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1.  Substrate Specificity and Structural Characteristics of the Novel Rieske Nonheme Iron Aromatic Ring-Hydroxylating Oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 
mBio  2010;1(2):e00135-10.
The Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 have been implicated in the initial oxidation of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), forming cis-dihydrodiols. To clarify how these two RHOs are functionally different with respect to the degradation of HMW PAHs, we investigated their substrate specificities to 13 representative aromatic substrates (toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene) by enzyme reconstitution studies of Escherichia coli. Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase. Metabolite profiles indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds, producing various isomeric dihydrodiol and phenolic compounds. NidAB and NidA3B3 showed the highest conversion rates for pyrene and fluoranthene, respectively, with high product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity. Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs. The NidAB and NidA3B3 systems showed the largest substrate-binding pockets in the active sites, which satisfies spatial requirements for accepting HMW PAHs. Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons.
Since the discovery of microbial ring-hydroxylating oxygenases (RHOs) in 1970, the sequences, structures, and enzyme biochemistry, including enantiospecific products, of RHOs have been studied and discussed extensively from the perspective of biodegradation, biotransformation, and biocatalysis processes. However, with all that effort to elucidate the enzymatic mechanisms of RHOs, little is known about the biochemistry and enzymology underlying high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) degradation. We used Mycobacterium vanbaalenii PYR-1 Nid enzymes, the first type V RHO members to display an apparent substrate preference for HMW PAHs. Here, we examine the mechanism of the RHO reaction by integrating structural information of the NidAB and NidA3B3 enzymes with substrate and product data. This study gives us an understanding of how the model RHO systems of M. vanbaalenii PYR-1 metabolize HMW PAHs. The information obtained would also be helpful for successful application of RHO enzymes to the production of industrially and medically important chiral chemicals and the development of PAH bioremediation technologies.
PMCID: PMC2921158  PMID: 20714442
2.  Chemical characterization and bioactivity of polycyclic aromatic hydrocarbons from non-oxidative thermal treatment of pyrene-contaminated soil at 250-1,000 degrees C. 
Environmental Health Perspectives  2000;108(8):709-717.
In this paper we report yields, identities, and mutagenicities of products from heating a polycyclic aromatic hydrocarbon (PAH)-contaminated, Superfund-related synthetic soil matrix without exogenous oxygen. We heated batch samples of soil pretreated with 5.08 wt% (by weight) pyrene in a tubular furnace under a constant flow of helium gas at 250, 500, 750, and 1,000 +/- 20 degrees C. Dichloromethane (DCM) extracts of cooled residues of heated soil and of volatiles condensed on a cold finger after 1 sec residence time at furnace temperature were assayed gravimetrically and analyzed for PAH by HPLC, HPLC coupled to mass spectrometry, and gas chromatography coupled to mass spectrometry. All four temperatures volatilized pyrene and generated other PAHs, including alkylated pyrenes. We detected bioactive PAHs in the product volatiles: cyclopenta[cd]pyrene (CPP) at 750 and 1,000 degrees C and benzo[a]pyrene (BaP) at 1,000 degrees C. We found a clean soil residue, i.e., no pyrene or other DCM extracts, only at 750 degrees C. Control experiments with uncontaminated soil, pyrene, and Ottawa sand plus 4.89 wt% pyrene revealed no CPP or BaP production from soil itself, but these experiments imply that pyrene interactions with soil, e.g., soil-bound silica, stimulate CPP and BaP production. We detected mutagenicity to human diploid lymphoblasts (in vitro) in volatiles from 1,000 degrees C heating of soil plus pyrene and sand plus pyrene, and in the residue from 500 degrees C heating of soil plus pyrene. Three plausible pathways for pyrene conversion to other PAHs are a) a reaction with light gas species, e.g., soil- or pyrene-derived acetylene; b) loss of C(2)-units followed by reaction with a PAH; and c) dimerization with further molecular weight growth via cyclodehydrogenation. This study shows that thermal treatment of PAH-polluted soil may generate toxic by-products that require further cleanup by oxidation or other measures.
PMCID: PMC1638299  PMID: 10964790
3.  Bacillus subtilis is a Potential Degrader of Pyrene and Benzo[a]pyrene 
Polycyclic Aromatic Hydrocarbons (PAHs) are a group of compounds that pose many health threats to human and animal life. They occur in nature as a result of incomplete combustion of organic matter, as well as from many anthropogenic sources including cigarette smoke and automobile exhaust. PAHs have been reported to cause liver damage, red blood cell damage and a variety of cancers. Because of this, methods to reduce the amount of PAHs in the environment are continuously being sought. The purpose of this study was to find soil bacteria capable of degrading high molecular weight PAHs, such as pyrene (Pyr) and benzo[a]pyrene (BaP), which contain more than three benzene rings and so persist in the environment. Bacillus subtilis, identified by fatty acid methyl ester (FAME) analysis, was isolated from PAH contaminated soil. Because it grew in the presence of 33μg/ml each of pyrene, 1-AP and 1-HP, its biodegradation capabilities were assessed. It was found that after a four-day incubation period at 30°C in 20μg/ml pyrene or benzo[a]pyrene, B. subtilis was able to transform approximately 40% and 50% pyrene and benzo[a]pyrene, respectively. This is the first report implicating B. subtilis in PAH degradation. Whether or not the intermediates resulting from the transformation are more toxic than their parent compounds, and whether B. subtilis is capable of mineralizing pyrene or benzo[a]pyrene to carbon dioxide and water, remains to be evaluated.
PMCID: PMC3810630  PMID: 16705827
Polycyclic Aromatic Hydrocarbons; bioremediation; Bacillus subtilis
4.  The influence of diesel exhaust on polycyclic aromatic hydrocarbon-induced DNA damage, gene expression and tumor initiation in Sencar mice in vivo 
Cancer letters  2008;265(1):135-147.
The carcinogenic effects of individual polycyclic aromatic hydrocarbons (PAH) are well established. However, their potency within an environmental complex mixture is uncertain. We evaluated the influence of diesel exhaust particulate matter on PAH-induced cytochrome P450 (CYP) activity, PAH-DNA adduct formation, expression of certain candidate genes and the frequency of tumor initiation in the two-stage Sencar mouse model. To this end, we monitored the effects of treatment of mice with diesel exhaust, benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP), or a combination of diesel exhaust with either carcinogenic PAH. The applied diesel particulate matter (SRM1975) altered the tumor initiating potency of DBP: a statistically significant decrease in overall tumor and carcinoma burden was observed following 25 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with DBP exposure alone. From those mice that were treated at the beginning of the observation period with 2 nmol DBP all survivors developed tumors (9 out of 9 animals, 100%). Among all tumors counted at the end, 9 carcinomas were detected and an overall tumor incidence of 2.6 tumors per tumor-bearing animal (TBA) was determined. By contrast, co-treatment of DBP with 50 mg SRM1975 led to a tumor rate of only 66% (19 out of 29 animals), occurrence of only 3 carcinomas in 29 animals and an overall rate of 2.1 tumors per TBA (P = 0.04). In contrast to the results with DBP, the tumor incidence induced by 200 nmol BP was found slightly increased when co-treatment with SRM1975 occurred (71% vs. 85% after 25 weeks). Despite this difference in tumor incidence, the numbers of carcinomas and tumors per TBA did not differ statistically significant between both treatment groups possibly due to the small size of the BP treatment group. Since bioactivation of DBP, but not BP, predominantly depends on CYP1B1 enzyme activity, SRM1975 affected PAH-induced carcinogenesis in an antagonistic manner when CYP1B1-mediated bioactivation was required. The explanation most likely lies in the much stronger inhibitory effects of certain PAHs present in diesel exhaust on CYP1B1 compared to CYP1A1. In the present study we also found molecular markers such as highly elevated AKR1C21 and TNFRSF21 gene expression levels in tumor tissue derived from animals co-treated with SRM1975 plus DBP. Therefore we validate microarray data as a source to uncover transcriptional signatures that may provide insights into molecular pathways affected following exposure to environmental complex mixtures such as diesel exhaust particulates.
PMCID: PMC2519885  PMID: 18353537
DNA adducts; carcinogenesis; diesel exhaust; PAH; cytochrome P450
5.  Communication: Synthesis of a Novel Triphenyltin(IV) Derivative of 2- Mercaptonicotinic Acid with Potent Cytotoxicity in vitro  
A novel triphenyltin(IV) derivative of 2-mercaptonicotinic acid (H2mna) of formula {[(C6H5)3Sn]2(mna).[(CH3)2CO]} (1) has been synthesized and characterized by elemental analysis and 1H, 13C-NMR, and FT-IR spectroscopic techniques. The crystal structure of complex (1) has been determined by single crystal X-ray diffraction analysis at 173(1) K. Compound (1) contains two triphenyltin moieties linked by a doubly de-protonated 2,mercaptonicotinic acid (H>2mna). It is an example of a pentacoordinated Ph3SnXY system with an axial-equatorial arrangement of the phenyl groups at Sn(1). Compound (1), exhibits potent, in vitro, cytotoxicity against sarcoma cancer cells (mesenchymal tissue) from the Wistar rat, polycyclic aromatic hydrocarbons (PAH, benzo[a]pyrene) carcinogenesis.
PMCID: PMC2267064  PMID: 18365056
6.  Estimation of individual dermal and respiratory uptake of polycyclic aromatic hydrocarbons in 12 coke oven workers. 
Twelve workers from a coke plant in The Netherlands participated in an intensive skin monitoring programme combined with personal air sampling and biological monitoring during five consecutive eight hour workshifts. The purpose of the study was to make a quantitative assessment of both the dermal and respiratory intake of polycyclic aromatic hydrocarbons (PAHs). Pyrene was used as a marker compound for both dermal and respiratory exposure to PAHs. The biological measure for the internal exposure to PAHs was urinary 1-OH-pyrene concentration. Measurements on exposure pads at six skin sites showed that mean total skin contamination of the 12 workers ranged between 21 and 166 micrograms pyrene a day. The dermal uptake of pyrene ranged between 4 and 34 micrograms/day, which was about 20% of the pyrene contamination on skin. The mean concentration of total pyrene in the breathing zone air of the 12 coke oven workers ranged from 0.1 to 5.4 micrograms/m3. The mean respiratory uptake of pyrene varied between 0.5 and 32.2 micrograms/day. Based on the estimates of the dermal and respiratory pyrene uptake it is concluded that an average 75% (range 28%-95%, n = 12) of the total absorbed amount of pyrene enters the body through the skin. Because of the difference in the pyrene:benzo(a)pyrene ratio between the air samples and the skin contamination samples, the dermal uptake of benzo(a)pyrene was also estimated. This was about 51% of the total absorbed amount (range 8%-92%, n = 12). The total excreted amount of urinary 1-OH-pyrene as a result of exposure to PAHs during the five consecutive workshifts varied between 36 and 239 nmol. A multiple regression model of the mass balance between pyrene dose (both dermal and respiratory) and 1-OH-pyrene excretion confirmed the relevance of the dermal exposure route. The variation in urinary 1-OH-pyrene excretion was determined more by the dermal pyrene dose than by the respiratory dose. The model showed an estimate of the percentage of the absorbed amount of pyrene that is metabolised and excreted as 1-OH-pyrene in urine. For the 12 workers this percentage varied between 13% and 49% depending on smoking habits and consumption of alcohol. The results of this study indicate that among coke oven workers, the skin is the main route of uptake of PAHs. Preventive measures to reduce exposure to PAHs should be focused more on the reduction of dermal contamination by PAHs than on the reduction of inhaled dose.
PMCID: PMC1035498  PMID: 8343423
7.  Exposure of iron foundry workers to polycyclic aromatic hydrocarbons: benzo(a)pyrene-albumin adducts and 1-hydroxypyrene as biomarkers for exposure. 
Exposure to polycyclic aromatic hydrocarbons (PAHs) in foundry workers has been evaluated by determination of benzo(a)pyrene-serum albumin adducts and urinary 1-hydroxypyrene. Benzo(a)pyrene binding to albumin and 1-hydroxypyrene were quantitatively measured by enzyme linked immunosorbent assay (ELISA) and reverse phase high performance liquid chromatography (HPLC), respectively. 70 male foundry workers and 68 matched controls were investigated. High and low exposure groups were defined from breathing zone hygienic samples, consisting of 16 PAH compounds in particulate and gaseous phase. Mean total PAH was 10.40 micrograms/m3 in the breathing zone, and mean dust adsorbed PAH was 0.15 microgram/m. All carcinogenic PAH was adsorbed to dust. Median benzo(a)pyrene-albumin adduct concentrations (10-90% percentiles) were similar in foundry workers (smokers 0.55 (0.27-1.00) and non-smokers 0.58 (0.17-1.15)) pmol/mg albumin and age matched controls (smokers 0.57 (0.16-1.45) and non-smokers 0.70 (0.19-1.55) pmol/mg albumin). Median 1-hydroxypyrene concentrations were significantly higher (P < 0.0001) in smoking and non-smoking foundry workers (0.022 (0.006-0.075) and 0.027 (0.006-0.164)) mumol/mol creatinine than in smoking and non-smoking controls (0 (0-0.022) and 0 (0-0.010) mumol/mol creatinine). Dose-response relations between total PAH, pyrene, carcinogenic PAHs, and 1-hydroxypyrene for smokers, and polycyclic aromatic hydrocarbons adsorbed to dust for non-smokers are suggested. Exposure to PAHs adsorbed to dust showed an additive effect. There was no correlation between the concentrations of 1-hydroxypyrene and benzo(a)pyrene-albumin adducts. The change in 1-hydroxypyrene over a weekend was also studied. Friday morning median 1-hydroxypyrene concentrations were significantly higher in both smokers and non-smokers (0.021 (0-0.075) and 0.027 (0.06-0.164)) mumol/mol creatinine than Monday morning median concentrations (0.007 (0-0.021) and 0.008 (0-0.021) mumol/mol creatinine). Smoking did not affect the concentrations of 1-hydroxypyrene or benzo(a)pyrene-albumin adducts. These data suggest that 1-hydroxypyrene is a sensitive biomarker for low dose PAH exposure. Exposure to PAHs may be aetiologically related to increased risk of lung cancer in foundry workers.
PMCID: PMC1128029  PMID: 7951774
8.  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.
PMCID: PMC91936  PMID: 10698765
9.  Isomeric differentiation of polycyclic aromatic hydrocarbons using silver nitrate reactive desorption electrospray ionization mass spectrometry 
Polycyclic aromatic hydrocarbons (PAHs) are nonpolar and difficult to detect by desorption electrospray ionization. We present a new detection method based on cationization with silver ions, which has the added advantage of being able to differentiate PAHs with the same mass but different structure.
9,10-Diphenylanthracene and triptycene, in addition to four different groups of PAH isomers: (1) anthracene and phenanthrene, (2) pyrene and fluoranthene, (3) benz[a]anthracene, benz[b]anthracene (tetracene), and chrysene (4) benzo[a]pyrene and benzo[k]fluoranthene, were deposited on a paper surface and bombarded with methanol droplets containing silver nitrate. The resulting microdroplets entered a quadruple mass spectrometer for mass analysis.
The mass spectrum shows [PAH]+, [Ag + OH + PAH]+, and [Ag(PAH)n]+ n (n = 1, 2) (and [PAH + O2]+ in the case of benz[b]anthracene) ions. PAHs having a bay structure, such as phenanthrene, showed a different tendency to interact with silver ions from those PAHs having a linear arrangement of the fused benzene rings, such as anthracene. The ratios of the [PAH]+ peak intensity to that of [Ag–PAH]+, [Ag + OH + PAH]+, [Ag(PAH)2]+, and [PAH + O2]+ were used to differentiate the PAH isomers sharing the same molecular formula with different structures. For isomeric mixtures the [PAH]+ to [Ag + OH + PAH]+ ratio was found to be the most useful parameter. The uncertainty in the mole fraction of an isomeric mixture was ±0.09, 0.13, ±0.25, and ±0.1 for phenanthrene-anthracene, fl benz[a] anthracene-chrysene, and benzo[a]pyrene-benzo[k]fluoranthene, respectively.
A simple method has been developed for the detection of PAHs in desorption electrospray ionization mass spectrometry based on Ag(I) cationization. The method showed a capability to differentiate PAHs isomers (having the same molecular mass) in isomeric mixture with an uncertainty in the mole fraction of about 0.1. At high inlet temperature and voltage, this method showed better sensitivity but less ability to differentiate between ± isomeric species.
PMCID: PMC4145873  PMID: 22847697
10.  Characterization of Solid Tumors Induced by Polycyclic Aromatic Hydrocarbons in Mice 
To assess the effects of single polycyclic aromatic hydrocarbons (PAHs) on solid tumor initiation, and investigate their roles in immune response regulation.
Mice (100) were randomly divided into 5 groups (n=20) to be intraperitoneally injected with 10 daily doses of DMSO (control), anthracene (50 mg/kg), benzo-(a)-pyrene (10 mg/kg), benzo-(a)-pyrene (20 mg/kg), and benzo-[G, H, I])-perylene (5 mg/kg), respectively. Three months later, serum IL-2 and IL-6 levels were assessed by ELISA; liver, kidney, stomach and lung tissues were subjected to histopathological examinations.
Liver cancer incidences after benzo-[G, H, I]-perylene, benzo-(a)-pyrene (10 mg/kg), benzo-(a)-pyrene (20 mg/kg), and anthracene were 21.1, 26.3, 35.3, and 27.8%, respectively; 21.1, 0, 41.2, and 0% showed stomach cancer, respectively; 0, 0, 11.8 and 0% displayed kidney cancer, respectively. The occurrences of precancerous liver lesions for benzo-[G, H, I]-perylene, benzo-(a)-pyrene (10 mg/kg), benzo-(a)-pyrene (20 mg/kg) and anthracene groups, respectively, were 68.4, 73.7, 64.7, and 55.6%; 78.9, 68.4, 29.4, and 27.8% showed precancerous stomach lesions, while 42.1, 47.4, 58.8, and 33.3% had precancerous kidney lesions; respectively. No obvious lung lesions were found in any group. Serum IL-2 and IL-6 levels in treatment groups were significantly lower compared with control values (P<0.01).
PAHs induce cancer and precancerous lesions in the liver, stomach, and kidney. Benzo (a) pyrene initiates gastric cancer in a dose-dependent manner, but does not induce precancerous lung lesions. Lower IL-2 and IL-6 levels in treatment groups compared with controls suggest that PAHs cause overt immune inhibition.
PMCID: PMC4435619  PMID: 25913077
Bay-Region, Polycyclic Aromatic Hydrocarbon; Hydrocarbons; Mice, 129 Strain; Carcinoma, Acinar Cell; Immunologic Factors
11.  Interaction of Polycyclic Aromatic Hydrocarbons with Human Cytochrome P450 1B1 in Inhibiting Catalytic Activity 
Chemical research in toxicology  2008;21(12):2313-2323.
Eleven polycyclic aromatic hydrocarbons (PAHs) and 14 acetylenic PAHs and biphenyls were used to analyze interactions with cytochrome P450 (P450) 1B1 in inhibiting catalytic activity, using 7-ethoxyresorufin O-deethylation (EROD) as a model reaction. Most of the chemicals examined were direct inhibitors of P450 1B1 except for 4-(1-propynyl)biphenyl, a mechanism-based inhibitor. In the case of direct inhibition of EROD activity {15 of 24 chemicals, e.g. benzo[a]pyrene, 1-(1-propynyl)pyrene, and 3-(1-propynyl)phenanthrene}, restoration of the EROD activity occurred with increasing incubation time, and kinetic analysis showed that EROD Km values were higher with these inhibitors at initial stages of incubation but became lower with increasing incubation time. With the other 9 chemicals, the Km values for P450 1B1-mediated EROD increased during the incubations. Acetylenic inhibitors, but not the 11 PAHs, induced reverse type I spectral changes with P450 1B1 and the low dissociation constants (Ks) suggested a role for such interaction in the inhibition of catalytic activity. Studies of quenching of P450 1B1-derived fluorescence with inhibitors demonstrated that acetylenic inhibitors and PAHs interacted rapidly with P450 1B1, with Kd values <10 μM. However, studies of quenching of inhibitor-derived fluorescence with P450 1B1 showed these interactions to be different, i.e. B[a]P interacted with P450 1B1 more slowly. Molecular docking of P450 1B1, based on P450 1A2 crystal structure, suggested that there are clear differences in the interaction of PAH inhibitors with P450 1B1 and 1A2 and that these differences may explain why PAH inhibitors inhibit P450 1 enzymes by different mechanisms. The results suggest that P450 1B1 interacts with synthetic polycyclic aromatic acetylenes and PAHs in different ways, depending on the chemicals, and that these differences in interactions may explain how these chemicals inhibit P450 activities by different mechanisms.
PMCID: PMC2772130  PMID: 19548353
12.  Sample preparation procedure for the determination of polycyclic aromatic hydrocarbons in petroleum vacuum residue and bitumen 
Analytical and Bioanalytical Chemistry  2011;401(3):1059-1069.
This paper describes a novel method of sample preparation for the determination of trace concentrations of polycyclic aromatic hydrocarbons (PAHs) in high-boiling petroleum products. Limits of quantitation of the investigated PAHs in materials of this type range from tens of nanograms per kilogram to <20 μg/kg. The studies revealed that in order to separate most of interferences from the analytes without a significant loss of PAHs, it is necessary to use size exclusion chromatography as the first step of sample preparation, followed by adsorption using normal-phase liquid chromatography. The use of orthogonal separation procedure described in the paper allows the isolation of only a group of unsubstituted and substituted aromatic hydrocarbons with a specific range of molar mass. The lower the required limit of quantitation of PAHs, the larger is the scale of preparative liquid chromatography in both steps of sample preparation needed. The use of internal standard allows quantitative results to be corrected for the degree of recovery of PAHs during the sample preparation step. Final determination can be carried out using HPLC-FLD, GC-MS, or HPLC-UV–VIS/DAD. The last technique provides a degree of identification through the acquired UV–VIS spectra.
FigureChromatograms obtained using UV-DAD detection with wavelength programming (A) and fluorimetric detection (B) for the separation of 18 PAH standards ((A) and (B)) and the fraction containing PAHs from road asphalt 50/70 prepared according to the procedure described in this work (C). Peak designation: 1 naphthalene, 2 acenaphthylene, 3 acenaphthene, 4 fluorene, 5 phenanthrene, 6 anthracene, 7 fluoranthene, 8 pyrene, 9 benzo[a]anthracene, 10 chrysene, 11 benzo[b]fluoranthene, 12 benzo[k]fluoranthene, 13 benzo[a]pyrene, 14 dibenzo[a,h]anthracene, 15 indeno[1,2,3-cd]pyrene, 16 benzo[ghi]perylene, 17 benzo[j]fluoranthene, 18 benzo[e]pyrene,19 highly polar components of road asphalt 50/70 eluted during backflush. BF backflush point
PMCID: PMC3140947  PMID: 21647802
Sample preparation techniques; Multidimensional liquid chromatography; Group separation; Size exclusion chromatography; Normal-phase adsorption chromatography; High-boiling petroleum products; Polycyclic aromatic hydrocarbons (PAHs); Trace analysis
13.  Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora 
White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. 14C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [14C]benzo[a]pyrene was recovered as 14CO2 in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of 14CO2 recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of 14CO2 recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in 14CO2 production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.
PMCID: PMC106782  PMID: 9687440
14.  Cancer risk assessment, indicators, and guidelines for polycyclic aromatic hydrocarbons in the ambient air. 
Environmental Health Perspectives  2002;110(Suppl 3):451-488.
Polycyclic aromatic hydrocarbons (PAHs) are formed during incomplete combustion. Domestic wood burning and road traffic are the major sources of PAHs in Sweden. In Stockholm, the sum of 14 different PAHs is 100-200 ng/m(3) at the street-level site, the most abundant being phenanthrene. Benzo[a]pyrene (B[a]P) varies between 1 and 2 ng/m(3). Exposure to PAH-containing substances increases the risk of cancer in humans. The carcinogenicity of PAHs is associated with the complexity of the molecule, i.e., increasing number of benzenoid rings, and with metabolic activation to reactive diol epoxide intermediates and their subsequent covalent binding to critical targets in DNA. B[a]P is the main indicator of carcinogenic PAHs. Fluoranthene is an important volatile PAH because it occurs at high concentrations in ambient air and because it is an experimental carcinogen in certain test systems. Thus, fluoranthene is suggested as a complementary indicator to B[a]P. The most carcinogenic PAH identified, dibenzo[a,l]pyrene, is also suggested as an indicator, although it occurs at very low concentrations. Quantitative cancer risk estimates of PAHs as air pollutants are very uncertain because of the lack of useful, good-quality data. According to the World Health Organization Air Quality Guidelines for Europe, the unit risk is 9 X 10(-5) per ng/m(3) of B[a]P as indicator of the total PAH content, namely, lifetime exposure to 0.1 ng/m(3) would theoretically lead to one extra cancer case in 100,000 exposed individuals. This concentration of 0.1 ng/m(3) of B[a]P is suggested as a health-based guideline. Because the carcinogenic potency of fluoranthene has been estimated to be approximately 20 times less than that of B[a]P, a tentative guideline value of 2 ng/m(3) is suggested for fluoranthene. Other significant PAHs are phenanthrene, methylated phenanthrenes/anthracenes and pyrene (high air concentrations), and large-molecule PAHs such as dibenz[a,h]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, and indeno[1,2,3-cd]pyrene (high carcinogenicity). Additional source-specific indicators are benzo[ghi]perylene for gasoline vehicles, retene for wood combustion, and dibenzothiophene and benzonaphthothiophene for sulfur-containing fuels.
PMCID: PMC1241197  PMID: 12060843
15.  Actions of Mycobacterium sp. Strain AP1 on the Saturated- and Aromatic-Hydrocarbon Fractions of Fuel Oil in a Marine Medium▿ †  
Applied and Environmental Microbiology  2009;75(19):6232-6239.
The pyrene-degrading Mycobacterium sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C12 to C40) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(a)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by Mycobacterium sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.
PMCID: PMC2753065  PMID: 19666730
16.  Octa­butyl­bis­(μ2-2-chloro-5-nitro­benzoato)bis­(2-chloro-5-nitro­benzoato)di-μ3-oxido-tetra­tin(IV) 
The title complex, [Sn4(C4H9)8(C7H3ClNO4)4O2], is a cluster formed by a crystallographic inversion center around the central Sn2O2 ring. Both of the two independent Sn atoms are five-coordinated, with distorted trigonal–bipyramidal SnC2O3 geometries. One Sn atom is coordinated by two butyl groups, one O atom of the benzoate anion and two bridging O atoms, whereas the other Sn atom is coordinated by two butyl groups, two O atoms of the benzoate anions and a bridging O atom. The O atoms of the bridging benzoate anion are disordered over two sites with an occupancy ratio of 0.862 (12):0.138 (12). One of the butyl groups coordinated to the Sn2O2 ring is disordered over two sites with an occupancy ratio of 0.780 (8):0.220 (8), whereas both of the two butyl groups coordinated to the other Sn atom are disordered over two sites with occupancy ratios of 0.788 (5):0.212 (5) and 0.827 (10):0.173 (10). All the butyl groups are equatorial with respect to the SnO3 trigonal plane. In the crystal, complex mol­ecules are stacked down [010] with weak inter­molecular C—H⋯π inter­actions stabilizing the crystal structure.
PMCID: PMC3011528  PMID: 21589351
17.  The role of CYP1A inhibition in the embryotoxic interactions between hypoxia and polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures in zebrafish (Danio rerio) 
Ecotoxicology (London, England)  2011;20(6):1300-1314.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with elevated concentrations in waters that may also experience hypoxia. Previous research has shown interactions between hypoxia and some PAHs (fluoranthene, α-naphthoflavone) but no interaction with others (benzo[a]pyrene (BaP), β-naphthoflavone). Here we examine how hypoxia (7.4% oxygen, ~35% of normoxia) affects the embryotoxicity of PAHs that act through different mechanisms and the role that CYP1A inhibition may play in these interactions. 500 μg/L BaP and 1-200 μg/L benzo[k]fluoranthene (BkF) interacted synergistically with hypoxia to induce pericardial edema in developing zebrafish (Danio rerio). Hypoxia protected from the embryotoxicity of pyrene (PY) and had no effect on the toxicity of polychlorinated biphenyl-126. Despite previous reports of other CYP1A inhibitors interacting with hypoxia, up to 2000 μg/L dibenzothiophene, 2-aminoanthracene (AA), and carbazole (CB) all failed to induce embryotoxicity under normoxic or hypoxic conditions. The toxicity of PAH mixtures—including binary mixtures of BaP/AA and BaP/CB and two environmentally relevant, complex mixtures—were exacerbated severely by hypoxia to induce or worsen pericardial edema and cause mortality. The interactions between hypoxia and BkF and PY were closely mimicked by morpholino knockdown of CYP1A, indicating a potential role for metabolism of these compounds in their toxicity. Our results indicate that various PAHs may exhibit synergistic, antagonistic or additive toxicity with hypoxia. The enhanced toxicity of environmental mixtures of PAHs under hypoxia suggests that risk assessments that do not take into account potential interactions with hypoxia may underestimate the threat of PAHs to fish in contaminated sites.
PMCID: PMC4018733  PMID: 21706407
Polycyclic aromatic hydrocarbons; hypoxia; zebrafish; multiple stressors; CYP1A
18.  Human Colon Microbiota Transform Polycyclic Aromatic Hydrocarbons to Estrogenic Metabolites 
Ingestion is an important exposure route for polycyclic aromatic hydrocarbons (PAHs) to enter the human body. Although the formation of hazardous PAH metabolites by human biotransformation enzymes is well documented, nothing is known about the PAH transformation potency of human intestinal microbiota. Using a gastrointestinal simulator, we show that human intestinal microbiota can also bioactivate PAHs, more in particular to estrogenic metabolites. PAH compounds are not estrogenic, and indeed, stomach and small intestine digestions of 62.5 nmol naphthalene, phenanthrene, pyrene, and benzo(a)pyrene showed no estrogenic effects in the human estrogen receptor bioassay. In contrast, colon digests of these PAH compounds displayed estrogenicity, equivalent to 0.31, 2.14, 2.70, and 1.48 nmol 17α-ethynylestradiol (EE2), respectively. Inactivating the colon microbiota eliminated these estrogenic effects. Liquid chromatography–mass spectrometry analysis confirmed the microbial PAH transformation by the detection of PAH metabolites 1-hydroxypyrene and 7-hydroxybenzo(a)pyrene in colon digests of pyrene and benzo(a)pyrene. Furthermore, we show that colon digests of a PAH-contaminated soil (simulated ingestion dose of 5 g/day) displayed estrogenic activity equivalent to 0.58 nmol EE2, whereas stomach or small intestine digests did not. Although the matrix in which PAHs are ingested may result in lower exposure concentrations in the gut, our results imply that the PAH bioactivation potency of colon microbiota is not eliminated by the presence of soil. Moreover, because PAH toxicity is also linked to estrogenicity of the compounds, the PAH bioactivation potency of colon microbiota suggests that current risk assessment may underestimate the risk from ingested PAHs.
PMCID: PMC1253702  PMID: 15626640
aryl hydrocarbon receptor; estrogen receptor; oral exposure; simulator of the human intestinal microbial ecosystem (SHIME)
19.  Specificity of Human Aldo-Keto Reductases, NAD(P)H: Quinone Oxidoreductase and Carbonyl Reductases to Redox-Cycle Polycyclic Aromatic Hydrocarbon Diones and 4-Hydroxyequilenin-o-Quinone 
Chemical research in toxicology  2011;24(12):2153-2166.
Polycyclic aromatic hydrocarbons (PAH) are suspect human lung carcinogens and can be metabolically activated to remote quinones, e.g. benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases and to non-K region o-quinones by aldo-keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS) and produce the mutagenic lesion 8-oxo-dGuo, and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1,000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion and hydrogen peroxide formation established that ROS were produced as a result of the redox-cycling. When compared with human recombinant NAD(P)H: quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and lastly CBR1 and CBR3. In A549 cells two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones.
PMCID: PMC3251162  PMID: 21910479
o-quinones; redox-cycling; reactive oxygen species; chemical carcinogenesis; hormonal carcinogenesis
20.  Linoleic Acid-Induced Ultra-Weak Photon Emission from Chlamydomonas reinhardtii as a Tool for Monitoring of Lipid Peroxidation in the Cell Membranes 
PLoS ONE  2011;6(7):e22345.
Reactive oxygen species formed as a response to various abiotic and biotic stresses cause an oxidative damage of cellular component such are lipids, proteins and nucleic acids. Lipid peroxidation is considered as one of the major processes responsible for the oxidative damage of the polyunsaturated fatty acid in the cell membranes. Various methods such as a loss of polyunsaturated fatty acids, amount of the primary and the secondary products are used to monitor the level of lipid peroxidation. To investigate the use of ultra-weak photon emission as a non-invasive tool for monitoring of lipid peroxidation, the involvement of lipid peroxidation in ultra-weak photon emission was studied in the unicellular green alga Chlamydomonas reinhardtii. Lipid peroxidation initiated by addition of exogenous linoleic acid to the cells was monitored by ultra-weak photon emission measured with the employment of highly sensitive charged couple device camera and photomultiplier tube. It was found that the addition of linoleic acid to the cells significantly increased the ultra-weak photon emission that correlates with the accumulation of lipid peroxidation product as measured using thiobarbituric acid assay. Scavenging of hydroxyl radical by mannitol, inhibition of intrinsic lipoxygenase by catechol and removal of molecular oxygen considerably suppressed ultra-weak photon emission measured after the addition of linoleic acid. The photon emission dominated at the red region of the spectrum with emission maximum at 680 nm. These observations reveal that the oxidation of linoleic acid by hydroxyl radical and intrinsic lipoxygenase results in the ultra-weak photon emission. Electronically excited species such as excited triplet carbonyls are the likely candidates for the primary excited species formed during the lipid peroxidation, whereas chlorophylls are the final emitters of photons. We propose here that the ultra-weak photon emission can be used as a non-invasive tool for the detection of lipid peroxidation in the cell membranes.
PMCID: PMC3143142  PMID: 21799835
21.  Interception of Benzo[a]pyrene-7,8-dione by UDP Glucuronosyltransferases (UGTs) in Human Lung Cells 
Chemical Research in Toxicology  2013;26(10):1570-1578.
Polycyclic aromatic hydrocarbons (PAHs) are environmental and tobacco carcinogens. Proximate carcinogenic PAH trans-dihydrodiols are activated by human aldo-keto reductases (AKRs) to yield electrophilic and redox-active o-quinones. Interconversion among benzo[a]pyrene (B[a]P)-7,8-dione, a representative PAH o-quinone, and its corresponding catechol generates a futile redox-cycle with the concomitant production of reactive oxygen species (ROS). We investigated whether glucuronidation of B[a]P-7,8-catechol by human UDP glucuronosyltransferases (UGTs) could intercept the catechol in three different human lung cells. RT-PCR showed that UGT1A1, 1A3, and 2B7 were only expressed in human lung adenocarcinoma A549 cells. The corresponding recombinant UGTs were examined for their kinetic constants and product profile using B[a]P-7,8-catechol as a substrate. B[a]P-7,8-dione was reduced to B[a]P-7,8-catechol by dithiothreitol under anaerobic conditions and then further glucuronidated by the UGTs in the presence of uridine-5′-diphosphoglucuronic acid as a glucuronic acid group donor. UGT1A1 catalyzed the glucuronidation of B[a]P-7,8-catechol and generated two isomeric O-monoglucuronsyl-B[a]P-7,8-catechol products that were identified by RP-HPLC and by LC-MS/MS. By contrast, UGT1A3 and 2B7 catalyzed the formation of only one monoglucuronide, which was identical to that formed in A549 cells. The kinetic profiles of three UGTs followed Michaelis–Menten kinetics. On the basis of the expression levels of UGT1A3 and UGT2B7 and the observation that a single monoglucuronide was produced in A549 cells, we suggest that the major UGT isoforms in A549 cells that can intercept B[a]P-7,8-catechol are UGT1A3 and 2B7.
PMCID: PMC3829198  PMID: 24047243
22.  A QM/MM Investigation of the Chemical Reaction in Dpo4 Reveals Water-Dependent Pathways and Requirements for Active Site Reorganization 
Journal of the American Chemical Society  2008;130(40):13240-13250.
The nucleotidyl-transfer reaction coupled with the conformational transitions in DNA polymerases is critical for maintaining the fidelity and efficiency of DNA synthesis. We examine here the possible reaction pathways of a Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4), for the correct insertion of dCTP opposite 8-oxoguanine using the quantum mechanics/molecular mechanics (QM/MM) approach, both from a chemistry-competent state and a crystal closed state. The latter examination is important for understanding pre-chemistry barriers to interpret the entire enzyme mechanism, since the crystal closed state is not an ideal state for initiating the chemical reaction. The most favorable reaction path involves initial deprotonation of O3′H via two bridging water molecules to O1A, overcoming an overall potential energy barrier of approximately 20.0 kcal/mol. The proton on O1A-Pα then migrates to the γ-phosphate oxygen of the incoming nucleotide as O3′ attacks Pα, and the Pα – O3A bond breaks. The other possible pathway in which the O3′H proton is transferred directly to O1A on Pα has an overall energy barrier of 25.0 kcal/mol. In both reaction paths, the rate-limiting step is the initial deprotonation, and the trigonal-bipyramidal configuration for Pα occurs during the concerted bond formation (O3′–Pα) and breaking (Pα–O3A), indicating the associative nature of the chemical reaction. In contrast, the Dpo4/DNA complex with an imperfect active-site geometry corresponding to the crystal state must overcome a much higher activation energy barrier (29.0 kcal/mol) to achieve a tightly organized site due to hindered O3′H deprotonation stemming from larger distances and distorted conformation of the proton acceptors. This significant difference demonstrates that the pre-chemistry reorganization in Dpo4 costs approximately 4.0 to 9.0 kcal/mol depending on the primer terminus environment. Compared to the higher fidelity DNA polymerase β from the X-family, Dpo4 has a higher chemical reaction barrier (20.0 vs. 15.0 kcal/mol) due to the more solvent-exposed active site.
PMCID: PMC3195406  PMID: 18785738
23.  Succession of Phenotypic, Genotypic, and Metabolic Community Characteristics during In Vitro Bioslurry Treatment of Polycyclic Aromatic Hydrocarbon-Contaminated Sediments 
Dredged harbor sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) was removed from the Milwaukee Confined Disposal Facility and examined for in situ biodegradative capacity. Molecular techniques were used to determine the successional characteristics of the indigenous microbiota during a 4-month bioslurry evaluation. Ester-linked phospholipid fatty acids (PLFA), multiplex PCR of targeted genes, and radiorespirometry techniques were used to define in situ microbial phenotypic, genotypic, and metabolic responses, respectively. Soxhlet extractions revealed a loss in total PAH concentrations of 52%. Individual PAHs showed reductions as great as 75% (i.e., acenapthene and fluorene). Rates of 14C-PAH mineralization (percent/day) were greatest for phenanthrene, followed by pyrene and then chrysene. There was no mineralization capacity for benzo[a]pyrene. Ester-linked phospholipid fatty acid analysis revealed a threefold increase in total microbial biomass and a dynamic microbial community composition that showed a strong correlation with observed changes in the PAH chemistry (canonical r2 of 0.999). Nucleic acid analyses showed copies of genes encoding PAH-degrading enzymes (extradiol dioxygenases, hydroxylases, and meta-cleavage enzymes) to increase by as much as 4 orders of magnitude. Shifts in gene copy numbers showed strong correlations with shifts in specific subsets of the extant microbial community. Specifically, declines in the concentrations of three-ring PAH moieties (i.e., phenanthrene) correlated with PLFA indicative of certain gram-negative bacteria (i.e., Rhodococcus spp. and/or actinomycetes) and genes encoding for naphthalene-, biphenyl-, and catechol-2,3-dioxygenase degradative enzymes. The results of this study suggest that the intrinsic biodegradative potential of an environmental site can be derived from the polyphasic characterization of the in situ microbial community.
PMCID: PMC92767  PMID: 11282603
24.  In Utero Exposure to Benzo[a]pyrene Increases Adiposity and Causes Hepatic Steatosis in Female Mice, and Glutathione Deficiency Is Protective 
Toxicology letters  2013;223(2):10.1016/j.toxlet.2013.09.017.
Polycyclic aromatic hydrocarbons (PAHs), including benzo[a]pyrene (BaP), are ubiquitous environmental pollutants found in tobacco smoke, air pollution, and grilled foods. Reactive metabolites and reactive oxygen species generated during PAH metabolism are detoxified by reactions involving glutathione (GSH). Early life exposures to tobacco smoke and air pollution have been linked to increased risk of obesity and metabolic syndrome. We investigated the independent and interactive effects of prenatal exposure to BaP and GSH deficiency due to deletion of the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, on adiposity and hepatic steatosis in adult female F1 offspring. We mated Gclm+/- dams with Gclm+/- males and treated the pregnant dams with 0, 2, or 10 mg/kg/day BaP in sesame oil by oral gavage daily from gestational day 7 through 16. We analyzed metabolic endpoints in female Gclm-/- and Gclm+/+ littermate F1 offspring. Prenatal BaP exposure significantly increased visceral adipose tissue weight, weight gain between 3 wks and 7.5 months of age, hepatic lipid content measured by oil red O staining, and hepatic fatty acid beta-oxidation gene expression in Gclm+/+, but not in Gclm-/-, female offspring. Hepatic expression of lipid biosynthesis and antioxidant genes were decreased and increased, respectively, in Gclm-/- mice. Our results suggest that reported effects of pre- and peri-natal air pollution and tobacco smoke exposure on obesity may be mediated in part by PAHs. GSH deficiency is protective against the metabolic effects of prenatal BaP exposure.
PMCID: PMC3856224  PMID: 24107266
polycyclic aromatic hydrocarbons; glutathione; fatty liver; glutamate cysteine ligase; obesity; prenatal programming
25.  Comparison of p53 Mutations Induced by PAH o-Quinones with Those Caused by anti-Benzo[a]pyrene Diol Epoxide In Vitro: Role of Reactive Oxygen and Biological Selection 
Chemical research in toxicology  2006;19(11):1441-1450.
Polycyclic aromatic hydrocarbons (PAH) are one of the major carcinogens in tobacco smoke. They are metabolically activated through different routes to form either diol-epoxides, PAH o-quinones, or radical cations and each of which has been proposed to be an ultimate carcinogen. To study how PAH metabolites mutate p53, we used a yeast reporter gene assay based on the p53 transcriptional activity. Colonies expressing wt p53 turn white (ADE +) and those expressing mutant p53 turn red (ADE −). We examined the mutagenicity of three o-quinones, benzo[a]pyrene-7,8-dione, benz[a]anthracene-3,4-dione and dimethylbenz[a]anthracene-3,4-dione, and compared them with (±) anti-benzo[a]pyrene diol epoxide ((±)-anti-BPDE) within the same system. The PAH o-quinones tested gave a dose-dependent increase in mutation frequency in the range of 0.160 – 0.375 μM quinone, provided redox-cycling conditions were used. The dominant mutations were G to T transversions (>42%), and the incidence of hotspot mutations in the DNA-binding domain was more than twice than would be expected by a random distribution. The dependence of G to T transversions on redox-cycling implicates 8-oxo-dGuo as the lesion responsible, which is produced under identical conditions (Chem. Res. Toxicol. (2005) 18: 1027). A dose-dependent mutation frequency was also observed with (±)-anti-BPDE but at micromolar concentrations (0–20 μM). The mutation pattern observed was G to C (63%) > G to A (18%) > G to T (15%) in umethylated p53 and was G to A (39%) > G to C (34%) > G to T (16%) in methylated p53. The preponderance of G mutations is consistent with the formation of anti-BPDE-N2-dGuo as the major adduct. The frequency of hotspots mutated by (±)-anti-BPDE was essentially random in umethylated and methylated p53, suggesting that 5′-CpG-3′ islands did not direct mutations in the assay. These data suggest that smoking may cause mutations in p53 by formation of PAH o-quinones which produce reactive oxygen species. The resultant 8-oxo-dGuo yields a pattern of mutations but not a spectrum consistent with that seen in lung cancer; we suggest that the emergence of the spectrum requires biological selection.
PMCID: PMC2366885  PMID: 17112231
Aldo-keto reductase; polycyclic aromatic hydrocarbon; ortho-quinones; reactive oxygen species

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