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.     

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.     

Contribution of polycyclic aromatic hydrocarbons and nitro-derivatives to the carcinogenic impact of diesel engine exhaust condensate evaluated by implantation into the lungs of rats

Diesel exhaust condensate was separated by a liquid-liquid distribution into a hydrophilic (I; about 25% by weight of the total condensate) and a hydrophobic part (II; about 75%-wt.). To evaluate the carcinogenicity, the proportionately dosed fractions have been implanted into the lungs of Osborne Mendel rats and compared with several doses of benzo[a]pyrene and the vehicle, a mixture of trioctanoin plus beeswax. Only the hydrophobic part which contained polycyclic aromatic compounds (PAC) resulted in 5 malignant tumors in a group of 35 animals. In addition, the hydrophobic part was separated by column chromatography on Sephadex LH 20 and subsequently on silica gel into several fractions, such as non-aromatic compounds plus PAC with 2 and 3 rings (IIa; 72%-wt of the total condensate), polycyclic aromatic compounds (PAH) with 4 and more rings (IIb; 0.8%-wt), polar PAC (IIc; 1.1%-wt) and nitro-PAH (IId; 0.7%-wt). PAH consisting of 4 and more rings (IIb) were found to be the most potent subfraction and provoked when proportionately dosed 6 carcinomas in a group of 35 rats. Only a low contribution to the carcinogenicity was observed by the subfraction of nitro-PAH (IId) which produced 1 carcinoma/35 rats. The polar PAC (IIc) and the fraction of non-aromatics plus PAC with 2 and 3 rings (IIa), although the main subfraction (72%-wt of the total condensate) did not provoke any tumors. The reconstitution of all hydrophobic subfractions (IIa-d) resulted in the same carcinogenic potency as the unfractionated hydrophobics (II), provoking 7 carcinoma in 35 rats. It may be concluded from these findings that most of the carcinogenicity of diesel exhaust originates from the PAH consisting of 4 or more rings.     

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.     

The contribution of polycyclic aromatic hydrocarbon fractions with different boiling ranges to the carcinogenic impact of emission condensate from coal fired residential furnaces as evaluated by topical application to the skin of mice

Flue gas condensate from briquet-fired residential furnaces was separated into a polycyclic aromatic compound (PAC)-free and a PAC-containing part, followed by a subfractionation of the PAC-containing fraction into 3 parts: PAC consisting predominantly of (a) 2 and 3 rings, (b) 4 and 5 rings and (c) 6 and more rings. To evaluate the carcinogenic potency of the condensate and its fractions, local application onto skin of mice in 2 or 3 doses was used. Since it was known from an earlier investigation that both the PAC-free fraction and the fraction containing PAC with 2 and 3 rings were almost ineffective, only PAC-fractions containing more than 3 rings were tested. The probit and Weibull analysis of the results showed that the condensate and the fractions containing PAC with 4 and 5 rings as well as 6 and more rings provoke local tumors after repeated application to the dorsal skin of mice. The tumor incidence exhibited a clear cut dose-response relationship. Fractions (b) and (c) were almost equally active, each contributing by about 50% to the total carcinogenicity. The content of benzo[a]pyrene (0.72 mg/g condensate) contributed by 10-11% to the total carcinogenicity of the emission.     

DNA adduct formation in human and mouse skin by mixtures of polycyclic aromatic hydrocarbons

32P-Postlabelling analysis has been used to detect the formation in vivo of DNA adducts by components of complex mixtures of polycyclic aromatic hydrocarbons (PAHs) in coal-tar, creosote, bitumen, juniper tar, used engine oils and fuel exhaust condensates. The presence of DNA adducts derived from these agents has been investigated in mouse skin, in human skin explants maintained in short-term organ culture and in human skin in vivo, and the formation of many different PAH-DNA adducts was observed. Similar levels and patterns of adducts were found in DNA from human skin to those in mouse skin, which is known to be susceptible to the carcinogenic activity of PAH mixtures, thus demonstrating the potential hazard to man of epidermal contact with these materials.     

Covalent binding of polycyclic aromatic hydrocarbons to adenine correlates with tumorigenesis in mouse skin.

Mouse epidermal homogenates were utilized to convert various polycyclic aromatic hydrocarbons to metabolites capable of binding covalently with nucleic acids. Poly(G) showed the highest capacity to bind covalently with the hydrocarbons; however, there was no correlation between binding to poly(G) and mouse skin tumorigenicity. On the other hand, covalent binding to poly(A) correlated well with values obtained for binding to DNA and mouse skin tumorigenicity. The order of binding to poly(A) was; 7,12-dimethylbenza[a]anthracene greater than benzo[a]pyrene greater than dibenz[a,h]anthracene greater than dibenz[a,c]anthracene.     

The optimization of sample preparation for the analysis of the content of carcinogenic polycyclic aromatic hydrocarbons]

Issues in preparation of samples of the environment for benzo(a)pyrene level assay are discussed. Certain procedures of sample preparation are described. A new method allowing better extraction of polycyclic aromatic hydrocarbons from solid samples is suggested.     

Enhancement of the skin tumor-initiating activity of polycyclic aromatic hydrocarbons by methyl-substitution at non-benzo 'bay-region' positions

The effect of substituting a methyl group at the non-benzo ‘bay-region’site of several polycyclic aromatic hydrocarbons on skin tumor-initiatingactivity was determined. A methyl group at this position enhancedthe tumor-initiating activity of dibenz[a,h]anthracene, 3-methylcholanthreneand 7-methyldibenz[a,j]anthracene but not of dibenz[a,c]anthracene.2,3,7,8-Tetrachlorodibenzo-p-dioxin was an effective inhibitorof skin tumor initiation by 7, 14-dimethyldibenz-[a,h]anthraceneand 3,6-dimethyicholanthrene. There appears to be a generalrule regarding methyl-substituted hydrocarbons: where a ‘bay-region’exists in a polycyclic aromatic hydrocarbon molecule, methyl-substitutionat the non-benzo ‘bay-region’ site results in enhancedtumor-initiating activity. The effect of methyl substitutionin this position can be most simply explained as due to enhancementof the reactivity of the benzo ring through distortion of thearomatic ring system from planarity. The consequences of thiseffect are discussed.     

Effect of extracellular calcium concentration on the metabolism of polycyclic aromatic hydrocarbons by cultured mouse keratinocytes

Cultures of adult mouse epidermal keratinocytes (MEKs) were utilized to determine whether the metabolism and metabolic activation of polycyclic aromatic hydrocarbons varied as a function of extracellular calcium (Ca2+) concentration. MEKs grown in low Ca2+-containing medium (0.05-0.10 mM) maintain basal cell morphology and proliferate while increasing the Ca2+ concentration in the medium to 1.2-1.4 mM signals the cells to undergo terminal differentiation. Relative to cultures of undifferentiated MEKs (low Ca2+), cultures of differentiated MEKs that had been switched to high Ca2+ medium 48 h prior to treatment with benzo(a)-pyrene [B(a)P] and 7,12-dimethylbenz(a)anthracene (DMBA) exhibited more rapid overall metabolism of both hydrocarbons. The greatest differences in the metabolism of B(a)P and DMBA between the two types of cultures occurred after a 3-6-h lag period. In addition, the levels of DNA-adducts formed from B(a)P and DMBA after a 24-h exposure to the hydrocarbon were 4- and 3-fold higher respectively, in cultures of differentiated MEKs (high Ca2+). Higher levels of mutagenesis and cytotoxicity were also observed in cocultures of Chinese hamster lung V-79 cells and MEKs that had been switched to high Ca2+-containing medium. In cocultures treated with the hydrocarbons at the time of Ca2+ shift, several hours elapsed before differences in mutagenesis were apparent between high and low Ca2+-containing cultures. This lag period was eliminated if the MEKs were switched to high Ca2+ medium 24 h prior to exposure to DMBA. Based on the present data, we propose that the expression and inducibility of certain enzyme activities involved in the metabolism of B(a)P and DMBA by cultured MEKs is regulated by the extracellular Ca2+ concentration and possibly the Ca2+-induced differentiation of MEKs.     

Genetic control of the regulation of cell susceptibility to carcinogenic polycyclic hydrocarbons by cyclic AMP

The metabolism of benzo (a) pyrene (BP) in normal golden hamster and BHK cells in culture was increased by treating the cells with dibutyryl cyclic AMP (dcAMP), prostaglandin E¹, theohylline or aminophylline. The largest increase, 6-fold for the normal cells and 20-fold for the BHK cells, was obtained by treatment with both dcAMP and aminophylline. Treatment with aminophylline also stimulated the metabolism of 20-methylcholanthrene and 7,12-dimethylbenz (a) anthracene. The increased metabolism of these three carcinogenic polycyclic hydrocarbons was associated with an increased cytotoxicity. Treatment with aminophylline increased the cytotoxicity of five other potent and weak carcinogenic polycyclic hydrocarbons, but not of two non-carcinogenic polycyclic hydrocarbons. The amount of BP metabolism in 27 different cell types from various mammals, including humans, ranged from less than 0.1 μg to 2.3 μg metabolized BP per 10⁶ cells. Treatment of these different cell types with aminophylline gave either an increase in BP metabolism, an induction of metabolism in cells that did not metabolize without aminophylline, or no induction after treatment with aminophylline and dcAMP. The existence of responding and non-responding cell lines indicates that the regulation of the level of polycyclic hydrocarbon metabolism by dcAMP is genetically controlled. The induction of metabolism in cells that did not metabolize without aminophylline resulted in the conversion of cell resistance to cell susceptibility to the cytotoxic effect of BP. Treatment with dcAMP and aminophylline can therefore be used to increase the sensitivity of screening tests for chemical carcinogens.     

of the carcinogenic mechanism for polycyclic aromatic hydrocarbons - extended bay region theory and its quantitative model

With the help of results on the metabolism and the carcinogenicactivities of polycyclic aromatic hydrocarbons (PAH), the comprehensivemetabolic process in vivo is discussed. It is thought that thecarcinogenic activity exhibited by a PAH is determined by thecompetition between the carcinogenesis and detoxification inwhich it participates. It is suggested that the essential agentof carcinogenesis should be, as a rule, the highest delocalizationenergy (ß unit) of the carbonium ion at the aromaticangular ring (A region), which is obtained by the perturbationalmolecular orbit (PMO) method. Since there are no essential distinctionsin the molecular geometry and delocalization energy states betweentwo carbonium ions of the aromatic angular ring and of the bayregion, the A region can be looked upon as the extended bayregion. On the basis of discussion of the overall metabolism,evaluation of the detoxification efficacy of each kind of thecompeting carcinogenic factors, including the biological factorB and three structural factors of the PAH molecule: K, A andL, was made. After making necessary approximation, K=0.228,A=0.5, L=1.22 and B=0.7 are obtained. It can be seen from thesevalues that the L region plays the most important role in detoxificationprocesses, and the K region plays the least important role.The effect of biological factor B is approximately the sum ofthe K region and the A region. This paper suggests the conceptof a carcinogenic constant. For the PAHs with the same numberof aromatic rings (N), C is a constant. The curve of functionC=f(N), including the extensional line, is an isosceles triangle.The author suggests that it should be called the ‘PyramidRule’. The final form of the quantitative equation is. The values for 50 PAHs which had been tested by animal experiments were calculated. Of these, 92% of thePAHs are in agreement with experiments on carcinogenic activities.     

Alteration of cholesterol biosynthetic pathways in the skin of mice administered polycyclic aromatic hydrocarbons

When polycyclic aromatic hydrocarbons were applied solely or together with a tumor promoter (12-O-tetradecanoylphorbol-13-acetate) to the skin of mice, a marked decrease in the level of lathosterol was observed, reflecting a significant change in the metabolism of sterols. Yet the total amount of cholesterol was not changed. When diazacholesterol (a metabolic inhibitor) was administered to mice, both desmosterol and 5 alpha-cholesta-7,24-dien-3 beta-ol accumulated in the skin, whereas the level of lathosterol decreased. These results seem to suggest that a significant portion of lathosterol is formed via 5 alpha-cholesta-7,24-dien-3 beta-ol in addition to the pathway through methostenol. When polycyclic aromatic hydrocarbon was applied to the skin of the mouse treated with diazacholesterol, a significant increase of desmosterol and a marked drop of the level of 5 alpha-cholesta-7,24-dien-3 beta-ol were observed. These results strongly suggest that polycyclic aromatic hydrocarbons perturb the metabolism of sterol in the skin of mice while keeping the total amount of cholesterol unchanged. A similar metabolism also seems to be operating in tumor tissue itself.     

Nitration of carcinogenic and non-carcinogenic polycyclic aromatic hydrocarbons results in products able to induce transformation of Syrian hamster cells

Five nitrated polycyclic aromatic hydrocarbons, synthesizedfrom benzo[a]pyrene, fluoranthene, pyrene, and chrysene induceddose-dependent transformation of Syrian hamster embryo cells.Benzo[a]pyrene, a known carcinogen, induced transformation whilethe other parental compounds, which are non-carcinogens, werenot effective. The transforming potential of the nitro derivativesvaried from compound to compound; on a molar basis, 1,8-dinitropyrenewas the most effective nitrated hydrocarbon followed in orderby 3-nitro-fluoranthene, 1-nitropyrene, 6-nitrochrysene, and6-nitro-benzo[a]pyrene. The ability to obtain dose-dependenttransformation frequencies indicates that hamster cell transformationrepresents a responsive model for elucidating the mechanismof action of nitrated carcinogens. Because of their ubiquitousdistribution and their ability to induce morphological transformationin mammalian cells, nitrated polycyclic aromatic hydrocarbonsmust be considered as potential carcinogens.