Gene expression of normal human epidermal keratinocytes modulated by trivalent arsenicals

Abstract

Chronic exposure to inorganic arsenic (iAs) is associated with the development of benign and malignant human skin lesions including nonmelanoma skin cancers. The precise arsenical form(s) responsible for this carcinogenic effect are unknown, although trivalent inorganic arsenic (iAs(III)) and two of its toxic metabolites, monomethylarsonous acid (MMA(III)) and methylarsinous acid (DMA(III)), are attractive candidates. In an effort to better understand and compare their toxic effects in the skin, we compared the global gene expression profiles of normal human epidermal keratinocytes (NHEKs) exposed to varying noncytotoxic/slightly cytotoxic concentrations of iAs(III), MMA(III), and DMA(III) for 24 h. Exposure to each arsenical treatment group exhibited a dose effect in the number of altered genes and the magnitude of expression change in NHEKs. The most significant gene expression changes associated with iAs(III) and MMA(III) exposure were consistent with several key events believed to be important to As-driven skin carcinogenesis, namely induction of oxidative stress, increased transcript levels of keratinocyte growth factors, and modulation of MAPK and NF-κB pathways. At both comparable arsenical concentrations and comparable NHEK toxicity, greater potential carcinogenic effects were observed in MMA(III)-exposed NHEKs than those exposed to iAs(III), including involvement of more proinflammatory signals and increased transcript levels of more growth factor genes. In contrast, none of these above-mentioned transcriptional trends were among the most significantly altered functions in the DMA(III) treatment group. This study suggests the relative capacity of each of the tested arsenicals to drive suspected key events in As-mediated skin carcinogenesis is MMA(III) > iAs(III) with little contribution from DMA(III).

Arsenic Induces p62 Expression to Form a Positive Feedback Loop with Nrf2 in Human Epidermal Keratinocytes: Implications for Preventing Arsenic-Induced Skin Cancer

“…Cells were treated with vehicle control or arsenic as described previously [8]. Arsenic concentrations of 4 and 8 μM were used since previous studies in keratinocytes and other cells indicated arsenic treatment in this dose range is relatively non-cytotoxic and induces signaling modulation consistent with arsenic induced skin carcinogenesis including oxidative stress and autophagy or p62 signaling [8,24,26,38]. …”

“…Cells positive for annexin V-fluorescein isothiocyanate (apoptotic cells) were quantified by flow cytometry using a BD Calibur (BD Biosciences, San Jose, CA, USA). Twenty-five-micromolar arsenic was used since similar concentrations have been indicated for arsenic-induced cytotoxicity in previous studies using in vitro models including keratinocytes [26,38,47]. …”

“…The CCK-8 analysis was performed following the manufacturer’s protocol. Twenty-micromolar arsenic was used since similar concentrations have been indicated for arsenic’s effect on viability in previous studies using in vitro models including keratinocytes [26,38,47]. …”

Radical Model of Arsenic(III) Toxicity: Theoretical and EPR Spin Trapping Studies

“…83 After analyzing trends in cell proliferation, apoptosis, and the expression of genes promoting cellular oxidative defense, apoptosis factors and growth factors, the study's authors concluded that MMA III was the most cytotoxic species (LC 50 = 2.8µM) , followed by DMA III (LC 50 = 5.6µM), and then iAs III (LC 50 = 17µM). In human bladder cancer cells, treatment with DMA III or DMMTA V (dimethylmonothioarsinic acid) drastically reduced GSH and p53 levels compared to control cells, while iAs also decreased p53 it was not as drastic as DMA III and treatment actually increased GSH levels compared to control.…”

Involvement of mtDNA Damage Elicited by Oxidative Stress in the Arsenical Skin Cancers

“…Although the in vitro experiments took place in a short period of time, the observed increase of ROS and antioxidant enzymes in arsenical cancers, but not in non-arsenical cancers, supports the notion that ROS has, at least in part, a role in the initiation and progression of arsenic carcinogenesis. A microarray analysis from arsenictreated keratinocytes also demonstrated that many genes involved in antioxidant responses, including glutathionerelated proteins (GCLC, GCLM, and SLC7A11), thioredoxin (TXNRD1), and the NADPH-dependent protein (PGD), were upregulated (Bailey et al, 2010). Recent studies also showed that selective knockdown of antioxidant enzymes significantly sensitized keratinocytes to arsenic cytotoxicity (Zhao et al, 2012).…”