Decreased in vivo acetaldehyde oxidation and hepatic aldehyde dehydrogenase inhibition in C57BL and DBA mice treated with carbon tetrachloride

Male $\text{C57BL}\text{6J}$ (C57) and $\text{DBA}\text{2J}$ (DBA) mice were dosed with 500 μl/kg ig CCl₄, 24 hr before a 3.25 g/kg ip dose of ethanol. The relative bioavailability of CCl₄ was similar in both mouse strains. CCl₄ pretreatment produced genotypically related decreases in blood ethanol elimination rates with DBA mice showing the greater decrease. Blood acetaldehyde concentrations were significantly elevated in the CCl₄-pretreated animals of both strains. DBA CCl₄-pretreated mice exhibited significantly higher blood acetaldehyde concentrations than C57 mice 1, 2, 3, and 4 hr after ethanol administration. CCl₄ pretreatment resulted in acetaldehyde elevations of approximately threefold in C57 mice four- to fivefold in DBA mice. Four hours after the ethanol dose, animals were sacrificed for enzymatic analyses of hepatic aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity. The specific activities and activity per gram of cytosolic and mitochondrial ALDH were decreased in both mouse strains. ADH activity was not significantly altered by CCl₄ pretreatment. Liver necrosis, as measured by serum alanine aminotransferase activity, was similar in both strains (approx fivefold increase). These data indicate that CCl₄ increased blood acetaldehyde concentrations by inhibiting hepatic cytosolic and mitochondrial ALDH and that the degree of blood acetaldehyde elevation is different in the two inbred mouse strains C57 and DBA.     

Metabolites and ketone body production following methyl n-butyl ketone exposure as possible indices of MnBK potentiation of carbon tetrachloride hepatotoxicity

While the biotransformation of methyl n-butyl ketone (MnBK) in animals is well characterized, little is known about the quantitative relationship between hepatic and plasma MnBK concentrations. This study provides such information and emphasizes the usefulness of MnBK metabolite quantification, as well as MnBK-induced metabolic ketosis for the biological monitoring of MnBK exposure in rats. Elimination of MnBK was followed 24 hr after oral administration (0.95, 1.90, and 5.70 mmol/kg in corn oil) to male Sprague-Dawley rats. Two metabolites [2-hexanol (2HOL), and 2,5-hexanedione (2,5HD)] were also monitored and their kinetics determined. These data were compared to ketone body (KB) concentrations found in plasma and liver during the same period. Plasma concentrations of MnBK and 2,5HD correlated well with those in the liver. This was not the case for 2HOL. MnBK, 2HOL, and 2,5HD were no longer detected in plasma and liver 18 hr after dosing. Meanwhile, a marked ketosis was observed from 12 to 24 hr. This ketotic state was due to an increase in beta-hydroxybutyrate (BOHB), while acetoacetate remained at its basal levels. These data indicate that MnBK can induce ketosis in rats and suggest that the resulting BOHB might be used as an alternative biological monitor of MnBK exposures at high concentrations.     

Ethanol decreases cadmium hepatotoxicity in rats: possible role of hepatic metallothionein induction

The present investigation examines the possibility that Cd and ethanol have a significant toxicological interaction. This examination was warranted as exposure to either chemical is known to compromise human health. Inasmuch as both chemicals affect the morphology, biochemistry, and physiology of liver, it seemed reasonable to consider liver as a possible site of interaction. Specifically, the hypothesis that ethanol alters the hepatotoxic action of Cd was evaluated. Accordingly, male rats were injected iv with hepatotoxic (3.0 mg/kg) or lethal (4.5 mg/kg) dosages of Cd, 24 hr after single-dose ethanol administration (7 g/kg, po). Cd-induced hepatotoxicity was assessed by measuring the activities of alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase in serum collected 10 hr after Cd injection. Lethality was assessed by recording the number of survivors over a 7-day period. Prior exposure to ethanol substantially reduced the lethal and hepatotoxic properties of Cd. Two mechanisms were evaluated in an effort to explain ethanol-induced suppression of Cd hepatotoxicity. Ethanol pretreatment was postulated to: (1) enhance Cd excretion in bile thereby decreasing hepatic Cd content and/or (2) reduce the interaction between Cd and target sites in liver such as organelles and cytosolic high-molecular-weight (HMW) proteins. The first proposed mechanism was incorrect as the biliary excretion of Cd was nearly abolished and the concentration of Cd in whole liver increased (33%) as a result of ethanol exposure. The second proposed mechanism was a plausible explanation of ethanol-induced suppression of Cd hepatotoxicity because ethanol pretreatment decreased (approximately 60%) the content of Cd in nuclei, mitochondria, and endoplasmic reticulum, and nearly eliminated the association of Cd with cytosolic HMW proteins. Reduction in the concentration of Cd in potential target sites of intoxication was caused by a metallothionein-promoted sequestration of Cd in cytosol.     

Lobar variation of carbon tetrachloride hepatotoxicity in the rat

The lobar variation m carbon tetrachloride-induced liver damage in the rat has been assessed by use of a systematic random sampling protocol and quantitative morphometry. A random inter-animal lobar variation in severity of damage was apparent, in contrast with the results obtained previously in which sampling bias, small animal numbers, and lack of fully quantitative measurement were apparent. The route of administration (oral vs. i.p.) did not influence these findings.     

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.     

Effect of hypoxia on carbon tetrachloride hepatotoxicity

The effect of hypoxia on carbon tetrachloride-induced hepatotoxicity was studied. Male rats were exposed to carbon tetrachloride for 2 hr in the presence of differing oxygen concentrations. Serum glutamate-pyruvate transaminase (SGPT) activities were measured 24 hr after the end of the exposure. Exposure of rats to 5000 ppm carbon tetrachloride in the presence of 100, 21,12, or 6% oxygen resulted in SGPT activities of 489, 420, 3768, and 1788 I.U./l respectively. Exposure of rats to air and 0, 1250, 2500, 5000, or 7500 ppm carbon tetrachloride gave SGPT activities of 35, 32, 69, 420, and 2188 I.U./l respectively; when 12% oxygen was used, the corresponding SGPT activities were 32, 665, 691, 3768, and 4200 I.U./l respectively. Exposure of rats to hypoxia produced histopathologically detectable condensation of hepatic cytoplasmic material, and exposure to 5000 ppm carbon tetrachloride in the presence of air produced mild centrilobular necrosis, which was much more severe when rats were exposed to 5000 pm carbon tetrachloride in the presence of 12% oxygen. Hepatic microsomal conjugated diene concentrations were increased by hypoxia and by exposure to carbon tetrachloride, but no synergistic interaction was observed. Hepatic microsomal cytochrome P-450 concentrations were decreased after exposure to carbon tetrachloride, but were the same after exposure to carbon tetrachloride and 12 or 21% oxygen. Hepatic carbon tetrachloride concentrations were the same in rats exposed to carbon tetrachloride in the presence of 12 or 21% oxygen; hepatic chloroform concentrations were higher in rats exposed to carbon tetrachloride in the presence of air than in the presence of 12% oxygen. The covalent binding of [¹⁴C]carbon tetrachloride metabolites to hepatic microsomal lipids and proteins was increased markedly by hypoxia as compared with normoxia. The covalent binding of metabolites of carbon tetrachloride to cellular macromolecules may play a role in the potentiation of carbon tetrachloride toxicity by hypoxia.     

Metformin protects against carbon tetrachloride hepatotoxicity in mice

In the present study, the hepatoprotective effect of metformin (Met), a dimethylbiguanide anti-hyperglycemic, was examined in a mouse model of liver damage induced by chronic repeated administration of carbon tetrachloride (CCl(4)) (5 microl/kg, twice a week for 12 weeks). Met, when given orally in drinking water at an estimated daily dose of 25 or 50 mg/kg for 10 weeks starting 2 weeks after CCl(4) challenge, protected against CCl(4) hepatotoxicity. The results indicate that the hepatoprotection afforded by Met treatment at a dose of 25 mg/kg against CCl(4) toxicity may at least in part be mediated by the enhancement of mitochondrial glutathione redox status.     

Temporal analysis of rat liver injury following potentiation of carbon tetrachloride hepatotoxicity with ketonic or ketogenic compounds

The administration of some ketonic or ketogenic compounds prior to a challenging dose of CCl4 potentiates the hepatic damage induced by this haloalkane. However, nothing is known about the recovery from the liver injury in these cases of chemically induced potentiation. To investigate this problem, we performed a temporal analysis of the hepatotoxic response of male Sprague-Dawley rats to CCl4 following a single pretreatment (p.o.) with: n-hexane, 2-hexanone, 2,5-hexanedione (15 mmol/kg in corn oil), isopropanol, acetone (33 and 34 mmol/kg in water, respectively); or the vehicle alone (10 ml/kg). They received, 18 h later, an i.p. injection of CCl4 (0.1, 0.75 or 1.0 ml/kg) and were killed 24-120 h later. Liver damage was assessed biochemically (ALT, OCT) and morphologically. A good correlation between biochemical and morphological results was observed. The ketonic or ketogenic compounds studied potentiated the liver injury produced by 0.1 ml/kg CCl4. Relative ranking orders regarding severity of maximal hepatic damage induced and time needed for complete recovery of liver injury were established; time of recovery was dependent on the maximal severity of the lesion, regardless of the potentiation. The results show that the temporal evolution of CCl4-induced liver injury is not markedly influenced by the administration of ketonic or ketogenic compounds as pretreatments, but rather depends on the severity of the maximal damage induced by the overall treatment.     

Effect of ethanol on carbon tetrachloride levels and hepatotoxicity after acute carbon tetrachloride poisoning

To study the effect of an acute dose of ethanol on carbon tetrachloride (CCl₄) concentration and hepatotoxicity, female rats received ethanol (2.5 ml/kg body wt.) either intragastrically or intraperitoneally following intragastric administration of CCl₄ (1.5 ml/kg body wt.). Three hours after acute CCl₄ intoxication there was a striking increase in CCl₄ concentration in animals treated simultaneously with ethanol intragastrically compared to those receiving ethanol intraperitoneally. This increase was significant (P<0.05) and amounted to 211% for blood, 236% for liver and 405% for fat tissue, whereas animals treated with CCl₄ alone showed CCl₄ concentrations in the range between the two other experimental groups. Serum activities of glutamate oxalacetate transaminase, glutamate pyruvate transaminase and glutamate dehydrogenase were found to be considerably higher in animals treated with the combination of CCl₄ and ethanol when compared to those receiving CCl₄ alone, showing that ethanol given intraperitoneally or intragastrically enhances CCl₄ hepatotoxicity. Since the intraperitoneal administration of ethanol led to a reduction rather than an increase in CCl₄ concentration in the early phase of intoxication, additional mechanisms independent of actual levels of CCl₄, such as direct effects of ethanol on the CCl₄ metabolizing enzyme of the membrane of the endoplasmic reticulum, have to be implicated in the pathogenesis of the potentiation of CCl₄ hepatotoxicity by ethanol.     

In vivo protective effect of Porphyra yezoensis polysaccharide against carbon tetrachloride induced hepatotoxicity in mice

To study the protective effect and possible mechanism of Porphyra yezoensis polysaccharide (PYP) in hepatotoxicity mice, acute liver injury was successfully induced by injecting 0.2% carbon tetrachloride (CCl(4)) intraperitoneally. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and liver homogenate, content of malondialdehyde (MDA), activities of total superoxide dismutase (T-SOD) in liver were measured by biochemical methods. Liver index was calculated and pathological changes of the liver tissue were observed microscopically. PYP was found to significantly decrease the activities of ALT and AST (P<0.05), to remarkably lower the liver indexes and MDA level in hepatical tissues in mice (P<0.05), and to upregulated the lower T-SOD level in liver homogenate (P<0.01). Furthermore, histologic examination showed that PYP could attenuate and the extent of necrosis, reduce the immigration of inflammatory cells. PYP plays a protective action against hepatotoxicity induced by CCl(4) in mice, and its mechanisms may be related to free radical scavenging, increasing SOD activities and anti-lipid peroxide.     

Modification of carbon tetrachloride hepatotoxicity by chemicals.

Cobaltous chloride (60 mg/kg, sc, daily for 2 days), which was found to effectively decrease the microsomal cytochrome P-450 content of mouse liver to approximately half of its normal value and which impaired the oxidative metabolism or hydroxylation of aminopyrine, ethylmorphine, and hexobarbital, offered no protection against CCl₄-induced liver damage. However, this hemoprotein inhibitor had no effect on the rate of reduction of cytochrome P-450 by NADPH and exerted a slight effect on aniline hydroxylation. SKF-525A (50 mg/kg, ip) also failed to protect against CCl₄ hepatotoxicity though it has been shown to inhibit the hydroxylation of a number of substrates. This inhibitor, a type I compound, was found to enhance cytochrome P-450 reduction by NADPH. Further studies revealed that CCl₄-induced hepatic injury could be prevented by phenazine methosulfate (2 mg/kg, ip, 5 doses at 0.5-hr intervals), which in vitro was found to inhibit NADPH-cytochrome c reductase noncompetitively. All of these findings are not satisfactorily explainable by electron transfer from NADPH-cytochrome c reductase to CCl₄ as the activation reaction for CCl₄ but are compatible with the hypothesis previously proposed by others that cytochrome P-450 is the critical site for CCl₄ activation.     

Endotoxin pretreatment protects against the hepatotoxicity of acetaminophen and carbon tetrachloride: role of cytochrome P450 suppression

Bacterial endotoxin (lipopolysaccharide, LPS) is known to potentiate the toxicity of many hepatotoxicants. However, exposure to a sublethal dose of LPS renders animals tolerant to a lethal dose of LPS, and protects against the toxicity of some chemicals. This study was designed to examine the effects of LPS pretreatment on acetaminophen- and carbon tetrachloride (CCl(4))-induced liver injury in LPS-sensitive C3H/OuJ and LPS-resistant C3H/HeJ mice. Pretreatment of male C3H/OuJ mice with a single injection of LPS (0. 1 mg/kg, ip, for 24 h) protected against the hepatotoxic effects of acetaminophen (400 mg/kg) and carbon tetrachloride (CCl(4), 30 mg/kg), as indicated by serum alanine aminotransferase activity. In contrast, pretreatment of C3H/HeJ mice with 0.1 or 10 mg/kg LPS afforded no protection against the hepatotoxic effects of acetaminophen and CCl(4). In an attempt to determine the mechanism of LPS-induced protection against acetaminophen- and CCl(4)-induced hepatotoxicity in C3H/OuJ mice, liver cytochrome P450 was determined 24 h after LPS injection. LPS treatment caused a 26% decrease in total P450 content in C3H/OuJ but not in C3H/HeJ mice. CYP3A-catalized testosterone 6 beta-, 2 beta-, and 15 beta-hydroxylation was decreased 40% by LPS only in C3H/OuJ mice. To determine whether the differences to LPS-response in the two stains of mice is mediated by a strain-related difference in the release of cytokines, mice were pretreated with interleukin-1 (IL-1 alpha, 5 x 10(5) U/mouse), and the hepatoprotection and hepatic P450 enzymes were examined. IL-1 alpha pretreatment equally protected against the hepatotoxicity of acetaminophen and CCl(4) in both strains, and suppressed the total microsomal P450 and P450 enzyme-catalyzed testosterone hydroxylation to a similar extent. In conclusion, LPS pretreatment suppressed hepatic cytochrome P450 enzymes and protected against the hepatotoxicity of acetaminophen and CCl(4) in LPS-sensitive C3H/OuJ mice, but not in LPS-refractory C3H/HeJ mice. This protective effect of LPS appears to be mediated through the release of cytokines such as IL-1 alpha, which in turn suppresses the cytochrome P450 responsible for the activation of acetaminophen and CCl(4) to reactive metabolites.