Biomedical subjects

D E Ryan

Publications and source records attributed to D E Ryan.

At least 37 records · Page 2Linked to original sources

PubMed1988

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b.

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.

Androstenedione↗

PubMed1988

Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g.

Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6 beta-hydroxyprogesterone and 16 alpha-hydroxyprogesterone), two secondary metabolites (6 beta,16 alpha-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16 alpha-hydroxyprogesterone). The Km,app for the formation of these products from progesterone was determined to be approximately 0.5 microM, while the Km,app for metabolism of 6 beta- and 16 alpha-hydroxyprogesterone was found to be 5-10 microM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 microM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6 beta,16 alpha-dihydroxyprogesterone and 6-keto-16 alpha-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6 beta-hydroxyprogesterone or 16 alpha-hydroxyprogesterone. Incubation of 6 beta-hydroxyprogesterone with a reconstituted system in an atmosphere of 18O2 resulted in greater than 90% incorporation of 18O in the 16 alpha-position of 6 beta,16 alpha-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.

Animals↗

PubMed1988

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase.

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.

Animals↗

PubMed1988

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats.

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.

Animals↗

PubMed1988

Hepatic cytochrome P-450j induction in the spontaneously diabetic BB rat.

Hepatic microsomal cytochrome P-450j has been studied using the male spontaneously diabetic BB rat as a model for insulin-dependent diabetes. This approach avoids any direct hepatotoxic effects from chemical diabetogenic agents. Both diabetic rats maintained on insulin and nondiabetic littermates were used as controls. Levels of cytochrome P-450j were increased approximately 3-fold in the diabetics 4 days after the cessation of insulin therapy. In addition, cytochrome P-450j-catalyzed enzymatic activities, aniline hydroxylation, and N-nitrosodimethylamine N-demethylation were increased by the diabetic state at this same time period. Cytochrome P-450f remained at control levels in all groups of animals. In order to test the hypothesis that ketone bodies are involved in the increase in cytochrome P-450j in the diabetic state, plasma beta-hydroxybutyrate levels were monitored. Hepatic aniline hydroxylation, N-nitrosodimethylamine N-demethylation, and cytochrome P-450j levels in individual animals were found to correlate with plasma beta-hydroxybutyrate levels (r = 0.59-0.71 p less than 0.001). In contrast, no significant correlation between levels of cytochrome P-450j and plasma glucose, insulin, or cholesterol was observed in individual animals (r = 0.07-0.23, p greater than 0.4). We conclude that cytochrome P-450j is induced in the livers of spontaneously diabetic rats, and that this induction may be associated directly or indirectly with elevated plasma ketone levels.

3-Hydroxybutyric Acid↗

PubMed1987

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes.

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.

Aging↗

PubMed1987

Isozyme specificity of testosterone 7 alpha-hydroxylation in rat hepatic microsomes: is cytochrome P-450a the sole catalyst?

In the present study we show that monospecific antibody against cytochrome P-450a completely inhibits testosterone 7 alpha-hydroxylation in hepatic microsomes of untreated male or female rats or rats of either sex treated with dexamethasone. These data are in contrast with those of K. Nagata et al. (1987, J. Biol. Chem. 262, 2787-2793) who recently reported that an antibody prepared against cytochrome P-450a completely inhibited testosterone 7 alpha-hydroxylase activity in microsomes from untreated or 3-methylcholanthrene-treated rats but only inhibited 50% of the activity in microsomes from dexamethasone-treated rats. They proposed that dexamethasone treatment of rats induced another testosterone 7 alpha-hydroxylase in rat liver. The discrepancy in the two sets of data was due, at least in part, to the use of a chromatography system by Nagata et al. that is incapable of resolving a number of testosterone metabolites. Dexamethasone treatment of rats leads to a marked increase in the production of several testosterone metabolites, including 15 beta-hydroxytestosterone which is cochromatographic with 7 alpha-hydroxytestosterone in their chromatography system. Our results indicate that cytochrome P-450a accounts for all of the testosterone 7 alpha-hydroxylase activity in microsomes from dexamethasone-treated rats, and that testosterone 7 alpha-hydroxylation continues to be a useful marker for monitoring cytochrome P-450a in rat hepatic microsomes.

Animals↗

PubMed1987

A simple, non-chromatographic purification procedure for monoclonal antibodies. Isolation of monoclonal antibodies against cytochrome P450 isozymes.

A simple, non-chromatographic purification procedure for monoclonal antibodies from mouse ascites fluid is described. This procedure, which is rapid, inexpensive, and has high capacity involves the precipitation of contaminating proteins with caprylic acid followed by precipitation of immunoglobulin using ammonium sulfate. This two-step procedure is shown to be effective for the purification of various immunoglobulins including IgG1, IgG2a and IgG2b. In the present report, more than 30 monoclonal antibodies directed against cytochrome P450 isozymes have been purified by this method and characterized.

Animals↗

PubMed1987

Stereoselective formation of benzo(c)phenanthrene (+)-(3S,4R) and (+)-(5S,6R)-oxides by cytochrome P450c in a highly purified and reconstituted system.

The principal oxidative metabolites formed from benzo(c)phenanthrene (B(c)Ph) by the cytochromes P450 in liver microsomes from control and treated rats are the 3,4- and 5,6-arene oxides. A procedure is described which allows determination of the enantiomer composition and absolute configuration of these arene oxides based on HPLC separation of isomeric thiolate adducts formed with N-acetyl-L-cysteine in base. Incubation of [3H]-B(c)Ph with highly purified cytochrome P450c in a reconstituted monooxygenase system followed by trapping of the metabolically formed arene oxides as above indicated that the 3,4-oxide was predominantly the (+)-(3S,4R)-enantiomer (90%) and that the 5,6-oxide consisted mainly of the (+)-(5S,6R)-enantiomer (76%). The results are discussed in terms of their implications about the catalytic binding site of cytochrome P450c.

Animals↗

PubMed1987

Regulation of cytochrome P-450j, a high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes.

Polyclonal antibodies were produced in rabbits against purified cytochrome P-450j isolated from isoniazid-treated adult male rats. The monospecificity of immunoadsorbed antibody to cytochrome P-450j was demonstrated by Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays, and immunoblots. Immunoquantitation results indicated that rat liver microsomal cytochrome P-450j content decreases between 3 and 6 weeks of age in both the male and female animal. Several xenobiotics, such as Aroclor 1254, mirex, and 3-methylcholanthrene, repressed cytochrome P-450j levels when administered to male rats. Isoniazid, dimethyl sulfoxide, pyrazole, 4-methylpyrazole, and ethanol were inducers of cytochrome P-450j in rat liver although these compounds showed different inducing potencies. Microsomes from adult male rats with chemically induced diabetes also contained elevated levels of cytochrome P-450j compared to untreated animals. Cytochrome P-450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries, and testes; however, extrahepatic cytochrome P-450j was inducible by isoniazid. Approximately 80-90% of microsomal N-nitrosodimethylamine demethylation was inhibited by antibody to cytochrome P-450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of cytochrome P-450j. The residual catalytic activity resistant to antibody inhibition may be a reflection of the inaccessibility of a certain amount of cytochrome P-450j due to interference by NADPH-cytochrome P-450 reductase based on results obtained with the reconstituted system. There was a good correlation (r2 = 0.87) between cytochrome P-450j content and N-nitrosodimethylamine demethylase activity in microsomes from rats of different ages and treated with various xenobiotics. The evidence presented indicates that cytochrome P-450j is the primary, and perhaps sole, microsomal catalyst of N-nitrosodimethylamine demethylation at substrate concentrations relevant to hepatocarcinogenesis induced by N-nitrosodimethylamine.

Animals↗

PubMed1987

Tissue biocompatibility of kevlar aramid fibers and polymethylmethacrylate, composites in rabbits.

Two groups of female NZW rabbits were implanted in the paravertebral muscles with aramid (du Pont Kevlar aramid 49) fibers and aramid-polymethylmethacrylate (PMMA) composites for 14 and 28 days. Rabbits were killed at these times periods, necropsies performed, sites scored for gross tissue response, and tissue specimens containing the implants removed for histopathological evaluation. A mild fibrous tissue reaction was observed around all implants containing aramid fiber similar to that observed around the silicone control implant. Some foreign body giant cells were also present adjacent to the fibers. An intense necrotic inflammatory reaction was present around the positive control material (PVC Y-78). The tissue response to implantation of aramid fiber and fiber-PMMA composites indicates that aramid is a biocompatible material.

Acrylic Resins↗

PubMed1987

A regulatory polymorphism for rat hepatic cytochrome P-450g.

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cis-acting.

Aging↗

PubMed1987

Purification and characterization of ethanol-inducible human hepatic cytochrome P-450HLj.

Human hepatic cytochrome P-450HLj was purified in a catalytically active state from microsomes obtained from an ethanol-intoxicated man. The electrophoretically homogeneous preparation of HLj was compared to rat P-450j and found to have a slightly different apparent molecular mass (54 vs 51.5 kDa) but highly similar immunochemical, spectral, and catalytic properties. Purified HLj exhibited high activity toward N-nitro-sodimethylamine (NDMA) and aniline metabolism, low but measurable activity toward benzphetamine and 7-ethoxycoumarin, and no detectable activity toward benzo[a]pyrene, testosterone, and progesterone. Antibody against rat P-450j reacted with HLj in immunoblot analyses and, when added directly to HLj before reconstitution with NADPH-cytochrome P-450 reductase and lipid, the antibody inhibited (96%) NDMA metabolism by HLj almost completely. However, if HLj was reconstituted with the other components before the addition of the anti-P-450j IgG, the ability of the antibody to inhibit the metabolism of NDMA was greatly diminished. This suggests that the interactions between reductase and HLj are similar to those previously observed between rat P-450j and reductase, and appear to prevent the complete access of anti-P-450j. The addition of cytochrome b5 to reconstitution systems containing HLj resulted in a small increase in the Vmax from NDMA demethylation accompanied by a decrease in Km,app (1.3 to 0.3 mM) as has been observed in reconstitution systems with rat P-450j. Therefore, in reconstituted systems, cytochrome b5 appears to play an important role in the biotransformations mediated by HLj and P-450j. In conclusion, this study demonstrates that HLj is functionally related to ethanol-inducible rat P-450j and rabbit LM3a.

Amino Acid Sequence↗

PubMed1987

Failed temporomandibular joint prostheses: MR imaging.

Magnetic resonance (MR) imaging with surface coils was performed on 43 temporomandibular joints with disk prostheses in 28 patients who had pain, dysfunction, or both after implantation of the prostheses. Fracture was diagnosed prospectively as a discontinuity of the prosthesis on the MR examination; the sensitivity and specificity of MR imaging for this diagnosis was determined on the basis of these studies. All patients underwent surgery, after which the final diagnosis was made. MR imaging was found to have a sensitivity of 0.81 and a specificity of 0.91. Other MR findings included an abnormal amount of soft tissue surrounding the disk, thinning of the disk, osteophytes, and erosion of the condyle. An as yet unexplained artifact was frequently seen and obscured the prostheses in four of the six false-negative studies. The one false-positive case resulted from the interpretation of a markedly thinned but intact region of the prosthesis as a fracture.

Female↗

PubMed1987

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes.

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.

Animals↗

PubMed1987

Comparison of high-resolution MRI and SPECT bone scintigraphy for noninvasive imaging of the temporomandibular joint.

Prospective evaluation by magnetic resonance imaging (MRI) and both single photon emission computed tomography (SPECT) and planar bone scintigraphy was undertaken in 31 temporomandibular joints (TMJs) of 21 symptomatic patients. When compared with the results of subsequent arthrography, MRI (0.88) was more sensitive than SPECT (0.76) or planar (0.56) scintigraphy for detection of internal derangement of the TMJ. A diagnostic sensitivity of 0.96 was achieved when the results of either MRI or SPECT was considered evidence of internal joint derangement. Five symptomatic TMJs, clinically thought to be abnormal, were positive on SPECT but showed no evidence of anterior disk displacement at the time of arthrography. In such instances, SPECT may be detecting functionally significant altered joint mechanics that are not evident on anatomic imaging of the TMJ.

Adolescent↗

PubMed1987

Antimony accumulation in hair during treatment of leishmaniasis.

We used instrumental neutron activation analysis (INAA) to determine antimony in small quantities of hair from individuals living in an area of endemic leishmaniasis. The groups studied were 12 patients undergoing treatment with N-methylglucamine antimoniate, 34 patients who had received such treatment a year ago, and 17 control subjects (untreated) who were living in the same area. Patients receiving treatment showed mean and median (12.13, 2.9 micrograms/g) values for antimony in hair that significantly exceeded those in either controls (1.01, 0.4 microgram/g) or patients one year after treatment (1.54, 1.0 microgram/g). We conclude that INAA can be useful in monitoring and differentiating such exposure to antimony in patients under treatment.

Antimony↗

PubMed1986

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone.

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.

Acetophenones↗