Trichloroaniline effects on renal function in vivo and in vitro

Toxicology Letters, 57 (1991) 319-328 @ 1991 Elsevier Science Publishers

319

B.V. 0378-4274/91/$3.50

ADON1~0378427491000914 TOXLET

02596

Trichloroaniline in vitro

effects on renal function in vivo and

Herng-Hsiang Lol, Patrick I. Brown2 and Gary 0. Rankin] Departments qf

‘Pharmacologyand 2Anatomy. Marshall University School of Medicine, Huntington, WV

(U.S.A.) (Received

22 December

1990)

(Accepted

15 February

199 1)

Keq’ words: Anilines;

Halogenated

hydrocarbons;

Kidney

SUMMARY Previous

studies have demonstrated

failure in vivo and altering study was to determine

organic

the nephrotoxic

ine their effects on organic (4 rats/group)

Renal function

2,3,4- and 3,4,5-TCA

effect on renal function nephrotoxic

of 4 trichloroaniline

in vitro.

was monitored

are capable

of inducing

the greatest

in vitro,

acute renal

slices in vitro. The purpose

In the in vivo experiments, intraperitoneally

male Fischer-344

reductions.

but that the isomers

tested are less potent

marked

tetraethylammon-

slices at bath concentrations

These results indicate

rats

(i.p.) or vehicle (sesame

at 24 and 48 h. None of the TCA isomers induced

by renal cortical

of this

(TCA) isomers in vivo and to exam-

In vitro, all TCA isomers were effective in decreasing

accumulation

inducing

by renal cortical

a TCA (0.8 or 1.5 mmol/kg)

renal effects at either time point. ium and p-aminohippurate

potential

ion accumulation

were administered

oil, 2.5 ml/kg).

that mono- and dichloroanilines ion accumulation

of lo-’ M with

that TCA can have a direct nephrotoxicants

than

the

mono- and dichloroanilines.

JNTRODUCTJON

Chlorinated aniline derivatives are commonly used for the manufacture of numerous dyes, drugs, pesticides, herbicides and a wide range of chemical intermediates [l&4]. Human exposure to chloroanilines occurs primarily in the industrial setting, but chloroanilines also are released following biotransformation of parent com-

Address.for correspondence: Gary School of Medicine,

Huntington,

0. Rankin,

Ph.D.,

WV 2575559310,

Department

U.S.A.

of Pharmacology,

Marshall

University

320

pounds in mammals or in the environment moved into the environment from industrial

[5-71. In addition, waste sites [S].

chloroanilines

have

In man, the most common acute toxicities following exposure to chloroanilines are methemoglobinemia and anemia [9,10], while chronic exposure in animals may result in neoplastic

and non-neoplastic

lesions in the spleen [I 1,121. Previous

studies in our

laboratory have demonstrated that mono- and dichloroanilines also induce acute nephrotoxicity in vivo and reduce organic anion and cation accumulation by renal cortical slices in vitro [13-l 51. Nephrotoxicity in vivo was characterized by oliguria, proteinuria, hematuria, decreased kidney weight and p-aminohippurate (PAH) accumulation by renal cortical slices, increased blood urea nitrogen (BUN) concentrations and tubular necrosis. Dichloroanilines (DCA) generally induced renal effects at lower doses in vivo or lower concentrations in vitro than the monochloroanilines (MCA). Also, the MCA possessed greater nephrotoxic potential than aniline, suggesting that increasing the number of chloro-groups on the aniline nucleus could result in aniline derivatives with enhanced nephrotoxic potential. The purpose of this study was to examine the effects of 4 trichloroaniline (TCA) isomers on renal function in vivo and in vitro using Fischer-344 rats as the animal model. Fischer-344 rats were selected because this rat strain is frequently more sensitive to nephrotoxicants than other rat strains [ 16.171. The TCA isomers were studied in vivo to monitor renal effects of the compounds in whole animals and in vitro to determine the nephrotoxic potential of each isomer in the absence of hepatic metabolism and methemoglobinemia-induced hypoxia. The four TCA isomers studied (2,3,4-TCA; 2,4,5-TCA; 2,4,6-TCA; and 3,4,5-TCA) were chosen based on their commerical availability. MATERIALS

AND METHODS

Male Fischer-344 rats (220-275 g) from Hilltop Lab Animals, Inc. (Scottdale, PA) were allowed a l-week acclimatization period and maintained in standard plastic cages until use. After the acclimatization period, rats (4 rats/group) were placed singly in stainless-steel metabolism cages for the in vivo studies and control data obtained as previously described [13]. Following the control day. rats received a single intraperitoneal (i.p.) injection of a TCA isomer (0.8 or 1.5 mmol/kg) or vehicle (sesame oil, 2.5 ml/kg). Urine volume was measured at 24 and 48 h. Urine contents were semiquantitatively analyzed at 6 and 30 h for the presence of protein, glucose and blood using Multistix’K’ (Ames Division, Miles Laboratories, Inc.). Food and water intake and body weight also were measured daily. At 48 h rats were killed by cervical dislocation. The left kidney was removed, renal cortical slices prepared freehand, and the accumulation of “C-labelled PAH and tetraethylammonium (TEA) by these slices determined [13]. The right kidney also was rapidly removed, weighed, cut into quarters and fixed in 10% neutral-buffered formalin solution for histological examination using light microscopy [I 31.

321

Prior to placing

the rats in metabolism

cages and again prior to killing,

sample was taken from the tail of each rat for the determination tration (Kit No. 640, Sigma Chemical Co.) In all in vivo experiments control rats were pair-fed

a blood

of the BUN concen-

to appropriate

treatment

groups to assure that renal effects were chemically-induced and not due to decreased food consumption. In the in vitro experiments, untreated rats were killed by cervical dislocation, both kidneys were removed, and renal cortical slices prepared. The accumulation of PAH and TEA was determined as previously described [13]. TCA solutions were prepared from the appropriate free bases in acetone such that the addition of 30 ~1 of the TCA solution would yield the desired final concentration in the 3 ml of incubation media. TCA or control solutions were added 30 min before the addition of PAH or TEA. Control samples had 30 ~1 of acetone only added to the incubation media. The data from the in vivo experiments were analyzed using one-way analysis of variance and/or Student’s t-tests. Data from the in vitro studies were analyzed using

2.3.4-TCA

DAY.9 POST-TREATYEM

2.4.5-TCA

3.4,5-TCA

DAYSPOST-TREATMENT

Fig. 1. Effect of TCA (treated) as means

DAYS POST-TREATYENT

or vehicle (control)

+ SE for n = 4 rats per group.

corresponding

An asterisk

day 0 value within a group. pair-fed

control

A dagger

administration indicates indicates

on urine volume. significantly

significantly

different different

group value for that day’s measurement.

Data (Pi

are expressed 0.05) from the

from the appropriate

322

one-way analysis of variance followed as the criterion for significance.

by a Dunnett’s

test. The 0.05 level was used

RESULTS

vivo renal ejjects Low-dose (0.8 mmol/kg) TCA treatment did not alter urine volume at 24 h in any treatment group, while 2,4,5-, 2,4,6- and 3,4,5-TCA injection reduced urine volume In

TABLE

I

EFFECT

OF TCA ADMINISTRATION

ON BUN CONCENTRATION

AND

KIDNEY

WEIGHT

AT 48 ha TCA

Dose

isomer

(mmol/kg)

Grouph

BUN concentration

(mgs)

Kidney (g/l00

0h

weight g body wt.)

4X h

___ 2.3.4

2.4. 5

2.4. 6

3,4. 5

rl Values are means h Control

Control

332 I

25*1*

Treated

295 i*

25*3

Control

26&l

23i1*

0.38*0.01

Treated

28k2

23*2*

0.36&0.01

Control

34* I

27+ 1*

0.38~0.01

Treated

32+2

26i

0.38t0.01

I*

Control

2x*3

Treated

x+2

22+1 _ 21*1

Control

24& I

23k I

0.37&0.01

Treated

23*4

75+1

0.36&0.01

0.36&0.01 0.38~0.01

Control

251 I

23&l

0.37*0.01

Treated

23&Z

24k2

0.38*0.01

Control

25+ I

25il

0.36kO.01

Treated

2813

24+2

0.39+0.01t

Control

26,

Treated

24* I

I

zl*l* 24i

0.37jo.01 l’r

0.42kO.Olt

+ SE for n = 4 rats per group

rats received

sesame oil (2.5 ml/kg)

dose indicated. *Significantly different

from 0 h value, P ~0.05.

t Significantly

from appropriate

different

only, while treated

pair-fed

control

rats received

value, P~0.05.

the test compound

at the

323

at 48 h (Fig. 1). High-dose (1.5 mmol/kg) TCA treatment induced mixed responses. 2,3,4-TCA treatment induced diuresis at 24 h and oliguria at 48 h, while 3,4,5-TCA only induced oliguria at 48 h (Fig. 1). Urine contents were not altered by high- or low-dose TCA treatment. BUN concentration was not increased at 48 h in any treatment group, although BUN concentration decreased in several groups (Table I). Kidney weight was increased at 48 h only in the 3,4,5-TCA (0.8 and 1.5 mmol/kg) groups and decreased in the 2,3,4-TCA (1.5 mmol/kg) group (Table I). In addition, few effects were induced by TCA on organic ion accumulation (Fig. 2). Basal and lactate-stimulated PAH accumulation were increased following 2,4,5- and 3,4,5-TCA (1.5 mmol/kg), while TEA uptake was decreased in the 2,4,5-TCA (0.8 mmol/kg), 2,4,6-TCA (1.5 mmol/kg) and 3,4,5-TCA (1.5 mmol/kg) treatment groups. None of the TCA isomers induced significant changes in renal morphology at 48 h with the doses tested. The results described above indicate that few renal effects are noted at 48 h following TCA administration at dose levels of 0.8 or 1.5 mmol/kg. To ascertain if renal

2,4,6GTCA

2.3,4-TCA

PAH

PAH + LACTATE

:m PAH

TEA

TEA

PAH + L4CTATE

slices at 48 h. Data significantly

Control

0 8 mmol/kg. I 5 mmol/kg. 1 5 mmol/kg.

Treated Control Treated

3,4,5STCA

Fig. 2. Effect of TCA (treated) cortical

0 6 rnrnol/kg,

m I [xl

PAH + LACTATE

2,4.5-TCA

PAH

0

or vehicle (control)

are expressed

different

TEA

PAH + LACTATE

as means

administration

on organic

_+ SE for n=4

(P < 0.05) from the appropriate

ion accumulation

rats per group.

pair-fed

control

A dagger

group value.

by renal indicates

324

toxicity Groups

had occurred earlier than 48 h, a second set of experiments was conducted. of rats were treated as described earlier, but only the 1.5 mmol/kg dose of

a TCA

isomer

changes

in kidney weight, BUN concentrations,

morphology.

or vehicle

was administered

In these animals,

no changes

and rats were evaluated organic in renal

at 24 h for

ion accumulation

function

and renal

or morphology

were

noted in the 2,3,4-, 2,4,5- or 2,4,6-TCA treatment groups (data not shown). The only changes in renal function noted were in the 3,4,5-TCA treatment group where lactate-stimulated PAH accumulation was increased (14.2 f 0.5, treated; 12.2 + 0.7, control) and TEA uptake was decreased (21.6 f 0.2, treated; 24.8 f 0.8, control). No change in BUN concentration or kidney weight was observed in the 3,4,5-TCA treatment group. In vitro @ects on PAH and TEA accumulation All four isomers markedly reduced basal and lactate-stimulated PAH and TEA accumulation at a TCA bath concentration of 10-s M with the 2,3,4- and 3,4,5-TCA isomers inducing the greatest reductions in uptake (Fig. 3). In addition, lactate-stimulated PAH accumulation was reduced by 2,4,5-TCA at a bath concentration of IO-”

MOLAR CONCENTRATION

MOLAR CONCENTRATION

MOLAR CONCENTRATION

MOLAR CONCENTRATION

Fig. 3. Effect of incubating TEA accumulation. slice to medium

TCA with renal cortical

Data are expressed (S/M) ratio.

A diamond

as means

slices from untreated

Fischer-344

* S.E. for at least 4 experiments

indicates significantly control value.

different

(P
rats on PAH and

and expressed

as the

from the appropriate

325

M, and TEA uptake

was reduced

by 3,4,5-TCA

at a bath concentration

of 10e6 M

or greater. DISCUSSION

Previous studies from our laboratory have demonstrated that MCA and DCA were capable of inducing acute nephrotoxicity in male Fischer-344 rats [13-151. In those studies, nephrotoxicity was evident at lower doses with nephrotoxicant DCA (0.8 mmol/kg or higher) than with aniline or its MCA isomers (1.0 mmol/kg or higher). In addition, several investigators have reported that increasing the number of chloro-groups on aniline from O-3 resulted in an increase in lethality or toxicity as the number of chloro-groups increased [16-181. These results might suggest that increasing the number of chloro-groups from 2 on the DCA to 3 on the TCA would also increase the nephrotoxic potential of this group of compounds. However, the results of the present study demonstrate that the TCA isomers tested in vivo in Fischer-344 rats do not exhibit an enhanced nephrotoxic potential when compared to DCA, since none of the TCA isomers was a nephrotoxicant at doses of 0.8 mmol/ kg and induced only minor changes in renal function at doses of 1.5 mmol/kg. The lack of nephrotoxic potential of the TCA as compared to the DCA could be due to a number of factors. One possible explanation for the lack of nephrotoxicity induced by the TCA might be that the addition of a third chloro-group retards formation of phenolic metabolites. Previous studies have demonstrated that p-aminophenol, a metabolite of aniline [19] and acetaminophen [20], is a nephrotoxicant in rats [21-231. However, Newton et al. also showed that or?ho- and meta-aminophenol were not nephrotoxicants in Fischer-344 rats [20]. The 4 TCA isomers examined in this study all contain a chloro-group in the para-position which would retard formation of potentially nephrotoxic para-aminophenol derivatives but, with the exception of 2,4,6-TCA, allow the formation of ordzoor meta-aminophenol metabolites. Therefore, one explanation for the lack of nephrotoxicity induced by the TCA isomers studied would be the inability of these compounds to be biotransformed to 4-aminophenol metabolites. A second possible explanation for the lack of nephrotoxic potential of the 4 TCA isomers might be related to the chemical form in which the TCA isomers were administered. In our previous studies [13-l 51 aniline, MCA and DCA isomers were administered as hydrochloride salts. The lack of basicity exhibited by the amino groups of the TCA isomers hampered efforts to prepare hydrochloride salts of these compounds and, as a consequence, the TCA isomers were administered in their free base form. Under these conditions, rats would not be exposed to the release of millimolar amounts of hydrochloric acid which occurs with MCA or DCA hydrochloride salts. This acid might be expected to potentiate the hemoglobin-induced acute renal failure associated with exposure to anilines, since hemoglobin alone does not seem to induce acute renal failure but requires acidosis, dehydration, shock or other conditions

326

which could lead to a reduction in renal blood flow [24]. Under these additional conditions, the hemoglobin released by aniline-induced hemolysis [25] could both exert a direct toxic effect on the tubules and alter renal hemodynamics [24]. These observations suggest that since acid would be expected to potentiate aniline-induced nephrotoxicity, exposure to TCA in the absence of added acid might not result in renal toxicity. Studies are currently underway to define more clearly the role of acid in the nephrotoxicity observed following intraperitoneal administration of hydrochloride salts of nephrotoxicants. In vitro, the DCA [ 151 and TCA isomers decrease organic ion transport at similar concentrations and exhibit similar structure-activity relationships. For example, among the DCA isomers, 3,4- and 3,5-DCA were the most potent isomers for decreasing TEA and basal and lactate-stimulated PAH accumulation. In the present study, the two isomers capable of inducing the largest decreases in organic ion accumulation contained the 3,4- and/or 3,5-dichloro substitution (2,3,4- and 3,4,5-TCA) (Fig. 3). In addition, the least effective DCA isomer at decreasing organic ion accumulation was 2,6-DCA [15], while the least effective TCA isomer was 2.4,6-TCA. Therefore, these results suggest that, like the DCA, the TCA have direct effects on renal function. It is also likely that in vitro aniline-compound-induced effects on organic ion accumulation represents a toxic response, since recent evidence has shown that 3,4- and 3,5-DCA, the most effective DCA isomers as inhibitors of organic ion accumulation in vitro, also markedly inhibit gluconeogenesis in vitro at concentrations capable of reducing organic ion transport (unpublished data). In summary, the 4 TCA isomers tested induced only minor renal effects in vivo. However, all TCA isomers were capable of reducing organic ion accumulation in vitro. These results demonstrate that addition of a third chloro-group does not enhance the nephrotoxic potential observed with the DCA isomers. The lack of nephrotoxic potential observed with the TCA isomers tested compared to the DCA isomers could be due to a lack of biotransformation of TCA to p-aminophenolic metabolites and/or the administration of the TCA in the free base form. ACKNOWLEDGEMENTS

This study was supported would like to thank

by NIH Grants

DK 31210 and ES 04954. The authors

Joyce Miner for her excellent

ryla for her help in the preparation

technical

assistance

and Darla Ku-

of this manuscript.

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