Modifying effects of Terminalia catappa on azoxymethane-induced colon carcinogenesis in male F344 rats.

The modifying effects of dietary administration of an herb, Terminalia catappa (TC), were investigated on rat colon carcinogenesis induced by a carcinogen azoxymethane (AOM). The number of aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCACs) in the colon, and proliferating cell nuclear antigen (PCNA) labelling index in the colonic epithelium were examined in a total of 36 male F344 rats. All animals were randomly divided into five experimental groups (4-10 rats in each group). At 6 weeks of age, rats in groups 1, 2 and 3 were given s.c. injections of AOM once a week for 2 weeks at a concentration of 20 mg/kg body weight. One week before the first injection of AOM, rats in groups 2 and 3 were fed a diet containing 0.02 and 0.1% TC, respectively, throughout the experiment. Rats in group 4 were fed a diet containing 0.1% TC. Rats in group 5 were served as untreated controls. All animals were sacrificed at the experimental week 5 after the start of the experiment. Oral administration of TC at both doses significantly decreased the numbers of both ACF/colon/rat (P<0.05 for 0.02% TC, P<0.005 for 0.1% TC) and BCAC/cm/rat (P<0.05 for both 0.02 and 0.1% TC), when compared with the control group (group 1). Colonic PCNA labelling index in groups 2 and 3 was also significantly lower than that in group 1 (P<0.001 for 0.02% TC, P<0.005 for 0.1% TC). These results suggest that TC has a potent short-term chemopreventive effect on biomarkers of colon carcinogenesis and this effect may be associated with the inhibition of the development of ACF and BCACs.

Dimerumic acid as an antioxidant of the mold, Monascus anka.

We previously reported that the mold Monascus anka, traditionally used for fermentation of food, showed antioxidant and hepatoprotective actions against chemically induced liver injuries. In the present study, the antioxidant component of M. anka was isolated and identified. The antioxidant was elucidated to be dimerumic acid. DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was significantly scavenged by the antioxidant whereas hydroxyl radical and superoxide anion were moderately scavenged. When the antioxidant (12 mg/kg) was given to mice prior to carbon tetrachloride (CCl(4), 20 microl/kg, ip) treatment, the CCl(4)-induced liver toxicity in mice seen in an elevation of serum aspartate aminotransferase and alanine aminotransferase activities was depressed, suggesting the hepatoprotective action of the antioxidant. The liver microsomal glutathione S-transferase activity, which is known to be activated by oxidative stress or active metabolites, was increased by CCl(4) treatment and the increase was also depressed by pretreatment with the mold antioxidant. Thus these data confirmed that the dimerumic acid isolated from M. anka is the potential antioxidant and protective against CCl(4)-induced liver injury.

Nitroglycerin relaxes coronary artery of the pig with no change in glutathione content or glutathione S-transferase activity.

1. The role of glutathione content and glutathione S-transferase activity in vascular relaxant responses to nitroglycerin was evaluated in potassium (30 mM)-contracted coronary artery strips of the pig by measuring changes in tension, glutathione content and glutathione S-transferase activity. 2. Prior exposure of coronary artery strips to nitroglycerin (10(-5)M or 10(-4)M for 20 min) resulted in tachyphylaxis to subsequent relaxation to nitroglycerin (10(-8)-10(-5)M). 3. The glutathione content and glutathione S-transferase activity of the arterial strips rendered tachyphylactic by prior exposure to nitroglycerin (10(-5)M for 20 min or 10(-3)M for 120 min) were not significantly different from those of control strips. 4. Treatment with diethyl maleate (10(-4)M or 10(-3)M for 60 min) markedly depleted arterial glutathione content in a concentration-dependent manner with no change in glutathione S-transferase activity. 5. The relaxant response of coronary artery strips to nitroglycerin (10(-8)-10(-5)M) was completely unaffected following treatment with diethyl maleate (10(-4)M or 10(-3)M for 60 min). 6. The results suggest that vascular glutathione content does not play an important role in vascular relaxation or tolerance development to nitroglycerin, at least in pig isolated coronary artery.

Oxidative stress-induced activation of microsomal glutathione S-transferase in isolated rat liver.

The activation of microsomal glutathione (GSH) S-transferase in isolated rat liver by oxidative stress was investigated using both ischemia/reperfusion and perfusion with hydrogen peroxide. When the isolated liver was reperfused for 30 min and 60 min after 90 min ischemia, microsomal GSH S-transferase activity, but not cytosolic transferase activity, was increased 1.2-fold and 1.3-fold, respectively. In addition, microsomal GSH peroxidase activity was also significantly increased after 60 min reperfusion following ischemia. The increase in microsomal GSH S-transferase activity by ischemia/reperfusion was reversed by dithiothreitol. When N-ethylmaleimide, which activates microsomal GSH S-transferase by covalent binding to the cysteine residue of the enzyme, was incubated with microsomes, transferase activity was increased to 526% in control microsomes and to 399% in liver that underwent ischemia/reperfusion liver. These data indicate that microsomal GSH S-transferase is activated by ischemia/reperfusion of the liver by means of disulfide bond formation. When rats were pretreated with a catalase inhibitor 3-amino-1,2,4-triazole for 6 weeks, microsomal GSH S-transferase activity was increased 1.4-fold by ischemia/reperfusion, corresponding to a 1.8-fold increase as compared to the non-perfused liver of untreated rats. Perfusion of the isolated liver with hydrogen peroxide (1 mM, 15 min) also caused a significant increase in microsomal GSH S-transferase activity with a concomitant decrease in GSH content, confirming that liver microsomal GSH S-transferase in rats was activated in vivo by oxidative stress.

Chloroform-induced alteration of glutathione S-transferase activity.

The effect of chloroform treatment on the hepatic glutathione S-transferases was studied in phenobarbital-treated rats. The apparent isozymic composition of glutathione S-transferases in hepatic cytosol was changed after chloroform treatment. Glutathione S-transferases AA, A, B, C, and D + E were observed in hepatic cytosol from untreated rats; in contrast, the catalytic activity associated with basic glutathione S-transferases, such as AA, A, B, and C, decreased with time after chloroform treatment. Glutathione S-transferase B was not detectable 2 hr after chloroform treatment, and glutathione S-transferases AA and C were scarcely detectable after 5 hr. Twenty-four hours after chloroform treatment, glutathione S-transferases A and C were clearly detectable as was D + E and a small amount of B. Hepatic cytosolic glutathione S-transferase activity was decreased by chloroform treatment, and reached a minimum at 5 hr after treatment. Corresponding to the decrease of hepatic cytosol glutathione S-transferase activity, serum glutathione S-transferase activity was elevated maximally 5 hr after chloroform treatment and returned to almost normal by 24 hr. Treatment of rats with SKF 525-A or cysteine inhibited the chloroform-induced elevation of serum glutathione S-transferase activity. The chromatographic properties of the glutathione S-transferases present in serum were similar to glutathione S-transferase D + E. Furthermore, after incubation of partially purified cytosolic glutathione S-transferases with chloroform in the presence of hepatic microsomes and NADPH, only transferase D + E was detected. The addition of bilirubin to partially purified cytosolic glutathione S-transferase decreased the basic character of glutathione S-transferases B and C. In conclusion, chloroform caused a release of hepatic cytosolic glutathione S-transferases into serum. Both the active metabolite of chloroform, which was produced by the microsomal cytochrome P-450 system, and bilirubin, which was increased by chloroform treatment, played roles in altering the properties of the glutathione S-transferases.

Alteration of hepatic glutathione S-transferases and release into serum after treatment with bromobenzene, carbon tetrachloride, or N-nitrosodimethylamine.

The effects of bromobenzene, carbon tetrachloride, and N-nitrosodimethylamine (DMN) on hepatic glutathione S-transferase activity were studied in untreated and in phenobarbital- or ethanol-treated rats. In phenobarbital-treated rats, the isozymic composition of the hepatic cytosolic glutathione S-transferases was changed after giving hepatotoxic chemicals; glutathione S-transferases 2-2(AA), 3-3(A), 1-2(B), 3-4(C), and 4-4 + 5-5(D + E) were present in cytosol from control rats, but only glutathione S-transferases cochromatographing with transferases 4-4 + 5-5(D + E) were detected in rats given carbon tetrachloride or bromobenzene. A marked decrease in hepatic and an increase in serum glutathione S-transferase activity were also observed after carbon tetrachloride or bromobenzene treatment, but little change was seen after giving DMN. On the contrary, in untreated or ethanol-treated rats, DMN administration decreased hepatic glutathione S-transferase activity and caused an elevation in serum glutathione S-transferase activity. The isozymic composition of the hepatic cytosolic glutathione S-transferases after giving DMN to untreated rats was also altered, but the alteration was much less than that observed after giving carbon tetrachloride or bromobenzene to phenobarbital-treated rats. The elevation in serum glutathione S-transferase activity was accompanied by an increase in both serum glutamate-pyruvate transaminase activity and serum bilirubin concentrations. Thus, hepatic glutathione S-transferase activity was altered and released into serum after giving hepatotoxic chemicals, and the alteration in glutathione S-transferase activity was dependent on treatment with phenobarbital or ethanol.

Increase in liver microsomal glutathione S-transferase activity by phenobarbital treatment of rats. Possible involvement of oxidative activation via cytochrome P450.

The possible involvement of oxidative activation of liver microsomal glutathione (GSH) S-transferase by the cytochrome P450 system was investigated. When rats were given phenobarbital (PB) intraperitoneally for 3 days, liver microsomal GSH S-transferase activity was stimulated 1.3-1.4-fold and the effect of PB on the transferase was potentiated by combination with a catalase inhibitor, 3-amino-1,2,4-triazole. Immunoblotting of microsomal proteins from PB-treated rats with anti-microsomal GSH S-transferase antibody after SDS-PAGE showed the presence of a dimer of the transferase. When microsomal suspensions prepared from PB-treated rats were placed on ice without GSH, the microsomal GSH S-transferase activity gradually increased with time and reached 200% of the initial level at 3 hr when activation of the transferase by N-ethylmaleimide was lost. The time-dependent increase in GSH S-transferase activity in PB-treated microsomes was prevented by addition of 0.1 mM GSH. The increase in microsomal GSH S-transferase activity by NADPH was depressed by cytochrome P450 inhibitors such as SKF 525-A (2-diethylaminoethyl-2,2-diphenylvalerate), metyrapone or isoniazid in agreement with the concomitant decrease in generation of hydrogen peroxide in microsomes. These results indicate that the increase in GSH S-transferase activity in liver microsomes by PB treatment of rats is due to the oxidative modification of the enzyme by reactive oxygen species which are concomitantly increased following induction of cytochrome P450.

Isozyme selective arylation of cytosolic glutathione S-transferase by [14C]bromobenzene metabolites.

[14C]Bromobenzene was incubated with NADPH-fortified liver homogenates from phenobarbital-treated rats, after which the glutathione S-transferases were isolated from the incubation mixture. Glutathione S-transferase activity, with 1-chloro-2,4-dinitrobenzene as the substrate, in the homogenate was unchanged after incubation with bromobenzene. Radioactivity derived from the [14C]bromobenzene remained associated with the cytosolic glutathione S-transferases after DE52 and Sephadex G-100 chromatography. Further purification of the cytosolic glutathione S-transferase by CM52 and hydroxylapatite chromatography showed that bromobenzene metabolites were bound to fractions containing glutathione S-transferase subunits 4, 5, and 1. The primary site of arylation appeared to be subunit 1, as indicated by autoradiography and hydroxylapatite chromatography. [14C]Bromobenzene metabolites were not bound to microsomal glutathione S-transferases. These data show that hepatic cytosolic glutathione S-transferases, especially glutathione S-transferases 4-4/5-5, 3-4, and 1-1 may act as trapping or scavenger proteins for reactive metabolites and that this effect is not associated with a loss of catalytic activity.

Glutathione S-transferases in rat testis microsomes: comparison with liver transferase.

Glutathione S-transferases in testis microsomes were purified from rats and compared with the liver microsomal transferase. When microsomal fractions were prepared from rat testis by the same method as used for liver microsomes, testis microsomal glutathione S-transferase activity was increased 2-fold by N-ethylmaleimide as compared to a 7-fold increase in that of the liver transferase. In contrast to the single glutathione S-transferase in liver microsomes, at least three isozymes of glutathione S-transferase were separated from testis microsomes on hydroxylapatite column chromatography. The major fraction exhibiting glutathione S-transferase activity from the testis microsomes was shown to contain a member of the Mu family. The second fraction with transferase activity contained one of the Alpha class, and the third and smallest fraction was found to contain the liver microsomal form of glutathione S-transferase. Since the GSH S-transferase of the Mu family is present in the cytosol, we isolated the GSH S-transferase from testis cytosol, it being suggested that the major GSH S-transferase in testis microsomes is the cytosolic transferase. These results indicate that testis microsomes contain mainly the cytosolic form of glutathione S-transferase, and that the activity of the liver microsomal form of the transferase is very low.

Effect of intracoronary captopril on coronary blood flow and regional myocardial function in dogs.

To evaluate the cardiac effect of an inhibitor of angiotensin-converting enzyme, the effect of intracoronary (i.c.) captopril on coronary blood flow and regional myocardial function was examined in the anesthetized open-chest dog. Blood flow of the left circumflex coronary artery (LCX), left ventricular pressure (LVP), aortic pressure (AoP) and regional myocardial segment length were measured continuously. Captopril i.v. (0.3 mg/kg) produced an immediate reduction in AoP and an increase in percent shortening of myocardial segments followed by a decrease in coronary vascular resistance and increases in heart rate and LVdP/dt. Reductions in LCX flow induced by i.c. angiotensin were attenuated and i.c. bradykinin-induced increases in LCX flow were augmented after captopril. On the contrary, i.c. infusion of captopril (0.01 mg/min) into the LCX caused no change in hemodynamic variables and myocardial shortening although responses to angiotensin I and bradykinin were markedly modified. These results suggest that captopril may have no direct cardiac effect.

Purification of anticoagulant factor from the spine venom of the crown-of-thorns starfish, Acanthaster planci.

The fraction with anticoagulant activity was purified from the spine venom of Acanthaster planci by fractionation with ammonium sulfate followed by column chromatography and designated plancinin. Its molecular weight determined by tricine-SDS polyacrylamide gel electrophoresis was about 7500 in native form and about 3000 in reduced conditions. Plancinin showed neither platelet aggregation nor an enhancement of vascular permeability. Fibrin formation time was prolonged by 25 micrograms of plancinin which was comparable to 0.08 units of heparin. 2-Mercaptoethanol inhibited the anticoagulant activity of plancinin with a 50% inhibition concentration of 5.6 x 10(-3) M. The bleeding time of mice was significantly prolonged by i.v. administration of plancinin and this effect was lost when plancinin was given orally or s.c. These data indicate that plancinin is a peptide with disulfide bond which is essential for the anticoagulant activity.

Effects of habu (Trimeresurus flavoviridis) venom on isolated and perfused hearts of rats.

Crude habu venom decreased coronary perfusion pressure and produced a small increase in myocardial tension of isolated and perfused rat hearts. Indomethacin infusion depressed the fall in perfusion pressure caused by the venom, without affecting the increase in tension. Heated venom decreased perfusion pressure, but did not increase myocardial tension. These results suggest that crude habu venom has coronary vasodilating and positive inotropic effects, possibly through actions of a phospholipase A2 and a heat-labile component, respectively.

Smooth muscle contractile action of the venom from the crown-of-thorns starfish, Acanthaster planci.

The fraction (venom B) of spine venom from the crown-of-thorns starfish (Acanthaster planci) caused contractions of the uterus of rats and enhanced vascular permeability in rabbits. The venom B-induced contraction of the smooth muscle was depressed by inhibitors of prostaglandin synthesis such as indomethacin or aspirin, but not by the anticholinergic agent, atropine. The fraction with the uterus contractile action was partially purified from venom B through column chromatography. This fraction contains phospholipase and proteinase activities and was different from the lethal factor in the venom. These results suggest that the uterine contractile action caused by venom B is mediated by prostaglandins and partly contributed by the activity of phospholipase in the venom.

Effect of the spine venom from the crown-of-thorns starfish, Acanthaster planci, on drug-metabolizing enzyme in rat liver.

The effect of spine venom from the crown-of-thorns starfish (Acanthaster planci) on drug-metabolizing enzymes in rat liver was studied. The spine venom was prepared by saturation of spine homogenate with ammonium sulfate and the protein fraction precipitating 50% saturation was used as venom B. Venom A was the protein precipitated between 50 and 100% saturation. When venom B (100-200 mg/kg) was given to rats, liver microsomal GSH S-transferase and cytochrome P450 activities decreased while cytosolic GSH S-transferase activity was not changed. The decrease in these microsomal enzyme activities was seen from 12 hr to 24 hr after giving 100 mg/kg of venom B. Rats given venom A died, suggesting an involvement of the lethal factor in venom A. The data showed that the spine venom B from A. planci depressed microsomal GSH S-transferase and cytochrome P450 activities in rat liver and that this venom was distinct from the lethal factor of the spine venom.

Inhibitory effects of Alpinia speciosa K. SCHUM on the porphyrin photooxidative reaction.

It is thought that the beta-carotene defense mechanism against photosensitivity involves the inhibition of singlet oxygen formation, a kind of active oxygen. When we screened chemical substances obtained from plants indigenous to Okinawa, known to have residents with the longest life span in Japan, we found that Alpinia speciosa K. SCHUM (Japanese name: gettou), which is used as a food preservative, has an activity similar to that of beta-carotene. We measured the amount of lipid peroxide (LPO) formed from a hematoporphyrin-containing rat liver microsomal suspension irradiated with visible light. The inhibitory effect of Alpinia speciosa on LPO formation was confirmed when the addition of increasing concentrations of Alpinia speciosa extract led to a decrease in the amount of LPO formed. Moreover, the reaction mechanism that affects the amount of singlet oxygen formed was measured, and the effect of the extract was determined by the ESR trapping technique. It was found that the extract effectively inhibited the formation of singlet oxygen. The extract of Alpinia speciosa contains dihydro-5,6-dehydrokawain. It was confirmed that dihydro-5,6-dehydrokawain, which is a water-soluble compound, has singlet oxygen quenching activity. We synthesized five derivatives of kawain and found that dimethyl [6-(2-phenylethyl)-2-oxo-2H-pyran-4-yl] phosphorothionate has the strongest singlet oxygen quenching activity. The use of the compound from Alpinia speciosa that exhibits singlet oxygen quenching activity as an inhibitory agent of the phototoxic reaction in porphyria is expected.

Medicinal herb, Thonningia sanguinea protects against aflatoxin B1 acute hepatotoxicity in Fischer 344 rats.

1. Thonningia sanguinea, a plant used prophylactically against bronchial asthma in Ghana was recently found to have antioxidative and hepatoprotective actions in our laboratory. 2. In this study, the effect of T. sanguinea extract on certain biochemical indices in serum and liver of Fischer 344 rats given a single intraperitoneal (i.p.) dose (1 mg/kg) of aflatoxin B1 (AFB1) was investigated. 3. Administration of AFB1 resulted in significant increases in serum alanine aminotransferase (ALT) and glutathione S-transferase (GST) levels and a significant decrease in aniline hydroxylase activity in liver microsomes. When T. sanguinea (5 ml/kg) was intraperitoneally administered to rats 12 h and 1 h before AFB1, liver injury was significantly reduced as seen in the decreased levels of serum ALT and serum GST. However, the decrease in aniline hydroxylase activity by AFB1 was not recovered but enhanced by T. sanguinea pre-treatment. 4. Kinetic analysis of cytochrome P450 activity of rat liver microsomes in vitro demonstrated that T. sanguinea inhibited aniline hydroxylase non-competitively suggesting depression of biotransformation of AFB1 to toxic metabolites. 5. The data indicate a hepatoprotective action of T. sanguinea against AFB1-induced liver injury.

Inhibitory effects of the medicinal herb, Thonningia sanguinea, on liver drug metabolizing enzymes of rats.

In this study we examined the effect of the aqueous extract of Thonningia sanguinea (T.S.) on 7-ethoxyresorufin O-deethylase (EROD, CYP1A1), 7-pentoxyresorufin O-dealkylase (PROD, CYP2B1/2), 7-methoxyresorufin O-demethylase (MROD, CYP1A2), aniline hydroxylase (aniline, CYP2E1), p-nitrophenol hydroxylase (PNPH, CYP2E1) and erythromycin N-demethylase (ERDM, CYP3A1) in rat liver in vitro and in vivo. Although T.S. extract increased ERDM activity in induced rat liver microsomes, it showed a dose-dependent inhibitory effect in vitro on other P450 monooxygenase activities particularly EROD and PROD, which are mediated primarily by CYP1A1 and CYP2B1/2, respectively. PROD, EROD and MROD activities were also decreased by 18%, 19% and 40%, respectively, in hepatic microsomes prepared from rats treated with T.S. extract for 3 days. Kinetic analysis of CYP activity of 3-methylchloranthrene-induced microsomes demonstrated that T.S. inhibited EROD and MROD activities by a noncompetitive and competitive mechanism, respectively. The analysis of alterations produced by T.S. on PROD kinetic parameters in phenobarbital-induced microsomes suggested that the inhibition is noncompetitive. Pretreatment of rats with T.S. prolonged pentobarbital and phenobarbital sleeping time; however, plasma phenobarbital concentration determined on awakening showed no significant difference between control and T.S.-treated rats. T.S. was also found to be a potent inhibitor of the liver cytosolic glutathione S-transferase. These data suggest that selective modulation of CYP isoenzymes by T.S. might contribute to protection of the liver from xenobiotic-induced intoxication or to alteration of the action of drug(s) concomitantly administered besides its antioxidative properties.

Antioxidant and hepatoprotective actions of medicinal herb, Terminalia catappa L. from Okinawa Island and its tannin corilagin.

The antioxidant and hepatoprotective actions of Terminalia catappa L. collected from Okinawa Island were evaluated in vitro and in vivo using leaves extract and isolated antioxidants. A water extract of the leaves of T. catappa showed a strong radical scavenging action for 1,1-diphenyl-2-picrylhydrazyl and superoxide (O(2)(.-)) anion. Chebulagic acid and corilagin were isolated as the active components from T. catappa. Both antioxidants showed a strong scavenging action for O(2)(.-) and peroxyl radicals and also inhibited reactive oxygen species production from leukocytes stimulated by phorbol-12-myristate acetate. Galactosamine (GalN, 600 mg/kg, s.c.,) and lipopolysaccharide (LPS, 0.5 microg/kg, i.p.)-induced hepatotoxicity of rats as seen by an elevation of serum alanine aminotransferase, aspartate aminotransferase and glutathione S-transferase (GST) activities was significantly reduced when the herb extract or corilagin was given intraperitoneally to rats prior to GalN/LPS treatment. Increase of free radical formation and lipid peroxidation in mitochondria caused by GalN/LPS treatment were also decreased by pretreatment with the herb/corilagin. In addition, apoptotic events such as DNA fragmentation and the increase in caspase-3 activity in the liver observed with GalN/LPS treatment were prevented by the pretreatment with the herb/corilagin. These results show that the extract of T. catappa and its antioxidant, corilagin are protective against GalN/LPS-induced liver injury through suppression of oxidative stress and apoptosis.

Activation of liver microsomal glutathione S-transferase activity by heating.

The effect of heating on rat liver microsomal glutathione S-transferase activity was investigated. The microsomal glutathione S-transferase activity increased with an elevation of temperature and reached a maximal level at 50 degrees C for 30 min, at which point the enhancement was 2.6-fold. The microsomal glutathione S-transferase activity had one low Km value for 1-chloro-2,4-dinitrobenzene at room temperature. However, two Km values were observed in heated microsomes. N-Ethylmaleimide increased microsomal glutathione S-transferase activity 7.7-fold and a 2.5-fold increase in the activity was observed even after heating of microsomes at 55 degrees C for 10 min. On the other hand, there was no additional activation in the heated-microsomal glutathione S-transferase by glutathione disulfide or diamide with glutathione. The increased activity of the microsomal glutathione S-transferase activity is activated by heating through a mechanism different from the activation caused by sulfhydryl reagents.