Ahmed N. Uddin

Arsenite-Induced Alterations of DNA Photodamage Repair and Apoptosis After Solar-Simulation UVR in Mouse Keratinocytes in Vitro

Our laboratory has shown that arsenite markedly increased the cancer rate caused by solar-simulation ultraviolet radiation (UVR) in the hairless mouse skin model. In the present study, we investigated how arsenite affected DNA photodamage repair and apoptosis after solar-simulation UVR in the mouse keratinocyte cell line 291.03C. The keratinocytes were treated with different concentrations of sodium arsenite (0.0, 2.5, 5.0 μM) for 24 hr and then were immediately irradiated with a single dose of 0.30 kJ/m2 UVR. At 24 hr after UVR, DNA photoproducts [cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (6-4PPs)] and apoptosis were measured using the enzyme-linked immunosorbent assay and the two-color TUNEL (terminal deoxynucleotide transferase dUTP nick end labeling) assay, respectively. The results showed that arsenite reduced the repair rate of 6-4PPs by about a factor of 2 at 5.0 μM and had no effect at 2.5 μM. UVR-induced apoptosis at 24 hr was decreased by 22.64% at 2.5 μM arsenite and by 61.90% at 5.0 μM arsenite. Arsenite decreased the UVR-induced caspase-3/7 activity in parallel with the inhibition of apoptosis. Colony survival assays of the 291.03C cells demonstrate a median lethal concentration (LC50) of arsenite of 0.9 μM and a median lethal dose (LD50) of UVR of 0.05 kJ/m2. If the present results are applicable in vivo, inhibition of UVR-induced apoptosis may contribute to arsenite’s enhancement of UVR-induced skin carcinogenesis.

Mechanism of Selenium-Induced Inhibition of Arsenic-Enhanced UVR Carcinogenesis in Mice

Background Hairless mice that ingested arsenite in drinking water exhibited more than a 5-fold enhancement of ultraviolet radiation (UVR) carcinogenesis, whereas arsenite alone was carcinogenically inactive. Dietary organoselenium blocked the cancer enhancement effect of arsenic but not cancer induction by UVR. Objective In this study we sought to explain selenium blockage of As enhancement by establishing the extent that As and Se tissue distributions are coincident or divergent. Methods We used the X-ray fluorescence microprobe at the Advanced Photon Source (Argonne National Laboratory) to probe sections of skin and liver from hairless mice exposed to a) UVR, b) UVR + As, c) UVR + organoselenium, or d) UVR + As + organoselenium. Results We found elevated levels of As in the skin epithelium (hair follicles and epidermis) and diffusely in the liver of mice exposed to UVR + As. Arsenic was entirely absent in skin in mice exposed to UVR + As + organoselenium, but a diffuse low level was seen in the liver. As and Se locations were consistently divergent in skin; As was more diffusely distributed, whereas Se was strongly associated with membranes. X-ray absorption near-edge spectra are consistent with the presence of the seleno-bis(S-glutathionyl) arsinium ion in the liver. Conclusions Supplemental Se was uncommonly effective at preventing even a trace of As in skin at 14 or 196 days of continuous exposure to As in drinking water. Traces of the seleno-bis(S-glutathionyl) arsinium ion in the liver suggested that formation of this compound was more likely to be responsible for the As-blocking effect of Se than was a mechanism based on antioxidation.

Variability in sensitivity to arsenite does not correlate with arsenic accumulation rate in normal human lymphoblasts.

Arsenic is a common environmental contaminant of our air, water and food, but not every individual who drinks arsenic-contaminated water shows clinical signs of toxicity. Large inter-individual variations are also found in arsenite-induced aneuploidy, chromosome aberrations and sister chromatid exchanges in peripheral blood lymphocytes from different human donors. Lymphoblasts are virally immortalized lymphocytes that retain most of the properties of lymphocytes. Individual lymphoblast cell lines retained their arsenite sensitivity after cryopreservation and subsequent revival. We measured the accumulation of 73[As]-arsenite into lymphoblast lines derived from 11 normal individuals. Arsenite accumulation rate varied 6.3 fold between the slowest and the fastest subjects. Assays in 14 lymphoblast lines showed variability to the toxic effects of arsenite, as measured by growth inhibition. Lymphoblast lines also vary with regard to their growth rates, but there is no relationship between growth rate and arsenite sensitivity. Surprisingly, we also found no correlation between arsenite accumulation rate and cellular sensitivity to growth inhibition, suggesting that the arsenite accumulation rate may not be the main determinant of cellular sensitivity to arsenic. We were also unable to detect evidence for a human homolog for the yeast arsenite efflux gene ACR3, using RT-PCR.

d-limonene prevents ultraviolet irradiation: Induced cyclobutane pyrimidine dimers in Skh1 mouse skin

d-limonene prevents ultraviolet irradiation: Induced cyclobutane pyrimidine dimers in Skh1 mouse skin

Abstract 4864: Aroma terpenes prevent UVB-induced sunburn in Skh1 mouse skin.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Understanding how aroma terpenes prevent sunburn and/or skin cancer in mice could lead to more effective and safer ways of blocking sun damage to human skin. In our previous study, β-damascenone, an aroma terpene, protected against sunburn by activating both keratinocyte and sebaceous gland pathways that fortified and thickened the cornified envelope and sebum layers of mouse epidermis. In the current study, the sunburn preventive activity of d-limonene was examined to confirm that these related compounds work similarly. In addition to sunburn, immunohistochemical analyses of proliferation (PCNA) and a damage-response gene N-Myc Down Regulated Gene 1 (NDRG1) were investigated in control versus d-limonene-treated Skh-1 mice. The mice were treated either topically on the dorsum or orally with different concentrations of d-limonene in liponate (100%, 10%, 1% and 0%) followed by exposure of the dorsum to solar simulation ultraviolet radiation (UVR) of 1.5 kJ/m2 from fluorescent ‘sunlamps’. Either 4 topical 5 μL doses or a single dose of 20 μL (0.95 μg/g body weight) of 100% d-limonene provided complete protection from UVR-induced sunburn. The protection was less but still significant at 10% d-limonene but was absent at 1% d-limonene in comparison to UVR alone. PCNA-labeling showed a marked proliferation increase of epidermal basal cells, outer root sheath cells of hair follicles and sebaceous gland cells by separate exposures to d-limonene and UVR relative to controls. The combined treatment of d-limonene followed by UVR produced fewer PCNA-positive cells in epithelial tissue relative to UVR-only mice. The NDRG1 protein (an indirect measure of DNA damage) was overwhelmingly (∼85% of keratinocytes) induced by UVR indicating heavy DNA damage. The UVR-induced NDRG1 index remained at ∼85% at 1% d -limonene, but was significantly reduced at 10% d-limonene to ∼24% and to less than 1% at 100% d -limonene indicating nearly complete elimination of cellular damage as a pure compound; a finding that is consistent with complete sunburn prevention. The thickness of the cornified envelope plus sebum layers nearly doubled within 5 days after administration of the d-limonene corresponding to the time of UVR application. Overall the results indicate that d-limonene protected against sunburn by activating keratinocyte/sebaceous gland-based pathways that fortified and thickened the cornified envelope plus sebum layers on the skin surface by releasing elevated levels of UVR-absorbing proteins that decreased UVR dose to underlying cutaneous tissues. Citation Format: Ahmed N. Uddin, Ivica Labuda, Feng Wu, KamMeng Tchou-Wong, Fredric J. Burns. Aroma terpenes prevent UVB-induced sunburn in Skh1 mouse skin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4864. doi:10.1158/1538-7445.AM2013-4864

Does arsenic require a carcinogenic partner

Publisher Summary Arsenic (As) contamination of drinking water is a worldwide problem. Chronic As exposure is of concern mainly because of its carcinogenic effects. A number of studies have shown synergy between arsenic and other environmental contaminants in the induction of cancers. The increase in cancer risk observed in epidemiological studies is attributed mainly to the presence of inorganic trivalent As. The association between skin cancer and As ingestion in drinking water was seen in studies in Taiwan, Chile, Argentina, and Mexico. The development of an animal model for As-related carcinogenesis now makes it possible to study the molecular mechanism of arsenics carcinogenicity in depth and develop chemopreventive strategies. In vitro studies have led to the hypothesis that arsenite acts as a cocarcinogen by blocking DNA repair and enhancing cell proliferation through unique signaling mechanisms. The blocking of DNA repair may occur via a signaling event that controls DNA repair or may result from the enhanced cell proliferation, thus forcing the cell to replicate damaged DNA.