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TOXICOLOGICAL SCIENCES 62, 140–147 (2001)
Copyright © 2001 by the Society of Toxicology
Polycystic Kidney Disease Induced in F1 Sprague-Dawley Rats Fed
para-Nonylphenol in a Soy-Free, Casein-Containing Diet
J. R. Latendresse,*,1 R. R. Newbold,‡ C. C. Weis,† and K. B. Delclos,†
*Pathology Associates International, National Center for Toxicological Research, Jefferson, Arkansas 72079; †Division of Biochemical Toxicology,
National Center for Toxicological Research, Jefferson, Arkansas; and ‡Developmental Endocrinology Section, Reproductive Toxicology Group, Laboratory
of Toxicology, Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received December 7, 2000; accepted February 20, 2001
Nonylphenol(NP;CAS#84852–15–3),analkylphenolwith
para-Nonylphenol (NP; CAS #84852–15–3), an alkylphenol a 9-carbon olefin side chain, is widely used in the manufacture
with a 9-carbon olefin side chain, is widely used in the manufac- of nonionic surfactants, lubricant additives, polymer stabiliz-
ture of nonionic surfactants, lubricant additives, polymer stabiliz- ers, and antioxidants (Gilbert et al., 1986; Naylor, 1992). NP
ers, and antioxidants. Due to its wide commercial use and putative has weakestrogenic activity (Dodds and Lawson, 1938; Shelby
endocrine activity in humans and wildlife, the NTP elected to et al., 1996; Soto et al., 1991). Due to its wide commercial use
assess its effects on reproduction in multigenerational studies. To and putative endocrine activity in humans and wildlife, the
avoid known estrogenic activity of phytoestrogens in soy and NTP elected to assess its effects on reproduction in a 5-gen-
alfalfa, a soy- and alfalfa-free, casein-containing diet was used in eration study designed to evaluate components of the “endo-
a range-finding study to determine the doses of NP to be tested crine disruptor hypothesis.” These include the potential for
further. NP was administered to Sprague-Dawley rats in the diet magnification of subtle reproductive effects over multiple gen-
at 0, 5, 25, 200, 500, 1000, or 2000 ppm to F0 dams beginning on erations, the importance of exposure windows, and whether
gestation-day 7. The F1 pups were weaned at postnatal day (PND)
21, and their exposure via diet was continued at the same dose effects are reversible or are imprinted to carry over across
level as their respective dams. Pup weights from birth through generations (Delclos and Newbold, 1997). We report here on a
weaning were not significantly different from controls in any dose range-finding study to determine the doses of NP to be further
group, but the average weight of both sexes was significantly less tested in the multigeneration assay. The focus of this range-
compared to controls, beginning with the PND 28 weighing. The finding study was on reproductive tract effects. One aspect of
F1 rats were sacrificed on PND 50 (n 5 15, 3 pups of each sex from this protocol that differs from the majority of other studies that
5 litters for all dose groups). Terminal body weights of males and have been conducted with nonylphenol and other endocrine-
females in the 2000-ppm dose group were 74% and 85% of con- active compounds is the use of a soy- and alfalfa-free diet to
trols, respectively. Severe polycystic kidney disease (PKD) was avoid known estrogenic activity of isoflavones in soy and other
present in 100% of the 2000 ppm-exposed male and female rats. At phytoestrogens such as coumestrol in alfalfa. While the recent
1000ppm,67%ofmalesand53%offemaleshadmildtomoderate studies of nonylphenol toxicity have been focused on endo-
PKD versus none of either sex in the control and lower-dose crine and reproductive endpoints, mild renal toxicity was re-
groups. The no-adverse-effect level (NOAEL) for PKD was deter- ported in a recent multigeneration assay utilizing dietary con-
mined to be 500 ppm. Previous studies with comparable duration centrations up to 2000 ppm (Chapin et al., 1999). We describe
and route of exposure, but using soy-containing diets, reported here unanticipated strong renal toxicity of nonylphenol en-
either no or only mild PKD at 2000 ppm NP. We conclude that the countered over a similar dose range under our experimental
renal toxicity of NP is highly dependent on the diet on which the
animals are maintained. The potential interaction of diet and test conditions.
compounds on nonreproductive as well as reproductive endpoints
should be considered when contemplating the use of special diets MATERIALS AND METHODS
formulated to minimize exogenous “hormone” content for the
study of the effects of putative endocrine disruptive chemicals. Test material. NP (CAS # 84852–15–3) was obtained from Schenectady
Key Words: polycystic kidney; nonylphenol; soy-free diet; endo- International, Inc. (Schenectady, NY). The purity of the compound was deter-
crine disruptor. mined by chromatography to be greater than 95%. Homogeneity and stability
of the NP in the 5-ppm mix were found to be within the tolerance limits (6
10% of target dose) for up to 30 days.
Feed. The genistein and daidzein levels in NIH-31 and 5K96 rations used
1 To whom correspondence should be addressed at Pathology Associates at NCTR were analyzed and reported by Doerge et al. (2000). The soy- and
International, P.O. Box 26, 3900 NCTR Road, Jefferson, AR 72079. Fax: (870) alfalfa-free diet, designated 5K96, was obtained from Purina Mills, Inc. (St.
543-7030. E-mail: jlatendresse@nctr.fda.gov. Louis, MO), and was sterilized by irradiation prior to shipment to NCTR. This
140
NONYLPHENOL AND POLYCYSTIC KIDNEY 141
TABLE 1
Fertility and Litter Data for Dams Treated with Dietary NP from GD 7 to Parturition
Dose (ppm)
0 5 25 200 500 1000 2000
Litters/plug-positive females 9/10 9/10 7/10 7/10 9/10 7/10 8/10
Gestation time (days) 22.7 6 0.17 22.4 6 0.17 22.5 6 0.19 22.5 6 0.19 23.0 6 0.00 22.9 6 0.26 23.0 6 0.00
Litter size 11.1 6 1.63 13.4 6 0.73 15.1 6 0.68 13.2 6 1.36 11.2 6 1.30 10.4 6 1.93 10.1 6 1.38
%Males 48.1 37.7 48.1 57 46 47 34.1
Pup birth wt (g) 6.50 6 0.31 6.31 6 0.24 5.78 6 0.14 6.42 6 0.34 6.85 6 0.19 6.33 6 0.38 6.65 6 0.27
LSMpup birth wt (g) 6.45 6 0.15 6.55 6 0.15 6.29 6 0.17 6.37 6 0.19 6.67 6 0.15 5.90 6 0.17 6.64 6 0.17
Note. Results are given as mean 6 SEM. Least squares mean (LSM) 6 SEM, adjusted for litter size and percent males.
soy- and alfalfa-supplemented diet, formulated for this study by Purina, was a tissues designated for microscopic examination from control and dose groups
modification of the NIH-31 diet used as the standard animal feed at NCTR, were trimmed, processed, embedded in Tissue Prep II, sectioned at 4–6
whichhadlevelsof31.963.9mg/gofgenisteinand30.463.1mg/gdaidzein, microns, mounted on glass slides, and stained with hematoxylin and eosin or
much lower than other widely used commercial chows. 5K96 had the soy and periodic acid-Schiff (PAS) and hematoxylin. As a result of the pathological
alfalfa proteins replaced by casein and the soy oil replaced by corn oil. The assessment of the high-dose group, the kidney was unexpectedly identified as
vitamin mix was adjusted to account for losses during irradiation. This diet a target tissue. Therefore, kidneys from the intermediate doses were also
meets the nutrient specifications for NIH-31, and has a nearly identical amino processed in a like manner and examined microscopically. Renal cysts were
acid composition and energy content. Assay of 5K96 indicated genistein and subjectively graded: 4 (severe), numerous large cystic tubules uniformly dis-
daidzein contents of 0.54 6 0.31 and 0.48 6 0.31 mg/g, respectively (Doerge tributed in the outer medulla and/or cortex (M/C) from one pole to the other in
et al., 2000), approximately 60-fold lower than NIH-31. a longitudinal section of kidneys; grade 3 (moderate), similar to grade 4, but
Animal husbandry. Sprague-Dawley (CD) rats (NCTR Strain Code 23) slightly fewer cysts; 2 (mild), approximately 4–10 cysts in the M/C; or 1
were utilized. Temperature and humidity in the animal room were maintained (minimal), 3 or fewer cysts.
at 23 6 3°C and 50 6 20%,respectively. A 12-h light/dark cycle was used, and Statistical analysis. Daily and weekly body weights and feed consumption
the room received 10–15 air changes per hour. Pregnant females were housed were analyzed by analysis of variance, using a mixed models approach to
singly with their litters in polycarbonate cages on standard hardwood-chip repeated measures. For the F1 generation, the statistical model included dose as
bedding (PJ Murphy, Montville, NJ). Weaned F1 pups were housed, 2 of the a fixed factor and litter nested within dose as a random factor to account for
same sex to a cage, until sacrifice at postnatal day (PND) 50. Dosed feed and possible litter effects. Litter weights were analyzed by analysis of covariance,
micropore-filtered tap water were provided ad libitum. with litter size and percent males as covariates. Dunnett’s test was used to
Experiment design. Ten date-mated, vaginal plug-positive females were makecomparisons between control and treatment groups. Histopathology data
assigned to each dose group to ensure 5 litters per treatment. Data on all litters were analyzed for NP effects on lesion incidence and severity by the Jonck-
(size, weight, percent alive, sex ratio) were collected after birth, and 5 dams heere-Terpstra test (Hollander and Wolfe, 1973). Williams’s modification of
and litters from each treatment group were randomly selected for continuation Shirley’s test (Williams, 1986) was used to compare dosed groups to control.
on the experiment. The females (F0) were started on the 5K96 ration 2 weeks All statistical tests were made at the p 5 0.05 level.
prior to breeding and randomly assigned to each group on day 6 of gestation
(GD 6). NP was fed in the diet at 0, 5, 25, 200, 500, 1000, or 2000 ppm, RESULTS
beginning on GD 7. Body weights and feed consumption of the dams were
measured daily from the start of dosing through parturition and weekly Body Weights and Feed Consumption
thereafter. Feed consumption measurements were based on feeder weights
before and after the exposure period, and they reflect both consumption and Damfeedconsumptionduringpregnancy showed a decreas-
spillage. F1 litters were randomly standardized to 4 males and females each on ing linear trend at early times (gestation days 8–10, 12–14, and
PND 2. Pup body weights were recorded on PND 4 and 7, and weekly 17) and Dunnett’s test showed only the 2000-ppm dose group
thereafter until PND 50. Feed consumption was measured weekly after wean-
ing. The F1 pups were weaned at PND 21, and their exposure was continued to be significantly lower (30–40%) than controls on gestation
at the same dose level in the feed as their respective dams received. The F1 rats days 8, 9, 12, and 13 (data not shown). Dam body weights
weresacrificed on PND50(n515,3animalspersexfromeachofthe5litters showed a significant decreasing linear trend from GD 12–21.
in each dose group). Only the 2000-ppm dose group differed significantly from the
Pathology. At study termination, the F1 rats were weighed, euthanized by control group (10% lower), and only on GD 21 (data not
exposure to carbon dioxide, and a complete necropsy was performed. All shown). Total feed consumption and body weight gains during
protocol-specified tissues (adrenal glands, bone and marrow, heart, kidneys,
liver, lung, mammary gland, spleen, thymus, thyroid glands, ureter, urethra, pregnancy were significantly lower than control in both the
urinary bladder, and reproductive tract and accessory glands, and gross lesions) 1000-ppm and 2000-ppm groups (data not shown). There were
were examined, removed, and preserved in either Bouin fixative or 10% no differences in the body weights or feed consumption of the
neutral-buffered formalin. All protocol-specified tissues were examined mi- dams after parturition, until sacrifice at the time that the litters
croscopically in the high-dose and control groups, initially, followed by were weaned. Nonylphenol treatment had no effect on gesta-
examination of the urogenital tissues and accessory sex glands for potential
endocrine disrupting effects, proceeding in descending order of dose. Thus, the tion time, litter weight at birth, or on other litter parameters
142 LATENDRESSE ET AL.
2000 ppm were 74 and 85% of controls, respectively (Fig. 3).
This represents a significantly reduced weight gain for both
sexes.
Microscopic Evaluation of Kidneys of F1 Animals
All the male and female rats in the 2000-ppm group had
severe polycystic kidney disease (PKD; Table 3, Figs. 4 and 5).
The disease of moderate to marked severity was characterized
by numerous large cystic tubules, often uniformly distributed
from one pole to the other. The outer stripe of the outer
medulla was most markedly affected, but cysts extended
deeper into the inner stripe of the outer medulla, and into the
superficial cortex, along the medullary rays. The tubules often
contained necrotic epithelial cells and/or neutrophils. There
were patchy areas of mild to marked chronic interstitial ne-
phritis and foci of degenerative and regenerative tubules. Five
of 15 male rats (33%) and 4 of 15 females (27%) in the
FIG. 1. Growth curves of F1 males (A) and females (B) fed the indicated
doses of p-nonylphenol. Asterisks mark samples significantly different from
controls at the same time point: *p , 0.05; **p , 0.01; ***p , 0.001. Control
line is bolded. Asterisks below the line are associated with the 2000-ppm dose
group, while those above the line are associated with the 200-ppm dose group.
(Table 1). Body weights of male and female pups did not differ
significantly from controls up to the time of weaning, after
which the high-dose group of both sexes had significantly
lower body weights than controls for the remainder of the
experiment (Fig. 1A and 1B). At later time points, both male
and female pups in the 200-ppm dose group had body weights
significantly greater than controls (Fig. 1). Pup feed consump-
tion was also generally lower than controls in the high-dose
group, with significant differences from controls in pairwise
comparisons, as shown in Figures 2A and 2B. Nonylphenol
intake at various stages of the experiment, as calculated from
feed consumption and body weight data, is shown in Table 2.
The mean daily dose of NP consumed by the dams at various
experimental stages was approximately 50% lower during
pregnancy when compared to the lactation period. The mean FIG. 2. Feed consumption of F1 males (A) and females (B) fed the
daily dose for both sexes of pups from PND 21 to 50 was indicated doses of p-nonylphenol. Asterisks mark samples significantly differ-
comparable to the NP intake of the dams during lactation. ent from controls at the same time point: *p , 0.05; **p , 0.01; ***p ,
0.001. Control line is bolded. All asterisks are associated with the 2000-ppm-
Terminal body weights of the males and females exposed to dose group.
NONYLPHENOL AND POLYCYSTIC KIDNEY 143
TABLE 2
Mean (range) Daily Dose of NP Consumed at Various Experiment Stages (mg/kg body weight/day)
Dose (ppm)
5 25 200 500 1000 2000
Pregnancy (GD7-21) 0.36 (0.24–0.46) 1.64 (1.24–2.07) 13.10 (7.79–16.44) 35.35 (30.52–44.59) 61.49 (56.29–67.60) 126.92 (108.44–175.73)
Lactation 0.62 (0.35–0.83) 2.95 (1.41–4.39) 21.72 (9.15–32.48) 60.38 (32.02–86.57) 103.41 (40.79–160.30) 243.35 (137.43–324.00)
Male pups (PND 21–50) 0.56 (0.48–0.68) 2.87 (2.30–3.52) 22.58 (18.88–29.05) 56.72 (47.27–68.13) 116.80 (101.64–137.65) 238.06 (196.89–291.34)
Female pups (PND 21–50) 0.58 (0.50–0.68) 3.05 (2.50–3.85) 22.72 (19.04–26.79) 59.60 (50.11–67.75) 116.66 (100.92–138.90) 251.33 (219.95–315.24)
1000-ppm groups manifested a moderate effect, while the ylphenol, a putative “estrogen effect,” and acquired renal dis-
incidence of mild PKD in male and female rats was 34% and ease. Further studies would be required to address each of these
26%, respectively. as potential contributors, incorporating such designs as paired
Nephrocalcinosis was also observed at high incidences (73– feeding and time-course exposures.
100%) in the female rats in both treatment and control groups There are reports in the literature where semisynthetic, soy-
alike. Minimal mineralization was also present in the male rats free diets fed to rats and mice genetically predisposed to PKD,
in the 500-ppm group (5/15), 1000-ppm group (12/15), and without further treatment, resulted in more severe PKD when
2000-ppm group (6/15). These incidences were significantly compared to rations containing soy (Ogborn et al., 1998;
different (p , 0.001) from the controls (0/15). Tomobe et al., 1998). To our knowledge, the Sprague-Dawley
rat has no recognized genetic or other predisposition for PKD.
DISCUSSION The control rats (males 53% and females 67%) in our study
Nonylphenol treatment decreased feed consumption and manifested one to a few tubular cysts (minimal) in or near the
body weight gain of dams during pregnancy in the 2000-ppm region of the outer medulla or corticomedullary junction. Some
dose group, and total body weight gain during pregnancy was rats in dose groups 5-ppm to 1000-ppm also had minimal cysts
also reduced in the 1000-ppm dose group, but litter parameters with incidences comparable to or lower than the control group.
were not affected by treatment. The pups showed decreased This raised concern that the soy-free diet alone in our study
bodyweightsandfeedconsumptionthatweresignificantinthe could have caused some PKD. After additional consideration,
high-dose group only after weaning. These results are gener- these cysts were interpreted as incidental, because the results of
ally consistent with results from a parallel feeding study with a 6-month feeding study comparing standard NIH-31 diet
nonylphenol that was designed to evaluate effects on sexually (soy-containing) with 5K96, which were not significantly dif-
dimorphic behaviors (Ferguson et al., 2000). Factors that could ferent, showed less than a 10% incidence of cysts in both the
have contributed to the pattern of decreased food consumption parental generation (at 6 months) and their offspring (at PND
and lower body weight observed include palatability of non- 65) fed 5K96 (unpublished data).
Recently, other toxicologic studies of NP administered at
comparable doses and longer exposure intervals were reported
with minimal to mild renal cyst formation (Chapin et al., 1999)
or no renal cyst formation (Cunny et al., 1997). Unlike our
study, in both of these reports rats were fed diets containing
soy. Earlier studies comparing the renal toxicity of butylated
hydroxytoluene (Meyer et al., 1978) and biphenyl (Sonder-
gaard and Blom, 1979) fed in either a semisynthetic diet
(casein as protein source) or commercial chow, reported sig-
nificantly less severe PKD when the compound was adminis-
tered in the commercial chow. The protein source of the
commercialdiets was not specified. The authors concluded that
diet has a significant impact on the renal toxicity of these
compounds. Soy-induced amelioration of PKD in rodent
strains used as models to study this disease has recently been
reported (Aukema et al., 1999; Ogborn et al., 1998; Tomobe et
al., 1998). These authors speculate on a number of factors that
FIG. 3. Terminal body weights. Means 6 SEM; *significantly different maycontribute to the apparent protective effect of soy, includ-
from control at p # 0.05; ***significantly different from control at p # 0.001. ing estrogenic effects, enzyme inhibition, fatty acid metabo-
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