Species differences in the biochemical properties of liver microsomal arylamine and arylamide N-hydroxylases

Cytochrome P-448 dependent microsomal N-hydroxylases are key enzymes in the metabolic activation of both arylamides and arylamines.Using 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF) as substrates, the present report compares the biochemical properties of rat, hamster and mouse liver N-hydroxylases. There are marked species differences both in terms of the affinity for the two substrates and in terms of maximum velocity of the enzymes. The rat and hamster liver arylamide N-hydroxylases are induced by pretreatment with 2-AAF which also significantly increases their affinity for the substrate. In mouse liver neither arylamide nor arylamine N-hydroxylases are modified or induced. With 2-AF as substrate, arylamide treatment never enhances N-hydroxylation but it reduces the K_m-value of the rat and hamster liver enzymes.Among the effectors tested in vitro, 3-methylcholanthrene (3-MC), 7,8-benzoflavone (BF), benzo[α]pyrene (B[α]P) and miconazole (MN) inhibit hepatic arylamide N-hydroxylase in the submicromolar range. Harman (H) and paraoxon (PX) act in a dose-dependent manner in the micromolar range and metyrapone (MP) is not an inhibitor even at 50-μM concentration.Among the position isomers, 1- and 3-AAF are inhibitors of the N-hydroxylating enzymes whereas 4-AAF is not.These data are discussed in relationship to the toxic effects (mutagenicity and hepatocarcinogenicity) of arylamides and arylamines with respect to the role and the complexity of their microsomal metabolism.     

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.     

Inhibition of the microsomal oxidation of ethanol and 1-butanol by the free-radical, spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide

The free-radical, spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), inhibited the microsomal oxidation of ethanol and 1-butanol as well as the metabolism of 2-keto-4-thiomethylbutyrate. DMPO also blocked the stimulation of ethanol oxidation caused by the addition of iron-EDTA to microsomes. Previous electron spin resonance studies with DMPO by others had shown that the free radical produced by rat liver microsomes and augmented by iron-EDTA was the hydroxyl radical. In the current study, DMPO had no effect on microsomal oxygen consumption with NADPH as substrate, nor on NADPH-cytochrome c reductase activity, nor on the demethylation of aminopyrine or the hydroxylation of aniline. Therefore, a general toxic action of DMPO on microsomes is excluded. DMPO also failed to inhibit the catalase-dependent pathway of ethanol oxidation by microsomes. Experiments with Chelex 100 resin and the chelating agent, diethylenetri-aminepentaacetic acid, rule out any major role for contaminating iron in the reagents. These results tend to identify the free radical responsible for microsomal metabolism of ethanol, 1-butanol, and 2-keto-4-thiomethylbutyrate as the radical trapped by DMPO, namely the hydroxyl radical.     

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 6: hydrolysis of 7,8-diacetoxy-4-methylcoumarin by a novel deacetylase in rat liver microsomes--a simple method for assay and characterisation

The existence of a novel microsomal deacetylase in rat liver catalysing deacetylation of diacetoxy 4-methylcoumarins has been reported. A simple method is outlined for the enzyme assay based upon the quantification of the dihydroxy derivative by measuring the UV absorption of its complex with ADP and Fe3+ at 600 nm. The enzyme can be routinely assayed using 7,8-diacetoxy-4-methylcoumarin (DAMC) as the substrate and demonstrated hyperbolic kinetics and yielded Km and vmax values of 1250 microM and 500 units, respectively. The pH optima was found to be 7.5 for the enzyme. No DAMC deacetylase activity was found in hepatic cytosol and the enzyme activity was not discernible in extrahepatic tissues.     

Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both phi X174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. We have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, we have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. phi X174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-AF. However, we have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed.     

Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.     

Modulation of the mutagenicity of three dinitropyrene isomers in vitro by rat-liver S9, cytosolic, and microsomal fractions following chronic ethanol ingestion.

The effects of chronic ethanol feeding of rats on the ability of liver fractions to modulate the bacterial mutagenicity of three dinitropyrene isomers (1,3-, 1,6- and 1,8-DNP), which require bacterial enzymes but not an exogenous enzyme source for activation, were studied. The mutagenicity of the DNP isomers toward S. typhimurium TA98 and TA100 was attenuated in the presence of post-mitochondrial supernatants (S9) from both ethanol-fed and pair-fed rats albeit, that from the ethanol-fed group was more efficient in lowering the mutagenicity. The cytosolic fraction from ethanol-fed rats enhanced the mutagenicity of all of the DNP isomers in TA100. The most notable enhancement was with 1,3-DNP in which a more than 4-fold enhancement was obtained. Cytosol from pair-fed rats enhanced only the mutagenicity of 1,3-DNP, this by 2.9-fold. Cytosolic NADPH-nitroreductase activity from ethanol-treated rats toward 1,6-, 1,8- and 1,3-DNP was increased 2.8-, 1.7- and 1.3-fold, respectively over pair-fed controls. Cytosolic NADH-nitroreductase from ethanol-fed rats was increased with 1,3-DNP (1.7-fold) and 1,8-DNP (1.4-fold) as substrates, but not with 1,6-DNP. Microsomes decreased the mutagenicity of DNP similarly to S9, i.e., fractions from ethanol-fed rats were more efficient than those of pair-fed rats in deactivating all the DNP isomers. Per mg of protein, detoxification of DNP by S9 was more efficient than with microsomes, thus both cytosolic and microsomal enzymes are required for maximal detoxification. In summary, ethanol feeding modulates both the augmented cytosolic activation of DNP to mutagens and the deactivation of the direct-acting mutagenicity of DNP by microsomes. In combination, as is the case with S9, the microsomal detoxifying activity outcompetes the cytosolic activation.     

Purification and characterization of a human liver arylacetamide deacetylase

Arylacetamide deacetylation is an important enzyme activity in the metabolic activation of arylamine substrates to ultimate carcinogens, best described as a carboxylesterase/amidase type of reaction. A 7-fold variation in the Vmax of 2-acetylaminofluorene deacetylation in 24 human livers was observed. An acetylaminofluorene deacetylase was purified 90 fold from human liver microsomes by PEG-fractionation, anion exchange and hydrophobic interaction chromatography. The purified 45kD protein showed no amino acid sequence homology to other carboxylesterases, neither in its N-terminus nor in tryptic peptides. Antibodies raised against the deacetylase recognized the protein with high specificity. This report thus describes the first arylacetamide deacetylase in human liver.     

Alterations in the enzyme activity and polypeptide composition of rat hepatic endoplasmic reticulum during acute exposure to 2-acetylaminofluorene

Studies have been made of the morphology, enzyme activity and protein composition of liver endoplasmic reticulum in rats exposed to acute doses of the carcinogen, 2-acetylaminofluorene (2-AAF). Electron microscopic examination revealed numerous ultrastructural changes in the hepatocyte; most consistent alterations were the disorganisation of endoplasmic reticulum system with apparent increase of smooth endoplasmic reticulum. Administration of 2-AAF to rats immediately depressed microsomal glucose-6-phosphatase activity and eventually induced epoxide hydratase activity 6–7-fold over control activity. The induction was time-dependent and maximal rates of induction were observed at dosages greater than 40 mg/kg body wt. The treatment also induced cytochrome b₅ content, NADH and NADPH cytochrome c reductase activities (1.0–1.5-fold). Only very small changes in the total content of cytochrome P-450 were noted. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of microsomal proteins from 2-AAF pretreated animals showed time-dependent induction of two polypeptides which differed slightly in migration, in the region of M_r = 48 000; the faster-migrating induced polypeptide has been identified as epoxide hydratase. Two-dimensional PAGE analysis of microsomal proteins from 2-AAF exposed rats showed a reproducible deletion of a protein with molecular weight in the region of 67 000. The basis for the alterations in the protein composition of endoplasmic reticulum in response to 2-AAF treatment is discussed.     

Comparative aspects of microsomal cytochrome P-450-dependent monooxygenase activities in musk shrew (Suncus murinus), Mongolian gerbil (Meriones unguiculatus), harvest mouse (Micromys minutus) and rat.

1. The cytochrome P-450 content (0.75 +/- 0.13 nmol/mg microsomal protein) in musk shrew (suncus, Suncus murinus) liver microsomes was lower than that (1.30 +/- 0.26) in rat liver microsomes, but it is approximately the same level as in the Mongolian gerbil (Meriones unguiculatus, 1.18 +/- 0.14), harvest mouse (Micromys minutus, 1.11 +/- 0.02) and rat. 2. The hydroxylation activity (based on cytochrome P-450) of medium-chain fatty acids (otanoic, decanoic, lauric and tridecanoic acids) is much higher in suncus, Mongolian gerbil and harvest mouse than in rat, with the exception of the activity of decanoic and tridecanoic acids in Mongolian gerbil. 3. This suggests that cytochrome P-450 species catalyzing the hydroxylation of medium-chain fatty acids are present in these laboratory animals in higher concentrations. 4. The aminopyrine N-demethylation activity based on microsomal protein or cytochrome P-450 in suncus is significantly lower than that in rat, but the N-demethylation activity in Mongolian gerbil and harvest mouse is approximately 1.7-2.0-fold greater than that in rat.     

Enantioselective aliphatic hydroxylations of racemic 1-hydroxy-3-methylcholanthrene by rat liver microsomes

Enantiomeric pairs of 1-hydroxy-3-hydroxymethylcholanthrene (1-OH-3-OHMC), 3-methylcholanthrene (3MC) trans- and cis-1,2-diols, and 1-hydroxy-3-methylcholanthrene (1-OH-3MC) were resolved by HPLC using a covalently bonded (R)-N-(3,5-dinitrobenzoyl)phenylglycine chiral stationary phase (Pirkle type 1A) column. The absolute configuration of an enantiomeric 3MC trans-1,2-diol was established by the exciton chirality CD method following conversion to a bis-p-N,N-dimethylaminobenzoate. Incubation of an enantiomeric 1-OH-3MC with rat liver microsomes resulted in the formation of enantiomeric 3MC trans- and cis-1,2-diols; the absolute configurations of the enantiomeric 1-OH-3MC and 3MC cis-1,2-diol were established on the basis of the absolute configuration of an enantiomeric 3MC trans-1,2-diol. Absolute configurations of enantiomeric 1-OH-3-OHMC were determined by comparing their CD spectra with those of enantiomeric 1-OH-3MC. The relative amount of three aliphatic hydroxylation products formed by rat liver microsomal metabolism of racemic 1-OH-3MC was 1-OH-3-OHMC greater than 3MC cis-1,2-diol greater than 3MC trans-1,2-diol. Enzymatic hydroxylation at C2 of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer over the 1R-enantiomer (approximately 3/1); hydroxylation at the C3-methyl group was enantioselective toward the 1R-enantiomer over the 1S-enantiomer (approximately 58/42). Rat liver microsomal C2-hydroxylation of racemic 1-OH-3MC resulted in a 3MC trans-1,2-diol with a (1S,2S)/(1R,2R) ratio of 63/37 and a 3MC cis-1,2-diol with a (1S,2R)/(1R,2S) ratio of 12/88, respectively.     

Microsomal cytochrome P450-dependent steroid metabolism in male sheep liver. Quantitative importance of 6 beta-hydroxylation and evidence for the involvement of a P450 from the IIIA subfamily in the pathway

The metabolism of testosterone (TEST), androstenedione (AD) and progesterone (PROG) was assessed in hepatic microsomal fractions from male sheep. Rates of total hydroxylation of each steroid were lower in sheep liver than in microsomes isolated from untreated male rat, guinea pig or human liver, 6 beta-Hydroxylation was the most important pathway of biotransformation of each of the three steroids (0.80, 0.89 and 0.43 nmol/min/mg protein for TEST, AD and PROG, respectively). Significant minor metabolites from TEST were the 2 beta-, 15 beta- and 15 alpha-alcohols (0.19, 0.22 and 0.17 nmol/min/mg microsomal protein, respectively). Apart from the 6 beta-hydroxysteroid, only the 21-hydroxy derivative was formed from PROG at a significant rate (0.27 nmol/min/mg protein). The 6 beta-alcohol was the only metabolite formed from AD at a rate greater than 0.1 nmol/min/mg protein. Antisera raised in rabbits to several rat hepatic microsomal P450s were assessed for their capacity to modulate sheep microsomal TEST hydroxylation. Anti-P450 IIIA isolated from phenobarbital-induced rat liver effectively inhibited TEST hydroxylation at the 2 beta-, 6 beta-, 15 alpha- and 15 beta-positions (by 31-56% when incubated with microsomes at a ratio of 5 mg IgG/mg protein). IgG raised against rat P450 IIC11 and IIB1 inhibited the formation of some of the minor hydroxysteroid metabolites but did not decrease the rate of TEST 6 beta-hydroxylation. Western immunoblot analysis confirmed the cross-reactivity of anti-rat P450 IIIA with an antigen in sheep hepatic microsomes; anti-IIC11 and anti-IIB1 exhibited only weak immunoreactivity with proteins in these fractions. Considered together, the present findings indicate that, as is the case in many mammalian species, 6 beta-hydroxylation is the principal steroid biotransformation pathway of male sheep liver. Evidence from immunoinhibition and Western immunoblot experiments strongly implicate the involvement of a P450 from the IIIA subfamily in ovine steroid 6 beta-hydroxylation.     

Purification and characterization of N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver.

N-Hydroxy-2-acetylaminofluorene (N-OH-2-AAF) sulfotransferase is an enzyme that catalyzes the sulfate transfer from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to N-OH-2-AAF to form a highly reactive product acetylaminofluorene N-sulfate. It has been purified about 2000-fold with a yield of over 12% from adult Sprague-Dawley male rat livers by an eight-step procedure. The final preparation was homogeneous on analytrical disc gel electrophoresis. The purified enzyme had activity toward p-nitrophenol with an approximately 1600-fold increase in specific activity over the crude homogenate, but it had almost no detectable activity toward steroids such as estrone, beta-estradiol, testosterone, dehydroisoandrosterone, and corticosterone. There was also very little sulfation activity toward serotonin and L-tyrosine methyl ester. The optimal pH for the enzyme activity is approximately 6.3 when measured in sodium phosphate buffer. Mg2+ at 6 to 9 mM could increase the enzyme activity up to 30%. Mn2+ activated the enzyme only slightly at very low concentrations. Zn2+, Co2+, Cu2+, and Ni2+ were all strongly inhibitory, but Ca2+ had very little effect. Thiol compounds were found to have a stabilizing effect and thiol-blocking reagents were potent inhibitors for this enzyme. The pure enzyme was very unstable especially in diluet solutions. The isoelectric point (pl) of the enzyme is 5.66 +/- 0.07. The molecular weight of the native enzyme was 68,000 +/- 500 as estimated by Sephadex G-100 and G-200 gel filtrations. A single component with molecular weight of 38,250 +/- 1,350 was observed on sodium dodecyl sulfate gel electrophoresis in the absence and presence of 2-mercaptoethanol. Comparison of the enzyme activity in mail and female rat livers at each stage of purification revealed that there was only a trace amount of N-OH-2-AAF sulfotransferase present in the female rat liver.     

Distinct site specificity of two pea histone deacetylase complexes

We report on the site specificity of two intact pea histone deacetylase complexes. HD1 deacetylates lysines 5 and 16 of H4 in the order K16 > K5, while in the case of H3 the preferred order is K4 > K18 approximately K9. The specificity of the HD2 complex is markedly different. The preferred residues in H4 are K8 approximately K5 > K16, while in H3 deacetylation, the complex HD2 prefers sites 4 and 18. To obtain these results, we have used a novel procedure based on the SPOT technique, a method to synthesize peptides on membrane supports. Different sets of membranes with sequentially overlapping histone peptides containing acetylated lysines in the sites corresponding to all in vivo acetylatable residues were incubated with the complexes. The acetyl groups removed by the deacetylase activity were then replaced by radioactive acetate by treating the membranes with labeled acetic anhydride. The subsequent counting of the membranes allows the quantification of the acetate removal in the histone deacetylase reaction in a way that circumvents some of the inconveniences of other available procedures.     

The effect of EDTA and iron on the oxidation of hydroxyl radical scavenging agents and ethanol by rat liver microsomes

Rat liver microsomes catalyzed an NADPH-dependent oxidation of dimethylsulfoxide, 2-keto-4-thiomethylbutyrate and ethanol. The addition of EDTA and iron (ferric)-EDTA increased the oxidation of the hydroxyl radical scavenging agents and ethanol. Unchelated iron had no effect; therefore, appropriately chelated iron is required to stimulate microsomal production of hydroxyl radicals. Catalase strongly inhibited control rates as well as EDTA or iron-EDTA stimulated rates of hydroxyl radical production whereas superoxide dismutase had no effect. The rate of ethanol oxidation was ten- to twenty-fold greater than the rate of oxidation of hydroxyl radical scavengers in the absence of EDTA or iron-EDTA, suggesting little contribution by hydroxyl radicals in the pathway of ethanol oxidation. In the presence of EDTA or iron-EDTA, the rate of ethanol oxidation increased, and under these conditions, hydroxyl radicals appear to play a more significant role in contributing toward the overall oxidation of ethanol.     

Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry

In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.     

N-Hydroxylation of arylamides by the rat and guinea pig: Evidence for substrate specificity and participation of cytochrome P1-450

The hypothesis that N-hydroxylation of arylamides is essential for carcinogenicity was examined in vivo and in vitro with N-2-fluorenylacetamide, a potent carcinogen, and with N-3-fluorenylacetamide, an isomer with marginal carcinogenicity. About 10–20% of 2-[9-¹⁴C]fluorenylacetamide administered intraperitoneally to the rat was excreted in the bile as the N-hydroxy-2-[9-¹⁴C]-derivative, whereas <0.1% of 3-[G-³H]fluorenylacetamide was found as the N-hydroxy metabolite in bile and urine. N-Hydroxylation of the 2- isomer by hepatic microsomes of untreated or 3-methylcholanthrene-treated rats was 40 to 50-fold greater than that of the 3- isomer. The role of cytochromes P-450 and P₁-450 in N-hydroxylation of arylamides by rat liver microsomes was shown by inhibition of the reaction with carbon monoxide and cobaltous chloride. Interaction of the arylamides with cytochrome P₁-450 was also demonstrated by binding spectra obtained on addition on 2- and 3-fluorenylacetamide to hepatic chromosomes of methylcholanthrene-treated rats. There appeared to be no correlation between the magnitude of the spectra and the extent of N-hydroxylation. N-Hydroxylation of the 2- isomer by hepatic microsomes of the guinea pig, a species resistant to the carcinogenecity of this compound, was markedly less than N-hydroxylation by rat liver microsomes, even though, as judged by the appearance of the binding spectra, both 2- and 3- isomers were bound by cytochrome P₁-450 of guinea pig-liver microsomes. The results are in agreement with the view that the microsomal N-hydroxylation of arylamides parallels their carcinogenicity.     

Structure-activity relationships in the deacetylation of O-glucosides of N-hydroxy-N-arylacylamides by mammalian liver microsomes

Structure-activity relationships in the deacylation of O-glucosides of N-hydroxy-N-aryl-acylamides were investigated to provide insights into the metabolic activation of carcinogenic/mutagenic O-glycosides of N-hydroxy-N-arylacylamides. In the subcellular fractions obtained from porcine liver, the deacetylation activity toward O-glucoside of N-hydroxyacetanilide (GAc) was mainly localized in the microsomes. Both the 2-chloro (2ClGAc) and 2-methyl (2MeGAc) derivatives of GAc were not deacetylated by the microsomes. Other compounds having either 3- or 4-substituent (chloro or methyl), however, were deacetylated and showed higher V(max)/K(m) values than that of GAc. 4-Methyl derivative (4MeGAc) was shown to competitively inhibit the deacetylation activity toward GAc, and the K(i) value of 4MeGAc was comparable with its K(m) value obtained in the microsome-catalyzed deacetylation. These apparent K(m) values were shown to correspond to their lipophilicities estimated from retention times on high-performance liquid chromatography (HPLC). As for the effect of acyl groups, the order of V(max)/K(m) values was N-propionyl derivative (GPr)>GAc>N-butyryl derivative (GBu). From the optimized structures and energy levels of the frontier orbitals of these compounds, calculated by the semi-empirical AM1 method, the effects of 2-substituents and acyl groups on the deacetylation activity is thought to be due to a steric factor. From the energy levels of the frontier orbitals of GAc and its 3- or 4-substituted derivatives, the compound having a lower level of LUMO was shown to be deacetylated effectively. There were marked species differences in the microsomal deacetylation activity toward GAc, and the highest activity was found in the rabbit, followed by the porcine, hamster, rat and then bovine liver microsomes.     

A simple radioisotope assay for microsomal aryl hydroxylase

A radioassay for liver microsomal aryl hydroxlase activity has been devised which depends on the liberation of tritiated water from generally tritiated benzo[α]pyrene during hydroxylation. The quantity of tritiated water has been shown to be proportional to the amount of 3-hydroxybenzo-[α]pyrene formed. Among the advantages of the radioassay are its speed, simplicity, and the fact that it essentially provides a cumulative measure of the hydroxylation of the benzo[α]pyrene ring. Investigation of a number of variables has made it possible to assay and obtain proportional results with as little as 3 μg of rat liver microsomes. Nucleic acids, but not their component mononucleotides, have been found capable of protecting the enzyme from product inhibition, presumably by interaction with benzo[α]-pyrene oxide, the primary product of benzo[α]pyrene hydroxylation.