Mutagenicity of hexachlorobutadiene, perchlorobutenoic acid and perchlorobutenoic acid chloride

Mutation Research, 137 (1984) 89-93

89

Elsevier MTR 00900

Mutagenicity of hexachlorobutadiene, perchlorobutenoic acid and perchlorobutenoic acid chloride D. Reichert *, T. Neudecker and S. Schhtz Institute of Pharmacology and Toxicology, University of W~rzburg, Versbacher Strasse 9, D- 8700 W'ftrzburg(F.R. G.)

(Received 1 December 1983) (Revision received 5 April 1984) (Accepted 10 April 1984)

Summary Hexachloro(1,3)butadiene (HCBD) is a well known environmental contaminant, The nephrocarcinogenic potential of H C B D has been shown in long-term studies with rats. Experiments were performed to assist in determining whether this effect is mediated by epigenetic or genotoxic mechanisms and to compare the mutagenic properties of H C B D with those of its monooxidation products, perchloro-3-butenoic acid (PCBA) and perchloro-3-butenoic acid chloride (PCBAC), which are conceivable metabolites of HCBD. All 3 compounds are mutagenic to the Salmonella typhimurium tester strain TA100. The mutagenic effect is dose-dependent and parallels the chemical reactivity of the compounds. H C B D is only mutagenic in the presence of drug-metabolizing enzymes ($9 mix) with an increased protein content. The mutagenic response after incubation with PCBAC and PCBA is 2-3-fold that of HCBD. Additionally, both PCBAC and PCBA exert a mutagenic response in the absence of $9 mix. The experiments support the assumption of a genotoxic potential of HCBD.

Chlorinated aliphatic hydrocarbons such as trichloroethylene and tetrachloroethylene are produced in large quantities. The production of these compounds is accompanied by the formation of hexachlorobutadiene (HCBD). This chemically unusually stable compound is released into the environment and has been detected in the tissues of plants, animals and man (Yip, 1976; Laska et al., 1976). Long-term ingestion of 20 m g / k g / d a y H C B D by rats for up to 2 years caused tubular adenomas and carcinomas of the kidney (Kociba et al., 1977). For human risk evaluation, a question of major interest is whether the carcinogenic effect of H C B D is mediated by genotoxicity or cyto* Mailing address: Priv.-Doz. Dr. Dieter Reichert, Institut fhr Pharmakologie und Toxikologie der Universit~it Wiirzburg, Versbacher Strasse 9, D-8700 Wi~rzburg, F.R.G. 0165-1218/$03.00 © 1984 Elsevier Science Publishers B.V.

toxicity. In fact, the kidney is the target organ of acute toxicity of HCBD. The cytotoxic effect of H C B D , i.e. tubular cell necrosis, could stimulate D N A replication and thereby cell division. The elevated rate of replication increases the possibility of the introduction of coding errors and therefore also increases the spontaneous mutation rate which possibly leads to the carcinogenic effect (epigenetic mechanism). In the series of structurally closdy related chlorinated aliphatic hydrocarbons, an epigenetic mechanism is discussed for the case of tetrachloroethylene, a compound which induces liver carcinomas in mice without being mutagenic in short-term test systems (Reichert, 1983a). The mutagenicity of H C B D has been tested with Salmonella typhimurium tester strains (Simmon, 1977; Reichert et al., 1983). The results of these studies do not concur and therefore cannot suffi-

90 ciently verify a genotoxic mechanism as a prerequisite for the nephrocarcinogenicity of HCBD. To answer this question finally, we carried out detailed experiments with Salmonella typhimurium tester strains using highly purified HCBD. In addition we generated conceivable metabolites of H C B D and compared their mutagenic potentials in order to provide insight into the biological activation mechanism and reactivity of HCBD. Materials and methods

Chemicals H C B D was purchased from Merck, Darmstadt (F.R.G.). The product was of analytical grade (98% purity) and was subjected for this study to the following purification procedure: a mixture (1 : 1, v : v) of H C B D and a 14% solution of sodium hydrogen sulfide in water was vigorously stirred at approximately 100 °C for about 4 h. The organic layer was separated, washed and treated for a further 2 h with a 10% solution of sodium hydroxide in water at about 100°C. The organic layer was washed and dried over potassium hydroxide. The liquid was fractionated twice using a Vigreux column. The purity of the distilled H C B D exceeded 99.5% as shown by gas chromatography. Perchloro-3-butenoic acid chloride (PCBAC) and perchloro-3-butenoic acid (PCBA) were prepared according to the method of Roedig and Bernemann (1956) from pentachloro-l-ethoxybutadiene, which was formed by refluxing H C B D with alcoholic potassium hydroxide. Further oxidation with chlorine gas led to the production of the acid chloride. Purification was achieved by distillation on a Vigreux column. PCBAC is a colorless liquid with a distasteful odor. The only detectable impurity after purification procedures was trace amounts of HCBD. PCBA was prepared from PCBAC and a solution of sodium hydrogencarbonate at 50 ° C, with stirring for 2 h. After acidification of the solution, an oily product was formed which was extracted with ether. The etherial solution was dried with sodium sulfate; PCBA crystallized after evaporation. Its purity was ascertained to be 99%. The physical and chemical properties of both compounds were in agreement with the literature. Furthermore the identities of PCBAC and the methyl

ester of PCBA were confirmed by gas chromatogr a p h y - m a s s spectrometry.

Mutagenicity testing The Salmonella typhimurium tester strain TA100 was kindly provided by Dr. B.N. Ames, Berkeley, CA, U.S.A. The sensitivity of this strain was routinely checked for each experiment by using NaN 3 and 2-aminoanthracene as positive controls in the absence or in the presence of $9 mix, respectively. $9 (protein content 38 m g / m l ) from Aroclor-1254-induced male Wistar rats and $9 mix were prepared according to Ames et al. (1975). The mutagenicity-testing system applied in this survey was a preincubation method similar to that described by Yahagi et al., (1975). The bacteria were grown overnight (16 h) in Oxoid medium. The test substances were diluted or dissolved in dimethyl sulfoxide (DMSO); 0.1 ml of the bacterial cell suspension plus 10 /L1 DMSO containing the test substances in the given amounts were added to 0.5 ml each of either phosphate buffer (0.1 M, p H 7.4) or $9 mix (protein concentration 3.8 m g / m l ) in small (7 cm x 1 cm) plastic vials which were then closed with air-tight caps and put into a shaker water-bath at 37 °C. After 120 rain, 2 ml of molten (45 °C) top agar were added to each vial and the mixture was poured onto petri plates containing Vogel-Bonner medium E (minimal agar) (Vogel and Bonner, 1956). Additionally, H C B D was tested with fortified incubation conditions: in this case $9 mix with a concentration of 11.4 mg p r o t e i n / m l was added to the test system. Revertant colonies were counted after 2 days of incubation at 37 o C. All determinations were made in duplicate in each experiment. Every experiment was performed at least twice. Differences in colony counts of analogous plates were always less than 30%. Results

H C B D was tested for mutagenic potential in

Salmonella typhimurium tester strain TA100. After metabolic activation, H C B D elicits a dose-dependent mutagenic effect at concentrations of 0.1-1.0 /~l/plate (Fig. 1). The mutagenic effect was achieved only by adding $9 mix with an increased protein content. Comparison of the chemical reac-

91

I

I

I

.~

o O.

,2_°,

Z

500 -

I

\

,

,

, /

/

,,i

200 ~ *m a~

I

~

\ g

200-

- -- ..O001

0.01

\ O,I

i

10

jut RESI~MGPER PLATE Fig. 1. Mutagenicityof perchloro-3-butenoicacidchloride(a), perchloro-3-butenoic acid (b) and hexachlorobutadiene (c) with (O) and without (O) $9 mix (3.8 mg protein/ml mix). Dosages of substances a and c are expressed i n / d / p l a t e , substance b is given in rag/plate. Substance c was also tested in the presence of $9 mix with an increased protein concentration of 11.4 m g / m l mix (I)

tivities and mutagenicities of the monooxidation products of HCBD, which are conceivable metabolities of HCBD, provides additional indication of a metabolic activation mechanism. Both PCBAC and PCBA are mutagenic in the absence of $9 mix (direct mutagens) and exert a mutagenic effect significantly higher than that of HCBD in the presence of $9 mix. Moreover, their direct mutagenicity is exerted at comparatively low doses which are 50-100 times lower than those necessary for the exertion of indirect mutagenicity. Thus both PCBAC and PCBA appear to be relatively strong direct mutagens which are considerably more cytotoxic in the absence than in the presence of $9 mix (Fig. 1). Discussion

The results dearly demonstrate a mutagenic effect of HCBD and its chemical oxidation products PCBAC and PCBA. The conversion of HCBD to these two oxidation products is readily explained by the assumption of a short-lived intermediate monoepoxide, which could spontaneously

rearrange to the corresponding acid chloride. The second step would be the hydrolyzation to the free PCBA (Fig. 2). This pathway has been demonstrated previously in chemical oxidation reactions of HCBD. It is suggested that HCBD undergoes a similar oxidation reaction in vivo, which could lead to the opening of one double bond. We know from our previous work (Reichert, 1983b) and from that of other groups (Wolf et al., 1983) that HCBD undergoes metabolic oxidation (and conjugation) reactions in the mammalian organism. The results of this mutagenicity study aid in the interpretation of the findings of Stott et al. (1981), who demonstrated renal DNA repair and alkylation in rats after HCBD administration, but no mutagenic effect in the S. typhimurium test system. Our experiments show that the mutagenicity testing of HCBD requires particular experimental conditions, due to the compound's high chemical stability. Only after modification of the bacterial test system by the addition of 3 times the normal concentration of drug-metabolizing enzymes to the incubation medium does the mutagenic property of HCBD become evident. The resistance of the HCBD molecule toward attack by drug-metabolizing enzymes is explained by its particular chemical

el

CI

CI

CI

Cl

HCBD

1°:,

Ct

Cl C|

)

• I

HCBD- monoepo×ide

Cl cl

Cll /

\

Cl Cl Pentachloro- 3- butenoic acid chloride Fig. 2. Scheme of the chemical and biological oxidation reaction of HCBD.

92

structure (Kogan, 1964) and its extremely low solubility in water. The presence of the 6 chlorine atoms determines the behavior of the diene: on the one hand, the chlorine atoms strongly attract the electrons of the symmetrical molecule thereby contributing to an electron loss around the C=C bonds, while on the other hand, the chlorine atoms sterically protect the double bonds against an electrophilic attack. If the stability of HCBD is altered by a chemical oxidation reaction resulting in the opening of one double bond, the resulting compound (or compounds) is chemically more reactive because of its lack of structural symmetry and loss of steric hindrance. The higher chemical reactivity is consistent with the higher biological reactivities shown in this mutagenicity study with PCBAC and PCBA. Both compounds exert mutagenic effects in the absence of a metabolic activation system and are considerably more cytotoxic for the test bacteria in the absence than in the presence of $9 mix. It is conceivable that two factors, i.e. reactions with nonenzymic constituents of the $9 mix and metabolic transformations to less toxic metabolites, may contribute to the toxicity-reducing effect of $9 mix. The conflicting results in mutagenicity testing of HCBD can be explained by the presence of contaminants (e.g. polychlorobutanes, polychlorobutenes, polychlorobutadienes, polychloroethanes and perchlorocyclopentadiene) in technical and even analytical grade HCBD. Such impurities are very difficult to remove because their boiling points are close to that of HCBD, and they form azeotropic mixtures. Some of these impurities are highly toxic to the bacterial tester strains; their extreme cytotoxicity could obviously prevent detection of a mutagenic effect of HCBD. Even analytical grade HCBD was totally bacteriocidal in our test system at doses as low as 0.003 #l/plate, proving that it is about 1000 times more toxic than its purified form (see Fig. 1). Furthermore the impurities render HCBD unstable on storage and also lead to further degradation products. The described purification of HCBD is based on the compound's stability in alkaline aqueous solutions. The results of this mutagenicity study support the assumption that HCBD interacts with bacterial DNA. The mutagenic effect is significantly enhanced when one double bond of the HCBD

molecule is opened by an oxidation reaction. Further studies in progress (Schiffmann et al., 1983) utilizing other short-term test systems show results which are consistent with these findings. The conclusion can be drawn that HCBD exerts genotoxic effects after metabolic activation. This fact is important for the evaluation of this compound's carcinogenic potential, as it is generally accepted that a minimum risk threshold for genotoxic substances cannot be assigned.

Acknowledgement This study was supported by the Deutsche Forschungsgemeinschaft (Grant No. Re 561/1-1).

References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Kociba, R.J., D.C. Keyes, G.C. Jersey, J.J. Ballard, D.A. Dittenber, J.F. Quast, C.E. Wade, C.G. Humiston and B.A. Schwetz (1977) Results of a two-year chronic toxicity study with hexachlorobutadiene (HCBD) in rats, Am. Ind. Hyg. Assoc. J., 38, 589-602. Kogan, L.M. (1964) Hexachlorobutadiene, Russ. Chem. Rev., 33, 176-188. Laska, A.L, C.K. Bartell and J.L. Laseter (1976) Distribution of hexachlorobenzene and hexachlorobutadiene in water, soil, and selected aquatic organisms along the lower Mississippi River, Louisiana, Bull. Environ. Contam. Toxicol., 15, 535-542. Reichert, D. (1983a) Biological actions and interactions of tetrachloroethylene, Mutation Res., 123, 411-429. Reichert, D. (1983b) Metabolism and disposition of hexachloro(1,3)butadiene, in: A.W. Hayes, R.C. Schnell and T.S. Miya (Eds.), Developments in the Science and Practice of Toxicology, pp. 411-414. Reichert, D., T. Neudecker, U. Spengler and D. Henschler (1983) Mutagenicity of dichloroacetylene and its degradation products trichloroacetyl chloride, trichloroacryloyl chloride and hexachlorobutadiene, Mutation Res., 117, 21-29. Roedig, A., and P. Bernemann (1956) Zur Kenntnis von hochchlorierten Vinyletheru, Liebigs Ann. Chem., 600, 1-11. Schiffmann, D., D. Reichert and D. Henschler (1983) Induction of morphological transformation and unscheduled DNA synthesis in Syrian hamster embryo fibroblasts by hexachlorobutadiene and pentachlorobutenoic acid, Cancer Lett., in press. Simmon, V.F. (1977) Structural correlations of carcinogenic and mutagenic alkyl halides, in: Proc. 2nd FDA Office of

93 Science Summer Symposium, U.S. Naval Academy, 31 August-2 September, pp. 163-171. Stott, W.T., J.F. Quast and P.G. Watanabe (1981) Differentiation of the mechanisms of oncogenicity of 1,4-dioxane and 1,3-hexachlorobutadiene in the rat, Toxicol. Appl. Pharmacol., 60, 287-300. Vogel, H.J., and D.M. Bonner (1956) Acetylornithinase of Escherichia coli: Partial purification and some properties, J. Biol. Chem., 218, 97-106. Wolf, C.R., J.B. Hook and E.A. Lock (1983) Differential destruction of cytochrome P-450-dependent mono-

oxygenases in rat and mouse kidney following hexachloro1 : 3-butadiene administration, Mol. Pharmacol., 23, 206-212. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96. Yip, G. (1976) Survey for hexachloro-l,3-butadiene in fish, eggs, milk and vegetables, Assoc. Offic. Anal. Chem., 59, 559-561.