c a t e d . 139 APPENDIX 4 OKANAGAN VALLEY ORCHARDISTS 140 APPENDIX 4A COMPILATION OF LIFESTYLE AND DIETARY INFORMATION FOR ORCHARDISTS FROM THE OKANAGAN VALLEY DURING THE DAYS OF URINE SAMPLING Subject Age Number Sex (Years) Smoking (Cigarettes/ Day) Coffee Consumption (Cups/Day) Month1 Alcohol (Litres/Day) Month v B C D E F Medication Month B C D E SI M 42 NS2 2 3 _3 _ 3 _ .3 0 _ _ .3 - 0 0 — — 0 _ S2 M 54 NS 2 3 - 1 0 1 .5 .3 - 0 .5 .3 0 0 - 0 0 0 S3 M 34 NS 3 4 4 3 4 3 .3 0 .5 .3 .3 .8 0 0 0 0 0 0 S4 M 43 NS 2 3 - 4 2 2 ' 0 0 - 0 0 0 0 0 - 0 0 0 S5 M 27 . NS 3 4 3 4 2 3 0 .3 0 0 .3 0 0 0 0 0 0 0 S6 M 39 NS 3 2 - 3 4 4 0 0 - 0 0 0 0 0 - 0 0 0 S7 M 43 NS 4 2 - 4 2 3 0 0 - 0 0 0 0 0 - 0 0 0 S8 M 41 NS - 4 - 4 4 3 - .3 - . 3 .8 .5 - 0 - 0 0 0 S9 M 19 NS 1 1 - - 0 0 0 0 - - 0 0 0 0 - - 0 0 S10 M 48 NS 4 2 4 2 3 2 .3 0 .3 .5 .8 1.0 0 0 0 0 0 S l l M 28 NS 0 0 0 2 0 1 0 .3 0 .5 .8 .5 0 0 MD MD 0 MD S12 M 26 NS 0 0 0 0 0 0 0 .5 1.0.5 .8 .8 0 0 0 0 0 0 S13 M 31 NS 1 1 1 1 0 1 .3 .5 0 .3 .5 .5 0 0 0 0 0 0 S14 M 37 NS 1 2 2 1 0 2 0 .3 .5 .8 .5 0 0 0 0 0 0 0 S15 M 53 NS 1 2 1 1 0 1 .8 .5 0 .5 .8 1.0 0 0 0 0 0 0 S16 M 59 NS 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 S17 M 46 NS - - 0 1 0 0 - - 0 0 0 0 - - 0 0 0 0 S18 M 40 NS - 0 - 1 0 - 0 0 - 0 0 - 0 0 - 0 0 -S19 M 34 NS 5 4 6 5 6 8 .5 .3 0 .8 .5 .8 0 0 0 0 0 0 S20 M 70 NS 5 7 - 9 10 9 .5 .3 - 1.3 .8 1.0 0 0 - 0 0 0 S21 M 28 NS - 1 - 0 1 1 - 0 - 0 0 0 - 0 - 0 0 0 1A, May 1984; B, June 1984;; C, July 1984; D, October 1984; E, March 1985; F, August 1985. 2 NS, non-smoker. indicates that no urine sample was collected for that month. 4 MD, medication: 2 antihistamine p i l l s per day. 141 APPENDIX 4B PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE FRACTIONS PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN MAY 1984 Percent Metaphases with Chromatid Aberrations Urine Parameters Creatinine Equivalence (mg/ml) Subject Volume Creatinine Fraction — Number (ml) pH (mg/ml) Number1 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 SI 245 6.25 1.54 1 1.0 o 5 0 0 0.0MI 3 n,4 T T T T 2 1.0 NT 0 0 0 5.6 13.6 T T - 3 1.0 NT 0 11.8 10.0 T T T T S2 310 6.41 1.63 1 6 0 NT 0 0 0 0 0 0 0 2 0 NT 1.0 11.1 T T T T T 3 0 NT 0 0 0 0 4.0 0.0MI T S3 155 5.18 1.21 1 0 NT 0 T T T T T T 2 0 NT 0 0 0 0 0 0 0 3 0 NT 0 0 20.0 T T T T S4 215 6.72 1.82 1 0 NT 0 0 0 0 0 0 0 2 0 NT 0 0 0 0 0 0 1.4 3 0 NT 0 0 1.4 0.0MI T T T S5 285 6.89 1.94 1 0 NT 0 0 0 0 0 0 2.0 2 0 NT 0 0 20.0 T T T T 3 0 NT 0 0 1.4 3.2 5.0MI T T S6 195 7.10 1.64 1 0 NT 0 0 0.0MI T T T T 2 0 NT 0 0 0 0 6.3 T T 3 0 NT 0 0 0 0 7.1 T T S7 200 6.92 1.28 1 0 NT 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 4.3 3 0 NT 0 0 0 0 0 0 1.4 S9 185 6.59 1.74 1 0 NT 0 0 0 2.9 4.3 5.8 12.9 2 0 NT 0 0 0 0 0 0 0 3 0 NT 0 0 0 0 0 0 1.4 S10 315 6.23 1.26 1 0 0 0 2.9 T T T T T 2 0 0 1.4 18.3 T T T T T 3 0 0 10.0 T T T T T T S l l 290 6.28 1.05 1 0 NT 0 0 0 0 0 0 1.0 2 0 NT 0 0 0 0 0 0 T 3 0 NT 0 0 0 0 0 0 T 142 APPENDIX 4 B (cont'd) Percent Metaphases with Chromatid Aberrations Urine Parameters Creatinine Equivalence (mg/ml) S u b j e c t Number Volume (ml) pH C r e a t i n i n e (mg/ml) F r a c t i o n Number 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 S12 305 5.91 1.64 1 0 1.0 T T T T T T T 2 0 NT 0 0 0 0 0 0 0 3 0 NT 0 0 0 0 4.3 T T S13 255 6.33 1.93 1 0 0 1.0 T T T T T T 2 0 NT 0 0 0 0 0 0 0 3 0 NT 0 0 0 0 4.0 T T S14 195 6.48 2.24 1 0 0 1.0 1.0 1.0 0.0MI 0.0MI T T 2 0 NT 0 0 0 0 0 2.0 8.0 3 0 NT 0 0 0 0 0 0 1.0 S15 210 6.15 1.32 1 1.0 0 1.0 T T T T T T 2 1.0 NT 0 0 0 0 0 0 2.2 3 1.0 NT 0 0 0 0 0 0 2.9 S16 180 6.47 1.79 1 1.0 NT 0 0 1.0 1.0 0.0MI T T 2 1.0 NT 0 0 0 0 1.4 T T 3 1.0 NT 0 0 0 4.0 T T T S19 235 7.01 1.58 1 1.0 NT 0 0 0 1.4 15.7 16.9 21.4 2 1.0 NT 0 1.4 4.3 10.0 22.7 T T 3 1.0 NT 7.5 14.3 16.0 28.0 26.3MI T T S20 220 6.42 1.70 1 1.0 NT 0 24.3 27.7 T T T T 2 1.0 NT 0 1.4 4.3 10.0 22.7 T T 3 1.0 NT 26.0 T T T T T T F r a c t i o n 1: 50% acetone e l u a t e ; f r a c t i o n 2: 15% acetone e l u a t e ; f r a c t i o n 3: 50% acetone e l u a t e . 2 70% d i s t i l l e d water c o n c e n t r a t e p e r 30% MEM ( v / v ) . 3 M I , m i t o t i c i n h i b i t i o n : l e s s than 40 metaphases obs e r v e d a t dose i n d i c a t e d . 4 T , t o x i c . ^NT, n o t t e s t e d . ^No d e t e c t a b l e a b e r r a t i o n s i n the metaphases examined. 143 A P P E N D I X 4 C P E R C E N T A G E O F A B E R R A N T M E T A P H A S E S I N D U C E D B Y U R I N E E X T R A C T S ( 5 0 * A C E T O N E E L U A T E ) P R E P A R E D F R O M U R I N E S A M P L E S C O L L E C T E D F R O M O R C H A R D I S T S I N J U N E 1 9 8 4 U r i n e P a r a m e t e r s Subject Number V o l u m e ( m l ) p H C r e a t i n i n e ( m g / m l ) P e r c e n t M e t a p h a s e s w i t h C h r o m a t i d A b e r r a t i o n s C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) L 1 . 0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0 S I 1 7 0 5 . 7 7 0 . 6 9 o 2 0 0 0 0 0 . 0 M I 3 T 4 T T S 2 4 3 5 5 . 8 9 0 . 6 9 0 0 0 0 0 0 7 . 0 T T S 3 3 0 5 5 . 4 0 1 . 5 8 0 0 0 1 . 0 1 . 0 2 . 0 2 . 2 T T S 4 3 6 5 5 . 7 3 1 . 2 3 0 0 0 5 . 0 5 . 0 T •T T T S 5 4 0 5 6 . 2 6 1 . 2 1 0 0 0 0 1 . 0 3 . 4 6 . 7 T T S 6 1 9 0 6 . 5 6 1 . 5 2 0 0 1 . 0 1 . 0 2 . 0 8 . 0 1 5 . 6 T T S 7 3 3 5 6 . 0 2 0 . 6 2 0 1 . 0 2 . 0 5 . 0 9 . 0 1 3 . 7 0 . 0 M I T T S 8 3 4 0 6 . 7 7 1 . 2 4 0 0 0 0 0 0 0 3 . 0 1 . 9 S 9 2 5 0 6 . 9 2 1 . 8 5 0 0 0 0 0 1 . 0 4 . 0 T T S 1 0 3 0 0 5 . 5 6 1 . 9 1 0 0 0 0 0 0 1 . 0 1 . 0 8 . 0 S l l 3 6 0 5 . 8 2 1 . 8 9 0 0 0 0 1 . 0 1 . 0 2 . 6 T T S 1 2 2 6 0 5 . 8 8 1 . 2 8 0 0 0 1 . 0 1 . 0 2 . 6 5 . 4 T T S 1 3 3 7 0 6 . 4 2 1 . 0 8 0 0 0 0 0 1 . 0 2 . 0 T T S 1 4 3 8 0 6 . 8 2 2 . 0 5 0 0 0 0 0 1 . 0 2 . 0 T T S 1 5 4 0 0 5 . 8 7 0 . 4 7 1 . 0 0 0 0 0 1 . 0 2 . 0 T T S 1 6 3 9 0 6 . 3 5 0 . 2 9 1 . 0 0 0 2 . 0 3 . 0 T T T T S 1 8 4 4 5 5 . 9 5 0 . 5 0 1 . 0 1 . 0 2 . 0 6 . 0 sa 8 . 3 M I T T T S 1 9 4 5 0 6 . 2 6 0 . 6 0 1 . 0 0 0 1 . 0 1 . 0 5 . 2 1 . 7 T T S 2 0 3 1 5 5 . 9 8 0 . 6 2 0 0 1 . 0 1 . 0 3 . 0 0 . 0 M I T T T 7 0 * d i s t i l l e d w a t e r c o n c e n t r a t e p e r 3 0 % MEM ( v / v ) . ' N o d e t e c t a b l e a b e r r a t i o n s i n t h e m e t a p h a s e s e x a m i n e d . 'MI, m i t o t i c i n h i b i t i o n : l e s s t h a n 4 0 m e t a p h a s e s o b s e r v e d a t d o s e i n d i c a t e d . 'T, t o x i c . 144 A P P E N D I X 4 0 E X T E N T O F C H R O M A T I D D A M A G E P E R M E T A P H A S E P L A T E I N D U C E D B Y U R I N E E X T R A C T S ( 5 0 % A C E T O N E E L U A T E ) P R E P A R E D F R O M U R I N E S A M P L E S C O L L E C T E D F R O M O R C H A R D I S T S I N J U N E 1 9 8 4 A v e r a g e N u m b e r o f C h r o m a t i d E x c h a n g e s p e r C e l l C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) S u b j e c t N u m b e r o1 1 . 0 . 2 : 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0 S I 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 M I 2 T 3 T T S 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 1 8 T T S 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 0 . 0 9 0 . 0 7 T T S 4 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 9 0 . 0 8 T T T T S 5 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 7 0 . 1 2 T T S 6 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 2 0 . 0 5 0 . 1 7 0 . 3 3 T T S 7 0 . 0 0 0 . 0 1 0 . 0 7 0 . 2 5 0 . 2 1 0 . 3 8 0 . 0 0 M I T T S 8 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 6 0 . 0 2 S 9 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 1 2 T T S 1 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 2 8 S l l 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 7 0 . 0 7 0 . 0 8 T T S 1 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 5 T T S 1 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 3 T T S 1 4 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 0 . 0 4 T T S I S 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 T T S 1 6 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 3 0 . 0 4 T T T T S 1 8 0 . 0 0 0 . 0 1 0 . 0 2 0 . 2 0 0 . 1 0 0 . 1 1 M I T T T S 1 9 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 2 3 0 . 1 2 T T S 2 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 9 0 . 0 0 M I T T T 1 7 0 % d i s t i l l e d w a t e r c o n c e n t r a t e p e r 3 0 % M E M ( v / v ) . 'MI, mitotic inhibition: less than 40 metaphases observed at dose Indicated. ' T , toxic. 145 APPENDIX 4E PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE EXTRACTS ( 5 0 % ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN JULY 1 9 8 4 Urine Parameters Percent Metaphases with Chromatid A b e r r a t i o n s C r e a t i n i n e Equivalence (mg/ml) Su b j e c t Volume Number (ml) p H C r e a t i n i n e (mg/ml) 1 . 0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0 S 3 2 5 5 6 . 7 3 1 . 3 8 o2 0 0 0 1 . 0 4 . 4 1 0 . 0 T 3 T S 5 2 9 5 6 . 0 1 1 . 3 2 0 0 0 0 2 . 0 5 . 0 2 . 0 T T S 1 0 3 4 0 6 . 0 4 1 . 4 6 1 . 0 0 0 0 1 . 0 1 . 4 2 . 1 1 . 5 2 . 0 S l l 3 7 0 6 . 9 2 3 . 5 2 1 . 0 0 0 0 0 0 3 . 0 T T S 1 2 3 8 0 5 . 9 2 0 . 9 9 1 . 0 0 0 0 3 . 0 4 . 0 8 . 3 M I 4 T T S 1 3 2 8 0 5 . 7 5 2 . 0 9 1 . 0 0 0 0 2 . 0 6 . 2 8 . 0 T T S 1 4 4 4 0 6 . 5 7 1 . 9 2 1 . 0 0 0 1 . 0 2 . 0 3 . 0 9 . 0 T T S 1 5 2 4 5 6 . 0 9 1 . 9 4 1 . 0 0 0 0 1 1 . 5 0 . 0 M I T T T S 1 6 3 3 0 6 . 4 4 0 . 6 6 0 0 0 0 0 0 1 . 0 T T S 1 7 4 8 5 5 . 5 9 1 . 6 1 0 0 0 0 0 2 . 0 2 . 0 9 . 7 5 . 9 S 1 9 3 5 0 5 . 9 5 3 . 1 0 0 0 0 0 0 0 2 . 0 T T 7 0 % d i s t i l l e d w a t e r c o n c e n t r a t e p e r 3 0 % M E M ( v / v ) . 2 N o d e t e c t a b l e a b e r r a t i o n s l n t h e m e t a p h a s e s e x a m i n e d . 3 T , t o x i c . 4 M I , m i t o t i c i n h i b i t i o n : l e s s t h a n 4 0 m e t a p h a s e s o b s e r v e d a t d o s e i n d i c a t e d . 146 APPENDIX 4F EXTENT OF CHROMATID DAMAGE PER METAPHASE PLATE INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN JULY 1984 Average Number o f Chromatid Exchanges per C e l l C r e a t i n i n e E q u i v a l e n c e (mg/ml) S u b j e c t Number o 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 S3 0.00 0.00 0.00 0.00 0.02 0.28 0.40 T 2 T S5 0.00 0.00 ' 0.00 0.00 0.10 0.32 0.10 T T S10 0.00 0.00 0.00 0.00 0.01 0.01 0.04 0.05 0.06 S l l 0.00 0.00 0.00 0.00 0.00 0.00 0.10 T T S12 0.00 0.00 0.00 0.00 0.01 0.09 0.25 T T S13 0.00 0.00 0.00 0.00 0.03 0.20 0.23 T T S14 0.00 0.00 0.00 0.00 0.10 0.07 0.23 T T S15 0.00 . 0.00 0.00 0.00 0.25 0.00 T T T S16 0.00 0.00 0.00 0.00 0.00 0.00 0.01 T T S17 0.00 0.00 0.00 0.00 0.00 0.02 0.08 0.24 0.11 S19 0.00 0.00 0.00 0.00 0.00 0.00 0.03 T T 1 7 0 % d i s t i l l e d w ater c o n c e n t r a t e p e r 30% MEM ( v / v ) . T, t o x i c . 147 APPENDIX 4G PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN OCTOBER 1984 Percent Metaphases with Chromatid Aberrations Urine Parameters Subject Number Volume (ml) Creatinine (mg/ml) Creatinine Equivalence (mg/ml) pH o 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 S2 430 5.77 0.96 o 2 0 0 0 0 0 0 1.0 2.0 S3 215 5.91 3.01 0 0 0 1.2 6.0 8.3 T 3 T T S4 380 6.21 0.95 0 0 1.0 4.0 5.0. T T T T S5 225 6.63 1.20 0 0 0 0 0 1.0 8.4 T T S6 400 6.36 1.05 0 0 0 0 1.0 2.0 T T T S7 180 6.08 1.29 0 0 0 1.0 4.0 1.4 T T T S8 325 6.26 1.46 0 0 0 0 0 0 0 1.0 5.2 S10 80 5.73 1.15 0 0 0 0 1.0 3.0 T T T S l l 410 6.13 1.02 1.0 0 0 0 0 0 2.0 T T S12 475 6.52 1.58 1.0 0 0 0 0 0 1.0 2.0 2.2 S13 200 5.77 1.51 1.0 0 0 0 1.0 5.0 6.2 5.6MI4 T S14 370 5.77 2.04 1.0 0 1.0 1.0 4.0 7.0 7.8 9.0 T S15 470 6.21 1.92 1.0 0 0 1.0 2.0 2.4 9.0 T T S16 395 6.90 1.82 1.0 0 0 0 0 2.0 7.0 6.2 7.8 S17 485 5.33 1.07 1.0 0 0 0 1.0 2.0 4.1 3.2 T S18 390 6.35 0.69 1.0 0 2.0 2.0 2-0 7.7 T T T S19 440 6.25 1.33 1.0 0 0 0 3.0 6.0 9.0 T T S20 315 5.69 1.28 1.0 0 0 1.0 4.0 6.2 7.0 T T '70* distilled water concentrate per 30* MEM (v/v). 'No detectable aberrations ln the metaphases examined. *T, toxic. 'MI, mitotic inhibition: less than 40 metaphases observed at dose indicated. 148 A P P E N D I X 4 H EXTENT OF CHROMATID DAMAGE PER METAPHASE PLATE INDUCED BY URINE EXTRACTS (SO* ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN OCTOBER 1 9 8 4 Average Number o f Chromatid Exchanges per C e l l C r e a t i n i n e Equivalence (mg/ml) 4UUJCI.L N u m b e r o l 1 . 0 2 . 0 3 . 0 4 . 0 5 . 0 6 . 0 7 . 0 8 . 0 S 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 2 S 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 9 0 . 1 5 T 2 T T S 4 0 . 0 0 0 . 0 0 0 . 0 5 0 . 1 2 0 . 1 4 T T T T S 5 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 2 9 T T S 6 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 2 T T T S 7 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 1 2 0 . 0 2 T T T S 6 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 1 2 S 1 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 3 T T T S l l 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 T T S 1 2 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 5 0 . 1 0 S 1 3 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 0 . 1 1 0 . 2 9 0 . 0 6 M I 3 T S 1 4 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 7 0 . 1 6 0 . 2 6 0 . 2 8 T S 1 5 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 6 0 . 0 3 0 . 2 0 T T S 1 6 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 1 6 0 . 1 2 0 . 1 0 S 1 7 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 1 0 . 0 1 0 . 0 3 0 . 0 9 T S 1 8 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 2 0 . 1 8 T T T S 1 9 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 6 0 . 0 9 0 . 1 6 T T S 2 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 0 0 . 0 6 0 . 0 8 0 . 2 2 T T l70\ d i s t i l l e d w a t e r c o n c e n t r a t e p e r 30% M E M ( v / v ) . T, t o x i c . M I , m i t o t i c i n h i b i t i o n : l e s s t h a n 4 0 m e t a p h a s e s o b s e r v e d a t d o s e I n d i c a t e d . 149 APPENDIX 41 PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN MARCH 1985 Percent Metaphases with Chromatid Aberrations Urine Parameters — Creatinine Equivalence (mg/ml) Subject Volume Creatinine — r — : •— Number (ml) pH (mg/ml) o 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 SI 375 7.33 0.49 o 2 0 0 0.0MI3 T 4 T T T T S2 445 5.48 0.29 0 0 0 0 0 0 0 0 1.0 S3 305 5.40 1.93 0 2.0 2.0 1.0 7.0 12.0 13.8 5.1MI T S4 275 5.84 1.37 0 0 0 0 8.0 1.0 T T T S5 295 6.01 0.96 0 0 0 0 4.0 8.1MI T T T S6 210 5.85 1.36 0 0 0 0 0 2.0 4.0 7.0 8.2 S7 115 6.29 1.18 0 0 0 0 0 0 0 0 2.0 S8 155 5.85 3.16 0 0 0 0 4.0 6.5 7.1 T T S9 240 5.82 2.33 0 0 0 0 0 0 1.0 2.0 9.0 S10 265 5.99 1.63 0 0 0 0 3.0 5.4 1.0 0.0MI T S l l 410 5.85 2.18 0 0 0 0 0 0 0 0 1.0 S12 240 5.82 2.06 0 0 0 1.0 3.0 1.0 T T T S13 430 5.48 0.24 0 0 0 0 0 0 1.0 1.0 1.0 S14 385 5.45 2.75 0 0 0 0 0 0 0 0 2.0 S15 245 5.67 2.85 0 0 0 0 0 1.0 2.0 3.0 4.0 S16 310 6.72 0.63 0 0 0 0 0 . 0 1.0 2.1 T S17 455 5.30 0.46 0 0 0 2.0 3.0 T T T T S18 350 6.10 1.35 0 0 0 0 1.0 2.0 T T T S19 320 5.74 1.43 0 1.0 2.0 3.0 8.1 T T T T S20 340 6.24 1.03 0 0 0 0 2.0 2.0 4.4 T T S21 375 7.32 0.82 0 - 0 0 0 1.0 1.0 5.0 3.3 5.9MI 70% d i s t i l l e d water concentrate per 30% MEM (v/v). 2 No detectable aberrations in the metaphases examined. 3MI, mitotic inhibition: less than 40 metaphases observed at dose indicated. 4T, toxic. 150 APPENDIX 4J EXTENT OF CHROMATID DAMAGE PER METAPHASE PLATE INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN MARCH 1985 Average Number of Chromatid Exchanges per C e l l Creatinine Equivalence (mg/ml) Subject Number 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 SI 0.00 0.00 0.00 0.00MI 2 a,3 T T T T T S2 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 S3 0.00 0.03 0.05 0.01 0.26 0.44 0.80 0.26MI T S4 0.00 0.00 0.00 0.00 0.28 0.02 T T T S5 0.00 0.00 0.00 0.00 0.09 0.11MI T T T S6 0.00 0.00 0.00 0.00 0.00 0.03 .0.10 0.10 0.17 S7 0.00 0.00 0.00 0.00 0.00 "0.00 0.00 0.00 0.00 S8 0.00 0.00 0.00 0.00 0.06 0.19 0.01 T T S9 0.00 0.00 0.00 0.00 0.00 0.00 0.02 0.05 0.20 S10 0.00 0.00 0.00 0.00 0.10 0.12 0.02 0.00MI T S l l 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 S12 0.00 0.00 0.00 0.02 0.03 0.01 T T T S13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 S14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 S15 0.00 0.00 0.00 0.00 0.00 0.00 0.05 0.08 0.06 S16 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.04 T S17 0.00 0.00 0.00 0.00 0.02 T T T T S18 0.00 0.00 0.00 0.00 0.01 0.02 T T T S19 0.00 0.01 0.06 0.04 0.16 T T T T S20 0.00 0.00 0.00 0.00 0.03 0.02 0.06 T T S21 0.00 0.00 0.00 0.00 0.07 0.05 0.09 0.04 0.09MI X70% d i s t i l l e d water concentrate per 30% MEM (v/v). MI, mitotic i n h i b i t i o n : less than 40 metaphases observed at dose indicated. T, toxic. 151 APPENDIX 4K PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN AUGUST 1985 Percent Metaphases with Chromatid A b e r r a t i o n s Urine Parameters C r e a t i n i n e Equivalence (mg/ml) S u b j e c t Volume C r e a t i n i n e — Number (ml) pH (mg/ml) 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 S2 225 6.33 1.21 o 2 0 5.0 18.0 26.1 3 T T T T S3 325 6.10 1.44 0 0 0 0 0 0 3.0 4.0 2.3 S4 400 5.92 1.85 0 0 1.0 3.0 16.2 33.3MI 4 T T T SS 435 5.76 1.89 0 0 0 0 5.0 8.0 7.2 21.0 3.3 S6 395 5.81 1.79 0 0 0 2.0 3.1 6.4 12.0 23.3 13.0MI S7 230 5.87 0.70 0 3.0 11.0 12.0 20.0 T T T T S8 390 6.82 1.22 0 1.0 6.0 8.0 30.0 42.3MI 0.0MI T T S9 255 6.54 0.46 0 0 0 0 5.0 6.0 18.2 T T S10 415 S.97 1.99 0 2.0 9.0 24.0 23.0 T T T T S l l 225 6.54 1.61 0 0 1.0 2.0 3.0 3.0 9.1 T T S12 330 6.50 1.43 0 0 0 0 1.0 1.0 1.2 1.5 3.0 S13 450 5.66 2.53 0 0 0 0 4.0 6.0 16.0 T T S14 485 5.42 2.13 0 0 0 2.0 17.0 21.7 26.0 T T SIS 480 6.23 1.44 0 0 0 1.0 3.0 7.0 5.4 T T S16 400 6.83 1.29 0 0 0 0 0 1.3 6.0 T T S17 370 6.47 1.00 0 0 0 2.0 15.0 22.5 28.0MI T T S19 365 S.51 1.72 0 0 0 2.0 4.0 7.0 8.0 13.2 7.7MI S20 435 6.4S 2.84 0 0 0 0 15.0 16.0 18.5 T T S21 375 6.06 1.65 0 2.0 9.0 21.0 30.0 T T T T 70% d i s t i l l e d water c o n c e n t r a t e per 30% MEM ( v / v ) . 'No d e t e c t a b l e a b e r r a t i o n s i n the metaphases examined. ' T , t o x i c . MI, m i t o t i c I n h i b i t i o n : l e s s than 40 metaphases observed a t dose i n d i c a t e d . 152 APPENDIX 4L EXTENT OF CHROMATID DAMAGE PER METAPHASE PLATE INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM ORCHARDISTS IN AUGUST 1985 Average Number o f Chromatid Exchanges per C e l l C r e a t i n i n e Equivalence (mg/ml) Su b j e c t Number o 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 S2 0.00 0.00 0.22 0.97 1.32 T 2 T T T S3 0.00 0.00 0.00 0.00 0.00 0.00 0.06 0.06 0.16 S4 0.00 0.00 0.01 0.03 0.46 1.14MI 3 T T T S5 0.00 0.00 0.00 0.00 0.07 0.19 0.18 0 . 5 5 0.17 S6 0.00 0.00 0.00 0.01 0.08 0.23 0.29 0.87 0.48MI S7 0.00 0.02 0.22 0.31 0.86 T T T T S8 0.00 0.01 0.24 0.24 2.07 0.80MI 0.00MI T T S9 0.00 0.00 0.00 0.00 0.09 0.20 0.25 T T S10 0.00 0.03 0.17 1.23 1.14 T T T T S l l 0.00 0.00 0.00 0.02 0.05 0.07 0.09 T T S12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.21 S13 0.00 0.00 0.00 0.00 0.08 0.10 0.47 T T S14 0.00 0.00 0.00 0.05 0.79 1.21 1.25 T T S15 0.00 0.00 0.00 0.03 0.08 0.24 0.17 T T S16 0.00 0.00 0.00 0.00 0.00 0.00 0.13 T T S17 0.00 0.00 0.00 0.03 0.38 0.41 0.44MI T T S19 0 . 0 0 0.00 0.00 0.03 0.12 0.10 0.18 0.15 0 . 0 0 M I S20 0.00 0.00 0.00 0.00 0.42 0.67 0.89 T T S21 0.00 0.04 0.43 1.13 1.20 T T T T X 7 0 % d i s t i l l e d water c o n c e n t r a t e p e r 30% MEM ( v / v ) . T, t o x i c . M I , m i t o t i c i n h i b i t i o n : l e s s than 4 0 metaphases observed a t dose i n d i c a t e d . 153 A P P E N D I X 4M PROTECTIVE CLOTHING AND SPRAYING EQUIPMENT USED BY ORCHARDISTS DURING THE SPRAYING PERIODS IN 1984 AND 1985 P r o t e c t i v e C l o t h i n g and Spraying Equipment Used 1 Spraying Month S u b j e c t — — - — — Number May 1984 June 1984 J u l y 1984 August 1985 SI B,C,D.E(X) B,C,D,E(X) - -S2 A,B,E(X) A,B,E(X) - A(X) S3 A,B,E(X) A,B.E(X,Y) A,B(X) A(X) S4 A,B,E(X) A,B,E(X) - A,E(X) S5 A.B.CJX) A,B,E(X) A,B,E(X) A,B,C,E(X) S6 A.B,C.D(X) A,B,E(X) - A,E(X) S7 A,B,E(Y) A,B,E(X) - A,B,C(X,Y) S8 - A.B.E(X) - A,B,E(X) S9 C,E(Y) A.C.E(X) - A,E(X,Y) S10 A,B,E(X) A,B(X) A,E(X) A,B(X) S l l A,B,E(X) NONE (X) E(X) E(X) S12 B,C,D,F(X) B,D(X) B,D(X) B,D,F(X) S13 D,E(X) D,E(X) A,D,E(X) D,E(X) S14 A,B,D(X) B,D,E(X) D,E(X) D,E(X,Y) S15 A,B,E(X) B.E(X.Y) A,E(Y) A,B(X) S16 A,D(X,Z) A,D(X) A,B,D(X) A,D(X) S17 - - A(X) A,B(X) S18 - A,E(X) - -S19 B,E(X) B,E(W) A,E(X) A,B(X) S20 NONE (W) B,C,E(X) B,E(X) B,E(X) S21 - B,E(X) - A(X) R17 2 - - - A,B,C.E(X) R18 2 - _ - A,B,E(X) A, c o v e r a l l s ; B, g l o v e s ; C, rubber boots; D, spray s u i t ; E, h a l f face r e s p i r a t o r s ; F, helmet; ( W ) , s p r a y gun; (X), a i r b l a s t s p r a y e r ; (Y). h e r b i c i d e a p p l i c a t o r ; ( Z ) , boom s p r a y e r . A g r i c u l t u r a l r e s e a r c h s t a t i o n s p r a y e r s . 154 APPENDIX 5 AGRICULTURAL RESEARCH STATION PERSONNEL FROM OKANAGAN VALLEY 155 APPENDIX SA COMPILATION OF LIFESTYLE AND DIETARY INFORMATION FOR AGRICULTURAL RESEARCH STATION PERSONNEL DURING THE DAYS OF URINE COLLECTION IN 1984 AND 1985 Smoking Coffee Alcohol (Cigarettes/ Consumption (Litres/ Day) (Cups/Day) Day) Medication Month1 Month Month Month Subject Age Number Sex (Years) A B c A B C A B C A B C Rl M 61 0 0 0 3 4 3 0 0 0 0 0 0 R2 M 40 - 5 8 5 5 6 0 0 0 0 0 0 R3 M 50 0 0 0 4 4 3 0 0 0 0 0 0 R4 M 43 - 13 15 2 3 3 .25 .25 .25 0 0 0 R5 M 49 - 18 20 6 5 8 .25 .25 .25 0 0 0 R6 M 49 0 0 0 1 4 3 0 0 0 0 0 0 R7 M 62 0 0 0 1 0 1 0 0 0 MD2 MD2 MD2 R8 M 50 0 0 0 3 2 4 0 0 0 MD3 MD3 MD3 R9 M 48 0 0 0 5. 3 4 0 0 0 0 0 0 RIO M 48 0 0 0 6 5 ' 6 0 0 0 0 0 0 Rll M 63 0 0 0 0 4 2 0 0 0 MD4 MD4 MD4 R12 M 54 - 12 20 4 2 3 0 0 0 0 0 0 R13 M 43 0 0 0 2 3 2 0 0 0 0 0 0 R14 M 32 0 0 0 3 2 3 0 0 0 0 0 0 R15 M 42 - 0 0 4 2 4 0 .25 .50 0 0 0 R16 M 35 16 17 15 4 4 5 0 0 0 0 0 0 R17 M 26 _ 0 0 9 8 10 0 .50 .75 0 0 0 R18 M 55 - 0 0 1 2 1 0 0 0 0 0 0 1k, May 1984; B, March 1985; c, August 1985. 'MD, medication: cortisone. 'MD, medication: chlortriplex. 'MD, medication: NPH insulin. 156 APPENDIX 5B PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE FRACTIONS PREPARED FROM URINE SAMPLES COLLECTED FROM NON-SMOKING AGRICULTURAL RESEARCH STATION PERSONNEL IN MAY 1984 P e r c e n t Metaphases w i t h Chromatid A b e r r a t i o n s U r i n e Parameters C r e a t i n i n e E q u i v a l e n c e (mg/ml) S u b j e c t Volume C r e a t i n i n e F r a c t i o n Number (ml) pH (mg/ml) Number 1 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 R l 310 6.82 1.42 1 o 3 4 NT 0 2.0 7.0 5 T T T T 2 0 0 1.0 1.0 4.0 T T T T 3 0 NT 0 1.0 2.0 6.0 T T T R3 240 6.65 1.29 1 0 NT 0 0 1.0 4.0 8.0 T T 2 0 NT 0 0 2.0 7.0 T T T 3 0 NT 0 1.0 2.0 9.0 T T T R7 345 7.05 1.87 1 0 NT 0 1.0 4.3 T T T T 2 0 NT 0 1.0 1.0 2.0 4.0 T T 3 0 NT 0 1.0 1.0 1.1 T T T R8 295 6.54 1.07 1 0 NT 0 3.6 T T T T T 2 0 NT 0 0 0 0 0 0 1.4 3 0 NT 0 0 0 0 0 0 0 R9 310 6.86 0.98 1 0 NT 0 0 0 0 fio 0 0 2 0 NT 0 0 5.7 12.0MI 6 T T T 3 0 NT 0 1.4 13.6 T T T T R10 320 6.19 1.97 1 0 NT 0 T T T T T T 2 0 NT 0 0.0MI T T T T T 3 0 NT 1.0 T T T T T T R l l 225 7.24 2.01 1 0 NT 0 0 0 1.0 0.0MI T T 2 0 NT 0 5.0 10.0 0.0MI ; T T T 3 0 NT 0 1.0 3.0 10.0 5.0 T T R13 315 6.75 1.66 1 0 NT 0 0 0 0 0 1.0 T 2 0 NT 0 1.0 2.0 2.0 4.0 T T 3 0 NT 0 0.0MI T T T T T R14 330 6.84 1.71 1 0 NT 0 0 0 0 0 0 5.7 2 0 NT 0 0 0 0 0 1.0 2.9 3 0 NT 0 0 0 4.3 8.8 8.3MI T R15 205 6.52 1.25 1 0 NT 0 0 0 0 1.3 T T 2 0 NT 0 0 0 0 1.4 T T 3 0 NT 2.9 4.1 7.1 16.1MI T T T F r a c t i o n 1: 50% acetone e l u a t e ; f r a c t i o n 2: 15% acetone e l u a t e ; f r a c t i o n 3: 250% acetone e l u a t e . 70% d i s t i l l e d water c o n c e n t r a t e p e r 30% MEM ( v / v ) . .No d e t e c t a b l e a b e r r a t i o n s i n the metaphases examined. 4 NT, not t e s t e d . ,T, t o x i c . MI, m i t o t i c i n h i b i t i o n : l e s s than 40 metaphases obs e r v e d a t dose i n d i c a t e d . 157 APPENDIX 5C PERCENTAGE OF ABERRANT METAPHASES INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM AGRICULTURAL RESEARCH STATION PERSONNEL IN MARCH 1985 Percent Metaphases with Chromatid A b e r r a t i o n s U r i n e Parameters C r e a t i n i n e E q u i v a l e n c e (mg/ml) Sub j e c t Volume C r e a t i n i n e — Number (ml) pH (mg/ml) 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 RI 300 6.74 1.21 1.0 1.0 1.0 1.0 2.0 4.0 6.0 T 2 T R2 200 5.98 1.42 1.0 0 0 1.0 2.0 5.0MI 3 T T T R3 250 5.75 2.01 1.0 0 1.0 2.0 4.0 6.0 T T T R4 325 5.93 1.75 1.0 1.0 2.0 7.0 12.0 14.0 T T T R5 425 5.42 1.21 1.0 1.0 2.1 6.0 9.0 12.2MI T T T R6 330 7.01 2.25 1.0 1.0 1.0 2.0 2.0 3.0 5.0 T T R7 260 6.24 1.14 1.0 0 0 0 1.0 1.0 0.0MI T T R8 220 5.96 1.63 1.0 0 0 0 3.0 4.0 7.0 T T R9 285 5.42 1.65 1.0 0 0 0 2.0 4.0 4.0 5.0 T R10 290 6.27 1.24 1.0 0 0 1.0 4.0 6.0 T T T R l l 355 6.11 1.55 1.0 0 0 0 6.0 7.0 0.0MI T T R12 275 5.51 1.74 o 4 0 1.0 2.0 11.0 13.0 17.0 T T R13 370 6.02 2.10 0 0 0 . 1.0 1.0 3.0 2.0 O.OMI T R14 280 6.86 1.86 0 0 1.0 2.0 4.0 8.0 O.OMI O.OMI T R15 430 6.49 1.51 0 0 1.0 1.0 2.0 5.0 T T T R16 290 5.87 1.69 0 0 0 1.0 5.0 12.0 5.0MI T T R17 310 5.82 1.51 0 0 1.0 3.0 6.0 8.0 T T T R18 285 6.21 1.64 0 0 0 1.0 3.0 5.0 T T T '70% d i s t i l l e d water c o n c e n t r a t e per 30% MEM ( v / v ) . > T, t o x i c . 'MI, m i t o t i c i n h i b i t i o n : l e s s than 40 metaphases observed a t dose i n d i c a t e d . No d e t e c t a b l e a b e r r a t i o n s i n the metaphases examined. 158 APPENDIX 5D EXTENT OF CHROMATID DAMAGE PER METAPHASE PLATE INDUCED BY URINE EXTRACTS (50% ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM AGRICULTURAL RESEARCH STATION PERSONNEL IN MARCH 1985 Average Number o f Chromatid Exchanges per C e l l C r e a t i n i n e E q u i v a l e n c e (mg/ml) Number o 1 1. 0 2.0 3. 0 4. 0 5. .0 6. 0 7.0 8.0 RI 0. 00 0. 00- 0.00 0. 01 0. 05 0. .12 0. ,18 T 2 T R2 0. 00 0. 00 0.00 0. 01 0. 04 0. .03MI 3 T T T R3 0. 00 0. 00 0.01 0. 05 0. 14 0. ,19 T T T R4 0. 00 0. 00 0.01 0. 14 0. 38 0. .47 T T T R5 0. 00 0. 00 0.02 0. 12 0. 28 0. • 42MI T T T R6 0. 00 0. 00 0.00 0. 01 0. 03 0. .06 0. .12 T T R7 0. 00 0. 00 0.00 0. 00 0. 01 0. .01 0. ,00MI T T R8 0. 00 0. 00 0.00 0. 00 0. 06 0. .09 0. ,18 T T R9 0. 00 0. 00 0.00 0. 00 0. 04 0. .06 0. 07 0.06 T RIO 0. 00 0. 00 0.00 0. 01 0. 06 0. .12 T T T R l l 0. 00 0. 00 0.00 0. 00 0. 12 0. .17 0. 00MI T T R12 0. 00 0. 00 0.01 0. 06 0. 32 0. ,38 0. 51 T T R13 0. 00 0. 00 0.00 0. 01 0. 01 0. ,02 0. ,02 0.00MI T R14 0. 00 0. 00 0.01 0. 01 0. 05 0. .07 0. 00MI 0.00MI T R15 0. 00 0. 00 0.00 0. 01 0. 03 0. .10 T T T R16 0. 00 0. 00 0.00 0. 01 0. 10 0. .44 0. 15MI T T R17 0. 00 0. 00 0.00 0. 02 0. 09 0. .16 T T T R18 0. 00 0. 00 0.00 0. 01 0. 05 0. .12 T T T L 7 0 % d i s t i l l e d water c o n c e n t r a t e p e r 30% MEM ( v / v ) . T, t o x i c . 'MI, m i t o t i c i n h i b i t i o n : l e s s than 40 metaphases observed a t dose i n d i c a t e d . 159 A P P E N D I X S E P E R C E N T A G E O F A B E R R A N T M E T A P H A S E S I N D U C E D BY U R I N E E X T R A C T S (50* A C E T O N E E L U A T E ) P R E P A R E D FROM U R I N E S A M P L E S C O L L E C T E D FROM A G R I C U L T U R A L R E S E A R C H S T A T I O N P E R S O N N E L I N A U G U S T 1985 P e r c e n t M e t a p h a s e s w i t h C h r o m a t i d A b e r r a t i o n s U r i n e P a r a m e t e r s C r e a t i n i n e E q u i v a l e n c e ( m g / m l ) ubject lumber Volume (ml) p H Creatinine (mg/ml) o 1 1.0 2.0 3.0 4.0 S.O 6.0 7.0 8.0 Rl 460 5.68 0.89 2 0 0 0 0 1.0 1.0 2.0 2.0 2.0 R2 345 6.10 0.84 0 0 0 0 1.0 2.0 6.0 11.0 A 4.8MI R3 455 6.45 1.26 0 0 0 2.0 4.0 5.0 2.1 H T T R4 500 5.56 1.10 0 0 1.0 11.0 17.1 13.3MI T T T RS 325 6.68 0. 59 0 0 1.0 13.0 15.0 8.6MI T T T R6 390 5.76 2.10 0 0 0 1.0 4.0 2.0 7.0 0.0MI T R7 520 6.35 0.60 0 0 0 0 0 0 0 0 1.0 R8 340 6.96 1.13 0 0 1.0 3.0 6.0 9.0 4.0 3.6 T R9 165 5.14 1.74 0 0 1.0 2.0 4.0 T T T T RIO 260 5.30 1.87 0 2.0 4.0 4.0 5.0 7.0 8.3 T T R l l 240 5.64 1.64 0 0 0 4.0 8.0 0.0MI T T T R12 220 6.03 1.09 0 0 1.0 3.0 8.0 13.0 T T T R13 220 5.93 3.30 0 0 0 0 1.0 2.0 4.0 8.0 11.2 R14 185 5.53 2.14 0 0 0 0 1.0 3.0 6.0 6.0 3.6MI R15 265 7.09 0.89 0 0 0 0 4.0 4.0 6.0 8.0 4.LMI R16 300 6.92 1.20 0 0 0 1.0 2.0 7.3 14.0 T T R17 5 400 6.42 1.52 0 0 4.0 14.0 24.0 27.0 11.4MI T T R18 5 310 6.11 1.82 0 0 1.0 9.0 22.0 19.2MI T T T 7 0 % d i s t i l l e d w a t e r c o n c e n t r a t e p e r 3 0 % MEM ( v / v ) . > ' N o d e t e c t a b l e a b e r r a t i o n s i n t h e m e t a p h a s e s e x a m i n e d . l M I , m i t o t i c i n h i b i t i o n : l e s s t h a n 4 0 m e t a p h a s e s o b s e r v e d a t d o s e i n d i c a t e d . ' T , t o x i c . ' A g r i c u l t u r a l r e s e a r c h s t a t i o n w o r k e r s . 160 APPENDIX SF EXTENT OF CHROMATID DAMAGE PER METAPHASE PLATE INDUCED BY URINE EXTRACTS (50* ACETONE ELUATE) PREPARED FROM URINE SAMPLES COLLECTED FROM AGRICULTURAL RESEARCH STATION PERSONNEL IN AUGUST 1985 Average Number of Chromatid Exchanges per C e l l C r e a t i n i n e Equivalence (mg/ml) S u b j e c t Number o 1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 RI 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.03 0.04 0.10MI 2 R2 0.00 0.00 0.00 0.00 0.00 0.00 0.12 0.26 R3 0.00 0.00 0.00 0.02 0.12 0.08 0.08 T 3 T R4 0.00 0.00 0.00 0.23 0.40 0.60MI T T T R5 0.00 0.00 0.00 0. 36 0.52 0.31MI T T T R6 0.00 0.00 0.00 0.05 0.09 0.05 0.14 0.00HI T R7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 R8 0.00 0.00 0.01 0.04 0.11 0.18 0.11 0.07 T R9 0.00 0.00 0.01 0.04 0.07 T T T T RIO 0.00 0.10 0.11 0.08 0.11 0.22 0.26 T T R l l 0.00 0.00 0.00 0.08 0.09 0.00MI T T T R12 0.00 0.00 0.03 0.10 0.24 0.36 T T T R13 0.00 0.00 0.00 0.00 0.00 0.01 0.12 0.20 0.30 R14 0.00 0.00 0.00 0.00 0.00 0.00 0.07 0.11 0.04MI R15 0.00 0.00 0.00 0.00 0.06 0.08 - 0.13 0.16 0.12MI R16 0.00 0.00 0.00 0.00 0.04 0.12 0.30 T T R17 0.00 0.00 0.00 0.02 0.09 0.16 T T T R18 0.00 0.00 0.00 0.01 0.05 0.12 T T T ' 7 0 % d i s t i l l e d w a t e r c o n c e n t r a t e p e r 30% M E M ( v / v ) . > " M I , m i t o t i c i n h i b i t i o n : l e s s t h a n 4 0 m e t a p h a s e s o b s e r v e d a t d o s e i n d i c a t e d . ' T , t o x i c . 161 

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