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Mouse skin tumor promotion by sodium arsenate is associated with enhanced PCNA expression
Cancer Letters 223 (2005) 27–35 www.elsevier.com/locate/canlet
Mouse skin tumor promotion by sodium arsenate is associated with enhanced PCNA expression L. Motiwalea, A.D. Ingleb, K.V.K. Raoa,* a
Chemical Carcinogenesis Group, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India b Animal Sciences Group, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India Received 20 April 2004; received in revised form 8 October 2004; accepted 12 October 2004
Abstract Drinking water contamination by arsenicals remains a major public health problem in many parts of the world more particularly in India and Bangladesh. Despite arsenic being a health hazard and implicated in human carcinogenesis, the experimental evidence available is much limited even now and the mechanisms involved during carcinogenesis and tumor promotions are not clear. Accordingly, in this study, we have studied the tumor promoter effects of sodium arsenate on mouse skin tumor promoter model system using 9,10-dimethyl-1,2-benzanthracene (DMBA) as a initiating carcinogen. Our studies showed development of papillomas on mice skin treated with only DMBA. However, mice treated with DMBA on skin and administered arsenate (As) in drinking water showed development of well differentiated squamous cell carcinomas. Further, both by immunohistochemistry and western blotting analysis studies higher levels of proliferating cell nuclear antigen (PCNA) was observed in mice treated with DMBA plus arsenate compared to only DMBA treated group. PCNA is known to be associated with S phase and DNA replication of the cell cycle. The plain controls and arsenate controls did not show significant difference either in tumor development or in PCNA levels. The present study demonstrates mouse skin tumor promoting effect of arsenate which seems to be associated with abnormal cell proliferation as indicated by higher levels of PCNA expression. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Sodium arsenate; Skin tumor promotion; Squamous cell carcinoma; DMBA; PCNA
1. Introduction
* Corresponding author. Tel.: C91 22 2740 5023; fax: C91 22 2741 2894. E-mail addresses: [email protected] (K.V.K. Rao), [email protected] (K.V.K. Rao), [email protected] (K.V.K. Rao).
Drinking water contamination by arsenic remains a major public health problem in many parts of the world. Of these, most severe arsenic contamination of drinking water was observed in Asia, especially in Bangladesh; West Bengal; India; Inner Mongolia, China; and Taiwan [1]. The world’s two biggest cases
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.10.020
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L. Motiwale et al. / Cancer Letters 223 (2005) 27–35
of groundwater contamination and those that affected the greatest number of people were in Bangladesh and West Bengal. Nine districts in West Bengal, India and 42 districts in Bangladesh have arsenic levels in ground water above the World Health Organization (WHO) maximum permissible limit of 50 mg/l. Chronic arsenic exposure at high doses has neurologic, dermatologic, vascular and carcinogenic effects. One hundred and fifty-one villages from Bangladesh and West Bengal were identified where people were suffering from arsenic induced skin lesions. More people are suffering from arsenical skin lesion in Bangladesh than in West Bengal. Arsenic is considered as a potent human carcinogen. Squamous cell carcinoma, basal cell carcinoma, bowen disease and carcinoma affecting the lung, uterus, bladder, genitourinary tract, or other sites are often seen in patients with advanced arsenical toxicity cases. Also, complications such as hyperkeratosis, liver enlargement (hepatomegaly), spleen enlargement (splenomegaly), and fluid in the abdomen (ascitis) are seen in severe cases [2,3]. Arsenic exists in many chemical forms with varying degrees of toxicity [4]. The more inorganic the form, the greater the virulence. For example arsenate and arsenite are known carcinogens. Methylated arsenic compounds, monomethyl arsinic acid and dimethyl arsinic acid are less toxic and organic arsenicals arsenobetaine and arsenocholine are relatively innocuous. So for causal factors in arsenic epidemiology, the focus has been on inorganic arsenite and arsenate, but experimental evidence was limited until recently [5]. Numerous experimental studies using laboratory animals have failed to show carcinogenic action of inorganic arsenics [6,7]. However recent research demonstrated a promotion potential for dimethyl arsinic acid (a main metabolite of inorganic arsenics in mammals) in the urinary bladder, kidney, liver and thyroid gland using a multiorgan carcinogenesis bioassay in rats and also for tumorigenesis in mice [8,9]. Furthermore it is well known that internationally, at least 1 million people are drinking arsenic contaminated water above WHO recommended value of 0.01 ppm and 200,000 people show skin lesions characteristic of arsenic poisoning [10]. Accordingly, we have made an attempt to investigate the tumor promoter effects of sodium arsenate; a form of inorganic arsenic, on DMBA
initiated skin carcinogenesis. The promoter activities of non-genotoxic and genotoxic chemicals generally related to an increase in cell proliferation [11,12] and accordingly we have studied PCNA expression. In the present study we would like to report tumor promoter effects of oral sodium arsenate using DMBA mouse skin tumor promoter model.
2. Materials and methods 2.1. Chemicals 9,10-Dimethyl-1,2-benzanthracene (DMBA) was purchased from Sigma Chemical Company (St Louis, MO, USA). PCNA (PC10) mouse monoclonal antibody was purchased from Santacruz Biotechnology (Santacruz, CA, USA). Sodium arsenate AR was from s.d. Fine Chemicals Ltd, (Boisar, India). All other chemicals were from Sigma, unless otherwise specified. 2.2. Animals A total of forty, 8 week old swiss-bald strain male hairless mice obtained from animal house of ACTREC were used. The animals were randomised and housed five per cage with rice husks for bedding. Food and water were provided ad libitum. 2.3. Experimental design The experimental protocol followed is shown in Fig. 1. The animals were divided into four groups (10 animals per group), with Group A serving as untreated control. Group B and D animals received topical application of 25 ml of DMBA in acetone (2 mg/ml) twice a week for 2 weeks. Group B served as DMBA control. Group C and D animals were given sodium arsenate at a concentration of 25 mg/l in drinking water ad libitum for a period of 25 weeks. Group C served as Arsenic control and group D served as group treated with DMBA and arsenic. The number of papillomas was counted weekly. The data are expressed as percentage of mice with papillomas and number of papilloma per mouse are plotted as a function of weeks on test.
L. Motiwale et al. / Cancer Letters 223 (2005) 27–35
Fig. 1. Experimental regimen for studying mouse skin tumor promoting effect of sodium arsenate.
At the end of the experiment, pictures of animals were taken for morphology and then the animals were sacrificed by cervical dislocation. The skin tumors and normal skin (for control groups) were excised and fixed with buffered 10% formalin for histopathology and PCNA staining and frozen at K70 8C for biochemical studies. To evaluate the histopathological grades of the tumors, paraffin sections of tumors were stained with hematoxylin and eosin. 2.4. Immunohistochemical staining Immunohistochemical analysis for PCNA was performed as per the method of Hall et al. [13]. Briefly, cut tissue sections were deparaffinized and endogenous peroxidase was quenched by incubation in 3% hydrogen peroxide in methanol for 30 min. Non-specific binding was blocked with normal sheep serum (5%) in PBS and then the tissue sections were incubated at 4 8C overnight with 1:50 diluted PCNA (PC-10) antibody. Immunostaining was performed using biotin-streptavidin peroxidase method. 2.5. Western blot analysis The method followed was as described by Towbin et al. [14]. Skin samples were homogenized directly in Nonidet P-40 buffer (NaCl 150 mM, EDTA 5 mM, Tris–HCl 50 mM (pH 7.5), Aprotinin 10 mg/ml,
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Leupeptin 1 mM, PMSF 1 mM, Sodium fluoride 5 mM, glycerophosphate 20 mM, Nonidet P-40 1% w/v). Lysate protein was quantified using modified Lowry’s method [15]. Equal amount of protein sample for each group was separated on 10% SDSPAGE and transferred to nitrocellulose membrane. Non-specific binding was blocked by incubation in blocking buffer (5% non-fat milk, 0.05% Tween-20 in 50 mM Tris pH 7.5, NaCl 150 mM) for 1 h at RT. The blot was incubated with monoclonal anti-PCNA (1 mg/ml) overnight at 4 8C. Secondary antibody antimouse HRPO (1:8000) was used. Signals were visualized with ECL detection method. Densitometric scannings were carried out as per Molecular Analyst 1 software (Version 1.4) from BIO-RAD Laboratories, CA, USA. 2.6. Statistical analysis All numeric data are presented as meansGstandard deviations (SD) and analysed using non-parametric Mann–Whitney U-test. A P value !0.05 is considered statistically significant.
3. Results 3.1. Survival and general conditions No treatment related direct effects of Sodium arsenate on survival and general conditions were observed in any of treated groups. The dose of Sodium arsenate utilised was apparently well tolerated. 3.2. Skin tumor development Representative gross lesions on the back skins of mice are shown in Fig. 2. No skin tumors developed in untreated mice and also in mice treated with As water (A and C). Small papillomas were found on mice treated with DMBA alone (B). Whereas large numbers of tumors were found on mice treated with DMBA plus As water (D). At the end of the experiment even the tumor volume in DMBA plus As water treated animals was much larger than tumor volume of DMBA treated animals. Fig. 6(A) shows the positive control of well developed squamous cell carcinoma of mouse.
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Fig. 2. Morphological appearance of mice during tumor promotion by sodium arsenate. (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As.
3.3. Incidence of skin tumors The number of mice bearing tumor in all groups at different weeks is shown in Fig. 3A and B. The onset of tumors (O2 mm) commenced at 9 weeks in DMBA plus As water treated mice and reached at 100% at 20 weeks, whereas in DMBA treated mice the onset of tumors was delayed by 1 week and only 70% of mice were having tumors even at the end of experiment. Similarly number of tumors per mouse at different weeks is shown in Fig. 3B. By week 25, the tumor incidence in DMBA plus As water treated animals was significantly higher than in DMBA treated animals (P!0.05). No tumor formation was seen in As water treated and untreated control animals. In addition, differences in the severity of tumor growth and the size distribution of papillomas among different groups were also recorded (Table 1). Papillomas observed in DMBA plus As treated group were significantly (PZ0.001) bigger in size (65% papillomas of R3 mm in diameter) as that of DMBA control group (16%). These results show that Sodium arsenate acts as a tumor promoter rather than an initiator. 3.4. Histopathological examination Histologically, the DMBA treated group (Fig. 4B) and DMBA plus As water treated group
(Fig. 4D) exhibited varying degrees of structural and cytological divergence as compared to untreated control group (Fig. 4A) and Arsenic treated group (Fig. 4C). Tumors of animals belonging to plain DMBA treated group showed intact basement membrane (arrow) as seen in control group and squamous cell hyperplasia (asterisk) at few places. This clearly characterises the benign nature of these tumors i.e. squamous cell papilloma. However tumors of animals belonging to DMBA plus As treated group (Fig. 4D) were composed of focal proliferation of squamous cells (arrow), presence of some necrotic cells (asterisk), keratinization (arrow head) and epithelial pearls (double arrow). This clearly indicates the malignant nature of tumors i.e. well differentiated squamous cell carcinoma. These results show that oral administration of Sodium arsenate leads in DMBA treated group to progression activity in skin tumorigenesis in mice. However Arsenic treated group (Fig. 4C) did not reveal any abnormalities indicating treatment of these mice with arsenic at 25 mg/l in drinking water has no effect on skin. Fig. 4E shows histological features of well developed squamous cell carcinoma. This figure clearly shows focal proliferation of squamous cells (arrow), keratinization (arrow head) and epithelial pearls (double arrow).
L. Motiwale et al. / Cancer Letters 223 (2005) 27–35
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shows immunohistochemical analysis of PCNA of well developed squamous cell carcinoma of mouse. A characteristic intense staining was observed (arrows). 3.6. Expression of PCNA during tumor promotion
Fig. 3. Tumor promoting effect of sodium arsenate on DMBA initiated mouse skin. (A) Percentage tumor bearing mice plotted as a function of weeks on test. Each value represents the mean incidence data of 10 animals. (B) Number of tumor/mouse plotted as a function of weeks on test. Each value represents the mean number of tumor per mouse.
3.5. Cell proliferation by PCNA immunohistochemistry PCNA immunohistochemical analysis was used to assess the proliferation activity in treated groups during tumor promotion (Fig. 5). PCNA is Proliferating Cell Nuclear Antigen associated with S phase of DNA replication. It was observed that PCNA labelling indices were higher in animals treated with DMBA plus As water as compared to DMBA treated animals. The representative control groups of animals did not show positive staining for PCNA. Fig. 6(B)
In order to confirm the possible relationship between tumor promotion by Sodium arsenate and associated abnormal cellular proliferation, we have looked at the levels of PCNA by Western blotting, a marker for cell proliferation (Fig. 7). An increase in the expression levels of PCNA in the group treated with DMBA plus As water was observed compared to only DMBA treated group. The PCNA expression level in DMBA treated group was much higher than the PCNA level in untreated control group. No PCNA expression was observed in plain As treated group (Fig. 7C) suggesting possible cytotoxic properties of As on normal skin. It is known that carcinogen administration induces resistance in cells which can proliferate even under cytotoxic conditions [16] as observed in DMBA and DMBA plus As treated groups. Fig. 7E shows the PCNA levels in positive control. These results indicate that Sodium arsenate acts as a skin tumor promoter by promoting abnormal cell proliferation. This abnormal cell proliferation activity appears to be responsible for the progression of squamous cell papilloma (as observed in DMBA treated group) to squamous cell carcinoma (as observed in DMBA plus As water treated group) of the skin. Table 1 Size distribution of papillomas Treatment group
Number of animals (n)
% Small papillomas (1–2 mm)a
% Big papillomas (R3 mm)a
Untreated control DMBA control Arsenic control DMBA plus Arsenic
10
Nil
Nil
10
84.0G1.95
16.0G1.34
10
Nil
Nil
10
35.0G0.78b
65.0G0.94b
a
MeanGSD. PZ0.001 compared with DMBA control group using nonparametric Mann-Whitney U-test. b
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Fig. 4. Histopathological changes of mouse skin during tumor promotion by sodium arsenate. (% 100) (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As; (E) Positive control of DMBA induced skin tumor.
Fig. 5. PCNA Immunostaining of mouse skin during tumor promotion by sodium arsenate. (% 244) (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As.
L. Motiwale et al. / Cancer Letters 223 (2005) 27–35
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Fig. 6. Positive control of DMBA induced skin tumor. (A) Morphological appearance; (B) PCNA immunostaining.
4. Discussion The metalloid arsenic found in rocks and mineral formation in the earth’s crust has long been associated with the development of the cancer in humans. In 1984 health assessment, the US Environmental Protection Agency (EPA) classified Arsenic as a class A human carcinogen based primarily on the epidemiologic evidence [17]. In certain parts of Bangladesh and West Bengal, India, as many as 5% of drinking water wells that are sampled had arsenic levels exceeding 1 mg/l and 27% of wells had levels exceeding 300 mg/l [18], which is 6 times higher than current US maximum contaminant level of 50 mg/l. Thus arsenic has become a serious environmental concern worldwide because of large number of known contaminated sites and millions of people at risk from drinking arsenic contaminated water. There has been substantial focus on the association between arsenic and skin cancer but evidences show that exposure of arsenic in drinking water increases mortality risk for several other internal cancers namely bladder, lung, kidney and liver [19]. Besides cancer it is also a risk factor for atherosclerosis, diabetes and peripheral neuropathy [20]. Arsenic, as trivalent arsenite (As3C) or pentavalent arsenate (As5C), is naturally occurring and ubiquitously present in the environment. Humans are exposed to these inorganic arsenic mainly through either oral or inhalation routes. Oral exposure occurs via consumption of contaminated water, food and drugs [21]. This oral exposure of arsenic for long period of time gives rise to arsenical skin lesions, which is a major concern for the people living in Bangladesh and West Bengal, India [1]. Some in vitro studies had been carried out to see the effect of this inorganic arsenic [22]. But because of
lack of suitable animal models as well as poor understanding of its carcinogenic/genotoxic mechanism, the in vivo studies using inorganic arsenic which is the major contaminant of ground water is still not reported. Furthermore orally administered inhibitors and suppressors, eg-curcumin [23], polyphenolic fraction isolated from grape seeds [24], tea extracts containing polyphenols [25], indomethacin [26] against skin tumor promotion have been reported for skin tumors. But orally effective skin tumor promoters are not much known. Yamanaka et al. reported the promoting effect of dimethyl arsinic acid, a main metabolite of inorganic arsenic on skin tumorigenesis in mice [27]. However, the mechanism underlying the carcinogenicity of arsenic remains unclear. In the present study we
Fig. 7. Expression and densitometric analysis of PCNA in mouse skin during tumor promotion by sodium arsenate. (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As; (E) Positive control.
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demonstrated tumor promoting and progressing action of Sodium arsenate, an inorganic form of arsenic, on skin tumorigenesis in hairless mice. The tumor promotion study has been carried out using PCNA, a marker for cell proliferation. In this study a significant increase in the expression of PCNA was observed in mice treated with DMBA and As water indicating increased proliferation of cells associated with abnormal tumor growth. Whereas there was no change in PCNA levels in mice treated with DMBA as compared to untreated control. Mice treated with As water alone showed decrease in PCNA levels suggesting that for normal cells As was toxic, however, the initiated/preneoplastic cells were resistant to these toxic effects and could proliferate. In summary, the present investigation provides evidence that sodium arsenate acts as a skin tumor promoter by increase in cellular proliferation, which in turn is responsible for the progression of lesion into malignancy and thus helping in tumor formation.
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgements
[12]
This work was supported in part by a CSIR Grant No. 37 (1141)/03/EMR-II, New Delhi, India to Dr K.V.K. Rao.
[13]
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L. Motiwale et al. / Cancer Letters 223 (2005) 27–35 [20] P.C. Chan, J. Huff, Arsenic carcinogenesis in animals and in humans: mechanistic, experimental, and epidemiological evidence, Environ. Carcinog. Ecotoxicol. Rev. C15 (1997) 83–122. [21] G.G. Garica-vargas, M.E. Cebrian, in: P. Cheng (Ed.), Toxicology of Metals, CRC press, Boca Raton, FL, 1996, pp. 423–438. [22] S.X. Liu, M. Athar, I. Lippai, C. Waldren, T.K. Hei, Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity, Proc. Natl Acad. Sci. USA 98 (2001) 1643–1648. [23] P. Limtrakul, S. Lipigorngoson, O. Namwong, A. Apisariyakul, F.W. Dunn, Inhibitory effect of dietary curcumin on skin carcinogenesis in mice, Cancer Lett. 116 (1997) 197–203. [24] J. Zhao, J. Wang, Y. Chen, R. Agarwal, Anti-tumor promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and
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identification of procyanidin B5-3 0 -gallate as the most effective antioxidant constituent, Carcinogenesis 20 (1999) 1737–1745. [25] Z.Y. Wang, M.T. Huang, Y.R. Lou, J.G. Xie, K.R. Reuhl, H.L. Newmark, et al., Inhibitory effect of black tea, green tea, decaffeinated black tea and decaffeinated green tea on ultraviolet B light-induced skin carcinogenesis in 7,12dimethylbenz[a]anthracene-initiated SKH-1 mice, Cancer Res. 54 (1994) 3428–3435. [26] V.E. Reeve, M.J. Matheson, C. Bosnic-Wilcox, The protective effect of indomethacin on photocarcinogenesis in hairless mice, Cancer Lett. 95 (1995) 213–219. [27] K. Yamanaka, K. Katsumata, K. Ikuma, A. Hasegawa, M. Nakano, S. Okada, 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 (2000) 79–85.
Mouse skin tumor promotion by sodium arsenate is associated with enhanced PCNA expression L. Motiwalea, A.D. Ingleb, K.V.K. Raoa,* a
Chemical Carcinogenesis Group, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India b Animal Sciences Group, Cancer Research Institute, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India Received 20 April 2004; received in revised form 8 October 2004; accepted 12 October 2004
Abstract Drinking water contamination by arsenicals remains a major public health problem in many parts of the world more particularly in India and Bangladesh. Despite arsenic being a health hazard and implicated in human carcinogenesis, the experimental evidence available is much limited even now and the mechanisms involved during carcinogenesis and tumor promotions are not clear. Accordingly, in this study, we have studied the tumor promoter effects of sodium arsenate on mouse skin tumor promoter model system using 9,10-dimethyl-1,2-benzanthracene (DMBA) as a initiating carcinogen. Our studies showed development of papillomas on mice skin treated with only DMBA. However, mice treated with DMBA on skin and administered arsenate (As) in drinking water showed development of well differentiated squamous cell carcinomas. Further, both by immunohistochemistry and western blotting analysis studies higher levels of proliferating cell nuclear antigen (PCNA) was observed in mice treated with DMBA plus arsenate compared to only DMBA treated group. PCNA is known to be associated with S phase and DNA replication of the cell cycle. The plain controls and arsenate controls did not show significant difference either in tumor development or in PCNA levels. The present study demonstrates mouse skin tumor promoting effect of arsenate which seems to be associated with abnormal cell proliferation as indicated by higher levels of PCNA expression. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Sodium arsenate; Skin tumor promotion; Squamous cell carcinoma; DMBA; PCNA
1. Introduction
* Corresponding author. Tel.: C91 22 2740 5023; fax: C91 22 2741 2894. E-mail addresses: [email protected] (K.V.K. Rao), [email protected] (K.V.K. Rao), [email protected] (K.V.K. Rao).
Drinking water contamination by arsenic remains a major public health problem in many parts of the world. Of these, most severe arsenic contamination of drinking water was observed in Asia, especially in Bangladesh; West Bengal; India; Inner Mongolia, China; and Taiwan [1]. The world’s two biggest cases
0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.10.020
28
L. Motiwale et al. / Cancer Letters 223 (2005) 27–35
of groundwater contamination and those that affected the greatest number of people were in Bangladesh and West Bengal. Nine districts in West Bengal, India and 42 districts in Bangladesh have arsenic levels in ground water above the World Health Organization (WHO) maximum permissible limit of 50 mg/l. Chronic arsenic exposure at high doses has neurologic, dermatologic, vascular and carcinogenic effects. One hundred and fifty-one villages from Bangladesh and West Bengal were identified where people were suffering from arsenic induced skin lesions. More people are suffering from arsenical skin lesion in Bangladesh than in West Bengal. Arsenic is considered as a potent human carcinogen. Squamous cell carcinoma, basal cell carcinoma, bowen disease and carcinoma affecting the lung, uterus, bladder, genitourinary tract, or other sites are often seen in patients with advanced arsenical toxicity cases. Also, complications such as hyperkeratosis, liver enlargement (hepatomegaly), spleen enlargement (splenomegaly), and fluid in the abdomen (ascitis) are seen in severe cases [2,3]. Arsenic exists in many chemical forms with varying degrees of toxicity [4]. The more inorganic the form, the greater the virulence. For example arsenate and arsenite are known carcinogens. Methylated arsenic compounds, monomethyl arsinic acid and dimethyl arsinic acid are less toxic and organic arsenicals arsenobetaine and arsenocholine are relatively innocuous. So for causal factors in arsenic epidemiology, the focus has been on inorganic arsenite and arsenate, but experimental evidence was limited until recently [5]. Numerous experimental studies using laboratory animals have failed to show carcinogenic action of inorganic arsenics [6,7]. However recent research demonstrated a promotion potential for dimethyl arsinic acid (a main metabolite of inorganic arsenics in mammals) in the urinary bladder, kidney, liver and thyroid gland using a multiorgan carcinogenesis bioassay in rats and also for tumorigenesis in mice [8,9]. Furthermore it is well known that internationally, at least 1 million people are drinking arsenic contaminated water above WHO recommended value of 0.01 ppm and 200,000 people show skin lesions characteristic of arsenic poisoning [10]. Accordingly, we have made an attempt to investigate the tumor promoter effects of sodium arsenate; a form of inorganic arsenic, on DMBA
initiated skin carcinogenesis. The promoter activities of non-genotoxic and genotoxic chemicals generally related to an increase in cell proliferation [11,12] and accordingly we have studied PCNA expression. In the present study we would like to report tumor promoter effects of oral sodium arsenate using DMBA mouse skin tumor promoter model.
2. Materials and methods 2.1. Chemicals 9,10-Dimethyl-1,2-benzanthracene (DMBA) was purchased from Sigma Chemical Company (St Louis, MO, USA). PCNA (PC10) mouse monoclonal antibody was purchased from Santacruz Biotechnology (Santacruz, CA, USA). Sodium arsenate AR was from s.d. Fine Chemicals Ltd, (Boisar, India). All other chemicals were from Sigma, unless otherwise specified. 2.2. Animals A total of forty, 8 week old swiss-bald strain male hairless mice obtained from animal house of ACTREC were used. The animals were randomised and housed five per cage with rice husks for bedding. Food and water were provided ad libitum. 2.3. Experimental design The experimental protocol followed is shown in Fig. 1. The animals were divided into four groups (10 animals per group), with Group A serving as untreated control. Group B and D animals received topical application of 25 ml of DMBA in acetone (2 mg/ml) twice a week for 2 weeks. Group B served as DMBA control. Group C and D animals were given sodium arsenate at a concentration of 25 mg/l in drinking water ad libitum for a period of 25 weeks. Group C served as Arsenic control and group D served as group treated with DMBA and arsenic. The number of papillomas was counted weekly. The data are expressed as percentage of mice with papillomas and number of papilloma per mouse are plotted as a function of weeks on test.
L. Motiwale et al. / Cancer Letters 223 (2005) 27–35
Fig. 1. Experimental regimen for studying mouse skin tumor promoting effect of sodium arsenate.
At the end of the experiment, pictures of animals were taken for morphology and then the animals were sacrificed by cervical dislocation. The skin tumors and normal skin (for control groups) were excised and fixed with buffered 10% formalin for histopathology and PCNA staining and frozen at K70 8C for biochemical studies. To evaluate the histopathological grades of the tumors, paraffin sections of tumors were stained with hematoxylin and eosin. 2.4. Immunohistochemical staining Immunohistochemical analysis for PCNA was performed as per the method of Hall et al. [13]. Briefly, cut tissue sections were deparaffinized and endogenous peroxidase was quenched by incubation in 3% hydrogen peroxide in methanol for 30 min. Non-specific binding was blocked with normal sheep serum (5%) in PBS and then the tissue sections were incubated at 4 8C overnight with 1:50 diluted PCNA (PC-10) antibody. Immunostaining was performed using biotin-streptavidin peroxidase method. 2.5. Western blot analysis The method followed was as described by Towbin et al. [14]. Skin samples were homogenized directly in Nonidet P-40 buffer (NaCl 150 mM, EDTA 5 mM, Tris–HCl 50 mM (pH 7.5), Aprotinin 10 mg/ml,
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Leupeptin 1 mM, PMSF 1 mM, Sodium fluoride 5 mM, glycerophosphate 20 mM, Nonidet P-40 1% w/v). Lysate protein was quantified using modified Lowry’s method [15]. Equal amount of protein sample for each group was separated on 10% SDSPAGE and transferred to nitrocellulose membrane. Non-specific binding was blocked by incubation in blocking buffer (5% non-fat milk, 0.05% Tween-20 in 50 mM Tris pH 7.5, NaCl 150 mM) for 1 h at RT. The blot was incubated with monoclonal anti-PCNA (1 mg/ml) overnight at 4 8C. Secondary antibody antimouse HRPO (1:8000) was used. Signals were visualized with ECL detection method. Densitometric scannings were carried out as per Molecular Analyst 1 software (Version 1.4) from BIO-RAD Laboratories, CA, USA. 2.6. Statistical analysis All numeric data are presented as meansGstandard deviations (SD) and analysed using non-parametric Mann–Whitney U-test. A P value !0.05 is considered statistically significant.
3. Results 3.1. Survival and general conditions No treatment related direct effects of Sodium arsenate on survival and general conditions were observed in any of treated groups. The dose of Sodium arsenate utilised was apparently well tolerated. 3.2. Skin tumor development Representative gross lesions on the back skins of mice are shown in Fig. 2. No skin tumors developed in untreated mice and also in mice treated with As water (A and C). Small papillomas were found on mice treated with DMBA alone (B). Whereas large numbers of tumors were found on mice treated with DMBA plus As water (D). At the end of the experiment even the tumor volume in DMBA plus As water treated animals was much larger than tumor volume of DMBA treated animals. Fig. 6(A) shows the positive control of well developed squamous cell carcinoma of mouse.
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Fig. 2. Morphological appearance of mice during tumor promotion by sodium arsenate. (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As.
3.3. Incidence of skin tumors The number of mice bearing tumor in all groups at different weeks is shown in Fig. 3A and B. The onset of tumors (O2 mm) commenced at 9 weeks in DMBA plus As water treated mice and reached at 100% at 20 weeks, whereas in DMBA treated mice the onset of tumors was delayed by 1 week and only 70% of mice were having tumors even at the end of experiment. Similarly number of tumors per mouse at different weeks is shown in Fig. 3B. By week 25, the tumor incidence in DMBA plus As water treated animals was significantly higher than in DMBA treated animals (P!0.05). No tumor formation was seen in As water treated and untreated control animals. In addition, differences in the severity of tumor growth and the size distribution of papillomas among different groups were also recorded (Table 1). Papillomas observed in DMBA plus As treated group were significantly (PZ0.001) bigger in size (65% papillomas of R3 mm in diameter) as that of DMBA control group (16%). These results show that Sodium arsenate acts as a tumor promoter rather than an initiator. 3.4. Histopathological examination Histologically, the DMBA treated group (Fig. 4B) and DMBA plus As water treated group
(Fig. 4D) exhibited varying degrees of structural and cytological divergence as compared to untreated control group (Fig. 4A) and Arsenic treated group (Fig. 4C). Tumors of animals belonging to plain DMBA treated group showed intact basement membrane (arrow) as seen in control group and squamous cell hyperplasia (asterisk) at few places. This clearly characterises the benign nature of these tumors i.e. squamous cell papilloma. However tumors of animals belonging to DMBA plus As treated group (Fig. 4D) were composed of focal proliferation of squamous cells (arrow), presence of some necrotic cells (asterisk), keratinization (arrow head) and epithelial pearls (double arrow). This clearly indicates the malignant nature of tumors i.e. well differentiated squamous cell carcinoma. These results show that oral administration of Sodium arsenate leads in DMBA treated group to progression activity in skin tumorigenesis in mice. However Arsenic treated group (Fig. 4C) did not reveal any abnormalities indicating treatment of these mice with arsenic at 25 mg/l in drinking water has no effect on skin. Fig. 4E shows histological features of well developed squamous cell carcinoma. This figure clearly shows focal proliferation of squamous cells (arrow), keratinization (arrow head) and epithelial pearls (double arrow).
L. Motiwale et al. / Cancer Letters 223 (2005) 27–35
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shows immunohistochemical analysis of PCNA of well developed squamous cell carcinoma of mouse. A characteristic intense staining was observed (arrows). 3.6. Expression of PCNA during tumor promotion
Fig. 3. Tumor promoting effect of sodium arsenate on DMBA initiated mouse skin. (A) Percentage tumor bearing mice plotted as a function of weeks on test. Each value represents the mean incidence data of 10 animals. (B) Number of tumor/mouse plotted as a function of weeks on test. Each value represents the mean number of tumor per mouse.
3.5. Cell proliferation by PCNA immunohistochemistry PCNA immunohistochemical analysis was used to assess the proliferation activity in treated groups during tumor promotion (Fig. 5). PCNA is Proliferating Cell Nuclear Antigen associated with S phase of DNA replication. It was observed that PCNA labelling indices were higher in animals treated with DMBA plus As water as compared to DMBA treated animals. The representative control groups of animals did not show positive staining for PCNA. Fig. 6(B)
In order to confirm the possible relationship between tumor promotion by Sodium arsenate and associated abnormal cellular proliferation, we have looked at the levels of PCNA by Western blotting, a marker for cell proliferation (Fig. 7). An increase in the expression levels of PCNA in the group treated with DMBA plus As water was observed compared to only DMBA treated group. The PCNA expression level in DMBA treated group was much higher than the PCNA level in untreated control group. No PCNA expression was observed in plain As treated group (Fig. 7C) suggesting possible cytotoxic properties of As on normal skin. It is known that carcinogen administration induces resistance in cells which can proliferate even under cytotoxic conditions [16] as observed in DMBA and DMBA plus As treated groups. Fig. 7E shows the PCNA levels in positive control. These results indicate that Sodium arsenate acts as a skin tumor promoter by promoting abnormal cell proliferation. This abnormal cell proliferation activity appears to be responsible for the progression of squamous cell papilloma (as observed in DMBA treated group) to squamous cell carcinoma (as observed in DMBA plus As water treated group) of the skin. Table 1 Size distribution of papillomas Treatment group
Number of animals (n)
% Small papillomas (1–2 mm)a
% Big papillomas (R3 mm)a
Untreated control DMBA control Arsenic control DMBA plus Arsenic
10
Nil
Nil
10
84.0G1.95
16.0G1.34
10
Nil
Nil
10
35.0G0.78b
65.0G0.94b
a
MeanGSD. PZ0.001 compared with DMBA control group using nonparametric Mann-Whitney U-test. b
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Fig. 4. Histopathological changes of mouse skin during tumor promotion by sodium arsenate. (% 100) (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As; (E) Positive control of DMBA induced skin tumor.
Fig. 5. PCNA Immunostaining of mouse skin during tumor promotion by sodium arsenate. (% 244) (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As.
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Fig. 6. Positive control of DMBA induced skin tumor. (A) Morphological appearance; (B) PCNA immunostaining.
4. Discussion The metalloid arsenic found in rocks and mineral formation in the earth’s crust has long been associated with the development of the cancer in humans. In 1984 health assessment, the US Environmental Protection Agency (EPA) classified Arsenic as a class A human carcinogen based primarily on the epidemiologic evidence [17]. In certain parts of Bangladesh and West Bengal, India, as many as 5% of drinking water wells that are sampled had arsenic levels exceeding 1 mg/l and 27% of wells had levels exceeding 300 mg/l [18], which is 6 times higher than current US maximum contaminant level of 50 mg/l. Thus arsenic has become a serious environmental concern worldwide because of large number of known contaminated sites and millions of people at risk from drinking arsenic contaminated water. There has been substantial focus on the association between arsenic and skin cancer but evidences show that exposure of arsenic in drinking water increases mortality risk for several other internal cancers namely bladder, lung, kidney and liver [19]. Besides cancer it is also a risk factor for atherosclerosis, diabetes and peripheral neuropathy [20]. Arsenic, as trivalent arsenite (As3C) or pentavalent arsenate (As5C), is naturally occurring and ubiquitously present in the environment. Humans are exposed to these inorganic arsenic mainly through either oral or inhalation routes. Oral exposure occurs via consumption of contaminated water, food and drugs [21]. This oral exposure of arsenic for long period of time gives rise to arsenical skin lesions, which is a major concern for the people living in Bangladesh and West Bengal, India [1]. Some in vitro studies had been carried out to see the effect of this inorganic arsenic [22]. But because of
lack of suitable animal models as well as poor understanding of its carcinogenic/genotoxic mechanism, the in vivo studies using inorganic arsenic which is the major contaminant of ground water is still not reported. Furthermore orally administered inhibitors and suppressors, eg-curcumin [23], polyphenolic fraction isolated from grape seeds [24], tea extracts containing polyphenols [25], indomethacin [26] against skin tumor promotion have been reported for skin tumors. But orally effective skin tumor promoters are not much known. Yamanaka et al. reported the promoting effect of dimethyl arsinic acid, a main metabolite of inorganic arsenic on skin tumorigenesis in mice [27]. However, the mechanism underlying the carcinogenicity of arsenic remains unclear. In the present study we
Fig. 7. Expression and densitometric analysis of PCNA in mouse skin during tumor promotion by sodium arsenate. (A) Control; (B) DMBA; (C) As control; (D) DMBA plus As; (E) Positive control.
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demonstrated tumor promoting and progressing action of Sodium arsenate, an inorganic form of arsenic, on skin tumorigenesis in hairless mice. The tumor promotion study has been carried out using PCNA, a marker for cell proliferation. In this study a significant increase in the expression of PCNA was observed in mice treated with DMBA and As water indicating increased proliferation of cells associated with abnormal tumor growth. Whereas there was no change in PCNA levels in mice treated with DMBA as compared to untreated control. Mice treated with As water alone showed decrease in PCNA levels suggesting that for normal cells As was toxic, however, the initiated/preneoplastic cells were resistant to these toxic effects and could proliferate. In summary, the present investigation provides evidence that sodium arsenate acts as a skin tumor promoter by increase in cellular proliferation, which in turn is responsible for the progression of lesion into malignancy and thus helping in tumor formation.
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgements
[12]
This work was supported in part by a CSIR Grant No. 37 (1141)/03/EMR-II, New Delhi, India to Dr K.V.K. Rao.
[13]
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