Hydroxylation Of Prostaglandins Research Articles

Sequence analysis of novel CYP4 transcripts from Mytilus galloprovincialis

Cytochrome P450 enzymes (CYPs) are essential components of cellular detoxification system. We identified and characterized seven new cytochrome P450 gene transcript clusters in the populations of bivalve mollusc Mytilus galloprovincialis from three different locations. The phylogenetic analysis identified all transcripts as clusters within the CYP4 branch. Identified clusters, each comprising a number of transcript variants, were designated CYP4Y1, Y2, Y3, Y4, Y5, Y6 and Y7. Transcript clusters CYP4Y2 and Y7, and CYP4Y5 and Y6 showed site specificity, while the transcript clusters CYP4Y1, Y3 and Y4 were present at all investigated locations. The comparison of transcripts deduced amino acid sequences with CYP4s from vertebrate and invertebrate species showed high conservation of the residues and domains essential to the putative function of the enzyme, as terminal ω-hydroxylation and prostaglandin hydroxylation. Our results suggest the great expansion of the CYP4Y cDNAs indicative of CYP4 proteins in the mussel M. galloprovincialis presumably as a response to different environmental conditions.

Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the ω-1 and ω-2 hydroxylation of prostaglandins

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the ω-2 hydroxylation of prostaglandin E 2 (PGE 2) by liver microsomes ( K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477–489 ). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the ω-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1,1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450 57K and P450 56K (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450 57K; and 1-36-1 binds solely P450 57K. MAb 1-7-1 inhibited ω-2 and ω-1 PGE 2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE 2 ω-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the ω-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the ω-2 hydroxylation and to a lesser extent the ω-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the ω-2 hydroxylating activity in intact MC-microsomes supported by H 2O 2, by demonstrating a much higher activity when H 2O 2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE 2 at the ω-2 and ω-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the ω-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some ω-2 hydroxylating activity is due to P450d was not ruled out. Certain purified P450s (RLM 5 and 5a) from untreated rat liver catalyze the ω-2 hydroxylation of PGE 2 (D. Kupfer et al. (1988) Arch. Biochem. Biophys. 261, 186–195). The current findings demonstrate that all the ω-2 hydroxylation in control microsomes and about 25% of the activity in MC-microsomes is by P450s which are not inhibited by MAb 1-7-1. It is not known whether these resistant P450s are RLM 5, 5a, or different constitutive P450s.

Isolation and biosynthesis of 18-hydroxyprostaglandins E 1 and E 2 in human seminal fluid

18-Hydroxy-PGE 1 and 18-hydroxy-PGE 2 were identified in human seminal fluid by capillary gas-liquid chromatography-mass spectrometry. The levels of these prostaglandins was 1–2% of the corresponding 19-hydroxy-PGE compounds in human semen. 18Hydroxy-PGE 1 and 18-hydroxy-PGE 2 are likely formed by cytochrome P-450 in seminal vesicles in analogy with the 19-hydroxy-PGE compounds. This was supported by the finding that microsome of seminal vesicles of the cynomolgus monkey, Macaca fascicularis, supplemented with 1 nM NADPH, metabolized PGE 1 to both 19-hydroxy-PGE 1 (92%) and 18-hydroxy-PGE 1 (8%). The hydroxylation of prostaglandins in seminal vesicles of primates may thus show a high but not absolute specificity for the penultimate carbon of prostaglandins.

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: Formation of a novel metabolite by a female-specific P-450

Previous studies demonstrated that liver microsomes from untreated rats catalyze the ω, ω-1, and ω-2 hydroxylation of prostaglandins [ K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477–489 ]. The current study examined the regioselectivity of hydroxylation of PGE 1 and PGE 2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE 1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH- P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b 5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE 1, RLM 5 catalyzed solely the ω-1 hydroxylation and 5a catalyzed primarily the ω-1 and little ω and ω-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE 1 and PGE 2 at the ω-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is ω-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE 1 hydroxylation demonstrated that the affinity of f4 for PGE 1 is approximately 100-fold higher than that of RLM 5; the K m values for f4, monitoring 19- and 17-hydroxylation of PGE 1, were about 10 μ m. Surprisingly, cytochrome b 5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE 2 by RLM 5 was at the ω, ω-1, and ω-2 sites, demonstrating a lesser regioselectivity than with PGE 1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b 5 requirement.

ω-1 and ω-2 hydroxylation of prostaglandins by rabbit hepatic microsomal cytochrome P-450 isozyme 6

Incubation of prostaglandin E 1 (PGE 1) with liver microsomes from control rabbits and from rabbits treated with ethanol or imidazole yielded 18-, 19-, and 20-hydroxy metabolites, representing hydroxylation at ω-2, ω-1, and ω carbons, respectively. The current investigation demonstrates that rabbit liver P-450 isozyme 6 effectively catalyzes the ω-1 andg w-2 hydroxylation of PGE 1 and PGE 2. Additionally, a small amount of product with Chromatographic characteristics of the corresponding 20-hydroxy metabolite has been detected. The incorporation of cytochrome b 5 into the reconstituted system did not enhance the rate of PGE 1 hydroxylation and had no effect on the ratio of products formed. The K m value for the ω-1 and ω-2 hydroxylation of PGE 1 with P-450 isozyme 6 from imidazole-treated rabbits was approximately 140 μ m; the V max's (nmol product min −1 nmol P-450 −1) were 2.1 and 1.1 for the ω-1 and ω-2 hydroxylations, respectively. These rates represent the highest activities by hepatic P-450 isozymes for hydroxylation of PGs, and suggest that isozyme 6 is responsible for the ω-2 hydroxylation of PGEs observed in rabbit liver microsomes.

Regioselectivity of hydroxylation of prostaglandins by liver microsomes supported by NADPH versus H 2O 2 in methylcholanthrene-treated and control rats: Formation of novel prostaglandin metabolites

The effects of methylcholanthrene (MC) treatment of male rats on the regioselectivity of hydroxylation of prostaglandins E 1 and E 2 (PGE 1 and PGE 2) by liver microsomes, supplemented with NADPH or H 2O 2, was examined. In the presence of NADPH, control microsomes catalyzed the hydroxylation at ω-1 (C 19) and at ω-(C 20) sites with minimal formation of novel monohydroxy metabolites of PGE 1 and PGE 2, referred to as compounds X 1 and X 2, respectively. Similarly, H 2O 2 supported the 19-hydroxylation and the formation of compounds X 1 and X 2, but yielded only minimal amounts of 20-hydroxy products. With NADPH, MC-treated microsomal incubations demonstrated only minor quantitative change in the 19- and 20-hydroxylation as compared with controls, but showed a 7- to 11-fold increase in formation of compound X 1 and a 10-fold increase in formation of X 2. By contrast with H 2O 2, MC-treatment increased by about 3-fold the 19- and 20-hydroxylation of PGE 1 and by 35- to 46-fold the formation of X 1; similarly, there was an approximate 2-fold increase in 19- and 20-hydroxylation of PGE 2 and a 10-fold increase in formation of X 2. These findings suggest that several monooxygenases are involved in catalyzing the hydroxylation at the various sites of the PGE molecule. Inhibitors of monooxygenases (SKF 525A, α-naphthoflavone, and imidazole derivatives) provided further evidence that the hydroxylation at the three sites of PGEs is catalyzed by different P-450 monooxygenases. It is striking that the inhibitors had a much lesser effect on the 20-hydroxylation of PGE 1 as compared with other sites of hydroxylation. Structural identification of compounds X 1 and X 2 was elucidated as follows. Resistance of the PGB derivative of X 1 to periodate oxidation and mass fragmentation analysis of the t-butyldimethylsilyl ether methyl ester, placed the hydroxylation at C 17 or C 18. Finally, mass fragmentation of trimethylsilyl ether methyl ester PGB derivatives of X 1 and X 2 provided conclusive evidence that X 1 and X 2 are 18-hydroxy-PGE 1 and 18-hydroxy-PGE 2, respectively. The above findings indicate that the high regioselectivity of hydroxylation of PGE 1 and PGE 2, resulting in the formation of 18-hydroxy-PGE 1 and 18-hydroxy-PGE 2, respectively, is catalyzed by P-450 isozyme(s) which are induced by MC, possibly by P-450 c.

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5.

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.

Evidence for different hepatic microsomal monooxygenases catalyzing omega- and (omega-1)-hydroxylations of prostaglandins E1 and E2. Effects of inducers of monooxygenase on the kinetic constants of prostaglandin hydroxylation.

Evidence for different hepatic microsomal monooxygenases catalyzing omega- and (omega-1)-hydroxylations of prostaglandins E1 and E2. Effects of inducers of monooxygenase on the kinetic constants of prostaglandin hydroxylation.

Metabolism of prostaglandins and xenobiotics by adrenal microsomal monooxygenase in the guinea pig

The possibility that prostaglandins could serve as substrates for the guinea pig adrenal microsomal monooxygenase was investigated. The binding of PGE 1 to adrenal microsomes was found to exhibit a reverse type I spectral change. Also PGE 1 diminished the magnitude of type I spectrum elicited by cortisol binding to adrenal microsomes. The incubation of [ 3H]PGE 1 or of [ 3H]PGE 2 with adrenal microsomes supplemented with NADPH yielded primarily the respective 19-hydroxy metabolite. The enzymatic activity catalyzing this hydroxylation appears to be a typical monooxygenase, requiring NADPH for activity and being strongly inhibited by metyrapone, SKF 525A, and cytochrome c. Carbon monoxide at a ratio of 9:1 to oxygen moderately inhibited the hydroxylation of PGE 1. Whereas the liver catalyzed the hydroxylation of PGE 1 and PGA 1 equally well, the adrenal microsomes preferentially catalyzed the hydroxylation of PGE 1. This finding and the observation that α-naphthoflavone is a weak inhibitor of the adrenal PGE 1 hydroxylation points to significant differences between the adrenal and liver prostaglandin hydroxylation activities. Cortisol, which is a substrate for adrenal monooxygenase, strongly inhibited PGE 1 and PGE 2 hydroxylation. By contrast, certain xenobiotics (ethylmorphine, hexobarbital, benzpyrene), which are also metabolized by adrenal microsomes, only slightly inhibited the hydroxylation of PGE 1. Similarly, PGE 1 only weakly inhibited ethylmorphine and benzphetamine demethylation and hexobarbital hydroxylation. These observations suggest that adrenal microsomes contain several monooxygenases with different affinities for prostaglandins and for the different xenobiotic substrates.

Effect of inducers and inhibitors of monooxygenase on the hydroxylation of prostaglandins in the guinea pig. Evidence for several monooxygenases catalyzing omega- and omega-1-hydroxylation.

The incubation of prostaglandins (PG's) with liver microsomes from guinea pigs treated with inducers of monooxygenase (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzo[alpha]pyrene (benzpyrene), or a mixture of chlorinated biphenyls (Aroclor 1254)) exhibited marked elevation of 19-hydroxylation of PGE1, PGE2, PGA1, and PGA2 without affecting significantly 20-hydroxylation. However, with respect to effects on hydroxylation of a variety of xenobiotics, benzpyrene and Aroclor treatments differed markedly; whereas Aroclor treatment elevated the demethylation of ethylmorphine, benzphetamine, and p-chloro-N-methylaniline (PCMA), benzpyrene treatment had no effect on demethylation of ethylmorphine and only a marginal effect on that of PCMA. Both inducers elevated benzpyrene hydroxylation. By contrast, treatment with phenobarbital did not affect the hepatic microsomal PG's hydroxylation, although the hydroxylation of benzpyrene and the demethylation of ethylmorphine, benzphetamine, and PCMA were enhanced. Also, the hydroxylation of PG's by kidney cortex microsomes was not affected by either benzpyrene or Aroclor treatment. Inhibitors of monooxygenase were used to help delineate the type of monooxygenases induced. At low levels of alpha-naphthoflavone (ANF), benzpyrene hydroxylation in control- and Aroclor-treated guinea pigs was only little affected; by contrast, the same concentration of ANF markedly inhibited benzpyrene hydroxylation in benzpyrene-treated guinea pigs. On the other hand, metyrapone was most inhibitory in control guinea pigs. Support for the conclusion that benzpyrene induces in the guinea pig a hepatic monooxygenase with different characteristics than that found in control animals was provided by the observation that ANF (10 MICROM) inhibited PGE1 hydroxylation more pronouncedly in liver microsomes from benzpyrene-treated than from Aroclor-treated guinea pigs or controls. In addition, in benzpyrene and Aroclor-treated guinea pigs, ANF inhibited the (omega-1)-hydroxylation more pronouncedly than that of omega-hydroxylation. By contrast, metyrapone appeared to inhibit omega-hydroxylation more effectively than (omega-1)-hydroxylation. These results indicate that in the guinea pig, hydroxylation of PG's at the omega (20-) and omega-1 (19-) positions is catalyzed by different monooxygenases and that the inducers tested affect several hepatic monooxygenases with different specificities toward xenobiotics; however, with respect to PG's only the enzyme(s) involved in the 19-hydroxylation is affected.