Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression

Abstract

Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida& Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.

Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection

“…The posttranscriptional regulation of Cyp2a5 identified to date, include mRNA stabilization and protein stabilization to increase expression and activity. The evidence of Cyp2a5 mRNA stabilization was first reported by Aida and Negishi (Aida and Negishi, 1991;Hahnemann et al, 1992). Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a multifunctional protein involved in transcriptional regulation and cell growth, has been identified as the RNA-binding protein responsible for the Cyp2a5 mRNA stabilization (Tilloy-ellul et al, 1999;Raffalli-Mathieu et al, 2002).…”

“…Furthermore, these conditions appear to be associated with increased serum bilirubin levels (Bonacini, 2004;Malaguarnera et al, 2005;Richardson et al, 2006b;Hansen, 2010). (Pellinen et al, 1993) Chloroform Anesthetic/Teflon (Camus et al, 1996) Cocaine Topical anesthetic (Pellinen et al, 1994a(Pellinen et al, , 1994b) Ethanol Fuel, beverage, and antiseptic (Lu et al, 2010) Phenobarbital Barbiturate and anticonvulsant (Hahnemann et al, 1992;Salonpää et al, 1994) Pyrazole Precursor for various medicines (Juvonen et al, 1985;Nichols and Kirby, 2008 (Cai et al, 2002) Buthionine sulfoximine Drug used in chemotherapy to reduce levels of glutathione (Gilmore et al, 2003) pregnenolone 16a-carbonitrile (PCN) Pregnane X receptor (PXR) agonist or antiglucocorticoid (Cai et al, 2002) Rifampicin Bactericidal antibiotics (Donato et al, 2000) Trans-4,5-dihydroxy-1,2-dithiane (DTTox) Intramolecular disulfide form of dithiothreitol; reducing agent (Gilmore and Kirby, 2004) (Montero et al, 1999) Bilirubin Bilirubin (BR) is the end product of heme catabolism, and heme is found in hemoglobin or other hemoproteins such as cytochromes, catalase, and a small pool of unbound free heme (Roy-Chowdhury et al, 2007). BR consists of a tetrapyrrole ( Figure D1).…”

“…It has been reported that CYP2A5 is upregulated following exposure to chemical hepatotoxins and during pathophysiological conditions in which the levels of most CYPs are either unchanged or down-regulated (Juvonen et al, 1985;Camus et al, 1996;Kojo et al, 1998;Kirby et al, 2011). The identified chemical inducers of CYP2A5 have varying structures and origin, and include various hepatotoxins, heavy metals, hormones, and porphyrinogenic compounds (Juvonen et al, 1985;Mangoura et al, 1989;Hahnemann et al, 1992;Viitala et al, 2001;Abu-Bakar et al, 2004). The increased CYP2A5 levels are also associated in various pathophysiological conditions such as hepatitis, hepatoma, malaria, fatty liver, and fasting (Kirby et al, 1994b;Chomarat et al, 1997;Sipowicz et al, 1997;Bauer et al, 2004;De-Oliveira et al, 2006Fisher et al, 2009).…”

Endoplasmic Reticulum Stress Due to Altered Cellular Redox Status Positively Regulates Murine Hepatic CYP2A5 Expression

“…Cytochrome P450 2a5 (CYP2A5) is induced by a diverse array of hepatotoxins, including nitrogen heterocycles (Kojo et al, 1998), chlorinated hydrocarbons (Camus-Randon et al, 1996), metal ions (Hahnemann et al, 1992), and porphyrinogenic agents (Donato et al, 2000), which are neither structurally similar nor generally considered to be inducers of P450s. Moreover, CYP2A5 is induced during viral (Kirby et al, 1994a), fulminant (Chemin et al, 1996), bacterial (Sipowicz et al, 1997), and parasitic hepatitis (Kirby et al, 1994b), pathophysiological conditions that are usually associated with suppression of P450s.…”

The Influence of Quinolines on Coumarin 7-Hydroxylation in Bovine Liver Microsomes and Human CYP2A6.

“…19) In the case of mouse, coumarin is 7-hydroxylated by CYP2A5. 20) During the course of investigation of the mechanism of selenocysteine synthesis with cytosol from bovine liver, 21) we reserved a large amount of bovine liver microsomes and found that bovine liver microsomes had the ability to catalyze coumarin to 7-hydroxycoumarin, which is generally dependent on CYP2A6 in human. We have also studied quinoline metabolism by the phase I reaction.…”