Date of Award

5-2018

Document Type

Dissertation

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Taosheng Chen, Ph.D.

Committee

Ying Kong, Ph.D. Richard E. Lee, Ph.D. David R. Nelson, Ph.D. John D. Schuetz, Ph.D.

Keywords

Drug-Induced Liver Injury, Heme Synthesis, Hepatotoxicity, Isoniazid, Rifampicin, Vitamin B6

Abstract

In a mouse model, rifampicin and isoniazid combination treatment results in cholestatic liver injury that is associated with an increase of protoporphyrin ix (PPIX), the penultimate heme precursor. Excess PPIX is believed to bind to bile acids, precipitate in bile canaliculi, and form bile plugs leading to cholestasis fol owed by liver injury. Both ferrochelatase (FECH/Fech) and aminolevulinic acid synthase 1 (ALAS1/Alas1) are crucial enzymes in regulating heme biosynthesis. Isoniazid has recently been reported to up-regulate Alas1 but down-regulate Fech protein levels in mice; however the mechanism of isoniazid mediated heme synthesis disruption has remained unclear. Interestingly, metabolites of isoniazid, pyridoxal isonicotinoyl hydrazone (PIH, the isoniazid and vitamin B6 conjugate) and hydrazine, have been detected in the urine of humans treated with isoniazid previously. Here I show that in primary human hepatocytes and the human hepatocellular carcinoma cell line HepG2/C3A: (1) the physiochemical properties of PPIX may contribute to toxicity (2) isoniazid treatment results in an increase of ALAS1 but a decrease in FECH protein levels by Western blot analysis; (3) hydrazine treatment up-regulates ALAS1 protein and mRNA levels; (4) PIH treatment decreases FECH protein levels; (5) PIH is detected by mass spectrometry analysis following isoniazid treatment with a further increase when exogenous vitamin B6 analogues are co-administrated. In addition, the (6) PIH mediated down-regulation of human FECH is dependent on iron levels. Proteomics profiling analysis suggests that in the livers of hPXR mice (7) rifampicin may induce CYP450 changes associated with increased hydrazine reactivity with cellular proteins, (8) multiple [2Fe-2S]-containing proteins and (9) [Fe-S] assembly machinery proteins may be down-regulated in mice due to isoniazid. Together these data demonstrate that hydrazine up-regulates ALAS1 while PIH down-regulates FECH, suggesting that the metabolites of isoniazid mediate its disruption of heme biosynthesis.

ORCID

http://orcid.org/ https://orcid.org/0000-0001-9697-3269

DOI

10.21007/etd.cghs.2018.0455

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