DNA adducts in skin biopsies and 1-hydroxypyrene in urine of psoriasis patients and healthy volunteers following treatment with coal tar.

Coal tar ointments (CTO) are frequently used in the treatment of psoriasis and eczema, but CTO contain carcinogenic polycyclic aromatic hydrocarbons (PAH). PAH are absorbed and metabolized in the skin. In psoriasis, the skin barrier is altered and therefore, absorption and metabolism of PAH may differ from healthy skin. In this study, levels of urinary 1-hydroxypyrene and PAH-DNA adducts in the skin were studied in psoriatic patients and healthy volunteers. Three punch biopsies were taken from the lower back of 10 male volunteers and from a psoriatic plaque in 10 male patients. A surface of 6.25 cm(2) was treated with CTO. After 96 h CTO was removed and another three skin biopsies were collected from the treated area. DNA was isolated from skin biopsies and urine was collected during and after the exposure period. After 24h, a twofold lower 1-hydroxypyrene urinary excretion was observed in patients compared to healthy volunteers and after 48 h, this difference reached statistical significance (p<0.05). Over 96 h the median level of the sum of PAH-DNA adducts, analyzed by (32)P-post-labeling, increased from 3.5 before CTO administration to 21.1 adducts per 10(8) nucleotides in volunteers, and from 1.0 to 3.6 adducts per 10(8) nucleotides in patients. At 96 h, PAH-DNA levels were higher in healthy volunteers than in patients (p<0.05). Biomarkers for uptake, bioavailability and bioactivation of PAH were lower in patients compared to volunteers. These data suggest a lower risk of carcinogenic effects of CTO in psoriatic skin compared to healthy skin.

Detection of carcinogenic aromatic amines in the urine of non-smokers.

Smoking is thought to be one of the most important anthropogenic risk factors involved in the development of urinary bladder cancer in humans. Tobacco smoke contains a complex mixture of chemicals including potent carcinogens such as aromatic amines. In the present study the amounts of several freebase aromatic amines including the potent carcinogens 2-aminonaphthalene and 4-aminobiphenyl have been analyzed in the urine of 48 German urban living smokers and non-smokers. The results indicate that (i) both groups excrete the identical set of four aromatic amines; (ii) smokers excrete approximately twice the total amount of these amines, but similar amounts of 2-aminonaphthalene and 4-aminobiphenyl are found in non-smokers; and (iii) the excreted aromatic amines are decomposed in the urine within a few hours thus, explaining why aromatic amines are difficult to detect in this matrix. Their decomposition could be prevented by adding small amounts of p-toluidine to the freshly collected urine. Unlike smokers the origin of aromatic amines detected in the urine of non-smokers is at present unknown. Based on the cotinine levels found in the urine of non-smokers environmental tobacco smoke can be excluded as a major source of aromatic amines. In addition, neither diesel exhaust-related nitroarenes nor the corresponding amino-derivatives, to which they may be metabolically converted, were found. The detected urinary levels of aromatic amines arising from sources other than tobacco smoke or diesel exhaust may play a role in the bladder cancer etiology of non-smokers.

Profile of urinary phenanthrene metabolites in smokers and non-smokers.

Phenanthrene metabolites (phenols and dihydrodiols) and 1-hydroxypyrene excreted in the 24-h urine of smokers, non-smokers and lung cancer patients, who after heavy smoking became light smokers, were determined and compared. In contrast to 1- hydroxypyrene, no significant differences of the absolute amounts of phenanthrene metabolites were found between smokers and non-smokers. A ratio phenanthrene metabolites/l-hydroxypyrene of 10.4 was observed for non-smokers and 9.9 for lung cancer patients, but 4.2 for smokers. Significantly different ratios for the regiospecific oxidation of phenanthrene were found for smokers when compared with non-smokers (1,2-oxidation vs 3,4-oxidation was 1.45 in the case of smokers, but 2.34 in the case of non-smokers) indicating a cigarette smoke - but not PAH - caused induction of CYP 1A2 in smokers. As a consequence of the degree of PAH exposure the ratio dihydrodiols/phenols depends on the total amount of metabolites excreted. Phenols predominate, equally in smokers and non-smokers after low exposure, while dihydrodiols become more prominent in highly exposed persons (coke plant workers). Both (i) the regiospecific oxidation of PAH and (ii) the ratio of dihydrodiol vs phenol formation may be recognized from the urinary phenanthrene metabolite profile. This pattern mirrors the enzymatic status (balance of the CYP isoforms and epoxide hydrolase) in individuals. Accordingly, more detailed information may be obtained from the urinary metabolite pattern than from 1- hydroxypyrene, commonly used in PAH biomonitoring.

DNA single strand breakage, DNA adducts, and sister chromatid exchange in lymphocytes and phenanthrene and pyrene metabolites in urine of coke oven workers.

OBJECTIVES: To investigate the specificity of biological monitoring variables (excretion of phenanthrene and pyrene metabolites in urine) and the usefulness of some biomarkers of effect (alkaline filter elution, 32P postlabelling assay, measurement of sister chromatid exchange) in workers exposed to polycyclic aromatic hydrocarbons (PAHs). METHODS: 29 coke oven workers and a standardised control group were investigated for frequencies of DNA single strand breakage, DNA protein cross links (alkaline filter elution assay), sister chromatid exchange, and DNA adducts (32P postlabelling assay) in lymphocytes. Phenanthrene and pyrene metabolites were measured in 24 hour urine samples. 19 different PAHs (including benzo(a)pyrene, pyrene, and phenanthrene) were measured at the workplace by personal air monitoring. The GSTT1 activity in erythrocytes and lymphocyte subpopulations in blood was also measured. RESULTS: Concentrations of phenanthrene, pyrene, and benzo(a)pyrene in air correlated well with the concentration of total PAHs in air; they could be used for comparisons of different workplaces if the emission compositions were known. The measurement of phenanthrene metabolites in urine proved to be a better biological monitoring variable than the measurement of 1-hydroxypyrene. Significantly more DNA strand breaks in lymphocytes of coke oven workers were found (alkaline filter elution assay); the DNA adduct rate was not significantly increased in workers, but correlated with exposure to PAHs in a semiquantitative manner. The number of sister chromatid exchanges was lower in coke oven workers but this was not significant; thus counting sister chromatid exchanges was not a good variable for biomonitoring of coke oven workers. Also, indications for immunotoxic influences (changes in lymphocyte subpopulations) were found. CONCLUSIONS: The measurement of phenanthrene metabolites in urine seems to be a better biological monitoring variable for exposure to PAHs than measurement of hydroxypyrene. The alkaline filter elution assay proved to be the most sensitive biomarker for genotoxic damage, whereas the postlabelling assay was the only one with some specificity for DNA alterations caused by known compounds.

Determination of urinary metabolites of polycyclic aromatic hydrocarbons (PAH) for the risk assessment of PAH-exposed workers.

A method for the simultaneous determination of urinary phenanthrene, fluoranthene, pyrene, chrysene and benzo[a]pyrene metabolites has been developed for individual risk assessment at polycyclic aromatic hydrocarbon (PAH)-burdened workplaces. The concentration of urinary metabolites as a measure for individual PAH exposure takes account not only of PAH masses resorbed by the respiratory tract but also those incorporated percutaneously. The method allows the determination of 25 different components with a low margin of error; the individual metabolite profiles thereby allow conclusions on the individual characteristics of PAH-oxidizing enzymes (monooxygenases). The coefficients of variation are lower than 10%. After enzymatic treatment of the urine with glucuronidase and arylsulfatase one part of the benzene or toluene extract is treated with diazomethane to convert phenols into methylethers, while another part is used to convert dihydrodiols into phenols. After further purification the metabolites are determined by means of a combination of gas chromatography and mass spectrometry. The PAH exposure of cock plant workers during several consecutive days resulted in fairly constant individual urinary metabolite profiles which, however, exhibited significant inter-individual variability. This held true also for Wistar rats exposed to tar pitch aerosol on 5 days during a period of 10 days. It was also demonstrated that in the case of coke plant workers there is a correlation between inhaled PAH and metabolites excreted. Mass relationships between inhaled PAH and metabolites excreted were found to differ from one individual to another.

Urinary metabolite profile of PAH as a potential mirror of the genetic disposition for cancer.

Polycyclic aromatic compounds such as polycyclic aromatic hydrocarbons or aromatic amines presently are considerably underestimated with regard to the formation of environmentally caused cancer diseases. The individual urinary metabolite profile raising from the PAH inhaled is invariant. This holds for tar-pitch aerosol exposed Wistar rats as well as for PAH-exposed workers. Significant individual differences of the urinary metabolite profile can be observed in different individuals. The differences reflect the different individual enzyme equipment. There is an individual correlation between the PAH-masses inhaled and the masses of their metabolites excreted in the urine; e.g. the excretion of phenanthrene varies from 5% to 20% for different coke workers. The PAH metabolite profile analysis appears to be a suitable tool to estimate the individual cancer.risk at PAH-exposed working places since the PAH-induced malign transformation is caused by specific PAH metabolites.

Biomonitoring of polycyclic aromatic hydrocarbons in highly exposed coke plant workers by measurement of urinary phenanthrene and pyrene metabolites (phenols and dihydrodiols).

A filter combination consisting of an impregnated glass fibre and a control filter was used for the collection of air samples in which gaseous and particulate polycyclic aromatic hydrocarbons (PAHs) were determined. To estimate the loss of lower boiling PAHs, d10-phenanthrene was applied as internal standard. A simple, well-producible method for the determination of 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, 1,2-, 3,4- and 9,10-dihydroxydihydrophenanthrene, 1-hydroxypyrene and 1,2-dihydroxy-1,2-dihydropyrene is described. By means of personal air samplers the exposure to PAHs of four coke plant employees working at different locations was measured over 4 days. Simultaneously the 24-h urine was collected and stored frozen until analysed. The main excretion product of pyrene is a 1-hydroxypyrene conjugate, whereas phenanthrene is excreted predominantly as dihydrodiol conjugate. As expected, workers on the battery topside were exposed the most and accordingly excreted by far the highest amounts of PAHs. Up to 34.0 micrograms phenanthrol conjugates (total of all isomeric phenols) and 195.5 micrograms dihydrodiol conjugates (total of all isomeric dihydrodiols) were excreted in the 24-h urine (mean of 4 days). The metabolite profiles of five isomeric phenanthrene phenols and three isomeric dihydrodiols exhibited only small percentage variations within one individual whereas significant interindividual differences were observed. These findings may indicate a genetically determined enzyme pattern responsible for the metabolic conversion of PAHs.

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.

Urinary and faecal excretion of chrysene and chrysene metabolites by rats after oral, intraperitoneal, intratracheal or intrapulmonary application.

The urinary and faecal excretion of chrysene and its phenolic metabolites after oral, intraperitoneal, intratracheal, and intrapulmonary administration to rats have been studied by means of gas chromatography/mass spectrometry. The metabolite profile was found to depend on the mode of excretion and on the route of administration. In all cases the oxidation of chrysene in the 1,2- or 3,4-position predominates, whereas oxidation in the 5,6-position (K-region) seems be a minor pathway.

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.

Gaschromatographic determination of polycyclic aromatic hydrocarbons, aza-arenes, aromatic amines in the particle and vapor phase of mainstream and sidestream smoke of cigarettes.

The present arrangement collects particles and semivolatiles of main- and sidestream smoke and allows a recovery of the trapped substances nearly quantitatively and without impurities. The fractionation procedure allows to separate various groups of carcinogens such as PAH, aza-arenes and aromatic amines for analytical and biological studies. Sidestream smoke contains ten times more polycyclic aromatic hydrocarbons (PAH) compared with mainstream smoke. This holds also true for aza-arenes and amines. PAH of the gaseous phases include only 1% of the particle-bound PAH.

Hydroxy-phenanthrenes in the urine of non-smokers and smokers.

Urinary hydroxy-phenanthrene (HO-PHE) excretion in non-smokers exposed to environmental tobacco smoke (ETS) is not increased. There is no significant difference in HO-PHE excretion between smokers (S) and non-smokers (NS), though excretion seems to be slightly elevated in smokers. A diet rich in polycyclic aromatic hydrocarbons leads to a rise in urinary HO-PHE excretion as compared to a diet low in polycyclic aromatic hydrocarbons (PAH), coming close to significance. HO-PHE excretion is not correlated with the mutagenic activity in urine.

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.

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.

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.

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.

Quantification of the carcinogenic effect of polycyclic aromatic hydrocarbons in used engine oil by topical application onto the skin of mice.

The purpose of this investigation was to identify the substances mainly responsible for the carcinogenic effect of used engine oil from gasoline engines using topical application as a carcinogen-specific bioassay. This was performed by comparison of the tumorigenic effect of single fractions with that of an unseparated sample of the lubricating oil. The probit analysis of the results shows: 1) The used engine oil, from gasoline-driven automobiles, investigated provoked local tumors after long-term application to the dorsal skin of mice. The incidence of carcinoma depended on the dose of the oil. 2) The fraction of the polycyclic aromatic hydrocarbons (PAH) containing more than three rings accounts for about 70% of the total carcinogenicity in the case of crankcase oil. This fraction constitutes only up to 1.14% by weight of the total oil sample. 3) The content of benzo(a)pyrene (216.8 mg/kg) accounts for 18% of the total carcinogenicity of the used oil. 4) Regarding the reduced carcinogenicity of the oil sample, which was reconstituted from all fractions, it seems possible that some of the carcinogenic substances were lost due to volatility, with evaporation of the solvents from the oil-fractionation processes. 5) Regarding the small effect of the PAH-free fraction, as well as the equal carcinogenic effects of the PAH-fraction (containing more than three rings) and the reconstituted oil sample, no hints for a co-carcinogenic activity were obtained.