Immunochemical quantitation of UV-induced oxidative and dimeric DNA damage to human keratinocytes

Polyaromatic hydrocarbons (PAHs) are prevalent, potent carcinogens, and 7,12-dimethylbenz[a]anthracene (DMBA) is a model PAH widely used to study tumorigenesis. Mice lacking Langerhans cells (LCs), a signatory epidermal dendritic cell (DC), are protected from cutaneous chemical carcinogenesis, independent of T cell immunity. Investigation of the underlying mechanism revealed that LC-deficient skin was relatively resistant to DMBA-induced DNA damage. LCs efficiently metabolized DMBA to DMBA-trans-3,4-diol, an intermediate proximal to oncogenic Hras mutation, and DMBA-treated LC-deficient skin contained significantly fewer Hras mutations. Moreover, DMBA-trans-3,4-diol application bypassed tumor resistance in LC-deficient mice. Additionally, the genotoxic impact of DMBA on human keratinocytes was significantly increased by prior incubation with human-derived LC. Thus, tissue-associated DC can enhance chemical carcinogenesis via PAH metabolism, highlighting the complex relation between immune cells and carcinogenesis.

Download Full-text

Opposed arsenite-mediated regulation of p53-survivin is involved in neoplastic transformation, DNA damage, or apoptosis in human keratinocytes

Toxicology ◽

10.1016/j.tox.2012.06.004 ◽

2012 ◽

Vol 300 (3) ◽

pp. 121-131 ◽

Cited By ~ 17

Author(s):

Yuan Li ◽

Rongrong Jiang ◽

Yue Zhao ◽

Yuan Xu ◽

Min Ling ◽

...

Keyword(s):

Dna Damage ◽

Neoplastic Transformation ◽

Human Keratinocytes

Download Full-text

Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2016.06.106 ◽

2016 ◽

Vol 477 (3) ◽

pp. 329-335 ◽

Cited By ~ 28

Author(s):

Tatsuya Hasegawa ◽

Masaya Nakashima ◽

Yoshiharu Suzuki

Keyword(s):

Dna Damage ◽

Nlrp3 Inflammasome ◽

Nuclear Dna ◽

Inflammatory Responses ◽

Human Keratinocytes ◽

Inflammasome Activation ◽

Nlrp3 Inflammasome Activation

Download Full-text

Genotoxic Effect of Arsenate and Arsenite in Human HaCat Cells in Culture Using Comet Assay

Journal of Advances in Medical and Pharmaceutical Sciences ◽

10.9734/jamps/2020/v22i1230211 ◽

2020 ◽

pp. 36-44

Author(s):

Ghazalla Benhusein ◽

Elaine Mutch ◽

Faith M. Williams

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Statistical Significance ◽

Human Keratinocytes ◽

Hacat Cells ◽

Environmental Chemical ◽

Viable Cells ◽

Buthionine Sulphoximine ◽

Ros Formation

Arsenic is an environmental chemical of toxicological concern today since it is a human genotoxin and chronic exposure is associated with development of cancers, including skin. Inorganic arsenate is metabolically reduced to arsenite by glutathione (GSH) prior to methylation. The aim of this study was to determine the relative toxic effects of arsenate and arsenite in HaCat cells (immortalized human keratinocytes) in vitro by measuring cytotoxicity, DNA damage, depletion of glutathione and apoptotic and necrotic events. HaCat cells were treated with arsenate and arsenite (10 μM) for DNA damage detection using Comet assay and cytotoxicity (10, 60 and 100 μM) all measured at 24 hr. In some experiment arsenate or arsenite (10 μM) was added at the same time as BSO 10 μM for 24 hr, and GSH levels were measured by HPLC with fluorescence detection. Flow cytometry was used to investigate apoptotic and necrotic events following arsenate and arsenite (10 μM) treatment for 24 hr. Arsenate and arsenite at 60 and 100 μM, but not 10 μM, reduced the number of adherent viable cells with time. Therefore, DNA damage could only be measured at 10 μM as at higher concentrations the cells did not produce classical Comets but showed fragmentation. DNA damage was significantly (p < 0.001) increased in cells treated for 24 hr with 10 μM arsenate and arsenite compared to control. GSH levels were significantly increased in HaCat cells treated with10 μM arsenate and arsenite (p < 0.05, p < 0.001, respectively) compared to control. Cells treated with buthionine sulphoximine (BSO) at the same time as arsenate had increased GSH levels (p < 0.001), but arsenite and BSO did not increase cellular GSH. Arsenate and arsenite increased apoptosis, and arsenate increased necrosis, although none of the values reached statistical significance. Arsenite was more cytotoxic than arsenate. Arsenate and arsenite are known to produce oxidative stress involving ROS formation and depletion of glutathione. The increase in GSH levels at low doses of arsenate and arsenite, and by arsenate even in the presence of BSO.

Download Full-text