Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue® Rat2 cell line

Mutation Research 445 Ž1999. 127–135 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres

Mutation spectrum of 4-nitroquinoline N-oxide in the lacI transgenic Big Blue w Rat2 cell line Jae-Chun Ryu a,) , Ji-Youn Youn a , Youn-Jung Kim a , Oh-Seung Kwon a , Yun-Seon Song a , Hyung-Tae Kim a , Kyung-Hae Cho b, Il-Moo Chang c a

Toxicology Laboratory, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, South Korea b Department of Biology, Seoul Woman’s UniÕersity, 126 Kongneung-dong, Nowon-ku, Seoul 139-744, South Korea c Natural Products Research Institute, Seoul National UniÕersity, 28 Yungun-dong, Chongro-ku, Seoul 110-460, South Korea Received 10 February 1999; received in revised form 30 June 1999; accepted 30 June 1999

Abstract This paper describes the spectrum of mutations induced by 4-nitroquinoline N-oxide Ž4-NQO. in the lacI target gene of the transgenic Big Blue w Rat2 cell line. There are only a few report for the mutational spectrum of 4-NQO in a mammalian system although its biological and genetic effects have been well studied. Big Blue w Rat2 cells were treated with 0.03125, 0.0625 or 0.125 mgrml of 4-NQO, the highest concentration giving 85% survival. Our results indicated that the mutant frequency ŽMF. induced by 4-NQO was dose-dependent with increases from three- to seven-fold. The DNA sequence analysis of lacI mutants from the control and 4-NQO treatment groups revealed an obvious difference in the spectra of mutations. In spontaneous mutants, transition Ž60%. mutations, especially G:C ™ A:T transition Ž45%., were most frequent. However, the major type of base substitution after treatment of 4-NQO was transversions Ž68.8%., especially G:C ™ T:A Ž43.8%., while only 25% of mutants were transitions. These results are consistent with those produced by 4-NQO in other systems and the transgenic assay system will be a powerful tool to postulate more accurately the mechanism of chemical carcinogenesis involved. q 1999 Elsevier Science B.V. All rights reserved. Keywords: 4-Nitroquinoline N-oxide; Big Blue w Rat2 cell line; lacI gene; Mutational spectrum

1. Introduction Mutagenesis studies are important to understand mechanisms of chemical carcinogenesis. Because specific carcinogens often have unique patterns of mutation w1x, and mutational spectrum provide a

) Corresponding author. Tel.: q82-2-958-5070; fax: q82-2958-5059; E-mail: [email protected]

basis for the study of cancer etiology and action mechanism of chemical carcinogens w2x, it is important to investigate mutational spectra w3x. Newly developed transgenic animals and cell lines permit the investigation of mutational spectra as well as mutant frequencies ŽMFs. of chemicals both in vivo and in vitro w4,5x. So, these models may provide considerable insight into the mechanisms of chemical mutagenesis and carcinogenesis. The transgenic Big Blue w mutagenesis assay system is the best

1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 3 6 - 9

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known example of such systems w2x. In cases of direct-acting chemicals which do not require exogenous metabolic activation, the MF and mutational spectrum in these transgenic mutagenesis assay systems show no differences between cell line and animal w6x. The Big Blue w system, transgenic mouse and Rat2 cell lines carry over 40 copies of lambda shuttle vector ŽBig Blue w shuttle vector. w7,8x containing lacI gene as a target w9–11x. The lacI gene as a mutational target is very useful for the study of the mutational characteristics of a carcinogen for several reasons. The relatively small size Ž1080 bp. facilitates sequence analysis, and the expression of repressor protein permits a rapid colorimetric assay to screen for mutations. Also, it is possible to accomplish subsequent comparison the large historical data base allowing investigation of the underlying mechanisms leading to mutations w12x. The mutations induced in the lacI gene can easily be quantified ŽMF. and the precise mutation type and distribution can quickly be identified by direct sequencing. Moreover, considering that mutations in lacI gene induced by chemicals reflect the effect on the other endogenous genes such as proto-oncogenes and tumor suppressor genes and that mutations occurred in these genes are most common event in many types of human cancer w12–14x, this assay may provide a powerful tool to postulate more accurately the mutation spectrum induced in cancer-related genes. 4-Nitroquinoline N-oxide Ž4-NQO. is a widely studied model mutagen and carcinogen and has used as a positive control in many mutagenic and carcinogenic studies in vivo, especially, in vitro without exogenous metabolic activation w15–17x. 4-NQO, after enzymatic reduction of its nitro group, binds covalently to DNA and its adducts induce misreplication events that results in mutations w1,18,19x. The previous studies using E. coli, Salmonella, CHO cells and at the hprt locus in monkey cells w19,35–38x indicate that 4-NQO is basically a base substitution mutagen acting at G residue. Therefore, in this present study, we investigated the mutational spectrum of 4-NQO in the lacI gene of the transgenic Big Blue w Rat2 cell line. We also speculate about the mutagenic effect of 4-NQO in several genes including cancer-related genes such as p53 tumor suppressor gene, ras oncogene from mutational spectrum in lacI gene in the 4-NQO induced mutant.

Part of this study has already been presented in a preliminary form w20x.

2. Materials and methods 2.1. Cell culture Transgenic Big Blue w Rat2 cell line was purchased from Stratagene ŽLa Jolla, CA, USA.. This cell line is derived from a Rat2 embryonic fibroblast cell line ŽATCC, Rockville, MD: CRL 1764. transfected with the Big Blue w shuttle vector Žlambda lacI r Z shuttle vector. and pSV2NEO plasmid, which provides an antibiotic selection marker w5x. This cell line was maintained in DMEM media ŽGibco BRL, USA. with 10% fetal bovine serum ŽGibco BRL. 200 mgrml geneticin ŽG418. ŽGibco BRL., 50 unitsrml penicillin and 50 mgrml streptomycin ŽGibco BRL.. The cells were grown at 378C, with 5% CO 2 and split every 3–4 days. 2.2. Treatment To determine the concentration of 4-NQO ŽSigma, St. Louis, MO, USA., we performed MTT w3-Ž4,5dimethylthiazol-2-yl.-2,5-diphenyl tetrazolium bromidex assay w21,22x on the basis of cell viability. This assay is based on the enzymatic activity of mitochondrial succinate dehydrogenase of live cell to reduce a tetrazolium-based compound, MTT, to a blue formazan product. The practical concentration was selected to have no significant alteration in cell growth. When cells were 30–40% confluent, they were treated with 0.03125, 0.0625 or 0.125 mgrml 4-NQO and incubated for 30 min at 378C. Control cells were grown in the presence of 1% DMSO ŽSigma. used as solvent for 4-NQO under same condition. After treatments, the cells were washed with PBS three times, added fresh medium and then the cells were allowed to grow to confluence. 2.3. Mutagenesis assay 2.3.1. Genomic DNA isolation When the cells were fully confluent, approximately 2–5 = 10 7 cells from each treatment were harvested by scraping. Then genomic DNA was

J.-C. Ryu et al.r Mutation Research 445 (1999) 127–135

prepared using Stratagene’s Genomic DNA Isolation Kit according to manufacturer’s recommendation.

129

Table 1 Sequence and location of sequencing primers Primer

Location

Sequence

2.3.2. In Õitro packaging and plating After DNA concentrations were adjusted, the purified genomic DNA was incubated with Transpacke packaging extract ŽStratagene. to recover the shuttle vector. The Transpacke packaging extract excises the lambda vector target and packages it into a lambda head. The phage stock produced as a result of in vitro packaging was used to infect host SCS-8 E. coli cells. The cells were plated on 25 = 25 cm2 assay tray ŽStratagene. containing 1.5 mgrml of X-gal ŽStratagene.. Total plaques were scored within 24 h after plating. The MFs were calculated as a ratio of blue plaques vs. total number of plaques Žclear q blue.. All blue mutant plaques containing sectored plaques were picked from the agar plates and were replated on the agar plate containing X-gal to verify the mutant. Forty-three mutant plaques from 0.0625 mgrml of 4-NQO exposure were selected for analysis of mutational spectrum. The verified and well isolated blue plaques were subjected for PCR amplification and sequencing.

ID a IID IIID IVU VU VIU

y51 to y34 347 to 361 549 to 563 385 to 401 983 to 1000 1185 to 1201

5 -CCCGACACCATCGAATG-3 X X 5 -TGTAAAGCGGTGCA-3 X X 5 -CTGGTCGCATTGGGTCA-3 X X 3 -CAGTGGGCTGATCATTA-5 X X 3 -TGCCCGTCTCACTGGTG-5 X X 3 -ATGTTGTGTGGAATTGT-5

2.4. Mutational spectrum

3.1. Mutant frequencies

Double-stranded 1300 bp fragments encompassing the lacI coding region were amplified using Big Blue w PCR primers obtained from Stratagene. Twenty microliters of crude boiled mutant phage lysate was used as a template. After initial denaturation step at 958C for 5 min and extension step at 728C for 5 min, 30 cycles of amplification were conducted as follows: denaturation at 958C for 90 s, annealing at 558C for 90 s, elongation at 728C for 150 s with a final extension at 728C for 10 min using thermocycler ŽRobocyler w 40 temperature cycler, Stratagene, CA, USA.. For effective sequencing, electrophoresis of PCR product was performed and then was recovered from low-melting temperature agarose gel ŽGibco BRL. using Geneclean II kit ŽBIO 101, La Jolla, CA, USA.. Sequencing was done using the dideoxynucleotide chain termination method with primers ŽStratagene. and ABI PRISMe dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA

To determine the test concentration of 4-NQO, we performed MTT assay based on estimation of enzyme activity of mitochondrial succinate dehydrogenase within viable cells w21,22x as described previously. For prevention of the bias, the toxic concentration was excluded which could impose the selection pressure on the cells and the concentrations which did not significantly alter the growth of the cells was selected. We determined treatment concentrations which induce 5, 10 and 15% of inhibition of cell growth as 0.03125, 0.0625 and 0.125 mgrml, respectively Ždata not shown.. The MF was estimated as a ratio blue plaques vs. total number of plaques. The MFs data are shown in Table 2. The MFs obtained from three independent experiments show dose-dependent manner. The average MFs Ž=10y4 . in 1% DMSO-treated group as a solvent control revealed 0.66 ranged from 0.21 to 1.1. The MFs Ž=10y4 . in 0.03125, 0.0625 and 0.125 mgrml 4-NQO-treated groups are 2.3 Ž2.0–

a

X

X

D: downstream; U: upstream.

polymerase ŽPerkin-Elmer, CA, USA. on Applied Biosystem 373 A DNA sequencer ŽPerkin-Elmer.. The primers used are shown in Table 1. The purified DNA sample corresponding to approximately 200 ng was added to 3.2 pmolrml primer and terminator dyes. The sequencing reactions were carried out 25 cycles as follows: 968C for 30 s; 508C for 15 s; 608C for 4 min, according to a protocol established by ABI.

3. Results and discussions

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130

Table 2 MF of 4-NQO in transgenic Big Blue w cell line Group

Concentration Žmgrml.

Number of total plaques Ž=10 3 .

Number of mutant plaques

MF Ž=10y4 .

Mean Ž=10y4 . "SD

Control

–a

47.50 94.50 103.23 55.30 85.06 67.39 55.20 112.03 108.24 – 103.47 76.57

5 2 9 13 19 13 31 36 40 – 52 41

1.10 0.21 0.87 2.40 2.20 2.00 5.60 3.20 3.70 – 5.00 5.40

0.66 " 0.20

4-NQO

0.03125

0.0625

0.125

a

2.30 " 0.01

4.70 " 0.40

5.20 " 0.01

1% DMSO.

2.4., 4.7 Ž3.2–5.6. and 5.2 Ž5–5.4., respectively. The MFs induced by 4-NQO increased about three- to seven-fold dose-dependently compared to control. This increase is meaningful because treatment concentration does not cause any effect on cell growth. According to the previous reports of 4-NQO, 10-fold increase of spontaneous MF in yeast w23x and 20% chromosomal aberration in CHO cells w24x was induced by 0.06 and 0.0003 mgrml of 4-NQO, respectively. Also, 80 mgrkg of 4-NQO induced micronucleus formation in mice w24x. There was a report that microgram quantities of 4-NQO is sufficient to in-

duce several kinds of cancer in mice, rabbit, rat and hamster etc. w18x. In this respect, the results of many reports and our MF data indicate that 4-NQO is a potent mutagen. 3.2. Mutational spectrum To clarify the type of mutations, all plaques of mutants were subjected for sequencing by automatic fluorescence sequencer. The spontaneous base substitution mutations in lacI gene of transgenic Big Blue w Rat2 cell line were shown in Table 3. Among

Table 3 Spontaneous base substitution mutations in lacI gene of transgenic Big Blue w cell line Base position

X

X

Sequence Ž5 ™ 3 .

149 GCG GCG ATG 168 AAT TAC ATT 267 ATT GT C GCG 341 GTC GAA GCC 398 ATC ATT AAC 406 AAC TAT CCG 421 GAC CAG GAT 785 GGC GCA ATG 805 ACC GAG TCC 838 TCG GTA GTG 842 GTG GGA TAC 848 TAC GAC GAT Total number of mutations

Base change

Amino acid change

Number of plaques

G™C A™G T™C G™T A™G T™A G™A G™C G™A A™T G™A G™A

Ala ™ Pro Tyr ™ Cys Val ™ Ala Glu ™ Stop Ile ™ Val Tyr ™ Stop Gln ™ Gln Ala ™ Pro Glu ™ Glu Val ™ Val Gly ™ Ser Asp ™ Asn

1 1 1 1 1 1 2 3 1 1 5 1 19

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19 spontaneous base substitution mutations, two mutations generated stop codon Žnonsense mutations. and four mutations revealed silent mutations with no change in amino acid sequence. The remaining mutations created missense mutations. Two of 20 mutants did not carry any mutations. Transition of G:C ™ A:T type of all base substitution mutations was the most frequent event. This trend is similar to the spectrum of spontaneous mutation in Big Blue w lambdarlacI mouse w25x.

131

The base substitution of 4-NQO induced lacI mutations summarized in Table 4. Only four out of 45 base substitution mutations revealed nonsense mutations and another four mutations were silent mutations with no change in sequence of amino acid. The remaining mutations created missense mutations as indicated in Table 4. Interestingly, two out of 20 spontaneous mutants Ž10%. carried multiple mutations as shown in Table 5. However, nine out of 43 mutants Ž21%. induced

Table 4 Base substitution mutations induced by 4-NQO in lacI gene of transgenic Big Blue w cell line Base position

X

X

Sequence Ž5 ™ 3 .

38 CCA GTA ACG 82 TAT CAG ACC 95 CGC GTG GTG 120 GTT TC T GCG 139 GAA AA A GTG 158 GCG GAG CTG 167 AAT TAC ATT 179 AAC CGC GTG 198 CTG GCG GGC 223 ATT GGC GTT 250 CTG CAC GCG 251 CAC GCG CCG 270 GTC GCG GCG 273 GCG GCG ATT 285 TCT C GC GCC 325 ATG GTA GAA 329 GAA CGA AGC 333 CGA AGC AGC 344 GAA GCC TGT 381 CAA C GC GTC 470 TTT C TT GAT 476 GAT GTC TCT 484 TCT GAC CAG 487 GAC CAG ACA 530 ACG CGA CTG 588 GCG GGC CCA 712 CAA CA A ACC 725 ATG C TG AAT 782 CTG GGC GCA 792 ATG C GC GCC 842 GTG GGA TAC 860 GAA GAC AGC 863 GAC AGC TCA 882 CCG C CG TTA 909 TTT C GC CTG 951 CTC TC T CAG 993 GTC TCA CTG 1056 TTG GCC GAT Total number of mutations

Base change

Amino acid change

Number of plaques

G™C G™C G™T C™A A™G G™T T™G C™A C™A C™T C™A G™C C™A C™A G™T T™A C™T G™A G™A G™C C™A G™T C™A G™C C™T G™T A™G C™A G™T G™T G™A G™A A™T C™T G™C C™T C™G C™G

Val ™ Leu Gln ™ His Val ™ Leu Ser ™ Tyr Lys ™ Lys Glu ™ Stop Tyr ™ Asp Arg ™ Ser Ala ™ Glu Gly ™ Gly His ™ Gln Ala ™ Pro Ala ™ Glu Ala ™ Glu Arg ™ Leu Val ™ Val Arg ™ Stop Arg ™ Asp Ala ™ Thr Arg ™ Pro Leu ™ Ile Asp ™ Phe Asp ™ Glu Gln ™ His Arg ™ Stop Gly ™ Val Gln ™ Gln Leu ™ Met Gly ™ Cys Arg ™ Leu Gly ™ Ser Asp ™ Asp Ser ™ Cys Pro ™ Leu Arg ™ Pro Ser ™ Phe Ser ™ Stop Ala ™ Gly

1 1 2 1 1 1 1 1 2 2 1 1 1 2 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 1 45

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132

Table 5 Spontaneous and 4-NQO induced multiple base substitution mutations in lacI gene of transgenic Big Blue w cell line Mutant Spontaneous aNC11-1 aC11

4-NQO induced a0–06 a0–16 a0–17 a0–20 a1–03 a1–09 a1–14 a1–31 a1–36

Base position 398 421 421 838 848

198 470 333 344 38 251 223 285 223 842 381 951 139 188–189 588 1056 476

X

X

Sequence Ž5 ™ 3 .

Base change

Amino acid change

ATC ATT AAC GAC CAG GAT GAC CAG GAT TCG GTA GTG TAC GAC GAT

A™G G™A G™A A™T G™A

Ile ™ Val Gln ™ Gln Gln ™ Gln Val ™ Val Asp ™ Asn

CTG GCG GGC TTT C TT GAT CGA AGC AGC GAA GCC TGT CCA GTA ACG CAC GCG CCG ATT GGC GTT TCT C GC GCC ATT GGC GTT GTG GGA TAC CAA C GC GTC CTC TC T CAG GAA AA A GTG GCA CAA CAA GCG GGC CCA TTG GCC GAT GAT GTC TCT

C™A C™A G™A G™A G™C G™C C™T G™T C™T G™A G™C C™T A™G CA ™ GG G™T C™G G™T

Ala ™ Glu Leu ™ Ile Arg ™ Asp Ala ™ Thr Val ™ Leu Ala ™ Pro Gly ™ Gly Arg ™ Leu Gly ™ Gly Gly ™ Ser Arg ™ Pro Ser ™ Phe Lys ™ Lys Gln ™ Gly Gly ™ Val Ala ™ Gly Asp ™ Phe

by 4-NQO carried multiple mutations ŽTable 5.. One of the double mutants Žnumbers 1–14. contained a 2-bp mutation. The incidence of multiple mutations induced by 4-NQO was increased about two times that of spontaneous mutation. This may be related to the structure of bulky adducts formed by 4-NQO. The DNA adduct formed by covalent binding to DNA after reduction of 4-NQO may disturb the DNA structure over long distances w2x. The altered structure of DNA may cause alteration of interaction between polymerase and DNA. Also, the stability and affinity of enzymes involved in proofreading, and DNA repair may be diminished and there are some perturbations in repair pathways operating for 4-NQO adduct w2x. Moreover, 4-NQO has often been referred to as an UV mimetic agent w26x. Thus multiple distant mutations may originate during the nucleotide excision repair like UV-induced repair. It is suggested that these are causes of high incidence of multiple mutation induced by 4-NQO. The other types of mutation found in both groups was frameshift by deletion of one or more bases as

shown in Table 6. This occurrence was less than 5% of total mutations as indicated in Table 7. The summary of sequence analysis is shown in Table 7. Total number of mutations of 43 mutants from 4-NQO-treated cells was 48. The differences between the number of mutations and that of total mutants reflect existence of multiple mutations. In both spontaneous and 4-NQO induced mutational spectra, the major type of mutation was single base substitution. The other types of mutation found in Table 6 Spontaneous and 4-NQO induced frameshift mutations in lacI gene of transgenic Big Blue w cell line Base position

Sequence Ž5X ™3X .

Change

Spontaneous Deletion 629–632

GGC TGG CAT

GGC AT

4-NQO induced Deletion 836 887

TCG GTA GTG TTA ACC ACC

TCG TAG TG TTA CCA CC

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Table 7 Summary of lacI mutational spectra from untreated and 4-NQO-treated transgenic Big Blue w cell line Mutation class

Number of occurrences Untreated Ž%.

Base substitution Transition G:C ™ A:T A:T ™ G:C Transversion A:T ™ T:A A:T ™ C:G G:C ™ T:A G:C ™ C:G Frameshifts Tandem double-base substitution Total mutations Total mutant plaques

19 Ž95. 12 Ž60. 9 Ž45. 3 Ž15. 9 Ž35. 2 Ž10. 0 Ž0. 1 Ž5. 4 Ž20. 1 Ž5. 0 Ž0. 20 Ž100. 20

both groups was frameshift by deletion of one base or more bases. The major spontaneous mutations were base substitution Ž95%. especially transition Ž60%. consistent with previously reported mutational spectra in untreated Big Blue w mice w12x and Big Blue w Rat2 cells w6x. Also, recently, Watson et al. w39x reported that G:C ™ A:T transitions are the most common in the spontaneous spectra of cII gene as well as lacI gene in Big Blue w Rat2 cells and Big Blue w animals. However, among total 48 mutations induced by 4-NQO, 45 mutations Ž93.9%. were base substitution consisted of 12 transitions Ž25%. and 33 transversions Ž68.8%.. Especially, transversion of G:C ™ T:A type was predominant representing 43.8% out of 93.9% of base substitutions. This indicate that in the lacI assay, 4-NQO is a base substitution mutagen which mainly acts at guanine residues. There have been several reports described that 4NQO predominantly induce G transition w17,36x. However, previous works in bacteria as well as in mammalian cells showed that G ™ T transversion were prevalent w35,38x. In particular, high occurrences of G transversion Ž64.7%. in 4-NQO sensitive ŽUV5. Chinese hamster ovary cells were also reported w37x. These reports w19,35–38x are in agreement with our results and may be caused by bulky adduct at guanosine. Also the comparison with other mutation spectra reported in Big Blue w Rat2 cell line revealed that mutation type induced by 4-NQO is distinctly different from the mutational spectra of

CpG

1

3

4-NQO-treated Ž%. 45 Ž93.9. 12 Ž25. 10 Ž20.8. 2 Ž4.2. 33 Ž68.8. 2 Ž4.2. 1 Ž2.1. 21 Ž43.8. 9 Ž18.8. 2 Ž4.2. 1 Ž2.1. 48 Ž100. 43

CpG

5

9 4

dimethylbenzw axanthracene ŽDMBA. w27x and ethylnitrosourea ŽENU. w6x. The common feature of mutation in three mutation spectra was G:C ™ A:T transition. In DMBA mutation spectrum, most frequently appeared mutation type was A:T ™ T:A transversion Ž38%. and second most frequent type of mutation was G:C ™ A:T transition Ž29%.. Mutational spectrum of ENU, an alkylating agent, was similar to DMBA. The most frequent mutation was G:C ™ A:T transition Ž40.5%. and second frequent mutation was A:T ™ T:A transversion Ž29.7%.. But the most frequent mutation type of 4-NQO was transversion of G:C ™ T:A type Ž43.8%., as described above. In both DMBA and ENU induced lacI mutation spectra, nearly 50% of base substitution occurred at A:T sites whereas mutations occurred at A:T site of all mutations induced by 4-NQO accounted for about 10%. This difference in mutation spectrum indicates that action mechanism of 4-NQO in mutagenesis and repair is different from alkylating agent, ENU, and polyaromatic hydrocarbons, DMBA. Compared with mutational spectrum of spontaneous mutants, 4-NQO exposure altered significantly the kinds of mutations observed in the lacI gene. This finding suggests that 4-NQO may alter the type of mutations sustained in the endogenous genes and transversion at CpG dinucleotide accounted for 47% of transversion of G:C ™ T:A type ŽTable 7.. The CpG dinucleotide was well known as a main DNA methylation site in mammal w28,29x. The methylation

134

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at CpG dinucleotide has been proposed to function in many ways including conformational change of DNA structure, maintenance of chromosome structure, regulation of gene expression, repair of DNA, development, establishment of preferred sites for mutation, protection of DNA against enzymatic degradation, aging and initiation of carcinogenesis w29,30x. Thus mutation at CpG site affects in various regulatory processes in cell. It has been demonstrated that CpG dinucleotide is a hotspot and transition at this site caused by an endogenous mechanism and spontaneous deamination of 5-methylcytosine to thymine occurs frequently in many cancers w31–34x. However, this trend may be reversed by an exogenous effect. From our results, we presume that 4NQO can induce alteration of mutation type at CpG site in p53 gene like mutational spectrum in lacI gene shows. In summary, we confirmed that 4-NQO is a potent mutagen in this study. No distinct differences in spontaneous MF and mutational spectrum at lacI gene observed between the transgenic Big Blue w cell line study and the transgenic animal study using Big Blue w mice and rat already reported w6,12,27x. Thus, it is suggested that transgenic Big Blue w cell line is useful to examine mutational spectra of direct-acting mutagens. Comparison of mutational spectra from spontaneous and 4-NQO-treated group revealed an obvious difference in the types of mutations ŽTable 7.. The major type of mutation occurred spontaneously in lacI gene was transition of G:C ™ A:T type. In contrast, the major type of mutation induced by 4-NQO was G:C ™ T:A transversion. From the results of DNA sequence analysis of 4-NQO induced lacI mutants confirmed that 4-NQO is a base substitution mutagen in the lacI assay, acting mainly at guanine residues. Also, 4-NQO has unique patterns of mutation from the comparison with the previously published mutational spectra in Big Blue w Rat2 cell w6,27,37x, although there are only a few published lacI mutational spectra in Big Blue w cell line. Mutational spectra of 4-NQO show a similarity between hprt in CHO cells w37x and lacI in Big Blue w Rat2 fibroblast cell line. These support that the Big Blue w Rat2 fibroblast cell line, harboring lacI gene as a target gene may provide an useful tool to elucidate the mechanism of mutagenesis in other endogenous genes. Recently, the usefulness of cII

locus as an alternative mutational target w39x and the possible use for routine cytogenetic study w40x of Big Blue w Rat2 fibroblast cell lines has been reported.

Acknowledgements This work was supported by Grant-in-Aid of the Ministry of Environment, Seoul, Republic of Korea and thanks to Dr. Michael D. Shelby and Dr. David E. Watson, NIEHS, USA for helpful assistance in manuscript preparation.

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