In vitro deacetylation of N-acetylserotonin by arylacetamide deacetylase.

Arylacetamide deacetylase (AADAC) is a deacetylation enzyme present in the mammalian liver, gastrointestinal tract, and brain. During our search for mammalian enzymes capable of metabolizing N-acetylserotonin (NAS), AADAC was identified as having the ability to convert NAS to serotonin. Both human and rodent recombinant AADAC proteins can deacetylate NAS in vitro, although the human AADAC shows markedly higher activity compared with rodent enzyme. The AADAC-mediated deacetylation reaction can be potently inhibited by eserine in vitro. In addition to NAS, recombinant hAADAC can deacetylate melatonin (to form 5-methoxytryptamine) and N-acetyltryptamine (NAT) (to form tryptamine). In addition to the in vitro deacetylation of NAS by the recombinant AADAC proteins, liver (mouse and human) and brain (human) extracts were able to deacetylate NAS; these activities were sensitive to eserine. Taken together, these results demonstrate a new role for AADAC and suggest a novel pathway for the AADAC-mediated metabolism of pineal indoles in mammals.

Arylacetamide deacetylase knockout mice are sensitive to ketoconazole-induced hepatotoxicity and adrenal insufficiency.

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.

Human superoxide dismutase 1 attenuates quinoneimine metabolite formation from mefenamic acid.

Mefenamic acid (MFA), one of the nonsteroidal anti-inflammatory drugs (NSAIDs), sometimes causes liver injury. Quinoneimines formed by cytochrome P450 (CYP)-mediated oxidation of MFA are considered to be causal metabolites of the toxicity and are detoxified by glutathione conjugation. A previous study reported that NAD(P)H:quinone oxidoreductase 1 (NQO1) can reduce the quinoneimines, but NQO1 is scarcely expressed in the human liver. The purpose is to identify enzyme(s) responsible for the decrease in MFA-quinoneimine formation in the human liver. The formation of MFA-quinoneimine by recombinant CYP1A2 and CYP2C9 was significantly decreased by the addition of human liver cytosol, and the extent of the decrease in the metabolite formed by CYP1A2 was larger than that by CYP2C9. By column chromatography, superoxide dismutase 1 (SOD1) was identified from the human liver cytosol as an enzyme decreasing MFA-quinoneimine formation. Addition of recombinant SOD1 into the reaction mixture decreased the formation of MFA-quinoneimine from MFA by recombinant CYP1A2. By a structure-activity relationship study, we found that SOD1 decreased the formation of quinoneimines from flufenamic acid and tolfenamic acid, but did not affect those produced from acetaminophen, amodiaquine, diclofenac, and lapatinib. Thus, SOD1 may selectively decrease the quinoneimine formation from fenamate-class NSAIDs. To examine whether SOD1 can attenuate cytotoxicity caused by MFA, siRNA for SOD1 was transfected into CYP1A2-overexpressed HepG2 cells. The leakage of lactate dehydrogenase caused by MFA treatment was significantly increased by knockdown of SOD1. In conclusion, we found that SOD1 can serve as a detoxification enzyme for quinoneimines to protect from drug-induced toxicity.

Substrate selectivity of human aldehyde oxidase 1 in reduction of nitroaromatic drugs.

Human aldehyde oxidase 1 (AOX1) catalyzes the oxidation of various drugs and endogenous compounds. Recently, we found that AOX1 catalyzed the reduction of drugs such as nitrazepam and dantrolene. In this study, we aimed to clarify the substrate selectivity of human AOX1 for the reduction of nitroaromatic drugs to obtain helpful information for drug development. We investigated whether 11 nitroaromatic drugs were reduced by AOX1 using recombinant AOX1 and human liver cytosol (HLC) in the presence of N1-methylnicotinamide, an electron donor to AOX1. We found that clonazepam, flunitrazepam, flutamide, nilutamide, nimesulide, and nimetazepam were substantially reduced by recombinant AOX1 and HLC, whereas azelnidipine, nifedipine, and nimodipine were slightly reduced and metronidazole and tolcapone were not reduced. Via structural analysis, we observed that nitroaromatic drugs reduced by AOX1 possessed a relatively electron-deficient nitro group. Since the addition of NADPH to human liver microsomes (HLM) did not increase the reductase activities of the drugs that were reduced by recombinant AOX1, it was determined that NADPH-dependent enzymes in microsomes, such as cytochrome P450, were not involved in this process. Inhibition studies using known AOX1 inhibitors supported the role of AOX1 in the reduction of drugs in HLC. In conclusion, this provides new information related to the substrate selectivity of human AOX1 for the reduction of nitroaromatic drugs.

In vitro approach to elucidate the relevance of carboxylesterase 2 and N-acetyltransferase 2 to flupirtine-induced liver injury.

The use of flupirtine, an analgesic, has been restricted in European countries because it causes liver injury in rare cases. Flupirtine is primarily metabolized to D-13223, an acetylamino form. In the process of D-13223 formation, it has been hypothesized that a reactive metabolite is formed which may be involved in flupirtine hepatotoxicity. The purpose of this study was to identify the potential reactive metabolite and the responsible enzymes in the human liver to get a clue to the mechanism of hepatotoxicity. Using recombinant enzymes, we found that D-13223 was formed from flupirtine via hydrolysis by carboxylesterase 2 (CES2) and subsequent acetylation by N-acetyltransferase (NAT) 2. A conjugate of N-acetyl-l-cysteine (NAC), a nucleophile, was detected by incubation of flupirtine with CES2, and the conjugate formation in human liver microsomes was inhibited by CES2 inhibitors, indicating that a reactive metabolite, which may be a quinone diimine, was produced in the process of CES2-mediated hydrolysis of flupirtine. The formation of the NAC conjugate in liver S9 samples from NAT2 slow acetylators was significantly higher than that from NAT2 rapid/intermediate acetylators, indicating that NAT2 could function as a detoxification enzyme for flupirtine. CES2-overexpressing HepG2 cells showed remarkable lactate dehydrogenase leakage under flupirtine treatment, while no cytotoxicity was observed in control cells, suggesting that the reactive metabolite formed by CES2-mediated hydrolysis of flupirtine would be a trigger of hepatotoxicity. NAT2 slow acetylators with high CES2 activity could be highly susceptible to flupirtine-induced liver injury.

Identification of enzymes responsible for nitrazepam metabolism and toxicity in human.

Nitrazepam (NZP) is a hypnotic agent that rarely causes liver injuries in humans and teratogenicity in rodents. In humans, NZP is primarily metabolized to 7-aminonitrazepam (ANZP) by reduction and subsequently to 7-acetylamino nitrazepam (AANZP) by acetylation. ANZP can be regenerated from AANZP by hydrolysis in rodents, but it is still unclear whether this reaction occurs in humans. In rodents, AANZP may be associated with teratogenicity, while in humans, it is known that drug-induced liver injuries may be caused by NZP reactive metabolite(s). In this study, we attempted to identify the enzymes responsible for NZP metabolism to obtain a basic understanding of this process and the associated metabolite toxicities. We found that the NZP reductase activity in human liver cytosol (HLC) was higher than that in human liver microsomes (HLM). We purified the responsible enzyme(s) from HLC and found that the NZP reductase was aldehyde oxidase 1 (AOX1). The role of AOX1 was confirmed by an observed increase in the NZP reductase activity upon addition of N1-methylnicotinamide, an electron donor of AOX1, as well as inhibition of this activity in HLC in the presence of AOX1 inhibitors. ANZP was acetylated to form AANZP by N-acetyltransferase (NAT) 2. An experiment using recombinant esterases in an inhibition study using HLM revealed that AANZP is hydrolyzed by arylacetamide deacetylase (AADAC) in the human liver. N-Hydroxylamino NZP, which is suspected to be a reactive metabolite, was detected as a conjugate with N-acetyl-l-cysteine through NZP reduction and ANZP hydroxylation reactions. In the latter reaction, the conjugate was readily formed by recombinant CYP3A4 among the various P450 isoforms tested. In sum, we found that AOX1, NAT2, AADAC, and CYP3A4 are the determinants for the pharmacokinetics of NZP and that they confer interindividual variability in sensitivity to NZP side effects.

Human arylacetamide deacetylase hydrolyzes ketoconazole to trigger hepatocellular toxicity.

Ketoconazole (KC), an antifungal agent, rarely causes severe liver injury when orally administered. It has been reported that KC is mainly hydrolyzed to N-deacetyl ketoconazole (DAK), followed by the N-hydroxylation of DAK by flavin-containing monooxygenase (FMO). Although the metabolism of KC has been considered to be associated with hepatotoxicity, the responsible enzyme(s) remain unknown. The purpose of this study was to identify the responsible enzyme(s) for KC hydrolysis in humans and to clarify their relevance to KC-induced toxicity. Kinetic analysis and inhibition studies using human liver microsomes (HLM) and recombinant enzymes revealed that human arylacetamide deacetylase (AADAC) is responsible for KC hydrolysis to form DAK, and confirmed that FMO3 is the enzyme responsible for DAK N-hydroxylation. In HLM, the clearance of KC hydrolysis occurred to the same extent as DAK N-hydroxylation, which indicates that both processes are not rate-limiting pathways. Cytotoxicity of KC and DAK was evaluated using HepaRG cells and human primary hepatocytes. Treatment of HepaRG cells with DAK for 24h showed cytotoxicity in a dose-dependent manner, whereas treatment with KC did not show due to the low expression of AADAC. Overexpression of AADAC in HepaRG cells with an adenovirus expression system elicited the cytotoxicity of KC. Cytotoxicity of KC in human primary hepatocytes was attenuated by diisopropylfluorophosphate, an AADAC inhibitor. In conclusion, the present study demonstrated that human AADAC hydrolyzes KC to trigger hepatocellular toxicity.