95
Cancer Letters, 60 (1991) 95 - 102 Elsevier Scientific Publishers Ireland Ltd.
Suppressive effect of geniposide on the hepatotoxicity hepatic DNA binding of aflatoxin B1 in rats
and
C.-J. Wanga, S.-W. Wangb and J.-K. Lin’ “Department ‘Institute
of Biochemistry,
of Biochemistry.
blnstitute
National
of Medicine,
Taiwan
Chung-Shan
University,
College
Medical
and Dental
of Medicine,
Taipei.
College, Taiwan
Taichung, (Republic
Taiwan
and
of China)
(Received 21 June 1991) (Revision received 28 July 1991) (Accepted 31 July 1991)
Summary
conditions,
The effects of geniposide pretreatment on both hepatic aflatoxin BI (AFBJ-DNA binding and AFBl hepatotoxicity in rats has been examined. For these studies, male SpragueDawley rats were treated with AFBl (2 mg/kg) by i.p. administration, and the different degrees of hepatic damage were revealed by the elevations of levels of serum marker enzymes such as aspartate aminotransferase alanine amino(AST), transferase (ALT) and y-glutamyltranspep-
tidase (y-GT). After pretreatment of animals with geniposide (10 mg/kg) daily for 3 consecutive days, the enzyme elevations were significantly suppressed. This suggested that the geniposide possessed chemopreventive effects on the early acute hepatic damage induced by AFBl. Under these experimental
Correspondence Biochemistry,
to: Dr.
Chau-Jong
Wang,
Medical
and Dental
Chung-Shan
Department College,
of No.
consistent elevation of the activities of glutathione S-transferase (GST) and yglutamylcysteine synthetase but not glutathione peroxidase (GSH-Px) and yglutamyltranspeptidase were observed. Treatment of rats with geniposide significantly lowered hepatic GSH and GSSG levels, but the ratio of GSH to GSSG was not changed. Geniposide treatment also decreased AFBI-DNA adduct formation in AFBI-treated animals. From these results, we suggest that the protective effect of geniposide on AFB, hepatotoxicity in rats might be due to the hepatic tissues’ defense mechanisms that inuolue the enhanced GST activity for AFB,
detoxication and induction y-glutamylcysteine synthetase for GSH biosynthesis.
Keywords: geniposide; aflatoxin B,; glutathione S-transferase; y-glutamylcysteine synthetase; hepatotoxicity; AFB1-DNA adduct
113, Section 2, Ta-Ching Street, Taichung, Taiwan, Republic of China. Abbreviations: glutathione
AFB,,
aflatoxin
S-transferase;
B,;
GSH-Px,
GSH,
glutathione;
glutathione
GST,
peroxidase;
GT.
y-glutamyl transpeptidase; AST, aspartate aminotransCDNB. aminotransferase; alanine ALT, ferase; 1-chloro-2.4-dinitrobenzene; DCNB, 3,4-dichloronitrobenzene; OPT,
0-phthaldialdehyde;
0304-3835/91/$03.50
COP,
0
cumene
hydroperoxide.
Introduction
y-
was Geniposide, an iridoid glycoside, isolated from the fruit of Gardenia jasminoides and co-workers [ 1,2]. Ellis. by Inouye
1991 Elsezier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
96
Gardenia jasminoides Ellis. f. grandiflora makino, a plant in the family of Rubiaceae, grows in the middle and southern parts of Taiwan. Its fruit, Gardenia, contains the dye crocin which has been widely used as a herbal medicine in the treatment of liver and gall bladder disorders, hepatitis and acute jaundice in the Far East [3]. Clinical studies show that the crude drug rapidly lowers serum bilirubin and transaminase levels in jaundice-induced acute hepatitis [4]. The antihyperbilirubinemic effect of Gardenia extracts has been demonstrated in animals [5,6]. Recently, Lau et al. [7] demonstrated that Gardenia or geniposide may facilitate the conjugation and biliary extraction of o-naphthylisothiocyanate and/or its toxic metabolite(s) . The hepato-protective effect of Gardenia jasminoides Ellis. has been demonstrated. In contrast, Hong et al. [8] demonstrated that Gardenia extract could induce hepatic pigmentation in pigs and mice. We have also studied the hepatotoxic effect induced by prolonged feeding of Gardenia extracts to rats [9]. Recently, the hepatotoxic effects of high doses of Gardenia extracts and geniposide were investigated in rats [lo, 111. Aflatoxin B1 (AFB1) is a potent hepatotoxin and hepatocarcinogen in certain species including rat [ 121 and man [ 131. The major toxic metabolite and ultimate carcinogen is thought to be the 8,9-epoxide 1141. The mechanism of the acute toxicity is not known but initiation of carcinogenesis is thought to follow the binding to DNA at the N-7 of guanine [ 151. Apart from binding to macromolecules, alternative fates for AFBr-8,9-oxide include hydrolysis to the 8,9-dihydrodiol [ 161 and conjugation with glutathione (GSH) to form &(S-glutathionyl)9-hydroxy-8,9-dihydro-AFB, (AFB1-SG) [17, 181. The AFB1-GSH conjugate formed by GSH S-transferase (GST) catalysis is considered to be a detoxication product. Resistance to the acute toxicity of AFBr has been correiated with the formation of AFB-GSH conjugates and activity of GST [19]. The GSH, and its related enzymes including GST, GSHPx and glutamylcysteine synthetase would therefore appear to provide an important detoxication pathway.
In the present study we describe the effect of pure geniposide on the potentiation of the AFBl detoxication pathway and the reduction of AFBl and DNA interactions. The suggestion is made that induction of GST and glutamylcysteine synthetase (a rate-limiting enzyme for GSH biosynthesis) [20] by pretreatment with geniposide may be important for protection from AFB1-induced hepatotoxicity. Materials
and Methods
Chemicals
[3H]AFB1 (spec. act. 15 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA) and checked for purity by reverse phase HPLC and showed one major peak containing more than 98% of the AFBr in the sample. Non-radioactive AFBr, GSH, NADPH, GSH reductase, DCNB, CDNB, calf thymus DNA, RNase A, proteinase K, BSA, OPT, COP, ATP, L-glutamate, L-o-aminobutyrate, EDTA, and L-Ydeoxycholic acid, glycylglycine glutamyl-p-nitroanilide, were purchased from Sigma Chemical Co. (St. Louis, MO), reagent kits for AST, ALT, and r-GT were purchased from E. Merck Co., Darmstadt, Germany. Geniposide was purchased from Wako Pure Chemical Co., Japan. Aquasolwas from New England Nuclear, Boston, MA. All other chemicals and reagents used were of the highest purity commerically available. Animals
and treatment
Male Wistar rats (150 - 180 g body wt.) purchased from National Taiwan University Hospital Animal Center were used for all experiments. The animals were housed three per cage in an environmentally controlled animal room. Food (Purina Lab Chow) and water were provided ad libitum. Geniposide and AFBr were dissolved in DMSO (10 mg/ml) and diluted with redistilled water. The final concentration of DMSO was 1% (v/v) and control rats received a total volume of 0.03 ml For protection against DMSO per dose. AFBr-induced hepatotoxicity studies, geniposide (5 mg and 10 mg/kg, respectively) were given daily by intubation for 3 con-
97
secutive days. On the fourth day, AFBr (2 mg/kg) was injected i.p. Twenty four hours later, the animals were killed by decapitation and blood samples were collected for assays of AST, ALT and y-GT, the liver was excised for determination of GSH content, activities of GST and GSH-peroxidase (GSH-Px), yglutamylcysteine synthetase and y-glutamyl transpeptidase (y-GT). Hepatotoxicity
assessment
Several hepatic enzymes - AST, ALT and y-GT were used as biochemical markers for early acute hepatic damage. The activities of AST and ALT were determined in serum according to the method of Reitman and Frankel [21]. The activity of y-GT in serum was measured according to the method of Persijin and Van der Slik [22]. GSH
and related
enzyme
assays
Rats were killed by decapitation and liver promptly removed. A small portion of liver was taken for GSH and GSSG determination [23] and the remaining tissues homogenized in 4 ~01s. (w/v) of a 50 mM Tris - buffer (pH 7.5) containing 0.25 M sucrose. Homogenates were centrifuged at 105 000 x g and the supernatant used for GST (CDNB and DCNB as substrates) [24] and GSH-Px (COP and HzOz as substrates) determinations [25]. Protein concentration was determined [26] using a standard commercial kit (Bio-Rad Lab. Ltd., Watford, England) with bovine serum albumin as standard. The activity of y-gluamylcysteine synthetase was measured as described by Sekure and Meister [27,28]. The enzyme activity (E.U.) was defined as 1 nmol P, released/mg protein per min. The activity of yGT in liver was measured by a modification of the method described by Tate and Meister [29]. The enzyme activity (E.U.) was defined as 1 nmol p-nitroaniline formed/mg protein per min. Isolation of DNA and analysis adducts
of AFBI-DNA
Geniposide was administrated at a dose of 6 mg and 10 mg/kg for 3 consecutive days. On
the fourth day, the labeled [‘H]AFBi (33.88 pg/kg, 15 Ci/ mmol) was injected i.p. Two hours later, rats were killed by decapitation, and the liver excised and minced. For isolation of DNA, the liver was ground into powder using a mortar and pestle on solid C02. Separation and quantitation of DNA was as described previously [30]. The covalent binding was expressed as pg/mg DNA. The radioactivity was determined by an Aloka LSC-900 liquid scintillation counter and the specific activity of [3H]AFB1 used here was 238703 dpm/ng AFB1. Results Several hepatic enzymes such as AST, ALT and y-GT were used as biochemical markers for early acute hepatic damage. When a single dose of hepatocarcinogen, AFBi, was given to rats by i.p. injection, striking acute hepatotoxic reaction appeared as revealed by the elevations of serum marker enzymes (Table I). The acute hepatotoxicity reaction was significantly suppressed by the geniposide (5 mg and 10 mg/kg) given for 3 days prior to AFBr administration. Most of these activities were highly significant with P values of 0.01-0.02 (Table I). The solvent DMSO (0.2%) and geniposide alone did not affect the activities of these serum enzymes. Geniposide itself was not toxic for liver at these doses. In order to demonstrate the protective effects of geniposide on the acute hepatic damage induced by AFB,, the effects of GSH, GSSG and relative liver weight (liver wt./body wt.) in AFB1-induced hepatotoxicity was observed. Treatment of rats with 10 mg/kg of geniposide (with or without AFBJ significantly lowered hepatic GSH levels (P < 0.05, Table II), but the ratio of GSH and GSSG was not significantly changed. A single dose of AFB, produced a 93% elevation in GSH level. This is in agreement with previous studies in which the effect of carcinogen treatment on the GSH level of the rat liver was estimated by the same procedure [31]. The GST and GSH-Px and other related enzymes play an important role ‘in AFB,-induced
98
Table 1.
Effect of geniposide
on the activities of serum ALT, AST and y-GT in rats.
Treatment’
ALT (I.U./I)
AST &U./I)
y-GT (I.U./I)
Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBr (2 mg/kg) AFB, + GPs (5 mg/kg) AFB, + GPs (10 mg/kg)
45.8 50.2 48.4 50.8
220 297 258 266
0.67 ziz 0.82 1.00 zt 0.89 0.20 f 0.40 0.33 f 0.52
f zt zt zt
6.2 4.0 3.9 4.6
+ 32 zt 38 zt 36 ztz 41
525 zt 208’
902 zt 272’
1.20 f 0.75
475 f 159 267 ztz 71’
810 zt 227 495 f 88”
0.75 0.75
f 0.74 f 0.74
aExperimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB, for the determination of serum ALT, AST and y-GT activities. Values are means f S.D.. n = 6. “P < 0.001 compared
‘P < 0.02,
l
with the DMSO control group. lP < 0.01 compared with the AFBr-treated
group.
hepatotoxicity. Thus, it seemed necessary to investigate whether these enzymes are affected by geniposide alone and in combination with AFBr as shown in Table III. Significant elevations were found in the activities of GST (P < 0.05-0.02; CDNB as substrate) and yglutamylcysteine synthetase (P < 0.05; Table IV), but the activities of GSH-Px and -y-GT
Table II.
Effect of geniposide
Treatmenta
were not affected by pretreatment with geniposide. The formation of covalently-bound adducts of AFB,-8,9-epoxide derivatives in rat liver DNA in rats treated with geniposide for 3 consecutive days was reduced with a concomitant decrease in .covalent 3H-radioactivitiy. Treatment of rats with geniposide (6 mg and 10
on the relative liver weight and the level of hepatic Relative liver wt.
GSH
(pg/g liver) Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBI (2 mg/kg) AFBr + GPs (5 mg/kg) AFBr + GPs (10 mg/kg)
3.84 3.40 3.65 3.43 3.84 3.89 3.76
f 0.21 f 0.11 f 0.27 l 0.12 zt 0.16’ f 0.18 zt 0.11
1934 2347 2114 1852 4519 4890 3709
l
zt zt f f zt f
334 340 467 198’ 590’ 468 449#
GSH and GSSG in rats.
GSSG (pg/g liver)
GSH/GSSG
368 431 478 371 691 621 495
5.26 5.44 4.42 4.99 6.54 7.87 7.49
+ zt f + f zt zt
36 69 100 25 121’ 40 72’
zt f zt + zt f f
1.04 1.18 1.35 0.63 1.43 0.91 1.42
“Experimental rats were pretreated with geniposide (GPs) by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB,. The livers were removed, weighted and homogenized for the determination of cytosol GSH and GSSG levels. Values are means f S.D., n = 6. lP < 0.05 compared with the DMSO control group. “P < 0.05 compared with the AFB,-treated group.
99 Table III.
Effect of geniposide
on the activities of GST and GSH-peroxidase GST (nmol/min
Treatment”
CDNB Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBl (2 r&kg) AFB, + GPs (5 mg/kg) AFB, + GPs (10 mg/kg)
128 128 134 136 122 130 132
f zt f zt f f zt
GSH-Px
per mg prot.)
12.1 11.3 12.9 11.5 14.2 13.4 13.7
(nmol/min
COP
DCNB 5 2 5’ 6” 7 5” 8’
in rats
f f z+ f +z f zt
1.4 1.0 1.4 0.8 0.7 0.3 0.8
19.0 19.0 19.7 18.4 17.2 16.7 19.0
per mg prot.) Hz&
zt f f zt zt zt f
2.3 1.4 3.1 3.4 3.2 3.2 3.7
10.2 8.9 7.6 8.1 10.2 12.4 12.0
f f f f f f *
1.3 1.6 1.0 2.0 2.2 1.5 2.1
aExperimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB, and the livers were minced and homogenized for the determination of enzyme activities. Values are means l S.D., n = 6. lP < 0.02 compared with the DMSO control group. lP < 0.05, “P < 0.05 compared with the AFB,-treated group. l
mg/kg) decreased the amount of AFBr-DNA it is adduct by - 50% (Table V). Therefore, likely that the decreased formation of these adducts after geniposide pretreatment resulted from the depletion of GSH for conjugation with AFBr-epoxide and was also consistent with reduced AFBr-induced hepatotoxicity.
Table IV.
Effect of geniposide
Discussion The results presented in this paper indicate that pretreatment of rats with geniposide leads to protection AFBr-induced against hepatotoxicity. This is supported by the fact that there was suppression of serum AST, ALT
on the activities of y-glutamylcysteine
synthetase
and y-glutamyltranspeptidase
Treatment”
y-glutamylcysteine synthetase (E.U.)b
y-glutamyltranspeptidase (E.U.)’
Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBr (2 mg/kg) AFB, + GPs (5 mg/kg) AFB, + GPs (10 mg/kg)
8.16 5.35 4.96 10.77 4.96 8.55 8.11
19.47 24.60 22.24 24.11 13.87 12.79 12.91
f zt zt f zt zt f
1.53 3.98 5.38 2.22’ 2.70 1.71” 0.55”
f 7.55 zt 8.40 zt 5.73 ztz 6.08 zt 2.56’ f 2.60 f 2.33
‘Experimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB, and the livers were minced and homogenized for the determination of enzyme activities. Values are means f S.D., n = 6. bThe enzyme activity (E.U.) was defined as 1 mmol Pi released/mg protein per min. ‘The enzyme activity (E.U.) was defined as 1 mmol p-nitroaniline formed/mg per min. lP < 0.05 compared with the DMSO group. 'P < 0.05 compared with the AFBr-treated group.
100 Table V. Effect of geniposide on the hepatic binding induced by AFB, in rats. Treatment=
AFB, (33.88 pg/kg) AFBt + GPs (6 mg/kg) AFB, + GPs (10 mg/kg)
AFB, bound
DNA-
(pg/mg DNA)
% of AFB, alone
3.31 f 2.10 1.70 + 0.28 1.80 ztz 0.42
100 52 54
aExperimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 2 h after the i.p. administration of AFB, and the livers were minced for the determination of AFBt-DNA adduct formation. Values are means f S.D., n = 3.
and -y-GT activities induced by AFBi in male rats. Previous reports have demonstrated a reduction of carbon tetrachloride(CCl&and cr-naphthylisothiocyanate(ANIT)-induced hepatotoxic effects by geniposide in experimental animals [32,33]. Similarly the marked reduction by pretreatment with geniposide in AFBi hepatotoxicity occurs when the AFBi was administrated 24 h after three daily doses of geniposide. The suppressive effect on AFBi-induced hepatotoxicity may be due to enhancement of the activities of certain drug-metabolizing enzymes by geniposide pretreatment leading to an increase in the conversion of AFBr to inactive metabolites. Single doses of AFBi produced an adaptive elevation in GSH levels. This is analogous to previous studies showing that after administration of some hepatocarcinogens there is a response of hepatic GSH [34]. However, the combination of geniposide with AFBi caused a significant reduction (P < 0.05) in hepatic GSH level (Table II)). Depletion by geniposide of liver GSH might increase hepatotoxicity [ 18,351, since conjugation with GSH is an important detoxifying pathway for AFBi [36]. However, our data show contrary results in that the marked reduction by a geniposide of AFBi hepatotoxicity occured when AFBl was administered. There is a
possibility that the accelerated reduction of GSH and GSSG resulted from direct interaction of geniposide [ll] and conjugation of AFBi-epoxide with GSH for AFBr detoxication. The initial depletion of GSH seen after geniposide administration did not affect the hepatic GSH pool size. It should be noted that the concentration of GSH in the hepatic cytosol fraction used for these studies was high. Therefore, the relatively minor depletion of GSH through conjugation with geniposide would be negligible. Other novel properties of geniposide are increases in the activities of yglutamylcysteine synthetase, the first and ratelimiting enzyme in GSH biosynthesis, and GST, which catalyzes the conjugation of AFBl with GSH. Hence, the depletion of GSH will be replenished by the induction of synthetase during geniposide pretreatment. The AFBi detoxication pathway will also be stimulated by the induction of GST. The lowering of GSH level which was found after geniposide and AFBl administration could, in part, possibly have resulted from the greater catalytic rate of conjugation by GST rather than through biosynthesis of GSH by y-glutamylcysteine synthetase. The formation of DNA adducts is regarded as the primary event in the carcinogenic process. Most of anticarcinogenic compounds have been tested for their ability to suppress the formation of AFBi-DNA adducts [37,38]. Therefore, the increased formation of conjugates after geniposide pretreatment is a likely result from a decreased reaction of AFBi-&g-oxide with DNA in rat liver in vivo. Consistent results were obtained from the induction of GST, and covalent binding to DNA was decreased by - 50% as compared with AFBi alone when geniposide was given at a dose of 6 mg and 10 mg/kg (Table V). our results indicate that In conclusion, geniposide is a modulator of AFBi-DNA adduct formation, and also demonstrate that geniposide supresses AFB,-induced hepatotoxicity by a mechanism that involves enhanced GST activity for AFBi detoxication and induction of GSH biosynthesis.
101
Acknowledgement
14
This work was supported by the National Science Council Grant NSC 80-0412B040-04, Republic of China.
15
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Cancer Letters, 60 (1991) 95 - 102 Elsevier Scientific Publishers Ireland Ltd.
Suppressive effect of geniposide on the hepatotoxicity hepatic DNA binding of aflatoxin B1 in rats
and
C.-J. Wanga, S.-W. Wangb and J.-K. Lin’ “Department ‘Institute
of Biochemistry,
of Biochemistry.
blnstitute
National
of Medicine,
Taiwan
Chung-Shan
University,
College
Medical
and Dental
of Medicine,
Taipei.
College, Taiwan
Taichung, (Republic
Taiwan
and
of China)
(Received 21 June 1991) (Revision received 28 July 1991) (Accepted 31 July 1991)
Summary
conditions,
The effects of geniposide pretreatment on both hepatic aflatoxin BI (AFBJ-DNA binding and AFBl hepatotoxicity in rats has been examined. For these studies, male SpragueDawley rats were treated with AFBl (2 mg/kg) by i.p. administration, and the different degrees of hepatic damage were revealed by the elevations of levels of serum marker enzymes such as aspartate aminotransferase alanine amino(AST), transferase (ALT) and y-glutamyltranspep-
tidase (y-GT). After pretreatment of animals with geniposide (10 mg/kg) daily for 3 consecutive days, the enzyme elevations were significantly suppressed. This suggested that the geniposide possessed chemopreventive effects on the early acute hepatic damage induced by AFBl. Under these experimental
Correspondence Biochemistry,
to: Dr.
Chau-Jong
Wang,
Medical
and Dental
Chung-Shan
Department College,
of No.
consistent elevation of the activities of glutathione S-transferase (GST) and yglutamylcysteine synthetase but not glutathione peroxidase (GSH-Px) and yglutamyltranspeptidase were observed. Treatment of rats with geniposide significantly lowered hepatic GSH and GSSG levels, but the ratio of GSH to GSSG was not changed. Geniposide treatment also decreased AFBI-DNA adduct formation in AFBI-treated animals. From these results, we suggest that the protective effect of geniposide on AFB, hepatotoxicity in rats might be due to the hepatic tissues’ defense mechanisms that inuolue the enhanced GST activity for AFB,
detoxication and induction y-glutamylcysteine synthetase for GSH biosynthesis.
Keywords: geniposide; aflatoxin B,; glutathione S-transferase; y-glutamylcysteine synthetase; hepatotoxicity; AFB1-DNA adduct
113, Section 2, Ta-Ching Street, Taichung, Taiwan, Republic of China. Abbreviations: glutathione
AFB,,
aflatoxin
S-transferase;
B,;
GSH-Px,
GSH,
glutathione;
glutathione
GST,
peroxidase;
GT.
y-glutamyl transpeptidase; AST, aspartate aminotransCDNB. aminotransferase; alanine ALT, ferase; 1-chloro-2.4-dinitrobenzene; DCNB, 3,4-dichloronitrobenzene; OPT,
0-phthaldialdehyde;
0304-3835/91/$03.50
COP,
0
cumene
hydroperoxide.
Introduction
y-
was Geniposide, an iridoid glycoside, isolated from the fruit of Gardenia jasminoides and co-workers [ 1,2]. Ellis. by Inouye
1991 Elsezier Scientific Publishers Ireland Ltd
Published and Printed in Ireland
96
Gardenia jasminoides Ellis. f. grandiflora makino, a plant in the family of Rubiaceae, grows in the middle and southern parts of Taiwan. Its fruit, Gardenia, contains the dye crocin which has been widely used as a herbal medicine in the treatment of liver and gall bladder disorders, hepatitis and acute jaundice in the Far East [3]. Clinical studies show that the crude drug rapidly lowers serum bilirubin and transaminase levels in jaundice-induced acute hepatitis [4]. The antihyperbilirubinemic effect of Gardenia extracts has been demonstrated in animals [5,6]. Recently, Lau et al. [7] demonstrated that Gardenia or geniposide may facilitate the conjugation and biliary extraction of o-naphthylisothiocyanate and/or its toxic metabolite(s) . The hepato-protective effect of Gardenia jasminoides Ellis. has been demonstrated. In contrast, Hong et al. [8] demonstrated that Gardenia extract could induce hepatic pigmentation in pigs and mice. We have also studied the hepatotoxic effect induced by prolonged feeding of Gardenia extracts to rats [9]. Recently, the hepatotoxic effects of high doses of Gardenia extracts and geniposide were investigated in rats [lo, 111. Aflatoxin B1 (AFB1) is a potent hepatotoxin and hepatocarcinogen in certain species including rat [ 121 and man [ 131. The major toxic metabolite and ultimate carcinogen is thought to be the 8,9-epoxide 1141. The mechanism of the acute toxicity is not known but initiation of carcinogenesis is thought to follow the binding to DNA at the N-7 of guanine [ 151. Apart from binding to macromolecules, alternative fates for AFBr-8,9-oxide include hydrolysis to the 8,9-dihydrodiol [ 161 and conjugation with glutathione (GSH) to form &(S-glutathionyl)9-hydroxy-8,9-dihydro-AFB, (AFB1-SG) [17, 181. The AFB1-GSH conjugate formed by GSH S-transferase (GST) catalysis is considered to be a detoxication product. Resistance to the acute toxicity of AFBr has been correiated with the formation of AFB-GSH conjugates and activity of GST [19]. The GSH, and its related enzymes including GST, GSHPx and glutamylcysteine synthetase would therefore appear to provide an important detoxication pathway.
In the present study we describe the effect of pure geniposide on the potentiation of the AFBl detoxication pathway and the reduction of AFBl and DNA interactions. The suggestion is made that induction of GST and glutamylcysteine synthetase (a rate-limiting enzyme for GSH biosynthesis) [20] by pretreatment with geniposide may be important for protection from AFB1-induced hepatotoxicity. Materials
and Methods
Chemicals
[3H]AFB1 (spec. act. 15 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA) and checked for purity by reverse phase HPLC and showed one major peak containing more than 98% of the AFBr in the sample. Non-radioactive AFBr, GSH, NADPH, GSH reductase, DCNB, CDNB, calf thymus DNA, RNase A, proteinase K, BSA, OPT, COP, ATP, L-glutamate, L-o-aminobutyrate, EDTA, and L-Ydeoxycholic acid, glycylglycine glutamyl-p-nitroanilide, were purchased from Sigma Chemical Co. (St. Louis, MO), reagent kits for AST, ALT, and r-GT were purchased from E. Merck Co., Darmstadt, Germany. Geniposide was purchased from Wako Pure Chemical Co., Japan. Aquasolwas from New England Nuclear, Boston, MA. All other chemicals and reagents used were of the highest purity commerically available. Animals
and treatment
Male Wistar rats (150 - 180 g body wt.) purchased from National Taiwan University Hospital Animal Center were used for all experiments. The animals were housed three per cage in an environmentally controlled animal room. Food (Purina Lab Chow) and water were provided ad libitum. Geniposide and AFBr were dissolved in DMSO (10 mg/ml) and diluted with redistilled water. The final concentration of DMSO was 1% (v/v) and control rats received a total volume of 0.03 ml For protection against DMSO per dose. AFBr-induced hepatotoxicity studies, geniposide (5 mg and 10 mg/kg, respectively) were given daily by intubation for 3 con-
97
secutive days. On the fourth day, AFBr (2 mg/kg) was injected i.p. Twenty four hours later, the animals were killed by decapitation and blood samples were collected for assays of AST, ALT and y-GT, the liver was excised for determination of GSH content, activities of GST and GSH-peroxidase (GSH-Px), yglutamylcysteine synthetase and y-glutamyl transpeptidase (y-GT). Hepatotoxicity
assessment
Several hepatic enzymes - AST, ALT and y-GT were used as biochemical markers for early acute hepatic damage. The activities of AST and ALT were determined in serum according to the method of Reitman and Frankel [21]. The activity of y-GT in serum was measured according to the method of Persijin and Van der Slik [22]. GSH
and related
enzyme
assays
Rats were killed by decapitation and liver promptly removed. A small portion of liver was taken for GSH and GSSG determination [23] and the remaining tissues homogenized in 4 ~01s. (w/v) of a 50 mM Tris - buffer (pH 7.5) containing 0.25 M sucrose. Homogenates were centrifuged at 105 000 x g and the supernatant used for GST (CDNB and DCNB as substrates) [24] and GSH-Px (COP and HzOz as substrates) determinations [25]. Protein concentration was determined [26] using a standard commercial kit (Bio-Rad Lab. Ltd., Watford, England) with bovine serum albumin as standard. The activity of y-gluamylcysteine synthetase was measured as described by Sekure and Meister [27,28]. The enzyme activity (E.U.) was defined as 1 nmol P, released/mg protein per min. The activity of yGT in liver was measured by a modification of the method described by Tate and Meister [29]. The enzyme activity (E.U.) was defined as 1 nmol p-nitroaniline formed/mg protein per min. Isolation of DNA and analysis adducts
of AFBI-DNA
Geniposide was administrated at a dose of 6 mg and 10 mg/kg for 3 consecutive days. On
the fourth day, the labeled [‘H]AFBi (33.88 pg/kg, 15 Ci/ mmol) was injected i.p. Two hours later, rats were killed by decapitation, and the liver excised and minced. For isolation of DNA, the liver was ground into powder using a mortar and pestle on solid C02. Separation and quantitation of DNA was as described previously [30]. The covalent binding was expressed as pg/mg DNA. The radioactivity was determined by an Aloka LSC-900 liquid scintillation counter and the specific activity of [3H]AFB1 used here was 238703 dpm/ng AFB1. Results Several hepatic enzymes such as AST, ALT and y-GT were used as biochemical markers for early acute hepatic damage. When a single dose of hepatocarcinogen, AFBi, was given to rats by i.p. injection, striking acute hepatotoxic reaction appeared as revealed by the elevations of serum marker enzymes (Table I). The acute hepatotoxicity reaction was significantly suppressed by the geniposide (5 mg and 10 mg/kg) given for 3 days prior to AFBr administration. Most of these activities were highly significant with P values of 0.01-0.02 (Table I). The solvent DMSO (0.2%) and geniposide alone did not affect the activities of these serum enzymes. Geniposide itself was not toxic for liver at these doses. In order to demonstrate the protective effects of geniposide on the acute hepatic damage induced by AFB,, the effects of GSH, GSSG and relative liver weight (liver wt./body wt.) in AFB1-induced hepatotoxicity was observed. Treatment of rats with 10 mg/kg of geniposide (with or without AFBJ significantly lowered hepatic GSH levels (P < 0.05, Table II), but the ratio of GSH and GSSG was not significantly changed. A single dose of AFB, produced a 93% elevation in GSH level. This is in agreement with previous studies in which the effect of carcinogen treatment on the GSH level of the rat liver was estimated by the same procedure [31]. The GST and GSH-Px and other related enzymes play an important role ‘in AFB,-induced
98
Table 1.
Effect of geniposide
on the activities of serum ALT, AST and y-GT in rats.
Treatment’
ALT (I.U./I)
AST &U./I)
y-GT (I.U./I)
Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBr (2 mg/kg) AFB, + GPs (5 mg/kg) AFB, + GPs (10 mg/kg)
45.8 50.2 48.4 50.8
220 297 258 266
0.67 ziz 0.82 1.00 zt 0.89 0.20 f 0.40 0.33 f 0.52
f zt zt zt
6.2 4.0 3.9 4.6
+ 32 zt 38 zt 36 ztz 41
525 zt 208’
902 zt 272’
1.20 f 0.75
475 f 159 267 ztz 71’
810 zt 227 495 f 88”
0.75 0.75
f 0.74 f 0.74
aExperimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB, for the determination of serum ALT, AST and y-GT activities. Values are means f S.D.. n = 6. “P < 0.001 compared
‘P < 0.02,
l
with the DMSO control group. lP < 0.01 compared with the AFBr-treated
group.
hepatotoxicity. Thus, it seemed necessary to investigate whether these enzymes are affected by geniposide alone and in combination with AFBr as shown in Table III. Significant elevations were found in the activities of GST (P < 0.05-0.02; CDNB as substrate) and yglutamylcysteine synthetase (P < 0.05; Table IV), but the activities of GSH-Px and -y-GT
Table II.
Effect of geniposide
Treatmenta
were not affected by pretreatment with geniposide. The formation of covalently-bound adducts of AFB,-8,9-epoxide derivatives in rat liver DNA in rats treated with geniposide for 3 consecutive days was reduced with a concomitant decrease in .covalent 3H-radioactivitiy. Treatment of rats with geniposide (6 mg and 10
on the relative liver weight and the level of hepatic Relative liver wt.
GSH
(pg/g liver) Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBI (2 mg/kg) AFBr + GPs (5 mg/kg) AFBr + GPs (10 mg/kg)
3.84 3.40 3.65 3.43 3.84 3.89 3.76
f 0.21 f 0.11 f 0.27 l 0.12 zt 0.16’ f 0.18 zt 0.11
1934 2347 2114 1852 4519 4890 3709
l
zt zt f f zt f
334 340 467 198’ 590’ 468 449#
GSH and GSSG in rats.
GSSG (pg/g liver)
GSH/GSSG
368 431 478 371 691 621 495
5.26 5.44 4.42 4.99 6.54 7.87 7.49
+ zt f + f zt zt
36 69 100 25 121’ 40 72’
zt f zt + zt f f
1.04 1.18 1.35 0.63 1.43 0.91 1.42
“Experimental rats were pretreated with geniposide (GPs) by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB,. The livers were removed, weighted and homogenized for the determination of cytosol GSH and GSSG levels. Values are means f S.D., n = 6. lP < 0.05 compared with the DMSO control group. “P < 0.05 compared with the AFB,-treated group.
99 Table III.
Effect of geniposide
on the activities of GST and GSH-peroxidase GST (nmol/min
Treatment”
CDNB Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBl (2 r&kg) AFB, + GPs (5 mg/kg) AFB, + GPs (10 mg/kg)
128 128 134 136 122 130 132
f zt f zt f f zt
GSH-Px
per mg prot.)
12.1 11.3 12.9 11.5 14.2 13.4 13.7
(nmol/min
COP
DCNB 5 2 5’ 6” 7 5” 8’
in rats
f f z+ f +z f zt
1.4 1.0 1.4 0.8 0.7 0.3 0.8
19.0 19.0 19.7 18.4 17.2 16.7 19.0
per mg prot.) Hz&
zt f f zt zt zt f
2.3 1.4 3.1 3.4 3.2 3.2 3.7
10.2 8.9 7.6 8.1 10.2 12.4 12.0
f f f f f f *
1.3 1.6 1.0 2.0 2.2 1.5 2.1
aExperimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB, and the livers were minced and homogenized for the determination of enzyme activities. Values are means l S.D., n = 6. lP < 0.02 compared with the DMSO control group. lP < 0.05, “P < 0.05 compared with the AFB,-treated group. l
mg/kg) decreased the amount of AFBr-DNA it is adduct by - 50% (Table V). Therefore, likely that the decreased formation of these adducts after geniposide pretreatment resulted from the depletion of GSH for conjugation with AFBr-epoxide and was also consistent with reduced AFBr-induced hepatotoxicity.
Table IV.
Effect of geniposide
Discussion The results presented in this paper indicate that pretreatment of rats with geniposide leads to protection AFBr-induced against hepatotoxicity. This is supported by the fact that there was suppression of serum AST, ALT
on the activities of y-glutamylcysteine
synthetase
and y-glutamyltranspeptidase
Treatment”
y-glutamylcysteine synthetase (E.U.)b
y-glutamyltranspeptidase (E.U.)’
Normal DMSO (0.2%) GPs (5 mg/kg) GPs (10 mg/kg) AFBr (2 mg/kg) AFB, + GPs (5 mg/kg) AFB, + GPs (10 mg/kg)
8.16 5.35 4.96 10.77 4.96 8.55 8.11
19.47 24.60 22.24 24.11 13.87 12.79 12.91
f zt zt f zt zt f
1.53 3.98 5.38 2.22’ 2.70 1.71” 0.55”
f 7.55 zt 8.40 zt 5.73 ztz 6.08 zt 2.56’ f 2.60 f 2.33
‘Experimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 24 h after the i.p. administration of AFB, and the livers were minced and homogenized for the determination of enzyme activities. Values are means f S.D., n = 6. bThe enzyme activity (E.U.) was defined as 1 mmol Pi released/mg protein per min. ‘The enzyme activity (E.U.) was defined as 1 mmol p-nitroaniline formed/mg per min. lP < 0.05 compared with the DMSO group. 'P < 0.05 compared with the AFBr-treated group.
100 Table V. Effect of geniposide on the hepatic binding induced by AFB, in rats. Treatment=
AFB, (33.88 pg/kg) AFBt + GPs (6 mg/kg) AFB, + GPs (10 mg/kg)
AFB, bound
DNA-
(pg/mg DNA)
% of AFB, alone
3.31 f 2.10 1.70 + 0.28 1.80 ztz 0.42
100 52 54
aExperimental rats were pretreated with geniposide by gastric intubation for 3 days. All rats were killed 2 h after the i.p. administration of AFB, and the livers were minced for the determination of AFBt-DNA adduct formation. Values are means f S.D., n = 3.
and -y-GT activities induced by AFBi in male rats. Previous reports have demonstrated a reduction of carbon tetrachloride(CCl&and cr-naphthylisothiocyanate(ANIT)-induced hepatotoxic effects by geniposide in experimental animals [32,33]. Similarly the marked reduction by pretreatment with geniposide in AFBi hepatotoxicity occurs when the AFBi was administrated 24 h after three daily doses of geniposide. The suppressive effect on AFBi-induced hepatotoxicity may be due to enhancement of the activities of certain drug-metabolizing enzymes by geniposide pretreatment leading to an increase in the conversion of AFBr to inactive metabolites. Single doses of AFBi produced an adaptive elevation in GSH levels. This is analogous to previous studies showing that after administration of some hepatocarcinogens there is a response of hepatic GSH [34]. However, the combination of geniposide with AFBi caused a significant reduction (P < 0.05) in hepatic GSH level (Table II)). Depletion by geniposide of liver GSH might increase hepatotoxicity [ 18,351, since conjugation with GSH is an important detoxifying pathway for AFBi [36]. However, our data show contrary results in that the marked reduction by a geniposide of AFBi hepatotoxicity occured when AFBl was administered. There is a
possibility that the accelerated reduction of GSH and GSSG resulted from direct interaction of geniposide [ll] and conjugation of AFBi-epoxide with GSH for AFBr detoxication. The initial depletion of GSH seen after geniposide administration did not affect the hepatic GSH pool size. It should be noted that the concentration of GSH in the hepatic cytosol fraction used for these studies was high. Therefore, the relatively minor depletion of GSH through conjugation with geniposide would be negligible. Other novel properties of geniposide are increases in the activities of yglutamylcysteine synthetase, the first and ratelimiting enzyme in GSH biosynthesis, and GST, which catalyzes the conjugation of AFBl with GSH. Hence, the depletion of GSH will be replenished by the induction of synthetase during geniposide pretreatment. The AFBi detoxication pathway will also be stimulated by the induction of GST. The lowering of GSH level which was found after geniposide and AFBl administration could, in part, possibly have resulted from the greater catalytic rate of conjugation by GST rather than through biosynthesis of GSH by y-glutamylcysteine synthetase. The formation of DNA adducts is regarded as the primary event in the carcinogenic process. Most of anticarcinogenic compounds have been tested for their ability to suppress the formation of AFBi-DNA adducts [37,38]. Therefore, the increased formation of conjugates after geniposide pretreatment is a likely result from a decreased reaction of AFBi-&g-oxide with DNA in rat liver in vivo. Consistent results were obtained from the induction of GST, and covalent binding to DNA was decreased by - 50% as compared with AFBi alone when geniposide was given at a dose of 6 mg and 10 mg/kg (Table V). our results indicate that In conclusion, geniposide is a modulator of AFBi-DNA adduct formation, and also demonstrate that geniposide supresses AFB,-induced hepatotoxicity by a mechanism that involves enhanced GST activity for AFBi detoxication and induction of GSH biosynthesis.
101
Acknowledgement
14
This work was supported by the National Science Council Grant NSC 80-0412B040-04, Republic of China.
15
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