Successive transformation of benzo[a]pyrene by laccase of Trametes versicolor and pyrene-degrading Mycobacterium strains

We previously hypothesized that polycyclic aromatic hydrocarbon (PAH)-degrading bacteria that produce laccase may enhance the degree of benzo[a]pyrene mineralization. However, whether the metabolites of benzo[a]pyrene oxidized by laccase can be further transformed by PAH degraders remains unknown. In this study, pyrene-degrading mycobacteria with diverse degradation properties were isolated and employed for investigating the subsequent transformation on the metabolites of benzo[a]pyrene oxidized by fungal laccase of Trametes versicolor. The results confirm the successive transformation of benzo[a]pyrene metabolites, 6-benzo[a]pyrenyl acetate, and quinones by Mycobacterium strains, and report the discovery of the involvement of a O-methylation mediated pathway in the process. In detail, the vast majority of metabolite 6-benzo[a]pyrenyl acetate was transformed into benzo[a]pyrene quinones or methoxybenzo[a]pyrene, via two distinct steps that were controlled by the catechol-O-methyltransferase mediated O-methylation, while quinones were reduced to dihydroxybenzo[a]pyrene and further transformed into dimethoxy derivatives.     

Comparative bacterial mutagenicity studies with 8-methoxypsoralen and 4,5′,8-trimethylpsoralen in the presence of near-ultraviolet light and in the dark

2 strains of S. typhimurium, TA98 and TA100, and 2 strains of E. coli, WP2(pKM101) and WP2uvrA⁻(pKM101) were used to study mutagenesis by 8-methoxypsoralen (8-MOP) and 4,5′,8-trimethylpsoralen (4,5′,8-TMP) in the dark and in the presence of near-ultraviolet (NUV) light both without metabolic activation and with rat-liver S9 at 3 levels (4, 10 and 30% in standard cofactors).The S9-independent base substitution mutagenic activity of 8-MOP plus NUV light was confirmed in WP2(pKM101), and a similar activity was seen for 4,5′,8-TMP, although neither substance was active in TA100. The frameshift mutagenic activity of 8-MOP in the dark in TA98 was not confirmed despite histidine levels which would ensure DNA replication, but this may be due to the lower concentrations of 8-MOP achieved in the common solvent system adopted.Both 8-MOP and 4,5′,8-TMP were mutagenic in WP2uvrA⁻(pKM101) after microsomal activation, and the responses were similar whether experiments were conducted in the dark or in NUV light. In view of the oral administration of 8-MOP to psoriasis patients, this finding may be of relevance in risk assessment, and tends to suggest that topical application of 4,5′,8-TMP to psoriatic patients may present reduced risk of malignant disease.     

Metabolic activation of polycyclic aromatic hydrocarbon trans-dihydrodiols by ubiquitously expressed aldehyde reductase (AKR1A1)

Polycyclic aromatic hydrocarbons (PAHs) are metabolized to trans-dihydrodiol proximate carcinogens by CYP1A1 and epoxide hydrolase (EH). CYP1A1 or aldo–keto reductases (AKRs) from the 1C subfamily can further activate the trans-dihydrodiols by forming either anti-diol-epoxides or reactive and redox active o-quinones, respectively. To determine whether other AKR superfamily members can divert trans-dihydrodiols to o-quinones, the cDNA encoding human aldehyde reductase (AKR1A1) was isolated from hepatoma HepG2 cells using RT-PCR, subcloned into a prokaryotic expression vector, overexpressed in E. coli and purified to homogeneity in milligram amounts. Studies revealed that AKR1A1 preferentially oxidized the metabolically relevant (−)-[3R,4R]-dihydroxy-3,4-dihydrobenz[a]anthracene. AKR1A1 also displayed high utilization ratios (V_max/K_m) for the following PAH trans-dihydrodiols: (±)trans-3,4-dihydroxy-3,4-dihydro-7-methylbenz[a]anthracene, (±)trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene and (±)trans-7,8-dihydroxy-7,8-dihydro-5-methylchrysene. Multiple tissue expression (MTE) arrays were used to measure the co-expressed of CYP1A1, EH and AKR1A1. All the three enzymes co-expressed to sites of PAH activation. The high catalytic efficiency of AKR1A1 for potent proximate carcinogen trans-dihydrodiols and its presence in tissues that contain CYP1A1 and EH suggests that it plays an important role in this alternative pathway of PAH activation (supported by CA39504).     

Enhancement of DNA binding,mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons after induction of cytochrome P-450 by ellipticines in rats and mice

Pretreatment of rats by ellipticines enhanced the microsomal concentration of cytochrome P-450, benzo[a]pyrene (BP) metabolism and activation and, to a smaller extent, ethoxycoumarin deethylation, but not acetanilide hydroxylation. This increased BP biotransformation was essentially due to the formation of bay-region metabolites, BP 9,10-diol, BP 7,8-diol and 9-hydroxy-BP, or to the formation of BP 7,8-diol-9,10-epoxide- and of 9-hydroxy-BP 4,5-oxide-DNA adducts. In the ellipticine series, 9-fluoroellipticine (9-FE) presents a slight inducing potency compared with the parent and 9-hydroxy molecules. Pretreatment of mice with 9-hydroxyellipticine (9-OHE) led also to an increased mutagenicity of BP and to an augmentation of skin carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA). These results clearly show that 9-OHE induces the biosynthesis of cytochrome P-450 which markedly stimulates the mutagenic and carcinogenic potentialities of polycyclic aromatic hydrocarbons (PAH).     

Benz[j]aceanthrylene: A novel polycyclic aromatic hydrocarbon with bacterial mutagenic activity

Initial studies on the mutagenicity and metabolism of a novel cyclopenta-PAH, benz[j]aceanthrylene, are reported in the Salmonella bacterial system. The spectrum of activity of benz[j]aceanthrylene over the 5 Ames tester strains is similar to that of benzo[a]pyrene, and the dose-response curves for strain TA98 are comparable. Like other biologically active PAH, benz[j]aceanthrylene is a frame-shift mutagen requiring metabolic activation. An interesting feature of the S9 dependence of activity is the low concentration (≅10-fold smaller than for benzo[a]pyrene) at which optimal activity is observed. The 1,2-dihydro-1,2-diol (product of metabolism of the cyclopenta-ring) appears to be the predominant metabolite, and implicates the 1,2-oxide as the ultimate mutagenic species.     

Mutagenic and chemical assay of extracts of airborne particulates

The mutagenic activity of extracts of airborne particulates was evaluated in the Salmonella system. The mutagenicity of airborne particulates was not always correlated with the content of benzo[a]pyrene (B[a]P) in the complex mixtures, especially when the samples were collected at different sites.Large-scale fractionation of extracts of airborne particulates was used to determine the content of specific mutagenic chemicals. The neutral fraction of material soluble in cyclohexane and nitromethane contained the polycyclic aromatic hydrocarbon (PAH) compounds, which accounted for 27.9% of the mutagenic activity of the whole extracts. 9 kinds of PAH compound were identified quantitatively by thin-layer chromatography. They included, per 1000 m³ of air, 12.6 μg of benzo[e]pyrene (B[e]P), 10.7 μg of chrysene (CHRY), 10.0 μg of fluoranthene (FL), 6.43 μg of benzo[ghi]perylene (B[ghi]P), 5.75 μg of benz[a]anthracene (B[a]A), 5.33 μg of B[a]P, 3.38 μg of pyrene (PYR), 1.83 μg of coronene (COR), and 1.34 μg of perylene (PERY).Mutagenicity of the ether-soluble acidic, basic and methanol-soluble neutral fractions accounted for 10.9, 9.71 and 6.78% of the total mutagenic activity of crude extract, respectively, when assayed in strain TA98 with liver S9 fraction. The total recovery of mutagenic activity after fractionation was 58%.Two acidic fractions (weak and strong ether-soluble acids) and the methanol-soluble neutral fraction reverted strain TA98 dramatically to prototrophy in the presence of rat-lung S9 fraction more than liver. But the mutagenic chemicals in these fractions remain to be clarified. Direct mutagens were present in essentially all fractions.The particulates, which had diameters ranging from 0.3 to 1.0 μm and were able to penetrate alveoli, contained a high content of mutagens.     

Degradation of eight highly condensed polycyclic aromatic hydrocarbons by Pleurotus sp. Florida in solid wheat straw substrate

The degradation of eight unlabeled highly condensed polycyclic aromatic hydrocarbons (PAH) and the mineralization of three ¹⁴C-labeled PAH by the white-rot fungus Pleurotus sp. Florida was investigated. Three concentrations containing 50, 250 or 1250 μg each unlabeled PAH/5 g straw were added to sterile sea sand. Selected treatments were added subsequently with ¹⁴C-labeled pyrene, benzo[a]anthracene or benzo[a]pyrene. The PAH-loaded sea sand was then mixed into straw substrate and incubated. The disappearance of the unlabeled four-to six-ring PAH: pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene and benzo[ghi]perylene, was determined by high-performance liquid chromatography. After 15 weeks of incubation, the recoveries were less than 25% for initial amounts of 50 μg (controls above 85%). The recoveries of unlabeled PAH increased in the inoculated samples with increasing concentrations applied. No correlation could be determined between the number of condensed rings of the PAH and the recoveries of added PAH. Pleurotus sp. Florida mineralized 53% [¹⁴C]pyrene, 25% [¹⁴C]benzo[a]anthracene and 39% [¹⁴C]benzo[a]pyrene to ¹⁴CO₂ in the presence of eight unlabeled PAH (50 μg applied) within 15 weeks. During the course of cultivation, Pleurotus sp. Florida degraded more than 40% of the wheat straw substrate. Variation of the initial concentration of PAH did not influence the extent of degradation of the organic matter. Received: 16 December 1996 / Received revision: 17 March 1997 / Accepted: 22 March 1997     

A sandwich enzyme-linked immunosorbent assay for adducts of polycyclic aromatic hydrocarbons with human serum albumin

Adducts of benzo[a]pyrene-diolepoxide (BPDE) with blood nucleophiles have been used as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs). The most popular such assay is a competitive enzyme-linked immunosorbent assay (ELISA) that employs monoclonal antibody 8E11 to detect benzo[a]pyrene tetrols following hydrolysis of BPDE adducts from lymphocyte DNA or human serum albumin (HSA). Here we used 8E11 as the capture antibody in a sandwich ELISA to detect BPDE-HSA adducts directly in 1-mg samples of HSA or 20 μl of serum/plasma. The assay employs an anti-HSA antibody for detection, and this is amplified by an avidin/biotinylated horseradish peroxidase complex. The sandwich ELISA has advantages of specificity and simplicity and is approximately 10 times more sensitive than the competitive ELISA. To validate the assay, HSA samples were assayed from three populations with known high PAH exposures (coke oven workers), medium PAH exposures (steel factory control workers), and low PAH exposures (volunteer subjects) (n = 30). The respective geometric mean levels of BPDE-HSA adducts—67.8, 14.7, and 1.93 ng/mg HSA (1010, 220, and 28.9 fmol BPDE equiv/mg HSA)—were significantly different (P < 0.05). The sandwich ELISA will be useful for screening PAH exposures in large epidemiologic studies and can be extended to other adducts for which capture antibodies are available.     

The role of base excision repair genes OGG1, APN1 and APN2 in benzo[a]pyrene-7,8-dione induced p53 mutagenesis

Lung cancer is primarily caused by exposure to tobacco smoke. Tobacco smoke contains numerous carcinogens, including polycyclic aromatic hydrocarbons (PAH). The most common PAH studied is benzo[a]pyrene (B[a]P). B[a]P is metabolically activated through multiple routes, one of which is catalyzed by aldo-keto reductase (AKR) to B[a]P-7,8-dione (BPQ). BPQ undergoes a futile redox cycle in the presence of NADPH to generate reactive oxygen species (ROS). ROS, in turn, damages DNA. Studies with a yeast p53 mutagenesis system found that the generation of ROS by PAH o-quinones may contribute to lung carcinogenesis because of similarities between the patterns (types of mutations) and spectra (location of mutations) and those seen in lung cancer. The patterns were dominated by G to T transversions, and the spectra in the experimental system have mutations at lung cancer hotspots. To address repair mechanisms that are responsible for BPQ induced damage we observed the effect of mutating two DNA repair genes OGG1 and APE1 (APN1 in yeast) and tested them in a yeast reporter system for p53 mutagenesis. There was an increase in both the mutant frequency and the number of G:C/T:A transversions in p53 treated with BPQ in ogg1 yeast but not in apn1 yeast. Knocking out APN2 increased mutagenesis in the apn1 cells. In addition, we did not find a strand bias on p53 treated with BPQ in ogg1 yeast. These studies suggest that Ogg1 is involved in repairing the oxidative damage caused by BPQ, Apn1 and Apn2 have redundant functions and that the stand bias seen in lung cancer may not be due to impaired repair of oxidative lesions.     

Phytochrome-mediated effects of near-ultraviolet radiation in the induction of flowering in etiolated Lemna paucicostata T-101, a short-day plant

Induction of flowering of etiolated Lemna paucicostata Hegelm. T-101, a short-day plant, was inhibited by far-red (FR) or blue light (BL) applied at the beginning of a 72-h inductive dark period which was followed by two short days. In either case the inhibition was reversed by a subsequent exposure of the plants to near-ultraviolet radiation (NUV), with a peak of effectiveness near 380 nm. Inhibition by BL or FR and its reversion by NUV are repeatable, i.e., NUV is acting in these photoresponses like red light although with much lower effectiveness. Thus, it is considered that NUV acts through phytochrome and no specific BL and NUV photoreceptor is involved in photocontrol of floral induction on this plant.Abbreviations BL blue light - FR far-red light - NUV near ultraviolet radiation - P red-absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - R red light     

Rat liver homogenate (S9)-mediated binding of benzo[a]pyren to DNA in V79 cells,V79 cell nuclei and aqueous solution

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[a]pyrene (B[a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[a]P (anti-B[a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[a]pyrene (9-OH-B[a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[a]PDE-DNA adduct to the 9-OH-B[a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[a]P in the presence of the S9 activation system.These results demonstrate that the relative abilities of various reactive bbenzo[a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.     

Aryl Hydrocarbon Receptor Facilitates DNA Strand Breaks and 8-Oxo-2��-deoxyguanosine Formation by the Aldo-Keto Reductase Product Benzo[a]pyrene-7,8-dione

Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by aldo-keto reductases are ligands for the aryl hydrocarbon receptor (AhR) (Burczynski, M. E., and Penning, T. M. (2000) Cancer Res. 60, 908–915). They induce oxidative DNA lesions (reactive oxygen species-mediated DNA strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo) formation) in human lung cells. We tested whether the AhR enhances PAH o-quinone-mediated oxidative DNA damage by translocating these ligands to the nucleus. Using the single cell gel electrophoresis (comet) assay to detect DNA strand breaks in murine hepatoma Hepa1c1c7 cells and its AhR- and aryl hydrocarbon receptor nuclear translocator-deficient variants, benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) produced fewer DNA strand breaks in AhR-deficient cells compared with aryl hydrocarbon receptor nuclear translocator-deficient and wild type Hepa1c1c7 cells. Decreased DNA strand breaks were also observed in human bronchoalveolar H358 cells in which the AhR was silenced by siRNA. The antioxidant α-tocopherol and the iron chelator/antioxidant desferal decreased the formation of B[a]P-7,8-dione-mediated DNA strand breaks indicating that they were reactive oxygen species-dependent. By coupling the comet assay to 8-oxoguanine glycosylase (hOGG1), which excises 8-oxo-Gua, strand breaks dependent upon this lesion were measured. hOGG1 treatment produced more DNA single strand breaks in B[a]P-7,8-dione-treated Hepa cells and H358 cells than in its absence. The levels of hOGG1-dependent DNA strand breaks mediated by B[a]P-7,8-dione were lower in AhR-deficient Hepa and AhR knockdown H358 cells. The AhR antagonist α-naphthoflavone also attenuated B[a]P-7,8-dione-mediated DNA strand breaks. The decrease in 8-oxo-dGuo levels in AhR-deficient Hepa cells and AhR knockdown H358 cells was validated by immunoaffinity capture stable isotope dilution ([¹⁵N₅]8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry. We conclude that the AhR shuttles PAH o-quinone genotoxins to the nucleus and enhances oxidative DNA damage.Polycyclic aromatic hydrocarbons (PAHs)² are ubiquitous environmental pollutants that include over 200 compounds with two or more fused benzene rings. PAHs are formed as a result of incomplete combustion of fossil fuels (e.g. coal and oil) and are present in car and diesel exhaust and smoked or charbroiled food (1–3). They are also found in cigarette smoke condensate and tobacco products and are suspect agents in the causation of human lung cancer (4, 5). PAHs must be metabolically activated to reactive genotoxins to cause their mutagenic and carcinogenic effects.Two major metabolic activation pathways are possible starting from the proximate PAH carcinogen (−)B[a]P-7,8-trans-dihydrodiol (Fig. 1). The P4501A1/1B1 pathway converts (−)B[a]P-7,8-trans-dihydrodiol to yield (+)-anti-7,8-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydroB[a]P (6–8). This diol epoxide forms stable N²-2′-deoxyguanosine (dGuo) adducts in vitro and in vivo (9, 10) and leads to mutation in H-ras (11) and may account for mutations in “hot spots” in p53 observed in lung cancer (12). The G to T transversions most often observed in these genes might arise because of the action of one or more trans-lesional by-pass DNA polymerases that read through stable diol-epoxide DNA adducts with low processivity and fidelity (13, 14).Open in a separate windowFIGURE 1.PAH activation by AKRs to cause oxidative DNA damage.As an alternative, human aldo-keto reductases (AKR1A1 and AKR1C1-AKR1C4) catalyze the NADP⁺-dependent oxidation of (±)B[a]P-7,8-trans-dihydrodiol to produce the electrophilic and redox-active B[a]P-7,8-dione (15, 16). In this pathway, AKRs convert B[a]P-7,8-trans-dihydrodiol to form a ketol that rearranges to a catechol. The catechol then undergoes two subsequent one-electron oxidations to yield the fully oxidized o-quinone. Once formed, B[a]P-7,8-dione amplifies reactive oxygen species (ROS) by entering futile redox cycles that deplete cellular reducing equivalents (e.g. NADPH) (17). PAH o-quinones can undergo 1,4- or 1,6-Michael addition with guanine and adenine bases to form stable N²-dGuo and N⁶-dAdo adducts in vitro (18–20). They can also react with the N7 position of guanine to yield depurinating adducts (21). It is possible that these covalent PAH o-quinone adducts could give to G to T transversion mutations.PAH o-quinones also cause oxidative DNA damage in vitro and in vivo (22–25). Nanomolar concentrations of PAH o-quinones under redox cycling conditions (NADPH and Cu(II)) lead to significant 8-oxo-dGuo formation in bulk DNA, and the responsible oxidant was found to be singlet oxygen (¹O₂) (24, 26). Under these conditions, PAH o-quinones produced 8-oxo-dGuo as the most dominant lesion among the three types of oxidative lesions measured (abasic sites, 8-oxo-dGuo, and oxidized pyrimidines) (26). In a yeast reporter gene assay, which scored loss-of-function mutations in p53, PAH o-quinones were found to be highly mutagenic but only under redox cycling conditions. The dominant mutation observed was a G to T transversion that was suppressed by ROS scavengers (27). Subsequent HPLC analysis coupled with electrochemical detection showed that there was a linear correlation between 8-oxo-dGuo formation in p53 and mutation frequency, indicating that 8-oxo-dGuo was the likely adduct responsible for the G to T transversions observed (28). These data suggest that oxidative DNA lesions caused by PAH o-quinones are more relevant in causing mutation than covalent PAH o-quinone-DNA adducts. In the latter case even if these adducts form, they do not appear to be mutagenic on p53.Recently, using either a hOGG1-coupled comet assay or an immunoaffinity capture-stable isotope dilution liquid chromatography/electrospray ionization/multiple reaction monitoring/mass spectrometry (LC/ESI/MRM/MS) assay, it was shown that both the AKR substrate (B[a]P-7,8-trans-dihydrodiol) and the AKR product (B[a]P-7,8-dione) caused significant DNA strand breaks and 8-oxo-dGuo formation in human lung adenocarcinoma A549 cells (25). Similar results were not observed with (+)-anti-7,8-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydroB[a]P or the regioisomer B[a]P-4,5-trans- dihydrodiol in these AKR-expressing cells. Subsequent use of the fluorescent dye dichlorofluorescein diacetate revealed that B[a]P-7,8-dione generated ROS in the nuclear compartment of the cells, suggesting that the PAH o-quinone was transported into the nucleus to increase the ROS-mediated DNA strand breaks and 8-oxo-dGuo (25). In addition, earlier disposition studies detected significant amounts of [³H]B[a]P-7,8-dione in the cell pellets of primary rat hepatocytes within 0.5 h, which caused extensive strand scission of the genomic DNA (29), suggesting that B[a]P-7,8-dione reached the nucleus. However, how PAH o-quinones gain entry into the nucleus and induce oxidative DNA damage is currently unknown.PAH o-quinones are ligands for the aryl hydrocarbon receptor (AhR) (30). These quinones can promote translocation of AhR to nucleus to induce P4501A1 expression. Upon binding with PAH o-quinones, the AhR dissociates from heat shock protein 90 and is rapidly translocated into nucleus where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) (31, 32). The quinone-bound AhR·ARNT complex then binds to the xenobiotic response element (XRE) and robustly activates the expression of AhR-regulated genes (30). These data raise the possibility that oxidative DNA damage caused by PAH o-quinones occurs because of their transportation and concentration in the nucleus mediated by the AhR. However, this hypothesis has not been formally tested.We now show that B[a]P-7,8-dione produces AhR-dependent DNA strand breaks and 8-oxo-dGuo formation using murine Hepa1c1c7 cells but not in its AhR-deficient variant. Similar results were obtained in human bronchoalveolar carcinoma H358 cells, but these effects were attenuated when the AhR was knocked down with siRNA. DNA lesions were measured by using the comet assay, which was coupled with hOGG1. These results were also confirmed by LC-ESI/MRM/MS assay for 8-oxo-dGuo. Our finding shows that PAH o-quinones produced by AKRs can cause ROS-mediated genotoxicity via an AhR-dependent mechanism, and this might contribute to PAH-mediated carcinogenesis.     

Seasonal Biotransformation of Naphthalene, Phenanthrene, and Benzo[a]pyrene in Surficial Estuarine Sediments

Transformation rates of naphthalene, phenanthrene, and benzo[a]pyrene in oxidized surficial sediments of a polluted urban estuary, Boston Harbor, Mass., were determined over a period of 15 months. Three sites characterized by muddy sediments were selected to represent a >300-fold range of ambient polycyclic aromatic hydrocarbon (PAH) concentration. Transformation rates were determined by a trace-level radiolabel PAH assay which accounted for PAH mineralization, the formation of polar metabolites, residue, and recovered parental PAHs in sediment slurries. Transformation rates of the model PAHs increased with increasing ambient PAH concentrations. However, turnover times for a given PAH were similar at all sites. The turnover times were as follows: naphthalene, 13.2 to 20.1 days; phenanthrene, 7.9 to 19.8 days, and benzo[a]pyrene, 53.7 to 82.3 days. At specific sites, rates were significantly affected by salinity, occasionally affected by temperature, but not affected by pH over the course of the study. Seasonal patterns of mineralization were observed for each of the PAHs at all sites. The timing of seasonal maxima of PAH mineralization varied from site to site. Seasonal potential heterotrophic activities as measured by acetate and glutamate mineralization rates did not always coincide with PAH mineralization maxima and minima, suggesting that the two processes are uncoupled in estuarine sediments.     

Predicting the Efficacy of Polycyclic Aromatic Hydrocarbon Bioremediation in Creosote-Contaminated Soil Using Bioavailability Assays

Nonexhaustive extraction (propanol, butanol, hydroxypropyl-β-cyclodextrin [HPCD]), persulfate oxidation and biodegradability assays were employed to determine the bioavailability of polycyclic aromatic hydrocarbons (PAHs) in creosote-contaminated soil. After 16 weeks incubation, greater than 89% of three-ring compounds (acenaphthene, anthracene, fluorene, and phenanthrene) and 21% to 79% of four-ring compounds (benz[a]anthracene, chrysene, fluoranthene, and pyrene) were degraded by the indigenous microorganisms under biopile conditions. No significant decrease in five- (benzo[a]pyrene, benzo[b+k]fluoranthene) and six-ring compounds (benz[g,h,i]perylene, indeno[1,2,3-c,d]pyrene) was observed. Desorption of PAHs using propanol or butanol could not predict PAH biodegradability: low-molecular-weight PAH biodegradability was underestimated whereas high-molecular-weight PAH biodegradability was overestimated. Persulfate oxidation and HPCD extraction of creosote-contaminated soil was able to predict three- and four-ring PAH biodegradability; however, the biodegradability of five-ring PAHs was overestimated. These results demonstrate that persulfate oxidation and HPCD extraction are good predictors of PAH biodegradability for compounds with octanol-water partitioning coefficients of < 6.     

Benz[a]anthracene Biotransformation and Production of Ring Fission Products by Sphingobium sp. Strain KK22

A soil bacterium, designated strain KK22, was isolated from a phenanthrene enrichment culture of a bacterial consortium that grew on diesel fuel, and it was found to biotransform the persistent environmental pollutant and high-molecular-weight polycyclic aromatic hydrocarbon (PAH) benz[a]anthracene. Nearly complete sequencing of the 16S rRNA gene of strain KK22 and phylogenetic analysis revealed that this organism is a new member of the genus Sphingobium. An 8-day time course study that consisted of whole-culture extractions followed by high-performance liquid chromatography (HPLC) analyses with fluorescence detection showed that 80 to 90% biodegradation of 2.5 mg liter⁻¹ benz[a]anthracene had occurred. Biodegradation assays where benz[a]anthracene was supplied in crystalline form (100 mg liter⁻¹) confirmed biodegradation and showed that strain KK22 cells precultured on glucose were equally capable of benz[a]anthracene biotransformation when precultured on glucose plus phenanthrene. Analyses of organic extracts from benz[a]anthracene biodegradation by liquid chromatography negative electrospray ionization tandem mass spectrometry [LC/ESI(−)-MS/MS] revealed 10 products, including two o-hydroxypolyaromatic acids and two hydroxy-naphthoic acids. 1-Hydroxy-2- and 2-hydroxy-3-naphthoic acids were unambiguously identified, and this indicated that oxidation of the benz[a]anthracene molecule occurred via both the linear kata and angular kata ends of the molecule. Other two- and single-aromatic-ring metabolites were also documented, including 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid and salicylic acid, and the proposed pathways for benz[a]anthracene biotransformation by a bacterium were extended.     

Biodegradation and sorption of polyaromatic hydrocarbons by Phanerochaete chrysosporium

The ability of the white-rot fungus Phanerochaete chrysosporium (INA-12) to degrade various polynuclear aromatic hydrocarbons (PAH) was investigated. Under static, non-nitrogen-limiting conditions, P. chrysosporium mineralized both phenanthrene and benzo[a]pyrene. Total mineralization, based on radioactive tracing, was limited to 1.8%–3% for phenanthrene and benzo[a]pyrene respectively. In both cases the pattern of mineralization did not correlate temporally with the production of lignin peroxidase activity. Sorption of radiolabelled material to the biomass was very significant with 22% and 40% of the total radioactivity being sorbed for benzo[a]pyrene and phenanthrene respectively. A number of models were examined to predict the sorption isotherms, the best performance being obtained with a three-parameter empirical model. It is apparent that lignin peroxidase is not necessarily involved in the biodegradation of all PAH and that a significant factor in PAH biodegradation and/or disappearance in cultures with the intact fungus may be attributed to sorption phenomena.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Degradation and Mineralization of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons by Defined Fungal-Bacterial Cocultures

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria (Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus (Penicillium janthinellum VUO 10,201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[a]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[a]anthracene, benzo[a]pyrene, and dibenz[a,h]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[a]pyrene was mineralized to CO₂ by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[a]pyrene mineralization (53% of added [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.     

Effect of rapeseed oil on the degradation of polycyclic aromatic hydrocarbons in soil by Rhodococcus wratislaviensis

The effect of rapeseed oil (0, 0.1 and 1% w/w) on the degradation of polycyclic aromatic hydrocarbons (PAH) by Rhodococcus wratislaviensis was studied in soils artificially contaminated with phenanthrene, anthracene, pyrene and benzo(a)pyrene (50 mg kg⁻¹ each), during 49 days at 30 °C. Without or with 0.1% of rapeseed oil, R. wratislaviensis degraded >90% of phenanthrene and anthracene in 14 days and mineralised approx. 23% of ¹⁴C-phenanthrene. The native microflora degraded pyrene (90% degradation; 75% mineralisation) and benzo(a)pyrene (30% degradation, no mineralisation). With 1% rapeseed oil, R. wratislaviensis degraded only 66% of the phenanthrene and mineralised 12.4%, and had no effect on other PAH, while degradation by the native microflora was inhibited. On the other hand, the addition of 1% oil promoted degradation of benzo(a)pyrene (75%) and anthracene (90%) and anthraquinone was produced at high concentrations and accumulated. Two distinct processes gave degradation of PAH, one biological and one abiotic. Biological processes mainly degraded phenanthrene and pyrene, either by R. wratislaviensis or by the indigenous microflora. Benzo(a)pyrene was degraded mainly by an abiotic process in the presence of 1% rapeseed oil. Anthracene was degraded by a combination of both processes.PAH are often found in contaminated soils and there is the need of developing techniques that can be applied in the remediation of these sites, where PAH, specially those with high molecular weight, pose health and environmental risks. There is a continuous search for efficient microorganisms able to degrade these pollutants and for methods to enhance their degradation and bioavailability, e.g. by the use of vegetable oils. This paper presents a novel process for the degradation of PAH by a combined biological/abiotic system.     

Effect of various substituents in the 6-position on the relative carcinogenic activity of a series of benzo[a]pyrene derivatives

To test the hypothesis that polycyclic aromatic hydrocarbons capable of being converted to a reactive ester of the mesohydroxymethyl metabolite would be carcinogenic, a series of 6-substituted derivatives of benzo[a]pyrene (B[a]P) were tested for carcinogenicity in Sprague-Dawley rats by subcutaneous injection of the compound in sesame oil on alternate days for 30 doses. At the 0.2-μmol dose level B[a]P, 6-acetoxymethyl(6-AcOCH₂)B[a]P, 6-hydroxymethyl(6-HOCH₂)B[a]P, 6-methyl(6-CH₃)B[a]P and 6-benzoyloxymethyl(6-BzOCH₂)B[a]P were nearly equipotent, 6-formyl(6-OCH)-and 6-chloromethyl(6-ClCH₂)B[a]P were less active, and 6-methoxymethyl (6-MeOCH₂)B[a]P was inactive. At lower doses the order of potency was estimated to be: 6-AcOCH₂- = 6-HOCH₂- = or > B[a]P > 6-CH₂- > 6-BzOCH₂- > 6-ClCH₂- > 6-OCH- > 6-BrCH₂B[a]P. Incubation of these compounds in the presence of cofactors or cofactors plus a microsomal preparation of rat subcutis indicated that enzymic activation was necessary for metabolism to highly polar products and for conversion of 6-AcOCH₂-, 6-BzOCH₂- and 6-OCHB[a]P to 6-HOCH₂B[a]P. The halomethyl compounds were converted to 6-HOCH₂B[a]P in the absence of enzyme by hydrolysis. 6-MeOCH₂B[a]P was unchanged in this system. These observations are consistent with the foregoing hypothesis with regard to derivatives of B[a]P and demonstrate that compounds of this series that are capable of conversion to the 6-HOCH₂-derivatives are carcinogenic.