Arsenic Impairs Wound Healing Processes in Dermal Fibroblasts and Mice.

Inorganic arsenic (NaAsO2) is a naturally occurring metalloid found in water resources globally and in the United States at concentrations exceeding the U.S. Environmental Protection Agency Maximum Contamination Level of 10 ppb. While exposure to arsenic has been linked to cancer, cardiovascular disease, and skin lesions, the impact of arsenic exposure on wound healing is not fully understood. Cultured dermal fibroblasts exposed to NaAsO2 displayed reduced migration (scratch closure), proliferation, and viability with a lowest observable effect level (LOEL) of 10 µM NaAsO2 following 24 h exposure. An enrichment of Matrix Metalloproteinase 1 (MMP1) transcripts was observed at a LOEL of 1 µM NaAsO2 and 24 h exposure. In vivo, C57BL/6 mice were exposed to 10 µM NaAsO2 in their drinking water for eight weeks, then subjected to two full thickness dorsal wounds. Wounds were evaluated for closure after 6 days. Female mice displayed a significant reduction in wound closure and higher erythema levels, while males showed no effects. Gene expression analysis from skin excised from the wound site revealed significant enrichment in Arsenic 3-Methyltransferase (As3mt) and Estrogen Receptor 2 (Esr2) mRNA in the skin of female mice. These results indicate that arsenic at environmentally relevant concentrations may negatively impact wound healing processes in a sex-specific manner.

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration.

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into changes in clinical standards used for treating wounds and decreasing morbidity and mortality for patients. Wound healing is a complex process that requires strategic cell and tissue interaction and function. One of the many critically important functions of wound healing is individual and collective cellular migration. Upon injury, various cells from the blood, surrounding connective, and epithelial tissues rapidly migrate to the wound site by way of chemical and/or physical stimuli. This migration response can largely dictate the outcomes and success of a healing wound. Understanding this specific cellular function is important for translational medicine that can lead to improved wound healing outcomes. Here, we describe a protocol used to better understand cellular migration as it pertains to wound healing, and how changes to the cellular environment can significantly alter this process. In this example study, dermal fibroblasts were grown in media supplemented with fetal bovine serum (FBS) as monolayer cultures in tissue culture flasks. Cells were aseptically transferred into tissue culture treated 12-well plates and grown to 100% confluence. Upon reaching confluence, the cells in the monolayer were vertically scratched using a p200 pipet tip. Arsenic diluted in culture media supplemented with FBS was added to individual wells at environmentally relevant doses ranging 0.1-10 M. Images were captured every 4 hours (h) over a 24 h period using an inverted light microscope to observe cellular migration (wound closure). Images were individually analyzed using image analysis software, and percent wound closure was calculated. Results demonstrate that arsenic slows down wound healing. This technique provides a rapid and inexpensive first screen for evaluation of the effects of contaminants on wound healing.

Estrogen Mitigates the Negative Effects of Arsenic Contamination in an In Vitro Wound Model.

Arsenic, a naturally occurring environmental contaminant, is harmful to humans at elevated concentrations. Increased levels of arsenic in the environment occur as a result of human activities and from natural geologically sourced leaching into ground and surface water. These sources pose an exposure risk above the USEPA standard to individuals whose food and water sources become contaminated. Arsenic exposure negatively impacts organ function and increases the risk for developing pathologies, including cancer. Some of the effects of arsenic on cancer translate to normal cell function in wound healing. To evaluate whether arsenic influences wound healing, an in vitro scratch assay was employed to study the effects of arsenic on cellular migration, which is a key component in the normal wound-healing process. In this study, skin cells were exposed to environmentally relevant concentrations of arsenic, and wound closure was evaluated. Results indicated that arsenic significantly decreased the rate of cellular migration in the scratch assay when compared with controls. In addition, estradiol, which has been shown to positively influence cellular and tissue processes involved in wound healing, reversed the slowing effects of arsenic on wound closure. These results suggest that arsenic contamination may inhibit, and estrogen may provide a therapeutic benefit for individuals with arsenic-contaminated wounds.

A Bench-Top In Vitro Wound Assay to Demonstrate the Effects of Platelet-Rich Plasma and Depleted Uranium on Dermal Fibroblast Migration.

Cellular migration assays are useful tools to investigate physiologic events on the bench top. Furthermore, this migration assay can be utilized to investigate wound healing therapeutics (those that encourage or accelerate wound closure) as well as deleterious agents (ones that mitigate or slow wound closure). The current study used an in vitro scratch assay to measure the effects of platelet-rich plasma (PRP) and depleted uranium (DU) in the form of uranyl acetate on cellular migration of human neonatal dermal fibroblasts in an in vitro simulation of wound healing. Data analyses included percent wound closure measured as the distance between cell margins, and rates of wound closure versus untreated controls. The highest doses of PRP (0.063, 0.125%) resulted in 50-65% wound closure after 4-8 hours relative to 38-44% in controls and the low-dose treatment group (0.031%). The high-dose treatments of PRP (0.125, 0.063%) reached 100% wound closure at 12 hours postwound versus 16 hours for controls and the low-dose treatment group (0.031%). Conversely, the higher doses of DU treatments (50 and 100 µM) resulted in <80% closure versus 100% closure in controls after 16 hours, with full closure observed at 20 hours. The highest dose of DU (1,000 µM) resulted in <20% closure versus 100% closure in controls after 16 hours. The use of the described scratch assay serves as a translatable bench-top model that has the potential to predict in vivo outcomes, and in many early studies can help to demonstrate proof-of-concept before moving into complex biological systems.