Metabolic activation by human arylacetamide deacetylase, CYP2E1, and CYP1A2 causes phenacetin-induce
Metabolic activation by human arylacetamide deacetylase,CYP2E1,and CYP1A2 causes phenacetin-induced methemoglobinemia
Yuki Kobayashi,Tatsuki Fukami,Ryota Higuchi,Miki Nakajima,Tsuyoshi Yokoi*
Drug Metabolism and Toxicology,Faculty of Pharmaceutical Sciences,Kanazawa University,Kakuma-machi,Kanazawa920-1192,Japan
1.Introduction
Phenacetin has been widely used as an analgesic antipyretic.
Although phenacetin had been developed as a prodrug of
acetaminophen(APAP),it was withdrawn from the market as it
caused methemoglobinemia and renal failure[1,2].Phenacetin is
primarily metabolized to APAP through O-deethylation by human
cytochrome(CYP)1A2as well as partially by CYP2E1and is also
metabolized to p-phenetidine through a hydrolysis reaction[3–5].
p-Phenetidine is considered to be further metabolized to N-
hydroxy-p-phenetidine,a metabolite thought to contribute
adverse effects[1,6].
Methemoglobin(Met-Hb)is formed when the iron of hemo-
globin is oxidized from the ferrous(Fe2+)to the ferric state(Fe3+).
Met-Hb cannot bind and transport oxygen;consequently,the
increased levels of Met-Hb are associated with clinically severe
symptoms[7].The normal level of Met-Hb is1.5–2%of the total
hemoglobin.As the Met-Hb level rises above10%of the total
hemoglobin levels,cyanosis usually develops.This is followed by
anxiety,fatigue,and tachycardia at Met-Hb levels between20%
and50%.Finally,under conditions of Met-Hb levels exceeding
50–70%of the total hemoglobin,coma and death may occur[8].
The metabolic pathway of phenacetin hydrolysis and subse-
quent metabolism is thought to contribute to phenacetin-induced
methemoglobinemia.However,this involvement has not been
experimentally shown.Contributing to this paucity in experimen-
tal evidence is the fact that the enzymes responsible for
phenacetin-induced methemoglobinemia have not been identi-
?ed.Previously,we have shown that human arylacetamide
deacetylase(AADAC)is the principal enzyme catalyzing the
hydrolysis of phenacetin[9].AADAC is one of the major serine
esterases expressed in human liver and the gastrointestinal tract
[10].AADAC is capable of hydrolyzing the antiandrogen drug
?utamide,the antituberculosis drug rifampicin[10,11],and
phenacetin.Nakayama and Masuda reported that an NADPH-
dependent enzyme(s)is associated with an increase in the
formation of Met-Hb formation induced by p-phenetidine[12].
Biochemical Pharmacology84(2012)1196–1206
A R T I C L E I N F O
Article history:
Received12July2012
Accepted17August2012
Available online23August2012
Keywords:
Arylacetamide deacetylase
Cytochrome P450
Phenacetin
Methemoglobinemia
A B S T R A C T
Phenacetin has been used as an analgesic antipyretic but has now been withdrawn from the market due
to adverse effects such as methemoglobinemia and renal failure.It has been suggested that metabolic
activation causes these adverse effects;yet,the precise mechanisms remain unknown.We previously
demonstrated that human arylacetamide deacetylase(AADAC)was the principal enzyme catalyzing the
hydrolysis of phenacetin.In this study,we assessed whether AADAC was involved in phenacetin-induced
methemoglobinemia.A high methemoglobin(Met-Hb)level in the blood was detected1h after
administration of phenacetin(250mg/kg,p.o.)to male C57BL/6mice.Pre-administration of tri-o-
tolylphosphate,a general esterase inhibitor,was found to decrease the levels of Met-Hb and the plasma
concentration of p-phenetidine,a hydrolyzed metabolite of phenacetin.An in vitro study using red blood
cells revealed that incubation of phenacetin or p-phenetidine with human liver microsomes(HLM)
increased the formation of Met-Hb.To identify the enzymes involved in the formation of Met-Hb,we
used recombinant enzymes and HLM treated with inhibitors in the measurement of the formation of
Met-Hb.High levels of Met-Hb were observed following incubation of human AADAC with either
cytochrome P450(CYP)1A2or CYP2E1.Furthermore,the increased Met-Hb formation by the incubation
of HLM with phenacetin was signi?cantly inhibited to25.1?0.7%of control by eserine,a potent AADAC
inhibitor.In conclusion,we found that the hydrolysis by AADAC and subsequent metabolism by CYP1A2and
CYP2E1play predominant roles in phenacetin-induced methemoglobinemia.
?2012Elsevier Inc.All rights reserved.
Abbreviations:AADAC,arylacetamide deacetylase;APAP,acetaminophen;BNPP,
bis-(p-nitrophenyl)phosphate;CES,carboxylesterase;CYP,cytochrome P450;FMO,
?avin-containing monooxygenase;HLM,human liver microsomes;i.p.,intraperi-
toneal;MLM,mouse liver microsomes;PNPA,p-nitrophenyl acetate;p.o.,per os;
RT-PCR,reverse transcription-polymerase chain reaction;TOTP,tri-o-tolyl phos-
phate.
*Corresponding author.Tel.:+81762344407;fax:+81762344407.
E-mail address:tyokoi@kenroku.kanazawa-u.ac.jp(T.Yokoi).
Contents lists available at SciVerse ScienceDirect
Biochemical Pharmacology
j o u rn a l h om e p a g e:w w w.e l s e v i e r.c o m/l o c a t e/bi o c h e m p h a r m
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https://www.wendangku.net/doc/d66929444.html,/10.1016/j.bcp.2012.08.015
Therefore,representative NADPH-dependent enzymes such as the CYP enzymes may play an important role in the metabolism of p-phenetidine and formation of Met-Hb.CYP enzymes are widely involved in drug metabolism(approximately75%of all clinically used drugs)and are sometimes involved in the development of toxicity.Building on this background information,we investigated with in vivo and in vitro studies whether AADAC and CYP enzymes are responsible for phenacetin hydrolysis and the subsequent metabolism,respectively,resulting in phenacetin-induced methe-moglobinemia.
2.Materials and methods
2.1.Chemicals and reagents
APAP,phenacetin,potassium cyanide,potassium hexacyano-ferrate(III),p-nitrophenol,and eserine were purchased from Wako Pure Chemical Industries(Osaka,Japan).p-Phenetidine,p-nitro-phenyl acetate(PNPA),chlorzoxazone,6-hydroxychlorzoxazone, and bis-(p-nitrophenyl)phosphate(BNPP)were obtained from Sigma–Aldrich(St.Louis,MO).Tri-o-tolyl phosphate(TOTP)was obtained from Kanto Chemical(Tokyo,Japan).Human liver microsomes(HLM)(pooled donors,n=50),recombinant human CYP1A2,CYP2A6,CYP2B6,CYP2C8,CYP2C9,CYP2C19,CYP2D6, CYP3A4,and CYP3A5that were expressed in baculovirus-infected insect cells,monoclonal mouse anti-human CYP1A2antibody, monoclonal mouse anti-human CYP2E1antibody,and monoclonal mouse anti-human CYP3A4antibody were purchased from BD Gentest(Woburn,MA).Primers were commercially synthesized at Hokkaido System Sciences(Sapporo,Japan).All other chemicals used in this study were of the highest or analytical quality that could be obtained commercially.
2.2.Animals
C57BL/6J mice(6-week old males,20–25g)were obtained from SLC Japan(Hamamatsu,Japan).Mice were housed in the institu-tional animal facility in a controlled environment(temperature 25?18C and12-h light/dark cycle)with access to food and water ad libitum.Mice were acclimated for one week prior to their use in our experiments.Animal maintenance and treatment were conducted in accordance with the National Institutes of Health Guide for Animal Welfare of Japan,and the protocols were approved by the Institutional Animal Care and Use Committee of Kanazawa University,Japan.
2.3.Preparation of mouse and human red blood cells
The use of human red blood cells was approved by the Ethics Committee of Kanazawa University(Kanazawa,Japan).Samples of human blood were obtained from5healthy male Japanese subjects (22–30years of age).Mouse and human blood samples were allowed to stand for30min and centrifuged3times at1000?g for 10min,following which the plasma and buffy coats were removed. The red blood cells precipitated were used in this study.All of the in vitro assays were performed immediately after separation of the red blood cells.
2.4.Administration of phenacetin and TOTP
Phenacetin(at250mg/kg and suspended in9%solutol,p.o.)was administered to mice that were not fasted.At various times(0, 0.25,0.5,1,3,6h)after administering phenacetin,blood was collected from the inferior vena cava for the measurement of Met-Hb levels,phenacetin,APAP,and p-phenetidine.To investi-gate the effect of TOTP on Aadac mRNA and protein expression and enzymatic activity in the liver,TOTP(125mg/kg,dissolved in corn oil,i.p.)was administered to mice,followed by the collection of liver samples at various time points(0,1,6,12h)immediately after administration of TOTP.To study the effect of TOTP on phenacetin-induced methemoglobinemia,mice were pretreated with TOTP (125mg/kg,i.p.)or vehicle,followed by administration of phenacetin(250mg/kg,p.o.)12h later.One hour after adminis-tration of phenacetin,blood samples were collected.
2.5.In vivo Met-Hb level
Met-Hb levels in blood were determined as a percent of the total hemoglobin according to the procedure used by Wang et al.
[13]with some modi?cations.A volume of100m l of blood was hemolyzed with8.0ml of distilled water followed by the addition of2.0ml of100mM sodium–potassium phosphate buffer(pH6.6). The hemolysate was centrifuged at1000?g for10min,and the supernatant was divided into4equal parts(2.5ml each).The absorbance of the?rst part was measured at630nm(A1)(the absorbance maximum for methemoglobin)using a U-2001 spectrophotometer(Hitachi,Tokyo,Japan).The sample was then measured again at630nm after the addition of25m l of5% potassium cyanide(A2).Potassium cyanide converts methemoglo-bin to cyanomethemoglobin,which does not absorb at630nm. Hence,the difference between the absorbance values of A1and A2 represents the absorbance due to methemoglobin.To measure the total hemoglobin levels,all of the hemoglobin was converted to methemoglobin using potassium hexacyanoferrate(III).Following the addition of25m l of20%potassium hexacyanoferrate(III)into the second part of the hemolysate,the sample was incubated for 30min at room temperature,and the absorbance was measured at 630nm(A3)and again at630nm after the addition of25m l of5% potassium cyanide(A4).The percentage of methemoglobin was calculated according to the following equation:
Met-Hbe%T?
A1àA2
A3àA4
?100
2.6.Plasma concentrations of phenacetin and its metabolites
The concentrations of phenacetin and p-phenetidine in the plasma were measured by adding50m l of10%HClO4to50m l of plasma in a reaction that precipitated the protein.The mixture was centrifuged at9500?g for10min,and a10m l sample of the supernatant was subjected to HPLC.The HPLC analysis was performed using an L-2130pump(Hitachi),an L-2200autosampler (Hitachi),an L-2400UV detector(Hitachi),and a D-2500chromato-integrator(Hitachi)equipped with a Mightysil RP-18C18GP column (5m m particle size,4.6mm i.d.?150mm:Kanto Chemical,Tokyo, Japan).The eluent was monitored at232nm with a noise-base clean Uni-3(Union,Gunma,Japan),which can reduce the noise by integrating the output and increase the signal3-fold by differenti-ating the output,and5-fold by further ampli?cation with an internal ampli?er,resulting in a maximum15-fold ampli?cation of the original signal.The mobile phase was25%methanol containing 50mM potassium dihydrogenphosphate.The?ow rate was1.0ml/ min.The column temperature was35?C.The quanti?cation of phenacetin and p-phenetidine was performed by comparing the HPLC peak heights with that of an authentic standard.
The concentration of APAP in the plasma was measured as follows:50m l of ice-cold acetonitrile was added to the50m l of the plasma sample to precipitate protein.The subsequent procedure was the same as that described for measuring phenacetin and p-phenetidine with the exception that the UV wavelength was 245nm and the mobile phase was10%methanol containing
Y.Kobayashi et al./Biochemical Pharmacology84(2012)1196–12061197
45mM potassium dihydrogenphosphate.The quanti?cation of APAP was performed by comparing its peak height following HPLC analysis with that of an authentic standard.
2.7.RNA preparation from TOTP-administered mouse liver and real-time reverse transcription-polymerase chain reaction(RT-PCR) analyses
Total RNA samples from the livers of TOTP-administered mice were prepared according to our previous study[14].Reverse transcription was described previously[15].Real-time RT-PCR was performed for quantitative determination of mRNA expression of mouse Aadac and Gapdh according to our previous studies[14,16], except for the PCR protocol for Gapdh mRNA(after an initial denaturation at958C for3min,the ampli?cation was performed by denaturation at948C for4s,annealing and extension at648C for20s for45cycles).
2.8.Preparation of mouse liver microsomes(MLM)
MLM were prepared according to our previous study[14].The protein concentrations were determined according to the method of Bradford[17]using g-globulin as the standard.
2.9.Immunoblot analysis
SDS-polyacrylamide gel electrophoresis and immunoblot anal-ysis were performed according to our previous study[14].MLM (5m g)were separated through10%polyacrylamide gels and electrotransferred onto a polyvinylidene di?uoride membrane (Immobilon-P,Millipore,Billerica,MA).The membrane was probed with monoclonal mouse anti-human AADAC antibody (Abnova,Taipei City,Taiwan)and the corresponding?uorescent dye-conjugated secondary antibody.An Odyssey infrared imaging system(LI-COR Biosciences,Lincoln,NE)was used for detection of protein bands.The band intensity was quanti?ed using Image-Quant TL Image Analysis software(GE Healthcare).The relative expression level of each protein band was determined by the intensity of the band.Analysis of the immunoblot was performed across the linear range of band intensity with respect to the amount of protein.
2.10.Phenacetin hydrolase and O-deethylase activities
The phenacetin hydrolase activity was determined according to the method of Watanabe et al.[9].Final concentrations of the phenacetin and the enzyme source(MLM)were0.1mM and 0.4mg/ml,respectively.
The phenacetin O-deethylase activity was determined accord-ing to the method of Fukami et al.[18]with slight modi?cations.A typical incubation mixture(?nal volume of0.2ml)contained 100mM potassium phosphate buffer(pH7.4),an NADPH-generating system(0.5mM NADP+,5mM glucose6-phosphate, 5mM MgCl2,and1U/ml glucose-6-phosphate dehydrogenase), and MLM(0.4mg/ml).The reaction was initiated by the addition of 0.1mM phenacetin after an initial2-min preincubation at378C. After the30-min incubation,the reaction was terminated by the addition of100m l of ice-cold methanol.The subsequent procedure was the same as that described for the measurement of the concentration of APAP in plasma.
2.11.Inhibitory effects of eserine and BNPP on human AADAC,CES1, and CES2enzyme activities
We previously constructed baculovirus expression systems for human AADAC,carboxylesterase(CES)1,and CES2[9,19].To compare the inhibitory effects of esterase inhibitors(eserine and BNPP)on human AADAC,CES1,and CES2,we undertook an inhibition analysis of the hydrolase activity of PNPA,which is a general esterase substrate.The concentrations of eserine and BNPP were10m M and they were dissolved in distilled water.The PNPA hydrolase activities of recombinant human AADAC,CES1,and CES2 (all at0.1mg/ml)were determined according to the method of Watanabe et al.[10],with the exception that the incubation time was1min.
2.12.Met-Hb formation in vitro
The formation of Met-Hb was assayed according to the method of Nakayama and Masuda[12]with slight modi?cations.A typical incubation mixture(with a?nal volume of0.2ml)contained a10% suspension of mouse red blood cells,100mM potassium phos-phate buffer(pH7.4),an NADPH-generating system,and various sources of enzyme.
(1)For the investigation of the time-dependent formation of Met-
Hb,HLM(1.0mg/ml)were used as a source of enzymes.The reaction was initiated by the addition of1mM phenacetin or p-phenetidine after an initial2-min preincubation at378C.After an incubation period of0to120min at378C,the reaction was terminated by placing the samples on ice.
(2)For the investigation of the substrate concentration-dependent
formation of Met-Hb,HLM(1.0mg/ml)were used as a source of enzymes.The reaction was initiated by the addition of phenacetin(0.01,0.1,1,and5mM),p-phenetidine(0.01,0.1, and1mM),or APAP(0.01,0.1,and1mM)after an initial2-min preincubation at378C.After an incubation period of60min at 378C,the reaction was terminated by placing the samples on ice.
(3)For the determination of the enzymes involved in p-pheneti-
dine-induced methemoglobinemia,recombinant human CYP enzymes(25pmol/ml)were used as the source enzymes.The reaction was initiated by the addition of p-phenetidine(1mM) after an initial2-min preincubation at378C.After an incubation period of60min at378C,the reaction was terminated by placing the samples on ice.
(4)For the determination of the enzymes involved in phenacetin-
induced methemoglobinemia,recombinant human esterases (AADAC,CES1,or CES2)(1.0mg/ml)and recombinant human CYP1A2or CYP2E1(25pmol/ml)were used as the sources of enzyme.The reaction was initiated by the addition of5mM phenacetin after an initial2-min preincubation at378C.After an incubation period of120-min at378C,the reaction was terminated by placing the samples on ice.
(5)In the investigation of the role played by AADAC in phenacetin-
induced methemoglobinemia,the inhibition analysis was performed using esterase inhibitors(eserine and BNPP).In the esterase inhibitor assays,HLM(1.0mg/ml)were used as a source of enzymes and the concentrations of the inhibitors were10m M.The reaction condition was the same as that described under(4)above.
(6)In the investigation of the involvement of CYP1A2and CYP2E1
in p-phenetidine-induced methemoglobinemia,the inhibition analysis was performed using anti-CYP antibodies.Recombi-nant CYP1A2and CYP2E1(25pmol/ml)and HLM(0.5mg/ml) were used as the sources of enzymes.Four microliters of monoclonal mouse anti-human CYP1A2,CYP2E1,or CYP3A4 antibodies were mixed with6m l of25mM Tris-buffer(pH7.5), followed by the addition of the sources of enzyme to this mixture,which was incubated on ice for30min.This typical incubation mixture(?nal volume of0.2ml),which included the antibody mixture described above,had the same reaction conditions as that described under(3)above.
Y.Kobayashi et al./Biochemical Pharmacology84(2012)1196–1206 1198
(7)In the investigation of the sensitivity of human red blood cells
to phenacetin-induced methemoglobinemia,the Met-Hb formation assay was conducted with human red blood cells instead of mouse red blood cells.Recombinant human AADAC
(1.0mg/ml),CYP1A2(25pmol/ml)and CYP2E1(25pmol/ml)
were used as sources of enzyme.The reaction conditions were the same as those described in(4)above.
The levels of Met-Hb in mouse or human red blood cells were determined as a percent of the total hemoglobin.Red blood cells in 50m l of incubation mixture were hemolyzed with800m l of distilled water followed by the addition of200m l of100mM sodium-potassium phosphate buffer(pH6.6).The hemolysate was centrifuged at1000?g for10min,and the supernatant was divided into4parts(0.25ml each).The experimental steps were the same as those described for the in vivo determination of the levels of Met-Hb.The exception here was that the volumes used for both5%potassium cyanide and20%potassium hexacyanoferrate (III),were2.5m l each.
In this preliminary study,we con?rmed that the omission of an enzyme source,an NADPH generating system,or any of the substrates from the incubation mixture did not cause an increase in the formation of Met-Hb.Phenacetin,p-phenetidine,and APAP were dissolved in acetonitrile,and the?nal concentration of acetonitrile in the incubation mixture was2.0%for phenacetin and
1.0%for p-phenetidine and APAP.
2.1
3.Statistical analysis
Statistical analyses for the in vivo study between two or multiple groups were performed using the nonparametric Mann-Whitney U test and nonparametric ANOVA,followed by a Dunn’s test,respectively.Statistical analyses for the in vitro study between multiple groups were performed using ANOVA,followed by the Tukey’s post hoc test.Data were considered statistically signi?cant at P<0.05.
3.Results
3.1.Changes of Met-Hb levels upon phenacetin administration in mice and plasma concentrations of phenacetin and its metabolites
After administration of phenacetin(250mg/kg,p.o.)to male C57BL/6mice,the levels of Met-Hb and the plasma concentrations of phenacetin,p-phenetidine,and APAP were measured at various time points over0–6h(Fig.1).In preliminary work,we could detect the formation of Met-Hb1h after the administration of either250mg/kg or500mg/kg phenacetin(16.7?2.1%and 32.3?3.5%,respectively),but not following the administration of 100mg/kg phenacetin(1.7?0.6%).Because high levels of Met-Hb formation were detected after the administration of250mg/kg phenacetin,the following in vivo studies were conducted at this dose. The highest levels of Met-Hb were detected1h after the administra-tion of phenacetin(Fig.1A).The highest concentration of phenacetin in the plasma was detected as early as15min,and these levels were substantially decreased by2h(Fig.1B).High levels of p-phenetidine and APAP in the plasma were detected over the time range of15min to1h(Fig.1C and D).Therefore,the time-dependent pro?les of altered plasma concentrations of p-phenetidine and APAP were comparable to those seen for the levels of Met-Hb.
3.2.Effects of TOTP on Aadac expression level and phenacetin-metabolizing activity
To investigate the effects of TOTP,an esterase inhibitor [20],on mRNA and protein expression levels of Aadac
and
Fig.1.Time-dependent changes in the levels of Met-Hb(A),plasma concentrations of phenacetin(B),p-phenetidine(C),and APAP(D)in phenacetin-administered mice.Mice were administered phenacetin(250mg/kg,p.o.),and blood was collected at0,0.25,0.5,1,2,3,and6h after administration.Each data point represents the mean?SD(n=3–5).
Y.Kobayashi et al./Biochemical Pharmacology84(2012)1196–12061199
phenacetin-metabolizing activity,mice were administered TOTP (125mg/kg,i.p.),and then,the livers were collected at various times (0–12h)after administration.Although expression levels of Aadac mRNA in the liver were slightly but signi?cantly decreased at 12h after administration of TOTP (%of control:71.6?9.4%)(Fig.2A),the protein expression levels of Aadac were unaffected (Fig.2B).In contrast to the mRNA and protein levels of Aadac,the phenacetin hydrolase activity in MLM was substantially and signi?cantly decreased at 12h after administration of TOTP (%of control:6.9?2.3%)(Fig.2C).Phenacetin O -deethylase activities were unchanged following TOTP administration (Fig.2D).Thus,it was con?rmed that TOTP markedly decreased the formation of p -phenetidine by inhibiting the esterase activity of mouse liver.
3.3.Effects of TOTP on phenacetin-induced methemoglobinemia in mice
To investigate the effect of TOTP on phenacetin-induced methemoglobinemia,mice were pretreated with TOTP (125mg/kg,i.p.)then administered with phenacetin (250mg/kg,p.o.)12h after administration of TOTP.One hour after treatment with phenacetin,the level of Met-Hb and the plasma concentrations of phenacetin,p -phenetidine,and APAP were determined.The level of Met-Hb markedly decreased from 25.0?6.5%to 1.2?0.2%following administration of TOTP (Fig.3A).The plasma concentration of p -phenetidine was also signi?cantly decreased from 81.7?52.1m M to 6.2?1.6m M following administration of TOTP,although the levels of phenacetin and APAP did not signi?cantly change (Fig.3B–D).Thus,these in vivo studies indicated that the hydrolysis pathway could be involved in phenacetin-induced methemoglobinemia.
3.4.Mechanism of Met-Hb formation in vitro
To investigate the formation of Met-Hb in vitro ,HLM were incubated with either phenacetin or p -phenetidine,an NADPH generating system,and mouse red blood cells for a period of 0–120min.The formation of Met-Hb was linear with respect to the incubation time (1mM phenacetin:<120min;1mM p -pheneti-dine:<60min)(Fig.4A).
To investigate the role played by the hydrolysis pathway in phenacetin-induced methemoglobinemia,HLM were incubated with phenacetin,p -phenetidine,and APAP,an NADPH generating system,and mouse red blood cells.Formation of Met-Hb was signi?cantly increased by both phenacetin (0.1–5mM)and p -phenetidine (0.01–1mM)in a concentration-dependent manner,while APAP (0.01–1mM)did not have any effect (Fig.4B).The formation of Met-Hb by p -phenetidine was detected at higher levels than that induced by phenacetin at the same concentrations (Fig.4B).The omission of an NADPH-generating system from the complete mixture did not cause any increase in the Met-Hb formation (data not shown).These results supported the in vivo studies described above where the hydrolysis pathway was shown to be involved in phenacetin-induced methemoglobinemia.
3.5.Met-Hb formation by the metabolic activation of p-phenetidine
The enzymes involved in the metabolism of p -phenetidine to the reactive metabolite(s)thought to cause methemoglobinemia were investigated.The studies described above suggested the involvement of NADPH-dependent CYP enzymes in the metabo-lism of p -phenetidine.p -Phenetidine (1mM)was incubated
with
Fig.2.Effects of in vivo treatment with TOTP on the expression levels of AADAC mRNA (A)and the protein content in mouse liver (B)and either phenacetin hydrolase activity (C)or phenacetin O -deethylase activity (D)in MLM.Mice were administered TOTP (125mg/kg,i.p.),and at 0,1,6,and 12h after administration,liver samples were collected.Each column represents the mean ?SD (n =4).*P <0.05as compared with 0h.
Y.Kobayashi et al./Biochemical Pharmacology 84(2012)1196–1206
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representative CYPs that were expressed in human liver,an NADPH-generating system,and mouse red blood cells.This resulted in a signi?cantly increased formation of Met-Hb by CYP1A2,CYP2C19,CYP2D6,and CYP2E1(Fig.5).Particularly high Met-Hb formation was detected in the presence of CYP1A2(66.0?1.8%)and CYP2E1(25.2?0.6%).These data suggest the involvement of both CYP1A2and CYP2E1in p -phenetidine metabo-lism leading to the formation of Met-Hb.
3.6.Involvement of hydrolysis and CYP-dependent metabolic pathway in Met-Hb formation induced by phenacetin
To investigate whether phenacetin-induced methemoglobine-mia was caused by hydrolysis and CYP-dependent metabolism,an in vitro study of methemoglobinemia was designed using the expression systems of human esterases and CYP enzymes.
For
Fig.3.Effects of TOTP treatment on phenacetin metabolism as assessed by Met-Hb formation (A)and plasma concentrations of phenacetin (B),p -phenetidine (C),and APAP (D)following phenacetin administration in mice.TOTP (125mg/kg,i.p.)was administered 12h before administering phenacetin (250mg/kg,p.o.).Plasma specimens were collected from the inferior vena cava 1h after administration of phenacetin.Each column represents the mean ?SD (n =4).*P <0.05as compared without TOTP
treatment.
Fig.4.(A)Time-dependent changes of phenacetin-and p -phenetidine-induced Met-Hb formation.HLM (1.0mg/ml),an NADPH-generating system,and mouse red blood cells were incubated for 0–120min with 1mM phenacetin and 1mM p -phenetidine.(B)Concentration-dependent changes in Met-Hb formation induced by phenacetin,p -phenetidine,and APAP.Phenacetin (0.01,0.1,1,and 5mM),p -phenetidine (0.01,0.1,and 1mM),and APAP (0.01,0.1,and 1mM)were incubated with HLM (1.0mg/ml),an NADPH-generating system,and mouse red blood cells.The incubation times were 60min.Each column represents the mean ?SD of triplicate determinations.*P <0.05and ***P <0.001as compared with 1%acetonitrile (p -phenetidine and APAP)or 2%acetonitrile (phenacetin).
Y.Kobayashi et al./Biochemical Pharmacology 84(2012)1196–1206
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esterases,the following esterases were chosen:AADAC,CES1,and CES2.Previously,our study showed that the CES enzymes contributed only marginally to phenacetin hydrolysis [9].Howev-er,CES enzymes were shown to be responsible for the hydrolysis of various drugs.The following CYP enzymes were also chosen:CYP1A2and CYP2E1.Phenacetin (5mM)was incubated with the expression systems of the above-listed enzymes,an NADPH-generating system,and mouse red blood cells.This resulted in signi?cantly high formation of Met-Hb when either CYP1A2and AADAC (9.0?0.4%),or CYP2E1and AADAC (17.2?0.3%)were used in combination.CYP1A2alone also promoted moderately high formation of Met-Hb (3.3?0.3%)as compared with no source of enzyme being present (1.3?0.3%).However,neither CES1nor CES2enhanced the formation of Met-Hb (4.1?0.3%and 3.8?0.1%,respectively).These results supported the idea that the metabolic pathway of hydrolysis by AADAC and hydroxylation by CYP1A2or CYP2E1would be involved in the formation of Met-Hb by phenacetin.
3.7.Inhibition analyses of esterase inhibitors and anti-CYP antibodies on Met-Hb formation induced by phenacetin
To further investigate the involvement of human AADAC in phenacetin-induced methemoglobinemia,inhibition analyses with esterase expression systems were performed using eserine and BNPP,both of which are potent inhibitors of AADAC and CES [10,21].The current study also con?rmed the potencies of these inhibitors when using 500m M PNPA as a substrate (Fig.7A).The hydrolase activity of AADAC was potently inhibited by 10m M eserine (%of control:6.8%)but not by 10m M BNPP (82.6%).The hydrolase activity of CES1was potently inhibited by 10m M BNPP (%of control:0.2%)but not by 10m M eserine (97.5%).The hydrolase activity of CES2was potently inhibited by both 10m M eserine (%of control:8.6%)and 10m M BNPP (1.0%).Thus,these observations con?rmed that eserine was a potent inhibitor of AADAC and CES2.In addition,BNPP was shown to be a potent inhibitor of both CES1and CES2.
Using the inhibitors described above,HLM were incubated with 5mM phenacetin,an NADPH generating system,and mouse red blood cells.The formation of Met-Hb was signi?cantly decreased by 10m M eserine (from 36.3?1.0%to 9.1?0.3%)(Fig.7B).Although Met-Hb formation was also decreased by 10m M BNPP (to 30.1?0.8%),the inhibitory potency seen with BNPP was much lower than that observed with 10m M eserine.Neither eserine nor BNPP altered the formation of Met-Hb (data not shown).This
inhibition study therefore suggested that AADAC would play a key role in the formation of Met-Hb in HLM.
To investigate the contributions of CYP1A2and CYP2E1to p -phenetidine-induced methemoglobinemia,inhibition studies were performed using CYP antibodies.p -Phenetidine at a concentration of 1mM was incubated with recombinant CYP1A2or CYP2E1,an NADPH generating system,and mouse red blood cells.The formation of Met-Hb induced by recombinant CYP1A2was decreased by an anti-CYP1A2antibody from 55.1%to 18.6%.By contrast,the formation of Met-Hb induced by recombinant CYP2E1was decreased by an anti-CYP2E1antibody from 21.2%to 8.6%(Fig.7C).The formation of Met-Hb induced by either CYP1A2or CYP2E1was unchanged by treatment with an anti-CYP3A4antibody used as a negative control.Thus,use of both anti-CYP1A2and anti-CYP2E1antibodies was con?rmed to speci?cally inhibit the corresponding CYP activities.
Using above anti-CYP antibodies,the contributions of CYP1A2and CYP2E1to Met-Hb formation induced by p -phenetidine in HLM were determined.The formation of Met-Hb was decreased by both anti-CYP1A2antibody and anti-CYP2E1antibody (from 25.9?2.7%to 20.9?1.8%and 11.8?0.8%,respectively).By contrast,the formation of Met-Hb was unaffected by anti-CYP3A4antibody (to 24.7?0.6%)(Fig.7D).Thus,the contributions of CYP1A2and especially CYP2E1to the formation of Met-Hb by p -phenetidine were indicated by these studies.
3.8.Phenacetin-induced Met-Hb formation in human red blood cells
In the in vitro studies described above,mouse red blood cells were used.Due to the possibility of species differences in the formation of Met-Hb seen in human or mouse red blood cells,formation of Met-Hb induced by phenacetin was investigated using human red blood cells.Phenacetin at 5mM was incubated with combinations of recombinant human AADAC,CYP1A2,and CYP2E1,and individual human red blood cells (from 5healthy Japanese subjects).Similar to the observations made for mouse red blood cells above (Fig.6),increased formation of Met-Hb was detected when combinations of AADAC and CYP1A2(8.6–9.6%),or AADAC and CYP2E1(12.2–13.6%)were used (Fig.8).In addition,inter-individual variability in the formation of Met-Hb was not observed among red blood cells taken from 5individuals.Thus,using human red blood cells,it was demonstrated that the metabolic pathway of hydrolysis by AADAC and the subsequent metabolism,probably hydroxylation,by CYP1A2or CYP2E1would likely be involved in phenacetin-induced methemoglobinemia.
4.Discussion
Phenacetin,an analgesic antipyretic,was withdrawn from the market because it caused methemoglobinemia and renal failure [1,2].It was suggested that phenacetin-induced methemoglobine-mia was associated with hydrolysis and subsequent N -hydroxyl-ation of phenacetin [1,22].Previously,our studies showed that AADAC was the principal enzyme catalyzing the hydrolysis of phenacetin [9].However,it has not been experimentally shown which enzymes play essential roles in phenacetin-induced methemoglobinemia.In this study,we ?rst found that hydrolysis of phenacetin by AADAC was highly involved in phenacetin-induced methemoglobinemia.
Administration of phenacetin (250mg/kg,p.o.)to mice resulted that the pro?les of the time-dependent changes in plasma concentrations of p -phenetidine and APAP were similar to the pro?les observed for Met-Hb (Fig.1).Pre-administration of TOTP (125mg/kg,i.p.)decreased the formation of Met-Hb induced by phenacetin,along with decreased plasma levels of p
-phenetidine
Fig.5.p -Phenetidine-induced Met-Hb formation following incubation with human CYP enzymes.Each recombinant human CYP enzyme (25pmol CYP/ml)was incubated with 1mM p -phenetidine,an NADPH-generating system,and mouse red blood cells.Each column represents the mean ?SD of triplicate determinations.*P <0.05and ***P <0.001as compared with the control (no source of enzymes).
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Fig.6.Phenacetin-induced Met-Hb formation following incubation with the combination of esterases and CYP enzymes.Recombinant human esterases (AADAC,CES1,and CES2,all at 1.0mg/ml)and recombinant human CYPs (CYP1A2and CYP2E1:both at 25pmol CYP/ml)were incubated with 5mM phenacetin,an NADPH-generating system,and mouse red blood cells.Each column represents the mean ?SD of triplicate determinations.***P <0.001as compared with CYP (à)and esterase (à).y P <0.05and yyy P <0.001as compared with CYP (+)and esterase (à
).
Fig.7.(A)Inhibitory effects of eserine and BNPP on the PNPA hydrolase activities of recombinant human AADAC,CES1,and CES2.The concentrations of PNPA and inhibitors were 500m M and 10m M,respectively.The control activities of recombinant AADAC,CES1,and CES2were 722.6nmol/min/mg,709.4nmol/min/mg,and 443.0nmol/min/mg,respectively.(B)The inhibitory effects of eserine and BNPP on phenacetin-induced Met-Hb formation following incubation with HLM.HLM (1.0mg/mL)were incubated with 5mM phenacetin,10m M eserine or 10m M BNPP,an NADPH-generating system,and mouse red blood cells.The control Met-Hb formation was 36.3?1.0%.(C)Inhibitory effects of anti-human CYP1A2,CYP2E1,and CYP3A4antibodies on p -phenetidine induced Met-Hb formation following incubation with recombinant human either CYP1A2or CYP2E1enzymes.(D)Inhibitory effects of anti-human CYP1A2,CYP2E1,and CYP3A4antibodies on p -phenetidine induced Met-Hb formation following incubation with HLM.Recombinant human CYP1A2,CYP2E1,or HLM were incubated with 1mM p -phenetidine,an NADPH-generating system,mouse red blood cells,and each CYP antibody.Each column represents the mean of duplicate determinations (A,C)or the mean ?SD of triplicate determinations (B,D).*P <0.01and ***P <0.001as compared with no chemical inhibitors or no antibodies.yyy P <0.001.
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(Fig.3).These results suggest that the hydrolysis pathway could be involved in phenacetin-induced methemoglobinemia.
Previously,we demonstrated that hydrolysis of phenacetin was predominantly catalyzed by AADAC in humans [9].We found that eserine was a potent inhibitor of human AADAC [9–11].However,when used at a concentration of 100m M,the hydrolysis of phenacetin by recombinant mouse Aadac was not inhibited by 1m M eserine (%of control:76.5%),whereas the hydrolysis of phenacetin by recombinant human AADAC was potently inhibited by 1m M eserine (%of control:8.9%)(data not shown).Because TOTP was frequently used as a general serine esterase inhibitor in rodents [23,24],this study also used TOTP (and not eserine)for the in vivo studies.In fact,the concentration of p -phenetidine in the plasma was signi?cantly decreased by pre-administration of TOTP (Fig.3C).Serine esterases,including Aadac,are inhibited by various organophosphates such as TOTP by covalently phosphor-ylation of the serine residue within the active site.Therefore,phenacetin hydrolase activity could be inhibited by TOTP without altering the levels of Aadac mRNA and protein (Fig.2A–C).In mice,we recently reported that phenacetin hydrolase activities in the microsomes of the liver,the small intestine,the kidney and the lung were re?ected by the expression levels of Aadac [14].Therefore,it is possible that Aadac is the principal enzyme catalyzing the hydrolysis of phenacetin in mice and in humans.However,species differences are generally found in drug-metabolizing enzymes.Thus,in vitro studies were performed to identify the enzymes involved in phenacetin-induced methemo-globinemia.
In vitro studies revealed concentration-dependent increases of Met-Hb formation by phenacetin or p -phenetidine.In contrast,the formation of Met-Hb remained unaltered with any concentrations of APAP (0.01–1mM)(Fig.4B).Additionally,higher levels of Met-Hb formation were detected following treatment with p -phene-tidine than by phenacetin.Thus,it is obvious that hydrolysis plays an important role in the phenacetin-induced methemoglobinemia.
Because omission of the NADPH-generating system did not cause an increase in the formation of Met-Hb (data not shown),the metabolism by an NADPH-dependent enzyme as well as the hydrolysis reaction would be required for the formation of Met-Hb induced by phenacetin.CYP enzymes account for approximately 75%of the metabolism of clinical drugs [25].In this study,we found that following incubation with p -phenetidine,high levels of Met-Hb formation were detected by the action of CYP1A2and CYP2E1(Fig.5).The formation of Met-Hb was also detected by CYP2C19
and CYP2D6,but to a lesser extent than that seen with either CYP1A2or CYP2E1.However,it has been reported that the expression levels of CYP1A2and CYP2E1(8.0%and 9.0%of total CYP,respectively)in human liver are higher than those of CYP2C19and CYP2D6(4.0%and 2.0%of total CYP,respectively)[26].Thus,both CYP1A2and CYP2E1would be mainly involved in the formation of Met-Hb induced by p -phenetidine.Flavin-containing monooxygenase (FMO)is also known as an NADPH-dependent enzyme.However,the formation of Met-Hb induced by p -phenetidine in HLM was not inhibited by 1mM methimazole,which is a known competitive FMO inhibitor [27](data not shown).Thus,FMO is unlikely to be involved in the metabolism of p -phenetidine.
From the data using recombinant esterases (AADAC,CES1,and CES2)and CYP enzymes (CYP1A2and CYP2E1)(Fig.6),it was clearly shown that high levels of Met-Hb formation were observed by incubating phenacetin with a combination of AADAC and either CYP1A2or CYP2E1.Therefore,it would seem increasingly more likely that the hydrolysis by AADAC and subsequent metabolism by CYP1A2or CYP2E1would contribute to phenacetin-induced methemoglobinemia.This result was supported by our previous study that AADAC,but not CES enzymes,is highly involved in phenacetin hydrolysis [9].
It is possible that hydrolysis by AADAC is followed by metabolism by the activities of CYP1A2or CYP2E1.However,we previously found that the catalytic ef?ciency of hydrolysis of APAP,which is a deethylated product of phenacetin by CYP1A2or CYP2E1,in HLM is over 50-fold lower than that of phenacetin [9].Therefore,the hydrolysis reaction would occur before the metabolism by CYPs in phenacetin metabolism that is associated with the formation of Met-Hb.
The formation of Met-Hb was increased by incubating phenacetin with CYP1A2alone,despite no involvement of AADAC (Fig.6).Therefore,the possible causes are as follows:(1)a small amount of p -phenetidine was non-enzymatically produced during incubation and the product might have been metabolized,probably hydroxylated,by CYP1A2.Although the incubation of phenacetin with CYP2E1alone did not show an increase in the levels of Met-Hb,this might be due to the lower activity of CYP2E1than CYP1A2.(2)N -hydroxyphenacetin might be involved in the formation of Met-Hb.Although the current study did not pursue the production of N -hydroxyphenacetin,it was previously reported that a negligible amount of N -hydroxyphenacetin was detected in human urine following an oral dose of phenacetin (10mg/kg)[28].
To further analyze the role played by AADAC and CYP1A2or CYP2E1in the formation of Met-Hb induced by phenacetin,inhibition studies using chemical inhibitors were performed with HLM.The formation of Met-Hb induced by phenacetin was signi?cantly decreased by eserine,a potent AADAC and CES2inhibitor,but not by BNPP,which is a potent inhibitor of both CES1and CES2(Fig.7B).Eserine can also inhibit butyrylcholinesterase,which is expressed in the liver and plasma [29,30].However,we previously reported that phenacetin hydrolase activity was not detected in human plasma [9].Additionally,the formation of Met-Hb induced by p -phenetidine was more markedly decreased when using an anti-CYP2E1antibody as compared with an anti-CYP1A2antibody (Fig.7D).Thus,CYP2E1appeared to highly contribute to formation of Met-Hb by p -phenetidine in HLM.In the study using recombinant CYP enzymes,CYP1A2played a signi?cant role in the formation of Met-Hb by p -phenetidine (Fig.5).To evaluate the expression of each CYP in HLM used in this study,we measured the activities of methoxyresoru?n O -demethylation and chlorzoxa-zone 6-hydroxylation,which are both marker activities of CYP1A2and CYP2E1,respectively (data not shown).Accordingly,CYP2E1(81.3pmol/mg)was estimated to show an
approximately
Fig.8.Phenacetin-induced Met-Hb formation assessed using human red blood cells.Human esterases (AADAC,CES1,and CES2,all at 1.0mg/ml)and CYPs (CYP1A2and CYP2E1,both at 25pmol CYP/ml)were incubated with 5mM phenacetin,an NADPH-generating system,and human red blood cells from 5healthy individuals.
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6.8-fold higher expression in HLM as compared with CYP1A2 (12.0pmol/mg).Thus,this study concluded that AADAC and CYP2E1(and partially CYP1A2)would play major roles in the formation of Met-Hb induced by phenacetin.
An additional in vitro assay for Met-Hb formation was investigated using human red blood cells obtained from5 individuals(Fig.8).Consistent with the results using mouse red blood cells(Fig.6),the formation of high levels of Met-Hb was observed following the combination of AADAC with CYP1A2or CYP2E1(Fig.8).Therefore,mouse red blood cells would be applicable for the assay of human Met-Hb formation in vitro.
Methemoglobinemia is reported to be induced by particular drugs,including the local analgesic benzocaine,the antileprosy drug dapsone,and the antibiotic drug sulfamethoxazole[31–33]. All of these agents possess an amine moiety in their chemical structures.As these drugs are considered to induce the formation of Met-Hb through N-hydroxylation,methemoglobinemia induced by phenacetin would be also due to a N-hydroxylated metabolite of p-phenetidine by CYP1A2and CYP2E1.In human urine following an oral dose of phenacetin(10mg/kg),2-hydroxy-p-phenetidine was also detected[28].Because it was reported on in vitro study that2-hydroxy-p-phenetidine could induce the formation of Met-Hb[34],2-hydroxy-p-phenetidine might be also a cause of the formation of Met-Hb induced by phenacetin.However,we could not obtain above metabolites,therefore we could not pursue the further investigation.
It is possible that Met-Hb formed by phenacetin is indirectly induced by the productions of free radical O2àor H2O2.If2-hydroxy-p-phenetidine is oxidized to an iminoquinone form,this iminoquinone can be reduced to semiiminoquinone.Semiimino-quinone generally reacts with O2to form oxygen free radicals. However,because O2àcan also reduce the Met-Hb to oxyhemo-globin[35],the involvement of O2àin the formation of Met-Hb is unclear.Semiquinone,which is characteristically similar to semiiminoquinone,itself can also convert Met-Hb to oxyhemo-globin[36].Therefore,this study did not seek the involvement of chemical species.
In addition to phenacetin,?utamide,which is suggested to induce methemoglobinemia[37],is hydrolyzed to the aromatic amine4-nitro-3-(tri?uoromethyl)phenylamine by AADAC[10]. Thus,AADAC might play an important role in the occurrence of methemoglobinemia through the hydrolysis of several drugs. Phenacetin was known to cause renal failure as well as methemoglobinemia[1,2].In addition,Jaenike[38]reported that renal dysfunction was caused by administration of Met-Hb in rats. Phenacetin-induced renal dysfunction might be induced by the formation of Met-Hb.Further study for the renal dysfunction is worth of investigation.
In conclusion,the current study clari?ed that the hydrolysis by AADAC and its subsequent metabolism,probably hydroxylation, by CYP1A2or CYP2E1were involved in phenacetin-induced methemoglobinemia in human.This is the?rst report to suggest the involvement of AADAC in an adverse drug reaction by experimental demonstration.If drugs are hydrolyzed to aromatic amines by AADAC,we would need to pay close attention to methemoglobinemia as an adverse drug reaction event. Acknowledgement
The Japan Society for the Promotion of Science supported this study[Grant-in-Aid for Young Scientists(B)21790148]. References
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