The Isoniazid Metabolites Hydrazine and Pyridoxal Isonicotinoyl Hydrazone Modulate Heme Biosynthesis

Abstract

In a mouse model, rifampicin and isoniazid combination treatment results in cholestatic liver injury that is associated with an increase in protoporphyrin IX, the penultimate heme precursor. Both ferrochelatase (FECH/Fech) and aminolevulinic acid synthase 1 (ALAS1/Alas1) are crucial enzymes in regulating heme biosynthesis. Isoniazid has recently been reported to upregulate Alas1 but downregulate Fech protein levels in mice; however, the mechanism by which isoniazid mediates disruption of heme synthesis has been unclear. Two metabolites of isoniazid, pyridoxal isonicotinoyl hydrazone (PIH, the isoniazid-vitamin B6 conjugate) and hydrazine, have been detected in the urine of humans treated with isoniazid. Here we show that, in primary human hepatocytes and the human hepatocellular carcinoma cell line HepG2/C3A, (1) isoniazid treatment increases Alas1 protein levels but decreases Fech levels; (2) hydrazine treatment upregulates Alas1 protein and Alas1 mRNA levels; (3) PIH treatment decreases Fech protein levels, but not Fech mRNA levels; and (4) PIH is detected after isoniazid treatment, with levels increasing further when exogenous vitamin B 6 analogs are coadministered. In addition, the PIH-mediated downregulation of human FECH is associated with iron chelation. Together, these data demonstrate that hydrazine upregulates ALAS1, whereas PIH downregulates FECH, suggesting that the metabolites of isoniazid mediate its disruption of heme biosynthesis by contributing to protoporphyrin IX accumulation.

Toxicoproteomic Profiling of hPXR Transgenic Mice Treated with Rifampicin and Isoniazid

“…Histologic examination revealed the presence of bile plugs ( Figure 3 C) and higher hepathopathologic (HP) scores ( Figure 3 A) in hPXR mice treated with both rifampicin and isoniazid. These changes are consistent with previous reports of hPXR -dependent hepatotoxicity in mice treated with isoniazid and rifampicin [ 27 , 39 , 40 ].…”

“…We purchased primary antibodies from the following sources: anti-FECH mouse monoclonal antibody (sc-377377) from Santa Cruz Biotechnology (Dallas, TX, USA; diluted 1:5000), anti-ALAS1 mouse monoclonal antibody (ab54758) from Abcam (Cambridge, UK; diluted 1:2000), and anti-CYP3A4 K03 mouse monoclonal antibody at a dilution of 1:1000 [ 29 , 30 , 31 ]. We purchased goat anti-mouse infrared dye (IRDye)-conjugated (680 RD and 800 CW) secondary antibodies from LI-COR (Cambridge, UK) and used them at a dilution of 1:10,000 to visualize proteins [ 27 ].…”

“…Then, we stripped the membranes with NewBlot Nitro stripping buffer (LI-COR), incubated them with an anti–β-actin primary antibody (a5441; Sigma-Aldrich, St. Louis, MO, USA; diluted 1:1000) overnight at 4°C, and then incubated them with a secondary antibody for 1 h at RT. We visualized the protein bands with an Odyssey infrared imager (LI-COR Biosciences) and determined the relative intensity of each band by normalizing their intensities to that of the actin band [ 27 , 33 ].…”

“…This hepatotoxicity is associated with increased protoporphyrin IX (PPIX), which is a precursor to heme suitable for quantification via high-throughput fluorescence imaging [ 26 ]. The iron-dependent modulation of ferrochelatase (FECH) and aminolevulinic acid synthase 1 (ALAS1) is also driven by metabolites of isoniazid [ 27 ]. Rifampicin is a well-known inducer of drug-metabolizing enzymes and transporters via hPXR.…”

Mitochondrial Heme Synthesis Enzymes as Therapeutic Targets in Vascular Diseases

“…It has anti-angiogenic effects in retinal ECs, blocking proliferation, migration, and tube formation in vitro and reducing neovascularization in vivo comparable to intraocular anti-VEGF treatment, in both OIR and L-CNV mouse models ( Figure 1B ) ( Basavarajappa et al, 2017 ; Pran Babu et al, 2020 ). Isoniazid, an anti-mycobacterial drug, decreases FECH expression while upregulating ALAS1 ( Brewer et al, 2019 ), and thus could be tested for potential anti-angiogenic activity in neovascularization models. Other inhibitors of heme synthesis used in vitro include succinylacetone and salicylic acid that block ALAD and FECH respectively ( Giger and Meyer, 1983 ; Gupta et al, 2013 ), however their use in preclinical angiogenesis models remains to be investigated.…”