Determination of styrene and styrene-7,8-oxide in human blood by gas chromatography-mass spectrometry.

Methods of isotope-dilution gas chromatography-mass spectrometry (GC-MS) are described for the determination of styrene and styrene-7,8-oxide (SO) in blood. Styrene and SO were directly measured in pentane extracts of blood from 35 reinforced plastics workers exposed to 4.7-97 ppm styrene. Using positive ion chemical ionization, styrene could be detected at levels greater than 2.5 microg/l blood and SO at levels greater than 0.05 microg/l blood. An alternative method for measurement of SO employed reaction with valine followed by derivatization with pentafluorophenyl isothiocyanate and analysis via negative ion chemical ionization GC-MS-MS (SO detection limit=0.025 microg/l blood). The detection limits for SO by these two methods were 10-20-fold lower than gas chromatographic assays reported earlier, based upon either electron impact MS or flame ionization detection. Excellent agreement between the two SO methods was observed for standard calibration curves while moderate to good agreement was observed among selected reinforced plastics workers (n = 10). Levels of styrene in blood were found to be proportional to the corresponding air exposures to styrene, in line with other published relationships. Although levels of SO in blood, measured by the direct method, were significantly correlated with air levels of either styrene or SO among the reinforced plastics workers, blood concentrations were much lower than previously reported at a given exposure to styrene. The two assays for SO in blood appear to be unbiased and to have sufficient sensitivity and specificity for applications involving workers exposed to styrene and SO during the manufacture of reinforced plastics.

Styrene oxide in blood, hemoglobin adducts, and urinary metabolites in human volunteers exposed to (13)C(8)-styrene vapors.

Styrene is used in the manufacture of plastics and polymers and in the boat-building industry. The major metabolic route for styrene in rats, mice, and humans involves conversion to styrene-7,8-oxide (SO). The purpose of this study was to evaluate blood SO, SO-hemoglobin (SO-Hb) adducts, and urinary metabolites in styrene-exposed human volunteers and to compare these results with data previously obtained for rodents. Four healthy male volunteers were exposed for 2 h during light physical exercise to 50 ppm (13)C(8)-styrene vapor via a face mask. Levels and time profiles of styrene in exhaled air, blood, and urine (analyzed by GC) and urinary excretion patterns of mandelic acid and phenylglyoxylic acid in urine (analyzed by HPLC) were comparable to previously published volunteer studies. Maximum levels of SO in blood (measured by GC-MS) of 2.5-12.2 (average 6.7) nM were seen after 2 h, i.e., in the first sample collected after exposure had ended. The styrene blood level in humans was about 1.5 to 2 times higher than in rats and 4 times higher than in mice for equivalent styrene exposures. In contrast the SO levels in human blood was approximately fourfold lower than in mice. The level of hydroxyphenethylvaline (determined by GC-MS-MS) in pooled blood collected after exposure was estimated as 0.3 pmol/g globin corresponding to a SO-Hb adduct increment of about 0.003 pmol/g and ppmh. NMR analyses of urine showed that a major portion (> 95%) of the excreted (13)C-derived metabolites was derived from hydrolysis of SO, while only a small percentage of the excreted metabolites (< 5%) was derived from metabolism via phenylacetaldehyde. Signals consistent with metabolites derived from other pathways of styrene metabolism in rodents (such as glutathione conjugation with SO or ring epoxidation) were not detected.

Biotransformation of the double bond in allyl glycidyl ether to an epoxide ring. Evidence from hemoglobin adducts in mice.

Allyl glycidyl ether (AGE) is used industrially in the production of various epoxy resins. The compound is mutagenic and evidence for carcinogenicity in mice and rats has been reported. A previous study in mice showed that AGE reacts directly, without metabolic activation, with N-terminal valine in hemoglobin to form adducts (AGEVal). Metabolism of AGE may lead to formation of diglycidyl ether (I) through epoxidation of the double bond or 1-allyloxy-2,3-dihydroxypropane (II) through hydrolysis of the epoxide ring. 2,3-Dihydroxypropyl glycidyl ether (III) may be formed either by hydrolysis of I or epoxidation of II. The main aim of the present study was to investigate if AGE is metabolized to the reactive epoxides I or III by analysis of adducts with hemoglobin. Nine male mice (C3H/Hej) were administered AGE dissolved in tricaprylin, 4 mg/mouse, by intraperitoneal (i.p.) injection. Eleven male mice were administered 4 mg/mouse of AGE dissolved in acetone, by skin application. Adducts of I or III with N-terminal valine, N-(2-hydroxy-3-(2,3-dihydroxy)propyloxy)propylvaline (diOHPrGEVal), were demonstrated in mice administered AGE by i.p. injection. The levels were in the range 1600-5600 pmol/g globin. The level of diOHPrGEVal in mice administered AGE by skin application (n = 5) was below the detection limit of the analytical method, 20 pmol/g globin. The level of AGEVal, analyzed in mice administered AGE by skin application (n = 6), was about 20 pmol/g globin (median value), as compared with 1600 pmol/g globin previously found in mice administered AGE by i.p. injection. Neither AGEVal nor diOHPrGEVal were detected in control animals. Both adducts were analyzed using a modified Edman method for derivatization and using gas chromatography/tandem mass spectrometry for detection. The hydroxyl groups of the Edman derivative of diOHPrGEVal were protected by acetylation.

32P-post-labelling of 7-(3-chloro-2-hydroxypropyl)guanine in white blood cells of workers occupationally exposed to epichlorohydrin.

Epichlorohydrin (ECH) is a simple 3-carbon epoxide of industrial importance. It has been shown to be genotoxic in several systems and carcinogenic in experimental animals. The aim of the present investigation was to study DNA adducts of ECH as a biomarker of occupational exposure to this chemical. 7-(3-Chloro-2-hydroxypropyl)guanine (7-CHP-guanine) was analysed in DNA from white blood cells using an anion exchange-based adduct enrichment protocol of the (32)P-post-labelling/HPLC-based assay. Blood samples were collected from seven workers handling ECH (exposed), nine workers not handling ECH but normally present in the premises where this chemical is used (potentially exposed) and 13 office and factory workers from locations in the plant where ECH is not handled (controls). 7-CHP-guanine was detected in five of the seven workers exposed to ECH (1.6-7.1 mol/10(9) mol nucleotides) and in two of the nine workers potentially exposed to ECH (0.8-1.5 mol/10(9) mol nucleotides). This adduct was not detected in any of the 13 controls. The difference in adduct levels between exposed workers and controls was statistically significant (Mann-Whitney test, P < 0.001), as was the difference between exposed workers and potentially exposed workers (P = 0.017). The recovery of 7-CHP-guanine in the (32)P-post-labelling assay was on average 48 +/- 7%, which is considerably higher than previously reported for other 7-alkylguanines. The method used had a limit of detection of approximately 0.4 mol adduct/10(9) mol nucleotides using 20 microg DNA. This study shows for the first time ECH-induced DNA adducts in humans and suggests that 7-CHP-guanine may be used as a biomarker of occupational exposure to ECH.

An evaluation of styrene genotoxicity using several biomarkers in a 3-year follow-up study of hand-lamination workers.

A study employing several biomarkers of styrene exposure and genotoxicity was carried out in a group of lamination (reinforced plastic) workers and controls, who had been repeatedly sampled during a 3-year period. Special attention will be paid to the last sampling (S.VI), reported here for the first time. Styrene concentration in the breathing zone, monitored by personal dosimeters, and urinary mandelic acid (MA) were measured as indicators of external exposure. Blood samples were assayed for styrene-specific O6-guanine adducts in DNA, N-terminal valine adducts of styrene in haemoglobin, DNA single-strand breaks (SSB), determined by use of the single cell gel electrophoresis (Comet) assay), and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutant frequencies (MF) in T-lymphocytes. O6-styrene guanine adduct levels were significantly higher in the exposed group (5.9 +/- 4.9 adducts/10(8) dNp) as compared to laboratory controls (0.7 +/- 0.8 adducts/10(8) dNp; P = 0.001). DNA adduct levels significantly correlated with haemoglobin adducts, SSB parameters and years of employment. Styrene-induced N-terminal valine adducts were detected in the lamination workers (1.7 +/- 1.1 pmol/g globin), but not in the control group (detection limit 0.1 pmol/g globin). N-terminal valine adducts correlated strongly with external exposure indicators, DNA adducts and HPRT MF. No significant correlation was found with SSB parameters. A statistically significant difference in HPRT MF was observed between the laminators (22.3 +/- 10.6/10(6)) and laboratory controls (14.2 +/- 6.5/10(6), P = 0.039). HPRT MF in the laminators significantly correlated with styrene concentration in air, MA and haemoglobin adducts, as well as with years of employment and age of the employees. No significant difference (P = 0.450) in MF between the laminators and the factory controls was observed. Surprisingly, we detected differences in MF between sexes. When data from all measurements were combined, women showed higher MF (geometric mean 15.4 vs. 11.2 in men, P = 0.020). The styrene-exposed group exhibited significantly higher SSB parameters (tail moment (TM), tail length (TL) and the percentage of DNA in the tail (TP)) than the control group (P < 0.001). SSB parameters correlated with indicators of external exposure and with O6-styrene guanine adducts. No significant correlation was found between SSB parameters and haemoglobin adducts or HPRT MF. The data encompassing biomarkers from repeated measurements of the same population over a 3-year period are discussed with respect to the mechanisms of genotoxic effects of styrene and the interrelationship of individual biomarkers.

Simultaneous analysis of hemoglobin adducts of acrylamide and glycidamide by gas chromatography-mass spectrometry.

Acrylamide (AA) is a carcinogen in experimental animals. Glycidamide (GA), formed by metabolic epoxidation of AA, is believed to be responsible for the carcinogenicity of AA. Occupational exposure to AA has been assessed earlier by measurement of its adducts with N-terminal valine in hemoglobin. A background of AA adducts [N-(2-carbamoylethyl)valine (AAVal), about 30 pmol/g globin] was found in individuals without known exposure to the compound. The method previously available for adducts of GA only allowed analysis of samples from highly exposed individuals and showed similar levels of AAVal and adducts of GA [N-(2-hydroxy-2-carbamoylethyl)valine (GAVal)]. We have developed a sensitive method for simultaneous quantification of adducts of GA and AA, which is suitable down to low exposure levels. The method is based on the so-called modified Edman method, where globin is reacted with pentafluorophenyl isothiocyanate under neutral conditions. The valine adducts are then extracted in the form of pentafluorophenylthiohydantoin (PFPTH) derivatives. The analytical procedure included reaction of the PFPTH derivatives with acetic anhydride in order to protect the hydroxyl group of GAVal. The PFPTH derivatives of AAVal and GAVal were analyzed by gas chromatography/tandem mass spectrometry. ((2)H(3))AAVal-PFPTH was used as the internal standard. The method was applied to samples from 11 workers at an AA production plant, 1 nonexposed nonsmoker, and a few participants of a smoking cessation program. AAVal levels were in the range 27-1854 pmol/g globin. Recorded levels of GAVal were 3-12% of those of AAVal, suggesting that previous measurements of GAVal overestimate GAVal at low levels of exposure to AA.

Adducts of acrylonitrile with hemoglobin in nonsmokers and in participants in a smoking cessation program.

Hemoglobin adducts have been used to assess exposure to carcinogenic compounds in tobacco smoke. However, because of background levels in nonsmokers, most adducts that have been studied are not useful for monitoring low-level exposure. Bergmark [(1997) Chem. Res. Toxicol. 10, 78-84] showed that the level of adducts of acrylonitrile (AN) with N-terminal valine (ANVal) increases with increasing cigarette consumption, and the increment from 1 cigarette/day was estimated to be 8 pmol/g of globin. The background level of ANVal in nonsmokers was not quantified (<2 pmol/g of globin). The objective of this study was to determine the background level of ANVal in hemoglobin and to study the stability of this adduct in vivo. Globin samples previously analyzed by Bergmark from 17 nonsmokers and 2 smokers were reanalyzed in the study presented here. Globin samples from 7 additional nonsmokers and from 10 participants in a smoking cessation program were also analyzed. Smoking habits and exposure to environmental tobacco smoke (ETS) were assessed by interview. Only two of the participants completed the program. The levels of ANVal in these 2 subjects decreased after quitting and were at background level by 126 days. The time course of the decrease was compatible with removal of stable adducts. The levels of ANVal in the nonsmokers were 0.76 +/- 0.36 (mean +/- SD) (n = 18; reporting no exposure ETS), 1.1 +/- 0.6 (n = 3; reporting exposure to ETS), and 1. 2 +/- 0.5 pmol/g of globin (n = 3; snuff users). Thus, the adduct level in nonsmokers corresponds to the adduct increment from about 0. 1 cigarette/day. Measurements of the level of ANVal could be used to distinguish between nonsmokers and low-level smokers on an individual level, but larger groups of individuals would be required to detect a possible contribution to the background from passive smoking.

Methods for biological monitoring of propylene oxide exposure in Fischer 344 rats.

Propylene oxide (PO) is used as an intermediate in the chemical industry. Human exposure to PO may occur in the work place. Propylene, an important industrial chemical and a component of, for example, car exhausts and cigarette smoke, is another source of PO exposure. Once taken up in the organism, this epoxide alkylates macromolecules, such as haemoglobin and DNA. The aim of the present investigation was to compare two methods for determination of in vivo dose, the steady state concentration of PO in blood of exposed rats and the level of haemoglobin adducts. Male Fischer 344 rats were exposed for 4 weeks (6 h/day, 5 days/week) to PO at a mean atmospheric concentration of 500 ppm (19.9 micromol/l). Immediately after the last exposure blood was collected in order to determine the steady state concentration of PO. Free PO was measured in blood samples of three animals by means of a head space method to be 37 +/- 2 micromol/l blood (mean +/- S.D.). Blood samples were also harvested for the measurement of haemoglobin adducts. N-2-Hydroxypropyl adducts with N-terminal valine in haemoglobin were quantified using the N-alkyl Edman method with globin containing adducts of deuterium-substituted PO as an internal standard and N-D,L-2-hydroxypropyl-Val-Leu-anilide as a reference compound. Tandem mass spectrometry was used for adduct quantification. The adduct levels were < 0.02 and 77.7 +/- 4.7 nmol/g globin (mean +/- S.D.) in control animals (n = 7) and in exposed animals (n = 34), respectively. The adduct levels expected at the end of exposure were calculated to be 71.7 +/- 4.1 nmol/g globin (mean +/- S.D.) using the measured steady state concentration of PO in blood and taking into account the growth of animals, the life span of erythrocytes, the exposure conditions and the second order rate constant for adduct formation. The good agreement between the estimated and measured adduct levels indicates that both end-points investigated are suitable for biological monitoring.

Adducts with haemoglobin and with DNA in epichlorohydrin-exposed rats.

Epichlorohydrin (1-chloro-2,3-epoxypropane; ECH) is an important industrial chemical and a carcinogen in experimental animals. The main aims of the present study were to characterize the adduct formation in female Wistar rats and to identify adducts that could potentially be used in human biomonitoring studies. The total binding of radioactivity to haemoglobin in rats administered 0, 0. 11, 0.22, 0.43, or 0.97 mmol [3H]ECH/kg body weight by i.p. injection, and sacrificed 24 h after treatment, was linearly related to a dose up to 0.43 mmol/kg body weight. The binding at the highest dose was higher than predicted by extrapolation from lower doses, indicating saturation of a metabolic process for elimination of ECH. Ion-exchange chromatography of a globin hydrolysate showed one major radioactivity peak corresponding to S-(3-chloro-2-hydroxypropyl)cysteine. The half-life of this adduct was estimated as about 4 days by analysis of globin from rats administered 0.43 mmol/kg body weight and sacrificed after 1, 2 and 9 days. Crosslinking of the adduct, presumably with glutathione, appeared to be the predominant secondary reaction. Hydrolysis of N-(3-chloro-2-hydroxypropyl)valine, the primary reaction product of ECH with N-terminal valine, would give N-(2,3-dihydroxypropyl)valine. A sensitive gas chromatography/mass spectrometry method for the dihydroxypropyl adduct was used to follow its formation and removal after administration of nonlabelled ECH (0.11 mmol/kg body weight). The level of this adduct reached a maximum of about 20 pmol/g globin after a few weeks, corresponding to about 0.1% of the initial binding of ECH to globin. N-7-(3-Chloro-2-hydroxypropyl)guanine was detected in rats administered 0.97 mmol [3H]ECH/kg body weight and sacrificed 6 h after treatment. The adduct levels in haemoglobin and DNA were compared with previously reported adduct levels in male Fischer 344 rats exposed to propylene oxide. Despite its higher chemical reactivity, the capacity of ECH to alkylate macromolecules in vivo was found to be somewhat lower than that of propylene oxide.

Epoxybutene-hemoglobin adducts in rats and mice: dose response for formation and persistence during and following long-term low-level exposure to butadiene.

Measurement of specific adducts to hemoglobin can be used to establish the dosimetry of electrophilic compounds and metabolites in experimental animals and in humans. The purpose of this study was to investigate the dose response for adduct formation and persistence in rats and mice during long-term low-level exposure to butadiene by inhalation. Adducts of 3,4-epoxy-1-butene, the primary metabolite of butadiene, with N-terminal valine in hemoglobin were determined in male B6C3F1 mice and male Sprague-Dawley rats following exposure to 0, 2, 10, or 100 ppm of 1,3-butadiene, 6 h/day, 5 days/week for 1, 2, 3, or 4 weeks. Blood samples were collected from groups of five mice and three rats at the end of each week during the 4 weeks of exposure and weekly for 3 weeks following the end of the 4-week exposure period. The increase and decrease, respectively, of the adduct levels during and following the end of the 4-week exposure followed closely the theoretical curve for adduct accumulation and removal for rats and mice, thereby demonstrating that the adducts are chemically stable in vivo and that the elimination follows the turnover of the red blood cells. The adduct level increased linearly with butadiene exposure concentration in the mice, whereas a deviation from linearity was observed in the rats. For example, after exposure to 100 ppm butadiene, the epoxybutene-hemoglobin adduct levels were about four times higher in mice than in rats; at lower concentrations of butadiene, the species difference was less pronounced. Blood concentrations of epoxybutene, estimated from hemoglobin adduct levels, were in general agreement with reported concentrations in mice and rats exposed by inhalation to 62.5 ppm. These studies show that adducts of epoxybutene with N-terminal valine in hemoglobin can be used to predict blood concentration of epoxybutene in experimental animals.

Genetic effects of 1,3-butadiene and associated risk for heritable damage.

A summary of the results of the studies conducted in the EU Project "Multi-endpoint analysis of genetic damage induced by 1,3-butadiene and its major metabolites in somatic and germ cells of mice, rats and man" is presented. Results of the project are summarized on the detection of DNA and hemoglobin adducts, on the cytotoxic and clastogenic effects in somatic and germinal cells of mice and rats, on the induction of somatic mutations at the hprt locus of experimental rodents and occupationally exposed workers, on the induction of dominant lethal mutations in mice and rats, and on heritable translocations induced in mice, after exposure to butadiene (BD) or its major metabolites, butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). The primary goal of this project was to collect experimental data on the genetic effects of BD in order to estimate the germ cell genetic risk to humans of exposure to BD. To achieve this, the butadiene exposure are based on data for heritable translocations and bone marrow micronuclei induced in mice and chromosome aberrations observed in lymphocytes of exposed workers. A doubling dose for heritable translocations in human germ cells of 4900 ppm/h is estimated, which, assuming cumulative BD exposure over the sensitive period of spermatogenesis, corresponds to 5-6 weeks of continuous exposure at the workplace to 20-25 ppm. Alternatively, the rate of heritable translocation induction per ppm/h of BD exposure is estimated to be approximately 0.8 per million live born, compared to a spontaneous incidence of balanced translocations in humans of approximately 800 per million live born. These estimates have large confidence intervals and are only intended to indicate orders of magnitude of human genetic risk. These risk estimates are based on data from germ cells of BD-exposed male mice. The demonstration that clastogenic damage was induced by DEB in preovulatory oocytes at doses which were not ovotoxic implies that additional studies on the response of mammalian female germ cells to BD and its metabolites are needed. The basic assumption of the above genetic risk estimates is that experimental mouse data obtained after BD exposure can be extrapolated to humans. Several points exist in the present report and in the literature which contradict this assumption: (1) the level of BMO-hemoglobin adducts was significantly elevated in BD-exposed workers; however, it was considerably lower than would have been predicted from comparable rat and mouse exposures; (2) the concentrations of the metabolites DEB and BMO were significantly higher in mouse than in rat blood after BD exposure. Thus, while metabolism of BD is qualitatively similar in the two species, it is quantitatively different; (3) no increase of HPRT mutations was shown in 19 workers exposed on average to 1.8 ppm of BD, while in a different population of workers from a US plant exposed on average to 3.5 ppm of BD, a significant increase of HPRT variants was detected; and (4) data from cancer bioassays and cancer epidemiology suggest that rat is a more appropriate model than mouse for human cancer risk from BD exposure. However, the dominant lethal study in rats gave a negative result. At present, we do not know which BD metabolite(s) may be responsible for the genetic effects even though the bifunctional alkylating agent DEB is the most likely candidate for the induction of clastogenic events. Unfortunately, methods to measure DEB adducts in hemoglobin or DNA are only presently being developed. Despite these several uncertainties the use of the mouse genetic data is regarded as a justifiable and conservative approach to human genetic risk estimation given the considerable heterogeneity observed in the biotransformation of BD in humans.

Haemoglobin adducts of epoxybutanediol from exposure to 1,3-butadiene or butadiene epoxides.

Epoxybutanediol is one of the reactive metabolites of butadiene. It is formed via hydrolysis followed by oxidation of the primary metabolite of butadiene, epoxybutene, or via hydrolysis of diepoxybutane, a secondary metabolite of butadiene. Groups of male Sprague Dawley rats were treated by intraperitoneal injection of epoxybutene, epoxybutanediol or diepoxybutane. N-(2,3,4-Trihydroxybutyl)valine adducts in haemoglobin, formed from epoxybutanediol in its reaction with N-terminal valine, were measured using the N-alkyl Edman method followed by acetylation of the Edman derivatives and analysis by gas chromatography mass spectrometry. The same adducts were also measured in male Wistar rats exposed to butadiene by inhalation and in a few workers with occupational exposure to butadiene. Haemoglobin binding indexes, HBI, (pmol adduct/g per mumol of alkylating agent, or, for butadiene, per ppm x h), were calculated. The HBI for epoxybutanediol (about 10) is comparable to that of ethylene oxide in the rat demonstrating a similar capacity of the two compounds to alkylate nucleophilic sites in vivo. The HBI of diepoxybutane (about 8) for epoxybutanediol adduct formation is approximately the same as that of epoxybutanediol itself. Epoxybutanediol adduct formation was nonlinearly related to exposure in butadiene exposed rats. The epoxybutanediol-haemoglobin adduct levels were substantially higher than those of epoxybutene in both butadiene-exposed rats and humans suggesting an important role of epoxybutanediol in the toxicity of butadiene. Adducts of epoxybutanediol are probably useful for biomonitoring of human exposure to butadiene.

Dosimetry of glycidyl ethers in mice by quantification of haemoglobin adducts.

Glycidyl ethers are used in epoxy resins. Epoxy resins are widely used in adhesives and coatings, as well as in electronics and structural composites, thus there is a potential of human exposure to glycidyl ethers. The aim of the present investigation was to explore the utility of haemoglobin adducts for biomonitoring of these types of compounds. Adducts to N-terminal valine were analysed by a modified Edman method with gas chromatographymass spectrometry (or tandem mass spectrometry) for adduct detection and quantification. Groups of three male mice (C3H/Hej) were administered 4 mg/mouse of allyl, butyl, phenyl or cresyl glycidyl ether (AGE, BGE, PGE or CGE) by i.p. injection. Blood samples were collected 24 h after treatment and assayed for haemoglobin adducts using the N-alkyl Edman method. Additional groups of AGE-treated mice were used to study dose response and adduct stability. The experiments with AGE indicate a linear dose response for adduct formation in the dose range studied (0, 2 and 4 mg/mouse). As expected for stable haemoglobin adducts, about 50% of the initial adduct level remained 21 days after exposure.

Ring test for the determination of N-terminal valine adducts of styrene 7,8-oxide with haemoglobin by the modified Edman degradation technique.

A ring-test was organised between three laboratories using different versions of the modified Edman degradation technique for the gas chromatographic-mass spectrometric determination of N-terminal valine adducts of styrene 7,8-oxide. The analyses were performed on a sample of human haemoglobin reacted in vitro with styrene 7,8-oxide and on a set of five haemoglobin samples from mice dosed by i.p. injection of styrene. Strong correlations between the haemoglobin adduct determinations of the different laboratories were observed. However, covariance analysis revealed different slopes for the dose-response curves, indicating differences for the calibration of the reference globin or reference peptide.

Biomonitoring of 1,3-butadiene and related compounds.

The 1990 Clean Air Act Amendments list several volatile organic chemicals as hazardous air pollutants, including ethylene oxide, butadiene, styrene, and acrylonitrile. The toxicology of many of these compounds shares several common elements such as carcinogenicity in laboratory animals, genotoxicity of the epoxide intermediates, involvement of cytochrome P450 for metabolic activation (except ethylene oxide), and involvement of at least two enzymes for detoxication of the epoxides (e.g., hydrolysis or conjugation with glutathione). These similarities facilitate research strategies for identifying and developing biomarkers of exposure. This article reviews the current knowledge about biomarkers of butadiene. Butadiene is carcinogenic in mice and rats, which raises concern for potential carcinogenicity in humans. Butadiene is metabolized to DNA-reactive metabolites, including 1,2-epoxy-3-butene and diepoxybutane. These epoxides are thought to play a critical role in butadiene carcinogenicity. Butadiene and some of its metabolites (e.g., epoxybutene) are volatile. Exhalation of unchanged butadiene and excretion of butadiene metabolites in urine represent major routes of elimination. Therefore, biomonitoring of butadiene exposure could be based on chemical analysis of butadiene in exhaled breath, blood levels of butadiene epoxides, excretion of butadiene metabolites in urine, or adducts of butadiene epoxides with DNA or blood proteins. Mutation induction in specific genes (e.g., HPRT) following butadiene exposure can be potentially used as a biomarker. Excretion of 1,2-dihydroxy-4-(N-acetylcysteinyl-S)butane or the product of epoxybutene with N-7 in guanine in urine, epoxybutene-hemoglobin adducts, and HPRT mutation have been used as biomarkers in recent studies of occupational exposure to butadiene. Data in laboratory animals suggest that diepoxybutane may be a more important genotoxic metabolite than epoxybutene. Biomonitoring methods need to be developed for diepoxybutane and other putative reactive butadiene metabolites. With butadiene and related compounds, the ultimate challenge is to identify useful biomarkers of exposure in which quantitative linkages between exposure and internal dose of the important DNA-reactive metabolites are established.

Assessment of exposure to butadiene in the process industry.

Occupational exposure levels to 1,3-butadiene (BD) are variable but generally below 1 ppm in the European process industry. A preliminary analysis showed that hemoglobin adduct levels of butadiene monoxide (BMO) were increased among the worker groups with higher potential exposure to BD (process work, bomb voiding, repair duties) than among less exposed workers in maintenance and laboratory or control persons. In the same workers no exposure related effects were seen in the cytogenetic parameters studied, i.e. chromosomal aberrations, sister chromatid exchanges or micronuclei in peripheral blood lymphocytes. However, the glutathione-S-transferase polymorphism in the T1 gene might play a role in determining interindividual sensitivity to BD-induced chromosomal aberrations. Chromosomal aberrations (gaps excluded) were significantly (P < 0.05) increased among the workers lacking the GSTT1 gene as compared to the BD workers with the gene, while the other polymorphic GSTM1 gene showed no association with the cytogenetic parameters. More work needs to be done to study the adducts by other active BD metabolites than BMO and the role of the genetic polymorphisms controlling the variability of individual responses.

Biomonitoring of epichlorohydrin by hemoglobin adducts.

A gas chromatographic mass spectrometric method for monitoring exposure to epichlorohydrin (ECH) by means of quantitative analysis of N-(2,3-dihydroxypropyl)valine in hemoglobin has been developed. The analysis is based on the modified Edman method for measurement of adducts to the N-terminal valine, one of the reactive sites of the globin chains. The presence of two hydroxyl groups in the adduct to be analyzed required special precautions to be introduced into the method, such as acetylation of the Edman derivative. An in vitro treated globin with [2H5]N-2,3-dihydroxypropyl adducts was used as internal standard. The limit of detection achieved is 4 fmol in analysis by tandem mass spectrometry. Adduct levels found in smokers (15 to 20 cigarettes per day) were between 6.5 and 11.2 pmol/g globin and for nonsmokers the adduct levels were close to the detection limit (about 2 pmol/g globin). In two rats, treated ip with 40 mg ECH/kg body wt and sacrificed after 30 days, the average adduct level was 44 pmol/g globin and that for two nonexposed rats was close to the detection level. The method will be useful for monitoring of exposure and for cancer risk estimation of ECH.

Haemoglobin adducts as biomarkers of occupational exposure to 1,3-butadiene.

Adducts of 1,2-epoxy-3-butene (EB) with haemoglobin were monitored in 17 workers from the 1,3-butadiene (BD) production unit of a petrochemical plant and in nine referents employed at the same factory but not exposed to BD. The air concentrations of BD were determined using stationary and personal monitoring. The ambient level of exposure of the plant workers handling butadiene containers (sampling and voiding) was on average 11.2 +/- 18.6 (mean +/- SD) mg/m3. Maintenance and laboratory workers were exposed to levels < or = 1.2 mg/m3. The particular haemoglobin adduct measured was 2-hydroxy-3-butenylvaline, formed by reaction of N-terminal valine with carbon 1 in EB. The adduct levels were increased (0.16 +/- 0.099 pmol/g; n = 10) in plant workers compared with the levels in maintenance and laboratory workers and controls (approximately 0.05 pmol/g; seven laboratory workers and nine controls evaluated). Thus, the method used for adduct determination--derivatization of 200-300 mg globin samples with penta-fluorophenyl isothiocyanate according to the N-alkyl Edman method and detection of the thiohydantoin derivatives by tandem mass spectrometry--is sufficiently sensitive to allow monitoring of exposure to BD down to the p.p.m. level.

Assessment of environmental and occupational exposures to butadiene as a model for risk estimation of petrochemical emissions.

1,3-Butadiene (BD) is an important industrial chemical and environmental contaminant, e.g. in urban air, traffic exhausts and tobacco smoke. It has been shown to be genotoxic in vitro and in vivo and carcinogenic in rodents, mice being more sensitive than rats. The present study confirmed this species difference. Using micronuclei in erythrocytes or bone marrow as a marker, mice responded at an effective level of 50 p.p.m., while the highest ineffective level in rats was 500 p.p.m. (inhalation of BD for 5 days). A dose-dependent increase in N-terminal valine haemoglobin adducts was seen in both rats and mice, but the adduct levels in the latter species were on average five times higher. For the first time, specific N6-alkyldeoxyadenosine adducts were identified in lung and liver DNA of rats exposed to BD by inhalation. No significant difference in DNA adduct level was seen in lung tissue of rats and mice at similar exposure levels. Occupational exposure levels to BD in the European Process industry are variable, but generally < 1 p.p.m. Haemoglobin adduct levels were seen to be increased among the worker groups with higher potential exposure to BD (process work, bomb voiding and repair duties) as compared with adduct levels in less exposed workers in maintenance and the laboratory or control personnel. However, the N-terminal valine haemoglobin adducts measured in the workers were one to two orders of magnitude lower than extrapolated for the same exposure dose in mice. In the same workers no exposure-related effects were seen in the cytogenetic parametres studied, i.e. chromosomal aberrations, sister chromatid exchanges or micronuclei in peripheral blood lymphocytes, or in the Ras oncoprotein levels of plasma samples. The studies so far conducted suggest that human exposure at the levels seen in the present day process industry can be documented at the biological dose level using haemoglobin adduct measurement, but not at the biological effect level using cytogenetic biomarkers. In order to quantitate the human genotoxic risk of BD exposure more work needs to be done on the role of other active BD metabolites than 1,2-epoxy-3-butene and on the genetic polymorphisms controlling the variability of individual responses.

1,3-Butadiene working group report.

During the Workshop in North Carolina, the in vivo metabolism, adduct formation and genotoxicity data available from rodent and human exposure to 1,3-butadiente (BD) were reviewed and they are summarized in the present report. BD is metabolized by cytochrome P-450-dependent monoxygenases to the primary metabolite 1,2-epoxybutene-3 (epoxybutene, EB). EB is subjected to further metabolism: oxidation to 1,2:3,4-diepoxybutane (DEB), hydrolysis to 3-butene-1,2-diol and conjugation to glutathione. The first pathway seems to prevail in mice while the latter is characteristic for rats and possibly for humans. Species differences exist in adduct formation of the monoepoxide to hemoglobin, for which the following pattern has been found: mice > rats > humans. Genotoxity of BD was found in mice with all applied tests; however, negative results were obtained in rats. In exposed humans, the cytogenetic studies in peripheral blood lymphocytes did not show genotoxic effects, although one report described elevated hprt variant levels in peripheral blood lymphocytes of exposed workers. It was concluded that the presently available data are insufficient for the application of the parallelogram model to estimate genetic risk for humans. As an alternative approach, a tentative estimate of the doubling dose for induction of hprt mutations in somatic cells of mice and men was performed and the calculated values were surprisingly similar, i.e. 9000 ppmh. However, this estimate is burdened with a number of caveats which were discussed in detail. The working group identified a series of urgent research needs to provide the appropriate data for the application of the parallelogram model, such as identification of metabolic pathways in different rodent species and humans, metabolic studies in mice, rats and humans considering metabolic polymorphisms, studies of adducts to DNA and hemoglobin especially of DEB and other butadiene metabolites in rodents and humans, studies of mutational spectra (mutational fingerprinting) in somatic and germinal cells, confirmation of the human hprt mutation data, conformation of the rodent malformation data, dose-response studies in rodent germ cell tests and studies on repair kinetics of mono-adducts induced by EB as opposed to repair of cross-links produced by DEB. Finally, it was suggested that the original parallelogram consisting of data from somatic cell studies in rodents and humans plus studies of heritable effects in rodents to extrapolate to germ cell risk for humans should be supplemented with studies in sperm of experimental animals and exposed men.