* Human Toxicology Research Group, Department of Environmental and Occupational Health, University of Montreal, P.O. Box 6128, Main Station, Montreal (QC), Canada H3C 3J7
1 To whom correspondence should be addressed. Fax: 1-514-343-2200. E-mail: claude.viau{at}umontreal.ca.
Received August 14, 2003; accepted November 30, 2003
ABSTRACT
During biological monitoring of exposure to a chemical, a possible source of interindividual variability in the measurement of a urinary metabolite that undergoes enterohepatic cycling is the presence of dietary fiber in the gastrointestinal tract. This study examined the effect of diets containing either the insoluble fiber Alphacel (nonnutritive bulk cellulose) or the soluble pectin (from citrus fruit, MW 20,00040,000). Five groups of male Sprague-Dawley rats received one of the following diets: poor (5% w/w) or rich (15% w/w) in Alphacel, poor (5% w/w) or rich (15% w/w) in pectin, or no fiber (NF). Five µmol/kg of pyrene was administered by iv injection immediately after feeding the animals with their respective diet, and urine and feces collections started for the determination of 1-hydroxypyrene (1-OHP), a metabolite of pyrene. The type of fiber had no influence on the results. The rats receiving diets both poor and rich in fiber excreted less 1-OHP (18 ± 8 and 15 ± 7 pmol per g of rat, respectively) in the 24-h urine samples than the NF group (28 ± 6 pmol/g). There was a nonstatistically significant trend towards increased fecal and total (urinary + fecal) 1-OHP excretion with increasing amount of fiber in the diet. An in vitro experiment showed an inverse correlation (r2 = 0.98) between the amount of Alphacel in suspension in a 1-OHP aqueous solution and the recovery of 1-OHP from the soluble fraction. The reduction in urinary output of the metabolite due to fiber reaching 40% may contribute to its interindividual variability observed in occupational and environmental studies.
Key Words: 1-hydroxypyrene; Alphacel; pectin; fiber; Sprague-Dawley rats.
Jongeneelen et al. (1988) first proposed the use of urinary 1-hydroxypyrene (1-OHP) as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAH). The variability in PAH composition in various workplaces (Bouchard and Viau, 1999
) and interindividual differences in the urinary excretion of 1-OHP in people exposed to the same PAH dose (Viau et al., 1995
, 2002
) contribute to the difficulty in setting up a single reliable Biological Exposure Index (BEI; ACGIH, 2003
).
It has been documented that the background urinary concentration of 1-OHP is different in various parts of the world (Levin, 1995). The differences can of course be largely ascribed to differences in the environmental PAH contamination in these various countries. Within a given population, part of the interindividual variability can be related to enzymatic polymorphism (Alexandrie et al., 2000
; Lee et al., 2002
). But there could be other reasons for the observed interindividual variability. Indeed, 1-OHP has been shown to undergo enterohepatic recycling (Withey et al., 1991
; Viau et al., 1999
). Rats in which the bile duct is canulated so that the bile does not flow into the intestine, excrete less urinary 1-OHP than sham-operated rats injected iv with an identical dose of pyrene (Bouchard and Viau, 1998
). The 1-OHP urine/bile ratio in canulated animals is lower than the urine/feces ratio of noncanulated ones (Viau et al., 1999
). By preventing the re-entry of 1-OHP, less is available for urinary excretion.
Dietary fibers are known to adsorb or interfere with the intestinal absorption of a number of organic molecules such as bile acids, drugs, and carcinogens (Harris et al., 1998; Jimenez-Escrig and Sanchez-Muniz, 2000
; Vahouny, 1982
). This can result in a shunt of the enterohepatic cycling (Fernandez, 2001
) causing an increased fecal elimination of the adsorbed molecules and reducing the systemic availability of the compounds. This phenomenon has been put to profit in some hypocholesterolemic treatments (Fernandez, 2001
). The increased excretion of bile acids was shown to be related to the type and source of dietary fiber (Jimenez-Escrig and Sanchez-Muniz, 2000
). The yield of urinary 1-OHP excretion could therefore be influenced by dietary habits and the time of urine collection with respect to food intake. This could affect the interpretation of urine 1-OHP analyzed for biological monitoring purposes.
It was hypothesized that 1-OHP secreted in the bile can be sequestered by dietary fiber and that the resulting impact of fiber on the urinary excretion of 1-OHP is related to both the nature and the amount of dietary fiber present in the gastrointestinal tract during metabolism and biliary excretion of 1-OHP following an iv administration of pyrene. The objective of this work was therefore to test this hypothesis in a well-characterized rat model used for over 10 years in our laboratory.
MATERIALS AND METHODS
Chemicals.
Pyrene was obtained from Aldrich Chemical Company Inc. (Oakville, ON, Canada). 1-OHP used as a standard in the analytical system was purchased from the NCI Chemical Carcinogens Reference Standards (Kansas City, MO). ß-Glucuronidase/arylsulfatase (100,000 Fishman U/ml and 800,000 Roy U/ml, respectively, from Helix Pomatia) used for the hydrolysis of the conjugated 1-OHP before analysis, was obtained from Boehringer Mannheim (Laval, QC, Canada). Emulphor (Alkamuls EL 620) was from Rhone-Poulenc and was used in the preparation of a soluble form of pyrene to allow its iv injection. C-18 Sep Pak were purchased from Waters (Dorval, QC, Canada). Methanol solvent, ethyl acetate, and sodium acetate were from Fisher Scientific (Nepean, ON, Canada), and ascorbic acid from Baker (Phillipsburg, NJ). The various constituents of the experimental diets were obtained from ICN Research Diets (Costa Mesa, CA): minerals were AIN 93M mineral mix; vitamins were Vitamin mixture AIN-76A; Alphacel is composed of finely ground cellulose; pectin is from citrus fruit.
Animals.
Male Sprague-Dawley rats (Charles River Canada Inc., St-Constant, QC, Canada) weighing between 200 and 250 g were used. The animals were kept in individual metabolism cages during the period required to acclimatize them to their respective diets. The room where the rats were housed was kept around 25°C with a 12-h light/dark cycle. The rats had free access to a mixture of D-glucose (40 g/l) and saccharin (1.5 g/l) to stimulate a polydipsic/polyuric behavior and improve quantitative recovery of the urinary metabolites, as previously described (Bouchard and Viau, 1996). All animals were treated in accordance with the guidelines of the Canadian Council on Animal Care.
Treatments, and urine and feces collection.
Twenty-six rats were randomly assigned to five experimental groups of five animals each, except for the reference group (n = 6). Two groups received diets that were poor in fiber (5%), two groups had diets rich in fiber (15%) and the reference group had no fiber in his diet. Two types of fiber were used: soluble (pectin, from citrus fruit, MW 20,00040,000) and insoluble (Alphacel, nonnutritive bulk cellulose). Table 1 gives the composition of the variable elements of the diets. In addition, each diet contained casein (20%), methionine (0.3%), choline (0.2%), minerals (3.5%), vitamins (1%), and soy oil (5%). All diets were balanced to be isocaloric. For a period of six consecutive days, the rats received their diet prepared as a paste made up of the solid ingredients and tap water. The paste was prepared by addition of just enough water to agglomerate the mixture. This reduced food wasting by the rats and thus urine contamination by food as preliminary data indicated this affected analytical recovery of 1-OHP. This six-day conditioning period was used to calculate the daily mean amount of food ingested by each group, which corresponded to 20 g dry weight. For the purpose of the study, it was mandatory that fiber be present in the digestive tract when pyrene was administered. Consequently, the following protocol was followed: after the six-day acclimatizing period, the rats were fasted for 24 h, after which they were given free access to 20 g of food for 2 h. The food was then withdrawn just before the iv administration of 5 µmol/kg of pyrene in a tail vein. Pyrene was dissolved in 20% Emulphor, 80% isotonic glucose and the injected volume was 2 ml/kg. Immediately after the injection, the rats were returned to the metabolism cages and urine and feces collection started. The remaining portion of the 20 g of food was returned to the animals after the first 8-h collection period. This compromise ensured that the initial 08 h urine collection was totally free from food contamination while keeping experimental conditions as close as possible to normal physiological conditions. For the remaining duration of urine collection, care was taken to avoid contamination of the urine by visible traces of food.
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Sample analyses.
1-OHP was determined in urine by HPLC with fluorescence detection following a protocol previously described (Bouchard et al., 1994). Analytical recovery of urinary 1-OHP was 81%. Feces were weighed and homogenized in sodium acetate buffer 0.1 M, pH 5 so as to constitute a 6% suspension based on the wet weight of feces. Ten µL of ß-glucuronidase/arylsulfatase solution was added and the mixture incubated at 37°C for 16 h. Two consecutive liquid-liquid extractions with ethyl acetate were then performed, followed by centrifugation and recovery of the organic phases. The solvent was evaporated to dryness and the residue dissolved in methanol and analysed by HPLC using the same protocol as for urine. Analytical recovery for fecal 1-OHP was 85%. Results presented in this article have been corrected for incomplete recovery. In addition, to reduce the interindividual variability resulting from variations in administered pyrene doses due to differences in body weight of the animals, the excretion results were normalized to a constant body weight of 250 g as follows:
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In vitro test on 1-OHP adsorption to fiber.
Since it was hypothesized that insoluble fiber could possibly adsorb 1-OHP, the following in vitro test was conducted. Four ml of a phosphate-buffered saline (0.01 M phosphate, pH 7.2) was added to 0, 10, 30, 50, and 100 mg of Alphacel and the suspension was agitated for 30 min at room temperature on an Eberbach agitator (180 cycles/min). A portion of the same buffer was subsequently spiked at a concentration of 1 nmol/ml of 1-OHP dissolved in methanol (100 nmol/ml). One ml of the latter solution was added to the fiber suspension and agitated under the same conditions for a further 30 min after which the suspension was centrifuged for 10 min at 2000 g. An aliquot of the clear supernatant was diluted with 1 to 3 volumes of methanol before injection in the HPLC system. In addition, another set of the 1-OHP-spiked 50 mg Alphacel suspension was also diluted with 3 volumes of methanol prior to centrifugation to verify the recovery of 1-OHP and ensure the stability of the analyte in the presence of fiber. The HPLC analysis was carried out as described for urine and feces. The assay was conducted in triplicate, except for the control sample (no Alphacel) for which n = 5.
Statistical analysis.
All results are presented as mean and SD. Kolmogorov-Smirnov test was performed to check normality of the distributions, acknowledging the small sample sizes. One-way ANOVA was used to test for differences between the groups followed by a Dunnett multiple comparisons post-hoc test to determine which groups differed from the group receiving no fiber. The level of significance was taken as p = 0.05.
RESULTS
The various groups consumed between 46 and 68% of the 20 g of food given during the 2-h period prior to iv injection of pyrene. Figure 1 shows the urinary excretion of 1-OHP for each of the three consecutive urine collection periods. Around 60% of the 24-h 1-OHP was collected during the initial 08 h period. ANOVA indicates a statistically significant difference (p < 0.05) between the groups only for the period 08 h. In the latter case, the Dunnett's post-hoc test indicates that both groups receiving diets rich in fiber are different from the group receiving no fiber.
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The results of the in vitro experiment aimed at verifying the adsorption of 1-OHP to Alphacel gave the following mean (± SD) 1-OHP fractions in the supernatant compared to control for the 10, 30, 50, and 100 mg alphacel suspensions: 48 ± 1.7%, 27 ± 1.8%, 19 ± 0.7%, and 11 ± 0.3%. The reduction is highly negatively correlated to the amount of Alphacel in the suspension on a semi-log plot (r2 = 0.99). The 50-mg suspension to which methanol was added prior to centrifugation gave a 100% recovery of 1-OHP compared with the control sample of 1-OHP-spiked buffer only. This suggests that 1-OHP adsorbs in a reversible manner to Alphacel fiber.
DISCUSSION
In this study, the iv administration route was chosen in order to free us from bioavailability differences that would increase the overall variability of the results. In doing so, the effect of fiber on the enterohepatic cycling can be better isolated than if other administration routes had been used. Furthermore, biological monitoring of exposure to industrial chemicals aims at assessing the internal dose to which individuals are exposed, no mater the route of exposure and differences in bioavailability between individuals. The iv administration ensured that all rats were exposed to the same internal dose of pyrene per kg body weight.
The current study demonstrated that urinary excretion of 1-OHP is reduced in rats immediately (08 h) following ingestion of food containing either soluble or insoluble dietary fiber, when compared to animals receiving a fiber-free diet. At the given fiber dosage, the percent reduction is not related to the nature of the fiber as both the insoluble Alphacel and soluble pectin had a similar impact, reducing excretion by about 4050%. There did not appear to be a marked difference due to the amounts of fiber given to the animals (5 and 15% w/w of the diet). However, even our "low" fiber diet corresponds to a 10-fold higher consumption per kg than what is considered a low fiber diet in humans (Ballesteros et al., 2001
). Because the nature of the fiber did not appear as a determinant of the urinary excretion of 1-OHP, the results of both low fiber, on the one hand, and high fiber diets, on the other hand, were grouped. This somewhat increased statistical power and indicated that both low fiber and high fiber groups excreted significantly less 1-OHP than the NF group for both the total 24-h collection period and for the initial 08 h period. Of course, human diet always contains fiber and it could be argued that the NF group of this study is not representative of an expected human situation. However, urine samples in workers occupationally exposed to PAH can be collected after fasting periods of many hours that would approach a "no fiber" situation.
Fecal excretion of 1-OHP was not statistically significantly affected by the diet. However, the fecal excretion results were highly variable, possibly in part owing to the fact that not all feces were voided at the end of the 24-h collection period. Nevertheless, it is noteworthy that fecal excretion increased in the a priori hypothesized order: no fiber < low fiber < high fiber. Similarly, the NF group is the only one in which urinary exceeds fecal excretion.
Acknowledging the fact that the differences between the groups were only statistically significant for the 1-OHP urinary output, possible mechanisms can be examined to explain the differences and trends in the data. Viscous fiber such as pectin is seen as hypocholesterolemic, possibly by reducing ileal bile acid absorption through impeding bulk diffusion to the luminal surface and so increasing fecal bile acid loss (Gallaher and Hassel, 1995; Jenkins et al., 2000
). It therefore appears reasonable to hypothesize that, following biliary excretion and intestinal hydrolysis of the glucurono-conjugated metabolite, intestinal absorption of a metabolite such as 1-OHP could similarly be impaired. Compared to a group receiving no fiber in his diet, this would result in a larger fecal excretion of the metabolite and a reduction in its urinary output by reducing the reentry of 1-OHP in the blood.
Contrary to the effects of soluble fiber, isolated insoluble fiber appears not to influence the absorption of nutrients in the small intestine (Edwards, 1995). Although it has been suggested that insoluble fiber can physically or chemically bind bile acids (Read and Eastwood, 1992
), the mechanism by which insoluble fiber could lower intestinal absorption of liposoluble material is less clear. However, our in vitro test suggests that reversible adsorption of 1-OHP to Alphacel might take place in the intestinal tract.
It has been shown that 1-OHP can be extensively biotransformed in vitro to other metabolites by liver post-mitochondrial fractions (Haddad et al., 1997). Therefore, enterohepatic cycling can have two opposite effects: increasing urinary 1-OHP while reducing the total urinary + fecal excretion. Indeed, as indicated above, enterohepatic cycling should increase the total available blood 1-OHP, with more 1-OHP being excreted in urine. But liver first pass effect linked to intestinal absorption of 1-OHP, initially secreted in the bile, will increase the likelihood that a larger fraction of 1-OHP will be further biotransformed into other metabolites. As a consequence, it would be expected that any reduction of the enterohepatic cycling of 1-OHP would reduce urinary 1-OHP, but increase the total urinary + fecal excretion of this metabolite. This corresponds to the nonstatistically significant trend observed. Of course, this does not take into account the possible biotransformation of 1-OHP in the gut and the potential influence of fiber on the gastrointestinal tract concentrations of key biotransformation enzymes. As one such example, dietary fiber can lower intestinal ß-glucuronidase activity, which in turn could partly prevent hydrolysis of the glucurono-conjugate of 1-OHP thus reducing its intestinal reabsorption (Manoj et al., 2001
). The enterohepatic circulation of many solutes is indeed considered to depend on the hydrolysis of the glucuronidated metabolites (Roberts et al., 2002
).
The influence of fiber on urinary 1-OHP can be put in perspective by comparing the influence of genetic polymorphism on this metabolite excretion. Alexandrie et al. (2000) found that CYP1A1 and GSTM1 polymorphism influenced the urinary excretion of 1-OHP in a control group and in aluminum potroom workers. In the cohorts studied by the authors, the median 1-OHP excretion (expressed in µmol/mol creatinine) was 4.3 and 7.5, respectively, in CYP1A1, exon 7, Ile642Val Ile/Ile, and Ile/Val individuals. Corresponding values for controls were 0.1 and 0.17 µmol/mol creatinine. In the same group the GSTM1 +/+ and +/- had a median excretion of 4.2 compared to 4.5 µmol/mol creatinine for the GSTM1 -/-. Corresponding values for controls were 0.10 and 0.12 µmol/mol creatinine. None of the other polymorphisms examined, i.e., GSTP1, GSTT1, and microsomal epoxide hydrolase had a significant influence on the urinary excretion of 1-OHP. By contrast, in coke oven workers, van Delft et al. (2001)
found no influence of GSTM1 polymorphism and confirmed the absence of GSTT1 polymorphism influence on urinary 1-OHP. Finally, in incineration workers, using a multivariate analysis, Lee et al. (2002)
reported a statistical association between GSTM1 polymorphism and urinary 1-OHP. However, the excretion value for the GSTM1 +/+, +/- was 0.22 ± 0.11 µmol/mol creatinine while GSTM1 -/- subjects excreted 0.19 ± 0.09. It should be noted that the difference is in the opposite direction compared to the findings of Alexandrie et al. reported above. It therefore appears that enzyme genetic polymorphism has a marginal, if any, effect for the glutathione transferases. As far as the CYP1A1, exon 7, Ile642Val polymorphism is concerned, the Ile/Val potroom workers have a 43% lower excretion than the Ile/Ile workers. This is the same relative difference as that observed in controls. It is also very similar to the relative difference observed between the no fiber and fiber groups in the current study. Thus dietary fiber could potentially exert as important an influence as enzymatic polymorphism on the urinary excretion of 1-OHP.
In conclusion, dietary fiber can substantially reduce the urinary yield of 1-OHP following an iv dose of pyrene, the reduction amounting to 40-50%. This observation may contribute to the interindividual variability in the urinary excretion of this metabolite observed in occupational and nonoccupational studies.
ACKNOWLEDGMENTS
The authors wish to thank Professor Beatriz Tuchweber from the Department of Nutrition of University of Montreal for useful recommendations in the preparation of the diets, and Ms. Suzanne Leroux and Mr. Ross Thuot for technical contribution to the study. This work was supported in part by the Canadian Institutes of Health Research. COI: The work described in the manuscript was supported in part by the Canadian Institutes of Health Research and by some money left over from doing chemical analyses of 1-hydroxypyrene for public and private organizations.
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