3,4-Dichlorophenylhydroxylamine cytotoxicity in renal cortical slices from Fischer 344 rats

Article abstract of DOI:10.1016/S0300-483X(01)00356-0

3,4-Dichlorophenylhydroxylamine (3,4-CPHA) is the N-hydroxyl metabolite of 3,4-dichloroaniline. 3,4-Dichloroaniline is a breakdown product of the herbicide Propanil. Previous work has shown that 3,4-dichloroaniline is acutely toxic to the kidney and bladder. The purpose of this study was to examine the in vitro toxicity of 3,4-dichlorophenylhydroxylamine. Renal cortical slices were prepared from male Fischer 344 rats (190-250 g) and were incubated with 0-0.5 mM 3,4-CPHA for 30-120 min under oxygen and constant shaking. 3,4-CPHA produced a concentration and time dependent alteration in lactate dehydrogenase (LDH) leakage, organic ion accumulation and pyruvate stimulated gluconeogenesis. Glutathione levels were diminished within 60 min below control values by 0.1 and 0.5 mM 3,4-CPHA. A 30 min pretreatment with 0.1 mM deferoxamine did not alter 3,4-CPHA toxicity. Alterations in pyruvate stimulated gluconeogenesis and LDH leakage were comparable between vehicle and deferoxamine pretreated tissues. Other studies examined the effect of (1 mM) glutathione, 2 mM ascorbic acid and 1 mM dithiothreitol (DTT) on toxicity. Pretreatment for 30 min with vehicle or 1 mM DTT induced comparable changes in LDH leakage and pyruvate stimulated gluconeogenesis. Pretreatment for 30 min with 1 mM glutathione or 2 mM ascorbic acid reduced 3,4-CPHA toxicity. LDH leakage was not elevated as markedly in renal slices pretreated with glutathione relative to slices pretreated with vehicle. These results indicate that 3,4-CPHA toxicity is through an iron independent mechanism. 3,4-CPHA cytotoxicity was reduced by pretreatment with glutathione or ascorbic acid suggesting formation of a reactive intermediate.

Full text of DOI:10.1016/S0300-483X(01)00356-0

Toxicology 162 (2001) 149–156
www.elsevier.com/locate/toxicol
3,4-Dichlorophenylhydroxylamine cytotoxicity in renal
cortical slices from Fischer 344 rats
Monica Valentovic *, John G. Ball, Syam Stoll, G.O. Rankin
Department of Pharmacology, Marshall Uni6ersity School of Medicine, 1542 Spring Valley Dri6e, Huntington,
WV 25704-9388, USA
Received 30 October 2000; accepted 5 February 2001
Abstract
3,4-Dichlorophenylhydroxylamine (3,4-CPHA) is the N-hydroxyl metabolite of 3,4-dichloroaniline. 3,4-
Dichloroaniline is a breakdown product of the herbicide Propanil. Previous work has shown that 3,4-dichloroaniline
is acutely toxic to the kidney and bladder. The purpose of this study was to examine the in vitro toxicity of
3,4-dichlorophenylhydroxylamine. Renal cortical slices were prepared from male Fischer 344 rats (190–250 g) and
were incubated with 0–0.5 mM 3,4-CPHA for 30–120 min under oxygen and constant shaking. 3,4-CPHA produced
a concentration and time dependent alteration in lactate dehydrogenase (LDH) leakage, organic ion accumulation
and pyruvate stimulated gluconeogenesis. Glutathione levels were diminished within 60 min below control values by
0.1 and 0.5 mM 3,4-CPHA. A 30 min pretreatment with 0.1 mM deferoxamine did not alter 3,4-CPHA toxicity.
Alterations in pyruvate stimulated gluconeogenesis and LDH leakage were comparable between vehicle and
deferoxamine pretreated tissues. Other studies examined the effect of (1 mM) glutathione, 2 mM ascorbic acid and
1 mM dithiothreitol (DTT) on toxicity. Pretreatment for 30 min with vehicle or 1 mM DTT induced comparable
changes in LDH leakage and pyruvate stimulated gluconeogenesis. Pretreatment for 30 min with 1 mM glutathione
or 2 mM ascorbic acid reduced 3,4-CPHA toxicity. LDH leakage was not elevated as markedly in renal slices
pretreated with glutathione relative to slices pretreated with vehicle. These results indicate that 3,4-CPHA toxicity is
through an iron independent mechanism. 3,4-CPHA cytotoxicity was reduced by pretreatment with glutathione or
ascorbic acid suggesting formation of a reactive intermediate. © 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Nephrotoxicity; Aromatic amines; Chlorophenylhydroxylamine; F344 rats; Renal cortical slices
1. Introduction
the nephrotoxicant, 3,4-dichloroaniline. 3,4-Di-
chloroaniline is an essential ingredient in the pro-
duction of the herbicide Propanil. Propanil is an
arylamide, post-emergent herbicide used to con-
trol broadleaf grasses in rice, wheat and potato
fields (Farm Chemicals Handbook, 1992; Hand-
book of Pesticide Toxicology, Stevens and Sum-
ner, 1991).
3,4-Dichlorophenylhydroxylamine (3,4-CPHA)
is an aromatic ring hydroxylated metabolite of
* Corresponding author. Tel.: +1-304-6967332; fax: +1-
304-6967391.
E-mail address: valentov@marshall.edu (M. Valentovic).
0300-483X/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0300-483X(01)00356-0

150
M. Valento6ic et al. / Toxicology 162 (2001) 149–156
The renal effects of 3,4-dichloroaniline have
tathione levels; and (c) examined whether
antioxidants or sulfhydryl containing agents could
been characterized in Fischer 344 rats (Valentovic
et al., 1997). 3,4-Dichloroaniline induced morpho-
logical changes to the proximal tubule. Renal
functional changes include diminished urine out-
put and hematuria. Proximal tubular function was
impaired since renal cortical slice uptake of p-
aminohippurate, an organic anion, was depressed
24 h after 3,4-dichloroaniline.
modify
3,4-dichlorophenyl
hydroxylamine
toxicity.
2. Methods and materials
2.1. Chemicals
Toxicity was also apparent following in vitro
exposure of renal cortical slices to 3,4-
dichloroaniline (Valentovic et al., 1995, 1997).
3,4-Dichloroaniline addition to renal cortical
slices induced a concentration dependent decrease
in pyruvate-stimulated gluconeogenesis. Incuba-
tion with 2 mM 3,4-dichloroaniline also elevated
lactate dehydrogenase (LDH) release above con-
trol values.
The mechanism for 3,4-dichloroaniline renal
toxicity is not yet clear. The toxic species that
induces 3,4-dichloroaniline nephrotoxicity also
still needs to be identified. 3,4-Dichloroaniline is
biotransformed in rat hepatic microsomes to two
major metabolites, 3,4-CPHA and 2-amino-4,5-
dichlorophenol (McMillan et al., 1990a).
Metabolites of another aromatic amine, aniline
have been attributed with generation of
methemoglobinemia and tissue hypoxia (Kiese,
1966; Gosselin et al., 1984). The phenyl hydroxy-
lamine and nitrosobenzene (Kiese, 1966) metabo-
lites induce oxidation of hemoglobin to
methemoglobin. The nitrosobenzene metabolite
can undergo reduction to the hydroxylamine and
further oxidize another molecule of hemoglobin
or nitrosobenzene can form hemoglobin adducts
(Kiese and Taeger, 1976).
Studies that have examined 3,4-dichloroaniline
toxicity to red blood cells indicate biotransforma-
tion is a necessary step for activation. 3,4-
Dichloroaniline induced methemoglobinemia is
associated with the metabolite 3,4-dichlorophenyl
hydroxylamine and its conversion within the red
blood cell to 3,4-dichloronitrosobenzene (McMil-
lan et al., 1990b).
3,4-CPHA was prepared by the method of
Rondestvedt and Johnson (1977). 3,4-CPHA was
prepared via reduction of 3,4-dichloronitroben-
zene with hydrazine-palladium on carbon. Recrys-
tallization from benzene-petroleum ether provided
pure product in a 60% yield (m.p. 73–75°C).
LDH, sulphosalicylic acid, glutathione, deferox-
amine and all other compounds were obtained
from Sigma-Aldrich (St. Louis, MO) and the
products obtained were of the highest quality and
purity.
2.2. Animals
Male Fischer 344 rats (weight 190–220 g) were
obtained from Hilltop Lab Animals Inc. (Scotts-
dale, PA). Animals were maintained under a con-
trolled ambient temperature (21–23°C), humidity
(40–55%) and light cycle (lights on 06:00–18:00
h). Animals were provided a 7 day acclimation
period prior to initiation of experiments.
2.3. Incubation of renal slices
Renal cortical slices were washed in 5 ml of
oxygenated Krebs buffer three times for 3 min at
25°C. The tissue was equilibrated for 5 min at
37°C under oxygen. Pyruvate (100 ml, final bath
concentration 10 mM) was added to all tissue. A
30 ml aliquot of 3,4-CPHA was added at a final
concentration of 0–0.5 mM and tissues were incu-
bated for up to 120 min. Media and tissue were
collected to measure LDH leakage, pyruvate stim-
ulated gluconeogenesis and glutathione levels.
Other experiments required a pretreatment of
renal slices for 30 min at 37°C with 1 mM reduced
glutathione (GSH), 1mM dithiothreitol (DTT), 2
mM ascorbic acid or 50 mM diphenylenediamine
The following studies investigated the in vitro
cytotoxicity of 3,4-CPHA. An emphasis was
placed on: (a) characterization of 3,4-CPHA toxi-
city in renal cortical slices; (b) alterations in glu-

M. Valento6ic et al. / Toxicology 162 (2001) 149–156
151
(DPPD). The potential role of an iron mediated
radical was examined using 0.1 mM deferoxamine
as a pretreatment of the renal slices. Renal slices
were pretreated for 30 min with a nonsulfhydryl
containing antioxidant, 2 mM ascorbic acid, to
examine differences in toxicity between ascorbic
acid and sulfhydryl containing antioxidants.
Upon completion of the pre-incubation period,
3,4-CPHA was added and tissues were incubated
for 90 min. Gluconeogenesis was stimulated by
addition of 100 ml of pyruvate (10 mM final
concentration, in Krebs) and the tissue was incu-
bated for a final 30 min.
Changes in glutathione were monitored as a func-
tion of time in renal slices incubated for 15–120
min with 0.1 mM 3,4-CPHA. Renal slices were
weighed, homogenized in 0.5 ml of 0.5%
sulphosalicylic acid and adjusted to 1 ml. Total
glutathione levels were measured using an
NADPH coupled reaction described by Andersen
(1985). Values were expressed as nmol/g tissue.
2.7. Statistical analysis
Values represent the mean9S.E.M., with N=
4/group. Differences between groups were deter-
mined using a one-way analysis of variance
followed by a Newman Keuls test. Differences
within groups were calculated using a one-way
analysis of variance followed by a Dunnett’s test.
All tests were conducted using a 95% confidence
interval.
2.4. Organic ion transport
Renal cortical slices were equilibrated for 15
min in 3 ml of oxygenated Krebs buffer pH 7.4 at
37°C in a Dubnoff metabolic shaker under oxy-
gen and constant shaking (100 cycles/min). Vehi-
cle (DMSO) or 3,4-CPHA were added (30 ml) to
yield a final concentration of 0–0.5 mM. The
slices were incubated for 30 min prior to the
addition of the organic anion p-aminohippurate
(14C-PAH, sp. activity 60 mCi/mmol; 0.07 mM,
0.02 mCi/ml) or the organic cation tetraethylam-
monium (14C-TEA, sp. activity 4.0 mCi/mmol;
0.01 mM, 0.02 mCi/ml) as previously described
(Valentovic et al., 1989). Renal slices were incu-
bated 60 min after the addition of PAH or TEA.
Upon completion of the incubation period, up-
take of organic ions was expressed as the ratio of
tissue slices to media (S/M) where S denotes dpm/
g tissue and M denotes dpm/ml of incubation
media.
3. Results
3,4-CPHA was cytotoxic to renal cortical slices.
3,4-CPHA (0.10–0.5 mM) diminished pyruvate
stimulated gluconeogenesis (Table 1) in a concen-
tration dependent manner. Glutathione levels
were diminished within 60 min as a function of
3,4-CPHA concentration. 3,4-CPHA induced al-
terations in membrane integrity were confirmed
by a rise in LDH leakage (Table 1) relative to
vehicle treated tissue. Renal cortical slice accumu-
lation of organic ions was diminished in a concen-
tration manner by 3,4-CPHA (Table 2). Uptake
of the organic anion, PAH was decreased (PB
0.05) relative to vehicle control at 0.5 mM. Up-
take of the organic cation, TEA was inhibited in a
concentration dependent manner relative to vehi-
cle controls at 3,4-CPHA concentrations of 0.01,
0.1 and 0.5 mM 3,4-CPHA. 3,4-CPHA (0.5 mM)
induced temporal changes in pyruvate stimulated
gluconeogenesis (Fig. 1), LDH leakage (Fig. 2)
and glutathione levels (Fig. 3).
2.5. Cytotoxicity assays
LDH release into the media was expressed as a
percentage of total LDH using a spectrophoto-
metric kinetic assay (Sigma, Kit c228). Media
glucose was measured using a glucose oxidase
enzymatic assay (Sigma, Kit c510).
Pretreatment with the iron chelating agent, de-
feroxamine, did not reduce 3,4-CPHA toxicity.
Alterations in pyruvate stimulated gluconeogene-
sis and LDH leakage (Table 3) were comparable
between vehicle and deferoxamine pretreated tis-
2.6. Glutathione determination
Glutathione levels were measured following a
60 min incubation with 0–0.5 mM 3,4-CPHA.

152
M. Valento6ic et al. / Toxicology 162 (2001) 149–156
Table 1
The effect of 3,4-CPHA on renal cortical slicesa
3,4-CPHA concentration (mM)
0
0.01
0.1
0.5
Glucose (mg/g tissue)
LDH leakage (% of total)
Glutathione (nmol/g tissue)
2.0390.11
11.690.64
793944
1.8790.09
11.991.2
713965*
1.4090.12*
16.591.9
565950*
0.5390.06*
31.792.9*
229943*
a Renal slices were incubated with 0–0.5 mM 3,4-CPHA. Cytotoxicity was assessed following 2 h exposure to 3,4-CPHA using
LDH leakage and pyruvate stimulated gluconeogenesis. Glutathione levels were measured after a 60 min exposure to the designated
concentration of 3,4-CPHA. Values represent mean9S.E.M. with N=4/group.
* Indicates statistical difference (PB0.05) when compared to control.
Table 2
In vitro effect of 3,4-CPHA on organic ion transport
3,4-CPHA concentration (mM)
0
0.005
0.01
0.05
0.10
0.50
PAH (S/M)
TEA (S/M)
3.1590.22a
10.0091.01
2.8590.25
9.2290.58
2.8690.25
8.1390.24*
3.0690.13
9.1791.02
2.9690.29
8.1190.58*
2.2190.29*
5.0391.17*
a Values represent mean9S.D. with N=4 different animals.
* Indicates statistical difference (PB0.05) from control (DMSO) treated tissue.
slices were exposed to 0.1 mM 3,4-CPHA when
compared to vehicle treated slices (Fig. 5). LDH
leakage was not reduced by ascorbic acid pretreat-
sues following exposure to 3,4-CPHA. These find-
ings suggest that 3,4-CPHA in vitro toxicity does
not require free iron for cytotoxicity to occur.
Pre-incubation with the sulfhydryl-containing
agent, DTT, did not reduce 3,4-CPHA toxicity.
3,4-CPHA associated effects on pyruvate stimu-
lated (Table 3) and LDH leakage were compara-
ble between vehicle and 1 mM DTT pretreated
slices.
Toxicity was reduced by pretreatment with 1
mM glutathione. Glutathione pretreatment pre-
vented 3,4-CPHA associated rise in LDH leakage
(Fig. 4). Glutathione did not totally protect renal
slices from 3,4-CPHA toxicity since gluconeogen-
esis (Table 3) was reduced in a similar manner
between vehicle and glutathione pretreated tissue
in the presence of 0.5 mM 3,4-CPHA. The success
of GSH pretreatment to reduce toxicity relative to
DTT may be due to differences in intracellular
concentration within the renal slices.
Fig. 1. Time dependent changes in pyruvate stimulated gluco-
neogenesis. Renal cortical slices were incubated for 30–90 min
with vehicle or 0.5 mM 3,4-CPHA. Pyruvate stimulated gluco-
neogenesis was expressed as mg glucose/g tissue. Values repre-
sent mean9S.E.M. with N=4/group. Pyruvate stimulated
gluconeogenesis was decreased (*PB0.05) below vehicle
treated tissue within 30 min.
Ascorbic acid pretreatment partially reduced
3,4-CPHA cytotoxicity. Pretreatment with ascor-
bic acid prevented a rise in LDH leakage when

M. Valento6ic et al. / Toxicology 162 (2001) 149–156
153
4. Discussion
Previously published studies have shown that
the parent compound, 3,4-dichloroaniline, in-
duced cytotoxicity to renal cortical slices (Valen-
tovic et al., 1995, 1997). Pyruvate stimulated
gluconeogenesis was inhibited by 0.5 mM 3,4-
dichloroaniline and LDH leakage was elevated
above control values in the presence of 2 mM
3,4-dichloroaniline (Valentovic et al., 1997). The
present study focussed on the in vitro cytotoxicity
of 3,4-CPHA, the N-hydroxyl metabolite of 3,4-
dichloroaniline. Exposure of renal cortical slices
to 3,4-CPHA resulted in a depletion of glu-
tathione and decreased organic ion transport.
Pyruvate stimulated gluconeogenesis was de-
creased in the presence of 0.1 mM 3,4-CPHA
while LDH leakage was apparent following expo-
sure to 0.5 mM 3,4-CPHA. These findings indi-
cate that the N-hydroxyl metabolite is more toxic
than its parent compound, 3,4-dichloroaniline
since cytotoxicity was detected in slices at a lower
level of 3,4-CPHA than had been previously ob-
served for 3,4-dichloroaniline.
Fig. 2. Time dependent effects of 3,4-CPHA on LDH leakage.
Renal cortical slices were incubated for 30–90 min with 0.5
mM 3,4-CPHA. LDH release was expressed as percent of
total. Values represent mean9S.E.M. Groups had N=4/
group. 3,4-CPHA induced an increase (*PB0.05) in LDH
leakage relative to vehicle treated tissue within 30 min.
Glutathione levels were rapidly reduced by ex-
posure to 3,4-CPHA and addition of glutathione
to the incubation media prevented 3,4-CPHA as-
sociated rise in LDH leakage from renal slices.
The reduction in glutathione may due to glu-
tathione acting as a radical scavenger (Reed,
1990). Total glutathione may be depleted by con-
version of the reduced form of glutathione (GSH)
to an oxidized GSSG form with donation of an
electron to neutralize a reactive intermediate of
3,4-CPHA. Glutathione may also interact with
3,4-CPHA or a metabolite to form a glutathione
conjugate. Further studies are necessary to deter-
mine the role of glutathione.
Pretreatment with deferoxamine prior to expo-
sure to 3,4-CPHA did not reduce cytotoxicity.
These results suggest that cytotoxicity mediated
by 3,4-CPHA occurs through an iron independent
mechanism. A possible alternate explanation is
that the deferoxamine did not have sufficient time
to chelate excess free iron or that the intracellular
concentration of deferoxamine was not sufficient
to chelate iron.
Fig. 3. Alterations in glutathione (GSH) levels as a function of
time following exposure to 0.5 mM 3,4-CPHA. Renal cortical
slices were incubated for 15–120 min with 3,4-CPHA or
vehicle for 60 or 90 min. The amount of glutathione was
determined and expressed as nmol/g tissue. Values represent
mean9S.E.M. with five animals/group. An asterisk (*) indi-
cates statistical difference (PB0.05) from respective vehicle
treated slices.
ment when slices were exposed to 0.5 mM 3,4-
CPHA. Consequently, ascorbic acid was only suc-
cessful at concentrations below 0.5 mM
3,4-CPHA.

154
M. Valento6ic et al. / Toxicology 162 (2001) 149–156
Table 3
The effect of deferoxamine, DTT and glutathione pretreatments on 3,4-CPHA toxicitya
Pretreatment
3,4-CPHA concentration (mM)
0
0.01
0.1
0.5
Glucose (nmol/g tissue)
Water
DFX
1.8090.35
1.4390.15
1.7190.16
1.6190.16
1.2890.13*
1.2890.11
0.5690.04*
0.5490.06*
Water
DTT
1.6890.70
1.6690.06
1.7590.14
1.6190.03
1.2290.09*
1.1490.03*
0.5490.13
0.4390.02*
Water
GSH
1.2590.31
1.9590.49
1.0590.30
1.9290.45
1.0790.30*
1.7090.44
0.2390.11*
0.2490.06*
LDH leakage (% of total)
Water
DFX
11.490.4
10.591.8
10.990.2
11.891.5
15.291.4
14.291.0
30.493.1*
31.892.5*
Water
DTT
16.390.9
18.290.2
17.190.8
18.490.7
20.190.5
23.590.9
34.892.1*
44.491.1*
a Renal cortical slices were preincubated with vehicle or 0.1 mM deferoxamine (DFX), 1 mM DTT or 1 mM glutathione (GSH)
for 30 min. Tissues were then incubated with varying concentrations of 3,4-CPHA for 90 min followed by a 30 min incubation with
10 mM pyruvate. Values represent mean9S.E.M. with N=4/group.
* Indicates different (PB0.05) from respective vehicle treated tissue.
cytotoxicity by preventing redox cycling to nitro-
sobenzene and benzoquinoneimine. Once
quinoneimines (Monks et al., 1992) are formed,
The ability of ascorbic acid to reduce 3,4-
CPHA in vitro toxicity would support the hy-
pothesis that toxicity involves formation of a
reactive intermediate. Ascorbic acid would be ex-
pected to reduce oxidation and lower toxicity of
3,4-CPHA. 3,4-CPHA could undergo oxidation to
a reactive intermediate through an enzymatic
pathway or via auto-oxidation. Phenylhydroxy-
lamine, the N-hydroxyl metabolite of aniline un-
dergoes oxidation to form nitrosobenzene in red
blood cells (Kiese, 1966; Eyer, 1988) and in rat
liver (Eyer et al., 1980). Ascorbic acid may pre-
vent the oxidation of 3,4-CPHA to 3,4-dichloroni-
trosobenzene and reduce toxicity mediated by the
one electron transfer.
Ascorbic acid has been shown to reduce the
toxicity of mono- and dihalo- aminochlorophenol.
Pre-treatment with ascorbic acid reduced the in
vivo nephrotoxicity of 4-amino-2,6-dichlorophe-
nol (Hong et al., 1997). Pretreatment with ascor-
bic acid was sufficient to prevent alterations in
renal function associated with 4-amino-2,6-
dichlorophenol. Ascorbic acid also reduced the in
vitro toxicity of 2-amino-5-chlorophenol (Valen-
tovic et al., 1999). Ascorbic acid may reduce
Fig. 4. The effect of glutathione pretreatment on 3,4-CPHA
toxicity. Renal cortical slices were pre-incubated for 30 min in
5 ml of oxygenated Krebs buffer with 0 or 1 mM reduced
glutathione (GSH). Renal slices were then incubated for 90
min with 3,4-CPHA at varying concentrations. The slices were
then incubated for 30 min with 10 mM pyruvate to stimulate
gluconeogenesis. Values represent mean9S.E.M. with N=4/
group. Groups that are different from each other (PB0.05)
are denoted by different superscripts.

M. Valento6ic et al. / Toxicology 162 (2001) 149–156
155
Acknowledgements
This work was supported by NIH grant
ES08834.
References
Andersen, M.E. 1985. Determination of glutathione and glu-
tathione disulfide in biological samples. Methods Enzymol.
113, 548-555.
Eyer, P., 1988. Detoxication of N-oxygenated arylamines in
erythrocytes: an overview. Xenobiotica 18, 1327–1333.
Eyer, P., Kampffmeyer, H., Maister, H., Rosche-Oehme, E.,
1980. Biotransformation of nitrosobenzne, phenylhydroxy-
lamine and aniline in the isolated perfused rat liver. Xeno-
biotica 10, 499–516.
Farm Chemicals Handbook. 1992. Meister Publishing Com-
pany, Willoughby, OH, p. C277.
Fowler, L.M., Foster, J.R., Lock, E.A., 1993. Effect of ascor-
bic acid, acivicin and probenecid on the nephrotoxicity of
4-aminophenol in the Fischer 344 rat. Arch. Toxicol. 67,
613–621.
Fig. 5. The effect of ascorbic acid pretreatment on 3,4-CPHA
toxicity. Renal cortical slices were pre-incubated for 30 min in
5 ml of oxygenated Krebs buffer with 0 or 1 mM reduced
glutathione (GSH). Renal slices were then incubated with
3,4-CPHA at varying concentrations for 90 min followed by
an additional 30 min incubation with 10 mM pyruvate to
stimulate gluconeogenesis. Values represent mean9S.E.M.
with N=4/group. Groups that are different from each other
(PB0.05) are denoted by different superscripts.
Gosselin, R.E., Smith, R.P. and Hodge, H.C. 1984. Aniline in
Clinical Toxicology of Commercial Products, 5th Edition,
William and Wilkins, New York, NY, III-31 to III-36.
Hong, S.K., Anestis, D.K., Ball, J.G., Valentovic, M.A.,
Brown, P.I., Rankin, G.O., 1997. 4-Amino-2,6-
dichlorophenol nephrotoxicity in the Fischer 344 rat: pro-
tection by ascorbic acid, AT-125 and aminooxyacetic acid.
Toxicol. Appl. Pharmacol. 147, 115–125.
Kiese, M., 1966. The biochemical production of ferri-
hemoglobin forming derivatives from aromatic amines and
mechanisms of ferrihemoglobin formation. Pharmacol.
Rev. 18, 1091–1161.
Kiese, M., Taeger, K., 1976. The fate of phenylhydroxylamine
in red blood cells. Arch. Pharmacol. 292, 59–66.
Lock, E.A., Cross, T.J., Schnellmann, R.G., 1993. Studies on
the mechanism of 4-aminophenol induced toxicity to renal
proximal tubules. Human Exp. Toxicol. 12, 383–388.
McMillan, D.C., Freeman, J.P., Hinson, J.A., 1990a.
Metabolism of the arylamide herbicide Propanil I. Micro-
somal metabolism and in vitro methemoglobinemia. Toxi-
col. Appl. Pharmacol. 103, 90–101.
McMillan, D.C., McRae, T.A., Hinson, J.A., 1990b. Propanil
induced methemoglobinemia and hemoglobin binding in
the rat. Toxicol. Appl. Pharmacol. 105, 503–507.
Monks, T.J., Lau, S.S., 1992. Toxicology of quinone-
thioethers. Crit. Rev. Toxicol. 22 (5/6), 243–270.
Monks, T.J., Hanzlik, R.P., Cohen, G.M., Ross, D., Graham,
D.G., 1992. Conteurporary issues in Toxicology: Quimone
Chemistry and Toxicology. Toxicol. Appl. Pharmacol. 112,
2–16.
these intermediates are highly reactive with intra-
cellular protein. Quinoneimines (Monks and Lau,
1992) and nitrosobenzene are also capable of un-
dergoing redox cycling that would induce oxida-
tive stress within the target tissue.
Ascorbic acid pretreatment reduces the renal
toxicity of 4-aminophenol or para-hydroxyaniline.
Fowler et al. (1993) reported ascorbic acid re-
duced in vivo 4-aminophenol toxicity in Fischer
344 rats. Pretreatment with ascorbic acid of rabbit
proximal tubules, greatly reduced the cytotoxicity
of 4-aminophenol (Lock et al., 1993). Ascorbic
acid pretreatment of rabbit proximal tubule was
able to maintain LDH leakage at levels compara-
ble to control values following exposure to 4-
aminophenol and prevented
intracellular glutathione levels.
a depletion in
Based on the present studies, 3,4-CPHA is di-
rectly toxic to renal cortical slices at a concentra-
tion that is lower than its parent compound,
3,4-dichloroaniline. The toxicity is associated with
a depletion of glutathione and diminished pyru-
vate stimulated gluconeogenesis. 3,4-CPHA toxic-
ity may be mediated by oxidation to form a
reactive intermediate that is prevented by the
presence of ascorbic acid.
Reed, D.J., 1990. Glutathione. Toxicological implications.
Annu. Rev. Pharmacol. Toxicol. 30, 603–631.

156
M. Valento6ic et al. / Toxicology 162 (2001) 149–156
Valentovic, M.A., Ball, J.G., Anestis, D.K., Rankin, G.O.,
1995. Comparison of the in vitro toxicity of dichloroaniline
structural isomers. Toxicol. In Vitro 9 (1), 75–81.
Valentovic, M.A., Tagouri, Y., Ball, J.G., Hong, S.K., Brown,
P.I., Rankin, G.O., 1997. 3,4-Dichloroaniline acute toxicity
in male Fischer 344 rats. Toxicology 124, 125–134.
Valentovic, M., Meadows, M.K., Harmon, R.C., Ball, J.G.,
Hong, S.K., Rankin, G.O., 1999. 2-Amino-5-chlorophenol
toxicity in renal cortical slices from Fischer 344 rats: effect
of antioxidants and sulfhydryl agents. Toxicol. Appl. Phar-
macol. 161, 1–9.
Rondestvedt, C.S., Johnson, T.A. 1977. Rapid preparation of
chloroarylhydroxylamine by hydrazine-palladium reduc-
tion of chloronitrobenzene. Synthesis, 850–851.
Stevens, J.T., Sumner, D.D., 1991. Herbicides. In: Hayes,
W.J., Laws, E.R. (Eds.), Handbook of Pesticide Toxicol-
ogy. Academic Press, New York, pp. 1345–1346.
Valentovic, M.A., Ball, J.G., Elliot, C.W., Madan, E., 1989.
Cephaloridine nephrotoxicity in streptozotocin (STZ)
induced diabetic Fischer 344 rats. Toxicology 57, 193–
207.
.