Biomedical subjects

V Longo

Publications and source records attributed to V Longo.

At least 19 recordsLinked to original sources

PubMed2007

In vivo inhibition of human hepatocellular carcinoma related angiogenesis by vinblastine and rapamycin.

This paper illustrates the use of the chick embryo chorioallantoic membrane (CAM) assay to determine the single and combined antiangiogenic effects of very low doses of vinblastine (VBL) and rapamycin (RAP) in human hepatocellular carcinoma (HCC). The angiogenic response induced by human HCC biopsy specimens was inhibited by each drug and sinergistically by their combination. Morever, immunohistochemical detection of microvessels with anti-CD31 mAB showed that their area was significantly lower in specimens treated with VBL and RAP in combination. Sinergy on the part of these well-known drugs when used in combination as antiangiogenics at very low doses may be of significance in the designing of new ways of treating HCC.

Angiogenesis Inhibitors↗

PubMed2006

Neridronate inhibits angiogenesis in vitro and in vivo.

The effects of the amino-bisphosphonate neridronate on endothelial cell functions involved in angiogenesis, namely, proliferation and morphogenesis on Matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo, by using the chick embryo chorioallantoic membrane (CAM) assay. In vitro, neridronate inhibited endothelial cell proliferation in a dose-dependent fashion, peaking at 30 microM. At the same concentration, neridronate inhibited fibroblast growth factor-2 (FGF-2)-induced capillary-like tube formation in the morphogenesis assay on Matrigel. In vivo, when tested in the CAM assay, neridronate again displayed the capability to inhibit FGF-2-induced angiogenesis. Overall, these results suggest that anti-angiogenesis by neridronate could be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases and cancer.

Angiogenesis Inhibitors↗

PubMed2004

Effects of dioxane on cytochrome P450 enzymes in liver, kidney, lung and nasal mucosa of rat.

The effect of acute and chronic dioxane administration on hepatic, renal, pulmonary and nasal mucosa P450 enzymes and liver toxicity were investigated in male rats. The acute treatment consisted of two doses (2 g/kg) of dioxane given for 2 days by gavage, whereas the chronic treatment consisted of 1.5% of dioxane in drinking water for 10 days. Both the acute and chronic dioxane treatments induced cytochrome P450 2B1/2- and P450 2E1-dependent microsomal monooxygenase activities (pentoxyresorufin O-depentylase and p-nitrophenol hydroxylase) in the liver, whereas in the kidney and nasal mucosa, only the 2E1 marker activities were enhanced. In addition in the liver, an induction of 2alpha-testosterone hydroxylase (associated with the constitutive and hormone-dependent P450 2C11) was also revealed, whereas the hepatic P450 4A-dependent omega-lauric acid hydroxylase was not enhanced by any dioxane treatment. These inductions were mostly confirmed by western blot analysis of liver, kidney and nasal mucosa microsomes. In the lung, no alteration of P450 activities was observed. To assess the mechanism of 2E1 induction, the hepatic, renal and nasal mucosa 2E1 mRNA levels were also examined. Following two kinds of dioxane administration, in the liver the 2E1 induction was not accompanied by a significant alteration of 2E1 mRNA levels, while both in the kidney and nasal mucosa the 2E1 mRNA increased about 2- to 3-fold, indicating an organ-specific regulation of this P450 isoform. Furthermore, dioxane was unable to alter the plasma alanine aminotransferase activity and hepatic glutathione (GSH) content, examined as an index of toxicity, when it was administered into rats with P450 2B1/2 and 2E1 preinduced by phenobarbital or fasting pretreatment. These results support the lack of or a poor formation of reactive and toxic intermediates during the biotrasformation of this solvent, even when its metabolism was enhanced by P450 inducers. The chronic administration of dioxane was also unable to induce the palmitoyl CoA oxidase, a marker of peroxisome proliferation, excluding this as a way to explain its toxicity. Thus, although the mechanism of dioxane carcinogenicity remains unclear, the present results suggest that the induction of 2E1 following a prolonged administration of dioxane might provide oxygen radical species, and thereby contribute to its organ-specific toxicity.

Administration, Oral↗

PubMed2004

Effects of beta-naphthoflavone, phenobarbital and dichlobenil on the drug-metabolizing system of liver and nasal mucosa of Italian water frogs.

In this study, we have examined the presence and inducibility of phase I and II drug-metabolizing enzymes in the liver and nasal mucosa of Italian water frogs of control and pretreated with beta-naphthoflavone, phenobarbital and dichlobenil by using typical substrates for these enzymes along with polyclonal antibodies mainly raised against mammalian enzymes. The CYP content and various monooxygenase and phase II enzyme activities in the liver of this frog were found similar, when reported, to those of largely aquatic and semiaquatic frogs. The treatment with beta-naphthoflavone resulted in an induction in the liver of a CYP1A and the induction was manifested by (a) immunoblot analysis using anti-rat CYP1A1, (b) an increase of CYP1A-mediated methoxyresorufin-O-demethylase and ethoxyresorufin-O-deethylase activities. The treatments with both phenobarbital and dichlobenil did not produce in the liver any effect on the assayed enzymes. When the nasal mucosa of water frogs was analyzed, various monooxygenase and phase II enzymatic activities, generally comparable to those of liver, were determined. However, by using antibodies anti-three GST different classes, we found a different reactivity into the cytosol of the two tissues indicating a differential tissue susceptibility to toxic effects of xenobiotics. In the nasal mucosa, a protein immunorelated to CYP2A and monooxygenase activities (i.e. ethoxycoumarin-O-deethylase and coumarin-7-hydroxylase) linked in mammals to this isoform have also been found. The treatment of water frogs with the herbicide dichlobenil decreased both the above-mentioned activities and the immunoreactive CYP2A apoprotein. The pretreatment with metyrapone, a CYP inhibitor, protected the CYP2A apoprotein and its linked activities from toxic effect of dichlobenil indicating a key role of this enzyme in the bioactivation of this herbicide. The findings of the present work suggest that the hepatic CYP1A induction and the nasal CYP2A-like inhibition profiles might provide two potential biomarkers of the Italian water frogs exposure to environmental and aquatic pollutants.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2003

Inhibition of cytochrome p450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax).

Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome p450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic p450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6beta-testosterone hydroxylase. In vitro experiments showed that EF at 10 microM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6beta-testosterone-hydroxylase, likely dependant on a p450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K(i) of 3.7 microM and a K(inact) of 0.045 min(-1). An immunoblot analysis with anti p450 3A27 of trout showed that the p450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the p450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the p450 enzymes. The mechanism by which EF or CF inactivate the p450 enzymes has not been studied but an attack of p450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of p450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.

Animals↗

PubMed2003

4-Biphenylaldehyde and 9-anthraldehyde: two fluorescent substrates for determining P450 enzyme activities in rat and human.

1. 4-Biphenylaldehyde (4-BA) and 9-anthraldehyde (9-AA) were examined as substrates for cytochrome P450 (CYPs) enzymes in rat and human. Both aldehydes were oxidized by CYPs to fluorescent carboxylic acids, which can be assayed with a high sensitivity by an easy fluorimetric method. 2. With liver microsomes from control and induced rats, the oxidation of both 9-AA and 4-BA followed simple Michaelis-Menten kinetics. Only microsomes from rats pretreated with phenobarbital (a strong inducer of P4502B1/2) could increase (about threefold) the oxidation rates (V(max)) of both aldehydes above the control values, which were 6.7+/-1.1 and 3.3+/-0.6 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively. On the other hand, the (K)(m)'s, which were similar for both aldehydes (about 25 micro M), did not change significantly with any inducer. The use of purified rat CYP1A1, 2E1, 2B1 and 2C11 in a reconstituted system showed that only 2B1 and 2C11 could oxidize both substrates with a high turnover. 3. In human liver microsomes, the oxidation rates of these aldehydes (1.6+/-0.2 and 0.42+/-0.1 nmol min(-1) mg(-1) protein for 4-BA and 9-AA, respectively) were lower than those of rat but with similar K(m)'s(20-26 microm). 4. The oxidation of these aldehydes was also determined with cDNA-expressed CYP1A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1 and 3A4 and with a characterized bank of 14 human liver microsomes. In a reconstituted system, only CYP2B6, 2A6, 3A4 and with a lower turnover 2C9 oxidized both substrates. 5. Among the CYP marker activities of the 14 human samples, good correlations were only observed between CYP3A-dependent 6 beta-testosterone hydroxylase and the oxidation of 4-BA (r = 0.74) or 9-AA (r = 0.80) and between the oxidation of 4-BA versus 9-AA (r = 0.74). Weak correlations were also found between the 2B6-linked S-mephenytoin N- demethylase and the oxidation of 4-BA (r = 0.58) or 9-AA (r = 0.65). 6. Inhibition experiments revealed that the oxidation of these aldehydes was inhibited by ketoconazole, 8-methoxypsoralene and sulphophenazole, selective inhibitors for P4503A6, 2A6 and 2C9, respectively. 7. In summary, based on the use of cDNA-expressed CYPs, correlation analysis and chemical inhibition, the metabolism in human liver microsomes of these aldehydes appears primarily catalysed by CYP3A, although CYP2A6, 2B6 and 2C9 may play a role. 9-AA and particularly 4-BA, owing to the high rate of its metabolism, may be two novel useful fluorescent probe substrates for assaying CYP activities in various species.

Adolescent↗

PubMed2001

Selective inactivation of rat and bovine olfactory cytochrome P450 by three haloethanes.

The effects of halothane, 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) and 1,1-dichloro-1-fluoroethane (HCFC-141b) on the P450 system in olfactory and hepatic microsomes of bovine and rat have been investigated. In the in vitro experiments, all three compounds decreased olfactory CYP-dependent activities in microsomes from both species, especially under anaerobic conditions, halothane showing the greatest effect. Hepatic activities were not affected. A selective olfactory CYP depletion was also observed in vivo after treatment with halothane, but not with HCFC-123 or HCFC-141b. A loss of olfactory ethoxycoumarin-O-deethylase activity was also found both in vitro and in vivo experiments, suggesting that a CYP2A isoform may be the main target of inactivation. The present results therefore suggest that CYP2A, the major isoform expressed in the olfactory tissue of mammals, may be particularly prone to catalyze the reductive metabolism of halothane both in anaerobic and aerobic conditions.

Anesthetics, Inhalation↗

PubMed2001

Oxidation of N,N-dimethylformamide and N,N-diethylformamide by human liver microsomes and human recombinant P450s.

N-N, dimethyl- (DMF) and N-N, diethyl-formamide (DEF) are two hepatotoxic solvents, whose metabolism has not been investigated in humans. To identify the P450 isoforms involved in the microsomal oxidation of these solvents we used (a) 12 human liver samples; (b) human recombinant P450 isoforms (1A1, 1A2, 2B6, 2C10, 2E1, 3A4); (c) chemical and immunological inhibitions. When correlation analyses were performed using enzymatic markers in human liver microsomes, the p-nitrophenol hydroxylation rate significantly correlated (r=0.87) with the dealkylation rate of DMF but not with that of DEF. Among the tested recombinant P450s only 2E1 oxidised DMF, while DEF was oxidised by 2E1, 2C10 and 3A4. 4-Methylpyrazole and anti human 2E1 IgG strongly inhibited the DMF demethylation but only partially the DEF deethylation. These findings indicate that, in the DMF metabolism, the role of 2E1 is crucial and its expression may be an important factor in determining the susceptibility of human to this solvent.

Cell Culture Techniques↗

PubMed2001

Effects of beta-naphthoflavone on the cytochrome P450 system, and phase II enzymes in gilthead seabream (Sparus aurata).

The effect of beta-naphthoflavone (beta-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, beta-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, beta-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg beta-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3-7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. beta-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose-response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg beta-NF and was the most sensitive measurement for CYP 1A-like induction. beta-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.

Animals↗

PubMed2001

Pharmacokinetics and microsomal oxidation of praziquantel and its effects on the P450 system in three-month-old lambs infested by Fasciola hepatica.

Praziquantel (PZQ) is a broadly effective trematocide and cestocide, widely employed in veterinary and human medicine. In view of several differences in both its pharmacokinetic profile in different animal species and in the cytochrome P450-dependent system between ruminant and nonruminant species, the present study was undertaken to determine the pharmacokinetics of this drug, its effects on the P450 system and the involvement of cytochrome P450 in its metabolism in 3-month-old lambs infested by Fasciola hepatica. Following both oral and i.m. administration, PZQ disposition was best described by a linear one-compartment open model with a rapid absorption and elimination. Although the PZQ dose used by the i.m. route was only half of that used by the oral route, the mean PZQ plasma concentration was higher after i.m. than after oral treatment. Oral treatment with 30 mg/kg/day of PZQ did not modify the mono-oxygenase activities tested, whilst the administration of PZQ at a dose of 60 mg/kg/day for 2 days caused a significant decrease in the P450 3A-dependent erythromycin N-demethylase and 6beta testosterone hydroxylase activities. From the incubation of microsomes from lambs not treated with PZQ, a single metabolite (PZQ 11b-OH or PZQ 1-OH) was identified by GC/MS analysis. By selective inhibition of the 3A subfamily performed with triacetyloleandromycin, the production of this metabolite declined by about 90% suggesting a prominent role of P450 3A isoforms in this oxidation. These features indicate that agents or drugs which are able to modulate P450 3A-dependent catalysis may interfere with the metabolism, bioavailability and therapeutic effects of PZQ.

Administration, Oral↗

PubMed2001

[Evaluation of oocyte maturity in patients undergoing ICSI and R-FSH treatment].

BACKGROUND: This study compares embryo quality, fertilisation rate and pregnancy rates after ICSI related with the quality of oocytes achieved after r-FSH stimulation. METHODS: We evaluated 955 oocytes from patients following r-FSH and 643 oocytes from patients stimulated with ultrapure urinary FSH. The oocytes were divided into: a) normal oocytes; b) ooctyes with extra cytoplasmatic abnormalities (dark zona pellucida, wide perivitelline space); c) oocytes with cytoplasmatic abnormalities (dark cytoplasm, granular cytoplasm, retractile body), d) oocytes with shape abnormalities; e) oocytes with double abnormalities; f) oocytes with triple abnormalities. The embryos were divided into: A) even and homogeneous blastomeres without fragmentation; B) even and homogeneous blastomeres with <20% fragmentation; C) uneven and non-homogeneous blastomeres with 20-50% fragmentation; D) uneven and non homogeneous blastomere with >50% fragmentation. RESULTS: 40.9% of oocytes from patients treated with r-FSH have a normal morphology vs 35.2% of control groups (p<0.04). Abnormalities have a similar frequency in the two groups, except for the presence of three combined abnormalities (7.7 vs 5.4%; p<0.04). Fertilisation rate, cleavage rate, oocyte quality and pregnancy rate do not appear to be influenced by oocyte morphology and the type of FSH used for stimulation. CONCLUSIONS: The administration of r-FSH allows a large number of oocytes to be rescued, with a high incidence of normal morphology. The fertilisation rate and the quality of embryos obtained from oocytes with structural abnormalities are similar to those observed in morphologically normal oocytes. Even the probability of pregnancy is similar in the two groups.

Adult↗

PubMed2000

Cytochrome P450-dependent monooxygenase activities and their inducibility by classic P450 inducers in the liver, kidney, and nasal mucosa of male adult ring-necked pheasants.

In this study, several P450-dependent monoxygenase activities in the liver, kidney, and nasal mucosa of ring-necked pheasants were examined. In addition, the presence and inducibility of P450 isoenzymes in the hepatic and renal tissues of pheasants were examined by using typical substrates and inducers of P450s along with polyclonal antibodies raised against mammalian isoforms. Anti-rat P450 1A1 recognized in microsomes of both pheasant liver and kidney a protein that was markedly induced by beta-naphthoflavone and accompanied by an increase of various monooxygenases, in particular, methoxyresorufin-O-demethylase (MROD) activity. Anti-rat P450 2E1 revealed in microsomes of the pheasant liver but not in kidney an immunoreactive protein that was slightly induced by acetone but not accompanied by an increase of para-nitrophenol hydroxylase activity. On the other hand, acetone treatment caused an induction of other hepatic monoxygenases including MROD, erythromycin N-demethylase, and 6beta-testosterone hydroxylase. These two latter activities, known to be markers for 3A isoenzymes in rodents, were also enhanced in pheasant liver by phenobarbital but not by dexamethasone. The treatment with these two inducers also lacked to point out hepatic and renal proteins immunorelated to P450 3A or 2B subfamily, suggesting that these isoforms may be not expressed in pheasant. On the other hand, anti-rat P450 2C11 recognized two immunorelated proteins in the liver of both control and treated pheasants. The treatment with clofibrate, a mammalian inducer of 4A subfamily, induced both in liver and kidney of pheasant: i) a protein that cross-reacted with anti rat P450 4A1 and ii) the (omega) and (omega-1) lauric acid hydroxylase activities, known to be associated in mammals to this P450 subfamily. In the nasal mucosa of pheasant, a protein immunorelated to P450 2A and some monooxygenase activities (i.e., 7-ethoxycoumarin O-deethylase) linked, in mammals, to this isoform have been found; by contrast a protein immunoreactive with anti P450 2G1 was not found. In conclusion, the immunochemical properties and monooxygenase activities of constitutive and inducible P450s in pheasants were different not only from those of mammals but also from those of chickens. The findings of the present work also suggest that the P450 induction profiles might provide a potential biomarker of pheasant exposure to chemicals or environmental pollutants in the wild-field or in the stock-farm.

Acetone↗

PubMed2000

Effect of starvation and chlormethiazole on cytochrome P450s of rat nasal mucosa.

Cytochrome P450 (CYP) enzymes of nasal tissue are relatively resistant to induction by classical inducers. In the present study, the effects of starvation on the expression of CYP1A, 2A, 2B, 2C, 2E, 2G, and 3A subfamilies in the nasal mucosa of rat were studied. Fasting for 72 hr caused an increase in 2E1-dependent p-nitrophenol hydroxylase and 1A-dependent ethoxy- (or methoxy) resorufin dealkylase activities, but did not affect either 2A-linked coumarin hydroxylase or the testosterone hydroxylase activity, the latter reaction being a marker of several CYPs including 2G1. Whereas increases in 2E1- and 1A- associated catalytic activities were accompanied by a concomitant increase in the corresponding apoproteins as determined by immunoblotting, immunoactive protein bands reactive with antibodies raised against rat 1A1, 2B1, 2C11, 3A1 or rabbit nasal 2A10/11 and 2G1 were not altered. Fasting also increased CYP2E1 and CYP1A2 on the mRNA level, but did not alter CYP1A1 mRNA as determined by hybridization with cDNA probes selective for these cytochromes. A reiterative administration of chlormethiazole, a specific inhibitor of 2E1 in liver, strongly inhibited many CYPs, including 2E1, 1A2, 2G1, and 2A in the nasal mucosa, but did not influence expression of 2B or 3A as determined by immunoblotting or catalytic activities. The chlormethiazole-mediated inhibition of 1A1 and 2E1 was demonstrated to be at the mRNA level. These results suggest that fasting induces the gene expression of 2E1 and 1A2 and that the mechanisms involved in the regulation of CYPs in the nasal mucosa are tissue-specific. The inducibility of the above-mentioned isoforms may have a significant role in the clearance of drugs and bioactivation of inhaled compounds.

Animals↗

PubMed2000

Heterogenous effects of anthraquinones on drug-metabolizing enzymes in the liver and small intestine of rat.

The induction of a variety of drug-metabolizing enzymes by six anthraquinones (AQs) has been investigated in the liver and small intestine of rat. In the liver, the intragastric administration for 3 days of 100 mg/kg 9,10-anthraquinone (9,10-AQ). 1-hydroxy-AQ, 1,4-dihydroxy-AQ, but not 1,2-dihydroxy-AQ and 2-carboxy-AQ, resulted in a significant induction of the UDP-GT, DT-diaphorase, P450 1A-linked monooxygenase activities and in particular the methoxyresorufin-O-demethylase (MEROD), an activity dependent on P450 1A2. Immunoblot analysis indicated that 1-hydroxy-AQ and 1,4-dihydroxy-AQ induced P450 1A2 but not 1A1 and 9,10-AQ induced both P4501A2 and P4502B. Northern blotanalysis, using a cDNA probe for CYP 1A1 and CYP 1A2, confirmed that the AQs induce CYP 1A2 but not 1A1 mRNA. In the mucosa of small intestine, none of the above-mentioned enzymatic activities were enhanced following AQ administration. The induction mechanism of the hepatic enzymes by AQs is not known and it deserves a further study as it might be independent from the activation of the Ah-receptor as reported for other tricyclic compounds. The results from inhibition experiments showed that the hydroxylated AQs were strong inhibitors of P450 1A2-dependent monooxygenases. This suggests that long-term ingestion of certain AQs, may affect the toxicity of other components present in the diet through the hepatic induction or inhibition of P450 1A2.

Animals↗

PubMed2000

Stabilization of cytochrome P4502E1 protein by ethanol in primary hamster hepatocyte cultures.

We report the effect of ethanol on monooxygenase activities in primary hamster hepatocyte cultures maintained on collagen-coated dishes. The addition of 50mM ethanol to cell cultures both from control and ethanol pretreated animals almost completely maintained, at least for 72hr, the P4502E1-dependent aniline hydroxylase (AnH) activity and the 2E1 immunodetectable apoprotein content at the levels of the corresponding 4-hr plated hepatocytes. On the contrary, other P450-dependent monooxygenase activities, as assayed by testosterone hydroxylases, kept decreasing falling-after 72hr of culture-to the levels of the 4-hr plated hepatocytes. In both cases, in the absence of ethanol, a rapid decline of AnH activities and 2E1 apoprotein contents were also observed, attaining undetectable levels at 72hr. The hybridizable 2E1 mRNA also rapidly declined in both cultures, but such decline was not significantly altered by the presence of 50mM ethanol in the culture medium. Furthermore, we show that P4502E1 in the liver possesses a rapid degradation phase with a half-life of about 6hr. Thus, in the hamster, P4502E1 appears regulated at post-translational level, as in rat, probably by a protein stabilization mechanism.

Aniline Hydroxylase↗

PubMed1999

Effect of clofibrate, a peroxisome proliferator, in sea bass (Dicentrarchus labrax), a marine fish.

The effects of 35 or 70 mg/kg clofibrate on some peroxisomal, mitochondrial, and microsomal enzymes (markers of peroxisome proliferation) in liver, gill, and kidney of sea bass following 2 weeks i.p. treatment have been studied. Induction of lauric acid hydroxylase, UDP-glucuronyl transferase, palmitoyl-CoA oxidase, carnitine-palmitoyl-transferase, p-nitrophenylacetate hydrolase, and benzaldehyde and propionaldehyde dehydrogenase activities was not observed in any case. The clofibrate administration at the dose of 70 mg/kg induced in the liver the glutathione S-tranferase and reduced epoxide hydrolase activities. These results demonstrate that a marine fish species, such as sea bass, is refractory to peroxisome proliferation.

Animals↗

PubMed1998

Expression and distribution of cytochrome P450 enzymes in male rat kidney: effects of ethanol, acetone and dietary conditions.

Ethanol, acetone, diet and starvation, known modulators of the hepatic cytochrome P450 (CYP)-dependent microsomal monooxygenase system, were assessed for their effects on cytochrome P450 isozyme content and monooxygenase activities in the male rat kidney. In acute experiments, rats were either treated with acetone, fasted or given a combination of the two treatments. Acetone treatment alone induced CYP2E1-dependent p-nitrophenol hydroxylase activity in kidney microsomes by 8-fold. This was accompanied by a 6-fold increase in CYP2E1 apoprotein as determined by Western blot analysis. There was, however, no significant increase in steady-state levels of CYP2E1 mRNA as measured by Northern blot analysis. Starvation also induced CYP2E1 apoprotein in the kidney and, as has been reported previously in the liver, had a synergistic inductive effect with acetone. CYP2B and CYP3A apoproteins were also induced by acetone, starvation and starvation/acetone combinations in the kidney. Immunohistochemical analysis revealed localization of CYP2E1 and CYP2B principally in the cortex associated with tubular cells. This distribution was maintained upon starvation/acetone treatment. Two induction experiments were performed in which the ethanol was administered as part of a system of total enteral nutrition (TEN). A short-term study was conducted in which ethanol was administered for 8 days in two liquid diets of different composition, and a chronic experiment was performed in which ethanol was administered for 35 days. A diet-independent 6-fold increase in CYP2E1 apoprotein was observed in the short-term experiment. Expression of CYP3A and CYP2A cross-reactive apoproteins in kidney microsomes appeared to be affected by alterations in diet but, were unaffected by ethanol treatment. In the chronic 35-day ethanol exposure experiment, CYP2E1 apoprotein was also elevated 6-fold and this was found to be accompanied by a significant 3-fold increase in CYP2E1 mRNA. In the same study, no ethanol effects were apparent on expression of CYP2B and CYP3A apoproteins. Thus, acetone induced a variety of renal cytochrome P450 forms in addition to CYP2E1, while ethanol appeared to be a much more specific renal CYP2E1 inducer. Furthermore, as reported in the liver, acetone and ethanol appeared to induce CYP2E1 in the kidney by different mechanisms.

Acetone↗

PubMed1998

Oxidation of methyl- and ethyl- tertiary-butyl ethers in rat liver microsomes: role of the cytochrome P450 isoforms.

Methyl t-butyl ether (MTBE) and ethyl t-butyl ether (ETBE) are commonly used in unleaded gasoline to increase the oxygen content of fuel and to reduce carbon monoxide emissions from motor vehicles. This study was undertaken to investigate: (1) the effect of administration to rats of ETBE and its metabolite, t-butanol, on the induction and/or inhibition of hepatic P450 isoenzymes; (2) the oxidative metabolism of MTBE and ETBE by liver microsomes from rats pretreated with selected P450 inducers and purified rat P450(s), (2B1, 2E1, 2C11, 1A1). ETBE administration by gavage at a dose of 2 ml/kg for 2 days induced hepatic microsomal P4502E1-linked p-nitrophenol hydroxylase and the P4502B1/2-associated PROD and 16beta-testosterone hydroxylase, verified by immunoblot experiments. t-Butanol treatments at doses of 200 and 400 mg/kg i.p. for 4 days did not alter any liver microsomal monoxygenases. Both MTBE and ETBE were substrates for rat liver microsomes and were oxidatively dealkylated to yield formaldehyde and acetaldehyde, respectively. The dealkylation rates of both MTBE and ETBE were increased c. fourfold in phenobarbital (PB)-treated rats. In rats pretreated with pyrazole, an inducer of 2E1, only the demethylation of MTBE was increased (c. twofold). When the oxidations of MTBE and ETBE were investigated with purified P450(s) in a reconstituted system, it was found that P4502B1 had the highest activities towards both solvents, whereas 1A1 and 2C1 were only slightly active; P4502E1 had an appreciable activity on MTBE but not against ETBE. Metyrapone, a potent inhibitor of P450 2B, consistently inhibited both the MTBE and ETBE dealkylations in microsomes from PB-treated rats. Furthermore, 4-methylpyrazole (a probe inhibitor of 2E1) and anti-P4502E1 IgG showed inhibition, though modest, only on MTBE demethylation, but not on ETBE deethylation. Inhibition experiments have also suggested that rat 2A1 may exert an important role in MTBE and ETBE oxidation. Taken together, these results indicate that 2B1, when expressed, is the major enzyme involved in the oxidation of these two solvents and that 2E1 may have a role, although minor, in MTBE demethylation. The implications of these data for MTBE and ETBE toxicity remain to be established.

Air Pollutants↗