* Corresponding author. Present address: Strategic and Exploratory Sciences, Abbott Laboratories, Department 463, AP13AD463, 100 Abbott Park Road, Abbott Park, IL 60064, USA. Tel.: +1-847-9389863; fax: +1-847-9383076; e-mail:[email protected].
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Fig. 1. Chemical structure of panadiplon. The oxadiazole ring can be cleaved by reduction to yield a bisamide, which may beoxidized to release cyclopropane carboxylic acid (CPCA).
1. Introduction
The quinoxalinone, panadiplon (3-[5-cyclo-propyl-1,2,4-oxadiazol-3yl]-5-[1-methylethyl]-imi-dazo{1,5-a}-quinoxalin-4[5H]-one; see Fig. 1) isan anxiolytic with a high affinity for benzodi-azepine receptors. It has been shown to possessboth agonist and antagonist activities (VonVoigt-lander et al., 1990; Piercey et al., 1990; Tang et al.,1991) and is thought to have minimal centralnervous system depression and thus fewer sideeffects compared to the full agonist benzodi-azepines (Tang et al., 1991). Phase I clinical trialswere terminated, however, when hepatic toxicitywas observed in some human volunteers. Toxicitywas not observed following oral administration torats, dogs or monkeys during preclinical safetyevaluation studies, though hepatic microvesicularlipid was observed in the monkey (T.A. Jackson,A.D. Hall, unpublished observations).
In subsequent studies, we observed a hepatictoxic syndrome in Dutch-belted rabbits followingmultiple-dose oral administration of panadiplon(Ulrich et al., 1995). Toxicity was evidenced byincreases in serum aminotransferase activities,hepatic microvesicular steatosis, and hepatic mul-tifocal centrilobular necrosis. The steatosis wasdue primarily to the hepatic accumulation oftriglyceride. Increases in serum triglyceride, alter-ations in serum glucose levels, and a depletion ofhepatic glycogen were also observed. The toxicresponse resembled a Reye’s syndrome-like toxic-ity (reviewed by Osterloh et al., 1989). Reye’ssyndrome-like toxicities have been observed inlaboratory animals and humans exposed to a
variety of xenobiotic carboxylic acids includingvalproate (Zimmerman and Kamal, 1982; Kester-son et al., 1984), pirprofen (Danan et al., 1985;Geneve et al., 1987) and the metabolites of hypo-glycin (Sherratt, 1986). These toxicities are char-acterized by a hepatic microvesicular steatosis andare thought to result from mitochondrial dysfunc-tion, including the inhibition of fatty acid b-oxi-dation. Though panadiplon is not a carboxylicacid, the oxadiazole ring can be cleaved by reduc-tion to yield a bisamide (Speed et al., 1993) whichmay be oxidized to release cyclopropane car-boxylic acid (CPCA, Fig. 1). The existence of theCPCA metabolite in rabbit plasma and urine hasbeen confirmed (Paul G. Pearson, personal com-munication). CPCA has been shown to have avariety of metabolic effects in rat and guinea pig,including inhibition of fatty acid oxidation (Dun-combe and Rising, 1972a; Buxton et al., 1983),inhibition of gluconeogenesis (Duncombe andRising, 1972b), and inhibition of pyruvatemetabolism (Buxton et al., 1983; Steinhelper and
Table 1Information regarding human tissue for hepatocyte isolation
13H6 Male Rhodamine 123 uptakeRhodamine 123 uptake,41 MaleH7fatty acid oxidation
R.G. Ulrich et al. / Toxicology 131 (1998) 33–47 35
Fig. 2. Concentration-response determinations in rabbit hepatocyte cultures showed that panadiplon did not produce cell lysis, asdetermined by lactate dehydrogenase release, at concentrations up to 160 mM for 48 h. A decrease in albumin secretion was observedat the higher concentrations. Data represent mean9S.E. (n=3; most error bars fall on the line).
Olson, 1985). We have observed CPCA to pro-duce toxicity in the rabbit that was essentiallyidentical to that produced by panadiplon (Ulrichet al., 1993).
In the studies reported here, we examined theeffects of panadiplon and the CPCA metaboliteon hepatic mitochondrial activities. Hepatocytesfor most experiments were obtained from Dutch-belted rabbits and from human donor tissue; insome experiments rat hepatocytes were also used.To determine if the parent compound and/ormetabolite have the potential to inhibit b-oxida-tion in rabbit or human hepatocytes, oxidation ofuniformly 14C-labelled palmitate to acid-solubleproducts was monitored. Acid-soluble productsinclude ketone bodies, acetyl-CoA, and tricar-boxylic acid cycle intermediates, and this tech-nique has been shown to provide an accuratemeasure of fatty acid oxidation (Otto et al., 1985).We also examined for the presence of an intactmitochondrial membrane potential in culturedhepatocytes (rabbit, human, and rat) using thefluorescent probe, rhodamine 123 (R123) andfluorescence imaging. The uptake of R123 intomitochondria is dependent on the presence of anintact proton gradient (Johnson et al., 1980, 1981;Ehrenberg et al., 1988). Several agents are known
to disrupt this gradient, which inevitably alterscellular functions (reviewed by Chen, 1988) andoften correlates with toxicity (Rahn et al., 1991).Potential effects on respiration were made usingmitochondria and digitonin-permeablized hepato-cytes isolated from liver of control andpanadiplon-treated rabbits; isolated control rabbitliver mitochondria were used to examine for anypotential direct respiratory inhibiting or uncou-pling effects of panadiplon.
2. Materials and methods
2.1. Panadiplon and CPCA
Stock solutions of panadiplon (lot cB11885-TGS-143, Pharmacia&Upjohn, Inc., Kalamazoo,MI) for in vitro experiments were prepared indimethylsulfoxide (DMSO, Sigma, St. Louis,MO) at a concentration of 100 mM. Aliquots ofstock solution were added to incubation media toobtain the desired final concentrations. For invivo drug administration, panadiplon was pre-pared as a suspension in vehicle (lot c21519-GDB-111, in 1.25% low viscosity, microcrystallinecellulose and sodium carboxymethylcellulose NF,
R.G. Ulrich et al. / Toxicology 131 (1998) 33–4736
Fig. 3. Inhibition of fatty acid b-oxidation in isolated rabbit hepatocytes by panadiplon and its carboxylic acid metabolite.Oxidation of [14C]palmitate to acid-soluble products was determined in vehicle controls and cells treated with 0.1–0.2 mMpanadiplon or 10 mM cyclopropane carboxylic acid (CPCA). Data represent parallel observations (n=2) in cells isolated from asingle rabbit.
0.2% polysorbate 80 NF food grade, and 0.1 Nsodium hydroxide in purified water USP withoutsorbic acid; vehicle lot c21519-GDB-110).CPCA was from Sigma (lot 86C-0180).
2.2. Hepatocyte isolation and culture
Rabbit hepatocytes were isolated using materi-als and methods previously described for monkey(Ulrich et al., 1990) with modifications as notedbelow. All animal procedures were conducted hu-manely in accordance with protocols approved bythe Corporate Animal Welfare Committee. Rab-bits (Dutch-belted; Langshaw Farms, Augusta,MI) were fasted 18–24 h prior to cell isolation. Asingle large lobe of liver was excised from pento-barbital-anesthetized animals and a catheter in-serted into a large portal vein for perfusion.Washout perfusion times were approximately 10min, and digestion required 10–15 min perfusiontime. For human, liver wedges (100–150 g) fromorgan donors were received on ice from the Inter-national Institute for the Advancement ofMedicine (Exton, PA). All donors were disease-
free with unremarkable health histories; addi-tional information is provided in Table 1.Isolation of human hepatocytes was conducted aspreviously described (Ulrich et al., 1998). Briefly,a large portal vein was cannulated for perfusion,and the organ wedge was allowed to warm to37°C during the washout period. Washout was for20 min at an initial flow rate of 75 ml/min and afinal rate of 95 ml/min, and digestion was forapproximately 30 min. Human hepatocytes wereused freshly isolated when possible, and wereotherwise cryopreserved (Ulrich et al., 1998). Cry-opresevation has no demonstrated effects on cellfunction or drug metabolizing capacity (Chesne etal., 1993, Strom et al., 1997) and preliminaryexperiments showed no effect on rhodamine 123staining. Rat hepatocytes were isolated by themethod of Seglan (1973) as modified by Elligetand Kolaja (1983) using male Sprague–Dawleyrats weighing 100–120 g. Cells were used as sus-pensions for b-oxidation studies (rabbit, human)or cultured for R123 studies (rabbit, human, andrat). Cells isolated from five rabbit livers, sevenhuman livers and two rat livers were used for
R.G. Ulrich et al. / Toxicology 131 (1998) 33–47 37
Fig. 4. Inhibition of fatty acid b-oxidation by cyclopropane carboxylic acid (CPCA) was concentration-dependent in isolated rabbithepatocytes, as determined by oxidation of [14C]palmitate to acid-soluble products. Data represent parallel observations (n=2) incells isolated from a single rabbit.
suspension and monolayer studies. Cells isolatedfrom an additional six rabbits were used for exvivo determinations (see below).
For cell culture, hepatocytes were suspended inmedium (Dulbecco’s modified Eagle’s medium,supplemented with 10% fetal bovine serum, 1.9mM L-glutamine, 4.6 mM D-glucose, 14 mM hy-drocortisone, 0.42 mg/ml insulin, and 2.2 g/lsodium bicarbonate) containing 100 U/ml gen-tamicin. All medium components were fromGIBCO (Grand Island, NY). Cells were plated ata density of 5.0×104 cells/cm2 on collagen coated25 mm diameter sterile glass coverslips insertedinto plastic 35 mm dishes (Falcon Plastics, Lin-coln Park, NJ) and allowed to attach for 4 h in ahumidified incubator (37°C with 95% air/5%CO2). Monolayers were washed once withmedium then overlaid with 2.0 ml of medium andincubated for 18 h prior to treatment.
2.3. b-Oxidation assay
Mitochondrial fatty acid b-oxidation was mea-sured in rabbit and human hepatocyte suspen-sions based on the method of Otto et al. (1985).
Hepatocytes were suspended at 2×106 cells/ml inKrebs–Henseleit buffer supplemented with 13.3mM glucose, 13.3 mM lactate, 2.7 mM pyruvateand 1.3 mM L-carnitine. Suspensions were thenpreincubated for 45 min with either 0.1%dimethylsulfoxide (DMSO), 100 mM panadiplon(100 mM stock in DMSO), or 10 mM CPCA(Sigma, lot c86C-0180) at 37oC with constantstirring in an atmosphere of 95% O2:5% CO2.Concentrations of panadiplon \100 mM couldnot be utilized due to drug solubility limitations inthe incubation buffer. A separate dose-responseexperiment in rabbit and human cells measuredb-oxidation activity in the presence of 0.01, 0.1, 1and 10 mM CPCA. During the preincubation,[14C(U)]palmitate (New England Nuclear) wasadded to a solution of 7.5 mM palmitate/20%bovine serum albumin (BSA) to a final specificactivity of 0.8 Ci/mol. At the end of the preincu-bation period, the [14C]palmitate/BSA complexwas added to the cell suspension for final concen-trations of 1.5 mM palmitate and 4% BSA. At 5,10, and 20 min of incubation, a 0.5 ml volumewas removed from the cell suspension and thereaction terminated by addition of 0.25 ml ice
R.G. Ulrich et al. / Toxicology 131 (1998) 33–4738
Fig. 5. Inhibition of fatty acid b-oxidation in isolated human hepatocytes by panadiplon and its carboxylic acid metabolite. Datarepresent parallel observations (n=2) in cells isolated from a single donor. Human donor tissue was from a 57-year-old female (H3).Oxidation of [14C]palmitate to acid-soluble products was determined in vehicle controls and cells treated with 0.1 mM panadiplonor 0.1–10 mM cyclopropane carboxylic acid (CPCA).
cold 60% perchloric acid to each aliquot; thesetime points were used to assure linearity. The acidextract was diluted with 3 vols. of hexane, mixedwith a vortex mixer, and centrifuging at 500×gfor 10 min. The hexane phase was discarded andthe acid extract was washed a second time. Analiquot of the washed extract was counted foracid-soluble radioactivity. After determiningCPCA to be inhibitory at 10 mM for all suspen-sions, it was routinely used as a positive controlfor b-oxidation inhibition. Viability of hepatocytesuspensions, determined by trypan blue exclusion,was recorded before and after each experiment.For protein determinations, a 50 ml aliquot wastaken from the hepatocyte suspension prior to theaddition of palmitate/BSA. Proteins were mea-sured according to the method by Lowry (1957).Data are presented as nmol palmitate oxidized/mgprotein.
2.4. Rhodamine 123 fluorescence imaging
The effects of panadiplon on mitochondrialtransport activity for R123 was determined in
cultured rabbit, human and rat hepatocyte cul-tures treated in vitro and in rabbit hepatocytes exvivo isolated from treated animals. Concentra-tion-response determinations indicated thatpanadiplon did not produce cytotoxicity at con-centrations up to 160 mg/ml (480 mM) for 48 h inrabbit hepatocyte cultures (Fig. 2), as determinedby lactate dehydrogenase release (Wroblewski andLaDue, 1955) and albumin secretion (Sun et al.,1990). Most subsequent experiments for fluores-cence imaging were done using final concentra-tions of 30 mM panadiplon in medium, whichapproximated the maximum in vivo hepatic expo-sure level in rabbits. Controls were 0.05% DMSOin medium. At intervals following addition ofdrug, control and treated cultures were labelledfor 1 h at 37°C with 1 mM R123 (Eastman Ko-dak, Rochester, NY). Cultures were then rinsedthree times with medium, and examined using aSPEX Fluorolog digital imaging system and Im-age 201 software (SPEX Industries, Edison, NJ)in conjunction with a Nikon Diaphot fluorescencemicroscope. The excitation monochrometer wasset at 480 nm, and a 510 nm dichroic mirror and
R.G. Ulrich et al. / Toxicology 131 (1998) 33–47 39
Fig. 6. Inhibition of fatty acid b-oxidation in isolated human hepatocytes by panadiplon and its carboxylic acid metabolite. Datarepresent parallel observations (n=2) in cells isolated from a single donor. Human donor tissue was from a 50-year-old female (H1).Oxidation of [14C]palmitate to acid-soluble products was determined in vehicle controls and cells treated with 0.1 mM panadiplonor 0.1 mM cyclopropane carboxylic acid (CPCA).
520 nm emission filter were used. Emitted fluores-cence was detected with a Dage SIT camera oper-ated in the manual mode. Experimental cellularfluorescence determinations were made relative tocontrols. In preliminary experiments, no acuteeffects (1–3 h exposure) were observed (data notshown). Subsequently, observations were made at18 h post-treatment, then at 24 h intervals follow-ing drug addition until inhibition of R123 uptakewas observed but not beyond 4 days post-isola-tion. In all cases, a minimum of two dishes perexperiment from each treatment and at each timepoint were examined. The ability of CPCA toblock R123 uptake was determined by incubatingrabbit and human hepatocyte monolayers with 10mM CPCA for 24 h followed by fluorescencedeterminations as described above.
For ex vivo mitochondrial fluorescence determi-nations, hepatocytes isolated from drug- and vehi-cle-treated rabbits were similarly labelled for 1 hwith R123 and viewed. For recovery experiments,these cells were also plated in 35 mm dishes asdescribed above and labelled with R123 at 3, 24and 48 h after isolation. Incubations were drug-free in medium.
2.5. Ex 6i6o experiments
For ex vivo examination of drug effects on livermitochondria (b-oxidation, R123 transport andrespiration), three rabbits were administeredpanadiplon (20 mg/kg per day) and three rabbitswere administered vehicle alone by oral gavagefor 7 days prior to sacrifice. For each animal, oneliver lobe was removed for hepatocyte isolation asabove, and a portion of the remainder was usedfor isolation of mitochondria. Mitochondrial res-piration and hepatocellular b-oxidation measure-ments were made from two control and twotreated animals; R123 uptake and hepatocellularrespiration were measured in all animals.
2.6. Rabbit li6er mitochondria and hepatocyterespiration
Rabbit liver mitochondria were isolated follow-ing a protocol used to prepare rat liver mitochon-dria (Hirsch et al., 1989). Mitochondria wereisolated from two panadiplon-treated and twovehicle control rabbits. A portion of the liver
R.G. Ulrich et al. / Toxicology 131 (1998) 33–4740
(15–20 g) was removed, rapidly minced with arazor blade into 1–2 mm pieces, and rinsed withice cold saline. Minced liver was suspended in 10ml of ice cold isolation buffer (250 mM sucrose, 1mM dipotassium EDTA, 1 mM EGTA, and 10mM MOPS; pH 7.4). The tissue was homogenizedby one pass with a longitudinally grooved Teflonpestle in a glass homogenizer. The homogenatevolume was adjusted to 10% liver w/v with icecold isolation buffer and centrifuged at 600×gfor 10 min at 4°C. The supernatant was trans-ferred to another tube and centrifuged at 1500×gfor 10 min. The supernatant was discarded andthe pellet was resuspended in 0.3–0.5 ml of icecold isolation buffer and kept on ice. The proteinconcentrations, determined using the Lowrymethod (Lowry, 1957), were 47–50 mg/ml. Forrespiration experiments, freshly isolated mito-chondria were suspended in medium (220 mMD-mannitol, 70 mM sucrose, 2 mM Hepes, 2.5mM KH2PO4, 0.5 mM EDTA, 2.5 mM MgCl2,and 1 mg/ml defatted BSA, pH 7.3) equilibratedwith air at 30°C. Oxygen consumption was mea-sured using a Clarke electrode at 30°C. L-gluta-mate and L-malate, 3 mM each, were provided assubstrates. State 3 respiration was initiated byinjection of 165 mM ADP. The ADP solution wasstandardized by HPLC (Meglasson et al., 1989).
Respiration experiments were also conductedusing hepatocytes isolated from three control andthree treated animals. For this, 1.2×106 cellswere added to 1.5 ml of an incubation mediumcontaining 110 mM KCl, 10 mM NaCl, 2 mMKH2PO4, MgSO4, 0.5 mM EGTA, 20 mMHEPES (pH 7.2). Following a brief oxygen elec-trode equilibration period, a 3.0 mM KHPO-HEPES solution (pH 7.2) was added to increasethe inorganic phosphate concentration to 5.0 mM.Then 7.5 mg of rotenone and 4.0 mM succinatewere added to establish the rate of succinateoxidation in the cell suspensions prior to digitonintreatment. This basal rate of respiration reflectedthe amount of damaged cells in the suspensions,since the plasma membrane in intact hepatocytesis not permeable to succinate. Treatment of cellswith digitonin to render the plasma membranepermeable led to an elevation of respiration in thesuspension. Subsequent addition of ADP (0.8
mM) stimulated state 3 respiration. ADP-inde-pendent respiration was estimated using the mito-chondrial ATPase inhibitor, oligomycin (10 mg ofoligomycin/1.2×106 cells). Measurements werepaired (one control and one treated evaluated inparallel).
2.7. Statistical analysis
Cytotoxicity (LDH) albumin data were ex-pressed as the mean9S.E. and were analyzed forstatistical significance using a one-way analysis ofvariance with a PB0.05 considered significant.Palmitate oxidation data are the average of twomeasurements at each point from individualdonors and were not subjected to statisticalevaluation.
3. Results
3.1. [14C]palmitate oxidation
Inhibited palmitate oxidation in panadiplon—or CPCA-treated hepatocyte suspensions was in-dicated by a decrease in the total amount of acidsoluble radioactivity present at the end of the20-min incubation period and a decreased rate(indicated by slope) as compared to control incu-bations. In rabbit hepatocyte suspensions,panadiplon inhibited [14C]palmitate oxidation incells isolated from two of five rabbits; no inhibi-tion was observed for the other three isolations.The oxidation rate in the presence of 100 mMpanadiplon was 44% of control (Fig. 3) and 43%(not illustrated) of control values for these twoisolations. 200 mM panadiplon further decreased[14C]palmitate oxidation to 25% of control rate(Fig. 3). CPCA was inhibitory in all rabbit hepa-tocyte isolations (Fig. 3), with [14C]palmitate oxi-dation rates ranging from 1 to 50% of controlvalues (mean rate was 29% of controls). Inhibitionby CPCA was concentration-dependent (Fig. 4).In human hepatocytes, panadiplon inhibited[14C]palmitate oxidation in cells from two of fourdonors; oxidation rates were 68 and 78% of con-trol values (Figs. 5 and 6, respectively). Undercomparable assay conditions, slight inhibition
R.G. Ulrich et al. / Toxicology 131 (1998) 33–47 41
Fig. 7. Inhibition of fatty acid b-oxidation in rabbit hepatocytes isolated from panadiplon-treated rabbits. Cells were isolated fromtwo control and two drug-treated rabbits (20 mg/kg per day for 7 days), and the rates of [14C]palmitate oxidation to acid-solubleproducts were determined ex vivo. Points represent the average of two observations, bars represent the range of measured values.
(92% of control) was observed in one other hu-man donor, and none was observed for the fourthdonor (not illustrated). CPCA was inhibitory inall human hepatocyte suspensions examined, andinhibition was concentration-dependent (Fig. 5)similar to rabbit. Oxidation rates in human hepa-tocytes exposed to 10 mM CPCA ranged from 1(Fig. 6) to 36% of control values (mean rate was17% of controls); 0.1 mM CPCA produced a rateapproximately 50% that of controls. In ex vivodeterminations (Fig. 7), [14C]palmitate oxidationrates in hepatocytes isolated from panadiplon-treated animals were 56 and 57% that of controlvalues. Cell viability, determined by trypan bluedye exclusion, was not effected in any experimentfor either rabbit or human hepatocytes.
3.2. Rhodamine 123 fluorescence imaging
R123 uptake into mitochondria was markedlyreduced in cultured rabbit (Fig. 8) and human(Fig. 9) hepatocytes treated with panadiplon. Rathepatocytes did not show inhibited R123 uptakein any experiment, regardless of exposure time orconcentration. The response in rabbit and human
cells was unambiguous; fluorescence was virtuallyabsent from responding treated cultures. Theoverall response was heterogeneous in that thetime required to observe inhibition at 30 mM wasapproximately 18 h or longer, but cultures fromsome rabbits and humans did not respond at allwithin a reasonable culture period (96 h). Thetime required for a response did not vary betweenculture dishes for any given individual. Acuteinhibition (B18 h) was not observed in any ex-periment. Cultures obtained from three of fiverabbits demonstrated inhibition within 18–48 h;two of five isolations showed little or no inhibi-tion. For human, three of four donor culturesdemonstrated reduced R123 transport. Resultsfrom experiments comparing various concentra-tions of panadiplon (5, 10, 20 and 40 mg/ml inhuman cells and 5 and 10 mg/ml in rabbit) indi-cated lack of a dose response; when inhibition wasobserved, it was at all concentrations (data notshown). R123 mitochondrial accumulation wasalso inhibited in both rabbit and human hepato-cytes by 10 mM CPCA (not illustrated). In exvivo experiments, inhibition of mitochondrialR123 accumulation was observed for hepatocytes
R.G. Ulrich et al. / Toxicology 131 (1998) 33–4742
Fig. 8. Fluorescence digital imaging of mitochondria in cultured rabbit hepatocytes labelled with rhodamine 123. Culturedhepatocytes were incubated with: (a) 0.05% DMSO; or (b) 30 mM panadiplon and 0.05% DMSO for 48 h. Drug-treated cells showreduced mitochondrial fluorescence. Magnification=630× .
isolated from all panadiplon-treated rabbits (Fig.10). At 24 h in culture, cells isolated from treatedrabbits and cultured drug-free had not yet recov-ered mitochondrial R123 transport activity; activ-ity was gradually restored between 48–72 h inculture.
3.3. Respiration by isolated rabbit li6ermitochondria and hepatocytes
The only effect observed in mitochondria iso-lated from panadiplon-treated rabbits (ex vivo,n=2) was a slight decrease in the P/O ratiocompared to controls (Table 2). State 4 and State3 respiration rates in isolated mitochondria werenot affected by any of the treatments, ex vivo orin vitro and the respiratory control ratios werenot systematically different, though this ratio waslower in one treated animal compared to thepaired control. No change in any value was ob-served when control rabbit mitochondria wereexposed directly to panadiplon (data not shown).For hepatocyte suspensions (Table 3), the respira-tory control ratios were lower for all treatedanimals compared to controls; mean respiratorycontrol ratios for cells from treated rabbits were68% that of control values. Also, the ratio ofsuccinate-stimulated to oligomycin-sensitive oxy-gen consumption (Vsucc/Voligo) was higher in hepa-tocytes from treated animals than in controls.
ATP synthesis rates and the ratio of the rate ofADP-stimulated oxygen consumption before andafter addition of oligomycin did not vary signifi-cantly between animals (data not shown).
4. Discussion
In these experiments, we have demonstratedthat the quinoxalinone anxiolytic, panadiplon, in-hibits various mitochondrial activities in rabbitand human hepatocytes. These activities includefatty acid b-oxidation, the mitochondrial mem-brane proton gradient, and mitochondrial respira-tion. Though likely related, the inhibition of thesevarious mitochondrial activities do not occursimultaneously. Fatty acid oxidation could be in-hibited acutely in rabbit and human hepatocytes,but effects on R123 transport and respirationrequired longer exposure. In no experiment wascytotoxicity (cell death) observed; this is in agree-ment with other studies indicating an additionalstress such as hypoxia is required to kill cellsfollowing panadiplon exposure (Bacon et al.,1996).
Fatty acid b-oxidation was acutely inhibited byboth panadiplon and CPCA in isolated rabbit andhuman hepatocytes. Minor variation in concen-tration responses and lack of response for someindividuals may have been due to individual vari-
R.G. Ulrich et al. / Toxicology 131 (1998) 33–47 43
Fig. 9. Fluorescence digital imaging of mitochondria in cultured human hepatocytes labelled with rhodamine 123. Hepatocytes wereisolated from a 41-year-old male donor and incubated with: (a) 0.05% DMSO; or (b) 30 mM panadiplon and 0.05% DMSO for 48h. Drug-treated cells show reduced mitochondrial fluorescence. Magnification=630× .
ations in transport or metabolism. The additionof CPCA to hepatocyte suspensions consistentlyinhibited palmitate oxidation in both rabbit andhuman hepatocytes, including those isolationsthat did not respond to panadiplon, suggestingthat the primary factor in these experiments mayhave been drug metabolism. The metabolism ofpanadiplon is somewhat unusual in that the open-ing of the oxadiazole ring leading to the release ofthe carboxylic acid is a reductive step (Speed etal., 1993; Steenwyk et al., 1994), which may evenoccur within the mitochondria. While the in vitroconcentrations for panadiplon approximated invivo concentrations in rabbits, the hepatic levelsof CPCA are not known and cannot be deter-mined from plasma or excreted values based onhepatic sequestration. The difference in the basalrates of b-oxidation in humans and rabbits mayalso have contributed to the varied responses.Studies have shown that fatty acid oxidation ratesfor a number of species can vary considerably(Agius et al., 1991). For our studies, rabbit hepa-tocytes were isolated from fasted animals whichmay have influenced inhibition since the levels offatty acid oxidation are dramatically altered dur-ing starvation (Mayes and Felts, 1967; Bremer et
al., 1978). No data as to nutritional status ofhuman donors were available, though in all casesseveral hours cold-storage of organs occurredprior to cell isolation. Finally, the composition ofthe incubation medium may also have influencedthe outcome of the b-oxidation experiments. Thepresence of 1.3 mM carnitine may somewhat re-verse the inhibitory effects of panadiplon; this issuggested by evidence showing carnitine pre-vented CPCA-induced inhibition of b-oxidationin isolated rat mitochondria (Duncombe and Ris-ing 1972a). Individual variations in carnitine and/or coenzyme A pools or ability to replenish thesepools may thus influence the rate or degree ofinhibition.
Other mitochondrial effects were also observedin this study including an inhibition of the mito-chondrial proton gradient, as evidenced by inhib-ited R123 uptake, and decreased respiratorycontrol ratios. Effects on the proton gradientlikely occurred in vivo, since R123 transport wasdecreased in cells isolated from treated animals.This effect was reversible, though 48–72 h wererequired for cells from treated rabbits to recoverto control levels of mitochondrial fluorescence.Rat hepatocyte mitochondrial R123 transport was
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not inhibited; this finding is consistent with thelack of toxicity in vivo for this species (T.A.
Table 2Respiration by liver mitochondria isolated from control andpanadiplon-treated rabbitsa
a Rabbits were treated with vehicle or 20 mg/kg per daypanadiplon for 7 days. Results are from two control and twotreated rabbits. Rates are shown as nmoles O2/min per mgprotein, and represent the average of two to five determina-tions per mitochondrial isolation. RCR, respiratory controlratio (State 3 respiration/State 4 respiration); P/O, ATP syn-thesized/oxygen consumed.
Fig. 10. Fluorescence digital imaging of rhodamine 123 (R123)fluorescence in hepatocytes isolated from rabbits administeredvehicle (a, c, e, g) or 20 mg/kg per day panadiplon in vehicle(b, d, f, h) for 7 days. (a) Mitochondrial fluorescence in cellsfreshly isolated from a control rabbit. (b) Mitochondrial up-take of R123 in hepatocytes freshly isolated from a drug-treated rabbit is greatly diminished relative to control. Cells insuspension after isolation were allowed to briefly attach to apetri dish prior to viewing. (c, d) Cells from control andtreated rabbits after 24 h in culture. (e, f) Cells from controland treated rabbits after 48 h in culture. (g, h) Cells fromcontrol and treated rabbits after 72 h in culture. Magnifica-tion=630× .
Jackson, A.D. Hall, unpublished observations).Respiration experiments with isolated mitochon-dria indicated that mitochondria from treatedrabbits were slightly uncoupled, as evidenced bythe decrease in the respiratory control ratios, butthere was no indication that oxidative phosphory-lation was directly inhibited. Since ATP produc-tion was not affected by panadiplon, an alternatesource such as glycolysis coupled with glycogenol-ysis was likely functioning. This is consistent withour earlier observation that livers frompanadiplon-treated rabbits were glycogen-de-pleted (Ulrich et al., 1995). Increased Vsucc/Voligo
suggested that cytoplasmic (non-mitochondrial)ATPases may be more active in hepatocytes fromtreated animals as compared to controls.
Compounds having similar effects topanadiplon in vivo include valproate (Zimmer-man and Kamal, 1982; Kesterson et al., 1984),pirprofen (Danan et al., 1985; Geneve et al., 1987)and the metabolites of hypoglycin (Sherratt,1986). The mitochondrial effects of these andother compounds have been reviewed (Fromentyand Pessayre, 1995). Drugs that inhibit b-oxida-tion are generally carboxylic acids or are metabo-lized to carboxylic acids. The two metabolites ofhypoglycin A, methylenecyclopropylacetate andmethylenecyclopropylformate, appear ultimatelyresponsible for metabolic inhibition. Thesemetabolites form CoA conjugates, which inhibitthe acyl-CoA dehydrogenase and 3-oxoacyl-CoA
R.G. Ulrich et al. / Toxicology 131 (1998) 33–47 45
Table 3Respiration by hepatocytes isolated from control and panadiplon-treated rabbitsa
Control Treated Control TreatedMeasurement Control Treated
a Rabbits were treated with vehicle or 20 mg/kg per day panadiplon for 7 days. Results are from three control and three treatedrabbits; measurements were paired (one control with one treated). Rates are shown as nmoles O2/min per mg protein. RCR,respiratory control ratio (state 3 respiration/state 4 respiration); Volig, oligomycin-sensitive, ADP-independent respiration; Vsucc/Volig, ratio of state 4 respiration to oligomycin-sensitive respiration.
thiolase enzymes of the b-oxidation pathway(Melde et al., 1991). The net results of this inhibi-tion include an accumulation of hepatic triglyce-ride, and in some cases hepatic necrosis andhypoglycemia, and death (Sherratt, 1986).Though panadiplon is not a carboxylic acid, sev-eral lines of evidence suggest that a metabolite,rather than the parent compound, mediates toxic-ity. In vitro, the inhibition of mitochondrial R123uptake was not immediate, but rather required 18h to become apparent. Also, direct exposure ofmitochondria isolated from untreated rabbits tothe parent compound did not produce an effecton respiration, though an effect was seen follow-ing in vivo exposure. Individual variation in re-sponses may have been due to different rates ofmetabolism or clearance. For example, minimalinhibition of palmitate oxidation was observedacutely in hepatocytes from human donor H7, butrhodamine transport was inhibited followingmore lengthy exposure. CPCA is a known in-hibitor of b-oxidation (Duncombe and Rising,1972a), and conjugates as an ester with bothcarnitine and coenzyme A (Steinhelper and Olson,1985; Steenwyk et al., 1994). The methylenecyclo-propylformate metabolite of hypoglycin A bears astructural similarity to the panadiplon metaboliteCPCA, and CPCA conjugates have been observedfollowing exposure of rabbits to panadiplon (Ul-rich et al., manuscript submitted). These conju-gates may be involved mechanistically in theinhibition of mitochondrial b-oxidation, but themolecular aspects are not known. CPCA adminis-tration significantly altered the acyl-CoA levels in
perfused rat livers (Steinhelper and Olson, 1985),and competition for carnitine and CoA by CPCAagainst the b-oxidation pathway likely con-tributed to the observed inhibition by panadiplonand CPCA. Impairment of mitochondrial b-oxi-dation can lead to severe consequences (reviewedby Fromenty and Pessayre, 1995). The net resultsof inhibited mitochondrial oxidation of fatty acidsinclude glycogen depletion, hepatic microvesicularsteatosis, cell death and liver failure; all of theseparameters have been observed in in vivo experi-ments with panadiplon and CPCA. Thus, celldeath in vivo (hepatic necrosis leading to serumtransaminase elevations) may have several con-tributing factors. Inhibition of mitochondrial b-oxidation would result in a reduced ability to rungluconeogenesis, switch cell energy production to-wards glycolysis, and lower cellular glycogenstores. In a healthy individual, this may have noconsequence. However, in an undernourished in-dividual this may lead to hypoglycemia; this hasbeen associated, for example, with hypoglycin Atoxicity (Sherratt, 1986). The inhibition of mito-chondrial activity in human hepatocytes bypanadiplon suggests that inhibition of b-oxida-tion may also have occurred in clinical patients,thus this activity should be avoided with futurepanadiplon analogues.
References
Agius, L., Peak, M., Sherratt, S.A., 1991. Differences betweenhuman, rat, and guinea pig hepatocyte cultures: A compar-ative study of their rates of b-oxidation and esterification
R.G. Ulrich et al. / Toxicology 131 (1998) 33–4746
of palmitate and their sensitivity to R-etomoxir. Biochem.Pharm. 42, 1711–1715.
Bacon, J.A., Cramer, C.T., Petrella, D.K., Sun, E.L., Ulrich,R.G., 1996. Potentiation of hypoxic injury in culturedrabbit hepatocytes by the quinoxalinone anxiolytic,panadiplon. Toxicology 108, 9–16.
Bremer, J., Christiansen, R.Z., Borreback, B., 1978. Regula-tion of partition of free fatty acids between triglyceridesynthesis and b-oxidation in liver. Reg. Mech. Carbohyd.Metab., V. Esmann: Pergamon Press, Oxford, pp. 161–170.
Buxton, D.B., Bahl, J.J., Olson, M.S., 1983. The effects ofcyclopropane carboxylic acid on hepatic pyruvatemetabolism. Metabolism 32, 736–744.
Chen, L.B., 1988. Mitochondrial membrane potential in livingcells. Ann. Rev. Cell Biol. 4, 155–182.
Chesne, C., Guyomard, C., Fautrel, A., Poullain, M.G., Fre-mond, B., De Jong, H., Guillouzo, A., 1993. Viability andfunction in primary culture of adult hepatocytes fromvarious animal species and human beings after cryopreser-vation. Hepatology 18, 406–414.
Duncombe, W.G., Rising, T.J., 1972. Studies on the hypogly-caemic compound cyclopropane carboxylic acid: Effects onfatty acid oxidation in vitro. Biochem. Pharm. 21, 1075–1088.
Duncombe, W.G., Rising, T.J., 1972. Studies on the hypogly-caemic compound cyclopropane carboxylic acid. Effects ongluconeogenesis in vitro. Biochem. Pharm. 21, 1089–1096.
Ehrenberg, B., Montana, V., Wei, M.D., Wuskell, J.P., Loew,L.M., 1988. Membrane potential can be determined inindividual cells from the Nernstian distribution of cationicdyes. Biophys. J. 53, 785–794.
Elliget, K.A., Kolaja, G.J., 1983. Preparation of primarycultures of rat hepatocytes suitable for in vitro toxicitytesting. J. Tissue Culture Methods 8, 1–6.
Fromenty, B., Pessayre, D., 1995. Inhibition of mitochondrialbeta-oxidation as a mechanism of hepatotoxicity. Pharma-col. Ther. 67, 101–154.
Geneve, J., Hayat-Bonan, B., Labbe, G., Degott, C., Letteron,P., Freneaux, E., Dinh, T., Larrey, D., Pessayre, D., 1987.Inhibition of mitochondrial b-oxidation of fatty acids bypirprofen. Role in microvesicular steatosis due to thisnonsteroidal anti-inflammatory drug. J. Pharm. Exp. Ther.242, 1133–1137.
Johnson, L.V., Walsh, M.L., Chen, L.B., 1980. Localization ofmitochondria in living cells with rhodamine 123. Proc.Natl. Acad. Sci. USA 77, 990–994.
Johnson, L.V., Walsh, M.L., Bockus, B.J., Chen, L.B., 1981.Monitoring of relative mitochondrial membrane potentialin living cells by fluorescence microscopy. J. Cell Biol. 88,526–532.
Kesterson, J.W., Granneman, G.R., Machinist, J.M., 1984.The hepatotoxicity of valproic acid and its metabolites inrats. I. Toxicologic, biochemical and histopathologic stud-ies. Hepatology 6, 1143–1152.
Lowry, O.H., 1957. Micromethods for assays of enzymes.Method. Enzymol. IV, 366–381.
Mayes, P.A., Felts, J.M., 1967. Regulation of fat metabolismin the liver. Nature 215, 716–718.
Meglasson, M.D., Smith, K.M., Nelson, D., Erecinska, M.,1989. a-Glycerophosphate shuttle in a clonal b-cell line.Am. J. Physiol. 256, E173–E178.
Melde, K., Jackson, S., Bartlett, K., Sherratt, H.S.A., Ghisla,S., 1991. Metabolic consequences of methylenecyclopropyl-glycine poisoning in rats. Biochem. J. 274, 395–400.
Osterloh, J., Cunningham, W., Dixon, A., Combest, D., 1989.Biochemical relationships between Reye’s and Reye’s-likemetabolic and toxicological syndromes. Med. Toxicol. Ad-verse Drug Exp. 4, 272–294.
Piercey, M.F., Nielsen, E.O., Honore, T., Hoffmann, W.E.,1990. Panadiplon is a benzodiazepine agonist in somebrain sites and an antagonist in others: a 2-DG autoradio-graphy study. Pharmacologist 32, 136.
Rahn, C.A., Bombick, D.W., Doolittle, D.J., 1991. Assess-ment of mitochondrial membrane potential as an indicatorof cytotoxicity. Fundam. Appl. Toxicol. 16, 435–448.
Seglan, P.O., 1973. Preparation of rat liver cells III. Enzymaticrequirements for tissue dispersion. Exp. Cell Res. 82, 391–398.
Sherratt, S.A., 1986. Hypoglycin, the famous toxin of theunripe Jamaican ackee fruit. TIPS, May, pp. 186–191.
Speed, W., Parton, A.H., Martin, I.J., Howard, M.R., 1993.The use of liquid chromatography/thermospray mass spec-trometry with on-line UV diode array and radiochemicaldetection: Characterisation of the putative metabolites ofU-78875 in female rat faeces. Biol. Mass Spectrom. 23,1–5.
Steenwyk, R.C., Sowell, C.G., Gammill, R.B., Pearson, P.G.,1994. Metabolic release of cyclopropane carboxylic acidfrom U-78875: Identification of cyclopropylcarbonylcar-nitine by gas chromatography tandem mass spectrometry(GC-MS-MS). Proc. 42nd ASMS Conf. Mass Spec., 29May–3 June, abstract 58.
Steinhelper, M.E., Olson, M.S., 1985. The effects of cyclo-propanecarboxylate on hepatic pyruvate metabolism.Arch. Bioch. Biophys. 243, 80–91.
Strom, S.C., Fisher, R.A., Thompson, M.T., Sanyal, A.J.,Cole, P.E., Ham, J.M., Posner, M.P., 1997. Hepatocytetransplantation as a bridge to orthotopic liver transplanta-tion in terminal liver failure. Transplantation 63, 559–569.
Sun, E.L., Aspar, D.G., Ulrich, R.G., Melchior, G.W., 1990.Cryopreservation of cynomolgus monkey (Macaca fascicu-laris) hepatocytes for subsequent culture and protein syn-thesis studies. In Vitro Cell. Dev. Biol. 25, 147–150.
R.G. Ulrich et al. / Toxicology 131 (1998) 33–47 47
Tang, A.H., Franklin, S.R., Himes, C.S., Ho, P.M., 1991.Behavioral effects of U-78875, a quinoxalinone anxiolyticwith potent benzodiazepine antagonist activity. J. Pharma-col. Exp. Ther. 259, 248–254.
Ulrich, R.G., Aspar, D.G., Cramer, C.T., Kletzien, R.F.,Ginsberg, L.G., 1990. Isolation and culture of hepatocytesfrom the cynomolgus monkey (Macaca fascicularis). InVitro Cell. Dev. Biol. 26, 815–823.
Ulrich, R.G., Petry, T.W., Bacon, J.A., Blakeman, D.P.,Cramer, C.T., Jolly, R.A., Petrella, D.K., Sun, E.L., 1993.Toxicity of a fatty acid analogue, cyclopropane carboxylicacid, in the Dutch-belted rabbit. Toxicologist 13, 199.
Ulrich, R.G., Bacon, J.A., Branstetter, D.G., Cramer, C.T.,Funk, G.M., Hunt, C.E., Petrella, D.K., Sun, E.L., 1995.Induction of an hepatic toxic syndrome in the Dutch-
belted rabbit by a quinoxalinone anxiolytic. Toxicology 98,187–198.
Ulrich, R.G., Cramer, C.T., Sun, E.L., Bacon, J.A., Petrella,D.K., 1998. A protocol for isolation, culture and cry-opreservation of hepatocytes from human liver. In VitroMol. Toxicol. 11, 23–33.
