Involvement of cytochrome P4502E1 in the toxicity of dichloropropanol to rat hepatocyte cultures

ELSEVIER

Toxicology 118 (1997) 171-179

Involvement of cytochrome P4502El in the toxicity of dichloropropanol to rat hepatocyte cultures Alison Department

H. Hammond*,

Jeffrey R. Fry

ofPhysiology and Pharmacology, Queen’s Medical Centre, Clifton Boulevard, Nottingham, NG7 2UH, UK Received 5 August 1996; accepted 10 December 1996

Abstract Hepatocytes were isolated and cultured from untreated rats and rats treated with isoniazid to induce cytochrome P4502El. Isoniazid selectively increased p-nitrophenol hydroxylase activity in 2-h cultures, and increased the toxicity of both 1,3- and 2,3-dichloropropanol. Isoniazid also increased the rate and extent of glutathione depletion by the dichloropropanols. There was no effect of isoniazid on the toxicity of 1,3-dichloroacetone, precocene II or ally1 alcohol. In addition, diethyldithiocarbamate selectively inhibited p-nitrophenol hydroxylase in 2-h cultures from untreated and isoniazid-treated rats, as well as abolishing toxicity of the dichloropropanols. In 24-h cultures from isoniazid-treated rats diethyldithiocarbamate inhibited high affinity MCOD activity by 55% and there was also a small but significant inhibition of precocene II toxicity. These results indicate that isoniazid-inducible P4502El can mediate the toxicity of dichloropropanol. 0 1997 Elsevier Science Ireland Ltd. Keywords:

Dichloropropanol;

Hepatocytes;

Hepatotoxicity;

Isoniazid;

Diethyldithiocarbamate

1. Introduction Abbreviations: 2E1, cytochrome P4502El; DCA, dichloroacetone; DCP, dichloropropanol; DEDC, diethyldithiocarbamate; EROD, ethoxyresorufin 0-deethylase; MCODhi, methoxycoumarin 0-demethylase high affinity form; MCODto,

total methoxycoumarin

0-demethylase;

marin PROD

PNPH, p-nitrophenol 0-depentylase; TC50,

0-depropylase; pentoxyresorufin

PCOD, propoxycouhydroxylase; toxic concen-

Halogenated hydrocarbons are used extensively in manufacturing industries, and therefore there is a large potential for environmental and/or occupational exposure to these chemicals (Raucy et al., 1993). fiichloropropanol is an industrial-solvent which has been associated with human liver

tration causing 50% cell death. * Corresponding author. Tel.: + 44 115 9709457; fax: + 44

dysfunction and lethal injury following inhalation exposure (Shiozaki et al., 1994); both 1,3- and

115 9709259.

2,3-dichloropropanol

0300-483X/97/$17.00

0 1997 Elsevier

PI1 SO300-483X(96)03604-9

Science

Ireland

Ltd. All rights

reserved.

were detected

in the serum

172

A.H. Hammond,

J.R. Fty i Toxicology

of one of the exposed men. Fulminant liver injury was also observed in the rat after i.p. administration of the 1,3- but not the 2,3-isomer (Haratake et al., 1993). 1,3-Dichloropropanol, but not 2,3-dichloropropanol, is toxic to rat hepatocyte cultures (Hammond et al., 1996), therefore the in vitro toxicity is similar to that seen after i.p. administration in vivo. The in vitro toxicity can be prevented by prior exposure of the cultures to the cytochrome P450 inhibitor, lnon-specific aminobenzotriazole and potentiated by prior dewith buthionine pletion of glutathione sulphoximine (Hammond et al., 1996). These experiments indicate that under appropriate conditions 1,3-dichloropropanol has considerable toxic potential. The isoforms responsible for the metabolism of dichloropropanol are not known, however, several haloalkanes have been shown to cause hepatic necrosis via metabolism by P4502E1, a constitutive isozyme which can also be induced by ethanol in humans and experimental animals 1993). If this isozyme can (Raucy et al., metabolise dichloropropanol, it may be an important determinant of human toxicity after occupational or environmental exposure to this chemical. Therefore, we investigated the effects of induction and inhibition of P4502El on the toxicities of 1,3- and 2,3-dichloropropanol. Isoniazid induces P4502El (Ryan et al., 1986) and diethyldithiocarbamate (DEDC) inhibits P4502El -mediated activwithout inhibiting alcohol ity and toxicity, dehydrogenase-mediated toxicity (Brady et al., 199 1; Lauriault et al., 1992). We also determined the toxicity of 1,3-dichloroacetone, a possible metabolite of 1,3-dichloropropanol; ally1 alcohol, which is an hepatotoxin metabolised by alcohol dehydrogenase (Ohno et al., 1985) and precocene II, a naturally occurring hepatotoxic benzopyrone (Hsia et al., 1981). Although precocene II is structurally unrelated to dichloropropanol, the in vitro toxicity does involve P450-mediated glutathione depletion (Hammond et al., 1995). The isoforms responsible for metabolism of precocene II are also unknown, although the toxicity is increased by induction of P450 by phenobarbitone and /?naphthoflavone (Hammond and Fry, 1991).

I18 (1997) 171~ 179

2. Materials

and methods

2.1. Muteriuls Details of the plasticware, chemicals, culture media and sera used in these studies have been reported elsewhere, as has the source and maintenance of the Wistar rats (Hammond and Fry, 1990b). Precocene II and DEDC were obtained from Sigma Chemical Co. (Poole, Dorset, UK); 1,3-dichloroacetone and the ally1 alcohol, dichloropropanols were obtained from Aldrich Chemical Co. (Gillingham, Dorset, UK). 2.2. Induction

and inhibition

of P4502El

Isoniazid (0.1%) in drinking water was administered to 6- to 7-week-old adult male rats for 10 days prior to hepatocyte isolation. DEDC was dissolved in water and diluted into culture medium. Cultures were exposed to 100 PM DEDC for 30 min, prior to exposure to toxin or determination of enzyme activity. This concentration was determined to be non-toxic and maximally effective in vitro in preliminary experiments (data not shown). 2.3. Preparation cultures

und treatment

of heputocyte

Hepatocytes were isolated from adult male rats by a two-step lobe perfusion, essentially as described by Reese and Byard (1981). After perfusion of the lobes with collagenase (100 Units/ml) for approximately 20 min, the liver was minced, strained and washed twice. The dead and damaged cells were removed using Percoll (Kreamer et al., 1986), and after a final wash hepatocytes were resuspended in modified Williams’ medium E (5 mM glutamine, 50 pug/ml gentamicin, 10 mu/ml insulin, 5 mM nicotinamide and 1 PM dexamethasone) containing 10% v/v foetal calf serum and plated onto Falcon PrimariaO plates or dishes at a seeding density of 0.17 million cells/ cm2. Plasma membrane integrity of all preparations used was 88-99X, as assessed by trypan blue exclusion.

A.H. Hammond, J.R. Fry / Toxicology 118 (1997) 171-l 79

After allowing 2 h for attachment, the serumcontaining medium was replaced with serum-free modified Williams’ E + inhibitor. After exposure to medium containing DEDC as described above, cultures were exposed to fresh serum-free medium containing toxin, or to Krebs buffer containing enzyme substrate. Toxins were dissolved in methanol (or water: ally1 alcohol) and diluted into the culture medium, the maximum final solvent concentration being 1% v/v. TC,, values (concentrations causing a 50% loss of LDH from the cells) were determined from dose-response curves obtained for each culture. TC,, values greater than 1 mM were judged to be non-toxic and were not included in the statistical tests. 2.4. Biochemical

analyses

Cell death was assessed by measuring retention of cellular LDH using a modification of the micromethod of Chen et al. (Chen et al., 1990). Protein content was determined using the Bradford assay, and glutathione by the Saville assay for non-protein sulphydryls ( > 90% of which are glutathione), both as previously described (Hammond and Fry, 1992). Reduced pyridine nuclestatus was determined using MTT, otide 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Shearman et al., 1994) as described by Fentem et al. (Fentem et al., 1992) except that the final concentration of MTT was 0.2 mg/ml and the exposure time was reduced to 20 min. P450-dependent enzyme activities were determined in intact cells. 2El activity was determined using p-nitrophenol hydroxylase (PNPH) at a concentration of 0.5 mM, as described by Dicker et al. (Dicker et al., 1990). 7-Ethoxy- and 7-pentoxyresorufin 0-dealkylase activities (EROD and PROD respectively) were determined essentially as described by Wortelboer (Wortelboer et al., 1990). These activities are thought to be mediated by the 2C6/11 isozymes in both untreated and ethanol-treated animals (Waxman et al., 1987; Nakajima et al., 1990).

173

7-Methoxyand 7-propoxycoumarin O-dealkylases (MCOD and PCOD respectively) were determined in a similar manner. Medium was removed and substrate was added to the cells in Krebs buffer, and the cultures incubated for 1 (PCOD) or 3 h at 37°C. The buffer was then removed and samples added to /?-glucuronidase in 0.2 M acetate buffer. These were then incubated at 37°C for 2 h or overnight. NaOH-glycine buffer (0.1 M, pH 10.4) was then added to stabilise the fluorescence of the 7-hydroxycoumarin. MCOD is biphasic in male rats (Boobis et al., 1986; Hammond and Fry, 1990b) and the high and low affinity forms appear to be dependent upon different P450 isozymes. Therefore, high affinity and total MCOD activity (MCODhi and MCODto) were determined at substrate concentrations of 20 and 500 ,uM respectively. Fluorescence measurements of resorufin and 7hydroxycoumarin concentrations were made on a Millipore Cytofluor 2350 plate reader. Cell protein was dissolved in 0.5 M NaOH and determined as described above. 2.5. Statistical

analysis

ANOVA and appropriate post hoc tests, t-tests were used as appropriate. A P-value 0.05 or less was determined to be significant.

or of

3. Results Isoniazid treatment of the rats markedly increased (9-fold) the amount of PNPH in 2-h hepatocyte cultures relative to cultures from untreated animals (Table 1). In 24-h cultures there was a 60-80% loss of PNPH, although 24-h cultures from isoniazid-treated rats contained 4.5 times more PNPH than 24-h cultures from untreated animals. Of the other activities measured, EROD and total MCOD were maintained in 24-h cultures from untreated animals and high affinity MCOD activity in 24-h cultures from isoniazidtreated rats. The effect of isoniazid treatment on toxicity in culture is shown in Table 2. 2,3-Dichloropropanol was toxic to hepatocytes cultured from isoniazid-

A.H. Hammond, J.R. Fry / Toxicology 118 (1997) I71 -179

174 Table 1 Effect of in vivo isoniazid vitro Enzyme

Activity

treatment

(pmol product/min/mg

2-h Cultures

EROD PROD MCODhi MCODto PCOD PNPH

on enzyme

activity

in

protein)

24-h Cultures

Untreated

Isoniazid

Untreated

Isoniazid

13k2 13* 1 17& 1 63 f 2 243 k 9 175 k42

13*5 12&2 21+2 98+4 153+8* 1619+98*

18k3 3* 1* 8k I* 29 + 2 73 * 14* 71 *24*

24 + 6+2 21* 62 + 104 + 320 k

6 lf 20 14 55t

Activity was determined in 2- and 24-h cultures from untreated or isoniazid-treated rats as described in the text. Values shown are mean f S.E.M. of three to six cultures. *Mean significantly different to untreated 2-h mean at P i 0.05, ANOVA and Bonferroni. TIndicates mean significantly different to 24-h untreated mean at P I 0.05, ANOVA and Bonferroni.

treated, but not untreated, rats. However, the toxicity of 1,3-dichloropropanol, which was moderately toxic to cultures from untreated rats, was markedly increased by isoniazid. Isoniazid treatment did not affect the toxicities of dichloroacetone, precocene II or ally1 alcohol. There was some loss of dichloropropanol toxicity in 24-h cultures from isoniazid-treated animals Table 2 Effect of in vivo isoniazid Compound

treatment TC,,

on toxicity

in vitro

OIM) 24-h Cultures

2-h Cultures

1,3-DCP 2,3-DCP 1,3-DCA Precocene II Ally1 alcohol

relative to the 2-h cultures, although there was still marked toxicity in these cultures. There was also some loss of precocene II toxicity in 24-h cultures, more so in cultures from untreated animals. The toxicities of dichloroacetone and ally1 alcohol were maintained in 24-h cultures from both untreated and isoniazid-treated animals. DEDC had no significant effects on EROD, PROD, total MCOD or PCOD (data not shown). In 2- and 24-h cultures from untreated rats, and 2-h cultures from isoniazid-treated rats, pre-treatment of cultures with DEDC almost totally inhibited PNPH (Fig. 1). However, there was detectable PNPH in 24-h cultures from isoniazidtreated rats after DEDC exposure, the activity being 42% of the control value. In 2-h cultures from untreated animals and 24-h cultures from isoniazid-treated rats DEDC also significantly decreased high affinity MCOD activity by 41-52%. DEDC abolished the toxicity of 1,3-dichloropropanol in cultures from untreated rats and abolished the toxicity of both dichloropropanols in cultures from isoniazid-treated rats (Table 3). Comparing the values in Table 2 with those in Table 3, it can be seen that DEDC had no effect on the toxicity of 1,3-dichloroacetone or the toxicity of ally1 alcohol, and that although DEDC did not alter the TC,, of precocene II in 2-h cultures from isoniazid-treated rats relative to cultures from untreated animals, there was a significant effect of DEDC in 24-h cultures from isoniazid-

Untreated

Isoniazid

Untreated

Isoniazid

564+24 > 1000 22 * 3 50&21 25 k 8

53+4* 270 + 30 18kl 56 k 1 26 * 10

489 f 26* > 1000 29 + 6 292 + 30* 18k8

95 * 1st 409 + 46 18+ 1 169f39f 24+ 10

Cultures were incubated with compound for 24 h. TC,,s were calculated from loss of intracellular lactate as described in the text. Values shown are mean k S.E.M. of three to eight cultures. *Mean significantly different to untreated 2-h mean at P10.05, ANOVA and Bonferroni. tIndicates mean significantly different to 24 h untreated mean at PsO.05, ANOVA and Bonferroni.

dehydrogenase,

measured

A.H. Hammond,

J.R. Fry / Toxicology

ti

175

PM), and was used in this study to allow direct comparison with cultures from untreated rats. A comparison of depletion in 24-h cultures from untreated and isoniazid-treated rats is shown in Fig. 3. At equimolar doses 1,3-dichloropropanol depletes glutathione faster, and 2,3dichloropropanol depletes glutathione more extensively, in cultures from isoniazid-treated rats compared to cultures from untreated rats.

?j 100

E 8 8 i? ,z s

118 (1997) 171-179

80

60

40

4. Discussion

20

0

PNPH

MCOD high a”!nW,

0

untreated

2hr

m

untreated 24hr

isoniazid

2hr

m

isoniazid

24hr

Fig. 1. Effect of DEDC on enzyme activity in 2- and 24-h cultures from untreated and isoniazid-treated rats. Values shown are mean PNPH and high affinity MCOD activities in DEDC-treated cultures expressed as a percentage of the mean activity in the control (no DEDC) cultures. Means were determined from three to six cultures. *The original DEDC mean value is significantly different to the control mean value at PI 0.05, paired r-test.

treated animals. DEDC pre-treatment of cultures did not alter the glutathione levels or the reduced nucleotide status of the cells relative to the control cultures (data not shown). Isoniazid pre-treatment itself did not significantly affect the glutathione content in 24-h cul(data shown), tures not however toxic concentrations of both dichloropropanols significantly depleted glutathione in 24-h cultures from isoniazid-treated rats in a dose-dependent manner following a 6-h exposure (Fig. 2). Depletion was more extensive with the more toxic 1,3-isomer, and was seen at lower concentrations. DEDC pre-treatment of cultures totally inhibited glutathione depletion by 2,3-dichloropropanol and low concentrations of 1,3-dichloropropanol. However, there was significant depletion after a 6-h exposure to 750 ,uM 1,3-dichloropropanol even after pre-treatment of the cultures with DEDC. 750 PM is a very toxic concentration in cultures from isoniazid-treated animals (TC,, = 95

Table 4 summarises the data obtained in this study. Isoniazid treatment produced a 9-fold increase in PNPH. Isoniazid, like ethanol, specifically induces P4502El (Ryan et al., 1986), and it has been estimated that 80-90% of induced PNPH activity is mediated by 2El in the rat (Koop et al., 1989). Previous results (Hammond and Fry, 1990a) taken together with those presented here show that isoniazid specifically increases PNPH in our rats, indicating a selective induction of P4502El. DEDC is a 2El inhibitor (Brady et al., 1991) and markedly decreased PNPH in cultures from both untreated and isoniazid-treated rats in this study. Although DEDC only inhibited 55% of the PNPH remaining in 24-h cultures from isoniazidtreated rats, the unaffected activity represented barely 10% of the activity measured in 2-h cultures in the absence of DEDC. This is consistent with lo-20% of PNPH activity being dependent upon non-2El P45Os in animals treated with 2El inducers (Koop et al., 1989). It was also reported that DEDC is not absolutely specific for 2El: the 2Al and 3A1/2 isozyme activities were inhibited by 25-30%, but this was far less pronounced than the effect on 2El activity (Brady et al., 1991). In our study, the only other activity sensitive to DEDC was high affinity MCOD, although this relative specificity may partially reflect the range of activities measured. Induction of 2El by isoniazid markedly increased the toxicity of 1,3-dichloropropanol, potentiated the toxicity of 2,3-dichloropropanol (which is not toxic to cultures from untreated

176

A.H.

Table 3 Effect of in vitro DEDC

treatment

Compound

TC,,

Hammond,

on toxicity

J.R. Fry 1 Toxicology

in cultures

from untreated

118 (1997) 171-179

and isoniazid-treated

(PM)

2-h Cultures

1,3-DCP 2,3-DCP 1,3-DCA Precocene II Ally1 alcohol

rats

24-h Cultures

Untreated

Isoniazid

Untreated

Isoniazid

> 1000 > 1000 23 f 2 46k 12 1X f 12

> 1000 > 1000 20 k 6 48 k I 15k2

> 1000 > 1000 30+4 334+41 18 k 10

> 1000 > 1000 24 i 9 344 f 23* 16k 1

Cultures from untreated and isoniazid-treated rats were incubated with 100 PM DEDC for 30 min prior to addition of the toxin. After 24 h incubation with toxin the TC,, was determined as described in the text. Values are mean _+ S.E.M. of three to eight cultures. *Mean significantly different to 24 h mean (Table 1) at P ~0.05, paired t-test.

animals) and increased the rate and extent of dichloropropanol-mediated glutathione depletion in rat hepatocyte cultures. In addition, DEDC abolished the toxicity of the dichloropropanols together with the associated glutathione depletion.

1.3~isomer

60

These effects were specific for dichloropropanol toxicity, as the toxicity of precocene II, which is also P450-mediated (Hammond et al., 1993, was unaffected by induction or inhibition of 2El (Table 4). Isoniazid also did not induce the toxicity of ally1 alcohol (Table 4) which is metabolised

2,3-isomer

1

0

0

500

750

150

225

750

PM dichloropropanol 0

control

0

I 1

0

I 2

I 3

exposure

, 4

I 5

time in hours

I 6

I 7

DEDC

Fig. 2. Glutathione depletion in 24-h cultures from isoniazidtreated rats following a 6 h exposure to dichloropropanol. Cultures were incubated with 100 PM DEDC as appropriate for 30 min prior to exposure to toxin. Values shown are mean k S.E.M. of three cultures. *DCP-treated mean is significantly different to appropriate (i.e. control or DEDC) methanol-treated mean at PI 0.05, ANOVA and Dunnett’s test.

0

untreated

l isoniazid

1.3-DCP 1.3.DCP

V v

untreated isoniazid

2,3-DCP 2,3-DCP

Fig. 3. Comparison of glutathione depletion in 24-h cultures from untreated and isoniazid-treated rats following exposure to dichloropropanol for l-6 h. Values shown are the mean glutathione values at each time-point expressed as a percentage of the mean 0 h value. Means were determined from three to five cultures.

A.H. Hammond, J.R. Fry / Toxicology 118 (1997) 171-l 79 Table 4 Summary

of effects of isoniazid

and DEDC Isoniazid

PNPH 1.3-DCP

on toxicity relative

and enzyme

to untreated

toxicity

2,3-DCP toxicity DCA toxicity MCODhi Precocene

toxicity

by alcohol dehydrogenase in untreated animals (Ohno et al., 1985), suggesting no induction of ADH by isoniazid and no P450-mediated metabolism of ally1 alcohol. Therefore, these results suggest that the toxicity of the dichloropropanols is predominantly, if not exclusively, mediated by P4502E1, as is the toxicity of other haloalkanes (Raucy et al., 1993). Since 2El is a major constitutive P450 inducible by ethanol in humans as well as in rats (Koop and Tierney, 1990), induction of this isozyme activity could potentiate human toxicity of the dichloropropanols following occupational and/or environmental exposure. It could theoretically also interfere with metabolism of other 2El substrates, both environmental pollutants (e.g. benzene) and drugs (e.g. isoniazid, chlorzoxazone, acetaminophen) since many 2El substrates can induce and/or inhibit this isozyme (Raucy et al., 1993). Although the rank order, in cultures f DEDC, of increasing 1,3-dichloropropanol toxicity was the same as the rank order of decreasing DEDCsensitive PNPH, the 60-80% loss of PNPH between 2 and 24 h in both treatment groups did not really match the small to negligible change in toxicity between these time points. It has been reported that only 40% of the PNPH activity in untreated animals is mediated by 2E1, therefore PNPH is not as good a marker for this isozyme in untreated animals as it is in inducer-treated animals (Koop et al., 1989). The loss of PNPH might then reflect greater loss of the non-2El PNPH, which could explain the maintenance of toxicity and inhibition by DEDC in the

activity

in vitro DEDC

relative

to control

Untreated

Isoniazid

if 0 0 L2h 0

11 0 1 24 h 1 24 h

face of loss of PNPH in cultures from untreated rats. It has been reported that 70% of the immunochemically detectable 2El is lost over 24 h in culture, but activity was not determined (Eliasson et al., 1988). In 24-h cultures from isoniazid-treated rats the loss of activity despite maintenance of dichloropropanol toxicity and rapid, DEDC-sensitive glutathione depletion suggests that either 2El does not mediate the toxicity in 24-h cultures, or that toxicity is not proportional to activity following isoniazid treatment. Induction and maintenance of 2El levels is effected primarily by post-translational stabilisation of the protein, not enhanced transcription, therefore although the amount of P4502El increases after induction due to inhibition of degradation, there is no new synthesis (Koop et al., 1989). This means that a small change in protein level could have a large effect on activity. Age-related changes in 2El activity have been shown to be greater than the changes in protein level: 2El enzyme activity decreased by more than 60% between the ages of 3 and 6 weeks in the male rat, whereas hepatic P4502El levels dropped (via changes in turnover) by only 30% (and were thereafter maintained) (Thomas et al., 1987). It seems plausible then that a small change in 2El content could produce a large change in activity, but not necessarily in toxicity: there may be enough stabilised enzyme remaining to activate the toxin, especially over a 24-h exposure period. 2,3-Dichloropropanol is approximately five times less toxic than the 1,3-isomer in cultures from isoniazid-treated animals, and glutathione

178

A.H.

Hammond,

J.R. Fry / Toxicology

depletion by the 2,3-isomer (which was inhibited by DEDC) was slower and less extensive than that seen with the 1,3-isomer. The reduced potential for glutathione depletion, which helps to explain the difference in ultimate toxicity between the two isomers, is presumably due either to different substrate affinities/rates of metabolism by P450 or to generation of different metabolites. Non-toxic monochlorodiols and 1,2-propanediol have been identified as metabolites of dichloropropanol in rats. However, these intermediates accounted for less than 15% of the total dose (Koga et al., 1992); the other metabolites are unknown. P450-mediated metabolism of 1,3dichloropropanol could theoretically produce 1,3dichloroacetone, a P450-generated metabolite of other chlorohydrocarbons, which is known to deplete glutathione and is also highly mutagenic (Eder and Dornbusch, 1988; Weber and Sipes, 1992). 1,3-Dichloroacetone was very toxic to rat hepatocyte cultures, the toxicity being unaffected by isoniazid or DEDC (Table 4) and was also very toxic to Chinese hamster ovary cells which possess little or no P450 activity: the TC,, was approximately 50 PM (unpublished results). In addition, 1,3-dichloroacetone has been shown to deplete glutathione in fortified rat liver microsomes, in a dose-dependent manner which did not require NADPH (M.J. Garle, personal communication). Taken together, this evidence suggests that 1,3dichloroacetone is a directly-acting cytotoxin, rather than a metabolism-mediated one, and its toxicity in vitro is similar to that of 1,3-dichloropropanol following isoniazid treatment. In summary, these results indicate that induction of P4502El markedly enhances the metabolism of the dichloropropanols to toxic, glutathione-depleting intermediates. This isozyme may also have a major role in mediating the toxicity of 1,3dichloropropanol in cultures from untreated rats. The marked toxicity of 1,3-dichloroacetone, a postulated P450-mediated metabolite of 1,3-dichloropropanol, was also shown to be independent of P450 induction or inhibition. Precocene II toxicity did not appear to be mediated by 2E1, but did seem to be associated with high affinity MCOD activity, an activity known to be age- and sex-dependent.

118 (1997) 171-179

Acknowledgements We gratefully acknowledge the generous financial support of the industrial sponsors of the Fund for the Replacement of Animals in Medical Experiments.

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A.H.

Hammond,

J.R. Fry / Toxicology

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