Dietary supplements are products taken orally, and they contain an ingredient intended to augment the diet. Many studies demonstrate clear alterations in microbe abundances and the production of microbiota-derived metabolites, such as short-chain fatty acids, following dietary changes. This review comprehensively explores the possible interactions among gut microbiota, lemon extracts, glycerin, and their mixture products. Lemon extracts/components are associated with a vast array of health benefits, including anti-inflammation, antioxidant, anti-atherosclerotic, and anti-diabetic effects. They are also associated with increased memory and decreased depression. Glycerin can reduce serum free fatty acids and mimic caloric restriction; its metabolites can function as a broad-spectrum antimicrobial. Additionally, glycerin has a dehydrating effect on the central nervous system and can reduce focal cerebral edema and improve performance by expanding plasma volume. However, it may also have side effects, such as hyperglycemia. Therefore, combined consumption of lemon extracts and glycerin may, in part, mitigate each other’s side effects while exerting their benefits. There is growing evidence that both lemon components and glycerin are metabolized by the gut microbiota and may modulate the intestinal microbiome composition. Therefore, gut microbiome alterations are also explored as an important mechanism in the gut–brain axis regulating various effects of these dietary supplements and their application in various noncommunicable neurological disorders.

Accurately evaluating chemical risk may benefit from the development of human-relevant models capable of capturing the effects of treatments that closely mimic real-world environmental and pharmaceutical exposures. Building on our previous work (Seo et al. Arch Toxicol 98:1919-1935),¹ where cytotoxicity compromised mutation detection following short-term treatments, this study investigated mutation accumulation in both 2D and 3D HepaRG cultures following 7- and 14-day exposures with relatively non-cytotoxic N-nitrosodimethylamine (NDMA) concentrations. A multi-endpoint approach was employed to assess NDMA-induced DNA damage, micronucleus formation, and mutagenesis. Seven-day NDMA treatments were not cytotoxic but produced concentration-dependent increases in DNA damage and mutations. After 14 days of exposure, the highest NDMA concentrations produced no more than 30% cytotoxicity, and induced greater mutation frequencies compared to the 7-day exposures. Overall, the mutation frequencies induced by NDMA exhibited concentration- and treatment-duration-dependent relationships in both 2D and 3D HepaRG cultures, with notably higher mutation frequencies in 3D spheroids than in 2D cultures. Quantitative analysis by benchmark concentration (BMC) modeling demonstrated lower BMC values in 3D spheroids compared to their 2D counterparts. The predominant mutation in NDMA-treated cultures was T→C transition. These findings indicate the value of extended exposure periods for conducting in vitro genotoxicity testing in HepaRG cells.