**3.1 Identification of gene polymorphisms relevant to the occupational exposure**

In our experience several biomonitoring studies have been promoted and carried out in industrial companies to assess the exposure risk of workers manipulating toxic and carcinogenic agents. Here we report the most recent results obtained during four different campaigns carried out in the last 6 years. In 2012, our research group analyzed the exposure of 315 workers to benzene in a petrochemical plant in Italy to evaluate the dose biomarkers S-phenylmercapturic acid (SPMA) and trans,transmuconic acid (t,t-MA) in the worker urine samples and the susceptibility biomarkers in the blood specimens [14]. Since the GST enzymes may influence the metabolite excretion, the research project analyzed the association between metabolite excretion and contribution of the glutathione S transferase T1 (GST-T1) and M1 (GST-M1) polymorphisms to the detoxification. The results confirmed the modulating effect on the excretion of SPMA metabolite in urine by the genetic polymorphism of GST-T1 after exposure to low benzene doses. The same modulating effect was caused by the GST-M1 polymorphism but only at higher benzene doses, like those produced by cigarette smoking. The genotype of the 315 workers has been characterized to identify groups with the highest susceptibility; the workers' gene frequency of GST-T1 positive/null genotype was 0.78 vs. 0.22, while the frequency of GST-M1 positive/null was 0.47 vs. 0.53. Taking into account the genotype frequency of both enzymes, it looks that the GST-M1 polymorphism positive/null is less efficient in the detoxification process than GST-T1. A second study on benzene exposure has been carried out on 301 employees in the oil refinery. The effect of polymorphic genes GST-T1, GST-M1, glutathione S transferase A1 (GST-A1), epoxide hydrolase 1 (EHPX1), NAD(P) H quinone dehydrogenase 1 (NQO1), cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1\*2A) and myeloperoxidase (MPO) involved in the detoxification of benzene has been analyzed. Here the dose biomarker was worked out as the ratio (R) between t,t-MA and S-PMA metabolites excreted in the urinary samples. The effect of smoking as confounding factor contributed to increase the relative production of S-PMA with respect to t,t-MA reducing the R value. This result was attributed to the higher levels of glutathione (GSH) in the red blood cells of smokers than in non-smokers. The analysis of susceptibility biomarkers showed a strong influence of GST-T1 positive polymorphism on the excretion of urinary S-PMA, reducing the conjugation rate of benzene epoxide with GSH in the GST-T1 null subjects. To a lesser extent, a similar effect was observed in individuals with GST-M1 null, GST-A1 and NQO1 mutant genotypes. It has been observed that in subjects with the double null GST-T1 and GST-M1 genotypes, an amplification of the t,t-MA biochemical pathway occurred with respect to the SPMA pathway [15]. However the activity of one GST is compensated by another in GST-M1 and GST-A1 defective subjects, but not in GST-T1 null genotypes, whose S-PMA average excretion is about 50% with respect to the positive genotypes for the same benzene exposure [16]. A further biomonitoring campaign has been conducted in two different manufacturing sites of central Italy on 30 styrene-exposed workers and 26 unexposed controls. In this work the authors

investigated the effects of polymorphic genes CYP2E1, EPHX1, GST-T1 and GST-M1 on the urinary concentrations of the styrene metabolites, i.e. mandelic acid (MA) and phenylglyoxylic acid (PGA), and on the ratio between MA and PGA. Here the concentrations of urinary styrene and of airborne styrene have been determined. In the exposed workers, a lower excretion of PGA and MA metabolites was detected in subjects with cytochrome P450 2E1\*5B (CYP2E1\*5B) and cytochrome P450 2E1\*6 (CYP2E1\*6) heterozygous genotype with respect to the homozygous wild type indicating the influence of SNPs on the dose biomarkers. Furthermore a reduced value of metabolite ratio (MA + PGA/urinary styrene) was observed in exposed workers with the EPHX1 Tyr113His slow/mutant allele in comparison to those with the wild-type allele. The results indicate a reduced excretion of MA + PGA, evidenced also by other authors only in association with other genotypes. This study confirmed the variability in the excretion of urinary styrene metabolites, strictly related to the individual gene polymorphisms, can significantly impact on the biological monitoring of styrene exposure. In the CYP2E1\*5B and CYP2E1\*6 heterozygous genotypes as well as in the EPHX1 slow mutant genotypes, the average excretion of MA + PGA is, respectively, reduced 20 and 35% in comparison to the wild-type population [17].
