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BRAZILIN PROTECTS CULTURED RAT HEPATOCYTES FROM BrCCI3-INDUCED TOXICITY a

a

a

Chang-Kiu Moon , Kwang-Sik Park , Seong-Gon Kim , a

Hyeon-Soon Won & Jin-Ho Chung

a

a

College of Pharmacy, Seoul National University, San 56-1, Sinlim-Dong, Kwariak-Gu, Seoul, 151, Korea Published online: 11 Apr 2015.

To cite this article: Chang-Kiu Moon, Kwang-Sik Park, Seong-Gon Kim, Hyeon-Soon Won & Jin-Ho Chung (1992) BRAZILIN PROTECTS CULTURED RAT HEPATOCYTES FROM BrCCI3-INDUCED TOXICITY, Drug and Chemical Toxicology, 15:1, 81-91 To link to this article: http://dx.doi.org/10.3109/01480549209035174

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DRUG A N D CHEMICAL TOXICOLOGY, 15(1), 81-91 (1992)

BRAZILIN PROTECTS CULTURED RAT HEPATOCYTES FROM BrCC1,-INDUCED TOXICITY

Chang-Kiu Moon, Kwang-Sik Park, Seong-Gon Kim, Hyeon-Soon Won and Jin-Ho Chung College of Pharmacy, Seoul National University, San 56-1, Sinlim-Dong, Kwanak-Gu, Seoul 151, Korea

ABSTRACT Brazilin, the main constituent of Caesalpinia sappan, is an antioxidative substance that has catechol moiety in its chemical structure. Considering the antioxidant-activity of brazilin, it was expected to have protective effects on the toxicities of radical generating chemicals. The incubation of rat hepatocytes with BrCCl, resulted in significant increase in lipid peroxidation, leakage of cytoplasmic enzymes and cytoplasmic glutathione depletion. The BrCC1,-induced toxicities on hepatocytes were reduced by the treatment of brazilin. Brazilin has been also proved to have a protective effect on the BrCC1,-induced depression of microsomal calcium sequestration activity. These results indicate that brazilin plays a protective role in BrCC1,-induced hepatocyte injury of the rat.

INTRODUCTION

Among the various biological activities of brazi1inl4 (Figure l),which is the main constituent of Caesalpinia sappan, the inhibition effect on lipid per~xidation’-~ suggested that brazilin might have preventive effects on the chemical-induced cell death. The mechanism of cell death has not yet been fully understood, but the evidence indicates that the perturbation of calcium homeostasis is the crucial and irreversible event leading to cell death in the

81 Copyright Q 1992 by Marcel Dekker, Inc.

MOON ET AL.

a2

OH

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HO

HO

OH FIGURE 1. Structure of brazilin

liver injury.&”

Based on these informations, we started to investigate the

effects of brazilin on the alteration of microsomal calcium sequestration activity which maintains the low level of cytoplasmic free calcium and the related biochemical factors, e.g., lipid peroxidation,” leakage of cytoplasmic and intracellular glutathione level14 in BrCC1,-intoxicated rat hepatocytes. MATERIALS AND METHODS

Materials:

SD male rats (200-250g) were obtained from Experimental Animal Breeding Center of Seoul National University and allowed food and water ad libitum. Brazilin was obtained from Aldrich Chemical Co., Milwaukee, WI. BrCC1, was obtained from Wako Chemical Co., Tokyo. Radiolabeled [45Ca]CaCI, was obtained from Amersham Corp., Arlington Heights, IL. Other reagents were obtained from Sigma Chemical Co., St. Louis, MO. Cell culture: Hepatocytes were isolated from SD male rats by a collagenase perfusion technique as described by Berry and Friend.” The isolated hepatocytes were

a3

BRAZILIN PROTECTS CULTURED RAT HEPATOCYTES

inoculated onto collagen coated culture dishes in CO, incubator and then, monolayer was formed within 4 hours. Several kinds of media were used according to the aims of the experiments.

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Determination of membrane damaee: Lipid peroxidation, AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were used as parameters of membrane damage. For the determination of lipid peroxidation, fresh isolated rat hepatocytes were used. Four milliliters of hepatocytes suspension (5x106 cells/ml, DMEM medium containing antibiotics, 2% bovine serum albumin) was incubated with BrCCl, (0.2 pl/ml) and brazilin (lo3, 10“ and lo5 M) dissolved in DMSO in a gas tight glass flask for 60 minutes. At the end of incubation, the cell suspension was withdrawn for the assay of lipid peroxides by TBA (thiobarbituric acid) method16. For the determination of the level of AST and ALT in medium, the cultured hepatocytes in Waymouth MB 752/1 were used.

After the

replacement of the medium with prewarmed HBSS (phenol red-free), BrCC13(0.2 pl/ml) and brazilin were added into the medium and the incubation was continued for one hour.

At the end of the incubation, the

levels of AST and ALT released into the medium were measured by the enzymatic reaction method.13 Determination of intracellular glutathione: After 24 hour culture of hepatocytes in William’s E medium (5% FBS), the medium was replaced with serum free William’s E medium. BrCCl, (0.2 pl/ml) and brazilin (lo3, lo4 and 10” M) were added directly to the culture medium and incubated for one hour.

At the end of incubation, the cells

were washed with ice cold phosphate buffered saline.

The washed

hepatocytes were collected with 0.5% picric acid (1 ml)into microcentrifuge tube.

After centrifugation, the supernatant was withdrawn for the

determination of total glutathione and oxidized glutathione.

To determine

the oxidized glutathione, reduced glutathione was derivatized with

a4

MOON ET AL.

2-vinylpyridine. The amount of glutathione was determined by DTNB(5,Sdithiobis-2-nitrobenoic acid)-GSSG(oxidized glutathione) reductase recycling method.”

Reduced glutathione (GSH) was calculated from the difference

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between the total glutathione and the oxidized glutathione. Determination of microsomal calcium sequestration activity: Microsomal fraction was prepared with the slight modification of the method previously described by Recknagel et a19 and calcium sequestering activity of microsomal fraction was measured by the membrane filtration technique?

In brief, microsomal fraction (0.3 mg/ml) was preincubated in a gas tight flask at 37°C for 10 minutes with the reagents for calcium uptake [lo0 pM CaCI,, 5 mM A”,

5 Mm ammonium oxalate, 10 mM MgCI,, 5 mM NaN,, and

[4sCa]CaC1, (0.4 pCi/ml)] and for NADPH generation [0.14 mM NADP, 2.5

mM

nicotine

amide,

3

mM

(D,L)-isocitrate, 0.07

unit

dehydrogenase/ml] in imidazole-histidine buffer (30 Mm, pH 6.8).

isocitric Then,

BrCCl, (0.2 pl/ml) and brazilin (lo”, lo4 and 10’ M) were added to the reaction mixture and the incubation was continued for 60 minutes. After the incubation, the reaction was stopped by standing the incubates in ice bath. The incubates were then filtered using filter holder (Hoefer Co., USA), on 0.45 p m cellulose triacetate filter (Metricel) wetted with 0.25 M sucrose

solution, and rinsed immediately with 10 ml of sucrose solution.

[4SCa]Ca

bound to the microsomal fraction on the filter was counted by li’quid scintillation counter and the amount of calcium was calculated. RESULTS AND DISCUSSION

Many of the damaging effects of BrCC1, on the liver require its metabolism to trichloromethyl radicals. One of the consequences of CCl, -production in the liver endoplasmic reticulum is the stimulation of lipid peroxidation, which

is usually measured by the formation of thiobarbituric acid-reactive substances.

BRAZILIN PROTECTS CULTURED RAT HEPATOCYTES

85

Malondialdehyde production was significantly increased by treatment of BrCCl, and was inhibited by brazilin in dose dependent manner (Table 1). Brazilin also reduced AST- and ALT-levels in the incubation media, which

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were used as parameters of membrane damage. No significant changes were observed both in lipid peroxidation and in enzyme leakage, followed by brazilin treatment alone in normal hepatocytes (data not shown).

In order to investigate the relationship between the membrane damage and the depletion of intracellular glutathione, the level of glutathione was determined after the treatment of BrCC1,. BrCCl, caused severe depletion of total glutathione primarily resulting from the decrease of reduced glutathione, and the ratio of glutathione redox state (GSSG/GSH) was not significantly changed (Table 2).

Since there is no increase in oxidized glutathione by

BrCC1, treatment, the main cause of glutathione depletion is supposed to be the conjugation with trichloromethyl radicals.

Simultaneous addition of

brazilin and BrCC1, into the medium resulted in the protection of glutathione-loss (Table 2). The effects of brazilin alone on total intracellular glutathione level were also investigated (Table 3). High concentration of brazilin (lo” M) caused a significant reduction of total glutathione.

The

mechanism of glutathione-depletion at higher concentration of brazilin (lo5

M) has not yet been elucidated, but it might be presumed that brazilin inhibited glutathione synthesis and/or consumed much more glutathione in the course of incubation. Perturbation of cellular calcium homeostasis has been documented in a number of diverse pathological phenomena.

The pathological excess of

intracellular free calcium has been implicated as a final common pathway of cell death produced by a wide range of toxins including the hepatotoxin, such as BrCCl,?

Many reports suggested that the loss of calcium sequestration

activity by BrCC1, or CC1, has been evaluated as initial events of cytotoxic mechanism and the calcium sequestration activity was more sensitive than any other cytotoxic parameters, such as lipid peroxidation, enzyme leakage and

3.0 f 0.2

50 f 10

456 f 93

BrCC1, Control

~

~~~~

+

48 f 8 2.4 k 0.1*

1 . 8 f 0.1*

1 . 5 k 0.1*

1*

17 f 4*

14

+-

437 k 100

BrCC1, BrX

1 5 6 f 40*

BrCC1, + BrX lo-'#

37 & 16*

BrCC1, + BrX 10q3M

The symbols represent the groups which are significantly different from BrCCl, group by one-way ANOVA/Duncan (p < 0.05) in each toxic parameter. Data for each treatment are the mean f S.D.from 5-6 samples.

MDA(nmol/mg protein) 1 . 2 f 0 . 2

13 f 3

ALT ( I U / L )

f 13

39

AST (IU/L)

Normal Control

Effects of brazilin (BrX) on the leakage of cytoplasmic enzymes and the lipid peroxidation in BrCCIJntoxicated rat hepatocytes.

TABLE 1.

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z

rj

m

0

87

BRAZILIN PROTECTS CULTURED RAT HEPATOCYTES

TABLE 2.

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Effects of brazilin (BrX) on the intracellular level of glutathione in cultured rat hepatocytes intoxicated with BrCCI,. Normal Control GSH+GSSG (nmol/mg protein)

4 0 . 8 -I 2.0

GSSG(nrnol/

mg protein)

1.0

BrCC13 Control

BrCC1, + BrCCl,+ BrX 10'3M BrX 1 0 - 4 M

2.5 f

32.6 i:

0.7

+-

1.4 f 0.2

0.4

GSH(nmol/mg 3 9 . 8 f protein) 2.0

1.3

0.4

32.7 k 18.5 2 5.3* 6.9*

4.0*

1.2 i:

0.2

BrCCl,+ BrX 10%

1.6 f 0.2

1.3

_+

0.3

31.2 2 31.1 f 17.2 f 3.9* 5.2* 6.9*

The symbols represent the groups which are significantly different from BrCCl, group by one-way ANOVA/Duncan (p < 0.0s) in each toxic parameter. Data for each treatment are the mean 2 S.D. from 5-6samples.

TABLE 3. Effect of brazilin (BrX) on the intracellular level of glutathione in cultured rat hepatocytes. ~

~~

~

BrX ~ o - ~ M B ~ XI O - ~ M BrX

Normal GSH+GSSG

(nmol/mg protein)

55.2

40.9 f 2.6*

f 1.4

~

GSSG(nmol/ mg protein)

6.0 f 0.2

GSH(nmol/mg protein)

49.2 2 3.3

~~

~

52.5 f 6.5 ~

6.9 i 0.5 34.2 i 2.6*

~~

~~~

52.1

-+ 5.5

~

~

~~

5.2 f 0.6

6.0 f 0.8

47.3 f 6.7

46.2 f 5.1

The symbols represent the groups which are significantly different from Normal group by one-way ANOVA/Duncan (p < 0.05). Data for each treatment are the mean ? S.D. from 5-6 samples.

MOON ET AL.

88

.-

E

P)

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C

CONTROL

BrCCl3

BrCCl3

BrCCl3

BrCCl

t

t

t

BRA$ILIN (10- M)

BRAZILIN (104M)

BRAZILIN (10%)

FIGURE 2. Effect of brazilin on the BrCCI,-induced depression of calcium sequestration activity of rat liver microsomes. Comparisons among the groups were made by a one-way analysis of variance followed by Duncan's test (p