Dependent Mixed Function Oxidase Research Articles

Effects of Sub Chronic Dermal Exposure of Gasoline Blended with Varying Concentration of Benzene on Hepatic Mixed Function Oxidases in Rats

Interaction of chemicals in a mixture, their biotransformation and adverse impact, if any, on environment and workroom atmosphere needs to be evaluated to identify the nature of potential toxic effects elicited by constituent chemical(s) in a mixture. The nature of chemical interaction(s) through which an individual chemical in a mixture undergoes metabolism and/or biotransformation in the target tissue could be helpful in evaluation of its toxic potential. Gasoline is a unique example of such chemical mixture where interaction of aromatics viz., benzene, toluene, ethyl benzene and xylene (BTEX) traverses through a common metabolic pathway involving cytochrome P-450 dependent mixed function oxidases. Travis et al. [1] has quantitatively evaluated that gasoline vapor has an antagonistic effect on benzene metabolism and its biotransformation products. It was reported that 1000 ppm gasoline vapor had the same inhibitory effect as 500 ppm toluene on the metabolism of 290 ppm benzene, indicating that there may be many confounding factors in gasoline which might affect the metabolism of benzene in turn affecting its toxicity at the molecular level. It has been reported that benzene causes myelotoxicity through its bioactive metabolites. Such biotransformation mechanisms involve cytochrome P-450 dependent Phase-I enzymes and the products are further detoxified through Phase-II enzymes. It was therefore considered pertinent to study the impact of gasoline blended with 1, 3 and 5% benzene concentrations. Female albino rats weighing 200–250 gm were exposed by dermal route to gasoline blended with varying benzene concentrations, @ 2 g/kg body wt., six days a week over a period of 90 days. The activities of hepatic mixed function oxidases, viz., aniline hydroxylase and aminopyrene demethylase as a monitor of Phase-I enzymes and Glutathione-S-Transferase for Phase-II enzyme was estimated along with free glutathione levels. Serum creatine kinase was also estimated to assess the extent of muscular dystrophy. The results of this sub chronic study will be discussed in the context of interaction of gasoline with varying concentration of benzene in experimental animals. Keywords: mixed function oxidases, benzene, toxicology, gasoline

Antiinflammatory and Analgesic Activities of Two Dicyclic Analogues of Acetaminophen and Its Regioisomer 3-Hydroxyacetanilide

Many of the nonsteroidal antiinflammatory drugs (NSAIDs) currently marketed produce severe gastrotoxic side effects. However, acetaminophen (4-hydroxyacetanilide), which is devoid of gastrotoxicity, is associated with hepatotoxicity and nephrotoxicity. Hepatotoxicity and nephrotoxicity of acetaminophen in man and experimental animals have been attributed to metabolic conversion of the drug into a reactive intermediate by the cytochrome P450 dependent mixed function oxidase system [1]. N-acetyl-p-benzoquinoneimine (NAPQI) is the most likely structure for the ultimate reactive intermediate that depletes cellular glutathione (GSH) and covalently binds to tissue macromolecules [2, 3]. Inasmuch as N-oxidation and attack by tissue nucleophiles on the oxidized aromatic ring of acetaminophen are features that correlate with hepatotoxicity, Harvison and coworkers prepared monomethylated acetaminophen analogues with the expectation that N-oxidation and or reaction with tissue nucleophiles would be hindered [4]. The reported results supported this hypothesis since N-methylation of acetaminophen completely blocks the hepatotoxic reaction [5]. Furthemore, methyl groups ortho to the amide function (2,6-dimethylacetaminophen) were found to block hepatotoxicity [3], whereas 2-methylacetaminophen was found to possess significantly lower hepatotoxicity when compared to acetaminophen [4]. However, the analgesic potency of the above-mentioned analogues seems to parallel their hepatotoxic potential, and both activities parallel the oxidation potentials in this series of compounds. In one sense this is unfortunate, since the authors of these studies had hoped to find a nonhepatotoxic analogue that was active as an analgesic. In research aimed at further understanding the mechanisms of acetaminophen toxicity, it was discovered that the regioisomer 3-hydroxyacetanilide was not hepatotoxic in mice [6 – 8] and also showed analgesic and antipyretic activity. On the other hand, a number of compounds, including the 3,4-dihydrocarbostyril [9], benzoxazolinone [10, 11], pyridinoxazolinone [12], 1,4-benzothiazine [13], pyridin1,4-thiazinone [14], and 1,4-benzoxazinone [15, 16] derivatives, have attracted attention as potential analgesic and or antiinflammatory agents. In view of the above, we considered that it might be of interest to evaluate the analgesic and anti-inflammatory activities of two derivatives of benzoxazine previously synthesized by us [17]. These compounds, since they can be viewed as cyclized versions of substituted 3and 4-hydroxyacetanilides, have frameworks similar to those of acetaminophen.

Bile acid structure and selective modulation of murine hepatic cytochrome P450-linked enzymes.

We examined the effects of the administration of different bile acids on in vivo hepatic murine cytochrome P450 (CYP) content, nicotinamide adenine dinucleotide phosphate (NADPH)-CYP-reductase, and individual mixed-function oxidases (MFOs). Neither CYP level nor reductase were appreciably affected by single intraperitoneal administration of taurodeoxycholic acid (TDCA) (12.2 or 24.4 mg x kg(-1) bw). MFO to various isoenzymes were slightly reduced 24 hours after treatment. Taurohyodeoxycholic acid (THDCA) and tauroursodeoxycholic acid (TUDCA) both induced CYP, reductase, and MFOs. CYP3A1/2-linked activity (i.e., testosterone 6beta-hydroxylase, and N-demethylation of aminopyrine) in a dose-dependent fashion was enhanced ( approximately 2-3-fold). CYP2E1- (hydroxylation of p-nitrophenol), CYP1A2-(O-demethylation of methoxyresorufin), CYP2A1/2- and CYP2B1/2-(6alpha-hydroxylase), and CYP2B9- (16alpha-hydroxylase) dependent MFOs, as well as 7alpha-, 16beta-, 2alpha-, and 2beta-hydroxylations, were all significantly induced by THDCA. Apart from alkoxyresorufin metabolism and a modest CYP2E1 increase, TUDCA behaved like THDCA. A generalized induction was also recorded after ursodeoxycholic acid (UDCA) administration. THDCA and TDCA did not show substantial differences in the N-demethylation of aminopyrine when different species (rat vs. mouse) and administration route (intraperitoneal vs. intravenous) were compared. Results on the most affected isoenzymes, CYP3A1/2 (THDCA, TUDCA, and UDCA) and CYP2E1 (UDCA), were sustained by means of Western immunoblotting. CYP3A induction was paralleled by a corresponding increase in mRNA. These data could partially explain the therapeutic mechanism of UDCA, TUDCA, and THDCA in chronic cholestatic liver disease. CYP3A induction, which is linked to P-glycoprotein (Pgp) family overexpression, may enhance hepatic metabolism, transport, and excretion of toxic endogenous lipophilic bile acids.

Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes.

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.

Assessment of Liver Function in Primary Cultures of Hepatocytes Using Diethoxy (5,6) Chloromethylfluorescein and Confocal Laser Scanning Microscopy

A method is presented which can be used to assess the function of hepatocytes in complex culture configurations without disrupting the integrity of the cell environment. It utilises a fluorescent probe for cytochrome P450 dependent mixed function oxidase (MFO) activity diethoxy (5,6) chloromethylfluorescein, and confocal laser scanning microscopy. The MFO activity of individual cells in primary cultures of intact hepatocytes can be detected in situ, and quantified by image analysis. This may be a valuable means of monitoring the effect of culture conditions on the function of bioartificial liver devices, and could be used to assess the need for effective oxygenation of cells, the influence of shear stress and of exposure to patient serum during clinical use.

IN-VITROTESTICULAR BIOACTIVATION OF ACRYLONITRILE

The present work examines the mechanism of testicular toxicity of acrylonitrile. In testicular centrifugal fractions from Sprague Dawley rats, the metabolism of VCN to cyanide (CN−) was highest in the microsomal fraction and required NADPH for maximum activity. This biotransformation of VCN to CN−was characterized with respect to time (30min), microsomal protein concentration (1.5mgml−1), pH (7.5) and temperature (37°C). The Vmaxof the reaction was 65.1pmol CN−mg protein−1min−1and Kmwas 88.6gmmol VCN. Flushing the microsomes with carbon monoxide (CO)(4:1, CO/O2v/v), addition of benzimidazole (1mM) or addition of SKF 525-A (5×10−4M) to incubation mixtures significantly inhibited VCN metabolism by 49%, 54% and 37.4% respectively. Activation of VCN to CN−was markedly increased in microsomes obtained from phenobarbital (PB)-treated rats (128.2%). Addition of glutathione (GSH),L-cysteine,D-penicillamine or 2-mercaptoethanol significantly enhanced the release of CN−from VCN 126%, 247%, 202% and 129% of the control value respectively. These findings indicate that VCN is metabolized in the testis via cytochrome P-450 dependent mixed function oxidase system.

Involvement of cytochrome P450 3A enzyme family in the major metabolic pathways of toremifene in human liver microsomes

The anti-estrogen toremifen-Fc-1157a or 4-chloro-1,2-diphenyl-1-[4-[2( N, N-dimethylamino) ethoxy]-phenyl]-1-butene is now used for the treatment of breast cancer. This drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-demethyl-(DMTOR), 4-hydroxy-(4OH-TOR) and deamino-hydroxy- (TOR III) toremifene metabolites. The specific forms of cytochrome P450 involved in these oxidation reactions were examined in 32 human liver microsomal preparations previously characterized with respect to their contents of several known P450 enzymes. Toremifene was demethylated with an apparent K m of 124 μM while it was hydroxylated with an apparent K m of 139 μm. The metabolic rates were 71 ± 56, 13 ± 9 and 15 ± 4 pmol/min/mg microsomal protein, respectively, for DMTOR, 4-OH-TOR and TOR III. The N-demethylation activity was strongly correlated with estradiol 2-hydroxylation ( r = 0.75), nifedipine oxidation ( r = 0.86), tamoxifen N-demethylation ( r = 0.73), testosterone 6β-hydroxylation ( r = 0.78) and erythromycin N-demethylation ( r = 0.84), all these monooxygenase activities known to be supported by CYP3A4 isoform. Furthermore, the CYP3A content of liver microsomal samples, measured by western blot analysis using a monoclonal anti-human CYP3A4 antibody, was strongly correlated with DMTOR formation ( r = 0.80). Compounds such as cyclosporin, triacetyl-oleandomycin and testosterone inhibited the N-demethylation of toremifene metabolism at 80, 89 and 56% vs control, respectively, while the formation of TOR III was inhibited at 78, 82 and 73% vs control and the 4-hydroxylation pathway was inhibited no more than about 50% vs control. Prior incubation of microsomes with 100 μM gestodene, known to be a selective mechanism-based inhibitor of CYP3A4 in the presence of NADPH, led to 76 ± 6 and 76 ± 5% (N = 5 samples) reductions in the N-demethylation and formation of TOR III, respectively. Polyclonal antibody directed against human CYP3A enzymes inhibited formation of DMTOR and TOR III by 60 and 46%, respectively. The metabolism of toremifene was not activated by α-naphthoflavone. Finally, the use of yeasts genetically engineered for expression of human P4501A1, 1A2, 2C9 and 3A4 allowed us to demonstrate that DMTOR and TOR III formations are mediated by P4501A and 3A4 enzymes and by contrast these enzymes are not involved in the 4-hydroxylation pathway. All these results taken together suggest that the major metabolic pathways of toremifene, namely N-demethylation and TOR III formation, are mediated mainly by cytochrome CYP3A4 enzyme in the human liver microsomes owing to the relative hepatic contents in P4501A and 3A.

Comparison of the hepatic effects of coumarin, 3,4-Dimethylcoumarin, dihydrocoumarin and 6-methylcoumarin in the rat

The mechanism of coumarin-induced hepatotoxicity in the rat has been investigated by comparing the effects of coumarin with those of three coumarin derivatives, namely 3,4-dihydrocoumarin (DHC), 3,4-dimethylcoumarin (3,4-DMC) and 6-methylcoumarin (6-MC). Male Sprague-Dawley rats were fed either control diet or diets containing 0.5 or 0.75% coumarin, 0.76% DHC, 0.6 or 0.9% 3,4-DMC or 0.82% 6-MC for 13 wk. The dietary levels of 0.5% coumarin and 0.6% 3,4-DMC, were equimolar (3.43mmol/100g diet), as were the dietary levels of 0.75% coumarin, 0.76% DHC, 0.9% 3,4-DMC and 0.82% 6-MC (5.14mmol/100g diet). All treatments resulted in an increase in relative liver weight, but only coumarin increased plasma alanine aminotransferase and aspartate aminotransferase activities. Morphological examination of liver sections from coumarin treated rats revealed vacuolation of centrilobular hepatocytes and bile duct hyperplasia. Cholangiofibrosis was also observed, particularly in rats given 0.75% coumarin. Treatment with DHC produced no abnormalities, whereas a slight hypertrophy of centrilobular hepatocytes was observed in some 3,4-DMC treated animals and a slight vacuolation of individual hepatocytes was noted in some 6-MC treated rats. DHC, 6-MC and particularly 3,4-DMC treatment resulted in an induction of cytochrome P-450 dependent mixed function oxidase enzyme activities. All treatments induced hepatic GSHS-transferase and γ-glutamyltransferase activities, induction being most marked in rats given coumarin and 6-MC. These results provide further evidence that coumarin-induced hepatotoxicity in the rat is due to the formation of a 3,4-epoxide intermediate.

Biochemical toxicology of argemone oil. I. effect on hepatic cytochrome P‐450 and xenobiotic metabolizing enzymes

The in vivo effect of argemone oil on hepatic xenobiotic metabolizing enzymes was investigated in albino rats following either a single (10 ml kg-1 body wt.) or multiple intraparenteral doses (5 ml kg-1 body wt.) for three days. Animals sacrificed 72 h after a single intraparenteral dose of argemone oil exhibited a significant loss of hepatic cytochrome P-450 (35%) and cytochrome b5 (34%) contents and inhibition of aminopyrine-N-demethylase (APD), aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin-O-deethylase (ECD) activities (21-39%). Three successive 24-hourly intraparenteral injections of argemone oil followed by sacrificing the animals after 24 h of the last injection, showed a greater degree of inhibition of the content of cytochrome P-450 (58%) and its dependent mixed-function oxidases (35-63%). Also, multiple treatment of argemone oil caused a depletion of endogenous hepatic glutathione (GSH) content (72%) with a concomitant increase in lipid peroxidation (177%) and decrease in glutathione-S-transferase (GST) activity (30%). A significant decrease in relative liver weight (39%) was observed in animals treated with multiple treatment of argemone oil. These results suggest that argemone oil can alter both membrane and cytosolic defences and destabilizes the hepatic cytochrome P-450 dependent mixed-function oxidase system, so that it tips in the direction of autooxidative peroxidation of lipids.

Identification of the cytochrome P450 IIIA family as the enzymes involved in the N-demethylation of tamoxifen in human liver microsomes

The antiestrogen tamoxifen (Tam or Nolvadex, ICI)—Z-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1,2-diphenyl-1-butene is widely used in treatment of hormone-dependent breast cancer. The drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-desmethyl metabolite (DMT). This study has been carried out to determine the P450 enzyme involved in the N-oxidative demethylation of Tam in microsomal samples from 25 human livers (23 adults, two children). This metabolic step was inhibited by carbon monoxide up to 75%. Tam was demethylated into DMT with an apparent K m of 98 ± 10 μM; rates varied between 37 and 446 pmol/min/mg microsomal protein. These metabolic rates were strongly correlated with 6β-hydroxylation of testosterone ( r = 0.83) and erythromycin N-demethylase ( r = 0.75), both activities known to be associated with P450 IIIA enzyme. To further assess whether or not the Tam demethylation pathway is catalysed by the same P450, the inhibitory effect of TST on this reaction was determined. The competitive inhibition had an apparent K i of 100 ± 10 μM. Drugs such as erythromycin, cyclosporin, nifedipine and diltiazem were shown to inhibit in vitro the metabolism of tamoxifen. Furthermore the P450 IIIA content of liver microsomal samples, measured by Western blot technique using a monoclonal P450NF (nifedipine) antibody, was strongly correlated with DMT formation ( r = 0.87). Tam N-demethylase activity was inhibited by more than 65% with polyclonal anti-human anti-P450NF. All these in vitro observations establish that a P450 enzyme of the IIIA sub-family is involved in the oxidative demethylation of tamoxifen in human liver.

The Effect of Tetrahydrofuran on Biological Systems: Does a Hepatotoxic Potential Exist?

Tetrahydrofuran (THF) is a widely used solvent in industry and research. THF is a weak toxin, with approximate acute LD50s in the range of 2 to 3 g/kg, 8 to 20 mg/L, and 800 mg/kg following oral, inhalation, and i.v. routes, respectively. The two primary signs of intoxication are narcosis and hepatocellular dysfunction, both occurring at doses of approximately one-half of the lethal dose. Little is known concerning the pharmacokinetics of THF. No evidence exists for genotoxicity due to THF. Ongoing carcinogenicity bioassay tests sponsored by the National Toxicology Program have not yet been completed. Two significant interactions of THF with cellular components have been studied; first, THF inhibits a number of enzymatic reactions at concentrations ranging from 10 to 100 mM. Most notably, THF is an inhibitor of a number of cytochrome P450 (P450) dependent mixed function oxidase activities, with a particular affinity for the alcohol-induced isozyme (P450IIE1). Second, THF has been noted to enhance the toxic action of a number of compounds (i.e. solvent effect), in particular stimulating the more rapid absorption of reactive metabolites. A third factor to be considered is THF's ability to form peroxides upon standing. Little is known concerning the toxicity of THF peroxides. Therefore, while there is little evidence to suggest that THF would be a direct (Type I) hepatotoxin at relatively low doses, its ability to inhibit drug-metabolizing reactions (perhaps more so in alcohol users), and enhance the absorbance of reactive metabolites, are relatively unexplored avenues for THF to contribute to a hepatotoxic response. As a great deal of the human hepatotoxicity (Type II) is "idiosyncratic" and cannot be readily reproduced in experimental animals, this potential route of hepatotoxicity should be considered in future evaluations of human exposure to THF.

Suppression of the hepatic microsomal cytochrome P-450 dependent mixed function oxidase. Activities in golden hamster during Leishmania donovani infection

Experimental infection of golden hamsters with Leishmania donovani caused significant alterations in the hepatic microsomal mixed function oxidase system. Gross examination of liver indicated hepatomegaly. Microsomal protein contents were only marginally elevated. Cytochrome P-450 as well as haem contents were significantly decreased and it directly correlated with the degree of infection. Cytochrome b 5 exhibited elevation at lower degrees of infection which came down to control levels at the peak infection. Concomitant suppression was also noticed in cytochrome P-450 dependent monooxygenase activities, viz. aniline hydroxylase, benzo[ a]pyrene hydroxylase and aminopyrine N-demethylase. No significant change was observed in NADH-cytochrome b 5 reductase and NADPH-cytochrome c reductase. The results indicate suppression of hepatic microsomal MFO activities during visceral leishmaniasis.

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal beta-oxidation and mixed-function oxidase activities in rats.

The effect of oral administration of a Prudhoe Bay crude oil (PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for 5-12 days on hepatic and renal microsomal monooxygenase activities and peroxisomal beta-oxidation has been investigated. PBCO administration leads to liver enlargement. This is associated with induction of microsomal cytochrome P-450 levels (1.6- to 2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-depentylase, representing cytochrome P-450I and cytochrome P-450IIB isoenzymes respectively, 9- to 15-fold; omega-oxidation of lauric acid representing the cytochrome P-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomal beta-oxidation (palmitoyl CoA oxidation, 2- to 5-fold). It was observed that rats exposed to PBCO showed an increase in renal microsomal cytochrome P-450 content (1.6- to 2.3-fold), cytochrome P-450I activity (5- to 8-fold) and omega-oxidation activity (1.3- to 1.4-fold). However, renal peroxisomal beta-oxidation was unaltered. Serum total triglycerides were lowered by 41-46% after PBCO exposure. These results suggest that induction of peroxisomal beta-oxidation and possibly mono-oxygenases may be related to the carcinogenic/tumorigenic potential of crude oil.

Effects of bile duct obstruction and decompression on hepatic microsomal mixed function oxidase system in rats

The effects of biliary obstruction and drainage on the hepatic microsomal mixed function oxidase system were studied in rats. Bile duct obstruction produced a significant reduction in the hepatic cytochrome P-450 dependent mixed function oxidase system. After release of the bile duct obstruction, the reduction in microsomal enzymes was practically reversible; however, the process of recovery was slow and differed with the microsomal enzymes in question. Increases in cytochrome b 5 content and NADH-cytochrome b 5 reductase activity were slower than increases in cytochrome P-450 content and NADPH-cytochrome c reductase activity. Aniline hydroxylase activity increased more rapidly and corresponded to cytochrome P-450 contents more so than did the aminopyrine demethylase activity. After the release of bile duct obstruction, however, the bile acids which had accumulated in the liver during cholestasis were reduced rapidly, to a normal range. These results suggest that there is a discrepancy between reductions in hepatic bile acids and those in the hepatic microsomal mixed function oxidase system after biliary decompression.

Biophysical and catalytic properties of the phenobarbital p-hydroxylase--a cytochrome P-450 dependent mixed function oxidase.

1. A sensitive method to detect and quantify the products of phenobarbital (PB) hydroxylation by model chemical systems and by biological systems has been developed. 2. Chemical model systems hydroxylate PB in the p-position of the phenyl ring and form one or two additional oxidation products, while in vitro and in vivo (bile fistula rats) biological systems hydroxylate PB only in the p-position. 3. Phenobarbital hydroxylation rates in vitro are of the order of 0.007 nmol/nmol cytochrome P-450 per min. These values are decreased by pretreatment of the rats with inducing doses of phenobarbital. 4. Enzymes catalysing the p-hydroxylation reaction of phenobarbital are localized in the microsomes and have the biophysical and chemical properties that are usually associated with cytochrome P-450-dependent mixed function oxidases.

Metabolic activation of olefins: Conversion of 1-octene to a putative reactive intermediate 1-octen-3-one: An alternative pathway to epoxidation

1. The enzymic activation of a model olefin oct-1-ene was studied in rat liver microsomal systems in vitro. 2. An active metabolite was trapped using N-acetylcysteine and identified by means of capillary GLC/ mass spectrometry and 360 MH z 1H NMR as S-3-oxo-octyl- N-acetylcysteine. A two step pathway for the formation of this adduct was proposed involving first the production of oct-1-en-3-ol by NADPH dependent mixed function oxidases and secondly a NADP or NAD linked oxidation, independent of cytochrome P-450, to yield the putative reactive intermediate oct-1-en-3-one. 3. Under physiological conditions, oct-1-en-3-one, prepared chemically, reacted non-enzymically with N-acetylcysteine with a t 1 2 of about 6 sec. 4. Enzymes catalysing the NADP-dependent oxidation of octen-3-ol were present in microsomal preparations from a number of organs apart from the liver, those from adrenal and intestinal epithelia showing the next highest levels of activity. Unlike the activation of octene, rates of hepatic activation of octen-3-ol were not induced by pretreatment of rats with phenobarbitone or 3-methylcholanthrene. 5. Using 1-octene as the substrate, comparisons were made of alternative routes of hepatic metabolism activation. Relative to the rate of formation of the 3-oxo intermediate trapped with N-acetylcysteine, epoxidation of octene and subsequent hydrolysis to octane-1,2-diol was over 40 times more rapid. The rate of formation of a presumptive oxirane precursor trapped with the haem of cytochrome P-450 as N-(2-hydroxyoctyl) protoporphyrin IX was about 17-fold lower.

Substrate-induced spectral changes in human normal and chronic myeloid leukemic granulocytes

Several drugs/chemicals were allowed to interact with the cytochrome P-450 dependent mixed function oxidase system in the postmitochrondrial supernatant fractions of Ficoll-Hypaque-separated granulocytes from human normal subjects and patients with chronic myeloid leukemia. The substrate-induced spectral changes were followed by recording the difference spectra. Compounds conventionally classified as type I and type II substrates, on addition to S 1 fractions of both normal and leukemic granulocytes, caused spectral changes that were reverse to those reported for the rat liver microsomes. Aminopyrine, phenobarbital, and Tween 80 evoked a reverse type I spectral change with a peak at 420–430 nm and a trough at 380–400 nm, whereas aniline and pyridine induced a modified type I (a reverse type II) spectral change characterized by a peak at 408 nm and a trough at 421 nm. These changes were found to be quantitatively proportional to the amounts of substrate added. However, the magnitude of the peaks and troughs was considerably less in the S 1 fraction of the leukemic granulocytes. Correspondingly, total heme content was significantly decreased in S 1 fractions of CML granulocytes as compared to similar fractions of normal granulocytes.

29 DECREASE IN CYTOCHROME P-450 DEPENDENT 3-N-DEMETHYLATION OF CAFFEINE MEASURED BY THE CAFFEINE 13CO2 BREATH TEST (CET) FOLLOWING GROWTH HORMONE THERAPY IN GROWTH HORMONE (GH) DEFICIENT CHILDREN

Significant changes in hepatic drug clearance occur in the transition from childhood to adulthood. These changes probably parallel the functional capacity of the hepatic cytochrome P-450 dependent mixed function oxidase (MFO) system. GH decreases hepatic MFO activity in animals and may act similarly in humans. Caffeine labelled with stable 13C has been shown to be a substrate for the MFO system. Five GH deficient children underwent CBT, with collections of expired air for 13CO2 enrichment analysis following ingestion of 13C-caffeine (3 mg/Kg) before, and 4-5 weeks following GH 0.1 u/Kg S.C. t.i.w. All children showed a decrease in % 13CO2 dose exhaled over 2 hours following GH administration: Pre 9.1±1.5, Post 7.3±2.9 (p < .05, paired t-test) The CBT is a safe effective technique for the measurement of the hepatic MFO system. N-demethylation of caffeine is decreased by GH therapy in GH deficient children. The capacity for N-demethylation is highest in prepubertal children. This may partially correlate with changing patterns of GH release.

Mechanism of DNA methylation by N-nitroso(2-oxopropyl)propylamine.

Using an in vitro assay system for measuring 7-methylguanine formation in DNA, it was demonstrated that N-nitroso(2-oxopropyl)propylamine (NOPPA) is converted into a methylating agent by a microsomal, cytochrome P-450 dependent mixed function oxidase from rat liver. Formation of propionaldehyde, but not formaldehyde, in this assay system, indicated that a single alpha-oxidation reaction on the propyl side chain of NOPPA leads to the formation of a methylating agent. A mechanism for this reaction is proposed.

The effect of nutritional factors on hepatic drug and toxicant metabolism

A variety of foreign agents, including environmental chemicals, food additives, natural food constituents, and pharmaceuticals, enter the body every day. Many of these compounds must undergo metabolism by specific enzymes within the body to facilitate their clearance. The most important of the enzyme systems, which is found in the liver, includes the cytochrome P-450 dependent mixed function oxidase, as well as several conjugation enzymes, including sulfotransferase, glucuronyl transferase, and glutathione-S-transferase. The balance of the reactions determines the rate of metabolism and clearance of the agents, as well as the degree of intracellular damage or toxicity. Specific dietary factors, such as proteins, carbohydrates, vitamins, and minerals, affect the efficiency of these reactions.