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.

The occupational exposure of dermatology nurses to polycyclic aromatic hydrocarbons - evaluating the effectiveness of better skin protection.

OBJECTIVES: We studied the uptake of polycyclic aromatic hydrocarbons (PAH) in nurses who apply ointments containing coal tar to patients and investigated the effectiveness of skin protection methods. METHODS: We determined gas-phase PAH on XAD-2 and particle-associated PAH on filters. We also used pads to determine PAH on the skin. Pyrene and benzo(a)pyrene were analyzed by gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry; their respective urinary metabolites 1-hydroxypyrene and 3-hydroxybenzo(a)pyrene were analyzed using high performance liquid chromatography with fluorescence detection. RESULTS: We ruled out the inhalation of airborne pyrene and benzo(a)pyrene as the sources of PAH exposure. However, substantial amounts of pyrene and benzo(a)pyrene were observed on the hands of the nurses (median 33.0 and 16.4 ng/cm (2), respectively). Excretion of urinary 1-hydroxypyrene indicated an increased uptake of pyrene in 8 out of 12 nurses. We asked 35 nurses to perform a treatment with gloves followed by a second treatment without gloves. The use of gloves changed the excretion of 1-hydroxypyrene by -0.58 mumol (range -5.1-1.0 mumol), corresponding to a median reduction of 51.5% (P<0.001). Based on this finding, a new protocol was adopted, involving the permanent use of vinyl gloves and Tyvek sleeves. The effectiveness of this protocol was tested against pre-existing work practices and showed a 97% reduction in skin contamination with pyrene and benzo(a)pyrene, and a lowering in urinary excretion of 1-hydroxypyrene of 57%. CONCLUSION: Protecting the skin more stringently reduced pyrene and benzo(a)pyrene contamination of the hands, and lowered urinary excretion of 1-hydroxypyrene.

Highly increased urinary 1-hydroxypyrene excretion rate in patients with atopic dermatitis treated with topical coal tar.

Coal tar preparations, as used in dermatological practice, contain numerous polycyclic aromatic hydrocarbons of which many are proven animal carcinogens. Increased urinary 1-hydroxypyrene excretion in patients with atopic dermatitis treated with topical coal tar preparations has been demonstrated. Little is known about the relationship between the dermal uptake of polycyclic aromatic hydrocarbons on the one hand and the amount of tar applied to the skin, the total body area affected, the condition of the epidermal barrier and the severity of the dermatitis on the other. We compared urinary 1-hydroxypyrene excretion rate with these variables. The urinary 1-hydroxypyrene excretion rate was highly dependent on the total amount of tar applied to the skin and the total body area affected, and less on the severity of the atopic dermatitis or the condition of the epidermal barrier. Exposure to therapeutic doses of coal tar leads to much higher rates of urinary 1-hydroxypyrene excretion than occupational exposure. Because of the potential carcinogenicity of coal tar, as clearly demonstrated both in animal studies and from occupational exposure, careful consideration should be given to the use of coal tar preparations in dermatological practice. However, the risk of short-term high exposure is unknown. Restriction of the use of coal tar should be based on epidemiological studies and/or appropriate risk models taking into account its relative safety established over many years of clinical use.

7H-benzo[c]fluorene: a major DNA adduct-forming component of coal tar.

Coal tar is a complex mixture that exhibits high carcinogenic potency in lungs of animals when administered in the diet. Studies have noted that lung tumor induction does not correlate with the benzo[a]pyrene content of coal tar, suggesting that other hydrocarbons may be involved in the observed tumorigenicity. Our previous studies have demonstrated that a major 'unknown' chemical-DNA adduct is formed in the lung of mice exposed to coal tar. We have used an in vitro rat microsomal activation system to generate the 'unknown' adduct with neat coal tar and fractions of coal tar obtained by chemical fractionation and HPLC. Chemical-DNA adduct formation was evaluated by (32)P-postlabeling using both multi-dimensional TLC and HPLC. GC-MS analysis of the coal tar fractions obtained from HPLC, which produced the 'unknown' adduct in vitro, demonstrated that the adducting hydrocarbon had a mass of 216. A careful evaluation of candidate hydrocarbons led to the conclusion that a benzofluorene derivative may be responsible for forming the 'unknown' chemical-DNA adduct. Comparative in vitro and in vivo studies on the adducting properties of all three isomers of benzofluorene indicated that 7H-benzo[c]fluorene is responsible for producing the 'unknown' adduct observed in the lung of mice ingesting coal tar. Animal feeding studies also demonstrated that 7H-benzo[c]fluorene formed considerably more lung DNA adducts than 11H-benzo[a]fluorene and 11H-benzo[b]fluorene. These data indicate that the four-ring polycyclic aromatic hydrocarbon 7H-benzo[c]fluorene, a hydrocarbon not previously shown to form DNA adducts in lung, is in fact a potent lung DNA adductor and is a candidate PAH for causing lung tumors in animals treated with coal tar.

Bioavailability of the genotoxic components in coal tar contaminated soils in Fischer 344 rats.

The effect of chemical aging on the bioavailability and subsequent genotoxicity of coal tar (CT)-contaminated soils was evaluated in a 17-day feeding study using Fischer 344 male rats. Rats consumed a control diet or diets amended with soil, 0.35% CT, or soil freshly prepared or aged for 9 months with 0.35% CT. Mild treatment-related microscopic lesions in liver tissue and elevated enzyme levels in serum were detected in all CT treatment groups. The (32)P-postlabeling assay was employed to determine DNA adduct formation in treated animals. All CT treatment groups induced DNA adducts in both the liver and lung. Adduct levels were 3-fold higher in lung DNA compared to hepatic DNA. After correcting adduct levels for total ingested polycyclic aromatic hydrocarbons (PAHs), a significant decrease (p < 0.05) in adduct levels was observed in both CT/soil treatment groups compared to CT control in liver and lung DNA. Adduct profiles of (32)P-postlabeled hepatic and lung DNA displayed several nonpolar DNA adducts that comigrated with PAH-adducted calf thymus DNA standards as determined through both thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). These results suggest that soil, but not aging of contaminants in soil, decreases the bioavailability of genotoxic components in CT, as evidenced by DNA adduct analysis.

Effects of heterocyclic PAHs (N, S, O) on the biodegradation of typical tar oil PAHs in a soil/compost mixture.

The interaction phenomena during the biodegradation of typical coal tar polycyclic aromatic hydrocarbons and their heterocyclic analogues (N, S, O) were investigated in an artificially contaminated AhA1-horizon/compost mixture. All compounds were partly or completely biodegraded. Degradation of two- to five-ring PAHs was inhibited by the presence of hetero-PAHs, whereas degradation of just some hetero-PAHs was inhibited by the presence of PAHs. Among the hetero-PAHs the sulphur-containing compounds were less susceptible to degradation than the corresponding oxygen- or nitrogen-containing analogues. The basic azaarene acridine showed an extreme persistence and strong sorption to the soil matrix proved by an increase of recovery after saponification of the soil matrix.

A novel skin penetration enhancer: evaluation by membrane diffusion and confocal microscopy.

PURPOSE: The aim of this study was to determine the in vitro transdermal efficacy of a Meyer Zall Laboratories (MZL) oil/water emulsion in two separate preparations containing the actives, coal tar and the non-steroidal anti-inflammatory drug, diclofenac sodium. METHOD: The release rate of the two active ingredients from MZL dermatological preparations, Exorex and Athru-Derm and four comparator products was determined using an enhancer cell system, whilst specific penetration characteristics of the MZL formulation were elucidated using confocal and electron microscopy. The latter properties were explored at both the organ level, using human skin, as well as at a cellular level using a melanoma cell line. RESULTS: While the in vitro release rates for all formulations was high, coal tar and diclofenac release from Exorex and Athru-Derm respectively was, at nearly all time intervals, significantly higher than from comparator products. Microscopy revealed the presence of spherical liposomal type structures in both the MZL lotion and a comparator gel. In the MZL lotion, the majority of these structures, referred to here as emzaloid particles, were in the order of magnitude of about 50 nm to 1 microm in diameter with a small minority exceeding these dimensions. After application of Athru-Derm to human skin, intact emzaloid particles of submicron dimensions were detected in the epidermis in association with the cell membranes. The affinity of the MZL lotion for cell membranes was further demonstrated with melanoma cells; in addition, the formulation was seen to penetrate even to the nucleus of viable cells. CONCLUSION: Overall the data suggest that the oil/water base in MZL formulations is a highly efficient transdermal vehicle able to transport a wide range of indication- specific actives to their site of action.

[Longitudinal study of the excretion of 1-hydroxypyrene in urine after external treatment with coal tar]. / Longitudinale Untersuchung zur Ausscheidung von 1-Hydroxypyren im Urin nach externer Behandlung mit Steinkohlenteer.

In order to study the dermal uptake, time course, and urinary excretion of polycyclic aromatic hydrocarbons, the concentration of 1-hydroxypyrene in urine was determined by means of high performance liquid chromatography with fluorescence detection before, during, and after the topical treatment with coal tar in 19 patients suffering from prurigo simplex subacuta, microbial eczema, atopic dermatitis, eczematization after scabies, exanthematous lichen ruber, pityriasis lichenoides and cutaneous sarcoidosis. Beginning with a value of 6.04 +/- 2.06 micrograms 1-hydroxypyrene/g creatinine before treatment, the urinary excretion significantly increased during the therapy with coal tar (p < 0.0001 at 3rd, 5th, and 6th day of therapy). A maximum was reached at day 8 of topical treatment with a value of 584.35 +/- 191.96 micrograms 1-hydroxypyrene/g creatinine (p < 0.002). Already during treatment at day 10 there was a beginning decrease of 1-hydroxypyrene to 361.63 +/- 170.13 micrograms/g creatinine. After the end of treatment, the excretion further decreased reaching a value of 5.31 +/- 2.85 micrograms 1-hydroxypyrene/g creatinine at the 10th day after therapy. Skin carcinomas due to therapeutical use of coal tar occur extremely rarely and only after vergoten, non-controlled use. We suggest that the duration of exposure is the most important factor for the carcinogenic effect of coal tar.

[A method of mutagenic activity testing for coal tar in atmospheric dust]. / Metoda oznaczania aktywnosci mutagennej substancji smolowych zawartych w pylach powietrza atmosferycznego.

For assessing of the environmental contamination with potentially carcinogenic mutagens the possibility of the measurement of the mutagenic activity of the substances absorbed on dust suspended in air would be important. These substances include at least several hundreds of organic compounds. Only a small part of them have been studied in detail, and, on the other hand, it is known that at least a score of them are carcinogenic. Additionally, the presence of some of them can change significantly the action of other ones. Because of that, the effects of a mixture of these substances should be studied as a whole. The fact that the discussed contaminants are present in the air and can be transported over great distances causes that they are dangerous to man both directly (when inhaled) and trough contamination of water, soil and food. Parallel use in the described method of two cross-bred strains of fruit flies made possible separate assessment of the activity of mutagens and promutagens requiring activation in presence of cytochrome P 450. The use of the eukaryotic organism in which mutations are observed in many cells in one individual (about 2500) enables reliable data to be obtained about the degree of human health risk.

Dermal absorption of polycyclic aromatic hydrocarbons in the blood-perfused pig ear.

Urinary 1-OH-pyrene, a metabolite of pyrene, is a sensitive biological marker for dermal absorption of pyrene in man. In order to determine whether this metabolite is a reliable biomarker of cutaneous absorption of other polycyclic aromatic hydrocarbons (PAHs), the blood-perfused pig ear model was used to compare the dermal absorption flux of pyrene with nine other PAHs after coal tar application. Cumulative absorption of PAHs into the perfusion blood, 200 min after application of an overdose of coal tar, ranged between 830 pmol cm-2 for phenanthrene to less than 4 pmol cm-2 for benzo[b]fluoroanthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[ah]anthracene and indeno[123-cd]pyrene. The results of this study show that when pyrene is used as a marker compound for PAH absorption through pig skin, the cumulative absorption of PAHs with a lower molecular weight will be underestimated: fluorene, tenfold; phenanthrene, 12-fold; anthracene and fluoranthene, ca. twofold. The percutaneous absorption of PAHs with a higher molecular weight than pyrene will be overestimated: e.g. benzo[a]pyrene, sevenfold; indeno [123-cd]pyrene, ca. 100-fold. It is likely that this conclusion is also valid for dermal PAH absorption in man.

Biochemical effects of manufactured gas plant residue following ingestion by B6C3F1 mice.

The toxic potential of manufactured gas plant residue (MGP) given in the diet to male and female B6C3F1 mice was evaluated. In addition, the bioavailability of chemical components of MGP were also investigated by monitoring polycyclic aromatic hydrocarbon (PAH) metabolites in urine and DNA adduct formation in forestomach and lung tissue. Basal gel diets containing 0.05, 0.25, 0.50% MGP or 0.005% benzo[a]pyrene (BaP) were fed to animals for 94 and 185 d. Mice readily consumed adulterated diets without any evidence of acute toxicity. The total amount of MGP and BaP consumed by mice ranged from 118 to 2604 mg and from 12 to 29 mg, respectively. Male mice fed a control or BaP diet and female mice fed a 0.05% MGP diet had the highest body weight gains. Male and female mice fed a 0.50% MGP diet had the lowest body weight gains. The bioavailability of chemical components of MGP was evaluated by monitoring the urinary excretion of PAH metabolites by male mice fed a 0.25% MGP diet. 1-Hydroxypyrene was determined by high-performance liquid chromatography analysis to be the major fluorescent metabolite excreted by mice throughout the 185 d of diet administration. At necropsy, no chemical-related gross lesions were detected. In addition, no treatment-related microscopic lesions were evident in tissues obtained from animals fed a 0.50% MGP- or BaP-adulterated diet. The 32P-postlabeling assay was used to evaluate MGP- and BaP-induced DNA adduct formation in lung and forestomach tissue. The level of DNA adducts formed from the chemical components of MGP paralleled the amount of material ingested by animals. Lung DNA adduct levels were considerably higher than forestomach levels when mice ingested a 0.25% or 0.50% MGP diet. These studies demonstrate that the continuous ingestion of MGP or BaP for 185 d does not result in acute toxicity or chemical-related lesions at doses up to 0.50% MGP or 0.005% BaP.

Quantitation of polycyclic aromatic hydrocarbons, 1-hydroxypyrene, and mutagenicity in urine of coal tar-treated psoriasis patients and untreated volunteers.

Coal tar-treated psoriasis patients were used as a model population to test a newly developed enzyme-linked immunosorbent assay (ELISA) for urinary excretion of benzo(a)pyrene and related polycyclic aromatic hydrocarbons (PAHs). The ability of the ELISA to detect exposure was also compared with that of two previously established biomonitoring methods, measurement of urinary 1-hydroxypyrene by high performance liquid chromatography with fluorescence detection and mutagenicity measured by the Salmonella typhimurium mutagenesis assay. Urine samples were collected from 57 patients and 53 untreated volunteers. Urinary excretion of PAH metabolites, measured by competitive ELISA with a monoclonal antibody (4D5), was elevated in patients (mean, 730 +/- 1370 mumol/mol creatinine) compared with untreated volunteers (110 +/- 90 mumol/mol creatinine; P < 0.0001). 1-Hydroxypyrene also was elevated in patients (mean, 547 +/- 928 mumol/mol creatinine) compared with volunteers (mean, 0.14 +/- 0.17 mumol/mol creatinine; P < 0.0001). Much larger differences between mean values in patients and volunteers were observed with the 1-hydroxypyrene assay compared with the PAH metabolite ELISA. No significant effect of smoking could be detected by either assay. Analysis by the Salmonella typhimurium mutagenesis assay indicated elevated mutagenicity in urine from patients (1410 +/- 2750 revertants/mmol creatinine) compared with volunteers (715 +/- 846 revertants/mmol creatinine; P = 0.072). In all subjects, there was a good correlation between the PAH metabolites and both 1-hydroxypyrene (r = 0.717; P < 0.0001) and urinary mutagenicity (r = 0.317; P = 0.004). These results suggest that the ELISA, which easily can be carried out on large numbers of samples, can be used for monitoring urinary excretion of PAHs in a high exposure population. Ongoing studies are designed to determine its applicability to lower exposure populations.

Absorption of polycyclic aromatic hydrocarbons through human skin: differences between anatomical sites and individuals.

In order to determine differences in absorption of polycyclic aromatic hydrocarbons (PAH) between anatomical sites and individuals, coal-tar ointment was applied to skin of volunteers at various sites. The surface disappearance of PAH and the excretion of urinary 1-OH-pyrene after skin application of coal-tar ointment were used as parameters for dermal PAH absorption. The surface disappearance was determined by the measurement of the fluorescence of PAH on skin. Surface disappearance measurements show low but significant differences in dermal PAH absorption between anatomical sites: shoulder > forehead, forearm, groin, > ankle, hand (palmar site). The average PAH absorption rate constant at different skin sites ranges from 0.036/h to 0.135/h (overall mean: 0.066/h). This indicates that after 6 h of exposure, 20-56% of a low dermal dose of PAH (e.g., about 1.0 ng pyrene/cm2) will be absorbed. The interindividual differences in PAH absorption are small (7%) in comparison with differences between anatomical sites (69%). Results based on the urinary excretion of 1-OH-pyrene are less clear. The site of application of the coal-tar ointment (dose: 2.5 mg/cm2 during 6 h) has no significant effect on the excreted amount of 1-OH-pyrene in urine. It is estimated that after coal-tar ointment application on skin, 0.3-1.4% of the pyrene dose (about 2 micrograms pyrene/cm2) becomes systemically available. For the accurate estimation of PAH uptake through skin of workers, it seems relevant to distinguish different body regions, not only because of the regional variation in percutaneous PAH absorption, but also because of the high dispersal of PAH contamination on skin of workers.

A comparative trial of Clinitar versus hydrocortisone cream in the treatment of atopic eczema.

Thirty patients with mild to moderate bilateral atopic eczema were treated on one side of the body with a new purified coal tar cream (Clinitar) and on the other side with 1% hydrocortisone cream, for 4 weeks. The degree of infiltration of the skin erythema, lichenification, excoriations and dryness were assessed. Both treatments were equally effective in reducing symptoms and there was also no difference in patient preference and cosmetic acceptability.