Impact of inhalational exposure to ethanol fuel on the pharmacokinetics of verapamil, ibuprofen and fluoxetine as in vivo probe drugs for CYP3A, CYP2C and CYP2D in rats

Highlights

• The effect of ethanol fuel inhalation on cytochrome P450 activity was investigated.

• Verapamil, ibuprofen and fluoxetine were used as probes of CYP3A, 2C and 2D.

• Inhalation of ethanol fuel at 2 times the TLV-STEL induced CYP3A and CYP2C, but inhibited CYP2D in rats.

Abstract

Occupational toxicology and clinical pharmacology integration will be useful to understand potential exposure-drug interaction and to shape risk assessment strategies in order to improve occupational health. The aim of the present study was to evaluate the effect of exposure to ethanol fuel on in vivo activities of cytochrome P450 (CYP) isoenzymes CYP3A, CYP2C and CYP2D by the oral administration of the probe drugs verapamil, ibuprofen and fluoxetine. Male Wistar rats exposed to filtered air or to 2000 ppm ethanol in a nose-only inhalation chamber during (6 h/day, 5 days/week, 6 weeks) received single oral doses of 10 mg/kg verapamil or 25 mg/kg ibuprofen or 10 mg/kg fluoxetine. The enantiomers of verapamil, norverapamil, ibuprofen and fluoxetine in plasma were analyzed by LC-MS/MS. The area under the curve plasma concentration versus time extrapolated to infinity (AUC^0–∞) was calculated using the Gauss–Laguerre quadrature. Inhalation exposure to ethanol reduces the AUC of both verapamil (approximately 2.7 fold) and norverapamil enantiomers (>2.5 fold), reduces the AUC^0–∞ of (+)-(S)-IBU (approximately 2 fold) and inhibits preferentially the metabolism of (−)-(R)-FLU. In conclusion, inhalation exposure of ethanol at a concentration of 2 TLV-STEL (6 h/day for 6 weeks) induces CYP3A and CYP2C but inhibits CYP2D in rats.

Introduction

Ethanol is among the chemicals of great importance for the human occupational exposure in terms of volume produced and the extent of its distribution. It is estimated that more than 650,000 workers are potentially exposed to ethanol mainly by inhalation but also by dermal route. Beyond the alcoholic beverages, the major industrial uses of ethanol are as solvent, as disinfectant, as intermediate in the synthesis of other chemicals and increasingly as biofuel (Anses, 2013, Bevan et al., 2009). Occupational exposure limits for ethanol are consistent around the world (Bevan et al., 2009). The American Conference of Governmental Industrial Hygienists (ACGIH) recommends a threshold limit value – short-term exposure limit (TLV-STEL) of 1880 mg/m³ (or 1000 ppm) to protect from respiratory, ocular irritation as well as long-term effects of ethanol vapor exposure (ACGIH, 2011). Ethanol is classified as a confirmed animal carcinogen with unknown relevance to humans (A3) by ACGIH. On the other hand, alcoholic beverages are classified as category 1 by the International Agency for Research on Cancer (IARC) due to sufficient evidence for carcinogenicity in humans. These different classifications are mainly based on different routes of exposure (inhalation versus oral), frequency and extent of exposure. It is estimated that exposure by inhalation to 1000 ppm for 8 h would be equivalent to the consumption of 12 g orally in 8 h in terms of amount absorbed, which means negligible increase of risk carcinogenesis (Bevan et al., 2009, American Conference, 2011).

Hepatic metabolism is responsible for the elimination of approximately 90% of ethanol, and is dependent mainly on alcohol dehydrogenase. Catalase and the microsomal cytochrome P450 (CYP) also contribute to ethanol metabolism, but in a lesser extent. The contribution of catalase and CYP isoforms to ethanol metabolism can increase in the presence of high amounts of H₂O₂ and after chronic alcohol consumption, respectively (Bruckner et al., 2013). Studies using human liver microsomes reported that CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2C8, CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4 and CYP4A11 convert ethanol in acetaldehyde with apparent K_m values around 10 mM. The selective inhibition of CYP2C9, CYP2C19, CYP2E1, CYP3A4 e CYP1A2 reduces ethanol oxidation in 8  ±  1.2%, 7.6  ±  1.6%, 11.9  ±  2.1%, 19.8  ±  1.9% and 16.3  ±  3.9%, respectively (Hamitouche et al., 2006).

The effect of ethanol exposure through inhalation on cytochrome P450 isoenzymes activity is not fully understood. In human lymphoblastoid microsomes, 0.1–1% ethanol inhibits CYP1A1 (19–69%), CYP2B6 (25–80%), CYP2C19 (28–72%) and CYP2D6 (11–59%) (Busby et al., 1999). Ethanol at 1% inhibited phenacetin O-deethylation (CYP1A) in rat liver microsomes by >20% (Li et al., 2010). Ethanol increased the K_m and decreased the intrinsic clearance of CYP2B6-mediated bupropion hydroxylation and of CYP2C8-mediated paclitaxel hydroxylation, in a concentration-dependent manner in human liver microsomes (Vuppugalla et al., 2007). Inhalation of ethanol vapors in high concentrations, enough to produces blood ethanol levels of 1.8 g/L, for 4 weeks, induced CYP2E1 in rats (Zerilli et al., 1995). The induction of CYP2E1 by chronically consumers of alcoholic beverages results in accelerated metabolism of many CYP2E1 substrates, such as paracetamol, halothane and chlorzoxazone. Thus, decreased drug efficacy of CYP2E1 substrates or the accumulation of its active metabolites are usually observed in patients chronically consuming ethanol (Klotz and Ammon, 1998).

The objective of the present study is to investigate the influence of exposure to ethanol fuel in a nose-only inhalation chamber on the pharmacokinetics of the verapamil, ibuprofen and fluoxetine enantiomers as biomarkers of in vivo activity of CYP3A, CYP2C and CYP2D, respectively, in rats. The integration of occupational toxicology with clinical pharmacology will be useful to understand the solvent–drug interaction and to shape risk assessment strategies in order to improve occupational health (Flack and Nylander-French, 2012).