Early molecular changes in arsenic exposed human urothelial cells depending on cellular uptake and biotransformation

Millions of people are highly exposed to arsenic, especially via contaminated drinking water. Once entered the human body, arsenic underlies (hepatic) metabolism leading to various methylated species, until it is either accumulated in skin and other tissues, or finally excreted via the renal pathway. Common health consequences are, among others, hyperkeratosis, vascular diseases, and cancer. Especially environmentally relevant doses of arsenic exhibit a highly carcinogenic potential. Arsenic-induced lung cancer, as well as skin, kidney, and bladder cancer have been reported. Bladder cancer in general was reported to occur very frequently with a high recurrence rate; it is in fifth place of the most commonly diagnosed cancer diseases and on second place of those observed in the urogenital tract (Zimbardi et al., 2012). Thus, this study was conducted to focus on the mechanisms of arsenic-induced bladder cancer. Since carcinogenesis is a very complex process with highly species- and tissue-specific mechanisms, an in vitro test system was selected to assay molecular mechanisms during arsenic-induced bladder carcinognesis, taking advantage of an isolated cellular system instead of the complexicity of a whole living organism. The aim of the study was to give an overview over some of the most relevant key events during arsenic-induced carcinogenesis, occurring as a consequence of short-term and chronic low-dose exposure by consolidating interdisciplinary research. To better understand the underlying mechanisms of arsenic carcinogenicity, studies were carried out to correlate arsenic metabolism and genotypic effects with epigenetic modifications and phenotypical alterations under chronic exposure conditions up to 90 weeks. Hence, it was important to detect and analyse intracellular arsenic species and their metabolic products. Therefore, the cellular uptake of arsenic species in non-methylating human urothelial cells (T24) in comparison to methylating human hepatic cells (HepG2) was investigated, and the intracellularly detected arsenic was speciated and quantified. Arsenic-induced genotoxic effects in T24 cells were measured by means of the Alkaline Comet Assay, and the malignant transformation after chronic arsenic treatment up to 90 weeks was assayed using the in vitro Mammalian Cell Transformation Assay, the Colony Formation Assay, and the Migration and Invasion Assay. MicroRNA analysis and examination of altered DNA methylation patterns were carried out to determine the arsenic-induced modulation of epigenetic regulation systems. Moreover, COX-2 protein, which is frequently reported to be increased in malignant tissues and therefore considerd to be an important molecula marker, was analysed. Since the various arsenic species emerging from the hepatic biotransformation of arsenic exhibit distinctly different toxicity, this study focussed especially on effects induced by the methylated metabolite monomethylarsonous acid (MMA(III)), which was recently shown to be one of the most cyto-and genotoxic metabolites. The present study aims to enhance the understanding of arsenic-induced toxicity and carcinogenesis in the urinary bladder epithelium. The experiments conducted within the present study revealed a rapid cellular uptake of MMA(III) in both T24 and HepG2 cells, followed by subsequent conjugation to proteins and other cellular structures. While in HepG2 cells MMA(III) was further methylated to dimethylated arsenic due to the metabolic capacity of these cells, no such biotransformation was observed in T24 cells. Nevertheless, in both cellular systems oxidation to pentavalent species was observed, leading to the assumption that autophagy of affected cellular structures by utilising oxidative processes occurred. This was further confirmed by the presence of unconjugated trivalent arsenic species, but to a minor degree. Genotoxicity of the trivalent arsenic species, as well as arsenate was detected already after 30 min of exposure. Longer exposure durations revealed cytotoxicity, overlapping genotoxic effects. Genotoxicity was observed to be not only dependent on the oxidation state, but also on the methylation state, as genotoxicity of trivalent arsenic species increased with the number of methyl groups. As a consequence and due to their biomethylation capacity, HepG2 cells appeared to be more sensitive towards the genotoxic potential of arsenic species when compared to T24 cells. Additionally, the study revealed that primary cells such as human urothelial epithelial cells (HUEPC) are more susceptible to arsenic-induced genotoxicity than permanent cell lines. Furthermore, to investigate whether MMA(III) is only a genotoxic carcinogen or if it also exhibits non-genotoxic molecular changes, the epigenetic effects on T24 cells after chronic low-dose treatment were investigated. Key events such as modification of DNA methylation and miRNA profiles, as well as COX-2 protein activation were analysed. The experiment revealed that in T24 cells, under the chosen test conditions, exposure to MMA(III) led to the alteration of miR-15a, -19a, 19b, 30a-3p, -126, -128a, -139-5p, -146a, -200a, and -429; which are known to correlate with the occurrence of cancer diseases (Gregory et al., 2008; Hurst et al., 2009; Ichimi et al., 2009; Lin te al., 2009). This indicates that altered miRNA profiles might play a pivotal role in MMA(III)-induced further malignant transformation of T24 cells, which was phenotypically verified in experiments described below. In contrast, altered DNA methylation was only observed for C1QTNF6 and CDH1. Nevertheless, since T24 cells are transitional cell carcinoma cells of the urinary bladder, they show a distinctly altered DNA methylation background. Hence, it cannot conclusively be excluded that those changes overlay the effects caused by MMA(III) exposure. Elevated COX-2 protein levels were observed throughout the chronic low-dose exposure to MMA(III). It can be assumed from the results that the regulation of the COX-2 protein is not accomplished by the amount of mRNA, but presumably by the interaction of COX-2 mRNA with miR-26a and -26b. Further experiments are necessary to confirm this and to unveil the respective mechanism. Among the molecular effects of chronic low-dose exposure to MMA(III) also the development of altered phenotypic appearance was investigated. The determination of the in vitro Mammalian Cell Transformation Test, the Colony Formation Assay, and the Migration and Invasion Assay revealed further malignant transformatioin of T24 cells after long-term exposure to MMA(III). In addition to morphological changes, the loss of contact inhibition and anchorage dependent growth were observed. Moreover, T24 cells chronically treated with MMA(III) developed the ability of migration and invasion, which can be concluded to be the basis for metastasizing properties in vivo. In summary, MMA(III) was shown to be rapidly taken up and to exhibit both genotoxicity, as well as tumour progression (i.e. non-genotoxic carcinogenic activities such as COX-2 protein activation, altered miRNA expression profiles, and altered DNA methylation), which resulted in a further malignant transformation of T24 carcinoma cells after chronic low-dose exposure up to 90 weeks. This study indicated various molecular modes of action of MMA(III) in vitro, which only could be analysed and correlated to each other by consolidating interdisciplinary research. Nevertheless, the results must be further confirmed in vivo to be conclusively correlated to the MMA(III)-induced development of bladder cancer.