Influence of metabolic genotype GSTM1 on levels of urinary mutagens in patients treated topically with coal tar.

Fifteen hospitalized, non-smoking, dermatological patients were treated with ointment containing 2% coal tar (CT) in order to assess the influence of metabolic genotype GSTM1 on urinary mutagen levels. Urinary 1-pyrenol, the main metabolite of pyrene, was used to check the high exposure to PAH of this population. The mean levels of urinary 1-pyrenol found in the 24-h urine of our patients were 467. 8+/-211.0 nmoles-24 h (range 94.6-890.1 nmoles-24 h). Mutagenicity was assessed on urine samples collected over a period of 24 h, after three consecutive days of topical application, using the bacterial mutagenesis test on Salmonella typhimurium strains TA98 and YG1024 in the presence of microsomal enzymes. The latter strain turned out to be more sensitive than the former in revealing urinary mutagens in these patients (42 693+/-30 867 vs. 6877+/-6040 net revertants-24 h). The mutagenicity on YG1024 strain and 1-pyrenol levels of urine samples were correlated (Spearman's rank correlation coefficient=0. 6678, P<0.01, z=2.795). The influence of genotype GSTM1 on urinary mutagen levels was assessed on strain YG1024. The values of urinary mutagenicity of subjects with genotype GSTM1-null (n=6) were on average higher than those of GSTM1-positive subjects (n=9) (55 498+/-45 957 vs. 34 156+/-11 933 net rev.-24 h), a non-significant statistical difference. The mean total excretion of mutagens corrected for PAH exposure (net rev./nmoles of urinary 1-pyrenol) in GSTM1-null patients was double that of GSTM1-positive ones (136. 8+/-34.7 vs. 70.8+/-23.3 net rev./nmoles of urinary 1-pyrenol; one-tailed Mann-Whitney U-test, U=11.5, P<0.05). These results indicate a greater body burden of promutagens, resulting from skin application of CT, in GSTM1-null subjects.

Urinary mutagenicity on TA98 and YG1024 Salmonella typhimurium strains after a hamburger meal: influence of GSTM1 and NAT2 genotypes.

Mutagenicity on TA98 and YG1024 Salmonella typhimurium strains of pan-fried hamburger extracts and of 24 h post-meal urine from 32 non-smoking volunteers was evaluated. Each participant in the study was GSTM1 and NAT2 genotyped. After cooking the meat showed mutagenic activity (mean +/- SD) on strains TA98 and YG1024 of 114 +/- 129 and 1437 +/- 1536 net revertants/g respectively. Twenty three of 32 urine samples showed clear mutagenic activity (i.e. caused at least a doubling of the number of spontaneous revertants) on the O-acetyltransferase over-producing strain YG1024, while none of the post-meal 24 h urine samples was clearly mutagenic on strain TA98. Total 24 h post-meal YG1024-active urinary mutagens were well correlated with the levels of mutagen intake with the meal (r2 = 0.5977, F = 44.58, P < 0.01). In the group under study GSTM1 genotypes did not influence urinary mutagenicity. Highly exposed subjects (n = 15) with the NAT2-ss genotype showed significantly increased levels of urinary mutagenicity on strain YG1024 in comparison with NAT2-R subjects (mutagen intake-adjusted total 24 h mutagen excretion = 1.00 +/- 0.29 versus 0.66 +/- 0.32, Mann-Whitney U test, U = 12.5, P < 0.05). Our results suggest that the levels of urinary mutagens derived from diets rich in heterocyclic aromatic amines, which are specifically detected by the YG1024 Salmonella strain, are modulated by NAT2-dependent enzyme activity, slow acetylators having higher levels of mutagens in their urine. Subjects with the rapid acetylator genotype, who are known to be at risk for colon cancer, seem to be partially protected with respect to the risk of bladder cancer.

GSTM1 and NAT2 genotypes and urinary mutagens in coke oven workers.

The influence of the metabolic genotypes GSTM1 and NAT2 on the urinary excretion of mutagens in 46 coke oven workers (27 of them smokers) was studied. Exposure to polycyclic aromatic hydrocarbons (PAH) was estimated from urinary 1-pyrenol levels, which varied from 0.23 to 5.59 micromol/mol creatinine. Fourteen urine samples (30.4%), all but one belonging to smokers, were positive for mutagenic activity (i.e. at least one of the assayed doses was able to double the number of spontaneous revertants). Nine of the urine-positive subjects were both GSTM1-null and NAT2-ss (64.3%), while the same combination of genotypes was found in nine out of 31 urine-negative subjects (29.0%) (P < 0.05). Significantly more smoking workers with the genotype combination GSTM1-null/NAT2-ss showed positive urine mutagenicity than the other subjects (75.0 versus 28.6%, P < 0.05). Smokers with the slow acetylator genotype showed a significantly higher frequency of positive urine samples than smoking fast acetylators (64.7 versus 22.2%, P < 0.05). Our results suggest that smoking coke oven workers with genotypes unfavourable for detoxification of aromatic amines (NAT2-ss) and PAH (GSTM1-null) may have an increased risk of developing bladder cancer.

Mutagenicity and contents of polycyclic aromatic hydrocarbons in used and recycled motor oils.

Thirteen samples of used motor oil and 33 recycled fractions, obtained in the laboratory by means of a recovery process similar to that currently used in Italy (vacuum distillation followed by thermal clay treatment) were examined. The Ames test (standard and modified version according to Blackburn) was used to determine the mutagenicity of the extracts and their contents of polyaromatic fraction (PAF) (IP346/80 method) and polycyclic aromatic hydrocarbons (PAH) (Grimmer's method). Used motor oils are mutagenic, both directly and indirectly. The highest values have been found in used oils from motor vehicles using leaded petrol (up to 118.8 revertants/mg). Samples from vehicles using unleaded petrol or diesel fuel are less mutagenic (up to 31.1 and 16.4 rev/mg, respectively). The enrichment in mutagens due to the use of oil in the three types of engine ranges from mean values of 6.2, 1.1 and 0.4 rev/mg per 1000 km, respectively. Recycled oils are almost completely devoid of direct mutagenic activity (33 samples: mean +/- SD = 1.6 +/- 1.5 rev/mg). Most recycled distillates show considerable mutagenic activity in the presence of microsomial enzymes (up to 82.5 rev/mg), although this is reduced with respect to the original oils (recycled, mean +/- SD = 13.8 +/- 15.5 rev/mg; original oils, mean +/- SD = 30.7 +/- 35.2, Mann-Whitney U-test, z = 1.793, p < 0.05). Both PAF and PAH contents are high in used oils from the two types of petrol engine but not in those from diesel engines. Recycling reduces PAF contents only in used oils from petrol engines, from a mean value of 13.91 +/- 7.32 to 4.23 +/- 2.90% (comparison with original used oils, Mann-Whitney U-test, U = 8, p < 0.01). The light distilled fractions have greater concentrations of indirect mutagens, PAF and PAH than the others. The increase in PAH in light recycled products with respect to the original used oils is significant (Wilcoxon's t-test, z = 2.306, p < 0.05). Benzo[a]pyrene (BaP) is found in appreciable quantities (> 10 ppm) in all used oils from petrol engines and in most of their recycled products. Recycling generally recovers 50% of mutagens and PAF and about 80% of PAH. Considered together, recycled products have in any case contents of mutagens and PAF which are significantly lower than those in the parent oils, but not of PAH (Wilcoxon's t-test; mutagens, z = 2.935, p < 0.01; PAF, z = 3.145, p < 0.01; PAH, z = 1.397, not significant). Lastly, many recycled oils have PAH concentrations which are equal to or higher than those of the original used oils. The health risks linked to professional exposure to these types of oils and the inadequate recycling process currently used (redistillation and thermal clay treatment) in reducing mutagenic and cancerogenic substances from used motor oils are stressed.

Excretion of mutagens, nicotine and its metabolites in urine of cigarette smokers.

Urine samples from 26 cigarette smokers on a restricted diet were collected in the late afternoon. Urine extracts on XAD-2 resin were tested for mutagenicity in the microsuspension assay using Salmonella typhimurium strain TA98 in the presence of metabolizing and deconjugating enzymes. Levels of urinary nicotine plus metabolites and cotinine were determined. Eighteen samples were clearly mutagenic, i.e. capable of doubling the number of spontaneous revertants at one of the assayed doses of urine. Urinary mutagenic activity ranged from 193 to 8462 net revertants/mmol of creatinine, while urinary nicotine plus metabolites and cotinine levels varied from 0.007 to 1.366 and from 0.011 to 0.297 mg/mmol creatinine. Urine samples with nicotine metabolite levels of < 0.33, 0.33- < 0.66 and > 0.66 mg/mmol creatinine had mean values +/- SD of mutagenic activity of 490 +/- 222 (n = 10), 964 +/- 560 (n = 9) and 2692 +/- 2807 (n = 7) revertants/mmol of creatinine, respectively, the statistical comparison between the groups being positive (Mann-Whitney U-test, P < 0.05). The mutagenic activity of urine samples from smokers correlated well with urinary nicotine plus metabolite levels (r = 0.658, P < 0.01). A less close correlation was found between urinary mutagenic activity and other indicators of tobacco smoke exposure, such as urinary cotinine (r = 0.504, P < 0.05), number of cigarettes smoked during the day of urine collection (r = 0.399, P < 0.05) and machine smoking-derived nicotine deliveries of the total number of cigarettes smoked (number of cigarettes multiplied by the nicotine content of each cigarette, as indicated by the manufacturer; r = 0.439, P < 0.05). These results suggest that the mutagenic activity of smokers' urine may be predicted by the urinary level of nicotine plus metabolites. The low degree of reliability of many presumptive indicators of exposure to tobacco smoke and the different urinary excretion kinetics of tobacco smoke mutagens with respect to cotinine (a frequently used biomarker for monitoring exposure to tobacco smoke) are both emphasized.

Mutagenicity and contents of polycyclic aromatic hydrocarbons in new high-viscosity naphthenic oils and used and recycled mineral oils.

Mutagenic activity on the Ames test was evaluated in 15 samples of naphthenic high-viscosity mineral oils and 12 samples of used lubricants (recovered and pooled) and their recycled products. Bacterial mutagenesis was assayed using both the standard technique and Blackburn's modification. The contents of polycyclic aromatic hydrocarbons (PAH) was also evaluated, as polynuclear aromatic fraction (PAF) and total PAH, determined respectively with the semi-quantitative dimethylsulphoxide-refractive index method and the Grimmer method. Only four samples (three acid-treated naphthenic oils and one recycled fraction of a used oil) showed mutagenic activity higher than 6 revertants/mg of oil, considered by Blackburn and coworkers as indicating a potential carcinogenic risk for these compounds. Limited mutagenicity was found in all used and recycled oils, but also in samples of acid- or solvent-treated oils. No hydrogen-treated naphthenic oils turned out to have any mutagenic activity. PAF contents of oils were closely correlated with those of total PAH (n = 15, r = 0.83; n = 12, r = 0.91; p < 0.01 for both naphthenic and used/recycled oils respectively). No recycled oil had high PAF contents. Eleven samples had PAF contents higher than 3%, the arbitrary danger threshold suggested by the CONCAWE (1988). Of these 11 samples, the majority were acid-treated products, although there was one hydrogen-treated oil and one used and recycled oil. No mutagenic activity could be demonstrated in almost half the oils with PAF > 3%. In this study, the presence of mutagens was not correlated wither with PAF or with total or mutagenic PAH. The difficulty of predicting the mutagenicity of mineral oils is stressed. Most naphthenic and some recycled oils clearly have components which inhibit the metabolizing system in the bacterial mutagenesis test, with consequent possible false negative results.

Urinary excretion of mutagens in coke oven workers.

The influence of occupational exposure to polycyclic aromatic hydrocarbons (PAHs) on urinary mutagenic activity was assessed in 75 coke oven workers, using a highly sensitive bacterial mutagen technique (extraction with C18 resin and liquid micro-preincubation test on strain TA98 of Salmonella typhimurium in the presence of metabolizing and deconjugating enzymes). Exposure to PAHs was assessed according to the urinary excretion of 1-pyrenol; the main confounding factors were checked by the number of cigarettes smoked per day and the levels of nicotine and its metabolites in urine, or by ascertaining whether recommended dietary restrictions had been followed. Of the 20 urine samples which turned out to be positive (producing at least double the number of spontaneous revertants), 19 (95%) belonged to smokers. Only one non-smoker had obvious urinary mutagenic activity, and was highly exposed occupationally to PAHs (urinary 1-pyrenol of 3.930 mumol/mol of creatinine). Of the five urine samples from subjects who had not followed the recommended diet, two (40%) were clearly mutagenic. Multiple regression analysis (n = 67) showed that the presence of samples positive for urinary mutagenic activity depended only on smoking habits, if this confounding factor was assessed according to the number of cigarettes smoked per day, while the significant influence of exposure to PAH could be shown when the confounding factor was objectively estimated according to the urinary levels of nicotine and its metabolites. Assessment of the mutagenic potency of urinary extracts (net revertants/mmol creatinine) confirmed the strong influence of smoking habits on urinary mutagenic activity (all smokers 2156 +/- 2691 versus non-smokers 939 +/- 947 net revertants/mmol creatinine; Mann-Whitney test: P < 0.01). In smokers highly exposed to PAHs, greater excretion of mutagens with respect to low-exposure smokers was revealed (3548 +/- 4009 versus 1552 +/- 1227 net revertants/mmol creatinine; Mann-Whitney test: P < 0.01). Multiple regression analysis showed that the mutagenic potency of urinary extracts of coke oven workers depended on exposure to PAHs, tobacco smoking habits, and consumption of fried, grilled or barbecued meat. Increased urinary mutagenic activity strengthens epidemiological evidence of the increased risk of renal and urinary tract tumours in these workers. The presence of mutagenic metabolites in urine as a result of occupational exposure to PAH may be demonstrated only by using highly sensitive techniques for assessing urinary mutagenic activity in studies which include careful checking of the main confounding factors.

Mutagens in indoor air particulate.

Twenty-seven extracts of airborne particulate from domestic environments, both in the absence of sources of pollution and during activities such as smoking tobacco, using a fireplace, and cooking using grills and barbecues, and eight control samples of outdoor particulate were tested using the Salmonella/microsome assay on strains TA98 and TA98NR. Dust levels and mutagenic activity in the indoor environments turned out to be very low in the absence of polluting sources, with highest mean values in winter of less than 0.1 mg/m3 and 6 and 12 revertants/m3, respectively without and with S9. The specific mutagenic activity of indoor dust ranged from 22 and 137 revertants/mg, with a contribution of nitroarene compounds of about 50%, indicating that, in city indoor air, the main cause of background particulate pollution is very probably penetration of traffic fumes from the outside. In contrast, in a country house far from traffic, very low dust and mutagenicity levels were found, without the influence of nitroarene compounds. The presence of autochthonous polluting sources, such as tobacco smoke and fumes from cooking and wood or charcoal burning, greatly increased indoor dust levels, especially during cooking operations, which reached 25.5 and 31.6 mg/m3. The particulate produced by the various indoor pollution sources showed varying specific mutagenic activities. The highest values were found for fumes produced by burning charcoal and wood, smoking tobacco, and cooking foods with high animal protein contents. Mutagens responsible were mainly direct-acting in the case of fumes from burning wood or charcoal, and required mammalian metabolic activation in the case of fumes from tobacco and meat, with a lower contribution (maximum 33%) of nitroarenes than in urban particulate.

Mutagens in urban air particulate.

Extracts of airborne particulate matter were demonstrated to be mutagenic in the Salmonella/microsome test. Urban airborne particulate was collected with high-volume samplers in an Italian town mainly polluted by traffic exhaust fumes. After being weighted for determination of total dust, the particulate was extracted with CH2Cl2/methanol and assayed by Salmonella/microsome assay on strains TA98, TA100 and TA98NR. All samples were mutagenic on strain TA98, with a mutagenic potency of 50 +/- 14 (-S9), 128 +/- 63 (+S9) and 104 +/- 51 (-S9), 211 +/- 97 (+S9) revertants/mg of particulate for summer (n = 23) and winter (n = 22) determinations, respectively. The mutagenic activity on strain TA98NR was about one-half that on strain TA98, indicating a large contribution of nitroaromatic mutagenic compounds. Mutagens from airborne particulate were less active on strain TA100. The summer and winter mean values of urban total dust were 0.15 +/- 0.07 and 0.35 +/- 0.18 mg/m3 respectively, and the mutagenicity of urban air on strain TA98 was 8 +/- 5 (-S9), 22 +/- 17 (+S9) and 30 +/- 11 (-S9), 61 +/- 21 (+S9) revertants/m3 in the two seasons, respectively. In winter, besides an increase in urban air mutagenicity, there also was a change in direct particulate activity per milligram, which was double that of summer.

Influence of smoking habit on the frequency of micronuclei in human lymphocytes by the cytokinesis block method.

To evaluate the frequency of micronucleated lymphocytes in a normal population, 45 healthy subjects were analysed by using a modified cytochalasin B block method; the influence of some confounding factors (sex, age, smoking, etc.) were taken into account. Using a stepwise regression test smoking habits were found to have a statistically significant influence on the frequency of micronucleated cells and micronuclei. In addition, the mitotic and proliferative indexes, and the frequency of micronucleated lymphocytes, at different culture times, were studied in four healthy subjects. Based on the results we suggest that cytochalasin B be added to cultures no later than the 42nd hour.