Background. Cutaneous photosensitivity disorders are diverse inflammatory skin conditions characterised by an abnormal sensitivity to sunlight. Drug-induced photosensitivity (DIP) occurs when people are exposed to ultraviolet radiation (UVR) after systemic or topical use of photosensitising drugs such as thiazide diuretics, non-steroidal anti-inflammatory drugs and quinine. DIP occurs mostly due to phototoxic reactions, although photoallergic reactions are also observed. During the phototoxic reaction, photosensitising drugs absorb UVR and can damage biomolecules through oxidative stress due to the generation of reactive oxygen species (ROS). Drug photosensitisation can have therapeutic applications, as shown in PUVA therapy (Psoralen + UVA) which is based on inducing phototoxic reactions to treat various skin conditions. Currently, the exact mechanisms underlying systemic drug sensitisation are not understood. Therefore, the only effective treatment is to stop taking the drug. However, in many cases this is not appropriate as the patient need the specific drug. Therefore, the treatments usually include management of the symptoms via photoprotection. Cutaneous lipids are potent signalling molecules that can be targeted and modified by ROS. As bioactive lipids are involved in inflammatory reactions and the skin's response to sunlight, it is possible that they are affected by DIP; to date, the cutaneous lipidome of DIP has not yet been explored. Aim. The project aims to explore the involvement of skin lipids in DIP and undertake a lipidomics approach to profile and measure cellular lipid changes caused by commonly reported systemic photosensitising drugs in vitro, using human skin cell lines as a model. Methods. The commonly used photosensitising drugs Hydrochlorothiazide (HCTZ), Naproxen (NAP), Quinine (QUIN) and 8-methoxypsoralen (8-MOP) were chosen to study phototoxicity. HaCaT epidermal keratinocytes and 1BR.3.G1 dermal fibroblasts were treated with drugs and UVA. Cell viability was assessed by the MTT assay to identify appropriate dose. Cellular lipids were extracted with organic solvents and analysed by ultrahigh performance supercritical fluid chromatography (UHPSFC) coupled to a quadrupole time-of-flight (Q-Tof) mass analyser with electrospray ionisation (ESI) (UHPSFC/ESI-MSE). Lipids were semi-quantified using class-specific deuterated internal standards. An in vitro study using phosphatidylcholine (PC) treated with UVA and HCTZ, was used to explore the formation of oxidised PC species (oxPC) and inform the lipidomic studies. A research ethics application to collect human skin samples for the development of a lipid mass spectrometry imaging (MSI) assay was submitted although the clinical study did not take place due to COVID-19 pandemic. Results and Conclusions. Lipid classes studied in drug and UVA-treated cells included: PC, lysophosphatidylcholine (LPC), long-chain oxidised PC (oxPC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylserine (PS), free fatty acid (FFA), cholesterol (CHL), cholesteryl ester (CE), diglycerol (DG) and triacylglycerol (TG) species. When cells were treated with UVA and the photosensitising drugs HCTZ and 8-MOP, an overall increase in most lipid classes and lipid species was observed. Interestingly, treatment with QUIN and UVA, had a less significant impact on cell lipids, while treatment with NAP and UVA, did not alter the lipids examined here. The oxPC species PC 34:1; O and PC 34:1; O2, were found to increase following UVA and drug treatment but their levels returned to baseline within 24h post-irradiation. Overall, the observed increased levels of cellular lipids could be part of a metabolic adaptation to maintain lipid homeostasis and survival under the increased oxidative stress caused by photosensitisation. The differences in the cellular lipid profiles following treatment with photosensitising drugs suggest that lipid changes could be explored to pursue disease and treatment stratification in DIP. A better understanding of how the observed lipid changes contribute to an adaptive and/or protective response might potentially help to identify effective targets for modulating the photosensitivity reactions.
Investigating the cutaneous lipidome to understand the molecular basis of photosensitivity disorders
Bashak, A. (Author). 17 Dec 2024
Student thesis: Phd