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Toxicology Letters 187 (2009) 131–136

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / tox le t

ytotoxicity of ginkgolic acid in HepG2 cells and primary rat hepatocytes

.H. Liu, S. Zeng ∗

epartment of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences,hejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China

r t i c l e i n f o

rticle history:eceived 10 September 2008eceived in revised form 4 February 2009ccepted 12 February 2009vailable online 21 February 2009

eywords:inkgolic acid

a b s t r a c t

Ginkgolic acids and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardouscompounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties. To determinewhether the phase I metabolism could contribute to their cytotoxicity, we investigated the cytotoxicity ofone model compound, ginkgolic acid (15:1), using in vitro bioassay systems. In the first step, cytochromeP450 enzymes involved in ginkgolic acid metabolism were investigated in rat liver microsomes; then, twoin vitro cell-based assay systems, primary rat hepatocytes and HepG2 cells, were used to study and themeasurement of MTT reduction was used to assess cell viability. Results indicated that the cytotoxicity of

at hepatocyteepG2 cellYP inhibitionYP inductionTT

ytotoxicity

ginkgolic acid in primary rat hepatocytes was lower than in HepG2 cells. Ginkgolic acid was demonstratedless cytotoxicity in four-day-cultured primary rat hepatocytes than in 20-h cultured ones. Co-incubationwith selective CYP inhibitors, �-naphthoflavone and ketoconazole, could decrease the cytotoxicity ofginkgolic acid in primary rat hepatocytes. In agreement, pretreatment with selective CYP inducers,�-naphthoflavone and rifampin, could increase the cytotoxicity of ginkgolic acid in HepG2 cells. Thesefindings suggest that HepG2 cells were more sensitive to the cytotoxicity of ginkgolic acid than primary

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rat hepatocytes, and CYP1

. Introduction

Ginkgo biloba has long been used in traditional Chinese medicineo treat circulatory disorders and enhance memory. Ginkgolic acids,mixture of structurally related n-alkyl phenolic acid compounds

n ginkgo biloba L., are strong allergens that could cause severellergic reactions (Hausen, 1998). Besides allergic properties, theyave been recognized to posses possible cytotoxic, mutagenic, car-inogenic and genotoxic properties (Siegers, 1999; Koch and Jaggy,000; Hecker et al., 2002; Baron-Ruppert and Luepke, 2001; Fuzzatit al., 2003; Westendorf and Regan, 2000). Ginkgolic acid (15:1, GA)Fig. 1), 2-hydroxy-6-[pentadec-8-enyl] benzoic acid, is selectedo study due to the fact that its structure is representative andt accounts for about 50% of the total ginkgolic acids (Yang et al.,002). Thus, the investigation of GA cytotoxicity could assist innderstanding the cytotoxicity of other ginkgolic acids.

The liver is mainly responsible for the biotransformation of theajority of xenobiotics. Test systems for hepatic toxicity should be

ble to assess whether the liver will be able to metabolize the testhemical either to a more or less toxic moiety. Rat liver microsomeslay an extensive role in drug discovery as a source of enzymes for

n vitro metabolism and inhibition studies. Because of the capacity

∗ Corresponding author. Tel.: +86 571 88208407.E-mail address: zengsu@zju.edu.cn (S. Zeng).

378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.toxlet.2009.02.012

d CYP3A could metabolize ginkgolic acid to more toxic compounds.© 2009 Elsevier Ireland Ltd. All rights reserved.

to maintain a sufficient level of xenobiotic metabolism, rat hepa-tocytes are frequently used in toxicity tests (Paillard et al., 1999).Primary cultures of rat hepatocytes have many advantages overthe use of whole animals in mechanistic studies (Hammond andFry, 1996; Melo et al., 2002). In contrast to rat hepatocytes, HepG2cells show only about 10% of the P450-dependent mono oxygenaseactivity of freshly isolated human adult hepatocytes (Rueff et al.,1996). HepG2 cells have also been shown to have lower levels ofNADPH-cytochrome P450 reductase and cytochrome b5 than thoseof human liver (Rodriguez-Antona et al., 2002; Yoshitomi et al.,2001). Pre-stimulating HepG2 cells with AhR, PXR, and CAR acti-vators before performing cytotoxicity assays might lead to a betterpredictivity of toxicity (Westerink and Schoonen, 2007a,b). In addi-tion, they have an obvious advantage of their ready availability andassurance of a certain reproducibility of experiments. Therefore,human-derived liver cells HepG2 have been extensively used asthe test system for the prediction of toxicity, carcinogenicity andcell mutagenicity in humans.

Many adverse drug reactions are caused by the cytochromeP450 (CYP) dependent activation of drugs into reactive metabolites.Although ginkgolic acids have shown cytotoxic effects, to date, there

have been no reported studies on the cytotoxicity related to theirmetabolism. The aims of this study were to investigate the cyto-toxicity of ginkgolic acid in primary rat hepatocytes and HepG2cells, and to determine whether the cytochrome P450-mediatedreactions could contribute to its cytotoxic effects.

132 Z.H. Liu, S. Zeng / Toxicology Let

Fig. 1. Chemical structure of ginkgolic acid (15:1).

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ity. In order to avoid possible effects of the solvent on metabolism,the organic solvent in the incubation mixture did not exceed 1%(v/v).The control contained the vehicle only.

As shown in Fig. 3, the results indicated that ketoconazoleand �-naphthoflavone were the potent inhibitors of CYP-mediated

ig. 2. The inhibitory effects of selective cytochrome P450 inhibitors, sul-aphenazole, quinine and 4-methylpyrazole, on GA metabolism in rat liver

icrosomes. The values are expressed as the mean percentage of control activity.

. Materials and methods

.1. Chemicals

Ginkgolic acid (15:1, GA) was prepared in our laboratory and determined by theC–MS method (purity > 99%). Dulbecco’s Modified Eagle’s Medium (DMEM) andon-essential amino acids were purchased from Gibco Zmitrogen (Life Technologies,aisley, Scotland, UK). Fetal calf serum (FCS) was a product from Sijiqing Biologicalngineering Materials Co., Ltd. (Hangzhou, CHN). 3,(4, 5-dimethylthiazol-2yl)-2,5-iphenyltetrazolium bromide (MTT) was obtained from Beijing Dingguo Biologicalechnology Co., Ltd. Trypsin, �-naphthoflavone, Sulfaphenazole, quinine, 4-methylyrazole, ketoconazole, �-naphthoflavone and rifampin were from Sigma (St. Louis,O, USA). All other chemicals used were of the highest grade commercially available.

.2. Preparation of rat liver microsomes

Sprague–Dawley rats (male, 180–210 g; age, 6–7weeks) were housed undertandard conditions and had ad libitum access to water and standard laboratoryodent diet. After no food was supplied for 12 h, the rats were sacrificed by decap-tation. Liver samples were excised, perfused with ice-cold physiological saline toemove blood, and homogenized in ice-cold 1.0 M Tris buffer. Liver microsomesere prepared by the calcium precipitation method (Gibson and Skett, 1994). Allanipulations were carried out in an ice-cold bath. Pellets were re-suspended in

ucrose–Tris buffer (pH 7.4) (95:5, w/v), and immediately stored at −80 ◦C. Micro-omal protein concentration was determined by the method of Lowry et al. (Lowryt al., 1951), using bovine serum albumin (BSA) as the standard.

.3. Isolation and primary culture of hepatocytes

Sprague–Dawley rats (male, 180–210 g; age, 6–7 weeks) were in the same condi-ions as those for microsomes preparation. Hepatocytes were isolated by a two-stepollagenase perfusion method (Moldeus et al., 1978). Cell viability was assessed byhe trypan blue exclusion test. Cells were seeded at a density of 5 × 105 cells/ml in6-well plates pre-coated with 0.2% gelatin. Dulbecco’s Modified Eagle’s MediumDMEM) supplemented with 10% heat-inactivated fetal calf serum, 0.05% strepto-ycin and 0.05% penicillin, was used for culture maintenance. Cells were incubated

n a humidified incubator at 37 ◦C containing 5% CO2 and 95% air. After cells attachedor 6 h, the medium was replaced with serum-free culture medium and the cells werereated as described below.

.4. HepG2 cells culture

The human hepatoma HepG2 cells (passage 26) were obtained from thehanghai Cellular Research Institute (CHN). HepG2 cells from one vial (containing

6 ◦

pproximately 10 cells) were thawed rapidly by immersing in a 37 C water bath.he cells were transferred to a 15 ml centrifuge tube containing 10 ml Dulbecco’sodified Eagle’s Medium (DMEM) and re-suspended by gentle aspiration with a

ipette. After centrifugation for 10 min at 1000 rpm, the supernatant was removednd the cells were re-suspended in complete medium supplemented with 10%eat-inactivated fetal calf serum, 1% non-essential amino acids (NEAA), pH 7.4. Cell

ters 187 (2009) 131–136

viability was assessed by Trypan blue exclusion test. Cell cultures were maintainedin 100 ml culture flasks at a density of 2 × 105 cells/ml in a humidified incubatorat 37 ◦C containing 5% CO2 and 95% air. The medium was refreshed every 2 or 3days and HepG2 cells were trypsinized by 0.25%Trypsin–0.02% EDTA when the cellsreached to 80–90% confluence. The well-grown cells of the third passage (passage28) were harvested and seeded into 96-well plates at a density of 2 × 105 cells/mlfor experiments.

2.5. MTT assay

After treatments, the cytotoxic effects of GA in primary rat hepatocytes andHepG2 cells were determined by the MTT assay using the method described byMossmann (Mosmann, 1983) with some modifications. Briefly, cells were washedonce with 37 ◦C PBS and then added 0.1 ml serum-free medium containing 0.05%MTT to each well. After incubation for 4 h, the culture medium was removed and0.1 ml of DMSO was added to each well to solubilize the formazan formed. The plateswere shaken gently for 10 min and the absorbance was measured at 570 nm. Theabsorbance of treated cells was compared with the absorbance of the controls, whichcells were exposed only to the vehicle and were considered as 100% viability value.

2.6. Statistical analysis

The results were expressed as means ± standard deviations. All calculations wereperformed using Microsoft Excel 2003. Data obtained from cytotoxicity studies werestatistically analyzed with the unpaired Student

′s t-test using the Prism software.

3. Results

3.1. Chemical inhibition studies in rat liver microsomes

To determine which cytochrome P450 isoforms would beinvolved in GA metabolism, the effects of specific CYP chemicalinhibitors such as �-naphthoflavone (CYP1A1/2) (Halpert et al.,1994; Murray and Reid, 1990), Sulfaphenazole (CYP2C6) (Ange’lineGradolatto et al., 2004), Quinine (CYP2D1) (Kobayashi et al., 1989.),4-methyl pyrazole (CYP2E1) (Deborah et al., 1994) and ketocona-zole (CYP3A2) (Baldwin et al., 1995) were investigated in rat livermicrosomes. Each inhibitor was tested in three randomly selectedrat liver samples. All incubations were carried out in a typical incu-bation mixture, which contained 0.5 mg/ml of microsomal protein,0.1 M Tris–HCl buffer (pH7.4), 15 mM MgCl2, 12 mM dl-isocitratetrisodium, 0.38 unit isocitrate dehydrogenase and 50 �M GA. Theconcentration range of inhibitors was 0–100 �M. The final volumewas 0.4 ml. After pre-incubation at 37 ◦C for 5 min, the reaction wasstarted by the addition of �-NADP and �- NADPH (0.9 mM/0.2 mM),and continued at 37 ◦C for 50 min in a shaking water bath (Yu et al.,2003). The metabolism of GA was analyzed by HPLC (Yang et al.,2002) and activities are expressed as a percentage of control activ-

Fig. 3. The inhibitory effects of selective cytochrome P450 inhibitors, �-naphthoflavone and ketoconazole, on GA metabolism in rat liver microsomes. Thevalues are expressed as the mean percentage of control activity.

Z.H. Liu, S. Zeng / Toxicology Letters 187 (2009) 131–136 133

Fig. 4. The dose- and time-dependent cytotoxic effects of GA on cell viability of pri-m(fw

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Fig. 6. The effects of cytochrome P450 specific chemical inhibitors on GA cytotoxicityin primary rat hepatocytes determined by the MTT assay. 0.5, 5 and 15 �M of �-

ary rat hepatocytes determined by the MTT assay. Hepatocytes were exposed to GA10–120 �M) for 1, 7 and 20 h, respectively. Results are presented as the means ± S.D.rom three independent experiments. *Statistically significant difference comparedith controls (p < 0.05).

etabolism of GA. At concentration of 24 �M, �-naphthoflavonenhibited about 25% of microsomes enzyme activity, and thenhibitory effect of ketoconazole was similar. Increasing concen-ration could help ketoconazole strongly enhance inhibitory effect100 �M, 80%), to a lesser extent by �-naphthoflavone (100 �M,0%). In contrast, no apparently inhibitory effects of Sulfaphenazole,uinine and 4-methyl pyrazole were observed (Fig. 2). Therefore,A might be mainly metabolized by CYP 3A2 and CYP1A1/2 iso-

orms in rat liver microsomes.

.2. Cytotoxicity studies in primary rat hepatocytes

After 20 h of culture, unattached cells were removed by gen-le agitation and the medium was changed to serum-free mediumontaining different concentrations of GA (final concentrations, 10,0, 40, 60, 80, 100 �M) or vehicle (DMSO) for control. The finaloncentration of DMSO in the test medium and controls was lesshan 1%. The cells were treated for 1,7 and 20 h, respectively. Eachoncentration was tested in three different experiments in foureplicates.

Fig. 4 shows that the addition of GA to primary culturef rat hepatocytes resulted in time- and dose-dependent cyto-oxicity. Compared with 1 h incubation or controls, 60–100 �MA could significantly decrease hepatocytes viability after 20 h

ncubation.In order to investigate the cytotoxicity in long-term-cultured

epatocytes, medium described above was supplemented with.1 �M insulin and changed every 24 h. At the fifth day, unattachedells were removed by gentle agitation and the medium washanged to serum-free medium containing different concentra-ions of GA (final concentrations 20, 40, 60, 80,100,120 �M).

After 24 h incubation at various concentrations of GA, as shownn Fig. 5, a dose-dependent cytotoxicity was also found in four-day-

ultured primary rat hepatocytes, whereas cells viability droppedlightly with increasing concentrations of GA. Compared with one-ay-cultured hepatocytes, viability changes are not statisticallyignificant, and only about 10% loss of viability was observed event 120 �M.

ig. 5. The cytotoxic effects of GA on the viability of four-day-cultured primaryat hepatocytes determined by the MTT assay. Hepatocytes were exposed to GA20–120 �M) for 24 h. Results are presented as the means ± S.D. from three inde-endent experiments.

naphthoflavone and 0.5, 5, 15 �M of ketoconazole co-incubated with 20 �M (C),40 �M (B), 60 �M (A) of GA in primary rat hepatocytes for 24 h, respectively. Resultsare presented as the means ± S.D. from three independent experiments. *Statisticallysignificant difference compared with incubations with no inhibitor (p < 0.05).

3.3. Effects of CYP inhibitors on GA cytotoxicity

To examine the contributions of specific cytochrome P450isoenzymes-mediated reactions on GA cytotoxicity, after 20 h ofculture, cell culture medium was replaced with test medium con-taining different concentrations of GA (final concentrations 20, 40,60 �M), and co-incubated with �-naphthoflavone (an inhibitor ofCYP1A1/2) or ketoconazole (an inhibitor of CYP3A2) (final con-centrations 0.5, 5, 15 �M) or vehicle (DMSO) for control. The finalconcentration of DMSO in the test medium was less than 1%. Eachconcentration was tested in three different experiments in fourreplicates.

The effects of cytochrome P450 specific chemical inhibitors ontoxicity of GA in primary rat hepatocytes are shown in Fig. 6. Both�-naphthoflavone and ketoconazole were observed to affect GAtoxicity in a dose dependent manner. At test concentrations of 40and 60 �M GA, 15 �M of both inhibitors could significantly enhancehepatocytes viability to over 90%. In contrast with ketoconazole,�-naphthoflavone could more efficiently inhibit the cytotoxicityof GA. No cytotoxicity was observed in the MTT assay when theinhibitors were tested in control experiments at these concentra-tions (data not shown).

In order to study whether the cytotoxic effects were indeed

caused by the formation of toxic metabolites, after the incuba-tion, the culture medium was collected, and the same volumeof acetonitrile was added. The mixture was then centrifuged at10,000 rpm for 20 min. The amount of GA was determined by a HPLCmethod (Yang et al., 2002). The inhibitory effects of cytochrome

134 Z.H. Liu, S. Zeng / Toxicology Letters 187 (2009) 131–136

Table 1The effects of cytochrome P450 specific chemical inhibitors on GA metabolism in primary rat hepatocytes determined by the HPLC analysis of GA remained in the culturemedium after the incubation.

Concentration (�M) GA remained in culture medium

Control �-Naphthoflavone ketoconazole

0.5 5 15 (�M) 0.5 5 15 (�M)

20 6.7 ± 0.6 7.8 ± 0.71 8.3 ± 0.6 9.4 ± 0.7 7.3 ± 0.7 7.7 ± 0.6 8.6 ± 0.840 13.9 ± 0.7 15.9 ± 0.8 17.0 ± 0.8 19.3 ± 0.9 14.7 ± 0.8 15.4 ± 0.7 16.9 ± 0.76 .0 31.5 ± 1.1 22.7 ± 0.9 24.5 ± 1.0 27.6 ± 0.9

0 with 20, 40, 60 �M of GA in primary rat hepatocytes for 24 h, respectively. Results arep

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.5, 5, 15 �M of �-naphthoflavone and 0.5, 5, 15 �M of ketoconazole co-incubatedresented as the means ± S.D. from three independent experiments.

450 specific chemical inhibitors on the metabolism of GA in pri-ary rat hepatocytes are shown in Table 1. The co-incubation with-naphthoflavone and ketoconazole could increase the amount ofA remained in the culture medium in a dose dependent man-er. In contrast with ketoconazole, �-naphthoflavone could morefficiently inhibit the metabolism of GA.

.4. Cytotoxicity studies in HepG2 cells

After about 24 h of culture when cells reached 60–70% conflu-nce, unattached cells were removed by gentle agitation and theedium was changed to serum-free medium containing various

oncentrations of GA (final concentrations: 5, 10, 20, 30, 40, 60 �M)r vehicle (DMSO) for control. The cells were treated for 24 h. Eachoncentration was tested in three different experiments in fiveeplicates. The final concentration of DMSO in the test medium andontrols was less than 1%.

As shown in Fig. 7, the addition of GA to HepG2 cell cul-ure resulted in dose-dependent cytotoxicity after 24 h exposure.lthough no apparent cytotoxic effect on cell viability was observedt lower concentrations (5 and 10 �M), at higher concentrations, GAas more toxic to HepG2 cells than to rat hepatocytes. Comparedith the control group, 60 �M of GA significantly decreased cell

iability by up to 90%.

.5. Effects of CYP inducers on GA cytotoxicity

To confirm the effects of certain cytochrome P450 isoforms-ediated reactions on GA cytotoxicity, after 24 h of culture, cell

ulture medium was replaced with induction medium contain-ng 25 �M �-naphthoflavone (an inducer of CYP1A2 and UGT1A),r 10 �M rifampin (an inducer of CYP2C9, CYP3A4 and UGT1A).he medium containing inducers was replaced every 24 h andnducers were present for 72 h. After pretreatment, the cells were

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ashed with warm (37 C) PBS and then incubated in serum-ree medium containing different concentrations of GA (finaloncentrations, 5, 10, 20, 30, 40, 60 �M) or vehicle (DMSO)or control. The cells were treated for 24 h. The final concen-ration of DMSO in the test medium was less than 1%. Each

ig. 7. The cytotoxic effects of GA on HepG2 cells determined by the MTT assay.epG2 cells were exposed to GA (5–60 �M) for 24 h. Results are presented as theeans ± S.D. from three independent experiments. *Statistically significant differ-

nce compared with controls (p < 0.05).

naphthoflavone (B) or 10 �M of rifampin (A) for 3 days. Thereafter, cells were exposedto GA (5–60 �M), respectively. Results are presented as the means ± S.D. from threeindependent experiments. *Statistically significant difference compared with con-trols (p < 0.05).

concentration was tested in three different experiments in fourreplicates.

Fig. 8 presents the GA cytotoxicity in HepG2 cells pretreatedwith �-naphthoflavone or rifampin. Both cytochrome P450 induc-ers increased sensitivity of HepG2 cells to GA cytotoxicity comparedwith unpretreated incubations. Exposed to 20 �M GA, HepG2 cellspretreated with BNF lost over 70% cell viability. In contrast, the cellviability loss was only about 44% observed at the same concen-tration in RIF treated cells. 60 �M GA was similarly toxic to eitherthe pretreated or unpretreated cells. No apparent cytotoxicity wasobserved in the MTT assay when the inducers were tested in controlexperiments at these concentrations (data not shown).

4. Discussion

Cytotoxicity assays are widely used in in vitro toxicology studies.One previous study showed that ginkgolic acids caused DNA strand-breaks in primary rat hepatocytes (Westendorf and Regan, 2000).Another study indicated that ginkgolic acids activated protein phos-phatase 2C to induce neurotoxic effects in cultured chick embryonicneurons (Barbara et al., 2001). In the present study, ginkgolic acidcytotoxicity was examined using human hepatoma cells and rathepatocytes from the viewpoint of phase I metabolism.

The LDH leakage assay, the protein assay, the neutral red, theATP assay and the MTT assay are the most common employed forthe detection of cytotoxicity or cell viability following exposureto toxic substances. For the detection of any effect due to toxicmetabolites, reproducible and sensitive endpoints are fundamental.

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Z.H. Liu, S. Zeng / Toxicolo

n vitro MTT assay is one of the most used for preliminary screen-ng since Mosmann (1983) developed. It determines the ability ofiable cells to convert a soluble yellow tetrazolium salt (MTT, 3-(4,-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) into

nsoluble purple formazan crystals by the mitochondrial dehydro-enase enzymes. The MTT assay is a rapid, versatile, quantitative,nd highly reproducible colorimetric assay for mammalian cell via-ility/metabolic activity.

The toxicity of GA in HepG2 cells was greater than that foundn primary rat hepatocytes. A difference in phase II metabolismetween HepG2 cells and primary rat hepatocytes might bene reason. The major phase II metabolizing enzymes are theDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs),lutathione S-transferases (GSTs), arylamine N-acetyltransferasesNATs), and epoxide hydrolases (EPHXs). Levels of SULT1A1, 1A2,E1, 1A2, and 2A1, microsomal GST 1, GST l1, NAT1, and EPHX1 inepG2 cells were almost similar to levels in primary human hep-tocytes. In contrast, levels of UGT1A1 and 1A6 transcripts wereetween 10- and more than 1000-fold higher in the primary hep-tocytes (Westerink and Schoonen, 2007a,b).Primary hepatocytesontain active glucuronyl transferases which could conjugate theydroxylated metabolites to more water soluble and less toxicorms (Smith et al., 2005; Westerink and Schoonen, 2007a,b). Theoxicity in HepG2 cell might probably represent a direct toxicity

ediated by the parent compound. This increased cytotoxicity pos-tively correlated with the increased concentrations of GA.

Hepatocytes closely reflect the metabolism in the liver. Viableepatocytes attach within 6 h to the culture dishes. After 20 h ofulture, cells could spread and develop cell–cell contact. It is wellnown that primary cultures of mammalian hepatocytes suffer aapid and gradual loss of cytochrome P450 content in the h/daysfter isolation (Paine, 1990), but the levels of the phase II enzymesecrease a little (Jover et al., 1992; Fry et al., 1995). For this rea-on, hepatocytes cultures of different ages (20 h and 4 days) weresed. Results showed that the toxicity of GA in aged hepatocytesultures (4 days) was lower, probably because either there were lit-le toxic phase I metabolites formed by the hepatocytes, or phaseI metabolism might mainly occur, which could produce nontoxicolar conjugates such as glucuronides and sulfate conjugates.

Differential induction or inhibition of xenobiotic metabolizingnzyme system can be used to provide information about mech-nisms by which xenobiotics exert their toxic effects. Factors ofnzyme inhibition in rat liver microsomes, such as enzyme con-entration, reaction time and GA concentration, were selectedccording to our previously enzyme kinetic studies (data not pub-ished). Ketoconazole, a known inhibitor of CYP3A2 in rat liver

icrosomes (Baldwin et al., 1995), showed CYP1A1 and 1A2 com-etition in human and rat supersomes at high concentrationsWesterink et al., 2008). In rat liver microsomes, we postulate thatYP1A1 and 1A2 might be also inhibited by 100 �M ketocona-ole, combining with the strong inhibition of CYP3A2, which ledo the significantly decrease of GA metabolism together. GA cyto-oxicity was decreased in the co-incubation with ketoconazole or-naphthoflavone in primary rat hepatocytes, probably because

hat the production of toxic metabolites mediated by CYP3A andYP1A isoforms were inhibited.

Rifampin (RIF) significantly upregulated CYP2B6 and CYP3A4 inepG2 cells, especially CYP3A4 (Matsuda et al., 2002; Martin et al.,008). Levels of CYP1A1 and CYP1A2 mRNAs in HepG2 cells were

ncreased in a concentration-dependent manner after treatmentith �-naphthoflavone (BNF) (Rika Ueda et al., 2006). The levels of

YP1A1 mRNA and CYP1A2 mRNA could reach over 20-fold higherhan controls after treatment with 10 �M BNF. To make sure thathe formation of CYP3A and CYP1A dependent reactive metabo-ites was toxic, HepG2 cells were pretreated with RIF and BNF forhree days. The results confirmed that the induction of CYP3A and

ters 187 (2009) 131–136 135

CYP1A enzymes could lead GA to more toxic compounds in HepG2cells.

There are many examples of metabolites or reactive inter-mediates of chemicals that have been shown to exert adversedrug reactions. Reactive metabolites are a common product ofphase I oxidation reactions mediated by cytochrome P450 (CYP)-dependent mixed function oxygenases, although also examplesof other phase I (e.g. flavin-mono oxygenases; FMOs) and phaseII drug metabolizing reactions have been described (Zhou et al.,2005). The generation of such reactive metabolites may produceadverse reactions via different inter-related process such as forma-tion of free radicals, oxidation of thiols and covalent binding withendogenous macromolecules, resulting in the oxidation of cellu-lar components or inhibition of normal cellular functions (Riley etal., 1988). The present study revealed that GA was more cytotoxicto human hepatoma cells HepG2 than to primary rat hepatocytes.Inhibition and induction of cytochrome P450 enzymes further indi-cated that the CYP-mediated reactions produced more cytotoxiccompounds than the parent compound of GA. Thus, it is very nec-essary to use cell-lines expressing CYP1A or CYP3A isoforms toconfirm the cytochrome P450-mediated cytotoxicity. Further studyshould be done to elucidate the mechanism of hepatic toxicity ofGA and its phase I reactive metabolites in rat liver or liver cells.In our laboratory, preliminary study of metabolic profiles of GA inHepG2 cells and primary rat hepatocytes revealed that there weretwo main metabolites. The negative ESI mass spectrum exhibitedone quasi-molecular ion peak [M−H]− at m/z 361 and the other atm/z 375. Compared with GA, there was an addition of 16 Daltons(the mass of one atom of oxygen-16) and 30 Daltons, respectively(mass spectrum not shown), indicating that the phase I metabolismindeed happened.

The species differences between rat and human might play a roleon the toxicity of GA. On the one hand, CYP1A shows a quite strongconservation among species (Mugford and Kedderis, 1998) withan identity to human higher that 80% in rat (83 and 80%, respec-tively for CYP1A1 and CYP1A2); on the other hand, CYP3A4 and itsrelated CYP3A5 are the most abundant CYP isoforms in human liver(Dresser et al., 2000), whereas CYP3A1 is the main CYP3A form inrat liver (Gonzalez et al., 1985). So to evaluate the hepatotoxicity,human liver hepatocytes could be the best type of cells in vitro.

Due to the adverse effects, guidelines of several regulatoryauthorities require the removal of ginkgolic acids from therapeuti-cally used Ginkgo extracts below a limit concentration of maximally5 ppm. In this study, just for the reason of the sensitivity of theexperiments, the concentrations of GA were much higher thanthat in a recommended normal diet. So the physiological relevanceshould be further evaluated. One result of this study, which theinduction of CYP1A and 3A enzymes could increase the toxicity ofGA, suggests that a combination of intake of CYP1A or 3A inducers(such as dexamethasone, omeprazole or rifampin) and GA mightcause the toxic events.

In conclusion, we investigated the cytotoxicity of GA in pri-mary rat hepatocytes and HepG2 cells, and its possible mechanismfrom the viewpoint of phase I metabolism. Results showed thatrat liver microsomes, rat hepatocytes and HepG2 cells were usefulas rapid and relatively inexpensive in vitro assays for the predic-tion of cytochrome P450-mediated toxicity. GA was less toxic in rathepatocytes than in HepG2 cells. The CYP3A and CYP1A-mediatedreactions could lead GA to more toxic compounds.

Conflict of interest

We declare that we have no financial and personal relationshipswith other people or organizations that can inappropriately influ-ence our work, there is no professional or other personal interestof any nature or kind in any product, service and/or company that

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36 Z.H. Liu, S. Zeng / Toxicolo

ould be construed as influencing the position presented in, or theeview of, the manuscript entitled.

cknowledgement

This project was supported by Key Scientific and Technical Foun-ation of Zhejiang (#2005C13026).

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