FUNDAMENTAL AND APPLIED TOXICOLOGY 37, 1 1 7 - 1 2 4 (1997)
ARTICLE NO. FA972321
Cytochrome P450-Mediated Metabolism and Nephrotoxicity of /V-(3,5Dichlorophenyl)succinimide in Fischer 344 Rats
Alexander K. Nyarko,* Ginny L. Kellner-Weibel.f and Peter J. Harvison:}:1
*Chemical Pathology Unit, Noguchi Memorial Institute, University of Ghana, Legon-Accra, Ghana; and
Departments of ^Chemistry and tPharmacology and Toxicology, Philadelphia College of Pharmacy and Science,
600 South Forty-third Street, Philadelphia, Pennsylvania 19104-4495
Received December 3, 1996; accepted April 21, 1997
icity studies indicated that NDPS induces a nephrotic synCytochrome P450-Mediated Metabolism and Nephrotoxicity of drome in rats that is similar to human interstitial nephritis
A/-(3,5-Dichlorophenyl)succinimide in Rats. Nyarko, A. K., Kell(Sugihara et al, 1975; Barrett et al, 1983). In acute toxicner-Weibel, G. L., and Harvison, P. J. (1997). Fundam. Appl. Toxity studies, NDPS produced diuresis, proteinuria, glucosicol. 37, 117-124.
uria, and elevated blood urea nitrogen; alterations in renal
The agricultural fungicide N-(3,5-dichlorophenyl)succinimide organic ion uptake and increased kidney weights were
(NDPS) is nephrotoxic in rats. Previous studies have suggested also reported (Rankin, 1982; Rankin et al., 1984, 1985;
that oxidative hepatic biotransformation is required for the induc- Kellner-Weibel et al, 1995). Sugihara et al. (1975) sugtion of kidney damage. The experiments described in this paper gested that NDPS is a potentially useful model compound
were designed to further investigate the relationship between
for studying chemically induced interstitial nephritis. In
NDPS metabolism and nephrotoxicity using various modulators
of cytochrome P450 activity. Male Fischer 344 rats were pretreated fact, the succinimide ring, which is important in NDPS
with the P450 inducers Aroclor 1254 (ARO), isoniazid (INH), 3- nephrotoxicity (see below), is also found in many other
methylcholanthrene (3-MC), and phenobarbital (PB), or the P450 compounds (including drugs, agricultural agents, and ininhibitor 1-aminobenzotriazole (ABT). Control animals received dustrial chemicals) to which humans and other species
vehicle only. NDPS metabolism was investigated using hepato- can be exposed.
cytes isolated from the various treatment groups. Separate experiPrevious studies (Ohkawa et al., 1974; Griffin and Harviments were also conducted to evaluate the effects of these pre- son, 1990) demonstrated that NDPS undergoes in vivo metabtreatments on NDPS-induced nephrotoxicity in rats. PB and ARO olism in rats (Fig. 1) to Af-(3,5-dichlorophenyl)succinamic
enhanced formation of the known nephrotoxic NDPS metabolites,
acid (NDPSA), A^-(3,5-dichlorophenyl)-2-hydroxysuccinamic
N-(3,5-dichlorophenyl)-2-hydroxysuccinimide, N-(3,5-dichlorophenyl>2-hydroxysuccinamic acid, and iV-(3,5-dichlorophenyl)-3- acid (2-NDHSA), /V-(3,5-dichlorophenyl)-3-hydroxysuccihydroxysuccinamic acid, by the hepatocytes. In contrast, ABT namic acid (3-NDHSA), and N-(3,5-dichlorophenyi)maloinhibited formation of the nephrotoxic metabolites, whereas INH namic acid (DMA). NDPSA, 2-NDHSA, 3-NDHSA, ^-(3,5and 3-MC did not alter NDPS biotransformation. NDPS-induced dichlorophenyl)-2-hydroxysuccinimide (NDHS), and /V-(3,5renal damage was potentiated by pretreating the rats with PB or dichloro-4-hydroxyphenyl)succinamic acid (NDHPSA) were
ARO and was attenuated by ABT. Compared with control ani- detected when NDPS was incubated with isolated rat hepatomals, toxicity was unaffected by INH or 3-MC pretreatments. cytes or rat liver homogenates (Nyarko and Harvison, 1995;
Thus, there was a correlation between pretreatments that induce Griffin et al., 1996). No oxidative metabolites were found in
P450-mediated NDPS metabolism and the effects that these com- incubations of NDPS with isolated rat kidney cells (Henesey
pounds have on NDPS-induced nephrotoxicity. The data indicate and Harvison, 1995).
that specific P450 isozymes metabolize NDPS to its hydroxylated
The actual mechanism leading to NDPS-induced nephroproducts and suggest that these metabolites mediate the nephrotoxicity
is not known, but metabolic activation by cytotoxicity induced by NDPS. © 1997 Sodtty of To
chrome P450 is strongly implicated. For example, deuterium
labeling of the succinimide ring attenuated the renal damage
associated with NDPS (Rankin et al, 1986). In addition,
iV-(3,5-Dichlorophenyl)succinimide (NDPS, Fig. 1) was modulation of NDPS toxicity by induction or inhibition of
originally synthesized in the early 1970s as an agricultural microsomal metabolism suggested that hepatic cytochrome
fungicide (Fujinami et al, 1972). Subsequent chronic tox- P450 was responsible for metabolic activation of this compound (Rankin etal, 1987). Finally, several hepatic metabolites (NDHS, 2-NDHSA, and 3-NDHSA) were found to be
' To whom correspondence should be addressed. Fax: 215-895-1161. Emore nephrotoxic than NDPS itself (Rankin et al, 1988,
mail: [email protected].
117
0272-0590/97 $25.00
Copyright © 1997 by the Society of Toxicology.
All rights of reproduction in any form reserved.
18
NYARKO, KELLNER-WEIBEL, AND HARVISON
NDPS
NDHS
COjH
NDPSA
3-NDHSA
2-NDHSA
FIG. 1. <V-(3,5-Dichlorophenyl)succinimide (NDPS) metabolic pathway.
1989). In contrast, the hydrolytic metabolite NDPSA was
markedly less toxic than NDPS at a dose of 0.4 mmol/kg
(Yang et al., 1985). NDHPSA, which could be produced by
hydroxylation in the aromatic ring, was nontoxic (Harvison
et al, 1992).
Despite the apparent role of hepatic cytochromes P450
in NDPS-induced nephrotoxicity, metabolism of the compound following modulation of P450 activity has not been
examined in detail. We hypothesized that specific P450
isozymes may be involved in the metabolic activation of
NDPS. In this paper, we therefore examine the effects of
cytochrome P450 activity modulation on in vitro metabolism and in vivo nephrotoxicity of NDPS. Metabolism
studies were conducted with isolated rat hepatocytes since
we previously showed that this system can convert NDPS
to most of the metabolites formed in vivo (Nyarko and
Harvison, 1995). These experiments may provide further
evidence on the role of cytochrome P450 isozymes in the
metabolic activation of NDPS.
METHODS
Reagents and animals. All reagents were of the highest purity commercially available. NDPS and [ U C]NDPS were synthesized according to the
method of Fujinami et al. (1972). Male Fischer 344 rats (175-200 g) were
purchased from Charles River Laboratories (Wilmington, MA) and were
housed singly in stajnless-steel cages under a 12-hr light-dark cycle at ca.
22°C and 4 5 - 5 0 % relative humidity. This species and strain were used for
comparison to our previous studies. A 1-week acclimatization period was
allotted before the animals were used in any experiments. All rats had free
access to food and water during this period. The studies described in this
paper were approved by the Institutional Animal Care and Use Committee
of the Philadelphia College of Pharmacy and Science.
In vivo pretreatments. Caution: Aroclor 1254 and 3-methylcholanthrene are suspected carcinogens and should be handled carefully. Personnel should wear lab coats, masks, and gloves when handling these chemicals. Dosing solutions should be prepared under a fume hood. Rats (4-8
animals/group) were pretreated prior to hepatocyte isolation or dosing with
NDPS as follows: phenobarbital (PB, 80 mg/kg, ip in saline, daily for 3
days), 3-methylcholanthrene (3-MC, 80 mg/kg, ip in corn oil, daily for 3
days), Aroclor 1254 (ARO, 300 mg/kg, ip in com oil, single dose 72 hr
prior to use), isoniazid (INH, 80 mg/kg/day, ip in saline, daily for 3 days),
or 1-aminobenzotriazole (ABT, 100 mg/kg, ip in saline, single dose 1 hr
prior to use). Control groups received saline or corn oil only.
Hepatocyte-mediated metabolism of NDPS. Following the appropriate pretreatments, liver cells were isolated by the method of Moldeus
et al. (1978). Initial cell viability was determined using trypan blue
exclusion or lactate dehydrogenase release (Moldeus, 1978; Jauregui et
at., 1981). Viabilities of the liver cells used in all experiments was
^ 9 0 % . Metabolism experiments with the hepatocytes were conducted
as previously described (Nyarko and Harvison, 1995). Briefly, [ I4 C]NDPS (2.0 mM, sp act 0.41 or 0.72 mCi/mmol) was incubated with
hepatocytes (5 X 106 cells/ml) in Krebs-Henseleit buffer (pH 7.4, 37°C)
for 3 hr. In some experiments, the P450 inhibitor SKF 525A (50 /XM)
was preincubated with the hepatocytes for 10 min prior to addition of
the substrate. After deproteinization with acetonitnle (v/v 2:1), aliquots
(20 iA) of centrifuged samples were analyzed by HPLC on a Beckman
C-18 column (4.2 mm X 25 cm) using a gradient (mobile phase flow
rate 1.7 ml/min) between water and acetonitnle (Nyarko and Harvison,
1995; Griffin et al., 1996). The HPLC system consisted of a Beckman
Model 421 controller, two Model 114M pumps, a Model 502 autosampler, a Model 165 variable-wavelength detector (set at 254 nm), and a
Raytest Ramona-5-LS radiochemical detector. EcoLume scintillation
fluid (ICN, Costa Mesa, CA) was pumped through the Ramona flow cell
at 5.1 ml/min. Metabolites were quantitated radiochemically using the
Chromasoft (RSM Analytische GmbH, Germany) software program. The
limit of detection for this assay is ca. 0.1 nmol and the overall recovery
of radioactivity (unreacted substrate plus metabolites) is greater than
98% (Nyarko and Harvison, 1995). Hepatocyte P450 content was determined by CO difference spectra (Aminco DW-2 spectrophotometer) us-
119
NDPS METABOLISM AND NEPHROTOXICITY
ing a molar absorptivity of 91 mM ' cm ' (Omura and Sato, 1964;
Moldeus et al., 1973).
Microsomal assays. Hepatic microsomes were isolated by differential
centrifugation (Lake, 1987). Microsomal cytochrome P450 content was
measured as described above. The success of P450 isozyme induction by
3-MC or INH was verified by measuring benzo[a]pyrene hydroxylation or
p-nitrophenol hydroxylation activities, respectively.
Microsomal benzo[a]pyrene (BP) hydroxylase activity was assayed as
previously described (Nebert and Gelboin, 1968). Incubations were conducted in a total volume of 1 ml Tns buffer (0.05 mM, pH 7.4, 37°C).
Reaction mixtures contained ca. 1 mg microsomal protein, 0.5 mg bovine
serum albumin, 0.4 mg NADPH, and 5.0 mM MgCl 2 . The reactions were
initiated by adding 100 nmol of BP and were allowed to proceed for 8 min
on a shaking water bath maintained at 37°C. Incubations were terminated
by the addition of 1 ml of cold acetone. The mixture were then shaken
with 3.25 ml hexane for 2 min. Aliquots (1 ml) of the organic layer were
removed and extracted with 3 ml 1 M NaOH in dim light. The fluorescence
of the product was immediately measured on a Hitachi F-3010 spectrofluorometer (excitation wavelength 395 nm, emission wavelength 522 nm)
against zero time and reagent blanks. Product formation was quantitated
by comparing the fluorescence of the samples with standards generated by
quinine sulfate prepared in 0.5 M HjSC^ (Nebert and Gelboin, 1968).
Hydroxylation of p-nitrophenol (PNP) to 4-nitrocatechol was determined
by the method of Reinke and Moyer (1985). Each incubation contained ca.
1 mg microsomal protein, 0.4 mg NADPH, and 5.0 mM MgCl2 in a total
volume of 1 ml Tris buffer (pH 7.4, 37°C). The reactions were initiated by
the addition of 100 nmol PNP and were terminated after 10 min by the
addition of 0.5 ml 0.6 N HC1O4 to each incubation. Precipitated proteins
were removed by centrifugation at 2500 rpm. Aliquots (1 ml) of the supernatants were then mixed with 0.1 ml of 10 M NaOH. The absorbance of each
sample was read at 546 nm (Hitachi U-3110 spectrophotometer) and the
amount of 4-nitrocatechol formed was calculated using a molar absorptivity
of 10.28 mM~'cnr' (Reinke and Moyer, 1985).
In vivo nephrotoxicity.
Toxicity studies were conducted as previously
described (Kellner-Weibel el al., 1995). Rats were transferred into plastic
metabolism cages for acclimatization prior to the in vivo toxicity studies.
Blood samples were collected by nicking the tails for determination of
initial blood urea nitrogen (BUN) levels. The animals were then pretreated
as described above. On Day 0 (control day) of the experiment, food and
water were removed for a 6-hr period to ensure collection of contaminantfree urine. This urine was semiquantitatively analyzed (N-Multistix SG,
Miles, Inc., Elkhart, IN) for the presence of protein, glucose, blood, and
ketones. Food and water were then returned to the rats. Following the
control day, the rats were injected with NDPS (0.2 or 0.4 mmol/kg, ip in
com oil) or corn oil only (2 ml/kg). After dosing, the rats were returned to
the metabolism cages for an additional 48 hr. Urine contents (analyzed as
above), total urine volume, body weight, and food and water consumption
were monitored each day of the experiment. A final blood sample was taken
by cardiac puncture under methoxyflurane anesthesia 48 hr postdosing.
While still anesthetized the animals were sacrificed by cervical dislocation.
The right kidney was then removed and weighed. BUN levels were determined using a commercially available kit (Kit 640-A, Sigma Chemical Co.,
St. l-nuis, MO).
Statistics. The data were analyzed by Student's t test, one way ANOVA,
or the corresponding nonparametric tests. Where significance was detected
in the ANOVA, the Student-Newman-Keuls, Dtinnett, or Dunn test was
used to isolate differences among the groups. A 5% level of significance
was used for all statistical analyses. Results are presented as means ±
standard errors of the mean (SE) with N = 4, unless otherwise indicated.
A larger group size was used in the in vivo toxicity studies due to the
variability in response to NDPS.
TABLE 1
Effect of Cytochrome P450 Modulation on N-(3,5-dichlorophenyT)succinimide (NDPS) Metabolism by Isolated Rat Hepatocytes
Metabolite production i
(nmol/10 6 cells/hr)**
Pre treatment
Inhibitor
N
Nephrotoxic
metabolites
Saline
ABT
INH
PB
PB
Corn oil
ARO
ARO
3-MC
None
None
None
None
SKF 525A
None
None
SKF 525A
None
4
4
4
4
4
4
4
4
4
12.0
1.2
20.0
72.3
33.3
15.7
53.7
7.3
9.1
±
±
±
±
±
±
±
±
±
0.5
0.4 c
5.2
5.1'
8.3*
1.1
2.2 r
1.2r>
2.6 r
Other
metabolites
85.5
90.3
77.9
60.5
76.8
84.3
20.6
62.1
59.2
±
±
±
±
±
±
±
±
±
4.5
3.7
5.4
9.0
1 5.8
5.1
3.4<2.3'''
2.2C
° [ I4 C]NDPS (2.0 mM) and hepatocytes (5 X 106 cells/ml) were incubated
in Krebs-Henseleit buffer (pH 7.4, 37°C) for 3 hr. Some incubations contained SKF 525A (50 //M).
* Results are expressed as means ± SE. "Nephrotoxic metabolites" represents the sum of 2-NDHSA, 3-NDHSA, and NDHS. "Other metabolites"
is the sum of NDPSA and NDHPSA.
c
Significantly different from the saline (ABT, INH, PB) or corn oilpretreated (ARO, 3-MC) groups (p < 0.05).
' Significantly different from the corresponding group without SKF 525A
(p < 0.05).
RESULTS
Production of the nephrotoxic metabolites (2-NDHSA, 3NDHSA, and NDHS) was enhanced 6-fold in the PB-induced cells when compared with the uninduced cells (Table
1). In contrast, formation of the other metabolites (NDPSA
and NDHPSA) was reduced, although not significantly (p
> 0.05), by PB induction. Preincubation of PB-induced cells
with the P450 inhibitor SKF 525A inhibited the production
of the nephrotoxic metabolites. Hepatocytes isolated from
ARO-pretreated rats produced a 3.5-fold greater amount of
the toxic metabolites than the uninduced cells (Table 1).
Formation of the other metabolites was significantly reduced
in the ARO-induced cells. In addition, ARO-induced cells
converted NDPS to a previously undetected metabolite (7.07
± 1.15 nmol/106 cells/hr) which eluted immediately after
the solvent front (retention time = 1.8 min). SKF 525A
reduced the production of the nephrotoxic metabolites (Table
1) and the unknown metabolite (not shown) by hepatocytes
isolated from ARO-pretreated rats. In contrast to the results
obtained with PB or ARO, conversion of NDPS to the toxic
metabolites was not enhanced by the 3-MC or INH pretreatment (Table 1). Hepatocytes isolated from rats pretreated
with ABT produced significantly less of the nephrotoxic
metabolites than control cells (Table 1).
When expressed as a percentage, the nephrotoxic metabo-
120
NYARKO, KELLNER-WBD3EL, AND HARV1SON
lites accounted for approximately 5 0 - 6 5 % of the total radioactivity (metabolites + unreacted substrate) in the incubations with the PB- or ARO-induced cells. In comparison,
the toxic metabolites accounted for only 10-15% of the total
using hepatocytes that were isolated from INH- or 3-MCpretreated rats. ABT pretreatment reduced formation of the
nephrotoxic metabolites to only 1% of the total.
To verify that enzyme induction had actually occurred
with PB, INH, ARO, and 3-MC, the cytochrome P450 content of the liver cells was determined by CO difference spectra. As shown in Fig. 2, hepatocyte P450 levels were increased 2.5- to 4-fold by the various inducers when compared with cells isolated from animals that were pretreated
with saline or corn oil (0.42-0.46 nmol P450/106 cells). To
prove that induction of specific P450 isozymes had occurred
with 3-MC and INH, experiments were conducted using liver
microsomes prepared from rats that were pretreated with
these two compounds. BP and PNP were used as substrates
of the 3-MC- and INH-inducible isozymes, respectively. Microsomal P450 levels were also assayed. As shown in Fig.
3A, the P450 content of microsomes isolated from salineor corn oil-pretreated (control) rats was 0.80 nmol/mg protein. This was increased ca. 220% by 3-MC induction. BP
hydroxylase activity (3.25-4.10 mg product/min/mg protein
in control microsomes) was elevated ca. 2-fold by 3-MC
pretreatment, but was unaltered by INH induction (Fig. 3B).
Hydroxylation of PNP in the uninduced liver microsomes
(0.98 nmol product/min/mg protein) was enhanced approximately 1.5-fold by pretreating rats with INH (Fig. 3C).
i- 0
Saline
INH
Corn
oil
3-MC
PRETREATMENT
FIG. 3. Effect of cytochrome P450 modulation on (A) microsomal P450
content, (B) benzo[a]pyrene (BP) hydroxylation activity, and (C) p-nitrophenol (PNP) hydroxylation. P450 levels were determined by carbon
monoxide difference spectra. Incubations contained BP or PNP (100 nmol
each), bovine serum albumin (0.5 mg, BP assay only), NADPH (0.4 mg),
MgCl2 (5 mM), and microsomes (ca. 1 mg) in 1 ml Tris buffer (0.05 mM,
pH 7.4, 37°C). Product formation was quantitated fluorometrically (BP
hydroxylation) or spectrophotometrically (PNP hydroxylation). Results are
expressed as means ± SE (N = 3). Asterisks indicate values that are significantly different (p < 0.05) from saline controls. Daggers indicate values
that are significandy different (p < 0.05) from com oil controls.
Saline
PB
INH
Corn 3-MC ARO
oil
PRETREATMENT
FIG. 2. Effect of cytochrome P450 modulation on hepatocyte P450
content. P450 levels were determined by carbon monoxide difference spectra. Results are expressed as means ± SE (N = 4). Asterisks indicate values
that are significantly different (p < 0.05) from saline controls. Daggers
indicate values that are significantly different (p < 0.05) from com oil
controls.
The effects of the various pretreatments on NDPS-induced
nephrotoxicity was evaluated in male Fischer 344 rats. Compared with animals (controls) that received corn oil instead
of NDPS, BUN levels were increased at least 6-fold in AROor PB-pretreated rats that received a nontoxic dose (0.2
mmol/kg) of NDPS or nonpretreated rats that received a
toxic dose (0.4 mmol/kg) of NDPS (Table 2). The elevations
in BUN for these three treatment groups were also greater
121
NDPS METABOLISM AND NEPHROTOXICITY
TABLE 2
Effect of Cytochrome P450 Modulation on N-(3,5-Dichlorophenyl)succinimide (NDPS)-Induced Changes in Blood Urea Nitrogen
(BUN) Levels and Kidney Weights in Male Fischer 344 Rats
BUN concentration (mg/dl)*
Pre treatment
None
ARO
PB
None
ARO
INH
3-MC
PB
None
ABT
NDPS dose
(mmol/kg)
N
0.0
0.0
0.0
0.2
0.2
0.2
0.2
0.2
0.4
0.4
4
4
4
8
7
4
7
7
4
4
Day 0
20.1
18.7
18.7
21.6
21.6
19.5
19.0
21.3
21.7
17.5
±
±
±
±
±
±
±
±
±
±
1.3
0.2
0.6
0.9
1.2
0.6
0.8
0.9
1.3
0.4
Kidney weight*
(g/100 g body wt)
Day 2
19.9 ± 0.7
21.9 ± 0.5'
24.4 ± O.^
49.7 ± 22.0*
124.6 ± 34.4'J
25.3 ± 1.6'
44.9 ± 22.6'
149.8 ± 35.4°*
155.2 ± lO.O'-'
16.5 ± 0.7
0.39
0.39
0.36
0.45
0.51
0.36
0.42
0.54
0.58
0.42
±
±
±
±
±
±
±
±
±
±
0.01
0.01
0.004
0.03
0.05
0.01
0.04
0.04
0.03
0.004
° NDPS was administered at two doses: 0.2 mmol/kg (nontoxic) and 0.4 mmol/kg (nephrotoxic). Control rats (0.0 mmol/kg NDPS) received corn oil
only (2 ml/kg).
* Results are expressed as means ± SE.
e
Significantly different from the Day 0 value within a group (p < 0.05).
* Significantly different from the nonpretreated control group (p < 0.05).
than the values obtained from the same animals before NDPS
was administered (Day 0 values). Relative to corn oil controls, 3-MC or INH induction had no effect on urea nitrogen
levels in rats that received 0.2 mmol/kg NDPS (Table 2).
Pretreatment with ABT reversed the increase in BUN that
was associated with the nephrotoxic dose of NDPS. The
ARO or PB pretreatments had no effect on BUN levels in
rats that received corn oil instead of NDPS. As shown in
Table 2, kidney weights were elevated in ARO- or PBpretreated animals that received 0.2 mmol/kg NDPS or nonpretreated rats that received 0.4 mmol/kg NDPS, although
these changes were not statistically significant (p > 0.05).
Kidney weights were normal in the other treatment groups,
including rats that received ABT prior to a nephrotoxic dose
of NDPS (Table 2).
Compared with Day 0, diuresis was present on Day 1 in
rats that were pretreated with ARO, 3-MC, or PB before
dosing with NDPS (0.2 mmol/kg) or in animals that received
a toxic dose of NDPS (Table 3). Diuresis was completely
prevented by pretreating rats with ABT 1 hr prior to administration of NDPS (0.4 mmol/kg). Urine output was not altered
by the other treatments and returned to normal levels in the
affected groups by Day 2.
Twenty-four hours after a nephrotoxic dose of NDPS (0.4
mmol/kg) was administered, there was a decrease (ca. 12%)
in body weight compared with corn oil controls (Table 4).
This decrease persisted into Day 2 and was prevented by
pretreating the animals with ABT prior to dosing with NDPS.
Relative to Day 0, decreases in body weight on Day 1 were
found in rats that received 0.2 mmol/kg NDPS (naive, AROor PB-pretreated) or 0.4 mmol/kg NDPS. Animals in these
treatment groups continued to lose weight on Day 2 of the
experiment. Body weights were not adversely affected in
any other treatment group. Compared with corn oil controls,
food intake on Days 1 or 2 was reduced in the 0.4 mmol/
kg NPDS, ARO + 0.2 mmol/kg NDPS, and PB + 0.2 mmol/
kg NDPS treatment groups (data not shown). In addition,
food intake in these groups after dosing with NDPS was
lower than on Day 0. Rats in the 0.2 mmol/kg NDPS and
TABLE 3
Effect of Cytochrome P450 Modulation on N-(3,5-Dichlorophenyl)succinimide (NDPS)-Induced Changes in Urine Volume
in Male Fischer 344 Rats
Urine volume (ml)'
Pretreatment
None
ARO
PB
None
ARO
INH
3-MC
PB
None
ABT
NDPS dose
(mmol/kg)
N
0.0
0.0
0.0
0.2
0.2
0.2
0.2
0.2
0.4
0.4
4
4
4
8
7
4
7
7
4
4
Day 0
12.3
10.5
16.0
13.3
14.8
13.8
9.4
15.6
11.3
11.0
±
±
±
±
±
±
±
±
±
±
1.8
0.9
1.4
0.9
1.3
0.5
0.7
1.0
1.5
0.4
Day 1
12.9 :fl.O
11.0 :t 0.4
13.5 :t 0.3
22.3 :t 3.4
28.6 :t 1.6'
15.0 :t 2.0
22.0 jt 3.7'
28.1 jt 2.4'
24.5 it i.r
9.3 :t 0.9
Day 2
14.8 ±
11.5 ±
13.8 ±
14.6 ±
17.7 ±
15.5 ±
14.9 ±
20.7 ±
15.8 ±
12.8 ±
1.3
0.9
1.7
3.2
2.4
1.5
3.0
2.1
3.3
1.8
" NDPS was administered at two doses: 0.2 mmol/kg (nontoxic) and 0.4
mmol/kg (nephrotoxic). Control rats received com oil only (2 ml/kg).
* Results are expressed as means ± SE.
' Significantly different from the Day 0 value within a group (p < 0.05).
122
NYARKO, KELLNER-WEIBEL, AND HARVISON
TABLE 4
Effect of Cytochrome P450 Modulation on N-(3,5-Dichlorophenyl)succinimide (NDPS)-Induced Changes in Body Weight
in Male Fischer 344 Rats
Body weight (g)*
Pretreatment
None
ARO
PB
None
ARO
INH
3-MC
PB
None
ABT
NDPS dose?
(mmol/kg) N
0.0
0.0
0.0
0.2
0.2
0.2
0.2
0.2
0.4
0.4
4
4
4
8
7
4
7
7
4
4
Day 1
Day 0
186.1 ±
176.7 1t
204.9 2t
207.1 ;t
215.1 =t
195.4 :t
195.3 ;t
208.9 ;t
179.4 2t
174.2 2t
3.8 184.7 ±4.8
3.3 180.6 ± 3.0
5.8 202.5 ± 5.8
2.3 194.6 ± \.T
2.7 195.5 ± 4.T
5.1 195.8 ± 53
3.6 184.8 ± 4.5
3.2 184.6 ± 3.8*
2.7 157.5 ± 22^
\5 170.8 ± 22
Day 2
1883 :t 5.4
184.4 :t 3.6
205.1 :t5.6
197.9 :t 3 . 4 '
190.4:t 6 . 4 '
197.0 :t5.2
188.0 ± 5.9
1763 :t5.5 c
146.6 ± 2.0^
1776 ± 2.5
° NDPS was administered at two doses: 0.2 mmol/kg (nontoxic) and 0.4
mmol/kg (nephrotoxic). Control rats received com oil only (2 ml/kg).
* Results are expressed as means ± SE.
c
Significantly different from the Day 0 value within a group (p < 0.05).
** Significantly different from the nonpretreated control group (p < 0.05).
ABT + 0.4 mmol/kg NDPS groups also ate less in the first 24
hr after dosing; however, food consumption in these animals
returned to normal on Day 2. Water consumption on Days
1 and 2 was not significantly different from that of corn oil
controls in any treatment group during the course of the
experiment (data not shown); however, rats in the 0.4 mmol/
kg NDPS and PB + 0.2 mmol/kg NDPS groups drank significantly less water for the first 24 hr after dosing. Water
intake in these animals returned to normal on Day 2.
Semiquantitative urinalysis showed that proteinuria
(>100 mg/dl) occurred within 6 hr in rats that received
0.2 mmol/kg NDPS (nonpretreated, ARO-, 3-MC-, and PBinduced) or 0.4 mmol/kg NDPS (data not shown). Urine
protein levels remained elevated in all of these groups on
Day 2. ABT pretreatment totally prevented the proteinuria
associated with the nephrotoxic dose of NDPS. Urine protein
levels were essentially normal in all other animals. Glucosuria (=*100 mg/dl) occurred in the 0.4 mmol/kg NDPS and
0.2 mmol/kg NDPS (ARO- and PB-induced) animals 6 hr
after administration of the compound (data not shown).
Urine glucose levels remained elevated in the PB + 0.2
mmol/kg NDPS and 0.4 mmol/kg NDPS treatment groups
on Day 2. The glucosuria associated with 0.4 mmol/kg
NDPS was prevented by ABT pretreatment. All other treatment groups had normal urine glucose levels. There was no
evidence of ketonuria or hematuria in any animals throughout the experiment (data not shown).
DISCUSSION
Microsomal enzyme inducers and inhibitors alter NDPS
toxicity (Rankin etal., 1987); however, little is known about
the effect that these pretreatments have on in vitro and in
vivo NDPS biotransformation. We were interested in determining if modulation of NDPS toxicity would correlate
with changes in metabolism. Male Fischer 344 rats were
therefore pretreated with a P450 inhibitor (ABT) or compounds (ARO, INH, 3-MC and PB) that induce different
P450 isozymes. The animals were then used to evaluate
NDPS toxicity and metabolism. NDPS was administered to
rats at two doses: 0.2 mmol/kg (nontoxic) and 0.4 mmol/kg
(toxic). These doses were chosen based on reports in the
literature (Rankin, 1982; Rankin etal., 1984, 1985; KellnerWeibel et al., 1995). Metabolism experiments were conducted using isolated rat liver cells as this system produces
most of the known, in vivo NDPS metabolites (Nyarko and
Harvison, 1995). Prior studies have shown that the biotransformation of other compounds, such as acetaminophen (Moldeus, 1978) and nicotine (Kyerematen et al., 1990), can be
modified in rat hepatocytes by enzyme induction.
The freshly isolated rat liver cells converted NDPS to
NDPSA, 2-NDHSA, 3-NDHSA, NDHS, and NDHPSA. Metabolite production by hepatocytes isolated from saline- or
corn oil-pretreated rats (indicative of constitutive P450 activity) was quantitatively similar to our prior results (Nyarko
and Harvison, 1995). Pretreatment of rats with ARO, INH,
3-MC, or PB significantly increased the cytochrome P450
content of the hepatocytes, indicating that enzyme induction
was successful. The P450 levels in our control and PB- and
3-MC-induced liver cell preparations were comparable to
those reported by Moldeus et al. (1973).
In agreement with Rankin et al. (1987), we found that PB
pretreatment potentiated NDPS (0.2 mmol/kg) nephrotoxicity in rats. PB is an inducer of the P450 2B1/2B2 isozymes
in rat liver (Guengerich et al., 1982), but has no effect on
rat renal P450 (Tarloff et al., 1990). Kidney damage in the
PB-pretreated rats was evident as increased diuresis, elevations in blood urea nitrogen levels and kidney weights, proteinuria and glucosuria, and decreased body weights and
food intake. We previously reported that these changes correlated with histological damage in rats that received a nephrotoxic dose (0.4 mmol/kg) of NDPS (Kellner-Weibel et al.,
1995). ARO, which is an inducer of the P450 1A and 2B
isozyme families (Guengerich et al., 1982), also enhanced
NDPS nephrotoxicity. The variability in BUN values obtained from rats that received 0.2 mmol/kg NDPS following
pretreatment with PB or ARO (Table 1) may be due to the
"steep" dose-response curve exhibited by NDPS in Fischer
344 rats (Rankin et al., 1985). Increasing the number of
animals in these experiments failed to reduce this variability.
Renal damage was not observed in PB- or ARO-pretreated
rats that received com oil instead of NDPS, indicating that
nephrotoxicity cannot be attributed to these pretreatments
alone. The potentiation of NDPS toxicity that occurred with
PB and ARO induction correlated with changes in the in
123
NDPS METABOLISM AND NEPHROTOXICITY
vitro metabolic profile of NDPS. In fact, these two pretreatments enhanced formation of the nephrotoxic metabolites
(2-NDHSA, NDHS, and 3-NDHSA) by the hepatocytes. A
similar increase in oxidative metabolism of NDPS by PBinduced rat liver homogenates was previously described by
Griffin et al. (1996). A novel, polar metabolite was produced
by the ARO-induced cells; however, the role (if any) of this
metabolite in NDPS-induced nephrotoxicity is not known.
The ability of SKF 525A to suppress formation of the unknown and nephrotoxic metabolites by the PB- and AROinduced cells indicates that cytochrome(s) P450 is involved
in their formation (Testa and Jenner, 1981). Overall, these
results indicate that the PB- and ARO-inducible isozymes
of P450 are capable of converting NDPS to nephrotoxic
metabolites.
Pretreatment with 3-MC or acetone was previously shown
to slightly attenuate NDPS-induced nephrotoxicity in rats
and it was suggested that this could be due to induction of
alternative metabolic pathways (Rankin et al, 1987; Lo et
al., 1987). 3-MC, a polycyclic aromatic hydrocarbon, induces P450 1A1/1A2 in rat liver (Guengerich et al, 1982),
whereas acetone is an inducer of P450 2E1 (Ryan et al.,
1985; Thomas et al., 1987). Aside from some diuresis and
proteinuria, we found little evidence of kidney damage in
rats that received a nontoxic dose (0.2 mmol/kg) of NDPS
following 3-MC pretreatment. These results are somewhat
different than those reported by Rankin et al. (1987) and
may be due to the fact that we used a different induction
protocol (three doses of 3-MC instead of one). Pretreatment
with INH, which, like acetone, is an inducer of P450 2E1
(Ryan et al., 1985; Thomas et al., 1987), also failed to potentiate NDPS nephrotoxicity. Furthermore, we found that
3-MC or INH pretreatment did not enhance the conversion
of NDPS to nephrotoxic metabolites in vitro. One possible
explanation for these observations is that induction of the
appropriate P450 isozymes was not achieved in the 3-MCor INH-pretreated rats; however, hepatocyte and microsomal
P450 levels were elevated following pretreatment with 3MC or INH. In addition, microsomal activities representative
of 3-MC- and INH-inducible P450 isozymes (BP hydroxylation and PNP hydroxylation, respectively) were significantly
higher than in control microsomes. Therefore, the absence
of increased NDPS metabolism or toxicity in the 3-MC- or
INH-pretreated rats was not due to lack of enzyme induction,
but rather a lack of specificity of the respective isozymes for
NDPS. Since the 3-MC-inducible isozymes have no apparent
role in the metabolic activation of NDPS, these results also
suggest that the effects seen with ARO (above) may be due
to the ability of this compound to increase levels of the PBinducible isozymes (i.e., 2B1/2B2) of P450.
Rankin et al. (1987) demonstrated that the P450 inhibitors
cobalt chloride and piperonyl butoxide inhibited NDPS-induced nephrotoxicity. We wanted to evaluate the ability of
ABT, a selective, suicide inhibitor of P450 (Mugford et al,
1992), to prevent NDPS metabolism and nephrotoxicity in
rats. BUN levels and kidney weights were elevated in nonpretreated rats that received a nephrotoxic dose (0.4 mmol/
kg) of NDPS. Diuresis, loss of body weight, proteinuria, and
glucosuria were also seen in animals that received this dose.
These changes are comparable to prior reports (Rankin,
1982; Rankin et al, 1984, 1985; Kellner-Weibel et al.,
1995). We also found that ABT pretreatment effectively
inhibited conversion of NDPS to nephrotoxic metabolites by
the liver cells and prevented NDPS toxicity in vivo. Thus,
there was an association between inhibition of P450-mediated NDPS metabolism in vitro and protection against nephrotoxicity.
In conclusion, we have found that modulation of NDPS
metabolism by isolated liver cells correlates closely with
changes in NDPS-induced renal damage in rats. P450 inducers that increase oxidative metabolism of this compound
in vitro also potentiate its nephrotoxicity in vivo. Inhibition
of P450-mediated NDPS metabolism protected against nephrotoxicity. The results obtained with the different enzyme
inducers suggest that only certain P450 isozymes (constitutive and 2B family) can convert NPDS to its nephrotoxic
metabolites.
ACKNOWLEDGMENTS
This publication was made possible by Grant ES05189 from the National
Institute for Environmental Health Sciences, NIH, and a Thomas Jefferson
Fellowship. The authors thank Dr. Bruce A. Mico (Hoffman-LaRoche,
Inc.) for providing the ABT.
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