Photomutagenicity of chlorpromazine and its N-demethylated metabolites assessed by NGS

Abstract

the human genome is constantly attacked by endogenous and exogenous agents (ultraviolet light, xenobiotics, reactive oxygen species), which can induce chemical transformations leading to DnA lesions. to combat DnA damage, cells have developed several repair mechanisms; however, if the repair is defective, DnA lesions lead to permanent mutations. Single-cell gel electrophoresis (coMet assay) is a sensitive and well-established technique for quantifying DnA damage in individual cells. nevertheless, this tool lacks relationship with mutagenesis. therefore, to identify errors that give rise to mutations it would be convenient to test an alternative known procedure, such as next generation sequencing (nGS). thus, the present work aims to evaluate the photomutagenicity of neuroleptic drug chlorpromazine (cpZ), and its N-demethylated metabolites using coMet assay and to test nGS as an alternative method to assess photomutagenesis. in this context, upon exposure to UVA radiation, coMet assay reveals cpZ-photosensitized DnA damage partially repaired by cells. conversely with this result, metabolites demethylchlorpromazine (DMcpZ) and didemethylchlorpromazine (DDMcpZ) promote extensive DnA-photodamage, hardly repaired under the same conditions. parallel assessment of mutagenesis by nGS is consistent with these results with minor discrepancies for DDMcpZ. to our knowledge, this is the first example demonstrating the utility of NGS for evaluating drug-induced photomutagenicity.

Cellular photo(geno)toxicity of gefitinib after biotransformation

“…The comet assay (single-cell gel electrophoresis) was performed as already detailed in Garcia-Lainez et al (2018) in order to detect both single and double strand breaks and alkaline labile sites on nuclear DNA. Briefly, HaCaT cells in exponential growth were harvested by trypsinization and kept in ice-cold PBS for 2 h to neutralize any DNA damage produced during trypsinization step, as has been previously demonstrated in FSK cells ( Agúndez et al, 2020 ). Then, 24-wells plates (1.0 × 10 4 cells/well) were seeded and treated with 100 μM of GFT or its metabolites (DMT-GFT or DMOR-GFT) for 30 min at 4°C in darkness to minimize cell aggregation and inhibit DNA repair.…”

“…This chemical change generates non-negligible modifications in the quinazoline chromophore, leading to a more efficient UV-light absorption ( Figure 1B ). Thus, although drug biotransformation is usually regarded to a decreased toxicity, in some cases metabolites may display more phototoxicity and photoreactivity than the parent drug ( Garcia-Lainez et al, 2018 ; Agúndez et al, 2020 ). Accordingly, investigating the photo(geno)toxicity of gefitinib and its metabolites is important for assessing drug safety, evaluating the risks associated with sunlight exposure during treatment.…”

Evaluation of phototoxicity induced by the anticancer drug rucaparib

“…Additionally, to determine if cell culture were able to repair the DNA damage promoted by compounds + UVA light, 5-OH DCF (100 µM) was used as recovery positive control 16 , 17 . Thus, before the cellular lysis, slides were embedded with DMEM, freed of supplements, and incubated at 37 °C for 6 h. Then, slides were immersed in the same lysis buffer for at least 2 h to allow cellular lysis and the same procedure was followed as describe above.…”

Drug-induced photosensitivity

“…Apart from acute phototoxicity, several phototoxic substances, like psoralens, chlorpromazine fluoroquinolones, and ketoprofen, also enhance chromosomal damage in the presence of UV light, both in vitro and in vivo [20][21][22] . These drugs can, therefore, behave as photogenotoxic and photomutagenic.…”