- Email: [email protected]
The adsorption of a range of dietary carcinogens by α-cellulose, a model insoluble dietary fiber
Mutation Research, 319 (1993) 257-266
257
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
MUTGEN 01934
The adsorption of a range of dietary carcinogens by a-cellulose, a model insoluble dietary fiber Lynnette R. Ferguson a, Anthony M. Roberton b, Mark E. Watson a, Philip Kestell a and Philip J. Harris b a Cancer Research Laboratory and b School of Biological Sciences, The University of Auckland, Auckland, New Zealand
(Received 6 January 1993) (Revision received31 March 1993) (Accepted 25 May 1993)
Keywords: Adsorption; t~-Cellulose;Dietary fiber; Heterocyclicaromatic amines; Hydrophobicity;C log P
Summary One of the ways dietary fibers may protect against colorectal cancer is by adsorbing carcinogens and carrying them out of the digestive tract, thus lessening interaction of the carcinogens with the colonic tissue. We investigated this mechanism of action by testing in vitro the abilities of a range of carcinogens, including known animal colon carcinogens, to adsorb to a-cellulose, which we have used as a model insoluble dietary fiber. The carcinogens were Nonitroso-N-methylurea (NMU), benzo[a]pyrene (B[a]P) and a number of heterocyclic aromatic amines which have been found in heated foods. It was found that the ability of a carcinogen to adsorb to a-cellulose is strongly related to the hydrophobicity of the carcinogen measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C log P). The hydrophilic carcinogen, NMU, (C log P = -0.204), adsorbed only poorly, whereas the very hydrophobic carcinogen, B[a]P, (C log P = 6.124), adsorbed strongly. Carcinogens with intermediate hydrophobicities showed intermediate abilities to adsorb.
It is not known which carcinogens in the diet are responsible for the initiating event in human colorectal cancer. This makes it difficult to test theories to explain the protective effects of particular types of dietary fiber against the development of colorectal cancer, especially theories that involve an interaction between the dietary fiber and the carcinogen.
Correspondence: Dr. L.R. Ferguson, Cancer Research Laboratory, The Universityof Auckland, Private Bag 92019, Auckland, New Zealand.
We investigated one of the hypotheses for the mode of action of dietary fiber: that dietary fiber adsorbs the carcinogens responsible for the initiating events and transports them from the body and so decreases the concentration of carcinogen to which the colon epithelium is exposed (Cummings and Branch, 1982). We developed a model in vitro system, using 1,8-dinitropyrene (DNP) (a known mutagen and carcinogen) as the test carcinogen, to investigate the adsorption of carcinogens to dietary fiber (Roberton et al., 1990). Firstly, we investigated the adsorption of DNP to a-cellulose, which we used as a model insoluble
258 dietary fiber because it is available commercially in large quantities. Cellulose has also been shown in animal experiments to protect against colorectal cancer (Heitman et al., 1989) and it has been
suggested (Heitman and Cameron, 1990) that it could be used as a human dietary supplement. We then investigated the adsorption of DNP to a variety of other dietary fiber preparations
C,H 3
CH 3
I tt
Ctt 3 Trp-P-1 3-amino-l,4-dimethyl-5H-pyrido[4,3-blindole
"
Trp-P-2 3-amino-l-methyl-5H-pyrido[4,3-b]indole
NH2
~
.--4N---CH 3
--CH3 -N-
IQ 2-amino-3-methylimidazo[4,5-.t]quinoline
~
"UH3
MeIQ 2-amino-3,4-dimethylimidazo[4,5-f]quinoline
NH 2
cH3 H3C,,~ .,N~ ~
,N'CH3
MelQx 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
PhIP 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine NO2
B[alP Benzolalpyrene
H2N\ IN//0 ~C--N O ~CH3 NMU N-nitroso-N-methylurea
DNP 1,8-dinitropyrene
H3CXN_.N/CH3 H/
xH
DMH 1,2-dimethylhydrazine
Fig. 1. Names, abbreviationsand structures of the carcinogensidentified in the text.
259
4000
~
o
300020001000-
~
0"
o
200
400
600
NMU (~g/plate)
a~ 4000 3000-
20001000-
o
n'-
0
50
1O0
150
200
TrpP1 (ng/plate)
Fig. 2. Dose-response curves for mutagenicity expressed as revertant colonies in Salmonella typhimurium strains. (A) NMU and bacterial strain TA98. (B) Trp-P-1 and bacterial strain TA100. A range of concentrations of each of the carcinogens was dissolved in DMSO and added to bacteria using standard plate-incorporation protocols (Maron and Ames, 1983). Regression lines, computed for the linear part of the curve, are also shown. (Roberton et al., 1991; Harris et al., 1991; Ferguson et al., 1992). In this paper we report an extension of our studies, using the in vitro model system, to a wider range of carcinogens which may be appropriate to animal or human colorectal cancer. The carcinogens we used a r e illustrated in Fig. 1. N-Nitroso-N-methylurea (NMU) is a direct-acting molecule which causes colon cancer in rats (Watanabe et al., 1979). Trp-P-1, Trp-P-2, IQ, MelQ, MelQx and PhlP are all heteroc-yclic aromatic amines (HAAs), a group of carcinogens identified in cooked meat, fish, chicken and grain products, the amounts depending on time and temperature (Wakabayashi et al., 1992; Overvik and Gustafsson, 1990). They are suspected of being involved in the initiation of various types of
human cancers. However, the target organs vary among animal species used as models and in humans the target organs are unknown. At least in rats, IQ, M e l Q and PhlP are colon carcinogens (Wakabayashi et al., 1992) and each requires a metabolic activation event to form the proximal carcinogen. B[a]P is probably the most widely studied carcinogen and also requires metabolic activation to form the proximal carcinogen (Conney, 1982). It causes colonic lesions in rats, although the colon is not the primary target organ (O'Neill et al., 1990; Bird et al., 1984). In our previous in vitro studies with DNP, we used the Salmonella typhimurium mutagenicity assay (Maron and Ames, 1983) to quantify the DNP. We have also used this rapid assay for part of the present study as it is convenient to carry out and overcomes some methodological problems. However, because carcinogens differ in their mutagenicities, the concentration range over which the assay gives a linear dose-response curve varies widely with carcinogen. Thus, this assay cannot be used to compare the abilities of a series of carcinogens to adsorb to a-cellulose using the same initial concentrations for each of the carcinogens. To enable this comparison to be made, we therefore developed assay techniques for the carcinogens using high performance liquid chromatography (HPLC). The logarithm of the partition coefficient of compounds (log P ) is frequently used as a measure of the hydrophobicities of these compounds, and has been successfully used in Quantitative Structure Actitivity Relationships (QSAR) in many fields, including drug design (Martin, 1978; Kuchar, 1984). The partition coefficient of a compound is the equilibrium concentration of that compound in a non-polar solvent (usually 1-octanol) divided by the concentration in a polar solvent, usually water (Medicinal Chemistry Project, 1987). Although partition coefficients can be determined experimentally, it is often more convenient to calculate log P (C log P ) from the structure of the compound by an additive-constitutive procedure using a computer program. Our preliminary experiments showed that the ability of a carcinogen to adsorb to a-cellulose appeared, at least partially, to be related to the hydrophobicity of the carcinogen (calculated as
260 its C log P value). We have therefore investigated this relationship in the present study. Materials and methods
Chemicals. The HAAs were the kind gift of Professor T. Sugimura, National Cancer Institute, Tokyo, Japan. Their chemical names, abbreviations and structures are shown in Fig. 1. NMU was a gift of Dr. G. Rewcastle, Cancer Research Laboratory, University of Auckland. B[a]P (Cat. No. B1,008-0) was obtained from Aldrich Chemical Co, Milwaukee, USA and a-cellulose (Cat. No. C8022 from Canadian bleached hardwood pulp) from Sigma Chemical Company, St. Louis, MO. HPLC grade chromatography solvents were purchased from Alphatech, Auckland, New Zealand. Hydrophobicities of the carcinogens. The calculated logarithm of the partition coefficient (C log P ) between 1-octanol and water was used as a measure of the hydrophobicity of each carcinogen and was calculated from the structure of the carcinogen by an additive-constitutive procedure using the computer program, CLOGP3 (Medicinal Chemistry Project, 1987).
Bacterial mutagenicity assays. The Salmonella typhimurium plate-incorporation assay (Maron and Ames, 1983) was carried out using strain TA98 for the HAAs and strain TA100 for NMU and B[a]P. To minimise day-to-day variability in the sensitivity of the tester strains to the mutagens, which is due largely to the exact growth phase of the culture, we grew cells for quantitative work as follows. A 1-ml vial (2 × 10 s cells) was removed from storage at - 8 0 ° 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. Samples from experiments with NMU were plated directly, but the HAAs and B[a]P had $9 mix added (prepared from Aroclor-induced Sprague-Dawley rats by standard procedures, as described by Maron and Ames, 1983). Each experimental point was performed in triplicate on
TABLE 1 CONCENTRATIONS AND C log P VALUES OF CARCINOGENS USED IN THE MUTAGENICITYASSAYS Carcinogen
DNP a MelQ Trp-P-2 IQ MeIQx Trp-P-1 PhIP B[alP NMU
Concentration Amount in solution per plate (/xg/ml) (/zg/plate) 0.05 0.0025 0.06 0.003 0.2 0.01 0.4 1 2 40 200 4000
0.02 0.05 0.1 2 20 200
C log P value 4.384 1.822 2.731 1.323 1.078 3.230 2.294 6.124 - 0.204
a Studied previously(Roberton et al., 1990).
at least two separate occasions. The background number of revertant colonies was approximately 65 (range 54-78). Positive controls containing 4nitro-o-phenylenediamine (100 /.~g/plate) were included in all experiments to test the bacterial strain response. The dose-response curves for each carcinogen were determined by plotting the amounts of each carcinogen added to the plates against the numbers of revertant colonies. For plate colony numbers less than 1000, the plates were counted by autocounter and one plate from each triplicate set was confirmed by hand counting. For plate counts higher than 1000, hand counting was used. The dose-response curves for NMU and Trp-P-1 are shown in Figs. 2A and 2B respectively. For the assays the amounts of each carcinogen added to each plate was adjusted so that all measurements were made on the linear part of the curve (Table 1). The amount of carcinogen chosen for experiments was selected to give about 1500 revertants per plate, in order to optimise sensitivity when the carcinogen was distributed between the supernatant, pellet and tube walls. Using the dose-response curves we converted the numbers of revertant colonies into amounts of carcinogen, as previously described (Harris et al., 1991), using linear regression analysis as described by Venitt and Crofton-Sleigh (1979). Results have been expressed as the percentage of added carcinogen
261 found at the end of the experiment in various fractions.
HPLC. Because mutagenicity assays cannot be used to compare the abilities of a series of carcinogens to adsorb to a-cellulose using the same initial concentrations for each of the carcinogens we developed high performance liquid chromatography (HPLC) methods to assay the carcinogens. HPLC of the carcinogens 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 l l l B UV detector (Gilson Medical Electronics, Middleton, WI, USA), a Shimadzu (Model RF530) fluorescence detector (Shimadzu, Kyoto, Japan), and a C18 4 ~ Novapak stainless steel column (150 x 3.9 mm, Phenomenex, Torrance, CA, USA). Most of the carcinogens 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-P1, Trp-P-2, 1 : 1 v/v). For MelQ, the mobile phase was the same as for PhlP except that the pH of the 25 mM sodium dihydrogen phosphate solution was 6.5. For B[a]P, the mobile phase was acetonitrile. The flow rates were 1-3 ml rain-1 for all carcinogens and detection was either by UV absorption (254 nm) or by fluorescence (TrpP-l, excitation 300 nm, emission 460 nm; Trp-P-2, excitation 356 nm, emission 485 nm). Data acquisition and integration were performed using a Unicam 4880 Chromatography Data Handling System (Unicam Ltd, Cambridge, UK). Calibration curves were constructed by plotting peak area against standard concentrations and the equations for the best-fit straight lines were determined by linear regression analysis. Quantitation of unknown samples was achieved by measuring the peak area in the unknown sample and using the equations obtained from the linear regression to calculate a concentration.
(PBS) (20 mM sodium phosphate buffer, pH 6.5, containing 130 mM sodium chloride) added to each tube. The PBS was added 30 min before each experiment to allow time for the a-cellulose to hydrate. At time zero, 40 /~I of a solution of the carcinogen in DMSO (for mutagenicity assays) or methanol (for HPLC assays) was added and the contents of the tubes mixed using a vortex mixer. The tubes were shaken (120 rpm) on an orbital shaker for 55 min at 37°C and centrifuged (2500 g for 2.5 min). For mutagenicity assay of the carcinogen, 3 aliquots (50 /zl unless otherwise specified) of the supernatant were taken and assayed as described above. For HPLC assay of the carcinogen, 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-/xl aliquots taken. Control incubations were also done in which the carcinogens were incubated with no a-cellulose. In experiments with B[a]P, this carcinogen was recovered from the a-cellulose and the tube walls after incubation. The a-cellulose pellets and the walls of the glass incubation tubes were washed twice with dimethyl sulfoxide (mutagenicity assays) or acetonitrile (HPLC assays). Results
Hydrophobicities of the carcinogens. The hydrophobicities of the carcinogens used in this study, calculated as C log P values, are shown in Table 1. Compared to the HAAs, which have C log P values ranging from 1.078 (MelQx) to 3.230 (Trp-P-1), NMU (C log P = - 0 . 2 0 4 ) is hydrophilic, and B[a]P (C log P = 6.124) is hydrophobic. We also included the C log P value for 1,8-dinitropyrene (DNP) in Table 1, as we studied this extensively in previous publications (Roberton et al., 1990, 1991; Harris et al., 1991; Ferguson et al., 1992).
Incubation of carcinogens with a-cellulose in PBS. All incubations were done in acid-washed
Effects of incubation time on the mutagenicity of carcinogen solutions. There were no changes
conical glass centrifuge tubes (capacity 12 ml). a-Cellulose (usually 10 rag) was weighed into the tubes and 1.96 ml of phosphate-buffered saline
in the mutagenicities of control solutions of the carcinogens in PBS in the absence of a-cellulose during incubation times of up to 2 h (results not
262
lulose had an effect, this was rapid and could be detected immediately after mixing the carcinogen with the a-cellulose. A complete comparison of the abilities of the carcinogens to adsorb to a-cellulose (10 mg) was done using the HPLC assay which enabled the same initial concentrations to be used for each of the carcinogens (see below).
100 80
~ 6o ~ 4o 20
~
0
0 0
I O0 Time (min)
200
Fig. 3. The effect of incubation time on the mutagenicity of a solution of NMU in the presence and absence of a-cellulose. NMU (4 mg m1-1) was incubated at 37°C in PBS in the presence or absence of a-cellulose (10 mg). Aliquots of supernatant (50/zl) from the incubations containing a-cellulose (e) or in its absence (o) were removed at intervals for mutagenicity assay. Values represent the number of revertant colonies (minus those of negative controls) expressed as a percentage of the number obtained for a comparable sample of NMU diluted into DMSO and plated immediately (in the presence of $9 mix). The% of NMU on the walls of the incubation tube in the presence and absence of a-cellulose is shown by • and • respectively.
shown), with the exception of B[a]P and NMU. After 2 h, most of the mutagenicity of the B[a]P solution had been lost, but almost all was recovered from the walls of the incubation tube by washing with DMSO. This showed that the loss of mutagenicity was due to the adsorption of B[a]P onto the walls of the tube and not due to decomposition or inactivation (results not shown). In contrast, NMU was unstable under these conditions, and over 70% of the mutagenicity disappeared from solution over 2 h but could not be recovered (Fig. 3, open circles). When the incubation temperature was lowered from 37°C to 22°C, less than 20% of the mutagenicity was lost (results not shown).
Effects of a-cellulose on the mutagenicity of carcinogen solutions. Addition of a-cellulose (10 mg) had a variable effect on the mutagenicity of the carcinogen solution depending on the carcinogen examined. For example, a-cellulose had no effect on the mutagenicity of a solution of NMU at 37°C (Fig. 3, closed circles) or at 22°C, but reduced the mutagenicity of a solution of Trp-P-1 by 38% (data not shown). Where a-cel-
Effect of increasing concentrations of a-cellulose on the distribution of the carcinogens. The effects varied depending on the carcinogen. In the absence of a-cellulose, most of the B[a]P adsorbed to the walls of the incubation tube. However, addition of increasing amounts of acellulose resulted in increasing proportions of the total mutagenicity added being recovered from the a-cellulose and decreasing proportions being recovered from the walls of the incubation tube (Fig. 4A). Thus, after incubation (1 h) in the absence of a-cellulose, 80% of the added mutagenicity was associated with the tube wall and only 20% remained in the supernatant. After incubation in the presence of 10 mg of a-cellulose, over 80% of the added mutagenicity was recovered from the a-cellulose, and equal amounts of the remainder were on the tube walls and in the supernatant. In contrast to the B[a]P, NMU showed no evidence of adsorption to a-cellulose even at the highest concentrations of a-cellulose (data not shown). Although the extent of adsorption varied, each of the HAAs had a similar pattern of adsorption to a-cellulose. With increasing concentrations of a-cellulose, an increasing proportion of each carcinogen adsorbed to the a-cellulose. Trp-P-1 was the most effectively adsorbed of this group of carcinogens (Fig. 4B). For example, in the presence of 10 mg of a-cellulose, 62% of the added mutagenicity remained in the supernatant and 38% was adsorbed to the a-cellulose. The least effectively adsorbed was PhIP (data not shown). In the presence of 10 mg of a-cellulose, only 5% of the added mutagenicity was adsorbed. Effects of a-cellulose on carcinogen concentration in the supernatant using the same initial concentrations for each carcinogen. The effects of a-cellulose (10 mg) on the carcinogen concentration in the supernatant was determined using the
263
3000" 100 0.
80~
2000'
6040"6
20. 0 ~ - :
0
1'0
1'5
0
20
a-cellulose (mg/2ml) B
100.
~
ao-
~
40-
"6
20O~
0
5 10 15 (z-cellulose (mg/2ml)
20
Fig. 4. Effect of increasing concentrations of a-cellulose on the distribution of (A) B[a]P or (B) Trp-P-1. B[a]P (200 /~g ml - t ) or Trp-P-1 (2 /zg m1-1) was incubated in PBS with various concentrations of a-cellulose. Aliquots (100 /zl for B[a]P or 50/zl for Trp-P-1) were taken for mutagenicity assays after 1 h. Values represent the number of revertant colonies (minus those of negative controls) expressed as a percentage of the number obtained for a comparable sample of carcinogen diluted into DMSO and plated immediately. ( zx) Carcinogen associated with the a-cellulose. (o) Carcinogen in the supernatant. ([]) Carcinogen adsorbed to the tube walls.
same initial concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5/zg m1-1) for each of the carcinogens. The carcinogens were assayed using HPLC. The detectors used gave a linear response with concentration for each carcinogen over the concentration range used. For each carcinogen, there was the same percentage reduction in concentration in the supernatant after incubation with a-cellulose (within experimental error) at each initial concentration. This was true for each of the carcinogens used and Fig. 5 shows data for Trp-P-1. The percentage reductions were calculated by comparing the regression lines relating initial carcinogen concentrations and peak areas from supernatant aliquots taken in the presence and
,
•
'
•
'
"
1 2 3 4 Initial Trp-P-1 conc. (p.g/ml)
5
Fig. 5. The effect of a-cellulose on the concentration of Trp-P-1 in the supernatant using various initial concentrations of Trp-P-1. Trp-P-1 at various concentrations was incubated in PBS in the presence (o) or absence (o) of a-cellulose (10 mg). Aliquots (100/~1) were taken for HPLC assay after 1 h. The data are expressed as peak areas.
absence of a-cellulose. This enabled the relative abilities of the carcinogens to adsorb to a-cellulose to be compared (Table 2). With B[a]P the carcinogen adsorbed to the walls of the incubation tube was washed off and included in the
TABLE 2 EFFECT OF a-CELLULOSE (10 mg) ON THE CONCENTRATION OF CARCINOGEN IN SOLUTION Carcinogen
Carcinogen concentration in supernatant as a % of control with no a-cellulose a
Standard error
NMU MelQx PhlP
98.2 95.7 91.1
1.27 3.36 2.64
MeIQ Trp-P-2 IQ
89.8 86.3 83.2
1.91 2.15 1.91
Trp-P-1 DNP b B[a]P
65.4 11.8 4.0
2.51 0.21 0.70
a Calculated by comparing the regression lines relating initial carcinogen concentrations and peak areas from supernatant aliquots taken in the presence and absence of a-cellulose. B[a]P and DNP adsorbed onto the walls of the incubation tube was washed off and included in the supernatant concentration. b Determined previously (Roberton et al., 1990) by mutagenicity assay.
264
Discussion
100 80 6O 4O 20 0
i
C log P value Fig. 6. The relationship between the calculated log partition coefficient (C log P) and the percentage of carcinogen remaining in the supernatant in the presence of a-cellulose. See
text and Tables 1 and 2 for details. (©) NMU (direct acting carcinogen), (z,) DNP (data for taken from Roberton et al., 1990), ([]) B[a]P; and the HAAs (o) Trp-P-1, ( • ) Trp P-2, ( v ) PhIP, (11) MelQ, ( 0 ) IQ and ( - ) MelQx.
supernatant concentration. Data for DNP, obtained previously using a mutagenicity assay (Roberton et al., 1990), are also included in Table 2. The initial concentrations of DNP were similar to those used for the carcinogens in the present study. The results confirmed those from the mutagenicity assay experiments in the present study, which were not carried out using the same concentrations of different carcinogens. The carcinogen with the least ability to adsorb to a-cellulose was NMU and the most effectively absorbed HAA was Trp-P-1.
Carcinogen hydrophobicities and adsorption to a-cellulose. The relationship between the hydrophobicities of the carcinogens, calculated as their C log P values, and the carcinogen concentration in the supernatant after incubation with a-cellulose expressed as a percentage of the control carcinogen concentration (from Table 2) is shown in Fig. 6. The curve is S-shaped: highly hydrophobic carcinogens, such as B[a]P and DNP adsorb strongly to a-cellulose, whereas hydrophilic carcinogens, such as NMU, adsorb poorly. Trp-P-1, which has an intermediate hydrophobicity, shows an intermediate degree of adsorption. However, the order of abilities of the carcinogens to adsorb to a-cellulose (Table 2) and the order of their hydrophobicites (calculated as C log P) (Table 1) are not quite identical.
We have previously reported (Roberton et al., 1990) that quantification of the in vitro adsorption of the hydrophobic mutagen, DNP, to a-cellulose presents special difficulties. This is because the DNP adsorbs to the walls of the incubation tube as well as to the a-cellulose. Thus the amount of DNP adsorbed to the a-cellulose cannot be estimated simply by measuring decreases in the concentration of DNP in solution. To account for all the carcinogen present, the amounts associated with the a-cellulose and the walls of the incubation tube also need to be measured. In the present study, B[a]P was the only carcinogen that adsorbed to the walls of the incubation tube in significant amounts and required the use of the methodology developed for DNP. The abilities of the other carcinogens to adsorb to a-cellulose could thus be estimated by measuring decreases in their concentration in solution. The ability of a carcinogen to adsorb to the walls of the incubation tube appears to be related to the carcinogen's hydrophobicity. Both DNP and B[a]P are highly hydrophobic with C log P values of 4.384 and 6.124 respectively. Trp-P-1 which is somewhat less hydrophobic (C log P value of 3.230) than DNP did not adsorb to the tube walls. Assay of the carcinogens by high performance liquid chromatography (HPLC) enabled us to compare the abilities of a series of carcinogens to adsorb to a-cellulose using the same initial concentrations for each of the carcinogens. However, for routine use when the same concentrations of different carcinogens are not required, mutagenicity assays have advantages over HPLC assays. Incubation tubes and dietary fibers can be washed with solvents, such as DMSO, to remove the carcinogen. Such solvents are incompatible with HPLC. Furthermore, mutagenicity assays enable replicate assays to be carried out much more quickly than by HPLC. Additionally, for strong mutagens such as DNP, mutagenicity assays have greater sensitivity than HPLC. In the present study we have shown that carcinogens vary widely in their ability to adsorb to a-cellulose. The failure of certain suspected colon carcinogens to adsorb effectively to the model fiber, a-cellulose, may suggest that adsorption is
265 not a mechanism for protection against these carcinogens and thus they may pose a greater human cancer risk than carcinogens that adsorb. The ability to adsorb to a-cellulose is closely related to the hydrophobicity of the carcinogen. However, the mechanism of adsorption to a-cellulose of the carcinogens used in this study is unknown. Polysaccharides, such as cellulose, are usually regarded as hydrophilic molecules and the adsorption of hydrophobic carcinogens onto them would not be expected. But an explanation can be advanced based on hydrogen bonding (Wiggins, 1990). Many internal hydrogen bonds occur between the hydroxyl groups on the surface of the cellulose and this bonding reduces the number of hydroxyl groups available to interact with water. Thus, hydrophobic domains on the surface of the a-cellulose can be envisaged in which there is nothing on the surface to form hydrogen bonds with water. The hydrophobic carcinogens may adsorb to these domains. The present study also highlights the differences between the hydrophilic carcinogens commonly used in animal model experiments on colorectal cancer and the much more hydrophobic H A A s that have been identified in human diets (Watanabe et al., 1979; Wakabayashi et al., 1992). N M U and other hydrophilic carcinogens, such as dimethylhydrazine ( D M H ) (Fig. 1), are commonly used in animal experiments on the effects of dietary fiber in preventing colorectal cancer, and such experiments have demonstrated that a-cellulose in the diet protects against colorectal cancer (Heitman et al., 1989). However, our present study shows that N M U is poorly adsorbed by a-cellulose and we predict from the calculated hydrophobicity of D M H (C log P value of - 1 . 3 6 8 ) that this will also adsorb only poorly to a-cellulose. It thus appears unlikely that when the inducing carcinogen is hydrophilic the protection against colorectal cancer given by a-cellulose will be a result of the carcinogen adsorbing to the a-cellulose. Protection is probably the result of other mechanisms which may or may not be relevant to humans. A further problem with many of the animal experiments designed to test the effects of the dietary fiber on the development of colorectal cancer is that the carcinogens are often adminis-
tered intra-rectally or by injection and thus there will be less opportunity for direct interaction between the carcinogen and the dietary fiber. It is unknown whether the results of such experiments can be extrapolated to humans where carcinogens are known to occur in the diet and are also produced endogenously. Thus it is unknown whether many of the animal experiments validly test the protective effects of the different types of fibers in the human diet.
Acknowledgements We thank Professor T. Sugimura for the gift of heterocyclic aromatic amines, Dr. G. Rewcastle for the gift of the NMU, Dr. C.M. Triggs for statistical advice, Dr. M. Hay for computing C log P values, Professor W.A. Denny for helpful discussions, and the Auckland Medical Research Foundation for their financial support of this work.
References Bird, R.P., E.A. McLellan and W.R. Bruce (1984) Aberrant crypts, putative precancerous lesions, in the study of diet in the etiologyof colon cancer, Cancer Surveys,8, 189-200. Conney, A.H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons, Cancer Res., 42, 4875-4917. Cummings, J.H., and W.J. Branch (1982) Postulated mechanisms whereby fibre may protect against bowel cancer, in: G.V. Vahouny and D. Kritchevsky(Eds.), Dietary Fiber in Health and Disease, Plenum, New York, pp. 313-325. Ferguson, L.R., A.M. Roberton, R.J. McKenzie, M.E. Watson and P.J. Harris (1992) Adsorption of a hydrophobic mutagen to dietary fiber from taro (Colocasia esculenta), an important food plant of the South Pacific,. Nutrition and Cancer, 17, 85-95. Harris, P.J., A.M. Roberton, H.J. Hollands and L.R. Ferguson (1991) Adsorption of a hydrophobic mutagen to dietary fibre from the skin and flesh of potato tubers, Mutation Res., 260, 203-213. Heitman, D.W., and I.L. Cameron (1990) Reduction of colon cancer risk by dietary cellulose supplementation, J. Natl. Cancer Inst., 82, 1154-1155. Heitman, D.W., V.A. Ord, K.E. Hunter and I.L. Cameron (1989) Effect of dietary cellulose on cell proliferation and progression of 1,2-dimethylhydrazine-induced colon carcinogenesis in rats, Cancer Res., 49, 5581-5585. Kuchar, M. (Ed.) (1984) QSAR in Design of bioactive compounds, Proceedings of the 1st International Telesymposium on Medicinal Chemistry, Prous, Barcelona, Spain.
266 Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Martin, Y. (1978) Quantitative drug design, Medicinal Research Series No. 8, Marcel Dekker, New York. Medicinal Chemistry Project (1987) MedChem Software Manual, Release 3.51, Pomona College, CA. O'Neill, I.K., A.C. Povey, S. Bingham and E. Cardis (1990) Systematic modulation by human diet levels of dietary fibre and beef on metabolism and disposition of benzo[a]pyrene in the gastrointestinal tract of Fischer F344 rats, Carcinogenesis, 11, 609-616. Overvik, E., and J. Gustafsson (1990) Cooked-food mutagens: current knowledge of formation and biological significance, Mutagenesis, 5, 437-446. 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.
Roberton, A.M., L.R. Ferguson, H.J. Hollands and P.J. Harris (1991) Adsorption of a hydrophobic mutagen to five contrasting dietary fiber preparations, Mutation Res., 262, 195-202. Venitt, S., and C. Crofton-Sieigh (1979) Bacterial mutagenicity tests of phenazine methosulphate and three tetrazolium salts, Mutation Res., 68, 107-116. Wakabayashi, K., M. Nagao, H. Esumi and T. Sugimura (1992) Food-derived mutagens and carcinogens, Cancer Res. (Suppl.) 52, 2092s-2098s. Watanabe, K., B.S. Reddy, J.H. Weisberger and D. Kritchevsky (1979) Effect of dietary alfafa, pectin and wheat bran on azoxymethane or methylnitrosourea-induced colon carcinogenesis in F-344 rats, J. Natl. Cancer Inst., 63, 141-145. Wiggins, P.M. (1990) Role of water in some biological processes, Microbiol. Rev., 54, 432-449.
257
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1218/93/$06.00
MUTGEN 01934
The adsorption of a range of dietary carcinogens by a-cellulose, a model insoluble dietary fiber Lynnette R. Ferguson a, Anthony M. Roberton b, Mark E. Watson a, Philip Kestell a and Philip J. Harris b a Cancer Research Laboratory and b School of Biological Sciences, The University of Auckland, Auckland, New Zealand
(Received 6 January 1993) (Revision received31 March 1993) (Accepted 25 May 1993)
Keywords: Adsorption; t~-Cellulose;Dietary fiber; Heterocyclicaromatic amines; Hydrophobicity;C log P
Summary One of the ways dietary fibers may protect against colorectal cancer is by adsorbing carcinogens and carrying them out of the digestive tract, thus lessening interaction of the carcinogens with the colonic tissue. We investigated this mechanism of action by testing in vitro the abilities of a range of carcinogens, including known animal colon carcinogens, to adsorb to a-cellulose, which we have used as a model insoluble dietary fiber. The carcinogens were Nonitroso-N-methylurea (NMU), benzo[a]pyrene (B[a]P) and a number of heterocyclic aromatic amines which have been found in heated foods. It was found that the ability of a carcinogen to adsorb to a-cellulose is strongly related to the hydrophobicity of the carcinogen measured as the calculated logarithm of the partition coefficient between 1-octanol and water (C log P). The hydrophilic carcinogen, NMU, (C log P = -0.204), adsorbed only poorly, whereas the very hydrophobic carcinogen, B[a]P, (C log P = 6.124), adsorbed strongly. Carcinogens with intermediate hydrophobicities showed intermediate abilities to adsorb.
It is not known which carcinogens in the diet are responsible for the initiating event in human colorectal cancer. This makes it difficult to test theories to explain the protective effects of particular types of dietary fiber against the development of colorectal cancer, especially theories that involve an interaction between the dietary fiber and the carcinogen.
Correspondence: Dr. L.R. Ferguson, Cancer Research Laboratory, The Universityof Auckland, Private Bag 92019, Auckland, New Zealand.
We investigated one of the hypotheses for the mode of action of dietary fiber: that dietary fiber adsorbs the carcinogens responsible for the initiating events and transports them from the body and so decreases the concentration of carcinogen to which the colon epithelium is exposed (Cummings and Branch, 1982). We developed a model in vitro system, using 1,8-dinitropyrene (DNP) (a known mutagen and carcinogen) as the test carcinogen, to investigate the adsorption of carcinogens to dietary fiber (Roberton et al., 1990). Firstly, we investigated the adsorption of DNP to a-cellulose, which we used as a model insoluble
258 dietary fiber because it is available commercially in large quantities. Cellulose has also been shown in animal experiments to protect against colorectal cancer (Heitman et al., 1989) and it has been
suggested (Heitman and Cameron, 1990) that it could be used as a human dietary supplement. We then investigated the adsorption of DNP to a variety of other dietary fiber preparations
C,H 3
CH 3
I tt
Ctt 3 Trp-P-1 3-amino-l,4-dimethyl-5H-pyrido[4,3-blindole
"
Trp-P-2 3-amino-l-methyl-5H-pyrido[4,3-b]indole
NH2
~
.--4N---CH 3
--CH3 -N-
IQ 2-amino-3-methylimidazo[4,5-.t]quinoline
~
"UH3
MeIQ 2-amino-3,4-dimethylimidazo[4,5-f]quinoline
NH 2
cH3 H3C,,~ .,N~ ~
,N'CH3
MelQx 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
PhIP 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine NO2
B[alP Benzolalpyrene
H2N\ IN//0 ~C--N O ~CH3 NMU N-nitroso-N-methylurea
DNP 1,8-dinitropyrene
H3CXN_.N/CH3 H/
xH
DMH 1,2-dimethylhydrazine
Fig. 1. Names, abbreviationsand structures of the carcinogensidentified in the text.
259
4000
~
o
300020001000-
~
0"
o
200
400
600
NMU (~g/plate)
a~ 4000 3000-
20001000-
o
n'-
0
50
1O0
150
200
TrpP1 (ng/plate)
Fig. 2. Dose-response curves for mutagenicity expressed as revertant colonies in Salmonella typhimurium strains. (A) NMU and bacterial strain TA98. (B) Trp-P-1 and bacterial strain TA100. A range of concentrations of each of the carcinogens was dissolved in DMSO and added to bacteria using standard plate-incorporation protocols (Maron and Ames, 1983). Regression lines, computed for the linear part of the curve, are also shown. (Roberton et al., 1991; Harris et al., 1991; Ferguson et al., 1992). In this paper we report an extension of our studies, using the in vitro model system, to a wider range of carcinogens which may be appropriate to animal or human colorectal cancer. The carcinogens we used a r e illustrated in Fig. 1. N-Nitroso-N-methylurea (NMU) is a direct-acting molecule which causes colon cancer in rats (Watanabe et al., 1979). Trp-P-1, Trp-P-2, IQ, MelQ, MelQx and PhlP are all heteroc-yclic aromatic amines (HAAs), a group of carcinogens identified in cooked meat, fish, chicken and grain products, the amounts depending on time and temperature (Wakabayashi et al., 1992; Overvik and Gustafsson, 1990). They are suspected of being involved in the initiation of various types of
human cancers. However, the target organs vary among animal species used as models and in humans the target organs are unknown. At least in rats, IQ, M e l Q and PhlP are colon carcinogens (Wakabayashi et al., 1992) and each requires a metabolic activation event to form the proximal carcinogen. B[a]P is probably the most widely studied carcinogen and also requires metabolic activation to form the proximal carcinogen (Conney, 1982). It causes colonic lesions in rats, although the colon is not the primary target organ (O'Neill et al., 1990; Bird et al., 1984). In our previous in vitro studies with DNP, we used the Salmonella typhimurium mutagenicity assay (Maron and Ames, 1983) to quantify the DNP. We have also used this rapid assay for part of the present study as it is convenient to carry out and overcomes some methodological problems. However, because carcinogens differ in their mutagenicities, the concentration range over which the assay gives a linear dose-response curve varies widely with carcinogen. Thus, this assay cannot be used to compare the abilities of a series of carcinogens to adsorb to a-cellulose using the same initial concentrations for each of the carcinogens. To enable this comparison to be made, we therefore developed assay techniques for the carcinogens using high performance liquid chromatography (HPLC). The logarithm of the partition coefficient of compounds (log P ) is frequently used as a measure of the hydrophobicities of these compounds, and has been successfully used in Quantitative Structure Actitivity Relationships (QSAR) in many fields, including drug design (Martin, 1978; Kuchar, 1984). The partition coefficient of a compound is the equilibrium concentration of that compound in a non-polar solvent (usually 1-octanol) divided by the concentration in a polar solvent, usually water (Medicinal Chemistry Project, 1987). Although partition coefficients can be determined experimentally, it is often more convenient to calculate log P (C log P ) from the structure of the compound by an additive-constitutive procedure using a computer program. Our preliminary experiments showed that the ability of a carcinogen to adsorb to a-cellulose appeared, at least partially, to be related to the hydrophobicity of the carcinogen (calculated as
260 its C log P value). We have therefore investigated this relationship in the present study. Materials and methods
Chemicals. The HAAs were the kind gift of Professor T. Sugimura, National Cancer Institute, Tokyo, Japan. Their chemical names, abbreviations and structures are shown in Fig. 1. NMU was a gift of Dr. G. Rewcastle, Cancer Research Laboratory, University of Auckland. B[a]P (Cat. No. B1,008-0) was obtained from Aldrich Chemical Co, Milwaukee, USA and a-cellulose (Cat. No. C8022 from Canadian bleached hardwood pulp) from Sigma Chemical Company, St. Louis, MO. HPLC grade chromatography solvents were purchased from Alphatech, Auckland, New Zealand. Hydrophobicities of the carcinogens. The calculated logarithm of the partition coefficient (C log P ) between 1-octanol and water was used as a measure of the hydrophobicity of each carcinogen and was calculated from the structure of the carcinogen by an additive-constitutive procedure using the computer program, CLOGP3 (Medicinal Chemistry Project, 1987).
Bacterial mutagenicity assays. The Salmonella typhimurium plate-incorporation assay (Maron and Ames, 1983) was carried out using strain TA98 for the HAAs and strain TA100 for NMU and B[a]P. To minimise day-to-day variability in the sensitivity of the tester strains to the mutagens, which is due largely to the exact growth phase of the culture, we grew cells for quantitative work as follows. A 1-ml vial (2 × 10 s cells) was removed from storage at - 8 0 ° 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. Samples from experiments with NMU were plated directly, but the HAAs and B[a]P had $9 mix added (prepared from Aroclor-induced Sprague-Dawley rats by standard procedures, as described by Maron and Ames, 1983). Each experimental point was performed in triplicate on
TABLE 1 CONCENTRATIONS AND C log P VALUES OF CARCINOGENS USED IN THE MUTAGENICITYASSAYS Carcinogen
DNP a MelQ Trp-P-2 IQ MeIQx Trp-P-1 PhIP B[alP NMU
Concentration Amount in solution per plate (/xg/ml) (/zg/plate) 0.05 0.0025 0.06 0.003 0.2 0.01 0.4 1 2 40 200 4000
0.02 0.05 0.1 2 20 200
C log P value 4.384 1.822 2.731 1.323 1.078 3.230 2.294 6.124 - 0.204
a Studied previously(Roberton et al., 1990).
at least two separate occasions. The background number of revertant colonies was approximately 65 (range 54-78). Positive controls containing 4nitro-o-phenylenediamine (100 /.~g/plate) were included in all experiments to test the bacterial strain response. The dose-response curves for each carcinogen were determined by plotting the amounts of each carcinogen added to the plates against the numbers of revertant colonies. For plate colony numbers less than 1000, the plates were counted by autocounter and one plate from each triplicate set was confirmed by hand counting. For plate counts higher than 1000, hand counting was used. The dose-response curves for NMU and Trp-P-1 are shown in Figs. 2A and 2B respectively. For the assays the amounts of each carcinogen added to each plate was adjusted so that all measurements were made on the linear part of the curve (Table 1). The amount of carcinogen chosen for experiments was selected to give about 1500 revertants per plate, in order to optimise sensitivity when the carcinogen was distributed between the supernatant, pellet and tube walls. Using the dose-response curves we converted the numbers of revertant colonies into amounts of carcinogen, as previously described (Harris et al., 1991), using linear regression analysis as described by Venitt and Crofton-Sleigh (1979). Results have been expressed as the percentage of added carcinogen
261 found at the end of the experiment in various fractions.
HPLC. Because mutagenicity assays cannot be used to compare the abilities of a series of carcinogens to adsorb to a-cellulose using the same initial concentrations for each of the carcinogens we developed high performance liquid chromatography (HPLC) methods to assay the carcinogens. HPLC of the carcinogens 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 l l l B UV detector (Gilson Medical Electronics, Middleton, WI, USA), a Shimadzu (Model RF530) fluorescence detector (Shimadzu, Kyoto, Japan), and a C18 4 ~ Novapak stainless steel column (150 x 3.9 mm, Phenomenex, Torrance, CA, USA). Most of the carcinogens 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-P1, Trp-P-2, 1 : 1 v/v). For MelQ, the mobile phase was the same as for PhlP except that the pH of the 25 mM sodium dihydrogen phosphate solution was 6.5. For B[a]P, the mobile phase was acetonitrile. The flow rates were 1-3 ml rain-1 for all carcinogens and detection was either by UV absorption (254 nm) or by fluorescence (TrpP-l, excitation 300 nm, emission 460 nm; Trp-P-2, excitation 356 nm, emission 485 nm). Data acquisition and integration were performed using a Unicam 4880 Chromatography Data Handling System (Unicam Ltd, Cambridge, UK). Calibration curves were constructed by plotting peak area against standard concentrations and the equations for the best-fit straight lines were determined by linear regression analysis. Quantitation of unknown samples was achieved by measuring the peak area in the unknown sample and using the equations obtained from the linear regression to calculate a concentration.
(PBS) (20 mM sodium phosphate buffer, pH 6.5, containing 130 mM sodium chloride) added to each tube. The PBS was added 30 min before each experiment to allow time for the a-cellulose to hydrate. At time zero, 40 /~I of a solution of the carcinogen in DMSO (for mutagenicity assays) or methanol (for HPLC assays) was added and the contents of the tubes mixed using a vortex mixer. The tubes were shaken (120 rpm) on an orbital shaker for 55 min at 37°C and centrifuged (2500 g for 2.5 min). For mutagenicity assay of the carcinogen, 3 aliquots (50 /zl unless otherwise specified) of the supernatant were taken and assayed as described above. For HPLC assay of the carcinogen, 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-/xl aliquots taken. Control incubations were also done in which the carcinogens were incubated with no a-cellulose. In experiments with B[a]P, this carcinogen was recovered from the a-cellulose and the tube walls after incubation. The a-cellulose pellets and the walls of the glass incubation tubes were washed twice with dimethyl sulfoxide (mutagenicity assays) or acetonitrile (HPLC assays). Results
Hydrophobicities of the carcinogens. The hydrophobicities of the carcinogens used in this study, calculated as C log P values, are shown in Table 1. Compared to the HAAs, which have C log P values ranging from 1.078 (MelQx) to 3.230 (Trp-P-1), NMU (C log P = - 0 . 2 0 4 ) is hydrophilic, and B[a]P (C log P = 6.124) is hydrophobic. We also included the C log P value for 1,8-dinitropyrene (DNP) in Table 1, as we studied this extensively in previous publications (Roberton et al., 1990, 1991; Harris et al., 1991; Ferguson et al., 1992).
Incubation of carcinogens with a-cellulose in PBS. All incubations were done in acid-washed
Effects of incubation time on the mutagenicity of carcinogen solutions. There were no changes
conical glass centrifuge tubes (capacity 12 ml). a-Cellulose (usually 10 rag) was weighed into the tubes and 1.96 ml of phosphate-buffered saline
in the mutagenicities of control solutions of the carcinogens in PBS in the absence of a-cellulose during incubation times of up to 2 h (results not
262
lulose had an effect, this was rapid and could be detected immediately after mixing the carcinogen with the a-cellulose. A complete comparison of the abilities of the carcinogens to adsorb to a-cellulose (10 mg) was done using the HPLC assay which enabled the same initial concentrations to be used for each of the carcinogens (see below).
100 80
~ 6o ~ 4o 20
~
0
0 0
I O0 Time (min)
200
Fig. 3. The effect of incubation time on the mutagenicity of a solution of NMU in the presence and absence of a-cellulose. NMU (4 mg m1-1) was incubated at 37°C in PBS in the presence or absence of a-cellulose (10 mg). Aliquots of supernatant (50/zl) from the incubations containing a-cellulose (e) or in its absence (o) were removed at intervals for mutagenicity assay. Values represent the number of revertant colonies (minus those of negative controls) expressed as a percentage of the number obtained for a comparable sample of NMU diluted into DMSO and plated immediately (in the presence of $9 mix). The% of NMU on the walls of the incubation tube in the presence and absence of a-cellulose is shown by • and • respectively.
shown), with the exception of B[a]P and NMU. After 2 h, most of the mutagenicity of the B[a]P solution had been lost, but almost all was recovered from the walls of the incubation tube by washing with DMSO. This showed that the loss of mutagenicity was due to the adsorption of B[a]P onto the walls of the tube and not due to decomposition or inactivation (results not shown). In contrast, NMU was unstable under these conditions, and over 70% of the mutagenicity disappeared from solution over 2 h but could not be recovered (Fig. 3, open circles). When the incubation temperature was lowered from 37°C to 22°C, less than 20% of the mutagenicity was lost (results not shown).
Effects of a-cellulose on the mutagenicity of carcinogen solutions. Addition of a-cellulose (10 mg) had a variable effect on the mutagenicity of the carcinogen solution depending on the carcinogen examined. For example, a-cellulose had no effect on the mutagenicity of a solution of NMU at 37°C (Fig. 3, closed circles) or at 22°C, but reduced the mutagenicity of a solution of Trp-P-1 by 38% (data not shown). Where a-cel-
Effect of increasing concentrations of a-cellulose on the distribution of the carcinogens. The effects varied depending on the carcinogen. In the absence of a-cellulose, most of the B[a]P adsorbed to the walls of the incubation tube. However, addition of increasing amounts of acellulose resulted in increasing proportions of the total mutagenicity added being recovered from the a-cellulose and decreasing proportions being recovered from the walls of the incubation tube (Fig. 4A). Thus, after incubation (1 h) in the absence of a-cellulose, 80% of the added mutagenicity was associated with the tube wall and only 20% remained in the supernatant. After incubation in the presence of 10 mg of a-cellulose, over 80% of the added mutagenicity was recovered from the a-cellulose, and equal amounts of the remainder were on the tube walls and in the supernatant. In contrast to the B[a]P, NMU showed no evidence of adsorption to a-cellulose even at the highest concentrations of a-cellulose (data not shown). Although the extent of adsorption varied, each of the HAAs had a similar pattern of adsorption to a-cellulose. With increasing concentrations of a-cellulose, an increasing proportion of each carcinogen adsorbed to the a-cellulose. Trp-P-1 was the most effectively adsorbed of this group of carcinogens (Fig. 4B). For example, in the presence of 10 mg of a-cellulose, 62% of the added mutagenicity remained in the supernatant and 38% was adsorbed to the a-cellulose. The least effectively adsorbed was PhIP (data not shown). In the presence of 10 mg of a-cellulose, only 5% of the added mutagenicity was adsorbed. Effects of a-cellulose on carcinogen concentration in the supernatant using the same initial concentrations for each carcinogen. The effects of a-cellulose (10 mg) on the carcinogen concentration in the supernatant was determined using the
263
3000" 100 0.
80~
2000'
6040"6
20. 0 ~ - :
0
1'0
1'5
0
20
a-cellulose (mg/2ml) B
100.
~
ao-
~
40-
"6
20O~
0
5 10 15 (z-cellulose (mg/2ml)
20
Fig. 4. Effect of increasing concentrations of a-cellulose on the distribution of (A) B[a]P or (B) Trp-P-1. B[a]P (200 /~g ml - t ) or Trp-P-1 (2 /zg m1-1) was incubated in PBS with various concentrations of a-cellulose. Aliquots (100 /zl for B[a]P or 50/zl for Trp-P-1) were taken for mutagenicity assays after 1 h. Values represent the number of revertant colonies (minus those of negative controls) expressed as a percentage of the number obtained for a comparable sample of carcinogen diluted into DMSO and plated immediately. ( zx) Carcinogen associated with the a-cellulose. (o) Carcinogen in the supernatant. ([]) Carcinogen adsorbed to the tube walls.
same initial concentrations (0.1, 0.25, 0.5, 1.0, 2.5 and 5/zg m1-1) for each of the carcinogens. The carcinogens were assayed using HPLC. The detectors used gave a linear response with concentration for each carcinogen over the concentration range used. For each carcinogen, there was the same percentage reduction in concentration in the supernatant after incubation with a-cellulose (within experimental error) at each initial concentration. This was true for each of the carcinogens used and Fig. 5 shows data for Trp-P-1. The percentage reductions were calculated by comparing the regression lines relating initial carcinogen concentrations and peak areas from supernatant aliquots taken in the presence and
,
•
'
•
'
"
1 2 3 4 Initial Trp-P-1 conc. (p.g/ml)
5
Fig. 5. The effect of a-cellulose on the concentration of Trp-P-1 in the supernatant using various initial concentrations of Trp-P-1. Trp-P-1 at various concentrations was incubated in PBS in the presence (o) or absence (o) of a-cellulose (10 mg). Aliquots (100/~1) were taken for HPLC assay after 1 h. The data are expressed as peak areas.
absence of a-cellulose. This enabled the relative abilities of the carcinogens to adsorb to a-cellulose to be compared (Table 2). With B[a]P the carcinogen adsorbed to the walls of the incubation tube was washed off and included in the
TABLE 2 EFFECT OF a-CELLULOSE (10 mg) ON THE CONCENTRATION OF CARCINOGEN IN SOLUTION Carcinogen
Carcinogen concentration in supernatant as a % of control with no a-cellulose a
Standard error
NMU MelQx PhlP
98.2 95.7 91.1
1.27 3.36 2.64
MeIQ Trp-P-2 IQ
89.8 86.3 83.2
1.91 2.15 1.91
Trp-P-1 DNP b B[a]P
65.4 11.8 4.0
2.51 0.21 0.70
a Calculated by comparing the regression lines relating initial carcinogen concentrations and peak areas from supernatant aliquots taken in the presence and absence of a-cellulose. B[a]P and DNP adsorbed onto the walls of the incubation tube was washed off and included in the supernatant concentration. b Determined previously (Roberton et al., 1990) by mutagenicity assay.
264
Discussion
100 80 6O 4O 20 0
i
C log P value Fig. 6. The relationship between the calculated log partition coefficient (C log P) and the percentage of carcinogen remaining in the supernatant in the presence of a-cellulose. See
text and Tables 1 and 2 for details. (©) NMU (direct acting carcinogen), (z,) DNP (data for taken from Roberton et al., 1990), ([]) B[a]P; and the HAAs (o) Trp-P-1, ( • ) Trp P-2, ( v ) PhIP, (11) MelQ, ( 0 ) IQ and ( - ) MelQx.
supernatant concentration. Data for DNP, obtained previously using a mutagenicity assay (Roberton et al., 1990), are also included in Table 2. The initial concentrations of DNP were similar to those used for the carcinogens in the present study. The results confirmed those from the mutagenicity assay experiments in the present study, which were not carried out using the same concentrations of different carcinogens. The carcinogen with the least ability to adsorb to a-cellulose was NMU and the most effectively absorbed HAA was Trp-P-1.
Carcinogen hydrophobicities and adsorption to a-cellulose. The relationship between the hydrophobicities of the carcinogens, calculated as their C log P values, and the carcinogen concentration in the supernatant after incubation with a-cellulose expressed as a percentage of the control carcinogen concentration (from Table 2) is shown in Fig. 6. The curve is S-shaped: highly hydrophobic carcinogens, such as B[a]P and DNP adsorb strongly to a-cellulose, whereas hydrophilic carcinogens, such as NMU, adsorb poorly. Trp-P-1, which has an intermediate hydrophobicity, shows an intermediate degree of adsorption. However, the order of abilities of the carcinogens to adsorb to a-cellulose (Table 2) and the order of their hydrophobicites (calculated as C log P) (Table 1) are not quite identical.
We have previously reported (Roberton et al., 1990) that quantification of the in vitro adsorption of the hydrophobic mutagen, DNP, to a-cellulose presents special difficulties. This is because the DNP adsorbs to the walls of the incubation tube as well as to the a-cellulose. Thus the amount of DNP adsorbed to the a-cellulose cannot be estimated simply by measuring decreases in the concentration of DNP in solution. To account for all the carcinogen present, the amounts associated with the a-cellulose and the walls of the incubation tube also need to be measured. In the present study, B[a]P was the only carcinogen that adsorbed to the walls of the incubation tube in significant amounts and required the use of the methodology developed for DNP. The abilities of the other carcinogens to adsorb to a-cellulose could thus be estimated by measuring decreases in their concentration in solution. The ability of a carcinogen to adsorb to the walls of the incubation tube appears to be related to the carcinogen's hydrophobicity. Both DNP and B[a]P are highly hydrophobic with C log P values of 4.384 and 6.124 respectively. Trp-P-1 which is somewhat less hydrophobic (C log P value of 3.230) than DNP did not adsorb to the tube walls. Assay of the carcinogens by high performance liquid chromatography (HPLC) enabled us to compare the abilities of a series of carcinogens to adsorb to a-cellulose using the same initial concentrations for each of the carcinogens. However, for routine use when the same concentrations of different carcinogens are not required, mutagenicity assays have advantages over HPLC assays. Incubation tubes and dietary fibers can be washed with solvents, such as DMSO, to remove the carcinogen. Such solvents are incompatible with HPLC. Furthermore, mutagenicity assays enable replicate assays to be carried out much more quickly than by HPLC. Additionally, for strong mutagens such as DNP, mutagenicity assays have greater sensitivity than HPLC. In the present study we have shown that carcinogens vary widely in their ability to adsorb to a-cellulose. The failure of certain suspected colon carcinogens to adsorb effectively to the model fiber, a-cellulose, may suggest that adsorption is
265 not a mechanism for protection against these carcinogens and thus they may pose a greater human cancer risk than carcinogens that adsorb. The ability to adsorb to a-cellulose is closely related to the hydrophobicity of the carcinogen. However, the mechanism of adsorption to a-cellulose of the carcinogens used in this study is unknown. Polysaccharides, such as cellulose, are usually regarded as hydrophilic molecules and the adsorption of hydrophobic carcinogens onto them would not be expected. But an explanation can be advanced based on hydrogen bonding (Wiggins, 1990). Many internal hydrogen bonds occur between the hydroxyl groups on the surface of the cellulose and this bonding reduces the number of hydroxyl groups available to interact with water. Thus, hydrophobic domains on the surface of the a-cellulose can be envisaged in which there is nothing on the surface to form hydrogen bonds with water. The hydrophobic carcinogens may adsorb to these domains. The present study also highlights the differences between the hydrophilic carcinogens commonly used in animal model experiments on colorectal cancer and the much more hydrophobic H A A s that have been identified in human diets (Watanabe et al., 1979; Wakabayashi et al., 1992). N M U and other hydrophilic carcinogens, such as dimethylhydrazine ( D M H ) (Fig. 1), are commonly used in animal experiments on the effects of dietary fiber in preventing colorectal cancer, and such experiments have demonstrated that a-cellulose in the diet protects against colorectal cancer (Heitman et al., 1989). However, our present study shows that N M U is poorly adsorbed by a-cellulose and we predict from the calculated hydrophobicity of D M H (C log P value of - 1 . 3 6 8 ) that this will also adsorb only poorly to a-cellulose. It thus appears unlikely that when the inducing carcinogen is hydrophilic the protection against colorectal cancer given by a-cellulose will be a result of the carcinogen adsorbing to the a-cellulose. Protection is probably the result of other mechanisms which may or may not be relevant to humans. A further problem with many of the animal experiments designed to test the effects of the dietary fiber on the development of colorectal cancer is that the carcinogens are often adminis-
tered intra-rectally or by injection and thus there will be less opportunity for direct interaction between the carcinogen and the dietary fiber. It is unknown whether the results of such experiments can be extrapolated to humans where carcinogens are known to occur in the diet and are also produced endogenously. Thus it is unknown whether many of the animal experiments validly test the protective effects of the different types of fibers in the human diet.
Acknowledgements We thank Professor T. Sugimura for the gift of heterocyclic aromatic amines, Dr. G. Rewcastle for the gift of the NMU, Dr. C.M. Triggs for statistical advice, Dr. M. Hay for computing C log P values, Professor W.A. Denny for helpful discussions, and the Auckland Medical Research Foundation for their financial support of this work.
References Bird, R.P., E.A. McLellan and W.R. Bruce (1984) Aberrant crypts, putative precancerous lesions, in the study of diet in the etiologyof colon cancer, Cancer Surveys,8, 189-200. Conney, A.H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons, Cancer Res., 42, 4875-4917. Cummings, J.H., and W.J. Branch (1982) Postulated mechanisms whereby fibre may protect against bowel cancer, in: G.V. Vahouny and D. Kritchevsky(Eds.), Dietary Fiber in Health and Disease, Plenum, New York, pp. 313-325. Ferguson, L.R., A.M. Roberton, R.J. McKenzie, M.E. Watson and P.J. Harris (1992) Adsorption of a hydrophobic mutagen to dietary fiber from taro (Colocasia esculenta), an important food plant of the South Pacific,. Nutrition and Cancer, 17, 85-95. Harris, P.J., A.M. Roberton, H.J. Hollands and L.R. Ferguson (1991) Adsorption of a hydrophobic mutagen to dietary fibre from the skin and flesh of potato tubers, Mutation Res., 260, 203-213. Heitman, D.W., and I.L. Cameron (1990) Reduction of colon cancer risk by dietary cellulose supplementation, J. Natl. Cancer Inst., 82, 1154-1155. Heitman, D.W., V.A. Ord, K.E. Hunter and I.L. Cameron (1989) Effect of dietary cellulose on cell proliferation and progression of 1,2-dimethylhydrazine-induced colon carcinogenesis in rats, Cancer Res., 49, 5581-5585. Kuchar, M. (Ed.) (1984) QSAR in Design of bioactive compounds, Proceedings of the 1st International Telesymposium on Medicinal Chemistry, Prous, Barcelona, Spain.
266 Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Martin, Y. (1978) Quantitative drug design, Medicinal Research Series No. 8, Marcel Dekker, New York. Medicinal Chemistry Project (1987) MedChem Software Manual, Release 3.51, Pomona College, CA. O'Neill, I.K., A.C. Povey, S. Bingham and E. Cardis (1990) Systematic modulation by human diet levels of dietary fibre and beef on metabolism and disposition of benzo[a]pyrene in the gastrointestinal tract of Fischer F344 rats, Carcinogenesis, 11, 609-616. Overvik, E., and J. Gustafsson (1990) Cooked-food mutagens: current knowledge of formation and biological significance, Mutagenesis, 5, 437-446. 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.
Roberton, A.M., L.R. Ferguson, H.J. Hollands and P.J. Harris (1991) Adsorption of a hydrophobic mutagen to five contrasting dietary fiber preparations, Mutation Res., 262, 195-202. Venitt, S., and C. Crofton-Sieigh (1979) Bacterial mutagenicity tests of phenazine methosulphate and three tetrazolium salts, Mutation Res., 68, 107-116. Wakabayashi, K., M. Nagao, H. Esumi and T. Sugimura (1992) Food-derived mutagens and carcinogens, Cancer Res. (Suppl.) 52, 2092s-2098s. Watanabe, K., B.S. Reddy, J.H. Weisberger and D. Kritchevsky (1979) Effect of dietary alfafa, pectin and wheat bran on azoxymethane or methylnitrosourea-induced colon carcinogenesis in F-344 rats, J. Natl. Cancer Inst., 63, 141-145. Wiggins, P.M. (1990) Role of water in some biological processes, Microbiol. Rev., 54, 432-449.