[Intoxication with beta-receptor blockers (author's transl)]. / Intoxikation mit Beta-Rezeptorenblockern.

Beta-blockers are increasingly often used with suicidal intent, but are also sometimes swallowed accidentally by small children. Beta-blockers available in the Federal Republic of Germany differ in their pharmacodynamics and pharmacokinetics. After analysing 49 cases of intoxication, no certain relationship was found between the different substances and specific symptoms. Cardiovascular signs such as sinus bradycardia, arrhythmia, hypotension (30 cases), as well as dizziness and drowziness (17) were the most frequent ones. Loss of consciousness and hallucination (13), as well as seizures (3), also occurred frequently. Hypoglycaemia or symptoms due to it (12) were noted especially in young children. In addition to primary removal of the drug, repeated administration of charcoal and sodium sulphate are recommended with most of the drugs for interrupting the enterohepatic circulation. Administration of atropine for bradycardia and hypotension was usually not effective. Dopamine is recommended; glucagon for definite signs of shock. Haemodialysis is indicated only in exceptional instances and is effective for only a few of the drugs. Forced diuresis should not be practised.

Teratogenic effects of cyproterone acetate and medroxyprogesterone treatment during the pre- and postimplantation period of mouse embryos. II. Cyproterone acetate and medroxyprogesterone acetate treatment before implantation in vivo and in vitro.

The study was performed to investigate direct embryotoxic effects of maternal progestin treatment during the preimplantation period. In the first experiment pregnant mice received a single subcutaneous injection of either cyproterone acetate (CA) or medroxyprogesterone acetate (MPA) on day 2 of pregnancy (5-600 mg/kg). In a second experiment four-cell embryos were exposed to CA or MPA in vitro (3 or 30 micrograms/ml medium). Our results revealed: (1) After maternal treatment the number of live embryos was reduced after the highest CA dose. Development into blastocysts was inhibited in a dose-related manner after CA but not after MPA. The number of cells in morulae, blastocysts, and of the inner cell mass (ICM) of late blastocysts was not affected. (2) When morulae and blastocysts were cultured in vitro after maternal treatment, hatching, attachment, and trophoblast outgrowth were inhibited after high doses, but development and differentiation of the ICM were inhibited even after low doses. (3) Application of 30 micrograms/ml of CA or MPA in vitro was directly embryolethal. Three micrograms/ml did not affect development into blastocysts, but ICM development and differentiation were again inhibited during subsequent culture in hormone-free medium. (4) Qualitative protein synthesis was altered in morulae and blastocysts 24 hours after maternal CA treatment. According to our results high doses of progestins are embryotoxic before implantation, low doses have delayed effects on embryonic development that are particularly evident after implantation in vitro.

[Intoxication by beta-blockers in children and adolescents (author's transl)]. / Vergiftungen mit beta-rezeptorenblockern bei Kindern und Jugendlichen.

After ingestion of beta-blocking agents 20 out of 77 children and adolescents developed clinical symptoms. 11 cases resulted from suicidal attempts. A 15 year old girl died. Neurological signs (12) predominated. Cardiovascular signs (10) were especially seen after suicidal ingestion of beta-blocking agents. Infants frequently showed hypoglycemia or symptoms based on hypoglycemia (6). After elimination from the gastrointestinal tract patients require intensive monitoring as well as symptomatic treatment. Treatment with atropin often gave insufficient results. However, treatment with glucagon was successful. Secondary detoxication generally is not required and must be evaluated after pharmacokinetic data. Forced diuresis is not indicated.

Teratogenic effects of cyproterone acetate and medroxyprogesterone treatment during the pre- and postimplantation period of mouse embryos. I.

Pregnant mice were treated with a single subcutaneous injection of either cyproterone acetate (CA) or medroxyprogesterone acetate (MPA). In the first experiment the animals received 5-900 mg/kg of the hormone before implantation (day 2 of pregnancy). CA treatment on day 2 caused a dose-dependent decrease in fetal weight and a significant dose-dependent increase in the rates of cleft palate and urinary tract abnormalities. Exencephaly and heart abnormalities were also significantly more frequent, but this increase was not dose-dependent. MPA treatment on day 2 was followed by sporadic increases in dead and resorbed fetuses, a decrease in fetal weight and an increase in the rates of cleft palate, and malformed or abnormally developed fetuses. None of these effects, however, was dose-dependent. In the second experiment the mice were given one single injection (30 mg/kg) of CA or MPA on any one of days 1-12 of gestation. Treatment with CA on one day between days 1 and 12 revealed that the specific sensitivity for abnormalities of the urinary tract was on days 5 and 6, for the respiratory tract on days 8 and 9, and for cleft palate on days 10 and 11. Treatment with MPA on one day between days 1 and 12 only revealed a high rate of respiratory and urinary tract abnormalities on day 9. After treatment with MPA cleft palate was again significantly more frequent in all treated groups, however, days of peak sensitivity were not detected. The long half-life of CA (60 hours) explains the teratogenic effect of high doses of this progestin after treatment on day 2 and also the pattern of abnormal development found after treatment with a single dose of CA on one of the days between day 1 and day 12.

Investigations on cyclophosphamide treatment during the preimplantation period. I. Differential sensitivity to maternal cyclophosphamide treatment.

To study the effect of cyclophosphamide (CPA) treatment of pregnant mice during the preimplantation period on different embryonic cells even before implantation, the cell numbers of the immunosurgically isolated inner cell mass (ICM) and the blastocyst total cell number (BTCN) were determined in late blastocysts. In animals treated at 2 P.M. on day 2 of pregnancy a dose-related reduction of the cell numbers was found for both ICM and BTCN in morphologically normal late blastocysts (day 4, 8 A.M.) at CAP doses of 20-60 mg/kg which did not induce embryonic death before implantation but did later during organogenesis. At identical CPA doses the reduction of the cell numbers of the ICM was significantly greater than that of the BTCN. These data provide evidence for a differential sensitivity of the two groups of cells in preimplantation mouse embryos (trophectoderm and ICM) to maternal CPA treatment even before implantation.

Decidual changes in the rat following cyclophosphamide treatment before implantation.

To explain the teratogenic action of cyclophosphamide treatment during the preimplantation period (day 3; 60 mg/kg), the decidual reaction of the uterus was studied in the rat by light microscopy and autoradiography on day 4 (preimplantation period) and on days 5 and 6 (implantation period). On day 4, 24 h after cyclophosphamide treatment, the 3H-thymidine labelling index of the stroma cells beneath the uterine epithelium was significantly increased (33.7 +/- 5.2%) as compared to untreated control animals (6.5 +/- 0.4%). In addition, the surface of the uterine lumina appeared smoother in treated animals than in controls. On day 5 of gestation no difference could be detected autoradiographically between the uteri and treated and untreated animals. Histologically, however, the decidual reaction in treated animals occupied a larger area than in controls. On day 6 of pregnancy the 3H-labelling index of stroma cells surrounding the implantation site of treated animals was considerably reduced (10.7 vs. 32.8% in controls) and necrotic stroma cells were found in the area around the implantation site. Mechanisms inducing the decidual changes in cyclophosphamide-treated animals are discussed.

In vitro methods for the study of the effects of teratogens on preimplantation embryos.

In vitro culture methods are described for mouse embryos during the preimplantation period (Whitten's medium) and for mouse and rat blastocysts during the time of implantation (NCTC-109 medium). Examples are given for the influence of the genetic background (strain differences) on in vitro development and for the detection of a DNA repair mechanism in cultured preimplantation embryos. S-shaped dose effect curves were obtained for the influence of UV-irradiation and cyclophosphamide (CPA) and its metabolites on development of pre implantation mouse embryos treated during in vitro culture. Similar curves were obtained for mouse blastocysts cultured at implantation after treatment of the mother with either CPA or the steroid sex hormones cyproterone acetate (CA) or medroxyprogesterone acetate (MPA). The embryo transplantation technique is described as a teratological method to test the viability of preimplantation mouse embryos which have incorporated the stable isotope 13C during in vitro culture. The importance of the in vitro culture techniques and of the transplantation method in experimental teratology during the earliest period of pregnancy is discussed.

Expression of lactate dehydrogenase isozyme 5 (LDH-5) in cultured mouse blastocysts in the absence of implantation and outgrowth.

Extensive extraction studies with Triton X-100 revealed only LDH-1 (B4) but no trace of LDH-5 (A4) in one-cell and two-cell mouse and rat embryos. The LDH isozyme pattern of preimplantation mouse embryos changes from the maternally inherited B subunit isozyme (LDH-1) to a pattern dominated by LDH-5 when mouse blastocysts are cultured under conditions that prevent hatching but allow trophoblast giant cell transformation. During differentiation of mouse blastocysts in vitro, implantation is therefore not essential for the appearance of the A subunit form of LDH (LDH-5) coded for by the embryonic genome. Mechanisms controlling the expression of LDH-5 in mouse blastocysts during in vivo development are discussed.

Recent progress in teratology. A survey of methods for the study of drug actions during the preimplantation period.

To evaluate the effects of drug treatment during the first days of pregnancy new approaches have been developed which allow the study of teratogenic effects already before and around implantation, during organogenesis, and at term. The procedures used for the culture of preimplantation mouse embryos that were either pretreated in vivo or previously untreated and exposed to a toxic agent in vitro are presented in particular detail. In addition, an example is given which shows that it is possible to detect a DNA repair mechanism in preimplantation embryos maintained in vitro. The techniques of embryo transplantation and in vitro cultivation of embryos beyond implantation are outlined. The importance of the two methods in teratological research on embryos pretreated either in vivo or in vitro is discussed. Also presented are a survey of the literature and recent data obtained with rats and mice from our laboratory which prove that the action of drugs on the embryo and mother during the preimplantation period are more complex than is generally assumed.

Effects of cyclophosphamide treatment before implantation on the development of rat embryos after implantation.

After treatment of pregnant rats 24 h before implantation with a single injection of cyclophosphamide (20--80 mg/kg), a dose-dependent increase in resorption was observed at term but no malformed fetuses could be found. The lowest cyclophosphamide dose that caused 100% resoprtion was 60 mg/kg. Somite number and wet weight indicated retardation of about 24 h during organogenesis. Determination of the time of implantation revealed that the developmental retardation in treated embryos was not due to delayed implantation. At implantation, 24 h after cyclophosphamide treatment, a significant and dose-dependent decrease of the cell number of blastocysts was found. Embryo transplantation experiments showed that early cyclophosphamide treatment interfered with the subsequent development of both the embryo and the mother. The decidual reaction seemed to be more affected by the treatment than the embryos. Most teratologists hold that mouse embryos after treatment in the preimplantation period either die before implantation or survive to term without being malformed. The present study, however, proves that the reaction of drugs at this early stage of pregnancy is more complex than is generally assumed.

Studies on the binding of antibody against mouse lactate dehydrogenase (isoenzyme X) by preimplantation mouse embryos.

The binding of antibodies against LDH-X by preimplantation mouse embryos was studied to detect LDH-X from spermatozoa in embryos after fertilization. Incubation of preimplantation mouse embryos with rabbit anti-mouse-LDH-X-IgG and then with peroxidase-labelled goat anti-rabbit IgG revealed a strong peroxidase staining of the zona pellucida of normal fertilized and unfertilized 1-cell ova. However, the reaction was significantly weaker with both fertilized and unfertilized 1-cell ova from females induced to superovulate and normal and superovulated blastocysts. Pure antibody against mouse LDH-X was obtained by affinity chromatography of the rabbit anti-mouse LDH-X-IgG on pure mouse LDH-X covalently bound to sepharose. The pure antibody against mouse LDH-X reacted immunochemically identically to anti-mouse LDH-X-IgG, but it was not bound by any stage of preimplantation mouse embryos. The IgG fractions which had passed through the affinity column during the purification procedure and which did not contain any anti-LDH-X activity were bound by the zonae of preimplantation mouse embryos in the same manner as was unpurified anti-mouse LDH-X-IgG. Histochemical studies indicated LDH activity only in the embryo proper, but not on the zona pellucida. It is concluded that LDH-X is not present in preimplantation mouse embryos.

Rapid purification of lactate dehydrogenase X from mouse testes by two steps of affinity chromatography on oxamate-sepharose.

At concentrations of 200 muM NADH and 0.5 M NaCl LDH-X is separated from the other LDH isozymes of mouse testes on oxamate-sepharose. In a second step LDH-X is bound to the same matrix at lower NADH and NaCl concentrations and the pure enzyme can subsequently be eluted.

PIP: Results of a method of rapid purification of lactate dehydrogenase X (LDH-X) from mouse testes by 2 stepts of affinity chromatography on oxamate-sepharose at different sodium chloride (NaCl) and NADH concentrations are discussed. At concentrations of 200 mcM NADH and .5 M NaCl, LDH-X is separated from other LDH isoenzymes. The binding of LDH-X to the column is then carried out on the unbound fractions containing LDH-X and contaminating proteins after a 1:5 dilution. The resulting NADH and NaCl concentrations allow the binding of LDH-X. The specific activity of purified mouse LDH-X from testes was 120 IU/mg. The main advantage of this technique is the rapidity and ease of purification.