Urinary excretion of mutagens in passive smokers

Six healthy young volunteers with no history of active smoking were asked to keep on their Western diets avoiding the consumption of alcoholic beverages, excess coffee, any sort of medicament, and the known pro- and/or anti-mutagen-containing foods and drinks, 24 h before and during the experiments. They were exposed passively to cigarette smoke produced by 4 habitual smokers in an unventilated 48.6 m3 room for 8 h. The carbon monoxide concentration was 18.85 +/- 7.3 ppm during the 8-h exposure. Frameshift mutagens were isolated from 10-h urine samples using chloroform and were tested for mutagenicity in the Salmonella/mammalian microsome assay employing Salmonella typhimurium TA98. Although clearly enhanced, no significant mutagenic activity could be found with 25 ml equivalent urine/plate after passive exposure to cigarette smoke. The weak mutagenicities found were highly significant when 50 ml equivalent urine/plate was tested. No direct correlation was observed between urine mutagenicity and the urinary cotinine concentration. The results obtained are discussed with reference to inconsistent reports in the literature concerning the mutagenicity of urine after passive smoking.     

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.     

Analysis of commercial bouillons for trace levels of mutagens

A new method, developed specifically for the extraction of heterocyclic aromatic amine (HAA) type mutagens from different food matrices, was applied to various forms of commercially available bouillons. This procedure is based on liquid-liquid extraction of the sample at different pH values. Recovery and reproducibility of the procedure was determined by processing spiked samples using a mutagenicity bioassay technique as an endpoint. The mutagenicity was tested in the Salmonella/microsome assay using strain TA98 with metabolic activation. 22 bouillon samples in liquid, cube or powder forms from seven manufacturers were extracted and tested for potential mutagenicity. The mutagenic activity of these samples varied and ranged from non-detectable to about 1200 induced revertants per gram of solid material, with a median value of approximately 250 revertants/g. The mutagenic response appeared to be dependent on the source rather than the type or form of the product tested. A negative response was obtained from only one chicken bouillon, and the highest positive response was obtained from a beef bouillon in cube form. It appears that the average beef sample, regardless of form, has a higher mutagenic potency than chicken or chicken and turkey samples. Overall, the intake of mutagens from commercial bouillons (obtained as cubes, concentrates or dry mixes) to prepare one serving (as bouillon, soup, casseroles, etc.) is considerably less than that reported in the literature for one serving of fried beef or pork. The extractability and mutagenic characteristics of these samples indicate the presence of HAA-type mutagens. Work is in progress to identify the mutagenic factors in bouillons.     

Mutagenicity of human urine after the consumption of fried salted salmon

Mutagenicity in the urine of four non-smoking individuals who had eaten salted salmon cooked at home for both lunch and supper was monitored by means of Salmonella/microsome mutagenicity tests. Extracts from fresh and salted salmon had the same level of mutagenicity after being cooked for 10 min at 200 degrees C, but no activity was detected before cooking. Salmonella strains TA98 and TA1538 were equally sensitive to the mutagens and required metabolic activation. No mutagenicity was shown with TA100 and TA1535. Urine samples were tested using a concentrate prepared by means of an XAD-2 resin column. Mutagenicity was detected mainly in urine excreted during 4-5 hr after the ingestion of cooked salmon, but only weak mutagenicity, or none at all, was detected in the urine after the ingestion of vegetables. The levels of urinary mutagenicity due to salmon consumption were not affected when cabbage was eaten simultaneously. The excretion of mutagenic substances was completed within about 20 hr, and there were almost no mutagens in the urine 24 hr after the ingestion of cooked salmon.     

Evaluation of genotoxic risk of handling cytostatic drugs in clinical pharmacy practice

The genotoxic risk of handling antineoplastic drugs was evaluated in fifteen women preparing chemotherapeutics in the Pharmacy Department of the University Hospital Maastricht. Twenty nurses of the same hospital, who were not exposed to cytostatics, acted as controls. Endogenous exposure to antineoplastic drugs was assessed by determination of urine mutagenicity, as well as by analysis of urinary methotrexate levels. As genotoxicological end-points, sister chromatid exchanges and hypoxanthine guanine phosphoribosyl transferase locus point mutations were studied in peripheral lymphocytes obtained via venous puncture. No differences in urine mutagenic activity, in sister chromatid exchange frequencies and in hypoxanthine guanine phosphoribosyl transferase point mutation frequencies between exposed and non-exposed groups were detected. Higher sister chromatid exchange frequency was observed in smokers as compared to non-smokers.     

Mutagen excretion and cytochrome P-450-dependent activity in germfree and conventional rats fed a diet containing fried meat

To evaluate a possible role of the intestinal microflora in the metabolism of the highly mutagenic compounds formed in fried meat, conventional and germfree male AGUS rats were fed a semi-synthetic diet containing fried meat. Changes in mutagen excretion in urine and faeces over time were studied using the Ames Salmonella assay. The faecal and urinary extracts were separated by means of high-performance liquid chromatography (HPLC), and the mutagenicity of the collected fractions was determined. Cytochrome P-450 IA (IA1 and/or IA2) were detected by the use of antibodies with the Western blot technique, and the corresponding enzyme activities were measured in microsomes from the small intestine and the liver. A quantitative as well as qualitative difference in excretion of mutagens between germfree and conventional rats was observed. The total excreted of mutagenicity was significantly higher for the conventional than for the germfree rats, as a result of a higher faecal excretion of mutagens in the conventional animals. The HPLC separations of urinary and faecal extracts showed a different mutagenic metabolite pattern between the germfree and conventional rats. An increased activity of the cytochrome P-450-dependent enzyme ethoxyresorufin-O-deethylase was observed in the small intestine of conventional rats on the fried meat diet, whereas no effect of this diet was observed in the germfree rats. Similar results were obtained in immunoblotting experiments using a P-450 IA antiserum. The present study indicates that the excretion pattern and thus also the metabolism of compounds present in fried meat are affected by the germfree status.     

Urinary mutagenicity after controlled exposure to environmental tobacco smoke (ETS)

20 non-smokers on a defined diet low in polycyclic aromatic hydrocarbons (PAH) were exposed to environmental tobacco smoke (ETS) in an unventilated room for 8 h. The urinary mutagenicity in the 24-h urine samples as tested with the Salmonella (TA98) microsome assay did not significantly increase after exposure to either 10 ppm CO or 20-25 ppm CO. We conclude that exposure of non-smokers to ETS does not lead to an increase in their urinary mutagenicity, provided the exposure conditions are within a realistic range.     

The effect of smoking on nicotine metabolism in vivo in man

Nicotine and a basic metabolite, cotinine, were determined in the urine by gas-liquid chromatography after intravenous administration of (—)-nicotine hydrogen (+)-tartrate to groups of male and female smokers and non-smokers in whom the urine was maintained at an acid pH. The urinary recoveries of nicotine and cotinine from male smokers fell in two groups. One showed a lower recovery of both alkaloids than was seen with male non-smokers. The other showed a similar recovery of nicotine but more cotinine than the male non-smokers. Female smokers excreted less nicotine but more cotinine than female non-smokers. More nicotine but less cotinine was excreted by female non-smokers than by male non-smokers. The results show sex dependent metabolism of nicotine occurs in non-smoking humans and that smoking causes alterations in nicotine metabolism.     

Increased urinary cadmium excretion and its relationship to urinary N-acetyl-β-d-glucosaminidase activity in smokers

To assess the renal effects of low-level exposure to cadmium due to smoking we examined blood and urinary levels of cadmium and urinary excretions of N-acetyl--d-glucosaminidase (NAG), ₂-microglobulin (BMG) and metallothionein in 94 male workers aged 18–55 years. Both blood and urinary cadmium levels indicated excess exposure to cadmium caused by smoking. The urinary cadmium concentration ranged between 0.1 and 5.0 g/g creatinine and increased significantly with age in the smokers. Neither urinary NAG nor BMG was increased in the smokers compared from non-smokers. A positive relationship between urinary cadmium and metallothionein was obtained not only in the smokers but also in the non-smokers. Furthermore, in the smokers urinary cadmium and metallothionein was positively related with urinary NAG. Since NAG in urine mostly originates from tubular cells by lysosomal exocytosis, the results may reflect an early cadmium effect on the lysosomal functions. Inhibitory effect of cadmium on the lysosomal degradation activities was discussed as a possible explanation of the positive relationship of urinary cadmium and metallothionein to urinary NAG.     

3-Hydroxybenzo[a]pyrene in the urine of smokers and non-smokers

The people studied were male volunteers without occupational and dietary exposure to PAH: 27 smokers (10 cigarettes or more) and 27 non-smokers matched for age and socio-professional category. For each person, all the 24h voided urine samples were reassembled in a single sample. 1-Hydroxypyrene (1-OHPy) and 3-hydroxybenzo[a]pyrene (3-OHBaP) were then determined by automated column-switching high-performance liquid chromatography. Urinary 1-OHPy ranged from 0.041 to 0.530 micromol/molCreatinine (arithmetic mean 0.144, median 0.115) for smokers and from 0.01 to 0.148 mmol/molCreatinine (arithmetic mean 0.044, median 0.032) for non-smokers. These values are close to those of some other studies. Urinary 3-OHBaP ranged from <0.01 to 0.084 nmol/molCreatinine (arithmetic mean 0.030, median 0.023) for smokers and from <0.01 to 0.045 nmol/molCreatinine (arithmetic mean 0.014, median 0.011) for non-smokers. Considering more particularly the urinary 3-OHBaP values, the influence of smoking could be important among workers exposed to low levels of BaP (<100 ng/m(3)) and the concentrations for smokers were equivalent to most of the preshift values of exposed workers. The dietary BaP intake was slightly lower than the BaP intake for an average smoker. From the present study, temporary basic reference levels may be proposed for urinary 3-OHBaP.     

Role of metabolic polymorphisms NAT2 and CYP1A2 on urinary mutagenicity after a pan-fried hamburger meal.

In this work the phenotyping approach was used to study the influence of metabolic polymorphisms NAT2 and CYP1A2 on S9-mediated urinary mutagenicity, detected with Salmonella strain YG1024, in 50 subjects after a meal of pan-fried hamburgers. All 50 post-meal samples, but not pre-meal ones, were clearly mutagenic (number of urine samples able to double number of spontaneous revertants was 50 to 0, respectively). CYP1A2 positively influences urinary mutagenicity: a rise in CYP1A2 activity increases levels of post-meal urinary mutagens (1.16+/-0.91 vs 1.72+/-1.19 7-h minimum mutagenic doses (MMDs)/intake), especially in NAT2 slow acetylators (2.18+/-1.33 vs 0.90+/-0.54 7-h MMDs/intake, Mann-Whitney U-test, P<0.05). NAT2 rapid acetylators exert lower post-meal urinary mutagenicity than slow ones (1.41+/-1.02 vs 1.77+/-2.45 7-h MMDs/intake) and even more if the latter are extensive CYP1A2 metabolizers (1.41+/-1.02 vs 2.18+/-1.33 7-h MMDs/intake), but the difference did not reach statistical significance. In conclusion, this study indicates that CYP1A2 and NAT2 activities influence the presence of urinary mutagens after a meal of pan-fried hamburger (rich in HHAs) and consequently their potential genotoxic risk.     

Urinary mutagenicity as an indicator of occupational exposure in a cohort of cosmetologists.

Epidemiological studies suggest a higher risk of hematopoietic disorders including lymphoma among cosmetologists. The etiology of these disorders among cosmetologists is unknown, but beauticians are exposed to a wide variety of chemicals in the workplace. In this study, the urinary mutagenicity of cosmetologists was studied as an indicator of occupational exposure. A microsuspension modification of the Ames assay with Salmonella typhimurium strain TA98 was used to detect direct-acting mutagens and promutagens in urine. A comparable group of teachers of similar age and gender, and living in the same geographic area was used as the control group. There was no elevated risk for urinary mutagenicity among the cosmetologists after controlling for a number of confounders including smoking. In a multivariate model, smoking regularly or within 24 h of sample collection was found to be positively associated with urinary mutagenicity among both groups. The number of cigarettes smoked daily, age, and length of employment were not associated with urinary mutagenicity. Analysis of urine samples collected successively from each participant showed a fair to good agreement between promutagens in samples, suggesting a fairly constant exposure to promutagens.     

URINARY MUTAGENICITY AS AN INDICATOR OF OCCUPATIONAL EXPOSURE IN A COHORT OF COSMETOLOGISTS

Epidemiological studies suggest a higher risk of hematopoietic disorders including lymphoma among cosmetologists. The etiology of these disorders among cosmetologists is unknown, but beauticians are exposed to a wide variety of chemicals in the workplace. In this study, the urinary mutagenicity of cosmetologists was studied as an indicator of occupational exposure. A microsuspension modification of the Ames assay with Salmonella typhimurium strain TA98 was used to detect direct-acting mutagens and promutagens in urine. A comparable group of teachers of similar age and gender, and living in the same geographic area was used as the control group. There was no elevated risk for urinary mutagenicity among the cosmetologists after controlling for a number of confounders including smoking. In a multivariate model, smoking regularly or within 24 h of sample collection was found to be positively associated with urinary mutagenicity among both groups. The number of cigarettes smoked daily, age, and length of employment were not associated with urinary mutagenicity. Analysis of urine samples collected successively from each participant showed a fair to good agreement between promutagens in samples, suggesting a fairly constant exposure to promutagens.     

Inhibition of protein pyrolysate mutagenicity by retinol (Vitamin A)

The mutagenicity in the Ames Salmonella-microsome test of four protein pyrolysate products, formed during the cooking of meat. (Trp-P-1, Trp-P-2, Glu-P-1 and Glu-P-2) was found to be inhibited by the addition of vitamin A in vitro in the form of retinol. The effect is interpreted as an inhibition of the metabolic activation of the mutagens to their respective ultimate mutagenic forms since retinol has been shown to have no effect on the survival of the Salmonella cells, no effect on directly acting mutagens and no effect on the formation of NADPH in the test system. The results demonstrate the need for an increased understanding of the interaction of dietary components in evaluating mutagenic/ carcinogenic risks from processed food.     

Urinary mutagens in municipal refuse incinerator workers and water treatment workers

Municipal refuse incineration workers may be exposed to mutagenic compounds from gaseous and particulate emissions and during ash removal operations. The frequency of urinary mutagens was measured by the Ames test among a sample of 104 refuse incinerator workers in seven incinerator plants during March-May 1988. The frequency was compared to that observed in 61 water treatment employees in 11 municipal water treatment facilities during the same period. Incinerator workers had a significantly higher risk for urinary mutagens and promutagens as compared to water plant workers after controlling for age. Among incinerator workers, increased risk of having urinary mutagens was associated with workers who wore protective clothing (defined as clothing other than masks or gloves) or whose job classification was equipment repair. It also showed a weak positive association with increasing age. There was an increased risk of urinary promutagens associated with not wearing gloves. The presence or absence of mutagenicity in workers' urine varied with plant location. Incinerator operating conditions affecting the production of toxicants and mutagens are discussed and the results of other studies involving toxicant exposure of humans near incinerators are cited.     

The Relative Importance of Mutagens and Carcinogens in the Diet

Known mutagens and carcinogens in the diet were compiled and the risk of cancer was estimated on the basis of average exposure levels in Switzerland and carcinogenic potencies from rodent bioassays. The analysis showed that, except for alcohol, the sum of all known dietary carcinogens could only explain a few percent of the cancer deaths attributed by epidemiologists to dietary factors. The discrepancy was explained by a “carcinogenicity” of excess macronutrients. This hypothesis was based on an evaluation of dietary restriction experiments in rats and mice, where a dramatic reducing effect on spontaneous tumour formation was seen. From these experiments, a "carcinogenic potency" was deduced for food in excess (TD₅₀ approximately 16 g/kg per day). Overnutrition in Switzerland was converted into excess food intake and the cancer risk estimated on the basis of the TD₅₀ value. The resulting risk of 60,000 cases per one million lives would allow to explain by overnutrition almost all "diet-related" cancer deaths in humans.     

Effects of smoking on spontaneous and induced sister chromatid exchanges in lymphocytes

Spontaneous and mitomycin C-induced sister chromatid exchanges (SCEs) in lymphocytes were analyzed in 24 non-smokers and 24 sex- and age-matched smokers. Mean spontaneous SCE frequency for non-smokers was 9.8 SCEs/cell, and that for smokers was 11.5 SCEs/cell. The difference was statistically significant (P less than 0.001 by t-test). These results suggest that spontaneous SCE frequency in lymphocytes is useful for evaluation of biological effects of environmental mutagens. However, we could not find any effects of smoking on the sensitivities of lymphocytes to mitomycin C in vitro. The effects of mutagens on humans may be independent of one another.     

Estimation of smoke dosage and mortality of non-smokers from environmental tobacco smoke

This paper shows how biochemical markers can be used to estimate smoke intake from passive smoking to complement epidemiological studies on the health risks and mortality to non-smokers. Using data from slow nicotine infusions given over 1 h, we estimated that the nicotine intake from passive smoking averages about 0.014 mg/h among urban non-smokers leading their usual daily lives. This compares with 0.23 mg/h in a smoke-filled public house, 0.36 mg/h during maximum exposure in an unventilated room, and 1.0 to 1.4 mg nicotine per cigarette taken in by active smokers. Data from several studies on urinary nicotine concentrations and those of cotinine in blood, urine and saliva were collated. The results show that the concentrations in non-smokers averaged about 0.7% (for nicotine) and 0.6% (for cotinine) of the levels found in smokers. From this we estimate that the mortality from passive smoking is about 1000 non-smokers per year in Britain and about 4000 per year in the United States, assuming that the relation of dose to risk is linear.     

Sources of mutagenicity in cooked Finnish foods

The mutagenic activities of the alkaline fractions derived from various heat-processed, ready-made meat, fish and poultry foods were studied using the Salmonella mutagenicity assay with strain TA98 and S-9 mix to provide information about the mutagenicity of everyday Finnish foods. The majority of the food samples tested were mutagenic. The mutagenic activities of various commercial ready-made products. Mutagenicity varied remarkably between different samples. The cooking temperature clearly affected the mutagenicities of fried or reheated food samples, mutagenicity increasing with increasing temperature. The results indicate that the main sources of cooked-food mutagens in everyday Finnish foods are grilled and broiled products and foods fried at restaurants and at home. By comparison, commercial ready-made fried foods are only a minor source of mutagenicity. Variations between equivalent food samples indicated that heat processing has a marked effect on the mutagenic activity of the product, which might therefore be reduced by modifying the cooking process.     

Urinary hydroxyproline excretion in smokers, non-smokers and passive smokers

The hydroxyproline/creatinine ratio in urine was investigated in 200 cigarette smokers, 199 pipe and/or cigar smokers and 24 non-smokers. For cigarette smokers a statistically significant positive correlation is found between this ratio and daily cigarette consumption, COHb, serum cotinine and nicotine excretion in urine. This smoking-related increase in the hydroxyproline/creatinine ratio is, for the most part or completely, due to the fact that creatinine urine concentrations inversely correlate with the smoke uptake variables. Neither pipe and/or cigar smoking nor passive smoke exposure of non-smokers seem to affect the hydroxyproline/creatinine ratio. A seasonal influence is found in these studies as well as in two experiments with limited numbers of subjects: the hydroxyproline/creatinine ratio is higher in winter than in summer for both smokers and non-smokers. Our data do not favour the idea that measuring hydroxyproline/creatinine ratios in urine is an accurate method of investigating early effects of smoking, passive smoking and air pollution in man.