Search

The effect of intratracheal administration of fly ash, its benzene-extracted residue and the benzene extract has been studied on the activities of hepatic mixed-function oxidases in the rat. Fly ash and its fractions significantly increased the levels of cytochrome P-450, cytochrome b5, cytochrome b5 reductase, NADPH-cytochrome c reductase, aminopyrine N-demethylase, aniline hydroxylase, and glutathione S-transferase in a dose-dependent manner. Phenobarbital or 3-methylcholanthrene treatment along with the administration of fly ash or its fractions showed an additive effect on the activities of the mixed-function oxidases. The observed effects were due to chemical component, i.e., organic and inorganic fractions of fly ash, and not due to its particulate nature. This was shown by the administration of glass beads which did not cause any alteration in the activities of hepatic mixed-function oxidases.

Effect of depot medroxyprogesterone acetate on the hepatic drug-metabolizing enzymes was studied in female protein-deficient and normal pair-fed rats. Treatment with this drug did not cause any change in organ weight, microsomal protein, and soluble protein yield per gram of tissue in both groups. MPA administration resulted in significant increases in the content of cytochrome P-450 and b5, and activities of benzo[a]pyrene hydroxylase, UDP-glucuronosyltransferase, and NADPH-Cyt c reductase in both pair-fed control and protein-deficient rats. However, the content of glutathione and activity of glutathione-S-transferase were not affected appreciably. The present study suggests that MPA treatment induces drug-metabolizing enzymes in liver to almost the same extent in both protein-deficient and normal pair-fed rats.

A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones. This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection. Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme. Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2'5'-ADP agarose chromatography. Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E. coli proteins. The final preparation exhibited considerable proteolytic degradation and only an estimated 5-10% of the immunoreactive protein was undegraded. Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase. High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000. Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 mumol of cytochrome c reduced/mg reductase/min at 22 degrees C). HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver. When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo[a]pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.

The effect of M7310U, a new non-steroidal analgesic anti-inflammatory agent, on liver microsomal drug-metabolizing enzymes was investigated. Rats were treated orally with M73101 (100, 200, 500 mg/kg), henylbutazone (PZ, 200 mg/kg), aminopyrine (AM, 100 mg/kg) or phenobarbital sodium (PB, 100 mg/kg) once daily for 2 weeks and then were observed for 2 weeks during which treatment was not given. On treatment with M73101, PZ, AM and PB, the liver enlarged but was restored to normal 1 week after the last administration. The rate of increase in the case of M73101 was lower than that seen with the reference compounds. M73101 markedly increased the content of microsomal protein, cytochrome P-450 or b5 and NADPH cytochrome C reductase, aniline hydroxylase and AM demethylase activity, but these increments returned to the normal level 1 week after the last administration. The serum concentration of M73101 after repeated administration (200 mg/kg, p.o.) for 1 week was lower than that after a single administration. Furthermore, M73101 increased Vmax for both aniline hydroxylase and AM demethylase, whereas it increased Km only for aniline hydroxylase. M73101 did not enhance the lipid peroxidation. Our observations suggest that the enlargement of rat liver seen with M73101 was due to the induction of drug-metabolizing enzymes and that this agent can probably be classified as a phenobarbital-type inducer.

The NADPH-cytochrome c (P-450) reductase induced in the filamentous fungus Rhizopus nigricans as a component of 11 alpha-hydroxylase of progesterone was resolved by DEAE-cellulose chromatography into two components. One of the components is an iron-sulfur protein (rhizoporedoxin), whereas the other component is a protein with reductase activity dependent on NADPH (rhizoporedoxin reductase). As shown in the reconstitution assay, the NADPH-cytochrome c (P-450) reductase activity was restored upon combination of these two proteins.

The potentiation of the expression of polycyclic aromatic hydrocarbon-inducible cytochromes P450IA1 and P450IA2, and phenobarbital-inducible cytochrome P450IIB1 and NADPH-cytochrome P-450 reductase by dexamethasone in vivo was investigated using adrenalectomized and sham-operated rats. Hepatic cytochrome P450IA1 activity (determined by ethoxyresorufin O-deethylase activity) was induced maximally (30-fold) in both sham-operated and adrenalectomized rats 24 hr after a single injection of 3-methylcholanthrene (10 mg/kg). Dexamethasone (10 mg/kg) increased hepatic tyrosine aminotransferase activity 3- to 4-fold, but had little or no effect on ethoxyresorufin O-deethylase activity. However, dexamethasone potentiated the 3-methylcholanthrene-dependent induction of hepatic P450IA1 activity 2- to 3-fold in adrenalectomized rats and 1.5- to 2-fold in sham-operated rats when administered concomitantly with 3-methylcholanthrene (P less than .05). The dose of dexamethasone required to potentiate 3-methylcholanthrene induction of P450IA1 activity (greater than 1 mg/kg) correlated well with the dose required to induce hepatic tyrosine aminotransferase activity; a marker of glucocorticoid action. Potentiation of 3-methylcholanthrene induction of P450IA1 activity in the rat appears to be tissue specific in that dexamethasone was found not to potentiate 3-methylcholanthrene induction of P450IA1 activity of rat lung or kidney. Cytochrome P450IA1 content induced by 3-methylcholanthrene in the liver was also potentiated 2- to 3-fold by dexamethasone. In contrast, potentiation of 3-methylcholanthrene induction of cytochrome P450IA2 content was not observed. Western blot analyses indicate that the increase in monooxygenase activity induced by 3-methylcholanthrene or 3-methylcholanthrene plus dexamethasone corresponds well only with the induction of cytochrome P450IA1 protein content.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the cnxH+ gene specified polypeptide in the formation and function of the NADPH-nitrate reductase in Aspergillus nidulans was examined with the use of two complementing mutant strains which were grown as forced heterocaryons in the presence of nitrate. The niaD-421 structural gene mutant and the cnxH-318 co-factor gene mutant produce two components of the NADPH-cytochrome c reductase co-activity which can be distinguished by their enzymatic and physical behavior. This combination enabled us to isolate the de novo synthesis of niaD+ gene specified protomers from the constitutively formed co-factor at two stages of development. The proportion of induced and constitutively formed protomers in the isolated holoenzyme was measured after pulsing with [3H]-histidine or [14C]-histidine prior to induction with nitrate. The newly formed nitrate reductase was resolved by agarose gel electrofocusing and activity staining. In vivo assembly of a 7.8s enzyme in the heterocaryotic mycelium of the above strains is apparently achieved by the convener action of the cnxH+ gene directed polypeptide from the niaD- strain on the niaD+ gene directed protomers of the cnxH- partner. This occurs with or without Mo as a co-factor.

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.

The effects of a new fluorinated macrolide (P-0501A) on drug metabolizing enzymes of rat liver were compared with three erythromycins--the base, the stearate and the estolate--after 7 days of dosing (1.36 mmol/kg po daily). The three erythromycins induced the synthesis of microsomal enzymes, but the products of their metabolism inactivated cytochrome P-450 in the order base less than or equal to stearate less than estolate. N-Demethylation of erythromycin and aminopyrine increased, while O-demethylation of 4-nitroanisole was reduced and hydroxylation of aniline was not changed after in vivo treatment. Pentobarbital sleeping time was prolonged and liver glutathione levels were lower in treated rats than in controls. In contrast to the three erythromycins, P-0501A did not induce the synthesis of microsomal enzymes, did not form an inactive complex with cytochrome P-450 and did not affect mono-oxygenase activities or pentobarbital narcosis.

Membrane-bound ribosomes were separated into two distinct classes (loosely-bound and tightly-bound ribosomes) by treatment with 0.6 M KCl, 1 mM puromycin, 0.05% DOC, or 10 mM EDTA. It was also confirmed that any one of these reagents except for EDTA dissociated the same class of ribosomes from the membrane. A population of lighter microsomal vesicles was formed from rough microsomes upon the dissociation of loosely-bound ribosomes by treatment with these chemicals. Rough microsomes were subfractionated into lighter and heavier fractions, L-rMs and H-rMs, by centrifugation using a discontinuous gradient of sucrose consisting of 1.3 M, 1.5 M, and 2.1 M solutions. It was found that L-rMs was rich in loosely-bound ribosomes, whereas H-rMs contained a high proportion of tightly-bound ribosomes. It is likely that loosely-bound and tightly-bound ribosomes are heterogeneously distributed among rough microsomal vesicles. Loosely-bound ribosomes and tightly-bound ribosomes synthesize different kinds of proteins. Two microsomal membrane proteins, NADPH-cytochrome c reductase and cytochrome b5, were exclusively synthesized by loosely-bound ribosomes, whereas serum albumin, which is a major component of the secretory proteins of hepatocytes, was synthesized only by tightly-bound ribosomes. Since the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 are released from bound ribosomes to the cytoplasmic surface of endoplasmic reticulum, while those of secretory proteins are discharged into the lumen across the membrane, the strength of the association between ribosomes and microsomal membrane seems to be correlated with the direction of release of nascent peptides.

The activities of the following enzymes in soybean roots were determined at early times after infection of the roots with zoospores of an incompatible or a compatible race of Phytophthora megasperma f.sp. glycinea: dimethylallyl-diphosphate : 3,6a,9-trihydroxypterocarpan dimethylallyltransferase (prenyltransferase), an enzyme specific for glyceollin biosynthesis; NADPH-cytochrome reductase and hydroxymethylglutaryl-CoA reductase, enzymes related to the glyceollin pathway; and isocitrate dehydrogenase. Already at 4 h after infection there was a higher activity of the prenyltransferase in the incompatible interaction than in the compatible interaction, and enzyme activity in the incompatible interaction increased considerably between 4 and 8 h after infection. In the compatible interaction prenyltransferase activity was only slightly higher than in uninfected roots. The activity of the other enzymes in infected roots was not significantly different from that in the uninfected roots. No qualitative differences could be detected between the two-dimensional patterns of unlabelled proteins or proteins labelled with L-[35S]methionine of infected and uninfected roots at early times after infection. We conclude from these and earlier results (A. Bonhoff et al. (1986) Arch. Biochem. Biophys. 246, 149-154) that infection of the soybean roots with an incompatible race of the fungus leads to selective induction of the phytoalexin pathway and presumably to induction of other as yet unknown defense mechanisms.

Alteration in content of enzymatic components was studied in microsomal oxygenases system of rats administered with 2-acetyl aminofluorene. Content of cytochrome R-450 was decreased beginning from the fourth week of the experiment. Activity of NADP cytochrome c reductase was higher in animals, treated with the drug as compared with controls. Inductors of the I and II type were capable to induce the enzymatic system up to the end of the experiment - within 16 weeks as far as liver tumors developed.

Nine nitroimidazo(2,1-b)thiazoles were tested for their ability to induce convertants, revertants and mitotic recombinants on growing and stationary phase cells of Saccharomyces cerevisiae D7 strain. All compounds were genetically active only on cells from the logarithmic growth phase (from a 20% glucose medium), inducing dose-related increases in conversion and reversion frequencies. The addition of S9 microsomal fraction to stationary phase cells gave positive results at levels of doses 10-100 times lower than that used with growing cells. NADPH cytochrome c (P-450) reductase activity was determined in order to compare the capability of hepatic and yeast microsomes in bioactivating the test compounds. Results showed that nitroreductive enzymes are inducible by conditions supporting growth. The greater sensitivity of the treatment with external metabolic activation could be due to the greater content (about 14-fold) of NADPH cytochrome c-reductase activity in hepatic microsomes.

Changes in body weight gain and in biochemical parameters of blood and liver were assessed in Sprague-Dawley rats after multiple oral administration of three test doses of an Alberta crude oil (ACO). Rats treated with ACO (1.25-5 ml/kg) did not show statistically significant (p greater than .05) differences from control, corn-oil treated (5 ml/kg) rats, in body weight gains, liver weight, and blood biochemical indicators of liver (alanine aminotransferase, gamma glutamyltransferase), kidney (blood urea nitrogen, creatinine), and erythrocyte (adenosine 5'-triphosphate, 2,3-diphosphoglyceric acid, reduced glutathione) cytotoxicity. Treatment with ACO, however, caused statistically significant (p less than .05) and dose-related increases from control in (1) microsomal protein and cytochrome P-450 content, and NADPH-cytochrome c reductase, aryl hydrocarbon hydroxylase (AHH), and 7-ethoxycoumarin-O-deethylase (7-ECOD) activities, and (2) cytosolic glutathione transferase activity of liver. The induction of hepatic cytochrome P-450 and xenobiotic-metabolizing enzymes in microsomes of ACO-treated rats was probably associated with dose-related changes in isozymic forms of cytochrome P-450, as evidenced by (1) appearance of a 448-nm spectral peak in microsomes of ACO-treated rats and (2) differences in the inhibition pattern of AHH and 7-ECOD activities in microsomes of control and ACO-treated rats upon treatment with metyrapone and 7,8-benzoflavone.

Male Sprague-Dawley rats were injected ip with benzene, toluene, or a mixture of xylene isomers at 20 mmol hydrocarbon/kg daily for 3 days. The effects of administration of these hydrocarbons upon their own in vitro metabolism, as well as upon cytochrome P-450, NADPH-cytochrome c reductase, aminopyrine N-demethylase, aniline hydroxylase, glutathione, glutathione S-transferase, and UDPglucuronyltransferase in liver were studied. Each hydrocarbon studied increased its own in vitro metabolism. Benzene had no effect on the metabolism of toluene or xylenes. Toluene and xylenes increased the metabolism of benzene, toluene, and xylenes. Cytochrome P-450 was elevated by toluene and xylenes, but was not affected by benzene. NADPH-cytochrome c reductase was induced by all three hydrocarbons. Aminopyrine N-demethylase and aniline hydroxylase were induced by toluene and xylenes and were not affected by benzene. Glutathione was elevated by benzene, decreased by xylenes, and not affected by toluene. Glutathione S-transferase was induced differentially by these hydrocarbons toward various substrates: toward 1-chloro-2,4-dinitrobenzene by benzene and toluene, toward 1,2-dichloro-4-nitrobenzene by benzene and xylenes, and no effect toward 1,2-epoxy-3-(p-nitrophenoxy)propane by any hydrocarbons. UDPglucuronyltransferase was induced by benzene and toluene when o-aminophenol and phenol were used as the substrate. Xylenes had no effect. Benzene was more effective at inducing conjugation enzymes. Xylenes were more effective at inducing cytochrome P-450 dependent enzymes. Toluene was equipotent at inducing both types of enzymes. The results indicate that the addition of methyl groups to the aromatic ring affects the inductive pattern of these monocyclic aromatic hydrocarbons.

Our previous studies have shown that 2,2-dimethyl-5-t-butyl-1,3-benzodioxole (DBBD), a methylenedioxyphenyl (MDP) analog in which the methylene hydrogens have been replaced by methyl groups, does not form an inhibitory complex with cytochrome P-450 nor induce this cytochrome. However, in the present experiments, DBBD-treated male Dub:ICR mice showed an increase in NADPH-dependent cytochrome c (P-450) reductase and epoxide hydrolase activity. This separation of cytochrome P-450 induction from the induction of epoxide hydrolase and NADPH-dependent cytochrome c (P-450) reductase appears to be unique among inducers of xenobiotic metabolizing enzymes. In similar experiments, mice were treated with phenobarbital + DBBD or 3-methylcholanthrene + DBBD and the following parameters were measured: cytochrome P-450 content; NADPH-dependent reduction of cytochrome c; ethylmorphine and benzphetamine N-demethylase; 7-ethoxycoumarin O-deethylase; benzo[a]pyrene hydroxylase; and ethoxyresorufin O-deethylase. The microsomal proteins were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Phenobarbital + DBBD treatment gave results which did not differ significantly from those obtained with phenobarbital alone. In contrast, cytochrome P-450 content and benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase activities were less in mice treated with 3-methylcholanthrene + DBBD than in animals treated with 3-methylcholanthrene alone. SDS-PAGE confirmed that induction of cytochrome P-450 by 3-methylcholanthrene was reduced by DBBD, suggesting that the latter compound may be an antagonist to the Ah cytosolic receptor.

The effects of saikosaponin-d extracted from the roots of Buplerum falcatum L. on increased toxicity of CCl4 and increased activities of microsomal enzymes induced by phenobarbitone have been examined. Saikosaponin-d showed protection against the CCl4-hepatotoxicity enhanced by phenobarbitone. It also inhibited increases in the content of cytochrome P450 and NADPH-cytochrome c reductase activity, which are induced by the phenobarbitone treatment, but the spectral characteristics of P450 were not altered. The rate of microsomal lipid peroxidation by NADPH and CCl4 was significantly lowered in-vitro in rats pretreated with phenobarbitone and saikosaponin-d compared with those pretreated with phenobarbitone alone.

Several reports have described the effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system (MFOS). These studies suggest that repeated TCE administration induces MFOS, especially cytochrome P-450 and NADPH-cytochrome c reductase. However, it is uncertain what isozymes are induced by TCE treatment, and it is not clear how microsomal enzymes or cytochrome P-450 isozymes are altered when TCE is administered for a duration longer than 28 days. We investigated the changes of MFOS by long-term TCE treatment. Male Wistar rats were injected with TCE, 1.0 g/kg body weight once a day for 5 continuous days or 2.0 g/kg body weight twice a week for 15 days. The mean body weight of the rats treated with TCE for 15 weeks was slightly, but not significantly, less than that of the control rats. Relative liver weights (liver wt/body wt) of the TCE-treated group were however significantly larger (21%) than those of the control group. The weights of the other organs were not changed by long-term TCE treatment. Trichloroethylene treatments for 5 days and 15 weeks caused significant increases in microsomal protein, cytochrome P-450, cytochrome b-5 and NADPH-cytochrome c reductase. TCE treatments produced an increase in a polypeptide band at 52,000 molecular weight range observed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increase in similar to, but less pronounced than that induced by phenobarbital (PB) treatment. There were no remarkable changes at 56,000 molecular weight range where a band appeared after the treatment with 3-methylcholanthrene (MC). It is likely that the induction of cytochrome P-450 by TCE is relatively similar to that by PB.