Development of a New Approach Method to Monitor and Modify Caffeine Metabolism Correlated to CYP1A2 Expression

New approach methodologies (NAMs) that seek to reduce reliance on animal testing require sensitive, mechanism-based assays to accurately predict human-specific metabolic responses. Caffeine, primarily metabolized by cytochrome P450 1A2 (CYP1A2), serves as an ideal probe substrate for evaluating CYP1A2 function. Here, we describe the development of an in vitro platform that combines high-sensitivity triple quadrupole multiple reaction monitoring (MRM) LC-MS analysis of paraxanthine with quantitative reverse-transcription PCR (qPCR) of CYP1A2 expression. Using human hepatocellular carcinoma-derived cell lines (HepG2 and Hep3B), we demonstrate that modulating CYP1A2 with known effectors, sulforaphane (a known CYP1A2 inhibitor), 3-methylcholanthrene (inducer), and galangin (moderate inducer), changes in both paraxanthine accumulation and CYP1A2 mRNA levels can be effectively monitored. The correlations observed between transcriptional responses and metabolic outputs validate paraxanthine as a sensitive readout of CYP1A2 function in these cell lines. Moreover, the assay remains robust across multiple experimental conditions and facilitates insights into enzyme induction or inhibition mechanisms. By providing a straightforward and scalable alternative to animal models, this approach expands the toolbox available for interrogating xenobiotic metabolism, and enzyme regulation. Ultimately, these findings highlight the utility of an integrated cell culture-based system for advancing studies of hepatic enzyme function. This platform enables investigators to readily screen and characterize compounds that influence CYP1A2-mediated metabolism, providing a straightforward, scalable, alternative to animal models.