PubMed Health⌕ Search

Biomedical subjects

V Longo

Publications and source records attributed to V Longo.

At least 37 records · Page 2Linked to original sources
PubMed1998

Xenobiotic-metabolizing enzymes in pig nasal and hepatic tissues.

1. A study of xenobiotic-metabolizing enzyme activity of the olfactory and respiratory epithelium in the pig was undertaken. The results indicated that porcine olfactory mucosa contains all the components of the P450 system. 2. Monooxygenase activities were much higher in olfactory than in respiratory microsomes, and the olfactory activities dependent on CYP2A were higher than those in the liver. By contrast, the olfactory monooxygenases associated with CYP2E1 were poorly or not detected, whereas CYP2G1 and a protein immunorelated to CYP1A2 were expressed in the olfactory epithelium. 3. The activities of several non oxidative enzymes (glutathione S-transferase, UDP-glucuronyl transferase, epoxide hydrolase, DT-diaphorase, benzaldehyde and propionaldehyde dehydrogenases, and various esterases) were also determined in porcine tissues and were found to be higher in the olfactory than in the respiratory mucosa, but lower or similar to those in liver. 4. An unexpected finding was a higher activity of olfactory UDP-GT compared with that of liver when 1-naphtol but not p-hydroxybiphenyl (a good substrate for a specific olfactory UDP-GT(olf) in bovine and rat) was used as substrate, suggesting a porcine specific expression of UDP-GT isoforms. 5. The results taken together indicate that the olfactory epithelium of mammals has a similar cytochrome P450 profile with the CYP2A and CYP2G1 as dominant isoforms, whereas the olfactory non-oxidative enzymes appear qualitatively and quantitatively expressed to different extents.

Animals↗
PubMed1998

Chlorzoxazone 6-hydroxylase and p-nitrophenol hydroxylase as the most suitable activities for assaying cytochrome P450 2E1 in cynomolgus monkey liver.

Western blot analyses of liver microsomes from 13 male and 12 female monkeys demonstrated that in each sample a variable amount of a cytochrome P450 (P450) protein, likely monkey P450 2E1, cross-reacted with anti-rat P450 2E1 antibodies. Therefore, the involvement of monkey 2E1 in the oxidation of typical substrates for 2E1 from other species, such as dimethylnitrosamine (DMN), p-nitrophenol (pNP), chlorzoxazone (CLZ), and aniline, was investigated. Kinetic studies using microsomes from five male and five female monkeys showed that CLZ and pNP hydroxylations were monophasic, with apparent KM values of 77 and 14 microgramsM, respectively, whereas aniline hydroxylation and DMN demethylation were multiphasic, suggesting that P450s other than 2E1 were involved in catalyzing the latter two reactions. When correlation analyses were performed using several monooxygenase activities determined in male and female monkey liver specimens, it was found that immunodetectable 2E1 contents were highly correlated (r >/= 0.75) with CLZ and pNP hydroxylations, weakly correlated (r = 0.6) with aniline hydroxylation, and not correlated with DMN demethylation or other monooxygenase activities; CLZ hydroxylation was strongly correlated with pNP hydroxylation, weakly correlated with aniline hydroxylation, and not correlated with DMN demethylation. Inhibition experiments showed that CLZ and pNP hydroxylations were immunoinhibited by 60-80% by anti-rat P450 2E1 and were inhibited by the prototypical 2E1 inhibitor 4-methylpyrazole with IC50 values of 1.5 and 13 microgramsM, respectively. In conclusion, the findings provide evidence that P450 2E1 is constitutively and equally expressed in male and female monkey liver and it exerts a major role only in hydroxylation of CLZ and pNP.

Animals↗
PubMed1997

Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium.

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals.

Amino Acid Sequence↗
PubMed1995

Effect of carrageenan-induced granuloma on hepatic cytochrome P-450 isozymes in rats.

Carrageenan-induced granuloma was used to study the apoprotein and RNA content, and catalytic activities of several cytochrome P-450 isozymes in liver. This model allowed discrimination between acute and chronic phases of experimental inflammation. The expression of most isozymes studied (CYP2D, CYP2E1, CYP3A1 and CYP4A) was reduced to 20% of the control level during the acute phase and partially recovered (30-60% of control group) during the chronic phase. CYP2B1 content was decreased to 65% of control during the acute and chronic phases of inflammation. RNA (CYP2B1 and CYP2E1) showed a strong depression during the acute phase and recovered during the chronic phase, without differences between isoenzymes. In most cases, there was a good correlation between the apoprotein content of isozymes and related activities. Our results show that the depletion of cytochrome P-450 induced by inflammation depends on the severity of the disease. Experimental inflammation equally affect the transcription of CYP2B1 and CYP2E1, so differences in apoprotein content and related activities between isozymes may due to differential posttranscriptional regulation.

Acute Disease↗
PubMed1994

Mutagenicity of hexahydrophenanthrenes and their oxirane derivatives.

The mutagenicity of some hexahydrophenanthrenes and their corresponding arene oxides was assayed in histidine-dependent mutants of Salmonella typhimurium TA98 and TA100. All the arene epoxides examined were devoid of mutagenic activity, although some of them could alkylate nicotinamide. By contrast, the 1,2,3,9,10, 10a-hexahydrophenanthrene, trans-1,2,3,4,4a,10a-hexahydrophenanthrene, 9-methyl-, 6-methoxy-trans-1,2,3,4,4a,10a-hexahydrophenanthrene, 7-bromo-trans-1,2,3,4,4a,10a-hexahydrophenanthrene and 9-methyl-trans-1,2,3,4,4a,10a-hexahydrophenanthrene were active as mutagens in the presence of S9 mix. A negative result was obtained with octahydrophenanthrene, suggesting that the benzylic double bond is a prerequisite for the mutagenic activities of hexahydrophenanthrenes. Thus, probably a very reactive intermediate (aryloxirane) formed by a secondary metabolism following the primary oxidation of the benzylic double bond by S9 mix could be responsible for the mutagenicity of the hexahydrophenanthrenes.

Epoxy Compounds↗
PubMed1993

Glutathione transferase isoenzymes in olfactory and respiratory epithelium of cattle.

Glutathione transferase (GST) was investigated in the olfactory and respiratory epithelium of cattle. A significantly more abundant GST in terms of either protein amount or activity was found in the olfactory rather than in the respiratory epithelium. No apparent qualitative differences in the isoelectric focusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC profiles were noted in the reduced glutathione (GSH) affinity purified GST pool of olfactory and respiratory epithelium. Both tissues have at least six GST isoenzymes with isoelectric point values of 4.9 (peak I), 5.3 (peak II), 5.95 (peak III), 6.5 (peak IV), 7.1 (peak V) and 9.3 (peak VI). From both tissues at least seven different GST subunits can be resolved by HPLC analysis. The GST isoenzymes having pI at 5.3 and 9.3 were predominantly expressed in the olfactory than in the respiratory epithelium. These latter forms conjugate GSH efficiently with alkenals and hydroperoxides, respectively. Kinetic, immunological and structural properties, including HPLC analysis and N-terminal region amino acid sequence seem to indicate that the bovine nasal mucosa tissue in addition to a GST subunit which is orthologue to rat subunit 8 (alpha class) express tissues specific subunits.

Animals↗
PubMed1993

Acetone-dependent regulation of cytochromes P4502E1 and P4502B1 in rat nasal mucosa.

The inducibility and molecular regulation of cytochrome P4502E1 (CYP2E1) has been examined in nasal mucosa of rats after acetone treatment and compared to that of cytochrome P4502B1 (CYP2B1). Twenty-four hours following treatment with acetone (5 mL/kg) for 2 days, the amount of CYP2E1 as well as the rate of microsomal 4-nitrophenol hydroxylase activity had increased by a factor of 2-3, in microsomes isolated from nasal mucosa. The increase in CYP2E1 was accompanied by a corresponding increase of CYP2E1 mRNA, as determined by northern and slot blot analyses. In contrast, hepatic and renal CYP2E1 mRNA, studied in the same rats, did not increase, despite the fact that the amount of CYP2E1 was increased 3- and 5-fold, respectively. The amount of CYP2B1, an isozyme known as acetone-inducible in other tissues, decreased significantly by acetone, as detected by immunoblot analysis. After 48 hr, the amount of CYP2E1 enzyme, the level of CYP2E1 mRNA and the rate of 4-nitrophenol hydroxylase activity had returned to normal levels, whereas in liver and kidneys the immunoreactive protein remained 3-4-fold higher than control. The results indicate that acetone does not regulate CYP2E1 in nasal mucosa by post-translational mechanisms, in contrast to the situation observed in liver and kidneys. This indicates a tissue-specific expression of post-translational regulatory systems responsible for P450 stabilization. Furthermore, nasal CYP2B1 also seems to be regulated in a tissue-specific manner by acetone.

Acetone↗
PubMed1992

Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.

A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species.

Acetone↗
PubMed1992

Drug-metabolizing enzymes in respiratory nasal mucosa and liver of cynomolgus monkey.

1. Microsomal and cytosolic drug-metabolizing enzyme activities of respiratory mucosa of male and female monkeys have been determined and compared to those of monkey liver. The results demonstrated that cytochrome P-450, NADPH-cytochrome P-450 reductase and some monooxygenase activities, especially ethoxycoumarin O-deethylase activity, were present in respiratory epithelium, although at lower levels than in liver. 2. Activities of non-oxidative enzymes--namely, epoxide hydrolase, UDP-glucuronyltransferase, glutathione S-transferase, DT-diaphorase, carbonyl reductase, benzaldehyde and propionaldehyde dehydrogenases--were also detected in respiratory tissue, some at higher levels than in liver. 3. The enzymic activities found in monkey nasal mucosa are not very similar to those in corresponding human tissue where, for example, UDP-glucuronyltransferase activity is not detectable. This indicates that monkey is not necessarily the best animal model for studies of the human upper respiratory tract.

Animals↗
PubMed1992

Depression of hepatic drug metabolism in endotoxin-treated and sarcoma-bearing mice.

The effect of a single dose of bacterial endotoxin (lipopolysaccharide, LPS) was compared with that of tumor implantation in mice on the activity of several hepatic cytochrome P-450-dependent monooxygenases. These included ethoxycoumarin O-deethylase, p-nitrophenol hydroxylase, aminopyrine N-demethylase, pentoxyresorufin O-depentylase, ethoxyresorufin O-deethylase and testosterone hydroxylase. For this purpose, mice were treated i.p. with 5 micrograms of LPS or implanted in the right paw with S 180 sarcoma. A comparable depression (30-50%) of total microsomal P-450 content as well as of the different P-450 monooxygenase activities tested was observed in LPS-treated mice (24 h after LPS) and in tumor bearing mice (12 days after implantation). The lack of differences in the pattern of depression of microsomal enzymes between LPS-treated and tumor-bearing mice suggests that a common mechanisms might be involved in the depression of P-450 by LPS or S-180 implantation.

Animals↗
PubMed1991

Xenobiotic-metabolizing enzymes in human respiratory nasal mucosa.

Study of oxidative and non-oxidative xenobiotic-metabolizing enzymes was undertaken in microsomal and cytosolic fractions of two human livers, 10 individual and several pooled samples of human respiratory nasal mucosa obtained by surgical operation of male and female patients affected by hypertrophy of the inferior turbinates. The purity of nasal microsomes was checked by electron microscopy and marker enzyme assay. The pooled samples of respiratory nasal epithelium contained, relative to liver, a low amount of cytochrome P450 (about 25 pmol/mg protein) and associated biotransformation activities, and a low level of other components of the mixed-function oxidase system such as cytochrome b5, NADH and NADPH-cytochrome c reductase however the NADH-cytochrome b5 reductase activity was comparable to that of liver. The P450-dependent monooxygenase activities such as ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase and the dimethylnitrosamine N-demethylase were found in nearly all nasal microsomal specimens. The aniline hydroxylase and the aminopyrine or hexamethylphosphoramide N-demethylases were detected only in the pooled nasal samples. With regard to the non-oxidative enzymes, the activities of glutathione S-transferase, DT-diaphorase, epoxide hydrolase, UDP-glucuronyl-transferase, carbonyl reductase, benzaldehyde and propionaldehyde dehydrogenases, were investigated both in the individual and pooled nasal tissues and livers. These activities were similar in nasal and liver tissue, except for UDP-glucuronyltransferase which was not detected in nasal mucosa. The present findings demonstrate that the respiratory section of human nose contains a wide array of oxidative and non-oxidative enzymes, which could play a crucial role in the bioactivation or detoxication in situ of inhaled xenobiotics.

Adolescent↗
PubMed1991

Drug-metabolizing enzymes in liver, olfactory, and respiratory epithelium of cattle.

The drug-metabolizing enzymes of olfactory and respiratory epithelium of cattle were determined. The data of nasal tissues were compared to those of bovine liver. Both oxidative and nonoxidative enzyme activities were investigated. Many compounds including testosterone were used as substrates for the P450-dependent monooxygenase activities. The results demonstrated that the P450 content and all the activities assayed including reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase were much higher in the olfactory than in the respiratory mucosa and for some activities (hexamethyl-phosphoramide and dimethylnitrosamine N-demethylase, aniline hydroxylase, and ethoxycoumarin O-deethylase) the values in the olfactory tissue were even markedly higher than those of liver. Also the activities of some nonoxidative enzymes such as glutathione S-transferase, uridine 5'-diphosphate (UDP)-glucuronyl-transferase, and epoxide hydrolase were higher in the olfactory than in the respiratory mucosa but lower than in liver. The results taken together suggest that the olfactory and respiratory epithelium of cattle, which contain in addition to a wide array of nonoxidative enzymes multiple forms of P450, can be useful and easily available tissues to study the biotransformation processes of odorants.

Animals↗
PubMed1990

Possible role of the acetone-inducible cytochrome P-450IIE1 in the metabolism and hepatotoxicity of thiobenzamide.

The effect of acetone pretreatment (5% in drinking water for 10 days on rat liver metabolism and toxicity of thiobenzamide (TB) was investigated. Hepatic microsomes from acetone-pretreated rats showed a significant increase of TB-S-oxidation rate which, on the basis of selective thermal inactivation of FAD-containing monooxygenase (FADM), appeared dependent only on cytochrome P-450. Furthermore, TB was able to competitively inhibit acetone hydroxylase (AcH), an enzymatic reaction highly specific for the P-450IIE1 isozyme. Acetone pretreatment of rats also produced an exacerbation of liver damage induced by acute administration of TB (150 mg/kg), as judged by the extent of liver necrosis and serum alanine-amino transferase (ALAT) activities. Coadministration of acetone with TB reduced on the other hand the extent of liver damage. The findings suggest that P-450 species induced by acetone, and in particular the P-450IIE1 isozyme, could be involved in the biotransformation of TB.

Acetone↗
PubMed1990

Effect of acetone administration on renal, pulmonary and hepatic monooxygenase activities in hamster.

Administration of acetone in drinking water to Syrian Golden hamsters for 9-10 days altered microsomal P-450 dependent monooxygenase activities in the liver and the kidney but not in the lung. While hepatic microsomal NADPH-cytochrome c reductase was unaffected, cytochrome b5 and P-450 content increased (about 100%) in liver but not in kidney. Furthermore acetone treatment resulted in an increase of microsomal reverse type I binding with DMSO and in an increase in the P-450IIE1-linked renal and hepatic activities such as aniline hydroxylase (AnH) and p-nitrophenol hydroxylase (pNPH). The SDS-PAGE analysis confirmed the induction in acetone-treated microsomes of a hepatic protein with the M.W. of ethanol inducible P-450IIE1 of hamster. The acetone treatment however, unlike ethanol, induced other activities such as benzphetamine N-demethylase and ethoxycoumarin O-deethylase in liver and aminopyrine N-demethylase in kidney. No change of ethoxyresorufin O-deethylase and pentoxyresorufin O-depentylase was observed in either renal or hepatic microsomes. Addition of acetone in vitro had an inhibitory effect on pNPH by hepatic microsomes from control or acetone induced hamsters, while AnH was not affected. Interruption of acetone administration for 24 h resulted in a return of AnH and pNPH activities to essentially basal levels in the liver suggesting a rapid turnover of the hamster P-450IIE1 (ham P-450j). Our results indicate that, as found in rat, acetone is a good inducer of the P-450IIE1 (ham P-450j) in hamster in both the liver and kidney. However other P-450 forms, such as, probably, the renal and hepatic P-450IIB1, are also induced. Thus acetone-treated hamsters, which, in certain respects, show a qualitatively different induction pattern from that reported for ethanol, can be used as an useful animal model to study the toxicity of certain xenobiotics.

Acetone↗
PubMed1990

Effects of acetone administration on drug-metabolizing enzymes in mice: presence of a high-affinity diethylnitrosamine de-ethylase.

Treatment of CD1 mice with acetone raised activities of hepatic microsomal p-nitrophenol hydroxylase, ethoxycoumarin de-ethylase, acetone hydroxylase and diethylnitrosamine de-ethylase (DENd) several-fold. P-450IIE1-linked acetone hydroxylase showed the highest inducibility. In microsomes from acetone-pretreated mice the cytochrome b5 and P-450 content was nearly doubled and their electrophoretic profile showed induction of a protein of Mr 53,000, probably P-450IIE1. Liver phase II enzymes were not affected by acetone treatment. Kinetic analyses of DENd were performed in control or acetone-induced microsomes and Km and Vmax were determined. Two distinctly apparent Km values (0.56 and 20.3 mM) were observed for DENd of control microsomes and at least 3 apparent Km values (0.05, 0.51, 8.4 mM) were observed in acetone-induced microsomes. Thus, acetone administration to mice induces a high-affinity form of DENd which can be important in vivo at low diethylnitrosamine (DEN) exposure as this enzyme functions when DEN concentration is below 0.1 mM.

Acetone↗
PubMed1990

Metabolism and mutagenicity of isoprene.

Liver microsomes of various rodents (mouse, rat, rabbit, and hamster) metabolize isoprene (2-methyl-1,3-butadiene) to the corresponding monoepoxides 3,4-epoxy-3-methyl-1-butene and 3,4-epoxy-2-methyl-1-butene. 3,4-Epoxy-3-methyl-1-butene (half-life 85 min) was found to be the main metabolite, although the stable 3,4-epoxy-2-methyl-1-butene was also formed (about 14-25% with respect to the main epoxide). The kinetic constants (Km and Vmax) for the formation of the major epoxide metabolite of isoprene were determined by gas-liquid chromatography. The minor epoxide was further epoxidized to the isoprene dioxide by the microsomes of all rodents studied. The Km and Vmax were determined and phenobarbital was found to be a good inducer for this epoxidation in all species. The mutagenic activity, using Salmonella typhimurium, and the chemical reactivity (alkylating power and half-life) of the epoxide metabolites of isoprene were investigated and compared to those of other structurally related epoxides. Isoprene and the monoepoxide intermediates of the isoprene biotransformation were not mutagenic in Salmonella typhimurium. However, the isoprene dioxide (2-methyl-1,2,3,4-diepoxybutane) was found to be mutagenic and have alkylating power towards nicotinamide, similar to the structurally corresponding 1,2,3,4-diepoxybutane. In conclusion, the metabolism of isoprene does not lead to the formation of mutagenic monoepoxide (in contrast to butadiene) but the formation of mutagenic and presumably carcinogenic isoprene diepoxide is possible, thereby a genotoxic effect of isoprene in rodents or other species cannot be ruled out.

Alkylation↗