Beneficial effect of nitric oxide synthase inhibitor on hepatotoxicity induced by allyl alcohol.

The effect of aminoguanidine (a selective inhibitor of inducible nitric oxide synthase) on allyl alcohol-induced liver injury was assessed by the measurement of serum ALT and AST activities and histopathological examination. When aminoguanidine (50-300 mg/kg, i.p.) was administered to mice 30 min before a toxic dose of allyl alcohol (75 microL/kg, i.p.), significant changes related to liver injury were observed. In the presence of aminoguanidine the level of ALT and AST enzymes were significantly decreased. All symptoms of liver necrosis produced by allyl alcohol toxicity almost completely disappeared when animals were pretreated with aminoguanidine at 300 mg/kg. Depletion of hepatic glutathione as a consequence of allyl alcohol metabolism was minimal in mice pretreated with aminoguanidine at 300 mg/kg. It was found that the inhibition of toxicity was not due to alteration in allyl alcohol metabolism since aminoguanidine did not effect alcohol dehydrogenase activity both in vivo and in vitro.

Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: a possible mechanism of protection.

Administration of thymoquinone (10 mg kg(-1)day(-1), p.o.) with drinking water starting 5 days before a single injection of doxorubicin (15 mg kg(-1)i.p.) and continuing during the experimental period ameliorated the doxorubicin-induced cardiotoxicity in rats. This protection was evidenced from the significant reduction in serum enzymes: lactate dehydrogenase elevated level, 24 h and creatine phosphokinase elevated levels, 24 h and 48 h after doxorubicin administration. The cardiotoxicity of doxorubicin has been suggested to result from the generation of superoxide free-radical. The protective action of thymoquinone was examined against superoxide anion radical either generated photochemically, biochemically or derived from calcium ionophore (A23187) stimulated polymorphonuclear leukocytes. The results indicate that thymoquinone is a potent superoxide radical scavenger, scavenging power being as effective as superoxide dismutase against superoxide. In addition thymoquinone has an inhibitory effect on lipid peroxidation induced by Fe(3+)/ascorbate using rat heart homogenate. The superoxide scavenging and anti-lipid peroxidation may explain, in part, the protective effect of thymoquinone against doxorubicin-induced cardiotoxicity. 2000 Academic Press@p$hr

Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice.

The present study was undertaken to evaluate the effect of aminoguanidine (AG) on carbon tetrachloride (CCl4)-induced hepatotoxicity. Treatment of mice with CCl4 (20 microl/kg, i.p.) resulted in damage to centrilobular regions of the liver, increase in serum aminotransferase and rise in lipid peroxides level 24 hours after CCl4 administration. Pretreatment of mice with AG (50 mg/kg, i.p.) 30 minutes before CCl4 was found to protect mice from the CCl4-induced hepatic toxicity. This protection was evident from the significant reduction in serum aminotransferase, inhibition of lipid peroxidation and prevention of CCl4-induced hepatic necrosis revealed by histopathology. Aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase, did not inhibit the in vitro lipid peroxidation. Taken together, these data suggest a potential role of nitric oxide as an important mediator of CCl4-induced hepatotoxicity.

Inhibition of benzo(a)pyrene-induced forestomach carcinogenesis in mice by thymoquinone.

The modulating effect of thymoquinone (TQ) on benzo(a)pyrene (BP)-induced forestomach tumours was investigated in female Swiss albino mice, receiving oral administration of BP at a dose of 1 mg twice weekly for 4 weeks. Administration of 0.01% of TQ in drinking water 1 week before, during and after BP treatment until the end of the experiment resulted in significant suppression of BP-induced tumourigenesis when compared with the group receiving BP alone. TQ inhibited both BP-induced forestomach tumour incidence and multiplicity by 70% and 67%, respectively. Lipid peroxide accumulation and decreased glutathione (GSH) content and glutathione-S-transferase (GST) and DT diaphorase activities were observed in the liver of BP-treated tumour-bearing mice. TQ alone showed a significant induction in the enzyme activities of hepatic GST and DT diaphorase. Mice treated with TQ along with BP showed almost normal hepatic lipid peroxides and GSH levels, and normal enzyme activities compared to the control group. The present data may indicate the potential of TQ, the main constituent of the volatile oil of Nigella sativa seed, as a powerful chemopreventive agent against BP-induced forestomach tumours in mice. The possible modes of action of TQ may be through its antioxidant and anti-inflammatory activities, coupled with enhancement of detoxification processes.

The protective action of thymol against carbon tetrachloride hepatotoxicity in mice.

The protective action of thymol (paramethyl-isopropyl-phenol) was investigated against carbon tetrachloride (CCl(4))-induced hepatotoxicity in male Swiss albino mice. The CCl(4)at a dose of 20 microl kg(-1)produced damage to liver cells and was followed by the significant increase (P<0.001) in serum alanine aminotransferase (ALT) activity and hepatic lipid peroxidation after 24 h. The hepatocellular necrosis was further confirmed by histopathological examination of liver section. Oral administration of thymol in a single dose (300 mg kg(-1)) resulted in significant (P<0.05) amelioration of CCl(4)-induced hepatotoxicity. Thymol also inhibited lipid peroxidation induced by CCl(4)in vivo. The protection offered by thymol was also evident from histopathology photomicrograph. In a separate in vitro assay, thymol inhibited the non-enzymatic lipid peroxidation of normal mice liver homogenate induced by Fe(3+)-ascorbate. The present study suggests that thymol protects the liver against CCl(4)-induced toxicity and the protection may be mediated through its ability to inhibit lipid peroxidation. However, other interactions between thymol and CCl(4)remains to be elucidated. 1999 Academic Press.

Thymoquinone protects against carbon tetrachloride hepatotoxicity in mice via an antioxidant mechanism.

Thymoquinone (TQ) is the major active component of the volatile oil of Nigella sativa seeds. The effects of TQ on carbon tetrachloride (CCl4)-induced hepatotoxicity was investigated in male Swiss albino mice. Carbon tetrachloride (20 microliters/Kg, i.p.) injected into mice, induced damage to liver cells and was followed by the increase in serum alanine aminotransferase (ALT) activity after 24 h. Oral administration of TQ in a single dose (100 mg/Kg) resulted in significant (p < 0.001) protection against the hepatotoxic effects of CCl4. TQ was tested as a substrate for mice hepatic DT-diaphorase in the presence of NADH. TQ appears to undergo reduction to dihydrothymoquinone (DHTQ). Reduction rates as a function of protein (liver homogenate) and substrate (TQ) concentrations are reported. An apparent K(m) of 0.1 mM and an apparent Vmax of 74 mumol/min/g liver were measured. TQ and DHTQ inhibited the in vitro non-enzymatic lipid peroxidation in liver homogenate (induced by Fe(3+)-ascorbate) in a dose dependent manner. In this in vitro model DHTQ was more potent in comparison with TQ and butylated hydroxytoluene (BHT). The IC50 for DHTQ, TQ and BHT were found to be 0.34, 0.87 and 0.58 microM respectively. The data suggest that the in vivo protective action of TQ against CCl4-induced hepatotoxicity may be mediated through the combined antioxidant properties of TQ and its metabolite DHTQ.

Protective effect of aminoguanidine against cardiovascular toxicity of chronic doxorubicin treatment in rats.

The cardiotoxicity-induced by chronic treatment of doxorubicin have recently be attributed to free radical formation and/or release of nitric oxide. In the present study, an already established rat model of doxorubicin -induced cardiotoxicity was used. Doxorubicin in a total cumulative dose of 15 mgkg(-1) I.P. given in six equal injections over two week period was administered. After three weeks of doxorubicin administration, the blood pressure, serum lactate dehydrogenase, lipid peroxides, asites fluid and mortality rate were significantly increased. Doxorubicin-induced cardiotoxicity was further confirmed by examining the histopathology of heart sections. Myocardial fibres necrosis with prominent acute inflammatory cells were observed in rats hearts treated with doxorubicin. Aminoguanidine, an inhibitor of nitric oxide synthase, 100 mgkg(-1) injected every other day for two week was given concurrently with doxorubicin. Aminoguanidine given concurrently with doxorubicin return blood pressure, lactate dehydrogenase and lipid peroxides to normal control values. Furthermore, aminoguanidine reduces the mortality rate, ascites fluid formation- induced by doxorubicin and improved the histopathology of rats hearts treated with doxorubicin. In conclusion, inhibition of nitric oxide formation may be beneficial in protecting rat hearts against doxorubicin- induced cardiotoxicity.

Thymoquinone protects against doxorubicin-induced cardiotoxicity without compromising its antitumor activity.

Doxorubicin (DOX) has a wide spectrum of antitumor activity with dose-related cardiotoxicity as a major side effect. This cardiotoxicity has been suggested to result from the generation of oxygen-free radicals. The objective of the present study was to investigate the influence of the antioxidant, thymoquinone (TQ) on cardiotoxicity and antitumor activity of DOX in mice. TQ (8 mg/kg/day, p.o.) administered with drinking water starting 5 days before a single i.p. injection of DOX (20 mg/kg) and continuing during the experimental period ameliorated the DOX-induced cardiotoxicity in mice. This finding was evidenced by significant reductions in serum lactate dehydrogenase and creatine kinase elevated levels and further supplemented by histopathological examination of cardiac tissue. TQ did not alter the plasma and heart DOX levels as monitored by fluorometric analysis. In in vivo study on mouse Ehrlich ascites carcinoma tumor, it could then be shown that TQ does not interfere with the antitumor activity of DOX. The current data support TQ as a potentially selective cytoprotective agent, which may ameliorate cardiotoxicity without decreasing DOX antitumor activity.

Effect of L-histidinol on cisplatin nephrotoxicity in the rat.

The effect of L-histidinol (LHL) on the acute nephrotoxicity produced by cisplatin (CDDP; 6 mg/kg, i.v.) was investigated in the rat. Intraperitoneal administration of LHL (100 mg/kg x 5 doses, 2 h apart) starting 2 h prior to CDDP single injection produced significant protection of renal function. The attenuation of nephrotoxicity was evidenced by significant reductions in serum urea and creatinine concentrations, decreased polyuria, reduction in body weight loss, marked reduction in urinary fractional sodium excretion and glutathione-S-transferase (GST) activity, and increased urine/serum creatinine ratio as well as increased creatinine clearance. LHL significantly ameliorated the toxic renal biochemical changes induced by CDDP. Renal lipid peroxides, glutathione levels and GST activity showed a marked tendency towards the normal values. Accumulation of platinum in renal tissues was significantly decreased in the presence of LHL. It is concluded that LHL can act as a nephroprotectant, and it is suggested that it would have beneficial effects on the kidney in clinical settings.

Thymoquinone ameliorates the nephrotoxicity induced by cisplatin in rodents and potentiates its antitumor activity.

The effects of thymoquinone (TQ) on cisplatin-induced nephrotoxicity in mice and rats were studied. Oral administration of TQ (50 mg/L in drinking water) for 5 days before and 5 days after single injections of cisplatin (5 mg/kg, i.v., in rats and 7 or 14 mg/kg, i.p., in mice) greatly ameliorated cisplatin-induced nephrotoxicity in both species. In rats, i.v. cisplatin caused 4- and 5-fold elevations in serum urea and creatinine, a 235% increase in urine volume, a 41% increase in kidney weight, 8.5-fold decrease in creatinine clearance, and extensive histological damage 5 days after treatment. In mice, similar alterations in kidney function were observed. TQ-induced amelioration of cisplatin nephrotoxicity was evident by significant reductions in serum urea and creatinine and significant improvement in polyuria, kidney weight, and creatinine clearance. The protective effects of TQ against cisplatin-induced nephrotoxicity in the rat were further confirmed by histopathological examination. To evaluate the possible modification of the antitumor activity of cisplatin by TQ, we studied their interaction in Ehrlich ascites carcinoma (EAC) bearing mice. The results revealed that TQ potentiated the antitumor activity of cisplatin. The current study suggests that TQ may improve the therapeutic index of cisplatin.

Spectrophotometric assay for superoxide dismutase based on the nitroblue tetrazolium reduction by glucose-glucose oxidase.

A new spectrophotometric assay of superoxide dismutase (SOD) is described. The assay is based on the SOD-mediated inhibition in the rate of nitroblue tetrazolium reduction to the blue formazan at alkaline pH. The optimized assay of SOD is performed in 50 mM glycine-NaOH buffer, pH 9.5, at 25 degrees C. The SOD concentration is determined from the V/v ratio of rates measured in the absence (V) or the presence (v) of SOD. One unit of SOD has been defined as the concentration that decrease the rate to 50% (V/v = 2). The assay is simple, sensitive, uses commercially available reagents, rapid and easy to perform and could be used routinely for monitoring superoxide dismutase levels in purified protein fractions.

Evidence for superoxide radical production by a simple flavoprotein: glucose oxidase.

Nitroblue tetrazolium was reduced to blue formazan during the oxidation of glucose by glucose oxidase. The rate of blue color formation was dependent on the concentrations of glucose, nitroblue tetrazolium and glucose oxidase. The rate of the reaction was negligible below pH 8.4, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, consistent with the participation of superoxide anion radical in the reaction.

Evidence that beta-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin.

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.

Depletion of rat hepatic glutathione and inhibition of microsomal trans-2-enoyl-CoA reductase activity following administration of a dec-2-ynol and dec-2-ynoic acid.

The effects of administration of dec-2-ynol and dec-2-ynoic acid on the hepatic glutathione (GSH) content and hepatic microsomal trans-2-enoyl-CoA reductase activity were examined in rat. Both compounds, when administered ip, caused a marked depletion of GSH levels and a corresponding inactivation of trans-2-enoyl-CoA reductase activity in both a time- and dose-dependent manner. The dec-2-ynoic acid caused greater hepatotoxicity than dec-2-ynol based on serum alanine transaminase activity. Based on the observations that (a) the alcohol did not interact with GSH in the presence or absence of cytosol, (b) the spectral manifestation of the interaction between GSH and the alcohol occurred only when NAD+ was added to the reaction mixture containing the cytosol and reactants, and (c) a similar absorbance spectrum was obtained following the interaction between aldehyde and GSH, it was concluded that dec-2-ynol is converted to an electrophile, dec-2-ynal, which causes depletion of GSH. The decrease in GSH content following administration of the acid appears to be due to activation of the acid to the electrophile, dec-2-ynoyl CoA, which then interacts with GSH, resulting in its depletion, based on the in vitro observations that (a) the acid did not interact with GSH in the presence or absence of cytosol, and (b) the spectral manifestation of interaction between GSH and dec-2-ynoyl CoA occurred both nonenzymatically and enzymatically in the presence of rat liver glutathione S-transferase (Sigma). Bovine serum albumin stimulated the enzymatic reaction. Comparable to the effects on GSH were the effects of dec-2-ynol, dec-2-ynal, dec-2-ynoic acid, and dec-2-ynoyl CoA on the microsomal trans-2-enoyl-CoA reductase activity in vitro. While the alcohol had no effect on the enzyme activity, its electrophilic product, the aldehyde, was a potent inhibitor. Similarly, the acid did not inhibit the enzyme activity unless the acid was present at high concentration; however, its electrophilic product, the CoA thioester, was a very potent inhibitor at very low concentration.

The fatty acid chain elongation system of mammalian endoplasmic reticulum.

Much has been learned about FACES of the endoplasmic reticulum since its discovery in the early 1960s. FACES consists of four component reactions, requires the fatty acid to be activated in the form of a CoA derivative, utilizes reducing equivalents in the form of NADH or NADPH, is induced by a fat-free diet, resides on the cytoplasmic surface of the endoplasmic reticulum, appears to function in concert with the desaturase system and appears to exist in multiple forms (either multiple condensing enzymes connected to a single pathway or multiple pathways). FACES has been found in all tissues investigated, namely, liver, brain, kidney, lung, adrenals, retina, testis, small intestine, blood cells (lymphocytes and neutrophils) and fibroblasts, with one exception--the heart has no measurable activity. Yet, much more needs to be learned. The critical, inducible and rate-limiting condensing enzyme has resisted solubilization and purification; the purification of the other components has met with limited success. We know nothing about the site of synthesis of each component of FACES. How is each component enzyme integrated into the endoplasmic reticulum membrane? Is there a single mRNA directing synthesis of all four components or are there four separate mRNAs? How are elongation and desaturation coordinated? What is (are) the physiological regulator(s) of FACES--ADP, AMP, IP3, G-proteins, phosphorylation, CoA, Ca2+, cAMP, none of these? The molecular biology of FACES is only in the fetal stage of development. We are only scratching the surface--it is an undiscovered country.

Decreased long-chain fatty acyl CoA elongation activity in quaking and jimpy mouse brain: deficiency in one enzyme or multiple enzyme activities?

Using long-chain fatty acyl CoAs (arachidoyl CoA and behenoyl CoA), a decrease in overall fatty acid chain elongation activity was observed in the quaking and jimpy mouse brain microsomes relative to controls. Arachidoyl CoA (20:0) and behenoyl CoA (22:0) elongation activities were depressed to about 50% and 80% of control values in quaking and jimpy mice, respectively. Measurement of the individual enzymatic activities of the elongation system revealed a single deficiency in enzyme activity; only the condensation activity was reduced to the same extent as total elongation in both quaking and jimpy mice. The activities of the other three enzymes, beta-ketoacyl CoA reductase, beta-hydroxyacyl CoA dehydrase, and trans-2-enoyl CoA reductase, in both mutants were similar to the activities present in the control mouse. In addition, the activities of these three enzymes were more than two to three orders of magnitude greater than the condensing enzyme activity in all three groups, establishing that the condensing enzyme catalyzes the rate-limiting reaction step of total elongation. When the elongation of palmitoyl CoA was measured, only a 25% decrease in total elongation occurred in both mutants; a similar percent decrease in the condensation of palmitoyl CoA also was observed. The activities of the other three enzymes were unaffected. These results support the concept of either multiple elongation pathways or multiple condensing enzymes.

Do rat kidney cortex microsomes possess the enzymatic machinery to desaturate and chain elongate fatty acyl-CoA derivatives?

Rat kidney cortex microsomal preparations were unable to catalyze delta 9, delta 6 and delta 5 desaturation of stearoyl-coenzyme A (CoA), linoleoyl-CoA and dihomo-gamma-linolenoyl-CoA, respectively. The kidney cortex microsomal fraction, however, did catalyze the malonyl-CoA dependent fatty acyl-CoA elongation. The biochemical properties of palmitoyl-CoA elongation were studied as a function of protein concentration, time, reduced nicotinamide adenine dinucleotide phosphate (NADPH), malonyl-CoA and substrate concentrations; of the substrates investigated, delta 6,9,12-18:3 was the most active. Unlike what was observed in the hepatic system, a high-carbohydrate, fat-free diet did not induce kidney fatty acid chain elongation. All intermediate kidney cortex microsomal reactions, i.e., beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase activities, were significantly higher (greater than one order of magnitude) than the condensing enzyme activity, suggesting that the rate-limiting step in total elongation is the initial condensation reaction. Contrary to other reports, the results suggest that the kidney cannot synthesize arachidonic acid needed for eicosanoid production.

Enzyme site-specific changes in hepatic microsomal fatty acid chain elongation in streptozotocin-induced diabetic rats.

The hepatic microsomal fatty acid chain elongation of palmitoyl-CoA and gamma-linolenoyl-CoA was diminished by 40-50% in male Sprague-Dawley rats made diabetic for 2 and 4 weeks following the intravenous administration of a single dose (65 mg/kg) of streptozotocin. Analysis of the activities of the four enzymatic components showed that only one enzyme, the condensing enzyme, which catalyzes the initial and rate-limiting step in chain elongation, was altered by the diabetic state. Both chain elongation and condensation activities were depressed to the same extent, whereas beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase activities were the same as the values obtained with non-diabetic controls. 2 week administration of 10 units of insulin per day to rats which were diabetic for a 2-week period resulted in the reversal of the reduced palmitoyl-CoA elongation and condensation activities to control values. However, neither the condensation nor the elongation of gamma-linolenoyl was reversed by the insulin treatment. These results support the notion of multiple condensing enzymes or chain elongation systems.

Evidence for two separate beta-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat.

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the beta-ketoacyl derivative. Employing beta-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent beta-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two beta-ketoacyl CoA reductases, one using NADH and the other NADPH.

Evidence for two separate beta-ketoacyl CoA reductase components of the hepatic microsomal fatty acid chain elongation system in the rat.

The hepatic microsomal fatty acid chain elongation system can utilize either NADPH or NADH. Elongation activity, measured as the rate of malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fat-free diet and by bovine serum albumin (BSA) when either cofactor was employed. When the intermediate products were determined, it was observed that in the presence of BSA and NADPH, the predominant product was the saturated elongated fatty acid, whereas in the presence of BSA and NADH, the major intermediate was the beta-ketoacyl derivative. Employing beta-ketostearoyl CoA as substrate, BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase activity and stimulated NADPH-supported activity. Furthermore, the sum of the NADH-dependent and NADPH-dependent beta-ketoreductase activities approximated the activity obtained when both cofactors were present in the incubation medium, suggesting the existence of two beta-ketoacyl CoA reductases, one using NADH and the other, NADPH.