Hispidulin protection against hepatotoxicity induced by bromobenzene in mice

Life Sciences, Vol. 55, No. 8, pp. PL 145.-150,1994 ~ t e 1994 Eigevier Science Lid Printed in the USA. All rights reserved

Pergamon

oo2~3~05/94$6.00 + .0o 0024-3205(94)00171-5 PHARMACOLOGY LETTERS Accelerated Communication

HISPIDULIN PROTECTION AGAINST HEPATOTOXICITY INDUCED BY BROMOBENZENE IN MICE M. L. Ferr~ndiz I , G. Bustos I , M. Pay~ 1 , R. Gunasegaran 2 and M. J. Alcaraz 1 1De[)artment of Pharmacology, University of Valencia, Spain. "Centre for Post Graduate Studies, Pondicherry, India.

(Submitted May 4,1994; accepted May 23, 1994; received in final form June 6, 1994) Abstract. The effects of the natural flavonoid hispidulin (6-methoxy-5,7,4'trihydroxyflavone) on bromobenzene-induced hepatotoxicity in mice were investigated. We found a correlation between liver injury and hepatic lipid peroxidation besides a strong liver glutathione depletion due to the toxicant. Hispidulin at doses between 50 and 150 mg/kg i.p. compared favourably with the reference compound N-acetyI-L-cysteine for inhibition of liver injury and lipid peroxidation. The flavonoid at the highest dose tested was also able to counteract reduced glutathione depletion induced by bromobenzene in starved mice. These hepatoprotective effects can be related to the antioxidant properties of hispidulin.

Key Words: hispidulln, flavonoid, bromobenzene, hepatotoxlcity, fipid peroxidation, glutathlone

Introduction Some flavonoids and other phenolic compounds such as phenolic acids are able to reduce xenobiotic-induced hepatotoxicity in animals (1-5). These compounds also exert antioxidant (1,6-9) or in vitro free radical scavenging effects (10,11 ), which can be provide a basis for some of their pharmacological properties. Those natural products could control reactions mediated by free radicals implicated in several patophysiological processes including toxicity induced by xenobioticso The inhibitory activity of flavonoids on free radical reactions can be related to their hepatoprotective effects since exogenous antioxidants may counteract the damaging effects of oxidative stress, cooperating with natural systems like reduced glutathione, ¢-tocopherol or protecting enzymes. Previous work from this laboratory has demonstrated that a number of flavonoids protect isolated rat hepatocytes against carbon tetrachloride-induced toxicity (12) or inhibit bromobenzene-induced hepatotoxicity in mice (5). In the present study we have selected the natural flavonoid hispidulin (6-methoxy-5,7,4'-trihydroxyflavone), isolated from the Indian plant, Helichrysum bracteatum var. bracteatum (Asteraceae), after the encouraging results obtained in preliminary experiments to assess its in vivo hepatoprotective activity.

Corresponding Author: Prof. M. J. Alcaraz. Department of Pharmacology, Faculty of Pharmacy. Avda. AndrOs Vicent Estell~s s/n, 46100 Burjassot, Valencia, Spain.

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Materials and methods

Reagents. Hispidulin was isolated from the flowers of Helichrysum bracteatum var. bracteaturn (Asteraceae) as previously described (13). 2-Thiobarbituric acid, 1,1,3,3tetramethoxypropane, reduced glutathione, 5-sulphosalicylic acid, NADPH, 5,5'-dithiobis(2nitrobenzoic) acid and N-acetyI-L-cysteine were purchased from Sigma Chemicals (St Louis, USA). All other chemicals were of analytical grade.

Bromobenzene intoxication. Male Swiss albino mice weighing 30-33 g were starved for 24 h with free access to water before receiving bromobenzene (9 mmol/kg p.o.) dissolved in olive oil (14). Test compounds were administered to mice at the doses of 50, 100 and 150 mg/kg i.p., suspended in vehicle (ethanol/Tween 80/H20, 5:5:90) 1 h before intoxication with bromobenzene. Two reference groups were used: non-intoxicated animals (V) which received the vehicles only and bromobenzene control (BB) which was intoxicated with bromobenzene and received the test compounds vehicle. 17 H after bromobenzene administration blood was taken by retro-orbital puncture and all animals were killed by cervical dislocation. The liver was excised, rinsed in ice-cold saline, weighed and homogenized in 5 % 5-sulphosalicylic acid and homogenates were centrifuged for 10 min at 10,000 g. In a set of experiments, animals were not starved previously to intoxication, to assess the influence of starvation on glutathione depletion. Liver necrosis was assessed by measuring the levels of serum alanine aminotransferase (SALT) activity, following the oxidation of NADH by the loss of absorbance at 340 nm using a diagnostic kit (ITC Diagnostics, Izasa S.A., Barcelona, Spain). Lipid peroxidation was estimated in tissue homogenates by measuring the hepatic content of thiobarbituric acid (TBA) reacting substances expressed as malondialdehyde (MDA) equivalents. Results were compared to a standard curve of MDA produced by acid hydrolysis of 1,1,3,3,-tetramethoxypropane (15). Protein was evaluated by the method of Lowry et a1.(16). Hepatic reduced glutathione levels (GSH) were determined in liver supernatants following the appearance of 5-thio-2nitrobenzoic acid at 412 nm (17). Results were expressed in nanomoles of reduced glutathione equivalents per mg of protein.

In vitro microsomal lipid peroxidation. Lipid peroxidation was induced in rat liver microsomes by Fe2 +/ascorbate, CCI4/NADPH or Fe3 +-ADP/NADPH as reported previously (18) and estimated by TBA reaction (15). Data analysis. All data are presented as means¢S.E.M. Statistical analysIs was performed using ANOVA followed by the Dunnett's t test for multiple comparisons. In correlation studies we calculated the Pearson correlation coefficient. Results Bromobenzene administration to mice did not affect the liver weight (1.6¢0.1 g, mean¢S.E.M., n = 2 2 in group BB versus 1.4+0.1 g, mean¢S.E.M., n = 1 0 in group V). In a similar way, drug treatment had no effect on this parameter (data not shown). As shown in figure 1 mice treated with bromobenzene after 24h starvation presented a high level of liver injury, assessed as SALT activity, which showed approximately a nine-fold increase in comparison with non-intoxicated controls (group V). Bromobenzene administration also stimulated lipid peroxidation estimated as MDA equivalents. A severe depletion in hepatic GSH levels (82 %) was observed in the intoxicated control (Figure 2). Thus, the development of hepatotoxicity was significant 17 h after bromobenzene intoxication in 24h-starved mice. Correlation analysis between those parameters revealed a significant relationship (r= 0.650, P<0.0001) between SALT levels and MDA equivalents, as reported by some authors (14) but no correlation could be demonstrated either between SALT and GSH, or MDA and GSH.

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Hispidulin Hepatoprotection

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FIG. 1 Effect of hispidulin and N-acetyI-L-cysteine on liver injury and hepatic lipid peroxidation induced by bromobenzene.

Results represent the mean+S.E.M, for 7-21 animals. H1 (hispidulin 50 rng/kg), H2 (hispidulin 100 mg/kg), H3 (hispidulin 150 mg/kg) N1 (N-acetyI-L-cysteine 50 mg/kg), N2 (N-acetyI-L-cysteine 100 mg/kg), N3 (N-acetyI-L-cysteine 150 mg/kg). ** P<0.01 with respect to the bromobenzene control group (BB). Open bars, SALT (U/I); closed bars, pmol MDA/mg protein.

Pretreatment with hispidulin significantly inhibited bromobenzene toxicity, with reduced SALT levels comparable to those caused by the reference compound, N-acetyI-Lcysteine at the same doses. Both compounds also significantly inhibited liver lipid peroxidation and interestingly hispidulin at the highest dose administered (150 mg/kg) suppressed MDA generation almost totally. The response of liver GSH levels to drug treatment was more difficult to establish, as prior observed in this experimental model (5). Only hispidulin at the highest dose tested (150 mg/kg) was able to significantly increase (112 %) the depleted levels of GSH, while N-acetyI-L-cysteine at the same dose did not affect this parameter. Other experiments were performed in a similar way but using mice maintained with free access to water and their normal diet previously to the administration of test compounds and toxicant. Comparison of the results presented in table I with those in figure 1 indicates that 24 h starvation had no influence on bromobenzene-induced hepatotoxicity or lipid peroxidation (P>0.05 when comparing SALT or MDA levels in BB groups from 24h-starved mice and non-starved mice).

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FIG. 2 Effect of hispidulin and N-acetyI-L-cysteine on liver glutathione depletion induced by bromobenzene, Results represent the mean+S.E.M, for 7-21 animals, H1 (hispidulin 50 mg/kg), H2 (hispidulin 100 mg/kg), H3 (hispidulin 150 mg/kg), N1 (N-acetyI-L-cysteine 50 mg/kg), N2 (N-acetyI-L-cysteine 100 mg/kg), N3 (N-acetyI-L-cysteine 150 mg/kg). • P
Nevertheless, as previously stated (19) starvation is an important factor in hepatic GSH~ depletion, since in non-starved animals GSH levels were higher than in the corresponding groups of 24h-starved mice. These last results also indicate that in the group V, GSH values were reduced by about 40% after 24 h starvation (Figure 2 and table I). Hispidulin was administered at the highest dose used in the first experiments (150 mg/kg) to assess its influence on non-starved mice. In these animals the fiavonoid reduced the elevation of SALT activity by 52 % and of MDA levels by 62 %, respectively, which were slightly below those values shown for starved mice and it was unable to modify GSH levels. Furthermore, hispidulin did not affect the glutathione status when administered to non-intoxicated animals nor it exerted any toxic effect. On the other hand, hispidulin was able to inhibit in vitro microsomal lipid peroxidation with inhibitory concentration 50 % values and confidence limits at P
m

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TaMe I Effect of hispidulin on SALT, lipid peroxidation and glutathione levels in non-starved mice Treatment Vehicle (V) Bromobenzene (BB) V + Hispidulin BB+ Hispidulin

SALT

Lipid peroxidation

GSH

(U/I)

(omol M D A I m a orotein)

(nmollma orotein)

65.3+7.3** 441.8 + 84.9 73.1 + 3 , 8 213.1 + 4 8 . 9 *

68.6¢2.8** 271.8 + 60.4 73.7+4.5** 104.1 :t:6.1 * *

42.0+3.1 **+ + 8.4 + 3.0 + 38.5+2,9 5.6¢0.6

Hispidulin was administered at 150 mg/kg i.p. Results represent the mean + S.E.M. for 6-9 animals. + P < 0 . 0 5 , + + P < 0 . 0 1 with respect to the corresponding groups in starved mice (results in Fig. 2 ) . . P < 0 . 0 5 , * * P<0.01 with respect to the bromobenzene control group (BB). Discussion Our results have demonstrated the hepatoprotective activity of hispidulin in bromobenzene-induced toxicity in mice. To avoid interferences in the absorption from the gut, different administration routes were used for toxicant and test compounds. Nevertheless previous preliminary results indicated that hispidulin was also able to counteract liver toxicity when administered orally at 150 mg/kg 4 h after bromobenzene intoxication (20). Bromobenzene induces severe glutathione depletion as a result of generation of electrophilic intermediates which react with cellular nucleophiles. When glutathione levels are decreased to a certain extent, lipid peroxidation appears resulting in a loss of protein thiols with alteration of calcium homeostasis and cell death (21). It has been suggested that during lipid peroxidation membrane protein thiols can be attacked by free radicals and reactive products of lipid peroxidation resulting in loss of protein thiols with alterations in membranes and cellular injury (22). In addition, covalent binding of the toxicant may be related to the cell necrosis present in later times (23). In bromobenzene-induced toxicity there is a relationship between liver necrosis and lipid peroxidation, a process which could play an important role in the pathogenesis of liver necrosis produced by this xenobiotic (21,24). It is likely that the loss of defence systems such as glutathione against oxidative stress leads to cellular damage. In the present experiments we have found a correlation between lipid peroxidation and liver injury, besides a strong depletion of glutathione levels induced by bromobenzene. Hispidulin and the reference compound, N-acetyI-L-cysteine at the same doses effectively protected against bromobenzene -induced liver toxicity with reduction in SALT activity and lipid peroxidation. N-acetyI-L-cysteine is a sulfhydryl donor which act as an antidote against the hepatotoxicity of acetaminophen and is able to increase glutathione synthesis in rats (25). Nevertheless this last effect was observed at a dose 8-24-fold higher than those we have used in our study and it is interesting to note that at our highest dose, 150 mg/kg only hispidulin reversed reduced glutathione depletion, thus acting on glutathione redox status during bromobenzene intoxication although it did not increase glutathione content in the liver of non-starved mice. It is likely that hispidulin does not directly increase glutathione, as it seems to be the case for silymarin (26) but in drastic situations of depletion it may protect this peptide which in its reduced form acts as an important antioxidant system (27). These observations are in line with the results we have obtained previously for another flavonoid, 7,8-dihydroxyflavone (5). Antioxidant agents can decrease bromobenzene hepatotoxicity in proportion to the reduction of lipid peroxidation (23) and it has been reported that in animals supplemented

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with ¢-tocopherol bromobenzene has a limited toxicity in spite of the presence of hepatic glutathione depletion (24). In fact, hispidulin is able to inhibit in vitro lipid peroxidation, suggesting that its antioxidant activity may participate in its hepatoprotective effects through the control of oxidative stress. References 1. C.G. FRAGA, V.S. MARTINO, G.E. FERRARO, J.D. COUSSIO AND A. BOVERIS, Biochem. Pharmacol. 3 6 7 1 7 - 7 2 0 (1987). 2. R. CAMPOS, A. GARRIDO, R. GUERRA AND A. VALENZUELA, Planta Med. 5~ 4174 1 9 (1989). 3. P. LETT6RON, G. LABBE, C. DEGOTT, A. BERSON, B. FROMENTY, M. DELAFORGE, D. LARREY AND D. PESSAYRE, Biochem. Pharmacol. 39 2027-2034 (1990). 4. R. CARINI, A. COMOGLIO, E. ALBANO AND G. POLl, Biochem. Pharmacol. 4 3 2 1 1 1 2115 (1992). 5. M. PAYS, M.L. FERR~NDIZ, M.J. SANZ AND M.J. ALCARAZ, Xenobiotica 2:~ 327-333 (1993). 6. A. VALENZUELA, R. GUERRA AND L.A. VlDELA, Planta Med. 52 438-440 (1986). 7. M. FAUR6, E. LlSSl, R. TORRES AND L.A. VlDELA, Phytochemistry 2~1 3773-3775 (1990). 8. A. MORA, M. PAYS, J.L. RfOS AND M.J. ALCARAZ, Biochem. Pharmacol. 40 793-797 (1990). 9. M.R. CHOLBI, M. PAY~ AND M.J. ALCARAZ, Experientia 4"7195-199 (1991). 10. W. BORS AND M. SARAN, Free Rad. Res. Comms. 2 2 8 9 - 2 9 4 (1987). 11. A.K. RATTY, J. SUNAMOTO AND N.P. DAS, Biochem. Pharmacol. :~7 989-995 (1988). 12. M.T. Afi6N, A. UBEDA AND M.J. ALCARAZ, Z. Naturforsch. 47~ 275-279 (1992). 13. R. GUNASEGARAN, A. UBEDA, M.J. ALCARAZ, R. JAYAPRAKASAM AND A.G. RAMACHANDRAN NAIR, Pharmazie 4~ 230-231 (1993). 14. M. COMPORTI, E. MAELLARO, B. DEL BELLO AND A.F. CASlNI, Xenobiotica 2__1 1067-1076 (1991). 15. H. OHKAWA, N. OHISHI AND K. YAGI, Anal. Biochem. 95 351-358 (1979). 16. O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR AND R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 17. J. SEDLAK AND R.H. LINDSAY, Anal. Biochem. ~25 192-205 (1968). 18. A. UBEDA, C. MONTESlNOS, M. PAYS, M.C. TERENCIO AND M.J. ALCARAZ, Free Rad. Res. Comms. 1 8 1 6 7 - 1 7 5 (1993). 19. M. COMPORTI, Chem. Biol. Interact. 7_2_21-56(1989). 20. M.C. MONTESlNOS, M.L. FERRc~NDIZ, M.J. SANZ, R. GUNASEGARAN, M. PAY~ AND M.J. ALCARAZ, Proceedings of the International Conference on critical Aspects of Free Radicals in Chemistry, Biochemistry and Medicine 278(1993).(Abstract) 21. A.F. CASlNI, E. MAELLARO, A. POMPELLA, M. FERRALI AND M. COMPORTI, Biochem. Pharmacol. 36 3689-3695 (1987). 22. A. POMPELLA, A. ROMANI, A. BENEDETTI AND M. COMPORTI, Biochem. Pharmacol. 411255-1259(1991). 23. A. CASlNI, M. GIORLI, R.J. HYLAND, A. SERRONI, D. GILFOR AND J.L. FARBER, J. Biol. Chem. 257 6721-6728 (1982). 24. E. MAELLARO, A.F. CASlNI, B. DEL BELLO AND M. COMPORTI, Biochem. Pharmacol. $~! 1513-1521 (1990). 25. H. LAUTERBURG, G.B. CORCORAN AND J.R. MITCHELL, J. Clin. Invest. 71 980-991 (1983). 26. A. VALENZUELA, M. ASPILLAGA, S. VIAL AND R. GUERRA, Planta Med. 55 420-422 (1989). 27. R. MUNDAY AND C.C. WlNTERBOURN, Biochem. Pharmacol. 38 4349-4352 (1989).