Fig 2: Characterization of POR cKO humanized liver mice. (A) Immunohistochemical staining of human Mitochondria (hMIT), CYP1A2, CYP2C9, and CYP3A4 proteins in liver tissue from POR cKO humanized liver mice. Rectangular area on top panel was enlarged in lower panel. Dotted circles indicate the region comprising mouse hepatocytes (m). P, portal tract, C, central vein. Scale bar, 5 mm (top panel) and 500 µm (lower panel). (B) Expression level of human pharmacokinetics-related genes in the liver from humanized liver TK-NOG (Normal Hu-liver) mice (n = 3) and POR cKO humanized liver (POR cKO Hu-liver) mice (n = 3) were measured via qRT-PCR. Data are presented as the mean ± SD. Statistically significant difference when compared with that in normal-Hu-liver mice; ****p < 0.0001. (C) Drug-metabolizing activity in liver microsomes from TK-NOG humanized liver mice (Normal Hu-liver), POR cKO mice, POR cKO humanized liver mice (POR cKO Hu-liver), and humans. Phenacetin (50 µM), diclofenac (40 µM), omeprazole (10 µM), metoprolol (5 µM), and midazolam (5 µM) were incubated with pooled liver microsomes (0.20 mg/mL) from normal Hu-liver mice (n = 4), POR cKO mice (n = 5), POR cKO Hu-liver mice (n = 4), and humans (n = 50) at 37 °C for 10–20 min. Data are presented as the mean ± SD.

Fig 3: Hyperacute APAP-induced liver injury by Ahr activation in hepatocytes depends on accumulation of toxic APAP adducts. Female Alb?/?Ahr mice and littermate control mice were treated with APAP+ITE (n = 9–11). Expression of the APAP- metabolizing enzymes Cyp1a2 and Cyp2e1 at (A, B) messenger RNA and (C, D) protein level 4 hours post APAP injection. (E) Total GSH levels in liver homogenate and (F) APAP adducts in serum 30 min post APAP injection (n = 4–6). (G) Hepatic expression of the inflammatory cytokines Il6, Il1b and Tnf 4 hours post APAP injection. Pictures were taken using a Biorevo Keyence BZ-9000 microscope with objective from Nikon (Plan Apo 10x/0,45 8/0.17 WD 4.0) and the Keyence BZ II Viewer and Analyzer software. Scale bar = 50 µm. Pooled data of 2 of 3 independent experiments are shown. For statistical analysis, the Mann-Whitney test was applied. Results are shown as mean ± SD. **P < .01, ***P < .001, ****P < .0001. KO, Alb?/?Ahr; L, littermate.

Fig 4: PARP inhibitor PJ34 attenuates APAP metabolism and liver toxicity.C57BL/6J mice were injected intraperitoneally with PJ34, and then administered with 300 mg/kg APAP injection. Blood and tissue were collected at indicated hours after APAP injection. a Representative H&E staining of liver sections from Vehicle, APAP and APAP plus PJ34-treated mice. b Quantification of liver necrosis area. Serum levels of (c) ALT and (d) AST activity. e Mice were treated with a lethal dose of APAP (1 g/kg). Survival was followed for 36 h post-administration. f Total hepatic GSH levels at indicated time points after APAP treatment. g Total hepatic H2O2 concentrations and h APAP-cysteine levels at 6 h after APAP treatment. i The hepatic expressions of Phase I (CYP1A2, CYP2E1 and CYP3A11) and Phase II enzyme genes (GSTa1, GSTp1, UGT1A1 and SULT2A1) were measured by real-time PCR assays. N = 10–12 for each group. *P < 0.01 vs. APAP

Fig 5: ITE-mediated exacerbation of APAP induced liver injury is similar in both sexes. (A–F) Male or (G–L) female C57Bl/6 mice (n = 6 each) were injected intraperitoneally on 2 consecutive days with 200 µg ITE or vehicle prior to intraperitoneal injection of 350 mg/kg APAP. (A, G) Disease severity, (B, H) ALT, and (C, I) AST serum levels, and gene expression of Ahr, Cyp1a2, and Cyp2e1 were analyzed. One representative experiment of 3 independent experiments is shown. For statistical analysis, the Mann-Whitney test was applied. Mean ± SD are shown. *P < .05, **P < .01.