- Email: [email protected]
The role of trivalent dimethylated arsenic in dimethylarsinic acid-promoted skin and lung tumorigenesis in mice: Tumor-promoting action through the induction of oxidative stress
Toxicology Letters 158 (2005) 87–94
The role of trivalent dimethylated arsenic in dimethylarsinic acid-promoted skin and lung tumorigenesis in mice: Tumor-promoting action through the induction of oxidative stress Mutsumi Mizoi a , Fumiyo Takabayashi b , Masayuki Nakano c , Yan An a , Yuko Sagesaka d , Koichi Kato a , Shoji Okada e , Kenzo Yamanaka a,∗ a
Department of Environmental Toxicology and Carcinogenesis, Nihon University College of Pharmacy, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan b University of Shizuoka College of Shizuoka, 2-2-1 Oshika, Shizuoka 422-8021, Japan c National Chiba Hospital, 4-1-2 Tsubakimori, Chuo-ku, Chiba 260-8606, Japan d Central Research Institute, Ito-En Ltd., Megami 21, Sagara-cho, Haibara, Shizuoka 421-0516, Japan e University of Shizuoka, 2-12-7 Mariko, Shizuoka 421-0103, Japan Received 7 December 2004; received in revised form 1 March 2005; accepted 1 March 2005 Available online 14 April 2005
Abstract We investigated the relationship between lung- and skin-tumor promotion and oxidative stress caused by administration of dimethylarsinic acid (DMA(V)) in mice. The incidence of lung tumors induced by lung tumor initiator (4NQO) and DMA(V) were, as well as 8-oxo-2 -deoxyguanosine (8-oxodG), suppressed by cotreatment with (−)epigallocatechin gallate (EGCG). When mice were topically treated with trivalent dimethylated arsenic (DMA(III)), a further reductive metabolite of DMA(V), not only an increase in skin tumors but also an elevation of 8-oxodG in epidermis were observed. These results suggest that tumor promotion due to DMA(V) administration is mediated by DMA(III) through the induction of oxidative stress. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Dimethylarsinic acid; Trivalent dimethylated arsenic; Tumor promotion; 8-Oxo-2 -deoxyguanosine; Oxidative stress
1. Introduction In the last decade, many researchers have pointed out that methylated metabolites of inorganic arsenics ∗ Corresponding author. Tel.: +81 47 465 6077; fax: +81 47 465 6077. E-mail address: [email protected] (K. Yamanaka).
play an important role in arsenic carcinogenesis. Concerning their tumorigenic actions, many reports have indicated that exposure to pentavalent dimethylarsinic acid [DMA(V), (CH3 )2 AsO(OH)] metabolically produced from inorganic arsenic promotes tumor production in the skin and lungs of mice (Yamanaka et al., 1996, 2000, 2001a) and in liver, kidney, thymus, and urinary bladder in rats (Morikawa et al., 2000;
0378-4274/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2005.03.009
88
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Yamamoto et al., 1995; Wanibuchi et al., 1997). Recent reviews have indicated the possibility that the genotoxicity and tumorigenic action of DMA(V) are caused by the induction of oxidative stress (Kitchin, 2001; Kitchin and Ahmad, 2003; Kenyon and Hughes, 2001; Styblo et al., 2002). Our previous study demonstrated that exposure to DMA(V) in mice brought about higher levels of 8-oxo-2 -deoxyguanosine (8-oxodG) in the skin (epidermis), lung, liver, and urinary bladder (Yamanaka et al., 2001b). Furthermore, our recent study elucidated the oxidative stress induced in skin carcinogenic process on arsenic-intoxicated patients (An et al., 2004) and in promotion process on lung tumorigenesis in mice (An et al., 2005). Recently, the concept that trivalent methylated arsenicals, particularly monomethylarsinous acid [CH3 As(OH)2 ] and dimethylarsinous acid [(CH3 )2 AsOH], which exist as intermediates in the metabolic methylation process of inorganic arsenic in humans (Aposhian et al., 2000; Le et al., 2000; Mandal et al., 2001), are more active than the parent inorganic arsenic with respect to enzyme inhibition (Styblo et al., 1997; Lin et al., 1999), cytotoxicity (Petrick et al., 2001), and genotoxicity (Mass et al., 2001; Ahmad et al., 2000, 2002; Nesnow et al., 2002) has been developed. Two hypotheses have been proposed concerning the genotoxic action of trivalent dimethylated arsenic compounds (DMA(III)), such as dimethylarsinous acid; one is our current hypothesis that dimethylated arsenic peroxide, which is produced by the reaction of DMA(III) and molecular oxygen, is responsible for the production of nuclear-base oxidation (Yamanaka et al., 2003). The other is a proposal that the genotoxic action of DMA(III) is mediated by reactive oxygen species (ROS) with or without iron release from ferritin (Ahmad et al., 2002; Nesnow et al., 2002). Based on these reports, we believe that the promotion of skin and lung tumors by oral DMA(V) administration in mice is induced via the induction of oxidative stress by DMA(III), a further reductive metabolite of DMA(V). In addition, there are no in vivo data for DMA(III) so far. Thus, DMA(III), which can be produced by the metabolic reduction of DMA(V), has also attracted considerable attention with respect to the relationship between tumor promotion and oxidative stress. The purpose of the present study was to examine the possibility that a candidate causal species of the
skin and lung tumor promotion induced by exposure to DMA(V) is DMA(III). Furthermore, we also investigated if the promoting action was caused by the induction of oxidative stress.
2. Materials and methods 2.1. Chemicals DMA(V) and dimethylbenz(a)anthracene were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Sigma–Aldrich Corp. (St. Louis, MO, USA), respectively; 4-nitroquinolin 1-oxide (4NQO) was obtained from Iwai Kagaku Yakuhin Co., Ltd. (Tokyo, Japan). Dimethylarsinous iodide [DMI(III), (CH3 )2 AsI] was synthesized as follows: DMA(V) was dissolved in 6 M HCl and then reduced by sulfur dioxide, which was generated from sodium bisulfide and concentrated H2 SO4 , in the presence of an excess of potassium iodide. The DMI(III) produced was extracted in chloroform and purified by distillation in vacuo (bp32 72–73 ◦ C). The DMI(III) synthesized was identified by mass spectrometry and gas chromatography (purity > 99%). (−)Epigallocatechin gallate (EGCG) was supplied by Ito En Ltd. (Shizuoka, Japan) as THEA–FLAN90S containing ca. 50% EGCG. 2.2. Animals Six-week-old male ddY and female Hos:HR-1 hairless mice were obtained from Japan SLC (Hamamatsu, Japan) and Sankyo Laboservice Corporation Inc. (Tokyo, Japan), respectively. Two to three mice were housed for 25 weeks in a cage under SPF conditions at 23 ◦ C and 55% humidity with a 12-h light/12-h dark cycle. The mice were given free access to food and drinking water. 2.3. Two-step tumorigenesis test Two experimental animal models were used to examine the effects of dimethylarsinic acid and its metabolite, dimethylarsinous acid, in skin and lung tumorigenesis in mice. A lung tumorigenesis assay using ddY mice that was based on the method described in our previous reports was used (Yamanaka et al., 1996).
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
The ddY mice were divided into six groups. Each group of 10 mice was given tap water or 400 ppm DMA solution as drinking water ad libitum for 25 weeks after injection of 4NQO (10 mg/kg, s.c.) suspended in a mixture of olive oil and cholesterol (20:1) with or without free access to powder chows containing 0.05% EGCG. The skin tumorigenesis assay using hairless mice was based on the method described previously (Yamanaka et al., 2001a). DMI(III) was used as a model compound of trivalent dimethylated arsenic. The hairless mice were divided into six groups. Each group of 10 mice was given tap water as drinking water ad libitum for 25 weeks after a single topical application of 400 nmol of DMBA dissolved in acetone (100 L). The DMBA-initiated mice were treated topically with DMI(III) (2 mg) or TPA (10 nmol) per mouse twice weekly for 25 weeks. All mice from each group were sacrificed at 25 weeks under nembutal anesthesia. The lung and skin tumors that had formed were counted, excised, and fixed with buffered 10% formalin. Paraffin sections of the tumors were stained with hematoxylin and eosin. 2.4. Measurement of 8-oxodG in lung and skin The 8-oxodG analysis of lungs and epidermis was based on the method described in our previous paper (Yamanaka et al., 2001b). Briefly, the mice were killed by nembutal anesthesia, and then, the lungs or skin was isolated. Dorsal skin epidermal samples obtained from the topical treatment with 10 mg DMI(III) in acetone (100 L) were prepared by removing the dorsal skin by heating at 55 ◦ C for 20 s according to the method described previously (O’Brien et al., 1997). The tissues were homogenized with 0.3 M sucrose (5 mL) and centrifuged at 3000 rpm for 20 min. The crude nuclear pellets were suspended in 1% SDS/1 mM EDTA (pH 8.0) and then 2.5 mg proteinase K (Merck, Darmstadt, Germany) was added. The mixture was incubated under anaerobic conditions for 1 h at 37 ◦ C. The DNA was withdrawn by twice sodium iodide/ethanolextractions. After washing with 70% ethanol, the DNA was suspended in 0.01× SCC buffer and then treated with RNase T1 (50 units, Sigma–Aldrich, St. Louis, USA) and RNase A (100 g, Sigma–Aldrich) for 30 min at 37 ◦ C. The DNA was extracted and washed with 70% ethanol once again. The DNA was dissolved in 0.5 mL of water treated with Chelex 100 Resin
89
(BIORAD, Hercules, CA). Five millilitres of 200 mM sodium acetate buffer (pH 4.8) and 5 L of nuclease P1 solution (1 mg/mL of 20 mM sodium acetate buffer) were added to the DNA (50 g/45 L water) and then the DNA was digested at 37 ◦ C for 1 h under anaerobic conditions. The digested mixture was further digested by adding 0.65 units of alkaline phosphatase type III (Sigma–Aldrich) and 5 L of 1 M Tris–HCl (pH 7.4). The practical analysis was performed according to the method described in our previous paper (Yamanaka et al., 2001b). The HPLC conditions were as follows: column, Pegasil ODS (particle size, 5 m; 4.6 mm × 150 mm, Senshu Scientific Co., Tokyo); ECD detector, ESA Coulechem II 5200 (Bedford, MA); HPLC system, Shimadzu LC-10 with UV detector (Tokyo); elutant, 0.6% methanol (pH 5.1) containing 12.5 mM citrate, 30 mM sodium hydroxide, 25 mM sodium acetate, and 10 mM acetic acid; flow rate, 1.4 mL/min; ECD-accelerated voltage, 350 mV. 2.5. Statistics The differences in the percentage of mice with tumors between the groups administered DMI(III) and the controls were analyzed by Fisher’s exact test. Differences in average number of lung tumors per mouse between six groups (4NQO + DMA(V) and EGCG) and five groups (4NQO and DMA(V)) were analyzed by the Cochran–Cox t-test. Differences in the amount of 8-oxodG in tissue samples were determined by Student’s t-test.
3. Results and discussion 3.1. Effects of DMA(V) and epigallocathechin gallate on 4NQO-initiated lung tumorigenesis in mice In the previous studies, we demonstrated that oral exposure of mice to DMA induced promotion in 4NQO-initiated lung (Yamanaka et al., 1996) and DMBA-initiated skin (Yamanaka et al., 2001a) tumorigenesis. We also found that oral administration of DMA in mice significantly increased 8-oxodG levels in lung and skin (Yamanaka et al., 2001b). To determine whether oxidative stress due to DMA(V) administration in mice mediates lung tumor
90
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Table 1 Effects of DMA(V) and EGCG on 4NQO-initiated lung tumorigenesis in mice Group No.
Initiator
Promoter
Diet
1 2 3 4 5 6
– 4NQO – – 4NQO 4NQO
– – DMA(V) – DMA(V) DMA(V)
– – – EGCG – EGCG
Total no. of tumors
Percentage of tumor-bearing mice (%)
Tumor no./mouse
0 31 0 0 40 10
0 70 0 0 100 70
0 3.10 ± 1.07 0 0 4.00 ± 0.82 0.89 ± 0.30*
Average size of tumor (mm)
0.97 ± 0.09 0 0 1.18 ± 0.08 1.13 ± 0.16
The ddY mice (6-week-old) were divided into six groups. Each group of 10 mice was given tap water or 400 ppm DMA solution as drinking water ad libitum for 25 weeks after s.c. injection, in the groups 2, 5, and 6, of 4NQO (10 mg/kg, s.c.) suspended in a mixture of olive oil and cholesterol (20:1) with or without free access to powder chows containing 0.05% EGCG. All mice from each group were sacrificed at 25 weeks under nembutal anesthesia. The lung tumors that had formed were counted, excised, and fixed with buffered 10% formalin. Paraffin sections of the tumors were stained with hematoxylin and eosin. * p < 0.01, significant difference from group 5.
promotion, the effect of EGCG in 4NQO and DMA(V)induced lung tumorigenesis in mice was examined. EGCG, an antioxidant extracted from green tea, is known to suppress the skin tumor-promoting actions of TPA (Stoner and Mukhtar, 1995), benzoyl peroxide (Katiyar et al., 1995), and other compounds. As shown in Table 1, although DMA(V) administration promoted lung tumorigenesis initiated by 4NQO, the treatment with EGCG significantly suppressed the incidence of skin tumors induced by 4NQO plus DMA(V). More than 40% of the tumors induced by 4NQO plus DMA(V) were diagnosed as malignant tumors, such as papillary adenomas and papillary adenomas with atypism, while most of the tumors induced by 4NQO alone were adenomas (data not shown), suggesting that the tumors induced by 4NQO plus DMA(V) are more malignant than those by 4NQO alone. On the other hand, in the case of treatment with EGCG, the incidence of lung tumors was less than that by 4NQO alone. Since the tumor-initiation process by 4NQO in the lungs will be completed within fourth weeks after injection of 4NQO, it can be believed that EGCG may suppress not only the tumor-promoting action of DMA(V), but also the tumor-initiating action of 4NQO. Each individual treatment groups of DMA(V) or EGCG and untreatment group had no incidence of lung tumors. Next, the levels of 8-oxodG in the lungs of mice after administration of 4NQO and DMA(V) with or without EGCG were examined (Table 2a). The levels with 4NQO alone did not increase, while those with DMA(V) alone or with 4NQO and DMA(V) did. How-
Table 2 The levels of 8-oxodG in lung and dorsal skin (epidermis) after treatment with DMA(V) and DMI(III), respectively, in mice lung Group No.
Initiator
Promoter
Diet
Lunga 1 2 3 4 5
– 4NQO – 4NQO 4NQO
– – DMA(V) DMA(V) DMA(V)
– – – – EGCG
8-oxodG/105 dG in lungs
1.11 ± 0.10 1.24 ± 0.35 1.58 ± 0.28* 1.53 ± 0.32** 1.21 ± 0.09***
Time after application of DMI(III) (h)
8-oxodG/105 dG in dorsal epidermis
Dorsal skinb 0 0.5 1 2 3
1.25 1.67 1.62 1.66 1.60
± ± ± ± ±
0.11 0.17* 0.12* 0.23* 0.15*
a The ddY-strain male mice (6-week-old) were given tap water or 400 ppm DMA(V) solution as drinking water for 4 weeks after s.c. injection, in the groups 2, 4, and 5, of 4NQO (10 mg/kg) with or without free access to powder chows containing 0.05% EGCG. Each value indicates mean ± S.D. (n = 4) of 8-oxodG levels in lungs. Asterisk indicates significant differences from groups 1 and 4, respectively. b The HR-1 female hairless mice (6-week-old) were topically treated with 10 mg DMI(III). Each value indicates mean ± S.D. (n = 5) of 8-oxodG levels in dorsal epidermis. Asterisk indicates significant difference from the control (0 h). * p < 0.01. ** p < 0.05. *** p < 0.05.
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
ever, the increase in 8-oxodG by 4NQO and DMA(V) administration significantly decreased to the control level by treatment with EGCG. These results suggest that the promoting action due to DMA administration may be induced via the induction of oxidative stress. DMA(V) is known to be metabolized to DMA(III) with reduced glutathione (GSH) (Thompson, 1993). Recently, it was reported that DMA(III) had more genotoxic action than DMA(V) (Mass et al., 2001; Ahmad et al., 2000, 2002; Nesnow et al., 2002; O’Brien et al., 1997). The possibility that the genotoxicity of DMA(III) is induced via the production of reactive oxygen species has been suggested (Ahmad et al., 2002; Nesnow et al., 2002). One of our recent studies demonstrated that nuclear-base oxidation by DMA(III) occurred by dimethylated arsenic peroxide produced by the reaction of DMA(III) and molecular oxygen (Yamanaka et al., 2003). These active species derived from DMA(III), in any case, may induce oxidative stress. However, a study of the effects of DMA(III) in animals has not been conducted thus far. Therefore, it should be important to further elucidate tumor promotion and oxidative stress due to DMA(III). 3.2. Oxidative stress and tumor promotion by DMA(III) in mouse skin Our previous studies have demonstrated that oral administration of DMA(V) induces skin tumor promotion in UV-initiated and DMBA-initiated skin tumorigenesis in mice (Yamanaka et al., 2000, 2001a). We also have suggested that the promotion action is due to DMA(III) produced metabolically from DMA. Thus, in the present study, we attempted to determine the likelihood of the induction of skin tumor promotion by DMA(III). Dimethylarsinous iodide was used as a model compound of DMA(III). DMI(III) has been widely used in studies on in vitro genotoxicity and cytotoxicity induced by DMA(III), because DMI(III) is considered to be easily hydrolyzed to dimethylarsinous acid, which is produced metabolically from DMA(V). When DMI(III) was topically dosed to hairless mice, the levels of 8-oxodG in the epidermis of dorsal skin were significantly elevated 0.5–3 h after dosing (Table 2b). The elevation of 8-oxodG was detected for the first time by the in vivo treatment of mice with DMA(III). This result, so far, suggests that the active species, includ-
91
Fig. 1. Promoting effect of trivalent dimethylated arsenic on 7,12dimethylbenz(a)anthracene (DMBA)-initiated skin tumorigenesis in female hairless mice. Female HR-1 hairless mice were divided into six groups of 10 mice each. The mice were treated topically with 400 nmol DMBA or 10 mg DMI(III)in 100 L of acetone. One week after DMBA treatment, the mice were treated topically with 2 mg DMI(III) or 10 nmol TPA in 100 L of acetone per mouse twice weekly for 25 weeks. Tumors greater than 1-mm in diameter were counted. The symbols are: DMBA alone (), DMBA plus DMI(III) (䊉), DMI(III) plus TPA (), DMI(III) alone (), TPA alone (). No tumors were observed in untreated (control) group, DMI alone and TPA alone group throughout the experimental period; * p < 0.05, significant difference from the group (DMBA alone group). The asterisk (*) is marked at 9–11 weeks.
ing hydroxyl radical and dimethylated arsenic peroxide, may be responsible for the elevation of 8-oxodG levels. The promotion of DMBA-initiated skin tumorigenesis in mice by DMA(III) was examined. As shown in Fig. 1, when DMBA-initiated hairless mice were topically treated with DMI(III) twice weekly for 25 weeks, the incidence of skin tumors was significantly more in the DMI(III)-treated group than in the DMBA alone group. On the other hand, when 10 mg DMI(III)initiated mice were treated with TPA twice weekly for 25 weeks, the incidence of skin tumor formation was extremely low. No skin tumors were observed in DMI(III) or TPA alone group, and untreated group (data not shown). Next, the tumors removed from mice treated with DMI(III) for 25 weeks were evaluated histopathologically. As shown in Table 3, the number of hyperkeratoses was 2.5-fold greater in the DMI(III)treated group than in the DMBA alone group, although
92
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Table 3 Histopathological evaluation of skin lesions induced by DMBA and DMI(III) in hairless mice Group No.
Initiator
Promoter
1 2 3 4 5 6
– DMBA – – DMBA DMI(III)
– – DMI(III) TPA DMI(III) TPA
No. of mice
Papillomas
Papillomas with hyperkeratoses
Hyperkeratoses
10 10 10 10 10 10
0 1 0 0 3 0
0 5 0 0 5 0
0 10 0 0 25 1
The Hos: HR-1 female hairless mice (6-week-old) were initiated by topical treatment with 400 nmol DMBA (groups 2 and 5), 10 mg DMI(III) (group 6) in 100 L of acetone or 100 L acetone alone (groups 1, 3 and 4). One week later, the mice were treated topically with 2 mg DMI (III) (groups 3 and 5) or 10 nmol TPA (groups 4 and 6) in 100 L of acetone as promoter or 100 L acetone alone (groups 1 and 2) twice weekly for 25 weeks. The skin tumors that had formed were counted weekly. All mice from each group were sacrificed at 25 weeks under nembutal anesthesia. The skin tumors were excised, and fixed with buffered 10% formalin. Paraffin sections of the tumors were stained with hematoxylin and eosin.
the number of papillomas was not different between these two groups. Moreover, when 10 mg DMI(III)initiated mice were treated with TPA twice weekly for 25 weeks, the incidence of skin tumor formation was only one case of hyperkeratosis. These results suggest that topical treatment with DMA(III) induces tumor promotion, not initiation, and oxidative stress in mouse skin. 3.3. Active arsenic species Fig. 2 summarizes the possible production pathway of active arsenic species by the metabolic re-
duction of DMA(V) and their genotoxic and tumorigenic actions. A previous report (Thompson, 1993) has proposed that DMA(V) reacts with GSH, resulting in the formation of DMA(III), such as dimethylarsinous acid. The present study has demonstrated that DMA(III) has a tumor-promoting action (Fig. 1) in skin tumorigenesis and the ability to form 8-oxodG (Table 2b). From the findings that dimethylarsinous acid formed cis-thymine glycol via the production of dimethylated arsenic peroxide (Yamanaka et al., 2003) and hydroxyl radical (Ahmad et al., 2002), DMA(III) may induce oxidative stress. Therefore, as a reason for tumor-promoting action induced by exposure of
Fig. 2. Possible tumorigenic role of active arsenic species produced by metabolic reduction of DMA(V).
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
DMA(V), this metabolite from DMA(V) seems to act as a tumor promoter by way of the induction of oxidative stress, because DMA(V) itself was considered to have no action of oxidative stress. However, DMA(III) has no tumor-initiating action, because treatment with DMA(III) and TPA in mice did not induce skin tumors (Table 3). On the other hand, we have already determined that the dimethylarsenic–glutathione conjugate, which is a species of DMA(III), is further reduced to dimethylarsine by two-electron reduction of GSH reductase. The dimethylarsine reacts with molecular oxygen and then forms dimethylarsenic and dimethylarsenic peroxy radicals (Yamanaka et al., 1990). A recent study (Andrewes et al., 2003) has demonstrated that dimethylarsine is a more potent genotoxin than DMA(III). Dimethylarsine, perhaps via these radicals formed by reaction with molecular oxygen (Yamanaka et al., 1990), might act as a tumor-initiating factor and be an ultimate substance, among any other ultimate substances unknown so far yet, in arsenic carcinogenesis. The in vivo production of dimethylarsine and its tumor initiation will be reported elsewhere in the near future. We would like to emphasize that molecular oxygen is required for the tumorigenic and genotoxic actions of these active arsenic species.
Acknowledgements This work was supported in part by a grant from “Academic Frontier” project for private universities, matching fund subsidy from Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 2002–2006, a joint research grant from Nihon University College of Pharmacy, and a Grant-in-Aid for Scientific Research (C) (no. 14572114) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References Ahmad, S., Kitchin, K.T., Cullen, W.R., 2000. Arsenic species that cause release of iron from ferritin and generation of activated oxygen. Arch. Biochem. Biophys. 382, 195–202. Ahmad, S., Kitchin, K.T., Cullen, W.R., 2002. Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferittin. Toxicol. Lett. 133, 47–57.
93
An, Y., Gao, Z., Wang, Z., Yang, S., Liang, J., Feng, Y., Kato, K., Nakano, M., Okada, S., Yamanaka, K., 2004. Immunohistochemical analysis of oxidative DNA damage in arsenic-related human skin samples from arsenic-contaminated area of china. Cancer Lett. 214, 11–18. An, Y., Kato, K., Nakano, M., Otsu, H., Okada, S., Yamanaka, K., 2005. Specific induction of oxidative stress in terminal bronchiolar Clara cells during dimethylarsenic-induced lung tumor promoting process in mice. Cancer Lett., in press. Andrewes, P., Kitchin, K.T., Wallace, K., 2003. Dimethylarsine and trimethylarsine are potent genotoxins in vitro. Chem. Res. Toxicol. 16, 994–1003. Aposhian, H.V., Gurzau, E.S., Le, X.C., Gurzau, A., Healy, S.M., Lu, X., Ma, M., Yip, L., Zakharyan, R.A., Maiorino, R.M., Dart, R.C., Tircus, M.G., Gonzalez-Ramirez, D., Morgan, D.L., Avram, D., Aposhian, M.M., 2000. Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic. Chem. Res. Toxicol. 13, 693–697. Katiyar, S.K., Rupp, C.O., Korman, N.J., Agarwal, R., Mukhtar, H., 1995. Inhibition of 12-O-tetradecanoylphorbol-13-acetate and other skin tumor-promoter-caused induction of epidermal interleukin-1 alpha mRNA and protein expression in SENCAR mice by green tea polyphenols. J. Investig. Dermatol. 105, 394–398. Kenyon, E.M., Hughes, M.F., 2001. A concise review of the toxicity and carcinogenicity of dimethylarsinic acid. Toxicology 160, 227–236. Kitchin, K.T., 2001. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. 172, 249–261. Kitchin, K.T., Ahmad, S., 2003. Oxidative stress as a possible mode of action for arsenic carcinogenesis. Toxicol. Lett. 137, 3–13. Le, X.C., Lu, X., Ma, M., Cullen, W.R., Aposhian, H.V., Zheng, B., 2000. Speciation of key arsenic metabolic intermediates in human urine. Anal. Chem. 72, 5172–5177. Lin, S., Cullen, W.R., Thomas, D.J., 1999. Methylarsenicals and arsinothiols are potent inhibitors of mouse liver thioredoxin reductase. Chem. Res. Toxicol. 12, 924–930. Mandal, B.K., Ogra, Y., Suzuki, K.T., 2001. Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal. India. Chem. Res. Toxicol. 14, 371–378. Mass, M.J., Tennant, A., Roop, B.C., Cullen, W.R., Styblo, M., Thomas, D.J., Kligerman, A.D., 2001. Methylated trivalent arsenic species are genotoxic. Chem. Res. Toxicol. 14, 355– 361. Morikawa, T., Wanibuchi, H., Morimura, K., Ogawa, M., Fukushima, S., 2000. Promotion of skin carcinogenesis by dimethylarsinic acid in keratin (K6)/ODC transgenic mice. Jpn. J. Cancer Res. 91, 579–581. Nesnow, S., Roop, B.C., Lambert, G., Kadiiska, M., Mason, R.P., Cullen, W.R., Mass, M.J., 2002. DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species. Chem. Res. Toxicol. 15, 1627–1634. O’Brien, T.G., Megosh, L.C., Gilliard, G., Peralta Soler, A., 1997. Ornithine decarboxylase overexpression is a sufficient condition for tumor promotion in mouse skin. Cancer Res. 57, 2630–2637.
94
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Petrick, J.S., Jagadish, B., Mash, E.A., Aposhian, H.V., 2001. Monomethylarsonous acid (MMA (III)) and arsenite: LD (50) in hamsters and in vitro inhibition of pyruvate dehydrogenase. Chem. Res. Toxicol. 14, 651–656. Stoner, G.D., Mukhtar, H., 1995. Polyphenols as cancer chemopreventive agents. J. Cell Biochem. Suppl. 22, 169–180. Styblo, M., Serves, S.V., Cullen, W.R., Thomas, D.J., 1997. Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem. Res. Toxicol. 10, 27–33. Styblo, M., Drobna, Z., Jaspers, I., Lin, S., Thomas, D.J., 2002. The role of biomethylation in toxicity and carcinogenicity of arsenic: a research update. Environ. Health Perspect. 110 (Suppl. 5), 767–771. Thompson, D.J., 1993. A chemical hypothesis for arsenic methylation in mammals. Chem. Biol. Interact. 88, 89–114. Wanibuchi, H., Hori, T., Meenakshi, V., Ichihara, T., Yamamoto, S., Yano, Y., Otani, S., Nakae, D., Konishi, Y., Fukushima, S., 1997. Promotion of rat hepatocarcinogenesis by dimethylarsinic acid: association with elevated ornithine decarboxylase activity and formation of 8-hydroxydeoxyguanosine in the liver. Jpn. J. Cancer Res. 88, 1149–1154. Yamamoto, S., Konishi, Y., Matsuda, T., Murai, T., Shibata, M.A., Matsui-Yuasa, I., Otani, S., Kuroda, K., Endo, G., Fukushima, S., 1995. Cancer induction by an organic arsenic compound, dimethylarsinic acid (cacodylic acid), in F344/DuCrj rats after pretreatment with five carcinogens. Cancer Res. 55, 1271– 1276. Yamanaka, K., Hoshino, M., Okamoto, M., Sawamura, R., Hasegawa, A., Okada, S., 1990. Induction of DNA damage by
dimethylarsine, a methabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical. Biochem. Biophys. Res. Commun. 168, 58–64. Yamanaka, K., Ohtsubo, K., Hasegawa, A., Hayashi, H., Ohji, H., Kanisawa, M., Okada, S., 1996. Exposure to dimethylarsinic acid, a main methabolite of inorganic arsenics, strongly promotes tumorigenesis initiated by 4-nitroquinolone 1-oxide in the lungs of mice. Carcinogenesis 17, 767–770. Yamanaka, K., Katsumata, K., Ikuma, K., Hasegawa, A., Nakano, M., Okada, S., 2000. 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 Lett. 152, 79–85. Yamanaka, K., Mizoi, M., Kato, K., Hasegawa, A., Nakano, M., Okada, S., 2001a. Oral administration of dimethylarsinic acid, a main metabolite of inorganic arsenic, in mice promotes skin tumorigenesis initiated by dimethylbenz(a)anthracene with or without ultraviolet B as a promoter. Biol. Pharm. Bull. 24, 510–514. Yamanaka, K., Takabayashi, F., Mizoi, M., Yan, A., Hasegawa, A., Okada, S., 2001b. Oral exposure of dimethylarsinic acid, a main metabolite of inorganic arsenics, in mice leads to an increase in 8-oxo-2 -deoxyguanosine level, specifically in the target organs for arsenic carcinogenesis. Biochem. Biophys. Res. Commun. 287, 66–70. Yamanaka, K., Mizoi, M., Tachikawa, M., Hasegawa, A., Hoshino, M., Okada, S., 2003. Oxidative DNA damage following exposure to dimethylarsinous iodide: the formation of cis-thymine glycol. Toxicol. Lett. 143, 145–153.
The role of trivalent dimethylated arsenic in dimethylarsinic acid-promoted skin and lung tumorigenesis in mice: Tumor-promoting action through the induction of oxidative stress Mutsumi Mizoi a , Fumiyo Takabayashi b , Masayuki Nakano c , Yan An a , Yuko Sagesaka d , Koichi Kato a , Shoji Okada e , Kenzo Yamanaka a,∗ a
Department of Environmental Toxicology and Carcinogenesis, Nihon University College of Pharmacy, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan b University of Shizuoka College of Shizuoka, 2-2-1 Oshika, Shizuoka 422-8021, Japan c National Chiba Hospital, 4-1-2 Tsubakimori, Chuo-ku, Chiba 260-8606, Japan d Central Research Institute, Ito-En Ltd., Megami 21, Sagara-cho, Haibara, Shizuoka 421-0516, Japan e University of Shizuoka, 2-12-7 Mariko, Shizuoka 421-0103, Japan Received 7 December 2004; received in revised form 1 March 2005; accepted 1 March 2005 Available online 14 April 2005
Abstract We investigated the relationship between lung- and skin-tumor promotion and oxidative stress caused by administration of dimethylarsinic acid (DMA(V)) in mice. The incidence of lung tumors induced by lung tumor initiator (4NQO) and DMA(V) were, as well as 8-oxo-2 -deoxyguanosine (8-oxodG), suppressed by cotreatment with (−)epigallocatechin gallate (EGCG). When mice were topically treated with trivalent dimethylated arsenic (DMA(III)), a further reductive metabolite of DMA(V), not only an increase in skin tumors but also an elevation of 8-oxodG in epidermis were observed. These results suggest that tumor promotion due to DMA(V) administration is mediated by DMA(III) through the induction of oxidative stress. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Dimethylarsinic acid; Trivalent dimethylated arsenic; Tumor promotion; 8-Oxo-2 -deoxyguanosine; Oxidative stress
1. Introduction In the last decade, many researchers have pointed out that methylated metabolites of inorganic arsenics ∗ Corresponding author. Tel.: +81 47 465 6077; fax: +81 47 465 6077. E-mail address: [email protected] (K. Yamanaka).
play an important role in arsenic carcinogenesis. Concerning their tumorigenic actions, many reports have indicated that exposure to pentavalent dimethylarsinic acid [DMA(V), (CH3 )2 AsO(OH)] metabolically produced from inorganic arsenic promotes tumor production in the skin and lungs of mice (Yamanaka et al., 1996, 2000, 2001a) and in liver, kidney, thymus, and urinary bladder in rats (Morikawa et al., 2000;
0378-4274/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2005.03.009
88
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Yamamoto et al., 1995; Wanibuchi et al., 1997). Recent reviews have indicated the possibility that the genotoxicity and tumorigenic action of DMA(V) are caused by the induction of oxidative stress (Kitchin, 2001; Kitchin and Ahmad, 2003; Kenyon and Hughes, 2001; Styblo et al., 2002). Our previous study demonstrated that exposure to DMA(V) in mice brought about higher levels of 8-oxo-2 -deoxyguanosine (8-oxodG) in the skin (epidermis), lung, liver, and urinary bladder (Yamanaka et al., 2001b). Furthermore, our recent study elucidated the oxidative stress induced in skin carcinogenic process on arsenic-intoxicated patients (An et al., 2004) and in promotion process on lung tumorigenesis in mice (An et al., 2005). Recently, the concept that trivalent methylated arsenicals, particularly monomethylarsinous acid [CH3 As(OH)2 ] and dimethylarsinous acid [(CH3 )2 AsOH], which exist as intermediates in the metabolic methylation process of inorganic arsenic in humans (Aposhian et al., 2000; Le et al., 2000; Mandal et al., 2001), are more active than the parent inorganic arsenic with respect to enzyme inhibition (Styblo et al., 1997; Lin et al., 1999), cytotoxicity (Petrick et al., 2001), and genotoxicity (Mass et al., 2001; Ahmad et al., 2000, 2002; Nesnow et al., 2002) has been developed. Two hypotheses have been proposed concerning the genotoxic action of trivalent dimethylated arsenic compounds (DMA(III)), such as dimethylarsinous acid; one is our current hypothesis that dimethylated arsenic peroxide, which is produced by the reaction of DMA(III) and molecular oxygen, is responsible for the production of nuclear-base oxidation (Yamanaka et al., 2003). The other is a proposal that the genotoxic action of DMA(III) is mediated by reactive oxygen species (ROS) with or without iron release from ferritin (Ahmad et al., 2002; Nesnow et al., 2002). Based on these reports, we believe that the promotion of skin and lung tumors by oral DMA(V) administration in mice is induced via the induction of oxidative stress by DMA(III), a further reductive metabolite of DMA(V). In addition, there are no in vivo data for DMA(III) so far. Thus, DMA(III), which can be produced by the metabolic reduction of DMA(V), has also attracted considerable attention with respect to the relationship between tumor promotion and oxidative stress. The purpose of the present study was to examine the possibility that a candidate causal species of the
skin and lung tumor promotion induced by exposure to DMA(V) is DMA(III). Furthermore, we also investigated if the promoting action was caused by the induction of oxidative stress.
2. Materials and methods 2.1. Chemicals DMA(V) and dimethylbenz(a)anthracene were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Sigma–Aldrich Corp. (St. Louis, MO, USA), respectively; 4-nitroquinolin 1-oxide (4NQO) was obtained from Iwai Kagaku Yakuhin Co., Ltd. (Tokyo, Japan). Dimethylarsinous iodide [DMI(III), (CH3 )2 AsI] was synthesized as follows: DMA(V) was dissolved in 6 M HCl and then reduced by sulfur dioxide, which was generated from sodium bisulfide and concentrated H2 SO4 , in the presence of an excess of potassium iodide. The DMI(III) produced was extracted in chloroform and purified by distillation in vacuo (bp32 72–73 ◦ C). The DMI(III) synthesized was identified by mass spectrometry and gas chromatography (purity > 99%). (−)Epigallocatechin gallate (EGCG) was supplied by Ito En Ltd. (Shizuoka, Japan) as THEA–FLAN90S containing ca. 50% EGCG. 2.2. Animals Six-week-old male ddY and female Hos:HR-1 hairless mice were obtained from Japan SLC (Hamamatsu, Japan) and Sankyo Laboservice Corporation Inc. (Tokyo, Japan), respectively. Two to three mice were housed for 25 weeks in a cage under SPF conditions at 23 ◦ C and 55% humidity with a 12-h light/12-h dark cycle. The mice were given free access to food and drinking water. 2.3. Two-step tumorigenesis test Two experimental animal models were used to examine the effects of dimethylarsinic acid and its metabolite, dimethylarsinous acid, in skin and lung tumorigenesis in mice. A lung tumorigenesis assay using ddY mice that was based on the method described in our previous reports was used (Yamanaka et al., 1996).
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
The ddY mice were divided into six groups. Each group of 10 mice was given tap water or 400 ppm DMA solution as drinking water ad libitum for 25 weeks after injection of 4NQO (10 mg/kg, s.c.) suspended in a mixture of olive oil and cholesterol (20:1) with or without free access to powder chows containing 0.05% EGCG. The skin tumorigenesis assay using hairless mice was based on the method described previously (Yamanaka et al., 2001a). DMI(III) was used as a model compound of trivalent dimethylated arsenic. The hairless mice were divided into six groups. Each group of 10 mice was given tap water as drinking water ad libitum for 25 weeks after a single topical application of 400 nmol of DMBA dissolved in acetone (100 L). The DMBA-initiated mice were treated topically with DMI(III) (2 mg) or TPA (10 nmol) per mouse twice weekly for 25 weeks. All mice from each group were sacrificed at 25 weeks under nembutal anesthesia. The lung and skin tumors that had formed were counted, excised, and fixed with buffered 10% formalin. Paraffin sections of the tumors were stained with hematoxylin and eosin. 2.4. Measurement of 8-oxodG in lung and skin The 8-oxodG analysis of lungs and epidermis was based on the method described in our previous paper (Yamanaka et al., 2001b). Briefly, the mice were killed by nembutal anesthesia, and then, the lungs or skin was isolated. Dorsal skin epidermal samples obtained from the topical treatment with 10 mg DMI(III) in acetone (100 L) were prepared by removing the dorsal skin by heating at 55 ◦ C for 20 s according to the method described previously (O’Brien et al., 1997). The tissues were homogenized with 0.3 M sucrose (5 mL) and centrifuged at 3000 rpm for 20 min. The crude nuclear pellets were suspended in 1% SDS/1 mM EDTA (pH 8.0) and then 2.5 mg proteinase K (Merck, Darmstadt, Germany) was added. The mixture was incubated under anaerobic conditions for 1 h at 37 ◦ C. The DNA was withdrawn by twice sodium iodide/ethanolextractions. After washing with 70% ethanol, the DNA was suspended in 0.01× SCC buffer and then treated with RNase T1 (50 units, Sigma–Aldrich, St. Louis, USA) and RNase A (100 g, Sigma–Aldrich) for 30 min at 37 ◦ C. The DNA was extracted and washed with 70% ethanol once again. The DNA was dissolved in 0.5 mL of water treated with Chelex 100 Resin
89
(BIORAD, Hercules, CA). Five millilitres of 200 mM sodium acetate buffer (pH 4.8) and 5 L of nuclease P1 solution (1 mg/mL of 20 mM sodium acetate buffer) were added to the DNA (50 g/45 L water) and then the DNA was digested at 37 ◦ C for 1 h under anaerobic conditions. The digested mixture was further digested by adding 0.65 units of alkaline phosphatase type III (Sigma–Aldrich) and 5 L of 1 M Tris–HCl (pH 7.4). The practical analysis was performed according to the method described in our previous paper (Yamanaka et al., 2001b). The HPLC conditions were as follows: column, Pegasil ODS (particle size, 5 m; 4.6 mm × 150 mm, Senshu Scientific Co., Tokyo); ECD detector, ESA Coulechem II 5200 (Bedford, MA); HPLC system, Shimadzu LC-10 with UV detector (Tokyo); elutant, 0.6% methanol (pH 5.1) containing 12.5 mM citrate, 30 mM sodium hydroxide, 25 mM sodium acetate, and 10 mM acetic acid; flow rate, 1.4 mL/min; ECD-accelerated voltage, 350 mV. 2.5. Statistics The differences in the percentage of mice with tumors between the groups administered DMI(III) and the controls were analyzed by Fisher’s exact test. Differences in average number of lung tumors per mouse between six groups (4NQO + DMA(V) and EGCG) and five groups (4NQO and DMA(V)) were analyzed by the Cochran–Cox t-test. Differences in the amount of 8-oxodG in tissue samples were determined by Student’s t-test.
3. Results and discussion 3.1. Effects of DMA(V) and epigallocathechin gallate on 4NQO-initiated lung tumorigenesis in mice In the previous studies, we demonstrated that oral exposure of mice to DMA induced promotion in 4NQO-initiated lung (Yamanaka et al., 1996) and DMBA-initiated skin (Yamanaka et al., 2001a) tumorigenesis. We also found that oral administration of DMA in mice significantly increased 8-oxodG levels in lung and skin (Yamanaka et al., 2001b). To determine whether oxidative stress due to DMA(V) administration in mice mediates lung tumor
90
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Table 1 Effects of DMA(V) and EGCG on 4NQO-initiated lung tumorigenesis in mice Group No.
Initiator
Promoter
Diet
1 2 3 4 5 6
– 4NQO – – 4NQO 4NQO
– – DMA(V) – DMA(V) DMA(V)
– – – EGCG – EGCG
Total no. of tumors
Percentage of tumor-bearing mice (%)
Tumor no./mouse
0 31 0 0 40 10
0 70 0 0 100 70
0 3.10 ± 1.07 0 0 4.00 ± 0.82 0.89 ± 0.30*
Average size of tumor (mm)
0.97 ± 0.09 0 0 1.18 ± 0.08 1.13 ± 0.16
The ddY mice (6-week-old) were divided into six groups. Each group of 10 mice was given tap water or 400 ppm DMA solution as drinking water ad libitum for 25 weeks after s.c. injection, in the groups 2, 5, and 6, of 4NQO (10 mg/kg, s.c.) suspended in a mixture of olive oil and cholesterol (20:1) with or without free access to powder chows containing 0.05% EGCG. All mice from each group were sacrificed at 25 weeks under nembutal anesthesia. The lung tumors that had formed were counted, excised, and fixed with buffered 10% formalin. Paraffin sections of the tumors were stained with hematoxylin and eosin. * p < 0.01, significant difference from group 5.
promotion, the effect of EGCG in 4NQO and DMA(V)induced lung tumorigenesis in mice was examined. EGCG, an antioxidant extracted from green tea, is known to suppress the skin tumor-promoting actions of TPA (Stoner and Mukhtar, 1995), benzoyl peroxide (Katiyar et al., 1995), and other compounds. As shown in Table 1, although DMA(V) administration promoted lung tumorigenesis initiated by 4NQO, the treatment with EGCG significantly suppressed the incidence of skin tumors induced by 4NQO plus DMA(V). More than 40% of the tumors induced by 4NQO plus DMA(V) were diagnosed as malignant tumors, such as papillary adenomas and papillary adenomas with atypism, while most of the tumors induced by 4NQO alone were adenomas (data not shown), suggesting that the tumors induced by 4NQO plus DMA(V) are more malignant than those by 4NQO alone. On the other hand, in the case of treatment with EGCG, the incidence of lung tumors was less than that by 4NQO alone. Since the tumor-initiation process by 4NQO in the lungs will be completed within fourth weeks after injection of 4NQO, it can be believed that EGCG may suppress not only the tumor-promoting action of DMA(V), but also the tumor-initiating action of 4NQO. Each individual treatment groups of DMA(V) or EGCG and untreatment group had no incidence of lung tumors. Next, the levels of 8-oxodG in the lungs of mice after administration of 4NQO and DMA(V) with or without EGCG were examined (Table 2a). The levels with 4NQO alone did not increase, while those with DMA(V) alone or with 4NQO and DMA(V) did. How-
Table 2 The levels of 8-oxodG in lung and dorsal skin (epidermis) after treatment with DMA(V) and DMI(III), respectively, in mice lung Group No.
Initiator
Promoter
Diet
Lunga 1 2 3 4 5
– 4NQO – 4NQO 4NQO
– – DMA(V) DMA(V) DMA(V)
– – – – EGCG
8-oxodG/105 dG in lungs
1.11 ± 0.10 1.24 ± 0.35 1.58 ± 0.28* 1.53 ± 0.32** 1.21 ± 0.09***
Time after application of DMI(III) (h)
8-oxodG/105 dG in dorsal epidermis
Dorsal skinb 0 0.5 1 2 3
1.25 1.67 1.62 1.66 1.60
± ± ± ± ±
0.11 0.17* 0.12* 0.23* 0.15*
a The ddY-strain male mice (6-week-old) were given tap water or 400 ppm DMA(V) solution as drinking water for 4 weeks after s.c. injection, in the groups 2, 4, and 5, of 4NQO (10 mg/kg) with or without free access to powder chows containing 0.05% EGCG. Each value indicates mean ± S.D. (n = 4) of 8-oxodG levels in lungs. Asterisk indicates significant differences from groups 1 and 4, respectively. b The HR-1 female hairless mice (6-week-old) were topically treated with 10 mg DMI(III). Each value indicates mean ± S.D. (n = 5) of 8-oxodG levels in dorsal epidermis. Asterisk indicates significant difference from the control (0 h). * p < 0.01. ** p < 0.05. *** p < 0.05.
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
ever, the increase in 8-oxodG by 4NQO and DMA(V) administration significantly decreased to the control level by treatment with EGCG. These results suggest that the promoting action due to DMA administration may be induced via the induction of oxidative stress. DMA(V) is known to be metabolized to DMA(III) with reduced glutathione (GSH) (Thompson, 1993). Recently, it was reported that DMA(III) had more genotoxic action than DMA(V) (Mass et al., 2001; Ahmad et al., 2000, 2002; Nesnow et al., 2002; O’Brien et al., 1997). The possibility that the genotoxicity of DMA(III) is induced via the production of reactive oxygen species has been suggested (Ahmad et al., 2002; Nesnow et al., 2002). One of our recent studies demonstrated that nuclear-base oxidation by DMA(III) occurred by dimethylated arsenic peroxide produced by the reaction of DMA(III) and molecular oxygen (Yamanaka et al., 2003). These active species derived from DMA(III), in any case, may induce oxidative stress. However, a study of the effects of DMA(III) in animals has not been conducted thus far. Therefore, it should be important to further elucidate tumor promotion and oxidative stress due to DMA(III). 3.2. Oxidative stress and tumor promotion by DMA(III) in mouse skin Our previous studies have demonstrated that oral administration of DMA(V) induces skin tumor promotion in UV-initiated and DMBA-initiated skin tumorigenesis in mice (Yamanaka et al., 2000, 2001a). We also have suggested that the promotion action is due to DMA(III) produced metabolically from DMA. Thus, in the present study, we attempted to determine the likelihood of the induction of skin tumor promotion by DMA(III). Dimethylarsinous iodide was used as a model compound of DMA(III). DMI(III) has been widely used in studies on in vitro genotoxicity and cytotoxicity induced by DMA(III), because DMI(III) is considered to be easily hydrolyzed to dimethylarsinous acid, which is produced metabolically from DMA(V). When DMI(III) was topically dosed to hairless mice, the levels of 8-oxodG in the epidermis of dorsal skin were significantly elevated 0.5–3 h after dosing (Table 2b). The elevation of 8-oxodG was detected for the first time by the in vivo treatment of mice with DMA(III). This result, so far, suggests that the active species, includ-
91
Fig. 1. Promoting effect of trivalent dimethylated arsenic on 7,12dimethylbenz(a)anthracene (DMBA)-initiated skin tumorigenesis in female hairless mice. Female HR-1 hairless mice were divided into six groups of 10 mice each. The mice were treated topically with 400 nmol DMBA or 10 mg DMI(III)in 100 L of acetone. One week after DMBA treatment, the mice were treated topically with 2 mg DMI(III) or 10 nmol TPA in 100 L of acetone per mouse twice weekly for 25 weeks. Tumors greater than 1-mm in diameter were counted. The symbols are: DMBA alone (), DMBA plus DMI(III) (䊉), DMI(III) plus TPA (), DMI(III) alone (), TPA alone (). No tumors were observed in untreated (control) group, DMI alone and TPA alone group throughout the experimental period; * p < 0.05, significant difference from the group (DMBA alone group). The asterisk (*) is marked at 9–11 weeks.
ing hydroxyl radical and dimethylated arsenic peroxide, may be responsible for the elevation of 8-oxodG levels. The promotion of DMBA-initiated skin tumorigenesis in mice by DMA(III) was examined. As shown in Fig. 1, when DMBA-initiated hairless mice were topically treated with DMI(III) twice weekly for 25 weeks, the incidence of skin tumors was significantly more in the DMI(III)-treated group than in the DMBA alone group. On the other hand, when 10 mg DMI(III)initiated mice were treated with TPA twice weekly for 25 weeks, the incidence of skin tumor formation was extremely low. No skin tumors were observed in DMI(III) or TPA alone group, and untreated group (data not shown). Next, the tumors removed from mice treated with DMI(III) for 25 weeks were evaluated histopathologically. As shown in Table 3, the number of hyperkeratoses was 2.5-fold greater in the DMI(III)treated group than in the DMBA alone group, although
92
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Table 3 Histopathological evaluation of skin lesions induced by DMBA and DMI(III) in hairless mice Group No.
Initiator
Promoter
1 2 3 4 5 6
– DMBA – – DMBA DMI(III)
– – DMI(III) TPA DMI(III) TPA
No. of mice
Papillomas
Papillomas with hyperkeratoses
Hyperkeratoses
10 10 10 10 10 10
0 1 0 0 3 0
0 5 0 0 5 0
0 10 0 0 25 1
The Hos: HR-1 female hairless mice (6-week-old) were initiated by topical treatment with 400 nmol DMBA (groups 2 and 5), 10 mg DMI(III) (group 6) in 100 L of acetone or 100 L acetone alone (groups 1, 3 and 4). One week later, the mice were treated topically with 2 mg DMI (III) (groups 3 and 5) or 10 nmol TPA (groups 4 and 6) in 100 L of acetone as promoter or 100 L acetone alone (groups 1 and 2) twice weekly for 25 weeks. The skin tumors that had formed were counted weekly. All mice from each group were sacrificed at 25 weeks under nembutal anesthesia. The skin tumors were excised, and fixed with buffered 10% formalin. Paraffin sections of the tumors were stained with hematoxylin and eosin.
the number of papillomas was not different between these two groups. Moreover, when 10 mg DMI(III)initiated mice were treated with TPA twice weekly for 25 weeks, the incidence of skin tumor formation was only one case of hyperkeratosis. These results suggest that topical treatment with DMA(III) induces tumor promotion, not initiation, and oxidative stress in mouse skin. 3.3. Active arsenic species Fig. 2 summarizes the possible production pathway of active arsenic species by the metabolic re-
duction of DMA(V) and their genotoxic and tumorigenic actions. A previous report (Thompson, 1993) has proposed that DMA(V) reacts with GSH, resulting in the formation of DMA(III), such as dimethylarsinous acid. The present study has demonstrated that DMA(III) has a tumor-promoting action (Fig. 1) in skin tumorigenesis and the ability to form 8-oxodG (Table 2b). From the findings that dimethylarsinous acid formed cis-thymine glycol via the production of dimethylated arsenic peroxide (Yamanaka et al., 2003) and hydroxyl radical (Ahmad et al., 2002), DMA(III) may induce oxidative stress. Therefore, as a reason for tumor-promoting action induced by exposure of
Fig. 2. Possible tumorigenic role of active arsenic species produced by metabolic reduction of DMA(V).
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
DMA(V), this metabolite from DMA(V) seems to act as a tumor promoter by way of the induction of oxidative stress, because DMA(V) itself was considered to have no action of oxidative stress. However, DMA(III) has no tumor-initiating action, because treatment with DMA(III) and TPA in mice did not induce skin tumors (Table 3). On the other hand, we have already determined that the dimethylarsenic–glutathione conjugate, which is a species of DMA(III), is further reduced to dimethylarsine by two-electron reduction of GSH reductase. The dimethylarsine reacts with molecular oxygen and then forms dimethylarsenic and dimethylarsenic peroxy radicals (Yamanaka et al., 1990). A recent study (Andrewes et al., 2003) has demonstrated that dimethylarsine is a more potent genotoxin than DMA(III). Dimethylarsine, perhaps via these radicals formed by reaction with molecular oxygen (Yamanaka et al., 1990), might act as a tumor-initiating factor and be an ultimate substance, among any other ultimate substances unknown so far yet, in arsenic carcinogenesis. The in vivo production of dimethylarsine and its tumor initiation will be reported elsewhere in the near future. We would like to emphasize that molecular oxygen is required for the tumorigenic and genotoxic actions of these active arsenic species.
Acknowledgements This work was supported in part by a grant from “Academic Frontier” project for private universities, matching fund subsidy from Ministry of Education, Culture, Sports, Science, and Technology (MEXT) 2002–2006, a joint research grant from Nihon University College of Pharmacy, and a Grant-in-Aid for Scientific Research (C) (no. 14572114) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
References Ahmad, S., Kitchin, K.T., Cullen, W.R., 2000. Arsenic species that cause release of iron from ferritin and generation of activated oxygen. Arch. Biochem. Biophys. 382, 195–202. Ahmad, S., Kitchin, K.T., Cullen, W.R., 2002. Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferittin. Toxicol. Lett. 133, 47–57.
93
An, Y., Gao, Z., Wang, Z., Yang, S., Liang, J., Feng, Y., Kato, K., Nakano, M., Okada, S., Yamanaka, K., 2004. Immunohistochemical analysis of oxidative DNA damage in arsenic-related human skin samples from arsenic-contaminated area of china. Cancer Lett. 214, 11–18. An, Y., Kato, K., Nakano, M., Otsu, H., Okada, S., Yamanaka, K., 2005. Specific induction of oxidative stress in terminal bronchiolar Clara cells during dimethylarsenic-induced lung tumor promoting process in mice. Cancer Lett., in press. Andrewes, P., Kitchin, K.T., Wallace, K., 2003. Dimethylarsine and trimethylarsine are potent genotoxins in vitro. Chem. Res. Toxicol. 16, 994–1003. Aposhian, H.V., Gurzau, E.S., Le, X.C., Gurzau, A., Healy, S.M., Lu, X., Ma, M., Yip, L., Zakharyan, R.A., Maiorino, R.M., Dart, R.C., Tircus, M.G., Gonzalez-Ramirez, D., Morgan, D.L., Avram, D., Aposhian, M.M., 2000. Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic. Chem. Res. Toxicol. 13, 693–697. Katiyar, S.K., Rupp, C.O., Korman, N.J., Agarwal, R., Mukhtar, H., 1995. Inhibition of 12-O-tetradecanoylphorbol-13-acetate and other skin tumor-promoter-caused induction of epidermal interleukin-1 alpha mRNA and protein expression in SENCAR mice by green tea polyphenols. J. Investig. Dermatol. 105, 394–398. Kenyon, E.M., Hughes, M.F., 2001. A concise review of the toxicity and carcinogenicity of dimethylarsinic acid. Toxicology 160, 227–236. Kitchin, K.T., 2001. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol. Appl. Pharmacol. 172, 249–261. Kitchin, K.T., Ahmad, S., 2003. Oxidative stress as a possible mode of action for arsenic carcinogenesis. Toxicol. Lett. 137, 3–13. Le, X.C., Lu, X., Ma, M., Cullen, W.R., Aposhian, H.V., Zheng, B., 2000. Speciation of key arsenic metabolic intermediates in human urine. Anal. Chem. 72, 5172–5177. Lin, S., Cullen, W.R., Thomas, D.J., 1999. Methylarsenicals and arsinothiols are potent inhibitors of mouse liver thioredoxin reductase. Chem. Res. Toxicol. 12, 924–930. Mandal, B.K., Ogra, Y., Suzuki, K.T., 2001. Identification of dimethylarsinous and monomethylarsonous acids in human urine of the arsenic-affected areas in West Bengal. India. Chem. Res. Toxicol. 14, 371–378. Mass, M.J., Tennant, A., Roop, B.C., Cullen, W.R., Styblo, M., Thomas, D.J., Kligerman, A.D., 2001. Methylated trivalent arsenic species are genotoxic. Chem. Res. Toxicol. 14, 355– 361. Morikawa, T., Wanibuchi, H., Morimura, K., Ogawa, M., Fukushima, S., 2000. Promotion of skin carcinogenesis by dimethylarsinic acid in keratin (K6)/ODC transgenic mice. Jpn. J. Cancer Res. 91, 579–581. Nesnow, S., Roop, B.C., Lambert, G., Kadiiska, M., Mason, R.P., Cullen, W.R., Mass, M.J., 2002. DNA damage induced by methylated trivalent arsenicals is mediated by reactive oxygen species. Chem. Res. Toxicol. 15, 1627–1634. O’Brien, T.G., Megosh, L.C., Gilliard, G., Peralta Soler, A., 1997. Ornithine decarboxylase overexpression is a sufficient condition for tumor promotion in mouse skin. Cancer Res. 57, 2630–2637.
94
M. Mizoi et al. / Toxicology Letters 158 (2005) 87–94
Petrick, J.S., Jagadish, B., Mash, E.A., Aposhian, H.V., 2001. Monomethylarsonous acid (MMA (III)) and arsenite: LD (50) in hamsters and in vitro inhibition of pyruvate dehydrogenase. Chem. Res. Toxicol. 14, 651–656. Stoner, G.D., Mukhtar, H., 1995. Polyphenols as cancer chemopreventive agents. J. Cell Biochem. Suppl. 22, 169–180. Styblo, M., Serves, S.V., Cullen, W.R., Thomas, D.J., 1997. Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem. Res. Toxicol. 10, 27–33. Styblo, M., Drobna, Z., Jaspers, I., Lin, S., Thomas, D.J., 2002. The role of biomethylation in toxicity and carcinogenicity of arsenic: a research update. Environ. Health Perspect. 110 (Suppl. 5), 767–771. Thompson, D.J., 1993. A chemical hypothesis for arsenic methylation in mammals. Chem. Biol. Interact. 88, 89–114. Wanibuchi, H., Hori, T., Meenakshi, V., Ichihara, T., Yamamoto, S., Yano, Y., Otani, S., Nakae, D., Konishi, Y., Fukushima, S., 1997. Promotion of rat hepatocarcinogenesis by dimethylarsinic acid: association with elevated ornithine decarboxylase activity and formation of 8-hydroxydeoxyguanosine in the liver. Jpn. J. Cancer Res. 88, 1149–1154. Yamamoto, S., Konishi, Y., Matsuda, T., Murai, T., Shibata, M.A., Matsui-Yuasa, I., Otani, S., Kuroda, K., Endo, G., Fukushima, S., 1995. Cancer induction by an organic arsenic compound, dimethylarsinic acid (cacodylic acid), in F344/DuCrj rats after pretreatment with five carcinogens. Cancer Res. 55, 1271– 1276. Yamanaka, K., Hoshino, M., Okamoto, M., Sawamura, R., Hasegawa, A., Okada, S., 1990. Induction of DNA damage by
dimethylarsine, a methabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical. Biochem. Biophys. Res. Commun. 168, 58–64. Yamanaka, K., Ohtsubo, K., Hasegawa, A., Hayashi, H., Ohji, H., Kanisawa, M., Okada, S., 1996. Exposure to dimethylarsinic acid, a main methabolite of inorganic arsenics, strongly promotes tumorigenesis initiated by 4-nitroquinolone 1-oxide in the lungs of mice. Carcinogenesis 17, 767–770. Yamanaka, K., Katsumata, K., Ikuma, K., Hasegawa, A., Nakano, M., Okada, S., 2000. 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 Lett. 152, 79–85. Yamanaka, K., Mizoi, M., Kato, K., Hasegawa, A., Nakano, M., Okada, S., 2001a. Oral administration of dimethylarsinic acid, a main metabolite of inorganic arsenic, in mice promotes skin tumorigenesis initiated by dimethylbenz(a)anthracene with or without ultraviolet B as a promoter. Biol. Pharm. Bull. 24, 510–514. Yamanaka, K., Takabayashi, F., Mizoi, M., Yan, A., Hasegawa, A., Okada, S., 2001b. Oral exposure of dimethylarsinic acid, a main metabolite of inorganic arsenics, in mice leads to an increase in 8-oxo-2 -deoxyguanosine level, specifically in the target organs for arsenic carcinogenesis. Biochem. Biophys. Res. Commun. 287, 66–70. Yamanaka, K., Mizoi, M., Tachikawa, M., Hasegawa, A., Hoshino, M., Okada, S., 2003. Oxidative DNA damage following exposure to dimethylarsinous iodide: the formation of cis-thymine glycol. Toxicol. Lett. 143, 145–153.