Glutathione S-transferases play a central role in drug detoxification and have been implicated in the sensitivity of tumour cells to anticancer drugs. In this study, glutathione S-transferase (GST) isozyme expression in normal and tumour tissue from human lung, colon, stomach, breast, kidney and liver tissue has been quantified using sensitive and subunit specific radioimmunoassays (RIA), together with Western blot analysis and measurement of substrate metabolism. Glutathione S-transferase pi was the predominant GST in the majority of the tumours examined. The concentration of this enzyme was increased significantly in tumour tissue relative to normal lung, colon, and stomach tissue. A strong correlation was observed (r = 0.77, P less than 0.01) between GST activity and GST pi levels in those tumour samples. The concentrations of the alpha class GST, the predominant isoenzymes in normal stomach, kidney and liver, decreased dramatically in tumour tissue from these organs. Western blot analysis revealed the presence of novel polypeptides that cross-reacted with antisera raised against alpha and mu class GST. Our data demonstrates that although GST pi is the predominant GST isoenzyme in many tumours, significant levels of the other GST subunits are also present and collectively can represent a significant proportion of the GST content. Therefore the properties of all the GST isoenzymes need consideration when assessing the role of these proteins in drug resistance. Selenium-dependent glutathione peroxidase, an enzyme activity also implicated in the mode of action of certain antitumour agents, was also studied and shown to be the predominant glutathione-dependent peroxidase in all tumours except the hepatoma.

Cytochrome P450s play a central role in the metabolism and disposition of an extremely wide range of drugs and chemical carcinogens. Individual differences in the expression of these enzymes may be an important determinant in susceptibility to adverse drug reactions, chemical toxins and mutagens. In this paper, we have measured the relative levels of expression of cytochrome P450 isoenzymes from eight gene families or subfamilies in a panel of twelve human liver samples in order to determine the individuality in their expression and whether any forms are co-regulated. Isoenzymes were identified in most cases on Western blots based on the mobility of authentic recombinant human cytochrome P450 standards. The levels of the following P450 proteins correlated with each other: CYP2A6, CYP2B6 and a protein from the CYP2C gene subfamily, CYP2E1 and a member of the CYP2A gene subfamily, CYP2C8, CYP3A3/A4 and total cytochrome P450 content. Also, the levels of two proteins in the CYP4A gene subfamily were highly correlated. These correlations are consistent with the relative regulation of members of these gene families in rats or mice. In addition, the level of expression of specific isoenzymes has also been compared with the rate of metabolism of a panel of drugs, carcinogens and model P450 substrates. These latter studies demonstrate and confirm that the correlations obtained in this manner represent a powerful approach towards the assignment of the metabolism of substrates by specific human P450 isoenzymes.

Liver cancer is a major cause of premature death in many areas of Africa and Asia and its incidence is strongly correlated with exposure to aflatoxin B1 (AFB1).Because AFB1 requires metabolic activation to achieve a biological response, there is a need for detailed knowledge of the mechanism of activation to assess individual risk. We have carried out an extensive study using a total of 19 human liver samples to determine the individual variability in the metabolism of the toxin to mutagenic or detoxification products and to identify the specific cytochrome P450 forms involved in these processes. Metabolism to the toxic 8,9-epoxide or to products mutagenic in the Ames test was found to exhibit very large individual variation. The rates of metabolic activation were highly correlated with both the level of proteins of the P450I1A gene family and with the total cytochrome P450 content of the microsomes. In agreement with this, antibodies reacting with P450IHA proteins were strong inhibitors of both

1. We have constructed a full-length human liver cytochrome P450IIA cDNA from a partial-length clone by oligonucleotide-directed mutagenesis, and subcloned it into the monkey kidney (COS-7) cell expression vector, pSVL. 2. The cDNA encodes a 49 kDa protein with coumarin 7-hydroxylase (COH) activity which cross-reacts with antisera to the mouse cytochrome P-450 isoenzyme responsible for COH activity and comigrates with a human liver microsomal protein. 3. Western blot analysis of a panel of human livers indicates that the level of the 49 kDa protein, detected using antisera to either the mouse COH P-450 or rat P450IIA1 protein, correlates very highly with COH activity. 4. Antisera to the rat P450IIA1 protein can inhibit COH activity in human liver microsomes. Taken together, these data indicate that a member of the P450IIA subfamily is responsible for most, if not all, of the COH activity in human liver.

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