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Chem. Res. Toxicol. 1996, 9, 374-381
Mutational Spectra of Salmonella typhimurium Revertants Induced by Chlorohydroxyfuranones, Byproducts of Chlorine Disinfection of Drinking Water Siegfried Knasmu¨ller,*,† Edith Zo¨hrer,† Leif Kronberg,‡ Michael Kundi,§ Robert Franze´n,‡ and Rolf Schulte-Hermann† Institute of Tumor Biology and Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria, Department of Organic Chemistry, Abo Akademi University, FIN-20500, Finland, and Institute of Environmental Hygienics, University of Vienna, A-1090 Vienna, Austria Received April 25, 1995X
The base substitution specificities of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), 3,4-dichloro-5-hydroxy2(5H)-furanone (MCA), and chloromalonaldehyde (CMA), a putative breakdown product of MCA, were examined in the hisG46 gene and in the hisG428 gene of Salmonella typhimurium using allele specific oligonucleotide hybridization. Although the compounds are structurally closely related, they induced substantially different mutation spectra: MCA and CMA caused primarily GC f AT transitions in the hisG46 allele (target sequence CCC), in particular, at the second position of the codon in strain TA100. In TA100 the mutation spectrum of MCA was similar to that of CMA. The mutational specificity of MCA can be explained as a consequence of misincorporation opposite to cyclic etheno adducts identical to those formed by the carcinogen vinyl chloride. The spectra induced by MX and CMCF in TA100 were almost identical but distinctively different from the spectra of MCA and CMA. Both compounds induced primarily GC f TA transversions, in particular, at the second position of the codon, and to a lesser extent in the first position of the codon. An identical site bias is induced by carcinogens such as polycyclic aromatic hydrocarbons and heterocyclic amines as a consequence of formation of (noncyclic) guanosine adducts. In hisG428 (target sequence TAA) MX induced again primarily GC f TA transversions in Tyr tRNA genes (supC/M) and, to a lesser extent, intragenic AT f TA transversions (TAA f AAA). The possible involvement of guanosine and adenosine adducts in the mutational specificity of MX is addressed.
Introduction Chlorohydroxyfuranones (CHFs)1 are produced as byproducts of chlorine disinfection of drinking water and of chlorine bleaching of pulp (1, 2). It has been shown that CHFs are bacterial mutagens, and one of these compounds, namely, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), is one of the most potent direct acting bacterial mutagens known (3-6). Although the concentration of MX in chlorine disinfected drinking water is extremely low (from a few ng/L up to 60 ng/L), the compound has been, due to its pronounced mutagenicity, estimated to account in most samples for about 30% of the total mutagenicity in drinking water extracts (7-10). The CHFs 3-chloro-4-(chloromethyl)-5-hydroxy2(5H)-furanone (CMCF) and 3,4-dichloro-5-hydroxy-2(5H)furanone (MCA) have been detected in water at approximately the same concentration as MX (1). The mutagenic potency of CMCF is lower than that of MX, and the compound accounts for at most 6% of the activity of water † Institute of Tumor Biology and Cancer Research, University of Vienna. ‡ Department of Organic Chemistry, Abo Akademi University, Finland. § Institute of Environmental Hygienics, University of Vienna. X Abstract published in Advance ACS Abstracts, January 1, 1996. 1 Abbreviations: MX, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone; CMCF, 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone; CMA, 3,4-dichloro-5-hydroxy-2(5H)-furanone; MCA; mucochloric acid; CMA, chloromalonaldehyde; CHF, chlorohydroxyfuranone; A, 1,N6ethenoadenosine; C, 3,N4-ethenocytidine; 1,N2-G, 1,N2-ethenoguanosine; N2,3-G, N2,3-ethenoguanosine; cC, ethenocarbaldehydocytidine; cA, ethenocarbaldehydoadenosine; rev, revertants.
0893-228x/96/2709-0374$12.00/0
extracts (1). MCA is still a weaker mutagen than CMCF (1, 11). On the other hand, recent studies have shown that MCA readily reacts with the purine and pyrimidine constituents of DNA (12, 13). During recent years, MX has been tested in numerous assays, and evidence is accumulating that the compound causes DNA damage in plants and in mammalian cells in vitro and in vivo as well (8, 14-21). It has been shown that the mutational specificities of several environmental carcinogens in Salmonella genes (hisG46, hisG428, hisD3052) are very similar to those induced in mammalian genes (22-25). The results obtained by mutational spectroscopy technologies can provide insights into the cause and relationships between environmental agents and genetic changes and improve the risk assessment of such agents (26). Although numerous people are exposed to CHFs and their genotoxic properties have been demonstrated in a variety of indicator organisms (for review see ref 27), the molecular basis for their mutagenic properties is not understood and their mutational specificities have not yet been determined. In the current work, we have investigated the mutation spectra of MX, CMCF, and MCA (Figure 1) in Salmonella typhimurium by use of a recently developed oligodeoxyribonucleotide hybridization technique (28, 29). The mutation spectra induced by these compounds were analyzed in cells carrying the hisG46 allele, a missense mutation (CTC f CCC) that can revert to histidine © 1996 American Chemical Society
Mutation Spectra of Chlorohydroxyfuranones
Figure 1. Structures of 3-chloro-4-(dichloromethyl)-5-hydroxy2(5H)-furanone (MX), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)furanone (CMCF), 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA, mucochloric acid), and chloromalonaldehyde (CMA).
prototrophy via five intragenic base substitutions at either of two adjacent G/C base pairs in the mutant gene or by extragenic supressor mutations resulting from AT f CG transversions (30-32). In order to obtain further information on the influence of the genetic background of the indicator cells on the mutational specificity of MX, the spectrum induced in strain TA100 (hisG46, ∆uvrB, rfa, pKM 101) was compared with the spectra induced in TA1535 (hisG46, ∆uvrB, rfa) and TA1950 (hisG46, ∆uvrB, LPS+). Kronberg et al. (13) have suggested that, in reaction mixtures of MCA and the base moieties of the genetic material (adenosine, guanosine, and cytidine), MCA is initially broken down to chloromalonaldehyde (CMA, Figure 1). Therefore, we analyzed also the spectrum of CMA in the same target sequence and compared it with that induced by MCA. Furthermore, the mutational specificity of MX was analyzed in the hisG428 allele (an ochre mutation, CAA f TAA) in strain TP2428 which has an identical genetic background as TA1950 (∆uvrB, LPS+). The hisG428 system is complementary to hisG46, in that, cells containing this sequence can revert by any of the seven base substitutions within the target in the mutant gene or by extragenic supressor mutations which result from GC f AT transitions, AT f TA transversions, or GC f TA transversions (22, 29, 34).
Experimental Section Bacterial Media and Strains. Salmonella typhimurium strains TA1535 (hisG46, ∆uvrB, rfa) and TA100 (hisG46, ∆uvrB, rfa, pKM 101) were obtained from the laboratory of B. N. Ames (University of California, Berkeley); strains TA1950 (hisG46, ∆uvrB, LPS+) and TP2428 (hisG428, ∆uvrB, LPS+) were a gift from T. Cebula (FDA, Washington). For a precise description of the construction of the later strain see ref 29. Test Compounds. Mucochloric acid, [MCA, 3,4-dichloro-5hydroxy-2(5H)-furanone] was obtained from commercial sources (Aldrich Chemie, Steinheim, FRG). MX [3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone] and CMCF [3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone] were synthesized according to the method described by Franze´n and Kronberg (35), and CMA (chloromalonaldehyde) was prepared from MCA as described by Dieckman and Platz (36). The purity of the compounds was at least 98% as determined by proton nuclear resonance (1H-NMR) spectrometry and by gas chromatography. Mutagenicity Assays and Isolation of His+ Revertants. Overnight cultures of the indicator strains were grown in Nutrient Broth No. 2 (Oxoid) at 37 °C in the dark. The experiments with the test compounds were carried out as plate incorporation assays as described by Maron and Ames (37). A total of (1.5-2) × 108 viable cells were plated with overlay agar and aqueous solutions of the test compounds (0.1 mL) on histidine free selective media plates. After 2 days of incubation
Chem. Res. Toxicol., Vol. 9, No. 2, 1996 375 (37 °C), the His+ revertants were counted and independent colonies streaked onto minimal agar supplemented with biotin (37). DNA Colony Hybridization. The purified revertants of strains carrying the hisG46 allele (TA1535, TA1950; TA100) were analyzed by the colony hybridization procedure initially described by Cebula and Koch (28) with 15-nucleotide oligomers with slight modifications (see also refs 25 and 38): The filters were hybridized for 24 h at 37 °C; then CAC, CTC, and CCC were washed in 3x SSC (saline sodium citrate) at 47 °C for 30 min, TCC at 47 °C for 60 min, ACC at 50 °C for 30, and GCC at 55 °C for 35 min. Recently, it has been shown that mutants that hybridize to the probe with the initial CCC sequence contain a supressor mutation in the anticodon region of either of two tRNAThr genes and represent TA f GC transversions (32). Intragenic and extragenic revertants of strain TP2428 (hisG428) were analyzed with 18-nucleotide oligomers essentially according to the procedure described by Prival and Cebula (29), but the washing times were in all cases extended to 24 h. Some of the intragenic revertants did not bind to any of the seven DNA probes used. It has been shown that such clones carry various short deletions that include the mutant site (29, 30). Statistical Analysis. Mutagenic potencies (rev/nmol) were calculated by regression analysis. Comparisons of the hisG46 and hisG428 spectra were performed with χ2 analysis.
Results Figure 2 shows the dose dependent induction of His+ revertants by MX, CMCF, and MCA in Salmonella strains TA100, TA1950, and TP2428. The strongest effects were observed in strain TA100 (Figure 2a): MX induced more than 2000 His+ revertants at a concentration of 0.11 µg/plate (4111 rev/nmol), CMCF generated about 1500 rev at 3.3 µg/plate (81 rev/nmol), and MCA formed about 700 revertants at 3.3 µg/plate (30 rev/nmol, Figure 2a). In derivatives lacking plasmid pKM 101 (TA1950, TA1535), the mutagenic response of the CHFs was considerably lower than in TA100. In strain TA1950 (Figure 2b), the highest revertant number of MX (60 rev/ plate) was induced at a dose of 1.1 µg (9.1 rev/nmol). CMCF induced about 30 revertants/plate at a dose of 11 µg (0.3 rev/nmol), and MCA was only marginally active at the highest dose tested (33 µg/plate). The mutagenic response of the compounds when tested under identical conditions in a rfa strain (TA1535) was more or less the same as those induced in TA1950 (data not shown). In an additional experiment, we tested CMA (a putative breakdown product of MCA) in TA100. The mutagenic activity of the dialdehyde was much weaker than that of MCA. Only at the highest dose tested (200 µg/ plate) was a moderate increase in revertant numbers over the background level found. Figure 2c depicts the induction of mutants by the CHFs in strains TP2428 which carries the hisG428 allele and is otherwise isogenic to TA1950. MX was the strongest mutagen also in this strain and induced about 93 rev/ nmol. CMCF caused only a marginal effect (approximately 0.4 rev/nmol), while MCA was not mutagenic at the highest concentration tested (33 µg/plate). Table 1 summarizes the base specificities of spontaneous and of MX, CMCF, MCA, and CMA induced mutants in strain TA100, and of spontaneous and MX induced revertants in TA1535 and TA1950. In TA1535 and TA1950 the most frequent mutations in the spontaneous spectra are GC f AT transitions (CCC f CTC). Partial loss of the lipopolysaccharide barrier (rfa character in TA1535) had no strong influence on the mutation spec-
376 Chem. Res. Toxicol., Vol. 9, No. 2, 1996
tester strain TA100
Knasmu¨ ller et al.
Table 1. Spontaneous and CHF Induced Mutation Spectra in the hisG46 Allelea total frequency of mutational events per class in %b genetic test mutants background conditions per plate GCC TCC CTC ACC CAC CCCc ∆uvrB; pKM 101; rfa
spontaneous MX (0.11 µg/pl) CMCF (3.3 µg/pl) MCA (3.3 µg/pl) CMA (3.3 µg/pl)
90 2060 1514 689 194
1.3 (1.1) 1.3 (25) 0 (0) 0 (0) 0 (0)
21 (19) 2.5 (51) 3.8 (56) 30 (206) 10 (20)
17 (15) 7.5 (154) 6.3 (94) 62 (430) 31 (60)
TA1535 ∆uvrB; rfa
spontaneous MX (0.3 µg/pl)
14 20
1.5 (0.21) 10 (1.4) 75 (10) 0 (0) 5.1 (1.0) 17 (3.5)
TA1950 ∆uvrB; LPS+
spontaneous MX (1.1 µg/pl)
12 57
1.7 (0.2) 0 (0)
8.3 (1.0) 65 (7.8) 6.7 (3.8) 8.3 (4.7)
16.25 (14.6) 20 (412) 16.25 (246.0) 1.25 (8.6) 12.5 (24.2) 9 (1.3) 7.6 (1.5) 15 (1.9) 3.3 (1.9)
negd
43 (39) 65 (1339) 68 (1040) 2.5 (17) 35 (69)
0 0 (0) 0 (0) 0 (0) 1.3 (2.4)
3 (0.42) 69 (13)
1.5 (0.21) 0 (0) 0 (0) 0 (0)
8.3 (1.0) 0.8 (0.1) 81 (46) 0 (0)
0 3.8 (77) 5 (75) 3.8 (25) 8.8 (17)
0 (0) 0 (0)
a For each experimental condition, 160 revertants were analyzed. b Numbers in parentheses give the revertant yield per class. c CCC is the target sequence of the hisG46 allele, and revertants containing this sequence represent extragenic suppressor mutations of either of two tRNAThr genes (32). d These revertants did not hybridize with any of the six probes; on the basis of earlier findings of Koch (23) and De Marini (41) it is likely that they represent multiple mutations.
a
b
c
Figure 2. Mutagenic activities of MX, CMCF, and MCA in three strains of Salmonella. (a) TA100 (hisG46, ∆uvrB, rfa, pKM 101); (b) TA1950 (hisG46, ∆uvrB, LPS+); (c) TP2428 (hisG428, ∆uvrB, LPS+). Arrows indicate concentrations at which revertants were picked for molecular analysis.
trum, although in the LPS+ strain (TA 1950) a somewhat higher number of GC f TA transversions (ACC and
CAC) was observed. The spontaneous spectrum of TA100 differs substantially from that of TA1535 (χ2 ) 377, p
