Contribution of polycyclic aromatic hydrocarbons and polar polycyclic aromatic compounds to the carcinogenic impact of flue gas condensate from coal-fired residential furnaces evaluated by implantation into the rat lung.

For identification of the substances chiefly responsible for the carcinogenic action of the emission condensate from coal-fired residential furnaces, the implantation method was used as a carcinogen-specific bioassay for comparison of the carcinogenic effect of various fractions with that of a total sample of flue gas condensate tested in 2 or 3 different doses. After implantation into the lungs of Osborne-Mendel rats, the condensate from coal-fired residential furnaces, a fraction containing polycyclic aromatic hydrocarbons (PAHs) and thiaarenes [sulfur-containing polycyclic aromatic compounds (S-PACs)] with 4-7 rings, as well as fraction containing more polar polycyclic aromatic compounds (PACs) and PAHs with higher molecular weight, induced lung carcinomas and sarcomas. According to probit analysis, the fraction containing PAHs plus S-PACs with 4-7 rings accounted for about 68.2% of the total carcinogenicity of flue gas condensate, whereas the fraction containing more polar PACs and higher PAHs accounted for about 54.6%. All other fractions, such as nonaromatic compounds and PACs with 2 and 3 rings, constituting about 70% of the weight of the total condensate, seemed not to be carcinogenic. Only 1.4% of the total carcinogenicity of the flue gas condensate was found to be attributable to the amount of benzo[a]pyrene (CAS: 50-32-8) present in the condensate (1.14 mg/g condensate). The contribution of more than 100% of both active fractions to the total carcinogenicity (68.2 and 54.6%) may suggest an interrelation of the fractions.

Contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of gasoline engine exhaust condensate evaluated by implantation into the lungs of rats.

An attempt was made to identify the substances chiefly responsible for the carcinogenicity of gasoline engine exhaust condensate. A carcinogen-specific bioassay was performed by a comparison of the carcinogenic effect of various fractions with that of a total sample of automobile exhaust condensate, tested in two or three different doses. The results were examined by Probit analysis. After implantation into the lungs of OM rats, the condensate emitted from a gasoline-driven automobile and the fraction of polycyclic aromatic compounds consisting of more than 3 rings induced lung carcinomas and sarcomas. The tumor incidence demonstrated a clear-cut dose-response relationship. The fraction of polycyclic aromatic hydrocarbons (PAH) consisting of more than 3 rings accounted for about 81% of the total carcinogenicity of automobile exhaust condensate. This fraction represented only 2.8% by weight of the condensate. The content of benzo[a]pyrene (CAS: 50-32-8; 0.483 mg/g condensate) accounted for 2.4% of the total carcinogenicity of automobile exhaust condensate. Regarding the minor effect of the PAH-free fraction (approximately equal to 87% by wt), no evidence of cocarcinogenic activity was observed, since the total condensate as well as the PAH fraction consisting of more than 3 rings applied proportionally caused about the same tumor incidence.

Experimental studies in rat lungs on the carcinogenicity and dose-response relationships of eight frequently occurring environmental polycyclic aromatic hydrocarbons.

The biologic activity of eight highly purified polycyclic aromatic hydrocarbons (PAH) widely distributed in the human environment was tested in the respiratory tracts of rats. These studies were performed for the examination of carcinogenic activity of the compounds and determination of a dose-response relationship. The lung implantation method was used in 3-month-old female OM rats. A dose-response relationship was obtained for benzo[a]pyrene (BaP), anthanthrene (ANT), benzo[b]fluoranthene (BbF), indeno[1,2,3-cd]pyrene (IND), benzo[j]fluoranthene (BjF), and benzo[k]fluoranthene (BkF). Benzo[e]pyrene and benzo[ghi]perylene showed no tumor-producing effect in this system when given at doses of 5 mg. The histologic and mathematical evaluations indicated that the investigated compounds had distinct carcinogenic potencies. After probit analysis of the results, the carcinogenic potencies of PAH investigated in the lung implantation model rank as follows: BaP, 1.00; ANT, 0.19; BbF, 0.11; IND, 0.08; BkF, 0.03; and BjF, 0.03.

Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples.

The identification of pollen plays an important role in ecology, palaeo-climatology, honey quality control and other areas. Currently, expert knowledge and reference collections are essential to identify pollen origin through light microscopy. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach, but the assessment of mixed pollen samples originating from multiple plant species is still a tedious and error-prone task. Next-generation sequencing has been proposed to avoid this hindrance. In this study we assessed mixed pollen probes through next-generation sequencing of amplicons from the highly variable, species-specific internal transcribed spacer 2 region of nuclear ribosomal DNA. Further, we developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. We assessed in total 16 mixed pollen samples, 14 originated from honeybee colonies and two from solitary bee nests. The sequencing technique resulted in higher taxon richness (deeper assignments and more identified taxa) compared to light microscopy. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Simulation analyses of taxon specificity and sensitivity indicate that 96% of taxa present in the database are correctly identifiable at the genus level and 70% at the species level. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialised palynological expert knowledge.

Profile analysis of polycyclic aromatic hydrocarbons and metal content in sediment layers of a lake.

The question is investigated whether polycyclic aromatic hydrocarbons (PAH) in the annual sediment layers of a lake mainly result from air dust pollution. Sample layers taken from drilling cores going back to 1915 show no significant differences at the forest shore (no buildings) during this period. In contrast, samples taken from a built-up area of the shore (with a highway and a main railway line) show today 5 times the amount of PAH compared with 1915. The same trend is observed in the content of Zn, whereas Pb, Fe, Cr, Ni, Cu and Mn levels are constant. Identification or characterization of PAH was accomplished by comparison of the retention times and mass spectrometry of authentic compounds. Sixty-four PAH are described. The results indicate that the burden of carcinogenic PAH air pollutants has increased 5-fold from 1915-1970.

Dose-dependent induction of rat liver microsomal aryl hydrocarbon monooxygenase by benzo[k]fluoranthene.

The environmentally widespread polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene is a potent AHH inducer. This has been proven by recording the benz[a]anthracene metabolite profile in the rat liver by means of gas chromatography/mass spectrometry (GC/MS) technique. Even a total dose of 3 times 50 micrograms/kg body wt increases the metabolism of benz[a]anthracene by a factor of 2. The formation of the 8,9- as well as the 5,6-dihydrodiol is stimulated to about the same extent, whereas the formation of the 10,11-dihydrodiol is suppressed. After comparatively low doses of the inducer, a metabolite is formed which corresponds in all parameters with the postulated ultimate carcinogen 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene. This metabolite and a number of other primary and secondary oxidation products could be identified after incubation with induced but not with normal microsomes. Therefore, it should be emphasised that metabolite profiles have to be recorded instead of measuring brutto conversions of PAH substrates to evaluate inducing effects.

The influence of polycyclic aromatic hydrocarbons as inducers of monooxygenases on the metabolite profile of benz[a]anthracene in rat liver microsomes.

Microsomal oxidation of benz[a]anthracene (BaA) in rat liver has been shown to occur at various positions (1,2-, 3,4-, 5,6-, 8,9- and 10,11-position) by means of gas chromatography/mass spectrometry (GC/MS) and comparison with synthetic reference substances. In normal rats trans-5,6-, 8,9- and, mainly, 10,11-dihydrodiols have been detected as primary metabolites. The induction of monooxygenases by polycyclic aromatic hydrocarbon (PAH) results in a considerable change in the metabolite profile, since the 5,6- and 8,9-isomers become the main metabolites while the amount of 10,11-isomer is not increased. Simultaneously, the secondary metabolism to form triols and tetrols is induced. Phenobarbital as well as 'moderately inducing' PAH (pyrene, benzo[ghi]perylene, benzo[e]pyrene) induced the oxidation at 5,6- and 8,9-position, whereas almost all other compounds investigated, especially the benzofluoranthenes, additionally induced the oxidation at the 3,4-position forming the precursor of the ultimate carcinogen of BaA, 3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene, which was detected as its isomerisation product, the 2,3,4-triol.

The predominant role of S-oxidation in rat liver metabolism of thiaarenes.

Thiaarenes are metabolized by liver microsomes of untreated rats predominantly to sulfones and sulfoxides. After pretreatment of rats with monooxygenase inducers, ring oxidation of thiaarenes is also observed. In case of benzo[b]naphtho[2,3-d]thiophene the formation of a p-quinone takes place. Rat liver microsomal metabolism of the thiaarenes tested as substrates did not resemble that of the polycyclic aromatic hydrocarbon (PAH) isosters at all.

Experimental studies on the carcinogenicity of five nitrogen containing polycyclic aromatic compounds directly injected into rat lungs.

Using a beeswax/tricaprylin mixture as vehicle, three doses each of acridine, benz[a]acridine (BaAC), benz[c]acridine (BcAC), dibenz[a,h]-acridine (DBa,hAC) and dibenz[a,j]acridine (DBa,jAC) were injected into the lungs of 35 female Osborne-Mendel rats per group. To compare the carcinogenic potency of the heterocycles, benzo[a]pyrene (BaP) was taken as reference substance. Dose-response relationships were obtained for DBa,hAC as well as for BaP. DBa,jAC and BcAC exhibited no carcinogenic effects in the dose range from 0.1 mg to 1 mg tested in the lung implantation model. From the results presented here, it cannot be excluded that tumor development may occur when testing higher doses. BaP, however, must be considered much more potent since it revealed, by far, higher tumor incidences and diminished life times.

Monooxygenase induction by various xenobiotics and its influence on rat liver microsomal metabolism of chrysene in comparison to benz[a]anthracene.

The potencies of various xenobiotics for induction of monooxygenases and their influence on the rat liver microsomal metabolite profile of the environmentally relevant weak carcinogen, chrysene, was determined. Among the widely distributed chemicals, polychlorinated biphenyls (PCB) and preferentially 3,3',4,4'-tetrachlorobiphenyl as well as PAHs and their heterocyclic analogues such as benzo[a]pyrene, benzo[b]- and -[j]fluoranthene, indeno[1,2,3-cd]pyrene, dibenz[a,h]acridine, benzo[b]naphtho-[2,1-d]thiophene, and 5,6-benzoflavone were found to be potent inducers stimulating the formation of the proximate, and some of them also the ultimate carcinogen of chrysene. Lindane, carbaryl, DDT, and pentachlorophenol were found to be inefficient or weak inducers. With the exception of phenobarbital no inducers were found among the pharmaceuticals investigated. Sex-dependent metabolism was found for Wistar-rats. No 1,2-oxidation was observed in females, and turnover rates were lower than in males. These findings confirm the results previously obtained with benz[a]anthracene as substrate. The inducing potencies of various compounds tested were similar for both of these substrates. It is interesting to note that in most cases the same effective xenobiotic induces the bay-region diolepoxide in both, chrysene and benz[a]anthracene.

On the metabolic activation of benz[a]acridine and benz[c]acridine by rat liver and lung microsomes.

The metabolism of benz[a]- and benz[c]acridine by liver and lung microsomes from untreated, phenobarbital (PB)-treated and benzo[k]fluoranthene (BkF)-treated rats has been studied by gas chromatography/mass spectrometry (GC/MS). Epoxidation and hydrolysis of the epoxides to dihydrodiols were found to be the predominant pathways for all substrates. N-Oxidation is likely to occur in the case of benz[c]acridine. However, no unequivocal evidence could be obtained for the formation of the ultimate carcinogens--the t-3,4-dihydrodiol-1,2-epoxides--in case of both benz[a]- and benz[c]acridine. K-Region oxidation was induced by phenobarbital, whereas the formation of non-K-region metabolites increased after BkF treatment in the case of benz[c]acridine.

Monooxygenase induction by various xenobiotics and its influence on the rat liver microsomal metabolite profile of benz[a]anthracene.

Several pesticides (lindane, carbaryl, pentachlorophenol, DDT), polycyclic aromatic hydrocarbons (PAH) and heterocyclic analogues (fluoranthene, dibenz[a,h]anthracene, dibenz[a,h]acridine, indeno[1,2,3-cd]pyrene, 10-azabenzo[a]pyrene) and pharmaceuticals (diphenylhydantoin, ethinylestradiol, levonorgestrel) were tested for their potencies to induce monooxygenase activities in the rat liver by means of recording the metabolite profile of benz[a]anthracene in rat liver microsomal incubations. Some of them were found to be weak or moderate inducers, but even less efficient ones altered the benz[a]anthracene metabolite profile significantly. Only indeno [1,2,3-cd]pyrene stimulated the bay-region oxidation of benz[a]anthracene. A sex-dependent metabolism was observed in both untreated and contraceptive-pretreated Wistar rats.

Local application to mouse skin as a carcinogen specific test system for non-volatile nitroso compounds.

Using the epicutaneous test as an experimental model for detecting carcinogenicity, 3 doses each of nitrosomethylurea (NMU), nitrosonornicotine (NNN) and nitrosocarbaryl (NC) were administered to the skin of 65 female CFLP mice/group. To compare the carcinogenic potency of the nitroso compounds, benzo[a]pyrene (BaP) was taken as reference substance. Dose-response relationships were obtained for NMU and NC as well as for BaP. NNN exhibited only a weak carcinogenic effect in the dose range from 12.5 micrograms to 200 micrograms tested in the skin painting model. It showed, however, no dose dependent activity. After probit analysis of the results, the carcinogenic potencies of the nitroso compounds investigated in this system rank as follows: NC, 0.18; NMU, 0.04; NNN, 0.008 (BaP, 1.00).

Induction of specific monooxygenases by isosteric heterocyclic compounds of benz[a]anthracene, benzo[c]phenanthrene and chrysene.

Benzo[c]phenanthrene and a series of heterocyclic compounds (benzo[b]naphtho(1,2-d)thiophene; benzo[b]naphtho(2,1-d)thiophene; benz[a]acridine and benz[c]acridine) were tested to their capacity of inducing monooxygenase activity in rat liver by means of recording the metabolite profile of benz[a]anthracene formed in rat liver microsomal incubations. Although all compounds tested were found to be weak monooxygenase inducers the pretreatment of rats with them resulted in significant changes of the microsomal metabolite profile of benz[a]anthracene. The thiophenes equally gave rise to oxidation at the 5,6- and the 8,9-positions, whereas the benzacridines being isosteric to benz[a]anthracene favoured the K-region oxidation (5,6-oxidation). A structure-dependent effect of monooxygenase inducers on the metabolite profile of benz[a]anthracene is discussed.

Contribution of polycyclic aromatic compounds to the carcinogenicity of sidestream smoke of cigarettes evaluated by implantation into the lungs of rats.

Particles and semivolatiles from sidestream smoke of cigarettes smoked on a smoking machine were collected by a filter combination consisting of a glass fibre filter and silanized polystyrene beads. The extract of the glass fibre filter was separated by a Sephadex LH-20 column chromatography into a fraction containing non-aromatic material plus polycyclic aromatic compounds (PAC) with 2 and 3 rings and a fraction consisting of PAC with 4 and more rings. To evaluate the carcinogenicity, both fractions as well as the semivolatiles were implanted into the lungs of Osborne-Mendel rats at a dose level of one cigarette per animal and compared with three dose levels of benzo[a]pyrene (BaP). The most pronounced carcinogenic effect of the sidestream smoke (100 ng BaP per cigarette) was caused by the fraction containing polycyclic aromatic hydrocarbons (PAH) with 4 and more rings (5 carcinomas of the lungs/35 rats). This fraction represents only 3.5% by weight of the total sidestream smoke condensate. By contrast, the semivolatile material did not provoke any tumors. Only a small contribution to the total carcinogenicity (1 carcinoma of the lungs/35 rats) was observed for the fraction containing non-aromatic material and 2- and 3-ring PAHs.

Investigation on the carcinogenicity of emission condensate from brown coal-fired residential furnaces applied to mouse skin.

Flue gas condensate emitted from brown coal-fired stoves was tested in 3 dosages applied chronically to the skin of female CFLP mice twice a week over a period of 104 weeks. To answer the question which portion of the total carcinogenicity results from benzo[a]pyrene (BaP), this compound was taken as reference substance. The probit and Weibull analysis of the results showed a linear dose-response relationship for both tumor incidences and tumor induction times. The amount of BaP in the emission condensate (0.593 mg/g condensate) contributes about 15% to the total carcinogenic effect of the brown coal flue gas condensate.

Comparison of chrysene metabolism in epithelial human bronchial and Syrian hamster lung cells.

Chrysene is metabolized to 1-, 2-, 3-, and 4-hydroxychrysene and trans-1,2- as well as trans-3,4-dihydroxydihydrochrysene in human and Syrian hamster epithelial lung cells as indicated by GC/MS analysis, whereas K-region oxidation is at most a very minor pathway. Cells of a permanent clonal line of fetal hamster lung metabolized 97% of the chrysene whereas fetal human bronchial epithelial cells converted 24% of the substrate within 8 days incubation. In human cells oxidation at the 3,4-position predominates, whereas oxidation at the 1,2-position is the major pathway in hamster cells. Indication for a bay-region oxidation of chrysene in hamster cells has been obtained.

DNA adduct formation in mice following treatment with used engine oil and identification of some of the major adducts by 32P-postlabelling.

Used engine oil from a petrol-powered vehicle was fractionated by column chromatography into seven parts for which the major polycyclic aromatic hydrocarbon (PAH) components were determined by GC. Topical treatment of mice with the fractions and 32P-postlabelling of the skin DNA resulted in the detection of multiple adduct spots on TLC for some, but not all, of the fractions. The majority of the DNA binding capacity of the used engine oil was possessed by the first three fractions, (equivalent to 25, 15 and 14.5%, respectively) of the adduct forming ability of the unfractionated oil. The chromatographic mobilities of the adduct spots induced by these fractions were compared to those produced by unfractionated used engine oil. In addition, mice were also treated topically with reference PAHs, either singly or as mixtures, dissolved in unused oil at the concentrations at which they were present in the used oil. Comparisons were made between the chromatographic mobilities of the adducts formed in mouse skin DNA by synthetic mixtures with those formed by the used oil. From these data, some of the major adducts produced by treatment with used engine oil are suggested to be formed by reactive metabolites of benzo[b]naphtho[1,2-d]thiophene, benzo[c]phenanthrene, benzo[g,h,i]fluoranthene, chrysene, benzo[a]pyrene and benzo[g,h,i]perylene.

On the contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of automobile exhaust condensate evaluated by local application onto mouse skin.

The objective of this investigation was to identify the substances chiefly responsible for the carcinogenicity of automobile exhaust condensate using topical application onto the skin of mice. This was performed by comparing the carcinogenic effect of various fractions with that of an unseparated sample of automobile exhaust condensate, tested in 3 different doses. The probit and Weibull analysis of the result shows: (a) The condensate, emitted from a gasoline-driven automobile provokes local tumors after long-term application to the dorsal skin of mice. The tumor incidence demonstrates a clear cut dose-response relationship. (b) The fraction of polycyclic aromatic hydrocarbons (PAH) containing more than 3 rings accounts for about 84-91% of the total carcinogenicity of automobile exhaust condensate. This fraction represents only about 3.5% by wt of the condensate. (c) The content of benzo[a]pyrene (BaP) (0.414 mg/g) accounts for 6-7.6% of the total carcinogenicity of automobile exhaust condensate, 15 selected PAHs for about 41%. (d) Regarding the minor effect of the PAH-free fraction (about 83% by wt), no hints for a cocarcinogenic activity were observed.

The contribution of polycyclic aromatic hydrocarbons to the carcinogenic impact of emission condensate from coal-fired residential furnaces evaluated by topical application to the skin of mice.

The objective of this investigation was to identify the substances chiefly responsible for the carcinogenicity of the emission condensate from coal-fired residential furnaces. To realize this, the carcinogenic effect of various fractions was compared with that of an unseparated sample of emission condensate, tested in different doses. The probit and Weibull analysis of the results showed: (1) The condensate emitted from a coal fired residential furnace as well as the reconstituted condensate combining all fractions, provoked local tumors after repeated application to the dorsal skin of mice. The tumor incidence exhibited a clear cut dose-response relationship. (2) The fraction of polycyclic aromatic hydrocarbons (PAH) and thiaarenes with more than three rings accounted for almost the total carcinogenicity (109-118% compared with the total condensate) of the emission condensate from the coal-fired residential furnace. (3) The fraction containing azaarenes and nitroarenes (NO2-PAH) accounted only for 4-7% of the total carcinoma incidence of the emission condensate. (4) The content of benzo[a]pyrene (0.702 mg/g condensate) contributes 10-11% to the total carcinogenicity of the emission condensate. (5) The PAH-free fraction and the fraction containing PAH with 2 and 3 rings (together about 77% by wt) were almost ineffective. No cocarcinogenic activity of this fraction was obtained, since the total condensate, as well as the PAH-fraction consisting of more than three rings applied proportionally provoked about the same carcinoma incidence.