J mice

Cancer Letters 125 (1998) 83–88

Dimethylarsinic acid, a main metabolite of inorganic arsenics, has tumorigenicity and progression effects in the pulmonary tumors of A/J mice Hiroyuki Hayashi a ,*, Masayoshi Kanisawa a, Kenzo Yamanaka b, Takaaki Ito a, Naoko Udaka a, Hiroshi Ohji a, Koji Okudela a, Shoji Okada c, Hitoshi Kitamura a a

Department of Pathology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan b Department of Biochemical Toxicology, Nihon University College of Pharmacy, Funabashi, Japan c Department of Radiobiochemistry, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka, Japan Received 26 September 1997; received in revised form 7 November 1997; accepted 7 November 1997

Abstract The pulmonary tumorigenicity of dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics, was examined in A/J mice fed with drinking water containing DMAA for 25 and 50 weeks. Mice fed with 400 ppm DMAA for 50 weeks produced more pulmonary tumors than untreated mice (mean number per animal 1.36 versus 0.50; P , 0.05). Histological examination revealed that the number of mice which bore adenocarcinomas or papillary adenomas correlated with the concentration of DMAA given (untreated versus 400 ppm; P = 0.002), suggesting that DMAA could promote tumorigenic processes. These results are consistent with the epidemiological studies on the pulmonary carcinogenesis of arsenics and suggest that DMAA alone can act as a carcinogen in mice.  1998 Elsevier Science Ireland Ltd. Keywords: Mouse; Pulmonary tumor; Dimethylarsinic acid; Tumorigenicity

1. Introduction Numerous epidemiological investigations have shown that inorganic arsenics are carcinogenic to humans, particularly in the skin and lungs [1,2]. High mortality rates for malignant neoplasms, including lung cancer, have been reported in an area on the southwest coast of Taiwan, where blackfoot disease is endemic and high-arsenic artesian wells were used as the drinking water source [3]. Occupational [4,5] and * Corresponding author. Tel.: +81 45 7872583; fax: +81 45 7890588.

iatrogenic arsenical exposures [6] have been reported to lead to various diseases, especially cancers of the respiratory tract and skin. Recent studies have shown adverse health effects due to serious arsenic contamination of the ground water in West Bengal State, India [7,8]. The arsenic-affected population in the State was estimated to be 4.4 million and the disorders so far identified include skin cancer and respiratory disease. However, experimental investigations using animals have not fully succeeded in proving the carcinogenicity of arsenic [1,2] and no promoting effect was demonstrated in spontaneous pulmonary tumorigenicity in mice [9].

0304-3835/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0484-9

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Dimethylarsinic acid (DMAA) is a main metabolite of inorganic arsenics and is eliminated from the kidney and excreted in urine [4]. Recent findings have revealed that DMAA induces lung-specific genetic damage in mice and in cultured cells. Oral administration of DMAA induced an increase of heterochromatin in venular endothelium of the lungs [10]. Also, single-strand DNA breaks in the lungs due to the actions of active oxygens and dimethylarsenic peroxyl radicals, both of which were produced in the metabolism of DMAA in mice [11–13], were induced. Using the human alveolar type II cell line (L-132), DMAA exposure causes DNA single-strand breaks, suppression of replicative DNA synthesis [14], DNA–protein crosslinks [15] and formation of apurinic/apyrimidinic sites in DNA [16]. Recently, Yamanaka et al. [17] reported that exposure of L-132 cells to DMAA

induced the activation of DNA repair due to the formation of DNA adducts. In the previous study, we showed that DMAA acts as a promoter in ddY mice lung tumorigenesis initiated by 4-nitroquinoline 1-oxide (4NQO) [18]. In the present study, we investigated lung tumorigenicity and progression activity by oral administration of DMAA on A/J mice which are susceptible to lung tumorigenesis [19].

2. Materials and methods 2.1. Animals Five-week-old male A/J mice were obtained from Japan SLC (Hamamatsu, Japan). Four or five mice

Fig. 1. Lung tumors induced in A/J mice by DMAA treatment for 50 weeks. (a) Hyperplasia (200 ppm DMAA). Intact alveolar septa are lined by proliferating alveolar type II cells. (b) Alveolar adenoma (200 ppm DMAA). Note the cords of cuboidal epithelial cells with mild swelling of the nuclei. (c) Papillary adenoma (400 ppm DMAA). (d) Adenocarcinoma (400 ppm DMAA). Alveolar spaces are obliterated by papillary growth of cuboidal to low columnar atypical epithelial cells in (c,d). Cancer cells in (d) show more pronounced atypism with prominent nucleoli than adenoma cells in (c). Note the mitotic figures (arrows) in adenocarcinoma (d). Hematoxylin–eosin stain. Magnification, ×350.

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were housed in a cage for 25 or 50 weeks and were maintained under conventional clean conditions with a 12 h light/dark cycle at 24 ± 1°C and 55 ± 5% relative humidity. Mice were fed with commercial pellets (Oriental Yeast, Tokyo, Japan) and were given free access to food and drinking water.

ing to a modification of Kimura’s criteria [20]. Sections were observed by two pathologists (H.H. and M.K.) independently without information about treatments.

2.2. Tumorigenesis experiments

The difference in the number of tumors per mouse was analyzed by the Mann–Whitney test and the difference in the incidence of tumor-bearing mice was analyzed by Fisher’s exact test.

2.4. Statistics

The mice were divided into eight groups of 24 each and given tap water (control), or a 50, 200 or 400 ppm DMAA solution (Nacalai tesque, Kyoto, Japan). DMAA was recrystallized with methanol twice before making solutions. Ten mice from each group were sacrificed at 25 weeks and the rest were sacrificed at 50 weeks. Under deep sodium pentobarbital anesthesia, the lungs were excised after fixation by intratracheal instillation of 10% buffered formalin. Pulmonary tumors were counted under a dissecting microscope and the size of each tumor was measured simultaneously.

3. Results 3.1. Incidence and size of the tumors Among the groups at 25 weeks, there were no significant differences in the percentage of tumor-bearing mice (20–40%), the total number of tumors, or the size of the tumors (Table 1). At 50 weeks, the percentage of tumor-bearing mice and the total number of tumors increased and the size of the tumors was enlarged compared to the mice at 25 weeks. Although significant differences were not observed in the percentage of tumor-bearing mice among the 50-week groups (50–78.6%), the number of tumors per mouse was higher in the DMAA-administered group than in the control group; some mice with DMAA administration had multiple tumors (a maximum of three), while the untreated mice had none or only a single tumor, if present. There was a significant dif-

2.3. Histology To evaluate the histological grades of tumors, paraffin sections of tumors were made and stained with hematoxylin and eosin and Alcian Blue-periodic acidSchiff. Two to five 4-mm sections were cut for each tumor at 100 mm intervals. The histology of the tumors was classified into four grades, i.e. hyperplasia (Fig. 1a), alveolar adenoma (Fig. 1b), papillary adenoma (Fig. 1c) and adenocarcinoma (Fig. 1d), accordTable 1 Effects of DMAA in drinking water on mice lung tumorigenesis Group (ppm DMAA) 25 weeks Untreated 50 200 400 50 weeks Untreated 50 200 400 a

No. of animals

No. of tumorbearing mice (%)

Total no. of tumors

No. of tumors/mouse (mean ± SD)

Size of tumor (average/ maximum) (mm)

10 10 10 10

2 3 4 3

(20.0) (30.0) (40.0) (30.0)

2 3 5 4

0.20 0.30 0.50 0.40

± ± ± ±

0.42 0.48 0.71 0.70

0.9/1.0 0.5/0.7 1.4/2.1 1.1/1.5

14 14 14 14

7 10 9 11

(50.0) (71.4) (64.3) (78.6)

7 15 15 19

0.50 1.07 1.07 1.36

± ± ± ±

0.52a 1.00 1.07 1.01a

1.0/1.8 1.2/3.0 1.4/3.7 1.5/6.3

Comparisons between values with the same letter were statistically significant: P , 0.05.

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ference in the number of tumors per mouse between the untreated group and the 400 ppm DMAA-administered group (P , 0.05). The size of the tumors tended to be larger in the DMAA-administered groups than in the untreated group. 3.2. Histology The histological grade of each tumor was variable among the groups at 25 weeks (Table 2). Among the groups at 50 weeks, papillary adenoma and adenocarcinoma tended to increase in the DMAA-administered groups. To see the tumor progression effects of DMAA, we classified mice by the possession of papillary adenoma or adenocarcinoma (Table 3). There were no differences among the 25-week groups, but among the 50-week groups, the ratio of mice with papillary adenoma or adenocarcinoma increased depending on the dosage of DMAA administered with statistical significance between the untreated group and the 400 ppm DMAA group (P = 0.002).

4. Discussion Yamamoto et al. [21] have proposed that DMAA acts as a tumor promoter in various organs except the lungs. In our previous study using ddY mice [18], we showed that the promoting effect of DMAA to pulmonary tumorigenesis was stronger than glycerol, a known potent promoter for lung tumors induced by Table 2 Histology of lung tumors Group (ppm DMAA) 25 weeks Untreated 50 200 400 50 weeks Untreated 50 200 400

HP

AA

PA

AC

NE

0 0 1 0

0 1 2 2

2 2 1 1

0 0 1 0

0 0 0 1

0 6 3 4

5 3 5 3

1 3 3 7

1 3 4 3

0 0 0 2

HP, hyperplasia; AA, alveolar adenoma; PA, papillary adenoma; AC, adenocarcinoma; NE, not examined.

Table 3 Progression effects of DMAA on mice lung tumorigenesis Group (ppm DMAA) 25 weeks Untreated 50 200 400 50 weeks Untreated 50 200 400a

No. of mice without either PA or AC

No. of mice with PA and/or AC

8 8 8 9

2 2 2 1

12 9 7 3

2b,c 5 7b 10c

PA, papillary adenoma; AC, adenocarcinoma. a A tumor in one mouse was not examined histologically. Comparisons between values with the same letter were statistically significant: bP = 0.052; cP = 0.002.

4NQO [22]. However, no increase in lung tumor incidence was observed by DMAA administration alone. In the present study, we demonstrated carcinogenic activity of DMAA in animal experiments for the first time, as lung tumor incidence increased by DMAA administration alone and there was a significant difference in the number of pulmonary tumors between the untreated group and the group with 400 ppm DMAA administration for 50 weeks. The interspecies difference in the tumorigenic effect of DMAA might be related to the difference in the susceptibility to carcinogenic agents between ddY and A/J mice. A/ J mice are known to be highly susceptible to the induction of lung tumors by chemical carcinogens compared to other mouse strains [19]. The carcinogenic effect of DMAA in the murine lung is most likely to be attributable to its capability to induce DNA damage in lung cells as reported, i.e. an increase in heterochromatin content [10], DNA single-strand breaks [11–14], DNA–protein crosslinks [15], the formation of apurinic/apyrimidinic sites in the DNA [16] and DNA adducts [17]. Although more tumors were induced by DMAA administration at 50 weeks, no differences were observed at 25 weeks. It can be considered reasonable that a 25-week period is not long enough for DMAA to exert its carcinogenic effect. Moreover, probable accumulation of DMAA may be needed to manifest pulmonary tumors. It is also conceivable that DMAAinduced DNA damage may be repairable, so a 25-

H. Hayashi et al. / Cancer Letters 125 (1998) 83–88

week period is not long enough to cause unrepairable damage. Evidence has been accumulated that shows that alveolar adenomas and papillary adenomas have different biological potentials and characteristics. Kimura [20] reported that papillary adenomas proliferate more rapidly than alveolar adenomas and that papillary adenomas have a potential to become malignant while alveolar adenomas rarely do so. Ultrastructural [23] and keratin-immunohistochemical [24] studies have shown that papillary adenomas consist of Clara cells while alveolar adenomas consist of alveolar type II cells. Moreover, enzyme histochemistry [25,26] and lectin histochemistry [27] have shown some differences in staining patterns between alveolar adenomas and papillary adenomas. In the histological observations made in this study, more than half of the lung tumors were papillary adenomas or adenocarcinomas in the 50-week group administered with 400 ppm DMAA. Consequently, the ratio of mice with papillary adenoma or adenocarcinoma increased in a dose-dependent manner. The size of the tumors tended to be larger in the DMAA-administered groups than in the untreated groups. These results suggest that DMAA causes tumor progression activity. Our previous study [18] showed that the tumors induced by 4NQO and DMAA tended to be more anaplastic than those induced by 4NQO alone. Furthermore, we observed an unusual case of adenosquamous carcinoma with intrapulmonary metastatic deposits. Thus, it seems likely that the tumor progression activity of DMAA was exerted not only in A/J mice but also in ddY mice, however, the effect was more pronounced in A/J mice. Accumulation of genetic changes caused by DMAA may be responsible for this tumor progression. The number of animals used in this study is not large enough to state definite conclusions. Further studies using a larger number of animals, including other strains of mice and other species, are required to conclusively demonstrate the carcinogenic potential of DMAA. K-ras gene mutations are commonly observed in lung tumors of A/J mice [28] and specific mutations induced by the administration of each carcinogen have been reported. For example, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone administration causes mainly G to A transitions [29], most benzo[b]-

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fluoranthene-induced tumors contain G to T transversions [30] and the majority of 7H-dibenzo[c,g] carbazole-induced mutations are A to T transversions [31]. To our knowledge, specific mutation of the Kras gene caused by DMAA is unknown. In the next study, molecular changes induced by DMAA, particularly in the K-ras gene, should be examined. Epidemiological studies have shown that arsenics are carcinogenic to human lungs [1,2]. Although its histological types have yet to be fully specified [5], Wicks et al. [32] reported a significantly higher incidence of adenocarcinoma. Thus, the present study using A/J mice, a useful model of lung adenocarcinoma in humans, is compatible with previous epidemiological investigations. In summary, we demonstrated for the first time that ingested DMAA from drinking water has pulmonary carcinogenic potential and a tumor progression effect in A/J mice. Our results described here are consistent with reports of DNA damage induced by DMAA in lung cells as well as the epidemiological evidence of pulmonary carcinogenesis by arsenics in humans.

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