Nickel(II) genotoxicity: potentiation of mutagenesis of simple alkylating agents

Mutation Research, 162 (1986) 187-199

187

Elsevier MTR 04218

Nickel(II) genotoxicity: potentiation of mutagenesis of simple alkylating agents Jeffrey S. Dubins * and James M. LaVelle ** Toxicology Program, Section of Pharmacology and Toxicology, School of Pharmacy, Box U-92, University of Connecticut, Storm, CT 06268 (U.S.A.) (Received 12 September 1985) (Revision received 5 March 1986) (Accepted 20 March 1986)

Summary Many metals have been shown to alter the function of a wide range of enzyme systems, including those involved in DNA repair and replication. To assess the impact in vivo of such metal actions a "Microtitre" fluctuation assay was used to examine the ability of Ni(II) to act as a comutagen with simple alkylating agents. In E. coli, Ni(II) chloride potentiated the mutagenicity of methyl methanesulfonate (MMS) in polymerase-proficient strains (WP2 + and WP2-), but not in poM- strains (WP6 and WP67) or in lexA(CM561) or recA- (CM571) strains. The absence of UV excision repair (WP2- and WP67) had little, if any, effect. An extended lag phase was seen at 2-4 h in the polA- strains following treatment with Ni(II) cMoride and MMS, but normal growth resumed thereafter. Results suggested that mutations induced by MMS were fixed during log phase growth and that more than 2 h of exposure were necessary for potentiation by Ni(II) to be observed. Thus, the extended lag phase probably cannot explain the lack of potentiation. RecA-dependence of the comutagenic effect was corroborated with S. typhymurium TA1535 and TA100. Only in the pKM101 containing strain, TA100, was potentiation of ethyl methanesulfonate (EMS) and MMS by Ni(II) chloride evident. The mucAB genes carried on pKM101 increase the sensitivity of TA100 to a variety of mutagens, providing there is a functional recA gene product. Taken together, the data suggest that Ni(II) acts indirectly, as a comutagen, in bacterial systems, possibly affecting processes involving recA- and/or poL4-dependent function(s).

Human epidemiologic and in vivo studies with experimental animals strongly suggest that Ni(II) is carcinogenic (Sunderman, 1979; Christie and Costa, 1983), yet mutagenicity in bacteria has not

* Current addresses: Department of Microbiology, University of Medicine and Dclltistry of New Jersey, 100 Bergen St., Newark NJ 07103 (U.S.A.). ** To whom reprint requests should be addressed. Send correspondence to James M. LaVelle.

been demonstrated (Sunderman, 1979). This anomaly might be explained if Ni(II) effects were indirect, involving enzymes necessary for repair of damage to DNA or enzymes involved in DNA synthesis. Divalent Ni(II) has been shown to substitute for other divalent metal ions, such as magnesium and zinc (Ciccarelli and Wetterhahn, 1984), thereby affecting a variety of enzymes. This may explain nickel's ability to decrease polymerase fidelity in vitro (Sirover and Loeb, 1976). A problem in studying this and similar effects

002%5107/86/$03.50 ©. 1986 Elsevier Science Publishers B.V. (Biomedical Division)

188

has been the lack of a convenient bacterial assay for studying compounds not directly genotoxic. Recent work by Rossman (1981a,b), LaVelle and Witmer (1984) and Mandel and Ryser (1984), however, demonstrates that indirect effects may be investigated by observing the interactions of metals with non-metal DNA-damaging agents in bacterial systems. Under these circumstances, one expects an indirect-acting agent (comutagen) to enhance the mutagenic consequences of DNA damage. For such experiments, a bacterial fluctuation assay was used. This method has been used successfully to monitor comutagenic effects of Cr(VI) in the absence of significant toxicity (LaVelle and Witmer, 1984). The test was applied to the investigation of Ni(II) in the hope that the sensitivity of the test would again obviate potential toxicity artifacts. Materials and methods

Bacterial strains All S. typhimurium strains were a gift from Dr. Bruce N. Ames, University of California, Berkeley, CA. E. coli strains WP6 and WP67 were a gift from Dr. Evelyn Witkin, Rutgers University, Piscataway, NJ and E. coli strains WP2 +, WP2-, CM561 and CM571 were supplied by Dr. Clifford Selsky, Stauffer Chemical Company, Farmington,

TABLE 1 GENOTYPES AND STRAINS USED Strain

SOURCES

Genotype

Escherichia coli WP2 + trp WP2trp, uvrA WP6 trp, polA WP67 CM561 CM571

trp, polA, uorA trp, lexA trp, recA

Salmonella typhimurium TA100 hisG46; AuvrB, chl, bio; rfa; pKM101 TA1535 hisG46; AuvrB, chl, bio;

rfa

FOR

BACTERIAL

Reference Green and Muriel (1976) Green and Muriel (1976) Witkin (personal communication) Green and Muriel (1976) Green and Muriel (1976) Green and Muriel (1976) Maron and Ames (1983) Maron and Ames (1983)

CT. Genotypes for all strains are provided in Table 1. Chemicals EMS (CAS No. 62-50-0, 99% pure) and MMS (CAS No. 66-27-3, 97% pure) were obtained from Sigma Chemical Co. (St. Louis, MO) and were stored in a desiccator at 4°C. Fresh solutions of these chemicals were prepared in DMSO just before use. Spectrophotometric grade DMSO (CAS No. 67-68-5) was obtained from EM Sciences. Nickel(II) chloride (CAS No. 7791-20-0, 99.9% pure) was obtained from Fisher Scientific (Certified ACS). Solutions of Ni(II) chloride were made in normal saline. All other chemicals were Fisher certified reagents. Bacterial cultures Bacterial cultures were inoculated from master plates as described by Maron and Ames (1983). Culture methods were taken from LaVelle and Witmer (1984). To summarize, bacterial cultures were grown in TS broth for 4 h at 37°C in a shaking water bath. To prepare selective media, Davis-Mingioli salt solution (DM salts) was supplemented to a final concentration of 1% glucose and 2.9/~M L-tryptophan for the E. coli strains. For the S. typhimurium strain TA1535, the medium was supplemented to a final concentration of 1% glucose, 4.8/~M D-biotin, and 4.8/~M L-histidine. For S. typhimurium strain TA100, the histidine concentration was reduced to 1.2/~M. This reduction was necessary because the high spontaneous mutation rate in TA100 gives too many positive wells in the controls at higher histidine concentrations, making it difficult to resolve comutagenic effects. While changing the overall response (number of positive well observed), reducing the histidine concentration did not noticeably affect mutagenic potency of either MMS or EMS and so had little effect on the sensitivity of the assay. Glucose, histidine, biotin and tryptophan were filter sterilized. Fluctuation tests Fluctuation tests were performed and analyzed by the 'micro' version methods of Gatehouse (1978) as adapted by LaVelle and Witmer (1984). Briefly, bacterial cultures (5 × 105 cells/ml) in

189 selective medium containing appropriate concentrations of test agent(s) were dispensed into a 96-well microtitre plate via an 8-port manifold (0.19 ml/well). These plates were then incubated at 37°C for 72-86 h. Bromocresyl purple (0.07 ml/well) was added at the end of the incubation period to monitor acidity produced by bacterial growth. Toxicity assays

Bacteria were incubated for 4 h in TS broth, centrifuged, resuspended in DM salts, and diluted in DM medium to a final concentration of 10 s cells/ml. Appropriate agents were added, a 0.5-ml aliquot was removed and the remaining culture incubated at 37°C in a shaking water bath. The 0.5-ml aliquot was diluted to a final concentration of approximately 102 cells/ml, plated on nutrient agar, incubated for 24 h at 37°C and the number of colony-forming units scored. This procedure was repeated at 2, 4, 8, 12 and 24 h. Results were compared with concurrently run controls to estimate relative survival and growth. Pretreatment assays

Bacteria were incubated for 4 h, centrifuged, resuspended in DM salts, and diluted in either DM salts or supplemented minimal media (SMM; 80 ml DM salts, 18 ml TS broth, 2 ml 40% glucose) to a final concentration of l0 s cells/ml. Appropriate agent(s) were added to bacterial cultures in either DM salts or SMM. Bacteria were then incubated either on ice, at room temperature, or in a 37°C shaking water bath. After 20-rain incubations, bacteria were centrifuged at 2400 g for 5 min at 4°C (to achieve rapid sedimentation of cells), resuspended in DM salts and dispensed into microtitre plates as previously described. After 2-h and 18-h incubations, bacteria were centrifuged at 1400 g for 15 min at 4°C, resuspended in DM salts and dispensed into microtitre plates. Data analysis

Raw data (numbers of positive wells) were used to calculate the average number of induced mutations per well (AIMW) for each treatment, using the control values to correct for spontaneous mutation. The significance of differences between individual treatments and controls was estimated

by calculation of a Chi-square statistic with 1 degree of freedom. For analysis of synergistic effects, AIMW values for the agents tested alone were combined giving an expected value. This was then compared, again using Chi-square, with the AIMW value actually observed when the combination of agents was tested. Complete details of these methods have been published (LaVelle and Witmer, 1984; Forster et al., 1980). Table 2 gives both the raw data and the transformed data as an example. The transformed data are omitted from the other tables for clarity. Results

Initial experiments with MMS and EMS alone were used to determine appropriate concentrations of these agents for the comutagenicity assays. Concentrations which produced consistent and significant increases in AIMW, but still left the majority of the test wells negative so that potentiation could be demonstrated, were selected. For MMS, appropriate concentrations ranged between 0.1 and 1.4 mM depending on the strain (Table 2-4). EMS concentrations of near 1 mM were used in all strains. Nickd(II) was not mutagenic in any of the stains (data not shown) and concentrations in comutagenicity assays were initially varied over a 10-fold range (3-30 #M) to determine effective concentrations. The highest Ni(II) concentrations used were just sufficient to reduce CFU in cultures treated with Ni(lI) relative to controls (data not shown). Using the above concentrations, Ni(II) chloride was able to potentiate mutagenicity of MMS in a dose-dependent manner in E. coli strains WP2 + and WP2-, which are polymerase I proficient (Table 2), but it had no effect on mutagenicity in polymerase I deficient strains, WP6 and WP67 (Table 3). Similarly, Ni(II) did not potentiate MMS mutagenicity in l e x A - (CM561) or recA - (CM571) strains (Table 4). In none of the above strains did Ni(II) chloride potentiate the mutagenicity of EMS. These results are consistent with a significant comutagenic action of Ni(II) dependent upon functional recA and poiA gene products. However, the lack of potentiation in several strains could also reflect increased toxicity of the MMS/Ni(II) combination. In fact, some toxicity

190

by MMS/Ni(II) cotreatment (as indicated by an extended lag phase and/or some cell killing) was seen in strains WP6 and WP67, the two polymerase-deficient strains, at 2 and 4 h after the

addition of agents. However, by 8 h, log phase growth returned (Fig. 1). No such toxicity was seen in either WP2 ÷ and WP2- (Fig. 2). Thus, it seemed possible that either toxicity was reducing

TABLE 2 T H E EFFECT OF Ni(II) C H L O R I D E ON THE M U T A G E N I C I T Y OF MMS IN E. coli STRAINS WP2 + A N D W P 2 Concentrations a Ni chloride

Positive wells b MMS

2.9

0.7

5.7

0.7

8.6

0.7

28.6

0.7

Ni

MMS

Chi-squares c

p

0.39)

> 0.50

4.23

< 0.05

10.9

< 0.001

36.3

< 0.001

2.53

> 0.10

4.02

< 0.05

4.15

< 0.05

5.57

< 0.02

1.30

> 0.25

1.61

> 0.20

1.06

> 0.30

6.21

< 0.02

13.1

< 0.001

20.4

< 0.001

Ni - MMS

WP2 + (parental) Spontaneous positive wells = 6 Spontaneous AMW = 0.06 (0.03) 6 8 5 [0.02 (0.05), 0.01 (0.03)1 6 8 20 [0.02 (0.05), 0.17 (0.05)] 3 8 25 [0.01 (0.05), 0.24 (0.06)] 2 8 50 [0.02 (0.05), 0.67 (0.11)]

O8

2.9

1.0

5.7

1.0

8.6

1.0

28.6

1.0

2.9

1.0

5.7

1.0

7.1

1.0

8.6

1.0

14.3

1.0

28.6

1.0

Spontaneous positive wells = 14 d Spontaneous AMW = 0.16 (0.04) 6 76 83 [1.32 (0.21), 1.84 (0.26)] 17 76 87 [1.45 (0.21), 2.21 (0.32)1 16 76 87 [1.44 (0.21), 2.21 (0.32)] 6 76 87 [1.32 (0.21), 2.21 (0.32)] W P 2 - (uvrA) Spontaneous positive wells = 10 e Spontaneous AMW = 0.05 (0.02) 5 39 46 f [0.42 (0.09), 0.58 (0.10)1 7 80 91 I0.47 (0.06), 0.59 (0.07)1 6 41 50 r [0.56 (0.09), 0.70 (0.11)] 6 80 103 I0.46 (0.06), 0.72 (0.08)1 2. 41 67 f [0.52 (0.09), 1.17 (0.15)] 4 39 72 r [0.41 (0.09), 1.31 (0.18)]

Ni(II) chloride in micromolar; MMS in millimolar. b Number of positive wells in fluctuation assays for comutagenesis. Numbers in [ ] are expected (first number) and observed (second number) AIMW's calculated for the data provided. Numbers in ( ) are standard deviations. c Two-tailed chi-square with a df. d Totals wells per treatment = 96. e Total wells per treatment = 192. f Total wells per treatment = 96, spontaneous positive wells = 5, spontaneous AMW = 0.05 (0.024).

191 TABLE 3 T H E EFFECT OF Ni(II) C H L O R I D E ON THE M U T A G E N I C I T Y OF MMS IN E. coli STRAINS WP6 A N D WP67 a Concentrations. Ni chloride

Positive wells" MMS

Ni

MMS

Chi-square"

P

Ni + MMS

wP6 ( poL4 ) 2.9 5.7 8.6 28.6

2.9 5.7 8.6 28.6

1.2 1.2 1.2 1.2

Spontaneous positive wells = 9 b 1 39 36 4 39 37 3 39 36 3 39 36

0.19 0.07 0.06 0.06

> > > >

0.50 0.75 0.80 0.80

1.0 1.0 1.0 1.0

WP67 (polA, uvrA) Spontaneous positive wells = 21 b 10 75 75 8 75 77 14 75 56 10 75 81

0.51 1.07 1.86 1.64

> = > =

0.50 0.30 0.20 0.20

a See Table 2 for concentrations, data analysis and statistics. b Total wells per treatment ffi 192.

the number of mutants arising early in the treatment or fewer mutants were arising during exponential growth due to increased repair of lesions during the extended "lag phase" or depletion of MMS in the medium. The former possibility seems ruled out by experiments in which WP6 cells were exposed to MMS in DM salts, then plated on medium containing no tryptophan so that only

preexisting mutants would be scored. After 4 h of incubation, no significant mutagenicity was observed. In contrast, exposure to MMS in SMM produced a large increase in the numbers of positive wells induced (Table 5). This indicates that few mutations are fixed prior to the onset of exponential growth in these experiments. Further, WP6 cells were incubated in medium containing

TABLE 4 T H E EFFECT OF Ni(II) C H L O R I D E ON THE M U T A G E N I C I T Y O F MMS IN E. coli STRAINS CM561 A N D CM571 a Concentrations" Ni chloride

2.9 5.7 8.6 28.6

2.9 5.7 8.7 28.6

Positive wells a

Chi-square a

p

1.4 1.4 1.4 1.4

CM561 ( lexA) Spontaneous positive wells ffi 5 b 5 23 19 3 23 21 6 23 25 5 23 26

0.35 0.001 0.02 0.18

> = > >

0.50 0.975 0.80 0.50

1.4 1.4 1.4 1.4

CM571 ( recA ) Spontaneous positive wells = 4 32 6 32 5 32 10 32

1.97 1.44 0.24 0.67

> > > >

0.10 0.25 0.50 0.30

MMS

Ni

MMS

a See Table 2 for concentrations, data analysis and statistics. b Total wells per treatment ffi 192.

Ni + MMS

6 b 20 23 35 29

192 Ni(II), M M S or a c o m b i n a t i o n for 4 h. The WP6 cells were removed by filtration and the filtrates innoculated with W P 2 ÷ cells and u s e d in a standard fluctuation assay. The results show significant mutagenicity in filtrates containing M M S alone (spontaneous positive wells = 3, M M S filtrate positive wells = 71, Chi-square = 21.7, p < 0.001). In addition, Ni(II) potentiation was still observed when expected versus observed A I M W ' s were calculated (expected = 1.31 (71 positive wells), observed = 4.53 (96 positive wells), Chisquare = 10.2, p < 0.001). (In this experiment, Ni(II) treatment p r o d u c e d only 3 positive wells. T h e A I M W calculated for the c o m b i n a t i o n treatm e n t is actually a m i n i m u m , since all of the wells were positive in this plate.) Thus, the exponential growth phase by itself seems sufficient for expression of comutagenesis b y Ni(II). T o corroborate the findings with E. coli, two S.

typhimurium strains were used. Nickel(II) chloride potentiated mutagenicity of both E M S and M M S in TA100 (Table 6). Such potentiation was not seen in TA1535 (Table 7). Since these two strains are isogenic, the presence of the p K M 1 0 1 plasmid in TA100 p r e s u m a b l y accounts for the difference observed. Further, it is TA100 that resembles the E. coil strains, WP2 ÷ and W P 2 - in its response to nickel. This m a y implicate plasmid-borne genes in the action of Ni(II). Interestingly, in TA100 it was necessary to use substantially higher concentrations of Ni(II) to observe potentiation than were required in WP2 ÷ and W P 2 - . This suggests some resistance in S. typhimurium to the effects of nickel, but no data are available on which to base a mechanism. T o further explore comutagenesis by Ni(II), it was deemed i m p o r t a n t to see whether Ni(II) and M M S had to be present simultaneously. W P 2 ÷

109

/J

107

/ 105l

I

I

I

I

I

I

4

8

12

16

20

24

I

l

I

4

8

I

12

I

I

I

16

20

24

T(h) Fig. 1. Effect of cotreatment with Ni(II) chloride and either MMS or EMS on the growth of E. coli strains WP6 (A) and WP67 (B). The closed squares represent control. The open squares represent bacteria treated with 28.6/~M Ni and 1.2 mM MMS (WP6) or 1.0 mM MMS (WP67). The stars represent cells treated with the same Ni(II) concentration and 0.9 mM EMS. Standard error bars are left off for clarity, hut never was the standard erro't greater than 10% of the mean.

193 A

109

10B

106

10 5

I

I

I

I

I

1

I

I

4

8

12

16

20

24

4

8

I

12

I

I

I

16

20

24

T(h) Fig. 2. Effect of cotreatment with Ni(II) chloride and either M M S or EMS on the growth of E. coli strains WP2 + (A) and W P 2 - (B). Symbols and concentrations are the same as for Fig. 1, except that the M M S concentration in all experiments was 1.2 raM.

was pretreated with MMS for 20 rain either at room temperature or while on ice (to slow enzymatic repair of the MMS-induced lesions). Ni(II) posttreatment had no effect on MMS mutagenesis TABLE 5 M U T A T I O N I N D U C E D BY M M S IN E. coli WP2 + D U R I N G A 4-h I N C U B A T I O N IN E I T H E R D M SALTS O R SMM a Concentration

Positive wells a

of M M S (mM)

D M salts

SMM

0.0 0.8 1.2 1.6 2.0

0 0 0 1 0

1 6 11 9 19

a

p

> < < < <

0.99 0.05 0.05 0.10 0.001

Bacteria were incubated in the presence of M M S in either D M salts or SMM, washed with D M salts and plated in medium containing no tryptophan. Chi-square analysis indicates significance levels for differences between D M salts and SMM incubated cells at each exposure level.

in these experiments (Table 8). Extending the preincubation time to 2 h did not alter these results (data not shown). However, it was also noted that incubation of WP2 + cells in the presence of Ni(II), MMS or both in either DM salts or SMM for 2 h followed by washing and plating in the standard assay was insufficient to demonstrate potentiation by Ni(II) (Table 9). This was true even through the concentrations of Ni(II) used were increased 3-fold over those used previously (Table 2). Since MMS exposure was clearly mutagenic by itself in these experiments, the possibility of some delay in the action of Ni(II) was hypothesized. To test this hypothesis, experiments with extended preincubations in the presence of Ni(II) were run. Because Ni(II) potentiated MMS mutagenicity in an 18-h coincubation experiment (Table 10), an overnight incubation in DM salts in the presence of Ni(II) was used for pretreatment. (The reduction in the mutant yield at 30 #M (Table 10) is assumed to be due to toxicity, since reductions in

194

TABLE 6 THE EFFECT OF Ni(II) CHLORIDE ON THE MUTAGENICITY Concentrations a N i chloride

O F M M S A N D E M S I N S. typhimurium S T R A I N T A 1 0 0 a

Positive wells a

Chi-square a

p

Mutagen

Ni

MMS

14.3 28.6 42.9 57.1

0.1 0.1 0.1 0.1

MMS Spontaneous 10 10 11 4

positive well = 9 b 28 39 28 43 28 47 28 52

1.98 3.76 5.55 7.81

> > < <

0.10 0.05 0.02 0.01

14.3 28.6 42.3 57.1 71.4

0.8 0.8 0.8 0.8 0.8

Spontaneous 7 6 7 4 7

positive wells = 6 b 84 81 84 88 84 92 84 92 84 92

0.42 0.87 3.61 4.0 3.77

> > > < >

0.5 0.3 0.05 0.05 0.05

1.1 1.1 1.1 1.1 1.1

EMS Spontaneous 41 40 45 35 22

positive wells'32 b 57 65 57 78 57 98 57 115 57 40

0.00 1.29 5.51 18.9 3.84

= > < < =

0.999 0.25 0.02 0.001 0.05

14.3 28.6 42.9 51.1 71.4

Ni + MMS

a E M S c o n c e n t r a t i o n in millimolar. See T a b l e 2 for other c o n c e n t r a t i o n s , d a t a analysis a n d statistics. b T o t a l wells per t r e a t m e n t = 96. ¢ T o t a l wells per t r e a t m e n t = 288.

TABLE 7 THE EFFECT OF Ni(II) CHLORIDE

ON THE MUTAGENICITY

Concentrations a

Positive wells a

N i chloride

O F M M S A N D E M S I N S. typhimurium S T R A I N TA1535 a Chi-square a

Ni

MMS

1.2 1.2 1.2 1.2

MMS Spontaneous 18 16 16 8

p o s i t i v e wells = 18 b 54 50 54 43 54 56 24 20

0.17 0.94 0.13 0.32

> > > >

0.50 0.30 0.70 0.50 c

14.3 28.6 42.9 51.7 71.4 85.7

0.9 0.9 0.9 0.9 0.9 0.9

EMS Spontaneous 14 13 17 13 8 9

p o s i t i v e wells = 10 ~ 30 28 30 30 30 22 30 23 30 23 30 24

0.50 0.10 3.52 1.71 0.60 0.54

> = > > > >

0.30 0.75 0.05 0.10 0.30 0.30

14.3 28.6 42.9 51.7 71.4 85.7

1.1 1.1 1.1 1.1 1.1 1.1

Spontaneous 4 4 5 4 7 4

p o s i t i v e wells = 5 c 37 33 37 33 37 35 37 31 37 23 37 15

0.23 0.23 0.08 0.59 4.93 10.5

> > > > < <

0.50 0.50 0.75 0.30 0.05 0.005

28.6 57.1 85.7 143.0

Ni + MMS

E M S c o n c e n t r a t i o n in millimolar. See T a b l e 2 for other c o n c e n t r a t i o n s , d a t a a n a l y s i s a n d statistics. b T o t a l wells per t r e a t m e n t = 192. c T o t a l wells per t r e a t m e n t = 96. a

p

Mutagen

195 TABLE 8 T H E E F F E C T OF Ni(II) C H L O R I D E ON M U T A G E N I C I T Y F O L L O W I N G P R E T R E A T M E N T O F E. coil S TR A IN WP2 + W I T H MMS a Concentrations a Ni chloride

Positive wells a

Chi-square a

p

ice 7• 16 18 23 18

0.44 0.01 0.03 0.04

> > > >

0.50 0.90 0.75 0.75

7c 20 17 13 24

0.001 0.06 2.01 0.34

> > > >

0.98 0.75 0.10 0.50

18 18 18 18

Pretreatment carded out at room temperature Spontaneous positive wells = 8 c 13 19 19 5 19 19 11 19 18 11 19 19

0.44 0.17 0.31 0.16

> > > >

0.50 0.50 0.50 0.50

24 24 24 24

Spontaneous 13 5 11 11

0.44 1.28 0.65 0.09

> > > >

0.75 0.25 0.30 0.75

MMS b

Ni

MMS

2.9 8.6 14.3 28.6

18 18 18 18

Pretreatments carried out on Spontaneous positive wells = 6 21 4 21 8 21 5 21

2.9 8.6 14.3 28.6

24 24 24 24

Spontaneous 6 4 8 6

2.8 8.6 14.3 28.6

2.9 8.6 14.3 28.6

positive wells = 21 21 21 21

positive wells = 13 13 12 13

Ni + MMS

8c 16 17 11 14

a See Table 1 for concentrations, data analysis and statistics. b Cells were exposed to MMS for twenty minutes either on ice or at room temperature. c Total wells per treatment = 96.

TABLE 9 E F F E C T O F Ni(II) ON T H E M U T A G E N I C I T Y OF MMS D U R I N G A 2-h E X P O S U R E IN E I T H E R D M SALTS O R S U P P L E M E N T E D M I N I M A L M E D I A U S I N G E. coli STRAIN WP2 + Concentrations a Ni chloride

30.0 60.0 100.0

30.0 60.0 100.0

Positive wells a

Chi-square a

p

MMS

Ni

47 47 47

D M Salts Spontaneous 13 6 5

positive wells = 6 c 36 42 36 40 36 42

0.04 0.32 0.89

>

47 47 47

SMM Spontaneous 11 11 5

positive wells = 8 c 65 62 65 60 65 59

0.37 0.82 0.52

> 0.50 > 0.30 > 0.30

MMS

a See Table 2 for concentrations, data analysis and statistics. b Total wells per treatment = 96.

N i + MMS

0.80 > 0.50 > 0.30

196 TABLE 10 T H E EFFECT OF 18-h Ni(II) C H L O R I D E PRETREATMENT ON T H E M U T A G E N I C I T Y OF MMS IN E. coil STRAIN WP2 ÷ Concentrations a Ni chloride b

15.0 30.0

15.0 30.0

15.0 30.0

Positive wells a

Chi-square ~

P

1.1 1.1

Combined Ni(II)/MMS in pretreatment' no posttreatment Spontaneous positive wells = 7 ¢ 9 16 92 9 16 34

36.3 5.56

< 0.001 < 0.02

0.7 0.7

Ni(II) only in pretreatment; MMS only in posttreatment Spontaneous positive wells'7c 9 73 73 7 73 86

0.008 5.27

> 0.90 < 0.025

0.7 0.7

Ni(II) only in pretreatment; nickel and MMS in posttreatment Spontaneous positive wells = 7 c 9 81 92 9 81 93

5.67 6.61

< 0.02 = 0.01

MMS

Ni

MMS

Ni + MMS

a See Table 2 for concentrations, data analysis and statistics. b Cells pretreated with Ni(II) for 18 h in DM salts at concentrations shown, then post-treated in the standard fluctuation assay as indicated. When nickel was present in fluctuation assay it was added at the same concentration used for the pretreatment. c Total wells per treatment = 96.

the MMS concentration and separation of Ni(II) and MMS exposure in subsequent experiments eliminated this effect). When WP2 ÷ was treated with Ni(II) alone for 18 h followed by washing and incubation in the presence of MMS in the standard assay, Ni(II) potentiated MMS mutagenesis at a pretreatment concentration of 30 /~M (Table 10). Secondly, WP2 ÷ was treated with Ni(II) for 18 h, washed and incubated with both Ni(II) and MMS in the standard assay. Ni(II) pretreatment further increased recovery of mutants from cells treated with the combination of N i / M M S (Table 10). It is unlikely that the potentiation was an artifact produced by growth of mutant cells during the long preincubation. First, cells were pretreated in D M salts (without a carbon source) and thus were growing slowly or not al all. Second, mutants would be observable at the end of the pretreatment period, yet pretreated cells showed no increase over controls when assayed in the absence of MMS and Ni(II). Cells receiving only the pretreatment produced 7 - 9 positive wells against a spontaneous background of 7 (Table 10).

Discussion The purpose of these experiments was to test a 'comutagen' hypothesis to explain the discrepancy between carcinogenicity of Ni(II) and its lack of mutagenicity in bacteria (Sunderman, 1979). Since standard bacterial mutagenicity assays are designed to detect agents which interact directly with D N A , they may be unable to detect agents acting as comutagens which do not cause direct damage. Based on t h e results presented in this paper, assaying for the ability of an agent to potentiate mutagenicity of D N A - d a m a g i n g agents m a y allow for the detection of comutagens. Similar results have been reported for comutagenic actions of arsenite (Rossman, 981), chromate (LaVelle and Witmer, 1984) and cadmium (Mandel and Ryser, 1984) using bacterial assays. The demonstration that Ni(II) potentiates MMS mutagenicity only in E. coli strains containing functional D N A polymerase I (Tables 2 and 3) is consistent with previous in vitro work suggesting that Ni(II) could affect D N A repair by decreasing

197

the fidelity of DNA synthesis via some action on DNA polymerases (Sirover and Loeb, 1976). Of course, the data presented here are not sufficient to differentiate effects directly on polymerases from effects on other enzymes involved in stepwise recognition and repair of lesions in DNA. However, some initial growth inhibition was seen in experiments with polA- strains (Fig. 1). This inhibition was not seen in polA ÷ backgrounds (Fig. 2). If substantial repair of lesions occurred during this early phase of incubation, or if substantial numbers of mutants were killed, comutagenic effects of Ni(II) might be masked. This seems unlikely for two reasons. First, mutants are not recovered in these assays when cells are treated with MMS in DM salts, then plated on unsupplemented minimal agar. This indicates that few, if any, mutants are produced in the absence of DNA replication in this system and that early mutant killing cannot fully explain the lack of potentiation. Second, MMS is not rapidly depleted in the medium during these assays. Even after 4 h of incubation with WP6, filtered medium still contained sufficient MMS for mutagenicity and potentiation by Ni(II) to be detected. Though these results do not conclusively rule out a toxicity artifact, it seems clear that MMS continues to exert mutagenic effects into the period of log phase growth where mutations are apparently 'fixed'. Since toxicity to pre-existing mutants is unlikely, it is difficult to understand how toxicity could reduce mutant yield during a time period when normal exponential growth is occurring and effective concentrations of agent(s) are shown to exist in the medium. Apparently, early toxic effects seen in WP6 and WP67 do not introduce a substantial artifact into the data. In other experiments, Ni(II) was unable to potentiate MMS mutagenesis in either lexA- or recA- strains (Table 3). In this case, growth curves of treated cells closely paralleled those of controls so that no suggestion of toxicity was seen which might complicate data interpretation (data not shown). This finding might be easily explained since MMS mutagenicity itself is strongly dependent on a functional recA gene product (Todd et al., 1981). However, in these experiments there was a significant component of MMS mutagenicity which was evident even in the recA and lecA

deficient strains (Table 3). The results from these strains thus suggest that recA-independent mutagenesis is not involved in potentiation by Ni(II). This finding seems to implicate recA/lexAdependent functions known to be involved in a variety of DNA-repair systems as potential targets for Ni(II) action(s). Consistent with this suggestion, potentiation of EMS and MMS mutagenesis by Ni(II) was seen only in S. typhimurium strain, TA100, which carries the pKM191 plasmid (Table 6) and not in the plasmid-lacking strain, RA1535 (Table 7). Because pKM101 carries analogues (mucAB) of the umuCD genes, it is thought that the presence of the plasmid enhances recA-dependent by-pass replication at sites of non-coding lesions (Walker, 1978; Schendel, 1981). Thus, the data presented may implicate translesion repair as a target for Ni(II) comutagenic action. An alternative explanation not excluded by the above results is that Ni(II) and MMS react abiotically to produce a unique mutagen which produces mutation in a recA-dependent manner. This is certainly not expected, since both agents are electrophilic and would be expected to seek electron rich centers rather than each other. However, a less direct reaction may be possible and should be considered. The first approach was to treat cells in DM salt solution with MMS to produce lesions in DNA, than transfer the bacteria into selective medium containing nickel. The idea was to expose resting cells to MMS to produce DNA lesions, then allow a few rounds of DNA replication in the presence of Ni(II) and look for potentiation of mutagenesis. In such experiments, no potentiation was seen suggesting that MMS and Ni(II) have to be present together to observe the interaction (Table 8). This is consistent with the concept of a reaction between MMS and nickel. However, it was also found that preincubation times of 2 h or less were not sufficient for demonstrating potentiation even when the two agents were present simultaneously (Table 9). This indicated that a time factor could be involved, perhaps due to poor penetration of Ni(II) into the bacterial cell, induction of enzymes or some other process. To test this possibility, experiments were carried out in which cells were treated with Ni(II) for 18 h. These cells were then exposed to MMS in the fluctuation assay. The experiments were con-

198 trolled to eliminate the possibility that growth of preexisting mutants during the prolonged pretreatment might invalidate the results (see Results section). At one dose level, Ni(II) pretreatment enhanced the mutant yield produced by MMS. This is taken as evidence that the two agents need not be present (in high concentration) simultaneously to observe potentiation. Further, at the two dose levels tested, Ni(II) pretreatment further enhanced the potentiation observed when Ni(II) and MMS are coincubated in the fluctuation assay. Again, it appears that Ni(II) can exert effects in the absence of MMS if a prolonged preincubation period is used. These findings, combined with the known chemical properties of Ni(II) and MMS, argue against the possibility of a direct reaction between Ni(II) and MMS and are thus consistent with the hypothesis that Ni(II) produces its effects by some action on repair or replication proteins. Nevertheless, it is not possible to completely separate Ni(II) and MMS exposure, since residual agent will always be present inside treated cells even after washing. Thus, an undefined reaction mediated by the chemistry a n d / o r enzymology of the bacterial cell must remain a theoretical possibility. Overall, to explain the results of these experiments, the following rationalization has been considered. The requirement for functional recA and lexA gene products might be explained in terms of potential adducts. The finding that mutagenesis by MMS was potentiated in the absence of pKM101, at least in the E. coil strains, but that EMS mutagenesis was potentiated only in the one strain harboring this plasmid is consistent with mutagenesis induced at apurinic sites formed by hydrolysis of N-7 adducts after MMS or EMS exposure (Lawley, 1979; Todd et al., 1981). N-7Ethylguanine is formed to a significantly smaller extent after exposure to EMS than is the analogous reaction product after exposure to MMS (Lawley, 1979; T o d d et al., 1981). EMS mutagenicity is much less dependent on recA gene function than is mutagenicity following MMS (Schendel and Defais, 1980; Todd et al., 1981). Thus, Ni(II) may act, for example, by inhibiting the error-free repair of MMS- or EMS-induced lesion(s). This might lead to an increase in the number of apurinic sites at unrepaired alkylated bases. Since mutagenesis at apurinic sites is an

SOS process (Schendel et al., 1978; Schendel, 1981; Ganesan, 1982; Little and Mount, 1982; Shank, 1984), any increase in yield of mutants would be recA / lexA --dependent. The above explanation does not assign a role for polymerase I, implicated in the experiments with strains WP6 and WP67. This is intentional since there is insufficient information on the role of this enzyme in recA-dependent responses to D N A damage to speculate on a specific mechanism. In fact, there is no reason a priori to rule out the possibility of multiple actions of Ni(II) ions in bacterial systems. For example, Rossman et al. (1984) report the induction of lambda prophage by Ni(II). This response may not be due to the induction of recA protein, since Ni(II) does not appear to induce the phenotype expected when recA-dependent processes are turned on. That is, strains with constitutive high levels of recA protein have an increased spontaneous mutation rate (Witkin et al., 1982), yet Ni(II) does not show 'mutagenicity' expected in bacterial systems where background reversion frequency has been increased by recA induction (Sunderman, 1979). Thus, conjecture on a role for polymerase I or for a pathway in which polymerase I plays a critical role must await more information.

Acknowledgements We thank Dr. F. William Sunderman Jr. for his helpful discussions of Ni(II) mutagenesis and carcinogenesis. The technical assistance of Mr. Steven Light is gratefully acknowledged. This study was supported by NIEHS grant ES 02972 to JML.

References Christie, N.T., and M. Costa (1983) In vitro assessment of the toxicity of metal compounds, III. Effects of metals on DNA structure and function is intact cells, Biol. Trace Elem. Res., 5, 55-71. Ciccarelli, R.B., and K.E. Wetterhahn (1984) Molecular basis for the activity of nickel, in F.W. Sunderman Jr. (Ed.), Nickel(II) in the Human Environment, Academic Press, New York, pp. 201-212. Forster, R., H:M.L. Green and A. Priestley (1980) Optimal levels of $9 fraction in the Ames and fluctuation tests: Apparent importance of diffusion of metabolites from top agar, Carcinogenesis,1, 337-346.

199 Ganesan, A.K., P.C. Cooper and C.A. Smith (1982) Biochemical mechanisms and genetic control of DNA repair, Progress Mutation Res., 4, 313-323. Gatehouse, D. (1978) Detection of mutagenic derivatives of cyclo-phosphamide and a variety of other mutagens in a ' Microtitre' fluctuation test without microsomal activation, Mutation Res., 53, 289-296. Gilbert, R.I. (1980) The analysis of fluctuation tests, Mutation Res., 74, 283-289. Green, M.H.L., and W.J. Muriel (1976) Mutagen testing using Trp reversion in Escherichia coil, Mutation Res., 38, 3-32. LaVeUe, J.M., and C.M. Witmer (1984) Chromium(VI) potentiates mutagenesis by sodium azide but not ethylmethane sulfonate, Environ. Mutagen., 6, 311-320. Lawley, P.D. (1979) Approaches to chemical dosimetry in mutagenesis and carcinogenesis: The relevance of reactions of chemical mutagens and carcinogens with DNA, in: P.L Grover (Ed.), Chemical Carcinogens and DNA, CRC Press, Boca Raton, FL, pp. 1°6. Little, J.W., and D.W. Mount (1982) The SOS regulatory system of Escherichia coil, Cell, 29, 11-22. Mandel, R., and H.J.P. Ryser (1984) Mutagenicity of cadmium in Salmonella typhimurium and its synergism with two nitroamines, Mutation Res., 138, 9-16. Maron, D.A., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Rossman, T.G. (1981a) Effect of metals on mutagenesis and DNA repair, Environ. Health Perspect., 40, 189-195. Rossman, T.G. (1981b) Enhancement of UV-mutagenesis by low concentrations of arsenite in E. coli, Mutation Res., 91, 207-211.

Rossman, T.G., M. Molina and L.W. Mayer (1984) The genetic toxicology of metal compounds, I. induction of prophage in E. coil WP2 (},), Environ. Mutagen., 6, 59-69. Schendel, P.F. (1978) Pathways of mutagenesis and repair in Escherichia coil exposed to low levels of simple alkylating agents, J. Bacteriol., 466-475. Schendel, P.F. (1981) Inducible repair systems and their implications for Toxicology, CRC Crit. Rev. Toxocol., 8(4), 311-362. Schendel, P.F., and M. Defals (1980) The role of umuC gene product in mutagenesis by simple alkylating agents, Mol. Gen. Genet., 177, 661-665. Shank, R.C. (1984) Toxicity-induced aberrant methylation of DNA and its repair, Pharm. Rev., 36(2), 19S-24S. Sirover, M.A., and L.A. Loeb (1976) Infidelity of DNA synthesis in vitro: Screening for potential metal mutagens or carcinogens, Science, 194, 1434-1436. Sunderman Jr., F.W. (1979) Mechanisms of metal mutagenesis, Biol. Trace Elem. Res., 1, 63-85. Todd, P.A., J. Brouwer and B.W. Glickman (1981) Influence of DNA-repair deficiencies on MMS- and EMS-induced mutagenesis in Escherichia coil K-12, Mutation Res., 82, 239-250. Walker, G.C. (1978) Isolation and characterization of mutants of the plasmid pKM101 deficient in their ability to enhance mutagenesis and repair, J. Bacteriol., 1203-1211. Witkin, E.M., J.O. McCall, M.R. Volkert and L.E. Wermundsen (1982) Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coil, Mol. Gen. Genet., 185, 43-50.