Phenanthrene Metabolites Research Articles

Biodegradation of phenanthrene by Pseudomonas sp. BZ-3, isolated from crude oil contaminated soil

A phenanthrene (PHE) degrading bacterium strain BZ-3 was isolated from the crude oil contaminated soil in Binzhou, China. The isolate was identified as Pseudomonas sp. BZ-3 on the basis of 16S rRNA gene sequence. Various experiments were conducted to investigate the effect of pH, salinity and PHE concentration on the degradation efficiency of PHE. The degradation efficiency and degradation metabolites of PHE were detected by using GC–MS and HPLC-MS analyses. The strain BZ-3 could degrade 75% of PHE at an initial concentration of 50 mg/L under 20 g/L salinity in 7 days. PHE degradation kinetics was estimated in a first-order degradation rate model and the rate coefficient was calculated as 0.108 d−1. On the basis of the identified degradation metabolites, the strain BZ-3 could degrade PHE in the salicylate metabolic pathway. In a mixture system consisting of PHE and other PAHs including naphthalene (NA), anthracene (ANTH), and pyrene (PYR), the strain BZ-3 showed an efficiently degradation capability. Further study showed that the strain BZ-3 could also use NA, ANTH, PYR, xylene, 1-hydroxy-2-naphthoic acid, and hexane as the sole carbon and energy source, but did not grow on nitrobenzene-containing medium.

Prevalence of External Skin Lesions and Polycyclic Aromatic Hydrocarbon Concentrations in Gulf of Mexico Fishes, Post‐Deepwater Horizon

AbstractWe surveyed offshore fish populations in the Gulf of Mexico in 2011 and 2012, following persistent reports of abnormal skin lesions and other pathologies in the aftermath of the Deepwater Horizon oil spill. The incidence of skin lesions in 2011 sampling was most frequent in some bottom‐dwelling species along the continental shelf edge north of the Deepwater Horizon site. Longline surveys revealed that by 2012 the overall frequency of lesions in northern Gulf of Mexico (NGM) fishes in the vicinity of the Deepwater Horizon had declined 53%, with severity also declining. Relatively high concentrations of polycyclic aromatic hydrocarbon (PAH) metabolites (up to 470,000 ng naphthalene equivalents/g bile wet weight), indicative of oil‐related pollution, were found in fish bile in 2011; concentrations of summed PAHs measured in fish liver and muscle were relatively low (<35 ng/g) due to the efficient metabolism of these compounds by teleost fish. Significant declines in bile concentrations of naphthalene and phenanthrene metabolites in Red Snapper Lutjanus campechanus between 2011 and 2012 indicate an episodic exposure to elevated levels of hydrocarbons of petrogenic origin. The composition of PAH parent compounds and alkylated homologs in Red Snapper liver samples was highly correlated with oil collected at the Deepwater Horizon wellhead but was less coherent with other PAH sources in the NGM. The elevated 2011 prevalence of skin lesions in some NGM species was unrelated to surface salinity or temperature anomalies and was not the result of an epizootic observable in our histopathology samples but was positively correlated with PAH concentration. Thus, we fail to reject the null hypothesis that elevated skin lesion frequency is unrelated to PAH exposure from the Deepwater Horizon oil spill.Received August 14, 2013; accepted March 26, 2014

Multiple degradation pathways of phenanthrene by Stenotrophomonas maltophilia C6

Stenotrophomonas maltophilia strain C6, capable of utilizing phenanthrene as a sole source of carbon and energy, was isolated from creosote-contaminated sites at Hilo, Hawaii. Twenty-two metabolites of phenanthrene, covering from dihydrodiol to protocatechuic acid, were isolated and characterized. Phenanthrene was degraded via an initial dioxygenation on 1,2-, 3,4-, and 9,10-C, where the 3,4-dioxygenation and subsequent metabolisms were most dominant. The metabolic pathways were further branched by ortho- and meta-cleavage of phenanthrene diols to produce 1-hydroxy-2-naphthoic acid, 2-hydroxy-1-naphthoic acid, and naphthalene-1,2-dicarboxylic acid. These intermediates were then transformed to naphthalene-1,2-diol. 1-Hydroxy-2-naphthoic acid was also degraded via a direct ring cleavage. Naphthalene-1,2-diol underwent primarily ortho-cleavage to produce trans-2-carboxycinnamic acid and then to form phthalic acid, 4,5-dihydroxyphthalic acid and protocatechuic acid. Accumulation of salicylic acid in prolonged incubation indicated that a limited extent of meta-cleavage of naphthalene-1,2-diol also occurred. This is the first study of detailed phenanthrene metabolic pathways by Stenotrophomonas maltophilia.

Formation and identification of PAHs metabolites in marine organisms

In this study, the development of the technique APCI+ LC/MS/MS allowed the detection of phenanthrene, pyrene and metabolites of alkyl homologs in fish bile (in situ) and in urine of crabs. Laboratory experiments were carried out exposing crabs from an unpolluted mangrove (Barra de Guaratiba) to phenanthrene, and to the alkylated homologs 1-methyl phenanthrene and 2,6,9-trimethyl phenanthrene. Urine samples were collected at 0, 24, 48, 72, and 96 h. Fishes were captured from strategic sites from Guanabara Bay. Hydroxylated metabolites of phenanthrene, epoxides, orthoquinone and glucoside conjugates were identified in both samples. The method APCI+ LC/MS/MS showed to be effective in a preliminary assessment of phenanthrene metabolite formation, although the low concentrations of 1-methyl phenanthene and 2,6,9-trimethyl phenanthrene did not allow a systematic evaluation of data. The method, however, proved to be excellent tool for studies of PAHs metabolites due to the high selectivity, sensitivity and separation attained.

Exposure to polycyclic aromatic hydrocarbons in women from Poland, Serbia and Italy – relation between PAH metabolite excretion, DNA damage, diet and genotype (the EU DIEPHY project)

Exposure of the general population to polycyclic aromatic hydrocarbons (PAH) is ubiquitous. The aim of this study was to analyze biomarkers associated with the uptake of PAH in 428 non-smoking women from Łodz (Poland), Viterbo (Italy), Belgrade (Serbia) and from the Pančevo area, where the petrochemical complex was destroyed by the air raids in 1999. Urinary excretion of PAH metabolites was lowest in Italian women, intermediary for Serbian and highest in Polish women, who predominantly excreted hydroxy phenanthrenes as metabolites of phenanthrene. Bulky DNA adduct levels were highest in Italian and Polish women. Genotype or PAH ambient air levels could not explain the dissimilarities between the study groups with respect to biomarker patterns, which probably reflected differences in life style-associated factors.

Determination of hydroxylated metabolites of polycyclic aromatic hydrocarbons in sediment samples by combining subcritical water extraction and dispersive liquid–liquid microextraction with derivatization

A sample preparation method for the determination of hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in sediment samples was developed using gas chromatography–mass spectrometry (GC–MS). Dispersive liquid–liquid microextraction (DLLME) with derivatization was performed following the subcritical water extraction (SWE) that provided which was provided by accelerated solvent extraction (ASE). Several important parameters that affected both SWE extraction and DLLME, such as the selection of organic modifier, its volume, extraction temperature, extraction pressure and extraction time were also investigated. High sensitivity of the hydroxylated PAHs derivatives by N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) could be achieved with the limits of detection (LODs) ranging from 0.0139 (2-OH-nap) to 0.2334μgkg−1 (3-OH-fluo) and the relative standard deviations (RSDs) between 2.81% (2-OH-phe) and 11.07% (1-OH-pyr). Moreover, the proposed method was compared with SWE coupled with solid phase extraction (SPE), and the results showed that ASE–DLLME was more promising with recoveries ranging from 57.63% to 91.07%. The proposed method was then applied to determine the hydroxylated metabolites of phenanthrene in contaminated sediments produced during the degradation by two PAH-degraders isolated from mangrove sediments.

Internal exposure to carcinogenic polycyclic aromatic hydrocarbons and DNA damage: a null result in brief

We previously reported DNA strand break frequencies and adduct levels of 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxo-dGuo) in 171 blood samples of coke oven, refractory, graphite electrode, and converter workers exposed to polycyclic aromatic hydrocarbons (PAH). Construction workers (n = 48) were used as controls (Marczynski et al. 2009). Internal dose was assessed by the determination of 1-hydroxypyrene (1-OHP) and the sum of five hydroxyphenanthrenes ( P OHPhe) in post-shift urine samples. Our results clearly indicated increased DNA damage in exposed workers on a group level. The results of the dose–response associations on the individual level, however, did not reveal quantitative correlations between measured exposures and effects. We have discussed several potential reasons for the observed results. In brief, internal exposure and effect was based on single time points (post-shift samples), and kinetics for the formation of 8-oxo-dGuo and DNA strand breaks after exposure to chemical carcinogens such as PAH are not known in detail in humans. The individual exposure circumstances such as varying routes of exposures (dermal vs. inhalation) and potential co-exposures to dust or other chemical compounds at the workplace may have contributed to the dose–response association rather than exposure to PAH alone (Courter et al. 2007). Although PAH can undergo redox cycling and are capable of inducing oxidative DNA damage (Park et al. 2008), DNA strand breaks and 8-oxo-dGuo in blood are general biomarkers of oxidative stress, thus being pathwayspecific rather than substance-specific. Finally, PAH are a complex mixture consisting of carcinogenic and non-carcinogenic compounds and exist in varying profiles in different industries and the environment (Rehwagen et al. 2005; Unwin et al. 2006). 1-OHP and P OHPhe are metabolites of pyrene and phenanthrene, thus representing noncarcinogenic PAH rather than carcinogenic PAH. In order to narrow down the reasons for the lacking association between exposures to PAH and DNA damage on the individual level, we re-analyzed the role of carcinogenic PAH. For this purpose, we focused on benzo[a]pyrene (B[a]P) and naphthalene. B[a]P is a complete carcinogen and often used as a positive control in bioassays when studying genotoxicity and carcinogenicity of chemical compounds (IARC 2010). In addition, B[a]P has been shown to have the highest enzyme inducing efficiency in the lung and liver of rodents, thus self-enhancing the formation of reactive electrophilic and carcinogenic intermediates during its metabolism (Elovaara et al. 2007; Skupinska et al. 2007). Naphthalene has been shown to be carcinogenic in rodents at high concentrations after inhalation (NTP 2000). Available biomarkers to assess H. U. Kafferlein (&) B. Marczynski J. Angerer H.-P. Rihs B. Pesch T. Bruning Institut fur Pravention und Arbeitsmedizin der Deutschen Gesetzlichen Unfallversicherung, Institut der Ruhr Universitat Bochum (IPA), Burkle-de-la-Camp Platz 1, 44789 Bochum, Germany e-mail: kaefferlein@ipa-dguv.de

Exposure to polycyclic aromatic hydrocarbons and serum inflammatory markers of cardiovascular disease

Polycyclic aromatic hydrocarbons (PAHs) are environmental and occupational carcinogens produced by the incomplete combustion of organic materials, such as coal and petroleum product combustion, tobacco smoking, and food cooking, that may be significant contributors to the burden of cardiovascular disease in human populations. The purpose of this study was to investigate associations between ten monohydroxy urinary metabolites of four PAHs and three serum biomarkers of cardiovascular disease (fibrinogen, homocysteine, and white blood cell count). Using data on 3219 participants aged 20 years and older from the National Health and Nutrition Examination Survey (NHANES) 2001–2004 dataset, the associations between PAH metabolites and serum inflammatory markers were analyzed using the Spearman correlations and multiple linear regression modeling. The PAH metabolites of naphthalene, fluorene, phenanthrene, and pyrene each showed both positive and negative correlations with homocysteine, fibrinogen, and white blood cell count (correlation coefficient range: −0.077–0.143) in nonsmoking participants. Using multiple linear regression models adjusted for age, gender, race/ethnicity, and body mass index, estimates of weighted geometric means of inflammatory marker levels were not significantly different between high and low levels (75th vs. 25th percentiles) for all PAH metabolites in nonsmoking subjects. The results of this study do not provide evidence for a relationship between PAH exposure (as measured by urinary levels of PAH metabolites) and serum biomarkers of cardiovascular disease after controlling for tobacco use.

The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB⁻ WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.

Biodegradation of polycyclic aromatic hydrocarbons by a halophilic microbial consortium

In this study we investigated the phenanthrene degradation by a halophilic consortium obtained from a saline soil sample. This consortium, named Qphe, could efficiently utilize phenanthrene in a wide range of NaCl concentrations, from 1% to 17% (w/v). Since none of the purified isolates could degrade phenanthrene, serial dilutions were performed and resulted in a simple polycyclic aromatic hydrocarbon (PAH)-degrading culture named Qphe-SubIV which was shown to contain one culturable Halomonas strain and one unculturable strain belonging to the genus Marinobacter. Qphe-SubIV was shown to grow on phenanthrene at salinities as high as 15% NaCl (w/v) and similarly to Qphe, at the optimal NaCl concentration of 5% (w/v), could degrade more than 90% of the amended phenanthrene in 6days. The comparison of the substrate range of the two consortiums showed that the simplified culture had lost the ability to degrade chrysene but still could grow on other polyaromatic substrates utilized by Qphe. Metabolite analysis by HPLC and GC-MS showed that 2-hydroxy 1-naphthoic acid and 2-naphthol were among the major metabolites accumulated in the Qphe-SubIV culture media, indicating that an initial dioxygenation step might proceed at C1 and C2 positions. By investigating the growth ability on various substrates along with the detection of catechol dioxygenase gene, it was postulated that the uncultured Marinobacter strain had the central role in phenanthrene degradation and the Halomonas strain played an auxiliary role in the culture by utilizing phenanthrene metabolites whose accumulation in the media could be toxic.

Metabolites of Polycyclic Aromatic Hydrocarbons (PAHs) in Bile as Biomarkers of Pollution in European Eel (Anguilla anguilla) from German Rivers

In the light of the alarming decline of the European eel (Anguilla anguilla L.) population, there is an urgent need to define ecological indicators for eel habitat quality. Due to an increasing shortage of glass eels available for local stock enhancement, the decision of whether restocking is a valuable management tool to increase high-quality silver eel escapement to the sea needs to be evaluated. Organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), are among the major threats to fish in their habitat. Therefore, the aim of the investigation presented here was to examine metabolites of PAHs in eel bile as one possible marker for habitat quality. In total, 170 yellow eels were collected in the rivers Rhine, Ems, Weser, Elbe, Havel, Schlei, Eider, Trave, Warnow, Peene, Uecker, and Oder in 2009. PAH metabolites in eel bile were analyzed using high-performance liquid chromatography with fluorescence detection. Metabolites of pyrene and phenanthrene were investigated. Concentrations of PAH metabolites in eel bile varied significantly between several rivers, with the highest mean concentrations of 1-hydroxypyrene and 1-hydroxyphenanthrene in eel bile from the river Trave (2421 and 632 ng/ml). Moreover, huge differences in the ratio of 1-hydroxypyrene to 1-hydroxyphenanthrene, with the highest mean value in eel bile from the river Ems (7.43) and the lowest mean value in eel bile from the river Uecker (0.70), indicate different sources of PAH contamination. A comparative analysis of PAH-metabolite contamination of eels in different river systems is seen as a first step toward a classification of freshwater habitats for restocking purposes.

Impact of the Improved Patsari Biomass Stove on Urinary Polycyclic Aromatic Hydrocarbon Biomarkers and Carbon Monoxide Exposures in Rural Mexican Women

Background: Cooking with biomass fuels on open fires results in exposure to health-damaging pollutants such as carbon monoxide (CO), polycyclic aromatic hydrocarbons (PAHs), and particulate matter.Objective: We compared CO exposures and urinary PAH biomarkers pre- and postintervention with an improved biomass stove, the Patsari stove.Methods: In a subsample of 63 women participating in a randomized controlled trial in central Mexico, we measured personal CO exposure for 8 hr during the day using continuous monitors and passive samplers. In addition, first-morning urine samples obtained the next day were analyzed for monohydroxylated PAH metabolites by gas chromatography/isotope dilution/high-resolution mass spectrometry. Exposure data were collected during the use of an open fire (preintervention) and after installation of the improved stove (postintervention) for 47 women, enabling paired comparisons.Results: Median pre- and postintervention values were 4 and 1 ppm for continuous personal CO and 3 and 1 ppm for passive sampler CO, respectively. Postintervention measurements indicated an average reduction of 42% for hydroxylated metabolites of naphthalene, fluorene, phenanthrene, and pyrene on a whole-weight concentration basis (micrograms per liter of urine), and a 34% reduction on a creatinine-adjusted basis (micrograms per gram of creatinine). Pre- and postintervention geometric mean values for 1-hydroxypyrene were 3.2 and 2.0 μg/g creatinine, respectively.Conclusion: Use of the Patsari stove significantly reduced CO and PAH exposures in women. However, levels of many PAH biomarkers remained higher than those reported among smokers.

DNA adducts and strand breaks in workers exposed to vapours and aerosols of bitumen: associations between exposure and effect

To study the associations between exposure to vapours and aerosols of bitumen and genotoxic effects, a cross-sectional and cross-shift study was conducted in 320 exposed workers and 118 non-exposed construction workers. Ambient air measurements were carried out to assess external exposure to vapours and aerosols of bitumen. Hydroxylated metabolites of naphthalene, phenanthrene and pyrene were measured in urine, whereas (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide ((+)-anti-BPDE), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodGuo) and DNA strand breaks were determined in blood. Significantly higher levels of 8-oxodGuo adducts and DNA strand breaks were found in both pre- and post-shift blood samples of exposed workers compared to those of the referents. No differences between exposed workers and referents were observed for (+)-anti-BPDE. Moreover, no positive associations between DNA damage and magnitude of airborne exposure to vapours and aerosols of bitumen could be observed in our study. Additionally, no relevant association between the urinary metabolites of PAH and the DNA damage in blood was observed. Overall, our results indicate increased oxidative DNA damage in workers exposed to vapours and aerosols of bitumen compared to non-exposed referents at the group level. However, increased DNA strand breaks in bitumen workers were still within the range of those found in non-exposed and healthy persons as reported earlier. Due to the lack of an association between oxidative DNA damage and exposure levels at the workplaces under study, the observed increase in genotoxic effects in bitumen workers cannot be attributed to vapours and aerosols of bitumen.

Phn and Nag-like dioxygenases metabolize polycyclic aromatic hydrocarbons in Burkholderia sp. C3

Burkholderia sp. C3 can transform polycyclic aromatic hydrocarbons (PAHs), a class of ubiquitous pollutants, through multiple pathways, indicating existence of multiple dioxygenases (Seo et al., in Biodegradation 18:123-131, 2006a). Both phn and nag-like genes in C3 were cloned and identified with the DNA sequence alignment and the gene organization in the clusters. When cloned and expressed in Escherichia coli, either the alpha- and beta-subunits of dioxygenase of the phn genes or the ferredoxin-, alpha- and beta-subunits of the nag-like genes transformed naphthalene, phenanthrene and dibenzothiophene but at different rates. The E. coli transformant containing the phn genes transformed phenanthrene faster than that containing the nag-like genes, which was consistent with higher transcription of the phnAc gene than the nagAc-like gene in C3 in response to phenanthrene. 1-Hydroxy-2-naphthanoic acid (1H2NA) and 2-hydroxy-1-naphthanoic acid (2H1NA) (3,4- and 1,2-dioxygenation metabolites of phenanthrene, respectively) were detected in the culture medium of the phn genes transformed E. coli. The concentration of 1H2NA was 262-fold higher than 2H1NA in the medium of the phn genes transformed E. coli. The results suggested that the phn genes play a major role in 1,2-/3,4-dioxygenation and 3,4-dioxygenation dominates. Twenty-eight PAH degradation-associated enzymes including those encoded by the nag-like and phn genes in phenanthrene-grown C3 cells were identified via alignment of amino acid sequences of the detected polypeptides with those in protein databases. The polypeptides were determined with nano liquid chromatography-ion trap mass spectrometry after tryptic in-gel digestion of the enzymes on 1D SDS-PAGE.

Multistressor interactions in the zebrafish ( Danio rerio): Concurrent phenanthrene exposure and Mycobacterium marinum infection

The simultaneous exposure of organisms to toxicants and disease causing agents poses a serious risk to important stocks. Worldwide, aquatic animal disease outbreaks have been increasing in both frequency and severity, and many have been associated with anthropogenic environmental change. Little is known about the complex interactions of the immune system and biotransformational pathways of vertebrates; however, urbanization and coastal development create a scenario in which a wide range of species are exposed to chemical pollutants in conjunction with a wide spectrum of ubiquitous, opportunistic pathogens. These interactions can severely compromise organismal health. Potential effects include decreased fitness, increased predation, decreased fecundity, reduced metabolic activity, suppressed immune function and mortality. Recent attention has been paid to immunomodulation in toxicant exposed fishes. In our current study we investigated the effects of the common polycyclic aromatic hydrocarbon phenanthrene in conjunction with Mycobacterium marinum infection in the zebrafish, Danio rerio. The goal of our study was to elucidate the interactions between stressors in the host organism. Fish were exposed to either a high or low dose of phenanthrene, infected with M. marinum or received a combination exposure of toxicant and bacteria. Results of our study were evaluated using survivorship analysis, toxicant body burden, and histology. Our data show an interaction between M. marinum infection and exposure to a high dose of phenanthrene in the zebrafish. Survivorship was significantly reduced for animals only exposed to the high dose of phenanthrene as compared to all other experimental groups. The increased survivorship for fish exposed to both Mycobacterium and a high dose of phenanthrene suggests an antagonistic interaction between stressors. Body burden data, which show significant differences in the ratio of phenanthrene:metabolites between experimental groups, suggests a disruption of the biotransformational pathway. We postulate that the inflammatory response, initiated by bacterial infection, is impeding the ability of the zebrafish to completely metabolize phenanthrene. In addition, the correlation between reduced metabolite production and increased survival indicates that phenanthrene metabolites are more toxic than the parent compound. Our study underscores the importance of investigating multiple stressor interactions as a way to better understand complex environmental interactions.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of phenolic polycyclic aromatic hydrocarbons (OH-PAH) in urine of non-smokers and smokers

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials and, therefore, occur ubiquitously in the environment and also in tobacco smoke. Since some PAH have been classified as carcinogens, it is important to have access to suitable analytical methods for biomarkers of exposure to this class of compounds. Past experience has shown that measuring a profile of PAH metabolites is more informative than metabolites of a single PAH. Assessment of environmental and smoking-related exposure levels requires analytical methods with high sensitivity and specificity. In addition, these methods should be fast enough to allow high throughput. With these pre-conditions in mind, we developed and validated a high-performance liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of phenolic metabolites of naphthalene, fluorene, phenanthrene and pyrene in urine of smokers and non-smokers. Sample work-up comprised enzymatic hydrolysis of urinary conjugates and solid-phase extraction on C18 cartridges. The method showed good specificity, sensitivity, and accuracy for the intended purpose and was also sufficiently rapid with a sample throughput of about 350 per week. Application to urine samples of 100 smokers and 50 non-smokers showed significant differences between both groups for all measured PAH metabolites, and strong correlations with markers of daily smoke exposure in smoker urine. Urinary levels were in good agreement with previously reported data using different methodologies. In conclusion, the developed LC-MS/MS method is suitable for the quantification of phenolic PAH metabolites of naphthalene, fluorene, phenanthrene, and pyrene in smoker and non-smoker urine.

Adhesion to the hydrocarbon phase increases phenanthrene degradation by Pseudomonas fluorescens LP6a

Microbial adhesion is an important factor that can influence biodegradation of poorly water soluble hydrocarbons such as phenanthrene. This study examined how adhesion to an oil-water interface, as mediated by 1-dodecanol, enhanced phenanthrene biodegradation by Pseudomonas fluorescens LP6a. Phenanthrene was dissolved in heptamethylnonane and added to the aerobic aqueous growth medium to form a two phase mixture. 1-Dodecanol was non-toxic and furthermore could be biodegraded slowly by this strain. The alcohol promoted adhesion of the bacterial cells to the oil-water interface without significantly changing the interfacial or surface tension. Introducing 1-dodecanol at concentrations from 217 to 4,100 mg l(-1) increased phenanthrene biodegradation by about 30% after 120 h incubation. After 100 h incubation, cultures initially containing 120 or 160 mg l(-1) 1-dodecanol had mineralized >10% of the phenanthrene whereas those incubated without 1-dodecanol had mineralized only 4.5%. The production and accumulation of putative phenanthrene metabolites in the aqueous phase of cultures likewise increased in response to the addition of 1-dodecanol. The results suggest that enhanced adhesion of bacterial cells to the oil-water interface was the main factor responsible for enhanced biodegradation of phenanthrene to presumed polar metabolites and to CO(2).

Does Switching to Reduced Ignition Propensity Cigarettes Alter Smoking Behavior or Exposure to Tobacco Smoke Constituents?

Since 2004, several jurisdictions have mandated that cigarettes show reduced ignition propensity (RIP) in laboratory testing. RIP cigarettes may limit fires caused by smoldering cigarettes, reducing fire-related deaths and injury. However, some evidence suggests that RIP cigarettes emit more carbon monoxide and polycyclic aromatic hydrocarbons, and smokers may alter their smoking patterns in response to RIP cigarettes. Both of these could increase smokers' exposures to harmful constituents in cigarettes. An 18-day switching study with a comparison group was conducted in Boston, MA (N = 77), and Buffalo, NY (N = 83), in 2006-2007. Current daily smokers completed 4 laboratory visits and two 48-hr field data collections. After a 4-day baseline, Boston participants switched to RIP cigarettes for 14 days, whereas Buffalo participants smoked RIP cigarettes throughout. Outcome measures included cigarettes smoked per day; smoking topography; salivary cotinine; breath CO; and hydroxylated metabolites of pyrene, naphthalene, phenanthrene, and fluorene. Because the groups differed demographically, analyses adjusted for race, age, and sex. We observed no significant changes in smoking topography or CO exposure among participants who switched to RIP cigarettes. Cigarette use decreased significantly in the switched group (37.7 cigarettes/48 hr vs. 32.6 cigarettes/48 hr, p = .031), while hydroxyphenanthrenes increased significantly (555 ng/g creatinine vs. 669 ng/g creatinine, p = .007). No other biomarkers were significantly affected. Small increases in exposure to phenanthrene among smokers who switched to RIP versions were observed, while other exposures and smoking topography were not significantly affected. Toxicological implications of these findings are unclear. These findings should be weighed against the potential public health benefits of adopting RIP design standards for cigarette products.

Effects of pyrene and fluoranthene on the degradation characteristics of phenanthrene in the cometabolism process by Sphingomonas sp. strain PheB4 isolated from mangrove sediments

Cometabolism has been suggested as an attractive approach to enhance the degradation rates of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). However, the effects of these recalcitrant PAHs on the degradation characteristics of low-molecular-weight PAHs are largely unknown. This study was conducted to investigate the effects of pyrene (PYR) and fluoranthene (FLA) on the degradation characteristics of phenanthrene (PHE) in the cometabolism process by Sphingomonas sp. strain PheB4 isolated from mangrove sediments. Based on the kinetics and characteristics of PHE metabolites, it was proposed that the transformation of hydroxylated PHE into 1-hydroxy-2-naphthoic acid was a rate-limiting step in the degradation of PHE by strain PheB4. Compared to a single addition of PYR or FLA, the presence of a mixture of PYR and FLA decreased the degradation rate of PHE to a larger extent, which was likely because it could inhibit the production of 1-hydroxy-2-naphthoic acid more effectively.

Studying associations between urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) and cardiovascular diseases in the United States

The association between background, enduring environmental exposure to polycyclic aromatic hydrocarbons (PAHs) and cardiovascular diseases has not been well studied in the general population. In this study, we used the National Health and Nutrition Examination Survey (NHANES) 2001–2004 to investigate the associations between eight monohydroxy PAHs (OH-PAHs) and self-report CVD. In a logistic regression model adjusting for cigarette smoking and other covariates, phenanthrene metabolite, 2-hydroxyphennathrene (2-PHEN), was significantly associated with self-report CVD. Compared to subjects within the lowest tertile of 2-PHEN, subjects within the middle and highest tertiles had higher self-report CVD (the 2nd tertile: AOR = 1.29, 95%CI: 0.97–1.72; the 3rd tertile: AOR = 1.45, 95%CI: 1.01–2.07; p for trend = 0.04). In addition, fluorene metabolite (i.e. 2-hydroxyfluorene) also showed a marginally significant linear trend with self-report CVD ( p for trend = 0.07). Further studies are necessary to explore the associations between these highly prevalent pollutants and CVD.