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R M Philpot

Publications and source records attributed to R M Philpot.

At least 91 records · Page 5Linked to original sources
PubMed1988

The distribution of cytochrome P-450 monooxygenase in cells of the rabbit lung: an ultrastructural immunocytochemical characterization.

The cytochrome P-450 monooxygenase system of the mammalian lung is known to be associated with the microsomal subcellular fraction and has been demonstrated in two pulmonary cell types rich in endoplasmic reticulum: Clara cells and type II pneumocytes. However, analysis of ultracellular fractions, isolated cell preparations, or light microscopic immunohistochemical studies of tissue sections has permitted only limited resolution of the distribution of this enzyme system within the 40 or more cell types of the lung. Therefore, we have used the greater resolving power of transmission electron microscopy and immunogold labeling to characterize the cellular and subcellular distribution of the cytochrome P-450 system in the lung. In Lowicryl-embedded sections of lung from adult rabbits, antisera (1:10,000) against the constitutive pulmonary microsomal cytochrome P-450 monooxygenase isozymes 2 and 5 and NADPH-cytochrome P-450 reductase (anti-2, anti-5 and anti-R) bound specifically to regions known to be rich in agranular endoplasmic reticulum (AER) in the cytoplasm of Clara cells. The plasma membranes of bronchiolar Clara cells, the tips of microvillae of ciliated cells, secretory granules of goblet cells, and the cell membrane and pinocytotic vesicles of endothelial cells were all intensely labeled with anti-2 and anti-5 but not with anti-R, even at a 10-fold higher concentration. The intensity of labeling of AER in Clara cells with anti-R and anti-2, but not anti-5, appeared to correlate positively with the cellular content of secretory granules. The Golgi membranes of ciliated cells were labeled intensely with anti-5 only. The plasma membrane of type II pneumocytes was not labeled by any of the antisera, but with anti-2 or anti-5 there was labeling of AER-associated vacuoles, the membranous residue of lamellar bodies, and, to some extent, mitochondria; at 1:5,000 but not 1:10,000 dilution, staining with anti-R was qualitatively similar. Type I pneumocytes, ciliated cell cytoplasm, and nuclei were essentially unlabeled. Immunoblots (Western) of tracheal homogenates yielded no evidence for epitopes other than those in microsomal fractions from whole lung. Contact blots of fresh whole trachea, before but not after lavage, bound anti-2 and anti-R. Thus, we have demonstrated for the first time that components of the pulmonary cytochrome P-450 monooxygenase, although localized in the AER-rich regions of the Clara cells and type II pneumocytes, are not restricted to these cell types or to the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Placental markers of human exposure to polychlorinated biphenyls and polychlorinated dibenzofurans.

Our studies have evaluated biochemical changes in placentae from humans exposed to rice oil contaminated with polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) in Taiwan. Placentae were obtained from nonsmoking women 4 to 5 years after the exposure had occurred. The exposed individuals ingested approximately 1 to 3 g PCBs and 5 mg PCDFs, and many exhibited symptoms characteristic of PCB poisoning. This disease was termed "Yu-Cheng" in Chinese. Based on data from experimental animal models, we examined a number of parameters in placentae from control and exposed women, including arylhydrocarbon hydroxylase (AHH) activity, cytochrome P-450 isozymes, epidermal growth factor (EGF) receptor binding properties and actions, and Ah receptor. We also quantified concentrations of various PCB and PCDF congeners known to be present in the contaminated rice oil. Our results revealed a dramatic elevation in placental AHH activity in samples from PCB/PCDF-exposed women. This increase in enzyme activity was associated with a parallel increase in placental microsomal protein immunochemically related to cytochrome P-450 form 6 [derived from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced rabbit lung]. No other cytochrome P-450 isozyme was detected in placental preparations, and the form 6 homolog was found only in placentae from exposed women. EGF receptor-mediated autophosphorylation capacity was significantly diminished in PCB/PCDF placentae, but this effect was not associated with changes in plasma membrane EGF receptor binding properties (Kd and Bmax). The EGF receptor autophosphorylation effect correlated well with the decrease in birthweight observed in offspring of exposed women, suggesting that this biochemical event might provide a good marker of effect for the toxic halogenated aromatics.

Adolescent↗
PubMed1987

Immunohistochemical demonstration of cytochrome P-450 monooxygenase in Clara cells throughout the tracheobronchial airways of the rabbit.

The nonciliated bronchiolar epithelial (or Clara) cell is considered the primary pulmonary site of cytochrome P-450-dependent monooxygenase activity. Despite the general conception that Clara cells are restricted to bronchioles, we have previously shown that a nonciliated cell with the cytological features of the Clara cell predominates throughout rabbit tracheobronchial airways. The present study was designed to determine if the cytochrome P-450 monooxygenase system has the same distribution. Trachea, terminal bronchioles and 3 generations of bronchi were selected by microdissection from fixed lungs of adult specific-pathogen-free rabbits. Serial sections of paraffin-embedded tissue were stained with either Alcian blue/periodic acid Schiff's (AB/PAS) or with antisera to one of the following: cytochrome P-450 form 2, form 5, or NADPH-dependent cytochrome P-450 reductase. The majority of nonciliated epithelial cells lining all 5 airway generations were PAS+ and AB-. Nonciliated cells in all 5 airway generations reacted positively with all three antisera. The primary deposition site was the apical portion of nonciliated cells. Other sites included ciliated surfaces and vascular endothelium. Reaction products from all three antisera had the same localization pattern. We conclude that (1) the cytochrome P-450 monooxygenase system is distributed throughout the tracheobronchial airways of the rabbit and (2) the Clara cells of the trachea and bronchi are functionally, as well as structurally, similar to those of the bronchioles.

Animals↗
PubMed1987

Species-dependent expression and induction of homologues of rabbit cytochrome P-450 isozyme 5 in liver and lung.

The presence of homologues of rabbit cytochrome P-450 isozyme 5 in pulmonary and hepatic microsomal preparations from guinea pig, mouse, monkey, hamster, and rat was examined by immunoblotting and inhibition of metabolism of 2-aminofluorene with antibodies to isozyme 5. Homologues to isozyme 5 were detected in pulmonary preparations from all five species. However, only hepatic preparations from hamster, in addition to those from rabbit, contained detectable levels of this isozyme. With the exception of induction by phenobarbital in rabbit liver, treatment of animals with phenobarbital or tetrachlorodibenzo-p-dioxin did not increase hepatic or pulmonary content of isozyme 5 homologues or the amount of 2-aminofluorene metabolism inhibited by antibodies to isozyme 5. Metabolism of 2-aminofluorene was measured both colorimetrically (formation of a reduced iron chelate from the N-hydroxyfluorene metabolite) and radiochemically (separation of 3H-metabolites by high performance liquid chromatography and quantitation by scintillation counting). A turnover number of 48 nmol of product X min-1 X nmol of enzyme-1 for isozyme 5-catalyzed metabolism of 2-aminofluorene was determined with incubations containing isozyme 5 purified from rabbit lung. A similar turnover number was calculated from the rabbit hepatic microsomal activity inhibited by antibodies to isozyme 5 and the microsomal isozyme 5 content measured by immunoquantitation. In other species, amounts of metabolism inhibited by antibodies to isozyme 5 agreed qualitatively with relative staining intensities on immunoblots. In all species except the hamster, rates of total and isozyme 5-catalyzed metabolism of 2-aminofluorene were greater with pulmonary than with hepatic microsomal preparations from untreated animals.

Animals↗
PubMed1987

Tissue- and species-dependent expression of multiple forms of mammalian microsomal flavin-containing monooxygenase.

The immunochemical relatedness, multiplicity, and expression of microsomal flavin-containing monooxygenases were assessed in hepatic, pulmonary, and renal microsomal preparations from adult, male rabbits, mice, rats, guinea pigs, and hamsters. Preparations from adult female (pregnant and nonpregnant) and immature male and female rabbits were also examined. Microsomes were analyzed by immunoblotting with polyclonal antibodies to flavin-containing monooxygenases purified from pig, mouse, and rabbit liver, and rabbit lung. Pulmonary flavin-containing monooxygenases, which differ from the major forms of the enzyme present in liver, were detected in pulmonary samples from all species. The major hepatic enzymes, or very closely related proteins, were also detected in pulmonary samples from every species except the rabbit. Three forms of pulmonary flavin-containing monooxygenase are found in rabbit and guinea pig. Differences in the expression of these three forms were observed with rabbits; all three were detected with some individuals and only two with others. The hepatic form of the flavin-containing monooxygenase is present in kidney of all species examined, and the pulmonary form is detected in kidney of rabbits, mice, and hamsters, but not rats or guinea pigs. Kidneys and lungs of individual rabbits were of the same phenotype with respect to the expression of the pulmonary forms of the enzyme. The findings show that the expression of flavin monooxygenase isozymes is tissue and species dependent.

Animals↗
PubMed1986

Formation of hydrogen peroxide and N-hydroxylated amines catalyzed by pulmonary flavin-containing monooxygenases in the presence of primary alkylamines.

In atypical reaction, incubation of purified rabbit pulmonary flavin-containing monooxygenase with certain primary alkylamines results in the oxidation of NADPH and the formation of hydrogen peroxide. In addition, significant amounts of N-hydroxylated primary amine are also generated, as determined by colorimetric assay and GC/MS analysis of n-octylamine metabolites. Similar reactions appear to be catalyzed by the mouse pulmonary enzyme. In contrast, incubation of primary alkylamines with hepatic flavin-containing monooxygenases from rabbit, mouse, or pig does not result in NADPH oxidation or metabolism. Another effect of primary alkylamines is marked activation of the mouse pulmonary and pig hepatic flavin-containing monooxygenases with some substrates. The structural requirements for primary alkylamines to elicit NADPH oxidation by the rabbit pulmonary enzyme or to activate the mouse pulmonary and pig hepatic enzymes are identical. This indicates that different flavin-containing monooxygenases probably have a conserved alkylamine-binding site of defined specificity. In the case of the rabbit pulmonary enzyme, this binding may occur very close to or at the catalytic site resulting in some N-hydroxylation of the alkylamine.

Amines↗
PubMed1986

The effect of substrate on the expression of activity catalyzed by cytochrome P-450: metabolism mediated by rabbit isozyme 6 in pulmonary microsomal and reconstituted monooxygenase systems.

The content of cytochrome P-450, isozyme 6, in the rabbit pulmonary microsomal fraction was estimated by immunochemical methods to be 1 to 3% of the total cytochrome P-450. Following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin, the pulmonary microsomal concentration of isozyme 6 increased 16-fold. Isozyme 6 was also detected by immunochemical methods, but not by electrophoresis and staining for protein, in preparations of isozyme 5 isolated from the pulmonary microsomal fraction of untreated rabbits. The metabolism of benzo[a]pyrene in these preparations was found to be catalyzed by isozyme 6, not by isozyme 5 as previously concluded. Cytochrome P-450, isozyme 4, was not detected in the pulmonary microsomal fraction from untreated or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rabbits. Although benzo[a]pyrene and 7-ethoxyresorufin are both substrates for isozyme 6, the pulmonary microsomal metabolism of these compounds was not increased to the same extent by treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (about 13-fold for 7-ethoxyresorufin and less than 2-fold for BP). However, lack of agreement between increases in isozyme 6 content and activity, and between the relative increases of the activities with the two substrates, was overcome by the addition of purified NADPH-cytochrome P-450 reductase to the microsomal incubations. When alpha-naphthoflavone, at the minimum concentration required for greater than 90% inhibition of isozyme 6 catalysis, was present in the incubations, no increases in activity were obtained by the addition of purified reductase. The turnover numbers of isozyme 6 in microsomal preparations incubated with purified reductase were similar to those of the purified isozyme in a reconstituted monooxygenase system. The relevance of our results to determinations of the substrate specificities and the microsomal concentrations and activities of isozymes of cytochrome P-450 is discussed. In addition, these parameters are used to assess the extent to which the catalytic potential of isozyme 6 is expressed in the rabbit pulmonary microsomal fraction.

Animals↗
PubMed1986

Unique properties of NADPH- and NADH-dependent metabolism of p-nitroanisole catalyzed by cytochrome P-450 isozyme 2 in pulmonary and hepatic microsomal preparations from rabbits.

Approximately 90% of the NADPH- and NADH-dependent O-demethylation of p-nitroanisole (PNA) in the hepatic microsomal fraction from phenobarbital (PB)-treated rabbits and in the pulmonary microsomal fraction from untreated rabbits is catalyzed by the same isozyme of cytochrome P-450. This isozyme of cytochrome P-450 catalyzes less than 60% of this reaction in the hepatic microsomal fraction from untreated rabbits. Antibodies to NADPH-cytochrome P-450 reductase inhibit NADPH-dependent metabolism of p-nitroanisole by about 90% but have no effect on NADH-dependent metabolism. Hepatic NADPH-dependent metabolism of pNA and reduction of cytochrome c are inhibited to the same extent with varying amounts of antibodies to NADPH cytochrome P-450 reductase. The same relationship between inhibition of monooxygenase and reductase activities is observed for the hepatic and pulmonary metabolism of benzphetamine and 7-ethoxycoumarin. In contrast, the relationship between inhibition of the pulmonary NADPH-dependent metabolism of pNA and reductase activity is biphasic; at 75% inhibition of reductase activity, metabolism of pNA is inhibited by less than 25%. For NADH-dependent metabolism of pNA, our results indicate that both electrons are transferred to cytochrome P-450 from cytochrome b5.

Animals↗
PubMed1986

Correlation of placental microsomal activities with protein detected by antibodies to rabbit cytochrome P-450 isozyme 6 in preparations from humans exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans.

Placental tissues were obtained from Chinese women in Taiwan who had been exposed to contaminated rice oils containing polychlorinated biphenyls and their thermal degradative products. Exposure via the diet occurred 4-5 years prior to pregnancy. Placental microsomal fractions from eight of the nine exposed subjects studied showed marked elevation of benzo(a)pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities related to control subjects. Placental microsomes from exposed subjects were found to contain a protein that cross-reacted with antibodies raised to rabbit cytochrome P-450 isozyme 6, an isozyme induced by polycyclic aromatic hydrocarbons. This protein was not observed with microsomal samples from control subjects. A significant correlation was found between the relative amounts of the immunoreactive protein and benzo(a)-pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities. The 7-ethoxyresorufin O-deethylation activities were inhibited by alpha-naphthoflavone, a compound known to inhibit activities of rabbit cytochrome P-450, isozyme 6.

Benzo(a)pyrene↗
PubMed1986

The cytochrome P-450 monooxygenase system of rabbit lung enzyme components, activities, and induction in the nonciliated bronchiolar epithelial (Clara) cell, alveolar type II cell, and alveolar macrophage.

Enzyme components and activities of the cytochrome P-450 monooxygenase system in microsomal preparations from the Clara cell, alveolar type II cell, and alveolar macrophage fractions isolated from lungs of untreated rabbits and rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin were examined. Results are compared to those obtained with microsomal preparations from whole lung. Concentrations of cytochrome P-450 isozymes 2 and 5 and NADPH-cytochrome P-450 reductase activities were higher in preparations from Clara cell fractions than in preparations from type II cell fractions or whole lung. For the most part, however, differences among these preparations were 2-fold or less. Microsomal preparations from the macrophage fraction contained low or undetectable levels of cytochrome P-450 isozymes but relatively high levels of cytochrome P-450 reductase activity. The concentration of cytochrome P-450 isozyme 6, in contrast to those of isozymes 2 and 5, was found to be highest in microsomal preparations from whole lung. Treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased the concentrations of isozyme 6 in preparations from the Clara and type II cell fractions and from whole lung about 20-fold. In contrast, the content of isozyme 6 in preparations from the macrophage fraction increased greater than 90-fold. In all cases, induction of isozyme 6 resulted in substantial increases in the O-deethylation of 7-ethoxyresorufin and only minor increases in the hydroxylation of benzo(a)pyrene. Activities per unit of isozyme 6, following induction, were similar in all preparations, and we estimate that less than 20% of the potential activity of isozyme 6 is expressed with benzo(a)pyrene and greater than 40% with 7-ethoxyresorufin. These similarities exist in spite of significant differences among the preparations from different fractions in the ratios of isozyme 6 to NADPH-cytochrome P-450 reductase.

Animals↗
PubMed1986

Rabbit pulmonary cytochrome P-450 monooxygenase system: isozyme differences in the rate and stereoselectivity of styrene oxidation.

The rate and stereoselectivity of the cytochrome P-450 (P-450)-dependent oxidation of styrene to styrene 7,8-oxide (SO) were determined in rabbit pulmonary microsomes and with purified rabbit pulmonary P-450 isozymes in reconstituted monooxygenase systems. Stereoselectivity was determined by separation of the diastereomeric SO-glutathione adducts by high-performance liquid chromatography; these four compounds accounted for more than 95% of the SO formed. Pulmonary microsomes preferentially formed (R)-SO [(R)-SO/(S)-SO = 1.6] at a rate of 7.5 nmol of SO formed/min/nmol of P-450. Antibodies to NADPH-P-450 reductase (antireductase) inhibited SO formation in pulmonary microsomes by greater than 98%. In the presence of antibodies to P-450 form 2 (anti-2), pulmonary microsomes oxidized styrene to equal amounts of (R)- and (S)-SO at a rate of 4.2 nmol of SO/min/nmol of total P-450; in the presence of antibodies to P-450 form 5 (anti-5), styrene was stereoselectively oxidized to (R)-SO [(R)-SO/(S)-SO = 2.0] at a rate of 6.5 nmol of SO/min/nmol of total P-450. In reconstituted monooxygenase systems, P-450 forms 2, 5 and 6 oxidized styrene to SO at rates of 10.0, 4.7 and 4.5 nmol of SO formed/min/nmol of P-450, respectively. The relative amounts of (R)-SO and (S)-SO produced were 2.0, 1.0 and 0.9, respectively. Predicted values for rate and stereoselectivity of styrene oxidation by pulmonary microsomes, calculated from the values obtained in the reconstitution experiments and the relative concentrations of the different P-450 isozymes, agreed well with experimentally determined values.

Animals↗
PubMed1985

Species susceptibility to the pulmonary toxicity of 3-furyl isoamyl ketone (perilla ketone): in vivo support for involvement of the lung monooxygenase system.

To explore a possible relationship between metabolism and lethality, the acute toxicity of naturally occurring perilla ketone (PK), 1-(3-furyl)-4-methyl-pentan-1-one, was examined in the uninduced mouse, hamster, rabbit, dog and pig. The LD50 (+/- SE), determined using intraperitoneal (ip) injection, for the mouse and hamster were low at 5.0 +/- .3 and 13.7 +/- .9 mg/kg, respectively. The rabbit died from an ip dosage of near 14 mg/kg and estimated ip LD50 dosages were quite high for the dog and pig, being 106 +/- 25 mg/kg and over 158 mg/kg, respectively. Dogs and the pig that died from ip injections of PK displayed varying degrees of midzonal and centrilobular liver damage and dogs also had elevated serum alkaline phosphatase and glutamic-pyruvic transaminase activities. In contrast, rodents and rabbits display only pulmonary toxicity from this agent. Cytochromes P-450 and b5 concentrations and NADPH-cytochrome c reductase activity were determined for the lung, liver and kidney of mice, hamsters, rabbits, dogs, swine, sheep and cattle. High correlation between lethality and enzyme concentration further supports the hypothesis that enzymatic bioactivation of PK is required for toxicity in all species.

Alanine Transaminase↗
PubMed1985

Cytochrome P-450 monooxygenase system. Localization in smooth muscle of rabbit aorta.

Cytochrome P-450 monooxygenase isozymes and NADPH-cytochrome P-450 reductase were detected in the microsomal fraction of rabbit aorta by immunoblotting and by enzymatic activity. The monomeric molecular weights of aortal proteins that cross-reacted with antibodies to cytochrome P-450 forms 2 or 6 and reductase were identical to those of the proteins purified from the liver. The induction of form 6 immunoreactive protein and O-deethylation of 7-ethoxyresorufin (a reaction catalyzed by form 6) was observed in aorta following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin or beta-naphthoflavone. The amount of reductase protein (equivalent to 22.4 +/- 3.2 activity units/mg of protein) correlated with the cytochrome c reductase activity (18.3 +/- 1.8 units/mg) and was the same for both treated and untreated rabbits. Consistent with immunoblot data, the amount of form 2 was insufficient for detection of activity (N-demethylation of benzphetamine). Significantly, removal of the endothelium, which was confirmed by light microscopy and by scanning electron microscopy, reduced by only 8 to 32% the specific enzymatic activity or content of immunoreactive proteins; only traces of protein or activity were recovered in the endothelial fraction. In studies of the vasculature, the potential of this metabolic pathway for the activation or detoxication of mutagens, carcinogens, toxins, or drugs and metabolism of endogenous substrates warrants consideration, especially in regard to the mutational events reported to be involved in the formation of atherosclerotic plaques.

Animals↗
PubMed1985

The cytochrome P-450 monooxygenase system of rabbit bladder mucosa: enzyme components and isozyme 5-dependent metabolism of 2-aminofluorene.

The microsomal fraction prepared from the mucosa of rabbit bladder was analyzed for the presence of enzymes and activities associated with the cytochrome P-450-dependent monooxygenase system. Reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (315 units/mg protein), reduced nicotinamide adenine dinucleotide:cytochrome b5 reductase (920 units/mg protein), cytochrome P-450 (0.22 nmol/mg protein), and cytochrome b5 (0.31 nmol/mg protein) were present in the microsomal preparation. Individual isozymes of cytochrome P-450, forms 2, 5, and 6, but not form 4, were detected by immunochemical methods. Treatment of rabbits with either phenobarbital or 2,3,7,8-tetrachlorodibenzo-p-dioxin did not alter the concentrations of these isozymes in the bladder preparation. Monooxygenase activities (pmol product/min/protein) in the bladder microsomal fraction were observed for benzphetamine N-demethylation (290), 7-ethoxyresorufin O-deethylation (29), 7-ethoxycoumarin O-deethylation (28), benzo(a)pyrene hydroxylation (10), and 2-aminofluorene hydroxylation (1400). The metabolism of 2-aminofluorene was determined by high performance liquid chromatography and scintillation counting; two products, 2-nitrosofluorene and 2,2'-azoxybisfluorene, were identified by chromatographic retention times, ultraviolet-visible spectroscopy, and mass spectrometry. Two additional metabolites were tentatively identified as N-hydroxy-2-aminofluorene and a ring-hydroxylated product. The metabolism of 2-aminofluorene was inhibited by antibodies to cytochrome P-450 form 5 or to reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase and by carbon monoxide (CO:O2, 4:1), but not by antibodies to cytochrome P-450 form 2. Acetylation of 2-aminofluorene in the presence of ethyl acetate (and deacetylation of 2-acetylaminofluorene) mediated by an enzyme sensitive to inhibition by either paraoxon or sodium fluoride was also observed.

2-Acetylaminofluorene↗
PubMed1985

Deacetylation to 2-aminofluorene as a major initial reaction in the microsomal metabolism of 2-acetylaminofluorene to mutagenic products in preparations from rabbit lung and liver.

The rabbit pulmonary and hepatic microsomal pathways for the metabolism of 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) to mutagenic products were investigated by means of high performance liquid chromatography and the Salmonella mutagenicity assay. Mutagenic activity approached a maximum with increasing concentrations of AAF incubated with hepatic microsomal preparations and Salmonella; with pulmonary microsomal preparations, mutagenic activity was proportional to the concentration of AAF over the range examined. The mutagenic activities of AF exhibited typical saturation kinetics with both hepatic and pulmonary microsomal preparations. Approximately 7 times more AF than N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF) was formed in incubations of AAF (0.5 mM) with hepatic microsomal preparations. When AAF was incubated with pulmonary microsomal preparations, formation of AF, but not N-hydroxy-AAF, was detected. The inclusion of paraoxon in the pulmonary incubations blocked the formation of AF but did not lead to the recovery of any N-hydroxy-AAF. We conclude that the metabolism of AAF to mutagenic products in pulmonary microsomal preparations from rabbits is initiated primarily, if not entirely, by deacetylation of AAF to AF. The mutagenic activity of AAF with the pulmonary microsomal preparations is limited by the deacetylase activity which, like mutagenic activity, exhibits a linear relationship with the concentration of AAF. On the basis of the rates of formation of AF and N-hydroxy-AAF and their mutagenic activities, we estimate that about 60% of the hepatic metabolism of AAF to mutagenic products is dependent upon deacetylation of AAF and subsequent oxidation of the AF formed.

2-Acetylaminofluorene↗
PubMed1984

Quantitation of rabbit cytochrome P-450, form 2, in microsomal preparations bound directly to nitrocellulose paper using a modified peroxidase-immunostaining procedure.

Rabbit hepatic microsomal suspensions were bound directly to nitrocellulose sheets using a "Hybridot" apparatus to ensure uniformity. Cytochrome P-450, form 2, was then detected by a modified immunochemical method wherein the nitrocellulose paper was incubated sequentially with antibody to form 2 for 1 h at 25 degrees C, rabbit anti-goat immunoglobulin G (IgG) at a 1:100 dilution for 15 min at 25 degrees C, goat peroxidase-antiperoxidase at a 1:2000 dilution for 15 min at 25 degrees C, and 3,3'-diaminobenzidine at 0.3 mg/ml plus 0.002% hydrogen peroxide for 30 min at 25 degrees C. These conditions, as opposed to those previously published, yielded less background staining. The density of the stain, scanned with a soft laser (Zeineh), increased linearly from 2 to 100 fmol for purified form 2. Cytochrome P-450, form 2, was detected and quantitated in microsomal samples containing 0.1 to 0.5 and 0.02 to 0.05 micrograms protein for preparations from untreated and phenobarbital-treated rabbits, respectively. The results agreed with those obtained by Western blotting and single radial immunodiffusion. This assay is more sensitive than either Western blotting or radial immunodiffusion and has significant advantages such as ease of operation, increased sample numbers, and reduced interference from extraneous proteins.

Animals↗
PubMed1984

Role of cytochrome P-450 and related enzymes in the pulmonary metabolism of xenobiotics.

The lung metabolizes a wide variety of xenobiotics and, in the process, forms products that may be more or less toxic than the parent compound. The consequence of metabolism, activation or detoxication, is a function of the nature of the substrate and of the characteristics and concentrations of the enzymes involved. As a result, the biotransformation of xenobiotics can lead to their excretion or to the formation of reactive products that produce deleterious effects by binding covalently to tissue macromolecules. Among the enzymes that metabolize xenobiotics, those associated with the cytochrome P-450-dependent monooxygenase system are probably the most important. The route by which a given substrate is metabolized in a tissue or cell is, to a great extent, determined by the types and concentrations of cytochrome P-450 isozymes present. We are just beginning to understand the distribution of these enzymes in lung and to appreciate the species and cellular differences that exist.

Animals↗