In vivo genotoxicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in lacI transgenic (Big Blue®) mice

Mutation Research 468 Ž2000. 19–25 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres

In vivo genotoxicity of 2-amino-3,8-dimethylimidazow4,5-f xquinoxaline in lacI transgenic žBig Blue w / mice q Toshiaki Itoh a,1, Takayoshi Suzuki a,) , Akiyoshi Nishikawa b, Fumio Furukawa b, Michihito Takahashi b, Wang Xue a , Toshio Sofuni a , Makoto Hayashi a a

DiÕision of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan b DiÕision of Pathology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Received 22 November 1999; received in revised form 29 February 2000; accepted 9 March 2000

Abstract 2-Amino-3,8-dimethylimidazow4,5-f xquinoxaline ŽMeIQx., a heterocyclic amine found in cooked meat, is a strong mutagen in the Salmonellarmicrosome assay and was proven to be a hepatocarcinogen in rodents. We used the lacI transgenic ŽBig Blue w . mouse to investigate MeIQx genotoxicity in vivo. lacI mutant frequencies were examined in liver and colon after single intragastric administration of MeIQx Žmales. or 12 weeks of feeding in the diet Žmales and females.. Micronucleus induction was monitored in the peripheral blood and cell proliferating activity was monitored by proliferating cell nuclear antigen ŽPCNA. immunostaining, but only after the intragastric administration. Intragastric treatment with MeIQx Ž100 mgrkg. did not increase mutant frequency ŽMF. in liver or colon but it did induce a slight but statistically significant increase in the incidence of micronucleated reticulocytes 48 h after the treatment. No apparent increase in PCNA-positive foci was observed in any of tissues analyzed 14 days after the treatment. Administration of MeIQx Ž300 ppm. in diet for 12 weeks, however, caused MF increases in liver and colon in male and female mice, with greater increases in the females. An increase was also obvious after 4 weeks, but only in females. The sex difference in MF is consistent with the fact that female mice are more susceptible to MeIQx carcinogenesis. These results demonstrated that in the transgenic mouse mutation assay, long-term feeding of MeIQx was more effective than single gastric exposures in revealing the compound’s mutagenicity in the target organs of carcinogenicity and that sex differences in susceptibility can also be observed. q 2000 Elsevier Science B.V. All rights reserved. Keywords: 2-Amino-3,8-dimethylimidazow4,5-f xquinoxaline ŽMeIQx.; lacI; Big Blue w mouse; Micronucleus assay; Cell proliferation

q

This article is communicated by the Mammalian Mutagenicity Study Group ŽMMS. of the Environmental Mutagen Society of Japan. Corresponding author. Tel.: q81-33700-9847; fax: q81-33700-2348. E-mail address: [email protected] ŽT. Suzuki.. 1 Present address: Department of Bacteriology, School of Medicine, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima, 770-8503, Japan; 463-10 Kagasuno Kawauchi-cho, Tokushima 771-0192, Japan. )

1383-5718r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 0 0 . 0 0 0 3 6 - X

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T. Itoh et al.r Mutation Research 468 (2000) 19–25

1. Introduction Various heterocyclic amines in pyrolysates of cooked meat and fish show strong mutagenicity in the Salmonellarmicrosome ŽAmes. assay w1,2x. Most require metabolic activation for expression of their genotoxicity. 2-Amino-3,8-dimethylimidazow4,5f xquinoxaline ŽMeIQx. induces hepatocellular carcinoma, adenoma, and neoplastic liver nodules in F344 rats and CDF1 mice w3–5x, lung cancer in female CDF1 mice, and lymphoma and leukemia in CDF1 mice of both sexes w4x. MeIQx mutagenicity has also been demonstrated in Chinese hamster cells w6,7x and Drosophila w8x, but the potency is far less than in the Salmonellarmicrosome test. Winton et al. w9x reported that MeIQx does not induce Dlb-1 mutations in mouse intestine. Furthermore, the clastogenic potential of MeIQx is very low in vitro and in vivo w10–12x despite its carcinogenic potential. Because of these inconsistencies, we examined the in vivo mutagenicity of these compounds in a newly developed transgenic system. There was no convenient way to detect gene mutations in vivo prior to the development of transgenic mice w13,14x. Two transgenic mouse systems with a l shuttle vector containing bacterial lacZ or lacI as reporter genes have been developed. They are commercially available as the MutaeMouse Ž lacZ transgenic. and the Big Blue w Ž lacI transgenic. mouse. Recently, another l-based system, named gpt-delta w37x, and the cII positive selection system w38x have been introduced. The accumulated data from these systems reveals their promising potential for standard mutation assays w15,16x. One of the advantages of the system is their capacity to analyze the organ specificity of mutagenesis. Previously, we combined the peripheral blood micronucleus assay with the transgenic mouse mutation assay in the same animal for concomitant detection of gene mutations and micronucleus induction in vivo w17x. We tested several mutagens, i.e. mitomycin C, ethylnitrosourea, ethyl methanesulfonate, diethylnitrosamine, dimethylnitrosamine, and MeIQ in the combined assay and showed their genotoxic specificity in vivo w17–20x. In the present study, we added a third endpoint, induction of cell proliferation, which we determined by histochemical analysis of proliferating cell nuclear antigen ŽPCNA. w21x in

organs recovered from the transgenic mice. Such data are important for understanding mechanisms of chemical carcinogenesis in relation to mutagenesis. In this paper, we report the results of two experiments Žone by intragastric administration, one by addition to diet. designed to determine whether MeIQx induces gene mutations in vivo and how such effects correlate with micronucleus induction in bone marrow and induction of cell proliferation in mutagenesis target organs. The sex difference in MeIQx mutagenicity was also examined in the feeding experiment. 2. Materials and methods 2.1. Animals and treatments Big Blue C57BLr6 transgenic mice 7 weeks of age were purchased from Stratagene ŽLa Jolla, CA, USA. and used after one week of acclimation. The cells of these mice carry 40 tandem copies of the transgene, lLIZa vector with the lacI target gene insertion, in a hemizygous way. MeIQx was obtained from Nard Institute ŽOsaka, Japan.. For the intragastric treatment experiment, randomized groups of six male mice were treated with 0, 0.1, 1, 10, or 100 mgrkg MeIQx in a volume of 10 mlrkg olive oil. The highest dose was set slightly above the dose Ž80 mgrkg. at which increase of GST-P positive foci was observed in a two-step carcinogenesis assay w40x. Blood Ž5 ml. was drawn from a tail vessel at 48 h for a micronucleus assay, and animals were sacrificed 14 days after the treatment. Tissues from three mice were shared for immunohistochemical analysis Žcell proliferation analysis. and the lacI mutation assay. Three mice per group were used for the lacI mutation assay. For the dietary treatment experiment, groups of three male and three female mice were fed 0 or 300 ppm MeIQx in a basal diet MF ŽOriental Yeast, Tokyo, Japan. for 4 or 12 weeks and subjected to the lacI gene mutation assay. The same dose as MeIQ in our previous report w20x was used. 2.2. Peripheral blood micronucleus assay The 5 ml of peripheral blood sample Žcollected without anticoagulant. was placed on an acridine

T. Itoh et al.r Mutation Research 468 (2000) 19–25 Table 1 Micronucleus frequency in peripheral blood of Big Blue w male mice 48 h after single intragstric treatment with MeIQx Ž ns6 per treatment group. Dose Žmgrkg. a

MNRETsb r1000RETs"SD

0 0.1 1 10 100

3.8"1.7 4.0"1.5 5.8"2.4 4.8"3.1 6.3"2.6 )

a

Vehicle: olive oil, 10 mlrkg. Micronucleated reticulocytes. ) ps 0.036 in Fisher’s exact test. b

orange-coated glass slide, covered with a coverslip, and allowed to be supravitally stained w23x. Within a few days, 1000 reticulocytes per animal were analyzed by fluorescence microscopy, and the number of cells with micronuclei was recorded. 2.3. Cell proliferation analysis Immediately after collection, tissues were fixed with Methacarn fixative Žmethanol:chloroform:acetic acid s 6:3:1., embedded in paraffin, and subjected to immunohistochemical staining with anti-PCNA antibody ŽDAKO Japan, Kyoto, Japan. followed by streptavidine–biotin complex formation and staining with diaminobenzidine. PCNA-positive cells were scored as the percentages of the total number of cells comprising the renal tubules, pancreatic acini, pan-

21

creatic ducts, islets of Langerhans, liver parenchyma Žhepatocytes., bile ducts, bronchi, alveoli, or splenic white pulp Žlymphocytes.. A total of 500 cells were analyzed for each tissue sample under the microscope with five independent regions. 2.4. Analysis of lacI gene mutations Analysis of lacI mutant frequency ŽMF. was performed according to the instruction manual ŽStratagene Big Bluee. and Rogers et al. w22x, with some modification. Total genomic DNA was isolated from liver and colon without phenol–chloroform, as previously described w19x. Briefly, nuclei were isolated from homogenized tissues by centrifugation in 0.5 M sucrose solution. The nuclear precipitate was treated with the proteinase K solution and mixed with an equal volume of 5 M ammonium acetate to precipitate sodium dodecyl sulfate and other contaminants. DNA was precipitated with 2-propanol. The genomic DNA was dissolved in TE buffer at a concentration of 0.5–1.0 mg DNArml, which was subjected to the in vitro packaging reaction. A total of 10 ml of the DNA solution was mixed with a l packaging extract ŽTranspack, Stratagene. twice for 90 min at 378C. The packaging reaction was terminated by the addition of SM buffer to give a final volume of 1 ml packaged sample. The integrated shuttle vector, lLIZa , was packaged into phage particles, which were then adsorbed onto Escherichia coli SCS-8 strain for 20 min at room

Fig. 1. PCNA-labeling indices in various organsrcells after single intragastric treatment with MeIQx Žerror bars represent SD..

T. Itoh et al.r Mutation Research 468 (2000) 19–25

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Table 2 lacI MF in liver and colon of Big Blue w mice 14 days after single intragastric treatment with MeIQx Ž100 mgrkg. Dose a

Mouse no.

Liver

Colon

Plaques

Mutants

MF = 10y6

Plaques

Mutants

MF = 10y6

0

1 2 3 Total

279,306 241,848 242,064 763,218

11 4 7 22

39.4 16.5 28.9 28.8

377,244 560,286 193,248 1,130,778

2 13 5 20

5.3 23.2 25.9 17.7

100 mgrkg

1 2 3 Total

315,540 192,456 232,641 740,637

6 2 5 13

19.0 10.4 21.5 17.6

496,170 213,678 325,269 1,035,117

9 8 10 27

18.1 37.4 30.7 26.1

a

Vehicle: olive oil, 10 mgrkg.

temperature. The lacI MF was determined by the incidence of blue plaques on 25 = 25 cm NZY agar plates Ž35 ml top agarose containing 0.7 mgrml of X-gal on 40 ml bottom agar.. The number of plaques per plate was adjusted to 15,000–20,000 by pre-titering the phage solution. This procedure for mutant screening was slightly modified from the original protocol w14x Žrefer to Ref. w19x in detail.. Three mice per group were used for mutation analysis and six for micronucleus assay.

3. Results

3.1. Micronucleus frequency At the highest dose, intragastric administration of MeIQx induced a slight but statistically significant increases in micronucleus reticulocytes ŽMNRET. frequency ŽTable 1..

Table 3 lacI mutant frequencies in liver and colon after 4 weeks of MeIQx feeding N.D.; no data because of a poor packaging efficiency. Dose

Colon 0

300 ppm

LiÕer 0

300 ppm

Male

Female

Mouse no.

Plaques

Mutants

MF = 10y6

Mouse no.

Plaques

6 7 8 Total 1 2 3 Total

258,642 315,711 247,482 821,835 248,733 231,588 279,801 760,122

8 9 10 27 7 6 9 22

30.9 28.5 40.4 32.9 28.1 25.9 32.2 28.9

16 17 18 Total 11 12 13 Total

188,964 N.D. 241,515 430,479 234,288 226,089 136,080 596,457

4

21.2

6 10 18 11 10 39

24.8 23.2 76.8 48.7 73.5 65.4

6 7 8 Total 1 2 3 Total

155,040 22,140 214,368 391,548 47,106 86,832 51,900 185,838

5 1 1 7 1 5 0 6

32.2 45.2 4.7 17.9 21.2 57.6 0.0 32.3

16 17 18 Total 11 12 13 Total

107,208 85,284 128,988 321,480 87,540 102,582 56,580 246,702

0 2 3 5 8 5 3 16

0.0 23.5 23.3 15.6 91.4 48.7 53.0 64.9

Mutants

MF = 10y6

T. Itoh et al.r Mutation Research 468 (2000) 19–25

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Table 4 lacI mutant frequencies in liver and colon after 12 weeks of MeIQx feeding Dose

Colon 0

300 ppm

LiÕer 0

300 ppm

Male

Female

Mouse no.

Plaques

Mutants

MF = 10y6

Mouse no.

Plaques

Mutants

MF = 10y6

26 27 28 Total 21 24 23 Total

46,080 154,305 98,400 298,785 196,794 79,584 177,750 454,128

1 3 1 5 16 5 10 31

21.7 19.4 10.2 16.7 81.3 62.8 56.3 68.3

36 37 38 Total 31 32 33 Total

64,416 278,424 79,056 421,896 213,084 73,440 194,580 481,104

1 13 4 18 25 4 19 48

15.5 46.7 50.6 42.7 117.3 54.5 97.6 99.8

26 27 28 Total 21 24 23 Total

106,464 243,810 136,284 486,558 177,390 117,312 200,070 494,772

1 9 1 11 17 3 15 35

9.4 36.9 7.3 22.6 95.8 25.6 75.0 70.7

36 37 38 Total 31 32 33 Total

110,304 290,390 114,432 515,126 227,205 79,776 113,715 420,696

1 8 3 12 47 8 32 87

9.1 27.5 26.2 23.3 206.9 100.3 281.4 206.8

3.2. Induction of cell proliferation Intragastric administration of MeIQx did not induced cell proliferation in any tissue analyzed ŽFig.1.. PCNA-positive foci in splenic lymphocytes were spontaneously high, reflecting their high proliferation rate. 3.3. Mutant frequency 3.3.1. Intragastic administration The lacI MF in liver and colon was analyzed only for the highest-dose group and did not differ significantly from that of the vehicle control group ŽTable 2.. 3.3.2. Dietary administration In the feeding experiment, lacI MFs were analyzed after 4 and 12 week of feeding with 300 ppm MeIQx ŽTables 3 and 4.. An increase in MF was observed in liver and colon in females after 4 weeks ŽTable 3. and in both sexes after 12 weeks ŽTable 4., with the female increase being greater. 4. Discussion The heterocyclic amines are strongly mutagenic in bacterial reverse mutation tests but less mutagenic in

the micronucleus test w20,24x. The main reasons for the weak genotoxic potential of heterocyclic amines in the micronucleus assay may be their requirement for metabolic activation and the high reactivity of their active metabolites, which prevent them from reaching the target tissue Žbone marrow.. The same scenario has been applied to nitrosamines w19,25x. On the other hand, in vivo mutagenicity of various heterocyclic amines has been detected in the transgenic mutation assays w20,26,27x with responses that are relatively high compared to other standard mutagens w28,29x. We initially tested the sensitivity of the transgenic mouse mutation assay to MeIQx by single intragastric administration because studies on the other heterocyclic amines were performed mainly with long-term exposures. At the highest dose Ž100 mgrkg., we observed a slight increase in MNRET frequency but no increase in lacI MF in colon and liver. DNA-adducts, however, do form in livers of rats fed MeIQx even at very low doses and in a dose-dependent manner w30–32x. These results suggest that the TG assay is not sensitive to acute mutagen exposures, but as confirmed in this study, chronic exposures increase the sensitivity w33x. The recent consensus protocol for TG assays recommends repeated dosing and a longer

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T. Itoh et al.r Mutation Research 468 (2000) 19–25

expression time to get the maximum response. The present results also support an efficacy of a long-term exposure. MeIQx increased MF in the liver, which is its main target organ for carcinogenesis. It also increased MF in colon, where tumor induction has not been reported. We do not consider this a false positive result Ži.e. MeIQx did induce mutations in colon., however, because MeIQx induces small intestinal tumors w4x and aberrant crypt foci Žpreneoplastic lesions. w34x in mice. The MF increase in colon suggests that colon tumors should be observed after the longer exposure periods used in carcinogenesis bioassays. The present results demonstrated a differential sensitivity of male and female mice to the genotoxicity of MeIQx. A sex difference in sensitivity to the carcinogenicty of MeIQx has also reported. In CDF1 mice, liver tumors were induced in 91% of females fed MeIQx but only 43% of males. In rats, in contrast, males are more susceptible than females to MeIQx hepatocarcinogenesis w5x. It would be interesting to test whether there is a sex difference in MeIQx mutagenicity in Big Blue rats. In contrast to the present results, Okonogi et al. w35x reported no sex difference in the induction of lacI MF by 2amino-1-methyl-6-phenylimidazow4,5-b xpyridine in the colon of the Big Blue rat whereas colon tumors are observed only in the male. The present results demonstrate that both sexes should be used in TG assays, especially for mutagens that are metabolized differently in males and females. A higher induction of P-450 enzymes in the female mice by heterocyclic amines w39x may account for a higher induction of mutations by MeIQx in the female. MeIQx caused no increase in cell-proliferation even in its target organ Žliver., although it is considered a complete carcinogen capable of initiation and promotion. The negative response might have been due to weak promotor activity, which was demonstrated in a short-term in vivo liver carcinogenesis assay w36x, or to inadequate testing time Žcell proliferation might be induced immediately after the treatment but diminished during 14 days.. In conclusion, we demonstrated genotoxicity of the ingested MeIQx in the liver and colon of Big Blue w mice and found the effect to be more pro-

found in females than in males. We are now studying the molecular nature of MeIQx-induced mutations.

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