Detection of free radical formation in various tissues after acute carbon tetrachloride administration in gerbil

alpha-phenyl N-tert-butyl nitrone (PBN) was used as a spin-trapping agent to search for free radical formation in various tissues of gerbils. A significantly large amount of free radicals was detected in liver, kidney, heart, lung and testis after a single intraperitoneal dose of CCl4. We have also detected free radical formation in the brain and blood, although the number of spins was considerably smaller than those found in the other tissues. Free radicals formed in brain tissue after an oxidative insult may give strong supporting evidence for the free radical theory of aging.     

Hepatic collagenolytic activity in rats after carbon tetrachloride poisoning

1. Collagenolytic activity towards acid-soluble collagen labelled with [(14)C]-proline was assayed in rat liver with and without carbon tetrachloride poisoning. The products of enzymic digestion were found to be free amino acids and peptides. 2. The hepatic collagenolytic activity increased under conditions of single-dose and subacute carbon tetrachloride poisoning, and correlated with hydroxyproline content. The highest activity was found during recovery from subacute poisoning. 3. Under the same experimental conditions, hepatic acid-proteinase activity changed independently of the collagenolytic activity and also of hepatic hydroxyproline content. 4. The increased collagenolytic activity during carbon tetrachloride poisoning was found mainly in the supernatant fraction. 5. The ratio of the collagenolytic activity to hepatic hydroxyproline content increased during recovery from single-dose and subacute poisoning, and decreased during subacute poisoning.     

Vinpocetine ameliorates acute hepatic damage caused by administration of carbon tetrachloride in rats

Vinpocetine is a widely used drug for the treatment of cerebrovascular and memory disorders. This study aimed to investigate the effect of vinpocetine on the acute hepatic injury caused in the rat by the administration of CCl4 in vivo. Vinpocetine (2.1, 4.2, 8.4 mg/kg) or silymarin (30 mg/kg) was given once daily orally simultaneously with CCl4 and for 15 days thereafter. Liver damage was assessed by determining serum enzyme activities and hepatic histopathology. Stained sections were subjected to morphometric evaluation using computerized image analyzer. The results showed that vinpocetine administered to CCl4-treated rats decreased the elevated alanine aminotransferase (ALT) by 49.3, 58.1 and 63.6%, aspartate aminotransferase (AST) by 10.5, 22.6 and 27.2% and alkaline phosphatase (ALP) by 52.5, 59.6 and 64.9%, respectively, and in a dose-dependent manner. Meanwhile, silymarin reduced elevated ALT, AST and ALP levels by 53.1, 26.9 and 66%, respectively. Histological examination of liver specimens revealed a marked reduction in liver cell necrosis in vinpocetine and silymarin-treated rats compared with vehicle-treated CCl4-treated rats. Quantitative analysis of the area of damage showed 85.3% reduction in the area of damage after silymarin and 72.2, 78.9 and 82.6% reduction after vinpocetine treatment at 2.1, 4.2, 8.4 mg/kg, respectively. It is concluded that administration of vinpocetine in a model of CCl4-induced liver injury in rats reduced liver damage. The reduction obtained by 4.2 mg/kg of vinpocetine was similar to that obtained by 30 mg/kg silymarin. Therefore, it is suggested that vinpocetine might be a good pharmacological agent in the treatment of liver disease besides its neuroprotective effects.     

Lipoperoxidation after carbon tetrachloride poisoning in rats previously treated with antioxidants

Propyl gallate (PG), reduced glutathione (GSH) and N,N′-diphenyl-p-phenylenediamine (DPPD), administered to rats prior to carbon tetrachloride, protect against hepatic fat infiltration until the fourth hour after poisoning. This effect does not seem to be mediated by a block in lipid mobilization from depot fat.A preliminary treatment with DPPD succeeds in inhibiting the double bond shifting in liver microsomal lipids within 30 min after dosing with CCl₄. The early peroxidative alteration occurs at the normal rate after the administration of either GSH or PG. The amount of lipid-bound radiocarbon and of ¹⁴CO₂ exhaled within 2 h after intragastric ¹⁴C-labelled carbon tetrachloride is not affected by the preliminary protection with the antioxidants.CCl₄ metabolites and/or lipoperoxides impair the in vitro combination of serum apoprotein with lipid. No changes are observed when lipoperoxidation is inhibited by antioxidants.These findings are interpreted as a support for the hypothesis that the possible contribution given by the enhancement of lipid peroxidation to the pathogenesis of CCl₄-induced fatty liver could depend on further structural and functional alterations occurring in the cytoplasmic environment of hepatocytes rather than the early radical attack onto the unsaturated lipids of liver microsomes. The functional integrity and the supply of the protein carrier for the triglyceride secretion mechanisms could be considered a target of the hepatotoxic action of CCl₄, at the molecular level.     

Effect of carbon tetrachloride on gonadal physiology in male rats.

Carbon tetrachloride was administered to male albino rats for varied intervals. It was found to have no significant adverse effect on spermatogenesis after 10 and 15 days of treatment. Severe damage to the spermatogenic cycle was observed after 20 days of treatment, leading to exfoliation of the germinal epithelium, depletion of germ cells, and shrinkage of the tubules.     

Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning

1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.     

L-tryptophan administration promotes the reversion of pre-established chronic liver injury in rats treated with carbon tetrachloride

We examined the effect of L-tryptophan (Trp) administration on the reversion of CCl(4)-induced chronic liver injury after hepatotoxicant withdrawal in rats. When rats treated with CCl(4) twice a week for 6 weeks were released from CCl(4) treatment for 2 weeks, there was an incomplete reversion of liver injury. The reversion was enhanced by 2 weeks of daily intraperitoneal administration of Trp (50 mg/kg body weight), starting just after CCl(4) withdrawal. There were increases in the levels of thiobarbituric acid reactive substances, an index of lipid peroxidation, Ca(2+), triglycerides, and Trp, and decreases in tryptophan 2,3-dioxygenase activity and serum triglyceride concentrations in the liver of rats treated with CCl(4) for 6 weeks. Serum albumin concentrations and in vitro hepatic protein synthesis activity did not change in the CCl(4)-treated rats. The changes in the CCl(4)-treated rats were partially attenuated 2 weeks after CCl(4) withdrawal. The attenuation was enhanced by 2 weeks of daily Trp administration. The increases in hepatic thiobarbituric acid reactive substances and triglycerides and the decreases in hepatic tryptophan 2,3-dioxygenase activity and serum triglyceride concentrations observed 2 weeks after CCl(4) withdrawal were almost completely attenuated by Trp administration. In vitro hepatic protein synthesis in CCl(4)-treated and untreated rats was increased by 2 weeks of daily Trp administration. These results indicate that Trp administration promotes the reversion of pre-established chronic liver injury in rats treated with CCl(4,) and suggest that Trp exerts this effect by enhancing the improvement of several parameters of liver dysfunction associated with chronic liver injury and by stimulating hepatic protein synthesis.     

Extrahepatic sites of metabolism of carbon tetrachloride in rats

Rats were injected i.v. and i.p. with [14C]carbon tetrachloride and the localization and binding of metabolites in the tissues were studied by autoradiography. Based on the autoradiographic findings, various tissues were tested for their capacity to form 14CO2 from [14C]carbon tetrachloride in vitro. Autoradiography in vitro was used to localize the sites of [14C]carbon tetrachloride metabolism under in vitro conditions. The results showed that several tissues accumulating metabolites in vivo had an ability to form 14CO2 in vitro, and accumulation of metabolites was observed also under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. The structures identified to have a marked carbon tetrachloride-metabolizing capacity were, besides the liver, the mucosa of the bronchial tree, the tracheal mucosa, the olfactory and respiratory nasal mucosa, the oesophageal mucosa, the mucosa of the larynx, the tongue and the cheeks, the lateral nasal gland and the kidney cortex. It is well established that the degradation of carbon tetrachloride involves the cytochrome P-450 system, and the metabolism of the substance in the mentioned tissues is probably correlated to high concentrations of cytochrome P-450. The nasal olfactory mucosa was found to be the tissue with the highest capacity to form 14CO2 from the [14C]carbon tetrachloride and microautoradiography indicated that in this tissue the cells of the subepithelial glands of the lamina propria mucosae are most actively engaged in the metabolism. It was also shown that cytochrome P-450 is present in the nasal olfactory mucosa.     

High-performance liquid chromatographic determination of phospholipid peroxidation products of rat liver after carbon tetrachloride administration

A method to detect and determine phospholipid peroxidation products in a biological system was developed using reversed-phase high performance liquid chromatography and normal-phase HPLC. Reversed-phase HPLC could separate phosphatidylcholine (PC) hydroperoxides and phosphatidylethanolamine (PE) hydroperoxides of rat liver from the respective phospholipids. A linear relationship was observed between these hydroperoxides and their peak areas on the chromatogram. In the experiment with rats administered CCl4, reversed-phase HPLC gave prominent, large peaks attributable to the peroxidation of phospholipids, and the peroxide level of the liver phospholipids was tentatively determined. Normal-phase HPLC analysis confirmed that both PC and PE in the liver phospholipids were peroxidized after CCl4 treatment. Neither the thiobarbituric acid value of the liver homogenate nor the fatty acid composition of the liver phospholipid fraction showed any significant difference between CCl4-treated and control rats. It is concluded that normal-phase HPLC and reversed-phase HPLC can complement each other to serve as a direct and sensitive method for the determination of lipid peroxide levels in a biological source. However, it was difficult to distinguish phospholipid hydroperoxides from their hydroxy derivatives.     

Enhanced hepatic chemiluminescence following carbon tetrachloride or hydrazine administration

Lipid peroxidation has been implicated as a mechanism of cellular injury due to a variety of hepatotoxic agents. In this study a scintillation spectrophotometer was employed to measure photon emission as an index of lipid peroxidation. Chemiluminescence (CL) from rat liver homogenates was significantly enhanced when animals were pretreated with hepatotoxic agents such as hydrazine or CCl₄. The enhanced CL was markedly reduced by the addition of antioxidants $\text{in}$$\text{vitro}$ or incubation with 100% nitrogen, events which have been demonstrated to decrease lipid peroxidation. Compared to results obtained employing the thiobarbituric acid assay, the CL procedure appears to be a more sensitive index of lipid peroxidation. The chemiluminescence procedure appears to merit further evaluation in the assessment of the role of lipid peroxidation in the pathogenesis of cell injury.     

Loss of blood and carbon tetrachloride poisoning]

In rats the induced enhancement of glutamic-pyruvic transaminase and leucine aminopeptidase activity in plasma to 5.2 mMol CC14/kg (per 05) is potentiated after repeated drawing of blood. The DL50 of CC14 following oral application in rats after loss of blood is reduced significant comparatively to controls and to animals with increased content of haemoglobin in blood.     

Detection of 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal after gavage of trans-4-hydroxy-2-nonenal or induction of lipid peroxidation with carbon tetrachloride in F344 rats

The 1,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal (HNE-dGp-adducts) were quantitated in tissues of rats treated with trans-4-hydroxy-2-nonenal (HNE) or carbon tetrachloride, respectively, using a 32P-postlabeling method. The method development was based on chemically synthesized HNE-1,N2-propanodeoxyguanosine adduct standard, which was characterized by NMR and mass spectra. The adducts were enriched by Nuclease P1. They were subsequently reacted with gamma-32P-ATP to give the respective 3'-5'-bisphosphates, which were two-directionally separated on PEI-cellulose-TLC and quantitated by autoradiography. The labeling efficiency for the adduct standard was 27%, and the recovery of spiked amounts of adduct standard in the enzymatical procedure was about 80%. Internal standard was used to eliminate methodological variations. The determination of the limit of quantitation in DNA from rat tissues by spiking of HNE-dGp-adduct standard revealed a sensitivity of about 20 HNE-dGp-adducts/10(9) normal nucleotides. Background levels of HNE-dGp-adducts in tissues of rats including liver, kidney, lung, colon and forestomach were found in the range of 18-158 adducts/10(9) nucleotides with relatively high adduct levels in the liver and low adduct levels in kidney, lung and colon. These background levels were statistically significantly increased by the factor of 2 in liver, lung, colon and forestomach after induction of lipid peroxidation by carbon tetrachloride. The finding that background HNE-dGp-adduct levels may be in context with different metabolic activities of the tissues and the increase of HNE-dGp-adduct levels after application of carbon tetrachloride indicate that HNE-dGp-adducts are an endogenous lesion and that they are probably formed from radical initiated lipid peroxidation.     

Studies on cytochrome oxidase in carbon tetrachloride treated rats

Cytochrome c oxidase was purified from control and CCl4 treated rats and its kinetic properties were studied. The activity of the enzyme was inhibited by 51% in CCl4 (4 g per kg body weight for 24 hr) treated rats. Studies on the kinetic properties showed that the K(m) of the enzyme increased by 60% while Vmax decreased by 44% in CCl4 treated rats compared to controls. The content of cytochrome aa3 was decreased by 34% while cytochrome b and c were not affected by CCl4 treatment. Phosphatidylcholine, phosphatidylethanolamine and cardiolipin were decreased significantly by 40%, 49% and 60% respectively in CCl4 treated rats. A decrease in the cytochrome aa3 content and a change in the lipid environment of the membrane are probably responsible for a decreased rate of electron transfer from cytochrome c to oxygen.     

Stabilities of hydroxyl radical spin adducts of PBN-type spin traps.

The stability of the hydroxyl spin adduct of nine different PBN-type spin traps has been examined in phosphate buffer solutions of various pH. The hydroxyl adduct is produced by short illumination of hydrogen peroxide with UV light in the presence of spin trap and the decay of its EPR signal followed. The stability measured by the half life of the first-order decay is strongly dependent on the pH of the solution and the structure of the aromatic ring used in the trap. All hydroxyl adducts are more stable in acidic media. tert-Butyl hydroaminoxyl is detected as a degradation product of the hydroxyl adduct from all spin traps.