Effects of ethanol and phenobarbital on hemoglobin adducts formation in rats exposed to benzidine and Direct Black 38

The objective of this study was to evaluate the effects of pretreatment of ethanol (EtOH) and phenobarbital (PB), which are known to affect the metabolism of xenobiotics, in the formation of hemoglobin adducts in rats administered with benzidine (BZ) and Direct Black 38 (DB38). The experimental rats were divided into BZ and DB38 groups; each group was subdivided into control, EtOH, and PB groups. Blood samples were separated into hemoglobin and plasma immediately after obtaining and basic hydrolysis was done to convert the adducts into aromatic amines. Hydrolyzed BZ, monoacetylbenzidine (MABZ), and 4-aminobiphenyl (4ABP) were separated by reversed-phase liquid chromatography without derivatization. Then, quantitative analyses were performed using a high performance liquid chromatograph equipped with an electrochemical detector. The amount of metabolites was expressed in the hemoglobin binding index (HBI). As a result, the formations of hemoglobin in BZ-, MABZ-, and 4ABP-HBI of BZ-EtOH and BZ-PB groups were increased compared with those of BZ-control group. In DB38 group, all of HBIs except for BZ-HBI were increased more than those of DB38-control group regardless of the pretreatment. These results are attributable to the fact that EtOH and PB induced N-hydroxylation is related to the formation of hemoglobin adducts. They indicate that EtOH not only increases the adduct formation by inducing N-hydroxylation but also induces N-acetylation. PB induced N-hydroxylation and increased the adduct formation in BZ group, but decreased the adduct formation in DB38 group due to decreasing azo reduction. These results suggest that the effects of EtOH or PB should be considered in biochemical monitoring of BZ and DB38 for the assessment of intermittent exposure to BZ and DB38.