TO STUDY THE EFFECT OF SALTS AND COMPLEXES OF LEAD AND ARSENIC METALS ON THE STATUS OF THIOLS IN BLOOD COMPONENTS, PHARMACOLOGICAL AND TOXICOLOGICAL PERSPECTIVES

Abstract

Heavy and transition metals have been widely known for their toxicities for centuries. Arsenic and Lead are highly valued in terms of toxicity to living systems due to their high affinity for sulfhydryl (-SH) containing residues. The chemical interactions of Arsenic and Lead with thiols (–SH rich molecules) results in numerous ill-health effects. As a part of this PhD thesis, the chemical status of GSH was determined in the presence of inorganic and organic complexes of lead and arsenic employing simple spectrophotometric analysis and 1H NMR methods. The behavior of Ellman’s reagent towards the metal-thiol chemistry was initially investigated to begin with the study of the changed status of thiols resulting from metal-thiol coordination. Chapter 2 describes the use of NMR methods to study the species in solutions. 1H NMR allowed us to identify additional components of the reaction mixture, their relative abundance and consequently the involvement of these additional Ellman’s based species in the spectrophotometric errors associated with the use of Ellman’s reagent. Using 1H NMR methods we are able to show that Ellman’s reagent can exchange with thiolates previously coordinated with lead and arsenic. In the context of this 1H NMR study, some limitations were found to be associated with the use of Ellman’s reagent in our study. Ellman’s reagent was found able to react with the thiols being previously attached with arsenic and lead, leading to an over estimation of the thiols in the solution systems. 4,4′-dithiodipyridine (DTDP) was adapted instead of Ellman’s reagent for the spectrophotometric determination of the mixtures involving metal-thiols complexes in aqueous solution and biological mixtures after establishing limitations associated with the use Ellman’s reagent in this capacity of the study. Chapter 3 describes the 1H NMR carried out to study the conditions and ratios of the complexed thiolates (complexes of both arsenic and lead with some important low molecular weight thiols (Glutathione, N. acetyl cysteine and D-Penicillamine). 1H NMR study presented in this chapter provides detailed information about the changes in the chemical status of GSH that might be the basis of this chemical change observed spectrophotometrically in the form of depletion. 1H NMR methods confirmed metal-thiol adducts (i.e., As-SG3 and Pb-SG2) to be the mechanism behind the GSH depletion in the presence of these metal compounds. After establishing the limitation associated with the use of Ellman’s reagent, 4,4′- dithiodipyridine was used for the spectrophotometric determination of the unbound thiols in the presence of complexed thiolates in aqueous solutions. In the course study presented in XII chapter 4, we have spectrophotometrically investigated the reactions of arsenic and lead with sulfhydryl containing thiols i.e., Glutathione, N-Acetyl cysteine and D-Penicillamine in aqueous solutions. In this chapter, the effects of arsenic (Sodium arsenite and Methylarsonous acid) and lead (Lead acetate and Lead acetyl acetonate) on low molecular weight thiols (Glutathione, N. acetyl cysteine and D-Penicillamine) have been spectrophotometerically quantitated in aqueous solutions. Employing influential variables (i.e., different metal concentrations, incubation times and pH ranges) to the study in aqueous solutions, prominent and regular decrease in the thiol levels were caused by each of the inorganic and organic compounds of arsenic and lead in spectrophotometric analysis. Chapter 5 describes the spectrophotometric estimation of important and most abundant low molecular weight thiol (Glutathione) in the whole blood and blood components (cytosolic Fraction and plasma) of human volunteer. In order to improve our understanding of the coordination chemistry of arsenic and lead in whole blood, cytosolic fraction and Plasma, a systematic study of the changes in glutathione (GSH) levels in these biological samples of healthy human volunteers, has been carried out. The effects of the inorganic and organic compounds of arsenic and lead on glutathione status in these biological samples have been spectrophotometerically evaluated by 4,4′-dithiodipyridine. Chapter 6 describes the spectrophotometric estimation of Glutathione in WBCs (Lymphocytes and Monocytes) isolated from human blood, while the study carried out in chapter 7 describes the estimation of Glutathione in liver isolated from rabbits. Chapter 8 describes the exchange of arsenic (AsIII) and lead (PbII) between Proteins (Albumin) and low molecular weight thiols (Glutathione, N. acetyl cysteine and D- Penicillamine). In addition to thiol disulfide exchange reactions, the exchange of arsenic and lead between Proteins (Albumin) and low molecular weight thiols (Glutathione, N. acetyl cysteine and D-Penicillamine) has also been investigated by Column chromatographic methods using Ellman’s reagent. The exchange behaviour of arsenic and lead between proteins and low molecular weight thiols have been analyzed spectrophotometrically by making use of Ellman’s reagent. Column chromatographic methods have been used to collect complexed proteins. The sulfhydryls present on the pure and complexed proteins have been estimated spectrophotometerically. The study regarding the thiol disulfide exchange reactions resulted that the low molecular weight thiolates (Reduced glutathione and N-acetylcysteine) take off As(III) and Pb(II) species which are attached to proteins. XIII Results from multiple studies presented in this PhD thesis are consistent to conclude and anticipate that lead and arsenic species are dynamic in nature as in the case of using Ellman’s reagent, thiolates could be removed from the coordination sphere of the arsenic and lead in As(SR)3 and Pb(SR)2 respectively and in the case involving albumin, the slow exchange of lead and arsenic species bound to cys-34 is the basis for a mechanism by which toxic species can become widely distributed around the body.