Regiospecific oxidation of polycyclic aromatic dihydrodiols by rat liver dihydrodiol dehydrogenase

Rat liver dihydrodiol dehydrogenase (DDH, E.C. 1.3.1.20) has recently been shown to oxidize the highly carcinogenic benz[a]anthracene-3,4- dihydrodiol in an NADP(+)-dependent reaction to its corresponding catechol. The present study is a systematic investigation of the substrate specificity of the purified enzyme towards synthetic trans-dihydrodiol metabolites of phenanthrene, benz[a]anthracene, chrysene, dibenz[a, h]anthracene and benzo[a]pyrene. DDH exhibited a remarkable regiospecificity of enzymatic catalysis with regard to the site of the dihydrodiol moiety of the parent hydrocarbon. M-region- and, with lower efficiency, bay-region dihydrodiols were found to be good substrates of the enzyme with maximal velocities between 20-80 nmol/min per mg enzyme and Km values in the micromolar range. K-region dihydrodiols were not accepted as substrates. Dihydrodiols situated at the terminal ring of an anthracene-type structure such as benz[a]anthracene-8,9-dihydrodiol as well as the corresponding dihydrodiol epoxides were also not oxidized by DDH at measurable rates. The results provide evidence for a detoxifying role of DDH in the metabolism of the chemical carcinogens benz[a]anthracene, chrysene and dibenz[a, h]anthracene.     

Stereoselectivity and regioselectivity of uridine-5'-diphosphoglucuronosyltransferase toward vicinal dihydrodiols of polycyclic aromatic hydrocarbons

The ability of a purified rat liver microsomal uridine-5'-diphosphoglucuronosyltransferase to catalyze the glucuronidation of stereoisomeric trans- and cis-9, 10-dihydroxy-9, 10-dihydrophenanthrenes and 4, 5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes is examined. The enzyme shows the ability to discriminate kinetically between the antipodes of trans-9, 10-dihydroxy-9, 10-dihydrophenanthrene with turnover numbers of 0.070 and 1.4 s-1 and kc/Kmapp values of 4.4 X 10(3) and 1.1 X 10(3) M-1 s-1 for the 9R, 10R and 9S, 10S stereoisomers. Glucuronidation of the nondissymmetric cis-9, 10-dihydroxy-9, 10-dihydrophenanthrene proceeds with a turnover number of 0.037 s-1 and kc/Kmapp of 18 X 10(3) M-1 s-1 to give a 60/40 mixture of the two possible diastereomeric products. Three of the four stereoisomers of 4,5-dihydroxy-4,5-dihydrobenzo[alpha] pyrene are regioselectively glucuronidated by the enzyme with a high degree of kinetic discrimination. Turnover numbers for the 4S,5S, 4R,5R, and 4S,5R stereoisomers are 4.1, 0.37, and 0.23 s-1 with kc/Kmapp values of 23.8 X 10(3), 0.23 X 10(3), and 3.15 X 10(3) M-1 s-1, respectively. The 4R,5S cis isomer is not a substrate. Enzyme-catalyzed reactions of the 4S,5S and 4S,5R isomers give exclusively (greater than or equal to 95%) the 4-glucuronide with the 4R,5R isomer giving the 5-glucuronide. The kinetic and regiochemical results indicate that the enzyme recognizes hydroxyl groups on the beta-face or bottom face of the 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioactivation of xenobiotics by formation of toxic glutathione conjugates

Evidence has been accumulating that several classes of compounds are converted by glutathione conjugate formation to toxic metabolites. The aim of this review is to summarize the current knowledge on the biosynthesis and toxicity of glutathione S-conjugates derived from halogenated alkanes, halogenated alkenes, and hydroquinones and quinones. Different types of toxic glutathione conjugates have been identified and will be discussed in detail: (i) conjugates which are transformed to electrophilic sulfur mustards, (ii) conjugates which are converted to toxic metabolites in an enzyme-catalyzed multistep mechanism, (iii) conjugates which serve as a transport form for toxic quinones and (iv) reversible glutathione conjugate formation and release of the toxic agent in cell types with lower glutathione concentrations. The kidney is the main, with some compounds the exclusive, target organ for compounds metabolized by pathways (i) to (iii). Selective toxicity to the kidney is easily explained due to the capability of the kidney to accumulate intermediates formed by processing of S-conjugates and to bioactivate these intermediates to toxic metabolites. The influences of other factors participating in the renal susceptibility are discussed.     

Purification and characterization of an arene cis-dihydrodiol dehydrogenase endowed with broad substrate specificity toward polycyclic aromatic hydrocarbon dihydrodiols

Initial reactions involved in the bacterial degradation of polycyclic aromatic hydrocarbons (PAHs) include a ring-dihydroxylation catalyzed by a dioxygenase and a subsequent oxidation of the dihydrodiol products by a dehydrogenase. In this study, the dihydrodiol dehydrogenase from the PAH-degrading Sphingomonas strain CHY-1 has been characterized. The bphB gene encoding PAH dihydrodiol dehydrogenase (PDDH) was cloned and overexpressed as a His-tagged protein. The recombinant protein was purified as a homotetramer with an apparent Mr of 110,000. PDDH oxidized the cis-dihydrodiols derived from biphenyl and eight polycyclic hydrocarbons, including chrysene, benz[a]anthracene, and benzo[a]pyrene, to corresponding catechols. Remarkably, the enzyme oxidized pyrene 4,5-dihydrodiol, whereas pyrene is not metabolized by strain CHY-1. The PAH catechols produced by PDDH rapidly auto-oxidized in air but were regenerated upon reaction of the o-quinones formed with NADH. Kinetic analyses performed under anoxic conditions revealed that the enzyme efficiently utilized two- to four-ring dihydrodiols, with Km values in the range of 1.4 to 7.1 microM, and exhibited a much higher Michaelis constant for NAD+ (Km of 160 microM). At pH 7.0, the specificity constant ranged from (1.3 +/- 0.1) x 10(6) M(-1) s(-1) with benz[a]anthracene 1,2-dihydrodiol to (20.0 +/- 0.8) x 10(6) M(-1) s(-1) with naphthalene 1,2-dihydrodiol. The catalytic activity of the enzyme was 13-fold higher at pH 9.5. PDDH was subjected to inhibition by NADH and by 3,4-dihydroxyphenanthrene, and the inhibition patterns suggested that the mechanism of the reaction was ordered Bi Bi. The regulation of PDDH activity appears as a means to prevent the accumulation of PAH catechols in bacterial cells.     

Peroxidase-mediated conjugation of glutathione to unsaturated phenylpropanoids. Evidence against glutathione S-transferase involvement

A number of plant species are thought to possess a glutathione S-transferase enzyme (GST: EC 2.5.1.18) that will conjugate glutathione (GSH) to trans -cinnamic acid (CA) and para -coumaric acid (4-CA). However, we present evidence that this activity is mediated by peroxidase enzymes and not GSTs. The N-terminal amino acid sequence of the GSH-conjugating enzyme purified from etiolated corn shoots exhibited a strong degree of homology to cytosolic ascorbate peroxidase enzymes (APX: EC 1.11.1.11) from a number of plant species. The GSH-conjugating and APX activities of corn could not be separated during chromatography on hydrophobic-interaction. anion-exchange, and gel filtration columns. Spectral analysis of the enzyme revealed that the protein had a Soret band at 405 nm. When the enzyme was reduced with dithionite, the peak was shifted to 423 nm with an additional peak at 554 nm. The spectrum of the dithionite-reduced enzyme in the presence of 0.1 m M KCN exhibited peaks at 430, 534 and 563 nm. These spectra are consistent with the presence of a heme moiety. The GSH-conjugating and APX activities of the enzyme were both inhibited by KCN. NaN₃, p -chloromercuribenzoate ( p CMB), and iodoacetate. The APX specific activity of the enzyme was 1.5-fold greater than the GSH-conjugating specific activity with 4-CA. In addition to the corn enzyme, a pea recombinant APX (rAPX) and horseradish peroxidase (HRP; EC 1.11.1.7) were also able to conjugate GSH to CA and 4-CA. The peroxidase enzymes may generate thiyl free radicals of GSH that react with the alkyl double bond of CA and 4-CA resulting in the formation of a GSH conjugate.     

Peroxidase-mediated formation of the fungal polyphenol 3,14'-bihispidinyl

Medicinal fungi, Phellinus linteus and Inonotus xeranticus, produce a cluster of yellow pigment in their fermentation broth that acts as an important element of biological activity. The pigment is composed of diverse polyphenols with a styrylpyrone moiety, mainly hispidin and its dimers, 3,14'-bihispidinyl, hypholomine B, and 1,1- distrylpyrylethan. Although dimeric hispidins were proposed to be biosynthesized from two molecules of monomer via oxidative coupling by ligninolytic enzymes, laccase and peroxidase, the details of this process remain unknown. In this preliminary study, we attempted to achieve enzymatic synthesis of the hispidin dimer from hispidin by using commercially available horseradish peroxidase (HRP). Consequently, a hispidin dimer, 3,14'-bihispidinyl, was synthesized, whereas the other dimers, hypholomine B and 1,1-distrylpyrylethan, were not produced. This result suggested that the oxidative coupling at the C-3 and C-14' positions of hispidins was dominant in the process of dimerization by HRP, and indicated that additional catalysts or substrates would be needed to synthesize other hispidin dimers present in the fungal metabolite.     

Photoproducts from DNA pyrimidine bases and polycyclic aromatic hydrocarbons

The major photoproduct formed between benzo[a]pyrene and thymine is identified as 1-(benzo[a]pyren-6-yl)-thymine by means of spectroscopic analysis and isotopic syntheses. Irradiation of 1-methylcytosine hydrochloride and anthracene gives two isolable photoproducts of which one is assigned the structure 5-(anthracen-9-yl)-1-methylcytosine.     

The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates

The metabolism of styrene by prostaglandin hydroperoxidase and horseradish peroxidase was examined. Ram seminal vesicle microsomes in the presence of arachidonic acid or hydrogen peroxide and glutathione converted styrene to glutathione adducts. Neither styrene 7,8-oxide nor styrene glycol was detected as a product in the incubation. Also, the addition of styrene 7,8-oxide and glutathione to ram seminal vesicle microsomes did not yield styrene glutathione adducts. The peroxidase-generated styrene glutathione adducts were isolated by high pressure liquid chromatography and characterized by NMR and tandem mass spectrometry as a mixture of (2R)- and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione. (1R)- and (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione were not formed by the peroxidase system. The addition of phenol or aminopyrine to incubations, which greatly enhances the oxidation of glutathione to a thiyl radical by peroxidases, increased the formation of styrene glutathione adducts. We propose a new mechanism for the formation of glutathione adducts that is independent of epoxide formation but dependent on the initial oxidation of glutathione to a thiyl radical by the peroxidase, and the subsequent reaction of the thiyl radical with a suitable substrate, such as styrene.     

Bacterial oxidation of chemical carcinogens: formation of polycyclic aromatic acids from benz[a]anthracene

A Beijerinckia strain designated strain B1 was shown to oxidize benz[a]anthracene after induction with biphenyl, m-xylene, and salicylate. Biotransformation experiments showed that after 14 h a maximum of 56% of the benz[a]anthracene was converted to an isomeric mixture of three o-hydroxypolyaromatic acids. Nuclear magnetic resonance and mass spectral analyses led to the identification of the major metabolite as 1-hydroxy-2-anthranoic acid. Two minor metabolites were also isolated and identified as 2-hydroxy-3-phenanthroic acid and 3-hydroxy-2-phenanthroic acid. Mineralization experiments with [12-14C]benz[a]anthracene led to the formation of 14CO2. These results show that the hydroxy acids can be further oxidized and that at least two rings of the benz[a]anthracene molecule can be degraded.     

Removal of polycyclic aromatic hydrocarbons from oil-contaminated Kuwaiti soil

In the uncontaminated farm soil, more than 80% of the supplemented acenaphthene, fluoranthene, and pyrene (100 mg/100 g soil) decreased in 90 days, while ratio of removal was about 20%, 30%, and 0%, respectively, in the Kuwaiti oil-contaminated soil. Simultaneous addition of naphthalene, phenanthrene, and anthrathene (100 mg of each compound/100 g soil) led the acenaphthene to a decrease of about 20% to 45% but not of fluoranthene and pyrene. Addition of the farm soil to the Kuwaiti soil did not enhance the decrease of these three PAHs.     

Biodegradation of polycyclic aromatic hydrocarbons

The intent of this review is to provide an outline of the microbial degradation of polycyclic aromatic hydrocarbons. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons are examined in some detail. The pathways of polycyclic aromatic hydrocarbon catabolism are discussed. Studies are presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.     

Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs)

The catalysis of glutathione (GSH) conjugation to epoxyeicosatrienoic acids (EETs) by various purified isozymes of glutathione S-transferase was studied. A GSH conjugate of 14,15-EET was isolated by HPLC and TLC; this metabolite contained one molecule of EET and one molecule of GSH. Fast atom bombardment mass spectrometry of the isolated metabolite confirmed the structure as a GSH conjugate of 14,15-EET. Studies designed to determine the isozyme specificity of this reaction demonstrated that two isozymes, 3-3, and 5-5, efficiently catalyzed this conjugation reaction. The Km values for 14,15-EET were approximately 10 microM and the Vmax values ranged from 25 to 60 nmol conjugate formed min-1 mg-1 purified transferase 3-3 and 5-5. The 5,6-, 8,9-, and 11,12-EETs were also substrates for the reaction, albeit at lower rates. These results demonstrate that the EETs can serve as substrates for the cytosolic glutathione S-transferases.     

Bioavailability of polycyclic aromatic hydrocarbons and formation of humic acid-like residues during bacterial PAH degradation

 The degradation of single polycyclic aromatic hydrocarbons (PAHs: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene and pyrene) and a mixture of all seven PAHs by a bacterial culture enriched from contaminated soil resulted in the formation of a dark-coloured residual fraction of dissolved (DOM) and particulate organic matter (POM). This fraction was highly resistant to bacterial degradation. Analysis of the DOM revealed a molecular-size-distribution similar to that of natural humic acids. A complete degradation of PAHs was apparently prevented by an irreversible incorporation of about 10% of the carbon from single PAHs or 20% of the carbon from the mixture of seven PAHs into the DOM- and POM- fraction. Some metabolites excreted during bacterial PAH-degradation were identified as known precursors for humification. Received: 1 June 1999 / Received revision: 16 July 1999 / Accepted: 1 August 1999     

Bacterial oxidation of chemical carcinogens: formation of polycyclic aromatic acids from benz[a]anthracene.

A Beijerinckia strain designated strain B1 was shown to oxidize benz[a]anthracene after induction with biphenyl, m-xylene, and salicylate. Biotransformation experiments showed that after 14 h a maximum of 56% of the benz[a]anthracene was converted to an isomeric mixture of three o-hydroxypolyaromatic acids. Nuclear magnetic resonance and mass spectral analyses led to the identification of the major metabolite as 1-hydroxy-2-anthranoic acid. Two minor metabolites were also isolated and identified as 2-hydroxy-3-phenanthroic acid and 3-hydroxy-2-phenanthroic acid. Mineralization experiments with [12-14C]benz[a]anthracene led to the formation of 14CO2. These results show that the hydroxy acids can be further oxidized and that at least two rings of the benz[a]anthracene molecule can be degraded.     

Formation and identification of glutathione conjugates from 2-nitrosofluorene and N-hydroxy-2-aminofluorene

2-Nitrosofluorene (NOF) and N-hydroxy-2-aminofluorene (N-HO-AF) are potent direct-acting mutagens, derived from metabolic activation of the carcinogen, N-acetyl-2-aminofluorene (AAF). To assess the ability of cellular glutathione (GSH) to detoxify these electrophilic derivatives, we examined the reaction of NOF and N-HO-AF with GSH in vitro. Two reaction products were isolated and identified as glutathionyl derivatives of 2-aminofluorene (AF) containing an N-S linkage. Amino acid analysis, infrared and NMR (500 MHz) spectroscopy, fast atom bombardment mass spectrometry and analysis of reaction characteristics and hydrolysis products established their structures as N-(glutathion-S-yl)-2-aminofluorene S-oxide (GS-AF_I) and N-(glutathion-S-yl)-2-aminofluorene (GS-AF_II).Ascorbic acid, which reduces NOF to N-HO-AF, was used to modify reaction yields. These results indicated that GS-AF_I was derived from reaction with NOF and that GS-AF_II could be formed from both NOF and N-HO-AF. A reaction scheme is proposed in which NOF reacts with GSH to form an intermediate addition product that can rearrange either to GS-AF_I or be reduced to GS-AF_II. The latter could also be formed by direct reaction with N-HO-AF.Similar reactions were also carried out with N-hydroxy-1- and N-hydroxy-2-naphthylamine and their nitroso derivatives; also, reaction characteristics and effects of ascorbic acid on product yields suggested an analogous scheme. The role of GSH in the detoxification or activation of nitrosoarenes and N-hydroxy arylamines is discussed.     

Selective oxidation of mitochondrial glutathione in cultured rat adrenal cells and its relation to polycyclic aromatic hydrocarbon-induced cytotoxicity

Primary cultures of rat adrenal cells, as well as rat adrenals in vivo, are sensitive to the potent carcinogen 7,12-dimethylbenz[a]anthracene and its liver metabolite 7-hydroxymethyl-12-methylbenz[a]anthracene, whereas unmethylated polycyclic aromatic hydrocarbons like benzo[a]pyrene or benzo[a]anthracene are ineffective. The adrenocorticolytic potencies of the hydrocarbons are affected by adrenocorticotrophic hormone and various steroids, cytochrome P450 inhibitors, and antioxidants. In the present investigation digitonin was used to fractionate cultured rat adrenal cells. It was found that the mitochondria and cytosol of the cells contained 3-5 nmol/10(6) cells (approximately 15%) and 20-30 nmol/10(6) cells (approximately 85%) of the total soluble cellular glutathione equivalents, respectively. After exposing the cells to 7-hydroxymethyl-12-methylbenz[a]anthracene in the culture medium, a time- and concentration-dependent selective oxidation of mitochondrial glutathione was observed, whereas the effect on the cytosolic glutathione was negligible. Under the same conditions, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene were unable to alter the redox levels of the subcellular pools of glutathione. Omission of adrenocorticotrophic hormone lowered the oxidation of mitochondrial glutathione induced by 7-hydroxymethyl-12-methylbenz[a]anthracene about twofold. The results suggest that rat adrenal cells contain two separate pools of glutathione, one cytosolic and one mitochondrial, of which the latter is selectively influenced by 7-hydroxymethyl-12-methylbenz[a]anthracene. Moreover, it is concluded that rat adrenal cells offer a unique model system for general studies of the effects of a selective oxidation of mitochondrial glutathione on various cell functions. These effects may constitute early changes in cytotoxicity, preceding, e.g., membrane damage and loss of cytosolic components.     

Optimized extraction of polycyclic aromatic hydrocarbons from contaminated soil samples

A comparison of Soxhlet extraction and a new extraction technique, fluidized-bed extraction, has been conducted. The extraction of polycyclic aromatic hydrocarbons (PAHs) by this new technique has been optimized considering as experimental variables the variation of the number of extraction cycles and the holding time after reaching the heating temperature by means of a surface response design. The significance of the operational parameters of the fluidized-bed extraction onto the performance characteristics has been investigated. For the determination of the analytes, a cleanup of the extracts followed by gas chromatography with mass spectrometric detection was used. The accuracy of the method was established by extraction and analysis of a reference material, supplied from the European Commission's Joint Research Centre.