Chronic Treatment with Isoniazid Causes Protoporphyrin IX Accumulation in Mouse Liver

Abstract

Isoniazid (INH) can cause hepatotoxicity. In addition, INH is contraindicated in patients suffering from porphyrias. Our metabolomic analysis revealed that chronic treatment with INH in mice causes a hepatic accumulation of protoporphyrin IX (PPIX). PPIX is an intermediate in the heme biosynthesis pathway, and it is also known as a hepatotoxin. We further found that INH induces delta-aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. We also found that INH downregulates ferrochelatase (FECH), the enzyme that converts PPIX to heme. In summary, this study illustrated that chronic treatment with INH causes PPIX accumulation in mouse liver in part through ALAS1 induction and FECH downregulation. This study also highlights that drugs can disrupt the metabolic pathways of endobiotics and increase the risk of liver damage.

Figure 1. Effect of INH on mouse liver metabolome

The mice were treated with 400 mg/L INH in drinking water for 0–28 days. Liver samples were analyzed by UPLC-QTOFMS in positive mode. (A) Separation of liver samples from the control and INH-treated mice in a PCA score plot. The t[1] and t[2] values represent the score of each sample in principle component 1 and 2, respectively. (B) Loading S plots of liver samples generated by OPLS-DA. The x-axis is a measure of the relative abundance of ions, and the y-axis is a measure of the correlation of each ion to the model. The number 1 ranking ion was identified as PPIX.

Figure 2. Identification and quantification of PPIX (I)

(A) Extracted chromatogram of PPIX. (B) MS/MS of PPIX. (C) Time-dependent accumulation of PPIX in the liver of mice treated with 400 mg/L INH in drinking water for 0–14 days. (D) Dose-dependent accumulation of PPIX in the liver of mice treated with 0, 100, 200, or 400 mg/L INH in drinking water for 14 days. *P < 0.05, ***P < 0.001 vs control (n = 3 or 4).

Figure 3. Effect of INH on PPIX levels in bone marrow cells (BMCs), serum and red blood cells (RBCs)

The mice were treated with 0, 100, 200, or 400 mg/L INH in drinking water for 14 days. All samples were analyzed by UPLC-QTOFMS in positive mode. (A) PPIX in BMCs. (B) PPIX in serum. (C) PPIX in RBCs. All data were expressed as mean ± SD (n = 4).

Figure 4. Effect of INH on FECH expression in the liver

FECH protein was analyzed by western blotting. GAPDH was used as loading control. (A and B) FECH expression in the liver of mice treated with 400 mg/L INH in drinking water for 0–14 days. (C and D) FECH expression in the liver of mice treated with 0, 100, 200 or 400 mg/L INH in drinking water for 14 days. All data were expressed as mean ± SD (n = 3). The data in the control group were set as 100%. ***P < 0.001, ****P < 0.0001 vs control.

Figure 5. Effect of INH on ALAS1 expression and activity in the liver

ALAS1 mRNA expression were analyzed by qPCR and ALAS1 protein was analyzed by western blotting. GAPDH was used as loading control for western blotting. (A) ALAS1 mRNA expression in the liver of mice treated with 400 mg/L INH in drinking water for 0–14 days. (B) ALAS1 mRNA expression in the liver of mice treated with 0, 100, 200 and 400 mg/L INH in drinking water for 14 days. (C and D) ALAS1 protein expression in the liver of mice treated with 400 mg/L INH in drinking water for 0–14 days. (E and F) ALAS1 protein expression in the liver of mice treated with 0, 100, 200 or 400 mg/L INH in drinking water for 14 days. (G) Effect of INH on ALAS1 activity in mouse liver. The mice were treated with vehicle or 400 mg/L INH in drinking water for 3 days. Liver mitochondria were used for analysis of ALAS1 activity. All data were expressed as mean ± SD (n = 3 or 4). The data in the control group were set as 1. *P < 0.05, **P < 0.01, ***P < 0.001 vs control.