Inhibition of diquat-induced lipid peroxidation and toxicity in precision-cut rat liver slices by novel antioxidants

The ability of the novel antioxidants U-74,006F and U-78,517G and a known antioxidant (N,N'-diphenyl-p-phenylenediamine, (DPPD)) to inhibit chemically induced (diquat dibromide) oxidative stress was examined in precision-cut liver slices. Previous studies in rat liver microsomes demonstrated the ability of these antioxidants to inhibit lipid peroxidation without preventing redox cycling of diquat. Diquat (1 mM) initiated lipid peroxidation in liver slices prepared from F344 rats. A 30-min preincubation with antioxidants inhibited formation of thiobarbituric acid reactive substances to control levels; ethane evolution, when elevated, was also inhibited by antioxidants. The toxicity of diquat (100 microM-3 mM) was evaluated in liver slices; 1 and 3 mM diquat caused decreases in intracellular K+ and intracellular LDH. Preincubation with antioxidants substantially decreased the toxicity of diquat as indicated by K+ and LDH. Diquat significantly decreased total glutathione levels in the slices; the antioxidants did not significantly inhibit this diquat-dependent effect. In summary, diquat, a compound which undergoes redox cycling and produces oxidative stress, was shown to produce lipid peroxidation, glutathione depletion, and toxicity in liver slices. Two experimental antioxidants, a 21-aminosteroid (U-74,006F) and a trolox-amine (U-78,517G) as well as a known antioxidant (DPPD) were shown to be effective in preventing lipid peroxidation and reducing the subsequent toxicity.     

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.     

Antioxidant-dependent inhibition of diquat-induced toxicity in vivo.

The abilities of two experimental antioxidants (U-74006F and U-78517G), as well as the model antioxidant, diphenyl-p-phenylenediamine (DPPD), to protect against diquat-induced toxicity in male Fischer-344 rats were examined. Both experimental compounds afforded near complete protection against diquat-induced hepatotoxicity, as measured by clinical chemistry and histopathological indices. When observed, diquat-induced nephrotoxicity was also inhibited. Minimal protection was afforded by the model compound, DPPD. In follow-up studies with U-78517G, no effect on diquat-induced biliary excretion of oxidized glutathione was observed, suggesting that a shift in the thiol:disulfide ratio is not responsible for diquat-induced hepatotoxicity. These data are consistent with those from previous in vitro studies in our laboratory and are in agreement with studies by others which suggest that lipid peroxidation is an important event in diquat-induced hepatotoxicity in vivo. The antioxidant effects were largely route-independent as either oral pre-treatment alone (200 mg/kg, 24 h before diquat), intravenous pre-treatment alone (6 mg/kg, 5 min before diquat) or the combination of both treatments produced a similar degree of protection. While pre-treatment with antioxidants was quite effective, no significant U-78517G-dependent inhibition of toxicity was observed when administration was delayed by as little as 10 min post diquat. These latter data suggest that initiation of diquat-induced hepatotoxicity is rapid and that these compounds would therefore be unlikely to have clinical utility in the treatment of diquat intoxication.     

Further examination of the selective toxicity of CCl4 in rat liver slices.

Lipid peroxidation and loss of enzymes located predominantly in either periportal or centrilobular hepatocytes were investigated in precision-cut liver slices from male Sprague-Dawley rats. Pretreatment of animals with 80 mg/kg phenobarbital for the site-specific enzyme studies enhanced and accelerated CCl4 toxicity in slices resulting from increased radical formation. Liver slices were exposed to 0.57 mM CCl4 by vaporization using a roller incubation system at 37 degrees C for a total of 9 hr. Conjugated diene formation, an index of lipid peroxidation, was detected 15 min following CCl4 administration and increased over time. Loss of cytochrome P450 occurred in a time-dependent manner relative to controls where levels in treated slices were 42% of controls at 9 hr. A 48-hr fast prior to termination increased intracellular K+ leakage relative to that present in slices from fed animals. Significant leakage of glucose-6-phosphate dehydrogenase and beta-glucuronidase from centrilobular hepatocytes occurred 9 hr following CCl4 administration. The content of the periportal enzymes (lactate dehydrogenase and sorbitol dehydrogenase) was unchanged in the same slices over the duration of the experiment. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a mitochondrial selective dye and indicator of viability, was significantly lower in treated slices from phenobarbital-treated animals at 9 hr relative to controls. These studies demonstrate that precision-cut slices are an ideal in vitro system for mechanistic studies and the investigation of site-specific toxicants since the integral architecture of the liver and cellular identity are maintained.     

Antioxidant and hepatoprotective actions of the medicinal herb Artemisia campestris from the Okinawa Islands

The antioxidant action of Artemisia campestris was examined in vitro and in vivo. A water extract of A. campestris showed a strong scavenging action of 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and superoxide anion radicals. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Depression of the elevation of serum enzyme levels after CCl4-treatment was also observed by oral administration of the extract. In that case, CCl4-derived lipid peroxidation in the liver was decreased by the extract treatment. These results suggest that the extract of A. campestris scavenges radicals formed by CCl4 treatment resulting in protection against CCl4-induced liver toxicity.     

Neuroprotective effects of 17beta-estradiol and nonfeminizing estrogens against H2O2 toxicity in human neuroblastoma SK-N-SH cells

Neuroprotective effects of estrogens have been shown in various in vitro and in vivo models, but the mechanisms underlying protection by estrogen are not clear. Mounting evidence suggests antioxidant effects contribute to the neuroprotective effects of estrogens. In the present study, we assessed the protective effects of estrogens against H2O2-induced toxicity in human neuroblastoma cells and the potential mechanisms involved in this protection. We demonstrate that 17beta-estradiol (17beta-E2) increases cell survival against H2O2 toxicity in human neuroblastoma cells. 17beta-E2 effectively reduced lipid peroxidation induced by 5-min H2O2 exposure. Furthermore, 17beta-E2 exerts the protective effects by maintaining intracellular Ca2+ homeostasis, attenuating ATP depletion, ablating mitochondrial calcium overloading, and preserving mitochondrial membrane potential. Two nonfeminizing estrogens, 17alpha- and ent-estradiol, were as effective as 17beta-E2 in increasing cell survival, alleviating lipid peroxidation, preserving mitochondrial function, and maintaining intracellular glutathione levels and Ca2+ homeostasis against H2O2 insult. Moreover, the estrogen receptor antagonist fulvestrant (ICI 182,780) did not block effects of 17beta-E2, but increased cell survival and blunted intracellular Ca2+ increases. However, these estrogens failed to reduce cytosolic reactive oxygen species, even at concentrations as high as 10 microM. In conclusion, estrogens exert protective effects against oxidative stress by inhibiting lipid peroxidation and subsequently preserving Ca2+ homeostasis, mitochondrial membrane potential, and ATP levels.     

Efficient amelioration of carbon tetrachloride induced toxicity in isolated rat hepatocytes by Syzygium cumini Skeels extract.

Syzygiumcumini, Indian black plum or Java plum, is a rich source for anthocyanins (230mg/100g DW) showing high antioxidant activity in vitro. In the following study it is further demonstrated that S. cumini peel extract rich in anthocyanins (SCA) offers considerable protection against carbon tetrachloride (CCl(4))-induced damage in rat hepatocytes. SCA itself being non-toxic to primary rat hepatocytes at concentrations ranging from 50 to 500ppm, was found to suppress CCl(4)-induced LDH leakage by 54% at 50ppm, thereby improving the cell viability by 39%. The SCA significantly reversed the CCl(4) induced changes in cellular glutathione (GSH) level, lipid peroxidation and activity of the antioxidant enzyme glutathione peroxidase. Exposure of hepatocytes to SCA after CCl(4) treatment was found to elevate GSH and GPx activities by 2-folds, whereas the activities of catalase and superoxide dismutase were not significantly affected. The fruit pulp extract (SPE) was less effective in offering protection to rat hepatocytes, particularly in terms of total GSH content and a consequent increase in lipid peroxidation although the higher GPx activity suggests the probable involvement of GSH as a substrate for GPx. These observations suggest that the fruit peel extract of S. cumini, is largely responsible for the reversal of CCl(4)-induced oxidative damage in rat hepatocytes. Both peel and pulp extract appear to offer protection to rat hepatocytes through GPx along with other biological pathways independent of catalase and superoxide dismutase.     

Toxicological evaluation and hepatoprotective potential of Clerodendron glandulosum.Coleb leaf extract

This inventory evaluates toxicological effects and hepatoprotective potential of Clerodendron glandulosum.Coleb (CG) aqueous extract. Acute and subchronic toxicity tests were performed using Swiss albino mice as per the guideline of Organisation for Economic Cooperation and Development (OECD). Also, hepatoprotective potential of CG extract was examined in experimental model of carbon tetrachloride (CCl( 4))-induced hepatotoxicity. Acute and subchronic toxicity tests revealed that CG extract is non-toxic and its median lethal dose (LD(50)) value is >5000 mg/kg bodyweight. Also, rats pretreated with CG extract followed by administration of CCl(4) recorded significant decrement in plasma marker enzymes of hepatic damage, total bilirubin content and hepatic lipid peroxidation. While, hepatic reduced glutathione, ascorbic acid content, activity levels of superoxide and catalase and plasma total protein content were significantly increased. Microscopic examination of liver showed that pretreatment with CG extract prevented CCl(4)-induced hepatic damage in CG + CCl( 4) group. CG extract has hepatoprotective potential by modulating activity levels of enzymes and metabolites governing liver function and by helping in maintaining cellular integrity of hepatocytes that is comparable with that of standard drug silymarin. CG extract exhibits potent hepatoprotective activity against CCl(4)-induced hepatic damage but does not exhibit any toxic manifestations.     

Protective effect of glycoprotein isolated from Ulmus davidiana Nakai on carbon tetrachloride-induced mouse liver injury

This study was carried out to evaluate the hepatoprotective activity of glycoprotein isolated from the stems of Ulmus davidiana Nakai (UDN), which has been used as an anti-inflammatory agent in folk medicine. We evaluated lipid peroxidation in glucose/glucose oxidase (G/GO)-induced BNL CL.2 cells and measured thiobarbituric acid reactive substances (TBARS), lactate dehydrogenase (LDH), nitric oxide (NO), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)), activity of cytotoxic-related signals (hepatic cytochrome c, nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1)) and levels of plasma lipids (triglyceride (TG) and total cholesterol (TC)) in carbon tetrachloride (CCl(4,) 1.0 mL kg(-1))-induced A/J mouse. The results in G/GO-induced BNL CL.2 cells showed that UDN glycoprotein had a dose-dependent inhibitory effect on lipid peroxidation. The results in carbon tetrachloride (CCl(4,) 1.0 mL kg(-1))-induced A/J mouse indicated that treatment with UDN glycoprotein (40 mg kg -1) lowered LDH activity and TBARS formation, and increased NO production and antioxidant enzymes activity, compared with control. Also, our finding from CCl(4)-treated mice after pretreatment with UDN glycoprotein demonstrated that the activity of cytotoxic-related signals decreased but the levels of plasma lipids increased, compared with CCl(4) treatment alone. Here, we speculate that UDN glycoprotein has a protective character to CCl(4)-induced mouse liver injury.     

Evaluation of amiodarone free radical toxicity in rat hepatocytes

The possible roles of free radicals and lipid peroxidation in the mechanism of toxicity of amiodarone (AD) [2-butyl-3-(3',5'-diiodo-4' alpha-diethylaminoethoxybenzoyl)benzofuran] and its principle metabolite, desethylamiodarone (DE), were examined in primary cultured Sprague-Dawley male rat hepatocytes. AD (20 and 40 micrograms/ml) and DE (10 and 25 micrograms/ml) killed hepatocytes in concentration- and time-dependent fashions. Several antioxidants [Cu,Zn-superoxide dismutase (200 U/ml), catalase (200 U/ml), N,N'-diphenylphenylenediamine (DPPD; 25 microM), butylated hydroxytoluene (0.1 mM), and N-acetylcysteine (5 mM)] were incapable of preventing AD and DE hepatocyte toxicity. Only vitamin E (VE, d,l-alpha-tocopherol acetate; 20-200 microM) prevented AD and DE toxicity. No correlation between the onset of hepatocyte death by AD and DE and hepatocyte lipid peroxidation was seen. Both drugs inhibited NADPH-dependent rat liver microsomal superoxide production. These results, excluding the preventive effects of VE, do not support a free radical/lipid peroxidation mechanism of hepatocyte toxicity by AD and DE. VE may have prevented hepatocyte toxicity through non-antioxidant effects.     

Effects of oxygen deficiency and calcium omission on carbon tetrachloride hepatotoxicity in isolated perfused livers from phenobarbital-pretreated rats

The effect of oxygen concentration and Ca2+ omission on CCl4-induced hepatotoxicity was studied in a non-recirculating and hemoglobin-free liver perfusion system using phenobarbital-pretreated rats. With 95% O2-saturated perfusate, infusion of 0.5 mM CCl4 caused an instantaneous increase of thiobarbituric acid reactive substances (TBA-RS) in the effluent perfusate, accompanied by only a slight leakage of K+ and lactate dehydrogenase (LDH). CBrCl3 produced a far greater increase in the TBA-RS level, but again with slight K+ and LDH leakage. With 20% O2-saturated perfusate, CCl4 caused a marked LDH leakage, which was preceded by an early and considerable increase in K+ leakage coupled with Na+ uptake, Ca2+ uptake was initially slight, being enhanced concurrently with the LDH leakage. The TBA-RS level changed biphasically with an initial moderate and a succeeding greater increase coupled with LDH leakage. N,N"-Diphenyl-p-phenylenediamine and promethazine suppressed the TBA-RS production, but improved neither K+ nor LDH leakage. Omission of the Ca2+ from the perfusate reduced the initial K+ leakage as well as the later TBA-RS release, and markedly delayed the LDH leakage. In retrograde perfusion under low oxygen supply with Ca2+, CCl4 produced essentially the same toxic manifestations as those observed in the anterograde perfusion. Hepatocytes of the periportal and pericentral areas were not stained with trypan blue in the antero- and retrograde perfusion systems respectively. Thus, oxygen deficiency, rather than lipid peroxidation by itself, and the essential role of extracellular Ca2+ may be important for CCl4-induced hepatic cell necrosis, in which plasma membrane permeability change may be an early and critical event.     

Effects of phospholipase A2 inhibitors on diethyl maleate-induced lipid peroxidation and cellular injury in isolated rat hepatocytes

Isolated hepatocytes provide a suitable system for investigation of various aspects of the mechanism of a toxic response. The mechanism by which most chemicals induce hepatotoxicity is still not known. Evidence that phospholipases may play a role in cellular injury has been reported. In the present study the effects of reported inhibitors of phospholipase A2 (quinacrine, chlorpromazine, dexamethasone, and dibutyryl cyclic AMP) on diethyl maleate (DEM)-induced lipid peroxidation, reduced glutathione (GSH) depletion, and cellular injury were examined in isolated hepatocyte suspensions. Hepatocytes were incubated for 7 h under control conditions or with (1) DEM (4 mM), (2) one of the inhibitors (quinacrine, 10, 50, or 150 microM; chlorpromazine, 50 microM; dexamethasone, 0.1, 0.5, 1, or 2.5 mM; dibutyryl cyclic AMP, 0.1, 0.5, 1, or 2.5 mM) or aspirin (500 microM), or (3) a combination of DEM and one of the inhibitors or aspirin to determine their effect on DEM toxicity. Samples were withdrawn at hourly intervals for estimation of cellular injury (loss of intracellular K+ and lactate dehydrogenase and trypan blue exclusion index), lipid peroxidation (thiobarbituric acid reactants assay), and GSH concentration. Quinacrine and chlorpromazine inhibited DEM-induced lipid peroxidation but not cellular injury or GSH loss. This suggests that phospholipase A2 may be involved in DEM-induced lipid peroxidation but not cell damage. However, dexamethasone and dibutyryl cyclic AMP enhanced both lipid peroxidation and loss of cell viability due to DEM, suggesting novel aspects of the biochemical mechanisms of chemically induced cytotoxicity.     

Effect of chitobiose and chitotriose on carbon tetrachloride-induced acute hepatotoxicity in rats

The purpose of the present study was to examine whether chitobiose and chitotriose can protect rats from CCl4-induced hepatic toxicity when given orally. We studied the effects of the 2 chitooligosaccharides given orally to rats on the acute hepatotoxicity induced by CCl4-dependent lipid peroxidation. The increase in the sum of malondialdehyde and 4-hydroxy-2-alkenals, a marker of lipid peroxidation, in both plasma and liver of CCl4-treated rats was suppressed by oral administration of chitobiose or chitotriose. The elevation in the levels of plasma aspartate transaminase and alanine transaminase activities, markers of hepatic injury, induced by CCl4 intoxication was also counteracted by oral administration of either chitooligosaccharide. The results indicate that chitobiose and chitotriose have the ability to exert a protective action against CCl4-induced acute hepatoxicity, probably by their antioxidant activity.     

Pharmacological Studies of Mentha longifolia Phenolic Extracts. II. Hepatoprotective Activity

The effects of Mentha longifolia crude ethanolic extract (F 1), rich in luteolin glycosides (F 2), apigenin glycosides (F 3) and phenolic acids (F 4), were studied on CCl 4 -induced liver injury in mice. Administration of CCl 4 -induced significant impairment in hepatic antioxidant status, decreasing the glutathione content and superoxide dismutase activity, and stimulating lipid peroxidation. Pretreatment with Mentha longifolia extracts significantly improved hepatic antioxidant status in mice, which was most pronounced in the reduction of CCl 4 -mediated lipid peroxidation. In addition, in pretreated animals, an increase in hepatic glutathione and superoxide dismutase activity, as well as a decrease of cytochrome P450, was found.     

Biochemical mechanism of metallothionein--carbon tetrachloride interaction in vitro

To elucidate the mechanism underlying the protective effect of metallothionein (MT) against carbon tetrachloride (CCl4) toxicity, in vitro experiments were carried out to study the interaction of metallothionein and CCl4. Results from this study showed that incubation of Cd,Zn-MT with CCl4 in the presence of hepatic microsomes and NADPH resulted in a time-dependent depletion of MT thiols with a concurrent reduction in the metal-binding sites of the protein. Moreover, this reaction also released Zn and Cd from MT. Results from experiments conducted to determine whether or not the CCl4-induced decrease in MT-thiol content was due to the scavenging of CCl4 metabolite(s) showed that the trichloromethyl radical, chloroform and phosgene as well as the products of CCl4-induced microsomal lipid peroxidation were not directly involved. Although covalent binding of 14CCl4 to MT was detected following incubation in the presence of a microsomal bioactivation system, it did not account for the CCl4-induced loss of MT thiol groups for the following reasons: (i) prior oxidation of sulfhydryl groups of MT by hydrogen peroxide did not alter the binding; and (ii) anaerobiosis did not alter the extent of covalent binding but obliterated the inhibitory effect of CCl4 on MT thiol content. Measurement of the thiol content of CCl4-treated MT after treatment with 1,4-dithiothreitol revealed that all the thiol groups that were lost subsequent to CCl4 treatment could be regenerated. These data suggest that CCl4-linked oxidation of MT, rather than the covalent binding of 14CCl4 metabolite(s), may be responsible for the CCl4-induced loss of metal binding sites of MT with the concurrent release of Zn and Cd. However, the precise role of the metal released during the oxidation of MT in CCl4 toxicity remains to be defined.     

3,3'-diselenodipropionic acid, an efficient peroxyl radical scavenger and a GPx mimic, protects erythrocytes (RBCs) from AAPH-induced hemolysis

3,3'-diselenodipropionic acid (DSePA), a derivative of selenocystine, has been synthesized and examined for antioxidant activity, glutathione peroxidase (GPx) activity, and cytotoxicity. The effect of DSePA on membrane lipid peroxidation, release of hemoglobin, and intracellular K+ ion as a consequence of erythrocyte (red blood cells or RBCs) oxidation by free radicals generated by 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) were used to evaluate the antioxidant ability. Lipid peroxidation, hemolysis, and K+ ion loss in RBCs were assessed, respectively, by formation of thiobarbituric acid reactive substances (TBARS), absorbance of hemoglobin at 532 nm and flame photometry. The IC50 values for lipid peroxidation, hemolysis, and K+ ion leakage were 45+/-5, 20+/-2, and 75+/-8 microM, respectively. DSePA treatment prevented the depletion of glutathione (GSH) levels in RBCs from free-radical-induced stress. DSePA is a good peroxyl radical scavenger and the bimolecular rate constant for the reaction of DSePA with a model peroxyl radical, trichloromethyl peroxyl radical (CCl 3O2*), was determined to be 2.7x10(8) M(-1) s(-1) using a pulse radiolysis technique. DSePA shows GPx activity with higher substrate specificity towards peroxides than thiols. The cytotoxicity of DSePA was studied in lymphocytes and EL4 tumor cells and the results showed that DSePA is nontoxic to these cells at the concentrations employed. These results when compared with two well-known selenium compounds, sodium selenite and ebselen, indicated that DSePA, although it shows lesser GPx activity, has higher free radical scavenging ability and lesser toxicity.     

Relationships between intracellular vitamin E, lipid peroxidation, and chemical toxicity in hepatocytes

The cellular content of vitamin E was measured in isolated rat hepatocytes exposed to various types of chemical injury. Vitamin E was determined as alpha-tocopherol by HPLC with in-line uv and electrochemical detection. The cytotoxicity of diquat, a redox cycling compound, was accompanied by a decrease in cellular alpha-tocopherol and a stimulation of lipid peroxidation. Both the loss of alpha-tocopherol and the accumulation of lipid peroxidation products could be prevented by addition of either the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) or the reducing agent dithiothreitol (DTT). DTT also prevented the oxidation of soluble and protein thiols and completely protected against cytotoxicity, while DPPD addition only delayed the onset of hepatocyte death. Cytotoxic doses of the naphthoquinone, menadione, and the pyridine compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-pyridinium ion did not deplete alpha-tocopherol levels, nor did they result in significant lipid peroxidation. On the other hand, a peroxidizing, but noncytotoxic dose of ADP-Fe3+ rapidly decreased cellular alpha-tocopherol levels. These data demonstrate that cellular alpha-tocopherol loss is neither a prerequisite for, nor a necessary consequence of toxicity. Moreover, a substantial depletion (ca. 50%) of alpha-tocopherol does not necessarily result in cell death. Although alpha-tocopherol protects against the oxidation of cellular lipids, the maintenance of hepatocyte alpha-tocopherol content does not prevent the oxidation of soluble and protein thiols. These other targets of oxidative damage seem to play a more critical role in hepatocyte toxicity.     

Effects of antioxidants on oxygen toxicity in vivo and lipid peroxidation in vitro.

Convulsions and pulmonary damage result when animals are exposed to hyperbaric oxygen at pressures above about 300 kPa. Several hydroxyl radical scavengers (namely dimethylsulphoxide, dimethylthiourea and mannitol), the iron chelator desferrioxamine and the lipid antioxidant butylated hydroxytoluene were tested for possible protection against such hyperbaric oxygen toxicity. Dimethylthiourea and dimethylsulphoxide prolonged the latency to the first convulsion, but, surprisingly, dimethylthiourea very significantly increased pulmonary damage at both pressures used (515 and 585 kPa). Desferrioxamine also slightly increased lung damage at 585 kPa. Other antioxidants did not alter neurotoxicity or pulmonary toxicity induced by hyperbaric oxygen at 515 or 585 kPa. The antioxidants were also tested for their ability to inhibit lipid peroxidation (TBARS formation) in vitro. Desferrioxamine (5 and 50 microM), and butylated hydroxytoluene (0.1 mM and 1 mM) greatly inhibited TBARS formation in brain and lung homogenates incubated at 37 degrees. None of the hydroxyl radical scavengers affected TBARS levels in homogenates. There was no correlation between in vitro inhibition of lipid peroxidation and in vivo protection against oxygen toxicity.     

Protection from CCl4 toxicity by prestimulation of hepatocellular regeneration in partially hepatectomized gerbils

The present investigation was undertaken to test our hypothesis that the slow responses of hepatocellular regeneration and tissue repair after CCl4-induced liver injury are responsible for the high sensitivity of gerbils to the hepatotoxic and lethal effects of CCl4. These studies were conducted in normal and actively regenerating livers using male gerbils 5 or 15 days after partial (2/3) hepatectomy (PH5 and PH15, respectively), or those undergoing sham operation (SH). An LD50 dose of CCl4 (80 microL/kg, i.p.) resulted in a mortality (21%) significantly (P less than 0.05) less than 50% in PH5 gerbils 48 hr after CCl4 administration, whereas the mortality observed in PH15 or SH gerbils was not significantly different from 50%. The elevations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were significantly (P less than 0.05) less in PH5 gerbils than in PH15 or SH groups after the administration of either the LD50 dose or a low dose (15 microL/kg) of CCl4. Histopathological and histomorphometric examinations also indicated that CCl4-induced liver injury was less severe in PH5 gerbils than in the PH15 and SH groups. The hepatic microsomal cytochrome P450 content measured before CCl4 administration in the PH5 gerbils was decreased (26%) significantly (P less than 0.05) as compared with the SH group, but was not significantly different from that of PH15 gerbils. In vivo metabolism of 14CCl4 and lipid peroxidation in liver tissue were not significantly different among the various groups. Therefore, the protection against CCl4 toxicity observed in PH5 gerbils is unlikely to be due to decreased bioactivation of CCl4 or lipid peroxidation in that group. [3H]Thymidine incorporation into hepatocellular nuclear DNA was 4- to 5-fold higher in PH5 gerbils than in the PH15 and SH groups, indicating active hepatocellular proliferation in PH5 gerbils. [3H]Thymidine incorporation was further increased significantly (P less than 0.05) 24 hr after challenge with a low dose of CCl4 in PH5 gerbils, whereas it remained low until 48 hr after the CCl4 injection in the PH15 or SH group. The protection against CCl4 toxicity afforded by partial hepatectomy was closely associated with active hepatocellular regeneration. The overall results confirm the concept that the high sensitivity of gerbils to CCl4 is due to very sluggish hepatocellular regeneration and tissue repair response to the CCl4-induced liver injury.     

Intracellular Ca2+ mediating biodefense signal on CCl4-induced autoprotection

To study a role of intracellular Ca2+ on protection effects in animals against intoxication, that is, autoprotection. When carbon tetrachloride (CCl4) of 0.2 ml/kg was i.p. administered daily to male Donryu rats, the serum alanine aminotransferase (ALT) activity rose to a maximal level one day later, and then decreased to the control level within three days. Thus, the animals were not received any intoxication effect of CCl4 due to second, third and fourth administrations. Thus, animals have a sensitive and non-sensitive periods against intoxication of CCl4. In non-sense period, the animals were protective against the poison. Such protection is an autoprotection. To study a role of intracellular Ca2+ on CCl4-induced autoprotection, we measured the intracellular Ca2+ in hepatocytes isolated from rats after daily CCl4 i.p. administration. Then, the peaks of the intracellular Ca2+ centered on three days and twelve hours after the first CCl4 administration from the first to fourth administration. These results show that the peak of the intracellular Ca2+ is perfectly corresponding with the trough of the serum ALT; that is, the protective period in animals against intoxication. Therefore, this result means that intracellular Ca2+ can mediate biodefense signal caused by CCl4 intoxication.