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H Raunio

Publications and source records attributed to H Raunio.

At least 73 records · Page 4Linked to original sources
PubMed1995

The role of CYP enzymes in cocaine-induced liver damage.

Cocaine is hepatotoxic in several species, including man. A high dose of cocaine produces metabolism-dependent, mainly pericentral, liver damage. At 24 h after a single dose of cocaine, mouse hepatic P450 content decreases but CYP2A activities; coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase increase concomitant with prominent diffuse cell necrosis. Repeated administration of cocaine for up to 5 days decreases CYP1A1/2, 2A4/5, 2Cx, and 2E1 related enzymatic activities. However, after five doses of cocaine, CYP2B10 increases in conjunction with the healing process. In the acute phase, the increased CYP2A activities do not participate in cocaine bioactivation. CYP3A enzymes are principally responsible for the cocaine N-demethylation in human and mouse liver microsomes. The hepatic metabolic CYP enzyme profile will change during prolonged cocaine intake, this being accompanied by altered cell morphology. Possible connections to cocaine toxicity in man are discussed.

Animals↗
PubMed1995

Individual expression of carcinogen-metabolizing enzymes: cytochrome P4502A.

Expression of enzymes metabolizing drugs, carcinogens, and other chemicals ("xenobiotics") is regulated by the interplay of genetic, host, and environmental factors, leading in human populations to a marked interindividual variability. On this basis it is speculated that individual differences in cancer susceptibility could be explained to a certain extent by interindividual differences in metabolic activation. CYP dependence of carcinogen activation is briefly reviewed; CYP2A6 as a more specific example and some consequences and corollaries are briefly discussed. At present, no consensus has been reached about the significance of interindividual differences (genetic or nongenetic) in carcinogen metabolism in cancer etiology. The likely reason is that chemically induced cancer is still a multifactorial, multistage disease involving numerous events before a clinically manifested disease develops. Molecular biological methods such as RFLP and PCR-based techniques as well as molecular dosimetry are making it possible to investigate the genetic background of individual subjects and environmental influences without biases caused by diseases, age, treatment, and other factors, which have plagued studies thus far.

Animals↗
PubMed1995

A genetic polymorphism in coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles.

A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes--CYP2A6, CYP2A7, and CYP2A13--plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6v2. Sequence differences in the CYP2A6v2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6v2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6v2 allele, and a variant--designated CYP2A6v1--that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6v1 and CYP2A6v2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.

Alleles↗
PubMed1994

Cytochrome P4502A5 expression and inducibility by phenobarbital is modulated by cAMP in mouse primary hepatocytes.

Factors involved in CYP2A5 expression were studied in mouse liver primary hepatocytes in culture. CYP2A5-mediated coumarin 7-hydroxylase (COH) activity was retained in simple culture conditions for at least 96 hours and the activity was inducible up to 33-fold by phenobarbital (PB). The constitutive activity and inducibility of COH was totally blocked by treatment of hepatocytes with actinomycin D, and short initial treatment with cycloheximide caused superinducibility when co-administered with PB. Treatment of hepatocytes with inhibitors of protein kinase C, tyrosine kinases and a generator of nitric oxide did not affect COH basal activity or inducibility. Administration of dibutyryl cAMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX) enhanced both basal and PB-induced COH activities and CYP2A5 mRNA levels, indicating that cAMP plays a major role in CYP2A5 expression.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Modification of hepatic cytochrome P450 profile by cocaine-induced hepatotoxicity in DBA/2 mouse.

Previous studies in our laboratory have shown that a hepatotoxic dose of cocaine increases coumarin 7-hydroxylase activity in male DBA/2 mouse liver. In the present study, the dose- and time-dependent responses of the hepatic CYP2A4/5 complex to cocaine-induced liver damage were studied. Cocaine increased CYP2A4/5 levels in a dose-dependent manner. The maximal increases in coumarin 7-hydroxylase activity (4-fold), microsomal CYP2A4/5 content (3-fold) and steady-state mRNA levels (10-fold) were observed at 24 h after administration of a single dose of 60 mg/kg cocaine coinciding with morphologically detectable diffuse liver damage, while the total P450 content was not changed. 3 and 5 days after the daily administration of cocaine severe, mainly pericentral (zone III of Rappaport), liver damage was apparent in parallel with a clear decline in CYP2A4/5 mRNA, protein content and coumarin 7-hydroxylase activity. After 5 days of treatment, CYP2A5 still remained at a very low level but an induction in CYP2B10 protein and related pentoxyresorufin O-dealkylase activity was observed. No marked changes in microsomal CYP2Cx and CYP1A1/2 contents or associated activities were observed. Dimethylnitrosamine N-demethylase activity, a marker for CYP2E1, decreased in parallel with increased cocaine dose and time and the severity of liver damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Metabolic interactions of methoxsalen and coumarin in humans and mice.

Methoxsalen (8-methoxypsoralen) is a very potent inhibitor of human cytochrome P450 2A6 (CYP2A6) and mouse Cyp2a-5-mediated coumarin 7-hydroxylation in vitro. To determine the effect of methoxsalen on coumarin 7-hydroxylation in humans in vivo, five subjects were given 45 mg of methoxsalen and 5 mg of coumarin. Methoxsalen inhibited in vivo coumarin metabolism by 47 +/- 9.2% (mean +/- SEM). Methoxsalen was metabolized in human liver microsomes at the rate of 50-100 pmol/mg protein/min (approx. 30% of the activity in mouse liver microsomes). Metabolism was not inhibited by the anti-Cyp2a-5 antibody in human liver microsomes. NIH 3T3 cells stably expressing catalytically active CYP2A6 enzyme did not metabolize methoxsalen, indicating that CYP2A6 does not accept methoxsalen as a substrate. In pyrazole-induced mouse liver microsomes, methoxsalen metabolism was inhibited by the anti-Cyp2a-5 antibody. Cyp2a-5 protein expressed in the yeast Saccharomyces cerevisiae was capable of metabolizing methoxsalen, indicating that methoxsalen is a substrate of Cyp2a-5. Although kinetic studies indicated that the inhibition of coumarin 7-hydroxylation by methoxsalen is competitive in human liver microsomes, methoxsalen does not appear to be a substrate for CYP2A6. Methoxsalen and coumarin have the potential of strong metabolic interactions in man.

Animals↗
PubMed1994

Expression of xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver.

Expression of human cytochrome P450 (CYP) genes in human adult and fetal liver were studied using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In adult liver mRNA of CYPs 1A1, 1A2, 2A6/2A7, 2B6/2B7, 2C8-19, 2D6, 2E1, 3A3/3A4 and 3A7 were detected while CYPs 2F1 and 4B1 were absent. In fetal liver mRNA of CYPs 2C8, 2D6, 3A3/3A4 and 3A7 were found but all other forms studied were undetectable. The results provide a comprehensive qualitative picture of the expression of CYP genes in families CYP1 through CYP4 in human adult and fetal liver.

Adult↗
PubMed1993

Expression of human placental cytochrome P450 aromatase (CYP19) cDNA in insect cells using a luciferase based baculovirus vector.

The cDNA encoding human placental cytochrome P450 aromatase (CYP19) was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system. The recombinant protein product was characterized by Northern and Western blot analyses as well as by direct measurement of aromatase activity. The expressed enzyme proved to be both catalytically active in the presence of P450 reductase and immunologically reactive with polyclonal antibodies raised against human placental aromatase. The activity of aromatase increased 10% after the addition of 0.1 microgram/ml hemin chloride to the culture medium. However, the level of aromatase protein decreased considerably when the concentration of hemin chloride reached 10 micrograms/ml indicating that hemin chloride has toxic effects on the lepidopteran insect cell line. In conclusion, the baculovirus system is suitable for high level expression of functional human placental CYP19.

Animals↗
PubMed1993

Retrovirus-mediated stable expression of human CYP2A6 in mammalian cells.

To study the pharmacological and toxicological significance of the human cytochrome P450 isoform CYP2A6, we expressed it in mammalian cells by retrovirus-mediated gene transfer. The LXSN vector and PA317 packaging cells were used to create amphotropic recombinant retroviruses containing CYP2A6 cDNA. NIH 3T3 and HeLa cells were infected with these retroviruses and cell clones expressing CYP2A6 were selected. The integration of the CYP2A6 construct was verified by PCR analysis and northern blot analysis showed that a 5 kb mRNA containing the CYP2A6 was present in the cells. The integrated cDNA directed the expression of catalytically active CYP2A6 enzyme which has remained stable over numerous cell passages. No oxidation of several other P450 substrates was detected. The promutagen aflatoxin B1 was metabolized to intermediates binding to the host cell genomic DNA by the 3T3 2A6 cells. These cell lines are thus well suited for the study of the catalytic profile and the biological consequences of promutagen activation by the human CYP2A6 isoform.

3T3 Cells↗
PubMed1993

Differential inhibition of coumarin 7-hydroxylase activity in mouse and human liver microsomes.

Coumarin is 7-hydroxylated by the P450 isoform Cyp2a-5 in mice and CYP2A6 in humans. Various drugs, endogenous substances, plant substances and carcinogens, altogether about 90 chemicals, were evaluated as possible inhibitors of coumarin 7-hydroxylase (COH) activity in mouse microsomes. The effects of selected compounds on COH activity in human liver microsomes were also tested. The furanocoumarin derivatives methoxsalen (8-methoxypsoralen) and psoralen proved to be the most potent inhibitors of mouse COH activity (IC50 values 1.0 and 3.1 microM, respectively). The furanocoumarins bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), imperatorin and sphondin also effectively inhibited mouse COH activity (IC50 values 19-40 microM). Methoxsalen, isopimpinellin and metyrapone were also inhibitors in mice in vivo. Methoxsalen was a potent inhibitor of COH activity also in human liver microsomes, (IC50 value 5.4 microM), whereas bergapten, isopimpinellin and imperatorin had no effect. The imidazole antimycotic miconazole was a potent but non-specific inhibitor of COH activity. Several known substrates and inhibitors of members in the CYP1A, CYP2B, CYP2C, CYP2D and CYP3A subfamilies were poor inhibitors of COH activity. These results suggest that (i) the coumarin-type compounds in particular interact with the active sites of Cyp2a-5 and CYP2A6, and (ii) the active sites of Cyp2a-5 and CYP2A6 are structurally different, since a number of compounds inhibited mouse, but not human COH activity.

Animals↗
PubMed1993

Cytochrome P450 isoforms in human fetal tissues related to phenobarbital-inducible forms in the mouse.

Four polyclonal antibodies raised against purified mouse liver cytochrome P450s representing Cyp1a, Cyp2a, Cyp2b and Cyp2c subfamilies were used to detect their related forms in human adult and fetal tissues. In immunoblot analysis, anti-Cyp2c antibody detected two to three proteins in adult livers and one to three proteins in 70% of the 18 fetal livers studied. Anti-Cyp2a-5 antibody recognized a 50-kDa protein in 50% of the fetal adrenals. Anti-Cyp1a-2 antibody reacted with a single protein (55 kDa) in adult liver. The anti-Cyp2b-10 antibody did not detect proteins in any of the tissues. No proteins were detected in fetal kidneys. There was no coumarin 7-hydroxylase activity (COH) in fetal liver or adrenals. The 7-ethoxycoumarin O-deethylase (ECOD) activities were slightly higher in fetal adrenals (mean 6.1 pmol/mg protein/min) vs livers. The fetal adrenal ECOD activity was not inhibited by the anti-Cyp2a-5 antibody. Aryl hydrocarbon hydroxylase (AHH) activities in fetal livers were about 5% of those in adult livers. AHH activity in fetal liver was not inhibited by the anti-Cyp2c antibody. Testosterone 6 beta-hydroxylase activity was much lower in fetal liver than in adult liver (about 20 and 1700 pmol/mg protein/min, respectively). No immunoinhibition occurred in fetal adrenal progesterone hydroxylation, hepatic benzphetamine N-demethylation and hepatic ethylmorphine N-demethylation. These data suggest that members of the P450 subfamilies 1A, 2A and 2B are expressed at a very low level in fetal liver, and that fetal liver may contain members of the 2C subfamily.

Adrenal Glands↗
PubMed1993

Cytochrome P4502A-mediated coumarin 7-hydroxylation and testosterone hydroxylation in mouse and rat lung.

Pulmonary coumarin 7-hydroxylase, testosterone hydroxylase and other P450-mediated activities were compared in the mouse and rat. Coumarin 7-hydroxylase activity was 20 pmol/mg/min. in mouse and 4 pmol/mg/min. in rat lung microsomes. Liver values were 180 (mouse) and 1 (rat) pmol/mg/min. Km values of rat and mouse lung coumarin 7-hydroxylase were about 1 microM whereas the rat liver Km value was > 100 microM. Phenobarbital and 3-methylcholanthrene did not affect rat lung (or liver) coumarin 7-hydroxylase activity. Anti-Cyp2a-5 antibody effectively inhibited mouse and rat lung coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylations but failed to block these activities in the rat liver. In immunoblot analysis anti-Cyp2a-5 antibody recognized the 50-kDa Cyp2a-4/5 protein in mouse lung microsomes. A P450 protein co-migrating with Cyp2a-5 was also detected in rat lung microsomes. Cyp2a-5 cDNA probe hybridized with a 1.8-kb mRNA species in rat lung RNA fraction. The hybridization signal was not increased by 3-methylcholanthrene or phenobarbital. These data suggest that the mouse lung expresses Cyp2a-5 which differs from the liver enzyme only in its regulation and that the rat lung contains a P450 isoform(s) belonging to the 2A subfamily which may be orthologous with the mouse Cyp2a-4/5 catalyzing coumarin 7-hydroxylase and testosterone 15 alpha-hydroxylase activities. The recently reported rat lung CYP2A3 (Kimura et al.) gene product is a candidate for the observed coumarin 7-hydroxylase activity in the rat lung.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1992

Cerium-induced strain-dependent increase in Cyp2a-4/5 (cytochrome P4502a-4/5) expression in the liver and kidneys of inbred mice.

The murine Cyp2a-4 and Cyp2a-5 genes encode P450 isoforms catalysing testosterone 15 alpha-hydroxylase and coumarin 7-hydroxylase (COH) activities, respectively. Two days after the administration of a hepatotoxic dose of cerium chloride (2 mg/kg i.v.), COH activity was increased 3.2-fold in the liver of DBA/2 mice. Three and 4 days after the cerium treatment, coinciding with the occurrence of overt liver damage, there was a dramatic decrease in COH activity. The activities of testosterone 15 alpha-hydroxylase and the Cyp1a-1-mediated 7-ethoxyresorufin O-deethylase (EROD) were decreased in response to cerium. Much less pronounced changes in the enzyme activities occurred in the C57BL/6 mouse liver. Northern blot analysis showed a 21-fold increase in the hepatic Cyp2a-4/5 mRNA in the DBA/2 mice at day 2, whereas no increase occurred in the C57BL/6 mice. Also in the kidneys the increase in COH activity and in Cyp2a-4/5 mRNA was marked only in the DBA/2 mice. A polymerase chain reaction-mediated analysis method utilizing a unique PstI restriction site in the Cyp2a-5 cDNA was used to differentiate between the highly homologous Cyp2a-4 and Cyp2a-5 mRNAs. Cerium was found to increase the amount of hepatic and renal Cyp2a-4 and Cyp2a-5 mRNA only in the DBA/2 mice. These data indicate that the Cyp2a-4/5 complex is regulated in a different way in DBA/2 and C57BL/6 mice and that some association exists between the development of liver damage and COH induction.

Animals↗
PubMed1992

Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression.

Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida & Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.

Animals↗
PubMed1991

Cloning and characterization of a member of the rat multidrug resistance (mdr) gene family.

The rat mdr gene family [genes encoding P-glycoprotein (Pgp)] was characterized and the complete sequence of a rat mdr cDNA was determined based on seven independent cDNA clones that correspond to the same gene. The longest of these clones contains a 4.3-kb insert which represents a full-length rat mdr cDNA. The longest open reading frame of this sequence is 3933 bp; the first ATG is at 103 bp, making the deduced protein 1277 amino acids long (141 kDa). This correlates well with previously identified Pgp. The sequence of this gene has a very high, greater than 90%, degree of identity to the mouse mdr1b gene (also known as the mdr1 gene) therefore, we designate it the rat mdr1b gene. Transcription of this gene begins at a single start point 151 nucleotides upstream from the start codon. We show here that the rat gene family is comprised of three members, which is consistent with previous data on other rodent species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Comparison between cobalt and pyrazole in the increased expression of coumarin 7-hydroxylase in mouse liver.

The data in this report show that administration of both cobalt and pyrazole results in an elevation in the amount of hepatic mRNA encoding for microsomal P45015 alpha/P450Coh, an increase in the amount of P450Coh protein, and an activation of COH and to a lesser extent testosterone 15 alpha-hydroxylase in two inbred strains of mice. Considerable quantitative differences between the two compounds and the two mouse strains in the response suggest that the effects of cobalt and pyrazole are mediated, at least partly, through different mechanisms. It is of interest that human hepatic COH resembles very closely that in the mouse liver.

Animals↗
PubMed1991

Distribution and effects on cytochrome P450 system of two hexachlorobiphenyl isomers in the rat.

Tissue distribution and effects induced by 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) on cytocrome P450 isozymes were compared with those of 2,2',3,3',6,6'-hexacholorobiphenyl (236-HCB). Male Wistar rats were given a single intragastric dose (23 mg/kg body wt) of either isomer, and killed after 72 h. At termination the tissue concentrations of 245-HCB were considerably higher than those of 236-HCB, suggesting a more effective metabolism of the latter. The binding affinity of 236-HCB to cytochrome P450 was higher and the magnitude of binding greater than of 245-HCB. 245-HCB-treatment elevated the hepatic concentration of cytochrome P450 and also the activities of 7-pentoxyresorufin O-depentylase (50-fold), aniline p-hydroxylase (2-fold) and 7-ethoxycoumarin O-deethylase (2-fold), a response typical of phenobarbital-type inducers. In the Western immunoblot of liver microsomes from 245-HCB treated rats, an increased amount of P450IIB 1/2 was detected by a monoclonal antibody 2-66-3, which specifically detects phenobarbital inducible isoenzymes. The minimum molecular mass of the P450 isozyme induced was 52 kDa. After 236-HCB administration, a weak inducing effect was observed.

Animals↗