THE SIGNIFICANCE OF POLYCYCLIC AROMATIC HYDROCARBONS AS ENVIRONMENTAL CARCINOGENS. 35 YEARS RESEARCH ON PAH—A RETROSPECTIVE

In vivo studies with laboratory animals as well as in vitro studies with bacteria and mammalian cell cultures have demonstrated that the mutagenic and/or carcinogenic properties of numerous PAHs require metabolic transformation. Metabolism of PAHs has been explored in vitro using cellular microsomal fractions, mammalian cell cultures and later genetically engineered cells expressing cytochromes P450 from several species including humans.

Balancing the carcinogenic potential of some environmental matrices (vehicle exhaust, condensate of hard coal combustion effluents, cigarette smoke condensate, used motor oil) after separation into sub-fractions evidenced that the carcinogenic effect may be attributed almost exclusively to PAH. Mixtures of well-known carcinogenic PAH in concentrations as present in these matrices, however, did not explain the total biological effect. Thus, it had been speculated that either very potent unknown carcinogens are still hidden in the PAH fraction, or that synergistic effects (enzyme induction) play a significant role.

In parallel to these carcinogenicity studies, the metabolism of various PAHs has been investigated in rat liver microsomes from untreated animals as well as from animals pre-treated with inducers of cytochrome P450. It was found that even non-carcinogenic PAHs possess a significant inducing potential. Moreover, in several mammals a highly species-specific metabolism of PAH could be observed allowing a critical view to the extrapolation from animal experiments to the human situation. This was further confirmed by experiments with mammalian cell cultures including human ones as well as by metabolic studies with genetically engineered Chinese hamster V79 cells singularly expressing various cytochrome P450 enzymes from a number of different species (human, rat, mouse, fish). With these cell lines metabolic studies were carried out with a larger number of PAHs as substrates including phenanthrene, pyrene, chrysene, benzo[a]anthracene, benzo[c]phenanthrene, benzo[a]pyrene, dibenzo[a,l]pyrene, and benzo[c]chrysene.

Based on the metabolism results, analytical methods have been developed to determine urinary biomarkers of human PAH exposure. Human biomonitoring studies have been performed with different occupationally exposed individuals as well as within smokers and non-smokers of the general population. Endogenous PAH exposure levels and changes in the urinary excreted metabolic profile depending on exposure level have been determined.     

POLYCYCLIC AROMATIC HYDROCARBONS (PAH) IN DANISH SMOKED FISH AND MEAT PRODUCTS

Twenty seven PAH were detected in 45 selected smoked food samples produced in Denmark, including mackerel, herring, trout, small sausages, salami, and bacon. The sum of PAH in smoked meat products ranged from 24 μg/kg for salami to 64 μg/kg in bacon, while those in fish products ranged from 22 μg/kg in smoked mackerel prepared in an electric oven to 1387 μg/kg in herring smoked by direct smoking. The concentration of benzo[a]pyrene for all sample types were below the maximum level of 5 μg/kg for smoked fish and meat set by the European Commission. Results from this survey confirm that the actual level of individual PAH in fish products is dependent on variables such as the type of wood used in the smoking process. Furthermore, the use of the benzo[a]pyrene approach for estimation of the carcinogenicity of PAH in food is confirmed. The Danish intake of benzo[a]pyrene from these smoked products is 2 to 4 ng/person/day.     

DETERMINATION OF DIBENZO[A,L]PYRENE AND OTHER FJORD-REGION PAH ISOMERS WITH MW 302 IN ENVIRONMENTAL SAMPLES

Polycyclic aromatic hydrocarbons (PAHs) occur in the environment as complex mixtures including compounds with mutagenic and carcinogenic activity. The PAH profile routinely determined in environmental samples at present encompasses isomers with molecular weight (MW) not greater than 300. However, PAHs with MW >300 have been demonstrated for several matrices to contribute up to 50% of the total activity when tested for carcinogenicity in experimental animals. Recent studies indicate that among the dibenzopyrenes with MW 302 dibenzo[a,l]pyrene, possessing a fjord region, is by far the most carcinogenic PAH hitherto identified. To further elucidate the environmental relevance of this compound we have applied the isotope dilution GC/MS technique as analytical procedure to determine this compound and the related fjord region PAH naphtho[1,2-a]- and naphtho[1,2-e]pyrene in various matrices. Identification was based on comparison of UV and mass spectra as well as retention times of authentic reference materials. Determination of these PAHs was achieved after clean-up by several chromatographic steps including fractionation on a modified TABA-silica gel column. Quantitative data for matrices such as two cigarette smoke condensates, motor vehicle exhaust condensate (Otto-type engines), and tar-cork are reported. Based on toxic equivalent factors the relative contribution of dibenzo[a,l]pyrene (5.4–42.3%) to the total carcinogenic activity of a PAH profile will be discussed comprising 14 selected isomers (benzo[b]naphtho[2,1-d]thiophene; cyclopenta[cd]pyrene; benz[a]anthracene; chrysene/triphenylene; sum of benzo[b]-, benzo[k]-, and benzo[j]fluoranthene; benzo[a]pyrene; indeno[1,2,3-cd]pyrene; dibenz[a,h]anthracene; benzo[ghi]perylene; anthanthrene; dibenzo[a,l]pyrene determined in these matrices.     

Approaches to the Estimation of Cancer Risk From Ingested PAH

The sum of eight carcinogenic polycyclic aromatic hydrocarbons (PAH) from food in three total diet studies has ranged from 1.45 to 3.50 μg/person/day. The effect of different approaches to the estimation of possible human cancer risk of food borne sources of PAH was illustrated by combining United Kingdom (UK) intake data with cancer potency information in four different ways. The lifetime cancer risk from this level of exposure, based on the sum of risk from individual PAH was estimated to be 5.1 × 10^?5or 8.2 × 10^?7for cancer potencies based on rodent studies and human inhalation data respectively. When benzo[a]pyrene was used as a surrogate for the cancer risk of a PAH containing mixture, the risk was estimated at 1.3 × 10^?3or 1.1 × 10^?5. Variation in dietary intake is small compared with the variation in cancer potency data presently available for risk assessment of PAH from dietary sources.     

Characterization of PM2.5-Bound Polycyclic Aromatic Hydrocarbons in the Ambient Air of Győr,Hungary

ABSTRACT

In Hungary, the nationwide monitoring of PM10-bound polycyclic aromatic hydrocarbons (PAHs) in ambient air is great importance for a number of reasons related to human health, the environment and compliance with European Union legislation. However, the measurement of PAH concentrations in PM2.5 aerosol fraction has not been carried out. Therefore, the concentration, distribution and sources of PM2.5-bound PAHs at different urban sites of Gy?r were investigated in a heating season. The total PAH concentrations (sum of 19 individual PAH compounds) ranged from 1.32 to 37.27 ng/m³ with the mean value of 10.54 ng/m³. The high molecular weight PAHs with 5 and 6 aromatic rings were the most abundant PAHs in PM2.5 aerosol samples, which averaged 82% of total PAHs. Using benzo(a)pyrene (BaP) equivalent approach on the concentration data of carcinogenic PAH species, BaP and indeno(1,2,3-cd)pyrene contributed the highest carcinogenic exposure equivalent (1.25 and 0.19 ng/m³ on average). However, the incremental lifetime cancer risk (ILCR) values for resident children and adults indicated low-potential cancer risk (ILCR < 10^?6). The source apportionment results reflected that the major sources of PAH compounds in the Gy?r atmosphere were fossil fuel combustion and vehicle emissions.     

Relationship Between Inhaled PAH and Urinary Excretion of Phenanthrene,Pyrene and Benzo[a]pyrene Metabolites in Coke Plant Workers

Abstract

By means of personal air samplers the exposure to polycyclic aromatic hydrocarbons (PAH) of four coke employees working at different locations was measured during 4 running days. Simultaneously the 24 hrs urines were collected. A simple, well reproducible method for the determination of 9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, 1-; 2-; 3-; 4- and 9-hydroxyphenanthrenes, 1,2-; 3,4-; and 9,10-dihydroxydihydrophenanthrenes as well as 1-hydroxypyrene and 1,2-dihydroxy-1,2-dihydropyrene was used. As expected, workers on the battery topside are most exposed and, accordingly, excrete by far the highest amount of PAH metabolites. A good correlation between the PAH inhaled during 8 hrs and the metabolites excreted in the 24 hrs urine is observed. 20% to 30% of the inhaled phenanthrene is excreted as dihydrodiols, but only 0.5% of the inhaled benzo[a]pyrene forms 9,10-dihydrodiol in the urine. The individual invariance of the metabolite profile of the isomeric phenols and dihydrodiols indicates a genetically caused enzyme pattern of oxygenases, which can or cannot be induced by exogenous factors. More investigations are necessary to clarify whether this metabolic profile may be suitable as a marker for carcinogenic risk.     

Route-Specific Urinary Biomarkers in the Risk Assessment of PAH Exposure

The route of exposure (oral or inhalatory) of the general population to polycyclic aromatic hydrocarbons (PAH) is toxicologically very important. From a literature survey on the toxicity of benzo[a]pyrene (BP) in relation to the route of exposure, it is concluded that although exposure by food is quantitatively the major route of exposure of B[a]P (94%), inhalation exposure is by far more adverse from a toxicological point of view.

Because of the different composition of the PAH mixture in food and air it is possible to distinguish between the route of exposure by monitoring specific metabolites in human urine. Exposure by food and air can be monitored independently by the determination of 1-hydroxypyrene and 1-hydroxynaphthalene in urine, respectively. A very sensitive GCMS method has been developed for urinary 1- and 2- hydroxynaphthalene, which has been applied to urines of non-occupationally exposed smokers and non-smokers. A large difference in urinary concentrations of hydroxynaphthalenes between both groups was observed being a factor 5.9 and 14 for 1- and 2-hydroxynaphthalene, respectively.     

Levels of 15 + 1 EU Priority Polycyclic Aromatic Hydrocarbons in Different Edible Oils Available in the Syrian Market

ABSTRACT

The levels of 15 + 1 EU priority polycyclic aromatic hydrocarbons (15 + 1 EU PAHs) have been determined in different edible oils (extra virgin olive oil, virgin olive oil, sunflower oil, corn oil, and soybean oil) available in the Syrian market. The samples have been prepared by donor–acceptor complex chromatography and subsequently characterized by high-pressure liquid chromatography coupled with fluorescence and ultraviolet detection for quantification purposes. Variable levels of contamination have been found within different kinds of edible oil samples, and only chrysene has been detected in all the studied samples. Moreover, the mean total sum of 15 + 1 EU PAHs has shown variation from 29.8 µg/kg (corn oil) to 63.7 µg/kg (virgin olive oil). A total of 11 samples out of 38 samples (28.9%) have not fulfilled the European Union (EU) food law requirements. Nine samples have exceeded the EU legislation limit of benzo[a]pyrene (BaP) (2 µg/kg) and only two samples have exceeded the EU legislation limit of PAH4 (10 µg/kg) and had acceptable level of BaP. Finally, the mean and maximum dietary exposures of PAHs through consumption of edible oils have been estimated.     

Determination of Averaged Long-term Air Concentrations of Semivolatile Polycyclic Aromatic Hydrocarbons: First Results

Abstract

Using our long-term sampling methodology presented previously, consecutive 14-day air samples of semivolatile polycyclic aromatic hydrocarbons (PAH) (from fluoranthene/pyrene to coronene) were collected over the time-span of nearly a whole year at a semi-rural site (Jülich) and quantified by GC-FID. As expected, concentrations were higher in winter [3 ng m^?3 for benzo[e]pyrene (BEP)] than in summer (0.4 ng m^?3 for BEP). However, normalized concentrations (to BEP =1) showed constant values for most PAH; thus, a largely stable PAH profile is obvious. Exceptions are benzo[a]pyrene and benz[a]anthracene: their concentrations normalized to BEP were lower in summer, indicating specific losses. Reasons for these losses are unknown as yet, although degradation reactions during atmospheric transport are a distinct possibility.     

Polycyclic Aromatic Hydrocarbons (PAHs) Occurrence and Toxicity in Camellia sinensis and Herbal Tea

ABSTRACT

This study describes a survey of polycyclic aromatic hydrocarbon (PAH) concentrations in 23 green, herbal, and black tea brands widely consumed in Nigeria by determining the levels of benzo[a]pyrene, chrysene (PAH2), benzo[a]pyrene, chrysene, benz[a]anthracene, benzo[b]fluoranthene (PAH4), benzo[a]pyrene, benz[a]anthracene, benzo[k]fluoranthene, chrysene, benzo[b]fluoranthene, dibenz[ah]anthracene, benzo[ghi]per-ylene and indeno[1,2,3-cd]pyrene (PA-H8). Toxic equivalence factor and mutagenic equivalence factor were applied to evaluate the toxic equivalence and mutagenic equivalence quotients relative to benzo[a]pyrene. The concentrations of PAHs indicate that Regulation 835/2011/EC was not fulfilled by benzo[a]anthracene, B[a]A, benzo[a]pyrene, B[a]P, benzo[b]fluoranthene, B[b]F, and chrysene, CHR. The PAH4 levels ranged from 1.28 to 44.57, 4.34 to 11.20, and 0.76 to 34.82 µg/kg in green, black, and herbal tea products, respectively. On the other hand, the PAH8 concentration varied between 1.63 and 65.73, 5.02 and 68.83, and 12.43 and 24.92 µg/kg in green, herbal, and black tea samples. The PAH4 and PAH8 provide more reliable indicators for determination of PAH contamination and risk characterization in food than PAH2.     

Relative Genotoxicities of PAH of Molecular Weight 252 AMU in Coal Tar-Contaminated Sediment

Bioassay-directed chemical fractionation methodology was used to calculate relative mutagenic potencies of polycyclic aromatic hydrocarbons (PAH) of molecular weight 252 amu in coal tarcontaminated sediment from Sydney Harbour, Nova Scotia. A normal phase HPLC technique was used to separate organic solvent extracts into fractions containing isomeric PAH of a single benzologue class. Bioassays with Salmonella typhimurium strain YG1025 with the addition of oxidative metabolism (S9) showed that approximately 50% of the mutagenic activity observed in the sediment extract was associated with PAH of molecular weight 252 amu. Further separation of the 252 PAH fraction using reversed phase HPLC yielded subfractions containing individual compounds; bioassay dose-response curves for these subfractions showed that benzo[a]pyrene was responsible for approximately 75% of the activity of the 252 PAH fraction.     

Very Large PAH Products from Pyrolysis of Mixtures of Smaller PAHS

The larger PAHs produced through pyrolysis of small PAHs as large as pyrene or perylene have been studied previously by several other research groups.^1.2 In the current work, three larger PAHs (benzo[ghi]perylene, coronene, and ovalene) were pyrolyzed at 450 degrees for 16 hours. The reactions used either each PAH alone or binary mixtures of each with the others or with pyrene.     

PAH in airborne particulate matter.: Carcinogenic character of PM10 samples and assessment of the energy generation impact

One of the main anthropogenic sources producing Polycyclic Aromatic Hydrocarbons (PAH) is related to combustion processes especially transport, power generation processes and other industrial activities. Therefore, the main cities constitute one of the main pollution sources for population. Due to the carcinogenic character of some of these pollutants, Directive 2004/107/EC established a target value of 1.0 ng/m³ with regard to Benzo(a)pyrene (BaP) for the total content in the particulate matter fraction averaged over a calendar year. Nevertheless, the consideration of only BaP can underestimate the carcinogenic character of the particulate matter.In this work, the carcinogenic character of the airborne PM10 of Zaragoza was studied during 2003-2004 by determining the concentration of BaP equivalents (BaP-eq), using toxic equivalent factors provided by Larsen and Larsen. Diagnostic ratios were used to discern regarding the main pollution sources in Zaragoza city in which the prevailing emission sources were related to diesel emissions and combustion sources. As PAH can travel long distances around the world, the impact of local pollution sources and long-range atmospheric transport on those samples exceeding 1.0 ng/m³ of BaP-eq that imply higher risk for human health were assessed by considering BaA/Chry and BaP/BeP ratios and by studying the origin of the air masses with the backward air trajectories according to the HYSPLIT model. Those samples were mainly produced during cold season. The local pollution sources were the dominant sources although one episode of long-range transport from European countries could be observed.     

OPTIMIZATION OF LARGE VOLUME INJECTION FOR IMPROVED DETECTION OF POLYCYCLIC AROMATIC HYDROCARBONS (PAH) IN MUSSELS

Detection of PAH of six benzene rings is somewhat troublesome and lowering the limits of detection (LODs) for these compounds in food is necessary. For this purpose, we optimized a Programmable-Temperature-Vaporisation (PTV) injection with Large Volume Injection (LVI) with regard to the GC-MS detection of anthracene, benz[a]anthracene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene and dibenzo[a,e]pyrene. The optimization of PTV-LVI for GC-MS analysis included the choice of liner, solvent venting, splitless time, split flow and initial inlet temperature for injection of 25 μ L standard solution and spiked mussel samples. Samples were extracted with Accelerated Solvent Extraction (ASE) followed by two semi-automatic clean-up steps; gel permeation chromatography (GPC) on S-X3 and solid phase extraction (SPE) on pre-packed silica columns, prior to gas chromatography-mass spectrometry (GC-MS) detection. In comparison to traditional splitless injection, LODs were lowered for eighteen PAHs by the use of PTV-LVI ranging from 0.05 μ g kg ^?1 to 1.0 μ g kg^?1 fresh weight. In particular, the LOD of dibenzo[a,e]pyrene was improved by a factor of ten when using the validated PTV-LVI method.     

IN VITRO INTESTINAL TRANSFER AND METABOLISM OF POLYCYCLIC AROMATIC HYDROCARBONS

Though polycyclic aromatic hydrocarbon (PAH) transfer through intestinal epithelium seems principally governed by transcellular diffusion, other mechanisms may interfere. Several studies suggest a PAH metabolism via CYP450, particularly in liver, but only few data are available regarding intestinal barrier. This in vitro work aimed at studying PAH metabolism and its consequences on the transfer in the intestinal epithelium according to molecule physicochemical properties. It used ¹⁴C-labeled benzo[a]pyrene, pyrene, and phenanthrene. Medium analysis proved a metabolism in intestinal cells for each PAH. Parallel metabolism appeared to play a positive role in the intestinal transfer of PAHs under different forms: ¹⁴C from benzo[a]pyrene, pyrene, and phenanthrene were respectively transferred 26, 4, and 2 times less when cells were incubated with CYP450 inhibitors. Generally speaking, the faster and the higher a PAH crosses intestinal barrier, the less metabolized it will be.     

UPLC MS/MS Quantification of Primary Metabolites of Benzo[a]pyrene and Fluoranthene Produced In Vitro by Sole (Solea solea) Liver Microsomal Activation

Biotransformation pathways of PAHs in a flat fish, Solea solea, living in nurseries close to coastal areas and estuaries constitute a great challenge for the environmental risk. Among PAHs, Fluoranthene (Fluo) has a different chemical structure than benzo[a]pyrene (BaP), and can influence its biotransformation pathways and genotoxic potential. The aim of this study was to bring some response elements about the in vitro metabolic activation of Fluo and BaP by sole liver microsomes. The quantification of several primary metabolites of BaP and Fluo produced in vitro, following a sole liver microsomal activation, was conducted using a sensitive analytical UPLC MS/MS method.

Four types of BaP metabolites (dihydrodiols, diones, OH-BaP, and 7,8-dihydro-epoxide), and three types of Fluo metabolites (2,3-dihydrodiol, 2,3-dione, and sum of OH-Fluo) were quantified. The concentration for which the metabolite production was maximal was of 40 μM and 100 μM for BaP and Fluo, respectively. The biotransformation rate was higher for Fluo (9.97%) than for BaP (6.69%).Whatever the PAH, dihydrodiols and phenols were the major metabolites produced accounting for, respectively, 50% and 30–40% for BaP, and 40 and 60% for Fluo. Despite some differences in conformation, both PAHs seem to be activated by similar pathways and the diol-epoxide appears as a predominant one.     

Thermodynamics of Multicomponent PAH Mixtures and Development of Tar-Like Behavior

This study explores the solid/liquid phase behavior of mixtures of polycyclic aromatic hydrocarbons (PAHs), exploring the transition from non-ideal solid mixtures to a relatively ideal liquid behavior characteristic of "tars". PAH mixtures have been studied using differential scanning calorimetry, melting point analysis and Knudsen effusion. Mixtures of anthracene, pyrene and fluoranthene show behavior that is consistent with other binary PAH mixtures; that is, the initially solid mixture exhibits a significant melting point depression, relative to the pure components, and in a certain range of composition, solid azeotrope behavior on vaporization. As the number of distinct PAH species is increased (by adding in benzo[a]pyrene, phenanthrene, fluorene and chrysene) this behavior gradually gives way to liquid phase character at even room temperature, and the vaporization behavior approaches that crudely predictable from ideal mixture theory.     

PAH Metabolism in Cultured Mammalian Lung Epithelial Cells

In vitro cultures of mammalian cells have an advantage over animal experiments in that human cells can be directly compared with various other mammalian cells. In this study we compared the metabolism of several polycyclic aromatic hydrocarbons (PAH) (benzo[a]pyrene, chrysene, pyrene, phenanthrene, benz[a]anthracene) in epithelial cells from hamster, rat and human lung.

The cells investigated in our system yielded more or less qualitatively similar metabolic profiles for all PAH mentioned above except benz[a]anthracene in the three species. Additionally, species-specific differences were prominent between human and rodent cells with regard to the ratio of phase I and phase II metabolites.

Indications have been obtained that monooxygenase induction is required in fetal cells prior to metabolic conversion of those PAH which lack an inductive potential for CYP450.     

PAH and other emissions from burning of JP-8 and diesel fuels in diffusion flames

Jet fuel JP-8 is of technical interest to the military aviation industry. JP-8 is now the single battlefield fuel for all US Army and Air Force equipment, replacing gasoline altogether and gradually replacing diesel fuel. Hence, emissions from the combustion of this fuel are the subject for this investigation. The emissions from the combustion of JP-8 fuel are examined and are compared to those from diesel fuel No. 2, burned under identical conditions. Combustion occurred inside a laboratory furnace in sooty diffusion flames, under adverse conditions that typically emit large amounts of products of incomplete combustion (PIC). Under such conditions, even compounds that otherwise might appear only in trace amounts were present in sufficient quantities for detection. The study reports on emissions of CO, light volatile organic compounds, semi-volatile organic compounds with an emphasis on polycyclic aromatic hydrocarbons (PAH), particulate emissions, oxides of nitrogen (NO_x) and oxides of sulfur (SO₂). Some PAH compounds are suspected of posing a threat to human health, benzo[a]pyrene being listed as a bio-accumulative toxin by the EPA. An afterburner was also used to examine the effects of longer furnace residence time. Results have demonstrated that PAH emissions from the combustion of diesel fuel were higher than those of JP-8, under most conditions examined. Moreover, as the temperature of the primary furnace was increased, in the range of 600–1000 °C, most of the emissions from both fuels increased. Particulate emissions were reduced by the afterburner, which was operated at 1000 °C, only when the primary furnace was operated at the lowest temperature (600 °C), but that condition increased the CO emissions. Overall, transient combustion of these two fuels, burning in laminar and sooty diffusion flames, did not reveal major differences in the emissions of the following PIC: C1–C4 light aliphatic hydrocarbons, PAH, CO and particulate matter.     

LONG TERM MONITORING OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHS) IN BLUE MUSSELS (Mytilus edulis) FROM A REMOTE SCOTTISH LOCATION

Farmed mussels have been collected on a monthly basis since 1999 from a remote site on the west coast of Scotland for polycyclic aromatic hydrocarbon (PAHs) analysis with the aim of establishing background concentrations as a benchmark against which to assess any environmental incident. Total PAH (2- to 6-ring parent and alkylated) concentrations ranged from 12.5 to 151.2 μg kg^?1 wet weight. Seasonal trends were evident with concentrations being significantly higher for samples collected between November and March compared to those collected between April and October. By taking the median of medians for each of these time periods two background concentrations are suggested for the total PAH concentrations (2- to 6-ring PAHs parent and alkylated); for April to October: 31.2 μg kg^?1 wet weight and for November to March: 62.9 μg kg^?1 wet weight. Individual PAH concentrations were mainly below the OSPAR Background Assessment Concentrations (BACs), where they are specified, and were only exceeded for the heavier 4- and 5-ring PAHs (fluoranthene, pyrene, benz[a]anthracene and benzo[a]pyrene) in samples collected between November and March. Differences were also seen in the PAH profiles with season. Mussels collected between November and March had a higher proportion of the heavier PAHs compared to mussels collected in the summer and autumn.