Protective effect of Phyllanthus fraternus against bromobenzene induced mitochondrial dysfunction in rat liver mitochondria.

The objective of the current study was to investigate the protective effect of an aqueous extract of Phyllanthus fraternus (AEPF) against bromobenzene induced mitochondrial dysfunction in rat liver mitochondria. Administration of bromobenzene (10 mmol/kg body wt.) significantly decreased the rate of respiration (with glutamate+malate or succinate as substrates), abolished respiratory control ratio (RCR) and P/O ratios completely. There was a significant increase in the levels of lipid peroxides and protein carbonyls and a significant decrease in the total sulphydryl groups. The activities of antioxidant enzymes like catalase, glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD) were decreased. The levels of antioxidants like reduced and oxidized glutathione were significantly decreased compared to control. Administration of rats with an AEPF (100mg/kg body wt.) prior to bromobenzene administration showed several beneficial effects like: (i) complete protection on mitochondrial respiration, RCR and P/O ratios (ii) lipid peroxides and protein carbonyl levels were significantly lowered (iii) increased the levels of sulphydryl groups and the activity of antioxidant enzymes and (iv) significant increase in the levels of reduced and oxidized glutathione. Vitamin E was used as positive control and bromobenzene induced mitochondrial dysfunction was protected better with AEPF compared to vitamin E.

Attenuation of bromobenzene-induced hepatotoxicity by poly(ADP-ribose) polymerase inhibitors.

Inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) have been demonstrated to attenuate pathophysiological conditions associated with toxicant-induced oxidative stress. This investigation evaluates Nicotinamide (NIC), a non-specific PARP inhibitor, and 6(5)-Phenanthridinone (Phen), a specific PARP-1 inhibitor, for their efficacy in blocking or attenuating bromobenzene (BB) induced hepatocellular toxicity. Male ICR mice were treated with an intraperitoneal injection of bromobenzene, followed by concomitant treatment with NIC or with NIC at 0.5, 1 and 2 hours after BB treatment, or with concomitant treatment of Phen at 10 mg/ml, 20 mg/ml, or 40 mg/ml solution concentration. Mice with only BB treatment displayed substantial hepatotoxicity as evidenced by a 3.5-fold increase in serum alanine transferase (ALT) compared to controls. Mice treated with 3 injections of NIC (at 0.5, 1 and 2 hours) after BB treatment demonstrated a 90% reduction in serum ALT at 24 hours after BB treatment (p < 0.05). Mice with concomitant BB and Phen treatment demonstrated a 75% reduction in ALT at 24 hours after treatment (p < 0.05). Histological evaluations of centrilobular hepatic tissue from treated animals confirm findings of reduced hepatotoxicity as indicated by the ALT results in the NIC and Phen treatment groups. Mortality after 7 days was reduced to levels near controls in the NIC and Phen treatment groups. The PARP-1 inhibitors evaluated in this investigation produce clinically significant attenuation of BB-induced liver injury in male ICR mice.

Modulation of halobenzene-induced hepatotoxicity by DT-diaphorase modulators, butylated hydroxyanisole and dicoumarol: evidence for possible involvement of quinone metabolites in the toxicity of halobenzenes.

Recent metabolic studies have demonstrated the importance of reactive intermediates like quinones or semiquinone radicals in the covalent binding of halobenzenes to liver protein. The current studies were designed to examine if quinone intermediates are involved in the toxicity of hepatotoxic halobenzenes, bromobenzene (BB) and 1,2,4-trichlorobenzene (1,2,4-TCB). Two-electron reduction of the quinone intermediates by DT-diaphorase is considered to be a detoxication pathway since the resulting hydroquinone may be readily conjugated and excreted. Mice were pretreated with butylated hydroxyanisole (BHA; 0.5% in the diet, for 3 days), an inducer of DT-diaphorase, or dicoumarol (0.3 mmol/kg, p.o.), an inhibitor of this enzyme. The mice were then given BB (2.5 or 3.5 mmol/kg, i.p.) or 1,2,4-TCB (0.75 or 1.5 mmol/kg, i.p.). Dietary BHA markedly suppressed the hepatotoxicity caused by both BB and 1,2,4-TCB while dicoumarol significantly enhanced it, as judged by serum alanine aminotransferase activity. When mice were treated with BB at different times after the end of dietary BHA exposure, the degree of the protection against the hepatotoxicity appears to correlate to the extent of the induction of DT-diaphorase activity by BHA pretreatment. BHA pretreatment failed to protect against carbon tetrachloride-induced hepatotoxicity. These results seem to provide evidence for the involvement of the quinone metabolites in BB- and 1,2,4-TCB-induced hepatotoxicity and for the protective role of DT-diaphorase against the toxicity.

Screening of hepatoprotective plant components using a HepG2 cell cytotoxicity assay.

Identification of the active components of plants with hepatoprotective properties requires screening of large numbers of samples during fractionation and purification. A screening assay has been developed based on protection of human liver-derived HepG2 cells against toxic damage. Various hepatotoxins were incubated with HepG2 cells in 96-well microtitre plates (30,000 cells well-1) for 1 h and viability was determined by metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS). Bromobenzene (10 mM) and 2,6-dimethyl-N-acetyl-p-quinoneimine (2,6-diMeNAPQI, 200 mM) had greater toxic effects than tert-butyl hydroperoxide (1.8 mM) or galactosamine (10 mM), reducing mean viability to 44.6 +/- 1.2% (s.e.m.) and 56.1 +/- 2.1% of control, respectively. Protection against toxic damage by these agents was tested using a crude extract of a known hepatoprotective Sri Lankan plant, Osbeckia aspera, and two pure established hepatoprotective plant compounds, (+)-catechin and silymarin (1 mg mL-1). Viability was significantly improved by Osbeckia (by 37.7 +/- 2.4%, P < 0.05, and 36.5 +/- 2.1%, P < 0.05, for bromobenzene and 2,6-diMeNAPQI toxicity, respectively). Comparable values for (+)-catechin were 68.6 +/- 2.9% and 63.5 +/- 1.1%, and for silymarin 24.9 +/- 1.4% and 25.0 +/- 1.6%. This rapid and reproducible assay should prove useful for the isolation and identification of active hepatoprotective compounds in crude plant extracts.

Protective effect of S-adenosyl-L-methionine on bromobenzene- and D-galactosamine-induced toxicity to isolated rat hepatocytes.

The protective effect of S-adenosyl-L-methionine (SAMe) on bromobenzene (BB)- or D-galactosamine (GalN)-induced damage to isolated rat hepatocytes and its effect on cellular glutathione (GSH) levels were investigated. SAMe at concentrations of 0.5 to 3.0 mmol/L significantly reduced lactate dehydrogenase leakage from cells exposed to 1.6 mmol/L BB (P < .05 to .001) during 2 hours' incubation. GalN at 25 to 50 mmol/L induced a marked increase of LDH leakage from the cells during the later stage of 24 hours' incubation and SAMe at 1.0 mmol/L clearly attenuated the LDH leakage in GalN (25 mmol/L)-exposed cells. The GSH content in the cells exposed to 2.4 mmol/L BB for 150 minutes was markedly decreased, and further decreased during 24 hours' incubation. SAMe (1.5 mmol/L) both reduced LDH leakage and corrected GSH depletion in cells exposed to 2.4 mmol/L BB. The GSH content in 25 and 50 mmol/L GalN-exposed cells was strikingly diminished to 51.2% and 32.8% of the controls, respectively, during 24 hours' of exposure. SAMe at 1.5 mmol/L significantly reduced the loss in GSH content in 25 mmol/L GalN-exposed cells. The findings show that SAMe has beneficial effects on both BB- and GalN-induced toxicity to rat hepatocytes. The main mechanism behind the protective effect of SAMe on BB and GalN toxicity seems to be associated with enhancement of GSH synthesis in the cells.

Effect of P-450 inducers on glutathione (GSH) depletion by bromobenzene in primary cultures of dog hepatocytes.

Primary cultures of dog hepatocytes sensitivity responded to various inducers of cytochrome P-450; phenobarbital (PB) significantly elevated the activity of 7-ethoxycoumarin O-deethylase (ECOD) and progesterone 6 beta-hydroxylase (6 beta-OHP). beta-Naphthoflavone (beta-NF) and rifampicin (Rif) elevated the levels of 7-ethoxyresorufin O-deethylase (EROD) and 6 beta-OHP, respectively. When primary cultures of dog hepatocytes were incubated with bromobenzene, cellular reduced glutathione (GSH) levels decreased time- and dose-dependently. Pretreatment of the cultures with PB enhanced GSH depletion by bromobenzene, while beta-NF and Rif had little effect, suggesting that the 2B type cytochrome P-450 is responsible for the primary oxidation of bromobenzene to GSH-reactive metabolite(s). Bromobenzene-dependent GSH depletion was completely prevented by 10 microM SKF-525A both in the control and PB-treated primary cultures. Treatment of dog primary cultures with PB analogues also elevated the level of drug-metabolizing activity leading the cells to be more susceptible to GSH depletion by bromobenzene.

Prevention of bromobenzene toxicity by N-acetylmethionine: correlation between toxicity and the impairment in O- and S-methylation of bromothiocatechols.

Bromobenzene (800 mg/kg, ip) caused severe liver necrosis with massive hemorrhage in the golden Syrian hamster within the first 24 hr. Kidney injury was also observed. Treatment with N-acetylmethionine (NAM) at an ip dose of 1200 mg/kg at 5 hr after bromobenzene administration significantly protected the liver and kidney against injuries. Plasma glutamate pyruvate transaminase and blood urea nitrogen levels were substantially decreased in the NAM-treated animals. Histological evaluations confirmed these results. When the urinary neutral and phenolic metabolites of bromobenzene from NAM-treated and untreated hamsters were isolated and compared by GC and GC/MS, a striking result was observed in terms of O- and S-methylated thiol-containing metabolite formation. The NAM-treated animals showed approximately a 8- to 14-fold increase in the excretion of the four isomeric O- and S-methylated bromothiocatechols. These thiocatechols, which are now known to be the 3,4-series metabolites of bromobenzene, can undergo methylation at either the thiol or the hydroxyl functional group. The excretion of 3-S- and 4-S-methylated bromodihydrobenzene thiolols was also increased significantly in the NAM-treated hamster, but other neutral and phenolic metabolites were relatively unchanged. These results suggest that bromobenzene toxicity in the Syrian hamster may be associated with impaired methylation capabilities, an impairment that could be due to methionine and glutathione depletion.

Protective effect of zinc in the hepatotoxicity of bromobenzene and acetaminophen.

Mice of the Balb'c strain were administered bromobenzene (BB) or acetaminophen (AA) i.p., in single doses of 400 and 300 mg/kg, respectively. In the blood activity of SGOT and SGPT as well as SDH was determined. In the liver the level of metallothionein (MT), malondialdehyde (MDA) and glutathione (GSH) was measured. The level of MT as well as GSH (determined as non-protein SH groups) showed a significant increase following administration of zinc alone. Joint action of zinc and either BB or AA resulted in a decrease of GSH which was less pronounced than expected for each of the xenobiotics alone. The protective effect of zinc reflected in the reduction of the increase of SGPT and SGOT activity was apparent shortly (4 h) after administration of AA. A day after injection of AA alone the activity of enzymes was lower and the rate of decline followed the sequence SGPT greater than SGOT greater than SDH. For BB, both the toxic effect and the protective influence of zinc were apparent 24 h following administration. At 4 h in a group receiving BB alone no changes of the indicatory enzymes in blood were noted.

Antagonism of bromobenzene-induced hepatotoxicity by phentolamine: evidence for a metabolism-independent intervention.

A previous study has revealed that phentolamine markedly antagonizes the bromobenzene-induced hepatotoxicity and lethality in B6C3F1 mice. One potential mechanism by which phentolamine may diminish the bromobenzene-induced hepatotoxicity is by a direct or indirect interference with the metabolism of bromobenzene to toxic metabolites. In the present study, phentolamine cotreatment failed to alter the elimination of bromobenzene from serum or the distribution of bromobenzene to liver. This suggests that phentolamine cotreatment does not indirectly interfere with bromobenzene bioactivation secondary to changes in bromobenzene absorption, distribution, or elimination. Further, a phentolamine concentration 10- to 20-fold greater than those measured in vivo failed to alter the in vitro metabolism of bromobenzene to its ortho- and para-phenolic metabolites. It is believed that para-bromophenol represents the rearrangement product of the hepatotoxic 3,4-epoxide and that ortho-bromophenol is a product of the nonhepatotoxic 2,3-epoxide pathway. Thus, it appears that phentolamine does not antagonize bromobenzene-induced hepatotoxicity by inhibiting the formation of hepatotoxic intermediates, nor by enhancing metabolism via the nonhepatotoxic pathway. On the basis of these studies, we conclude that phentolamine antagonism of bromobenzene-induced hepatotoxicity occurs through a mechanism independent of bromobenzene bioactivation.

Antagonism of bromobenzene-induced hepatotoxicity by the alpha-adrenergic blocking agents, phentolamine and idazoxan.

The coadministration of phentolamine, an alpha-adrenoreceptor antagonist, was found to be effective in antagonizing the hepatotoxicity produced by bromobenzene in B6C3F1 mice. Multiple doses of phentolamine, administered in dosages of 10 mg/kg, attenuated almost completely the acute lethality resulting from a 0.5 ml/kg dosage of bromobenzene. Consistent with this decline in lethality, the coadministration of phentolamine significantly altered the magnitude of hepatocellular necrosis, the elevation of serum alanine aminotransferase activity, and the glutathione depression normally produced by this dose of bromobenzene. These protective effects were not limited to phentolamine. Idazoxan, an adrenergic antagonist more specific for alpha 2-receptors, was equally effective in antagonizing the bromobenzene-induced hepatotoxicity. Measurements of serum catecholamine levels revealed that the administration of hepatotoxic doses of bromobenzene elevates serum epinephrine levels. Furthermore, the phentolamine antagonism of the bromobenzene hepatotoxicity could be correlated to elevated serum epinephrine levels in both a temporal and dose-dependent manner. Although the mechanism of the phentolamine antagonism remains to be established, one promising hypothesis involves its prevention of an epinephrine-mediated compromise in the glutathione-dependent detoxification of bromobenzene.

Effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the metabolism and toxicity of bromobenzene: an acute study.

The effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on certain aspects of the metabolism and toxicity of bromobenzene administered acutely to mice was investigated by (i) characterizing the influence of OTCA on the metabolic profile of low and high bromobenzene dose at 0-6, 6-12, and 12-24 h, (ii) determining the effective doses range and administration time for OTCA, as well as the optimum period for urine sampling; and (iii) measuring the efficacy of OTCA for protection against bromobenzene induced toxicity. Coadministration of OTCA and bromobenzene enhanced the urinary excretion of mercapturic acid and phenolic metabolites, during 6-12 h, by approximately 152 and 193%, respectively. Maximum efficacy was observed when OTCA (16.0 mmol/kg) was administered concomitantly with bromobenzene (4.0 mmol/kg). Finally, OTCA administration was found to afford substantial protection against elevation of plasma transaminases used as indices of bromobenzene-induced hepatotoxicity. N-acetylcysteine, another cysteine prodrug, had essentially similar effects on the metabolism and toxicity of bromobenzene. Thus, administration of cysteine prodrugs enhances the urinary excretion of several metabolites of bromobenzene and affords protection against bromobenzene-induced hepatotoxicity.

Effect of sodium selenite upon bromobenzene toxicity in rats. I. Hepatotoxicity.

The effects of sodium selenite on bromobenzene hepatotoxicity were examined in male rats. Rats pretreated with sodium selenite (12.5 or 30 mumol/kg, ip) 72 hr prior to injection of bromobenzene (7.5 mmol/kg, ip) showed a marked reduction in bromobenzene-induced liver injury as evidenced by decreased plasma alanine and aspartate transaminase values, sorbitol dehydrogenase activity, and reduced histologic damage. Administration of bromobenzene did not affect the selenium content of blood or liver. At 72 hr after treatment with selenite, hepatic reduced (GSH) and oxidized (GSSG) glutathione values or GSH synthetic and degradation enzyme activities were not altered. However, from 3 to 12 hr following bromobenzene administration, hepatic GSH and cysteine amounts declined less rapidly in selenite-treated rats compared to control. Thus, acute selenite treatment ameliorated bromobenzene hepatotoxicity in a manner suggesting facilitation of hepatic GSH production by selenite for use in bromobenzene detoxication.

Cytoprotective effect of the prostacyclin derivative iloprost against liver cell death induced by the hepatotoxins carbon tetrachloride and bromobenzene.

It was investigated whether the prostacyclin derivative Iloprost (Schering, Berlin) protects rat hepatocytes against lethal damage induced by carbon tetrachloride (CCl4) and bromobenzene (BB). Iloprost was tested in whole animal experiments (intoxication with 2 ml CCl4/kg) and with primary hepatocyte cultures (intoxication with 1.6 mM BB). Cell damage was estimated by light microscopic examination of hepatocellular morphology and by the release of hepatocellular enzymes (glutamic-pyruvic transaminase, GPT; glutamic-oxalacetic transaminase, GOT; lactic dehydrogenase, LDH) into the blood or culture medium. In both experimental set-ups, Iloprost (0.1 micrograms/kg/min in whole animal experiments and 10(-9)-10(-12) M in primary hepatocyte cultures) largely preserved normal hepatocellular morphology after intoxication. Furthermore, the toxin-induced release of hepatocellular enzymes into the blood (GOT, GPT) or into the culture medium (LDH) was reduced by 50%-70% in the presence of Iloprost. It is concluded that the prostacyclin derivative Iloprost possesses cytoprotective activity on rat hepatocytes against lethal injury by CCl4 or BB.

Amelioration of bromobenzene hepatotoxicity in the male rat by zinc.

Experiments were conducted to examine the role of zinc in the prevention of bromobenzene hepatoxicity in male rats. Bromobenzene (BB) (7.5 mmol/kg, ip) produced a marked hepatotoxicity as evidenced by increases in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and a marked depression in hepatic glutathione (GSH) content 24 hr after administration. The administration of zinc (92 mumol Zn/kg, ip, at 48 and 24 hr prior to the bromobenzene) ameliorated the bromobenzene elevations in plasma AST (25%) and plasma ALT (50%) but did not alter the decreases in hepatic GSH. Following administration of [14C]BB, the radioactive label was distributed primarily in the cytosolic and lipid fractions derived from liver homogenates. Furthermore, the subcellular distribution of [14C]BB was not altered by zinc pretreatment. The extent of covalent binding of [14C]BB metabolites to hepatic tissue was significantly depressed in zinc-treated rats. Zinc induced the hepatic levels of metallothionein but [14C]BB did not bind to this sulfhydryl rich protein. Further experiments showed that zinc treatment depressed cytochrome P-450 content, the activity of NADPH cytochrome c reductase, and the metabolism of aniline, but not that of ethylmorphine. These studies suggest that the hepatoprotective effect of zinc against bromobenzene toxicity does not involve altered binding of the reactive toxic metabolite to glutathione or metallothionein, but it may be mediated by the inhibitory effect of zinc on the microsomal cytochrome P-450-dependent drug metabolizing system.

Antidotal effects of dimethyl sulphoxide against paracetamol-, bromobenzene-, and thioacetamide-induced hepatotoxicity.

In mice the hepatotoxic effects of paracetamol (0.5-1.0 g kg-1, orally) as evidenced by increased serum enzyme activities of the aminotransferases and sorbitol dehydrogenase were dose-dependently inhibited by simultaneous treatment with dimethyl sulphoxide (DMSO 0,25-1.0 g kg-1, i.p.). DMSO was also active against bromobenzene- and thioacetamide-induced hepatotoxicity, but failed to protect mice against carbon tetrachloride-induced liver damage. Hepatic glutathione depletion in mice amounting to 94% after paracetamol (0.5 g kg-1, orally) and to 60% after bromobezene (0.25 ml kg-1, orally) was dose-dependently reduced by the simultaneous administration of DMSO(0.25--1.0 G KG-1, I.P.). This indicates less conjugation of the toxic metabolites of paracetamol and bromobenzene to liver glutathione (G-SH) in the presence of DMSO.