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The potential for exposing humans and wildlife to environmental polycyclic aromatic hydrocarbons (PAHs) has increased. Risk assessments describing how PAHs disturb lipid metabolism and induce hepatotoxicity have only received limited attention. In the present study, seven-week-old male ICR mice received intraperitoneal injections of 0, 0.01, 0.1 or 1mg/kg body weight 3-methylcholanthrene (3MC) per week for 10 weeks. A high-fat diet was provided during the exposure. Histopathological lipid accumulation and lipid metabolism-related genes were measured. We observed that sub-chronic 3MC exposure significantly increased lipid droplet and triacylglycerol (TG) levels in the livers. A low dose of 3MC activated the aryl hydrocarbon receptor, which negatively regulated lipid synthesis in the livers. The primary genes including acetyl-CoA carboxylase (Acc), fatty acid synthase (Fas) and stearoyl-CoA desaturase 1 (Scd1) decreased significantly when compared with those in the control group, indicating that de novo fatty acid synthesis in the hepatocytes was significantly inhibited by the sub-chronic 3MC exposure. However, the free fatty acid (FFA) synthesis in the adipose tissue was greatly enhanced by up-regulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulatory element binding protein-1c (SREBP1C) and target genes including Acc, Fas and Scd1. The synthesized FFA was released into the blood and then transported into the liver by the up-regulation of Fat and Fatp2, which resulted in the gradual accumulation of lipids in the liver. In conclusion, histological examinations and molecular level analyses highlighted the development of lipid accumulation and confirmed that 3MC significantly impaired lipid metabolism in mice., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Berberine, an alkaloid isolated from the plant Berberis aristata, has been found to inhibit significantly the carcinogenesis induced by 20-methylcholanthrene (200 microg/0.1 mL/mouse) or N-nitrosodiethylamine (NDEA; 0.02% NDEA in distilled water, 2.5 mL/animal by gavage, five days a week for 20 weeks) in a dose-dependent manner in small animals. Administration of berberine (0.5, 2.5 or 5.0 mg kg(-1)) could reduce significantly the incidence of tumour in animals after an injection of 20-methylcholanthrene and increased their life span compared with the control. When berberine (10, 25 or 50 mg kg(-1)) was administered simultaneously with NDEA, the markers of liver injury (liver weight, gamma-glutamyl transpeptidase activity and glutathione S-transferase level) were reduced significantly compared with animals treated with NDEA only, which resulted in all the values being elevated. A similar decrease was noted in the serum levels of lipid peroxide, bilirubin and glutamate pyruvate transaminase. Morphology of liver tissue and levels of marker enzymes indicated that berberine offered protection against chemical carcinogenesis.

Induction of UDP-glucuronosyltransferases (UGTs) toward thyroxine (T4) and p-nitrophenol (pNP) by 3-methylcholanthrene (MC), dexamethasone (DEX), clofibrate (Cl), and MC combined with DEX or Cl was studied in rat hepatocyte culture. We have developed a sensitive method for the measurement of glucuronide conjugates of the two substrates based on HPLC analysis of culture medium. MC, Cl, or DEX increased the activity of T4 UGT. Combination of MC and Cl showed additive effect, enzyme activity was enhanced compared with either MC or Cl treatment alone (617, 441, and 217% of the control, respectively). Combination of MC and DEX did not result in higher T4 UGT activity than MC treatment alone. Both MC and DEX enhanced the pNP UGT activity (182 and 162% of the control, respectively). Combination of MC with DEX resulted in additive effect. Cl treatment did not affect pNP conjugation either alone or in combination with MC. Western blot analysis revealed that only the amount of UGT1A1 was elevated by Cl and DEX. In contrast, concentration of UGT1A6 was increased by MC. Previous studies demonstrated that UGT1A1 inducers like phenobarbital have no effect on T4 conjugation (). Our results suggest that Cl, a known inducer of UGT1A1, enhances the activity of other enzyme(s) involved in T4 glucuronidation as well. It is well documented that DEX potentiates the inductory effect of polycyclic aromatic hydrocarbon on UGT1A6 (). In our study, MC increased the rate of T4 glucuronidation, and DEX had no additional effect on this reaction, suggesting that UGT1A6 is not the only enzyme inducible by MC that can catalyze T4 conjugation.

The effect of 16 d intake of 300 mg carotenoids/kg diet (beta-carotene (beta C), bixin (BX), lycopene (LY), lutein (LU), canthaxanthin (CX) or astaxanthin (AX) on xenobiotic metabolizing enzymes in the liver, lung, kidney and small intestine of male Wistar rats was assessed. A control group received the basal diet (AIN-76) without carotenoids and a positive control group for enzyme induction received 3-methylcholanthrene (3-MC) at 666 mg/kg diet. Cytochrome P450 activity was assessed using the substrates ethoxyresorufin for P450 1A1, methoxyresorufin for P450 1A2, pentoxyresorufin for P450 2B1/2 and benzyloxyresorufin for P450 types 1A1/2, 2B1/2 and 3A. Glutathione-S-transferase (EC 2.5.1.18) and reduced glutathione status were assessed. Carotenoid uptake by the tissues was also determined. 3-MC and the carotenoids BX, CX and AX led to significant increases compared with control in liver, lung and kidney ethoxyresorufin-O-deethylation. Methoxyresorufin-O-demethylation activity was significantly increased in liver and lung by BX, CX and AX but only CX and AX significantly increased activity in kidney. Pentoxyresorufin-O-depentylation and benzyloxyresorufin-O-dearylation increased in liver of 3-MC-, BX-, CX- and AX-treated rats, but to a much lesser degree than for the other two substrates. Benzyloxyresorufin-O-dearylation in lung was significantly decreased by all carotenoids. Activities of any of the measured enzymes in the small intestine were undetectable in all treatment groups except the 3-MC group. Glutathione status was unaffected by any of the treatments. This is the first study identifying the carotenoids BX, CX and AX as inducers of rat lung and kidney xenobiotic metabolizing enzymes.

The carcinogenic potential of phenobarbital (PB) and 3-methylcholanthrene (3-MC) was assayed in a new medium-term carcinogenicity bioassay using D-galactosamine (DGA) as a nonsurgical method to induce liver cell regeneration in place of partial hepatectomy (PH). Rats were initially given a single ip injection (200 mg/kg) of diethylnitrosamine (DEN) and after 2 wk on basal diet received 2 ip injections of DGA (300 mg/kg) at the end of wk 2 and 5. They were treated with one of the test compounds PB or 3-MC in the diet or fed basal diet for wk 3-8 Carcinogenic potential was assessed by comparing the numbers and areas per square centimeter of glutathione S-transferase placental form-positive (GST-P+) foci in the livers of test chemical-treated animals with those of the control animals given DEN/DGA alone. Positive estimations of carcinogenicity were obtained for PB, which is a nongenotoxic liver tumor promoter, and for 3-MC, which is a genotoxic nonliver carcinogen. Increases of liver/body weight ratios and serum total cholesterol were observed in rats treated with PB or 3-MC. Interestingly, interlobe differences were found on the development of GST-P+ liver cell foci. Our results thus confirm that the present bioassay protocol with repeated administration of DGA instead of PH may offer a new and sensitive method to screen large-numbers of environmental liver and nonliver carcinogens.

The mechanism of metaldehyde toxicity is unclear. It may be due to the compound itself or, at least in part, to acetaldehyde resulting from the hydrolysis of metaldehyde in the stomach. In this study, we orally dosed rats with twice the LD50 of metaldehyde following no pretreatment (control) or pretreatment with 1 of 3 different cytochrome P-450 inducers either phenobarbital or o,p'-DDD (inducers of cytochromes P-450 IIB and IIIA) or 3-methylcholanthrene (an inducer of P-450 IA). Our results show strong protection against metaldehyde poisoning afforded by the phenobarbital-DDD P-450 inducers, but only weak protection with 3-methylcholanthrene pretreatment. Acetaldehyde administered at the same molarity failed to produce the clinical signs of metaldehyde toxicity and no clinical differences were observed between any of the pretreated groups.

The liver probably plays a major role in the metabolic activation of the bladder carcinogen 2-naphthylamine (2-NA) and in the inactivation of the non-carcinogenic isomer 1-naphthylamine (1-NA). However, metabolic profiles of these compounds (including primary metabolites and directly determined conjugates) in hepatocytes are not available. Therefore metabolism of 1- and 2-NA was compared in freshly isolated hepatocytes from 3-methylcholanthrene (MC)-treated and untreated rats. At 10 microM, 2-NA was found to be mainly N-acetylated (66% of total metabolites after 1 h incubation) and N-glucuronidated (19%). Minor pathways led to C-oxidation (7%) and N-oxidation (3%; 2% present as the N-glucuronide). In hepatocytes from MC-treated rats total metabolism was slightly affected (1.5-fold increase). However, C- and N-oxidation were markedly increased (63 and 18% respectively), while N-acetylation and N-glucuronidation were diminished (5 and 2% respectively). Similar experiments were carried out with 1-NA. Its N-glucuronide was the predominant metabolite (68%) followed by the N-acetylated compound (15%) while C-oxidation was low and N-oxidized metabolites could not be detected, even after induction. The results demonstrate that MC treatment markedly shifted 2-NA metabolism from N-acetylation and N-glucuronidation to N- and C-oxidation. In the case of 1-NA metabolism extensive N-glucuronidation together with the lack of N-oxidation may prevent carcinogenesis.

The effect of inducing the rat liver nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene on NADPH- and NADH-dependent production of reactive oxygen intermediates was evaluated. The inducing agents produced a 2-fold increase in cytochrome P-450, a 50 to 70% increase in NADPH-cytochrome c reductase activity, and a 20 to 30% increase in NADH-cytochrome c reductase activity. Associated with these increases was a corresponding increase in NADPH- and NADH-dependent production of hydroxyl radical (.OH)-like species and of H2O2. Rates of .OH production were inhibited by catalase and partially sensitive to superoxide dismutase. The increase in nuclear production of .OH-like species after drug treatment appears to be due a corresponding increase in H2O2 generation. In contrast to H2O2 and .OH generation, production of thiobarbituric acid-reactive material by nuclei was not increased by the phenobarbital or 3-methylcholanthrene treatment. Redox cycling agents such as menadione and paraquat increased oxygen radical generation to similar extents in the control and the induced nuclei. These results indicate that induction of the nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene can result in a subsequent increase in production of reactive oxygen intermediates in the presence of either NADPH or NADH.

It was observed, that following an injection of 3-methylcholanthrene (MC), the tissue redox-state potential is modified expressively both in liver and in red muscles. In the liver in the first day an oxidosis develops, which is followed by redosis, but in the muscle a redosis can be observed already in the first day. It is a meaningful fact, that MC influences biochemical processes in the early phase of its effect not only in the liver but also in the red muscle. By reason of this data the possibility of a prevention of the MC influence by adequate redox agents might also be arised.

The relationship between the oxidative stress and inactivation of hepatic enzymes was examined in rats. An intake of lipid peroxidation products or pro-oxidative drugs provokes oxidative stress in the living body. Secondary peroxidation products of linoleic acid were administered orally, and the oxidative stress was evaluated by thiobarbituric acid (TBA) and haemoglobin-methylene blue (HMB) tests, and by the reduction in tocopherol level. A specific decrease in hepatic phosphoglucomutase activity was found following the oxidative stress caused by the dose with secondary products. Then, ten pro-oxidative drugs were administered intraperitoneally and the effects on the enzymatic activity were determined. Among the ten drugs, CCl4, alcohol, paraquat, phenobarbital, thiopental and methylcholanthrene caused the TBA values to increase, and the phosphoglucomutase activity to decrease, in the liver 24 h after the doses. It was attempted to clarify the inactivation mechanism by using parenchymal hepatocytes. When the cells were cultured in medium containing aldehydic products originating from lipid peroxidation, these aldehydes significantly suppressed the induction of phosphoglucomutase by dexamethasone as compared with the cells in aldehyde-free medium. We consider that aldehydes inhibit the hormonal induction of phosphoglucomutase in the rat liver.

A rapid and inexpensive method has been developed to estimate rates of benzo(a)pyrene phenol production by perfused rat liver. This method is based on the measurement of benzo(a)pyrene phenols utilizing a simple fluorometric procedure. Within 2 to 3 min after infusion of benzo(a)pyrene bound to serum albumin, phenols are excreted into the perfusate, primarily as glucuronide and sulfate conjugates. Maximal rates of phenol release were 8 to 10 nmol/g/hr in livers from control rats and 40 to 42 nmol/g/hr in livers from 3-methylcholanthrene-treated rats. Fasting of 3-methylcholanthrene-treated rats for 24 hr prior to perfusion experiments did not affect either the rate of phenol production or the extent of their conjugation. Ethanol (20 mM) inhibited rates of phenol formation by 50% in livers from fasted, 3-methylcholanthrene-treated rats but had no effect on benzo(a)pyrene hydroxylase activity in isolated hepatic microsomes. These data indicate that ethanol inhibits phenol formation from benzo(a)pyrene in intact liver, probably by diminishing the supply of the cofactor reduced nicotinamide adenine dinucleotide phosphate.

The ability of isolated rat livers and lungs to clear circulating benzo(a)pyrene was examined under conditions of altered perfusion and pretreatment with 3-methylcholanthrene (3-MC). In isolated organs from control rats, pulmonary benzo(a)pyrene clearance was low (approximately 1 ml/min) and independent of flow between 10 and 45 ml/min, whereas hepatic clearance was greater and flow dependent, increasing from 3.2 +/- 0.2 ml/min at a flow of 7 ml/min to 13.2 +/- 0.7 ml/min at a flow of 20 ml/min. 3-MC pretreatment increased markedly the arylhydrocarbon hydroxylase activity of broken cell preparations from both organs. However, clearance of benzo(a)pyrene was not increased in isolated livers by 3-MC pretreatment. On the other hand, clearance did increase and became flow dependent in isolated lungs of 3-MC-pretreated rats. In fact, when organs from these animals were perfused at flows occurring in vivo, hepatic and pulmonary clearance were about equal. These results indicate that flow and metabolic capacity are important determinants of both hepatic and pulmonary clearance of xenobiotic compounds and suggest that alterations in either factor may affect the relative roles of these organs in the total body disposition of xenobiotic compounds.

The binding of 4-dimethylaminoazobenzene (DAB) metabolites to liver total protein and nuclear DNA was studied in rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC), then fed with a (-14C)DAB diet (0.06%) for 3 days. Three consecutive injections of PB (80mg-kg) led to a 50% decrease of DAB metabolites bound to total protein and nuclear DNA. A single injection of 3-MC(20 mg-kg) roughly doubled the amount of metabolites bound to nuclear DNA but did not modify those bound to protein. When given in the diet (0.005%) for 8 days before the (-14C)-DAB diet, 3-MC did not change the amount of DAB metabolites bound either to protein, or to DNA. The opposite effect of PB and 3-MC injections on azodye binding to cellular constituents might be relevant to the action of these drugs on carcinogenesis. Moreover the route of 3-MC administration might be meaningful since DAB metabolite binding to DNA was only increased by intraperitoneal injection.

The metabolism of [3H]-3-methylcholanthrene (3-MC) was studied in the isolated, perfused rat liver. Following addition of 250 mug to the perfusion fluid, 3-MC disappeared rapidly. After 2 hr, approximately 34% of the radioactivity was excreted in the bile, 6% remained in the perfusate, and 60% was found in the liver. Of the liver radioactivity, 80% was unchanged 3-MC, 11% was conjugated metabolites, 4% was free hydroxymetabolites, and 4% was nonextractable, presumably bound to macromolecules. Of the perfusate radioactivity, 82% was conjugated metabolites, 3% was free hydroxymetabolites, and 15% was unchanged 3-MG. A similar distribution was observed in intact, bile-cannulated rats, but biliary excretion was about one-fourth as high with double the i.v.-injected dose. Biliary excretion in perfused livers rose rapidly during the first 30 to 40 min, then decreased steadily. It was nearly twice as high in male as in female rat livers. Pretreatment of rats with 3-MC more than doubled the biliary excretion rate over the first 20 to 30 min in livers of both sexes and raised that of the female to that of the male rat liver. Neither retinol acetate nor 7,8-benzoflavone had any appreciable effect on biliary excretion of 3-MC metabolites. 2-Diethylaminoethyl-2,2-diphenylvalerate, a well-known microsomal oxygenase inhibitor, lowered excretion by 80 to 90% and lengthened the lag period, and dibutyryl-3',5'-cyclic adenosine monophosphate markedly increased the rate of excretion of 3-MC metabolites. Fractionation of bile by chromatography on Sephadex LH-20 revealed six well-defined peaks of radioactivity. In contrast, bile of intact rats given 3-MC gave a pattern on Sephadex chromatography consisting of only three peaks. Preliminary data suggest that these consist of conjugates of dihydroxymetabolites as well as more highly hydroxylate derivatives. The data obtained indicate that the perfused liver is an appropriate experimental model for studies on the hepatobiliary metabolism of carcinogens.

By use of a high-performance liquid chromatographic procedure, phenacetin (acetophenetidin) and its O-de-ethylated metabolite paracetamol (acetaminophen), after hydrolysis of paracetamol conjugates, were simultaneously quantified in arterial plasma of both control and 3-methylcholanthrene-pretreated rats after the i.v., portal vein and intraduodenal administration of phenacetin. 3-Methylcholanthrene pretreatment resulted in enhanced phenacetin disposition as was shown from decreased plasma half-life time, decreased oral availability, increased clearance and a raise in metabolite levels. By constructing plasma concentration-time curves and determining the areas under the curves, it was possible to assess liver and gut wall first-pass metabolism. It is concluded that in 3-methylcholanthrene-pretreated rats the intestine contributes significantly, and predominantly over the liver, to phenacetin first-pass metabolism. In contrast, gut wall metabolism in control rats could not be demonstrated.

The effect of feeding and lighting on the regulation of aryl hydrocarbon hydroxylase activity in liver, small intestine, and lung from C3H/He, DBA/2, and Swiss albino mice was investigated. A circadian rhythm of aryl hydrocarbon hydroxylase activity in the liver from DBA/2 mice was observed under the controlled lighting conditions, showing higher activity at the end of the light period of a day and lower activity in the dark period. Contrasting to the change of the enzyme activity in mice fed freely in the continuous dark period, inconsistent and variable results of the enzyme activity and its induction by 3-methylcholanthrene were observed in the mice that were fed during the continuous light period, depending on the sex and strain of the mice used. Different responses in the induction of aryl hydrocarbon hydroxylase to different routes of 3-methylcholanthrene administration were found between the liver and small intestine of the mice, but not in the lung. The higher levels of the enzyme activity induced in the liver and small intestine were detected after the intraperitoneal and intragastric administration of 3-methylcholanthrene. The significance of controlled feeding schedules for obtaining reproducible experimental results in the regulation of aryl hydrocarbon hydroxylase activity in the mice was discussed.