Identification of cellular biomarkers induced by light sensitive chemicals in an in vitro model

Abstract

This study aims to develop novel in vitro methodology, currently unavailable, to discriminate between photoirritant and photoallergic reactions induced by chemical compounds with established evidence of photoirritation and/or photoallergic activity. The proposed approach is based on the quantification of specific biomarkers release using ELISA. Analyzed biomarkers were the interleukin-6 (IL-6), interleukin-8 (IL-8), and matrix metalloproteinase-1 (MMP-1), which are known to play key roles in inflammatory and tissue-remodeling responses in the skin. To achieve this objective and to ensure the reliability of the selected biomarkers, two experimental human cell lines were employed and evaluated under irradiated and non-irradiated conditions. The HaCaT immortalized human keratinocyte cell line was used as a dermal model, while the THP-1 human monocytic cell line mimic cutaneous dendritic cells and the immune-related responses involved in photoirritation and photoallergic mechanisms. Five chemical compounds with well-known phototoxic reactions were analyzed: as photoirritant and photoallergic was used chlorpromazine (CPZ), as non-phototoxic lactic acid (LA), as photoallergic 6-methylcoumarin (6-MC) and ketoprofen (KP) and as photoirritant retinoic acid (RA). Prior to biomarker evaluation, cell viability was assessed using LDH release and MTT reduction assays in both cell lines following exposure to each compound in irradiated and non-irradiated conditions. These preliminary assays ensured the use of subtoxic concentrations in subsequent ELISA experiments to detect the levels of the biomarkers proposed, thereby preventing cytotoxicity-related interference and enabling an accurate interpretation of biomarker modulation. Results showed that MMP-1 does not appear to be a suitable biomarker for identifying or discriminating photoirritant or photoallergenic compounds in HaCaT cells, as no significant differences were observed between irradiated and non-irradiated conditions. In addition, no notable differences were detected in the secretion of IL-6 in HaCaT cells for any of the tested compounds, further limiting its usefulness as a discriminative biomarker under the experimental conditions employed. In the case of IL-8, results showed that IL-8 cannot be considered a reliable biomarker in THP-1 cells at low lactic acid concentrations within the tested range. Under irradiated conditions, these concentrations reduced the expression of IL-8, while under non-irradiated conditions, no significant differences were observed. Additionally, when attempting to perform the LDH cytotoxicity assay with the same compound in THP-1 cells, the assay could not be successfully executed because the acidic environment interfered with the method.