Assessment of urinary trans, trans-muconic acid as a biomarker of exposure to benzene

OBJECTIVE: To assess the use of trans, trans-muconic acid as a biomarker of occupational exposure to benzene. METHODS: Trans, trans-muconic acid in urine samples of exposed (exposed group, n=36) and non-exposed (non-exposed group, n=116) workers to benzene. Urinary levels of trans, trans-muconic acid were quantified by high-performance liquid chromatography. The study sample consisted of subjects exposed to benzene in an oil refinery in Belo Horizonte, Brazil. Non-parametric statistical analysis was carried out using Kruskall-Wallis test, Mann-Whitney test and Spearman correlation at p<0.05. RESULTS: Workers were exposed on average to benzene levels of 0.15 +/- 0.05 mg/m3 (0.05 ppm) and they showed a urinary trans, trans-muconic acid mean value of 0.19 +/- 0.04 mg/g of creatinine. The reference value range of trans, trans-muconic acid in non-exposed subjects was 0.03 to 0.26 mg/g of creatinine (mean 0.10 +/- 0.08 mg/g of creatinine). There was seen a statistical difference between trans, trans-muconic acid levels in urine samples from exposed and non-exposed groups. There was no correlation between urinary trans, trans-muconic acid and air benzene levels. There was no correlation between urinary trans, trans-muconic acid levels in the exposed group and smoking. Alcohol consumption up to 48 hours before sampling procedure showed no effect on trans, trans-muconic acid levels in both exposed and non-exposed groups. There was however a correlation between age (range 18 to 25 years) and urinary metabolite levels in the latter group. CONCLUSIONS: The results show that it is important to evaluate the effect of age and smoking habits on urinary trans, trans-muconic acid levels.     

Analysis of urinary S-phenylmercapturic acid and trans, trans-muconic acid as exposure biomarkers of benzene in petrochemical and industrial areas of Korea

OBJECTIVES: Recently, S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA) in urine have been proposed as reliable biomarkers for monitoring occupational exposure to benzene. The aim of this study was to test the applicability of S-PMA and t,t-MA as exposure biomarkers and to monitor the occupational exposure level and the extent of environmental contamination from benzene in Korea. METHODS: The urinary excretion of S-PMA and t,t-MA in rats after the intraperitoneal administration of benzene (0.88-800 mg/kg body weight, 7 days) was examined. These biomarkers were also validated in human urine samples collected from elementary schoolchildren in several industrial areas including chemical manufacturing plants, oil refineries, and natural gas-producing installations in Korea. Urine was collected from elementary schoolchildren in a mountain village with no known occupational exposure to benzene and air pollution as the reference group. RESULTS: In rats, there was a significant relationship between the benzene concentration and the excretion of the urinary S-PMA and t,t-MA as a function of concentration, and the excretion of benzene metabolites peaked on the first day after intraperitoneal administration. In human urine, higher levels of S-PMA and t,t-MA were detected more frequently in petrochemical industrial areas than in areas with no known occupational exposure to benzene. CONCLUSIONS: These results show that the quantitative determination of S-PMA and t,t-MA in urine can be used as a reliable exposure biomarker for benzene, and they also suggest that extensive attention to benzene exposure is needed for maintaining the health of the population in Korea.     

职业性苯暴露反-反式粘糠酸生物接触限值研究

目的研究职业性苯暴露反．反式粘糠酸（t,t—MA）生物接触限值。方法实验室建立生产环境空气中苯浓度的气相色谱检测方法及作业工人尿中t,t—MA含量的高效液相色谱检测方法,并通过检测苯暴露现场工人8h苯暴露水平及班前、班后尿中t,t．MA含量,研究其相关性。结果苯暴露者班前、班后尿中t,t—MA含量与其苯暴露水平有明显的相关关系。班前y（mg／gCr）=0．924＋0．108X（me／m^3）,r=0．62,P〈0．01;班后y（mg／gCr）=2．103＋0．177X（mg／m^3）,r=0．791,P〈0．01。结论根据我国作业场所空气中苯的国家卫生标准,按回归方程推导出职业接触苯生物接触限值,推荐职业暴露苯的生物接触限值为工作班班后尿t,t．MA含量为3．0mg／gCr,下一班班前尿t,t．MA含量为1．5mg／gGr。     

Improvement in HPLC analysis of urinary trans,trans-muconic acid,a promising substitute for phenol in the assessment of benzene exposure

Summary Urinary trans, trans-muconic acid (t,t-MA), a minor metabolite of benzene, is a potential candidate for biological monitoring of benzene. A clean-up procedure using SPE extraction cartridges was applied to urinary samples in order to improve the reliability of t,t-MA determinations by HPLC-UV greatly and to carry out convenient analyses on a routine scale, particularly at low levels of t,t-MA concentrations. The detection limit of the method is low enough to measure urinary t,t-MA at a concentration of 00.05–0.1 mgg/l.The recovery rates and relative standard deviations from spiked urines (1 mg/l to 20 mg/l) were about 90% and 5%, respectively. t,t-MA was found to be rapidly excreted by rats and humans. In rats the background range never exceeded 0.5 mg/l with a mean concentration around 0.3 mg/l. In 49 human blank urines, t,t-MA average and median-value were respectively around 0.2 and < 0.1 mg/l with a range of < 0.1 to 0.5 mg/l. Experimental exposure of rats for 1 h to 10.2 ppm of benzene induced urinary excretion of 13 mg/l of t,t-MA during a 6-h post-exposure period while occupational exposures to 2.6 ppm (mean exposure level during 5d–8 h) and 7 ppm (4 h) of benzene resulted in urinary excretion of 2.1 (mean excretion level) and 6.5 mg/l respectively at the end of the exposure. In humans, t,t-MA has a similar half-time as phenol. Analysis of urinary t,t-MA seems to be a better indicator than phenol for the assessment of exposure to low levels of benzene. Ingestion of 200 mg of sorbic acid, the only other known precursor of t,t-MA, interfered minimally with the background excretion of t,t-MA.     

苯暴露大鼠尿中反-反式黏糠酸变化研究

目的观察苯动态染毒大鼠模型尿中反-反式黏糠酸(t,t-MA)的变化情况,探讨尿t,t-MA作为苯职业暴露水平生物标志物的可行性。方法48只成年Wistar大鼠,随机分为对照组、低浓度组、中浓度组和高浓度组,每组数量相同,雌雄各半;纯苯动态染毒28d(分4个时段,每时段染毒5d后停2d)。监测苯浓度,每个时段染毒后立即取5h尿,反相高效液相色谱-紫外检测法检测大鼠尿中t,t-MA浓度,并用尿肌酐校正。结果在不同染毒时段内,对照组、低、中、高浓度组间尿t,t-MA含量差异有统计学意义(P<0.05),鼠尿中t,t-MA浓度随着环境中苯浓度增高而升高,且没有随染毒时间延长而变化(P>0.05)。结论动物模型研究说明尿中t,t-MA是反映苯接触水平比较敏感的生物标志物。     

Lack of sensitivity of urinary trans,trans-muconic acid in determining low-level (ppb) benzene exposure in children

Benzene is a widespread pollutant of which the main source in the outside environment is automotive traffic. Benzene is also present in cigarette smoke, and small quantities exist in drinking water and food; all of these sources contribute to pollution of indoor environments. Benzene exposure may be studied with biologic indicators. In the present study, the authors evaluated whether differences in urinary concentrations of trans,transmuconic acid (t,t-MA) were detectable in a sample of 150 children and if the chemical was correlated with environmental exposures to low levels of benzene. The children attended primary schools that had significantly different-but low-environmental benzene levels. Analysis of urinary t,t-MA was achieved with high-performance liquid chromatography (photodiode array detector), and analysis of passive air samplers for benzene was performed with gas chromatography-mass spectrometry. Statistical analysis (Kruskal-Wallis test) indicated that differences in urinary levels of t,t-MA in children from urban and rural areas were not statistically significant (p = .07), nor were there significant differences between children with and without relatives who smoked (p = .69). As has been shown in other studies of children and adults, results of our study evidenced (1) the difficulty of correlating concentrations of urinary biomarkers with environmental exposure to benzene at a parts-per-billion level (i.e., traffic and environmental tobacco smoke) and, consequently, (2) the lack of specificity of t,t-MA as a biological indicator for the study of a population's exposure.     

Comparison of benzene exposure in drivers and petrol stations workers by urinary trans,trans-muconic acid in west of Iran

Motor vehicle traffic is the main emission source of benzene. We undertook this study in order to compare benzene exposure and urinary levels of trans,trans-muconic acid (t,t-MA) in taxi drivers and petrol station workers. Air benzene levels were analyzed with gas chromatography using a Flame Ionization Detector. t,t-MA was extracted from urine and analyzed using high performance liquid chromatography. Significant differences in levels of urinary t,t-MA were found in drivers and petrol station workers when compared to a control group (p<0.05). Correlation coefficients between benzene in air and t,t-MA for petrol station workers and drivers were 0.65 and 0.30, respectively. The concentration of benzene in the breathing zone of petrol station workers was 2-3 times higher than drivers, and also 3 times greater than a threshold level (0.5 ppm) recommended by the American Conference of Governmental Industrial Hygienists (ACGIH). The lowest benzene concentration at which urinary t,t-MA increased to a measurable level was approximately 0.17 ppm. In conclusion our results suggested that high benzene levels are emitted in petrol stations in west Iran. t,t-MA analysis was able to separate those exposed from the non-exposed benzene group when benzene in the breathing zone of subjects was greater than 0.17 ppm.     

苯接触标志物尿中苯巯基尿酸的分析与评价

尿中苯巯基尿酸(SPMA)与苯接触存在良好相关性,是低浓度苯接触特异和敏感的生物标志物。SPMA可用高效液相色谱法(HPLC)、液质联谱(LC/MS)、气质联谱(GC/MS)和酶联免疫吸附试验(ELISA)等方法检测。本文详细介绍了国外检测尿SPMA的方法,评价了尿SPMA作为职业苯接触生物标志物的有效性和应用。     

trans,trans-Muconic acid as a biomarker of non-occupational environmental exposure to benzene

 Excretion of trans,trans-muconic acid (2,4-hexadienedioic acid; t,t-MA), a potential biomarker of low-level exposure to benzene, was determined in 32 smokers and 82 nonsmokers. In smokers the median background excretion of t,t-MA was 0.13 (0.06–0.39) mg/g creatinine and was significantly higher (P<0.05) than the value of 0.065 (0.02–0.59) mg/g creatinine in nonsmokers. For nonsmokers, the correlation between t,t-MA excretion and environmental exposure to benzene in ambient air, which was determined during the 8-day study period by personal diffusion samplers, was not significant (r=0.164, P=0.18). Nonsmokers living in the city tended to have higher t,t-MA excretion rates than nonsmokers living in the suburbs. However, the difference was only significant for nonsmokers from nonsmoking homes. For the same location (suburb or city), smoking at home leads to a marginal increase in t,t-MA excretion of the nonsmoking members of the household. In a further study with eight nonsmokers we found that dietary supplementation with 500 mg sorbic acid significantly increased (P<0.001) the mean urinary t,t-MA excretion from 0.08 (0.04–0.12) to 0.88 (0.57–1.48) mg/24 h. Under study conditions 0.12% of the sorbic acid dose was excreted in urine as t,t-MA, thereby indicating that a typical dietary intake of 6–30 mg/day sorbic acid accounts for 10–50% of the background t,t-MA excretion in nonsmokers, and for 5–25% in smokers. As a consequence, sorbic acid in the diet is a significant confounding factor in assessing low-level benzene exposure if t,t-MA excretion in urine is used as a biomarker. Received: 10 October 1995 / Accepted: 26 February 1996     

Biological monitoring of occupational exposure to benzene and toluene

This study was carried out to evaluate the biological monitoring of occupational exposure to benzene and toluene in a total number of 31 male exposed workers and 30 control subjects. The present study showed a statistically significant higher level of biological indices of exposure (p < 0.01) of phenol and hippuric acid in urine of workers exposed to benzene and toluene than control subjects. Significant changes (p < 0.05, 0.01) in the levels of hematological and biochemical findings have been observed among exposed workers and control group. In addition, statistically significant higher levels of Mg, Mn and Ca were found among workers exposed to benzene and toluene while statistically significant lower levels of serum iron (p < 0.05) have been observed. No significant variations could be detected in the level of Zn and Cu between exposed and control subjects.     

Biological monitoring of occupational exposure to low levels of benzene.

To obtain reference values for the biological monitoring of benzene, the kinetics of benzene were studied in volunteers. Benzene in blood and expired air could easily be followed until the next morning after a 4-h exposure to a benzene concentration of 10 cm3.m-3. Even after exposure to 1.7 cm3.m-3 the benzene levels in the morning blood and expired air samples differed from those in unexposed subjects. One hour after exposure to 10 and 1.7 cm3.m-3 the mean levels of benzene were 238 and 25 nmol.l-1 in blood and 13.2 and 2.5 mumol.m-3 in exhaled air, respectively. It was concluded that, at high benzene levels (approximately 10 cm3.m-3), samples collected 16 h after exposure reflect the body burden of benzene, while at low exposure (< 1 cm3.m-3) samples collected 1 h after exposure may be used to estimate the exposure over the preceding few hours. Exposure to benzene from smoking is a potential confounder in estimating occupational exposure to low levels of benzene.     

尿中苯巯基尿酸的高效液相色谱-质谱测定方法

目的 研制我国职业接触苯工人尿中苯巯基尿酸(SPMA)的生物限值.方法 在苯作业车间选择空气中苯浓度在32.5 mg/m~3以下接苯工人55人,应用个体采样器采集空气样品,用气相色谱法检测作业者个体苯接触水平,同时采集当日工人班后尿,应用高压液相色谱/质谱法(HPLC/MS)测定尿中SPMA含量以评价苯接触者的内暴露水平,内外暴露水平的比较用相对内暴露指数(RIE)加以评定.结果 接苯工人工作环境空气苯浓度范围为0.71～32.17 mg/m~3,几何平均浓度为6.98 mg/m~3,中位数为7.50 mg/m~3,尿中SPMA与个体苯暴露量存在良好的线性关系Y(μg/g Cr)=-8.625+18.367 X(mg/m~3),r=0.8035(P<0.01);将我国职业苯接触限值时间加权平均容许浓度(PC-TWA=6 mg/m~3)代入回归方程,推算工作班后尿中SPMA含量为101.58μg/g Cr.计算的RIE指数显示,接触1 mg/m~3苯可平均代谢转化为尿中SPMA 18.23 μg/g Cr,并且随接苯水平的上升其代谢转化效率呈下降趋势.结论 参考国外SPMA生物限值标准,建议我国职业接触苯生物限值班后尿中SPMA为100μg/gCr(47μmol/mol Cr).     

Application of the urinary S-phenylmercapturic acid test as a biomarker for low levels of exposure to benzene in industry.

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 studied were the urinary excretion characteristics of S-PMA, the specificity and the sensitivity of the assay, and the relations between exposures to airborne benzene and urinary S-PMA concentrations and between urinary phenol and S-PMA concentrations. The range of exposures to benzene was highest in workers in chemical manufacturing plants and in workers cleaning tanks or installations containing benzene as a component of natural gas condensate. Urinary S-PMA concentrations were measured up to 543 micrograms/g creatinine. Workers' exposures to benzene were lowest in oil refineries and S-PMA concentrations were comparable with those in smoking or nonsmoking control persons (most below the detection limit of 1 to 5 micrograms/g creatinine). In most workers S-PMA was excreted in a single phase and the highest S-PMA concentrations were at the end of an eight hour shift. The average half life of elimination was 9.0 (SD 4.5) hours (31 workers). Tentatively, in five workers a second phase of elimination was found with an average half life of 45 (SD 4) hours. A strong correlation was found between 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 micrograms/g creatinine (95% confidence interval 41-50 micrograms/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/m3 (10 ppm), the average urinary S-PMA concentration was 383 micrograms/g creatinine. In conclusion, with the current sensitivity of the test, eight hour time weighted average benzene exposures of 1 mg/m3 (0.3 ppm) and higher can be measured.     

Benzene exposure, assessed by urinary trans,trans-muconic acid, in urban children with elevated blood lead levels.

A pilot study was performed to evaluate the feasibility of using trans,trans-muconic acid (MA) as a biomarker of environmental benzene exposure. A secondary aim was to provide data on the extent of exposure to selected toxicants in a unique population consisting of inner-city children who were already overexposed to one urban hazard, lead. Potential sources of benzene were assessed by a questionnaire. Exposure biomarkers included urinary MA and cotinine and blood lead. Mean MA was 176.6 +/- 341.7 ng/mg creatinine in the 79 children who participated. A wide range of values was found with as many as 10.1%, depending on the comparison study, above the highest levels reported in adults not exposed by occupation. Mean MA was increased in children evaluated in the afternoon compared to morning, those at or above the median for time spent playing near the street, and those studied in the first half of the investigation. MA levels were not associated with blood lead or, consistently, with either questionnaire environmental tobacco smoke (ETS) data or cotinine. As expected, the mean blood lead level was elevated (23.6 micrograms/dl). Mean cotinine was also increased at 79.2 ng/mg creatinine. We conclude that the use of MA as a biomarker for environmental benzene exposure is feasible since it was detectable in 72% of subjects with a wide range of values present. In future studies, correlation of MA with personal air sampling in environmental exposure will be essential to fully interpret the significance of these findings. In addition, these inner-city children comprise a high risk group for exposure to environmental toxicants including ETS, lead, and probably benzene, based on questionnaire sources and its presence in ETS.     

Biological monitoring of exposure to benzene in petrol pump workers and dry cleaners.

Exposure to benzene has been monitored in petrol-pump workers and dry cleaners of Meerut City (India) by measuring phenol content of their urine samples. Average values for phenol in urine were higher in petrol-pump workers than dry cleaners. Alcoholic subjects excreted more phenol than smokers and non-vegetarians. It is concluded that alcohol can alter the susceptibility of man to benzene toxicity by affecting its metabolism.     

低苯暴露人群尿中t，t-MA及S-PMA的生物监测

目的分析职业低苯和环境低苯接触与人体尿液中t,t-MA和S-PMA浓度的相关性。方法选取广州市某制鞋厂钳帮和刷胶工人等苯职业接触人员作为职业低苯暴露人群（职业组）,选取非职业苯接触且家庭1年内装修过并已入住半年以上的人员作为环境低苯接触人群（环境组）。采用超高效液相串联质谱联用（UPLC—MS／MS）内标法测定尿中t,t-反式粘糠酸（t,t-MA）及苯巯基尿酸（S-PMA）含量,采用气相色谱法检测空气中苯浓度。结果职业组个体空气暴露的苯浓度（均值±标准差）为（0．16±0．06）mg／m^3,尿中t,t-MA及S-PMA含量分别为（42．7±39．2）和（0．28±0．19）μg／gCr;环境组个体空气暴露的苯浓度中位数（四分位间距）为0．01（0．02）mg／m^3,尿中t,t-MA及S-PMA含量的中位数（四分位间距）分别为20．5（16．2）和0．03（0．04）μg/gCr;经非参数Mann—WhitneyU—test检验分析发现：职业组的个体空气暴露苯浓度及尿中t,t-MA、S-PMA含量均高于环境组（均P〈0．01）。相关性分析结果显示,当空气中苯浓度为0．16mg／m^3时,尿中t,t—MA和S-PMA与空气中苯浓度存在良好的相关性（r=0．499、0．715）。结论t,t-MA及S-PMA可作为生物标记物用于职业低苯和环境低苯暴露的生物监测。     

苯接触与尿中反-反式黏糠酸和苯巯基尿酸关系研究

目的探讨职业苯接触与尿中反-反式黏糠酸(ttMA)和苯巯基尿酸(SPMA)的相关性,评定两接触标志物作为生物监测指标的适用性。方法对44名制鞋厂接苯工人进行个体苯暴露水平的作业环境监测,采集当日班前与班后尿样,分别用高效液相色谱和液质联谱测定尿中ttMA和SPMA含量。结果个体苯接触浓度为2.57~146.11 mg/m3,几何平均浓度为(27.91±3.29)mg/m3。班后尿中ttMA和SPMA含量均较班前增高,差异有统计学意义(P0.01),班后ttMA和SPMA与空气苯浓度的相关系数分别为0.905(P0.01)和0.537(P0.01),个体苯接触代谢转化为ttMA和SPMA的相对内暴露指数(RIE)随苯接触浓度的增高而下降。结论在中、高浓度的苯接触时,班后尿ttMA与空气苯浓度的相关性优于SPMA。     

Biological monitoring of exposure to benzene in traffic policemen of north India

Occupational health of traffic policemen employed at six major towns of north India was monitored during these investigations. Traffic controllers face the risk of exposure to benzene present in the ambient air as a component of fuel exhaust. Inhaled benzene is metabolized and excreted as phenol. Our observations on urinary phenol show much higher values than prescribed by ACGIH. Furthermore, social habits like alcohol consumption and cigarette smoke were found to modulate benzene metabolism. It was noticed that cigarette smoke synergizes the effect of benzene whereas antagonistic effects of alcohol were observed.     

Elevated urinary phenol levels not related to benzene exposure.

Two common over-the-counter medications may elevate urinary phenol to levels exceeding 75 mg/liter (ppm). This study presents data indicating that it is invalid to correct urinary phenol levels to specific gravity of 1.024. Animal studies indicate that having elevated urinary phenol levels secondary to phenyl salicylate ingestion are not harmful.