Mutation Research, 245 (1990) 111-117
111
Elsevier MUTLET 0406
Effects of bile salts on the adsorption of a hydrophobic mutagen to dietary fiber Lynnette R. Ferguson 1, Philip J. Harris 2, H. John Hollands 2 and Anthony M. Roberton a 1Cancer Research Laboratory, and Departments of 2Botany and 3Biochemistry, The University of .4uckland, Auckland (New Zealand)
(Received 16 March 1990) (Revision received18 May 1990) (Accepted29 May 1990)
Keywords: Adsorption;~t-Cellulose;Bilesalts; Dietary fibre; 1,8-Dinitropyrene
Summary The effect o f the bile salts, sodium cholate, deoxycholate, glycocholate and taurocholate, on the solubility in aqueous solution o f the hydrophobic, environmental mutagen, 1,8-dinitropyrene (DNP), was examined. In the absence o f bile salts, the DNP appeared to precipitate out o f solution, whereas bile salts at a concentration o f _ 4 m M maintained the DNP in solution. In the presence o f the model dietary fiber, a-cellulose, the D N P adsorbed to this preferentially. Bile salts reduced this adsorption at low o~-cellulose levels, but had little effect at high o~-cellulose levels. The implication of these results is that bile salts have solubilising properties that could affect the distribution o f hydrophobic molecules, including mutagens, in the digestive tract.
Epidemiological studies indicate that certain types of dietary fiber may protect against the development of colon cancer (Burkitt, 1978; WiIlett, 1989). Although the factors involved are complex and not yet understood, one suggested mechanism to explain these protective properties is that dietary fiber may adsorb certain mutagens or cancer promoters in the digestive tract and these are carried out o f the body adsorbed onto undigested dietary fiber. Thus the effective concentrations of these substances available to initiate or Correspondence: Dr. L.R. Ferguson, Cancer Research Laboratory, The University of Auckland, Private Bag, Auckland (New Zealand).
promote cancerous changes in the gut mucosal cells are lowered. Certain mutagens and cancer promoters have been shown to adsorb onto a range o f dietary fibers in vitro (e.g. Barnes et al., 1983; Kada et al., 1984; Smith-Barbaro et al., 1981; Takeuchi et al., 1988; Roberton et al., 1990). We have previously studied the adsorption o f 1,8-dinitropyrene (DNP) to o~-cellulose as a model in vitro system for examining the adsorption of hydrophobic, environmental mutagens to dietary fiber (Roberton et al., 1990). We found that the D N P partitioned between the aqueous medium, the or-cellulose and the glass walls o f the tube. We also found that the solvent, dimethyl sulfoxide (DMSO), at final concentrations o f 25O7o (v/v) or
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
112
greater, maintained the DNP in solution and prevented it adsorbing to the a-cellulose. In the digestive tract, bile salts play a role in maintaining hydrophobic molecules, such as lipids, in solution. Thus, they could affect the partitioning o f hydrophobic mutagens, such as DNP, between the lumen, dietary fiber and surface of the tract. In this paper we report a study on the effect of bile salts on the partitioning of DNP in our model in vitro system (Roberton et al., 1990). Materials and methods
Chemicals. Cholic acid, deoxycholic acid, glycocholic acid and sodium taurocholate of > 98070 purity were obtained from Sigma Chemical Company, St. Louis, MO. DNP and acelluose (cat. No. C8022 from Canadian bleached hardwood pulp) were obtained from the same source. A solution of DNP (1 mg m l - 1) in DMSO was stored at - 80°C and diluted in DMSO before use. The concentration o f DMSO in the incubation medium was 2070 (v/v). Bacterial mutagenicity assay. The Salmonella typhimurium plate-incorporation assay was done using strain TA98 as described by Maron and Ames (1983) with each experimental point performed in triplicate on at least two separate occasions. In order to minimize day-to-day variability in tester strain sensitivity to mutagen, which is largely due to the exact growth phase of the culture, we grew cells for quantitative work as follows. A 1 ml vial (2 × 108 cells) was removed from storage at -80°C and inoculated into bacterial complete medium (20 ml). It was grown until a 1:10 dilution in fresh medium gave an absorbance of 0.11-0.12 at 654 nm, which took approximately 3 h. DNP levels in experiments were measured by assaying mutagenicity, and in the interpretation of data we have assumed a quantitative relationship between D N P concentration and revertant colonies. Positive controls containing 4-nitro-o-phenylene diamine (100 ng per plate) were included in all experiments.
Incubation o f DNP with bile salts. To prepare a solution of bile salts, bile acids were first solubilised with sodium hydroxide, and the pH adjusted back to 6.5 with HCI. A solution of phosphatebuffered saline (PBS) was added to give final concentrations of 20 mM sodium phosphate buffer (pH 6.5) and 130 mM NaC1. A series of solutions of the bile salts (0-8 mM) in PBS was prepared in acid-washed, conical glass centrifuge tubes (capacity 12 ml). The bile salt concentrations in the incubations were chosen to encompass their critical micellar concentrations (O'Connor and Wallace, 1985). Each bile salt dilution (1.96 ml) was prewarmed to 37°C for 30 min and at zero time DNP (200 ng) in DMSO (40 #l) was added and incubated with shaking (300 rev/min, 37°C). After 1 h, aliquots (3 x 50 /~l) were removed for mutagenicity assay. DNP associated with the tube walls was measured as described below. Incubation of DNP and bile salts with acellulose. At 30 min before zero time, the solution of bile salt in PBS (1.96 ml) was added to acellulose and pre-warmed to 37°C. At zero time D N P (200 ng) in DMSO (40 ~l) was added and incubated as above. After 55 min the tube was centrifuged (2500 x g, 2.5 min) and aliquots (3 x 50 ~l) of the supernatant were removed for the mutagenicity assay at 1 h. The DNP associated with the a-cellulose and the tube walls was recovered and measured as described below. Control incubations in which (a) a-cellulose was omitted or (b) bile salts were omitted, were carried out simultaneously as appropriate. Recovery of DNP associated with tube walls and of-cellulose. In those experiments in which no acellulose was added, the solution after incubation and sampling was vortex mixed (10 sec), discarded, and the tubes shaken to remove any remaining liquid. DMSO (2 ml) was then added, the contents vortex mixed (2 x 10 sec), and aliquots (3 x 50/tl) removed for the mutagenicity assay. This procedure measures the DNP associated with the tube walls. In experiments in which a-cellulose was added,
113
the incubation, centrifuging and sampling of the supernatant were performed as above. The pellet was resuspended by vortex mixing (1 x 10 sec) and the contents then tipped into a second tube. The first tube was rinsed with PBS (2 ml) and the contents added to the second tube. DMSO was added to the first tube as described above to measure the DNP associated with the tube walls. The second tube was centrifuged (2500xg, 5 min) and the supernatant discarded. DMSO (2 ml) was added to the pellet, the contents vortex mixed (2 x 10 sec), and the tube recentrifuged (2500 x g, 5 min). Aliquots (3 x 50 t~l) were removed for the mutagenicity assay. This procedure measures the DNP associated with the s-cellulose.
Data comparisons. The experiments were designed to examine the effect of a dose range of a bile salt at constant u-cellulose concentration, or of a dose range of a-cellulose at constant bile salt (glycocholate) concentration. Data from several experiments have been combined after normalising the data to compensate for minor variations in tester strain sensitivity. This was done by diluting the stock solution of DNP (40 #1 containing 200 ng)
1O0
_ o ~
in DMSO (1.96 ml) and assaying its mutagenicity. The DNP content of each experimental sample was then expressed as a percentage of the DNP added initially. Results
The effect o f bile salts on the mutagenic activity o f DNP. In preliminary experiments, it was found that the mutagenic activity of DNP was not altered when it was added with bile salts in a standard Ames mutagenicity test. The effect o f bile salts on the concentration o f D N P in solution. No more than 25% of the DNP remained in the PBS after 1 h incubation in the absence of bile salts (Fig. 1A). However, after incubation for 1 h in the presence of bile salts, the proportion of DNP maintained in solution increased with increasing bile salt concentration (Fig. 1A) and the DNP associated with the tube walls decreased (Fig. 1B). At a concentration for each bile salt of 4 mM or above, more than 75°7o of the DNP remained in solution. In general, all the bile salts used had a similar effect at the same concen-
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Fig. IA. The effect of increasing bile salt concentration on the mutagenic activity of DNP in PBS. DNP (200 ng/2 ml) was incubated in PBS containing various concentrations of bile salts: sodium cholate (O), deoxycholate (~7), glycocholate ([]), and taurocholate (A). Supernatant samples (50 p.1) were taken after 1 h for mutagenicity assays. Values represent total numbers of revertant colonies (mimis those for negative controls) expressed as a percentage of the numbers seen from a comparable sample of DNP diluted into DMSO and plated immediately. Fig. lB. The DNP adsorbed to walls of the glass tubes, from the experiment reported in Fig. 1A, was assayed as mutagenic activity in the DMSO washings.
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Fig. 2A. The effect of increasing bile salt concentration on the mutagenic activity of DNP in PBS, in the presence of a-cellulose (10 mg/2 ml). The experiment was similar to that in Fig. IA except that t~-cellulose was present in all tubes. Symbols as in Fig. 1. Fig. 2B. The D N P adsorbed to the walls of the glass tubes, from the experiment reported in Fig. 2A, was assayed as mutagenic activity in DMSO washings. Fig. 2C. The D N P adsorbed to ~-cellulose from the experiment reported in Fig. 2A was assayed as mutagenic activity in the DMSO washings.
tration. Greater than 95% of the added DNP was accounted for when the amount of DNP recovered from the tube wall was added to that measured in the supernatant.
The effect o f a-cellulose and bile salts on the D N P content o f the supernatant. The previous experiment was repeated in the presence of c~cellulose (10 mg). The presence of ct-cdlulose decreased the amount of DNP found in solution at all bile salt concentrations (cf. Fig. 1A and Fig. 2A). Only small amounts of DNP were now
associated with the tube walls (Fig. 2B). In the absence of bile salts, 60-80% of the DNP was associated with the t~-cellulose. Increasing concentrations of all the bile salts decreased the amount of DNP associated with the c~-cellulose (Fig. 2C).
The effect o f increasing a-cellulose amounts on the D N P content o f a solution containing glycocholate. At a constant glycocholate concentration (4 mM), the proportion o f D N P in solution decreased from > 90% to < 20% as the s-cellulose was increased from 0 to 50 mg (Fig. 3A). Only a
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Fig, 3A. The effect of increasing a-cellulose concentrations on the mutagenic activity of DNP in PBS, in the presence (E3) or absence (m) of 4 mM glycocholate. DNP (200 ng/2 ml) was incubated in PBS containing various concentrations of a-cellulose. Supernatant samples (50 pl) were taken after 1 h for mutagenicity assays. Values represent total numbers of revertant colonies (minus the negative control) expressed as a percentage of the numbers seen from a comparable sample of DNP diluted into DMSO and plated immediately. Fig. 3B. The DNP adsorbed to walls of the glass tubes from the experiment reported in Fig. 3A was assayed as mutagenic activity in the DMSO washings. Fig, 3C. The DNP adsorbed to a-cellulose from the experiment reported in Fig. 3A was assayed as mutagenic activity in the DMSO washings.
small amount of DNP was associated with the tube walls at all measured a-cellulose concentrations (Fig. 3B). The DNP lost from solution was mainly associated with the at-cellulose and could be recovered (Fig. 3C). At low at-cellulose levels (5 and 10 mg/2 nil) much less DNP was associated with the at-cellulose in the presence of glycocholate, whereas, at high at-cellulose levels (25 mg/2 ml or more) glycocholate had no effect.
Discussion In a previous study we found that more than 60°70 of the mutagenic activity of a solution of DNP in PBS was lost after 1 h. Almost all of the DNP lost could be recovered in a DMSO wash of the tube walls. We interpreted this result as showing that most of the DNP precipitated out of solution and became adsorbed to the tube walls. When
116
o~-cellulose, used as a model dietary fiber, was added to the incubation tubes, the DNP became almost completely associated with the o~-cellulose. In the present study, we used a similar experimental approach to examine the influence of the bile salts, sodium cholate, deoxycholate, glycocholate and taurocholate on the maintenance of DNP in aqueous solutions and its adsorption to o~-cellulose. In the presence of bile salts the mutagenic activity of a DNP aqueous solution, after incubation for 1 h, was substantially higher than that in a comparable solution which did not contain bile salts. Our evidence indicates that this was not a co-mutagenic effect as described by Wilpart et al. (1983), but was due to the solubilising properties of the bile salts maintaining the DNP in solution. Furthermore, the adsorption of this hydrophobic compound to the model dietary fiber was reduced by bile salts. The extent of this reduction depended on the concentration of the bile salts, but there was little difference between the different bile salts. The reduction was most apparent at low levels of o~-cellulose; at high levels, there was little effect. If the hypothesis is correct that dietary fiber protects the bowel by adsorbing cancer initiating mutagens, the results of this study suggest that in vivo intraluminal concentrations of endogenous bile salts and exogenous dietary fibers may have a pivotal role in determining the distribution of mutagens and hence the development of colonic carcinoma. The concentration of bile salts in the small intestine is determined in part by the fat content of the diet. Most of the micellar bile salts are reabsorbed in the distal small intestine, but a small proportion escapes into the colon where the bile salts can be deconjugated by bacteria. The concentration of bile salts in the small intestine is therefore much higher than in the colon. The situation is further complicated because bile salts themselves can adsorb to dietary fibers (Kritchevsky and Story, 1986) and some dietary fibers can be digested by colonic bacteria. The bile salts adsorbed to dietary fiber may escape the absorptive mechanism in the distal small intestine, and could be released in the colon if the type of fiber with
which they are associated is subsequently extensively digested. Thus the partitioning of hydrophobic environmental mutagens, such as DNP, between the lumen, dietary fiber and the surface of the digestive tract is likely to be different in the small intestine and colon, and to depend in each location on the concentration and types of dietary fibers and bile salts present. All these factors may have a role in explaining differences in the incidence of colon cancer within the population and the distribution of cancers within the intestines.
Acknowledgements We wish to thank Professors W.A. Denny, C.J. O'Connor and C. Tasman-Jones for critically reading the manuscript, and Professor O'Connor for supplying us with some of the bile salts. This work was supported by a grant from the Auckland Medical Research Foundation. We also thank the Provincial Grand Lodge of New Zealand for an equipment grant to A.M.R. to purchase a shaking water bath.
References Barnes, W.S., J. Maiello and J.H. Weisburger (1983) In vitro binding of the food mutagen 2-amino-3-methylimidazo[4,5J]quinoline to dietary fibers, J. Nat. Cancer Inst., 70, 757-760. Burkitt, D.P. (1978) Colonic-rectal cancer: fiber and other dietary factors, Am. J. Clin. Nutr., 31, S58-S64. Kada, T., M. Kato, K. Aikawa and S. Kiriyama (1984) Adsorption of pyrolysate mutagens by vegetable fibers, Mutation Res., 141, 149-152. Kritchevsky, D., and J.A. Story (1986) Influence of dietary fiber on cholesterol metabolism in experimental animals, in: G.A. Spiller fed.), Handbook of Dietary Fiber in Human Nutrition, CRC Press, Boca Baton, FL, pp. 129-142. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. O'Connor, C.J., and R.G. Wallace (1985) Physico-chemical behaviour of bile salts, Adv. Colloid Interface Sci., 22, 1-111. Roberton, A.M., P.J. Harris, H.J. Hollands and L.R. Ferguson (1990) A model system for studying the adsorption of a hydrophobic mutagen to dietary fiber, Mutation Res., 244, 173-178.
117 Smith-Barbaro, P., D. Hanson and B.S. Reddy (1981) Carcinogen binding to various types of dietary fiber, J. Nat. Cancer Inst., 67, 495-497. Takeuchi, M., M. Hara, T. Inoue and T. Kada (1988) Adsorption of mutagens by refined corn bran, Mutation Res., 204, 263 -267. Willett, W. (1989) The search for the causes of breast and colon cancer, Nature (London), 338, 389-394.
Wilpart, M., P. Mainguet, A. Maskens and M. Roberfroid (1983) Mutagenicity of 1,2-dimethylhydrazine towards Salmonella typhimurium, co-mutagenic effect of secondary biliary acids, Carcinogenesis, 4, 45-48. Communicated by J.M. Gentile
111
Elsevier MUTLET 0406
Effects of bile salts on the adsorption of a hydrophobic mutagen to dietary fiber Lynnette R. Ferguson 1, Philip J. Harris 2, H. John Hollands 2 and Anthony M. Roberton a 1Cancer Research Laboratory, and Departments of 2Botany and 3Biochemistry, The University of .4uckland, Auckland (New Zealand)
(Received 16 March 1990) (Revision received18 May 1990) (Accepted29 May 1990)
Keywords: Adsorption;~t-Cellulose;Bilesalts; Dietary fibre; 1,8-Dinitropyrene
Summary The effect o f the bile salts, sodium cholate, deoxycholate, glycocholate and taurocholate, on the solubility in aqueous solution o f the hydrophobic, environmental mutagen, 1,8-dinitropyrene (DNP), was examined. In the absence o f bile salts, the DNP appeared to precipitate out o f solution, whereas bile salts at a concentration o f _ 4 m M maintained the DNP in solution. In the presence o f the model dietary fiber, a-cellulose, the D N P adsorbed to this preferentially. Bile salts reduced this adsorption at low o~-cellulose levels, but had little effect at high o~-cellulose levels. The implication of these results is that bile salts have solubilising properties that could affect the distribution o f hydrophobic molecules, including mutagens, in the digestive tract.
Epidemiological studies indicate that certain types of dietary fiber may protect against the development of colon cancer (Burkitt, 1978; WiIlett, 1989). Although the factors involved are complex and not yet understood, one suggested mechanism to explain these protective properties is that dietary fiber may adsorb certain mutagens or cancer promoters in the digestive tract and these are carried out o f the body adsorbed onto undigested dietary fiber. Thus the effective concentrations of these substances available to initiate or Correspondence: Dr. L.R. Ferguson, Cancer Research Laboratory, The University of Auckland, Private Bag, Auckland (New Zealand).
promote cancerous changes in the gut mucosal cells are lowered. Certain mutagens and cancer promoters have been shown to adsorb onto a range o f dietary fibers in vitro (e.g. Barnes et al., 1983; Kada et al., 1984; Smith-Barbaro et al., 1981; Takeuchi et al., 1988; Roberton et al., 1990). We have previously studied the adsorption o f 1,8-dinitropyrene (DNP) to o~-cellulose as a model in vitro system for examining the adsorption of hydrophobic, environmental mutagens to dietary fiber (Roberton et al., 1990). We found that the D N P partitioned between the aqueous medium, the or-cellulose and the glass walls o f the tube. We also found that the solvent, dimethyl sulfoxide (DMSO), at final concentrations o f 25O7o (v/v) or
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
112
greater, maintained the DNP in solution and prevented it adsorbing to the a-cellulose. In the digestive tract, bile salts play a role in maintaining hydrophobic molecules, such as lipids, in solution. Thus, they could affect the partitioning o f hydrophobic mutagens, such as DNP, between the lumen, dietary fiber and surface of the tract. In this paper we report a study on the effect of bile salts on the partitioning of DNP in our model in vitro system (Roberton et al., 1990). Materials and methods
Chemicals. Cholic acid, deoxycholic acid, glycocholic acid and sodium taurocholate of > 98070 purity were obtained from Sigma Chemical Company, St. Louis, MO. DNP and acelluose (cat. No. C8022 from Canadian bleached hardwood pulp) were obtained from the same source. A solution of DNP (1 mg m l - 1) in DMSO was stored at - 80°C and diluted in DMSO before use. The concentration o f DMSO in the incubation medium was 2070 (v/v). Bacterial mutagenicity assay. The Salmonella typhimurium plate-incorporation assay was done using strain TA98 as described by Maron and Ames (1983) with each experimental point performed in triplicate on at least two separate occasions. In order to minimize day-to-day variability in tester strain sensitivity to mutagen, which is largely due to the exact growth phase of the culture, we grew cells for quantitative work as follows. A 1 ml vial (2 × 108 cells) was removed from storage at -80°C and inoculated into bacterial complete medium (20 ml). It was grown until a 1:10 dilution in fresh medium gave an absorbance of 0.11-0.12 at 654 nm, which took approximately 3 h. DNP levels in experiments were measured by assaying mutagenicity, and in the interpretation of data we have assumed a quantitative relationship between D N P concentration and revertant colonies. Positive controls containing 4-nitro-o-phenylene diamine (100 ng per plate) were included in all experiments.
Incubation o f DNP with bile salts. To prepare a solution of bile salts, bile acids were first solubilised with sodium hydroxide, and the pH adjusted back to 6.5 with HCI. A solution of phosphatebuffered saline (PBS) was added to give final concentrations of 20 mM sodium phosphate buffer (pH 6.5) and 130 mM NaC1. A series of solutions of the bile salts (0-8 mM) in PBS was prepared in acid-washed, conical glass centrifuge tubes (capacity 12 ml). The bile salt concentrations in the incubations were chosen to encompass their critical micellar concentrations (O'Connor and Wallace, 1985). Each bile salt dilution (1.96 ml) was prewarmed to 37°C for 30 min and at zero time DNP (200 ng) in DMSO (40 #l) was added and incubated with shaking (300 rev/min, 37°C). After 1 h, aliquots (3 x 50 /~l) were removed for mutagenicity assay. DNP associated with the tube walls was measured as described below. Incubation of DNP and bile salts with acellulose. At 30 min before zero time, the solution of bile salt in PBS (1.96 ml) was added to acellulose and pre-warmed to 37°C. At zero time D N P (200 ng) in DMSO (40 ~l) was added and incubated as above. After 55 min the tube was centrifuged (2500 x g, 2.5 min) and aliquots (3 x 50 ~l) of the supernatant were removed for the mutagenicity assay at 1 h. The DNP associated with the a-cellulose and the tube walls was recovered and measured as described below. Control incubations in which (a) a-cellulose was omitted or (b) bile salts were omitted, were carried out simultaneously as appropriate. Recovery of DNP associated with tube walls and of-cellulose. In those experiments in which no acellulose was added, the solution after incubation and sampling was vortex mixed (10 sec), discarded, and the tubes shaken to remove any remaining liquid. DMSO (2 ml) was then added, the contents vortex mixed (2 x 10 sec), and aliquots (3 x 50/tl) removed for the mutagenicity assay. This procedure measures the DNP associated with the tube walls. In experiments in which a-cellulose was added,
113
the incubation, centrifuging and sampling of the supernatant were performed as above. The pellet was resuspended by vortex mixing (1 x 10 sec) and the contents then tipped into a second tube. The first tube was rinsed with PBS (2 ml) and the contents added to the second tube. DMSO was added to the first tube as described above to measure the DNP associated with the tube walls. The second tube was centrifuged (2500xg, 5 min) and the supernatant discarded. DMSO (2 ml) was added to the pellet, the contents vortex mixed (2 x 10 sec), and the tube recentrifuged (2500 x g, 5 min). Aliquots (3 x 50 t~l) were removed for the mutagenicity assay. This procedure measures the DNP associated with the s-cellulose.
Data comparisons. The experiments were designed to examine the effect of a dose range of a bile salt at constant u-cellulose concentration, or of a dose range of a-cellulose at constant bile salt (glycocholate) concentration. Data from several experiments have been combined after normalising the data to compensate for minor variations in tester strain sensitivity. This was done by diluting the stock solution of DNP (40 #1 containing 200 ng)
1O0
_ o ~
in DMSO (1.96 ml) and assaying its mutagenicity. The DNP content of each experimental sample was then expressed as a percentage of the DNP added initially. Results
The effect o f bile salts on the mutagenic activity o f DNP. In preliminary experiments, it was found that the mutagenic activity of DNP was not altered when it was added with bile salts in a standard Ames mutagenicity test. The effect o f bile salts on the concentration o f D N P in solution. No more than 25% of the DNP remained in the PBS after 1 h incubation in the absence of bile salts (Fig. 1A). However, after incubation for 1 h in the presence of bile salts, the proportion of DNP maintained in solution increased with increasing bile salt concentration (Fig. 1A) and the DNP associated with the tube walls decreased (Fig. 1B). At a concentration for each bile salt of 4 mM or above, more than 75°7o of the DNP remained in solution. In general, all the bile salts used had a similar effect at the same concen-
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Fig. IA. The effect of increasing bile salt concentration on the mutagenic activity of DNP in PBS. DNP (200 ng/2 ml) was incubated in PBS containing various concentrations of bile salts: sodium cholate (O), deoxycholate (~7), glycocholate ([]), and taurocholate (A). Supernatant samples (50 p.1) were taken after 1 h for mutagenicity assays. Values represent total numbers of revertant colonies (mimis those for negative controls) expressed as a percentage of the numbers seen from a comparable sample of DNP diluted into DMSO and plated immediately. Fig. lB. The DNP adsorbed to walls of the glass tubes, from the experiment reported in Fig. 1A, was assayed as mutagenic activity in the DMSO washings.
114
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Fig. 2A. The effect of increasing bile salt concentration on the mutagenic activity of DNP in PBS, in the presence of a-cellulose (10 mg/2 ml). The experiment was similar to that in Fig. IA except that t~-cellulose was present in all tubes. Symbols as in Fig. 1. Fig. 2B. The D N P adsorbed to the walls of the glass tubes, from the experiment reported in Fig. 2A, was assayed as mutagenic activity in DMSO washings. Fig. 2C. The D N P adsorbed to ~-cellulose from the experiment reported in Fig. 2A was assayed as mutagenic activity in the DMSO washings.
tration. Greater than 95% of the added DNP was accounted for when the amount of DNP recovered from the tube wall was added to that measured in the supernatant.
The effect o f a-cellulose and bile salts on the D N P content o f the supernatant. The previous experiment was repeated in the presence of c~cellulose (10 mg). The presence of ct-cdlulose decreased the amount of DNP found in solution at all bile salt concentrations (cf. Fig. 1A and Fig. 2A). Only small amounts of DNP were now
associated with the tube walls (Fig. 2B). In the absence of bile salts, 60-80% of the DNP was associated with the t~-cellulose. Increasing concentrations of all the bile salts decreased the amount of DNP associated with the c~-cellulose (Fig. 2C).
The effect o f increasing a-cellulose amounts on the D N P content o f a solution containing glycocholate. At a constant glycocholate concentration (4 mM), the proportion o f D N P in solution decreased from > 90% to < 20% as the s-cellulose was increased from 0 to 50 mg (Fig. 3A). Only a
115
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50
Fig, 3A. The effect of increasing a-cellulose concentrations on the mutagenic activity of DNP in PBS, in the presence (E3) or absence (m) of 4 mM glycocholate. DNP (200 ng/2 ml) was incubated in PBS containing various concentrations of a-cellulose. Supernatant samples (50 pl) were taken after 1 h for mutagenicity assays. Values represent total numbers of revertant colonies (minus the negative control) expressed as a percentage of the numbers seen from a comparable sample of DNP diluted into DMSO and plated immediately. Fig. 3B. The DNP adsorbed to walls of the glass tubes from the experiment reported in Fig. 3A was assayed as mutagenic activity in the DMSO washings. Fig, 3C. The DNP adsorbed to a-cellulose from the experiment reported in Fig. 3A was assayed as mutagenic activity in the DMSO washings.
small amount of DNP was associated with the tube walls at all measured a-cellulose concentrations (Fig. 3B). The DNP lost from solution was mainly associated with the at-cellulose and could be recovered (Fig. 3C). At low at-cellulose levels (5 and 10 mg/2 nil) much less DNP was associated with the at-cellulose in the presence of glycocholate, whereas, at high at-cellulose levels (25 mg/2 ml or more) glycocholate had no effect.
Discussion In a previous study we found that more than 60°70 of the mutagenic activity of a solution of DNP in PBS was lost after 1 h. Almost all of the DNP lost could be recovered in a DMSO wash of the tube walls. We interpreted this result as showing that most of the DNP precipitated out of solution and became adsorbed to the tube walls. When
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o~-cellulose, used as a model dietary fiber, was added to the incubation tubes, the DNP became almost completely associated with the o~-cellulose. In the present study, we used a similar experimental approach to examine the influence of the bile salts, sodium cholate, deoxycholate, glycocholate and taurocholate on the maintenance of DNP in aqueous solutions and its adsorption to o~-cellulose. In the presence of bile salts the mutagenic activity of a DNP aqueous solution, after incubation for 1 h, was substantially higher than that in a comparable solution which did not contain bile salts. Our evidence indicates that this was not a co-mutagenic effect as described by Wilpart et al. (1983), but was due to the solubilising properties of the bile salts maintaining the DNP in solution. Furthermore, the adsorption of this hydrophobic compound to the model dietary fiber was reduced by bile salts. The extent of this reduction depended on the concentration of the bile salts, but there was little difference between the different bile salts. The reduction was most apparent at low levels of o~-cellulose; at high levels, there was little effect. If the hypothesis is correct that dietary fiber protects the bowel by adsorbing cancer initiating mutagens, the results of this study suggest that in vivo intraluminal concentrations of endogenous bile salts and exogenous dietary fibers may have a pivotal role in determining the distribution of mutagens and hence the development of colonic carcinoma. The concentration of bile salts in the small intestine is determined in part by the fat content of the diet. Most of the micellar bile salts are reabsorbed in the distal small intestine, but a small proportion escapes into the colon where the bile salts can be deconjugated by bacteria. The concentration of bile salts in the small intestine is therefore much higher than in the colon. The situation is further complicated because bile salts themselves can adsorb to dietary fibers (Kritchevsky and Story, 1986) and some dietary fibers can be digested by colonic bacteria. The bile salts adsorbed to dietary fiber may escape the absorptive mechanism in the distal small intestine, and could be released in the colon if the type of fiber with
which they are associated is subsequently extensively digested. Thus the partitioning of hydrophobic environmental mutagens, such as DNP, between the lumen, dietary fiber and the surface of the digestive tract is likely to be different in the small intestine and colon, and to depend in each location on the concentration and types of dietary fibers and bile salts present. All these factors may have a role in explaining differences in the incidence of colon cancer within the population and the distribution of cancers within the intestines.
Acknowledgements We wish to thank Professors W.A. Denny, C.J. O'Connor and C. Tasman-Jones for critically reading the manuscript, and Professor O'Connor for supplying us with some of the bile salts. This work was supported by a grant from the Auckland Medical Research Foundation. We also thank the Provincial Grand Lodge of New Zealand for an equipment grant to A.M.R. to purchase a shaking water bath.
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