Ine). (b) Pathway enrichment analyses with feature lists containing raw p values Caspase 2 Gene ID identified 2, 1, and three affected metabolic pathways for PCB exposures of two, eight, and 24 h, respectively (p 0.05). Pathways with much less than four substantial capabilities weren’t presented. A metabolite was incorporated in the pathway analysis only if the primary molecular ion ([M-H]-) was statistically substantial involving groups. The number of attributes altered by PCB3 exposure is listed as overlap/total characteristics for each pathway. (c) Tryptophan metabolism was identified as significantly affected by PCB3 exposure at the 24 h time point. Metabolites with yellow, red, and green backgrounds decreased, increased, or did not modify due to PCB3 exposure, respectively. Metabolites in white boxes couldn’t be identified with acceptable self-confidence scores. (d) Modifications in the tryptophan metabolism-kynurenine pathway following exposure of HepG2 cells to PCB3 with levels of 5-hydroxyindoleacetaldehyde, indolepyruvate, kynurenine, serotonin, 5-hydroxytryptophan, and 6-hydroxymelatonin decreasing and levels of methylserotonin, formylkynurenine, and formyl-acetyl-5-methoxykynurenamine growing. Data are shown as normalized raw intensity, with p 0.05 () or p 0.01 (). The accurate m/z, retention occasions, Coccidia Storage & Stability adducts, significances, and self-confidence scores from the metabolite annotations in the tryptophan metabolism pathway are listed in Table S5. For information about the pathway enrichment analyses with a looser parameter setting, see Figure S14.characterize the possible toxicities connected together with the formation of 3,4-di-OH-3 in extra human-like models, including key hepatocytes. Modifications in Endogenous Metabolites Following PCB3 Exposure in HepG2 Cells. We performed metabolomic analyses using the LC-Orbitrap MS data to investigate changes in endogenous metabolic pathways in HepG2 cells following PCB3 exposure. In the univariate analyses, we identified 555, 534, and 1929 metabolic functions (p 0.05) and 10, 20, and 966 options having a false discovery price (FDR) 0.05 that drastically differed amongst handle and PCB3-exposed media at the 2, eight, and 24 h time points (Figure 4a). Metabolicpathways enriched in these important options had been identified using mummichog having a human pathway library. Two, one particular, and 3 metabolic pathways had been significantly affected in the two, eight, and 24 h time points (p 0.05) (Figure 4b). Pathway enrichment analyses having a looser parameter setting identified an overlap in pathways affected in the 2 and 8 h but not the 24 h time point (i.e., linoleate metabolism and fatty acid metabolism, Figure S13). It’s not surprising that the effects of PCB3 around the metabolome within the experimental system modify over time on account of adaptive responses in the cells and time-dependent alterations within the PCB3 and the PCB3 metabolite mixture present inside the cells. These adjustments reflecthttps://doi.org/10.1021/acs.est.1c01076 Environ. Sci. Technol. 2021, 55, 9052-Environmental Science Technologypubs.acs.org/estArticleFigure 5. Metabolome-wide association evaluation suggests that PCB3 metabolite classes formed in HepG2 cells are substantially related with various metabolic pathways. The size of circles is proportional for the overlap size (quantity of considerable attributes) of the pathway enrichment. Circles with black borders are key pathways with 5 considerably connected attributes. Metabolome-wide association analyses had been performed on 18 samples incubated with and with out PCB3. Peak locations o.