Cong. Anom., 31: 41-45, 1991

Short Communication

Induction of Cardiovascular Malformations by Leupeptin in the Rat

Koichiro MIYATA, Akihiko KODAMA, Shen-Fang CHEN, Toshio TACHIKURA, Shozo OKU, Takatoshi YAMASAKI and Makoto NAKAMURA Department of Pediatrics, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890, Japan

ABSTRACT The teratogenic effects of leupeptin on the rat embryonic heart were exa- mined. Wistar rats were injected with 30 mg/kg of leupeptin, 50 mg/kg of leupeptin, and 2.5 ml/kg of normal saline (control group) on days 9 and 10 of gestation. Term fetal weight was significantly lower in the leupeptin groups than in controls. At leupep- tin 30 mg/kg, the resorption rate, incidence of malformation, and incidence of cardi- ovascular (CV) malformation were 13.2070, 34.8'70, 16.7'70, respectively. At leupeptin 50 mg/kg, the resorption rate, incidence of malformation, and incidence of CV malfor- mation were 19.7%, 81.4070, 36.3'70, respectively. The most common malformations were hydrocephaly, anophthalmia, microphthalmia, VSD, ASD, hydronephrosis, and di- aphragmatic hernia. Multiple organ systems were affected. Malformation types were similar to those induced by anti-kidney antiserum (AKS) and trypan blue (TB). Key words: protease inhibitor, cardiovascular malformation, rat fetus

Leupeptin is an inhibitor of serine and thiol protease groups, and therefore specifically inhibits the lysosomal proteolytic enzymes cathepsin B, H, and L. Its teratogenicity has been reported by Tanaka (Tana- ka, 1982) and Freeman et al. (Freeman and Lloyd, 1983b). In their studies, multiple organ system defects were induced. The most common defects were microphthalmia, anophthalmia, hydrocephalus, and kid- ney malformations. However its induction of CV malformations was not specifically described. In this study, we examined CV malformation induced by leupeptin, and hypothesize that in the rat it inhibits certain reactions of protein metabolism.

MATERIALS AND METHODS

Animals Wistar rats aged 12 weeks were used. They were purchased from Kyudo Company Ltd. (Kumamoto,

Japan) at 5 weeks old and kept in an animal room maintained at 22?2"C. They were given a standard rat chow (CE-2, Clea Japan) and tap water ad libitum. The experiment complied with the Guide for Animal Experimentation of the Faculty of Medicine, Kagoshima University.

42 K . Miyata et al.

Chemical Leupeptin was purchased from Peptide Institute Inc. (Osaka, Japan). For administration, 100 mg of

leupeptin was dissolved in 5 ml of normal saline (NS).

Administration regimen Twenty-seven pregnant rats were divided into three groups. On day 9 of gestation (plug day = day 0),

2.5 ml/kg of NS was given to group Saline, 30 mg/kg of leupeptin to group L (30), and 50 mg/kg of leupeptin to group L (50) by the intraperitoneal injection. Rats were again injected on day 10 with the same dose of NS or leupeptin as on day 9. The amount of NS injected in control group is equal to that of leupeptin solution injected in L (50) group.

Malformations The rats were sacrificed on day 21 of gestation and the number of resorbed and live fetuses recorded.

Fetuses were examined for gross external malformation and weighed. Each fetus was fixed in Bouin's fixa- tive and dissected according to the technique of Barrow and Taylor (Barrow and Taylor, 1969). Statistical comparison of resorption rates and incidence of malformation were made by the chi-square test, and statistical comparison of term fetal weight by the F test and Student's t-test.

RESULTS

Resorption, Fetal Weight, and Malformation Production Table 1 shows the resorption rate, term fetal weight, incidence of malformation, and incidence of C V

malformation in each of the three groups. A significant difference in resorption rate between group Saline and both group L (30) and group L (50) was identified (p<0.05, p<O.Ol, respectively). Term fetal weight significantly decreased when leupeptin dose was increased (p < 0.001). The incidences of malformation and CV malformation significantly increased (p < 0.01) when leupeptin dose was increased.

Types of Malformation Table 2 shows the types, numbers, and incidence (To) of malformation in each group. In group L (30),

Table 1. The effect of leupeptin on rat embryonic development

Number No. of Resorption NO. of Malformation CV malformation Treatment Term fetal weight (g)')

of embryos term litters on day 9 No. (TO) fetuses No. ("lo) No. (070) ( m g / k g 1

Saline 8 97 5.10k0.28 lo2

( 4 '9) 'b i 102 3.95k0.46 Leupeptin (30) 11 Leupeptin (50) 8 127 25 (19.7)

a Significantly different at P<0.01 by xz test b Significantly different at P<0.05 by xZ test c Significantly different at P<O.O01 by F test and Student's t-test I' M e a n 2 S D

152 20 (13.2) a 132 4.52+-0.58Ic

Cardiovascular malformations induced by leupeptin 43

Table 2. Types, numbers and incidence (70) of malformation in each group

Treatment (mg/kg)

Saline Leupeptin (30) Leupeptin (50)

Number of term fetuses Brain anomalies

Exencephaly Hydrocephaly

Eye anomalies Anophthalmia Microphthalmia

Cleft palate Total aortic arch anomalies Heart anomalies

VSD ASD PS ECD TOF Truncus arteriosus

Diaphragmatic hernia Renal anomalies

Hydronephrosis Renal hypoplasia Renal agenesis

Short, kinked tail Omphalocele Maxillar hypoplasia Limb anomalies

99 0 0 0 I (1.0) 0 1 (1.0) 0 0 1 (1.0) 0 0 1 (1.0) 0 0 0 0 0 0 0 0 0 0 0 0

132 13 ( 9.8) 0

13 ( 9.8) 20 (15.2) 16 (12.1) 5 ( 3.8) 1 ( 0.8) 1 ( 0.8)

22 (16.7) 15 (11.4) 5 ( 3.8) 6 ( 4.5) 2 ( 1.5) 1 ( 0.8) 0 3 ( 2.3)

11 ( 8.3) 9 ( 6.8) 1 ( 0.8) 1 ( 0.8) 1 ( 0.8) 0 0 0

1 i02 43 (42.2)* 6 ( 5.9)*

39 (38.2)* 59 (57.8)* 49 (48.0)* 21 (20.6)*

1 ( 1.0) 2 ( 2.0)

37 (36.3)* 24 (23.5)** 14 (13.7)* 11 (10.8)*

1 ( 1.0) 1 ( 1.0) 2 ( 2.0)

35 (34.3)* 11 (10.8)**

25 (24.5)* 7 ( 6.9)** 7 ( 6.9)** 3 ( 2.9) 4 ( 3.9)** 2 ( 2.0) 2 ( 2.0)

Statistical analysis between the leupeptin (30) and leupeptin (50) groups. * P<O.01 ** P<0.05

VSD: ventricular septal defect ASD: atrial septal defect PS : pulmonary stenosis

ECD: endocardia1 cushion defect TOF: tetralogy of Fallot

the incidence of CV malformation was 17.5%, higher than anomalies of the eye, brain, and kidney. The most common heart defects were VSD, PS, and ASD. In group L (50), the incidence of CV malformation was 38.3%, lower than those of eye and brain anomalies, but higher than that of renal anomaly. The most common heart defects were VSD, ASD, and PS. In fetuses with VSD, the incidence of isolated VSD was 61.5070, and 7 fetuses (17.9%) had VSD beneath the pulmonary valve. PS was limited to those without concomitant overiding aorta and dilatation of ventricular infundibulum. In fetuses with truncus arterio- sus, bilateral pulmonary arteries arised from the ascending aorta. 3 fetuses with total aortic arch anoma- lies were all double aortic arch.

44 K . Miyata et al.

DISCUSSION

Teratogens may act before the development of the chorioallantoic placenta (Lloyd, Williams and Beck,

1974). At this time, the embryo depends mainly on histiotrophic nutrition for its growth (Beck, 1981). Through the process of pinocytosis, endodermal cells of the yolk sac incorporate maternally-derived pro- tein into their lysosomes, wherein proteolysis yields amino acids used by the conceptus for protein synthe- sis (Freeman, Beck and Lloyd, 1981; Freeman and Lloyd, 1983a). Yolk sac placental function in the rat is known to be very active on the 8th to about the 12th day (Barrow and Taylor, 1971).

Leupeptin is an inhibitor of serine and thiol protease groups. It is found in culture filtrates of Strep-

tomyces species (Aoyagi, 1978). It specifically inhibits the lysosomal proteolytic enzymes cathepsin B, H, and L, and therefore prevents normal yolk sac function (Beck and Lowy, 1982). In 1983, Freeman et al.

hypothesized that leupeptin exerts its teratogenic action by inhibiting proteolysis in the yolk sac lysosomes, thereby depriving the developing embryo of its amino acid supply at a critical stage of development (Free- man and Lloyd, 1983b).

In our study, the incidence of malformation increased in a dose-dependent manner. In group L (30),

CV malformation was more common than those of other organ systems; however, in group L (50), brain and eye anomalies were more common than CV malformation. The incidence of complicated anomalies

in group L (30) was 50.0%, and was 78.3% in group L (50), indicating that leupeptin induces multiple

organ system defects. Fetal weight decreased when leupeptin dose increased. The average term fetal weight of malformed fetuses nas lower than that of fetuses in rats receiving the same dose. The spectrum of defects is similar to that reported by Tanaka (Tanaka, 1982) and Freeman et a1 (Freeman and Lloyd, 1983b), ex- cept that induction of CV malformations was not specifically described in their reports.

The wide spectrum of CV, central nervous system, eye, and renal defects induced in this study is similar to those seen after AKS and TB administration. Animal studies using various anti-tissue antisera as terato- gens have been reported (Brent, Averich and Drapiewski, 1961; Brent, 1964; Brent, 1967; Brent, Johnson and Jensen, 1971). Among antisera, only AKS and anti-placenta antiserum were teratogenic. Barrow and Taylor noted a high incidence of CV malformation induced by AKS and anti-placenta antiserum (Barrow

and Taylor, 1971). Miyata et al. described high incidence of CV malformations with combined use of an- tisera to kidney and fetal heart (Miyata et al, 1984). It has been hypothesized that antisera induce birth

defects by inhibiting pinocytosis by the endodermal cells of the yolk sac, thereby depriving the embryo of available nutrients (Freeman, Brent and Lloyd, 1982). TR induces defects of CV, central nervous sys- tem and extremities (Fox and Goss, 1956; Wilson et al, 1959; Ema and Kanoh, 1984). In 1967, Beck et al. proposed that TB induces congenital malformation by inhibiting the degradative enzymes within the lysosomes (Beck, Lloyd and Griffith, 1967a; Beck, Lloyd and Griffith, 1967b). In 1976, Williams et al. reported that TB inhibits pinocytosis in the rat yolk sac (Williams, Roberts and Kidston, 1976).

Both AKS and TB are supposed to induce multiple organ system defects by inhibition of histiotrophic nutrition. In this study, the wide spectrum of defects (similar to those induced by AKS and TB) suggests

that leupeptin’s mechanism of action may also be due to inhibition of histiotrophic nutrition, it is consis- tent with Freeman’s hypothesis that leupeptin exerts its teratogenic action by inhibiting proteolysis in the yolk sac lysosomes.

Cardiovascular malformations induced by leupeptin 45

ACKNOWLEDGEMENTS

This s tudy was suppor ted in par t by a grant (No. 02454272) from t h e Ministry o f Educat ion , Science

a n d Culture , J a p a n .

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