Metabolomic Reprogramming Induced by Benzo[a]pyene in Skin Keratinocytes and Protective Effects of Glutathione Amino Acid Precursors

Abstract

Background

Pollutant particles can penetrate and accumulate in skin, leading to excessive oxidative stress, inflammation, and skin disorders. Reduced glutathione (GSH) is considered as “the master antioxidant” and major detoxification agent.

Aims

To characterize the metabolomic changes of skin keratinocytes under the pollutant benzo[a]pyrene (BaP) challenge and investigate the interventional effects of glutathione amino acid precursors (GAP).

Methods

Normal human epidermal keratinocytes (NHEKs) were challenged with BaP with or without GAP treatment. GSH/GSSG levels were measured by UPLC–MS/MS. Non-targeted metabolome analysis was conducted with UPLC-QTOF mass spectrometry. Transcriptomics analysis was performed using RNA-seq. DNA damage biomarker γ-H2AX was analyzed by western blot. Reconstructed pigmented skin equivalent models (pLSE) were used for evaluating phenotypical changes.

Results

One micromolar BaP exposure induced widespread metabolic reprogramming in in vitro NHEKs with over-represented differential metabolites in pathways including purine and pyrimidine nucleotide metabolism, xenobiotic metabolism, methylation, and RNA modification, etc. GAP co-treatment improved GSH/GSSG ratio, reduced reactive BaP metabolites, and partially reversed BaP induced metabolic and transcriptomic alterations. Western blotting further confirmed that GAP treated samples showed reduced γ-H2AX staining. In pLSE models, GAP treatment significantly ameliorated BaP induced skin darkness and hyperpigmentation.

Conclusions

In summary, GAP shows in vitro protective effects against BaP by maintaining GSH homeostasis, helping metabolic detoxification, reducing DNA damage, and is effective in preventing hyperpigmentation of skin models under pollution challenge.