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Biomedical subjects

R M Philpot

Publications and source records attributed to R M Philpot.

At least 109 records · Page 6Linked to original sources
PubMed1983

Interactions between xenobiotics that increase or decrease the levels of cytochrome P-450 isozymes in rabbit lung and liver.

Isozyme-specific enzymatic activities, radial immunodiffusion, and Western blotting were used to study the effects of phenobarbital, Aroclor 1260, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on microsomal levels of cytochrome P-450 forms 2, 5, and 6. The pulmonary concentration of form 2 was decreased to trace levels by the administration of Aroclor 1260 but not TCDD; co-administration of phenobarbital did not mitigate the decrease. The pulmonary concentration of form 5 was not significantly changed following treatment with Aroclor 1260, phenobarbital, or TCDD. The pulmonary content of form 6 was increased 5- and 10-fold by administration of Aroclor 1260 and TCDD, respectively, and decreased 2-fold by treatment with phenobarbital. Further, phenobarbital antagonized the induction of form 6 by Aroclor 1260 but not by TCDD. In the liver, increases and decreases in form 2-mediated benzphetamine N-demethylation were observed following treatment with Aroclor 1260 depending on the time after dose and individual rabbit response. Hepatic induction of form 2 by phenobarbital was significantly decreased by co-administration of Aroclor 1260. However, form 5 was induced in the liver by either Aroclor 1260 or phenobarbital or both. Thus, the effect of Aroclor 1260 on the control of the concentration of form 2 is independent of that of form 5 in the lung and liver. Fractionation of the Aroclor 1260 to remove co-planar polychlorinated biphenyls and dibenzofurans was useful in demonstrating that the pulmonary induction of form 6 was dependent on these components and unrelated to the repression of form 2 in the lung and liver. The apparent repressive effect of phenobarbital on form 6 in the lung is interesting as it suggests an interaction between phenobarbital and polycyclic hydrocarbon-mediated induction.

Animals↗
PubMed1983

Hepatic microsomal NADPH-cytochrome P-450 reductase from little skate, Raja erinacea. Comparison of thermolability and other molecular properties with a mammalian enzyme.

Components of little skate (an elasmobranch) and rabbit hepatic microsomal cytochrome P-450 dependent monooxygenase systems were examined for differences which might explain the decreasing xenobiotic-metabolizing activity of little skate microsomes assayed at temperatures above 30 degrees C. The proportion of saturated fatty acids in microsomal lipids and the habitat temperature are both lower in skate as compared to rabbit, which is consistent with the known adaptive pattern. The more thermolabile enzyme of the skate system in microsomal preparations is NADPH-cytochrome P-450 reductase. The optimal assay temperature for purified skate reductase (30 degrees C) is 10 degrees C lower than that for the purified rabbit reductase. The purified skate reductase differs from rabbit reductase in monomeric molecular weight, in peptides produced by partial proteolysis, in immunochemical properties, but not in flavin content.

Animals↗
PubMed1983

Cytodifferentiation of the nonciliated bronchiolar epithelial (Clara) cell during rabbit lung maturation: an ultrastructural and morphometric study.

The nonciliated bronchiolar epithelial (Clara) cell of adult lung is commonly defined by two cellular components: abundant agranular endoplasmic reticulum (AER) and electron-dense ovoid secretory granules. These reflect the Clara cell's proposed functions as the source of bronchiolar surface secretions and the site of xenobiotic metabolism via the cytochrome P-450 monooxygenase system. Since previous studies have indicated that Clara cells may not attain a fully functional state until some weeks after birth, the present study was undertaken to characterize systematically the differentiation of this cell type during lung maturation. Lungs were fixed by airway infusion with glutaraldehyde/paraformaldehyde (550 mOsm, pH 7.4) from at least three male rabbits at each of the following ages: 24, 27, and 30 days fetal, and 0-1 day, 3-4 days, 1, 2, 3, 4, 5, 8, 12, 15, 17, and 25 weeks postnatal; and pieces were processed for transmission electron microscopy by a selective embedding procedure. Quantitation was performed on electron micrographs (at 15,750 X) of cell profiles, which included the base, apex, and nucleus. Volume fractions of constituents of a minimum of 30 cells per animal (8 weeks and younger) and 10 per animal in older groups, were estimated by point counting with a Weibel 168-point test grid. Cell and nuclear size were estimated with a computerized digitizer (Zeiss Videoplan). Nonciliated cells of prenatal animals had large amounts of cytoplasmic glycogen (over 60% of the cell cytoplasm), few mitochondria (less than 15%), little granular endoplasmic reticulum (GER) (20%), minimal AER (less than 5%), and no granules. Postnatal animals 2 weeks of age and younger were similar, except for the presence of secretory granules and slightly more abundant AER (5 to 20%). By 4 weeks postnatal age, nonciliated cells resembled that of older animals with abundant apical AER (over 40%), secretory granules, little glycogen (11%), and GER (10%). We concluded that (1) the Clara cell is immature at birth; (2) differentiation occurs primarily during weeks 3 and 4 of postnatal life; (3) vast amounts of cytoplasmic glycogen are characteristic of the undifferentiated cell; and (4) four cellular constituents, AER, glycogen, mitochondria, and GER, undergo significant shifts in abundance during differentiation. These shifts appear to be in the sequence expected of a cell type undergoing the initiation of biosynthesis of secretory products and biogenesis of agranular endoplasmic reticulum.

Animals↗
PubMed1983

Characterization of NADPH-cytochrome P-450 reductase in a mouse hepatoma cell line.

NADPH-cytochrome c reductase in Hepa-1 cells was induced 2-fold by phenobarbital, but was not induced by benz[a]anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The apparent Km of the enzyme for NADPH was 0.57 microM; the activity was inhibitable by NADP; and segregated primarily to the microsomal fraction. Cytoplasm of Hepa-1 cells bound antibody to rabbit cytochrome P-450 reductase. 3T3 cells, which possessed one sixth of the cytochrome c reductase activity of Hepa-1 cells, bound correspondingly less cytochrome P-450 reductase antibody. This supports the notion that cytochrome P-450 reductase was responsible for the cytochrome c reductase activity that was measured.

Animals↗
PubMed1983

Covalent binding of metabolites of 4-ipomeanol to rabbit pulmonary and hepatic microsomal proteins and to the enzymes of the pulmonary cytochrome P-450-dependent monooxygenase system.

The covalent binding of metabolites of 4-ipomeanol, a potent lung toxin, to proteins in rabbit pulmonary and hepatic microsomal preparations and in purified monooxygenase systems was investigated. The rate of binding was 12-fold greater in pulmonary preparations than in hepatic preparations. Covalent binding in pulmonary microsomal fractions was inhibited 39 to 49% by antibodies to rabbit pulmonary cytochrome P-450II or P-450I and 90% by antibodies to cytochrome P-450 reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scintillation autoradiography of pulmonary microsomal proteins revealed the presence of heavily labeled bands throughout the molecular weight range (Mr) examined. Two of these bands corresponded in mobility to pulmonary cytochrome P-450I (Mr 52,000) and P-450II (Mr 58,000). In addition, there was a great deal of binding associated with very high molecular weight proteins, probably in the form of cross-linked aggregates which were unable to penetrate the gel matrix. In the absence of cofactor, no binding was observed. Binding was decreased by the addition of the following: antireductase greater than glutathione = NADH (without NADPH) greater than anti-II greater than anti-I. The electrophoretic patterns of the proteins from incubation of [3H]-4-ipomeanol with purified pulmonary P-450-dependent monooxygenase enzymes were also examined. In the complete system, the majority of the binding was associated with high molecular weight species located at the origin and with low molecular weight species that migrated with the tracking dye. In the absence of cofactor, some binding to proteins that corresponded with cytochrome P-450 and P-450 reductase was observed. Protease digestion of incubation mixtures resulted in the migration of all bound material at the dye front.

Animals↗
PubMed1983

The relationship between increases in the hepatic content of cytochrome P-450, form 5, and in the metabolism of aromatic amines to mutagenic products following treatment of rabbits with phenobarbital.

Treatment of rabbits with phenobarbital is followed by increases in the hepatic microsomal concentration of cytochrome P-450, form 5, and in the hepatic microsomal metabolism of aromatic amines to mutagenic products. Inhibition by antibodies to form 5 of the activation of 2-aminoanthracene and 2-aminofluorene demonstrates that these increases are directly related. The extent of the apparent induction of form 5 by phenobarbital is determined from single radial immunodiffusion, immunostaining of form 5 on nitrocellulose sheets containing microsomal proteins transferred from polyacrylamide gels, and the amount of antibody required for 50% of maximal inhibition of activity. The results indicate that phenobarbital increases the hepatic microsomal concentration of cytochrome P-450, form 5, to the same extent that it increases form 5-mediated metabolism of aromatic amines to mutagenic products: 10- to 12-fold. In contrast to the effects of phenobarbital, treatment of rabbits with beta-naphthoflavone decreases the hepatic microsomal concentration of cytochrome P-450, form 5, to less than detectable levels and has little effect on the metabolism of aromatic amines to mutagenic products. Our findings, along with the known effects of phenobarbital on cytochrome P-450, form 2, and the known catalytic activity of cytochrome P-450, form 4, lead to the following conclusions: (a) treatment of rabbits with phenobarbital results in increases in the hepatic microsomal concentrations of at least two structurally, immunochemically, and catalytically distinct isozymes of cytochrome P-450, forms 2 and 5; (b) the metabolism of aromatic amines to mutagenic products in rabbit hepatic microsomal preparations depends on the relative concentrations of at least two isozymes of cytochrome P-450, forms 4 and 5, that change in response to different inducers.

Amines↗
PubMed1982

The relationship between the catalytic activities of rabbit pulmonary cytochrome P-450 isozymes and the lung-specific toxicity of the furan derivative, 4-ipomeanol.

Metabolism of the pulmonary toxin, 4-ipomeanol, in microsomal preparations from rabbit liver and lung and in purified cytochrome P-450-dependent monooxygenase systems was investigated. The rate of formation of reactive electrophilic products from 4-ipomeanol was estimated by measuring covalent binding to protein or glutathione. Pulmonary microsomal preparations were much more active than hepatic preparations in mediating these reactions. Both of the rabbit pulmonary cytochrome P-450 isozymes, P-450I and P-450II, were active in the metabolism of 4-ipomeanol. In the assay for covalent binding to protein, P-450I was slightly more active than P-450II at high substrate concentrations and significantly more active at low substrate concentrations. Incubation of either isozyme with 4-ipomeanol produced two glutathione conjugates but at quite different ratios. Rates of metabolism determined by conjugate formation in the purified systems were about 3 times the rates determined by covalent binding to protein, whereas both determinations gave the same values in the microsomal preparations. The relationship between the activities of P-450I and P-450II in the metabolism of 4-ipomeanol and the pulmonary toxicity of 4-ipomeanol is discussed.

Animals↗
PubMed1981

The rabbit pulmonary monooxygenase system. partial structural characterization of the cytochrome P-450 components and comparison to the hepatic cytochrome P-450.

Two forms of rabbit pulmonary cytochrome P-450, P-450I and P-450II, are distinguished by unique peptides observed upon electrophoresis of the products of limited proteolysis (with papain or chymotrypsin) in the absence or presence of sodium dodecyl sulfate. In contrast, the peptides from the proteolytic digestions of pulmonary cytochrome P-450I are indistinguishable from those of the major form of hepatic cytochrome P-450 induced by phenobarbital (P-450PB). Electrophoresis of the fragments of P-450, and P-450II produced by treatment with cyanogen bromide also shows different peptides, whereas the peptides produced from P-450I and P-450PB are the same. The amino acid composition and P-450II is different from that of P-450I or P-450PB. P-450II is distinguished further from the other two cytochromes on the basis of amino acid composition and subunit molecular weight (either as calculated from the amino acid composition or as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis). The sequence of the first five residues at the NH2-terminal segment of P-450I and P-450PB are identical, while that of P-450II is different.

Amino Acids↗
PubMed1981

Metabolism of benzo(a)pyrene and benzo(a)pyrene 4,5-oxide in rabbit lung.

For several years our laboratory has been investigating the biotransformation of various environmental pollutants by lung. Studies have been performed with pulmonary subcellular fractions, purified monooxygenase and glutathione transferase enzymes, and preparations having intact cellular structure including the isolated perfused lung and cell fractions enriched in alveolar macrophages, Clara cells and alveolar type II cells. Collectively, these investigations have identified several metabolic factors which may contribute to the pulmonary toxicity mediated by certain polycyclic aromatic hydrocarbons (PAH). First, although lung has low overall cytochrome P-450-dependent monooxygenase activity for many substrates, relative to liver, this activity is localized in only a few cell types and specific activity in certain cell types, such as the non-ciliated bronchiolar epithelial (Clara) cell, can be high. Second, oxidative metabolites of benzo(a)pyrene tend to accumulate in pulmonary tissue due, at least in part, to the low ability of lung (relative to liver) to conjugate and detoxify phenolic, dihydrodiol and epoxide metabolites. Thus, products such as benzo(a)pyrene 7,8-dihydrodiol are available for further cytochrome P-450-dependent oxidation to ultimate carcinogens and cytotoxins. Moreover, the lung is efficient in removing benzo(a)pyrene 4,5-oxide and presumably other oxidized PAH metabolites, from the bloodstream. Consequently, the uptake of relatively stable electrophilic metabolites released by the liver may also contribute to pulmonary toxicity.

Animals↗
PubMed1981

Identification of cytochrome P-450 isozymes in nonciliated bronchiolar epithelial (Clara) and alveolar type II cells isolated from rabbit lung.

Two forms of cytochrome P-450 (P-450I and P-450II) have been shown by several techniques to be present in both nonciliated bronchiolar cells (Clara) and alveolar type II cells isolated from rabbit lung. In contrast, the alveolar macrophage contains little or none of these cytochromes. Cross-reactivity between antibodies to cytochrome P-450I or P-450II and detergent-digested microsomes prepared from 80% type II or 70% Clara cell fractions was shown by Ouchterlony double immunodiffusion. The presence of both cytochromes was also demonstrated by histochemical immunofluorescence in smears of type II cells stained by a modified Papanicolaou procedure and Clara cells stained with nitroblue tetrazolium. However, this same fluorescent antibody technique used for localization of rabbit pulmonary cytochromes P-450I and P-450II in tissue sections showed most of the immunofluorescence in the Clara cells of the bronchiolar epithelium. SDS-polyacrylamide gel electrophoresis of microsomes from either the type II or Clara cell fractions produced bands which corresponded to cytochrome P-450I (52,000 daltons) and cytochrome P-450II (58,000 daltons).

Animals↗
PubMed1980

Purification and structural comparison of pulmonary and hepatic cytochrome P-450 from rabbits.

A procedure is described for the purification of a major form of cytochrome P-450 from the livrs of rabbits treated with phenobarbital and a major form of the cytochrome from the lungs of untreated rabbits. Preparations in good yield (13--17%) and of high purity (up to 21 nmol of cytochrome per mg of protein) that were free of lipid and contained minimal amounts of non-ionic detergent were obtained from either tissue. The two cytochromes cannot be distinguished from each other on the basis of absorption spectra, extinction coefficients, apparent molecular weights (52 000), amino acid compositions, or peptide fragments produced by treatment of the proteins with cyanogen bromide. These data are consistent with a major indigenous form of rabbit pulmonary cytochrome P-450 being the same as the major form of hepatic cytochrome induced by phenobarbital.

Amino Acids↗
PubMed1980

Cytochrome p-450: localization in rabbit lung.

Cytochrome P-450-dependent monooxygenase systems, which metabolize endogenous as well as foriegn compounds, are found in hepatic and several extrahepatic tissues of mammals, including humans. A form of cytochrome P-450 is localized in the nonciliated bronchiolar epithelial cells (Clara cells) of the small airways of rabbit lung. The apparent high concentration of the cytochrome in this pulmonary cell type compared to liver may be an important determinant in the susceptibility of the lung to a number of toxic chemicals that undergo metabolic activation.

Animals↗
PubMed1980

Cytochrome P-450 mediated genetic activity and cytotoxicity of seven halogenated aliphatic hydrocarbons in Saccharomyces cerevisiae.

Cells of Saccharomyces cerevisiae, harvested from log-phase cultures, contain cytochrome P-450 and are capable of metabolizing promutagens to genetically active products. The activities of 7 halogenated aliphatic hydrocarbons in the yeast system have been investigated. All of the compounds tested (methylene chloride, halothane, chloroform, carbon tetrachloride, trichloroethylene, tetrachloroethylene and s-tetrachloroethane) induced mitotic gene convertants and recombinants and, to a lesser extent, gene revertants when incubated with log-phase cells of the yeast strain D7. An examination of the difference spectra observed upon the addition of carbon tetrachloride, halothane and trichloroethylene to whole-cell or microsomal suspensions of yeast suggested that cytochrome P-450 mediated the metabolism of the hydrocarbons tested to cytotoxic and genetically active compounds.

Biotransformation↗