The influence of co-exposure to dimethyldithiocarbamate on butadiene metabolism

Chemico-Biological Interactions 135–136 (2001) 585–598

The influence of co-exposure todimethyldithiocarbamate on butadiene

metabolism

Trevor Green *, Alison Toghill, Richard MooreZeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK

Abstract

Treatment of rats and mice with a single oral dose of dimethyldithiocarbamate (DMDTC;250 mg/kg) had a marked effect on hepatic CYP2E1 and aldehyde dehydrogenase activities,measured in vitro, for up to 24 h after dosing. The same treatment did not affect CYP2A6,glutathione S-transferase, epoxide hydrolase, alcohol dehydrogenase activities or hepaticglutathione levels. As a consequence of the loss of CYP2E1 activity, butadiene metabolismin liver fractions from DMDTC treated rats and mice was markedly reduced, as was themetabolism of the mono-epoxide to the di-epoxide in mouse liver. The conversion of themono-epoxide to the diol by epoxide hydrolases was not affected by DMDTC treatment.Urinary excretion of radioactivity, following dosing with DMDTC and exposure to 200 ppmC-14 butadiene for 6 h, was markedly reduced in rats, but increased in mice. The profiles ofurinary metabolites were qualitatively similar from mice exposed to butadiene to thoseexposed after dosing with DMDTC. In the rat, pre-dosing with DMDTC resulted in theformation of three additional urinary metabolites following exposure to butadiene. Overall,DMDTC appears to impact qualitatively and quantitatively on the metabolism of butadiene.The nature and full significance of these changes has yet to be characterised. © 2001 ElsevierScience Ireland Ltd. All rights reserved.

Keywords: Butadiene; Dimethyldithiocarbamate; Metabolism

www.elsevier.com/locate/chembiont

* Corresponding author. Tel.: +44-1625-515458; fax: +44-1625-586396.E-mail address: [email protected] (T. Green).

0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0009 -2797 (01 )00198 -3

T. Green et al. / Chemico-Biological Interactions 135–136 (2001) 585–598586

1. Introduction

Epidemiology studies associated with exposure to butadiene have looked atcohorts exposed to butadiene alone and those exposed within the styrene-butadienerubber (SBR) industry [1]. Conflicting results have come from these studies withrespect to increases in hematopoietic or lymphoid cancers. Of the more recentstudies, the Delzell et al. [2] study is one of the largest and most comprehensiveassessments of this industry. Of the various cancers considered, the only exposurerelated increases were in leukemias. These increases were seen only in the SBRindustry, mainly in workers employed between 1950 and 1965. No such increaseswere seen in those workers exposed to butadiene alone. Within the SBR industry,the increases in leukemia were associated with certain activities, namely laboratoryworkers, polymerisation, coagulation and maintenance.

The lack of effect in workers exposed to butadiene alone is consistent with theoutcome of the studies comparing the metabolic activation of butadiene in mice,rats and humans [3–5]. The risk of developing cancer following exposure tobutadiene appears to be associated with the metabolism of butadiene to itsepoxides. Thus mice, the most sensitive species, extensively metabolise butadiene toits monoepoxide and convert most of that to the diepoxide. Rats, which are largelyresistant to butadiene induced cancer, form less monoepoxide and convert verylittle of that to the diepoxide, tending to form glutathione conjugates of themonoepoxide or its hydrolysis product, butenediol. Primates and humans converteven less to the monoepoxide and all of that appears to be converted to thebutenediol.

The mechanistic and pharmacokinetic studies are therefore in agreement with theepidemiology studies in predicting that workers exposed to butadiene alone are notat risk of developing leukemia. This implies that the leukemias seen in the SBRindustry are caused either by another agent alone or as a result of co-exposure tobutadiene and another agent used in that industry. One potential candidate isdimethyldithiocarbamate (DMDTC) which was added in aqueous solution to theSBR latex where it acted as a stopping agent for the polymerisation reaction.Dermal exposure occurred during handling of the latex. The use of this material inthe SBR industry from the early 1950s until 1965 is coincident with the increase inleukemia and a positive correlation exists for leukemia and co-exposure to butadi-ene and DMDTC [2]. Evidence for significant exposure to DMDTC comes from theobservation of an ‘Antabuse’ effect in SBR workers consuming alcohol. DMDTCis a close analogue to disulfiram or antabuse that is used in the treatment ofalcoholism. Typical clinical dose levels of antabuse are in the range 200–500 mg perday [6].

DMDTC and other related dithiocarbamates have a vast range of chemical andbiological properties that have led to their use in industry, as fungicides and drugs.These properties and their possible influence on the outcome of the butadieneepidemiology studies, were reviewed by Irons and Pyatt [7]. Of their variousbiological properties, these chemicals are known to interfere with a wide range ofmetabolic enzymes, including many of those involved in the metabolism of butadi-

T. Green et al. / Chemico-Biological Interactions 135–136 (2001) 585–598 587

ene. These include inhibition of aldehyde dehydrogenases (the antabuse reaction),cytochromes P-450, including CYP2E1, and glutathione S-transferases. It is entirelypossible, therefore, that co-exposure to DMDTC could alter the metabolism ofbutadiene in humans in a manner that could increase the risk of developingleukemia in those populations exposed to both chemicals. In this study, we haveinvestigated the effects, in rats and mice, of DMDTC exposure on the activities ofall of the major enzymes involved in the metabolism of butadiene. Subsequently,the metabolism of butadiene has been compared in vivo in rats and mice exposedto butadiene with and without prior treatment with DMDTC and in vitro in liverfractions from control and DMDTC treated animals.

2. Materials and methods

2.1. Chemicals

1,3-Butadiene (�99%), butadiene monoepoxide (98%), 1,3-butadiene diepoxide(97%), 3-butene-1,2-diol and dimethyldithiocarbamate sodium salt (98%) wereobtained from Sigma-Aldrich Co., Dorset, UK. C-14 1,3-butadiene (�99%),specific activity 20 �Ci/mmol, and S-35 glutathione, specific activity 701 Ci/mmol,were obtained from Amersham-Pharmacia, Cardiff, UK.

2.2. Animals

Male F344 rats (180–200 g b.w.) and male B6C3F1 mice (20–25 g b.w.) wereobtained from Harlan Olac, UK. The animals received Rat and Mouse Number 1(RM1) pelleted diet from Special Diet Services, Witham, Essex, UK and mainswater ad libitum. The environment of the animal room was controlled, with thetemperature maintained within a target range of 19–23°C, relative humidity withina target range of 40–70%, an artificial light cycle of 12:12 h light:dark and between25 and 30 air changes per hour.

2.3. The effects of DMDTC on hepatic enzyme acti�ities

2.3.1. Preparation of tissue fractionsBoth species were given single oral doses of 250 mg/kg DMDTC in water

(control animals were dosed with water) and groups of three animals were sacrificed2, 6, 12 and 24 h post-dosing by asphyxiation with a rising concentration of CO2,followed by cardiac puncture. The livers were removed, pooled by treatment andtime point and thoroughly washed in ice cold 1.15% potassium chloride. The tissueswere scissors minced and a 30% (w/v) homogenate was prepared in 0.25 M sucrose,20 mM Tris, 5 mM EDTA pH 7.4 using a Potter Elvehjem homogeniser. Thehomogenates were centrifuged at 16 900×g for 15 min at 4°C, the supernatantstransferred to fresh tubes and centrifuged at 105 000×g for 70 min at 4°C. Thesupernatant (cytosol) was removed from the microsomal pellet and stored at


−70°C. The microsomal pellet was resuspended in 20 mM Tris, 1.15% potas-sium chloride pH 7.4, centrifuged at 105 000×g for 70 min at 4°C and the pelletresuspended in 0.1 M sodium phosphate pH 7.4 (2:1 w/v original tissue wetweight to buffer). The microsomes were stored at −70°C until required. Proteinconcentrations in the tissue fractions was determined using the method of Lowryet al. [10].

2.3.2. Enzyme assaysCYP2E1 activity was assayed by measuring the hydroxylation of p-nitrophe-

nol, as described by Reinke and Moyer [11]; CYP2A6 was measured as cou-marin 7-hydroxylase activity, as described by Juvonen et al. [12] and epoxidehydrolase activity was measured with styrene oxide as the substrate, as describedby Carlson [13]. Alcohol and aldehyde dehydrogenases were determined as de-scribed by Koivisto and Eriksson [14].

Glutathione S-transferase activity was determined using 1-chloro-2,4-dini-trobenzene (broad spectrum substrate), 3,4-dichloronitrobenzene (m� class) andethacrynic acid (� class), as described by Habig et al. [15]. Cumene hydroperox-ide (� class) was determined as described by Reddy et al. [16]. Non-proteinsulphydryl (NPSH) levels were determined using the method of Sedlak and Lind-say [17].

2.4. In �itro metabolism studies

Hydroxymethyl vinylketone (HMVK-1-hydroxy-3-butene-2-one) was preparedas described by Kaptein et al. [8]. HMVK (3 mmoles) was reacted with anequimolar amount of glutathione in 20 ml water for 18 h. The crystallineproduct was characterised by NMR spectroscopy and mass spectroscopy.

The metabolism of butadiene to its mono-, di-epoxide and 3-butene-1,2-diolwas determined as described by Csanady et al. [3].

2.5. In �i�o metabolism

Rats (n=3 per group) and mice (n=10 per group) were given single oraldoses of 250 mg/kg DMDTC either 6 or 24 h before being exposed to atmo-spheres of 200 ppm C-14 butadiene (250 �Ci/mmole) for 6 h. The atmosphereswere prepared by mixing radiolabelled butadiene with air to give a concentrationof 200 ppm that was passed through the 20-l glass exposure chambers at a flowrate of 1.5 l/min. C-14 butadiene leaving the chamber was trapped on charcoal.Urine and faeces were collected during exposure and for 48 h post-exposure.Food was withdrawn during the exposure period.

Urine samples were analysed by high performance liquid chromatography us-ing the method described by Richardson et al. [9] with the exception that theacetonitrile content of the eluting buffer was reduced to 2%.


Table 1Cytochrome P-450 and epoxide hydrolase activities in mouse and rat liver microsomal fractionsfollowing a single oral dose of 250 mg/kg DMDTCa

CYP2A6CYP2E1Time (h) Epoxide hydrolase(pmol/min/mg) (nmol/min/mg)(nmol/min/mg)

Mouse0.67�0.280 1.16�0.28 19.1�4.20.48�0.355.4�2.3b2 0.20�0.07b

6 0.62�0.180.21�0.04b 11.4�4.00.53�0.1730.8�13.90.55�0.10b12

17.4�4.20.79�0.08 0.56�0.0924

Rat3.02�0.670.000 0.48�0.05

0.08�0.02b 0.00 2.22�0.7522.39�0.410.000.04�0.00b6

0.000.10�0.05b 3.00�0.43120.000.22�0.03b 2.74�0.3924

a CYP2E1 activity was measured with p-nitrophenol and CYP2A6 with coumarin. Epoxide hydrolaseactivity was measured with styrene oxide.

b P�0.01.

Table 2Alcohol and aldehyde dehydrogenase activities in mouse and rat liver cytosol fractions following asingle oral does of 250 mg/kg DMDTC

Alcohol dehydrogenaseTime (h) Aldehyde dehydrogenase(nmol/min/mg) (nmol/min/mg protein)

Mouse0 0.89�0.070.87�0.152 0.74�0.08 0.17�0.05a

0.28�0.04a0.71�0.03612 0.33�0.06a0.52�0.08a

24 0.27�0.03a0.84�0.04

Rat0.20�0.041.21�0.220

2 0.14�0.020.80�0.066 1.13�0.27 0.15�0.10

0.94�0.22 0.07�0.01a121.02�0.35 0.02�0.02a24

a P�0.01.


Fig. 1. NMR spectrum of the conjugate formed by the reaction of HMVK with glutathione in aqueoussolution.

3. Results

3.1. The effects of DMDTC on hepatic enzyme acti�ities

The results of the assays of cytochromes P-450, glutathione S-transferases,epoxide hydrolase and the dehydrogenases are shown in Tables 1–4. Of the variousenzymes assayed, only CYP2E1 and aldehyde dehydrogenase was significantlyaffected by treatment with DMDTC. In both rat and mouse, CYP2E1 activity wasrapidly reduced 2 h after dosing. The activity had largely recovered by 24 h in themouse, but remained depressed in the rat (Table 1). As with CYP2E1, aldehydedehydrogenase activity was rapidly reduced in the mouse and although it recoveredslightly, the activity was still significantly reduced 24 h after dosing. In the rat, thetime course was longer, the maximum reduction in activity being achieved 24 hafter dosing (Table 2).

Of the other enzymes assayed, there was a transient reduction in CYP2A6activity in mouse liver 2 h after dosing and an increase at 12 h. GlutathioneS-transferase activities were unaffected by DMDTC treatment when assayed withthe broad spectrum substrate CDNB or with substrates more specific for the mainglutathione S-transferase classes (Tables 3 and 4). Epoxide hydrolase and alcoholdehydrogenase activities were also unchanged following DMDTC treatment (Table1). Hepatic NPSH (glutathione) levels were slightly reduced (Tables 3 and 4).

3.2. In �itro metabolism

The product formed by the reaction of HMVK and glutathione gave a massspectrum (m/e M-1, 392) and NMR spectrum (Fig. 1) consistent with the productbeing the glutathione conjugate S-(glutathionyl)-4-hydroxybutan-3-one.

T.

Green

etal./

Chem

ico-B

iologicalInteractions

135–

136(2001)

585–

598591

Table 3Glutathione S-transferase activities and sulphydryl levels in mouse liver cytosol fraction following a single oral dose of 250 mg/kg DMDTCa

GST � (nmol/min/mg)Time (h) GST m� (nmol/min/mg) GST � (nmol/min/mg) Total GST (�mol/min/mg) NPSH (nmol/mg)

17.9�1.6 35.9�1.90 78.8�6.1 2.6�0.1 75.6�4.319.3�2.5 36.5�6.42 69.0�20.7 2.9�0.8 62.1�17.318.1�2.9 36.7�3.66 73.7�11.2 3.7�0.4 68.1�3.921.7�2.7 35.8�7.9 87.0�4.5 2.6�0.412 72.0�8.620.4�6.8 34.7�5.9 98.4�21.3 2.8�1.124 56.8�8.2*

a Activities were measured with the following substrates: �, cumene hydroperoxide; m�, 3,4-dichloronitrobenzene; �, ethacrynic acid; total, 1-chloro-2,4-dinitrobenzene.

T.

Green

etal./

Chem

ico-B

iologicalInteractions

135–

136(2001)

585–

598592

Table 4Glutathione S-transferase activities and sulphydryl levels in rat liver cytosol fraction following a single oral dose of 250 mg/kg DMDTCa

GST � (nmol/min/mg)Time (h) GST m� (nmol/min/mg) GST � (nmol/min/mg) Total GST (�mol/min/mg) NPSH (nmol/mg)

15.0�4.9 40.8�2.20 58.7�24.6 1.4�0.5 59.4�8.612.4�4.9 35.5�5.0 45.2�14.92 0.7�0.2 41.4�3.3b

16.0�1.9 39.4�3.46 56.7�5.1 1.3�0.2 51.0�5.08.0�1.6 34.2�4.5 38.8�5.5 0.5�0.3 39.1�6.5b12

11.2�1.4 37.1�4.2 43.2�10.7 1.2�0.1 45.6�5.024

a Activities were measured with the following substrates: �, cumene hydroperoxide; m�, 3,4-dichloronitrobenzene; �, ethacrynic acid; total, 1-chloro-2,4-dinitrobenzene.

b P�0.01.


The metabolism of butadiene to its oxides and 3-butene-1,2-diol was compared inliver fractions from control and DMDTC treated rats and mice. The results wereconsistent with the results of the enzyme assays above. Conversion of butadiene toits mono-epoxide was markedly reduced in both mouse and rat liver by DMDTCtreatment, as was the metabolism of the mono-epoxide to the di-epoxide in mouseliver (Table 5). The latter reaction could not be detected in rat liver fractions.Conversion of the mono-epoxide to the diol by epoxide hydrolases was not affectedby DMDTC treatment.

3.3. In �i�o metabolism

The atmospheric concentrations of butadiene in the exposure chambers over the6-h exposure periods were; control rats, 193.4�4.5 ppm; rats dosed with DMDTC6 h before exposure, 208�4.0 ppm; rats dosed with DMDTC 24 h before exposure,188�10.5 ppm; control mice, 190�8.7 ppm, mice dosed with DMDTC 6 h beforeexposure, 189�9.5 ppm; and mice dosed with DMDTC 24 h before exposure,194�7.4 ppm.

The levels of radioactivity in 0–48 h urine samples from rats and mice dosed withDMDTC prior to exposure to butadiene are shown in Table 6. In mice, urinaryexcretion was increased by treatment with DMDTC, to 134% of control whenDMDTC was given 6 h before exposure and to 152% when given 24 h before. Inthe rat, excretion of radioactivity in urine was significantly decreased to 10% ofcontrol, in rats given DMDTC 6 h before exposure. Urinary excretion in rats dosedwith DMDTC 24 h before exposure to butadiene was comparable with controls.

Urinary metabolite profiles from rats and mice exposed to 200 ppm C-14butadiene for 6 h with and without a prior dose of DMDTC are shown in Fig. 2.In the mouse, metabolism appeared to be largely unaffected by DMDTC treatmentother than an increase in the metabolite eluting after 13 min. In the rat, three peakswere observed eluting between 15 and 20 min in DMDTC treated animals that werenot present in rats exposed to butadiene alone.

Table 5The in vitro metabolism of butadiene and butadiene mono-epoxide in hepatic microsomal fractionsfrom control and DMDTC (250 mg/kg) treated rats and micea

Mouse Rat

DMDTCbControlDMDTCbControl

246 24 6

Metabolism of:0.41 0.05 0.23 0.06 0.01BD to EB (ppm/mg/min) 0.03

0.30.00.3BO to DEB (pmol/min/mg) 0.00.00.0BO to EBD (ppm/min/mg) 1.581.661.390.600.73 0.73

a BD, butadiene; BO, epoxybutene; DEB, diepoxybutane; EBD, epoxybutene diol.b Measured in livers taken from animals dosed with DMDTC either 6 or 24 h before sacrifice.


Table 6The excretion of radioactivity in rats and mice pretreated with DMDTC (250 mg/kg) 6 or 24 h beforeexposure to 200 ppm C-14 1,3-butadiene for 6 h

Time post exposure Radioactivity excreted in urine(h)

DMDTC 24 h beforeDMDTC 6 h beforeControlexposureexposure

�CI�CI % �CI %%

Mouse368.850–24 97.9236.52 414.87 85.583.9

7.9924–48 2.145.22 13.29 14.516.1376.84 100.0 428.16100.0 100.0Total % 281.74

% Control 133.8100.0 – 152.0 ––

Rat54.520–24 98.3518.24 505.56 97.998.40.9424–48 1.78.60 10.84 2.11.6

55.46 100.0 516.40100 100.0Total % 526.8410.33 – 98.0% Control –100.0 –

4. Discussion

Butadiene metabolism differs between species in the rate of metabolism to themono-epoxide and in the fate of that epoxide [3–5]. Epoxidation of butadiene is fargreater in mice than rats, primates or humans. Additionally, a far greater propor-tion of the mono-epoxide is oxidised to the di-epoxide in mice. In rats, themono-epoxide is detoxified by glutathione conjugation and metabolised by epoxidehydrolases and in primates and humans, the mono-epoxide is predominantlymetabolised by epoxide hydrolases to 3-butene-1,2-diol. Further metabolism of thebutenediol leads to the major urinary metabolite, 1,2-dihydroxy-4-(N-acetylcys-teinyl)butane [18]. The mechanism involved in the conjugation of the 3-butene-1,2-diol with glutathione, the precursor of this mercapturate, has been a source ofspeculation for a number of years. Addition of glutathione to the double bond of3-butene-1,2-diol is not an energetically favoured reaction and requires an oxidationby alcohol dehydrogenases and possibly cytochrome P-450, to form the vinylketone (HMVK), thereby activating the double bond before conjugation withglutathione can occur [19,20]. Following conjugation, it is proposed that the ketoneis reduced by dehydrogenases to give the diol form of the conjugate which appearsin urine as its mercapturate (Fig. 3).

DMDTC primarily inhibited cytochrome P-450 2E1 (CYP2E1) and aldehydedehydrogenase in rats and mice following a single oral dose, inhibition of the latterenzyme being consistent with the enzyme inhibition reported in humans dosed withdisulfiram [21–23]. The reduction in CYP2E1 activity significantly reduced themetabolism of butadiene to its oxides in liver fractions from DMDTC treated


animals compared to controls (Table 5). It seems unlikely that this reduction inCYP2E1 activity and the conversion of butadiene to its oxides would lead to anincreased carcinogenic risk.

In vivo, the effects of DMDTC were more complex with a marked speciesdifference between rats and mice. In rats, as expected from the in vitro results,

Fig. 2. Urinary metabolites from rats and mice exposed to 200 ppm C-14 butadiene for 6 h with andwithout a pre-exposure dose of 250 mg/kg DMDTC.


Fig. 3. The proposed metabolism of 3-butene-1,2-diol.

urinary excretion of radioactivity was markedly reduced to 10% of control in thoserats given DMDTC 6 h before exposure. In contrast, when DMDTC was given 24h before exposure, urinary excretion was comparable with controls (Table 6). Thissuggests that the reduction in urinary excretion is caused by the inhibition ofCYP2E1, since this is markedly reduced 6 h after dosing, but had recoveredsignificantly in the experiments where exposure to butadiene started 24 h after asingle dose of DMDTC (Table 1). Aldehyde dehydrogenase, on the other hand, isnot reduced in the rat until 12–24 h after dosing (Table 2) suggesting that thisenzyme does not influence the excretion of radioactivity in rats exposed to butadi-ene. In the mouse, butadiene metabolism is significantly stimulated by priortreatment with DMDTC, either 6 or 24 h before exposure (Table 6). Since bothCYP2E1 and aldehyde dehydrogenase are both markedly reduced in mice dosedwith DMDTC (Tables 1 and 2), this result suggests that these enzymes are notcritical to the metabolism of butadiene. It seems probable that in the absence ofthese enzymes, other isoforms, particularly of cytochromes P-450, are able tometabolise butadiene in the mouse. Further characterisation of the dose responsefor DMDTC (studies in progress) may shed further light on these interestingchanges, particularly at lower dose levels approaching occupational exposures toDMDTC.

A role for aldehyde dehydrogenase in the metabolism of butene diol has yet to beestablished. HMVK was found to react chemically with glutathione, as expected, toform S-(glutathionyl)-4-hydroxybutan-3-one, the immediate precursor of the bu-tane diol (Fig. 3), which is excreted in urine as its mercapturate. In vivo, the urinarybutadiene metabolite profiles changed following DMDTC treatment, qualitativelyin the mouse and quantitatively in the rat. At the present time it is not knownwhether any of these changes are a consequence of aldehyde dehydrogenaseinhibition, nor are the toxicological consequences of these changes known.


In conclusion, pre-treatment of rats and mice with DMDTC has an impact onbutadiene metabolism, both qualitatively and quantitatively, in vitro and in vivo.The overall metabolism of butadiene in vivo is significantly reduced by DMDTC inrats, but increased in mice. In the mouse, the proportions of the urinary metaboliteschanged and in the rat, three new metabolites were apparent. The exact nature ofthese changes has yet to be characterised.

Acknowledgements

These studies were sponsored by the American Chemistry Council.

References

[1] IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 71 (Part 1), Lyon,France, pp 109–225.

[2] E. Delzell, N. Sathiakumar, M. Hovinga, M. Macaluso, J. Julian, R. Larson, P. Cole, D.C.F. Muir,A follow-up study of synthetic rubber workers, Toxicology 113 (1996) 182–189.

[3] G.A. Csanady, F.P. Guengerich, J.A. Bond, Comparison of the biotransformation of 1,3-butadieneand its metabolite, butadiene monoepoxide, by hepatic and pulmonary tissues from humans, ratsand mice, Carcinogenesis 13 (1992) 1143–1153.

[4] W.E. Bechtold, M.R. Strunk, Y.C. Chang, J.B. Ward, R.F. Henderson, Species differences inurinary butadiene metabolites: Comparisons of metabolite ratios between mice, rats and humans,Toxicol. Appl. Pharmacol. 127 (1994) 44–49.

[5] R.F. Henderson, J.R. Thorton-Manning, W.E. Bechtold, A.R. Dahl, Metabolism of 1,3-butadi-ene:species differences, Toxicology 113 (1996) 17–22.

[6] C. Brewer, Supervised disulfiram in alcoholism, Br. J. Hosp. Med. 2 (1986) 116–119.[7] R.D. Irons, D.W. Pyatt, Dithiocarbamates as potential confounders in butadiene epidemiology,

Carcinogenesis 19 (1998) 539–542.[8] B. Kaptein, G. Barf, R.M. Kellogg, F. Van Bolhuis, Synthesis and coordinating properties of

ligands designed for modeling of the active site zinc of liver alcohol dehydrogenase, J. Org. Chem.55 (1990) 1890–1901.

[9] K.A. Richardson, M.M.C.G. Peters, R.H.J.J.J. Megens, P.A. van Elburg, B.T. Golding, P.J.Boogaard, W.P. Watson, N.J. van Sittert, Identification of novel metabolites of butadiene monoe-poxide in rats and mice, Chem. Res. Toxicol. 11 (1998) 1543–1555.

[10] O. Lowry, N. Rosebrough, A. Farr, R. Randall, Protein measurement with the Folin phenolreagent, J. Biol. Chem. 193 (1951) 265.

[11] L.A. Reinke, M.J. Moyer, p-Nitrophenol hydroxylation. A microsomal oxidation which is highlyinducible by ethanol, Drug Metab. Dispos. 13 (1985) 548–552.

[12] R.O. Juvonen, P.K. Kaipainen, M.A. Lang, Selective induction of coumarin 7-hydroxylase bypyrazole in mice, Eur. J. Biochem. 152 (1985) 3–8.

[13] G.P. Carlson, Metabolism of styrene oxide to styrene glycol by mouse liver and lung, J. Toxicol.Environ. Hlth. 53 (1998) 19–27.

[14] T. Koivistio, C.J.P. Eriksson, Hepatic aldehyde and alcohol dehydrogenases in alcohol preferringand alcohol avoiding rat lines, Biochem. Pharmacol. 48 (1994) 1551–1558.

[15] W.H. Habig, J.P. Pabst, W.B. Jakoby, Glutathione S-transferases, J. Biol. Chem. 249 (1974)7130–7139.

[16] C.C. Reddy, C.-P.D. Tui, J.R. Burgess, C.-Y. Ho, R.W. Scholz, E.J. Massaro, Evidence for theoccurrence of a selenium-independent glutathione peroxidase activity in rat liver microsomes,Biochem. Biophys. Res. Comm. 101 (1981) 970–974.


[17] J. Sedlak, R.H. Lindsay, Estimation of total, protein bound and nonprotein sulphydryl groups intissue with Ellmans reagent, Anal. Biochem. 25 (1982) 192–205.

[18] P.J. Sabourin, L.T. Burka, W.E. Bechtold, A.R. Dahl, M.D. Hoover, I.Y. Chang, R.F. Henderson,Species differences in urinary butadiene metabolites; identification of 1,2-dihydroxy-4-(N-acetylcys-teinyl)butane, a novel metabolite of butadiene, Carcinogenesis 13 (1992) 1633–1638.

[19] R.A. Kemper, A.A. Elfarra, Oxidation of 3-butene-1,2-diol by alcohol dehydrogenase, Chem. Res.Toxicol. 9 (1996) 1127–1134.

[20] R.A. Kemper, A.A. Elfarra, S.R. Myers, Metabolism of 3-butene-1,2-diol in B6C3F1 mice, DrugMetab. Dispos. 26 (1998) 914–920.

[21] E.D. Kharasch, K.E. Thummel, J. Mhyre, J. Lillibridge, Single dose disulfiram inhibition ofchlorzoxazone metabolism: A clinical probe for P-450 2E1, Clin. Pharmacol. Ther. 53 (1993)643–650.

[22] E.D. Kharasch, D.C. Hankins, P.J. Baxter, K.E. Thummel, Single dose disulfiram does not inhibitCYP2A6 activity, Clin. Pharmacol. Ther. 64 (1998) 39–45.

[23] E.D. Kharasch, D.C. Hankins, C. Jubert, K.E. Thummel, J.K. Taraday, Lack of single dosedisulfiram effects on cytochrome P-450 2C9, 2C19, 2D6 and 3A4 activities: Evidence for specificitytoward P-450 2E1, Drug Metab. Dispos. 27 (1999) 717–723.

.

Documents

The influence of co-exposure to dimethyldithiocarbamate on butadiene metabolism