Nephrotoxic and hepatotoxic potential of imidazolidinedione-, oxazolidinedione- and thiazolidinedione-containing analogues of N-(3,5-dichlorophenyl)succinimide (NDPS) in Fischer 344 rats

Article abstract of DOI:10.1016/S0300-483X(02)00692-3

Nephrotoxicity of the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) in rats is believed to involve metabolism on the succinimide ring. To further investigate this hypothesis, we synthesized and tested the following NDPS analogues, which contain other cyclic imide rings and may therefore be metabolized differently than NDPS: 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO), 3-(3,5-dichlorophenyl)-2,4-imidazolidinedione (DCPI), 3-(3,5-dichlorophenyl)-1-methyl-2,4-imidazolidinedione (DCPM) and 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Male Fischer 344 rats were administered DCPO, DCPI, DCPM, DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil), NDPS (0.6 mmol/kg, i.p. in corn oil) or corn oil (4 ml/kg). As evidenced by diuresis, proteinuria, elevated blood urea nitrogen levels, increased kidney weights and proximal tubular damage, NDPS produced severe nephrotoxicity in the rats. In contrast, DCPO, DCPI, DCPM and DCPT were mild nephrotoxicants. None of the compounds elevated serum alanine transferase activity or liver weights in the rats, however DCPT produced centrilobular necrosis. These experiments confirm that NDPS-induced nephrotoxicity is critically dependent on the presence of the succinimide ring. Furthermore, replacement of the succinimide ring with a thiazolidinedione ring produced a more pronounced effect on the liver than on the kidney. Liver damage has been reported in type II diabetic patients taking troglitazone, rosiglitazone and pioglitazone. Since these compounds also contain a thiazolidinedione ring, DCPT may be useful for investigating the role of this structural feature in hepatotoxicity.

Full text of DOI:10.1016/S0300-483X(02)00692-3

Toxicology 186 (2003) 79ꢀ91
/
www.elsevier.com/locate/toxicol
Nephrotoxic and hepatotoxic potential of imidazolidinedione-,
oxazolidinedione- and thiazolidinedione-containing analogues
of N-(3,5-dichlorophenyl)succinimide (NDPS) in Fischer 344
ratsꢀ
Erica L. Kennedy, Ruy Tchao, Peter J. Harvison *
Department of Pharmaceutical Sciences, University of the Sciences in Philadelphia, 600 South Forty-Third Street, Philadelphia,
PA 19104-4495, USA
Received 27 August 2002; received in revised form 5 November 2002; accepted 10 November 2002
Abstract
Nephrotoxicity of the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) in rats is believed to involve
metabolism on the succinimide ring. To further investigate this hypothesis, we synthesized and tested the following
NDPS analogues, which contain other cyclic imide rings and may therefore be metabolized differently than NDPS: 3-
(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO), 3-(3,5-dichlorophenyl)-2,4-imidazolidinedione (DCPI), 3-(3,5-di-
chlorophenyl)-1-methyl-2,4-imidazolidinedione (DCPM) and 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT).
Male Fischer 344 rats were administered DCPO, DCPI, DCPM, DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil), NDPS
(0.6 mmol/kg, i.p. in corn oil) or corn oil (4 ml/kg). As evidenced by diuresis, proteinuria, elevated blood urea nitrogen
levels, increased kidney weights and proximal tubular damage, NDPS produced severe nephrotoxicity in the rats. In
contrast, DCPO, DCPI, DCPM and DCPT were mild nephrotoxicants. None of the compounds elevated serum alanine
transferase activity or liver weights in the rats, however DCPT produced centrilobular necrosis. These experiments
confirm that NDPS-induced nephrotoxicity is critically dependent on the presence of the succinimide ring.
Furthermore, replacement of the succinimide ring with a thiazolidinedione ring produced a more pronounced effect
on the liver than on the kidney. Liver damage has been reported in type II diabetic patients taking troglitazone,
rosiglitazone and pioglitazone. Since these compounds also contain a thiazolidinedione ring, DCPT may be useful for
investigating the role of this structural feature in hepatotoxicity.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Nephrotoxicity; Hepatotoxicity; N-(3,5-Dichlorophenyl)succinimide; 3-(3,5-Dichlorophenyl)-2,4-oxazolidinedione; 3-(3,5-
Dichlorophenyl)-2,4-imidazolidinedione;
thiazolidinedione
3-(3,5-Dichlorophenyl)-1-methyl-2,4-imidazolidinedione;
3-(3,5-Dichlorophenyl)-2,4-
ꢀ Presented in part at the Fortieth Annual Meeting of the Society of Toxicology, 25ꢀ
* Corresponding author. Tel.: ꢁ1-215-596-8979; fax: ꢁ1-215-895-1161.
E-mail address: p.harvis@usip.edu (P.J. Harvison).
/29 March, 2001, in San Francisco, CA, USA.
/
/
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 6 9 2 - 3

80
E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ91
/
1. Introduction
tion or glucuronidation of NDHS, followed by
elimination, could yield the highly electrophilic
N-(3,5-Dichlorophenyl)succinimide
(NDPS,
species
N -(3,5-dichlorophenyl)maleimide
Fig. 1) was originally synthesized as an agricul-
tural antifungal and antimicrobial agent and
exhibited activity against a wide range of plant
pathogenic microorganisms (Fujinami et al.,
1972a). In spite of its promising activity, NDPS
is not currently used in the US due to health
concerns (Rankin et al., 1992). Characteristic signs
of kidney damage caused by acute exposure to
NDPS include diuresis, proteinuria, glucosuria,
hematuria and elevations in blood urea nitrogen
(BUN) levels and kidney weight (Rankin, 1982;
Rankin et al., 1984, 1985a; Yang et al., 1985a).
Clinical chemistry analyses and morphological
evidence have indicated that NDPS is a selective
nephrotoxicant in rats. For example, there were no
reported effects on the liver (i.e. no elevations in
serum alanine aminotransferase levels or liver
weights) or other organs in rats when NDPS was
administered chronically at a dose of 5000 ppm
(Ito et al., 1974; Sugihara et al., 1975) or acutely at
a dose of 0.8 mmol/kg (Rankin et al., 1995). Due
to the selective nature of its nephrotoxicity, NDPS
has been used as a model compound to investigate
chemically induced kidney damage (Rankin et al.,
1985b; Yang et al., 1985a; Kellner-Weibel et al.,
1997).
(NDPM) (Aleo et al., 1991). In fact, NDPM and
a sulfate conjugate of NDHS were shown to be
cytotoxic to isolated rat renal cortical cells (Ran-
kin et al., 2001). Furthermore, covalent binding of
14C-NDPS-derived radioactivity to rat renal and
hepatic proteins in vivo was partly metabolism-
dependent, which suggests that a reactive inter-
mediate was generated (Henesey and Harvison,
2002).
Studies investigating the nephrotoxicity of
NDPS have shown that it is important to have
specific substituents on the phenyl ring of the
parent compound, N-phenylsuccinimide, for ne-
phrotoxicity to occur (Rankin et al., 1985b, 1992;
Yang et al., 1985b, 1987). For example, N-(3,5-
diiodophenyl)succinimide was equally as toxic as
the 3,5-dichloro compound, whereas N-(3,5-di-
fluorophenyl)succinimide was non-nephrotoxic
(Yang et al., 1987; Kellner-Weibel et al., 1997).
Replacement of the 3,5-dichloro substituents with
other functional groups eliminated toxicity (Ran-
kin et al., 1992). Another important structural
feature in NDPS-induced nephrotoxicity is the
presence of an intact, unsubstituted succinimide
ring. Hydrolysis or methylation of the succinimide
ring, or replacement of one or both carbonyl
groups in the succinimide ring with methylene
groups significantly attenuated NDPS nephrotoxi-
city (Yang et al., 1985a). NDPS analogues con-
taining other cyclic imides were also investigated.
Iprodione and vinclozolin, which contain substi-
tuted imidazolidinedione and oxazolidinedione
rings, respectively, were non-nephrotoxic (Rankin
et al., 1989). However, the presence of bulky
substituents on the cyclic imide rings in iprodione
Although the mechanism of NDPS toxicity is
unknown, it has been demonstrated that there is
an essential bioactivation step necessary for the
production of the nephrotoxic metabolite (Rankin
et al., 1987; Nyarko et al., 1997). This occurs via
initial hydroxylation of the succinimide ring of
NDPS by hepatic cytochromes P-450 to yield
N -(3,5-dichlorophenyl)-2-hydroxysuccinimide
(NDHS) (Rankin et al., 1987). Subsequent sulfa-
Fig. 1. Structures of NDPS and analogues.

E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ
/
91
81
and vinclozolin could conceivably interfere with
the essential bioactivation step necessary to pro-
duce renal damage. Furthermore, replacing the
succinimide ring of NDPS with an unsubstituted,
six-membered glutarimide ring also abolished ne-
phrotoxicity (Kellner-Weibel et al., 1995).
kit (BUN, No. 640-5) and urine/blood glucose
assay kit (No. 510-DA) were all products of Sigma
Chemical Co. (St. Louis, MO). Ethyl 2-mercap-
toacetate and glycine were obtained from Aldrich
Chemical Co. (Milwaukee, WI). Bio-Rad protein
assay kit was obtained from Bio-Rad Laboratories
(Hercules, CA). Labstix were purchased from
Bayer Corporation (Elkhart, IN).
The present work was undertaken to study the
effect of replacing the succinimide ring with other
five-membered cyclic imide ring systems on
NDPS-induced toxicity. Therefore, the following
NDPS analogues were synthesized and evaluated
for their nephrotoxic and hepatotoxic potential in
male Fischer 344 rats (Fig. 1): 3-(3,5-dichlorophe-
nyl)-2,4-oxazolidinedione (DCPO), 3-(3,5-dichlor-
ophenyl)-2,4-imidazolidinedione (DCPI), 3-(3,5-
dichlorophenyl)-1-methyl-2,4-imidazolidinedione
(DCPM) and 3-(3,5-dichlorophenyl)-2,4-thiazoli-
dinedione (DCPT). These compounds retain the
3,5-dichloro-substitution pattern in the phenyl
ring, but their unsymmetrical cyclic imide rings
could potentially be metabolized differently than a
succinimide ring. For example, the presence of the
oxygen, nitrogen or sulfur atoms in the cyclic
imide rings of the NDPS analogues could affect
cytochrome P450-dependent hydroxylation at the
adjacent methylene group, which is a critical step
in NDPS metabolic activation. Furthermore, the
nitrogen and sulfur atoms of DCPI and DCPT,
respectively or the methyl group of DCPM,
represent alternative sites where competing oxida-
tive metabolic pathways may occur. Determining
the nephrotoxic potential of DCPO, DCPI,
DCPM and DCPT may indicate if cyclic imides
other than a succinimide ring can also induce
kidney damage. These studies are relevant to
human health since NDPS and the other com-
pounds have structural features in common with
drugs that are used to treat epilepsy and diabetes.
2.2. Animals
Male Fischer 344 rats (210ꢀ225 g) from Charles
/
River Laboratories (Wilmington, MA) were used
in all experiments. The animals were housed in
standard stainless steel hanging cages under a
12:12 h light/dark cycle at :
/
22 8C and 45ꢀ50%
/
relative humidity. Rats were given a 1-week
acclimation period prior to use in any experiments.
Food (laboratory rodent diet No. 5001, PMI
Foods, Inc., St. Louis, MO) and water were freely
available during this period. The Institutional
Animal Care and Use Committee of the University
of the Sciences in Philadelphia approved all
experiments involving rats.
2.3. Syntheses
Melting points were determined with
a
ThomasꢀHoover capillary melting point appara-
/
tus. TLC (Silica Gel GF plates, Analtech, Inc.,
Newark, DE) was used to monitor progress of the
1
reactions. H-NMR and 13C-NMR spectra were
recorded at 90 and 22 MHz, respectively, on an
Anasazi Eft-90 spectrometer. Chemical shifts (d)
are reported in ppm relative to the internal
standard tetramethylsilane (TMS). The abbrevia-
tions used in reporting the data are as follows: s
(singlet), d (doublet), t (triplet), q (quartet).
Coupling constants (J) are reported in Hertz.
Galbraith Laboratories (Knoxville, TN) or Mi-
cro-Analysis, Inc (Wilmington, DE) performed the
elemental analyses.
2. Methods
2.1. Chemicals/reagents
3-(3,5-Dichlorophenyl)-2,4-oxazolidinedione
(DCPO) was prepared from 3,5-dichlorophenyl
isocyanate and ethyl glycolate using a method
similar to that of Fujinami et al. (1971). DCPO
was recrystallized from absolute ethanol until
3,5-Dichlorophenyl isocyanate and ethyl glyco-
late were obtained from Alfa Aesar (Ward Hill,
MA). Sarcosine, alanine aminotransferase assay
kit (ALT, No. 505-P), blood urea nitrogen assay
pure, m.p. 169ꢀ
/
170 8C (lit. m.p. 165.5ꢀ166.5 8C
/

82
E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ91
/
1
(Fujinami et al., 1971)). H-NMR (DMSO-d6): d
7.79 (t, 1H, Jꢂ1.8, Ar-H4), 7.58 (d, 2H, Jꢂ1.8,
Ar-H2,6) and 5.01 (s, 2H, CH2). 13C-NMR
(DMSO-d6): d 170.5 (CꢂO), 154.9 (Cꢂ0),
169.4 (R?CH2CO2R), 165.2 (R’NHCOSR), 147.3
(Ar-C1), 134.6 (Ar-C3,5), 123.2 (Ar-C4), 117.2 (Ar-
¯
/
/
C
2,6), 61.3 (RCO2CH2CH3), 31.6 (R?SCH2R), 13.9
¯
/
/
(CH3).
134.6 (Ar-C3,5), 133.9 (Ar-C1), 128.8 (Ar-C4),
125.6 (Ar-C2,6) and 68.7 (CH2). Anal. Calc. for
C9H5Cl2NO3 (246.04): C, 43.93; H, 2.05; N, 5.69;
Cl, 28.82. Found: C, 44.04; H, 2.07; N, 5.64; Cl,
29.20.
3-(3,5-Dichlorophenyl)-2,4-thiazolidinedione
(DCPT) was prepared from crude ethoxycarbo-
nylmethyl N-(3,5-dichlorophenyl)thiolcarbamate
using a method similar to that of Fujinami et al.
(1971). DCPT was recrystallized from absolute
3-(3,5-Dichlorophenyl)-2,4-imidazolidinedione
(DCPI) was synthesized from 3,5-dichlorophenyl
isocyanate and glycine using a variation on the
method of Fujinami et al. (1972b). DCPI was
recrystallized twice from absolute ethanol until
ethanol until pure, m.p. 169.5ꢀ
164.5ꢀ
165.5 8C (Fujinami et al., 1971)). 1H-NMR
(DMSO-d6): d 7.79 (t, 1H, Jꢂ1.9, Ar-H4), 7.54
(d, 2H, Jꢂ
1.9, Ar-H2,6), 4.31 (s, 2H, CH2). 13C-
NMR (DMSO-d6): d 170.1 (CꢂO), 134.5 (Ar-
3,5), 133.2 (Ar-C1), 127.9 (Ar-C4), 126.2 (Ar-
2,6), 30.1 (CH2). Anal. Calc. for C9H5Cl2NO2S
(262.11): C, 41.24; H, 1.92; N, 5.34; Cl, 27.05; S,
12.23. Found: C, 41.16; H, 1.80; N, 5.16; Cl, 27.42;
S, 12.33.
/
170.5 8C (lit. m.p.
/
/
/
/
pure, m.p. 194ꢀ
(Fujinami et al., 1972b)). 1H-NMR (DMSO-d6): d
8.48 (s, 1H, NH), 7.68 (t, 1H, Jꢂ1.9, Ar-H4), 7.54
(d, 2H, Jꢂ
1.9, Ar-H2,6), 4.08 (s, 2H, CH2). 13C-
NMR (DMSO-d6): d 171.4 (CꢂO), 156.3 (Cꢂ
/
196 8C (lit. m.p. 196.5ꢀ
/
197 8C
C
C
/
/
/
/
O), 135.2 (Ar-C3,5), 134.4 (Ar-C1), 127.7 (Ar-C4),
125.6 (Ar-C2,6), 46.2 (CH2). Anal. Calc. for
C9H6Cl2N2O2 (245.06): C, 44.11; H, 2.47; N,
11.43; Cl, 28.93. Found: C, 43.93; H, 2.30; N,
11.20; Cl, 29.57.
3-(3,5-Dichlorophenyl)-1-methyl-2,4-imidazoli-
dinedione (DCPM) was prepared similarly to
DCPI using sarcosine instead of glycine. DCPM
was recrystallized from absolute ethanol until
2.4. In vivo toxicity
The experimental design is a modification of a
literature method (Rankin, 1982). Before any
treatments began, a blood sample was obtained
by amputating the tip of the tail. Toxicity was
assessed at 24 and 48 h after each animal received
a single dose of the appropriate test compound
(0.6 or 1.0 mmol/kg, i.p. in corn oil), NDPS (0.6
mmol/kg) or corn oil only (4 ml/kg). The dose of
0.6 mmol/kg NDPS was chosen for comparison,
since this is reproducibly nephrotoxic in rats
pure, m.p. 202ꢀ
158.5 8C (Fujinami et al., 1972b)). H-NMR: d
7.62 (t, 1H, Jꢂ2.0, Ar-H4), 7.46 (d, 2H, Jꢂ2.0,
/
203.5 8C (lit. m.p. 157.5ꢀ
/
1
/
/
Ar-H2,6), 4.05 (s, 2H, CH2), 2.88 (s, 3H, CH3). Due
to poor solubility, a 13C-NMR spectrum could not
be obtained for this compound. Anal. Calc. for
C10H8Cl2N2O2 (259.09): C, 46.36; H, 3.11; N,
10.81; Cl, 27.37. Found: C, 45.81; H, 3.07; N,
10.65; Cl, 27.74.
Ethoxycarbonylmethyl N-(3,5-dichlorophenyl)-
thiolcarbamate was prepared from 3,5-dichloro-
phenyl isocyanate and ethyl 2-mercaptoacetate
using a method similar to that of Kolbezen et al.
(1954). The product was then filtered and dried in
(Griffin and Harvison, 1998). Dosages ꢀ1.0
/
mmol/kg are impractical due to solubility issues.
On all days of the experiment, urine was collected
for 6 h to measure baseline urine contents. Food
and water were removed during this period to
avoid dilution and contamination of the urine
sample. Labstix were used to semi-quantitatively
analyze urine for pH and the presence of protein,
glucose, ketones and blood. After dosing (48 h), a
blood sample was obtained by cardiac puncture
under general anesthesia (methoxyflurane) for
assessment of post-treatment BUN and blood
glucose levels. ALT levels were analyzed on fresh
serum. While still anesthetized, the rats were
euthanized by cervical dislocation. After euthana-
sia, the kidneys and liver were removed, weighed
a vacuum oven at 50 8C for 4 h, m.p. 134ꢀ
(lit. m.p. 134.5ꢀ135.5 8C (Kolbezen et al., 1954)).
1H-NMR (acetone-d6): d 9.82 (s, 1H, NH), 7.63
(d, 2H, Jꢂ1.9, Ar-H2,6), 7.21 (t, 2H, Jꢂ1.9, Ar-
H4), 4.19 (q, 2H, Jꢂ7.2, CH2), 3.82 (s, 2H, CH2),
1.25 (t, 3H, Jꢂ
7.2, CH3). 13C-NMR (DMSO-d6):
/
136 8C
/
/
/
/
/

E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ
/
91
83
and the right kidney and a section of liver were
fixed in formalin for histological analysis. Tissue
sections were prepared and stained with hematox-
ylin and eosin by American Histolabs, Inc.
(Gaithersburg, MD). Slides were coded and read
by a person blind to the code. Urine protein
concentrations were measured using the method
of Bradford (1976).
elevated urine protein levels at any time during
the experimental period. Fig. 3 indicates that
glucosuria was seen on Day 2 of the experiment,
following administration of DCPT (0.6 and 1.0
mmol/kg). However, animals treated with DCPT
(1.0 mmol/kg) showed a reduction in glucose levels
on Day 1 before any signs of glucosuria on Day 2
of the experiment. The NDPS (0.6 mmol/kg)
treatment group exhibited an elevation in urine
glucose values, but these were not significantly
different from controls. Urine glucose values were
not significantly elevated by treatment with
DCPO, DCPI or DCPM at any time point.
DCPT (0.6 and 1.0 mmol/kg) produced persistent
ketonuria (small to moderate, semi-quantitative
analysis) throughout the experiment (data not
shown). There was no significant evidence of
ketonuria in any of the other treatment groups.
Kidney weights were significantly increased in
animals treated with NDPS (0.6 mmol/kg) and
DCPT (1.0 mmol/kg only) (Table 2). Compared to
rats treated with corn oil, kidney weights in the
DCPO, DCPI and DCPM treatment groups were
not significantly increased. Only animals treated
with NDPS (0.6 mmol/kg) exhibited a toxicologi-
cally significant increase in BUN levels (Table 2).
None of the treatment groups showed any
significant change in liver weights compared to
the corn oil controls (data not shown). ALT values
were also not significantly different from the
control animals (data not shown).
Most treatment groups, except DCPI (0.6
mmol/kg) and DCPM (0.6 and 1.0 mmol/kg),
exhibited a significant decrease in food intake on
Day 1, but only NDPS (0.6 mmol/kg) treated
animals showed a persistent decrease in food
intake throughout the experiment (data not
shown). By Day 2, most treatment groups returned
to normal feeding patterns. Treatment with
DCPM (0.6 mmol/kg) caused a significant de-
crease in water consumption on Day 1, but values
returned to normal by 48 h. However, DCPT (0.6
mmol/kg) caused a significant increase on Day 2
(data not shown). NDPS (0.6 mmol/kg) and
DCPT (0.6 and 1.0 mmol/kg) caused a persistent
loss of body weight throughout the experiment
(data not shown).
2.5. Statistical analyses
Results are expressed as means9
/
S.E. (Nꢂ
/
3ꢀ8).
/
The data were analyzed by a t-test or one-way
analysis of variance (ANOVA) followed by a
Studentꢀ
the normality and/or equal variance tests failed,
the data were analyzed by the KruskalꢀWallis
/
Newmanꢀ/Keuls post hoc test. When
/
one-way analysis of variance on ranks (ANOVA
on Ranks) test. Differences between means were
considered significant when P B0.05.
/
3. Results
3.1. Effect of NDPS analogues on rat morbidity
Rats treated with DCPI (1.0 mmol/kg only)
appeared to go into circulatory shock within 24 h
of dosing, showing signs of cyanosis and low body
temperature. The animals were euthanized a day
early; therefore Day 2 values are not included in
the figures and tables. All other experiments were
conducted for the full 48 h.
3.2. Nephrotoxicity/hepatotoxicity measurements
Table 1 indicates that diuresis occurred within 6
h (Day 1) following administration of NDPS (0.6
mmol/kg) and DCPT (0.6 and 1.0 mmol/kg) to the
rats. Diuresis persisted in the DCPT-treated rats
(both doses) for the duration of the experiment.
Urine volumes in the NDPS-treated rats on Day 2
or the other treatment groups on either day were
not significantly elevated from corn oil controls.
Animals treated with NDPS (0.6 mmol/kg) ex-
hibited proteinuria on Day 1 of the experiment
(Fig. 2), but values returned to normal by Day 2.
No other treatment groups showed signs of

84
E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ91
/
Table 1
Effect of NDPS analogues on urine volume
Compound
Dose (mmol/kg)
Urine volume (ml)a
Control Day
Day 1
Day 2
Corn oil
NDPS
10.69
13.49
10.29
12.99
9.59
11.89
13.09
10.49
11.39
13.89
/
2.1
7.89
18.69
12.19
13.19
7.59
3.29
5.09
6.49
17.69
20.69
/
0.5
7.19
14.19
8.69
11.09
6.99
/
0.5
0.6
/1.4
/
2.1b
/3.0
DCPO
0.6
1.0
/0.7
/
1.1
2.0
/1.2
/2.1
/
/5.0
DCPI
0.6
1.0
/1.3
/
1.7
1.1c
/
1.2
/1.6
/
n.d.d
DCPM
DCPT
0.6
1.0
/3.9
/
1.3
1.4
4.89
/
0.7
/0.8
/
7.69
/
1.7
0.6
1.0
/0.6
/
1.0b,c
1.0b
25.69
/
3.3b,c
3.1b,c
/1.9
/
26.09
/
a
Values are reported as means9
/
S.E. (Nꢂ
/
4ꢀ/8 rats).
b
Significantly different from corn oil control value.
Significantly different from the Control Day within that treatment group.
Not determined (n.d.) because animals were sacrificed a day early due to complications.
c
d
Fig. 2. Effect of NDPS analogues (0.6 mmol/kg, A; 1.0 mmol/kg, B) on urine protein levels (Nꢂ
/
3ꢀ/8). Asterisks indicate values that
are significantly different (P B0.05) from corn oil controls; daggers indicate values that are significantly different (P B
/
/0.05) from the
Control Day within that treatment group. The Day 2 values for animals treated with DCPI (1.0 mmol/kg) are not included because
they were sacrificed a day early due to complications.

E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ
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91
85
Fig. 3. Effect of NDPS analogues (0.6 mmol/kg, A; 1.0 mmol/kg, B) on urine glucose levels (Nꢂ/3ꢀ/8). Asterisks indicate values that
are significantly different (P B
/
0.05) from corn oil controls; daggers indicate values that are significantly different (P B0.05) from the
/
Control Day within that treatment group. The Day 2 values for animals treated with DCPI (1.0 mmol/kg) are not included because
they were sacrificed a day early due to complications.
Table 2
Effect of NDPS analogues on kidney weights and BUN levels
Compound
Dose (mmol/kg)
Kidney weighta (g/100% BW)
BUN concentrationa (mg/dl)
Pre-dose value 48 h post-dosing
Corn oil
NDPS
0.3509
0.469
0.379
0.379
0.389
/
0.002
19.29
21.49
15.99
20.09
18.09
37.39
19.19
17.09
18.19
17.69
/
1.6
19.79
118.49
16.29
19.99
19.79
/
0.7
0.6
/
0.03b
/
2.0
/
42.9c
DCPO
0.6
1.0
/
0.01
0.02
/
1.2
2.4
/
1.1
1.2
/
/
/
DCPI
DCPM
DCPT
a
0.6
1.0
/
0.01
/
1.1
2.9b
/
1.7
n.d.d
/
n.d.d
0.6
1.0
0.389
/
0.01
/
0.3
0.7
20.19
/
2.0
1.8
0.389
/
0.01
/
20.69
/
0.6
1.0
0.409
/
0.01
0.01b
/
1.5
0.3
21.59
/
1.3
2.7c
0.469
/
/
24.59
/
Values are reported as means9
/
S.E. (Nꢂ
/
3ꢀ/8 rats).
b
c
Significantly different from corn oil control value.
Significantly different from the pre-dose value within that treatment group.
Not determined (n.d.) because animals were sacrificed a day early due to complications.
d

86
E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ91
/
3.3. Kidney and liver histology
Cells lining the proximal tubules exhibited severe
swelling with occlusion of many proximal tubular
lumina. Edema in the interstitium was also ob-
served. Swelling of some vasculature caused accu-
mulation of blood in the interstitial space. Cell
casts and debris were seen scattered throughout
the proximal tubules indicating that cell death had
occurred and that proteinaceous material could
appear in the urine. Occasional mitotic figures
were observed. The glomeruli appeared normal.
Treatment with DCPO, DCPI and DCPM pro-
duced minor renal morphological changes com-
parable to those observed in corn oil-treated
animals (data not shown). DCPT (0.6 and 1.0
mmol/kg) treatment caused swelling of the prox-
imal and distal tubules with evidence of minor cell
damage and death (Fig. 4C). There were signs of
cells breaking down, such as loss of the cell brush
border. Proteinaceous debris also appeared in the
proximal and collecting tubules. Occasional mito-
tic figures appeared in the lower part of the
convoluted tubules, distal tubules and collecting
ducts.
Slight swelling of some proximal convoluted
tubules was noted in the rats that received corn oil
(Fig. 4A). NDPS treatment (0.6 mmol/kg) caused
distention in some of the distal tubules, with
damage to the cells lining the tubules (Fig. 4B).
Treatment with corn oil (Fig. 5A) did not cause
any noticeable injury to the liver. NDPS (Fig. 5B),
DCPO, DCPI and DCPM (data not shown)
produced minimal damage to the hepatocytes.
There were signs of occasional swelling of the
nuclei and loss of proteinaceous material in the
cytoplasm. The damaged cells appeared to radiate
out from the central veins. DCPT treatment (0.6
and 1.0 mmol/kg) caused swelling of the hepato-
cytes and the appearance of large areas of cell
death around the centrilobular veins (Fig. 5C).
The swelling forced vacuolization to occur in the
cytoplasm. Some cells contained condensed nuclei.
In many instances, the nuclear membrane had
dissolved and the nuclear material had fragmen-
ted, causing irregular staining of the cytoplasm.
The bile canaliculi appeared normal. DCPT treat-
ment produced mostly nuclear, rather than cyto-
plasmic changes.
4. Discussion
Fig. 4. Effect of corn oil (4 ml/kg, A), NDPS (0.6 mmol/kg, B)
or DCPT (0.6 mmol/kg, C) on kidney morphology. Tissue
In an attempt to further understand the im-
portance of the succinimide ring towards the
sections were stained with H&E. Magnification is 400ꢃ.
/

E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ
/
91
87
The analogues were tested in vivo at dosages of
0.6 and 1.0 mmol/kg, which are known nephro-
toxic doses of NDPS (Rankin et al., 1984; Griffin
and Harvison, 1998). NDPS (0.6 mmol/kg) caused
diuresis, proteinuria, persistent loss of body weight
due to reductions in food consumption, and
increases in kidney weight and blood urea nitrogen
(BUN) levels. Although urine glucose values were
elevated in these animals, they were not signifi-
cantly different from the control group. These data
are consistent with previous studies and indicate
that kidney damage had occurred (Rankin, 1982;
Rankin et al., 1984, 1985a). The histology data
supported the toxicological findings in that NDPS
(0.6 mmol/kg) treatment caused severe damage to
the proximal tubular cells. In contrast to NDPS
treatment, corn oil produced a very slight effect on
the kidneys. The glomeruli, proximal, distal and
collecting tubules were essentially normal in these
animals. DCPO, DCPI, DCPM and DCPT also
did not produce the same severity of damage to the
kidneys as occurred with NDPS. The histology
data was generally consistent with the minor
toxicological changes and alterations in kidney
function that were seen. Mitotic figures were
present in the kidneys of rats that received
NDPS or DCPT, but not the other NDPS
analogues. Since the mitotic figures were not
observed in the corn oil-treated animals either,
they are probably a consequence of NDPS or
DCPT treatment. One possible explanation for the
appearance of the mitotic figures in the kidneys of
these animals could be localized recovery of some
damaged tubular cells.
The glucosuria seen on Day 2 following treat-
ment with NDPS (0.6 mmol/kg) and DCPT (0.6
and 1.0 mmol/kg) may be attributed to slight
swelling and minor damage to the cells of the
proximal tubules. Since glucose is absorbed along
the proximal tubule (Vander, 1985), damage to
this area may result in glucosuria. There may also
be more subtle damage that cannot be observed by
light microscopy to account for the decrease in
glucose reabsorption. DCPM (1.0 mmol/kg) treat-
ment also caused hyperglycemia on Day 2. This
increase in blood glucose could be a result of the
minor damage caused to the liver, as seen in the
histology data (see below). This minor, focal
Fig. 5. Effect of corn oil (4 ml/kg, A), NDPS (0.6 mmol/kg, B)
or DCPT (0.6 mmol/kg, C) on liver morphology. Tissue
sections were stained with H&E. Magnification is 400ꢃ.
/
mechanism of NDPS toxicity, we conducted a
structureꢀactivity relationship study. A series of
/
NDPS analogues (Fig. 1) were synthesized and
evaluated for their nephrotoxic and hepatotoxic
potential in male Fischer 344 rats. The selection of
these compounds was based on the fact that they
each contain an oxygen, nitrogen, substituted
nitrogen or sulfur atom in place of one of the
methylene groups in the NDPS succinimide ring.

88
E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ91
/
damage may cause a breakdown of glycogen,
resulting in hyperglycemia.
These results are consistent with previous findings
that NDPS-induced kidney damage is critically
dependent on the presence of an intact, unsubsti-
tuted succinimide ring (Rankin et al., 1985b, 1992;
Yang et al., 1985a,b, 1987; Kellner-Weibel et al.,
1995, 1997).
DCPT-treated (0.6 and 1.0 mmol/kg) animals
consumed a large amount of water on Day 2,
which was not seen with any of the other treatment
groups. This could also be a factor in the diuresis
that was observed in these rats. The histology data
of animals treated with DCPT (0.6 and 1.0 mmol/
kg) showed some swelling of the proximal and
distal tubules, which is also consistent with the
observed diuresis.
Treatment with DCPT (0.6 and 1.0 mmol/kg)
caused persistent, mild to moderate ketonuria.
Similar to NDPS-treated rats, food consumption
in the animals treated with DCPT was reduced on
Day 1. Ketonuria could be a result of fasting or
abnormalities of carbohydrate and lipid metabo-
lism (Wilson et al., 1987). Under conditions of
fasting or a diabetic environment, oxaloacetate is
unavailable for condensation with acetyl CoA into
the citric acid cycle and is therefore consumed and
converted to glucose. Therefore, acetyl CoA is
converted to acetoacetate. Acetoacetate can also
be converted to acetone and b-hydroxybutyric
acid, but the ketone test on the Labstix only reacts
with the acetoacetic acid in urine, which is
produced by the metabolism of fatty acids. How-
ever, since ketonuria was not seen in rats that
received NDPS, decreased food consumption is
unlikely to be a reason for the increase in urine
ketone levels in the DCPT-treated animals. An-
other possibility could be a reaction between
component(s) in the urine with the Labstix. In
In the present work, we have shown that
replacement of the succinimide ring with other
five-membered cyclic imides (oxazolidinedione,
imidazolidinedione or thiazolidinedione) signifi-
cantly attenuates nephrotoxicity. Iprodione and
vinclozolin, that also contain imidazolidinedione
and oxazolidinedione rings, respectively, were
previously found to be non-nephrotoxic (Rankin
et al., 1989). However, unlike the analogues
described here, iprodione and vinclozolin have
relatively bulky substituents present on their cyclic
imide rings, which could interfere with metabo-
lism. NDPS-induced nephrotoxicity is critically
dependent on an initial hydroxylation step on the
succinimide ring (Rankin et al., 1987; Nyarko et
al., 1997). Furthermore, metabolism could yield a
reactive intermediate, such as NDPM (Aleo et al.,
1991; Rankin et al., 2001; Henesey and Harvison,
2002). In fact, each of the NDPS analogues
contains an unsubstituted methylene group, where
hydroxylation could still occur. However, the
presence of a heteroatom adjacent to the methyl-
ene group could affect biotransformation at this
critical site. Furthermore, DCPI, DCPM and
DCPT each contain an additional site (nitrogen
atom, methyl group or sulfur atom, respectively)
for oxidation, which is not present in the succini-
mide ring of NDPS. Therefore, alternative meta-
bolic pathways that do not result in the formation
of a reactive intermediate may occur with the
NDPS analogues or they may not be metabolized
at all.
fact, falseꢀpositive ketonuria has been reported in
/
patients taking thiol-containing drugs, such as
mesna or captopril (Cantwell et al., 1986; Hinberg
and Poon, 1987) and is due to a chemical reaction
between the thiol group on the drug and the
nitroprusside reagent on the dipstick (Csako,
1987). Although DCPT does not contain a free
thiol group, it is conceivable that one might be
generated by hydrolysis or metabolism of the
thiazolidinedione ring in rats. These possibilities
will require further investigation.
The potential hepatotoxicity of DCPO, DCPI,
DCPM and DCPT was also evaluated. Liver
weights and alanine aminotransferase (ALT) levels
were normal in all treatment groups. The histolo-
gical findings show that NDPS and the analogues,
except for DCPT, produced only minor effects on
liver morphology and that these effects were
concentrated around some of the centrilobular
veins. The minor damage consisted of swelling of
some hepatocytes, condensation of some nuclei
Collectively, the data suggest that DCPO,
DCPI, DCPM and DCPT are mild nephrotoxi-
cants in male Fischer 344 rats when compared to
NDPS, which is a potent, nephrotoxic agent.

E.L. Kennedy et al. / Toxicology 186 (2003) 79ꢀ
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91
89
and the appearance of vacuolization in the cyto-
plasm. These toxicological findings suggest that
DCPO, DCPI, and DCPM are mild hepatotox-
icants.
factor in the toxicity that was observed in these
animals. Whether or not DCPI actually produced
methemoglobinemia in the rats would require
further investigation.
Treatment of animals with DCPT (0.6 and 1.0
mmol/kg) produced more widespread liver damage
(that primarily radiated out from the centrilobular
veins) than was observed with NDPS or the other
analogues. The severe lesions consisted of swelling
of the hepatocytes, which in many cases caused
vacuolization to appear in the cytoplasm and
karyolysis to occur. In spite of this damage, ALT
levels and liver weights were normal at 48 h post-
administration of DCPT. Severe cell death oc-
curred at this time point, but was not accompanied
by an inflammatory response. The DCPT-induced
hepatotoxicity that we observed in rats is interest-
ing since severe liver damage has been reported in
diabetic patients treated with troglitazone, which,
like DCPT, also contains a thiazolidinedione ring
(Kohlroser et al., 2000; Graham et al., 2001).
Furthermore, there have been several cases of
hepatic injury reported in patients taking other
thiazolidinedione-containing drugs, such as rosi-
glitazone (Forman et al., 2000; Gouda et al., 2001)
and pioglitazone (Maeda, 2001; May et al., 2002).
Rats treated with DCPI (1.0 mmol/kg only)
appeared to go into circulatory shock, however
this compound did not produce any serious kidney
or liver damage. One possible explanation for this
finding could be the presence of an unsubstituted
nitrogen atom, a potential site for N-oxidation, in
the imidazolidinedione ring. N-Hydroxylation re-
actions have been associated with the induction of
methemoglobinemia (Nohl and Stolze, 1998),
which would be consistent with the symptoms
observed in the DCPI-treated rats. We did not
observe this effect with DCPM, the N-methylated
analogue of DCPI, where direct oxidation of the
nitrogen atom could not occur. Similarly, methe-
moglobinemia was not reported in rats that
received iprodione, which like DCPM also con-
tains an N-substituted imidazolidinedione ring
(Rankin et al., 1989). Furthermore, phenacetin,
but not its N-methylated derivative, produced
methemoglobinemia in rats (Nelson et al., 1978).
Thus, it is possible that the presence of an
unsubstituted nitrogen atom in DCPI could be a
In conclusion, DCPO, DCPI, DCPM and
DCPT were mild nephrotoxicants in rats. This
indicates that replacement of one of the methylene
groups in the succinimide ring of NDPS with a
heteroatom significantly reduces the adverse renal
effects that are associated with this compound.
Our findings extend previous results that NDPS-
induced nephrotoxicity is critically dependent
upon the presence of the succinimide ring.
DCPO, DCPI and DCPM were mild hepatotox-
icants. However, DCPT produced severe liver
damage in rats. These results are interesting
because thiazolinedione-containing drugs, such as
troglitazone, rosiglitazone and pioglitazone, are
associated with hepatotoxicity in humans.
Although the mechanism of troglitazone-induced
liver damage is controversial, metabolites derived
from bioactivation of the thiazolidinedione ring
have been detected in rats (Kassahun et al., 2001;
Tettey et al., 2001). DCPT may therefore be a
useful model compound for further investigating
the role of the thiazolidinedione ring in hepato-
toxicity.
Acknowledgements
The authors would like to thank Dr Joan B.
Tarloff, Dr Charles J. Thoman and Dr Guillermo
Moyna, University of the Sciences in Philadelphia,
for their advice and expertise.
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