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Risks Assessment of Environmental Exposure to Certain Organochemicals in Male Rats: The Possible Modulatory Effect of

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 ElNabarawy Ass. Prof. Dr./ Samia K. Ayad Prof. of RadioBiochemistry, 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 ElNabarawy 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 , 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 and rarely through food. The main symptoms of exposure are headache, dizziness, and confusion, beyond the effects on the , work place exposure to TCE has been associated with toxic effects in many organs including , kidney and testes in addition to attenuation to the . The present study aims to investigate the possible modulatory effect of certain micronutrients such as C and zinc alone and in combination on the damage of liver, kidney and testes of male rats intoxicated with for 20 and 105 days. The results showed significant decrease in body and testes weight and increase in liver and kidney weights after long 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 TCEintoxicated rat groups for the two periods of treatment. Also results revealed significant decrease of total in TCEintoxicated 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 was recorded after 105 days of the treatment. Withdrawal 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 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 and its role in toxicity………..…...... ….....… 7 Liver Toxicity………………………...... …………..….…. 9 Kidney Toxicity……………...... ……………….…….....….. 10 Reproductive Toxicity……………………….....………..……. 11 Immunotoxicity………………………...... ………..…….... 12 IIApoptosis ………………………………………………...…... 14 Measurement of apoptosis……………………………………… 14 Effect of trichloroethylene on apoptosis…………..………..…... 15 IIIMicronutrients …………………...... ……...... ….... 16 1 Vitamin C ……………………...... ………………...... ……….. 17 Physiological Role……………………...... …...... ….…….... 19 …………………...... ….….... 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 IAnimals ……………………………………….………………. 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 …………….………………..….. 36 Determination of transaminase (ALT) and aspartate transaminase (AST) activities in serum ………..……….….… 37 Determinayion of alkaline phosphatase activity………...….… 38 Determination of total bilirubin…………………….…..….…. 39 BRenal Function Tests ………………………………………... 40 Determination of serum urea……………………….…...…..… 40 Estimation of serum uric …………………….………....… 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 TCEintoxicated 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 TCEintoxicated 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 in serum of normal and 75 TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Table (10) Liver function biomarkers in serum of normal and iii TCEintoxicated 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 TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Table (12) Renal function biomarkers in serum of normal and 78 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated rats . Fig. (12) Serum aspartate transaminase (AST) activity of different groups of normal and TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 106 rats supplemented with vitamin C& zinc for 20 days [A], and 105 days [B]. Fig. (18) AST /ALT ratios of normal and TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 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 TCEintoxicated

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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated rats. Fig. (46) T4/T3 ratios of normal and TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated rats. Fig. (54) Percentage change of total testosterone of normal and TCEintoxicated 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 TCEintoxicated rats supplemented with vitamin 146 C for 20 days. Fig. (58) Histopathological section of the liver tissue of TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated 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 TCEintoxicated rats supplemented with zinc 155 (gpΠ,) for 20 days. Fig. (69) Histopathological section of the testes tissue of TCEintoxicated rats supplemented with vitamin 156 C& zinc (gpΠ,) for 20 days. Fig. (70) Histopathological sections of the liver tissue of 157 TCEintoxicated rats (gpΙV,) for 105 days. Fig. (71) Histopathological section of the liver tissue of TCEintoxicated rats supplemented with vitamin 158 C (gpΙV,) for 105 days. Fig. (72) Histopathological section of the liver tissue of TCEintoxicated 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 TCEintoxicated rats (gpΙV,) for 105 days. Fig. (75) Histopathological section of the kidney tissue of 161

xii TCEintoxicated rats supplemented with vitamin C (gpΙV,) for 105 days. Fig. (76) Histopathol ogical section of the kidney tissue of TCEintoxicated 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 TCEintoxicated rats (gpΙV,) for 105 days. Fig. (79) Histopathological section of the testes tissue of TCEintoxicated 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 TCEintoxicated rats supplemented with vitamin 164 C& zinc (gpΙV,) for 105 days. Fig. (82) Histopathological section of the liver tissue of 165 TCEintoxicated 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 TCEintoxicated 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 δaminolevulinic acid ALAD δaminolevulinate dehydratase DCAC Dichloro acetyl chloride DCVC 1, 2 DichlorovinylL 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 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 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 (shortterm) and chronic (longterm) 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, , 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 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 metabolitesinduced 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 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 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 (A, E, C, and B complex) and minerals (, iodine, zinc, ) 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 , 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 longterms 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 , 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 atom. Their wide structural variety and divergent chemical properties 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 , flavonoids, , 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 lightningignited fires that predate synthetic dioxins (Gribble, 1994). In addition, a variety of simple chlorinated hydrocarbons including dichloromethane, , and tetrachloride have been isolated from marine (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 nonpolar 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 , 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 . 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 longterm exposure levels of trichloroethylene may lead to heart abnormalities, compromised immunity, liver and kidney damage .

The symptoms of acute nonmedical exposure are similar to those of 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 nonHodgkin 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 . 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 lupuslike 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 (FlindtHansen 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 drinkingwater 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, 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 p450 activity. Second, conjugation reactions with (GSH) also account for significant metabolism of TCE and forms the primary metabolite, 1,2, dichlorovinylLcysteine (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 speciesspecific and organspecific 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 TCEexposure 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 trichloroethylenemediated 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 , 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 Sconjugate formation, then enzymatic hydrolysis of the glutathione Sconjugates to cysteine Sconjugates, followed by renal uptake of cysteine Sconjugates. It is generally accepted that the cysteine S conjugate S(1,2dichlorovinyl)Lcysteine is the penultimate nephrotoxicant.

S(1,2Dichlorovinyl)Lcysteine can undergo bioactivation by renal cysteine Sconjugate βlyase to reactive species, whose reaction with cellular proteins is associated with cell damage and death (Phoon et al., 1984). A second pathway of haloalkene Sconjugates 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+ also appear to be a key step in DCVCinduced 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 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 precursors (cholesterol and ascorbic acid) or testosterone plays a role in trichloroethyleneinduced 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 glucose6phosphate 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 Blymphocytes (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 Tlymphocytes (thymes derived lymphocytes) mediate the cellular immune responses.

Memory T lymphocytes (effector T lymphocytes) a Cytotoxic Tcells: 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 lymphocytes d Helper T cells: this helps the function of the cytotoxic T&B lymphocytes.

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Immunotoxicity can be divided into two areas depending on whether the immune system is activated (such as in allergies or in chemicalinduced autoimmune diseases) or suppressed by xenobiotics (foreign or nonendogenous chemicals, including drugs and environmental chemicals). Mammalian immune systems have innate and adaptive components that play important roles in resistance to infections and cancer.

The immune systems of mammals are formed by primary lymphoid organs, including sac, fetal liver, bone marrow, and thymus. Secondary lymphoid organs (e.g., lymph nodes, spleen, mucosa associated lymphoid tissues) store differentiated cells that await activation by environmental antigens or undergo endogenous selection processes to discriminate self from non self. T and B cells are activated in clonally restricted (antigenspecific) ways, and they demonstrate a memory response. One feature of innate immunity is that the responding cells (macrophages, natural killer cells, ) do not demonstrate clonal specificity. However, families of receptors have been identified (such as tolllike receptors) that allow innate cells to respond to certain families of environmental molecules or toxins (e.g., endotoxin) (Wright et al., 1991)

Xenobiotics may interfere with normal immune system homeostasis by affecting the formation of immune cells; modifying celltocell interactions; modifying cell activation, proliferation, or differentiation; altering cell selection; and enhancing or suppressing the release of immune products such as cytokines, chemokines, antibodies, and complement factors (Aranyi et al., 1986).

The immunotoxicity of chemicals is evaluated in animal models, studies, and occasionally in humans after occupational or environmental exposures. Environmental epidemiology studies are often conducted to determine whether xenobiotic exposures are associated with disease. Because of the complexity of the innate and adaptive immune systems, no single assay can be used to study the potential toxicity of xenobiotics.

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Instead, a tiered approach has been developed and validated by several laboratories for studies in animals (Luster et al., 1988, 1992).

Although there is no single immune assay or parameter that can be used to determine whether a xenobiotic exerts a toxic effect on the immune system, certain combinations of markers and functional assays can predict immunotoxicity (Aranyi et al., 1986).

Various studies indicate that exposures to moderate or high concentrations of trichloroethylene over long periods have the potential to produce immunosuppression in animal models. There are important differences in the amounts and types of immunosuppression depending on species and gender. II Apoptosis Apoptosis is a form of cell death in which a programmed sequence of events leads to the elimination of unnecessary and unhealthy cells without releasing harmful substances into the surrounding area.The human body replaces perhaps a million cells per second by apoptosis.

When programmed cell death does not work right, cells that should be eliminated may hang around and become immortal. So apoptosis is called programmed cell death or cell suicide, and programmed cell death refers to the complete underlying process (Alberts et al., 2008)

Measurement of Apoptosis :

There are many techniques available for the detection of apoptotic cells; some are based on morphological changes, others on biochemical events. However, electrophoretic detection of the systematic cleavage of DNA into oligonucleosomal multimers of 180 200 bp remains the "hallmark" of apoptosis. Agarose gel electrophoresis of DNA from apoptotic cells can be used to resolve the multimers into the characteristic DNA ladders indicative of

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apoptotic cell death according to molecular weight of fragments. (Allen and Newland, 1998).

Visualizing these fragments can aid in characterizing an apoptotic event. This method should always be combined with more quantitative methods in order to compare the degree of apoptosis among the experimental samples ( Shailaja et al., 2006).

Apoptosis has been characterized biochemically by the production of 180200 bp internucleosomal DNA fragments resulting from the activation of an endonucleases. The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern ( Oberhammer et al., 1993).

DNA fragmentation during apoptosis proceeds through an ordered series of stages commencing with the production of DNA fragments of 300 kbp, which are then degraded to fragments of 50 kbp. The 50kbp fragments are further degraded, in some but not all cells, to smaller fragments (1040 kbp) and release the small oligonucleosome fragments that are recognized as the characteristic DNA ladder on conventional agarose gels. Methodology is presented for the detection of the initial stages of DNA fragmentation using pulsedfield gel electrophoresis or a combination of pulsedfield gel electrophoresis and conventional agarose gel electrophoresis that allows detection of the DNA ladder in the same sample (sikorska et al., 1993).

Effect of Trichloroethylene on Apoptosis:

The cancer inducing effect of trichloroethylene (TCE) was studied by various methods. DNA complexing activity and apoptosis inhibition were found to be the key elements of the carcinogenicity of TCE and its metabolites. The ability of TCE to interact with DNA was low, but its incorporation into the RNA and DNA of the ,

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testes, pancreas, kidney, liver, lung and spleen, cannot be excluded. Exposure to TCE and its metabolites provides a selective growth advantage to spontaneously occurring mutations in some K and H ras oncogenes (as non specific results of secondary DNA or RNA damage). Dichloroacetic and trichloroacetic acid were found to be hepatic carcinogens in mice, and the specificity depends on peroxisome proliferation induction. Possibly, TCE and related compounds down regulated apoptosis in mouse liver, and the reduced ability to remove initiated cells by apoptosis could be responsible for liver cancer induction by TCE (Motohashi et al., 1999).

Chloral hydrate (CH) is widely used as a sedative and hypnotic in pediatric medicine. It is also a byproduct of water chlorination and a metabolite of trichloroethylene. Examination of the toxicological effects and cell death mechanisms of CH in rats and human, change liver cells and lymphocytes, found that CH caused a greater cytotoxic effect in human. After treatment with CH, apoptosis features were observed in human lymphocytes, but not change liver cells. CH induced cell damage in lymphocytes may offer signals for the induction of caspases activation (Ho YS et al., 2003).

III Micronutrients Called micronutrients because they are needed only in minuscule amounts, these substances are the “magic wands” that enable the body to produce enzymes, hormones and other substances essential for proper growth and development. The importance of nutrition in protecting the living organism against the potentially lethal effects of reactive oxygen species and toxic environmental chemicals has recently been realized (Lall et al., 1999). Increasing micronutrient status is interpreted in terms more suitable than the more presence or absence of classical deficiency diseases. Low micronutrient intake cause suboptimal status across the whole range of micronutrients and interact with infections and other pathologies. Recently, general awareness has developed about the importance of such as vitamin C and zinc in the protection against free 16 Review of Literature

radical species capable of initiating and propagating lipid peroxidation (WHO, 2011). can mimic radiation (or chemicals) in damaging DNA by causing singleand double strand breaks, oxidative lesions or both. These micronutrients are folic acid, B12, B6, niacin, vitamin C and E, iron and zinc.

Inadequate dietary intake of vitamin C and zinc leads to DNA damage and a wide spectrum of chemical manifestations. Clinical expression of vitamin C deficiency, scurvy, is a lethal condition unless appropriately treated. Thus humans must ingest vitamin C to survive.

Micromineral, including zinc, is indispensable components of certain enzymes responsible for various metabolic processes in different tissues including the brain. Therefore, the present study was designated to investigate the protective effect of vitamin C and/ or zinc supplementation upon TCEinduced toxicity in male Wister rats throughout the assay of some biochemical parameters, histopathological and detection of apoptosis that expected to be disturbed in relation to TCE exposure trace it with vitamin C and/ or zinc supplementation. 1 Vitamin C Vitamin C referred to as ascorbic acid or ascorbate, belongs to the watersoluble class of vitamins. Humans are one of the few species who lack the that convert glucose to vitamin C (Groff et al., 1995). Ascorbic acid (AA) is an odorless, white having the chemical formula C6H8O6. The vitamin is easily oxidized to form dehydroascorbic acid (DHAA), and thus oxidation is readily reversible.

A wide variety of food exists contains vitamin C, the best sources of vitamin C are fruits and their juices. Fruits with high vitamin C content include oranges, , , , and . A wide variety of other foods also contain sufficient quantities of vitamin C. , , , leaf , 17 Review of Literature

tomatoes, potatoes, and beans also have relatively high (7 mg/100 g to 163 mg/100g) vitamin C content (Jacob, 1999).

Absorption of vitamin C is greater when several individual doses of vitamin C, in quantities less than one gram, are taken throughout the day rather than one megadose (Jacob, 1999). Eighty to ninetyfive percent of the vitamin C found in foods is absorbed (Groff et al., 1995). Vitamin C absorption can be impaired by a number of factors. A single large dose saturates the enzyme kinetics for vitamin C, leading to excess AA in the intestinal lumen, which causes numerous gastrointestinal problems. A high iron concentration in the may cause oxidative destruction and in turn impair uptake (Groff et al., 1995). Active transport is the main mechanism of vitamin C distribution within the body. Simple diffusion may occur in the mouth and stomach but accounts for only a very small percentage of uptake (Groff et al., 1995). independent transport systems shuttle vitamin C across the basolateral membrane of the intestinal cells. In the plasma absorbed ascorbic and dehydroascorbate (DHAA) can either be transported freely or be bound to albumin. Ascorbate can also move into body cells and tissues (Groff et al., 1995). As previously mentioned DHAA is the primary form of vitamin C that crosses cellular membranes. The adrenal and pituitary glands, red blood cells, lymphocytes, and neutrophils all receive vitamin C in the form of DHAA (Groff et al., 1995). Vitamin C is stored throughout body tissues and blood. Ascorbic acid content of blood components, fluid, and tissue varies widely on an individual basis. (Howald et al., 1975). Other tissues with intermediate levels of vitamin C include the kidneys, brain, liver, lungs, and thyroid. The watersoluble properties of vitamin C prevent it from being stored in the adipose tissue of the body.

The halflife of AA is believed to be between 16 and 20 days (Jacob , 1999). Its halflife is inversely related to intake. The watersoluble properties of vitamin C lead to urinary excretion of the vitamin. 18 Review of Literature

Metabolites of vitamin C including dehydroascorbate (DHAA), oxalic acid, 2Omethyl ascorbate, and 2ketoascorbitol are also excreted from the body via the urinary system (Groff et al., 1995). The kidneys play a major role in vitamin C excretion and retention. DHAA and AA can be reabsorbed by the kidney tubules as long as body pool levels are equal to or less than 1500 mg.

Physiological Role:

Vitamin C has been participates in numerous biochemical reactions, suggesting that vitamin C is important for every body process from bone formation to scar tissue repair (Groff et al., 1995). The only established role of the vitamin appears to be in curing or preventing scurvy. Vitamin C is the major watersoluble within the body. The vitamin readily donates to break the chain reaction of lipid peroxidation.

The watersoluble properties of vitamin C allow for the quenching of free radicals before they reach the cellular membrane. Tocopherol and glutathione also rely on AA for regeneration back to their active isoforms. The relationship between AA and glutathione is unique. Vitamin C reduces glutathione back to the active form. Once reduced, glutathione will regenerate vitamin C from its DHAA or oxidized state.

The prophylactic effects of vitamin C as an antioxidant during exercise, when free radical formation is high. A wellknown function of AA is the role it plays in reactions that are essential for the formation of collagen. Vitamin C is important in collagen formation as it allows for a tight crosslinking of the triple helix, thereby resulting in stabilization of the peptide. Evidence also suggests that AA may be involved in collagen . However, this mechanism is not well understood. synthesis prefers to use vitamin C as the (Groff et al., 1995).

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Carnitine facilitates the betaoxidation of fat, through its role transporting long chain fatty acids from the cytoplasm into the mitochondrial matrix of cardiac and skeletal muscle. Vitamin C is directly involved in the enzyme activity of two dependent monooxygenases, which are important in the formation of norepinephrine and serotonin (Groff et al., 1995). Furthermore, AA regulates the activity of some within the brain. Some of these functions include membrane receptor synthesis, and neurotransmitter dynamics. Indirectly, AA plays important regulatory roles throughout the entire body due to its involvement in the synthesis of hormones, hormonereleasing factors, and (Groff et al., 1995).

Animal models have also shown that AA is an important factor in development of the nervous system, specifically in the maturation of glial cells and myelin (Jacob , 1999). Vitamin C is important to a host of numerous other functions within the body. The vitamin is an important aid in the absorption and conversion of iron to its storage form. Bile acid formation, and hence cholesterol degradation are highly dependent on AA. Some hypothesize that vitamin C may have a hypocholesterolemic effect. This because the enzyme needed for the first step in bile acid synthesis, cholesterol 7alpha hydroxylase, is dependent upon the presence of vitamin C. Ascorbic acid may also has vasodilatory and anticlotting effects within the body by stimulating nitric release. Physiological effects such as an antihistamine modified bronchial tone, and responses have been linked to AA.

The protection of neural and endothelial tissue, along with effects on cellular tone can also be attribute to vitamin C. Possible other functions for vitamin C include regulation of cellular concentrations, immune function, and the endocrine system. Vitamin C has been proposed by some to have pharmacological benefits in

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preventing cancer, infections, and the . However, these benefits have yet to be reported in the scientific literature.

Dietary Reference Intake (DRI): The Recommended Daily Allowance (RDA) has been replaced by a DRI for vitamin C in the year 2000. In 1989 the RDA was established at 60 mg for adults. This level was believed to be sufficient enough to maintain body pool levels at 1500 mg and does not differ from that established in 1980. A RDA for smokers was established in 1989 at 100 mg/day. This greater RDA was established because smokers have a higher turnover rate of vitamin C versus non smokers (Groff et al., 1995). The DRI does not differentiate the need between smokers and nonsmokers. Dietary reference intakes for vitamin C have been established at 90 mg for men and 75 mg for women (NAS, 2000).

Toxicity: The saturable kinetics of vitamin C makes toxicity more likely when multiple large doses (~1gram) are consumed throughout a day versus one single dose. A common symptom of unabsorbed vitamin C left in the gastrointestinal tract is osmotic (Groff et al., 1995). Vitamin C can be transformed in the body to , which is a common constituent of kidney stones. Doses up to 10 grams have shown to be associated with a higher prevalence of oxalate excretion, but the level does not fall outside of the normal range. As a precaution, people who are prone to kidney stones may want to avoid large doses (10 times the DRI or greater) of the vitamin (Groff et al., 1995). People who lack the control to regulate iron uptake should also avoid large doses of the vitamin. As stated earlier vitamin C enhances iron absorption which, can lead to toxicity of iron in some people. Furthermore, excess ascorbate in the urine and feces can falsify lab tests such as glucose in the urine and fecal occult blood test.

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Efficacy of Vitamin C :

It removes chlorine contents from tap water. Chlorine in tap water weakens the skin immunity, accelerates the appearance of ageing in skin, makes skin dry, damages hair, and triggers atopic dermatitis. When vitamin C meets with chlorine that is used to sterilize tap water, a chemical reaction takes place, completely removing the chlorine. This is a sort of purification process, which can greatly reduce skin problems. Vitamin C makes hair and skin more supple and elastic. Beauty ingredients and chitosan contained in the vitamin C filter keep skin moisturized while removing harmful matter. This, in turn, will strengthen the immunity of your skin and hair, making them look and feel healthier. 2 Zinc Zinc is widely distributed in nature. Grain, vegetables and fruits are poor in zinc. While , fish and marine organisms are rich in zinc ( Elinder and Piscator, 1979 ). The bioavailability of zinc depends on different factors that inhibit or facilitate it. , for instance, are rich in zinc, but the absorption is poor due to the phytate that are also present ( Reinhold et al., 1976). The body content in a 70 kg man is about 2.5 g zinc, which is mainly in muscles (60%) and skeleton (30 %), the remaining 10 % is distributed in all tissues with highest concentrations in eyes, , and hair. All tissues levels depend on age ( Jackson, 1989). The mean daily requirement for humans is about 1015 mg taking into account a bioavailability in food of approximately 20 % but this may fluctuate according to circumstances. Higher requirements are needed for children (300 g/kg/day), during pregnancy (1520 mg/day), and lactation (up to 25 mg /day) (Agett, 1985).

The absorption of Zn 2+ mainly occurs in the small intestine and especially in the jejunum. The absorption in the duodenum and in the stomach seems to be very low Zn 2+ is absorbed at the level of the

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intestinal epithelial cell, possibly in the form of complexes with amino acids, citrate, etc. Then, inside it combines with metalloenzymes, membrane proteins, and , the later probably being a regulator of the homeostasis of Zn 2+ (Kirchgessner and Weigand, 1983). In blood the absorbed zinc is distributed between red blood cells (80 %), plasma (17 %) and white blood cells (3 %) .

Animal proteins and amino acids present in a meal increase the absorption of zinc, which is normally about 20% . Intake of emphasizes this phenomenon. Zinc solutions taken on an empty stomach are absorbed to about 60% . Phytates seem to be powerful antagonists of zinc absorption (Sandstorm and Lonnerdal, 1989).

Unsaturated fatty acids increase zinc absorption by their action on prostaglandin synthesis, aspirin, perhaps by inhibition of the synthesis of prostaglandin, decreases it ( Kirchgessner and Weigand, 1983 ). also decrease the absorption of Zn 2+ by formation of insoluble complexes (Sandstorm and Lonnerdal, 1989 ). Copper at high levels, ferrous compounds and (Sandstorm and Lonnerdal, 1989 ) compete with zinc for absorption. The capability to absorb Zn 2+ decrease with age (Spencer et al., 1976). The intestine is the major excretion organ of zinc (Jackson et al., 1984). It loss through epithelial skin cells, . The sperms are other minor route of excretion (Cousins and Smith, 1980).

Physiological Role:

Zinc is necessary for various physiological functions including the growth and multiplication of cells (enzymes responsible for DNA and RNA synthesis, growth hormones, etc.), , skin integrity (synthesis of protein, especially collagen (Elinder and Piscator, 1979 ), bone metabolism (diminution of alkaline phosphatases, osteoblasts and chandrocytes), functioning of male reproductive organs and fertility (Bunce, 1989).

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One of the main biochemical roles of zinc is its influence on the activity of over 300 enzymes, which are distributed into six classes; oxidoreductases, transferases, hydrolases, layases, isomerases, and ligases. Zinc can be essential for the structure, regulation and / or the catalytic action of an enzyme Zn 2+ occurs in enzymes that realize the synthesis and metabolism of DNA and RNA (Riordan and Vallee, 1975), it influences the metabolism and the synthesis of proteins, it participates in glycolysis and gluconeogenesis and in prostaglandin synthesis and cholesterol metabolism, it prevents lipid peroxidation and maintains membrane structures (Elinder and Piscator, 1979 ). Other roles of zinc include its effect on the storage, secretion, polymerization, and receptive fixation of insulin. It influences the biosynthesis of adrenocorticotropic hormone (ACTH), it occurs in biologically active proteins such as growth factor (Kirchgessner and Roth, 1983) or gustin (Henkin et al., 1988).

Biological Function of Zinc:

Zinc is present in every cell of the body, and participates in all major metabolic pathways. It is a essential to the function of over 160 enzymes including those required for DNA and RNA synthesis (Simmer, 1986). Over 40 metalloenzymes exist in which zinc is bound lightly to the apoenzyme in specific stoichiometric ratios, and in which it serves one or more structural, regulatory or catalytic functions (Riordan and Vallee, 1976). Mammalian metalloenzymes including , carbxypeptidase, , alkaline phosphatase, alcohol retinal, malate, lactate, glutamate, and glyceraldehyde 3phosphate dehydro genase. Some metaloenzymes require additional for activity; cytosolic has a requirement for copper as well as zinc.

Zinc is essential for all phases of the cell cycle. Deficiency of zinc is associated with RNA disaggregation and increase ribonuclease activity (Prasad and Oberieas, 1973). The ability of zinc to inhibit

24 Review of Literature

adenylate cyclase and phosphodiestrase may indicate a role in cell functions and genetic expression by regulating the relative intracellular concentrations of cyclic AMP (Mc Machon, 1974). In human lymphocytes, zinc acts as mitogen, but the mechanism is unclear but appears to be mediated by monocytes. Zinc stabilizes plasma and subcellular membranes (Chvapile, 1973), as well as nucleic acids and microtubules (Nickolsen and Veldstra, 1970), also it stabilizes lysosomes. Membrane lipid peroxidation is increased in states.

Toxicity:

Zinc is relatively nontoxic. Nevertheless, high doses (1g) or repetitive doses of 100 mg/ day during several months may lead to disorders. Among these are gastrointestinal tract symptoms (Solomons and Cousins, 1984), a decrease in heme synthesis due to an induced , diminution of high lipoproteins (HDLs), hyperglycemia, reduction of serum and copper (Fox, 1989), and an increase of intestinal and alkaline phosphatase activity. Exposure to ZnO fumes and dust may induce chemical pneumonia and then sever pulmonary , with fever, hypernea, coughing, pains in legs and chest, vomiting, etc. this is called the zinc fume fever (Elinder and Piscator, 1979 ).

Deficiency:

Zinc deficiency in humans may have different origins e.g. by diminution of intake (phytates, fibers), increased needs (pregnancy, lactation, growth), or increased losses (burns, chelating agents). Sever deficiency may lead to achrodermatitis enteropathica. The main clinical manifestations of this are skin injury (parakeratosis, keratitis, and roughened hyper pigmented skin), alopecia, poor wound healing (susceptibility to infections), anorexia, loss of senses (taste, smell, and eyesight), growth retardation, impaired development and function of

25 Review of Literature

male reproductive organs, and immunodeficiencies. There are also indications that Zn 2+ deficiency is teratogenic in humans during pregnancy (Elinder and Piscator, 1979 ).

Zn has been shown to have an antioxidant role(s) in defined chemical systems. Two mechanisms have been elucidated; the protection of sulfhydryl groups against oxidation and the inhibition of the production of reactive by transition metals. Supraphysiological concentrations of Zn have antioxidantlike effects in organellebased systems and isolated cellbased systems in vitro. Administration of pharmacological doses of Zn in vivo has a protective effect against general and liverspecific prooxidants (Tammy and William, 1990).

Zinc treatment in liver cirrhosis is known to prevent a number of clinical symptoms. Previous studies have also indicated that Zn has a protective effect on the development of the clinical, biochemical and morphological manifestations of hepatic injury if administered simultaneously with the noxious agent. The protective effects of zinc treatment against the development of liver cirrhosis have been tested in cirrhotic rats treated by intragastric administration of carbontetrachloride (CCl4) ( Gaudio et al., 1993).

26 Matrials and methodes

MATERIALS

І Animals:

The present study was performed on 102 male Wister albino rats weighing 120 150 grams. They were purchased from the modern veterinary office, Elharam, Giza. All animals were kept in cages with controlled temperature (2530°C) at the animal house, Radioisotopes Department, Atomic Energy Authority. All animals were freely fed on normal rodent pellets and clean water offered adlibitum throughout the experimental period. They were acclimatized to their environment for two weeks before starting the experiment and treatment.

Experiment design :

Rats were classified into four groups according to the following protocol:

Group I : The animals of this group were considered as normal control. They were divided into four subgroups each 6 rats and treated as follows:

normal control: given normal saline in volume of 1ml/100gm.

normal control+ vit.C: supplemented with vit.C, 50 mg/kg body weight/ day, according to Sebastian et al.(2008) per orally (p.o.).

normal control +Zn: supplemented with zinc sulphat 200 mg/kg body weight/ day, according to Unsal et al.(2003) p.o.

27 Matrials and methodes

normal control + vit.C &zinc: supplemented with vit.C 50 mg/kg body weight/ day, followed by zinc sulphat 200 mg/kg body weight/ day, p.o.

Group II : The animals of this group were treated with TCE at dose level of 750 mg/kg body weight /day according to khan et al. (2008) P.o. They were divided into 5 subgroups each 6 rats and treated as follows:

TCEintoxicated control: given normal saline in volume of 1ml/100g.

TCEintoxicated rats + vit.C: supplemented with vit.C, 50 mg/kg body weight/ day, p.o.

TCEintoxicated rats+ zinc: supplemented with zinc sulphat 200 mg/kg body weight/ day, p.o.

TCEintoxicated rats+ vit.C & Zn: supplemented with vit.C 50 mg/kg body weight/ day, followed by zinc sulphat 200 mg/kg body weight/ day, p.o.

Withdrawal of TCE: treated with TCE for 20 days at the start of the experiment and withdrawn for recovery until the end of the experiment.

The treatment of group I & II were continued for 20 days (short term).

Group III and IV : The animals of this group were classified as group I and II but the treatment of TCE and the supplementation of vitamin C and zinc continued to 105 days (long term).

28 Matrials and methodes

Blood Sampling:

After termination of the treatments, the tested rats were sacrificed succeively under anesthesia. Blood samples were withdrawn from the heart carefully avoiding hemolysis; one part of blood samples was received in separate tubes containing EDTA as anticoagulant to be used for determination of the hematological parameters. The other part of the blood samples were incubated for 30 minutes in water bath at 37° C, and then centrifuged at 4000 rpm for15 minutes. The separated sera were transferred into clean polypropylene tubes and kept at 18° C until the assays of biochemical determinations.

Tissue Sampling:

Liver, kidney and testes tissues were quickly excised and weighed. Tissue portions were collected and preserved in 10% formal saline for histopathological examination, and other portions of liver, kidney and testes were preserved frozen in liquid and stored at 80°C until apoptosis assay using DNA laddering.

II Chemicals and Analytical Reagents:

The sourcesof the chemicals and test reagent kits used in this study are included in the following list:

29 Matrials and methodes

Chemicals and test reagent kits source Alanine transaminase (ALT). Quimica Clinica Aplicapa, SPAIN Albumin. Stanbio laboratory, Texas – USA. Alkalin phosphatase (ALP). Quimica Clinica Aplicapa, SPAIN Ascorbic acid (vit. C). Elgomhoria Company, Egypt. Aspartate transaminase (AST). Quimica Clinica Aplicapa, SPAIN Billirubin. Quimica Clinica Aplicapa, SPAIN Creatinine. Stanbio laboratory, Texas – USA. Free triiodothyronine (FT3). Seimens Healthcare, USA. Free tetraiodothyronine (FT4). Seimens Healthcare, USA. Immunoglobulin G. Biocientifca S.A. Argentina. Immunoglobulin M. Biocientifca S.A. Argentina Thyroid stimulating hormone DPC'S Technical srvices, USA. (TSH). Total ptotein. Stanbio laboratory, Texas – USA. Total testosterone. Seimens Healthcare, USA. Trichloroethylene (TCE). Elgomhoria Company, Egypt. Urea. Quimica Clinica Aplicapa, SPAIN Uric acid. Stanbio laboratory, Texas – USA. Zinc sulphat. Elgomhoria Company, Egypt.

METHODS I Biochemical Marker Determinations: 1 Hematological Parameters: The hematological parameters included; red blood cells count (RBC’s), white blood cells count (WBC’s), Platelets count (Pt), hemoglobin content (Hb), total and differential leucocytic count. Red blood cells count: The red blood cells were counted by visual means using neubauer hemocytometer as described by Dacie and Lewis (1995).

30 Matrials and methodes

Assay procedure: The blood in formal citrate solution was diluted to (1: 200). This was done by washing 20 l of blood taken into a micropipette into 4 ml of diluting fluid contained in a glass or plastic tube. After sealing the tube with a tightly fitting rubber or plastic bung, the diluted blood was mixed in a mechanical mixer or by hand for at least 2 min. by tilting the tube through an angle of about 120° combined with rotation, thus allowing the air bubble to mix the suspension. A clean dry counting chamber was filled with its cover glass already in position, without delay. This was accomplished with the aid of a Pasteur pipette. The chamber was left undisturbed on a bench for at least 2 min. for the cells to settle, but not much longer, for drying at the edges of the preparation may initiate currents which cause movement of the cells after they have settled. It is important that the cover glass should be of a special thick glass and perfectly flat, so that when laid on the counting chamber, diffraction rings were seen. The cover glass should be of such a size that when placed correctly on the counting chamber, the central ruled areas lie in the center of the rectangle to be filled with the cell suspension. The cells were counted using a 4 mm dry objective and × 6 or × 10 eyepieces. Calculation: No. of cells counted 6 6 Red blood cells count (10 /L) = × Dilution × 10 Volume counted (L)

White blood cells count: White blood cells were counted by visual means using Neubauer hemocytometer as described by Dacie and Lewis (1995).

31 Matrials and methodes

Assay procedure: The blood was diluted (1 in 20 dilution) by adding 20 L of blood to 0.38 ml of diluting fluid [it consist of 2 % (20 ml) colored pale violet with gentian violet] in a glass or plastic tube. After tightly crooking the tube, the suspension was mixed by rotating in a cell suspension mixer for at least 1 min. The counting chamber was filled by means of a Pasteur pipette or stout glass capillary, as for red – cell counts. The red cells were lysed by the diluting fluid but the leucocytes remained intact, their nuclei were stained deep violet–black. The preparation was viewed using a 4 mm objective and ×6 eyepieces or a 16 mm objective and ×10 eyepieces. At least 100 cells were counted in as many 1 mm² area (0.1 L in volume) as may be necessary, the ruled area in an improved Neubauer chamber consists of 9 of these areas.

Calculation: No. of cells counted Count of WBCs (10 3 /L) = × Dilution × 10 3 Volume counted (L)

Platelets count: Platelet was counted by using haemocytometer as described by Lewis et al. (1979).

Reagents: 0.15% glutaraldehyde in isoton II (coulter). 3 ml of 50% glutaraldehyde was added to 1L of isoton II and mixed well. This reagent should be prepared and used immediately. 0.15% glutaraldehyde in glycerol / isoton II. To each 600 ml of glycerol 400 ml of isoton II and 3 ml of 50% glutaraldehyde were added and mixed well.

32 Matrials and methodes

Assay procedure: Mixed fresh blood was centrifuged for 10 minutes, and platelet rich plasma was collected. 100ml volumes of glutaraldehyde isoton II was dispensed into a series of clean dry 150 ml screwcapped glass bottles and to each 15 ml of the plateletrich plasma. This capped and mixed on a roller mixer for 20 minutes.

The suspensions was pooled in a 2 L round bottomed flask and rotated at 4°C. 50 ml volume of glutaraldehyde glycerolisoton II solution was dispensed into another set of 150 ml screwcapped bottles; carefully 50 ml of filtered fixed platelet suspension was layered on to this solution then caped, stand and allowed platelets to drift through glycerol based mixture where yellowish supernatant became clear and lower layer milky. Platelet was resuspended by shaking and roller mixing for 20 minutes. 1 vial of streptomycin was added to every 500 ml of platelet suspension, and with continuous mixing 510 ml volumes was dispensed into sterile glass containers and caped tightly. Values were assigned and measured by haemocytometer.

Calculation: No. of cells counted Count (10 3 /L) = × Dilution × 10 3 Volume counted (L)

Determination of hemoglobin content (Hb): Principle: Blood hemoglobin content (g/dl blood) was determined by cyanmethemoglobin method using Drabkin´s solution as described by Dacie and Lewis (1984).

33 Matrials and methodes

Reagents: Diluent: The original Drabkin reagent has a pH of 8.6. The following modified solution, which has a pH of 9.6, is less likely to cause turbidity from precipitation of plasma proteins, it consist of potassium ferricyanide 200 mg, potassium 50 mg, water to 11. Assay procedure: 20 L of blood was added to 4 ml of diluent and the tube containing the solution was inverted several times. After being allowed to stand at room temperature for a sufficient period of time to ensure the completion of the reaction (310 min.), the solution is ready to be compared with the standard and reagent blank in a spectrophotometer at 540 nm. The absorbance of the test sample must be measured within 6 hours of its being diluted Calculation: A sample Hb (g/dL) = × Standard

A standard

A sample is the absorbance of the sample. A standard is the absorbance of standard.

Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), and Mean Corpuscular Hemoglobin Concentration (MCHC). Were calculated according to following formulas:

PCV × 10 MCV = Erythrocyte count (millions/microliter)

Hemoglobin (g/dl) × 10 MCH =

Erythrocyte count (millions/microliter)

34 Matrials and methodes

Hemoglobin (g/dl) × 100 MCHC= PCV

2 Serum Biochemical Determinations Laboratory investigations were carried out in the Central Laboratory of Analysis Services, Radioisotopes Department, Atomic Energy Authority .

A Liver function tests: The liver function test included: alanine transaminase (ALT) activity, aspartate transaminase (AST) activity, alkaline phosphatase activity, and other liver functions as albumin, globulin, total protein, and total bilirubin.

Determination of serum total protein: Total protein was determined colorimetrically according to biuret method (Gornal et al., 1949). Reagent Total protein reagents: Copper Sulphate pentahydrate 0.3 g/dL 0.8 g/dL Potassium iodide 30 mmol Potassium Sodium tartrate 32 mmol Total Protein Standard 10 g/dL

Assay procedure: In a blank, standard and sample tubes 1 ml of total protein reagent was added, and 10 L standard solution was added to standard tube, then 10 L of serum was added to the sample tube. All tubes were mixed and left for at least 5 minutes at room temperature. The absorbance was measured using colorimetric technique at wavelength 550 nm. 35 Matrials and methodes

Calculation: The concentration of total protein (g/dL) was measured using the following equation:

A sample Serum total protein (g/dL) = ×10 A standard

A sample is the absorbance of sample. A standard is the absorbance of standard. 10 is the concentration of standard (g/dL).

Estimation of serum albumin: Albumin was determined colorimetrically in serum according to the method of Dumas et al. (1971). Reagents: Bromocresol green 18.8 mg/dl Citrate buffer, pH 4.2 and Surfactant. Albumin standard 6 g/dl

Assay procedure: In a blank, standard and sample tubes, 1 ml of albumin reagent was added, and 10 L standard solution was added to standard tube, then 10 L of serum was added to the sample tube. All tubes were mixed and the color was developed immediately. The absorbance was measured using colorimetric technique at wave length 550 nm within 15 minutes. Calculation: The concentration of albumin (g/dL) was measured by the following equation.

A sample Serum albumin (g/dL) = × 6 .0 A standard A sample is the absorbance of sample. A standard is the absorbance of standard. 6 is the concentration of standard.

36 Matrials and methodes

Determination of alanine transaminase (ALT) and aspartate transaminase (AST) activities in serum: Colorimetric determination of ALT and AST activities were carried out according to Reitman and Frankel (1957) method.

Principle: Colorimetric determination of ALT or AST activities according to the following reaction: AST Aspartate + αketoglutarate oxaloacetate + Glutamate.

ALT Alanine + αketoglutarate pyruvate + Glutamate.

The pyruvate or oxaloacetate formed is measured in its derivative form, 2,4 dinitrophenyl hydrazone.

Reagents: Reagent 1: AST Substrate This reagent contains phosphate buffer pH 7.4, 100mmol / L, aspartate 100 mmol / L and αketoglutarate 2 mmol /L. Reagent 2: ALT Substrate This contain phosphate buffer pH 7.4, 95 mmol /L, alanine 200 mmol /L and αketoglutarate 2 mmol /L. Reagent 3: Color reagent This composed of 2,4dinitrophenyl hydrazine 1mmol /L. Reagent 4: Standard This contain pyruvate, NaOH 0.4 N. All of these reagents stored at 28°C, and regent 3 stored in dark.

Assay Procedure: In two test tubes 0.5 ml of reagent 1 (AST Substrate) and also 0.5 ml of reagent 2 (ALT Substrate) were added to each tube and the both were incubated for 5 min. at 37°C, then 0.1 ml of 37 Matrials and methodes

serum was added to the tubes and mixed well. Incubation was carried out at 37° C for exactly one hour in case of AST and exactly 30 min. in case of ALT. After incubation, 0.5ml of reagent 3 was added to each tube, then the tubes mixed and left stand for 20 minutes at room temperature. 5 ml of 0.4 N NaOH was added to each tube, then mixed and waited 5 minutes before measured. The measurement was carried out at wavelength 505 nm.

Calculation: The activities of ALT and AST were estimated using the calibration curve.

- Determination of alkaline phosphatase activity: Alkaline phosphatase activity was estimated according to Szasz and Rautenburg (1971).

Principle: Kinetic determination of alkaline phosphatase activity was carried according to the following reaction. ALP p Nitrophenyl phosphate +H 2O p Nitrphenol + Phosphate

The liberated was measured in the presence of amino4antipyrine and potassium ferricyanide.

Reagents: Reagent 1 (Buffer solution) Reagent 2 (Substrate tablets)

Working reagent: One tablet of the substrate was dissolved in 3ml of buffer solution, and let stand 35 min. and mixed well until completely dissolved, the concentration of working reagent were: diethanolamine/HCl buffer pH 9.8 1M

38 Matrials and methodes

pNitrophenylphosphate 10mM

MgCl 2 0.5mM Assay procedure: In each serum samples and blank tubes 3 ml of working reagent was added. They were incubated in water bath at 37° C for 5 min, and then 50 •L of serum was added to the tube of serum sample, mixed well and let stand for 4min. then the absorbance was measured at wave length 405 nm. Reagent blank was zero adjustment.

Determination of total bilirubin: Principle: Colorimetric method for the quantitative determination of total bilirubin was estimated according to Jendrassik and Grof (1938).

Total bilirubin is determined in the presence of , which release albumin bound bilirubin, by the reaction with diazotized sulphanilic acid. Reagents:

Reagent 1: Sulphanilic acid 29 mmol/l 0.17 N

Reagent 2: Sodium Nitrite 25 mmol/l

Reagent 3: Caffeine 0.26 mol/l Sodium benzoate 0.52 mol/l Reagent 4: Tartrate 0.93 mol/l Sodium Hydroxide 1.9 N Assay procedure: In each sample blank tube and sample tube, 0.2 ml of reagent 1 was pipetted, then 0.05 ml of reagent 2 was added to sample tube and 1 ml of reagent 3 was added to both tubes. The 39 Matrials and methodes

tubes were mixed and allowed to stand for 10 min. at 2025°C, then 1 ml of reagent 4 was added to both tubes, the tubes were mixed and allowed to stand for 530 minutes at 2025° C, and then read. The absorbance of the sample against sample blank was recorded at 578 nm. Calculation: Total bilirubin (mg/dl) = 10.8 × ATB ATB: Absorbance of total bilirubin.

B Renal function tests The kidney function parameters included: urea, uric acid and creatinine. Determination of serum urea: Enzymatic colorimeteic method was used for determination of urea as described by Foster and Hochholzer (1979). Reagents:

Reagent A: This reagent contains phosphate buffer 20 mmol/L, sodium salicylate 61 mmol/L, sodium nitroprusside 3.4 mmol/L, EDTA 1.34 mmol/L, urease 23 U/ml. The contents were dissolved 1:100 ml with deionized water.

Reagent B: Phosphate buffer 20 mmol/Lsodium hydroxide 160 mmol/L sodium hypochloride 7.5 mmol/L. The contents were diluted up to 500 ml with deionized water.

Standard (40 mg/dL): It contains aqueous solution of urea with sodium azide used as stabilizer.

40 Matrials and methodes

Assay procedure: In sample and standard tubes 10 L of serum sample and standard were added respectively, then 1.0 ml of reagent A was added to blank, sample and standard tubes respectively. All tubes were mixed and incubated for 5 minutes at room temperature, and then 1.0 ml of reagent B were added to each tube then mixed and incubated for 5 minutes at 37°C. The absorbance was measured using kinetic technique at wavelength 578 nm.

Calculation: The concentration of urea was calculated using the following equation : As Serum urea (mg/dl) = × 40 A st

As is the absorbance of the sample. Ast is the absorbance of the standard. 40 is the standard concentration.

Determination of serum uric acid: Quantitative enzymatic colorimetric determination in serum, carried out according to Fossati et al. (1980).

Principle: Colorimetric determination of serum uric acid according to the following reaction: uricase

Uric acid + H 2O H 2O2 + CO 2 + Allantoin.

Peroxidase

H 2O2+DCHB+4aminophenazone N(4anipyryl)3 Chloro 5 sulfonate p benzo – quinonemonoimine + H 2O

41 Matrials and methodes

Reagents: Enzymatic uric acid reagent : Phosphate buffer PH 7.0 50 mmol/L 3,5Dichloro2hydroxybenzenesulfonic 4 mmol/L 4Aminophenazone 0.3 mmol/L Peroxidase >1000U/L Uricase >200 U/ml Stabilizers and activators in a buffered solution.

Standard (8 mg/dL): It contains aqueous solution of uric acid with stabilizer.

Assay procedure: In sample and standard tubes 20 L of serum sample and standard were added respectively, then 1.0 ml of reagent was added to blank, sample and standard tubes respectively. All tubes were mixed and incubated at 37°C for 5 minutes, The absorbance was measured within 15min. at wavelength 520 nm.

Calculation: The concentration of uric acid was calculated using the following equation: As Serum uric acid (mg/dl) = × 8 A st

As is the absorbance of the sample. Ast is the absorbance of the standard. 8 is the standard concentration.

Estimation of serum creatinine: Creatinine was estimated according to the kinetic method of Larsen (1972).

42 Matrials and methodes

Principle: The principle of the measurements of creatinine are based on the following equation: Creatinine + picric acid → Creatinine +picrate complex

Reagents: Creatinine picric acid reagent: Saturated aqueous solution of picric acid 3.6 mmol/L. Creatinine sodium hydroxide reagent: Aqueous solution of sodium hydroxide 240mmol/L. Creatinine standard 5 mg/dl Aqueous solution of creatinine . Reagent preparation: One part creatinine acid reagent was mixed with one part creatinine base reagent to prepare working reagent.

Assay Procedure: Spectrophotometer was adjusted at zero with deionized water at 510 nm. 1.0 ml of working reagent was added to corresponding label tube of standard and unknown then incubated at 37 °C for 3 minutes. 50 •L of standard and samples were pipeted to respective tubes, and immediately placed in measuring cuvette well. After exactly 20 seconds absorbance was recorded

(A 1), at exactly 60 sec. after measuring (A1) the absorbance (A 2) was measured.

Calculation: The concentration of creatinine was calculated according to the equation: As

Serum creatinine (mg/dl) = × St. Concentration A st A s is the absorbance change (A 2 A 1) of the sample. A st is the absorbance change (A 2 A 1) of the standard. St. Concentration is the standard concentration. 43 Matrials and methodes

C Determination of immunoglobulins Immunoglobulins were estimated by radialimmunodiffusion (RID) according to Palmer and Woods (1972).

Principle of the procedure: The procedure consists in an immunoprecipitation in agarose between an antigen and its homologous antibody. It was performed by incorporating one of the two immune reactants (usually antibody) uniformly throughout a layer of agarose gel, and then introducing the other reactants (usually antigen) into wells duly punched in the gel. Antigen diffuses radially out of the well into the surrounding gelantibody mixture, and a visible ring if precipitation formed where the antigen and antibody reacted. A quantitative relationship does exist between ring diameter and antigen concentration. Reagents: RID plate for tests packed in foil pouch. Each plate contains monospecific antiserum directed against immunoglobulins (IgG or IgM).

Equipment required: 1 Micropipettes capable of accurately measuring and delivering 5l. 2 Ruler capable of measuring with 0.1 mm precision. 3 Reference table showing the correspondence between diameter and concentrations.

Assay procedure: The plate was opened and stayed for about 5 minutes at room temperature, and allowed to stay any possible condensation to evaporate. The wells were filled with 5l of serum and control samples. Wet cotton was put in the plate center to avoid agarose dehydration, and then the plate was closed tightly. The plate

44 Matrials and methodes

allowed staying flat at room temperature along 48 hours for IgG and 72 hours for IgM.

Reading the results: End point of diffusion was indicated by a sharp precipitation ring. It was achieved when incubation time was finished. Reading was done at this time. The rings were measured with 0.1 mm precision by ruler, and the results were obtained directly from the reference table.

D Determination of Serum Hormonal Level Estimation of FT4in serum: FT4 was estimated according to Bigos (1979). CoatA count free T4 is a solid 125 I radioimmunoassay designated for the quantitative measurement of non protein bound thyroxin (freeT4) levels in serum.

Principle of the procedure: The coatAcount free T4 procedure is a solidphase radioimmunoassay , where in 125 I labeled T4 analog competes for a fixed time with free T4 in the sample for sites on T4 specific antibody. Because the antibody is immobilized to the wall of a polypropylene tube, simply decanting the supernatant to terminate the competition. Counting the tube in a gamma counter yields a number which converts by way of calibration curve to a measure of the free T4 present in the sample. Reagents:

Free T4 Abcoated tubes (TF41). Polypropylene tubes coated with antibodies to thyroxin and packed in ziplock plastic bags. Stable at 28°C for 6 months or until the expiration date.

45 Matrials and methodes

125 I free T4 (TF42). Lyophilized tracer, consisting of an iodinated thyroxin analog. Reconstituting each vial by adding a measured 110 ml distilled water let stand for 10 min. then mixed by gentle inversion. Stable at 28°C for 30 days after opening, or until the expiration date.

Free T4 calibrators (F4C39) Seven vials, of lyophilized processed human serum, labeled A through G. At least 30 minutes before use, reconstitute the zero calibrator A with 2.0 ml of distilled or deionized water and the remaining calibrators B through G with 1.0 ml each. Mixed by gentle swirling or inversion, stable at 28°C for 30 days after reconstitution. The calibrators contain respectively, 0, 0.1, 0.05, 1.3, 2.2, 4.8 and 10 ng/dl of free thyroxin.

Materials required: Gamma Counter, distilled or deionized water, graduated cylinder, micropipettes: 50 L and 200Ldispenser, foam

decanting rack, volumetric pipettes 1.0 ml and 2.0 ml.

Radioimmunoassay: The assay was performed in the Central Laboratory of Analysis Services, Radioisotopes Department, Atomic Energy Authority. All components must be at room temperature (15 28° C) before use. Four (uncoated) polypropylene tubes T (total counts) and NSB (non specific binding) in duplicate were prepared. Fourteen free T4 Abcoated tubes were labeled A (maximum binding) and B through G in duplicate. 50 L of each calibrator, control and patient serum sample was pipetted directly to the bottom into the tubes prepared, then 1.0 ml 125 I free T4 was added to every tube, and vortexed, (except T tube). All the tubes were incubated for 60 minutes at 37°C. Then the tubes were decanted thoroughly; (all visible moisture were removed). Count

46 Matrials and methodes

was recorded for 1 minute in a gamma counter. The total counts tube should be separated from the remaining assay tubes by at least one space.

Calculation: Free T4 concentrations was calculated from a logitlog representation of the calibration curve Net counts = (Average CPM) – (Average NSB counts) Then percent binding was calculated for each standard and unknown as follows: Percent pound = (Net counts/Net MB counts) × 100 3cycle loglog graph paper was used and percent pound versus concentration was plotted.

Estimation of FT3 in serum: FT3 was estimated according to Albertini and Ekins (1982). CoatAcount free T3 is a solidphase 125 I radioimmunoassay designated for the quantitative measurement of freeT3 levels in serum. Principle of the procedure: The coatAcount free T3 procedure is a solidphase radioimmunoassay , where in 125 I labeled T3 analog competes for a fixed time with free T3 in the sample for sites on T3 specific antibody. Because the antibody is immobilized to the wall of a polypropylene tube, simply decanting the supernatant to terminate the competition. Counting the tube in a gamma counter yields a number which converts by way of calibration curve to a measure of the free T3 present in the sample. Reagents: Free T3 Abcoated tubes(TF31). Polypropylene tubes coated with antibodies to triiodothyroxin (T3) and packed in ziplock plastic bags. Stable at 28°C for 6 months or until the expiration date. 47 Matrials and methodes

125 I free T3 (TF32). Lyophilized tracer, consisting of an iodinated T3 analog. Reconstituting each vial by adding a measured 110 ml distilled water let stand for 10 min. then mixed by gentle inversion. Stable at 28°C for 30 days after opening, or until the expiration date.

Free T3 calibrators (F3C39) Seven vials, of lyophilized processed human serum, labeled A through G. At least 30 minutes before use, reconstitute the zero calibrator A with 2.0 ml of distilled or deionized water and the remaining calibrators B through G with 1.0 ml each. Then mixed by gentle swirling or inversion, stable at 28°C for 30 days after reconstitution. The calibrators contain respectively, 0, 0.5, 1.6, 3.5, 7.0, 21 and 42 pg/ml of free T3.

Materials required: Gamma Counter, distilled or deionized water, graduated cylinder, micropipettes: 100 L and 1000Ldispenser, foam decanting rack, volumetric pipettes 1.0 ml and 2.0 ml.

Radioimmunoassay: All components must be at room temperature (15 28° C) before use. Four (uncoated) polypropylene tubes T (total counts) and NSB (non specific binding) in duplicate were prepared. Fourteen free T3 Abcoated tubes were labeled A (maximum binding) and B through G in duplicate. 100 L of each calibrator, control and patient serum sample was pipetted directly to the bottom into the tubes prepared, then 1.0 ml 125 I free T3 was added to every tube, and vortexed, (except T tube). All the tubes were incubated for 3hours at 37°C. All the tubes were decanted thoroughly; (all visible moisture was removed). Count was recorded for 1 minute in a gamma counter. The total counts tube should be separated from the remaining assay tubes by at least one space. 48 Matrials and methodes

Calculation: For calculation free T3 concentrations, from a logitlog representation of the calibration curve Net counts = (Average CPM) – (Average NSB counts) Then percent binding was calculated for each standard and unknown as follows: Percent pound = (Net counts/Net MB counts) × 100 3cycle loglog graph paper was used and percent pound versus concentration was plotted.

Estimation of TSH in serum: TSH was estimated according to Bayer et al. (1985). Coat Acount TSH IRMA is an immunoradiometric assay designated for the quantitative measurement of thyroid stimulating hormone (thyrotropin, TSH) levels in serum.

Principle of the procedure: The coatAcount TSH IRMA procedure is a solidphase immunoradiometric assay based on monoclonal and polyclonal antiTSH antibodies: one 125 I labeled antiTSH polyclonal antibody in liquid phase, and monoclonal antiTSH antibodies immobilized to the wall of tube. TSH is captured between monoclonal antiTSH antibodies immobilized to the wall of polystyrene tube and the radiolabeled polyclonal antiTSH tracer. Unbound 125 I labeled antiTSH antibody is removed by decanting the reaction mixture and washing the tube. The TSH concentration is directly proportional to the radioactivity present in the tube, the radioactivity is counted using a gamma counter, after which the concentration of TSH in the sample is obtained by comparing the counts per minute with those obtained for the set of calibrators provided. 49 Matrials and methodes

Reagents: TSH Abcoated tubes(ITS1). Polypropylene tubes coated with murine monoclonal antibodies to TSH and packed in ziplock plastic bags. Stable at 2 8°C for one year from the date of manufacture.

125 I TSH Ab (ITS2). Iodinated antiTSH goat polyclonal antibody, with . The reagent is supplied in liquid form, ready to use each vial contains 5.5ml. Stable at 28°C for 30 days after opening, or until the expiration date.

TSH calibrators (F3C39) Eight vials, labeled A through H, of lyophilized TSH calibrators in an equine serum/buffer matrix. 30 minutes before use, reconstitute the zero calibrator A with 6.0 ml of distilled water and the remaining calibrators B through H with 3.0 ml each. Mix by gentle swirling or inversion, stable at 28°C for 30 days after reconstitution. The calibrators contain respectively, 0, 0.15, 0.5, 1.5, 4.0, 15,30 and 60 IU/ml of TSH.

Materials required: Gamma Counter, distilled or deionized water, graduated cylinder, micropipettes: 100 L and 200Ldispenser, foam decanting rack, volumetric pipettes 3.0 ml and 6.0 ml rack shaker, buffer wash solution.

Radioimmunoassay: All components must be at room temperature (15 28° C) before use. Sixteen TSH Abcoated tubes were labeled A (nonspecific binding) and B through H in duplicate. 200 L of each calibrator, control and patient serum sample was pipetted 50 Matrials and methodes

directly to the bottom into the tubes prepared, then 100 L 125 I TSH to every tube. All the tubes were shaked for 2hours at room temperature on a rack shaker. All the tubes were decanted thoroughly; 2ml of buffered wash solution was added to all tubes and wait for 12 minutes, then decant thoroughly. Then 2ml of buffered wash solution was added and wait for 12 minutes, then decant thoroughly. All visible moisture was removed; Count was recorded for 1 minute in a gamma counter. The total counts tube should be separated from the remaining assay tubes by at least one space.

Calculation: For calculation TSH concentrations, from a loglog representation of the calibration curve.

Net counts = (Average CPM) – (Average NSB CPM) Then percent binding was calculated for each standard and unknown as follows:

Percent pound = (Net counts/Net MB counts) × 100

Estimation of total testosterone in serum: Total testosterone was estimated according to Abraham (1977). CoatAcount total testosterone is a solidphase 125 I radioimmunoassay designated for the quantitative measurement of total testosterone levels in serum.

Principle of the procedure: The coatAcount procedure is a solidphase radioimmunoassay , based on testosterone specificantibody immobilized to the wall of a polypropylene tube, 125 I labeled testosterone competes for a fixed time with testosterone in the sample for antibody sites. The 51 Matrials and methodes

tubes is then decanted to separate the bound from free, and counted in a gamma counter. The amount of testosterone present in samples is determined from a calibration curve.

Reagents: Total testosterone Abcoated tubes (TTT1): Polypropylene tubes coated with antibodies to testosterone and packed in ziplock plastic bags. Stable at 28°C for 6 months or until the expiration date.

125 I Total testosterone (TTT2): 105 ml of iodinated testosterone. Stable at 28°C for 30 days after opening, or until the expiration date.

Total testosterone calibrators (TTC38): Six vials of testosterone calibrators, ready to use. The zero calibrator A contains 4.0 ml and the remaining calibrators B through F with 1.0 ml each. Stable at 28°C for 30 days after opening. The calibrators contain respectively, 0, 20, 100, 400, 800 and 1600 ng/dl.

Materials required: Gamma Counter, distilled or deionized water, graduated cylinder, micropipettes: 50 L and 1mLdispenser, foam decanting rack.

Radioimmunoassay: All components must be at room temperature (15 28° C) before use. Four (uncoated) polypropylene tubes T (total counts) and NSB (non specific binding) in duplicate were prepared. Twelve total testosterone Abcoated tubes were labeled A (maximum binding) and B through F in duplicate. 50 L of each calibrator, control and patient serum sample was pipetted directly 52 Matrials and methodes

to the bottom into the tubes prepared, then 1.0 ml 125 I total testosterone was added to every tube, then vortexed. All the tubes were incubated for 3hours at 37°C. All the tubes were decanted thoroughly; (all visible moisture was removed). Count was recorded for 1 minute in a gamma counter. The total counts tube should be separated from the remaining assay tubes by at least one space.

Calculation: For calculation total testosterone concentrations, from a logitlog representation of the calibration curve Net counts = (Average CPM) – (Average NSB counts) Then percent binding was calculated for each standard and unknown as follows: Percent pound = (Net counts/Net MB counts) × 100

II Histopathological Examination:

Autopsy samples were taken from the livers, kidneys, and testes of scarified rats from each subgroup. They were fixed in 10% formal saline solution for 24 hours at least. They washed in tap water for 12 hours. Serial (methyl, ethyl and absolute) were used for dehydration of the tissue samples. Tissue specimens were cleared in and embedded in paraffin. The paraffin blocks were sectioned at 4 micro thickness by slidge microtome. The obtained tissue sections were collected on the glass slides and stained by hematoxylin and easin stain (1) for histopathological examination by the light microscope. A minimum of 10 fields for each organ slide were examined and assigned for severity of changes using scores in scale of non (), mild (+) moderate (+ +) and sever (+++) damage, according to Bancraft, et al. (1996).

53 Matrials and methodes

III Detection of DNA Fragmentation: Nucleic acids extraction and apoptotic detection was done according to Aljanabi and Martinez (1997).

DNA extraction: The sample tissues of 100mg of each of the liver, Kidney and testes were homogenized. The tissues were lysed in centrifuge tubes by adding 120 l of a 0.5 M EDTA solution (PH 8.0) and 500 l of Nuclei Lysis solution for each tube and chilled on ice. About 0.5 to 1 ml of fresh or thawed stool was added, then 600 l of the EDTA/ Nuclei Lysis solution was added to each tube followed by 17.5 l of 20 mg /ml proteinas K and the mixture was then incubated overnight at 55˚Cwith gentle shaking. 3 l of R nase was added to the nuclear lysate and the sample was mixed by inverting the tube 25 times. The mixture was incubated for 1530 minutes at 37˚C and left to cool at room temperature for 5 minutes before proceeding. 200 l of protein precipitation solution was added to each tube and then the tubes vortexed vigorously at high speed for 20 seconds, then the samples were chilled an ice for 5 minutes. The mix was inverted several times till fine fibers appear, and then centrifuged for 4 minutes at 15,000 rpm. The supernatant containing the DNA was removed and transferred to a clean 1.5 micro centrifuge tube containing 600 l of isopropanol. The solution was gently mixed by inversion several time till the white the thread – like strands of DNA formed a visible mass, then the tubes were centrifuged again for 1 minute at 15,000 rpm at room temperature. The supernatant was removed and the pellets were washed with 600 l of 70% ethanol, centrifuged at 15,000 rpm for 1 minute. After centrifugation the alcohol was aspirated using a sequencing pipette tip and the tubes were blotted on clean absorbent paper, till the pellets appeared to be dry. The pellets were rehydrated in 100 l of DNA Rehydrtion solution (10m M tis, 1m M EDTA, PH. 8).

54 Matrials and methodes

The solution was periodically mixed by gently tapping the tube, and then looted directly into the gel – wells. The extracted DNA was measured by spectrophotometer at 260 nm to determine its concentration.

Detection of DNA fragmentation on agarose gel electrophoresis DNA fragmentation was determined according to Hale et al. method (1996).

Reagent: 1. Agarose (Ultrapure agarose, electrophoresis grade). 2. TrisAcetate EDTA buffer (TAE) was prepared by adding 20 ml of 50 x TAE buffer to 980 ml distilled water. The 50 x TAE buffer contains: 121 g Tris base, 28.55 ml glacial acetic acid, 50 ml of 0.5 EDTA pH: 8 was completed to 500 ml with distilled water. 3. Ethidium Bromide (EB): The stain was prepared as a stock solution of 10 mg/ml by adding 10 mg of EB powder to 1 ml distilled water and mixing well by vortex. EB was stored in a dark tube wrapped in aluminum foil and stored at approximately 4 ْ C. This was incorporated into the gel at a concentration of 0.5 ug/ml. 4. Gel control DNA marker was used in this test. 5. Gel loading buffer (10ml): This buffer was prepared as follows: 2.5 ml of bromophenol blue of stock solution (prepared by adding 100 mg bromophenol blue powder to 10 ml distilled water). 2.5 ml xylene cyanol of stock solution (prepared by adding 100 mg xylene cyanol powder to 10 ml distilled water). 1 ml of glycerol in water of stock solution (prepared by adding 3 ml glycerol to 7 ml distilled water). 4 ml water was added to reach 10 ml.

55 Matrials and methodes

The loading buffer serves three purposes, increases the density of the sample, ensures that the DNA drops evenly into the well, and adds color to the sample, thereby simplifying the loading process, and contains that in an electric field move toward the at predictable rates

Equipments required: 1. Hybrid horizontal gel tank: (Biometra). 2. UV transilluminator. 3. Polaroid camera.

Assay Procedure: Gel preparation: 2% agarose gel was prepared by dissolving one gram of agarose in 50 ml of TAE buffer in a flask covered with aluminum foil for 5 minutes in a microwave adjustable to medium temperature. When the agarose solution cooled to 60° C, 2.5 ul of EB was added to allow subsequent visualization of the DNA. Gloves were used when working with solutions containing EB dye because it is a powerful mutagen and is moderately toxic. The gel was poured into a clean and dry gel mold. When the agarose had set completely (2030 min), the comb was carefully removed and the gel was placed in the electrophoresis chamber. The sample wells were positioned at the negative electrode end of the gel tank so that DNA migrates through the gel towards the positive electrode. An amount of TAE buffer was added to sufficiently cover the gel. For loading the gel, 9 ul from the second amplified DNA was added with 3 ul of loading dye in a 0.5 ml Eppendorf tube. Samples were loaded carefully into the wells without hitting the gel to avoid diffusion. The gel control marker was also loaded to determine the size of PCR product and 5 ul of the marker and 3 ul of the loading dye were used.

56 Matrials and methodes

The lid of electrophoresis chamber was closed, the gel run at 100 , 250 Ampere for 20 minutes. The DNA was visualized by placing the gel on an transilluminator. The EB intercalated into DNA and gave bright pink bands.

Statistical Analysis: All values were expressed as mean ±SE. Statistical analysis was performed with one way analysis of variance (ANOVA) followed by Duncan's test using SPSS program version 17.0. P values < 0.05 were considered to be statistically significant

57 Results

I Assessment of Body Weight The results of current study showed that rats gradually recovered their weights during the period of experiment. TCE intoxicated rats (750mg/kg/day) for short and long terms of treatment (TCE intoxicated control rats of gp. II & IV) resulted in a significant decrease in the body weight by 12.19% and 49.72%; respectively lower than those in the normal control groups (tables 1&2 and figs.1&2). Vitamin C and zinc supplemented to TCE intoxicated rats in group II and IV produced improvement in the body weight gain by 16.66% , 13.88% (for short term), 2.92% and 6.71% (for long term); respectively when compared to the TCEintoxicated control. While the mixture of both (vitamin C and zinc) produced marked increase by 19.44 % and 20.62 %; respectively. It was noticed that withdrawal of the TCE (table2) recorded gain in body weight by 18.90% when compared to the TCEintoxicated control rats treated for 20 days. Organs weights were represented as mean value tables 3&4. TCEintoxicated rats for short period of treatment (20 days) induced changes in livers and kidneys weights by 16.11% and 4.7%; respectively (table 3). While the treatment of TCE for long period (105 days) recorded significant elevation in the livers and kidneys weights by 64.12% and 60.74%; respectively when compared to normal control group (table 4). On the other hand TCE seems to have little effect on testes weight percentage (11.54%) for short term of treatment, but the weights of testes were decreased by 33.81% in the TCEintoxicated rats for long term than that in normal control. The results reveled that the organs of TCE intoxicated rats supplemented with both vitamin C and/ or zinc for short term gained weights nearly as the control group (table 3), while withdrawal of TCE and intoxicated rats supplemented with vitamin C and/ or zinc for long term showed significant

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improvement in the recorded weights of rat organs compared to TCE intoxicated control group (table 4). The general effect of the previous micronutrients in between groups on liver weight /body weight revealed decrease in the percentage as compared to the intoxicated groups as indicated in tables 5&6. II Assessment of Hematological Parameters Statistical analysis was performed using the statistical program for analyzing the resulted data. The different hematological parameters were summarized in tables 7&8. There were significant decreases (P<0.05) in erythrocytic counts, hemoglobin, platelets count, mean corpuscular volume (MCV), mean corpuscular hemoglobin Hb/RBCs (MCH), and mean corpuscular hemoglobin concentration Hb/ PCV (MCHC) in TCE intoxicated rat groups (group II and IV) as compared to that of normal control group, whereas no significant changes were noticed in the same parameters for the normal controls of group I and III. TCE treatments induced highly significant (P<0.05) increase in white blood cell count (WBCs), lymphocytes, monocytes, esinophils and staff neutrophils as compared to the corresponding levels of both control and supplemented groups with vitamin C and /or zinc. There were significant changes in the most of the hematological parameters between the withdrawal TCE rats and the groups of normal and TCE intoxicated rats. The pronounced effect of TCE for several days and few months was reduced by supplemented the animals with vitamin C and/ or zinc, as showen in tables 7&8.

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III Assessment of Biochemical and Immunological parameters

A Liver function biomarkers: TCEinduced significant alteration in the liver function tests including total protein, albumin, globulin, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and total billirubin in the groups of rats that treated with TCE for short and long period (group II and IV) (tables 910 & figs.324), without significant changes (P<0.05) in the A/G ratio for the same groups.

Rats intoxicated with TCE showed significant increase (P<0.05) in serum AST, ALT, ALP and total bilirubin values as compared to the normal control values. On the contrary, animals supplemented with vitamin C and /or zinc showed significant improvement in their liver function tests as compared to the TCE intoxicated control groups. However the mean values of these parameters showed mild improvement in the withdrawal group than the corresponding values of normal control group. AST/ALT ratio was almost normalized in between the rats of group I &II, while there was a significant decrease (P<0.05) in AST/ALT ratio of TCE intoxicated rats (group IV) as compared to that of normal control group (group III).

Regarding to group II for short term and group IV for long term, there were significant decrease (p<0.05) in total protein, albumin and globulin in TCEintoxicated rats as compared to that of normal control rats, and the mean values of these parameters significantly increased (p<0.05) in groups of rats intoxicated with TCE and supplemented with vitamin C and /or zinc as compared to that of TCEintoxicated control group as shown in tables 78 &figs.324; respectively.

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B Renal function biomarkers: There were significant increase (p<0.05) in the mean values of urea, uric acid and creatinine in TCEintoxicated rats as compared to that of normal control groups (group II and IV), and the mean values were significantly decreased (p<0.05) in group of rats intoxicated with TCE and supplemented with vitamin C and/ or zinc as compared to that of TCEintoxicated control group. Mean values of urea, uric acid and creatinine of withdrawal group were significantly decreased (p<0.05) as compared to that of TCE intoxicated control group as indicated in tables 1112& figs.2533.

C Assessment of serum IgG and IgM levels: Serum IgG and IgM were significantly (p<0.05) elevated in both conditions of treatment with TCE as compared to that of normal control group, with slight reduction in the mean values of both parameters for the groups supplemented with vitamin C and/ or zinc and withdrawal group as compared to that of TCE intoxicated control group as presented in tables1314 & figs. 34 39.

D Assessment of serum hormones: The results revealed significant decrease (P<0.05) in free tetraiodothyronine (FT4) in TCEintoxicated rats (group II & IV) as compared to that of normal control rats (group I & III), with percent change 37.08 % and 52.60 %; respectively, and return to increase significantly (p<0.05) in groups of rats intoxicated with TCE and supplemented with vitamin C with percent change 27.36 % and 20.73 %. It was noticed that significant increase (p<0.05) in FT4 levels in group of TCEintoxicated rats and supplemented with zinc with percent change 40.00 % and 31.70 % for group II and IV; respectively. Also it was found significant increase (p<0.05) in FT4 levels in group of rats intoxicated with TCE and

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supplemented with vitamin C and zinc (group II & IV) with percent change 42.10 % and 69.51 %; respectively as compared to that of TCEintoxicated control groups (tables 1516& figs.40 54).

On other hand the results revealed significant increase (P<0.05) in free triiodothyronine (FT3) levels for TCEintoxicated rats of group II & IV as compared to that of normal control group with percent change 76.66 % and 64.39 %; respectively, and return to decrease significantly (p<0.05) with percent change 5.18 %, 6.60 % and 16.98 % in groups of rats intoxicated with TCE and supplemented with vitamin C and/ or zinc respectively in group II, and also decrease significantly (p<0.05) with percent change 9.21 %, 11.05 % and 13.36 % in group of rats intoxicated with TCE and supplemented vitamin C and/ or zinc respectively in group IV.

There was significant decrease (p<0.05) in T4/T3 ratio in TCEintoxicated rats (groups II & IV) and the groups of rats supplemented with vitamin C and/ or zinc as compared to that of normal control group, and insignificant changes in groups I and III were recorded (fig. 46).

Thyrotropin (TSH) concentration showed significant increase (P<0.05) in TCEintoxicated groups as compared to that of normal control group with percent change 1400.00 % and 1555.55 % for group II and IV; respectively, and return to decrease significantly (p<0.05) with percent change 7.40 %, 37.04 % and 82.56 % in groups of rats intoxicated with TCE and supplemented with vitamin C and or zinc; respectively in group II, and also significant decrease was noticed in TSH levels with percent change 31.54 %, 42.95 % and 48.99 % in groups of rats intoxicated with TCE and supplemented with vitamin C and or/ zinc respectively in group IV.

62 Results

The results revealed significant decrease (P<0.05) in the mean values of total testosterone for TCEintoxicated rats (group II &IV) as compared to that of normal control groups (group I & III) with percent change 39.51 % and 49.25 %; respectively, and return to increase significantly (p<0.05) with percent change 9.49 %, 12.19 % and 41.16 % in groups of rats intoxicated with TCE and supplemented with vitamin C and/ or zinc respectively (group II), and increase insignificantly (p<0.05) with percent change 23.02 % and 25.73 % in group of rats intoxicated with TCE and supplemented vitamin C or zinc respectively (group IV), but significant increase (p<0.05) in group of rats intoxicated with TCE and supplemented both vitamin C and zinc with percent change 54.02 % was recorded.

Mean values of FT4 and total testosterone of withdrawal group were significantly increased (p<0.05), and significantly decreased (p<0.05) in FT3 and TSH levels as compared to that of TCEintoxicated control group.

IV Histopathological Assessment of Liver, Kidney and Testicular Tissues The histological changes in the liver, kidney and testes tissues of all groups were graded and the results were scored and described in tables (1722) and figures (5584).

Figure 55 illustrated the normal histological structure of liver section of normal rats of groups I &III for short term and long term of treatment with vitamin C and /or zinc. It consists of numerous hepatic lobule and connective tissue septa. It was noticed that normal histological structure of the central vein (c) and surrounding hepatocytes (h). Figure 56 illustrated

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histopathological changes in the livers sections of TCE intoxicated rats. There was dilatation and hyperemia in the central vein (c) and the portal vein (p) associated with inflammatory cells infiltration (m), as in [A] with diffuse proliferation of kupffer cells (arrow) between the hepatocytes [B], in addition to the degenerated hepatocytes (d) as in [C].

Congestion in the central vein (c) with degeneration in the hepatocytes (h) and apoptotic bodies in some of the hepatocytes were observed (fig. 70), in the liver sections of group IV. Prominent improvements in the liver histology were observed in rats supplemented with vitamin C and /or zinc in addition to withdrawal TCE intoxicated rats, (fig 57, 58, 59, 71, 72 & 73).

The histological structure of the kidney tissue of normal rats of group I &III for short and long terms of supplementation with vitamin C and /or zinc were illustrated in figure 60. It showed the normal histological structure of the glomeruli (g) and surrounding tubules(r), in the cortex [A], and normal histological structure of the tubules(r) at medullary portion [B].

In the kidney tissues of TCE intoxicated rats for short period of treatment (fig. 61) showed focal inflammatory cells infiltration inbetween and surrounding the glomeruli and tubules at the cortex with swelling in the tubula epithelium(s) [A], also it showed focal haemorrage (h) in the corricomedullary portion [B].

After long period of treatment kidney tissues sections showed severe congestion in sclerotic blood vessels with focal haemorrage, also showed focal inflammatory cells infiltration inbetween the glomeruli and tubules (fig 74). Supplementation of rats with vitamin C and/ or zinc for long period of intoxication with TCE showed very mild focal tubular

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necrosis and normalized TCE induced histological changes in comparison to TCE intoxicated animals (fig 75, 76 &77). Also supplementation of micronutrients demonstrated less interstitial cellular infiltration, apparent renal corpuscle as well as some tubules are relatively dilated and contained epithelial debris.

Histopathological examination of testicular tissues showed no abnormal microscopical alterations for the normal rats that received vitamin C and /or zinc (fig. 65). On the other hand there were marked structure alteration observed in the testes tissue of TCEintoxicated rats (gpΠ,) for 20 days. Figure 66 showed degenerated spermatogonial cells in the lumen of the seminiferous tubules with appearance of homogenous eosinophilic albuminous material inbetween, and multinumber of sertoli cells in the lumen of the degenerated tubules, and also mitosis in the spermatogonial cells of some seminiferous tubules was observed. In addition to appearance of azospermia in most seminiferous tubular lumen was noticed after long period of treatment (fig.78).

Supplementation of vitamin C and /or zinc nearly normalized the TCE induced histopathological changes (fig. 78, 79, 80 & 81). The histopathological assessment of liver, kidney and testicular tissues of withdrawal group summarized in tables18, 20, 22 and figures 82, 83, 84 respectively.

V Detection of DNA Fragmentation DNA Fragmentation was checked in this study by DNA electrophoresis laddering technique with separation of DNA fragments according to length of multiples of base pairs (bp) compared to control liver, kidney and testes tissue. As showed in table 23 and fig. 85, liver of TCE intoxicated rats recorded an obvious increase in the intensity of DNA fragments after 20 days of treatment with TCE; it gained 20 % 65 Results

higher percentage of DNA fragmentation when compared to normal control rats. The intensity of released DNA fragments in the liver tissue was increased after 105 days with the concentration of DNA fragments 60% when compared to the normal control 15%. Observable decrease in fragmentation of DNA in the liver samples were detected in rats supplemented with vitamin C and /or zinc 41%, 37 % & 22 %; respectively when compared to the control of TCE intoxicated rats 45 %. The present results of DNA fragments extracted from the tissue of kidney of TCE intoxicated rats for short term of treatment recorded increase in the optical density of the DNA fragments 26 % when compared to the DNA fragments of control. The long term of treatment with TCE recorded high value of density of DNA fragmentation 42 % when compared to the normal control. The supplement of TCE intoxicated rats with vitamin C and /or zinc induced marked amelioration of DNA fragments 40 %, 37 % & 32 %; respectively as showed in table 23 & fig. 85. As shown in table 23& fig. 85, testes of rats treated with TCE showed increase in optical density at both periods of treatment with the value of 32% and 63%; respectively when compared to the normal control 25%. The supplementation of vitamin C and /or zinc declined the value of DNA fragments concentration 28 %, 27 % and 21%; respectively when compared to +ve control of intoxicated rates 38 %. The fragments of DNA extracted from liver, kidney and testes of the withdrawal group recorded mild decrease in the concentration of DNA fragment 13 %, 14 % and 3 %; respectively.

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Table (1): Effect of supplementation with vitamin C and /or zinc on body weight change of normal and TCEintoxicated rats for short term (20 days). Group I Group II Groups (Normal rats) (TCEintoxicated rats)

Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn

Rat body weight in (g)

Initial weight 137.00 ±4.24 135.00±4.67 144.00±3.30 140.00±3.47 138.40±10.21 123.00±1.56 140.83±2.36 135.00±1.78

weight after 10 days 158.00±3.25 162.25±2.79 169.05±2.01 164.44±3.11 154.16±3.15 150.50±2.18 164.00±3.02 157.75±3.25

178. 00 ±6.23 184.00±5.10 189.00±3.68 185.00±4.54 174.00±16.42 165.00±2.94 181.00±4.65 178.00±0.96 Final weight

41.00 49.00 45.00 45.00 36.00 42.00 41.00 43.00 Net weight gain

%of body weight 19.51 9.75 9.75 12.19 16.66 13.88 19.44 change Data are means of 6 replicates ± SE % change of subgroups normal+ vit.C and /or Zn are compared to normal subgroup. % change of subgroups TCE + vit.C and /or Zn are compared to TCE subgroup.

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Table (2): Effect of supplementation with vitamin C and /or zinc on body weight change of normal and TCEintoxicated rats for long term (105 days). Group III Group IV Groups (Normal rats) (TCEintoxicated rats)

Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn vit.C&Zn Withdrawal Rat body weight in (g) Initial weight 130.25±6.41 121.25±1.25 137.75±3.07 126.00 ± 6.62 143.20±3.55 135.10±2.44 142.50±3.4 4 125.60±3.14 139.80±11.22 weight after 15 days 153.00±5.92 147.75±2.87 170.50±7.77 152.50±9.61 177.30±3.89 166.20±3.13 173.80±4.25 153.20±4.76 150.20±9.76 weight after 30 days 177.25±3.97 172.50±4.73 195.00±2.86 176.75±11.38 202.80±3.94 195.40±5.02 201.30±6.85 189.30±4.83 161.40±10.91 weight after 45 days 187.00±3.65 193.00±5.05 207.75±2.78 192.00±13.34 205.70±3.83 206.90±5.34 218.80±7.42 195.80±5.13 175.20±8.80 weight after 60days 201.50±3.28 205.25±3.75 227.50±7.89 214.75±14.02 216.00±3.63 204.50±5.59 231.90±7.18 218.70±6.01 191.40±7.18 weight after 75 days 210.50±3.52 217.75±8.76 245.50±12.88 228.00±16.23 230.90±5.78 217.20±6.21 220.80±5.49 209.70±6.30 199.80±7.77 weight after 90days 226.25±7.70 249.25±13.76 268.50±10.55 247.00±21.92 210.20±4.76 202.00±4.83 212.10±5.42 202.80±6.26 204.60±9.11 weight after 105 days 246.00±6.62 271.50±11.27 288.75±7.19 278.25±19.94 201.40±5.39 195.00±4.51 204.60±5.65 195.80±4.24 209.00±11.39

Net weight gain 115.75 150.25 151.00 152.25 58.20 59.90 62.10 70.20 69.20 % of body weight - 29.81 30.45 31.53 49.72 2.92 6.71 20.62 18.90 change Data are means of 6 replicates ± SE % change of subgroups normal+ vit.C and /or Zn are compared to normal subgroup. % change of subgroups TCE + vit.C and /or Zn and withdrawal are compared to TCE subgroup.

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Table (3): Variation in the organs weight ratios of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group I Group II

(Normal rats) (TCEintoxicated rats)

Organs Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn weight in (g)

7.28 ±0.30 7.51 ±0.32 8.36 ±0.33 7.36 ±0.10 Liver weight 7.20 ±0.48 7.21 ±0.42 7.91 ±0.43 7.53 ±0.198

1.11 4.30 16.11 5.38 9.93 11.96 % of weight change - 0.13

1.68 ±0.11 1.71 ±0.06 1.62 ±0.06 1.63 ±0.04 1.68 ±0.10 Kidney weight 1.70 ±0.06 1.72 ±0.14 1.67 ±0.02

1.17 0.59 4.70 0.62 3.70 % of weight change - 1.17 3.08

2.34 ±0.09 2.37±0.19 2.38 ±0.11 2.35±0.07 2.61 ±0.13 2.57±0.13 2.52±0.08 2.50±0.126 Tests weight

1.71 0.43 11.54 3.45 4.21 % of weight change - 1.28 -1.53 Data are means of 6 replicates ± SE % change of subgroups normal+ vit.C and /or Zn are compared to normal subgroup. % change of subgroups TCE + vit.C and /or Zn are compared to TCE subgroup.

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Table (4): Variation in the organs weight ratios of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Groups Group III Group IV

(Normal rats) (TCEintoxicated rats)

Organs Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal weight in (g )

8.16 ±0.14 8.05 ±0.35 8.20 ±0.20 13.13±0.60 9.99 ±0.68 8.63 ±0.18 8.42±0.18 Liver weight 8.00 ±0.49 9.50 ±0.73

2.00 0.63 2.50 64.12 27.65 23.91 34.27 35.87 % of weight change -

Kidney weight 1.35 ±0.04 1.44 ±0.06 1.48±0.03 1.39±0.06 2.17±0.24 1.84±0.10 1.53±0.03 1.48±0.08 1.64±0.05

% of weight change - 6.66 9.63 2.96 60.74 15.20 29.49 31.80 24.42

Tests weight 2.75 ±0.17 2.75±0.07 2.79±0.19 2.79±0.12 1.82±0.16 2.32±0.07 2.35±0.21 2.45 ±0.06 1.95 ±0.234

% of weight change - 00.00 1.45 1.45 33.81 27.47 29.12 34.61 7.14 Data are means of 6 replicates ± SE % change of subgroups normal+ vit.C and /or Zn are compared to normal subgroup. % change of subgroups TCE + vit.C and /or Zn and withdrawal are compared to TCE subgroup.

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Table (5): Effect of supplementation with vitamin C and/ or Zn on % body weight change and % of liver weight / body weight of normal and TCEintoxicated rats for short term (20 days). change of body weight Liver weight / body weight Subgroups Mean % of change Mean % of change

Control 41.00 0.040

+vit.C 49.00 19.51 0.039 2.50

+Zn 45.00 9.75 0.038 5.00

+vit.C&Zn 45.00 9.75 0.040 00.00

TCE 36.00 12.19 0.047 17.50

+vit.C 42.00 16.66 0.047 00.00

+Zn 41.00 13.88 0.041 12.76

+vit.C&Zn 43.00 19.44 0.041 12.76 % change of subgroups normal+ vit.C and /or Zn are compared to normal subgroup. % change of subgroups TCE + vit.C and /or Zn are compared to TCE subgroup.

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Table (6): Effect of supplementation with vitamin C and/ or Zn on % body weight change and % of liver weight / body weight of normal and TCEintoxicated rats for long term (105days). change of body weight Liver weight / body weight Subgroups Mean % of change Mean % of change Control 115.75 0.032

+vit.C 150.25 29.81 0.030 6.25

+Zn 151.00 30.45 0.028 12.50 +vit.C&Zn 152.25 31.53 0.028 12.50 TCE 58.20 49.72 0.065 103.12 +vit.C 59.90 2.92 0.049 24.61 +Zn 62.10 6.71 0.049 24.61 +vit.C&Zn 70.20 20.62 0.044 32.30

Withdrawal 69.20 18.90 0.040 38.46 % change of subgroups normal+ vit.C and /or Zn are compared to normal subgroup. % change of subgroups TCE + vit.C and /or Zn and withdrawal are compared to TCE subgroup.

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Table (7): Descriptive and comparative analysis of hematological parameters of normal and TCEntoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group Group (Normal rats) (TCEintoxicated rats)

Variables Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal

Red blood cell a a a a c bc bc b ab counts(×10 6l) 5.26 ±0.34 5.33 ±0.29 5.55 ±0.29 5.60±0.26 3.60 ±0.11 3.79 ±0.08 4.16 ±0.06 4.31 ±0.10 4.93±0.28 Hemoglobin (g/dl) 13.04 ±0.11 a 13.08 ±0.21 a 13.08 ±0.26 a 13.34 ±0.27 a 11.13 ±0.18 d 11.45 ±0.18 cd 11.99 ±0.22 bc 12.25 ±0.14 b 12.51±0.15 b

P.C.V % 36.50±0.65 a 39.50±0.65 a 37.75±0.48 a 37.25±0.85 a 36.25±2.28 a 36.25±0.48 a 37.00±0.41 a 38.25±0.85 a 37.57±1.14 a M.C.V Fl 85.03±0.37 a 84.83±0.15 a 84.28±0.11 a 84.10±0.18 a 82.37±0.85 b 83.73±0.75bb 83.53±0.19 b 85.30±0.16 a 84.00±1.08 a M.C.H Pg 28.80±00.15 a 29.25±0.12 a 29.78±0.63 a 29.25±0.12 a 27.67±0.23 c 28.15±0.17 ab 28.18±0.85 ab 28.27±0.19 a 28.65±0.55 a M.C.H.C % 36.35±0.13 a 36..20±0.09 a 36.65±0.09 a 36.75±0.65 a 33.10±0.07 c 34.62±0.12 b 35.37±0.19 ab 35.38±0.23 ab 36.40±0.13 a Platelets count (×10³l) 265.00±8.66 a 265.60±8.78 a 272.50±15.66 a 270.00±15.58 a 189.13±9.46 c 201.25±6.11 bc 205.00±8.02 bc 225.63±8.83 b 262.50±8.81 a

White blood cell b b b b a a a a b 4.64 ±0.19 4.65 ±0.17 4.59 ±0.16 4.50 ±0.12 7.75±0.24 7.03±0.66 7.00 ±0.24 6.10±0.21 5.40±0.37 count(×10³l) Monocytes % 4.00 ±0.46 a 4.00±0.50 a 4.00±0.53 a 3.88 ±0.44 a 5.00±0.46 a 4.00±0.57 a 5.00±0.46 a 5.00±0.42 a 3.00±0.19 a Lymphocytes % 34.00 ±1.78 c 33.00±1.70 c 32.38 ±1.55 c 32.50±1.50 c 53.00±1.56 a 44.13 ±1.51 b 42.63±1.61 b 37.50±2.17 c 38.37±2.18 c Basophils % 1.00±0.00 a 1.00±0.00 a 1.00±0.00 a 1.00±0.00 a 1.00±0.00 a 1.00±0.00 a 1.00±0.00 a 1.00±0.00 a 1.00±0.00 a Esinophils % 2.00±0.32 b 3.00±0.42 b 2.00±0.27 b 2.00±0.27 b 5.00±0.56 a 5.00±0.26 a 3.00±0.53 b 2.50±0.42 b 2.00±0.00 b Segmented neutrophils % 57.87±2.32 a 58.00±2.04 a 59.63±1.63 a 59.88±1.69 a 35.37±2.06 c 43.63 ±1.44 c 45.13±0.99 b 54.25±1.26 ab 53.37±2.36 a Staff neutrophils % 1.00±0.26 b 1.00±0.27 b 1.00±0.00 b 0.75±0.00 b 1.88 ±0.22 a 2.25±0.31 a 2.00±0.00 a 1.00±0.19 b 1.25±0.31 b

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p<0.05

73 Results

Table (8): Descriptive and comparative analysis of hematological parameters of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Groups Group III Group IV (Normal rats) (TCEintoxicated rats)

Variables Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn

6 a a a d cd cd Red blood cell counts(×10 l) 5.35±0.32 a 5.35±0.30 5.65±0.32 5.63±0.23 3.47±0.09 3.71±0.09 4.12±0.06 4.33±0.14 bc a a a d cd Hemoglobin (g/dl) 13.17±0.09 a 13.27±0.20 13.40±0.29 13.27±0.15 10.91±0.09 11.15±0.09 11.55±0.1 12.32±0.18 b a a c bc ab P.C.V % 44.02±1.06 a 43.50±1.19 44.05±1.31 a 45.00±1.05 39.50±1.55 42.05±1.09 43.42±1.53 43.10±0.31 ab a a b b b M.C.V Fl 87.75±1.88 a 86.37±0.24 86.52±1.68 a 88.17±2.26 76.07±3.05 76.35±1.87 76.82±2.30 78.32±1.47 b a a a c c b ab M.C.H Pg 30.15±0.71 a 30.22±0.40 30.70±0.66 30.00±0.81 27.35±0.89 27.70±0.24 28.05±1.03 29.67±0.52 a a c bc b M.C.H.C % 36.52±0.18 a 36.50±0.17 36.92±0.13 36.50±0.17 a 30.67±0.22 32.32±0.16 33.70±0.12 34.60±0.11 ab a a d d cd Platelets count (×10³l) 288.75±10.76 a 293.12±9.67 295.25±7.66 a 296.87 ±4.52 183.75±9.80 188.75±9.19 201.25±7.48 220.62±9.93 c d d d a ab bc White blood cell count(×10³l) 5.21±0.29 d 5.08±0.35 5.05±0.38 5.07±0.39 14.05±0.74 13.50±0.65 12.16±0.66 10.90±0.37 c c c a ab ab Monocytes % 2.87±0.39 c 3.00±0.53 3.00±0.42 2.00±0.26 c 5.00±0.46 4.12±0.44 4.00±0.37 3.25±0.37 bc c c a a b Lymphpcytes % 36.00±1.33 c 36.13±2.24 35.63±1.66 35.50±1.21 c 67.00±1.89 65.38±1.58 60.75±2.21 56.38±1.73 b a a a a a a Basophils % 1.00±0.00 a 1.00±0.00 1.00±0.00 a 1.00±0.00 2.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 d d d a b c Eosinophils % 2.00±0.00 d 2.25±0.25 2.00±0.19 2.00±0.00 5.00±0.33 4.00±0.42 3.00±0.57 3.00±0.33 c a d d c a 55.62±2.39 a a 18.75±1.71 22.00±1.79 28.25±3.00 b Segmented neutrophils % 55.87±1.64 56.37±1.93 53.50±1.32 34.13±1.58 c c c a ab ab Staff neutrophils % 1.00±0.26 c 2.00±0.37 2.00±0.42 2.00±0.19 3.50±0.33 3.00±0.32 3.00±0.37 2.25±0.26 bc Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p< 0.05.

74 Results

Table (9): Liver function biomarkers in serum of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group I Group II

(Normal rats) (TCEintoxicated rats)

Parameters Control +vit.C +Zn +vt.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal

a a a a d c b ab b 12.14±0.33 12.24±0.54 12.16±0.31 12.31±0.34 8.90±0.27 9.99±0.16 11.02±0.39 11.71±0.30 10.83 ±0.29 T protein (g/dl) d cd bc a a a a 3.76± 0.18 4.16±0.20 4.57±0.20 ab ab Albumin(g/dl) (A) 5.68 ±0.13 5.69 ±0.19 5.52±0.42 5.61±0.22 4.94 ±0.27 5.00 ±0.13

ab ab ab ab b ab ab a Globulin (g/dl) (G) 6.46 ±0.60 6.53±0.61 6.52±0.61 6.69±0.48 5.14±0.35 5.82±0.24 6.44±0.53 6.77±0.47 5.58±0.26 ab

A/G ratio 0.88±0.07 a 0.87±0.07 a 0.85±0.03 a 0.83±0.07 a 0.73±0.05 a 0.71±0.06 a 0.71±0.09 a 0.73±0.07 a 0.89±0.05 a

AST (U/L) 159.50±5.44 b 158.12±4.90 b 151.38±4.22 b 154.75±8.04 b 188.75±9.18 a 174.38±10.30 ab 170.00±6.19 ab 164.00±6.72 b 162.50±2.87 b

ALT (U/L) 46.50±3.01 c 46.87±1.82 c 47.62±1.80 c 47.50±1.74 c 62.88 ±2.08 a 57.88 ±4.16 ab 57.38 ±3.55 ab 54.62 ±2.03 bc 53.63±4.36 bc

AST/ ALT ratio 3.46±0.30 a 3.39±0.13 a 3.18±0.06 a 3.24±0.09 a 3.03±0.20 a 3.13±0.30 a 3.06±0.24 a 3.02±0.15 a 3.22±0.31 a

ALP (U/L) 320.12±15.68 c 301.75±26.03 c 321.37±13.65 c 304.63±10.28 c 485.13±40.97 a 409.37±13.27 ab 383.12±11.18 b 339.62±10.57 b 350.50±14.12 c

TBilirubin(mg/dl) 0.21 ±0.06 a 0.22 ±0.03 a 0.21 ±0.02 a 0.22 ±0.02 a 0.30 ±0.07 a 0.25 ±0.07 a 0.25 ±0.05 a 0.23 ±0.04 a 0.21±0.03 a

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p< 0.05.

75 Results

Table (10): Liver function biomarkers in serum of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Group III Group IV Groups (Normal rats) (TCEintoxicated rats)

Parameters Control +vit.C +Zn +vit.C&Zn +vit.C +Zn +vit.C&Zn Control

T protein (g/dl) a a a a c b b b 12.21±0.23 12.29±0.55 12.38±0.66 12.81±0.15 7.80±0.28 10.61±0.18 11.00±0.28 11.24±0.29

Albumin(g/dl) (A) 6.28±0.17 a 6.30±0.17 a 6.28±0.07 a 6.28±0.12 a 3.73±0.12 d 4.90±0.38 c 5.33±0.14 bc 5.37 ±0.32 bc

Globulin (g/dl) (G) 5.92±0.34 a 5.98±0.60 a 6.10±0.18 a 6.04±0.43 a 4.70±0.67 a 5.71±0.50 a 5.90±0.29 a 5.63 ±0.44 a

A/G ratio 1.06±0.05 a 1.05±0.07 a 1.03 ±0.04 a 1.04±0.06 a 0.79±0.04 a 0.86±0.07 a 0.90±0.06 a 0.95±0.09 a

d d d d 197.62±7.6 b bc bc AST (U/L) 154.87±3.86 152.25±3.36 153.00±3.97 153.12±4.81 a 181.12±6.21 171.12±5.13 167.37±4.47

d d d d 75.87±1.31 b bc cd ALT (U/L) 43.50±4.93 42.38±1.83 40.00±2.73 40.50± 2.13 a 61.62±1.90 54.12±2.24 48.00±1.61

AST/ ALT ratio 3.97±0.52 a 3.66±0.25 a 3.97±0.32 a 3.82±0.16 a 2.58±0.79 c 2.94±0.10 bc 3.19±0.13 abc 3.52±0.19 ab d d d d a b b bc ALP (U/L) 319.25±7.58 313.12±9.83 315.37±20.29 321.75±15.08 511.25±20. 454.87±19.36 453.00±15.55 387.50±20.03 b b b b a ab b b TBilirubin(mg/dl) 0.20 ±0.02 0.20 ±0.01 0.20 ±0.02 0.21 ±0.03 0.34 ±0.03 0.26 ±0.02 0.23±0.02 0.22 ±0.02

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p<0.05

76 Results

Table (11): Renal function biomarkers in serum of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group I Group II

(Normal rats) TCEintoxicated rats)

Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal Parameters

c c c c a ab ab bc b Urea (mg/dl) 37.88±2.18 38.75±1.25 39.62±1.62 37.37 ±2.78 51.87 ±2.28 48.63±2.92 48.75 ±4.86 42.50±3.21 45.75 ±5.15

b b b b a ab ab ab b Uric acid(mg/dl) 3.80±0.09 3.64±0.10 3.82±0.36 3.41 ±0.34 4.98 ±0.27 4.48±0.43 4.51±0.50 4.36 ±0.38 3.88±0.20

b b b b a a ab b b Creatinine(mg/dl) 0.59±0.03 0.52±0.03 0.56±0.03 0.57±0.02 0.74±0.05 0.74±0.07 0.62 ±0.04 0.60 ±0.01 0.58±0.02

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p< 0.05.

77 Results

Table (12): Renal function biomarkers in serum of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Groups Group III Group IV

(Normal rats) (TCEintoxicated rats)

Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Parameters

Urea (mg/dl) b b b b a ab ab ab 42.50 ±1.08 41.25 ±4.48 42.25±2.26 42.75±4.46 56.00±3.40 53.25±1.86 49.87 ±4.51 48. 37 ±2.87

Uric acid (mg/dl) b b b b a a ab ab 3.22±0.10 3.21 ±0.10 3.24±0.10 3.22±0.27 4.38±0.34 4.14±0.22 4.05±0.35 3.94±0.20

Creatinine (mg/dl) b b b b a a ab ab 0.53±0.03 0.51±0.03 0.53±0.03 0.51±0.02 0.73±0.06 0.67±0.02 0.62±0.04 0.59±0.04

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p< 0.05

78 Results

Table (13): Changes in serum IgG and IgM levels of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Group I Group II Groups (Normal rats) (TCEintoxicated rats)

Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal Parameters

c c c c a ab ab b b IgG (mg/dl) 278.15±18.06 286.01±21.40 261.02±33.42 275.86±23.51 561.88±24.80 512.48±28.38 518.30±31.98 463.07±24.04 438.38±34.14

c c c c a a ab b b IgM (mg/dl) 36.60±3.32 37.50±2.84 35.50±2.65 37.76±3.34 65.68±2.31 64.25±2.02 59.18±1.84 55.70±2.82 53.50±2.13

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p<0.05.

79 Results

Table (14): Changes in serum IgG and IgM of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Group III Group IV Groups

(Normal rats) (TCEintoxicated rats)

Parameters Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn

d d d d a ab ab bc IgG (mg/dl) 295.68±19.68 273.66±21.20 266.05±20.91 283.37±24.40 594.81±14.10 529.55±34.99 534.38±28.00 500.82±25.17

c c c c a a ab b IgM (mg/dl) 37.21±2.97 38.17±3.08 37.77±2.84 38.95±2.97 67.15±2.17 65.00±1.52 59.92±1.64 53.70±2.82

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p<0.05.

80 Results

Table (15): Changes in serum free thyroxine, free triiodothyronine, thyrotropin and testosterone levels of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group I Group II

(Normal rats) (TCEintoxicated rats)

Parameters +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawl Control

a a a a b ab ab a 1.16±0.12 ab FT4 (ng/dl) 1.51±0.15 1.52±0.12 1.52±0.16 1.59±0.07 0.95±0.08 1.21 ±0.13 1.33±0.10 1.35±0.09

% change 0.66 0.66 5.29 37.08 27.36 40.00 42.10 22.10 c c c c a ab ab b FT3 (pg/ml) 1.20±0.07 1.35 ±0.05 1.43±0.06 1.43±0.08 2.12±0.20 2.01±0.07 1.98±0.10 1.76±0.10 1.61±0.04 bc

% change 12.50 19.16 19.16 76.66 5.18 6.60 16.98 24.05

a a a a c c c bc bc T4/ T3 1.33±0.19 1.15±0.11 1.08±0.1 1.14±0.09 0.46±0.05 0.60±0.06 0.69±0.06 0.78±0.06 0.72±0.07

0.23 ±0.01 c c c c a a b b TSH(IU/ml) 0.11 ±0.05 0.09 ±0.01 0.10 ±0.01 1.35 ±0.02 1.25 ±0.07 0.85 ±0.01 b 0.25±0.07 0.09±0.01

% change 22.22 00.00 11.11 1400 7.40 37.04 81.48 82.56

TTestosterone b a e de cd b a bc bc 141.91±6.19 171.32±3.08 85.55±5.76 87.66±5.76 110.29±9.15 267.18±16.51 (ng/dl) 129.18±12.68 132.87±13.09 78.13±10.15

9.85 39.51 41.16 241.96 % change 2.85 32.62 9.49 2.19 Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p<0.05.

81 Results

Table (16): Changes in serum free thyroxine, free triiodothyronine, thyrotropin and testosterone levels of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Groups Group III Group IV

(Normal rats) (TCEintoxicated rats)

Parameter Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn

a a a a c bc bc ab FT4 (ng/dl) 1.73 ±0.09 1.83 ±0.21 1.87 ±0.25 1.86±0.24 0.82±0.08 0.99±0.04 1.08 ±0.12 1.39 ±0.22

% change 5.78 8.09 7.51 52.60 20.73 31.70 69.51 c c c c a a ab ab FT3 (pg/ml) 1.32 ±0.17 1.42±0.07 1.41±0.13 1.46±0.11 2.17 ±0.08 1.97±0.17 1.93±0.10 1.88±0.07

% change 7.75 6.81 10.60 64.39 9.21 11.05 13.36

a a a a b b b b T4/ T3 1.55±0.21 1.32 ±0.18 1.43 ±0.26 1.36 ±0.21 0.39 ±0.04 0.52 ±0.03 0.56 ±0.05 0.74±0.11

c c c c a ab b b TSH(IU/ml) 0.09±0.01 0.10 ±0.03 0.11 ±0.03 0.08 ±0.03 1.49 ±0.02 1.02 ±0.01 0.85 ±0.02 0.76 ±0.01

% change 11.11 22.22 11.11 1555.55 31.54 42.95 48.99 a a a c c c b T Testosterone (ng/dl) 324.27±16. 348.46±3.50 345.33±9.72 337.00±7.90 164.55±21.88 202.44±12.36 206.90±25.48 253.44±16.02

% change 7.46 6.49 3.92 49.25 23.02 25.73 54.02

Data are means of 6 replicates ± SE, means in the same row have different symbols are significantly different at p< 0.05.

82 Results

Table (17): Grading of histopathological changes in the liver tissues of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group I Group II

(Normal rats) (TCEintoxicated rats)

Parameters Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn

Inflammatory cells + + + infiltration. Degeneration of hepatocytes. + +

Fibroblast cells. + +

Edema.

Bile duct proliferation and hyperplasia. Vascular congestion. + Scoring was done as follows: () absent, (+) mild.

83 Results

Table (18): Grading of histopathologyical changes in the liver tissues of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Groups Group III Group IV

(Normal rats) (TCEintoxicated rats)

Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal Parameters

+ ++ + + + + + + Inflammatory cells

Infiltration.

Vascular congestion. + ++ + + + + +

Degeneration of + + + + + + hepatocytes.

Fibroblast cells. + + + +

++ + + Edema.

Bile duct proliferation ++ + + and hyperplasia. Scoring was done as follows: ( ) absent,( + ) mild, ( ++ )moderate, ( +++ )severe.

84 Results

Table (19): Grading of histopathological changes in the kidney tissues of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group I Group II

(Normal rats) (TCEintoxicated rats) Parameters Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn

Inflammatory cells

infiltration. + + + +

Glomeular changes ++ + + +

Tubular dilation + + + + + +

Vascular congestion +++ + + +

Degeneration of renal tubules. ++ + + +

Fibroblast cells + + +

Edema. + + Scoring was done as follows: ( ) absent, ( + ) mild, ( ++ )moderate, ( +++ )severe.

85 Results

Table (20): Grading of histopathological changes in the kidney tissues of normal and TCEintoxicated rat supplemented with vitamin C and /or zinc for long term (105 days). Groups Group III Group IV

(Normal rats) (TCEintoxicated rats)

Parameters Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal

Inflammatory cells + + + + ++ + + + ++ infiltration.

+ ++ + + + + + + Glomeular changes.

Tubular dilation. + + + + + + + ++

Vascular congestion. + + + + + + + +

Degeneration of renal +++ + + + + + tubules.

Fibroblast cells . + + + +

Edema. ++ + + +

Scoring was done as follows: ( ) absent, ( + ) mild, ( ++ )moderate, ( +++ )severe. 86 Results

Table (21): Grading of histopathological changes in the testicular tissues of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short term (20 days). Groups Group I Group II

(Normal rats) (TCEintoxicated rats)

Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Parameters Morphological changes. + + +

Congestion in blood vessels + + + + and thickening of their walls.

+ + + + Infiltration of interstitial cells.

Interstitial fibrosis. + ++ ++ +++ +++ + Round spermatides. Degeneration in + + + spermatogonial cells.

Leyding cell degeneration.

Scoring was done as follows: ( ) absent,( + ) mild, ( ++ )moderate, ( +++ )severe.

87 Results

Table (22): Grading of histopathological changes in the testicular tissues of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for long term (105 days). Groups Group III Group IV

(Normal rats) (TCEintoxicated rats) Parameters Control +vit.C +Zn +vit.C&Zn Control +vit.C +Zn +vit.C&Zn Withdrawal

Morphological changes. ++ + + + + ++

Congestion in blood vessels and + ++ ++ + + + + + ++ thickening of their walls.

Infiltration of interstitial cells. ++ + + + + + + + + +++

Interstitial fibrosis. + + + + + + ++

Round spermatides. ++ ++ +++ +++ + + Degeneration in Spermatogonial +++ ++ + + + ++ cells.

Leyding cell degeneration. ++ ++ + + +

Scoring was done as follows :( ) absent, (+) mild, (++) moderate, (+++) severe.

88 Results

Table (23): DNA denity in liver, kidney and testes tissues of normal and TCEintoxicated rats supplemented with vitamin C and /or zinc for short and long terms of treatment. TCEintoxicated rats (long term) TCEintoxicated Organs Normal control Withdrawal short term Control +Vit.C +Zn +C+Zn

35 60 56 52 37 28 15 Liver +20 + 45 + 41 + 37 + 22 + 13 change% -

39 55 53 13 50 45 27 Kidney +26 +42 +40 +37 +32 +14 change% -

Testes 25 32 63 53 52 46 28 change% - + 7 +38 +28 + 27 + 21 +3

89 Results

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn

200 190 180 170 160 150 Weight Weight in gm 140 130 120 0 10 20 Time in days

[A]

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn withdrawal

300 290 280 270 260 250 240 230 220 210 200

Weight in gm 190 180 170 160 150 140 130 120 0 15 30 45 60 75 90 105 Time in days

[B] Fig.(1): Body weight for different groups of normal and TCEintoxicated rats supplemented with vitamin C and/or zinc for 20 days [A], and 105 days [B].

90 Results

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn

22 20 18 16 14 12 10 8 6 4 2 0 -2 -4 1 % of weight change -6 -8 -10 -12 -14

[A]

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn Withdrawal

40 30 20 10 0 -10 1 -20 -30 % of weight change weight of % -40 -50 -60

[B]

Fig.(2): percentage of body weight change for different groups of normal and TCE intoxicated rats supplement with vitamin C and/or zinc for 20 days [A], and 105 days [B].

91 Results

Normal control Normal+vit.C TCE control TCE+vit. C

14 a a 12 c 10 d

8

6 T proteinT (g/dl) 4

2

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

14 a a 12 b 10 c 8

6 Tprotien (g/dl) Tprotien 4

2

0

[B]

Fig.(3): Effect of supplementation with vitamin C for 20 days [A], and 105 days[B]on serum total protein level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

92 Results

Normal control Normal+Zn TCE control TCE+Zn

14 a a 12 b

10 d 8

6

Tprotien (g/dl) 4

2

0

[A]

Normal control Normal+Zn TCE control TCE+Zn

14 a a 12 b

10 c 8

6 Tprotien (g/dl) 4

2

0

[B]

Fig. (4): Effect of supplementation with zinc for 20 days [A], and 105 days [B]on serum total protein level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

93 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

14 a a ab 12 b 10 d

8

6

Tprotien (g/dl) 4

2

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

14 a a b 12

10 c 8

6

Tprotien (g/dl) 4

2

0

[B]

Fig. (5): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B]on serum total protein level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

94 Results

Normal control Normal+vit.C TCE control TCE+vit. C

7 a a 6

5 cd d 4

3

Albumin (g/dl) Albumin 2

1

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

7 a a 6 c 5

4 d

3

Albumin (g/dl) 2

1

0

[B]

Fig. (6): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B]on serum albumin level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

95 Results

Normal control Normal+Zn TCE control TCE+Zn

7 a 6 a

5 bc d 4

3

Albumin (g/dl) 2

1

0

[A]

Normal control Normal+Zn TCE control TCE+Zn

7 a a 6 c 5

4 d 3

Albumin (g/dl) Albumin 2

1

0

[B]

Fig. (7): Effect of supplementation with zinc for 20 days [A], and 105 days [B]on serum albumin level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05

96 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

7 a 6 a ab ab 5 d 4

3

Albumin (g/dl) 2

1

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

7 a a 6 bc

5

4 d 3

Albumin (g/dl) 2

1

0

[B]

Fig. (8): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B]on serum albumin level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

97 Results

Normal control Normal+vit.C TCE control TCE+vit. C

8 ab ab 7 ab 6 b

5 4 3 Globulin (g/dl) 2 1 0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

7 a a a 6 a 5

4

3

Globulin (g/dl) Globulin 2

1

0

[B]

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 TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

98 Results

Normal control Normal+Zn TCE control TCE+Zn

8 ab ab ab 7

6 b 5

4

3 Globulin (g/dl) 2

1 0

[A]

Normal control Normal+Zn TCE control TCE+Zn

7 a a a 6 a 5

4

3

Globulin (g/dl) 2

1

0

[B]

Fig. (10): Effect of supplementation with zinc for 20 days [A], and 105 days [B]on serum globulin level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

99 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

8 ab ab a 7 ab 6 b 5 4

3

Globulin2 (g/dl) 1

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

7 a a a 6 a 5

4

3

Globulin (g/dl) 2

1

0

[B]

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 TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

100 Results

Normal control Normal+vit.C TCE control TCE+vit. C

220 210 a 200 190 ab 180 170 b b 160 150 AST (U/L) 140 130 120 110 100

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

220 210 a 200 b 190 180 170 d d 160 150 AST (U/L) 140 130 120 110 100

[B] Fig. (12): Serum aspartate transaminase (AST) activity of different groups of normal and TCEintoxicated rats supplemented with vitamin C for 20 days [A], and 105 days [B].

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

101 Results

Normal control Normal+Zn TCE control TCE+Zn

210 200 a 190 ab 180

170 b 160 b 150 AST AST (U/L) 140 130 120 110 100

[A]

Normal control Normal+Zn TCE control TCE+Zn

220 210 a 200 190 180 bc 170 d d 160 150 AST (U/L) 140 130 120 110 100

[B]

Fig. (13): Serum aspartate transaminase (AST) activity of different groups of normal and TCEintoxicated rats supplemented with zinc for 20 days [A], and 105 days [B].

Values are represented as mean ±SE, and different symbols means significantly different at p < 0.05.

102 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

220

200 a

180 b b b b 160

AST (U/L) 140

120

100

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

220 a 200

180 bc d d 160 AST AST (U/L) 140

120

100

[B]

Fig.(14): Serum aspartate transaminase (AST) activity of different groups of normal and TCEintoxicated rats supplemented with vitamin C& zinc for 20 days [A], and 105 days [B].

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

103 Results

Normal control Normal+vit.C TCE control TCE+vit. C

70 a ab 60 c c 50

40

30 ALT ALT (U/L) 20

10

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

90 a 80

70 b 60 d 50 d

40 ]= ALT (U/L) ALT 30 20 10 0

[ [ B]

Fig.(15): Serum alanine transaminase(ALT) activity of different groups of normal and TCE intoxicated rats supplemented with vitamin C for 20 days [A], and 105 days [B].

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

104 Results

Normal control Normal+Zn TCE control TCE+Zn

70 a ab 60 c c 50

40

30 ALT ALT (U/L) 20

10

0

[A]

Normal control Normal+Zn TCE control TCE+Zn

90 a 80 70 60 bc d 50 d 40

ALT ALT (U/L) 30 20 10 0

[B]

Fig.(16): Serum alanine transaminase (ALT) activity of different groups of normal and TCEintoxicated rats supplemented with zinc for 20 days [A], and 105 days [B].

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

105 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

70 a 60 bc bc c c 50

40

30 ALT (U/L) 20

10

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

70 a 60 bc bc c c 50

40

30 ALT (U/L) 20

10

0

[ [ B]

Fig.(17): Serum alanine transaminase (ALT) activity of different groups of normal and TCE intoxicated rats supplemented with vitamin C& zinc for 20 days [A], and 105 days [B].

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

106 Results

Normal Normal supplemented TCE TCE supplemented Withdrawal

3.5

3.4

3.3

3.2

3.1 AST/ALT ratios 3

2.9

2.8 Vit.C Zn Vit.C&Zn

[A]

Normal Normal supplemented TCE TCE supplemented

4.2 4 3.8 3.6 3.4 3.2 3 2.8 AST/ALT ratios 2.6 2.4 2.2 2 Vit.C Zn Vit.C&Zn

[ [ B]

Fig.(18): AST /ALT ratios of normal and TCEintoxicated rats supplemented with vitamin C and/ or zinc for 20 days [A], and 105 days [B] .

107 Results

Normal control Normal+vit.C TCE control TCE+vit. C

550 a

500

450 ab

400

c 350 c ALP (U/L) 300

250

200

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

550 a

500 b

450

400

d 350 d ALP (U/L) 300

250

200

[B]

Fig. (19): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum alkaline phosphatase (ALP) concentration for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

108 Results

Normal control Normal+Zn TCE control TCE+Zn

550 a

500

450 b 400

350 c c ALP (U/L) 300

250

200

[A]

Normal control Normal+Zn TCE control TCE+Zn

550 a

500 b 450

400 d 350 d ALP(U/L)

300

250

200

[ [ B]

Fig. (20): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum alkaline phosphatase (ALP) concentration for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p<0.05.

109 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

550 a 500

450

400 b c c 350 c ALP (U/L) 300

250

200

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

550 a

500

450 bc 400 d 350 d ALP (U/L) 300

250

200

[B]

Fig. (21): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum alkaline phosphatase (ALP) concentration for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

110 Results

Normal control Normal+vit.C TCE control TCE+vit. C

0.4 a

0.35 a

0.3 a a 0.25

0.2

0.15

Tbilirubin (mg/dl) 0.1

0.05

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

0.4 a 0.35

0.3 ab

0.25 b b 0.2

0.15

Tbilirubin (mg/dl) 0.1

0.05

0

[B]

Fig. (22): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum total billirubin level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

111 Results

Normal control Normal+Zn TCE control TCE+Zn

0.4 a 0.35 a 0.3 a 0.25 a

0.2

0.15

Tbilirubin (mg/dl) 0.1

0.05

0

[A]

Normal control Normal+Zn TCE control TCE+Zn

0.4 a 0.35

0.3 b 0.25 b b 0.2

0.15

Tbilirubin (mg/dl) 0.1

0.05

0

[B]

Fig. (23): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum total billirubin level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

112 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

0.4 a 0.35

0.3 a a a 0.25 a

0.2

0.15

Tbilirubin (mg/dl) 0.1

0.05

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

0.4 a 0.35

0.3 b b 0.25 b

0.2

0.15

Tbilirubin(mg/dl) 0.1

0.05

0

[B]

Fig.(24): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days[B] on serum total billirubin level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

113 Results

Normal control Normal+vit.C TCE control TCE+vit. C

60 a ab 50 c c 40

30

Urea (mg/dl) Urea 20

10

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

70 a 60 ab

50 b b 40

30

Urea (mg/dl) 20

10

0

[B]

Fig.(25): Effect of supplementation with vitamin C for 20 days [A], and 105 days[B] on serum urea level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

114 Results

Normal control Normal+Zn TCE control TCE+Zn

60 a ab 50 c c 40

30

Urea (mg/dl) Urea 20

10

0

[A]

Normal control Normal+Zn TCE control TCE+Zn

70 a 60 ab 50 b b

40

30

Urea (mg/dl) Urea 20

10

0

[B]

Fig.(26): Effect of supplementation with zinc for 20 days [A], and 105 days[B] on serum urea level or different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

115 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

60 a b 50 bc c c 40

30

Urea20 (m g/dl)

10

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

70 a 60 ab b 50 b

40

30

Urea (mg/dl) 20

10

0

[B]

Fig.(27): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days[B] on serum urea level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

116 Results

Normal control Normal+vit.C TCE control TCE+vit. C

6 5.5 a ab 5 4.5 b b 4 3.5 3 2.5 2

Uric acid1.5 (m g/dl) 1 0.5 0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

5 a 4.5 a 4 b b 3.5 3 2.5 2 1.5 Uric acid (mg/dl) 1 0.5 0

[ [ B]

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 TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

117 Results

Normal control Normal+Zn TCE control TCE+Zn

6 5.5 a 5 ab 4.5 b b 4 3.5 3 2.5 2

Uric acid1.5 (m g/dl) 1 0.5 0

[A]

Normal control Normal+Zn TCE control TCE+Zn

5 a ab 4.5 4 b b 3.5 3 2.5 2 1.5 Uric acid (mg/dl) 1 0.5 0

[B]

Fig. (29): Effect of s supplementation with zinc for 20 days [A], and 105 days [B] on serum uric acid level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

118 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

6 5.5 a 5 ab 4.5 b b 4 b 3.5 3 2.5 2

Uric1.5 acid (m g/dl) 1 0.5 0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

5 a

4.5 ab 4 b b 3.5 3 2.5 2

Uric acid (mg/dl) 1.5 1 0.5 0

[B]

Fig.(30): Effect of supplementation with vitamin C & zinc for 20 days [A], and 105 days [B] on serum uric acid level for different groups of normal and TCEintoxicated rats

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

119 Results

Normal control Normal+vit.C TCE control TCE+vit. C

0.9 a a 0.8 0.7 b 0.6 b 0.5 0.4 0.3

Creatinine (mg/dl) Creatinine 0.2 0.1 0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

0.9 a 0.8 a 0.7 b 0.6 b 0.5 0.4 0.3

Creatinine (mg/dl) 0.2 0.1 0

[ [ B]

Fig. (31): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum creatinine level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

120 Results

Normal control Normal+Zn TCE control TCE+Zn

0.9 a 0.8 0.7 ab b 0.6 b 0.5 0.4 0.3

Creatinine Creatinine (mg/dl) 0.2 0.1 0

[A]

Normal control Normal+Zn TCE control TCE+Zn

0.9 a 0.8 0.7 ab 0.6 b b 0.5 0.4 0.3

Creatinine (mg/dl) 0.2 0.1 0

[B]

Fig. (32): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum creatinine level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

121 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

0.9 a 0.8 0.7 b b b b 0.6 0.5 0.4 0.3

Creatinine (mg/dl) Creatinine 0.2 0.1 0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

0.9 a 0.8 0.7 ab 0.6 b b 0.5 0.4 0.3

Creatinine (mg/dl) 0.2 0.1 0

[ B]

Fig. (33): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum creatinine level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

122 Results

Normal control Normal+vit.C TCE control TCE+vit. C

700

600 a ab 500

400 c c 300 IgG (m g/dl) 200

100

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

700 a 600 ab

500

400 d d 300 IgG (mg/dl) 200

100

0

[ [ B]

Fig. (34): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum immunoglobulin IgG level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

123 Results

Normal control Normal+Zn TCE control TCE+Zn

700

600 a ab

500

400 c c 300 IgG (mg/dl) 200

100

0

[A]

Normal control Normal+Zn TCE control TCE+Zn

700 a 600 ab 500

400 d 300 d IgG (mg/dl) 200

100

0

[B]

Fig. (35): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum immunoglobulin IgG level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

124 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

700

600 a

500 b b

400 c c 300 IgG (m g/dl) 200

100

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

700 a 600 bc 500

400 d d 300 IgG (mg/dl) 200

100

0

[B] Fig.(36): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum immunoglobulin IgG level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

125 Results

Normal control Normal+vit.C TCE control TCE+vit. C

80 70 a a 60 50 c 40 c 30 IgM (m g/dl 20 10 0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

80 a 70 a 60 50 c c 40 30 IgM (mg/dl) 20 10 0

[B] Fig. (37): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum immunoglobulin IgM level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

126 Results

Normal control Normal+Zn TCE control TCE+Zn

80

70 a ab 60

50 40 c c

30 IgM (mg/dl) 20

10 0

[A]

Normal control Normal+Zn TCE control TCE+Zn

80 a 70 ab 60

50 c c 40 30 IgM (mg/dl) 20 10 0

[ [ B]

Fig. (38): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum immunoglobulin IgM level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

127 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

80 70 a

60 b b 50 c c 40 30 IgM (mg/dl) 20 10 0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

80 a 70

60 b 50 c c 40 30 IgM (mg/dl) 20

10 0

[ [ B]

Fig. (39): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum immunoglobulin IgM level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

128 Results

Normal control Normal+vit.C TCE control TCE+vit. C

1.8 a a 1.6 1.4 ab 1.2 b 1 0.8

FT40.6 (ng/dl) 0.4 0.2 0

[ [ A]

Normal control Normal+vit.C TCE control TCE+vit. C

2.5

a 2 a

1.5 bc 1 c FT4 (ng/dl)

0.5

0

[ [ B]

Fig. (40): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum free tetraiodothyronine (FT4) level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

129 Results

Normal control Normal+Zn TCE control TCE+Zn

1.8 a a 1.6 ab 1.4 1.2 b 1 0.8

FT4 0.6(ng/dl) 0.4 0.2 0

[ [ A]

Normal control Normal+Zn TCE control TCE+Zn

2.5 a 2 a

1.5 bc

1 c FT4 (ng/dl)

0.5

0

[B]

Fig. (41): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum free tetraiodothyronine (FT4) level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

130 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

1.8 a a 1.6 a 1.4 ab 1.2 b 1 0.8

FT4 0.6(ng/dl) 0.4 0.2 0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

2.5 a 2 a ab 1.5

c 1 FT4 (ng/dl)

0.5

0

[B]

Fig. (42): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum free tetraiodothyronine (FT4) level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

131 Results

Normal control Normal+vit.C TCE control TCE+vit. C

2.5 a ab 2

c 1.5 c

1 FT3 FT3 (pg/ml)

0.5

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

2.5 a a 2

c c 1.5

1 FT3 (pg/ml) FT3

0.5

0

[B]

Fig. (43): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum free triiodothyronine (FT3) level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

132 Results

Normal control Normal+Zn TCE control TCE+Zn

2.5 a ab 2 c 1.5 c

1 FT3 FT3 (pg/ml)

0.5

0

[ [ A]

Normal control Normal+Zn TCE control TCE+Zn

2.5 a ab 2 c c 1.5

1 FT3 (ng/ml)

0.5

0

[ [ B]

Fig. (44): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum free triiodothyronine (FT3) level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

133 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

2.5 a

2 b bc c 1.5 c

1 FT3 FT3 (pg/ml)

0.5

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

2.5 a ab 2 c c 1.5

1 FT3 FT3 (pg/ml)

0.5

0

[ [ B]

Fig. (45): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum free triiodothyronine (FT3) level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

134 Results

Normal Normal supplemented TCE TCE supplemented Withdrawal

1.4

1.2

1 0.8

0.6 T4/T3 ratios 0.4

0.2

0 Vit.C Zn Vit.C&Zn

[A]

Normal Normal supplemented TCE TCE supplemented

1.8 1.6 1.4 1.2 1 0.8

T4/T3 ratios 0.6 0.4 0.2 0 Vit.C Zn Vit.C&Zn

[ [ B]

Fig. (46): T4/T3 ratios of normal and TCEintoxicated rats supplemented with vitamin C and/ or zinc for 20 days [A], and 105 days [B].

135 Results

Normal control Normal+vit.C TCE control TCE+vit. C

1.6 a 1.4 a 1.2

1 0.8

0.6 TSH (IU/ml) 0.4 0.2 c c 0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

1.6 a 1.4 1.2 b 1 0.8 0.6 TSH (IU/ml) 0.4 c c 0.2 0 1

[ [ B]

Fig. (47): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum thyrotropin (TSH) level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

136 Results

Normal control Normal+Zn TCE control TCE+Zn

1.6 a 1.4 1.2

1 b 0.8 0.6 TSH (IU/ml) 0.4

0.2 c c 0

[A]

Normal control Normal+Zn TCE control TCE+Zn

1.6 a

1.4 1.2

1 b 0.8

0.6 TSH (IU/ml) 0.4

0.2 c c 0

[B]

Fig. (48): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum thyrotropin (TSH) level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

137 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

1.6 a 1.4 1.2

1 0.8

0.6 TSH (IU/ml) 0.4 b b 0.2 c c 0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

1.6 a 1.4 1.2 1 b 0.8 0.6 TSH (IU/ml) 0.4

0.2 c c 0

[B]

Fig. (49): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum thyrotropin (TSH) level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

138 Results

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn Withdrawal

1600 1400 1200 1000 800 600

%change %change of TSH 400 200 0 1

[A]

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn

1800 1600 1400 1200 1000 800 600 400 % change of TSH 200 0 -200 1

[B]

Fig.(50): Percentage change of TSH of normal and TCEintoxicated rats supplemented with vitamin C and/ or zinc for 20 days [`A], and 105 days [B] .

139 Results

Normal control Normal+vit.C TCE control TCE+vit. C

160 bc bc 140

120 de 100 e 80

60

40 Ttestosterone(ng/dl) 20

0

[A]

Normal control Normal+vit.C TCE control TCE+vit. C

400 a 350 a

300

250 c c 200

150

100 Ttestosterone (ng/dl) Ttestosterone 50

0

[B]

Fig. (51): Effect of supplementation with vitamin C for 20 days [A], and 105 days [B] on serum total testosterone level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

140 Results

Normal control Normal+Zn TCE control TCE+Zn

160 b bc 140 120 cd 100 e 80 60 40 Ttestosterone (ng/dl) 20 0

[A]

Normal control Normal+Zn TCE control TCE+Zn

400 a a 350 300

250 c 200 c

150 100 Ttestosterone (ng/dl) 50 0

[ [ B] Fig. (52): Effect of supplementation with zinc for 20 days [A], and 105 days [B] on serum total testosterone level for different groups of normal and TCEintoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

141 Results

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn Withdrawal

300 a

250

200 a

150 bc bc 100 e

Ttestosterone50 (ng/dl)

0

[A]

Normal control Normal+vit.C&Zn TCE control TCE+vit. C&Zn

400 a 350 a 300 b 250 200 c 150 100 Ttestosterone (ng/dl) 50 0

[ [ B]

Fig. (53): Effect of supplementation with vitamin C& zinc for 20 days [A], and 105 days [B] on serum total testosterone level for different groups of normal and TCE intoxicated rats.

Values are represented as mean ±SE, and different symbols means significantly different at p< 0.05.

142 Results

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn Withdrawal

250

200

150

100

50

0 1

% change of testosterone -50

-100

[A]

Normal control Normal+vit.C Normal+ZN Normal+vit.C&ZN TCE control TCE+vit. C TCE+ZN TCE+vit. C&Zn

20

10

0 1 -10

-20 -30

-40

%Change of testosterone -50 -60

[B]

Fig. (54): Percentage change of total testosterone of normal and TCEintoxicated rats treated with vitamin C and/ or zinc for 20 days [A], and 105 days [B] .

143 Results

Fig.(55): Histopathological section (stained with H& E) of the liver tissue of normal rats (gpΙ) supplemented with vitamin C and /or zinc for 20 days showing the normal histological structure of the central vein (c) and surrounding hepatocytes (h), (x 64).

144 Results

[A]

[B]

[C]

Fig.(56): Histopathological sections (stained with H& E) of the liver tissue of TCE intoxicated rats (gpΠ, sub gp TCE) for 20 days showing the dilatation in the central vein (c) and the portal vein (p) with inflammatory cells infiltration (m) in the portal area [A], also showing the inflammatory cells in the portal area(m) with diffuse kupffer cells proliferation (arrow) between the hepatocytes (x 80), [B], in addition to the degenerated hepatocytes (d), (x160), [C].

145 Results

[A]

[B]

[C]

Fig.(57): Histopathological section (stained with H& E) of the liver tissue of TCE intoxicated rats supplemented with vitamin C for 20 days of treatment showing the congestion in central vein (c ), (x40), [A] & [B] and in sinsoids (s) (x64), [C].

146 Results

Fig.(58): Histopathological section (stained with H& E) of the liver tissue of TCE intoxicated rats supplemented with zinc for 20 days showing focal inflammatory cells infiltration(m ) and diffuse kupffer cells proliferation (arrow) in between the degenerated hepatocytes (d)(x160).

Fig.(59): Histopathological section (stained with H& E) of the liver tissue of TCE intoxicated rats supplemented with vitamin C& zinc for 20 days showing the normal histological structure of the central vein (c ) and surrounding hepatocytes (h), (x 40).

147 Results

[A]

[B]

Fig.(60): Histopathological sections (stained with H& E) of the kidney tissue of normal rats (gpΙ) supplemented with vitamin C and/ or zinc for 20 days showing the normal histological structure of the glomeruli (g) and surrounding tubules(r), in the cortex (x 80) [A],and normal histological structure of the tubules(r) at medullary portion[B](x80).

148 Results

[A]

[B]

Fig.(61): Histopathological sections (stained with H& E) of the kidney tissue of TCE intoxicated rats for 20 days showing focal inflammatory cells infiltration inbetween and surrounding the glomeruli& tubules at the cortex with swelling in the tubula epithelium(s) (x 80) [A], also showing focal haemorrage (h) in the corricomedullary portion (x80), [B].

149 Results

[A]

[B]

Fig.(62): Histopathological section (stained with H& E) of the kidney tissue of TCE intoxicated rats supplemented with vitamin C for 20 days showing the congestion in stromal blood vessels of the cortex (v ), (x64), [A] and also showing focal inflammatory cells infiltration inbetween the tubules at the cortex (m) (x80), [B].

150 Results

[A]

[B]

Fig.(63): Histopathological section (stained with H& E) of the kidney tissue of TCE intoxicated rats supplemented with zinc for 20 days showing swelling and vacuolization of the endothelial cells lining the glomeruli (g), (x160) [A], also showing few inflammatory cells infiltration inbetween the renal tubules in the cortex (m)(x80), [B].

151 Results

Fig.(64): Histopathological section (stained with H& E) of the kidney tissue of TCE intoxicated rats supplemented with vitamin C& zinc (gpΠ,) for 20 days showing congestion in the cortical blood vessels (v), (x 40).

152 Results

[A]

[B]

Fig.(65): Histopathological section (stained with H& E) of the testes tissue of normal rats (gpΙ) supplemented with vitamin C and /or zinc for 20 days showing the normal histological structure of mature active seminiferous tubules with complete spermatogenic series(s)(x 40) [A], also showing normal histological structure of the tubules with spermatozoa in the tubular lumen (t)(x40) [B].

153 Results

[A]

[B]

[C]

Fig.(66): Histopathological sections (stained with H& E) of the testes tissue of TCE intoxicated rats (gpΠ,) for 20 days, showing degenerated spermatogonial cells (n) in the lumen of the seminiferous tubules with appearance of homogenous eosinophilic albuminous material in between (h),(x80)[A], also showing multinumber of sertoli cells in the lumen of the degenerated tubules (arrow) (x 80)[B], and showing mitosis in the spermatogonial cells of some seminiferous tubules (arrow) (x160), [C].

154 Results

Fig.(67): Histopathological section (stained with H& E) of the testes tissue of TCE intoxicated rats supplemented with vitamin C (gpΠ,) for 20 days showing azospermia in the lumen of some individual seminiferous tubules (s)(x40).

Fig.(68): Histopathological section (stained with H& E) of the testes tissue of TCE intoxicated rats supplemented with zinc (gpΠ,) for 20 days of treatment showing normal histological structure of the seminiferous tubules with complete spermatogenic series(s)(x 40).

155 Results

[A]

[B]

[C]

Fig.(69): Histopathological section (stained with H& E) of the testes tissue of TCE intoxicated rats supplemented with vitamin C& zinc (gpΠ,) for 20 days showing azospermia in the lumen of some seminiferous tubules(s)(x40) [A],and showing the sertoli cells as predominant cells in seminiferous tubules(arrow)(x80) [B], It also showing the sertoli cells as a most and predominant cells in the lumen of some individual seminiferous tubules(arrow)(x160) [C]

156 Results

[A]

[B]

a

[C] Fig. (70): Histopathological sections (stained with H& E) of the liver tissue of TCE intoxicated rats (gpΙV,) for 105 days showing congestion in the central vein (c) with degeneration in the hepatocytes (h), (x64) [A], and magnification of fig. (A) to identify the congestion in central vein (c) and degeneration in the hepatocytes (h),(x80) [B], in addition apoptosis in some of the hepatocytes (a) with diffuse kupffer cells proliferation (arrow), (x160), [C].

157 Results

Fig.(71): Histopathological section (stained with H& E) of the liver tissue of TCE intoxicated rats supplemented with vitamin C (gpΙV,) for 105 days showing degeneration in the hepatocytes (h), (x80).

Fig.(72): Histopathological section (stained with H& E) of the liver tissue of TCE intoxicated rats supplemented with zinc (gpΙV,) for 105 days of treatment showing diffuse kupffer cells proliferation in between the hepatocytes (arrow)(x80).

158 Results

Fig.(73): Histopathological section (stained with H& E) of the liver tissue of TCE intoxicated rats supplemented with vitamin C & zinc (gpΙV) for 105 days showing mild degeneration in the hepatocytes (h), (x 80).

159 Results

[A]

[B]

Fig.(74): Histopathological sections (stained with H& E) of the kidney tissue of TCE intoxicated rats (gpΙV,) for 105 days showing severe congestion in sclerotic blood vessels(v) with focal haemorrage (h) (x 40) [A], also showing focal inflammatory cells infiltration (g) inbetween the glomeruli& tubules (x80), [B].

160 Results

Fig.(75): Histopathological section (stained with H& E) of the kidney tissue of TCE intoxicated rats supplemented with vitamin C (gpΙV,) for 105 days showing hyperemic glomeruli tuft (g), (x80).

Fig.(76): Histopathological section (stained with H& E) of the kidney tissue of TCE intoxicated rats supplemented with zinc (gpΙV,) after 105 days showing congestion of the glomerular tuft (g), and intertubular capillaries (r)(x80).

161 Results

Fig.(77): Histopathological section (stained with H& E) of the kidney tissue of TCE intoxicated rats supplemented with vitamin C& zinc (gpΙV,) for 105 days showing hyperemic glomeruli tuft (g), (x80).

Fig. (78): Histopathological sections (stained with H& E) of the testes tissue of TCE intoxicated rats (gpΙV,) for 105 days, showing azospermia in most seminiferous tubular lumen (s),(x40).

162 Results

Fig.(79): Histopathological section (stained with H& E) of the testes tissue of TCE intoxicated rats supplemented with vitamin C (gpΙV,) for 105 days showing intact histological structure (s)(x40).

163 Results

Fig.(80): Histopathological section (stained with H& E) of the testes tissue of TCE intoxicated rats supplemented with zinc (gpΙV,) for 105 days showing intact histological structure (s) (x40).

Fig.(81): Histopathological section (stained with H& E) of the testes tissue of TCE intoxicated rats supplemented with vitamin C& zinc (gpΙV,) for 105 days showing intact histological structure (s)(x40).

164 Results

Fig.(82): Histopathological section (stained with H& E) of the liver tissue of TCE intoxicated rats for 20 days and TCEwithdrawal showing, diffuse kupffer cells proliferation inbetween the hepatocytes (arrow), (x 80).

Fig.(83): Histopathological section (stained with H& E) of the kidney tissue of TCE intoxicated rats for 20 days and TCEwithdrawal showing, intact histological structure (g)(x80).

165 Results

Fig.(84): Histopathological section (stained with H& E) of the testes tissue of TCE intoxicated rats for 20 days and TCEwithdrawal showing, degeneration in the seminiferous tubules (s) with giant spermatogonial cells formation(arrow), (40).

166 Results

[A]

[B]

[C] Figure (85): Apoptotic laddering pattern showed by Agarose Gel Electrophoresis of DNA fragments of liver [A], kidney [B] and testes [C] tissues of normal and TCE intoxicated rats supplemented with vitamin C and /or zinc. Lane 1 &2: DNA fragments extracted from the normal rats. Lane 3 &4: DNA fragments extracted from TCE intoxicated rats for short term of treatment. Lane 5 &6: DNA fragments extracted from TCE intoxicated rats for long term of treatment. Lane 7 : DNA fragments extracted from TCE intoxicated rats supplemented with vitamin C. Lane 8 : DNA fragments extracted from TCE intoxicated rats supplementewith zinc for long period of treatment Lane 9&10: DNA fragments extracted from TCE intoxicated rats supplemented with vitamin C and zinc for long of treatment Lane 11: DNA fragments extracted from the withdrawal rats. 167 Discussion

Trichloroethylene has been shown to generate free radicals and induces lipid peroxidation both in vivo and in vitro ( Ogino et al., 1991; Channel et al., 1998; Khan et al., 2001). However, role of TCEinduced lipid peroxidation in the induction/acceleration of an autoimmune response remains unknown.

TCE is an environmental and industrial pollutant whose toxicity and carcinogenicity have been demonstrated in several animal species, including humans ( Davidson and Beliles, 1991).

TCE, a volatile organic compound widely used as an industrial solvent and a degreasing agent, is involved in the development of autoimmune disorders and immune system dysfunction both in human and animal studies (Kilburn et al., 1992; Yanez et al., 1992; Khan et al., 1995 andGriffin et al., 2000a). Autoimmune disorders were observed in humans following exposure to TCE through inhalation and contaminated drinking water or food (Kilburn et al., 1992) or from occupational exposures ( Flindt Hansen et al., 1987 and Nietert et al., 1998).

Environmental exposure to TCE is associated with several types of immune disorders including systemic lupus erythematosus (Kilburn et al., 1992), systemic sclerosis and fasciitis ( Kilburn et al., 1992 ), whereas occupational exposure is linked to the development of scleroderma ( FlindtHansen et al., 1987 and Nietert et al., 1998).

Evaluation of the risk for humans potentially exposed to TCE in either the workplace or due to environmental contamination is complicated by the existence of multiple metabolic pathways, multiple target organs, poor or incomplete exposure data for many epidemiology studies, and sex and

168 Discussion

speciesdependent differences in sensitivity to toxic effects (Lash et al., 2000).

The results of the present work revealed a significant change in all parameters studied; body and organs weights, biochemical parameters, histopathological examination and apoptosis for normal and TCE intoxicated animals supplemented with vitamin C and/ or zinc

І Body and Organs Weights:

Body weight is frequently the most sensitive indicator of the adverse effects of xenobiotics, so it is considered as a determined parameter of toxicity testing.

The present study indicated that administration of trichloroethylene to rats for short and long periods significantly affecting on body and organs weights, however, significant decrease in body weight gain at the end of long period of the experiment was noticed and these results were agreed with Zenick et al. (1984) who reported that TCErelated effects were seen primarily as reduced body weight gain and elevated liver/body weight ratios.

In contrast, Merrick et al. (1989) stated that body weights were not altered by TCE but liver/body weight ratios were uniformly increased by TCE administered in either vehicle in both sexes. However Kjellstrand et al. (1981) study indicated that an age dependent decrease in body weight gain was observed in female rats exposed to TCE.

Data of the present study showed that TCE administration produced significant inhibition in the normal relative testes weight and variation in testicular enzyme activity ( Kumar et al., 2001).

169 Discussion

These findings are in agreement with a bundle of literatures which pointed out the toxic effects of TCE on the testicular tissue.

In the present study the relative weights of liver and kidney were significantly increased in manner dependent on the dose and period of administration of TCE, these results fit with data of Geol et al. (1992) who found that significant increase in liver weight, and increase in kidney weight, indicating the sensitivity of liver and kidney as target tissues in TCEtoxicity.

Also Kjellstrand et al. (1984) reported that liver weight increased in a time and concentration dependent manner and both sexes exhibited significant liver enlargement, and decrease in body weight gain was seen in both sexes after administration of TCE and the kidney weight was slightly increased. Moreover increased liver weight (175% of control) was reported by Elcombe et al. (1985) after administration of TCE where it may contributed to hypertrophy.

Carter et al. (1995) reported that enlarged liver was due to significant glycogen accumulation and focal areas with necrosis was seen in the liver with chronic exposure to TCE, these was due to inactivation of enzymes that responsible for glycogen metabolism.

In this study there was significant reduction in testicular weight after long exposure to TCE, these result was agreed with Kumar et al. (2001) who stated that significant reduction in absolute testicular weight and altered marker testicular enzyme activity.

TCE has been shown to generate free radicals and induces lipid peroxidation both in vivo and in vitro (Ogino et al., 1991; Channel et al., 1998; Khan et al., 2001 ). However, role of TCE

170 Discussion

induced lipid peroxidation in the induction/acceleration of an autoimmune response remains unknown.

Several dietary micronutrients contribute greatly to the protective system. Based on the growing interest in free radical biology and the lack of effective therapies for many of the chronic diseases ( Machlin and Bendich, 1987).

The present study revealed that the supplement of TCE intoxicated animals with vitamin C and/or zinc induced marked amelioration of pathological lesions induced in liver, kidney and testicular tissues manifested by improvement in body and organs weight toward the normal, this may contributed to the potential antioxidant role of vitamin C in regeneration of vitamin E to keep its role as antioxidant and halt peroxidation of cellular membrane to maintain and repair tissue damage this is in agree with the results recorded by ElMissiry, (1999).

The supplementation of pharmacological doses of zinc in vivo has a protective effect against general and liverspecific prooxidants. Dietary zinc deficiency causes increased susceptibility to oxidative damage in membrane fractions from some tissues suggesting that increased oxidative stress may be a mild but significant. ( Tammy and William, 2003).

ІІ Biochemical Parameters:

A Hematological Parameters:

The current results demonstrate that TCE induced oxidative stress and inflammatory response in rats as verified by biochemical determinations.

The results of the present study revealed significant reduction in erythrocytic count, hemoglobin, platelets,

171 Discussion

hematocrit, MCH, MCV, MCHV and segmented neutrophils and significant increase in WBC count, lymphocytes, monocytes, basophils eosinophils and staff nutrophils, it may be due to the increase in erythrothetic destruction as a result of abnormalities in the environment contaminated with TCE. These results were coincide with Bond, (1996) who found significant increase in number of typical lymphocytes, and white blood cell count was 10,100mm 3 with 27% eosinophilia and this is due to sensitization to trichloroethylene or more likely to one of its metabolites.

On the contrary the hematological studies by Geol et al. (1992) showed a significant increase in RBC counts and a reduction in WBC counts without any statistically significant change in the hemoglobin in the blood of TCEexposed mice.

The present findings indicated a significant reduction in platelet counts (thrombocytopenia) at long period of treatment, and decreased by 189.13 and 183.75x10 3l for short and long period respectively at dose level (750mg/kg body weight /day) than that of normal control (288.75 x10 3l). TCE treatment inhibits adenylate cyclase and stimulates phosphodiesterase activity with a concomitant increase in the rate of platelet aggregation. Also, TCE significantly reduced cytosolic cAMP level, which is a regulatory molecule in the event platelet aggregation and disruption of its homeostasis is directly correlated to xenobiotic effects of TCE. Kumar et al. (2001) reported that TCE appears to be one of the most toxic xenobiotic to platelet function.

It was evident from the present study that a dietary vitamin C and zinc may have curative effects on modulatory long term TCE toxicity in rats.

172 Discussion

The observations recorded in the present work were significantly correlated to the previous work of Geol et al. (1992) who noticed that a doserelated increase in cell density and acid phosphatase activity with parallel significant decrease in the activity of deltaALAD (deltaaminolevulinic acid dehydratase) were observed in the bone marrow, which appear to be responsible for hematological alterations in TCEexposed mice. The results suggested that early metabolic, pathological and hematological perturbations following a shortterm exposure of TCE in mice can provide the basis for its documented potential for chronic effects like blood dyscrasia and cancer.

Zinc protoprophyrin, a sensitive indicator of iron deficiency and impairment of heme biosynthesis, showed a significant increase in TCE exposure. These changes were accompanied by significant alterations in almost hematological parameters with an increase period and dose of TCE exposure. The hemoglobin damage induced by TCE may lead to hemolysis and contributes to peroxide production and oxygenated hemoglobin

(HbO 2) to produce methemoglobin (metHb) and eventually, degraded hemoglobin (Hb) characterized by gross precipitation of the protein. The main Pathway for TCE reduction in RBCs is sulfhydryl dependent.

Fontanella et al. (2002) showed that the key enzyme which involved in the synthesis of heme were: (1) deltaaminolevulenic acid synthetase (deltaALAS), a mitochondorial enzyme that catalyzes the function of deltaaminolevulenic acid (deltaALA) and (2) deltaALA dehydratase (deltaALAD) a cytosolic enzyme that catalyzes formation of prophobilinogen. Through several of steps, coproprophyrin and protoprophyrin are formed from prophobilinogen and finally the mitochondorial enzyme ferrocheelatase catalyzes the insertion if iron into protoprophyrin to form heme. When erythrocyte deltaALAD activity is inhibited, 173 Discussion

aminolevulenic acid (ALA) levels in blood are increased, leading to elevated levels of ALA in urine. The results showed that delta ALAD potentially restoring its activity after vitamin C supplementation.

One of the acute effects of zinc is an apparent stabilization of sulfhydryls, i.e., zinc protects certain enzyme sulfhydryls from oxidation. The enzyme that has been most extensively studied is δaminolevulinate dehydratase, which catalyzes the formation of the pyrrole porphobilinogen from two molecules of δ aminolevulinic acid ( Anderson and Desnick, 1979 ). δ aminolevulinate dehydratase are sulfhydryl dependent, and there is a strong correlation between thiol and enzyme activity ( Gibbs et al.,1985).

B Liver Function Biomarkers:

When liver cell plasma membrane is damaged a variety of enzymes normally located in the cytosol are released to the blood stream including AST, ALT and ALP. Thus, estimation of their blood levels is considered as indicators of liver function. Xu et al. (2009) reported that transaminases showed increased activity in experimental animals after exposure to TCE. This increase reflected tissue damage and release of tissue enzymes which related to the TCE induced hepatic necrosis in liver.

The current study recorded that short and long period of exposure to TCE revealed significant elevation of liver trasaminases enzymes ( ALT and AST), alkalin phosphatase (ALP) and total bilirubin (p<0.05) particularly when compared to the normal control, these data of liver was agreed with Xu et al. (2009 ). Khan et al. (2009 ) reported that TCE caused severe damage to liver and alter carbohydrate metabolism and suppressed antioxidant 174 Discussion

defense system, causing reactive oxygen species (ROS) production that lead to membrane lipid peroxidation and liver injury with released transaminases enzymes into the circulation. Joseph et al. (2000) reported that accelerated autoimmune response induced by treatment with occupationally relevant doses of trichloroethylene for 32 weeks was accompanied by an increase in serum ALT and AST levels, indicating lowlevel hepatotoxicity. Cai et al. (2008) and Glinda et al. (2009) reported that long period of exposure to TCE induced liver injury that are measured by serum levels of ALT and AST. On the other hand reduction in total protein, albumin and globulin was observed as result of short and long term of exposure to TCE. It was suggested that long period of exposure to TCE impair liver synthetic function, these results were coincide with Khan et al. (2009) who hypothesized that TCE induces oxidative stress to various rat tissues and alter their metabolic functions indicating that TCE caused severe damage to liver. Throughout vitamin C and/or zinc supplementation the mean AST, ALT and ALP values were decreased as compared with intoxicated control group. That means the dietary zinc and ascorbic acid significantly counteracts the hepatotoxic effect of TCE, the serum level of total protein, albumin and total bilirubin were almost normalized at short period of treatment as compared with the control group. C Renal Function Biomarkers: The kidneys are main target organ for TRI, although much controversy exists about its importance for humans, as significant species differences exist in susceptibility (Lash et al., 2000a).

Renal effects of TRI are generally attributed to its conjugation with GSH and subsequent metabolism within the proximal tubules to generate DCVC, which is further metabolized

175 Discussion

to a reactive intermediate ( Lash et al., 2000b). Thus, the renal disposition and toxicity of TRI are dependent on GSH status (Lash et al., 1995a, 1998b). Mitochondria are a prominent and early target for DCVCinduced cytotoxicity in renal proximal tubules ( Lash et al., 1995a, 2001a). The results obtained in the present work showed administration of TCE for short and long period of exposure resulted in significant rise of serum urea, uric acid and creatinine (p<0.05) as compared to that of control, these data were in accordance with the finding of Khan et al. (2009), who reported that TCE administration increased blood urea and serum creatinine indicating toxicity and severe damage to kidney . In contrast Geol et al. (1992) reported no statistical significant change in the urea nitrogen, creatinine and uric acid levels in the blood of TCEexposed mice.

In the current investigation, administration of vitamin C or zinc renders rats less susceptible to kidney damage induced by treatment with TCE. This protection was evidenced by significant lowering of urea and creatinine levels in the serum of intoxicated rats. This result consistent with those reported by Upadhyay et al. (2009). Moreover Jacob, (1999) demonstrated that vitamin C ameliorated TCE induced renal injury in terms of LDH leakage from a renal epithelial cell line.

The watersoluble properties of vitamin C allow for the quenching of free radicals before they reach the cellular membrane. Tocopherol and glutathione also rely on ascorbic acid for regeneration back to their active isoforms. The relationship between AA and glutathione is unique. Vitamin C reduces glutathione back to the active form. Once reduced, glutathione will regenerate vitamin C from its DHAA or oxidized state (Jacob, 1999).

176 Discussion

The ability of zinc to retard oxidative processes has been recognized for many years. In general, the mechanism of antioxidation can be divided into acute and chronic effects. Chronic effects involve exposure of an organism to zinc on a longterm basis, resulting in induction of some other substance that is the ultimate antioxidant, such as the .

The acute effects involve two mechanisms: protection of protein sulfhydryls or reduction of OH formation from H 2O2 through the antagonism of active transition metals, such as iron and copper (Ternay and Sorokin 1997).

In the present work liver and kidney biomarkers show slight improvement when the animal supplemented with one micronutrient (vitamin C or zinc) but highly improved toward normal control if coadministrated and this may be due to the antioxidant potential of both micronutrients that scavenging free radicals and stopping liver and kidney injury, the data are in harmony with Upadhyay et al. (2009) who found zinc and ascorbic acid treatment show moderate therapeutic efficacy when administrated individually, where more pronounced protective effects were observed after combined therapy of zinc and ascorbic acid and may be useful in restoration of toxicity induced biochemical alterations.

D Immunological Biomarkers:

Immunological response in the current study showed that exposure to TCE for short and long period causing significant activation in the immune system with elevation in the immune response that lead to significant increase (p<0.05) in the circulating humoral antibodies IgG and IgM than that of normal control, these results may be contributed to that exposure to TCE causing alteration in the immune system that lead to Tcell

177 Discussion

activation which may in turn promote the development of autoimmunity and increased circulating antibodies, these results were in agreement with Ping Cai et al. (2008) who hypothesize that TCE reactive metabolites bind to selfproteins to form protein adducts, which then escape normal tolerance to selfproteins and act as neoantigens, elicit autoreactive T cells resulting in B cell activation, induce autoimmune responses, and eventually lead to autoimmune diseases.

Also provided evidence that TCE induces or accelerates autoimmune responses by Khan et al. (1995), and that treatment with a reactive TCE metabolite, dichloroacetyl chloride (DCAC), increases serum levels of total IgG, DCACspecific antibodies, and antinuclear antibodies, which is a measure of systemic autoimmunity (Khan et al., 1997; Cai et al., 2006).

Bloemen et al. (2001 ) reported that TCE can be metabolized either oxidatively or through glutathione conjugation pathways. The glutathionemediated metabolism is of minor significance in humans and, he focused on the oxidative metabolic pathway. TCE can be oxidized to TCEoxide, which can either rearrange to DCAC or be hydrolyzed to dichloroacetic acid. In aqueous solution, TCEoxide forms lysine adducts with proteins (Cai and Guengerich, 1999, 2000 ). As small molecules, TCE or its metabolites will not be antigenic by themselves, but may haptenize to selfproteins, and thus result in structural modifications that render selfproteins antigenic, potentially leading to autoimmune responses. Increasing evidence suggests that autoimmune diseases are multifactorial, and could involve genetic, hormonal and environmental influences. In addition to bacteria and viruses, other environmental factors including such chemicals as trichloroethene (TCE) ( Kilburn et al., 1992; Khan et al., 1995).

178 Discussion

TCE is involved in the development of autoimmune disorders and immune system dysfunction both in human and animal studies ( Kilburn et al., 1992; and Griffin et al., 2000a). Autoimmune disorders were observed in humans following exposure to TCE through contaminated drinking water ( Kilburn et al., 1992 ) or from occupational exposures ( FlindtHansen et al., 1987 ). Environmental exposure to TCE is associated with several types of immune disorders.

In the present work the supplementation of dietary micronutrients as vitamin C and/or zinc to the intoxicated rats for short and long terms showed significant decrease of IgG & IgM than that of the intoxicated control group (gp IV) these findings indicate that the improvement of the immunological status with alleviation in Tcell activation and reduction in humoral antibodies to the normal levels. Orally gavage with both micronutrients together was more effective in the improvement of immunological system and antioxidant potential effect than if micronutrients taken separately. Also vitamin C and zinc developing a particular complementary role in supporting immune functions and combating infections ( Maggini et al., 2010).

E Hormonal Biomarkers:

Effects on the thyroid functions in the present study as a result of acute and chronic exposure to TCE were obvious in decreasing in the serum levels of FT4 values and increasing in both FT3 and TSH and these may be contributed to that exposure to TCE activate immune response and Tcell activation that lead to production of cytotoxic cells causing thyroid disruption and imbalance the thyroid hormones also may be due to destruction of the pituitary gland or hybothalamus according to Kjellstrand et al. (1985). As a result the FT4/FT3 ratio decreased in the treated groups compared to the normal controls. The results could 179 Discussion

possibly from the consequence of a changed serum binding capacity (TBG and TBPA), altered TSH stimulation, extrathyroidal conversion of T4 to T3 caused by factors regulated by negative feedback mechanism involving TSH, also a change to a lower ratio between the number of FT4/FT3 molecules on the thyroglobulin protein (snyder,1973).

The results mentioned before were in harmony with (Villanger et al., 2011) who reported that exposure to organochlorine may influence on thyroid hormone levels by acting on hybothalamic – pituitary –thyroid axis, so organochlorine may affect on circulating thyroid hormone levels and may interfere with thyroid homeostasis ( weisglas et al., 2000) reported that exposure to organochlorines can lead to adverse effect on immune, reproductive, neurobehavioral and endocrine functions.

Short and long terms of toxicity in this study have been conducted on the male rats via oral exposure to TCE show a significant (p<0.05) decrease in serum testosterone level, these results were in agreement with Kumar et al. (2001) who reported that inhalation of TCE may bring about testicular toxic effects, and the results indicated significant reduction in absolute testicular weight, and alters marker testicular enzymes activity associated with spermatogenesis and germ cell maturation.

The observation of higher levels of serum testosterone in long term group than the short term group might be due to seasonal change as observed by Schopper, et al. (1984). His results indicate that the seasonal variation in testicular steroid production by the wild boar, regulated by photoperiod. Or this observed higher level of serum testosterone may be an indicative of maturity of the pituitary–gonadal axis in male rats, and TCE could be considered as endocrimne chemical disrupting and it`s mode of action on the thyroid ant testes is most probably through

180 Discussion

receptor and / or post recsptor mechanism (Lunn et al., 1994, Kelnar et al., 2002).

Also Kumar et al. (2000) reported significant decrease (p<0.05) in total epididymal sperm count, sperm motility, specific activities of enzymes Glucose 6pdehydrogenase (G6pDH) and 17 betahydroxy steroid dehydrogenase (17beta HSD) with concomitant decrease in serum testosterone concentrations in TCE inhaled rats showing reduced male reproductive efficiency.

Kjellstrand et al. (1985) stated that depletion of testosterone through castration or destruction of the pituitary gland or hypothalamu s as a result of exposure to trichloroethylene

Thyroid function in the current study were improved by supplementation with dietary vitamin C and zinc or mixture of both, these may be due to antioxidant potential and protective effect of both micronutrients with improvement in the thyroid hormone released axis (Gilbert et al., 2004 and Blossom and Gilbert. 2006).

Also testosterone level in the current study were improved by supplementation with dietary vitamin C and zinc due to the effect of each in repairing damage occur.

ElMissiry, (1999) stated that toxic effects were accompanied by significant elevation of testicular lipid peroxidation, decreased plasma testosterone level and a drop in copper and zinc concentrations in testes. The administration of ascorbic acid after toxic treatment blunted the increased testicular lipid peroxidation and the decreased plasma testosterone level probably by protecting antioxidants and the loss of copper and zinc from testes. The data suggested that ascorbic acid has a protective effect on toxic induced damage by maintaining the activity of cellular antioxidants. 181 Discussion

ІІІ Histopathological Examination : The current results demonstrated that TCE induced oxidative stress and inflammatory response in rats as verified by biochemical observations. Hence the kidney has an affinity for TCE and certain cytochrom P450 predominantly in the cortex; it is thought that the mechanism of TCE nephrotoxicity is probably the same as that of the liver. The significant change of hepatic and renal biomarker in serum of TCE intoxicated rats verified by histopathology finding. Histopathological examination of liver tissues of short and long term TCE intoxicated rats showed inflammatory cells in the portal area, degeneration of hepatocytes with diffuse kuppffer cells proliferation. Apoptosis and DNA damage in the liver were appeared. These findings were in agreement with Geol et al. (1992) who reported that degeneration/necrosis of hepatocytes and characteristics proliferation of endothelial cells of hepatic sinusoids.

Also TCE exposure to different doses in MRL +/+ mice for 32 weeks induced histopathological changes similar to autoimmune hepatitis (Griffin et al., 2000).

TCE exposure for longterm altered liver histopathology in MRL +/+ mice. Exposure to TCE for 36 and 48 weeks caused hepatocyte necrosis and leukocyte infiltration into liver lobules according to Cai et al. (2008).

These may be attributed to those TCE metabolites capable of triggering an immune response with activation of Tcells that lead to induce cytotoxic cells and outoimmune hepatitis. In other side exposure to TCE may induce liver injury with morphological and hepatocytes alteration which may be due to the production of 182 Discussion

ROS that lead to membrane peroxidation and hepatocellular injury (Wang et al., 2008). The data were in agreement with Joanne et al. (2009) who reported that interaction between TCE exposure and immune gene variation given the observed effects of TCE on the immune system and inflammatory response genes. This study, could suggested that certain micronutrients such as vitamin C and zinc may protect liver and kidney by their antioxidant and anti inflammatory effects on TCE intoxicated injury, various investigator have demonstrated an inflammatory action of vitamin C, zinc and both in vitro and in vivo ( Jacob, 1999 and El Missiry, 1999 ). It has been reported to be specific inhibitors of nuclear factor kappa (BNF Kb), which may account for some of its –inflammatory properties. The NFkb pathway is a key mediator of gens involved in cellular proliferation, apoptosis and cytokine production. Hence, hepatic tissue of vitamin C and /or zinc supplemented rats showed remarkable rednation in inflammatory signs, absence of periportal fibrosis, decrease in vascular degeneration and appearance of mitotic figures in nucleus with regeneration of hepatic cells as well as absence of necrotic foci in comparison with TCE intoxicated ones these findings are in agreement with the studies of (Merino et al., 1996)

It was suggested that dietary vitamin C and zinc have a hepatoprotective effect on hepatic injury, which may be explained by inhibition of phase I enzymes and induction of phase II enzymes.

The ability of ascorbic acid to protect from prooxidant induced toxic injury was investigated in isolated, intact rat hepatocytes, whose ascorbic acid content had been restored by means of exogenous supplementation to rats exposed to organochlorine.

The antioxidant effect appears primarily to involve membrane lipids, and can be independent from the cellular content

183 Discussion

of vitamin E, thus suggesting that ascorbic acid can play a direct and independent role in the intact cell ( Maellaro et al., 1994 ). (Unsal et al., 2008) reported a reduction in liver damage in zinc treated group of rats exposed to organochlorine, and conclude that zinc has the potential to alleviate the toxic effects of organochlorine in rat liver. The kidney histopathology, biochemical determinations and the morphology of the kidney by using the light microscopy were evaluated as end points of renal damage of TCEintoxicated rats for long period of treatment. The examination of renal sections showed focal inflammatory cells infiltration in between and surrounding the glomeruli and tubules at the cortex with swelling in the tubula epithelium. Also focal haemorrage in the corricomedullary portion and severe congestion in sclerotic blood vessels were appeared with TCE – administration for the both period of treatment. These results were in agreement with Geol et al. (1992) who found that increase in kidney weight, glomerular nephrosis, degeneration/ desquamation of tubular epithelium and characteristic amyloid deposition in glomeruli. The data revealed that the histopathological examination of kidney slices of TCE rats supplemented with vitamin C and /or zinc illustrated moderate atrophy of glomerular interstitial hemorrhage and lymphocytic infiltration. Renal tissues exhibited mild congestion in its blood vessels with mild dilatation in some renal tubules and normal glomerular structure. These findings are consistent with the study of (liu et al., 2006). Ascorbic acid supplementation reversed all the changes in biochemical indices, as well as histopathological alterations normally induced by toxicity. The findings imply that reactive oxygen species play a causal role in I/Rinduced renal injury, and that AA exerts renoprotective effects, probably by radical scavenging and antioxidant activities according to ( Korkmaz and Kolankaya., 2009).

184 Discussion

Different histological studies established the existence of a definite relationship between TCE intoxication and testicular damage (Kumar et al., 2000). The findings showed decreased activity of the testicular marker (LDHx) after TCE administration. Several investigators reported decreased LDHx activity after exposure to different testicular toxicants.

Histological examination of testicular tissue of TCE intoxicated rats for short and long periods of treatment showed degenerated spermatogonial cells in the lumen of the seminiferous tubules with appearance of homogenous eosinophilic albuminous material in between also showing multinumber of sertoli cells in the lumen of the degenerated tubules and showing mitosis in the spermatogonial cells of some seminiferous tubules and azospermia in most seminiferous tubular lumen. These results were in harmony with Kumar et al. (2001) who reported that inhalation of TCE by male rats for 12 and 24 weeks brings about significant reduction in absolute testicular weight, and alters marker testicular enzymes activity associated with spermatogenesis and germ cell maturation, along with marked histopathplogical changes showing depletion in germ cells and spermatogenic arrest.

Lamb and Hentz, (2006) studies showed various organ effects in the male reproductive system. Enzyme induction and oxidative metabolism appear to be important in the systemic toxicity and may play role in the reproductive toxicity of TCE. Oxidative metabolites of TCE are formed in the mouse epididymis resulting in epididymal damage, and at systematically toxic high doses, TCE may adversely affect the maturation of sperm and decreasing sperm motility. Short term TCE – treatment and after its withdrawal in this study induced deleterious effects as oligospermia secondary to germ cells damage combined with testicular atrophy and interstitial fibroblasts cells proliferation.

185 Discussion

Many investigators reported nearly similar results and attributed their findings to disturbance in endocrine function (weisglas et al., 2000).

This study revealed that the treatment of the TCE intoxicated animals with vitamin C and/ or zinc induced marked amelioration of pathological lesions induced in testicular tissue. Ascorbic acid blunted the increased testicular lipid peroxidation and the decreased plasma testosterone level probably by protecting antioxidants and the loss of copper and zinc from testes. The data suggested that ascorbic acid has a protective effect on toxic induced damage by maintaining the activity of cellular antioxidants (ElMissiry, 1999). IV Detection of DNA Fragmentation: Apoptosis is genetically programmed series of events that allow for the removal of unneeded, senescent or infected cells from the body. Unlike necrosis, apoptosis or programmed cell death in the tissue of an organism is not associated with inflammation or scarring ( Van wangenheim and Peterson , 1998 ). It is important and inevitable events in the remodeling of tissue during development and aging ( Searle et al., 1982 ). This phenomenon occurs in cells injured by certain toxic agents. It is also a crucial process for eliminating cancer cells ( Guchelaar et al., 1997). The TCE intoxication induced apoptosis and DNA damage in liver, kidney and testes of treated rats for short and long terms. TCE intoxicated rats supplemented with vitamin C and / or zinc and the withdrawal of the TCE for long time induced antiapoptotic effect. DNA fragmentation detected by agarose gel electrophoresis (Wyllie et al., 1980 ) is used to demonstrated the ladder pattern of DNA (a hallmark of apoptosis) which is generated by endonucleolytic cleavage of genomic DNA into nucleosomal size

186 Discussion

DNA of approximately 180bp long (monomers) or oligonucleotides, which are multiple of 180bp (oligomers). The present study revealed that TCE has slight DNA damage effect on liver, kidney and testicular tissues for short period of treatment, but induced apoptosis after long period of treatment. On the other hand vitamin C and / or zinc supplementation for the treated rats with TCE resulted in antiapoptotic potentials in the prevention of DNA damage in TCE – intoxicated rats.

Kupffer cells play a key role in the liver phagocytic activity which includes the phagocytosis of apoptotic cells ( Dini et al., 2002 ). The apoptogenic action of kupffer cells could be exerted by releasing factors and /or cytokines. In addition, kupffer cells may induce hepatic apoptosis in synergy with other cells such as the endothelial cells ( Ruttinger et al., 1996 ). The obtained apoptotic effect of trichloroethylene may be attributed to their cytotoxic consequence ( Feng et al., 2008 ). On the similar ground, TCE significantly potentiate the MPP (+) induced cell death associated with observed DNA fragmentation, which is one of the hallmarks of apoptosis. In addition, TCE markedly reduced the efflux of MPP (+) from pc12 cells(3(3(2(7 chloro2 quinolinyl ) ethenyl) phenyl )(3 dimethyl amino 3oxo propyl )thio) methyl) proponoic acid (MK571),which in an inhibitor of multidrug resistance proteins (MRPs),mimicked the NSAIDs induced effects, increasing cell toxicity and promoting the accumulation of MPP(+). These results suggest that TCE might cause a significant increase in the intracellular accumulation of MPP (+) via the suppression of by the blockade of MRP, resulting in the potentiation of MPP (+) induced cell death (Colotta et al., 1992 ).

From the present work, it can be concluded that TCE exerts harmful effect on pituitary –thyroid axis, liver kidney and

187 Discussion

testicular tissues of the treated animals for any period of time. The biochemical and histopathology finding showed that TCEimposes an oxidative stress and inflammatory response to hepatic tissue vitamin C and zinc confer a beneficial therapeutic effect against the oxidative damage and inflammation associated with toxic substance. The supplementation of vitamin C and/or zinc to the TCE intoxicated rats for the recommended period is advisable. It can be assumed that the results are valid for humans too.

188 Summary and conclusion

Trichloroethylene, one of several xenobiotics, is an industrial organic volatile solvent most often used for cleaning and degreasing of fabricated metal parts. Because of its widespread commercial use, TCE has also become a major environmental pollutant. TCE is the most frequently reported organic contaminant in groundwater, the source of 93% of public water systems.

Although most TCE exposures occur in the occupational setting, the general population can be exposed via environmental contamination through drinking water, air, or food. TCE is a common contaminant found at Superfund sites and has been identified in at least 852 of the 1416 hazardous waste sites proposed for inclusion on the Environmental Protection Agency National Priorities List. As a result, it has been estimated that 10% of the non occupationally exposed population has detectable levels of TCE in the blood.

The present study focused on potential health risks associated with the administration of TCE which is important indoor contaminant used in various household products. The clinical signs of acute trichloroethylene overdose are commonly coma, cardiac conduction disturbances, diarrhea, and vomiting leading to multiple organ failure.

TCE intoxicated rats were supplemented vitamin C and/or zinc for short and long terms to evaluate the possible modulatory effect of these micronutrients against TCE induced toxicity and to assess their possible .

The present study was performed on 102 male Wister albino rats weighing 120 150 grams. Rats were classified into four groups as follows:

189 Summary and conclusion

Group I : normal control divided into four subgroups each 6 rats and treated as follows:

Normal control, normal control+ vit.C (50mg/kg body weight/ day) normal control +Zn (zinc sulphat 200 mg/kg body weight/ day) and normal control + vit.C & zinc (50 and 200 mg/kg body weight/ day, respectively) per orally (p.o.)

Group II : the animals of this group treated with TCE at dose level of 750 mg/kg body weight /day. P.o. They were divided into 5 subgroups each 6 rats and treated as follows:

TCEintoxicated control, TCEintoxicated rats + vit.C (50 mg/kg body weight/ day), TCEintoxicated rats+ zinc (zinc sulphat 200 mg/kg body weight/ day) and TCE intoxicated rats+ vit.C & Zn (50 and zinc sulphat 200 mg/kg body weight/ day, p.o.).

Withdrawal group: treated with TCE for 20 days at the start of the experiment and withdrawn for recovery until the end of the experiment (105 days). The treatment of group I & II were continued for 20 days (short term).

Group III and IV : The animals of this group were classified as group I and II but the treatment of TCE and the supplementation of vitamin C and zinc continued to 105 days (long term).

The results of the current study showed a significant decrease in the body weight after the treatment of TCE for long period (105 days), also recorded significant elevation in the liver and kidney weights and decrease of testes weight in the TCEintoxicated rats for long term than that in normal control.

190 Summary and conclusion

Hematological parameters were evaluated such as RBC’s, WBC’s, Pt and Hb and differential leucocyte. The selected biochemical determinations such as ALT, AST, ALP, albumin, globulin, total protein and total bilirubin for all groups were estimated. Moreover, some hormones were evaluated, such as FT3, FT4 and total testosterone by radioimmunoassay assay (RIA) technique and TSH by immunoradiometric assay (IRMA) technique. Determination of some immunoglobulins such as IgG and IgM were estimated by radialimmunodiffusion (RID), also histopathological examination and detection of apoptosis in solid tissues were conducted on normal and TCE intoxicated rats for long and short terms.

Some of the selected hematological and biochemical parameters in serum of the treated rats significantly changed after short and long period of treatment. The results revealed significant decrease (P<0.05) in FT4 and significant increase in free FT3 and TSH in TCEintoxicated rat groups for the two period of treatment as compared to that of normal control group. The current results revealed significant decrease (P<0.05) in the mean values of total testosterone in TCEintoxicated rat group as compared to that of normal control.

Histopathological examination of liver tissues showed congestion with degeneration in the hepatocytes and apoptosis were observed in some of the hepatocytes after long period of treatment, In the kidney tissues, it was showed marked focal inflammatory cells infiltration inbetween and surrounding the glomeruli& tubules at the cortex with swelling in the tubula epithelium, and severe congestion in sclerotic blood vessels with focal haemorrage, also vaculation and focal necrosis of tubular lining and interstitial cell infiltration and desquamation of the tubular epithelial cells in the renal cortex after long period of treatment. 191 Summary and conclusion

Marked structure alteration was observed in the testes tissue of TCEintoxicated rats for 20 days showing degenerated spermatogonial cells in the lumen of the seminiferous tubules with appearance of homogenous eosinophilic albuminous material in between, and multinumber of sertoli cells in the lumen of the degenerated tubules. In addition to appearance of azospermia in most seminiferous tubular lumn after long period of treatment.

The DNA damage with apoptosis was detected by agarose gel electrophorasis, liver, kidney and testes tissues in intoxicated rats showed an increase in percentage of DNA fragmentation followed administration of TCE for 20 and 105 days but the percentage of DNA damage was obvious in case of intoxicated group for long period.

Withdrawal group recorded mild improvement in all changed parameters and the damaged tissues also slightly improved and recorded mild decrease in the apoptotic bands of DNA fragments.

The results of the present work revealed that body and organs weight, hematological parameters, biochemical biomarkers, and damaged tissues showed slight improvement when supplementation occur by one micronutrient vitamin C or zinc, but highly improved toward normal or control if the two micronutrients coadministrated and this due to the antioxidant potential of both micronutrients that scavenging free radicals and stopping liver, kidney and testes injury.

Accordingly, 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 vitamin C and zinc together to compensate the TCE hazardous effects.

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ﺍﻝﻤﻠﺨﺹ ﺍﻝﻌﺭﺒﻲ

ﺍﻝﻤﻘﺩﻤﺔ: ﻝﻘﺩ ﻝﻭﺤﻅ ﻓﻲ ﺍﻷﻭﻨﻪ ﺍﻷﺨﻴﺭﺓ ﺍﻨﺘﺸﺎﺭ ﺍﻝﻤﻠﻭﺜﺎﺕ ﺍﻝﺒﻴﺌﻴﺔ ﺒﺼﻭﺭﺓ ﻜﺒﻴﺭﺓ ﻭﺯﻴﺎﺩﺓ ﻅﻬـﻭﺭ ﺍﻷﻤﺭﺍﺽ ﺍﻝﻤﺘﺭﺘﺒﻪ ﻋﻠﻲ ﻫﺫﻩ ﺍﻝﻤﻠﻭﺜﺎﺕ ﺍﻝﺒﻴﺌﻴﺔ- ﻭﺘﻌﺘﺒﺭ ﻤﺭﻜﺒﺎﺕ ﺍﻷﻭﺭﺠـﺎﻨﻭﻜﻠﻭﺭﻴﻥ ( ﻫـﻲ ﻤﺭﻜﺒﺎﺕ ﻋﻀﻭﻴﻪ ﻤﺭﺘﺒﻁﻪ ﺘﺴﺎﻫﻤﻴﺎﹰ ﻋﻠﻲ ﺍﻷﻗل ﺒﺫﺭﺓ ﻜﻠﻭﺭ ) ﻫـﻲ ﺇﺤـﺩﻱ ﻫـﺫﻩ ﺍﻝﻤﻠﻭﺜـﺎﺕ ﺍﻝﻤﻨﺘﺸﺭﺓ ﺍﻨﺘﺸﺎﺭﻭﺍﺴﻌﺎﹰ ﺒﺴﺒﺏ ﺇﺴﺘﺨﺩﺍﻤﺎﺘﻬﺎ ﺍﻝﻜ ﺜﻴﺭﺓ ﻭﺍﻝﻤﺘﻌﺩﺩﺓ . . ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻫﻭ ﺍﺤﺩ ﻤﺭﻜﺒﺎﺕ ﺍﻷﻭﺭﺠﺎﻨﻭﻜﻠﻭﺭﻴﻥ- ﻭﻫﻭ ﻤﺭﻜﺏ ﻤﺘﻁﺎﻴﺭ ﻝﻪ ﺍﺴﺘﺨﺩﺍﻤﺎﺕ ﻭﺍﺴﻌﻪ ﻓﻲ ﺤﻴﺎﺘﻨﺎ ﺍﻝﻴﻭﻤﻴﺔ ﻤﻨﻬﺎ ﺇﺯﺍﻝﻪ ﺼﺩﺍﺀ ﻭﺘﻠﻤﻴـﻊ ﺍﻝﻤﻌـﺎﺩﻥ ﻭﻴﺴـﺘﺨﺩﻡ ﻓـﻲ ﺍﻝﺘﻨﻅﻴﻑ ﺍﻝﺠﺎﻑ ﻜﻤﺎ ﺍﻨﻪ ﻴﺩﺨل ﻀﻤﻥ ﺘﺭﻜﻴﺏ ﻤﻭﺍﺩ ﺍﻝﻁﻼﺀ ﻝﺫﻝﻙ ﻓﻬﻭ ﺃﺼﺒﺢ ﻤﻠﻭﺙ ﺍﺴﺎﺴـﻲ ﻝﻠﺒﻴﺌﺔ ﺍﻜ. ﺜﺭ ﻤﻥ ٣,٥ ﻤﻠﻴﻭﻥ ﺸﺨﺹ ﻴﺘﻌﺭﻀﻭﻥ ﻝﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﺴﻨﻭﻴﺎﹰ ﻤـﻥ ﺨـﻼل ﺍﻝﻌﻤل ﻭ ١٠ % ﻤﻥ ﺍﻝﻌﺎﻤﻪ ﻴﺘﻌﺭﻀﻭﻥ ﻝﻪ ﻋﻥ ﻁﺭﻴﻕ ﺍﻝﺘﻠﻭﺙ ﺍﻝﺒﻴﺌﻲ ﻤﻥ ﺨﻼل ﻤﻴﺎﻩ ﺍﻝﺸﺭﺏ ﻭ ﺍﻝﻬﻭﺍﺀ ﻭ ﻜﺫﻝﻙ ﺍﻝﻁﻌﺎﻡ ﻭﺒﺎﻝﻔﺤﺹ ﺘﺒﻴﻥ ﺍﻥ ﻤﻌﻅﻡ ﺍﻝﻌﺎﻤﻪ ﺍﻝﻠﺫﻴﻥ ﻴﺘﻌﺭﻀﻭﻥ ﻝﻬﺫﺍ ﺍﻝﻤﺭﻜﺏ ﻜـﺎﻥ ﻝﺩﻴﻬﻡ ﻤﺴﺘﻭﻱ ﻤﻠﺤﻭﻅ ﻤﻥ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻓﻲ ﺍﻝﺩﻡ . . ﺍﻝﻬﺩﻑ ﻤﻥ ﺍﻝﺩﺭﺍﺴﺔ: ﺍﺴﺘﻬﺩﻑ ﻫﺫﺍ ﺍﻝﺒﺤﺙ ﻤﺤﺎﻭﻝﺔ ﻝﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭﻤﺭﻜﺏ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻋﻠﻲ ﻤﻌﺩﻻﺕ ﻨﻤـﻭ ﺍﺠﺴﺎﻡ ﺫﻜﻭﺭ ﺍﻝﻔﺌﺭﺍﻥ ﻭ ﻜﺫﻝﻙ ﺍﻋﻀﺎﺌﻬﺎ ﺍﻝﺩﺍﺨﻠﻴﺔ ﻭﺍﻴﻀﺎ ﺘﻘﻴﻴﻡ ﺍﻝﺘﻐﻴﻴﺭ ﻓﻲ ﻤﻌﺩﻻﺕ ﻫﺭﻤﻭﻨـﺎﺕ ﺍﻝﻐﺩﺓ ﺍﻝﺩﺭﻗﻴﺔ ﻭﻫﺭﻤﻭﻥ ﺍﻝﺘﻴﺴﺘﻭﺴﺘﻴﺭﻭﻥ ﻭﻜﺫﻝﻙ ﺩﺭﺍﺴﺔ ﺍﻝﺘـﺄﺜﻴﺭﺍﺕ ﺍ ﻝﺒﻴﻭﻜﻴﻤﻴﺎﺌﻴـﺔ ﻭﺍﻝﻤﻨﺎﻋﻴـﺔ ﻭﻜﺫﻝﻙ ﺍﻝﺘﺄﺜﻴﺭﺍﺕ ﺍﻝﺒﻴﻭﻝﻭﺠﻴﺔ ﺍﻝﺠﺯﻴﺌﻴﺔ ﺍﻝﻤﺭﺘﺒﻁﺔ ﺒﺘﻌﺎﻁﻲ ﺫﻜﻭﺭ ﺍﻝﻔﺌﺭﺍﻥ ﺒﺠﺭﻋﺎﺕ ﻤﻥ ﺜﻼﺜـﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻝﻔﺘﺭﺍﺕ ﻗﺼﻴﺭﺓ ( ٢٠ ﻴﻭﻡ ) ﻭ ﻁﻭﻴﻠﺔ ( ١٠٥ ﻴﻭﻡ ). ﻭﺫﻝﻙ ﻤﻥ ﺨﻼل ﺩﺭﺍﺴـﺔ ﺍﻝﺘﻐﻴﻴﺭﺍﺕ ﺍﻝﺒﻴﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﺍﻝﺠﻭﻫﺭﻴﺔ ﻝﻤﻌﺩﻻﺕ ﻨﻤـﻭ ﺍﻝﺠﺴـﻡ ﻭﺍﻷﻋﻀـﺎﺀ ﻭﻜـﺫﻝﻙ ﻤﻌـﺩﻻﺕ ﻫﺭﻤ ﻭﻨﺎﺕ ﺍﻝﻐﺩﺓ ﺍﻝﺩﺭﻗﻴﺔ ﻭﺍﻝﻬﺭﻤﻭﻥ ﺍﻝﻤﻨﺸﻁ ﻝﻠﻐﺩﺓ ﺍﻝﺩﺭﻗﻴﺔ ﻭ ﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻴﺔ ﻋﻠﻰ ﺍﻝﺠﻬﺎﺯ ﺍﻝﻤﻨﺎﻋﻰ ﻤﻥ ﺨﻼل ﺍﻝﻘﻴﺎﺴﺎﺕ ﺍﻝﺤﻴﻭﻴﺔ ﺍﻝﻤﻨﺎﻋﻴﺔ، ﻭﻓﺤﺹ ﻤﻜﻭﻨﺎﺕ ﺍﻝﺩﻡ . ﻭﻜﺫﻝﻙ ﺩﺭﺍﺴﺔ ﺘـﺄﺜﻴﺭ ﺴﻤﻴﺘﺔ ﻋﻠﻰ ﺇﺤﺩﺍﺙ ﺍﻝﻤﻭﺕ ﺍﻝﺨﻠﻭﻯ ﺍﻝﻤﺒﺭﻤﺞ ( ﺍﻷﺒﻭﺒﺘﻭﺯﻴﺱ ) ﻓـﻰ ﺨﻼﻴـﺎ ﺍﻝﻜﺒـﺩ ﻭﺍﻝﻜﻠـﻰ ﻭﺍﻝﺨﺼﻰ ﻓﻰ ﺫﻜﻭﺭ ﺍﻝﻔﺌﺭﺍﻥ . .

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ﻜﻤﺎ ﺘﻡ ﺩﺭﺍﺴﺔ ﺘﻘﻴﻴﻡ ﺘﻌﺎﻁﻲ ﺒﻌﺽ ﺍﻝﻤﻐﺫﻴﺎﺕ ﺍﻝﺩﻗﻴﻘﺔ ﻤﺜل ﻓﻴﺘﺎﻤﻴﻥ ﺴـﻲ ﻭﻋﻨﺼـﺭ ﺍﻝﺯﻨـﻙ ﻤﻨﻔﺭﺩﻴﻥ ﺍﻭ ﻤﺸﺘﺭﻜﻴﻥ ﻤﻌﺎﹰ ﻝﻤﻌﺭﻓﺔ ﺍﺜﺎﺭ ﺘﻠﻙ ﺍﻝﻤﻐﺫﻴﺎﺕ ﻓﻰ ﺘﺨﻔﻴﻑ ﺴـﻤﻴﺔ ﻤﺭﻜـﺏ ﺜﻼﺜـﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻭﺒﻴﺎﻥ ﻤﺩﻱ ﻗﺩﺭﺘﻬﺎ ﻋﻠﻰ ﺘﺤﺴﻴﻥ ﺍﻝﻭﻅﺎﺌﻑ ﺍﻝﻤﺨﺘﻠﻔﺔ ﻝﻠﺠﺴﻡ ﻭﺍﻝﻘـﺎﺀ ﺍﻝﻀـﻭﺀ ﻋﻠﻰ ﻜﻴﻔﻴﺔ ﺍﻝﺤﻤﺎﻴﺔ ﻤﻥ ﺍﻝﺘﻠﻭ ﺙ ﻝﻤﺜل ﻫﺫﻩ ﺍﻝﻤﺭﻜﺒﺎﺕ ﺍﻝﻤﻨﺘﺸﺭﺓ ﺤﻭﻝﻨﺎ . . ﺨﻁﺔ ﺍﻝﺒﺤﺙ: ﺍﺸﺘﻤﻠﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﻋﻠﻲ ١٠٢ ﻤﻥ ﺫﻜﻭﺭ ﺍﻝﻔﺌﺭﺍﻥ ﺫﺍﺕ ﺍﻷﻭﺯﺍﻥ ١٢٠- ١٥٠ ﺠـﺭﺍﻡ ﺘـﻡ ﺘﻘﺴﻴﻤﻬﺎ ﻜﺎﻷﺘﻲ : : ١ - ﻤﺠﻤﻭﻋﺔ ﻀﺎﺒﻁﺔ ﻁﺒﻴﻌﻴﺔ ﻗﺴﻤﺕ ﻋﻠﻲ ﺍﺭﺒﻊ ﻤﺠﻤﻭﻋﺎﺕ ﻓﺭﻋﻴﺔ ﻜل ﻤﻨﻬـﺎ ٦ ﻓﺌـﺭﺍﻥ ، ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺭﻋﻴﺔ ﺍﻷﻭﻝـﻲ ﺘﻐـﺫﺕ ﻋﻠـﻲ ﻭﺠﺒـﺎﺕ ﻋﺎﺩﻴـﺔ ، ﻭﺍﻝﺜﺎﻨﻴـ ﺔ ﺍﻋﻁﻴـﺕ ٥٠ ﻤﺠﻡ/ ﻜﺠﻡ/ ﻴﻭﻡ ﻤﻥ ﻓﻴﺘﺎﻤﻴﻥ ﺴﻲ ، ﻭﺍﻝﺜﺎﻝﺜﺔ ﺍﻋﻁﻴﺕ ٢٠٠ ﻤﺠﻡ/ ﻜﺠﻡ/ ﻴﻭﻡ ﻤﻥ ﻜﺒﺭﻴﺘـﺎﺕ ﺍﻝﺯﻨﻙ ، ﻭﺍﻝﺭﺍﺒﻌﺔ ﺍﻋﻁﻴﺕ ﻜﻼﻫﻤﺎ ﻤﺠﺘﻤﻌﻴﻥ ﺒﻨﻔﺱ ﺍﻝﺘﺭﻜﻴﺯ ﺍﻝﺴﺎﺒﻕ ﻤﻊ ﺍﻝﺘﻐﺫﻴﺔ ﺍﻝﻴﻭﻤﻴـﺔ ﺍﻝﻌﺎﺩﻴﺔ . . ٢ - ﻤﺠﻤﻭﻋــﺔ ﺍﻝﺘﻌــﺎﻁﻲ ﻝﺜﻼﺜــﻲ ﻜﻠﻭﺭﻴــﺩ ﺍﻹﻴﺜﻴﻠــﻴﻥ ﻭﺍﻋﻁﻴــﺕ ﺍﻝﻔﺌــﺭﺍﻥ ﺠﻤﻴﻌﻬﺎ ٧٥٠ ﻤﺠﻡ/ ﻜﺠﻡ/ ﻴﻭﻡ ﻭﻗﺩ ﻗﺴﻤﺕ ﺇﻝﻲ ﺨﻤﺴﺔ ﻤﺠﻤﻭﻋﺎﺕ ﻓﺭﻋﻴﺔ ﻜـل ﻤﻨﻬـﺎ ٦ ٦ ﻓﺌﺭﺍﻥ ، ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺭﻋﻴﺔ ﺍﻷﻭﻝﻲ ﺍﻋﻁﻴﺕ ٧٥٠ ﻤﺠﻡ/ ﻜﺠﻡ/ ﻴﻭﻡ ﻤﻥ TCE ﻭﺃﻋﺘﺒـﺭﺕ ﻫﺫﻩ ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺭﻋﻴﺔ ﻀﺎﺒﻁﺔ ﻝﻤﺎ ﺒﻌﺩﻫﺎ ﻤﻥ ﺍﻝﻤﺠﻤﻭﻋﺎﺕ ﺍﻝﻔﺭﻋﻴﺔ ﺍﻷﺨﺭﻱ ﺍﻝﻤﻌﺎﻝﺠﺔ ، ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺭﻋﻴﺔ ﺍﻝﺜﺎﻨﻴﺔ ﺍﻋﻁﻴﺕ ٥٠ ﻤﺠﻡ/ ﻜﺠﻡ/ ﻴﻭﻡ ﻤﻥ ﻓﻴﺘﺎﻤﻴﻥ ﺴﻲ ، ﻭﺍﻝﺜﺎﻝﺜﺔ ﺍﻋﻁﻴﺕ ٢٠٠ ﻤﺠﻡ/ ﻜﺠﻡ/ ﻴﻭﻡ ﻤﻥ ﻜﺒﺭﻴﺘﺎﺕ ﺍﻝﺯﻨﻙ ، ﻭﺍﻝﺭﺍﺒﻌـﺔ ﺍﻋﻁﻴـﺕ ﻜﻼﻫﻤـﺎ ﻤﺠﺘﻤﻌـﻴﻥ ، ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺭﻋﻴﺔ ﺍﻝﺨﺎﻤﺴﺔ ﻫﻲ ﻤﺠﻤﻭﻋﺔ ﺍﻷﻨﺴﺤﺎﺏ ﻭ ﺍﻝﺘﻲ ﻋﻭﻤﻠﺕ ﻤﺜل ﺍﻝﻤﺠﻤﻭﻋـﺔ ﺍﻝﻔﺭﻋﻴﺔ ﺍﻷﻭﻝﻲ ( ﺍﻝﺘﻌﺎﻁﻲ ﺍﺴﺘﻤﺭ ﻝﻔﺘﺭﺓ ﻗﺼﻴﺭﺓ ﺍﻝﻤﺩﻱ ) ، ﺃﺴﺘﻤﺭﺕ ﺍﻝﻤﻌﺎﻝﺠﺔ ﻋﻠﻰ ﻨﺤـﻭ ﻤﺎ ﺴﺒﻕ ﻝﻤﺩﺓ ٢٠ ﻴﻭﻡ ﻤﺘﺘﺎﻝﻴﻴﻥ ( ﻤﺩﻱ ﻗﺼﻴﺭ ﻓﻲ ﺍﻝﺘﺠﺭﺒﺔ ). ﺒﻴﻨﻤﺎ ﺍ ﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻤﺴـﺤﻭﺒﺔ ﺘﺭﻜﺕ ﻝﻤﺩﺓ ١٠٥ ﻴﻭﻡ ﻝﻠﺘﻌﺎﻓﻲ ﻤﻥ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ . . ٣- ﻤﺜل ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻷﻭﻝﻲ ﻭﻝﻜﻥ ﻋﻭﻝﺠﺕ ﻝﻤﺩﺓ ١٠٥ ﻴﻭﻡ ( ﻤﺩﻱ ﻁﻭﻴل ﻓﻲ ﺍﻝﺘﺠﺭﺒﺔ ). ٤- ﻤﺜل ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﺜﺎﻝﺜﺔ ﻭﻝﻜﻥ ﻋﻭﻝﺠﺕ ﻝﻤﺩﺓ ١٠٥ ﻴﻭﻡ ( ﻤﺩﻱ ﻁﻭﻴل ﻓﻲ ﺍﻝﺘﺠﺭﺒﺔ ). ).

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ﻤﻊ ﻨﻬﺎﻴﺔ ﻓﺘﺭﺓ ﺍﻝﻤﺩﻱ ﺍﻝﻘﺼﻴﺭ ﺘﻡ ﺍﻝﺤﺼﻭل ﻋﻠﻲ ﺍﻤﺼ ﺎل ﺍﻝﻔﺌﺭﺍﻥ ﻭﻜـﺫﻝﻙ ﺍﻨﺴـﺠﺔ ﺍﻝﻜﺒـﺩ ﻭﺍﻝﻜﻠﻲ ﻭﺍﻝﺨﺼﻲ ﺒﻴﻨﻤﺎ ﺘﺭﻜﺕ ﻤﺠﻤﻭﻋﺔ ﺍﻷﻨﺴﺤﺎﺏ ﺤﺘﻲ ﻨﻬﺎﻴﺔ ﻓﺘﺭﺓ ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﺜﺎﻝﺜـﺔ ﻭﺍﻝﺭﺍﺒﻌﺔ ﻭ ﺘﻡ ﺍﻝﺤﺼﻭل ﻋﻠﻲ ﺍﻤﺼﺎل ﺍﻝﻔﺌﺭﺍﻥ ﻭﻜﺫﻝﻙ ﺍﻷ ﻨﺴﺠﺔ ﺍﻝﻤﺫﻜﻭﺭﺓ ﺍﻋﻼﻩ . ﻭﻝﻘـﺩ ﺴﺠﻠﺕ ﺍﻭﺯﺍﻥ ﺍﻝﻔﺌﺭﺍﻥ ﻋﻠﻲ ﻓﺘﺭﺍﺕ ﻤﻥ ﺍﻝﻤﻌﺎﻝﺠﺔ ﺍﻝﻤﺴﺘﻤﺭﺓ ﻭﺴﺠﻠﺕ ﺍﻴﻀﺎ ﺍﻭﺯﺍﻥ ﻜـل ﻤﻥ ﺍﻝﻜﺒﺩ ﻭ ﺍﻝﻜﻠﻲ ﻭﺍﻝﺨﺼﻲ ﻝﻜل ﻓﺄﺭ ﻋﻨﺩ ﺍﻝﺫﺒﺢ . . ﺍﻝﺩﺭﺍﺴﺎﺕ ﻭﺍﻝﺘﺠﺎﺭﺏ ﺍﻝﻘﺎﺌﻤﺔ ﻓﻲ ﻫﺫﺍ ﺍﻝﺒﺤﺙ: ﺘﻀﻤﻨﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﻤﺎﻴﻠﻲ -: -: ١- ﻓﺤﺹ ﻤﻜﻭﻨﺎﺕ ﺍﻝﺩﻡ ﻤﺜل ﻋﺩﺩ ﻜﺭﺍﺕ ﺍﻝﺩﻡ ﺍﻝﺤﻤـﺭﺍﺀ ، ﻋـﺩﺩ ﻜـﺭﺍﺕ ﺍﻝـﺩﻡ ﺍﻝﺒﻴﻀـﺎﺀ ، ﺍﻝﻬﻴﻤﻭﺠﻠﻭﺒﻴﻥ ﻭﺍﻝﺼﻔﺎﺌﺢ ﺍﻝﺩﻤﻭﻴﺔ ﻭﻤﻘﺎﺭﻨﺘﻬﺎ ﺒﺎﻝﻤﺠﻤﻭﻋﺎﺕ ﺍﻝﻀﺎﺒﻁﺔ . . ٢- ﺘﻘﻴﻴﻡ ﺒﻌﺽ ﺍﻝﻘﻴﺎﺴﺎﺕ ﺍﻝﺒﻴﻭ ﻜﻴﻤﻴﺎﺌﻴﺔ ﻤﺜل ﺇﻨﺯﻴﻤﺎﺕ ﺍﻝﻜﺒـﺩ ، ﺍﻻﻝﺒﻴـﻭﻤﻴﻥ ، ﺍﻝﺠﻠﻭﺒﻴـﻭﻝﻴﻥ ، ﻭﺍﻝﺒﺭﻭﺘﻴﻨﺎﺕ ﺍﻝﻜﻠﻴﺔ ، ﺍﻝﺒﻠﻴﺭﻭﺒﻴﻥ ﺍﻝﻜﻠﻲ ، ﺍﻝﻴﻭﺭﻴﺎ ، ﺍﻝﻜﺭﻴﺎﺘﻴﻨﻴﻥ ﻭﺤﻤﺽ ﺍﻝﺒﻭﻝﻴﻙ . . ٣- ﺘﻘﻴﻴﻡ ﻤﻌﺩﻻﺕ ﻫﺭﻤﻭﻨﺎﺕ ﺍﻝﻐﺩﺓ ﺍﻝﺩﺭﻗﻴﺔ ﺍﻝﺤﺭﺓ ﻭﺍﻝﻬﺭﻤﻭﻥ ﺍﻝﻤﻨﺸﻁ ﻝﻠﻐﺩﺓ ﺍﻝﺩﺭﻗﻴﺔ ﻭﻫﺭﻤﻭﻥ ﺍﻝﺘﻴﺴﺘﻭﺴﺘﻴﺭﻭﻥ . . ٤- ﺩﺭﺍﺴﺔ ﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻴﺔ ﻋﻠﻰ ﺍﻝﺠﻬﺎﺯ ﺍﻝﻤﻨﺎﻋﻰ ﻤﻥ ﺨﻼل ﺍﻝﻘﻴﺎﺴﺎﺕ ﺍﻝﺤﻴﻭﻴﺔ ﺍﻝﻤﻨﺎﻋﻴﺔ . . ٥- ﺇﺠﺭﺍﺀ ﺘﺼﻭﻴﺭ ﻭﻓﺤﺹ ﺤﺎﻝﺔ ﺍﻷﻨﺴﺠﺔ ﺍﻝﻤﺼﺒﻭﻏﺔ ﻤﺠﻬﺭﻴﺎﹰ ﻝﻜل ﻤـﻥ ﺍﻝﻜﺒـﺩ ، ﺍﻝﻜﻠـﻲ ﻭﺍﻝﺨﺼﻴﺔ ﻝﺫﻜﻭﺭ ﺍﻝﻔﺌﺭﺍﻥ ﺒﻐﻴﻪ ﻓﺤﺹ ﻭﺘﻘﺩﻴﺭ ﻤﺩﻱ ﺍﻝﺘﻐﻴﺭ ﺍﻝﻤﺠﻬﺭﻱ ﺍﻝﺫﻱ ﻝﺤﻕ ﺒﺎﻝﻔﺌﺭﺍﻥ ﻤﻥ ﺠﺭﺍﺀ ﺘﻌﺎﻁﻲ ﻫﺫﺍ ﺍﻝﻤﺭﻜﺏ ﻭﻤﺩﻱ ﻋﻼﻗﺘﻪ ﺒﺎﻝﻤﺅﺸﺭﺍﺕ ﺍﻝﻜﻴﻤﻴﺎﺌﻴﺔ ﺍﻝﺤﻴﻭﻴﺔ ﺍ ﻝﺘﻲ ﺘـﻡ ﺘﻘﺩﻴﺭﻫﺎ ﻨﺴﻴﺠﻴﺎﹰ . . ٦- ﺘﻘﻴﻴﻡ ﺘﺄﺜﻴﺭ ﺍﻝﺴﻤﻴﺔ ﻋﻠﻲ ﺇﺤﺩﺍﺙ ﺍﻝﻤﻭﺕ ﺍﻝﻤﺒﺭﻤﺞ ﻝﻜل ﻤﻥ ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻀﺎﺒﻁﺔ ﻭﺒـﺎﻗﻲ ﺍﻝﻤﺠﻤﻭﻋﺎﺕ ﻤﻥ ﺨﻼل ﺘﻘﺩﻴﺭ ﻜﻤﻴﺔ ﺍﻝﺘﺠﺯﺌﺔ ﻓﻲ ﺍﻝﺤﺎﻤﺽ ﺍﻝﻨﻭﻭﻱ . . ٧- ﺘﻘﻴﻴﻡ ﺘﺄﺜﻴﺭ ﺍﻝﻤﻐﺫﻴﺎﺕ ﺍﻝﺩﻗﻴﻘﺔ ﻓﻲ ﺍﻝﺘﻐﻠﺏ ﻋﻠﻲ ﺍﻝﺘـﺄﺜﻴﺭﺍﺕ ﺍﻝﻀـﺎﺭﺓ ﻝ ﺜﻼﺜـﻲ ﻜﻠﻭﺭﻴـﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﺍﻝﻤﺤﺩﺜﺔ ﻓﻲ ﺫﻜﻭﺭ ﺍﻝﻔﺌﺭﺍﻥ . . ﻨﺘﺎﺌﺞ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ: ١- ﺃﻭﻀﺤﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺍﻨﺨﻔﺎﺽ ﺍﻭﺯﺍﻥ ﺍﻝﻔﺌﺭﺍﻥ ﻭﺍﻝﺘﻲ ﺍﻅﻬﺭﺕ ﺍﻨﺨﻔﺎﻀـﺎﹰ ﻤﻌﻨﻭﻴـﺎﹰ ﻓـﻲ ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﺘﻲ ﺘﻌﺎﻁﺕ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻋﻠﻰ ﺍﻝﻤﺩﻱ ﺍﻝﻁﻭﻴل

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٢- ﻝﻭﺤﻅ ﺯﻴﺎﺩﺓﹰ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻭﺯﻥ ﺍﻝﻜﺒﺩ ﻭﺍﻝﻜﻠﻰ ﺒﻴﻨﻤﺎ ﺍﻨﺨﻔﺎﻀﺎﹰ ﻤﻌﻨﻭﻴﺎﹰ ًﻓﻲ ﻭﺯﻥ ﺍﻝﺨﺼﻲ ﻓﻲ ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﺘﻲ ﺘﻌﺎﻁﺕ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻋﻠﻰ ﺍﻝﻤﺩﻱ ﺍﻝﻁﻭﻴـل ﻭﺘﺤﺴـﻥ ﻫﺫﻩ ﺍﻷﻭﺯﺍﻥ ﻓﻲ ﺍﻝﻤﺠﻤﻭﻋﺎﺕ ﺍﻝﺘﻲ ﻋﻭﻝﺠﺕ ﺒﻔﻴﺘﺎﻤﻴﻥ ﺴﻲ ﻭﻋﻨﺼﺭ ﺍﻝﺯﻨﻙ . . ٣- ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻨﻘﺹ ﻤﻌﻨﻭﻱ ﻓﻲ ﻜل ﻤﻥ ﻋﺩ ﻜﺭﺍﺕ ﺍﻝﺩﻡ ﺍﻝﺤﻤﺭﺍﺀ- ﻤﺴـﺘﻭﻱ ﺍﻝﻬﻴﻤﻭﺠﻠﻭﺒﻴﻥ- ﻋﺩ ﺍﻝﺼﻔﺎﺌﺢ ﻭﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻋﺩ ﻜﺭﺍﺕ ﺍﻝﺩﻡ ﺍﻝﺒﻴﻀﺎﺀ. ٤- ﺍﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌ ﺞ ﻨﻘﺹ ﻤﻌﻨﻭﻱ ﻓﻲ ﺍﻻﻝﺒﻴﻭﻤﻴﻥ- ﺍﻝﺠﻠﻭﺒﻴـﻭﻝﻴﻥ ﻭﺍﻝﺒﺭﻭﺘﻴﻨـﺎﺕ ﺍﻝﻜﻠﻴـﺔ ﻭﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻤﺴﺘﻭﻱ ﺍﻨﺯﻴﻤﺎﺕ ﺍﻝﻜﺒﺩ ﻭﺍﻝﺒﻠﻴﺭﻭﺒﻴﻥ ﺍﻝﻜﻠﻲ ﻭ ﻤﺴﺘﻭﻱ ﻜـل ﻤـﻥ ﺍﻝﻴﻭﺭﻴﺎ ﻭﺍﻝﻜﺭﻴﺎﺘﻴﻨﻴﻥ ﻭﺤﻤﺽ ﺍﻝﺒﻭﻝﻴﻙ . ﻜﻤﺎ ﺍﺤﺩﺜﺕ ﺍﻝﻤﻐﺫﻴﺎﺕ ﺍﻝﺩﻗﻴﻘﺔ ﺘﻐﻴﺭﺍﺕ ﻁﻔﻴﻔـﺔ ﻓﻲ ﺼﻭﺭﺓ ﻤﻜﻭﻨﺎﺕ ﺍﻝﺩﻡ ﻭﺒﺎﻗﻲ ﺍﻝﻤﻜﻭﻨﺎﺕ ﻭﺘﻨﺎﺴﺒﺕ ﺍﻝﺩﻻﻻﺕ ﺍﻝ ﻤﻌﻨﻭﻴﺔ ﻝﻠﻨﺘﺎﺌﺞ ﺘﻨﺎﺴﺒﺎﹰ ﻁﺭﺩﻴﺎﹰ ﻤﻊ ﺯﻴﺎﺩﺓ ﻤﺩﻱ ﺍﻝﺘﻌﺭﺽ . ٥- ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﻨﻘﺹ ﻤﻌﻨﻭﻱ ﻓﻲ ﻫﺭﻤﻭﻥ ﺍﻝﺘﻴﺴﺘﻭﺴﺘﻴﺭﻭﻥ ﻭﻫﺭﻤﻭﻥFT4 ﻭﺯﻴـﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻫﺭﻤﻭﻥ FT3 ﻭ TSH ﻋﻠﻰ ﺍﻝﻤﺩﻱ ﺍﻝﻘﺼﻴﺭﻭ ﺍﻝﻁﻭﻴـل ﻭﺫﻝـﻙ ﻓـﻲ ﺍﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻤﻌﺭﻀﺔ ﻝﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﺇﺫﺍ ﻤﺎ ﻗﻭﺭﻨﺕ ﺒﺎﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻀـﺎﺒﻁﺔ ، ﻭﻜﺎﻨﺕ ﺃﻗل ﻨﺴﺒﺔ ل T4/T3 ﻓﻲ ﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺌﺭﺍﻥ ﺍﻝﺘﻲ ﺘﻠﻘﺕ ﺍﻝﺘﻌﺎﻁﻲ ﻝﻤـﺩﺓ ١٠٥ ﻴﻭﻡ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻝﻤﺠﻤﻭﻋﺔ ﺍﻝﻀﺎﺒﻁﺔ . ﻭﻜﺫﻝﻙ ﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓـﻲ ﻤﺴـﺘﻭﻱ ﺍﻷﺠﺴـﺎﻡ ﺍﻝﻤﻨﺎﻋﻴﺔ ( IgG ﻭ IgM) . ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺤﺩﻭﺙ ﺘﻠﻑ ﻓﻲ ﺍﻷﻨﺴﺠﺔ ﻭ ﺘﺠﺯ ﺌﺔ ﻓﻲ ﺍﻝﺤﺎﻤﺽ ﺍﻝﻨﻭﻭﻱ ( DNA ) ﻝﻜل ﻤﻥ ﺍﻝﻜﺒﺩ ﻭﺍﻝﻜﻠـﻲ ﻭﺍﻝﺨﺼـﻴﺔ ﻋﻠـﻲ ﺍﻝﻤـﺩﻱ ﺍﻝﻘﺼﻴﺭﻭ ﺍﻝﻁﻭﻴل ﻝﻠﺘﻌﺭﺽ. ﻭﻗﺩ ﺘﺒﻴﻥ ﺘﺤﺴﻥ ﻨﺴﺒﻲ ﻓﻲ ﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺌﺭﺍﻥ ﺍﻝﻤﺴﺤﻭﺒﺔ ﻝﻠﺘﻌﺎﻓﻲ . ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺘﺤﺴﻥ ﻤﻌﻨﻭﻱ ﻭﻏﻴﺭ ﻤﻌﻨﻭﻱ ﻓﻲ ﺍﺜﺎﺭ ﺍﻝﺘﻠﻑ ﻭﺍﻝﻀﺭﺭ ﺍﻝﻨﺎﺘﺠﺔ ﻤـﻥ ﺍﻝﺘﻌـﺭﺽ ﻝ ﺜﻼﺜـﻲ ﻜﻠﻭﺭﻴـﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻓﻲ ﻤﺠﻤﻭﻋﺎﺕ ﺍﻝﻔﺌﺭﺍﻥ ﺍﻝﻤﻌﺎﻝﺠﺔ ﺒﻔﻴﺘﺎﻤﻴﻥ ﺴﻲ ﺍﻭﻋﻨﺼﺭ ﺍﻝﺯﻨﻙ ﺍﻭ ﺒﻜﻼﻫﻤﺎ ﻤﻌـﺎﹰ . ﻭﻝﺫﻝﻙ ﻴﻤﻜﻨﻨﺎ ﺍﻝﺘﻭﺼﻴﺔ ﺒﺄﻥ ﺍﻝﻠﺫﻴﻥ ﻴﺘﻌﺭﻀﻭﻥ ﻝ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻓﻲ ﺍﻝﻌﻤـل ﺍﻭ ﻓـﻲ ﺍﻝﻤﻨﺎﺯل ﻴﻤﻜﻥ ﺍﻥ ﻴﺘﻨﺎﻭﻝﻭﺍ ﻓﻴﺘﺎﻤﻴﻥ ﺴﻲ ﻭﻋﻨﺼﺭ ﺍﻝﺯﻨﻙ ﻤﻌﺎﹰ ﻭﺫﻝـﻙ ﻝﺘﻌـﻭﻴﺽ ﺍﻝﺘـﺄﺜﻴﺭﺍﺕ ﺍﻝﻤﺩﻤﺭﺓ ﻝ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﺩﺍﺨل ﺍﻝﺠﺴﻡ . . ﻭﻤﻥ ﻫﻨﺎ ﻴﻤﻜﻥ ﺃﻥ ﻨﺴﺘﺨﻠﺹ ﺍﻷﺘﻲ:

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١- ﺃﻅﻬﺭ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﺘﺴﻤﻤﺎﹰ ﻝﻠﻔﺌﺭﺍﻥ ﻋﻨﺩﻤﺎ ﺃﻋﻁﻲ ﻋﻠﻲ ﺍﻝﻤـﺩﻱ ﺍﻝﻘﺼـﻴﺭ ﻭﺍﻝﻁﻭﻴل ، ﺘﻤﻴﺯ ﺒﺘﻐﻴﺭﺍﺕ ﺠﻭﻫﺭﻴﺔ ﻓﻲ ﻤﻌﺩﻻﺕ ﻨﻤﻭ ﺍﻝﺠﺴﻡ ﻭﺍﻷﻋﻀـﺎﺀ ﺍﻝﺘـﻲ ﺘـﻡ ﻭﺯﻨﻬﺎ . . ٢- ﺃﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺍﻥ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻝﻪ ﺘﺄﺜﻴﺭ ﻤﻀﺎﺩ ﻝﻨﺸﺎﻁ ﺍﻝﻐـﺩﺓ ﺍﻝﺩﺭﻗﻴـﺔ ﻴﺤ ﺙ ﺍﻨﻪ ﻝﻭﺤﻅ ﺘﻐﻴﺭ ﻓﻲ ﻤﺴﺘﻭﻱ ﻜل ﻤﻥ FT4,FT3 . . - ﻋﺯﺯﺕ ﻨﺘﺎﺌﺞ ﺩﺭﺍﺴﺔ ﺍﻷﻨﺴﺠﺔ ﻫﺫﻩ ﺍﻝﺘﻐﻴﺭﺍﺕ ﺍﻝﻜﻴﻤﻭﺤﻴﻭﻴﺔ ﻭﺃﻴﻀﺎ ﻤﻜﻭﻨـﺎﺕ ﺍﻝـﺩﻡ ﺒﻭﺠﻭﺩ ﺍﻝﻤﻭﺕ ﺍﻝﻤﺒﺭﻤﺞ ﺍﻝﻤﺘﻤﺜل ﻓﻲ ﺍﺨﺘﻔﺎﺀ ﺍﻷﻨﺒﻭﺒﻴﺎﺕ ﺍﻝﻜﻠﻭﻴﺔ ﻭ ﺍﻷﻨﺴﺠﺔ ﺍﻝﺩﺍﺨﻠﻴـﺔ ﻝﻠﻜﻠﻲ ﻤﻊ ﺘﺩﻤﻴﺭ ﺍﻝﻘﺸﺭﺓ . . - ﺃﺩﻱ ﺍﻝﺘﻌﺎﻁﻲ ﻓﻲ ﺍﻝﻤﺩﻱ ﺍﻝﻘﺼﻴﺭ ﻝﻠﻤﺭﻜﺏ ﺍﻝﻌﻀﻭﻱ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠـﻴﻥ ﺇﻝـﻲ ﺍﻷﺤﺘﻘﺎﻥ ﻤﻊ ﺘﻠﻑ ﺒﺴﻴﻁ ﻓﻲ ﺨﻼﻴﺎ ﺍﻝﺨﺼﻴﺔ ﺃﻤﺎ ﺍﻝﺘﻌﺎﻁﻲ ﻓﻲ ﺍﻝﻤﺩﻱ ﺍﻝﻁﻭﻴل ( ﻤﺯﻤﻥ ) ) ﻓﻘﺩ ﻜﺎﻥ ﺘﺄﺜﻴ ﺭﺓ ﺃﺨﻁﺭ ﺤﻴﺙ ﺴﺒﺏ ﺘﻠﻴﻑ ﺒﻴﻥ ﺨﻼﻴﺎ ﺍﻝﺨﺼﻴﺔ ﻤﺼﺤﻭﺒﺎﹰ ﺒﻌﺩﻡ ﻭﺠـﻭﺩ ﺤﻴﻭﺍﻨﺎﺕ ﻤﻨﻭﻴﺔ ﻨﺎﻀﺠﺔ ﻤﻊ ﺇﺭﺘﺸﺎﺡ ﺨﻼﻴﺎ ﺍﻝﺩﻡ ﺍﻝﺒﻴﻀﺎﺀ . ﻭﺒﻌـﺩ ﺍﻷﻨﺴـﺤﺎﺏ ﻤـﻥ ﺘﻌﺎﻁﻲ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻭﺠﺩﺕ ﺒﻌﺽ ﺍﻷﻭﻋﻴﺔ ﺍﻝﺩﻤﻭﻴﺔ ﺍﻝﻤﺤﺘﻘﻨـﺔ ﺴـﻤﻴﻜﺔ ﺍﻝﺠﺩﺭ ﻤﻊ ﺒﻌﺽ ﺃﺜﺎﺭ ﺍﻝﺘﻌﺎﻁﻲ ﺍﻝﻤﺯﻤﻥ ﺍﻝﺴﺎﺒﻕ ﺫﻜﺭﻫﺎ . - ﺃﺜﺒﺘﺕ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻝﻜﻔﺎﺀﺓ ﺍﻝﻌﺎﻝﻴﺔ ﻝﻠﺘﺄﺜﻴﺭ ﺍﻝﻤﺸﺘﺭﻙ ﻤﻥ ﺍﻝﺠﺭﻋﺎﺕ ﺍﻝﻤﺴﺘﺨﺩﻤﺔ ﻝﺒﻌﺽ ﺍﻝﻤﻐﺫﻴﺎﺕ ﺍﻝﺩﻗﻴﻘﺔ ﻤﺜل ﻓﻴﺘﺎﻤﻴﻥ ﺴﻲ ﻭﻜﺫﻝﻙ ﺍﻝﺯﻨﻙ ﻜل ﻋﻠﻲ ﺤﺩﻩ ﺍﻭ ﻤﺸﺘﺭﻜﻴﻥ ﻤﻌﺎﹰ ﻓﻲ ﺘﺤﺴﻥ ﺃﺜﺎﺭ ﺘﻌﺎﻁﻲ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ . - ﻭ ﻴﺴﺘﺨﻠﺹ ﻤﻥ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻥ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﺫﻭ ﺍﻷﻨﺘﺸﺎﺭ ﺍﻝﻭﺍﺴﻊ ﺤﺘـﻲ ﺩﺍﺨل ﻤﻨﺎﺯﻝﻨﺎ ﻫﻭ ﺍﻝﺴﺒﺏ ﻓﻰ ﻜل ﺍﻝﺘﺄﺜﻴﺭﺍﺕ ﺍﻷﻴﻀﻴﺔ ﻭﺍﻝﺘﻐﻴﺭﺍﺕ ﺍﻝﺨﻠﻭﻴﺔ ﺍﻝﻤﺭﻀـﻴﺔ ﺍﻝﻨﺎﺠﻤﺔ ﻋﻥ ﺃﺴﺘﺨﺩﺍﻤﺔ ﻓﻲ ﺫﻜﻭﺭ ﺍﻝﻔﺌﺭﺍﻥ . - ﻭﻨﺴﺘﻨﺘﺞ ﻤﻥ ﻫﺫﻩ ﺍﻝﺩﺭﺍﺴﺔ ﺍﻥ ﻤﻥ ﺍﻝﻤﻤﻜﻥ ﺃﺴﺘﺨﺩﺍﻡ ﺍﻝﻤﻐ ﺫﻴﺎﺕ ﺍﻝﺩﻗﻴﻘﺔ ﻜﺤﻤﺎﻴﺔ ﻭﻭﻗﺎﻴـﺔ ﻤﻥ ﺍﻝﺘﺄﺜﻴﺭﺍﺕ ﺍﻝﻀﺎﺭﺓ ﻝ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ . ﻭﻜﺫﻝﻙ ﺤﻤﺎﻴﺔ ﺃﻨﺴﺠﺔ ﺍﻝﻜﺒﺩ ﻭﺍﻝﻜﻠـﻰ ﻭﺍﻝﺨﺼﻰ ﻤﻥ ﻤﻭﺕ ﺍﻝﺨﻼﻴﺎ ﺍﻝﻤﺒﺭﻤﺞ ﺍﻝﻤﺤﺩﺙ ﻓﻲ ﺍﻝﻔﺌﺭﺍﻥ ﺍﻝﻤﺘﻌﺎﻁﻴﺔ ﻝ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴـﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ.

٥ ا

ﺍﻝﻤﺴﺘﺨﻠﺹ

ﻴﺴﺘﺨﺩﻡ ﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻋﻠﻲ ﻨﻁﺎﻕ ﻭﺍﺴﻊ ﻓﻲ ﺤﻴﺎﺘﻨﺎ ﺍﻝﻴﻭﻤﻴﺔ ﻭ ﺤﻴـﺙ ﺍﻨـﻪ ﻤﺭﻜـﺏ ﻋﻀﻭﻱ ﻤﺘﻁﺎﻴﺭﻓﺈﻥ ﺍﻋﺩﺍﺩ ﻜﺒﻴﺭﺓ ﻤﻥ ﺍﻝﺒﺸﺭ ﺘﺘﻌﺭﺽ ﻝﻪ ﻤﻥ ﺨﻼل ﺍﻝﺘﻨﻔﺱ ﻜﻤﺎ ﺍﻨﻪ ﻴﺩﺨل ﺍﻝﺠﺴﻡ ﺍﻴﻀﺎ ﺒﻁﺭﻴﻘﺔ ﺍﻻﻤﺘﺼﺎﺹ ﻤﻥ ﺍﻝﺠﻠﺩ ﺍﻭ ﺒﻭﺍﺴﻁﺔ ﻤﻴﺎﺓ ﺍﻝﺸﺭﺏ ﻭ ﻓﻲ ﺤﺎﻻﺕ ﺃﺨﺭﻱ ﺒﻭﺍﺴﻁﺔ ﺍﻝﻁﻌﺎﻡ . .

ﻭﻴﻨﺸﺄ ﻋﻥ ﺍﻝﺘﻌﺭﺽ ﻝﻪ ﺍﻝﺼﺩﺍﻉ ﻭ ﺍﻝﺩﻭﺨﺔ ﺒﺠﺎﻨﺏ ﺍﻝﺘﺄﺜﻴﺭ ﻋﻠﻲ ﺍﻝﺠﻬﺎﺯ ﺍﻝﻌ ﺼﺒﻲ ﺍﻝﻤﺭﻜﺯﻱ ، ﺍﻝﺘﻌﺭﺽ ﻝﻪ ﻴﺼﺤﺒﻪ ﺤﺎﻻﺕ ﺘﺴﻤﻡ ﻝﻸﻋﻀﺎﺀ ﺍﻝﺩﺍﺨﻠﻴﺔ ﻭ ﻤﻨﻬﺎ ﺍﻝﻜﺒﺩ ﻭﺍﻝﻜﻠﻲ ﻭﺍﻝﺨﺼﻴﺔ ﺒﺎﻷﻀﺎﻓﺔ ﺇﻝـﻲ ﺇﻀـﻌﺎﻑ ﺍﻝﺠﻬﺎﺯ ﺍﻝﻤﻨﺎﻋﻲ . ﻴﻬﺩﻑ ﻫﺫﺍ ﺍﻝﻌﻤل ﺇﻝﻲ ﺍﻝﻘﻴﺎﻡ ﺒﺩﺭﺍﺴﺔ ﺩﻭﺭﺒﻌﺽ ﺍﻝﻤﻐﺫﻴﺎﺕ ﺍﻝﺩﻗﻴﻘﺔ ﻤﺜـل ﻓﻴﺘـﺎﻤﻴﻥ ﺝ ﺝ ﻭﻋﻨﺼﺭ ﺍﻝﺯﻨﻙ ﻤﻨﻔﺭﺩﻴﻥ ﺍﻭ ﻤﺠﺘﻤﻌﻴﻥ ﻀﺩ ﺍﻷﺜﺎﺭ ﺍﻝﻤﺩﻤﺭﺓ ﺍﻝﻨﺎﺘﺠﺔ ﻋﻥ ﺍﻝﺘﻌ ﺎﻁﻲ ﺍﻝﻴﻭﻤﻲ ﺒﺎﻝﻔﻡ ﻝﺜﻼﺜـﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻭ ﺫﻝﻙ ﻋﻠﻲ ﻤﺩﻱ ﻗﺼﻴﺭ ( ٢٠ ﻴﻭﻡ ) ﻭﻤﺩﻱ ﻁﻭﻴل ( ١٠٥ ﻴﻭﻡ ) ﻭﺫﻝﻙ ﻤﻥ ﺨﻼل ﻓﺤﺹ ﺍﻝﺘﻐﻴﺭﺍﺕ ﺍﻝﺩﻤﻭﻴﺔ ﻭﺍﻝﺒﻴﻭﻜﻴﻤﻴﺎﺌﻴﺔ ﻭﺍﻝﻨﺴﻴﺠﻴﺔ ﻭﻜﺫﻝﻙ ﺩﺭﺍﺴﺔ ﺍﻝﺤﻤﺽ ﺍﻝﻨﻭﻭﻯ( DNA ) ﻝﻜل ﻤﻥ ﺍﻝﻜﺒـﺩ ﻭﺍﻝﻜﻠﻲ ﻭﺍﻝﺨﺼﻴﺔ . ﻭﻗﺩ ﺍﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺍﻥ ﺍﻝﺘﻌﺭﺽ ﻝﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴ ﻥ ﻋﻠﻰ ﺍﻝﻤـﺩﻱ ﺍﻝﻁﻭﻴـل ﻴﺅﺜﺭﻋﻠﻲ ﻭﺯﻥ ﺍﻝﺠﺴﻡ ﻭﺍﻝﺨﺼﻴﺔ ﺒﺎﻝﻨﻘﺼﺎﻥ ﻤﻊ ﺯﻴﺎﺩﺓ ﺍﻭﺯﺍﻥ ﺍﻝﻜﺒﺩ ﻭﺍﻝﻜﻠﻲ ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﻨﻘـﺹ ﻤﻌﻨﻭﻱ ﻓﻲ ﻜل ﻤﻥ ﻋﺩ ﻜﺭﺍﺕ ﺍﻝﺩﻡ ﺍﻝﺤﻤﺭﺍﺀ- ﻤﺴﺘﻭﻱ ﺍﻝﻬﻴﻤﻭﺠﻠﻭﺒﻴﻥ- ﻋﺩ ﺍﻝﺼـﻔﺎﺌﺢ – ﺍﻻﻝﺒﻴـﻭﻤﻴﻥ- ﺍﻝﺠﻠﻭﺒﻴﻭﻝﻴﻥ ﻭﺍﻝﺒﺭﻭﺘﻴﻨﺎﺕ ﺍﻝﻜﻠﻴﺔ ﻭﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻤﺴﺘﻭﻱ ﺍﻨﺯﻴﻤﺎﺕ ﺍﻝﻜﺒﺩ ﻭ ﻤ ﺴﺘﻭﻱ ﻜل ﻤﻥ ﺍﻝﻴﻭﺭﻴﺎ ﻭﺍﻝﻜﺭﻴﺎﺘﻴﻨﻴﻥ ﻭﺤﻤﺽ ﺍﻝﺒﻭﻝﻴﻙ . ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﻨﻘﺹ ﻤﻌﻨﻭﻱ ﻓﻲ ﻫﺭﻤﻭﻥ ﺍﻝﺘﻴﺴﺘﻭﺴـﺘﻴﺭﻭﻥ ﻭﻫﺭﻤـﻭﻥ FT4 ﻭﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻫﺭﻤﻭﻥ FT3 ﻭ TSH ﻋﻠﻰ ﺍﻝﻤﺩﻱ ﺍﻝﻘﺼﻴﺭﻭ ﺍﻝﻁﻭﻴل ﻭﻜﺫﻝﻙ ﺯﻴﺎﺩﺓ ﻤﻌﻨﻭﻴﺔ ﻓﻲ ﻤﺴﺘﻭﻱ ﺍﻷﺠﺴﺎﻡ ﺍﻝﻤﻨﺎﻋﻴﺔ ( IgG ﻭ IgM). ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﺍﻝﺩﺭﺍﺴﺔ ﺤﺩﻭﺙ ﺘﻠﻑ ﻓﻲ ﺍﻷ ﻨﺴـﺠﺔ ﻭ ﺘﺠﺯﺌﺔ ﻓﻲ ﺍﻝﺤﺎﻤﺽ ﺍﻝﻨﻭﻭﻱ ( DNA ) ﻝﻜل ﻤﻥ ﺍﻝﻜﺒﺩ ﻭﺍﻝﻜﻠﻲ ﻭﺍﻝﺨﺼﻴﺔ ﻋﻠـﻲ ﺍﻝﻤـﺩﻱ ﺍﻝﻘﺼـﻴﺭﻭ ﺍﻝﻁﻭﻴل ﻋﻨﺩ ﺍﻝﺘﻌﺎﻁﻲ ﻝﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻻﻴﺜﻴﻠﻴﻥ . .

ﻭﻗﺩ ﺘﺒﻴﻥ ﺘﺤﺴﻥ ﻨﺴﺒﻲ ﻓﻲ ﻤﺠﻤﻭﻋﺔ ﺍﻝﻔﺌﺭﺍﻥ ﺍﻝﻤﺴﺤﻭﺒﺔ ﻝﻠﺘﻌﺎﻓﻲ . ﻜﻤﺎ ﺍﻅﻬﺭﺕ ﺍﻝﻨﺘﺎﺌﺞ ﺘﺤﺴﻥ ﻤﻌﻨـﻭﻱ ﻭﻏﻴﺭ ﻤﻌﻨﻭﻱ ﻓﻲ ﺍﺜﺎﺭ ﺍﻝﺘﻠﻑ ﻭﺍﻝﻀﺭﺭ ﺍﻝﻨﺎﺘﺠﺔ ﻤﻥ ﺍﻝﻤ ﻌﺎﻝﺠﻪ ﺒﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻓﻲ ﻤﺠﻤﻭﻋـﺎﺕ ﺍﻝﻔﺌﺭﺍﻥ ﺍﻝﻤﻌﺎﻝﺠﺔ ﺒﻔﻴﺘﺎﻤﻴﻥ ﺴﻲ ﺍﻭﻋﻨﺼﺭ ﺍﻝﺯﻨﻙ ﺍﻭ ﺒﻜﻼﻫﻤﺎ ﻤﻌﺎﹰ . ﻭﻝﺫﻝﻙ ﻴﻤﻜﻨﻨﺎ ﺍﻝﺘﻭﺼﻴﺔ ﺒﺄﻥ ﺍﻝﻠـﺫﻴﻥ ﻴﺘﻌﺭﻀﻭﻥ ﻝﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﻤﻥ ﺨﻼل ﺍﻷﺴﺘﻨﺸﺎﻕ ﺃﻭ ﺍﻝﺸﺭﺏ ﺃﻭ ﺍﻷﺴﺘﺤﻤﺎﻡ ﺒﺎﻝﻤﻴـﺎﻩ ﺍﻝﺴـﺎﺨﻨﻪ ﻝﻔﺘﺭﺍﺕ ﻁﻭﻴﻠﻪ ﻤﺘﺘﺎﻝﻴﻪ ﻓﻲ ﺍﻝﻤﻨﺎﺯل ﺍﻭ ﻓ ﻲ ﺍﻝﻤﺼﺎﻨﻊ ﻴﻤﻜﻨﻬﻡ ﺘﻨﺎﻭل ﻓﻴﺘﺎﻤﻴﻥ ﺴﻲ ﻭﻋﻨﺼﺭ ﺍﻝﺯﻨﻙ ﻤﻌـﺎﹰ ﻭﺫﻝﻙ ﻝﻠﺘﻐﻠﺏ ﻋﻠﻲ ﺍﻝﺘﺄﺜﻴﺭﺍﺕ ﺍﻝﻤﺩﻤﺭﺓ ﻝﺜﻼﺜﻲ ﻜﻠﻭﺭﻴﺩ ﺍﻹﻴﺜﻴﻠﻴﻥ ﺩﺍﺨل ﺍﻷﻋﻀـﺎﺀ ﺍﻝﻤﺨﺘﻠﻔـﻪ ﻝﺠﺴـﻡ ﺍﻝﻸﻨﺴﺎﻥ .

ﺭﺴﺎﻝﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ﺭﺸﺎ ﻴﻭﺴﻑ ﻤﺤﻤﺩ ﺍﺒﺭﺍﻫﻴﻡ ﻤﺩﺭﺱ ﻤﺴﺎﻋﺩ ﺍﻝﻜﻴﻤﻴﺎﺀ ﺍﻝﺤﻴﻭﻴﺔ – ﺍﻝﻁﺎﻗﺔ ﺍﻝﺯﺭﻴﺔ ﺒﻜﺎﻝﻭﺭﻴﻭﺱ ﻜﻴﻤﻴﺎﺀ – ﻜﻴﻤﻴﺎﺀ ﺤﻴﻭﻴﺔ – ﺠﺎﻤﻌﺔ ﺤﻠﻭﺍﻥ ﻤﺎﺠﺴﺘﻴﺭ ﻓﻲ ﺍﻝﻜﻴﻤﻴﺎﺀ ﺍﻝﺤﻴﻭﻴﺔ- ﻜﻠﻴﺔ ﺍﻝﻌﻠﻭﻡ- ﺠﺎﻤﻌﺔ ﺤﻠﻭﺍﻥ

ﻝﻠﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺩﻜﺘﻭﺭﺍﻩ ﺍﻝ ﻔﻠﺴﻔﺔ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝ( ﻜﻴﻤﻴﺎﺀ ﺍﻝﺤﻴﻭﻴﺔ ) )

ﺘﺤﺕ ﺇﺸﺭﺍﻑ

ﺩ.ﺃ / ﺤﻴﺎﺓ ﻤﺤﻤﺩ ﺸﺭﺍﺩﺓ ﺩ.ﺃ / ﻤﻬﺠﻪ ﺸﻔﻴﻕ ﻋﺒﺩ ﺍﷲ ﺃﺴﺘﺎﺫ ﺍﻝﻜﻴﻤﻴﺎﺀ ﺍﻝﺤﻴﻭﻴﺔ - ﻜﻠﻴﺔ ﺍﻝﻌﻠﻭﻡ ﺃﺴﺘﺎﺫ ﺍﻝﻜﻴﻤﻴﺎﺀ ﺍﻝﺤﻴﻭﻴﺔ - ﻜﻠﻴﺔ ﺍﻝﻌﻠﻭﻡ ﺠﺎﻤﻌﺔ ﺤﻠﻭﺍﻥ ﺠﺎﻤﻌﺔ ﺤﻠﻭﺍﻥ ﺩ.ﺃ / ﻓﺎﻁﻤﺔ ﺍﻝﺴﻴﺩ ﺍﻝﻨﺒﺭﺍﻭﻯ ﺩ.ﻡ.ﺃ / ﺴﺎﻤﻴﺔ ﻜﺎﻤل ﻋﻴﺎ ﺩ ﺩ ﺃﺴﺘﺎﺫ ﺍﻝﻜﻴﻤﻴﺎﺀ ﺍﻝﺤﻴﻭﻴﺔ ﺍﻹﺸﻌﺎﻋﻴﺔ ﺃﺴﺘﺎﺫ ﻤﺴﺎﻋﺩ ﺍﻝﻜﻴﻤﻴﺎﺀ ﺍﻝﺤﻴﻭﻴﺔ ﺍﻹﺸﻌﺎﻋﻴﺔ ﺍﻝﻁﺎﻗﺔ ﺍﻝﺫﺭﻴـﺔ ﺍﻝﻁﺎﻗﺔ ﺍﻝﺫﺭﻴـﺔ

ﺠﺎﻤﻌـﺔ ﺤـﻠﻭﺍﻥ ﻜﻠﻴـﺔ ﺍﻝﻌـﻠﻭﻡ ٢٠١١