Detection of acute toxicity of aflatoxin B1 to human hepatocytes in vitro and in vivo using chimeric mice with humanized livers

Abstract

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes. Chimeric mice with highly humanized livers and SCID mice received daily injections of vehicle (corn oil), AFB1 (3 mg/kg), and carbon tetrachloride (50 mg/kg) for 2 days. Histological analysis revealed that AFB1 promoted hepatocyte vacuolation and inflammatory cell infiltration in the area containing human hepatocytes. A novel human alanine aminotransferase 1 specific enzyme-linked immunosorbent assay demonstrated the acute toxicity of AFB1 to human hepatocytes in the chimeric mouse livers. The sensitivity of cultured fresh human hepatocytes isolated from the humanized liver mice for AFB1 cytotoxicity was comparable to that of primary human hepatocytes. Long-term exposure to AFB1 (6 or 14 days) produced a more severe cytotoxicity. The half-maximal lethal concentration was 10 times lower in the 2-week treatment than after 2 days of exposure. Lastly, the significant reduction of AFB1 cytotoxicity by a pan-CYP inhibitor or transfection with CYP3A4 specific siRNA clearly suggested that bioactivation of AFB1 catalyzed by CYPs was essential for AFB1 cytotoxicity to the human hepatocytes in our mouse model. Collectively, our results implicate the humanized liver mice and derived fresh human hepatocytes are useful models for studies of AFB1 cytotoxicity to human hepatocytes.

Fig 1. Summary of in vivo and in vitro experiments for AFB1-induced human hepatotoxicity with PXB-mice and PXB-cells.

Fig 2. AFB1-induced human hepatotoxicity in PXB-mice.

Male PXB-mice and SCID mice received daily intraperitoneal administration of corn oil (n = 5), AFB1 (n = 5), or CCl₄ (n = 3) for 2 days. All animals were necropsied 24 h after the final injection. (A) Relative body weight to initial value. Values are means ± S.D. *p <0.01 (Tukey-Kramer test). n.s., not significant (p >0.05, Steel-Dwass test). (B) Liver sections stained with hematoxylin and eosin. Each animal was treated with the indicated chemical. Upper, middle, and lower panel represents human and mouse hepatocyte area in the PXB-mice, and in the SCID mouse livers, respectively. Arrows and arrowheads represent vacuolated hepatocytes and infiltrated inflammatory cells, respectively. Scale bars, 100 μm. (C) Representative images of Gr-1 stained liver sections collected from PXB-mice injected with AFB1. White arrowheads indicate Gr-1-positive cells. Neg; Negative control. Scale bar, 100 μm.

Fig 3. Quantification of plasma total ALT activity and hALT1 levels.

(A) Plasma total ALT activity levels in PXB-mice and SCID mice were measured with a conventional method detecting both human- and mouse-derived ALTs. (B) Reactivity of the presently developed hALT1-specific ELISA to rhALT1 and rhALT2. Serially diluted rhALT1 and rhALT2 were measured using the hALT1-specific ELISA. (C) Reactivity of the hALT1-specific ELISA to samples derived from human or mouse. Levels of hALT1 and total ALT activity in serially diluted human patient sera with liver disease and mouse sera collected from HDI-SCID mice were measured with the conventional method (x-axis) and the presently developed hALT1-specific ELISA (y-axis), respectively. (D) Correlation of serum ALT activity and hALT1 levels. Total ALT activity and hALT1 levels in serially diluted gold standard serum were measured with the conventional method (x-axis) and the present hALT1-specific ELISA (y-axis). (E) Serum hALT1 levels in the PXB-mice were measured with the hALT1-specific ELISA. Values are means ± S.D. **; p <0.01 (Tukey-Kramer test).

Fig 4. Dose-dependent cytotoxicity of AFB1 on PXB-cells.

(A) Representative images of PXB-cells treated with AFB1 indicated concentrations for 5 days. Scale bar; 100 μm. (B) Cytotoxicity curves of PXB-cells exposed to AFB1 at different doses for 150 h. Cell index curves were generated with the iCELLigence software. (C) Dose-response curves of PXB-cells exposed to AFB1 for 2, 6, and 14 days. Cell viabilities were measured with the WST-1 assay.

Fig 5. Effects of ABT on CYP3A activity and cell viability in AFB1-treated PXB-cells.

(A) PXB-cells were treated with ABT at indicated dose for 5 days and CYP3A activities were measured with HPLC. (B) PXB-cells were treated with AFB1 in the presence or absence of ABT for 8 days and cell viabilities were analyzed with the WST-1 assay. Values are means ± S.D. *p <0.05, **p <0.01 (Tukey-Kramer test). n.s., not significant (p >0.05, Tukey-Kramer test).

Fig 6. Effects of CYP3A4-specific suppression on cytotoxicity of AFB1.

Expression levels of CYP3A4 mRNA (A) and CYP3A activities (B) in PXB-cells were measured by RT-qPCR and HPLC, respectively, 7 days after transfection of NC or CYP3A4-specific siRNAs (3A4). (C) Effects of CYP3A4 knock down on viabilities in PXB-cells treated with AFB1 were analyzed with the WST-1 assay. PXB-cells were treated with AFB1 at the indicated dose beginning 7 days after transfection for 6 days. Values are means ± S.D. **p<0.01 (Student’s t test or Welch’s t test). n.s.; not significant (p>0.05, Student’s t test).