Protective effect of butylated hydroxytoluene (BHT) against the clastogenic acitivity of cadmium chloride and potassium dichromate in hamster ovary cells

Abstracts

The effect of butylated hydroxytoluene (BHT), a widely used food additive, on chromosomal alterations induced by cadmium chloride (CC) and potassium dichromate (PD) in Chinese hamster ovary (CHO) cells was studied both at metaphase and anaphase-telophase. CHO cells were cultured for 15-16 h in the presence of PD (6.0, 9.0 or 12.0 <FONT FACE="Symbol">m</font>M), BHT (1.0 <FONT FACE="Symbol">m</font>g/ml), or PD plus BHT as well as CC (0.5, 1.0 and 2.0 <FONT FACE="Symbol">m</font>M), BHT or CC plus BHT for the analysis of chromosomal aberrations. To perform the anaphase-telophase test, cells were cultured in cover glasses and treated 8 h before fixation with the same chemicals. An extra dose of CC (4 <FONT FACE="Symbol">m</font>M) was used in this test. Both metal salts significantly increased chromosomal aberration frequencies in relation to untreated controls, and to DMSO- and BHT-treated cells. Post-treatment with BHT decreased the yield of chromosomal damage in relation to treatments performed with CC and PD. However, chromosomal aberration frequencies were significantly higher than those of the controls. In the anaphase-telophase test, CC significantly increased the yield of lagging chromosomes with the four doses employed and the frequency of lagging fragments with the highest dose. In combined treatments of CC and BHT, frequencies of the two types of alterations decreased significantly in relation to the cells treated with CC alone. No significant variation was found in the frequencies of chromatin bridges. Significant increases of numbers of chromatin bridges, lagging chromosomes and lagging fragments were found in cells treated with PD. The protective effect of BHT in combined treatments was evidenced by the significant decrease of chromatid bridges and lagging chromosomes in relation to PD-treated cells. Whereas BHT is able to induce chromosomal damage, it can also protect against oxidative damage induced by other genotoxicants.

Estudou-se o efeito de hidroxitolueno butilado (BHT) sobre as alterações cromossômicas induzidas por cloreto de cádmio (CC) e dicromato de potássio (PD), tanto na metáfase como em anáfase-telófase de células do ovário de hamster chinês (CHO). Para a análise de aberrações cromossômicas, células CHO foram cultivadas por 15-16 h na presença de CC (6, 9 ou 12 <FONT FACE="Symbol">m</font>M), BHT (1 <FONT FACE="Symbol">m</font>g/ml) ou CC mais BHT, assim como PD (0,5, 1,0 e 2,0 <FONT FACE="Symbol">m</font>M), BHT ou PD mais BHT. Para o teste em anáfase-telófase, células foram cultivadas em lamínulas de vidro e tratadas 8 h antes da fixação com as mesmas substâncias. Uma dose extra de CC (4 <FONT FACE="Symbol">m</font>M) foi usada neste teste. Os resultados mostraram que ambos os sais metálicos aumentaram significativamente a freqüência de aberrações cromossômicas em relação a controles não tratados ou células tratadas com DMSO e BHT. O pós-tratamento com BHT diminuiu a ocorrência de danos cromossômicos em relação aos tratamentos com CC e PD. Contudo, a freqüência de aberrações cromossômicas foi significativamente maior do que a dos controles. No teste em anáfase-telófase, CC aumentou significativamente a ocorrência de cromossomos "lagging" com as 4 doses empregadas e a freqüência de fragmentos "lagging" com a dose maior. Em tratamentos combinados de CC e BHT, as freqüências dos dois tipos de alterações diminuíram em relação às células tratadas com CC. Não se encontrou variação significativa na freqüência de pontes de cromatina. Em células tratadas com PD, encontraram-se aumentos significativos nas pontes de cromatina, cromossomos "lagging" e fragmentos "lagging". O efeito protetor do BHT em tratamentos combinados foi evidenciado pela diminuição significativa das pontes de cromatina e dos cromossomos "lagging" em relação às células tratadas com PD. Esses resultados podem ser explicados assumindo-se que CC e PD induziram dano cromossômico por pelo menos dois mecanismos: 1) a ligação de cátions metálicos ao DNA e a subseqüente indução de efeitos conformacionais; 2) a geração de oxigênio reativo. O pós-tratamento com BHT diminuiu a ocorrência de dano cromosômico possivelmente ao afetar a geração de oxigênio reativo, mas o efeito clastogênico do próprio BHT deve ser levado em consideração. Conseqüentemente, a ocorrência de aberrações cromossômicas observadas após o pós-tratamento com BHT poderia resultar das lesões cromossômicas induzidas diretamente pela ligação de cada cátion ao DNA e também pelo dano induzido pelo BHT. Esta freqüência é menor que a causada pelos sais metálicos devido ao efeito protetor do BHT. Como BHT é amplamente usado como aditivo em alimentos e também em várias indústrias, seu duplo efeito é um ponto de interesse. Ao mesmo tempo que o composto pode induzir danos cromossômicos, ele pode proteger contra o dano oxidativo causado por outros produtos genotóxicos.

Protective effect of butylated hydroxytoluene (BHT) against the clastogenic acitivity of cadmium chloride and potassium dichromate in chinese hamster ovary cells

Claudia A. Grillo, Analía I. Seoane and Fernando N. Dulout

Centro de Investigaciones en Genética Básica y Aplicada (CIGEBA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, 60 y 118 - CC 296 - 1900 La Plata, Argentina. Send correspondence to F.N.D. E-mail: [e-mail]

ABSTRACT

The effect of butylated hydroxytoluene (BHT), a widely used food additive, on chromosomal alterations induced by cadmium chloride (CC) and potassium dichromate (PD) in Chinese hamster ovary (CHO) cells was studied both at metaphase and anaphase-telophase. CHO cells were cultured for 15-16 h in the presence of PD (6.0, 9.0 or 12.0 mM), BHT (1.0 mg/ml), or PD plus BHT as well as CC (0.5, 1.0 and 2.0 mM), BHT or CC plus BHT for the analysis of chromosomal aberrations. To perform the anaphase-telophase test, cells were cultured in cover glasses and treated 8 h before fixation with the same chemicals. An extra dose of CC (4 mM) was used in this test. Both metal salts significantly increased chromosomal aberration frequencies in relation to untreated controls, and to DMSO- and BHT-treated cells. Post-treatment with BHT decreased the yield of chromosomal damage in relation to treatments performed with CC and PD. However, chromosomal aberration frequencies were significantly higher than those of the controls. In the anaphase-telophase test, CC significantly increased the yield of lagging chromosomes with the four doses employed and the frequency of lagging fragments with the highest dose. In combined treatments of CC and BHT, frequencies of the two types of alterations decreased significantly in relation to the cells treated with CC alone. No significant variation was found in the frequencies of chromatin bridges. Significant increases of numbers of chromatin bridges, lagging chromosomes and lagging fragments were found in cells treated with PD. The protective effect of BHT in combined treatments was evidenced by the significant decrease of chromatid bridges and lagging chromosomes in relation to PD-treated cells. Whereas BHT is able to induce chromosomal damage, it can also protect against oxidative damage induced by other genotoxicants.

INTRODUCTION

Though it is well known that metal compounds can be carcinogenic in humans, the mechanisms of metal carcinogenesis are not well understood. Most metal ions and their derivatives are able to induce point mutations either in bacteria or mammalian cells as well as chromosomal aberrations in eukaryotic organisms. These genotoxic effects have often been related with carcinogenesis, assuming that neoplastic transformation is a consequence of DNA damage. Among genotoxic/carcinogenic ions, chromium and cadmium can directly and indirectly cause a variety of DNA lesions by means of oxygen radicals and reactive intermediates (Snow, 1994).

Whereas chromium is an essential trace element involved in glucose and lipid metabolism (Morris et al., 1988; Anderson, 1990), over-exposure produces a wide spectrum of effects. Epidemiological studies carried out in exposed workers demonstrated a high incidence of respiratory cancers (IARC, 1990). In experimental animals chromium compounds induced tumors (Langard, 1988), nephrotoxicity (Laborda et al., 1986; Appenroth and Braunlich, H., 1988) and hepatotoxicity (Laborda et al., 1986). Chromium compounds also showed mutagenic activity in bacteria (Nishioka, 1975), induced chromosomal aberrations, sister chromatid exchanges as well as mutations and transformation in cultured mammalian cells (Fradkin et al., 1975; Tsuda and Kato, 1977; Majone and Levis, 1979; Rainaldi, G., 1982; Sen and Costa, 1986; Sen et al., 1987; Patierno et al., 1988).

The most toxically active form of chromium is the hexavalent oxidation state which exists as an oxyanion at physiological pH (Levis and Bianchi, 1982; Heck and Costa, 1982). Oxyanions, such as chromate, are actively transported into cells by the sulfate transport system, resulting in high intracellular accumulation. Chromium (VI) reacts with a number of reducing agents in cells, including glutathione, hydrogen peroxide, microsomal enzymes and ribonucleotides. Chromium (VI) is eventually reduced to the kinetically inert and stable chromium (III). The reduction process itself, as well as the formation of intermediate states of chromium, is thought to influence their genotoxicity due to the induction of oxidative DNA damage (Snow, 1994).

The mutagenic activity of cadmium has been demonstrated in hypoxanthine guanine phosphoribosyl transferase and thymidine kinase assay systems in cultured mammalian cells (Amacher and Paillet, 1980; Oberly et al., 1982; Ochi and Ohsawa, 1983). Cadmium has also been shown to be clastogenic in human lymphocytes when given as cadmium sulfide (Shiraishi et al., 1972) or cadmium chloride (Cea et al., 1983). Ochi et al. (1984) found structural and numerical changes in cultured Chinese hamster V79 cells treated with cadmium chloride. Using the anaphase-telophase test, Seoane and Dulout (1994) detected the aneugenic effect of cadmium chloride by an increase of lagging chromosomes in CHO cells. Ochi et al. (1983) showed that cadmium chloride induced DNA single-strand scissions by means of active oxygen species.

The ability of different chemical compounds to reduce the mutagenic/clastogenic activity of other chemicals has been demonstrated. An important antimutagenic compound is butylated hydroxytoluene (BHT), a phenolic antioxidant able to modify the mutagenic effect of most standard mutagens in different in vitro and in vivo assays (Waters et al., 1990; Grillo and Dulout, 1997). As the formation of active oxygen species seems to be one of the most important mechanisms of DNA damage induction by chromium and cadmium, the protective effect of BHT on chromosomal damage using different cytogenetic tests has been studied. The present study summarizes the results obtained.

MATERIAL AND METHODS

Cells

Chinese hamster ovary (CHO) cells were originally obtained from the American Type Culture Collection (ATCC). Cells were grown in Ham F10 medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum, penicillin and streptomycin sulfate in a humidified atmosphere with 5% CO₂ at 37ºC. CHO cell cycle duration under these culture conditions varies between 12 and 15 h (Grillo and Dulout, 1995).

Chemicals

Cadmium chloride (CC) and potassium dichromate (PD) were obtained from Sigma. Both compounds were dissolved in distilled water. Doses of 0.5, 1.0, 2.0 mM of CC and 6.0, 9.0, 12 mM of PD were employed. These doses were selected after pilot experiments carried out with a wide range of dosages.

BHT (Sigma) was diluted in dimethyl sulfoxide (DMSO) (Sigma) before treatment. A dose of 1.0 µg/ml was selected based on previous studies (Grillo and Dulout, 1997).

Solutions were made and 100 µl was added to each culture in order to obtain the desired concentration.

Structural chromosome aberration test

CHO cells were cultured for 15-16 h in the presence of PD, BHT or PD plus BHT as well as CC, BHT or CC plus BHT. Two hours before harvesting, colchicine was added to the cultures (0.1 µg/ml final concentration). Air dried slides were prepared following routine protocols (Tjio and Levan, 1956). Each treatment was repeated five times and 500 metaphases were scored in coded slides per treatment (100 per repetition). Statistical analysis was performed using the c² test.

Anaphase-telophase test

CHO cells were cultured as a monolayer in 24 x 36-mm cover glasses attached with a small drop of siliconized grease to the bottom of 90-mm Petri dishes. Three cover glasses were placed in each Petri dish. Each cover glass was seeded with 1.5-ml culture medium containing about 50,000 cells. After 1 h, 8.5 ml of culture medium was added to each Petri dish. Cultures were incubated at 37ºC in a humidified atmosphere of 5% CO₂. The set of cultures for each experiment was treated simultaneously for 8 h before fixation to avoid the detachment of cells from cover slides. One extra dose of CC (4 mM) was used in this test because lower doses did not induce significant increments of chromosomal damage. Each treatment was repeated five times. Cell harvesting was accomplished by adding an equal volume of fixative (methanol-acetic 3:1) to the culture medium. After 10 min, two changes of fixative were made. Cover glasses were stained with carbolfuchsin (Carr and Walker, 1961) and attached with DPX mounting medium to coded slides. Statistical comparisons were made by means of Sokal and Rohlf G method (Sokal, 1979). Regression analyses were performed to evaluate mitotic index variations.

In all experiments, untreated cultures and DMSO-treated cultures (0.1 ml DMSO per 10 ml culture medium) were used as controls.

RESULTS

Structural chromosome aberration test

During cytogenetic analyses three types of aberrant metaphases were found: 1) with chromatid-type aberrations (chromatid and isochromatid breaks and chromatid exchanges), 2) with chromosome-type aberrations (fragments, dicentric and ring chromosomes) and 3) with both types of aberrations.

Treatment with BHT alone induced a slight but significant increase of chromatid-type aberrations (chromatid and isochromatid breaks) (P < 0.025; Table I). Treatments with CC significantly increased frequencies of chromatid- and chromosome-type aberrations (P < 0.001). In combined treatments with CC and BHT the frequencies of chromatid-type aberrations decreased significantly compared to those induced in treatments with CC alone (P < 0.001). Also the frequencies of chromosome-type aberrations induced by CC decreased after post-treatment with BHT (P < 0.001).

Table I
- Mean frequencies in percent (± standard error) of structural chromosome aberrations in CHO cells (N = 500) treated with cadmium chloride (cc) and butylated hydroxytoluene (BHT).

Treatment	Abnormal metaphases	Chromosomal aberrations
		AL	B’	B"	RB’	FRG	DIC
---	1.4	1.6 ± 0.12	1.0 ± 0.09	---	---	0.4 ± 0.06	---
DMSO	2.6	3.4 ± 0.17	2.2 ± 0.14	0.2 ± 0.04	---	---	---
BHT	4.8	1.6 ± 0.12	5.0 ± 0.22	0.2 ± 0.04	---	0.2 ± 0.04	---
0.5 mM CC1	10.4	1.6 ± 0.12	9.8 ± 0.29	0.6 ± 0.07	0.2 ± 0.04	1.2 ± 0.10	0.4 ± 0.06
1.0 mM CC2	13.8	1.2 ± 0.10	11.4 ± 0.32	3.6 ± 0.18	0.2 ± 0.04	0.6 ± 0.07	---
2.0mM CC3	16.4	1.2 ± 0.10	17.2 ± 0.38	3.2 ± 0.17	0.2 ± 0.04	1.2 ± 0.10	0.2 ± 0.04
CC1 + BHT	4.6	1.2 ± 0.10	4.4 ± 0.20	0.2 ± 0.04	---	0.2 ± 0.04	---
CC2 + BHT	7.0	1.8 ± 0.13	6.8 ± 0.25	0.4 ± 0.06	---	0.4 ± 0.06	---
CC3 + BHT	8.6	1.2 ± 0.10	9.6 ± 0.29	1.0 ± 0.09	0.4 ± 0.06	0.2 ± 0.04	---

Abnormal metaphases: metaphases with at least one chromosomal aberration. Metaphases exhibiting only achromatic lesions were not scored as abnormal. AL, Achromatic lesions. B', Chromatid breaks. B", Isochromatid breaks. RB', Chromatid exchanges. FRG, Chromosome fragments. DIC, Dicentric chromosomes.

Table II summarizes the results obtained after treatment of CHO with different doses of PD and BHT during one cell cycle. As in the previous experiment, BHT alone induced a significant increase of chromatid and isochromatid breaks (P < 0.001). Treatment with the three doses of PD significantly increased the frequency of chromatid-type aberrations in a dose-dependent way (P < 0.001). Chromosome-type aberrations (chromosome fragments, dicentrics) were also induced, though at a lower proportion, but the corresponding frequencies were not correlated with the doses employed. In combined treatments with PD and BHT the frequencies of chromatid-type aberrations decreased significantly (P < 0.001), but the frequencies of chromosome-type aberrations were not modified.

Table II
- Mean frequencies in percent (± standard error) of structural chromosome aberrations in CHO cells (N = 500) treated with potassium dichromate (PD) and butylated hydroxytoluene (BHT).

Treatment	Abnormal metaphases	Chromosomal aberrations
		AL	B’	B"	RB’	FRG	DIC
---	1.0	1.2 ± 0.10	0 1.2 ± 0.10	---	---	---	---
DMSO	2.0	1.4 ± 0.12	0 1.8 ± 0.13	0.2 ± 0.04	---	---	---
BHT	4.8	1.4 ± 0.12	0 4.8 ± 0.21	---	---	0.4 ± 0.06	---
6.0 mM PD1	17.4	1.0 ± 0.09	16.2 ± 0.36	3.8 ± 0.19	0.4 ± 0.06	---	0.2 ± 0.04
9.0 mM PD2	21.4	0.8 ± 0.09	21.8 ± 0.41	4.0 ± 0.19	---	0.6 ± 0.07	---
12.0 mM PD3	28.4	0.8 ± 0.09	28.8 ± 0.45	4.2 ± 0.20	2.6 ± 0.20	0.8 ± 0.09	0.6 ± 0.07
PD1 + BHT	10.6	1.2 ± 0.10	10.2 ± 0.30	1.0 ± 0.09	0.6 ± 0.07	0.2 ± 0.04	---
PD2 + BHT	12.4	1.4 ± 0.12	12.2 ± 0.33	1.8 ± 0.13	0.2 ± 0.04	---	0.2 ± 0.04
PD3 + BHT	14.8	1.0 ± 0.09	16.6 ± 0.37	2.2 ± 0.15	1.4 ± 0.12	0.4 ± 0.06	---

For abbreviations see legend to Table I

Cells exhibiting chromatid- and chromosome-type aberrations were found after treatments with CC, PD or CC plus BHT and PD plus BHT. However, frequencies of such cells were not significantly higher than controls.

Anaphase-telophase test

Three types of alterations were analyzed in cells at late anaphase-early telophase: 1) chromatin bridges, 2) lagging chromosomes and 3) lagging fragments.

Table III shows the results obtained when the CHO cells were treated with CC and BHT. Frequencies of anaphase-telophase alterations in DMSO- and BHT-treated cells did not differ from those of untreated cells. Treatments with CC increased significantly the yield of lagging chromosomes with the four doses employed (P < 0.025) and the frequency of lagging fragments with the highest dose (P < 0.01). In combined treatments with CC and BHT the frequencies of the two types of alterations decreased in comparison to the CC-treated cells (P < 0.05). No significant variation was found in the frequencies of chromatin bridges.

Table III
- Mean frequencies in percent (± standard error) of anaphase-telophase alterations in CHO cells (N = 750) treated with cadmium chloride (CC) and butylated hydroxytoluene (BHT).

Treatment	Anaphase-telophase alterations			Mitotic index (%)
	CB	LC	LF
---	0.40 ± 0.06	0.66 ± 0.08	0.26 ± 0.05	2.3
DMSO	0.66 ± 0.08	0.66 ± 0.08	0.13 ± 0.03	3.3
BHT	0.66 ± 0.08	0.66 ± 0.08	0.26 ± 0.05	2.6
0.5 mM CC1	0.53 ± 0.07	0.93 ± 0.09	0.13 ± 0.03	2.2
1.0 mM CC2	0.93 ± 0.09	0.93 ± 0.09	0.13 ± 0.03	2.0
2.0 mM CC3	0.93 ± 0.09	1.06 ± 0.10	0.13 ± 0.03	2.0
4.0 mM CC4	1.06 ± 0.10	2.13 ± 0.14	1.33 ± 0.11	1.9
CC1 + BHT	0.66 ± 0.08	0.40 ± 0.06	0.26 ± 0.05	2.8
CC2 + BHT	0.66 ± 0.08	0.66 ± 0.08	0.26 ± 0.05	3.2
CC3 + BHT	0.93 ± 0.09	0.66 ± 0.08	0.26 ± 0.05	2.2
CC4 + BHT	1.20 ± 0.10	1.06 ± 0.10	0.53 ± 0.07	3.0

CB,Chromatin bridges.LC, Lagging chromosomes.LF, Lagging fragments.

Table IV summarizes the results obtained using PD plus BHT. As in the previous experiment, frequencies of anaphase-telophase alterations were not increased by DMSO and BHT. In cells treated with PD, significant increase in the frequencies of chromatin bridges, lagging chromosomes and lagging fragments was found. However, compared with untreated controls significant differences for chromatin bridges and lagging chromosomes (P < 0.001) were found, as well as poorly significant differences in lagging fragments (P < 0.05). The protective effect of BHT in combined treatments was evidenced by the significant decrease of chromatid bridges (P < 0.001) and lagging chromosomes (P < 0.05) in relation to PD-treated cells.

Table IV
- Mean frequencies in percent (± standard error) of anaphase-telophase alterations in CHO cells (N = 750) treated with potassium dichromate (PD) and butylated hydroxytoluene (BHT).

Treatment	Anaphase-telophase alterations			Mitotic index (%)
	CB	LC	LF
—	0.13 ± 0.03	0.13 ± 0.03	0.13 ± 0.03	5.2
DMSO	0.13 ± 0.03	0.13 ± 0.05	0.26 ± 0.03	5.3
BHT	0.26 ± 0.51	0.26 ± 0.05	0.26 ± 0.05	6.1
6.0 mM PD1	2.13 ± 0.14	1.60 ± 0.12	0.80 ± 0.08	4.1
9.0 mM PD2	3.46 ± 0.18	1.86 ± 0.13	1.20 ± 0.10	3.1
12.0 mM PD3	2.80 ± 0.16	1.73 ± 0.13	0.66 ± 0.08	1.9
PD1 + BHT	1.20 ± 0.10	1.20 ± 0.10	1.20 ± 0.10	7.2
PD2 + BHT	1.20 ± 0.10	0.93 ± 0.09	0.93 ± 0.09	6.8
PD3 + BHT	2.13 ± 0.14	1.06 ± 0.10	0.53 ± 0.07	6.2

CB, Chromatin bridges.LC, Lagging chromosomes. LF, Lagging fragments.

The cytotoxic effect of both compounds was evidenced by the decrease of the mitotic index. However, the decrease induced by CC was not significant, although a tendency inversely proportional to the doses employed was observed (r = 0.75, P > 0.1). The decrease induced by PD was significant (P < 0.001) and inversely correlated with the doses employed (r = 0.95, P < 0.05).

DISCUSSION

The underlying mechanisms of metal carcinogenesis remain unclear. However, two hypotheses have been considered to explain the induction of DNA damage by metals: the binding of cations to DNA and other constituents of cell nuclei such as DNA polymerases and repair enzymes or the induction of oxidative damage (Kasprzak, 1995). The binding of metal cations to DNA through ionic and coordination bonds is reversible and, as such, can not produce all the lesions observed in the chromatin of cells exposed to carcinogenic metal compounds. These DNA lesions, such as strand scission, depurination, crosslinking, and base modifications, found in experimental animals as well as in a variety of mammalian cell lines (Sunderman, 1986; Kasprzak et al., 1995; Kasprzak, 1996), result from either breakage of existing covalent bonds or formation of new covalent bonds in DNA and proteins. Hence, not only the direct, mostly conformational effects of metal binding, but also some other, obviously indirect effects of metals on nuclear chromatin, must be considered. These effects can be produced by reactive oxygen species (Kasprzak, 1996; Oleinick et al., 1987; Snow, 1994).

Under the experimental conditions employed, results described above clearly show that CC and PD induced chromosomal damage by at least two mechanisms. Both compounds induced chromosomal aberrations and anaphase-telophase alterations in Chinese hamster ovary cells, and post-treatment with BHT decreased the yield of chromosomal damage. Nevertheless, the effect of the antioxidant was not enough to attain complete protection, since after post-treatment, the frequencies of the different alterations, either in metaphase or in anaphase-telophase, were still significantly higher than in the controls. However, the clastogenic effect of BHT itself must be taken into account. Consequently, the yield of chromosomal aberrations observed after post-treatment with BHT could arise from the chromosomal lesions induced directly by the binding of each cation to DNA plus the damage induced by BHT. This yield is lower than that induced by metal salts alone due to the protective effect of BHT.

The effects of BHT, a non-protein natural antioxidant, on chromium- and cadmium-induced damage can be explained assuming that BHT presumably affects the generation of chromium (V) or (III) during chromium (VI) reduction. In addition, chromium (VI), in the absence of chelators, could interact with H₂O₂ and superoxide (O₂^-) through Fenton/Haber-Weiss chemistry (Haber and Weiss, 1934). On the other hand, BHT decreased the clastogenic effect of cadmium. This effect is in accordance with the results of Ochi and Ohsawa (1985), who found that BHT protected Chinese V79 hamster cells from induction of chromosomal aberrations with a higher concentration of CC (50 mM) than that employed in our experiments.

As BHT is widely used as a food additive as well as in several industries, its dual effect is an interesting point. Whereas the compound is able to induce chromosomal damage (Grillo and Dulout, 1995), it can protect against oxidative damage induced by other genotoxicants (Grillo and Dulout, 1997).

ACKNOWLEDGMENTS

This work was supported by grants from the "Consejo Nacional de Investigaciones Científicas y Técnicas" (PID No. 3018/92), the "Comisión de Investigaciones Científicas de la Provincia de Buenos Aires" and the "Escuela de Postgrado en Ambiente y Patología Ambiental" (Universidad Nacional de La Plata-Universita degli Studi di Siena M.A.E.-D.G.C.S). This study is a part of the "Research Project of the Faculty of Veterinary Sciences, the National University of La Plata" between Japan and Argentina supported by JICA (Japan International Cooperation Agency). The authors are thankful to Susana J. Barani for her helpful collaboration in the preparation of the manuscript.

RESUMO

Estudou-se o efeito de hidroxitolueno butilado (BHT) sobre as alterações cromossômicas induzidas por cloreto de cádmio (CC) e dicromato de potássio (PD), tanto na metáfase como em anáfase-telófase de células do ovário de hamster chinês (CHO). Para a análise de aberrações cromossômicas, células CHO foram cultivadas por 15-16 h na presença de CC (6, 9 ou 12 mM), BHT (1 mg/ml) ou CC mais BHT, assim como PD (0,5, 1,0 e 2,0 mM), BHT ou PD mais BHT. Para o teste em anáfase-telófase, células foram cultivadas em lamínulas de vidro e tratadas 8 h antes da fixação com as mesmas substâncias. Uma dose extra de CC (4 mM) foi usada neste teste. Os resultados mostraram que ambos os sais metálicos aumentaram significativamente a freqüência de aberrações cromossômicas em relação a controles não tratados ou células tratadas com DMSO e BHT. O pós-tratamento com BHT diminuiu a ocorrência de danos cromossômicos em relação aos tratamentos com CC e PD. Contudo, a freqüência de aberrações cromossômicas foi significativamente maior do que a dos controles. No teste em anáfase-telófase, CC aumentou significativamente a ocorrência de cromossomos "lagging" com as 4 doses empregadas e a freqüência de fragmentos "lagging" com a dose maior. Em tratamentos combinados de CC e BHT, as freqüências dos dois tipos de alterações diminuíram em relação às células tratadas com CC. Não se encontrou variação significativa na freqüência de pontes de cromatina. Em células tratadas com PD, encontraram-se aumentos significativos nas pontes de cromatina, cromossomos "lagging" e fragmentos "lagging". O efeito protetor do BHT em tratamentos combinados foi evidenciado pela diminuição significativa das pontes de cromatina e dos cromossomos "lagging" em relação às células tratadas com PD. Esses resultados podem ser explicados assumindo-se que CC e PD induziram dano cromossômico por pelo menos dois mecanismos: 1) a ligação de cátions metálicos ao DNA e a subseqüente indução de efeitos conformacionais; 2) a geração de oxigênio reativo. O pós-tratamento com BHT diminuiu a ocorrência de dano cromosômico possivelmente ao afetar a geração de oxigênio reativo, mas o efeito clastogênico do próprio BHT deve ser levado em consideração. Conseqüentemente, a ocorrência de aberrações cromossômicas observadas após o pós-tratamento com BHT poderia resultar das lesões cromossômicas induzidas diretamente pela ligação de cada cátion ao DNA e também pelo dano induzido pelo BHT. Esta freqüência é menor que a causada pelos sais metálicos devido ao efeito protetor do BHT. Como BHT é amplamente usado como aditivo em alimentos e também em várias indústrias, seu duplo efeito é um ponto de interesse. Ao mesmo tempo que o composto pode induzir danos cromossômicos, ele pode proteger contra o dano oxidativo causado por outros produtos genotóxicos.

(Received November 21, 1997)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  21 Nov 1997