Human benzene metabolism following occupational and environmental exposures

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Abstract

We previously reported evidence that humans metabolize benzene via two enzymes, including a hitherto unrecognized high-affinity enzyme that was responsible for an estimated 73% of total urinary metabolites [sum of phenol (PH), hydroquinone (HQ), catechol (CA), E,E-muconic acid (MA), and S-phenylmercapturic acid (SPMA)] in nonsmoking females exposed to benzene at sub-saturating (ppb) air concentrations. Here, we used the same Michaelis–Menten-like kinetic models to individually analyze urinary levels of PH, HQ, CA and MA from 263 nonsmoking Chinese women (179 benzene-exposed workers and 84 control workers) with estimated benzene air concentrations ranging from less than 0.001–299 ppm. One model depicted benzene metabolism as a single enzymatic process (1-enzyme model) and the other as two enzymatic processes which competed for access to benzene (2-enzyme model). We evaluated model fits based upon the difference in values of Akaike's Information Criterion (ΔAIC), and we gauged the weights of evidence favoring the two models based upon the associated Akaike weights and Evidence Ratios. For each metabolite, the 2-enzyme model provided a better fit than the 1-enzyme model with ΔAIC values decreasing in the order 9.511 for MA, 7.379 for PH, 1.417 for CA, and 0.193 for HQ. The corresponding weights of evidence favoring the 2-enzyme model (Evidence Ratios) were: 116.2:1 for MA, 40.0:1 for PH, 2.0:1 for CA and 1.1:1 for HQ. These results indicate that our earlier findings from models of total metabolites were driven largely by MA, representing the ring-opening pathway, and by PH, representing the ring-hydroxylation pathway. The predicted percentage of benzene metabolized by the putative high-affinity enzyme at an air concentration of 0.001 ppm was 88% based upon urinary MA and was 80% based upon urinary PH. As benzene concentrations increased, the respective percentages of benzene metabolized to MA and PH by the high-affinity enzyme decreased successively to 66 and 77% at 0.1 ppm, 20 and 58% at 1 ppm, and 2.7 and 17% at 10 ppm. This indicates that the putative high-affinity enzyme was active primarily below 1 ppm and favored the ring-opening pathway.

Introduction

Benzene is an important environmental contaminant that is present worldwide at air concentrations ranging from ppb in rural and urban settings to ppm in some workplaces [1], [2]. Mounting scientific evidence has shown that benzene causes leukemia and probably other lymphohematopoietic cancers in humans [3], [4], [5] and that benzene alters blood cell counts in persons exposed below 1 ppm (3.2 mg/m3) [6].

The toxicity of benzene has been related to its metabolism, which is summarized in Fig. 1[7], [8], [9]. The initial metabolic step involves CYP oxidation of benzene to benzene oxide, which exists in equilibrium with its tautomer oxepin. Most benzene oxide spontaneously rearranges to phenol (PH), which is either excreted or further metabolized to hydroquinone (HQ) and 1,4-benzoquinone. The remaining benzene oxide is either hydrolyzed to produce catechol (CA) and 1,2-benzoquinone or reacts with glutathione to produce S-phenylmercapturic acid (SPMA). Metabolism of oxepin is thought to open the aromatic ring, yielding the reactive muconaldehydes and E,E-muconic acid (MA) [10]. Human exposures to benzene at air concentrations between 0.1 and 10 ppm, result in urinary metabolite profiles with 70–85% PH, 5–10% each of HQ, MA and CA, and less than 1% of SPMA [11], [12].

Because essentially all humans are exposed to benzene and because benzene must be metabolized to exert toxic effects, the relationship between levels of benzene exposure and benzene metabolites is important to our understanding of potential human health risks. We used studies of benzene-exposed and control workers in China to investigate benzene metabolism over a wide range of air concentrations. Interestingly, our studies showed supralinear production of benzene-related albumin adducts at air concentrations below 1 ppm (i.e. increased exposure-specific adduct levels below 1 ppm) [13], [14]. We followed up the adduct studies by modeling urinary levels of PH, HQ, CA and MA from the same benzene-exposed and control workers; again we observed supralinear effects at benzene air concentrations below 1 ppm [11], [15], [16]. Since the observed relationships between metabolite levels and benzene exposure were inconsistent with single-enzyme kinetics of benzene metabolism, we tested whether a hitherto unrecognized second enzyme might be responsible for most benzene metabolism below 1 ppm. Using Michaelis–Menten-like models, we investigated levels of total urinary benzene metabolites (the sum of PH, HQ, CA, MA, and SPMA) in a subset of workers represented by 263 nonsmoking females exposed to benzene at air concentrations between less than 0.001 and 299 ppm [17]. Results provided strong statistical evidence favoring two metabolizing enzymes and indicated that the higher-affinity enzyme was responsible for about 73% of all benzene metabolism at nonsaturating (ppb) air concentrations.

In the current study, we investigated the fits of the same Michaelis–Menten-like models to the major individual metabolites PH, HQ, CA and MA in the same sample of 263 nonsmoking females (because the minor metabolite SPMA showed no effect of saturable formation over the full range of benzene exposures in this population [11], [15], [16], kinetic models were not fit to levels of urinary SPMA). The 2-enzyme model provided much better fits for levels of PH and MA but only marginally better fits for levels of HQ and CA. Furthermore, the putative high-affinity enzyme favored the ring-opening pathway, leading to production of MA, to a much greater extent than the low-affinity enzyme.

Section snippets

Study population and sampling

Subjects were from two cross-sectional studies of Chinese benzene-exposed and control workers carried out in Shanghai (1992) [18], [19], [20] and in Tianjin (2000–2001) [6], [15], [21]. Subject enrolment and interview procedures, exposure assessment methods, and urinary metabolite measurements in these two studies were carried out by the same group of investigators using the same procedures. Workers with occupational exposure to benzene were employed in factories where benzene was present, and

Scatter plots

Fig. 2 shows scatter plots of individual benzene metabolite levels for the 263 subjects versus the air concentration of benzene on the day of urine collection. At benzene concentrations below about 0.1 ppm, levels of PH, HQ and CA primarily reflected background sources while those of MA showed evidence of both benzene exposure and background sources. The effect of benzene exposure on production of each metabolite became increasingly apparent at air concentrations above about 1 ppm, and above 100 

Evidence for two metabolizing enzymes

Our previous analyses of total benzene metabolites (the sum of PH, HQ, CA, MA and SPMA) provided statistical evidence favoring two (rather than one) metabolizing enzymes (Evidence Ratio = 15.4) [17]. Furthermore, the level of total benzene metabolites predicted from the 2-enzyme model (194 μM total metabolites/ppm benzene) was quite close to the value predicted from the overall rate of benzene metabolism (184 μM total metabolites/ppm benzene), based upon independent rates of benzene uptake and

Conclusions

In conclusion, our results provide extremely strong statistical evidence that benzene is metabolized to PH and MA via two enzymes rather than one enzyme, and that the putative high-affinity enzyme is active primarily below 1 ppm. Model predictions further suggest that the ring-opening pathway, which leads to MA, is favored by the high-affinity enzyme.

Conflict of interest

S.M.R. has received consulting and expert testimony fees from law firms representing plaintiffs’ cases involving exposure to benzene and has received research support from the American Petroleum Institute and the American Chemistry Council. G.L. has received funds from the American Petroleum Institute for consulting on benzene-related health research. M.T.S. has received consulting and expert testimony fees from law firms representing both plaintiffs and defendants in cases involving exposure

Acknowledgements

The authors are indebted to Dr. Mustafa Dosemici, who compiled some of the benzene exposure data used in this investigation, to Dr. Min Shin, who helped assemble a portion of the database, and to Dr. Richard Hayes who assisted with conduct of the Shanghai portion of our study.

Funding sources: This research was supported by the National Institute for Environmental Health Sciences through grants P42ES05948 and P30ES10126 (S.M.R.) and R01ES06721 and P42ES04705 (M.T.S.) and by funds from the

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