Hepatoprotective effects of Tagetes lucida root extract in carbon tetrachloride-induced hepatotoxicity in Wistar albino rats through amelioration of oxidative stress.

CONTEXT: The roots of Tagetes lucida Cav. (Asteraceae) have antioxidant and antimicrobial properties. OBJECTIVE: This study aimed to examine the hepatoprotective effects of T. lucida roots ethanol extract (TLRE) using carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. MATERIALS AND METHODS: The active ingredients of TLRE were identified by high-performance liquid chromatography, infra-red spectrum, and mass spectrometric procedures. Ninety rats were distributed into four main groups: positive, therapeutic, protective, and negative group. The therapeutic group was implemented using CCl4 (a single dose of 2 mL/kg) before TLRE or silymarin administration. Meanwhile, the protective group was implemented by administering CCl4 (a single dose of 2 mL/kg) after force-feeding TLRE or silymarin. Each therapeutic and protective group was divided into three subgroups: force-fed with saline, TLRE (500 mg/kg), and silymarin (25 mg/kg). The positive group was split into two subgroups that were force-fed TLRE and silymarin. Positive, therapeutic, and protective groups were compared to the negative group (untreated rats). CCl4, TLRE, and silymarin were orally administrated using a gastric tube. RESULTS: In the therapeutic and protective groups, TLRE significantly reduced liver enzymes, i.e., aspartate aminotransferase (12.47 and 6.29%), alanine aminotransferase (30.48 and 11.39%), alkaline phosphatase (17.28 and 15.90%), and cytochrome P450-2E1 (39.04 and 48.24%), and tumour necrosis factor-α (53.72 and 53.72%) in comparison with CCl4-induced hepatotoxicity controls. CONCLUSIONS: TLRE has a potent hepatoprotective effect with a good safety margin. After a repeated study on another type of small experimental animal, their offspring, and an experiment with a large animal, this study may lead to clinical trials.

Análisis fitofarmacológico de los frutos de Luffa acutangula para determinar su actividad antihepatotóxica / Phytopharmacological screening of Luffa acutangula fruits for its antihepatotoxic activity

Se examinó el extracto acuoso y etanólico (100 mg/kg) de Luffa acutangula Linn (frutos) para determinar la actividadantihepatotóxica en ratas druckrey mediante la hepatotoxicidad inducida por tetracloruro de carbono (CCl4)y paracetamol (PCM). Se demostró que el extracto posee un efecto hepatoprotector signifi cativo, ya que reduce losniveles séricos de transaminasas (SGPT y SGOT), fosfatasa alcalina (ALP) y bilirrubina. La signifi cativa actividadhepatoprotectora de Luffa acutangula es comparable a la de la silimarina, agente hepatoprotector estándar, lo quejustifi ca su uso en afecciones del hígado

The ethanolic and aqueous extract (100mg/kg) of Luffa acutangula Linn(fruits) was examined for antihepatotoxic activityin druckrey rats by inducing hepatotoxicity with Carbon tetrachloride(CCl4) and Paracetamol(PCM). The extracthas shown to posses signifi cant hepatoprotective effect by lowering the serum level of transaminases (SGPT & SGOT),Alkaline phosphatase (ALP) and bilirubin. The signifi cant hepatoprotective activity of Luffa acutangula is comparableto that standard hepatoprotective agent silymarin, which justify its use in liver affection

Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCI4 by cytochrome P450 and effects on lipid homeostasis.

The CCl4-induced development of liver damage was studied in monolayer cultures of primary rat hepatocytes: (1) CCl4 caused accumulation of triglycerides in hepatocytes following cytochrome P450 induction with beta-naphthoflavone or metyrapone. Ethanol or a high dose of insulin plus triiodothyronine had the same effect. (2) CCl4 increased the synthesis of fatty acids and triglycerides and the rate of lipid esterification. Cholesterol and phospholipid synthesis from acetate was also increased. (3) CCl4 reduced beta-oxidation of fatty acids as assessed by CO2-release and ketone body formation. Hydrolysis of triglycerides was also reduced. (4) The content of unsaturated fatty acids in microsomal lipids was decreased by almost 50% after incubation with CCl4, while saturated fatty acids increased slightly. (5) CCl4 exerted a pronounced inhibitory effect on the exocytosis of macromolecules (albumin), but did not affect secretion of bile acids from hepatocytes.

Effect of Emulphor, an emulsifier, on the pharmacokinetics and hepatotoxicity of oral carbon tetrachloride in the rat.

Emulphor, a polyethoxylated vegetable oil, is now being used widely to incorporate volatile organic compounds (VOCs) and other lipophilic compounds into aqueous solutions for biochemical, pharmacokinetic, and toxicological studies. Previous work in this laboratory demonstrated that 0.25% Emulphor did not alter the kinetics or hepatotoxicity of low doses of CCl4 compared to when the halocarbon was given to rats orally in water. The present study was undertaken as there was concern that higher concentrations of Emulphor (necessary to maintain lipophilic VOCs in stable aqueous emulsions for extended periods) might alter the VOCs' absorption, disposition, and/or toxicity. Dosages of 10 and 180 mg CCl4/kg bw were given, as an aqueous emulsion using 1, 2.5, 5, or 10% Emulphor, by gavage to fasted male Sprague-Dawley rats. Serial microsamples of blood were collected from an indwelling cannula in unanesthetized, freely moving rats at intervals of 2-60 min for up to 12 hr. The samples' CCl4 content was measured by headspace gas chromatography. Thereby, it was possible to obtain blood CCl4 concentration-versus-time profiles. Animals were euthanized 24 hr postdosing and blood was collected for measurement of serum enzymes as indices of hepatotoxicity. No toxicologically significant differences in pharmacokinetic parameters as a function of Emulphor concentration were found. Similarly the hepatotoxic potency of 10 and 180 mg/kg CCl4, as reflected by elevation in serum enzyme activities, did not vary significantly with the concentration of Emulphor utilized. Hence, it can be concluded that Emulphor, in concentrations as high as 10% (equivalent to 260 mg Emulphor/kg bw) in aqueous emulsions, does not significantly affect the absorption, disposition, or acute hepatotoxicity of CCl4 in male Sprague-Dawley rats.

A physiological and system analysis hybrid pharmacokinetic model to characterize carbon tetrachloride blood concentrations following administration in different oral vehicles.

Oral absorption of chemicals can be influenced significantly by the administration vehicle or diluent. It has been observed that the oral absorption of carbon tetrachloride (CCl4) and other volatile organic chemicals is markedly affected by the dosing vehicle, with administration in oils producing erratic blood concentration-time profiles with multiple peaks. Analysis of this type of data by a compartmental modeling approach can be difficult, and requires numerous assumptions about the absorption processes. Alternatively, a system analysis method with few assumptions may provide a more accurate description of the observed data. In the current investigations, a nonlinear system analysis approach was applied to blood CCl4 concentration-time data obtained following iv and oral administration. The oral regimens consisted of 25 mg CCl4/kg body wt given as an aqueous emulsion, in water, as pure chemicals, and in corn oil. The system analysis procedure, based upon a disposition decomposition method, provided an absorption input rate function, F, for each regimen. A physiological pharmacokinetic model, based primarily on parameters available in the literature, and the F input functions, formed a hybrid model that adequately described the observed blood CCl4 concentration-time data. The same physiological pharmacokinetic model, employing conventional first-order absorption input schemes, did not predict the data as well. Overall, the system analysis approach allowed the oral absorption of CCl4 to be characterized accurately, regardless of the vehicle. Though system analysis is based on general mathematical properties of a system's behavior rather than on its causal mechanisms, this work demonstrates that it can be a useful adjunct to physiological pharmacokinetic models.

Effects of medium and trace metals on kinetics of carbon tetrachloride transformation by Pseudomonas sp. strain KC.

Under denitrifying conditions, Pseudomonas sp. strain KC transforms carbon tetrachloride (CT) to carbon dioxide via a complex but as yet undetermined mechanism. Transformation rates were first order with respect to CT concentration over the CT concentration range examined (0 to 100 micrograms/liter) and proportional to protein concentration, giving pseudo-second-order kinetics overall. Addition of ferric iron (1 to 20 microM) to an actively transforming culture inhibited CT transformation, and the degree of inhibition increased with increasing iron concentration. By removing iron from the trace metals solution or by removing iron-containing precipitate from the growth medium, higher second-order rate coefficients were obtained. Copper also plays a role in CT transformation. Copper was toxic at neutral pH. By adjusting the medium pH to 8.2, soluble iron and copper levels decreased as a precipitate formed, and CT transformation rates increased. However, cultures grown at high pH without any added trace copper (1 microM) exhibited slower growth rates and greatly reduced rates of CT transformation, indicating that copper is required for CT transformation. The use of pH adjustment to decrease iron solubility, to avoid copper toxicity, and to provide a selective advantage for strain KC was evaluated by using soil slurries and groundwater containing high levels of iron. In samples adjusted to pH 8.2 and inoculated with strain KC, CT disappeared rapidly in the absence or presence of acetate or nitrate supplements. CT did not disappear in pH-adjusted controls that were not inoculated with strain KC.

Reactive oxygen species in the progression of CCl4-induced liver injury.

Pretreatment of rats with large doses of vitamin A (retinol) dramatically increased the hepatotoxicity of carbon tetrachloride (CCl4). Experiments were performed to elucidate the mechanism of this potentiation. Hypervitaminosis A was produced by oral administration of retinol, 250,000 IU/kg for seven days. CCl4 was then administered at a dose of 0.15 ml/kg, ip. This large dose of vitamin A did not enhance the biotransformation of CCl4, but did produce a 4-fold increase in CCl4-induced lipid peroxidation, as assessed by ethane exhalation. Because vitamin A has been shown to activate macrophages, it was hypothesized that this increased lipid peroxidation and liver injury resulted from the release of reactive oxygen species from activated Kupffer cells. By using a chemiluminescence assay, an enhanced release of free radicals was detected in Kupffer cells isolated from vitamin A pretreated rats. In addition, Kupffer cells from vitamin A pretreated rats displayed enhanced phagocytic activity in vitro, towards sheep red blood cells. In vivo, vitamin A pretreated rats cleared carbon particles from the blood 2-3 times faster than non-pretreated rats. In vivo administration of superoxide dismutase (SOD) 2 hr after CCl4 exposure did not influence CCl4 toxicity in control rats but did block the enhanced ethane exhalation and also the potentiation of CCl4 liver injury in vitamin A treated rats. Administration of methyl palmitate, an inhibitor of Kupffer cell function, did not inhibit CCl4 toxicity in control rats, but did effectively block enhanced ethane exhalation and potentiation of CCl4 injury in vitamin A treated rats. We conclude that potentiation of CCl4 hepatotoxicity by hypervitaminosis A is mediated in part by reactive oxygen species released from activated Kupffer cells.

Effect of oral dosing vehicles on the acute hepatotoxicity of carbon tetrachloride in rats.

Although carbon tetrachloride (CCl4) is of concern as a drinking water contaminant, it has been necessary in most oral toxicity studies to give CCl4 in an oil vehicle due to its limited water solubility. The primary objective of our study was to assess the influence of dosing vehicles on the acute hepatotoxicity of CCl4. Fasted 200- to 230-g rats were generally found to be more susceptible to CCl4 hepatotoxicity than fasted 300- to 330-g rats. A time-course study revealed that corn oil did not delay the onset or time of maximal liver injury by an oral 100 mg/kg dose of CCl4, but did reduce the extent of injury relative to that when the chemical was given undiluted or as an aqueous emulsion. Fasted 200- to 230-g male Sprague-Dawley rats were given 0, 10, 25, 50, 100, 250, 500, or 1000 mg CCl4/kg body wt by gavage: in corn oil; as an aqueous emulsion; as the undiluted chemical; and in the 10 and 25 mg/kg doses only, in water. Blood and liver samples were taken 24 hr after dosing for measurement of serum and microsomal enzymes. Pathological examination of liver samples was also conducted. Dose-dependent increases in serum enzyme levels and pathological changes and dose-dependent decreases in microsomal P450 and glucose-6-phosphatase activity were observed in each vehicle group. Both the 10 and 25 mg/kg oral doses of CCl4 in water caused significant elevations in serum enzymes and hepatic centrolobular vacuolation. The study revealed that acute hepatotoxicity was less pronounced at each dosage level in rats given CCl4 in corn oil than in other vehicle groups. These findings demonstrate that dosing vehicles can significantly influence the acute hepatotoxicity of CCl4 in rats and are a cause for additional consideration and review of the practice of routinely using vegetable oils as a diluent in studies of volatile organic compound (VOC) toxicity. The use of aqueous Emulphor emulsions appears more appropriate in acute toxicity studies of VOC drinking water contaminants such as CCl4, in that the emulsion did not substantially alter the toxicity of CCl4 from that of undiluted CCl4 or CCl4 ingested in water.

Effect of dosing vehicles on the pharmacokinetics of orally administered carbon tetrachloride in rats.

The primary objectives of this investigation were to determine whether oil and aqueous dosage vehicles alter the pharmacokinetics of orally administered carbon tetrachloride (CCl4) in rats, and to relate vehicle effects on CCl4 absorption and bioavailability to alterations of the acute hepatotoxicity of CCl4 seen in a companion study (H.J. Kim, S. Odend'hal, and J. V. Bruckner, 1990, Toxicol. Appl. Pharmacol. 102, 34-49). Fasted 200- to 230-g male Sprague-Dawley rats with indwelling arterial cannulas received 25 mg CCl4/kg body wt by gavage: in corn oil; as an Emulphor aqueous emulsion; in water; and as pure undiluted chemical. The 25 mg/kg dose was also given iv in PEG 400 through an indwelling jugular cannula. Serial blood samples were taken from the iv and gavage animals and analyzed for CCl4 content to obtain blood concentration-versus-time profiles. CCl4 was absorbed very rapidly from the GI tract, as peak concentrations of CCl4 in the blood were reached within 3-6 min of dosing in the aqueous emulsion and water groups. These peak levels were higher than those in the undiluted CCl4 group and substantially higher than those in the corn oil group. Corn oil markedly delayed the absorption of CCl4 from the GI tract and produced secondary peaks in the blood concentration-versus-time profiles. Elimination of CCl4 from the bloodstream of the iv group followed a triexponential pattern. CCl4 was eliminated from the blood at approximately the same rate in the iv and po groups, as reflected by similar elimination rate constant and half-life values. There was a high degree of correlation of both Cmax and AUC0(120) with hepatotoxicity. CCl4 was apparently less acutely hepatotoxic in corn oil due to delay and prolongation of CCl4 absorption, resulting in a marked decrease in the concentration of the chemical in the arterial blood. These findings suggest that corn oil has sufficient effect on the pharmacokinetics of orally administered CCl4 to require an appraisal of its use in studies of the acute oral toxicity of CCl4 and other volatile organic chemicals (VOCs). The use of aqueous Emulphor emulsions appears appropriate in studies of VOC contaminants of drinking water, in that the emulsion did not substantially alter the pharmacokinetics or hepatotoxicity of CCl4 from that ingested in water.