Mutagenicity of different fractions of extract of human feces

Mutation Research, 119 (1983) 151-160 Elsevier Biomedical Press

151

Mutagenicity of different fractions of extracts of human feces P. Dion and W . R . Bruce* Ludwig Institute f o r Cancer Research, Toronto Branch, 9 Earl Street, Toronto, Ont. M 4 Y IM4 (Canada) (Accepted 21 October 1982)

Summary An extraction-fractionation scheme for the isolation of non-volatile fecal mutagens is described. Extraction of feces was with acetone, and a 2-step fractionation scheme employing silica gel SepPak cartridges and normal-phase highpressure liquid chromatography was used. Assay of mutagens was with the standard plate mutagenicity assay with Salmonella typhimurium tester strains TA98 and TA100, with and without the Aroclor induced $9 microsomal activation system. Single feces samples from 24 donors from a wide socioeconomic spectrum were tested. It was found that most fecal mutagenicity extracted by aqueous acetone could be attributed to a lipid soluble mutagen active on both TA98 and TA100 that has been previously reported.

In a search for etiological agents responsible for large bowel cancer, many investigators have examined human feces for the presence of putative carcinogens, co-carcinogens and tumor promoters. In particular, several investigators have applies extracts of human feces to mutagenesis assays. It has been shown that organic extracts of samples from approx. 30070 of individuals on Western diets are mutagenic to Salmonella typhimurium tester strains TA100 by plate assay (Bruce et al., 1977, 1979; Wilkins et al., 1980, 1981). Aqueous extracts from other donors were active on the Salmonella strains when the fluctuation test was used (Kuhnlein

Supported in part by the National Cancer Institute of Canada. * To whom requests for reprints should be addressed. Abbreviations: DCM, dicldoromethane; NADP, nicotinamide adenine dinucleotide phosphate; HPLC, high performance liquid chromatography. 0165-7992/83/0000-0000/$03.00 © Elsevier Biomedical Press

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and Kuhnlein, 1980). Extracts have also been found to contain inhibitors of mutagenicity (Bruce et al., 1977; Hayatsu et al., 1981) and co-mutagens (Reddy et al., 1980a). It thus seems likely that feces contain many factors that affect mutagenicity and that these factors may interact in complex ways. Hence, it seems important to attempt to assess the relative mutagenic potency of different fractions of partially purified feces. Here we describe an extraction and fractionation scheme utilizing acetone extraction of wet feces followed by silica gel fractionation. We have used the method with feces from 24 donors on Western diets and have assessed mutagenicity with the Salmonella tester strains TA98 and TA100 with and without microsomal activation in the standard plate assay. We have found that most of the mutagenicity extracted from the frozen feces by this improved extraction method was derived from a single fraction, and could be attributed to one particular mutagen that has been previously reported.

Materials and methods

Donors. Single feces samples were collected from 24 donors aged 19-86. There were 19 females and 5 males chosen from a wide socio-economic spectrum. Most were causasians on Western diets. Feces samples were collected at home in plastic cups, immediately frozen in dry ice, and removed to our laboratory ( - 76°C) until analysis, conditions under which we have found mutagenicity to be preserved (Dion et al., 1982). Extraction and silica SecPak fraction of feces A 25-g aliquot of thawed well-mixed feces was combined with 125 ml of glassdistilled acetone in a 250-ml screw-capped centrifuge tube. The tube was agitated vigorously in an electrically driven mechanical shaker for 15 rain, then centrifuged at 250 g for 5 min. The supernatant was decanted, brought to near dryness with a rotary evaporator at 38°C, and transferred with less than 10 ml of a dichloromethane-methanol 1:1 (v:v) solution into a test-tube. The solution was dried under nitrogen to 1 ml, transferred with washings by Pasteur pipette to a small cellulose column prepared by packing 6 ml granular cellulose into a 12-cc syringe barrel, and then freeze-dried overnight on the column. The contents of each cellulose column was eluted from the cellulose column directly through a silica SepPak cartridge (Waters Associates, Inc., Milford, MA). With this procedure it was found possible to fractionate substantially larger samples of extracts without overloading the silica cartridge. The combined column-cartridge apparatus was eluted with the following solvents in turn: 10 ml of hexane/dichloromethane 1:1 (v:v), 5 ml dichloromethane, 10 ml acetone, 10 ml methanol and 10 ml water. Each of the 5 resulting extract fractions was split, for

153

mutagenicity and for high pressure chromatography (HPLC). Each fraction was then dried under nitrogen.

High-pressure liquid chromatography fractionation Some silica SepPak fractions were further fractionated by high-pressure liquid chromatography. Fractions corresponding to 5 g feces were dissolved in 1 ml acetone, and 2 x 250/zl injected onto a Radial-Pak B column in a RCM-100 Radial Compression Module (Waters Associates). The eluting solvent was dichloromethane-isopropanol 3:1 (v:v) at a flow rate of 2 ml/min. Peaks were detected with a UV absorbance detector set at 340 nm. The eluted sample was collected in 3 adjacent fractions; the first (I) from the solvent front to the beginning of an absorbance peak eluted at 5.5 ml, the second (II) was the absorbance peak 5.5

A

Inject_~

Inject

1

B

I i

I r

I

D

r~

~rs~

Fig. 1. H P L C tracingsa o f UV absorbance (340 nm) for extracts o f typical active (A) and inactive (B) fecal samples. The eluted extract was collected in fractions I, II and III as indicated. T h e m u t a g e n peak is labelled with an M.

154

ml (to 6.5 ml), and the third (III) consisted of the eluant from 6.5 ml to 22 ml (Fig. 1). The height of the absorbance peak corresponding to fraction II was measured. All 3 fractions were dried under nitrogen and dissolved in dimethyl sulfoxide for the mutagenicity assay. The silica SepPak fractions from which these chromatography fractions were derived were also re-assayed, on the same day.

Assay for mutagenicity Doses corresponding to 0.5, 1 and 2 g of feces for each of the silica SepPak fractions, dissolved in dimethyl sulfoxide at a concentration corresponding to 5 g feces/ml, were assayed with one plate per dose by the pour-plate method of Ames (Ames et al., 1975) with tester strains TA98 and TA100, with and without ($9) microsomal activation. For the high-pressure chromatography fraction doses corresponding to 0.3, 0.6 and 1.2 g of feces were plated with TA100 without microsomal activation in most cases. Reproducibility of the extraction and assay was tested with homogenized samples from 3 donors and were found to show a similar degree of variability ( S E - 16o70) to that reported in a previous study (Dion et al., 1982). The $9 mix was prepared from Aroclor 1254 induced Sprague-Dawley rat livers as per the procedure of Ames (Ames et al., 1975). Each ml of $9 mix contained 0.1 ml of liver homogenate ($9), 8 #moles MgCI2, 33 #moles KC1, 5 #moles glucose 6-phosphate, and 4 #moles NADP in a sodium phosphate buffer at pH 7.4, and 0.2 ml of the mix was added to each plate. Negative control plates were employed for strains TA98 and TA100, with the bacteria alone and with dimethyl sulfoxide added. Positive control plates employed N-methyl-N'-nitro-N-nitrosoguanidine (3 #g) for TA100, p-nitro-o-phenylene diamine (2 #g) for TA98, and 2-aminofluorene (2 #g) with and without the $9 mix. Average spontaneous revertants rates were 170, 159, 39 and 29 per plate respectively for TA100 and TA98 with and without $9 activation, and mutagenically active fractions were taken to be those with revertant counts above 150°70 of background. Revertant colonies were counted by hand and the mutagenicity of active fractions calculated from the (least squares linear regression) slope of the dose-response curve and expressed as revertants/g feces. In some cases, extract fractions were toxic to the bacteria as indicated by bacterial lawns or dose-response curves, or the number of colonies were too numerous to count. In such cases the fractions were diluted in dimethyl sulfoxide and re-assayed.

Results

Extraction and silica SepPak fractionation In preliminary studies extracts of feces were prepared with a wide range of solvents (hexane, dichloromethane, chloroform, acetone, methanol, and water) and

155

TABLE

1

MUTAGENICITY

OF SILICA

SepPak FRACTIONS

(A) Assayed with tester strain TA100 Solvent

Hexane/DCM

DCM

-

-

Acetone

MeOH

H20

fraction $9

+

+

-

+

-

+

-

+

Donor 2

- 34

5

6

- 16

26

- 38

8

-37

-62

13

- 46

-

3

- 29

1770

- 22

16

-11

-56

9

-

7

8008

1738

-31

63

46

22100

5530

18

19

-23

-

- 23

- 13

4717

677

14

32

- ll

- 31

l0

- 13

17

20

4

26

8

-12

-

4

21

2

22

- 16

- 36

-

31

-58

1

-27

10

-27

3

-32

60

-

8

4

-56

0

-

9

526 a

423

7

3480

2750

3

576

282

-14

-

5

-16

- 58

- 12

32

-

- 17

14

3

-13 48 -62

21

- 14 34

-39

-34

-19

- 12

- 20

- 30

-20

-22

16 9

- 11

-30

-43

-16

-48

10

-68

-39

-13

23

5,7,9,10,11,12,14,15,17, 18,19,20,23,24

y i e l d e d s i m i l a r s c o r e s t o 1, 3 a n d 4

(B) A s s a y e d w i t h t e s t e r s t r a i n T A 9 8

Solvent

Hexane/DCM

DCM

-

-

Acetone

MeOH

H20

fraction $9

+

+

-

+

-

+

-

+

Donor 2

-

1

-

3

6

-

6

-

2

6

-26

13

-

8

-

16

-

6

-14

21

-12

8

22

-4 2 -2

8

-

l

0

4

-

1

150

88

- 10 2 3

-

-2

-

-3

-11

4

-15

-7

-

-

3

6

3

-

4

4

-7

17

12

-7

2

5

-13

5,7,9,10,11,12,14,15,17, 18,19,20,23,24

-

-

0

y i e l d e d s i m i l a r s c o r e s t o 1, 3 a n d 4

aUnderlined figures are deemed significant.

-

6

6

8

8

-

4 -

28

8

8

4

10

155

5

-

- 13

1

9

2

-

-

5

14 -

-

-5

2

0

0

3

3

l

290

-

-

10

5

500

0

-

9

-

5

-5

1

-

88

1

3

1

1 8

4

-

-16

-4

6

-

3

-

-

2

2 -

5

3

3

-

8

2

23

-

2

-

1

-

2

-20

3

0

4

15

156

combinations of solvents in the presence and absence of added acid and base. Acetone alone yielded a rapid, efficient reproducable extract with all samples tested. Thus the feces samples from the 24 donors were extracted with acetone and the extracts applied to cellulose columns as described in methods. The columns were eluted with progressively more polar solvents through silica gel SepPak cartridges to yield 5 fractions. The mutagenicity of each fraction was assessed by the Salmonella typhimurium tester strains TA98 and TA100 with and without microsomal activation. The results (Table 1) show that all 5 fractions of the fecal extract from 17 of the donors were inactive under the conditions with which we measured mutagenicity. 7 donors were active and in each case the mutagenicity on TA100 without $9 activation resided in the acetone fraction. Mutagenicity was also seen with tester strain TA98 in 4 of these cases; in all cases the presence of $9 reduced the levels of mutagenicity (data not detailed).

High-pressure liquid chromatography Since all mutagenicity was observed in the acetone fractions from the SepPak, this fraction was further fractionated by HPLC. The H PL C was carried out with the knowledge that a frequent fecal mutagen had a characteristic elution on the/~-porasil column and a 340-nm UV absorption (Wilkins et al., 1980). Thus 3 fractions were collected (Fig. 1), the second (II) corresponding to that of the previously described compound, and the fractions were tested on tester strain TA100 without activation. The results (Table 2) show that all HPLC fractions from the 17 inactive acetone TABLE 2 M U T A G E N I C I T Y A N D UV A B S O R B A N C E OF H P L C F R A C T I O N S Mutagenicity TA100 (induced revertants/g feces) Acetone fraction

H P L C fractions I

2 6 8 13 16 21 22

699+48 3635 + 393 368 ± 24 1912±197 10736 ±.977 35280 ± 1359 3032 ± 125

628 ± 36 1382 ± 77 125 ± 8

1 3

18:t=45 -37+60

20+46 -154-38

4

-

6+5

35 + 4 2 20 + 29 218 ± 25 -

59+6

1,3,4,5,7,9,10, 11,12,14,15,17, 18,19,20,23,24, yielded similar scores to 1, 3 and 4 Underlined figures are deemed significant.

II

Ill

982+9 4826 + 413 1832 ± 21 1083 ± 7 5 4835 ± 273 18638 + 1937 1895 ± 63

- 3 0 + 23 14 ± 17 - 49 + 26 20+32 744 ± 92 - 2 ± 22 - 25 ± 13

UV absorbance (units/g) 0.128 0.260 0.128 0.128 0.350 1.227 0.150

-13+46 -144-40

-22+43 1+29

<0.004 <0.004

12+30

-35+129

<0.004

157

SepPak fractions were negative for mutagenicity. The 7 active acetone fractions identified in Table 1 all had activity in HPLC fraction II. No mutagenicity was found in I or III without much higher activity being found in fraction II and the mutagenicity of fraction II and that of the acetone fraction from which it was derived were highly correlated (R = 0.984), although in some cases (donors 13, 16, 21, 33) the further purification led to a decrease in activity while in others (donors, 2, 6, 8) it led to an increase. A comparison of the UV absorption corresponding to fraction II and mutagenicity of this fraction for each of the samples (Fig. 2) shows that these measurements are highly correlated (R = 0.996). This result provides additional evidence that the major source of mutagenicity on both the SepPak fraction and HPLC fraction is attributable to a single mutagenic compound or several closely related compounds. 20,000

i

i

./

~5,ooo ~3 ~3

~

I0,000

5,000

/, i

05 1.0 Abs0rbence 340nm(units) Fig. 2. Relationship between the UV absorbance and the mutagenicactivity of the HPLC fraction II of fecal extracts of samples from 24 donors. The large dot near the origin represents 16 samples which cannot be individually plotted.

158

TABLE 3 M U T A G E N I C I T Y (Induced Reverants/G) OF SAMPLE 21 Tester

$9

Acetone SepPak

strain

H P L C fractions I

TAI00

-

30830±5447

59±55

TAI00

+

14342± 1383

TA98

-

905±265

-

TA98

+

423± 105

- 16+4

0 ± 12

6±6

II

III

19386+ 1971

4±51

3723

6±4

281

8±7

- 10± 15

3±8

Underlined figures deemed significant.

It is unlikely that the H P L C fractions in Table 2 contain mutagens active on TA98 other than the major TA100 mutagen seen in fraction II. In all acetone SepPak fractions, activity on TA98 was never seen without corresponding activity on TA100. Also, it is known that the TA100 mutagen seen in H P L C fraction II is also active on TA98 (Bruce et al., 1977; Wilkins et al., 1980). To illustrate this, we have re-extracted and re-chromatographed sample 21 and have assayed the acetone SepPak and H P L C fractions with both tester strains with and without activation (Table 3). It is clear that TA98 activity is seen only with corresponding TA100 activity. As described before (Ehrich et al., 1981) the $9 system inactivates the mutagen.

Discussion

An early extraction scheme for fecal mutagens employed diethyl ether with aqueous phases incorporating strong acid and base (Bruce et al., 1977). With this method a mutagen was detected in feces from a relatively few donors. Chromatographic separation showed that this mutagen had a characteristic UV absorption spectrum, was active on both Salmonella typhimurium TA100 and TA98, and was partially inactivated by the microsomal activation system (Wilkins et al., 1980; Ehrich et al., 1981). In the present study we have tested a large number o f samples from a wide range o f donors using a more complete acetone extraction and a chromatographic separation and have obtained similar results. We have found only one mutagen fraction in feces, and this fraction has a UV absorption spectrum with a maximum at 340 nm. Because this extraction-fractionation method was designed to be a generalized one, it is a rather cumbersome procedure for extraction o f any specific compound. For the extraction and detection o f the lipid-soluble mutagen alone we have described a much easier and more rapid method (Dion et al., 1981).

159

Other investigators using different extraction methods have obtained different results. Reddy et al. (1980b), in their study of mutagen prevalence in 3 populations, found 2 ether extracts with activity on TA98 without activity on TA100. However, we have found, on several occasions, fecal samples that were active on TA100 when extracted with dichloromethane (or acetone) were not active when extracted with diethyl ether. This was the case even when the ether was distilled before use, and the antioxidant, butylated hydroxytoluene, was added (P. Dion, unpublished data). Kuhnlein and Kuhnlein (1980), in their study of water-soluble mutagens, used the sensitive fluctuation test and found activity amongst there polar compounds. We have found that the mutagen we have detected is extracted very inefficiently by the method they have described (data not presented) and thus it is possible that they were measuring small quantities of this mutagen with the sensitive assay. Alternatively, there may be relatively weak water-soluble mutagens not detectable with the standard plate assay. It is of course possible that the extraction and fractionation scheme here described could miss certain mutagens. First, this method would fail to detect volatile or reactive compounds which could be lost in the evaporation steps or lost in the period between evacuation and extraction. Second, it is possible that very polar compounds, such as those extracted by water, might not be eluted from silica gel by methanol or water. Third, there is the possibility that mutagens infrequently present could be missed in a screen of only 24 individual samples. Statistically there is a small probability (p -- 0.05) that we would miss a strong mutagen present in 12V0 of feces. Fourth, there is the possibility that inhibitors of mutagenicity (Bruce et al., 1977; Hayatsu et al., 1981) have been fractionated together with mutagens which are therefore not detected. We have observed here that the mutagen we have described is detected at different efficiencies depending on purity. And fifth and probably most importantly, the results we have reported are based on the Salmonella tester system. The fractions could be also assessed on other systems and other active compounds might well be found. Nevertheless, it is remarkable that the mutagenicity of feces judged in this way is so dominated by one major compound, or group of compounds. It is not known whether or not this mutagen represents a hazard to man (Venitt, 1982), but is does appear to be a rather unique compound. Further research may tell us the properties and structure of this compound, what effect if any that diet may have on fecal levels of this mutagen, and if exposure of this compound is related to any disease.

Acknowledgements The authors wish to thank the donors who took part in these studies, and Mrs. N. Siddiqui and Mr. D. Block for technical assistance. Dr. Dion was supported by a Medical Research Council of Canada Fellowship which is gratefully acknowledged.

160

References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. Bruce, W.R., A.J. Varghese, R. Furrer and P.C. Land (1977) A mutagen in human feces, in: H.H. Hiatt, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor, New York, pp. 1641-1644. Bruce, W.R., A.J. Varghese, S. Wang and P. Dion (1979) The endogenous production of nitroso compounds in the colon and cancer at that site, in: E.C. Miller et al. (Eds.), Naturally Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis, Jpn. Sci. Soc. Press, Tokyo/Univ. Park Press, Baltimore, pp. 221-228. Dion, P.W., E.B. Bright-See, C.C. Smith and W.R. Bruce (1982) The effect of dietary ascorbic acid and tx-tocopherol on fecal mutagenicity, Mutation Res., 102, 27-37. Ehrich, M., J.E. Aswell and T.D. Wilkins (1981) Alternation of the mutagenicity of human fecal extracts by hepatic microsomal enzymes, J. Toxicol. Environ. Hlth., 7, 107-115. Hayatsu, H., S. Arimoto, K. Togawa and M. Makita (1981) Inhibitory effect of the ether extract of human feces on activities of mutagens: Inhibition by oleic and linoleic acids, Mutation Res., 81, 287-293. Kuhlein, H.V., and U. Kuhnlein (1980) Mutagens in feces from subjects on controlled formula diets, Nutrition and Cancer, 2, 119-124. Reddy, B.S., C. Sharma and E. Wynder (1980a) Fecal factors which modify the formation of fecal comutagens in high-and low-risk population for colon cancer, Cancer Lett., 10, 123-132. Reddy, B.S., C. Sharma, L. Darby, K. Laakso and E.L. Wynder (1980b). Metabolic epidemiology of large bowel cancer, Fecal mutagens in high and low risk population for colon cancer, A preliminary report, Mutation Res., 72, 511-522. Venitt, S. (1982) Mutagens in faeces, Are they relevant to cancer of the large bowel? Mutation Res., 98, 265-286. Wilkins, T.D., M. Lederman and R.L. Van Tassell (1981) Isolation of a mutagen produced in the human colon by bacterial action, in: W.R. Bruce, P. Correa, M. Lipkin, S.R. Tannenbaum and T.D. Wilkins (Eds.), Gastrointestinal Cancer, Endogenous Factors, Banbury Report 7, Cold Spring Harbor, New York, pp. 205-214. Wilkins, T.D., M. Lederman, R.L. Van Tassell, D.G.I. Kingston and J. Menion (1980) Characterization of a mutagenic bacterial product in human feces, Am. J. Clin. Nutr., 33, (Suppl. 11), 2413-2520.