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The adsorption of heterocyclic aromatic amines by model dietary fibres with contrasting compositions
Ch~k:o-~ ~aetk~t ELSEVIER
Chemico-Biological Interactions 100 (1996) 13-25
The adsorption of heterocyclic aromatic amines by model dietary fibres with contrasting compositions Philip J. Harris *a, Christopher M. Triggs b, Anthony M. Roberton a, Mark E. Watson c, Lynnette R. Ferguson c aSchool of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand bDepartment of Statistics, The University of Auckland, Private Bag 92019, Auckland, New Zealand CCancer Research Laboratory, The University of Auckland, Private Bag 92019, Auckland, New Zealand
Received 4 July 1995; revision received 13 November 1995; accepted 16 November 1995
Abstract
It is often recommended that consumption of dietary fibre should be increased to protect against colorectal cancer. However, although more than 95% of dietary fibre is contributed by whole plant cell walls, very little experimental work has been done using whole plant cell walls. These may protect by adsorbing carcinogens, thus lowering their effective concentration in the alimentary tract, and by carrying the carcinogens out of the body in the faeces. However, plant cell walls vary widely in their composition and physical properties, and not all cell walls will necessarily have protective properties. We therefore isolated 4 plant cell-wall preparations with contrasting compositions as models of the types of cell walls that occur in the diet. We investigated the abilities of these preparations to adsorb in vitro 6 heterocyclic aromatic amines (HAAs). HAAs occur in the human diet and several are colon carcinogens, at least in rats. We found that the ability of the HAAs to adsorb to the plant cell walls increased with increasing hydrophobicity of the HAA, measured as the calculated logarithm of the partition coefficient between l-octanol and water (C logP). A cell-wall preparation containing
Abbreviations: AOAC, Association of Official Analytical Chemists; C logP, calculated logarithm of the partition coefficient; DNP, 1,8-dinitropyrene; HAA, heterocyclic aromatic amine; IQ, 2-amino-3methylimidazo[4,5-/]quinoline; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-j]quinoline; MeIQx, 2-amino3,8-dimethylimidazo[4,5-/]quinoxaline; PhIP, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine; Trp-P1, 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3-amino-l-methyl-5H-pyrido[4,3-b]indole. * Corresponding author.
0009-2797/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(95)03682-C
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mainly the walls of parenchyma cells (the most common cell type in food plants) had only poor adsorptive ability. A cell-wall preparation from commercial cork had the best adsorptive ability. This preparation was the most hydrophobic of those examined because the cell walls contained the polymer, suberin, together with associated waxes. The preparation modelled suberized cell walls which occur in the diet, for example in potato skins. The other two cellwall preparations contained another hydrophobic polymer, lignin, and had intermediate adsorptive abilities which were not significantly different from one another. These preparations modelled lignified cell walls which occur in the diet, for example in wheat bran. Our results indicate that suberized and lignified cell walls may be important in protecting against colorectal cancer.
Keywords: Carcinogens; Colorectal cancer; Dietary fibres; Heterocyclic aromatic amines; Hydrophobicity; Plant cell walls
1. Introduction
In attempts to reduce the incidence of colorectal cancer and a range of other diseases, many Western countries run public health campaigns which recommend eating more dietary fibre. However, this generalised recommendation frequently does not recognise that the term dietary fibre encompasses a wide range of heterogeneous and complex materials with widely different properties. Some of these materials almost certainly protect against colorectal cancer, while others probably do not. We have recently defined dietary fibre as follows: 'dietary fibre is composed of plant cell walls and components obtained from these walls. It also includes nonstarch polysaccharides from sources other than plant cell walls' [1]. Two broad categories of dietary fibres, soluble and insoluble, are often determined analytically, depending on their solubility in water and buffer solutions. Except for cellulose, many of the polysaecharides in plant cell walls are soluble in water after they have been extracted from the walls (soluble dietary fibres). However, because of the crosslinks between the cell-wall components, these polysaccharides are usually waterinsoluble when within the cell wall, and make the whole cell walls mainly waterinsoluble under the physiological conditions in the gastrointestinal tract (insoluble dietary fibres) [2]. Soluble dietary fibres (soluble-fibre polysaccharides) are widely used as food additives (thickeners, stabilizers, emulsifiers and gelling agents) and, as well as being extracted from plant cell walls (e.g. pectin preparations), they are also obtained from microorganisms, from the cell walls of seaweeds, and from plant sources other than cell walls (e.g. the outer layers of some seeds) [1]. In Western diets more than 95% of the dietary fibre is contributed by whole plant cell walls [3]. Food plants contain a range of different cell types, the walls of which differ in their structure, composition and physical properties. Furthermore, the composition of the walls of a particular cell type can be quite different in different species of food plants [4]. It is unlikely that these different types of plant cell walls are equally effective in protecting against colorectal cancer. Animal experiments designed to test the effects of dietary fibres on the development of colorectal cancer have mostly not tested the effects of preparations of whole
P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
15
plant cell walls. Instead, these experiments have frequently tested dietary fibres that usually occur in only small amounts in Western diets [1]. These dietary fibres include soluble-fibre polysaccharides from a variety of sources, and preparations of cellulose. But although some of these dietary fibres tested are extracted from plant cell walls, it is unlikely that they will have the same effects as when they are part of the whole cell walls. In addition, the effects of cereal brans have also been tested frequently. Cereal brans are often erroneously described as dietary fibres, but in fact are only sources of dietary fibres. For example, a reference wheat bran contained only 43.9% dietary fibre determined by the AOAC method [1]. Thus components other than dietary fibres could be responsible for any protective properties shown by such brans. One mechanism by which plant cell walls may prevent the development of colorectal cancer is by adsorbing carcinogens in the digestive tract [5-8]. If these cell walls are not significantly degraded by bacterial enzymes in the colon, the carcinogens may be carried out of the body adsorbed onto the cell walls, thus lowering the effective concentrations available to initiate or promote cancerous changes in the gut mucosal cells. The most common cell type in food plants is the parenchyma cell which has walls composed mainly of polysaccharides. We have previously obtained cell-wall preparations from this cell type from potato tubers [6] and cabbage leaves [7] and tested in vitro the ability of the hydrophobic, environmental carcinogen 1,8dinitropyrene (DNP) to adsorb to these cell walls. We found that DNP adsorbed only weakly to these cell walls. In the present study, we used a parenchyma cell-wall preparation from potato tubers as a model for parenchyma cell walls in the diet. In contrast to parenchyma cell walls, the walls of some cell types from food plants contain, in addition to polysaccharides, the hydrophobic polymers lignin or suberin which make the cell walls hydrophobic. Because these cell types are present in food plants in only small proportions it is technically much easier to obtain preparations composed mainly of lignified or suberized cell walls from model plant material that is not usually eaten by humans. We previously showed that model cell-wall preparations which contained mainly lignified cell walls (obtained from wheat straw and mature cabbage stems) and model cell-wall preparations that contained mainly suberized cell walls (obtained from potato skins and commercial cork) adsorbed DNP in vitro much more effectively than did parenchyma cell walls [6,7]. Commercial cork is an excellent source of suberized cell walls without the contamination from parenchyma cell walls that occurs to some extent in cell-wall preparations from potato skins. We used cell-wall preparations from these plant materials as models of lignified and suberized cell walls which do occur in the diet (see Discussion). These models are important in enabling us to test experimentally the role which lignified and suberized cell walls may play in the prevention of colorectal cancer. We do not imply that wheat straw, mature cabbage stems, or commercial cork are normal dietary components. We have now extended these studies to test the effectiveness of model preparations of parenchyma cell walls, lignified cell walls, and suberized cell walls to adsorb in vitro 6 heterocyclic aromatic amines (HAAs) which occur in the human diet. At least
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P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
in rats, IQ, MelQ and PhlP are colon carcinogens [9]. We have also related the abilities of HAAs to adsorb to the model cell-wall preparations to the hydrophobicities Of the HAAs. Although HAAs are known to be converted to reactive products by N-oxidation [9], we consider that adsorption of the HAAs to cell walls very likely occurs at an early point in the passage through the gastrointestinal tract and before the HAAs are metabolized. We have previously shown [51 that the ability of a carcinogen to adsorb to a preparation of a-cellulose was related to the hydrophobicity of the carcinogen. In the present study we have selected HAAs to span a range of hydrophobicities. 2. Materials and methods 2.1. Chemicals
The HAAs, IQ, MelQx, PhlP and Trp-P-1 were obtained from Toronto Research Chemicals Inc., Downsview, Ontario, Canada, and MelQ and Trp-P-2 were the kind gift of Professor T. Sugimura, National Cancer Center Research Institute, Tokyo, Japan. Their full chemical names and structures are shown in Fig. 1. HPLC grade chromatography solvents were purchased from Alphatech, Auckland, New Zealand. CH3
CH3
~
~
/ H.
N
NH2 CH3
Trp-P-1 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole NH2
~
~
/
N
NH2
H
TrpLP-2 3-amino-1-methyl-5H-pyrido[4,3-b]indole NH2
N@'--CH3
IQ 2-amino-3-methylhnidazo[4,5-J]quinoline
MelQ 2-amino-3,4-dimethylimidazo[4,5-J]quinoline
NH2 H3C~ .,N,~ ~
IN~CH3
-N'LJ
MeIQx 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
PhlP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
Fig. 1. The names,abbreviationsand structuresof the heterocyclicaromaticaminesidentifiedin the text.
P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
17
2.2. Hydrophobicities o f the H A A s
The calculated logarithm of the partition coefficient (C logP) between 1-octanol and water was used as a measure of the hydrophobicity of each HAA and was calculated from the structure of the HAA by an additive-constitutive procedure using the computer program, CLOGP3 [5,10,11]. 2.3. Model cell-wall preparations
Model cell-wall preprations from commercial cork, from the stem internodes of wheat straw, and from the woody secondary xylem cylinder of mature cabbage stems were obtained as described by Roberton et al. [7]. The preparation of parenchyma cell walls from the flesh of potato tubers was obtained as described by Harris et al. [61. 2.4. H P L C
HPLC of the HAAs was performed using a system which comprised a Waters 510 pump (Waters Assoc., Milford, MA, USA), a Gilson Model 231-401 auto-sampling injector, a UV absorption detector (Model 111B, Gilson Medical Electronics, Middleton, WI, USA), a fluorescence detector (Model RF-530, Shimadzu, Kyoto, Japan), a diode array detector (Model 4120 Philips, Cambridge, UK), and a Waters Cis 4 # Novapak stainless steel column (150 x 3.9 mm, Phenomenex, Torrance, CA, USA). The HAAs were eluted with a mobile phase consisting of 25 mM sodium dihydrogen phosphate (adjusted to pH 3.0 with 25 mM orthophosphoric acid) and methanol (IQ, 9:1 v/v; MelQx, 4:1 v/v; PhlP, 3:2 v/v; Trp-P-l, and Trp-P-2, 1:1 v/v). For MelQ, the mobile phase was the same as for the PhlP except that the pH of the 25 mM sodium dihydrogen phosphate was adjusted to 6.5. The flow rates were 1-3 ml/min for all HAAs. The HAAs were detected using the UV absorption detector (at 254 nm), the fluorescence detector (Trp-P-l, excitation 300 nm, emission 460 nm; Trp-P-2, excitation 356 nm, emission 485 nm). The diode array detector was used (at wavelengths from 190-390 nm) for all experiments involving the cell-wall preparation from commercial cork. The use of the diode array detector with the cork cell-wall preparation was necessary because material that absorbed in the UV leached from the preparation. The HAAs were detected at wavelengths that minimised interference from this material. Data acquisition and integration were performed using a Unicam 4880 Chromatography Data Handling System (Unicam Ltd, Cambridge, UK). We have previously shown [5,10] that the UV absorption, diode array and fluorescence detectors gave a linear response curve for the HAAs over the concentration range used. 2.5. Incubation o f H A A s with model cell-wall preparations in P B S
All incubations were done in acid-washed conical glass centrifuge tubes (capacity 12 ml). The model cell-wall preparations (10 rag) were weighed into the tubes and
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P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
1.96 ml of phosphate-buffered saline (PBS) (20 mM sodium phosphate buffer, pH 6.5, containing 130 mM sodium chloride) added to each tube. Each tube stood for 30 rnin to allow the cell-wall preparations to hydrate, then 40 ttl of a solution of the HAA in methanol was added, giving final HAA concentrations of 0.5, 1.0, 2.5 and 5/zg/ml (0.1, 0.2, 0.5 and 1.0/~g/mg of cell-wall preparation), and the contents of the tubes were mixed using a vortex mixer. The tubes were shaken (120 rev./min ) on an orbital shaker for 55 min at 37°C and centrifuged (2500 x g for 5 min). The supernatants were filtered using an Acrodisc (Polysulfone) disposable filter unit (diameter 13 mm, pore size 0.2 #m; Gelman Sciences, Ann Arbor, MI) and 3 x 100 /zl aliquots taken. Control incubations were also done in which the HAAs were incubated with no cell-wall preparations. In these control incubations aliquots were also taken at zero time. There was no evidence for degradation of any of the HAAs during incubation. The amounts of HAAs adsorbed to the cell-wall preparations were determined from the difference between the concentration of HAAs in the supernatants in the presence of the cell-wall preparations and the supernatant concentrations measured in control tubes in the absence of cell-wall preparations. The amounts adsorbed by the cell walls are expressed as percentages of the total amounts present in control experiments without cell walls. The amounts of HAAs adsorbed on the tube walls were determined as we have previously described, except that acetonitrile was used to recover the HAAs [12]. However, preliminary experiments showed that none of the HAAs were adsorbed to the walls of the glass tubes, and thus in subsequent experiments only the supernatant was sampled.
2.6. Statistical methods The percentages of the HAAs that were adsorbed by the model cell-wall preparations were plotted on a logistic scale to stabilize variance. The percentages were plotted against the initial HAA concentrations (~g HAA/mg cell-wall preparation) plotted on a logarithmic scale. The resulting scatterplots showed that the relationship between these two variables may be approximated closely by a straight line and so (logit) percentage adsorbed was regressed on (log) initial HAA concentration. 3. Results
3.1. Hydrophobicities of the HAAs The hydrophobicities of the HAAs used in this study, calculated as C logP values, are shown in Table 1.
3.2. Effect of initial HAA concentration on the percentage of HAAs adsorbed by the model cell-wall preparations The possible effect that the initial HAA concentration had on the total percentage of the HAAs adsorbed by the cell-wall preparations was examined. This was done by plotting the (logit) percentages adsorbed by the cell-wall preparations against the
P.J. Harris et aL /Chemico-Biological Interactions 100 (1996) 13-25
19
Table 1 Hydrophobicities of HAAs and their estimated percentage adsorption to cell-wall preparations HAA
MelQx IQ MelQ PhlP Trp-P-2 Trp-P-I
C logP
Estimated percentage adsorption of HAAs by cell-wall preparations
1.078 1.323 1.822 2.294 2.731 3.230
Potato flesh
Wheat straw
Cabbage stem
Commercial cork
7 (3.5) 6 (1.5) 14 (3.2) 25 (3.9) 28 (1.5) 17 (10.1)
30 (2.7) 31 (1.9) 97 (1.8) 61 (5.0) 64 (1.2) 84 (0.48)
18 (4.0) 45 (5.9) 54 (I.1) 56 (3.9) 86 (1.8) 62 (0.42)*
74 75 96 85 91 92
(6.0)* (4.6)* (2.6) (3.6) (3.6) (0.56)*
*Estimated percentage of HAAs adsorbed at a HAA concentration of 0.3 #g/mg cell-wall preparation obtained from the fitted regression line when the slope is significantly different from zero (see Fig. 2). All other percentages are mean values calculated over the four HAA concentrations used. For these combinations of HAAs and ceil-wall preparations the slope of the fitted regression line does not differ from zero (P > 0.05) (see Fig. 2). Standard errors are shown in parentheses. All calculations were done on a Iogit scale to stabilis¢ variance and the results were back-transformed to a percentage scale.
log o f the initial c o n c e n t r a t i o n o f H A A s (/~g H A A / m g cell-wall p r e p a r a t i o n ) . Fig. 2 s h o w s the p e r c e n t a g e o f I Q a d s o r b e d by the cell-wall p r e p a r a t i o n s p l o t t e d against the initial c o n c e n t r a t i o n o f IQ. A d s o r p t i o n o n l y to the c o r k cell-wall p r e p a r a t i o n was significantly affected by the initial I Q c o n c e n t r a t i o n . H e r e , the p e r c e n t a g e o f I Q ad-
90 '0
............ ....................0 ................
75 ........................................................................ =:::::::::Z'O.................................. 0 ....
"'0 0
50
"'0
0
Ii ~
25 10 5
-V-
- - - %
A -----%.%------
A- .............
A
2.5 O. 10
0.20 0.50 ,ug IQ/mg cell wall
1.0
Fig. 2. The effect of the concentration of IQ (t~g/mgcell-wall preparation) at the beginning of the incubation on the percentage of IQ that was adsorbed by the model cell-wall preparations at the end of the incubation. For each cell-wall preparation, the fitted regression line and the line through the mean percentage (logit %) are plotted. A, potato; V, wheat straw; 13, cabbage; O, cork.
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P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
sorbed decreased with increasing initial IQ concentration (P = 0.03). For most of the other combinations of HAAs and cell-wall preparations, the percentage of HAA adsorbed by the cell-wall preparations was also unaffected by the initial concentration of the HAA. Thus, the slopes of the fitted regression lines were mostly not significantly different from zero (i.e. the data can be adequately summarized by a horizontal line). With the other HAAs, a significant effect of initial HAA concentration on the percentage of HAA adsorbed by the cell-wall preparations was found only for the adsorption of MelQx and Trp-P-1 to the cork cell-wall preparation (P = 0.04 and 0.03, respectively), and for the adsorption of Trp-P-1 to the cabbage cell-wall preparation (P = 0.01). For the adsorption of Trp-P-1 to the cork cell-wall preparation there was increased adsorption with increasing initial Trp-P-1 concentrations. For the other combinations of HAAs and cell-wall preparations where there was a significant effect of initial HAA concentration, there was decreased adsorption with increasing initial HAA concentrations. In addition, for the cabbage cell-wall preparation there was some evidence that the adsorption of MelQ and Trp-P-2 decreased with increasing initial concentration (P = 0.046 and 0.059, respectively). Similarly, for the cork cell-wall preparation there was weak evidence that the adsorption of PhlP and TrpP-2 decreased with increasing initial concentration (P = 0.059 and 0.075). However, even in combinations where the percentage of HAA adsorbed by the cell-wall preparations was significantly affected by the initial HAA concentration, the differences between the percentages of HAA adsorbed at initial concentrations of 0.1 and 1.0 t~g HAA/mg cell wall were small compared to the differences in percentages adsorbed between the different cell-wall preparations. Effectively, we could regard the percentages of HAAs adsorbed by the cell-wall preparations as being independent of the initial HAA concentrations. 3.3. Estimated percentage adsorption of HAAs at a single HAA concentration To enable a comparison to be made of the adsorptive abilities of the four model cell-wall preparations, the percentage of each HAA adsorbed to each cell-wall preparation was estimated at a HAA concentration approximately half way along the range used (0.3 #g HAA/mg cell wall). The mean percentage adsorptions over the four HAA concentrations were also calculated (Table 1). These mean percentages were indistinguishable from the estimated percentages at 0.3/~g HAA/mg cellwall preparation, even for those combinations of HAA and cell-wall preparation where the initial HAA concentration had a significant effect on the percentage adsorbed. The percentage adsorptions ranged from 6% (for the adsorption of IQ by the potato flesh cell-wall preparation) to 97% (for the adsorption of MelQ by the wheat straw cell-wall preparation). 3.4. The relationship between hydrophobicities of the HAAs and estimated percentage adsorption to the model cell-wall preparations A similar statistical approach was used to that shown above to describe the rela-
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P.J. Harris et aL / Chemico-Biological Interactions 1190(1996) 13-25
95 t (b) 7 o--£14
.
1.0
1.5
.
. 2.0
V i
. 2.5
3.0
1.0
1.5
C log P
95
~
2.0
2.5
3.0
C log P
(c) .~ 75 I~)- 0
1.0
1.5
2.0
2.5
C log P
3.0
1.0
1.5
2.0
2.5
3.0
C log P
Fig. 3. The effectof the hydrophobicityof the HA.&(expressedas the calculatedlogarithmof the partition coefficientbetween 1-octanoland water) (C log/') on the percentageof the HAA adsorbed by modelcellwall preparations(at an initial concentrationof 0.3 t~g HAA/mgcell-wall preparation)from: (a) potato flesh, (b) wheat straw, (c) cabbagestem, and (d) commercialcork, with fitted regressionlinessuperimposed. The valuefor the adsorptionof MelQ (C logP = 1.822)by the cell-wallpreparation from wheat straw was regarded as an outlier and was excludedfrom the determination of the regressionline.
tionship between the estimated percentage of HAA adsorbed and the initial HAA concentration. Fig. 3 (a, b, c, and d) shows the estimated (logit) percentage adsorptions (at an initial concentration of 0.3/~g HAA/mg cell-wall preparation) of these HAAs by potato, wheat straw, cabbage, and cork cell-wall preparations plotted against, and regressed on the hydrophobicities (C logP values) of the HAAs. With all the model cell-wall preparations, the estimated percentage adsorptions increased with increasing hydrophobicity. The slopes of the lines were compared and found not to differ (P = 0.62). However, the positions of the lines varied. Even hydrophobic HAAs adsorbed only weakly to the potato cell-wall preparation. At the other extreme, all HAAs, including hydrophilic ones, adsorbed strongly to cork cell-wall preparations. 3.5. Comparison o f the adsorptive abilities o f the four model ceil-wall preparations
The relative adsorptive abilities of the four model cell-wall preparations were compared by calculating the predicted percentage adsorptions of hypothetical carcinogens with C logP values of 2.15 (which is the mid-value of the HAAs used) and 0.0. The predicted percentage adsorptions are shown in Table 2. For both hypothetical carcinogens, the cell-wall preparation from commercial cork had a significantly
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P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
Table 2 Predicted percentage adsorption of hypothetical carcinogens Cell-wall preparation
Potato flesh Wheat straw Cabbage stem Commercial cork
C IogP = 0
C logP = 2.15
%
logit(%)
%
lo~t(%)
3 17 17 55 S.E.D.
(-3.51) (-1.58) ( - 1.57) (0.19) (0.36)
15 56 56 88 S.E.D.
(-!.71) (0.22) (0.23) (1.99) (0.36)
All calculations were done on percentages transformed to the logit scale and then back transformed to give predicted percentages. Values in parentheses are the predicted logit values.
greater adsorptive ability than did the preparations from cabbage stem and wheat straw (P < 0.001). The cell-wall preparations from cabbage stem and wheat straw were not significantly different in their adsorptive abilities (P > 0.05). However, both of these preparations had significantly greater adsorptive abilities than had the cell-wall preparation from potato flesh (P < 0.001). 4. Discussion
The present study shows that the ability of HAAs to adsorb to plant cell walls depends on the hydrophobicity of the HAA (measured as C logP) and the hydrophobicity of the cell walls. The ability of a HAA to adsorb to each of the model cell-wall preparations increased with increasing hydrophobicity. We have also previously shown that this was so for the adsorption of HAAs to an a-cellulose preparation which we used as a model insoluble dietary fibre [5]. We were able to compare the adsorptive abilities of the four model cell-wall preparations by calculating the predicted adsorptions at particular C logP values. These calculations showed that the cell-wall preparation from commercial cork had the best adsorptive ability. Commercial cork is from the outer bark of the cork oak (Quercus suber) and consists of cork cells. Because the walls of these cells contain the complex hydrophobic polymer, suberin, together with associated waxes, the cellwall preparation from commercial cork was the most hydrophobic we examined. Suberin consists of aliphatic polyester domains covalently attached to phenolic, lignin-like domains [13]. We have previously shown that cell-wall preparations from commercial cork and from potato skin (which is rich in cork cells) strongly adsorbed DNP [6,7]. The model cell-wall preparations from the woody secondary xylem cylinder of mature cabbage stems and from the stem internodes of wheat straw contain mainly lignified cell walls. Lignin is a complex hydrophobic polymer, and in lignified cell walls of flowering plants the main non-cellulosic polysaccharides are heteroxylans which are insoluble in water [4]. These two model cell-wall preparations had similar adsorptive abilities, but were less effective than the model cell-wall preparation from
P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
23
commercial cork. Our previous work showed that these preparations also adsorbed DNP strongly, but were not as effective as the cell-wall preparation from commercial cork [7]. It is likely that lignified or suberized cell walls, as well as being effective at adsorbing carcinogens, are not degraded to any extent in the colon [1]. Thus it is likely that carcinogens adsorbed on these cell walls will be carried out of the body on the cell walls. The model cell-wall preparation from the flesh of potato tubers, which contained mainly parenchyma cell walls, had the least adsorptive ability of the four cell-wall preparations tested. This is consistent with our previous finding that a parenchyma cell-wall preparation from potato tubers (and a similar parenchyma cell-wall preparation from cabbage leaves) adsorbed DNP only weakly [6,7]. The present study shows that a parenchyma cell-wall preparation from potato tubers also adsorbed HAAs only weakly. These cell walls are rich in pectic polysaccharides, a complex class of polysaccharides comprising a family of acidic polysaccharides (rhamnogalacturonans) and several neutral polysaccharides (arabinans, galactans, and arabinogalactans) [4]. Pectic polysaccharides are partially soluble in water and we previously found that a phosphate-buffered saline extract of potato parenchyma cell walls containing such polysaccharides held DNP in solution [6]. In aqueous solutions in the absence of such soluble polysaccharides, the DNP came out of solution. We suggested that the soluble pectic polysaccharides reduced the adsorption of DNP to the cell walls. In further studies we showed that a preparation of pectic polysaccharides from citrus fruits, and other soluble-fibre polysaccharides, reduced the adsorption of DNP to c~-cellulose [14]. However, unlike DNP (C logP---4.384), Trp-P-1 (C logP = 3.230), which was the most hydrophobic of the HAAs that we examined, was sufficiently hydrophilic to remain in solution in phosphate-buffered saline. Furthermore, the soluble-fibre polysaccharide, gum arabic, did not reduce the adsorption of Trp-P-1 to a-cellulose [10]. This suggests that the weak adsorption of HAAs to the model parenchyma cell-wall preparation from potato tubers may simply be because the cell walls contain mainly hydrophilic components. If carcinogens are adsorbed by cell walls and these cell walls are subsequently degraded by bacterial enzymes in the colon, the carcinogens will not be carried out of the body adsorbed in this way. Thus cell walls that are degraded in the colon will not protect against colorectal cancer by this mechanism. There is good evidence that parenchyma cell walls are extensively degraded in the colon [3,15,16]. This indicates that if parenchyma cell walls protect against colorectal cancer, they do so by a different mechanism. It should be noted, however, that there is no evidence available that they do protect. The relationship between the hydrophobicity of HAAs and their ability to adsorb to dietary fibres, which we have reported previously [5], has been further demonstrated here. The techniques we have used in this paper for analysing the results enable the results to be extended to other classes of carcinogens. The abilities of these other carcinogens to adsorb to the four model cell-wall preparations can be predicted from their hydrophobicities (C logP values). Compared to the HAAs which we have used in the present study (and which have been identified in Western diets [5,9]), many of the carcinogens used in animal exper-
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P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
iments to test the ability of dietary fibres to protect against colorectal cancer are much more hydrophilic (e.g. [17]). These carcinogens include N-nitroso-Nmethylurea (NMU) and 1,2-dimethylhydrazine (DMH) which have C logP values of -0,204 and -1.368, respectively. Table 2 shows that the predicted percentage adsorptions for a carcinogen with a C logP value of zero are 55, 17, 17, and 3 for the model cell-wall preparations from commercial cork, cabbage stem, wheat straw and potato flesh respectively. We have found it difficult to determine experimentally the adsorption of NMU to the four model cell-wall preparations because it is unstable, especially at 37°C [5]. However, even when the experiments were carried out at 22°C, at which temperature the extent of breakdown was reduced, we found that NMU adsorbed significantly ( - 9 % ) only to the cell-wall preparation from commercial cork. We have also previously found that NMU was poorly adsorbed by a-cellulose [5]. From its C logP value, we also predict that DMH will adsorb to the model cell-wall preparations to only a small extent. Thus protection by dietary fibres in animal experiments in which such hydrophilic carcinogens have been used is probably the result of mechanisms other than adsorption and may or may not be relevant to naturally occurring and food-derived carcinogens in humans. The present study, in which model cell-wall preparations were examined, indicates that it may be desirable to increase consumption of food plants that contain lignified or suberized cell walls and which are the most effective absorbers of hydrophobic carcinogens, including HAAs. Such cell walls should be beneficial in removing these carcinogens from potential contact with colonic mucosal cells. In Western diets these cell walls usually occur in only small amounts [1]. However, sclereids or stone cells, which have thick lignified cell wails, occur in a number of species of fruits, for example in pears [18] and in feijoas [19]. A number of cell types with lignified cell walls also occur in some cereal brans, e.g. in wheat bran. Indeed, wheat bran is an important source of lignified cell walls in Western diets. Cell types that have lignified walls sometimes develop as the food plant matures, but lignified cell walls can make the plants less palatable. For example, sclerenchyma fibre cells with thick lignified walls, which develop in asparagus shoots as they mature, give the shoots a tough, stringy texture. Suberin, found in the walls of cork cells, occurs not only in the skins of potatoes, but also in the skins of many other tubers and root vegetables. Thus, increased consumption of foods that contain suberized or lignified cell walls (e.g. potato skins or wheat bran) may be particularly important in the prevention of colorectal cancer.
Acknowledgements We are grateful to the Auckland Medical Research Foundation, the Cancer Society of New Zealand, the Provincial Grand Rights of New Zealand, and the New Zealand Lottery Grants Board for financial support during the course of this work. We thank J.H. Bentley for critically reading the manuscript.
References [i] P.J. Harris and L.R. Ferguson, Dietary fibre: its composition and role in protection against colorectal cancer, Mutat. Rcs., 290 (1993) 97-110.
P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
25
[2] S.C. Fry, Analysis of cross-links in the growingcell walls of higher plants, in: H.-F. Linskens and J.F. Jackson, (Eds.), Plant Fibers, Springer, Berlin, 1989, pp. 12-36. [3] B.J.H. Stevens, R.R. Selvendran, C.E. Bayliss and R. Turner, Degradation of cell wall material of apple and wheat bran by human faecal bacteria in vitro, J. Sci. Food Agric., 44 (1988) 151-156. [4] A. Bacic, P.J. Harris and B.A. Stone, Structure and function of plant cell walls, in: J. Preiss, (Ed.), The Biochemistry of Plants, Vol. 14, Carbohydrates, Academic Press, San Diego, 1988, pp. 297-371. [5] L.R. Ferguson, A.M. Roberton, M.E. Watson, P. Kesteli and P.J. Harris, The adsorption of a range of dietary carcinogens by a-cellulose, a model insoluble dietary fiber, Mutat. Res., 319 (1993) 257-266. [6] P.J. Harris, A.M. Roberton, H.J. Hollands and L.R. Ferguson, Adsorption ofa hydrophobic mutagen to dietary fibre from the skin and flesh of potato tubers, Mutat. Res., 260 (1991) 203-213. [7] A.M. Roberton, L.R. Ferguson, H.J. Hollands and P.J. Harris, Adsorption ofa hydrophebic mutagen to five contrasting dietary fiber preparations, Mutat. Res., 262 (1991) 195-202. [8] P.D. Smith-Barbaro, D. Hansen and B.S. Reddy, Carcinogen binding to various types of dietary fiber, J. Natl. Cancer Inst., 67 (1981) 495-497. [9] K. Wakabayashi, M. Nagao, H. Esumi and T. Sugimura, Food-derived mutagens and carcinogens, Cancer Res. (Suppl.), 52 (1992) 2092s-2098s. [10] L.R. Ferguson, A.M. Roberton, M.E. Watson, C.M. Triggs and P.J. Harris, The effects of a soluble-fibre polysaccharide on the adsorption of carcinogens to insoluble dietary fibres, Chem.Biol. Interact., 95 (1995) 245-256. [11] Medicinal Chemistry Project, MedChem Software Manual, Release 3.51. Pomona College, CA, 1987. [12] L.R. Ferguson, P.J. Harris, H.J. Hollands and A.M. Roberton, Effects of bile salts on the adsorption of a hydrophobic mutagen to dietary fiber, Mutat. Res., 245 (1990) 111-117. [13] P.E. Kolattukudy and K.E. Espelie, Biosynthesis of cutin, suberin, and associated waxes, in" T. Higuchi, (Ed.), Biosynthesis and Biodegradation of Wood Components, Academic Press, Orlando, 1985, pp. 161-207. [14] P.J. Harris, A.M. Roberton, M.E. Watson, C.M. Triggs and L.R. Ferguson, The effects of solublefiber polysac~harides on the adsorption of a hydrophobic carcinogen to an insoluble dietary fiber, Nutr. Cancer, 19 (1993) 43-54. [15] D.F. Gray, M.A. Eastwood, W.G. Brydon and S.C. Fry, Fermentation and subsequent disposition of 14C-labelled plant cell wall material in the rat, Br. J. Nutr, 69 (1993) 189-197. [16] A.M. Stephen and J.H. Cummings, Mechanism of action of dietary fibre in the human colon, Nature (London), 284 (1980) 283-284. [17] K. Watanabe, B.S. Reddy, J.H. Weisberger and D. Kritchevsky, Effect of dietary alfalfa, pectin and wheat bran on azomethane or methylnitrosourea-induced colon carcinogenesis in F-344 rats, J. Natl. Cancer Inst., 63 (1979) 141-145. [18] C. Sterling, Sclereid development and the texture of Bartlett pears, Food Res., 19 (1954) 433-443. [19] J.E. Harman, Feijoa fruit: growth and chemical composition during development, NZ J. Exp. Agric., 15 (1987) 209-215.
Chemico-Biological Interactions 100 (1996) 13-25
The adsorption of heterocyclic aromatic amines by model dietary fibres with contrasting compositions Philip J. Harris *a, Christopher M. Triggs b, Anthony M. Roberton a, Mark E. Watson c, Lynnette R. Ferguson c aSchool of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand bDepartment of Statistics, The University of Auckland, Private Bag 92019, Auckland, New Zealand CCancer Research Laboratory, The University of Auckland, Private Bag 92019, Auckland, New Zealand
Received 4 July 1995; revision received 13 November 1995; accepted 16 November 1995
Abstract
It is often recommended that consumption of dietary fibre should be increased to protect against colorectal cancer. However, although more than 95% of dietary fibre is contributed by whole plant cell walls, very little experimental work has been done using whole plant cell walls. These may protect by adsorbing carcinogens, thus lowering their effective concentration in the alimentary tract, and by carrying the carcinogens out of the body in the faeces. However, plant cell walls vary widely in their composition and physical properties, and not all cell walls will necessarily have protective properties. We therefore isolated 4 plant cell-wall preparations with contrasting compositions as models of the types of cell walls that occur in the diet. We investigated the abilities of these preparations to adsorb in vitro 6 heterocyclic aromatic amines (HAAs). HAAs occur in the human diet and several are colon carcinogens, at least in rats. We found that the ability of the HAAs to adsorb to the plant cell walls increased with increasing hydrophobicity of the HAA, measured as the calculated logarithm of the partition coefficient between l-octanol and water (C logP). A cell-wall preparation containing
Abbreviations: AOAC, Association of Official Analytical Chemists; C logP, calculated logarithm of the partition coefficient; DNP, 1,8-dinitropyrene; HAA, heterocyclic aromatic amine; IQ, 2-amino-3methylimidazo[4,5-/]quinoline; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-j]quinoline; MeIQx, 2-amino3,8-dimethylimidazo[4,5-/]quinoxaline; PhIP, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine; Trp-P1, 3-amino-l,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2, 3-amino-l-methyl-5H-pyrido[4,3-b]indole. * Corresponding author.
0009-2797/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(95)03682-C
14
P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
mainly the walls of parenchyma cells (the most common cell type in food plants) had only poor adsorptive ability. A cell-wall preparation from commercial cork had the best adsorptive ability. This preparation was the most hydrophobic of those examined because the cell walls contained the polymer, suberin, together with associated waxes. The preparation modelled suberized cell walls which occur in the diet, for example in potato skins. The other two cellwall preparations contained another hydrophobic polymer, lignin, and had intermediate adsorptive abilities which were not significantly different from one another. These preparations modelled lignified cell walls which occur in the diet, for example in wheat bran. Our results indicate that suberized and lignified cell walls may be important in protecting against colorectal cancer.
Keywords: Carcinogens; Colorectal cancer; Dietary fibres; Heterocyclic aromatic amines; Hydrophobicity; Plant cell walls
1. Introduction
In attempts to reduce the incidence of colorectal cancer and a range of other diseases, many Western countries run public health campaigns which recommend eating more dietary fibre. However, this generalised recommendation frequently does not recognise that the term dietary fibre encompasses a wide range of heterogeneous and complex materials with widely different properties. Some of these materials almost certainly protect against colorectal cancer, while others probably do not. We have recently defined dietary fibre as follows: 'dietary fibre is composed of plant cell walls and components obtained from these walls. It also includes nonstarch polysaccharides from sources other than plant cell walls' [1]. Two broad categories of dietary fibres, soluble and insoluble, are often determined analytically, depending on their solubility in water and buffer solutions. Except for cellulose, many of the polysaecharides in plant cell walls are soluble in water after they have been extracted from the walls (soluble dietary fibres). However, because of the crosslinks between the cell-wall components, these polysaccharides are usually waterinsoluble when within the cell wall, and make the whole cell walls mainly waterinsoluble under the physiological conditions in the gastrointestinal tract (insoluble dietary fibres) [2]. Soluble dietary fibres (soluble-fibre polysaccharides) are widely used as food additives (thickeners, stabilizers, emulsifiers and gelling agents) and, as well as being extracted from plant cell walls (e.g. pectin preparations), they are also obtained from microorganisms, from the cell walls of seaweeds, and from plant sources other than cell walls (e.g. the outer layers of some seeds) [1]. In Western diets more than 95% of the dietary fibre is contributed by whole plant cell walls [3]. Food plants contain a range of different cell types, the walls of which differ in their structure, composition and physical properties. Furthermore, the composition of the walls of a particular cell type can be quite different in different species of food plants [4]. It is unlikely that these different types of plant cell walls are equally effective in protecting against colorectal cancer. Animal experiments designed to test the effects of dietary fibres on the development of colorectal cancer have mostly not tested the effects of preparations of whole
P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
15
plant cell walls. Instead, these experiments have frequently tested dietary fibres that usually occur in only small amounts in Western diets [1]. These dietary fibres include soluble-fibre polysaccharides from a variety of sources, and preparations of cellulose. But although some of these dietary fibres tested are extracted from plant cell walls, it is unlikely that they will have the same effects as when they are part of the whole cell walls. In addition, the effects of cereal brans have also been tested frequently. Cereal brans are often erroneously described as dietary fibres, but in fact are only sources of dietary fibres. For example, a reference wheat bran contained only 43.9% dietary fibre determined by the AOAC method [1]. Thus components other than dietary fibres could be responsible for any protective properties shown by such brans. One mechanism by which plant cell walls may prevent the development of colorectal cancer is by adsorbing carcinogens in the digestive tract [5-8]. If these cell walls are not significantly degraded by bacterial enzymes in the colon, the carcinogens may be carried out of the body adsorbed onto the cell walls, thus lowering the effective concentrations available to initiate or promote cancerous changes in the gut mucosal cells. The most common cell type in food plants is the parenchyma cell which has walls composed mainly of polysaccharides. We have previously obtained cell-wall preparations from this cell type from potato tubers [6] and cabbage leaves [7] and tested in vitro the ability of the hydrophobic, environmental carcinogen 1,8dinitropyrene (DNP) to adsorb to these cell walls. We found that DNP adsorbed only weakly to these cell walls. In the present study, we used a parenchyma cell-wall preparation from potato tubers as a model for parenchyma cell walls in the diet. In contrast to parenchyma cell walls, the walls of some cell types from food plants contain, in addition to polysaccharides, the hydrophobic polymers lignin or suberin which make the cell walls hydrophobic. Because these cell types are present in food plants in only small proportions it is technically much easier to obtain preparations composed mainly of lignified or suberized cell walls from model plant material that is not usually eaten by humans. We previously showed that model cell-wall preparations which contained mainly lignified cell walls (obtained from wheat straw and mature cabbage stems) and model cell-wall preparations that contained mainly suberized cell walls (obtained from potato skins and commercial cork) adsorbed DNP in vitro much more effectively than did parenchyma cell walls [6,7]. Commercial cork is an excellent source of suberized cell walls without the contamination from parenchyma cell walls that occurs to some extent in cell-wall preparations from potato skins. We used cell-wall preparations from these plant materials as models of lignified and suberized cell walls which do occur in the diet (see Discussion). These models are important in enabling us to test experimentally the role which lignified and suberized cell walls may play in the prevention of colorectal cancer. We do not imply that wheat straw, mature cabbage stems, or commercial cork are normal dietary components. We have now extended these studies to test the effectiveness of model preparations of parenchyma cell walls, lignified cell walls, and suberized cell walls to adsorb in vitro 6 heterocyclic aromatic amines (HAAs) which occur in the human diet. At least
16
P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
in rats, IQ, MelQ and PhlP are colon carcinogens [9]. We have also related the abilities of HAAs to adsorb to the model cell-wall preparations to the hydrophobicities Of the HAAs. Although HAAs are known to be converted to reactive products by N-oxidation [9], we consider that adsorption of the HAAs to cell walls very likely occurs at an early point in the passage through the gastrointestinal tract and before the HAAs are metabolized. We have previously shown [51 that the ability of a carcinogen to adsorb to a preparation of a-cellulose was related to the hydrophobicity of the carcinogen. In the present study we have selected HAAs to span a range of hydrophobicities. 2. Materials and methods 2.1. Chemicals
The HAAs, IQ, MelQx, PhlP and Trp-P-1 were obtained from Toronto Research Chemicals Inc., Downsview, Ontario, Canada, and MelQ and Trp-P-2 were the kind gift of Professor T. Sugimura, National Cancer Center Research Institute, Tokyo, Japan. Their full chemical names and structures are shown in Fig. 1. HPLC grade chromatography solvents were purchased from Alphatech, Auckland, New Zealand. CH3
CH3
~
~
/ H.
N
NH2 CH3
Trp-P-1 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole NH2
~
~
/
N
NH2
H
TrpLP-2 3-amino-1-methyl-5H-pyrido[4,3-b]indole NH2
N@'--CH3
IQ 2-amino-3-methylhnidazo[4,5-J]quinoline
MelQ 2-amino-3,4-dimethylimidazo[4,5-J]quinoline
NH2 H3C~ .,N,~ ~
IN~CH3
-N'LJ
MeIQx 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
PhlP 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
Fig. 1. The names,abbreviationsand structuresof the heterocyclicaromaticaminesidentifiedin the text.
P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
17
2.2. Hydrophobicities o f the H A A s
The calculated logarithm of the partition coefficient (C logP) between 1-octanol and water was used as a measure of the hydrophobicity of each HAA and was calculated from the structure of the HAA by an additive-constitutive procedure using the computer program, CLOGP3 [5,10,11]. 2.3. Model cell-wall preparations
Model cell-wall preprations from commercial cork, from the stem internodes of wheat straw, and from the woody secondary xylem cylinder of mature cabbage stems were obtained as described by Roberton et al. [7]. The preparation of parenchyma cell walls from the flesh of potato tubers was obtained as described by Harris et al. [61. 2.4. H P L C
HPLC of the HAAs was performed using a system which comprised a Waters 510 pump (Waters Assoc., Milford, MA, USA), a Gilson Model 231-401 auto-sampling injector, a UV absorption detector (Model 111B, Gilson Medical Electronics, Middleton, WI, USA), a fluorescence detector (Model RF-530, Shimadzu, Kyoto, Japan), a diode array detector (Model 4120 Philips, Cambridge, UK), and a Waters Cis 4 # Novapak stainless steel column (150 x 3.9 mm, Phenomenex, Torrance, CA, USA). The HAAs were eluted with a mobile phase consisting of 25 mM sodium dihydrogen phosphate (adjusted to pH 3.0 with 25 mM orthophosphoric acid) and methanol (IQ, 9:1 v/v; MelQx, 4:1 v/v; PhlP, 3:2 v/v; Trp-P-l, and Trp-P-2, 1:1 v/v). For MelQ, the mobile phase was the same as for the PhlP except that the pH of the 25 mM sodium dihydrogen phosphate was adjusted to 6.5. The flow rates were 1-3 ml/min for all HAAs. The HAAs were detected using the UV absorption detector (at 254 nm), the fluorescence detector (Trp-P-l, excitation 300 nm, emission 460 nm; Trp-P-2, excitation 356 nm, emission 485 nm). The diode array detector was used (at wavelengths from 190-390 nm) for all experiments involving the cell-wall preparation from commercial cork. The use of the diode array detector with the cork cell-wall preparation was necessary because material that absorbed in the UV leached from the preparation. The HAAs were detected at wavelengths that minimised interference from this material. Data acquisition and integration were performed using a Unicam 4880 Chromatography Data Handling System (Unicam Ltd, Cambridge, UK). We have previously shown [5,10] that the UV absorption, diode array and fluorescence detectors gave a linear response curve for the HAAs over the concentration range used. 2.5. Incubation o f H A A s with model cell-wall preparations in P B S
All incubations were done in acid-washed conical glass centrifuge tubes (capacity 12 ml). The model cell-wall preparations (10 rag) were weighed into the tubes and
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P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
1.96 ml of phosphate-buffered saline (PBS) (20 mM sodium phosphate buffer, pH 6.5, containing 130 mM sodium chloride) added to each tube. Each tube stood for 30 rnin to allow the cell-wall preparations to hydrate, then 40 ttl of a solution of the HAA in methanol was added, giving final HAA concentrations of 0.5, 1.0, 2.5 and 5/zg/ml (0.1, 0.2, 0.5 and 1.0/~g/mg of cell-wall preparation), and the contents of the tubes were mixed using a vortex mixer. The tubes were shaken (120 rev./min ) on an orbital shaker for 55 min at 37°C and centrifuged (2500 x g for 5 min). The supernatants were filtered using an Acrodisc (Polysulfone) disposable filter unit (diameter 13 mm, pore size 0.2 #m; Gelman Sciences, Ann Arbor, MI) and 3 x 100 /zl aliquots taken. Control incubations were also done in which the HAAs were incubated with no cell-wall preparations. In these control incubations aliquots were also taken at zero time. There was no evidence for degradation of any of the HAAs during incubation. The amounts of HAAs adsorbed to the cell-wall preparations were determined from the difference between the concentration of HAAs in the supernatants in the presence of the cell-wall preparations and the supernatant concentrations measured in control tubes in the absence of cell-wall preparations. The amounts adsorbed by the cell walls are expressed as percentages of the total amounts present in control experiments without cell walls. The amounts of HAAs adsorbed on the tube walls were determined as we have previously described, except that acetonitrile was used to recover the HAAs [12]. However, preliminary experiments showed that none of the HAAs were adsorbed to the walls of the glass tubes, and thus in subsequent experiments only the supernatant was sampled.
2.6. Statistical methods The percentages of the HAAs that were adsorbed by the model cell-wall preparations were plotted on a logistic scale to stabilize variance. The percentages were plotted against the initial HAA concentrations (~g HAA/mg cell-wall preparation) plotted on a logarithmic scale. The resulting scatterplots showed that the relationship between these two variables may be approximated closely by a straight line and so (logit) percentage adsorbed was regressed on (log) initial HAA concentration. 3. Results
3.1. Hydrophobicities of the HAAs The hydrophobicities of the HAAs used in this study, calculated as C logP values, are shown in Table 1.
3.2. Effect of initial HAA concentration on the percentage of HAAs adsorbed by the model cell-wall preparations The possible effect that the initial HAA concentration had on the total percentage of the HAAs adsorbed by the cell-wall preparations was examined. This was done by plotting the (logit) percentages adsorbed by the cell-wall preparations against the
P.J. Harris et aL /Chemico-Biological Interactions 100 (1996) 13-25
19
Table 1 Hydrophobicities of HAAs and their estimated percentage adsorption to cell-wall preparations HAA
MelQx IQ MelQ PhlP Trp-P-2 Trp-P-I
C logP
Estimated percentage adsorption of HAAs by cell-wall preparations
1.078 1.323 1.822 2.294 2.731 3.230
Potato flesh
Wheat straw
Cabbage stem
Commercial cork
7 (3.5) 6 (1.5) 14 (3.2) 25 (3.9) 28 (1.5) 17 (10.1)
30 (2.7) 31 (1.9) 97 (1.8) 61 (5.0) 64 (1.2) 84 (0.48)
18 (4.0) 45 (5.9) 54 (I.1) 56 (3.9) 86 (1.8) 62 (0.42)*
74 75 96 85 91 92
(6.0)* (4.6)* (2.6) (3.6) (3.6) (0.56)*
*Estimated percentage of HAAs adsorbed at a HAA concentration of 0.3 #g/mg cell-wall preparation obtained from the fitted regression line when the slope is significantly different from zero (see Fig. 2). All other percentages are mean values calculated over the four HAA concentrations used. For these combinations of HAAs and ceil-wall preparations the slope of the fitted regression line does not differ from zero (P > 0.05) (see Fig. 2). Standard errors are shown in parentheses. All calculations were done on a Iogit scale to stabilis¢ variance and the results were back-transformed to a percentage scale.
log o f the initial c o n c e n t r a t i o n o f H A A s (/~g H A A / m g cell-wall p r e p a r a t i o n ) . Fig. 2 s h o w s the p e r c e n t a g e o f I Q a d s o r b e d by the cell-wall p r e p a r a t i o n s p l o t t e d against the initial c o n c e n t r a t i o n o f IQ. A d s o r p t i o n o n l y to the c o r k cell-wall p r e p a r a t i o n was significantly affected by the initial I Q c o n c e n t r a t i o n . H e r e , the p e r c e n t a g e o f I Q ad-
90 '0
............ ....................0 ................
75 ........................................................................ =:::::::::Z'O.................................. 0 ....
"'0 0
50
"'0
0
Ii ~
25 10 5
-V-
- - - %
A -----%.%------
A- .............
A
2.5 O. 10
0.20 0.50 ,ug IQ/mg cell wall
1.0
Fig. 2. The effect of the concentration of IQ (t~g/mgcell-wall preparation) at the beginning of the incubation on the percentage of IQ that was adsorbed by the model cell-wall preparations at the end of the incubation. For each cell-wall preparation, the fitted regression line and the line through the mean percentage (logit %) are plotted. A, potato; V, wheat straw; 13, cabbage; O, cork.
20
P.J. Harris et al. /Chemico-Biological Interactions 100 (1996) 13-25
sorbed decreased with increasing initial IQ concentration (P = 0.03). For most of the other combinations of HAAs and cell-wall preparations, the percentage of HAA adsorbed by the cell-wall preparations was also unaffected by the initial concentration of the HAA. Thus, the slopes of the fitted regression lines were mostly not significantly different from zero (i.e. the data can be adequately summarized by a horizontal line). With the other HAAs, a significant effect of initial HAA concentration on the percentage of HAA adsorbed by the cell-wall preparations was found only for the adsorption of MelQx and Trp-P-1 to the cork cell-wall preparation (P = 0.04 and 0.03, respectively), and for the adsorption of Trp-P-1 to the cabbage cell-wall preparation (P = 0.01). For the adsorption of Trp-P-1 to the cork cell-wall preparation there was increased adsorption with increasing initial Trp-P-1 concentrations. For the other combinations of HAAs and cell-wall preparations where there was a significant effect of initial HAA concentration, there was decreased adsorption with increasing initial HAA concentrations. In addition, for the cabbage cell-wall preparation there was some evidence that the adsorption of MelQ and Trp-P-2 decreased with increasing initial concentration (P = 0.046 and 0.059, respectively). Similarly, for the cork cell-wall preparation there was weak evidence that the adsorption of PhlP and TrpP-2 decreased with increasing initial concentration (P = 0.059 and 0.075). However, even in combinations where the percentage of HAA adsorbed by the cell-wall preparations was significantly affected by the initial HAA concentration, the differences between the percentages of HAA adsorbed at initial concentrations of 0.1 and 1.0 t~g HAA/mg cell wall were small compared to the differences in percentages adsorbed between the different cell-wall preparations. Effectively, we could regard the percentages of HAAs adsorbed by the cell-wall preparations as being independent of the initial HAA concentrations. 3.3. Estimated percentage adsorption of HAAs at a single HAA concentration To enable a comparison to be made of the adsorptive abilities of the four model cell-wall preparations, the percentage of each HAA adsorbed to each cell-wall preparation was estimated at a HAA concentration approximately half way along the range used (0.3 #g HAA/mg cell wall). The mean percentage adsorptions over the four HAA concentrations were also calculated (Table 1). These mean percentages were indistinguishable from the estimated percentages at 0.3/~g HAA/mg cellwall preparation, even for those combinations of HAA and cell-wall preparation where the initial HAA concentration had a significant effect on the percentage adsorbed. The percentage adsorptions ranged from 6% (for the adsorption of IQ by the potato flesh cell-wall preparation) to 97% (for the adsorption of MelQ by the wheat straw cell-wall preparation). 3.4. The relationship between hydrophobicities of the HAAs and estimated percentage adsorption to the model cell-wall preparations A similar statistical approach was used to that shown above to describe the rela-
21
P.J. Harris et aL / Chemico-Biological Interactions 1190(1996) 13-25
95 t (b) 7 o--£14
.
1.0
1.5
.
. 2.0
V i
. 2.5
3.0
1.0
1.5
C log P
95
~
2.0
2.5
3.0
C log P
(c) .~ 75 I~)- 0
1.0
1.5
2.0
2.5
C log P
3.0
1.0
1.5
2.0
2.5
3.0
C log P
Fig. 3. The effectof the hydrophobicityof the HA.&(expressedas the calculatedlogarithmof the partition coefficientbetween 1-octanoland water) (C log/') on the percentageof the HAA adsorbed by modelcellwall preparations(at an initial concentrationof 0.3 t~g HAA/mgcell-wall preparation)from: (a) potato flesh, (b) wheat straw, (c) cabbagestem, and (d) commercialcork, with fitted regressionlinessuperimposed. The valuefor the adsorptionof MelQ (C logP = 1.822)by the cell-wallpreparation from wheat straw was regarded as an outlier and was excludedfrom the determination of the regressionline.
tionship between the estimated percentage of HAA adsorbed and the initial HAA concentration. Fig. 3 (a, b, c, and d) shows the estimated (logit) percentage adsorptions (at an initial concentration of 0.3/~g HAA/mg cell-wall preparation) of these HAAs by potato, wheat straw, cabbage, and cork cell-wall preparations plotted against, and regressed on the hydrophobicities (C logP values) of the HAAs. With all the model cell-wall preparations, the estimated percentage adsorptions increased with increasing hydrophobicity. The slopes of the lines were compared and found not to differ (P = 0.62). However, the positions of the lines varied. Even hydrophobic HAAs adsorbed only weakly to the potato cell-wall preparation. At the other extreme, all HAAs, including hydrophilic ones, adsorbed strongly to cork cell-wall preparations. 3.5. Comparison o f the adsorptive abilities o f the four model ceil-wall preparations
The relative adsorptive abilities of the four model cell-wall preparations were compared by calculating the predicted percentage adsorptions of hypothetical carcinogens with C logP values of 2.15 (which is the mid-value of the HAAs used) and 0.0. The predicted percentage adsorptions are shown in Table 2. For both hypothetical carcinogens, the cell-wall preparation from commercial cork had a significantly
22
P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
Table 2 Predicted percentage adsorption of hypothetical carcinogens Cell-wall preparation
Potato flesh Wheat straw Cabbage stem Commercial cork
C IogP = 0
C logP = 2.15
%
logit(%)
%
lo~t(%)
3 17 17 55 S.E.D.
(-3.51) (-1.58) ( - 1.57) (0.19) (0.36)
15 56 56 88 S.E.D.
(-!.71) (0.22) (0.23) (1.99) (0.36)
All calculations were done on percentages transformed to the logit scale and then back transformed to give predicted percentages. Values in parentheses are the predicted logit values.
greater adsorptive ability than did the preparations from cabbage stem and wheat straw (P < 0.001). The cell-wall preparations from cabbage stem and wheat straw were not significantly different in their adsorptive abilities (P > 0.05). However, both of these preparations had significantly greater adsorptive abilities than had the cell-wall preparation from potato flesh (P < 0.001). 4. Discussion
The present study shows that the ability of HAAs to adsorb to plant cell walls depends on the hydrophobicity of the HAA (measured as C logP) and the hydrophobicity of the cell walls. The ability of a HAA to adsorb to each of the model cell-wall preparations increased with increasing hydrophobicity. We have also previously shown that this was so for the adsorption of HAAs to an a-cellulose preparation which we used as a model insoluble dietary fibre [5]. We were able to compare the adsorptive abilities of the four model cell-wall preparations by calculating the predicted adsorptions at particular C logP values. These calculations showed that the cell-wall preparation from commercial cork had the best adsorptive ability. Commercial cork is from the outer bark of the cork oak (Quercus suber) and consists of cork cells. Because the walls of these cells contain the complex hydrophobic polymer, suberin, together with associated waxes, the cellwall preparation from commercial cork was the most hydrophobic we examined. Suberin consists of aliphatic polyester domains covalently attached to phenolic, lignin-like domains [13]. We have previously shown that cell-wall preparations from commercial cork and from potato skin (which is rich in cork cells) strongly adsorbed DNP [6,7]. The model cell-wall preparations from the woody secondary xylem cylinder of mature cabbage stems and from the stem internodes of wheat straw contain mainly lignified cell walls. Lignin is a complex hydrophobic polymer, and in lignified cell walls of flowering plants the main non-cellulosic polysaccharides are heteroxylans which are insoluble in water [4]. These two model cell-wall preparations had similar adsorptive abilities, but were less effective than the model cell-wall preparation from
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23
commercial cork. Our previous work showed that these preparations also adsorbed DNP strongly, but were not as effective as the cell-wall preparation from commercial cork [7]. It is likely that lignified or suberized cell walls, as well as being effective at adsorbing carcinogens, are not degraded to any extent in the colon [1]. Thus it is likely that carcinogens adsorbed on these cell walls will be carried out of the body on the cell walls. The model cell-wall preparation from the flesh of potato tubers, which contained mainly parenchyma cell walls, had the least adsorptive ability of the four cell-wall preparations tested. This is consistent with our previous finding that a parenchyma cell-wall preparation from potato tubers (and a similar parenchyma cell-wall preparation from cabbage leaves) adsorbed DNP only weakly [6,7]. The present study shows that a parenchyma cell-wall preparation from potato tubers also adsorbed HAAs only weakly. These cell walls are rich in pectic polysaccharides, a complex class of polysaccharides comprising a family of acidic polysaccharides (rhamnogalacturonans) and several neutral polysaccharides (arabinans, galactans, and arabinogalactans) [4]. Pectic polysaccharides are partially soluble in water and we previously found that a phosphate-buffered saline extract of potato parenchyma cell walls containing such polysaccharides held DNP in solution [6]. In aqueous solutions in the absence of such soluble polysaccharides, the DNP came out of solution. We suggested that the soluble pectic polysaccharides reduced the adsorption of DNP to the cell walls. In further studies we showed that a preparation of pectic polysaccharides from citrus fruits, and other soluble-fibre polysaccharides, reduced the adsorption of DNP to c~-cellulose [14]. However, unlike DNP (C logP---4.384), Trp-P-1 (C logP = 3.230), which was the most hydrophobic of the HAAs that we examined, was sufficiently hydrophilic to remain in solution in phosphate-buffered saline. Furthermore, the soluble-fibre polysaccharide, gum arabic, did not reduce the adsorption of Trp-P-1 to a-cellulose [10]. This suggests that the weak adsorption of HAAs to the model parenchyma cell-wall preparation from potato tubers may simply be because the cell walls contain mainly hydrophilic components. If carcinogens are adsorbed by cell walls and these cell walls are subsequently degraded by bacterial enzymes in the colon, the carcinogens will not be carried out of the body adsorbed in this way. Thus cell walls that are degraded in the colon will not protect against colorectal cancer by this mechanism. There is good evidence that parenchyma cell walls are extensively degraded in the colon [3,15,16]. This indicates that if parenchyma cell walls protect against colorectal cancer, they do so by a different mechanism. It should be noted, however, that there is no evidence available that they do protect. The relationship between the hydrophobicity of HAAs and their ability to adsorb to dietary fibres, which we have reported previously [5], has been further demonstrated here. The techniques we have used in this paper for analysing the results enable the results to be extended to other classes of carcinogens. The abilities of these other carcinogens to adsorb to the four model cell-wall preparations can be predicted from their hydrophobicities (C logP values). Compared to the HAAs which we have used in the present study (and which have been identified in Western diets [5,9]), many of the carcinogens used in animal exper-
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P.J. Harris et al. / Chemico-Biological Interactions 100 (1996) 13-25
iments to test the ability of dietary fibres to protect against colorectal cancer are much more hydrophilic (e.g. [17]). These carcinogens include N-nitroso-Nmethylurea (NMU) and 1,2-dimethylhydrazine (DMH) which have C logP values of -0,204 and -1.368, respectively. Table 2 shows that the predicted percentage adsorptions for a carcinogen with a C logP value of zero are 55, 17, 17, and 3 for the model cell-wall preparations from commercial cork, cabbage stem, wheat straw and potato flesh respectively. We have found it difficult to determine experimentally the adsorption of NMU to the four model cell-wall preparations because it is unstable, especially at 37°C [5]. However, even when the experiments were carried out at 22°C, at which temperature the extent of breakdown was reduced, we found that NMU adsorbed significantly ( - 9 % ) only to the cell-wall preparation from commercial cork. We have also previously found that NMU was poorly adsorbed by a-cellulose [5]. From its C logP value, we also predict that DMH will adsorb to the model cell-wall preparations to only a small extent. Thus protection by dietary fibres in animal experiments in which such hydrophilic carcinogens have been used is probably the result of mechanisms other than adsorption and may or may not be relevant to naturally occurring and food-derived carcinogens in humans. The present study, in which model cell-wall preparations were examined, indicates that it may be desirable to increase consumption of food plants that contain lignified or suberized cell walls and which are the most effective absorbers of hydrophobic carcinogens, including HAAs. Such cell walls should be beneficial in removing these carcinogens from potential contact with colonic mucosal cells. In Western diets these cell walls usually occur in only small amounts [1]. However, sclereids or stone cells, which have thick lignified cell wails, occur in a number of species of fruits, for example in pears [18] and in feijoas [19]. A number of cell types with lignified cell walls also occur in some cereal brans, e.g. in wheat bran. Indeed, wheat bran is an important source of lignified cell walls in Western diets. Cell types that have lignified walls sometimes develop as the food plant matures, but lignified cell walls can make the plants less palatable. For example, sclerenchyma fibre cells with thick lignified walls, which develop in asparagus shoots as they mature, give the shoots a tough, stringy texture. Suberin, found in the walls of cork cells, occurs not only in the skins of potatoes, but also in the skins of many other tubers and root vegetables. Thus, increased consumption of foods that contain suberized or lignified cell walls (e.g. potato skins or wheat bran) may be particularly important in the prevention of colorectal cancer.
Acknowledgements We are grateful to the Auckland Medical Research Foundation, the Cancer Society of New Zealand, the Provincial Grand Rights of New Zealand, and the New Zealand Lottery Grants Board for financial support during the course of this work. We thank J.H. Bentley for critically reading the manuscript.
References [i] P.J. Harris and L.R. Ferguson, Dietary fibre: its composition and role in protection against colorectal cancer, Mutat. Rcs., 290 (1993) 97-110.
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[2] S.C. Fry, Analysis of cross-links in the growingcell walls of higher plants, in: H.-F. Linskens and J.F. Jackson, (Eds.), Plant Fibers, Springer, Berlin, 1989, pp. 12-36. [3] B.J.H. Stevens, R.R. Selvendran, C.E. Bayliss and R. Turner, Degradation of cell wall material of apple and wheat bran by human faecal bacteria in vitro, J. Sci. Food Agric., 44 (1988) 151-156. [4] A. Bacic, P.J. Harris and B.A. Stone, Structure and function of plant cell walls, in: J. Preiss, (Ed.), The Biochemistry of Plants, Vol. 14, Carbohydrates, Academic Press, San Diego, 1988, pp. 297-371. [5] L.R. Ferguson, A.M. Roberton, M.E. Watson, P. Kesteli and P.J. Harris, The adsorption of a range of dietary carcinogens by a-cellulose, a model insoluble dietary fiber, Mutat. Res., 319 (1993) 257-266. [6] P.J. Harris, A.M. Roberton, H.J. Hollands and L.R. Ferguson, Adsorption ofa hydrophobic mutagen to dietary fibre from the skin and flesh of potato tubers, Mutat. Res., 260 (1991) 203-213. [7] A.M. Roberton, L.R. Ferguson, H.J. Hollands and P.J. Harris, Adsorption ofa hydrophebic mutagen to five contrasting dietary fiber preparations, Mutat. Res., 262 (1991) 195-202. [8] P.D. Smith-Barbaro, D. Hansen and B.S. Reddy, Carcinogen binding to various types of dietary fiber, J. Natl. Cancer Inst., 67 (1981) 495-497. [9] K. Wakabayashi, M. Nagao, H. Esumi and T. Sugimura, Food-derived mutagens and carcinogens, Cancer Res. (Suppl.), 52 (1992) 2092s-2098s. [10] L.R. Ferguson, A.M. Roberton, M.E. Watson, C.M. Triggs and P.J. Harris, The effects of a soluble-fibre polysaccharide on the adsorption of carcinogens to insoluble dietary fibres, Chem.Biol. Interact., 95 (1995) 245-256. [11] Medicinal Chemistry Project, MedChem Software Manual, Release 3.51. Pomona College, CA, 1987. [12] L.R. Ferguson, P.J. Harris, H.J. Hollands and A.M. Roberton, Effects of bile salts on the adsorption of a hydrophobic mutagen to dietary fiber, Mutat. Res., 245 (1990) 111-117. [13] P.E. Kolattukudy and K.E. Espelie, Biosynthesis of cutin, suberin, and associated waxes, in" T. Higuchi, (Ed.), Biosynthesis and Biodegradation of Wood Components, Academic Press, Orlando, 1985, pp. 161-207. [14] P.J. Harris, A.M. Roberton, M.E. Watson, C.M. Triggs and L.R. Ferguson, The effects of solublefiber polysac~harides on the adsorption of a hydrophobic carcinogen to an insoluble dietary fiber, Nutr. Cancer, 19 (1993) 43-54. [15] D.F. Gray, M.A. Eastwood, W.G. Brydon and S.C. Fry, Fermentation and subsequent disposition of 14C-labelled plant cell wall material in the rat, Br. J. Nutr, 69 (1993) 189-197. [16] A.M. Stephen and J.H. Cummings, Mechanism of action of dietary fibre in the human colon, Nature (London), 284 (1980) 283-284. [17] K. Watanabe, B.S. Reddy, J.H. Weisberger and D. Kritchevsky, Effect of dietary alfalfa, pectin and wheat bran on azomethane or methylnitrosourea-induced colon carcinogenesis in F-344 rats, J. Natl. Cancer Inst., 63 (1979) 141-145. [18] C. Sterling, Sclereid development and the texture of Bartlett pears, Food Res., 19 (1954) 433-443. [19] J.E. Harman, Feijoa fruit: growth and chemical composition during development, NZ J. Exp. Agric., 15 (1987) 209-215.