So far, biological monitoring methods and biological limit values applied in occupation- tonal and environmental medicine have been developed on the assumption that individuals do not differ in their biotransformation capacities. It has become clear, however, that this is not the case but wide inter-individual differences exist in the metabolism of xenobiotics. Integration of the data on individual biotransformation capacity in bolo- goal monitoring studies is anticipated to represent a significant refinement of the currently used methods. Consequently, the main goal of the present project is to clarity the potential role of the polymorphisms in CYP2EI, EPHX, GSTM1, GSTP1, and GSTT1 genes in modifying individual responses to styrene, as measured by urine metabolites, adducts in blood macromolecules, and cytogenesis alterations in lymphocytes. It also aims to de- flop alternative methods to assess CYP2E1 phenotype and to clarify the still unclear correlation between peroxide hydrolyse genotypes and phenotypes.
The results achieved so far clearly indicate that GSTM1 genotype is an important modifier of urinary PHEMAs excretion, and that the mechanism by which GSTs catalyze GSH-conjugation of SO in humans is regio- and stereo-selective. The [R,R]-M1 is the main mercapturate affected by the GSTM1 genotype and it accounts, in the GSTM1 positive subjects, for 56% of total PHEMAs excreted during 24 hours following styrene exposure. Although less significant than that of GSTM1, an involvement of GSTT1 in the formation of PHEMAs is also possible. The influence of this genotype was specifically noted for the [R,R]-M2 metabolite, further illustrating the regio- and stereo-selectivity of GST activity on SO. A modifying role of GSTM1 and GSTT1 was also found regarding the induction of sister chromatid exchanges (SCEs) by styrene. Although glutathione conjugation is a minor route in human detoxification in vivo, individual sensitivity associated with the GSTM1 and GSTT1 null genotypes may be important locally in blood circulation and in blood-forming organs. In contrast to GSTM1 and GSTT1, we were unable to demonstrate an effect of GSTP1 polymorphism on the urinary excretion of PHEMAs.
The observed significant relationship between urinary excretion of MA during 24 hours following styrene exposure and CYP2E1 mRNA in PBLs demonstrates the importance of CYP2E1 in the metabolism of styrene in humans. On the other hand, based on the biotransformation pathway of styrene in man, a combined effect of CYP2E1 phenotype and GSTM1 genotype could be hypothesised to explain inter-individual variability in urinary PHEMA excretion. This could not be verified in the field study using the classic chlorzoxazone (CZX) metabolic ratio (CMR) to assess CYP2E1 activity, but could be verified in the voluntary study where the CYP2E1 phenotype was assessed by CYP2E1 mRNA levels in PBLs. This highlights the potential value of CYP2E1 phenotyping, through measurement of CYP2E1 mRNA in PBLs, in GSTM1 positive subjects. Therefore, while CZX may be considered as an 'acceptable' probe substrate for CYP2E1, a possibility exist that the CMR does not provide measure accurate enough to finely reflect the CYP2E1 catalytic activity. To conclude, although the present study shows that genotyping and/or phenotyping of relevant XMEs does not significantly improve the interpretation of urinary levels of the main metabolites of styrene in general, these analyses might greatly improve the interpretation of urinary concentrations of minor, but more specific, metabolites. In this respect PHEMA determination can now be considered as a useful tool for biological monitoring of styrene exposure in occupational and environmental settings.
Funding SchemeCSC - Cost-sharing contracts
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