Cosette J. Serabjit-Singh

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).

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 Schiffs (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.

Female-predominant expression of testosterone 16α-hydroxylase (“I”-P-45016α) and its repression in strain 129/J

Abstract Using specific testosterone 16α-hydroxylase activity as the basis for selection of fractions during purification, the cytochrome P-450 ( “ I ”-P-450 16α ) 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 16α was used to determine the amount of hepatic microsomal 16α-hydroxylase activity due to “ I ”-P-450 16α 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-45016α-dependent activity was detected in the microsomes from males of either mouse strain. Immunoblotting of microsomal proteins with the antibody to “ I ”-P-450 16α 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/μg protein, respectively). A cDNA clone (R17) for phenobarbital-inducible rat cytochrome P-450 selected “ I ”-P-450 16α mRNA of mice, indicating a high degree of homology between the mRNAs of mouse “ I ”-P-450 16α 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 16α 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-45016α-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.

Tissue and cellular differences in the expression of cytochrome P-450 isozymes

Metabolism of exogenous chemicals by the cytochrome P-450 mono-oxygenase system (P-450 system) of various tissues and cells is necessarily dependent upon the contents and specificities of the individual isozymes of cytochrome P-450 present. Therefore, we should be able to relate differences in tissue or cellular metabolism of xenobiotics to constitutive isozyme profiles and to differential changes in profiles due to the inductive and/or repressive effects of chemical modulators of P-450 synthesis. The complexity of P-450 isozyme profiles requires that sensitive methods for detection and quantitation be employed and that these methods be applicable to different tissues and cell types under both native and altered conditions. Our past studies of rabbit liver and lung have pointed out a number of marked differences and similarities between the P-450 systems of these two tissues (Philpot & Wolf, 1981). With the development of highly sensitive immunochemical methods, we have now been able to examine P-450 systems in greater detail and have extended our observations to other tissues and species.

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.

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.

THERMOLABILITY OF MIXED-FUNCTION OXIDASE ACTIVITY IN HEPATIC MICROSOMES FROM LITTLE SKATE, Raja erinacea, AND RABBIT

We have identified several differences between rabbit and skate hepatic mixed-function oxidase (MFO) components which could contribute to the observed difference in temperature “optimum” for in vitro assay of MFO activity. Particularly involved is NADPH-cytochrome c reductase; the skate enzyme is more temperature labile than is the rabbit enzyme in microsomes and in a reconstituted system.

PURIFICATION OF RABBIT PULMONARY AND HEPATIC CYTOCHROME P-450 BY HYDROPHOBIC COLUMN CHROMATOGRAPHY

Procedures have been developed for the purification of two forms of rabbit pulmonary cytochrome P-450 and the hepatic form induced by phenobarbital. The procedures take advantage of the hydrophobic nature of cytochrome P-450 through the utilization of octyl- and phenyl-Sepharose 4B. Pulmonary cytochrome P-450I and the hepatic cytochrome can be purified to 21 nmol/mg protein by the reported method and the final preparations of these hemoproteins contain up to 17% of the microsomal P-450 content. This suggests a yield of up to 33% for the form of the cytochrome purified. Pulmonary cytochrome P-450I and the hepatic form of P-450 induced by phenobarbital appear to be the same enzyme as determined by a number of criteria. Pulmonary cytochromes P-450I and P-450II are clearly different proteins.