Toxicology, 75 (1992) 121-131 Elsevier Scientific Publishers Ireland Ltd.
121
Acute renal and hepatic toxicity of 2-haloanilines in Fischer 344 rats M o n i c a A. V a l e n t o v i c a, J o h n G. Ball a, D i a n n e K. A n e s t i s a, Kelly W. Beers a, Elio M a d a n b, J o h n L. H u b b a r d c a n d G a r y O. R a n k i n a Departments of aPharmacology and bpathology, Marshall University School of Medicine and CDepartment of Chemistry, Marshall University, Huntington, West Virginia 25755-9310 (USA) (Received January 23rd, 1992; accepted July 6th, 1992)
Summary Aniline and its halogenated derivatives are widely used as chemical intermediates. The purpose of this study was to determine the hepatotoxic and nephrotoxic potential of the 2-haloanilines. Male Fischer 344 rats (n >_ 4) were injected (i.p.) with 1.0 or 1.25 mmol/kg of: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-CIA), 2-bromoaniline (2-BrA), 2-iodoaniline (2-IA) or vehicle (0.9% saline, 2.5 ml/kg). All compounds were injected as hydrochloride salts. Renal and hepatic function was monitored 24 h after treatment. All of the 2-haloanilines induced oliguria, diminished kidney weight, tubular casts and decreased renal cortical slice accumulation of organic anions. Blood urea nitrogen (BUN) levels were increased (P < 0.05) by treatment with 1.0 or 1.25 mmol/kg of 2-FA, 2-CIA or 2-BrA. Hepatic alterations were also observed and characterized by elevated plasma ALT/GPT activity and altered morphology in the centrilobular region. The nephrotoxic and hepatotoxic potentials were similar among the 2-haloanilines but aniline was less toxic than its 2-halo derivatives. These results demonstrated that halogen substitution at the 2-position of aniline increased hepatic and renal toxicity. However, the severity of toxicity was not influenced by the nature of the halogen substituent.
Key words: Anilines; Halogenated hydrocarbons; Nephrotoxicity; Hepatotoxicity; Rats
Introduction Aniline (aminobenzene) serves as the parent compound for a common group of chemical intermediates, the halogenated anilines. Haloanilines are widely used in the manufacture of antioxidants, dyes, pharmaceuticals, agricultural chemicals and many products [1-3]. Previous studies from our laboratory have demonstrated that intraperitoneal (i.p.) administration of ring chlorinated derivatives of aniline can induce acute nephrotoxicity characterized by oliguria, increased proteinuria, hematuria, decreased kidney Correspondence to." Monica A. Valentovic, Department of Pharmacology, Marshall University School of Medicine, Huntington, WV 25755-9310, USA. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
122 weight and p-aminohippurate (PAH) accumulation by renal cortical slices, increased blood urea nitrogen (BUN) concentration and proximal tubular cell damage [4-61. Among the three monochloroaniline isomers, 2-chloroaniline (2-C1A) proved to be the most potent nephrotoxicant [4], while among the six dichloroaniline isomers, 3,5-dichloroaniline (3,5-DCA) was the most potent nephrotoxicant [6]. Structure-nephrotoxicity relationship studies conducted on mono- or dihalophenyl derivatives of N-phenylsuccinimide, an antifungal derivative of aniline [3], demonstrated that the nature of the halogen atom could influence the nephrotoxic potential of the resulting succinimide [7,8]. In addition, there are numerous reports which indicate that the nature of the halogen atom(s) on an aniline derivative can have a marked influence on the resulting toxicity or therapeutic efficacy of the compound [9-12]. Therefore, the position and nature of halogen atoms on an aniline derivative can influence the biological activity of the compound. The purpose of this study was to examine the importance of halogen type for the renal and hepatic toxicity induced by the 2-haloanilines. These studies were part of our ongoing studies on the structure-toxicity relationships among the halogenated anilines. The 2-haloaniline compounds were selected because we had previously demonstrated that among the monochloroanilines, 2-chloroaniline (2-C1A) was the most potent nephrotoxicant [4]. Toxicity was monitored 24 h after administration of the designated compounds since alterations in renal function were comparable between previously published work [4] conducted at 48 h and preliminary work conducted 24 h after administration of 2-haloaniline. Materials and methods
Mater&Is Anilines were purchased from Aldrich Chemical Co. (Milwaukee, WI) in the highest purity available and converted to hydrochloride salts using etherealhydrogen chloride. All aniline hydrochloride salts were dissolved in 0.9% saline for injection. [14C]-p-aminohippurate (PAH) and [14C]tetraethylammonium (TEA) were obtained from New England Nuclear (Wilmington, DE). ScintiVerse BD scintillation cocktail was purchased from Fisher Scientific (Pittsburgh, PA). All other reagents were of the highest commercial grade obtainable. Animals Male Fischer 344 rats (185-260 g) were obtained from Hilltop Laboratory Animals (Scottsdale, PA) and were housed at a constant ambient temperature (21-23°C) and light cycle (lights on 06:00-18:00 h). The animals were provided food and water ad libitum during a minimum of a 5-day acclimation period prior to initiation of any experiments. Aniline administration The animals were randomly divided into treated and control groups (at least 4 rats per group) and individually housed in stainless steel metabolism cages in order to separate urine and feces. Rats were permitted free access to food and water during an initial 24-h acclimation period. Baseline values for body weight, food and water
123 intake, urine output and urine contents were obtained during a 24-h period running from 09:00 h to 09:00 h the following morning. All data collected during this 24-h period were designated day 0. Food was withheld between 09:00-13:00 h in order to provide urine free of food contamination. Urine was collected on ice, centrifuged and stored at -10°C until assayed for protein and/or glucose. Rats were injected (i.p.) with vehicle (0.9% saline, 2.5 ml/kg) or a hydrochloride salt of aniline or 2-haloaniline (1.0 or 1.25 mmol/kg). The vehicle group was pair fed (PFC) to treated animals in order to eliminate variability in data due to food intake. Urine volume and contents, BUN concentration, alanine aminotransferase (ALT/GPT) activity, liver and kidney weight, histology and renal cortical slice accumulation of organic ions were determined at 24 h post-treatment.
BUN concentration and A L T / G P T activity determinations Rats were anesthetized with diethyl ether and blood was collected into heparinized syringes from the abdominal aorta. The plasma was stored at -10°C. BUN concentration was quantitated using a urease assay (Sigma, no. 640) while ALT/GPT activity was quantitated using a spectrophotometric enzymatic assay (Sigma, no. 505-P) and expressed as Sigma Ferkel units/ml. Organic ion accumulation The left kidney was excised, decapsulated and placed in 3 ml cold Krebs-Ringer buffer (pH 7.4). Renal cortical slices were prepared freehand and the accumulation of PAH and TEA was quantitated as outlined previously [5]. Briefly renal cortical slices (70-100 mg) were equilibrated in 3 ml Krebs-Ringer for 15 min at 25°C in a 100% oxygen atmosphere with constant shaking. The tissue was further incubated for 90 min with [IaC]PAH (0.07 mM; specific activity 50 mCi/mmol) or [IaC]TEA (0.01 mM; specific activity 4.8 mCi/mmol). PAH uptake was quantitated in the presence of 0 mM or 10 mM lactate. Renal cortical slice accumulation of PAH and TEA was expressed as the slice to media (S/M) ratio where S denotes radioactivity in dprn/g tissue and M denotes radioactivity in dpm/ml of media.
Histological examination The right kidney was weighed, cut into quarters and placed in 10% neutral buffered formalin solution. Fixed tissues were embedded, sectioned at a maximal thickness of 6 ~m and stained with hematoxylin and eosin (H&E) prior to examination by light microscopy.
Statistical analysis Values were reported as mean ± S.E.M. and all groups contained a minimum of 4 different animals. Differences within groups were quantitated using an analysis of variance (ANOVA) test followed by a Dunnett's test at a 95% confidence interval. Differences between treated and PFC groups were determined using an ANOVA test followed by a Newman-Keuls test.
124
Results
Renal toxicity of hydrochloride salts of aniline and 2-haloanilines Renal
function
was
monitored
by
quantitating
functional
and
biochemical
c h a n g e s i n t h e k i d n e y . U r i n e o u t p u t ( T a b l e I) w a s d e c r e a s e d w i t h i n 24 h o f t h e a c u t e administration
of the 2-haloaniline
compounds.
relative to the PFC group, 24 h after treatment
BUN
concentration
was elevated,
with 2-FA, 2-C1A or 2-BrA (Table
II). D i f f e r e n c e s w i t h i n a g r o u p b e t w e e n d a y 0 a n d d a y 1 B U N c o n c e n t r a t i o n w e r e observed in animals treated with the low dose of 2-BrA or 2-FA. Aniline hydrochloride
administration
did not alter BUN concentration
weight (Table III). Administration
( T a b l e II) o r k i d n e y
o f 2 - I A (1.0 o r 1.25 m m o l / k g )
failed to increase
TABLE I 24 h-URINE OUTPUT F O L O W l N G 2-HALOANILINE ADMINISTRATION a Compound
Dose (mmol/kg)
Group
Urine output (ml/24 h) Day 0
Day 1
Aniline
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11 11 10 8
± ± ± ±
1 1 1 1
12 ± 1 8 ± 1b 10 ± 1 8 ± 1
2-FA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
10 9 12 12
± ± ± ±
1 1 1 1
14 8 12 9
± ± ± ±
2 1b,c 2 1b
2-CIA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
7 9 12 11
± ± ± 4-
2 1 1 1
3 5 13 5
± ± 44-
l 1 1 lb,C
2-BrA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11 10 10 10
± ± ± ±
1 1 1 1
11 6 8 5
± ± 4±
1 1b,c 1 2
2-IA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11 11 9 11
± ± 4±
1 1 1 2
12 4 9 4
4± ± ±
2 1b,c 1 1
aValues represent mean ± S.E.M. for n = 4 rats per group. Treated rats received an aniline hydrochloride at the dose indicated, while pair-fed control (PFC) rats were administered vehicle only. bDenotes statistical difference (P < 0.05) from day 0 value within a group. CDenotes statistical difference (P < 0.05) from appropriate PFC value on day 1.
125
T A B L E II BUN LEVEL FOLLOWING
Compound
2-HALOANILINE
Dose (mmol/kg)
Group
ADMINISTRATION
a
BUN Concentration (mg/dl) Day 0
Day 1
Aniline
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
16.8 21.7 12.9 16.8
4. 444-
1.6 1.5 1.6 1.3
19.6 19.2 10.7 14.9
4444-
1.2 0.5 1.1 0.7
2-FA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
13.5 9.8 17.4 18.1
4. 4. 4±
1.1 0.6 c 3.3 1.I
17.2 33.6 16.1 25.6
4. 444.
1.5 5.0 b,c 0.7 3.3 c
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
14.7 14.8 17.1 20.3
444±
1.4 2.2 0.2 0.4 c
14.0 33.1 14.5 32.8
± 444.
1.3 7.1 c
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
17.1 15.1 18.3 17.9
4444-
0.2 0.6 c 1.1 0.3
14.4 25.9 17.4 32.6
4444-
4.3 b,c 1.7 5.6 c
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11.4 15.9 17.3 20.3
4+ 44-
1.0 1.0 c 2.1 2.8
19.7 27.1 18.0 25.0
4± 44-
2.2 8.2 1.4 2.7
2-C1A
2-BrA
2-IA
1.1 5.7 c
1.1
aValues represent mean 4 - S . E . M . f o r n = 4 r a t s p e r group. Treated rats received an aniline hydrochloride at the dose indicated, while PFC rats were administered vehicle only. bDenotes statistical difference ( P < 0.05) from day 0 value within a group. CDenotes statistical difference ( P < 0.05) from the appropriate PFC value.
BUN concentration (Table II) but kidney weight (Table III) was diminished (P < 0.05) following treatment with 2-IA (1.0 mmol/kg). All 2-haloaniline compounds (1.0 and 1.25 mmol/kg) decreased basal and lactatestimulated PAH uptake by renal cortical slices (Figs. 1 and 2). Treatment with aniline (1.25 mmol/kg) diminished basal and lactate-stimulated PAH accumulation by renal cortical slices. Renal cortical slice accumulation of TEA was diminished by acute exposure to 1.25 mmol/kg of aniline or 2-CIA hydrochloride salts (Fig. 3). Morphological changes in kidney were minor and observed as interstitial perivascular edema and the presence of casts in some proximal tubules. Despite the presence of hematuria, the bladder integrity remained intact following 2-haloaniline injection. Observed alterations in the bladder included the presence of areas of submucosal hemorrhage and slight edema.
L I V E R A N D KIDNI~,Y WEItJI-I.I 24 n A F I E K / ' - H A L U A I N I L I l N L A I O M I I N I ~ I R A I I O N * Dose (mmol/kg)
Group
Compound Aniline
2-FA
2-CIA
2-BrA
2-IA
4- 4 4- 11 4- 7 + 10"
186 204 245 250
4-2 4- 4 4- 3 + 6
198 211 172 188
206 216 176 187
Body weight (g) 1.0 1.0 1.25 1.25
PFC TR PFC TR
212 231 171 191
444+
1 4 4 1
212 221 243 238
+2 4- 7 4- 3 4- 8
4-6 4- 4 -1- 5 4- 4
PFC TR PFC TR
6.864- 0.14 8.51 4- 0.09* 5.69 4- 0.12 5.90 4- 0.15
7.54 7.08 7.68 7.95
4+ 4+
0.30 0.39 0.35 0.26
6.16 7.05 8.62 8.20
44+ +
0.18 0.22 0.28 0.31
6.52 7.15 5.30 7.00
44± 4-
0.21 0.29 0.20 0.36
6.72 7.14 6.18 6.90
+ 444-
0.31 0.15 0.14 0.20*
3.25 3.68 3.14 3.34
4-4-0.05 4- 0.04* 4- 0.06 4- 0.09
3.34 3.43 3.14 3.34
4-4-0.12 + 0.04 4- 0.06 4- 0.09
3.31 3.45 3.51 3.27
4- 0.06 + 0.05 -4- 0.13 4- 0.05
3.30 3.37 3.09 3.76
+ 444-
0.09 1.07 0.11 0.19"
3.26 3.29 3.52 3.68
+ 44+
0.06* 0.04 0.17 0.06
0.76 0.84 0.72 0.65
4+ 44-
0.01 0.02* 0.02 0.01"
0.77 0.72 0.83 0.78
4- 0.04 4-4-0.02 + 0.03 4- 0.03
0.66 0.67 0.89 0.78
+ 44+
0.02 0.04 0.03 0.03
0.76 0.72 0.65 0.67
+ + 4+
0.04 0.02 0.03 0.04
0.76 0.70 0.71 0.66
+ + 44-
0.04 0.02 0.04 0.02
0.36 0.36 0.34 0.32
± 44+
0.01 0.01 0.01 0.01
0.36 0.33 0.34 0.32
4+ 44-
0.35 0.32 0.36 0.31
+ 4+ 4-
0.01 0.01 0.01 0.01*
0.36 0.34 0.38 0.36
± + 44-
0.01 0.01 0.01 0.01
0.37 0.32 0.40 0.35
4+ 4+
0.01 0.01" 0.02 0.01
Liver weight (g) 1.0 1.0 1.25 1.25
Liver weight (g/lO0 g body weight) 1.0 1.0 1.25 1.25
PFC TR PFC TR
Kidney weight (g) 1.0 1.0 1.25 1.25
PFC TR PFC TR
Kidney weight (g/lO0 g body weight) 1.0 1.0 1.25 1.25
PFC TR PFC TR
0.01 0.01 0.01 0.01
aValues represent m e a n + S.E.M. for n = 4 r a t s p e r g r o u p . T r e a t e d ( T R ) rats received a n aniline h y d r o c h l o r i d e at the d o s e i n d i c a t e d , while p a i r - f e d c o n t r o l
127
PFC; 1.0 rnmol/kg TR;
6~
sj o
I
PAH
1.0 mmol/kg
PFC; 1.25 mmol/kg TR;
1.25 mrnol/kg
4
n,-
3-
:~
4 rats per group. An asterisk indicated different (P < 0.05) from the appropriate PFC group.
[ZZZ] PFC; 1.0 mmol/kg TR; 18
PAH
+
I
LAC
TR;
O -I-< FF
(/3
1.0 mmol/kg
PFC; 1.25 mmol/kg 1,25 mrnol/kg
12
9
6 3
0
A
2-FA
2-CIA
2-BrA
2-1A
Fig. 2. Effect of 2-haloaniline administration on lactate-stimulated PAH accumulation by renal cortical slices 24 h after treatment. The treated (TR) group was injected with a hydrochloride salt of: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-CIA), 2-bromoaniline (2-BrA) or 2-iodoaniline (2-1A) at the designated dose. The pair-fed control (PFC) animals were injected with vehicle. Uptake of PAH was calculated as a ratio of slice to media (S/M) where S denotes dpm/g tissue and M denotes dpm/ml of media. Values were expressed as mean :~ S.E.M. for n ~ 4 rats per group. An asterisk denoted different (P < 0.05) from the appropriate PFC group.
128
I~]
PFC; 1.0 mmol/kg TR;
25 30-
1.0 mmol/kg
PFC; 1.25 rnrnol/kg
TEA
K2ET] TR;
1.25 rnrnol/kg
020 I l l I
~-I~
15 1o
i
A
2-FA
2-CIA
2-BrA
2-1A
Fig. 3. Renal cortical slice accumulation of TEA 24 h after treatment with 2-haloaniline hydrochloride salt. The treated (TR) group was injected with: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-CIA), 2-bromoaniline (2-BrA) or 2-iodoaniline (2-IA). The pair-fed control (PFC) animals were injected with vehicle. Uptake of TEA was calculated as a ratio of slice to media (S/M) where S denotes dpm/g tissue and M denotes dpm/ml of media. Values were expressed as mean + S.E.M. for n _> 4 rats per group. An asterisk indicated different (P < 0.05) from appropriate PFC group.
PFC; 1.0 mmol/kg I~
1.0 mmol/kg
PFC; 1.25 mmol/kg
175-
~g
1501
E
TR; TR;
1.25 mmol/kg
I×
125 ~
×
1001 I--n I.-_J
4 per group. An asterisk indicated different (P < 0.05) from the appropriate PFC group.
129
Hepatic toxicity of hydrochloride salts of aniline and 2-haloanilines Hepatic toxicity was assessed by measuring liver weight, plasma ALT/GPT activity and histologic examination by light microscopy. Liver weight tended to be increased following treatment with a 2-haloaniline but significant increases were detected only in the aniline (1.0 mmol/kg) and 2-BrA (1.25 mmol/kg) treatment groups (Table III). ALT/GPT activity was increased by all 2-haloaniline hydrochloride salts (Fig. 4). However, no differences in ALT/GPT activity were detected in the groups treated with aniline hydrochloride. Livers from treated rats exhibited dose-dependent congestion, hepatic centrilobular degeneration and the presence of reactive nuclei following the administration of all 2-haloanilines (1.0 or 1.25 mmol/kg). However, these effects were localized and minor in nature. Aniline hydrochloride administration did not induce hepatic morphological changes. Discussion
Aniline is an important industrial chemical which serves as the parent compound for the widely-used halogenated anilines [1-3]. Methemoglobinemia [13,14] is the predominant adverse effect associated with human exposure to aniline. Aniline poisoning is also associated with toxicity directed toward the liver and kidney [13-16]. The results of the present study demonstrate that addition of a halogen atom to the 2-position of aniline produces compounds with enhanced hepatotoxic and nephrotoxic potential. The nature of the halogen substituent seemed to be less important for enhancing the toxic potential of the aniline derivative than the presence of a halogen atom. Although 2-C1A produced slightly larger changes in hepatic and renal function than the other 2-haloanilines, these differences were not remarkable. This observation would suggest that the electronic nature or steric properties of the halogen substituent were not critical in the toxicity of these compounds. In other words, the electronegative nature was the greatest for the fluoro-group (strongest electron withdrawing group) and the lowest for the iodo-group (weakest electron withdrawing group), yet 2-FA and 2-IA had similar hepatotoxic and nephrotoxic potentials. In addition, the iodo-group has a large atomic radius, while the fluoro-group has the smallest radius and is comparable to a hydrogen substituent. The reason why 2-haloanilines are more toxic to the kidney than aniline is not known but might be related to differences in distribution to the target organs or alterations in biotransformation. McCarthy et al. [17] demonstrated that in Fischer 344 rats the major urinary metabolites of aniline were 4-aminophenyl sulfate (10.0%), 2-aminophenyl sulfate (9.4%) and 4-acetamidophenyl sulfate (71.8%). Substitution of aniline at the 2-position by a halogen group would be expected to reduce formation of ortho phenolic metabolites by having a halogen group occupying one of the ortho positions (2- or 6- positions) and reduce acetylation of the amino group via steric hindrance. Although the metabolic pathway for each of the 2-haloanilines has not been identified, metabolites identified following 2-FA administration included 4-amino-3-fluorophenol (29% of urinary radioactivity) and 4-acetamido-3-fluorophenol (50% of urinary radioactivity) and their respective conjugates [18]. No 2-amino-3-fluorophenolic metabolites were detected. These results
130
demonstrate an increase in 4-hydroxylation and a decrease in ortho hydroxylation and N-acetylation from the values obtained with aniline [17]. The mechanism has yet to be identified for 2-haloaniline-induced hepatotoxicity. The findings that hepatic changes occurred predominantly in the centrilobular region suggested that hepatotoxicity might be partially attributed to a cytochrome P-450 generated metabolite. Cytochrome P-450 enzyme activities in the liver tend to be present in greatest concentrations near the central vein and lowest near the periportal sites. Other indirect evidence of the possible involvement of a metabolite can be derived from the metabolism of aniline. The major urinary metabolite of aniline has been identified as 4-acetamidophenol, better known as acetaminophen [17]. The hepatic toxicity of acetaminophen has been characterized as centrilobular [19,20]. Acetaminophen is further biotransformed by hepatic microsomal enzymes to the ultimate hepatotoxicant species N-acetyl-4-benzoquinoneimine [19]. In summary, 2-haloanilines are more potent nephrotoxicants and hepatotoxicants than aniline. However, the hepatotoxic and nephrotoxic potentials for all 2haloanilines were similar. These conclusions were based on the greater increase in BUN concentration, alterations in renal cortical slice accumulation of organic ions and elevated ALT/GPT activity. The mechanism(s) by which the 2-haloanilines induce liver and kidney damage is unknown.
Acknowledgments The authors would like to thank Darla Kuryla for her excellent assistance in the preparation of the manuscript and Beverley Pofahl for her excellent technical assistance. This work was supported by NIH grant ES04954.
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121
Acute renal and hepatic toxicity of 2-haloanilines in Fischer 344 rats M o n i c a A. V a l e n t o v i c a, J o h n G. Ball a, D i a n n e K. A n e s t i s a, Kelly W. Beers a, Elio M a d a n b, J o h n L. H u b b a r d c a n d G a r y O. R a n k i n a Departments of aPharmacology and bpathology, Marshall University School of Medicine and CDepartment of Chemistry, Marshall University, Huntington, West Virginia 25755-9310 (USA) (Received January 23rd, 1992; accepted July 6th, 1992)
Summary Aniline and its halogenated derivatives are widely used as chemical intermediates. The purpose of this study was to determine the hepatotoxic and nephrotoxic potential of the 2-haloanilines. Male Fischer 344 rats (n >_ 4) were injected (i.p.) with 1.0 or 1.25 mmol/kg of: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-CIA), 2-bromoaniline (2-BrA), 2-iodoaniline (2-IA) or vehicle (0.9% saline, 2.5 ml/kg). All compounds were injected as hydrochloride salts. Renal and hepatic function was monitored 24 h after treatment. All of the 2-haloanilines induced oliguria, diminished kidney weight, tubular casts and decreased renal cortical slice accumulation of organic anions. Blood urea nitrogen (BUN) levels were increased (P < 0.05) by treatment with 1.0 or 1.25 mmol/kg of 2-FA, 2-CIA or 2-BrA. Hepatic alterations were also observed and characterized by elevated plasma ALT/GPT activity and altered morphology in the centrilobular region. The nephrotoxic and hepatotoxic potentials were similar among the 2-haloanilines but aniline was less toxic than its 2-halo derivatives. These results demonstrated that halogen substitution at the 2-position of aniline increased hepatic and renal toxicity. However, the severity of toxicity was not influenced by the nature of the halogen substituent.
Key words: Anilines; Halogenated hydrocarbons; Nephrotoxicity; Hepatotoxicity; Rats
Introduction Aniline (aminobenzene) serves as the parent compound for a common group of chemical intermediates, the halogenated anilines. Haloanilines are widely used in the manufacture of antioxidants, dyes, pharmaceuticals, agricultural chemicals and many products [1-3]. Previous studies from our laboratory have demonstrated that intraperitoneal (i.p.) administration of ring chlorinated derivatives of aniline can induce acute nephrotoxicity characterized by oliguria, increased proteinuria, hematuria, decreased kidney Correspondence to." Monica A. Valentovic, Department of Pharmacology, Marshall University School of Medicine, Huntington, WV 25755-9310, USA. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
122 weight and p-aminohippurate (PAH) accumulation by renal cortical slices, increased blood urea nitrogen (BUN) concentration and proximal tubular cell damage [4-61. Among the three monochloroaniline isomers, 2-chloroaniline (2-C1A) proved to be the most potent nephrotoxicant [4], while among the six dichloroaniline isomers, 3,5-dichloroaniline (3,5-DCA) was the most potent nephrotoxicant [6]. Structure-nephrotoxicity relationship studies conducted on mono- or dihalophenyl derivatives of N-phenylsuccinimide, an antifungal derivative of aniline [3], demonstrated that the nature of the halogen atom could influence the nephrotoxic potential of the resulting succinimide [7,8]. In addition, there are numerous reports which indicate that the nature of the halogen atom(s) on an aniline derivative can have a marked influence on the resulting toxicity or therapeutic efficacy of the compound [9-12]. Therefore, the position and nature of halogen atoms on an aniline derivative can influence the biological activity of the compound. The purpose of this study was to examine the importance of halogen type for the renal and hepatic toxicity induced by the 2-haloanilines. These studies were part of our ongoing studies on the structure-toxicity relationships among the halogenated anilines. The 2-haloaniline compounds were selected because we had previously demonstrated that among the monochloroanilines, 2-chloroaniline (2-C1A) was the most potent nephrotoxicant [4]. Toxicity was monitored 24 h after administration of the designated compounds since alterations in renal function were comparable between previously published work [4] conducted at 48 h and preliminary work conducted 24 h after administration of 2-haloaniline. Materials and methods
Mater&Is Anilines were purchased from Aldrich Chemical Co. (Milwaukee, WI) in the highest purity available and converted to hydrochloride salts using etherealhydrogen chloride. All aniline hydrochloride salts were dissolved in 0.9% saline for injection. [14C]-p-aminohippurate (PAH) and [14C]tetraethylammonium (TEA) were obtained from New England Nuclear (Wilmington, DE). ScintiVerse BD scintillation cocktail was purchased from Fisher Scientific (Pittsburgh, PA). All other reagents were of the highest commercial grade obtainable. Animals Male Fischer 344 rats (185-260 g) were obtained from Hilltop Laboratory Animals (Scottsdale, PA) and were housed at a constant ambient temperature (21-23°C) and light cycle (lights on 06:00-18:00 h). The animals were provided food and water ad libitum during a minimum of a 5-day acclimation period prior to initiation of any experiments. Aniline administration The animals were randomly divided into treated and control groups (at least 4 rats per group) and individually housed in stainless steel metabolism cages in order to separate urine and feces. Rats were permitted free access to food and water during an initial 24-h acclimation period. Baseline values for body weight, food and water
123 intake, urine output and urine contents were obtained during a 24-h period running from 09:00 h to 09:00 h the following morning. All data collected during this 24-h period were designated day 0. Food was withheld between 09:00-13:00 h in order to provide urine free of food contamination. Urine was collected on ice, centrifuged and stored at -10°C until assayed for protein and/or glucose. Rats were injected (i.p.) with vehicle (0.9% saline, 2.5 ml/kg) or a hydrochloride salt of aniline or 2-haloaniline (1.0 or 1.25 mmol/kg). The vehicle group was pair fed (PFC) to treated animals in order to eliminate variability in data due to food intake. Urine volume and contents, BUN concentration, alanine aminotransferase (ALT/GPT) activity, liver and kidney weight, histology and renal cortical slice accumulation of organic ions were determined at 24 h post-treatment.
BUN concentration and A L T / G P T activity determinations Rats were anesthetized with diethyl ether and blood was collected into heparinized syringes from the abdominal aorta. The plasma was stored at -10°C. BUN concentration was quantitated using a urease assay (Sigma, no. 640) while ALT/GPT activity was quantitated using a spectrophotometric enzymatic assay (Sigma, no. 505-P) and expressed as Sigma Ferkel units/ml. Organic ion accumulation The left kidney was excised, decapsulated and placed in 3 ml cold Krebs-Ringer buffer (pH 7.4). Renal cortical slices were prepared freehand and the accumulation of PAH and TEA was quantitated as outlined previously [5]. Briefly renal cortical slices (70-100 mg) were equilibrated in 3 ml Krebs-Ringer for 15 min at 25°C in a 100% oxygen atmosphere with constant shaking. The tissue was further incubated for 90 min with [IaC]PAH (0.07 mM; specific activity 50 mCi/mmol) or [IaC]TEA (0.01 mM; specific activity 4.8 mCi/mmol). PAH uptake was quantitated in the presence of 0 mM or 10 mM lactate. Renal cortical slice accumulation of PAH and TEA was expressed as the slice to media (S/M) ratio where S denotes radioactivity in dprn/g tissue and M denotes radioactivity in dpm/ml of media.
Histological examination The right kidney was weighed, cut into quarters and placed in 10% neutral buffered formalin solution. Fixed tissues were embedded, sectioned at a maximal thickness of 6 ~m and stained with hematoxylin and eosin (H&E) prior to examination by light microscopy.
Statistical analysis Values were reported as mean ± S.E.M. and all groups contained a minimum of 4 different animals. Differences within groups were quantitated using an analysis of variance (ANOVA) test followed by a Dunnett's test at a 95% confidence interval. Differences between treated and PFC groups were determined using an ANOVA test followed by a Newman-Keuls test.
124
Results
Renal toxicity of hydrochloride salts of aniline and 2-haloanilines Renal
function
was
monitored
by
quantitating
functional
and
biochemical
c h a n g e s i n t h e k i d n e y . U r i n e o u t p u t ( T a b l e I) w a s d e c r e a s e d w i t h i n 24 h o f t h e a c u t e administration
of the 2-haloaniline
compounds.
relative to the PFC group, 24 h after treatment
BUN
concentration
was elevated,
with 2-FA, 2-C1A or 2-BrA (Table
II). D i f f e r e n c e s w i t h i n a g r o u p b e t w e e n d a y 0 a n d d a y 1 B U N c o n c e n t r a t i o n w e r e observed in animals treated with the low dose of 2-BrA or 2-FA. Aniline hydrochloride
administration
did not alter BUN concentration
weight (Table III). Administration
( T a b l e II) o r k i d n e y
o f 2 - I A (1.0 o r 1.25 m m o l / k g )
failed to increase
TABLE I 24 h-URINE OUTPUT F O L O W l N G 2-HALOANILINE ADMINISTRATION a Compound
Dose (mmol/kg)
Group
Urine output (ml/24 h) Day 0
Day 1
Aniline
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11 11 10 8
± ± ± ±
1 1 1 1
12 ± 1 8 ± 1b 10 ± 1 8 ± 1
2-FA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
10 9 12 12
± ± ± ±
1 1 1 1
14 8 12 9
± ± ± ±
2 1b,c 2 1b
2-CIA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
7 9 12 11
± ± ± 4-
2 1 1 1
3 5 13 5
± ± 44-
l 1 1 lb,C
2-BrA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11 10 10 10
± ± ± ±
1 1 1 1
11 6 8 5
± ± 4±
1 1b,c 1 2
2-IA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11 11 9 11
± ± 4±
1 1 1 2
12 4 9 4
4± ± ±
2 1b,c 1 1
aValues represent mean ± S.E.M. for n = 4 rats per group. Treated rats received an aniline hydrochloride at the dose indicated, while pair-fed control (PFC) rats were administered vehicle only. bDenotes statistical difference (P < 0.05) from day 0 value within a group. CDenotes statistical difference (P < 0.05) from appropriate PFC value on day 1.
125
T A B L E II BUN LEVEL FOLLOWING
Compound
2-HALOANILINE
Dose (mmol/kg)
Group
ADMINISTRATION
a
BUN Concentration (mg/dl) Day 0
Day 1
Aniline
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
16.8 21.7 12.9 16.8
4. 444-
1.6 1.5 1.6 1.3
19.6 19.2 10.7 14.9
4444-
1.2 0.5 1.1 0.7
2-FA
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
13.5 9.8 17.4 18.1
4. 4. 4±
1.1 0.6 c 3.3 1.I
17.2 33.6 16.1 25.6
4. 444.
1.5 5.0 b,c 0.7 3.3 c
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
14.7 14.8 17.1 20.3
444±
1.4 2.2 0.2 0.4 c
14.0 33.1 14.5 32.8
± 444.
1.3 7.1 c
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
17.1 15.1 18.3 17.9
4444-
0.2 0.6 c 1.1 0.3
14.4 25.9 17.4 32.6
4444-
4.3 b,c 1.7 5.6 c
1.0 1.0 1.25 1.25
PFC Treated PFC Treated
11.4 15.9 17.3 20.3
4+ 44-
1.0 1.0 c 2.1 2.8
19.7 27.1 18.0 25.0
4± 44-
2.2 8.2 1.4 2.7
2-C1A
2-BrA
2-IA
1.1 5.7 c
1.1
aValues represent mean 4 - S . E . M . f o r n = 4 r a t s p e r group. Treated rats received an aniline hydrochloride at the dose indicated, while PFC rats were administered vehicle only. bDenotes statistical difference ( P < 0.05) from day 0 value within a group. CDenotes statistical difference ( P < 0.05) from the appropriate PFC value.
BUN concentration (Table II) but kidney weight (Table III) was diminished (P < 0.05) following treatment with 2-IA (1.0 mmol/kg). All 2-haloaniline compounds (1.0 and 1.25 mmol/kg) decreased basal and lactatestimulated PAH uptake by renal cortical slices (Figs. 1 and 2). Treatment with aniline (1.25 mmol/kg) diminished basal and lactate-stimulated PAH accumulation by renal cortical slices. Renal cortical slice accumulation of TEA was diminished by acute exposure to 1.25 mmol/kg of aniline or 2-CIA hydrochloride salts (Fig. 3). Morphological changes in kidney were minor and observed as interstitial perivascular edema and the presence of casts in some proximal tubules. Despite the presence of hematuria, the bladder integrity remained intact following 2-haloaniline injection. Observed alterations in the bladder included the presence of areas of submucosal hemorrhage and slight edema.
L I V E R A N D KIDNI~,Y WEItJI-I.I 24 n A F I E K / ' - H A L U A I N I L I l N L A I O M I I N I ~ I R A I I O N * Dose (mmol/kg)
Group
Compound Aniline
2-FA
2-CIA
2-BrA
2-IA
4- 4 4- 11 4- 7 + 10"
186 204 245 250
4-2 4- 4 4- 3 + 6
198 211 172 188
206 216 176 187
Body weight (g) 1.0 1.0 1.25 1.25
PFC TR PFC TR
212 231 171 191
444+
1 4 4 1
212 221 243 238
+2 4- 7 4- 3 4- 8
4-6 4- 4 -1- 5 4- 4
PFC TR PFC TR
6.864- 0.14 8.51 4- 0.09* 5.69 4- 0.12 5.90 4- 0.15
7.54 7.08 7.68 7.95
4+ 4+
0.30 0.39 0.35 0.26
6.16 7.05 8.62 8.20
44+ +
0.18 0.22 0.28 0.31
6.52 7.15 5.30 7.00
44± 4-
0.21 0.29 0.20 0.36
6.72 7.14 6.18 6.90
+ 444-
0.31 0.15 0.14 0.20*
3.25 3.68 3.14 3.34
4-4-0.05 4- 0.04* 4- 0.06 4- 0.09
3.34 3.43 3.14 3.34
4-4-0.12 + 0.04 4- 0.06 4- 0.09
3.31 3.45 3.51 3.27
4- 0.06 + 0.05 -4- 0.13 4- 0.05
3.30 3.37 3.09 3.76
+ 444-
0.09 1.07 0.11 0.19"
3.26 3.29 3.52 3.68
+ 44+
0.06* 0.04 0.17 0.06
0.76 0.84 0.72 0.65
4+ 44-
0.01 0.02* 0.02 0.01"
0.77 0.72 0.83 0.78
4- 0.04 4-4-0.02 + 0.03 4- 0.03
0.66 0.67 0.89 0.78
+ 44+
0.02 0.04 0.03 0.03
0.76 0.72 0.65 0.67
+ + 4+
0.04 0.02 0.03 0.04
0.76 0.70 0.71 0.66
+ + 44-
0.04 0.02 0.04 0.02
0.36 0.36 0.34 0.32
± 44+
0.01 0.01 0.01 0.01
0.36 0.33 0.34 0.32
4+ 44-
0.35 0.32 0.36 0.31
+ 4+ 4-
0.01 0.01 0.01 0.01*
0.36 0.34 0.38 0.36
± + 44-
0.01 0.01 0.01 0.01
0.37 0.32 0.40 0.35
4+ 4+
0.01 0.01" 0.02 0.01
Liver weight (g) 1.0 1.0 1.25 1.25
Liver weight (g/lO0 g body weight) 1.0 1.0 1.25 1.25
PFC TR PFC TR
Kidney weight (g) 1.0 1.0 1.25 1.25
PFC TR PFC TR
Kidney weight (g/lO0 g body weight) 1.0 1.0 1.25 1.25
PFC TR PFC TR
0.01 0.01 0.01 0.01
aValues represent m e a n + S.E.M. for n = 4 r a t s p e r g r o u p . T r e a t e d ( T R ) rats received a n aniline h y d r o c h l o r i d e at the d o s e i n d i c a t e d , while p a i r - f e d c o n t r o l
127
PFC; 1.0 rnmol/kg TR;
6~
sj o
I
PAH
1.0 mmol/kg
PFC; 1.25 mmol/kg TR;
1.25 mrnol/kg
4
n,-
3-
:~
4 rats per group. An asterisk indicated different (P < 0.05) from the appropriate PFC group.
[ZZZ] PFC; 1.0 mmol/kg TR; 18
PAH
+
I
LAC
TR;
O -I-< FF
(/3
1.0 mmol/kg
PFC; 1.25 mmol/kg 1,25 mrnol/kg
12
9
6 3
0
A
2-FA
2-CIA
2-BrA
2-1A
Fig. 2. Effect of 2-haloaniline administration on lactate-stimulated PAH accumulation by renal cortical slices 24 h after treatment. The treated (TR) group was injected with a hydrochloride salt of: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-CIA), 2-bromoaniline (2-BrA) or 2-iodoaniline (2-1A) at the designated dose. The pair-fed control (PFC) animals were injected with vehicle. Uptake of PAH was calculated as a ratio of slice to media (S/M) where S denotes dpm/g tissue and M denotes dpm/ml of media. Values were expressed as mean :~ S.E.M. for n ~ 4 rats per group. An asterisk denoted different (P < 0.05) from the appropriate PFC group.
128
I~]
PFC; 1.0 mmol/kg TR;
25 30-
1.0 mmol/kg
PFC; 1.25 rnrnol/kg
TEA
K2ET] TR;
1.25 rnrnol/kg
020 I l l I
~-I~
15 1o
i
A
2-FA
2-CIA
2-BrA
2-1A
Fig. 3. Renal cortical slice accumulation of TEA 24 h after treatment with 2-haloaniline hydrochloride salt. The treated (TR) group was injected with: aniline (A), 2-fluoroaniline (2-FA), 2-chloroaniline (2-CIA), 2-bromoaniline (2-BrA) or 2-iodoaniline (2-IA). The pair-fed control (PFC) animals were injected with vehicle. Uptake of TEA was calculated as a ratio of slice to media (S/M) where S denotes dpm/g tissue and M denotes dpm/ml of media. Values were expressed as mean + S.E.M. for n _> 4 rats per group. An asterisk indicated different (P < 0.05) from appropriate PFC group.
PFC; 1.0 mmol/kg I~
1.0 mmol/kg
PFC; 1.25 mmol/kg
175-
~g
1501
E
TR; TR;
1.25 mmol/kg
I×
125 ~
×
1001 I--n I.-_J
4 per group. An asterisk indicated different (P < 0.05) from the appropriate PFC group.
129
Hepatic toxicity of hydrochloride salts of aniline and 2-haloanilines Hepatic toxicity was assessed by measuring liver weight, plasma ALT/GPT activity and histologic examination by light microscopy. Liver weight tended to be increased following treatment with a 2-haloaniline but significant increases were detected only in the aniline (1.0 mmol/kg) and 2-BrA (1.25 mmol/kg) treatment groups (Table III). ALT/GPT activity was increased by all 2-haloaniline hydrochloride salts (Fig. 4). However, no differences in ALT/GPT activity were detected in the groups treated with aniline hydrochloride. Livers from treated rats exhibited dose-dependent congestion, hepatic centrilobular degeneration and the presence of reactive nuclei following the administration of all 2-haloanilines (1.0 or 1.25 mmol/kg). However, these effects were localized and minor in nature. Aniline hydrochloride administration did not induce hepatic morphological changes. Discussion
Aniline is an important industrial chemical which serves as the parent compound for the widely-used halogenated anilines [1-3]. Methemoglobinemia [13,14] is the predominant adverse effect associated with human exposure to aniline. Aniline poisoning is also associated with toxicity directed toward the liver and kidney [13-16]. The results of the present study demonstrate that addition of a halogen atom to the 2-position of aniline produces compounds with enhanced hepatotoxic and nephrotoxic potential. The nature of the halogen substituent seemed to be less important for enhancing the toxic potential of the aniline derivative than the presence of a halogen atom. Although 2-C1A produced slightly larger changes in hepatic and renal function than the other 2-haloanilines, these differences were not remarkable. This observation would suggest that the electronic nature or steric properties of the halogen substituent were not critical in the toxicity of these compounds. In other words, the electronegative nature was the greatest for the fluoro-group (strongest electron withdrawing group) and the lowest for the iodo-group (weakest electron withdrawing group), yet 2-FA and 2-IA had similar hepatotoxic and nephrotoxic potentials. In addition, the iodo-group has a large atomic radius, while the fluoro-group has the smallest radius and is comparable to a hydrogen substituent. The reason why 2-haloanilines are more toxic to the kidney than aniline is not known but might be related to differences in distribution to the target organs or alterations in biotransformation. McCarthy et al. [17] demonstrated that in Fischer 344 rats the major urinary metabolites of aniline were 4-aminophenyl sulfate (10.0%), 2-aminophenyl sulfate (9.4%) and 4-acetamidophenyl sulfate (71.8%). Substitution of aniline at the 2-position by a halogen group would be expected to reduce formation of ortho phenolic metabolites by having a halogen group occupying one of the ortho positions (2- or 6- positions) and reduce acetylation of the amino group via steric hindrance. Although the metabolic pathway for each of the 2-haloanilines has not been identified, metabolites identified following 2-FA administration included 4-amino-3-fluorophenol (29% of urinary radioactivity) and 4-acetamido-3-fluorophenol (50% of urinary radioactivity) and their respective conjugates [18]. No 2-amino-3-fluorophenolic metabolites were detected. These results
130
demonstrate an increase in 4-hydroxylation and a decrease in ortho hydroxylation and N-acetylation from the values obtained with aniline [17]. The mechanism has yet to be identified for 2-haloaniline-induced hepatotoxicity. The findings that hepatic changes occurred predominantly in the centrilobular region suggested that hepatotoxicity might be partially attributed to a cytochrome P-450 generated metabolite. Cytochrome P-450 enzyme activities in the liver tend to be present in greatest concentrations near the central vein and lowest near the periportal sites. Other indirect evidence of the possible involvement of a metabolite can be derived from the metabolism of aniline. The major urinary metabolite of aniline has been identified as 4-acetamidophenol, better known as acetaminophen [17]. The hepatic toxicity of acetaminophen has been characterized as centrilobular [19,20]. Acetaminophen is further biotransformed by hepatic microsomal enzymes to the ultimate hepatotoxicant species N-acetyl-4-benzoquinoneimine [19]. In summary, 2-haloanilines are more potent nephrotoxicants and hepatotoxicants than aniline. However, the hepatotoxic and nephrotoxic potentials for all 2haloanilines were similar. These conclusions were based on the greater increase in BUN concentration, alterations in renal cortical slice accumulation of organic ions and elevated ALT/GPT activity. The mechanism(s) by which the 2-haloanilines induce liver and kidney damage is unknown.
Acknowledgments The authors would like to thank Darla Kuryla for her excellent assistance in the preparation of the manuscript and Beverley Pofahl for her excellent technical assistance. This work was supported by NIH grant ES04954.
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