Risks Assessment of Environmental Exposure to Certain Organochemicals in Male Rats: The Possible Modulatory Effect of Micronutrients
THESIS
Submitted In Partial Fulfillment For The Requirement Of Degree Of Doctor Of Philosophy In Science (Biochemistry)
By
Rasha Youssef Mohammed Ibrahim Ass. Lecturer of Biochemistry Atomic Energy B.Sc. (in Biochemistry), Faculty of Science, Helwan University M.Sc. (in Biochemistry), Faculty of Science, Helwan University
Under the Supervision of
Prof. Dr./ Hayat M. Sharada Prof. Dr./ Mohga S. Abdallah Prof. of. Biochemistry, Prof. of. Biochemistry, Faculty of Science, Faculty of Science, Helwan University. Helwan University.
Prof. Dr./ Fatma El Nabarawy Ass. Prof. Dr./ Samia K. Ayad Prof. of Radio Biochemistry, Ass. Prof. of Radio Biochemistry, Atomic Energy. Atomic Energy.
Faculty of Science Helwan University 2011
ACKNOWLEDGMENT
I wish to express my profound gratitude to Prof. Dr. Fatma El Nabarawy Professor of Radiobiochemistry, Radioisotopes Department, Atomic Energy Authority, for her wisdom guidance, assistance and efforts in planning and revising this work. She did her best for achieving the study. I am really indebted to her and will never forget her generosity.
I am very grateful to Prof. Dr. Hayat Mohammed Sharada Professor of Biochemistry, faculty of science, Helwan University, for her sustained encouragement, continuous support, great assistance and kind supervision throughout the work.
I am greatly thankful to Prof. Dr. Mohga Shafik Abdallah Professor of Biochemistry, faculty of science, Helwan University, for her great guidance in achieving this work.
I would like to express my deep thanks to Dr. Samia Kamel Ayad Assistant Professor of Radiobiochemistry, Radioisotopes Department, Atomic Energy Authority, for her great assistance in learning and advising throughout this work.
I am so thankful to Dr. Mona Ibrahim, lecturer of Biochemistry, Radioisotopes Department, Atomic Energy Authority, for her great assistance throughout this work.
I would like to thank DR. Adel Baker Professor of Pathology – cairo university and DR. Laila Rashed Professor of clinical Biochemistry faculty of medicine – cairo university for help in achieving the practical part of the study.
I would like to thank my colleagues in Radioisotopes Department, Atomic Energy Authority, for help in achieving the practical part of the study.
Rasha Youssef
RISKS ASSESSMENT OF ENVIRONMENTAL EXPOSURE TO CERTAIN ORGANOCHEMICALS IN MALE RATS: THE POSSIBLE MODULATORY EFFECT OF MICRONUTRIENTS By
Rasha Youssef Mohammed Ibrahim
ABSTRACT
Trichloroethylene (TCE) is a widely volatile organic compound, because of its widespread commercial use. So, TCE become a major environmental pollutant. It is the most frequently reported organic contaminant in ground water, so a considerable numbers of people are exposed to TCE via inhalation, through the skin or through drinking water and rarely through food. The main symptoms of exposure are headache, dizziness, and confusion, beyond the effects on the central nervous system, work place exposure to TCE has been associated with toxic effects in many organs including liver, kidney and testes in addition to attenuation to the immune system. The present study aims to investigate the possible modulatory effect of certain micronutrients such as vitamin C and zinc alone and in combination on the damage of liver, kidney and testes of male rats intoxicated with trichloroethylene for 20 and 105 days. The results showed significant decrease in body and testes weight and increase in liver and kidney weights after long period of treatment with TCE. Some of the selected hematological and biochemical parameters of the rats intoxicated by TCE for short and long period significantly changed. The results revealed significant decrease in free tetraiodothyronine (thyroxine) (FT4) and significant increase in free triiodothyronine (FT3) and thyroid stimulating hormone (Thyrotropin) (TSH) in TCE intoxicated rat groups for the two periods of treatment. Also results revealed significant decrease of total testosterone in TCE intoxicated rat groups as compared to that of normal control. Also significant changes were detected in the level of immunoglobulins IgG and IgM.
Histopathological examination of liver, kidney and testicular tissues showed significant alteration. The DNA damage was observed in both period of treatment and increased DNA damage with apoptosis was recorded after 105 days of the treatment. Withdrawal group recorded mild improvement in all changed parameters and the damaged tissues also slightly improved and recorded mild decrease in the damage of DNA fragments. The results of the present work showed slight improvement when supplementation was by one micronutrient such as vitamin C or zinc, but highly improved toward normal control if the two micronutrients coadministrated.
In conclusion, we recommend that there must be a threshold limit of dose and time for exposure to trichloroethylene for occupational workers, and recommend that workers and normal people whom may be exposed to TCE can supplement with vitamin C and zinc together to compensate the TCE hazardous effects.
Key words : Organochlorene, trichloroethylene, toxicity, liver, kidney, testes, apoptosis, vitamin C, zinc.
CONTENTS
Page List of Tables List of Figures List of Abbreviations INTRODUCTION AND AIM OF THE WORK 1 REVIEW OF LITERATURE 4 I Trichloroethylene ……………………………………………... 4 Human exposure…………………………………….….…… 6 Metabolism and its role in toxicity………..…...... ….....… 7 Liver Toxicity………………………...... …………..….…. 9 Kidney Toxicity……………...... ……………….…….....….. 10 Reproductive Toxicity……………………….....………..……. 11 Immunotoxicity………………………...... ………..…….... 12 II Apoptosis ………………………………………………...…... 14 Measurement of apoptosis……………………………………… 14 Effect of trichloroethylene on apoptosis…………..………..…... 15 III Micronutrients …………………...... ……...... ….... 16 1 Vitamin C ……………………...... ………………...... ……….. 17 Physiological Role……………………...... …...... ….…….... 19 Dietary Reference Intake…………………...... ….….... 22 Toxicity………………………………………………...……… 22 Efficacy of vitamin C………………………………...……….. 22 2 ZINC …………………………………………….….…….…… 22 PhysiologicalRole…………………………….………….…….. 23 Biological function of zinc……………………..…..………… 24 Toxicity…………………………………….…...... ……… 25 Deficiency………………………………….……...……..…… 25 MATRIALS AND METHODS ……………………………..…… 27 Materials ...... 27 I Animals ……………………………………….………………. 27 Experiment design ………...... ……………..…...…...……... 27 Blood Sampling ...... ………………………………….. 29 Tissue Sampling ...... ……………………..…….. 29 II Chemicals and Analytical Reagents ……………..... ……… 29 Methods …………………………………………………………... I Biochemical Marker Determination …………………..…. 30 i 1 Hematological Parameters ...... …………….……….……….. 30 Red blood cells count (RBC’s)………………..……………… 30 White blood cells count (WBC’s)……………… ……………. 31 Platelets count (Pt. )………………………...... ….…………... 32 Haemoglobin content (Hb)………………………..……...... 33 2 Serum Biochemical Determinations ……………….………. 35 A Liver function tests ………………………………………... 35 Determination of serum total protein………………..……..…. 35 Estimation of serum albumin…………….………………..….. 36 Determination of alanine transaminase (ALT) and aspartate transaminase (AST) activities in serum ………..……….….… 37 Determinayion of alkaline phosphatase activity………...….… 38 Determination of total bilirubin…………………….…..….…. 39 B Renal Function Tests ………………………………………... 40 Determination of serum urea……………………….…...…..… 40 Estimation of serum uric acid…………………….………....… 41 Estimation of creatinine………….……………..……...…....… 42 C Determination of immunoglobulins ……………………….… 44 D Determination of serum hormonal levels ……………..….. 45 Estimation of FT4in serum ……………………..……………. 45 Estimation of FT3in serum………………………………..….. 47 Estimation of TSH in serum………………………………..…. 49 Estimation of Total testosterone in serum…………………….. 51 II Histopathological examination …………………………...... 53 III Detection of DNA fragmentation …………………………… 54 DNA Extraction ………………….………………...... ……… 54 Detection of DNA fragmentation on agarose gel electrophoresis. 55 Statistical Analysis ………………………………………….…... 57 RESULTS ……………………………………………………..….. 58 DISCUSSION ……………………………………………….….… 168 SUMMARY ………………………………………………….…… 189 REFERENCES ……………………………………..………..…… 193 ١ …………………………………………… ARABIC SUMMARY
ii List of Tables
Table (1) Effect of supplementation with vitamin C and /or 67 zinc on body weight change of normal and TCE intoxicated rats for short term (20 days). Table (2) Effect of supplementation with vitamin C and /or 68 zinc on body weight change of normal and TCE intoxicated rats for long term (105 days). Table (3) Variation in the organs weight ratios of normal 69 and TCE intoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Table (4) Variation in the organs weight ratios of normal 70 and TCE intoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Table (5) Effect of supplementation with vitamin C and/ or 71 Zn on % body weight change and % of liver weight / body weight of normal and TCE intoxicated rats for short term (20days). Table (6) Effect of supplementation with vitamin C and/ or 72 Zn on % body weight change and % of liver weight / body weight of normal and TCE intoxicated rats for long term (105days). Table (7) Descriptive and comparative analysis of 73 hematological parameters of normal and TCE intoxicated rats supplemented with vitamin C and/ or zinc for short term (20 days). Table (8) Descriptive and comparative analysis of 74 hematological parameters of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Table (9) Liver function biomarkers in serum of normal and 75 TCE intoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Table (10) Liver function biomarkers in serum of normal and iii TCE intoxicated rats supplemented with vitamin C 76 and /or zinc for long term (105 days). Table (11) Renal function biomarkers in serum of normal and 77 TCE intoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Table (12) Renal function biomarkers in serum of normal and 78 TCE intoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Table (13) Changes in serum IgG and IgM levels of normal 79 and TCE intoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Table (14) Changes in serum IgG and IgM levels of normal and TCE80 intoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Table (15) Changes in serum free thyroxine, free 81 triiodothyronine, thyrotropin and testosterone levels of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for short term (20 days ). Table (16) Changes in serum free thyroxine, free 82 triiodothyronine, thyrotropin and testosterone levels of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Table (17) Grading of histopathological changes in the liver 83 tissues of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for short term (20days). Table (18) Grading of histopathologyical changes in the liver 84 tissues of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for long
iv term (105days). Table (19) Grading of histopathological changes in the 85 kidney tissues of normal and TCE intoxicated rat supplemented with vitamin C and /or zinc for short term (20days). Table (20) Grading of histopathological changes in the 86 kidney tissues of normal and TCE intoxicated rat supplemented with vitamin C and /or zinc for long term (105days). Table (21) Grading of histopathological changes in the 87 testicular tissues of normal and TCE intoxicated rats treated with vitamin C and /or zinc for short term (20 days). Table (22) Grading of histopathological changes in the 88 testicular tissues of normal and TCE ntoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Table (23) DNA dencity in liver, kidney and testes tissues of 89 normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for short and long terms of treatment.
v List of Figures
Fig. (1) Body weight for different groups of normal and TCE intoxicated rats supplemented with vitamin 90 C and/or zinc for 20 days [A], and 105 days [B]. Fig. (2) percentage of body weight change for different groups of normal and TCE intoxicated rats 91 supplemented with vitamin C and/or zinc for 20 days [A], and 105 days [B]. Fig. (3) Effect of supplementation with vitamin C for 20 days [A], and 105 days[B]on serum total protein 92 level for different groups of normal and TCE intoxicated rats. Fig. (4) Effect of supplementation with zinc for 20 days [A], and 105 days [B]on serum total protein level 93 for different groups of normal and TCE intoxicated rats. Fig. (5) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B]on serum total 94 protein level for different groups of normal and TCE intoxicated rats. Fig. (6) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B]on serum albumin 95 level for different groups of normal and TCE intoxicated rats. Fig. (7) Effect of supplementation with zinc for 20 days [A], and 105 days[B]on serum albumin level for 96 different groups of normal and TCE intoxicated rats. Fig. (8) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B]on serum total 97 protein level for different groups of normal and TCE intoxicated rats. Fig. (9) Effect of supplementation with vitamin C for 20 days [A], and 105 days[B]on serum globulin level for different groups of normal and TCE intoxicated rats. 98
Fig. (10) Effect of supplementation with zinc for 20 days 99 vi [A], and 105 days[B]on serum globulin level for different groups of normal and TCE intoxicated rats. Fig. (11) Effect of supplementation with vitamin C &zinc for 20 days [A], and 105 days [B]on serum globulin level or different groups of normal and 100 TCE intoxicated rats . Fig. (12) Serum aspartate transaminase (AST) activity of different groups of normal and TCE intoxicated 101 rats supplemented with vitamin C for 20 days [A], and 105 days [B]. Fig. (13) Serum aspartate transaminase (AST) activity of different groups of normal and TCE intoxicated rats supplemented with zinc for 20 days [A], and 102 105 days [B]. Fig. (14) Serum aspartate transaminase (AST) activity of different groups of normal and TCE intoxicated 103 rats supplemented with vitamin C& zinc for 20 days [A], and 105 days [B]. Fig. (15) Serum alanine transaminase(ALT) activity of different groups of normal and TCE intoxicated 104 rats supplemented with vitamin C for 20 days [A], and 105 days [B]. Fig. (16) Serum alanine transaminase (ALT) activity of different groups of normal and TCE intoxicated 105 rats supplemented with zinc for 20 days [A], and 105 days [B]. Fig. (17) Serum alanine transaminase (ALT) activity of different groups of normal and TCE intoxicated 106 rats supplemented with vitamin C& zinc for 20 days [A], and 105 days [B]. Fig. (18) AST /ALT ratios of normal and TCE intoxicated rats supplemented with vitamin C and/ or zinc for 107 20 days [A], and 105 days [B] . Fig. (19) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum alkaline 108 phosphatase (ALP) concentration for different groups of normal and TCE intoxicated rats. Fig. (20) Effect of supplementation with zinc for 20 days 109 vii [A], and 105 days [B] on serum alkaline phosphatase (ALP) concentration for different groups of normal and TCE intoxicated rats. Fig. (21) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum alkaline phosphatase (ALP) concentration for 110 different groups of normal and TCE intoxicated rats. Fig. (22) Effect of supplementation with vitamin C for 20 days [A], and 105 days[B] on serum total 111 billirubin level for different groups of normal and TCE intoxicated rats. Fig. (23) Effect of supplementation with zinc for 20 days [A], and 105 days[B] on serum total billirubin 112 level for different groups of normal and TCE intoxicated rats. Fig. (24) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days[B] on serum total 113 billirubin level for different groups of normal and TCE intoxicated rats. Fig. (25) Effect of supplementation with vitamin C for 20 days [A], and 105 days[B] on serum urea level 114 for different groups of normal and TCE intoxicated rats. Fig. (26) Effect of supplementation with zinc for 20 days [A], and 105 days[B] on serum urea level or 115 different groups of normal and TCE intoxicated rats. Fig. (27) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum urea 116 level for different groups of normal and TCE intoxicated rats. Fig. (28) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum uric acid level for different groups of normal and TCE intoxicated rats. 117 Fig. (29) Effect of s supplementation with zinc for 20 days [A], and 105 days[B] on serum uric acid level for 118 different groups of normal and TCE intoxicated
viii rats. Fig. (30) Effect of supplementation with vitamin C & zinc for 20 days [A], and 105 days [B] on serum uric 119 acid level for different groups of normal and TCE intoxicated rats. Fig. (31) Effect of supplementation with vitamin C for 20 days [A], and 105 days[B] on serum creatinine 120 level for different groups of normal and TCE intoxicated rats. Fig. (32) Effect of supplementation with zinc for 20 days [A], and 105 days[B] on serum creatinine level 121 for different groups of normal and TCE intoxicated rats. Fig. (33) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum 122 creatinine level for different groups of normal and TCE intoxicated rats. Fig. (34) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum 123 immunoglobulin IgG level for different groups of normal and TCE intoxicated rats. Fig. (35) Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum immunoglobulin 124 IgG level for different groups of normal and TCE intoxicated rats. Fig. (36) Effect supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum 125 immunoglobulin IgG level for different groups of normal and TCE intoxicated rats. Fig. (37) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum 126 immunoglobulin IgM level for different groups of normal and TCE intoxicated rats. Fig. (38) Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum immunoglobulin 127 IgM level for different groups of normal and TCE intoxicated rats. Fig. (39) Effect of supplementation with vitamin C& zinc 128 for 20 days [A], and 105 days [B] on serum
ix immunoglobulin IgM level for different groups of normal and TCE intoxicated rats. Fig. (40) Effect of supplementation with vitamin C for 20 days [A], and 105 days[B] on serum free 129 tetraiodothyronine (FT4) level for different groups of normal and TCE intoxicated rats. Fig. (41) Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum free 130 tetraiodothyronine (FT4) level for different groups of normal and TCE intoxicated rats. Fig. (42) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum free 131 tetraiodothyronine (FT4) level for different groups of normal and TCE intoxicated rats. Fig. (43) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B]on serum free 132 triiodothyronine (FT3) level for different groups of normal and TCE intoxicated rats. Fig. (44) Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum free 133 triiodothyronine (FT3) level for different groups of normal and TCE intoxicated rats. Fig. (45) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum free 134 triiodothyronine (FT3) level for different groups of normal and TCE intoxicated rats. Fig. (46) T4/T3 ratios of normal and TCE intoxicated rats supplemented with vitamin C and/ or zinc for 20 135 days [A], and 105 days [B] . Fig. (47) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B]on serum thyrotropin 136 (TSH) level for different groups of normal and TCE intoxicated rats. Fig. (48) Effect of supplement with zinc for 20 days [A], and 105 days [B] on serum thyrotropin (TSH) 137 level for different groups of normal and TCE intoxicated rats. Fig. (49) Effect of supplementation with vitamin C& zinc 138 for 20 days [A], and 105 days[B] on serum
x thyrotropin (TSH) level for different groups of normal and TCE intoxicated rats. Fig. (50) Percentage change of TSH of normal and TCE intoxicated rats supplemented with vitamin C and/ 139 or Zinc for 20 days [A], and 105 days [B]. Fig. (51) Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum total 140 testosterone level for different groups of normal and TCE intoxicated rats. Fig. (52) Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum total testosterone 141 level for different groups of normal and TCE intoxicated rats. Fig. (53) Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum total 142 testosterone level for different groups of normal and TCE intoxicated rats. Fig. (54) Percentage change of total testosterone of normal and TCE intoxicated rats supplemented with 143 vitamin C and/ or Zinc for 20 days [A], and 105 days [B]. Fig. (55) Histopathological section of the liver tissue of normal rats (gpΙ) supplemented with vitamin C 144 and /or zinc for 20 days. Fig. (56) Histopathological sections of the liver tissue of TCE intoxicated rats (gpΠ, sub gp TCE) for 20 145 days. Fig. (57) Histopathological section of the liver tissue of TCE intoxicated rats supplemented with vitamin 146 C for 20 days. Fig. (58) Histopathological section of the liver tissue of TCE intoxicated rats supplemented with zinc for 147 20 days. Fig. (59) Histopathological section of the liver tissue of TCE intoxicated rats supplemented with vitamin 147 C& zinc for 20 days. Fig. (60) Histopathological sections of the kidney tissue of normal rats (gpΙ) supplemented with vitamin C 148 and/ or zinc for 20 days.
xi Fig. (61) Histopathological sections of the kidney tissue of 149 TCE intoxicated rats for 20. Fig. (62) Histopathological section of the kidney tissue of TCE intoxicated rats supplemented with vitamin 150 C for 20 days. Fig. (63) Histopathological section of the kidney tissue of TCE intoxicated rats supplemented with zinc for 151 20 days. Fig. (64) Histopathological section of the kidney tissue of TCE intoxicated rats supplemented with vitamin 152 C& zinc (gpΠ,) for 20 days. Fig. (65) Histopathological section of the testes tissue of normal rats (gpΙ) supplemented with vitamin C 153 and /or zinc for 20 days . Fig. (66) Histopathological sections of the testes tissue of 154 TCE intoxicated rats (gpΠ,) for 20 days. Fig. (67) Histopathological section of the testes tissue of TCE intoxicated rats supplemented with vitamin 155 C (gpΠ,) for 20 days. Fig. (68) Histopathological section of the testes tissue of TCE intoxicated rats supplemented with zinc 155 (gpΠ,) for 20 days. Fig. (69) Histopathological section of the testes tissue of TCE intoxicated rats supplemented with vitamin 156 C& zinc (gpΠ,) for 20 days. Fig. (70) Histopathological sections of the liver tissue of 157 TCE intoxicated rats (gpΙV,) for 105 days. Fig. (71) Histopathological section of the liver tissue of TCE intoxicated rats supplemented with vitamin 158 C (gpΙV,) for 105 days. Fig. (72) Histopathological section of the liver tissue of TCE intoxicated rats supplemented with zinc 158 (gpΙV,) for 105 days. Fig. (73) Histopathological section of the liver tissue of TCE intoxicated rats supplemented with vitamin 159 C& zinc (gpΙV,) for 105 days. Fig. (74) Histopathological sections of the kidney tissue of 160 TCE intoxicated rats (gpΙV,) for 105 days. Fig. (75) Histopathological section of the kidney tissue of 161
xii TCE intoxicated rats supplemented with vitamin C (gpΙV,) for 105 days. Fig. (76) Histopathol ogical section of the kidney tissue of TCE intoxicated rats supplemented with zinc 161 (gpΙV,) after 105 days. Fig. (77) Histopathological section of the kidney tissue of TCE intoxicated rats supplemented with vitamin 162 C& zinc (gpΙV,) for 105 days. Fig. (78) Histopathological sections of the testes tissue of 162 TCE intoxicated rats (gpΙV,) for 105 days. Fig. (79) Histopathological section of the testes tissue of TCE intoxicated rats supplemented with vitamin 163 C (gpΙV,) for 105 days. Fig. (80) Histopathological section of the testes tissue of TCE intoxicated rats supplemented with zinc 164 (gpΙV,) for 105 days. Fig. (81) Histopathological section of the testes tissue of TCE intoxicated rats supplemented with vitamin 164 C& zinc (gpΙV,) for 105 days. Fig. (82) Histopathological section of the liver tissue of 165 TCE intoxicated rats for 20 days and withdrawn. Fig. (83) Histopathological section of the kidney tissue of 165 TCE intoxicated rats for 20 days and withdrawn. Fig. (84) Histopat hological section of the testes tissue of 166 TCE intoxicated rats for 20 days and withdrawan Fig. (85) Apoptotic laddering pattern showed by Agarose Gel Electrophore sis of DNA fragments of liver, kidney 167 and tests tissues of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc.
xiii List of Abbreviations AA Ascorbic Acid ACTH Adrenocorticotropic homone ALP Alkalin phosphatase ALT Alanin transaminase AST Aspartate transaminase CCL4 Carbontetrachloride CH Chloral hyolnate CNS Central nervous system ALA δ aminolevulinic acid ALAD δ aminolevulinate dehydratase DCAC Dichloro acetyl chloride DCVC 1, 2 Dichlorovinyl L cysteine DHAA dehydro ascorbic acid DNA Deoxyribonucleic acid DRI Dietary reference intake FT3 Free triiodothyronine FT4 Free tetraiodothyronin GSH Glutathion Hb hemoglobin IgG Immunoglobulin G IgM Immunoglobulin M LDH Lactate dehydrogenase MCH Mean corpuscular hemoglobin MCHC Mean corpuscular hemoglobin concentration MCV Mean corpuscular volum P.O Per orall Pt platelets RBC's Red blood cells RNA Ribonucleic acid ROS Reactive oxygen species SLE Systemic lupus erythematosus TCE Trichloroethylene TRI Trichloroethylene TSH white blood cells WBC's Thyroid stimulating hormone Zn Zinc
xiv Introduction & Aim of the work
Several environmental pollutants are called xenobiotics causing the development of autoimmune disease as a consequence of interaction between exposure to xenobiotic and immunoalteration and inflammatory genes (Joanne et al., 2009). Organochlorine compounds are one of xenobiotics that are unique pollutants of human exposure. The pollutant enter the body either by inhalation, dermal contact or ingestion, then carried by the circulation to be accumulated in the liver, kidneys, reproductive system, RBCs, bones and lungs causing chronic long term health problem ( Jarup L. 2003 and Curl , et al., 2002 ).
Trichloroethylene (TCE) is one of several organochlorine compounds that used as an industrial organic solvent, used for cleaning and degreasing of fabricated metal parts. Most people are occupationally exposed to TCE because of its widespread commercial use and improper disposal, TCE has also become a major environmental pollutant, and is the most frequently reported organic contaminant in groundwater (ATSDR, 1997 ).
Most of the trichloroethylene used in industry is released into the atmosphere from industrial degreasing operations. Acute (short term) and chronic (long term) inhalation exposure to trichloroethylene can affect the human central nervous system (CNS), with symptoms such as dizziness, headaches, confusion, euphoria, facial numbness, and weakness. Liver, kidney, immunological, endocrine, and developmental effects have also been reported in humans. A recent analysis of available epidemiological studies reports trichloroethylene exposure to be associated with several types of cancers in humans, especially kidney, liver, cervix, and lymphatic system (ATSDR, 2000 ).
In addition, TCE is found in air emissions, soil, and a wide variety of food, it is readily absorbed into the circulation via 1 Introduction & Aim of the work
oral, dermal, or inhalation exposure due to its high lipophilic property. Considering the multiple routes of exposure, it is not surprising that the Third National Health and Nutrition Examination Survey revealed that about 10% of the general, non occupationally exposed population had detectable levels of TCE in their blood ( Ashley et al., 1994 ).
Organochlorine compounds may disrupt the endocrine system. These compounds have been called endocrine disruptors, because they inhibit the action of natural hormones, and alter the normal regulatory function of the immune, nervous and endocrine systems ( Peterlli et al., 2003 ). Also exert adverse effects include thyroid dysfunction and alteration of immune behavioral function in mammals and rodents (Harvey and Johnson, 2002).
Chronic exposure to TCE and its metabolites induced T cell activation with induced cytotoxic cells causing pathological alteration in kidney proximal tubules of rats and humans which were manifested by presence of necrosis and apoptotic bodies with biochemical and immunological alterations ( Lash et al., 1995a, 2001a).
Exposure to TCE causing DNA faulty that leads to activation of apoptosis. Apoptosis, or programmed cell death, is a process in which cells play an active role in their own death (suicidal cells). Upon receiving specific signals instructing the cells to undergo apoptosis, a number of distinctive changes occur in the cell. Similarly, degradation of enzymes such as DNases, which begin to cleave the DNA in the nucleus ( Dini et al., 2002).
Environmental and occupational exposure to TCE has been linked to the development of a specific form of liver and kidney diseases, potentially to serious health problems. Effects of TCE on male reproduction and fertility have been studied in mice and rats, and assessed in workers exposed to TCE. 2 Introduction & Aim of the work
Micronutrients such as vitamins (A, E, C, and B complex) and minerals (iron, iodine, zinc, selenium) are essential dietary components required in minute amounts for healthy physiological function. Common micronutrient deficiencies are likely to damage DNA by the same mechanism as radiation and many chemicals. Several toxicological studies indicate that micronutrients such as vitamin A, E and C may have either a direct or indirect endocrine modulating activity. Therefore, exogenous supplementation with micronutrients may reduce the damaging effects of organochemicals on the critical biomolecules of the biological systems ( Atti et al., 2003 ).
The present study is undertaken to investigate the potential health risks associated with administration of trichloroethylene for a short or long terms to male rats throughout the determination assays of some hematological, biochemical and immunological parameters that were expected to be disturbed in relation to TCE exposure and costing the light on the histopathologyical changes and apoptotic effects of TCE as contaminants on the liver, kidney and testicular tissues of treated rats by using DNA agarose gel electrophoresis.
Based on these evidences, the present study was an attempt to elucidate the possible modulatory effect of certain micronutrients such as vitamin C and zinc alone and in combination on the damage of livers, kidneys and testes of male rats intoxicated with trichloroethylene, and to assess their possible mechanisms of action.
3 Review of Literature
I Trichloroethylene Organochlorine compounds are organic compounds containing at least one covalently bonded chlorine atom. Their wide structural variety and divergent chemical properties lead to a broad range of applications. These compounds have been isolated from natural sources ranging from bacteria to humans (Gordon and Gribble, 1999).
Chlorinated organic compounds are found in nearly every class of biomolecules including alkaloids, terpenes, amino acids, flavonoids, steroids, and fatty acids ( Kjeld and Engvild, 1986). Organochlorides, including dioxins, are produced in the high temperature environment of forest fires, and dioxins have been found in the preserved ashes of lightning ignited fires that predate synthetic dioxins (Gribble, 1994). In addition, a variety of simple chlorinated hydrocarbons including dichloromethane, chloroform, and carbon tetrachloride have been isolated from marine algae (Gribble, 1996).
A majority of the chloromethane in the environment is produced naturally by biological decomposition, forest fires, and volcanoes (Gribble, 1996). The natural organochloride epibatidine, an alkaloid isolated from tree frogs, has potent analgesic effects and has stimulated research into new pain medication.
The most important of organochlorine is chloromethane such as chloroform, dichloromethane, dichloroethene, and trichloroethane, because of their low molecular weight they are useful solvents. These solvents tend to be relatively non polar therefore they were immiscible with water and effective in cleaning applications such as degreasing and dry cleaning. (Rossberg et al., 2006).
Trichloroethylene is a chemical compound classified as a chlorinated hydrocarbon. It is a nonflammable and colorless liquid, but is characterized by a sweet odor. It was first used in extraction of vegetable oils from plant sources, such as coconut, soy, and palm. Trichloroethylene was also commonly used to extract flavoring agents
4 Review of Literature
from certain spices and herbs, such as hops, in addition to decaffeinating coffee beans. From the 1930s through the early 1960s, the vaporized form of trichloroethylene served as a gas anesthetic substitute for chloroform and ether. Later it was determined that trichloroethylene is a neurotoxin, its application in the food industry or as an anesthetic was discontinued.
Information about the adverse effects on human health from exposure to trichloroethylene has been largely obtained from workplace incidences where exposure has surpassed occupational air standards. Since trichloroethylene is not completely soluble in water, it tends to evaporate quickly from surface water and remain in the air as vapor, when inhaled; TCE can suppress the central nervous system and produce symptoms similar to being intoxicated. In addition, high or long term exposure levels of trichloroethylene may lead to heart abnormalities, compromised immunity, liver and kidney damage .
The symptoms of acute non medical exposure are similar to those of alcohol intoxication, beginning with headache, dizziness, and confusion and progressing with increasing exposure to unconsciousness. Respiratory and circulatory depression can result in death (Orkin, 1986).
Stevens and Kingston (1989 ), reported that exposure to trichloroethylene has been associated with toxic effects on the liver and kidney.
Many research from Cancer bioassays performed by the National Cancer Institute showed that exposure to trichloroethylene is carcinogenic in animals, producing liver cancer in mice, and kidney cancer in rats, with incidence of leukemia and non Hodgkin lymphoma in populations exposed to TCE in their drinking water (ATSDR, 1997 ).
Recent studies in experimental animals and observations in human populations suggest that exposure to trichloroethylene might be associated with congenital heart defects (Lyne and McLachlan, 1949). While it is not clear what levels of exposure are associated 5 Review of Literature
with cardiac defects in humans, there is consistency between the cardiac defects observed in studies of communities exposed to trichloroethylene contamination in groundwater, and the effects observed in laboratory animals.
The health risks of trichloroethylene have been studied extensively. The U.S. Environmental Protection Agency (EPA) sponsored a "state of the science" review of the health effects associated with exposure to trichloroethylene. (EHP, 2000)
The National Academy of Sciences concluded that evidence on the carcinogenic risk and other potential health hazards from exposure to TCE has strengthened since EPA released their toxicological assessment of TCE, and encourages federal agencies to finalize the risk assessment for TCE using currently available information, so that risk management decisions for this chemical can be expedited. Human Exposure:
There is abundant evidence that trichloroethylene has become systemic in the environment. It is found in soil and groundwater samples where it binds to water particles and can reside for long periods of time. In the home, trichloroethylene exposure can occur from the frequent use of typewriter correction fluid or spot removers, in addition to drinking, bathing, or swimming in water that has been contaminated with trichloroethylene. Some are exposed to TCE through contaminated drinking water (Lyne and McLachlan , 1949). Because of its widespread commercial use and improper disposal, TCE has also become a major environmental pollutant. TCE is the most frequently reported organic contaminant in groundwater (ATSDR, 1997).
In addition, TCE is found in air emissions, soil, and a wide variety of food in the United States. TCE is highly lipophilic and is readily absorbed into the circulation via oral, dermal, or inhalation exposure. Considering the multiple routes of exposure, so it is not surprising that the Third National Health and 6 Review of Literature
Nutrition Examination Survey revealed that about 10% of the general, nonoccupationally exposed, population in the United States had detectable levels of TCE in the blood (Ashley et al., 1994).
Other studies reported that chronic exposure to a domestic water supply contaminated with TCE was associated with lupus like symptoms including increased numbers of T cells and increased serum levels of antinuclear antibodies (Abs) (Gist and Burg, 1995; Kilburn and Warshaw, 1992; Nietert et al., 1998 ).
Occupational exposure to trichloroethylene has recently been linked to the development of scleroderma ( Nietert et al., 1998). In addition, environmental exposure to trichloroethylene has been associated with systemic lupus erythematosus (Clark et al., 1994; Kilburn and Warshaw, 1992), systemic sclerosis (Flindt Hansen and Isager, 1987; Lockey et al., 1987; Yanez Diaz et al., 1992) and fasciitis (Waller et al., 1994).
Chloroform (Trihalomethane) and trichloroethylen are two highly volatile toxic chemicals that have been identified in many municipal drinking water supplies, may causing death to many people in the U.S. each year from cancers caused by ingesting these contaminants in water. Chlorine which is present in water combines with organic substances forming trihalomethanes including chloroform. Metabolism and its Role in Toxicity: TCE is metabolized in mice, rats and humans by two primary pathways (Lash et al. 2000, Green et al. 1997). First, oxidative metabolism occurs very rapidly once TCE has entered into the blood stream and forms various metabolites such as chloral, trichloroacetic acid and trichloroethanol.
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The major steps in the metabolic pathway for TCE are illustrated according to Barton et al. (1996).
Oxidative metabolism is considered the major metabolic pathway for TCE, which predominately occurs in the liver as a result of significant cytochrome p 450 activity. Second, conjugation reactions with glutathione (GSH) also account for significant metabolism of TCE and forms the primary metabolite, 1,2, dichlorovinyl L cysteine (DCVC).
Studies on TCE in mouse, rat and human have illustrated substantial differences in the relative importance of the metabolic pathways. Various investigators have related them to specific adverse effects. Green et al. (1997) described the relationship between the accumulation of a TCE metabolite, chloral, in the Clara cells of the lung and the production of lung tumors. Analysis of the metabolites and accumulation of toxicologically significant metabolites such as 8 Review of Literature
chloral and trichloroethanol and their glucuronides in mice were critical observations in assessing the cause of this species specific and organ specific toxicity. The authors concluded that despite positive animal data, humans are "highly unlikely" to be at risk of TCE induced lung damage and subsequent lung tumor development.
Metabolic pathways in mice, rat and human in reproductive systems are likely to play a similarly important role and high risk from TCE exposure on male reproductive structure and function. Liver Toxicity:
It is well documented that trichloroethylene produces hepatotoxicity in experimental animals and humans (ATSDR, 1997a). Rodents exposed to high doses of trichloroethylene or some of its metabolites develop hepatocellular necrosis ( Buben and O’Flaherty, 1985).
Features of hepatotoxicity reference increases in serum glutamic pyruvic transaminase, and the histologic findings includes swollen hepatocytes and minimal evidence of necrosis. (Buben and O’Flaherty, 1985). More recent evidence from mouse studies suggests that an autoimmune response might play a role in trichloroethylene mediated liver disease (Griffin et al., 2000a). There is some evidence that occupational exposure to trichloroethylene results in several forms of non cancer liver disease such as hepatic necrosis, fatty liver, and cirrhosis. It is well established that acute occupational exposure to trichloroethylene does not produce liver injury, whereas chronic exposure does. Case reports have linked occupational exposure to trichloroethylene with Stevens Johnson syndrome (erythema multiform major) of abrupt onset (Phoon et al., 1984).
All these cases demonstrated liver involvement ranging from mild jaundice to fatal liver failure. Another case report documented
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that repeated exposure to trichloroethylene in the work setting resulted in chronic cirrhosis and portal hypertension ( Thiele et al., 1982).
Genetic and environmental factors that influence xenobiotic metabolizing enzymes can favor the formation of trichloroethylene metabolites capable of triggering an immune response against the liver. Contribution of metabolites to hepatotoxicity Chloral hydrate, a metabolic intermediate of trichloroethylene, has been reported to contribute to the hepatotoxic potential of this solvent. Kidney Toxicity: The kidneys are one target organ for TCE, although much controversy exists about its importance for humans, as significant species differences exist in susceptibility ( Lash et al., 2000a).
Renal effects of TCE are generally attributed to its conjugation with GSH and subsequent metabolism within the proximal tubules to generate DCVC, which is further metabolized to a reactive intermediate ( Lash et al., 2000b).
Trichloroethylene causing nephrotoxicity is associated with a multistep metabolic pathway that includes hepatic or renal glutathione S conjugate formation, then enzymatic hydrolysis of the glutathione S conjugates to cysteine S conjugates, followed by renal uptake of cysteine S conjugates. It is generally accepted that the cysteine S conjugate S (1,2 dichlorovinyl) L cysteine is the penultimate nephrotoxicant.
S (1,2 Dichlorovinyl) L cysteine can undergo bioactivation by renal cysteine S conjugate β lyase to reactive species, whose reaction with cellular proteins is associated with cell damage and death (Phoon et al., 1984). A second pathway of haloalkene S conjugates bioactivation and toxification involving sulfoxidation of haloalkene cysteine and mercapturic acid conjugates has been identified (Lash et al., 2000).
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Changes in mitochondrial Ca 2+ ion homeostasis also appear to be a key step in DCVC induced renal toxicity (Lash et al., 1987). Reproductive Toxicity: Zenick et al. (1984) reported that trichloroethylene at an oral dose of 1000 mg/kg/day (5 days/week for 6 weeks) inhibited copulatory behavior in male rats. Because the effects occurred during the first few weeks of exposure and returned to normal after 5 weeks, the narcotic properties of trichloroethylene were suspected to be responsible for the initial changes in copulatory behavior. No effects were observed on semen plug weights or on sperm counts, motility, or morphology. A study of male mice exposed to trichloroethylene via inhalation found significantly increased percentages of abnormal sperm at the highest test concentration.
Investigations of whether testicular steroid precursors (cholesterol and ascorbic acid) or testosterone plays a role in trichloroethylene induced effects showed that total cholesterol content was greater in the testes of rats exposed to trichloroethylene than in controls ( Kumar et al., 2000). The authors concluded that the findings indicated possible impairment of testicular testosterone biosynthesis, which might explain, at least in part, the reproductive inefficiency initially reported. Another study by Kumar et al. (2001) investigates the histomorphology of the testes, sperm count and motility, and marker testicular enzymes involved in sperm maturation and spermatogenesis. They found significant reductions in body and testes weights, total sperm count, and percent motile sperm after 12 and 24 weeks of exposure. Histologically, at 12 weeks, the testes exhibited fewer spermatogenic cells and spermatids in the seminiferous tubules, with some of the spermatogenic cells appearing necrotic. After 24 weeks, the testes were atrophied and harder, with smaller seminiferous tubules. Leydig cells were hyperplastic. Testicular dehydrogenase and glucose 6 phosphate dehydrogenase were significantly reduced, and glutamyltransferase and β glucuronidase were significantly increased. The authors concluded that postmeiotic stages of spermatogenesis in rats were susceptible to 11 Review of Literature
trichloroethylene induced insult. They suggested that exposure to trichloroethylene “may cause testicular toxicity, which in turn affects postmeiotic cells of spermatogenesis, Sertoli cells, and Leydig cell functions” (Kumar et al., 2001). Immunotoxicity: Immunity is the resistance of body to pathologic microbes and their toxins or to other kinds of foreign substances (immune= save from something).
Types of immune response: 1 Humoral responses resulting in the production of specific circulating plasma proteins termed antibodies or immunoglobulins. The cells responsible for production of these antibodies are B lymphocytes (bone marrow derived lymphocytes).
2 Cellular responses leading to the production of specifically reactive small lymphocytes which behaved as if they carried antibody like molecules on their surfaces. The T lymphocytes (thymes derived lymphocytes) mediate the cellular immune responses.
Memory T lymphocytes (effector T lymphocytes) a Cytotoxic T cells: secrete toxic substance called lymphokines that destroying the antigen. b Natural killer T cells: secrete a chemical substance called interferon which interferes with the division of cancer cells and viruses. c Suppressor T cells: which suppress the function of T&B