32 Congenital Anomalies 2002; 42,32-35

ORIGINAL ARTICLE

Postnatal survival of rat offspring prenatally exposed to pure 2,4-Dichlorophenoxyacetic acid (2,4-D)

Djibril Fofana, Hidehiko Kobae, Kiyoko Samesbima, and Koichiro Miyata Department of Pediatrics, Faculty of Medicine, Kagoshima University, Kagoshima, Japan

ABSTRACT In previous report on prenatal devel- opmental effects of pure 2,4-dichlorophenoxyacetic acid (2,4-D) in rat, we found that this chemical was maternally toxic, embryolethal, and that it induced urogenital mal- formations in the fetuses. In the present report, we inves- tigated the postnatal survival of the offspring prenatally exposed to 2,4-D during organogenesis, to determine the participation of urogenital malformations on postnatal survival. We used doses of 70 mg, 110 mg and 150 mg, which were each found to induce significant urogenital malformations, when administered in different periods of organogenesis: GD 6 to 15, GD 6 to 10, and GD 11 to 15. We found that 2,4-D has a significant influence on prog- eny viability by increasing the postnatal death. The kid- ney and urinary tract malformations induced in the fe- tuses might be the cause of the increased rate of postnatal death. 2,4-D did not impair the postnatal growth of the unaffected offspring.

Key words: 2,4-D, rat offspring, urogenital malformations, postnatal survival

INTRODUCTION A prenatal exposure to a chemical can result in an acute or chronic toxic effect on the offspring. The effects of pesticides exposure during prenatal development may be permanent or reversible. The transgenerational effect can also occur (by mutation, enzyme inhibitions, mitotic interference, etc.) at anytime in an offspring's life (Colbom et al., 1993). It must be important to investigate the postnatal survival of the off- spring exposed prenatally.

The high teratogenicity of the phenoxyacetic acid, the world- wide used herbicides, has been attributed to the derivative

Corresponding Author : Fofana Djibril, M.D., Department of Pediatrics, Fac- ulty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: <fofana@med3.kufm.kagoshima-u.ac.jp>

Received October 18,2001; revised and accepted December 3,2001.

2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and the contami- nant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In the pre- vious report we found that the pure 2,4-D itself was embryotoxic, and induced urogenital malformations in the fetuses when administered to pregnant rats during organogen- esis (Fofana et al., 2001). As pointed out in the prenatal study, many investigators dealing with 2,4-D and its derivatives in the herbicide formulations reported different conclusions due to the test products, the experimental species, the study proto- col and design used. Khera et al. (1 97 1) using the 2,4-D and 2,4,5-T derivatives without dioxin contaminant in the Wistar rat to investigate pre and postnatal effects, reported reduced litters size and weight for some derivatives at 100 mg and 150 mg/kg. Meanwhile, Courtney et al. ( 197 1) reported that these compounds were not teratogenic in the SD rat.

We undertook this study to evaluate the postnatal survival of the rat offspring exposed to pure 2,4-D during gestational period, to investigate the participation of the malformations induced during embryogenesis to their postnatal survival.

MATERIALS AND METHODS We mated female rats with male overnight (12-weeks-old or older Wistar rats, weighing 200 to 300g, from Kyodo Co., Ltd., Kumamoto, Japan); the following day was designated as day 0 of pregnancy. The pregnant rats were individually caged under laboratory conditions with controlled room tem- perature (22 f l0C), humidity (55 f 10%) and lightldark cycle (12/12 hours). This study was carried out in accordance with the Guidelines for Animal Experimentation, Faculty of Medi- cine, Kagoshima University.

2,4-D with 99.9% purity (from Sigma Aldrich Chemical Co., St Louis, MO, USA) was diluted in distilled water and ad- ministered by gavage once a day at 70, 1 10 and 150 mgkg bodyweight. The chemical was administered according to the organogenesis period (Steele et al., 1990). We randomly dis- tributed the pregnant rats among the dose groups into three main groups: Group 1 (gestational Day (GD) 6 to 15), group

33 Terarogenic effect ofpure 2,4-0 in rats

2 (GD6 to 10) for 70 mg and 1 10 mg, and group 3 (GDl1 to 15) for 150 mg. We used 24 pregnant rats, 3 for each dose and in each main group. We adopted control group for each main group. Control groups were given an identical volume of sol- vent (distilled water: lmU100g). Pregnant rats that did not deliver on day 21 were sacrificed 48 hours later to c o n f m the pregnancy failure. The neonates were examined for exter- nal gross defects. We recorded their viability, and weight gain twice a week from delivery until they were sacrificed on day 28 (4 weeks). We kept the offspring with their mothers and without any adjustment throughout the evaluation time. We recorded postnatal deaths by noting the differences in the num- bers of living fetuses twice a week. On day 28, we sacrificed all the living rats, then examined the internal organs under a light dissecting microscope.

The parameters selected for evaluation were the total num- ber of delivered litter sizes, the number of living rats at the end of observation time, the number of postnatal deaths and the weight gain. We compared the above parameters in the 2,4-D-treated groups with those in the control groups. The data were analysed using the Chi-square test and the nonpara- metric t test.

RESULTS We observed three cases of pregnancy failure, one case at 70 mg in group 1, and two cases at 110 mg in groups 1 and 2. The data of the postnatal survival and postnatal death are sum- marized in Table 1.

The proportions of postnatal deaths in the 2,4-D-treated groups were significantly different from those in control in

group 1 (p < O.Ol), group 2 (p < 0.02) and group 3 (p c 0.001). We did not find any significant difference in the proportions of the postnatal deaths among the three main groups. The pro- portions of postnatal deaths were significantly different at 70 mg in group 1 (p < 0.01), and in group 2 (p c 0.05). These proportions were also significantly different at 1 10 mg in group 1 (p c 0.05) and group 2 (p < 0.01). The proportions of the postnatal deaths at 70 mg, when compared to 1 lOmg were sigmfkantly different in neither group 1 nor group 2 (p > 0.05). The occurrence of the postnatal deaths during the 4 weeks is showed in Table 2.

The body weight gain from delivery to weaning, in 2,4-D- treated and in control groups were not significantly different (Table 3). Eye-opening occurred on day 13 to 14 in all the offspring in both the control and 2,CD-treated groups. We did not observe any gross malformations (external or inter- nal) in the surviving rats at 4 weeks in either the 2A-D-treated groups or in the control.

DISCUSSION We used 70 mg, 110 mg and 150 mg of pure 2,4-D in this study which were each found to induce significant urogenital malformations in the rat fetuses when administered to the preg- nant mothers during organogenesis. We have reproduced all that we previously reported about maternal toxicity (maternal weight loss and skeletal myopathy) in this study (Fofana et al., 2001). The pregnancy failures indicated the massive embryolethality caused by 2,4-D, which was confirmed by maternal necropsy.

In all three main groups, the significant differences in the

Table 1 Postnatal survival of rat offspring exposed to pure 2,4-D prenatally

No. of neonates Postnatal death Live rats after Number of Fealure of pregnant rat pregnancy 4weeks (%)

Main groups Doses recorded after within 4 weeks delivery

control 3 0 27 24 (89)

Group 1 (GD 6 to 15)

70mg 3 1 20 10 (50)** 10 (50)

1 lOmn 3 1 21 7 (33)* 14 (67) control 3 0 27 1 (4) 26 (96)

Group 2 (GD 6 to 10) 70mg 3 0 28 7 (25)* 21 (75)

1 lOmg 3 1 15 5 (33)** 10 (67) control 3 0 35 0 35 (loo)

Group 3 (GD11 to 15) 15Omg 3 0 22 6 (27)*** 16 (73)

Positif from Chi-square test: *p < 0.05; **p < 0.01; ***p < 0.001

34 D. Fofana et al.

Table 2 Number of death of offspring from birth to 4th week

Main groups Doses 1st week 2nd week 3rd week 4th week 3 0 0 0

(n = 27) (n = 24) (n = 24) (n = 24) control

6 4 0 0 (n = 10) 70mg Group 1

(GD 6 to 15) (n = 20) (n = 14) (n = 10) 5 2 0 0

(n = 14) 1 lOmg (n = 21) (n = 16) (n = 14)

control 1 (n = 27)

0 (n = 26)

0 (n = 26)

0 (n = 26)

4 3 0 0 Group 2 70mg (GD 6 to 10) (n = 28) (n = 24) (n = 21) (n = 21)

1 lOmg 5 (n = 15)

0 (n = 10)

0 (n = 10)

0 (n = 10)

Group 3 control 0 (n = 35)

0 (n = 35)

0 (n = 35)

0 (n = 35)

4 2 0 0 (n = 22) (n = 18) (n = 16) (n = 16) 150mg (GD11 to 15)

Table 3 Postnatal body weight gain of rat offspring exposed prenatally to pure 2,4-D

Main groups Body weight (g) after delivery 1st week 2nd week 3rd week 4th week Doses

(Mean f SD) (Mean f SD) (Mean f SD) (Mean f SD) (Mean f SD) 6 rt 0.3 13 f 1.0 34 f 2.1 47 f 3.3 75 f 6.0

(n = 27) (n = 24) (n = 24) (n = 24) (n = 24) 32 f 3.0 47 A 5.0 86 f 5.0 6 f 0.2

(n = 20) (n= 14) (n = 10) (n = 10) (n = 10) 5 f 0.2 11 f 1.1 24 i 2.1 45 +- 4.0 84 f 3.0

(n = 21) ( n = 16) (n = 14) (n = 14) (n = 14)

control

15 f 1.2 7omg

Group 1 (GD 6 to 15)

1 lOmg

6 f 1.0 13 f 1.0 27 f 2.1 54 f 6.0 81 f 2.0 (n = 27) (n = 26) (n = 26) (n = 26) (n = 26) 5 f 1.0 12f 1.0 30 i 0.2 50 f 0.2 83 f 1.0

(GD 6 to 10) (n = 28) (n = 24) (n = 21) (n = 21) (n = 21) 5 f 0.0 13 f 0.3 30 f 0.3 52 f 5.0 74 f 5.0

(n= 10) (n = 10) (n = 10) (n = 10) (n = 15) 5 f 0.0 11 f 0.4 22 f 1.0 43 f 1.0 71 f 3.0

Group 3 (n = 35) (n = 35) (n = 35) (n = 35) (n = 35) 6 f 0.2 1 4 f 1.0 33 f 1.0 55 f 1.0 85 ~t 1.3

(n = 22) ( n = 18) (n = 16) (n = 16) ( n = 16)

control

70mg Group 2

1 lomg

control

150mg (GD11 to 15)

35 Teratogenic effect ofpure 2,4-0 in rats

proportions of the postnatal deaths were 2,4-D-related. The fact that there was no significant difference in the proportions of the postnatal deaths in the 2,4-D-treated groups among the three main groups indicated that the occurrence of the fetal malformations was not related to the administration period of the 2,4-D during organogenesis.

Necropsy on the dead neonates has not been attainable be- cause of the difficulty of recovering them. We know that it is common for mother rats to eat suffering or malformed neo- nates. We did not exclude that the mothers could eat some babies just after delivery, but we could not add the number to our count. In spite of these obscure issues, the proportions of postnatal deaths were sufficient enough in the 2,4-D-treated groups comparing to the control. We did not observe any gross malformations in the living rats, in either 2,4-D-treated or control groups at 4 weeks.

The weight of the surviving rats in the 2P-D-treated groups was not significantly different from those in the control, as also reported with the 2,4-D herbicide form and some of its esthers by Khera et al. (1972). This suggests that the postna- tal growth of the prenatally exposed offspring is unrelated to the teratogenic potential of the chemical.

The increased postnatal death can be attributed to the kid- ney and urinary tract malformations induced by 2,4-D in the fetuses as we reported previously (Fofana et al., 2001). Ad- equate elimination of soluble wastes and toxins from the body .through the urinary tract is essential to survival. The obstruc- tive uropathy we described previously in ours cases are sec- ondary manifestation of the direct effect of 2,4-D on the epi- thelial structure, which occurs from the renal pelvis to the tip of the ureters. Urinary tract obstruction results in profound alteration of renal hemodynamics. The evolution of these pro- cesses depend on the duration, the degree and whether or not the obstruction is unilateral or bilateral, as well as on the pro- portion of glomeruli actually functioning.

In experimental animals, the potential for recovery of renal function depends on the duration, the completeness of the obstruction, the extent to which the obstruction has been re- lieved or remains in effect, the glomerular filtration rate (GFR) recovery and the species studied (Yarger, 199 1; Curhan et al., 1994). In dogs subject to one week of complete unilateral ure- teral obstruction, GFR was 25% of normal upon release of the obstruction, with gradual partial restoration of function up to 50% of normal levels at two years after the obstruction (Kerr, 1954). In rat, unilateral obstruction for 7 and 14 days resulted in residual GFR of 17% and 9% of normal, respectively, if the contralateral kidney was left in place, and 31% and 14%, re- spectively, if the contralateral kidney was removed at the time the obstruction was removed (Prevoost et al., 1981; Jaenike, 1972; Tanner, 1979). In the present experiment, all postnatal

deaths took place within the first and second weeks after birth. Conclusion: Administration of pure 2,4-D during organogen- esis to the pregnant rat has a significant influence on the vi- ability of the offspring by inducing significant rate of postna- tal death. The kidney and urinary tract malformations induced on the fetuses by 2,4-D (as we reported previously) might be a probable cause of increased postnatal death. The prenatal exposure to 2,4-D does not impair the growth of the unaf- fected litters postnatally.

ACKNOWLEDGMENTS The authors express their gratitude to Miss Sakaemura M. and Miss Wakimoto N. for their laboratory help.

REFERENCES Colborn T, Svom Sod F, Soto AM (1993) Developmental

effects of Endocrine-disrupting chemicals in wildlife and humans. Environmental Health Perspective, lOl(5): 378- 384.

Courtney KD, More JA (1971) Teratology studies with 2,4,5- Trichloro-phenoxyacetic acid and 2,3,7,8-Tetrachloro- dibenzo-p-dioxin. Toxicol Appl P h a m c o l , 2 0 396-403.

Curhan GC, Mak LZ (1994) Urinary tract obstruction. .In: The Kidney, 5th edn. pp 1936-1957.

Fofana D, Kobae H, Oh S, Nishi JI, Miyata K (2001) Prena- tal Developmental effects of pure 2,4-Dichlorophenoxyace- tic acid (2,4-D) on the rat. Cong Anom, 40: 287-296.

Jaeniike JR (1972) The renal functional defect of postobstructive nephropathy: The effect of bilateral ureteral obstruction in the rat. J Clin Invest, 51: 2999-3006.

Ken Ws Jr (1954) Effect of complete ureteral obstruction for one week on kidney function. JAppl Physiol, 6: 762-772.

Khera KS, McKinley WP (1972) Pre and postnatal studies on 2,4,5-Trichlorophenoxyacetiac acid, 2,4-Dichlorophenoxy- acetic acid and their derivatives in rats. Toxicol Appl

Prevoost Ap, Molenaar (1981) Renal function during and af- ter a temporary Complete unilateral ureteral obstruction in rat. Invest Urol, 18: 242-246.

Steele CE, Copping GP (1990) Teratogen testing. In: Copp AJ and Cockroft DL (eds) Post-implantation Mammalian Embryos, A Partical Approach. pp 22 1-233.

Tanner GA (1979) Effects of kidney tubule obstruction on glomerular function in rats. Am J Physiol, 237: F379-F385.

Yarger WE (1991), Urinary tract obstruction. In: Brenner BM, Rector FC (eds) The Kidney, 4th edn. WB Sauders, Phila- delphia, pp 1768- 1808.

Phamcol , 22: 14-28.