Objectives-Comparison of the suitability of two minor urinary metabolites of benzene, trans,trans-muconic acid (tt-MA) and S-phenylmercapturic acid (S-PMA), as biomarkers for low levels of benzene exposure. Methods-The sensitivity of analytical methods of measuring tt-MA and S-PMA were improved and applied to 434 urine samples collected from 188 workers in 12 studies in different petrochemical industries and from 52 control workers with no occupational exposure to benzene. In nine studies airborne benzene concentrations were assessed by personal air monitoring. Results-Strong correlations were found between tt-MA and S-PMA concentrations in samples from the end of the shift and between either of these variables and airborne benzene concentrations. It was calculated that exposure to 1 ppm (8 hour time weighted average (TWA)) benzene leads to an average concentration of 1'7 mg tt-MA and 47 jg S-PMA/g creatinine in samples from the end of the shift. It was estimated that, on average, 3 9%/6 (range 1.90/o-7.3%) of an inhaled dose of benzene was excreted as tt-MA with an apparent elimination half life of 5*0 (SD 2.3) hours and 0*11% (range 0-050/%0-26%) as S-PMA with a half life of 9 1 (SD 3.7) hours. The mean urinary S-PMA in 14 moderate smokers and 38 non-smokers was 3*61 and 1*99 uglg creatinine, respectively and the mean urinary tt-MA was 0 058 and 0 037 mglg creatinine, respectively. S-PMA proved to be more specific and more sensitive (P = 0-030, Fisher's exact test) than tt-MA. S-PMA, but not tt-MA, was always detectable in the urine of smokers who were not occupationally exposed. S-PMA was also detectable in 20 of the 38 nonsmokers from the control group whereas tt-MA was detectable in only nine of these samples. The inferior specificity of tt-MA is due to relatively high background values (up to 0'71 mglg creatinine in this study) that may be found in non-occupationally exposed people. Conclusions-Although both tt-MA and S-PMA are sensitive biomarkers, only S-PMA allows reliable determination of benzene exposures down to 0 3 ppm (8 h TWA) due to its superior specificity. Because it has a longer elimination half life S-PMA is also a more reliable biomarker than tt-MA for benzene exposures during 12 hour shifts. For biological monitoring of exposure to benzene concentrations higher than 1 ppm (8 h TWA) tt-MA is also suitable and may even be preferred due to its greater ease of measurement. (Occup Environ Med 1995;52:611-620)

The possibilities and limitations of using mercapturic acids and protein and DNA adducts for the assessment of internal and effective doses of electrophilic chemicals are reviewed. Electrophilic chemicals may be considered as potential mutagens and/or carcinogens. Mercapturic acids and protein and DNA adducts are considered as selective biomarkers because they reflect the chemical structure of the parent compounds or the reactive electrophilic metabolites formed during biotransformation. In general, mercapturic acids are used for the assessment of recent exposure, whereas protein and DNA adducts are used for the assessment of semichronic or chronic exposure. 2-Hydroxyethyl mercapturic acid has been shown to be the urinary excretion product of five different reactive electrophilic intermediates. Classification of these electrophiles according to their acid-base properties might provide a tool to predict their preference to conjugate with either glutathione and proteins or with DNA. Constant relationships appear to exist in the cases of 1,2-dibromoethane and ethylene oxide between urinary mercapturic acid excretion and DNA and protein adduct concentrations. This suggests that mercapturic acids in some cases may also play a role as a biomarker of effective dose. It is concluded that simultaneous determination of mercapturic acids, protein and DNA adducts, and other metabolites can greatly increase our knowledge of the specific roles these biomarkers play in internal and effective dose assessment. If the relationship between exposure and effect is known, similar to protein and DNA adducts, mercapturic acids might also be helpful in (individual) health risk assessment.

Phenol is not reliable as a biomarker for exposure to benzene at concentrations below 5 ppm (8-hr time-weighted average [TWA]). S-Phenylmercapturic acid (S-PMA) and trans-trans-muconic acid (tt-MA), two minor urinary metabolites of benzene, have been proposed as biomarkers for low-level exposures. The aim of this study was to compare their suitability as biomarkers. S-PMA and tt-MA were determined in 434 urine samples collected from 188 workers in various settings in the petrochemical industry and from 52 control workers with no occupational exposure to benzene. Benzene concentrations in the breathing zone of the potentially exposed workers were assessed by personal air monitoring. Strong correlations were found between S-PMA and tt-MA concentrations in end-of-shift samples and between either of these parameters and airborne benzene concentrations. Exposure to 1 ppm benzene (8-hr TWA) leads to an average concentration in end-of-shift samples of 21 mol S-PMA and 1.5 mmol tt-MA per mol creatinine. Of an inhaled dose of benzene, on average 0.11% (range 0.05-0.26%) was excreted as S-PMA with an apparent elimination half-life of 9.1 (standard error [SE] 0.7) hr and 3.9% (range 1.9-7.3%) as tt-MA with a half-life of 5.0 (SE 0.5) hr. Due to its longer elimination half-life, S-PMA proved a more reliable biomarker than tt-MA for benzene exposures during 12-hr shifts. Specificity of S-PMA, but not tt-MA, was sufficient to discriminate between the 14 moderate smokers and the 38 nonsmokers from the control group. The mean urinary S-PMA was 1.71 (SE 0.27) in smokers and 0.94 (SE 0.15) mol/mol creatinine in nonsmokers (p = 0.013). The mean urinary tt-MA was 0.046 (SE 0.010) in smokers and 0.029 (SE 0.013) mmol/mol creatinine in nonsmokers (p = 0.436). The inferior specificity of tt-MA was due to relatively high background values of up to 0.56 mmol/mol creatinine, which may be found in nonexposed individuals and limits the use of tt-MA to concentrations of benzene over 1 ppm (8-hr TWA). We conclude that S-PMA is superior to tt-MA as a biomarker for low-level benzene exposures because it is more specific, enabling reliable determination of benzene exposures down to 0.3 ppm (8-hr TWA), and because its longer half-life makes it more suited for biological monitoring of operators working in shifts longer than 8 hr.

1,3-Butadiene (BD) is carcinogenic in mice and rats, with mice being considerably more sensitive than rats. Urine metabolites are 1, 2-dihydroxybutyl mercapturic acid (DHBMA) and a mixture of monohydroxy-3-butenyl mercapturic acids (MHBMA). The reactive metabolite 1,2-epoxy-3-butene forms 1- and 2-hydroxy-3-butenyl valine adducts in hemoglobin (MHBVal). The objectives of the study were (1) to compare the suitability of MHBMA, DHBMA, and MHBVal as biomarkers for low levels of exposure to BD, and (2) to explore relative pathways of metabolism of BD in humans for comparison with mice and rats, which is important in relation to cancer risk assessment in man. Analytical methods of measuring MHBMA, DHBMA, and MHBVal were modified and applied in 2 studies to workers engaged in the manufacture and use of BD. Airborne BD concentrations were assessed by personal air monitoring. MHBMA in urine was more sensitive for monitoring recent exposures to BD when compared to DHBMA and could measure 8-h time weighted average exposures as low as 0.13 ppm. Relatively high natural background levels in urine restricted the sensitivity of DHBMA. The origin of this background is currently unknown. The measurement of MHBVal adducts in hemoglobin was a sensitive method for monitoring cumulative exposures to BD at or above 0.35 ppm. Statistically significant relationships were found between urinary MHBMA and DHBMA concentrations, between either of these variables and 8-h airborne BD levels and between MHBVal adducts and average airborne BD levels over 60 days. The data on biomarkers demonstrated a much higher rate of hydrolytic metabolism of 1,2-epoxy-3-butene in humans compared to mice and rats, which was reflected in a much higher DHBMA/(DHBMA + MHBMA) ratio and in much lower levels of MHBVal in humans. Assuming a genotoxic mechanism, the data of this study, coupled with other published data on DNA and hemoglobin binding in mice and rats, suggest that the cancer risk for man from exposure to BD is expected to be less than for the rat and much less than for the mouse.

Recently, the determination of S-phenylmercapturic acid (S-PMA) in urine has been proposed as a suitable biomarker for the monitoring of low level exposures to benzene. In the study reported here, the test has been validated in 12 separate studies in chemical manufacturing plants, oil refineries, and natural gas production plants. Parameters eight hour exposure to airborne benzene of 1 mg/m3 (0 3 ppm) and higher and urinary S-PMA concentrations in end of shift samples. It was calculated that an eight hour benzene exposure of 3-25 mg/m3 (1 ppm) corresponds to an average S-PMA concentration of 46 pg/g creatinine (95% confidence interval 41-50 pg/g creatinine). A strong correlation was also found between urinary phenol and S-PMA concentrations. At a urinary phenol concentration of 50 mg/g creatinine, corresponding to an eight hour benzene exposure of 32-5 mg/m' (10 ppm), the average urinary S-PMA concentration was 383 ug/g creatinine. In conclusion, with the current sensitivity of the test, eight hour time weighted average benzene exposures of 1 mg/m' (0 3 ppm) and higher can be measured.