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Year : 1977  |  Volume : 25  |  Issue : 4  |  Page : 21-25

Eye malformations induced by cyclophosphamide in chick embryos

Department of Anatomy, Medical College, Simla, India

Correspondence Address:
Shamer Singh
Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varansi 221005
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Source of Support: None, Conflict of Interest: None

PMID: 96026

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How to cite this article:
Gupta P K, Singh S. Eye malformations induced by cyclophosphamide in chick embryos. Indian J Ophthalmol 1977;25:21-5

How to cite this URL:
Gupta P K, Singh S. Eye malformations induced by cyclophosphamide in chick embryos. Indian J Ophthalmol [serial online] 1977 [cited 2021 Mar 6];25:21-5. Available from: https://www.ijo.in/text.asp?1977/25/4/21/34608

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Table 1

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Polyfunctional alkylating agents are known to interfere with the process of cell division and hence check the growth of rapidly proliferating normal or maligant tissues. Cyclophosphamide, a potent antitumour agent, has been shown to be teratogenic in Drosophila, Ambystoma, chick, rat and rabbit [20],[27] Although chick embryos were first to be tested for teratological effect in 1963, [10] there seems to be no subsequent report on the teratogenicity of cyclophosphamide in chicks till 1971 when Singh et al. [32] in a prelimi­nary communication described skeletal defects induced by this drug. Besides a report on general teratogenicity of cyclophosphamide in chicks, there have been several subsequent reports by Singh and his associates describing the effects of this drug on developing chick brain. [13],[17],[18],[25],[26],[29] However, there has been no report on the eye anomalies induced in chicks by cyclophosphamide though eye mal­formations by this drug in rat fetuses have been reported. [28] The present paper concerns the eye malformations induced by cyclophosphamide in chick embryos.

  Materials and Methods Top

Fertile eggs of white leghorn chicken were obtained from a Government poultry farm. They were incubated at 37°C-38°C, the relative humidity ranging from 65% to 80% and were turned twice a day by a mechanical device built in the incubators, throughout the period of incubation. Freshly prepared solution of the cyclo­phosphamide in normal saline was injected into the yolk sac of the eggs on different days of incubation by a s mple technique described by Singh and Sinha [31] . Ex­perimental eggs (1472) received varying amounts of the cyclophosphamide in 0.04 ml of normal saline on 3rd to 6th days of incubation [Table - 1] while the control eggs (226) run separately with each experimental group received the same quantity of normal saline without the drug. Some of the controls were given no injection at all. Dead embryos detected on candling were removed periodically. All other eggs were opened on the 19th day of incubation. Gross malformations of eyes besides other malformations were observed and recorded.

  Observations Top

Besides malformations of the eyes induced by cyclophosphamide injections, other defects included defects of beak, limbs, toes, spine, exencephaly, ectopia viscerum, rudimentary wings and stunting of growth. Cyclophospha­mide was found to be more lethal than terato­genie when given during 3rd to 6th day of incubation in 0.035 mg-0.08 mg doses [Table - 1]. Lethal effect increased proportionately with the higher dosage of the drug on each day of treatment.

The malformations of the eyes included absence of eyelids, bleb formation over the eyes, exophthalmos, unilateral anophthalmia and ectopia lentis [Table - 2],[Figure - 1],[Figure 2],[Figure 3],[Figure 4]. Unilateral ectopic lens was found in one embryo treated with 0.04 mg on 4th day of incubation [Figure - 1],[Figure 2],[Figure 3]. Maximum number of embryos with eye malformations were induced by 0.05 mg of cyclophosphamide given on 3rd day of incubation as compared to 4th and 5th day. No eye anomaly was noted in the 6th day treated group. The same dose (0.05 mg) was also found to be most lethal on 3rd and 4th day. On analysis of all dead embryos, it was found that lethal effect was almost instant causing immediate death on the day of injection or just after that in most of the cases during this investigation. All dead embryos (early and late) were found grossly deformed having all types of malformations. None of the embryos in control groups showed any abnormality.

  Discussion Top

The malformations of eye in chicks have been produced by a vast array of substances. These include alcohols, [9] e.g. methyl. ethyl, propyl, benzyl and amyl alcohols; hypoxia, [11] elevated temperature [4],[21] hormones, [1],[2],[5],[6],[7],[14],[16],[19],[23],[33] e.g. cortisone acetate, insulin; thalidomide [3],[8],[12] acriflavin [30] and chlorambu­cil. [31]

The embryo may be regarded as an increas­ingly elaborate set of chemical reactions, all of which stem from the genes of the fertilized egg. By innumerable steps of enzymatically control­led synthesis, three major stages of differentia­tion are achieved, first chemical, then cellular and tissue, and finally organ and functional differentiation. At all stages, nutrients, oxygen and other materials needed for synthesis must be available from the environment. Ordinarily the maternal organism, which constitutes the immediate environment, provides these require­ments and also protects the embryo from deleterious physical and chemical influences. Occasionally, however, this is not the case and an essential metabolite is withheld, or an enzyme is poisoned, or a nucleoprotein is destroyed by the presence of a teratogenic agent in the environment. In this study cyclo­phosphamide was used as a teratogen which is supposed to have destructive interference with the cellular divisions at some susceptible phase, presumably early in mitosis or in the immediate premitotic interval. Because of its affinity to intracellular enzymes, the active alkylating form of the drug is more easily taken up by the rapidly growing cells, e.g. in the embryo or neoplastic tissue. The inhibition of deoxyri­bonucleic acid (DNA) synthesis produced by it, when prolonged, probably leads to localized cell deaths, sufficient to upset their proliferative rates within the embryos, therefore resulting in malformations. [22]

In chick embryos the differentiation begins almost simultaneously with incubation, the undifferentiated stage being passed within the genital tract of the mother. Period of 30 hours to 55 hours is the critical period for the orga­nogenesis of eye in chicks for the formation of optic vesicle, optic cup, and extension of vesi­cular invagination within the optic cup for the lens. For this reason major malformations of eye were not observed by injection of cyclo­phosphamide on 3rd, 4th and 5th day of incub­ation except a few, i.e. unilateral anophthalmia and ectopic lens. Absence of lids and exoph­thalmos were more frequent because lid form­ation occurs in later part of the development. Absence of lids in most cases added to the appearance of exophthalmos. Maximum num­ber of malformations of eye were found in the 3rd day injected group which conforms to the fact that most organs pass through their period of greatest susceptibility, relatively soon after cellular differentiation begins. Cyclophospha­mide apparently is acutely toxic to the embryo which die almost immediately. Many of the surviving embryos show malformations. In this respect cyclophosphamide differs from radiation, which produces consistent develop­mental defect in chick embryos treated on the 4th day. [15] This difference is possibly related to more rapid recovery after cyclophosphamide than after radiation.

  Summary Top

Cyclphosphamide, a widely used anticancer drug when injected into the yolk sac of 1472 chick embryos on 3rd to 6th day of incubation caused anomalies of the eyes (27%) in the 3rd day injected group while the 6th day group showed no eye defect. Malformations of the eyes included absence of lids, blebs over eyes, exophthalmos, ectopia lentis and unilateral anophthalmia. Genesis of eye anomalies in­duced by cyclophosphamide is discussed.

  References Top

Agarwal, I.P. and Monga, J.N., 1958, Ind J. Med. Res. 46, 647.  Back to cited text no. 1
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Duraiswami, P.K., 1950, Brit. J. Med. 2,384-390.  Back to cited text no. 6
Duraiswami, P.K, (1955) J. Bone Joint Surg. 37A, 277.  Back to cited text no. 7
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Grabowski, C.T. 1964, Exp. Zool. 157,307­  Back to cited text no. 11
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Kar, A.K., Singh, S. and Sanyal, A.K., 1974, Ind. J. Med. Res. 62, 905.  Back to cited text no. 13
Karnofsky, D.A., Ridgway, L. P., Patterson, P.A., 1951, Endocrinology 48, 596-.  Back to cited text no. 14
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Malik, M.R. and Singh, S., Cong. Anom. 16, 29.   Back to cited text no. 17
Malik, M.R. and Singh, S. 1976., Neurol Ind. 24, 171  Back to cited text no. 18
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Singh, S., Kar, A.K. and Sanyal, A.K., 1973, Neurol. Ind., 21, 104.  Back to cited text no. 25
Singh, S. and Malik, M.R., 1977, J. Anat.'Soc. India, (in press).  Back to cited text no. 26
Singh, S. and 4anyal, A.K., 1972, J. Anat. Soc. India, 21, 10.  Back to cited text no. 27
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Zwilling, E., 1948, J. Exp. Zool., 109,197.  Back to cited text no. 33


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  [Table - 1], [Table - 2]


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