Decreased utilization of [2-14C]orotic acid for the synthesis of cytidine nucleotides in rat liver after administration of alpha-hexachlorocyclohexane.

Administration of alpha-1,2,3,4,5,6-hexachlorocyclohexane (alpha-HCH) to rats decreased the utilization of [2-14C]orotic acid for the synthesis of liver cytidine nucleotides. The specific radioactivities of uridine components of the acid-soluble pool and rRNA increased during the first hours of treatment with the drug. Later on the specific radioactivities of uridine nucleotides remained unchanged, while those of cytidine components decreased gradually. Administration of hydrocortisone increased the incorporation of labelled orotic acid into rRNA cytidylic acid.     

Effect of butylated hydroxytoluene (E321) pretreatment versus L-arginine on liver injury after sub-lethal dose of endotoxin administration

Aim of this study was to compare the effects of L-arginine (L-arg) and food-antioxidant butylated hydroxytoluene (BHT) against oxidative stress of Escherichia coli endotoxin (LPS) in liver. Ninety Wistar albino rats were assigned in three groups. Rats received one of the following pre-treatment previous to 5mg/kg LPS intraperitoneally: saline, L-arg (NO donor, 100mg/kg) or BHT (250 mg/kg/day), for 3 days. At second, fourth and sixth hours, plasma nitrite-plus-nitrate, circulating liver enzymes, glutathione levels, superoxide dismutase, glutathione peroxidase activities were measured. The most remarkable liver injury was evident in BHT pre-treated animals at all time points compared to L-arg pre-treated rats. While BHT enhanced superoxide dismutase activities following LPS, glutathione decreased simultaneously compared to L-arg group. Although the risk associated with the use of BHT alone in subthreshold doses appeared to be low, higher risk of liver toxicity should be considered when over-consuming this food additive in endotoxemic settings.     

Changes in polyamine metabolism in rat liver after oral administration of alpha-naphthylisothiocyanate

Changes in the levels of urea cycle enzymes and polyamine metabolism in the liver of rats treated with alpha-naphthylisothiocyanate (ANIT), an inducer of experimental cholestasis, were studied. Activities of arginase increased approximately two-fold compared to the control values during the period of 24-72 hours after oral administration of ANIT (100 mg/kg), while activities of ornithine carbamyltransferase and ornithine aminotransferase decreased. The activity of ornithine decarboxylase was elevated by approximately 20- and 10-fold at 12 and 60 hours, respectively, after ANIT administration. Putrescine concentration doubled 24-48 hours after the ANIT administration, but spermidine level rose more slowly and reached the level of 1.5-fold of the control level in 36-72 hours. Spermine concentration decreased initially but increased in 96 hours. These results suggest that the increased activity of urea cycle accounts for the increase in the ornithine content and that the putrescine and spermidine acts as the initiator of recovery of the liver damaged by ANIT treatment.     

Toxicologic studies of N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide (captan): its metabolism by rat liver drug-metabolizing enzyme system

The degree of toxicity caused in rats by captan (N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide) administered intraperitoneally is greater than that induced by orally administered captan. With regard to its effect on the drug-metabolizing enzymes of rat liver, the activity of aniline hydroxylase and the level of cytochrome P-450 were found to decrease in the treated rats 24 h after a single oral dose (650 mg/kg). The loss was even greater in the animals receiving diethyl maleate 1 h prior to captan. Furthermore, usual increase in the activity of drug biotransformation enzymes seen after phenobarbital treatment appears to decrease in rats dosed with this funaicide. In vitro incubations of rat liver microsomes with captan resulted in a profound loss of cytochrome P-450 and the acitivty of benzphetamine N-demethylase as well as aniline hydroxylase. Although the inhibition of drug-metabolizing enzyme activity by captan was observed in microsomal incubations with or without NADPH, a detectable amount of carbonyl sulfide (COS) was found only in the incubations that contained captan plus NADPH. Carbonyl sulfide appears to arise from a captan-derived metabolite, thiophosgene (CSCl2), which decomposes to COS in aqueous solutions and in the presence of NADPH inhibits the activity of drug biotransformation enzymes.     

Dose--response study of hemorrhagic death by dietary butylated hydroxytoluene (BHT) in male rats

Male Sprague-Dawley rats were fed a 24% casein diet containing 0.58, 0.69, 0.83, 1.00, 1.20, or 1.44% butylated hydroxytoluene (BHT) ad libitum for 40 days. The rate of death was dose dependent and death occurred when BHT was given in amounts over 526 mg/kg/day. The LD50_{(40 days)} was 760 (669–864) mg/kg/day. In dead animals, a massive hemorrhage was found in the pleural and peritoneal cavities, and external hemorrhage was also observed in some animals. Furthermore, hemorrhage was observed in some organs such as epididymis, testis, and pancreas of the survivors in all groups given BHT, but it was not observed in the control group. The prothrombin index was decreased with all survivors given BHT. The extent of decrease was dependent on the daily dose of BHT. These observations suggest that BHT and/or its metabolite cause hemorrhage in rats.     

The biliary metabolism of butylated hydroxytoluene (3,5-di-t-butyl-4-hydroxytoluene) and its derivatives in the rat

The biliary metabolism of 3, 5-di-t-butyl-4-hydroxybenzyl alcohol (BHT-CH₂OH), 3, 5-di-t-butyl-4-hydroxybenzaldehyde (BHT-CHO) and 3, 5-di-t-butyl-4-hydroxybenzoic acid (BHT-COOH) after parenteral administration has been examined in the rat and compared to that of 3, 5-di-t-butyl-4-hydroxytoluene. Quantitative excretion and chemical examination of bile showed that in the enterohepatic circulation BHT-COOH or its ester glucuronide is the recirculating compound from the four compounds studied. Biliary excretion data are also presented for 1, 2-bis(3, 5-di-t-butyl-4-hydroxyphenyl)ethane.     

The effects of harman and norharman on the metabolism of benzo(a)pyrene in isolated perfused rat lung and in rat lung microsomes.

The effects of harman and norharman, nitrogen-containing pyrolysis products of amino acids present in cigarette smoke, on the metabolism of benzo(a)pyrene in rat lung microsomes in vitro and in isolated perfused rat lung were studied. In rat lung microsomes, both harman and norharman inhibited the metabolism of benzo(a)pyrene (BP) to dihydrodiols, phenols and quinones at concentrations over approximately 0.05 mM. The formation of BP-7, 8-dihydrodiol and BP-9, 10-dihydrodiol was inhibited more than that of BP-4, 5-dihydrodiol. No appreciable differences in inhibition were seen between microsomes from control or 3-methylcholanthrene-pretreated rats. In isolated perfused rat lung, 1 mM of harman in the perfusion fluid inhibited the formation of ethyl acetate-soluble metabolites of benzo(a)pyrene except BP-9, 10-dihydrodiol, and inhibited the total covalent binding of benzo(a)pyrene metabolites to lung tissue macromolecules. 0.03 mM of harman seemed to increase other metabolites than BP-7,8-dihydrodiol without changing the total covalent binding. These results suggest that at most concentrations both β-carboline derivatives, harman and norharman, inhibit benzo(a)pyrene metabolism and covalent binding both in lung microsomes in vitro and in isolated perfused rat lung.     

Decrease in blood coagulation factors II (prothrombin), VII, IX and X in the rat after a single oral dose of butylated hydroxytoluene

Male Sprague-Dawley rats were given butylated hydroxytoluene (BHT) in a dose of 800 mg/kg body weight orally, and 0.5-72 hr later plasma concentrations of factors II, VII, IX and X and hepatic levels of BHT and BHT quinone methide (2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone) were determined. Levels of factors II, VII, X and IX were reduced 36-60 hr after BHT treatment, but by 72 hr, those most affected (VII and IX) showed some recovery and X had returned to normal. Hepatic levels of BHT reached a maximum 3 hr (a major peak) and 24 hr after BHT dosing and BHT quinone methide reached a maximum at 6 and 24 hr (a major peak). In rats given BHT orally in doses of 200, 400 and 800 mg/kg, factors II, VII and X decreased after 48 hr only in rats given the highest dosage, but factor IX was more susceptible to BHT and showed a dose-dependent decrease. Phylloquinone (1 mg/rat) injected ip 24 hr after the administration of 800 mg BHT/kg maintained normal levels of factors VII and X and an almost normal level of factor IX, but had little effect on the level of factor II. In studies of the effects of drug-metabolizing-enzyme modifiers, neither ip pretreatment with 75 mg phenobarbital sodium/kg for 3 days nor the feeding of 1% cysteine in the diet throughout the experiment prevented the decrease in vitamin-K-dependent factors by 800 mg BHT/kg, but 2-day ip pretreatment with 60 mg cobaltous chloride/kg/day maintained normal levels of factors II and VII and reduced the BHT effect on factors IX and X. SKF 525A (50 mg/kg) injected ip either 30 min before or 12 hr after BHT treatment partially prevented the decrease in factors II, VII and X, or in all four factors, respectively. Thus the decrease in vitamin K-dependent factors may be the same with a single oral dose of BHT as with dietary BHT, and the anticoagulant effect may require the metabolic activation of BHT.     

Stereochemical aspects of parachloroamphetamine metabolism: Rabbit liver microsomal metabolism of RS-, R(-)-, and S(+)-para-chloroamphetamine

With the aid of a chiral derivatizing reagent and a sensitive and specific nitrogen-phosphorus gas Chromatographic assay, metabolism of para-chloroamphetamine (PCA) enantiomers has been studied following incubation of racemic (RS)-, R(?) and S(+)-PCA with rabbit liver microsomal preparations. Significant metabolism of racemic PCA and the individual enantiomers was observed following incubation in rabbit liver microsomal preparations. Metabolism required viable microsomes, NADPH and molecular oxygen, and the rate of metabolism increased following pretreatment with phenobarbital. Incubation of racemic PCA resulted in more rapid metabolism of the S(+) enantiomer than of the R(?) enantiomer. When the enantiomers were incubated individually, each enantiomer was metabolized more rapidly than when incubated as part of a racemic mixture, and the R(?) enantiomer was metabolized more rapidly than the S(+) enantiomer.     

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [³²P]orthophosphate, [Me?¹⁴ C]choline or [2?³H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [³²P] and [¹⁴C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [³²P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2?³-H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [³²P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.     

Alterations in function,enzyme activities,and histopathology of the liver of the rat after administration of phytohemagglutinins (Lectins)

The administration of phytohemagglutinins (lectins) to rats induced impairment of liver function, activation of some dehydrogenase enzymes connected with carbohydrate metabolism, and finally coagulative necrosis of the liver. Some of the lectins elicited more severe biochemical changes than others. However, the histopathological alterations were similar in all cases.     

Modifications of carcinogen metabolism in hepatic microsomes of suckling young by 3-methylcholanthrene or β-naphthoflavone administered to lactating rats

The effects of treating lactating rats with 3-methylcholanthrene (3-MC) or β-naphthoflavone (β-NF) (three i.p. injections of 20 or 40 mg compound/kg of body weight) on hepatic microsomal enzymes of their suckling young were examined. This treatment had no apparent effect on the contents of cytochromes P-450 and b₅ or on the activities of NADH- and NADPH-cytochrome c reductases in hepatic microsomes of the pups. However, these microsomes had 8- and 6-fold increased capacities for hydroxylations of benzo[a]pyrene (B[a]P) and N-2-fluorenylacetamide (2-FAA) respectively. These increases were about 5-fold greater in the hepatic microsomes of the dams, in which they were inhibited by 0.1 mM α-naphthoflavone (α-NF) invitro 72–81 and 89–95% and by 0.1 mM β-NF in vitro 12–41 and 60–76% respectively. In the pups, the induced activities were also inhibited, whereas the basal hydroxylations of B[a]P and 2-FAA were stimulated by α-NF 2.7- and 5.0-fold and by β-NF 1.4- and 2.4-fold respectively. The inhibition of the induced hydroxylations by α-NF and β-NF may be explained by their higher affinities (K_s, 0.14 and 0.28 μM, respectively) than those of B[a]P and 2-FAA (K_s 4.4 to 8.8 and 2.4 to 3.1 μM, respectively) for cytochrome P-450. Whereas β-NF gave a type I binding spectrum, α-NF gave a spectrum composed of type I and reverse-type I elements. Analysis of metabolites of 2-FAA showed differences in their type and amounts formed by hepatic microsomes of β-NF-treated lactating rats and their pups. Thus, in the dams the formation of 1-, 3-, 5-, 7-, 9- and N-hydroxy-2-FAA was increased by 9-, 30-, 40-, 5-, 20- and 5-fold respectively. In the pups, the formation of 1-, 3-, 5-, 7- and N-hydroxy-2-FAA was increased by 2-, 30-, 18-, 4- and 27-fold respectively. All these increased hydroxylations were inhibited by 0.1 mM α-NF in vitro. In the hepatic microsomes of pups from the corn oil-treated dams, α-NF stimulated all ring-hydroxylations, but not N-hydroxylation of 2-FAA. The results support earlier findings that microsomal enzymes differ in immature and mature rat liver and suggest that N-hydroxylation of 2-FAA, the activation required for carcinogenesis, and specific ring-hydroxylations are catalyzed by different cytochrome P-450 isozymes. Our studies showed that 3-MC and β-NF and/or their metabolites were transferred with milk of dams to their suckling pups in which they modified metabolism of carcinogens.     

Effects of inducers and inhibitors of rat liver mono-oxygenases on digitoxin metabolism

In rats, microsomal mono-oxygenases are involved in the cleavage and hydroxylation of digitoxin (dt-3).2 Previous to hydrolytic cleavage the terminal digitoxosyl has to be oxidized to the corresponding dehydro-digitoxosyl. The microsomal formation rate of 15'-dehydro-dt-3 could be enhanced 3 to 7 fold by pretreatment with pregnenolone-16α-carbonitril, spironolactone, cancrenoate and cyproterone. Phenobarbital and polychlorinated biphenyls had no effect, whereas polyaromatic hydrocarbons caused a significant decrease of both C12-β-hydroxylation and sugar oxidation. The inducing effects of the steroids were less pronounced in the formation of 9'-dehydro-dt-2 and 3'-dehydro-dt-1. For the latter, phenobarbital evoked an inducing effect by factor 1.7. The sugar chain oxidation could be inhibited by metyrapone, spironolactone, cancrenoate and digitoxigenin when added in vitro. a-Naphthoflavone was a weak inhibitor only. The results indicate that the terminal digitoxosyl oxidation is due to a specific isocytochrome P450 (or pattern of isocytochromes) inducible by some synthetic steroids.     

Actions of juglone on energy metabolism in the rat liver

Juglone is a phenolic compound used in popular medicine as a phytotherapic to treat inflammatory and infectious diseases. However, it also acts as an uncoupler of oxidative phosphorylation in isolated liver mitochondria and, thus, may interfere with the hepatic energy metabolism. The purpose of this work was to evaluate the effect of juglone on several metabolic parameters in the isolated perfused rat liver. Juglone, in the concentration range of 5 to 50 μM, stimulated glycogenolysis, glycolysis and oxygen uptake. Gluconeogenesis from both lactate and alanine was inhibited with half-maximal effects at the concentrations of 14.9 and 15.7 μM, respectively. The overall alanine transformation was increased by juglone, as indicated by the stimulated release of ammonia, urea, l-glutamate, lactate and pyruvate. A great increase (9-fold) in the tissue content of α-ketoglutarate was found, without a similar change in the l-glutamate content. The tissue contents of ATP were decreased, but those of ADP and AMP were increased. Experiments with isolated mitochondria fully confirmed previous notions about the uncoupling action of juglone. It can be concluded that juglone is active on metabolism at relatively low concentrations. In this particular it resembles more closely the classical uncoupler 2,4-dinitrophenol. Ingestion of high doses of juglone, thus, presents the same risks as the ingestion of 2,4-dinitrophenol which comprise excessive compromising of ATP production, hyperthermia and even death. Low doses, i.e., moderate consumption of natural products containing juglone, however, could be beneficial to health if one considers recent reports about the consequences of chronic mild uncoupling.     

A comparative study on the effects of alpha-hexachlorocyclohexane and its metabolite beta-pentachlorocyclohexene on growth and monooxygenase activities in rat liver

The present study adds support to the hypothesis that β-pentachlorocyclohexene (β-PCH) is a primary intermediate in α-hexachlorocyclohexane (α-HCH)4 metabolism in the rat. Degradation of α-HCH to β-PCH was shown to occur in vitro and in vivo, partially by non-enzymic catalysis. β-PCH accumulated in liver and adipose tissue of α-HCH treated rats, which had received the glutathione-lowering agent diethyl maleate. β-PCH disappears from the body much more rapidly than the parent compound α-HCH: about 50 per cent of a single i.p. dose were degraded within 2.5 hr, while half-life of α-HCH is known to be approximately 130 hr. To maintain equimolar liver concentrations, β-PCH must be given in doses 100-fold higher than α-HCH. β-PCH and α-HCH were fed for a period of ten days at various dose levels to give steady-state liver concentrations. It was found that β-PCH has similar hepatic effects to α-HCH: both agents induced liver growth and a phenobarbital-type pattern of monooxygenase activities, as measured by the following substrates: aminopyrine, ethylmorphine, benzphetamine, 4-nitroanisole, aniline, benzo[α]pyrene, ethoxyresorufin and 2,5-diphenyl-oxazole. Threshold doses for these effects were 30–43 μmoles/kg/day for β-PCH and 1.0–1.7 μmoles/kg/day for α-HCH. However, on the basis of molar hepatic concentrations β-PCH was a more potent inducer than α-HCH (2–10 times). Threshold concentrations ranged from 0.4 to 0.6 nmoles β-PCH/g liver and from 0.7 to 1.5 nmoles α-HCH/g liver. β-PCH concentrations in livers of rats treated even with high doses of α-HCH were below the threshold for induction of liver growth and of monooxygenase increases. It is, therefore, highly unlikely that β-PCH is responsible for the effect of α-HCH on rat livers.     

Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration.

The metabolic fate and pathway of the imidazole fungicide prochloraz (1-[N-propyl-N-2-(2,4,6-trichlorophenoxy) ethyl carbamoyl] imidazole) were investigated in the rat after administration of oral single doses with radiolabelled molecules. At both dose levels (50 and 250 mg/kg body weight), virtually all of the ingested [14C-phenyl]prochloraz was excreted in the urine or faeces within 96 hr, the bulk of excretion occurring between 24 and 48 hr after dosing. Urinary elimination accounted for 61 and 68% of the respective initial doses. Urinary metabolic products were isolated and identified by thin-layer chromatography, gas chromatography or gas chromatography coupled with mass spectrometry analysis. Prochloraz was completely metabolized with no unchanged compound being excreted in the urine. The main biotransformation products in rat urine were 2,4,6-trichlorophenoxyacetic acid and its corresponding alcohol, the latter as a glucuronic acid conjugate. Ring hydroxylation also occurred, with the hydroxy-2,4,6-trichlorophenoxyethanol and hydroxy-2,4,6-trichlorophenoxyacetic acid metabolites excreted in small amounts in the urine. 2,4,6-Trichlorophenol and unconjugated 2,4,6-trichlorophenoxyethanol were identified as minor urinary metabolites.