BIOLOGICAL T R A C E ELEMENT RESEARCH 2, 81~87 0980)

REVIEW

Evidence for the Antimutagenicity and the Mutagenicity of Selenium R A Y M O N D J. S H A M B E R G E R

Department of Biochemistry, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, Ohio 44106 Received August, 10, 1979; Accepted August 29, 1979

Abstract Evidence is summarized for the antimutagenicity as well as the mutagenicity of selenium. In general, antimutagenicity predominates at physiological levels, while mutagenicity occurs at 3 to 1000 times normal physiological levels. Index Entries: Antimutagenicity, of selenium; mutagenicity of selenium; selenium, mutagenicity and antimutagenicity of.

Introduction Selenium at nutritional levels has numerous anticarcinogenic effects against carcinogen-induced breast, colon, liver, and skin cancer in animals. The numerous anticarcinogenic effects have been recently summarized (1). Mutagenic test systems are used to predict carcinogenicity because it is thought that certain types of mutations lead to cancer. One popular mutagenic testing system is that of Ames, which is known to be about 90% effective in predicting whether a substance is carcinogenic (2). The ultimate percentage of predictions may be even greater when some of the 10% of the noncarcinogens are tested more extensively in animals. Several other mutagenesis test systems are also effective in predicting carcinogenicity. Because selenium has a marked anticarciogenic effect, selenium may also be antimutagenic. The object of this review is to summarize experimental evidence both for the antimutagenicity and the mutagenicity of selenium. If selenium is mutagenic or antimutagenic, some of the short term mutagenic assays might be used to predict whether or not selenium or another antioxidant will have an effect on a carcinogenic test system. Secondly, if an effect were found in a mutagenic system that is known to mutate in a certain 9 1980 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163 ~4984 / 80 / 0300 4)081 $02.00

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way, some insight might be gained into the mechanism of mutagenesis or even of cancer.

Evidence for the Antimutagenicity of Selenium Shamberger et al. (3) found that human blood leukocyte cultures incubated with antioxidants in different combination and the suspected carcinogen sodium cyclamate and the carcinogen 7,12-dimethylbenzanthracene (DMBA) had fewer chromosomal breaks. There were 17.4% more chromosomal breaks in the group of ceils treated with DMBA only, than the untreated controls. The reductions in the chromosomal breaks by the antioxidants were as follows: ascorbic acid, 31.7%; butylated hydroxytoluene, 63.8%; Na2SeO2, 42%; and dl-~-tocopherol, 63.2% (Table 1). Multiple chromosomal breaks were distributed equally throughout the experimental groups. Sodium cyclamate had only slightly more chromosomal breaks than the controls (11.6 compared to 10.9%). More acrocentrictype chromosomal breaks (21.7%) were seen in the untreated cells than the cells treated with cyclamate (3.4%) or DM BA alone (4.8%). The carcinogentreated groups had a higher percentage of meta breaks than the untreated controls. Jacobs et al. (4) found that selenium decreased the mutagenicity of 2acetylaminofluorene (AAF), N-hydroxy-2-acetylaminofluorene (N-OHAAF) and N-hydroxyaminofluorene (N-OH-AF) in the Salmonella typhlI murium TA 1538 bacterial tester system. Selenium decreased the mutagenicity of AAF, N-OH-AAF and N-OH-AF to 65, 68, and 61% of their respective controls with mutagen alone. Metabolism of AAF and N-OHAAF to the active mutagen, N-OH-AF, was accomplished with rat liver extracts, which may have introduced some natural antioxidants, such as TABLE 1 Effect of Antioxidants on Chromosomal Breakage Induced by 1.6 #M 7,12Dimethylbenzanthracene (3) Antioxidant + DMBA None (DMBA only) Ascorbic acid (10 #M) BHT (0.21 #M) Na2SeO~ (0.20 #M) d/-a-Tocophero! None

Cells

Breaks, %

Breaks minus control, %

Reduction, %

290 127

82 (28.3) 30 (23.6)

(17.4) (11.9)

-(31.7)

157 171 156 211

29 (18.4) 37 (21.6) 28 (17.9) 23 (10.9)

(6.3) (10.1) (6.4) --

(63.8) (42.0) (63.2) --

SELENIUM: A MUTAGEN AND AN ANT1MUTAGEN

83

vitamin E and selenium, thereby confounding the experiment. This testing system uses the Ames testing principle. The Salmonella bacteria do not grow on a histidine deficient media. However, once they are mutated they are able to grow on the histidine deficient media. The inhibition of mutagenicity by selenium in addition to previous reports of anticarcinogenicity seems to indicate that the Ames system might also be used for carcinogenesis. In another group of experiments Shamberger et al. (5) have also used the Ames testing procedure, but in contrast to Jacobs et al. (4) they used direct acting mutagens rather than those needing activation by liver extracts. In these experiments, increasing concentrations of malonaldehyde and flpropiolactone were increasingly mutagenic with seven mutants of Salmonella typhimurium, five of which mutated by a frameshift mechanism and two of which mutated through base-pair substitution (Figs. 1, 2). The tester strains of Salmonella typhimurium used in these experiments and the types of mutagens they detect are listed in Table 2. The antioxidants vitamin C, vitamin E, selenium, and butylated hydroxytoluene (BHT) at three logarithmic concentrations reduced mutagenesis in those strains that mutated by the frameshift mechanism (Figs. 3, 4). The results seem to indicate that inhibition of mutagenesis may be a direct effect of blocking the carcinogen from reacting with DNA, rather than an effect of blocking 1644

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MICROMOLES OF MALONALDEHYDE a ,0 ~b'13.9~c'27.Tid, 41.6~e" 55.4~ f-110.8

FIG. 1. Micromoles of malonaldehyde and the number of mutations per plate: a = 0; b = 13.85; c = 27.7; d = 41.6; e = 55.4, and f = 110.8.

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FIG. 2. Millimoles of fl-propiolactone and the number of mutations per plate: a = O; b = 0.390; c = 0.790; d = 1.18; e = 1.58; and f = 3.20.

TABLE 2 Tester Strains of Salmonella typhimurium Used in Shamberger et al. (5) Experiments and the Types of Mutagens They Detect Additional mutations Type hisG46 TA 1975 hisC207 hisC3076 TA 1977 hisD3052 TA1978

Mutagen type

Excision repair

Cell wall

Base-pair Base-pair Frameshift Frameshift Frameshift Frameshift Frameshift

Normal Normal Normal Normal Normal Normal Normal

+" rFa b + + + + rFa

'q~Iormal polysaccharide. bDeep rough.

SELENIUM: A MUTAGEN AND AN ANTIMUTAGEN

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ANTIOXIDANT CONCENTRATIONS o b c d

9 9 9 9

FMOLES 053,53,33 A~orbk:0cid 0.007,0.07,0.7 BHT 0.0067,0.067,0.67 Se 0.33,3.3,33 Vit. E

FIG. 3. Antioxidant concentrations and the percentage reduction in the number of mutants induced by 41.6 micromoles of malonaldehyde: (a) from left to right is

ascorbic acid 0.33; 3.3 and 33/.tmoles, (b) BHT 0.007; 0.07; and 0.7/,tmoles, (c) selenium 0.0067; 0.067 and 0.67/,tmoles, (d) vitamin E 0.33; 3.3; and 33 ~moles.

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CONCENTRATIONS F Mol~

a b r d

9 , 9 9

0.33,33.33 0.007,0.07,0.7 0.0067,0.067,0.67 0.33.3.3.33

Amx'bir BHT Se Vit.E

FIG. 4. Antioxidant concentrations and the percentage reduction in the number of mutants induced by 1.18 millimoles of fl-propiolactone: (a) from left to right is ascorbic acid 0.33; 3.3, and 33/,tmol; (b) BHT 0.007; 0.07; 0.7 #mol; (c) selenium 0.0067, 0.067, and 0.67/.tmol, (d) vitamin E 0.33, 3.33, and 33 ~tmol.

activation of the carcinogen, because neither malonaldehyde nor flpropiolactone need activation. Rosin and Stich (6) have studied sodium selenite as well as cysteamine, disulfiram, butylated hydroxyanisole, propyl gallate, sodium bisulfite, t~tocopherol succinate, and sodium ascorbate using the Salmonella tester strain TA 1535 and the mutagens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which induces base-substitutions mutations and N-acetoxy-2acetylaminofluorene (N-acetoxy-AAF), a frameshift mutagen. The muta-

86

SHAMBERGER

genic activity of MNNG was most effectively inhibited by cysteamine and sodium bisulfite with selenite, propyl gallate and sodium ascorbate being only slightly less effective. Propyl gallate, selenite, and cysteamine had a similar ability to inhibit N-acetoxy-AAF-induced mutagenesis, whereas the addition of sodium bisulfite, sodium ascorbate, and disulfiram had no detectable inhibitory effect on this carcinogen. Butylated hydroxyanisole and ot-tocopherol succinate did not affect the mutagenic activity of either carcinogen. The authors recommend studying an antioxidant's effect on numerous carcinogens prior to drawing any conclusions as to the potential use of the antioxidant as a carcinogen inhibitor.

Evidence for the Mutagenicity of Selenium Nakamuro et al. (7) tested five selenium compounds for their capacity to induce chromosome aberrations in cultured human leukocytes and for their reactivity with DNA by a rec-assay system and inactivation of transforming activity in Bacillis subtilis. The rec-assay, developed by Kada et al. (8), uses a B. subtilis recombination-deficient mutant, rec" [Cultures of wild and rec" strains are streaked on broth agar plates from one point, and filter paper soaked in a solution of the test chemical was placed on that point. Compounds causing DNA damage showed more inhibition along the streaked line of rec-strain than along that of the wild strain. Chromosome breaking activity was significantly higher for the compounds with fourvalent than with six-valent selenium, the efficiency being in the decreasing order H2SeO3 > Na2SeO3 > SeO2 > H2SeO4 ~ Na2SeO4. Rec-assay using B. subtilis with different recombination capacities suggested that damage to DNA was produced by selenites, but not by selenates. The reactivity of selenites with DNA was also indicated by a significant loss of transformation of the tryptophan marker of B. subtilis DNA treated with H2SeO3 and SeO2. In general, the test doses of 13, 26, and 53 • 10-5 M were quite likely cytotoxic. Sodium selenate actually showed a protective effect in one group of experiments. Blood in the United States ordinarily contains about 20 ~gSe/100 mL, or 2.53 • 10 -6 M. The test doses used by Nakamuro et al. (7) were 100-150 times normal and should not be considered to be in the physiological range. In another experiment (8) sodium selenite (Na2SeO3) at doses varying from 8 • 10-5 to 3 • 10-3 M (30-1000 times normal blood levels) induced DNA fragmentation, DNA-repair synthesis, chromosome aberrations, and a mitotic inhibition in cultured human fibroblasts. Incubation with mouse liver S-9 microsomal fraction increased the capacity of selenite to induce chromosome aberrations, DNA-repair synthesis, and a lethal effect. Sodium selenate (Na2SeO4) at doses ranging from 8 • 10-5 to 3 • 10-3 M could not be activated by incubating with a S-9 preparation.Selenate had the capacity to induce a small but significant DNA-repair synthesis. The

SELENIUM: A MUTAGEN AND AN ANTIMUTAGEN

87

greater mutagenic effect by sodium selenite than by sodium selenate in these experiments may reflect the known greater cytotoxicity of selenite. In another observation (9) sodium selenite (Na2SeO3) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl-aminofluorene (N-OH-AAF) on SCE frequency. High Na2SeO3 concentrations (7.90 • 10 -6 and 1.19 • 10-SM) (3-27 times normal blood levels) resulted in a threefold increase in the SCE frequency above background level (6-7 SCE / cell). Exposure of lymphocytes to 1 • 10-4M MMS for the last 19 h of culture yielded a'n average SCE frequency of 30.17 + 0.75, while a similar exposure to 2.7 • 10-5 M N-OHAFF resulted in 13.61 + 0.43 SCE cell. Simultaneous addition of the high Na2SeO3 concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25-30%, respectively, below the sum of the SCE frequencies produced by the individual compounds.

Discussion The different mutagenic and antimutagenic effects by selenium compounds may relate to the physiological and the toxic effects that selenium can exhibit. Numerous antimutagenic effects are evident. On the other hand, mutagenic effects seem to be related to toxicity. The cytotoxicity induced mutagenic effects nonetheless can be carried into the next generation. Because mutagenicity and carcinogenicity are closely related, similar patterns of carcinogenicity and anticarcinogenicity are evident for selenium. There is now considerable evidence that selenium is an anticarcinogen. The organs mainly protected are the breast, colon, skin, and liver. On the other hand, selenium at very toxic levels has been claimed to cause some low grade liver tumors in two experiments. The two experiments either lacked controls or were poorly controlled. In a properly controlled experiment selenium at an extremely toxic dose could prove to be a carcinogen, but that this effect is certainly nonphysiological. The numerous antimutagenic effects of selenium seem to parallel the anticarcinogenic effects of selenium. The Ames system or other mutagenic systems could be used for carcinogenesis studies, but caution should be used in the interpretation of the results.

References . (a) R. J. Shamberger, in Geochemistry and the Environment, Vol. III, pp. 105-113, The National Research Council, Natl. Acad. Sciences, Washington D.C., 1978. (b) G. N. Schrauzer, in Advances in Nutritional Research, Vol. II, H. H. Draper, ed. Plenum Press, New York, 1979, p. 220-244.

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2. J. McCann, E. Choi, E. Jamasaki, and B. N. Ames, in Proc. Natl. Acad. Sci. 72, 5135 (1975). 3. R. J. Shamberger, F. F. Baugham, S. L. Kalchert, C. E. Willis, and G.C. Hoffman, Proc. Natl. Acad. Sci. 70, 1461 (1973). 4. M. M. Jacobs, T. S. Matney, and A. C. Griffin, Cancer Letters 2, 319 (1977). 5. R.J. Shamberger, C. L. Corlett, K. D. Meamon, and B. L. Kasten, Mutation Res. 66, 349 (1979). 6. M. P. Rosin and H. F. Stich, Internatl. J. Cancer 23, 722 (1979). 7. K, Nakamuro, Y. Yoshikawa, Y. Sayato, H. Kurata, M. Tonomura, and T. Tonomura, Mutation Res. 40, 177 (1976). 8. L. W. Lo, J. Koropatnick, and H. F. Stich, Mutation Res. 49, 305 (1978). 9. J. H. Ray, J. L. Altenburg, and M. M. Jacobs, Mutation Res. 57, 359 (1978).

Evidence for the antimutagenicity and the mutagenicity of selenium.

Evidence is summarized for the antimutagenicity as well as the mutagenicity of selenium. In general, antimutagenicity predominates at physiological le...
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