Non-substituted Polycyclic Aromatic Hydrocarbons Research Articles

Naphthalene Carboxylation in the Sulfate-Reducing Enrichment Culture N47 Is Proposed to Proceed via 1,3-Dipolar Cycloaddition to the Cofactor Prenylated Flavin Mononucleotide.

Polycyclic aromatic hydrocarbons are persistent pollutants of anthropogenic or natural origin in the environment and accumulate in anoxic habitats. In this study, we investigated the mechanism of the enzyme naphthalene carboxylase as a model reaction for polycyclic aromatic hydrocarbon activation by carboxylation. An enzyme assay was established with cell extracts of the highly enriched culture N47. In assays without addition of ATP, naphthalene carboxylase catalyzed a stable isotope exchange of the carboxyl group of naphthoate with 13C-labeled bicarbonate buffer, which can only occur via a partial backwards reaction of the naphthalene carboxylase reaction to an intermediate that does not include the carboxyl group. Hence, a new carboxyl group from the labeled bicarbonate is added upon forward reaction to the naphthoate. This indicates that the reaction mechanism consists of two or more steps and that at least the latter steps are reversible and ATP independent. Naphthalene carboxylation assays were carried out in deuterated buffer and revealed the incorporation of 0, 1, 2, or 3 deuterium atoms in the final product naphthoyl-coenzyme A, indicating that the reaction is fully reversible. Putative reaction mechanisms were tested by quantum mechanical calculations. The proposed mechanism of the reaction consists of three steps: the activation of the naphthalene by 1,3-dipolar cycloaddition of the cofactor prFMN to naphthalene, release of a proton and rearomatization producing a stable intermediate, and a carboxylation with a reverse 1,3-dipolar cycloaddition and cleavage of the bond to the cofactor producing 2-naphthoate. IMPORTANCE Pollution with polycyclic aromatic hydrocarbons poses a great hazard to humans and animals, with considerable long-term effects. The anaerobic degradation of polycyclic aromatic hydrocarbons in anoxic zones and anaerobic growth of such organisms is very slow, leading to only poor investigation of the degradation pathways, so far. In this work, we elucidated the mechanism of naphthalene carboxylase, a key enzyme in anaerobic naphthalene degradation. This is the first mechanism proposed for a carboxylase targeting nonsubstituted (polycyclic) aromatic compounds and can serve as a model for the initial activation reaction in the anaerobic degradation of benzene or nonsubstituted polycyclic aromatic hydrocarbons, as well as similar enzymatic reactions from the expanding class of UbiD-like (de)carboxylases.

Similar polycyclic aromatic hydrocarbon and genotoxicity profiles of atmospheric particulate matter from cities on three different continents.

The extractable organic material (EOM) from atmospheric total suspended particles (TSP) contains several organic compounds including non-substituted polycyclic aromatic hydrocarbons (PAHs), alkyl-PAHs, and nitro-PAHs. These chemicals seem to be among the key drivers of TSP genotoxicity. We have shown previously that the mutagenic potencies of the EOM from Limeira, Stockholm, and Kyoto, cities with markedly different meteorological conditions and pollution sources are similar. Here we compare the profiles of non-substituted PAHs (27 congeners), alkyl-PAHs (15 congeners), and nitro-PAHs (7 congeners) from the same EOM samples from these cities. We also compared the genotoxicity profiles using comet and micronucleus assays in human bronchial epithelial cells. The profiles of PAHs, as well as the cytotoxic and genotoxic potencies when expressed in EOM, were quite similar among the studied cities. It seems that despite the differences in meteorological conditions and pollution sources of the cities, removal, mixing, and different atmospheric transformation processes may be contributing to the similarity of the PAHs composition and genotoxicity profiles. More studies are required to verify if this would be a general rule applicable to other cities. Although these profiles were similar for all three cities, the EOM concentration in the atmospheres is markedly different. Thus, the population of Limeira (∼10-fold more EOM/m3 than Stockholm and ∼6-fold more than Kyoto) is exposed to higher concentrations of genotoxic pollutants, and Kyoto's population is 1.5-fold more exposed than Stockholm's. Therefore, to reduce the risk of human exposure to TSP genotoxins, the volume of emissions needs to be reduced.

Identification of naphthalene carboxylase subunits of the sulfate-reducing culture N47

Expanding industrialization and the associated usage and production of mineral oil products has caused a worldwide spread of polycyclic aromatic hydrocarbons. These pollutants accumulate and persist under anoxic conditions but little is known about the biochemical reactions catalyzing their anaerobic degradation. Recently, carboxylation of naphthalene was demonstrated for the sulfate-reducing culture N47. Proteogenomic studies on N47 allowed the identification of a gene cluster with products suggested to be involved in the initial reaction of naphthalene degradation. Here, we performed comparative proteomic studies with N47 proteins extracted from naphthalene versus 2-methylnapththalene-grown cells on blue native PAGE. The analysis led to the identification of subunits of the naphthalene carboxylase of N47. Moreover, we show that the identified subunits are encoded in an operon structure within the previously mentioned naphthalene carboxylase gene cluster. These findings were supported by a pull-down experiment revealing in vitro interaction partners of a heterologously produced GST-tagged naphthalene carboxylase subunit. Based on these lines of evidence, naphthalene carboxylase is proposed to be a complex of about 750 kDa. Naphthalene carboxylase can be seen as a prototype of a new enzyme family of UbiD like de-/carboxylases catalyzing the anaerobic activation of non-substituted polycyclic aromatic hydrocarbons.

Enhanced crude oil biodegradative potential of natural phytoplankton-associated hydrocarbonoclastic bacteria.

Phytoplankton have been shown to harbour a diversity of hydrocarbonoclastic bacteria (HCB), yet it is not understood how these phytoplankton-associated HCB would respond in the event of an oil spill at sea. Here, we assess the diversity and dynamics of the bacterial community associated with a natural population of marine phytoplankton under oil spill-simulated conditions, and compare it to that of the free-living (non phytoplankton-associated) bacterial community. While the crude oil severely impacted the phytoplankton population and was likely conducive to marine oil snow formation, analysis of the MiSeq-derived 16S rRNA data revealed dramatic and differential shifts in the oil-amended communities that included blooms of recognized HCB (e.g., Thalassospira, Cycloclasticus), including putative novel phyla, as well as other groups with previously unqualified oil-degrading potential (Olleya, Winogradskyella, and members of the inconspicuous BD7-3 phylum). Notably, the oil biodegradation potential of the phytoplankton-associated community exceeded that of the free-living community, and it showed a preference to degrade substituted and non-substituted polycyclic aromatic hydrocarbons. Our study provides evidence of compartmentalization of hydrocarbon-degrading capacity in the marine water column, wherein HCB associated with phytoplankton are better tuned to degrading crude oil hydrocarbons than that by the community of planktonic free-living bacteria.

Response of the bacterial community associated with a cosmopolitan marine diatom to crude oil shows a preference for the biodegradation of aromatic hydrocarbons.

Emerging evidence shows that hydrocarbonoclastic bacteria (HCB) may be commonly found associated with phytoplankton in the ocean, but the ecology of these bacteria and how they respond to crude oil remains poorly understood. Here, we used a natural diatom-bacterial assemblage to investigate the diversity and response of HCB associated with a cosmopolitan marine diatom, Skeletonema costatum, to crude oil. Pyrosequencing analysis and qPCR revealed a dramatic transition in the diatom-associated bacterial community, defined initially by a short-lived bloom of Methylophaga (putative oil degraders) that was subsequently succeeded by distinct groups of HCB (Marinobacter, Polycyclovorans, Arenibacter, Parvibaculum, Roseobacter clade), including putative novel phyla, as well as other groups with previously unqualified oil-degrading potential. Interestingly, these oil-enriched organisms contributed to the apparent and exclusive biodegradation of substituted and non-substituted polycyclic aromatic hydrocarbons (PAHs), thereby suggesting that the HCB community associated with the diatom is tuned to specializing in the degradation of PAHs. Furthermore, the formation of marine oil snow (MOS) in oil-amended incubations was consistent with its formation during the Deepwater Horizon oil spill. This work highlights the phycosphere of phytoplankton as an underexplored biotope in the ocean where HCB may contribute importantly to the biodegradation of hydrocarbon contaminants in marine surface waters.

Effects of Nereis diversicolor on the Transformation of 1-Methylpyrene and Pyrene: Transformation Efficiency and Identification of Phase I and II Products

Transformation of nonsubstituted and alkyl-substituted polycyclic aromatic hydrocarbons (PAHs) by the benthic invertebrate Nereis diversicolor was compared in this study. Pyrene and 1-methylpyrene were used as model compounds for nonsubstituted and alkyl-substituted PAHs, respectively. Qualitative and quantitative analyses of metabolites and parent compounds in worm tissue, water, and sediment were performed. Transformation of 1-methylpyrene generated the benzylic hydroxylated phase I product, 1-pyrenecarboxylic acid that comprised 90% of the total metabolites of 1-methylpyrene, and was mainly found in water extracts. We tentatively identified 1-methylpyrene glucuronides and 1-carbonylpyrene glycine as phase II metabolites not previously reported in literature. Pyrene was biotransformed to 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1-glucuronide, and pyrene glucoside sulfate, with pyrene-1-glucuronide as the most prominent metabolite. Transformation of 1-methylpyrene (21% transformed) was more than 3 times as efficient as pyrene transformation (5.6% transformed). Because crude oils contain larger amounts of C₁-C₄-substituted PAHs than nonsubstituted PAHs, the rapid and efficient transformation of sediment-associated 1-methylpyrene may result in a high exposure of water-living organisms to metabolites of alkyl-substituted PAHs, whose toxicities are unknown. This study demonstrates the need to consider fate and effects of substituted PAHs and their metabolites in risk assessments.

Hydroxylation of non-substituted polycyclic aromatic hydrocarbons by cytochrome P450 BM3 engineered by directed evolution

Chrysene and pyrene are known toxic compounds recalcitrant to biodegradation. Here directed evolution allowed us to identify two new mutants of cytochrome P450 BM3 that are able to hydroxylate both compounds. Random mutagenesis has been used to generate libraries of mutants of P450 BM3 active toward polycyclic aromatic hydrocarbons (PAHs) PAHs. After two rounds of error-prone PCR and backcross with parental DNA, three mutants were identified for improved activity toward pyrene and for the first time a new activity toward chrysene in comparison to the wild type enzyme. The mutants show higher affinity and coupling efficiency for chrysene with faster rates of product formation compared to the wild type. Furthermore, the mutants are able to hydroxylate chrysene in different positions, producing four metabolites, 1-, 3-, 4-, and 6-hydroxychrysene, and to hydroxylate pyrene to 1-hydroxypyrene.The majority of the mutation sites are found to be far from the active site, demonstrating the power of directed evolution in identifying mutations difficult to predict with a rational design approach.The different product profiles obtained for the different P450 BM3 mutants indicate that substrate orientation in the catalytic pocket of the protein can be modified by protein engineering. The mutants can be used for metabolic engineering for safe and cost-effective sustainable production of hydroxylated PAHs for industrial purposes as well as for the assessment of their carcinogenic activity in mammals.

Micellar solid-phase microextraction for determining partition coefficients of substituted polycyclic aromatic hydrocarbons in micellar media: possible prediction of hydrocarbon–micelle behaviour

Micellar solid-phase microextraction (MSPME) coupled to gas chromatography-mass spectrometry (GC-MS) has been used to obtain partition coefficients of a group of 18 substituted aromatic hydrocarbons to ionic and nonionic micelles. Statistical and factor analyses have been utilized to establish some general equations relating molecular descriptors of non-substituted polycyclic aromatic hydrocarbons and their partition coefficients obtained by MSPME. The obtained equations have correlation coefficients higher than 0.94. They are used to predict hydrocarbon-micelle partition coefficients for a group of hydrocarbons with reported literature values giving a correlation coefficient of 0.98 and a standard deviation of the prediction of 0.182. The predictive model was also applied to substituted polycyclic aromatic hydrocarbons with partition coefficient values obtained by MSPME, with a 69% level of success.

Human Aryl Hydrocarbon Receptor Ligand Activity of 31 Non-substituted Polycyclic Aromatic Hydrocarbons as Soil Contaminants

Toxic equivalency factors (TEFs) of 31 non-substituted polycyclic aromatic hydrocarbons (PAHs) for the human aryl hydrocarbon receptor (AhR) ligand activity were measured using the yeast recombinant reporter gene assay established by Miller et al., in order to estimate the toxic equivalents of individual PAHs in environmental samples. Ten PAHs showed sigmoid-shape concentration-response curves. An effective concentration to achieve 50% activity obtained with 1 μmol/l benzo[a]pyrene (BaP) (EC50BaP) was calculated by curve fitting, and TEFs were calculated from the ratio of EC50BaP for BaP to that for the chemical studied. The highest TEF was found for naphthacene (NPC) (TEF=35) followed by benzo[b]fluorene (BbFl) (19), benzo[k]fluoranthene (BkF) (11), benz[a]anthracene (BaA) (7.0), benzo[j]fluoranthene (BjF) (4.0), benzo[a]fluorene (BaFl) (1.9), benzo[b]fluoranthene (BbF) (1.4), chrysene (CHR) (1.1), and BaP (1.0). Concentration dependent activities were also observed for indeno[1,2,3-cd]pyrene (IP) and fluoranthene (FLT). The TEFs of IP and FLT were about 0.2 and 0.02, respectively. The other 19 PAHs, naphthalene (NPT), acenaphtylene (ACL), acenaphthene (ACT), fluorene (FLU), anthracene (ANT), phenanthrene (PHN), pyrene (PYR), triphenylene (TRI), benzo[e]pyrene (BeP), perylene (PER), benzo[ghi]perylene (BgP), dibenz[a, h]anthracene (DahA), picene (PIC), coronene (COR), dibenzo[a, e]pyrene (DaeP), dibenzo[a, h]pyrene (DahP), dibenzo[a, i]pyrene (DaiP), dibenzo[a, l]pyrene (DalP) and naphtho[2,3-a]pyrene (NaP), showed little or no activity in the 0.1-1,000 nmol/l range. The toxic equivalents (TEQs) of surface soil were calculated by multiplication of the TEFs with the concentrations in soil samples collected from Kyoto, Japan. The TEQ of BaA showed the highest value of the 16 US Environmental Protection Agency Priority Pollutant PAHs.

Contamination of polycyclic aromatic hydrocarbons and their implication for environmental hazard

Studies concerning the contamination and pollution sources of polycyclic aromatic hydrocarbons (PAHs) in the Yodo River System, Japan, are summarized in this review. Mutagenicity and genotoxicity have been frequently detected in the tributary rivers of Yodo River, and novel contaminants such as phenylbenzotriazole type mutagens, nitrated PAHs, and heterocyclic amines have been suggested to be the causative agents. The contribution of nonsubstituted PAHs to the toxicity is considered to be low since only few ng/l of phenanthrene, fluoranthene, and pyrene have been found in the river waters. The application of an aryl hydrocarbon receptor (AhR) dependent bioassay in evaluating the combined toxicity caused by various pollutants in environmental samples is also introduced. Point and non-point sources of pollution discharging into the Lake Biwa-Yodo River System, including treated sewage effluents and deposited road particles, have been investigated using the AhR-dependent reporter gene assay. The results suggested the presence of newly identified AhR ligands such as anthraquinone dyes or unidentified AhR ligands in the treated sewage effluents and the road particles, respectively. Finally, toxic effects of PAHs that are mediated via AhR binding are also summarized.

Genotoxicity related to transfer of oil spill pollutants from mussels to mammals via food.

Heavy fuel oils containing high levels of polycyclic aromatic hydrocarbons (PAHs) were released into the marine environment after the Erika oil spill on the Atlantic coast. As highly condensed PAH pollutants can bioaccumulate in invertebrates, their transfer to vertebrates through the food chain was of concern. This study aimed to estimate potential genotoxic effects in rats fed for 2 or 4 weeks with the marine mussel Mytilus edulis contaminated by oil pollutants. Two levels of PAH contamination were studied, around 100 and 500 microg of total PAHs/kg dry weight (d.w.) in mussels. Genotoxic damage in rats was investigated by single-cell gel electrophoresis (Comet assay) and micronucleus assays in liver, bone marrow, and peripheral blood. DNA damage was observed in the liver of rats fed with the most contaminated mussels (500 microg PAHs/kg d.w.).DNA damage also was observed in the bone marrow but less than that in the liver. A small increase in micronuclei frequency was registered as well. This work underlines the bioavailability of pollutants in fuel-oil-contaminated mussels to consumers and the usefulness of the Comet assay as a sensitive tool in biomonitoring to analyze responses to PAH transfer in food. The occurrence of substituted PAHs and related compounds such as benzothiophenes in addition to nonsubstituted PAHs in fuel oils and mussels raised the question of whether they were implicated in the genotoxic effects registered in rats.

Electrospray mass spectrometric determination of 1-nitropyrene and non-substituted polycyclic aromatic hydrocarbons using tropylium cation as a post-column HPLC reagent

A method is presented for the electrospray mass spectrometric determination of 1-nitropyrene and non-substituted polycyclic aromatic hydrocarbons (PAHs) using tropylium cation as a post-column HPLC reagent. In the method, the analytes form a π–π complex with the tropylium cation by mixing with the tropylium solution after the separation of 1-nitropyrene and PAHs by reversed-phase liquid chromatography. The complexes then transfer the cations of the polycyclic aromatic compounds (PACs) and the tropylium compounds by collision-induced dissociation (CID) at the ion transfer region of the electrospray system. The generated PAC cations are detected by mass spectrometry. With the proposed method, 1-nitropyrene and PAHs could be simultaneously determined, and the detection limits (S/N=3) in the selected-ion monitoring mode were 0.83 ng of the injected 1-nitropyrene and 0.67–1.24 ng of PAHs.