Assessment of Oxidative Stress-Induced Oral Epithelial Toxicity

Abstract

Reactive oxygen species (ROS) are highly reactive molecules generated in living organisms and an excessive production of ROS culminates in oxidative stress and cellular damage. Notably, oxidative stress plays a critical role in the pathogenesis of a number of oral mucosal diseases, including oral mucositis, which remains one of cancer treatments’ most common side effects. We have shown previously that oral keratinocytes are remarkably sensitive to oxidative stress, and this may hinder the development and reproducibility of epithelial cell-based models of oral disease. Here, we examined the oxidative stress signatures that parallel oral toxicity by reproducing the initial events taking place during cancer treatment-induced oral mucositis. We used three oral epithelial cell lines (an immortalized normal human oral keratinocyte cell line, OKF6, and malignant oral keratinocytes, H357 and H400), as well as a mouse model of mucositis. The cells were subjected to increasing oxidative stress by incubation with hydrogen peroxide (H2O2) at concentrations of 100 μM up to 1200 μM, for up to 24 h, and ROS production and real-time kinetics of oxidative stress were investigated using fluorescent dye-based probes. Cell viability was assessed using a trypan blue exclusion assay, a fluorescence-based live–dead assay, and a fluorometric cytotoxicity assay (FCA), while morphological changes were analyzed by means of a phase-contrast inverted microscope. Static and dynamic real-time detection of the redox changes in keratinocytes showed a time-dependent increase of ROS production during oxidative stress-induced epithelial injury. The survival rates of oral epithelial cells were significantly affected after exposure to oxidative stress in a dose- and cell line-dependent manner. Values of TC50 of 800 μM, 800 μM, and 400 μM were reported for H400 cells (54.21 ± 9.04, p < 0.01), H357 cells (53.48 ± 4.01, p < 0.01), and OKF6 cells (48.64 ± 3.09, p < 0.01), respectively. Oxidative stress markers (MPO and MDA) were also significantly increased in oral tissues in our dual mouse model of chemotherapy-induced mucositis. In summary, we characterized and validated an oxidative stress model in human oral keratinocytes and identified optimal experimental conditions for the study of oxidative stress-induced oral epithelial toxicity.

Inflammation Can Be a High-Risk Factor for Mucosal Nonunion of MRONJ by Regulating SIRT1 Signaling When Treated with an Oncologic Dose of Zoledronate

“… 53 It was commonly recognized that elevated oxidative stress was the primary indicator of an inflammatory environment and that oxidative stress was crucial for tissue healing. 54 Given the diverse effects of oxidative stress on keratinocytes reported in various studies, 55–57 we hypothesized that oxidative stress might be a crucial factor linking inflammation to MRONJ pathogenesis. To test this hypothesis, we employed NAC, a synthetic derivative of the endogenous amino acid L-cysteine and a precursor of glutathione.…”

A Curcumin-Based Oral Gel Has Potential Protective Efficacy against Oral Mucositis: In Vitro Study

“…Such a model is not novel and has been adopted and validated in several previous studies [22,28]. The literature includes several in vitro models that employed various approaches to simulate oxidative stress damage, such as ultraviolet (UV) radiation and ionising radiation [29,30]. However, inducing oxidative stress through H 2 O 2 offers several advantages over other models.…”