Biomedical subjects
Publications and source records attributed to N A Chebotar'.
Embryotoxic effect of serotonin in direct (intra-amniotic administration) and indirect (subcutaneous administration) treatment was studied. In both cases serotonin causes embryo death and different abnormalities in development in small number of embryos. Mechanism of serotonin embryotoxic effect is under discussion.
Vasopressin administration induced a right-sided bias in the rat litter, the asymmetry being similar to a right-sided bias in the turning behaviour of adult male rats in the T-maze. The findings suggest the nigrostriatal system to be responsible for the lateralising effect of vasopressin in the brain.
The effect of alimentary administration of silver salts upon embryogenesis in rats has been studied. Feeding of AgCl to pregnant female rats throughout gestation did not result in any alterations in their physiological functions, although the active copper-containing ceruloplasmin (Cp) was eliminated from the blood stream. However, anomalous development of embryos, their prenatal death or total mortality of newborn rats within the first 24 hours after birth were evidenced. The copper content in the placenta and embryonic tissues decreased appreciably. The Cu,Zn-superoxide dismutase (SOD) activity was diminished in the cytoplasm of embryonic cells alongside with its drop, though less pronounced, in the tissues of the pregnant females. The embryotoxicity of AgCl was considerably reduced by repetitive injections of native Cp to pregnant rats. Such treatment caused an increase in the SOD activity in the placenta and embryonic tissues. Mortality of the newborns also went down. It is suggested that the embryotoxic effect of AgCl is due to its ability to interfere with copper metabolism, by altering the copper-transporting function of Cp.
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Some peculiarities of rat milk ceruloplasmin (CP) biosynthesis have been analysed. Electrophoretic and immunoelectrophoretic data suggest that rat milk contains up to 20 mg/100 ml of CP. Rat milk CP is represented by a single molecular form whose molecular mass, enzymatic activity and antigenic properties are fully identical with those of serumal CP. According to the dot hybridization date, the CP-mRNA content in rat mammary cells is about ten times as low as that in adult rat liver. Analysis of polyribosomal RNA by blot hybridization of RNA-cDNA revealed that rat mammary CP-mRNA is represented by a single molecular form, 3.5 kb in length, whose molecular mass is identical with that of the predominant CP-mRNA of rat liver. The dynamics of secretion of the de novo synthesized CP into rat milk indicates that the CP synthesis and secretion occur independently of the serumal CP. In vitro and in vivo pulse labelling studies demonstrated that rat milk CP is synthesized and secreted by rat mammary cells.
The exposure of rat fetuses to ethanol by direct intra-amniotic injection in the afternoon of the 14th day of embryogenesis resulted in developmental anomalies (34%), the defects located mostly in the left extremities. Intraperitoneal injections of ethanol at the same period of pregnancy resulted in analogous injuries but only in occasional fetuses. There was no embryotoxic effect after exposure to high doses of ethanol by per os. It was concluded that even a single dose of ethanol in certain periods of pregnancy and in certain homeostasis disorders in the maternal organism could induce the fetal alcohol syndrome (FAS) in the newborns.
A combined administration of sodium salicylate and ethanol to female rats in threshold embryotoxic doses on the 9th, 10th, 12th and 13th days of pregnancy results in a sharp increase of embryolethal and teratogenic effects. The embryotoxic effect of sodium salicylate and ethanol at their combined injection exceeds the summational embryotoxic effect at the separate action of the preparations. The spectrum of malformations after their combined effect does not differ from that induced with sodium salicylate.
In the experiments performed in chick embryos, using the chick embryo screening test (CEST)-1 and CEST-2 technique, 100 various classes of chemical substances have been tested for teratogenic activity. Estimation of three morphogenetic systems (caudal part of the body, facial skull and extremities) has demonstrated that some of the substances do not produce any damaging effect, other produce some lesions in selected germs, the third influence two or three germs simultaneously. Similar picture is also specific for the effect of the chemical substances to the rat intrauterine development. Owing to data obtained, we come to the conclusion that for screening of embryotoxicity of various xenobiotics under in vivo and in vitro experimental conditions one should not orient to one single germ.
The effect of pyrimethamine (chloridine) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on embryo survival, cleavage rate and frequency of chromosome aberrations when injected into female rats just before ovulation was studied. Pyrimethamine induced 70% of embryonic death at a dose of 5 mg/kg and 27%--at a dose of 2 mg/kg. 200 mg/kg of 2,4,5-T had no embryotoxic effect, while 400 mg/kg increased the embryo loss up to 48%. Pyrimethamine (5 mg/kg) and 2,4,5-T (400 mg/kg), injected intragastrically just before ovulation, significantly affected the cleavage rate in the four day rat embryos. The frequency of chromosome aberrations in ovulated oocytes at metaphase, II subjected to pyrimethamine and 2,4,5-T, reached 6--8% and exceeded the control value in 5--10 times.
Cytogenetic analysis was carried out for the ovulated oocytes and preimplantation embryos at the age of 3 and 4 days in the mice heterozygous by 2 Robertsonian translocations Rb (8; 17) 1 IEM and Rb (16; 17)7 Bnr. Out of 171 ovulated oocytes, 101 had chromosome aberrations, 44 nullisomy and 19 disomy by autosome 17. On the 3rd day of development the frequency of aneuploid embryos attained 41%; the same types of chromosome aberrations were found as in the oocytes and in about the same ratios: 15% of embryos had trisomy and 11.6% monosomy by autosome 17; the average number of blastomeres in these embryos did not differ from that in the diplod embryos; micronuclei were, as a rule, found in the cells of embryos with monosomy 17. Out of 100 karyotyped 4 days old embryos, 19 had trisomy by autosome 17, no monosomy by this chromosome was found; 20% of embryos contained 4 to 16 blastomeres with numerous micronuclei and picnoses. The trisomy by autosome 17 did not affect the preimplantation mouse development, whereas the embryos with monosomy died at the stage of 8 to 16 blastomeres.
One day delay of ovulation in rats, caused by the injection of medinal, resulted in the marked slowing down of the cleavage rate. The delay is due to additive effect of both the drug itself and preovulatory over-ripeness of the oocytes. Upon two days medinal delay of ovulation, the toxic effect of over-ripeness was not stronger than upon the twofold injection of medinal; two days hormonal delay of ovulation resulted in the distinct inhibition of cleavage.
The injection of adrenocorticotropic hormone (ACTH) of prolonged effect at the doses of 4 and 10 M. U. to the intact rats from the 11th till the 15th day of pregnancy resulted in the twofold increase of protein content in the brain and its decrease in the liver of 15 days old embryos, as compared with the control ones. The content of DNA, RNA and proteins in the placenta of experimental animals increased as well. The rate of incorporation of 3H-thymidine in the liver DNA and 14C-leucine in the liver and brain acid-soluble protein decreased within small intervals of time following the treatment. The total radioactivity of proteins in the liver, brain and placenta calculated per DNA unit was similar to the control one whereas the specific radioactivity of total protein in the liver of experimental embryos was higher than in the control.
328 oocytes from 874 studied were found suitable for counting chromosomes. 263 oocytes (80.18%) had a normal chromosome number, while aneuploidy was observed in 18.3% of oocytes (61). Among then 46 (14.02%) were with hypoploidy and 14 oocytes (4.26%) were hyperploid with 21 chromosomes and over. Structural chromosome aberrations were found in 24 oocytes (7.31%).
The chromosome number in 174 zygotes on the stage of the 1st cleavage division was counted. In 113 zygotes (64.94%) euploidy (metaphase with 42 chromosomes) and in 57 zygotes (32.77%) aneuploidy was found. Frequencies of spontaneous chromosomal aberration were 14.93%. 33 zygotes were hypoploid, 6 (3.44%)--hyperploid (with 43 and 46 chromosomes), and 3 (5.76%)--polyploid, 4 zygotes (2.29%) had structural anomalies of chromosomes. Hypoploidy may be often due to artifacts. Superovulation induced by gonadotrophin results in an increased polyspermia.
White female rats were injected intragastrically with 2,4,5-trichlorphenoxyacetic acid (2,4,5-T) in the dose of 400 mg/kg, commercial sample of butyl ester in the dose of 50, 100, 200 mg/kg and purified ester of 2,4,5-T in the dose of 800 mg/kg every day on the 9, 10, 11, 12, 13 and 14th day of gestation. Stress reaction (immobilization) was produced by fixation of the animals on a board modified by S. I. Renaud (1959) for 2 h after the injection of corresponding substances. The rats were killed on the 20th day and the number of alive and dead fetuses was registered. Under the conditions of immobilization embryotoxic effect of 2,4,5-T increased for certain on the 9th and 10th days (52.7% and 33.64%, respectively), that of butyl ester of 2,4,5-T--on the 10th day (55.66%). Immobilization increased teratogenic effect of 2,4,5-T on the 9th, 10th, and 11th days, that of butyl ester of 2,4,5-T on the 13th, 14th days. Purified butyl ester of 2,4,5-T proved to be 4 times less toxic and produced a severe embryotoxic and a mild teratogenic effect.
The maximal rate of incorporation of 32P-phosphate, 14C-formate and 14C-thymidine in DNA was recorded on the 13th day of development in the rat embryos and that of 14C-formate and 32P-phosphate in RNA and nucleotides of the acid-soluble fraction on the 12th day. The maximal incorporation of 14C-formate was recorded later: on the 15-16th days. Chloridin inhibited the incorporation of 14C-formate in DNA at all developmental stages, irrespective of the sensitivity of embryos to its teratogenic effect. The period of the maximal rate of DNA synthesis coincides with that of highest teratogenic activity of the drug. A suggestion is put forward to the effect that quantitative differences in the intensity of DNA synthesis at different stages of organgenesis provide one of the main causes of differential sensitivity of embryos to the teratogenic effect of inhibitors of nucleic acid synthesis.
Teratogenic doses of antimalaria preparation, 2,4-diamino-5-p-chlorophenyl-6-ethylpyrimidine (chloridine), known in biochemistry as a specific inhibitor of dihydrofolate reductase, result in the decrease of 32P-phosphate incorporation into ATP and ADP, coupled with the inhibitions of DNA synthesis, in rat embryo and placenta on 13th day of development, while the incorporation of 14C-formiate into free nucleotides of acid soluble embryo and placenta fractions (at the same intervals after teratogen injections), is the same as in the control. These data show that the primary blocking of folate cycle and DNA biosynthesis in 13 days old embryos with induced anomalies of development is some way coupled with disturbances of ATP metabolism.
The patterns of DNA synthesis and kinetics of cell population in the rat and mouse embryos were studied by means of 3H-thymidine autoradiography and cytofluorimetry. The rat and mouse embryos during the period of cleavage consist of a heterogenous population of blastomeres. At all the stages under study, all phases of the cell cycle occur in the blastomeres: G1, S, G2 and mitosis. The embryonic cells were distributed into groups containing 2c, 3c, 4c and more DNA. The ratio of cell number in these groups differed in the mouse and rat embryos. The mouse embryos are characterized by the appearance of a considerable amount of polyploid cells in S phase at the morula stage. The stage and species specific quantitative and qualitative patterns were established for DNA synthesis and kinetics of the cell population of blastomeres.