Effect of pineal indoles on the chick embryo

General Pharmacology 32 (1999) 219–224

Effect of pineal indoles on the chick embryo Vincent E.C. Ooi a,*, Dorothy W.M. Lee a, T.B. Ng

b

a Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, People’s Republic of China Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, People’s Republic of China Manuscript received March 1, 1998; accepted manuscript June 6, 1998

b

Abstract In a study on the embryotoxicity of pineal indoles on developing chick embryos in vivo, the pineal indoles—namely, melatonin (MEL), methoxytryptamine (MTA) and methoxytryptophol (MTP)—were injected into the yolk sacs of the chick embryos through the air chambers of the eggs on the 4th day of incubation. The eggs were opened and the embryos examined after 6, 10 or 14/15 days of incubation. Abnormalities were found to occur mainly in the 6- and 10-day-old embryos, which exhibited external malformations such as twisted vertebral column, abdominal hernia, exteriorization of heart and viscera, defects of eye, beak and limb. From the results obtained from embryos on the 14th or 15th day of incubation, MEL was found to be the most toxic indole in regard to the mortality induced, whereas MTA had the highest teratogenicity because of the frequent incidence of abnormal embryos. Effect of MTP treatment on the development of chick embryos varied greatly between doses, and there were no abnormal embryos found on the 14th or l5th day of incubation.  1999 Elsevier Science Inc. All rights reserved. Keywords: Embryotoxicity; Melatonin; Methoxytryptamine; Pineal indoles; Chick embryo

The pineal gland appears to be essential for the regulation of photoperiodic response and functional development of the reproductive system in some mammals (Edmonds and Stetson, 1993; Goldman and Nelson, 1993; Ooi et al., 1989; Reiter, 1985; Stetson and WatsonWhitmyre, l984). Administration of pineal indoles such as melatonin (MEL), methoxytryptamine (MTA) and methoxytryptophol (MTP) causes testicular regression (Ng and Chan, 1993; Ng and Ooi, 1989; Ooi and Ng, 1989; Petterborg and Reiter, 1980; Pevet et al., 1981; Tamarkin et al., 1976) and inhibits steroidogenesis (Ng, 1987; Ng and Lo, 1988). Chan and Ng (1994a) examined the effects of pineal indoles on the development of preimplantation mouse embryos. In the in vitro system, methoxytryptamine exerted the most prominent embryotoxic effect on mouse embryos at the four-cell stage, but its effect on embryos at the eight-cell to compacting stage was much milder. Melatonin and methoxytryptamine did not exert a retarding effect. In the in vivo system, only methoxytryptamine and methoxytryptophol caused embryo resorption (Chan and Ng, 1994a). McElhinny et al. (1996) found that melatonin had no adverse effect on the cleavage rate of C57BL/6 and CBA/Ca mouse preimplantation embryos cultured * Corresponding author. Tel.: (852) 2609-6353; Fax: (852) 26035646; E-mail: [email protected].

in vitro. An investigation on the actions of pineal indoles on postimplantation mouse embryos showed that melatonin, methoxytryptamine and methoxytryptophol produced an embryotoxic effect at the early somite stage in vitro. The number of abnormal embryos and the incidence of abnormal yolk sac circulation, body axis, optic and otic placodes, branchial apparatus, forelimb buds and cranial neural tube were increased (Chan and Ng, 1995). However, only methoxytryptamine has an adverse effect on embryonic development in vivo (Chan and Ng, 1995). A structural analog of melatonin, 6-methoxy-2-benzoxazolinone, exhibits a similar embryotoxic effect in vitro (Chan and Ng, 1994b). To our knowledge, there is no available information concerning the effects of pineal indoles on avian embryos, which offer an alternative attractive embryo model with enclosed developing system. The aim of the present study was to examine the effects of three pineal indoles—namely, melatonin and two related indoleamines, 5-methoxytryptamine and 5-methoxytryptophol—on the development of chick embryos. 1. Materials and methods The pineal indoles used in this study—melatonin, 5-methoxytryptamine and 5-methoxytryptophol—were purchased from Sigma (St. Louis, MO, USA).

0306-3623/99/$–see front matter  1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00182-7

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V.E.C. Ooi et al./General Pharmacology 32 (1999) 219–224 Table 1 Abnormality and mortality in the chick embryos observed on the 6th day of incubation

Treatment (mg) Untreated EtOH (ml) MEL

MTA

MTP

a

8 32 3.2 12.5 50 200 800 50 100 200 400 800 50 200 800

Total number of embryos 12 4 3 2 2 2 4 2 2 2 6 2 2 2 4 2

Type of abnormality

Number of deaths

Number of abnormal embryos

Twisted body axis

Ext. of hrt. & vis.a

0 0 2 2 0 0 0 0 0 0 0 1 1 0 0 2

0 1 0 0 0 0 1 0 0 1 3 0 1 0 0 0

— 1 — — — — 1 — — 1 3 — 1 — — —

— — — — — — — — — — 2 — — — — —

Exteriorization of heart and viscera.

Fresh white Leghorn eggs weighing about 50 g were obtained from Kadoorie Farm, Hong Kong. The incubators used contained an egg rotator, a thermometer, an electric heating coil to maintain the temperature at 37–388C and a trough of water to keep the relative humidity at 65–85%. Each of the eggs occupied a “seat” on the automatic egg rotator with its blunt end up and was rotated six times per day. On the 4th day of incubation, the eggs were taken out and pineal indoles were injected. The method of administration applied in this study was the yolk sac injection method (Karnofsky, 1965). A stock solution of pineal indoles was prepared by dissolving 25 mg of the indole in 1 ml of absolute etha-

nol (EtOH). The stock solution was diluted with distilled water, and a series of solutions with different concentrations were prepared for each indole. Using a scalpel, a tiny hole was made at the blunt end of each egg. The pineal indole solution (0.05 ml) was then injected into the yolk sac with the use of a 1-ml tuberculin syringe with a 23-G needle. Two types of control were prepared, the first type being untreated and the second one treated with the vehicle, absolute ethanol, diluted with distilled water. After injection, the hole on the egg was sealed with molten wax. The egg was then put back into the incubator and incubation resumed. The entire injection process was done under sterile conditions. On the 6th, 10th, or 14/15th day of incubation, the eggs

Table 2 Abnormality and mortality in the chick embryos observed on the 10th day of incubation

Treatment (mg) Untreated EtOH (ml) MEL

MTA

MTP

a

8 32 3.2 12.5 50 200 800 50 100 200 400 50 200 800

Type of abnormality

Total number of embryos

Number of deaths

Number of abnormalities

Twisted body axis

Ext. of hrt. & vis.a

Eye defect

Limb defect

8 4 3 2 2 4 4 2 2 2 3 2 2 4 2

0 1 1 0 1 4 1 1 0 0 0 1 2 0 1

2 0 0 0 0 0 0 0 0 0 1 0 0 1 0

1 — — — — — — — — — 1 — — 1 —

1 — — — — — — — — — 1 — — — —

1 — — — — — — — — — — — — — —

1 — — — — — — — — — — — — — —

Exteriorization of heart and viscera.

V.E.C. Ooi et al./General Pharmacology 32 (1999) 219–224

221

Fig. 1. An untreated embryo examined on the 6th day of incubation.

were opened for inspection of external malformations and assessment of mortality (incidence of death). After examination, the embryos were fixed in Bouin’s fluid or 10% buffered formalin for preservation and storage. 2. Results 2.1. Observations on the 6th and 10th days of incubation From Tables 1 and 2, abnormalities can be seen to have occurred at early stages of chick embryonic development. A twisted body axis was found in EtOH-, MEL-, and MTA-treated embryos examined on the 6th day of incubation (Figs. 1 and 2). Besides a twisted body, malformations such as exteriorization of the heart and viscera, as well as defects of the eye, beak and limb, were observed in the untreated, MTA-treated, and MTP-treated embryos examined on the 10th day of incubation (Fig. 3).

Fig. 2. Two MTA-treated embryos (200 mg/injection) examined on the 6th day of incubation. Note the curvature (C) near the tail end of the body.

Fig. 3. MTA-treated embryo (200 mg/injection) examined on the 10th day of incubation. Exteriorization of heart and viscera (HV) was found. The embryo had no left eye (E) and an abnormal beak (B).

2.2. Observations on the 14/15th day of incubations 2.2.1. MEL-treated embryos. A significantly higher mortality was found in the MEL-treated embryos when compared with the two controls. The toxic effect was obvious even at a very low dose, that is, 3.2 mg/injection (Table 3). On the other hand, abnormalities could be observed only in the embryo which was treated with 800 mg of MEL per injection (Fig. 4). That embryo had a deformed left leg, and a similar malformation was not found in EtOH-treated embryos. 2.2.2. MTA-treated embryos. In comparison with the controls, a significantly higher mortality was found in chick embryos when the dosage of MTA was increased to 400 mg/injection. Malformations were observed with the treated embryos, and the number of affected embryos was high. Compared with the EtOH-treated embryos, in which abnormalities could also be detected, MTA-treated embryos manifested different types of malformation; that is, twisted body axis, exteriorized heart and viscera, and defects of the limb and eye (Fig. 5). Although abdominal hernia and exteriorization of the heart and viscera wereas found in both types of embryos, a greater incidence of the abnormality was detected in the MTA-treated embryos, especially in those that had received 200 mg of indole per injection (Fig. 6).

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V.E.C. Ooi et al./General Pharmacology 32 (1999) 219–224

Fig. 4. MEL-treated embryo (800 mg/injection) with a deformed leg (800(g/injection) (L) examined on the 14th/15th 14/15th day of incubation.

Fig. 5. MTA-treated embryos (400 mg/injection) inspected on the 14/ 15th day of incubation. The embryo had not developed feathers. Its legs (L) curved backwards during development. Exteriorization of heart and viscera (HV) was observed.

2.2.3. MTP-treated embryos. The results obtained from the MTP-treated embryos varied because the mortality of chick embryos fluctuated with the dosage of MTP administered. A high mortality resulted when 50 mg was given per injection, but fewer dead embryos were found when the dose was 200 mg/injection for those examined on the 14th or 15th day of incubation. When the dose was increased to 800 mg/ injection, the mortality rate rose again. No abnormal embryos were found on the 14th/15th day, but one embryo with twisted body axis was observed on the 10th day of incubation (Tables 2 and 3). 3. Discussion In the chick embryos examined on the 14/15th day of incubation, melatonin (MEL) was found to be the most toxic among the three indoles tested. Even at a very low dose (3.2 mg/injection), MEL was able to induce a high rate of mortality in the chick embryos. However, the teratogenic effect of MEL appeared to be only minimal. Compared with MEL, methoxytryptamine (MTA) was weaker in toxicity, causing a high mortality only at a high dose. The toxic effect of methoxytryptophol (MTP) was only very mild and varied considerably. The observations that MEL, MTA and MTP had an embryonic effect on chick development were similar to those obtained on mouse preimplantation embryos cultured in vitro (Chan and Ng, 1995), but were different from the

Fig. 6. MTA-treated embryo (200 mg/injection) examined on the 14/ 15th day of incubation. Both eyes (E) were absent in this abnormal embryo. Exteriorization of heart and viscera (HV) was also apparent.

V.E.C. Ooi et al./General Pharmacology 32 (1999) 219–224

223

Table 3 Abnormality and mortality in the chick embryos observed on the 14/15th day of incubation

Treatment (mg) Untreated EtOH (ml) MEL

MTA

MTP

a

8 32 3.2 12.5 50 200 800 50 100 200 400 800 50 200 800

Type of abnormality

Total number of embryos

Number of deaths (%)

Number of abnormalities (%)

Twisted body axis

Ext. of hrt. & vis.a

Limb defect

Eye defect

Beak defect

30 13 15 9 9 16 11 9 11 11 12 10 10 9 13 8

3 3 10 8 8 16 10 5 4 5 7 9 7 8 2 5

0 3 0 0 0 0 0 1 1 4 6 2 0 0 0 0

— 2 — — — — — 0 1 1 2 2 — — — —

— 1 — — — — — 0 1 3 5 2 — — — —

— 0 — — — — — 1 0 0 1 1 — — — —

— 0 — — — — — 0 0 1 2 0 — — — —

— 0 — — — — — 0 0 0 1 0 — — — —

(10) (23) (67) (89) (89) (100) (91) (56) (36) (45) (58) (90) (70) (89) (15) (63)

(13)

(11) (9) (36) (50) (20)

Exteriorization of heart and viscera.

results observed in the in vivo mouse model system, which showed that only MTA and MTP could cause embryo resorption (Chan and Ng, 1994a). That MEL exerted an embryotoxic effect on chick embryos developed in an enclosed living system but did not have an adverse effect on in vivo mouse embryos was probably due to local interference of exogenous MEL, which acted to modify some vital processes of developing chick embryos. Although MTA was found to be less toxic than MEL, its teratogenic effect was stronger. MTP appeared to be the least teratogenic indole, because no abnormalities were observed in the MTP-treated embryos on the 14/ 15th day of incubation. A series of external malformations were observed in the MTA-treated embryos examined on the 14/15th day of incubation, including a twisted vertebral column, abdominal hernia, exteriorization of heart and viscera, and defects of the eye, beak and limb. The most common form of malformations was abdominal hernia and exteriorization of the heart and viscera, and the incidence was much higher than that in the EtOH-treated controls. One possible cause of this abnormality was that the cells on the two sides of the chest and abdomen failed to join during development, and consequently the heart and viscera remained outside the body. Another possible factor causing this malformation was the twisting of the body axis. There was a high coincidence of twisted body axis and herniation of the abdominal and thoracic contents, which could result in exteriorization of heart and viscera in abnormal embryos—for example, 6 out of 13 in 14/15-dayold abnormal MTA-treated embryos. It is likely that, when the vertebral column became curved, the heart and viscera were forced out of the abdominal and tho-

racic hernia and remained exposed. It is noteworthy that the effects of pineal indoles on chick embryos resemble those of hypoxia (Grabowski, 1964), calcium salts (Grabowski, 1966), and dithiocarbamates (Korhonen et al., 1983) in which many similar abnormalities are produced. The embryotoxic effects of pineal indoles might be related in some way to the fact that these agents cause some degree of disturbance of fluid and electrolyte balance. It is also apparent that interferences in physiological conditions of the embryos exposed to these agents can lead to malformations. Pineal indoles have been shown to inhibit enzymatic activities of the steroid biosynthetic pathway in isolated Leydig cells (Ng and Lo, 1988), and reduce incorporation of radioactive thymidine, uridine and leucine in tumor cell lines (Sze, et al., 1993). Hence it is possible that some important biosynthetic enzymes and uptake processes essential to embryonic development are suppressed in the presence of pineal indoles.

Acknowledgment The investigation is partly supported by an Earmarked Grant from the Research Grants Council of Hong Kong.

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