Fundamental and Molecular Mechanisms of Mutagenesis
Mutation Research 332 (1995) 9-15
Detection of oxidative mutagens in strains of Escherichia coli deficient in the OxyR or MutY functions: dependence on SOS mutagenesis Amparo Urios, Guadalupe Herrera, Manuel Blanc0
Abstract The Ewherichiu co/i strain IC3811. a 3ouyR derivative of WP2 ul,rA rrpE65. was more sensitive to mutagenicity promoted by t-butyl hydroperoxide and cumene hydroperoxide than the isogenic oxyR+ control. Mutagenicity of menadione. a redox cycling quinone, was clearly detected in the 3o.ryR strain. whereas only a slight mutagenic response was observed in the o.qR+ strain. Plumbagin, another quinone structurally similar to menadione, was not mutagenic to any of the strains. These mutagenic responses appeared to involve the SOS processing of oxidative DNA lesions and were mediated by MucA/B proteins more efficiently than by UmuD/C. In cells lacking mutagenesis proteins, induction of SOS-independent mutations by the two alkyl hydroperoxides required a deficiency in the MutY DNA glycosylase and was increased by the presence of the Jo.,:\R mutation. In contrast, the two quinones assayed were unable to induce SOS-independent mutations in the MutY-deficient strains. Ke,wrw~l.~ Oxidative
mutagen: OxyR: MutY; SOS mutagerwis;
1. Introduction Reactive oxygen species such as superoxide anion radicals ( * 0;). hydrogen peroxide (H,O, ). hydroxyl radicals ( - OH) and singlet oxygen (‘O?) have been implicated in mutagenesis and carcinogenesis. as well as a variety of degenerative diseases (Ames and Gold, 1991; Halliwell and Aruoma, 1991). Salntorlellu tyhimurium tester strains TA IO2 and TA104, carrying the hisG428 ochre mutation, effi-
* Tel.: (6) 3608500:
Alkyl hydroperoxide: Naphthoquinone
ciently detect oxidative mutagens (Levin et al.. 1982). including reactive oxygen species (De Flora et al., 1989). Oxidative mutagens can also be detected with Escherichia coli WP2 derivatives carrying the trpE65 ochre mutation (Wilcox et al., 1990; Kato et al., 1994). However. since aerobic organisms are protected against oxidative stress by scavenging and antioxidant defence systems (for reviews, see Demple, 1991; Farr and Kogoma, 199 I), it is often difficult to prove the mutagenicity of a variety of oxidants (see Farr et al., 1985; Touati and Farr. 1990). To overcome this problem, bacteria defective
Q 1995 Elaevier Science B.V. All rights reserved
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A. Urios et al. /Mutation
in some of the detoxifying enzymes have been used as tester cells in mutagenicity assays. These bacteria, which should be highly sensitive to the mutagenic effects of oxidants, include sodA / B mutants (Touati and Farr, 19901, as well as mutants defective in the mu&t, m&Y, or so.xR/S loci (Kato et al., 1994). In S. typhimurium and E. coli, the OxyR protein positively regulates the expression of several genes encoding antioxidant enzymes such as catalase, alkyl hydroperoxide reductase and glutathione reductase (Christman et al., 1985). According to the results of Storz et al. (1987). OxyR-deficient bacteria would not be useful for mutagenesis assays because of their high spontaneous mutation rate. However, as we have shown recently, the spontaneous mutability exhibited by the oqR_ mutants in plate reversion assays is similar to that of oqR+ cells (Blanc0 et al., 1995). This fact, together with the increased mutagenicity of oxidants such as t-butyl hydroperoxide, could make o.yvR bacteria practical for mutagen screening. The mutagenicity of oxidants appears to be mediated in part by the mutagenic SOS system and therefore involves the bypass of replication-blocking DNA lesions (Levin et al., 1982, 1984). SOS mutagenesis is induced by DNA damage as part of the SOS response and depends on the activity of either UmuD/C proteins or their plasmid-encoded analogs MucA/B proteins (reviewed by Woodgate and Sedgwick, 1992). In addition to SOS-dependent mutations, oxidative stress has been shown to promote a SOS-independent mutagenesis in sodA /B mutants (Farr et al., 1986). Also, oxidatively damaged guanines are known to cause a mutator effect in mutY mutants (Nghiem et al., 1988) which occurs independently of the SOS processing (Urios et al., 1994). It is therefore of interest to know the different mechanisms that can convert the oxidative DNA lesions to mutations. This paper describes a further study of the induction of mutations by chemical oxidants in E. coli WP2 derivatives carrying the AoxyR30 mutation. We examined the mutagenicity of two alkyl hydroperoxides, t-butyl hydroperoxide and cumene hydroperoxide, and two naphthoquinones, menadione and plumbagin. Moreover, the induction of SOS-independent mutations by these oxidants was screened using a AoxyR30 mutY double mutant.
Research 332 (1995) 9- 15
2.1. Bacterial strains and plasmids Bacterial strains are listed in Table 1. Strain IC382 I, carrying the AoqR30 mutation (see Blanc0 et al., 19951, was a tetracycline-sensitive derivative of strain IC381 1 (btuB::Tn10) isolated by the Tn IO-mediated deletion method (Malay and Nunn, 1981). Plasmids were the low copy number pSC 101 (Spc’) derivatives pRWl44 (mucA/B) and pRWl54 (urn&/C) (Ho et al., 1993). 2.2. Media Nutrient broth was Oxoid Nutrient Broth No. 2. Solid minimal E4 medium contained 15 g Difco agar and 4 g glucose per litre of Vogel-Bonner E buffer (Maron and Ames, 1983). Solid ET4 medium was solid minimal E4 medium supplemented with 0.5 mg tryptophan per litre. Top agar contained 6 g Difco agar and 5 g NaCl per litre. 2.3. Plate reversion mutagenesis
To 2 ml of molten top agar at 45°C were added: 100 ~1 of a fresh overnight culture grown in nutrient
Table I Bacterial
trvE65 oxvR+ t&A23 ox.vR+ mutY68::kun trpE65 oxyR+ pRW144 tmucA/B) trpE65 oxyR+ mutY68::kan trpE65 o,yyR+ htuB::Tn IO/ pRWl44(mucA/B) trpE65 d o.q~R30 / pRW144 (mucA/B) trpE65 doxyR30 trpE65 AqyR30/ pRW154 (umuD/C) trpE65 Ao.xyR30 mutY68::karz
IC3789 IC3793 IC3811
IC382 IC3841 IC3844 IC3894
*All strains sulA1.
Source or reference Urios et al. (1994) Urios et al. (1994) IC2869 PI. IC3742
IC3789. Blanc0 et al. (1995) IC38 I I _spont. Tc
IC382 I cured of pRW 144 I(33841 PI. IC3742 X IC3841
A. Urios et al. /Mutation
luted in DMSO and then in distilied oxidants were obtained from Sigma.
broth at 37°C and 100 ~1 of the suitable dilution of the test compound. The mixture was poured on minimal ET4 plates. These were incubated 2 days at strains are prone to a high 37°C. Since the oxyR spontaneous mutability during growth on nonselective media (Blanc0 et al., 1995). it is important that the overnight cultures of these strains are prepared from frozen permanents (at -80°C) known to contain a low revertant level. Moreover, the zero dose in the mutagenesis assays must be screened on unsupplemented E4 plates to verify that preplating mutants had not been originated during the overnight growth. Experiments were carried out at least three times, and three plates per dose were employed. r-Butyl hydroperoxide was diluted in distilled water, whereas cumene hydroperoxide, menadione (2methyl-l .6naphthoquinone) and plumbagin (5hydroxy-2-methyl-l,4-naphthoquinone) were first di-
Research 332 f 1995) 9-15
3. Results We analyzed the effect of the AqvR30 mutation on the reversion of the trpE65 ochre allele by four selected oxidative mutagens. The strains carried a full deletion of the chromosomal umuD / C operon and contained plasmid pRW144 (mucA /B), so that only one class of mutagenesis proteins was present in them. The dose-response curves in Fig. 1 indicated that the number of Trp’ revertants induced by t-butyl hydroperoxide (BHP) and cumene hydroperoxide (CHP) in the AoqR strain IC3821 was higher than in the oxyR+ control IC3789. The mutagenicity of menadione was clearly detected in the AqvR
800 al xi a , CA F (II r
z L 400
Fig. 1. Effect of the Ao.qR30 mutation on the reversion of the trpE65 ochre mutation by t-butyl hydroperoxide (left panel) and cumene hydroperoxide (right panel). Squares. IC3789 (oxyR+): triangles, IC3821 (Aox~1R30). Each point represents the mean ( f SEM) of three independent experiments.
Table 2 Mutability proteina
strain but, in contrast, only a slight mutagenic response was observed in the o.pR+ strain (Fig. 2). Plumbagin was not mutagenic to any of the strains (Fig. 2). Note in Figs. 1 and 2 that the lethal toxicity of the oxidants was enhanced in the OxyR-deficient cells, causing a decrease in the number of revertants at the highest concentrations. The effects of the AqvR30 mutation on cell mutability were also analyzed in strains containing UmuD/C proteins instead of MucA/B. As shown in Table 2, UmuD/C proteins promoted the mutagenicity of BHP and menadione with lower efficiency than MucA/B. To detect the induction of SOS-independent mutations we used strains containing no mutagenesis proteins. As shown in Table 3, a weak mutability after treatment with BHP or CHP was exhibited by strains IC3841 ( AoxyR) and IC2869 (rnyR+ 1.
~ +BHP + Menadione
20+3.3 381+ 26.0 308? 15.1
+ BHP + Menadione
70+ 13.6 IO66 f 24.3 750 + 18.4
’ The BHP dose was 100 pg/plate:
Trp’ colonies per plate (mean + SEM)
dose was 30
Derivatives of these strains carrying the tnutY6K mutation were also used since a deficiency in the MutY DNA-glycosylase appears to improve the de-
loo/~ , 01
Fig. 2. Effect of the do.r.vR30 mutation on the reversion of the trpE65 ochre mutation by menadione (left panel) and plumbagin (right of three independent panel). Squares. IC3789 (o.ryR+); triangles, IC3821 ( ilo.~~R30). Each point represents the mean (+SEM) experiments.
Table 3 Effect of a MutY deficiency on the mutability of strains lacking mutagenesis proteins Compound
Dose ( PcLg Per plate)
_ BHP CHP Menadione Plumhagin
Trp+ colonies per plate (mean f SEM)
IC384 I (Jm:vR
( o.\vR +
39 f 9.0
37 * 5.4
33 i 2.6
173 & 14.0
55 f 5.0
23 f 7.8
56 + 5.0
I8 + 2.2
I57 + 15.0
76 + 3.7
33 * I.9
335 * 28.7
98 f 7.6
38 i 5.7
46 + 2.8
9 + 1.6
51 * 3.7
34+37 _ _._
I3 + 7.4
34 + 3.0
33 * 2.0
IO + 2.4
I2 * 2.0 -
35 + 3.3
34 i 4.0
tection of SOS-independent mutations (Urios et al., 1994). The results indicated the occurrence of a significant induction of Trp+ revertants by both BHP and CHP in the m~ltY strains (Table 3). Moreover, the Aox.vR mutY double mutant strain IC3894 was more sensitive to the SOS-independent mutagenicity of alkyl hydroperoxides than the oxyR+ mutY single mutant IC3793. The comparison of the level of mutants induced by BHP or CHP in strains IC3821 (Jco.qR/pRW 144) (Fig. I) and IC3894 ( AmyR tnurY) (Table 3) indicated that, on average, the yield of mutations dependent on the SOS processing mediated by MucA/B proteins was about 4 times the yield of SOS-independent mutations. Mutagenic activity of either menadione or plumbagin was not detected with strain IC3894 ( AmyR mutY) (Table 3). Since menadione was mutagenic to IC3821 (Fig. 2), it seems that reversion of the trpE6.5 mutation by this quinone can only occur via the SOS processing mechanism. However, menadione failed to induce the SOS response. assayed by measuring the expression of the wnd-llac fusion gene on plasmid pSKl002 (Oda et al., 198.5). in o.ryR+ cells (Kato et al., 1994), and in the AoxyR30 mutant (data not shown).
4. Discussion In this paper we have shown that bacteria carrying a deletion of the o,yvR gene exhibit an increased sensitivity in detecting oxidative mutagens. The Ao.yvR cells have two other advantages in obtaining
31 I f
86 f 6.3 I I9 _t 13.0 67 + 4.5
quantitative results in mutagenesis assays. First, they grow similarly to the corresponding o.pR+ cells. This may be due to the fact that the Ao.r_vRmutation affects a gene encoding a regulatory protein but not genes encoding antioxidant enzymes. The constitutive level of these enzymes might protect cells against the endogenous reactive oxygen species generated during normal growth. After treatment with oxidants, defence genes would not be induced in the OxyR-deficient bacteria, which would then appear hypersensitive to the oxidative damage. The second advantage of the AmyR cells is their normal spontaneous mutability when tested in the plate reversion assay (Blanc0 et al., 1995). However, since that mutability may be enhanced during growth on nonselective media (Blanc0 et al., 1995). problems derived from this fact can be avoided by using cultures which contain a low number of preexisting mutants. The increase in both mutagenic and lethal effects of BHP and CHP observed in the Ao.qR mutants could account for their inability to overexpress the nhpC/F operon encoding the alkyl hydroperoxide reductase (Storz et al., 1989). Such effects are attributed to the highly reactive alkoxy radicals produced by reduction of hydroperoxides in the presence of a suitable transition metal such as Fe”. Alkoxy radicals may damage DNA by causing strand breaks and by forming base adducts (Simic et al., 1989). In OxyR-deficient strains. high levels of strand breaks could increase the cell killing by BHP and CHP, whereas the enhanced mutagenicity of these oxidants would probably be promoted by abasic sites generated from the adducted bases.
A. Urios et al. /Mutation
Menadione and plumbagin have been shown to be mutagenic in E. coli strains deficient in either superoxide dismutase or catalase (Prieto-Alamo et al., 1993). Our results show that cells proficient in these detoxifying enzymes but defective in the OxyR function exhibit a significant mutability by menadione. Treatment with this quinone of cells containing superoxide dismutase must lead to high levels of . 0; radical able to generate HZOZ by dismutation. In the AoxyR mutants, since there is not stimulation of hydroperoxidase I synthesis (Christman et al., 1985), H20Z will accumulate and generate - OH radicals by metal-catalyzed reduction. This could explain the enhanced mutagenicity of menadione in the Ao~R strain. Note that the absence of mutability by menadione has been reported by Kato et al. (19941 in a oxyR+ strain similar to IC3789, thus confirming the requirement for a deficiency of OxyR in order to detect the reversion of the trpE65 mutation by menadione. In contrast to this result, the OxyR deficiency was unable to allow the detection of Trp’ revertants induced by plumbagin. Since a similar absence of mutagenicity to strain IC3821 has been observed for two other quinones, 1,4_naphthoquinone and 5-hydroxy- 1,4_naphthoquinone (unpublished results), we suggest that there are differences in the reactive oxygen species produced during the autoxidation of menadione and that of the other quinones assayed. It would confirm the difficulty to draw a general scheme applying to the cellular metabolism of quinones (see Hassan and Fridovich, 1979; Cadenas, 1989). The weak mutability exhibited by strains lacking mutagenesis proteins (Table 3) indicated that the mutagenic responses obtained with the correspondent strains containing either AoqR30 and oxyR+ MucA/B ( Figs. 1 and 2) or UmuD/C proteins (Table 2) were mainly mediated by the SOS processing of the oxidative DNA lesions. Moreover, the results of Table 3 confirmed the usefulness of cells deficient in the MutY function to detect SOS-independent mutations (see Urios et al., 1994). MutY is a DNA-glycosylase that removes adenine misincorporated opposite S-oxoguanine (Au et al., 1989). This altered base results from the attack of hydroxyl radicals or singlet oxygen on guanine (reviewed in Tchou and Grollman, 1993). Treatment with alkyl hydroperoxides might cause a high frequency of 8-oxoG . A mispairs which would promote the oc-
Research 332 f 1995) 9- 15
currence of revertants of the trpE65 mutation in MutY-deficient cells. In contrast, it seems that low levels of 8-oxoG . A mispairs are originated by menadione or plumbagin, since none of these quinones caused a significant induction of Trpf revertants in the mutY cells. In conclusion, our results have demonstrated that the use of OxyR-deficient tester strains may improve the quantitative evaluation of the mutagenicity of oxidants in the standard plate mutagenesis assay. The ability of the OxyR deficiency to enhance the mutagenicity of a given compound might suggest that reactive oxygen species participate in that mutagenicity. The oxyR- mutations could have an effect on the sensitivity to oxidative mutagens similar to that of utir- mutations on the activity of mutagens that make bulky DNA adducts. Moreover, the OxyR deficiency can be combined with other functional changes such as the presence or absence of different mutagenesis proteins and/or deficiencies in repair processes (e.g. absence of MutY) in order to obtain a better understanding of the mechanisms involved in mutagenesis.
Acknowledgements This work was supported by Comisidn Interministerial para la Ciencia y Tecnologia grant SAF930056. A.U. is the recipient of a Forrnacidn de Postgrado Fellowship from the Ministerio de Education y Ciencia.
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