Immediately after recovery from hexobarbital anesthesia, mice were injected intraperitoneally with one of the following interferons: natural mouse alpha/beta, recombinant mouse (rmouse gamma IFN-A) or human alpha A, alpha D, alpha AD interferon (rHu alpha IFN-A, rHu alpha IFN-D, rHu alpha IFN-AD). All of these interferons, except rHu alpha IFN-A induced unconsciousness ("sleep"); all produced stimulatory effects that mimicked those produced by morphine in the mouse. Quantification of the duration of sleep, induced by rmouse gamma IFN, was investigated and found to be dose-related. Only 3 of the 5 interferons (mouse alpha/beta IFN; rmouse gamma IFN, rHu alpha IFN-AD) possesses antiviral activity and depresses the cytochrome P-450 system in the mouse, yet all 5 of the interferons produced CNS effects. This partition of effects, together with the very short latency of the interferon-induced CNS effects, shows that the CNS effects were mechanistically independent of the anti-viral and anti-cytochrome P-450 effects. This disparity of the actions of the interferons suggests the possibility that selected morphine antagonists could be used to counter some of the dose-limiting CNS effects of the large doses of interferons used in clinical situations.

The duration and intensity of drug action depend not only on the dose of the drug but also on the rates at which drugs are transformed to products that can be excreted readily by the kidney. Two general categories of drug metabolism occur in the liver: phase 1 reactions (oxidations-reductions and hydrolyses) and phase 2 reactions (synthetic conjugations). Phase 1 reactions produce functional groups that can participate in phase 2 reactions. Phase 1 reactions are almost nonexistent in the fetuses of laboratory animals; however, many appear in primates during the first trimester of gestation. Phase 2 reactions are deficient prenatally in both rodents and primates. Parturition triggers a surge of both phase 1 and phase 2 reactions. The lack of uniformity of the development of phase 1 oxidative reactions during the early neonatal period reflects the multiplicity of cytochrome P-450 hemoproteins, the terminal oxidases responsible for most hepatic oxidative biotransformations. The rate of recovery of chemically induced losses of cytochrome P-450 systems is age dependent.

The hepatic monooxygenase systems largely responsible for the biotransformation of drugs and other xenobiotics are comprised of NADPH-cytochrome P-450 reductase and multiple forms of cytochrome P-450. Optimal temperatures for these systems in the trout and rat are 26 degrees and 37 degrees, respectively. Purified trout and rat reductases are optimally functional at 26 degrees and 37 degrees, respectively, when added to trout and rat microsomes. However, rat reductase was shown to function optimally at 26 degrees when added to trout microsomes and trout reductase functioned optimally at 37 degrees when added to rat microsomes. Corresponding shifts in optimal temperatures of cytochrome P-450-linked 0-deethylation of 7-ethoxycoumarin occurred when these reductases were added to rat or trout microsomes. It is proposed that the phospholipid annulus surrounding the active site of membrane-bound cytochrome P-450 determines the optimal temperature of cytochrome P-450 systems.

Double-stranded polyriboinosinic acid·polyribocytidylic acid is a potent interferon inducing agent and depressant of hepatic cytochrome P-450 monooxygenase systems. Single-stranded polyriboinosinic acid or polyribocytidylic acid are not. However, it is known that interferon is induced in mice when the administration of polyriboinosinic acid is followed shortly thereafter by the administration of polyribocytidylic acid. The current study demonstrates that this sequential administration of single-stranded polynucleotides induces serum and hepatic interferon and depresses the cytochrome P-450 systems. Neither of these effects were seen when the order of administration of these polynucleotides was reversed.

Almost all drugs and environmental xenobiotics are metabolized by hepatic cytochrome P-450-dependent mixed function oxidase (MFO) systems. When administered to animals, most of these chemicals either induce or have little or no effect on MFO systems, but a few produce marked losses. Allylisopropylacetamide (AIA), CCl4, CS2 and polyriboinosinic acid·polyribocytidylic acid (poly rI·rC) are prototypes of xenobiotics which destroy MFO systems by different mechanisms. With the exception of poly rI·rC, these agents are believed to produce their effect via “suicidal” reactions, i.e., only after they have been converted by cytochrome P-450 to reactive metabolites (reviewed by De Matteis 1978). The metabolites of AIA, and those of many other compounds that possess terminal olefin groups, destroy cytochrome P-450 by reacting with its heme to form green pigments (De Matteis 1971). Cytochrome P-450 destroys itself by converting CS2 to a product which combines covalently with apocytochrome P-450 (Bond and De Matteis 1969; Catignani and Neal 1975; Dalvi et al. 1975; De Matteis 1978). CCl4 is believed by many to exert its toxic effect through the cytochrome P-450-mediated formation of free radical intermediates (Hrycay and O’Brien 1971), but there is less agreement as to the mechanism involved. Lipid peroxidation of the microsomal membrane is a major consequence of CCl4 intoxication (reviewed by De Matteis 1978). Destruction of cytochrome P-450 probably occurs both indirectly as a result of disruption of the membrane and more directly by the hydroperoxides produced by lipid peroxidation. The mechanism of action of poly rI·rC and other interferon inducing agents is not well understood.

A study was undertaken to determine the mechanism by which fatty acyl CoA (stearoyl or oleoyl CoA) inhibits NADPH-supported hepatic cytochrome P-450-dependent monooxygenase systems. About half of the inhibitory effect of fatty acyl CoA on ethylmorphine N-demethylase activity, and all of the inhibitory effect on aniline p-hydroxylase activity, was shown to be due to stimulation of lipid peroxidation. The remaining half of the inhibition of ethylmorphine demethylation was shown not to be due to a) competition for electrons from NADPH by the fatty acyl CoA desaturase system, b) inhibition of NADPH-cytochrome c reductase or NADPH-cytochrome P-450 reductase, c) 3959 ADP, which might have been formed from CoA through the action of nucleotide pyrophosphatase, d) a shortage of electrons from NADPH due to an inhibitory effect of fatty acyl CoA on glucose 6-phosphate dehydrogenase, or e) fatty acyl CoA or its products acting as substrate inhibitors. Stearoyl CoA prevented the stimulation of NADPH-cytochrome P-450 reductase by ethylmorphine and caused a small but consistent loss of the type I binding spectrum elicited by ethylmorphine. These observations and the kinetics of the inhibition of ethylmorphine N-demethylase by stearoyl CoA suggest that fatty acyl CoA may inhibit hepatic monooxygenase reactions by acting as a detergent.

Previous studies from our laboratory have shown that a variety of interferon-inducing agents depress cytochrome P-450-dependent monooxygenase systems when administered to rats. With the expectation that the use of cultured hepatocytes would provide a more accessible means of studying the mechanism by which interferon-inducing agents depress these enzyme systems, measurements were made of the effects of the interferon inducer, poly rI·rC and a crude preparation of mouse interferon on the cytochrome P-450 content and aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities of primary, nonreplicating mouse hepatocytes maintained on floating collagen membranes. During the first 24 hr of culture, hepatocytes lost about 80% of their cytochrome P-450, 97% of their aminopyrine N-demethylase activity, and 90% of their benzo[a]pyrene hydroxylase activity. The specific activity of cytochrome P-450 (nanomoles of [14C]formaldehyde formed from the N-demethylation of aminopyrine per nanomole of P-450 per minute) was lowered from 4.6 to as little as [unknown] this value. Exposure of the cultures to poly rI·rC (5 µg/ml of culture) during the second 24 hr of culture caused a 40% increase in the cytochrome P-450 content of the hepatocytes. The specific activity of cytochrome P-450 de novo relative to the N-demethylation of aminopyrine was restored to that of the cytochrome P-450 of freshly isolated hepatocytes or the cytochrome P-450 of microsomes isolated from liver homogenates. The mouse interferon preparation (1000 units/ml of culture medium) was considerably less potent as an inducer of cytochrome P-450 in cultured hepatocytes, but the specific activity of the cytochrome P-450 de novo induced by mouse interferon was as high as that of the cytochrome P-450 induced by poly rI·rC. Relative to benzo[a]pyrene hydroxylase activity, the specific activity of the cytochrome P-450 that survived the first 24 hr of culture was about the same as that for the cytochrome P-450 of microsomes isolated from liver homogenates. Both poly rI·rC and mouse interferon preparation induced the hydroxylase activity in 24-hr-old cultures of hepatocytes. Poly rI·rC did not induce the hydroxylase activity in cultured Reuber hepatoma cells. Poly rI·rC was shown to induce interferon activity in cultured hepatocytes. Methods are described for the determination of aminopyrine N-demethylase and benzo[a]pyrene hydroxylase activities and cytochrome P-450, cytochrome b5, and DNA contents of cultured hepatocytes from one mouse.

The induction of hepatic P-450 hemoprotein-dependent mono-oxygenase systems was studied in fetal and neonatal rats. The fetal liver was refractive to phenobarbital induction of aminopyrine and ethylmorphine N-demethylase. which are cytochrome P-450-dependent mono-oxygenases, but was not refractive to the 3-methylcholanthrene induction of benzo[a]pyrene hydroxylase. a cytochrome P1-450-dependent mono-oxygenase. After parturition, all three enzyme activities were inducible. These and other observations suggest that a control mechanism operates in the fetal rat which selectively suppresses the induction of cytochrome P-450. but allows induction of cytochrome P1-450. This selective suppression of phenobarbital induction in the fetus was reversed in part by the simultaneous administration of 3-methylcholanthrene. Several other inducing agents also partially reversed the suppression of phonobarbital induction in Fetal livers: dibenz-[a,c]anthracene, 2-diethylaminoethyl-2.2-diphenyl-valerate (SKF 525-A), and 3β-hydroxy-20-oxopregn-5-ene-16α-carbonitrile (PCN). Other inducing agents were inactive: α-naphthoflavone, β-naphthoflavone. 1,1-bis[p-chlorophenyl]2,2,2-trichloroethane (DDT). 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). dieldrin. chlordane and chlorpromazine.

Cytochrome P-450-dependent monooxygenase activities and cytochrome P-450 levels were depressed in hepatic microsomes from rats treated with 12 interferon inducing agents of various types: small molecules (e.g. tilorone), an RNA virus (Mengo), a fungal mycophage (statolon), liver RNA, a synthetic double-stranded polynucleotide (poly rI · poly rC), a bacterial lipopolysaccharide (E.coli endotoxin) and an attenuated bacteria (B.pertussis vaccine). The results suggest that the depression of hepatic cytochrome P-450-dependent monooxygenase systems may be a general property of interferon inducing agents.

AbstractAls Metabolit von Levallorphan (I) erhält man die Hydroxy‐Verbindung (II), die über ihr Röntgenspektrum identifiziert wird.

Studies of the DPNH synergism of the TPNH-dependent mixed-function oxidase system of hepatic microscomes using a variety of substrates revealed that synergism occurred when the substrates were type I binding compounds (aminopyrine, benzphetamine, codeine, ethylmorphine, norcodeine), but not when the substrates were type II binding compounds (aniline, p -chloro- N -methylaniline). The role of type I binding in DPNH synergism of drug metabolism was investigated by employing microsomes which varied in their abilities to produce a type I binding spectrum with ethylmorphine. This was accomplished by selecting microsomes from different animal sources (male rats of different ages, female rats, 3-methyl cholanthrene-treated rats) or by subjecting microsomes from a given source to treatments known to diminish type I binding (treatment with SKF 525-A or with phospholipase C, storage). Using ethylmorphine as the substrate, type I binding was shown to be directly correlated with DPNH synergism and with DPNH utilization. The mean ratio of DPNH utilized to ethylmorphine metabolized by the various microsomes used in the study was 1.15. No correlation was seen between DPNH utilization and rate of hydroxylation of the type II compound, aniline. No increase in DPNH utilization above that seen in the absence of substrate was observed during aniline hydroxylation. The mechanism whereby type I substrates elicit DPNH synergism is postulated to occur as follows. The first of the 2 electrons required for drug oxidation is derived from TPNH and is utilized in the reduction of the oxidized cytochrome P-450-substrate complex. The second electron is derived from either TPNH or DPNH and is transported through cytochrome b 5 to the oxygenated, reduced cytochrome P-450-substrate complex, although the possibility remains that second electrons from TPNH may be contributed by a route that circumvents cytochrome b 5 . When type I substrates are introduced into the system, the rate of entry of first electrons is accelerated and second electrons from the electron pool provided by DPNH are drawn via cytochrome b 5 into the system to balance the elevated input of first electrons. When type II substrates are introduced into the system, the rate of entry of first electrons is not accelerated and there is neither the need nor the means for the system to utilize excess second electrons provided by DPNH. When only TPNH is present, the electron pool created at cytochrome b 5 is not large enough to match the pool of first electrons created by the addition of the type I substrate, and the over-all reaction is therefore slower than the DPNH-synergized reaction.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIsolation, x-ray analysis, and synthesis of a metabolite of (-)-3-hydroxy-N-allylmorphinanJohn F. Blount, Erno Mohacsi, and Floie M. VaneCite this: J. Med. Chem. 1973, 16, 4, 352–355Publication Date (Print):April 1, 1973Publication History Published online1 May 2002Published inissue 1 April 1973https://pubs.acs.org/doi/10.1021/jm00262a009https://doi.org/10.1021/jm00262a009research-articleACS PublicationsRequest reuse permissionsArticle Views66Altmetric-Citations10LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information Get e-Alerts

Cytochromes P-450 and P 1 -450 (P-448), found predominantly in hepatic microsomes from untreated and from 3-methylcholanthrene-treated rats, respectively, are reported to be distinct molecular entities. To test this hypothesis, a comparison was made of soluble P-420 hemoproteins obtained from membrane-bound P-450 hemoproteins by digesting microsomes with steapsin. Partially purified, soluble cytochromes P-420 and P 1 -420 from microsomes from untreated and 3-methylcholanthrene-treated rats, respectively, were found to differ in their electrophoretic mobilities and in the molar absorbances of their carbon monoxide complexes (P-420, 110 mM -1 cm -1 ; P 1 -420, 134 mM -1 cm -1 ); when caused to aggregate, cytochrome P-420 exhibited both type I (hexobarbital) and type II (aniline difference spectra, but aggregated cytochrome P 1 -420 exhibited a type II difference spectrum only. That cytochrome P 1 -450 is not simply a complex of cytochrome P-450 with 3-methylcholanthrene or its metabolites was demonstrated by the failure of soluble, purified cytochrome P 1 -420 from rats treated with tritiated 3-methylcholanthrene to exhibit radioactivity. These studies support the view that cytochromes P-450 and P 1 -450 are distinct molecular entities.

Components of the hepatic microsomal P-450 hemoprotein electron transfer system (TPNH-cytochrome c reductase and P-450 hemoprotein) and the microsomal cytochrome b5 electron transfer system (DPNH-cytochrome b5 reductase and cytochrome b5) were solubilized using Triton N-101 and glycerol, and separated using DEAE-cellulose chromatography. Microsomal aniline hydroxylase activity was reconstituted when the TPNH-cytochrome c reductase fractions and the P-450 hemoprotein fraction were combined. Cross-activities of the TPNH reductases and P-450 hemoproteins were obtained with preparations from phenobarbital- and 3-methylcholanthrene-treated rats. The reconstituted aniline hydroxylase system resembled the native system with respect to turnover number, reducibility of the P-450 hemoprotein component with TPNH, and inhibition with carbon monoxide, p-chloromercuribenzoate, and vitamin K3, but differed in its ability to react with type I substrates such as ethylmorphine and aminopyrine.

Evidence is presented to show that the administration of polycyclic hydrocarbons causes the appearance in hepatic microsomes of a P-450 hemoprotein which differs in certain of its physical and biochemical properties from that found normally. This previously unrecognized hemoprotein, which has been named cytochrome P1-450, does not result from the combination of polycyclic hydrocarbons or their metabolites with native cytochrome P-450, but is formed as a specific molecular entity as a result of de novo hemoprotein biosynthesis. The possibility that cytochrome P1-450 may be an aberrant hemoprotein is considered.