ToxSci Advance Access originally published online on March 22, 2006
Toxicological Sciences 2006 91(2):631-642; doi:10.1093/toxsci/kfj171
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Published by Oxford University Press 2006.
Dietary Modulation of p-Nonylphenol–Induced Polycystic Kidneys in Male Sprague-Dawley Rats
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* Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079; Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and Toxicologic Pathology Associates, Jefferson, Arkansas 72079
1 To whom correspondence should be addressed at Division of Biochemical Toxicology, National Center for Toxicological Research, 3900 NCTR Road, HFT-110, Jefferson, AR 72079. Fax: (870) 543-7136. E-mail: Barry.Delclos{at}fda.hhs.gov.
Received December 19, 2005; accepted March 20, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We had previously found that p-nonylphenol (NP) at 1000–2000 ppm in a soy- and alfalfa-free diet induced severe polycystic kidney disease (PKD) in both male and female pups exposed from gestation day 7 through postnatal day (PND) 50 and hypothesized that differences in dietary components contributed to the severity of lesions relative to those reported in other studies using similar doses of NP. The present study investigated the dietary modulation of NP-induced PKD using the same exposure regimen with 2000 ppm NP in four different diets: the natural ingredient soy- and alfalfa-free diet that had been used in the earlier study, Purina 5K96; two defined diets AIN-93G, designated AIN-CAS, and a modified AIN-93G with soy protein isolate replacing casein as the protein source (AIN-SPI); and the commonly used natural ingredient diet Purina 5001 (P5001). Serum isoflavone levels were negligible in animals fed the soy-free AIN-CAS and 5K96 diets and were 2- to 18-fold higher in animals fed P5001 than in those fed AIN-SPI. Consumption of P5001 was significantly greater than consumption of the other diets, and those animals fed P5001 were generally significantly heavier than animals receiving the other diets. NP significantly reduced body weight gain in male pups regardless of the diet fed. There was no evidence of NP-induced kidney toxicity in male pups at PND 2, 14, or 21 or in the dams. In PND 50 male pups, serum blood urea nitrogen was significantly elevated by NP in all diet groups. Urine volume and urinary N-acetyl ß-glucuronidase were significantly increased by NP in the soy-free 5K96 and AIN-CAS diet groups. Relative kidney weights were increased by NP in all diet groups except P5001, with the greatest increase in AIN-CAS and 5K96 diet groups. Microscopic evaluation of kidneys from the PND 50 males showed that NP induced PKD in all diet groups but with marked variation in the severity depending on the diet. PKD was severe in 100% of the NP-treated animals in the AIN-CAS and 5K96 groups, moderate in 88% of the AIN-SPI diet group, and mild in only 40% of the P5001 diet group. Thus, diet can significantly modulate the development of PKD induced by dietary NP in rats. Soy components, as well as other complex dietary factors, may account for the level of protection afforded by the P5001 diet.
Key Words: p-nonylphenol; kidney; polycystic kidney; soy; AIN-93G purified diet.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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p-Nonylphenol (NP) is widely used as an intermediate in the synthesis of alkylphenol polyethoxylate surfactants and other derivatives in lubricants, polymer stabilizers, plasticizers, and antioxidants (Gilbert et al., 1986
; Naylor, 1992). Degradation of alkylphenol polyethoxylates and other nonylphenol derivatives in the environment leads to the release of NP and short chain ethoxylates that have been demonstrated to be weak estrogens (Inoue et al., 2002; Kim et al., 2003; Naylor, 1992; Routledge and Sumpter, 1997), although the activity has been proposed to vary depending on the target tissues evaluated (Watanabe et al., 2004). NP has become a focus of toxicological research in recent years, primarily because of potential endocrine disrupting activity in wildlife and humans due to this weak estrogenic activity.
Several studies in rodents have also identified the kidney as a target organ for the toxicity of NP. Alterations in adult kidneys including increased kidney weights, tubular dilatation, and isolated cyst formation were found with dietary NP exposure greater than 200 ppm (aproximately 20 mg/kg/day) in a multigenerational study with Sprague-Dawley rats (Chapin et al., 1999). In another multigenerational study where NP was administered daily to Sprague-Dawley rats by gavage, Nagao et al. (2001) found increased absolute and relative kidney weights in adult males of the parental generation treated with 50 mg/kg/day. No morphological alterations were observed in the kidneys at that dose, and no significant renal effects were noted in the F1 generation. In the same report, a separate set of adult male and female rats orally treated with 250 mg NP/kg/day developed a variety of effects, including dilatation, necrosis, and regeneration of renal tubules (Nagao et al., 2001). Han et al. (2004) reported significant increases in relative kidney weights in rats that were gavaged with 50 mg/kg/day for 50 days. Cunny et al. (1997) exposed Sprague-Dawley rats to NP in doses up to 2000 ppm (approximately 150 mg/kg/day) in diet for 90 days beginning at 6 weeks of age. Male rats had a dose-related increase in kidney weights that was significant in the 2000-ppm group, but the kidney effect did not persist after a 4-week recovery period (Cunny et al., 1997). In a study from our laboratory in which Sprague-Dawley rats were exposed to dietary doses of NP up to 2000 ppm from gestation day (GD) 7 through termination at postnatal day (PND) 50, both males and females in the 1000- and 2000-ppm dose groups were observed to have a significant incidence of polycystic kidney disease (PKD), with 100% of the animals of both sexes severely affected at the high dose (Latendresse et al., 2001). We hypothesized that the use of a soy-free diet contributed to the pronounced renal toxicity observed in that study.
PKD is most commonly associated with genetic disorders, but acquired PKD is also observed in humans and animals (Gagnon et al., 2000; Gretz et al., 1996; Ito et al., 1998). It has been demonstrated that soy-containing diets are protective against cyst formation in genetic models of PKD in rodents (Aukema et al., 1999; Ogborn et al., 1998; Philbrick et al., 2003; Tomobe et al., 1998). Other studies have reported findings suggesting soy to be protective against chemically induced renal cysts (Meyer et al., 1978). While the mechanisms of soy's protective effects have not been defined, both differences in soy protein relative to casein, the protein to which it has been most often compared, and soy isoflavones have been suggested to be beneficial in modulating chronic renal disease (Aukema and Housini, 2001; Ogborn et al., 1998; Ranich et al., 2001). Estrogenic effects of soy isoflavones as well as antioxidant and protein tyrosine kinase and topoisomerase inhibitory activities are among the mechanisms that have been proposed as contributing to the effects of genistein in the kidney (Ranich et al., 2001). However, a single study of the isoflavone genistein in a mouse model of PKD suggested that it did not alter disease development at a dietary concentration of 500 ppm (Tomobe et al., 1998) and other soy components, including soyasaponins, possibly play roles in the modulation of renal toxicity (Philbrick et al., 2003). In addition to soy, other dietary factors may modulate toxic responses in the kidney and other organs (Ogborn et al., 2002, 2003; Sankaran et al., 2004). The purpose of the present study was to investigate the dietary modulation of NP-induced PKD.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Test chemical and dose formulation.
NP, branched (CAS # 84852-15-3), was obtained from Schenectady International (Schenectady, NY). Its identity and purity were established using gas chromatography/mass spectrometry and 1H- and 13C-NMR. The test compound was determined to contain 95% branched side chain isomers of NP (4-NP) with the remaining 5% being 2-NP and trace amounts of decylphenol isomers. Dosed feed formulations (2000 ppm) were prepared by direct injection of a solution of 160 g NP in one l of 95% ethanol into an 80-kg capacity blender equipped with an intensifier bar (Patterson-Kelly, Stroudsberg, PA) and blending for 30 min. Control diets were mixed with an equivalent quantity of 95% ethanol. Ethanol was removed under vacuum before the feed was removed from the blender. This mixing procedure had been shown to provide homogeneous blends that were within 10% of the target dose and stable under refrigerated storage conditions (2–8°C) for up to 42 days and under rat cage conditions for up to 14 days (Delclos and Weis, 2003
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Diets.
Four meal diets (Table 1) that were similar in protein, fat, vitamin, and caloric contents were used in this experiment. Two soy protein–free diets, Purina 5K96 and AIN-93G (Dyets, Inc., Bethlehem, PA, Product #110750, referred to herein as AIN-CAS), were used along with two soy-containing diets, Purina 5001 (Purina Mills Inc., St. Louis, MO) and AIN-93G with soy protein isolate substituted for casein ([AIN-SPI], Dyets, Inc., Product #110751). 5K96 diet is a modified NIH-31 diet from Purina Mills, Inc., from which the soy protein and alfalfa have been removed and replaced with casein and the soy oil with corn oil. This diet has been shown to have greatly reduced isoflavone content, typically measuring less than 0.5 ppm for both genistein and daidzein (Doerge et al., 2000). AIN-93G (AIN-CAS) (Dyets Inc.) is a purified protein diet with casein as its sole source of protein and is, thus, isoflavone free. AIN-CAS does contain soy oil, but soy oil does not contain isoflavones. Despite the presence of soy oil in AIN-CAS, both this diet and 5K96 are referred to as "soy free" in this article as neither contains soy protein or soy-derived isoflavones.
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The soy-containing diets differed in the source of soy (AIN-SPI with soy protein isolate or P5001 with soy meal). Isoflavone levels in the diets used were not determined in this study; however, serum levels of isoflavones in blood taken at the time of necropsy were determined (see below). The isoflavone content of soybeans and soy-containing products including soy protein isolate and soy meal is highly variable due to such factors as the particular cultivar used and growing conditions (Setchell and Cole, 2003
). The AIN-SPI diet was produced by Dyets, Inc., using soy protein isolate obtained from the Solae Company (St. Louis, MO). The isoflavone content of this soy protein isolate is reported to range from 1 to 3 mg/g (L. Wagner, Dyets, Inc., personal communication), which would give an isoflavone level in the range of 200–600 ppm in the AIN-SPI diet consistent with the 394- to 430-ppm isoflavone levels that have been reported in similarly formulated diets in the literature (Dave et al., 2005; Hakkak et al., 2000). The P5001 diet contains soy meal and dehydrated alfalfa meal, although the percentages of these components in this closed formula diet are not available. P5001 has been reported to have an isoflavone content ranging from near 300 ppm to 800 ppm (Brown and Setchell, 2001; Degen et al., 2002; Odum et al., 2001; Thigpen et al., 2003).
The AIN diets are defined ingredient, purified protein diets (Reeves et al., 1993) that differ solely in the protein source. In contrast, the closed formula natural ingredient diets, 5K96 and P5001, have multiple and variable differences in composition and protein sources. More complete information on these diets is available from the Purina Mills catalog or Web site (http://www.labdiet.com/indexlabdiethome.htm). Single lots of each of the diets were utilized in this study.
Experimental design.
The exposure protocol used in this experiment mirrored that previously showed to induce PKD in Sprague-Dawley rats (Latendresse et al., 2001). Male and female pups (96 of each) were obtained at weaning from the National Center for Toxicological Research (NCTR) Sprague-Dawley (CD) rat breeding colony, and equal numbers were assigned to each of the four diets by a stratified randomization procedure so that the mean starting body weights of each group were approximately equivalent. Food consumption and body weights were monitored on a weekly basis throughout the study. Food consumption was monitored by weighing feeders weekly, and values were not corrected for spillage. Males and females were paired (one male with one female) in wire-bottomed breeding cages when they were 70–84 days old. Females were checked daily for vaginal plugs and were removed from the breeding cage when plug detection was positive (designated GD 0), housed individually, and maintained on the same diet that they had received since weaning. Breeder males, which were not exposed to NP, were euthanized with carbon dioxide. On GD 7, half of the pregnant females (n = 12) in each diet group were placed on diet containing 2000 ppm NP, and half were continued on respective control diets (n = 12). The dams were maintained on these diets until they were euthanized at the time that their litters were weaned, and the pups continued on the same diet and treatment regimen as their respective dams.
Although both sexes had been found to be equally susceptible to NP-induced PKD (Latendresse et al., 2001), since there is evidence in several models of genetic PKD that the male disease progresses more rapidly (reviewed in Aukema and Housini, 2001) and use of males would avoid the need to consider estrous cycle effects in future biochemical studies, male pups were selected as the primary focus of the current study. Following parturition, on PND 2, litters were culled to eight pups consisting of up to six males with the remainder females when numbers allowed. All excess pups of both sexes remaining after the culling at PND 2 were euthanized, and tissues were collected. Two males in each litter were maintained to PND 50, with the remaining pups being euthanized at either PND 14 or 21. Dams that were euthanized when their litters were weaned were evaluated to determine if exposure to NP in adult females could induce PKD as it did in young animals (Latendresse et al., 2001). At each sampling interval, terminal body weights and organ weights were recorded. Blood, kidneys, and livers were collected immediately after the animals were euthanized. One intact kidney (PND 2 animals) or a center quarter of a kidney (PNDs 14, 21, and 50) from one pup per sex per litter was fixed in 10% neutral buffered formalin (NBF), and the remaining kidney tissue and livers were snap frozen in liquid nitrogen and stored at –80°C for future analyses. Blood samples were allowed to clot for 30 min and centrifuged at 1500 rpm at 4°C. Serum was collected, frozen on dry ice, and stored at –80°C. Kidneys were removed from NBF after 48 h, embedded in Formula R (Surgipath, Richmond, IL), sectioned, and stained with hemotoxylin and eosin (H&E) for microscopic evaluation.
Total area of the kidney (cortex and outer medulla) in five randomly selected H&E–stained slides from each group and the total area occupied by cysts in these regions were determined using an Optimas image analysis system (Optimas Corp., Seattle, WA).
Serum isoflavones.
Serum samples were analyzed for isoflavones (genistein and daidzein) and equol, using a previously validated liquid chromatography–electrospray tandem mass spectrometry method (Twaddle et al., 2002). The limit of quantification from the analysis of 10 µl of serum was 0.005 µM for genistein, daidzein, and equol. The precision of isoflavone measurements (coefficient of variation) was in the range of 3–13% across the concentration ranges previously reported, and the corresponding accuracies were 88–99%.
Renal pathophysiology.
One PND 50 male pup per litter was transferred to a metabolic cage the day before termination, and 24-h urine samples were collected and preserved with 1 ml 1% Na-azide. After collection, urine volumes were measured, and samples were briefly centrifuged to remove particulates (900 x g for 10 min), which were discarded. Urinary N-acetyl ß-glucosaminidase (NAG), a lysosomal enzyme involved in the catabolism of glycoproteins that is released in response to renal injury, was measured with reagents from Diazyme (La Jolla, CA) according to the manufacturer's directions. Blood urea nitrogen (BUN) and serum and urine creatinine were measured with a Cobas Mira Plus analyzer (Roche Diagnostic Systems, Indianapolis, IN) with Roche Diagnostic's reagents.
Statistical analysis.
For purposes of statistical analysis, the litter was the unit of measure, and litter means are reported for end points where data were collected from more than one pup per litter. For the histopathology and renal function data, only one animal per litter was evaluated. Continuous end points (terminal body weights, absolute and relative organ weights, absolute and relative cyst areas) were analyzed by two-way ANOVA, with diet and NP treatment as factors. For comparisons of ingested NP doses, diet and phase of the experiment (pregnant dams, dams with pups during lactation, or pups after weaning) were the factors in the two-way ANOVA. Data that were not normally distributed or showed unequal variance were log transformed prior to analysis. This was necessary only for cyst area and relative cyst area data. Pairwise comparisons of means were conducted using Tukey's test.
Histopathology data (incidence and severity of polycystic kidney) were analyzed by nonparametric techniques. In order to consider the multiway structure of the data, a relative effects (generalized mean) analysis following the method outlined by Brunner and Puri (1996) was conducted. A three-way design with NP dose, dietary soy, and diet nested within soy was conducted. In addition, a more traditional Kruskal-Wallis analysis was run on the 2000-ppm NP data with contrasts used to compare the various diets. A Hochberg-Holms adjustment was applied to the resulting p-values to control for the multiple comparisons. The conclusions of both analyses were comparable. A value of p 
0.05 was considered significant for all statistical tests.
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RESULTS |
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Body Weight, Food Consumption, and Organ Weights
The ingested doses of NP, calculated from food consumption and body weight data, are shown in Table 2. Two-way ANOVA indicated that the ingested dose differed significantly among diets (p < 0.001) and stages of the experiment (pregnant dams, dams with pups during lactation, or pups after weaning) (p < 0.001) but that the differences among diets did not depend on the stage of the experiment (i.e., there was no significant interaction). As is evident from the terminal body weights shown in Table 3, the base diet affected body weight gain, as both control and NP-exposed animals in the P5001 groups weighed more than animals consuming any other diet. This was also true for dams (data not shown). For both dams and male pups, consumption of P5001 was significantly greater (40–100%, p < 0.001) than consumption of any of the other diets regardless of the presence of NP (data not shown). In addition, consumption of 5K96 diet was significantly greater than consumption of either of the AIN diets by approximately 30%, but there was no significant difference between the amounts of AIN-CAS and AIN-SPI consumed.
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For male pups (Table 3), body and organ weight data are shown for PNDs 14, 21, and 50. In general, although not all of the pairwise comparisons are significant, the PND 14 and 21 data indicate higher body and absolute organ weights in the P5001 diet group and the depression of these values by NP treatment. Relative organ weights did not differ at these time points except that the relative liver weights were lower in NP-treated groups than controls in the 5K96 and P5001 diet groups at PND 14.
At the final time point, PND 50, body weights were significantly elevated in the P5001 group relative to the other diets, and the NP-treated groups had lower mean body weights than their respective controls in all diet groups. There were significant diet x NP treatment interactions for relative liver and kidney weights, indicating that the effect of NP was dependent on diet. NP treatment significantly elevated relative liver weights in the AIN-CAS (17%), AIN-SPI (15%), and P5001 (8%) diet groups but not in the 5K96 group. The most dramatic effects were the NP-induced elevations of relative kidney weights in the 5K96 (80%), AIN-CAS (34%), and AIN-SPI (23%) diet groups. NP treatment did not cause a significant change in relative kidney weight in the P5001 diet group.
Histopathology
PND 50 males.
Consistent with previously reported data (Latendresse et al., 2001), PKD was diagnosed in NP-dosed animals at PND 50 (Fig. 1 and Table 4). PKD was diagnosed in all NP-treated animals in the soy-free 5K96 (8 of 8 animals) and AIN-CAS (8 of 8 animals) diet groups, 7 of 8 NP-treated pups in the AIN-SPI diet group, and 4 of 10 animals in the P5001 diet group. Among the control groups, only one animal in the 5K96 diet group (of 39 examined across all diet groups) was diagnosed with minimal PKD (PKD score = 1). There was a clear difference in the severity of NP-induced PKD in the various diet groups, with the highest severity in the 5K96 and AIN-CAS diet groups, moderate severity in the AIN-SPI group, and minimal to mild severity in the P5001 group (see Table 4). A relative effects analysis (Brunner and Puri, 1996) with NP treatment, dietary soy, and diet type within dietary soy as factors was conducted, and the results are summarized graphically in Figure 2. Within the 2000-ppm NP dose groups, the PKD response in the P5001 diet group differed from 5K96, AIN-CAS, and AIN-SPI (Holm's adjusted p < 0.0001 for all three comparisons), the response in the AIN-SPI group differed from the 5K96 and AIN-CAS groups (Holm's adjusted p = 0.008 for both comparisons), and the response in the 5K96 and AIN-CAS groups did not differ from one another (Holm's adjusted p = 0.95).
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Focal areas of nephropathy consisting of tubular degeneration and associated regeneration in the outer cortex were also diagnosed in the PND 50 male kidneys. These data are not reported in Table 4 since this diagnosis could not be definitively made (i.e., it could not be dissociated from the lesions resulting from PKD) in cases of severe PKD that involved numerous tubules and extended into the outer cortex. In control animals, minimal to mild nephropathy was observed in 100% (10 of 10) of the 5K96 group, 40% (4 of 10) of the AIN-CAS group, 38% (3 of 8) of the AIN-SPI group, and 45% (5 of 11) of the P5001 group. Kruskal-Wallis analysis of the incidence and severity data indicated a marginally significant diet effect (asymptotic p = 0.06, exact p = 0.04) on nephropathy in the control groups, suggestive of a possible higher incidence in the 5K96 diet group.
Absolute cyst area and the proportion of renal cortex and medulla area occupied by cysts in five randomly selected kidney sections from each treatment group are also shown in Table 4. The results are consistent with the histopathology in that NP exposure increased the proportion of area occupied by cysts in the 5K96, AIN-CAS, and AIN-SPI diet groups, compared to the respective controls, with the greatest increases in the soy-free diets (5K96 and AIN-CAS). The mean relative cyst area in the P5001 diet group did not differ significantly from the controls.
PND 14 and 21 male pups and dams.
Male pups examined at PND 14 and PND 21 showed no evidence of PKD. Focal areas of minimal to mild nephropathy were observed, with no clear relationship to diet or treatment (data not shown). In the kidneys of dams examined at the time that litters were weaned (after 35 days of NP exposure), PKD was not evident as an NP treatment–related lesion, with only two dams (one in the P5001 control group and one in the P5001 NP group) diagnosed with mild PKD (PKD score = 2). As with the PND 14 and 21 male pups, local areas of nephropathy were diagnosed in the dams, with no clear relationship to diet or NP treatment. Minimal mineralization of the tubules was observed in approximately 90% of the dams in the 5K96 diet groups (control and NP treated) but was virtually absent in the dams fed other diets (2 of 16 in the AIN-SPI diet and 0 of 41 in the other two diets), suggesting that the 5K96 diet was involved in the development of this lesion.
Renal Pathophysiology
While there was a significant overall effect of NP treatment on BUN in the dams, there were no significant differences between treatment and control groups in any diet group in pairwise comparisons. In PND 14 and 21 male pups, NP significantly elevated BUN only in the AIN-SPI diet group on PND 14 (data not shown). In the PND 50 male pups, however, BUN was significantly increased by NP treatment in all diet groups by 28–67% (Table 5). There was no significant effect of diet or NP treatment on serum creatinine levels in the dams (data not shown) or in the PND 50 male pups.
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Urine was collected and analyzed only for PND 50 male pups. There was a significant diet x NP interaction for the 24-h urine volumes of the PND 50 male pups (p < 0.001). In the control animals, the urine volume was the highest in the pups fed the P5001 diet. Urine volumes were not altered by NP treatment in animals fed the soy-containing diets, P5001 and AIN-SPI; however, pups fed the soy-free diets, 5K96 and AIN-CAS, had urine volumes that increased significantly (approximately three- and twofold, respectively) in response to NP treatment (Table 5).
There was a significant diet x NP interaction (p < 0.001) on urinary creatinine concentrations, with NP-treated soy-free diet groups, 5K96 and AIN-CAS, having 2.6- and 2.1-fold lower creatinine levels, respectively, than the control groups. This effect was consistent with the increase in urinary volume seen in NP-treated animals of these diet groups. While urinary creatinine concentrations in the NP-treated soy-containing diet groups were slightly lowered (by 20% in AIN-SPI and 13% in P5001), these differences were not significant (Table 5). Urinary NAG, a lysosomal enzyme released in renal injury that is relatively specific to the proximal convoluted tubule, was increased significantly by NP treatment in the soy-free diet groups. NP increased NAG by 2.9-fold in the 5K96 diet group and by 2.6-fold in the AIN-CAS diet group. The pups fed the soy-containing AIN-SPI and P5001 diets had only minimal and nonsignificant increases of urinary NAG of about 20 and 40%, respectively, after NP treatment (Table 5).
Using the urine volume along with the serum and urinary creatinine concentration measurements of the PND 50 pups, creatinine clearance was calculated as an estimate of glomerular filtration rates (GFRs). Diet affected creatinine clearance, with the value in pups fed the natural ingredient 5K96 and P5001 diets, regardless of NP exposure, being approximately twice that of pups fed the two purified protein AIN diets (Table 5). NP significantly affected GFR only in animals fed P5001, where GFR was approximately 29% lower in controls than in NP-treated animals.
Serum Isoflavone Levels
The mean serum levels of the soy isoflavones genistein, daidzein, and equol, a metabolite of daidzein, in dams and in PND 2, 14, 21, and 50 male pups are reported in Table 6. As expected, the serum levels of the isoflavones were negligible in animals consuming the soy-free 5K96 and AIN-CAS diets. Levels of the isoflavones increased with age in the soy-containing diet groups and were consistently higher (2.4- to 17.6-fold) in animals in the P5001 diet group relative to those in the AIN-SPI diet group. Equol was the major isoflavone in the serum of samples containing isoflavones, except those of the PND 2 and 14 AIN-SPI groups, and it accounted for 47–85% of the total isoflavones.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The potential role of diet, particularly the phytoestrogen content of diet, in affecting the results of studies conducted with endocrine-active agents in rodents is a subject that has received considerable attention in recent years (Ashby et al., 2000
; Boettger-Tong et al., 1998; Brown and Setchell, 2001; Casanova et al., 1999; Naciff et al., 2004; Owens et al., 2003; Thigpen et al., 2004; Wade et al., 2003). However, the potential effect of dietary composition on study outcomes is a subject that deserves broader consideration in biomedical research. Modulation by diet of inherited PKD (Cowley et al., 1996; Ogborn et al., 1998; Tomobe et al., 1998), sexual development (Odum et al., 2001), exencephaly (Harris and Juriloff, 2005), liver toxicity (Shertzer et al., 1988), kidney toxicity (Meyer et al., 1978), taste solution preferences (Tordoff et al., 2002), embryo implantation (Wang et al., 2005a), anxiety and stress hormone release (Hartley et al., 2003), ultrasonic vocalizations of neonates (Becker et al., 2005), and activities of xenobiotic metabolizing enzymes (Appelt and Reicks, 1997; Ronis et al., 2004; Stott et al., 2004) are among the effects that have been reported in rodent models.
Our present study examined the dietary modulation and time of onset of the renal toxicity of NP using 2000 ppm NP administered in four different diets. Based on published reports of the inhibition of cyst development in rodent genetic models of PKD by soy protein (Aukema et al., 1999; Ogborn et al., 1998; Philbrick et al., 2003; Tomobe et al., 1998), we had hypothesized that the lack of soy in the 5K96 diet had contributed to the severe PKD observed in our earlier NP study (Latendresse et al., 2001). To test this hypothesis, we compared two diets (AIN-CAS and AIN-SPI) that differed only in their protein source (casein and soy protein isolate, respectively). In addition, we tested the Purina 5001 diet because it has long been one of the most common laboratory rodent diets used in the United States and it has been reported to have a high soy isoflavone content (Brown and Setchell, 2001; Thigpen et al., 2004). The fourth diet was the soy-free 5K96 diet used in our earlier study. As expected, animals consuming the soy-free diets had negligible concentrations of circulating isoflavones. As has been previously established (Brown and Setchell, 2001), equol was the major circulating isoflavone in rats consuming the soy-containing diets P5001, with similar results being obtained with AIN-SPI. The serum levels of the isoflavones equol, genistein, and daidzein were consistent with the expectations based on the amounts of the total isoflavones reported in these two diets in the literature (Brown and Setchell, 2001; Dave et al., 2005; Hakkak et al., 2000).
Consistent with our earlier report, all of the PND 50 animals fed 2000 ppm NP in 5K96 were diagnosed with severe PKD. The kidney effects of NP in animals fed AIN-CAS (significantly elevated relative kidney weights, urine output, urinary NAG, elevated cyst area, and severe PKD) were nearly indistinguishable from those in the NP-treated 5K96 diet group. On the other hand, NP-treated animals in the AIN-SPI and P5001 diet groups developed PKD at a lower incidence and/or severity than observed in the two soy-free diet groups, with a significantly greater protective effect provided by P5001. Neither urine output nor NAG was significantly elevated by NP treatment in AIN-SPI or P5001 groups as they were in the 5K96 and AIN-CAS groups. Collectively, these data imply some level of protection afforded by the dietary soy and suggest much less biological significance to the cysts observed in the soy-based diets. The protective effect of the diets seemed to correlate with the measured serum isoflavone levels, but while the soy and phytoestrogen contents of the diets are potential contributors to the differential responses observed, there are multiple other differences in the composition of the diets, some of which are summarized in Table 1. For example, the use of animal fat in the P5001 diet leads to a much higher cholesterol content in this diet (200 ppm) than in 5K96 (0.02 ppm) or the two AIN diets (0 ppm). Despite the generally higher ingested dose of NP in the P5001 diet group, those pups showed the least severe effects of NP. Effects of the diet on lowering NP uptake and metabolism cannot be ruled out as potentially contributing to the low severity of the renal effects of NP, but the fact that body weight gain suppression in the pups by NP did not differ significantly among the diets suggests that consistent and effective internal dose levels of NP were achieved in all diet groups.
While NP did significantly elevate serum BUN in all diet groups, other markers of renal function, particularly creatinine clearance (measured as an indicator of GFR), were not significantly affected by NP despite the severe PKD observed in some of the diet groups. This was undoubtedly due to the extensive reserve capacity of the kidney that precludes manifestation of clinical signs or laboratory abnormalities until progression to more significant renal disease. Diet alone did significantly affect GFR, with the control natural ingredient diets (5K96 and P5001) showing GFRs ranging from 1.8- to 2.6-fold higher than the control AIN diets. The 24-h urine volume also differed significantly among diet groups, with the output in the P5001 diet group approximately twofold greater than the output in the other diet groups, greater than the difference that would have been predicted based on body size alone. While water consumption was not measured in this study and diet-related changes in fluid intake cannot be ruled out, both genistein and equol have been shown to be diuretics as a result of inhibition of Na-K-Cl cotransport in the kidney (Alda et al., 1996; Martinez et al., 1998). The high levels of equol in the animals consuming P5001 could thus contribute to the twofold greater output of urine and increased GFR observed in that group. NP also significantly increased urine output in the soy-free diet groups. In a previous study under similar conditions with NP and 5K96 diet, 2000 ppm NP did not significantly increase water intake except when the animals were simultaneously offered a 3% sodium chloride solution (Ferguson et al., 2000). Coupled with the absence of an effect of NP on GFR, the increased urine output in the soy-free diet groups suggests an alteration in the process of tubular reabsorption of water and thereby implicates the proximal convoluted tubules as an impacted region of the nephron in NP toxicity.
In our earlier study (Latendresse et al., 2001), PKD was observed in PND 50 pups of both sexes treated with 1000 or 2000 ppm dietary NP from GD 7 through PND 50. In the present study, kidneys of male pups were examined at PNDs 14 and 21 as well as on PND 50, but there were no indications from organ weights or histopathology of developing PKD at the earlier time points. It is possible that the lack of evident lesions at these younger ages was due to a lower dose of NP delivered to the pups in utero and during lactation than they are exposed to by direct consumption of feed after weaning. In utero exposure to NP following NP administration has been measured in our experimental model (Doerge et al., 2002) and found to be approximately 30–40% of the levels found in the dams. Lactational exposure has been indirectly demonstrated by increases in calbindin-D9k mRNA expression in the uterus of pups after injection of dams with NP (Hong et al., 2004), but the extent of transfer has not been quantified.
It is also likely that there is a critical exposure period for the induction of PKD by NP. A recent study of Fukuda et al. (2004) found that tetrabromobisphenol caused polycystic lesions in the kidneys of rats treated during development (PND 3 to PND 21) but not in older rats when given 10-fold higher doses for an equivalent length of time. Dams in the present study did not develop PKD after 35 days exposure to 2000 ppm NP as adults. Renal lesions have been reported in both males and females dosed orally as adults with 250 mg/kg NP (Nagao et al., 2001), but PKD was not observed. As nephrogenesis is a developmental process beginning in the third week of gestation and continuing into the second week postnatally (Chevalier, 1998), it seems possible that some of the renal toxicity and PKD observed in the PND 50 male pups may have been initiated during development and manifested at the later date. While our studies suggest the importance of early exposure to NP in the development of PKD, further experiments would be required to establish the sensitive exposure window.
A high incidence of mild nephrocalcinosis was observed in dams in the 5K96 control and NP-treated groups. Diet and sex are known to play significant roles in the development of nephrocalcinosis, with females more susceptible and both the level and the ratio of calcium to phosphorous important dietary factors (Cockell et al., 2002; Rao, 2002). The 5K96 diet does have a higher concentration of calcium and phosphorous than the other diets tested in this study, and the Ca2+:P molar ratio is lower than in the other diets (5K96, 1.0; AIN-CAS and AIN-SPI, 1.3; P5001, 1.1). NP was found to induce mild nephrocalcinosis in males at doses above 500 ppm in our previous study using the 5K96 diet (Latendresse et al., 2001). Mineralization of the renal tubules at the 2000-ppm dose level in the soy-free diets in the present study could not be distinguished from the consequences of severe PKD, but nephrocalcinosis was not observed in NP-treated males in the soy-containing diet groups that showed less severe PKD. This suggests that the earlier observation of mineralization induced by NP in males may be dependent on diet. Neither the mineralization nor the minimal to mild nephropathy observed in animals in this study appeared to be associated with the development of PKD.
The results of the present study clearly indicate that dietary factors have a strong influence on the induction of PKD by NP. Although serum isoflavone levels correlated with dietary protection, the specific dietary factors responsible for modulating NP's renal toxicity cannot be established from this study design. The mechanism whereby NP induces renal toxicity is also not known. While the focus of much of the interest in NP has been on its weak estrogenic activity, NP also has been shown to affect the expression of genes not regulated by estrogen (Watanabe et al., 2004) and to affect cellular calcium regulation (Hughes et al., 2000; Wang et al., 2005b). The development of PKD can involve multiple complex pathways (Calvet and Grantham, 2001; Cowley, 2004; Murcia et al., 1999; Sullivan et al., 1998), and the impact of cystogens, such as NP, and diet on these pathways remains to be elucidated.
The importance of diet in influencing the outcome of animal experiments has been a subject of discussion and research for years and led to the formulation of the purified AIN-76 diet (American Institute of Nutrition, 1977, 1980) to control variability in experimental diets. While adequate for many types of studies, AIN-76 is not optimal for breeding and lactation (Odum et al., 2001), and earlier concerns about nutritional deficiencies and pathologies related to the formulation, such as nephrocalcinosis, led to the formulation of the AIN-98G (for growth, pregnancy, and lactation) and AIN-98M (for adult maintenance) diets (Reeves et al., 1993). Still, both cereal-based (or natural ingredient) and the AIN diets are widely used in animal toxicology studies, and the selection of the diet used is often made on the basis of price or familiarity rather than on the appropriateness of the diet for the animal model being used. The results of the present study underline the importance of giving careful consideration to the diet when designing and interpreting toxicological studies in animals. The importance of minimizing diet variability, of using the diet formulation best suited for the experimental model, and of clearly identifying the diet when reporting the results are general issues that need consideration.
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ACKNOWLEDGMENTS |
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We thank the following individuals for their invaluable contributions to this study: Tina Glover and the animal care staff and Andy Matson and the diet preparation staff of Bionetics at NCTR; Don Hussen and Stephanie Green of Z-Tech, Inc., for data management support; Brett Thorn of Z-Tech, Inc., for suggesting and conducting the relative effects analysis of the histopathology data; and Alan Warbritton and Levan Muskhelishvili of Toxicologic Pathology Associates for guidance and advice and for producing the micrographs and measurements of renal sections used in this report. This research was made possible in part by funding from the Oak Ridge Institute for Science and Education (Oak Ridge, TN 37831).
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