Metabolism of benz[a]anthracene by human liver microsomes

The metabolism of benz[a]anthracene (BA) by human hepatic microsomes was investigated. Only dihydrodiols were observed when BA was the substrate. No tetrahydrotetrols were detected, indicating lack of diol epoxide formation. The BA-dihydrodiols identified by GCMS analysis and comparison to authentic standards were BA-8,9-dihydrodiol (42.4% of total metabolites), BA-5,6-dihydrodiol (25%), BA-10,11-dihydrodiol (24.8%), BA-3,4-dihydrodiol (5.3%), and BA-1,2-dihydrodiol (< 1.5%). BA-dihydrodiols were also used individually as substrates. Only BA-1,2-dihydrodiol, the least abundant isomer produced from BA, was converted efficiently to a tetrahydrotetrol (> 72% conversion). BA-10,11-dihydrodiol was converted to BA-8,9,10,11-tetrahydrotetrols in < 12% yield. BA-10,11- and BA-3,4-dihydrodiols were not converted to tetrahydrotetrols.     

Identification of hepatic metabolites of two highly carcinogenic polycyclic aza-aromatic compounds, 7,9-dimethylbenz[c]acridine and 7,10-dimethylbenz[c]acridine

The hepatic microsomal metabolites of the highly carcinogenic dimethylbenzacridines, 7,9-dimethylbenz[c]acridine (7,9-DMBAC), and 7,10-dimethylbenz[c]acridine (7,10-DMBAC) were obtained with preparations from 3-methylcholanthrene-pretreated rats. Metabolites were separated by reversed-phase HPLC and characterized using UV spectral data and chemical ionization-mass spectrometry after trimethylsilylation and GC. Comparisons with products formed in the presence of the epoxide hydrolase inhibitor, 1,1,1-trichloropropane 2,3-oxide and with those formed from the three synthetic alcohol derivatives of each parent compound, aided the assignment of firm or tentative structures to 16 products from 7,9-DMBAC found in 22 reversed-phase chromatographic peaks, and for 17 products of 7,10-DMBAC found in 19 chromatographic peaks. The more abundant metabolites were derived from oxidation of the methyl groups. Other metabolites were dihydrodiols, epoxides, phenols and secondary metabolites. The 9-methyl group prevented dihydrodiol formation at the 8,9-position from 7,9-DMBAC, and for each carcinogen, the 3,4-dihydrodiol was formed. As well, 3,4-dihydrodiols of methyl oxidized compounds were found.     

Structure, tumorigenicity, microsomal metabolism, and DNA binding of 7-Nitrodibenz[a,h]anthracene

It has been previously proposed that a nitropolycyclic aromatic hydrocarbon (nitro-PAH) with its nitro functional group perpendicular or nearly perpendicular to the aromatic moiety exhibits lower tumorigenicity than the corresponding parent aromatic hydrocarbon. We also hypothesized that reduction of the nitro group is not involved, or contributed less significantly in the metabolic activation of this class of nitro-PAHs. To verify this hypothesis, we selected 7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A) for study. The X-ray crystallographic structure of 7-NDB[a,h]A was determined and indicated that the dihedral angle between the nitro functional group and the aromatic dibenz[a,h]anthracenyl moiety was 80.6 degrees, indicating the nitro group preferentially adopts a nearly perpendicular orientation. The tumorigenicity of 7-NDB[a,h]A and dibenz[a,h]anthracene (DB[a,h]A) was determined in the male B6C3F1 neonatal mouse. Mice were administered ip injections of 1/7, 2/7, and 4/7 of the total dose of 7-NDB[a,h]A (400 nmol in 35 microL of DMSO per mouse) within 24 h of birth and at 8 and 15 days of age, respectively, and sacrificed at 12 months of age. DB[a,h]A induced 78 and 96% hepatocellular adenomas and carcinomas, respectively. However, 7-NDB[a,h]A induced only 50 and 8% hepatocellular adenomas and carcinomas compared with the 8 and 4% hepatocellular adenomas and carcinomas induced by the solvent vehicle, DMSO. Aerobic metabolism of 7-NDB[a,h]A by liver microsomes of 15-day old male B6C3F1 neonatal mice resulted in trans-3,4-dihydroxy-3, 4-dihydro-7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A trans-3, 4-dihydrodiol) and trans-10,11-dihydroxy-10, 11-dihydro-7-nitrodibenz[a,h]anthracene (7-NDB[a,h]A trans-10, 11-dihydrodiol) as predominant metabolites. Under anaerobic conditions, 7-NDB[a,h]A was not metabolized (nitroreduced). The DNA adduct levels in liver and lung tissues of male B6C3F1 mice treated with 7-NDB[a,h]A and sacrificed 24 h and 6 days after final dosing were determined by 32P-postlabeling/TLC. In all cases, the DNA adducts derived from 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a, h]A trans-10,11-dihydrodiol were formed. These results suggest that both of the metabolites, 7-NDB[a,h]A trans-3,4-dihydrodiol and 7-NDB[a,h]A trans-10,11-dihydrodiol, are involved in the metabolic activation of 7-NDB[a,h]A, leading to tumor induction in the neonatal mouse. Thus, our results described in this paper support our hypotheses that a nitro-PAH with a perpendicular nitro orientation exhibits lower tumorigenicity than the corresponding parent PAH and that nitroreduction contributes less significantly in the metabolic activation.     

Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1

A human cytochrome P-450 (P450) 1B1 cDNA was expressed in Saccharomyces cerevisiae and the microsomes containing P450 1B1 were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also determined and compared these activities of P450 1B1 with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli. The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, N-nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the three P450 enzymes examined here, P450 lB1 was found to have the highest catalytic activities for the activation of 11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene, 1,2-dihydroxy-1,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 11,12-dihydroxy-11,12-dihydrobenzo[g]chrysene, 3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 2-aminoanthracene, 3-methoxy-4-aminoazobenzene, and 2-nitropyrene. P450 1B1 also catalyzed the activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-aminofluorene, 6-aminochrysene and its 1,2-dihydrodiol, (-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 1,2-dihydroxy-1,2-dihydrochrysene, 1,2-dihydroxy-1,2-dihydro-5,6-dimethylchrysene, 2,3-dihydroxy-2,3-dihydrofluoranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene, and 6-nitrochrysene to appreciable extents. However, P450 1B1 did not produce genotoxic products from benzo[a]pyrene, trans- 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenzo[a]anthracene, 7,12-dimethylbenz[a]anthracene and its cis-5,6-dihydrodiol, 5-methylchrysene, 11,12-dihydroxy-11,12-dihydro-3-methylcholanthrene, 1,2-dihydroxy-1,2-dihydro-6-methylchrysene, benzo[c]phenanthrene, 2-amino-6-methyldipyridol[1,2-a:3',2'-d]imidazole, 2-acetylaminofluorene, benzidine, 2-naphthylamine, aflatoxin B1, aflatoxin G1, sterigmatocystin, N-nitrosodimethylamine, vinyl carbamate, or acrylonitrile in this assay system. P450 1B1 is expressed constitutively in extrahepatic organs, including fetal tissue samples, and is highly inducible in various organs by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in experimental animal models. Thus, activation of procarcinogens by P450 lB1 may contribute to human tumors of extrahepatic origin.     

Isolation and identification of new rapamycin dihydrodiol metabolites from dexamethasone-induced rat liver microsomes

1. Rapamycin is metabolically transformed in rat liver microsomes to 3,4- and 5,6-dihydrodiol metabolites under the influence of the cytochrome P-450 mixed function oxygenase system. These metabolites were produced from dexamethasone-induced as well as from non-induced rat liver microsomes. The comparison of the ion spray mass spectra of the 5,6-dihydrodiol with the 3,4-dihydrodiol of rapamycin shows clearly that dihydrodiols were formed in two distinct positions of rapamycin. 2. FAB mass spectra as well as electrospray mass spectra of two additional peaks isolated from the same chromatographic run confirm the presence of a 3,4-dihydrodiol metabolite of rapamycin as also strongly suggested by UV spectra. Hplc reinjection of each individual peak always resulted in chromatograms showing a combination of the same three peaks and therefore are to be considered as tautomers of the 3,4-dihydrodiol of rapamycin. 3. These tautomeric conformations were found to have no immunosuppressive potency, most probably due to important structural and stereochemical modifications of the rapamycin binding domain to the binding protein (FKBP-12) and/or to important metabolic structural modifications of rapamycin effector domain.     

Effect of polycyclic aromatic hydrocarbons on the elimination kinetics of pyrene and the urinary excretion profile of 1-hydroxypyrene in the rat

Pyrene was chosen as a noncarcinogen model of polycyclic aromatic hydrocarbons (PAHs). Groups of male Wistar rats were dosed with pyrene and with mixture of pyrene and fluoranthene, pyrene and benz[a]anthracene, or pyrene, fluoranthene, and benz[a]anthracene at 20 mg/kg by intravenous or oral routes. Blood samples were taken at 0.25, 0.5, 1, 2, 3, 4, and 5 h after administration. The concentration of pyrene was determined by gas chromatography. The toxicokinetic parameters for pyrene were determined from the time course of blood concentration. A significant increase in the bioavailability of pyrene after treatment with other PAHs was observed. Urinary 1-hydroxypyrene excretion was analyzed after pretreatment with acenaphthene, naphthalene, chrysene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. The urine from rats was collected for 3 d and the concentration of 1-hydroxypyrene was determined using high-performance liquid chromatography (HPLC). Most compounds examined caused a decrease in the urinary excretion of the metabolite of pyrene.     

Epoxide-diol pathway in the metabolism of 5H-dibenzo[b,f]-azepine (iminostilbene)

5H-Dibenzol[b,f]azepine-10,11-epoxide and 10,11-dihydro-10,11-dihydroxy-5H-dibenzo[b,f]azepine were identified by gas chromatography-mass spectrometry in rat urine as the main biotransformation products in the metabolism of 5H-dibenzo[b,f]azepine (iminostilbene). The presence of these metabolites was confirmed in vitro by incubating iminostilbene with rat liver microsomal enzymes.     

Lack of correlation between in vitro and in vivo replication of precisely defined benz-a-anthracene adducted DNAs

Like other polycyclic aromatic hydrocarbons, certain metabolites of benz[a]anthracene have been implicated as potent carcinogens. These effects are thought to be caused by the covalent binding of these species to nucleophilic groups on the bases of DNA. To address the molecular mechanisms by which these molecules induce mutations, this study employed oligonucleotides containing four site-specific N6 adenine-benz[a]anthracene diol epoxide adducts. Using a prokaryotic in vivo replication system, we have shown that both non-bay region anti-trans-benz[a]anthracene adducts are essentially nonmutagenic. In contrast, the bay region anti-trans-benz[a]anthracene lesions do induce point mutations at the adduct site. The mutagenic frequency of these bay region lesions is dependent on the stereochemistry about the adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated. The ability of the bacterial replication machinery to bypass the lesions does not correlate with the differences observed in their mutagenesis. While both non-bay region adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree. In vitro studies of the interactions of E. coli DNA polymerase III with these adducts have also been undertaken to further dissect the relationship between adduct structure and biological activity.     

Mutagenic and carcinogenic metabolites of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one

Microsomal metabolites of the carcinogen 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (Structure I) were separated by high-pressure liquid chromatography, and their structures were established on the basis of their ultraviolet and mass spectra, together with considerations of their general chemical properties. This was assisted by comparisons with metabolites formed in the same way from the synthetic 15-hydroxy (Structure III), 16-hydroxy (Structure II), and 11-hydroxymethyl (Structure IV) derivatives, which themselves occur as metabolites of Structural I. Products derived from attack at the two benzo-ring double bonds occurred, but no K-region products were found. Only metabolites having a non-bay region 3,4-dihydrodiol system were mutagenic and bound to DNA after in vitro microsomal activation, and it was concluded that the 3,4-dihydro-3,4-diol (Metabolite e) was the main form and that the 3,4-diols of the monools (Structure II to IV) were minor proximate forms of this carcinogen. In a two-stage experiment, the synthetic 16-ol (Structure II) was shown to be almost as carcinogenic as was Structure I itself in mice; the 15-ol (Structure III) and 11-hydroxymethyl derivative (Structure IV) were much less active. The same order was also observed in the mutagenicity of these compounds in the Ames test.     

Polycyclic aromatic hydrocarbon-degrading capabilities of Phanerochaete laevis HHB-1625 and its extracellular ligninolytic enzymes

The ability of Phanerochaete laevis HHB-1625 to transform polycyclic aromatic hydrocarbons (PAHs) in liquid culture was studied in relation to its complement of extracellular ligninolytic enzymes. In nitrogen-limited liquid medium, P. laevis produced high levels of manganese peroxidase (MnP). MnP activity was strongly regulated by the amount of Mn2+ in the culture medium, as has been previously shown for several other white rot species. Low levels of laccase were also detected. No lignin peroxidase (LiP) was found in the culture medium, either by spectrophotometric assay or by Western blotting (immunoblotting). Despite the apparent reliance of the strain primarily on MnP, liquid cultures of P. laevis were capable of extensive transformation of anthracene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Crude extracellular peroxidases from P. laevis transformed all of the above PAHs, either in MnP-Mn2+ reactions or in MnP-based lipid peroxidation systems. In contrast to previously published studies with Phanerochaete chrysosporium, metabolism of each of the four PAHs yielded predominantly polar products, with no significant accumulation of quinones. Further studies with benz[a]anthracene and its 7,12-dione indicated that only small amounts of quinone products were ever present in P. laevis cultures and that quinone intermediates of PAH metabolism were degraded faster and more extensively by P. laevis than by P. chrysosporium.     

Estrogenic sensitivity of alpha-receptors in the urethra musculature

Metabolism of 7-methylbenz[a]anthracene (7MeBA) by 3-methylcholanthrene-induced rat liver microsomes in the presence of added native or denatured DNA resulted in covalent binding of the hydrocarbon to the nucleic acid. Enzymatic degradation and column chromatographic fractionation showed that the hydrocarbon-deoxyribonucleoside products were separable from the products similarly obtained from DNA having 7MeBA bound following treatment of mouse embryo cells in culture with this hydrocarbon. Comparison of the microsome catalysed hydrocarbon-deoxyribonucleoside products with those obtained by reaction with DNA of 7MeBA-5,6-oxide suggested that this K-region epoxide made a significant contribution to the liver microsome-induced DNA binding.     

Interindividual variation in binding of benzo[a]pyrene to DNA in cultured human bronchi

The binding of benzo[a]pyrene to DNA in cultured human bronchus was measured in specimens from 37 patients. The binding values ranged from 2 to 151 picomoles of benzo[a]pyrene per milligram of DNA with an overall mean +/- standard error of 34.2 +/- 5.2. This 75-fold interindividual variation in the binding of benzo[a]pyrene to DNA is similar in magnitude to that found in pharmacogenetic studies of drug metabolism. Aryl hydrocarbon hydroxylase is also inducible by benz[a]anthracene in the bronchial mucosa.     

The evidence of clonal evolution with monosomy 7 in aplastic anemia following granulocyte colony-stimulating factor using the polymerase chain reaction

The polycyclic aromatic hydrocarbons (PAH) containing fractions of smoked and charcoal-broiled foods, namely, Sheat fish (Kytopterus apogon), Mimrow (Crossocheilus reba), Freshwater catfish (Clarias batrachus), chicken wings, rice pork sausage and pork, in addition to naphthalene, acenaphthene, anthracene, phenanthrene, fluoranthene, pyrene, benz[a]anthracene, naphthacene, benzo[a]pyrene, benzo[e]pyrene, 9,10-dimethyl-1,2-benzanthracene, dibenz[ah]anthracene, benzo[ghi]perylene and coronene, were evaluated for their mutagenic potential using Salmonella typhimurium strains TA98 and TA100 in the absence of metabolic activation after being treated with nitrite (500 mM) for 4 hr at 37 degrees C and in acid solution pH 3.0-3.5. The presence of N-nitroso compounds was also determined. Results showed that nitrite could convert most samples to direct-acting mutagens towards both strains except for fluoranthene and benzo[ghi]perylene, which exhibit mutagenicity only with TA98. It was demonstrated that treatment of PAHs with nitrite in acid solution produced some non-N-nitroso direct-acting mutagens, suggesting that they might belong to nitro-PAHs. Therefore, the consumption of charcoal-broiled and smoked foods simultaneously with nitrite is not recommended.     

Bone marrow stromal cells constitutively express high levels of cytochrome P4501B1 that metabolize 7,12-dimethylbenz[a]anthracene

The polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA) is a potent carcinogen that produces immunotoxic effects in bone marrow. Here, we show that bone marrow stromal cells metabolize DMBA to such products as 3,4-dihydrodiol, the precursor to the most mutagenic DMBA metabolite. The BMS2 bone marrow stromal cell line constitutively expressed higher levels of CYP1B1 protein and mRNA than C3H10T1/2 mouse embryo fibroblasts. BMS2 cells also produced a DMBA metabolite profile that was consistent with CYP1B1 activity. Treatment with the potent aryl hydrocarbon receptor (AhR) ligand 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced a approximately 2-fold increase in CYP1B1 mRNA, protein, and activity in BMS2 cells. Two forms of the AhR (97 and 104 kDa) and the AhR nuclear translocator were detected in BMS2 cells. The AhR translocated to the nucleus after treatment with TCDD or DMBA but was approximately 5 times slower with DMBA. Primary bone marrow stromal (BMS) cell cultures established from AhR-/- mice showed similar basal CYP1B1 expression and activity as cell cultures established from heterozygous littermates or C57BL/6 mice. However, primary BMS cells from AhR-/- mice did not exhibit increased CYP1B1 protein expression after incubation with TCDD. BMS cells therefore constitutively express functional CYP1B1 that is not dependent on the AhR. This contrasts with embryo fibroblasts from the same mouse strain, in which basal CYP1B1 expression is AhR dependent. We therefore conclude that bone marrow toxicity may be mediated by CYP1B1-dependent DMBA metabolism, which is regulated by factors other than the AhR.     

K-region oxides and imines derived from alkylated benz[a]anthracene congeners: synthesis, stability in aqueous media and mutagenicity

The K-region oxides and imines of benz[a]anthracene, 1-methylbenz[a]anthracene, 7-methylbenz[a]anthracene, 7-ethylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene were synthesized and characterized (melting point, 1H-NMR and electron impact mass spectra, elemental analysis, IR spectroscopy). All 10 compounds showed high mutagenic activity in Salmonella typhimurium (reversion of his- strains TA97, TA98, TA100 and TA104). The arene imines were more potent than the corresponding arene oxides. Alkyl substitutions strongly influenced the activities. Furthermore, all compounds were more active when exposure took place in the absence of inorganic ions than when KCl (125 mM) was present. The influence of the exposure medium was more pronounced with strain TA98 than with strain TA100. The half-lives of the test compounds were determined from mutagenicity experiments in which the compound was added to the exposure medium at varying times before the bacteria. In dilute sodium phosphate buffer (10 mM, pH 7.4), the half-lives of these chemicals (or their biological activity) varied from 0.5 to 110 min. Addition of KCl (150 mM) did not measurably affect the half-lives of some test compounds and appeared to slightly shorten those of others. Therefore, it is unlikely that the strong effect of KCl on mutagenicity and the dependence of this effect on the bacterial strain used can be explained by influences of KCl on the test compounds. Rather, it appears more likely than an effect of KCl on the bacteria may be an important factor. This study provides further examples of strong influences of unobtrusive media components on mutagenicity. It also demonstrates that small structural changes (alkyl substituents at diverse positions of the aromatic system) may play an important role in chemical reactivity and biological activity.     

An automated liquid chromatographic plasma analysis of amino acids used in combination with positron emission tomography (PET) for determination of in vivo plasma kinetics

Quantification of physiological processes measured with positron emission tomography (PET) requires an "input" function which can be the concentration of administered radio-tracer in plasma. Radioactive nuclides used in PET have short half lives (2-20 min) and a limited time is available for the PET investigation including analysis of the composition of the radioactive signal in plasma. Therefore, an automated method for the analysis and separation of beta-[11C]-L-5-hydroxy-tryptophan ([11C]-L-5-HTP), beta-[11C]-L-3,4-dihydroxy-phenylalanine ([11C]-L-DOPA) and L-[methyl-11C]-methionine and their respective metabolites in plasma was developed. A size exclusion exclusion column was used for isolation of the low molecular weight fraction. In the case of [11C]-L-5-HTP and [11C]-L-DOPA, the low molecular weight fraction was injected onto a liquid chromatographic system for separation of radioactive tracer from in vivo formed radio-labelled metabolites. The elution volume from the size exclusion column was 7.0, 5.0 and 3.5 ml for [11C]-L-5-HTP, [11C]-L-DOPA and L-[methyl-11C]-methionine, respectively. An interaction with the column matrix and the solutes was observed for both [11C]-L-5-HTP and [11C]-L-DOPA. The yield in the isolation step was > 98%. Separation of [11C]-L-5-HTP and [11C]-L-DOPA from their respective metabolites was performed with high-performance liquid chromatography with automated collection of fractions of the eluate corresponding to those of administered tracer and metabolites. The fractions were measured for radioactivity in a well counter.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-performance liquid chromatography study of stereospecific microsomal enzymes catalysing the reduction of a potential cytostatic drug, oracin. Interspecies comparison

One of the main metabolites of oracin (I) ?6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2- c] isoquinoline?, a potential cytostatic drug, is 11-dihydrooracin (II) ?(+),(-)-6-[2-(2-hydroxyethyl)aminoethyl]-5-oxo-11-hydroxy-5,6-dihydro-1 1H- indeno[1,2-c]isoquinoline?, a metabolite formed by the reduction of oracin's pro-chiral centre on C 11. This metabolite has been found in all laboratory species in vitro and in vivo and it constitutes the main metabolite in man. The stereospecificity of reducing enzymes participating in the oracin biotransformation pathway was investigated using microsomal preparations from standard laboratory animals. Enzyme stereospecificity has been defined as preferential formation by the enzyme of the (+) or (-) stereoisomer of II. Significant interspecies differences were observed in the stereospecificity of the respective biotransformation enzymes. HPLC quantitative determinations of both enantiomers were performed using a Chiralcel OD-R column as chiral stationary phase with excellent resolution and stability.     

Metabolic activation of aromatic hydrocarbons in purified rat liver nuclei: induction of enzyme activities and binding to DNA with and without monooxygenase-catalyzed formation of active oxygen

Purified rat liver nuclei covalently bound low levels of seven aromatic [14C]hydrocarbons to nuclear DNA. Induction with 3-methylcholanthrene increased the binding of six carcinogenic hydorcarbons, but did not raise the level of binding of noncarcinogenic anthracence. Removal of the nuclear envelope by Triton N-101 eliminated binding and aryl hydrocarbon hydroxylase activities and cytochrome P-450 from the nuclei. Binding of two of two strong carcinogens, benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene, to nuclear DNA was compared to the levels of aryl hydrocarbon hydroxylase and cytochrome P-450 in nuclei from uninduced and benz[a]anthracene-, 3-methylcholanthrene-, and phenobarbital-induced rats. Microsomal hydroxylase and cytochrome P-450 were also assayed. Induction with 3-methylcholanthrene gave the largest increases in nuclear activities: 11 times as much hydroxylase, 6 times as much cytochrome P-450, and 4 times as much binding of both hydrocarbons. Benz[a]anthracene and phenobarbital induced these nuclear activities 0- to 4-fold. In the presence of added NADPH, binding of benzol[a]pyrene to DNA by nuclei increased rapidly for at least 20 min. When NADPH was not added, the reaction stopped at a low level in 5 min. When CO was bubbled through the reaction mixture with or without added NADPH, binding of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene was partially inhibited, indicating that cytochrome P-450 plays a role in this activation. Since no nuclear hydroxylase activity was seen without added NADPH or in the presence of CO, activation and subsequent binding of hydrocarbons to nuclear DNA, at least in part, does not require the activated oxygen used in monooxygenase reactions.     

Occurrence of Gasterophilus intestinalis and some parasitic nematodes of horses in Sweden

The biological accuracy of a nonlinear compartmental model describing the in vivo kinetics of L-3,4-dihydroxy-6-[18F]fluorophenylalanine ([18F]FDOPA) metabolism was investigated. Tissue activities for [18F]FDOPA and its labeled metabolites 3-O-methyl-[18F]FDOPA ([18F]OMFD), 6-[18F]fluorodopamine ([18F]FDA), L-3,4-dihydroxy-6-[18F]fluorophenylacetic acid ([18F]FDOPAC), and 6-[18F]fluorohomovanillic acid ([18F]FHVA) were calculated using a plasma [18F]FDOPA input function, and kinetic constants estimated previously by chromatographic fractionation of 18F-labeled compounds in plasma and brain extracts from rat. Present data accurately reflected the measured radiochemical composition in rat brain for tracer circulation times past 10 min. We formulated the hypothesis that the discrepancy between calculated and measured fractions of [18F]FDOPA and the deaminated metabolite [18F]FDOPAC at times earlier than 10 min reflected storage of [18F]FDA in vesicles without monoamine oxidase. This hypothesis explained the initially rapid appearance of [18F]FDOPAC in striatum by delayed transfer of [18F]FDA from cytosol into vesicles. We conclude that the simpler model of [18F]FDOPA compartmentation is accurate when the cytosolic and vesicular fractions of [18F]FDA are at steady-state; the approach to equilibrium has a time constant of 15-30 min. The present model is valid for positron emission tomography studies of [18F]FDOPA metabolism in living brain.     

cDNA cloning, sequence analysis, and induction by aryl hydrocarbons of a murine cytochrome P450 gene, Cyp1b1

C3H mouse embryo fibroblast cells, designated 10T1/2, can be transformed by physical and chemical agents including polycyclic aromatic hydrocarbons. In a previous report (Shen et al., Proc. Natl. Acad. Sci. USA 90, 11483-11487, 1993), we identified a cytochrome P450 gene induced by polycyclic aromatic hydrocarbons (PAHs) that is different from 1A1 or 1A2, and which we tentatively named P450CMEF. Here, we report the entire cDNA sequence of P450CMEF (5,128 bp) and the amino acid sequence deduced from it (543 residues). A comparison of the latter sequence with known cytochrome P450s indicates that P450CMEF is in a new subfamily of family 1 of the P450 superfamily. Accordingly, the Committee on Standardized Cytochrome P450 Nomenclature designated the gene Cyp1b1. Exposure to various aryl hydrocarbons (2.5 hr) induced Cyp1b1 mRNA in 10T1/2 cells to different degrees: 2,3,7,8-tetrachlorodibenzo-p-dioxin, 7,12-dimethylbenz[a]anthracene, benz[a]anthracene, benzo[a]pyrene, and beta-naphthoflavone were strong inducers; alpha-naphthoflavone and 3-methylcholanthrene, were moderate inducers; and benzo[e]pyrene was a weak inducer.