62, 140 –147 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Polycystic Kidney Disease Induced in F 1 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
para-Nonylphenol (NP; CAS #84852–15–3), an alkylphenol
with a 9-carbon olefin side chain, is widely used in the manufacture of nonionic surfactants, lubricant additives, polymer stabilizers, and antioxidants. Due to its wide commercial use and putative
endocrine activity in humans and wildlife, the NTP elected to
assess its effects on reproduction in multigenerational studies. To
avoid known estrogenic activity of phytoestrogens in soy and
alfalfa, a soy- and alfalfa-free, casein-containing diet was used in
a range-finding study to determine the doses of NP to be tested
further. NP was administered to Sprague-Dawley rats in the diet
at 0, 5, 25, 200, 500, 1000, or 2000 ppm to F 0 dams beginning on
gestation-day 7. The F 1 pups were weaned at postnatal day (PND)
21, and their exposure via diet was continued at the same dose
level as their respective dams. Pup weights from birth through
weaning were not significantly different from controls in any dose
group, but the average weight of both sexes was significantly less
compared to controls, beginning with the PND 28 weighing. The
F 1 rats were sacrificed on PND 50 (n ⴝ 15, 3 pups of each sex from
5 litters for all dose groups). Terminal body weights of males and
females in the 2000-ppm dose group were 74% and 85% of controls, respectively. Severe polycystic kidney disease (PKD) was
present in 100% of the 2000 ppm-exposed male and female rats. At
1000 ppm, 67% of males and 53% of females had mild to moderate
PKD versus none of either sex in the control and lower-dose
groups. The no-adverse-effect level (NOAEL) for PKD was determined to be 500 ppm. Previous studies with comparable duration
and route of exposure, but using soy-containing diets, reported
either no or only mild PKD at 2000 ppm NP. We conclude that the
renal toxicity of NP is highly dependent on the diet on which the
animals are maintained. The potential interaction of diet and test
compounds on nonreproductive as well as reproductive endpoints
should be considered when contemplating the use of special diets
formulated to minimize exogenous “hormone” content for the
study of the effects of putative endocrine disruptive chemicals.
Key Words: polycystic kidney; nonylphenol; soy-free diet; endocrine disruptor.
1
To whom correspondence should be addressed at Pathology Associates
International, P.O. Box 26, 3900 NCTR Road, Jefferson, AR 72079. Fax: (870)
543-7030. E-mail: [email protected].
Nonylphenol (NP; CAS # 84852–15–3), an alkylphenol with
a 9-carbon olefin side chain, is widely used in the manufacture
of nonionic surfactants, lubricant additives, polymer stabilizers, and antioxidants (Gilbert et al., 1986; Naylor, 1992). NP
has weak estrogenic activity (Dodds and Lawson, 1938; Shelby
et al., 1996; Soto et al., 1991). Due to its wide commercial use
and putative endocrine activity in humans and wildlife, the
NTP elected to assess its effects on reproduction in a 5-generation study designed to evaluate components of the “endocrine disruptor hypothesis.” These include the potential for
magnification of subtle reproductive effects over multiple generations, the importance of exposure windows, and whether
effects are reversible or are imprinted to carry over across
generations (Delclos and Newbold, 1997). We report here on a
range-finding study to determine the doses of NP to be further
tested in the multigeneration assay. The focus of this rangefinding study was on reproductive tract effects. One aspect of
this protocol that differs from the majority of other studies that
have been conducted with nonylphenol and other endocrineactive compounds is the use of a soy- and alfalfa-free diet to
avoid known estrogenic activity of isoflavones in soy and other
phytoestrogens such as coumestrol in alfalfa. While the recent
studies of nonylphenol toxicity have been focused on endocrine and reproductive endpoints, mild renal toxicity was reported in a recent multigeneration assay utilizing dietary concentrations up to 2000 ppm (Chapin et al., 1999). We describe
here unanticipated strong renal toxicity of nonylphenol encountered over a similar dose range under our experimental
conditions.
MATERIALS AND METHODS
Test material. NP (CAS # 84852–15–3) was obtained from Schenectady
International, Inc. (Schenectady, NY). The purity of the compound was determined 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 (⫾
10% of target dose) for up to 30 days.
Feed. The genistein and daidzein levels in NIH-31 and 5K96 rations used
at NCTR were analyzed and reported by Doerge et al. (2000). The soy- and
alfalfa-free diet, designated 5K96, was obtained from Purina Mills, Inc. (St.
Louis, MO), and was sterilized by irradiation prior to shipment to NCTR. This
140
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NONYLPHENOL AND POLYCYSTIC KIDNEY
TABLE 1
Fertility and Litter Data for Dams Treated with Dietary NP from GD 7 to Parturition
Dose (ppm)
Litters/plug-positive females
Gestation time (days)
Litter size
% Males
Pup birth wt (g)
LSM pup birth wt (g)
0
5
25
200
500
1000
2000
9/10
22.7 ⫾ 0.17
11.1 ⫾ 1.63
48.1
6.50 ⫾ 0.31
6.45 ⫾ 0.15
9/10
22.4 ⫾ 0.17
13.4 ⫾ 0.73
37.7
6.31 ⫾ 0.24
6.55 ⫾ 0.15
7/10
22.5 ⫾ 0.19
15.1 ⫾ 0.68
48.1
5.78 ⫾ 0.14
6.29 ⫾ 0.17
7/10
22.5 ⫾ 0.19
13.2 ⫾ 1.36
57
6.42 ⫾ 0.34
6.37 ⫾ 0.19
9/10
23.0 ⫾ 0.00
11.2 ⫾ 1.30
46
6.85 ⫾ 0.19
6.67 ⫾ 0.15
7/10
22.9 ⫾ 0.26
10.4 ⫾ 1.93
47
6.33 ⫾ 0.38
5.90 ⫾ 0.17
8/10
23.0 ⫾ 0.00
10.1 ⫾ 1.38
34.1
6.65 ⫾ 0.27
6.64 ⫾ 0.17
Note. Results are given as mean ⫾ SEM. Least squares mean (LSM) ⫾ SEM, adjusted for litter size and percent males.
soy- and alfalfa-supplemented diet, formulated for this study by Purina, was a
modification of the NIH-31 diet used as the standard animal feed at NCTR,
which had levels of 31.9 ⫾ 3.9 ␮g/g of genistein and 30.4 ⫾ 3.1 ␮g/g daidzein,
much lower than other widely used commercial chows. 5K96 had the soy and
alfalfa proteins replaced by casein and the soy oil replaced by corn oil. The
vitamin mix was adjusted to account for losses during irradiation. This diet
meets the nutrient specifications for NIH-31, and has a nearly identical amino
acid composition and energy content. Assay of 5K96 indicated genistein and
daidzein contents of 0.54 ⫾ 0.31 and 0.48 ⫾ 0.31 ␮g/g, respectively (Doerge
et al., 2000), approximately 60-fold lower than NIH-31.
Animal husbandry. Sprague-Dawley (CD) rats (NCTR Strain Code 23)
were utilized. Temperature and humidity in the animal room were maintained
at 23 ⫾ 3°C and 50 ⫾ 20%, respectively. A 12-h light/dark cycle was used, and
the room received 10 –15 air changes per hour. Pregnant females were housed
singly with their litters in polycarbonate cages on standard hardwood-chip
bedding (PJ Murphy, Montville, NJ). Weaned F 1 pups were housed, 2 of the
same sex to a cage, until sacrifice at postnatal day (PND) 50. Dosed feed and
micropore-filtered tap water were provided ad libitum.
Experiment design. Ten date-mated, vaginal plug-positive females were
assigned to each dose group to ensure 5 litters per treatment. Data on all litters
(size, weight, percent alive, sex ratio) were collected after birth, and 5 dams
and litters from each treatment group were randomly selected for continuation
on the experiment. The females (F 0) were started on the 5K96 ration 2 weeks
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,
beginning on GD 7. Body weights and feed consumption of the dams were
measured daily from the start of dosing through parturition and weekly
thereafter. Feed consumption measurements were based on feeder weights
before and after the exposure period, and they reflect both consumption and
spillage. F 1 litters were randomly standardized to 4 males and females each on
PND 2. Pup body weights were recorded on PND 4 and 7, and weekly
thereafter until PND 50. Feed consumption was measured weekly after weaning. The F 1 pups were weaned at PND 21, and their exposure was continued
at the same dose level in the feed as their respective dams received. The F 1 rats
were sacrificed on PND 50 (n ⫽ 15, 3 animals per sex from each of the 5 litters
in each dose group).
Pathology. At study termination, the F 1 rats were weighed, euthanized by
exposure to carbon dioxide, and a complete necropsy was performed. All
protocol-specified tissues (adrenal glands, bone and marrow, heart, kidneys,
liver, lung, mammary gland, spleen, thymus, thyroid glands, ureter, urethra,
urinary bladder, and reproductive tract and accessory glands, and gross lesions)
were examined, removed, and preserved in either Bouin fixative or 10%
neutral-buffered formalin. All protocol-specified tissues were examined microscopically in the high-dose and control groups, initially, followed by
examination of the urogenital tissues and accessory sex glands for potential
endocrine disrupting effects, proceeding in descending order of dose. Thus, the
tissues designated for microscopic examination from control and dose groups
were trimmed, processed, embedded in Tissue Prep II, sectioned at 4 – 6
microns, mounted on glass slides, and stained with hematoxylin and eosin or
periodic acid-Schiff (PAS) and hematoxylin. As a result of the pathological
assessment of the high-dose group, the kidney was unexpectedly identified as
a target tissue. Therefore, kidneys from the intermediate doses were also
processed in a like manner and examined microscopically. Renal cysts were
subjectively graded: 4 (severe), numerous large cystic tubules uniformly distributed in the outer medulla and/or cortex (M/C) from one pole to the other in
a longitudinal section of kidneys; grade 3 (moderate), similar to grade 4, but
slightly fewer cysts; 2 (mild), approximately 4 –10 cysts in the M/C; or 1
(minimal), 3 or fewer cysts.
Statistical analysis. Daily and weekly body weights and feed consumption
were analyzed by analysis of variance, using a mixed models approach to
repeated measures. For the F 1 generation, the statistical model included dose as
a fixed factor and litter nested within dose as a random factor to account for
possible litter effects. Litter weights were analyzed by analysis of covariance,
with litter size and percent males as covariates. Dunnett’s test was used to
make comparisons between control and treatment groups. Histopathology data
were analyzed for NP effects on lesion incidence and severity by the Jonckheere-Terpstra test (Hollander and Wolfe, 1973). Williams’s modification of
Shirley’s test (Williams, 1986) was used to compare dosed groups to control.
All statistical tests were made at the p ⫽ 0.05 level.
RESULTS
Body Weights and Feed Consumption
Dam feed consumption during pregnancy showed a decreasing linear trend at early times (gestation days 8 –10, 12–14, and
17) and Dunnett’s test showed only the 2000-ppm dose group
to be significantly lower (30 – 40%) than controls on gestation
days 8, 9, 12, and 13 (data not shown). Dam body weights
showed a significant decreasing linear trend from GD 12–21.
Only the 2000-ppm dose group differed significantly from the
control group (10% lower), and only on GD 21 (data not
shown). Total feed consumption and body weight gains during
pregnancy were significantly lower than control in both the
1000-ppm and 2000-ppm groups (data not shown). There were
no differences in the body weights or feed consumption of the
dams after parturition, until sacrifice at the time that the litters
were weaned. Nonylphenol treatment had no effect on gestation time, litter weight at birth, or on other litter parameters
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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 F 1 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 nephritis 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 F 1 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 consumption 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
daily dose for both sexes of pups from PND 21 to 50 was
comparable to the NP intake of the dams during lactation.
Terminal body weights of the males and females exposed to
FIG. 2. Feed consumption of F 1 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. All asterisks are associated with the 2000-ppmdose group.
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NONYLPHENOL AND POLYCYSTIC KIDNEY
TABLE 2
Mean (range) Daily Dose of NP Consumed at Various Experiment Stages (mg/kg body weight/day)
Dose (ppm)
5
Pregnancy (GD7-21)
Lactation
Male pups (PND 21–50)
Female pups (PND 21–50)
0.36
0.62
0.56
0.58
(0.24–0.46)
(0.35–0.83)
(0.48–0.68)
(0.50–0.68)
25
1.64
2.95
2.87
3.05
(1.24–2.07)
(1.41–4.39)
(2.30–3.52)
(2.50–3.85)
200
500
13.10 (7.79–16.44)
21.72 (9.15–32.48)
22.58 (18.88–29.05)
22.72 (19.04–26.79)
1000-ppm groups manifested a moderate effect, while the
incidence of mild PKD in male and female rats was 34% and
26%, respectively.
Nephrocalcinosis was also observed at high incidences (73–
100%) in the female rats in both treatment and control groups
alike. Minimal mineralization was also present in the male rats
in the 500-ppm group (5/15), 1000-ppm group (12/15), and
2000-ppm group (6/15). These incidences were significantly
different (p ⬍ 0.001) from the controls (0/15).
DISCUSSION
Nonylphenol treatment decreased feed consumption and
body weight gain of dams during pregnancy in the 2000-ppm
dose group, and total body weight gain during pregnancy was
also reduced in the 1000-ppm dose group, but litter parameters
were not affected by treatment. The pups showed decreased
body weights and feed consumption that were significant in the
high-dose group only after weaning. These results are generally consistent with results from a parallel feeding study with
nonylphenol that was designed to evaluate effects on sexually
dimorphic behaviors (Ferguson et al., 2000). Factors that could
have contributed to the pattern of decreased food consumption
and lower body weight observed include palatability of non-
FIG. 3. Terminal body weights. Means ⫾ SEM; *significantly different
from control at p ⱕ 0.05; ***significantly different from control at p ⱕ 0.001.
35.35
60.38
56.72
59.60
1000
(30.52–44.59) 61.49 (56.29–67.60) 126.92
(32.02–86.57) 103.41 (40.79–160.30) 243.35
(47.27–68.13) 116.80 (101.64–137.65) 238.06
(50.11–67.75) 116.66 (100.92–138.90) 251.33
2000
(108.44–175.73)
(137.43–324.00)
(196.89–291.34)
(219.95–315.24)
ylphenol, a putative “estrogen effect,” and acquired renal disease. Further studies would be required to address each of these
as potential contributors, incorporating such designs as paired
feeding and time-course exposures.
There are reports in the literature where semisynthetic, soyfree diets fed to rats and mice genetically predisposed to PKD,
without further treatment, resulted in more severe PKD when
compared to rations containing soy (Ogborn et al., 1998;
Tomobe et al., 1998). To our knowledge, the Sprague-Dawley
rat has no recognized genetic or other predisposition for PKD.
The control rats (males 53% and females 67%) in our study
manifested one to a few tubular cysts (minimal) in or near the
region of the outer medulla or corticomedullary junction. Some
rats in dose groups 5-ppm to 1000-ppm also had minimal cysts
with incidences comparable to or lower than the control group.
This raised concern that the soy-free diet alone in our study
could have caused some PKD. After additional consideration,
these cysts were interpreted as incidental, because the results of
a 6-month feeding study comparing standard NIH-31 diet
(soy-containing) with 5K96, which were not significantly different, showed less than a 10% incidence of cysts in both the
parental generation (at 6 months) and their offspring (at PND
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 (Sondergaard and Blom, 1979) fed in either a semisynthetic diet
(casein as protein source) or commercial chow, reported significantly less severe PKD when the compound was administered in the commercial chow. The protein source of the
commercial diets 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
may contribute to the apparent protective effect of soy, including estrogenic effects, enzyme inhibition, fatty acid metabo-
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TABLE 3
Incidence of Polycystic Kidney Disease (PKD)
Dose (ppm)
Sex
0
5
25
200
500 a
1000
2000
Male
Female
0/15 (0%)
0/15 (0%)
0/15 (0%)
0/15 (0%)
0/15 (0%)
0/15 (0%)
0/15 (0%)
0/15 (0%)
0/15 (0%)
0/15 (0%)
10/15 (67%)⫹⫹
8/15 (53%)⫹⫹
15/15 (100%)⫹⫹⫹
15/15 (100%)⫹⫹⫹
Note. Severity: ⫹⫹, mild to moderate; ⫹⫹⫹, severe.
a
No observed-adverse-effect level (NOAEL) as determined by Jonckheere-Terpstra test (see Materials and Methods).
lism, and amino acid profiles. Interestingly, supplementation
(500 ppm) of a casein-based diet with genistein, an estrogenactive isoflavone, did not reduce PKD in a mouse model
(Tomobe et al., 1998). Recent feeding studies in our laboratory
using an essentially identical experiment design and the same
diet (5K96) used to test NP, but testing genistein (up to 1250
ppm) or ethinyl estradiol (up to 200 ppb) did not cause PKD in
CD rats (unpublished data). These results underline the fact
that the soy-free diet itself does not induce PKD in the CD rats
used in our studies, which are not genetically predisposed to
PKD.
NP and/or its metabolites may be able to cause some renal
cysts with little or no dietary influence, which may have more
relevance to human exposure. Nonylphenol has been reported
as an air pollutant (2.2–70 ng/m 3) in the coastal atmosphere of
New York and New Jersey (Dachs et al., 1999). Furthermore,
alkylphenolic chemicals approximating 1 ␮g/l have been detected in drinking water (Clark et al., 1992), close to the
National Research Council (NRC) estimate of potential human
exposure of 0.7 ␮g nonylphenol through drinking water (NRC,
1999). However, in the present study, the dietary doses that
FIG. 4. Kidney from a male F 1 SD
rat exposed to 2000 ppm of p-nonylphenol in a soy-free, casein-containing diet.
Severe polycystic kidney disease; magnification ⫻9.5, H&E stain.
caused PKD in Sprague-Dawley rats were much higher than
these likely human exposures.
Chapin et al. (1999) have observed mild renal cyst formation
in rats fed NP in a more conventional soy-containing diet. NP
and some of its derivatives are excreted by the kidney (Drotman, 1980; Knaak et al., 1966) which may deliver a significant
dose to the target site, potentially from both blood and urine.
Kidney in humans (Concolino et al., 1993) and experimental
animals have estrogen receptors (Hamilton et al., 1975; Li and
Li, 1996). An estrogen receptor-mediated effect on tubular
epithelial cell proliferation has been proposed as a potential
cause of acquired cystic kidney disease (Concolino et al.,
1993). It is conceivable that receptor binding by NP could
activate epithelial estrogen receptors, secondarily causing cystic tubules, due to cell proliferation resulting in tubular blockage. However, as noted above, neither genistein nor ethinyl
estradiol induced PKD under identical experimental conditions
where body weight gain depression and estrogenic actions of
these compounds were clearly evident (unpublished data).
Mineralization of the renal tubule epithelium is a well
known estrogen-induced effect in rats. This condition has
NONYLPHENOL AND POLYCYSTIC KIDNEY
145
FIG. 5. Kidney from a female F 1 SD
rat exposed to 2000 ppm of p-nonylphenol in a soy-free, casein-containing diet.
Severe polycystic kidney disease; magnification ⫻9.5, H&E stain.
been reviewed (Ritskes-Hoitinga and Beynen, 1992). In contrast to females, nephrocalcinosis is an uncommon spontaneous
lesion in young male CD rats. In females, endogenous estrogens cause the condition, in part. Exogenous estrogen has been
reported to cause similar mineralization in males, apparently
resulting from an alteration in calcium and phosphorus homeostasis (Ritskes-Hoitinga and Beynen, 1992), and we have
observed similar effects of genistein and ethinyl estradiol in
experiments identical in design to those described here (unpublished data). Because of the reported weak estrogenic activity of NP, it is possible that the minimal mineralization
observed in the 3 male groups exposed to the highest doses was
an “estrogenic” effect of NP on kidney tubules. This seems
more plausible than the possibility that it was a sequela of
tubular epithelial necrosis associated with the toxicity of the
NP-dietary interaction (e.g., PKD), because severe PKD occurred in 100% of the 2000-ppm group, but mineralization was
observed in only 40% of the same group. Furthermore, mineralization was present the 500-ppm group that, like the control
and the 3 other lower-dose groups, did not have PKD. In
addition, Chapin et al. (1999) also noted mineralization of
renal tubules in males at doses as low as 200-ppm NP under
conditions where cysts or tubular dilatation were not reported.
In addition to the previously noted difference in diets, the study
of Chapin et al. (1999) differed from ours in that animals were
older at the time of necropsy and had been exposed to treatment for a longer period.
NP has other effects that are apparently not mediated by
cytosolic or nuclear estrogen receptors. NP and other alkylphenols have been shown to inhibit intracellular calcium (Ca)
pumps in skeletal and smooth muscle and rat testis (Michelan-
geli et al., 1990, 1996; Orlowski and Champeil, 1991; Ruehlmann et al., 1998). Alterations of normal cytosolic Ca homeostasis can potentially have significant consequences on
molecular signal transduction and other vital cellular functions,
including ion pumps regulating electrolyte and water exchange
(Chapin et al., 1999; Michelangeli et al., 1996). Zhang and
O’Neil (1996) have reported the presence of an L-type calcium
channel in renal epithelial cells. Chapin et al. (1999) have
hypothesized an NP-induced ion pump defect as a potential
mechanism of renal cyst formation. NP has also been reported
to act via induction of oxidative stress (Okai et al., 2000) and
as a genotoxin (Roy et al., 1997). It is plausible that NP or its
metabolites may induce some renal tubular cysts through these
estrogen receptor- and non-estrogen receptor-mediated potential mechanisms or others, but currently available data are
insufficient for any conclusions to be drawn. The complex
exposure regimen used in the present protocol, which involves
indirect developmental exposure as well as direct dietary exposure of the pups, also does not allow conclusions to be drawn
as to critical windows of exposure for the induction of renal
cysts. Chapin et al. (1999) reported renal cysts only in generations that received NP developmentally, and not in the F 0
generation that were exposed only as adults. Likewise, Cunny
et al. (1997) did not report renal toxicity in a 90-day adult-only
dietary exposure. We did not initiate the present experiment to
address the issue of renal toxicity, and dams were not necropsied. However, the ongoing multigeneration experiment includes animals that are exposed only as adults as well as
animals that are exposed only developmentally (i.e., in utero
and preweaning), although the highest dose used is 750 ppm.
Comparing previous studies with this one (where the dose
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LATENDRESSE ET AL.
and route of exposure of NP are the same, but the diet is not),
the striking difference in the severity of PKD observed leads to
the conclusion that the renal toxicity of NP is highly dependent
on the diet on which the animals are maintained. Furthermore,
there appear to be some protective effects associated with
soy-meal supplementation, although the dietary factors responsible are unknown. Considerable research is ongoing to test the
endocrine-disruptor hypothesis that certain exogenous hormones (phytoestrogens) or hormone-active industrial pollutants may alter the development and function of the reproductive system in humans and animals. Recent studies have shown
that the levels of phytoestrogens widely vary in diets used in
experimental animal studies (Boettger-Tong et al., 1998; Thigpen et al., 1999a). As a result, some researchers advocate
modifications of conventional diets to minimize the content of
constituents known to have hormone action that could confound animal studies testing putative endocrine disruptors
(Thigpen et al., 1999b). Although this is a rational approach,
such alterations of the diet to eliminate exogenous “hormones”
may have unanticipated consequences, such as the exacerbation of the renal toxicity of NP reported here. More research is
needed to identify and characterize the specific dietary factors
that modulate the susceptibility to renal cyst formation.
ACKNOWLEDGMENTS
This work was supported by Interagency Agreement 224 –93– 0001 between
the National Institute for Environmental Health Sciences and the United States
Food and Drug Administration. We appreciate the considerable efforts of the
experimental liaison and biostatistical support groups of ROW Sciences, the
animal care and diet preparation staffs of Bionetics, the staff of Pathology
Associates International, and the analytical group of Division of Chemistry at
NCTR during the planning and conduct of this experiment.
REFERENCES
Aukema, H. M., Housini, I., and Rawling, J. R. (1999). Dietary soy protein
effects on inherited polycystic kidney disease are influenced by gender and
protein level. J. Am. Soc. Nephrol. 10, 300 –308.
Boettger-Tong, H., Murthy, L., Chiappetta, C., Kirkland, J. L., Goodwin, B.,
Adlercreutz, H., Stancel, G. M., and Makela, S. (1998). A case of a
laboratory animal feed with high estrogenic activity and its impact on in vivo
responses to exogenously administered estrogens. Environ. Health Perspect.
106, 369 –373.
Chapin, R. E., Delaney, J., Wang, Y., Lanning, L., Davis, B., Collins, B.,
Mintz, N., and Wolfe, G. (1999). The effects of 4-nonylphenol in rats: A
multigeneration reproduction study. Toxicol. Sci. 52, 80 –91.
Clark, L. B., Rosen, R. T., Hartman, T. G., Louis, J. B., Suffet, I. H.,
Lippincott, R. L., and Rosen, J. D. (1992). Detemination of alkylphenolethoxylates and their acetic acid derivatives in drinking water by particlebeam liquid chromatography/mass spectrometry. Int. J. Environ. Anal.
Chem. 147, 167–180.
Dachs, J., Van Ry, D. A., Eisenreich, S. J. (1999). Occurrence of estrogenic
nonylphenols in the urban and coastal atmosphere of the lower Hudson
River estuary. Environ. Sci. Tech. 33, 2676 –2679.
Delclos, K. B., and Newbold, R. R. (1997). A comprehensive protocol for the
evaluation of reproductive, neurological, behavioral, immunological, and
carcinogenic effects of potential endocrine disrupting chemicals. Presented
at Estrogens in the Environment IV, Washington, DC, July 21, 1997.
Dodds, E. C., and Lawson, W. (1938). Molecular structure in relation to
oestrogenic activity. Compounds without a phenanthrene nucleus. Proc.
Royal Soc. B 125, 222–232.
Doerge, D. R., Churchwell, M. I., and Delclos, K. B. (2000). On-line sample
preparation using restricted-access media in the analysis of the soy isoflavones, genistein and daidzein, in rat serum using liquid chromatography
electrospray mass spectrometry. Rapid Commun. Mass Spectrom. 14, 673–
678.
Drotman, R. B. (1980). The absorption, distribution, and excretion of alkylpolyethoxylates by rats and humans. Toxicol. Appl. Pharmacol. 52, 38 – 44.
Ferguson, S. A., Flynn, K. M., Delclos, K. B., and Newbold, R. R. (2000).
Maternal and offspring toxicity but few sexually dimorphic behavioral
alterations result from nonylphenol exposure. Neurotoxicol. Teratol. 22,
583–591.
Gilbert, J., Startin, J. R., and McGuinness, J. D. (1986). Compositional analysis
of commercial PVC bottles and studies of aspects of specific and overall
migration into foods and stimulants. Food Addit. Contam. 3,133–143.
Hamilton, J. M., Flaks, A., Saluja, P. G., and Maguire, S. (1975). Hormonally
induced renal neoplasia in the male Syrian hamster and the inhibitory effect
of 2-bromo-alpha-ergocryptine methane-sulfonate. J. Natl. Cancer Inst. 54,
1385–1400.
Hollander, M., and Wolfe, D. A. (1973). Nonparametric Statistical Methods. J.
Wiley, New York.
Knaak, J. B., Eldridge, J. M., and Sullivan, L. J. (1966). Excretion of certain
polyethylene glycol ether adducts of nonylphenol by the rat. Toxicol. Appl.
Pharmacol. 9, 331–340.
Li, J. J., and Li, S. A. (1996). Estrogen carcinogenesis in the hamster kidney:
A hormone-driven, multistep process. In Cellular and Molecular Mechanisms of Hormonal Carcinogenesis (J. Huff, J. Boyd, and J.C. Barrett, Eds.),
pp. 255–268. Wiley-Liss, New York.
Meyer, O., Blom, L., and Olsen, P. (1978). Influence of diet and strain of rat
on kidney damage observed in toxicity studies. Arch. Toxicol.. 1(Suppl.),
355–358.
Michelangeli, F. Orlowski, S., Champeil, P., East, J. M., and Lee, A. G. (1990).
Mechanism of inhibition of the (Ca(⫹)-Mg2⫹)-ATPase by nonylphenol.
Biochemistry 29, 3091–3101.
Michelangeli, F., Tovey, S., Lowes, D. A., Tien, R. F. I., Mezna, M., McLellan, H., and Hughes, P. (1996). Can phenolic plasticizing agents affect
testicular development by disturbing intracellular calcium homeostasis?
Biochem. Soc. Trans. 24, 2938.
National Research Council. (1999). Hormonally Active Agents in the Environment. National Academy Press, Washington, DC.
Naylor, C. G. (1992). Environmental fate of alkylphenol ethoxylates. Soap
Cosmet. Chem. Spec. 68, 27–31, 72.
Ogborn, M.R., Bankovic-Calic, N., Shoesmith, C., Buist, R., and Peeling, J.
(1998) Soy protein modification of rat polycystic kidney disease. Am. J.
Physiol. 274 (Renal Physiol. 43), F541–549.
Concolino, G., Lubrano, C., Ombres, M., Santonati, A., Flammia, G. P., and Di
Silverio, F. (1993). Acquired cystic kidney disease: The hormonal hypothesis. Urology 41, 170 –175.
Okai, Y., Higashi-Okai, K., Machida, K., Nakamura, H., Nakayama, K., Fujita,
K., Tanaka, T., Otani, S., and Taniguchi, M. (2000). Protective effects of
antioxidants against para-nonylphenol-induced inhibition of cell growth in
Saccharomyces cerevisiae. FEMS Microbiol. Lett. 185, 65–70.
Cunny, H. C., Mayes, B. A., Rosica, K. A., Trutter, J. A., and Van Miller, J. P.
(1997). Subchronic toxicity (90-day) study with para-nonylphenol in rats.
Regul. Toxicol. Pharmacol. 26, 172–178.
Orlowski, S., and Champeil, P. (1991). Kinetics of calcium dissociation from
its high-affinity transport sites on sarcoplasmic reticulum ATPase. Biochemistry 30, 352–361.
NONYLPHENOL AND POLYCYSTIC KIDNEY
Ritskes-Hoitinga, J., and Beynen, A. C. (1992). Nephrocalcinosis in the rat: A
literature review. Prog. Food Nutri. Sci. 16, 85–124.
Roy, D., Palangat, M., Chen, C. W., Thomas, R. D., Colerangle, J., Atkinson,
A., and Yan, Z. J. (1997). Biochemical and molecular changes at the cellular
level in response to exposure to environmental estrogen-like chemicals. J.
Toxicol. Environ. Health 50, 1–29.
Ruehlmann, D. O., Steinert, J. R., Valverde, M. A., Jacob, R., and Mann, G. E.
(1998). Environmental estrogenic pollutants induce acute vascular relaxation by inhibiting L-type Ca 2⫹ channels in smooth muscle cells. FASEB J.
12, 613– 619.
Shelby, M., Newbold, R., Tully D., Chae, K., and Davis, V. (1996). Assessing
environmental chemicals for estrogenicity using a combination of in vitro
and in vivo assays. Environ. Health Perspect. 104, 1296 –1300.
Sondergaard, K., and Blom, L. (1979). Polycystic changes in rat kidney
induced by biphenyl fed in different diets. Arch. Toxicol. 2(Suppl.), 499 –
502.
147
Soto, A. M., Justicia, H., Wray, J. W., and Sonnenchein, C. (1991). pNonylphenol: An estrogenic xenobiotic released from “modified” polystyrene. Environ. Health Perspect. 92, 167–173.
Thigpen, J. E., Setchell, K. D. R., Ahlmark, K. B., Locklear, J., Spahr, T.,
Caviness, G. F., Goelz, M. F., Haseman, J. K., Newbold, R. R., and
Forsythe, D. B. (1999a). Phytoestrogen content of purified, open- and
closed-formula laboratory animal diets. Lab. Anim. Sci. 49, 530 –536.
Thigpen, J. E., Setchell, K. D. R., Goelz, M. F., and Forsythe, D. B. (1999b). The
phytoestrogen content of rodent diets. Environ. Health Pespect. 107, A182–183.
Tomobe, K., Philbrick, D. J., Ogborn, M. R., Takahashi, H., and Holub, B. J.
(1998). Effect of dietary soy protein and genistein on disease progression in
mice with polycystic kidney disease. Am. J. Kidney Dis. 31, 55– 61.
Williams, D. A. (1986). A note on Shirley’s nonparametric test for comparing
several dose levels with a zero-dose control. Biometrics 42, 183–186.
Zhang, M. I. N., and O’Neil, R. G. (1996). An L-type calcium channel in renal
epithelial cells. J. Membr. Biol. 154, 259 –266.