A novel DNA adduct, originating from 1,2-epoxy-3,4-butanediol, is the major DNA adduct after exposure to [2,3-(14)C]-1,3-butadiene,[4-(14)C]-1,2-epoxy-3-butane.

1,3-Butadiene is a rodent carcinogen and its epoxide metabolites, 1,2-epoxy-3-butene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2:3,4-diepoxybutane (DEB) have been suggested as ultimate carcinogens. This study aimed at identification and quantification of DNA adducts in rats and mice following exposure to BD and its major metabolite EB to identify the reactive epoxide(s) in target tissues. Reaction of [4-(14)C]-EB with 2'-deoxyguanosine (dG) or DNA gave equal amounts of N7-(2-hydroxy-3-butenyl)guanine (G1) and N7-(1-(hydroxymethyl)-2-propenyl)guanine (G2). Reaction of DEB stereomers with dG yielded N7-(2,3,4-trihydroxybutyl)guanine (G3) as major adduct and novel, minor adduct (G4) that was tentatively identified as N7-(1-(hydroxymethyl)-2,3-dihydroxypropyl)guanine. For the stereomers of EBD, the opposite was found: reaction with dG led to G4 as major and G3 as minor adduct. 2D-Total correlation 1H-NMR spectroscopy of G4 indicated that the N7-alkyl group was in a virtually fixed conformational state and might interact with the O(6) of guanine, which might imply a higher degree of mutagenicity of G4, due to G-C mispairing, than of any other adducts observed in this study. At 48 h following exposure to [4-(14)C]-EB (1-50 mg/kg), DNA adduct profiles in rat and mouse were qualitatively similar, with G1 and G2 as main, and G4 as minor adduct. Following nose-only exposure to 200 ppm [2,3-(14)C]-BD for 6 h, G1 and G2 were minor adducts in liver (1.9 and 8.0 per 10(8) nucleotides) and lung (1.6 and 6.6 per 10(8) nucleotides, for rats and mice, respectively). G3 was absent in rats, but present in mouse liver and lung, at 20 and 12 adducts/10(8) nucleotides. The major adduct was G4 accounting for 13 and 90 (liver) and 11 and 139 (lung) adducts/10(8) nucleotides in rats and mice, respectively. Forty-two hours later, these adduct levels had only little changed. Our recent biomarker studies confirm that following exposure to BD, but not BDO, EBD is the major epoxide available for macromolecular binding in humans and rodents. Most probably because EBD is in contrast to EB and DEB, a poor substrate for epoxide hydrolases. In conclusion, the major DNA adduct following exposure to BD is G4, originating from EBD, and not from EB or BDE. It is concluded that EBD and G4 should be taken into account for human risk assessment for exposure to BD.

Urinary metabolites and haemoglobin adducts as biomarkers of exposure to 1,3-butadiene: a basis for 1,3-butadiene cancer risk assessment.

Since 1,3-butadiene (BD) is a suspected human carcinogen, exposure to BD should be minimised and controlled. This study aimed at comparing the suitability of biomarkers for low levels of exposure to BD, and at exploration of the relative pathways of human metabolism of BD for comparison with experimental animals. Potentially sensitive biomarkers for BD are its urinary metabolites 1,2-dihydroxybutyl mercapturic acid (DHBMA, also referred to as MI) and 1- and 2-monohydroxy-3-butenyl mercapturic acid (MHBMA, also referred to as MII) and its haemoglobin (Hb) adducts 1- and 2-hydroxy-3-butenyl valine (MHBVal). In two field studies in BD-workers, airborne BD, MHBMA, DHBMA and MHBVal were determined. MHBMA proved more sensitive than DHBMA for monitoring recent exposures to BD and could measure 8-h time weighted average exposures as low as 0.13 ppm (0.29 mg/m(3)). The sensitivity of DHBMA was restricted by relatively high natural background levels in urine, of which the origin is currently unknown. MHBVal proved a sensitive method for monitoring cumulative exposures to BD at or above 0.35 ppm (0.77 mg/m(3)). Statistically significant relationships were found between either MHBMA or DHBMA and 8-h airborne BD levels, and between MHBVal adducts and average airborne BD levels over 60 days. The data showed a much higher rate of hydrolytic metabolism of BD in humans compared to animals, which was reflected in a much higher DHBMA/(MHBMA+DHBMA) ratio, and in much lower levels of MHBVal in humans, confirming in vitro results. Assuming a genotoxic mechanism, the data of this study coupled with our recent data on DNA and Hb binding in rodents, suggest that the cancer risk for humans from exposure to BD will be less than for the rat, and much less than for the mouse.

Quantification of DNA adducts formed in liver, lungs, and isolated lung cells of rats and mice exposed to (14)C-styrene by nose-only inhalation.

Bronchiolo-alveolar tumors were observed in mice exposed chronically to 160 ppm styrene, whereas no tumors were seen in rats up to concentrations of 1000 ppm. Clara cells, which are predominant in the bronchiolo-alveolar region in mouse lungs but less numerous in rat and human lung, contain various cytochrome P450s, which may oxidize styrene to the rodent carcinogen styrene-7,8-oxide (SO) and other reactive metabolites. Reactive metabolites may form specific DNA adducts and induce the tumors observed in mice. To determine DNA adducts in specific tissues and cell types, rats and mice were exposed to 160 ppm [ring-U-(14)C]styrene by nose-only inhalation for 6 h in a recirculating exposure system. Liver and lungs were isolated 0 and 42 h after exposure. Fractions enriched in Type II cells and Clara cells were isolated from rat and mouse lung, respectively. DNA adduct profiles differed quantitatively and qualitatively in liver, total lung, and enriched lung cell fractions. At 0 and 42 h after exposure, the two isomeric N:7-guanine adducts of SO (measured together, HPEG) were present in liver at 3.0 +/- 0.2 and 1.9 +/- 0.3 (rat) and 1.2 +/- 0.2 and 3.2 +/- 0.5 (mouse) per 10(8) bases. Several other, unidentified adducts were present at two to three times higher concentrations in mouse, but not in rat liver. In both rat and mouse lung, HPEG was the major adduct at approximately 1 per 10(8) bases at 0 h, and these levels halved at 42 h. In both rat Type II and non-Type II cells, HPEG was the major adduct and was about three times higher in Type II cells than in total lung. For mice, DNA adduct levels in Clara cells and non-Clara cells were similar to total lung. The hepatic covalent binding index (CBI) at 0 and 42 h was 0.19 +/- 0.06 and 0.14 +/- 0.03 (rat) and 0. 25 +/- 0.11 and 0.44 +/- 0.23 (mouse), respectively. The pulmonary CBIs, based on tissues combined for 0 and 42 h, were 0.17 +/- 0.04 (rat) and 0.24 +/- 0.04 (mouse). Compared with CBIs for other genotoxicants, these values indicate that styrene has only very weak adduct-forming potency. The overall results of this study indicate that DNA adduct formation does not play an important role in styrene tumorigenicity in chronically exposed mice.

Dermal penetration and metabolism of five glycidyl ethers in human, rat and mouse skin.

1. Glycidyl ethers (GE), an important class of industrial chemicals, are considered to be potentially mutagenic in vivo because some GE have been shown to be direct mutagens in short-term in vitro tests. 2. The percutaneous penetration and metabolism of representatives of different classes of GE was studied in the fresh, full-thickness C3H mouse, and dermatomed human and Fisher 344 rat skin to determine the apparent permeability constants, lag times and metabolic profiles. 3. Five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C12GE) and o-cresol (o-CGE), were synthesized by reaction of their alcohols with epichlorohydrin. Their radiolabelled analogues were synthesized with a 14C-label using [U-14C]-epichlorohydrin. 4. There was a large variation (four orders of magnitude) in percutaneous penetration between the five GE. In general, penetration through full-thickness mouse skin was higher than through dermatomed rat skin, whereas dermatomed human skin was the least permeable. The permeability increased in the order YX4000 < BADGE < C12GE < o-CGE < HDDGE. 5. The relative skin permeability of the five GE could be explained for a significant part by the lipophilicity, expressed as log P(o/w), in combination with the molecular weight of the compounds. 6. During skin penetration, all GE were extensively metabolized to their corresponding (bis-)diols. Virtually no YX4000, and only very small amounts of C12GE and BADGE, penetrated the skin unchanged, but significant amounts of HDDGE and o-CGE penetrated the skin unchanged. For o-CGE, but none of the other GE, the percentage of the applied dose that penetrated the skin unchanged increased over time. 7. The large variation in response observed with the five selected GE indicates that GE should not be considered as a single class of compounds but rather on the basis of their individual properties.

Metabolic inactivation of five glycidyl ethers in lung and liver of humans, rats and mice in vitro.

1. Some glycidyl ethers (GE) have been shown to be direct mutagens in short-term in vitro tests and consequently GE are considered to be potentially mutagenic in vivo. However, GE may be metabolically inactivated in the body by two different enzymatic routes: conjugation of the epoxide moiety with the endogenous tripeptide glutathione (GSH) catalysed by glutathione S-transferase (GST) or hydrolysis of the epoxide moiety catalysed by epoxide hydrolase (EH). 2. The metabolic inactivation of five different GE, the diglycidyl ethers of bisphenol A (BADGE), 4,4'-dihydroxy-3,3',5,5'-tetramethylbiphenyl (Epikote YX4000) and 1,6-hexanediol (HDDGE) and the GE of 1-dodecanol (C12GE) and o-cresol (o-CGE), has been studied in subcellular fractions of human, C3H mouse and F344 rat liver and lung. 3. All GE were chemically very stable and resistant to aqueous hydrolysis, but were rapidly hydrolysed by EH in cytosolic and microsomal fractions of liver and lung. The aromatic GE were very good substrates for EH. In general, microsomal EH is more efficient than cytosolic EH in hydrolysis of GE, and human microsomes are more efficient than rodent microsomes. 4. The more water-soluble GE, o-CGE and HDDGE, were good substrates for GST whereas the more lipophilic GE, YX4000 and C12GE, were poor substrates for GST. In general, rodents are more efficient in GSH conjugation of GE than humans. 5. In general, the epoxide groups of YX4000 are the most and those of HDDGE the least efficiently inactivated of the five GE under study. For the other three GE no general trend was observed: the relative efficiency of inactivation varied with organ and species. 6. The large variation in metabolism observed with five representative GE indicate that GE have variable individual properties and should not be considered as a single, homogenous class of compounds.

Biomarkers of exposure to 1,3-butadiene as a basis for cancer risk assessment.

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.

Formation of DNA adducts and induction of mutagenic effects in rats following 4 weeks inhalation exposure to ethylene oxide as a basis for cancer risk assessment.

Ethylene oxide (EO) is mutagenic in various in vitro and in vivo test systems and carcinogenic in rodents. EO forms different adducts upon reaction with DNA, N7-(2-hydroxyethyl)guanine (N7-HEG) being the main adduct. The major objectives of this study were: (a) to determine the formation and persistence of N7-HEG adducts in liver DNA of adult male rats exposed to 0, 50, 100 and 200 ppm by inhalation (4 weeks, 5 days/week, 6 h/day) and (b) to assess dose-response relationships for Hprt gene mutations and various types of chromosomal changes in splenic lymphocytes.N7-HEG adducts were measured 5, 21, 35 and 49 days after cessation of exposure. By extrapolation, the mean concentrations of N7-HEG immediately after cessation of exposure ('day 0') to 50, 100 and 200 ppm were calculated as 310, 558 and 1202 adducts/10(8) nucleotides, respectively, while the mean concentration in control rats was 2.6 adducts/10(8) nucleotides. At 49 days, N7-HEG values had returned close to background levels. The mean levels of N-(2-hydroxyethylvaline) adducts in haemoglobin were also determined and amounted 61.7, 114 and 247 nmol/g globin, respectively. Statistically significant linear relationships were found between mean N7-HEG levels ('day 0') and Hprt mutant frequencies at expression times 21/22 and 49/50 days and between mean N7-HEG ('day 0') and sister-chromatid exchanges (SCEs) or high frequency cells (HFC) measured 5 days post-exposure. At day 21 post-exposure, SCEs and HFCs in-part persisted and were significantly correlated with persistent N7-HEG adducts. No statistically significant dose effect relationships were observed for induction of micronuclei, nor for chromosome breaks or translocations. In conclusion, this study indicates that following sub-chronic exposure, EO is only weakly mutagenic in adult rats. Using the data of this study to predict cancer risk in man resulting from low level EO exposures in conjunction with other published data, i.e., those on (a) genotoxic effects of EO in humans and rats, (b) DNA binding of other carcinogens, (c) natural background DNA binding and (d) genotoxic potency of low energy transfer (LET) radiation, it is not expected that long term occupational exposure to airborne concentrations of EO at or below 1 ppm EO produces an unacceptable increased risk in man.

Metabolic inactivation of 2-oxiranylmethyl 2-ethyl-2,5-dimethylhexanoate (C10GE) in skin, lung and liver of human, rat and mouse.

The inactivation of 2-oxiranylmethyl 2-ethyl-2,5-dimethylhexanoate (C10GE), one of the most abundant isomers of the epoxy-resin Carduras E-10 glycidyl ester, was studied in subcellular fractions of human, C3H mouse and F344 rat liver, lung and skin. C10GE is chemically very stable and resistant to aqueous hydrolysis, but it was rapidly metabolized in both cytosolic and microsomal fractions of all organs by epoxide hydrolase (EH)-catalysed hydrolysis of the epoxide moiety as well as carboxylesterase (CE)-catalysed hydrolysis of the ester bond. In cytosol the epoxide group was also efficiently conjugated with glutathione, catalysed by glutathione S-transferase (GST), but this conjugation was much less important than hydrolysis in human as well as rodent samples. Although CE-catalysed hydrolysis of C10GE would theoretically give rise to the formation of glycidol, a directly acting mutagen, it is highly unlikely that any significant level of glycidol would occur in vivo since reported rates of inactivation of glycidol exceed the total rate of hydrolysis of C10GE. The overall rates of inactivation in vitro decreased in the following order: mouse > rat > human. Scaling of the data in vitro to clearances in vivo suggests that the detoxifying capacity in the rodents is similar and about an order of magnitude greater than in human. Nevertheless, the rate of inactivation is 2-3 orders of magnitude greater than for simple epoxides such as butadiene monoxide and about one order of magnitude higher than for the diglycidyl ether of bisphenol A (BADGE). The transdermal penetration and metabolism of [14C]-C10GE was studied in fresh full-thickness mouse, and dermatomized human and rat skin. Of the total radioactivity applied on the skin, only 0.24+/-0.06 (SD), 1.8+/-0.2 and 6.8+/-0.6% penetrated through human, mouse and rat skin respectively. The corresponding apparent skin permeability constants were 0.81, 6.42 and 26.4 x 10(-6) cm/h. During transdermal penetration, [14C]-C10GE was extensively hydrolysed to the corresponding diol and the free acid. Only 0.01, 0.11 and 0.21]% of the applied dose was absorbed unchanged through the human, mouse and rat skin respectively.

Quantitative and qualitative differences in the metabolism of 14C-1,3-butadiene in rats and mice: relevance to cancer susceptibility.

1,3-Butadiene (butadiene) is a potent carcinogen in mice, but not in rats. Metabolic studies may provide an explanation of these species differences and their relevance to humans. Male Sprague-Dawley rats and B6C3F1 mice were exposed for 6 h to 200 ppm [2,3-14C]-butadiene (specific radioactivity [sa] 20 mCi/mmol) in a Cannon nose-only system. Radioactivity in urine, feces, exhaled volatiles and 14C-CO2 were measured during and up to 42 h after exposure. The total uptake of butadiene by rats and mice under these experimental conditions was 0.19 and 0.38 mmol (equivalent to 3.8 and 7.5 mCi) per kg body weight, respectively. In the rat, 40% of the recovered radioactivity was exhaled as 14C-CO2, 70% of which was trapped during the 6-h exposure period. In contrast, only 6% was exhaled as 14C-CO2 by mice, 3% during the 6-h exposure and 97% in the 42 h following cessation of exposure. The formation of 14C-CO2 from [2,3-14C]-labeled butadiene indicated a ready biodegradability of butadiene. Radioactivity excreted in urine accounted for 42% of the recovered radioactivity from rats and 71% from mice. Small amounts of radioactivity were recovered in feces, exhaled volatiles and carcasses. Although there was a large measure of commonality, the exposure to butadiene also led to the formation of different metabolites in rats and mice. These metabolites were not found after administration of [4-14C]-1,2-epoxy-3-butene to animals by i.p. injection. The results show that the species differences in the metabolism of butadiene are not simply confined to the quantitative formation of epoxides, but also reflect a species-dependent selection of metabolic pathways. No metabolites other than those formed via an epoxide intermediate were identified in the urine of rats or mice after exposure to 14C-butadiene. These findings may have relevance for the prediction of butadiene toxicity and provide a basis for a revision of the existing physiologically based pharmacokinetic models.

Biomonitoring of exposure to ethylene oxide and propylene oxide by determination of hemoglobin adducts: correlations between airborne exposure and adduct levels.

OBJECTIVES: Ethylene oxide (EO) and propylene oxide (PO) are important industrial chemicals. Exposure to these directly acting mutagens may be monitored by determination of their adducts to hemoglobin (Hb). This study establishes correlations between airborne concentrations of EO and PO and their Hb adducts in petrochemical workers. METHODS: In three different studies conducted during maintenance shutdown of petrochemical plants the external occupational exposure to EO and PO was assessed by personal air monitoring (PAM). The internal exposure to EO and PO was concomitantly assessed by determination of N-(2-hydroxyethyl)valine (HOEtVal) and N-(3-hydroxypropyl)valine (HOPrVal) in blood samples of the operators using the N-alkyl-Edman degradation method. RESULTS: In the first study, PAM was applied once a month at random over a period of 4 months. Blood samples for Hb-adduct determination were collected at the end of this period. No significant correlation was found between PAM and Hb-adduct data. In the next two studies, PAM was applied to the operators during the entire shift on every working day during the shutdown. Blood samples were collected before and immediately after the shutdown period. Highly significant correlations were found between the increment in the concentration of HOEtVal and HOPrVal over this period and the total exposure to EO and PO, respectively. CONCLUSIONS: Time-integrated exposure to EO or PO can be readily and reliably assessed by measurement of the concentration of HOEtVal or HOPrVal in a small blood sample. In workers occupationally exposed to low concentrations of EO or PO, good correlations were found between these Hb adducts and the airborne concentrations of EO and PO. These correlations allow the calculation of tentative biological exposure limits (BELs) for EO and PO. At the current Dutch occupational exposure limit (OEL) for EO (0.84 mg m(-3), 8-h TWA) the BEL is 3.2 nmol HOEtVal/g globin. At the value of 10 mg m(-3) (8-h TWA), which is currently being investigated as the new Dutch OEL for PO, the corresponding BEL is 5.3 nmol HOPrVal/g globin.

Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide.

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.

Identification of novel metabolites of butadiene monoepoxide in rats and mice.

Differences in the metabolism of 1,3-butadiene (Bd) in rats and mice may account for the observed species difference in carcinogenicity. Previous studies of the metabolic fate of Bd have identified epoxide formation as a key metabolic transformation which gives 1, 2-epoxy-3-butene (BMO), although some evidence of aldehyde metabolites is reported. In this study, male Sprague-Dawley rats and male B6C3F1 mice received single doses of [4-14C]BMO at 1, 5, 20, and 50 mg/kg of body weight (0.014, 0.071, 0.286, and 0.714 mmol/kg of body weight). Analysis of urinary metabolites indicated that both species preferentially metabolize BMO by direct reaction with GSH when given by ip administration. The excretion of (R)-2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (IIa), 1-(N-acetyl-L-cystein-S-yl)-2-(S)-hydroxybut-3-ene (IIb), 1-(N-acetyl-L-cystein-S-yl)-2-(R)-hydroxybut-3-ene (IIc), and (S)-2-(N-acetyl-L-cystein-S-yl)-1-hydroxybut-3-ene (IId) accounted for 48-64% of urinary radioactivity in rats and 46-54% in mice. The metabolites originating from the R-stereoisomer of BMO (IIc and IId) predominated over those arising from the S-stereoisomer (IIa and IIb) in both species. IIc was formed preferentially in mice and IId in rats. The corresponding mercaptoacetic acids, S-(1-hydroxybut-3-en-2-yl)mercaptoacetic acid (IIf) and S-(2-hydroxybut-3-en-1-yl)mercaptoacetic acid (IIg), were identified only in mouse urine (ca. 20% of the recovered radioactivity). 4-(N-Acetyl-L-cystein-S-yl)-1,2-dihydroxybutane (Ia), a metabolite derived from hydrolysis of BMO, accounted for 10-17% of the radioactivity in rat and 6-10% in mouse urine. 4-(N-Acetyl-L-cystein-S-yl)-2-hydroxybutanoic acid (Ib), 3-(N-acetyl-L-cystein-S-yl)propan-1-ol (Ic), and 3-(N-acetyl-L-cystein-S-yl)propanoic acid (Id), also derived from the hydrolysis of BMO, were only present in the rat. Metabolites of 1,2,3,4-diepoxybutane (DEB) were not detected after administration of BMO in rat or mouse urine. This study showed both quantitative and qualitative differences in the metabolism of BMO with varying doses and between species. The data aid in the safety evaluation of Bd and contribute to the interpretation of mathematical models developed for quantitative risk assessment and extrapolation of animals to humans.

Allylmercapturic acid as urinary biomarker of human exposure to allyl chloride.

OBJECTIVE: To evaluate the use of urinary mercapturic acids as a biomarker of human exposure to allyl chloride (3-chloropropene) (AC). During three regular shut down periods in a production factory for AC, both types of variables were measured in 136 workers involved in maintenance operations. METHODS: Potential airborne exposure to AC was measured by personal air monitoring in the breathing zone. In total 205 workshifts were evaluated. During 99 workshifts no respiratory protection equipment was used. Mercapturic acid metabolites were measured in urinary extracts by gas chromatography-mass spectrometry (GC-MS). RESULTS: During 86 work shifts when no respiratory protection was used the air concentrations of AC were below the Dutch eight hour time weighted average (8 h-TWA) occupational exposure limit (OEL) of AC (3 mg/m3), whereas in 13 workshifts the potential exposure, as measured by personal air monitoring, exceeded the OEL (3.3 to 17 mg/m3). With the aid of GC-MS, 3-hydroxypropylmercapturic acid (HPMA) was identified as a minor and allylmercapturic acid (ALMA) as a major metabolite of AC in urine samples from the maintenance workers exposed to AC. The concentrations of ALMA excreted were in a range from < 25 micrograms/l (detection limit) to 3550 micrograms/l. The increases in urinary ALMA concentrations during the workshifts correlated well with the 8h-TWA air concentrations of AC (r = 0.816, P = 0.0001, n = 39). Based on this correlation, for AC a biological exposure index (BEI) of 352 micrograms ALMA/g creatinine during an eight hour workshift is proposed. In some urine samples unexpectedly high concentrations of ALMA were found. Some of these could definitely be attributed to dermal exposure to AC. In other cases garlic consumption was identified as a confounding factor. CONCLUSION: The mercapturic acid ALMA was identified in urine of workers occupationally exposed to airborne AC and the increase in ALMA concentrations in urine during a workshift correlated well with the 8 h-TWA exposure to AC. Garlic consumption, but not smoking, is a potential confounding factor for this biomarker of human exposure to AC.

Suitability of S-phenyl mercapturic acid and trans-trans-muconic acid as biomarkers for exposure to low concentrations of benzene.

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.

Biological monitoring of butadiene exposure by measurement of haemoglobin adducts.

The measurement of haemoglobin (Hb) adducts is an important tool for monitoring exposures to industrial chemicals such as ethylene, ethylene oxide and propylene oxide. The aim of the present study was to use this method to monitor occupational exposure to butadiene. The methodology was evaluated with Hb samples obtained by reacting butadiene monoepoxide (BMO), the primary reactive metabolite of 1,3-butadiene, with blood and by dosing BMO to rats and mice. The Hb adducts: N-(2-hydroxy-3-buten-1-yl)valine and N-(1-hydroxy-3-buten-2-yl)valine were determined as the pentaflurophenylthiohydantoin derivatives using the modified Edman degradation procedure and gas chromatography with negative ion chemical ionisation mass spectrometry. The adducts were quantified using d4-N-(2-hydroxyethyl)valine as an internal reference chemical. Relatively high background signals were observed which made accurate quantitation difficult. The lower limit of detection was estimated as 10-20 pmol/g globin. The experiments have demonstrated that appropriate reference samples are required for accurate quantitation and the method requires further refinement to improve the sensitivity of the analysis.

Effects of exposure to elemental mercury on the nervous system and the kidneys of workers producing natural gas.

Early signs of alterations in renal and neurological functions were studied in three groups of workers who were exposed to different levels of mercury that were below the current biological exposure index of 35 microg/g creatinine. There were no differences among the three study groups with respect to either motor nerve conduction velocity or tremor frequency spectra of physiological tremors. Also, no significant correlations were found between the results of the neurological tests and any of the present or historical biological monitoring data. In contrast, N-acetyl-beta-D-glucosaminidase was increased significantly in the group with the higher exposure, compared with either the lower-exposure or control groups. N-acetyl-beta-D-glucosaminidase was correlated strongly with mercury concentration in urine and was correlated weakly with historical biological monitoring data; however, there was no correlation with duration of exposure. These results suggest that after exposure to mercury at levels below the biological exposure index, a transient increase in N-acetyl-beta-D-glucosaminidase can be observed, but is not an early indicator of developing renal dysfunction.

Biological monitoring of exposure to benzene: a comparison between S-phenylmercapturic acid, trans,trans-muconic acid, and phenol.

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 micrograms S-PMA/g creatinine in samples from the end of the shift. It was estimated that, on average, 3.9% (range 1.9%-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 0.05%-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 micrograms/g creatinine, respectively and the mean urinary tt-MA was 0.058 and 0.037 mg/g 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 non-smokers 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 mg/g 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.

Biological effect monitoring in industrial workers following incidental exposure to high concentrations of ethylene oxide.

Peripheral blood from four groups of seven workers from a chemical manufacturing plant in The Netherlands was analyzed for hemoglobin adducts in erythrocytes and for hprt mutants, micronuclei and SCEs in lymphocytes. Group I workers were incidentally exposed to acute high doses of ethylene oxide ranging from 52 to 785 mg/m3. Group II and III workers were chronically exposed to low doses of ethylene oxide for < 5 years or > 15 years respectively. Group IV workers served as unexposed controls and came from the Occupational Health Department. Hemoglobin adduct levels in group I workers were very high and ranged from 1461 to 19913 pmol HOEtVal/g Hb approximately 1 month after the accident. HOEtVal values for group II and III workers fluctuated between 0 and 190 pmol/g Hb corresponding with average EtO exposure levels in the range of < 0.01 and 0.06 mg/m3 EtO. The statistical analysis of the genetic data did not reveal any statistically significant differences between any combination of worker groups. The genetic tests for group I workers were performed on blood samples collected 89-180 days after the incidental exposure. The absence of enhanced frequencies of mutations, micronuclei and SCEs suggests that significant induction of hprt mutations in vivo did not occur and that persistent preclastogenic lesions were not present in significant amounts when the exposed lymphocytes were put in culture to visualize any induced cytogenetic damage. This finding implies that the incidental exposure to high concentrations of EtO did not cause any measurable permanent mutational/cytogenetic damage in exposed lymphocytes.

Urinary 1-hydroxypyrene as biomarker of exposure to polycyclic aromatic hydrocarbons in workers in petrochemical industries: baseline values and dermal uptake.

The suitability of urinary 1-hydroxypyrene as a biomarker for the assessment of exposure to polycyclic aromatic hydrocarbons (PAH) in petrochemical industries was evaluated in 562 workers involved in various operations in petrochemical industries. The median 1-hydroxypyrene concentration in 121 of these workers (both smokers and non-smokers) who had had no recent occupational exposure to PAH was 0.11 mumol/mol creatinine. The upper limit of the 95% confidence interval was 0.51 mumol/mol creatinine. During activities with a low potential exposure to PAH, such as loading bitumen and the handling of clarified slurry oils and furfural extracts, 1-hydroxypyrene concentrations were only marginally increased compared with the values measured in the 121 workers with no recent occupational exposure to PAH. Despite the substantially higher potential exposure to PAH during clean-out operations of various oil refinery installations, the concentrations of 1-hydroxypyrene in the workers involved were in the same range. This suggests that personal protection equipment was generally adequate to prevent excessive exposure. However, in workers digging PAH-contaminated soil and workers engaged in the production of needle coke from ethylene cracker residue, significantly increased urinary 1-hydroxypyrene concentrations were measured. A major decrease in urinary 1-hydroxypyrene following the application of dermal protective equipment in the ground workers suggested that skin absorption plays a major role in occupational exposure to PAH. The excretion of 1-hydroxypyrene by the workers of the needle coke plant was investigated in relation to potential determinants of exposure to PAH. It was indeed found that not only inhalatory but also dermal exposure was a significant determinant of occupational exposure to PAH.

Biological monitoring of exposure to 3-chloro-4-fluoroaniline by determination of a urinary metabolite and a hemoglobin adduct.

In two studies, involving 75 and 72 workers, potential exposure to 3-chloro-4-fluoroaniline (CFA) was biologically monitored by determination of its main urinary metabolite 2-amino-4-chloro-5-fluorophenol sulfate (CFA-S). As this method only allows the detection of recent exposure, analysis of CFA adducts bound to hemoglobin (Hb) was investigated as a method that allows biological monitoring of exposure to CFA over longer periods. The median CFA-S concentration in 67 samples from the first study was 0.14 mumole/g creatinine (range < 0.05-2.82) and in 201 samples from the second study 0.21 mumole/g creatinine (range < 0.05-6.05). In addition, urine samples, collected after shifts with supposed incidental exposure, slightly higher concentrations were measured: 0.27 mumole/g creatinine (range < 0.05-122; 18 samples) and 0.76 mumole/g creatinine (range < 0.05-18.5; 46 samples), respectively. The median Hb adduct concentration in 75 samples from the first study was 9 pmoles CFA/g Hb (range < 5-640) and in 46 samples from the second study 12 pmoles/g Hb (range 3-24). In 24 blood samples collected after incidents, a median concentration of 13 pmoles CFA/g Hb (range < 5-52) was found. Urinary CFA-S and Hb adducts correlated well in samples collected shortly after incidental exposures. However, in 25% of the operators, no CFA-S was detected during routine biological monitoring while Hb adduct analysis showed clear evidence of exposure. This indicates that because of the stability of Hb adducts of CFA in blood, intermittent exposure to CFA is more reliably biologically monitored by determination of Hb adducts of CFA than by assessment of urinary CFA-S.