CCL4 INDUCED GENOTOXICITY AND PROTECTIVE EFFECT OF ANTIOXIDANTS AFTER IN VIVO ADMINISTRATION TO SHEEP

Dianovsk ̆ J . , K. ·iviková: CCl4 Induced Genotoxicity and Protective Effect of Antioxidants After in vivo Administration to Sheep. Acta Vet. Brno 2001, 70: 467-472. Protective effect against carbon tetrachloride-induced genotoxicity was tested in sheep after antioxidative supplementation. Ten 3-4-year-old sheep were treated by carbon tetrachloride, orally. The total dose of 0.05 ml/kg b/w. was corresponding to the former recommended antihelminthic usage in the seventies. Five such sheep were pre-treated by injection of vitamin E (400 mg) and selenium (12 mg) subcutaneously 24 h prior to CCl4 administration. Sister chromatid exchanges (as a DNA repair error detection technique) were used for genotoxic influences estimation. The technique was applied 16 days after CCl4 administration in cultured peripheral lymphocytes. SCEs were significantly increased in both tested groups, i.e. with and without of antioxidative pre-treatment. However, the first group exhibited the significance of p < 0.05, contrary to the second untreated group in which the statistical significance reached p < 0.001 when using Student’s t-test. This difference supports the protective genotoxic influence of the studied antioxidants in peripheral lymphocytes. In neither of the groups, a significant decrease in the proliferation index was recorded. Thus no cytotoxic effect on the investigated tissue has been proved. As our study showed, administration of a single concurrent treatment of vitamin E and selenium could reduce the genotoxic effect exhibited by the SCE frequencies in comparison to the non-protected individuals, remarkable. Carbon tetrachloride, genotoxicity, SCE, vitamin E, selenium, protective effect In ruminants, carbon tetrachloride CCl4 has been widely used as an antihelmintic drug for a long time. Intoxication occurred in animals as a result of misuse of this substance by application of an overdose. Carbon tetrachloride as well as the other aliphatic hydrochlorides is actually used as a very effective organic solvent in various industrial branches. Its distribution and properties may lead to subsequent environmental contamination including the soil, the surface and surrounding waters (Rabergh and Lipsky 1996). Efficacy of the compound including various toxic influences, namely hepatotoxicity for the patients treated, has been demonstrated in different mammalian species, i.e. humans, guinea pigs and rabbits, such as skin irritation reactions (Wahlberg 1984), dose-related maternal toxicity and litter resorption in rodents (Narotsky and Kavlock 1995), immunosuppression due to a decrease of T lymphocyte populations in rodents or in sheep (Vajdovich et al. 1995). With respect to cytotoxic, genotoxic or mutagenic activity of chlorinated aliphatic hydrocarbons the data showed that the DNA reactivity such of compounds increased with increasing degree of halogenisation (Tafazol i and Kirsch-Volders 1996). The carcinogenic potential of carbon tetrachloride has been early shown by Weisburger (1977) in mice in which liver and adrenal tumors were induced, and in rats in which disseminated neoplastic nodules were observed after oral exposure. The positive clastogenic effect has been demonstrated by in vitro conditions in metabolic competent.MCL-5 and h2E1 human cell lines (Doherty et al. 1996) and in workers ACTA VET. BRNO 2001, 70: 467–472 Address for correspondence: Doc. MVDr. Ján Dianovsk ̆, PhD. Department of Veterinary Genetics, University of Veterinary Medicine, Komenského 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 95 63 21 706 Fax: +421 95 63 236 666 E-mail: dianov@ uvm.sk1 http://www.vfu.cz/acta-vet/actavet.htm

In ruminants, carbon tetrachloride CCl 4 has been widely used as an antihelmintic drug for a long time.Intoxication occurred in animals as a result of misuse of this substance by application of an overdose.Carbon tetrachloride as well as the other aliphatic hydrochlorides is actually used as a very effective organic solvent in various industrial branches.Its distribution and properties may lead to subsequent environmental contamination including the soil, the surface and surrounding waters (Rabergh and Lipsky 1996).Efficacy of the compound including various toxic influences, namely hepatotoxicity for the patients treated, has been demonstrated in different mammalian species, i.e. humans, guinea pigs and rabbits, such as skin irritation reactions (Wahlberg 1984), dose-related maternal toxicity and litter resorption in rodents (Narotsky and Kavlock 1995), immunosuppression due to a decrease of T lymphocyte populations in rodents or in sheep (Vajdovich et al. 1995).
With respect to cytotoxic, genotoxic or mutagenic activity of chlorinated aliphatic hydrocarbons the data showed that the DNA reactivity such of compounds increased with increasing degree of halogenisation (Tafazoli and Kirsch-Volders 1996).The carcinogenic potential of carbon tetrachloride has been early shown by W eisburger (1977) in mice in which liver and adrenal tumors were induced, and in rats in which disseminated neoplastic nodules were observed after oral exposure.
The positive clastogenic effect has been demonstrated by in vitro conditions in metabolic competent.MCL-5 and h2E1 human cell lines (Doherty et al. 1996) and in workers occupationally exposed to mixture of chlorinated solvents or carbon tetrachloride in comparison to the group that was not exposed (da Silva et al. 1997).
Because much of cellular or sub-cellular damage may originate due to lipid peroxidationinduced free radicals the particular effective mechanisms based in order to minimalize these nucleinic acid damaging reactions (Bell and Mehendale 1987).These changes could be moderated by the cytochrome P 450 inhibitors and antioxidants.Vitamin E as well as selenium influence is one of the most important free radical decreasing factors and corresponding health protective effects.Pre-treatment of experimental animals with vitamin E was shown to be effective against CCl 4 induced acute liver necrosis (Biasi et al. 1991;Parola et al. 1992).
In this paper we report the influence of antioxidants (vitamin E and selenium) on carbon tetrachloride induced sister chromatid exchanges ( SCE ) in sheep peripheral lymphocytes in vivo.

Materials and Methods
Healthy, 3-4-year-old non-pregnant Merino ewes were treated with non-lethal dose of 0.05ml/kg b.w. of carbon tetrachloride (99.8 % Microchem Bratislava, Slovak Republic) in 1:1 v/v dilution with paraffin oil via stomach tube into the rumen.However, one group of five sheep 24 was injected s.c. with E 400 mg s.c. as tocopherylacetatate and selenium 12 mg as a sodium selenite 24 hours prior to CCl 4 administration (vitamin E + selenium, Bremer Pharma GmbH , Bremerhaven, Germany).The serum concentrations of vitamin E and selenium ranged levels 2.82 ± 0.56 (spectrophotometric assay) and 0.173 ± 0.022 (determined by AAS model 410, respectively) prior to their experimental supplementation; thus the animals were not suffering from their latent insufficiency.
The experiments were aimed to detect clinically the protective effect of the antioxidants mentioned against the biochemical changes and health disturbances induced by CCl 4 .Both experimental groups were housed at school farm at the standard conditions.Before and during the experiment, the same diet consisted of meadow hay ad libitum and BAK (concentrate for sheep) and fresh water was available ad libitum.

Cultivation of cells
Blood samples were obtained by jugular venipuncture into heparinized test tubes before, and on the 16 th day following the administration of vitamin E and selenium to the second experimental group.
Bromodeoxyuridine (BrdUrd, Sigma) was added to all cultures at a final dose of 8 µg/ml 48h before the harvest.To achieve cytostatic block, colchicine (Merck, Darmstadt, Germany) was added at a concentration of 5 µg/ml 2h before the harvest.

Evaluation and statistical methods
Chromosome preparations were obtained by the standard method.Slides were stained with FPG technique to differentiate sister chromatids and cell cycles (P e rry and W olff 1974) for the SCE delumination and cell cycle kinetics.Fifty differentially stained metaphases per donor or total 250 metaphases per concentration were examined for the SCE.The proliferation indices (PI) were calculated according to Lamberti et al. (1983), thus frequencies of mitotic divisions (M 1 , M 2 and M 3+ ).
Statistical analysis of results was performed using a simple analysis of variance (ANOVA).Then Student's t-test was applied to evaluate the SCEs significance for the both of the tested groups before and after the CCl 4 exposure.The same statistical procedure has been used with respect to the distribution of SCE among donors to evaluate the significant differences between treated with vitamin E and selenium, and untreated group.

Results
Results both of experiments focused on the CCl 4 in vivo genotoxicity in sheep are summarised in Table 1.Prior to statistic verification the ANOVA test was applied to detect the clastogenic effect of the compound tested.In all sheep treated with CCl 4 those missing the protective agent administration, the remarkable increasing of induced SCE was detected; p < 0.01 using the Student's t-test, for one individual minimally.The more significant performance of p < 0.001 has been obtained for a whole group of animals to compare to control level prior to CCl 4 administration.
Contrary to this, none of the animals belonging to the second experimental group (i.e., pre-treated with vitamin E and selenium), exhibited a significant increase of SCE.The same criterion calculated for the experimental group, however was significant (p < 0.05).Kinetics both of experiments with respect to the individual response to CCl 4 administration is seen in Table 2.
According to Student's t-test, the protective effect for vitamin E and selenium against CCl 4 induced clastogenicity has been verified at the significance level of p < 0.01.
A slight, non-significant decrease in proliferation indices was also observed (Table 1).In both experiments, the proportions of M 1 , M 2 and M 3 were not significantly different from those seen in controls.The difference in induced mitotic delay between the basic and postexperimental levels reflected by PI was not thus verified.

Discussion
In mammals, CCl 4 has been widely used as a model chemical in the study of acute lever injury and it is known to cause fat accumulation and necrosis in centrilobullar hepatocytes (Clawson 1989).In vivo studies with CCl 4 have indicated a species specific difference in the toxicity of this compound with mice being more sensitive than rats, and birds being least sensitive (Diaz Gomez et al. 1975).
With respect to its molecular characteristics CCl 4 does induce an increase of unsaturated fatty acid, lipoperoxide and free peroxide radical concentrations with the corresponding health disturbances.In our experiment, one hour after CCl 4 exposure several sheep became anorectic with muscular fasciculation, convulsions and with increased respiratory rate.Within three hours, however, all sheep recovered clinically (Milad et al. 2000).
Genotoxic activities of CCl 4 have been reported in various assays.As for clastogenic effect of CCl 4 the micronucleus assay in vivo was widely employed.No increases in micronuclei was obtained in mouse bone marrow and mouse peripheral lymphocytes in comparison to the control after administration a single intraperitoneal injection of carbon tetrachloride (Suzuki et al. 1997;Crebeli et al. 1999).However, this substance was confirmed to cause cancer in rodents after prolonged exposure to CCl 4 (Westbrook-Collins et al. 1990).Positive clastogenic or aneugenic results after treatment with of CCl 4 were found in human metabolically competent cell lines; MCL-5 CYP which expresses cDNA encoding the human CYP1A1, and h2E1 cell lines containing cDNA for CYP2E1 (Doherty et al. 1996).Carbon tetrachloride in combination with chlordecone was genotoxic using the in vivo -in vitro animal model and a battery of biochemical assays to measure the DNA repair in the rat hepatocytes (Ikegwuonu and Mehendale 1991).
Because of long-lived tissue contacting different organs and organic systems, and that they have a potential to accumulate DNA lesions (Carrano and Natarajan 1988) the peripheral lymphocytes are considered to be suitable for in vivo assays (Rojas et al. 1992).
SCEs are very sensitive, although non-specific markers (Tucker and Preston 1996) efficiently induced by chemical substances that form covalent adducts to the DNA or otherwise interfere with DNA metabolism repair (Morimoto et al. 1985).
Results obtained by us showed a significant increase of SCE (p < 0.05; p < 0.001 ) in sheep peripheral lymphocytes after CCl 4 treatment in both assays i.e. with and without protective agent administration.However, comparison between groups of animals treated, hint at the effective influence of vitamin E and selenium on the level of SCE induced (p < 0.01).Tafazoli et al. (1998) pointed out in their report that mutagenicity of CCl 4 occurred in absence of significant cytotoxicity.
As our study showed, the administration of a single dose of 0.05ml/kg b.w. to sheep in vivo failed to exert of detectable cytotoxic effect in peripheral lymphocytes.Contrary to the positive influence of CCl 4 on SCE frequencies in the present study, neither of experimental groups did exhibit a significant decrease of proliferation indices.
Antioxidants such as vitamin E and selenium have a fundamental role in the prevention of oxidative damage.Vitamin E is present in the membrane components of the cell and prevents free radicals formation after CCl 4 influence (Fariss et al. 1993), while selenium functions throughout the cytoplasm to destroy peroxides mainly due to its involvement to the glutathione peroxidase activity (Ianas et al. 1995).
Under conditions of the present study, the simultaneous treatment of vitamin E and selenium with CCl 4 was confirmed.They could reduce the SCE frequencies in comparison to the non-protected individuals remarkably.