Polycyclic aromatic hydrocarbon oxidation during prostaglandin biosynthesis

Polycyclic hydrocarbons and other xenobiotics are cooxygenated during the oxidative metabolism of polyunsaturated fatty acids. The products of polycyclic hydrocarbon cooxygenation depend upon the hydrocarbon but they appear to be formed by radical (or electron transfer) mechanisms. The cooxygenations are hydroperoxide-dependent oxidations catalyzed by peroxidases and may be triggered by enzymes which biosynthesize fatty acid hydroperoxides. Prostaglandin endoperoxide synthetase is the principal hydroperoxide-generating system which has been studied to date. The extent to which polyunsaturated fatty acid-dependent cooxygenation operates $\text{in vivo}$ is uncertain but preliminary studies suggest it does occur.     

Stereospecificity of the hydrogen transfer catalyzed by human placental aldose reductase.

Placental aldose reductase (EC 1.1.1.21) was incubated with glucose in the presence of [4A-2H] NADPH prepared in the oxidation of [2-2H] isocitrate by isocitrate dehydrogenase (EC 1.1.1.42) or [4B-2H] NADPH prepared in the oxidation of [1-2H] glucose-6-phosphate dehydrogenase (EC 1.1.1.49). The sorbitol formed from [4A-2H] NADPH contained deuterium and from [4B-2H] NADPH it did not. Therefore, aldose reductase in an A-type enzyme.     

Polycyclic aromatic hydrocarbon degradation by biosurfactant-producing Pseudomonas sp. IR1

We characterized a newly isolated bacterium, designated as IR1, with respect to its ability to degrade polycyclic aromatic hydrocarbons (PAHs) and to produce biosurfactants. Isolated IR1 was identified as Pseudomonas putida by analysis of 16S rRNA sequences (99.6% homology). It was capable of utilizing two-, three- and four-ring PAHs but not hexadecane and octadecane as a sole carbon and energy source. PCR and DNA hybridization studies showed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by IR1 during growth on both water miscible and immiscible substrates. The biosurfactants lowered the surface tension of medium from 54.9 dN cm(-1) to 35.4 dN cm(-1) and formed a stable and compact emulsion with an emulsifying activity of 74% with diesel oil, when grown on dextrose. These findings indicate that this isolate may be useful for bioremediation of sites contaminated with aromatic hydrocarbons.     

Polycyclic aromatic hydrocarbon quinones may be either substrates for or irreversible inhibitors of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase.

Under aerobic conditions, 9,10-phenanthrenequinone and 5,6-chyrsenequinone undergo oxidation-reduction cycling in the presence of NADH and the NAD-linked 15-hydroxyprostaglandin dehydrogenase. This results in the formation of potentially hazardous semiquinones, the superoxide anion, and H2O2. Superoxide dismutase inhibits this cycling by destroying the free radical chain propagator, the superoxide anion. Four other polycyclic aromatic hydrocarbon quinones are not substrates of the enzyme and they cause it to undergo a time-dependent inactivation. This presumably results from alkylation of the enzyme. Glutathione fully protects the enzyme against inactivation by 1,2-naphthoquinone but is only partially effective against 7,8-benzo[a]pyrenequinone. These results suggest that in tissues which contain the NAD-linked 15-hydroxyprostaglandin dehydrogenase some polycyclic aromatic hydrocarbon quinones might produce deleterious effects by undergoing redox cycling. Others might cause such effects by irreversibly inhibiting the enzyme which catalyzes the first step in prostaglandin catabolism.     

Polycyclic aromatic hydrocarbon quinones and glutathione thioethers as substrates and inhibitors of the human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase

The human placental NADP-linked 15-hydroxyprostaglandin dehydrogenase catalyzes oxidoreduction at the 9- and 15-positions of many prostaglandins, but its catalytic efficiency (i.e. kcat/Km) for these reactions is low (Jarabak, J., Luncsford, A., and Berkowitz, D. (1983) Prostaglandins 26, 849-868). In the present study, we demonstrate that both K-region and non-K-region o-quinones of polycyclic aromatic hydrocarbons are excellent substrates for this enzyme. These compounds are reduced with kcat/Km values ranging from 3 to 20 X 10(6) S-1 M-1. The glutathione thioethers of menadione and toluquinone are reduced with similar catalytic efficiencies. Furthermore, these substances and certain other glutathione thioethers are potent inhibitors of prostaglandin B1 oxidation ([I50] = 7 X 10(-8) to 5 X 10(-6) M); while several glutathione thioethers also inhibit polycyclic aromatic hydrocarbon quinone reduction ([I50] = 1.7-6.5 microM). These findings raise the possibility that the potential toxicity of quinones of polycyclic aromatic hyrocarbons and other xenobiotic substances may be altered in the placenta by an oxidoreductase for which prostaglandins are relatively poor substrates. They also suggest that the presence in placental tissue of certain glutathione thioethers could influence the reduction of these quinones and other xenobiotic substances by this enzyme.     

Polycyclic aromatic hydrocarbon oxidation by the white-rot fungus Bjerkandera sp. strain BOS55 in the presence of nonionic surfactants

The effect of nonionic surfactants on the polycyclic aromatic hydrocarbon (PAH) oxidation rates by the extracellular ligninolytic enzyme system of the white-rot fungus Bjerkandera sp. strain BOS55 was investigated. Various surfactants increased the rate of anthracene, pyrene, and benzo[a]pyrene oxidation by two to fivefold. The stimulating effect of surfactants was found to be solely due to the increased bioavailability of PAH, indicating that the oxidation of PAH by the extracellular ligninolytic enzymes is limited by low compound bioavailability. The surfactants were shown to improve PAH dissolution rates by increasing their aqueous solubility and by decreasing the PAH precipitate particle size. The surfactant Tween 80 was mineralized by Bjerkandera sp. strain BOS55; as a result both the PAH solubilizing activity of Tween 80 and its stimulatory effect on anthracene and pyrene oxidation rates were lost within 24 h after addition to 6-day-old cultures. It was observed that the surfactant dispersed anthracene precipitates recrystallized into larger particles after Tween 80 was metabolized. However, benzo[a]pyrene precipitates remained dispersed, accounting for a prolonged enhancement of the benzo[a]pyrene oxidation rates. Because the endogenous production of H2O2 is also known to be rate limiting for PAH oxidation, the combined effect of adding surfactants and glucose oxidase was studied. The combined treatment resulted in anthracene and benzo[a]pyrene oxidation rates as high as 1450 and 450 mg L-1 d-1, respectively, by the extracellular fluid of 6-day-old fungal cultures.     

Polycyclic aromatic hydrocarbon degradation by a Mycobacterium sp. in microcosms containing sediment and water from a pristine ecosystem.

Microcosm studies were conducted to evaluate the survival and performance of a recently discovered polycyclic aromatic hydrocarbon (PAH)-degrading Mycobacterium sp. when this organism was added to sediment and water from a pristine ecosystem. Microcosms inoculated with the Mycobacterium sp. showed enhanced mineralization, singly and as components in a mixture, of 2-methylnaphthalene, phenanthrene, pyrene, and benzo[alpha]pyrene. Studies utilizing pyrene as the sole added PAH showed that the Mycobacterium sp. survived in microcosms for 6 weeks both with and without preexposure to PAH and mineralized multiple doses of pyrene. Pyrene mineralization rates for sterilized microcosms inoculated with the Mycobacterium sp. showed that competition with indigenous microorganisms did not adversely affect survival of or pyrene degradation by the Mycobacterium sp. Pyrene mineralization by the Mycobacterium sp. was not enhanced by inorganic nutrient enrichment and was hindered by organic nutrient enrichment, which appeared to result from overgrowth of indigenous bacteria. This study demonstrates the versatility of the PAH-degrading Mycobacterium sp. and expands its potential applications to include the degradation of two-, three-, four-, and five-ringed PAHs in sediments.     

Polycyclic aromatic hydrocarbon degradation by a Mycobacterium sp. in microcosms containing sediment and water from a pristine ecosystem

Microcosm studies were conducted to evaluate the survival and performance of a recently discovered polycyclic aromatic hydrocarbon (PAH)-degrading Mycobacterium sp. when this organism was added to sediment and water from a pristine ecosystem. Microcosms inoculated with the Mycobacterium sp. showed enhanced mineralization, singly and as components in a mixture, of 2-methylnaphthalene, phenanthrene, pyrene, and benzo[alpha]pyrene. Studies utilizing pyrene as the sole added PAH showed that the Mycobacterium sp. survived in microcosms for 6 weeks both with and without preexposure to PAH and mineralized multiple doses of pyrene. Pyrene mineralization rates for sterilized microcosms inoculated with the Mycobacterium sp. showed that competition with indigenous microorganisms did not adversely affect survival of or pyrene degradation by the Mycobacterium sp. Pyrene mineralization by the Mycobacterium sp. was not enhanced by inorganic nutrient enrichment and was hindered by organic nutrient enrichment, which appeared to result from overgrowth of indigenous bacteria. This study demonstrates the versatility of the PAH-degrading Mycobacterium sp. and expands its potential applications to include the degradation of two-, three-, four-, and five-ringed PAHs in sediments.     

Kinetics of carbonyl reductase from human brain.

Initial-rate analysis of the carbonyl reductase-catalysed reduction of menadione by NADPH gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. The fluorescence of NADPH was increased up to 7-fold with a concomitant shift of the emission maximum towards lower wavelength in the presence of carbonyl reductase, and both NADPH and NADP+ caused quenching of the enzyme fluorescence, indicating formation of a binary enzyme-coenzyme complex. Deuterium isotope effects on the apparent V/Km values decreased with increasing concentrations of menadione but were independent of the NADPH concentration. The results, together with data from product inhibition studies, are consistent with carbonyl reductase obeying a compulsory-order mechanism, NADPH binding first and NADP+ leaving last. No significant differences in the kinetic properties of three molecular forms of carbonyl reductase were detectable.     

Siderophore reduction catalyzed by higher plant NADH:nitrate reductase

Squash cotyledon NADH:nitrate reductase catalyzes the reduction of the siderophore ferrioxamine B. The enzyme also reduced ferric ion in a buffer system containing the chelators oxalate and maleate. Ferrioxamine B reduction was maximal at pH 4; ferric ion reduction was maximal at pH 8. The present study indicates that iron assimilation by higher plants may occur with microbial siderophores serving as ferric ion sources and nitrate reductase functioning as the siderophore reductase.     

Immunohistochemical localization of carbonyl reductase in human tissues.

Carbonyl reductase, an NADPH-dependent oxidoreductase of broad specificity, is present in many human tissues. Its precise localization, however, has remained unclear, as well as its physiological and possible pathophysiological significance. The present study reports the immunohistochemical localization of the enzyme in normal human tissues. Immunostaining was detectable in all organs investigated. The highest concentrations were found in the parenchymal cells of the liver, the epithelial cells of the stomach and small intestine, the epidermis, the proximal tubules of the kidney, neuronal and glial cells of the central nervous system, and certain cells of the anterior lobe of the pituitary gland. Consistently pronounced staining was also observed in smooth muscle fibers and the endothelium of blood vessels. The results are in agreement with a housekeeping function of carbonyl reductase in the elimination of reactive carbonyl compounds.     

Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed of 1:1:1 relationship between the amount of NADPH and O2 consumed and the amount of H2O2 formed (measured fluorometrically). The amount of O2 consumed during the redox cycling of resorufin decreased approximately 50% in the presence of catalase, whereas the rate of O2 consumption decreased in the presence of superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O2 is converted to H2O2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O2 to H2O2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O2 to superoxide anion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polycyclic aromatic hydrocarbon residues in blood serum and human milk in Egypt,A pilot case study

This study was performed to monitor residues of polycyclic aromatic hydrocarbons (PAHs) in samples of human blood serum and human milk taken from volunteers from one rural area of Egypt. Extraction and clean-up processes were conducted using the Quick Easy Cheap Effective Safe methodology. PAH residue analysis was performed by Gas chromatography-flame ioninzation detector (GC-FID) and High performance liquid chromatography-fluorescence detector (HPLC-FLD) for blood and milk samples, respectively. Some confirmatory work was conducted using a mass spectrometer. The concentrations of PAH residues in blood samples were between 0.007 and 0.407 mg/l, with many of the congeners below detection limits. Residues of the most carcinogenic PAH congeners including benzo (a) pyrene were below the limits of detection in all blood samples, and total PAH concentrations have ranged between 0.156 and 3.61 mg/l. Regarding human milk samples, the sum of PAH concentrations ranged from 95.23 to 229.26 (µg/kg f.w.) with a mean of 154.35 (µg/kg f.w.). Benzo[a]pyrene was detected in concentrations ranging from 0.348 to 15.4, with a mean of 7.872 (µg/kg f.w.). Acenaphthylene, dibenzo [a,h]anthracene, acenaphthene, and naphthalene were the most abundant congeners in milk samples. Results indicated that the sources of PAHs in blood serum and human milk are of pyrogenic and petrogenic origin, respectively.     

Mechanistic basis for nonlinear kinetics of aldehyde reduction catalyzed by aldose reductase

Bovine kidney aldose reductase (ALR2) displays substrate inhibition by aldehyde substrates that is uncompetitive versus NADPH when allowance is made for nonenzymic reaction of the aldehyde with the adenine moiety of NADPH. A time-dependent increase in substrate inhibition observed in product versus time plots for reduction of short-chain aldoses containing an enolizable alpha-proton, but not for p-nitrobenzaldehyde, is shown to be consistent with a model in which rapidly reversible inhibition due to formation of the dead-end E-NADP-glycolaldehyde complex is combined with the formation at the enzyme active site of a tightly-bound covalent NADP-glycolaldehyde adduct. Quantitative analysis of reaction time courses for ALR2-catalyzed reduction of glycolaldehyde using NADPH or the 3-acetylpyridine analogue, (AP)ADPH, yields values of the forward and reverse rate constants for ALR2-mediated adduct formation that agree with the values determined in the absence of glycolaldehyde turnover. Substrate inhibition is only partial, indicating that reaction can occur via an alternate pathway at high [glycolaldehyde]. Kinetic evidence for a slow isomerization of the E-NADP complex at pH 8.0 is used to explain the similar V/Et values observed for glycolaldehyde reduction at pH 7.0 using NADPH, (AP)ADPH, and the hypoxanthine analogue N(Hx)DPH. The practical implications of these results for kinetics studies of aldose reductase are discussed.     

Inactivation of carbonyl reductase from human brain by phenylglyoxal and 2,3-butanedione: a comparison with aldehyde reductase and aldose reductase

Aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and carbonyl reductase (secondary-alcohol:NADP+ oxidoreductase, EC 1.1.1.184) constitute the enzyme family of the aldo-keto reductases, a classification based on similar physicochemical properties and substrate specificities. The present study was undertaken in order to obtain information about the structural relationships between the three enzymes. Treatment of human aldehyde and carbonyl reductase with phenylglyoxal and 2,3-butanedione caused a complete and irreversible loss of enzyme activity, the rate of loss being proportional to the concentration of the dicarbonyl reagents. The inactivation of aldehyde reductase followed pseudo-first-order kinetics, whereas carbonyl reductase showed a more complex behavior, consistent with protein modification cooperativity. NADP+ partially prevented the loss of activity of both enzymes, and an even better protection of aldehyde reductase was afforded by the combination of coenzyme and substrate. Aldose reductase was partially inactivated by phenylglyoxal, but insensitive to 2,3-butanedione. The degree of inactivation with respect to the phenylglyoxal concentration showed saturation behavior. NADP+ partially protected the enzyme at low phenylglyoxal concentrations (0.5 mM), but showed no effect at high concentrations (5 mM). These findings suggest the presence of an essential arginine residue in the substrate-binding domain of aldehyde reductase and the coenzyme-binding site of carbonyl reductase. The effect of phenylglyoxal on aldose reductase may be explained by the modification of a reactive thiol or lysine rather than an arginine residue.     

Acid-volatile selenium formation catalyzed by glutathione reductase.

The production of acid-volatile selenide (apparently H2Se) was catalyzed by glutathione reductase in an anaerobic system containing 20 mM glutathione, 0.05 mM sodium selenite, a TPNH-generating system, and microgram quantities of highly purified yeast glutathione reductase. H2Se production in this system was proportional to glutathione reductase concentration and was maximal at pH 7. Significant nonenzymic H2Se production occurred in the system lacking glutathione reductase and TNPH. A concentration of arsenite (0.1 mM) which does not inhibit glutathione reductase inhibited selenide volatilization, as did bovine serum albumin (1.67 mg/ml). Both appear to inhibit Se volatilization by reacting with the selenide product(s). The selenotrisulfide derivative of glutathione (GSSeSG) was readily converted to H2Se by glutathione reductase and TPNH without the addition of glutathione. These results suggest that GSSeSG formed nonenzymically from glutathione and selenic undergoes stepwise reduction by glutathione reductase (or excess GSH) to GSSeH and finally to H2Se. The same pathway operates when glutathione is used as the reducing agent but to a lesser extent.     

Anaerobic oxidation of the aromatic plant hydrocarbon p-cymene by newly isolated denitrifying bacteria

The capability of nitrate-reducing bacteria to degrade alkyltoluenes in the absence of molecular oxygen was investigated with the three isomers of xylene, ethyltoluene, and isopropyltoluene (cymene) in enrichment cultures inoculated with freshwater mud. Denitrifying enrichment cultures developed most readily (within 4 weeks) with p-cymene, a natural aromatic hydrocarbon occurring in plants, and with m-xylene (within 6 weeks). Enrichment of denitrifiers that utilized m-ethyltoluene and p-ethyltoluene was slow (within 8 and 12 weeks, respectively); no enrichment cultures were obtained with the other alkylbenzenes within 6 months. Anaerobic degradation of p-cymene, which has not been reported before, was studied in more detail. Two new types of denitrifying bacteria with oval cells, strains pCyN1 and pCyN2, were isolated; they grew on p-cymene (diluted in an inert carrier phase) and nitrate with doubling times of 12 and 16 h, respectively. Strain pCyN1, but not strain pCyN2, also utilized p-ethyltoluene and toluene. Both strains grew with some alkenoic monoterpenes structurally related to p-cymene, e.g., α-terpinene. In addition, the isolates utilized p-isopropylbenzoate, and mono- and dicarboxylic aliphatic acids. Determination of the degradation balance of p-cymene and growth with acetate and nitrate indicated the capacity for complete oxidation of organic substrates under anoxic conditions. Adaptation studies with cells of strain pCyN1 suggest the existence of at least two enzyme systems for anaerobic alkylbenzene utilization, one metabolizing p-cymene and p-ethyltoluene, and the other metabolizing toluene. Excretion of p-isopropylbenzoate during growth on p-cymene indicated that the methyl group is the site of initial enzymatic attack. Although both strains were facultatively aerobic, as revealed by growth on acetate under air, growth on p-cymene under oxic conditions was observed only with strain pCyN1. Strains pCyN1 and pCyN2 are closely related to members of the Azoarcus-Thauera cluster within the β-subclass of the Proteobacteria, as revealed by 16S rRNA gene sequence analysis. This cluster encompasses several described denitrifiers that oxidize toluene and other alkylbenzenes. Received: 15 July 1998 / Revision received: 29 July 1999 / Accepted: 2 August 1999     

Induction of a human carbonyl reductase gene located on chromosome 21

Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunorubicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of Mr 30,375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably coded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 mouse/human somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10 microM 2,(3)-t-butyl-4-hydroxyanisole (BHA), beta-naphthoflavone or Sudan 1.