The World Health Organization defines an adverse drug reaction (ADR) as “a response to a drug that is harmful and unintended and occurs at doses normally used in humans for the prevention, diagnosis, or treatment of disease, or for the modification of physiological function” (Edwards and Aronson, 2000). Cutaneous ADRs are a global problem in the healthcare system, occurring in approximately 10% of hospitalizations and requiring additional medical costs (Al Aboud et al., 2022; Kowalska et al., 2021). One of the most frequently occurring skin ADRs is phototoxicity, which results from the interaction between ultraviolet radiation (UVR) and a photosensitizing drug or its metabolite (Kim et al., 2015). Phototoxicity is considered to be one of the most common nonimmunologic photosensitivity reactions, and its characteristic features include the occurrence of several minutes to a few hours after skin exposure to the radiation, high concentration of used drug, and dose-dependent effect, as well as manifestation after the first exposure to the photosensitizing agent (Kowalska et al., 2021).

The phototoxic reaction mechanism involves the generation of large amounts of reactive oxygen species (ROS), such as hydrogen peroxide, singlet oxygen, and hydroxyl radical, which can induce oxidative stress (OS) in cells (Verrugio et al., 2017). OS is caused by an imbalance between the antioxidant activity of specific enzymes, such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), and the amount of ROS generated (Roy et al., 2017). While ROS play a regulatory and mediator role in cellular signaling, proliferation, migration, differentiation, and apoptosis, they may also cause oxidative damage to important cellular biomolecules, such as lipids, proteins, and nucleic acids, resulting in a cytotoxic effect (Roy et al., 2017; Sies and Jones, 2020). DNA damage induces upregulation of the p53 protein, which results in the inhibition of normal cell proliferation and induction of the apoptosis process. In abnormal conditions, mutations may occur within the gene encoding the p53 protein, which is associated with uncontrolled cell division and carcinogenesis (Kulesza et al., 2019). Enzymatic and structural proteins exposed to ROS may undergo inactivation, aggregation, dimerization, or fragmentation, resulting in the loss of their original properties. Peroxidation of cellular lipids leads to a change in their permeability and fluidity within cell membranes, and additionally, it can cause the formation of lipid-derived radicals, leading to cellular damage (Kowalska et al., 2021). Furthermore, OS and ROS are pathophysiologic basics of many diseases, including cancer, diabetes, cardiovascular and neurodegenerative disorders, and ADRs (Sies and Jones,2020; Sies et al., 2022; Zhang et al., 2022).

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most popular therapeutics, mainly due to their high effectiveness in treating pain, fever, and inflammation (Bindu et al., 2020). NSAIDs make up >5–10% of all therapeutics prescribed each year (Abdu et al., 2020). Their mechanism of action is based on inhibiting cyclooxygenase (COX) activity, which prevents the synthesis of prostaglandins and the development of inflammation (Onder et al., 2004).

Meloxicam (MLX) is a derivative of NSAIDs that belongs to the class of enolic acids (Jianmin and Yunhua, 2016). MLX, as a preferential COX-2 isoenzyme inhibitor, is widely used in the treatment of musculoskeletal disorders, such as rheumatoid arthritis, osteoarthritis, juvenile idiopathic arthritis, and ankylosing spondylitis (Bekker et al., 2018). Even though MLX has a greater safety profile than other NSAIDs, gastrointestinal side effects are a disturbing problem during long-term treatment with high drug doses. To avoid ADRs, it was necessary to develop an alternative route for MLX delivery. One of the most promising methods of MLX application is through topical formulations, which may allow for the maximization of local therapeutic effects (Bekker et al., 2018; Khalil and Aldosari, 2020). Due to the fact that MLX has the ability to absorb UVA radiation, it is highly possible that side effects associated with the new method of drug application, such as phototoxic skin reactions, may occur after UVA iradiation (Arabi et al., 2020; Nikolaychuk, 2023).

The skin is a multilayered, self-regulating organ whose homeostasis is based on effective interaction between different cell types. The basic task of the skin is to protect the deeper tissues against harmful external factors, e.g. ultraviolet radiation type A (UVAR) capable of inducing pathological reactions such as carcinogenesis, autoimmune responses, or aging (Slominski et al., 2018). It should be noted that the physiological functions of skin cells are regulated and coordinated by the neuroendocrine system. Mediators, cytokines and neuropeptides released from the immune and endocrine systems acting in a para- or autocrine manner play a key role in the response of cells to stress factors. This protection also involves keratinocytes, the pigment system, an elements of the immune and hormonal systems and the microbiome, which interact with appropriate systems in the deeper skin tissues in order to maintain the skin and systemic homeostasis (Slominski et al., 2022a, Slominski et al., 2022b).

There are two types of melanin pigments - eumelanin and pheomelanin produced by melanocytes. Eumelanin is known to be a heterogeneous polymer formed in various proportions by 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI) (d'Ischia et al., 2013). The activity of dopachrome tautomerase and the availability of copper ions influence the relative ratio of DHI to DHICA, which impact on the antioxidant capacity of eumelanin. The much greater radical scavenging properties of DHICA compared to DHI result from the different chemical, structural and aggregation properties of eumelanin components. DHI is composed of planar, stacked components, while DHICA is characterized by a lower degree of stabilization and aggregation by electronic delocalization, becoming more susceptible to reactions with ROS (d'Ischia et al., 2015). In the process of pheomelanin synthesis, a gradual structural modification involving the transformation of benzothiazine into a benzothiazole moiety is observed (Shosuke et al., 2017). The predominance of the benzothiazine moiety in pheomelanin is considered a dye with a pro-oxidant effect (Shosuke et al., 2017). Additionally, the susceptibility of pheomelanin to photodegradation, during which superoxide anions and hydrogen peroxide are formed, contributes to the damaging effects caused by exposure to UV radiation and may lead to the induction of damage to the genetic material of melanocytes (d'Ischia et al., 2015). The presence of melanins allows melanocytes to perform many unique functions such as absorbing UVR, chelating metal ions, and being redox buffers play a key role in the defensive reaction against environmental stressors (Slominski et al., 2022a, Slominski et al., 2022b). On the one hand, melanin biopolymers may play a protective role against oxidative stress induction due to the eumelanin antioxidant properties, but on the other hand, the binding of drugs to melanin may increase the intracellular drug concentration and intensify its toxic effects (Rimpela et al., 2018). Moreover, the content of melanin biopolymers as well as the ratio between eumelanin and pheomelanin in the skin is considered to be one of the main factors predisposing to the occurrence of a phototoxic reaction. Caucasians, whose skin is characterized by a low content of melanin pigments, are almost 70 times more susceptible to the development of melanoma and the occurrence of a phototoxic reaction compared to the Negroid race characterized by a high content of melanin in the skin (d'Ischia et al., 2015).

Previous studies have shown that MLX combined with UVAR shows a phototoxic effect on human normal skin cells by inducing changes in the cell number, viability, intracellular level of reduced thiols, mitochondrial potential, and the cell cycle. Furthermore, MLX was shown to cause a significant increase in intracellular ROS in fibroblasts (HDF) and melanocytes (HEMn-LP) which were confirmed by H2DCFDA analysis (Karkoszka et al., 2022). Because that phototoxicity pathomechanism involves many various types of skin cells with different melanin content, we would like to analyze the oxido-reduction homeostasis of fibroblasts and melanocytes which allow to compare the diversity of the antioxidant response and study the activity and expression of individual enzymes in detail.

This study was performed as a continuation of the MLX-induced redox homeostasis disorders, to verify in detail its influence on antioxidant enzyme activity in human normal skin cells.