A number of epidemiological studies have found that persistent exposure of Arsenic to the human population can lead to different types of cancers including that of bladder, lung, skin, liver, and kidney. Even though the mechanism of action of Arsenic toxicity is not well understood, oxidative stress caused by Arsenic exposure is predicted to be a contributing factor.
The levels of Reactive oxygen species (ROS) play a key role in normal cell signaling and its alteration can result in aberrant expression of genes that are activated by redox mechanisms.
Notably, genes associated with redox mechanisms include those regulating cellular proliferation, differentiation, and apoptosis. ROS generation can also influence cell proliferation by deregulating the expression of cell cycle proteins like NF-kB, c-Myc, and HO-1. Arsenite can lead to caspase activation and cell cycle alteration in MCF-7 cells thereby inducing apoptosis. The consequences of ROS production can further lead to DNA damage which typically involves the conversion of 2-deoxyguanine to 8-hydroxyl-2-deoxyguanine (8-OHdG) and has been used as a marker of DNA oxidative stress. Therefore increased production of ROS can lead to the development of tumors and cancerous processes.
In humans, arsenic is known to target the liver and its exposure can cause the development of liver lesions, fatty infiltration, and hepato-cellular carcinomas. Hepato-cellular lesions including neoplasia were reported in mice after repeated injections of Arsenate. In addition to this, organic arsenic exposure to pregnant mice caused high occurrence of hepatocellular carcinomas in adult offspring. Therefore we can say that Arsenic has the potential to induce liver carcinogenesis in both human and non-human species.
In mammalian liver, Arsenic methylation by an incompletely characterized methyltransferase using S adenosylmethionine (SAM) as a methyl donor to MMA and DMA occurs at a high level. This can lead to depletion of SAM in normal cellular reactions. SAM is a global methyl donor, required for DNA methylations and its depletion can lead to hypomethylation of DNA resulting in alteration of gene expression like c myc, c met, cyclin D1 and induction of carcinogenesis.
DNA methylation is an epigenetic modification that plays an important role in controlling the expression of various genes. Methylation generally occurs at cytosine residues located in symmetrical CpG nucleotide sequences and its alteration, both in the global and regional levels has been associated with oncogenesis. Methylation of CpG islands in the promoter region suppresses gene expression, as 5-methylcytosine interferes with the binding of transcription factors or other DNA-binding proteins causing reduced transcription. On the other hand, promoter hypomethylation causes over-expression of associated genes. Therefore aberrant DNA methylation could be an underlying epigenetic mechanism that causes altered gene expression that contributes towards the formation of liver cancers. Therefore to summarize, chronic arsenic exposure induces hepatic DNA hypomethylation, which can potentially lead to aberrant gene expression and oncogenic growth in the liver, therefore suggesting a plausible mechanism of hepatocarcinogenesis.
Estrogens are considered to be liver carcinogens in rodents and are suspected to cause carcinogenesis in humans. Evidence suggests that they cause hepatocellular proliferation and aberrant mitogenesis through ER-mediated mechanisms in addition to the proposal that they confer epigenetic modifications. Moreover, arsenic exposure is reported to cause hypomethylation of ER-a promoter region and ER-a over-expression along with the associated formation of proliferative lesions and hepatocellular carcinogenesis. Therefore chronic arsenic exposure causes overexpression of ER-a creating hypersensitivity of hepatic cells to endogenous steroids.
As evidenced by microarray analysis, various cell cycle regulating genes like cyclin D1, cyclin D2, and cyclin D3 were over-expressed by arsenic. Liver cells that acquired malignant properties upon arsenic treatment also showed cyclin D1 over-expression. In addition, this over-expression had a direct effect on the observed malignant transformation as selective cyclin D1 overexpression in the liver was sufficient enough to initiate hepatocellular carcinogenesis. Cyclin D1 can, therefore, be considered as a hepatic oncogene.
Cyclin D1 is also known to be upregulated transcriptionally by various growth factors which potentially includes estrogens. In estrogen-responsive tissues like the liver and uterus, proliferative lesions and co-overexpression of ER-a and cyclin D1 after chronic arsenic exposure is reported. Cyclin D1 activation by arsenic may be a secondary effect to ER-a over-expression as cyclin D1 is potentially an ER-a-linked gene. Therefore we can expect that aberrant expression of cyclin D1 along with that of other oncogenes leads to carcinogenic transformation. Altogether, cyclin D1 overexpression was seen upon arsenic exposure in multiple in vitro and in vivo model systems of arsenic carcinogenesis, which includes skin and bladder cancers in rodents. Thus, under conditions of arsenic-induced carcinogenesis, over-expression of cyclin D1 is observed consistently.
Glutathione and other aminothiols such as cysteine and cysteamine comprise the nonprotein sulfhydryls (NPSCs) in a cell and have significant free radical scavenging abilities. Therefore depletion of intracellular glutathione levels is known have an effect on arsenic mutagenesis.
Studies have shown that pre-treatment of cells with buthionine sulphoximine (BSO), which inhibits the biosynthesis of glutathione reduces NPSHs levels in the cell and enhances both the cytotoxicity and mutagenicity of arsenic. In contrast, glutathione and cysteine pre-treatment are capable of protecting mammalian cells against the toxic effects of arsenite.
In a similar way, various antioxidants also have a significant effect on arsenic-induced genotoxicity. The balance between the rate of generation of free radicals and the rate of their removal by various antioxidant enzymes such as superoxide dismutase and catalase dictates the deleterious effect of oxidative stress. Enzymes like superoxide dismutase and catalase are capable of partially suppressing both the toxicity and the mutagenic potential of sodium arsenite by catalyzing the dismutation of superoxide anions and by preventing the formation of hydroxyl radicals via removal of hydrogen peroxide respectively. On the other hand, no protection was offered by heat-inactivated catalase treatment.
Therefore catalase and SOD are capable of reducing the mutagenic potential of arsenic. This is also consistent with other data obtained including the ability of sodium arsenite to induce heme oxygenase, an oxidative stress protein, and peroxidase in various human cell lines. Moreover, the arsenite-induced occurrence of sister chromatid exchanges was reduced by antioxidant enzymes such as SOD in cultured human lymphocytes.
It is known that arsenic induces hydrogen peroxide which is a precursor of hydroxyl radicals in AL cells. Even though SOD (30 kDa) and catalase (250 kDa) cannot diffuse across the membrane without being phagocytosed due to the relatively large size, hydrogen peroxide can. Even though the exact pathway is not known, the addition of extracellular antioxidants can reduce the intracellular oxidative stress induced by arsenite treatment and suppress the mutagenicity of arsenic in mammalian cells and reduced subsequent genotoxic damage.
Arsenic was capable of inducing specific DNA lesions consistent with oxidative damage like 8-hydroxy-2V-deoxyguanosine (8-OHdG) generation. Arsenic treatment (4 Ag/ml for 24 h) was shown to increases the level of 8-OHdG in AL cells by more than 2-fold when compared to non-treated controls as evidenced by antibody staining. Moreover, the addition of SOD and catalase was capable of reducing this increase by 75%. Moreover, 8-OHdG has also been detected in the skin of patients with arsenic-related Bowen’s disease and in the liver of rats exposed to DMAV. These results indicate that ROS generation is a major pathway for Arsenic-mediated genotoxicity in mammalian cells.
In mouse lung tissue, reduced expression of proteins associated with cellular migration was observed when exposed to low dose of arsenic, as evidenced by high-throughput protein screening experiments. In addition to this alteration of a specific wound repair protein, marker was also observed in mouse bronchoalveolar fluid. On lung tissue of mice fed low-dose arsenic, changes in extracellular matrix (ECM) protein expression and a large increase in matrix metalloproteinase (MMP)-9 expression was revealed as seen in microarray experiments. MMPs are responsible for ECM degradation among other proteolysis. MMP-9 is the most prominently studied MMP in the lung and has been associated with a variety of lung diseases. An increase in the ratio of MMP-9 to tissue inhibitor of matrix metalloproteinase (TIMP)-1 in collected sputum samples of humans were observed under low-level arsenic exposure. This imbalance between MMP-9 and TIMP-1 can cause changes in epithelial wound response, thereby contributing to the progression of airway remodeling. Altered wound response is partly due to increased secretion and activity, as arsenic concentration increases. Moreover, an increase in arsenic concentration inhibits the ability for 16HBE14o- cells to repair monolayers in culture. To conclude, arsenic is capable of causing or exacerbating lung diseases by directly affecting signaling pathways involved in cell migration, and remodeling of the airway. Therefore arsenic ingestion may alter wound response and specifically, MMP-9/TIMP-1 ratios in the lung.
Following scrapewounds of monolayer cultures, Arsenic (30–290 ppb) was capable of inhibiting the reformation of the epithelial monolayer. An increase in activity and expression ofMMP-9 without increases of TIMP-1 protein expression was also observed along with this alteration in wound repair. Furthermore, an improvement in epithelial cell wound repair response was seen after inhibition of MMP-9 even though the cells were exposed to 290 ppb arsenic. To conclude, arsenic is capable of altering the airway epithelial barrier as arsenic induced increase in MMP-9/TIMP-1 ratio in lung epithelial cells can restrict proper wound repair.
MMP-9 expression has been associated with airway epithelial wound repair in primary cultured cells and in vivo. Moreover, neutralizing MMP-9 using antibodies inhibited migration of HRECs indicating that MMP-9 was important in cell migration during respiratory wound repair. Moreover, MMP-9 expression directly coincided with the speed of migration in HBECs. Therefore, MMP-9 is normally upregulated in cells near the wound edge and in the presence of arsenic, dysregulated wound repair was observed due to MMP-9 overexpression in airway epithelial cells.
In the fibroblast model, alterations in focal adhesion kinases, without a significant effect on actin cytoskeleton rearrangements were observed at about 200 ppb arsenite. This resulted in altered cell migration independent of MMP-9. There are also contrasting reports showing that 750 ppb arsenic of unknown form alters actin cytoskeleton and can lead to superoxide production and limit cell migration in an endothelial cell line. In contrast with observations that arsenite can inhibit migration,there are also reports that show arsenic (37.5–375 ppb) in the form of arsenic trioxide (As2O3) can reduce carcinogenic cell invasiveness in carcinogenic cell lines in part by downregulating MMP-9 (15, 46, 53)