Luciano Maestri

Evaluation of occupational exposure to benzene by urinalysis

Urinary phenol determinations have traditionally been used to monitor high levels of occupational benzene exposure. However, urinary phenol cannot be used to monitor low-level exposures. New biological indexes for exposure to low levels of benzene are thus needed. The aim of this study was to investigate the relations between exposure to benzene (Abenzene, ppm), as measured by personal air sampling, and the excretion of benzene (U-benzene, ng/l),trans,trans-muconic acid (MA, mg/g creatinine), andS-phenylmercapturic acid (PMA, μg/g creatinine) in urine. The subjects of the study were 145 workers exposed to benzene in a chemical plant. The geometric mean exposure level was 0.1 ppm (geometric standard deviation = 4.16). After logarithmic transformation of the data the following linear regressions were found: log (U-benzene, ng/l) = 0.681 log (A-benzene ppm) + 4.018; log (MA, mg/g creatinine) = 0.429 log (A-benzen ppm) − 0.304; and log (PMA, μg/g creatinine) = 0.712 log (A-benzene ppm) + 1.664. The correlation coefficients were, respectively, 0.66, 0.58, and 0.74. On the basis of the equations it was possible to establish tentative biological limit values corresponding to the respective occupational exposure limit values. In conclusion, the concentrations of benzene, mercapturic acid, and muconic acid in urine proved to be good parameters for monitoring low benzene exposure at the workplace.

Trans, trans-muconic acid, a biological indicator to low levels of environmental benzene: Some aspects of its specificity

BACKGROUND The specificity of trans,trans-muconic acid (MA) as a biomarker of exposure to low benzene levels and the role of sorbic acid (SA) as a confounding factor were evaluated. MA, a urinary ring-opened metabolite of benzene, has been recently proposed for the biological monitoring of populations exposed to low levels of this chemical. The usual presence of MA in urine of non-occupationally exposed people is generally attributed to benzene world-wide contamination (mainly by smoking habits, urban pollution, and maybe by food contamination). However, the scientific literature reveals that the common food preservative and fungistatic agent SA is converted into MA though in trace amounts. METHODS Urinary benzene and MA before and after administration of SA were measured in smokers and non-smokers. Benzene dissolved in urine was analyzed injecting a headspace sample in a gas-chromatografic system. Urinary MA was measured by means of a HPLC apparatus. RESULTS The mean background values of MA were about 60 mg/L (or 50 mg/g creat.); after experimental administration of SA (447 mg), the mean urinary MA concentration became more than 20 times higher. The biotransformation rates of SA into MA after ingestion of 447 mg of SA ranged from 0.05 to 0.51%. The ratio between unmetabolized benzene in the two groups of smokers and non-smokers was significantly different from the ratio between MA in the same two groups. DISCUSSION Other sources of MA excretion, different from benzene, influence the urinary concentration of the metabolite: only 25% of MA background values can be attributed to benzene. The urinary MA induced by 100 mg of ingested MA is 77% of that expected after an 8-hour benzene exposure to 0.5 ppm (current threshold limit value according to ACGIH). In conclusion, MA is not a sufficiently specific biomarker of low benzene exposure; a significant effect of SA ingestion is predictable.

Determination of S-phenylmercapturic acid in urine as an indicator of exposure to benzene

S-phenylmercapturic acid (S-PMA) was measured in urine from 145 subjects exposed to low benzene concentrations in the air (C(I), benzene). The 8-h, time-weighted exposure intensity of individual workers was monitored by means of charcoal tubes and subsequent gas-chromatographic analysis after desorption with CS2. S-PMA excretion level in urine was determined by high-performance liquid chromatography with fluorescence detection. The following linear correlation was found between S-PMA concentrations in urine and benzene concentrations in the breathing zone: log(S-PMA, microg/g creatinine) = 0.712 log (C(I)-benzene, ppm) + 1.644 (n = 145, r = 0.74, P < 0.001). The geometric mean (GSD) of S-PMA concentrations in urine from 45 subjects occupationally not exposed to benzene but smoking more than 20 cigarettes/day was 7.8 microg/g creatinine (2.11), the corresponding value among non-smokers being 1.0 microg/g creatinine (2.18). It is concluded that the urinary level of S-PMA can be regarded as a useful indicator of exposure to benzene.

Urinary excretion of specific mercapturic acids in workers exposed to styrene

Styrene is an important chemical of wide industrial use, particularly in the manufacture of polymers and reinforced plastics. Environmental and occupational exposures to styrene occur predominantly via inhalation. Styrene undergoes biotransformation mainly by side chain oxidation catalyzed by cytochrome P-450 enzymes to its reactive metabolite, styrene oxide. The (R)- and (S)-enantiomers of styrene oxide can be conjugated with glutathione to both (R)- and (S)-diastereoisomers of specific mercapturic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1) and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). We conducted this biomonitoring study with the aim of evaluating the association between excretion of specific mercapturic acids (M1 and M2) and level of exposure to styrene among occupationally exposed people. The mean time-weighted average (TWA) exposure was about one-half the current threshold limit value, the range of the values varied from 44 to 228 mg/m3. Geometric mean (GM) concentrations of 650, 1,084, and 31.8 micrograms/g creatinine were measured, respectively, for M1-S, M2, and M1-R. The environmental styrene concentration exhibited a significant correlation with total specific mercapturic acid (Mtot = sum of M1-R, M1-S, and M2), making it possible for the first time to calculate the approximate relationship between styrene uptake and excretion of these substances. The M2 mercapturic acid had a better correlation (r = 0.56) with respect to M1-R and M1-S. Significant correlations were found also between the excretion of specific mercapturic acids and biological exposure indices (i.e., mandelic and phenylglyoxylic acids and urinary styrene).

The Specificity of trans,trans- Muconic Acid as a Biological Indicator for Low Levels of Environmental Benzene

trans, trans -Muconic acid (MA), a urinary ring-opened metabolite of benzene, is a newly proposed biological indicator of benzene exposure which should enable the assessment of low levels of exposure to this ubiquitous environmen tal pollutant. The presence of MA in the urine of non-occupationally exposed people is generally attributed to world-wide contamination of the environ ment by benzene (arising from such sources as smoking and other combustion, urban pollution from vehicles and maybe by food contamination). But, exami nation of the scientific literature reveals that a common food preservative and fungistatic agent, sorbic acid (SA), is also converted into MA, although in trace amounts. The permitted maximum concentration of SA in food ranges from 0.2 to 2 g·kg -1 and the acceptable daily intake (ADI-FAO/WHO) is 25 mg. kg -1 of body weight. The question, therefore, is whether the amount of MA excreted as a metabolite of SA can make a significant contribution to the total MA excreted. If so, the specificity of MA as a biological indicator for benzene exposure assessment is in doubt. Four experiments in 2 subjects were set up to try to clarify the effect on the urinary excretion of MA of the experimental ingestion of SA (2 doses of 447 and 44.7 mg); urine samples were collected, in fractions as voided, for 24 h before and 48 (or 60 h) after SA ingestion. The total amounts of MA excreted during the 24 h before ingestion of SA were respectively 79 and 34 mg in the 2 subjects; after ingestion of 447 or 44.7 mg of SA the total excretion of MA was at least respectively 20 and 2 times the background levels (when the single dose of SA was ingested) and at least 10 and 3 times (when the SA was given in divided doses). The elimination of MA after ingestion of SA was complete in 24 h; the peak concentrations always appeared in the first urine sample after the ingestion of the substance and they were more than 90 times and more than 3 times the basal levels respectively for a single ingestion of 447 and 44.7 mg of SA. The rate of biotransformation of SA into MA was, on average, 0.34 and 0.21 % in the 2 subjects; on the basis of these levels of biotransformation, it appears that 0.3-0.5 g of ingested SA would suffice to lead to excretion of 1 mg of MA, a quantity corresponding to that believed to derive from benzene exposure at levels greater than 1 ppm. Only in urine collected many hours after the last meal can a possible additive effect of MA from SA be ignored.

Biological monitoring of workers exposed to carbon disulfide (CS2) in a viscose rayon fibers factory

The exposure-excretion relationship to carbon disulfide (CS2) vapor in 407 exposed workers was studied during the second half of the working week. Carbon disulfide concentrations were also determined in 50 nonexposed subjects. The geometric mean value for CS2 in urine samples from the latter was: 0.23 microgram/l (95% upper limit = 0.52 microgram/l) when log-normal distribution was assumed. Among the exposed workers, the CS2 level in urine samples collected after the first half shift exceeded the 95% upper limit of nonexposed subjects in every case. The time-weighted average intensity of exposure to CS2 vapor was measured using personal diffusive samplers (in which carbon cloth served as an adsorbent). CS2 concentrations in urine were determined in samples collected at the end of the first half shift from the 407 exposed cases as well as from 50 nonexposed controls. There was a significant correlation (p < 0.0001) between the exposure to CS2 vapor at concentrations of up to 64 mg/m3 and the levels of CS2 measured in the urine samples after four hours of exposure. The correlation indicated that a mean level of 15.5 micrograms CS2/l urine (95% confidence range, 13.8-17.1 micrograms/l) was excreted following an exposure to CS2 at 31 mg/m3 (the current occupational exposure limit).

The Determination of Trans, Trans-Muconic Acid in Urine as an Indicator of Occupational Exposure to Benzene

Abstract Recently the determination of muconic acid (MA; milligrams/gram creatinine) in urine has been proposed as a suitable biomarker for the monitoring of low level exposure to benzene. A sample of 171 subjects employed in a factory was studied for personal exposure to benzene [CI, benzene (parts per million)] and excretion of AM. The 8-hour time-weighted exposure intensity of individual workers was monitored by means of charcoal tubes. MA excretion level in urine was determined by high performance liquid chromatography with ultraviolet detection. The following linear correlation was found between MA concentrations in urine and benzene concentrations in the breathing zone log MA(mg/g creatinine) = 0.549 log CI, benzene (ppm) – 0.18 (n = 171, r = 0.614, p 20 cigarettes/day and 50 nonsmokers) were also determined [geometric mean of 0.207 mg/g creatinine (1.5) and 0.067 mg/g creatinine (2.1), respectively]. In the current study...

Effects of ethanol administration on cerebral non-protein sulfhydryl content in rats exposed to styrene vapour

Glutathione (GSH) and other non-protein sulfhydryls (NPS) are known to protect cells from oxidative stress and from potentially toxic electrophiles formed by biotransformation of xenobiotics. This study examined the effect of a simultaneous administration of styrene and ethanol on NPS content and lipid peroxidation in rat liver and brain. Hepatic cytochrome P450 and cytochrome b5 content, aniline hydroxylase and aminopyrine N-demethylase activities as well as the two major urinary metabolites of styrene, mandelic and phenylglyoxylic acids were also measured. Groups of rats given ethanol for 3 weeks in a liquid diet were exposed, starting from the second week, to 326 ppm of styrene (6 h daily, 5 days a week, for 2 weeks). In control pair-fed animals, styrene produced about 30% depletion of brain NPS and 50% depletion of hepatic NPS. Subchronic ethanol treatment did not affect hepatic NPS levels, but caused 23% depletion of brain NPS. Concomitant administration of ethanol and styrene caused a NPS depletion in brain tissue in the order of 60%. These results suggest that in the rat, simultaneous exposure to ethanol and styrene may lead to considerable depletion of brain NPS. This effect is seen when both compounds are given on a subchronic basis, a situation which better resembles possible human exposure.

Determination of 2,5-hexandione by high-performance liquid chromatography after derivatization with dansylhydrazine

A sensitive method for the determination of free and total urinary 2,5-hexandione (2,5-HD) using high-performance liquid chromatography with fluorescence detection was developed. After purification of urine with a disposable C18 cartridge, 2,5-HD was derivatized with dansylhydrazine; 1,3-diacetyl benzene (1,3-DAB) was added to the samples, as internal standard, prior to extraction. The resulting fluorescent adducts were separated on a reversed-phase column with a gradient mobile phase of 25 mM phosphate buffer (pH 6.4) and acetonitrile. The retention times of the 2,5-HD and 1,3-DAB derivatives were 9.4 and 13.7 min, respectively. The derivatives were detected by a fluorescence detector (excitation 340 nm, emission 525 nm). The mean recoveries of 2,5-HD and 1,3-DAB were 92.0 and 94.0%, respectively; the detection limit of 2,5-HD (signal-to-noise ratio of 3) was 5 micrograms/l in urine without hydrolysis and ca. 12 micrograms/l in hydrolyzed samples. The method was applied to 39 urine samples from workers exposed to n-hexane; the mean values were 2.597 mg/l (S.D. = +/- 0.758) for total 2,5-HD and 0.179 mg/l (S.D. = +/- 0.086) for free 2,5-HD. Urine samples of 22 non-exposed subjects showed a mean concentration of 0.437 mg/l (S.D. = +/- 0.109) and 0.022 mg/l (S.D. = +/- 0.011) for total and free 2,5-HD, respectively.

Measurement of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine by high-performance liquid chromatography with direct ultraviolet detection.

A new high-performance liquid chromatographic (HPLC) method is described for the determination of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), the final product of the conjugation reaction between a metabolic intermediate of N,N-dimethylformamide (DMF) and glutathione. Urine samples were purified by C(18) solid-phase extraction and then directly analysed by HPLC with an Aminex Ion Exclusion HPX-87H column maintained at 25 degrees C and a UV detector set at 196 nm. Under isocratic conditions (2.4 mM sulphuric acid, flow-rate=0.6 ml/min) AMCC eluted at 20.2 min. The reproducibility (C.V.%) was 1.3-2.7% (intra- and inter-assay, N = 5); the accuracy was 98.0+/-1.7% at 10 mg/l and 101.9+/-1.5% at 800 mg/l (mean+/-SD, N = 3). AMCC was measured in urine from 22 exposed subjects. A strong correlation was found between AMCC and environmental DMF [AMCC (mg/g creatinine)=3.40xDMF (mg/m(3)) + 3.07; r=0.95], while in the urine of 20 unexposed subjects the concentration of AMCC was constantly below the detection limit of the method (0.9 mg/l in urine). The method described appears to be useful for the biological monitoring of DMF exposure.