Mutation Research, 229 (1990) 11-15 Elsevier
Induction of umu gene expression in Salmonella typhimurium TA1535/pSK1002 by dimethyl sulfoxide (DMSO) Sei-ichi Nakamura, Yoshimitsu Oda and Masahiro Ugawa Osaka Prefectural Institute of Public Health, Nakamichi-1, Higashinari-ku, Osaka 537 (Japan) (Received 16 June 1989) (Revision received 5 September 1989) (Accepted 6 October 1989)
Keywords: SOS response; umu gene; Genotoxicity; Dimethyl sulfoxide
Summary The genotoxicity of dimethyl sulfoxide (DMSO) was demonstrated by the umu test using Salmonella typhimurium TA1535/pSK1002 carrying the u m u C - l a c Z fusion gene. The level of/3-galactosidase activity which shows umu gene expression in the test system was dependent on the concentration of DMSO in the culture medium. The maximum /3-galactosidase activity was approximately 3.5 times as high as the background level with 10% of DMSO in the culture medium. The lowest concentration of DMSO required for a response of over twice the background level was approximately 5%. Four structurally related chemicals (acetone, di-n-butylsulfoxide, dimethylsulfide, methylphenylsulfoxide) did not show umu gene expression at their non-toxic doses.
In mutagenicity assays, dimethyl sulfoxide (DMSO) is very frequently used as the solvent for the test compound (Ames et al., 1975) because of its efficient solvent properties for a wide range of chemicals, low toxicity to bacteria, lack of influence on the action of the activating enzyme system ($9), and miscibility with the test medium (Maron et al., 1981). DMSO is generally considered to be genetically inactive in the Salmonella microsome assay (McCann et aL, 1975) and in Drosophila melanogaster (Mollet, 1976). To detect the DNA-damaging activity of chemicals, we have developed a novel system, the umu
Correspondence: Dr. S. Nakamura, Osaka Prefectural Institute of Public Health, Nakamichi-1, Higashinari-ku, Osaka 537 (Japan).
test (Oda et al., 1985; Nakamura et al., 1987). This system is based on the ability of a chemical to induce umu gene expression in a new tester strain Salmonella typhimurium TA1535/pSK1002 into which a plasmid-borne u m u C - l a c Z fused gene has been introduced. A high degree of correlation was seen between mutagenic potency as measured by the Ames Salmonella test and level of induction of SOS responses as measured by the umu test (Nakamura et al., 1987). However, some chemicals gave conflicting results in the 2 tests. One of them was DMSO which induced SOS responses in the umu test but did not exert mutagenicity in the Salmonella/microsome assay system (McCann et al., 1975). The present paper describes detailed studies concerning the genotoxicity of DMSO and structurally related compounds (Fig. 1) examined with
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(a) (b) (c) CH3-S-CH3 CIL,-!-CH3 CIL,-,S,-~ 0 0 0 (d~
CH3-S-CHa CH3(CH,)3-S-(CH,~CH3 0 Fig. 1. Structures of the chemicals used in the genotoxicity assay. (a) Dimethyl sulfoxide (CAS No. 67-68-5); (b) acetone (CAS No. 67-64-1); (c) rnethylphenyl sulfoxide; (d) dimethyl sulfide (CAS No. 75-18-3); (e) di-n-butylsulfoxide (CAS No. 2168-93-6).
the umu test and also discusses the possibility of DMSO having DNA-damaging properties. Materials and methods Chemicals
DMSO (special reagent grade), acetone, dimethyl sulfide and o-nitrophenyi-fl-D-galactopyranoside (ONPG) were purchased from Wako Pure Chemical Co. (Osaka). Di-n-butylsulfoxide and methylphenyl sulfoxide were purchased from Aldrich Chemical Co. (Milwaukee) and Tokyo Kasei Chemical Co. (Tokyo), respectively. In tests of DMSO from different manufacturers, the source is indicated in the text. Bacterial strain Salmonella typhimurium TA1535/pSK1002 was
used. The bacterial strain was transformed with plasmid pSK1002 which is a derivative of pMC1403 carrying the promoter of the umu operon, the umuD and umuC-lacZ fusion genes (Shinagawa et al., 1983). Lethafity tests
Bacteria in logarithmic phase were suspended in 0.1 M phosphate-buffered saline (PBS) at a concentration of about 10 9 cells/ml after washing twice with PBS. Various doses of DMSO were added to the suspension. After incubation for 120 min at 37 °C, the mixture was diluted with PBS and spread on bouillon plates. The number of colonies was counted after incubation for 20 h at 37°C.
SOS-inducing activity The umu test for detecting SOS-inducing activity ( u m u gene expression) was carried out essen-
tially as described previously (Oda et al., 1985), Briefly, an overnight culture of the tester bacterial strain in Luria broth (1% bactotryptone, 0.5% NaCI and 0.5% yeast extract) was diluted 50-fold with fresh T G A medium (1% bactotryptone, 0.5% NaC1 and 0.2% glucose; supplemented with 20 mg/1 of ampicilin), and incubated at 3 7 ° C until the bacterial density at 600 nm reached 0.25-0.30. The culture was divided into 2-ml portions in test tubes, and 0.5 ml of the appropriate concentration of DMSO solution in distilled water was added to each tube. After 2 h incubation at 3 7 ° C with shaking, the culture was centrifuged to collect cells, which were resuspended in 2.5 ml of PBS and the cell density was read at 600 nm with one portion of the suspension. Using the other portion, the level of /3-galactosidase activity in the cells was assayed by the method of Miller (1972). All tests were performed in duplicate. The tests for acetone and dimethyl sulfide were carried out in screw-cap test tubes to prevent evaporation. Results
First, we examined the toxicity of DMSO and its 4 structurally related compounds (acetone, din-butylsulfoxide, dimethyl sulfide and methylphenyl sulfoxide) for S. typhimurium T A 1 5 3 5 / pSK1002. As shown in Fig. 2, di-n-butylsulfoxide and methylphenyl sulfoxide were highly toxic, and the 50% killing concentrations were approximately 0.3 and 0.4%, respectively. The toxicities of dimethyl sulfide and acetone were lower; the 50% killing concentrations were 0.8% for dimethyl sulfide and 1.2% for acetone. D M S O showed quite low toxicity and bacterial survival was over 50% even at a concentration of 15%. We then examined the SOS-inducing activity of DMSO and related compounds in several doses which did not exceed toxic levels to the bacteria. As shown in Table 1, umu gene expression was observed with DMSO. The other 4 chemicals, despite their high toxicity, did not show umu gene expression at non-toxic doses. The time course of induction of/3-galactosidase activity by D M S O was studied at several doses. As
Fig. 2. Survival curves of S. typhimurium TA1535/pSK1002 after treatment with DMSO or other chemicals. The number of surviving cells was measured. ( - O - ) Dimethyl sulfoxide; (-zx-) acetone; (-12]-) methylphenyl sulfoxide; ( - v - ) dimethyl sulfide; ( - e - ) di-n-butylsulfoxide.
I 2 incubation
I 3 time
Fig. 3. Time course of the induction of fl-galactosidase activity after incubation with several doses of DMSO. DMSO concentration in the incubation medium is (a) 8%, (b) 4%, (c) 2% and (d) 0%.
TABLE 1 SOS-INDUCING ACTIVITY OF DMSO AND STRUCTURALLY RELATED COMPOUNDS Chemical
Control Furylfuramide (0.02 p,g/ml)
fl-Galactosidase activity (units)
2 4 8
0.821 0.698 0.416
0.193 0.221 0.218
0.025 0.023 0.013
121 173 313
2 3 4
0,802 0,657 0,465
0.160 0.135 0.086
0.026 0.021 0,015
95 100 86
0.15 0.25 0.35
0.996 0.846 0.553
0.145 0.162 0.149
0,029 0.025 0.017
75 80 102
0.2 0.4 0.6
0.791 0.580 0,460
0.183 0.141 0.085
0.022 0.014 0.008
122 134 103
0.2 0.4 0.5
0,889 0.703 0.480
0.177 0.120 0.082
0.034 0.022 0.013
88 77 82
shown in Fig. 3, fl-galactosidase activity increased with incubation time in a dose-dependent manner. In order to clarify the mode of SOS induction by DMSO, the dose-response relationship was examined further (Fig. 4). The level of u m u gene expression was increased with the dose of DMSO in the medium, and maximum/3-galactosidase activity was found to be 310 units (approximately 3.5 times as high as the background level) with 10% DMSO in the medium. The lowest concentration of DMSO required for a response over twice the background level was approximately 5%. The possibility that the induction of u m u gene expression results from contaminants or adducts in some batches of DMSO was considered. Therefore, the SOS-inducing activity of DMSO was confirmed for 3 types of freshly bottled DMSO (special grade, Wako Pure Chemical Co.; Uvasole, Kanto Chemical Co.; Dotait-Spectrosol, Dojin Chemical Lab.). The results of the kinetics and the dose-response relationship for u r n u gene expression with each type of DMSO were siinilar (Table 2). This suggests that the u m u gene expression was caused by DMSO and not by some adducts or contaminants in some batches of DMSO. The effect of DMSO on measurement of /3-galactosidase activity was tested to confirm that
TABLE 2 S O S - I N D U C I N G ACTIVITY OF 3 TYPES OF D M S O IN T H E umu TEST Dose
/~-galactosidase activity (units)
DMSO ] b
DMSO 2 b
DMSO 3 b
2 4 8
110+ 10 132_+12 247_+16
108_+16 134_+14 257_+17
109_+ 8 143_+13 233__+14
Control Furylfuramide (0.02 # g / m l )
94 -+ 12 431 -+ 72
Mean _+SD (n = 4). b DMSO 1, special reagent grade (Wako Pure Chemical Co., Osaka); D M S O 2, Dotait-Spectrosol (Dojin Chemical Lab., Kumamoto); DMSO 3, Uvasol (Kanto Chemical Co., Tokyo).
DMSO did not react with O N P G ; if it did, a false-positive result could be obtained. After 2 h incubation of the bacterial culture, several doses of DMSO were added and the B-galactosidase activity in the bacteria was assayed without washing them. The level of/3-galactosidase was found to be equal to that of the control cells not treated with DMSO. Therefore, D M S O did not have any effects on the colorimetric assay of fl-galactosidase (data not shown). Discussion
2~oo u m
Fig. 4. Dose-response relationship between fl-galactosidase activity (units) and amount of DMSO in S. typhirnurium TA1535/pSK1002.
The present study indicates that genotoxicity of DMSO can be detected by the u m u test using S. typhimurium T A 1 5 3 5 / p S K 1 0 0 2 carrying the umuC-lacZ fusion gene, although it should be noted that u m u gene expression was detected only at high doses, i.e., 5-15%. The level of /3-galactosidase activity which reflects u m u gene expression was dependent on the concentration of D M S O in the culture medium, However, acetone, di-nbutylsulfoxide, dimethyl sulfide and methylphenyl sulfoxide did not induce the SOS response at their (individual) non-toxic doses. Due to their high toxicity relative to DMSO, we could not examine the SOS-inducing activity of these 4 chemicals at the much higher doses used for DMSO. For the effect of DMSO on D N A or D N A synthesis, there are several reports which suggest the induction of mutagenicity or abnormal D N A synthesis. Hayes et al. (1984) reported the muta-
15 genicity of D M S O based o n the R K bacterial test. F u l t o n a n d Bond (1984) showed D M S O - i n d u c e d a n e u p l o i d y in a fungal test system where D M S O i n d u c e d segregational errors at both the first a n d the second meiotic division. D M S O is also k n o w n to affect the i n d u c t i o n of differentiation in cells such as erythroleukemic cells, pro-myelocytic cells a n d n e u r o b l a s t o m a cells ( F r i e n d et al., 1971), although these effects are also only seen at high doses. The effect o n the i n d u c t i o n of differentiation seemed not to be caused by D N A lesions ( D N A damage) b u t by the results of some interactions of D M S O with the cell m e m b r a n e or some d i s t u r b a n c e of D N A synthesis in the bacteria. D M S O is generally considered to be inactive in the Ames Salmonella test. Quillardet et al. (1985) also reported that D M S O was inactive in the SOS chromotest, which is an SOS-function assay a n d is essentially similar to the present u m u test system. A l t h o u g h the reason for these discrepancies is obscure at present, it should be pointed out that different tester strains were used in these test systems. First, the m e m b r a n e permeability of D M S O may differ between S. t y p h i m u r i u m in the u m u test a n d E. coli in the SOS chromotest. Second, the u m u gene in the u m u test was carried on a plasmid, while the s f i A gene in the SOS chromotest was carried o n the c h r o m o s o m e of the host bacteria. Finally, the gene whose expression is regulated by the SOS system differs for the test systems, being the u m u gene in the u m u test a n d the s f i A gene in the SOS chromotest. Since the u m u gene is related more directly to mutagenesis than other k n o w n SOS genes in bacteria, the u m u test may more directly reflect mutagenicity ( K a t o et al., 1982; Kitagawa et al., 1985). I n any case, further studies are required to elucidate the mecha n i s m of SOS i n d u c t i o n by D M S O .
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