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In vivo and in vitro nephrotoxicity of aniline and its monochlorophenyl derivatives in the fischer 344 rat
Toxicology, 38 (1986) 269--283 Elsevier Scientific Publishers Ireland Ltd.
IN VIVO AND IN VITRO NEPHROTOXICITY OF ANILINE AND ITS MONOCHLOROPHENYL DERIVATIVES IN THE FISCHER 344 RAT*,**
G.O. RANKINa, D.J. YANGa, K. CRESSEY-VENEZIANOa, S. CASTOa, R.T. WANGb and P.I. BROWNb Departments of Pharmacology a and Anatomy b, Marshall University School of Medicine, Huntington, West Virginia 25704-2901 (U.S.A.) (Received July 8th, 1985) (Accepted September 12th, 1985)
SUMMARY
Aniline (A) and its monochlorophenyl derivatives (2-CA, 3-CA and 4-CA) are widely-used chemical intermediates. In the present study, the in vivo and in vitro nephrotoxic potential of these compounds was assessed in Fischer 344 rats. In the in vivo experiments, rats were administered a single intraperitoneal (i.p.) injection of an aniline (0.4, 1.0 or 1.5 mmol/kg) or 0.9% saline (2.0 ml/kg, i.p.), and renal function monitored at 24 and 48 h° 2-CA was the only compound tested which decreased urine volume, elevated the blood urea nitrogen (BUN) concentration and depressed both basal and lactate° stimulated p-aminohippurate (PAH) accumulation by renal cortical slices at the 1.0 retool/ kg dose. Similar results were produced following 3- and 4-CA administration, but these compounds required a dose of 1.5 mmol/kg. Aniline had little effect on renal function at the doses used in this study. In the in vitro experiments, 2-CA (10 -4 M or greater) depressed basal PAH accumulation. Tetraethylammonium {TEA) uptake was decreased by all compounds with an incubate concentration of the aniline at 10 -3 M. Lactatestimulated PAH uptake was not decreased by any test compound. These results indicate that chlorine substitution on the phenyl ring of aniline enhances nephrotoxic potential, and that 2-substitution produces the greatest increase.
Key words: Anilines; Halogenated hydrocarbons; Nephrotoxicity; Rats *Presented in part at the fall meeting of the American Society for Pharmacology and Experimental Therapeutics, Indianapolis, Indiana, August 1984. **Supported by NIH Grants AM 31210 and RR 05870. Address all correspondence to: Gary O. Rankin, Ph.D., Department of Pharmacology Marshall University School of Medicine, Huntington, WV 25704-2901, U.S.A. 0300-483X/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
269
INTRODUCTION
Aniline and its monochlorophenyl derivatives are widely used industrial compounds and are produced by most industrialized nations. In the United States alone over 400 million pounds of aniline were produced in 1975 [1]. These compounds are important chemical intermediates in the synthesis of many organic chemicals used as antioxidants, dyestuffs, pharmaceuticals, agricultural chemicals, and in numerous other industrial capacities [2]. Human exposure to aniline and its derivatives generally occurs in the industrial setting, and much of our knowledge concerning the toxicity of aniline compounds in man has come from industrial exposure [3--5]. Following acute exposure to aniline compounds, the primary effect is the production of methemoglobinemia with resulting cyanosis and tissue hypoxia [2,6]. Chronic exposure to anilines can result in anemia, hypoxia, cutaneous lesions and possible bladder and/or spleenic tumors [2,7]. The production of tumors is a particularly worrisome problem since populations worldwide could be exposed to low to moderate levels of anilines from environmental exposure to industrial wastes [8--11] or following breakdown of agricultural chemicals [12--14]. Although many aniline derivatives produce symptoms or renal damage in man [6,15,16], detailed studies describing the acute effects of many of these compounds on renal function have not been conducted. In addition, since many aliphatic and aromatic halogenated hydrocarbons have been shown to produce nephrotoxicity in man and animals [17--21], it is possible that ring halogenated derivatives of aniline might also be nephrotoxic. The lack of detailed studies investigating the nephrotoxic potential of these widely-used halogenated aniline derivatives suggests that further studies should be conducted to deliniate more clearly the potential risk to man from exposure to these chemicals. The purpose of this study was to investigate the acute nephrotoxic potential of aniline (A) and its monochlorophenyl derivatives: 2~chloroaniline (2-CA), 3-chloroaniline (3-CA) and 4¢hloroaniline (442A) in male Fischer 344 rats. Fischer 344 rats were used because this rat strain is frequently more sensitive to nephrotoxins than other rat strains [22--24]. The renal effects of these compounds were studied in vivo to monitor whole animal effects and in vitro to determine the nephrotoxic potential of each compound in the absence of hepatic metabolism and methemoglobinemia-induced hypoxia. METHODS
Animals
Male Fischer 344 rats (200--300 g) were used in all experiments. Rats were purchased from Hilltop Lab Animals, Inc. (Scottdale, PA) and housed in rooms with controlled temperature (21--23°C), humidity and light period (on 0600 h to 1800 h). Rats were housed singly in stainless steel metabolism cages during the
270
in vivo experiments. At all other times rats were housed in groups of not more than 6 in standard animal cages. In vivo studies A blood sample was taken from the tail of each rat 3 days prior to placem e n t in metabolism cages for the determination of the basal blood urea nitrogen (BUN) concentration (Sigma Kit No. 640). Rats were allowed free access to food (powdered Purina R o d e n t Chow) and water on the first day of individual housing. The next day (control day), food was removed at 0900 h and withheld for 6 h to assure collection of a urine specimen w i t h o u t food contamination. The 6-h urine specimens were semiquantitatively analyzed (Multistix®, Ames Division, Miles Laboratories, Inc.) for the presence of protein, glucose, ketones and blood. Body weight, food and water intake and urine volume were determined at 24-h intervals. After 1 control day, rats (4 rats/group) were administered a single intraperitoneal injection of aniline or one of its monochlorophenyl derivatives {0.4, 1.0 or 1.5 mmol/kg} or vehicle only (0.9% saline, 2.0 ml/kg) at 0900 h and monitored as described above. At 48-h post-injection blood samples were taken from the tail of each rat for BUN concentration determinations. Rats were then killed by cervical dislocation. Both kidneys were rapidly removed with the left kidney being used for the renal slice studies as described below. The right kidney was weighed and examined for histological changes. No animals died among the vehicle groups or among the groups that received A or 4-CA. However, among rats receiving 2-CA {1.0 mmol/kg) 2 of 6 animals died, and within the group receiving 3-CA (1.5 mmol]kg) 8 of 12 rats died. Data from animals t h a t died are not included in the results. In addition, since lethality was high and nephrotoxicity evident with 2-CA administration at a dose o f 1.0 mmol/kg, higher doses of 2-CA were not tested. Control rats were pair-fed for their appropriate treatment group. This assured that any observed nephrotoxic response was due to chemically-induced damage rather than decreased food intake. In vitro studies In the in vitro studies, untreated rats were killed by cervical dislocation around 0900 h and renal slices were prepared and processed as described below. Tissue slices were incubated for 30 min with vehicle or an aniline prior to PAH or TEA addition and for 90 min with PAH and TEA. Aniline solutions were prepared from the appropraiate hydrochloride salts in 0.9% saline such that the addition of 30 ~l of the aniline solution would yield the desired final concentration in the 3 ml of incubation media. Control samples had 30 ~1 of 0.9% saline only added to the incubation media. S/M ratios for each experiment were determined as described below. Renal slice studies Renal slice studies were performed as described earlier [20] with a slight
271
modification. Renal cortical slices were prepared freehand and approximately 100 mg of tissue were incubated with shaking for 90 min in Krebs-Ringer phosphate buffer (pH 7.4) containing 5.0 mM potassium and 1.0 mM calcium at 25°C under a 100% oxygen atmosphere in a Dubnoff metabolic shaker. The uptake of p-[14C] aminohippurate (PAH) and [14C] tetraethylammonium (TEA) was studied. Sodium lactate (10 -2 M) was added in some PAH experiments. Accumulation of PAH and TEA by renal cortical slices was expressed as the slice to medium (S/M) ratio, where S equalled radioactivity (dpm)/g of tissue and M equalled the radioactivity (dpm)/ml of incubation media. Histology Following cervical dislocation, the right kidney was removed, weighed, cut in half lengthwise and placed in 10% neutral buffered formalin. Fixed tissues were embedded in paraffin, sectioned at 6--8 pm and stained with hematoxylin and eosin (HE) or periodic-acid Schiff (PAS)reagent before histological examination of the thick sections. In some cases, rats were anesthetized with sodium pentobarbital (50 rag/ kg, i.p.), and both kidneys perfused in situ with sodium cacodylate buffer (0.18 M, pH 7.2) followed by fixative (2.0% paraformaldehyde and 3.0% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) via the descending aorta. Tissues were post-fixed (2% osmium tetroxide) for 2 h, rinsed (buffer) and en bloc stained with 0.5% uranyl acetate (30 min). Following dehydration (ethanol), tissues were embedded (Spurt medium, Polysciences, Inc.) and 2-~m sections (thin sections) cut using a glass knife (MT-5000 ultramicrotome, Dupont). Sections were stained (1% toluidine blue) prior to examination for histopathology. Statistical analysis The data were analyzed using one-way analysis of variance and/or Students t-tests. The 0.05 level was used as the criterion for significance. RESULTS
Effects on food and water intake and body weight Food and water intake were markedly decreased by A and its monochlorophenyl derivatives (Table I). At comparable doses, 2-CA produced the most pronounced decreases in food and water intake of the compounds tested. Body weight decreased in all treatment groups (data not shown) in parallel with the decreases in food and water intake. Effects on urine volume and content The effects of A and its monochlorophenyl derivatives on urine volume are presented in Fig. 1. Both 2-CA and 4-CA decreased urine volume on treatment day 1 following a 1.0 mmol/kg dose of the aniline, but urine volume remained depressed on day 2 only in the 2-CA treatment group at
272
TABLE I EFFECT OF ANILINE AND MONOCHLOROANILINE WATER INTAKE a
TREATMENT ON FOOD AND
Treatment (mmol/kg)
W a t e r i n t a k e (ml)
F o o d i n t a k e (g) Day 0
Day 1
Day 2
Day 0
Day 1
Day 2
A 1.0 1.5
19.9 ± 2.1 16.0 ± 0.9
6.2 ± 2.4 b 1.4 ± 0.4 b
13.0 ± 0.7 b 7.6 _+ 2.4 b
32 ± 3 29 ± 1
14 ± 4 b 11 ± 1 b
27 ± 19 ±
1 5
2-CA 0.4 1.0
17.5 ± 0.6 16.4 ± 0.8
1.3 _+ 0.9 b 0.5 ± 0.1 b
5.6 ± 1.6 b 0.3 ± 0.1 b
32 ± 2 29 ± 2
5 ± 2b 6 ± 1b
19 ± 6 ±
3b 1b
342A 1.0 1.5
16.5 +_ 0.2 19.7 ± 1.1
1.2 ± 0.5 b 0.2 ± 0.1 b
8.8 ± 0.8 b 0 . 6 ± 0,3 b
33 _'!-1 33 ± 2
23 ± 2 b 13 ± 7
15 _+ I b 18 ± 6
442A 1.0 1.5
19.2 ± 1.1 18.6 ± 0.8
0.7 ± 0.1 b 0b
5.5 ± 0.4 b 6.4 ± 0.2 b
38 + 1 36 ± 2
13 ± 1 b 0b
18 ± 3 b 14 ± 10
aValues are m e a n s _+ S.E. f o r at least 4 r a t s / g r o u p . b S i g n i f i c a n t l y d i f f e r e n t f r o m d a y 0, P < 0.05.
this dose. Urine volume also was decreased by administration of A, 3-CA and 4-CA at a dose of 1.5 mmol/kg with 4-CA producing the greatest decline on day 1. The major urinary c o n t e n t abnormalities produced by administration of an aniline were hematuria and increased proteinuria. Administration of 2-CA {1.0 mmol/kg) produced hematuria ( + + + ) and an enhanced proteinuria ( + + ) , which were maintained on day 2 (Table II). These abnormalities were more pronounced and of longer duration than those produced by the other c o m p o u n d s studied. Hematuria ( + + + ) also was the most prominent urinary finding following 442A treatment (day 2). Although 3-CA (1.0 mmol/kg) administration produced hematuria {++) on both post-treatment days, this finding was not as marked following treatment with 1.5 mmol/kg 342A. Glucosuria {trace) was seen only in the 3-CA treatment groups on day 1. Ketonuria was not noted in any group studied. Effects on BUN concentration
The BUN concentration was increased by 2-CA {1.0 mmol/kg) from a pretreatment value of 21.7 + 1.4 to 72.2 + 16.0 mg/dl at 48 h (Fig. 2). Higher doses of 3-CA and 4 ~ A were required to significantly elevate the BUN concentration to 93.4 + 0.4 and 171.6 -+ 18.5 mg/dl, respectively. BUN concentration was not altered following administration of either dose of
273
!1:
A
2-CA
t5
~5 o o
I
TIME(days)
2
TIME(days)
3-CA
IS_
0
is
4-CA
A ~10-
~ 10-
5-
o'
i
2
o
0
!
2
TIME(days) Tim(days) Fig. 1• Effect of aniline (A) and its monochlorophenyl derivatives (2-, 3- and 4-CA) on urine volume. Rats were housed singly, and some rats received a single i.p. injection of an aniline (u, 0.4 mmol/kg; o, 1.0 mmol/kg; A, 1.5 mmol/kg). Other groups (open symbols) received vehicle only and were pair-fed for their appropriate treated group. An asterisk indicates significantly different from 0 h (P < 0.05). A star indicates significantly different from the appropriate control group (P < 0.05).
A studied. T h u s , 2-CA is m o r e p o t e n t in elevating t h e B U N c o n c e n t r a t i o n t h a n t h e o t h e r c o m p o u n d s studied•
Effects on organic ion accumulation Lactate~stimulated P A H a c c u m u l a t i o n and T E A a c c u m u l a t i o n w e r e r e d u c e d b y 2~:~A (0.4 m m o l / k g ) at 48 h post-injection (Fig. 3). F u r t h e r r e d u c t i o n s in basal a n d l a c t a t e - s t i m u l a t e d P A H a c c u m u l a t i o n w e r e seen in t h e 2 ~ A (1.0 m m o l / k g ) t r e a t m e n t g r o u p . A d m i n i s t r a t i o n o f 3 ~ A (1.5 m m o l / k g ) also r e s u l t e d in d e c r e a s e d basal and l a c t a t e - s t i m u l a t e d PAH u p t a k e w i t h o u t altered T E A a c c u m u l a t i o n . T r e a t m e n t o f rats w i t h 4-CA (1.5 m m o l ] k g ) resulted in d e c r e a s e d T E A a c c u m u l a t i o n while A (1.0 or 1.5 m m o l / k g ) p r o d u c e d a decrease o n l y in n e t basal PAH u p t a k e . T h e lack o f a dose-related decrease in P A H + lactate u p t a k e f o l l o w i n g 4-CA administ r a t i o n is m o s t p r o b a b l y d u e t o t h e variability o f d a t a in t h e high dose group. T h e effects o f in vitro i n c u b a t i o n o f anilines w i t h renal cortical slices o n organic ion a c c u m u l a t i o n are s h o w n in Fig. 4. All 4 anilines depressed T E A a c c u m u l a t i o n w h e n p r e s e n t at a c o n c e n t r a t i o n o f 10 -3 M in the incub a t i o n m e d i a . L a c t a t e - s t i m u l a t e d P A H a c c u m u l a t i o n was n o t decreased b y 274
TABLE II EFFECT Treatment (mmol/kg)
OF ANILINE TREATMENT
ON URINE CONTENT a
Protein b
Blood
Day 0
Day 1
Day 2
Control c
+
+
+
A 1.0 1.5
+ +
~- + + ÷
+ +
Day 0
Day 1
Day 2
--
trace
trace trace
+ ++ +
++ +
2~A 0.4
trace
+ +
+
-
1.0
trace
~- +
+ +
--
trace +
trace ++
+
--
+
--
+ + trace
+ + --
+ trace
+ d
+ + +
_
+ _d
+++ ++ +
3-CA 1.0
1.5 4-CA 1 .0 1.5
aValues most frequently observed for at least 4 rats/group. b S e m i q u a n t i t a t i v e v a l u e s f o r p r o t e i n a r e : t r a c e --- < 3 0 m g / d l ; + = 3 0 m g / d l ; + + mg/dl. CAll p a i r - f e d c o n t r o l r a t s h a d s i m i l a r v a l u e s s o t h e i r r e s u l t s a r e c o m b i n e d . d N o 6-h u r i n e w a s p r o d u c e d o n d a y 1.
= 100
any test c o m p o u n d , b u t basal PAH accumulation was decreased by 2-CA at incubate concentrations o f 10-4 M or higher.
Effects on morphology Renal morphological effects observed in thick sections (6 #m) were seen primarily in the renal cortex of treated animals. These alterations consisted of random apical sloughing of the brush border from some proximal tubular cells. However, many proximal tubular cells appeared to be unaffected. Distal tubular cells appeared normal. Marked accumulation of erythrocytes was noted in several cortical blood vessels. Although clear functional differences were noted between treated groups, morphological changes were similar regardless of dose or c o m p o u n d tested. Examination of thin sections (2 ~m) of perfused kidneys revealed differences in morphology among the treatment groups. The most severe m o ~ h o l o g i c a l changes were observed in the 2-CA (1.0 mmol/kg) treatment group (Fig. 5) where both proximal and distal tubular cells exhibited marked degenerative changes. Most proximal tubular cells had luminal protuberances and extensive vacuolization which caused clumping of microvilli. Nuclei of these cells were reduced in size and often crenated. Sloughing of some cells 275
1200-
[ ] 0.4umllkl(Ulbol ) [ ] 0.4mul/il(tml~l) [ ] 1.1lid/kl(Ceetml ) " ___~l.luud/.l'l(trelhld) [ ] 1.5nUlill(Cutrol ) • 1.5mmollkl (tllited)
!:2 0 0 ~ 600-
.,
"
i l l
-i
Fig. 2. Effect of aniline (A) and its monochlorophenyl derivatives (2-, 3- and 4-CA) on the blood urea nitrogen (BUN) concentration at 48 h post-injection. Blood samples were obtained from the tail prior to administration of test compounds or vehicle (0 h) and at 48 h post-injection. Data are expressed as the mean percent of the appropriate pretreatment control value (0 h) +- S.E. for 4 rats/group. BUN concentration control values (0 h) were typically between 15.0 and 20.0 rag% (range = 14.2 ± 0.8 to 22.0 ± 1.1). An asterisk indicates significantly different from the 0 h value (P < 0.05). A diamond indicates significantly different from the appropriate control group (P < 0.05).
resulted in lumenal occlusion. Distal tubular cells also exhibited extensive vacuolization, distensions of basal infoldings and swelling. Occlusion of distal tubular segments also was common. Blood vessels and glomeruli appeared normal. Morphological changes produced by 3-CA (1.5 mmol/kg) were similar to but less severe than those produced by 2-CA (1.0 mmol/kg). Treatment with 4 ~ A (1.5 mmol/kg) also produced renal morphological changes (Fig. 6). These changes were characterized by hypertrophic alterations in both proximal and distal tubular cells with lumens frequently occluded. Increased numbers of lysosomal granules were evident in many tubular segments. Treatment with A had little effect on renal morphology. However, in the 1.5 mmol/kg treatment group vacuolization was evident in some proximal tubular cells near blood vessels. Cells furthest from the blood vessels appeared normal. DISCUSSION
The results of this study demonstrate that the decreasing order of nephrotoxic potential for A and its 3 monochlorophenyl derivatives in Fischer 344 rats is: 2-CA > 4 ~ A > 3 ~ A > A. At 1.0 mmol/kg, 2 ~ A was the only aniline which elevated the BUN concentration and decreased both basal and
276
A
lO0
7-
•75-
2-CA
~ 0 . 4 m mol'k| ~ l . O m m e l kg ~ L 5 m tool ~'g
SO ~ 2S-
25
PAH
PAH
- TEA
o
LAdAte
PAH LACTATE
TEA
4-CA
100
3-CA i
~75
PAH
~- Z5
PAH
PAH LACTATE
TEA
eAu
eA+H
TFA
LACTATE
Fig. 3. Effect o f aniline (A) and its m o n o c h l o r o p h e n y l derivatives (2-, 3- and 4-CA) administration o n t h e a c c u m u l a t i o n o f PAH and T E A by renal cortical slices. Rats were killed b y cervical dislocation at 48 h post-injection o f an aniline or vehicle, and kidneys rapidly r e m o v e d for preparation o f renal cortical slices (see Methods for details). Each bar is the m e a n and each vertical line the standard error for 4 rats. Data are expressed as t h e percent o f the m e a n o f the appropriate pair-fed c o n t r o l group. Typical S/M ratios f r o m pair-fed control groups were b e t w e e n 3.0 and 4.0 (range = 2.62 ~ 0.23 to 3.93 + 0.38) for P A H uptake, b e t w e e n 8.0 and 10.0 (range = 7.18 -+ 0.46 to 9.56 ± 0.43) for PAH + lactate u p t a k e and b e t w e e n 16.0 and 20.0 (range = 15.41 + 0.58 to 20.13 +0.57) for T E A uptake. An asterisk indicates significantly different f r o m the appropriate pair-fed c o n t r o l group (P < 0.05).
lactate-stimulated P A H accumulation. 2 ~ A also was the only aniline to depress basal P A H accumulation in the in vitro studies. Higher doses (1.5 mmol/kg) of 4-CA and 3-CA were required to elevate the B U N concentration, while A had no effect on B U N concentration following administration of the 1.5 mmol/kg dose. The decrease in the B U N concentration seen in the pair-fed control groups is not unexpected. Food intake was markedly reduced by the chlorophenyl derivatives to less than 1.5 g on day 1 (Table I). Animals in the low dose groups consumed between 5.0 and 9.0 g on day 2, while rats in the higher dose groups generally ate less than 1.0 g. Marked reduction in food intake has previously been reported to decrease B U N concentration. For example, Traina and Sather [25] reported that fasted Sprague--Dawley rats have decreased B U N concentrations for up to 7 days. Thus, the depressed control B U N concentrations likely are due to decreased food intake.
277
A
2015~
c~
~
~
2-CA
2o
,
cD 15e'~l 0.
10-
! ,L
.L
,,t
.d.
.L
lo -°
io -~
1o-.
,'o-~
CONCENTRATION (M) 20
3-CA
5 1o'-~
lo '~
1o'-'
Io-~
CONCENTRATION (M) 2o
4-CA
15o
t--o~ 10-
v~ 5-
lo-~ 1~~ lo-' io-:
CONCENTRATION (M)
lo-~ lo-° i~-" lo-o
CONCENTRATION (M)
Fig. 4. Effect of the i n c u b a t i o n of renal cortical slices w i t h various c o n c e n t r a t i o n s of aniline (A) or one o f its m o n o c h l o r o p h e n y l derivatives (2-, 3-, or 4-CA) o n PAH and T E A a c c u m u l a t i o n . Basal (A) and lactate-stimulated (o) PAH and ( o ) T E A a c c u m u l a t i o n were measured. Data are expressed a s t h e m e a n S/M ratio +- S.E. for at least 4 experiments. An asterisk indicates significantly d i f f e r e n t (P < 0.05) f r o m control experiments.
Species sensitivity to the induction of methemoglobin b y aromatic amines varies markedly and is a well k n o w n phenomenon. The cat is the most sensitive species (100%) followed b y man (60%), dog (30%) and the rat (5%) [ 2 ] . Although species sensitivity is variable, the rank order of the methemoglobin formation produced b y a series of amines is similar among species. For example, McLean et al. [26] have reported that in cats the decreasing order of methemoglobin-forming activity of A and its monochlorophenyl derivatives is 4-CA > 2-CA > A ~ 3-CA. Kiese [27] found similar results in dogs. Similar studies with the chloroanilines have not been c o n d u c t e d in the Fisher 344 rat. However, it is probable that a similar rank order of methemoglobin generation would exist in this species. While the magnitude of the formation of methemoglobin within a species is dependent on proportional metabolite generation [2] and methemoglobin reductase activity [ 2 8 ] , it is unlikely that these events would significantly change the rank order of a series of related chemicals. This conclusion is supported by the fact that aniline, acetanilide and acetophenetidin, among other aromatic amines, retain their rank order of methemoglobin-inducing properties in cat, dog, man and rat [ 4 ] . Thus, comparison of the methemoglobin-forming properties of a series of amines should remain similar between species. Since 2-CA was the most nephrotoxic aniline derivative in Fischer 344 rats, it appears that
278
Fig. 5. Renal corticalsections (X 250) from rats treated with either a single intraperitoneal injection of 2-CA (1.0 mmol/kg) (B) or vehicle (0.9% saline, 1.0 ml/kg) (A) and killed 48 h post-treatment. Glomeruli (G), proximal tubules (PT) and distal tubules (DT) are indicated. Note severe vacuolization present in most distal tubules with congestion of m a n y distal tubule lumen (5B, arrows). Tissue was stained with 1% toluidine blue stain.
279
Fig. 6. Renal cortical sections from rats treated with a single intraperitoneal injection of 4-CA (1.5 mmol/kg) (B) o r vehicle (0.9% saline, 1.0 ml/kg) (A) and killed 48 h posttreatment. Glomeruli (G), proximal tubules (PT) and distal tubules (DT) are indicated. Note the damage t o proximal tubules characterized b y the congestion of many lumen and the increased accumulation o f dense lysosomal granules in the cytoplasm (6B). Tissue was stained with 1% toluidine blue stain. Magnifications used were x 250 (A) and × 500
(B).
280
nephrotoxicity does n o t result from tissue hypoxia alone. This conclusion is supported b y the observation that 2 ~ A (> 10 -~ M) reduced PAH accumulation in the in vitro organic ion accumulation studies. Thus, renal toxicity can occur in the absence of hypoxia. However, it is likely that the renal hypoxia which follows administration of an aniline exacerbates the developing renal lesions. Studies are currently underway to clarify this point. The renal effects produced b y administration of some anilines might be due at least in part to a metabolite of extracortical origin. For example, 3-CA (1.5 mmol/kg) elevated the BUN concentration and depressed organic ion accumulation, b u t incubation of renal cortical slices with 3-CA had little effect on PAH or TEA accumulation. Similar results were seen with 2-CA, although basal PAH accumulation was reduced in the in vitro studies by 2-CA bath concentrations of 10 -4 M or greater. Since nephrotoxic aromatic amines are frequently substrates for the cooxidation p a t h w a y of the renal medulla [ 2 8 ] , a toxic species could be generated within the renal medulla rather than in the renal cortex following administration of an aniline. In addition, studies with another halogenated phenyl c o m p o u n d , bromobenzene, have indicated that this chemical produces nephrotoxicity via a metabolite of hepatic rather than renal origin [ 2 9 , 3 0 ] . Therefore, it appears that a metabolite originating from a site other than the renal cortex might contribute to aniline-induced renal effects. However, the lack of decreased organic ion accumulation following incubation of renal cortical slices with an aniline c o m p o u n d could be due to other factors. For example, using in vitro incubation techniques similar to those used in this study, Goldstein et al. [31] showed that cisplatin, a direct-acting nephrotoxin in vivo, had no effect on PAH or TEA accumulation b y renal cortical slices when they used in vitro incubation concentrations of cisplatin similar to renal cortical platinum concentrations obtained in vivo. In addition, incubating kidney slices at 25°C might alter the formation of toxic aniline metabolites which form at 37°C. Further studies are required to more clearly define the role of extracortical or extrarenal metabolites in the nephrotoxicity which aniline compounds exhibit. In summary, the nephrotoxic potential of A and its 3 monochlorophenyl derivatives has been studied in Fischer 344 rats. 2-CA was the most nephrotoxic c o m p o u n d and A was the least nephrotoxic compound. Nephrotoxicity did not follow methemoglobin-producing activity. The relative lack o f direct effects of anilines on organic ion accumulation in the in vitro studies suggests that a metabolite of extracortical or extrarenal origin might be responsible for the in vivo effects o f these c o m p o u n d s on renal function. REFERENCES I U.S. International Trade Commission, Synthetic organic chemicals, U.S. production and sales, 1975. USITC Publication 804, U.S. Government Printing Office, Washington, DC. (1977).
281
2 J.L. Radomski, The primary aromatic amines: Their biological properties and structure-activity relationships. Annu. Rev. Pharmacol. Toxicol., 19 (1979) 129. 3 H.C. Halsted, Industrial methemog]obinemia. J. Occup, Med., 2 (1960) 591. 4 R.R. Beard and J.T. Noe. Aromatic nitro and amino compounds, in G.D. Clayton and F.E. Clayton (Eds.), Party's Industrial Hygiene and Toxicology, 3rd edn., Interscience Publishers, Inc., New York, 1981, p. 2413. 5 I.M. Wetherhold, A.L. Linch and R.C. Chareka, Chemical cyanosis: Causes, effects and prevention. Arch. Environ. Health, 1 (1960) 353. 6 R.E. Gosselin, H.C. Hodge, R.P. Smith and M.N. Gleason, Aniline, in Clinical Toxicology of Commercial Products: Acute Poisoning, 4th ed., Section If, Williams and Wilkins, Baltimore, MD, 1976, p. 29. 7 J.M. Ward, G. Reznik and F.M. Garner, Proliferative lesions of the spleen in male F 344 rats fed diets containing p-chloroaniline. Vet. Pathol., 17 (1980) 200. 8 G.E. Parris, G.W. Diachenko, R.C. Entz, J.A. Poppitti, P. Lombardo, T.K. Rohrer and J.L. Hesse, Waterborne methylene bis(2-chloroaniline) and 2-chloroaniline contamination around Adrian, Michigan, Bull. Environ. Contam. Toxicol., 24 (1980) 497. 9 L.M. Games and R.A. Hites, Composition, treatment efficiency, and environmental significance of dye manufacturing plant effluents. Anal. Chem., 49 (1977) 1433. 10 J.C. Dacre, Potential health hazards of toxic organic residues in sludge, in G. Bitton (Ed.), Sludge: Health Risks of Land Application, Ann Arbor Science Publishers, Ann Arbor, MI, 1980, p. 85. 11 D.D. Ellis, C.M. Jone, R.A. Larson and D.J. Schaeffer, Organic constituents of mutagenic secondary effluents from wastewater treatment plants. Arch. Environ. Contain. Toxicol., 11 (1982) 373. 12 D.D. Kaufman, Degradation of carbamate herbicides in soil. J. Agric. Food Chem., 15 (1967) 582. 13 D.D. Kaufman and P.C. Kearney, Microbial degradation of isopropyl-n-3-chlorophenylcarbamate and 2-chloroethyl-n-3-chlorophenylcarbamate. Appl. Microbiol., 13 (1965) 443. 14 P.C. Kearney, Enzyme hydrolysis, purification, and properties of an enzyme responsible for hydrolyzing phenylcarbamates. J. Agric. Food Chem., 13 (1965) 561. 15 J. Graubarth, C.J. Bloom, F.C. Coleman and H.N. Solomon, Dye poisoning in the nursery. J.Am. Med. Assoc., 128 (1945) 1155. 16 A.D. Casciano, Acute methemogloblnemia due to aniline. J. Med. Soc. N.J., 49 (1952) 141. 17 K.M. McCormack, W.M. Kluwe, D.E. Rickert, V.L. Sanger and J.B. Hook, Renal and hepatic microsomal enzyme stimulation and renal function following three months of dietary exposure to polybrominated biphenyls. Toxicol. Appl. Pharmacol., 44 (1978) 539. 18 W.M. Kluwe and J.B. Hook, Effects of environmental chemicals on kidney metabolism and function. Kidney Int., 18 (1980) 648. 19 W.M. Kluwe, The nephrotoxicity of low molecular weight ha|ogenated alkane solvents, pesticides, and chemical intermediates, in J.B. Hook (Ed.), Toxicology of the Kidney, Raven Press, New York, 1981, p. 179. 20 G.O. Rankin, Nephrotoxicity following acute administration of N-(3,5-dichlorophenyl) succinimide in rats. Toxicology, 23 (1982) 21. 21 G.O. Rankin, D.J. Yang, E.P. Lahoda, K. Cressey-Veneziano, M.L. Bailey and P.I. Brown, Acute nephrotoxicity of N-phenyl and N-(monochlorophenyl) succinimides in Fischer 344 and Sprague--Dawley rats. Toxicology, 34 (1985) 299. 22 R.I. Mazze, M.J. Cousins and J.C. Kosek, Strain differences in metabolism and susceptibility to the nephrotoxic effects of methoxyflurane in rats. J. Pharmacol. Exp. Ther., 184 (1973) 481. 23 J.C. Kosek, R.I. Mazze and M.J. Cousins, Nephrotoxicity of gentamicin. Lab. Invest., 30 (1974) 48.
282
24 R.J. McMurtry, W.R. Snodgrass and J.R. Mitchell, Renal necrosis, glutathione depletion, and covalent binding after acetaminophen. Toxicol. Appl. Pharmaco]., 46 (1978) 87. 25 V.M. Traina and B.T. Sather, Effect of starvation on renal function. Lancet, 2 (1973) 620. 26 S. McLean, G.A. Starmer and J. Thomas, Methaemoglobin formation by aromatic amines. J. Pharm. Pharmacol., 21 (1969) 441. 27 M. Kiese, The effect of certain substituents upon the N-oxidation of aniline in vivo. Naunyn Schmiedeberg Arch. Exp. Pathol., 244 (1963) 387. 28 B.B. Davis, M.B. Mattammal and T.V. Zenser, Renal metabolism of drugs and xenobioties. Nephron, 27 (1981) 187. 29 W~D. Reid, Mechanism of renal necrosis induced by bromobenzene or chlorobenzcne. Exp. Mol. Pathol., 19 (1973) 197. 30 S.S. Lau, T.J. Monks, K.E. Greene and J.R. Gillette, The role of ortho-bromophenol in the nephrotoxicity of bromobenzene in rats. Toxicol. Appl. Pharmacol., 72 (1984) 539. 31 R.S. Goldstein, B. Noordewier, J.T. Bond, J.B. Hook and G.H. Mayor, cis-Dichlorodiammineplatinum nephrotoxicity: Time course and dose response of renal functional impairment. Toxicol. Appl. Pharmacol., 60 (1981) 163.
283
IN VIVO AND IN VITRO NEPHROTOXICITY OF ANILINE AND ITS MONOCHLOROPHENYL DERIVATIVES IN THE FISCHER 344 RAT*,**
G.O. RANKINa, D.J. YANGa, K. CRESSEY-VENEZIANOa, S. CASTOa, R.T. WANGb and P.I. BROWNb Departments of Pharmacology a and Anatomy b, Marshall University School of Medicine, Huntington, West Virginia 25704-2901 (U.S.A.) (Received July 8th, 1985) (Accepted September 12th, 1985)
SUMMARY
Aniline (A) and its monochlorophenyl derivatives (2-CA, 3-CA and 4-CA) are widely-used chemical intermediates. In the present study, the in vivo and in vitro nephrotoxic potential of these compounds was assessed in Fischer 344 rats. In the in vivo experiments, rats were administered a single intraperitoneal (i.p.) injection of an aniline (0.4, 1.0 or 1.5 mmol/kg) or 0.9% saline (2.0 ml/kg, i.p.), and renal function monitored at 24 and 48 h° 2-CA was the only compound tested which decreased urine volume, elevated the blood urea nitrogen (BUN) concentration and depressed both basal and lactate° stimulated p-aminohippurate (PAH) accumulation by renal cortical slices at the 1.0 retool/ kg dose. Similar results were produced following 3- and 4-CA administration, but these compounds required a dose of 1.5 mmol/kg. Aniline had little effect on renal function at the doses used in this study. In the in vitro experiments, 2-CA (10 -4 M or greater) depressed basal PAH accumulation. Tetraethylammonium {TEA) uptake was decreased by all compounds with an incubate concentration of the aniline at 10 -3 M. Lactatestimulated PAH uptake was not decreased by any test compound. These results indicate that chlorine substitution on the phenyl ring of aniline enhances nephrotoxic potential, and that 2-substitution produces the greatest increase.
Key words: Anilines; Halogenated hydrocarbons; Nephrotoxicity; Rats *Presented in part at the fall meeting of the American Society for Pharmacology and Experimental Therapeutics, Indianapolis, Indiana, August 1984. **Supported by NIH Grants AM 31210 and RR 05870. Address all correspondence to: Gary O. Rankin, Ph.D., Department of Pharmacology Marshall University School of Medicine, Huntington, WV 25704-2901, U.S.A. 0300-483X/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
269
INTRODUCTION
Aniline and its monochlorophenyl derivatives are widely used industrial compounds and are produced by most industrialized nations. In the United States alone over 400 million pounds of aniline were produced in 1975 [1]. These compounds are important chemical intermediates in the synthesis of many organic chemicals used as antioxidants, dyestuffs, pharmaceuticals, agricultural chemicals, and in numerous other industrial capacities [2]. Human exposure to aniline and its derivatives generally occurs in the industrial setting, and much of our knowledge concerning the toxicity of aniline compounds in man has come from industrial exposure [3--5]. Following acute exposure to aniline compounds, the primary effect is the production of methemoglobinemia with resulting cyanosis and tissue hypoxia [2,6]. Chronic exposure to anilines can result in anemia, hypoxia, cutaneous lesions and possible bladder and/or spleenic tumors [2,7]. The production of tumors is a particularly worrisome problem since populations worldwide could be exposed to low to moderate levels of anilines from environmental exposure to industrial wastes [8--11] or following breakdown of agricultural chemicals [12--14]. Although many aniline derivatives produce symptoms or renal damage in man [6,15,16], detailed studies describing the acute effects of many of these compounds on renal function have not been conducted. In addition, since many aliphatic and aromatic halogenated hydrocarbons have been shown to produce nephrotoxicity in man and animals [17--21], it is possible that ring halogenated derivatives of aniline might also be nephrotoxic. The lack of detailed studies investigating the nephrotoxic potential of these widely-used halogenated aniline derivatives suggests that further studies should be conducted to deliniate more clearly the potential risk to man from exposure to these chemicals. The purpose of this study was to investigate the acute nephrotoxic potential of aniline (A) and its monochlorophenyl derivatives: 2~chloroaniline (2-CA), 3-chloroaniline (3-CA) and 4¢hloroaniline (442A) in male Fischer 344 rats. Fischer 344 rats were used because this rat strain is frequently more sensitive to nephrotoxins than other rat strains [22--24]. The renal effects of these compounds were studied in vivo to monitor whole animal effects and in vitro to determine the nephrotoxic potential of each compound in the absence of hepatic metabolism and methemoglobinemia-induced hypoxia. METHODS
Animals
Male Fischer 344 rats (200--300 g) were used in all experiments. Rats were purchased from Hilltop Lab Animals, Inc. (Scottdale, PA) and housed in rooms with controlled temperature (21--23°C), humidity and light period (on 0600 h to 1800 h). Rats were housed singly in stainless steel metabolism cages during the
270
in vivo experiments. At all other times rats were housed in groups of not more than 6 in standard animal cages. In vivo studies A blood sample was taken from the tail of each rat 3 days prior to placem e n t in metabolism cages for the determination of the basal blood urea nitrogen (BUN) concentration (Sigma Kit No. 640). Rats were allowed free access to food (powdered Purina R o d e n t Chow) and water on the first day of individual housing. The next day (control day), food was removed at 0900 h and withheld for 6 h to assure collection of a urine specimen w i t h o u t food contamination. The 6-h urine specimens were semiquantitatively analyzed (Multistix®, Ames Division, Miles Laboratories, Inc.) for the presence of protein, glucose, ketones and blood. Body weight, food and water intake and urine volume were determined at 24-h intervals. After 1 control day, rats (4 rats/group) were administered a single intraperitoneal injection of aniline or one of its monochlorophenyl derivatives {0.4, 1.0 or 1.5 mmol/kg} or vehicle only (0.9% saline, 2.0 ml/kg) at 0900 h and monitored as described above. At 48-h post-injection blood samples were taken from the tail of each rat for BUN concentration determinations. Rats were then killed by cervical dislocation. Both kidneys were rapidly removed with the left kidney being used for the renal slice studies as described below. The right kidney was weighed and examined for histological changes. No animals died among the vehicle groups or among the groups that received A or 4-CA. However, among rats receiving 2-CA {1.0 mmol/kg) 2 of 6 animals died, and within the group receiving 3-CA (1.5 mmol]kg) 8 of 12 rats died. Data from animals t h a t died are not included in the results. In addition, since lethality was high and nephrotoxicity evident with 2-CA administration at a dose o f 1.0 mmol/kg, higher doses of 2-CA were not tested. Control rats were pair-fed for their appropriate treatment group. This assured that any observed nephrotoxic response was due to chemically-induced damage rather than decreased food intake. In vitro studies In the in vitro studies, untreated rats were killed by cervical dislocation around 0900 h and renal slices were prepared and processed as described below. Tissue slices were incubated for 30 min with vehicle or an aniline prior to PAH or TEA addition and for 90 min with PAH and TEA. Aniline solutions were prepared from the appropraiate hydrochloride salts in 0.9% saline such that the addition of 30 ~l of the aniline solution would yield the desired final concentration in the 3 ml of incubation media. Control samples had 30 ~1 of 0.9% saline only added to the incubation media. S/M ratios for each experiment were determined as described below. Renal slice studies Renal slice studies were performed as described earlier [20] with a slight
271
modification. Renal cortical slices were prepared freehand and approximately 100 mg of tissue were incubated with shaking for 90 min in Krebs-Ringer phosphate buffer (pH 7.4) containing 5.0 mM potassium and 1.0 mM calcium at 25°C under a 100% oxygen atmosphere in a Dubnoff metabolic shaker. The uptake of p-[14C] aminohippurate (PAH) and [14C] tetraethylammonium (TEA) was studied. Sodium lactate (10 -2 M) was added in some PAH experiments. Accumulation of PAH and TEA by renal cortical slices was expressed as the slice to medium (S/M) ratio, where S equalled radioactivity (dpm)/g of tissue and M equalled the radioactivity (dpm)/ml of incubation media. Histology Following cervical dislocation, the right kidney was removed, weighed, cut in half lengthwise and placed in 10% neutral buffered formalin. Fixed tissues were embedded in paraffin, sectioned at 6--8 pm and stained with hematoxylin and eosin (HE) or periodic-acid Schiff (PAS)reagent before histological examination of the thick sections. In some cases, rats were anesthetized with sodium pentobarbital (50 rag/ kg, i.p.), and both kidneys perfused in situ with sodium cacodylate buffer (0.18 M, pH 7.2) followed by fixative (2.0% paraformaldehyde and 3.0% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) via the descending aorta. Tissues were post-fixed (2% osmium tetroxide) for 2 h, rinsed (buffer) and en bloc stained with 0.5% uranyl acetate (30 min). Following dehydration (ethanol), tissues were embedded (Spurt medium, Polysciences, Inc.) and 2-~m sections (thin sections) cut using a glass knife (MT-5000 ultramicrotome, Dupont). Sections were stained (1% toluidine blue) prior to examination for histopathology. Statistical analysis The data were analyzed using one-way analysis of variance and/or Students t-tests. The 0.05 level was used as the criterion for significance. RESULTS
Effects on food and water intake and body weight Food and water intake were markedly decreased by A and its monochlorophenyl derivatives (Table I). At comparable doses, 2-CA produced the most pronounced decreases in food and water intake of the compounds tested. Body weight decreased in all treatment groups (data not shown) in parallel with the decreases in food and water intake. Effects on urine volume and content The effects of A and its monochlorophenyl derivatives on urine volume are presented in Fig. 1. Both 2-CA and 4-CA decreased urine volume on treatment day 1 following a 1.0 mmol/kg dose of the aniline, but urine volume remained depressed on day 2 only in the 2-CA treatment group at
272
TABLE I EFFECT OF ANILINE AND MONOCHLOROANILINE WATER INTAKE a
TREATMENT ON FOOD AND
Treatment (mmol/kg)
W a t e r i n t a k e (ml)
F o o d i n t a k e (g) Day 0
Day 1
Day 2
Day 0
Day 1
Day 2
A 1.0 1.5
19.9 ± 2.1 16.0 ± 0.9
6.2 ± 2.4 b 1.4 ± 0.4 b
13.0 ± 0.7 b 7.6 _+ 2.4 b
32 ± 3 29 ± 1
14 ± 4 b 11 ± 1 b
27 ± 19 ±
1 5
2-CA 0.4 1.0
17.5 ± 0.6 16.4 ± 0.8
1.3 _+ 0.9 b 0.5 ± 0.1 b
5.6 ± 1.6 b 0.3 ± 0.1 b
32 ± 2 29 ± 2
5 ± 2b 6 ± 1b
19 ± 6 ±
3b 1b
342A 1.0 1.5
16.5 +_ 0.2 19.7 ± 1.1
1.2 ± 0.5 b 0.2 ± 0.1 b
8.8 ± 0.8 b 0 . 6 ± 0,3 b
33 _'!-1 33 ± 2
23 ± 2 b 13 ± 7
15 _+ I b 18 ± 6
442A 1.0 1.5
19.2 ± 1.1 18.6 ± 0.8
0.7 ± 0.1 b 0b
5.5 ± 0.4 b 6.4 ± 0.2 b
38 + 1 36 ± 2
13 ± 1 b 0b
18 ± 3 b 14 ± 10
aValues are m e a n s _+ S.E. f o r at least 4 r a t s / g r o u p . b S i g n i f i c a n t l y d i f f e r e n t f r o m d a y 0, P < 0.05.
this dose. Urine volume also was decreased by administration of A, 3-CA and 4-CA at a dose of 1.5 mmol/kg with 4-CA producing the greatest decline on day 1. The major urinary c o n t e n t abnormalities produced by administration of an aniline were hematuria and increased proteinuria. Administration of 2-CA {1.0 mmol/kg) produced hematuria ( + + + ) and an enhanced proteinuria ( + + ) , which were maintained on day 2 (Table II). These abnormalities were more pronounced and of longer duration than those produced by the other c o m p o u n d s studied. Hematuria ( + + + ) also was the most prominent urinary finding following 442A treatment (day 2). Although 3-CA (1.0 mmol/kg) administration produced hematuria {++) on both post-treatment days, this finding was not as marked following treatment with 1.5 mmol/kg 342A. Glucosuria {trace) was seen only in the 3-CA treatment groups on day 1. Ketonuria was not noted in any group studied. Effects on BUN concentration
The BUN concentration was increased by 2-CA {1.0 mmol/kg) from a pretreatment value of 21.7 + 1.4 to 72.2 + 16.0 mg/dl at 48 h (Fig. 2). Higher doses of 3-CA and 4 ~ A were required to significantly elevate the BUN concentration to 93.4 + 0.4 and 171.6 -+ 18.5 mg/dl, respectively. BUN concentration was not altered following administration of either dose of
273
!1:
A
2-CA
t5
~5 o o
I
TIME(days)
2
TIME(days)
3-CA
IS_
0
is
4-CA
A ~10-
~ 10-
5-
o'
i
2
o
0
!
2
TIME(days) Tim(days) Fig. 1• Effect of aniline (A) and its monochlorophenyl derivatives (2-, 3- and 4-CA) on urine volume. Rats were housed singly, and some rats received a single i.p. injection of an aniline (u, 0.4 mmol/kg; o, 1.0 mmol/kg; A, 1.5 mmol/kg). Other groups (open symbols) received vehicle only and were pair-fed for their appropriate treated group. An asterisk indicates significantly different from 0 h (P < 0.05). A star indicates significantly different from the appropriate control group (P < 0.05).
A studied. T h u s , 2-CA is m o r e p o t e n t in elevating t h e B U N c o n c e n t r a t i o n t h a n t h e o t h e r c o m p o u n d s studied•
Effects on organic ion accumulation Lactate~stimulated P A H a c c u m u l a t i o n and T E A a c c u m u l a t i o n w e r e r e d u c e d b y 2~:~A (0.4 m m o l / k g ) at 48 h post-injection (Fig. 3). F u r t h e r r e d u c t i o n s in basal a n d l a c t a t e - s t i m u l a t e d P A H a c c u m u l a t i o n w e r e seen in t h e 2 ~ A (1.0 m m o l / k g ) t r e a t m e n t g r o u p . A d m i n i s t r a t i o n o f 3 ~ A (1.5 m m o l / k g ) also r e s u l t e d in d e c r e a s e d basal and l a c t a t e - s t i m u l a t e d PAH u p t a k e w i t h o u t altered T E A a c c u m u l a t i o n . T r e a t m e n t o f rats w i t h 4-CA (1.5 m m o l ] k g ) resulted in d e c r e a s e d T E A a c c u m u l a t i o n while A (1.0 or 1.5 m m o l / k g ) p r o d u c e d a decrease o n l y in n e t basal PAH u p t a k e . T h e lack o f a dose-related decrease in P A H + lactate u p t a k e f o l l o w i n g 4-CA administ r a t i o n is m o s t p r o b a b l y d u e t o t h e variability o f d a t a in t h e high dose group. T h e effects o f in vitro i n c u b a t i o n o f anilines w i t h renal cortical slices o n organic ion a c c u m u l a t i o n are s h o w n in Fig. 4. All 4 anilines depressed T E A a c c u m u l a t i o n w h e n p r e s e n t at a c o n c e n t r a t i o n o f 10 -3 M in the incub a t i o n m e d i a . L a c t a t e - s t i m u l a t e d P A H a c c u m u l a t i o n was n o t decreased b y 274
TABLE II EFFECT Treatment (mmol/kg)
OF ANILINE TREATMENT
ON URINE CONTENT a
Protein b
Blood
Day 0
Day 1
Day 2
Control c
+
+
+
A 1.0 1.5
+ +
~- + + ÷
+ +
Day 0
Day 1
Day 2
--
trace
trace trace
+ ++ +
++ +
2~A 0.4
trace
+ +
+
-
1.0
trace
~- +
+ +
--
trace +
trace ++
+
--
+
--
+ + trace
+ + --
+ trace
+ d
+ + +
_
+ _d
+++ ++ +
3-CA 1.0
1.5 4-CA 1 .0 1.5
aValues most frequently observed for at least 4 rats/group. b S e m i q u a n t i t a t i v e v a l u e s f o r p r o t e i n a r e : t r a c e --- < 3 0 m g / d l ; + = 3 0 m g / d l ; + + mg/dl. CAll p a i r - f e d c o n t r o l r a t s h a d s i m i l a r v a l u e s s o t h e i r r e s u l t s a r e c o m b i n e d . d N o 6-h u r i n e w a s p r o d u c e d o n d a y 1.
= 100
any test c o m p o u n d , b u t basal PAH accumulation was decreased by 2-CA at incubate concentrations o f 10-4 M or higher.
Effects on morphology Renal morphological effects observed in thick sections (6 #m) were seen primarily in the renal cortex of treated animals. These alterations consisted of random apical sloughing of the brush border from some proximal tubular cells. However, many proximal tubular cells appeared to be unaffected. Distal tubular cells appeared normal. Marked accumulation of erythrocytes was noted in several cortical blood vessels. Although clear functional differences were noted between treated groups, morphological changes were similar regardless of dose or c o m p o u n d tested. Examination of thin sections (2 ~m) of perfused kidneys revealed differences in morphology among the treatment groups. The most severe m o ~ h o l o g i c a l changes were observed in the 2-CA (1.0 mmol/kg) treatment group (Fig. 5) where both proximal and distal tubular cells exhibited marked degenerative changes. Most proximal tubular cells had luminal protuberances and extensive vacuolization which caused clumping of microvilli. Nuclei of these cells were reduced in size and often crenated. Sloughing of some cells 275
1200-
[ ] 0.4umllkl(Ulbol ) [ ] 0.4mul/il(tml~l) [ ] 1.1lid/kl(Ceetml ) " ___~l.luud/.l'l(trelhld) [ ] 1.5nUlill(Cutrol ) • 1.5mmollkl (tllited)
!:2 0 0 ~ 600-
.,
"
i l l
-i
Fig. 2. Effect of aniline (A) and its monochlorophenyl derivatives (2-, 3- and 4-CA) on the blood urea nitrogen (BUN) concentration at 48 h post-injection. Blood samples were obtained from the tail prior to administration of test compounds or vehicle (0 h) and at 48 h post-injection. Data are expressed as the mean percent of the appropriate pretreatment control value (0 h) +- S.E. for 4 rats/group. BUN concentration control values (0 h) were typically between 15.0 and 20.0 rag% (range = 14.2 ± 0.8 to 22.0 ± 1.1). An asterisk indicates significantly different from the 0 h value (P < 0.05). A diamond indicates significantly different from the appropriate control group (P < 0.05).
resulted in lumenal occlusion. Distal tubular cells also exhibited extensive vacuolization, distensions of basal infoldings and swelling. Occlusion of distal tubular segments also was common. Blood vessels and glomeruli appeared normal. Morphological changes produced by 3-CA (1.5 mmol/kg) were similar to but less severe than those produced by 2-CA (1.0 mmol/kg). Treatment with 4 ~ A (1.5 mmol/kg) also produced renal morphological changes (Fig. 6). These changes were characterized by hypertrophic alterations in both proximal and distal tubular cells with lumens frequently occluded. Increased numbers of lysosomal granules were evident in many tubular segments. Treatment with A had little effect on renal morphology. However, in the 1.5 mmol/kg treatment group vacuolization was evident in some proximal tubular cells near blood vessels. Cells furthest from the blood vessels appeared normal. DISCUSSION
The results of this study demonstrate that the decreasing order of nephrotoxic potential for A and its 3 monochlorophenyl derivatives in Fischer 344 rats is: 2-CA > 4 ~ A > 3 ~ A > A. At 1.0 mmol/kg, 2 ~ A was the only aniline which elevated the BUN concentration and decreased both basal and
276
A
lO0
7-
•75-
2-CA
~ 0 . 4 m mol'k| ~ l . O m m e l kg ~ L 5 m tool ~'g
SO ~ 2S-
25
PAH
PAH
- TEA
o
LAdAte
PAH LACTATE
TEA
4-CA
100
3-CA i
~75
PAH
~- Z5
PAH
PAH LACTATE
TEA
eAu
eA+H
TFA
LACTATE
Fig. 3. Effect o f aniline (A) and its m o n o c h l o r o p h e n y l derivatives (2-, 3- and 4-CA) administration o n t h e a c c u m u l a t i o n o f PAH and T E A by renal cortical slices. Rats were killed b y cervical dislocation at 48 h post-injection o f an aniline or vehicle, and kidneys rapidly r e m o v e d for preparation o f renal cortical slices (see Methods for details). Each bar is the m e a n and each vertical line the standard error for 4 rats. Data are expressed as t h e percent o f the m e a n o f the appropriate pair-fed c o n t r o l group. Typical S/M ratios f r o m pair-fed control groups were b e t w e e n 3.0 and 4.0 (range = 2.62 ~ 0.23 to 3.93 + 0.38) for P A H uptake, b e t w e e n 8.0 and 10.0 (range = 7.18 -+ 0.46 to 9.56 ± 0.43) for PAH + lactate u p t a k e and b e t w e e n 16.0 and 20.0 (range = 15.41 + 0.58 to 20.13 +0.57) for T E A uptake. An asterisk indicates significantly different f r o m the appropriate pair-fed c o n t r o l group (P < 0.05).
lactate-stimulated P A H accumulation. 2 ~ A also was the only aniline to depress basal P A H accumulation in the in vitro studies. Higher doses (1.5 mmol/kg) of 4-CA and 3-CA were required to elevate the B U N concentration, while A had no effect on B U N concentration following administration of the 1.5 mmol/kg dose. The decrease in the B U N concentration seen in the pair-fed control groups is not unexpected. Food intake was markedly reduced by the chlorophenyl derivatives to less than 1.5 g on day 1 (Table I). Animals in the low dose groups consumed between 5.0 and 9.0 g on day 2, while rats in the higher dose groups generally ate less than 1.0 g. Marked reduction in food intake has previously been reported to decrease B U N concentration. For example, Traina and Sather [25] reported that fasted Sprague--Dawley rats have decreased B U N concentrations for up to 7 days. Thus, the depressed control B U N concentrations likely are due to decreased food intake.
277
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Fig. 4. Effect of the i n c u b a t i o n of renal cortical slices w i t h various c o n c e n t r a t i o n s of aniline (A) or one o f its m o n o c h l o r o p h e n y l derivatives (2-, 3-, or 4-CA) o n PAH and T E A a c c u m u l a t i o n . Basal (A) and lactate-stimulated (o) PAH and ( o ) T E A a c c u m u l a t i o n were measured. Data are expressed a s t h e m e a n S/M ratio +- S.E. for at least 4 experiments. An asterisk indicates significantly d i f f e r e n t (P < 0.05) f r o m control experiments.
Species sensitivity to the induction of methemoglobin b y aromatic amines varies markedly and is a well k n o w n phenomenon. The cat is the most sensitive species (100%) followed b y man (60%), dog (30%) and the rat (5%) [ 2 ] . Although species sensitivity is variable, the rank order of the methemoglobin formation produced b y a series of amines is similar among species. For example, McLean et al. [26] have reported that in cats the decreasing order of methemoglobin-forming activity of A and its monochlorophenyl derivatives is 4-CA > 2-CA > A ~ 3-CA. Kiese [27] found similar results in dogs. Similar studies with the chloroanilines have not been c o n d u c t e d in the Fisher 344 rat. However, it is probable that a similar rank order of methemoglobin generation would exist in this species. While the magnitude of the formation of methemoglobin within a species is dependent on proportional metabolite generation [2] and methemoglobin reductase activity [ 2 8 ] , it is unlikely that these events would significantly change the rank order of a series of related chemicals. This conclusion is supported by the fact that aniline, acetanilide and acetophenetidin, among other aromatic amines, retain their rank order of methemoglobin-inducing properties in cat, dog, man and rat [ 4 ] . Thus, comparison of the methemoglobin-forming properties of a series of amines should remain similar between species. Since 2-CA was the most nephrotoxic aniline derivative in Fischer 344 rats, it appears that
278
Fig. 5. Renal corticalsections (X 250) from rats treated with either a single intraperitoneal injection of 2-CA (1.0 mmol/kg) (B) or vehicle (0.9% saline, 1.0 ml/kg) (A) and killed 48 h post-treatment. Glomeruli (G), proximal tubules (PT) and distal tubules (DT) are indicated. Note severe vacuolization present in most distal tubules with congestion of m a n y distal tubule lumen (5B, arrows). Tissue was stained with 1% toluidine blue stain.
279
Fig. 6. Renal cortical sections from rats treated with a single intraperitoneal injection of 4-CA (1.5 mmol/kg) (B) o r vehicle (0.9% saline, 1.0 ml/kg) (A) and killed 48 h posttreatment. Glomeruli (G), proximal tubules (PT) and distal tubules (DT) are indicated. Note the damage t o proximal tubules characterized b y the congestion of many lumen and the increased accumulation o f dense lysosomal granules in the cytoplasm (6B). Tissue was stained with 1% toluidine blue stain. Magnifications used were x 250 (A) and × 500
(B).
280
nephrotoxicity does n o t result from tissue hypoxia alone. This conclusion is supported b y the observation that 2 ~ A (> 10 -~ M) reduced PAH accumulation in the in vitro organic ion accumulation studies. Thus, renal toxicity can occur in the absence of hypoxia. However, it is likely that the renal hypoxia which follows administration of an aniline exacerbates the developing renal lesions. Studies are currently underway to clarify this point. The renal effects produced b y administration of some anilines might be due at least in part to a metabolite of extracortical origin. For example, 3-CA (1.5 mmol/kg) elevated the BUN concentration and depressed organic ion accumulation, b u t incubation of renal cortical slices with 3-CA had little effect on PAH or TEA accumulation. Similar results were seen with 2-CA, although basal PAH accumulation was reduced in the in vitro studies by 2-CA bath concentrations of 10 -4 M or greater. Since nephrotoxic aromatic amines are frequently substrates for the cooxidation p a t h w a y of the renal medulla [ 2 8 ] , a toxic species could be generated within the renal medulla rather than in the renal cortex following administration of an aniline. In addition, studies with another halogenated phenyl c o m p o u n d , bromobenzene, have indicated that this chemical produces nephrotoxicity via a metabolite of hepatic rather than renal origin [ 2 9 , 3 0 ] . Therefore, it appears that a metabolite originating from a site other than the renal cortex might contribute to aniline-induced renal effects. However, the lack of decreased organic ion accumulation following incubation of renal cortical slices with an aniline c o m p o u n d could be due to other factors. For example, using in vitro incubation techniques similar to those used in this study, Goldstein et al. [31] showed that cisplatin, a direct-acting nephrotoxin in vivo, had no effect on PAH or TEA accumulation b y renal cortical slices when they used in vitro incubation concentrations of cisplatin similar to renal cortical platinum concentrations obtained in vivo. In addition, incubating kidney slices at 25°C might alter the formation of toxic aniline metabolites which form at 37°C. Further studies are required to more clearly define the role of extracortical or extrarenal metabolites in the nephrotoxicity which aniline compounds exhibit. In summary, the nephrotoxic potential of A and its 3 monochlorophenyl derivatives has been studied in Fischer 344 rats. 2-CA was the most nephrotoxic c o m p o u n d and A was the least nephrotoxic compound. Nephrotoxicity did not follow methemoglobin-producing activity. The relative lack o f direct effects of anilines on organic ion accumulation in the in vitro studies suggests that a metabolite of extracortical or extrarenal origin might be responsible for the in vivo effects o f these c o m p o u n d s on renal function. REFERENCES I U.S. International Trade Commission, Synthetic organic chemicals, U.S. production and sales, 1975. USITC Publication 804, U.S. Government Printing Office, Washington, DC. (1977).
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