Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. I. Chromosomal determination of some cytochrome P-450-dependent reactions

The genetic regulation of some cytochrome P-450-dependent enzyme activities has been studied in adult Drosophila. Strains having genetically determined high or low enzyme activities were crossed with a marker strain and the metabolism was analyzed in microsomes from hybrids carrying different combinations of chromosomes from the strain under test. High p-nitroanisole (PNA) N-demethylation, biphenyl 3-hydroxylation and an increased amount of a protein with an apparent mol. wt. of 54 000, after SDS-gel electrophoresis of the microsomes in insecticide-resistant Drosophila strains, are inherited as dominant second chromosome traits. A low capacity for benzo[a]pyrene (BP) hydroxylation and 7-ethoxycoumarin O-deethylation in the Hikone R strain is semidominantly inherited in both cases and determined by gene(s) on the third chromosome. A semidominantly inherited high 4-hydroxylation of biphenyl and a high amount of a protein with an apparent mol. wt. of 56 000 in the Oregon R strain are also localized to the second chromosome. The results indicate that several other cytochrome P-450-dependent activities are not regulated by the genes mentioned above. In conclusion, at least three genes regulating the cytochrome P-450 system in Drosophila have been identified.     

Induction of cytochrome P-450-dependent sulfonylurea metabolism in Streptomyces griseolus

Inducible cometabolism of several sulfonylurea herbicides by Streptomyces griseolus has been shown to occur by hydroxylation, O-dealkylation, or deesterification reactions. Only after growth of the bacterium in the presence of sulfonylurea did cell-free extracts exhibit NAD(P)H-dependent sulfonylurea metabolism. These extracts were shown to contain elevated levels of soluble cytochrome P-450 and exhibit sulfonylurea induced difference spectra consistent with binding of substrate to cytochrome(s) P-450. These results establish the presence of an inducible cytochrome P-450-dependent sulfonylurea metabolizing system in S. griseolus.     

Microtubule-associated proteins present in different developmental stages of Drosophila melanogaster

Microtubule-associated proteins (MAPs) have been isolated from different development stages of Drosophila melanogaster and characterized by their association to tubulin, but not to tubulin lacking its 4-kD carboxy terminal region (S-tubulin), and by their ability to promote tubulin polymerization. Following these criteria some peptides of Mr 255, 205, and 180 kD were identified as MAPs. By means of immunological analogy we have identified a peptide related to mammalian brain MAP known as tau factor.     

Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. II. Localization of some genes regulating cytochrome P-450 activity

The localization of some genes determining the capacity for some cytochrome P-450 -dependent reactions have been studied in adult Drosophila. Strains with genetically determined high or low enzyme activities were crossed with strains carrying recessive visible markers on the chromosomes, and enzyme activities were measured in microsomes from recombinant F2 progeny. A dominantly inherited high p-nitroanisole (PNA) demethylation and biphenyl 3-hydroxylation in insecticide-resistant strains were both shown to be located around 65 cM on the second chromosome, regulated by one gene or closely linked genes. This localizes these activities to the same region as the gene responsible for the cross resistance to several classes of insecticides and a high metabolism of vinyl chloride in resistant strains. The occurrence of a regulatory gene mutation as a basis for the insecticide resistance is proposed. Hydroxylation of benzo[a]pyrene (BP) and deethylation of 7-ethoxy-coumarin seems to be determined by two third chromosome genes, at approx. 51 and 58 cM, respectively. The capacity for biphenyl 4-hydroxylation was shown to be determined by two genes on the second chromosome, one at or to the left of the gene black (48 cM) responsible for a low metabolism in strain Berlin K, and one at about 63 cM giving high formation of this metabolite in Oregon R. The latter could not be separated from the gene in insecticide-resistant strains at c:a 65 cM discussed above on the basis of the genetic localization, but observations supporting the occurrence of two closely linked genes regulating these different activities are available. In conclusion, 4-5 genes determining the capacity for several reactions, being a part of the genetic regulation of the cytochrome P-450 system in Drosophila melanogaster were indicated.     

NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.     

DNA polymerase activity in developmental stages of Drosophila melanogaster

An assay procedure was developed that allowed the first reproducible measurement of DNA polymerase activity in all developmental stages of Drosophila melanogaster. Evidence is presented that the same enzymatic species is present in extracts of embryos, pupae, and adults of both sexes and that this activity has many properties similar to vertebrate α-polymerases. Polymerase activity per individual is low in embryos and rises steadily through larval instars, reaches a peak in early pupae, declines through the late pupal period, and remains low in newly eclosed adults of both sexes. A dramatic increase is observed in adult females as mature oocytes are formed. This pattern of enzyme activity is completely coincident with changes in DNA levels during development, and suggests that the Drosophila enzyme, like vertebrate α-polymerases, functions in cellular DNA replication. Two mutagen-sensitive mutants, deficient in both replication on undamaged templates and postreplication repair, were found to have normal levels of this α-polymerase activity. Our results suggest that a single enzymatic species of α-polymerase holoenzyme exists in Drosophila and is common to all developmental stages of this organism.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

Phenobarbital induces cytochrome P-450- and cytochrome P-448-dependent monooxygenases in rat hepatoma cells

The induction by phenobarbital (PB) of aldrin epoxidase (AE) and aryl hydrocarbon hydroxylase (AHH), markers of cytochrome P-450- and cytochrome P-448-dependent monooxygenases, was studied in cell lines derived from Reuber H35 rat hepatoma which differ widely in their degree of differentiation. The following results were obtained: (1) PB induced AE 2-6-fold and AHH 2-4-fold in the differentiated clones, Fao, 2sFou, and C2Rev7 during an exposure period of 72 h. The barbiturate increased AHH but not AE in the dedifferentiated clone H5, the poorly differentiated line H4IIEC3/T, and in the well differentiated line H4IIEC3/G-. (2) Continuous presence of the barbiturate was required for maintaining the induction of the two monooxygenase activities in C2Rev7 cells. (3) Maximum induction of AE was observed at a PB concentration of 1.5-3.0 mM. (4) The effects of 7,8-benzoflavone on AHH-activities induced by phenobarbital in C2Rev7 and H5 cells suggested that they are mediated by cytochrome P-450- and cytochrome P-448-dependent monooxygenase forms, respectively. Thus, the flavonoid had only a slight inhibitory effect on PB-induced AHH in C2Rev7 cells, but strongly inhibited PB-induced AHH in H5 cells. The induction of AE and of 7,8-benzoflavone-inhibitable AHH in 2sFou cells indicated that PB is capable of inducing cytochromes P-450 and cytochrome P-448 in the same cell.     

Alteration of cytochrome P-450 2C11-dependent testosterone metabolism in Gunn rat liver.

Cytochrome P-450 2C11 (CYP2C11) is the main isozyme present in adult male rat liver and specifically hydroxylates testosterone in positions 2 alpha and 16 alpha. In Gunn rats, this isozyme is recognized by the anti-CYP2C11 antibody but its activity towards testosterone is dramatically decreased. Moreover, peptide mapping, after digestion of microsomal fractions by V8 protease and probing with anti-CYP2C11 antibody, exhibit a pattern which differs from that obtained with Wistar rats. Taken together, data suggest that the protein sequence of CYP2C11 from the Gunn rat differs from that of the Wistar rat.     

Induction of cytochrome P-450-dependent drug metabolism by juvenile hormone mimics in mammalian cell culture

The monooxygenase activity of fetal hepatocytes in culture shows a differential response toward juvenile hormone I and analogs. Juvenile hormone I, R-20458, and Methoprene increase the deethyiation of 7-ethoxyresorufin while not affecting or even inhibiting the N-demethylation of p-chloro-N-methylaniline. RO-203600, a 1,3-benzodioxole-containing analog, increases both the deethylase and the N-demethylase, whereas Hydroprene does not affect either activity. The inductive effect with juvenile hormone I is obtained with exposure periods of at least 30 min and is maximum when the concentration of the hormone is 14 μM in the medium. This amount results in the covalent binding to cellular macromolecules of 1.3 × 19^?18 moles/cell. The induction requires continuous protein synthesis but RNA synthesis only for a short initial period. It is concluded that juvenile hormone and mimics induce specific cytochrome P-450 species in fetal liver cells even if the culture conditions are not optimal. The toxicological implications of these results are briefly discussed.     

Presence of cytochrome P-450- and cytochrome P-448-dependent monooxygenase functions in hepatoma cell lines

Cell lines derived from Reuber H-4-II-E hepatoma cells and their hybrids that differ in the expression of liver-specific functions are shown to contain different forms of monooxygenases. According to 1) the specificity toward the substrates benzo(a)pyrene, aldrin and chenodexycholic acid, 2) the kinetics of the epoxidation of aldrin, 3) the response to inducers, such as benz(a)anthracene and dexamethasone, and 4) the $\text{in}$$\text{vitro}$ modifier 7,8-benzoflavone, the monooxygenases predominating in differentiated cell lines belong to the cytochrome P-450-dependent enzyme(s), those in the less differentiated lines belong to the cytochrome P-448-dependent form(s).     

Styrene metabolism in Exophiala jeanselmei and involvement of a cytochrome P-450-dependent styrene monooxygenase.

The yeast-like fungus Exophiala jeanselmei degrades styrene via initial oxidation of the vinyl side chain to phenylacetic acid, which is subsequently hydroxylated to homogentisic acid. The initial reactions are catalyzed by a NADPH- and flavin adenine dinucleotide-dependent styrene monooxygenase, a styrene oxide isomerase, and a NAD(+)-dependent phenylacetaldehyde dehydrogenase. The reduced CO-difference spectrum of microsomal preparations of styrene-grown cells shows a characteristic absorption maximum at 450 nm, which strongly suggests the involvement of a cytochrome P-450-dependent styrene monooxygenase. Inhibition of styrene monooxygenase activity in cell extracts by cytochrome P-450 inhibitors SKF-525-A, metyrapone, and CO confirms this assumption.     

Comparison of Drosophila cytochrome P450 metabolism of natural and model substrates

Metabolism of some insecticides and toxic natural plant compounds is known to involve cytochrome P450 enzymes. Correlations between insecticide resistance and deethylation of the model substrate, 7-ethoxycoumarin, have prompted its use in screens for potentially resistant insect populations. The applicability of this model substrate as an indicator of the enzyme activities and inductive responsiveness of cytochrome P450 isoforms involved in the metabolism of carnegine was investigated. This toxic isoquinoline alkaloid is found in the host-plants of some species of cactophilic Drosophila. The results show that the ethoxycoumarin (ECOD) assay does not accurately predict carnegine metabolism either quantitatively or with respect to the overall pattern of activity. Therefore, the ECOD assay may be as isozyme-specific in insects as has already been demonstrated in mammals and its use as an indicator of general P450 activity is questionable.     

Excess nitric oxide decreases cytochrome P-450 2J4 content and P-450-dependent arachidonic acid metabolism in lungs of rats with acute pneumonia

Recently, we demonstrated that pulmonary CYP2J4 content, a prominent source of EETs and HETEs formation in rat lungs, is reduced in pneumonia. Therefore, the purpose of this study was to determine the role of iNOS-derived NO in reduced pulmonary CYP2J4 protein content and decreased CYP metabolites in pneumonia. Rats were randomized to control, control plus 1400W (iNOS inhibitor), pneumonia, and pneumonia plus 1400W groups. Pseudomonas organisms were injected into lungs of pneumonia rats. At 40 h after surgery, rats were treated with either saline or 1400W for 4 h before death. Venous plasma samples were obtained for measuring nitrites/nitrates (NOx). There was no significant effect of 1400W on blood pressure measured in control or pneumonia rats, whereas 1400W reduced the elevated plasma NOx levels in pneumonia rats by half. CYP primary metabolites of AA formed at significantly lower rates in pulmonary microsomes from pneumonia rats compared with control rats. Treatment of pneumonia rats with 1400W resulted in a significant increase in the rate of formation of pulmonary EETs and omega-terminal HETEs compared with untreated pneumonia rats. The reduction in CYP2J4 protein content in pneumonia lung microsomes was also partially prevented by 1400W. Therefore, excess NO from iNOS decreases the pulmonary production of EETs and omega-HETEs in acute pneumonia. Inhibition of iNOS restores CYP2J4 protein content and CYP activity in acute pneumonia, indicating an important NO-CYP interaction in pulmonary responses to infection. We speculate CYP2J4 and its AA metabolites are involved in the modulation of pulmonary function in health and disease.