A dietary ethanol protocol for Cyp2e1 induction in CD-1 mice and its application to studies on the genotoxicity, immunotoxicity and metabolism of low-level benzene

Humans are exposed to multiple potentially toxic xenobiotics. However, there is a paucity of information about consequences of exposure to multiple chemicals at environmentally-relevant levels. The overall goal of the research conducted for this dissertation project was to test the hypothesis that dietary ethanol would enhance the genotoxicity and immunotoxicity of inhalation exposure to environmentally-relevant concentrations of benzene. The rationale for this chemical combination is that ethanol is a potent inducer of Cytochrome P450 2E1 (Cyp2e1), an enzyme with critical roles in the biotransformation of benzene to its proximate toxicants. My first aim was to develop a dietary ethanol protocol for inducing Cyp2e1 in female CD-1 mice. After screening several concentrations, I found that a 4.1% ethanol diet produced a stable ∼4-fold induction of hepatic Cyp2e1 without altering other biotransformation enzymes. This well-tolerated protocol also produced no hepatotoxicity or genotoxicity to the bone marrow or spleen lymphocytes, the target organs for benzene toxicity. My second aim was to apply this protocol towards the study of benzene toxicity. Mice maintained on the ethanol diet were exposed by inhalation to benzene concentrations of 0.44 or 4.40 ppm for 6 to 11 weeks. I detected no evidence for a benzene-exposure effect with or without Cyp2e1 induction on immunotoxicity, as measured by a battery of Tier I and II, or genetic damage, as measured by frequency of hprt-mutant spleen lymphocytes and DNA-protein crosslinks in the bone marrow. This study demonstrated the application of this ethanol protocol towards investigations on Cyp2e1 influence on xenobiotic toxicity. My third aim was to develop a simple HPLC method for the quantitation of urinary benzene metabolites that was not dependent on the use of radiolabeled benzene. I found that coupling an ultraviolet detector to an electrochemical detector would efficiently detect six major benzene metabolites in urine as validated by analysis of urine samples spiked with specific hydroxylated and ring-opened metabolites. Peaks from phenol, hydroquinone and an unknown metabolite were detected in urine from benzene-treated animals. Observations from this project are important for risk assessment because the doses of ethanol and benzene evaluated are within the range of human exposure.