Elsevier

Toxicology Letters

Volume 195, Issues 2–3, 2 June 2010, Pages 147-154
Toxicology Letters

The effect of β-naphthoflavone on the metabolism of amiodarone by hepatic and extra-hepatic microsomes

https://doi.org/10.1016/j.toxlet.2010.03.019Get rights and content

Abstract

Amiodarone is a potent antiarrhythmic drug with several limiting side effects, some of which have been correlated with increased levels of its more toxic metabolite, desethylamiodarone. Elevated serum desethylamiodarone to amiodarone ratios are associated with a risk of amiodarone-induced pulmonary toxicity. Polycyclic aromatic hydrocarbons such as β-naphthoflavone are known to increase desethylamiodarone levels in rat in vivo. In this article we investigated if this increase was solely due to increased formation as a result of cytochrome P450 (CYP) 1A1 and 1A2 induction in different rat hepatic and extra-hepatic tissues. Additionally, the effect of amiodarone treatment on CYP1A1 and 1A2 gene expression and activity was investigated. In rats, β-naphthoflavone was found to increase desethylamiodarone forming activity in lung and kidney microsomes. Amiodarone increased β-naphthoflavone mediated induction of CYP1A1 gene expression in liver, lung and kidney. However, there was no significant change in CYP1A activity. As expected, the data indicated that the increase in desethylamiodarone levels in vivo was partly due to increased formation through CYP1A1 induction, although increased formation was only evident in some extra-hepatic tissues. Amiodarone treatment did not affect basal or induced CYP1A activity.

Introduction

Amiodarone (AM) is a benzofuran derivative commonly prescribed for the treatment of ventricular and supraventricular arrhythmias (Naccarelli et al., 2000, Piccini et al., 2009, Snider et al., 2009). It has a large volume of distribution, being extensively sequestered by tissues such as lung, liver and adipose tissues (Brien et al., 1987, Holt et al., 1983, Shayeganpour et al., 2008). Metabolic transformation in the liver is the main route for AM elimination (Fabre et al., 1993, Trivier et al., 1993). To date, there is evidence of several metabolites being formed from AM biotransformation (Ha et al., 2001, Young and Mehendale, 1986). The main circulating metabolite, however, is mono-N-desethylamiodarone (DEA), which is detectable at relatively high levels in serum and/or plasma and tissues of human and preclinical animal species (Brien et al., 1987, Elsherbiny and Brocks, 2010, Hamdy and Brocks, 2009, Kannan et al., 1989, Meng et al., 2001, Shayeganpour et al., 2008, Stark et al., 1991). Notably, DEA also possesses electrophysiological activity similar to that of its parent drug (Stark et al., 1991). Several cytochrome P450 (CYP) isoenzymes are involved in the biotransformation of AM to DEA. For example, CYP3A4, 1A1/2, 2D6 and 2C8 were found to be involved in DEA formation in human whereas CYP3A1/2, 1A1, 2C11 and 2D1 were found to mediate DEA formation in rat (Elsherbiny et al., 2008, Ohyama et al., 2000a, Shayeganpour et al., 2006).

Despite the beneficial use of AM in the treatment of arrhythmias, AM use is sometimes discontinued because of some serious side effects. These adverse effects can involve liver, thyroid, skin, pancreas and lung (Batcher et al., 2007, Chen et al., 2007, Jarand et al., 2007, Puli et al., 2005). In several organs, DEA has been shown to be more toxic than AM. For example, DEA caused a decrease in the mitochondrial membrane potential and cell death in human and rat pulmonary alveolar epithelial cells at lower concentrations than those of AM (Bargout et al., 2000, Bolt et al., 2001). The concentrations in human alveolar epithelial cell lines (A549) that resulted in 50% cell death were recently reported to be 50 and 12 μM for AM and DEA, respectively (Seki et al., 2008). Furthermore, this more potent cytotoxic effect of DEA was consistently observed in human hepatocellular carcinoma (HepG2) and immortalized thyrocytes cell line (SGHTL-34 cells) (Beddows et al., 1989, Waldhauser et al., 2006) with similar observations being documented in animal models such as the rat (Somani et al., 1990).

Exposure to polycyclic aromatic hydrocarbons (PAH), potent CYP1A inducers, can lead to significant increases in the DEA levels in the plasma, liver, heart and lung tissues of AM treated rats (Elsherbiny and Brocks, 2010). Although these compounds are considered as ubiquitous contaminants that occur naturally in the environment, a more intense exposure can occur in some people due to occupational or lifestyle reasons. For example, coke-oven workers, roofers, pavers, chimney sweepers and cigarette smokers are all exposed to PAH up to several 1000-fold higher than the background levels normally present in the environment (Anonymous, 1998, Straif et al., 2005).

Although the previous study (Elsherbiny and Brocks, 2010) in rats established that DEA tissue and plasma concentrations were increased with CYP1A induction, metabolic induction of CYP1A1/2 within tissues was not examined. Unlike some other CYP isoenzymes, this consideration extends beyond that of liver for CYP1A1 because it is expressed and is inducible in several important extra-hepatic organs. One of these tissues, the lung, is known to express CYP1A1 (Shimada et al., 1996), and is the focus of pulmonary fibrosis, one of the most serious side effects of AM treatment (Jarand et al., 2007, Thum et al., 2006). Although some associative patient factors have been identified for the onset of this side effect (e.g. older age, long duration of therapy, pre-existing lung injury, high patient body mass index), most patients with these same factors do not develop the toxicity. An increased DEA:AM serum concentration ratio has also been shown to be associated with the side effect, indicating that the drug's metabolism may play a role in its development (Ernawati et al., 2008, Okayasu et al., 2006, Pollak, 1999).

To further explore the possible relationship of amiodarone metabolism to its toxicity, here we describe the effect of exposure to a representative PAH, β-naphthoflavone (BNF), on the gene expression of CYP1A1 and 1A2 in several key organs. Additionally, its effect on CYP1A marker activity and DEA formation by microsomes collected from induced and non-induced rats were studied. It was hypothesized that induction of CYP1A could increase not only local tissue levels of enzyme, but also its functional activity both in terms of a specific CYP1A substrate and DEA formation from AM.

TRIzol reagent was purchased from Invitrogen (Carlsbad, CA). High-Capacity cDNA Reverse Transcription Kit, SYBR Green SuperMix and 96-well optical reaction plates with optical adhesive films were purchased from Applied Biosystems (Foster City, CA). Real-time PCR primers were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) according to previously published sequences. AM, ethopropazine HCL, β-nicotinamide adenine dinucleotide phosphate tetrasodium (NADPH), β-naphthoflavone (BNF), 7-ethoxyresorufin were purchased from Sigma (St. Louis, MO, USA). Resorufin was purchased from ICN Biochemicals Canada (Toronto, ON). DEA was obtained as a gift from Wyeth Ayerst (Research Monmouth Junction, NJ, USA). Methanol, acetonitrile, hexane (all HPLC grade), triethylamine and sulfuric acid (both analytical grade) were purchased from EM Science (Gibstown, NJ, USA). Potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate, potassium chloride, magnesium chloride hexahydrate, sucrose and calcium chloride dihydrate (all analytical grade) were obtained from BDH (Toronto, ON, Canada). Isoflurane BP was purchased from Benson Medical Industries (Ontario, Canada). Supersomes expressing rat CYP1A1 with supplementation of CYP reductase were purchased from BD Gentest (Woburn, MA).

Section snippets

Animal treatment

The study was approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. Male Sprague–Dawley rats (Charles River, Quebec, Canada) were used in the study. The average ± SD values for their body weights were 302 ± 55 g. At the beginning of the study, there were no significant differences in the body weights of rats assigned to different treatment groups. All the rats were housed in temperature controlled rooms with 12 h light per day. The animals were fed a standard

Results

BNF treatment was associated with significant increases in liver mass compared to the Co treated rats. The liver weights were 17 ± 4.1 and 13 ± 2.8 g in the BNF and Co treated rats, respectively. When the liver weight was normalized to body weight measured at the time of collection, the ratios were 0.040 ± 0.0036 and 0.051 ± 0.0050 in the control and BNF treated rats, respectively (p < 0.05). The rest of the collected organs showed no significant change in response to BNF treatment.

In terms of the

Discussion

As expected, at the level of gene expression, it was observed that BNF resulted in significant increases in CYP1A1 expression in each of the tested tissues. These results are consistent with previous reports which showed that CYP1A1 gene expression was significantly induced in almost all of the tested tissues by similar BNF dose (Sinal et al., 1999). CYP1A2 mRNA, in contrast, was only induced in liver and intestine. Although BNF is considered as a prototypical inducer of CYP1A1 and 1A2, the

Conflict of interest statement

The authors declare that there are no conflicts of interest.

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