Department of Molecular Toxicology, Shell International Chemicals BV, SRTCA, PO Box 38000, 1030 BN Amsterdam, The Netherlands
Received February 7, 2000; accepted April 6, 2000
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ABSTRACT |
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Key Words: 1,3-butadiene; risk assessment; biomarker; biological monitoring; carcinogenesis; urinary metabolites; Hb adducts; DNA adducts.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Synthesis of Standards for Urinary Mercapturic Acids
Reference standards N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA) and an isomeric mixture of (R)/(S)-N-acetyl-S-(1-(hydroxymethyl)-2-propenyl)-L-cysteine and (R)/(S)-N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (MHBMA) were prepared as described by Sabourin et al. (1992) and Elfarra et al. (1995), respectively. N-Acetyl-S-(2,3-dihydroxypropyl)-L-cysteine (DHPMA) was used as an internal standard for urinary DHBMA and prepared as described by Jones (1975). A deuterium [d6]-labeled isomeric mixture of MHBMA was used as an internal standard for urinary MHBMA and prepared as described by Elfarra et al. (1995). An isomeric mixture of N-acetyl-S-(2,3,4-trihydroxybutyl)-L-cysteine and N-acetyl-S-(1-(hydroxymethyl)-2,3-dihydroxypropyl)-L-cysteine (THBMA) was prepared as described previously (Richardson et al., 1998c). A deuterium [d6]-labeled isomeric mixture of THBMA was prepared similarly from [d6]-labeled 3,4-epoxy-1,2-butanediol and used as internal standard. Reference and internal standard solutions were prepared in methanol in concentrations of 100 µg/l MHBMA or [d6]-MHBMA, 10 mg/l DHBMA or DHPMA and 100 µg/l THBMA or [d6]-THBMA and stored at 20°C.
Synthesis of Standards for Hydroxybutenylvaline in Hb
An isomeric mixture of N-(1-(hydroxymethyl)-2-propenyl)valine and N-(2-hydroxy-3-butenyl)valine (MHBVal) in globin was used as reference standard and prepared as described by Osterman-Golkar et al. (1996). An isomeric mixture of [d6]-MHBVal in globin was used as an internal standard and prepared as follows: an aliquot (70 µl) of a solution of [d6]-labeled BMO in DMSO (30 mg BMO in 300 µl) was added to washed human erythrocytes and then placed in a shaker incubator at 35°C for 16 h. [d6]-BMO was prepared from [d6]-1,3-butadiene as described by Handley (1994). Globin was extracted from the erythrocytes and purified as described previously (Törnqvist et al., 1986). The level of [d6]-MHBVal in globin was determined using [14C]-labelled MHBVal in globin as a reference in the Edman degradation method (see below). The amount of [14C]-MHBVal in globin was measured by protein hydrolysis and chromatography. Using this procedure, the level of [d6]-MHBVal in globin was determined to be 951 nmol/g globin. Reference and internal standard solutions were prepared in formamide and stored at 200C.
Study Populations and Collection of Biological and Airborne Samples
Studies were carried out in 2 locations on workers who were potentially exposed to BD during loading and manufacturing operations. Study 1 was performed in 1995 at a chemical manufacturing site in The Netherlands where BD monomer is produced (Van Sittert and Van Vliet, 1994). Blood samples for MHBVal in Hb measurements were collected in heparinized vacutainers from 44 male workers who were engaged in the loading of BD in ships and in road and rail tankers. For the determination of normal values, MHBVal was also measured in Hb of 28 male administrative workers from the same plant who were not knowingly occupationally exposed to BD. Erythrocytes were isolated, washed with isotonic saline and frozen at 20°C. Frozen samples were sent to the Amsterdam laboratory and stored at 20°C until analyzed. No BD exposure measurements were carried out in 1995, but in 1994 and 1997 personal air sampling had been performed during loading activities on 4 and 7 workers respectively, using passive dosimeters (3M gas diffusion badges, type 3520).
Study 2 took place in 1998 in a BD monomer and styrene-butadiene rubber (SBR) production facility in Prague, Czech Republic. This study was part of a larger transitional epidemiological study designed to examine biomarkers of exposure, dose, effect and susceptibility in workers exposed to low levels of BD (R. Albertini et al., in preparation). The study population comprised 24 workers of the BD monomer plant, 34 workers of the SBR plant, and 25 control subjects without occupational BD exposure. All study subjects were males. Personal air sampling was carried out during 10 full 8-h shifts on each of the 58 exposed workers over a 60-day period. Air samples were also collected from the control group. The measurements were made using passive thermal badges. Badges were sent to the UK for analysis in the Health and Safety Executive Laboratory (Lynch et al., 1999). Blood samples were obtained on the last day of the survey. Blood of each worker was collected in heparinized vacutainers and erythrocytes were isolated, washed using isotonic saline, and frozen at 70°C. Frozen samples were shipped to Amsterdam and stored at 20°C until analyzed. Urine spot samples were obtained at the beginning and the end of the 8-h shift after a 2-day period without BD exposure to allow a complete excretion of retained metabolite prior to the pre-shift sample. In 21 workers, personal air sampling was also carried out during the day of urine collection. Urine samples (50 ml) from each worker were frozen (-70°C), sent to Amsterdam, and stored at 20°C until analysis. Urinary concentration of a metabolite may greatly depend on the rate of urine production, and its measurement in either too dilute or too concentrated urine specimens can lead to misinterpretation. Therefore, urinary creatinine was determined in all urine samples. Samples with creatinine concentrations outside the range of 430 mmol/l were not included in the statistical analyses (Hoet, 1996
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Measurement of Urinary MHBMA and DHBMA
Aliquots of 0.1-ml internal standard solutions [d6]-MHBMA and DHPMA were added to 1-ml urine samples (pH 2.5) containing 200 mg of sodium chloride, and the metabolites were extracted with ethyl acetate containing 20% v/v methanol. After evaporation of the organic phase, metabolites were derivatized by methylation in 1 ml methanol/anhydrous HCl (1:10) for 15 min at room temperature, followed by pentafluorobenzoylation by reaction with pentafluorobenzoylchloride (7 µl) in toluene (400 µl) and pyridine (2 µl) for 60 min at 60°C. The methylated and pentafluorobenzoylated metabolites were dried and the residue dissolved in toluene (100 µl). Derivatized metabolites were analyzed by gas chromatography with negative electron capture ionization tandem mass spectrometry (GC-NECI-MS-MS) and multiple-reaction monitoring (MRM) on a Finnigan TSQ700 triple quadrupole mass spectrometer equipped with a Varian 3400 gas chromatograph. The analyses were performed by a 1-µl cold on-column injection using a DB-1 column (15 m x 0.32 mm, 0.25 µm film thickness; J&W Scientific) and helium as a carrier gas at a flow rate of 1 ml/min. The initial oven temperature was 80°C. After 1 min, the temperature was increased at a rate of 10°C/min to 320°C and held for 5 min. Methane was used as the moderator gas and argon as the collision gas at a source temperature of 120°C and a source pressure of 5000 mTorr. Ions of the derivatives were monitored at m/z 653 167, 211 (DHBMA), 639
167, 211 (DHPMA), 441
175, 176 (MHBMA) and 447
175, 176 ((d6)-MHBMA). Calibration curves were prepared using control urines spiked with known quantities of MHBMA (0200 µg/l) and DHBMA (020 mg/l) reference standards. The peak areas of MHBMA and DHBMA derivatives increased linearly with MHBMA and DHBMA concentrations over the entire range.
Measurement of Urinary THBMA
Aliquots of 0.5 ml of the methanolic internal standard solution of [d6]-THBMA were pipetted into test tubes and evaporated to dryness. Urine samples (1 ml) were brought into these tubes and diluted with 4 volumes of purified water. After thorough mixing, the solutions were applied to solid-phase extraction columns (1000 mg SAX cartridges; Baker) which were preconditioned by triple washing with 2 ml of diluted formic acid (pH = 3). After application of the samples, the columns were washed with purified water (3 x 2 ml) and the mercapturates were subsequently eluted with a 1 M aqueous formic acid solution (3 x 1 ml). The eluted fractions were evaporated to dryness at 50°C with a gentle stream of N2 and stored at 20°C until analysis. The dry residue was dissolved in 100 µl dimethylformamide and 25 µl pentafluorobenzyl bromide, and 50 µl diisopropylethylamine was added. The mixture was allowed to react for 15 min at 60°C and subsequently evaporated to dryness at 50°C with a gentle stream of N2. The pentafluorbenzoylated samples were dissolved in 1.0 ml of acetylating agent, consisting of 1 volume acetic anhydride and 2 volumes ethyltriamine dissolved in 5 volumes acetone, and was allowed to react for 60 min at 60°C. The pentafluorobenzylated and acetylated metabolites were evaporated to dryness at 50°C with a gentle stream of N2 and the residues were dissolved in toluene (1.00 ml). Derivatized metabolites were analyzed by GC-NECI-MS-MS and MRM on a Finnigan TSQ700 triple quadrupole mass spectrometer equipped with a Varian 3400 gas chromatograph. The analyses were performed by a 1 ml cold on-column injection using a BPX-50 fused silica capillary column (30 m x 0.25 mm, 0.25 µm film thickness; SGE) and helium as a carrier gas at a constant gas pressure of 30 psi. The initial oven temperature was 90°C. After 1 min, the temperature was increased at a rate of 10°C/min to 320°C and held for 5 min. Methane was used as the moderator gas and argon as the collision gas (800 mTorr) at a source temperature of 120°C and a source pressure of 5000 mTorr. Ions of the derivatives were monitored at m/z 392 143 (THBMA) and 398
147 ([d6]-THBMA). Calibration curves were prepared using control urine samples spiked with known quantities of THBMA (050 mg/l). The peak areas of THBMA derivatives increased linearly with THBMA concentrations over the entire range.
Measurement of MHBVal in Hb
The amount of MHBVal was measured as described by Richardson et al. (1996), except that [d6]-MHBVal was used as an internal standard. In brief, globin was isolated and internal standard solution (10 pmol/g globin) was added to 200 mg globin samples dissolved in formamide (6 ml). The pH was adjusted to between pH 6.6 and 7, and pentafluorophenylisothiocyanate (PFPITC, 30 ml) was added, followed by incubation (ca 16 h) in the dark at room temperature. The reaction mixture was then heated to 45°C for 90 min, and after cooling to room temperature, the substituted phenylthiohydantoin (PFPTH) derivatives were extracted by partition between water and diethyl ether and then cleaned up by partition between aqueous sodium carbonate (0.1 M) and toluene. After evaporation of the organic phase, the dry samples were stored at 20°C until required for analysis. The samples were redissolved in toluene (50 ml) and the substituted pentafluorophenylthiohydantoin (PFPTH) of the alkylated N-terminal valine adduct was analyzed by GC-NECI-MS-MS and MRM (see above). The analyses were performed by a 1-ml cold on-column injection using a DB5 MS column (30 m 0.25 mm, 0.25 µm film thickness; J&W Scientific) and helium as a carrier gas at a flow rate of 1 ml/min. The initial oven temperature was 80°C. After 1 min, the temperature was increased at a rate of 30°C/min to 190°C followed by an increase of 3°C/min to 320°C. Methane was used as the moderator gas and argon as the collision gas at a source temperature of 120°C and a source pressure of 5000 mTorr. Ions of the derivatives at m/z 374
304, 318 (MHBVal-PFPTH) and m/z 380
304, 320 ([d6]-MHBVal-PFPTH) were measured. Calibration curves were prepared using control globins spiked with a known quantity of MHBVal (050 pmol/g globin). The peak area of MHBVal-PFPTH increased linearly with MHBVal in globin concentrations over the entire range.
Statistical Analysis
One way analysis of variance was applied to assess differences in:
Significance of differences was tested at the 5% level. Regression analysis was used to quantify the relationships between:
Linear regression models were produced following logarithmic transformation of the variables. Significance of tests was performed at a 5% level. Because BD air exposures were sometimes reported as zero ppm, an offset was applied to both BD air (1 day) and BD air (average 60 days). The size of the offsets was chosen to optimize the r2 of the models. The offsets were 0.007 ppm for BD air (1 day) and 0.016 ppm for BD air (average 60 days). Logarithmic transformation of variables in the relationships between BD air exposure and MHBMA, DHBMA or MHBVal was carried because the between-subjects variation of each of the biomarkers increases with increasing BD exposure and because BD air (1 day) exposures of 4 persons were much higher than exposures of the other 16 persons in the group. Application of linear regression to the original variables would attribute a far too large influence to the measurements of high-exposed compared to low-exposed subjects. The logarithmic transformation makes the variation between the low-exposed subjects visible and balances the influence of data points. Multiple regression was used to examine the effect of smoking; in none of the models was smoking found to have a significant effect (p > 0.05).
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RESULTS |
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Airborne BD Measurements
Table 1 shows that worker 8-h, time-weighted average (TWA) exposures to BD during BD loading operations (Study 1) were comparable to exposures during manufacture of SBR in Study 2. Much lower airborne levels were measured in workers engaged in the manufacture of the BD monomer (Study 2). Further details of airborne measurements in Study 2 will be published elsewhere (R. Albertini et al., in preparation).
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In samples collected at the beginning of the shift, MHBMA levels in SBR workers, but not in monomer workers, were statistically significantly higher (p < 0.05) compared to the control group. DHBMA concentrations were not significantly different between the 3 groups. In both SBR and monomer workers, urinary MHBMA and DHBMA concentrations had increased in end-of-shift samples compared to samples collected at the beginning of the shift. These differences were statistically significant (p < 0.01) for SBR workers but not for monomer workers.
In samples provided at the end of the shift, MHBMA concentrations exceeded the upper-normal-limit value of the control group in 23 of 30 samples (77%) from SBR workers compared to 9 of 23 samples (39%) from monomer workers. DHBMA concentrations exceeded the upper normal limit value of the control group in 25 of 30 samples (83%) from SBR workers and in 6 of 23 samples (26%) from monomer workers. MHBMA and DHBMA concentrations in urine provided at the end of the shift by workers of the SBR plant were higher than the monomer plant, which were higher than the controls. The differences in MHBMA and DHBMA levels were statistically significant among all 3 groups (Table 2). Maximum urinary MHBMA and DHBMA concentrations of 234 µg/l and 26,207 µg/l, respectively, were measured in SBR plant workers, which is about 40-fold higher than their 97.5 percentile levels in non-occupationally exposed control subjects.
Five of 21 subjects from whom air samples were collected had been exposed to BD levels in the range of 0.7212.5 ppm ("high-exposure" group) and 16 of 21 subjects to levels in the range of 0.000.20 ppm ("low-exposure" group) (Table 3). MHBMA concentrations exceeded the upper normal limit value in 5 of 5 urine samples (100%) from the high-exposure group compared to 6 of 21 samples (29%) from the low-exposure group. DHBMA concentrations were above the upper normal limit in 4 of 5 samples (80%) from the high-exposure group compared to 7 of 21 samples (33%) from the low-exposure group. Both MHBMA and DHBMA concentrations were statistically significantly higher (p < 0.01) in the high-exposure group compared to the low-exposure group. Levels in both groups were statistically significantly (p < 0.01) higher than in the control group.
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MHBVal in Hb of Workers with No Occupational BD Exposure
In 16 control subjects of Study 1, the 2.597.5 percentile levels of MHBVal in Hb were < 0.11.20 pmol/g Hb and in 25 control subjects of Study 2 these percentiles were 0.10.76 pmol/g Hb. Smoking had no influence on MHBVal levels in these subjects (Table 4).
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Relation between Airborne BD (1 Day) and Urinary Mercapturic Acids
The relation between 8 h TWA exposures to airborne BD (ppm) and urinary mercapturic acid concentrations (µg/l) in samples collected at the end of the shift was determined from data sets obtained from 5 subjects in the `high' and 16 subjects in the low-exposure groups. Personal air sampling and urine collection took place on the same day. In one worker in the high-exposure group, who had the greatest exposure to BD (12.5 ppm), unusually low levels of MHBMA and DHBMA were found, and this worker was eliminated from the statistics. Statistically significant correlations were found between BD exposure and urinary MHBMA (r2 = 0.542; p < 0.001) or urinary DHBMA (r2 = 0.325; p < 0.01). The following linear regression equations were obtained (SE of the regression coefficients in parentheses):
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and
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The regression lines are shown in Figures 4 and 5. From the regression lines, calculations were made of urinary MHBMA or DHBMA concentrations (mean and 95% confidence intervals) at 8 h TWA BD exposures, which have been adopted by several national and international organizations. Results are shown in Table 5
. For example, at an 8-h TWA BD exposure of 1.0 ppm (2.2 mg/m3), the average MHBMA and DHBMA concentrations in end-of-shift urine samples amounted to 39 and 2213 µg/l, respectively, which is a 26-fold and 6-fold increase, compared to their average natural background concentrations. The 95% confidence intervals (CI) for both average MHBMA and DHBMA concentrations are quite wide, in particular at the higher BD exposures. This is mainly due to the relatively small number of workers (4 of 21) who had BD exposures between 0.7 and 4.8 ppm, while the majority of workers had exposures between 0.0 and 0.2 ppm. At BD exposures greater than 5 ppm, MHBMA and DHBMA concentrations were obtained by extrapolation of the regression lines and are less reliable compared to concentrations calculated at low level BD exposures.
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The regression line (Fig. 6) shows a cluster of data points connecting low airborne BD concentrations with relatively high MHBVal levels in Hb. These adduct levels might have been produced by BD exposures which have been missed in the 60-day personal air monitoring survey. Table 5
lists the MHBVal levels (means and 95% confidence intervals) at BD exposures, which have been adopted by several national and international organizations. The average level of MHBVal adducts in Hb following cumulative exposures to 1.0 ppm BD for several months is 1.7 pmol/g Hb, which is an 8.5-fold increase compared to the average natural background level.
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Relation between Urinary Mercapturic Acids and MHBVal in Hb
The relations between MHBMA or DHBMA concentrations (µg/l) in urine samples collected at end-of- shift times and MHBVal levels in Hb (pmol/g Hb) were determined from data sets obtained from the same groups as above. Highly significant correlations were obtained and the regression equations were the following (SE of regression coefficients in parentheses):
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and
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The regression lines are shown in Figures 8 and 9. The 95% CIs were much narrower compared to urinary MHBMA or DHBMA because 4 times more data is available.
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The regression line is shown in Figure 10.
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DISCUSSION |
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Investigations on these potential biomarkers were carried out in different types of operations: loading of BD in ships or road and rail tankers (Study 1), and manufacture of BD monomer and of SBR (Study 2). Results of both urinary biomarkers and Hb adducts were consistent with airborne measurements. In samples of SBR workers collected at the beginning of the first working day MHBMA, but not DHBMA, concentrations were significantly higher than controls, which indicates that elimination of MHBMA from previous BD exposure is not yet complete after the presumed 48-h period without occupational BD exposure. This suggests that the average urinary half life of elimination (t) of MHBMA is somewhat protracted compared to the t
of other mercapturic acids, such as the benzene metabolite S-phenylmercapturic acid, which has an average t
of 9 h (Boogaard and Van Sittert, 1995
; 1996). Urinary concentrations were generally higher at the end of the shift when compared to the beginning, and for routine biological monitoring, spot samples should be collected at the end of the shift.
Linear regression models of the relations between BD air exposure (1 day) and urinary MHBMA or DHBMA and between BD air (average 60-days) and MHBVal in Hb fitted best following logarithmic transformation of the variables. This demonstrates that the proportion of inhaled BD that is excreted as MHBMA or DHBMA in urine or that binds with Hb to form MHBVal decreases at increasing BD exposures. From the regression equations, urinary MHBMA or DHBMA concentrations in end of shift samples or MHBVal adduct levels in Hb can be calculated that are equivalent to BD airborne exposures. Table 5 lists BD exposures at existing national OELs (ECETOC, 1997) and the corresponding levels of MHBMA or DHBMA in urine and of MHBVal adducts in Hb. Urinary concentrations greater than 5.5 µg/l MHBMA or 700 µg/l DHBMA (97.5% percentile levels in the control group) are considered to result from exposures to BD and from the regression lines it can be calculated that these concentrations correspond to 8-h TWA BD exposures of 0.13 ppm and 0.25 ppm respectively. Adduct levels greater than 0.76 pmol/g Hb are considered to result from exposures to BD and from the regression line it can be calculated that this adduct level corresponds to an average cumulative BD exposure of 0.35 ppm.
In conclusion, the present study has demonstrated that all 3 biomarkers investigated can be used for monitoring exposure of groups of workers to low levels of BD. The measurement of MHBMA in urine is a more sensitive test for monitoring recent exposures to low levels of BD compared to DHBMA in urine, which is due to the relatively high natural background levels of DHBMA. The measurement of MHBVal adducts in Hb is a sensitive and accurate test for monitoring average cumulative exposures to BD at or above 0.35 ppm. Significant correlations were found between urinary MHBMA (recent exposure) and MHBVal adducts (cumulative exposure), indicating that one-day exposures of workers to BD in Study 2 were representative of average exposures during a 60-day period. Swenberg and co-workers (1999) measured THBVal adducts on the same blood samples used for MHBVal in Study 2. Mean THBVal adduct levels in monomer and SBR workers were 381-fold and 326-fold higher compared to mean MHBVal levels. The mean THBVal level in the control group was 450-fold higher compared to the mean MHBVal level. Due to the high natural background level of THBVal adducts in Hb compared to MHBVal, the determination of MHBVal is recommended for monitoring cumulative exposure of individual workers to BD. The finding of relatively high natural background levels of DHBMA in urine and of THBVal adducts in Hb from individuals with no known exposures to BD demonstrates that both DHBMA and THBVal are not only produced following exposure to BD, but also from other sources. Following BD exposure, DHBMA is formed by hydrolysis of BMO to DHB followed by conjugation with GSH. THBVal adducts are formed by reaction of BDO2 and/or EBD with Hb (Fig. 2). The latter metabolite can be formed either by hydrolysis of BDO2 or by cytochrome P450-mediated oxidation of DHB (Csanády et al., 1992
; Kemper et al., 1996). It is unlikely that background levels of DHBMA originate from BMO and background levels of THBVal from BDO2 and/or EBD as metabolites of environmental or endogenous BD exposure. Therefore, it may be postulated that both endogenous DHBMA and THBVal originate from endogenous DHB, the former by conjugation of DHB with GSH and the latter by oxidation of endogenous DHB to EBD. The implications of these natural backgrounds for cancer risk of BD exposure are currently unknown.
The data on biomarkers obtained in this study can be used to explore relative pathways of metabolism of BD in humans, which can be compared with known data in rats and mice. Table 6 summarizes the excretion of urinary metabolites in rat, mouse, and human following exposure to BD. By comparison of the ratio DHBMA/(DHBMA + MHBMA) between the species, the importance of hydrolytic metabolism between the species can be assessed. The ratio was of the rank order: man |L: rat > mouse (Table 6
), which is consistent with findings of Bechtold and co-workers (1994). Following exposure to [2,314C]-BD for 6 h, the ratio of "total metabolites formed via hydrolytic pathway/total metabolites formed via hydrolytic plus GSH pathways" was even greater in rats and lower in mice than the ratio DHBMA/DHBMA + MHBMA (Richardson et al., 1999
). These findings indicate that in humans a much greater proportion of BMO is metabolized by hydrolysis and a much smaller proportion detoxified by direct conjugation with GSH compared to rats and mice. In man, the DHBMA/(DHBMA + MHBMA) ratio decreased at increasing BD exposures (Tables 2, 3, 5
), but the changes were small. Nevertheless, these data indicate that a somewhat smaller proportion of BMO is hydrolyzed into DHB and a somewhat greater proportion detoxified by direct conjugation with GSH with increasing BD exposure. The high rate of hydrolytic metabolism of BMO in humans is also reflected in the very low levels of MHBVal adducts (Table 4
). Following metabolic conversion of BD to BMO, the GSH conjugation of BMO and conversion into harmless mercapturic acids is so efficient that only a small proportion of the BMO is bioavailable to react with N-terminal valine in Hb. The amount of MHBVal formed in Hb is determined by the BMO dose in blood and the reaction rate constant for the reaction of BMO with N-terminal valine in Hb. This is represented by: Acum = kval x DBMO, in which Acum is the cumulative amount of adduct formed over the exposure period and DBMO the integral of the BMO concentration in blood over that period. The kval for the mouse is roughly 2-fold greater than that for the rat (Moll and Elfarra, 1999
). From the results of studies with EO it is known that kval for human Hb is of the same order as for rat Hb (Segerbäck, 1990
). On the basis of the MHBVal adduct levels shown in Table 7
, it can be demonstrated that at equivalent exposures to BD (ppm x h), the DBMO levels are in the rank order: mouse
rat |L: human.
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The basis for the carcinogenicity in rodents exposed to BD is considered to be the reactivity of its epoxide metabolites with DNA. From MHBVal levels in Hb, derived from BMO, (Table 7), it is concluded that, following exposure to equivalent doses of BD, the amount of total MHBGua in humans is much lower than in the mouse or rat. Therefore, the genotoxic risk from BMO, as a reactive intermediate of BD, is expected to be much lower in the human compared to the mouse or the rat. Since it is currently believed that BDO2 is the critical metabolite in BD carcinogenesis, being 50- to > 120-fold more mutagenic than BMO and 125-fold more mutagenic than EBD (Cochrane and Skopek, 1994
; Murg et al., 1999
), the critical question that needs to be answered is to what extent humans metabolize BD to BDO2. The detection of relatively high amounts of THBVal adducts in humans exposed to BD (Table 4
) provides evidence of the bioavailability of BDO2 or EBD in the human body following BD exposure (Swenberg et al., 1999
). Although rate constants (kval) for the reactions of BDO2 and EBD with N-terminal valine in Hb have not yet been determined, it can be inferred from the values of THBVal in Table 7
that DBDO2 or DEBD levels at equivalent exposures to BD (ppm x h) are in the rank order: mouse > rat
human.
From the above findings on the metabolism of BMO in humans, rats and mice following exposure to BD, it may be expected that the proportion of BMO that undergoes further oxidation to BDO2 is of the rank order mouse > rat |L: human. Even if BDO2 is formed in humans, it is expected that it would be rapidly hydrolyzed to EBD and/or erythritol or would be further metabolized by direct conjugation with GSH (Boogaard et al., 1996; Boogaard and Bond, 1996
). Thus the extent to which BDO2 would be available, if at all, to react with either DNA or Hb is very small.
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ACKNOWLEDGMENTS |
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NOTES |
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