Search PubMed⌕ Search

Biomedical subjects

R T Mayer

Publications and source records attributed to R T Mayer.

At least 37 records · Page 2Linked to original sources
PubMed1986

Major differences between lung, skin and liver in the microsomal metabolism of homologous series of resorufin and coumarin ethers.

Phenoxazone and a homologous series of its ethers (methoxy to octoxy plus benzyloxy), and coumarin and a series of its ethers (methoxy to propoxy), were metabolized by liver, lung and skin microsomes of normal adult female BALB/c mice. For each series of substrates, and with each tissue, clear structure-activity relationships were seen, relating metabolic activity to the length of the ether side-chain. With the coumarin series of substrates the structure-activity relationships were almost identical in the three tissues, with liver more active than lung and lung more active than skin. Liver, lung and skin microsomes each showed very different structure-activity relationships, however, for metabolism of the phenoxazone series of substrates. Benzyloxyphenoxazone was metabolized almost twice as fast in lung as in liver, but for the other phenoxazone substrates the activities were much greater in liver than in lung or skin. Liver, lung and skin microsomal propoxy- and benzyloxyphenoxazone dealkylase activities differed in their sensitivities to inhibition by metyrapone and alpha-naphthoflavone. The structure-activity relationship and inhibitor data for the phenoxazone substrates are consistent with a view that mouse lung and sking cyt. P-450 are predominantly similar to phenobarbitone-induced and 3-methylcholanthrene-induced forms of hepatic cyt. P-450 respectively. The results also show that the pattern of microsomal metabolism of xenobiotics in lung and skin cannot be reliably predicted from that in liver.

Animals↗
PubMed1986

Effects of chitin synthesis inhibitors on incorporation of nucleosides into DNA and RNA in a cell line from Manduca sexta (L).

Five putative chitin synthesis inhibitors (CSI) were tested to determine if they inhibited nucleoside incorporation into acid precipitable material in a cell line from Manduca sexta (L.). The results varied. Diflubenzuron (DFB) (100 micron) inhibited cytidine incorporation by 38%; EL-494 (100 micron) inhibited adenosine incorporation by 43%; Bay Sir 8514 (100 micron) inhibited uridine incorporation by 24%. Superdiflubenzuron (100 micron) was the worst inhibitor overall (18-22%) for the benzoylphenyl urea CSI. The triazine CSI, CGA 19255, was the best inhibitor tested with 60% inhibition for cytidine and 49% for adenosine incorporation into DNA and RNA. Examination of cells incubated with diflubenzuron by scanning electron microscopy revealed distinct external morphological changes. Transmission electron microscopy showed that crystalline structures accumulated in the cytoplasm of cells treated with DFB. The crystalline structures were assumed to be diflubenzuron and they persisted even after diflubenzuron was removed from the medium.

Animals↗
PubMed1986

Alkoxyresorufin O-dealkylases: association with the murine Ah locus.

The hepatic microsomal dealkylation of a series of alkoxyresorufins and the oxidation of phenoxazone to resorufin were investigated in C57BL/6 and DBA/2 mice of both sexes. In both strains of mice and in both sexes the dealkylation rate decreased with increasing length of the alkyl chain. With all alkoxyresorufins the dealkylation rates were higher in the C57BL mice than the DBA mice, whereas the rate of phenoxazone hydroxylation was higher in the latter. In the C57BL mice, and to a lesser extent in the DBA mice, females were more efficient in dealkylating the resorufin ethers. Treatment with 3-methylcholanthrene (3MC) enhanced the rates of dealkylation of all alkoxyresorufins in the C57BL mice but not in the DBA mice, the extent of stimulation being highest for the propoxy- and butoxyresorufins and least for pentoxy-, heptoxy- and benzyloxyresorufins. The same treatment had no effect on the oxidation of phenoxazone in either strain of mice. It is concluded that the dealkylation of alkoxyresorufins, not the oxidation of phenoxazone, is associated with the murine Ah locus.

Animals↗
PubMed1985

Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450.

The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were O-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of O-dealkylation to the length and type of the ether side-chain. Phenobarbitone (PB), 3-methylcholanthrene (MC), Aroclor 1254 (ARO), isosafrole (ISO) and SKF-525A each induced preferentially the O-dealkylation of different members of the homologous series, resulting in the appearance of 5 different SAR plots, which characterized and differentiated between the 5 different inducers. beta-Napthoflavone (BNF) had a similar effect to MC, whereas pregnenolone 16 alpha-carbonitrile treatment caused no large change in the metabolism of any of the substrates tested. For characterizing the effects of the different inducers it was largely sufficient to compare the O-dealkylations of just 4 of the ethers: methoxy-, ethoxy-, pentoxy- and benzyloxyphenoxazone. Very high degrees of induction were seen. MC and ARO each induced preferentially the O-dealkylation of ethoxyphenoxazone (51- and 61-fold respectively). PB and SKF-525A each induced preferentially the O-dealkylation of pentoxyphenoxazone (283- and 324-fold respectively). ISO induced preferentially the O-dealkylation of benzyloxyphenoxazone (43-fold). For any particular induced type of microsomes the substrate with the fastest metabolism was not necessarily the substrate whose metabolism was induced the most, so that in order to characterize each of the 5 different inducers (PB, MC/BNF, ARO, ISO, SKF) it was necessary to compare both the degrees of induction and the specific activities of the reactions. Experiments with purified cyt. P-450 isozymes showed that ethoxyphenoxazone and pentoxyphenoxazone were highly selective substrates for the major isozymes induced by MC and PB respectively, whilst benzyloxyphenoxazone was a good substrate for both isozymes. Experiments using the organic inhibitors metyrapone and alpha-naphthoflavone and inhibitory antibodies against individual cyt. P-450 isozymes indicated that similar substrate selectivities occurred with the monooxygenase system in the microsomal membrane. It is suggested that the use of some or all of these homologous phenoxazone ethers will provide both a simple routine test for the characterization of several types of inducing agents and a powerful tool for investigating the biochemical basis for cyt. P-450 isozyme substrate selectivity.

Animals↗
PubMed1985

Stereoselective and regioselective hydroxylation of warfarin and selective O-dealkylation of phenoxazone ethers in human placenta.

The oxidative metabolism of warfarin and a series of phenoxazone ethers was studied in two groups of human placentas which exhibited high or low levels of aryl hydrocarbon hydroxylase (AHH). Warfarin metabolism was stereoselective (mean R/S = 2.48) for the R-enantiomer and regioselective for the 6- and 8- positions in the high AHH group whereas warfarin metabolism in the low AHH group displayed no significant overall stereoselectivity (mean R/S = 1.24) and was regioselective for the 7- position. The high AHH group metabolized the methyl, ethyl, propyl and butyl ethers of phenoxazone rapidly, while the low AHH group catalyzed their biotransformation at very low or negligible rates. Neither group detectably metabolized phenoxazone or pentyloxyphenoxazone whereas both groups metabolized benzyloxyphenoxazone at low but similar rates. Rates of warfarin R-6 and R-8 hydroxylation were highly correlated with metabolism of benzo(alpha)pyrene (r = 0.99) and the C1-C4 phenoxazone ethers (r greater than 0.87), but poorly correlated with metabolism of benzyloxyphenoxazone (r less than 0.50). These data support the use of warfarin and the phenoxazone ethers as sensitive biochemical probes for P-450 isozymes in human extrahepatic tissues. They indicate the presence of a multiplicity of xenobiotic metabolizing P-450's in placental tissue which has not been exposed to inducing agents that elevate AHH.

Aryl Hydrocarbon Hydroxylases↗
PubMed1985

Dealkylation of pentoxyresorufin: a rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat.

The O-dealkylation of pentoxyresorufin (7-pentoxyphenoxazone) by rat liver microsomes was examined. The reaction appeared highly specific for certain phenobarbital inducible forms of cytochrome P-450 and was increased 95- to 140-fold by animal pretreatment with phenobarbital (75 mg/kg/day, four ip injections) and approximately 50-fold by Aroclor 1254 (500 mg/kg, one ip injection) while animal pretreatment with 3-methylcholanthrene (50 mg/kg/day, three ip injections) resulted in less than a 2-fold increase over the rate detected in control microsomes. It was observed that this activity, in microsomes for Aroclor-pretreated rats, was dependent on O2 and was inhibited by metyrapone and SKF 525-A, indicative of cytochrome(s) P-450 mediation in the reaction. When antibodies directed against purified cytochrome(s) P-450s were employed to inhibit the pentoxyresorufin O-dealkylation reaction, antibodies to P-450PB-B greatly inhibited the reaction (greater than 90%), while antibodies to P-450PB-C or P-450PB/PCN-E had minimal effects. Assay of hepatic microsomes from rats which were pretreated with varying doses of phenobarbital (0.9-75 mg/kg/day, four ip injections) indicated that while aminopyrine-N-demethylase activity was induced only 2-fold at the maximum dose (75 mg/kg/day), pentoxyresorufin O-dealkylase activity was induced approximately 140-fold at this dose and approximately 4-fold by a dose of phenobarbital as low as 0.9 mg/kg.

Aminopyrine N-Demethylase↗
PubMed1984

Inhibition of the uptake of nucleosides in cultured Harding-Passey melanoma cells by diflubenzuron.

Diflubenzuron (DFB) significantly inhibited the uptake of uridine, adenosine, and cytidine but not thymidine, in cultured Harding-Passey melanoma cells. Inhibition of nucleoside uptake was rapid (i.e., less than or equal to 5 min) and could not be reversed by washing. These results suggest that DFB may affect membrane properties and - as shown by in vivo tests - growth of melanoma cells.

Animals↗
PubMed1984

Effects of diflubenzuron on growth of malignant melanoma and skin carcinoma tumors in mice.

The insect growth regulator diflubenzuron (DFB), which may also inhibit growth of imaginal epidermal cells in insects, was studied for antitumor activity in two mouse tumor models of epidermal origin. DFB inhibits chitin deposition, but the mechanisms by which DFB controls chitin deposition or regulates growth of insect epidermal cells are unknown. A single injection of 20 mg (800 mg/kg) of DFB into C57BL/6 mice with B16 malignant melanomas or AKR mice with skin tumors (CA 1025) induced a rapid (24 h) decrease in tumor volume in 78% and 66% of the tumors, respectively. In contrast, 85% of the melanomas and 91% of skin tumors in control mice increased in volume during the same 24-h period. Tumor volume decreased by as much as 55% for about 1% of the tumors, but the median decrease was 20% for both types of tumors. Since control tumors concommitantly increased, DFB-treated tumors decreased, relatively, to 60% of the volume of matched control tumors. After the initial volume decrease, both types of tumors resumed exponential growth resulting in an average growth curve delay, calculated for 12-14 days, of about 2.0 days. Subsequent treatment of melanomas with DFB 24 h after the initial treatment resulted in a further decrease in relative tumor volume to 40-50% of control tumor volume and a growth curve delay of 2.6 days. The most effective regimen used was 5 daily, 20-mg doses of DFB. Melanomas decreased to 40% of control tumor volume after the third injection and the mean growth curve delay was extended to 4.3 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins).

The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed greatly in activity for the metabolism and binding of the various substrate homologues. The most rapidly metabolised substrates for control microsomes were phenoxazone and its shortest-chain ethers, for PB microsomes phenoxazone and the pentyl ether, and for 3MC microsomes the ethyl and propyl ethers. The variations in activity occurred in Vmax rather than in the apparent Km-value. All the ethers gave Type I cytochrome P-450-binding spectra. The substrates giving the largest Type I spectra were the same for all microsomes--the ethyl, propyl and butyl ethers--but the magnitudes of the spectra differed in the order 3MC- greater than PB- greater than control microsomes. Phenoxazone and resorufin gave Modified Type II cytochrome P-450-binding spectra. PB-induction was most marked for the depentylation reaction (increased 101-fold), whereas 3MC-induction was most marked for depropylation and debutylation (88- and 96-fold). The intermicrosomal differences were interpreted as reflecting the different metabolic specificities of variant forms of cytochrome P-450. Substrate lipophilicity increased with increasing ether chain length and was not a major influence on specificity. The main substrate influence on specificity was steric, due to the presence and length of the ether side chain. The preeminent effect of ether chain length was considered to be on the rate of substrate transformation rather than on substrate interaction with cytochrome P-450.

Animals↗
PubMed1982

Characterization of house fly microsomal mixed function oxidases: inhibition by juvenile hormone i and piperonyl butoxide.

The microsomal mixed function oxidase system of the house fly (Musca domestica [L.]) was characterized with respect to N-demethylation of p-chloromethylaniline, O-demethylation of methoxyresorufin, epoxidation of aldrin and the formation of a metabolite-cytochrome P-450 complex during oxidation of piperonyl butoxide (PB). The inhibition of these reactions by juvenile hormone I (E, E cis methyl 10,11-epoxy-7-ethyl-3, 11-dimethyl-2, 6-tridecadienoate, JH-I) was competitive for the N-demethylase, epoxidase and the formation of the PB metabolite-complex. Non-competitive inhibition was observed for the O-demethylase. The inhibition of these reactions by JH-I provides evidence that the mixed function oxidase system participates in the degradation of juvenile hormone, and that the juvenile hormone-like properties of PB and other methylenedioxyphenyl compounds are derived from their inhibition of this degradation. The PB metabolite-cytochrome P-450 complex has 2 absorbance maxima in the reduced form (427 and 455 nm), and a single absorbance maximum in the oxidized form (438 nm). pH affected the extinction of the 427 and 455 nm absorbance bands. The pH equilibrium point was 8.3 for the reduced PB metabolite complex, and 9.5 for ethylisocyanide. In addition, the PB metabolite complex could be generated in vivo.

Animals↗
PubMed1979

Preparation of homogenous NADPH cytochrome c (P-450) reductase from house flies using affinity chromatography techniques.

NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.

Animals↗
PubMed1979

Ecdysone 3-epimerase from the midgut of Manduca sexta (L.).

Ecdysone 3-epimerase was partially purified by ammonium sulfate fractionation from the 100,000 g supernate of Manduca sexta midguts. The enzyme converts ecdysone and 20-hydroxyecdysone to their respective 3-epimers, requires NADH or NADPH and O2 for this reaction, and has the following kinetic parameters: for ecdysone, Km = 17.0 +/- 1.4 microM, Vmax = 110.6 +/- 14.6 pmol min-1 mg-1; for 20-hydroxyecdysone, Km = 47.3 +/- 7.5 microM, Vmax = 131.0 +/- 3.5 pmol min-1 mg-1: for NADPH, Km = 85.4 +/- 10.6 microM; for NADH, Km = 51.3 +/- 1.3 microM. The reaction is irreversible and can be inhibited by various ecdysteroids.

Animals↗
PubMed1978

Thin-layer chromatographic in situ analysis of insect ecdysones via fluorescence-quenching.

The possibility of quantitating insect ecdysones in situ on thin-layer chromatographic plates was examined. Two approaches were evaluated: 1) the induction of ecdysone fluorescence by sulfuric acid treatments and 2) the fluorescence-quenching of fluorescent thin-layer chromatographic plates by ecdysones. The fluorescence-quenching method was found to be most suitable and had a linear response range from 0.5 to 3 microgram for alpha-ecdysone and 20-hydroxyecdysone. Fluorescence-quenching and high pressure liquid chromatographic analyses obtained from extracts of alpha-ecdysone 20-hydroxylase incubations gave similar results. New data concerning the acid-induced fluorescence of ecdysones showed alpha-ecdysone to be twice as fluorescent as 20-hydroxyecdysone.

Animals↗
PubMed1978

Ecdysone 20-hydroxylase in midgut mitochondria of Manduca sexta (L.).

The conversion of alpha-ecdysone to 20-hydroxyecdysone in the midgut of Manduca sexta (L.) was found to be catalyzed by a mitochondrial, cytochrome P450-mediated monooxygenase. The reaction required oxygen and was inhibited by the presence of carbon monoxide. Tricarboxylic acid cycle intermediates, such as succinate, malate, and isocitrate, supported the hydroxylation as did NADPH, NADH, ATP, and ADP. Temperature and pH optima were 30 degrees C and 8.5, respectively. The apparent Km and V values for the ecdysone 20-hydroxylase were 18.3 +/- 6.8 micronM and 46.6 +/- 14.2 pmol per min per mg protein. The midgut mitochondria were found to contain malate dehydrogenase and NAD(P) transhydrogenase. The presence of these enzymes suggests that the tricarboxylic acid cycle intermediates and NADH support the ecdysone hydroxylation indirectly by providing NADPH for the cytochrome P450 system. The content of cytochromes a + a3, b, c + c1, and P450 in midgut mitochondria was determined.

Animals↗