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Mutagens in human urine: effects of cigarette smoking and diet
Mutation Research, 158 (1985) 149-157 Elsevier
149
MTR 01028
Mutagens in human urine: effects of cigarette smoking and diet I s a a c M. Sasson, D a n i e l T. C o l e m a n , E d m o n d J. L a V o i e , D i e t r i c h H o f f m a n n a n d E r n s t L. W y n d e r Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, N Y 10595 (U.S.A.)
(Received 1 October 1984) (Revision received 5 July 1985) (Accepted 19 July 1985)
Summary Human urine from smokers and nonsmokers on strictly controlled diets was assayed for mutagenic activity. Two distinct diets were employed in this study. Diet study A consisted of a high-meat, high-fat diet, observed for 5 days, followed by a vegan diet, adhered to for the next 5 days. The vegan diet contained no meat, fish, eggs, or dairy products. It was comprised of soy products, prepackaged vegan dinners, seeds, nuts, fruits, vegetables, beans and herbal teas. Diet study B consisted of 3 days on a typical western diet followed by a macrobiotic diet of grains and fresh vegetables for 5 days. Portions of 24-h urine samples were assayed in Salmonella typhimurium TA1538. The levels of urinary creatinine and cotinine were measured. Mutagenic activity was observed in the urine of most smokers. However, the levels of mutagens in the urine of light smokers were similar to those of nonsmokers. For both nonsmokers and smokers there was a significant increase in urine mutagenicity when volunteers were on the vegan diet. Several nonsmokers on the vegan diet in diet study A had pronounced mutagenic activity in their urine samples, in some instances at higher levels than that in the urine of smokers on a meat diet. In diet study B no clear differences were observed between the meat diet and the macrobiotic diet. In diet studies A and B the mutagenic potency of smokers' urine could not be correlated with COfirfine levels alone or with urinary pH. These data suggest that dietary factors can play a dominant role in the mutagenicity of urine concentrates.
The correlation between cigarette smoking and bladder cancer has been documented by numerous epidemiological studies (U.S. Department of Health and Human Services, 1982). The/etiologic agents associated with the increased incidence of bladder cancer among smokers are believed to be present in the urine. Yamasaki and Ames (1977) first demonstrated the presence of mutagens in the urine of cigarette smokers, thus suggesting a correlation between mutagens in smokers' urine and increased risk for bladder cancer. Since publication of these data, other studies have reported an
association between mutagens in urine and cigarette smoking (Sirtori et al. 1978; Van Doom et al., 1979; Aeschbacher and Chappuis, 1981; Recio et al., 1982). Many reseachers have, in addition, sought to quantify hazards of occupational environments and lifestyles by measuring the levels of excreted urinary mutagens (Burnett et al., 1979; Falck et al., 1980; Dolara et al., 1981; Bos et al., 1982; Pasquini et al., 1982; Recio et al., 1984). Conclusions from these studies have not been consistent. The smokers participating in these studies had
0165-1218/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
150 mutagenic urine, but a direct correlation between theurinary excretion of mutagens and the levels of cigarette consumption alone (or the cigarette smoke yields alone) could not be established (Jaffe et al., 1983). In some studies, nonsmokers were found to have mutagenic urine. Some researchers have held that passive smoke exposure is involved in the observed urine mutagenicity among nonsmokers (Puzrath et al., 1981; Bos et al., 1983). Mutagenic urine was also found in patients suffering from cirrhosis (Gelbart and Sontag, 1980), and from bladder cancer (Garner et al., 1982). None of the above studies included an adequate diet control, nor did they take into account the effects of dietary variables which we believe to be of major importance. Baker et al. (1982) had detected mutagenic activity in human urine following the ingestion of fried pork or bacon; no urine mutagenicity was observed when the pork or bacon was microwave-cooked. We investigated the effects of diet and smoking on the detection of mutagenic agents in human urine. We have taken into account variables such as individual differences in inhalation of cigarette smoke, by measuring the urinary excretion of cotinine, the major metabolite of nicotine. Other monitored variables were urinary pH and creatinine concentration. Urine samples consisted of a complete 24-h specimen. Diet protocols were strictly defined. Materials and methods
Diet Diet study A 5 smokers and 5 nonsmokers (men and women over 18 years of age, not taking any medication, and in good health) ate high-meat, high-fat meals for 5 days. These volunteers were then switched to a vegan diet for 5 days. Urine (24-h specimens) was collected on days 3, 4, and 5 and on days 8, 9, and 10. All participants followed the identical strictly defined daily diets which were preselected from the following:
Meat diet (days 1-5) Breakfasts: eggs, ham, sausages, waffles, butter, toast, juice, coffee, tea. Lunches: tuna fish, roast beef, pastrami, turkey, chicken, rye bread, lettuce and tomatoes, soda, coffee, tea.
Dinners:
steak, veal cutlet, lamb chop, pork chop, chicken, rice, potatoes, string beans, carrots, broccoli, ice cream, coffee, tea.
Vegan diet (days 6-10) Breakfasts: granola, raisins, corn or bran muffin, preserves, soy milk, juice, herbal teas, coffee substitute. Lunches: peanut butter, tofu, sprouts, sunflower or sesame seeds, soyburgers, canned fruits, packaged tabboule (burgul or cracked wheat), lettuce, fruits, rye or whole wheat bread or pita, herbal teas, coffee substitute. Dinners: packaged or canned dinners (frozen tofu lasagna, vegetable patties, legumes tofu ravioli, macaroni, soyburgers), frozen broccoli, spinach, string beans, cauliflower, rye or whole wheat bread or pita, sunflower or sesame seeds, iceberg lettuce, tomatoes, cucumbers, carrots, sprouts, radishes, chickpeas, dried fruits, jello, granola bars, nondairy ice cream, herbal teas, coffee substitute.
Diet study B 5 smokers and 5 nonsmokers ate a normal western diet for 3 days, providing a 24-h urine sample on the third day. The participants then switched to a macrobiotic diet for 5 days and provided 24-h urine samples on the last 2 days. In addition, 3 smokers and 6 nonsmokers who had customarily adhered to macrobiotic diets for at least 1 year, maintained the identical macrobiotic diet plan and also provided 24-h urine samples on the last 2 days. The dietary regimens were explicitly defined and selected from the following:
Meat diet (days 1-3) Breakfasts: eggs, cereal, bread rolls, milk, juice, coffee, tea. Lunches: tuna fish, roast beef, turkey, bread rolls, juice, coffee, tea. Dinners: steak, lamb chop, chicken, rice, baked potatoes, string beans, broccoli, ice cream, coffee, tea
Macrobiotic diet (prepared fresh daily," days 4-8) Breakfasts: oatmeal, butternut squash, brown rice, cream, baked apple, sourdough bread, barley tea.
151 Lunches and dinners:
Brown rice, beans, millet, barley, seaweed, steamed fresh carrots, broccoli, onions, scallions, squash, turnips, romaine lettuce, spinach, tofu, rice cakes, barley tea.
Urine collection, extraction and concentration All samples were collected in polyethylene bottles and stored in a freezer prior to extraction (within 2 weeks of collection). Thawed urine was then gravity-filtered, pH-adjusted to 7.0, and concentrated on Glass Econo columns (BioRad Laboratories, Richmond, CA) packed with washed Amberlite XAD-2 resins (Aldrich Chemicals), following a modified procedure of Yamasaki and Ames (1977). 3 g of resin was used per 100 ml urine. In diet study A, 250-ml urine aliquots were used. In diet study B, the entire 24-h specimens were concentrated. The columns were washed with distilled water (10 ml water/100 ml urine), and eluted with acetone (10 ml/100 ml urine). To insure that histidine and other highly water-soluble components of urine were completely removed, the acetone eluate was concentrated and partitioned between methylene chloride/brine, the organic layer was washed with brine, dried with Na2SO4, and concentrated to dryness. Distilled water (1000 ml) was also passed through a XAD-2 resin column and simultaneously concentrated, to check for artefactual contamination. Urinalysis Urinary cotinine levels were determined by radioimmunoassay (RIA) using a procedure modified from that of Langone et al. (1973). Urinary creatinine levels were established by using a colorimetric method for the red creatinine-picrate complex (Tietz, 1982). The pH was measured, and all urine samples were routinely tested for nitrite, and for total bacterial andGram-negative counts using Microstix-3 (Ames 3009) strips. Strips of N-Multistix-C (Ames 2862) were also routinely employed to determine ascorbic acid, nitrite, protein, glucose, bilirubin, occult blood, and urobihnogen present. Mutagenicity assays The Ames assay (Ames et al., 1975) was carried out using S. typhimurium TA1538, and required
activation with Aroclor-induced Fischer rat liver $9 mix. TA1538 (along with TA98) is a widely used frameshift mutagen indicator, more responsive to urine mutagens than the base-pair mutagen indicator TA100 (Connor et al., 1983). The $9 mix was prepared following established procedure (Ames et al., 1975). In diet study A, the concentrate of the 250-ml urine sample was dissolved in 500 /~1 dimethyl sulfoxide (DMSO) and tested in duplicate at 25 ml, 12.5 ml, 6.25 ml, and 0 ml (control) urine equivalent per plate. In diet study B, one-fourth of each 24-h urine concentrate was dissolved in 500 #1 DMSO and tested in duplicate at 2.5%, 1.25, 0.62, 0.31, and 0% (control) equivalent of total urine sample per plate. The plates were incubated at 37°C for 72 h and were counted after 48 and 72 h. The data presented are based on the 72-h reading. Controls with and without metabolic activation were run in duplicate using DMSO, picrolonic acid (Pic; 250 #g), 4-aminobiphenyl (4ABP; 50 #g), 2-aminofluorene (2AF; 20 #g), and 1-methyl-3-nitro-l-nitrosoguanidine (MNNG; 5 #g). The histidine revertants for the controls were: without $9 activation: DMSO, 19; Pic, 1150; MNNG, 30, with $9 activation: DMSO, 35; 4ABP, 500; 2AF, 1230; distilled water, 15. A 500-ml distilled water sample that contained 200 #g 2AF was worked-up as above, and dissolved in 500 #1 DMSO. This control sample, tested in duplicate, had values of: 175 (6.25 ml equivalent), 450 (12.5 ml), and 325 (25 ml). Evaluation of data The method of linear regression was used to determine the mutagenicity of urine samples. For diet study A, the mean urinary mutagen levels are expressed as histidine revertants per 20-ml sample. The linear portion of the dose-response curves for the control (0 ml), 6.25 ml, 12.5 ml and 25 ml urine equivalents per average of 2 plates each was used to determine the mutagenicity of a 20-ml sample. After subtracting the control values, the results are divided by the creatinine contents of 1 ml urine to account for the differences in osmolality. These data are illustrated in Fig. 1 For diet study B, the linear portion of the dose-response curves for the total urine sample per average of 2 plates each was used to determine the mutagenicity of 1% of the total 24-h urine
* • a b ¢ a
1500 700 750 1050 1000 700 1200 1250 750 1800
Day3a
2000 1500 950 1100 1000 750 2 800 1050 1200 800
Day5a 1300 1600 1100 1650 1100 800 1550 850 800 1650
Day8b 1500 850 550 1100 1150 600 1 350 600 650 1 250
DaylOb
Smoker. Macrobiotic adherent for at least 1 year. Meat regimen, diet study A. Vegan regimen, diet study A. Meat regimen, diet study B. Macrobiotic regimen, diet study B.
1 2 3 4 5 6* 7* 8* 9* 10 *
Participant Diet study A No. 24-h urine (ml)
1.0 0.82 1.8 0.63 1.9 0.92 1.4 1.2 0.83 0.87
1.1 1.0 1.1 0.86 1.3 0.85 1.8 0.85 0.90 1.0
1000 1 800 1500 1700 . 850 1600 700 . .
1,4 1.2 t.2 0.87 1.6 0.79 1.7 0.95 0.76 1.0
1.4 1.1 1.2 0.69 2.0 1.2 1.8 1.0 0.80 1.5
100 650 1100 1600 . . 600 900 600 . . . . 1
Day8d
Day3C
Day3a DaySa Day8b Dayl0b
Diet study B 24-h urine (ml)
Creatinine (g/24 h)
24-h U R I N E COLLECTION: VOLUME A N D C R E A T I N I N E VALUES
TABLE 1
1,0 1.8 1,7 1.5 . 1.2 1,9 0.90 . . 1.2 1.4 0.62
1.2 1.1 1.1 1,1
Day3C D a y 8 d
Creatinine ( g / 2 4 h)
h)
Creatinine (g/24
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Day3 c Day8 d Day3 c Day8 d
24-h urine (ml)
Participant Diet study B No.
b.a
153 sample. After subtracting the solvent control ( D M S O + $9 mix, 35 and 33 for diet studies A and B, respectively), the data are plotted, expressed as histidine revertants in 1% of 24-h urine (Fig. 2). All the baseline values were reproducible, with an error margin of less than 10%. A sample with twice the histidine revertants of the control was considered mutagenic. Results
The results of the mutagenicity tests of the urine concentrates from smokers and nonsmokers in diet study A are shown in Fig. l a - d and Table 1. The n o n s m o k i n g status of the latter group of
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Fig. 1. Mutagenicity of urine on diet A. The number of histidine revertants of S. typhimurium TA1538 in 20 ml urine are normalized for urinary creatinine contents. The data are taken from the linear portion of the dose-response curve for each urine sample, a and b represent the mean urinary mutagen levels on days 3 and 8, and 5 and .10, respectively, of nonsmokers, c and d show the smokers' mean urinary mutagen levels on days 3 and 8, and 5 and 10, respectively. Data from the following samples are not based on the full dose-response curves; deviation from linearity at the highest dose tested was observ~l (M, meat diet; V, vegan diet). (a) 2M, 3V, 4M; (b) 2M, 3V; (c) 6M, 9M; (d) 10V.
participants was confirmed by the absence of cotinine in their urine. Fig. l a represents the urine mutagenicity of nonsmokers on the third day of the meat and of the vegan components of diet study A (i.e., days 3 and 8). Fig. l b represents the mutagenicity of urine excreted by nonsmokers on the fifth day of the meat and of the vegan components of diet study A (i.e., days 5 and 10). The data for the urine concentrates of the smokers in diet study A are summarized in Fig. l c and d: Fig. l c represents days 3 and 8, Fig. l d represents those corresponding to days 5 and 10. The results are expressed in revertants per 20 ml urine, and normalized for urinary creatinine content. Most of the smokers in this study had mutagenic urine. P r o n o u n c e d mutagenic activity was detected in the urine collected from the nonsmoking participants when they adhered to the vegan diet portion of diet study A. In comparing the two diets, we found that for the nonsmokers, the mean regimen resulted in combined (days 3 and 5) mean his + revertants of 14.7 + 12.9 (standard deviation) and standard error of 4.1. The vegan regimen had a mean urine mutagenicity of 40.3 + 31.4 (standard deviation) and standard error of 4.1 ( p < 0.01). In some cases, levels of mutagenic activity detected in the urine of nonsmokers on the vegan diet were equal to or higher than those detected in the urine collected of smokers on the meat-containing portion in diet study A (see Fig. l a - d ) . * There is a general pattern of increasing mutagenic potency in the urine of cigarette smokers with an increase in the smoking habit, as measured by the urinary cotinine levels; however, it is not a linear dose response. W h e n cotinine levels are taken into account, most smokers on the vegan portion of diet study A exhibited an increase in urine mutagenic activity as c o m p a r e d to those on the meat portion of diet study A. The dietary * The urine from days 4 and 9 was collected in bottles containing sulfamic acid as a preservative. These samples were used for quantitating N-nitrosoproline (NPRO) in the urine. As expected, the vegan diet did not yield any detectable amount of NPRO (less than 0.5 ng/ml urine), when measured as the methyl ester by a gas chromatograph interfaced with a Thermal Energy Analyzer. In the absence of any supplemental administration of proline, there were no significant differences in NPRO contents in the urine of smokers versus that of nonsmokers when both groups were on the meat diet.
154 TABLE 2 URINE MUTAGENICITY OF 'HEAVY' SMOKERS ON DIET STUDY A a Participant No.
Equivalent per plate volume
Cotinine
12.5 ml
25 mi
(ng/ml)
Creatinine (mg/nal urine)
67 51 75 70
79 68 117 101
118 96 213 171
3152 1960 2300 2930
1.52 0.58 0.93 1.37
6.25 ml 7 Day Day Day Day
3Mb 5M 8V 10 V
8 Day Day Day Day
3M 5M 8V 10 V
73 63 116 77
102 88 127 99
111 121 215 170
2460 2118 2736 2668
0.78 0.90 1.41 1.29
9 Day Day Day Day
3M 5M 8V 10 V
83 54 67 66
91 62 77 71
67 82 100 78
3500 1316 2120 1448
1.05 0.63 1.04 1.39
a Average per 2 plates. Values not adjusted for creatinine contents. DMSO ~ $9 control: 35. b M. meat regimen: V, vegan regimen.
100
MEAT DiET • DAY 3 MACROBIOTIC DIET [ ] DAY 8 PRACTICING ( over one year) MACROBIOTIC [ ]
BO 60 ,o o2o
14. 0
1
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MEAT DIET • DAY :5 I MACROBIOTIC DIET I'-] DAY 8 JPRACTICING (aver one year) MACROBIOTIC Jng COTININE/rag CREATININE ( )
2
3
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12 13 14 15 16 17
factors that are responsible for the smokers' urine mutagenicity cannot be isolated, since cigarette smoking habits are not duplicated on a daily basis. We can, however, compare and contrast the dose-response pattern of the 3 heavy smokers ( v o l u n t e e r s 7, 8, 9 i n Fig. 1), as s h o w n i n T a b l e 2. A l l 3 s m o k e r s h a d c o t i n i n e levels e x c e e d i n g 1 0 0 0 n g / m l u r i n e f o r e a c h d a y o f t h e s t u d y . T h e r e is a h i g h e r level o f m u t a g e n i c r e s p o n s e s w h e n c o m p a r i n g t h e h i g h e s t d o s e t e s t e d , 25 m l e q u i v a l e n t p e r p l a t e , f o r all 3 s m o k e r s o n t h e v e g a n diet. B u t it is difficult to separate the effects of changes in diet from the effects of intake of differing amounts of tobacco smoke.
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Fig, 2. Mean urinary mutagen levels on diet B, expressed as histidine revertants of S. typhimurium TA1538 in 1% of 24-h urine. The data are taken from the linear portion of the dose-response curve for each urine sample, a represents the mean urinary mutagen levels of nonsmokers, and b the mean urinary mutagen levels of smokers. The black bars show the mutagenic responses on the 3rd day, when the participants ate a meat diet. The white bars give the mutagenic response, on the 8th day of diet B, when the volunteers were eating macrobiotic meals. Dotted bars show customary macrobiotic levels. Data from the following samples are not based on the full dose-response curves; deviation from linearity at the highest dose tested was observed (M, meat regimen; Ma, macrobiotic regimen). (a) 1M, 4M, 4Ma, 12, 17; (b) 6Ma, 7M, 18Ma.
155 The mean urinary pH ranged from 5.9 to 6.5, with individual variations from 5.4 to 7.2. The urinary pH did not seem to be related to mutagenic activities. The urine of most smokers in diet study B was mutagenic (Fig. 2b). Some of the nonsmokers had mutagenic urine (Fig. 2a) as did some of the participants who had been customarily adhering to a macrobiotic diet for more than 1 year (Fig. 2b). There were no consistent differences in this diet study in the mutagenic activity of urine excreted by those on the meat diet versus those on the macrobiotic diet (p > 0.2). The limited number of individuals in each controlled diet study, however, could preclude the detection of subtle but significant differences in the mutagenicity of such urine concentrates. Discussion
Many studies have reported that nonsmokers' urine may contain mutagens, but no convincing etiological explanations have been offered. While demonstrating that bladder cancer patients had mutagenic urine, one study showed that 32 out of 72 nonsmokers who constituted the control group, also had mutagenic urine (Garner et al., 1982). Other examples of nonsmokers having mutagenic urine are seen in studies by Recio et al. (1984) and by others who were attempting to correlate workrelated risks to mutagenic principles in the urine of employees (Falck et al., 1979, 1980; Gelbart and Sontag, 1980; Dolara et al., 1981; Bos et al., 1982; Pasquini et al., 1982). Our studies indicate that diet alone could account for much of the observed urine mutagenicity among nonsmokers (Fig. la and b). Fig. lc and d show that diet may also be a confounding factor of the observed mutagenic activity in the urine of smokers. Also, the effect of diet may explain the findings that have attempted to relate environmental (passive) smoke exposure to urine mutagenicity, Puzrath et al. (1981), have attributed the presence of mutagens in the urine of a nonsmoking spouse of a cigarette smoker to the related passive smoke exposure. Examination of the pattern of mutagenic activity of urine samples as profiled by HPLC from the smoker and the spouse reveals that the curves had the same shape, albeit at different mutagen levels. Bos et al. (1983)
also attributed to passive smoke exposure findings of urinary mutagenicity obtained with 8 nonsmokers exposed to smoke from 10 cigarette smokers. Warren and Rogers (1982), in a study that measured the urine mutagenicity of college students, found no urinary mutagens in subjects smoking < 10 low-tar and low-nicotine cigarettes per day. In our studies, 'light' smoking paticipants (15 medium-tar cigarettes/day or less) had levels of mutagenic urine that could not be distinguished from those of nonsmoking participants on the same diet (e.g., smokers Nos. 6 and 10 on the meat diet in Fig. lc and d, and smokers Nos. 6, 20, 21, 22 on the macrobiotic diet in Fig. 2b). The efficacy of smoking, i.e. frequency and depth of inhalation, retention of smoke, and individual metabolic variations d o play an important role in regard to uptake of smoke components. It is clear from dosimetry studies, however, that the uptake of environmental smoke compounds by nonsmokers generally corresponds to merely a few percent of the doses measured in physiological fluids of active cigarette smokers (Hoffmann et al., 1984). Thus, a measurable impact of environmental smoke exposure on urinary mutagenicity levels would not be expected. Studies by several investigators (Sugimura et al., 1977; Nagao et al., 1977; Wakabayashi et al., 1978) have shown that pyrolysates of meat produce mutagens. In addition, it has been shown (Baker et al., 1982) that the ingestion of fried pork or fried bacon results in mutagenic urine. Our diet study A included meat that needed broiling or frying, and some soy products that were broiled or fried. Results from our diet study A, however, did not reveal the presence of excessively high levels of urinary mutagens among nonsmoking participants on a diet consisting of both high meat and high fat. The possible appearance of such mutagens in the feces, however, cannot be discounted. Mutagenicity was detected in the urine after the nonsmokers had switched to the vegan diet. It is not as yet known whether the comparatively smaller amounts of fried or broiled of soy products that were ingested would cause the observed urine mutagenicity. The identity and the significance of these urinary mutagens in regard to human health is not known. Our data indicate that the effect of diet on mutagens in human urine is complex and
156 may be difficult to relate to a single environmental or lifestyle factor. Cigarette smoke condensate (CSC) contains mutagenic and carcinogenic components (Sato et al., 1977). The genotoxicity, and the types of genetic damage that CSC can induce, have recently been reviewed (DeMarini, 1983). Our study shows that smokers with increased amounts of urinary cotinine clearly have higher levels of mutagenic urine, although there is no linear correlation. Connor et al. (1983) have reported minor quantities of 2-aminonaphthalene, along with consider. able amounts of its possible metabolite, 2-amino7-naphthol, to be present in the urine of a cigarette smoker. However, the proportion of these compounds reported present in the urine is so high that it is difficult to find a rational explanation for their origin: a commercial, 85-mm nonfilter cigarette contains an average of 1.0 ng of 2-naphthylamine (Patrianakos and Hoffmann, 1979). The agents responsible for the mutagenicity of urine observed in the vegan portion of diet study A are not known. Many processed foods and food additives have been found to contain mutagens (Pariza et al., 1979; Sugimura and Sato, 1983). ;~ recent report shows that many heat-processed. canned foods display mutagenic activity in strai~. TA1538 with $9 activation (Krone and Iwaoka, 1984). Also, plants contain flavonoids, some of which are mutagens (White et al., 1973; MacGregor and Jurd, 1978; Brown and Dietrich, 1979; Ames, 1983; Stavric, 1984). It should be mentioned that quercetin, a flavonoid widely found in ingested foods of plant origin, is mutagenic in the Ames Salmonella microsomal test. However, while quercetin was judged a bladder carcinogen in a study on rats (Pamukcu et al., 1980), in-depth studies failed to verify that quercetin is, in fact, an animal carcinogen in tests on rats, mice and hamsters (Morino et al., 1982; Saito et al., 1980). There are, in addition, other as yet unidentified mutagens in food products derived from plants (Van der Hoeven et al., 1982). Puzrath et al. (1981) have shown by H P L C fractionation of smokers' urine that the mutagens responsible for the observed responses are a complex mixture of relatively non-polar compounds. We are in the process of isolating and identifying the mutagens derived from various diets and from
cigarette smoking. Our studies have now demonstrated that diet has a pronounced effect upon the level of mutagenic activity measured in S. typhimurium present in human urine. Several studies have attempted to relate mutagenic activity in human urine to environmental or occupational factors. Our data indicate that in any such study strict dietary controls are essential.
Acknowledgements We would like to thank Ms. Caryn Axelrad, Mr. Steve Colosimo and Mrs. Katherine Tilton for assistance in the biochemical studies. This study is supported by American Cancer Society Grant SIG-8.
References Aeschbacher, H.U., and C. Chappuis (1981) Non-mutagenicity of urine from coffee drinkers compared with that from cigarette smokers, Mutation Res., 89, 161-177. Ames, B.N. (1983) Dietary carcinogens and anticarcinogens, Science, 221, 1256-1263. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsomemutagenicity test, Mutation Res., 31,347-364. Baker, R., A. Arlauskas, A. Bonin and D. Angus (1982) Detection of mutagenic activity in human urine following fried pork or bacon meals, Cancer Lett., 16, 81-89. Bos, R.P., A.O. Leenaars, J.L.G. Theuws and P.Th. Henderson (1982) Mutagenicity of urine from nurses handling cytostatic drugs; infuence of smoking, Int. Arch. Occup. Environ., 5, 359-369. Bos, R.P., J.L.G. Theuws and P.Th. Henderson (1983) Excretion of mutagens in human urine after passive smoking, Cancer Lett., 19,. 85-90. Brown, J.P., and P.S. Dietrich (1979) Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test, Mutation Res., 66, 223-240. Burnett, C.M., C.M. Fuchs and J.F. Corbett (1979) Mutagenicity studies on urine concentrates from female users of dark hair color products, Drugs Chem. Toxicol., 2, 283-293. Connor, T.H., V.M.S. Ramanujan, J.B. Ward Jr. and M.S. Legator (1983) The identification and characterization of a urinary mutagen resulting from cigarette smoke, Mutation Res., 113, 161-172. DeMarini, D.M. (1983) Genotoxicity of tobacco smoke and tobacco smoke condensate, Mutation Res., 114, 59-89. Dolara, P., S. Mazzoli, D. Rosi, E. Buiatti, S. Baccetti, A. Turchi and V. Vanucci (1981) Exposure to carcinogenic chemicals and smoking increases urinary excretion of mutagens in humans, J. Toxicol. Environ. Health, 8, 95-103. Falck, K., P. GriShn, M. Sorsa, H. Vainio, E. Heinonen and
157 L.R. Holsti (1979) Mutagenicity in urine of nurses handling cytostatic drugs, Lancet, 1, 1250-1251. Falck, K., M. Sorsa and H. Vainio (1980) Mutagenicity in urine of workers in rubber industry, Mutation Re*., 79, 45-52. Garner, R.C., A.J. Mould, V. Lindsay-Smith, R.A. Cartwright and B. Richards (1982) Mutagenic urine from bladder cancer patients, Lancet, 2, 389. Gelbart, S.M., and S.J. Sontag (1980) Mutagenic urine in cirrhosis, Lancet, 1, 894-896. Hoffmann, D., N.J. Haley, J.D. Adams and K.D. Brunnemann (1984) Tobacco sidestream smoke: Uptake by nonsmokers, Prev. Med., 13, 608-617. Jaffe, R.L., W.J. Nicholson and A.J. Garro (1983) Urinary mutagen levels in smokers, Cancer Lett., 20, 37-42. Krone, C.A., and W.T. Iwaoka (1984) Occurrence of mutagens in canned foods, Mutation Re*., 141, 131-134. Langone, J.J., H.B. Gjika and H. Van Vunakis (1973) Radioimmunoassay for nicotine and cotinine, Biochemistry, 12, 5025-5030. MacGregor, J.T., and L. Jurd (1978) Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium, Mutation Re*., 54, 297-309. Morino, K., N. Matsukura, H. Ohgaki, T. Kawashi, T. Sugimura and I. Hirono (1982) Carcinogenicity test of quercetin and rutin in golden hamsters by oral administration, Carcinogenesis, 3, 93-97. Nagao, M., M. Honda, Y. Seino, T. Yahagi, T. Kawachi and T. Sugimura (1977) Mutagenicities of protein pyrolysates, Cancer Lett., 2, 335-340. Pamukcu, A.M., S. Yaiciner, J.F. Hatcher and G.T. Bryan (1980) Quercetin, a rat intestinal and bladder carcinogen present in bracken fern (Pteridium aquilinum), Cancer Re*., 40, 3468-3471. Pariza, M.W., S.H. Ashoor and F.S. Chu (1979) Mutagens in heat-processod meat, bakery, and cereal products, Food Cosmet. Toxicol., 17, 429-430. Pasquini, R., S. Monarca, G.S. Sforzoline, R. Conti and F. Fagiolo (1982) Mutagens in urine of carbon electrode workers, Int. Arch. Occup. Environ. Health, 50, 387-395. Patrianakos, C., and D. Hoffmann (1979) Chemical studies on tobacco smoke, LXIV. On the analysis of aromatic amines in cigarette smoke, J. Anal. Toxicol., 3, 150-154. Puzrath, R.M., D. Langley and E. Eisenstadt (1981) Analysis of mutagenic activity in cigarette smokers' urine by high performance liquid chromatography, Mutation Re*., 85, Q'I_ 1 ~ o
Recio, L., H.G. Enoch and M.A. Hannan (1982) Parameters affecting the mutagenic activity of cigarette smokers' urine, J. Appl. Toxicol., 2, 241-246. Recio, L., H.G. Enoch, M.A. Hannan and R.H. Hill (1984) Application of urine mutagenicity to monitor coal liquefaction workers, Mutation Res., 136, 201-207. Saito, D., A. Shirai, T. Matsushima, T. Sugimura and I. Hirono (1980) Test of carcinogenicity of quercetin, a widely distributed mutagen in foods, Teratogen. Carcinogen. Mutagen., 1, 213-221.
Sato, S.Y., Y. Seins, T. Ohka, T. Yahagi, M. Nagao, T. Matsushima and T. Sugimura (1977) Mutagenicity of smoke components from cigarettes, cigars and pipe tobacco, Cancer Lett., 3, 1-8. Sirtori, C., M. Paganuzzi, C. Lombardo, S. Scalese and T. Ruzzon (1978) Pre*enza di sostanze mutagene nelle urine di fumatori, Gaslini, 10, 18-20. Stavric, B. (1984) Mutagenic food flavonoids, Fed. Proc., 43, 2454-2458. Sugimura, T., and S. Sato (1983) Bacterial mutagenicity of natural materials, pyrolysis products, and additives in foodstuffs and their association with genotoxic effects in mammals, in: A.W. Hayes, R.C. Schnell and T.S. Miya (Eds.), Developments in the Science and Practice of Toxicology, Proceedings 3rd Int. Congr. on Toxicology, San Diego, CA, 28 Aug.-3 Sept. 1983, Elsevier, Amsterdam, pp. 115-133. Sugimura, T., T. Kawachi, M. Nagao, T. Yahagi, Y. Seino, T. Okamoto et al. (1977) Mutagenic principle(s) in tryptophan and phenylalanine pyrolysis products, Proc. Jpn. Acad., 53, 56-61. Tietz, N.W. (1982) Fundamentals of Clinical Chemistry, Saunders, Philadelphia, PA, pp. 981 and 997-999. U.S. Department of Health and Human Services (1982) The Health Consequences of Smoking: A Report of the Surgeon General, Publ. No. 82-50179, Government Printing Office, Washington, DC, p. 322. Van der Hoeven, J.C.M., W.J. Lagerwey, C.A.J.M. Meeuwissen, P.C.M. Hauwert, A.G.J. Voragen and J.H. Koeman (1982) Mutagens in food products of plant origin, in: Mutagens in our Environment, Liss, New York, pp. 327-338. Van Doom, R., R.P. Box, Ch.-M. Leijdekkers, M.A.P. Wagenaar-Zegers, J.L.G. Theuws and P.Th. Henderson (1979) Thioether concentration and mutagenicity of urine from cigarette smokers, Int. Arch. Occup. Environ. Health, 43, 159-166. Wakabayashi, K., K. Tsuji, T. Kosuge, K. Takeda, K. Yamaguchi, K. Shudo, Y. Iitaka, T. Okamoto, T. Yahagi, M. Nagao and T. Sugimura-(1978) Isolation and structure determination of a mutagenic substance in L-lysine pyrolysate, Proc. Jpn. Acad., 54B, 569-571. Warren, G.R., and S.J. Rogers (1982) Urine mutagen screening as a population monitoring technique: Children in an isolated, high lung cancer mortality area, in: B.A. Bridges, B.E. Butterworth and 1.B. Weinstein (Eds.), Banbury Rep. 13, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 39-48. White, R.D., P.H. Krumperman, P.R. Cheeke and D.R. Buhler (1973) An evaluation of acetone extracts from six plants in the Ames mutagenicity tests, Toxicol. Lett., 15, 25-31. Yamasaki, E., and B.N. Ames (1977) Concentration of mutao gens from urine by adsorption with the non-polar resin XADo2; cigarette smokers have mutagenic urine, Proc. Natl. Acad. Sci. (U.S.A.), 74, 3555-3559.
149
MTR 01028
Mutagens in human urine: effects of cigarette smoking and diet I s a a c M. Sasson, D a n i e l T. C o l e m a n , E d m o n d J. L a V o i e , D i e t r i c h H o f f m a n n a n d E r n s t L. W y n d e r Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, N Y 10595 (U.S.A.)
(Received 1 October 1984) (Revision received 5 July 1985) (Accepted 19 July 1985)
Summary Human urine from smokers and nonsmokers on strictly controlled diets was assayed for mutagenic activity. Two distinct diets were employed in this study. Diet study A consisted of a high-meat, high-fat diet, observed for 5 days, followed by a vegan diet, adhered to for the next 5 days. The vegan diet contained no meat, fish, eggs, or dairy products. It was comprised of soy products, prepackaged vegan dinners, seeds, nuts, fruits, vegetables, beans and herbal teas. Diet study B consisted of 3 days on a typical western diet followed by a macrobiotic diet of grains and fresh vegetables for 5 days. Portions of 24-h urine samples were assayed in Salmonella typhimurium TA1538. The levels of urinary creatinine and cotinine were measured. Mutagenic activity was observed in the urine of most smokers. However, the levels of mutagens in the urine of light smokers were similar to those of nonsmokers. For both nonsmokers and smokers there was a significant increase in urine mutagenicity when volunteers were on the vegan diet. Several nonsmokers on the vegan diet in diet study A had pronounced mutagenic activity in their urine samples, in some instances at higher levels than that in the urine of smokers on a meat diet. In diet study B no clear differences were observed between the meat diet and the macrobiotic diet. In diet studies A and B the mutagenic potency of smokers' urine could not be correlated with COfirfine levels alone or with urinary pH. These data suggest that dietary factors can play a dominant role in the mutagenicity of urine concentrates.
The correlation between cigarette smoking and bladder cancer has been documented by numerous epidemiological studies (U.S. Department of Health and Human Services, 1982). The/etiologic agents associated with the increased incidence of bladder cancer among smokers are believed to be present in the urine. Yamasaki and Ames (1977) first demonstrated the presence of mutagens in the urine of cigarette smokers, thus suggesting a correlation between mutagens in smokers' urine and increased risk for bladder cancer. Since publication of these data, other studies have reported an
association between mutagens in urine and cigarette smoking (Sirtori et al. 1978; Van Doom et al., 1979; Aeschbacher and Chappuis, 1981; Recio et al., 1982). Many reseachers have, in addition, sought to quantify hazards of occupational environments and lifestyles by measuring the levels of excreted urinary mutagens (Burnett et al., 1979; Falck et al., 1980; Dolara et al., 1981; Bos et al., 1982; Pasquini et al., 1982; Recio et al., 1984). Conclusions from these studies have not been consistent. The smokers participating in these studies had
0165-1218/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
150 mutagenic urine, but a direct correlation between theurinary excretion of mutagens and the levels of cigarette consumption alone (or the cigarette smoke yields alone) could not be established (Jaffe et al., 1983). In some studies, nonsmokers were found to have mutagenic urine. Some researchers have held that passive smoke exposure is involved in the observed urine mutagenicity among nonsmokers (Puzrath et al., 1981; Bos et al., 1983). Mutagenic urine was also found in patients suffering from cirrhosis (Gelbart and Sontag, 1980), and from bladder cancer (Garner et al., 1982). None of the above studies included an adequate diet control, nor did they take into account the effects of dietary variables which we believe to be of major importance. Baker et al. (1982) had detected mutagenic activity in human urine following the ingestion of fried pork or bacon; no urine mutagenicity was observed when the pork or bacon was microwave-cooked. We investigated the effects of diet and smoking on the detection of mutagenic agents in human urine. We have taken into account variables such as individual differences in inhalation of cigarette smoke, by measuring the urinary excretion of cotinine, the major metabolite of nicotine. Other monitored variables were urinary pH and creatinine concentration. Urine samples consisted of a complete 24-h specimen. Diet protocols were strictly defined. Materials and methods
Diet Diet study A 5 smokers and 5 nonsmokers (men and women over 18 years of age, not taking any medication, and in good health) ate high-meat, high-fat meals for 5 days. These volunteers were then switched to a vegan diet for 5 days. Urine (24-h specimens) was collected on days 3, 4, and 5 and on days 8, 9, and 10. All participants followed the identical strictly defined daily diets which were preselected from the following:
Meat diet (days 1-5) Breakfasts: eggs, ham, sausages, waffles, butter, toast, juice, coffee, tea. Lunches: tuna fish, roast beef, pastrami, turkey, chicken, rye bread, lettuce and tomatoes, soda, coffee, tea.
Dinners:
steak, veal cutlet, lamb chop, pork chop, chicken, rice, potatoes, string beans, carrots, broccoli, ice cream, coffee, tea.
Vegan diet (days 6-10) Breakfasts: granola, raisins, corn or bran muffin, preserves, soy milk, juice, herbal teas, coffee substitute. Lunches: peanut butter, tofu, sprouts, sunflower or sesame seeds, soyburgers, canned fruits, packaged tabboule (burgul or cracked wheat), lettuce, fruits, rye or whole wheat bread or pita, herbal teas, coffee substitute. Dinners: packaged or canned dinners (frozen tofu lasagna, vegetable patties, legumes tofu ravioli, macaroni, soyburgers), frozen broccoli, spinach, string beans, cauliflower, rye or whole wheat bread or pita, sunflower or sesame seeds, iceberg lettuce, tomatoes, cucumbers, carrots, sprouts, radishes, chickpeas, dried fruits, jello, granola bars, nondairy ice cream, herbal teas, coffee substitute.
Diet study B 5 smokers and 5 nonsmokers ate a normal western diet for 3 days, providing a 24-h urine sample on the third day. The participants then switched to a macrobiotic diet for 5 days and provided 24-h urine samples on the last 2 days. In addition, 3 smokers and 6 nonsmokers who had customarily adhered to macrobiotic diets for at least 1 year, maintained the identical macrobiotic diet plan and also provided 24-h urine samples on the last 2 days. The dietary regimens were explicitly defined and selected from the following:
Meat diet (days 1-3) Breakfasts: eggs, cereal, bread rolls, milk, juice, coffee, tea. Lunches: tuna fish, roast beef, turkey, bread rolls, juice, coffee, tea. Dinners: steak, lamb chop, chicken, rice, baked potatoes, string beans, broccoli, ice cream, coffee, tea
Macrobiotic diet (prepared fresh daily," days 4-8) Breakfasts: oatmeal, butternut squash, brown rice, cream, baked apple, sourdough bread, barley tea.
151 Lunches and dinners:
Brown rice, beans, millet, barley, seaweed, steamed fresh carrots, broccoli, onions, scallions, squash, turnips, romaine lettuce, spinach, tofu, rice cakes, barley tea.
Urine collection, extraction and concentration All samples were collected in polyethylene bottles and stored in a freezer prior to extraction (within 2 weeks of collection). Thawed urine was then gravity-filtered, pH-adjusted to 7.0, and concentrated on Glass Econo columns (BioRad Laboratories, Richmond, CA) packed with washed Amberlite XAD-2 resins (Aldrich Chemicals), following a modified procedure of Yamasaki and Ames (1977). 3 g of resin was used per 100 ml urine. In diet study A, 250-ml urine aliquots were used. In diet study B, the entire 24-h specimens were concentrated. The columns were washed with distilled water (10 ml water/100 ml urine), and eluted with acetone (10 ml/100 ml urine). To insure that histidine and other highly water-soluble components of urine were completely removed, the acetone eluate was concentrated and partitioned between methylene chloride/brine, the organic layer was washed with brine, dried with Na2SO4, and concentrated to dryness. Distilled water (1000 ml) was also passed through a XAD-2 resin column and simultaneously concentrated, to check for artefactual contamination. Urinalysis Urinary cotinine levels were determined by radioimmunoassay (RIA) using a procedure modified from that of Langone et al. (1973). Urinary creatinine levels were established by using a colorimetric method for the red creatinine-picrate complex (Tietz, 1982). The pH was measured, and all urine samples were routinely tested for nitrite, and for total bacterial andGram-negative counts using Microstix-3 (Ames 3009) strips. Strips of N-Multistix-C (Ames 2862) were also routinely employed to determine ascorbic acid, nitrite, protein, glucose, bilirubin, occult blood, and urobihnogen present. Mutagenicity assays The Ames assay (Ames et al., 1975) was carried out using S. typhimurium TA1538, and required
activation with Aroclor-induced Fischer rat liver $9 mix. TA1538 (along with TA98) is a widely used frameshift mutagen indicator, more responsive to urine mutagens than the base-pair mutagen indicator TA100 (Connor et al., 1983). The $9 mix was prepared following established procedure (Ames et al., 1975). In diet study A, the concentrate of the 250-ml urine sample was dissolved in 500 /~1 dimethyl sulfoxide (DMSO) and tested in duplicate at 25 ml, 12.5 ml, 6.25 ml, and 0 ml (control) urine equivalent per plate. In diet study B, one-fourth of each 24-h urine concentrate was dissolved in 500 #1 DMSO and tested in duplicate at 2.5%, 1.25, 0.62, 0.31, and 0% (control) equivalent of total urine sample per plate. The plates were incubated at 37°C for 72 h and were counted after 48 and 72 h. The data presented are based on the 72-h reading. Controls with and without metabolic activation were run in duplicate using DMSO, picrolonic acid (Pic; 250 #g), 4-aminobiphenyl (4ABP; 50 #g), 2-aminofluorene (2AF; 20 #g), and 1-methyl-3-nitro-l-nitrosoguanidine (MNNG; 5 #g). The histidine revertants for the controls were: without $9 activation: DMSO, 19; Pic, 1150; MNNG, 30, with $9 activation: DMSO, 35; 4ABP, 500; 2AF, 1230; distilled water, 15. A 500-ml distilled water sample that contained 200 #g 2AF was worked-up as above, and dissolved in 500 #1 DMSO. This control sample, tested in duplicate, had values of: 175 (6.25 ml equivalent), 450 (12.5 ml), and 325 (25 ml). Evaluation of data The method of linear regression was used to determine the mutagenicity of urine samples. For diet study A, the mean urinary mutagen levels are expressed as histidine revertants per 20-ml sample. The linear portion of the dose-response curves for the control (0 ml), 6.25 ml, 12.5 ml and 25 ml urine equivalents per average of 2 plates each was used to determine the mutagenicity of a 20-ml sample. After subtracting the control values, the results are divided by the creatinine contents of 1 ml urine to account for the differences in osmolality. These data are illustrated in Fig. 1 For diet study B, the linear portion of the dose-response curves for the total urine sample per average of 2 plates each was used to determine the mutagenicity of 1% of the total 24-h urine
* • a b ¢ a
1500 700 750 1050 1000 700 1200 1250 750 1800
Day3a
2000 1500 950 1100 1000 750 2 800 1050 1200 800
Day5a 1300 1600 1100 1650 1100 800 1550 850 800 1650
Day8b 1500 850 550 1100 1150 600 1 350 600 650 1 250
DaylOb
Smoker. Macrobiotic adherent for at least 1 year. Meat regimen, diet study A. Vegan regimen, diet study A. Meat regimen, diet study B. Macrobiotic regimen, diet study B.
1 2 3 4 5 6* 7* 8* 9* 10 *
Participant Diet study A No. 24-h urine (ml)
1.0 0.82 1.8 0.63 1.9 0.92 1.4 1.2 0.83 0.87
1.1 1.0 1.1 0.86 1.3 0.85 1.8 0.85 0.90 1.0
1000 1 800 1500 1700 . 850 1600 700 . .
1,4 1.2 t.2 0.87 1.6 0.79 1.7 0.95 0.76 1.0
1.4 1.1 1.2 0.69 2.0 1.2 1.8 1.0 0.80 1.5
100 650 1100 1600 . . 600 900 600 . . . . 1
Day8d
Day3C
Day3a DaySa Day8b Dayl0b
Diet study B 24-h urine (ml)
Creatinine (g/24 h)
24-h U R I N E COLLECTION: VOLUME A N D C R E A T I N I N E VALUES
TABLE 1
1,0 1.8 1,7 1.5 . 1.2 1,9 0.90 . . 1.2 1.4 0.62
1.2 1.1 1.1 1,1
Day3C D a y 8 d
Creatinine ( g / 2 4 h)
h)
Creatinine (g/24
-
-
20*.
21 * 22 *.
500 400
1100
1200 1300 700 1600 1000
1000
-
1900 1250
1000 1200 650 11 12 13 14 15 16 17 18 19"
700 -
-
-
2.1 1.3
~
1.1 -
0,81 1,1
1.4
0.97 0,90 1.1 2,0 1.0 1.0 0.88 1.4 1.1
Day3 c Day8 d Day3 c Day8 d
24-h urine (ml)
Participant Diet study B No.
b.a
153 sample. After subtracting the solvent control ( D M S O + $9 mix, 35 and 33 for diet studies A and B, respectively), the data are plotted, expressed as histidine revertants in 1% of 24-h urine (Fig. 2). All the baseline values were reproducible, with an error margin of less than 10%. A sample with twice the histidine revertants of the control was considered mutagenic. Results
The results of the mutagenicity tests of the urine concentrates from smokers and nonsmokers in diet study A are shown in Fig. l a - d and Table 1. The n o n s m o k i n g status of the latter group of
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Fig. 1. Mutagenicity of urine on diet A. The number of histidine revertants of S. typhimurium TA1538 in 20 ml urine are normalized for urinary creatinine contents. The data are taken from the linear portion of the dose-response curve for each urine sample, a and b represent the mean urinary mutagen levels on days 3 and 8, and 5 and .10, respectively, of nonsmokers, c and d show the smokers' mean urinary mutagen levels on days 3 and 8, and 5 and 10, respectively. Data from the following samples are not based on the full dose-response curves; deviation from linearity at the highest dose tested was observ~l (M, meat diet; V, vegan diet). (a) 2M, 3V, 4M; (b) 2M, 3V; (c) 6M, 9M; (d) 10V.
participants was confirmed by the absence of cotinine in their urine. Fig. l a represents the urine mutagenicity of nonsmokers on the third day of the meat and of the vegan components of diet study A (i.e., days 3 and 8). Fig. l b represents the mutagenicity of urine excreted by nonsmokers on the fifth day of the meat and of the vegan components of diet study A (i.e., days 5 and 10). The data for the urine concentrates of the smokers in diet study A are summarized in Fig. l c and d: Fig. l c represents days 3 and 8, Fig. l d represents those corresponding to days 5 and 10. The results are expressed in revertants per 20 ml urine, and normalized for urinary creatinine content. Most of the smokers in this study had mutagenic urine. P r o n o u n c e d mutagenic activity was detected in the urine collected from the nonsmoking participants when they adhered to the vegan diet portion of diet study A. In comparing the two diets, we found that for the nonsmokers, the mean regimen resulted in combined (days 3 and 5) mean his + revertants of 14.7 + 12.9 (standard deviation) and standard error of 4.1. The vegan regimen had a mean urine mutagenicity of 40.3 + 31.4 (standard deviation) and standard error of 4.1 ( p < 0.01). In some cases, levels of mutagenic activity detected in the urine of nonsmokers on the vegan diet were equal to or higher than those detected in the urine collected of smokers on the meat-containing portion in diet study A (see Fig. l a - d ) . * There is a general pattern of increasing mutagenic potency in the urine of cigarette smokers with an increase in the smoking habit, as measured by the urinary cotinine levels; however, it is not a linear dose response. W h e n cotinine levels are taken into account, most smokers on the vegan portion of diet study A exhibited an increase in urine mutagenic activity as c o m p a r e d to those on the meat portion of diet study A. The dietary * The urine from days 4 and 9 was collected in bottles containing sulfamic acid as a preservative. These samples were used for quantitating N-nitrosoproline (NPRO) in the urine. As expected, the vegan diet did not yield any detectable amount of NPRO (less than 0.5 ng/ml urine), when measured as the methyl ester by a gas chromatograph interfaced with a Thermal Energy Analyzer. In the absence of any supplemental administration of proline, there were no significant differences in NPRO contents in the urine of smokers versus that of nonsmokers when both groups were on the meat diet.
154 TABLE 2 URINE MUTAGENICITY OF 'HEAVY' SMOKERS ON DIET STUDY A a Participant No.
Equivalent per plate volume
Cotinine
12.5 ml
25 mi
(ng/ml)
Creatinine (mg/nal urine)
67 51 75 70
79 68 117 101
118 96 213 171
3152 1960 2300 2930
1.52 0.58 0.93 1.37
6.25 ml 7 Day Day Day Day
3Mb 5M 8V 10 V
8 Day Day Day Day
3M 5M 8V 10 V
73 63 116 77
102 88 127 99
111 121 215 170
2460 2118 2736 2668
0.78 0.90 1.41 1.29
9 Day Day Day Day
3M 5M 8V 10 V
83 54 67 66
91 62 77 71
67 82 100 78
3500 1316 2120 1448
1.05 0.63 1.04 1.39
a Average per 2 plates. Values not adjusted for creatinine contents. DMSO ~ $9 control: 35. b M. meat regimen: V, vegan regimen.
100
MEAT DiET • DAY 3 MACROBIOTIC DIET [ ] DAY 8 PRACTICING ( over one year) MACROBIOTIC [ ]
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MEAT DIET • DAY :5 I MACROBIOTIC DIET I'-] DAY 8 JPRACTICING (aver one year) MACROBIOTIC Jng COTININE/rag CREATININE ( )
2
3
4
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12 13 14 15 16 17
factors that are responsible for the smokers' urine mutagenicity cannot be isolated, since cigarette smoking habits are not duplicated on a daily basis. We can, however, compare and contrast the dose-response pattern of the 3 heavy smokers ( v o l u n t e e r s 7, 8, 9 i n Fig. 1), as s h o w n i n T a b l e 2. A l l 3 s m o k e r s h a d c o t i n i n e levels e x c e e d i n g 1 0 0 0 n g / m l u r i n e f o r e a c h d a y o f t h e s t u d y . T h e r e is a h i g h e r level o f m u t a g e n i c r e s p o n s e s w h e n c o m p a r i n g t h e h i g h e s t d o s e t e s t e d , 25 m l e q u i v a l e n t p e r p l a t e , f o r all 3 s m o k e r s o n t h e v e g a n diet. B u t it is difficult to separate the effects of changes in diet from the effects of intake of differing amounts of tobacco smoke.
,,o,~
,~,,0; ,,'°,
eo
(Z4SO)
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I,-~ \
6
7
8
(Z3ZO)
~,,50)
18
19
20 21 22
Fig, 2. Mean urinary mutagen levels on diet B, expressed as histidine revertants of S. typhimurium TA1538 in 1% of 24-h urine. The data are taken from the linear portion of the dose-response curve for each urine sample, a represents the mean urinary mutagen levels of nonsmokers, and b the mean urinary mutagen levels of smokers. The black bars show the mutagenic responses on the 3rd day, when the participants ate a meat diet. The white bars give the mutagenic response, on the 8th day of diet B, when the volunteers were eating macrobiotic meals. Dotted bars show customary macrobiotic levels. Data from the following samples are not based on the full dose-response curves; deviation from linearity at the highest dose tested was observed (M, meat regimen; Ma, macrobiotic regimen). (a) 1M, 4M, 4Ma, 12, 17; (b) 6Ma, 7M, 18Ma.
155 The mean urinary pH ranged from 5.9 to 6.5, with individual variations from 5.4 to 7.2. The urinary pH did not seem to be related to mutagenic activities. The urine of most smokers in diet study B was mutagenic (Fig. 2b). Some of the nonsmokers had mutagenic urine (Fig. 2a) as did some of the participants who had been customarily adhering to a macrobiotic diet for more than 1 year (Fig. 2b). There were no consistent differences in this diet study in the mutagenic activity of urine excreted by those on the meat diet versus those on the macrobiotic diet (p > 0.2). The limited number of individuals in each controlled diet study, however, could preclude the detection of subtle but significant differences in the mutagenicity of such urine concentrates. Discussion
Many studies have reported that nonsmokers' urine may contain mutagens, but no convincing etiological explanations have been offered. While demonstrating that bladder cancer patients had mutagenic urine, one study showed that 32 out of 72 nonsmokers who constituted the control group, also had mutagenic urine (Garner et al., 1982). Other examples of nonsmokers having mutagenic urine are seen in studies by Recio et al. (1984) and by others who were attempting to correlate workrelated risks to mutagenic principles in the urine of employees (Falck et al., 1979, 1980; Gelbart and Sontag, 1980; Dolara et al., 1981; Bos et al., 1982; Pasquini et al., 1982). Our studies indicate that diet alone could account for much of the observed urine mutagenicity among nonsmokers (Fig. la and b). Fig. lc and d show that diet may also be a confounding factor of the observed mutagenic activity in the urine of smokers. Also, the effect of diet may explain the findings that have attempted to relate environmental (passive) smoke exposure to urine mutagenicity, Puzrath et al. (1981), have attributed the presence of mutagens in the urine of a nonsmoking spouse of a cigarette smoker to the related passive smoke exposure. Examination of the pattern of mutagenic activity of urine samples as profiled by HPLC from the smoker and the spouse reveals that the curves had the same shape, albeit at different mutagen levels. Bos et al. (1983)
also attributed to passive smoke exposure findings of urinary mutagenicity obtained with 8 nonsmokers exposed to smoke from 10 cigarette smokers. Warren and Rogers (1982), in a study that measured the urine mutagenicity of college students, found no urinary mutagens in subjects smoking < 10 low-tar and low-nicotine cigarettes per day. In our studies, 'light' smoking paticipants (15 medium-tar cigarettes/day or less) had levels of mutagenic urine that could not be distinguished from those of nonsmoking participants on the same diet (e.g., smokers Nos. 6 and 10 on the meat diet in Fig. lc and d, and smokers Nos. 6, 20, 21, 22 on the macrobiotic diet in Fig. 2b). The efficacy of smoking, i.e. frequency and depth of inhalation, retention of smoke, and individual metabolic variations d o play an important role in regard to uptake of smoke components. It is clear from dosimetry studies, however, that the uptake of environmental smoke compounds by nonsmokers generally corresponds to merely a few percent of the doses measured in physiological fluids of active cigarette smokers (Hoffmann et al., 1984). Thus, a measurable impact of environmental smoke exposure on urinary mutagenicity levels would not be expected. Studies by several investigators (Sugimura et al., 1977; Nagao et al., 1977; Wakabayashi et al., 1978) have shown that pyrolysates of meat produce mutagens. In addition, it has been shown (Baker et al., 1982) that the ingestion of fried pork or fried bacon results in mutagenic urine. Our diet study A included meat that needed broiling or frying, and some soy products that were broiled or fried. Results from our diet study A, however, did not reveal the presence of excessively high levels of urinary mutagens among nonsmoking participants on a diet consisting of both high meat and high fat. The possible appearance of such mutagens in the feces, however, cannot be discounted. Mutagenicity was detected in the urine after the nonsmokers had switched to the vegan diet. It is not as yet known whether the comparatively smaller amounts of fried or broiled of soy products that were ingested would cause the observed urine mutagenicity. The identity and the significance of these urinary mutagens in regard to human health is not known. Our data indicate that the effect of diet on mutagens in human urine is complex and
156 may be difficult to relate to a single environmental or lifestyle factor. Cigarette smoke condensate (CSC) contains mutagenic and carcinogenic components (Sato et al., 1977). The genotoxicity, and the types of genetic damage that CSC can induce, have recently been reviewed (DeMarini, 1983). Our study shows that smokers with increased amounts of urinary cotinine clearly have higher levels of mutagenic urine, although there is no linear correlation. Connor et al. (1983) have reported minor quantities of 2-aminonaphthalene, along with consider. able amounts of its possible metabolite, 2-amino7-naphthol, to be present in the urine of a cigarette smoker. However, the proportion of these compounds reported present in the urine is so high that it is difficult to find a rational explanation for their origin: a commercial, 85-mm nonfilter cigarette contains an average of 1.0 ng of 2-naphthylamine (Patrianakos and Hoffmann, 1979). The agents responsible for the mutagenicity of urine observed in the vegan portion of diet study A are not known. Many processed foods and food additives have been found to contain mutagens (Pariza et al., 1979; Sugimura and Sato, 1983). ;~ recent report shows that many heat-processed. canned foods display mutagenic activity in strai~. TA1538 with $9 activation (Krone and Iwaoka, 1984). Also, plants contain flavonoids, some of which are mutagens (White et al., 1973; MacGregor and Jurd, 1978; Brown and Dietrich, 1979; Ames, 1983; Stavric, 1984). It should be mentioned that quercetin, a flavonoid widely found in ingested foods of plant origin, is mutagenic in the Ames Salmonella microsomal test. However, while quercetin was judged a bladder carcinogen in a study on rats (Pamukcu et al., 1980), in-depth studies failed to verify that quercetin is, in fact, an animal carcinogen in tests on rats, mice and hamsters (Morino et al., 1982; Saito et al., 1980). There are, in addition, other as yet unidentified mutagens in food products derived from plants (Van der Hoeven et al., 1982). Puzrath et al. (1981) have shown by H P L C fractionation of smokers' urine that the mutagens responsible for the observed responses are a complex mixture of relatively non-polar compounds. We are in the process of isolating and identifying the mutagens derived from various diets and from
cigarette smoking. Our studies have now demonstrated that diet has a pronounced effect upon the level of mutagenic activity measured in S. typhimurium present in human urine. Several studies have attempted to relate mutagenic activity in human urine to environmental or occupational factors. Our data indicate that in any such study strict dietary controls are essential.
Acknowledgements We would like to thank Ms. Caryn Axelrad, Mr. Steve Colosimo and Mrs. Katherine Tilton for assistance in the biochemical studies. This study is supported by American Cancer Society Grant SIG-8.
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