Ability of various alkylating agents to induce adaptive and SOS responses: A study with lacZ fusion

149

Mutation Research, 146 (1985) 149-154 DNA Repair Reports Elsevier

MTR 06108

Ability of various alkylating agents to induce adaptive and SOS responses: A study with lacZ fusion M a s a n o r i O t s u k a 1, Y u s a k u N a k a b e p p u 2 a n d M u t s u o Sekiguchi 2 i Hita Research Laboratories, Chemical Biotesting Center, Chemical Inspection and Testing Institute, Ishii-cho 3, Hita, Ohita 877, and 2 Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812 (Japan)

(Received 13 March 1985) (Revision received 15 April 1985) (Accepted 17 April 1985)

Summary We used alkA'-lacZ' and umuC'-lacZ' fused genes and determined the ability of various alkylating agents to induce adaptive and SOS responses. The degree of induction of expression of these genes was quantitatively measured by a simple colorimetric assay of/3-galactosidase activity. SN1 type methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, were more effective inducers for the alkA than for the umuC system, while SN1 type ethylating agents, such as N-ethyl-N'nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea, were more potent inducers for the umuC than for the alkA system. Similar but less striking effects on the two systems were obtained with SN2 type alkylating agents.

Alkylating agents are potent mutagens and carcinogens, and organisms respond in a complex manner to these agents. In Escherichia coli there are at least two pathways inducible by alkylating agents; namely, adaptive and SOS responses. The adaptive response is induced solely by simple alkylating agents and two genes, alkA and ada, are involved in the process (Samson and Cairns, 1977; Karran et al., 1982; Evensen and Seeberg, 1982). The SOS response, on the other hand, is induced by a wide variety of agents, including UV and chemicals that damage DNA or inhibit DNA rep-

Abbreviations: Ap, ampicillin; ONPG, O-nitrophenyl-fl-Dgalactopyranoside; kb, kilobase; MNNG, N-methyl-N'-nitroN-nitrosoguanidine; ENNG, N-ethyl-N'-nitro-N-nJtrosoguarfidine; MNU, N-methyl-N-nitrosourea; ENU, N-ethyl-Nnitrosourea; MMS, methyl methanesulfonate; EMS, ethyl ethanesulfonate; DMS, dimethyl sulfate; DES, diethyl sulfate. sulfate.

lication, and more than 15 genes are concerned (Witkin, 1976; Little and Mount, 1982). umuC, which controls induced mutagenesis, is one of this class of genes (Kato and Shinoura, 1977). By placing lacZ, the structural gene for /3galactosidase, under control of one of the genes involved in the adaptive or the SOS response, the degree of response can be measured quantitatively by colorimetric assay. Thus, we constructed an alkA'-lacZ" fused gene and investigated the effects of various agents on induction of the adaptive response (Nakabeppu et al., 1984). lacZ fusions of genes associated with the SOS response, sfiA and umuC, were also made (Quillardet et al., 1982; Shinagawa et al., 1983). Using these fused genes, we investigated the ability of various alkylating agents to induce the two processes. We report herein the results of these experiments in which we obtained a basis for assaying the genotoxicity of alkylating agents.

0167-8817/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

150 Materials and methods

Medium and buffer

Plasmids and bacteria

Bacteria were grown in M9 medium supplemented with 50/~g/ml of ampicillin. The Z buffer used was as described by Miller (1972).

pMCP1000 is a pBR322-derived plasmid, carrying an alkA'-lacZ' fusion, with the proper control region for alkA expression (Nakabeppu et al., 1984). An EcoRI/SalI fragment carrying the alkA'-lacZ', derived from pMCP1000, was joined with EcoRI/SalI fragments of pUC9 and pMF3 (Manis and Kline, 1977) to yield plasmid pYN1200 and pYN1300, respectively (Fig. 1). Plasmid pSK1002, carrying the umuC'-lacZ', was constructed by Shinagawa et al. (1983) and was provided by Dr. S. Nakamura. Strain CSH26 (ara, A(lac-pro), thi), which is a derivative of E. coli K12 (Miller, 1972), was transformed with each one of these plasmids and the transformants were used as tester strains.

Chemicals Alkylating agents used were purchased from the following sources: methyl methanesulfonate (MMS), N-methyl-N-nitrosourea (MNU), Nethyl-N-nitrosourea (ENU), diethyl sulfate (DES) and N-methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) from Nakarai Chemicals (Kyoto); ethyl methanesulfonate (EMS) from Eastman Kodak Co. (New York); dimethyl sulfate (DMS) from Tokyo Kasei Kogyo (Tokyo); N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) from Iwai Kagaku Yakuhin (Tokyo). O-Nitrophenyl-fl-D-galactopyranoside (ONPG), the substrate for fl-galactosidase, was obtained from Nakarai Chemicals (Kyoto).

PalkA

Test procedure The test procedure consisted of a colorimetric assay of fl-galactosidase activity after incubation of the tester strains in the presence of various amounts of alkylating agents. Bacteria were grown in M9 supplemented with 50/~g/ml of ampicillin at 37°C and, at OD660 =0.5 (about 5 × 108 cells/ml), the culture was diluted to I : 5 with fresh medium containing the alkylating agents to be tested. After incubation with shaking at 37°C for 2 h, fl-galactosidase activity was assayed. I EcoRI/Sall

T4 D N A L i g a s e

PalkA

Assay of fl-galactosidase activity

i EcoRI/Sal[

T4 D N A k i g a s e

PalkA •

Fig. 1. Construction of plasmids carrying the alkA'-lacZ' fused gene. D N A of pMCP1000 was digested with EcoRI and Sail and ligated with pUC9 or pMF3 D N A digested with the same enzymes. B, BamHt; H, HindIII; E, EcoRI; S, SalI. The arrows indicate the directions of transcription. P (I) denotes promoter.

The assay was based on the method of Miller (1972), with minor modification. To the culture, 0.05-0.5 ml of Z buffer was added to give a final volume of I ml. To disrupt the cell membrane, one drop of 0.1% sodium dodecyl sulfate and two drops of chloroform were added, and the preparation was shaken vigorously. After equilibration at 28°C for 5 min, the reaction was started by adding 0.2 ml of O N P G solution (4 m g / m l in 0.1 M phosphate buffer). Incubation was carried out at 28°C after which the reaction was terminated with the addition of 0.5 ml of 1 M Na2CO 3. After the bacterial cells were removed by centrifugation at 3000 rpm for 10 rain, the color intensity of the supernatant was measured spectrophotometricaIly at 420 nm. Enzyme units were calculated according to an equation of Miller (1972).

151

Results

Effect of gene dosage on expression alkA'-lacZ' fused gene

of the o

To establish a system .for measuring the extents of the adaptive response to alkylating agents by a direct colorimetric assay, we first examined the effects of gene dosage on the basal and induced levels of fl-galactosidase activity. For this purpose, a 7-kb E c o R I / S a l I fragment carrying an alkA'lacZ' fused gene, derived from p M C P 1 0 0 0 ( N a k a b e p p u et al., 1984), was recloned with lowand high-copy plasmid vectors, p M F 3 and pUC9, to construct p Y N 1 3 0 0 and pYN1200, respectively (see Fig. 1). p M F 3 is an F-derived plasmid, thus, the copy n u m b e r of p Y N 1 3 0 0 was approximately 1 - 2 per cell. p U C 9 has a r u n a w a y character, and the resulting plasmid, pYN1200, showed an increasing n u m b e r of copies at elevating temperatures. In addition, the original alkA'-lacZ' plasmid, pMCP1000, was used, t h e c o p y n u m b e r of which was about 20 per cell. Levels of fl-galactosidase in cells harboring either one of these plasmids, with or without treatm e n t with MMS, are shown in Table 1. The highest ratio for the induced level to the basal level was obtained with cells bearing pMCP1000. Thus, in the following experiments, this system was used to measure the degree of adaptive response to the alkylating agents. TABLE 1 INDUCTION OF fl-GALACTOSIDASE IN CELLS HARBORING 3 DIFFERENT PLASMIDS WITH THE alkA'-lacZ' FUSION GENE Plasmid

TemCopy /3-Galactosidase " perature number/ Basal Induced Ratio (°C) cell level (A) level(B) (B/A)

pYN1300 pMCP1000 pYN1200 pYN1200

37 37 30 37

1-2 20 ND b ND b

24 102 220 23

305 4023 2344 255

12.7 39.4 10.7 11.1

a An exponentially growing culture of CSH26 cells harboring one of the plasmids indicated was divided into two portions, one was incubated with 0.01% MMS while the other was incubated without this addition, fl-Galactosidase activity was determined after 6 h of incubation. b Not determined. It was difficult to determine the precise copy number as plasmids in the recA+cells were re-arranged.

O

~ 2~ ENN~r "~

'

~

'

,

Concentrationof Inducer(M) Fig. 2. Dose-response curves for induction of fl-galactosidase

in cells carrying pMCP1000 or pSK1002 with MNNG or ENNG. An exponentially growing culture of tester strains was divided into several portions, and various concentrations of the compounds indicated were added to each one of the portions. The cultures were incubated at 37°C for 2 h, and fl-galactosidase activity was determined. O, CSH26/pMCP1000 (alkA'lacZ'); O, CSH26/pSK1002 (umuC'- lacZ').

As a reference for the SOS response, we used plasmid pSK1002 which carries the umuC'-lacZ' fused gene. The copy n u m b e r of pSK1002 was approximately 20 per cell, which is almost equal that of pMCP1000.

Induction of fl-galactosidase by various alkylating agents Simple alkylating agents are electrophilic as such or are converted to electrophilic reactants metabolically, and interact with nucleophiles in D N A , yielding phosphate triester or various alkylated bases (Hemminki, 1983). In' accordance with the different reaction kinetics, these agents are classified as using SN1 mechanism and SN2 mechanism (Lawley, 1974). M N N G , E N N G , M N U and E N U belong to the former and MMS, EMS, D M S and D E S to the latter. We first tested the former group of chemicals for induction of fl-galactosidase activity. D o s e - r e s p o n s e curves for induction with M N N G and E N N G are shown in Fig. 2. For induction of the alkA-associated activity, M N N G was more effective than E N N G ; concentrations of M N N G and E N N G required for the highest levels of induction were 2 × 10 -6 M and 3 × 10 -5 M, respectively. O n the other hand, umuC-associated activity was induced by the two agents at m u c h the

152

I M.O %

/

i 05 o

B ,~

I

DES

{ o-o "

~oConcentration of

,'o-,

Inducer(M)

,'o-,

Fig. 3. Dose-response curves for induction of fl-galactosidase with M N U or ENU. The procedure was as described in Fig. 2. o, C S H 2 6 / p M C P 1 0 0 0 (alkA'-IacZ'); O, CSH26/pSK1002

/,'/

Concentration of Induce r (M) Fig. 5. Dose-response curves for induction of fl-galactosidase with DMS or DES. The procedure was as described in Fig. 2. o, C S H 2 6 / p M C P 1 0 0 0 (alkA'-lacZ'); O, CSH26/pSK1002

( umuC'- lacZ').

( umuC'- lacZ').

same concentrations. A similar result was obtained with M N U and ENU, although these agents required higher concentrations for induction (Fig. 3). It seems, therefore, that methylating but not ethylating agents, are more effective inducers for the alkA system than for the umuC system. We next tested the effects of SN2 type alkylating agents on the induction of the two systems and these results are shown in Figs. 4 and 5. All the agents tested induced the two systems and a similar tendency was observed with the SNI type comlaounds, despite the differential effects of methylating and ethylating agents being less significant. To compare the effects of different compounds

on the two systems, we computed two parameters, potency and sensitivity, as adopted by Quillardet et al. (1982); potency ( U / / ~ m o l e / m l ) is the reciprocal of the amount of the compound required to induce the half-maximal fl-galactosidase activity while sensitivity (/~M) is defined as the lowest point at which the response is systematically over twice the background. From Table 2 it is evident that M N N G and M N U had higher potency values for the alkA than for the umuC, whereas the corresponding ethylating agents responded in the opposite manner. With the SN2-type compounds, TABLE 2 P O T E N C Y A N D SENSITIVITY OF V A R I O U S A L K Y L A T ING AGENTS

!

OS

MMS

~ R Type

Compounds

SNI

MNNG ENNG MNU ENU

SN2

MMS EMS DMS DES

EMS

10-4

10-3

Concentration of

10-2 Inducer(M)

10-t

Fig. 4. Dose-response curves for induction of fl-galactosidase with MMS or EMS. The procedure was as described in Fig. 2. e, C S H 2 6 / p M C P 1 0 0 0 (alkA'-IaeZ'); O, CSH26/pSK1002

( umuc'- lacZ').

a

Potency a

Sensitivity b

alkA

umuC

alkA

980 43.5 1.92 0.38

131 222 0.91 1.59

1.7 0.03 0.71 0.12

0.56 0.06 0.25 0.16

0.07 2.7 16 900 190 15 000 210 2100

umuC 0.5 0.3 250 100 90 5 200 150 1200

Potency ( U / t t m o l e / m l ) is the reciprocal amount of the compound required for induction of the half-maximal fl-galactosidase activity. b Sensitivity (/~M) is the lowest concentration of the compound at which the response is systematically over twice the background.

153

a similar but less significant tendency was obtained.

Discussion In a colorimetric assay of the induction of an alkA function involved in the adaptive response in E. coli, we found that the alkA gene expression

was most effectively induced by SNl-type methylating agents, such as M N N G and MNU. It is well documented that while both SN1 and SN2 type methylating agents alkylate N-7 of guanine in DNA primarily, SN1 methylating agents react with DNA at its phosphate group or 0-6 of guanine more efficiently than do SN2 methylating agents (Lawley, 1974; Hemminki, 1983). This is in accord with the current model for induction of the adaptive response; namely that the methyl group of O6-methylguanine in alkylated DNA is transferred to a cysteine residue of Ada protein, which is coded by the ada gene and is itself the methyltransferase, and that the methylated form of Ada protein acts as a positive regulator for expression of the alkA and also of the ada gene, per se (Nakabeppu et al., 1985a, b). Thus, fl-galactosidase activity induced in cells carrying the alkA'-lacZ' fused gene would reflect the content of O6-methylguanine in the alkylated DNA. Ethylating agents also induced the alkA-associated activity although their potency was 10 times less than that of corresponding methylating agents. This seems to correlate with the finding that the ada-coded methyltransferase transfers the ethyl group of O6-ethylguanine at a rate 10 times less than that for the methyl transfer (Olsson and Lindahl, 1980). The role of O6-methylguanine in mutagenesis and carcinogenesis has well been established (Goth and Rajewsky, 1974; Schendel and Robins, 1978; Newbold et al., 1980; Eadie et al., 1984). O6-Meth ylguanine frequently pairs with thymine during replication and as a result causes a G : C to A : T transition (Snow et al., 1984). Such changes were evident in the H-ras-1 oncogene of tumors induced by a single injection of MNU into rats (Sukumar et al., 1983). In this regard, a sensitive and simple colorimetric assay for alkylating agents that produce such lesions may be of great value in detecting possible carcinogens. The present report pro-

vides a basis for establishing such an assay. By comparing the potency of a compound for the alkA and for the umuC system, it can be determined whether or not the activity is due to methylating agents because the alkA system is specifically sensitive to methylating agents while the umuC system responds to a wide variety of chemicals that produce DNA lesions. If both alkA'-lacZ' and u m u C ' - l a c Z ' genes are present in the same cells, we can expect a higher sensitivity to a wide variety of chemicals. This may be achieved by recloning these genes in two plasmids belonging to different compatibility groups, or by placing the umuC control region in front of the alkA'-lacZ' gene, on a single plasmid.

Acknowledgements We are grateful to Drs. H. Shinagawa and S. Nakamura for supplying pSK1002 and pMF3, and M. Ohara for comments on the manuscript. This study was supported by Grants for Scientific and Cancer Research and a Grant-in-Aid for Special Project Research (Cancer-Bioscience) from the Ministry of Education, Science and Culture of Japan.

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