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.

Metabolism of aflatoxin B1 in the upper airways of the rabbit: role of the nonciliated tracheal epithelial cell.

Short-term tracheal explant cultures from the rabbit were used to study the metabolism of the carcinogen aflatoxin B1 (AFB1) and to determine the cell types that are susceptible to damage by AFB1 and their relative contents of monooxygenase enzymes. Tracheas were cultured in serum-free medium for 0.5-24 h with 0.7 microM [3H]AFB1, and metabolism was measured by determining the level of binding of the carcinogen to DNA and by the release of metabolites into the medium. The binding of aflatoxin B1 was time dependent and appeared to peak at 12 h in culture. In addition, the metabolites aflatoxicol, aflatoxin M1, and aflatoxin Q1 were produced by the explants. Ultrastructural evaluation of cultured tracheas showed degenerative changes exclusively in nonciliated secretory cells after 4 h in culture. Extensive nonciliated secretory cell necrosis was evident by 12 h. Ciliated cells did not show degenerative changes until 12 h and appeared much more viable after 24-h exposure to AFB1 relative to the nonciliated cells. Tracheal sections stained to demonstrate rabbit lung cytochrome P-450, Forms 2 and 5, and cytochrome P-450 reduced nicotinamide adenine dinucleotide phosphate reductase by an immunoperoxidase technique showed intense staining selectively within nonciliated cells. In total, the data revealed that: (a) rabbit tracheal explants are able to metabolize aflatoxin B1; (b) the nonciliated secretory cell population in this tissue is the target cell for cytotoxicity of this carcinogen; and (c) as is the case in the more distal airways, the nonciliated epithelial cells appear to have a high content of components of the pulmonary cytochrome P-450 monooxygenase system, which may be an important factor in the susceptibility of these cells and this region of the airways to suspected airborne carcinogens.

Diminished drug metabolism due to hormonal effects of cobalt mesoporphyrin on flavoproteins.

Substituted metalloporphyrins, in addition to their use as pharmacological agents, are used to investigate metabolic pathways by inhibiting cytochrome P-450. We have examined the specificity of this approach with cobalt mesoporphyrin (CoMP). In vivo, CoMP (50 mumol/kg, s.c.) decreased rat hepatic microsomal cytochrome P-450, NADPH cytochrome P-450 reductase, benzphetamine N-demethylase (BZPH) activity, and thyroid hormones by > 50%, all of which returned to control levels after 45 days; testosterone levels were also reduced at this dose. The half-life of CoMP was 18 days, which is consistent with this sustained effect. At 10 mumol/kg of CoMP, the reductase activity was decreased, but cytochrome P-450 was unchanged. An effect of residual CoMP on the reductase was ruled out as the CoMP content of tissue fractions was not high enough to inhibit directly the reductase activity (even after 50 mumol CoMP/kg). However, immunoblots indicated a lower level of immunoreactive reductase protein following treatment. After 8 weekly doses of 1 mumol CoMP/kg, BZPH activity was 39% less than control but neither P-450 content nor reductase activity was significantly changed. The P-450 content and reductase activity in rabbits were much less affected by CoMP, perhaps due to differences in the disposition of CoMP. Thyroidectomy decreased reductase activity in rats to an extent that was seen with CoMP at 50 mumol/kg; CoMP treatment of thyroidectomised rats did not further decrease reductase activity. Supplementation with thyroid hormone blocked the CoMP-related decrease. The flavin-containing monooxygenase was decreased by CoMP and by castration, and the decrease was not blocked by the thyroid hormone supplement. Thus in the rat, the CoMP-related decreases in thyroid hormone and testosterone decrease flavoproteins that support or mediate monooxygenase activities. This is contrary to the reported specificity of this class of compound as inhibitors of cytochrome P-450.

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.

Shorter development of immunoassay for drugs: application of the novel RIMMS technique enables rapid production of monoclonal antibodies to ranitidine.

High affinity, specific murine monoclonal antibodies have been produced for ranitidine using the novel RIMMS (repetitive immunizations, multiple sites) technique. We demonstrate that this technique can be employed to produce high affinity monoclonal antibodies to drug haptens in approximately 1 month; whereas, conventional techniques typically require 3-9 months. Polyclonal antiserum development typically requires at least 6 months. Consequently, RIMMS has a clear impact allowing reagent antibodies to be available earlier in the drug development process. Isotyping studies demonstrated that the developed antibodies are either IgG1 or IgG2b immunoglobulins which confirms that the technique produces class-switched, affinity matured reagent antibodies. The most promising monoclonal antibody for quantitative applications afforded similar sensitivity, by competitive ELISA, to the established sheep polyclonal anti-ranitidine sera. The calibration range, estimated as the limits between the asymptotic regions of calibration graphs, is 0.5-41.2 ng ranitidine per well. Specificity studies indicated that the monoclonal antibody afforded superior selectivity, yielding only 4.1% cross-reactivity with the ranitidine sulphoxide metabolite; the corresponding value for the antiserum was 8.6%. Both reagents had similar cross-reactivities with the N-oxide metabolite.

Purification and biochemical characterization of the rabbit pulmonary glutathione S-transferase: stereoselectivity and activity toward pyrene 4,5-oxide.

Two homodimeric isozymes, glutathione S-transferase (GST) 25 kDa and GST 27 kDa, in equal proportion comprise the majority (greater than 75%) of the pulmonary cytosolic GST of untreated rabbits. The subunits of GST 25 kDa and GST 27 kDa are distinguishable by electrophoretic mobility (25 and 27 kDa, respectively), apparent isoelectric points (pI 7.4 and pI 9.1, respectively), and immunoreactivity. Immunoblots indicated that these subunits may be minor components in hepatic cytosol. The pulmonary isozymes could not be distinguished by their activities toward chloro-2,4-dinitrobenzene (CDNB) or activity and stereoselectivity toward pyrene 4,5-oxide (PyO). The purified GST fractions represented less than or equal to 16% of the PyO activity for pulmonary cytosol. The stereoselectivity of the cytosolic GST for the pro-S-configured oxirane carbon of PyO was not maintained in the purified preparations which were virtually nonstereoselective. Immunoprecipitation of pulmonary cytosolic GST with anti-GST 27 kDa and anti-GST 25 kDa indicated that at least 84 and 60% of the activity toward CDNB and PyO, respectively, is mediated by the two isozymes. The specific PyO activities of GST 27 kDa, GST 25 kDa, and the rabbit hepatic preparations (approximately 0.2 unit/mg) were similar to that of hepatic GST purified from horse, cow, and pig, and to human placental GST pi (0.02-0.5 unit/mg) but one-tenth that of rat hepatic GST or human hepatic GST mu. However, the activity of the hepatic cytosol from rat and human was similar to that of rabbit. Thus, some GST isozymes may be particularly susceptible to modulation of activity/stereoselectivity that can be discerned with arene oxide substrates such as PyO.

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.

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)

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.

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.

Female-predominant expression of testosterone 16 alpha-hydroxylase ("I"-P-450(16)alpha) and its repression in strain 129/J.

Using specific testosterone 16 alpha-hydroxylase activity as the basis for selection of fractions during purification, the cytochrome P-450 ("I"-P-450(16)alpha) has been isolated from livers of phenobarbital-treated 129/J female mice [K. Devore, N. Harada, and M. Negishi (1985) Biochemistry, 24, 5632-5637]. An antibody elicited in rabbits to "I"-P-450(16)alpha was used to determine the amount of hepatic microsomal 16 alpha-hydroxylase activity due to "I"-P-450(16)alpha in untreated females and males of the two mouse strains, 129/J and BALB/cJ. The activities inhibited were 0.03 and 0.3 nmol/min/mg protein in the 129/J and BALB/cJ females, respectively. No significant level of "I"-P-450(16)alpha-dependent activity was detected in the microsomes from males of either mouse strain. Immunoblotting of microsomal proteins with the antibody to "I"-P-450(16)alpha revealed approximately a 10-fold greater amount of a 54-kDa protein in the microsomes from BALB/cJ than from 129/J females (0.03 and 0.26 pmol/micrograms protein, respectively). A cDNA clone (R17) for phenobarbital-inducible rat cytochrome P-450 selected "I"-P-450(16)alpha mRNA of mice, indicating a high degree of homology between the mRNAs of mouse "I"-P-450(16)alpha and phenobarbital-inducible rat cytochrome P-450s. Northern and dot hybridization of total mouse liver poly(A)+ RNA with the R17 cDNA probe indicated that the specific content of the hybridizable mRNA was more than 10 times higher in BALB/cJ females than in males, and that the mRNA level in female 129/J mice was very similar to that of 129/J and BALB/cJ males. The repression of "I"-P-450(16)alpha in 129/J females was inherited as an autosomal recessive trait in 129/J and BALB/cJ pairs as indicated by the levels of mRNA in female F1 offspring and the "I"-P-450(16)alpha-dependent hydroxylase activity. Female and male mice of eight more inbred strains (AKR/J, DBA/2J, C57BL/6J, C3H/HeJ, NZB/J, A/J, CBA/CaJ, and P/J) were tested for levels of mRNA. The results showed that the levels of mRNA were always 5- to 10-fold greater in the females than in the corresponding males, although there was some variation in the mRNA content in the males from the different strains. 129/J females appear to be a genetic variant where the female-predominant expression of the mRNA is repressed.

The major alpha-class glutathione S-transferases of rabbit lung and liver. Primary sequences, expression, and regulation.

The complete primary structures of two distinct rabbit alpha-class glutathione S-transferase (GST) subunits, rbGST alpha I and rbGST alpha II, have been derived from cDNA sequences. Clones encoding rbGST alpha I were isolated from both hepatic and pulmonary cDNA libraries, whereas clones encoding rbGST alpha II were isolated only from the hepatic library. Immunochemical and peptide sequence data confirmed that rbGST alpha I corresponds to the 27-kDa alpha-class subunit purified from rabbit lung (Serabjit-Singh, C. J., and Bend, J. R. (1988) Arch. Bioch. Biophys. 267, 184-194). Expression of rbGST alpha II in liver but not in lung and expression of rbGST alpha I in both liver and lung was substantiated by Northern and immunochemical analyses. rbGST alpha I and rbGST alpha II are composed of 223 and 221 amino acids, respectively, and are 78% identical in amino acid sequence. Compared to published GST sequences, both proteins are most closely related to the human Ha subunit (greater than 80% identity). On the basis of sequence comparison and Northern and Southern analyses, we conclude that rbGST alpha I and rbGST alpha II are products of different genes that are independently regulated. Further, the regulatory elements of the alpha-class GST genes may be significantly different in the rabbit as compared to the rat, as evidenced by the lack of induction by phenobarbital of rabbit hepatic or pulmonary alpha-class GST subunits, enzymatic activity, or mRNA. This tissue- and species-dependent expression of the predominant class of cytosolic GST implies unique functions for each isozyme and may contribute to the differential susceptibility of tissues and animals to toxicants.

CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4.

Human cytochrome CYP3A4 is the most abundant of all the P450s in human liver and is involved in the metabolism of many environmental toxicants and drugs. Kinetic studies with CYP3A4 have been hampered due to low activity of this enzyme obtained from recombinant gene expression systems or difficulty in reconstituting activity with the native enzyme purified from human liver. To overcome these obstacles, we have expressed high levels of catalytically active CYP3A4 and human NADPH-cytochrome P450 reductase (CYPOR) together in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T.ni), via a single recombinant baculovirus carrying both cDNAs (CYP3A4-OR). Microsomes containing recombinant CYP3A4-OR from these cell lines were up to 50-times more active in testosterone 6 beta-hydroxylase activity than recombinant CYP3A4 expressed alone and supplemented with purified rabbit CYPOR. The spectral P450 content of CYP3A4-OR T.ni microsomes was 107 pmol/mg microsomal protein and the cytochrome c reductase activity was 3904 units/mg. Recombinant CYP3A4-OR was catalytically similar to human liver CYP3A4 based on similarities in the testosterone metabolite profile, time course of metabolite formation, Vmax and Km values (for CYP3A4-OR, Vmax was 8.8 nmol/min/mg microsomal protein [70 nmol/min/nmol CYP3A4] and Km was 33 microM), the extent of inhibition by 100 microM troleandomycin (> 75%) in the presence of 25 microM testosterone, and the degree of P450 activation in the presence of 20 microM 7,8-benzoflavone. The coexpression of recombinant cytochrome b5 with CYP3A4-OR did not result in an additional increase in CYP3A4-OR activity.

Unique distribution profiles of glutathione S-transferases in regions of kidney, ureter, and bladder of rabbit.

BACKGROUND: Glutathione S-transferases detoxify a broad range of exogenous compounds, but are important also in the metabolism of endogenous compounds. Physiologically relevant substrates are the endoperoxide and hydroperoxide metabolites of arachidonic acid that play important roles in many tissues including the kidney. EXPERIMENTAL DESIGN: We used immunohistochemical and immunoblotting techniques in a systematic study of renal localization of four rabbit enzymes that represent three major mammalian cytosolic glutathione S-transferase classes, alpha, pi, and mu. RESULTS: The two alpha-class enzymes (rbGST alpha I, rbGST alpha II) were distributed discretely in kidney, ureter, and bladder, while pi and mu were widely distributed in the renal system. Immunohistochemical localization in paraffin sections with antibodies specific for rbGST alpha I or rbGST alpha II demonstrated that no compartment of the renal system contained both enzymes. Collecting ducts of the inner medulla and all epithelial cells of the kidney pelvis, ureter, and bladder contained rbGST alpha I. All cells lining proximal tubules contained rbGST alpha II. No other compartment of the renal system exhibited immunoreactivity with anti-rbGST alpha II. Antibody specific for pi reacted with cells lining nephrons, ureter, and bladder and with endothelial cells throughout the renal system. Localization of pi was most prominent in the collecting ducts of medulla and in the epithelial cells lining the kidney pelvis, ureter, and bladder. As anti-mu did not react in tissue sections, distribution of mu was determined by immunoblotting. Immunoblots of cytosolic preparations from whole kidney, cortex, medulla, and epithelia of ureter, bladder, and kidney pelvis were prepared and tested with each of the 4 antibodies. This second localization method confirmed the distribution data from tissue sections for rbGST alpha I, rb GST alpha II, and pi; also, it demonstrated that the staining observed in tissue was specifically for each enzyme. mu was detected in all the renal cytosolic preparations except those from the epithelium of the kidney pelvis. CONCLUSIONS: The discrete renal distribution of rbGST alpha I and rbGST alpha II and their distinct catalytic activities with prostaglandin substrates suggest important roles for these enzymes in prostaglandin-dependent renal functions.

The rabbit pulmonary monooxygenase system. Immunochemical and biochemical characterization of enzyme components.

The enzymatic components of the rabbit pulmonary monooxygenase system, cytochromes P-450I and P-450II and NADPH-cytochrome P-450 reductase, are immunochemically distinct proteins. In pulmonary microsomes, the N-demethylation of benzphetamine, amino-pyrine, and ethylmorphine, and the O-deethylation of 7-ethoxycoumarin are dependent only on cytochrome P-450I, and the hydroxylation of coumarin is apparently catalyzed by both cytochromes. Cytochrome P-450II is immunochemically distinct from the major forms of hepatic cytochrome P-450 induced by phenobarbital or 3-methylcholanthrene, whereas cytochrome P-450I is indistinguishable from the former on the basis of physical and catalytic as well as immunochemical characteristics. Pulmonary and hepatic NADPH-cytochrome P-450 reductases also have identical physical, catalytic, and immunochemical properties. The lack of response of the lung monooxygenase system to phenobarbital, therefore, is apparently not due to an inability of the lung to synthesize the enzymes induced by phenobarbital in the liver. The relatively high proportion of cytochrome P-450I in the lung appears to be responsible for the higher rates (per nmol of P-450) of N-demethylation that have been observed in rabbit pulmonary as compared to hepatic microsomal fractions.

Glutathione S-transferase-catalyzed conjugation of bioactivated aflatoxin B1 in rabbit lung and liver.

Aflatoxin B1 (AFB1) requires bioactivation to AFB1-8,9-epoxide for carcinogenicity, and glutathione S-transferase (GST)-catalyzed conjugation of activated AFB1 with glutathione (GSH) is a critical determinant of susceptibility to the mycotoxin. Incubations containing [3H]AFB1, rabbit liver microsomes, an NADPH-generating system, 1 mM GSH, and GST-containing lung or liver cytosol were performed to assess the abilities of lung and liver GSTs to conjugate AFB1-8,9-epoxide. [3H]AFB1-GSH was isolated by isocratic reverse-phase high-performance liquid chromatography (HPLC) and quantitated by liquid scintillation spectroscopy. Maximal [3H]AFB1-GSH formation rates were significantly lower for lung than for liver (0.3 +/- 0.1 and 1.7 +/- 0.4 nmol/mg/hr, respectively). Immunoprecipitation of rabbit pulmonary cytosolic GSTs with anti-alpha or anti-mu GST antisera decreased [3H]AFB1-GSH production by approximately 45 and 51%, respectively, indicating that alpha-class and mu-class GSTs are of similar importance in catalyzing this reaction in the lung. Because mu-class GSTs comprise only a small proportion of total lung GST content, these enzymes have high specific activity toward AFB1-8,9-epoxide. In contrast, the pi-class GST appeared to play a negligible role. Using a rat liver microsomal system to generate both AFB1 exo- and endoepoxide isomers, and analysis based on chiral HPLC, we found that rabbit liver cytosolic GSTs catalyzed formation of both AFB1 exo- and endo-epoxide-GSH conjugates, whereas pulmonary cytosolic GSTs catalyzed formation of only the exo stereoisomer at detectable levels. Despite a preference for conjugating the more mutagenic AFB1 exo-epoxide isomer, the relatively low capacity for GST-catalyzed detoxification of bioactivated AFB1 in lung may be an important factor in the susceptibility of the lung to AFB1 toxicity.

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.

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.

Distribution of cytochrome P-450 monoxygenase enzymes in the nasal mucosa of hamster and rat.

Deposition of inhaled particulates onto the respiratory mucosa is relatively great in that portion of the nasal cavity unprotected by ciliated, goblet, or keratinized superficial cells. The cytochrome P-450 system is an important enzyme system involved in the biotransformation of xenobiotics into metabolites that are more readily absorbed. To examine the transitional region caudal to the nasal vestibule, nasal tissues of hamster and rat were prepared for immunocytochemistry. Blocks of tissue representing four levels along the long axis of the nasal cavity were examined. Paraffin sections were processed through the avidin-biotin peroxidase procedure, with diaminobenzidine tetrahydrochloride as the chromagen. Enzyme localization was accomplished through the use of antibodies for three rabbit cytochrome P-450 isozymes; 2, 5, and 6 (subfamilies IIB, IVB, and IA, respectively); and for rabbit NADPH-cytochrome P-450 reductase. Enzyme distribution was similar in both hamster and rat nasal tissues except in cells of striated and intercalated ducts of nasal glands and in cells of the nasolacrimal duct where immunoreactivity was greater in the hamster. Immunoreactivity for reductase and isozyme 2 was intense in nonciliated cells lining the nonolfactory epithelium, in sustentacular cells of the olfactory epithelium, and in acinar cells of olfactory glands. Distribution of reaction products to isozyme 5 and 6 were similar to but not so intense as those of reductase and isozyme 2. Reaction products for reductase and isozyme 2 occurred generally in the same cellular and intracellular regions with the following exceptions: isozyme 2 was more concentrated in cells of striated ducts and of the nasolacrimal duct, and reductase was more abundant in intercalated ducts of nasal glands.(ABSTRACT TRUNCATED AT 250 WORDS)