133
Cancer Letters, 65 (1992) 133 - 137 ElsevterScientiftcPublishersIrelandLtd.
Inhibitory effect of geniposide on aflatoxin B1-induced DNA repair synthesis in primary cultured rat hepatocytes Shao-Win %stitute
Wang”,
of Medicine,
Chung-Yih bDepartment
and Dental College, Taichung,
Laib and Chau-Jong
of Medico1 Technology,
Taiwan
Wang’
and ‘Department
of Biochemistry,
Chung-Shan
Medical
(ROC)
(Received 17 February 1992) (Revision received 6 May 1992) (Accepted 8 May 1992)
Summary We have previously demonstrated that geniposide (GP) inhibits the aflatoxin BI (AFB,) induced-hepatotoxicity and hepatic DNA binding in rats. To address the mechanism of action, the effects of GP on AFBI-induced DNA repair synthesis and AFBl biotransformation in cultured rat hepatocytes were investigated. By evaluation of unscheduled DNA synthesis (LIDS), GP reduced AFBI-induced DNA repair synthesis in a dose-dependent manner in hepatocyte cultures. GP elevates the metabolism of AFBl and decreases the formation of AFMl. The
enzyme activities of glutathione S-transferase (GST) and GSH-peroxidase (GSH-Px) in AFB,-treated hepatocyte cultures are enhanced in the presence of GP. GP reduces AFBIinduced DNA repair synthesis through an increased AFBl detoxication metabolism. It provides one possible mechanism for the chemopreventive activity of GP. Correspondence to: Chau-Jong Wang, Department of Biochemistry, Chung-Shan Medical and Dental College, No. 113, Section 2, Ta-Ching Street, Taichung, Taiwan, Republic of China. Abbreviations: GP, geniposide; APB,, aflatoxin B,; GSH. glutathione; GST, glutathione S-transferase; GSH-Px, glutathione peroxidase; CDNB, 1-chloro-2,4-dinitrobenzene; UDS, unscheduled DNA synthesis.
K-o&: geniposide; aflatoxin B,; unscheduled DNA synthesis; glutathione Stransferase; glutathione peroxidase IXktrodUCtiOD
Geniposide (GP), an iridoid glycoside, was isolated from the fruit of Gardenia jasminoides Ellis by Inouye et al. [1,2]. Gardenia jasminoides Ellis. f. frandiflora makino, a plant in the family of Rubiaccase, grows widely in the middle and southern parts of Taiwan. Its fruit, gardenia, has been used as a herbal medicine to treat liver and gall bladder disorders, hepatitis and acute jaundice [3]. The crude extract rapidly lowers serum bilirubin and transaminase levels in acute hepatitis [4]. The antihyperbilirubinemic effect of Gardenia extracts has been reported in an animal model [5,6]. Lau et al. [7] proposed that gardenia or GP may facilitate the billiary extraction of cy-naphthyisothiocyanate and/or its toxic metabolite(s). Recently, we have demonstrated the suppressive effect of GP on the hepatotoxicity and hepatic DNA binding of aflatoxin B1 in rats [8]. Aflatoxin B1 (AFBJ is a potent hepatotoxin and hepatocarcinogen in rats and humans [9, lo]. The toxic and carcinogenic effects of AFBl are seen only after it is metabolized by cytochrome P-450 enzymes. These enzymes
0 1992 Ekevier Scientific Publishers Ireland Ltd. 0304-3835/92/$05.00 Printed and Published in Ireland
134
catalyze the formation of various oxidative derivatives, including an unstable, highlyreactive 8,9-epoxide that is presumed to be the ultimate carcinogenic species of AFBl [ll]. This reactive electrophilic epoxide covalently interacts with nucleophilic centers in DNA and protein. Apart from binding to macromolecules, alternative fates for AFB1-&g-oxide include hydrolysis to 8,9-dihydrodiol [12] and conjugation with glutathione (GSH) to form 8-(S-gIutathiony)-9-hydroxy-8,9-dihydroAFBl (AFB1-SG) [13,14]. The formation of the AFB1-GSH conjugate, through the GSH S-transferase (GST) catalysis, is a detoxication reaction. Resistance to the acute toxicity of AFBl has been correlated with the formation of AFB1-GSH conjugates and activity of GST [ 151. GSH and its related enzymes including GST and GSH-peroxidase GSH-Px) would therefore provide an important detoxication pathway. In the present study, the effect of GP on AFB1-induced unscheduled DNA synthesis and AFBl biotransformation is investigated. The possible mechanism of the chemopreventive action of GP on AFBl induced hepatic damage is discussed. Materials
and Methods
Chemicals [Methyl-3H]thymidine (spec. act. 20 Ci/ mmol) was purchased from ICN Radiochemicals (Irvine, CA, USA). Non-radioactive thymidine , AFBl , AFM1, collagenase , calf thymus DNA, DABA, Tris, proteinase K, GSH, NADPH, GSH reductase, CDNB, sodium azide, OPT, ATP, L-glutamate, L-W aminobutyrate and EDTA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). GP was purchased from Wako Pure Chemical Co. Japan. Aquasol- was obtained from New England Nuclear (Boston, MA, USA) and the protein assay kit from Bio-Rad Lab. Ltd. (Watford, Herts, UK). All other chemicals and reagents used were of the highest purity commerically available.
Culture
of rut hepatocytes
Hepatocytes were isolated from adult female Sprague - Dawley rats (Taichung Veterans General Hospital, Taiwan, 150 - 200 g, fed ad Iibitum) by the collagenase perfusion method [16]. They were plated at a cell density of 3.75 x lo5 cells/ml in 4 ml of Williams E medium (Gibco) supplemented with 10% calf bovine serum, 1% PSN (Gibco) antibiotic mixture and nicotinamide (1 mg/l) . The medium was changed 3 h after plating to remove nonadherent cells and the culture was maintained for the time indicated. For treatment of culture with test agents, stock solutions of chemicals in dimethylsulfoxide (Sigma) were added to the cultures so that the final concentration of DMSO was less than 0.2%.
Measurement of DNA repair synthesis The effect of GP on AFBl induced DNA repair synthesis was measured by determinating the amount of [methyl-3H]thymidine incorporated into nuclear DNA in the presence of hydroxyurea (15 mM) [17]. Hepatocyte cultures were treated with 0.01 mM AFBl and GP at desired concentrations following preincubation with hydroxyurea for 1 h. A l-&i quantity of [3H]thymidine/ml was added to the culture medium for an additional 20 h. The cell monolayer was washed twice with PBS and harvested at the end of culturing. Following harvesting the cells were loaded onto a 25mm, 2-pm pore size PC filter (Nucleopore) through a Swinnex polyethylene filter holder to lyse the cells with 10 ml lysing buffer containing 2% SDS, 0.025 M EDTA, 0.5 mg/ml thymidine, 0.1 M glycine (pH 10) plus 0.15 mg/ml proteinase K and washed with 3 ml of the same buffer without proteinase K. The filter was carefully transfered to a scintillation vial. A l-ml quantity of 0.5 N HC104 was added to the vial and heated at 60°C in a water bath for 1 h. An aliquot was taken for the determination of radioactivity by liquid scintillation spectrometry and DNA content, expressed as dpm/pg DNA. DNA was measured in a cell lysate using the DABA method [18].
135
Eualution of less-toxic metubolites of AFBl The effect of GP on AFBr metabolism was measured by adding 0.01 mM AFBr and GP to the culture medium for 6 h, when the medium was removed and an equal volume of ice-cold methanol added. This mixture was extracted three times with CHCls. The CHCIS phase was washed with distilled water and concentrated to analyze all known metabolites of AFBr by HPLC [12]. On a Zorbax ODS column AFBr and its metabolite were eluted with 15% dimethylfomamide in 0.01% phosphoric acid at 1.5 ml min - ’ and a column temperature of 69OC. Enzyme actiuities of GST and GSH-Px To examine the effects of GP on the activities of GST and GSH-Px in hepatocyte cultures treated with AFBr, the cells were harvest at the end of experiment to determine the enzyme activities as previously described [8]. After harvest the cells were homogenized with a tight-fitting polytron pestle (20 strokes) in Tris - sucrose buffer (pH 7.5) and centrifuged. The supernatant was used for the determination of GST [19] and GSH-Px [ZO] activities. Protein concentration was measured by using a standard commercial kit (Bio-Rad. Lab. Ltd., Watford, Herts, UK) with BSA as standard [Zl].
Effects of GP on Table 1. damage in cultured hepatocytes. Treatment”
dpm/pg
AFBl (0.01 mM) AFB, (0.01 mM) plus: GP (0.02 mM) GP (0.05 mM) GP (0.10 mM)
97.75
AFB1-induced
DNA
% of control
DNA
f 9.34b
100
74.54 zt 5.58’ 69.26 zt 9.37’ 56.70 z!z5.81”
76 71 58
“Hepatocytes (lo6 cells) were cultured with William E medium for 3 h and incubated with hydroxyurea for the other 1 h. AFB, with or without GP was then added into the culture medium. A l-&i quantity of [methyl3H]thymidine (20 Ci/mmol) was added to the culture medium immediately after the test chemical for an additional 20 h. At the end of culture, the cells were harvested and lysed for radioactivity counting and DNA deter- mination. DNA damage was determined by UPS and expressed as dpm/pg DNA. bMean f S.D., values are the average of triplicate determinations. ‘P < 0.05, lP < 0.01, compared with the AFB,treated group. l
Table U. Effects cultured hepatocytes. Treatment”
of
GP
on
AFMl
formation
in
AFBl residue
AFM, kg(s)
(c(g)
Effect of GP on AFBr induced DNA repair synthesis The effect of GP on AFBr induced DNA repair synthesis was measured as unscheduled DNA synthesis (171. GP significantly inhibited AFBr induced DNA repair synthesis in a dose response manner (P < 0.05 in 0.02 and 0.05 mM, P < 0.01 in 0.1 mM) (Table I). Addition of GP to the culture medium reduced AFBrinduced DNA repair synthesis in cultured hepatocytes. It indicates that GP inhibited AFBl induced DNA damage in primary culture hepatocytes.
1.043 * 0.208
AFBl (0.01 mM) 0.937 f 0.076b AFB, (0.01 mM) plus: GP (0.02 mM) 0.898 f 0.114 GP (0.05 mM) 0.720 zt 0.113 GP (0.10 mM)
0.692 zt 0.042’
l
0.231 0.185 .*
f 0.058’ f 0.035
0.197 .
zt 0.076
l
‘Hepatocyte cultures were treated with AFBr and GP with designated concentrations for 6 h. The medium was collected for the determination of AFM, formation. bMean f S.D., values are the average of triplicate determinations. ‘P < 0.01 compared with AFB, treated ‘P < 0.02, l
Qroup
136
Effect of GP on AFBl
Discussion
metabolites
The effect of GP on AFBl metabolism was measured by analyzing AFBr metabolite formation. In Table II, during the 6-h incubation of hepatocytes with AFBi, most of AFBi was metabolized. AFMi was the only CHClsextractable metabolite seen by HPLC analysis. The amounts of AFBi residue and AFMi were significantly decreased in culture medium in the presence of GP (0.1 mM). This indicates that the inhibitory effect of GP on AFBl induced DNA repair synthesis was not due to increased conversion of AFBi into the less toxic metabolite AFMi. Effect of GP on the actioities of GST Px induced by AFBl
and GSH-
The activities of GST and GSH-Px were slightly increased but not significantly by adding AFBi (Table III). When GP (0.1 mM) was added in combination with AFB1, there was a significant elevation of GST and GSH-Px activities (P < 0.01). The inhibitory effect of GP on AFBi induced DNA damage may be due to the increased detoxication of AFBi.
Table 111. Effects of GP on the activities of GST and GSH-Px induced by AFBr in cultured hepatocytes. Treatment”
GST (nmol/min per mg protein)
DMSO (0.2%) 273 l 14 AFBr (0.01 mM) 294 l 12 AFBl (0.01 mM) plus: GP (0.02 mM) 307 f 3 GP (0.05 mM) 307 f 2 GP (0.10 mM) 346 zt 4’
GSH-Px (nmol/min per mg protein) 286 zt 11 291 f 17 294 f 13 310 f 7 375 f 31’
“Hepatocyte cultures were treated with AFBl and GP with designated concentration for 6 h. Cells were harvested, homogenized and centrifuged for enzyme activity assays at the end of culture. bMean f SD., values are the average of triplicate determinations. lP < 0.01, compared with AFB, treated group.
The results presented in this paper show the chemopreventive effect of GP on AFBi induced DNA damage in cultured hepatocytes. This is supported by the reduction of AFBiinduced DNA repair synthesis in the presence of GP. Previous reports have demonstrated a reduction of the hepatotoxic effects of carbon tetrachloride and cr-naphthylisothiocyanate by GP [23,24]. We have also reported the suppressive effect of GP on the hepatotoxicity and hepatic DNA binding of AFBi in rats [8]. Similarly, there is an inhibition of AFBiinduced DNA damage with the presence of GP in cultured hepatocytes. The inhibitory effect of GP on AFBi induced DNA damage may be due to the enhancement of the activities of certain drug-metabolizing enzymes. This results in an increased conversion of AFBi to a less toxic metabolite. Protection against AFBiDNA binding may result from either or both of the following two mechanisms (a) the conversion of AFBi into less toxic metabolites, such as AFMi 1251, (b) induction of detoxication pathways via the conjugation with GSH [15]. The present studies show that GP elevates the metabolism of AFBi and decreases the formation of AFMr (Table II). It indicates that the inhibitory effect of GP was not caused by the conversion of AFBi into the less toxic metabolite such as AFMi. Since conjugation with GSH plays an important role on AFBi detoxication, the effect of GP on the activities of GST in AFBt-treated hepatocyte cultures is also analysed. When GP is added to the culture medium in combination with AFBi there is a significant elevation of GST activity (Table III). The increased activity of GST induced by GP may increase the AFBi exclusion by conjugation with GSH. Thus, the inhibitory effect of GP on AFBi induced DNA damage may be mediated by increased GST activity. The GP also induced GSH-Px activity. This phenomenon possibly occurs through an increased utilization of GSH, which can facilitate the reduction of peroxides or scavenging of free radicals
137
generated from AFBr minor metabolism via the co-oxidative pathway [26]. In conclusion, the possible mechanisms of GP inhibits AFBr induced DNA repair synthesis were investigated. The inhibitory effects of geniposide on AFBr-induced hepatic DNA damage possibly involve enhancing GST and GSH-Px activities for AFBr detoxication.
C.E. 12
13
14
Acknowledgement 15
This work was supported by the National Science Council Grant NSC 81-0421-B-040OlZ, National Science Council Grant, Republic of China. References
16
17
1
Inouye, H. and Saito, S. (1969) Zwei neue iridoidglucoside aus Gardenia jasminoides: gardenosid und geniposide. Tetrahedron Len., 28, 2347 - 2350. H. (1973) The constituents of 2 Endo, T. and Taguchi, Gardenia jasminoides: geniposide and genipin-gentiobioside. Chem. Pharm. Bull., 21, 2684-2688. 3 Miyasita, S. (1976) A historical study of Chinese drugs for the treatment of jaundice. Am. J. Chin. Med., 4, 239 - 243. 4 Wu, Y .Y. (1956) Clinical efficiency of capillaris combination as main therapy for jaundice. Chin. Med. J., 10, 930 - 932. 5 Miwa, T. (1954) Study on Gardenia Florida as remedy for icterus (IV). Jpn. J. Pharmacol., 4, 69-810. 6 Kong, Y.C., Che, C.T., Yip, T.T. and Chang, H.M. (1977) Effects of Fructs Gardenia extract on hepatic function. Comp. Med. East West, 5, 241- 255. (1986) Effects 7 Lau, F.T.K., Chang, H.M. andPak,R.C.K. of Gardenia jasminoides and geniposide on hepatic drugmetaboliiing enzyme activities: implications for yhepatotoxtcity. naphthylisothiocyanate-induced Asia Pacific J. Pharmacol., 1, 91-98. 8 Wang, C.J., Wang, SW. and Lin, J.K. (1991) Suppressive effect of geniposide on the hepatotoxicity and hepatic DNA binding of aflatoxin Bt in rats. Cancer Len., 60, 95- 102. P.M. and Butoer, W.H. (1969) Acute and 9 Newberne, chronic effects of aflatoxin Br on the liver of domestic and laboratory animals: a review. Cancer Res., 29, 236 - 250. R.C., Bhamarapravati, N., Cordon, J.E. and 10 Shank, Wogan, G.N. (1972) Dietary aflatoxins and human liver cancer, N. Incidence of primary liver cancer in two municipal populations in Thailand. Food Cosmet. Toxicol., 10, 171-179. 11 Busbyr, W.F. and Wogan, G.N. (1985) Aflatoxins. In: Chemical Carcinogens, 2nd edn., pp. 945 - 1136. Editor:
18 19
20
21
22
23
24
25
26
Searle.
American
Chemical
Society,
Washington,
DC. Neal, G.E. and Colley, D.J. (1979) The formation of 2,3dihydro-2,3-dihydroxy-aflatoxin B, by the metabolism of aflatoxin B1 in vitro by rat liver microsomes. FEBS Len., 101. 382-386. Degan, G.H. and Neuman, H.G. (1978) The major metabolites of aflatoxin Br in the rat is a glatathione conjugate. Chem.-Biol. Interact., 22, 239 - 255. Emerole, G.O., Neskovic, N. and Dioxn, R.L. (1979) The detoxication of aflatoxin B, with glutathione in the rat. Xenobiotica, 9, 737 - 743. Neal, G.E., Metcalf, S.A., Legg, R.F., Judah, A.J. and Green, J.A. (1981) Mechanism of the resistence to cytotoxicity which precedes aflatoxin B, hepatocarcinogenesis. Carcinogenesis, 2, 457 - 461. Bonney, V.R., Becker, J.E., Walker, P.R. and Potter, V.R. (1974) Primary monolayer cultures of adult rat liver parenchymal cells suitable for study of the regulation of enzyme synthesis. In Vitro (Rochville), 9, 399 -413. Hsia, M.T.S., Kreamerand, B.L. and Dolara, P. (1983) A rapid and simple method to quantitate chemically induced unscheduled DNA synthesis in freshly isolated rat hepatocytes facilitated by DNA retention of membrane filters. Mutat. Res., 122, 177- 185. Vytasek, R. (1982) A sensitive fluorometric assay for the determination of DNA. Anal. Biochem., 120, 243- 248. Habig, W.H., Pabst, M.J. and Jakoby, W.B. (1974) Glutathion S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem., 219. 7130- 7139. Paglia, D.E. and Valentive, W.N. (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathion peroxidase. J. Lab. Clin. Med., 70, 158 - 169. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254. Decad, Gary M., Hsieh, D.P.H. and Byard. James L. (1977) Maintenance of cytochrome P-450 and metabolism of aflatoxin Br in primary hepatocyte cultures. Biochem. Biophys. Res. Commun., 78, 279- 287. Chen, C.F., Yu, S., Yang, H.Y. and Chuang, C.Y. (1986) Hepatoprotective effects of geniposide. an iridoid glycoside from Gardenia jasminoides Ellis. Abstract of The First Joint Conference of Biomed. Sci. Taipei, ROC, pp. 103. Cheng, Y.Y., Chart, Y.S., Choang-Tai, K.F. and Chang, H.M. (1986) Effect of geniposide on acute jaundice in rats caused by ANIT poisoning. Acta Pharmacol. Sinica., 7, 69-72. Wong, Jeffrey J. and Hsieh, Dennis P.H. (1976) Mutagenicity of aflatoxins related to their metabolism and carcinogenic potential. Proc. Natl. Acad. Sci. USA, 73, 2241- 2244. Batttsta, T.R. and Marnett, L.J. (1985) Prostaglandin H synthase-dependent epoxidation of aflatoxin 8,. Carcinogenesis, 8, 1227 - 1229.
Cancer Letters, 65 (1992) 133 - 137 ElsevterScientiftcPublishersIrelandLtd.
Inhibitory effect of geniposide on aflatoxin B1-induced DNA repair synthesis in primary cultured rat hepatocytes Shao-Win %stitute
Wang”,
of Medicine,
Chung-Yih bDepartment
and Dental College, Taichung,
Laib and Chau-Jong
of Medico1 Technology,
Taiwan
Wang’
and ‘Department
of Biochemistry,
Chung-Shan
Medical
(ROC)
(Received 17 February 1992) (Revision received 6 May 1992) (Accepted 8 May 1992)
Summary We have previously demonstrated that geniposide (GP) inhibits the aflatoxin BI (AFB,) induced-hepatotoxicity and hepatic DNA binding in rats. To address the mechanism of action, the effects of GP on AFBI-induced DNA repair synthesis and AFBl biotransformation in cultured rat hepatocytes were investigated. By evaluation of unscheduled DNA synthesis (LIDS), GP reduced AFBI-induced DNA repair synthesis in a dose-dependent manner in hepatocyte cultures. GP elevates the metabolism of AFBl and decreases the formation of AFMl. The
enzyme activities of glutathione S-transferase (GST) and GSH-peroxidase (GSH-Px) in AFB,-treated hepatocyte cultures are enhanced in the presence of GP. GP reduces AFBIinduced DNA repair synthesis through an increased AFBl detoxication metabolism. It provides one possible mechanism for the chemopreventive activity of GP. Correspondence to: Chau-Jong Wang, Department of Biochemistry, Chung-Shan Medical and Dental College, No. 113, Section 2, Ta-Ching Street, Taichung, Taiwan, Republic of China. Abbreviations: GP, geniposide; APB,, aflatoxin B,; GSH. glutathione; GST, glutathione S-transferase; GSH-Px, glutathione peroxidase; CDNB, 1-chloro-2,4-dinitrobenzene; UDS, unscheduled DNA synthesis.
K-o&: geniposide; aflatoxin B,; unscheduled DNA synthesis; glutathione Stransferase; glutathione peroxidase IXktrodUCtiOD
Geniposide (GP), an iridoid glycoside, was isolated from the fruit of Gardenia jasminoides Ellis by Inouye et al. [1,2]. Gardenia jasminoides Ellis. f. frandiflora makino, a plant in the family of Rubiaccase, grows widely in the middle and southern parts of Taiwan. Its fruit, gardenia, has been used as a herbal medicine to treat liver and gall bladder disorders, hepatitis and acute jaundice [3]. The crude extract rapidly lowers serum bilirubin and transaminase levels in acute hepatitis [4]. The antihyperbilirubinemic effect of Gardenia extracts has been reported in an animal model [5,6]. Lau et al. [7] proposed that gardenia or GP may facilitate the billiary extraction of cy-naphthyisothiocyanate and/or its toxic metabolite(s). Recently, we have demonstrated the suppressive effect of GP on the hepatotoxicity and hepatic DNA binding of aflatoxin B1 in rats [8]. Aflatoxin B1 (AFBJ is a potent hepatotoxin and hepatocarcinogen in rats and humans [9, lo]. The toxic and carcinogenic effects of AFBl are seen only after it is metabolized by cytochrome P-450 enzymes. These enzymes
0 1992 Ekevier Scientific Publishers Ireland Ltd. 0304-3835/92/$05.00 Printed and Published in Ireland
134
catalyze the formation of various oxidative derivatives, including an unstable, highlyreactive 8,9-epoxide that is presumed to be the ultimate carcinogenic species of AFBl [ll]. This reactive electrophilic epoxide covalently interacts with nucleophilic centers in DNA and protein. Apart from binding to macromolecules, alternative fates for AFB1-&g-oxide include hydrolysis to 8,9-dihydrodiol [12] and conjugation with glutathione (GSH) to form 8-(S-gIutathiony)-9-hydroxy-8,9-dihydroAFBl (AFB1-SG) [13,14]. The formation of the AFB1-GSH conjugate, through the GSH S-transferase (GST) catalysis, is a detoxication reaction. Resistance to the acute toxicity of AFBl has been correlated with the formation of AFB1-GSH conjugates and activity of GST [ 151. GSH and its related enzymes including GST and GSH-peroxidase GSH-Px) would therefore provide an important detoxication pathway. In the present study, the effect of GP on AFB1-induced unscheduled DNA synthesis and AFBl biotransformation is investigated. The possible mechanism of the chemopreventive action of GP on AFBl induced hepatic damage is discussed. Materials
and Methods
Chemicals [Methyl-3H]thymidine (spec. act. 20 Ci/ mmol) was purchased from ICN Radiochemicals (Irvine, CA, USA). Non-radioactive thymidine , AFBl , AFM1, collagenase , calf thymus DNA, DABA, Tris, proteinase K, GSH, NADPH, GSH reductase, CDNB, sodium azide, OPT, ATP, L-glutamate, L-W aminobutyrate and EDTA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). GP was purchased from Wako Pure Chemical Co. Japan. Aquasol- was obtained from New England Nuclear (Boston, MA, USA) and the protein assay kit from Bio-Rad Lab. Ltd. (Watford, Herts, UK). All other chemicals and reagents used were of the highest purity commerically available.
Culture
of rut hepatocytes
Hepatocytes were isolated from adult female Sprague - Dawley rats (Taichung Veterans General Hospital, Taiwan, 150 - 200 g, fed ad Iibitum) by the collagenase perfusion method [16]. They were plated at a cell density of 3.75 x lo5 cells/ml in 4 ml of Williams E medium (Gibco) supplemented with 10% calf bovine serum, 1% PSN (Gibco) antibiotic mixture and nicotinamide (1 mg/l) . The medium was changed 3 h after plating to remove nonadherent cells and the culture was maintained for the time indicated. For treatment of culture with test agents, stock solutions of chemicals in dimethylsulfoxide (Sigma) were added to the cultures so that the final concentration of DMSO was less than 0.2%.
Measurement of DNA repair synthesis The effect of GP on AFBl induced DNA repair synthesis was measured by determinating the amount of [methyl-3H]thymidine incorporated into nuclear DNA in the presence of hydroxyurea (15 mM) [17]. Hepatocyte cultures were treated with 0.01 mM AFBl and GP at desired concentrations following preincubation with hydroxyurea for 1 h. A l-&i quantity of [3H]thymidine/ml was added to the culture medium for an additional 20 h. The cell monolayer was washed twice with PBS and harvested at the end of culturing. Following harvesting the cells were loaded onto a 25mm, 2-pm pore size PC filter (Nucleopore) through a Swinnex polyethylene filter holder to lyse the cells with 10 ml lysing buffer containing 2% SDS, 0.025 M EDTA, 0.5 mg/ml thymidine, 0.1 M glycine (pH 10) plus 0.15 mg/ml proteinase K and washed with 3 ml of the same buffer without proteinase K. The filter was carefully transfered to a scintillation vial. A l-ml quantity of 0.5 N HC104 was added to the vial and heated at 60°C in a water bath for 1 h. An aliquot was taken for the determination of radioactivity by liquid scintillation spectrometry and DNA content, expressed as dpm/pg DNA. DNA was measured in a cell lysate using the DABA method [18].
135
Eualution of less-toxic metubolites of AFBl The effect of GP on AFBr metabolism was measured by adding 0.01 mM AFBr and GP to the culture medium for 6 h, when the medium was removed and an equal volume of ice-cold methanol added. This mixture was extracted three times with CHCls. The CHCIS phase was washed with distilled water and concentrated to analyze all known metabolites of AFBr by HPLC [12]. On a Zorbax ODS column AFBr and its metabolite were eluted with 15% dimethylfomamide in 0.01% phosphoric acid at 1.5 ml min - ’ and a column temperature of 69OC. Enzyme actiuities of GST and GSH-Px To examine the effects of GP on the activities of GST and GSH-Px in hepatocyte cultures treated with AFBr, the cells were harvest at the end of experiment to determine the enzyme activities as previously described [8]. After harvest the cells were homogenized with a tight-fitting polytron pestle (20 strokes) in Tris - sucrose buffer (pH 7.5) and centrifuged. The supernatant was used for the determination of GST [19] and GSH-Px [ZO] activities. Protein concentration was measured by using a standard commercial kit (Bio-Rad. Lab. Ltd., Watford, Herts, UK) with BSA as standard [Zl].
Effects of GP on Table 1. damage in cultured hepatocytes. Treatment”
dpm/pg
AFBl (0.01 mM) AFB, (0.01 mM) plus: GP (0.02 mM) GP (0.05 mM) GP (0.10 mM)
97.75
AFB1-induced
DNA
% of control
DNA
f 9.34b
100
74.54 zt 5.58’ 69.26 zt 9.37’ 56.70 z!z5.81”
76 71 58
“Hepatocytes (lo6 cells) were cultured with William E medium for 3 h and incubated with hydroxyurea for the other 1 h. AFB, with or without GP was then added into the culture medium. A l-&i quantity of [methyl3H]thymidine (20 Ci/mmol) was added to the culture medium immediately after the test chemical for an additional 20 h. At the end of culture, the cells were harvested and lysed for radioactivity counting and DNA deter- mination. DNA damage was determined by UPS and expressed as dpm/pg DNA. bMean f S.D., values are the average of triplicate determinations. ‘P < 0.05, lP < 0.01, compared with the AFB,treated group. l
Table U. Effects cultured hepatocytes. Treatment”
of
GP
on
AFMl
formation
in
AFBl residue
AFM, kg(s)
(c(g)
Effect of GP on AFBr induced DNA repair synthesis The effect of GP on AFBr induced DNA repair synthesis was measured as unscheduled DNA synthesis (171. GP significantly inhibited AFBr induced DNA repair synthesis in a dose response manner (P < 0.05 in 0.02 and 0.05 mM, P < 0.01 in 0.1 mM) (Table I). Addition of GP to the culture medium reduced AFBrinduced DNA repair synthesis in cultured hepatocytes. It indicates that GP inhibited AFBl induced DNA damage in primary culture hepatocytes.
1.043 * 0.208
AFBl (0.01 mM) 0.937 f 0.076b AFB, (0.01 mM) plus: GP (0.02 mM) 0.898 f 0.114 GP (0.05 mM) 0.720 zt 0.113 GP (0.10 mM)
0.692 zt 0.042’
l
0.231 0.185 .*
f 0.058’ f 0.035
0.197 .
zt 0.076
l
‘Hepatocyte cultures were treated with AFBr and GP with designated concentrations for 6 h. The medium was collected for the determination of AFM, formation. bMean f S.D., values are the average of triplicate determinations. ‘P < 0.01 compared with AFB, treated ‘P < 0.02, l
Qroup
136
Effect of GP on AFBl
Discussion
metabolites
The effect of GP on AFBl metabolism was measured by analyzing AFBr metabolite formation. In Table II, during the 6-h incubation of hepatocytes with AFBi, most of AFBi was metabolized. AFMi was the only CHClsextractable metabolite seen by HPLC analysis. The amounts of AFBi residue and AFMi were significantly decreased in culture medium in the presence of GP (0.1 mM). This indicates that the inhibitory effect of GP on AFBl induced DNA repair synthesis was not due to increased conversion of AFBi into the less toxic metabolite AFMi. Effect of GP on the actioities of GST Px induced by AFBl
and GSH-
The activities of GST and GSH-Px were slightly increased but not significantly by adding AFBi (Table III). When GP (0.1 mM) was added in combination with AFB1, there was a significant elevation of GST and GSH-Px activities (P < 0.01). The inhibitory effect of GP on AFBi induced DNA damage may be due to the increased detoxication of AFBi.
Table 111. Effects of GP on the activities of GST and GSH-Px induced by AFBr in cultured hepatocytes. Treatment”
GST (nmol/min per mg protein)
DMSO (0.2%) 273 l 14 AFBr (0.01 mM) 294 l 12 AFBl (0.01 mM) plus: GP (0.02 mM) 307 f 3 GP (0.05 mM) 307 f 2 GP (0.10 mM) 346 zt 4’
GSH-Px (nmol/min per mg protein) 286 zt 11 291 f 17 294 f 13 310 f 7 375 f 31’
“Hepatocyte cultures were treated with AFBl and GP with designated concentration for 6 h. Cells were harvested, homogenized and centrifuged for enzyme activity assays at the end of culture. bMean f SD., values are the average of triplicate determinations. lP < 0.01, compared with AFB, treated group.
The results presented in this paper show the chemopreventive effect of GP on AFBi induced DNA damage in cultured hepatocytes. This is supported by the reduction of AFBiinduced DNA repair synthesis in the presence of GP. Previous reports have demonstrated a reduction of the hepatotoxic effects of carbon tetrachloride and cr-naphthylisothiocyanate by GP [23,24]. We have also reported the suppressive effect of GP on the hepatotoxicity and hepatic DNA binding of AFBi in rats [8]. Similarly, there is an inhibition of AFBiinduced DNA damage with the presence of GP in cultured hepatocytes. The inhibitory effect of GP on AFBi induced DNA damage may be due to the enhancement of the activities of certain drug-metabolizing enzymes. This results in an increased conversion of AFBi to a less toxic metabolite. Protection against AFBiDNA binding may result from either or both of the following two mechanisms (a) the conversion of AFBi into less toxic metabolites, such as AFMi 1251, (b) induction of detoxication pathways via the conjugation with GSH [15]. The present studies show that GP elevates the metabolism of AFBi and decreases the formation of AFMr (Table II). It indicates that the inhibitory effect of GP was not caused by the conversion of AFBi into the less toxic metabolite such as AFMi. Since conjugation with GSH plays an important role on AFBi detoxication, the effect of GP on the activities of GST in AFBt-treated hepatocyte cultures is also analysed. When GP is added to the culture medium in combination with AFBi there is a significant elevation of GST activity (Table III). The increased activity of GST induced by GP may increase the AFBi exclusion by conjugation with GSH. Thus, the inhibitory effect of GP on AFBi induced DNA damage may be mediated by increased GST activity. The GP also induced GSH-Px activity. This phenomenon possibly occurs through an increased utilization of GSH, which can facilitate the reduction of peroxides or scavenging of free radicals
137
generated from AFBr minor metabolism via the co-oxidative pathway [26]. In conclusion, the possible mechanisms of GP inhibits AFBr induced DNA repair synthesis were investigated. The inhibitory effects of geniposide on AFBr-induced hepatic DNA damage possibly involve enhancing GST and GSH-Px activities for AFBr detoxication.
C.E. 12
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Acknowledgement 15
This work was supported by the National Science Council Grant NSC 81-0421-B-040OlZ, National Science Council Grant, Republic of China. References
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