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The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice
Cancer Letters 152 (2000) 79±85 www.elsevier.com/locate/canlet
The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice Kenzo Yamanaka a,*, Koichi Katsumata a, Kazuhiro Ikuma a, Akira Hasegawa a, Masayuki Nakano b, Shoji Okada c b
a Nihon University College of Pharmacy, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan Division of Pathology, Chiba University Hospital School of Medicine, 1-8-1 Inohana, Chyuoh-ku, Chiba 260-8677, Japan c University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka 422-8526, Japan
Received 24 September 1999; received in revised form 30 November 1999; accepted 2 December 1999
Abstract The effect of dimethylarsinic acid (DMA) on skin tumorigenesis by UVB irradiation was examined. Hairless mice (Hos: HR1) irradiated with UVB at a dose of 2 kJ/m 2 twice weekly, were fed with drinking water containing 1000 ppm DMA, a main metabolite of inorganic arsenics, produced more skin tumors than DMA-untreated mice. Histopathological examination revealed that the mouse malignant tumors with severe atypism appeared only in the treatment group of UVB plus 1000 ppm DMA. These positive results point out the importance of dimethylated metabolites of inorganic arsenic in the process of skin carcinogenesis. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dimethylarsinic acid; Arsenic; UVB; Skin tumor; Promotion activity; Progression activity
1. Introduction Epidemiological investigations have demonstrated that inorganic arsenics are carcinogenic for skin, lung, and bladder [1,2]. Arsenic-contamination in drinking water is widespread, particularly in Asia, causing a serious health problem. High morbidity rates of malignant neoplasms due to arsenic uptake have been reported in areas of the southwest coast of Taiwan [3,4], Inner Mongolia in China [5], and the West Bengal in India [6] etc. Arsenics in underground water in the West Bengal have been found at higher * Corresponding author. Tel./fax: 1 81-47-465-6077. E-mail address: [email protected] (K. Yamanaka)
level than the WHO maximum permissible limit of 0.05 ppm. Internationally, at least one million people are drinking arsenic-contaminated water above the WHO recommended value of 0.01 ppm and 200 000 people show skin lesions characteristic of arsenic poisoning [6]. Numerous experimental studies using laboratory animals, on the other hand, have failed to show the carcinogenic action of inorganic arsenics [1,2]. However, recent reports indicated that the oral administration of dimethylarsinic acid (DMA), a main metabolite of inorganic arsenics in mammals, to mice [7] and rats [8,9] promoted chemical carcinogen-initiated tumorigenesis in the lungs of mice and in the bladder, kidney, liver, and thyroid of rats. Furthermore, our
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00440-1
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K. Yamanaka et al. / Cancer Letters 152 (2000) 79±85
previous study indicated that oral administration of only DMA to mice for 1 year induced lung cancer [10]. We thought that the dimethylarsenic peroxyl radical [(CH3)2AsOO´] produced in the further metabolic processing of DMA was responsible for the pulmonary tumorigenic activity. These results suggest that dimethylarsenics have a potential for cancer induction. However, positive animal experimental studies of skin carcinogenesis have not yet been reported for arsenics. Using the animal experimental model of two-stage skin carcinogenesis, which characterizes papillomas as premalignant lesions, benzoyl peroxide [11±14] and chrysarobin [15] are known to have strong promotion activity and are suggested to induce reactive oxygen species (ROS). Benzoyl peroxide, an enhancer for the progression of papillomas to squamous cell carcinomas [11], has been reported to produce benzoyloxyl radicals, phenyl radicals, and hydroxyl radicals, both in vivo [14] and in vitro [16±18], and to induce DNA damage, such as DNA strand breaks, DNA±protein crosslinks, and base modi®cations [19± 21] in the presence of Cu 1 and Fe 21. We previously estimated that dimethylarsenic peroxyl radical [(CH3)2AsOO´] induced DNA damage such as DNA single- and double-strand breaks, DNA±protein crosslinks and nucleic base-modi®cations [22±29]. From the standpoint of skin carcinogenesis of arsenics, it appeared desirable to investigate the effects of dimethylarsenics in UV-induced skin tumorigenesis. In this paper we describe the actions of tumor promotion and progression effects of oral dimethylarsenics on UVB-irradiated mouse skin.
2. Materials and methods 2.1. Experimental animals Six-week-old female Hos: HR-1 hairless mice were obtained from Sankyo Laboservice Corporation, Inc. (Tokyo, Japan). Five mice were housed in a cage for 25 weeks and were maintained under the speci®c pathogen-free (SPF) conditions with a 12 h-light/ dark cycle at 238C ^ 18C and 55% relative humidity. Mice were given water and g-ray-sterilized chow pellets from the Funabashi Farm Co. (Funabashi, Japan) ad libitum.
2.2. Chemicals and UVB light DMA was purchased from Nakarai Tesque (Kyoto, Japan), and was recrystallized twice from methanol before use. Treatment of mice with UVB was made as follows: each cage (20 £ 15 £ 5 cm) containing ®ve mice was placed under an overhead UVB lamp (HP15M, 280±380 nm, max. 312 nm; Atto Co., Tokyo, Japan) at an irradiation distance of 9 cm. The exposure time for a 2 kJ/m 2 dose of UVB was 222 s. No detectable heat was produced during irradiation. The UVB doses were determined at 302 nm with an radiometer (UVX31: UVP, Inc., CA). 2.3. UVB-induced tumorigenesis protocol The method described by Kobayashi et al. [30] was principally employed. Brie¯y, the mice were divided into three groups of 10±11 animals each and given tap water (control, group 1), a 400 ppm DMA (group 2) or 1000 ppm DMA (group 3) solution (pH 3.4) ad libitum, and were irradiated twice weekly with UVB at a dose of 2 kJ/m 2. The mice from each group were sacri®ced at 25 weeks. Under sodium pentobarbital anesthesia, the skin tumors greater than 2 mm in diameter were counted, and then were excised and ®xed with buffered 10% formalin. To evaluate the histopathological grades of the tumors, paraf®n sections of tumors were stained with hematoxylin and eosin. 2.4. Statistics The difference in the number of tumors per mouse between the groups administered DMA and the controls was analyzed by the non-parametric Mann± Whitney U-test at 2-week intervals. The difference in percentage tumor-bearing mice between the groups administered DMA and the controls was analyzed by the Fisher's exact test at 1-week intervals. 3. Results 3.1. Incidence of skin tumors The dose of DMA used was neither lethal nor apparently toxic. As shown in Fig. 1a, no signi®cant difference was seen in body weight gain between DMA-treated groups (groups 2 and 3) and control
K. Yamanaka et al. / Cancer Letters 152 (2000) 79±85
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Fig. 1. Promoting effects of oral administration of dimethylarsinic acid (DMA) on the formation of UVB-induced skin tumors in hairless mice. Female HR-1 mice were irradiated with UVB (2 kJ/m 2) twice weekly for 25 weeks. DMA solution (400 or 1000 ppm) was used as the drinking ¯uid throughout the entire study. Group 1 (W), UVB alone (control); Group 2 (X), mice treated 400 ppm DMA solution with UVB irradiation; Group 3 (K), mice treated 1000 ppm DMA solution with UVB irradiation. Each value in (a) and (b) represents the mean ^ SEM for 10 or 11. (b): signi®cant difference for group 3 versus group 1 using the non-parametric Mann±Whitney U-test (control) (*P , 0:05, **P , 0:01). (c): signi®cant difference for group 3 versus group 1 (control) using the Fisher's exact test (*P , 0:05).
group (group 1). The average DMA intake in groups 2 and 3 was 2077 and 5771 mg/mouse per day, respectively, based on the weekly measurements. In the skins of HR-1 hairless mice irradiated with UVB at a dose of 2 kJ/m 2 twice a week, tumors larger than 2-mm in diameter were counted every week. The size of tumors became larger as the UVB-irradiation period continued. The skin tumors ®rst appeared at 12 weeks after the start of the experiment and were present at 16 weeks in all the control mice exposed to UVB only. Although the tumor number of group 2 (400 ppm DMA-treated) showed no signi®cant difference from group 1 (control), the tumor number per mouse in group 3 (1000 ppm DMA-treated) was signi®cantly
more than that in group 1 for 13±19 weeks after the start of UVB irradiation (Fig. 1b). Furthermore, percentage tumor-bearing mice in group 3 was signi®cantly higher than group 1 (Fig. 1c). However, there are no differences among group 2 as similar to that shown in Fig. 1b,c. These results show that a higher dose of oral DMA promoted skin tumorigenesis in UVBinitiated mice. 3.2. Histopathological examination To see the tumor progression effect of dimethylarsenics, histopathological evaluations for the tissues of skin tumors (acanthomas) induced by the treatment
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with UVB and DMA were performed. The tumors were composed of focal proliferation of squamous cells. The tumors were classi®ed into four degrees of atypism by the criterion of nuclear hyperchromasia, irregular cell arrangement, and more solid growth (Fig. 2). The two mice with the malignant tumors (severe atypism, 111) showing apparent proceeding to carcinomas (Fig. 2d) were observed only in the 1000 ppm DMAtreated (group 3 of Table 1). The degree of atypism was not agreement with the size of tumors. This result shows that oral administration of DMA leads to progression activity in skin tumorigenesis of mice. 4. Discussion Our previous study demonstrated that oral administration of DMA caused the promotion and, further,
progression of 4-nitroquinoline 1-oxide (4-NQO)initiated lung tumorigenesis in A/J male mice [7]. Furthermore, Yamamoto et al. reported that oral administration of DMA promotes multi-organ tumorigenesis F344 female rats initiated with ®ve different carcinogens [8]. These ®ndings support the scienti®c theory that dimethylated arsenic may act as an unique promoter for various tissues, differently from a tissuespeci®c promoter such as TPA (12-O-tetradecanoylphorbol-13 acetate). In the present study using UVB irradiation and hairless mice, we demonstrated tumor promoting and progressing action of dimethylarsenic on skin tumorigenesis. Orally administered inhibitors and suppressors, e.g. tea extracts containing polyphenols [31±34], l-selenomethionine [35], chlorpromazine [36], and indomethacin [37] against skin-tumor promotion have been reported for skin tumors. However orally effective promoters are so far
Fig. 2. Micrograph of acanthomas lesions induced by treatment with UVB and oral administration of 1000 ppm DMA. Representative four degrees (2, 1, 11 and 111) of atypism are shown in (a), (b), (c), and (d), respectively (hematoxylin and eosin, £ 125).
K. Yamanaka et al. / Cancer Letters 152 (2000) 79±85 Table 1 Histopathological evaluation of the skin tumors (acanthomas) induced by the treatment with UVB and oral administration of DMA a Group
UVB alone (group 1) UVB 1 400 ppm DMA (group 2) UVB 1 1000 ppm DMA (group 3)
Atypism 2
1
11
111
7 5
2 3
2 2
0 0
1
2
5
2
a Six-week-old female hairless mice (Hos: HR-1) were divided into three groups. Ten to 11 mice of each group were irradiated twice weekly with UVB at a dose of 2 kJ/m 2, and were given tap water (group 1, control), 400 ppm (group 2) or 1000 ppm (group 3) DMA solution as drinking water ad libitum for 25 weeks. The tumors (acanthomas) greater than 2-mm in diameter were excised and ®xed with 10% buffered formalin. The histopathological evaluations were classi®ed into four lesions according to the degree of atypism of acanthomas; (2) none of atypism, (1) mild atypism, (11) moderate atypism, (111) severe atypism. Each value represents no. of mice having tumors classi®ed into each lesion.
unknown. Our ®nding that the oral administration of DMA has substantial promoting effect on UVBinduced skin tumorigenesis in mice may contribute to the further development of mouse skin carcinogenesis models. Some reports show that organic peroxide such as benzoyl peroxide promotes 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin tumorigenesis in mouse in vivo [11,12,14] and X-ray-initiated malignant transformation in vitro [18]. Benzoyl peroxide is a well-known free radical-generating compound; benzoyloxyl radicals, phenyl radicals, and hydroxyl radicals are produced in the presence of copper or ferrous ions [16±21]. Skin tumor promoting-activity by benzoyl peroxide in the two-stage carcinogenesis in mice might be caused by the production of these free radicals. Benzoyl peroxide, however, did not promote UV-induced skin tumorigenesis in hairless mice, when present on the skin either before, during, or after repetitive UV irradiation [13]. In the present study, an appreciable enhancement of skin tumor number in mice was detected in repetitive irradiation with UVB and oral administration with DMA for 25 weeks. On the other hand, our previous study indicated that free radicals such as dimethylar-
83
senic peroxyl radical and superoxide anion radical were produced via the reductive metabolism of DMA [(CH3)2As(O)OH] to dimethylarsine [(CH3)2AsH] [24]. Thus, free radicals, particularly dimethylarsenic peroxyl radical [(CH3)2AsOO´], may be responsible for the tumor-promoting action by DMA administration in UVB-induced skin tumors in mice. Furthermore, the free radicals caused genetic damage such as DNA single-strand breaks [22,23], DNA-protein cross-linking [26], and DNA modi®cations [26±29]. Thus, accumulation of genetic changes caused by oral administration of DMA may be responsible for the tumor progression. We observed in mouse skin regard to the skin-tumor promotion by dimethylarsenics, two possibilities for the mechanism may be considered: (a) dimethylarsenics and/or free radicals produced in the metabolic processing of DMA may have promoting activity or (b) they may enhance the promoting activity of UVB. Our preliminary studies support interpretation (a), that is, the oral administration of DMA in mice promotes skin tumorigenesis of not only UVB-initiated but also DMBA-initiated mice. In contrast, one report indicated that UVB irradiation promoted tumorigenesis of DMBA-initiated on mouse skin [32] Acknowledgements This work was in part supported by a Grant-in-Aid (No. 10672113) from the Ministry of Education, Science, Sports and Culture of Japan. We thank Ms Yoko Fukui of Nihon University College of Pharmacy for technical assistance. References [1] International Agency Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 23: Some Metals and Metallic Compounds, IARC, Lyon, 1980, pp. 39±141. [2] International Agency Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 1±42, (Suppl. 7): Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs, IARC, Lyon, 1987, pp. 100±106. [3] C.-J. Chen, Y.-C. Chuang, T.-M. Lin, M.-M. Wu, Malignant neoplasms among residents of a Blackfoot disease-endemic area in Taiwan: High-arsenic artesian well water and cancers, Cancer Res. 45 (1985) 5895±5899. [4] C.-J. Chen, C.-W. Chen, M.-M. Wu, T.-L. Kuo, Cancer poten-
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K. Yamanaka et al. / Cancer Letters 152 (2000) 79±85 Lou, K. Reuhl, M. Iatropoulos, C.S. Yang, A.H. Conney, Inhibitory effect of green tea in the drinking water on tumorigenesis by ultraviolet light and 12-o-tetradecanoylphorbol13-acetate in the skin of SKH-1 mice, Cancer Res. 52 (1992) 1162±1170. [33] Z.Y. Wang, M.-T. Huang, Y.-R. Lou, J.-G. Xie, K.R. Reuhl, H.L. Newmark, C.-T. Ho, C.S. Yang, A.H. Conney, Inhibitory effects of black tea, green tea, decaffeinated black tea, and decaffeinated green tea on ultraviolet B light-induced skin carcinogenesis in 7, 12-dimethylbenz[a]anthracene-initiated SKH-1 mice, Cancer Res. 54 (1994) 3428±3435. [34] M.-T. Huang, J.-G. Xie, Z.Y. Wang, C.T. Ho, Y.R. Lou, C.-X. Wang, G.C. Hard, A.H. Conney, Effects of tea, decaffeinated tea, and caffeine on UVB light-induced complete carcinogen-
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esis in SKH-1 mice: Demonstration of caffeine as a biologically important constituent of tea, Cancer Res. 57 (1997) 2623±2629. [35] K.E. Burke, G.F. Combs Jr., E.G. Gross, K.C. Bhuyan, H.A. Libdeh, The effects of topical and oral L-selenomethionine on pigmentation and skin cancer induced by ultraviolet irradiation, Nutr. Cancer 17 (1992) 123±137. [36] M.J. Peak, M. Pfaff, C. Peraino, Chlorpromazine reduces UVinduced squamous cell carcinogenesis in hairless mice and enhances UV-induced DNA damage in cultured cells, Br. J. Cancer 60 (1989) 220±222. [37] V.E. Reeve, M.J. Matheson, M. Bosnic, C. Boehm-Wilcox, The protective effect of indomethacin on photocarcinogenesis in hairless mice, Cancer Lett. 95 (1995) 213±219.
The role of orally administered dimethylarsinic acid, a main metabolite of inorganic arsenics, in the promotion and progression of UVB-induced skin tumorigenesis in hairless mice Kenzo Yamanaka a,*, Koichi Katsumata a, Kazuhiro Ikuma a, Akira Hasegawa a, Masayuki Nakano b, Shoji Okada c b
a Nihon University College of Pharmacy, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan Division of Pathology, Chiba University Hospital School of Medicine, 1-8-1 Inohana, Chyuoh-ku, Chiba 260-8677, Japan c University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka 422-8526, Japan
Received 24 September 1999; received in revised form 30 November 1999; accepted 2 December 1999
Abstract The effect of dimethylarsinic acid (DMA) on skin tumorigenesis by UVB irradiation was examined. Hairless mice (Hos: HR1) irradiated with UVB at a dose of 2 kJ/m 2 twice weekly, were fed with drinking water containing 1000 ppm DMA, a main metabolite of inorganic arsenics, produced more skin tumors than DMA-untreated mice. Histopathological examination revealed that the mouse malignant tumors with severe atypism appeared only in the treatment group of UVB plus 1000 ppm DMA. These positive results point out the importance of dimethylated metabolites of inorganic arsenic in the process of skin carcinogenesis. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Dimethylarsinic acid; Arsenic; UVB; Skin tumor; Promotion activity; Progression activity
1. Introduction Epidemiological investigations have demonstrated that inorganic arsenics are carcinogenic for skin, lung, and bladder [1,2]. Arsenic-contamination in drinking water is widespread, particularly in Asia, causing a serious health problem. High morbidity rates of malignant neoplasms due to arsenic uptake have been reported in areas of the southwest coast of Taiwan [3,4], Inner Mongolia in China [5], and the West Bengal in India [6] etc. Arsenics in underground water in the West Bengal have been found at higher * Corresponding author. Tel./fax: 1 81-47-465-6077. E-mail address: [email protected] (K. Yamanaka)
level than the WHO maximum permissible limit of 0.05 ppm. Internationally, at least one million people are drinking arsenic-contaminated water above the WHO recommended value of 0.01 ppm and 200 000 people show skin lesions characteristic of arsenic poisoning [6]. Numerous experimental studies using laboratory animals, on the other hand, have failed to show the carcinogenic action of inorganic arsenics [1,2]. However, recent reports indicated that the oral administration of dimethylarsinic acid (DMA), a main metabolite of inorganic arsenics in mammals, to mice [7] and rats [8,9] promoted chemical carcinogen-initiated tumorigenesis in the lungs of mice and in the bladder, kidney, liver, and thyroid of rats. Furthermore, our
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00440-1
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K. Yamanaka et al. / Cancer Letters 152 (2000) 79±85
previous study indicated that oral administration of only DMA to mice for 1 year induced lung cancer [10]. We thought that the dimethylarsenic peroxyl radical [(CH3)2AsOO´] produced in the further metabolic processing of DMA was responsible for the pulmonary tumorigenic activity. These results suggest that dimethylarsenics have a potential for cancer induction. However, positive animal experimental studies of skin carcinogenesis have not yet been reported for arsenics. Using the animal experimental model of two-stage skin carcinogenesis, which characterizes papillomas as premalignant lesions, benzoyl peroxide [11±14] and chrysarobin [15] are known to have strong promotion activity and are suggested to induce reactive oxygen species (ROS). Benzoyl peroxide, an enhancer for the progression of papillomas to squamous cell carcinomas [11], has been reported to produce benzoyloxyl radicals, phenyl radicals, and hydroxyl radicals, both in vivo [14] and in vitro [16±18], and to induce DNA damage, such as DNA strand breaks, DNA±protein crosslinks, and base modi®cations [19± 21] in the presence of Cu 1 and Fe 21. We previously estimated that dimethylarsenic peroxyl radical [(CH3)2AsOO´] induced DNA damage such as DNA single- and double-strand breaks, DNA±protein crosslinks and nucleic base-modi®cations [22±29]. From the standpoint of skin carcinogenesis of arsenics, it appeared desirable to investigate the effects of dimethylarsenics in UV-induced skin tumorigenesis. In this paper we describe the actions of tumor promotion and progression effects of oral dimethylarsenics on UVB-irradiated mouse skin.
2. Materials and methods 2.1. Experimental animals Six-week-old female Hos: HR-1 hairless mice were obtained from Sankyo Laboservice Corporation, Inc. (Tokyo, Japan). Five mice were housed in a cage for 25 weeks and were maintained under the speci®c pathogen-free (SPF) conditions with a 12 h-light/ dark cycle at 238C ^ 18C and 55% relative humidity. Mice were given water and g-ray-sterilized chow pellets from the Funabashi Farm Co. (Funabashi, Japan) ad libitum.
2.2. Chemicals and UVB light DMA was purchased from Nakarai Tesque (Kyoto, Japan), and was recrystallized twice from methanol before use. Treatment of mice with UVB was made as follows: each cage (20 £ 15 £ 5 cm) containing ®ve mice was placed under an overhead UVB lamp (HP15M, 280±380 nm, max. 312 nm; Atto Co., Tokyo, Japan) at an irradiation distance of 9 cm. The exposure time for a 2 kJ/m 2 dose of UVB was 222 s. No detectable heat was produced during irradiation. The UVB doses were determined at 302 nm with an radiometer (UVX31: UVP, Inc., CA). 2.3. UVB-induced tumorigenesis protocol The method described by Kobayashi et al. [30] was principally employed. Brie¯y, the mice were divided into three groups of 10±11 animals each and given tap water (control, group 1), a 400 ppm DMA (group 2) or 1000 ppm DMA (group 3) solution (pH 3.4) ad libitum, and were irradiated twice weekly with UVB at a dose of 2 kJ/m 2. The mice from each group were sacri®ced at 25 weeks. Under sodium pentobarbital anesthesia, the skin tumors greater than 2 mm in diameter were counted, and then were excised and ®xed with buffered 10% formalin. To evaluate the histopathological grades of the tumors, paraf®n sections of tumors were stained with hematoxylin and eosin. 2.4. Statistics The difference in the number of tumors per mouse between the groups administered DMA and the controls was analyzed by the non-parametric Mann± Whitney U-test at 2-week intervals. The difference in percentage tumor-bearing mice between the groups administered DMA and the controls was analyzed by the Fisher's exact test at 1-week intervals. 3. Results 3.1. Incidence of skin tumors The dose of DMA used was neither lethal nor apparently toxic. As shown in Fig. 1a, no signi®cant difference was seen in body weight gain between DMA-treated groups (groups 2 and 3) and control
K. Yamanaka et al. / Cancer Letters 152 (2000) 79±85
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Fig. 1. Promoting effects of oral administration of dimethylarsinic acid (DMA) on the formation of UVB-induced skin tumors in hairless mice. Female HR-1 mice were irradiated with UVB (2 kJ/m 2) twice weekly for 25 weeks. DMA solution (400 or 1000 ppm) was used as the drinking ¯uid throughout the entire study. Group 1 (W), UVB alone (control); Group 2 (X), mice treated 400 ppm DMA solution with UVB irradiation; Group 3 (K), mice treated 1000 ppm DMA solution with UVB irradiation. Each value in (a) and (b) represents the mean ^ SEM for 10 or 11. (b): signi®cant difference for group 3 versus group 1 using the non-parametric Mann±Whitney U-test (control) (*P , 0:05, **P , 0:01). (c): signi®cant difference for group 3 versus group 1 (control) using the Fisher's exact test (*P , 0:05).
group (group 1). The average DMA intake in groups 2 and 3 was 2077 and 5771 mg/mouse per day, respectively, based on the weekly measurements. In the skins of HR-1 hairless mice irradiated with UVB at a dose of 2 kJ/m 2 twice a week, tumors larger than 2-mm in diameter were counted every week. The size of tumors became larger as the UVB-irradiation period continued. The skin tumors ®rst appeared at 12 weeks after the start of the experiment and were present at 16 weeks in all the control mice exposed to UVB only. Although the tumor number of group 2 (400 ppm DMA-treated) showed no signi®cant difference from group 1 (control), the tumor number per mouse in group 3 (1000 ppm DMA-treated) was signi®cantly
more than that in group 1 for 13±19 weeks after the start of UVB irradiation (Fig. 1b). Furthermore, percentage tumor-bearing mice in group 3 was signi®cantly higher than group 1 (Fig. 1c). However, there are no differences among group 2 as similar to that shown in Fig. 1b,c. These results show that a higher dose of oral DMA promoted skin tumorigenesis in UVBinitiated mice. 3.2. Histopathological examination To see the tumor progression effect of dimethylarsenics, histopathological evaluations for the tissues of skin tumors (acanthomas) induced by the treatment
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with UVB and DMA were performed. The tumors were composed of focal proliferation of squamous cells. The tumors were classi®ed into four degrees of atypism by the criterion of nuclear hyperchromasia, irregular cell arrangement, and more solid growth (Fig. 2). The two mice with the malignant tumors (severe atypism, 111) showing apparent proceeding to carcinomas (Fig. 2d) were observed only in the 1000 ppm DMAtreated (group 3 of Table 1). The degree of atypism was not agreement with the size of tumors. This result shows that oral administration of DMA leads to progression activity in skin tumorigenesis of mice. 4. Discussion Our previous study demonstrated that oral administration of DMA caused the promotion and, further,
progression of 4-nitroquinoline 1-oxide (4-NQO)initiated lung tumorigenesis in A/J male mice [7]. Furthermore, Yamamoto et al. reported that oral administration of DMA promotes multi-organ tumorigenesis F344 female rats initiated with ®ve different carcinogens [8]. These ®ndings support the scienti®c theory that dimethylated arsenic may act as an unique promoter for various tissues, differently from a tissuespeci®c promoter such as TPA (12-O-tetradecanoylphorbol-13 acetate). In the present study using UVB irradiation and hairless mice, we demonstrated tumor promoting and progressing action of dimethylarsenic on skin tumorigenesis. Orally administered inhibitors and suppressors, e.g. tea extracts containing polyphenols [31±34], l-selenomethionine [35], chlorpromazine [36], and indomethacin [37] against skin-tumor promotion have been reported for skin tumors. However orally effective promoters are so far
Fig. 2. Micrograph of acanthomas lesions induced by treatment with UVB and oral administration of 1000 ppm DMA. Representative four degrees (2, 1, 11 and 111) of atypism are shown in (a), (b), (c), and (d), respectively (hematoxylin and eosin, £ 125).
K. Yamanaka et al. / Cancer Letters 152 (2000) 79±85 Table 1 Histopathological evaluation of the skin tumors (acanthomas) induced by the treatment with UVB and oral administration of DMA a Group
UVB alone (group 1) UVB 1 400 ppm DMA (group 2) UVB 1 1000 ppm DMA (group 3)
Atypism 2
1
11
111
7 5
2 3
2 2
0 0
1
2
5
2
a Six-week-old female hairless mice (Hos: HR-1) were divided into three groups. Ten to 11 mice of each group were irradiated twice weekly with UVB at a dose of 2 kJ/m 2, and were given tap water (group 1, control), 400 ppm (group 2) or 1000 ppm (group 3) DMA solution as drinking water ad libitum for 25 weeks. The tumors (acanthomas) greater than 2-mm in diameter were excised and ®xed with 10% buffered formalin. The histopathological evaluations were classi®ed into four lesions according to the degree of atypism of acanthomas; (2) none of atypism, (1) mild atypism, (11) moderate atypism, (111) severe atypism. Each value represents no. of mice having tumors classi®ed into each lesion.
unknown. Our ®nding that the oral administration of DMA has substantial promoting effect on UVBinduced skin tumorigenesis in mice may contribute to the further development of mouse skin carcinogenesis models. Some reports show that organic peroxide such as benzoyl peroxide promotes 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin tumorigenesis in mouse in vivo [11,12,14] and X-ray-initiated malignant transformation in vitro [18]. Benzoyl peroxide is a well-known free radical-generating compound; benzoyloxyl radicals, phenyl radicals, and hydroxyl radicals are produced in the presence of copper or ferrous ions [16±21]. Skin tumor promoting-activity by benzoyl peroxide in the two-stage carcinogenesis in mice might be caused by the production of these free radicals. Benzoyl peroxide, however, did not promote UV-induced skin tumorigenesis in hairless mice, when present on the skin either before, during, or after repetitive UV irradiation [13]. In the present study, an appreciable enhancement of skin tumor number in mice was detected in repetitive irradiation with UVB and oral administration with DMA for 25 weeks. On the other hand, our previous study indicated that free radicals such as dimethylar-
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senic peroxyl radical and superoxide anion radical were produced via the reductive metabolism of DMA [(CH3)2As(O)OH] to dimethylarsine [(CH3)2AsH] [24]. Thus, free radicals, particularly dimethylarsenic peroxyl radical [(CH3)2AsOO´], may be responsible for the tumor-promoting action by DMA administration in UVB-induced skin tumors in mice. Furthermore, the free radicals caused genetic damage such as DNA single-strand breaks [22,23], DNA-protein cross-linking [26], and DNA modi®cations [26±29]. Thus, accumulation of genetic changes caused by oral administration of DMA may be responsible for the tumor progression. We observed in mouse skin regard to the skin-tumor promotion by dimethylarsenics, two possibilities for the mechanism may be considered: (a) dimethylarsenics and/or free radicals produced in the metabolic processing of DMA may have promoting activity or (b) they may enhance the promoting activity of UVB. Our preliminary studies support interpretation (a), that is, the oral administration of DMA in mice promotes skin tumorigenesis of not only UVB-initiated but also DMBA-initiated mice. In contrast, one report indicated that UVB irradiation promoted tumorigenesis of DMBA-initiated on mouse skin [32] Acknowledgements This work was in part supported by a Grant-in-Aid (No. 10672113) from the Ministry of Education, Science, Sports and Culture of Japan. We thank Ms Yoko Fukui of Nihon University College of Pharmacy for technical assistance. References [1] International Agency Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 23: Some Metals and Metallic Compounds, IARC, Lyon, 1980, pp. 39±141. [2] International Agency Research on Cancer, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 1±42, (Suppl. 7): Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs, IARC, Lyon, 1987, pp. 100±106. [3] C.-J. Chen, Y.-C. Chuang, T.-M. Lin, M.-M. Wu, Malignant neoplasms among residents of a Blackfoot disease-endemic area in Taiwan: High-arsenic artesian well water and cancers, Cancer Res. 45 (1985) 5895±5899. [4] C.-J. Chen, C.-W. Chen, M.-M. Wu, T.-L. Kuo, Cancer poten-
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