Time-Integrated DNA Adduct Levels and Relative Tumorigenic Potencies of Six Polycyclic Aromatic Hydrocarbons in Strain A/J Mouse Lung

We have investigated the induction of DNA adducts and adenomas in the lungs of strain A/J mice following the i.p. administration of several polycyclic aromatic hydrocarbons (PAH): pyrene, dibenz[a,h]anthracene (DBA), benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), 5-methylchrysene (5-MeC), 3-methylcholanthrene (3-MC), and cyclopenta[cd]pyrene (CPP). All of the PAH induced lung adenomas, with relative tumor potency rankings as a function of administered dose: DBA = 3-MC > 5-MeC > CPP > B[a]P > B[b]F. DNA adducts reached maximal levels between 3 and 7 days after injection, followed by a gradual decrease. The time-integrated DNA adduct level (TIDAL) was calculated by numerically integrating the areas under the adduct persistence curves extrapolated out to 240 days for each PAH at each dose level. Tumorigenic potencies as a function of TIDAL values for 5-MeC, B[a]P, B[b]F, and CPP were all equal, while 3-MC was 2.6-fold more potent and DBA was 25.8-fold more potent.     

Protein engineering of cytochrome P450cam (CYP101) for the oxidation of polycyclic aromatic hydrocarbons

Mutations of the active site residues F87 and Y96 greatly enhancedthe activity of cytochrome P450_cam (CYP101) from Pseudomonasputida for the oxidation of the polycyclic aromatic hydrocarbonsphenanthrene, fluoranthene, pyrene and benzo[a]pyrene. Wild-typeP450_cam had low (<0.01 min^–1) activity with these substrates.Phenanthrene was oxidized to 1-, 2-, 3- and 4-phenanthrol, whilefluoranthene gave mainly 3-fluoranthol. Pyrene was oxidizedto 1-pyrenol and then to 1,6- and 1,8-pyrenequinone, with smallamounts of 2-pyrenol also formed with the Y96A mutant. Benzo[a]pyrenegave 3-hydroxybenzo[a]pyrene as the major product. The NADHoxidation rate of the mutants with phenanthrene was as highas 374 min^–1, which was 31% of the camphor oxidation rateby wild-type P450_cam, and with fluoranthene the fastest ratewas 144 min^–1. The oxidation of phenanthrene and fluoranthenewere highly uncoupled, with highest couplings of 1.3 and 3.1%,respectively. The highest coupling efficiency for pyrene oxidationwas a reasonable 23%, but the NADH turnover rate was slow. Theproduct distributions varied significantly between mutants,suggesting that substrate binding orientations can be manipulatedby protein engineering, and that genetic variants of P450_cammay be useful for studying the oxidation of polycyclic aromatichydrocarbons by P450 enzymes.     

Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.     

Release of carcinogenic hydrocarbons in the carbonization of coal pitch

The carbonization of industrial coal-pitch samples is studied, with particular attention to the liberation and conversion of benz[a]pyrene and other carcinogenic polycyclic aromatic hydrocarbons. The hydrocarbon composition of pitch sublimates and the benz[a]pyrene concentration in the smokestack gases are determined during regular and high-speed carbonization. The maximum benz[a]pyrene content in the smokestack gases is seen at 750–800°C, regardless of its content in the initial pitch. The benz[a]pyrene content basically determines the carcinogenic hazards of pitch processing.     

Comparison of the Morphological Transforming Activities of Fjord-Region PAHs with Dibenzo[a,e]Pyrene and Benzo[a]Pyrene

The morphological transforming activities in mouse embryo C3H10T1/2CL8 (C3H10T1/2) cells were examined for six PAHs: benzo[c]chrysene (B[c]C); benzo[g]chrysene (B[g]C); benzo[c]phenanthrene (B[c]P); dibenzo[a, l]pyrene (DB[a, l]P); dibenzo[a,e]pyrene (DB[a,e]P) and benzo[a]pyrene (B[a]P). C3H10T1/2 cells treated with B[c]P or B[g]C at concentrations of 0–3 μg/ml did not produce any transformed Type II or III foci after 24 hr of exposure. Concurrent cytotoxicity was observed. Under the same conditions, B[a]P and B[c]C were active, with B[c]C approximately one-half the activity of B[a]P. However, after a 48-hr treatment, B[c]P and B[g]C gave significant activity measured as both foci/dish or the number of dishes exhibiting foci. After a 24-hr treatment, comparison of B[a]P with two dibenzopyrenes, DB[a, l]P and DB[a,e]P, gave activities in the order: DB[a, l]P > B[a]P > DB[a,e]P. After 48 hr of treatment, both B[a]P and DB[a,e]P had similar activities.     

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.     

Polycyclic aromatic hydrocarbons in coke-plant wastewater

The content of polycyclic aromatic compounds—including the strong carcinogens benz[a]pyrene and dibenz[a,h]anthracene—in coke-plant wastewater is investigated. Biochemical purification permits the removal of the following polycyclic aromatic compounds: naphthalene, acenaphthylene, fluorene, acenaphthene, phenanthrene, anthracene, fluoranthene, pyrene, and benz[k]fluoranthene (79.6–99.9% removal); and benz[a]pyrene (65.7% removal). By contrast, biochemical treatment increases the content of the following compounds in the wastewater: benz[b]fluoranthene, benz[g,h,i]perylene, and indeno[1,2,3-cd]pyrene.     

Reducing the carcinogenic impact of pitch processing

Diverse opinions exist regarding the properties of benz[a]pyrene and other polycyclic aromatic hydrocarbons in coal pitch and their carcinogenic impact. Current concepts regarding chemical carcinogenesis and the development of occupational sickness are outlined. Information is presented relating to the reduction in the carcinogenic impact of coal pitch and its processing products. It is shown that the content of benz[a]pyrene and other polycyclic aromatic hydrocarbons in coal pitch, its sublimates, and atmospheric emissions may be reduced by means of various additives. However, it is probably impossible to eliminate benz[a]pyrene emission completely, since it is an unavoidable product of the high-temperature pyrolysis of organic materials.     

Interaction Analyses of Binary Mixtures of Carcinogenic PAHs Using Morphological Cell Transformation of C3H10T1/2CL8 Mouse Embryo Fibroblasts in Culture

Studies of defined mixtures of carcinogenic polycyclic aromatic hydrocarbons (PAH) have identified three major categories of interactions: antagonism; synergism; and additivity depending on the biological model, tissue, route of exposure, and specific PAH. To understand the bases of these interactions we studied binary mixtures of benzo[a]pyrene (B[a]P) and dibenz[a,h]anthracene (DBA) in transformable C3H10T1/2C18 (C3H10T1/2) mouse embryo fibroblast cells in culture. C3H10T1/2 cells treated with binary mixtures of B[a]P and DBA gave less than additive morphological cell transformation based on response additivity. These results were consistent with those reported in mice and rats on the antagonistic effects of B[a]P and DBA on tumorigenesis. ³²P-Postlabeling DNA adduct studies revealed that DBA reduced B[a]P-DNA adduct levels by 47% with no effect on DBA-DNA adduct levels. This suggests that one mechanism for the inhibition of morphological cell transformation of binary mixtures of B[a]P and DBA is due to alterations in the metabolic activation of B[a]P.     

Carbonization of coal pitch with additives

The ability of organic and inorganic additives (polyethylene terephthalate, titanium dioxide, finely disperse carbon, petroleum bitumen) to reduce the carcinogenic impact of coal-pitch carbonization is studied. Additives may reduce the quantity of pitch sublimates and their content of carcinogenic polycyclic aromatic hydrocarbons. Some additives are able to reduce the benz[a]pyrene content in the exhaust gases, but its complete elimination is impossible, since benz[a]pyrene is a natural product of the high-temperature pyrolysis of organic materials. For this reason, additions of petroleum products to coal pitch cannot reduce the benz[a]pyrene emissions in the exhaust gases.     

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes

Intracellular free zinc ([Zn²⁺]_i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn²⁺]_i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn²⁺ have been identified, proteins that are involved in Zn²⁺ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn²⁺ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn²⁺]_i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn²⁺]_i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca²⁺ oscillations. Assays with fluorescent Zn²⁺ indicators revealed that the basal [Zn²⁺]_i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn²⁺]_i level in the presence of 1 μM Zn²⁺ in inflammatory FLSs. Among the 17 out of 24 known Zn²⁺ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn²⁺]_i level. Taken together, Zn²⁺ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn²⁺]_i in inflammatory FLSs.     

Assessing the carcinogenicity of carbon-bearing binders

Methods of determining the carcinogenicity of carbon-bearing binders in terms of their benzo[a]pyrene content and the benzo[a]pyrene emissions in binder carbonization at temperatures up to 850°C are considered. In the laboratory, CARBORES-T and CARBORES-P binders with reduced benzo[a]pyrene content are studied, as well as the industrial carbon pitch from which they are derived. It is found that predicting the carcinogenicity of carbon-bearing binders in terms of their benzo[a]pyrene content is unsatisfactory, largely because the formation and emission of benzo[a]pyrene on binder heating is ignored. Quantitative determination of the benzo[a]pyrene emission in the carbonization of the binders may be regarded as a more reliable and more universal method. It may be used to compare the carcinogenicity of products derived from coal and petroleum, as well as binders that do not contain benzo[a]pyrene.     

Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri,Chordata)

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher’s) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name “B. belcheri EAS/AOS” (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.     

Major gaseous and PAH emissions from a fluidized-bed combustor firing rice husk with high combustion efficiency

This experimental work investigated major gaseous (CO and NO_x) and PAH emissions from a 400 kW_th fluidized-bed combustor with a cone-shaped bed (referred to as ‘conical FBC’) firing rice husk with high, over 99%, combustion efficiency. Experimental tests were carried out at the fuel feed rate of 80 kg/h for different values of excess air (EA). As revealed by the experimental results, EA had substantial effects on the axial CO and NO_x concentration profiles and corresponding emissions from the combustor. The concentration (mg/kg-ash) and specific emission (μg/kW h) of twelve polycyclic aromatic hydrocarbons (PAHs), acenaphthylene, fluorene, phenanthrene, fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene and indeno[1,2,3-cd]pyrene, were quantified in this work for different size fractions of ash emitted from the conical FBC firing rice husk at EA = 20.9%. The total PAHs emission was found to be predominant for the coarsest ash particles, due to the effects of a highly developed internal surface in a particle volume. The highest emission was shown by acenaphthylene, 4.1 μg/kW h, when the total yield of PAHs via fly ash was about 10 μg/kW h.     

Characterization of molecular markers in smoke from residential coal combustion in China

The organic constituents and distributions of molecular markers emitted from a residential coal-stove burning honeycomb coal briquettes were determined in this study. The major organic components emitted directly in smoke particles were polycyclic aromatic hydrocarbons (PAHs), with abundant hydroxy-polycyclic aromatic hydrocarbons (OH-PAHs), i.e., thermally altered derivative compounds from coal combustion, UCM (unresolved complex mixture of branched and cyclic compounds), n-alkanes and n-alkanoic acids. Other compounds present as minor components included n-alkenes, phenols, alkylbenzenes and n-alkanols. The distributions of the organic compounds in the coal smoke samples were highly variable and dependent on combustion temperature, flame aeration, fire duration, and coal rank. Coal smoke emissions may be identified by some indicators including: (1) presence of hydroxy-PAHs, (2) the decrease in carbon preference index (CPI) of n-alkanoic acids with increasing rank, (3) the decrease of the ratios of 17α(H),21β(H)-29-norhopane to 22R-17α(H),21β(H)-homohopane and 17α(H),21β(H)-29-norhopane to 17α(H),21β(H)-hopane with increasing rank, (4) the increases in the homohopane index [22S/(22S + 22R)] and the 17α(H),21β(H)-hopane to 17β(H), 21α(H)-hopane ratio with increasing rank, and (5) the increase of benzo[e]pyrene/(benzo[e]pyrene + benzo[a]pyrene) with increasing rank. In addition, the diagnostic ratios among PAHs and between PAHs and the corresponding hydroxy-PAHs, such as benz[a]anthracene/(benz[a]anthracene + chrysene), indeno[1,2,3-cd]pyrene/(indeno[1,2,3-cd]pyrene + benzo[ghi]perylene), pyrene/OH-pyrene, and chrysene/OH-chrysene can be used to distinguish bituminous from anthracite coal smoke emissions.     

Responses of Human Cells to PAH-Induced DNA Damage

Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 (CYP) CYP1A1 and CYP1B1, which metabolize these polycyclic aromatic hydrocarbons (PAHs) into DNA-binding species. In order to detail roles of CYP1A1 and CYP1B1 in activation of DB[ a,l ]P to the diol epoxide, we here report the inhibition of CYP1A1 in human MCF-7 cells with phosphorodiamidate morpholino antisense oligomers (morpholinos). PAH-DNA adduct formation was also determined after treatment with morpholinos and B[ a ]P or DB[ a,l ]P. p53 is involved in DNA repair, cell cycle arrest, and apoptosis. Cells with normal p53 protein arrest in the G1 phase of the cell cycle on exposure to DNA-damaging agents (presumably allowing the cell sufficient time to repair damaged DNA prior to replication). Previous studies in human MCF-7 cells indicate that cells with PAH-DNA adducts escape cell cycle arrest and accumulate in the S phase. In the present study the effect of PAH-DNA adducts on the cell cycle were observed in human diploid fibroblasts (HDF). We found that treatment of HDF with the diol epoxide of DB[ a,l ]P causes cell cycle arrest in G 1 . An increase in DNA adduct formation with increase in concentration of dibenzo[ a,l ]pyrene diol epoxide {( m )- anti -DB[ a,l ]PDE} was also observed.     

MOLECULAR GEOMETRY,CYP1A GENE INDUCTION AND CLASTOGENIC ACTIVITY OF CYCLOPENTA[c]PHENANTHRENE IN RAINBOW TROUT

Cyclopenta[c]phenanthrene (CP[c]Ph) is a PAH member that shows similarities to bay-and fjord region possessing PAHs. On the basis of X-ray measurements it was found that the molecule of this hydrocarbon is planar. In this case, intramolecular strains caused by repulsion between protons in the pseudo fjord-region are balanced by both the shortening of some bonds which acquire more double bond character, and by the enlargement of exocyclic angles within the pseudo fjord-region. The activity of CP[c]Ph was investigated in vivo in rainbow trout, Oncorhynchus mykiss. The CYP1A mRNA levels following 48h-treatment with CP[c]Ph or benzo[a]pyrene (B[a]P; positive control) were determined and compared with incidences of clastogenic changes observed in the peripheral blood erythrocytes. We have found that the ability to induce CYP1A by these PAH compounds is positively correlated with the incidences of clastogenic changes in rainbow trout erythrocytes.