Microsomal Cytochrome P-450 Levels Research Articles

Dietary accumulation of tetrabromobisphenol A and its effects on the scallop Chlamys farreri

Tetrabromobisphenol A (TBBPA) is currently the most widely-used brominated flame retardant (BFR) and has been proven to have a very high toxicity to aquatic organisms including bivalves. In this study, a laboratory experiment was conducted to obtain a better understanding of the role of algae food on the bioaccumulation of TBBPA and its effects on the suspension-feeding bivalve, the scallop, Chlamys farreri. Scallops were exposed to TBBPA via algae food alone or food+water for 10days. Results showed that TBBPA was accumulated rapidly by scallops, reaching an approximate steady state in soft tissues within 3days. The primary route of TBBPA accumulation was via the water, while dietary uptake was relatively minor. TBBPA exposure led to significant inhibition on microsomal cytochrome P450 and cytochrome b5 levels in gills and digestive gland, whereas GST activity and GSH level increased significantly, which indicated that TBBPA could be a suitable substrate to directly participate in phase II metabolism. TBBPA clearly induced the activity of SOD, suggesting the oxidant stress induced by TBBPA. This study suggests that dietary uptake was not the predominant uptake route for TBBPA in bivalves and also provides a preliminary basis for studying the detoxification and antioxidant responses of marine bivalves upon exposure to TBBPA.

Selenium and/or iodine deficiency alters hepatic xenobiotic metabolizing enzyme activities in rats

The objective of this study was to investigate the effects of iodine (I2) and/or selenium (Se) deficiency on thyroid hormones and hepatic xenobiotic metabolizing enzyme systems using a triple animal model. Three-week-old male Wistar rats were fed for seven weeks. Se deficiency was introduced by a diet containing <0.005mg/kg Se, and I2 deficiency was produced by sodium perchlorate containing drinking water. The levels of plasma thyroid hormones [total T4 (TT4), total T3 (TT3)], thyroid stimulating hormone (TSH); total microsomal cytochrome P450 (CYP450) and cytochrome b5 (CYP b5) levels; activities of microsomal NADPH-cytochrome P450 reductase (P450R), microsomal aniline hydroxylase (CYP2E1), microsomal 7-ethoxyresorufin O-deethylase (EROD), microsomal 7-pentoxyresorufin O-depentylase (PROD) and cytosolic glutathione S-transferase (GST) were determined. In I2 deficiency total CYP450 levels, activities of CYP2E1, EROD and GST decreased, and CYP b5 content increased significantly. In Se-deficient rats, total CYP450 level and CYP2E1 activity increased, and EROD and GST activities and CYP b5 level decreased significantly. In combined I2 and Se deficiency, except for CYP450 content and CYP2E1 activity, all enzyme activities and CYP b5 content decreased significantly compared to control group. Overall results of this study have suggested that metabolism of xenobiotics as well as endogenous compounds is affected by Se and I2 status.

The effect of antidepressants on ethylmorphine and imipramine N-demethylation in rat liver microsomes.

The effects of single and multiple doses of desipramine, amitriptyline or citalopram on the rat liver microsomal cytochrome P-450 level and on the rate of ethylmorphine and imipramine demethylation in-vitro have been investigated. Desipramine, amitriptyline or citalopram when given to rats as a single dose, did not affect the level of cytochrome P-450 in the liver microsomes, however, there was a tendency towards acceleration of imipramine, and particularly ethylmorphine, demethylation. Prolonged administration of desipramine and citalopram, but not amitriptyline, elevated the microsomal level of cytochrome P-450 and accelerated the rate of ethylmorphine demethylation. All the drugs investigated, when given chronically, inhibited the rate of imipramine demethylation. Since demethylation of ethylmorphine and imipramine in a CO atmosphere was inhibited by ca 90% for the former and only by 58% for the latter, it can be assumed that prolonged administration of the drugs investigated has two different effects on the oxygenase systems in rat liver microsomes: on the one hand they stimulate the cytochrome P450 oxygenase system involved in ethylmorphine demethylation and, on the other, they inhibit the other microsomal oxygenase system involved in demethylation of imipramine.

Effect of cigarette smoke on mutagenic activation of environmental carcinogens by cytochrome P450 2A8 and inactivation by glucuronidation in hamster liver

To elucidate the mechanism underlying suppression of N-nitrosobis(2-oxopropyl)amine (BOP)-induced hamster pancreatic carcinogenesis by cigarette smoke (CS), hepatic levels of microsomal cytochrome P450 (CYP) enzymes, mutagenic activation of environmental carcinogens and three types of uridine diphosphate-glucuronyltransferase (UDPGT) and sulphotransferase (ST) activities were assayed in male Syrian golden hamsters and F344 rats exposed to CS. Immunoblot analyses of microsomal CYP proteins revealed induction of constitutive CYP1A2 (2.6-fold increase) and 2A8 (4.0-fold increase) and induction of CYP1A1 and constitutive CYP1A2 (3.9-fold increase) in rats following exposure to CS for 4 weeks using a Hamburg type II smoking machine. CS exposure enhanced mutagenicities of four heterocyclic amines in the presence of liver S9 in both species, whereas the mutagenicities of aflatoxin B(1) (AFB(1)), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were significantly increased by CS in hamsters but not in rats. However, no CS-induced alterations in the mutagenic activities of other carcinogens, including BOP and other pancreatic carcinogens, were observed in either species. Application of several CYP inhibitors revealed that the mutagenic activities of MeAαC, AFB(1) and NNK in the hamster liver S9 were partly associated with CYP2A8, whereas those of the three pancreatic carcinogens were selectively associated with CYP2B. CS enhanced UDPGT activities towards 4-nitrophenol (4-NP) (1.9- to 2.0-fold) but did not affect those of bilirubin, testosterone UDPGTs and three STs in both species. Together with the previous findings that BOP does not induce tumourigenesis in rats and that the glucuronidation of β-oxypropylnitrosamines is higher in rats than in hamsters, suppression of BOP-induced pancreatic carcinogenesis by CS might be attributed to increased detoxification by 4-NP UDPGT and not decreased CYP2B activation. This is the first demonstration of the induction of CYP2A protein by CS; CYP2A protein polymorphisms have been associated with oral and pulmonary carcinogenesis in smokers.

Kava hepatotoxicity: pathogenetic aspects and prospective considerations

Kava hepatotoxicity is a well-defined herb-induced liver injury, caused by the use of commercial anxyolytic ethanolic and acetonic kava extracts, and of traditional recreational aqueous kava extracts. The aim of this review is to elucidate possible pathogenetic factors for the development of kava-induced liver injury, considering also confounding variables. In patients with liver disease in a causal relation to kava ± comedication, confounding factors include non-adherence to therapy recommendations and comedication consisting of synthetic and herbal drugs and dietary supplements including herbal ones and herbs-kava mixtures. Various possible pathogenetic factors have to be discussed and comprise metabolic interactions with exogenous compounds at the hepatic microsomal cytochrome P450 level; genetic enzyme deficiencies; toxic constituents and metabolites derived from the kava extract including impurities and adulterations; cyclooxygenase inhibition; P-glycoprotein alterations; hepatic glutathione depletion; solvents and solubilizers of the extracts; and kava raw material of poor quality. In particular, inappropriate kava plant parts and unsuitable kava cultivars may have been used sometimes for manufacturing the kava extracts instead of the rhizome of a noble cultivar of the kava plant (Piper methysticum G. Forster). In conclusion, kava hepatotoxicity occurred independently of the extraction medium used for the kava extracts and may primarily be attributed to daily overdose, prolonged treatment and to a few kava extract batches of poor quality; by improving kava quality and adherence to therapy recommendation under avoidance of comedication, liver injury by kava should be a preventable disease, at least to a major extent.

Pharmacokinetics of 5‐fluorouracil in mutant Nagase analbuminemic rats: faster metabolism of 5‐fluorouracil via CYP1A

It has been reported that plasma albumin concentrations were significantly lower in cancer patients than those in the healthy volunteers, and the expression and mRNA level of hepatic microsomal cytochrome P450 (CYP) 1A2 increased in mutant Nagase analbuminemic rats (NARs). After intravenous administration of 5-fluorouracil at a dose of 30 mg/kg to NARs, the time-averaged nonrenal clearance (Clnr) of the drug was significantly faster than the controls (51.3 versus 28.8 ml/min/kg), possibly due to an increase in the expression and mRNA level of CYP1A2 in NARs. In order to determine whether 5-fluorouracil is metabolized via CYP1A2 in male Sprague-Dawley rats, the rats were pretreated with 3-methylcholanthrene (a main inducer of CYP1A1/2 in rats). The Clnr of 5-fluorouracil was significantly faster (34.3 versus 27.3 ml/min/kg) in rats pretreated with 3-methylcholanthrene. The aforementioned data indicate that CYP1A is involved in the metabolism of 5-fluorouracil in rats.

Activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation in mice fed citrus flavonoids

Objective We investigated the physiologic activity of citrus flavonoids, naringenin, and hesperetin in affecting the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and serum and liver lipid levels in male ICR mice. Methods Six-week-old male ICR mice were fed experimental diets containing 1% naringenin or hesperetin and a control diet free of citrus flavonoids for 21 d. Results Naringenin caused a significant 56.1% increase in the hepatic cyanide-insensitive palmitoyl–coenzyme A oxidation rate. This compound also caused significant increases ranging from 9.8% to 55.6% in activity levels of various enzymes involved in hepatic fatty acid oxidation. These increases were accompanied by upregulation of gene expression of enzymes involved in peroxisomal fatty acid oxidation including increases of 46.8% to 88.7% of carnitine octanoyltransferase, acyl–coenzyme A oxidase, bifunctional enzyme, and 3-ketoacyl–coenzyme A thiolase. Messenger RNA levels of mitochondrial proteins involved in fatty acid oxidation except for trifunctional enzyme subunit-β were not affected by naringenin. Naringenin also significantly increased more than five-fold the mRNA level of microsomal cytochrome P-450 IV A1 involved in ω-oxidation of fatty acids. However, hesperetin did not affect any parameter of hepatic fatty acid oxidation. Naringenin, but not hesperetin, significantly lowered serum levels of triacylglycerol, cholesterol, phospholipids, and free fatty acid. Conclusion We demonstrated that naringenin has the physiologic effect of increasing hepatic fatty acid oxidation through upregulation of gene expression of enzymes involved in peroxisomal β-oxidation in mice. The change may account for the propensity of this compound to lower serum lipid levels.

Combined supplementation of vanadium and 1α,25-dihydroxyvitamin D 3 inhibit diethylnitrosamine-induced rat liver carcinogenesis

A combination of a differentiation-inducing agent like 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] with a compound that blocks entry of calcium into cells like vanadium (V) may offer a new approach to differentiation therapy and address the problem of hypercalcemia. Initiation of hepatocarcinogenesis was performed by a single intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg b.wt.) in male Sprague–Dawley rats. Supplementation of V, 1,25(OH) 2D 3, or both V and 1,25(OH) 2D 3 were started 4 weeks prior to DEN injection and continued thereafter till 20th week. It was observed that supplementation of V (0.5 ppm) in drinking water ad libitum or 1,25(OH) 2D 3 (3 μg/ml propylene glycol) per os twice weekly for the entire period of the experiment significantly reduces the number and size of hyperplastic nodules while the combination treatment offered an additive effect in reducing it to 37.5% from 83.3%. V-1,25(OH) 2D 3 combination was also effective in elevating the level of hepatic microsomal cytochrome P-450 (Cyt. P-450) ( P<0.001). Moreover, A significant reduced level of cytosolic glutathione (GSH) ( P<0.001) and glutathione S-transferase (GST) ( P<0.001) activity as well as reduction in the appearance of γ-glutamyltranspeptidase (GGT)-positive foci ( P<0.001) as compared to carcinogen control were observed in V plus 1,25(OH) 2D 3 treated group. These results suggest that V may be useful in combination with 1,25(OH) 2D 3 in the inhibition of experimental rat hepatocarcinogenesis.

Comparison of hepatic and renal drug-metabolising enzyme activities in sheep given single or two-fold challenge infections with Fasciola hepatica

The activity of drug-metabolising enzymes was compared in liver and kidneys of adult sheep given single or two-fold fluke infection. Fascioliasis was induced by oral administration of 200 metacercariae of Fasciola hepatica to female sheep either 10 or 20 weeks (mono-infections) or 10 and 20 weeks (bi-infection) before killing. The parasitic pathology was ascertained at autopsy and by clinical observation of animals. In the liver of both mono- and bi-infected animals, significant decreases ( P<0.05) (17–44%) were observed in the microsomal content of cytochrome P450 and in the two measured P450-dependent monooxygenase activities, benzphetamine and ethylmorphine N-demethylations. Moreover, Western blot analysis of microsomes demonstrated a decrease in the expression of cytochrome P4503A subfamily correlative with that of its presumed corresponding activity ethylmorphine N-demethylase. By contrast, the conjugation of chloro-dinitrobenzene to glutathione remained unchanged in liver cytosolic fractions prepared from all these animals. In kidneys, a significant decrease ( P<0.05) (30%) in microsomal cytochrome P450 level of 10-week mono-infected sheep was observed whereas there was no change in the other groups of animals. The inflammatory origin and the consequences in terms of pathology and animal productivity of the fascioliasis-induced decreases in tissue-oxidative drug metabolism are discussed, particularly in the case of adult sheep suffering repetitive infections.

Studies on Hepatic Mitochondrial Cytochrome P-450 during Plasmodium yoelii Infection and Pyrimethamine Treatment in Mice

Hepatic mitochondrial cytochrome P-450 and b5 activities were significantly depressed, whereas heme and hemozoin were increased during Plasmodium yoelii infection. Type II, aniline–HCl binding efficacy and polyacrylamide gel electrophoretic profile also depicted impairment of cytochrome P-450 during infection. However, the above alterations were more pronounced in the infected hepatic mitochondria, compared to microsomes. Oral treatment of pyrimethamine (10 mg/kg body weight×4 days) to P. yoelii-infected mice brought the altered levels of mitochondrial and microsomal cytochrome P-450 and b5 to almost normal, whereas heme and hemozoin levels remained unchanged.

Cytochrome P-450 levels and EROD activity in the Adriatic sturgeon (Acipenser naccarii, Chondrostei) and its induction by ?-Naphthoflavone

Summary The cytochrome P-450 enzyme system was studied in juvenile Acipenser naccarii (317.6±77 g initial live weight) reared at 23°C and at a standard diet over a period of two month before using for experiments (final weight 546.3±148.8 g). While determining normal levels of cytochrome P-450 in liver tissue its induction was also studied under exposure to P-naphthoflavone (BNF; single dose at 80 mg/Kg body weight 52 hrs prior to sampling). Control fish showed total cytochrome P-450 levels in liver microsomes around 0.40±0.12 nmoles/mg proteins (n=12) while in BNF exposed fish the values increased significantly (0.55±0.11nmoles/mg proteins; n=12). Ethoxyresorufin-O-deethylase (EROD) in control fish was 0.015±0.02 nmoles/mg proteins/min (n=12); it was found to be NADPH dependent and inducible by BNF. Western Immunoblotting and enzyme-linked-immunosorbent (ELISA) analyses of liver microsomes from untreated and BNF-exposed fish, using an antibody to rat cytochrome P-450 1A1 isoform (P-450 1A1) (Nelson et al.,1996) suggested the presence of a constitutive and BNF-inducible cytochrome P-450 homologous to rat P-450 protein. Measurement of cytochrome P-450-dependent EROD activity and P-450 1A1 expression levels in A. naccarii may be a useful parameter in monitoring environmental pollution.

Induction of cytochrome P450 3A and heat shock protein by tributyltin in blue crab, Callinectes sapidus

Tributyltin (TBT), a toxic contaminant in aquatic environments, breaks down cytochrome P450 enzymes (P450s) in vitro. To determine in vivo effects of long-term TBT-exposure in aquatic invertebrates, respiration rates, heat shock protein induction, microsomal cytochrome P450 levels and metabolism of [ 14C]testosterone were examined in the hepatopancreas of blue crabs fed TBT. Four groups of crabs were fed fish injected with ethanol or 2, 4 or 8 μg TBT Cl dissolved in ethanol/g meat; doses of 0, 125, 250, or 500 μg kg −1 respectively. Crabs were fed TBT-treated meat every other day for 16 days. Respiration rates were significantly decreased after TBT exposure. Hepatopancreas microsomes and cytosol were prepared 24 h after the last feeding. Heat shock protein was induced at the two highest TBT exposure concentrations. Total P450 levels were similar in all samples; however, protein cross-reacting with anti-scup CYP 3A, an isozyme responsible for 6 β-hydroxylation of testosterone in vertebrates, increased at the 250 μg and 500 μg TBT Cl kg −1 dose. EROD activity showed that CYP 1A was not elevated. Hydroxylation of [ 14C]testosterone at the 6 β, 6 α, 7 α, and 16 α positions by hepatopancreas microsomes increased significantly in crabs exposed to 250 μg TBT Cl kg −1. The lack of increase in [ 14C]testosterone hydroxylation at the 500 μg kg −1 dosage may result from TBT Cl's disruption of P450 activity at high concentrations. Incubation of control microsomes with TBT significantly reduced spectrally quantified P450 as well as P450 activity after 2 h, showing that TBT Cl interferes with crab microsomal P450 proteins. Immunoinhibition of testosterone 6 β-hydroxylation was accomplished by using scup CYP 3A antibody, showing that the antibody specifically binds to a protein with 6 β-hydroxylase activity. These results indicate that blue crabs are stressed by TBT-exposure, and upregulate P450 isozymes possibly to aid in metabolism and elimination of TBT.

Studies of the hepatic mitochondrial and microsomal mixed-function oxidase system during Plasmodium yoelii infection and inducer treatment in Swiss albino mice.

Plasmodium yoelii infection resulted in depression of hepatic mitochondrial and microsomal mixed-function oxidase system indices, e.g. cytochrome P-450, cytochrome b5 and phase II detoxification enzyme glutathione-S-transferase, while heam and haemozoin registered a marked increase in Swiss albino mice. Phenobarbitone (inducer) treatment showed induced levels of hepatic mitochondrial and microsomal cytochrome P-450 and glutathione-S-transferase in normal as well as in infected mice. The induced cytochrome P-450 and glutathione-S-transferase activities were similar in normal and infected mice. The findings were further supported by the isoenzymic profile and drug-binding properties of the terminal monoxygenase, cytochrome P-450.

Cytochrome P4501A immunoassay in freshwater turtles and exposure to PCBs and environmental pollutants

AbstractThis is the result of a comparative study of cytochrome P4501A (CYP1A) induction in liver microsomes from three species of freshwater turtles. CYP1A induction in turtle hepatic microsomes was compared to CYP1A induction in microsomes from the alligator, Alligator mississippiensis. Treatment of two species of freshwater turtles with four consecutive intraperitoneal injections of 100 mg/kg Aroclor 1254 caused a four‐ to five‐fold increase in P4501A in hepatic microsomes of Chrysemys picta picta and Chrysemys picta elegans. The same treatment administered to another freshwater turtle, Mauremys caspica rivulata, resulted in a very low but significant (p &lt; 0.01) induction of P4501A in hepatic microsomes. Specimens of M. caspica rivulata collected from an organic waste oxidation pond near the petrochemical industry area of the city of Ashdod exhibited normal levels of total hepatic microsomal cytochrome P450 but no detectable level of induction of cytochrome P4501A. The lack of P4501A1 induction could have resulted from two possible reasons. The first possibility is that the turtles were not exposed to residues of petrochemical waste in the pond. More likely, the apparent lack of induction resulted from the low response to CYP1A inducers found in this species. Induction of cytochrome P4501A was evaluated immunohistochemically in liver tissue of C. picta picta pretreated with Aroclor 1254 (400 mg/kg) or (124 mg/kg) 3,3′,4,4′‐tetrachlorobiphenyl. The most intensive staining was exhibited by sections of liver from a 3,3′,4,4′‐tetrachlorobiphenyl‐treated turtle. Staining of P4501A in liver sections from Aroclor 1254‐treated turtles was relatively moderate. In induced turtles, staining of the hepatocytes concentrated near the cell membranes and nuclear membranes, but stained granules were observed throughout the cytoplasm. The presence of inducible CYP1A enzymes in turtles is of importance from an evolutionary point of view and has potential ecological relevance.

Ascorbic Acid Protects Guinea Pigs from Acute Aflatoxin Toxicity

These studies were conducted to investigate whether ascorbic acid protected guinea pigs from aflatoxin B1 (AFB1) toxicity. Young guinea pigs, fed either 0 (AA) or 25 mg (25 AA) or gavaged 300 mg ascorbic acid (300 AA) per day for 21 days, were gavaged with the LD50 dose of AFB1on the 22nd day. Seven out of 10 animals in the AA group died within 72 hr of AFB1administration. The livers of the animals showed regional massive necrosis and multilobular degeneration. There was no mortality in the 25 AA group. Their livers, however, showed changes similar to those seen in AA group. Serum alanine amino transferase (ALAT) and aspartate amino transferase (ASAT) levels were elevated. There was neither mortality nor pathological changes in livers in the 300 AA group. Their ALAT and ASAT levels were unaffected.In vitroproduction of AFM1by liver microsomes tended to be higher than that in the other two groups. Three animals saved from the 300 AA group and continued with their supplementation were administered a second, intraperitoneal (ip) LD50 dose of AFB11 month after the first AFB1dose. One animal died. Livers of the animals showed centrilobular degeneration and moderate necrosis in scattered hepatocytes. Liver microsomal cytochrome P450 and cytosolic glutathioneS-transferase (GST) levels and AFM1production were drastically reduced. ALAT and ASAT activities were raised. The results indicated that intake of 300 mg of ascorbic acid almost protected the animals from acute toxicity of AFB1when given by gavage, but not when administered as a second dose ip.

The Use of Macroporous Microcarriers for the Large-Scale Growth of V79 Cells Genetically Designed to Express Single Human Cytochrome P450 Isoenzymes and for the Characterization of the Expressed Cytochrome P450

In this study, macroporous microcarriers were used for the large-scale growth of parental V79 cells and V79 cells genetically engineered to express a single human cytochrome P4501A1 isoenzyme (V79h1A1). Starting from 2 × 105cells/ml, approximately 1 × 107cells/ml could easily be harvested after 6 days in the case of the parental V79 cells, or after 11 days in the case of the V79h1A1 cells, resulting in a total of 3.6 × 1010cells. For the first time, the presence of cytochrome P450 (CYP) in the expressed V79 cells could be demonstrated by CO difference spectra with a Soret maximum around 450 nm. CYP levels in microsomes derived from the V79h1A1 cells of 14 pmol/mg protein were achieved. Importantly, no CYP was detected in microsomal fractions of the parental V79 cells. Cytochrome b5 levels could also be measured by difference spectrophotometry. No significant differences were found between cytochrome b5 levels in microsomes derived from the large-scale growth of V79h1A1 cells and parental V79 cells, i.e., 16.7 ± 7.9 vs 14.5 ± 7.6 pmol/mg protein. The presence of human cytochrome P4501A1 (CYPh1A1) in microsomal fractions derived from the large-scale growth of V79h1A1 cells was further substantiated by measuring 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-dealkylase (ECOD), and testosterone-6β-hydroxylation activities. EROD, ECOD, and testosterone-6β-hydroxylation activities of the V79h1A1 microsomes were 40 pmol resorufin/min/pmol CYPh1A1, 13 pmol hydroxy-coumarin/min/pmol CYPh1A1, and 0.16 pmol 6β-hydroxytestosterone/min/pmol CYPh1A1, respectively, indicating the presence of a highly active human CYP1A1 enzyme system. Further confirmation that the CYP protein was correctly expressed was obtained by Western blotting. In conclusion, the use of macroporous microcarriers is suitable for large-scale growth of V79 cells expressing human CYP isoenzymes. The present method may provide an easy and rather inexpensive tool in obtaining large quantities of microsomes containing human CYP isoenzymes, which are involved in the bioactivation and bioinactivation of xenobiotics. High yields of microsomes containing human CYP isoenzymes may substantially facilitate the production of sufficient quantities of human metabolites to allow isolation and identification in an early stage of development of pharmacologically interesting drugs.

Interaction between xenoestrogens and low-affinity glucocorticoids binding sites in rat liver microsomes

We investigated the physiologic activity of citrus flavonoids, naringenin, and hesperetin in affecting the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and serum and liver lipid levels in male ICR mice.Six-week-old male ICR mice were fed experimental diets containing 1% naringenin or hesperetin and a control diet free of citrus flavonoids for 21 d.Naringenin caused a significant 56.1% increase in the hepatic cyanide-insensitive palmitoyl–coenzyme A oxidation rate. This compound also caused significant increases ranging from 9.8% to 55.6% in activity levels of various enzymes involved in hepatic fatty acid oxidation. These increases were accompanied by upregulation of gene expression of enzymes involved in peroxisomal fatty acid oxidation including increases of 46.8% to 88.7% of carnitine octanoyltransferase, acyl–coenzyme A oxidase, bifunctional enzyme, and 3-ketoacyl–coenzyme A thiolase. Messenger RNA levels of mitochondrial proteins involved in fatty acid oxidation except for trifunctional enzyme subunit-β were not affected by naringenin. Naringenin also significantly increased more than five-fold the mRNA level of microsomal cytochrome P-450 IV A1 involved in ω-oxidation of fatty acids. However, hesperetin did not affect any parameter of hepatic fatty acid oxidation. Naringenin, but not hesperetin, significantly lowered serum levels of triacylglycerol, cholesterol, phospholipids, and free fatty acid.We demonstrated that naringenin has the physiologic effect of increasing hepatic fatty acid oxidation through upregulation of gene expression of enzymes involved in peroxisomal β-oxidation in mice. The change may account for the propensity of this compound to lower serum lipid levels.

Bile acids produce a generalized reduction of the catalytic activity of cytochromes P450 and other hepatic microsomal enzymes in vitro: relevance to drug metabolism in experimental cholestasis.

In bile duct-ligated male rats, there is a reduction of total hepatic microsomal cytochrome P450 (P450) levels and of NADPH-cytochrome P450 reductase (P450-reductase) activity, but the changes in activity of individual microsomal enzymes are nonuniform. We have proposed that the initial effect of cholestasis on microsomal proteins is a non-specific reduction caused by bile acid-mediated destruction, whereas the disproportionate lowering of male-specific P450 enzymes results from secondary down-regulation of some cytochrome P450 (CYP) genes. We report herein the results of experiments to test the first part of this hypothesis, at least as indicated by enzyme inhibition. Hepatic microsomal fractions from normal male rats were incubated at 37 degrees C with increasing concentrations of a range of bile acids selected for their varying physicochemical properties. The endpoints were catalytic activity of three individual CYP proteins, CYP 2A1 (measured as testosterone 7 alpha-hydroxylase activity), 2C11 (testosterone 2 alpha-hydroxylase and 16 alpha-hydroxylase) and 3A2 (testosterone 6 beta-hydroxylase), and the non-CYP enzymes, steroid 17 beta-dehydrogenase and P450-reductase. With 0.25 mmol/L cholic acid, a concentration exceeded in serum following bile duct ligation, there was a significant reduction in the activity of all enzymes at 4 h. Cholic acid-mediated inhibition was dose-dependent and there was no difference in inhibitory activity towards the male sex-dependent CYP 2C11 and 3A2 and the non-sex-dependent CYP 2A1 and other microsomal enzymes. Taurocholic acid was twice as potent an inhibitor as unconjugated cholic acid, the respective apparent I50 values being approximately 0.6 mmol/L compared with approximately 1.2 mmol/L. The dihydroxy bile acids, chenodeoxycholic acid and deoxycholic acid, were also more potent inhibitors than cholic acid, exhibiting I50 values in the range of 0.3-0.5 mmol/L, but the monohydroxy bile acid, lithocholic acid, was the most potent inhibitor (I50 approximately 0.2 mmol/L). Thus, the inhibitory potential of bile acids towards microsomal enzymes was inversely related to their extent of hydroxylation, while taurine conjugation enhanced the inhibitory potential of cholic acid. These data confirm the potential of bile acids to inhibit the activity of microsomal enzymes in livers of bile ductligated rats and indicate that such changes can occur with concentrations of bile acids that are physiologically relevant. Further, the results are consistent with the proposal that the disproportionately greater reduction of the male sex-dependent CYP, 2C11 and 3A2, is not explained by a destructive mechanism.

Reduction in Hepatic Microsomal P-450 and Related Catalytic Activity in Farm-Raised Red Drum

Hepatic microsomal factors were investigated in red drum Sciaenops ocellatus, an important aquaculture and sportfish species. Constitutive and induced levels of microsomal cytochrome P-450 and P-420, catalytic activities of 7-ethoxyresorufin O-deethylase (EROD) and NADPH–cytochrome-c reductase, and the extent of membrane lipid peroxidation were compared in juvenile farm-raised and wild fish. We detected smaller amounts of active cytochrome P.450 and more cytochrome P-420, the denatured form of cytochrome P-450, in farm-raised than in wild red drum. Lipid peroxide formation was significantly higher and EROD catalytic activity was decreased in farm-raised red drum, whereas wild fish exhibited significantly higher levels of cytochrome P-450 and EROD activity. No significant alterations in NADPH–cytochrome-c reductase activity were observed in farm-raised fish. The livers from farm-raised red drum had higher levels of cellular lipid, which correlated with higher levels of lipid peroxides and were apparently associated with the loss of active cytochrome P-450. These data indicate that farm-raised fish fed a commercial, potentially lipogenic, diet may be less capable of metabolizing endobiotics, drugs, and hydrocarbon pollutants, and suggest that substantially increased hepatic lipid deposition may have potentially dangerous toxicological implications for these animals.

Effect of Route and Pattern of Exposure on the Pharmacokinetics and Acute Hepatotoxicity of Carbon Tetrachloride

The objectives of this study were to evaluate the influence of both route and pattern of exposure on the pharmacokinetics (PK) and target organ toxicity of a common volatile organic chemical, carbon tetrachloride (CCl4). Male Sprague-Dawley rats, 325-375 g, inhaled 100 or 1000 ppm CCl4 for 2 hr through a one-way breathing valve. The total amount of CCl4 retained by each rat (i.e., the systemically absorbed dose) during the 2-hr period was determined to be 17.5 and 179 mg CCl4/kg body wt, respectively. CCl4, in doses of 17.5 and 179 mg/kg body wt, was administered in an aqueous emulsion by bolus gavage or by constant gastric infusion over 2 hr. Serial micro blood samples from the animals were analyzed for CCl4, in order to delineate blood concentration-versus-time profiles. Serum enzyme activities and total liver microsomal cytochrome P450 level and glucose 6-phosphatase activity were measured 24 hr postdosing as indices of CCl4 hepatotoxicity. The pattern of oral exposure, or dosage regimen, had a significant effect on the PK and acute the hepatotoxicity of CCl4. Arterial blood levels in the gastric infusion group were much lower than in the oral bolus group with both doses. Since CCl4 is quickly and extensively absorbed from the GI tract, large amounts of CCl4 in the portal blood following the oral bolus apparently exceeded the capacity of the liver to metabolize the chemical. Thus, substantially higher Cmax and AUC0∫∝ values were manifest. Hepatotoxicity was also significantly greater in these animals. The route of exposure also had a significant effect on the PK of CCl4. Levels of CCl4 in the arterial blood were much higher during inhalation than during gastric infusion. However, AUC0 ∫∝ values for the two groups were not significantly different, due to relatively slow elimination after gastric infusion. There was little difference between the two groups in hepatotoxicity indices 24 hr postdosing.