evaluation of epichlorohydrin (ech) genotoxicity
TRANSCRIPT
Muta~on Research, 109 (1983) 41-52 41 Elsevier Biomedical Press
Evaluation of epichlorohydrin (ECH) genotoxicity
Microsomal epoxide hydrolase-dependent deactivation of ECH mutagenicity in Schizosaccharomyces pornbe in vitro
A.M. Rossi a,,, L. Migliore a, N. Loprieno a, M. Romano b and M. Salmona b
"Istituto di Biochimica, Biofisica e Genetica, Universitit di Pisa, Via S. Maria 53, 56100 Pisa (Italy) and h Laboratory for Enzyme Research, lstituto di Ricerche Farmacologiche "Mario Negri', Via Eritrea 62,
20157 Milano (Italy)
(Received 30 July 1982) (Revision received 5 November 1982)
(Accepted 19 November 1982)
Summary
The mutagenic effect of epichlorohydrin (ECH) on the yeast Schizosaccharomyces pombe was studied in vitro in the presence of mouse-liver $9 mix and microsomal and cytosolic fractions. The incubations were always performed in the absence of NADPH-generating systems. $9 mix and microsomes from phenobarbital-pretreated mice significantly reduced ECH mutagenicity, whereas the cytosol did not result in any deactivating effect.
The various protein contents of the subcellular fractions were not involved in any scavenger effect as regards ECH mutagenic activity. Moreover, the addition of reduced glutathione to the incubation mixtures indicated that it did not play an important role, either per seor through the enzyme(s) glutathione-S-epoxide trans- ferase(s), in preventing ECH genotoxicity.
Our results suggest that microsomal epoxide hydrolase(s) represents the major step in the detoxifying pathway of ECH.
These observations were supported by measurements of the specific epoxide hydrolase'activity in the various fractions on the same substrate.
* To whom correspondence should be addressed.
Abbreviations: ECH, epichlorohydrin; GSH, glutathione; NADPH, reduced nicotinamide-adenine di- nucleotide phosphate; PB, phenobarbital.
0027-5107/83/0000-0000/$03.00 © 1983 Elsevier Science Publishers
42
Epichlorohydrin (ECH) has been commercially produced on a large scale for over 30 years. In 1975, production was estimated at around 250 million kg. Its applica- tions include preparations of synthetic glycerins, epoxy resins and elastomers, manufacture of pesticides, paper, textiles and pharmaceuticals (IARC, 1976). Be- cause of its wide production, the occupational exposure is considerable. Epidemio- logical studies, although not conclusive (Rose and Lane, 1979), and experimental carcinogenesis work in mice (Van Duuren et al., 1974) and rats (Laskin et al., 1980), have provided evidence of a potential carcinogenic effect, though weak, in man. Considerable evidence for the genotoxic activity of ECH, consistent with its alkylat- ing properties, has been gathered in several systems, implying that ECH can act as a gene mutagen (Andersen et al., 1978; Bartsch et al., 1980; Bridges, 1978; Ehremberg and Hussain, 1981; Fishbein, 1976; Stolzenberg and Hine, 1979; Voogd et al., 198 l; Wade et al., 1978; and refs. therein) as well as a clastogen and SCE-inducing compound (Dean and Hodson-Walker, 1979; Kucerova et al., 1976; Kucerova and Polivkova, 1976; Norppa et al., 1981; Sram et al., 1976, 1981; White, 1980), without metabolic conversion. Furthermore, in several epidemiological studies on occupa- tionally exposed persons, a significant increase of chromosomal aberrations in peripheral lymphocytes has been reported (Kucerova et al., 1977; Picciano, 1979; Sram et al., 1980).
In assays in vitro, the presence of mammalian metabolizing systems ($9 mix) reduces the induction of his reversions in S. typhimurium (Andersen et al., 1978; Stolzenberg and Hine, 1979; Voogd et al., 1981) and sister-chromatid exchanges in human lymphocytes (White, 1980) exposed to ECH. In a previous paper, we described a similar deactivation found on studying the mutagenic action of a series of aliphatic epoxides on the yeast Schizosaccharomyces pombe (Migliore et al., 1982). This deactivation has been interpreted as a process of conversion to mutagenically less active metabolites mediated by hepatic enzymes present in the post-mitochondrial fraction ($9), such as soluble and particulate epoxide hydrolases (Gill and Ham- mock, 1979, 1980; Lu et al., 1977; Oesch, 1973; Oesch et al., 1971, 1973, Ota and Hammock, 1980) and glutathione-S-epoxide transferases (Fjellstedt et al., 1973; Friedberg et al., 1979; Habig et al., 1974; Hayakawa et al., 1975).
In this paper we present evidence that the liver microsomal epoxide hydrolase represents the committed step in ECH detoxication as regards its mutagenic effect, in vitro, on the yeast Schizosaccharomyces pombe.
Materials and methods
Chemicals Epichlorohydrin (ECH), with its GC-MS (flame-ionizing detector) analysis, was
kindly supplied by the Division of Occupational Health, Montedison (Milano, Italy). The analysis gave the following composition: epichlorohydrin, 99.82%; dichloropro- pene, 0.11%; others, 0.07%. Chromatographic areas are expressed as percentages.
Glutathione (reduced for biochemistry), albumin (from bovine blood for bio- chemistry), 1-chloro-2,4-dinitrobenzene (for analysis), styrene-7,8-oxide were
43
purchased from Merck (Darmstadt, West-Germany). 3-Chloro-1,2-propanediol from Egachemie (Steinheim, Albuck, West-Germany), ethyl acetate from Farmitalia Carlo Erba (Milano, Italy) and 2-bromoethylbenzene from Aldrich Europe (Beerse, Bel- gium) were of analytical reagent grade.
Yeast strain The yeast Schizosaccharomyces pombe, strain P1 (ade6-60, radiO.198, h-), con-
tains a missense mutation at the ade6 locus and a mutation in the radlO locus. Forward mutations occurring at 5 loci (ade 1, 3, 4, 5, 9) of the adenine pathway can easily be scored by a phenotypic change of the colonies (purple ~ white) on complete medium (Loprieno et al., 1974).
Mutagenicity assay For mutagenic treatment in vitro 1 ml of a growing cell suspension (4 × 10 6
cells/ml) was incubated in liquid complete medium with 1 ml of a metabolizing mixture (2 ml final volume) for 6 h at 32°C in a shaker, When the metabolizing system was omitted, 1 ml of 0.02 M phosphate buffer (pH 7.4) was added. The test substance, dissolved in distilled water, was added at less than 0.1 ml to the cell suspension. Test tubes were sealed. After the treatment, during which 2-3 cell divisions may have occurred, the cells were washed twice, counted, appropriately diluted and plated on complete medium. The colonies were allowed to grow for 5 days at 32°C, then stored for 1-2 days at 4°C to allow their purple pigmentation to increase.
Treatment of mice and preparation of liver homogenate fractions Swiss albino male mice (25-30 g b.w.) were given i.p. injections of phenobarbi-
tone, 80 m g / k g / d a y for 4 days, and fasted for 24 h before they were killed. The livers were washed and homogenized in a Potter apparatus with 0.01 M phosphate buffer (pH 7.4). The homogenate (1 g / 4 ml in buffer) was centrifuged at 10000 X g for 20 min. The supernatants ($9) post-mitochondrial fraction was collected. Subcel- lular fractions (i.e. microsomes and cytosol) were obtained by further centrifugation of the $9 at 105000 x g for 1 h. The supernatant was used as the crude soluble fraction (cytosol). The pellet (microsomes) was washed and resuspended in 0.02 M phosphate buffer (pH 7.4) containing MgC12 and KC1 (5 mM and 200 mM, respectively) to the original volume. Each procedure was carried out at 4°C. The collected fractions were stored at -70°C.
The metabolizing mixtures were prepared as follows and added to 1 ml of the cell suspension.
(1) $9 mix (1 ml). 200 #1 phosphate buffer 0.1 M (pH 7.4), 10 /~l MgC12 0.5 M + KCI 2 M solution, 500 or 250 or 125 /~l $9 (25.0 mg of proteins/ml), distilled water to 1 ml.
(2) Glutathione mix (1 ml). To the above mix, or to 1 ml of 0.02 M phosphate buffer, a buffered GSH solution was added to a final concentration of 5 or l0 mM.
(3) Albumin mix (l ml). As for $9 mix, but an albumin solution, 25 mg/ml, replaced $9.
44
(4) Cytosol. 1 ml of cytosolic fraction (13.0 mg of proteins/ml) was added. (5) Microsome mix (1 ml). 200/~1 phosphate buffer 0.1 (pH 7.4), 300, 100 or 30/xl
of purified microsomes (7.0 mg of proteins), distilled water to 1 ml.
Determination of protein content and enzymatic activity The protein content of the various subcellular fractions was determined by the
method described by Lowry et al. (1951) with bovine serum albumin as standard. The glutathione-S-transferase activity was determined according to Habig et al. (1974) with 1-chloro-2,4-dinitrobenzene as substrate.
The hydration of styrene-7,8-oxide in the microsomal and cytosolic fractions was determined according to Gazzotti et al. (1980).
The hydration of ECH epoxide hydrolase was determined as follows. The incubation mixture, in a final volume of 1 ml, contained about 1 mg of protein, 1.2 mM ECH, 5 mM MgC12 and 150 mM KC1 all dissolved in 5 mM phosphate buffer (pH 7.4). After 15 min of incubation at 37°C the reaction was stopped by adding 1 ml of cold n-butanol. The samples were gently shaken for 10 min and centrifuged at 3000 x g for 20 min. To 100/tl of the supernatant butanolic fractions, 25 /~1 of butylboronic acid solution (20 mg/ml in dimethylformamide) and 20/~1 of internal standard 2-bromoethylbenzene solution (1.67 x 10 -8 mg/ml in butanol) were ad- ded.
1/tl of each sample was injected into a Carlo Erba Fractovap mod 2300 gas chromatograph equipped with an electron-captor detector. The stationary phase was 3% OV-17 on Gas Chrom Q (100-120 mesh) packed into a 2-m long column, 2 mm c.d.; the oven temperature was 180°C, the injector temperature 260°C and the flow rate of carrier gas (nitrogen) was 30 ml/min.
The recovery of ECH from biological samples was over 70%, and the overall sensitivity of the method 5/~g/ml. All the experiments were performed in a linearity range from 10 to 100 /~g/ml. The amounts of diol formed during the enzymatic reaction were linear up to incubation times of 20 min and in the ranges of protein from 0.5 to 2.0 mg/ml. The coefficient of variation of the method calculated for 6 concentrations (10, 20, 40, 60, 80, 100 t~g/ml) was about 10%.
Results
In a previous study, we tested the ECH mutagenicity on the yeast S. pombe at various doses in buffer and in the presence of the $9 fraction from PB-pretreated mouse liver. In the latter case the activity was markedly reduced (Migliore et al., 1982).
In the present study we took into account two concentrations of ECH, chosen on the basis of the results reported in the above paper: 0.8 mM, where the effect of $9 mix was only slightly appreciable and 3.2 mM, where the presence of $9 mix considerably reduced the mutagenic activity of ECH.
Because the $9 effect was independent of NADPH (data not reported), in accord with reports of other authors (Andersen et al., 1978; White, 1980), this coenzyme was omitted.
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49
TABLE 5 DETERMINATION OF MOUSE-LIVER CYTOSOLIC AND MICROSOMAL EPOXIDE HYDRO- LASE ACTIVITY TOWARDS ECH AND STYRENE-7,8-OXIDE (nmoles/min X mg protein)
Treatment Cytosol Microsomes
ECH Styrene- ECH Styrene- 7,8-oxide 7,8-oxide
Control n.d. 2.42 + 0.09 12.80 + 0.43 3.42 + 0.23
Phenobarbital n.d. 4.20___ 0.04 ** 15.5 + 0.70 ** 4.72 + 0.19 **
Each value is the mean _+ S.E. of 4 determinations. ** P < 0.01 according to Student's test. n.d., not detectable.
Table 1 reports the mutagenicity of ECH in the absence or presence of various proportions of $9 mix from untreated and PB-pretreated mouse-liver homogenates. The latter preparation significantly reduced the ECH genotoxicity in a way related to the amount of protein used only at 3.2 mM ECH, whereas $9 mix from untreated animals was much less effective and its action was not related to the protein content for either concentration.
'Hea t inactivated $9' (100°C × 10 min) and bovine serum albumin added in amounts comparable to the protein of the active $9 fraction did not influence the mutagenic effect of ECH (Table 2).
The action of purified microsomes was similar to that of the $9 m i x when comparisons were made on the basis of a calculation of the tissue-weight equivalents. The cytosolic fraction alone was virtually ineffective, even when the amount of preparation added was twice the amount of fresh liver used for the highest level of $9 mix (Table 3).
In our subcellular fractions, when GSH-transferase activity was assayed, the endogenous GSH was completely oxidized, so that the reaction could start only after the addition of reduced GSH. Addition to the incubation mixture of reduced GSH alone or with the cytosolic fraction did not affect ECH's mutagenic action. Moreover the addition of 5 mM GSH with $9 mix did not modify the detoxifying power of this fraction. Only at 10 mM GSH concentration was a complete inhibition of mutagenic activity observed (Table 4).
Table 5 shows the epoxide hydrolase specific activity towards ECH and styrene- 7,8-oxide in the presence of mouse-liver cytosolic or microsomal fractions. Strangely enough, the cytosolic activity of both control and phenobarbital-pretreated mfice were completely devoid of ECH-hydrating capacity. On the other hand, the micro- somal enzyme was active on this substrate and was 1.2 times induced by pheno- barbital pretreatment. The presence of the soluble epoxide hydrolase, activity was checked in the same cytosolic preparations which appeared to be active on the second substrate used as a marker.
50
Discussion
The aim of this study was to investigate the role of all the known active and passive effectors that could modulate ECH mutagenicity in vitro. The possible detoxifying pathways of ECH should include: spontaneous hydration and/or bind- ing to macromolecular cellular constituents, enzymatic hydration by cytosolic and microsomal epoxide hydrolase (Gill and Hammock, 1979; Oesch, 1973; Oesch et al., 1971, 1973; Ota and Hammock, 1980) and spontaneous and/or glutathione-S- epoxide transferase-mediated conjugation with GSH (Fjellstedt et al., 1973; Fried- berg et al., 1979; Habig et al., 1974; Hayakawa et al., 1975).
From the present study we can draw the following conclusions. (a) The detoxica- tion mechanism is NADPH-independent, suggesting that no oxidative reaction dependent on cytochrome P-450 is involved. (b) Because heat-inactivated $9 and albumin did not modify ECH mutagenicity, it can be reasonably excluded that the observed deactivation was due to the scavenger role of protein rather than to specific enzymatic action. In these conditions, protein nucleophilic sites are available to drain away the mutagenic potential of an electrophilic compound, but do not have any catalytic property. (c) The microsomal fraction has the greatest detoxifying ability, attributable to particulate epoxide hydrolase(s). The lack of epoxide hydro- lase activity towards ECH in the cytosolic fraction is probably related to a difference in the substrate specificity of microsomal and cytosolic epoxide hydrolases. In fact, if styrene oxide is used as substrate in the same preparation the cytosolic fraction is much more powerful than microsomes in forming the diol derivative.
Microsomal epoxide hydrolase(s) was sensitive to PB pretreatment, and the observed increase of its activity seemed important in the effectiveness of the protective action of microsomes and $9 mix. This is in good agreement with the known enzyme induction after pretreatment of animals with PB (Bresnick et al., 1977; Oesch et al., 1973; Salmona et al., 1976), which has become a standard practice in mutagenicity assays in vitro using mammalian metabolizing extracts.
(d) The lack of any important detoxifying effect of cytosolic fraction with added GSH suggested that the enzymatic conjugation of ECH with GSH has only an ancillary role if any. Spontaneous conjugation did not occur to an extent that could modify its biological activity. This ancillary role of GSH may be due to the relatively high concentrations of ECH used and thus we cannot exclude that, at lower substrate concentrations, GSH may play a more relevant protective role.
Acknowledgements
This study has been supported by the Commission of the European Community Research, Contract ENV/1/267-80, by the National Research Council of Italy (Environmental Mutagenesis Program; Control of Cancer Growth Program) and by the International Atomic Energy Agency, Vienna (Contract 2005/R3/RB).
Part of this work has represented the theses of Drs. T. Aglietti and A. Graziani. The Authors express to them their thankful appreciation for their interest and help during the study.
51
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