Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.     

Molecular orbital study of porphyrin-substrate interactions in cytochrome P450 catalysed aromatic hydroxylation of substituted anilines

The reaction mechanism for the primary reaction step of the hydroxylation of 3-fluoro-6-methylaniline, attacked at different positions (oxygen attack across a C-C bond and direct attack at positions para and ortho with respect to the NH(2)-group) catalysed by a high-valent ferryl-oxo porphyrin a(2u)-cation complex with H(3)CS(-) as an axial ligand, has been investigated on the basis of electronic structure calculations in local spin-density approximation. Non-repulsive potential curves are obtained only in cases of direct attack at the para- and ortho-positions with respect to NH(2), but not for epoxide formation. Comparing the potential curves for the hydroxylation at the positions para and ortho to the NH(2)-group, an attack at the para-position is more likely. The relative orientation of the substrate towards the porphyrin is essentially determined by the interaction between the substituents of the substrate and the porphyrin. Consequently, different geometrical orientations of the substrate are obtained for hydroxylation at the para- and ortho-positions. In both cases of direct attack the substrate plane is not parallel to the porphyrin plane. The decisive role of sulphur in the hydroxylation is demonstrated by the participation of the S(3p)-orbitals in all molecular orbitals involved in the reaction.     

Hydroxylation of biphenyl by the yeast Trichosporon mucoides

Hydroxylation of biphenyl by the dibenzofuran-degrading yeast Trichosporon mucoides SBUG 801 was studied. Glucose-grown cells degraded 40% of the biphenyl added within the first 24 h of incubation. The first step in the biotransformation pathway was the monohydroxylation of the biaryl compound to produce 2-, 3-, and 4-hydroxybiphenyl. Further oxidation produced seven dihydroxylated intermediates; the second hydroxyl group was added either on the aromatic ring already hydroxylated or on the second ring. Of all metabolites, 2,5-dihydroxybiphenyl accumulated in the supernatant in the highest concentration. The initial hydroxylation favors the 4-position to produce 4-hydroxybiphenyl, which is subsequently hydroxylated to form 3,4-dihydroxybiphenyl. When biphenyl was replaced as a substrate by 4-hydroxybiphenyl, further hydroxylation of the intermediate 3,4-dihydroxybiphenyl resulted in 3,4,4'-trihydroxybiphenyl. Incubation of T. mucoides with biphenyl and 18O2 indicated a monooxygenase-catalyzed reaction in the oxidation of biphenyl. The hydroxylation was inhibited by 1-aminobenzotriazole and metyrapone, known cytochrome P450 inhibitors. These results are very similar to those observed in the biotransformation of biphenyl in mammals.     

Microsomal hydroxylation of specifically deuterated monosubstituted benzenes. Evidence for direct aromatic hydroxylation

The aromatic hydroxylation of six pairs of selectively deuterated monosubstituted benzenes was investigated with rat liver microsomes of various induction states. The substrates studied included 3,5-D2C6H3X (1a-6a) and 2,4,6-D3C6H2X (1b-6b), where X = Br, CN, NO2, OCH3, CH3, or Ph, respectively. The deuterium content of the ortho, meta, and para hydroxylated metabolites, as well as side chain oxidation products from 4 and 5, was determined by capillary gas chromatography-mass spectroscopy. These data were analyzed according to a hypothetical model in which a molecule of substrate can undergo either direct aromatic hydroxylation (defined as obligatory and complete loss of deuterium from the site of hydroxylation) or indirect aromatic hydroxylation (defined as the obligatory and complete shift of deuterium to an adjacent position, followed by its partial loss as governed by a kinetic deuterium isotope effect). From this and other analyses of the data the following conclusions were reached. (1) The relative extent of meta hydroxylation increased and the total yield of metabolites decreased as the substituents X became more electron withdrawing. (2) The induction state of the microsomes altered the regioselectivity of hydroxylation (2, 3, 4, or side chain) noticeably and predictably but had little or no effect on the retention or loss of deuterium during each hydroxylation. (3) With each substrate and at each ring position hydroxylation was found to occur by a combination of direct and indirect mechanisms. (4) The relative importance of direct vs. indirect mechanisms did not vary in a simple manner with either the position of hydroxylation or the nature of the substituent X.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp. LB400

Abstract In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. LB400, recombinant E. coli strains expressing the respective genes were constructed. Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho -dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour. Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.     

Biotransformation of Biphenyl by Paecilomyces lilacinus and Characterization of Ring Cleavage Products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4′-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Metabolism of radioactive 17 beta-estradiol 3-methyl ether by humans.

A mixture of 2-3H and 4-14C-17beta-estradiol 3-methyl ether was administered orally to a man and to a woman. 34 and 35 percent of the 3H was liberated into the body water of the man and of the woman, respectively, reflecting reactions involving position 2. The metabolism of estradiol methyl ether was qualitatively similar to that observed previously for radioactive estradiol administered intravenously to the same subjects, as judged by the measurement of various urinary metabolites by reverse isotope dilution. Evidence was obtained for hydroxylation at position 2 without demethylation by the isolation of urinary 2-hydroxyestrone 3-methyl ether which retained 33% of the original 3H. This 3H was presumably at position 1, resulted from an NIH shift which does not occur during hydroxylation of estrone or estradiol. This was confirmed by subsequent administration of a mixture of 4-14C and 3H-(methoxyl)-estradiol 3-methyl ether to the man. There was no evidence (by reverse isotope dilution) for 1-hydroxyestrone, 1-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether or 4-hydroxyestradiol 3-methyl ether as urinary metabolites of estradiol 3-methyl ether.     

Microsomal monooxygenation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Synthesis and identification of cis and trans monohydroxylated products.

Liver microsomal hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur on the cyclohexyl ring at positions 3 and 4. Four metabolites were isolated by selective solvent extraction and purifed by high-pressure liquid chromatography. cis-4-, trans-4-, cis-3-, and trans-3-OH derivatives of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea were synthesized and their chromatographic, mass spectral, and nuclear magnetic resonance characteristics matched those of the metabolites. The position of ring hydroxylation and the identity of each geometric isomer were established by nuclear magnetic resonance using a shift reagent in conjunction with spin decoupling techniques. Microsomes from rats pretreated with phenobarbital showed a sixfold increase in hydroxylation rate (19.5 vs. 3.3 nmol per mg per min). The induction was quite selective for cis-4 hydroxylation (19-fold); however, induction of trans-4 (threefold), cis-3 (threefold), and trans-3 (twofold) hydroxylation did occur. Quantitatively the cis-4-hydroxy metabolite was 67of the total product by phenobarbital-induced microsomes and 21% for normal microsomes. Microsomes from animals pretreated wit- 3-methyl-cholanthrene gave about the same rate and product distribution that normal microsomes gave. A mixture of 80% carbon monoxide-20% oxygen inhibited formation of all four hydroxy metabolites with the inhibition ranging from 55 to 78%.     

Studies on anabolic steroids--11. 18-hydroxylated metabolites of mesterolone, methenolone and stenbolone: new steroids isolated from human urine.

New metabolites of mesterolone, methenolone and stenbolone bearing a C18 hydroxyl group were isolated from the steroid glucuronide fraction of urine specimens collected after administration of single 50 mg doses of these steroids to human subjects. Mesterolone gave rise to four metabolites which were identified by gas chromatography/mass spectrometry as 18-hydroxy-1 alpha-methyl-5 alpha-androstan-3,17-dione 1, 3 alpha,18-dihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 2, 3 beta,18-dihydroxy-1-alpha-methyl-5 alpha-androstan-17-one 3 and 3 alpha,6 xi,18-trihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 4. These data suggest that mesterolone itself was not hydroxylated at C18, but rather 1 alpha-methyl-5 alpha-androstan-3,17-dione, an intermediate metabolite which results from oxidation of mesterolone 17-hydroxyl group. In addition to hydroxylation at C18, reduction of the 3-keto group and further hydroxylation at C6 were other reactions that led to the formation of these metabolites. It is of interest to note that in the case of both methenolone and stenbolone, only one 18-hydroxylated urinary metabolite namely 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 5 and 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 6 were both detected in post-administration urine specimens. These data indicate that the presence of a methyl group at the C1 or C2 positions in the steroids studied is a structural feature that seems to favor interaction of hepatic 18-hydroxylases with these steroids. These data provide further evidence that 18-hydroxylation of endogenous steroids can also occur in extra-adrenal sites in man.     

Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.     

Reductive debromination of the commercial polybrominated biphenyl mixture firemaster BP6 by anaerobic microorganisms from sediments.

Anaerobic microorganisms eluted from three sediments, one contaminated with polybrominated biphenyls (PBBs) and two contaminated with polychlorinated biphenyls, were compared for their ability to debrominate the commercial PBB mixture Firemaster. These microorganisms were incubated with reduced anaerobic mineral medium and noncontaminated sediment amended with Firemaster. Firemaster averages six bromines per biphenyl molecule; four of the bromines are substituted in the meta or para position. The inocula from all three sources were able to debrominate the meta and para positions. Microorganisms from the Pine River (St. Louis, Mich.) contaminated with Firemaster, the Hudson River (Hudson Falls, N.Y.) contaminated with Aroclor 1242, and Silver Lake (Pittsfield, Mass.) contaminated with Aroclor 1260 removed 32, 12, and 3% of the meta plus para bromines, respectively, after 32 weeks of incubation. This suggests that previous environmental exposure to PBBs enhances the debromination capability of the sediment microbial community through selection for different strains of microorganisms. The Pine River inoculum removed an average of 1.25 bromines per biphenyl molecule during a 32-week incubation period, resulting in a mixture potentially more accessible to aerobic degradation processes. No ortho bromine removal was observed. However, when Firemaster was incubated with Hudson River microorganisms that had been repeatedly transferred on a pyruvate medium amended with Aroclor 1242, 17% of the meta and para bromines were removed after 16 weeks of incubation and additional debromination products, including 2-bromobiphenyl and biphenyl, were detected. This suggests the possibility for ortho debromination, since all components of the Firemaster mixture have at least one ortho-substituted bromine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reductive debromination of the commercial polybrominated biphenyl mixture firemaster BP6 by anaerobic microorganisms from sediments.

Anaerobic microorganisms eluted from three sediments, one contaminated with polybrominated biphenyls (PBBs) and two contaminated with polychlorinated biphenyls, were compared for their ability to debrominate the commercial PBB mixture Firemaster. These microorganisms were incubated with reduced anaerobic mineral medium and noncontaminated sediment amended with Firemaster. Firemaster averages six bromines per biphenyl molecule; four of the bromines are substituted in the meta or para position. The inocula from all three sources were able to debrominate the meta and para positions. Microorganisms from the Pine River (St. Louis, Mich.) contaminated with Firemaster, the Hudson River (Hudson Falls, N.Y.) contaminated with Aroclor 1242, and Silver Lake (Pittsfield, Mass.) contaminated with Aroclor 1260 removed 32, 12, and 3% of the meta plus para bromines, respectively, after 32 weeks of incubation. This suggests that previous environmental exposure to PBBs enhances the debromination capability of the sediment microbial community through selection for different strains of microorganisms. The Pine River inoculum removed an average of 1.25 bromines per biphenyl molecule during a 32-week incubation period, resulting in a mixture potentially more accessible to aerobic degradation processes. No ortho bromine removal was observed. However, when Firemaster was incubated with Hudson River microorganisms that had been repeatedly transferred on a pyruvate medium amended with Aroclor 1242, 17% of the meta and para bromines were removed after 16 weeks of incubation and additional debromination products, including 2-bromobiphenyl and biphenyl, were detected. This suggests the possibility for ortho debromination, since all components of the Firemaster mixture have at least one ortho-substituted bromine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity

Toluene 4-monooxygenase (T4MO) is a diiron hydroxylase that exhibits high regiospecificity for para hydroxylation. This fidelity provides the basis for an assessment of the interplay between active site residues and protein complex formation in producing an essential biological outcome. The function of the T4MO catalytic complex (hydroxylase, T4moH, and effector protein T4moD) is evaluated with respect to effector protein concentration, the presence of T4MO electron-transfer components (Rieske ferredoxin, T4moC, and NADH oxidoreductase), and use of mutated T4moH isoforms with different hydroxylation regiospecificities. Steady-state kinetic analyses indicate that T4moC and T4moD form complexes of similar affinity with T4moH. At low T4moD concentrations, the steady-state hydroxylation rate is linearly dependent on T4moD-T4moH complex formation, whereas regiospecificity and the coupling efficiency between NADH consumption and hydroxylation are associated with intrinsic properties of the T4moD-T4moH complex. The optimized complex gives both efficient coupling and high regiospecificity with p-cresol representing >96% of total products from toluene. Similar coupling and regiospecificity for para hydroxylation are obtained with T3buV (an effector protein from a toluene 3-monooxygenase), demonstrating that effector protein binding does not uniquely determine or alter the regiospecificity of toluene hydroxylation. The omission of T4moD causes an approximately 20-fold decrease in hydroxylation rate, nearly complete uncoupling, and a decrease in regiospecificity so that p-cresol represents approximately 60% of total products. Similar shifts in regiospecificity are observed in oxidations of alternative substrates in the absence or upon the partial removal of either T4moD or T3buV from toluene oxidations. The mutated T4moH isoforms studied have apparent V(max)/K(M) specificities differing by approximately 2-4-fold and coupling efficiencies ranging from 88% to 95%, indicating comparable catalytic function, but also exhibit unique regiospecificity patterns for all substrates tested, suggesting unique substrate binding preferences within the active site. The G103L isoform has enhanced selectivity for ortho hydroxylation with all substrates tested except nitrobenzene, which gives only m-nitrophenol. The regiospecificity of the G103L isoform is comparable to that observed from naturally occurring variants of the toluene/benzene/o-xylene monooxygenase subfamily. Evolutionary and mechanistic implications of these findings are considered.     

Biotransformation of terodiline I. Identification of metabolites in dog urine by mass spectrometry

Nine metabolites of terodiline (N-tert-butyl-4,4-diphenyl-2-butylamine) have been identified in dog urine by various chromatographic techniques and mass spectrometry. The main metabolic pathway is aromatic hydroxylation, leading to the quantitatively most important metabolite, N-tert-butyl-4-(4-hydroxyphenyl)-4-phenyl-2-butylamine, and to two dihydroxylated metabolites, one mono substituted in both rings (N-tert-butyl-4,4'-bis(4-hydroxyphenyl)-2-butylamine), and one disubstituted in one ring (N-tert-butyl-4-(3,4-dihydroxyphenyl)-4-phenyl-2-butylamine). The latter is further metabolized by methylation, forming N-tert-butyl-4-(4-hydroxy-3-methoxyphenyl)-4-phenyl-2-butylamine, the second most abundant metabolite. Still another metabolite is formed by hydroxylation in the tert-butyl group to N-(2-hydroxymethyl-2-propyl)-4,4-diphenyl-2-butylamine. A very minor dihydroxylated metabolite results from oxidation both in an aromatic ring and in the tert-butyl group, giving N-(2-hydroxymethyl-2-propyl)-4-(4-hydroxyphenyl)-4-phenyl-2-butylamine. Oxidation of the carbon adjacent to the nitrogen and subsequent deamination gives the two ketones 4-(4-hydroxyphenyl)-4-phenyl-2-butanone and 4-(4-hydroxy-3-methoxyphenyl)-4-phenyl-2-butanone. Reduction of the carbonyl function in the former yields the corresponding alcohol, 4-(4-hydroxyphenyl)-4-phenyl-2-butanol. Some unchanged terodiline is also present. All metabolites formed by functionalization appear to be extensively conjugated, presumably with glucuronic acid.     

Regioselectivity of hydroxylation of prostaglandins by liver microsomes supported by NADPH versus H2O2 in methylcholanthrene-treated and control rats: formation of novel prostaglandin metabolites

The effects of methylcholanthrene (MC) treatment of male rats on the regioselectivity of hydroxylation of prostaglandins E1 and E2 (PGE1 and PGE2) by liver microsomes, supplemented with NADPH or H2O2, was examined. In the presence of NADPH, control microsomes catalyzed the hydroxylation at omega-1 (C19) and at omega-(C20) sites with minimal formation of novel monohydroxy metabolites of PGE1 and PGE2, referred to as compounds X1 and X2, respectively. Similarly, H2O2 supported the 19-hydroxylation and the formation of compounds X1 and X2, but yielded only minimal amounts of 20-hydroxy products. With NADPH, MC-treated microsomal incubations demonstrated only minor quantitative change in the 19- and 20-hydroxylation as compared with controls, but showed a 7- to 11-fold increase in formation of compound X1 and a 10-fold increase in formation of X2. By contrast with H2O2, MC-treatment increased by about 3-fold the 19- and 20-hydroxylation of PGE1 and by 35- to 46-fold the formation of X1; similarly, there was an approximate 2-fold increase in 19- and 20-hydroxylation of PGE2 and a 10-fold increase in formation of X2. These findings suggest that several monooxygenases are involved in catalyzing the hydroxylation at the various sites of the PGE molecule. Inhibitors of monooxygenases (SKF 525A, alpha-naphthoflavone, and imidazole derivatives) provided further evidence that the hydroxylation at the three sites of PGEs is catalyzed by different P-450 monooxygenases. It is striking that the inhibitors had a much lesser effect on the 20-hydroxylation of PGE1 as compared with other sites of hydroxylation. Structural identification of compounds X1 and X2 was elucidated as follows. Resistance of the PGB derivative of X1 to periodate oxidation and mass fragmentation analysis of the t-butyldimethylsilyl ether methyl ester, placed the hydroxylation at C17 or C18. Finally, mass fragmentation of trimethylsilyl ether methyl ester PGB derivatives of X1 and X2 provided conclusive evidence that X1 and X2 are 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively. The above findings indicate that the high regioselectivity of hydroxylation of PGE1 and PGE2, resulting in the formation of 18-hydroxy-PGE1 and 18-hydroxy-PGE2, respectively, is catalyzed by P-450 isozyme(s) which are induced by MC, possibly by P-450c.     

Characterisation of metabolites of 3-ethyl-3-(4-pyridyl)-piperidine-2,6-dione, a potential breast cancer drug

The identification of metabolites from the pyridylglutarimide 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione (PG, Rogletimide) was achieved using liquid chromatography—mass spectrometry with a thermospray interface (LC—TSP—MS). The urinary metabolites include PG N-oxide, the products of 4- and 5-hydroxylation in the piperidine residue (4- and 5-hydroxy-PG) and a γ-butyrolactone derived via terminal hydroxylation in the ethyl residue. In addition to the above metabolites, several products of glutarimide ring-opening could be detected in the plasma extracts after multiple-dose treatment. Thus LC—TSP—MS is potentially a simple and rapid technique in studies of drug metabolism for the important glutarimide class of drug.     

Synthesis and pharmacological evaluation of imidazoline sites I1 and I2 selective ligands

Several series of 2-aryl or heterocyclic-imidazoline compounds have been prepared and evaluated in vitro as imidazoline sites (I1 and I2) and alpha-adrenergic (alpha1 and alpha2) receptor ligands. Their pKi values indicate that linkage of the imidazoline moiety at the 2-position with an aromatic substituent dramatically decreases alpha-adrenergic affinity. I1 sites are more accessible by phenyl imidazolines substituted by a methyl or a methoxy group at the ortho or meta position. Indeed, 2-(2'-methoxyphenyl)-imidazoline (17) is one of the best I1 ligands ever reported (pKi = 8.53 and I1/I2 > 3388). On the other hand, I2 selectivity increases in the presence of a methyl group in the para position. The original compound, 2-(3'-fluoro-4'-tolyl)-imidazoline (31) is a new potent ligand for the I2 sites with high selectivity (pKi = 8.53 and I2/I1 > 3388).     

Alkylation and carbamoylation intermediates from the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl -1-nitrosourea (CCNU)

Evidence is presented which shows that 1-(2-chloroethyl) -3-cyclohexyl-1-nitrosourea (CCNU) upon degradation provides a 2-chloroethyl alkylating intermediate, possibly 2-chloroethyl carbonium ion, and 2-chloroethanol. Thiol alkylation occurs $\text{in vivo}$ and a major urinary metabolite of CCNU is thiodiacetic acid. A rapid microsomal hydroxylation of the cyclohexyl ring occurs which yields varying ratios of at least five metabolites: cis or trans 2-hydroxy, trans- 3-hydroxy, cis-3-hydroxy, cis-4-hydroxy and trans-4- hydroxy-CCNU. $\text{In vivo}$ carbamoylation appears to not be due to cyclohexylisocyanate but to the various hydroxy-cyclohexylisocyanates which are formed from hydroxy CCNU metabolites.     

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.