N-Deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes

doi:10.1016/S0300-483X(96)03560-3

Toxicology

Volume 117, Issues 2–3, 28 February 1997, Pages 123-131

https://doi.org/10.1016/S0300-483X(96)03560-3 Get rights and content

Abstract

Ketoconazole (KT) is an azole antifungal agent that has been associated with hepatotoxicity. The mechanism of its hepatotoxicity has not yet been resolved. It has been suggested that a reactive metabolite may be the cause of toxicity because the hepatic injury does not appear to be mediated through an immunoallergic mechanism. Several metabolites of KT have been reported in the literature of which the deacetylated metabolite, N-deacetyl ketoconazole (DAK), is the major metabolite which undergoes further metabolism by the flavin-containing monooxygenases (FMO) to form a potentially toxic dialdehyde. The objective of this study was to evaluate DAK's cytotoxicity and the role of FMO in a primary culture system of rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). The cultures were exposed to various concentrations of DAK (20–160 μM) for 0.5–4 h. There was a significant increase (P < 0.05) in LDH leakage and an immediate decrease in MTT reduction (P < 0.05) as early as 0.5 h. The MTT reduction assay appeared to be more sensitive than the LDH assay in that lower concentrations were needed to observe a 50% reduction of MTT (107, 90, 75, 58 μM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively). The concentrations to observe 50% LDH leakage from the hepatocytes were 155, 133, 100, 70 μM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively. Moreover, co-treatment with methimazole, a competitive substrate for FMO, produced a significant decrease (P < 0.05) in % LDH leakage as early as 0.5 h, when compared to cells treated solely with DAK. Also, the toxicity was significantly (P < 0.05) enhanced as early as 0.5 h by n-octylamine, a known positive effector for FMO. These results demonstrate that DAK is a more potent cytotoxicant than its parent compound, KT, as reported previously by our laboratory (Rodriguez and Acosta, Toxicology, 96: 83–92, 1995) and its toxicity was expressed in a dose- and time-dependent manner. Furthermore, DAK's cytotoxicity was enhanced with n-octylamine and suppressed with methimazole, suggesting a role for FMO in the toxicity of the metabolite.

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Ketoconazole is a strong inhibitor of cytochrome P450 3A4 (CYP3A4) and of P-glycoprotein (P-gp) and is often used as an index inhibitor especially for CYP3A4-mediated drug metabolism. A preliminary physiologically based pharmacokinetic (PBPK) model for drug-drug interactions indicated possible involvement of a metabolite to the perpetrator potential of ketoconazole. Still unknown for humans, in rodents, N-deacetyl ketoconazole (DAK) has been identified as the major ketoconazole metabolite. We therefore investigated in vitro, whether DAK also inhibits the human CYPs and drug transporters targeted by ketoconazole and quantified DAK in human plasma from healthy volunteers after receiving a single oral dose of 400 mg ketoconazole. Our data demonstrated that DAK also inhibits CYP3A4 (2.4-fold less potent than ketoconazole), CYP2D6 (13-fold more potent than ketoconazole), CYP2C19 (equally potent), P-gp (3.4-fold less potent than ketoconazole), breast cancer resistance protein (more potent than ketoconazole) and organic anion transporting polypeptide 1B1 and 1B3 (7.8-fold and 2.6-fold less potent than ketoconazole). After a single oral dose of 400 mg ketoconazole, maximum concentrations of DAK in human plasma were only 3.1 ‰ of the parent compound. However, assuming that DAK also highly accumulates in the human liver as demonstrated for rodents, inhibition of the proteins investigated could also be conceivable in vivo. In conclusion, DAK inhibits several CYPs and drug transporters, which might contribute to the perpetrator potential of ketoconazole.
Arylacetamide deacetylase knockout mice are sensitive to ketoconazole-induced hepatotoxicity and adrenal insufficiency
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Wewering et al. (2017) reported that DAK showed higher cytotoxicity (LC50: 13.3 μM) than KC (43.6 μM) against HepG2 cells after 24 hr of treatment [13]. In addition, Rodriguez et al. (1997b) reported that the cytotoxicity of DAK was enhanced by n-octylamine, an activator of FMO, whereas it was attenuated by methimazole, an inhibitor of FMO in rat primary hepatocytes [18]. Supporting these findings, we previously revealed that DAK showed higher cytotoxicity (LC50: 22.8 μM) than KC (over 50 μM) in HepaRG cells after 24 hr of treatment, that the overexpression of AADAC resulted in increased cytotoxicity of KC, and that treatment with diisopropylfluorophosphate, an inhibitor of AADAC, decreased KC-induced leakage of lactate dehydrogenase in human primary hepatocytes [16].
Orally administered ketoconazole may rarely induce liver injury and adrenal insufficiency. A metabolite formed by arylacetamide deacetylase (AADAC)-mediated hydrolysis has been observed in cellulo studies, and it is relevant to ketoconazole-induced cytotoxicity. This study tried to examine the significance of AADAC in ketoconazole-induced toxicity in vivo using Aadac knockout mice. Oral administration of 150 mg/kg ketoconazole resulted in the area under the plasma concentration-time curve values of ketoconazole and N-deacetylketoconazole, a hydrolyzed metabolite of ketoconazole, in Aadac knockout mice being significantly higher and lower than those in wild-type mice, respectively. With the administration of ketoconazole (300 mg/kg/day) for 7 days, Aadac knockout mice showed higher mortality (100%) than wild-type mice (42.9%), and they also showed significantly higher plasma alanine transaminase and lower corticosterone levels, thus representing liver injury and steroidogenesis inhibition, respectively. It was suggested that a higher plasma ketoconazole concentration likely accounts for the inhibition of the synthesis of corticosterone, which has anti-inflammatory effects, in the adrenal gland in Aadac KO mice. In Aadac knockout mice, hepatic mRNA levels of immune- and inflammation-related factors were increased by the administration of 300 mg/kg ketoconazole, and the increase was restored by the replenishment of corticosterone (40 mg/kg, s.c.) along with recoveries of plasma alanine transaminase levels. In conclusion, Aadac defects exacerbate ketoconazole-induced liver injury by inhibiting glucocorticoid synthesis and enhancing the inflammatory response. This in vivo study revealed that the hydrolysis of ketoconazole by AADAC can mitigate ketoconazole-induced toxicities.
Kinetic study and structural elucidation of the main ketoconazole metabolite
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Furthermore, DAK is recognized to be the main toxic KTZ detivative in different hepatic cell lineages [10]. Concerning the biological degradation pathway of KTZ, an esterase would catalyze deacetylation of KTZ into DAK [1,4], despite any enzyme responsible for such reaction are not still reported [4]. Studies also report that such initial KTZ metabolism is performed by hepatic microsomal enzymes [1,11].
Ketoconazole (KTZ) is a commonly used drug worldwide for both skin and systemic treatment of mycotic infections. A major concern regarding the oral use of this drug is the possibility of severe hepatotoxicity caused mainly by its major metabolite, namely, N-deacetyl-KTZ (DAK), which is also found as a KTZ impurity. Based on the toxicological relevance of DAK, here we assessed the time-dependent KTZ concentration in pH 2 and 37 °C by quantitative ¹H nuclear magnetic resonance (NMR) with high specificity and sensitivity, and obtained for the first time a crystal form of DAK, as a dihydrochloride salt. The overall conformation of DAK resembles that found in the crystal structure of racemic KTZ and of its pure 2S,4R-enantiomer. The molecules are line-shaped, with the imidazole ring laid back on itself due to an intramolecular CH … π interaction between one methylene group and the imidazole ring. Centrossymetric dimers made up of two DAKH₂Cl₂ units held together by four classical NH⋯Cl-hydrogen bonds build the three-dimensional network. Besides such structural knowledge, its crystal structure provides an analytical pattern in the solid state for this relevant toxic metabolite and impurity from KTZ.
Monoamine Oxidases and Flavin-Containing Monooxygenases
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Progress in structural biology and related disciplines has advanced our knowledge of the physical and mechanistic properties of flavoproteins. This article will focus on two classes of flavoproteins (i.e., monoamine oxidase A and B (MAO A and B) and the flavin-containing monooxygenases (FMOs)) that are involved in the biotransformation of chemicals, drugs, pesticides, and xenobiotics in both small and large animals. Emphasis will be placed on the human enzymes, but comparison with animal forms will highlight the differences. MAO and FMO enzymes have largely been associated with detoxication of chemicals to more polar and generally less toxic metabolites that are usually efficiently excreted. Sometimes, however, MAOs or FMOs convert less toxic chemicals to more toxic materials. This article will attempt to present a view of the balance between these dual roles of MAO and FMO flavoenzymes. Before a detailed discussion of each enzyme is considered, however, a few general comments about the manner that MAO A and B and the FMOs fit into the overall view of the family of flavoproteins that exist in nature will be presented.
Development of a quantitative cytotoxicity assay using mouse lymphoma TK cells
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We describe a new, quantitative, cytotoxicity method using mouse lymphoma L5178Y thymidine kinase +/− (TK) suspension cells for medical device safety assessment that has equivalent sensitivity, and greater efficiency compared to the colony formation assay (CFA) using adherent V79-4 cells. Assays were carried out in 24-well plates. TK cells were seeded on concave side of six types of contact lenses and cell growth rate after 72 h determined using the Vi-Cell XR. Five of six contact lenses studied demonstrated the same results between the TK and the CFA methods. One lens showed a difference between the CFA and TK suspension cell methods (79% colony formation rate vs. 17% cell growth rate). Although colony size is not a CFA test criterion, colonies were smaller than control indicating a cytotoxic effect that is reflected in the TK suspension cell assay which measures cell number. The mouse lymphoma TK suspension cell assay performs as well as the CFA direct contact method with distinct advantages. This quantitative, automated method is not susceptible to mechanical damage from test materials, does not depend on cell adherence to test materials and represents a valuable tool to evaluate medical device cytotoxicity.
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Studies in rat postnatal hepatocytes showed that KET was cytotoxic in a dose and time-dependent manner.91 Furthermore, KET is metabolized by flavin monooxygenases and other enzymes to N-deacetyl- KET, which is more toxic than the parent drug and can undergo further metabolism to generate reactive aldehyde that can induce toxicity.92 In vivo studies in rats also showed that KET can lead to the depletion of glutathione and covalently bind to hepatic proteins in microsomes.93

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N-Deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes

Abstract

J. Biol. Chem.

J. Am. Acad. Dermatol.

Lancet

J. Immunol. Methods

Biochem. Pharmacol.

Toxicology

Biochem. Pharmacol

J. Hepatol.

J. Infect.

J. Pharm. Biomed. Anal.

J. Pharm. Biomed. Anal.

Toxicology

Preparation of primary monolayer cultures of postnatal rat liver cells

J. Tissue Cult. Methods

An in vitro approach to the study of target organ toxicity of drugs and chemicals

In Vitro Cell. Dev. Biol.

Prolonged jaundice following ketoconazole-induced hepatic injury

Dig. Dis. Sci.

Ketoconazole-induced fulminate hepatitis

Gut

Azole antifungal agents

Clin. Infect. Dis.

Disposition of ketoconazole, an oral antifungal, in humans

Antimicrob. Agents Chemother.

Ketoconazole hepatotoxicity in a patient treated for environmental illness and systemic candidiasis

Ann. Pharmacother.

Contribution of N-oxygenation to the metabolism of MPTP (l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) by various liver preparations

Mol. Pharmacol.

Safety aspects of ketoconazole, the most commonly used systemic antifungal

Mycoses