Hepatotoxic interactions of ethanol with allyl alcohol or carbon tetrachloride in rats.

To assess whether potential toxic interactions occur between ethanol and allyl alcohol or carbon tetrachloride following subacute, concurrent chemical exposure, male Fischer 344 rats, approximately 70 d of age, were given ethanol at 0, 0.05, 0.1, 0.2, or 0.5 ml/kg in corn oil daily by gavage for 14 d (ETOH group), or the same levels of ethanol with 21 mg allyl alcohol/kg (ALAC group), or the same levels of ethanol with 20 mg carbon tetrachloride/kg (CCL4 group). Hepatic response was assessed 24 h after the last dose. Interactions were evaluated by comparing the ETOH group with either the ALAC group or the CCL4 group using multivariate analysis of variance procedures. No statistically significant interaction was seen between the ETOH group and the ALAC group at the dosages used. Although an interaction between ethanol and carbon tetrachloride given simultaneously was not statistically significant, a small interactive effect on weight gain from d 0 to termination was apparent (p = .057). Exposure to ethanol alone resulted in a concentration-dependent decrease in absolute and relative liver weight, with a threshold between 0.05 and 0.1 ml/kg. There was no histopathological evidence of hepatic damage with ethanol alone, and no effect on hepatic cytochrome P-450 and glutathione levels or on serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALK). Exposure to allyl alcohol alone resulted in significant increases in absolute and relative liver weights, liver glutathione, and periportal hepatocellular vacuolar degeneration. Exposure to carbon tetrachloride alone resulted in significant increases in absolute and relative liver weight, serum levels of ALT, AST, and ALK, and centrilobular hepatocellular vacuolar degeneration and necrosis. These observations indicate that subacute, concurrent exposure of ethanol with carbon tetrachloride or allyl alcohol at ethanol levels comparable to those reported in gavage vehicles did not result in interactive toxicity.     

The antihepatotoxic activity of dithiocarb as compared with six other thio compounds in mice

In mice, diethyldithiocarbamate (dithiocarb, 100 mg/kg i.p.) completely prevented the increments of serum enzyme activities (GOT, GPT, SDH) induced by oral administration of carbon tetrachloride (0.1 ml/kg), allyl alcohol (0.05 ml/kg), bromobenzene (0.25 ml/kg), and thioacetamide (50 mg/kg). In this respect, cysteine (200 mg/kg i.p.) was active against CCl₄ and bromobenzene, cysteamine (100 mg/kg i.p.) against CCl₄ and allyl alcohol, penicillamine (100 mg/kg i.p.) against allyl alcohol, thiazolidine carbonic acid (100 mg/kg i.p.) against bromobenzene, and thioctic acid (100 mg/kg i.p.) against allyl alcohol and thioacetamide. Dimercaprol (100 mg/kg i.p.) had a weak antidotal effect only against allyl alcohol poisoning. None of the tested antidotes inhibited serum enzyme elevations evoked by dimethyl nitrosamine (100 mg/kg p.o.). These findings prove the antihepatotoxic activity of diethyldithiocarbamate to be superior to that of all other thio compounds under observation.The lowest dose of dithiocarb active against carbon tetrachloride was 25 mg/kg i.p. The dose-response curves for serum-enzyme elevations induced by carbon tetrachloride (0.1–4 ml/kg p.o.) were shifted to the right under the influence of dithiocarb indicating a competitive antagonism. Dithiocarb (100 mg/kg i.p.) depressed the p-hydroxylation of aniline in the 9000 x g liver homogenate supernatant of mice by about 55%. Thus, the antihepatotoxic activity of dithiocarb seems to be the consequence of a decreased oxidase activity.     

Pentoxifylline attenuates bacterial lipopolysaccharide-induced enhancement of allyl alcohol hepatotoxicity.

Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-100 microg/kg) enhance the hepatotoxicity of allyl alcohol in male Sprague-Dawley rats. This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown. Since Kupffer cells produce tumor necrosis factor-alpha (TNF alpha) upon exposure to LPS, and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF alpha contributes to LPS enhancement of allyl alcohol hepatotoxicity. Rats were treated with LPS (10-100 microg/kg iv) 2 h before allyl alcohol (30 mg/kg ip). Co-treatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma. Treatment with LPS caused an increase in plasma TNF alpha concentration, which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF alpha serum (1 ml/rat iv) one h prior to LPS. Only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF alpha serum had no effect. Exposure of cultured hepatocytes to LPS (1-10 microg/ml) or to TNF alpha (15-150 ng/ml) for 2 h did not increase the cytotoxicity of allyl alcohol (0.01-200 microM). These data suggest that neither LPS nor TNF alpha alone was sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol. Furthermore, hepatocytes isolated from rats treated 2 h earlier with LPS (i.e., hepatocytes which were exposed in vivo to TNF alpha and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from na?ve rats. These data suggest that circulating TNF alpha is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects.     

Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced liver injuries.

The antioxidant action of medicinal herbs used in Ghana for treating various ailments was evaluated in vitro and in vivo. Five plants, Desmodium adscendens, Indigofera arrecta, Trema occidentalis, Caparis erythrocarpus, and Thonningia sanguinea were tested for their free radical scavenging action by their interaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH). Of these five plants, only Thonningia sanguinea was found to scavenge the DPPH radical. Lipid peroxidation in liver microsomes induced by H2O2 was also inhibited by T. sanguinea. The hepatoprotective effect of T. sanguinea was studied on acute hepatitis induced in rats by a single dose of galactosamine (GalN, 400 mg/kg, IP) and in mice by carbon tetrachloride (CCl4, 25 microl/kg, IP). GalN induced hepatotoxicity in rats as evidenced by an increase in alanine aminotransferase (ALT) and glutathione (GSH) S-transferase activities in serum was significantly inhibited when T. sanguinea extract (5 ml/kg, IP) was given to rats 12 hr and 1 hr before GalN treatment. The activity of liver microsomal GSH S-transferase, which is known to be activated by oxidative stress, was increased by the GaIN treatment and this increase was blocked by T. sanguinea pretreatment. Similarly, T. sanguinea pretreatment also inhibited CCl4-induced hepatotoxicity in mice. These data indicate that T. sanguinea is a potent antioxidant and can offer protection against GalN- or CCl4-induced hepatotoxicity.     

Primary cultures of human hepatocytes as a tool in cytotoxicity studies: cell protection against model toxins by flavonolignans obtained from Silybum marianum

The aim of this study was to evaluate the cytoprotective effects upon primary human hepatocytes of silymarin extract and its main flavonolignans following exposure to the cytotoxic actions of model toxins. The conditions for the hepatocyte intoxication were optimised for allyl alcohol, carbon tetrachloride, D-galactosamine and paracetamol. Silymarin extract, silychristin and silydianin did not exert cytotoxicity (10-100 microM), whereas silybin and isosilybin at higher concentrations and after longer incubation periods were cytotoxic. All main flavonolignans of silymarin tested displayed concentration-dependent cytoprotection against the toxic effects of both allyl alcohol and carbon tetrachloride but neither paracetamol nor galactosamine. The best protection was achieved by silydianin and silychristin and to a lesser degree by silymarin, while silybin and isosilybin were less effective. It is concluded that these differing outcomes result from the varying abilities of the Silybum marianum substances tested to stabilize the cell membrane, exert antioxidant properties and exhibit intrinsic toxicity.     

Preventive effect of neutropenia on carbon tetrachloride-induced hepatotoxicity in rats

The preventive effect of neutropenia on carbon tetrachloride (CCl4)-induced hepatotoxicity was examined in rats. In rats treated once with CCl4 (1 ml kg(-1), i.p.), the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indices of liver cell damage, and the hepatic activity of myeloperoxidase (MPO), an index of tissue neutrophil infiltration, increased at 6 h after the intoxication and further increased at 24 h. The liver of CCl4 -treated rats showed an increase in the concentration of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, and decreases in superoxide dismutase (SOD) activity and reduced glutathione (GSH) concentration at 6 h after the intoxication followed by a further increase in TBARS concentration and further decreases in SOD activity and GSH concentration at 24 h with increased xanthine oxidase (XO) activity at 24 h. Neutropenic treatment with anti-rat neutrophil antiserum (2 ml kg(-1), i.p.) at 0.5 h after CCl4 intoxication attenuated the increases in serum ALT and AST activities and hepatic MPO activity and TBARS concentration and the decreases in hepatic SOD activity and GSH concentration found at 6 and 24 h after CCl4 intoxication and the increase in hepatic XO activity found at 24 h after the intoxication. This neutropenia reduced the necrotic and degenerative changes with inflammatory cell infiltration in the liver cell of CCl4 -treated rats. These results indicate that neutropenia prevents CCl4 -induced hepatotoxicity in rats by attenuating the disruption of hepatic reactive oxygen species metabolism mediated by neutrophils accumulating in the liver tissue.     

Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells.

The modulation of garlic oil (GO) and three allyl compounds, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), on the antioxidation system in rat livers and red blood cells was examined. Rats were orally administered GO (200 mg/kg body weight), DAS (20, 80 mg/kg body weight), DADS (80 mg/kg body weight) or DATS (70 mg/kg body weight) three times a week for 6 weeks. Control rats received corn oil (2 ml/kg body weight) alone. GO, DADS and DATS treatment significantly increased the glutathione (GSH) content (48-84%) in red blood cells (P < 0.05). DATS displayed a greater enhancement than GO and DADS (P < 0.05). Hemolysis induced by tert-butyl hydroperoxide was not suppressed by GO or allyl compound treatment although higher GSH content was evident. Hepatic GSH was not influenced by garlic components. In rat livers, DADS and DATS significantly increased the activity of GSH reductase (46 and 54%, respectively) and of GSH S-transferase (GST) (63 and 103%, respectively), but decreased the GSH peroxidase activity (27 and 28%, respectively). In contrast, GSH reductase and GST activities in the DAS group, either 20 or 80 mg/kg body weight, were similar to the control group. A decrease of GSH peroxidase activity was observed in rats dosed with 80 mg/kg body weight (P < 0.05). An increase in GST activity and a decrease in GSH peroxidase activities were also noted in GO-treated rats (P < 0.05). In red blood cells, three GSH-related antioxidant enzyme activities were not affected by garlic oil and its organosulfur components. Immunoblot assay showed that, accompanying the increase in hepatic GST activity, GO, DADS, DAS (80 mg/kg body weight) and DATS increased the expression of GST Ya, Yb1 and Yc proteins. Results indicate that GO and three allyl compounds play a differential role in modulation of the GSH-related antioxidant system in rat livers and red blood cells.     

Acetaminophen hepatotoxicity in aging rats

Severity of liver damage 24 hr after i.p. administration of acetaminophen in doses of 0.4 and 0.8 g/kg was evaluated in male Fischer 344 rats at 4, 14 and 25 months of age. Both doses of acetaminophen produced significant elevations of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities in 4-month-old rats. Enzyme release was somewhat diminished in old age. Hepatic glutathione (GSH) and microsomal cytochrome P-450 concentrations were decreased in rats that received 0.8 g/kg of acetaminophen. The decreases occurred in young-adult and middle-aged rats, but not in old rats. The results demonstrated that old age does not enhance the hepatotoxic effects of acetaminophen in male Fischer 344 rats.     

Chemically-induced alteration of UDP-glucuronic acid concentration in rat liver

Since many xenobiotics alter hepatic UDP-glucuronosyltransferase activity, their effect on UDPGA concentration was determined. Rats were pretreated with: 1) microsomal enzyme inducers (7,8-benzoflavone, benzo(a)pyrene, butylated hydroxyanisole, isosafrole, 3-methylcholanthrene, phenobarbital, pregnenolone-16 alpha-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin, trans-stilbene oxide); 2) inhibitors of microsomal enzymes (cobaltous chloride, piperonyl butoxide, SKF 525-A, borneol, galactosamine); 3) hepatotoxins (allyl alcohol, aflatoxin B1, alpha-naphthylisothiocyanate, bromobenzene, cadmium chloride, carbon tetrachloride, 1,1-dichloroethylene), and 4) commonly used anesthetics (pentobarbital, urethane, diethyl ether, halothane, enflurane, methoxyflurane). Rats were decapitated before removal of the liver. All inducers except PCN and isosafrole increased UDPGA 36-85% above control. Mixed-function oxidase inhibitors had no effect whereas borneol and galactosamine reduced UDPGA 85-90%. Aflatoxin B1 and cadmium produced decreases of 59 and 25%, respectively. Hepatic UDPGA content was diminished 70-95% after exposure to the inhalation anesthetics, whereas the other anesthetics reduced UDPGA about 25%. Thus, numerous xenobiotics alter the concentration of UDPGA in rat liver, which may influence the rate of glucoronidation.     

Hepatotoxin-induced hypertaurinuria: a proton NMR study

The urinary excretion of taurine by rats after dosing with various hepatotoxins has been investigated by¹H NMR spectroscopy. After single hepatotoxic doses of hydrazine, carbon tetrachloride, 1-naphthylisothiocyanate, or thioacetamide there was biochemical and histopathological evidence of hepatic damage. Proton NMR spectroscopy of the urine collected for 24 h after dosing from these animals revealed a marked elevation in taurine (control 11.9 mole/h/kg) after dosing with thioacetamide (42.2 mole/h/kg), carbon tetrachloride (52.5 gmmole/ h/kg), 1-naphthyl-isothiocyanate (80.4 mole/h/kg) and hydrazine (52.9 mmole/h/kg). After allyl alcohol administration there was no increase in taurine excretion (7.5 mol/h/kg). The excretion of taurine after hydrazine administration was dose related. High resolution proton NMR spectroscopic analysis of urine also revealed resonances from several metabolites of hydrazine, an N-acetylcysteine conjugate of allyl alcohol, and acetamide as a metabolite of thioacetamide after dosing with the respective compounds. Changes in endogenous substances that may be related to the pathological events were also detected, such as a decrease in the excretion of 2-oxoglutarate and citrate after both hydrazine and carbon tetrachloride administration. The results confirm that proton NMR spectroscopic analysis of urine is a powerful analytical tool for the evaluation and study of toxic substances. Furthermore, measurement of urinary taurine may provide a non-invasive indicator of acute hepatic damage with certain classes of hepatotoxins.     

Rat hepatic mitochondria are more sensitive to allyl alcohol than are those of mice

The hepatotoxic effects of allyl alcohol with particular reference to mitochondrial morphology and function were compared in male CD1 mice and male CD rats 24 h after 0.05 ml/kg i.p. in corn oil. As already noted by others, allyl alcohol-treated rats usually showed histologic evidence of tissue necrosis when hematoxylin-eosin-stained tissue sections were examined whereas mouse tissue sections did not. The serum glutamic pyruvic transaminase (SGPT) activities were significantly elevated in both mice and rats but to a much greater extent in the latter. Pentobarbital sleeping time was significantly increased over that of corn oil control groups in rats but decreased in mice. In rats electron microscopy showed mitochondria which contained flocculent densities. State 4 respiration was not altered by allyl alcohol in rats, but state 3 respiration was significantly depressed indicating an absence of respiratory control and an inability to perform energy coupling. In allyl alcohol-treated mice the isolated mitochondria were found to be primarily in a condensed form. Except for the effect on pentobarbital sleeping time and SGPT, no other findings were different from those in control groups given only corn oil. When the dose of allyl alcohol in mice was increased to 0.15 ml/kg in an attempt to elicit more severe signs of hepatotoxicity, there was a high mortality in the first 24 h period without histologic evidence of liver necrosis. Thus, we confirm that at equivalent doses, male rats are more sensitive to the hepatotoxic effects of allyl alcohol than are male mice, and extend the generalization to the liver mitochondria of the 2 species.     

A novel biologically active seleno-organic compound--VI. Protection by ebselen (PZ 51) against galactosamine/endotoxin-induced hepatitis in mice

Male albino NMRI mice were given 700 mg/kg galactosamine and 33 micrograms/kg salmonella endotoxin intraperitoneally. After 9 hr, serum sorbitol dehydrogenase activity had risen from 60 to 7320 U/l, SGOT from 90 to 5580, and SGPT from 70 to 10,440. When a similar dose of galactosamine alone or endotoxin alone was given, no significant liver injury was found. Animals pre-treated with an oral dose of ebselen (600 mg/kg 1-3 hr before galactosamine/endotoxin administration) were fully protected against this type of hepatitis. When pretreated 1 hr before intoxication with different doses of ebselen, significant dose-dependent reduction of serum enzyme activities was observed at doses higher than 1 mg/kg. After pre-treatment with 6 mg/kg ebselen, no biochemical or histological signs of liver lesions were detectable 36 hr after intoxication. In order to comparatively evaluate the model used, several established anti-inflammatory drugs were administered at doses which showed 50% effectiveness in preventing carageenan paw edema. A dose of 200 micrograms/kg dexamethasone, or 9 mg/kg indomethacin abolished galactosamine/endotoxin-induced enzyme release in our animals, as did the lipoxygenase pathway inhibitor diethylcarbamazine (78 mg/kg). In contrast, administration of cyclooxygenase pathway inhibitors such as aspirin (220 mg/kg) or ibuprofen (45 mg/kg) failed to prevent hepatitis. The effect of ebselen was also investigated in four different models of acute drug-induced liver damage. A dose of 600 mg/kg of the organic selenium compound was ineffective or weakly active in benzo(alpha)pyrene- or phenobarbital-treated mice which were intoxicated by intraperitoneal administration of 350 or 400 mg/kg body weight of paracetamol. Similarly negative results were obtained against bromobenzene-induced hepatotoxicity (520 mg/kg bromobenzene i.p.), carbon tetrachloride intoxication (3.2 g/kg), or allyl alcohol-induced liver damage (60 mg/kg). The selective efficacy of ebselen against galactosamine/endotoxin induced liver damage is interpreted in terms of its recently recognized ability to inhibit the formation of leukotrienes.     

Dose-dependent biliary and renal excretion of paracetamol in the rat.

In rats, the biliary and renal excretion of paracetamol changed in a dose-dependent manner. After an oral nonhepatotoxic dose of 200 mg/kg paractamol, the drug excreted in the bile amonted to only 5.5% within 24 h, whereas 72% of the dose were excreted into the urine. Following an oral hepatoxic dose (1,000 mg/kg), the biliary excretion of thotal paracetamol was enhanced to 13.5% whereas the renal elimination was reduced to 51% of the dose. After a nontoxic intravenous treatment with paracetamol (25-100-400 mg/kg), both the excretion of paracetamol conjugates into the bile and the elimination of free paracetamol into the urine were augmented in a dose-dependent manner. Hepatic damage due to carbon tetrachloride pretreatment (0.5 ml/kg i.p. 24 h before 100 mg/kg paracetamol i.v.) diminished both the bile flow and the biliary excretion of paracetamol conjugates, whereas the renal elimination was not affected.     

Activation of the microsomal glutathione-S-transferase and reduction of the glutathione dependent protection against lipid peroxidation by acrolein

Allyl alcohol is hepatotoxic. It is generally believed that acrolein, generated out of allyl alcohol by cytosolic alcohol dehydrogenase, is responsible for this toxicity. The effect of acrolein in vitro and in vivo on the glutathione (GSH) dependent protection of liver microsomes against lipid peroxidation, and on the microsomal GSH-S-transferase (GSH-tr) in the rat was determined. In vitro incubation of liver microsomes with 5 mM acrolein for 30 sec resulted in a 2-fold activation of the GSH-tr. This activation probably proceeds via alkylation of the thiol group of the GSH-tr. In vivo administration of 1.1 mmol allyl alcohol/kg to rats did also result in a 2-fold stimulation of the GSH-tr activity. Administration of 375 mg pyrazole/kg, an inhibitor of the alcohol dehydrogenase, thus reducing the acrolein formation, prevented the in vivo stimulation of GSH-tr by allyl alcohol. This indicates that the activation of GSH-tr in vivo by allyl alcohol probably also proceeds via alkylation of the thiol group of the GSH-tr by acrolein. GSH protects liver microsomes against lipid peroxidation, probably via a free radical reductase that reduces vitamin E radicals at the expense of GSH. Incubating liver microsomes for 30 min with 0.1 mM acrolein reduced the GSH dependent protection against lipid peroxidation, probably because an essential thiol group(s) on the free radical reductase is alkylated. In vivo administration of allyl alcohol did not reduce the GSH dependent protection of the microsomes. Probably the thiol group(s) located on the free radical reductase is less accessible or less reactive than the thiol group on the GSH-tr. After administration of allyl alcohol we found no evidence for in vivo lipid peroxidation. Therefore we could not evaluate the importance of the GSH dependent protection against lipid peroxidation in vivo.     

Protective effect of (+)cyanidanol-3 in acute liver injury induced by galactosamine or carbon tetrachloride in the rat

Summary Pretreatment of rats with (+)cyanidanol-3 decreases the alterations in liver function tests (transaminase and bilirubin) as well as the accumulation of hepatic triglycerides induced by acute doses of galactosamine or carbon tetrachloride.This action was related to the dose of (+)cyanidanol-3 administered. The lowest effective doses were 3 intraperitoneal injections of 125 mg/kg administered before galactosamine or carbon tetrachloride.In the case of carbon tetrachloride intoxication, in vivo administration of (+)cyanidanol-3 reduced to a slight extent the absorption of conjugated dienes produced in liver microsomal lipid.This observation confirms that (+)cyanidanol-3 is able to prevent lipid peroxidation in vivo. As the protective effect of (+)cyanidanol-3 becomes apparent in two types of intoxication which are very different in their primary mechanisms of action, it is suggested that, the flavonoid also acts on a later stage of the process leading to necrosis and steatosis of the liver.     

Involvement of nonparenchymal cells in oxygen-dependent hepatic injury by allyl alcohol.

Allyl alcohol injury to hepatocytes in the perfused liver is oxygen-dependent. It is not known if this injury involves direct action of allyl alcohol on hepatocytes or requires participation of other cell types (e.g., Kupffer cells) present in the liver. Accordingly, the action of allyl alcohol (100-500 microM) on isolated hepatocytes was studied using cells maintained at either 95 or 21% O2. Allyl alcohol toxicity, as indexed by trypan blue uptake, lactate dehydrogenase release, and ATP content, did not differ in the two groups of cells, suggesting that O2 dependency of allyl alcohol toxicity involves other cell types. Administration of allyl alcohol (30 or 40 mg/kg, ip) to rats caused extensive hepatic necrosis localized primarily to periportal regions. To test the involvement of Kupffer cells in the genesis of this injury, male rats (200-350 g) were treated with gadolinium chloride (GdCl3, 10 mg/kg, iv) which diminishes Kupffer cell function and number. The extent of hepatic damage assessed by light microscopy and serum enzymes, aspartate aminotransferase and alanine aminotransferase, was markedly attenuated by pretreatment of rats with GdCl3 24 hr prior to allyl alcohol injection. Thus, O2-dependent hepatic necrosis caused by allyl alcohol involves the presence of Kupffer cells. Since GdCl3 did not prevent toxicity in the perfused liver, circulating blood elements may also contribute to injury of the liver by allyl alcohol in vivo.     

Effect of phenobarbital treatment on carbon tetrachloride-mediated cytochrome P-450 loss and diene conjugate formation.

The effect of phenobarbital treatment on the linkage between carbon tetrachloride-mediated cytochrome P-450 loss and lipid peroxidation in rat liver microsomes was studied. Male Sprague-Dawley rats, pretreated with 3 daily i.p. doses of phenobarbital (50 mg/kg) or saline, were orally dosed with carbon tetrachloride (0.01-2.5 ml/kg), with liver microsomes prepared at 7.5-180 min after carbon tetrachloride treatment. In vivo cytochrome P-450 loss displayed pseudo-first-order kinetics, and the initial rates of diene conjugate formation were saturable with dose. Phenobarbital pretreatment decreased the in vivo t0.5,max from 27.0 to 15.6 min, and increased the Kd,app from 0.78 to 1.30 ml/kg for carbon tetrachloride mediated cytochrome P-450 loss. Phenobarbital had no effect on the in vivo Vmax (1.03 to 1.04 delta OD232 nm/min/mg phospholipid) for carbon tetrachloride mediated diene conjugate formation, but decreased the Km,app from 0.22 to 0.10 ml/kg. These results are consistent with destruction of cytochrome P-450 heme resulting from a metabolite which does not leave the site of generation, and with phenobarbital pretreatment enhancing the initiation of lipid peroxidation.     

Potentiation of carbon tetrachloride hepatotoxicity by phenylpropanolamine

Hepatic necrosis produced by carbon tetrachloride (0.02, 0.06, or 0.20 ml/kg, ip) in mice was found to be potentiated by simultaneous cotreatment with phenylpropanolamine (200 mg/kg, ip), a drug with catecholamine-like pharmacologic effects. The ability to potentiate carbon tetrachloride-induced hepatic necrosis was shared by a compound with agonist effects relatively selective for alpha 2-adrenoreceptors (clonidine, 5 mg/kg, ip), but not by specific alpha 1-adrenoreceptor agonists (phenylephrine, up to 100 mg/kg, ip and methoxamine, up to 50 mg/kg, ip) or by the beta-adrenoreceptor agonist isoproterenol (up to 100 mg/kg, ip). Yohimbine (5 mg/kg, ip), a selective alpha 2-adrenoreceptor antagonist, completely blocked the potentiating effect of phenylpropanolamine on carbon tetrachloride hepatotoxicity, providing further evidence that the increased hepatotoxic response with phenylpropanolamine cotreatment was mediated through alpha 2-adrenoreceptor stimulation. Four potential mechanisms for phenylpropanolamine potentiation of liver injury from carbon tetrachloride were examined: (1) increased concentrations of carbon tetrachloride in the liver from greater absorption or altered distribution; (2) diminished food consumption leading to a starvation-like increase in responsiveness to carbon tetrachloride; (3) impaired detoxification through a depletion of hepatic glutathione content; and (4) enhanced toxicity produced by elevated core body temperature. None of these potential mechanisms was supported by the experimental results. It is concluded that phenylpropanolamine and related compounds potentiate carbon tetrachloride hepatotoxicity through a mechanism involving alpha 2-adrenoreceptor stimulation that has yet to be identified.     

The influence of ethanol pretreatment on the effects of nine hepatotoxic agents

The hepatotoxic effects of carbon tetrachloride (0.01 ml/kg i.p.), thioacetamide (50 mg/kg intraperitoneally), paracetamol (0.5 g/kg intraperitoneally), and allyl alcohol (0.05 ml/kg intraperitoneally) as estimated by determination of serum enzyme activities (GOT, GPT, SDH) were enhanced in mice treated with one oral dose of 4.8 g/kg ethanol 16 hrs. previously. Pretreatment of mice with ethanol did not increase the hepatotoxic actions of bromobenzene (0.25 ml/kg intraperitoneally), phalloidin (1.5 mg/kg intraperitoneally), alpha-amanitin (0.75 mg/kg intraperitoneally), and praseodymium (12 mg/kg intravenously) though there was a trend to higher enzyme activities in the case of bromobenzene. In guinea-pigs ethanol also aggravated CCl4-induced liver damage, but only strengthened the hepatotoxic activity of D-galactosamine (150 mg/kg intraperitoneally). Treatment with 4.8 g/kg ethanol did not influence liver glutathione levels in mice but increased aniline hydroxylation in the 9000 x g liver homogenate supernatant of mice and guinea-pigs. A dose of 2.4 g/kg ethanol, on the other hand, neither increased aniline hydroxylase activity nor enhanced carbon tetrachloride-induced hepatotoxicity in mice. It is assumed that the enhanced sensitivity to hepatotoxic agents after treatment with ethanol may be due to an enhanced microsomal activation of these substances.     

A comparative 90-day toxicity study of allyl acetate, allyl alcohol and acrolein

Allyl acetate (AAC), allyl alcohol (AAL), and acrolein (ACR) are used in the manufacture of detergents, plastics, pharmaceuticals, and chemicals and as agricultural agents. A metabolic relationship exists between these chemicals in which allyl acetate is metabolized to allyl alcohol and subsequently to the highly reactive, alpha,beta-unsaturated aldehyde, acrolein. Due to the weaker reactivity of the protoxicants, allyl acetate and allyl alcohol, relative to acrolien we hypothesized the protoxicants would attain greater systemic exposure and therefore deliver higher doses of acrolein to the internal organs. By extension, the higher systemic exposure to acrolein we hypothesized should lead to more internal organ toxicity in the allyl acetate and allyl alcohol treated animals relative to those treated with acrolein. To address our hypothesis we compared the range of toxicities produced by all three chemicals in male and female Fischer 344/N rats and B6C3F1 mice exposed 5 days a week for 3 months by gavage in 0.5% methylcellulose. Rats (10/group) were dosed with 0-100mg/kg allyl acetate, 0-25mg/kg allyl alcohol, or 0-10mg/kg acrolein. Mice (10/group) were dosed with 0-125mg/kg allyl acetate, 0-50mg/kg allyl alcohol, or 0-20mg/kg acrolein. The highest dose of allyl acetate and acrolein decreased survival in both mice and rats. The primary target organ for the toxicity of all three chemicals in both species and sexes was the forestomach; squamous epithelial hyperplasia was observed following exposure to each chemical. In both species the highest allyl acetate dose group exhibited forestomach epithelium necrosis and hemorrhage and the highest dose of acrolein led to glandular stomach hemorrhage. Liver histopathology was the most apparent with allyl acetate, was also observed with allyl alcohol, but was not observed with acrolein. All chemicals had effects on the hematopoietic system with allyl acetate having the most pronounced effect. When dosed at quantities limited by toxicity, allyl acetate and allyl alcohol produce higher levels of urinary mercapturic acids than the minimally toxic dose of acrolein. This observation is likely due to biotransformation of allyl acetate and ally alcohol to acrolein that occurs after absorption and suggests that these chemicals are protoxicants that increase systemic exposure of acrolein. Increased systemic exposure to acrolein is likely responsible for the differences in hepatic toxicological profile observed with these chemicals.