Abstract
Many halogenated xenobiotics such as the environmental contaminant trichloroethylene are detoxified by conjugation to glutathione. Subsequent metabolic processing yields the cysteine conjugate of the original compound which may then serve as a substrate for cysteine conjugate β-lyase (glutamine transaminase K, GTK). In rats bioactivation of certain cysteine conjugates by β-lyase results in toxicity specific to the P3 segment of the renal proximal tubule whereas in humans, the nephrotoxicity is less acute, however, excessive exposure to halogenated alkenes can result in specific neurotoxicity and neurodegeneration. As an approach to studying the species difference toxicity we have isolated and transiently expressed in COS-1 cells, full length cDNAs encoding rat and human cysteine conjugate β-lyase (Perry et al., 1993 & 1995). In this thesis I examine the differences in enzyme activity observed with the transiently expressed rat and human β-lyase cDNAs. Rat β-lyase expressed in COS-1 cells showed lower activity than the human enzyme, it also possessed a 5’ non-coding region containing an inverted repeat, the removal of which did not increase the activity of the expressed truncated rat cDNA. This would suggest that the inverted repeat was not interfering with mRNA translation and that differences in activity between rat and human β-lyase were due to some other reason. In order to assess the structure-activity relationships of rat and human cysteine conjugate β-lyase, their full length cDNAs in the expression vector pUS1000 were stably incorporated into the pig kidney epithelial cell line, LLC-PK1. Confirmation of the stable retention of the transfected cDNAs and the continuing high level expression was obtained using Southern blot analysis and enzyme activity assays. The cell lines stably expressing rat and human cysteine conjugate β-lyase were then used to assess the in vitro toxicity of sixteen different cysteine conjugates using a tetrazolium salt (XTT -sodium 3’-[l-(phenylamino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulphonic acid hydrate) method to assess mitochondrial integrity. The kinetics of the metabolism of several cysteine conjugates by cytosolic extracts from the stable cell lines expressing rat and human cysteine conjugate β-lyase were examined. It would appear that those cysteine conjugates which are good substrates for cytosolic cysteine conjugate β-lyase are not necessarily the most toxic. Again, those haloalkenes containing fluorine groups appear to be better substrates for cysteine conjugate β-lyase than those containing chlorine groups. The cysteine conjugates DCVC and PCBC would appear to be preferentially metabolised by a mitochondrial enzyme, since they exhibit greater cytotoxicity in the wild type LLC-PK1 cells than in the cell lines stably transfected with rat and human cytosolic cysteine conjugate β-lyase. This data would suggest that the cytosolic β-lyase in the stable cell lines is preventing some of the DCVC and PCBC from getting to its mitochondrial target. Those conjugates which are metabolised within the mitochondria have a far greater toxic effect than those which do not. These data suggest that the site of metabolism of the cysteine conjugate determines the degree of cytotoxicity observed.