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.     

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.     

Experimental results with percutaneous applications of automobile exhaust condensates in mice.

The induction of squamous-cell carcinoma and papillomas in mice after 80 weeks' skin painting with different doses of automobile exhaust condensates and their fractions is described. The results demonstrate that the carcinogenic activity of the condensate resides in its polycyclic aromatic hydrocarbon fractions. Benzo[a]pyrene accounts for approximately 9% of the activity of the condensate. The sebaceous gland suppression test, used as a screening method, is predictive of carcinogenicity and is positive for those condensate subfractions which contain carcinogenic PAH.     

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.     

Analysis of automobile exhaust condensates.

1. On the basis of figures for the production of PAH during the Europa drive cycle by 100 passenger cars and those for the consumption of petrol in the Federal Republic of Germany, an annual emission of 1,850 kg benzo[a]pyrene from petrol engine vehicles has been calculated. 2. The carcinogenic effect of benzo[a]pyrene accounts for only 9% of the total activity of exhaust condensates. 3. The amounts of other known carcinogenic PAH, such as benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene and dibenzo[a,h]anthracene are shown in Table 2. (table: see text). Assuming that there is no significant promoting or hyper-additive effect, it can be estimated that these six known carcinogenic PAH contribute about 10-15% of the total carcinogenicity. 4. Six unknown PAH were found: cyclopenteno[cd]pyrene (mol wt 226), methylenebenzo[a]pyrene (mol 264), methylenebenzo[e]pyrene (mol wt 264), methylenebenzo[ghi]perylene (mol wt 288), PAH mol wt 300A and PAH mol wt 300B. It is reasonable to assume that these unknown PAH account for the predominant part of the carcinogenic effect. Biological tests with these pure substances are being undertaken by Drs Pott, Pfeiffer and Habs. 5. It has been shown that almost all of the carcinogenic effect of automobile exhaust condensates is due to PAH. To support this claim, the carcinogenic effects of the exhaust condensate should be compared with those of a mixture of the known and unknwon PAH in the same proportions as are found in the exhaust condensate. The gas chromatogram of such a mixture is shown in Figure 6.     

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.     

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.     

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.     

32P-postlabelling analysis of DNA adducts in the skin of mice treated with petrol and diesel engine lubricating oils and exhaust condensates

Samples of unused or used petrol and diesel engine lubricating oils were applied to the shaved dorsal skin of 4- to 6-week-old male Parkes mice, either as a single treatment (50 microliters/mouse) or as four consecutive daily treatments (50 microliters/application). DNA isolated from the skin 24 h after the final treatment was digested to 3'-mononucleotides and analysed by 32P-postlabelling for the presence of aromatic adducts. Enhancement of sensitivity using butanol extraction or nuclease P1 digestion of the DNA hydrolysates led to the detection of up to eight adduct spots on polyethyleneimine-cellulose thin-layer chromatograms with samples of DNA from skin treated with used engine oils, at levels of 40-150 amol total adducts/micrograms DNA. Multiple treatments with the used oils gave rise to similar patterns of adducts in lung DNA. A single treatment of mouse skin with petrol engine exhaust condensate (50 microliters), or diesel engine exhaust condensate (50 microliters), containing 20 and 46 micrograms benzo[a]pyrene (BaP)/g respectively, gave rise to approximately 75 amol total adducts/micrograms DNA in skin. A significant proportion, 31 and 48% respectively, of the adducts formed by the petrol and diesel engine exhaust condensates co-chromatographed with the major BaP-DNA adduct, but with the used engine oils, only petrol engine oil, and not diesel engine oil, produced significant amounts of an adduct (22% of total) that corresponded to the BaP-DNA adduct.     

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.     

Effect of glycerol on local and systemic carcinogenicity of topically applied tobacco condensate

When glycerol was added to tobacco smoke condensate in acetone solvent, the topical carcinogenicity and the ability to produce epithelial hyperplasia in mice was reduced. Two doses of condensate were applied, combined with 2 concentrations of added glycerol. Age-standardized results show that glycerol reduced the incidence of tumours and malignant tumours and of hyperplasia in animals not developing skin tumours. The relative incidences of malignant tumours, benign tumours, hyperplasia and unaffected skin suggest that there is a sequential relationship (i.e. normal skin to hyperplasia to benign neoplasia to malignant neoplasia) which is impeded by glycerol. There was no systemic effect attributable to the condensate.     

The mouse-skin carcinogenicity of a mutagenic fraction from beech wood dusts

A life-time mouse-skin carcinogenicity assay was conducted usingfemale NMRI mice to evaluate the possible direct carcinogenicactivity of a mutagenic fraction isolated from beech wood dusts.The samples of untreated beech wood dusts were extracted withmethanol at pH3 and were purified from the inhibitory compoundstoxic to bacteria, using silica-gel cohimn chromatography. Thefraction obtained after passing through the column was testedfor mutagenicity in the Ames assay employing Salmonella typhimuriumTA100 in the presence of Aroclor-treated rat-liver-S9. Usingacetone as the vehicle, this mutagenic fraction was tested forcarcinogenicity on an area of 1 - 1.5 cm shaved skin of miceon the lower back. The mice were treated with half of each dose,twice a week, for only 3 months. The total doses applied perweek were 2.5, 5, 7.5 or 10 g equivalent dust/mouse. No substancewas used as promoter. No statistically significant differencewas found when the life spans of treated and untreated animalswere compared. TTie observed carcinogenic effect was based ontumours and lesions found only on the ate of application ofthe test material. Of 210 mice (effective number, 129) servingas the negative controls, three developed skin lesions but notumours. Of 280 treated animats (effective number, 188) 34 developeddifferent types of tumours and 20 had a uniform type of precancerousskin lesion. Of 34 tumours observed 21 were originated fromthe skin, 12 from the mammary glands beneath the site of application,and one was a lymphoma. Comparing the negative controls withthe treated animals, the overall carcinogenic effect observedwas dose-dependent and statistically significant. Excludingthe mammary tumours and a lymphoma found beneath the site oftreatment, the overall induction of skin tumours was still significant.However, the dose-dependent increase in the number of skin tumoursalone was not statistically significant. These results suggestthat beech wood dust contains mutagenic and carcinogenic constituent(s).     

Pulmonary tumorigenesis in Syrian golden hamsters after intratracheal instillations with automobile exhaust condensate.

Syrian golden hamsters were intratracheally instilled once every two weeks for life with automobile exhaust condensate (AEC) at two dose levels. The condensate was prepared from the most common German passenger car driven in the Europa-Test cycle (simulated city driving) and contained 340 microgram/g benzo(a)pyrene (B(a)P). Despite the very low total dose of B(a)P received, all animals of both dosage groups developed multiple pulmonary adenomas. Electron microscopy revealed these tumors to resemble B(a)P induced pulmonary adenomas in their ultrastructure. Numerous condensate-laden macrophages in tumor and peripheral lung tissues indicated that little of the instilled condensate had been removed from the airways through mucociliary action.     

Synergistic mechanisms in carcinogenesis by polycyclic aromatic hydrocarbons and by tobacco smoke: a bio-historical perspective with updates.

B[a]P (benzo[a]pyrene) has been used as a prototype carcinogenic PAH since its isolation from coal tar in the 1930's. One of its diol epoxides, BPDE-2, is considered its ultimate carcinogen on the basis of its binding to DNA, mutagenicity and extreme pulmonary carcinogenicity in newborn mice. However, BPDE-1 has a similar binding to DNA and mutagenicity but it is not carcinogenic. In addition, BPDE-2 is a weak carcinogen relative to B[a]P when repeatedly applied to mouse skin, the conventional assay site. Its carcinogenicity is increased when applied once as an initiator followed repeatedly by a promoter. This indicates a major role for promotion in carcinogenesis by PAHs. Promotion itself is a 2-stage process, the second of which is selective propagation of the initiated cells. Persistent hyperplasia underlies selection by promoters. The non-carcinogenicity of BPDE-1 has yet to be resolved. PAHs have long been considered the main carcinogens of cigarette smoke but their concentration in the condensate is far too low to account by themselves for the production of skin tumors. The phenolic fraction does however have strong promotional activity when repeatedly applied to initiated mouse skin. Several constituents of cigarette smoke are co-carcinogenic when applied simultaneously with repeated applications of PAHs. Catechol is co-carcinogenic at concentrations found in the condensate. Since cigarette smoking involves protracted exposure to all the smoke constituents, co-carcinogenesis simulates its effects. Both procedures, however, indicate a major role for selection in carcinogenesis by cigarette smoke. That selection may operate on endogenous mutations as well as those induced by PAHs. There are indications that the nicotine-derived NNK which is a specific pulmonary carcinogen in animals contributes to smoking-induced lung cancer in man. Lung adenoma development by inhalation has been induced in mice by the gas phase of cigarette smoke. The role of selection has not been evaluated in either of these cases.     

Fluorine substitution as a probe for the role of the 6-position of benzo[a]pyrene in carcinogenesis

The tumorigenic activities of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-F-BP) were compared to determine whether an unsubstituted 6-position is important for the carcinogenic effect of BP. Highly purified samples of 6-F-BP and BP had similar activities for the induction of lung adenomas in Swiss Webster mice treated before weaning. The 6-fluoro derivative, however, had about one-half as much activity as BP for the initiation of skin papillomas in CD-1 mice. Similarly, 6-F-BP (approximately equal to 90% purity) had about one-half the activity of BP for the induction of skin tumors in C57BL/6J mice given repetitive treatments of the hydrocarbons and for the induction of sarcomas in C3H/fCum mice given a single sc injection. 6-F-BP (approximately equal to 90% purity) had activity similar to that of BP for induction of sarcomas at the sc injection site in Fischer 344 rats. These results and related data indicate the need for detailed metabolic studies whenever fluorine substitution is used as a probe to assess the role of the unsubstituted position in the carcinogenicity of the parent compound.     

Skin carcinogenesis tests in mice of derivatives of 7,12-dimethyl- benz[a]anthracene substituted in the 'A' ring

Seven derivatives of 7,12-dimethylbenz[a]anthracene (DMBA) havingsubstituents in the ‘A’ ring have been tested forcarcinogenic activity by chronic application to the skin offemale Swiss mice. Saturation of the A ring, as in 1,2,3,4-tetrahydro-DMBA,resulted in a very potent skin carcinogen which induced almost100% of skin carcinomas within 26 weeks. The 2-bromo-derivativewas apparently not carcinogenic. Respectively, 35% and 40% ofthe mice treated with 2-methoxy and 4-bromo-DMBA developed skintumors, mainly squamous cell carcinomas with the former andmainly neurosarcomas for the latter. The remaining compounds,3-methoxy-, 4-methoxy- and 3-bromo-DMBA each induced skin tumorson 3–5 animals. There were no quantitative correlationsbetween bacterial mutagenicity of the substituted DMBA's andtheir carcinogenic potencies in mouse skin painting. The reasonsfor the differences in carcinogenic activity are not clear,but the effects, particularly the strong carcinogenicity ofthe tetrahydro-derivative, suggest the possibility that mechanismsother than formation of a dihydrodiol epoxide in the A ringmight be involved.     

Response of rat lung to tobacco smoke condensate or fractions derived from it administered repeatedly by intratracheal instillation.

The repeated intratracheal instillation of cigarette smoke condensate (SWS) in rats at close to maximum tolerated dose levels failed to induce squamous neoplasms in the lungs although such treatment was associated with an increased incidence of cuboidal/columnar metaplasia (CCM) and squamous metaplasia (Sq.M) of alveolar epithelium. With one exception, various fractions of SWS had no effect on lung tumour incidence though some were more effective than SWS in increasing the incidence of CCM and Sq.M. The exceptional fraction, Fraction P, which contains most of the polycyclic aromatic hydrocarbons of smoke and is the most effective of the fractions tested in producing tumours in mouse skin, gave rise to 4 squamous tumours of doubtful malignancy and one metastasizing squanmous carcinoma among 3 groups of 18 animals exposed at 3 different dose levels. The results are discussed in relation to the possible development of a method for comparing condensates for relative lung carcinogenicity.     

Selective clonal expansion and microenvironmental permissiveness in tobacco carcinogenesis

Historically our knowledge about the direct carcinogenic activity of cigarette smoke and its constituents grew from painting experiments on the skin of mice to produce papillomas and carcinomas. The neutral fraction of cigarette smoke condensate had most of the carcinogenic activity in this test and was rich in carcinogenic polycyclic aromatic hydrocarbons (PAHs), the most abundant by far being BP. However, the concentration of BP in the condensate was only about 2% the amount of pure BP required to cause skin tumors. In other fractions there were non-carcinogenic constituents that promoted tumor formation when applied repeatedly to mouse skin that had been initiated by a single subcarcinogenic application of BP. There were also constituents of cigarette smoke that acted as co-carcinogens when applied simultaneously with repeated applications of BP. BP was effective as an initiator at lower concentrations than as a complete carcinogen, and some non-carcinogenic PAHs in the condensate were also active initiators. It was concluded from these studies that cigarette smoke condensate is primarily a tumor-promoting and co-carcinogenic agent with weak activity as a complete carcinogen. A major effect of promoters, and possibly of co-carcinogens, is a diffuse hyperplasia which includes selective expansion of clones carrying endogenous mutations and/or mutations induced by PAHs and other carcinogens such as NNK. The induced mutations as well as damaged cells would occur throughout the exposed region and, along with the hyperplasia, increase the permissiveness of the cellular microenvironment for neoplastic expression of any potential tumor cell in its midst. Since neither the promoters nor co-carcinogens in tobacco smoke are known to interact directly with DNA, their effects can be considered epigenetic processes that act upon genetically altered cells. Examples are cited from studies of experimental skin carcinogenesis, smoking-induced histopathological changes in human lung and spontaneous transformation in cell culture to illustrate the genetic and epigenetic interactions of neoplastic development in general and their significance for smoking-induced lung cancer in particular. Certain dietary modifications that appear to be effective in moderating the promotional phase of animal and human carcinogenesis are suggested for trial in managing lung cancer.     

Studies on the local and systemic carcinogenicity of topically applied smoke condensate from a substitute smoking material.

The topical carcinogenicity to mouse skin of smoke condensates obtained from a tobacco substitute (NSM), alone or in combination with tobacco, has been compared with condensate from tobacco and with acetone, the solvent used. Sixteen different types of cigarette were used to make the condensates, and the age-standardized results have been analysed according to the Weibull distribution model. The results show that NSM condensate has less than 25% of the potency of tobacco condensate (37% at 95% upper confidence limit), and that condensates from blends of NSM and tobacco are similarly reduced in activity. General pathology analysis failed to reveal abnormalities due to NSM.     

A study of the dose response of mouse skin to cigarette smoke condensate

Smoke condensate from two types of cigarette, dissolved in two solvents, has been applied regularly to the backs of mice at each of seven different dose levels. Treatment was continued 3 times weekly for up to 110 weeks, by which time 509 of the 1428 treated mice had developed skin tumours. The dependence of tumour incidence on age was adequately described by the Weibull distribution. The relation - ship between dose of smoke condensate and tumour incidence rate was, however, erratic. It was less regular than the simple relationship which has in previous work been found to obtain when the pure carcinogen benzo(a)pyrene is applied to mouse skin.