Since Ames and his colleagues. (Ames et al. 1973) introduced the bacterial system to detect mutagens and carcinogens, a large number of environmental ...
Bull. Environ. Contam. Toxicol. (1988) 40:597-603 9 1988 Springer-Verlag New York Inc.
Environmental j Contamination i a n d Toxicology
Mutagenicity of Heavy Metals P. K. Wong Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
S i n c e A m e s and his c o l l e a g u e s ( A m e s et al. 1 9 7 3 ) introduced the bacterial system to detect mutagens and carcinogens, a large number of environmental pollutants such as o r g a n i c c o m p o u n d s ( M c C a n n et al. 1 9 7 5 ) , d y e s (McCann et al. 1975) as well as pesticides (Waters et al. 1981) were found to be mutagenic or c a r c i n o g e n i c by using this S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e mutagenicity tests (Ames et al. 1975). Certain heavy metals are required, as trace elements for normal cellular functions. However, heavy metals are t o x i c to c e l l s o n c e t h e i r l e v e l s e x c e e d t h e i r low physiological values. The toxicity of heavy metals on microorganisms (Gadd and G r i f f i t h 1 9 7 8 ) , on p l a n t s ( I c h i k u r a et al. 1 9 7 0 ) , and on a n i m a l s ( E i s l e r and Hennekey 1977) has been w e l l - d o c u m e n t e d . Eichhorn (1975) d e s c r i b e d the b i n d i n g of h e a v y m e t a l s to t h e phosphate, d e o x y r i b o s e and heterocyclic base residues of DNA. These interactions may induce the alteration of the primary as well as secondary structures of the DNA and result in mutation(s).
Though the rec assay with Bacillus subtilis and t h e reversion assay with E~cher~chia coli were used to assess the mutagenicity o f some h e a v y m e t a l s ( G r e e n e t al. 1976, Nishioka, 1975), the present communication reports the results in determining the mutagenicity and carcinogenicity of ten heavy metals commonly f o u n d i n polluted areas by u s i n g the Salmonella/mammalian-
microsome mutagenicity t e s t . MATERIALS
AND METHODS
Heavy m e t a l
stock
solutions
(i0 597
parts
per thousand,
ppt)
were prepared by dissolving chloride salts of different heavy metals in distilled water containing 0.i mM citric acid and sterilized by autoclaving. Nicotinamide adenine dinucleotide phosphate (NADP), g l u c o s e - 6 - p h o s p h a t e (G-6-P), L-histidine, D-biotin and 2 - a m i n o f l u o r e n e were purchased from Sigma Chemical Co. USA. Bacterial media (except nutrient broth no.2 from Oxoid Co. USA) were purchased from Difco Co. USA. Other chemicals used in this study were of reagent grade. Salmonella t v o h i m u r i u m TA98, TAI02, TA1535, and TA1537 tester strains were k i n d l y provided by Dr. Bruce Ames (Department of Biochemistry, University of C a l i f o r n i a at Berkeley). The liquid culture, storage and preparing the b a c t e r i a l cultures for m u t a g e n i c i t y tests were carried out according to Maron and Ames (1983). The toxicity of heavy metals on the growth of the tester strains were determined by g r o w i n g the bacterial cells in VBE medium (Vogel and Bonner 1956) supplemented with 0.4% g l u c o s e , 0.25 mM L-histidine and 3 JIM D-biotin in the presence of different c o n c e n t r a t i o n s (in parts per m i l l i o n , ppm) of h e a v y m e t a l s . The g r o w t h of the bacteria cells was monitored by a K l e t t - S u m m e r s o n p h o t o e l e c t r i c c o l o r i m e t e r with a red filter (wavelength of 640-700 nm). The 50% toxic doses (the concentrations of h e a v y m e t a l s t h a t e x h i b i t a 50% inhibition in g r o w t h ) and 9 0 % t o x i c doses (the concentrations of h e a v y m e t a l s t h a t e x h i b i t a 90% inhibition in g r o w t h ) to t h e t e s t e r strains were obtained from these growth inhibition experiments. The plate-incorporation method was used for the mutagenicity test (Maron and A m e s 1983). The c o n c e n t r a t i o n s of heavy metals used in the m u t a g e n i c i t y t e s t s w e r e t h o s e b e t w e e n the 50% t o x i c d o s e and 90% toxic dose determined in t h e growth inhibition experiments. $9 preparation and the m u t a g e n i c i t y tests in the presence of $9 were performed as described (Lin et al. 1986). The metabolic activity of Sg-mix was determined by assessing the activation of the m u t a g e n i c i t y of 2 - a m i n o f l u o r e n e to the tester strains (Maron and Ames 1983). RESULTS
AND D I S C U S S I O N
Figure i shows the toxicity of different c o n c e n t r a t i o n s of cadmium (0 to 120 ppm) to the tester strain TA98. The 50% toxic dose of cadmium to strain TA98 was 20 ppm, and growth of strain TA98 was about 90% inhibited by 120 ppm cadmium (this was 90% toxic dose and the high concentration of c a d m i u m u s e d in t h e m u t a g e n i c i t y 598
~ f
200-
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150-
100-
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