Biomedical subjects

H Raunio

Publications and source records attributed to H Raunio.

At least 91 records · Page 5Linked to original sources

PubMed1991

Metabolic interaction and disposition of methyl ethyl ketone and m-xylene in rats at single and repeated inhalation exposures.

1. Rats were exposed to m-xylene (300 ppm) and methyl ethyl ketone (MEK, 600 ppm) vapour, separately and in combination. 2. Repeated exposures to m-xylene enhanced liver drug-metabolizing capacity, whereas MEK showed no effects. After mixed exposure the cytochrome P-450-dependent monooxygenase activities were additively or synergistically induced. 3. In the presence of MEK the overall metabolism of xylene was strongly inhibited both after single and repeated exposures, an effect accompanied by elevation of xylene concentration in blood (18-29%) and fat (25-32%). 4. The 24-h excretion of the urine metabolites of m-xylene was decreased by 22-24% in mixed exposures: the excretion of methylhippuric acid was decreased (29%), but that of 2,4-dimethylphenol increased (9-35%). 5. After repeated inhalation exposures the excretion of xylene metabolites in urine was consistently higher, whereas the concentrations of xylene in fat (but not the concentration of MEK) were lower than after a single treatment, conceivably due to accelerated metabolic clearance of xylene. 6. Thioether excretion in urine was enhanced in xylene-treated rats (7-13-fold), but was not influenced by the induced changes in the metabolism of xylene. Xylene inhalation caused liver GSH to decrease slightly (10%), as did inhalation of MEK, but the latter did not enhance the excretion of thioethers. 7. MEK is a potent inhibitor of the side-chain oxidation of m-xylene producing methylhippuric acid, but not of its ring oxidation to 2,4-dimethylphenol, and exhibits a synergistic inducing effect on liver enzymes responsible for the oxidation of m-xylene. The increased ring oxidation of m-xylene was not associated with increased production of reactive metabolites indicated by GSH-depletion or thioether formation.

Administration, Inhalation↗

PubMed1990

Immunochemical detection of human liver cytochrome P450 forms related to phenobarbital-inducible forms in the mouse.

Polyclonal antibodies generated to four distinct mouse liver phenobarbital-inducible cytochrome P450 isoforms were used to analyse related forms in human liver. N-terminal sequence analysis and biochemical properties of the P450s used as antigens suggest that they belong to P450 subfamilies IIB (P450PBI), IA (P450PBII), IIC (P450PBIII) and IIA (P450Coh). In immunoblot analysis, anti-P450PBII detected a single protein presumed to be P450IA2 in all the human livers tested. No proteins corresponding with P450IA1 could be detected. Anti-PBIII and anti-P450Coh antibodies each detected one band (54 and 48 kDa, respectively) in the liver samples. No bands were revealed by anti-P450PBI antibody. Protein dot-immunobinding analysis showed that P450s immunodetectable by anti-P450PBII, anti-P450PBIII and anti-P450Coh antibodies are expressed in human liver (range 9 to 69 pmol P450/mg protein). In immunoinhibition experiments the activity of 7-ethoxyresorutin O-deethylase (EROD) was blocked up to 90% by the anti-P450PBII antibody. Aryl hydrocarbon hydroxylase (AHH) was inhibited only by anti-P450PBIII, and coumarin 7-hydroxylase (COH) only by anti-P450Coh antibody. Testosterone hydroxylations in positions 6 beta, 7 alpha, 15 alpha and 16 alpha were not affected significantly by any of the antibodies. These data suggest that the human liver P450IA2 is responsible for most of the elevated EROD activity, P450s in the IIC subfamily for constitutive AHH and P450s in the IIA subfamily for all of COH activity.

Adult↗

PubMed1990

Immunochemical and molecular biological studies on human placental cigarette smoke-inducible cytochrome P-450-dependent monooxygenase activities.

The induction of specific forms of cytochrome P-450 and P-450-associated xenobiotic-metabolizing monooxygenase activities by maternal cigarette smoking was characterized in human placenta employing polyclonal and monoclonal antibodies and recombinant DNA probes. The anti-BNF-B2 (prepared against rat liver P-450 induced by beta-naphthoflavone) inhibited about 60 per cent of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase activities (ERDE) in placental tissues from smoking mothers, whereas the anti-PB-B2 (to phenobarbital-induced rat liver P-450) was without significant inhibitory effect. Inhibition of 7-ethoxycoumarin O-deethylase (ECDE) by the anti-BNF-B2 was dependent on maternal smoking: the enzyme from non-smokers was not significantly inhibited, whereas the enzyme from smokers was variably inhibited by 15-60 per cent. The monoclonal antibodies towards the major 3-methylcholanthrene-inducible and phenobarbital-inducible rat liver P-450s (Mab 1-7-1 and 2-66-3, respectively) behaved similarly, except the inhibition was somewhat stronger if present. Antibody raised against rat liver NADPH-cytochrome P-450 oxido-reductase did not inhibit any activity studied. In immunoblotting experiments, the anti-reductase recognized the protein in human placental microsomes. However, neither anti-BNF-B2, anti-PB-B2 or Mab 1-7-1 or Mab 2-66-3 detected any proteins in human placental microsomes, regardless of smoking status. Northern blot hybridization analysis of placental RNA samples showed that only P-450IA1 mRNA existed in the placentas of smoking mothers with detectable ERDE activity. Despite the discrepancy between protein blotting and immunoinhibition data all other findings support the conclusion that maternal cigarette smoking induces the expression of the CYPIA1 gene (and not CYPIA2), resulting in an increased synthesis of P-450IA1 protein and increased AHH, ERDE and ECDE activities in human placenta.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1990

Cytochrome P450 isozyme induction by methyl ethyl ketone and m-xylene in rat liver.

The rat hepatic cytochrome P450 induction pattern caused by administration of a high peroral dose of methyl ethyl ketone (MEK, 1.4 ml/kg once daily for 3 consecutive days) and m-xylene (1.0 ml/kg X 3) was studied by catalytic activity and immunoblotting techniques. MEK caused a marked increase in the amount of P450 isozymes belonging to the phenobarbital- and ethanol-inducible P450 subfamilies P450IIB and P450IIE, respectively. Catalytic activities linked with these isozymes, pentoxyresorufin O-depentylase (P450IIB), aniline hydroxylase, and N-nitrosodimethylamine N-demethylase (P450IIE), were also increased (18.0-, 5.4-, and 2.4-fold, respectively). The activity of ethoxyresorufin O-deethylase, which is predominantly linked with the polycyclic aromatic hydrocarbon-inducible P450 isozymes, was also increased 2.3-fold without an apparent increase in the amount of the respective P450 protein (P450IA). m-Xylene caused a similar induction pattern with less effect on P450IIE. Simultaneous administration of MEK and m-xylene resulted in an additive or, in the case of pentoxyresorufin O-depentylase, a potentiating effect on P450-linked catalytic activities. These data indicate that MEK and m-xylene elicit a qualitatively similar induction of P450 isozymes, which may play a role in the metabolic interactions of these compounds.

Animals↗

PubMed1990

[Cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria and human placenta: immunochemical properties and structural characteristics].

An immunochemical comparison of components of cholesterol side chain cleavage system from bovine adrenocortical and human placental mitochondria has been carried out. Antibodies against cytochrome P-450scc, adrenodoxin reductase and adrenodoxin from bovine adrenocortical mitochondria were shown to cross-react with corresponding antigens of human placental mitochondria. A highly sensitive immunochemical method for cytochrome P-450scc determination has been developed. Limited proteolysis of cytochrome P-450scc of human placental mitochondria was studied, and the products of trypsinolysis were identified using antibodies against cytochrome P-450scc and fragments of its polypeptide chain: F1, F2 and F3. Immunochemical relatedness of ferredoxins from bovine adrenocortical and human placental mitochondria allowed one to develop a fast and efficient method for cytochrome P-450scc purification from human placental mitochondria by affinity chromatography on adrenodoxin-Sepharose.

Adrenal Cortex↗

PubMed1989

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex.

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.

Adrenal Glands↗

PubMed1989

Mouse liver P450Coh: genetic regulation of the pyrazole-inducible enzyme and comparison with other P450 isoenzymes.

Genetic experiments with two inbred strains of mice, AKR/J and DBA/2N, show a single major gene inheritance of additive mode for pyrazole-inducible coumarin 7-hydroxylase. Intragroup variation in the enzyme activity further suggests the contribution of minor modifying genes to the final enzyme activity. Western blot analysis with a polyclonal antibody raised against the purified isozyme P450Coh (highly active in the 7-hydroxylation of coumarin) showed that a difference in the amounts of P450Coh protein between the D2 and AKR mice is the reason for the differences in the enzyme activity between the two mouse strains. Accordingly, changes at the regulatory level rather than at the structural gene would explain the genetic difference in the activity of coumarin 7-hydroxylase. This hypothesis is further supported by the identical Km values of the basal and induced enzyme. The inducibility of coumarin 7-hydroxylase by phenobarbital (PB) and its genetic regulation have been previously studied by A. W. Wood and colleagues ((1974) Science 185, 612-614; (1979); J. Biol. Chem. 254, 5641-5646 and 5647-5651). Our present experiments show that the regulation is the same for the pyrazole-inducible enzyme. Furthermore the experiments with anti-P450Coh antibody show that the PB- and pyrazole-inducible proteins have the same molecular weight and are immunologically indistinguishable. This suggests that PB and pyrazole may induce the same enzyme. Immunoinhibition of microsomal coumarin 7-hydroxylase is practically 100% for control animals and after pretreatment with pyrazole or PB. This suggests that in each case the same or immunologically closely related proteins are metabolizing coumarin and that the P450Coh may be the only P450 isoenzyme in mouse liver microsomes catalyzing the 7-hydroxylation of coumarin. The N-terminal amino acid sequence of P450Coh was found to be identical with those from Type I and Type II genes of the mouse P45015 alpha family for the first 21 amino acids. With rat PB-inducible P450b the homology is only 33%. Also the immunological properties of P450Coh are different from those of P450b. This may suggest that P450Coh has a closer association to the steroid 15 alpha-hydroxylase gene family than to the P450IIB subfamily of phenobarbital-inducible isoenzymes.

Animals↗

PubMed1989

Induction of cytochrome P450IA1 in mouse hepatoma cells by several chemicals. Phenobarbital and TCDD induce the same form of cytochrome P450.

The mouse hepatoma cell line Hepa-1 was studied for aryl hydrocarbon hydroxylase (AHH) inducibility by sixteen compounds known to be inducers of cytochrome P450 of different "classes". Both 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and sodium phenobarbital induced AHH activity. A cytochrome P450IA1-specific (P1-450) mouse cDNA probe was used to quantitate mRNA induction. There was a good correlation between the amount of cytochrome P450IA1 mRNA induced and AHH activity. Immunoblots with monoclonal antibody 1-7-1, which recognizes rat liver P450IA1 and P450IA2 (P450c and P450d, respectively), showed that both phenobarbital and TCDD increase the amount of a P450 isozyme immunorelated to P450IA1 in this cell line. Hepa-1 mutants with no AHH inducibility (no functional P450IA1 structural gene; no Ah receptor; no nuclear translocation of the inducer-receptor complex; and presence of dominant repressor) did not respond to phenobarbital. The cytosolic receptor for TCDD (Ah receptor) was characterized to see if phenobarbital induced cytochrome P450IA1 mRNA and the hydroxylase enzyme through the same mechanism as TCDD. 20 mM Phenobarbital almost completely abolished the binding of 3H-TCDD to the cytosolic receptor. These data indicate that phenobarbital can be a weak ligand for the Ah receptor and thus induce cytochrome P450IA1 and AHH activity. The observation increases the list of different P450 forms inducible by phenobarbital.

Animals↗

PubMed1989

2-(4'-chlorophenyl)benzothiazole is a potent inducer of cytochrome P450IA1 in a human and a mouse cell line. Anomalous correlation between protein and mRNA induction.

Two benzothiazole derivatives, 2-(4'-chlorophenyl)benzothiazole (CPBT) and 2-(4'-formylphenyl)benzothiazole (FPBT) were studied for their ability to induce aryl hydrocarbon hydroxylase activity in a mouse and a human cell line. In both the mouse hepatoma cell line, Hepa-1, and the human choriocarcinoma cell line, JEG-3, a high aryl hydrocarbon hydroxylase activity was observed after treatment with CPBT. In contrast, FPBT had a very weak inducing capacity in both cell lines. The maximal induction by CPBT was several times (e.g. in Hepa-1 about fourfold on average) greater than that observed with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific cDNA probe for mouse cytochrome P4501A1 gene was used to quantify mRNA levels in Hepa-1 cells. CPBT increased cytochrome P450IA1 mRNA to a level of 88% of that induced by TCDD. Immunoblot analysis with monoclonal antibody 1-7-1, directed against rat liver cytochrome P450IA1 and P450IA2, showed that the amount of P450IA1 is substantially increased in Hepa-1 cells after treatment with CPBT. The observation that CPBT competed TCDD off its specific cytosolic binding site suggests a receptor-mediated induction of cytochrome P450IA1 mRNA. An in vitro activation effect did not explain the exceptionally high hydroxylase activity. The results show that CPBT is a more efficient inducer of aryl hydrocarbon hydroxylase than TCDD in Hepa-1 and JEG-3 cells and that the induction is supported by P450IA1. The discordant effect of CPBT on mRNA and aryl hydrocarbon hydroxylase activity suggests that post-translational modifications of P450IA1 account for a major part of the increased enzyme activity.

Animals↗

PubMed1989

Comparison of the formation of benzo[a]pyrene diolepoxide-DNA adducts in vitro by rat and human microsomes: evidence for the involvement of P-450IA1 and P-450IA2.

The involvement of cytochrome P-450 isozymes in the activation of benzo[a]pyrene (BP) by human placental and liver microsomes was studied in vitro using monoclonal antibodies (Mab) toward the major 3-methylcholanthrene (MC)-inducible and phenobarbital-inductible rat liver P-450 isozymes (Mab 1-7-1 and Mab 2-66-3, respectively). Microsomes from human placenta and liver and rat liver were incubated with BP and DNA, and BP-diolepoxide-DNA (BPDE-DNA) adducts were measured by synchronous fluorescence spectrophotometry (SFS). The only BP metabolite giving the same fluorescence peak as chemically modified BPDE-DNA was BP-7,8-dihydrodiol. Five (smokers) out of 29 human placentas (smokers and nonsmokers), and five out of nine human livers were able to metabolically activate BP to BPDE-DNA adducts in this system. The Mab 1-7-1 totally inhibited the formation of BPDE-DNA adducts in placental microsomal incubations. Inhibition using rat or human liver microsomes was 50-60% and about 90%, respectively. The Mab 2-66-3 had no effect in any of the microsome types. Adduct formation was inhibited more strongly and at lower concentrations of Mab 1-7-1 compared with the inhibition of AHH activity. This study is a clear indication of the major role of P-450IA1 (P-450c) in human placenta and probably P-450IA2 (P-450d) in human liver in BP activation, while other isozymes also take part in the activation in rat liver. Furthermore, this clearly indicates that AHH activity and BP activation are not necessarily associated.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1989

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate.

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1988

Mouse hepatic cytochrome P-450 isozyme induction by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, pyrazole, and phenobarbital.

The effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pyrazole on mouse hepatic cytochrome P-450 isozyme expression were compared to the P-450 induction pattern elicited by phenobarbital. TCPOBOP and PB administration caused a similar induction profile by increasing microsomal protein and cytochrome P-450 content and the catalytic activities of several monooxygenases in DBA/2N and AKR/J mice. There were, however, several quantitative and some qualitative differences in the induction profile caused by phenobarbital and TCPOBOP. A few strain-related differences were also observed. Immunoblot analysis with polyclonal anti-coumarin hydroxylase (P-450Coh) antibody and epitope-specific monoclonal antibodies 1-7-1 and 2-66-3 showed that both phenobarbital and TCPOBOP increase the amount of P450IIB and P-450Coh. TCPOBOP caused a more pronounced increase in the amount of P-450IIB than phenobarbital, and TCPOBOP also caused an increase in the amount of P-450IA2. These data suggest that in the mouse, TCPOBOP increases mainly the expression of P-450 isozymes responsive to phenobarbital. The effects of pyrazole differed greatly from those caused by TCPOBOP and phenobarbital. In the DBA/2N mice, pyrazole increased coumarin 7-hydroxylation 9.4-fold, whereas in the AKR/J mice the activity was induced only to a level equivalent to the DBA/2N basal level. In immunoblot experiments with anti-P-450Coh antibody, the amount of P-450Coh was considerably higher in DBA/2N mice treated with phenobarbital, TCPOBOP, or pyrazole in comparison with the AKR/J mice, indicating a strain specificity in the inducibility of coumarin 7-hydroxylase by pyrazole.

Animals↗

PubMed1988

Immunochemical and catalytical studies on hepatic coumarin 7-hydroxylase in man, rat, and mouse.

The cytochrome P-450-mediated coumarin 7-hydroxylase (COH) was studied in microsomal preparations from Wistar rat, DBA/2N mouse, and human liver. Human liver contained the highest constitutive COH activity of up to about 500 pmol/mg microsomal protein/min. The rat liver contained low levels of COH (about 3-5 pmol/mg protein/min) which could be demonstrated only with high substrate concentrations. Rabbit polyclonal antibody generated against P-450Coh (a P-450 isozyme purified from pyrazole-treated DBA/2N mouse liver showing high activity for coumarin 7-hydroxylation) inhibited COH activity by almost 100% in human liver microsomes and 86-99% in mouse liver microsomes. Also the deethylation of 7-ethoxycoumarin was inhibited somewhat by the antibody, whereas no inhibition was obtained in ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. None of these enzyme activities was affected by the antibody in the rat liver microsomes. In Ouchterlony immunodiffusion analysis precipitin lines were obtained with human, mouse and rat liver microsomes. Complex coalescence patterns were obtained suggesting full identity between human and pyrazole-treated mouse antigens, partial identity between mouse and rat antigens, and no identity between human and rat antigens. Western blot analysis with the anti-P-450Coh antibody revealed a distinct 48-kDa protein in all four human samples tested. A 50-kDa protein comigrating exactly with P-450Coh was observed in microsomes from PB and pyrazole-treated mouse liver microsomes. No distinct protein bands appeared in rat liver samples. These data suggest that despite slightly differing molecular masses, the human and mouse P-450s supporting COH are structurally conserved at their active centers. The corresponding rat P-450 appears to differ from that of mouse and man.

Animals↗

PubMed1988

Hepatic mitochondrial coumarin 7-hydroxylase: comparison with the microsomal enzyme.

The specific activity of cytochrome P450-linked coumarin 7-hydroxylase (COH) of hepatic mitoplasts from DBA/2N mice is up to 55% as great as the microsomal activity. According to Western blot and immunodiffusion analysis and inhibition studies with anti-P450Coh and metyrapone, the mitoplastic P450Coh had the same molecular weight and immunochemical and catalytic properties as the corresponding microsomal enzyme. The inducibility of the two proteins by pyrazole and their genetic regulation, as studied with DBA/2N and AKR/J mice, appears to be similar. However, the mitochondrial electron transfer system was not able to support the COH activity of reconstituted microsomal P450Coh although the enzyme was fully active with the microsomal NADPH-cytochrome P450 reductase. This indicates some differences between the two proteins with respect to their interaction with the electron transfer system. This was confirmed by the ability of anti-adrenodoxin reductase antibody to effectively inhibit the mitoplastic COH but not the COH reconstituted with purified microsomal P450Coh and NADPH-P450 reductase. We have previously found that P450Coh does not react with anti-P450b or anti-P450c antibodies, which recognize respective isoforms in rat liver mitoplasts. While P450Coh from microsomes and mitoplasts possess a number of properties in common, the mitoplast P450Coh represents a new subspecies of mitochondrial P450. Some characteristics of mitoplast P450Coh may be the result of post-translational modifications necessary for processing and translocation into the mitochondria.

Animals↗

PubMed1988

Pyrazole is different from acetone and ethanol as an inducer of the polysubstrate monooxygenase system in mice: evidence that pyrazole-inducible P450Coh is distinct from acetone-inducible P450ac.

The induction of liver microsomal monooxygenase activities elicited by pyrazole, ethanol, and acetone, all shown to be inducers of rat P450j and rabbit P450LM3a, has been compared in inbred strains of DBA/2N, AKR/J, and Balb/c mouse. Pyrazole strongly increases coumarin 7-hydroxylase (COH) activity in DBA/2N but much less in other strains. The effect of pyrazole on aniline p-hydroxylase and ethanol oxidase activities is also strain dependent: an increase was seen only in the DBA/2N strain. Ethanol and acetone were unable to induce COH, whereas aniline p-hydroxylase and ethanol oxidase were elevated about 1.4- to 3.3-fold in all strains. No strain difference could be detected in aniline p-hydroxylase or ethanol oxidase inducibility. There was a strong correlation between aniline p-hydroxylase and ethanol oxidase activities in every strain, whereas no positive correlation could be found between COH and aniline p-hydroxylase activities. Immunoinhibition experiments showed that a polyclonal antibody against purified pyrazole-inducible COH (P450Coh) blocked about 90% of COH activity, but only about 10% of aniline p-hydroxylase or ethanol oxidase in mouse liver microsomes. Monoclonal antibody 1-91-3 (raised against rat acetone-inducible P450ac) did not inhibit COH, whereas aniline p-hydroxylase was blocked 46-76% and ethanol oxidase 25-70%, depending on the source of microsomes. In immunoblots, anti-P450Coh recognized only its own antigen but not the P450ac, whereas monoclonal antibody 1-98-1 against P450ac detected P450ac and a corresponding form in the D2 mouse liver, but not the P450Coh. The purified P450ac and P450Coh had molecular masses of 52 and 50 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These antigens were expressed differentially in response to pyrazole, ethanol, and acetone: P450Coh was increased only after pyrazole treatment, but 1-98-1-detectable protein was elevated in D2 mouse liver microsomes by ethanol and acetone, but not by pyrazole. We conclude that mouse P450Coh and rat P450ac are not corresponding forms of the same isozyme, and that a P450ac-like protein, responsible for most of aniline p-hydroxylation and ethanol oxidation, is present in the D2 mouse liver. These two P450 isozymes are also dissimilarly expressed in the mouse liver in response to inducer administration.

Acetone↗

PubMed1988

Hepatic Ah-receptor levels and the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic microsomal monooxygenase activities in a TCDD-susceptible and -resistant rat strain.

Previous studies have shown that in two inbred strains of mice, straightforward correlations exist among the number of hepatic Ah-receptors, enzyme inducibility by TCDD, and lethality of TCDD. Here, studies were conducted in two strains of rats (Han/Wistar and Long-Evans) which differ widely in susceptibility to the lethal effects of TCDD, to determine if these are general phenomenona in TCDD toxicity. The total number of specific binding sites (Ah-receptors) for [3H]TCDD proved to be approximately equal in the livers of both rat strains. Likewise, no notable difference was detected in the effect of TCDD on the activities of 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin O-deethylase, and ethylmorphine N-demethylase or on the amount of cytochrome P-450 in hepatic microsomal fractions. Immunoblot analysis was carried out with monoclonal antibodies (Mabs). Mab 1-7-1 directed against rat liver 3-methylcholanthrene (MC)-inducible P-450 recognized forms P-450c and P-450d in TCDD-treated rats in a dose-dependent fashion and to a similar extent in both strains. In contrast, Mab 2-66-3 (against phenobarbital-inducible P-450) did not recognize any proteins in either strain, confirming the conclusion that TCDD elicits a MC-type induction of hepatic cytochrome P-450 in both strains of rats. Thus, it seems that the correlations observed in mice do not hold in rats and therefore should not be generalized. The parameters measured in the present study are causally unrelated to the mechanism of lethal action of TCDD in these rat strains.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1987

Heterogeneity of intercellular adhesion in rat liver cells in culture.

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.

Aneuploidy↗

PubMed1987

Altered cardiac polyamine biosynthesis in spontaneously hypertensive rats.

To evaluate the role of polyamine biosynthesis in myocardial growth, the activity of ornithine decarboxylase (ODC) and the levels of the polyamines, spermidine, spermine, and putrescine, were measured in the hearts of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats between birth and 30 wk of age and in 30-wk-old rats after 6 wk treatment with either minoxidil (0.08 mg/ml) or methyldopa (5 mg/ml drinking water). ODC activity was initially high in the developing heart (150 pmol X mg protein-1 X 30 min-1) and decreased with age. In the SHR after 4 wk of age, spermidine content was consistently raised in both ventricles compared with the WKY rats. Spermine was reduced in the left ventricle of the SHR, resulting in high spermidine-to-spermine ratios characteristic of rapidly growing systems. Ventricular ODC activity and putrescine levels were also slightly but less consistently elevated in the ventricles of the SHR compared with respective WKY rats. Minoxidil treatment increased heart weight and left ventricular spermidine and spermine content in both SHR and WKY rats. Methyldopa also caused a significant increase in left ventricular spermidine content despite a marked reduction in ventricular mass. Thus ventricular hypertrophy in SHR is accompanied by an enhanced synthesis and accumulation of spermidine. The fact that myocardial spermidine content increased during methyldopa treatment, which reduced the myocardial mass, suggests that regression of ventricular hypertrophy can occur independently of changes in polyamine content.

Animals↗