GOMAL UNIVERSITY D.I.KHAN

To Study the Effect of Salts and Complexes of Palladium and Vanadium Metals on the Status of Thiols in Blood Components  Pharmacological and Toxicological Perspectives

BY: Mohammad Mukhtiar

Thesis Submitted To the Department of Pharmaceutical Chemistry Faculty of Pharmacy in Partial Fulfillment of Requirements for the Degree of DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY, FACULTY OF PHARMACY GOMAL UNIVERSITY DERA ISMAIL KHAN (KPK) PAKISTAN

Jun 2013

DEDICATION

To my Parents Who took Pains to Make the Way Easy for me towards this Success

The work presented in this dissertation, has been carried out by me at the Laboratory of

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gomal University D.I.

Khan and Department of pure and Applied Chemistry University of Strathclyde,

Glasgow Scotland UK

Under the supervision of

PROF. DR. MUHAMMAD FARID KHAN

Mohammad Mukhtiar [email protected]

CERTIFICATE OF APPROVAL

“To Study the Effect of Salts and Complexes of Palladium and Vanadium Metals on the Status of Thiols in Blood Components . Pharmacological and Toxicological Perspectives

Thesis prepared by Mr. Mohammad Mukhtiar under my guidance in partial fulfillment of the requirements of the degree DOCTOR OF PHILOSOPHY IN PHARMACY is hereby approved for the submission to the Faculty of Pharmacy, Gomal University

D.I.Khan.

PROF. DR. MUHAMMAD FARID KHAN Supervisor Department of Pharmaceutical Chemistry Faculty of Pharmacy, Gomal University Dera Ismail Khan

ROLL NO 20 SESSION 2008-2012

RESEARCH SUPERVISOR AND PROF. DR. MUHAMMAD FARID KHAN INTERNAL EXAMINER Department of Pharmaceutical Chemistry Faculty of Pharmacy

CHAIRMAN Mr. Ghulam Mustafa Department of Pharmaceutical Chemistry Faculty of Pharmacy

DEAN Mr. NUSRAT ULLAH Dean Faculty of Pharmacy

EXTERNAL EXAMINER ______

Ph.D Scholar Mohammad Mukhtiar (B. Pharm.)

Table of Contents

S.No Contents Page Acknowledgment 1 Abstract 2 Chapter:1 General introduction 1.1. Background 4 1.2. Thiols 4 1.3. Glutathione 6 1.3.1. History of Glutathione 6 1.3.2. Introduction 6 1.3.3. Protective functions of glutathione 9 1.3.5. Glutathione synthesis 14 1.3.6. Pharmacokinetics 17 1.3.7. Measurement of Glutathione 22 1.4. N‐ Acetyl cysteine (NAC) 22 1.4.1. Introduction 22 1.4.2 Biochemistry and pharmacokinetics 23 1.4.3. Clinical indications 23 1.5. D‐penicillamine (DPA) 24 1.5.1. Pharmacological profile 25 1.5.2. Biological role of D‐penicillamine 25 1.6. Albumin 26 1.6.1. Introduction 26 1.6.2. Medical uses 26 1.7.1. Vanadium 26 1.7.2. Chemistry and compounds 26 1.7.3. Natural occurrence of Vanadium 27 1.7.4. Biochemistry 28 1.7.5. Pharmacokinetics 28 1.7.6. Biological role of Vanadium 28 1.7.7. Pharmacological and therapeutic importance of Vanadium 29 1.7.8. Toxicological profile of Vanadium compounds 30 1.8.1. Palladium 31 1.8.2. Natural Occurrence and Environmental level 32 1.8.3. Palladium Compounds 32 1.8.4. Pharmacokinetics 33 1.8.5 Therapeutic Uses 34 1.8.6 Toxicology of Palladium 34 1.9.1. Scientific objectives 35

CHAPTER: 2

UV‐ visible Spectrophotometric Study of Effect of Palladium and Vanadium compounds on The Status of Thiols (Glutathione, N‐ Acetyle cysteine and D‐penicillamine) in Aqueous Medium 2.1. Introduction 36 2.2. Methodology 37 2.2.1. Materials 37 2.2.2. Method 37 2.2.2.1. Preparation of stock solutions 37 2.2.2.2. Estimation of GSH by Ellman’s (Modified) Method 39 2.2.2.3. Standard Calibration Curve 39 2.2.2.4. Experimental Protocol 40 2.3. Results 41 2.3.1. Standard Curve of Glutathione N‐Acetyle Cysteine and D‐Penicillamine 41 2.3.2. Palladium Results 43 2.3.3. Effect of various Conc. (6.7 to 67µM) of PDN/BBNPDC on the Chemical 43 Status Glutathione and With Time (0 to90 Minutes) 2.3.4. Effect of Two Conc. (6.7µM and 67µM) of PDN on the Chemical Status 45 Glutathione with pH (7.0, 7.6, 8.0) 2.3.5. Effect of various Conc. (6.7µM to 67µM) of PDN/BBNPDC on the 45 Chemical Status N‐ Acetylcysteine and With Time (0 to90 Minutes) 2.3.6. Effect of Two Conc. (6.7 and 67µM) of PDN on the Chemical Status N‐ 47 Acetylcysteine with PH (7.0, 7.6, 8.0) 2.3.7. Effect of various Conc. (6.7 to 67uM) Conc. PDN/ BBNPDC on the 47 Chemical Status D‐Penicillamine and With Time (0 to 90 Minutes) 2.3.8. Effect of Two Conc. (6.7 and 67uM) of PDN on the Chemical Status of D‐ 49 Penicillamine with pH (7.0, 7.6 and 8.0) 2.4. Results of Vanadium Metal 49 2.4.1. Effect of various Conc. (6.7 to 67µM) of AMV/VOTEO on the Chemical 49 Status Glutathione and with time (0 ‐90 Minutes) 2.4.2. Effect of Two Conc. (6.7 and 67uM) (AMV) on the Chemical Status 51 Glutathione, with pH (7.0, 7.6 and 8.0) 2.4.3. Effect of various Conc. (6.7µM to 67µM) of AMV /VOTEO on the Chemical 52 Status N‐ Acetylcysteine, and with Time (0 to 90 Minutes) 2.4.4. Effect Two Conc. of (6.7 to 67µM) of AMV on The Chemical Status N‐ 53 Acetyle Cysteine (NAC) with pH (7.0, 7.6, 8.0) 2.4.5. Effect of Various Conc. (6.7µM to 67µM) of AMV/ VOTEO on the Chemical 54 Status D‐Penicillamine, and with time (0 to 90 Minutes) 2.4.6. Effect of Two conc. (6.7 and 67uM) of (AMV) on the Chemical Status D‐ 55 Penicillamine, with pH (7.0, 7.6, 8.0) 2.5. Discussion 56 CHAPTER: 3

UV‐ visible Spectrophotometric Study of Effect of Palladium and Vanadium on The Status of Thiols (Glutathione) in whole Blood

(plasma and Cytosolic fraction)

3.1. Introduction 60 3.2. Methodology 61 3.2.1. Materials 61 3.2.2. Method 61 3.2.2.1. Preparation of Stock Solutions 61 3.2.2.2. Isolation of Blood components 63 3.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method 63 3.2.2.4. Experimental Protocol 64 3.3. Results 65 3.3.1. Results in Blood Plasma 65 3.3.1.1. Effect of various Conc. (6.7 to 67µM) of PDN/ BBNPDC on the Chemical 65 Status Plasma Glutathione, and with time (0 to90 Minutes) 3.3.1.2. Effect of Two Conc. (6.7 and 67µM) PDN on The Chemical Status Plasma 68 Glutathione, with pH (7.0, 7.6, 8.0) 3.3.1.3. Effect of Various Conc. (6.7 to 67µM) of AMV/VOTEO on the Chemical 68 Status of Plasma Glutathione, and With Time (0 to 90) Minutes 3.3.1.4. Effect of Two Conc. (6.7 to 67µM) of AMV on the Chemical Status of 71 Plasma Glutathione with pH (7.0, 7.6, 8.0) 3.3.2. Results in Cytosolic Fraction (C.F) 72 3.3.2.1. Effect of Various Conc. (6.7 to 67µM) of (PDN / (BBNPDC) on the 72 Chemical Status of C.F Glutathione, and With Time (AT 0 ‐90 Minutes) 3.3.2.2. Effect of Two Conc. (6.7µM and 67µM) Of PDN on the Chemical Status of 75 C.F Glutathione with pH (7, 7.6, 8.0) 3.3.2.3. Effect of various Conc. (6.7 to 67µM) Of (AMV)/ (VOTEO) On the Chemical 76 Status C.F Glutathione, and With Time (At 0 ‐90 Minutes) 3.3.2.4. Effect of Two Conc. (6.7 to 67µM) of AMV on the Chemical Status C.F. 79 Glutathione with pH (7.0, 7.6, 8.0) 3.4. DISCUSSION 80 CHAPTER: 4

UV‐ visible Spectrophotometric Study of Effect of Palladium and Vanadium on the Status of Thiols (Glutathione) in WBC, S

4.1. Introduction 82 4.2. Methodology 83 4.2.1. Materials 83 4.2.2. Method 83 4.2.2.1. Preparation of stock solutions 83 4.2.2.2. Isolation of WBC Component (Lymphocytes (T‐cell and B‐cells) and 85 Neutrophils 4.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method 87 4.2.2.4. Reaction Protocol 87 4.3. Results 88 4.3.1. Results of Lymphocytes with Palladium and Vanadium Compounds 88 4.3.1.1. Effect of Various Conc (6.7 to 67µM) Of PDN / BBNPDC on the Chemical 88 Status of Lymphocytes Glutathione, and with Time (0 to ‐90 Minutes) 4.3.1.2. Effect of Two Conc. (6.7 and 67µM) PDN on the Chemical Status 89 Lymphocytes Glutathione with PH (7, 7.6, 8.0) 4.3.1.3. Effect of Various Conc. (6.7 to 67µM) of PDN/BBNPDC on the Chemical 90 Status of B‐Cells Glutathione, and with time (0 ‐90) Minutes 4.3.1.4. Effect of Two Conc. (6.7 to 67uM) PDN on the Chemical Status of B‐Cells 91 Glutathione with pH (7, 7.6, 8.0) 4.3.1.5. Effect of Various Conc. (6.7 to 67uM) PDN on the Chemical Status of T‐ 92 Cells Glutathione, and with Time (at 0 ‐90 Minutes), 4.3.1.6. Effect of Two Conc. (6.7 and 67uM) PDN on the T‐Cells Chemical Status 94 Glutathione with pH (7, 7.6, 8.0) 4.3.1.7. Effect of various Conc. (6.7 to 67µM) AMV on The Chemical Status of 94 Lymphocytes Glutathione and with Time (0‐90) 4.3.1.8. Effect of Two Conc. (6.7 to 67µM) AMV on the Chemical Status 96 lymphocytes Glutathione with pH (7, 7.6, 8.0) 4.3.1.9. Effect of Various Conc. (6.7 to 67uM) AMV/VOTEO on The Chemical 97 Status B‐Cells Glutathione, with, Time (0, 90) Minutes 4.3.1.10. Effect of Two Conc. (6.7 and 67uM) of AMV and on the Chemical Status B‐ 99 Cells Glutathione, with, pH (7, 7.6, 8.0) 4.3.1.11. Effect of Various Conc. (6.7 to 67uM) of AMV/VOTEO on the Chemical 99 Status T‐Cells Glutathione, With, Time (0, 90) Minutes 4.3.1.12. Effect of Two Conc. (6.7 and 67uM) Of AMV on the Chemical Status T‐ 101 Cells Glutathione, with, pH (7.0, 7.6, 8.0) 4.3.2. Results of Neutrophils 101 4.3.2.1. Effect of Various Conc. (6.7 to 67uM) PDN/BBNPDC on the Chemical 101 Status of Neutrophils GSH, and with Time (0, 90) Minutes 4.3.2.2. Effect of Two Conc. (6.7 and 67µM) PDN on The Chemical Status of 103 Neutrophils GSH with pH (7.0, 7.6, 8.0) 4.3.2.3. Effect of Various Conc. (6.7 to 67uM) AMV/VOTEO on the Chemical Status 104 of Neutrophils GSH, and with Time (0, 90) Minutes 4.3.2.4. Effect of Two Conc. (6.7 and 67µM) AMV on The Chemical Status of 106 Neutrophils GSH with pH (7.0, 7.6, 8.0) 4.4. Discussion 106 Chapter: 5

UV‐ visible Spectrophotometric Study of Effect of Palladium and

Vanadium on The Status of Thiols (Glutathione) in Liver Homogenate

5.1. Introduction 108 5.2. Methodology 108 5.2.1. Materials 108 5.2.2. Method 109 5.2.2.1. Preparation of stock solutions 109 5.2.2.2. Preparation Liver Homogenate sample 110 5.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method 111 5.2.2.5. Reaction Protocol 111 5.3. Results 112 5.3.1. Results of Liver Homogenate GSH wit Palladium compounds 112 5.3.1.1. Effect of Various Conc. (6.7 to 67uM) PDN/ BBNPDC on Liver Homogenate 112 GSH content, and With Time at (0 ‐90) Minutes), 5.3.1.2. Effect of Two Concentrations (6.7 and 67uM) PDN on Glutathione content 114 of Liver Homogenates with pH at (7, 7.6, 8.0) 5.3.2.1. Effect of Various Conc. (6.7 to 67uM) AMV/VOTEO on the Chemical Status 114 of Liver Hepatocytes GSH, and With Time (At 0 ‐90 Minutes) 5.3.2.2. Effect of Two Conc. (6.7 to 67uM) AMV on The Chemical Status of Liver 116 Homogenate GSH With pH (7.0, 7.6, 8.0) 5.4. DISCUSSION 117 CHAPTER: 6

Proton Nuclear Magnetic Resonances (1HNMR) Study of Palladium

and Vanadium effect on Thiols (GSH, NAC and D‐Pen)

6.1. Introduction 119 6.2. Materials and Methods 120 6.2.1. NMR assignments for Ellman’s reagent, Ellman’s anion and the thiolate 120 mixed disulfides of Ellman’s reagent 6.2.2. Calibration of the thiol disulfide exchange by NMR methods 121 6.2.3. Interaction of palladium with thiolates 121 6.3. Results and discussion 121 6.3.1. Effect of PH on the chemical status of Elman’s reagent 121 6.3.2. Exchange reaction of thiolates (N‐ Acetylcysteine Glutathione and D‐ 122 penicillamine), with Elman’s reagent (ESSE) 6.3.3. Interaction of palladium with Glutathione 126 6.3.4. Interaction of palladium with N‐Acetylcysteine 128 6.3.5. Interaction of palladium with (D) ‐penicillamine 130

Chapter: 7 Effect of Palladium and Vanadium Compounds on the Exchange Reaction between Bovine Serium Albumin and Thiols Containing Compounds Using UV‐ Visible Spectrophotometric Method

7.1. Introduction 133 7.2. Experimental Procedure 133 7.2.1. Preparation of Standard curve for BSA solution 134 7.2.2. The calculation of free thiolate content of BSA, 134 7.2.3. Preparation of Elman’s modified BSA (BSA‐SSE) 134 7.2.4. Treatment of BSA‐SSE with thiolates 134 7.2.5. Preparation of Elman’s modified BSA with palladium and vanadium 135 7.2.6. Treatment of BSA‐Pd and BSA‐V with thiolates 135 7.3.1. Results and discussion 135 7.3.2. Calibration curve for the calculation of unknown concentration of 138 Albumin in Palladium or vanadium BSA mixture 7.3.3. The exchange reactions of either palladium /or vanadium with either 138 Glutathione, or N‐Acetylcysteine, or D‐penicillamine 8. Conclusion 143 9. References 144

List of Abbreviations

AMV: Ammonium Vanadate ARE: antioxidant response element BBNPDC: Bis‐benzonitrile palladium (II) chloride C.F.: Cytosolic fraction CFTR: Cystic fibrosis transport receptor Conc. Concentration C‐raf: Proto‐oncogene Serine/threonine‐protein kinase D‐pen: D‐Penicillamine DTNB: 5, 5’‐dithiobis (2‐nitrobenzoic acid EpRE: Electrophile response elements GCL: Cysteine‐ligase GCLC: Glutamate cysteine ligase catalytic subunit GCLM: Glutamate cysteine ligase GPx: Glutathione peroxidase enzymes GSH: Glutathione GSSG, Glutathione Di Sulfide HNE: 4‐Hydroxy‐2‐nonenal) IFN: interferon alpha JUN and FOS : Jun and FOS family of transcription factors are cellular immediate‐early genes L.HG: Liver Homogenate. LOO: Hydroperoxide radical Lymph. Lymphocytes MAPK: Mitogen‐activated Protein Kinase NAC: N‐Acetylcysteine Neut. Neutrophils NF_kB: Nuclear factor NMR: Nuclear Magnetic Resonance Spectrophotometer. NOX: NADPH oxidase, ONOO: peroxynitrite PDI: Protein disulfide‐isomerase PDN: Palladium Nitrate PSI: Protein disulfide isomerase Ras: Rat Sarcoma SOD: Superoxide dismutases TNB: Thiolate, 5‐thio‐2‐nitrobenzoic acid VOTEO: Vanadium oxi Tri ethoxide List of Figures and Tables Figure and Title Page Table No Figure 1.1 Structure of GSH 6 Figure 1.2 Redox cycling of 1, 4‐naphthoquinones 7 Figure 1.3 Reaction of GSH and HOCl 10 Figure 1.4 Schematic Diagram of Metabolism of GSH 18 Figure 1.5 The oxidation‐reduction pathways of GSH 19 Figure 1.6 Recycling Of GSH 20 Figure 2.1 standard curve of GSH 42 Figure 2.2 Standard curve of NAC 42 Figure 2.3 Standard curve of D‐Pen 42 Figure 2.4 Conc. dependent effect of (6.7 and 67 µM) of PDN/ BBNPC on the 44 chemical status of GSH Figure 2.5 Time dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN / 44 BBNDC on the chemical status of GSH Figure 2.6 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN 45 on the chemical status of GSH Figure 2.7 Conc. dependent effect of (6.7 and 67 µM) of PDN) / BBNPC on the 46 chemical status of NAC Figure 2.8 Time dependent effect of lowest and highest Conc. (6.7 to 67µM) of PDN) 46 / BBNDC on the chemical status of NAC Figure 2.9 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN 47 on the Chemical status of NAC Figure 2.10 Conc. Dependent effect of (6.7 to 67 µM) PDN / BBNPC on the chemical 48 status of D‐Pen Figure 2.11 Time dependent effect of Lowest and Highest Conc (6.7 to 67µM) of PDN / 48 BBNDC on the chemical status of D‐pen Figure 2.12 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN 49 on the chemical status of D‐Pen Figure 2.13 Conc. dependent effect of (6.7 to 67 µM) AMV / VOTEO on the chemical 50 status of GSH. Figure 2.14 Time dependent effect of Lowest and Highest Conc. (6.7 and 67µM) AMV) 51 /VOTEO on the chemical status of GSH Figure 2.15 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV 51 on the chemical status of GSH Figure 2.16 Conc. dependent effect of 6.7 to 67 µM) AMV / VOTEO on the chemical 52 status of NAC Figure 2.17 Time Dependent effect of Lowest and Highest Conc. (6.7 and 67µM) of 53 AMV / VOTEO on the chemical status of NAC. Figure 2.18 pH dependent effect of lowest and highest Conc. (6.7 and 67µM) of AMV 54 on the chemical status of NAC Figure 2.19 Conc. dependent effect of (6.7 to 67µM) of AMV / VOTEO on the chemical 55 status of D‐Pen Figure 2.20 Time Dependent effect of lowest and highest conc. (6.7 and 67µM) of 55 AMV / VOTEO on the chemical status of D‐Pen. Figure 2.21 pH Dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV / 56 VOTEO on the chemical status of D‐Pen. Figure 3.1 Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPC on the chemical 66 status of plasma GSH before its isolation from Blood Figure 3.2 Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPC on the chemical 66 status of plasma GSH after its isolation from Blood Figure 3.3 Time dependent effect of Lowest and highest conc. of PDN / BBNPC on 67 the chemical status of plasma GSH before its isolation from blood. Figure 3.4 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) PDN / 67 BBNPC on the chemical status of plasma GSH after isolation of Blood. Figure 3.5 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN / 68 BBNPC on the chemical status of plasma GSH after isolation of Blood Figure 3.6 Conc. dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical 69 status of plasma GSH before its isolation from blood. Figure 3.7 Conc. dpendent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical 70 status of plasma GSH after its isolation from blood. Figure 3.8 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 70 AMV / VOTEO on the chemical status of plasma GSH before its isolation from blood. Figure 3.9 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 71 AMV/ VOTEO on the chemical status of plasma GSH after its isolation from blood. Figure 3.10 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV 72 on the chemical status of plasma GSH after its isolation from Blood. Figure 3.11 Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPDC on the 73 chemical status of GSH in C.f. before its isolation from blood Figure 3.12 Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPDC on the 74 chemical status of GSH in C.f. after its isolation from blood Figure 3.13 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 74 PDN/BBNPDC on the chemical status of C.F GSH before its isolation from blood Figure 3.14 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) 75 PDN/BBNPDC on the chemical status of C.F GSH after its isolation from blood Figure 3.15 pH dependent effects of Lowest and highest conc. (6.7 and 67 µM) of PDN 76 on the chemical status of C.f. GSH after isolation from blood Figure 3.16 conc. dependent effect of AMV/ VOTEO on the chemical status of GSH in 77 Cytosolic fraction (CF) before isolation of CF from blood. Figure 3.17 conc. dependent effect of (6.7 to 67 µM) of AMV/ VOTEO on the chemical 78 status of GSH in Cytosolic fraction (CF) after isolation from blood) Figure 3.18 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 78 AMV/ VOTEO on the chemical status of GSH of Cytosolic fraction before isolation from blood Figure 3.19 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 79 AMV/ VOTEO on the chemical status of GSH in Cytosolic fraction after its isolation from Blood Figure 3.20 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV 80 on the chemical status of GSH of Cytosolic fraction after its isolation from blood Figure 4.1 Conc. dependent effect of (6.7 and 67 µM) of PDN / BBNPC on the 88 chemical status of lymphocytes GSH Figure 4.2 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 89 PDN / BBNPC on the chemical status of lymphocytes GSH Figure 4.3 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN 90 on the chemical status of lymphocytes GSH Figure 4.4 Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on the chemical 91 status of B‐cells GSH Figure 4.5 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 91 PDN/BBNPDC on the chemical status of B‐cells GSH Figure 4.6 pH dependent effect of lowest and highest conc. (6.7 and 67µM) PDN on 92 the chemical status of B‐cells GSH Figure 4.7 Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on the chemical 93 status of GSH in T‐cells after isolation of T‐cells. Figure 4.8 Time dependent effect of Lowest and highest Conc. (6.7 and 67 µM) of 93 PDN/BBNPDC on the chemical status of T‐cells GSH Figure 4.9 pH dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN on 94 the chemical status of T‐cells GSH Figure 4.10 Conc. dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical 95 status of lymphocytes GSH Figure 4.11 Time dependent effect of lowest and highest conc. (6.7 and 67 µM) of 96 AMV / VOTEO on the chemical status of lymphocytes GSH Figure4.12 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV 97 on the chemical status of lymphocytes GSH Figure 4.13 Conc. dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical 98 status of B‐cells GSH Figure 4.14 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 98 AMV / VOTEO on the chemical status of B‐cells GSH Figure 4.15 pH dependent effects of Lowest and highest conc. (6.7 to 67 µM) of AMV 99 on the chemical status of B‐cells GSH Figure 4.16 Conc. dependent effect of (6.7 and 67 µM) of AMV /VOTEO on the 100 chemical status of T‐cells GSH Figure 4.17 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of 100 AMV / VOTEO on the chemical status of T‐cells GSH Figure 4.18 pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV 101 on the chemical status of T‐cells GSH Figure 4.19 Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on the chemical 102 status of Neutrophils GSH Figure 4.20 Time dependent effect of Lowest and highest Conc. (6.7 and 67 µM) of 103 PDN on the chemical status of Neutrophils GSH Figure 4.21 pH dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN on 104 the chemical status of Neutrophils GSH Figure 4.22 Conc. Dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the 105 chemical status of Neutrophils GSH Figure 4.23 Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) AMV 105 / VOTEO on the chemical status of Neutrophils GSH Figure 4.24 pH dependent effect of lowest and highest conc. (6.7 and 67µM) AMV on 106 GSH content of Neutrophils Figure 5.1 Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on GSH content 113 of liver homogenate Figure 5.2 Time dependent effect of Lowest and highest Conc. (6.7 and 67 µM) of 113 PDN / BBNDC on GSH content of liver Homogenate Figure 5.3 pH dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN on 114 the chemical status of GSH content Liver Homogenate Figure 5.4 Conc. Dependent effect of (6.7 to 67 µM) of AMV/ VOTEO on the chemical 115 status of GSH content liver homogenate Figure 5.5 Time effect of Lowest and highest conc. (6.7 and 67 µM) of AMV/ VOTEO 115 on the chemical status of GSH of liver Homogenate. Figure 5.6 pH dependent effect of lowest and highest conc. (6.7 and 67µM) lowest 116 and highest conc. (6.7 and 67µM) AMV on the chemical status of GSH of Liver Homogenate Figure 6.1 The 400 MHz 1H NMR spectra of (top) 1mg/ml Ellman’s reagent treated 122 with NaOD solution and (bottom) the same solution treated with hydrogen peroxide. Trace amounts of Ellman’s reagent can be observed in the baseline of the peroxide treated solution (bottom).

Figure 6.2 The 400 MHz 1H NMR spectras obtained by titrating a solution (300 μl) of 124 2 Ellman’s reagent (10 mg/1.5 mL, 0.1 M KH2PO4 in H2O at pH 7.4) with Top: N‐acetylcysteine. (a) Ellman’s reagent, (b) 0.4 mg N‐acetylcysteine, (c) 0.8 mg N‐acetylcysteine, (d) 1.2 mg N‐acetylcysteine, (e) 1.6 mg N‐ acetylcysteine and (f) 2.0 mg N‐acetylcysteine. Figure 6.3 The 400 MHz 1H NMR spectras obtained by titrating a solution (300 μl) of 124 2 Ellman’s reagent (10 mg/1.5 mL, 0.1 M KH2PO4 in H2O at pH 7.4) with GSH) (a) (0‐mg) GSH, (b) 0‐mg) GSH, (c) 0‐mg) GSH, (d) 0‐mg GSH, (e) (0‐ mg) GSH. Figure 6.4 The 400 MHz 1H NMR spectras obtained by titrating a solution (300μl) of 125 2 Ellman’s reagent (10 mg/1.5 mL, 0.1 M KH2PO4 in H2O at pH 7.4) with D‐ Pen. (a) 0.15 mg D‐Pen, (b) 0.30 mg D‐Pen, (c) 0.45 mg D‐Pen, (d) 0.60 mg D‐Pen and (e) 1.12 mg D‐Pen. Figure 6.5 The calibration graph for the reaction of GSH with Ellman’s reagent 125

obtained using the relative areas of the integrals derived from Hc. Figure 6.6 The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of 127 2 GSH 0.1 M KH2PO4 in H2O at pH 7.4) with a Pd (NO3)2 (forming Pd (SGH) 2 Complex in situ Figure 6.7 The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of 128 2 GSH 0.1 M KH2PO4 in H2O at pH 7.4) with a [Pd (NO3)2 (forming Pd (SGH) 2 Complex in situ. Figure 6.8 Structure of N‐Acetyle cysteine 129 Figure 6.9 The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of 129 N‐ acetylcysteine 0.1 M KH2PO4 in 2H2O at pH 7.4) with a Palladium Nitrate (2:1) formed in‐situ. Figure 6.10 The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of 130 N‐ acetylcysteine 0.1 M KH2PO4 in 2H2O at pH 7.4) with a Palladium Nitrate1:1 formed in‐situ. Figure 6.11 The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of 131 D‐Pen with Pd (NO3)2 0.1 M KH2PO4 in 2H2O at pH 7.4) with a Pd (D‐pen) 2 and formed in‐situ Figure 6.12 The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of 132 Glutathione 0.1 M KH2PO4 in 2H2O at pH 7.4) with a [Pd (D‐pen)] n, and formed in‐situ. Figure 7.1 The titration of BSA (13.2 mg/3 ml) with Ellman’s reagent (1.98 mg/ml 5 136 µl).The expected deviation from linear behaviour at high Ellman’s reagent concentrations (150, 1.98mg/ 5µl) is evident. An intercept is found which is consistent with residual absorbance by BSA at 412 nm. Figure 7.2 Top: The titration of BSA‐SSE 200uM) with GSH solution (200uM) the 137 formation of Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm. Figure 7.3 Calibration curve of BSA at 280 nm 138 Figure 7.4 The titration of BSA‐Pd 50uM) with GSH solution (50uM) the formation of 139 No Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm. Figure 7.5 The titration of BSA‐Pd 50uM) with N‐ Acetylcysteine solution (50uM) the 139 formation of No Ellman’s anion (equation 3) is evident from the appearance of a band at 412 nm. Figure 7.6 The titration of BSA‐Pd 50uM) with D‐Pen (50uM) the formation of No 140 Ellman’s anion (equation 4) is evident from the appearance of a band at 412 nm. Figure 7.7 The titration of BSA‐V 50uM) with GSH solution (50uM) the formation of 140 No Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm. Figure 7.8 The titration of BSA‐Pd 50uM) with N‐Acetylcysteine solution (50uM) the 141 formation of No Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm. Figure 7.9 The titration of BSA‐Pd 50uM) with D‐Pen solution (50uM) the formation 141 of No Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm. Table 6.1 The chemical shifts of the Ellman’s based species formed in the thiol‐ 123 disulfide equilibrium (equation 1) using N‐acetylcysteine and its alkaline hydrolysis. The data is obtained from the spectra shown in figure 1 to 3. Typical coupling constants are given Table 6.2: Summary of Distribution of chemical shifts for the 1H‐NMR spectrum of 127 GSH Table 6.3 Summary of Distribution of chemical shifts for the 1H‐NMR spectrum of N‐ 129 acetylcysteine Table.6.4 Summary of Distribution of chemical shifts for the 1H‐NMR spectrum of 131 (D) ‐penicillamine

Acknowledgement In the name of Allah Almighty, the most merciful, the most beneficent, a torch of guidance and knowledge. The book you are looking at is the abstract of many desperate attempts, during which, I was guided, motivated and supported by many people whom I feel to acknowledge.

First of all, I wish to record my sincere gratitude to my Supervisor Prof. Dr. Muhammad Farid Khan for his valuable guidance, advice and for conveying a spirit of commitment to research. With his valuable comments, I was able to finalize the writing process for my thesis. I would like to pay my gratitude to Prof. John Reglinski not only for letting me to use the facilities of the department of Pure and Applied Chemistry but also for providing guidance and a helping hand during many of the critical experiments, during the visit to University of Strathclyde Glasgow Scotland UK in the last year of my PhD. I am grateful to the Higher Education Commission of Pakistan for providing financial support. I enjoyed the company of the wonderful PhD colleagues and other members of the research lab during my PhD study. I am thankful to Prof. Dr. Gul Majid Khan, former dean and to Mr. Nusrat Ullah, present dean of the Faculty of Pharmacy for their continuous support throughout my research work in the laboratories of this faculty. I am indebted to the chairman Department of Pharmaceutical Chemistry Mr. Ghulam Mustafa and all other faculty members of this faculty. I am also grateful to all of my M. Phil and Ph.D colleagues who have extended their cooperation to me in a number of ways during carrying out this piece of work. I do not have words at my command to express my gratitude and profound admiration to my affectionate parents and family members. They provided me with their continuous support and love during my entire life. I would also like to appreciate my family-in-law for their moral support. Lastly, I would like to express my gratitude towards my wife, who supported me during my PhD. The best possible words I can say to all of them in return are Jazak-a-Allah (Allah will reward you).

Mohammad Mukhtiar PhD Scholar Department of pharmaceutical chemistry Faculty of Pharmacy Gomal University D.I. khan

1

Abstract The selected metalloelements i.e. Vanadium and Palladium have a number of potential Pharmaco-clinical advantages. Vanadium decreases the level of glucose and cholesterol, improves the function of hemoglobin and myoglobin and has anti-cancerous and diuretic functions. Similarly, Palladium compounds have antiviral, antibacterial, neuroprotective and antitumor properties. However studies have also indicated some mild to serious toxic effects of these metalloelements. Biothiols are important antioxidant that provides protection against metals toxicity. The interaction of metalloelements with biothiols can provide valuable information about the level of toxicity of the metalloelements and about the protective role of biothiols thereof. In this piece of work the effect of salts and complexes of Vanadium and Palladium on the status of different thiols (GSH, NAC, D-Pen and albumin) in aqueous medium, blood components and liver homogenate . The thiol quantification was carried out using Elman’s method through UV-visible spectrophotometry and 1H- NMR. Results of the study performed in aqueous medium, as shown in chapter 2, showed that level of different thiols depleted after the addition of the inorganic salts and organic complexes of Vanadium and Palladium. Such depletion was further enhanced with increasing concentrations of the metalloelements and with time incubation. We also observed a maximum depletion in the levels of different thiols at pH 7.6 which is near to physiological pH. Similar observations were also made in blood components as mention in chapter 3. We observed a decrease in the level of Glutathione under the effect of the said metalloelements in whole blood as well as in separated plasma and cytosolic fraction. Observations were also made under different concentrations of the metalloelements, time and pH parameters. Results were showing that the effect of these metalloelements on the level of GSH in blood components is high at pH 7.6 and increases with increasing concentrations of the metalloelements and with time elapse. Such depletion in cytosolic fraction, in particular, is indicative of the fact that the anions of both the metals can cross the membrane of the erythrocytes. Effect of metalloelements Vanadium and Palladium on the status of Glutathione in WBCs (Lymphocytes, B-cells, T-cells and Neutrophils) was also investigated as mention in chapter 4. The results showed that the level of Glutathione in the selected types of WBC’s was depleted as a result of interaction with Vanadium and Palladium. This depletion was further aggravated with increasing concentrations of Palladium and Vanadium, time elapse as well as at

2

pH 7.6. The effect these metalloelements on the concentration of Glutathione in liver homogenate, under different parameters was also observed as mention in chapter 5.for this purpose liver homogenate was prepared according to the protocol established by Schiefer. We observed that the metalloelements decreases the level of Glutathione in the liver homogenate sample which was further enhanced with elevated concentrations of the metals, time incubation and was high at pH7.6. The mechanism of interaction of Palladium with thiols was examined using H-NMR as mentioned in chapter 6. The results revealed that five species are produced during the quantification of thiols with Elman’s reagents (ESSE). These species were ES-, ESSE, ESSR, RSSR and RSH. The results further indicate that the depletion in the level of thiols may be due to 1:1 or 1:2 conjugation of Palladium with thiols respectively. The bonding strength of both the elements with albumin and exchange reaction by low molecular weight thiols were also examined as mentioned in chapter 7. The UV- visible spectrophotometric observation was made at wavelength ranging from 240nm to 500nm. A negligible absorbance was observed at 412nm which suggested that there was no exchange reaction between albumin metal complexes and low molecular weight thiols. The finding of the study suggests that the metalloelements Vanadium and Palladium conjugate with different thiols in aqueous medium, blood components and liver homogenate. The chances of such assumed conjugation reactions further increases with time elapse, with increasing concentration of the metalloelements and in suitable conditions of pH 7.6. These conjugation reactions further suggest that the metalloelements Vanadium and Palladium have xenobiotic nature causing oxidative stress and thiols play their role in detoxification and biotransformation of these metalloelements. This study perform in situ can be used as a model of in vivo study.

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Chapter: 1

1.1. Background Metallo-elements is a wide area of studies focused on the interactions of metal ions with biomolecules, transport and behavior of metals in organisms as well as the general impact of the exposure to the metal ions has a great share of the current state of knowledge in the field of biochemistry and biomedicine. Organo-metalloelement complexes and metal based drugs have attracted the attention of scientists in general and biomedical scientists in particular for the diagnosis, investigation, research and treatment of a number of diseases. Since Glutathione (GSH), a sulfhydryl (-SH) containing low molecular weight biological compound is present almost in all cells of the body at low and high concentrations and also involved in a number of biological processes and found perturbed in disease state and with its changed status. GSH conjugation is a remarkable process between GSH and others compounds, drugs and metals base drugs and thus plays an important role in detoxification and excretion. The mechanism of action and the formation of conjugates is an important area of research. The detoxification of organo- metallic complexes / drugs or derivatives form bound –metals with GSH and other Thiols in living system. We have studied the interaction of Vanadium, Palladium salts/ organo-metals derivatives with GSH in aqueous, blood components (Plasma, Cytosolic fraction, neutrophils T- cells and B-cells), liver homogenate Bovine serum albumin using Ellman’s modified method using UV spectrophotometer and NMR spectrophotometer.

1.2. Thiols

1.2.1. Introduction to Thiols

Thiols an organo sulpher compound is a combination of two words “thio” and “alcohols” with the first world derived from Greek world “thion” which mean “sulfur”. Thiols are characterized by the presence of sulphydryl group. Thiols are analogue of alcohols i.e. oxygen of hydroxyl group is replaced by sulfur (R-SH) (Liddell et al., 1994). Chemically Thiols are called “mercaptan” derived from “mercurium captans” due to its strong affinity for mercury and biologically it is called biothiols (Patai, 1974; Cremlyn, 1996 & Thiol, 2011). Thiols can be

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Chapter: 1

divided into low molecular weight free Thiols and high molecular weight protein Thiols (Chandan et al., 2000).

1.2.2. Preparation of Thiols

Thiols can be prepared on both laboratories as well as on industrial scale.

On industrial scale Thiols can be reacting alcohol with hydrogen sulfide in the presence of acidic catalyst.

CH3OH + H2S CH3SH + H2O

Thiols can also be obtained by reacting hydrogen sulfide with alkenes in the presence metal catalyst (Kathrin et al., 2002).

In Laboratory different methods are employed for the preparation of thiol on such method involves reaction of Grignard reagents with organo- lithium (Jones et al., 1990).

RLi + S RS Li

RSLi + HCl RSH + LiCl

1.2.3. Thiols Radicals

In organic chemistry as well as biochemistry, a free radical which often takes parts in chemical reaction is called Thiols radical or thiyl radical or mercaptans with general formula RS•(Cremlyn, 1996).

1.2.4. Chemical Reaction of Thiols

Alkylation: Thiols on reaction with alkyl halide produce thio ether (R—S—R)

Acidity: Thiols Shows more acidic behaviours, than alcohols.

Redox reaction: Thiols oxidized in the presence of hydrogen peroxide results in the formation of sulfonic acid (RSO3H) (Akhmadullina et al., 1993

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Chapter: 1

1.3. Glutathione 1.3.1. History of Glutathione Glutathione was first discovered in 1888 by J.de Rey- pailhade when he noticed the antioxidant properties of a water extract of Backer’s yeast (pailhade, 1888). He initially named the substance philothione (sulfur loving in Greek) based on how it reacted towards sulfur (Pailhade. 1888). This discovery was followed in 1908 by heffter, s demonstrations of thiol in the living cells by means of calorimetric sodium nitroprusside assay for mercaptans (Weiland, 1954). Later, after further observation were made about its structure between1921-1941, Gowland Hopkins, the “Father of biochemistry” of Cambridge, England, renamed the substance “Glutathione”. Pure Glutathione (GSH) was not defined, until the synthetically experiments of Harington &mead (Harington et al., 1935). The correct structure of Glutathione (GSH) was published as early as 1935 (Colowick and Keplen, 1985). Harington &mead synthesized Glutathione (GSH) from a reaction combining n-benzyl-carbonylglcystiene & glycine ester to yield cysteinylglycine ester followed by the addition of n-benzyl-carbonylglutamic acid chloride. The resultant tripeptide (GSH) was isolated following alkaline hydrolyses & phosphonium iodide (ph4i) reduction. Platt first reported the presence of Glutathione (GSH) in white blood cell W.B.C (Platt, 1931)

1.3.2. Introduction The tripeptide, c- L-glutamyl- L -cysteinyl-glycine known as Glutathione (GSH) (as shown in Figure1.1) (Irwin & Jakoby, 1975).

HS O O O

NH HO NH OH

NH2 O Glutathione ( GSH Reduced Form of Glutathione)

Figure 1.1: Structure of GSH

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Chapter: 1

GSH is the most important low molecular weight antioxidant synthesized in cells. Its molecular formula is C10 H17N3O6S and its molecular weight is 307.33. Glutathione disulfide is also known as GSSG. Its molecular formula is C20H32N6O12S2.The Glutathione in reduced form is crystalline power, odorless or with a slightly specific order and sour taste. It is freely soluble water but particularly insoluble in methanol or ethanol. It is synthesized by the sequential addition of Cysteine to glutamate followed by the addition of glycine. The sulfhydryl group (–SH) of the Cysteine is involved in reduction and conjugation reactions that are usually considered as the most important functions of GSH. These reactions provide the means for removal of peroxides and many xenobiotic compounds; however, GSH is also involved in regulation of the cell cycle (Meister 1992).

Sources of oxidants GSH play a very essential role in the removal of various reactive oxygen species from the body. But before highlighting of removal of these species it is important to explore all those aspects that lead to the generation of these reactive oxygen species and their pathological consequences that are avoided by Glutathione. One class of Redox cycling molecules also including some xenobiotic compounds and drugs are quinones. In these species the redox cycling leads to the ability to cycle between in reduced and oxidized forms and results in the production of reactive oxygen species, such as superoxide (O2) and Hydrogen peroxide (H2O2).

Diagram

Figure 1.2: Redox cycling of 1, 4-naphthoquinon

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Chapter: 1

In the above Figure Quinone reduced into semiquinone by one electron transport reaction, which is a free radical that has the ability to initiate the reaction further and produce O2. A Naphthoquinone is also generated with two variable species during reaction which is also reduced enzymatically by NADPH or NADP resulting in the generation of semiquinone radical, which react further with oxygen to produced superoxide and restore naphthoquinone (Forman et al., 2009). There are some other spots in the cells where reactive oxygen species can also be produced. It is the reactive oxygen species generation mechanism that results in the killing of microorganism during phagocytosis (Forman and Thomas, 1986). NADPH oxidase (NOX) is the first enzymes involved in the production of O2, which is now known to be a class of enzyme usually present in all forms of cells (Vignais, 2002). Once oxygen is generated it is converted into (H2O2) by two consecutive pathways: by a relatively fast non enzymatic reaction and by a very fast enzymatic reaction usually catalyzed by superoxide dismutases (SOD). Some phagocytes also have the ability to secrete one of the enzyme myeleoperoxidases that have the ability to convert Hydrogen peroxide and chloride or bromide into HOCl and HOBr (Bakkenist et al., 1980).These acids can kill bacteria but also harm host cells by causing inflammation reaction in normal tissue. H2O2 produced in the cells is potentially hazardous and can react with any radical present nearby in the cell. It can also react with iron in ferrous form results in the generation of hydroxyl radical (OH- ) which have the capability to react with teflon or fluorine or any other at near diffusion limited rate and thus oxidized them. Oxygen has also the ability to reduced Fe+3 to Fe+2 suggesting its two important roles in the production of OH- ; However Ferric iron (Fe+3) is also reduced by other antioxidants like ascorbic acid (Vitamin C). Hydroxyl radical is highly dangerous when produced near a membrane because it oxidizes the lipids of the membranes, results in a chain reaction and thus damage the membrane. Lipid peroxidation reaction initiation by OH radical can lead to the conversion of reduced lipid specie into a lipid radical and water molecule which can then react with oxygen (O2) to produced hydroperoxide radical (LOO),which then can react with another lipid molecule generating (LOOH) and another lipid radical and thus results in initiation of a chain reaction. Besides the cell membrane damage during lipid peroxidation it can also lead into the generation of other by-products e.g. 4- Hydroxy-2-nonenal (HNE). Arachidonic acid which is polyunsaturated fatty acid present in the membrane of all cells when oxidized, results in breakdown of large number of products

8

Chapter: 1

including a,b-unsaturated aldehydes (Poli et al., 1987).These species are considered to be toxic because they have the potential to react with tissue proteins especially at the site of cysteine, lysine or histidine either by the mechanism of Michael addition (c-c) or by the mechanism of Schiff base formation ( ) ( Esterbauer et al., 1991; Eckl, 2003 & Schaur, 2003). These reactions can lead to inactivation of proteins; for instance the reaction at the active site of cysteine can destroy the activity of enzyme. The final components of the oxidative damage consider here is peroxynitrite (ONOO), produced during the reaction of O2 and Nitrogen oxide (NO). These two radicals then can react at such a fastest rate which is only fastest reaction known to occur in biology, also consider being fastest from dismutation reaction of O2 catalysis by enzymes superoxide dismutases. Peroxynitrite (ONOO) when exist in its basic form does not react with organic molecule but it split into nitrite NO2 and nitrate NO3. The protonation of peroxynitrite results in production of highly reactive species peroxynitrous acid (ONOOH). That has the reactivity similar to that of NO2, the most toxic specie usually present as a core component of smoke and cigarette smoke (Esterbauer et al., 1991).

1.3.3. Protective functions of Glutathione a. Reduction

Glutathione is present almost in the range of 1-10mM in all cells of living organisms usually found in the cytosol of the cells (Meister, 1988). The concentration of Glutathione varies from cells to cells, in erythrocytes its concentration is usually found in the range of 1-2 mM, in plasma and extracellular fluids its concentration is in the range of 2-50uM, its concentration in the extracellular fluids is usually very low i.e. in micromolar rang but in some extra-cellular fluids its concentration is higher like in the fluids of the lining of the lungs due its secretion by the epithelial cells. It is present in almost in highest concentration in the liver ranges from 5-10mM (Sutherland et al., 1985 & Cantin et al., 1987). During smoking or inhalation oxidants; there is a chance of potential inflammation that involves the invasion of neutrophils from the blood through the epithelial and endothelial cells into the air spaces. As the neutrophils squeeze among the cells, they release Hypochlorous acid which then can react with the Glutathione normally secreted from the lining of epithelial cells and thus protect them from being foreign invaded species (Venglarik et al., 2003). In cystic fibrosis patients where there is a secretion of lower GSH than from normal individuals into the lining fluid covering their alveoli and in smokers,

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Chapter: 1

Whose lungs are exposed to many oxidants including nitrogen dioxide and H2O2, there is both deficiency of Glutathione and chronic inflammation (Roum et al., 1993). The proteins found in the lining of fluids or on the surface of the epithelial cells are also oxidized by HOCl. The Hypochlorous acid can also react with lipids resulted in the generation of even more dangerous compounds than are produced from lipid by its own lipid peroxidation (Pullar et al., 2000).

(GSH + HOCl GSCl + H2O

GSCl + GSH GSSG + HCl

GSCl + 2 HOCl GSO2Cl + 2HCl

GSO2Cl GSO2NH (Formed with an amino group of

GSH itself) + GSO2Cl + GSH GSO2SG + GSO2SG + HCl)

Figure 1.3: Reaction of GSH and HOCl

Figure: 1.3 shows how Glutathione (GSH) reacts with HOCl and removes it (Winterbourn and Brennan, 1997). While many studies on the Glutathione during inflammation of lungs have been done it is sought out that these interaction can occur in any part of the body. In patient of cystic fibrosis where there is a mutation of proteins also known as cystic fibrosis transport receptor (CFTR) the secretion of Glutathione into the air spaces of the lungs is depressed (Roum et al.,1993).The lining of CFTR, derived from patient of cystic fibrosis ,has lower secretion rate of Glutathione apical region( air spaces). If the wild type of CFTR is transfected into the cells, the secretion rate of Glutathione is enhanced to such a level as seen in normal cells (Gao et al., 1993). The generation of hypochlorous acid (HOCl) in the surface fluid covering of the normal epithelial to enhance the simulated neutrophils action, can lower in the electrical resistance of that epithelial cell layers, however the presence of Glutathione in such a high concentration as that of normal fluid lining can protect it against the loss of electrical resistance (Venglarik et al., 2003). Usually the internal environment of the cells is quite different from external environment; still Glutathione plays an essential role in various cellular comportments, e.g. its plays central role in regulating apoptosis versus necrosis in mitochondria (Yuan and Kaplowitz, 2009). Glutathione also plays a key role in regulation of cell division in nucleus (Pallardó et al., 2009).

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Chapter: 1

The lower level of Glutathione both extracellularly and intracellularly can adversely affect the lungs. Liver is the main organs where majority of studies related to pathologies involving Glutathione pharmacodynamics has been done (Biswas and Rahman 2009; Yuan and Kaplowitz, 2009 and Fraternate et al., 2009). GPx is a three member of Glutathione peroxidase enzyme which utilizes most of the Glutathione antioxidant defense role in the cells (Brigelius-Flohe, 1999) and one of the peroxiredoxin (predx6). The reduction of Hydrogen peroxide by Glutathione into water and Glutathione disulphide (GSSG) formation is usually catalyzed by the three enzymes. Prdx also needed Glutathione transferase Pi in order to be in active form (Ralat et al., 2006). (GPx IV) is an enzyme also known as phosphatide hydroperoxide Glutathione peroxidase having the potential to reduce lipid peroxide into lipid alcohols (Imai and Nakagawa 2003). Glutathione disulphide (GSSG) is potentially toxic to almost all the cells but the cells normally have a protective mechanism in the form of Glutathione reductase which maintains most of the Glutathione in reduced form. Some of the Glutathione disulphide is also secreted from the cells. During oxidative stress, GS-SG could react with sulphydryl group of proteins by the disulfide exchange mechanism resulting in production of protein mixed disulphide which then can react with another sulphydryl group of protein to produced protein-disulphide (Huang and Huang, 2002). Normally the exchange reaction is very slow, unless catalyzed by an enzyme like protein disulfide isomerase (PSI) which is an important enzyme especially found in the endoplasmic reticulum, where proteins folding occur. G-SH/GS-SG is found to be relatively high in the cysternae of the endoplasmic reticulum. In cytosol the PS-SG formation is transient except during oxidative stress. The PSSG formation along with some enzymes may play a role in signal transduction although the exact mechanism of their formation is unclear. During the thiol exchange with protein, catalyzed by PDI, some proteins may have microenvironment in which thiolate (--S) which is much more reactive than Isa thiol in both the reaction with H2O2 or Disulphide exchange is formed. The microenvironment has required to be composed in part by basic amino acids in a close association with cysteine to allow dissociation of thiols having normally a PKa value of around (8.3). Glutathione- disulphide (GS-SG) production is catalyzed by GSH peroxidase, which forms a mixed disulphide by the ability of potentially with thiolate. But in the cytosol the G-SH/ GS-SG ratio remain very high even during oxidative stress, making that exchange reaction unfavorable. The rate of reaction can be enhanced by enzyme PDI but like any other it cannot change the position of, equilibrium. Instead it has been suggested that during

11

Chapter: 1

physiological signaling when H2O2 is used as a second messenger some of thiolates on the proteins have the potential to react and form sulfenic acid PSOH; however the rate non- enzymatic reaction is too slow to account for the inactivation of the enzyme by most of the thiolate , also including Glutathione (Forman, 2007). It is the active site of peroxiredoxins where the reaction between thiolate and H2O2 can occur 6 times faster than the reaction of Glutathione in its thiolate form itself. A protein sulfinate once form rapidly reacts with GSH resulting in the production of mixed disulfide. It may be one of the mechanisms of formation of PSSG for some proteins in the cytosol during oxidative stress when H2O2 is high enough to overcome a slow rate constant.

(2GSH + H2O2 GSSG + 2H2O

PSH + GSSG PSSG + GSH

But GSH/GSSG is very high in the cytosol

- PS + H2O2 PSSG + GSH

But the rate is very slow except for peroxiredoxins

PSO + GSH PSSG + OH-)

Equation: Formation of protein mixed disulfide.

Both Glutathione peroxidases and peroxiredoxin 6 can catalyze the oxidation of Glutathione by hydrogen peroxide to Glutathione disulfide and water. GSSG can then undergo an exchange reaction with protein sulfhydryl to form PSSG, which is usually catalyzed by a protein disulfide isomerase.

An alternate mechanism is the oxidation of a proteins thiolate to a sulfenic acid which then will react with GSH to form PSSG and water (Forman et al., 2009). b. Conjugation

GSH eliminates many xenobiotic from the body by a process of conjugation followed by excretion of adduct from the body (Boyland and Chasseaud 1969). GSH is the most abundant cellular non-protein thiol, function is, to help in the transport, metabolism and protection of the

12

Chapter: 1

cells against the toxicological impacts of various oxygen species and many other exogenous agents like heavy metals (Meister and Anderson 1983 & Dolphin 1989). GSH react non- enzymatically with Metals and results in the formation of an adduct with them (Ballatori 1994). Glutathione has six potential sites for the coordination with metals namely cysteinyl, glutamyl- amino, sulfhydryl, two peptide bonds, glycyl and glutamyl carboxylic groups. The sulfhydryl group is the most is the most reactive site for metal binding, particularly, cadmium, copper, zinc, silver, mercury, Arsenic and lead among all six metal binding sites (Wang and Ballatori, 1998). The interaction sulfhydryl group of Glutathione with heavy metals can be stabilized by coordination with other potential binding sites. Divalent cations have the ability to produce the most stable complexes in 1 to 2 ratios with Glutathione. As the reaction is thermodynamically stable so a spontaneous complexation takes place, resulting in a stable mercaptides. For these metals GSH complexes numerous metabolic functions have been proposed. (i) They have the ability to mobilize and metabolize the cations between the ligands. (ii) They have the ability to serve as metal transportation across membrane. (iii) They can source of cysteine; play a vital role in metal homeostasis. (iv)They also play also play a major role in the form of cofactor in various redox reactions, generating numerous speciation or biochemical forms of metal compounds (Wang and Ballatori, 1998 and Rannug et al., 1978, 1980 and Van Bladeren et al., 1981). GSH also play an important role in the metabolism and excretion of chromium, copper, and iron ions in various organisms (Lushchak, 2011 and Valko et al., 2005). GSH –dependent reduction of Cr result in the formation of less toxic anionic or cationic form of chromium (Cr) (Lushchak, 2011 and Coudray, 1992). Chromium in Cr+6 attached with oxygen in its anionic forms is readily transported into the cells with help of non-specific anion carriers , but Cr+3 cation is consider to be less toxic its interactions with various cellular ligands and thus pharmacokinetic ally less or not available. Therefore the reduction of Cr+6 into Cr+3 is considered to be a decrease in chromium toxicity (Nickens et al., 2010 and Velma et al., 2009). Chromium reduction by a non- enzymatic way also suggested that in the cells GSH and GSH-dependent enzymes either alone or in combination with cysteine or ascorbic acid play a central role in chromium detoxification (Valko et al., 2005; Gunaratnam et al., 2008 and Lushchak et al., 2008). One example is inhibition of GR by carmustine prevented Cr+6 reduction in isolated rat hepatocytes (Gunaratnam and Grant 2001). The non-toxic biological impacts of chromium are generally associated with biotransformation of Cr+6 by Glutathione. Although it is still unclear

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That how it takes place however the effect of chromium on the metabolism carbohydrates, Cr+6 reduction capacity of biological system in association with Glutathione may be used to deliver chromium into the biological system. GSH plays a more essential role and well documented central role in the metabolism copper, because it is that GSH help in the transportation and mobilization of copper ion for the biosynthesis of copper containing proteins (Wang and Ballatori, 1998). In this case GSH plays a key role Reduction of cu+ from Cu+2, transportation of copper from its stores and delivery of cu+ during the formation of mature proteins, which need copper in its cu+1 form before incorporation of copper into apoprotein (freedman et al., 1989). Interestingly GSH not only associated with Cu+ as a carrier but also but also involved in the mobilization of Cu+ from metallothionein in a reversible manner. Cu-GS complex is employed for the incorporation of copper into Cu-Zn superoxide dismutase (Cu-Zn-SOD) form bovine erythrocytes (Ciriolo et al., 1990), lobster apohemocynanin (Brouwer and Brouwer- Hoexum, 1992). And blood plasma albumin (Suzuki et al., 1989). c. Interaction of Glutathione with other Non-enzymatic antioxidant

Glutathione is consider to be the highly essential low molecular weight thiol present in the cells, however there are numerous other low molecular weights antioxidant present in diet such as vitamin-C and Vitamin-E .the lipid hydroxyl radical and lipid peroxidase produced from poly – unsaturated fatty acid can be reduced by Vitamin- E, the oxidized vitamin E is then reduced by Vitamin-c by a non-enzymatic rapid reaction. The oxidized Vitamin –C is then reduced by Glutathione in catalyzed by specific enzyme in the body.

1.3.5. Glutathione synthesis Glutathione Synthesis takes place in two steps in 1st step Glutamate combines with cysteine and produces C-glutamyl cysteine. The enzyme responsible for this step is called Glutamate- cysteine-ligase (GCL) or C-glutamylcysteine synthetase. In the first step an amide bond is formed between the amino group of cysteine and the C-carboxyl group of Glutamate (Huang et al., 1993).

In the next step Glycine is added to the dipeptide and thus Glutathione (GSH) is produced. This step takes place in the presence of an enzyme called Glutathione synthetase (Meister, 1974). GSH can transports out of cells. This transportation provides the opportunity of presence of GSH

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in the plasma coming from the hepatocytes. This GSH acts as a source of Cysteine for Glutathione synthesis in cells other than hepatocytes (Anderson et al., 1980). Plasma GSH concentration remains very low because of this cellular uptake (Sies and Graf, 1985 & Hirota et al., 1986). This process of cellular metabolism of Plasma GSH is regulated by two enzymes. First Glutamate is transferred to other amino acids by Enzyme C-glutamyl transpeptidase and to release cysteinylglycine. Then a dipeptidase can break the cysteinylglycine into Glycine and cysteine (Hirota et al., 1986 & Kozak and Tate, 1982). Then specific amino acid transporters moves Glycine and cysteine along with C-glutamyl amino acid into the cells where they are used in biosynthesis of Glutathione (GSH) (Meister, 1991). The enzyme Glutamate cysteine ligase (GCL) has two levels of regulation, first at the level of its activity and second at the level of expression of its two subunits. The activity of high molecular weight (73KDa) also known as Glutamate cysteine ligase catalytic subunit (GCLC), is inhibited by GSH (Huang et al., 1993), while this feedback inhibition by GSH is reduced by the low molecular weight (28KDa) also known as Glutamate cysteine ligase (GCLM) (Huang et al, 1993 and Choi et al,. 2000). GCLM subunit which is well known for its modulatory action may also show its effect on the steady state level of the Glutathione content in the cells if any change occurs in GCLM /GCLC expression (Richman and Meister, 1975; Choi et al., 2000 & Krzywanski et al., 2004). The increased expression of GCLM /GCLC may lead to high level of Glutathione while High level of GCLM /GCLC may further enhance the production of GSH. For Example in HIV Tat protein suppresses GCLM expression resulting in decreased GCLM/GCLC and thus decreased GSH (Choi et al., 2000). Similarly the phosphorylation of these subunits also regulates GCL’s kinetics (Sun et al., 1996). The regulation of GCL also takes place at many levels. The transcription of both the subunits of GCL can also be increased by electrophile and oxidant species (Shi et al., 1994a; Rahman et al., 1996; Tian et al., 1997 & Lu et al., 2009). Usually it is activation of signal transduction pathways that take part in controlling the transcription of GCLM and GCLC genes but some electrophile and oxidants may also involve in the stabilization of mRNA (Liu et al., 1998). It is well known for almost 20 years that sublethal concentration of electrophiles can enhance production of GSH (Ogino et al., 1989 and Darley –Usmar et al., 1991). However it is still unknown whether increased production is due to increase in the reduction of GSSG level or due to increase Kinetic or transcriptional level of GCLC subunit. But by using the technique of nuclear run analysis for measuring transcription level of GCLC and also using the redox cycling

15

Chapter: 1

analysis assay of quinone for increase production of H2O2, it is now evident that an increase production in GCLC level can generate a sustained increase in the level of GSH in the cells (Shi et al., 1994a). Similarly various other labs also provided the evidence that a variety of other substances can also cause oxidative stress by the generation of H2O2, elevated concentration of electrophiles and Nitric oxides that can affect GCLC or GCLM subunit or both (Rahman et al., 1996; Wild and Mulcahy., 1999 and Moellering et al., 1999). The substances that can affect the GCLC and GCLM subunits were first discovered in human and the in others animals like rodents (Gipp et al., 1995; Hudson and Kavanagh, 2000 and yang and Wang., 2001). There are some similar cis elements or promoters in both humans and rodents but they can produce different effect on regulation of genes in both humans and rodents (Iles and Liu 2005).

As for human GCL genes are concerned the two genes promoter enhancer regions contained several elements having the ability to respond to various electrophiles and oxidants (Gipp et al., 1995; yang et al., 2001 and Dickinson et al 2002). TRE (Transcriptional factor binding sites) elements bind sites also known as AP1 bonding site is one of the important oxidant responsive cis elements which regulate the GCL genes. TRE have the ability to bind the members of JUN and FOS family of transcription factor (Ofir et al., 1990 and Binetruy et al., 1991). Electrophile response elements (EpRE) are other important elements of GCL genes promoters in Human that responds to electrophile in the cells and enhances genes expression (Rushmore et 1991; Jaiswal 1994 and Vasiliou et al 1995). Human GCLM and GCLC promoters also comprise EpRE elements (Gipp et al., 1995). Initially EpRE was referred as antioxidant response element (ARE) because the first compound appeared that activate the ARE was a so called antioxidant shown, subsequently, to generate H2O2 through redox cycling (Pinkus et al., 1996). The EpRE elements have the ability to bind proteins members of small Maf family, Jun family, Nrf family (Venugopal and Jaiswal., 1998; Kong and Owuor, 2001; Moran at al., 2002 and Itoh et al 2004). Nrf2 is another transcription factor to establish having the ability to bind EpRE elements located in the cytosol through the inhibitory interaction with Keapl and resting cells. When Nrf2 stimulated at translocated into the nucleus after dissociation from the keapl (Itoh et al., 1991). While the redox and electrophilic responsive elements have been identified less has been done to identify the signaling mechanism that activates the transcription factors that binds to those elements. Darley –Usmar and his co-worker have identified that HNE directly modified Keapl

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Chapter: 1

which help Nrf2 to inhibit degradation and transferred into nucleus where it can bind to EpRE elements and the promoters of the GCLC and GCLM genes (Levonen et al., 2004). Different amino acids also regulate GSH biosynthesis among these amino acids cysteine is consider as the rate limiting factor (Lyons et al, 2000). As the cellular uptake of cysteine is enhanced by insulin and growth factors thus insulin and growth factors also play their role in GSH synthesis (Lu, 2000). Injection of Cysteine precursors is thus recommended in pathological conditions which results in GSH deficiency (Townsend et al., 2003).

Another amino acid that regulates biosynthesis of GSH is Glutamic acid. This amino acid exerts its action through two mechanisms it block the cysteine uptakes by cells when its concentration is high in extracellular fluids resulting in reduction of GSH synthesis (Tapiero et al., 2002). Similarly high intracellular glutamate level Block the feedback inhibitory activity of GSH resulting in enhancement of GSH synthesis (Griffith, 1999).

When GSH oxidation is triggered by Glucagon in the liver in that case glycine acts as a rate limiting factor in GSH synthesis (Mabrouk et al., 1998). In other conditions like burning and severe malnutrition glycine becomes the rate limiting factor, as well (Persaud et al 1996 and Yu et al., 2002). Dietary supplementation of glycine can improve GSH synthesis (Grimble et al., 1992).

1.3.6. Pharmacokinetics The pharmacokinetics of oral Glutathione (GSH) in humans is not well understood. It appears that in some animals (rats, mice, guinea pigs), Serum Glutathione (GSH) levels do increase following its oral administration. Most human studies of Glutathione (GSH) have not found this to be the case. It appears that oral Glutathione (GSH) is hydrolyzed in the intestine via the intestinal gamma-glutamyl transferase enzyme. A small amount of orally administered Glutathione (GSH) may reach the portal circulation, but apparently this is also rapidly metabolized by the gamma-glutamyltransferase. Thus most studies have not observed a significant increase in circulating Glutathione (GSH) following its oral administration. However, there is an occasional study that does show an increase in circulating Glutathione (GSH) after oral administration. Further, there is some evidence that Glutathione (GSH) may be observed a significant increase in circulating Glutathione (GSH) following its oral administration. However

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Chapter: 1

There is an occasional study that does show an increase in circulating Glutathione (GSH) may be absorbed into the enterocytes following ingestion, but may not be released by these cells into the circulation. Research is needed to resolve the issue of Glutathione GSH) absorption. Glutathione (GSH) metabolism and utilization The metabolism of GSH has been worked out to such an extent that cannot be shown herein: late Alton Meister and his co-worker’s publications have provided greater detail (Meister and Larsson, 1995 & Meister, 1995). Glutathione (GSH) status is homeostatically controlled, being continually self-adjusting with respect to balance between GSH synthesis (by GSH synthetase enzymes), It’s recycling from GSSG by (GSH reductase), and its utilization (by peroxidase, transferase, transhydrogenases and transpeptidases). The overall picture of GSH metabolism is summarized by way of the gamma-glutamyl cycle in figure1.4.

Figure 1.4 Schematic Diagram of Metabolism of Glutathione taken as a courtesy Meister,

Glutathione (GSH) synthesis occurs within cells in two closely linked, enzymatically controlled reactions that utilize ATP and draw non-essential amino acids as substrates. First cystein and glutamate are combined (by the enzyme gamma-glutamyl cysteinyl synthetase, as shown in reaction 1 in figure 1.4, with availability of Cysteine usually being the rate-limiting factor. Cysteine, produced from essential amino acid methionine, dietary protein degradation, or from turnover of endogenous proteins. The built up of GSH acts to feedback-inhibit this enzymes, thereby helping to ensure homeostatic control over GSH synthesis. The second GSH synthesis reaction combines gamma-glutamyl Cysteine with glycine to generate GSH (catalyzed by GSH synthetase reaction 2 in figure 1.4). Excessive accumulation of gamma-glutamylcysteine in the

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Chapter: 1

absence of its conversion to GSH can lead to its conversion to 5-oxoprolin by the enzyme gamma-glutamyl cyclotransferase (reaction 4 in figure 1.4) buildup of 5-oxoproline can have adverse consequences due to metabolic acidosis. The GSH pool is drawn on for 3 major applications: (a) as cofactor for the GSGS-transferases in the detoxicative pathways (reaction 7 in Figure.1.4); (b) as substrat for the gamma-glutamyl transpeptidases, enzymes which are located on the outer cell surface and which transfer the glutamine moiety from GSH to other amino acids for subsequent uptake into the cell ( reaction 3 in Figure.1.4); and (c) for direct free radical scavenging and as an antioxidant enzymes that conjugate GSH with fat soluble substances as the major feature of liver detoxification . For further details of the gamma- glutamyl cycle, the reader is referred to Meister (Meister, Larsson, 1995 and Meister, 1994) and Anderson (Anderson, 1997).

Figure 1.5: The Oxidation-reduction pathways of GSH

Is taken as a courtesy Anderson

Glutathione is an essential cofactor for antioxidant enzymes, namely the GSH peroxidases (both Se-dependent and non –Se-dependent forms exist) and the more recently described phospholipid hydroperoxidase GSH peroxidases (Zhang, 1989). The GSH peroxidase serve to detoxify peroxides (hydrogen peroxide, other peroxides) in the water phase, by reacting them with GSH; the latter enzymes use GSH to detoxify peroxides generated in the cell membranes and other lipophilic cell phases (cathcart, 1985). This is one instance of the water-soluble GSH providing collectively known as GSH transhydrogenase use GSH as a cofactor to reconvert dehydroascorbate to ascorbate, ribo nucleotides to deoxyribonucleotides, and for the variety of S- S-> -SH inter conversion (figure 1.5). After GSH has been oxidized to GSSG, the recycling of GSSG to GSH is accomplished mainly by the enzymes Glutathione (GSH) reductase. This enzyme uses as its source of electrons the coenzymes NADPH nicotinamide adenine

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Chapter: 1

dinucleotide phosphate, reduced). Therefore NADPH, coming mainly from the pentose phosphate shunt, is the predominant source of GSH reducing power. Cathcart used this to explain why subjects unable to make adequate NADPH may be at increased risk of Oxidative Damage from GSH insufficiency (Cathcart, 1985). Through its significant reducing power, GSH also makes major contributions to the recycling of other antioxidants that have become oxidized. As shown in Figure 1.6.

Figure 1.6: Recycling of GSH

This picture is taken as courtesy (Cathcart)

This could be the basis by which GSH helps to conserve lipid-phase anti-oxidants such as alpha- tocopherol (vitamin E), and perhaps also the carotenoids. Meister and his group used buthionine sulfoximine (BSO) to inhibit GSH synthesis in rodents, and concluded from their findings that GSH almost certainly plays such a role in vivo (Meister, 1994; Meister 1995). The liver seems to have two pools of GSH; one has a fast turnover (half-life of 2-4 hours), while the other is avidly retained with a half-life of about 30 hours (Meister, 1995). The first corresponds to Cytosolic GSH, the second mainly to mitochondrial GSH which is more tightly held. Though this pool represents a minor portion of the total GSH, the mitochondria are normally under high oxidative stress (Richter et al., 1995) and thus conserving their GSH. With regard to the essentiality of GSH for the survival of the whole organism, substantial information is available from studies on hereditary GSH depletion in the human, and from experimental depletion and repletion of GSH in animal models and cells cultures (Meister and Larson, 1995 and Beutler, 1989). Inherited deficiency of the enzymes gamma-glutamyl Cysteine synthetase, the first of the two enzymes

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Chapter: 1

necessary for GSH synthesis, has been described in two human siblings. They exhibited generalized GSH deficiency, hemolytic anemia, spincerebellar degeneration, peripheral neuropathy, myopathy, and aminoaciduria, and serve neurological complications as they moved into their fourth decade of life (Meister and Larson, 1995). Their red cell GSH was less than 3% of normal, their muscle GSH less than 25%, and their white cells GSH less than 50% normal. One of them may have hypersensitive to antibiotics, having developed psychosis after a single dose of sulfonamide for a urinary tract infection. Deficiency in GSH synthetase the second enzyme of GSH synthesis also is associated with hemolytic tendency and defective central nervous system function. This condition is complicated by the metabolic consequences of an excess of 5-oxoproline , formed as a “spillover” from the accumulation of gamma-glutamyl Cysteine after its normal synthesis by the first enzymes and its lack of conversion to GSH by the second enzyme ( Meister and Larson,1995 and Beutler, 1989). Human hereditary GSH deficiency states are not necessarily lethal, probably because some GSH is obtained directly from the diet. Mister’s group set dietary GSH at zero for their experimental animals, and simultaneously blocked endogenous GSH synthesis (at the first step, using buthionine sulfoximine) (Anderson, 1997). They observed that GSH levels decreased in the plasma, liver, kidney and other tissues of these animals; in guinea pigs and newborn rat’s death ensued in few days. At the cell level, the damage mostly involved the mitochondria, but nuclear changes were also observed. Lung Type 2 cells showed damage to their lamellar bodies, the vesicles that package lung surfactant and release it to the cell exterior. This damage from GSH depletion could be ameliorated by simultaneously administering precursors of GSH; thus the cataracts newborn rats were blocked using orally administering precursors of GSH monoesters. Meister, Anderson, and collaborators reasonably assumed that the damage produced in their test animals from inhibition of GSH synthesis was endogenous, since they had not applied any exogenous sources of oxidative challenge. The mitochondria appeared to be the most susceptible foci in the GSH –depleted tissues. This finding was consistent with the mitochondria assuming the bulk of the endogenous oxygen radical burden, yet being unable to make their own GSH; they must import it from the cell cytosol. The investigators found that dietary ascorbate can protect against the tissue damage that typically results from depletion of GSH (Anderson, 1997), in animals such as adult rats and mice that are able to make adequate ascorbate on their own, GSH depletion was not lethal. By contrast, in those animals that could not make their own ascorbate (newborn rats, guinea pigs), GSH depletion was lethal. Supplementation of the diet with ascorbat protected these animals against GSH depletion and saved their lives. Interestingly, this story has a

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“flipside” guinea pigs placed on an ascorbate deficient diet was salvaged by dietary administration of GSH and its precursors (Meister, 1995). Thus these two water phase antioxidants are tightly linked: GSH can conserve ascorbate in vivo, and ascorbate can converse GSH (Winkler et al., 1994).

1.3.7. Measurement of Glutathione Glutathione can be measured by the following ways

1. GSH can react with Di-thiobis-nitro benzoic acid (DTNB) Resulting in the formation GS-TNB conjugate and a free TNB anion which can be measured spectrophotometrically as it give absorbance at 412 nm in UV-visible region (Akerboom and Sies, 1981).

2. The total Glutathione can also be measured by enzyme recycling assay method in which the rate of TNB production is measured which is proportional to the initial concentration of Glutathione. Glutathione is first react with DTNB and GS-TNB complex is produced which then can further react with GSH and further TNB and GSSG are produced, the GSSG is then reduced to GSH by an enzyme Glutathione reductase and thus Glutathione is measured ( Forman et al.,2009).

3. Other method for measuring GSH include well-known HPLC method, Nitroso Glutathione reaction with orthophthaldehyde (OPT)) to produced fluorescent compounds which are then measured spectrophotometrically and also by GSNO labeling by N-15 and measuring through mass spectroscopy (Fariss and Reed, 1987; Tsikas et al., 1991; Gladwin et al., 2006 and kluge et al., 1997).

1.4. N- Acetyl cysteine (NAC) 1.4.1. Introduction It is the acetylated derivative of the amino acid L-cysteine. It has been used in respiratory diseases as a mucolytic agent and acetaminophen antidote in Hepatic diseases. Studies have established that NAC is a powerful anti-oxidant and have the potential to be used in treatment of different ailments like HIV, cancer, metal toxicity and Heart diseases. Moreover it can also be used in conditions of oxidative depression like hepatitis c influenza myoclonus epilepsy and Sjogren’s syndrome.

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1.4.2. Biochemistry and Pharmacokinetics

NAC contains sulfhydryl group. Its absorption is rapid into various tissues after oral administration. It is d-acetylated and metabolized in intestine and liver. Its metabolites bind with peptides and proteins. Its peak plasma level is reached in about one hour. Its bioavailability is only 4% to 10 % after overall intake which is however considered clinically effective (Borgstrom et al., 1986). The sulfhydryl group of NAC is actually responsible for the biological activity. The acetyl substituted amino group protects NAC from metabolic and oxidative degradation (Bonanomi and Gazzaniga, 1980 and Sjodin et al., 1989).

Mechanism of Action

NAC effectively reduces cystine in the extra cellular fluid to cysteine. It also acts as a source of sulfhydryl group and stimulates Glutathione synthesis. It increases the activity of Glutathione transferase. It inhibits biotransformation of xenobiotics and thus enhances liver detoxification. NAC is a nucleophile and captures free radicals (Vries and Flora et al., 1993 and Flora et al., 1985). Its Sulfhydryl group interacts with disulphide bond of mucoproteins and thus act as a mucolytic agent. It stimulates the gastro-pulmonary vagal reflex, clears the airways for the mucus and thus exerts its expectorant action (Zimet, 1988). It dissociates disulfide bonds lowering lipoproteins and homocysteine, thus, is considered beneficial in heart diseases (Gavish and Breslow, 1996; Wiklund et al., 1996). It refinishes Glutathione redox system thus provide protection against reperfusion damage and ischemia (Horowitz and Henry, 1988).

1.4.3. Clinical indications a. Respiratory Illness

Studies have shown that NAC decreases cough severity and acts as an expectorant (Jackson et al., 1984). It decreases Diaphragm fatigue (Hida et al., 1996). In a study the subjects were suffering from alveolitis, a condition characterized by low level of Glutathione. They were given 600mg NAC three times daily for twelve weeks which improved their pulmonary function (Behr et al., 1997). Some further studies also revealed that exacerbation in patients with chronic bronchitis were decreased after NAC administration (Gotz et al., 1980).

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b. HIV Infection

NAC triggers colony formation of T-cells and enhances immunity (Wu et al., 1989). It also increases the lymphocytes cell counts and has a positive impact on the level of cysteine in plasma (Akerlund et al., 1996).

c. Cancer / Chemoprevention

NAC is potentially capable to treat certain cancer conditions like skin, nick, lung, liver and breast cancers (Flora De et al., 1992).

In vitro studies have confirmed the anti-carcinogenic and anti-mutagenic effect of NAC (Flora De et al., 1986). It protects Normal cells from radiation and chemotherapy, selectively (Flora De et al., 1996). Some other studies have suggested that NAC inhibit proliferation and cell growth and astrocytoma, melanoma and prostate cancer (Chiao et al., 2000; Redondo et al., 2000 and Arora- kuruganti et al., 1996).

NAC has been used as Acetaminophen antidote historically. Metabolites of acetaminophen are hepatotoxic, decreases hepatic Glutathione and can cause death. This condition of acetaminophen toxicity can be smoothly reversed if NAC is administered within 24 hours (Smilkstein et al., 1988; Wang et al., 1997 and Perry and Shannon, 1998).

d. Viral Hepatitis

The interferon alpha (IFN) is used in the treatment of Hepatitis C but in cases of IFN resistant NAC supplementation can be used to improve the IFN therapy. This activity of NAC is because it normalizes serum alanine aminotransferase (Beloqui et al., 1993 and Neri et al., 2000).

e. Heart Diseases

Studies have shown that NAC reduces plasma lipoproteins and homocysteine levels (Wiklund et al., 1996). It increases tissue GSH level and thus can be useful in treatment of reperfusion and ischemia in acute myocardial infection. It also potentiates the anti-platelet and dilating properties of nitroglycerine. In cases of myocardial infarction and unstable angina pectoris, NAC may be useful as a combination therapy (Winniford et al., 1986).

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1.5. D-penicillamine (DPA) The chemical name of DPA is ß-ß- dimethylcysteine or 3-mercapto-D-Valine. It is an amino acid containing degradation product of penicillin and sulfhydryl group. Its D-isomer is useful while the L-Isomer can cause optic neuritis. DPA chelates heavy metals and decreases their toxicity (Roussaeux and MacNabb, 1992).

1.5.1. Pharmacological profile DPA can be administered through intravenous and oral route. Its absorption from Gastro intestinal tract is sufficient. It is distributed into extracellular fluid after oral administration within 1to 4 hours; its peak plasma concentration is reached. Some of its fraction is metabolized to Disulfide in liver but most of it excretes unchanged in urine (Gupta et al., 1980). Its elimination half-life is 1 to 7 hours. Although use of DPA is mostly safe however its chronic use may cause nausea vomiting and anorexia (Grasedyck, 1988).

DPA may stimulate the Histaminergic receptors and thus have being found ulcerogenic in rates (Gupta et al., 1980). DPA is contraindicated in patients having allergy to penicillin and also in case of renal insufficiency. It is also contraindicated in combination with cytotoxic drugs antimalarial and phenyl butazone (Gupta et al., 1980).

1.5.2. Biological role of D-penicillamine a. In Wilson’s diseases

A hereditary disease characterized by accumulation of Cu in different organs is called Wilson’s diseases (Gaffney et al., 2000). D-penicillamine chelates and removes the copper out of the body of the patient

b. In Metal toxicity

D-penicillamine can be used in treatment of the toxicity of different metals like Gold, Lead, Copper and Arsenic (Master, 2008).

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c. In Prevention of retinopathy

Retinopathy, a retinal vascular disorder, common in premature infants, can be prevented by D- penicillamine (Palmer et al., 1991).

d. In scleroderma

It is a condition of harden skin and may also affect other organs. It has been proven that D- penicillamine can effectively treat scleroderma (Arcangelo et al., 2006).

e. In rheumatoid arthritis

D-penicillamine is effective in the treatment of rheumatoid arthritis (Brewer et al., 2005).

f. In cystinuria

D-penicillamine, like other thiols can be used in treatment of cystinuria (Dahlberg et al., 1997).

1.6. Albumin 1.6.1. Introduction Albumin refers to any protein that is water soluble, moderately soluble in salts solutions and undergoes heat denaturation. They are found in plasma and do not glycosylate. This property makes them different from other drug proteins. Substance, for example egg white, containing albumin are known as albuminoids (Schoentgen et al., 1986 and Lichenstein et al., 1994).

1.6.2. Medical uses Albumin has the potentials to be given to patient with low blood volume. However its medical uses has not been fully recommended and requires clinical trials (Cochrane Database 2011).

Functions

It is an important binding protein of the plasma and can binds different cations, hormones, fatty acids, bilirubin drugs and water. It regulates the blood’s colloidal osmatic pressure (He and Carter, 1992).

1.7.1. Vanadium In the periodic table Vanadium belongs to group V-B (Morinville et al., 1998). A Swedish Nils Sefstrom discovered Vanadium in 1930. It is soft, ductile and Bright sliver white metal

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Chapter: 1

(Almedeida et al., 2001). It resists corrosion, strong acids and alkalis (Holleman et al., 1985). It has been discovered that Vanadium is present as an essential element in certain marine species. This discovery has increased interest of scientists to study the nutritive role of Vanadium (Almedeida, et al., 2001). The major source of Human exposer to Vanadium is the food; though most foods contain Vanadium in minute’s quantities (Barceloux, 1999). Vanadium is present in high quantity in foods like shellfish, mushrooms, black pepper parsley and dill seed etc. (Barceloux, 1999).

1.7.2. Chemistry and compounds The most common oxidation states of Vanadium are +2, +3, +4 and +5. Compounds of Vanadium (II) acts as reducing agents while that of Vanadium (V) are oxidizing agents. Similarly compounds of Vanadium IV occur as Vanadyl derivatives containing VO2+ (Holleman et al., 1985). Different colours of Vanadium in these common oxidation states can be obtained by reducing Ammonium Vanadate (NH4VO3) with Zinc (Holleman et al., 1985). Vanadium pentoxide is commercially the most important Vanadium compound that is used as catalyst in the production of Sulfuric acid (Holleman et al., 1985). Vanadium pentoxide is reduced from +5 to

+3 and oxidizes sulfur from +4 to +6 and converts Sulfur dioxide (SO2) to sulfur trioxide (SO3). Vanadium can react with hydrogen peroxide to produced different Per-Oxo-complexes. For 2+ + example Oxo-Vanadium (V) ions (VO ) produces Per-Oxo-Vanadium (V) ions [VO (O2)2 ] if treated with hydrogen peroxide (Strukul and Giorgio, 1992). Vanadium also exist in different halides with oxidation states +2, +3 and +4, among which VCl4 is commercially important (Greenwood et al., 1997).

1.7.3. Natural occurrence of Vanadium Vanadium does not exist in free metallic form in nature; however it is found in upto 65 various minerals. Some of its minerals that have economic significance include Vanadinite (Pb5

(VO4)3Cl), patronite (VS4) and carnotite (K2 (UO2)2(VO4)2·3H2O). The most important source of Vanadium production is its magnetite. Mostly, Vanadium is mined in China, South Africa and Russia (Magyar & Michael, 2011).

Another source of Vanadium is its Bauxite, found in coal, crude oil, tar sand and oil shale. The oil, containing Vanadium, when burnt, can cause corrosion in boilers and motors (Pearson and Green, 1993). It is estimated that burning of the fossil fuels emit about 110,000 tonnes of Vanadium to the atmosphere, yearly (Anke and Manfred: 2004). 27

Chapter: 1

1.7.4. Biochemistry Vanadium has been listed as essential element in some animals (Sakurai et al., 2006). For instance, its deficiency in chickens may adversely affect their feathers, bones and blood. However, it is yet to decide, whether Vanadium is an essential nutrient in human (Sakurai et al., 2006).

Some species of mushroom, e.g. Amanita muscaria contain a natural Vanadium compound called Vanadium amavadine dipropionic acid. It is considered that this compound act as mediator in the oxidation of thiols with esters or carboxylic acid (Sakurai et al., 2006).

It is estimated that 5% of the ingested Vanadium is absorbed from the gastrointestinal tract (GIT). It is converted to vanadyl cations in vivo. The highest amounts of Vanadium is found in kidney, liver and bones (Jellin et al., 2006).

1.7.5. Pharmacokinetics Vanadium is poorly absorbed from the GIT (Nriagu, 1998 and Poucheret et al., 1998). Before its absorption in the small intestine, most of the Vanadium is converted to its vanadyl cations in the stomach (Hirano and Suzuki, 1996).

However, anionic Vanadate form of Vanadium absorbs more than the Vanadyl form (Hirano and Suzuki, 1996). The vanadyl form of Vanadium spontaneously oxidizes to Vanadate form in vivo (Li et al., 1996). In blood, most of the vanadate reduces back to vanadyl and both these forms are transported by the blood protein into various tissues of the body (Fantus et al., 1995). Vanadium concentrates in different tissues, including bone, liver, kidney and spleen, if taken chronically (Hamel and Duckworth, 1995 & Ramandham et al., 1991). Excretion of Vanadium takes place through urine and bile. But the major route of elimination is the bile through feces (Alimonti et al., 2000).

1.7.6. Biological role of Vanadium Vanadium is required for the action of bromoperoxidase in the production of organobromine compounds in a number of marine algae (Butler et al., 2004). Studies have shown that Vanadium has very limited role in the biology of land species. However Vanadium is found to have essential role in marine species. For example Vanadium is present as Vanabin in bluebell Tunicate and in Sea Squirts (Ascidiacea), and as Amavadin in Amanita Muscaria (Henze, 1911and Michibata et al., 2002). Certain microorganism e.g. Azotobacter uses Vanadium

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Nitrogenase in Nitrogen fixing (Robson et al., 1986). Vanadium is also essential in small amount for growth and reproduction in rats and chicken (Schwarz et al., 1971). Vanadyl sulfate is considered to improve insulin sensitivity in cases of diabetes, the finding is however controversial (Goldwaser et al., 1999 and Goldfine et al., 2000). Similarly Vanadium is considered to enhance efficiency of athletes.

However this is not proven, as well (Talbott et al., 2007). Certain forms of Vanadium like Oxovanadates and Decavanadate are considered to be biologically active in a number of biochemical processes (Aureliano et al., 2009).

a. Impact of Vanadium deficiency in higher animals

Vanadium deficiency results in decreased milk production and abortion in goats (Badmaev et al., 1999). Such a deficiency in goats may also alter the biochemistry in a number of ways, like decreasing the level of lactate dehydrogenase, beta-lipoproteins, and isocitrate dehydrogenase and serium creatinine. Vanadium deficiency can also cause tarsal joints, swollen forehead, deform forelegs and altered thyroid metabolism (Badmaev et al., 1999). Vanadium deficiency may cause a number of biological impairment in species like chicken and rats and may lead to disturbances in lipid and glucose metabolism in certain other higher animals (Nakai et al., 1995 and Nriagu, 1998).

b. Vanadium in Genetic modulation

It is reported that Vanadium compounds activates several genes. It increases levels of macrophages and also the binding activity of NF-kB (Chong et al., 2000a). It is also known that Vanadate induces interlukin-8, tumor necrosis factor-alpha and activator protein-1 gene expression (Ding et al., 1999; Jaspers et al., 1999 and Ye et al., 1999). Compounds of Vanadium also increase the levels of C-raf-1, P70s6k, ras and MAPK in insulin receptor over-expressing cells (Pandey et al., 1999).

1.7.7. Pharmacological and therapeutic importance of Vanadium Vanadium is used to decrease the level of glucose and cholesterol. It has natriuretic, diuretic and anti-carcinogenic properties. It contracts blood vessels and improves the oxygen affinity of hemoglobin and myoglobin (Poucheret et al., 1998; Rehder, 1992 and Thompson et al., 1993). In

29

Chapter: 1

a study it has been shown that Vanadyl sulfate has caused a decreased in blood pressure of rates (Bhanot and McNeill, 1994). Oxo-Vanadium has the potential to be used in obesity as it decreases appetite and thus body weight (Wilsky et al., 2001 and Wang et al., 2001). Certain enzymes contain Vanadium (Almedeida et al., 2001 and Badmaev et al., 1991). Such enzymes can perform activities of haloperoxidase, nitrogenase and bromoperoxidase. Vanadium sulfate is indicated as muscle builder in athletes (Clarkson and Rawson, 1999 & Fawcett et al., 1997). Reportedly, Vanadium compounds induce calcium signaling in T-lymphocytes and basophils in rates (Ehring et al., 2000). These compounds are also reported to induce NO synthase improving the level of nitrate in mice blood (Matte et al., 2000). Vanadyl reduces the chances of urinary stones and off renal and testicular tumors in rats (Alimonti et al., 2000; Shi et al., 1996). Vanadium compounds can normalize blood level of glucose in diabetic patients (Cam et al., 2000). It is suggested that Vanadium provides insulin mimetic effect through an unknown complicated pathway (Pandey et al., 1999). It has been shown that Vanadium compounds have anti-cancerous effect against liver tumor in rats (Bishayee and Chatterjee, 1995a). Vanadium compound have also been reported to inhibit the growth of tumor cells in human (Hanauske et al., 1987).

1.7.8. Toxicological profile of Vanadium compounds Vanadium compounds decrease the activity of different enzymes e.g. ATPases, ribonuclease, protein kinases and phosphatases (Sabbioni et al., 1991; Bollen et al., 1990; Lau et al., 1993 and Tracey, 2000). They also induce mutagenic and genotoxic effect through causing alteration in the activity of DNA or RNA enzymes (Stemmler and Burrows, 2001). The levels of LD50 in mice have been established to be 0.2 to 0.3 mmol /kg (Venugopal and Luckey, 1978). A study has demonstrated that a dose of 50mg twice a day for four weeks in humans was well tolerated (Boden et al., 1996). Certain other studies have also confirmed the similar findings (Dai et al., 1994 and Fawcett et al., 1997). Vanadium scientists suggest that this is due to the poor absorption and rapid proteins binding of Vanadium, if absorbed (Hirano and Suzuki, 1996 and Wilsky et al., 2001).

Complexes of Glutathione and related thiols

Reduced GSH is apparently seemed to have a primary role in metabolism of Vanadium. It is proven that GSH stabilizes and transport the VO+2, formed as a result of reduction of Vanadate by GSH in cells (Baran, 1997 and Costa et al., 2002). Another agent that reduces vanadate is L-

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Chapter: 1

cysteine. Studies shows that reduction of Vanadate in VO3-/cysteine system is rapid (Sakurai, 1981). Similarly interaction of Oxo-Vanadium with cysteine has also been confirmed (Ferrer and Williams, 1998).

a. Oxo-Vanadium (IV) complexes of L-Ascorbic Acid and of its Oxidation products

Another potential reducing agent for Oxo-Vanadium as well as vanadates is the l- ascorbic acid (Vitamin-C). The acid and its oxidation products can interact with the reduced species (Baran, 2000 and Ferrer et al., 1998, 2001). A number of complexes are produced as a result of interaction between VO2+ cation and ascorbic acid. Similarly the VO2+ may also interact with certain species of the acid generated after its oxidation (Ferrer et al., 1998). For example VO2+ interacts rapidly with de-hydroascorbic acid and produces 2:1 ligand-to-metal complex VO2+/ de-hydroascorbate complex (Ferrer et al., 1998).

b. Transferrin and serum albumin complexes

The +3, +4 and +5 oxidation states of Vanadium tightly bind with transferrin (Rehder and Angew, 1991; Butler and Carrano, 1991 and Chasteen et al., 1995). As a result of this Vanadium modified transferrin are formed which helps in transport of Vanadium (Cantley et al., 1978). Studies show that Vanadium (V) binds reversibly at two binding sites with serum transferrin in human (Harris and Carrano 1984). A detailed study has confirmed that VO2+ binds with serum albumin in bovine at a binding site specific for copper (CuII) (Purcell et al., 2001).

1.8.1. Palladium Palladium is a transition metal. Its atomic mass is 106.42. Its atomic number is 46. Its colour is steel-white and it occurs in earth’s crust in a low concentration (Degussa, 1995; Kothny, 1979; Aldrich, 1996 and Kroschwitz, 1996). The alloys of Palladium are extensively used in dentistry (Stumke, 1992; Zinke, 1992 and Daunderer, 1993). Palladium often occurs as insoluble metal or its oxides and resists chemical reactions caused by radiations or oxidative radicals (Renner and Schmuckler, 1991). Palladium is a platinum group metal (PGM) of the earth crust and is assumed to find with humic acid, peptides or with fulvic acid in marine environment (Morgan and Stumm, 1991; Fishbein, 1976 and Johnson et al., 1976).

31

Chapter: 1

1.8.2. Natural Occurrence and Environmental level Palladium like other PGMs is in small concentration in the environment. Palladium makes 20% of the total fraction of PGMs (Renner, 1992 & Renner and Schmuckler, 1991). Concentration of Palladium is considered to be in the range of 19 to 70pg/l in salt water and 0.4 to 22ng/l in fresh water (Eller et 1989 and Shah and Wai, 1985). Reports suggest that its concentration is up to 47µg/kg in soil, 18 to 260µg/kg in sewage sludge (Lottermoser, 1995). Palladium does not occur, normally, in drinking water or may occur only in ng/l, thereof (Johnson et al., 1976). Palladium may occur in food, like fishes, aquatic invertebrates, meet, bread and certain plants, in very small quantities (Yang, 1989; Whyte and Boutillier, 1991). The chances of human exposer to Palladium are generally through jewellery, dental alloys, food and emission from automobile converters (Wataha et al., 1991a). Exposer of gums to Palladium in dental alloys is the most important one. Similarly skin may expose to Palladium containing jewellery (Wirz et al., 1993). Workers in Palladium refineries may also exposed to Palladium directly and there are reports about manifestation of 0.006µg/l Palladium in their urine (Johnson et al., 1975b, 1976). Palladium, usually, accumulates in leaves and roots of marine plants (Farago and parsons, 1994). Water hyacinth contains 133µg/kg while leaves of marine plants contain 785µg/kg of Palladium (Abbasi, 1987).

1.8.3. Palladium Compounds There are hundreds of Palladium compounds that contain Palladium in different oxidation states, however only a few of them are important economically. Generally Palladium occurs in its +2 oxidation state in these compounds. Compounds containing Palladium in +4 oxidation state are only few and less stable. Palladium has a characteristic to form coordination complexes and a number of organic complexes (Kroschwitz, 1996).

Uses of Important Palladium compounds

Amine complexes of Palladium have their importance in industry for Palladium production. Acetate of Palladium is an important chemical catalyst (Budavari et al., 1996). Palladium chloride is used in parts of watches and clocks, photography, manufacture of ink, detection of leaks of carbon monoxide and in preparation of metal intended to be used as catalyst (Budavari et al., 1996). Nitrate of Palladium is used in separation of iodine and chlorine and also as a catalyst in synthesis reactions (Budavari et al., 1996). Hydrogen tetrachloropalladate is generally used in preparation of Palladium catalysts and other Palladium compounds (Renner, 1992).

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Chapter: 1

Another Palladium compound the tetra-ammine Palladium hydrogen carbonate is used in preparations of automobile catalysts (Johnson Matthey, 2000).

1.8.4. Pharmacokinetics Data on pharmacokinetic of Palladium is not very established. A finding has shown that Palladium dust, which is insoluble in water, dissolves appreciably in biological media (Roshchin et al., 1984). Palladium was also found to be soluble in aqueous solution containing biogenic compounds (Freiesleben et al., 1993). Absorption of Palladium compound from GIT is poor (Moore et al., 1974, 1975). Absorption of Palladium given through intra venous or through inhalation to rats was founds higher (Moore et al., 1974, 1975). In several laboratory animals like rabbits, dogs and rats it was found that intravenous Palladium concentrates in liver, kidney ,lymph nodes spleen, lungs, adrenal glands and bones (Moore et al., 1974, 1975 and Kolpakov et al., 1980). It was observed that after intravenous administration of Palladium compounds to rats small amounts of Palladium transfers through placenta and milk as (Moore et al., 1974). Reports show that Palladium forms stable organometalic compounds in organisms (Wood et al., 1978). In a study about 3400ug/kg of Palladium was found in Bladder Papilloma of a patient who had exposed chronically to Palladium and dental alloys (Daunderer, 1993). Experiments aiming to know about the elimination of Palladium have shown that Palladium is eliminated in both urine and faeces (Moore et al., 1974). In a study the elimination of 95% of orally administered Palladium through faeces in rats was actually manifesting the poor absorption of Palladium from the digestive tract (Moore et al., 1974). Palladium was found to be excreted in urine of rabbits and guinea pigs (Roshchin et al., 1984; Taubler, 1977). In another study on rats it was found that the half-life of Palladium is 5-12 days (Estler, 1992).

Palladium Complexes with Biological Molecules

Palladium ions bind to different acids like L-cysteine, L-cystine, L-Methionine (Akerfeldt and Lovgren, 1964). It also forms complexes with proteins like silk fibroin, casein and many enzymes (Spikes and Dodgson, 1969). In a study it was found that Palladium too binds with metallothionein of rat liver (Nielson et al., 1985). Palladium also forms complexes with DNA and other macromolecules like Vitamin B 6 (Jain et al., 1994; Matilla et al., 1994).

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Chapter: 1

1.8.5. Therapeutic Uses

Palladium compounds suppress virus entry, cell to cell spread, capsid assembly and transactivation of virus genome and thus show antiviral activity (Petia et al., 2004 & Genova et al., 2004). Palladium compounds can also act as a neuroprotective agent in cases of cardiac arrest, ischemic attack, drowning or anesthetic accidents (Francis et al., 2004). In case of human breast cancer complexes of Palladium have shown a better cytotoxic effect (Engin et al., 2011). Similarly Palladium (II) complexes of 2-Acetylpyridin and 2-formylpyridin have shown excellent antitumor activity (Matesanz et al., 1999). Several other compounds of Palladium have antibacterial, antiviral and /or fungicidal activity (Graham and Williams, 1979). Palladium hydroxide has been used in the treatment of obesity (Kauffman, 1913). Palladium chloride can be used safely for cosmetic tattooing and as a topical germicide (Meek et al., 1943).

1.8.6. Toxicity of Palladium

a. Toxicity in laboratory animals

Toxicity of Palladium varies from its compound to compound and also along with the route of administration (Wiester, 1975). Palladium administered through oral route is less toxic was found that Palladium produces acute toxic manifestations like anorexia, convulsions, peritonitis, cardiovascular effects, biochemical changes and even death (Orestano, 1993 and Wiester, 1975). If taken orally, Palladium can change the histology and physiology of kidney, liver, spleen and gastric mucosa of rats. Such changes results in haematoma, weight loss and anemia (Johnson Matthey, 1997b and Moore et al., 1975). In a study it was found that Palladium compounds causes alterations in parameters of urine and serum which are indicative for kidney and liver damage (Roshchin et al., 1984). Exposure of rates to Palladium dust through inhalation has been observed to cause lungs inflammation and alteration in the biochemistry of urine and serum (Roshchin et al., 1984). In a number of tests it was found that Palladium compounds cause various dermal reactions in rabbits (Campbell et al., 1975 and Johnson Matthey, 1995b). Palladium compounds cause eye irritation (Johnson Matthey, 1995b). Palladium compounds also affect reproductive and developmental processes in mice (Fisher et al., 1975). Palladium salts inhibit several enzymes like creatine kinase (Liu et al., 1979b).

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Chapter: 1

b. Toxicity in Human

Studies show that Palladium containing dental alloys causes clinical symptoms in oral mucosa. Similarly some others effects of Palladium in humans include haemolysis, fever, necrosis at injection site, oedema and erythema (Marcusson, 1996; Richter and Geier, 1996; Schaffran et al., 1999). Palladium can cause allergic reactions in humans (Kranke et al., 1995).

1.9.1. Scientific objectives Interaction of metalloelements and their complexes with thiols as a biomarker of detoxification is receiving clinical interest. The study performed to determine the effect of selected metalloelements on the chemical status of thiols, under different parameters, can encompass a variety of objectives. However we focused on some specific scientific objectives of this study, so as to ensure the relevancy of this piece of work with the intended objectives.

The major scientific objectives are mentioned below

1. Quantification of thiols in presence of the selected metalloelements is useful to guide the biochemical scientists about the value of such metalloelements for pharmaceutical purposes.

2. Such studies about the interaction of metalloelements with biothiols are helpful to explore the protective role of thiols against the selected metalloelements.

3. Thiols conjugation reactions have gained importance in metabolism and excretion of toxic metalloelements. In this context this study is beneficial to determine the role of thiols in the biotransformation of salts and complexes of Vanadium and Palladium.

4. This study will also elaborate the usefulness of simple, modified and inexpensive spectrophotometric method determination of toxicity of the selected metalloelements.

5. The study will provide a base for the exploration of different Pharmaco-therapeutic parameters of the selected metalloelements, if they were intended to be used for clinical purposes

35

Chapter: 2

2.1. Introduction Oxidative stress leads to oxidative damage by reactive oxygen species (ROS) and is related with pathological processes such as atherosclerosis, cancer, and neurodegenerative disorders (Moran et al., 2001). Due to their capacity of being easily oxidized, sulfhydryl groups are vulnerable to oxidative stress. This impairs the sulfhydryl-disulfide balance which is a key player in redox- sensitive processes (Winterboum & Hampton, 2008). Sulfhydryl groups occur as non-protein compounds (Glutathione and free cysteine) and, in protein such as thioredoxins, glutaredoxine and albumin, which is the chief protein constituent of blood plasma (Moran et al., 2001, Winterboum & Hampton, 2008 and Biswas et al., 2006). The total sulfhydryl group (TSH) content of a biological sample is a valuable indicator of oxidative stress and of oxidative protein damage, Quantification of protein thiols relies on use of thiol-reactive reagents, such as 5,5’- dithiobis (2-nitrobenzoic acid) (DTNB, Ellman’s Reagent) (Ying et al, 2007). DTNB is the most commonly used reagent for the measurement of total sulfhydryl groups spectrophotometrically due to its simplicity and the validity of obtained results (Elman’s, 1959). Reaction between sulfhydryl groups and DTNB produces a mixed disulfide and a yellow colored thiolate, 5-thio-2- nitrobenzoic acid (TNB) with maximal absorbance at 412nm (Elman’s, 1959; Hu, 1994). We have applied the DTNB-based assay to transition inorganic/organometalic complexes of Palladium and Vanadium format. This permits to study with reduced amount of sample and reagents with satisfactory results in short period of time. Transition metals act as catalysts in the oxidative deterioration of biological macromolecules, and therefore, the toxicities associated with these metals may be due at least in part to oxidative tissue damage (currie, 1995). Recent studies have shown that metals such as Palladium and Vanadium exhibit the ability to produce reactive oxygen species, resulting in lipid peroxidation. Enhanced peroxidation of lipids may results in the damage to the cells, tissues and organs. The response of the biota to exposure to individual metals may differ from its response to other metals, as metals may interact with the biological system or may be chemically inert are low effinity for biological system resultant in sever toxicity or may be low toxic or showing no toxicity. Thus in the present study we intent to analysis and compare for the first time, the aqueous, the blood and hepatic antioxidant responses induced by different doses of two different toxic metals (Vanadium and Palladium) by treating alone with the thiols (Glutathione, N- Acetylcysteine and D-penicillamine) in aqueous medium in order to compare their toxicity. Physiological thiols vary substantially in their reactivity, and

36

Chapter: 2

on this basis, thiol groups would be preferred cellular targets for various oxidants. In this chapter we set out to determine different parameters relating to the oxidative status of various thiols like Glutathione N- Acetylcysteine and d-penicillamine verses Palladium and Vanadium like concentration and pH.

2.2. Methodology 2.2.1. Materials Reagents

All reagents were commercially obtained. Ellman’s reagent (5, 5 di-thiobis 2 nitro benzoic acid i.e. DTNB), Bis benzo-nitrile Palladium ii chloride, Ammonium Vanadate, Palladium Nitrate, were purchased from Sigma Aldrich. Sodium Dihydrogen Phosphate (Merck) Sodium Hydroxide, L. Glutathione (GSH), N-Acetylcysteine, D-Pencillamine, HCl 35% (Kolchlight) and Vanadium (V) oxytriethoxide, 95% were purchased from (fluka), (10M Perchloric Acid 70% (fluka), Sodium chloride (Merck), Distilled Water (Double Refined) and Potassium Dihydrogen Phosphate (Merck). PH Meter (NOV-210, Nova Scientific Company Ltd. Korea), UV. Visible Spectrophotometer (Shimadzue, 1601Japan, Magnetic Stirrer, hot plate 400(England), Oven: Memmert Model U-30,854 Schwabach (Germany), Micropipettes 200 µl, 500 µl, 1000 µl (Socorex Swiss Finland), Siliconized Glass test tubes, Disposable rubber gloves.

Glass wares

Glassware of Pyrex were used to carry out experiments, they were properly washed using detergent, washing powder, chromic mixture, distilled water and organic solvents etc. After washing all the apparatus were dried at 110◦C for two hours in oven of Memmert Ltd. (Germany).

2.2.2. Method 2.2.2.1. Preparation of stock solutions 1. 0.1M Phosphate buffer PH 7.6 Stock Solution: - 200 ml of phosphate buffer pH 7.6

was prepared by first dissolving 27.218g of Monobasic Potassium Phosphate (KH2PO4) in 1000ml of distilled water to get 0.2M Monobasic Potassium Phosphate solution and then mixing 42.4ml of 0.2M NaOH solution with 50ml of already prepared 0.2M Monobasic Potassium Phosphate solution and diluted to 200ml with distilled water. The pH was then adjusted with 0.2M NaOH or 0.2M HCl solutions. 37

Chapter: 2

2. 2mM Glutathione (GSH) Stock solution: - 30.75mg (Mol. weight 307.4) of Glutathione (GSH) was prepared by dissolving 30.75mg of Glutathione sufficient quantity of .1M Phosphate buffer in a 50ml volumetric flask, the final volume was adjusted to 50ml by adding further 0.1m Phosphate buffer pH7.6. This stock solution was then kept in refrigerator till further use.

3. 2mM N- Acetylcysteine (NAC) Stock Solution: - N- Acetyle cysteine (NAC) was prepared by dissolving 16.23mg mg of N- Acetylcysteine (Mol. weight 162.3 a.m.u) in sufficient quantity of 0.1M Phosphate Buffer in a 50ml volumetric flask, the final volume was adjusted to 50ml by adding further 0.1M Phosphate buffer PH 7.6. This stock solution was then kept in refrigerator till further use.

4. 2mM D-pen Stock solution: - 2mM of D-pencillamine(D-pen) was prepared by dissolving 14.9mg of D-pencillamine (Mol. weight 149.2) in sufficient quantity of 0.1M phosphate Buffer pH 7.6 in a 50ml volumetric flask, the final volume was adjusted to 50ml by adding further 0.1M phosphate buffer pH 7.6. This stock solution was then kept in refrigerator till further use.

5. 1mM 5, 5-dithiobis (2-nitrobenzoic acid), DTNB/ Ellman’s Reagent:-1mM of 5, 5- dithiobis, 2-Nitrobenzoic Acid (DTNB) (M.W 396.35) was prepared by dissolving 39.64mg (DTNB) in sufficient quantity of 0.1M phosphate buffer pH 7.6 to make 100ml of 1mM DTNB solution. This stock solution was then kept in refrigerator till further use.

6. 0.1M (Perchloric acid 70%): 0.1M (Perchloric acid purity70%) was prepared by mixing 1ml of 10M (Perchloric acid purity70%) in sufficient quantity of Distle water to make the final volume 100ml.

7. 2mM of Palladium Nitrate (PDN) and Ammonium Vanadate(AMV) stock solutions were prepared by dissolving 26.6 mg , 11.6mg, of Palladium Nitrate (PDN Mol. weight 266.3 g/mol), and Ammonium Vanadate (AMV Mol. weight 116.36 g/mol) respectively in sufficient quantity of distilled water to make the whole volume 50ml.

8. 2mM Bis-benzonitrile) Palladium ii chloride: 2mM Bis-benzonitrile Palladium II chloride stock solution prepared by dissolving 38.36 mg of benzonitrile Palladium II chloride (BNPDC, Mol. weight 383.57 g/mol in sufficient quantity of .1M (Perchloric acid 70%) to make final volume 50ml.

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Chapter: 2

9. 2mM Vanadium Oxi-Tri-ethoxide (VOTEO) was prepared by mixing 177µl of Vanadium Oxi-Tri-ethoxide (VOTEO) (mol.wt. 202.3g/mol and density 1.139g/ml) in sufficient quantity of phosphate buffer pH 7.6 to make the final volume 50 ml.

2.2.2.2. Estimation of GSH by Ellman’s (Modified) Method

Method of Elman’s modified (1962) was followed to estimate thiols (GSH, NAC and D-pen). The colored complex formed by reaction of SH group of thiols blank and (mixtures of thiols with metals after treatment with different concentrations of Palladium and Vanadium salts and complexes) with Ellman’s reagent i.e.5, 5’dithio bis- 2 nitro benzoic acid (DTNB) in aqueous solution, which give absorbance at 412nm which intern shows the concentration of corresponding thiol

The detail methodology is given below.

Thiols (GSH, NAC and D-pen) standard solutions (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6, 1.8 and 2 mM) were prepared in 0.1M Phosphate buffer PH 7.6. Then 0.5 ml of DTNB was taken in test tubes and was mixed with 0.2ml of thiols (GSH, NAC and D-pen)either standard solution or (mixture of thiols and metals) and then the volume is adjusted with 2.3ml of phosphate buffer pH 7.6 and mixed it thoroughly, incubate it for 5 minutes at room temperature. Absorbances are recorded on spectrophotometer at 412nm (λmax for thiolate anion TNB). Finally the thiols concentration is to be determined from thiols GSH, NAC and D-pen) standard curves as prescribed below. 2.2.2.3. Standard Calibration Curve A series of five different dilution i.e. 0.2, 0.4, 0.6, 0.8 and 1mM GSH was prepared by adding further 0.1M Phosphate buffer PH 7.6 to the above 2mM GSH stock solution. Then 0.5ml (500µl) of 5, 5-dithiobis, 2-Nitrobenzoic acid (DTNB) was taken separately from1mM stock solution in five different test tubes. 0.2 ml (200µl) of each of the above diluted solutions of GSH was added to the above test tubes and the final volume was raised to 3ml with further addition of 2.3 ml (2300µl) of Phosphate buffer PH 7.6. The mixture was then shaken thoroughly and incubated for five minutes. Then using phosphate buffer pH 7.6 as a reference solution (or blank), the UV absorbance of each of the above each thiol GSH, NAC and D-pen solution was determined at fixed wavelength of 412nm. The absorbance of (GSH, NAC and D-pen) was then

39

Chapter: 2

plotted as a function of final concentration of each thiol in mixture to produce a standard curve as Shown in figure (2.1) , 2.2 and 2.3.

Linear regression analysis was performed on this curve using Microsoft Excel® 2010.The spectrophotometric analysis was determined by performing a linear regression analysis of the absorbance versus each thiol concentration plot (standard curve). The correlation coefficient (R2) with a Value of 0.999 indicates a good regression within the given range of concentrations that will be analyzed in this study. The data of the standard curve is best described by a linear equation:

Y = m c + b

Where,

Y = Absorbance at 412 nm.

m = Slope of thiols (Glutathione,) standard curve of known concentration.

b = Intercept.

c = Concentration of thiols (Glutathione) standard curve of known concentration.

c = Y - b/m

If, Y, b and m are known, “c” unknown concentration of Glutathione can be determined.

The above equation was used to calculate the concentration of each thiol GSH, NAC and D-pen in aqueous solution after treatment with different concentrations of Palladium and Vanadium inorganic/organometalic complexes.

2.2.2.4. Experimental Protocol Preparation of Reaction Mixture, Reading Sample for Either Palladium Nitrate with either Glutathione, or N- Acetylcysteine or D-Penicillamine for Aqueous Media

1.0ml of different concentrations (.2, .4, .6, .8, 1, 1.2, 1.4, 1.6, 1.8 to 2mM) of Palladium Nitrate, solution was added to 1.0 ml of 2mM Glutathione, solution taken in ten separate test tubes, shacked well. Final concentration of Glutathione in each of the above test tube was 1mM (1000µM) and that of Palladium Nitrate was 0.1mM(100µM), 0.2mM (200µM), 0.3mM (300µM), 0.4mM (400µM), 0.5mM (500µM) and .6mM(600µM) , 0.7mM(700µM),

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Chapter: 2

.8mM(800µM), 0.9mM(900µM),1mM (1000µM) respectively. Then a series of ten samples cuvettes were prepared by taking 0.5ml of 1mM DTNB from stock solution in 10 separate test tubes. To it added 0.2ml of Palladium Nitrate Glutathione mixture from each one of the above made test tubes, diluted with 2.3ml of phosphate buffer pH 7.6 and incubated for five minutes. The final concentration of Glutathione in each of these test tubes was 0.03333mM (33.33µM) and of Palladium Nitrate was (6.7µM 13.4µM, 20.1µM, µM, 26.8µM, 33.5µM), 40.2µM (46.9µM), 53.6µM (60.3µM) and 67.0µM respectively. For control sample 0.2ml of 2mM Glutathione stock solution was added to a separated test tube already containing 0.5ml 1mm DTNB stock solution and the final volume was adjusted to 3ml by the addition 2.3 ml of phosphate buffer pH 7.6.The reaction mixture and reading sample for the other metals salts like bis-benzonitrile Palladium (II) chloride, Ammonium Vanadate, Vanadium oxi–tri-ethoxide with either Glutathione or N-acetylcysteine or d-penicillamine respectively were prepared in similar way as for the preparation of reaction mixtures and reading samples for Palladium Nitrate and Glutathione. Absorbances for each of the above mentioned sample of all the metal salts and complexes were recorded on UV- visible spectrophotometer at fixed wavelength at 412nm.

2.3. Results 2.3.1. Standard Curve of Glutathione N-Acetyle Cysteine and D-Penicillamine The absorbance of each dilution in each tube was recorded on UV-Visible spectrophotometer at fixed wavelength 412nm. The Absorbance of each dilution of GSH, NAC and D-pen were then plotted as a function of final concentration of GSH, NAC and D-pen to produce a standard curves as shown in [Figure 2.1, 2.2 and 2.3 respectively. The concentration of GSH, NAC and D-pen were calculated using regression equation (Y = m c + b) above.

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Chapter: 2

Standard curve of Glutathione 2

1.5 1.49 1.17 1 0.86 y = 0.2991x - 0.0229 0.5 0.57 R² = 0.999 0.26 Absorbance 0 0 0123456 ‐0.5 Concentration µM

Figure 2.1: Standard curve of GSH

STANDARD CURVE of N‐ACETYLECYSTIENE 2

1.5

1 y = 0.0233x + 0.1211

Absorbance R² = 0.999 0.5

0 0 10203040506070 Concentration (µM)

Figure 2.2: Standard curve of NAC

standard curve of D-penicillamine 1.8 1.6 1.4 1.2 1 0.8 0.6 Absorbance 0.4 0.2 0 0123456 concentration(µM)

Figure 2.3: Standard curve of D-Pen

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Chapter: 2

2.3.2. Palladium Results Two different salts of Palladium metal were studied for their effect on the levels of Glutathione, D.Pen and NAC in aqueous media. These salts were Palladium Nitrate (PDN) and Bi- Benzonitrile Palladium II chloride (BBNPDC). Readings were taken various concentrations of the salts, time intervals and pH.

2.3.3. Effect of various Conc. (6.7 to 67µM) of PDN/BBNPDC on the Chemical Status Glutathione and With Time (0 to90 Minutes) Reduced Glutathione (GSH) of reaction mixture of either Palladium salts and/ or its organometalic complex was measured in each tube by Ellman’s method. The absorbances were recorded at 412nm on a UV- Visible spectrophotometer and were converted into concentrations. The unknown conc. of GSH in the Palladium and GSH mixture was then calculated using the above known standard curve. When GSH was exposed to Lowest and Highest concentrations of either Palladium Nitrate/or bis-benzonitrile Palladium (ii) chloride (6.7µM to 67μM) respectively in aqueous media, it was observed that the level of GSH was depleted significantly (p<0.001) as compared to control GSH from (44.1% to 75%) and (34.0 to 65.0 %) as shown in figure (2.4). During this study time dependent effect of Lowest and Highest concentrations (6.7µM to 67µM) of either Palladium Nitrate/ and or Bisbenzonitrile Palladium (II) Chloride on the chemical status of GSH was also investigated and it was found that there was further depletion from (51% to 83%) and (41 to 72.0 %) in level of GSH for the time interval (0-90) minutes as shown figure (2.5).

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Chapter: 2

60

*** 40

20 GSH Control(µM) Conc. of GSH uM Conc.PDN(µM) Conc. of BBNPDC (µM) 0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 2.4:Conc. dependent effect of (6.7 and 67 µM) of PDN/ BBNPC on the chemical status of GSH Results are the mean ±SE of n=3 experiments of GSH. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

60 Control GSH (µM) PDN 6.7 (µM) PDN 67 (µM) *** 40 BBNPDC 6.7 (µM) BBNPDC 67 (µM)

20 Conc. of GSH (uM) GSH of Conc.

0 0 30 60 90 Time of Incubation (Minutes)

Figure 2.5: Time dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN / BBNDC on the chemical status of GSH Results are the mean ±se of 3 experiments of Glutathione. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

44

Chapter: 2

2.3.4. Effect of Two Conc. (6.7µM and 67µM) of PDN on the Chemical Status Glutathione with pH (7.0, 7.6, 8.0) The effect of pH (7.0, 7.6, 8.0) on the chemical status of GSH was also investigated and the result showed that the level GSH content was dropped from (3.25 to 4.6%) and was elevated from (4.4 to 4.7%), when pH was lowered to 7.0 with addition o.2N HCl and raised to 8.0 with (0.2M NaOH) as compared to pH 7.6 (nearly Body pH) with Palladium Nitrate as shown in the figures (2.6)

Figure 2.6: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN on the chemical status of GSH Results are the mean ±se of 3 experiments of Glutathione. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

2.3.5. Effect of various Conc. (6.7µM to 67µM) of PDN/BBNPDC on the Chemical Status N- Acetylcysteine and With Time (0 to90 Minutes) N- Acetylcysteine (NAC) was also measured in each tube by Ellman’s method the absorbances were recorded at 412nm and were converted into concentration as mention in standard curve. Again when NAC was exposed to different concentrations (6.7µM to 67µM) of either Palladium Nitrate /or Bis-Benzonitrile Palladium (II) Chloride respectively in aqueous media, the level of NAC was also significantly decreased (p<0.001) as compared to control NAC from (42.39 % to 74%) and (37.5% to 67%) as shown in figure (2.7). During this study time dependent effect of 45

Chapter: 2

lowest and highest concentrations (6.7µM to 67µM) of either Palladium Nitrate / or Bis- Benzonitrile Palladium (II) Chloride on the chemical status of NAC was also investigated and it was found that there was further dropped from (46.95 to 80%) and (44.2 to 69.5%) in NAC concentration with either Palladium Nitrate / or Benzonitrile Palladium (II) Chloride respectively for the time interval (0-90) minutes as shown in figure (2.8).

60

48

36 ***

24

Conc. of(uM) NAC NAC Control(µM) 12 Conc.PdN (µM) Conc. of BBNPdC (µM) 0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 2.7:Conc. dependent effect of (6.7 and 67 µM) of PDN) / BBNPC on the chemical status of NAC Results are the mean ±se of 3 experiments of NAC. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

65 NAC Control (µM) 52 PDN 6.7 (µM) PDN 67 (µM) 39 *** BBNPDC 6.7 (µM) BBNPDC 67 (µM) 26

Conc. of (uM)NAC 13

0 0 30 60 90 Time of Incubation (Minutes)

Figure 2.8: Time dependent effect of lowest and highest Conc. (6.7 to 67µM) of PDN) / BBNDC on the chemical status of NAC Results are the mean ±se of 3 experiments of NAC. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

46

Chapter: 2

2.3.6. Effect of Two Conc. (6.7 and 67µM) of PDN on the Chemical Status N- Acetylcysteine with PH (7.0, 7.6, 8.0) The effect of pH (7.0, 7.6, 8.0) on the chemical status of NAC was also studied and it was found that the level of NAC was increased from (3.54 to 4%), and was decreased from (3.7 to 4.3%), when pH was lowered to 7.0 with addition o.2N HCl and raised to 8.0 with (0.2M NaOH) as compared to pH 7.6 (nearly normal physiological pH of the blood) with Palladium Nitrate as shown in the figures (2.9).

Figure 2.9: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN on the Chemical status of NAC Results are the mean ±SE of n= 3 experiments of (NAC) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

2.3.7. Effect of various Conc. (6.7 to 67uM) Conc. PDN/ BBNPDC on the Chemical Status D-Penicillamine and With Time (0 to 90 Minutes) D-penicillamine (D-pen) was measured in each tube by Ellman’s method, again when D-pen was exposed to different concentrations (6.7µM to 67µM) of either Palladium Nitrate / or Bis (benzonitrile) Palladium (II) chloride respectively in aqueous media. The level of D- penicillamine was decreased significantly (p<0.001) as compared to control D-pen from (32.6 to 66.56%) and (26% to 60%) by Palladium Nitrate and Bis (benzonitrile) Palladium (ii) chloride treatment respectively, as shown in figure (2.10). During this study time dependent effect of lowest and highest concentrations (6.7µM to 67µM) of either Palladium Nitrate/ or Bis(benzonitrile)Palladium(II) chloride on the chemical status of D-pencillamine was also 47

Chapter: 2

investigated and it was found that there was further decreased from (44% to 79%) and 34% to 69 %) in D-pen concentration with either Palladium Nitrate/or Bis(benzonitrile)Palladium(II)chloride for the time interval (0-90) minutes as shown in figure (2.11).

65

52 *** M) u 39

26 Dpen Control (µM)

Conc. D-Pen of ( Conc.PdN(µM) 13 Conc. of BBNPdC (µM)

0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM) Figure 2.10:Conc. Dependent effect of (6.7 to 67 µM) PDN / BBNPC on the chemical status of D-Pen Results are the mean ±SE of 3 experiments of (D-pen). Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

65 Control D-pen (µM) 52 PDN 6.7 (µM) *** PDN 67 (µM) 39 BBNPDC 6.7 (µM) BBNPDC 67 (µM) 26 Conc.Dpen(uM) 13

0 0 30 60 90 Time of Incubation (Minutes)

Figure 2.11: Time dependent effect of Lowest and Highest Conc (6.7 to 67µM) of PDN / BBNDC on the chemical status of D-pen Results are the mean ±SE of 3 experiments of (D-pen). Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

48

Chapter: 2

2.3.8. Effect of Two Conc. (6.7 and 67uM) of PDN on the Chemical Status of D-Penicillamine with pH (7.0, 7.6 and 8.0) The effect of pH (7.0, 7.6, 8.0) on the chemical status of GSH was also studied and it was found that the level of control GSH was increased from (3.9 to 4.0%) and an decreased from (3.7 to 3.9%) when pH was decreased to 7.0 with 0.2N HCl and raised to 8.0 with (0.2M NaOH) respectively as compared to pH 7.6 (nearly normal physiological PH of the blood) with the highest and lowest concentration of Palladium Nitrate as shown in the figure (2.12).

Figure 2.12: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN on the chemical status of D-Pen Results are the mean ±SE of n=3 experiments of D-Pen. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

2.4. Results of Vanadium Metal Two different salts of Vanadium metal were studied for their effect on the levels of Glutathione, D.Pen and NAC in aqueous media. These salts were Ammonium Vanadate (AMV) and Vanadium Oxi-tri ethoxide (VOTEO). Readings were taken at various concentrations of the salts, time intervals (0-90minute and pH (7.0, 7.6, and 8.0).

2.4.1. Effect of various Conc. (6.7 to 67µM) of AMV/VOTEO on the Chemical Status Glutathione and with time (0 -90 Minutes) Reduced Glutathione (GSH) was also measured in each tube by Ellman’s method for Vanadium compounds. Again when GSH was exposed to different concentrations (6.7µM to 67μM) of

49

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either Ammonium Vanadate/or Vanadium Oxi-Tri-ethoxide respectively in aqueous media and the absorbances were recorded on UV-Visible spectrophotometer at 412nm. The absorbances were then converted into conc. of GSH using known standard curve. Then the level of GSH was decreased significantly (p<0.001) as compared to control GSH from (25% to 48%) and (10.2% to 40.1 %), as shown in figure (2.13) with the Palladium inorganic salt and its organic complex respectively. During this study time dependent effect of two concentrations (6.7µM to 67µM) of either Ammonium Vanadate / or Vanadium Oxi-Tri-ethoxide on the chemical status of GSH was also studied and it was found that there was further decreased from (10% to 40 %) and (21% to 56 %) in GSH concentration with either Ammonium Vanadate /or Vanadium oxi-tri-ethoxide, for the time interval (0-90) minutes as shown in figure (2.14).

60

50 ***

40 Conc. of GSH uM 30 GSH Control (µM) Conc. of AVM (µM) Conc. of VOTEO (µM) 20 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 2.13:Conc. dependent effect of (6.7 to 67 µM) AMV / VOTEO on the chemical status of GSH. Results are the mean ±SE of n=3 experiments of (GSH) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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Chapter: 2

60 GSH Control (M) AMV 6.7 (M) AMV 67 (M) 40 VOTEO 6.7 (M) VOTEO 6.7 (M) 20 **

Conc. of GSH (uM) *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 2.14: Time dependent effect of Lowest and Highest Conc. (6.7 and 67µM) AMV) /VOTEO on the chemical status of GSH Results are the mean ±se of 3 experiments of GSH. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P, 0. 01 ,***P, 0.001, versus control. 2.4.2. Effect of Two Conc. (6.7 and 67uM) (AMV) on the Chemical Status Glutathione, with pH (7.0, 7.6 and 8.0) The effect of pH ranged from (7.0, 7.6, 8.0) on the chemical status of GSH was also studied and it was found that the level of GSH was increased (3.9%, to 4.2%) with Ammonium Vanadate when pH was decreased to 7.0 with (0.2NHCl) and was decreased from (4.1%, 4.3%) when pH was raised to 8.0 with 0.2MNaOH as compared to pH 7.6 (nearly normal physiological pH of blood) as shown in the figures (2.15).

Figure 2.15: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV on the chemical status of GSH Results are the mean ±se of 3 experiments of GSH. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P, 0. 01, ***P, 0.001, versus control. 51

Chapter: 2

2.4.3. Effect of various Conc. (6.7µM to 67µM) of AMV /VOTEO on the Chemical Status N- Acetylcysteine, and with Time (0 to 90 Minutes) N- Acetylcysteine (NAC) was also measured in each tube by Ellman’s method, again when NAC was exposed to different concentrations (6.7µM to 67µM) of either Ammonium Vanadate /or Vanadium Oxi-Tri-ethoxide respectively in aqueous media and the results were compared with control sample of NAC. Then level of NAC was significantly (p<0.001) decreased from (20.34 to 50.34%) and (11.45 to 40 %) by either Ammonium Vanadate /or Vanadium Oxi-Tri-ethoxide treatment respectively as shown in figure (2.16). During this study time dependent effect of lowest and highest concentrations (6.7µM to 67µM) of either Ammonium Vanadate /or Vanadium Oxi-Tri-ethoxide on the chemical status of NAC was also investigated and it was found that there was further dropped from (24 % to 65%) and (19% to 51.28%) in NAC concentration as compare to control, with either Ammonium Vanadate /or Vanadium Oxi-Tri- ethoxide, for the time interval (0-90) minutes as shown in figure (2.17).

60

45 ***

30

NAC Control (µM) 15 Conc. of AVM (µM) Conc. of NAC (uM) NAC of Conc. Conc. of VOTEO (µM)

0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 2.16:Conc. dependent effect of 6.7 to 67 µM) AMV / VOTEO on the chemical status of NAC Results are the mean ±SE of 3 experiments of NAC. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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60 Control NAC (M) 48 AMV 6.7 (M) AMV 67 (M) 36 VOTEO 6.7 (M) VOTEO 67 (M) 24

Conc. of(uM) GSH 12 ***

0 0 30 60 90 Time of Incubation (Minutes)

Figure 2.17: Time Dependent effect of Lowest and Highest Conc. (6.7 and 67µM) of AMV / VOTEO on the chemical status of NAC. Results are the mean ±SE of n=3 experiments of (NAC) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

2.4.4. Effect Two Conc. of (6.7 to 67µM) of AMV on The Chemical Status N- Acetyle Cysteine (NAC) with pH (7.0, 7.6, 8.0)

The effect of pH ranged from (7.0, 7.6, 8.0) on the chemical status of NAC was also studied and it was found that the level of NAC was slightly increased from (3.6, 3.8%) and decreased (4.6 to 4.8%), with the Highest and lowest concentration of Ammonium Vanadate when pH was lowered to 7.0 with (0.2NHCl) and was decreased from (4.31%, 4.12%) when pH was elevated upto 8.0 with 0.2MNaOH as compared to pH 7.6 (nearly normal physiological pH of blood) as shown in figure (2.18).

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Figure 2.18: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV on the chemical status of NAC Results are the mean ±SE of n= 3 experiments of (NAC). Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

2.4.5. Effect of Various Conc. (6.7µM to 67µM) of AMV/ VOTEO on the Chemical Status D-Penicillamine, and with time (0 to 90 Minutes) D-penicillamine (D-pen) was also measured in each tube by Ellman’s method for Vanadium compounds , again when D-pen was exposed to different concentrations (6.7µM to 67µM) of either Ammonium Vanadate or Vanadium Oxytriethoxide) respectively in aqueous media, the level of D-pen was decreased significantly (p<0.001) as compared to control D-pen from 14.9% to 42.9%) and (7% to 34 %) by either Ammonium Vanadate or Vanadium oxytriethoxide treatment respectively, as shown in in figure (2.19). During this study time dependent effect of different concentrations (6.7uM to 67uM) of either Ammonium Vanadate or Vanadium oxytriethoxide on the chemical status of D-pen was also investigated and it was found that there was further depletion from (21% to 60%) and (11% to 43.8%) in D-pen concentration with either Ammonium Vanadate or Vanadium oxytriethoxide, for the time interval (0-90) minutes as shown in figure (2.20).

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65

52 ***

39 D-pen Control (µM) Conc. of D-Pen (uM) D-Pen of Conc. Conc. of AVM (µM) Conc. of VOTEO (µM)

0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 2.19:Conc. dependent effect of (6.7 to 67µM) of AMV / VOTEO on the chemical status of D-Pen Results are the mean ±SE of n= 3 experiments of (D-pen) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

65 Control Dpen (M) 52 AMV 6.7 (M) AMV 67 (M) 39 VOTEO 6.7 (M) VOTEO 67 (M) 26 *

Conc.of (uM) GSH 13 *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 2.20: Time Dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV / VOTEO on the chemical status of D-Pen. Results are the mean ±SE of n= 3 experiments of (D-pen) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

2.4.6. Effect of Two conc. (6.7 and 67uM) of (AMV) on the Chemical Status D-Penicillamine, with pH (7.0, 7.6, 8.0) The effect of pH ranged from (7.0, 7.6, 8.0) on the chemical status of (D-pen) was also studied and it was found that the level of (D-pen) was slightly increased from (3.7 to 4.2%) and dropped from (4.9 to 5.2%) with the Highest and lowest concentration Ammonium Vanadate when pH was lowered to 7.0 with (0.2NHCl) and was decreased from (4.4% and 4.6%) when pH was elevated upto 8.0 with 0.2MNaOH as compared to pH 7.6 (nearly normal physiological pH of blood) as shown figure (2.21).

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Figure 2.21: pH Dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV / VOTEO on the chemical status of D-Pen. Results are the mean ±SE of 3 experiments of D-pencillamine. Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

2.5. Discussion Interactions between and Pd (II) and Vanadium V (+V) complexes with sulfur-containing biomolecules are very important from a biological and medical point of view. For instance Despite the clinical anti-cancer utility of cis-platin, carboplatin, oxaliplatin, and several other complexes in clinical trials, there is a continued interest in the design of new complexes that shows anti-tumor activities equivalent or better than these agents (Torshizi Mahboube I- Moghaddam et al., 2008; Kwonet al., 2003 and Alverdi et al., 2004). The continued interest in platinum-based antitumor compounds is stimulated by the fact that certain tumors are resistant to the clinically used drugs cis-platin and carboplatin. The similarity between the coordination chemistry of platinum (II) and Palladium (II) compounds supports the theory that Palladium complexes can act successfully as antitumor drugs and show fewer side effects relative to other heavy metal anticancer compounds (Divsalar et al., 2007). Pd (II) analogues of Pt(II) complexes are well suited for kinetic and mechanistic studies by application of rapid-mixing techniques. Recently interactions of Pt(II) anticancer drugs and their Pd(II) analogues with sulfur containing amino acid side chains have attracted much attention in studies on the biological activity of cis- platin (Rau et al., 1998 and Pettit et al.,1994) and carboplatin (Barnham et al.,1996). In addition, it is widely accepted that sulfur donor ligands are usually co-administered with Palladium drugs

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owing to their important roles in the biological processing of anticancer Palladium drugs as protecting agents to reduce the toxicity. In the present study two transitional metals Palladium and Vanadium, and their inorganic/organometalic salts were treated with biologically important low molecular thiols (Glutathione, and N-acetylcysteine) a synthetically active cystien containing residue D-penicillamine) spectrophotometrically in ordered to tested which salts showing more toxicity toward biologically active ligand and also to explore new agent like D- pencillamine in reducing their toxicity while using these Metallo-element as a therapeutic agents, when various concentration(6.7µM to 67µM) of Palladium/ or ,Vanadium inorganic and organometalic complex were treated with different thiols (Glutathione, N-Acetylcysteine and D- penicillamine ) in aqueous media and results were compared to the control thiol (Glutathione ,N- Acetylcysteine and D-penicillamine) then the results showed that there was a concentration as well as time dependent depletions of all the three thiols by either of the metals Palladium/ or Vanadium and or their organometalic complexes, in case of Palladium the depletion level of thiols(Glutathione, N- Acetylcysteine and D-penicillamine) were higher than its organometalic complexes and also from Vanadium and its Vanadium Oxytriethoxide, when the time of incubation was increased from 0 to 90 minutes there was a further drop in the levels of the three thiols. The results of our this research suggested that Palladium and Vanadium depleted the level of Glutathione in aqueous medium both in dose dependent manner as well as time dependent manner, a possible explanation of the depletion level of all the thiols (Glutathione, N- acetylcysteine and D-penicillamine ) in aqueous media may be due to the Pd -(SR)2 or Pd(SR) , V-(SR)2 or (V-SR) conjugation or the oxidation of the thiols into its Mixed disulfide i.e.RS-SR. the conjugate formation is then further confirmed later in our NMR study (chapter No:6). Our this study is also supported by literature stated elseware that Vanadium treatment decreased the Glutathione content, the decreased Glutathione content could be due to its involvement in the Mechanisms of detoxification of various xenobiotics (Meister & Anderson, 1983) in another study elseware, showed that Sulfhydryl groups of protein, Glutathione and free cysteine are readily oxidized to disulfides by oxidants, and are important indicators of oxidative stress. (Ebru Taylan, Halil Resmi several other papers have reported decreased levels of GSH after exposure to arsenic, It was reported that one hour after exposure to arsenic (15.86 mg/kg body wt.), the GSH concentration was significantly decreased in the liver of male (Maiti S and Chatterjee 2001). A decrease in GSH content by Vanadium has also been reported by Scibior and Zaporowska (Scibior & Zaporowska, 2010). This study also supported by another data in literature that vanadate can form complexes with thiols without undergoing reduction even

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though the same thiol under other conditions will induce redox chemistry. A complex that forms between aqueous Vanadium (IV) and 2-mercaptoethanol has a (1:2) stoichiometry and is sufficiently stable to be observable at high 2-mercaptoethanol concentration (Poubaix M, 1966). These results are also in agreement with other reports that showed that smaller thiol species like D-pencillamine form Pd (D-pen)1, Pd (D-pen)2 Pd-(D-pen)3 complexes with Palladium in which the S, N,O of D-penicillamine co-ordinates with Palladium resulting in a mono, di and tridentate species (Cervantes G et al.,1998). These results were also nearly correlated with that of other another author who stated that Cis-platin-Glutathione conjugates have been shown to be formed intracellularly and actively pumped out of the cell by the MRP/Gx pump (ishikawa and Ali-Osman 1993). When the depletion level of the three thiols were compared then the results also showed that the depletion level in Glutathione and N Acetylcysteine was almost the same while both showed more than from D-pencillamine . A possible explanation of the depletion deference among three thiols is that Glutathione and N- Acetyle cysteine are structure analog and thus showing nearly same affinity towards the metals while the lower depletion of the d- penicillamine may be due steric hindrances because of the two methyl groups attached to the cysteinyl group of D-penicillamine. This study is also supported by literature elsewhere that the reactivity of the thiol nucleophile follows the sequence D-penicillamine < L-cysteine < Glutathione (Zivadin et al., 2001). In another study it is stated that Glutathione is considerably more reactive when treated with Pd+2 and Pt+2 complexes treated with thiols (Glutathione N- acetylcysteine) due to an appreciable anchimeric (neighbouring group) effect capable of reducing the activation barrier of the substitution reaction, arising from hydrogen bonding interactions between the acidic group located in a suitable position of the nucleophile (Wilkins, et al., 1991). The anchimeric (neighbouring group) effect has been reported for other reactions at Pt(II) complexes and is well known for organic reactions (Wilkins, et al., 1991). Compare to Glutathione, d-pencillamine showed strong steric interactions due its two methyl bulky groups on the carbon center near to the sulfur atom, making the entering thiols into its lower nucleophilicity (Wilkins, 1991). When the depletion level of both the metals and their metallic complexes were mutually compared it was observed that the depletion level of Palladium was more pronounced as compared to Vanadium and its organometalic complexes suggesting that Palladium is strong electrophile compared to Vanadium and showing strong affinity towards the thiols and thus may be readily attached to the thiols sulphydryl group and form a strong complex as compared to Vanadium and thus may showing more toxicity’s compare to Vanadium by depleting the level of thiols especially Glutathione in living organism. When the results of the

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inorganic and organic salt were compared with control, then the inorganic salts show more decrease level as compare to organic salt of the same metal suggesting that the inorganic salt produced more toxicity compared to its organic complexes. This finding could be due to the rapid dissociation of metal inorganic salt into free radical formation in solution. And hence may easily form complexes with thiols sulphydryl group. The result of the different PH range (7.0 7.6. 8.0) showed that the depletion level with either Palladium or Vanadium salts or at organic complex at PH 7.0 was slight lower than at (pH 7.6), at pH 8 the depletion was slightly higher than pH. 7.6 respectively suggesting that there was negligible effect of pH and temperature in these ranges, on the conjugation of the metals with thiols. This study Suggests that most of depletion of thiols takes place nearly physiological pH. It is also concluding from this finding that D-pencillamine and N- Acetylcysteine could be proven as effective chelating agent in protecting the redox status of Glutathione in highly oxidative stress condition.

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Chapter: 3

3.1. Introduction GSH is found virtually in all cells, and its concentration varies depending upon the number and kind of metabolic needs requiring GSH in these cells and tissues (Friedman, 1973). Blood is also one of the most major cite of GSH content, Erythrocyte contain a large amount of GSH approximately 2-3mM (Hagenfeldt and Larsson, 1928) and leukocyte seven time more (14- 21mm) than that of erythrocyte (Hardin et al., 1954) and thought to be involved in protecting cells against oxidative damage. The metabolism of thiols in general and GSH in particular can be altered by endogenous and exogenous substances or certain physiological changes and certain selected chemicals and /or compounds have important and toxicological applications. Blood cells rich in GSH have not been given much attention with regards to chemicals or drugs induced toxicity. Also little attention have been paid to capacity of blood to the metabolize xenobiotics although these cells are equipped with a variety of enzymes that are required for biotransformation of chemicals and drugs. GSH serving as a cofactor for some 20 enzymes is often the 1st line of biological defense against tissue injury and the major cellular non protein thiol. Glutathione is the most sensitive indicator of the cell and it is its ability to resist toxic challenge. Glutathione depletion leads to suicide of cell by a well-known apoptosis process (Slater et al., 1995).

Human erythrocytes are among the first cell systems to be affected by the heavy metals (Vanadium) compounds after they are absorbed in the GI tract. Also erythrocytes have a simple morphology and physiology, together with well-known membrane transport systems, having a well-defined metabolic mechanism for the maintenance of reduced Glutathione levels during oxidative stress. Thus, erythrocytes allow both the study of cell uptake of Vanadium compounds and their effects on intracellular metabolism and redox state. (Teresa C et al., 2005.) GSH is also released at high rates across the sinusoidal membrane into blood plasma, for delivery to other tissues (Nazzareno et al., 2009). In this chapter we also set out to determine different parameters relating to the oxidative status of plasma and erythrocytes Glutathione verses Palladium and Vanadium like concentration, time of incubation and pH. In the present study we investigated the effects of heavy metals Vanadium and Palladium on erythrocytes antioxidant status i.e. (the level of Glutathione (GSH)) and its ability to detoxifying and reducing these metals.

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3.2. Methodology 3.2.1. Materials Reagents

All reagents were commercially obtained. Ellman’s reagent (5, 5 di-thiobis 2 nitro benzoic acid i.e. DTNB), Bis benzo-nitrile Palladium ii chloride, Ammonium Vanadate, Palladium Nitrate, were purchased from Sigma Aldrich. Sodium Dihydrogen Phosphate (Merck) Sodium Hydroxide, HCl 35% (Kolchlight) and Vanadium (V) oxytriethoxide, 95% were purchased from (fluka),(10M Perchloric Acid 70% (fluka),Sodium chloride (Merck), Potassium Dihydrogen Phosphate (Merck), Sodium hydroxide(NaOH), Sodium Edetate (Riedel Dehean AG Sleeze Hannover), Dextrose were purchased from (Merck). Distilled Water (Double Refined) ,Chloroform (Merck), Ethanol (Merck). PH Meter (NOV-210, Nova Scientific Company Ltd. Korea), UV. Visible Spectrophotometer (Shimadzue, 1601Japan, Magnetic Stirrer, hot plate 400(England), Oven: Memmert Model U-30,854 Schwabach (Germany), Micropipettes 200 µl, 500 µl, 1000 µl (Socorex Swiss Finland) Centrifuge(H-200,Kokusan Ensink company Japan), Eppendolf’s tubes (Plastic, 10l) , Siliconized Glass test tubes, sterile pyrogen free disposable syringes (B.D), Disposable rubber gloves.

Glass wares

Glassware of Pyrex were used to carry out experiments, they were properly washed using detergent, washing powder, chromic mixture, distilled water and organic solvents etc. After washing all the apparatus were dried at 110◦C for two hours in oven of Memmert Ltd. (Germany).

3.2.2.Method 3.2.2.1. Preparation of Stock Solutions

1. Normal Saline (0.9% w/v NaCl Solution (0.154 M) :-100 ml of 0.9% NaCl solution was prepared by dissolving 0.90 g of pharmaceutical grade sodium chloride in sufficient quantity of water for injection to make the whole volume 100 ml.

2. 2mM Isotonic Solutions of Vanadium and Palladium: Isotonicity of solution is important from physiological point of view and therefore all solutions of metals used during experiments were made to have same osmotic pressure as that of blood except those used in

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observing the effect of metals on the chemical status of GSH in aqueous media. Therefore all the metals stock solutions and phosphate buffer were made isotonic with the addition of 0.9 percent solution of sodium chloride or a five percent solution of dextrose, before processing the metals with the blood components.

3. 0.1M Phosphate buffer PH 7.6 Stock Solution: - 200 ml of phosphate buffer pH 7.6 was freshly prepared in similar way as mention in chapter: 2. and was made isotonic by the addition of 0.9 percent solution of sodium chloride.

4. 1mM 5, 5-dithiobis (2-nitrobenzoic acid), DTNB/ Ellman’s Reagent:-1mM of 5, 5- dithiobis, 2-Nitrobenzoic Acid (DTNB) (M.W 396.35) was prepared in similar way as mention in chapter: 2. This stock solution was then kept in refrigerator till further use.

5. 0.1M (Perchloric acid 70%): A 50ml of 0.1M (Perchloric acid purity70%) was prepared in similar way as mention in chapter: 2.

6. 2mM of Palladium Nitrate (PDN) and Ammonium Vanadate (AMV) stock solutions were prepared in similar way as mention in chapter: 2, and was made isotonic by the addition of 0.9 percent solution of sodium chloride.

7. 2mM Bis-benzonitrile) Palladium ii chloride: 50ml of 2mM (Bis-benzonitrile) Palladium II chloride stock solution was prepared in similar way as mention in chapter 2 and was made isotonic by the addition of 0.9 percent solution of sodium chloride.

8. 2mM of Vanadium Oxi-Tri-ethoxide (VOTEO): 50ml of 0.2mM of Vanadium Oxi- Tri-ethoxide (VOTEO) solution was prepared in similar way as mention in chapter1 and was made isotonic by the addition of 0.9 percent solution of sodium chloride.

9. 0.5M and 5mM Disodium Edetate Solutions:-10ml of 0.5M disodium Edetate (Molecular weight =372.2) solution was prepared by dissolving 1.861g of disodium Edetate (EDTA-2Na) in 10ml of distilled water. Then from this solution 200 ml of 5mM disodium Edetate Solution was prepared by diluting 2ml of the above solution to 200ml with distilled water.

10. Chloroform: Ethanol Mixture:-160ml of Chloroform and Ethanol mixture in a ratio of 3:5 was prepared by mixing 60ml of chloroform to 100ml of ethanol, shacked and then placed in refrigerator till use.

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11. 0.5M and 5mM Disodium Edetate Solutions:-10ml of 0.5M disodium Edetate (Molecular weight =372.2) solution was prepared by dissolving 1.861g of disodium Edetate (EDTA-2Na) in 10ml of distilled water. Then from this solution, 200 ml of 5mM disodium Edetate Solution was prepared by diluting 2ml of the above solution to 200ml with distilled water.

3.2.2.2. Isolation of Blood components a. Isolation of Plasma

The Experimental protocol for blood sampling was approved by the ethical committee of the Gomal University of DIK KPK Pakistan. 200ml venous blood from human healthy volunteer was collected in using a heparinized bag. The blood was centrifuged at 3000rpm for 10 minutes at 4•C. The top yellow colour plasma layer was collected in another test tube without disturbing the white buffy layer. The plasma was then deprotonated by adding 5% tri acetic Acid solution and then centrifuged 3000rpm for 5 minutes and then 50 µl HCl (0.1N) was added to maintain the GSH content in reduced form then store below 20c in refrigerator till use.

b. Isolation of Cytosolic fraction

The red blood cell fractions left after isolation of plasma in the above test tubes was then gently taken and washed with 0.9% NaCl solution. This fraction of blood was then centrifuged softly for 5 minutes. The supernatant layer was then discarded and 0.5ml of red blood cell fraction thus obtained was then mixed with 0.5ml of diluted water and placed in a refrigerator for 1hour. After which 0.6ml of chloroform: ethanol (3:5) mixture was added to each of the above test tubes to precipitate hemoglobin. It was then mixed softly with addition of 0.1ml of water. These mixtures were then centrifuged hard for 10 minutes at 10000-12000 rpm. The pale yellow supernatant layer (cytosolic fraction) from all test tubes were then collected and was added 50 µl (0.1N)HCl to maintain the GSH content in reduced form and kept in refrigerator till used.

3.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method Method of Elman’s modified (1962) was followed for estimation Glutathione as mention in Chapter No: 2.

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3.2.2.4. Experimental Protocol Preparation of reaction and reading sample for biological fluids ( Human whole blood, components ) with Palladium, Vanadium salts and organometalic complexes

Two set of experiments were performed for whole blood plasma and cytosolic fraction with either Palladium or Vanadium inorganic salts or its organo-metallic Complexes.

(1) In First set of Experiment 10 ml of blood was first mixed with 10 ml of different concentrations (0.2-2mM) of Palladium Nitrate, the final concentration of Palladium Nitrate, (10 to 1000µM) respectively. The blood content of each tube was then processed for (plasma, and cytosolic fraction, by centrifugation according to above mention procedure. The reading samples were then prepared from each components of the blood in such a way that a series of ten samples cuvettes were prepared by taking 0.5ml of 1mM DTNB from stock solution in 10 separate test tubes. To it added 0.2ml of Palladium Nitrate Glutathione mixture from each one of the above made test tubes, diluted with 2.3ml of phosphate buffer saline pH 7.6 and incubated for five minutes. The final concentration of Palladium Nitrate was (6.7µM 13.4µM, 20.1µM, µM, 26.8µM, 33.5µM), 40.2µM (46.9µM), 53.6µM (60.3µM) and 67.0µM respectively. For control sample 0.2ml of Glutathione content of pure blood components (either plasma or cytosolic fraction) was added to a separated test tube already containing 0.5ml 1mm DTNB stock solution and the final volume was adjusted to 3ml by the addition 2.3 ml of phosphate buffer saline pH 7.6. The reaction mixture and reading sample for the other metals salts like bis- benzonitrile Palladium (II) chloride, Ammonium Vanadate, Vanadium Oxi–Tri-ethoxide with either Glutathione content plasma or cytosolic fraction respectively were prepared in similar way as for the preparation of reaction mixtures and reading samples for Palladium Nitrate and Glutathione content of each blood components. Readings (absorbances) for each of the above mentioned sample of all the metal salts and complexes were then taken on UV- visible spectrophotometer at fixed wavelength at 412nm.

2. In 2nd set of experiment the blood components were first isolated by centrifugation according to above procedure, then 10 ml of either plasma, or cytosolic fraction were taken in 10 separate test tubes and was mixed with (0.2 to 2mM) of either Palladium Nitrate or , Bisbenzonitrile Palladium (II) chloride or Ammonium Vanadate or Vanadium Oxy-Tri- ethoxide and incubated for 10 minutes. Then for the determination of GSH content, the same procedure was repeated as for the preparation of reading sample for blood components before isolation of blood. Absorbances were then recorded on 300 UV-Visible spectrophotometer at 64

Chapter: 3

fixed wavelength 412 nm for each dilution .The absorbance were then converted into concentration using known standard curve of Glutathione already prepared mention chapter :2 above.

3.3. Results 3.3.1. Results in Blood Plasma 3.3.1.1. Effect of various Conc. (6.7 to 67µM) of PDN/ BBNPDC on the Chemical Status Plasma Glutathione, and with time (0 to90 Minutes) Reduced Glutathione (GSH) content of Plasma of the human whole blood was also measured in each tube by Ellman’s method in two different sets of experiments. In one set either Palladium Nitrate / or Bisbenzonitrile Palladium (II) Chloride was added before separation of blood and in second set of experiment either Palladium or Bisbenzonitrile Palladium (II) Chloride was added to the isolated plasma after its separation from blood as shown in the methodology. Then absorbances were recorded at 412nm and were converted into conc. using known standard curve as shown in Chapter: 2. Again when various concentration (6.7 to 67µM) of either Palladium Nitrate / or Bisbenzonitrile Palladium (II) Chloride was exposed to Plasma of whole blood and isolated Plasma the level of GSH in Plasma before separation was decreased significantly (p<0.01) from (20.1% to 58.4%) with highest and lowest concentration of Palladium Nitrate and was dropped from (6.2% to 54.1%) with lowest and highest concentration of (Bis – Benzonitrile Palladium (II) Chloride) as compared to control sample of plasma as shown in (3.1). After separation of plasma and addition of Palladium Nitrate the level of GSH content was dropped from (24.4% to 61.72 % ) and the level dropped from (10.26 % to 55.5 %) with Bisbenzonitrile Palladium (II) Chloride respectively as compared to control sample of GSH content of plasma, as shown in figure (3.2). The time dependent effect on the GSH content of plasma were also investigated, again when various concentration (6.7 to 67µM) of either Palladium Nitrate/ or Bisbenzonitrile Palladium (II) Chloride mixtures with plasma were incubated (0 to 90) minutes and the result were compared with control then there was further dropped in the level of GSH content of Plasma in first set of experiment from (30.71% to 70.12%) with lowest and highest conc. of Palladium Nitrate and from (19.0 % to 59%) with Bisbenzonitrile Palladium (II) Chloride respectively as shown in figure (3.3). In 2nd set of experiment the level GSH content of plasma was dropped from (39.5.0 % to 75.16% ) with Palladium Nitrate and was dropped (22 % to 65.8 %) with Bisbenzonitrile Palladium (II) Chloride as shown in the figure (3.4). 65

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21

*** 14

Conc. of GSH uM 7 Control GSH of PLasma(µM) Conc. of PDN (µM) Conc. of BBNPDC (µM)

0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM) Figure 3.1:Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPC on the chemical status of plasma GSH before its isolation from Blood Results are the mean ±SE of (n= 3) experiments of plasma GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

21.3

14.2 ***

7.1 GSH Control of Plasma (µM) Conc. ofGSH uM Conc. Of PDN (µM) Conc. Of BBNPdC(µM)

0.0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 3.2:Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPC on the chemical status of plasma GSH after its isolation from Blood Results are the mean ±SE of (n= 3) experiments of plasma GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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21 Control GSH of Plasma (µM) PDN 6.7 (µM) PDN 67 (µM) 14 BBNPDC 6.7 (µM) BBNPDC 67 (µM)

7 Conc. of GSH (uM) GSH of Conc. *** **

0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.3: Time dependent effect of Lowest and highest conc. of PDN / BBNPC on the chemical status of plasma GSH before its isolation from blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P, 0. 01***P, 0.001, versus control.

21 Control GSH of Plasma (µM)

PDN 6.7 (µM)

14 PDN 67 (µM) BBNPDC 6.7 (µM) BBNPDC 67 (µM) 7

Conc. of GSH (uM) GSH of Conc. ** *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.4: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) PDN / BBNPC on the chemical status of plasma GSH after isolation of Blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P, 0. 01, ***P, 0.001, versus control.

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3.3.1.2. Effect of Two Conc. (6.7 and 67µM) PDN on The Chemical Status Plasma Glutathione, with pH (7.0, 7.6, 8.0) The pH dependent effect on the interaction of plasma GSH content with Palladium Nitrate was also investigated and when two concentration of Palladium Nitrate were incubated with isolated plasma at various pH (7.0 7.6 and 8.0) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ) then there was a ( 3.4 to 4.5 % )increased and ( 4.2 to 4.7% ) decreased in the level of GSH content of isolated plasma at pH 7 and 8 as compared to pH 7.6 (nearly equal to normal physiological pH of the blood) as shown in the figure (3.5.).

Figure 3.5: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN / BBNPC on the chemical status of plasma GSH after isolation of Blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

3.3.1.3. Effect of Various Conc. (6.7 to 67µM) of AMV/VOTEO on the Chemical Status of Plasma Glutathione, and With Time (0 to 90) Minutes Reduced Glutathione (GSH) content of Plasma of the human whole blood was also measured in each tube by Ellman’s method in two different sets of experiments. In one set either Ammonium Vanadate / or Vanadium Oxi-Tri-ethoxide was added before separation of blood and in second set of experiment either Ammonium Vanadate or Vanadium Oxi-Tri-ethoxide was added to the isolated Plasma after its separation from blood as shown in the methodology. Again when

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various concentration (6.7 to 67µM) of either Ammonium Vanadate / or Vanadium Oxi-Tri- ethoxide was exposed to Plasma of whole blood and isolated Plasma, the level of GSH in Plasma before separation was dropped significantly (p<0.05) from (16.3% to 51.2%) with lowest and highest concentration Ammonium Vanadate and was depleted from (5.04 to 45.02 %) with highest and lowest concentration of Vanadium Oxi-Tri-ethoxide as compared to control sample of plasma. As shown in Figure (3.6). After separation of Plasma and addition of Ammonium Vanadate the level of GSH content was dropped from (20.6% to 56.4%) and with Vanadium Oxi-Tri-ethoxide from (8.04 to 47.02 %) as compared to control sample of GSH content of plasma, as shown in figure (3.7). The time dependent effect on the GSH content of plasma were also investigated, again when various concentration (6.7 to 67µM) of either Ammonium Vanadate / or Vanadium Oxi-Tri-ethoxide mixtures with plasma were incubated (0 to 90) minutes and the result were compared with control , then there was further dropped in the level of GSH content of plasma in first set of experiment from ((30.0 % to 62.33%) with lowest and highest conc. of Ammonium Vanadate and from (21.0 % to 56.33%) with Vanadium Oxi-Tri- ethoxide respectively as shown in figure (3.8). In 2nd set of experiment the level GSH content of Plasma was dropped from (32.0 % to 67.33%) with Ammonium Vanadate and was dropped from (24.5 % to 58.16%) with Vanadium Oxi-Tri-ethoxide as shown in the figure (3.9).

25

20

15 ***

10 Control GSH of Plasma (µM)

Conc. of GSH (uM) 5 Conc. of AMV (µM) Conc. of VOTEO (µM) 0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 3.6:Conc. dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical status of plasma GSH before its isolation from blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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21.3

14.2 ***

7.1 Control GSH of Plasma(µM) Conc. of GSH uM GSH of Conc. Conc. of AMV (µM) Conc. of VOTEO µM) 0.0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 3.7: dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical status of plasma GSH after its isolation from blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

21 Control GSH of Plasma (µM) AMV 6.7 (M) AMV 67 (M) 14 VOTEO 6.7 (M) VOTEO 67 (M)

7 * Conc. of GSH (uM) *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.8: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of AMV / VOTEO on the chemical status of plasma GSH before its isolation from blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. *P, 0.05 ***P, 0.001, versus control.

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21 GSH Controle of Plasma (M) AMV 6.7 (M) 14 AMV 67 (M) VOTEO 6.7 (M) VOTEO 67 (M) 7

Conc. of GSH (uM) GSH of Conc. ** *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.9: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of AMV/ VOTEO on the chemical status of plasma GSH after its isolation from blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P, 0.01, ***P, 0.001, versus control.

3.3.1.4. Effect of Two Conc. (6.7 to 67µM) of AMV on the Chemical Status of Plasma Glutathione with pH (7.0, 7.6, 8.0) The pH dependent effect on the interaction of GSH with Vanadium was also investigated and when two concentration of Ammonium Vanadate were incubated with isolated plasma at various pH (7.0 and 8.0) and the result were compared with pH 7.6 (nearly equal to normal physiological pH of blood) then there was a an increased in the level of GSH content of Plasma from (4.18 to 4.3% ) and, decreased from ( 4.63 to 5.3%) in the level of GSH content of isolated plasma at pH 7 and 8 respectively as shown in the figure (3.10).

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Figure 3.10: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV on the chemical status of plasma GSH after its isolation from Blood. Results are the mean ±SE of (n= 3) experiments of plasma GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

3.3.2. Results in Cytosolic Fraction (C.F) 3.3.2.1. Effect of Various Conc. (6.7 to 67µM) of (PDN / (BBNPDC) on the Chemical Status of C.F Glutathione, and With Time (AT 0 -90 Minutes) Reduced Glutathione (GSH) content of Cytosolic fraction of the human whole blood was measured in each tube by Ellman’s method in two different sets of experiments. In one set either Palladium Nitrate / or Bisbenzonitrile Palladium (II) Chloride was added before separation of blood and in second set of experiment either Palladium or Bisbenzonitrile Palladium (II) Chloride was added to the isolated cytosolic fraction after its separation from blood as shown in the methodology. Again when various concentration (6.7 to 67µM) of either Palladium Nitrate/ or Bisbenzonitrile Palladium (II) Chloride was treated with cytosolic fraction of whole blood and isolated cytosolic fraction, the level of GSH in cytosolic fraction before separation was depleted significantly (p<0.001) from (21.2% to 57.83%) with highest and lowest concentration Palladium Nitrate and was dropped from (6.0 to 46.45 % ) with highest and lowest concentration of (Bis –Benzonitrile Palladium (II) Chloride) as compared to control sample of Cytosolic fraction as shown in figure (3.11). After separation of cytosolic fraction and addition of Palladium Nitrate the level of GSH content was dropped from (25.74% to 59.82 %) and (10 % to 49.8 %) with either Palladium Nitrate / or Bisbenzonitrile Palladium(II) Chloride as compared to control sample of GSH content of cytosolic fraction, as shown in figure (3.12). The

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time dependent effect on the GSH content of cytosolic fraction were also investigated, again when various concentration (6.7 to 67µM) of either Palladium Nitrate / or Bis –Benzonitrile Palladium (II)Chloride mixtures with cytosolic fraction were incubated (0 to 90) minutes and the result were compared with control, then there was further drop in the level of GSH content of cytosolic fraction in first set of experiment from (40% to 66.21 % ) with lowest and highest conc. of Palladium Nitrate and from (26.5 % to 55.19%) with Bisbenzonitrile Palladium (II) Chloride respectively as shown in figure (3.13). In 2nd set of experiment the level GSH content of cytosolic fraction was dropped from (45.26 % to 68.0 8 %) with Palladium Nitrate and was dropped (32.4.0 % to 59.7%), with Bisbenzonitrile Palladium (II) Chloride respectively as shown in the figure (3.14).

20

15 ***

10

conc.of GSHuM GSH Control of CF (µM). 5 Conc. of PDN (µM) Conc. of BBNPdC ( µM)

0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 3.11:Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPDC on the chemical status of GSH in C.f. before its isolation from blood Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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20

15 ***

10

Conc. of PDN (uM)

conc. of GSH uM Conc.of BBNPDC(uM) 5 Controle GSH of C.F(uM)

0 0 14 28 42 56 70 Conc. of PDN/PDBBC in (uM)

Figure 3.12:Conc. dependent effect of (6.7 to 67 µM) of PDN / BBNPDC on the chemical status of GSH in C.f. after its isolation from blood Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

20 Control GSH C.F (µM) PDN 6.7 (µM) 15 PDN 67 (µM) BBNPDC 6.7 (µM) 10 BBNPDC 67 (µM)

5

Conc.of (uM) GSH ** *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.13: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of PDN/BBNPDC on the chemical status of C.F GSH before its isolation from blood Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P, 0.01 ***P, 0.001, versus control.

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20

Control GSH C.F(µM) 15 PDN 6.7 (µM) PDN 67 (µM) 10 BBNPDC 6.7 (µM) BBNPDC 67 (µM)

5 Conc. of GSH (uM) *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.14: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) PDN/BBNPDC on the chemical status of C.F GSH after its isolation from blood Results are the mean ±SE of (n= 3) experiments of cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

3.3.2.2. Effect of Two Conc. (6.7µM and 67µM) Of PDN on the Chemical Status of C.F Glutathione with pH (7, 7.6, 8.0) The pH dependent effect on the interaction of GSH content of Cytosolic fraction with Palladium Nitrate was also investigated and when twos concentrations (6.7 and 67uM) of Palladium Nitrate were incubated with isolated Cytosolic fraction at various pH (7.0 , 7.6 and 8.0 ) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ) then there was a (3.09 to 4.3% ) increased and (4.02 to 5.01%) decreased in the level of GSH content of isolated Cytosolic fraction at pH 7 and 8 respectively as compared to pH 7.6 (nearly equal to normal physiological pH of the blood, as shown in the figure (3.15).

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Figure 3.15: pH dependent effects of Lowest and highest conc. (6.7 and 67 µM) of PDN on the chemical status of C.f. GSH after isolation from blood

Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

3.3.2.3. Effect of various Conc. (6.7 to 67µM) Of (AMV)/ (VOTEO) On the Chemical Status C.F Glutathione, and With Time (At 0 -90 Minutes) Reduced Glutathione (GSH) content of Cytosolic fraction of the human whole blood was measured in each tube by Ellman’s method in two different sets of experiments. In one set, either Ammonium Vanadate / or Vanadium Oxi-Tri ethoxide was added before separation of blood and in second set of experiment either Ammonium Vanadate / or Vanadium Oxi-Tri ethoxide was added to the isolated Cytosolic fraction after its separation from blood as shown in the methodology. Again when various concentration (6.7 to 67µM) of either Ammonium Vanadate/ or Vanadium Oxi-Tri ethoxide was exposed to whole blood and isolated Cytosolic fraction the level of GSH in Cytosolic fraction before separation was decreased significantly (p<0.001) from (18.9% to 54.57%) with highest and lowest concentration of Ammonium Vanadate and was dropped from (6.30% to 44.0 %) with highest and lowest concentration of Vanadium Oxi-Tri- ethoxide as compared to control as shown in figure (3.16). After separation of Cytosolic fraction and addition of Ammonium Vanadate the level of GSH content was dropped from (22.7% to 57.22 %) and (8 % to 47.56 %) with Vanadium Oxi Tri-ethoxide as compared to control sample of GSH content of Cytosolic fraction, as shown in figure (3.17).The time dependent effect on the GSH content of Cytosolic fraction were also investigated. Again when Lowest and Highest concentrations (6.7 to 67µM) of either Ammonium Vanadate/ or Vanadium Oxi-Tri-

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ethoxide mixtures with Cytosolic fraction were incubated from (0 to 90) minutes and the result were compared with control sample, then there was further dropped in the level of GSH content Cytosolic fraction in first set of experiment from (33.3 % to 60.23%) with lowest and highest conc. of Ammonium Vanadate and from (20.2% to 53.2 %) with Vanadium Oxi-Tri-ethoxide respectively as shown in figure (3.18) . In 2nd set of experiment the level GSH content of Cytosolic fraction was dropped from (37.6 % to 65.6 %) with Ammonium Vanadate and was dropped (24.5 % to 55.9 %) with Vanadium Oxi-Tri-ethoxide as shown in the figure (3.19).

20

15 *** 10

Control GSH of CF. (µM)

Conc. of GSH uM 5 Conc. of AMV (µM) Conc. of VOTEO µM)

0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 3.16:Conc. dependent effects of AMV/ VOTEO on the chemical status of GSH in Cytosolic fraction (CF) before isolation of CF from blood. Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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20

15 ***

10

Conc. of GSH uM 5 Control GSH of C.F (µM) Conc. of AMV (µM) Conc. of VOTEO (µM) 0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 3.17:Conc. dependent effect of (6.7 to 67 µM) of AMV/ VOTEO on the chemical status of GSH in Cytosolic fraction (CF) after isolation from blood) Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

18 GSH Control of CF. (M)

AMV 6.7 (M)

12 AMV 67 (M) VOTEO 6.7 (M)

VOTEO 67 (M) 6 ** Conc.of GSH (uM) *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.18: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of AMV/ VOTEO on the chemical status of GSH of Cytosolic fraction before isolation from blood Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P,0.01, ***P, 0.001, versus control.

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20 GSH Control of CF. (M) AMV 6.7 (M) 15 AMV 67 (M) VOTEO 6.7 (M) 10 VOTEO 67 (M)

5

Conc.of (uM) GSH ** *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 3.19: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of AMV/ VOTEO on the chemical status of GSH in Cytosolic fraction after its isolation from Blood. Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content). Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P,0.01, ***P, 0.001, versus control. 3.3.2.4. Effect of Two Conc. (6.7 to 67µM) of AMV on the Chemical Status C.F Glutathione with pH (7.0, 7.6, 8.0) The pH dependent effect on the interaction of GSH with Ammonium Vanadate was also investigated and when two concentration of Ammonium Vanadate were incubated with isolated Cytosolic fraction at various pH (7.0,7.6, 8.0) and the result were compared with pH 7.6 (nearly equal to normal physiological pH of blood) then there was a (3.9% to 5.8%) increase and (4.2 to 5.94%) decrease in level of GSH content of cytosolic fraction of blood after isolation and treating Ammonium Vanadate at pH 7.0 and 8 respectively as compared to pH 7.6 (nearly equal to normal physiological pH of the blood) as shown in the figure (3.20).

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Figure 3.20: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV on the chemical status of GSH of Cytosolic fraction after its isolation from blood Results are the mean ±SE of (n= 3) experiments of Cytosolic fraction GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

3.4. DISCUSSION

When either Palladium and Vanadium was treated with the whole blood before isolation and after isolation into plasma and cytosolic fraction and the results were compared with the control samples of Plasma and cytosolic fraction, then the result of our study showed that level of Glutathione content of the plasma and erythrocytes was deferentially depleted with both metals salts and its complexes and also before and after its isolation into plasma and erythrocytes both in dose dependent and as well as time dependent. Our this result suggested that both plasma and cytosolic Glutathione render itself to by either oxidize or making complex formation with the free radicals during heavy metals like Palladium and Vanadium in order to reduced its toxicity . When the pH effect was observed then it was found from our study that most of oxidation takes place near physiological pH. As there was no significant difference of low pH (7.0) and high pH (8) on the oxidation of Glutathione content of both the component blood, plasma and cytosolic fraction as compared to pH 7.6 (Which are the nearly normal Physiological pH of the blood). This scientific data about the interaction and effect of Palladium and Vanadium on the chemical modulation of GSH in whole blood before separation and after isolation of plasma and cytosolic fraction will enable us to understand further the role of Palladium,

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Vanadium, and GSH and also strengthen our knowledge about their therapeutic uses in much disease. The Palladium and Vanadium induced the depletion of GSH, and thus has interesting physiological as well as pharmacological uses like detoxification by participation in redox system, activation of SH- coenzyme, Co-enzyme action and conjugation. Thus it was of interest to further study the interaction of these metals in vivo to establish further the scientific data. In case of whole blood the results obtained from the plasma and cytosolic fraction of whole blood for the effect of Palladium and Vanadium on whole blood was positively correlated between duration of Palladium and Vanadium presence in whole blood and (the depletion of reduced (GSH). The results obtained from the cytosolic fraction part of whole blood for the both Palladium and Vanadium effect on whole blood was promising and showed that the Palladium and Vanadium can cross the semi permeable membrane of the RBCs, though not too much extent but can induce a change in the Chemical status of GSH and brought reduction in level of reduced GSH. It is also concluding from this finding that, Palladium showed more toxicity as compared to Vanadium, as it depleted more the level of GSH content of Plasma and cytosolic fraction as compared to Vanadium. It is further concluded that the inorganic salt of Palladium and Vanadium are more toxic as compared to their organometalic complexes, as inorganic salts showed more depletion as compared to organic salts of both metals, suggesting that the inorganic salts are readily dissociated into free radicals and easily available for GSH as compared to organic complexes of both the metals. Further the effect of both the metals Palladium and Vanadium for their Pharmacological actions like Vanadium may be used as anti-diabetic agent because of its insulin mimetic action and like Palladium as a dental alloy, this study Provides a caution in the use of both metals Palladium and Vanadium as it depletes the reduced Glutathione which is our main antioxidant material found in the body and due to increased use of Palladium and Vanadium without measuring its effect on reduced GSH. It can enhance other complications related to presence of free radicals in the body. This study also provides the verification of studies conducted for the passage of metals across RBCs membrane.

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4.1. Introduction The immune system is an elegant and complex group of components that combine to fight against disease, infections, and various pathogens. A healthy immune system differentiates organisms in the body as natural, which are untouched, or invaders that are rapidly destroyed. A healthy immune system is able to combat the attack, whereas a compromised immune system allows invading organisms and other foreign agent like heavy metals to flourish. The natural immune response relies on various white blood cells and barriers to block any attack from foreign invader. Antioxidants are invaluable because they limit damage to the cells. The WBC’s (Leukocytes) are the mobile units of the body defense system. They are formed partially in the Bone marrow [Granulocytes (Neutrophils) and Agranulocytes (Lymphocytes especially T-cell and B-cells)]. After formation they are transported into the blood to the different parts of the body, where they are to be used. The real value of WBCs is that most of them are specifically transported to the areas of serious infections and inflammation, thereby providing rapid and potent defense against any infectious agent that might be present. The Granulocytes and Monocytes have the special capability of “seek and destroy” the foreign invader. The normal physiological role of the lymphocytes is guaranteed by various integrating functions, White Blood Cells (Leukocytes) being individually performed by the numerous biomolecules. The physiological function of a particular biomolecule originates generally from the chemical composition and more specifically from the functional group in its chemical structure. Studies have shown that Glutathione is food for the immune cells, boosting the strength of lymphocytes. B-cell lymphocytes identify the unwanted pathogen while that of the T-cells then attack. T-cells also shut down the immune response when the job is done. Reduced Glutathione (GSH) provides a major intracellular defense against oxidative injury. On the other hand neutrophils generate large amounts of oxidants; they might need GSH to protect themselves against injury while performing their antimicrobial or inflammatory activities. Neutrophils defective in Glutathione metabolism have impaired function (Roos et al., 1979 and Spielberg et al., 1979). Stimulation of neutrophils causes a decrease in their GSH concentration (Voetman et al., 1980). Heavy metals have the ability to attach to these sulfhydryl groups rendering GSH levels in the physiological compartments like lymphocytes to be modulated. In cases of GSH modulation the vitality of lymphocytes is compromised and eventual cell death may occur. It was thought of worth to study lymphocytes and neutrophils for GSH modulation after exposure to heavy metals like Palladium

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and Vanadium. That is why we extended our study to WBC’s in order to investigate that what changes occur during the interaction of Palladium and Vanadium while treating with the components the GSH Content of WBC’s. 4.2. Methodology 4.2.1. Materials a. Reagents

All reagents were commercially obtained. Ellman’s reagent (5, 5 di-thiobis 2 nitro benzoic acid i.e. DTNB), Bis benzo-nitrile Palladium ii chloride, Ammonium Vanadate, Palladium Nitrate, RPMI-1640, Histopaque, (>98%; agarose gel electrophoresis lyophilized) were purchased from Sigma Aldrich. Sodium Dihydrogen Phosphate (Merk) Sodium Hydroxide, HCl 35% (Kolchlight) and Vanadium (V) oxytriethoxide, 95% were purchased from (fluka),(10M Phercloric Acid 70% (fluka),Sodium chloride (Merck), Potassium Dihydrogen Phosphate (Merck), Sodium hydroxide(NaOH), Sodium Edetate (Riedel Dehean AG Sleeze Hannover), Dextrose were purchased from (Merck). Distilled Water (Double Refined) ,Chloroform (Merck), Ethanol (Merck). PH Meter (NOV-210, Nova Scientific Company Ltd. Korea), UV. Visible Spectrophotometer (Schimadzu, 1601 Japan, Magnetic Stirrer, hot plate 400(England), Oven: Memmert Model U-30,854 Schwabach (Germany), Potter-eveljhem homogenizer (japan), Micropipettes 200 µl, 500 µl, 1000 µl (Socorex Swiss Finland), Centrifuge (H-200,Kokusan Ensink company Japan), Eppendolf’s tubes (Plastic, 10l) , Siliconized Glass test tubes, sterile pyrogen free disposable syringes (B.D), Disposable rubber gloves.

a. Glass wares

Glassware of Pyrex were used to carry out experiments, they were properly washed using detergent, washing powder, chromic mixture, distilled water and organic solvents etc. After washing all the apparatus were dried at 110◦C for two hours in oven of Memmert Ltd. (Germany).

4.2.2. Method 4.2.2.1. Preparation of stock solutions

1.Normal Saline (0.9% w/v NaCl Solution (0.154 M) :-100 ml of 0.9% NaCl solution was prepared by dissolving 0.90 g of pharmaceutical grade sodium chloride in sufficient quantity of water for injection to make the whole volume 100 ml.

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2.2mM Isotonic Solutions of Vanadium and Palladium: Isotonicity of solution is important from physiological point of view and therefore all solutions of metals used during experiments were made to have same osmotic pressure as that of blood except those used in observing the effect of metals on the chemical status of GSH in aqueous media. Therefore all the metals stock solutions and phosphate buffer were made isotonic with the addition of 0.9 percent solution of sodium chloride or a five percent solution of dextrose, before processing the metals with the blood components.

3.0.1M Phosphate buffer PH 7.6 Stock Solution: - 200 ml of phosphate buffer pH 7.6 was freshly prepared in similar way as mention in chapter 2. And was made isotonic by the addition of 0.9 percent solution of sodium chloride.

4.1mM 5, 5-dithiobis (2-nitrobenzoic acid), DTNB/ Ellman’s Reagent:-1mM of 5, 5- dithiobis, 2-Nitrobenzoic Acid (DTNB) (M.W 396.35) was prepared in similar way as mention in chapter 2. This stock solution was then kept in refrigerator till further use.

5.0.1M (Perchloric acid 70%): A 50ml of 0.1M (Perchloric acid purity70%) was prepared in similar way as mention in chapter 2.

6. 2mM of Palladium Nitrate (PDN) and Ammonium Vanadate (AMV) stock solutions were prepared in similar way as mention in chapter 1, and was made isotonic by the addition of 0.9 percent solution of sodium chloride.

7.2mM Bis-benzonitrile) Palladium ii chloride: 50ml of 2mM (Bis-benzonitrile) Palladium II chloride stock solution was prepared in similar way as mention in chapter 2 and was made isotonic by the addition of 0.9 percent solution of sodium chloride.

8. 2mM of Vanadium Oxi-Tri-ethoxide (VOTEO): 50ml of 0.2mM of Vanadium Oxi-Tri- ethoxide (VOTEO) solution was prepared in similar way as mention in chapter1 and was made isotonic by the addition of 0.9 percent solution of sodium chloride.

9. Mediums used in the isolation of WBC’S

I. Histopaque (lymphocyte separation medium):- It is a sterile filtered solution which contains 6.2g ficoll and 9.4g sodium metrizoate per 100ml.

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II. RPMI: - it is a balanced salt nutrition media, RPMI stand for Roswell park memorial institute.

III. Sucrose: - it is used in 20% concentration. It is of pure grade intended for laboratory use.

IV. Foetal calf serum

4.2.2.2. Isolation of WBC Component (Lymphocytes (T-cell and B-cells) and Neutrophils The components of WBC’s were isolated from blood by a technique developed by a scientist terasaki and titled as terasaki technique. This is a density gradient separation technique carried out with a suitable separation mediums creating density-wise separation of WBCs components through centrifugation.

1. Isolation of lymphocytes

10ml of fresh venous blood from healthy human volunteer was taken in heparinized tube. Transformed 4ml of this venous blood slowly to two test tubes separately each containing 4ml of Histopaque, centrifuged at 1800 rpm for 30 minutes. After centrifugation, five distinct layers were produced in the test tubes (Plasma, lymphocytes, Histopaque, neutrophils and RBC’s). The upper layer of plasma was discarded using pipette without disturbing the white cloudy layer of lymphocytes (Leaving behind about 2mm of plasma above the lymphocyte layer). The lymphocytes layer was collected along with half of Histopaque layer in another two centrifuge tubes. Added enough of RPMI to each of these tubes to makeup the final volume 8ml. The lymphocytes layer was further centrifuged at 1800 rpm for 15 minutes, Lymphocytes sedimented at the base of test tubes. The upper layer was discarded .lymphocytes sediment was suspended with 5 ml of RPMI in each test tube. Further 3ml of 20% sucrose was added to the base of each test tube with the help of pipette to remove the platelets from the lymphocytes. Again centrifuged at 700 rpm for 15 minutes, lymphocytes were sedimented. Then the upper layer was discarded. To the remaining clump of lymphocytes, 0.5-1ml RPMI was added in each test tube. Further, added 0.5 ml D/W to the test tubes to lyse RBC’s if any by slightly shaking the test tubes (tapping). The final volume of the tubes was makeup by adding 5-8ml of RPMI to both tubes and centrifuged at 700 rpm for 5 minutes. After centrifugation the upper layer above the

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lymphocytes clump was discarded, lymphocytes sediment were collected, re-suspended with 1- 2ml of RPMI and place on ice both for further use.

Isolation of lymphocytes into its type’s t-cells and b-cells

a. Isolation of t-cells

The lymphocytes obtained were further isolated into its types t-cells and b-cells. For separation of t-cells, upright column was constructed using 1 ml syringe containing nylon wool already incubated at 370C for 30 minutes. The lymphocytes sample was poured slowly into the column and T-cells were collected below the column drop wise in a test tube. The remaining t-cells were collected in the test tube by adding slowly 10ml of RPMI to the column, 50 µl HCl (0.1N) was added to each of this tube to keep GSH intact form (reduced form i.e. GSH). Sealed it tightly and placed on ice bath till further use.

b. Isolation of B-Cells

5-10ml RPMI was added slowly to the above mentioned column. The b-cells had stuck to the nylon wool because of their irregular shape. The b-cells were Collected drop wise in another tube by squeezing the column with the help of a glass rod to an extent until all drops from the column have been collected in the tube. To these collected drops containing the b-cells, 50 µl HCl (0.1N) were added in test tube. Sealed it tightly and placed in refrigerator till further use.

2. Isolation of Neutrophils

The five layers (plasma, lymphocytes, Histopaque, neutrophils and RBC’s) mention above were also obtained for neutrophils and processed in such a way that discarded the upper three layer i.e. plasma, lymphocytes and Histopaque. The neutrophils layer was collected very carefully without disturbing the RBC’s with the help of pasture pipette in two test tubes, each containing about 8ml of RPMI and centrifuged at 700 rpm for 15 minutes. Neutrophils were sediment, discarded upper layer. Further added 0.5-1 ml D/W to the same clump of neutrophils in each of this tube to lyse RBCs, if any, made up the total volume of the tubes equal with RPMI. Then centrifuge at 700 rpm for 5 minutes, discarded the upper layer above the neutrophils clump, combined the neutrophils content of the two tubes with the addition of 50 µl HCl (0.1 N) in order to keep GSH reduced form). Placed it on ice bath till further used.

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4.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method Method of Elman’s modified (1962) was added to estimate Glutathione content of WBCs.

4.2.2.4. Reaction Protocol Preparation of Reaction and Reading Sample for Biological Fluids (WBC’s) With Palladium, Vanadium Salts and Organometalic Complexes

In this section of experiment WBC’s were first isolated into lymphocytes , T-cells, B-cells and neutrophils by centrifugation according to above procedure, then 10 ml of either whole lymphocytes , or T-cells, or , or B-cells or neutrophils were taken in 10 separate test tubes and was mixed with (0.2 to 2mM) of Palladium Nitrate and incubated for 10 minutes. The final concentration of Palladium Nitrate was (10 to 1000µM) respectively. The reading samples were then prepared from each components of the WBC,S in such a way that a series of ten samples cuvettes were prepared by taking 0.5ml of 1mM DTNB from stock solution in 10 separate test tubes. To it added 0.2ml mixture of Palladium Nitrate with either whole lymphocytes , or T- cells, or , or B-cells or neutrophils from each one of the above made test tubes, diluted with 2.3ml of phosphate buffer saline pH 7.6 and incubated for five minutes. The final concentration of Palladium Nitrate was reduced to (6.7µM 13.4µM, 20.1µM, µM, 26.8µM, 33.5µM), 40.2µM (46.9µM), 53.6µM (60.3µM) and 67.0µM) respectively. For control sample 0.2ml of Glutathione content of pure WBC’s components (either whole Lymphocytes or T-cells or B-cells or neutrophils) was added to a separated test tube already containing 0.5ml 1mm DTNB stock solution and the final volume was adjusted to 3ml by the addition 2.3 ml of phosphate buffer saline pH 7.6. The reaction mixture and reading sample for the other metals salts like bis- benzonitrile Palladium (II) Chloride, Ammonium Vanadate, Vanadium Oxi–Tri-ethoxide with Glutathione content either whole lymphocytes or T-cells or B-cells or Neutrophils respectively were prepared in similar way as for the preparation of reaction mixtures and reading samples for Palladium Nitrate and Glutathione content of each plasma and erythrocytes samples. Readings (absorbances) for each of the above mentioned sample of all the metal salts and complexes were then measured on UV- visible spectrophotometer at fixed wavelength at 412nm.The absorbance were then converted into concentration using known standard curve of Glutathione already prepared as mention above chapter: 2.

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4.3. Results 4.3.1. Results of Lymphocytes 4.3.1.1. Effect of Various Conc (6.7 to 67µM) Of PDN / BBNPDC on the Chemical Status of Lymphocytes Glutathione, and with Time (0 to -90 Minutes) Reduced Glutathione (GSH) content of Lymphocytes of the human whole blood was also measured in each tube by Ellman’s method by treating with either Palladium Nitrate/or Bisbenzonitrile Palladium (II) Chloride after the isolation of lymphocytes from blood as shown in the methodology. Again when various concentration (6.7 to 67µM) of either Palladium Nitrate / or Bisbenzonitrile Palladium (II) Chloride was exposed to lymphocytes after their isolation from blood. The level of GSH in lymphocytes before separation was decreased significantly (p<0.001) from (43.5% to 73.8 %) with highest and lowest concentration Palladium Nitrate and was depleted from (28.1% to 64.9 %) with highest and lowest concentration of (Bis – Benzonitrile Palladium (II) Chloride) as compared to control sample of GSH content of lymphocytes, as shown in figure (4.1). The time dependent effect on the GSH content of lymphocytes were also investigated, again when lowest and highest concentrations (6.7 to 67µM) of either Palladium Nitrate/ or its organometallic complex mixtures with lymphocytes were incubated from (0 to 90) minutes and the result were compared with control, then there was further drop in the level of GSH content lymphocytes from (52.15 % to 85.36 %) with lowest and highest conc. of Palladium Nitrate and from (43.15% to 75.36 %) with Bisbenzonitrile Palladium(II) Chloride respectively as shown in Figure (4.2).

30

24

18 ***

12 Conc. of GSH uM GSH Control of Lymph. (µM) 6 Conc. of PDN (µM) Conc. of BBNPDC (µM)

0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 4.1:Conc. dependent effect of (6.7 and 67 µM) of PDN / BBNPC on the chemical status of lymphocytes GSH Results are the mean ±SE of (n= 3) experiments of lymphocytes GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control. 88

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30 Control GSH of Lymph. (µM) PdN 6.7 (µM) PdN 67 (µM) 20 BBNPDC 6.7 (µM) BBNPDC 67 (µM) 10 Conc. of GSH uM *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.2: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of PDN / BBNPC on the chemical status of lymphocytes GSH Results are the mean ±SE of (n= 3) experiments of lymphocytes GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

4.3.1.2. Effect of Two Conc. (6.7 and 67µM) PDN on the Chemical Status Lymphocytes Glutathione with PH (7, 7.6, 8.0) The pH dependent effect on the interaction of lymphocytes GSH content with Palladium was also investigated and when two concentration of Palladium Nitrate was incubated with isolated lymphocytes at various pH (7.0 ,7.6 and 8.0 ) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood then there was an increase in the level of GSH content of lymphocytes from ( 3.4 % to 3.9 % ) and an decrease in the level of GSH content of isolated Lymphocytes from (3.79 % to 4.45 %) with Palladium Nitrate at pH 7 and pH 8 respectively as compared to pH 7.6 (nearly equal to normal physiological pH of the blood), as shown in the figure (4.3).

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Figure 4.3: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of PDN on the chemical status of lymphocytes GSH Results are the mean ±SE of (n= 3) experiments of lymphocytes GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

4.3.1.3. Effect of Various Conc. (6.7 to 67µM) of PDN/BBNPDC on the Chemical Status of B-Cells Glutathione, and with time (0 -90) Minutes

Glutathione (GSH) content of B-Cells was also measured in each tube by Ellman’s method. When various concentration (6.7 to 67µM) of either Palladium Nitrate / or Bis- Benzonitrile Palladium (II) Chloride by treating with the isolated B-cells respectively, the level of GSH in B-cells was decreased significantly (p<0.001) (35.65.7% to 63.58%) and (22 to 55.7 %) with either Palladium Nitrate or Bisbenzonitrile Palladium (II) Chloride as compared to control GSH as shown in the figure (4.4). When Lowest and Highest concentrations were incubated for time interval from (0 to 90) minutes the level of GSH in isolated B-cells was further decreased (48.9% to 82.02 %) and (39.7 % to 69.02%) with either Palladium Nitrate or its Bisbenzonitrile Palladium (II) Chloride respectively as compared to control sample of B-cells, as shown in Figure(4.5).

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18.0

13.5

*** 9.0

Conc. ofGSH uM 4.5 GSH Control of B-cells (µM) Conc. of PDN (µM) Conc. of ( BBNPdC) µM) 0.0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 4.4:Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on the chemical status of B-cells GSH Results are the mean ±SE of (n= 3) experiments of B-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

16

Control GSH of B-Cells (µM) 12 PdN 6.7 (µM) PdN 67 (µM)

8 BBNPDC 6.7 (µM) BBNPDC 67 (µM)

Conc. of GSH (uM) of GSH Conc. 4

*** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.5: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of PDN/BBNPDC on the chemical status of B-cells GSH Results are the mean ±SE of (n= 3) experiments of B-cells GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

4.3.1.4. Effect of Two Conc. (6.7 to 67uM) PDN on the Chemical Status of B- Cells Glutathione with pH (7, 7.6, 8.0) The pH dependent effect on the interaction of GSH content of B-cells with Palladium was also investigated and when various concentration of Palladium Nitrate was incubated with isolated B-cells at various pH (7.0 , 7.6 and 8.0 ) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ), then there was an increased in the

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level of GSH content of B-cells from (3.25% to 4.31% ) with Palladium Nitrate and a drop in the level of GSH content of isolated B-cells from (4.38% to 5.52% ) with Palladium Nitrate at pH 7 and 8 respectively as compared to pH 7.6 (nearly equal to normal physiological pH of the blood), as shown in the figure (4.6).

Figure 4.6: pH dependent effect of lowest and highest conc. (6.7 and 67µM) PDN on the chemical status of B-cells GSH Results are the mean ±SE of (n= 3) experiments of B-cells GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

4.3.1.5. Effect of Various Conc. (6.7 to 67uM) PDN/ BBNPDC on the Chemical Status of T-Cells Glutathione, and with Time (at 0 -90 Minutes), Glutathione (GSH) content of T-Cells was measured in each tube by Ellman’s method. The Absorbances were recorded UV- Visible spectrophotometer. The absorbances were then converted into concentration using Known Standard curve of GSH as shown in Chapter No2. The concentrations were then plotted against control as shown in (figures 4.7 and 4.8). When various concentrations (6.7 to 67µM) of either Palladium Nitrate / or Bis- benzonitrile Palladium (ii) Chloride were exposed to isolated T-cells respectively, the level of GSH content of T-cells was also depleted significantly (p<0.001) from (34.2% to 67.95%) and (18. to 58.87 % ) with either Palladium Nitrate or Bisbenzonitrile Palladium (II) Chloride as compared to control GSH as shown in the figure (4.7). When lowest and Highest concentrations were

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incubated for (0 to 90) minutes the level of GSH in isolated T-cells was further depleted (49.2% to 81.1 %) and (29.0 % to 70.33%) with either Palladium inorganic or its organometalic complexes respectively as compared to control sample of T-cells as shown in the Figure (4.8).

20

16 ***

12

8 GSH Control of T-cells (µM) Conc.of GSH uM 4 Conc. of PDN (µM) Conc. of BBNPdC (µM) 0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 4.7:Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on the chemical status of GSH in T-cells after isolation of T-cells. Results are the mean ±SE of (n= 3) experiments of T-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

20 Control GSH of T-Cells (µM) PDN 6.7 (µM) 15 PDN 67 (µM) BBNPDC 6.7 (µM) 10 BBNPDC 67 (µM)

Conc. of GSH (uM) GSH of Conc. 5 *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.8: Time dependent effect of Lowest and highest Conc. (6.7 and 67 µM) of PDN/BBNPDC on the chemical status of T-cells GSH Results are the mean ±SE of (n= 3) experiments of T-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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4.3.1.6. Effect of Two Conc. (6.7 and 67uM) PDN on the T-Cells Chemical Status Glutathione with pH (7, 7.6, 8.0) The pH dependent effect on the interaction of GSH content T-cells with Palladium was also investigated and when two concentrations of Palladium Nitrate (6.7 and 67µM) were incubated with isolated T-cells at various pH (7.0 , 7.6 and 8.0 ), and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ). Then there was an increase in the level of GSH content of T-cells from (3.74 to 3.93%) and a drop in level of GSH from ( 3.09 % to 4.5% ) respectively at pH 7 and 8 as compared to pH 7.6 (nearly equal to normal physiological pH of the blood) as shown in the figure (4.9).

Figure 4.9: pH dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN on the chemical status of T-cells GSH Results are the mean ±SE of (n= 3) experiments of T-cells GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control. 4.3.1.7. Effect of various Conc. (6.7 to 67µM) AMV on The Chemical Status of Lymphocytes Glutathione and with Time (0-90) Reduced Glutathione (GSH) content of lymphocytes of the human whole blood was measured in each tube by Ellman’s method for Vanadium salt and its organo metallic complex. Again when various concentrations (6.7 to 67µM) of either Ammonium Vanadate/ or its organometallic complexes were exposed to lymphocytes of whole blood after their isolation from whole blood. The level of GSH in lymphocytes was decreased significantly (p<0.001) from (20.5 % to 55%) with highest and lowest concentration of Ammonium Vanadate and was dropped from (5 to 49.2%) with highest and lowest concentration of (Vanadium oxytriethoxide) as compared to

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control sample of GSH content of lymphocytes respectively, as shown in figure (4.10). The time dependent effect on the GSH content of lymphocytes were also investigated again when Lowest and Highest concentrations (6.7 to 67µM) of either Ammonium Vanadate/ or its organometallic complexes mixtures with lymphocytes were incubated (0 to 90) minutes and the results were compared with control sample, then there was further dropped in the level of GSH content of lymphocytes from (29.2% to 74.81 %) with lowest and highest conc. of Ammonium Vanadate and from (29.2% to 74.81 %) with Vanadium Oxytriethoxide as shown in figure (4.11) .

35

28

21 ***

14 ControleGSH of Lymph. (µM)

Conc. of GSH (uM) GSH of Conc. 7 Conc. of AMV (µM) Conc. of VOTEO µM) 0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 4.10:Conc. dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical status of lymphocytes GSH Results are the mean ±SE of (n= 3) experiments of lymphocytes GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. ***P, 0.001, versus control.

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30 Controle GSH of Lymph.(M) 24 AMV 6.7 (M) AMV 67 (M) 18 VOTEO 6.7 (M) VOTEO 67 (M) 12 *

Conc. ofGSH (uM) 6 *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.11: Time dependent effect of lowest and highest conc. (6.7 and 67 µM) of AMV / VOTEO on the chemical status of lymphocytes GSH Results are the mean ±SE of (n= 3) experiments of lymphocytes GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean± SE. **P, 0.001,***P, 0.001, versus control.

4.3.1.8. Effect of Two Conc. (6.7 to 67µM) AMV on the Chemical Status lymphocytes Glutathione with pH (7, 7.6, 8.0) The pH dependent effect on the interaction of lymphocytes GSH content with Vanadium was also investigated and when two concentrations of Ammonium Vanadate was incubated with isolated lymphocytes at various pH (7.0 7.6 and 8.0 ) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ), then there was an elevation in the level of GSH content of lymphocytes from ( 2.87% to 3.9 % ) and an drop in the level from (3.59% to 3.73%) with Ammonium Vanadate at pH 7 and 8 respectively as compared to pH 7.6 (nearly equal to normal physiological pH of the blood) as shown in the figure (4.12).

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Figure 4.12: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV on the chemical status of lymphocytes GSH Results are the mean ±SE of (n= 3) experiments of lymphocytes GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. *P , 0.05, ***P , 0.001, versus control GSH

4.3.1.9. Effect of Various Conc. (6.7 to 67uM) AMV/VOTEO on the Chemical Status B-Cells Glutathione, with, Time (0, 90) Minutes

Reduced Glutathione (GSH) was also measured in each tube by Ellman’s method for Isolated B- Cells. When various concentration (6.7 to 67µM) of either Ammonium Vanadate / or Vanadium Oxi-Tri-ethoxide was exposed to isolated B-cells respectively .The level of GSH in B-cells was decreased significantly (p<0.001) (13.8% to 55.9%) and (9.4 % to 44.3%) with either Ammonium Vanadate or Vanadium Oxytriethoxide as compared to control GSH content of B- cells as shown in the figure (4.13). When various concentration (6.7 to 67uM) Ammonium Vanadate / or Vanadium Oxi-Tri-ethoxide were further incubated for (0-to 90) minutes the level of GSH in isolated B-cells was further decreased (27.6% to 66.5 %) and (21.38 % to 55.63%) with Ammonium Vanadate and Vanadium Oxy-Tri-Ethoxide respectively as compared to control sample of B-cells, as shown in the Figure (4.14).

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16

12 ***

8

Control GSH of B-Cells (µM)

Conc. of GSH (uM) 4 Conc. of AMV (µM) Conc. of VOTEO µM) 0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 4.13:Conc. dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical status of B- cells GSH Results are the mean ±SE of (n= 3) experiments of B-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P<0.001, versus control GSH

18 ControlGSH of B-Cells (µM) AMV 6.7 (M) 12 AMV 67 (M) VOTEO 6.7 (M) VOTEO 67 (M) 6 Conc. of GSH (uM) GSH of Conc. *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.14: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of AMV / VOTEO on the chemical status of B-cells GSH Results are the mean ±SE of (n= 3) experiments of B-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P value 0.001, versus control .

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4.3.1.10. Effect of Two Conc. (6.7 and 67uM) of AMV and on the Chemical Status B-Cells Glutathione, with, pH (7, 7.6, 8.0) The pH dependent effect on the interaction of (GSH) content of B-cells with Vanadium was also studied and when both lowest and highest concentrations of Ammonium Vanadate were incubated with isolated B-cells at various pH (7.0 7.6 and 8.0 ), and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ). Then there was slight elevation in the level of GSH content of B-cells from (3.19% to 4.2 %) and a depletion in the level GSH content of B-cell from (3.43 % to 4.15 %) with Ammonium Vanadate at pH 7 and 8 as compared to pH 7.6 (nearly equal to normal physiological pH of the blood), as shown in the figure (4.15).

Figure 4.15: pH dependent effects of Lowest and highest conc. (6.7 to 67 µM) of AMV on the chemical status of B-cells GSH Results are the mean ±SE of (n= 3) experiments of B-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. *P < 0.05, ***P < 0.01, versus control.

4.3.1.11. Effect of Various Conc. (6.7 to 67uM) of AMV/VOTEO on the Chemical Status T-Cells Glutathione, With, Time (0, 90) Minutes

Reduced Glutathione (GSH) was measured in each tube by Ellman’s method for T-Cells. When various concentration (6.7 to 67µM) of either Ammonium Vanadate or Vanadium Oxi-Tri- ethoxide was exposed to isolated T-cells respectively, the level of GSH in T-cells was decreased significantly (p<0.01) (19.78% to 59.97% ) and (10.41% to 46.87 %) with either Ammonium Vanadate or Vanadium Oxytriethoxide as compared to control GSH as shown in the figure

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(4.16). When various concentration were incubated for (0 to 90) minutes the level of GSH in isolated T-cells was further decreased (28.64% to 76.31 %) and (25.7 % to 67.73%) with either Ammonium Vanadate and or Vanadium Oxytriethoxide respectively as compared to control sample of T-cells as shown in the Figure (4.17).

21

14 ***

Control GSH of T-Cells (µM) 7 Conc. of AMV (µM) Conc. of GSH (uM) GSH of Conc. Conc. of VOTEO µM) 0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 4.16:Conc. dependent effect of (6.7 and 67 µM) of AMV /VOTEO on the chemical status of T- cells GSH Results are the mean ±SE of (n= 3) experiments of T-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

20 Control GSH of T-cells(M) AMV 6.7 (M) 15 AMV 67 (M) VOTEO 6.7 (M) 10 VOTEO 67 (M)

5 Conc. of GSH (uM) ** *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.17: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) of AMV / VOTEO on the chemical status of T-cells GSH Results are the mean ±SE of (n= 3) experiments of T-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. *P < 0.05, ***P < 0.001, versus control.

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4.3.1.12. Effect of Two Conc. (6.7 and 67uM) Of AMV on the Chemical Status T-Cells Glutathione, with, pH (7.0, 7.6, 8.0) The pH dependent effect on the interaction of GSH content of T-cells with Vanadium was also investigated and when two concentrations (6.7 and 67uM) of Ammonium Vanadate was incubated with isolated T-cells at various pH (7.0 7.6and 8.0 ). Again when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ), then there was an increase in the level of GSH content of T-cells from (3.91% to 4.72 % ) and a decrease in the level of GSH content of isolated T-cells from (4.52% to4.93%) with Ammonium Vanadate at pH 7 and 8 as compared to pH 7.6 (nearly equal to normal physiological pH of the blood) as shown in the figure (4.18).

Figure 4.18: pH dependent effect of lowest and highest conc. (6.7 and 67µM) of AMV on the chemical status of T-cells GSH Results are the mean ±SE of (n= 3) experiments of T-cells GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. *P < 0.05, ***P < 0.001, versus control.

4.3.2. Results of Neutrophils

4.3.2.1. Effect of Various Conc. (6.7 to 67µM) PDN /BBNPDC on the Chemical Status Neutrophils GSH, and with Time (At 0 -90 Minutes)

Reduced Glutathione (GSH) content of neutrophils of the human whole blood was also measured in each tube by Ellman’s method, by treating neutrophils with either Palladium /or Bisbenzonitrile Palladium (II) Chloride after their isolation from blood as shown in the methodology. The Absorbances were recorded for each sample and was then converted into

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concentration using standard curve of known concentration as mention in chapter: 2. Again when various concentration (6.7 to 67µM) of either Palladium Nitrate/ or its organometallic complexes was exposed to neutrophils of isolated neutrophils, the level of GSH content of neutrophils was decreased significantly (p<0.001) from (39.4 to 70.8 %) with highest and lowest concentration Palladium Nitrate and was dropped from (31.6 % to 62.6 %) with highest and lowest concentration of (Bis –Benzonitrile Palladium (II) Chloride) respectively as compared to control sample of GSH content of neutrophils as shown in figure (4.19). The time dependent effect on the GSH content of neutrophils was also investigated. Again when two concentration (6.7 to 67µM) of either Palladium Nitrate / or its organometallic complexes mixtures with neutrophils were incubated for time interval (0 to 90) minutes and the result were compared with control, then there was further dropped in the level of GSH content of neutrophils from (48.63.2% to 83.025 %) with lowest and highest conc. of Palladium Nitrate and from (42.0 % to 74.4%) with Bisbenzonitrile Palladium(II) Chloride respectively as compared to control sample of Neutrophils as shown in figure (4.20).

35

28

*** 21

14

Conc. of GSH uM GSH Control of Neut. (µM) 7 Conc. of PDN (µM) Conc. of BBNPDC (µM)

0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 4.19:Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on the chemical status of Neutrophils GSH Results are the mean ±SE of (n= 3) experiments of Neutrophils GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control

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35 Control GSH of Neut. (µM) 28 PdN 6.7 (µM) PdN 67 (µM) 21 BBNPDC 6.7 (µM) BBNPDC 67 (µM) 14

Conc. of GSH (uM) GSH of Conc. 7 *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.20: Time dependent effect of Lowest and highest Conc. (6.7 and 67 µM) of PDN on the chemical status of Neutrophils GSH Results are the mean ±SE of (n= 3) experiments of Neutrophils GSH content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

4.3.2.2. Effect of Two Conc. (6.7 to 67µM) PDN on Neutrophils GSH content with pH (7, 7.6, 8.0).

The pH dependent effect on the interaction of GSH content of neutrophils with Palladium was also investigated. Again when two concentrations of Palladium Nitrate were incubated with isolated neutrophils at various pH (7.0 , 7.6 and 8.0 ) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ), then there was an increase in the level of GSH content of neutrophils from ( 3.34% to 5.39% ) and a drop from (3.49% to 4.35% with lowest and highest concentration Palladium Nitrate at pH 7 and 8 as compared to pH 7.6 (nearly equal to normal physiological pH of the blood), as shown in the figure (4.21).

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Figure 4.21: pH dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN on the chemical status of Neutrophils GSH Results are the mean ±SE of (n= 3) experiments of Neutrophils GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

4.3.2.3. Effect of Various Conc. (6.7 to 67uM) AMV/VOTEO on the Chemical Status of Neutrophils GSH, and with Time (0, 90) Minutes Reduced Glutathione (GSH) content of neutrophils of the human whole blood was measured in each tube by Ellman’s method for neutrophils. By adding either Ammonium Vanadate /or Vanadium oxytriethoxide to neutrophils after their separation from blood as shown in the methodology. Again when various concentration (6.7 to 67µM) of either Ammonium Vanadate/ or its organometallic complexes was exposed to isolated neutrophils. The level of GSH contents of neutrophils was decreased significantly (p<0.001) from (18.78% to 60.75%) with the highest and lowest concentration Ammonium Vanadate and was dropped from (7.4 % to 52.4 %) with highest and lowest concentrations of (Vanadium oxytriethoxide) as compared to control sample of GSH content of neutrophils respectively as shown in figure (4.22). The time dependent effect on the GSH content of neutrophils was also investigated for both inorganic and organometalic salt of Vanadium. Again when lowest and highest concentrations (6.7 to 67µM) of either Ammonium Vanadate/ or its organometallic complexes mixtures with neutrophils were incubated for (0 to 90) minutes, and the results were compared with control sample of Neutrophils. Then there was further drop in the level of GSH content of neutrophils from (25.90

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% to 71.33%) with lowest and highest conc. of Ammonium Vanadate and from (15.5.0 % to 64.16%) with Vanadium oxytriethoxide as shown in figure (4.23).

35

28

21 ***

14 Control GSH of Neut. (µM) Conc. of AMV (µM) Conc. of GSH (uM) GSH of Conc. 7 Conc. of VOTEO (µM) 0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 4.22:Conc. Dependent effect of (6.7 to 67 µM) of AMV / VOTEO on the chemical status of Neutrophils GSH Results are the mean ±SE of (n= 3) experiments of Neutrophils GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

35 Control GSH of Neut.(M) 28 AMV 6.7 (M) AMV 67 (M) 21 VOTEO 6.7 (M) VOTEO 67 (M) 14

Conc. of GSH (uM) 7 ** *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 4.23: Time dependent effect of Lowest and highest conc. (6.7 and 67 µM) AMV / VOTEO on the chemical status of Neutrophils GSH Results are the mean ±SE of (n= 3) experiments of Neutrophils GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. **P < 0.01, ***P < 0.001, versus control.

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4.3.2.4. Effect of Two Conc. (6.7 and 67µM) AMV on The Chemical Status of Neutrophils GSH with pH (7.0, 7.6, 8.0) The pH dependent effect on the interaction of GSH content neutrophils with Vanadium was also investigated. Again when two concentrations of Ammonium Vanadate were incubated with isolated neutrophils at various pH (7.0 , 7.6 and 8.0 ) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ) then there was an elevation in the level of GSH content of neutrophils from ( 3.54% to 4.1% ) and a drop in the level of GSH content of isolated neutrophils from (4.49% to 5.63% with Ammonium Vanadate, at pH 7 and 8 as compared to pH 7.6 (nearly equal to normal physiological pH of the blood), as shown in the figure (4. 24).

Figure 4.24: pH dependent effect of lowest and highest conc. (6.7 and 67µM) AMV on GSH content of Neutrophils Results are the mean ±SE of (n= 3) experiments of Neutrophils GSH content Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. *P < 0.05, ***P < 0.001, versus control.

4.4. Discussion After treating Palladium inorganic and organic salts of palladium and vanadium with WBCS (Lymphocytes T-Cell and B-Cells and Neutrophils our finding showed that there was a depletion in the GSH Content of all components of WBC,S (Lymphocytes T-Cell and B-Cells and Neutrophils) both Concentration wise as well as time wise. The pH study showed that almost optimum depletion takes place at near physiological pH 7.6 as there was a negligible changes ( 3 to 5%) in the depletion level at near Physiological pH 7.6 with respect to maximum and

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Minimum pH (7 and 8). The results of the combine Lymphocytes, (T-cells and B-Cell) showed that both the Lymphocytes T- cells and B-cells offer its Glutathione contents for combating the foreign invaders. It’s also evident from this finding that both the metals and their inorganic/organo metallic complexes can pass through the membrane of the cells and may form conjugate or oxidized the GSH and thus can be removed by the immune system. Our result is also in agreement with other authors who also reported that the level of Glutathione has been decreased in blood by attacking various foreign agents and other diseases including cancer (Dellarovere et al., 2000). The Results from our finding also showed that GSH provides protection of the body by making a complex with Palladium and Vanadium species, thus decreasing the availability of both the metals for toxic effects. The decrease in GSH Contents of lymphocytes and neutrophils after exposure to Palladium and Vanadium species may result in decreased antioxidant capacity. This study further suggested that GSH provides a first line of defense against Palladium and Vanadium induced toxicities. It can be concluded from this study that Glutathione which is an important antioxidant of the body immune system may boosts up white blood cell production to fight infection, particularly the T-cells, and B-cells which are combinely called lymphocytes. It’s also concluded from this study that wbc’s especially T-cells B-Cells and Neutrophils are at the core of our immunity, and tailor the body's immune response to not only pathogens, viral and bacterial infections but also to Heavy metals the cells recognize as being invasive. And thus help the immune system (WBC, s) which may directly attack and destroy foreign agents and guard the body against heavy metals attacks. The results derived from chemical modulation as well as alteration in GSH status in lymphocytes and Neutrophils of human blood caused by heavy metals can be applied for human safety evaluations.

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5.1. Introduction The liver is one of the hardest working and important organs in the body. The highest tissue levels of Glutathione are found in the liver (Monograph GSH, 2002). Here it plays huge roles in deactivating and escorting a wide range of toxins from the body, including the breakdown products from excessive alcohol consumption (Kiefer D, 2005). Of the many jobs the liver performs, detoxification is the liver’s most important. The most essential element used by the liver in this process is Glutathione (GSH). The liver is our ultimate filter and has two critical functions: to process nutrients and eliminate toxins from the body. It does this by cleansing the blood at about two quarts a minute. Large quantities of Glutathione are found the liver where a two-stage detoxification process takes place. Phase One begins the chemical conversion of harmful compounds that are mainly fat-soluble into intermediate forms. But Phase two is where the final transformation takes place to help convert the intermediate toxins into water-soluble substances that can be excreted through the bowel or kidneys. Only water-soluble substances can be excreted. If there is not enough Glutathione to generate the Phase two enzymes, toxins will build to dangerous levels in the liver (Koch D, (2011). In this chapter we set out to investigate the effects of heavy metals Vanadium and Palladium on Liver antioxidant status (the level of Glutathione (GSH)) and its ability to detoxifying and reducing these metals.

5.2. Methodology 5.2.1. Materials Reagents

All reagents were commercially obtained. Ellman’s reagent (5, 5 di-thiobis 2 nitro benzoic acid i.e. DTNB), Bis benzo-nitrile Palladium ii chloride, Ammonium Vanadate, Palladium Nitrate, were purchased from Sigma Aldrich. Sodium Dihydrogen Phosphate (Merck) Sodium Hydroxide, and Vanadium (V) oxytriethoxide, 95% were purchased from (fluka),(10M Perchloric Acid 70% (fluka),Sodium chloride (Merck), Potassium Dihydrogen Phosphate (Merck), Sodium hydroxide(NaOH), Sodium Edetate (Riedel Dehean AG Sleeze Hannover), Dextrose were purchased from (Merck). Distilled Water (Double Refined), PH Meter (NOV-210, Nova Scientific Company Ltd. Korea), UV. Visible Spectrophotometer (Shimadzue, 1601Japan, Magnetic Stirrer, hot plate 400(England), Oven: Memmert Model U-30,854 Schwabach (Germany), Potter-eveljhem homogenizer (japan), Micropipettes 200 µl, 500 µl, 1000 µl 108

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(Socorex Swiss Finland) Centrifuge(H-200,Kokusan Ensink company Japan), Eppendolf’s tubes (Plastic, 10l) , Siliconized Glass test tubes, sterile pyrogen free disposable syringes (B.D), Disposable rubber gloves.

Glass wares

Glassware of Pyrex were used to carry out experiments, they were properly washed using detergent, washing powder, chromic mixture, distilled water and organic solvents etc. After washing all the apparatus were dried at 110◦C for two hours in oven of Memmert Ltd. (Germany).

5.2.2. Method 5.2.2.1. Preparation of stock solutions 1. Normal Saline (0.9% w/v NaCl Solution (0.154 M) :-100 ml of 0.9% NaCl solution was prepared by dissolving 0.90 g of pharmaceutical grade sodium chloride in sufficient quantity of water for injection to make the whole volume 100 ml.

2. 0.1M Phosphate buffer PH 7.6 Stock Solution: - 100 ml of freshly 0.1M phosphate buffer pH 7.6 was prepared by similar way as mention in chapter No2 and was made isotonic with help of 0.9% NaCl solution.

3. 1mM 5, -di-thiobis (2-nitrobenzoic acid), DTNB/ Ellman’s Reagent:-1mM of 5, 5- dithiobis, 2-Nitrobenzoic Acid (DTNB) (M.W 396.35) was prepared by similar way as mention in chapter 2 .This stock solution was then kept in refrigerator till further use.

4. 0.1M (Perchloric acid 70%): 0.1M (Perchloric acid purity70%) was prepared by similar way as mention in chapter: 2.

5. 2mM of Palladium Nitrate (PDN) and Ammonium Vanadate (AMV): 50ml of 2mM Palladium Nitrate (PDN) and Ammonium Vanadate (AMV) stock solutions were prepared by similar way as mention in chapter: 2 and were made isotonic with help of 0.9% NaCl solution.

6. 2mM Bis-benzonitrile) Palladium ii chloride: 50ml of 2mMBis-benzonitrile) Palladium ii chloride stock solution was prepared by similar way as mention in chapter 2 and was made isotonic with help of 0.9% NaCl solution.

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7. 2mM Vanadium Oxi-Tri-ethoxide (VOTEO):50ml of 2mM Vanadium Oxi-Tri- ethoxide (VOTEO) was prepared by similar way as mention in chapter 2 and was made isotonic with help of 0.9% NaCl solution.

8. 2mM Isotonic Solutions of Vanadium and Palladium: - Isotonicity of solution is important from physiological point of view and therefore all solutions of metals used during experiments were made to have same osmotic pressure as that of blood except those used in observing the effect of metals on the chemical status of GSH in aqueous media. Therefore all the metals stock solutions and phosphate buffer were made isotonic with the addition of 0.9 percent solution of sodium chloride or a five percent solution of dextrose, before processing the metals with the blood components.

9. 0.5M and 5mM Disodium Edetate Solutions:-10ml of 0.5M disodium Edetate (Molecular weight =372.2) solution was prepared by dissolving 1.861g of disodium Edetate (EDTA-2Na) in 10ml of distilled water. Then from this solution 200 ml of 5mM disodium Edetate Solution was prepared by diluting 2ml of the above solution to 200ml with distilled water.

10. 0.5M and 5mM Disodium Edetate Solutions:-10ml of 0.5M disodium Edetate (Molecular weight =372.2) solution was prepared by dissolving 1.861g of disodium Edetate (EDTA-2Na) in 10ml of distilled water. Then from this solution, 200 ml of 5mM disodium Edetate Solution was prepared by diluting 2ml of the above solution to 200ml with distilled water.

5.2.2.2. Preparation Liver Homogenate sample a. Isolation of whole liver

The rat was anesthetized with diethyl ether. The fully anesthetized Rabbit was decapitated with gluiton, the abdominal cavities was opened with scissors to expose large area. The liver was carefully cut and washed with 0.9% saline to remove blood and blotted. After washing, dried and weighed the livers. Three rabbit were used to gain average weight of livers. The weight obtained was 150mg. b. Isolation liver homogenate (schiefer, 1985)

20 % (w/v) homogenate was prepared of the above collected liver in 5% TCA (trichloro-acetic acid) solution and 1mm EDTA, using a potter-eveljhem homogenizer with motor driven Teflon 110

Chapter:5

pestle. Centrifuged homogenate at 2,000*g for 15 minutes and collected protein free supernatant. Added 50 µl HCl (0.1 N) to the supernatant to keep GSH in reduced form. Sealed it tightly and placed it on ice till further use. To obtain clear spectra of absorbance upto 1.6 the homogenate obtained was diluted four times with phosphate buffer saline pH 7.6.

5.2.2.3. Estimation of GSH by Ellman’s (DTNB Modified) Method Method of Elman’s modified (1962) was followed to estimate Glutathione of liver Homogenate. The detail procedure is mention in chapter 2.

5.2.2.4. Standard Calibration Curve

Standard curve of known concentration of Glutathione was constructed as shown in chapter: 2

5.2.2.5. Reaction Protocol Preparation of reaction mixture and reading sample for liver homogenate

In this section sample preparation for treating Liver Homogenate Sample with either Palladium or Vanadium inorganic salts or its organo-metallic Complexes was prescribed in detailed as follow

Liver homogenate was first prepared, and then GSH content of liver homogenate was isolated by centrifugation according to the above mention procedure. Then 10 ml of GSH content of liver homogenate was mixed with 10 ml of different concentrations (0.2-2mM) of Palladium Nitrate, the final concentration of Palladium Nitrate, (10 to 1000µM) respectively. The reading samples were then prepared from each tube in such a way that a series of ten samples cuvettes were prepared by taking 0.5ml of 1mM DTNB from stock solution in 10 separate test tubes. To it added 0.2ml of mixture of Palladium Nitrate Glutathione contents of liver homogenate from each one of the above obtained test tubes, diluted with 2.3ml of phosphate buffer saline pH 7.6 and incubated for five minutes. The final concentration of Palladium Nitrate was (6.7µM, 13.4µM, 20.1µM, µM, 26.8µM, 33.5µM), 40.2µM (46.9µM), 53.6µM (60.3µM) and 67.0µM respectively. For control sample 0.2ml of Glutathione content of pure liver homogenate was added to a separated test tube already containing 0.5ml 1mm DTNB stock solution and the final volume was adjusted to 3ml by the addition 2.3 ml of phosphate buffer saline pH 7.6. The reaction mixture and reading sample for the other metals salts like bis-benzonitrile Palladium (ii) chloride, Ammonium Vanadate, Vanadium Oxi–Tri-ethoxide with Glutathione content liver homogenate were prepared in similar way as for the preparation of reaction mixtures and reading 111

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samples for Palladium Nitrate and Glutathione content of Liver homogenate samples. Readings (absorbances) for each of the above mentioned sample of all the metal salts and complexes were then measured on UV- visible spectrophotometer at fixed wavelength at 412nm.

The absorbances were then converted into concentrations using known standard curve of Glutathione already prepared mention in chapter: 2.

5.3. Results 5.3.1. Results of Liver Homogenate GSH wit Palladium Compounds 5.3.1.1. Effect of Various Conc. (6.7 to 67uM) PDN/ BBNPDC on Liver Homogenate GSH content, and With Time at (0 -90) Minutes), The standard Elman’s protocol was applied to liver homogenate sample according to methodology. Again when GSH content of liver homogenate was exposed to different concentrations (6.7µM to 67µM) of either Palladium Nitrate /or Bisbenzonitrile Palladium (II) Chloride respectively in liver Homogenate after centrifugation and the results were compared with control sample GSH content of liver homogenate (without metal salt and complexes) the level of GSH content was also decreased significantly (p<0.001) from (43.6% to 72.62%) with Palladium Nitrate and from (24.09 to 59.5%) with Bis benzonitrile Palladium II Chloride as compared to control as Shown in figure (5.1). A further decreased was observed in the level of GSH content when time of incubation was increased from 0 to 90 minutes from (49.7 to 87.1%), with Palladium Nitrate and from (29.3% to 67.6%) by addition Palladium organometalic complexes respectively after centrifugation, as compared to control as shown in figure (5.2) .

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65

52

39 ***

26

Conc.of uM GSH GSH Control of L.HG. (µM) 13 Conc. of PDN (µM) Conc. of BBNPDC (µM)

0 0 14 28 42 56 70 Final Conc. of PDN/PDBBC in (uM)

Figure 5.1:Conc. dependent effect of (6.7 to 67 µM) of PDN/BBNPDC on GSH content of liver homogenate Results are the mean ±SE of 3 experiments of (GSH) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

65 Control GSH of L.HG (uM)

52 PdN 6.7 (µM)

PdN 67 (µM) 39 BBNPDC 6.7 (µM) 26 BBNPDC 67 (µM) Conc. of GSH uM GSH of Conc. 13

0 *** 0 30 60 90 Time of Incubation (Minutes)

Figure 5.2: Time dependent effect of Lowest and highest Conc. (6.7 and 67 µM) of PDN / BBNDC on GSH content of liver Homogenate Results are the mean ±SE of 3 experiments of (GSH) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

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5.3.1.2. Effect of Two Concentrations (6.7 and 67uM) PDN on Glutathione content of Liver Homogenates with pH at (7, 7.6, 8.0) The pH dependent effect on the interaction of liver homogenate GSH content with Palladium was also investigated and when various concentration of Palladium Nitrate was incubated with liver homogenate ( after centrifugation) at various pH (7.0 7.6and 8.0 ) and when the results were compared with pH 7.6 (which is nearly equal to normal physiological pH of blood ) then there was elevation of GSH content of liver homogenate from (4.32% to 5.4 %), and depletion from (4.6 % to 5.8 %) with Palladium Nitrate at pH 7 and 8 as compared to pH 7.6 (nearly equal to normal physiological pH of the blood) as shown in the figure (5.3) .

Figure 5.3: pH dependent effect of lowest and highest Conc. (6.7 and 67µM) PDN on the chemical status of GSH content Liver Homogenate Results are the mean ±SE of 3 experiments of (GSH liver Homogenate content) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

5.3.2. Results of Liver homogenates GSH with Vanadium compound

5.3.2.1. Effect of Various Conc. (6.7 to 67uM) AMV/VOTEO on the Chemical Status of Liver Hepatocytes GSH, and With Time (At 0 -90 Minutes) Again when GSH content of liver homogenate was exposed to different concentrations (6.7uM to 67uM) of to either Ammonium Vanadate or Vanadium Oxy-Tri-ethoxide respectively in liver Homogenate after centrifugation and the results were compared with control sample of liver homogenate GSH content (without metal salt and complexes), the level of GSH content was also decreased significantly (p<0.001 from (11.5% to 53.2%) with Ammonium Vanadate, while the

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level of GSH content was dropped from (6.2% to 46.0 %) by the addition Vanadium Oxi-Tri- ethoxide as compared to control GSH content of liver homogenate respectively as shown in (figure:5.4). A further decreased was observed in the level of GSH content when time of incubation was increased from (0 to 90) minutes from ( 25.6 to 71.4%) by Ammonium Vanadate and from (16.4% to 60.6%) by addition of Vanadium Oxi-Tri –Ethoxide respectively as compared to control after centrifugation as shown in figure (5.5).

60

*** 40

GSH Control of L.HG (µM) 20 Conc. of AMV (µM) Conc. of GSH uM GSH of Conc. Conc. of VOTEO µM)

0 0 14 28 42 56 70 Final Conc. of AMV/VOTEO in (uM)

Figure 5.4:Conc. Dependent effect of (6.7 to 67 µM) of AMV/ VOTEO on the chemical status of GSH content liver homogenate Results are the mean ±SE of 3 experiments of (GSH Liver Homogenate) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control.

65 Control GSH of L.HG (M) 52 AMV 6.7 (M) AMV 67 (M) 39 VOTEO 6.7 (M) VOTEO 67 (M) 26

Conc.of (uM) GSH 13 * *** 0 0 30 60 90 Time of Incubation (Minutes)

Figure 5.5: Time effect of Lowest and highest conc. (6.7 and 67 µM) of AMV/ VOTEO on the chemical status of GSH of liver Homogenate. Results are the mean ±SE of 3 experiments of (GSH Liver Homogenate) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. ***P < 0.001, versus control. 115

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5.3.2.2. Effect of Two Conc. (6.7 to 67uM) AMV on The Chemical Status of Liver Homogenate GSH With pH (7.0, 7.6, 8.0) The effect of pH (7.0, 7.6, and 8.0) on the chemical status of GSH was also investigated. Again it was found that minute increase i.e. (3.7% to 4.72%) in the content of GSH of liver homogenate at pH. 7.0 as compared to PH 7.6 (which is near to the normal physiological pH of the body) in the mixture sample of liver homogenate GSH content with Ammonium Vanadate occurred. While a negligible decrease i.e. (3.1 to 4.9% ) in the content of liver homogenate GSH content occurred, when the sample mixtures of liver homogenate, with Ammonium Vanadate was incubated at pH 8 as compared to pH 7.6 (which is near to normal physiological pH of the body) as shown in figure (5.6).

Figure 5.6: pH dependent effect of lowest and highest conc. (6.7 and 67µM) lowest and highest conc. (6.7 and 67µM) AMV on the chemical status of GSH of Liver Homogenate Results are the mean ±SE of n= 3 experiments of (GSH content of liver Homogenate) Values were compared statistically by using One way ANOVA followed by dunnett test. The error bars indicate the mean SE. *P < 0.01, ***P < 0.001, versus control.

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5.4. DISCUSSION GSH forms metal complexes via non-enzymatic reactions (Ballatori, 1994). GSH is one of the most versatile and pervasive metal binding ligands and plays an important role in metal transport, storage, and metabolism. GSH works (a) in the mobilization and delivery of metals between ligands, (b) in the transport of metal across cell membranes, (c) as a source of cysteine for metal binding, and (d) as a reductants or cofactor in redox reactions involving metals. The sulfhydryl group of the cysteine moiety of GSH has a high affinity for metals, forming thermodynamically stable but kinetically labile mercaptides with several metals, including mercury, silver, cadmium, arsenic, lead, gold, zinc, and copper (Rannug, 1980; Van Bladeren et al., 1981; Rannug et al., 1978).

The concentration, time and pH dependent effect of Palladium and Vanadium was also extended to liver homogenate. Liver homogenate was isolated from rabbit liver through centrifugation and both metals were treated as mention in experimental procedure. When various concentration (6.7to 67uM) of the Palladium and Vanadium were treated with the liver homogenate GSH content a consistent decrease in the level of Glutathione was observed both concentration wise as well as time wise for (0 to 90). The results obtained when compared to plasma, Cytosolic fraction and wbc’s it was observed that prominent change in the depletion of Glutathione content in liver homogenate was also seen compared with control of Liver homogenate, suggesting that these metals have the same affinity for thiols in all the biological samples. It was also revealed from the result that both metals have affinity for SH group of proteins, but the affinity of Palladium is higher than Vanadium. Also when the samples were examined at various pH, Ranging from (7.0, 7.6 and 8), there was also a negligible differences in the depletion level at pH 7 ,8 and 7.6 nearly physiological pH of the blood. It further suggested that most of the depletion takes place at the physiological pH of the blood. The depletion of in the GSH content of Liver Homogenate may be attributed to the fact that Palladium and Vanadium being electrophilic compounds, having the property to facilitates them to react with vital cellular nucleophiles possessing SH, NH2 and OH groups and GSH being a cellular non-protein sulfhydryl molecule, which on treating with Palladium and Vanadium is accompanied by its significant depletion in cells by reacting with SH group of Glutathione. This may be resulted in formation of Glutathione S-conjugates or conversion of GSH into GSSG which is the initial step in the biotransformation of electrophiles. On the basis of the results obtained we conclude that both Vanadium and Palladium interactions are involved in the induction of oxidative stress under the exposure to

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these substances. The disturbances in the oxidative status observed in our experimental model may indicate a risk of liver damage during exposure to Vanadium and/or Palladium via the free radical mechanisms. It appears that our metal compounds lower hepatic GSH level via extra hepatic action. So our primary experiments and the subsequent available results suggested that liver homogenate suspension may be a useful model for Metallo-elements toxicity in the future. These observations also showed that Metallo-elements may cause or induce toxicities to various organs of the body including hepatotoxicity. As our Metallo-element complexes are structurally and oxi-datively different and they cause depletion of hepatic GSH by different extents. Suggesting that they were potentially more are less toxic. Also the detoxification mechanism of Glutathione of heavy metals like Palladium and Vanadium metalloelements is an important tool that may contribute to counteract metal induced toxicity. Therefore the study of Palladium and Vanadium interaction with Liver homogenate content may provide a new concept in toxicology that may attract future research in several chemical induced injuries, also these finding may aid in the development of better therapeutic approach to manage chemical exposure in certain situations.

On the bases of the results obtained from aqueous medium, blood components and liver homogenate we proposed the following reaction of GSH with Palladium and Vanadium.

+2 Pd + 2GSH Pd (GS) 2 + 2H+

Or

Pd +2 + 2 G SH Pd + GSSG oxidized Glutathione)

+ VO2 + GSH V –(SG)+ H2O

Or

+ VO2 + 2GSH GSSG + VO + H20

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6.1. Introduction Ellman’s reagent (5, 5’-dithiobis (2-nitrobenzoic acid)) has been an important reagent for the spectrophotometric assay of sulfhydryl groups in biology for many years (Elman’s 1962; Riddles et al., 1979 and Riddles et al., 1983). The accuracy of the analysis is based on controlling the two related equilibria (equation 1) which form when treating Ellman’s reagent (ESSE) with thiolate (RS-). Great care must be taken to avoid the involvement of the second exchange reaction and consequently the assay is typically conducted using an excess of Ellman’s reagent. However, if Ellman’s reagent is present at too high a concentration during the analysis, the - deconvolution of the bands in the spectra (ESSE, λmax 325 nm: ES , λmax 412 nm) becomes difficult and the accuracy of the analysis is compromised. The solubility of Ellman’s reagent and the pKa of the sulfhydryl groups also require some attention. Ellman’s reagent is only sparingly soluble in water below a pH of 7. Furthermore, if the pH rises above 8.5, the solution develops an intense colouration which cannot be reversed (Danehy et al., 1971). Fortuitously, the important thiolate species typically studied (e.g. Glutathione, human serum albumin) have an appropriate pKa (~8.5) which makes analysis at physiological pH possible.

K1 K2

ESSE + 2RS-  ESSR + RS- + ES-  RSSR + 2ES- -(1)

The application of the spectrophotometric Ellman’s assay to more complex mixtures, specifically where there is the potential for competition for the sulfhydryl groups, can be problematic. This situation arises with the speciation of metals in biological mixtures, where there is the possibility of dynamic exchange between species bound to the sulfhydryl groups with the parent ligand, solvent (water) or buffer (Rejlinski et al., 1988). In these instances there is concern that the Ellman’s reagent can perturb the equilibrium and compete for the coordinated thiolate as well as the “free” sulfhydryl present in solution. The consequences of this will be an over-estimation of the amount of free sulfhydryl present in the system.

We design two phases of experiment in NMR analyses

1st Phase of NMR analysis: In first phase of NMR analysis we were interested to confirm that what types of species are produced during the interaction of thiols with Elman’s reagent during quantification of the thiols with Ellman’s reagent. 119

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2nd phase of NMR analysis: As in our Spectrophotometric analysis of thiols, with either Palladium and or Vanadium interaction we observe the depletion of thiols with the addition of transition metals, so in second phase of NMR analysis, we plane to confirm that the depletion in the level of thiol, is whether due to the thiol metal complexation, or the metals convert the thiols into its thiols mixed disulphide.

We procedue our observation with Palladium, as Palladium is known to form a range of metals chelate complexes with biologically relevant thiolates (Cervantes et al., 1998; Stypinski-Mis and Anderegg, 2000; Yoshinari et al.,2009 and Freeman et al., 1974) in this metal the coordination sphere of the metal is made up from a sulpher and nitrogen from the ligands

6.2. Materials and Methods All reagents were commercially obtained. Ellman’s reagent was purchased from Sigma Aldrich. 1H NMR spectra were obtained using a Bruker AVANCE 3 spectrometer operating at 400.12 MHz. Samples were maintained at 300 K during spectral acquisition. The free induction decay was generated by a 3.13 s pulse width corresponding to a 30o pulse with a 2 sec delay between pulses. Each data set was collected in 32 k of memory. A 1 Hz line broadening function was applied before Fourier transformation to reduce the effect of the baseline noise.

6.2.1. NMR assignments for Ellman’s reagent, Ellman’s anion and the thiolate mixed disulfides of Ellman’s reagent N-acetylcysteine: Ellman’s reagent (10 mg, 25.2 μmoles) was dissolved in 1.5 ml of buffer (0.1 2 M KH2PO4 in H2O at pH 7.4). 300 μL of this solution were placed in an NMR tube (0.5 mm diameter). Solutions (500 μl) containing 0.4 mg (2.45 μmoles), 0.8 mg (5.90 μmoles), 1.2 mg (7.35 μmoles), 1.6 mg (11.8 μmoles)and 2.0 mg (12.26 μmoles) of N-acetylcysteine were prepared. These were added to sequentially to the NMR tubes containing the Ellman’s reagent. The solutions were mixed and the NMR spectra recorded. An additional sample of Ellman’s reagent 1 mg in 800 μL was prepared as a reference sample.

D-penicillamine: Ellman’s reagent (12 mg, 30 μmoles) was dissolved in 1.8 ml of buffer (0.1 M 2 KH2PO4 in H2O at pH 7.4). 300 uL (5 μmoles) of this solution were placed in an NMR tube (0.5mm diameter). Solutions (500 μL) containing 0.19 mg (1.27 μmoles), 0.38 mg (2.54 μmoles), 0.57 mg (3.81 μmoles), 0.76 mg (5.08 μmoles), 1.13 mg (7.57 μmoles), and 1.5 mg (10

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μmoles) of D-penicillamine were prepared. These were added to sequentially to the NMR tubes containing the Ellman’s reagent. The solutions were mixed and the NMR spectra recorded.

6.2.2. Calibration of the thiol disulfide exchange by NMR methods

2 Ellman’s reagent (1.0 mg/300 μL, 2.5 μmole, 0.1 M KH2PO4 in H2O at pH 7.4) was placed in

0.5 mm diameter NMR tube. A series of Glutathione solutions were prepared in 0.1 M KH2PO4 2 in H2O at pH 7.40 viz 0.155 mg (0.2 μmole), 0.31 mg (0.4 μmoles), 0.462 (0.6 μmoles) 0.62 (0.8 μmoles and 0.775 mg (1.0 μmoles) each in 500 μL. These were added sequentially to the NMR tubes and the NMR spectra recorded. An additional sample of Ellman’s reagent 1 mg in 800 μL was prepared as a reference sample. The spectra obtained were processed as described above. The integrals of the key resonances were used to calculate the relative amount of Ellman’s reagent, Ellman’s anion and the Glutathione- Ellman’s mixed disulfide present in the samples.

6.2.3. Interaction of Palladium with thiolates A series of mono and Bis thiolate-Palladium (Glutathione, D-penicillamine, N-acetylcysteine) solutions were prepared in-situ 0.1MKH2PO4 in H2O at PH 7.4 using Palladium Nitrate dehydrate and either one or two equivalents of corresponding thiolate. By dissolving1.33mg of Palladium Nitrate in 5 ml volumetric flask and 1.54mg of Glutathione, .082mg of NAC and .76mg of D-penicillamine and then these solution were mixed in 1:2 and 1:1 Palladium Nitrate and thiols in 0.8ml NMR Test tube respectively and the 1HNMR spectras were recorded.

6.3. Results and discussion 6.3.1. Effect of PH on the chemical status of Elman’s reagent PH of the buffer in which the Ellman’s reagent is prepared is also known to be critical. At high pH’s the Ellman’s reagent is known to disproportionate to form a mixture of Ellman’s anion and Ellman’s sulfinate (equation 2). This process was investigated using NMR methods by allowing the pH of the solution to rise above 8 by replacing the buffer with deuterated sodium hydroxide. The spectrum reveals the formation of two Ellman’s based species in a ratio of 3:1 as predicted by (Danehy et al) The dominant resonances to high field (7.2, 7.3 and 7.75) are assigned to Ellman’s anion consistent with the data in figure (6.1). The remaining resonances which come to lower field (7.15, 7.65, and 8.15) are thus assigned to the corresponding sulfinate. Treating the solution with hydrogen peroxide

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we can engineer the consumption of the Ellman’s anion and force the reaction to completion - thus generating a solution which contains only the sulfinate (ESO2 ). The ability to identify the presence of the sulfinate in the solution and thus the integrity of the solution to be used for analysis is another distinct advantage of NMR over spectrophotometry.

2ESSE + 4OH-  3ES- + ESO2- + 2H2O – (2)

Figure 6.1:The 400 MHz 1H NMR spectra of (top) 1mg/ml Ellman’s reagent treated with NaOD solution and (bottom) the same solution treated with hydrogen peroxide. Trace amounts of Ellman’s reagent can be observed in the baseline of the peroxide treated solution (bottom).

6.3.2. Exchange reaction of thiolates (N- Acetylcysteine Glutathione and D- penicillamine), with Elman’s reagent (ESSE) The NMR analysis of solutions generated by titrating thiolate; N-acetylcysteine (NAC), figure 6.2, ) reduced Glutathione (GSH) figure (6.3) and D-penicillamine (PEN) figure (6.4) into Ellman’s reagent clearly allows the identification of Ellman’s anion (ES-), Ellman’s reagent (ESSE) and the mixed disulfide (ESSR) in the 1H NMR spectra. The resonances from Ellman’s anion appear to higher field due to the effect of the distributed charge on the ring protons (figure 6.2, table 6.1). As expected the resonances from the reagent itself and the mixed disulfide appear in a similar region of the spectrum. There is a slight overlap of certain resonances from each Ellman’s based species but, in the main, the assigned resonances are distinct and can be used to calculate the relative concentration of the three species in solution. Thus a calibration curve can be generated by titrating a thiolate solution into a solution of Ellman’s reagent. By plotting the relative integrals of the resonance assigned to Hc (or Hb) in Ellman’s anion ( 6.3.2) against the amount of thiolate used we obtain a graph (figure 6.5) which is linear and which is consistent with what is observed spectrophotometrically. The intrinsic error in the integrals derived from NMR spectra is much higher than those derived from optical methods and it is unlikely that NMR will replace spectrophotometry for quantitative measurements. The mixed disulfide

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(ESSR, R = thiolate) and homoleptic disulfides (RSSR) are also dominant features in the high field region ( 4.2 -  2.0) of the spectra (figure 6.2, 6.3) making it possible to simultaneously - observe the five components (ES , ESSE, ESSR, RSSR and RSH) of the complex equilibria (K1,

K2: equation 1). The spectra clearly show that, at higher thiolate concentrations, the reaction of

Ellman’s reagent with N-acetylcysteine and Glutathione extends into the second equilibrium (K2, equation 1). Thus, once all the Ellman’s reagent is consumed these thiolates react further with their corresponding mixed disulfides to form the homoleptic disulfide (RSSR) and a further aliquot of Ellman’s anion. In contrast D-penicillamine, due to the steric problems with the methyl groups adjacent to the thiol, is only capable of participating in the first equilibrium (K1, equation 1) and even at high D-penicillamine concentrations the mixed disulfide persists (figure 6.4). Thus, it would seem that NMR spectroscopy can provide a more complete and detailed view of the reaction under study.

O O - - O N ES ESSE ESSR ESO2

Hc Ha Doublet J1 = 2 Hz  7.20  7.56  7.57  7.65 HO

Hb Quartet J1 = 2 Hz  7.31  7.62  7.68  7.74 Ha Hb

S J2 = 8.8 Hz

Hc Doublet J2 = 8.8 Hz  7.75  8.00  8.03  8.15 Table 6.1: The chemical shifts of the Ellman’s based species formed in the thiol- disulfide equilibrium (equation 1) using N-acetylcysteine and its alkaline hydrolysis. The data is obtained from the spectra shown in figure 1 to 3. Typical coupling constants are given

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Figure 6.2: The 400 MHz 1H NMR spectras obtained by titrating a solution (300 μl) of Ellman’s reagent (10

2 mg/1.5 mL, 0.1 M KH2PO4 in H2O at pH 7.4) with Top: N-acetylcysteine. (a) Ellman’s reagent, (b) 0.4 mg N- acetylcysteine, (c) 0.8 mg N-acetylcysteine, (d) 1.2 mg N-acetylcysteine, (e) 1.6 mg N-acetylcysteine and (f) 2.0 mg N-acetylcysteine

Figure 6.3: The 400 MHz 1H NMR spectras obtained by titrating a solution (300 μl) of Ellman’s reagent (10 2 mg/1.5 mL, 0.1 M KH2PO4 in H2O at pH 7.4) with Glutathione) (a) (0-mg) Glutathione, (b) 0-mg) Glutathione, (c) 0-mg) Glutathione, (d) 0-mg Glutathione, (e) (0-mg) Glutathione.

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Figure 6.4: The 400 MHz 1H NMR spectras obtained by titrating a solution (300 μl) of Ellman’s reagent (10 2 mg/1.5 mL, 0.1 M KH2PO4 in H2O at pH 7.4) with D-penicillamine. (a) 0.15 mg D-penicillamine, (b) 0.30 mg D- penicillamine, (c) 0.45 mg D-penicillamine, (d) 0.60 mg D-penicillamine and (e) 1.12 mg D-penicillamine.

Figure 6.5: The calibration graph for the reaction of Glutathione with Ellman’s reagent obtained

using the relative areas of the integrals derived from Hc.

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6.3.3. Interaction of Palladium with Glutathione In order to confirm the interaction of Palladium with Glutathione again 1:2 and 1:1 Complexation of Palladium Nitrate with Glutathione were generated in situ and the H1NMR spectras were generated on H-NMR 400Hz. The NMR Spectra of solutions of GSSG and Plane GSH in 7.4 pH 0.1M phosphate buffer was also generated in order to compare the structural differences in the reduced and oxidize Glutathione with thiolate Palladium mixture spectras. In H-NMR, the amino acids protons of the reduced Glutathione give signals at 6 different chemical shifts. The cysteine residue of the reduced Glutathione shows signals at 3.0ppm and 4.6 ppm in case of oxidized Glutathione, β-cysteinyl residue split in such away, that two identical quadrates are produced due to chiral center giving signals at (3 to 3.5ppm), while the ά-cysteinyl residue of oxidized Glutathione disappeared due to water of oxidation. In case of Palladium GSH complex the β-cysteinyl residue also split into two quadrate between (2.6ppm and 3.5ppm, but not identical like homoleptic disulphide of oxidized Glutathione as shown in table (6.2), and figure (6.6) and (6.7). From this finding it is suggested that Palladium combine with the cysteine residue of the Glutathione and Palladium cysteinyl complex is formed which bring changes in the resonances of the βcysteinyl residual proton environment and thus split into new peaks, which is different from both cysteine, residue of reduced and oxidized Glutathione, this finding is in agreement with another data stated by other author who showed that arsenic Glutathione complexes AS(GS)3 displays a very familiar distributed eight line pattern between(3.2 and 3.35) indicative of two cysteinyl connected as a disulphide or cis-metal thiolate(Scott et al.,1993, Raab et al., 1993&percy et al.,2008). In 1:1 Palladium GSH reaction again we see splitting of signals of cysteinyl residue to some complex peak and also changes in the all other peaks of GSH, suggesting that Palladium may form some complex structure in which both sulfur as well as nitrogen of two different carbons are bonded which bring conformational changes in the environments of all the residual protons of the entire species figure (6.7). This study is also supported by literature that Palladium may form 1:2 as well as more complex structures with thiols (D-penicillamine) in which Palladium is attached to sulfur of carbon of one amino acid and nitrogen of the next in cluster form (Cervantes et al., 1998). From this finding it is concluded that Palladium may deplete the level of Glutathione by making complex with it.

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SNo Chemical shift, No of protons Assignment of species ppm ()

1 4.6 2 Cys (CάH2)

2 3.0 2 Cys (CβH2)

3 3.91 2 Glu (CάH2)

4 2.5 2 glu (CɤH2)

5 2.1 2 Glu (CβH2

6 4.09 2 gly (CH2)

Table (6.2): Summary of Distribution of chemical shifts for the 1H‐NMR spectrum of Glutathione

Figure 6.6: The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of Glutathione 0.1 2 M KH2PO4 in H2O at pH 7.4) with a Pd (NO3)2 (forming Pd (SGH) 2 Complex in situ

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Figure 6.7: The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of Glutathione 0.1 2 M KH2PO4 in H2O at pH 7.4) with a [Pd (NO3)2 (forming Pd (SGH) 2 Complex in situ.

6.3.4. Interaction of Palladium with N-Acetylcysteine Again N-Acetylcysteine plane solution and 1:2 and 1:1 Complexation of Palladium Nitrate and

N-Acetylcysteine spectras were generated on in phosphate buffer in D2O pH 7.4 on a Bruker 400MHz H-NMR instrument. The resulting spectrum are shown in table (6.3) and Figures (6.9,) and (6.10) that showed that a singlet band was observed at chemical shifts of 1.37ppm integrated for three protons, of the methyl groups, attached to the carbon of N- acetyl group. A singlet at 4.4 ppm, integrated for one proton, of the CH attached carboxylic function group. A singlet at 2.9 ppm, integrated for one proton, of the CH attached SH- functional group. Results showed that the Palladium 1:2 and 1:1 exhibited the same pattern of splitting of the cysteinyl residue of the N-acetylcysteine as in Glutathione because in 1:2 reaction the cysteinyl residue at 2.9ppm split into two quadrates and in the 1:1 Palladium N-Acetylcysteine reaction, a more complex peak was observed also the Acetyle peak showed changes due to complex structure, produced during 1:1 reaction of Palladium and N-Acetyle cysteine. From this finding it is suggested that Palladium form both 1:1 and 2:1 complex with N-acetylcysteine. The pattern of splitting of cysteinyl residue was almost similar as in Glutathione.

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Figure 6.8: Structure of N-Acetyle cysteine

S.No Chemical shift, Number of Assignment ppm protons

1.37 3 Actyl-CH3

2.9 2 Cystenyl-CH2

4.4 1 Carboxyl-CH

Table: (6.3.) Summary of Distribution of chemical shifts for the 1H‐NMR spectrum of N-acetylcysteine

Figure 6.9: The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of N- acetylcysteine 0.1 M

2 KH2PO4 in H2O at pH 7.4) with a Palladium Nitrate (2:1) formed in-situ.

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Figure 6.10: The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of N- acetylcysteine 0.1 M

2 KH2PO4 in H2O at pH 7.4) with a Palladium Nitrate1:1 formed in-situ.

6.3.5. Interaction of Palladium with (D) ‐penicillamine

1 The H‐NMR spectrum of (D) ‐penicillamine in D2O Phosphate buffer pH7.4 was obtained on a Bruker 400 MHz instrument. The resulting spectrum is shown in Table 6.4 and Fig 6.11 and 6.12) that showed that two singlet bands were observed at chemical shifts of 1.37 and 1.55 ppm, each of which integrated for three protons, of the two methyl groups, attached to the carbon of cysteinyl group. A singlet at 3.59ppm integrated for one proton, of the CH function group. When the thiolate Palladium spectra was compared with the control D-pencillamine then a shifting of the resonances of CH3 group from 1.37ppm to lower ppm and splitting of the resonance of CH3 group attached to the SH group at 1.45ppm into two resonance at (1.5 and 1.6ppm) occurs. Also the resonances of the CH group split into two resonance at 3.59 ppm into (3.7ppm, 2.2ppm) occur in both 1:1 and 1:2 reactions. When the 1:1 and 1:2 resonances were compared then there were differences of the shifting in the entire signal in both spectras of 1:1 and1:2 complexes, it is confirmed from this results that Palladium making both 1:1 and 1:2 complexation with D-pen.

As the Pd (D-pen) and Pd (D-pen) 2 have different proton environment so the observed signals of both species were different. This study is in agreement with the study of an another author who showed that Palladium makes both simple 1:2 as well as multiple dentate complexes with D- pencillamine anion, in which Palladium atoms form trimer of an isosceles triangle with thiols in which Each pair of metal atoms in the trimer is bridged by a penicillamine anion which is linked via its amino group to one Palladium atom via its carboxylate group to the other and via the Sulfur atom to both. From theses finding we concluded that NMR spectroscopy is a valuable tool

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in studying thiol-disulphide exchange process using Ellman’s reagent. Although not as accurate as the spectrophotometric assay, NMR spectroscopy has value for the study of complex mixtures as it can identify additional species in solution. Also NMR is a valuable tool in identifying the species during the interaction of thiols with metals, in biological fluids and aqueous solutions of the thiols and metals like Palladium, complexation of the thiols with Palladium occur instead of conversion of the thiols into a mixed disulfide.

S.No Chemical shift, Number of protons Assignment ppm

1 1.37 3 CH3

2 1.55 3 CH3

3 3.59 1 CH

Table.6.4: Summary of Distribution of chemical shifts for the 1H‐NMR spectrum of (D) ‐ penicillamine

Figure 6.11: The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of D-penicillamine with Pd

2 (NO3)2 0.1 M KH2PO4 in H2O at pH 7.4) with a Pd (D-pen) 2 and formed in-situ

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Figure 6.12: The 400 MHz 1H NMR spectra (ns = 64) obtained by titrating solutions of Glutathione 0.1 M

2 KH2PO4 in H2O at pH 7.4) with a [Pd (D-pen)] n, and formed in-sit

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7.1. Introduction Sulfhydryl groups are widely distributed across biological systems being found in range of small molecules (e.g. Glutathione, Homocysteine) and proteins (e.g. albumin, haemoglobin). In the circulating plasma albumin is consider to be the major thiol containing protein. Albumin contains a single thiolate group positioned at cysteine-34(cys-34) in the first domin of the protein. While a large proportion of the protein circulates in the thiolate from a significant amount is found in a disulfide form; bound to either Glutathione or cysteine. Many studies, however, focus on the thiolate form of the protein and the status of cys-34) is routinely assayed in clinical studies of diseases involving oxidative stress. Albumin also binds a wide variety of metals and metals complex is at various sites around the protein. Those metals which are classified as soft (e.g. gold) are known to preferentially bind to cys-34. Consequently this site is seen as a sink for both toxic and therapeutic heavy metal compounds.

The binding of soft metals at cys-34 provides a mechanism by which the residence time of potentially toxic species in the body can be increased. However, while many studies have sought to study the oxidative modification of, and metal binding capacity of cys-34 few studies have studied the ease with which it is possible to effect disulfide-thiol exchange at this sites/or remove a metal bound at this position. The premise is that the slow exchange of species bound to cys-34 is the basis for a mechanism by which toxic species can become widely distributed around the body. In this study we have sought to briefly investigate these issues. Albumin labelled using Ellman’s reagent has been prepared and used to demonstrate the cleavage of protein mixed disulphide. In the second phase of study we have metalated albumin with soft metals (Palladium and Vanadium). These modified proteins have subsequently been challenged with thiolate (Glutathione, N-Acetylcysteine, D-penicillamine) in an attempt to remove the metal and regenerate cys-34.

7.2. Experimental Procedure All reagents were commercially obtained. Elman’s Reagent, Bovine serum Albumin (>98%) agarose gel electrophoresis lyophilized) and Sephadex (G25 coarse) were purchased from Sigma Aldrich. U.V-visible spectra were recorded on a Unicam U.V.300 spectrophotometer.

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7.2.1. Preparation of Standard curve for BSA solution A 50uM solution of Bovine Serum albumin (BSA) was prepared by dissolving 16.75mg of BSA in 5ml of (0.1 M KH2PO4, pH 7.4), then 5 different dilution i.e. 10,20,30,40,50 from this stock solution were prepared by the addition of further (0.1 M KH2PO4, pH 7.4) to stock solution. UV spectrum (200-600nm) of the solution was recorded after each addition. Plotting the absorbance of the solution at λ280 nm as a function of BSA concentration gives a straight line (R2=.993) with an intercept (0.0911) derived from the natural background of fluorescence of the albumin solution. The unknown concentration of BSA in the BSA –Pd and BSA-V mixtures were then calculated on the bases of molecular weight, extent coeffecient of BSA and using beer lambert law as shown in Figure 7.1.

7.2.2. The calculation of free thiolate content of BSA, Elman’s reagent (13.2mg, 3ml in BPS was titrated into solution of Albumin (1.98mg, 50µl) (the UV spectrum (200-600nm) of the solution were recorded after each addition. Plotting the absorbance of the solution at λ412 nm as a function of Ellman’s reagent concentration gives a straight line (R2=.993) with an intercept (0.0911) derived from the natural background of fluorescence of the albumin solution. The thiolate content of albumin is obtained from the concentration of the Ellman’s anion released for BSA based on a molecular weight of 66,000.

7.2.3. Preparation of Elman’s modified BSA (BSA-SSE) Solutions of BSA (200mg, 1mL) and Ellman’s reagent (1mg, 1ml) in PBS (0.1 M KH2PO4, pH 7.4) were mixed and allowed to react overnight. The solution was carefully applied to a column (10 cm x 2 cm) packed with swollen Sephadex (G25 coarse). The mixture was eluted with PBS. The appearance of the protein in the eluent was identified by testing the liquors with tri- chloroacetic acid (which precipitates denatured protein) whereupon collection commenced. Periodic sampling identified when the eluent was protein free. The residual Ellman’s reagent and anion (identified as a yellow band) eluted second and were washed from the column with further aliquots of PBS. The concentration of protein in solution was calculated using Beers Law at (max max = 280 nm;  = 43,824 cm-1M-1) (Peters, 1975.)

7.2.4. Treatment of BSA-SSE with thiolates The protein solution collected above was diluted by taking 1ml of elute and adding 4ml of ml (0.1 M KH2PO4, pH 7.4) in order to generate a solution of known concentration (typically 60µM) which produced a solution with an absorbance at 280 nm of approximately 2.5. The 134

Chapter: 7

spectrum (200 – 600nm) was recorded. This solution was titrated with thiolate (reduced Glutathione, N-acetyl cysteine and penicillamine) with spectra being recoded after each addition.

The release of Ellman’s anion is assess at max max = 412 nm ( = 14,150 cm-1M-1).

7.2.5. Preparation of Elman’s modified BSA with Palladium and Vanadium Solutions of BSA (200mg/1mL) and Palladium Nitrate (1mg /1ml), and 1mg /1ml) Ammonium Vanadate in PBS (0.1 M KH2PO4, pH 7.4) was mixed and allowed to react overnight respectively. The solutions were carefully applied to a column (10 cm x 2 cm) packed with swollen Sephadex (G25 coarse). The mixture was eluted with PBS. The appearance of the protein in the eluent was identified by testing the liquors with tri-chloroacetic acid (which precipitates denatured protein) whereupon collection commenced. Periodic sampling identified when the eluent was protein free. The residual free Palladium Nitrate, Ammonium Vanadate eluted second due to their smaller molecular sizes and was washed from the column with further aliquots of PBS. The concentration of protein in solution was calculated using Beers Law at

(max max = 280 nm;  = 43,824 cm-1M-1) (Peters, 1975).

7.2.6. Treatment of BSA-Pd and BSA-V with thiolates The modified Palladium Vanadium BSA protein were freeze dried in order to reduce the volume of water content up to 1.5 ml and the mixed with thiolate ((reduced Glutathione, N-acetyl cysteine and penicillamine). Again pass through the column in order to remove the free thiols if any.

The modified protein (BSA-Pd, BSA-V) solutions collected above were diluted by taking 1ml of elute and adding 4ml of ml (0.1 M KH2PO4, pH 7.4) in order to generate a solution of known concentration (typically 60µM) which produced a solution with an absorbance at 280 nm of approximately 2.5 The spectrum (200 – 600 nm) was recorded. These solutions were then titrated with Elman’s reagent the spectras were being recoded after each addition. The release of

Ellman’s anion is assess at max max = 412 nm ( = 14,150 cm-1M-1).

7.3.1. Results and discussion The thiolate form of bovine serum albumin (BSA) typically comprises ~30 - 50% of the protein in the commercially available material. This value varies from batch to batch necessitating the calculation of the relative amount of thiolate present on the protein before the study can commence. The thiolate status of the BSA was assessed by titrating a solution of Ellman’s

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reagent (13.2mg/3ml) with BSA (1.98mg/ml in 5ul aliquots). The Ellman’s anion released was measured spectrophotometrically at 412 nm ( = 14,150 cm-1M-1). Plotting the amount of Ellman’s anion released as the BSA concentration increases allows us to compensate for the natural absorbance of albumin at 412 nm (figure 7.1) and hence gain an accurate value for its thiolate status. Using this approach the BSA used here was found to be 30 % in the thiolate form

 BSA-S- + ESSE   BSA-SSE + ES- -(1)

Figure7. 1: The titration of BSA (13.2 mg/3 ml) with Ellman’s reagent (1.98 mg/ml 5 ul).The expected deviation from linear behaviour at high Ellman’s reagent concentrations (150, 1.98mg/ 5µl) is evident. An intercept is found which is consistent with residual absorbance by BSA at 412 nm. The assay method not only releases Ellman’s anion but generates a stoichiometric amount of BSA labelled with an Ellman’s moiety at cysteine-34 (BSA-SSE, equation 1). Taking advantage of this reaction it is possible to synthesis significant amounts of BSA which has been capped with an Ellman’s moiety. Thus incubating Albumin with a small excess of Ellman’s reagent overnight (based on the thiolate assay, figure 7.1) followed by chromatographic separation (Sephadex G25) we obtained a solution of Ellman’s modified BSA in PBS. The solution was desalted and freeze dried to give BSA-SSE (~50% ES form). Alternatively, the molar absorbtivity of the solution at 280 nm was to be used to give a suitable estimate of the protein concentration in the eluent sample. We were interested here in the ability of small thiolate species (Glutathione, D-penicillamine, N-acetylcysteine) to react with cys-34 in its disulfide form and as such we opted to work with the protein solutions.

Treating BSA-SSE with either Glutathione or N-acetylcysteine we observed the rapid release of a commensurate amount of Ellman’s anion (equation 2, figure 7.2) indicating that an exchange has taken place. In contrast the reaction with D-penicillamine produced no Ellman’s anion.

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Studies reported elsewhere have shown that D-penicillamine does not exchange with its mixed disulfide of Ellman’s reagent to form penicillamine disulphide due to steric problems. This observation suggests that the nature of the protein pocket acts to prevent the release of entities bound at cys-34. This observation, however, is also an important control for the reactions involving Glutathione and N-acetylcysteine. A result similar to that shown in figure 7.2 might be expected from an exchange of thiolate with Ellman’s reagent loosely bound (H-bonded, or hydrophobically associated) to the protein or which remains in solution as a result of poor chromatographic separation. If either of these situations were present an exchange reaction D- penicillamine would be expected.

Figure 7.2 :( Top): The titration of BSA-SSE 200uM) with Glutathione solution (200uM) the formation of Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm. Middle: The addition of a single aliquot of D-penicillamine (200uM), Glutathione (200uM) and N- acetylcysteine (200uM) to a BSA-SSE solution (200uM). Bottom: Ellman’s anion is not released from the protein by incubation with D- penicillamine.

 (BSA-SSE + GS-  BSA-SSG + ES- - (2)

 BSA-SSE + NACS-  BSA-SSENAC + ES- - (2)

 BSA-SSE + D-pen-  No exchange - (2)

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7.3.2. Calibration curve for the calculation of unknown concentration of Albumin in Palladium or Vanadium BSA mixture

Figure 7.3: Calibration curve of BSA at 280 nm

7.3.3. The exchange reactions of either Palladium /or Vanadium with either Glutathione, or N-Acetylcysteine, or D-penicillamine The exchange reactions of Palladium and Vanadium with Glutathione, N-Acetylcysteine- penicillamine are shown in figure 7.4, to 7.9 respectively. From the figures it was evident that Albumin gave absorbance at 280nm, Elman’s reagent gave absorbance at 325nm while there was almost no absorbances at 412nm at which TNB anions give absorbances usually produced during the interaction of free SH group of Albumin with Elman’s reagent (ESSE). The result suggested that both Palladium and Vanadium form stable complexes with the thiol group of the albumin which could not be broken by smaller thiols like GSH, NAC, and D-penicillamine as shown from figure 7.4 to 7.9). The data in this study also suggested that Palladium and Vanadium showed more toxicity on protein level by forming somewhat strong coordination which could not be exchanged by free thiolate group like Glutathione N-acetylcysteine and D-penicillamine usually present in the biological fluids. On the other hand it can be anticipated from this model of the study that besides the propound toxicity of either Palladium or Vanadium towards GSH, Vanadium or Palladium can also render the albumin malfunctioning. Albumin being present in rich concentration in plasma may not play its critical physiological roles in the living system in high Palladium and Vanadium environment.

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3 2.5 Alb‐pd + GSH 50uM 2 +ESSE50uM 1.5 Alb‐pd + GSH 40uM +ESSE40uM 1 Absorbances Alb‐pd + GSH 30uM 0.5 +ESSE20uM 0 Alb‐pd + GSH 20uM 250 350 450 +ESSE20uM wave length nm

Figure 7.4: The titration of BSA-Pd 50uM) with Glutathione solution (50uM) the formation of No Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm.

2.5 2 Alb‐pd + NAC 20uM + ESSE 20uM 1.5 Alb‐pd + NAC 30uM 1 + ESSE 30uM Absorbance 0.5 Alb‐pd + NAC 40uM + ESSE 40uM 0 Alb‐pd + NAC 50uM 260 360 460 + ESSE 50uM wavelength

Figure 7.5: The titration of BSA-Pd 50uM) with N- Acetylcysteine solution (50uM) the formation of No Ellman’s anion (equation 3) is evident from the appearance of a band at 412 nm.

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2 1.8 1.6 Alb‐Pd +D‐pen 50uM 1.4 + ESSE 50uM 1.2 1 Alb‐Pd +D‐pen 40uM 0.8 + ESSE 40uM

Absorbances 0.6 Alb‐Pd +D‐pen 30uM 0.4 + ESSE 30uM 0.2 Alb‐Pd +D‐pen 20uM 0 + ESSE 20uM 250 350 450 Wavelength nm

Figure 7.6: The titration of BSA-Pd 50uM) with D-penicillamine (50uM) the formation of No Ellman’s anion (equation 4) is evident from the appearance of a band at 412 nm.

3

2.5 Alb‐v + GSH 30uM + 2 ESSE 30uM Alb‐v + GSH 40uM + 1.5 ESSE 40uM

Absorbance 1 Alb‐v + GSH 50uM + ESSE 50uM 0.5 Alb‐v + GSH 60uM + 0 ESSE 60uM 240 340 440 Wavelength nm

Figure 7.7: The titration of BSA-V 50uM) with Glutathione solution (50uM) the formation of No Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm.

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3 Alb‐V + NAC (30uM) + ESSE (30uM)

Alb‐V + NAC (40uM) + ESSE (40uM)

2 Alb‐V + NAC (50uM) + ESSE (50uM)

Alb‐V + NAC (60uM) + ESSE (60uM)

Absorbance 1

0 250 300 350 400 450 500 wavelength (nm)

Figure 7.8: The titration of BSA-Pd 50uM) with N-Acetylcysteine solution (50uM) the formation of No Ellman’s anion (equation 2) is evident from the appearance of a band at 412 nm.

3.000

2.500 Alb‐V + D‐pen 2.000 20uM + ESSE 20uM

1.500 Alb‐V + D‐pen 30uM + ESSE 30uM

Absorbance 1.000 Alb‐V + D‐pen 0.500 40uM + ESSE 40uM

0.000 Alb‐V + D‐pen 250 350 450 50uM + ESSE 50uM λmax (nm)

Figure 7.9: The titration of BSA-Pd 50uM) with D-penicillamine solution (50uM) the formation of No Ellman’s anion (equation 2) is evident from the appearance of a band at 412nm

141

Albumin Palladium exchange reaction

 +2  + (BSA-SH)+ Pd (BSA-S)2-Pd + 2H - (3)

 -  (BSA-S)2-Pd + NACS No exchange - (3)

 -  (BSA-S) 2-Pd + NACS No exchange - (3)

 (BSA-S) 2-Pd + D-pen-  No exchange - (3)

Albumin Vanadium exchange reaction

 +  2(BSA-SH) +VO2 2(BSA-S)-V + H2O - (4)

 (BSA-S)-V+ GSH  No exchange - (4)

 (BSA-S)-V + NAC  No exchange - (4)

 (BSA-S) -V + D-pen-  No exchange - (4)

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8. Conclusion Humans are exposed to Vanadium mainly through the polluted atmosphere from combustion products of Vanadium bearing fuels fumes and dust food contain a very low contents of and Vanadium usually below in ng/g. This metal enters the organisms mainly through inhalation, skin and bone saesand to be lesser extent also through lungs. In case of Palladium general population is primarily exposed to Palladium through dental alloys, jewellery, food (present in tissues of small aquatic invertebrates, different types of Meat, fish, bread and plants) and emissions from automobile catalytic converters. Several in vivo and in vitro studies suggested that exposure of experimental animals to inorganic or organic forms of Vanadate and Palladium accompanied by the induction of oxidative stress. Our findings provide an insight into the role of Palladium, Vanadium metal-induced toxicity and carcinogenesis. The results provide evidence that toxic and carcinogenic metals are capable of interacting with nuclear proteins and DNA causing site-specific damage. The “direct” damage may involve Conformational changes to biomolecules due to the coordinated metal while “indirect” damage is its consequence. The current study of thiols with Palladium and Vanadium revealed that the primary route for their toxicity is depletion of Glutathione and bonding to sulfhydryl groups of proteins. Glutathione has been shown to be a significant factor in heavy metal mobilization and excretion, specifically with application to Palladium and Vanadium. Glutathione depletion and Glutathione supplementation have specific effects on vanadate toxicity, both by altering antioxidant status in the body and by directly affecting excretion of vanadate and other heavy metals in the bile. Thus Glutathione is an important antioxidant in biological fluids which makes conjugation with Palladium and Vanadium and detoxifies these metals. On the basis of the results obtained we conclude that Vanadium and Palladium interactions with thiols are involved in the induction of oxidative stress under exposure to these substances. The disturbances in the oxidative status may be a result of an independent effect of Vanadium and /or Palladium. Since the human blood components and liver homogenate of rabbits GSH content treated with Vanadium and /or Palladium had reduced their level of GSH content by making complexation that might result oxidative stress. The disturbances in the oxidative status observed in our experimental model can be present as a model of in-vivo.

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List of Research Publications

1. Muhammad Muhthiar, Mohammad Farid Khan, Haroon Khan, Naseem Ullah, Asim.ur.Rehman, Evaluation of interaction of Vanadium with Glutathione in blood components. Pak. J. Pharm. Sc. (2012). (Impact factor 1.13)

2. Muhammad Mukhtiar, Muhammad Farid Khan, Naseem Ullah, Seyed Umer Jan, Haroon Khan. Evaluation of the Interaction of Vanadium with Glutathione in Human Blood Components Pak. J. Pharm. Sc. (2012).(Impact factor 1.13).

3. Naseem Ullah, Muhammad Farid Khan, Muhammad Mukhtiar. Human Blood GSH, As a Tool for Arsenic Detoxification Afr. J. Pharm. Pharmacol 6(1)17-23. (2012). (Impact factor 0.667).

4. Haroon Khan, Mohammad Farid Khan, Naseem Ullah, Muhammad Mukhtiar, Concentration and time dependent effects of Silver metal on the chemical status of Glutathione in plasma of Human Blood., African Journal of Biotechnology (2012).

5. Arshad farid , Abdul Haleem Shah, Mohammad Mukhtiar Protective role of Glutathione in Tungsten induced toxicity in blood components , pharmacological and Toxicological perspective., IJRAP (2011)

6. Naseem Ullah, Muhammad Farid Khan, Muhammad Mukhtiar Metabolic Modulation of Glutathione in Whole Blood Components against Lead Induced Toxicity A. Jour. Biotech 10(77), 17853-17858 (2011).

7. Haroon Khan, Mohammad Farid Khan, Naseem Ullah, Muhammad Mukhtiar, Naheed Haque, Barkat Ali, Abdul Wahab, Arshad Farid, Kamal Shah. Effect of Aluminium Acetyl Acetonate on the Chemical Status of Glutathione by Influential Parameters in Aqueous Medium. IJBMSP, 1(1);23-26 (2011)

8. Javed Khan, Muhammad Farid Khan, Haroon Khan, Barkat Ali Khan, Naseem Ullah, Naheed Haque, Muhammad Mukhtiar and Arshad Farid Complexation of BaCl2 with Glutathione (GSH) in blood components, AJPP (2012) (Impact Factor 0.667).

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9. Haroon Khan, Muhammad Farid Khan, Barkat Ali Khan, Naseem Ullah, Muhammad Mukhtiar and Arshad Farid Metabolic changes of Glutathione in human T and B lymphocytes induced by organo-aluminum complex, Afr. J. Pharm. Pharmacol. (2013).(Impact Factor 0.667).

Submitted Research Papers in International/ Local Journal

1. Evaluation of the Interaction of Vanadium Oxi-Tri-Ethoxide with Glutathione in Human Blood Components (Plasma and Erythrocytes).

2. The Interaction of Bis benzonitrile Palladium (II) Chloride with GSH in Human Blood Components (Plasma and Erythrocytes).

3. Formation of an L-γ-glutamyl-L-cysteinyl-glycine-Vanadium Organometallic Complex in Isolated T-cells and B-cells of Human Blood.

4. Palladium Inor/ Organic Complex formed Conjugated Product with a tripeptide GSH in Neutrophils of Leukocytes.

5. Vanadium Inor/ Organic Complex formed Conjugated Product with a tripeptide GSH in Neutrophils of WBC, S.

6. Lead organic and inorganic complex changed the metabolic Status of GSH through Conjugation in Neutrophils.

7. The exchange reactions of either Palladium /or Vanadium with either Glutathione, or N- Acetylcysteine, or D-penicillamine

8. Reactivity of Ellman’s reagent with Complexed thiolates.

9. Interaction of Vanadium with Biological molecule (Glutathione) in Liver Homogenate.

10. Palladium Interaction with Glutathione content of biological fluid Liver Homogenate.

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