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Efflux Transporters Regulate Arsenite-Induced Genotoxicity in Double Negative and Double Positive T Cells

Huan Xu¹, Sebastian Medina¹, Fredine T Lauer¹

¹Department of Pharmaceutical Sciences, The University of New Mexico College of Pharmacy, Albuquerque, New Mexico 87131.

Toxicological Sciences : an Official Journal of the Society of Toxicology

|May 5, 2017

Abstract:

Arsenite (As+3) exposure is known to cause immunotoxicity in human and animal models. Our previous studies demonstrated that As+3 at 50-500 nM concentrations induced both genotoxicity and nongenotoxicity in mouse thymus cells. Developing T cells at CD4-CD8- double negative (DN) stage, the first stage after early T cells are transported from bone marrow to thymus, were found to be more sensitive to As+3 toxicity than the T cells at CD4 + CD8 + double positive (DP) stage in vitro. Induction of Mdr1 (Abcb1) and Mrp1 (Abcc1), 2 multidrug resistance transporters and exporters of As+3, was associated with the reversal of As+3-induced double strand breaks and DNA damage. In order to confirm that the thymus cell populations have different sensitivity to As+3in vivo, male C57BL/6J mice were exposed to 0, 100, and 500 ppb As+3 in drinking water for 30 d. A significant decrease in DN cell percentage was observed with exposure to 500 ppb As+3. Low to moderate concentrations of As+3 were shown to induce higher genotoxicity in sorted DN than DP cells in vitro. Calcein AM uptake and Mdr1/Mrp1 mRNA quantification results revealed that DN cells not only had limited As+3 exporter activity, but also lacked the ability to activate these exporters with As+3 treatments, resulting in a higher accumulation of intracellular As+3. Knockdown study of As+3 exporters in the DN thymic cell line, D1 using siRNA, demonstrated that Mdr1 and Mrp1 regulate intracellular As+3 accumulation and genotoxicity. Taken together, the results indicate that transporter regulation is an important mechanism for differential genotoxicity induced by As+3 in thymocytes at different developmental stages.

Insights

Arsenite exposure harms developing T cells. Double negative (DN) T cells are more sensitive to arsenite toxicity due to limited transporter activity, leading to higher DNA damage.

Area of Science:

Immunotoxicology
Environmental Health
Molecular Toxicology

Background:

Arsenite (As+3) exposure is a known immunotoxin.
Previous studies showed As+3 induces genotoxicity and non-genotoxicity in mouse thymus cells.
Developing T cells at the double-negative (DN) stage are more sensitive to As+3 than double-positive (DP) T cells.

Purpose of the Study:

To investigate the in vivo sensitivity of thymus cell populations to As+3.
To elucidate the role of multidrug resistance transporters in differential As+3 genotoxicity.
To understand the mechanism behind heightened As+3 toxicity in DN T cells.

Main Methods:

Exposure of C57BL/6J mice to varying concentrations of As+3 in drinking water for 30 days.
Flow cytometry analysis to assess thymus cell population percentages.
In vitro genotoxicity assays, Calcein AM uptake, and Mdr1/Mrp1 mRNA quantification in sorted DN and DP cells.
siRNA-mediated knockdown of Mdr1 and Mrp1 in a DN thymic cell line.

Main Results:

Significant decrease in DN cell percentage observed with 500 ppb As+3 exposure in vivo.
DN cells exhibited higher genotoxicity than DP cells in vitro.
DN cells demonstrated limited As+3 exporter activity and impaired exporter activation, leading to increased intracellular As+3 accumulation.
Mdr1 and Mrp1 knockdown confirmed their role in regulating intracellular As+3 accumulation and genotoxicity.

Conclusions:

Transporter regulation is a key mechanism underlying differential arsenite-induced genotoxicity in thymocytes at various developmental stages.
DN T cells are particularly vulnerable to arsenite toxicity due to deficient efflux transporter function.
These findings highlight specific cellular mechanisms contributing to arsenite immunotoxicity.

Read full analysis

Keywords:

arsenite differential sensitivity double negative T cells double positive T cells genotoxicity multidrug resistance-associated transporters

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