Toxic. in Vitro Vol. I, No. 1, pp. 55-57, 1987 Printed in Great Britain
0887-233/87 $3.00+ 0.00 Pergamon Journals Ltd
INCREASE IN CHROMOSOMAL ABNORMALITIES IN CHINESE HAMSTER OVARY CELLS TREATED WITH BUTYLATED H Y D R O X Y T O L U E N E IN VITRO R. M. PATTERSON,L. A. KEITH and J. STEWART Department of Biology, Atlanta University, Atlanta, GA 30314, USA (Recewed 12 March 1986)
Abstract--Cytogenetic studies showed that treatment of Chinese hamster ovary (CHO) cells with 1, 5 or l0 #g BHT/ml for 48 hr increased the number of cells with chromosomal abnormalities. CHO cultures treated with 1.0#g BHT/ml demonstrated a 35% level of abnormal cell types. These abnormalities included chromosome gaps, triradials, polycentrics, endoreduplications and polyploids. When CHO cells were exposed to 5.0 #g BHT/ml, 36% of the cells were found to be abnormal. However, in cells treated with l0 #g BHT/ml, a 20% level of cell abnormalities was noted. The incidence of polyploid cells was dose dependent, but the dose dependency ceased at l0 #g BHT/ml, at which a decrease in polyploids as well as in the total percentage of abnormal cells was observed compared with the cells exposed to lower concentrations of BHT.
After treatment with one of the specific concentrations of BHT, the experimental cells together with the untreated control and ethanol control cells were prepared for cytogenetic analysis. Briefly, the cells in culture were treated with 0.1/~gcolcemid/ml for 2.5 hr at 37°C. The cells were then harvested, treated with a hypotonic solution (1% citrate), fixed in acetic acid-methanol (l:3, v/v) and stained in diluted Giemsa. For experimental observation and data collection, the slides were labelled with an experimental number and code. In order to reduce subjectivity in data recording, the experimental number and code were covered with a slip of paper, taped and recoded by an assistant. The slides were not decoded until chromosome aberrations and abnormalities were counted on at least 100 well-spread metaphase cells/slide. All data were analysed statistically by Student's t test for comparison of individual sample means.
Butylated hydroxytoluene (BHT) is an antioxidant that is commonly added to such products as biscuits, chips, cereals, cooking oils and margarine to prevent rancidity (Kermode, 1972). BHT has been reported in the literature to have both adverse and beneficial effects. For example, it has been reported to reduce chromosomal breaks in blood leucocyte cultures treated with sodium cyclamate and 7,12-dimethylbenz[a]anthracene (Shamberger et al. 1973). The antioxidant has been shown to enhance the metabolism of aflatoxin B 1 (AFBI) to aflatoxin M t (AFM~), to increase the rate of A F M l conjugation and to protect against the carcinogenic effect of AFB~ (Fukayama et al. 1984; Salocks et al. 1984). BHT protects against 1,2-dimethylhydrazine carcinogenesis in BALB/c mice (Clapp et al. 1979) but has been reported to be a promoter of adenoma formation in the lungs of Swiss-Webster mice initiated with urethane (Witschi & Lock, 1979). BHT has also been reported to cause lung damage (Marino & Mitchell, 1972) and to increase pulmonary glucose-6phosphate dehydrogenase (G6PDHL D N A polymerase, thymidine kinase activity (Witschi et aL 1976) and superoxide dismutase (Omaye et al. 1977). In the investigation reported here, a study was made of the effects of BHT on Chinese hamster ovary (CHO) cells, as revealed by cytogenetic methods.
MATERIALS AND METHODS
Ovary cells of Cricetulus griseus, the Chinese hamster (American Type Culture Collection, Rockville, MD), were grown in Ham's Nutrient Medium F-10 supplemented with 10% foetal bovine serum and kanamycin (GIBCO, Grand Island, NY) at l0 #g/ml. The CHO cells were treated with BHT (Calb]ochemBehring Corp., La JoUa, CA) in a concentration of 1, 5 or 10#g/ml for 48hr at 37°C. The BHT was dissolved in purified absolute ethanol. 55
In CHO cells treated with 1.0/~g BHT/ml for 48 hr, 35% of the cells showed chromosomal abnormalities, compared with 6% in the untreated control group and 11% in the ethanol control (Table 1). In the experimental group, 14% of the cells were polyploids. The experimental group also showed a significant increase (P < 0.05) in chromatid breaks (4%) and polycentrics (5%) compared with untreated controls. As the concentration of BHT was increased from !.0 to 5.0/~g/ml, the total percentage of polyploids nearly doubled (to 23%), but the percentage of polycentrics (4%) was relatively constant. The other specific aberrations observed were 1% chromosome gaps, 1% chromatid gaps, 1% chromatid breaks, 3% rings, 1% triradials, 1% pulverized chromosomes and 1% endoreduplications (Table 1). When the concentration of BHT was increased to 10/~g/ml, the total percentage of cytogenetically abnormal cells decreased significantly (P < 0.05). The
R. M. PATTERSON et al. Table 1. Effects on CHO chromosomes of exposure to 1.0, 5.0 or 10tag BHT/ml for 48 hr Occurrence of affected cells* Untreated control
0 1.0 2.0 0 1.0 2.0 0 0 0 0 5.0
1.0 1.0 4.0 1.0 2.0 5.0 2.0 1.0 1.0 3.0 14.0
1.0 pg BHT/ml
Chromosome gaps Chromatid gaps Chromatid breaks Chromatid fusions Rings Polycentrics Pulverized chromosomes Complex interchanges Fragments Endoreduplications Polyploids
0 0 0 0 0 0 0 0 0 0 6.0
Total Mean + SEM/metaphase
11.0 0.11 + 0.0395 NSD~ at 0.05 level
35.0 0.35 + 0.0536 Significant increasea'b at 0.01 level
5.0 # g BHT/ml
Chromosome gaps Chromatid gaps Chromatid breaks Rings Triradials Polycentrics Pulverized chromosomes Endoreduplications Polyploids
0 0 1.0 0 0 0 0 0 3.0
Total Mean + SEM/metaphase
0 2.0 3.0 0 0 0 0 0 3.0 8.0 0.08 + 0.0336 NSDa at 0.05 level
1.0 1.0 1.0 3.0 1.0 4.0 1.0 1.0 23.0 36.0 0.36 + 0.0521 Significant increasea'b at 0.01 level
Chromosome gaps Chromosome breaks Chromatid breaks Chromatid gaps Triradials Polycentrics Fragments Polyploids
0 0 0 2.0 0 0 0 3.0
Total Mean + SEM/metaphase
5.0 -t testt...
0 0 1.0 1.0 1.0 0 1.0 8.0 12.0 0.12 + 0.0393 NSDa at 0.05 level
l.O 1.0 0 0 0 1.0 5.0 12.0 20.0 0.20 + 0.0458 Significant increasea'b at 0.01 level; NSDb at 0.05 level
CHO = Chinese hamster ovary NSD = No significant difference *In each case, 100 metaphases were examined. tt test: aLevel of significance for increase/decrease in abnormal cells compared with mean value for untreated control; b level of significance for increase/decrease in abnormal cells compared with mean value for ethanol control.
effects o f 1 0 # g B H T / m l o n C H O cells are also r e c o r d e d in T a b l e 1. T h e total p e r c e n t a g e o f a b n o r m a l cells o b s e r v e d in the u n t r e a t e d c o n t r o l cells w a s 5 % . T h e specific a b n o r m a l i t i e s were 2 % c h r o m a t i d g a p s a n d 3 % p o l y p l o i d s . T h e total p e r c e n t a g e o f a b n o r m a l cells o b s e r v e d in e x p e r i m e n t a l c u l t u r e s t r e a t e d w i t h 10/zg B H T / m l w a s 2 0 % . T h e t y p e s o f a b n o r m a l i t i e s o b s e r v e d in the B H T e x p e r i m e n t a l g r o u p s were c h r o m o s o m e g a p s ( 1 % ) , c h r o m o s o m e breaks (1%), polycentrics (1%), fragments (5%) and polyploids (12%).
DISCUSSION It w a s o b s e r v e d in these B H T studies t h a t the h i g h e s t p e r c e n t a g e o f total a b n o r m a l i t i e s o c c u r r e d in cells t r e a t e d w i t h 5.0 # g B H T / m l . T h e r e w a s a significant decrease in the p e r c e n t a g e o f c h r o m o s o m a l a b n o r m a l i t i e s in C H O cells t r e a t e d w i t h I0 # g / m l . It is p o s s i b l e t h a t the latter d o s e m a y h a v e b e e n toxic to the cells, t h u s r e d u c i n g the n u m b e r o f cells t h a t c o n t a i n e d c h r o m o s o m a l a b n o r m a l i t i e s . T h i s m a y be related to the fact t h a t 20 # g B H T / m l c a u s e d 100% cell d e a t h in the C H O cultures.
Cytogenetic effects of BHT In these studies, BHT, at specific concentrations, significantly increased the percentage of polyploids, compared with the occurrence in untreated controls. The occurrence of polyploid cells in experimental cultures treated with BHT may be correlated with previous studies, which have shown that BHT increases the DNA content of pulmonary cells in vivo (Saheb & Witschi, 1975). These investigators observed that the total lung weight in Swiss-Webster mice increased significantly after BHT treatment; even 9 days after a BHT injection, the lung weight in treated animals was significantly increased over that in the controls. Omaye et al. (1977) provided further evidence with the observation that BHT (62.5, 215 and 500mg/kg) significantly increased the lung weight in a dose-dependent manner. The increase in lung weight was accompanied by increases in lung DNA and non-protein sulphydryl levels. There were also corresponding increases in the activities of glutathione (GSH) peroxidase, GSH reductase, G6PDH and superoxide dismutase. Witschi & Lock (1978) reported a 1.7-fold increase in the total amount of DNA in the lungs of male Swiss-Webster mice that had been given 400 mg BHT/kg by stomach tube. This increase in the quantity of DNA was associated with extensive cell proliferation and promotion of adenoma development. Sciorra et al. (1974) have reported that concentrations of BHT up to 50 gg/ml caused a shortening of the cell cycle for phytohaemagglutinin-stimulated human leucocytes; this effect was dose dependent. BHT was also observed to induce uncoiling of the chromosomes and damage to the cell membrane. Conclusion
The cytogenetic effects of BHT are dose dependent and the compound may act as a weak mutagenic agent in CHO cells in vitro. The studies presented here cannot distinguish between the mutagenic and the toxic effects of BHT, as related to the decrease in the percentage of chromosomal aberrations observed when cells were treated with 10 #g BHT/ml. BHT has been reported to act as an antioxidant by reducing chromosomal breaks in blood leucocyte cultures treated with sodium cyclamate and 7,12-dimethylbenz[a]anthracene (Shamberger et al. 1973). Moreover, the incidence of polyploid cells suggests an
alteration in the cell cycle that may be related to the mutagenic effects of BHT. Acknowledgements--This research was supported by NIH Grant 5S06-RR08006-14. We wish to thank Mrs Joyce Lockhart for assistance in the preparation of the manuscript.
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