EFFECTS OF NICOTINAMIDE RIBOSIDE ON METABOLIC ALTERATIONS INDUCED BY BENZO[A]PYRENE IN A THREE-DIMENSIONAL CULTURE MODEL

Resumo

Introduction: Benzo[a]pyrene (B[a]P), classified by IARC as a human carcinogen, is formed through the incomplete combustion of organic matter. Upon biotransformation, B[a]P generates reactive metabolites capable of inducing damage to DNA and RNA, contributing to carcinogenesis. B[a]P also induces changes in cellular metabolism with characteristics that fit the Warburg effect. Nicotinamide riboside (NR) is a precursor of NAD⁺, which plays critical roles in metabolic pathways, regulation of gene expression, and DNA repair. We hypothesize that NR has a role in preventing or mitigating genetic damage and cellular transformation induced by carcinogenic agents like B[a]P. Objective: To investigate the effects of NR on metabolic alterations induced by B[a]P using a spheroid model. Methodology: A three-dimensional culture model (spheroid) was established using BEAS-2B cells. The spheroids were divided into 4 experimental groups (N = 4 in each group): control (CTRL), 2 μM B[a]P, 5 μM NR, and simultaneously 2 μM B[a]P and 5 μM NR. Each group was subjected to exposure for seven days with daily renewal of the exposure medium. Lactate, malate, aspartate, citrate, nicotinamide mononucleotide, nicotinamide, ATP, ADP, AMP, NAD + , glutamine, glutamate, arginine and adenosine were quantified by HPLC-ESI-MS/MS. Results: Targeted metabolomics analysis revealed differences in NAD + metabolism between spheroids exposed to B[a]P and B[a]P+NR, suggesting that NAD + utilization and recycling were higher in spheroids exposed to B[a]P+NR. The higher NAD + utilization in cells exposed to B[a]P+NR may impact B[a]P-induced transformation, which will be further investigated. Metabolite analysis by pmol/μg protein as lactate (120 h of exposure: CTRL, 63.2 + 23.9; NR, 104.9 + 11.9; B[a]P, 314.1 + 47.0; B[a]P+NR, 135.0 + 35.9), glutamate (168 h of exposure: CTRL, 14.2 + 3.1; NR, 11.8 + 1.7; B[a]P, 24.1 + 2.5; B[a]P+NR, 16.6 + 2.9), adenosine (168 h of exposure: CTRL, 0.14 + 0.07; NR, 0.11 + 0.04; B[a]P, 0.90 + 0.02; B[a]P+NR, 0.07 + 0.04), aspartate (120 h of exposure: CTRL, 4.8 + 0.9; NR, 1.9 + 0.9; B[a]P, 25.1 + 9.6; B[a]P+NR, 5.4 + 3.0) and citrate (168 h of exposure: CTRL, 57.5 + 13.1; NR, 51.6 + 17.9; B[a]P, 122.5 + 9.1; B[a]P+NR, 27.0 + 9.4) increased in spheroids exposed to B[a]P, while they remained at normal concentrations in spheroids exposed to B[a]P+NR. In both groups, energetic stress was observed, indicated by decreased ATP/ADP and ATP/AMP ratios, but it is possible that the sources of energetic stress are distinct between groups. Conclusion: These results suggest that NR may play an important role in modulating the impact of B[a]P on cells.