Adaptation to a chemically challenging environment is a critical aspect of the evolutionary process. As a result a wide variety of systems involved in xenobiotic metabolism have evolved. Two such systems are the phase I (drug toxification) Cytochrome P-450 monooxygenases and the phase II (drug detoxification) glutathione S-transferases. In order to evaluate the role of specific gene products in chemical toxicity and mutagenicity members of each of these groups have been expressed in the lower eukaryote <i>S.cerevisiae</i> and the mutation frequency and sensitivity to a wide variety of cytotoxic agents examined. A cDNA encoding the rat P-450IIB1 protein has been expressed to a level of between 0.1-0.2% of total yeast protein. The protein was localised to the microsomal fraction and found to be functional as determined by activity towards the model substrate, benzyloxyresorufin. An attempt was made to develop this P450IIB1 expressing strain as a viable short term test (STT) for the screening of potential mutagens. To this end the strain was exposed to a series of known mutagens all requiring metabolic activation by cytochrome P-450 to exert their effects. An increase in mutation frequency, as determined by resistance to L-canavanine, was obtained when exposed to the anticancer drug, cyclophosphamide and the mycotoxin, sterigmatocystin. The PB-inducible cytochrome P-450's have been implicated in the metabolism of these chemicals. The specificity and potential of this P450IIB1 expressing strain and the possibilities for the expression of cytochrome P-450's in <i>S.cerevisiae</i> as an STT is discussed. The overexpression of the glutathione S-transferases (GST) has been associated with the drug resistance. In order to directly evaluate this possibility the human alpha (B<SUB>1</SUB>B<SUB>1</SUB>) and pi class GST have been expressed in <i>S.cerevisiae</i> either singly or in tandem. These strains were then exposed to a variety of alkylating agents, anticancer drugs and organic hydroperoxides to determine if GST overexpression modulates sensitivity to cytotoxic insult. In all cases GST expression resulted in a significant reduction in the cytotoxic effects of these agents. These data provide evidence that the overexpression of GST observed in cells resistant to anticancer drugs is directly involved in the resistance mechanism.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:641664 |
| Date | January 1990 |
| Creators | Black, Stephen M. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/23730 |
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