Mutagens in feces from vegetarians and non-vegetarians

Mutation Research, 85 (1981) 1--12 © Elsevier/North-Holland Biomedical Press

MUTAGENS IN FECES FROM VEGETARIANS AND NON-VEGETARIANS

URS KUHNLEIN, DANIELLE BERGSTROM 1 and HARRIET KUHNLEIN 1

Department of Medical Genetics, and 1 Division of Human Nutrition, University of British Columbia, Vancouver, B.C. (Canada)

(Received 20 January 1980) (Revision received 15 June 1980) (Accepted 24 July 1980)

Summary

Mutagens in water extracts from feces of persons in 3 different diet groups were measured with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. The 3 diet groups were ovo- lacto vegetarians (N = 6), strict vegetarians (N = 11) and non-vegetarians (N = 12). All subjects were from the urban area of Vancouver, British Colum- bia, Canada. On TA100 ovo-lacto vegetarians and strict vegetarians had signifi- icantly lower levels of fecal mutagens than non-vegetarians (P ~< 0.025 and P < 0.010, resp.). The same pattern, although less significant, was obtained with TA98. Correlation studies between mutagenicity on TA100 and TA98 and between the pH of the fecal homogenate and mutagenicity indicate the presence of 2 or more major fecal mutagens.

Human feces contain mutagens (Bruce et al., 1977; Ehrich et al., 1979; Varghese et al., 1977). Since the majority of mutagens are also carcinogens (McCann and Ames, 1975; Poirier and Simmon, 1976), it is possible that fecal mutagens are directly involved in the etiology of colon cancer. If this hypothesis is true, one woUld expect that population groups at low risk for cancer of the colon also have low levels of fecal mutagens.

Such a study was carried out by Ehrich et al. (1979) who compared fecal mutagens in 3 South African populations at different levels of risk for colon cancer (Kenda, 1976; Walker and Burkitt, 1976). It was found that 19% of the samples from urban white South Africans, a population at high risk, were mutagenic using the standard Ames test with Salmonella typhimurium TA100 (Ames et al., 1975). In contrast only 2% of the samples from urban blacks and none of the samples from rural blacks, both low-risk populations, were found to contain mutagens. Similar, although less significant, differences were found with Salmonella typhimurium TA98.

Several epidemiological studies indicate that diets low in meat and/or high in fiber are correlated with a low risk for colon cancer (Doll, 1979). We therefore compared mutagens in feces from a group of vegetarians with mutagens in feces from persons on "typical North American" diets containing meat. In our survey we used the fluctuation test of Green and Muriel (1976) for the detection of mutagens. This test is very sensitive and can be used to detect mutagens at concentrations of less than 1/100th of the concentrations required in the standard Ames test (Hollstein and McCann, 1979). It was therefore possible to measure fecal mutagens in diluted aqueous extracts which were prepared by homogenization of the fecal samples with water followed by high speed cen- trifugation.

Materials and Methods

Preparation of ex tracts All subjects were provided with containers for sample collection and styro-

foam boxes with dry ice. The subjects were requested to collect the fecal mat- ter of an entire bowel movement and to place the sample containers into the dry ice boxes immediately after sample collection. In the laboratory, the samples were thawed and homogenized in a blender with an equal weight of water and centrifuged at 4 ° for 50 min at 150 000 g. The supernatant was sterilized by successive passage through filters with 0.4 pm pore size and 0.2 pm pore size (Acrodisc, Gelman Sciences Inc.). The extracts were stored at --15°C.

Media Nutrient broth: 8 g of Difco Bacto Nutrient broth, 5 g of NaC1 and 1 1 of

distilled deionized water. Autoclave. Davis-Mingioli salts: 7 g of K2HPO4, 2 g of KH2PO4, 1 g of (NH4)2SO4, 0.25 g

of sodium citrate, 0.1 g MgSO4 • 7H:O, 5 mg of bromcresol purple and 1 1 of distilled deionized water. Autoclave.

Fluctuation test Stocks of TA100 and TA98 were prepared as follows: An overnight culture

grown in nutr ient broth was diluted 10S-fold into nutr ient broth and again grown overnight with aeration. Dimethylsulfoxide was added to a concentration of 10% (v/v) and 1-ml portions were frozen in liquid nitrogen. The same batch of subcultures was used in all Expts.

For the fluctuation test a 1-ml aliquot of bacteria was thawed, diluted with 10 ml of nutr ient broth and incubated overnight with aeration. For the fluctuation test 120 ml of Davis Mingioli salts were mixed with 2.4 ml of 20% D-glu- cose, 0.48 ml of 0.1 mg/ml biotin, 30 pl of I mg/ml L-histidine, 1 ml of a solu- tion of 10S/ml bacteria and 1 ml or less of fecal extract. From this mixture 2-ml aliquots were dispensed into 50 tubes and incubated for 5 days at 37 ° .

The amount of histidine present allows the his- bacteria to grow to about 10 s per ml. Tubes with revertants, however, reach a several-fold higher concentration of bacteria and can be recognized by their turbidity or by the acid- induced color change of the bromcresol purple contained in the medium. An increase in revertant tubes over those due to spontaneous mutat ions (blank)

indicates the presence of a mutagen. A test was rated positive when the increase in the frequency of revertant tubes was statistically significant (P <(0.01), using the X 2 test as outlined by Green and Muriel (1976).

The original procedure of Green and Muriel (1976) with a t ryptophan- requiring E. coli tester strain prescribes 3 days of incubation at 37 ° before scoring. With the TA100 and TA98 strains it was found that the number of revertant tubes was still increasing between the 3rd and 4th day of incubation. A 5-day incubation period was therefore chosen in our Expts.

Other methods The per cent dry weight was determined by drying an aliquot of fecal homo-

genate at 45°C for 36--48 h and measuring the weight loss. The pH of the homogenates was determined with an Orion pH meter model 601A and an Orion combination electrode No. 91-05.

Subjects were selected to have no history of digestive problems or recent use of internal bactericidal medication. They were young adult males and females ranging in age from 19--44 years. The female/male ratios were: 4/2 ovo-lacto vegetarians, 7/4 strict vegetarians and 6/6 non-vegetarians. Characterization of the subjects ' diets was made by a nutritionist/dietitian using dietary records and a food frequency questionnaire before the samples were taken.

Statistical evaluations were carried out as described by Siegel (1956).

Results

(a) Reproducibility of the fluctuation test The fluctuation test of Green and Muriel (1976) is a sensitive and reprodu-

cible assay for fecal mutagens. Table 1 shows the number of revertant tubes in dependence of the concentration of fecal extract. 2 extracts were tested with TA100 at intervals of 15 days storage a t - -15 ° . Concentrations as low as 2/~l/ml of extract increased the number of revertant tubes in the 50-tube fluctuation test

T A B L E 1

R E P R O D U C I B I L I T Y OF T H E F L U C T U A T I O N T E S T

C o n c e n t r a t i o n N u m b e r o f revcrtant tu b e s o u t of 50 of e x t r a c t (# l /ml ) T A 1 0 0 T A 9 8

Extr ac t 1 Ex t r a c t 2

Det , 1 Det . 2 Det . 1 Det . 2

Ex tra c t 3

Det . 1 Det . 2

0 22 21 23 23 16 13 0.5 32 33 25 22 26 24 1.0 37 36 23 25 23 26 2.1 47 46 49 46 27 31 4.2 46 44 49 49 35 39 8.3 32 35 33 31 45 42

The 2 d e t e r m i n a t i o n s using T A I 0 0 were m a d e 2 we e ks apart and the 2 d e t e r m i n a t i o n s using T A 9 8 were m a d e 23 weeks apar t . Th e e x t r a c t s w e r e m a d e f r o m d i f f erent samples and s tored at - - 1 5 ° C . Extrac t s 1 and 3 w e r e f r o m f eca l sam p le s o f non-vegetar ians . Ex tr ac t 2 wa s f r o m a fecal sample of a s t r ic t vege ta r ian .

from 22 to 47 tubes. This increase was significant at the 0.01 level (×2 test for 2 independent samples). The duplicate determinations with TA100 differed by less than 4 tubes for both extracts, indicating that the assay was reproducible and that the mutagens revealed in the fluctuation test were stable, at least for this period of time.

A similar sensitivity and reproducibility was obtained with TA98 as a tester strain (Table 1).

(b) Comparison of extracts from vegetarians and non-vegetarians Tables 2 and 3 list the number of revertant tubes obtained at different

extract concentrations from 6 ovo-lacto vegetarians (consumers of egg and dairy products, but no other animal food), 11 strict vegetarians (consumers of

T A B L E 2

N U M B E R O F R E V E R T A N T T U B E S O U T O F 50 IN D E P E N D E N C E O F T H E F E C A L E X T R A C T CON-

C E N T R A T I O N F O R T A 1 0 0

D i e t S u b j e c t

g r o u p n u m b e r

E x t x a c t c o n c e n t r a t i o n (tzl/ml)

0 a 0 .5 1.0 2.1 4 .2 8.3 R a n k b

O v o - l a c t o v e g e t a r i a n s

S t r i c t v e g e t a r i a n s

N o n - v e g e t a r i a n s

1 22 21 28 35 39 38 5 2 20 15 22 31 37 31 4

3 22 39 49 49 48 50 29

4 22 21 32 46 45 41 15

5 17 11 18 31 33 23 2

6 16 17 24 34 35 27 6

7 16 17 19 28 39 42 8

8 16 23 25 37 37 24 9

9 24 26 40 47 40 34 10 10 21 24 36 47 40 36 16

11 16 17 28 41 42 31 17 .5 12 17 18 28 33 30 50 13.5

13 17 10 i0 15 25 36 1

14 21 25 23 49 49 33 13.5

15 16 24 24 41 46 49 25

16 24 36 41 46 34 34 I i

17 17 11 26 20 34 29 3

18 22 26 41 45 47 46 24 19 20 23 26 43 44 50 22

20 21 27 36 46 36 33 12

21 16 20 33 43 44 22 21 22 20 24 38 50 49 48 28 23 17 18 22 38 43 43 17 .5

24 24 29 47 49 50 50 26 25 21 32 37 47 46 32 23 26 24 27 31 45 50 47 19 .5 27 20 21 28 39 46 46 19 .5

28 20 26 23 31 45 39 7 29 20 26 41 46 47 48 27

a F o r e a c h s u b j e c t t e s t e d t h e n u m b e r o f r e v e r t a n t t u b e s w h e n n o e x t r a c t w a s a d d e d w a s a l so d e t e r m i n e d .

T h e b l a n k s f r o m t h e s a m e E x p t . w e r e a v e r a g e d . b T h e n u m b e r o f r e v e r t a n t t u b e s w e r e c o r r e c t e d f o r t h e b l a n k s a n d s u m m e d f o r e a c h s u b j e c t . T h e s u m s

w e r e t h e n r a n k e d .

T A B L E 3

N U M B E R OF R E V E R T A N T TUBES IN D E P E N D E N C E OF T H E F E C A L E X T R A C T C O N C E N T R A -

T I O N F O R T A 9 8

Diet Subjec t Ex tr ac t c o n c e n t r a t i o n (# l /ml ) R a n k group n u m b e r

0 0.5 1.0 2.1 4.2 8.3

Ovo- lacto vegetarians

Str ict vegetarians

Non-vegetar ians

1 15 15 18 19 17 33 2 2 15 18 24 30 23 29 12.5 3 15 27 32 32 34 50 29

4 16 21 26 25 26 26 6.5 5 15 26 27 22 28 30 15 6 15 21 32 29 30 32 18.5

7 15 17 20 27 31 28 11 8 17 13 20 30 22 25 1 9 17 18 25 29 27 26 3

10 16 23 23 24 25 30 8 11 16 25 26 24 20 32 10 12 16 28 33 25 31 35 22

13 15 19 28 28 25 33 15 14 17 18 27 31 31 20 5 15 15 22 32 31 30 29 18.5

16 16 21 30 24 25 38 15 17 15 33 25 31 26 36 25

18 15 26 29 26 32 50 27 19 15 27 30 31 29 50 28

20 16 29 22 23 20 35 12.5 21 15 17 23 24 27 25 4 22 16 24 28 21 33 50 25 23 16 24 26 27 38 36 20.5

24 17 16 34 33 32 41 20.5 25 15 23 18 19 30 29 6.5 26 17 21 31 29 33 34 17

27 16 25 29 36 31 33 23 28 16 21 24 22 29 30 9 29 16 27 29 31 39 30 25

The b lank values w e r e averaged and th e ranks d e t e r m i n e d as in Table 2.

only small accidental amounts of animal foods), and 12 non-vegetarians (consumers of the mix o f animal and plant foods typical for North Americans). The raw data is given to permit different types of statistical analysis.

The highest mutagenic activity with TA100 was observed at a concentration of 4.2 /~l/ml of extract where 27 of the 29 subjects were positive (X 2 test, P < 0.01). With TA98 the highest mutagenic activity was at 8.3 #l /ml where 24 subjects were positive.

For an initial analysis the number of revertant tubes were averaged at each extract concentration. Fig. 1 indicates that, when assayed with TA100, the

non-vegetarians had a higher average number if revertants tubes than both the strict vegetarians and the ovo-lacto vegetarians. No differences were observed between strict vegetarians and ovo-lacto vegetarians. With TA98, the average number o f revertants tubes were similar for all 3 groups at low concentration of

(/) 50

~ 4o

a : 30

©

c~

z

N 10 <

u.l

o

~ TA 100

c Ovo-lacto vegetarians Strict vegetarians

o Non-vegetarians

co 5o

~- ~ 98 p- z 40

o

w 3 0 e

© o /

rn

o Ovo-lacto vegetarians z

10 ~ Strict vegetarians LIJ o Non-vegetarians

2 4 6 8 2 4 6 8

EXTRACT CONCENTRATION ( p l / m l ) EXTRACT CONCENTRATION ( p l / m l )

Fig. 1. Average n u m b e r o f revertant tubes o u t o f 5 0 in d e p e n d e n c e o f the ex tra c t c o n c e n t r a t i o n using Salmonel la t y p h i m u r i u m T A 1 0 0 .

Fig. 2. Average n u m b e r o f revertant tubes ou t o f 5 0 in d e p e n d e n c e o f the ex tra c t c o n c e n t r a t i o n using Salmonel la t y p h i m u r i u m T A 9 8 .

extract (Fig. 2). At high concentrations of extract, however, the non-vegetarians were again higher than the strict vegetarians or the ovo-lacto vegetarians, whereas the ovo-lacto and the strict vegetarians were identical.

The shape of the dose--response curve was similar for the different diet groups and also for most of the individual extracts. With TA100, the number of revertant tubes increased with the amount of extract, levelled off at 2 #l /ml of extract and decreased at higher concentrations (Fig. 1). With TA98 the number of revertant tubes increased sharply at low concentrations, levelled off between 1 and 2 pl/ml of extract and then again increased at higher concentrations (Fig. 2). Because of the complex dose--response curves it is not possible to define a mutation rate.

To analyze the differences between vegetarians and non-vegetarians for significance, the number of revertant tubes at each extract concentration was corrected for the blank (number of revertant tubes without extract) and summed for every subject. The ranks of these sums were then analyzed with the Mann-Whitney U test (Siegel, 1956). Since we specifically asked if vegetarians had lower levels of fecal mutagens than non-vegetarians, the one-tailed test was used. The results are summarized in Table 4. Vegetarians ranked significantly lower than non-vegetarians (P < 0.05) with both TA100 and TA98. This was also the case when just the strict vegetarians were compared with the non- vegetarians. When the ovo-lacto vegetarians were compared with the non-vegetarians, a significant difference was only observed with TA100.

As shown above, measurements of fecal mutagens with the fluctuation test were very reproducible and the activities were stable. However, it is possible that experimental errors, like an error in histidine concentration, could influence the result o f our analysis. Using the values listed in Tables 2 and 3 it was found that with both TA100 and TA98, low ranks of mutagenicity did not cor- relate significantly with low blank values (TA100: Spearman rank correlation

T A B L E 4

C O M P A R I S O N OF D I E T G R O U P S W I T H T H E M A N N W H I T N E Y U T E S T ( O N E - T A I L E D )

Die t group N u m b e r Average rank in the S igni f icance o f Mann-Whitney U tes t subjects T A 1 0 0 T A 9 8

T A I O 0 T A 9 8

Vegetar ians vs. 17 11.1 12.8 <:0.010 d 0 . 0 5 0 non-vegetar ians 12 20.5 18.2

Str ic t vege ta r ians vs. 11 8.0 9.5 <:0.010 ~<0.050 non-vegetar ians 12 15.7 14.3

Ovo- lac to vege tar ians vs. 6 5.8 7.8 ~ 0 . 0 2 5 n.s. a non-vegetar ians 12 11.3 10.3

Ovo- lac to vege ta r ians vs. 6 7.8 9.6 n.s. n.s. strict vegetarians 11 9.6 8.7

The ranks o f Tables 2 and 3 were ana lyzed wi th the Mann-Whitney U tes t as descr ibed by Siegel (1956) . The s igni f icance is the probabi l i ty that th e observed d i f f e r e nces are due to cha nce . a N o t s ignif icant (P ~ 0 .20) .

coefficient rs = 0.236, P > 0.20; TA98: rs = 0.230, P > 0.20). Since the time between sample collection and assay varied from subject to subject, the correlation between storage time of the sample and mutagenicity on TA100 and TA98 was also determined. The Spearman rank correlation coefficients were --0.295 for TA100 and 0.255 for TA98, both values not being significant (P > 0.20).

Since differences in mutagenicity between vegetarians and non-vegetarians were found for TA100 and TA98, it was of interest to establish if high levels of mutagenicity with TAI00 correlated with high levels of mutagenicity with TA98 (Table 5). A statistically significant correlation (P ~< 0.02) was found for all subjects. When separate diet groups were analyzed, the Spearman coefficient indicated a similar correlation among the non-vegetarians; however, the small number of subjects made the correlation less significant (P < 0.10).

(c) Correlation of mutagenicity with other parameters of the fecal samples In addition to mutagenicty, the wet weight of the samples, the per cent dry

weight of the samples and the pH of the homogenates were determined. The average wet weight for all subjects was 122.5 + 71.4 g, the average per cent dry weight was 25.2 + 6.3%, and the average pH was 6.4 + 0.37. The vegetarian and

T A B L E 5

S P E A R M A N R A N K C O R R E L A T I O N BETW EEN M U T A G E N I C I T Y W I T H T A 1 0 0 A N D T A 9 S

Die t group Spearman rank S igni f icance corre la t ion c o e f f i c i e n t

All subjects 0 . 436 <:0.02 Vegetar ians - - 0 . 0 2 5 n.s. a Non-vegetar ians 0 . 496 <:0.10

The corre lat ion c o e f f i c i e n t s w e r e d e t e r m i n e d f r o m the ranks l i s ted in Tables 2 and 3 as descr ibed by Siegel (1956) . a N o t s ignif icant (P ~> 0 .20) .

8

T A B L E 6

S P E A R M A N R A N K C O R R E L A T I O N B E T W E E N M U T A G E N I C I T Y AND p H OF T H E F E C A L HOMO- G E N A T E S

Diet g roup pH of h o m o g e n a t e

R a n k cor re la t ion coef f ic ien t Signif icance

T A 1 0 0 T A 9 8 T A 1 0 0 T A 9 8

All subjects ---0.425 - -0 .387 ~ 0 . 0 2 < 0 . 0 5 Vege ta r ians - 0 . 6 4 1 - 0 . 1 3 2 ~ 0 . 0 1 n.s. Non-vege ta r i ans - - 0 . 3 9 6 - 0 . 6 8 6 n.s. <:0.02

non-vegetarian samples did not vary significantly. When the Spearman rank correlation coefficients between these parameters

and the ranks in the fluctuation test (Table 2 and 3) were calculated, no significant correlation was found between mutagenicity on TA100 or TA98 with the wet weight or the per cent dry weight. However, there was a negative correlation between the pH of the homogenates and mutagenicty which was significant for TA100 and TA98 when all subjects were analyzed (Table 6). Sur- prisingly, when the vegetarians alone were analyzed, the correlation was only significant with TA100, whereas for the non-vegetarians it was only significant with TA98.

The average number of bowel movements per day was 0.93 + 0.48 for non- vegetarians and 1.62 + 0.72 for vegetarians. The Mann-Whitney U test indicated that the difference in the frequency of bowel movements was significant (P < 0.02, two-tailed test). However, Spearman rank correlation analysis with all subjects or with the diet subgroups did not indicate a significant correlation between the frequency of bowel movements and mutagenicity on TA100 or TA98. When all subjects were included, the correlation coefficients were r s =- -0 .305 , P > 0.10 (two-tailed test) and rs =- -0 .008 , P > 0.20 (two-tailed test) for TA100 and TA98, resp.

Discuss ion

Using the fluctuation test of Green and Muriel (1976) we detected fecal mutagens at concentrations as low as 2 pl of extract per ml of culture suspension, a concentrat ion which is equivalent to 1 mg of feces per ml. At this concentration 24 of the 29 subjects tested were positive (P < 0.01) with TA100, an indi- cator for base-substitution mutagens. Mutagenic activity with TA98, an indi- cator for frame-shift mutagens was lower. Only 12 subjects were positive at 2 #l/ml of extract.

The method developed by Bruce et al. (1977) and used in the survey of low- and high-risk populations in South Africa by Ehrich et al. (1979) is less sensitive. In their method ether extracts from freeze-dried fecal samples are assayed with the standard Salmonella/Ames test. With extract from 200 mg freeze-dried sample (600 mg wet weight) per plate and using TA100, Ehrich et al. (1979) found 20% of the samples from urban white population positive and Bruce et

al. (1977) found 15% of all samples from subjects on average North American diet positive. Similar to our study, Ehrich et al. (1979) found less activity for TA98 than for TA100.

The low percentage of positive samples in the Salmonella/Ames test might be due to the presence of inhibitors of mutagenesis present in the fecal extract. Bruce et al. (1977) has shown that such an inhibitor is present in the ether extract, but that it can be separated from the main mutagen by additional extraction procedures. We have tried to detect fecal mutagens in aqueous extracts with the Salmonella/Ames test using liquid suspension treatment but could not detect any activity. Since concentrations of extract are used in the fluctuation test which are roughly 100-fold lower than those used by Bruce et al. (1977), one might expect that toxic or inhibitory substances are less likely to interfere with the assay. However, with TA100 there is a decrease in the number of revertant tubes at high concentration of extract. This was not asso- ciated with a decrease of the concentration of bacteria in tubes not containing revertants and is therefore not a general " tox ic" effect. It is possible that with TA100, high concentrations of extract have an inhibitory effect on metabolic pathways involved in mutagenesis. An apparent inhibitory effect due to selec- tive killing of the fast growing revertant cells is not likely, since a decrease of the number of revertant tubes at high extract concentration was infrequently observed with TA98. An influence of the fecal extract on bacterial metabolism is also indicated by the frequent occurrence of filamentous bacteria in tubes containing high levels of extract.

In this s tudy it was shown that a group of 17 Vancouver vegetarians had significantly lower fecal mutagenicity levels than a group of 12 Vancouver non- vegetarians. Epidemiological studies associate vegetarian or predominantly vegetarian diets with a low risk of cancer of the colon (Doll, 1979; Haenszel et al., 1973; Phillips, 1975). Our results therefore support the hypothesis that fecal mutagens are involved in the etiology of cancer of the colon, as do the studies on South African populations reported by Ehrich et al. (1979). How- ever, more high- and low-risk population groups have to be analyzed in order to esablish the role of fecal mutagens in the etiology of colon cancer. Particularly interesting would be the analysis of fecal mutagens of the Mormon population in Utah, which, despite a high meat consumption, has a low frequency of colon cancer (Lyon et al., 1976).

The correlation studies between mutagenicity levels on TA100 and TA98 and between the pH of the fecal samples and the mutagenicity indicate the presence of several fecal mutagens. Thus, among the vegetarians, fecal mutagenicity on TA98 and TA100 was not correlated, indicating that at least 2 separate compounds were present. This is supported by the observation that the mutagenicity on only 1 of the 2 tester strains was correlated with the pH of the fecal homogenate. The non-vegetarian samples, on the other hand, had mutagenic activity for TA100 and TA9S which correlated. However, only mutagenicity for TA98 was correlated with the pH, indicating again the presence of at least 2 mutagens.

The fluctuation of the mutagenicity among individuals in the 2 diet groups are considerable. Thus, the vegetarian group contains both the subject with the highest and the subject with the lowest level of fecal mutagens. The variations

10

clearly indicate that diet items or diet habits other than vegetarianism influence the level of fecal mutagens. Our studies included detailed diet records taken prior to sample collection and a questionnaire on dietary habits. These are now being assessed for relevance to the levels of fecal mutagens reported here.

Acknowledgements

Supported by the National Cancer Institute of Canada. U. Kuhnlein is a Re- search Scholar of the N.C.I. We thank Dr. M.P. Rosin, N. Aiama and P. Bell for their help, as well as Dr. F.P. Glick from the Dept. of Health Care and Epi- demiology (University of British Columbia) for his advice on statistical methods.

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L y o n , J .L . , M.R. K l a u b e r , T.W. G a r d n e r a n d C .R . S m a r t ( 1 9 7 6 ) C a n c e r i n c i d e n c e in M o r m o n s a n d n o n - M o r m o n s in U t a h , 1 9 6 6 - - 1 9 7 0 , N. Engl . J . Med . , 2 9 4 , 1 2 9 - - 1 3 3 .

M c C a n n , J . , a n d B.N. A m e s ( 1 9 7 5 ) D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t he S a l m o n e l l a / m i c r o s o m e tes t : A s s a y o f 3 0 0 c h e m i c a l s , P roc . Nat l . A c a d . Sci. (U .S .A. ) , 72, 5 1 3 5 - - 5 1 3 9 .

Phi l l ips , R .L . ( 1 9 7 5 ) Ro le o f l ife s ty l e a n d d i e t a r y h a b i t s in r i sk o f c a n c e r a m o n g S e v e n t h D a y A d v e n t i s t s , C a n c e r Res. , 35 , 3 5 1 5 - - 3 5 2 2 .

Po i r i e r , L .A . , a n d V . F . S i m m o n ( 1 9 7 6 ) M u t a g e n i c ~ c a r c i n o g e n i c r e l a t i o n s h i p s a n d the ro le of m u t a g e n i c s c r e e n i n g t e s t s f o r c a r c i n o g e n i c i t y , Clin. P h a r m a c o l . T o x i c o l . , 9 , 7 6 1 - - 7 7 1 .

Siegel , S. ( 1 9 5 6 ) N o n - p a r a m e t r i c s t a t i s t i c s f o r t he b e h a v i o r a l sc iences , M c G r a w - H i l l B o o k C o m p . , Inc. L ib r . o f C o n g r . , ca t . c a r d No . 5 6 - - 8 1 8 5 .

Va rghese , A .J . , P. L a n d , R. F u r r e r a n d W.R. Bruce ( 1 9 7 7 ) N - N i t r o s o c o m p o u n d s in t he h u m a n b o d y , P roc . A m . Ass. C a n c e r Res . , 18 , 80 .

Walke r , A .R .P . , a n d D .R . B u r k i t t ( 1 9 7 6 ) C o l o n c a n c e r : h y p o t h e s e s of c a u s a t i o n , d i e t a r y p r o p h y l a x i s a n d f u t u r e r e sea rch , A m . J . Dig. Dis. , 2 1 , 9 1 2 - - 9 1 7 .

APPENDIX: INFLUENCE OF HISTIDINE ON THE FLUCTUATION TEST

The question arises whether fecal extracts contain enough histidine (or histidine precursors) to account for some of the apparent mutagenicity of the extracts and if variations of such compounds might explain the difference in mutagenicity between vegetarians and non-vegetarians.

Histidine and histidine precursors in fecal samples would be expected to originate from protein contained in the diet, although processes of digestion, absorption, endogenous secretion and bacterial metabolism complicate the

II

T A B L E 7

C O N C E N T R A T I O N OF C O L O N Y - F O R M I N G B A C T E R I A (m1-1 × 10 -7 ) IN TUBES C O N T A I N I N G NO R E V E R T A N T S IN D E P E N D E N C E OF H I S T I D I N E A N D E X T R A C T C O N C E N T R A T I O N

His t id ine Ex t r a c t T u b e concentrat ion concentrat ion n u m b e r (/.tg/ml) (#al/ml)

T i m e of i n c u b a t i o n (days)

1 2 3

0 .167 0 1 3.2 7.0 ~ 0 . 2 2 2.7 6.4 < 0 . 2

4.2 1 1.8 3.2 ~ 0 . 2 2 1.7 5.8 ~ 0 . 2

0 . 2 5 0 a 0 1 3.6 12 .0 ~ 0 . 2 2 2.6 _ b 0.5

4.2 1 1.4 7.0 0.4 2 1.7 - - - -

0 . 333 0 1 5.1 15 .2 ~ 0 . 2 2 4.6 14 .4 6.7

4.2 1 2.3 7.5 5.1 2 2.4 7.4 6.4

Al iquo t s f r o m tubes con ta in ing no r eve r t an t s were d i lu ted wi th n u t r i e n t b ro th , spread on nutr ient agar plates and incubated overnight at 37°C. The bac ter ia l t i te r a t the beg inn ing of the f luctuat ion test was 1.4 × 103 /ml . The extract was f r o m a fecal sample of a non-vege ta r ian .

a S t anda rd c o n c e n t r a t i o n of hist idine. b T u b e con t a i ned r eve r t an t s 24 h a f t e r t i t r a t ion .

issue. In any event, comparison of the dally intake of total protein computed from 5-day diet records indicates that there is a relatively small variation among the diet groups (ovo-lacto vegetarians, 71 + 14 g; strict vegetarians, 66 + 23 g; non-vegetarians, 90 + 21 g); hence it is unlikely that substantially differing levels of free fecal histidine result from those levels of dietary protein. Further- more, Spearman rank correlation analysis revealed that mutagenicity of the extract and protein intake were not related (unpublished results). Estimation of dietary histidine was not possible because analytical values for this amino acid in many foods are no t available.

In order to directly assess the influence of fecal histidine, we carried out a fluctuation test with TA100 at different histidine concentrations. The concentration of bacteria in tubes containing no revertants was then determined in dependence of the time of incubation {Table 7). The bacterial titer increased with the time of incubation up to 2 days and then decreased upon further incubation. At all incubation times and histidine concentrations the bacterial titer in tubes containing extract was lower than in tubes wi thout extract. Thus the higher number of revertant tubes in the presence of extracts cannot be at tr ibuted to a stimulation of bacterial growth by histidine present in the fecal extract.

The concentration of histidine in one of the fecal extracts was estimated from the dependence of the mutat ion rate on the amount of histidine added to the fluctuation test (Fig. 3). The mutat ion rate (average number of mutat ion events per tube) was calculated by assuming a Poisson distribution according to the equation # = --ln P0 (P0 = fraction of tubes without revertants). A linear dose--response curve was obtained with and without added extract. The

1 2

° / }.-

a: 2

¢D

o_1

0.1 02 0.3 HISTIDINE CONCENTRATION (pg/ml)

F i g . 3. A v e r a g e f r e q u e n c y o f m u t a t i o n e v e n t s p e r t u b e in d e p e n d e n c e o f t h e h i s t i d i n e a n d e x t r a c t c o n c e n -

t r a t i o n . T h e f r e q u e n c i e s o f m u t a t i o n e v e n t s p e r t u b e w e r e c a l c u l a t e d f r o m t h e n u m b e r o f r e v e r t a n t t u b e s b y a s s u m i n g a P o i s s o n d i s t r i b u t i o n . I n c u b a t i o n w a s 5 d a y s a t 3 ' I ° . T h e e x t r a c t w a s t h e s a m e as in T a b l e 1. o, n o e x t r a c t ; ~, e x t r a c t ( 1 .0 t t l /ml) ; a , e x t r a c t (4 .2 t t l /ml) .

straight line obtained in the absence of extract intercepts the abscissa at 0.080 pg/ml (0.075 pg/ml in a duplicate Expt.) This concentration might represent the minimal amount of histidine required to induce cell division of the station- ary cells used for inoculation in the fluctuation test. The straight lines obtained in the presence of extract intercept the abscissa at an earlier histidine concentration, indicating that the cells start to divide at a lower concentration of added histidine. It is reasonable to assume that this lower threshold is due to the histidine contained in the extract. Using the difference of the intercepts one can estimate that the particular fecal extract examined contained 8.8 pg/ml of histidine or histidine precursor.

Using the dose dependence of Fig. 3, one can estimate that removal of all the histidine present in the fecal sample would, at an extract concentration of 4.2 pg/ml, lower the mutat ion frequency from 2.02 to 1.65 mutat ion events per tube. This difference corresponds to a decrease from 43.4 to 40.4 revertant tubes and is small when compared with the range of revertant tubes obtained at the same extract concentration with non-vegetarians (36--50) and vegetarians (17--49).

The fact that the differences in protein consumption among the diet groups is small, that there is no correlation between protein consumption and mutagenicity, and that the free histidine concentration in fecal extracts is relatively small indicate that the increased mutagenicity observed in fecal samples of non- vegetarians is not due to an increased level of histidine or histidine precursors.

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Mutagens in feces from vegetarians and non-vegetarians