Metabolism Of Benzphetamine Research Articles

The enhancement of cytochrome P-450 catalyzed methoxyflurane metabolism by a heat-stable microsomal protein

We report the existence of a microsomal, heat-stable, trypsin-sensitive factor that stimulates the O-demethylation of methoxyflurane (CHCl 2CF 2OCH 3) by partially purified preparations of rabbit hepatic cytochrome P-450. The factor is able to stimulate by five to twelve-fold the methoxyflurane metabolizing activity of cytochrome P-450. In contrast, the metabolism of benzphetamine is not affected by the presence of the factor. The factor is inactivated by extraction with methanol, chloroform, butanol and ethanol. It remains intact after treatment with 6M guanidine hydrochloride and is soluble in trifluoroethanol. Thus, the weight of evidence indicates that this factor is a rather hydrophobic protein.

Purification and characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450 e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450 b and P-450 c, have also been highly purified during the isolation of cytochrome P-450 e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450 b, highly purified cytochrome P-450 e is immunochemically identical to cytochrome P-450 b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 ( P-450 a, P-450 c, P-450 d) or epoxide hydrolase. Purified cytochrome P-450 e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450 b or P-450 d (52,000) but clearly distinct from cytochromes P-450 a (48,000) and P-450 c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450 e is at 450.6 nm, whereas the peak of cytochrome P-450 b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450 e and P-450 b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450 b and P-450 e, respectively. Metyrapone binds to reduced cytochromes P-450 e and P-450 b (absorption maximum at 445–446 nm) but not cytochromes P-450 a, P-450 c, or P-450 d. Metabolism of several substrates catalyzed by cytochrome P-450 e or P-450 b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450 e usually paralleled that of cytochrome P-450 b except that the rate of metabolism of benzphetamine, benzo[ a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450 e was only 15–25% that of cytochrome P-450 b. In contrast, cytochrome P-450 e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450 b. Cytochrome P-450 d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450 a, P-450 b, P-450 c, or P-450 e. The peptide fragments of cytochromes P-450 e and P-450 b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.

Induction of polysubstrate monooxygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus NRRL 3240

The presence of the components of polysubstrate monooxygenase (PSMO) activity, viz., cytochrome P-450 and NADPH cytochrome P-450 reductase has been established for the first time in the microsomes of Aspergillus parasiticus . The microsomes were able to metabolize benzphetamine. NADPH cytochrome P-450 reductase, benzphetamine metabolism and aflatoxin production was increased by the presence of phenobarbitone (PB, 2mg/ml) in the medium. These results demonstrate that induction of PSMO activity could be a prerequisite for increased production of aflatoxins, since hydroxylation of intermediates is an obligatory step in aflatoxin biosynthesis.

In vivo and in vitro effect of chelating agents on drug metabolizing enzymes of the rat

Rats given calcium disodium ethylenediaminetetraacetic acid (CaNa 2EDTA), diethylenetriaminepentaacetic acid (DTPA), dimercaprol, p-aminosalicylic acid (PAS), or d-penicillamine (penicillamine) i.p. for 7 successive days showed a significant decrease in the activity of hepatic microsomal benzphetamine N-demethylase. There was no appreciable change in the microsomal cytochrome P-450 concentration. In vitro incubation of the chelating drugs with liver microsomes isolated from rats pre-treated with phenobarbital caused no significant loss of the hemoprotein. The decreased rate of benzphetamine metabolism in microsomal preparations from rats, pretreated with the chelating drugs, may be attributed partly to hepatic depletion of essential trace elements by the chelating drugs.

Production of hydroxyl radicals and their role in the oxidation of ethanol by a reconstituted microsomal system containing cytochrome P-450 purified from phenobarbital-treated rats

Ethanol oxidation by a reconstituted system composed of cytochrome P-450 purified from liver microsomes of phenobarbital-treated rats, NADPH-cytochrome c reductase, phospholipid and NADPH was inhibited by a series of hydroxyl radical scavenging agents. Inhibition was competitive with respect to ethanol and was specific in the sense that the metabolism of aminopyrine or benzphetamine by the reconstituted system was not affected by the scavengers. The generation of ethylene gas from 2-keto-4-thiomethylbutyric acid in an ethanol-sensitive manner provided chemical evidence consistent with the ability of the reconstituted system to generate hydroxyl radicals. These results suggest that the oxidation of ethanol by the reconstituted system reflects the interaction of ethanol with hydroxyl radicals generated during NADPH oxidation.

Separation and characterization of highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, and 3-methylcholanthrene.

Highly purified cytochromes P-450, designated P450,, P-450b, and P-450,, have been isolated from hepatic microsomes of rats treated with the polychlorinated biphenyl mixture Aroclor 1254. Each form shows a single protein-staining band on sodium dodecyl sulfate-polyacrylamide gels with minimum molecular weights of 48,000 (P-450,), 52,000 (P-450& and 56,000 (P-450,). Cytochromes P-450, and P-450,, have been obtained from phenobarbital-treated rats, and cytochromes P-450, and P-450, were isolated from 3-methylcholanthrene-treated rats. Cytochrome P-450,, is the major hemeprotein inducible by phenobarbital, and cytochrome P-450, is the predominant hemeprotein induced by 3-methylcholanthrene. The CO-reduced difference spectral peak of cytochrome P-450, is at 452 nm, P-45Ob is at 450 nm, and P-450, is at 447 nm. Cytochrome P-450, preferentially hydroxylates testosterone at the 7a: position but has low catalytic activity for the metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, and zoxazolamine. Cytochrome P-450,, catalyzes the hydroxylation of testosterone at the 16a: position and the demethylation of benzphetamine most efficiently. Cytochrome P-450, has very high catalytic activity for benzo[a]pyrene and zoxazolamine hydroxylation and 7-ethoxycoumarin O-dealkylation. Aniline is hydroxylated by the three forms of cytochrome P-450 at comparable rates. Antisera prepared against cytochrome P-450,, P-450,,, or P-450, do not cross-react with the heterologous antigens in Ouchterlony double diffusion plates. When cytochromes P-450,, P-450b, and P-450,, were subjected to limited proteolysis in the presence of sodium dodecyl sulfate, the resulting peptide maps provided additional evidence that these hemeproteins are structurally distinct. The forms of cytochrome P-450 obtained from phenobarbitaland 3-methylcholanthrene-treated rats are indistinguishable from the corresponding forms from Aroclor 1254~treated rats by all criteria examined.

Short Communication: Metabolism and Excretion of Benzphetamine: Sources of Error in Reporting Results

Benzphetamine (Didrex | ) is prescribed as an anorectic to be taken in 25or 50-mg doses, one to three times per day (1). It is a central nervous system stimulant similar in structure and action to amphetamine (1) and, therefore, subject to occasional abuse. Benzphetamine is a weak base and as such, very little is excreted unchanged in the urine (2). Studies have shown that it is extensively metabolized to amphetamine and methamphetamine in rats (2,3). An effort was made to determine the extent of metabolism of benzphetamine into amphetamine and methamphetamine in man. A patient (male, 29 years old, 210 lbs.) with no history of amphetamine, methampbetamine, or benzphetamine use ingested 20 mg of benzphetamine. Twelve urine specimens taken at intervals during the following 44 hours were analyzed for amphetamine, methamphetamine, and benzphetamine by a combination of enzyme-multiplied immunoassay technique and gas-liquid chromatography (4). The minimum detectable levels using 40 ml of urine sample for each amphetamine and methamphetamine were 0.05 mg/l and for benzphetamine 0.15 rag/1. Table I shows that even from this low dosage of benzphetamine, detectable levels of both amphetamine and methamphetamine were produced for at least 22 hours. Urine testing results for a single oral dose of 20 mg of benzphetamine would thus be indistinguishable from results obtained following ingestion of metbamphetamine and amphetamine together or methamphetamine alone (which metabolizes to amphetamine), since detectable levels of benzphetamine itself are not excreted. A urine sample from a chronic benzphetamine user, however, was found to contain trace amounts of unchanged benzphetamine. The small quantities excreted and its relatively long gas chromatographic retention time--20 minutes on a 1.8-m x 2-ram i.d. glass column packed with 10% Apiezon L-10% KOH run at 200~ compared to about 1 minute for methamphetamine under the same conditions (4)--make the detection of even chronic benzphetamine use difficult. Thus, it is generally not possible to distinguish the use of benzphetamine from the use of methamphetamine or both amphetamine and methamphetamine based on only urine results.

Effect of ethylenebisdiisothiocyanato sulfide (EBIS) on hepatic monooxygenase activity

The effect of ethylenebisdiisothiocyanato sulfide (EBIS), a breakdown product of the ethylenebisdithiocarbamate fungicides, on the heptic cytochrome P-450-containing monooxygenase system of rats and mice has been examined. In vivo administration of EBIS to rats and mice causes a dose-dependent decrease of hepatic cytochrome P-450 and an inhibition of the cytochrome P-450 monooxygenase-catalyzed metabolism of aniline and benzphetamine. Incubation of EBIS with hepatic microsomes from rats and mice in both the presence and absence of NADPH also leads to a decrease in the level of cytochrome P-450 in these microsomes and a decrease in the monooxygenase activity toward aniline or benzphetamine. The in vitro but not the in vivo decrease in cytochrome P-450 is accompanied by an increase in cytochrome P-420, an inactive form of cytochrome P-450. The decrease in cytochrome P-450 and the inhibition of monooxygenase activity seen on incubation of EBIS with hepatic microsomes appears to be a result of the nonenzymatic reaction of EBIS with sulfhydryl groups of cysteine residues in cytochrome P-450. The inhibition of hepatic monooxygenase activity and decrease in hepatic cytochrome P-450 concentration seen on administration of EBIS in vivo appear to result from a mechanism other than reaction of EBIS with cysteine residues in hepatic cytochrome P-450.

Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containing compounds

Ten thiono-sulfur-containing compounds of varying structure were administered by intraperitoneal injection to untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated adult male rats. Six hr later, the concentration of hepatic cytochrome P-450 and the ability of the hepatic microsomes to metabolize benzphetamine were examined. In the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated groups, two, four and four compounds, respectively, significantly decreased the concentration of cytochrome P-450 in the hepatic microsomes. A similar effect on benzphetamine metabolism was also seen. When examined 48 hr after the administration of the ten thiono-sulfurcontaining compounds, four, five and seven of the compounds decreased both the levels of hepatic cytochrome P-450 and the rate of benzphetamine metabolism in the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated animals respectively. Eight of the thiono-sulfur-containing compounds were incubated in the presence of NADPH with hepatic microsomes isolated from untreated, phenobarbital-pretreated or 3-methylcholanthrene-pretreated animals. All of the compounds examined significantly decreased the concentration of cytochrome P-450 in the microsomes from each treatment group. Similar reductions in benzphetamine metabolism were also seen. When these same compounds were incubated with microsomes in the absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen. When the oxygen analogs of six of the thiono-sulfur compounds were administered in vivo or incubated with hepatic microsomes either in the presence or absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen.

Toxicological implications op the mixed-function oxidase catalyzed metabolism of carbon disulfide

The results of these studies have indicated that the decrease in the activity of the hepatic mixed-function oxidase enzyme system and the concentration of cytochrome P-450 seen on incubation of carbon disulfide (CS2) with rat liver microsomes in the presence of NADPH is the result of the binding of the sulfur atom released in the mixed-function oxidase catalyzed metabolism of CS2 to carbonyl sulfide (COS). Moreover, it appears that COS is further metabolized by the mixed-function oxidase enzyme system to CO2 and that, analogous to the metabolism of CS2 to COS, the sulfur atom released in this reaction also binds to the microsomes and inhibits benzphetamine metabolism and decreases the concentration of cytochrome P-450 detectable as its carbon monoxide complex. The results of these studies also suggest that the decrease in the concentration of cytochrome P-450 and the liver damage seen on in vivo administration of CS2 to phenobarbital pretreated rats, is due to the mixed-function oxidase catalyzed release and binding of the sulfur atoms of CS2. The decrease in the concentration of cytochrome P-450 seen on incubation of CS2 with rat liver microsomes in the presence of NADPH does not appear to be the result of destruction of the heme group or its dissociation from the apoenzyme since the total amount of protoheme is unchanged in microsomes which have been incubated with CS2 and NADPH as compared to those not incubated with these compounds.

Heterogeneity of liver microsomal cytochrome P-450: The spectral characterization of reactants with reduced cytochrome P-450

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.

Role of Cytochrome b5 in Hydroxylation by a Reconstituted Cytochrome P-450-containing System

1. The role of cytochrome b5 in the oxidative metabolism of benzphetamine and 3,4-benzpyrene in the presence of NADPH was studied with the reconstituted hydroxylation system isolated from rat liver microsomes whose components were cytochrome P-450 or P-448, NADPH-cytochrome c reductase, and lipid. In the absence of cytochrome b5, the turnover number for benzphetamine and 3,4-benzpyrene (nanomoles per min per nmole of P-450 or P-448) in the reconstituted system was at least as good as that in liver microsomes, indicating that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation system. 2. The addition of cytochrome b5 at a ratio of cytochrome P-450 to b5 normally found in microsomes inhibited NADPH-dependent benzphetamine N-demethylation by the three components (cytochrome P-450, NADPH-cytochrome c reductase, and lipid). This inhibition was shown to be due to competition between cytochrome b5 and cytochrome P-450 for NADPH-cytochrome c reductase. The addition of NADH and cytochrome b5 reductase to the reconstituted system reversed the inhibition of NADPH-dependent benzphetamine N-demethylation by cytochrome b5. Thus, the NADH synergistic effect observed in liver microsomes with benzphetamine as the substrate and NADPH as electron donor could be due to NADH and cytochrome b5 reductase preventing the inhibition by cytochrome b5. 3. The addition of cytochrome b5 at a ratio of cytochrome P-448 to b5 normally found in microsomes did not inhibit the NADPH-dependent hydroxylation of 3,4-benzpyrene by the three components (cytochrome P-448, NADPH-cytochrome c reductase, and lipid). In the presence of NADH, NADPH, cytochrome b5, cytochrome b5 reductase, and the three components, the rate of 3,4-benzpyrene hydroxylation was significantly increased. This increased rate was due to a NADH-dependent hydroxylation pathway via cytochrome b5 and cytochrome b5 reductase. 4. It is concluded that cytochrome b5 is not an obligatory component in the NADPH-dependent benzphetamine or 3,4-benzpyrene hydroxylation systems. However, this cytochrome plays an important role in regulating NADPH-dependent hydroxylation of these substrates and is also an essential component of the NADH-dependent hydroxylation pathway.

Studies of the binding of sulfur released in the mixed-function oxidase-catalyzed metabolism of diethyl p-nitrophenyl phosphorothionate(parathion) to diethyl p-nitrophenyl phosphate (paraoxon)

Incubation of [ 35S]labeled parathion with rat liver microsomes in the presence of an NADPH-generating system leads to the covalent binding of sulfur to the macromolecules of the microsomal membrane. The maximal binding of sulfur to microsomes requires the presence of NADPH, is increased using microsomes from phenobarbital and 3-methyl-cholanthrene-treated animals, and is inhibited by carbon monoxide. The majority, if not all, of the sulfur bound is in a form free of the remainder of the parathion molecule. These findings, coupled with the fact that the apparent K m and V max for sulfur binding are not statistically different from the apparent K m and V max for metabolism of parathion to paraoxon, indicate the sulfur bound is that released in the mixed-function oxidase-catalyzed metabolism of parathion to paraoxon. Under the conditions of the experiments described in this report, the binding of sulfur to microsomes decreases the concentration of cytochrome P-450 in the microsomes and inhibits slightly the rate of the mixed-function oxidase-catalyzed metabolism of benzphetamine.

Effects on drug metabolism of carbaryl and 1-naphthol in the mouse

The effects of carbyl and 1-naphthol on hepatic microsomal drug-metabolizing enzyme systems were investigated. The agents were fed at a level of 25 mmol/kg of feed to groups of young male Swiss-Webster mice for 14-day periods. Body weight was depressed by carbaryl, but not by 1-naphthol. The rates of in vitro metabolism of aniline and benzphetamine were greater than control rates in livers of mice fed carbaryl, but the rate of in vivo hydrolysis of the carbamate insecticide Zectran was decreased by carbaryl feeding. Administration of 1-naphthol did not change the rates of in vitro metabolism of either aniline or benzphetamine. Hepatic microsomal concentrations of cytochromes P-450 and b 5 were increased by carbaryl, but feeding of 1-naphthol did not affect levels of either cytochrome. Radiolabeled pentobarbital disappeared from the blood of carbaryl-fed mice more rapidly than from the blood of control animals, and carbaryl-fed mice slept a shorter period of time than controls following pentobarbital administration. The LD50 of an acute oral dose of carbaryl was increased two-fold by feeding carbaryl for 14 days. It was concluded that carbaryl is a weak inducer of hepatic microsomal drug-metabolizing activity, and that the effects observed are not likely due to 1-naphthol.

Evidence for an inhibitory product · cytochrome P-450 complex generated during benzphetamine metabolism by liver microsomes

Spectrophotometric observations of hepatic microsomal pigments during the aerobic metabolism of dl-benzphetamine have revealed the appearance of a spectral species presumed to be associated with the generation of a product · cytochrome P-450 complex. This spectral species exhibits an absorption band maximum at 456 nm in the difference spectrum. The velocity and extent of formation of this absorption band are maximal using limiting concentrations of dl-benzphetamine in the reaction mixture and its development is suppressed in the presence of an excess of a second hydroxylatable substrate. The generation of this presumed enzyme · product complex modifies the carbon monoxide difference spectrum of reduced cytochrome P-450 and efficiently decreases the subsequent oxidation of other drug substrates. The implication of these studies to possible side effects observed during multiple drug therapy is discussed.

Pregnenolone-16α-carbonitrile: A new type of inducer of drug-metabolizing enzymes

Pretreatment of female rats with pregnenolone-16α-carbonitrile (PCN) for 2 1 2 days resulted in a significant increase in cytochrome P-450 content and NADPH-cytochrome c reductase activity in hepatic microsomes. The spectral characteristics of the microsomal hemoprotein from PCN-treated rats were similar to those obtained from untreated and phenobarbital (PB)-treated rats but different from 3-methylcholanthrene (3-MC)-treated animals. However, the specificity of the induction effect of PCN on the oxidative metabolism of benzphetamine, ethylmorphine, and 3,4-benzpyrene differed from the specificity of either PB or 3-MC. Studies on the induction of enzymes that metabolize the above three substrates revealed that PB, PCN, and 3-MC exerted their greatest stimulatory effect on benzphetamine N-demethylation, ethylmorphine N-demethylation, and 3,4-benzpyrene hydroxylation, respectively.

A comparison of the effects of benzpyrene administration on some hepatic microsomal mixed-function oxidases of rats and mice

There were marked differences in the responses of the hepatic microsomal enzymes of rats and mice after treatment of these animals with benzpyrene. Benzpyrene treatment caused qualitative as well as quantitative changes in the hepatic microsomal mixed-function oxidases of rats. Qualitative changes included: a decrease in the ratio of the peaks at 430 and 455 mμ in the pH-dependent ethyl isocyanide difference spectrum; an increase in the pH optimum of aniline p-hydroxylation; and changes in the apparent Michaelis-Menten kinetics of aniline, (+)-benzphetamine and benzpyrene metabolism. In addition, there were marked alterations in the substrate concentration-dependent “kinetics” of the difference spectra produced by the interaction of aniline and (+)-benzphetamine with rat liver microsomal P-450. Treatment of rats with benzpyrene increased both the apparent V max and apparent K m of aniline p-hydroxylation, and the degree of change was dependent upon substrate concentration and pH of the incubation systems. Benzpyrene treatment also increased the apparent ΔA max of the aniline-induced microsomal difference spectrum, although the apparent spectral dissociation constant ( K s ) was decreased. Both the rate of (+)-benzphetamine metabolism and the magnitude of the (+)-benzphetamine-induced microsomal difference spectrum were decreased after benzpyrene treatment. Treatment of rats with benzpyrene also increased the apparent V max and decreased the apparent K m of benzpyrene hydroxylation several-fold. In identical experiments performed concomitantly with mice, benzpyrene treatment produced no measurable effect on any of these characteristics of the mouse hepatic microsomal mixed-function oxidases, except for a decrease in the magnitude of the (+)-benzphetamine-induced microsomal difference spectrum. Although the administration of benzpyrene produced no stimulatory effects on the drug-metabolizing enzymes of the mouse, treatment with 3-methylcholanthrene did enhance the benzpyrene hydroxylase activity of mouse liver microsomes. Studies showed that the apparent V max of this reaction was increased more than 2-fold.