Superfund Research Program
Development and Application of Biomarkers of Exposure
Project Leader: Stephen M. Rappaport (University of California-Berkeley)
Grant Number: P42ES005948
Funding Period: 1995 - 2011
Through analysis of biological samples from highly exposed benzene workers in Shanghai, China, project investigators compared a comprehensive collection of biomarkers including urinary benzene and its metabolites and protein adducts of two electrophilic benzene metabolites, namely benzene oxide (BO) and 1,4-benzoquinone (1,4-BQ). These experiments support the basic hypothesis that these biomarkers will allow us to quantify the relationship between benzene exposure and metabolism in humans.
Project investigators developed a method for measuring urinary benzene (UB) and applied the method to highly exposed workers and unexposed subjects in Shanghai. Measurements were performed via headspace solid-phase microextraction of 0.5-ml urine specimens followed by gas chromatography-mass spectrometry. This assay is more sensitive than other methods (detection limit = 0.016 mg benzene/l of urine). A linear relationship was observed between the logarithm of UB and the logarithm of benzene exposure in exposed subjects. Considering all subjects, linear relationships were also observed between UB and the corresponding logarithms of four urinary metabolites of benzene, namely, t,t-muconic acid, phenol, catechol, and hydroquinone. Ratios of individual metabolite levels to total metabolites vs. UB provide evidence of competitive inhibition of cytochrome P-450 (CYP450) enzymes leading to increased production of phenol and catechol at the expense of hydroquinone and muconic acid. Among control subjects UB was readily detected in workers exposed to benzene below 10 ppm; this suggests that UB is a good biomarker for exposure to low levels of benzene.
Hemoglobin (Hb) and albumin (Alb) adducts of the benzene metabolites, benzene oxide (BO) and 1,4-benzoquinone (1,4-BQ) were analyzed by gas chromatography-mass spectrometry (GC/MS) in 43 exposed workers and 44 unexposed controls from Shanghai. The measure of cigarette smoking, urinary cotinine, was significantly correlated with 1,4-BQ-Alb but not with the BO adducts or with the urinary biomarkers of benzene. This relationship between adducts and cigarette smoking clearly delineated additive effects of benzene exposure and cigarette smoking upon production of 1,4-BQ-Alb. Thus the researchers conclude that cigarette smoke is a significant source of 1,4-BQ-Alb in humans. In a separate analysis of these same data, levels of BO-Alb were marginally greater among female controls than male controls.
To test the influence of radical-mediated pathways on production of these BO-protein adducts, the project investigators employed Fenton chemistry to generate free radicals in vitro. Incubations containing human Hb, ascorbate, and H2O2 produced levels of BO-Hb that were approximately 13 times higher than control incubations. Researchers then tested the radical-mediated production of BO-protein adducts in vivo by administering a single dose of 0 to 1600 mg of carbon tetrachloride (CCl4) (a known producer of free radicals in vivo) per kg body weight to 24 male F344 rats, with and without co-exposure to sodium benzoate. Comparison of adduct concentration by dose of CCl4 using an analysis of variance (ANOVA) procedure suggests that there was no significant increase in the concentration of BO-Alb or BO-Hb as a function of CCl4 dose. Simultaneous treatment with CCl4 and aqueous sodium benzoate (5 mmol/kg body weight) also failed to significantly increase levels of BO-Alb or BO-Hb as a function of CCl4 dose. Thus, the scientists were unable to demonstrate radical-mediated production of background BO-Alb and BO-Hb in vivo.
Levels of benzene oxide-albumin adducts (BO-Alb), reported previously among a sample of Chinese workers, were analyzed for evidence of nonlinear exposure-rate effects and interindividual variability in benzene metabolism. Because the control subjects' blood contained significant amounts of BO-Alb, originating from unknown background (non-benzene) sources, exposed subjects' adduct levels were adjusted for control levels prior to statistical analysis. Multiple linear regression of these background-adjusted adduct levels revealed strong effects of benzene exposure and alcohol consumption. Project investigators infer that the rate of benzene oxide production diminished with increasing benzene exposure above about 10 ppm of benzene in air. Since benzene is a substrate for CYP4502E1, these results are consistent with saturation of CYP450 metabolism by benzene. Unexpectedly, daily alcohol consumption significantly diminished levels of BO-Alb at a given level of benzene exposure by 17% per drink.