Exp Toxic Pathol 1992; 44: 399-405 Gustav Fischer Verlag Jena
Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Gennany
Inftuence of bile acids on stimulated lipid peroxidation and hydrogen peroxide production in rat liver microsomes A. BARTH and M. BERNST With 3 figures and 2 tables Recei ved: December 2, 1991; Accepted: December 28, 1991 Address for correspondence: Dr. sc. med. A. BARTH, Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, LobderstraBe 1, lena, D-O-6900, Gennany. Key words: Lipid peroxidation; Hydrogen peroxide Production; Bile acids; Liver microsomes; Microsomes, liver; Antioxidants; Peroxidation, lipid.
Summary Bile acids were found to be effective antioxidants in bile and intestine. The influence of different bile acids on the NADPH-Fe + +-stimulated lipid peroxidation (LPO) and cytochrome P-4S0 dependent hydrogen peroxide production (H 2 0 2) in rat liver microsomes was investigated in vitro. LPO was detennined as production of thiobarbituric acid reactants (TBAR). Different tri-, di- and monohydroxylated bile acids and cholesterol were given to the incubation mixture in concentrations ranging from 10- 5 to 1O- 3 M. Sodium salts of cholic, tauroglycocholic and deoxycholic acids as well as cheno-deoxycholic, ursodeoxycholic, lithocholic acids and cholesterol did not alter the microsomal production of TBAR. H20 2 fonnation was significantly decreased by sodium deoxycholate whereas cholesterol increased H2 0 2 production up to 4 times. These results show that bile acids were not able to protect microsomal membrane lipids against peroxidative damage. Cholesterol mediated H20 2 fonnation as a source of hydroxyl radicals had no toxic effect concerning LPO, TBAR were not enhanced significantly.
on lipid concentration, bile acids can also act as enhancers of lipid peroxidation (DE LANGE and GLAZER 1990). We investigated the in vitro influence of sodium cholate, sodium tauroglycocholate, sodium deoxycholate, chenodeoxycholic, ursodeoxycholic and lithocholic acids and cholesterol in increasing concentrations on NADPHFe++ -stimulated lipid peroxidation (LPO) and hydrogen peroxide production (H 20 2 ) in rat liver microsomes. The cytochrome P-450 system in the liver is responsible for H2 0 2 and radical production. The toxic effects of H 2 0 2 may be mediated by the hydroxyl radical generated in vivo by a Fenton reaction (JONAS et al. 1989, MINOTTI 1990). It was the aim of these investigations to find out possible protective or enhancing effects of bile acids on iron stimulated LPO and oxidase function of cytochrome P-450.
Material and methods Introduction Oxidation and radical reactions in living cells and tissues can be generated by the reduction of oxygen to superoxide radical, hydrogen peroxide or hydroxyl radical. Different antioxidant mechanisms minimize free radical damage in the living organism. These mechanisms include enzymes and small molecules such as glutathione, vitamins C and E, and bilirubin, which react directly with free radicals (FEHER et al. 1987). DE LANGE and GLAZER (1990) reported that bile acids at physiologic concentration are also effective antioxidants by scavenging peroxyl radicals via direct oxidation to keto derivatives. But depending
6O-day-old male Wistar rats were used. The animals were kept under conventional conditions with pellet diet (Versuchstierproduktion Schonwalde, GDR, prescription Rehbriicke). The rats were decapitated in ether anesthesia, the livers were removed and homogenized in 0.1 M sodium phosphate buffer pH 7.4 (l g liver + 2 ml buffer). The homogenate was centrifuged at 9000 g for 20 min at 0 DC. The 9000 g supernatant was mixed with 25 mM magnesium chloride solution (1 ml supernatant + 2.5 ml MgCh) and centrifuged at 9000 g for 20 min at 0 DC. The sediment was washed and rehomogenized in sodium phosphate buffer at 4 DC (SILMAN et al. 1965). The protein content of the microsomal suspension was determined by the Biuret Exp Toxic Pathol 44 (1992) 7
399
METHOD ACCORDING TO KLINGER and MULLER (1974). The microsomal suspension was kept at - 20°C for not more than one week. The enzymatic LPO was stimulated in the following system: 0.1 M sodium phosphate buffer (pH 7.4), 0.5 mM Fe++ as FeS04 X 7H2 0, about 0.25 mg protienlml, 0.5 mM NADPH. Microsomes were incubated at 37°C for 10 min using a metabolic shaker (Vibrotherm). The reaction was stopped with trichloroacetic acid (30%). LPO was measured using thiobarbituric acid assay (BUEGE and AUST 1978). An extinction coefficient of 1.56 X 105 1 mol-1cm- 1 was employed in the calculation of the resulting concentration of malondialdehyde equivalents. Controls included incubation mixtures to which trichloroacetic acid was added before NADPH. The thiobarbituric acid assay is the method most commonly used to study lipid peroxidation. In the NADPH system, melondialdehyde represents the major thiobarbituric acid reactive substance (TBAR) in the oxidized microsomal lipid as seen by HPLC and thiobarbituric acid methods (ESTERBAUER and ZOLLNER 1989, TOMITA et al. 1990). H2 0 2 formation was measured with the ferroammonium sulfate potassium thiocyanate method in the presence of NaN 3 to block the peroxidative activity (HILDEBRANDT et al. 1978). The incubation mixture of 1.5 ml contained 1 mg protein, 50 mM tris-chloride buffer pH 7.4, NADPH O. 75j.tmol. The microsomes were incubated 20 min at 30°C under continuous shaking. Controls included incubation mixtures, to which trichloroacetic acid was added before microsomal suspension. Rats were pretreated with 1 mllkg b.m. carbon tetrachloride in sunflower oil (lOmllkg b.m.) intraperitoneally. Twelve hours later the rats were anesthetized with ether and decapitated. Liver microsomes were prepared, and LPO and H20 2 were measured as described above. Bile acids were added to the incubation medium reading final concentrations of 10- 5 to 10- 3 M. Sodium salts of cholic, tauroglycocholic and deoxycholic acids were dissolved in sodium phosphate buffer 0.1 M, pH 7.4. Chenodeoxycholic, ursodeoxycholic, lithocholic acids and cholesterol in ethanol were dried by room air overnight in individual assay tubes prior to the addition of other assay components.
Statistics Statistical evaluation of the data was performed by the Student's t-test. The level of significance was chosen as p :::; 0.05.
Results The effects of a single 1 mllkg b.m. dose of CCl 4 given to the rats intraperitoneally 12 hours prior to examination on LPO and H2 0 2 formation in rat liver microsomes are demonstrated in tab. 1. TBAR were significantly enhanced, whereas H 20 2 formation was significantly diminished in microsomes obtained from CC1 4 treated rats. 400
Exp Toxic Pathol 44 (1992) 7
The in vitro influence of bile acids on iron stimulated LPO in liver microsomes of untreated rats is shown in tab. 2. Neither trihydroxylated nor di- or monohydroxylated bile acids changed the production rate of TBAR. Table 1. Lipid peroxidation (TBAR) and H2 0 2 formation in
liver micro somes of adult male Wistar rats 12 hours after carbon tetrachloride treatment (CCI4, 1 mglkg b.m. intraperitoneally). Controls (C) recieved sunflower oil (10 ml! kg b.m. intraperitoneally). Arithmetic means ± S.E.M., n = 5, * significant differences to controls, p :::; 0.05, STUDENT'S t-test. TBAR [nmollmg X min]
H 20 2
3.02 ± 0.26 5.32 ± 0.30*
1.21 ± 0.17 0.53 ± 0.07*
[nmollmg X min]
Cholesterol had no influence on microsomal LPO, either (tab. 2). Figures 1- 3 demonstrate the in vitro effect of bile acids and cholesterol on hepatic microsomal H2 0 2 production. The sodium salt of free or conjugated trihydroxylated cholic acid did not alter the microsomal H20 2 production. Among the dihydroxylated bile acids only sodium deoxycholate reduced the H 20 2 production in a dose dependent manner. Whereas monohydroxylated lithochoic acid showed only a tendency to increase cholesterol significantly enhanced the microsomal H 2 0 2 formation in all three concentrations (fig. 3).
Discussion The use of many drugs is limited by their toxic side effects, very often mediated by lipid peroxidation or free radical production. Efforts were made to prevent the resulting toxic effects by radical scavengers such as tocopherol, glutathione, superoxide dismutase or superoxide dismutase - like constitutents of Ginkgo biloba leaves (HILL and BURK 1984; CASINI et al. 1985; BARTH et al. 1991). The aim of the presented study was to find out possible radical scavenging properties of bile acids. Bile acids were investigated in rat liver microsomes incuated with iron as effective catalyst of the production of potent oxidizing species (BACON and BRITTON 1989; MINOTTI and AUST 1989). Carbon tetrachloride (CCI4) was used as a well-known model substance producing lipid peroxidation and liver damage (RECKNAGEL et al. 1989, COMPORTI 1989; WOLFGANG et al. 1990). As shown by the results, CCl4 enh~ced susceptibility to in vitro lipid peroxidation 12 hours after in vivo treatment of the rats (tab. 1). The inhibition of the microsomal H2 0 2 production may be related to the toxic effect of CCl4 on cytochrome P-450 (COMPORTI 1989). The microsomal inducer pregnenolone16 cx-carbonitril enhanced, whereas the microsomal
Table 2. Lipid peroxidation (TBAR) in liver microsomes of adult male Wi star rats 10 minutes incubated with bile acids and cholesterol. Arithmetic means ± S.E.M., n = 4-6. Control
TBAR [nmollmg x min] bile acids [mollI]
10- 5
10- 4
10- 3
2.83 ± 0.38 3.00 ± 0 .38
Na-cholate Na-tauroglycocholate
2.39 ±0.50 2.34 ± 0.69
2.99 ± 0.45 2.82 ± 0.55
3.00 ± 0.45 1.89 ± 0.34
2.97 ± 0.36 2.94 ± 0 .35 2.72 ± 0.31
Na-desoxycholate ursodeoxycholic acid chenodeoxycholic acid
2.87 ± 0 .76 2.58 ± 0.55 2.07 ± 0.43
3.46 ± 0.63 3.11 ± 0.46 2.90 ± 0.44
2.76 ± 0.50 2.86 ± 0 .59 2.85 ± 0.40
2.88 ± 0.32
lithocholic acid
2.87 ± 0.25
3.33 ± 0.40
3.07 ± 0.39
2.73 ± 0.31
cholesterol (molll)
2.15 ± 0 .50
3.02 ± 0.51
3.71 ± 0.32
2 r-~~~~----------------------
__~
1.G ,
;
10 N.~(nMIVI'
2,G r-~~~------------------------
______~
2
Fig. 1. Hydrogen peroxide (H 20 2) production in liver microsomes of adult male Wi star rats 20 minutes incubated with different concentrations of trihydroxylated bile acids. Data expressed as arithmetic means ± S.E.M., n = 4-5.
Exp Toxic Pathol 44 (1992) 7
401
2~rl"-~~~~~------------------------------~ 2
2~~r----~~~~----------------------------~ 2
1
0,5
O~~~~~--~~~~L-~~
control
10-
10
10
chenodeo,x,ahollo IIOId (1ftOIII)
'Arl"-~~~~~------------------------------~ ',8
,,,,
',2
1
OA 0,_ 0,4
0.2 O~~~~~--~~~~--~~~~
10- 4
control
__-L~~~LJ
Na-deoxwoholate (moll!)
402
Exp Toxic Pathol 44 (1992) 7
10
Fig. 2. Hydrogen peroxide (H 2 0 2 ) production in liver microsomes of adult male Wi star rats 20 minutes incubated with different concentrations of dihydroxylated bile acids. Data expressed as arithmetic means ± S.E.M., n = 4- 5, asterisk indicates significant difference to control, p :5 0.05, STUDENT'S t-test.
2~~~~----------------~ 1,5
0,5
10
Ithoohollo MId (mot/I)
.lrn_.aH ___~~I.~PG~.._~_I______________________________--,
•
3 2
Fig. 3. Hydrogen peroxide (H 20 2) proliuction in liver microsomes of adult male Wistar rats 20 minutes incubated with different concentrations of monohydroxylated bile acid and cholesterol. Data expressed as arithmetic means ± S.E.M., n = 4-5, asterisks indicate significant differences to controls, p ::; 0.05, STUDENT'S t-test.
inhibitor metyrapone diminished the cytochrome P-450 dependent H2 0 2 formation (BAST et al. 1989), Phenobarbital and [3-naphthoflavone were without effect (HAUFE and KLINGER 1990). Bile acids have the capacity to induce both choleresis and cholestasis, depending on concentrations and hydrophobic or hydrophilic properties of the individual bile acids. Monohydroxylated and dihydroxylated bile acids were found to be more toxic than trihydroxylated ones (SCHOLMERICH et al. 1984). Transported through the hepatocytes bile acids interact with the endoplasmic reticulum and the Golgi apparatus, especially at higher bile acid loads and higher intracellular concentrations (ERUNGER 1990). According to DE LANGE and GLAZER (1990) bile acids can function as effective antioxidants in bile and the intestine. Determining the rate of linoleate oxidation by peroxyl radicals, glycocholate was
1·: ;.
able to promote lipid peroxidation if bile acidllinoleate concentration ratio was changed to smaller values (DE LANG and GLAZER 1990). The data presented here show no influence of bile acids in vitro on microsomal lipid peroxidation (tab. 2). Neither conjugated nor free, neither hydrophobic nor hydrophilic bile acids altered the iron stimulated production of TBAR. The highly cholestatic lithocholic acid (TAKIKAW A et al. 1991) as well as cholesterol as endogenous and exogenous source for bile acid synthesis (Ay AKI et al. 1981) did not influence the microsomal lipid peroxidation (tab. 2). DE LANGE and GLAZER (1990) used bile acid concentrations ranging from 10- 1 to 1O- 3 M, normally found in the gallbladder and in the duodenum. According to FISHER et al. (1976) only 2% ofthe bile acid pool is situated in the liver in adult Wistar rats, accordingly 2 X 1O- 4 M. The bile Exp Toxic Pathol 44 (1992) 7
403
acid concentrations used in our incubation mixture never exceeded 10- 3 M. Bile acids were also unable to influence the microsomal HzO z formation. One exception is sodium deoxycholate, which inhibited the HzO z production (fig. 2). The detergent potency of deoxycholate may be responsible for dose dependent damage of cytochrome P-450 (SCHOLMERICH et al. 1984). Surprinsingly, other dihydroxylated bile acids tested had no effect on HzO z formation. Different effect of dihydroxylated bile acids are known according to the position of the OH-groups. For example the cholestatic potency and the detergent power of deoxycholate is higher than that of chenodeoxycholate whereas ursodeoxycholate can prevent bile acid induced cholestasis and hepatotoxicity (DREW and PRIESTLY 1979; KITANI and KANAI 1982; ATTILI et al. 1986; GALLE et al. 1990). HzO z production is associated with the oxidation of NADPH in liver microsomes. Some substrates of cytochrome P450 such as hexobarbital stimulate the rate of HzO z formation concomitantly with the oxidative biotransformation of this substrate (KLINGER et al. 1986). 7-Ethoxycoumarin acts as an uncoupler for the cytochrome P-450 reaction system, thereby enhancing the HzO z production (ESTABROOK et al. 1979). The presence of cholesterol in our incubation mixture markedly enhanced the formation of HzO z (fig. 3). The 7ex-hydroxylation of cholesterol is the major rate limiting step in the overall conversion of cholesterol into bile acids (SHEFER et al. 1970; HEUMAN et al. 1988). The cholesterol7 ex-hydroxylation as well as some other hydroxylations on the steroid nucleus appear to be cytochrome P-450-dependent (BJORKHEM et al. 1975; ZIMNIAK et al. 1991). Although the hydroxylations in the bile acid metabolism differ in some respects from those involved in the drug metabolism, cholesterol may act as an uncoupler for the cyclic function of cytochrome P-450, thereby increasing the HzO z formation. Surprisingly, the up to 4 times higher HzO z formation in the microsomes did not stimulate lipid peroxidation, the TBAR were not enhanced significantly in incubation mixtures without sodium acid (tab. 2). With unblocked peroxidative acitivity of cytochrome P-450 cholesterol mediated HzO z may be decomposed in H 20 and hydroxylated substrate (HILDEBRANDT et al. 1978). Another possibility is that cholesterol itself acts as hydroxyl radical scavenger as seen in studies with ultrasoundinduced lipid peroxidation and in liposomes (JAN Aet al. 1990, HERNANDEZ-CASELLES et al. 1990). Also LETKO et al. (1990) did not find a correlation between cell lysis and elevation of TBAR afterincubation of pancreatic acinar cells with H 20 2 . On the other hand, ISLAM et al. (1991) tested steroids, structurally related to cholesterol, and revealed mutagenic activity which appears to involve the formation of H Z0 2 • According to BHADRA et al. (1991) cholesterol-alpha-epoxide is a major product of cholesterol peroxidation in the presence of endothelial cells, playing a key role in atherogenesis. In conclusion bile acids were not able to protect microsomal membrane lipids against peroxidative damage. Cholesterol enhanced the cytochrome P-450-dependent H 2 0 2 formation without affecting the microsomal lipid peroxidation significantly. 404
Exp Toxic Pathol 44 (1992) 7
References ATT!LI AF, ANGELICO M, CANTAFORA D, et al.: Bile acidinduced liver toxicity: relation to the hydrophobic-hydrophilic balance ofbile acids. Medical Hypotheses 1986; 19: 57-69. AYAKI Y, TSUMA-DATE T, ENDO S, et al.: Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats. Steroids 1981; 38: 495-509. BACON BR, BRITTON RS: Hepatic injury in chronic iron overload. Role oflipid peroxidation. Chern BiolInteractions 1989; 70: 183-226. BARTH SA, INSELMANN G, ENGEMANN R, et al.: Influences of Ginkgo Biloba on Cyclosporin A induced lipid peroxidation in human liver micro somes in comparison to vitamine E, glutathione and N-Acetylcysteine. Biochem Pharmacol1991; 41: 1521-1526. BAST A, GOOSSENS PAL, SAVENIJE-CHAPEL EM: Dependence of hydrogen peroxide formation in rat liver microsomes on the molecular structure of cytochrome P-450 substrates: a study with barbiturates and !)-adrenoreceptor antagonists. Europ J Drug Metab Pharmacokin 1989; 14: 93-100. BHADRA S, ARSHAD MA, RYMASZEWSKI Z, et al.: Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu + + ions: identification of major products and their effects. Biochem Biophys Res Commun 1991; 176: 431-440. BJORKHEM I, DANIELSSON H, WIKVALL K: Cytochrome P-450dependent hydroxylations in biosynthesis and metaolism of bile acids. Biochem Soc Transact 1975; 3: 825-828. BUEGE JKA, AUST SD: Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302-310. CASINI AF, POMPELLA A, COMPORT! M: Liver glutathione depletion induced by bromobenzene, iodobenzene, and dietylmaleate poisoning and its relation to lipid peroxidation and necrosis. Am J Pathol 1985; 118: 225-237. COMPORT! M: Three models of free radical-induced cell injury. Chern BioI Interactions 1989; 72: 1-56. DE LANGE RJ, GLAZER AN: Bile acids: antioxidants or enhancers of peroxidation depending on lipid concentrations. Archs Biochem Biophys 1990; 276: 19-25. DREW R, PRIESTLY BG: Choleretic and cholestatic effects of infused bile salts in the rat. Experientia 1979; 35: 809-810. ERLINGER S: Role of intracellular organelles in the hepatic transport of bile acids. Biomed Pharmacother 1990; 44: 409-416. ESTABROOK RW, KAWANO S, WERRINGLOER J, et al. : Oxycytochrome P450: its breakdown to superoxide for the formation of hydrogen peroxide. Acta bioI med germ 1979; 38: 423-434. EsTERBAUER H, ZOLLNER H: Methods for determination of aldehydic lipid peroxidation products. Free Rad BioI Med 1989; 7: 197-203. FEHER J, CSOMOS G, VERECKEI A: Free radical reactions in medicine. Springer-Verlag Berlin-Heidelberg-New YorkLondon-Paris-Tokyo 1987; pp. 11-17. FISHERMM, KAKIS G, YOUSEFIM: Bile acid pool in Wistarrats. Lipids 1976; 11: 93-96. GALLE PR, THEILMANN L, RAEDSCH R: Ursodeoxycholate reduces hepatotoxicity of bile salts in primary human hepatocytes. Hepatology 1990; 12: 486-491. HAUFE S, KLINGER W: Der EinfluB verschiedener Wirkstoffklassen auf die H20z-Bildung durch Rattenlebermikrosomen. Tag.-Ber., Akad. Landwirtsch.-Wiss. DDR, Berlin 1990; 285: 107 - 109.
HERNANDEZ-CASELLES, VILLALAIN J, GOMEZ-FERNANDEZ JC: Stability of liposomes on long term storage. J Pharm Pharmacol 1990; 42: 397-400. HEUMAN OM, VLAHCEVIC ZR, BAILEY ML, et al.: Regulation of bile acid synthesis. n. Effect of bile acid feeding on enzymes regulating hepatic cholesterol and bile acid synthesis in the rat. Hepatology 1988; 8: 892-897. HILDEBRANDT AG, ROOTS I, TJOE M, et al.: Hydrogen peroxide in hepatic microsomes. Methods Enzymol 1978; 52 : 342-350. HILL KE, BURK RF: Influence of vitamin E and selenium on glutathione-dependent protection against microsomal lipid peroxidation. Biochem Pharmacol 1984; 33: 1065-1068. ISLAM S, SHAFIULLAH A, AHMAD M: Mutagenic activity of certain synthetic steroids : structural requirement for mutagenic activity in Salmonella and E. coli. Mutat Res 1991; 259: 177-187. JANA AK, AGARWAL S, CHATTERJEE SN: The induction of lipid .peroxidation in liposomal membrane by ultrasound and the role of hydroxyl radicals. Radiat Res 1990; 124: 7-14. JONAS SK, RILEY PA, WILLSON RL: Hydrogen peroxide cytotoxicity. Biochem J 1989; 264: 651-655. KlTANI K, KANAl S : Tauroursodeoxycholate prevents taurocholate induced cholestasis. Life Sci 1982; 30: 515-523. KLINGER W, FREYTAG A, SCHMITT W: Influence of age, hexobarbital, and aniline on NADPHINADH dependent hydrogen peroxide production in rat hepatic microsomes. Arch Toxicol 1986; Suppl9: 382-385. KLINGER W, MULLER 0: The influence of age on the protein concentration in serum, liver and kidney of rats determined by various methods. Z Versuchstierk 1974; 16: 146-153. LETKO G, WINKLER b, MATTHIAS R, et al.: Studies on lipid peroxidation in pancreatic tissue. In vitro formation of thiobarbituric-acid-reactive substances (TBRS). Exp Pathol 1991 ; 42: 151-157.
MINOTTI G: NADPH- and Adriamycin-dependent microsomal release of iron and lipid peroxidation. Archs Biochem Biophys 1990; 227: 268-276. MINOTTI G, AUST SO: The role of iron in oxygen radical mediated lipid peroxidation. Chern BioI Interactions 1989; 71:1-19. RECKNAGEL RO, GLENDE EA, DOLAK JA, et al.: Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther 1989; 43: 139-154. SCHOLMERICH J, BECHER MS, SCHMIDT K, et al.: Influence of hydroxylation and conjugation of bile salts on their membrane-damaging properties - studies on isolated hepatocytes and lipid membrane vesicles. Hepatology 1984; 4: 661-666. SHEFER S, HAUSER S, BEKERSKY J, et al.: Biochemical site of regulation of bile acid biosynthesis in the rat. J Lipid Res 1970; 11: 404-411. SILMAN N, ARTMAN M, ENGELBERG H: Effect of magnesium and spermine on the aggregation of bacterial and mammalian ribosomes. Biochim Biophys Acta 1965; 103: 231-240. TAKlHAWA H, OHKI H, SANO N, et al.: Cholestasis induced by lithocholate and its glucuronide: their biliary excretion and metabolism. Biochim Biophys Acta 1991; 1081: 39-44. TOMITA M, OKUYAMA T, KAWAI S: Determination ofmalonaldehyde in oxidized biological materials by high performance liquid chromatography. J Chromatography 1990; 515 : 391-397. WOLFGANG GHJ, DONARSKY WJ, PETRY TW: Effects of novel antioxidants on carbon tetrachloride-induced lipid peroxidation and toxicity in precision-cut rat liver slices. Toxicol Appl Pharmacol 1990; 106: 63-70. ZIMNIAK P, HOLSZTYNSKA EJ, RADOMINSKA A, et al.: Distinct forms of cytochrome P-450 are responsible for 6 ~-hydroxy lation of bile acids and of neutral steroids. Biochem J 1991; 275 : 105-111.
Exp Toxic Pathol44 (1992) 7
405
Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Gennany
Inftuence of bile acids on stimulated lipid peroxidation and hydrogen peroxide production in rat liver microsomes A. BARTH and M. BERNST With 3 figures and 2 tables Recei ved: December 2, 1991; Accepted: December 28, 1991 Address for correspondence: Dr. sc. med. A. BARTH, Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, LobderstraBe 1, lena, D-O-6900, Gennany. Key words: Lipid peroxidation; Hydrogen peroxide Production; Bile acids; Liver microsomes; Microsomes, liver; Antioxidants; Peroxidation, lipid.
Summary Bile acids were found to be effective antioxidants in bile and intestine. The influence of different bile acids on the NADPH-Fe + +-stimulated lipid peroxidation (LPO) and cytochrome P-4S0 dependent hydrogen peroxide production (H 2 0 2) in rat liver microsomes was investigated in vitro. LPO was detennined as production of thiobarbituric acid reactants (TBAR). Different tri-, di- and monohydroxylated bile acids and cholesterol were given to the incubation mixture in concentrations ranging from 10- 5 to 1O- 3 M. Sodium salts of cholic, tauroglycocholic and deoxycholic acids as well as cheno-deoxycholic, ursodeoxycholic, lithocholic acids and cholesterol did not alter the microsomal production of TBAR. H20 2 fonnation was significantly decreased by sodium deoxycholate whereas cholesterol increased H2 0 2 production up to 4 times. These results show that bile acids were not able to protect microsomal membrane lipids against peroxidative damage. Cholesterol mediated H20 2 fonnation as a source of hydroxyl radicals had no toxic effect concerning LPO, TBAR were not enhanced significantly.
on lipid concentration, bile acids can also act as enhancers of lipid peroxidation (DE LANGE and GLAZER 1990). We investigated the in vitro influence of sodium cholate, sodium tauroglycocholate, sodium deoxycholate, chenodeoxycholic, ursodeoxycholic and lithocholic acids and cholesterol in increasing concentrations on NADPHFe++ -stimulated lipid peroxidation (LPO) and hydrogen peroxide production (H 20 2 ) in rat liver microsomes. The cytochrome P-450 system in the liver is responsible for H2 0 2 and radical production. The toxic effects of H 2 0 2 may be mediated by the hydroxyl radical generated in vivo by a Fenton reaction (JONAS et al. 1989, MINOTTI 1990). It was the aim of these investigations to find out possible protective or enhancing effects of bile acids on iron stimulated LPO and oxidase function of cytochrome P-450.
Material and methods Introduction Oxidation and radical reactions in living cells and tissues can be generated by the reduction of oxygen to superoxide radical, hydrogen peroxide or hydroxyl radical. Different antioxidant mechanisms minimize free radical damage in the living organism. These mechanisms include enzymes and small molecules such as glutathione, vitamins C and E, and bilirubin, which react directly with free radicals (FEHER et al. 1987). DE LANGE and GLAZER (1990) reported that bile acids at physiologic concentration are also effective antioxidants by scavenging peroxyl radicals via direct oxidation to keto derivatives. But depending
6O-day-old male Wistar rats were used. The animals were kept under conventional conditions with pellet diet (Versuchstierproduktion Schonwalde, GDR, prescription Rehbriicke). The rats were decapitated in ether anesthesia, the livers were removed and homogenized in 0.1 M sodium phosphate buffer pH 7.4 (l g liver + 2 ml buffer). The homogenate was centrifuged at 9000 g for 20 min at 0 DC. The 9000 g supernatant was mixed with 25 mM magnesium chloride solution (1 ml supernatant + 2.5 ml MgCh) and centrifuged at 9000 g for 20 min at 0 DC. The sediment was washed and rehomogenized in sodium phosphate buffer at 4 DC (SILMAN et al. 1965). The protein content of the microsomal suspension was determined by the Biuret Exp Toxic Pathol 44 (1992) 7
399
METHOD ACCORDING TO KLINGER and MULLER (1974). The microsomal suspension was kept at - 20°C for not more than one week. The enzymatic LPO was stimulated in the following system: 0.1 M sodium phosphate buffer (pH 7.4), 0.5 mM Fe++ as FeS04 X 7H2 0, about 0.25 mg protienlml, 0.5 mM NADPH. Microsomes were incubated at 37°C for 10 min using a metabolic shaker (Vibrotherm). The reaction was stopped with trichloroacetic acid (30%). LPO was measured using thiobarbituric acid assay (BUEGE and AUST 1978). An extinction coefficient of 1.56 X 105 1 mol-1cm- 1 was employed in the calculation of the resulting concentration of malondialdehyde equivalents. Controls included incubation mixtures to which trichloroacetic acid was added before NADPH. The thiobarbituric acid assay is the method most commonly used to study lipid peroxidation. In the NADPH system, melondialdehyde represents the major thiobarbituric acid reactive substance (TBAR) in the oxidized microsomal lipid as seen by HPLC and thiobarbituric acid methods (ESTERBAUER and ZOLLNER 1989, TOMITA et al. 1990). H2 0 2 formation was measured with the ferroammonium sulfate potassium thiocyanate method in the presence of NaN 3 to block the peroxidative activity (HILDEBRANDT et al. 1978). The incubation mixture of 1.5 ml contained 1 mg protein, 50 mM tris-chloride buffer pH 7.4, NADPH O. 75j.tmol. The microsomes were incubated 20 min at 30°C under continuous shaking. Controls included incubation mixtures, to which trichloroacetic acid was added before microsomal suspension. Rats were pretreated with 1 mllkg b.m. carbon tetrachloride in sunflower oil (lOmllkg b.m.) intraperitoneally. Twelve hours later the rats were anesthetized with ether and decapitated. Liver microsomes were prepared, and LPO and H20 2 were measured as described above. Bile acids were added to the incubation medium reading final concentrations of 10- 5 to 10- 3 M. Sodium salts of cholic, tauroglycocholic and deoxycholic acids were dissolved in sodium phosphate buffer 0.1 M, pH 7.4. Chenodeoxycholic, ursodeoxycholic, lithocholic acids and cholesterol in ethanol were dried by room air overnight in individual assay tubes prior to the addition of other assay components.
Statistics Statistical evaluation of the data was performed by the Student's t-test. The level of significance was chosen as p :::; 0.05.
Results The effects of a single 1 mllkg b.m. dose of CCl 4 given to the rats intraperitoneally 12 hours prior to examination on LPO and H2 0 2 formation in rat liver microsomes are demonstrated in tab. 1. TBAR were significantly enhanced, whereas H 20 2 formation was significantly diminished in microsomes obtained from CC1 4 treated rats. 400
Exp Toxic Pathol 44 (1992) 7
The in vitro influence of bile acids on iron stimulated LPO in liver microsomes of untreated rats is shown in tab. 2. Neither trihydroxylated nor di- or monohydroxylated bile acids changed the production rate of TBAR. Table 1. Lipid peroxidation (TBAR) and H2 0 2 formation in
liver micro somes of adult male Wistar rats 12 hours after carbon tetrachloride treatment (CCI4, 1 mglkg b.m. intraperitoneally). Controls (C) recieved sunflower oil (10 ml! kg b.m. intraperitoneally). Arithmetic means ± S.E.M., n = 5, * significant differences to controls, p :::; 0.05, STUDENT'S t-test. TBAR [nmollmg X min]
H 20 2
3.02 ± 0.26 5.32 ± 0.30*
1.21 ± 0.17 0.53 ± 0.07*
[nmollmg X min]
Cholesterol had no influence on microsomal LPO, either (tab. 2). Figures 1- 3 demonstrate the in vitro effect of bile acids and cholesterol on hepatic microsomal H2 0 2 production. The sodium salt of free or conjugated trihydroxylated cholic acid did not alter the microsomal H20 2 production. Among the dihydroxylated bile acids only sodium deoxycholate reduced the H 20 2 production in a dose dependent manner. Whereas monohydroxylated lithochoic acid showed only a tendency to increase cholesterol significantly enhanced the microsomal H 2 0 2 formation in all three concentrations (fig. 3).
Discussion The use of many drugs is limited by their toxic side effects, very often mediated by lipid peroxidation or free radical production. Efforts were made to prevent the resulting toxic effects by radical scavengers such as tocopherol, glutathione, superoxide dismutase or superoxide dismutase - like constitutents of Ginkgo biloba leaves (HILL and BURK 1984; CASINI et al. 1985; BARTH et al. 1991). The aim of the presented study was to find out possible radical scavenging properties of bile acids. Bile acids were investigated in rat liver microsomes incuated with iron as effective catalyst of the production of potent oxidizing species (BACON and BRITTON 1989; MINOTTI and AUST 1989). Carbon tetrachloride (CCI4) was used as a well-known model substance producing lipid peroxidation and liver damage (RECKNAGEL et al. 1989, COMPORTI 1989; WOLFGANG et al. 1990). As shown by the results, CCl4 enh~ced susceptibility to in vitro lipid peroxidation 12 hours after in vivo treatment of the rats (tab. 1). The inhibition of the microsomal H2 0 2 production may be related to the toxic effect of CCl4 on cytochrome P-450 (COMPORTI 1989). The microsomal inducer pregnenolone16 cx-carbonitril enhanced, whereas the microsomal
Table 2. Lipid peroxidation (TBAR) in liver microsomes of adult male Wi star rats 10 minutes incubated with bile acids and cholesterol. Arithmetic means ± S.E.M., n = 4-6. Control
TBAR [nmollmg x min] bile acids [mollI]
10- 5
10- 4
10- 3
2.83 ± 0.38 3.00 ± 0 .38
Na-cholate Na-tauroglycocholate
2.39 ±0.50 2.34 ± 0.69
2.99 ± 0.45 2.82 ± 0.55
3.00 ± 0.45 1.89 ± 0.34
2.97 ± 0.36 2.94 ± 0 .35 2.72 ± 0.31
Na-desoxycholate ursodeoxycholic acid chenodeoxycholic acid
2.87 ± 0 .76 2.58 ± 0.55 2.07 ± 0.43
3.46 ± 0.63 3.11 ± 0.46 2.90 ± 0.44
2.76 ± 0.50 2.86 ± 0 .59 2.85 ± 0.40
2.88 ± 0.32
lithocholic acid
2.87 ± 0.25
3.33 ± 0.40
3.07 ± 0.39
2.73 ± 0.31
cholesterol (molll)
2.15 ± 0 .50
3.02 ± 0.51
3.71 ± 0.32
2 r-~~~~----------------------
__~
1.G ,
;
10 N.~(nMIVI'
2,G r-~~~------------------------
______~
2
Fig. 1. Hydrogen peroxide (H 20 2) production in liver microsomes of adult male Wi star rats 20 minutes incubated with different concentrations of trihydroxylated bile acids. Data expressed as arithmetic means ± S.E.M., n = 4-5.
Exp Toxic Pathol 44 (1992) 7
401
2~rl"-~~~~~------------------------------~ 2
2~~r----~~~~----------------------------~ 2
1
0,5
O~~~~~--~~~~L-~~
control
10-
10
10
chenodeo,x,ahollo IIOId (1ftOIII)
'Arl"-~~~~~------------------------------~ ',8
,,,,
',2
1
OA 0,_ 0,4
0.2 O~~~~~--~~~~--~~~~
10- 4
control
__-L~~~LJ
Na-deoxwoholate (moll!)
402
Exp Toxic Pathol 44 (1992) 7
10
Fig. 2. Hydrogen peroxide (H 2 0 2 ) production in liver microsomes of adult male Wi star rats 20 minutes incubated with different concentrations of dihydroxylated bile acids. Data expressed as arithmetic means ± S.E.M., n = 4- 5, asterisk indicates significant difference to control, p :5 0.05, STUDENT'S t-test.
2~~~~----------------~ 1,5
0,5
10
Ithoohollo MId (mot/I)
.lrn_.aH ___~~I.~PG~.._~_I______________________________--,
•
3 2
Fig. 3. Hydrogen peroxide (H 20 2) proliuction in liver microsomes of adult male Wistar rats 20 minutes incubated with different concentrations of monohydroxylated bile acid and cholesterol. Data expressed as arithmetic means ± S.E.M., n = 4-5, asterisks indicate significant differences to controls, p ::; 0.05, STUDENT'S t-test.
inhibitor metyrapone diminished the cytochrome P-450 dependent H2 0 2 formation (BAST et al. 1989), Phenobarbital and [3-naphthoflavone were without effect (HAUFE and KLINGER 1990). Bile acids have the capacity to induce both choleresis and cholestasis, depending on concentrations and hydrophobic or hydrophilic properties of the individual bile acids. Monohydroxylated and dihydroxylated bile acids were found to be more toxic than trihydroxylated ones (SCHOLMERICH et al. 1984). Transported through the hepatocytes bile acids interact with the endoplasmic reticulum and the Golgi apparatus, especially at higher bile acid loads and higher intracellular concentrations (ERUNGER 1990). According to DE LANGE and GLAZER (1990) bile acids can function as effective antioxidants in bile and the intestine. Determining the rate of linoleate oxidation by peroxyl radicals, glycocholate was
1·: ;.
able to promote lipid peroxidation if bile acidllinoleate concentration ratio was changed to smaller values (DE LANG and GLAZER 1990). The data presented here show no influence of bile acids in vitro on microsomal lipid peroxidation (tab. 2). Neither conjugated nor free, neither hydrophobic nor hydrophilic bile acids altered the iron stimulated production of TBAR. The highly cholestatic lithocholic acid (TAKIKAW A et al. 1991) as well as cholesterol as endogenous and exogenous source for bile acid synthesis (Ay AKI et al. 1981) did not influence the microsomal lipid peroxidation (tab. 2). DE LANGE and GLAZER (1990) used bile acid concentrations ranging from 10- 1 to 1O- 3 M, normally found in the gallbladder and in the duodenum. According to FISHER et al. (1976) only 2% ofthe bile acid pool is situated in the liver in adult Wistar rats, accordingly 2 X 1O- 4 M. The bile Exp Toxic Pathol 44 (1992) 7
403
acid concentrations used in our incubation mixture never exceeded 10- 3 M. Bile acids were also unable to influence the microsomal HzO z formation. One exception is sodium deoxycholate, which inhibited the HzO z production (fig. 2). The detergent potency of deoxycholate may be responsible for dose dependent damage of cytochrome P-450 (SCHOLMERICH et al. 1984). Surprinsingly, other dihydroxylated bile acids tested had no effect on HzO z formation. Different effect of dihydroxylated bile acids are known according to the position of the OH-groups. For example the cholestatic potency and the detergent power of deoxycholate is higher than that of chenodeoxycholate whereas ursodeoxycholate can prevent bile acid induced cholestasis and hepatotoxicity (DREW and PRIESTLY 1979; KITANI and KANAI 1982; ATTILI et al. 1986; GALLE et al. 1990). HzO z production is associated with the oxidation of NADPH in liver microsomes. Some substrates of cytochrome P450 such as hexobarbital stimulate the rate of HzO z formation concomitantly with the oxidative biotransformation of this substrate (KLINGER et al. 1986). 7-Ethoxycoumarin acts as an uncoupler for the cytochrome P-450 reaction system, thereby enhancing the HzO z production (ESTABROOK et al. 1979). The presence of cholesterol in our incubation mixture markedly enhanced the formation of HzO z (fig. 3). The 7ex-hydroxylation of cholesterol is the major rate limiting step in the overall conversion of cholesterol into bile acids (SHEFER et al. 1970; HEUMAN et al. 1988). The cholesterol7 ex-hydroxylation as well as some other hydroxylations on the steroid nucleus appear to be cytochrome P-450-dependent (BJORKHEM et al. 1975; ZIMNIAK et al. 1991). Although the hydroxylations in the bile acid metabolism differ in some respects from those involved in the drug metabolism, cholesterol may act as an uncoupler for the cyclic function of cytochrome P-450, thereby increasing the HzO z formation. Surprisingly, the up to 4 times higher HzO z formation in the microsomes did not stimulate lipid peroxidation, the TBAR were not enhanced significantly in incubation mixtures without sodium acid (tab. 2). With unblocked peroxidative acitivity of cytochrome P-450 cholesterol mediated HzO z may be decomposed in H 20 and hydroxylated substrate (HILDEBRANDT et al. 1978). Another possibility is that cholesterol itself acts as hydroxyl radical scavenger as seen in studies with ultrasoundinduced lipid peroxidation and in liposomes (JAN Aet al. 1990, HERNANDEZ-CASELLES et al. 1990). Also LETKO et al. (1990) did not find a correlation between cell lysis and elevation of TBAR afterincubation of pancreatic acinar cells with H 20 2 . On the other hand, ISLAM et al. (1991) tested steroids, structurally related to cholesterol, and revealed mutagenic activity which appears to involve the formation of H Z0 2 • According to BHADRA et al. (1991) cholesterol-alpha-epoxide is a major product of cholesterol peroxidation in the presence of endothelial cells, playing a key role in atherogenesis. In conclusion bile acids were not able to protect microsomal membrane lipids against peroxidative damage. Cholesterol enhanced the cytochrome P-450-dependent H 2 0 2 formation without affecting the microsomal lipid peroxidation significantly. 404
Exp Toxic Pathol 44 (1992) 7
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