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Inorganic arsenic (iAs) is a human urinary bladder, skin and lung carcinogen. iAs is metabolized to methylated arsenicals, with trivalent arsenicals more cytotoxic than pentavalent forms in vitro. In this study, cytotoxicity and gene expression changes for arsenite (iAs(III)), monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) were evaluated in three human cell types, urothelial (1T1), keratinocyte (HEK001) and bronchial epithelial (HBE) cells, corresponding to target organs for iAs-induced cancer. Cells were exposed to arsenicals to determine cytotoxicity and to study gene expression changes. Affymetrix chips were used to determine differentially expressed genes (DEGs) by statistical analysis. Lethal concentrations (LC50) for trivalent arsenicals in all cells ranged from 1.6 to 10μM. MMA(III) and DMA(III) had 4-12-fold greater potency compared to iAs. Increasing concentrations of iAs(III) induced more genes and additional signaling pathways in HBE cells. At equivalent cytotoxic concentrations, greater numbers of DEGs were induced in 1T1 cells compared to the other cells. Each arsenical altered slightly different signaling pathways within and between cell types, but when altered pathways from all three arsenicals were combined, they were similar between cell types. The major signaling pathways altered included NRF2-mediated stress response, interferon, p53, cell cycle regulation and lipid peroxidation. These results show a similar process qualitatively and quantitatively for all three cell types, and support a mode of action involving cytotoxicity and regenerative proliferation.

Inorganic arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. 2,3-Dimercaptopropane-1-sulfonic acid (DMPS), a chelating agent, is capable of reducing pentavalent arsenicals to the trivalent state and binding to the trivalent species, and it has been used in the treatment of heavy metal poisoning in humans. Therefore, we investigated the ability of DMPS to inhibit the cytotoxicity and regenerative urothelial cell proliferation induced by arsenate administration in vivo. Female rats were treated for 4 weeks with 100 ppm AsV. DMPS (2800 ppm) co-administered in the diet significantly reduced the AsV-induced cytotoxicity of superficial cells detected by scanning electron microscopy (SEM), and the incidence of simple hyperplasia observed by light microscopy and the bromodeoxyuridine (BrdU) labeling index. It also reduced the total concentration of arsenicals in the urine and the methylation of arsenic. There were no differences in oxidative stress as assessed by immunohistochemical staining for 8-hydroxy-2′-deoxyguanosine (8OHdG) of the bladder urothelium. No differences were detected in urine sediments between groups. These data suggest that DMPS has the ability to inhibit both arsenate-induced acute toxicity and regenerative proliferation of the rat bladder epithelium, most likely by decreasing exposure of the urothelium to trivalent arsenicals excreted in the urine. These data provide additional evidence that the effects of arsenate exposure in vivo do not appear to be related to oxidative effects on dG in DNA.

The PPARγ agonist troglitazone (TG) induced an increased incidence of hemangiosarcomas in mice but was not carcinogenic in rats. In contrast, pioglitazone (PIO) did not induce hemangiosarcomas or any other tumors in mice. We previously demonstrated that TG increased the proliferation of endothelial cells (ECs) in liver and adipose tissue in mice, and acted as a mitogenic stimulant and an inhibitor of apoptosis in vitro in mouse, but not human, ECs. In the present study, we investigated whether TG had any effect on the proliferation of ECs in rats. We also evaluated the in vivo and in vitro effects of PIO on ECs in mice. In rats, TG did not increase the Ki-67 labeling index (LI) of ECs in liver or adipose tissue at doses used in the two-year bioassay, and did not increase hepatocyte proliferation. PIO administered to mice did not increase the Ki-67 LI of hepatocytes or ECs in liver or white adipose tissue, but slightly increased the EC proliferation in brown adipose tissue. PIO was slightly mitogenic on cultured mouse ECs after 3 days of treatment but not after 6 days, and there was no inhibition of apoptosis, in contrast to what was seen with TG. The data support the conclusion that sustained EC proliferation in mice is necessary, for the induction of hemangiosarcomas by TG, and these short-term and long-term effects are not seen with TG in the rat or with PIO in mice, treatments that also are not related to the induction of hemangiosarcomas in two-year bioassays.

Arsenite (As(III)), an inorganic arsenical, is a known human carcinogen, inducing tumors of the skin, urinary bladder and lung. It is metabolized to organic methylated arsenicals. Oxidative stress has been suggested as a mechanism for arsenic-induced carcinogenesis. Reactive oxygen species (ROS) can be important factors for carcinogenesis and tumor progression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is known to produce intracellular ROS, therefore, we investigated the ability of apocynin (acetovanillone), an NADPH oxidase inhibitor, to inhibit the cytotoxicity and regenerative cell proliferation of arsenic in vitro and in vivo. Apocynin had similar effects in reducing the cytotoxicity of As(III) and dimethylarsinous acid (DMA(III)) in rat urothelial cells in vitro. When tested at the same concentrations as apocynin, other antioxidants, such as l-ascorbate and N-acetylcysteine, did not inhibit As(III)-induced cytotoxicity but they were more effective at inhibiting DMA(III)-induced cytotoxicity compared with apocynin. In vivo, female rats were treated for 3 weeks with 100ppm As(III). Immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) showed that apocynin reduced oxidative stress partially induced by As(III) treatment on rat urothelium, and significantly reduced the cytotoxicity of superficial cells detected by scanning electron microscopy (SEM). However, based on the incidence of simple hyperplasia and the bromodeoxyuridine (BrdU) labeling index, apocynin did not inhibit As(III)-induced urothelial cell proliferation. These data suggest that the NADPH oxidase inhibitor, apocynin, may have the ability to partially inhibit arsenic-induced oxidative stress and cytotoxicity of the rat bladder epithelium in vitro and in vivo. However, apocynin did not inhibit the regenerative cell proliferation induced by arsenite in a short-term study.