Constanze Lewandowski

Modification and standardization of the culture of early postimplantation embryos for toxicological studies.

Abstract1.The method of culturing “whole” rat embryos (days 9.5–11.5 of gestation, i.e. at the early stage of organogenesis) as modified and standardized in our laboratory is presented;2.We have succeeded in using bovine serum as culture medium instead of rat serum as recommended in the original procedure. Experimental conditions are described for obtaining reproducible results;3.An improved scoring system was developed which, in connection with a computerized documentation, greatly facilitates the evaluation of the data.

Pharmacokinetic aspects of drug effects in vitro: effects of serum protein binding on concentration and teratogenicity of valproic acid and 2-en-valproic acid in whole embryos in culture

Pharmacokinetic studies were performed in connection with culture experiments. Using the technique of cultivating whole rat embryos of the early postimplantation stage, we measured the concentration of valproic acid (VPA) and 2-en-VPA in the culture medium (free and protein-bound form) and in embryonic tissue. The following results were obtained:1.The concentrations of VPA and 2-en-VPA reached in the embryos were lower than corresponding total concentrations added to the culture medium, but exceeded the free concentrations in the medium.2.The concentrations of 2-en-VPA found in the embryo were lower than the comparable VPA total levels because of the more extensive protein binding of 2-en-VPA in the culture serum.3.The percentage of binding to serum proteins decreased with increasing total drug concentratrations in the medium; the concentration of the free drug in the medium increased overproportionally with increasing total drug concentrations. Therefore, the free drug concentrations in the medium were not proportional to the dose of the drug dissolved in the medium (for a drug bound to plasma proteins).4.The concentrations of VPA and 2-en-VPA found in the embryos after incubation in vitro were not proportional to the drug concentrations dissolved in the medium. This result has to be taken into account when dose-response relationships are evaluated.5.VPA concentrations of 40 μg/g wet weight and above in the embryos clearly induced abnormal development in about 30% of the embryos, while 2-en-VPA concentrations as high as 200 μg/g embryo (wet weight) were inactive. Thus, differences in the embryotoxic potencies — in vivo and in vitro — were not due to pharmacokinetic differences.6.The results of these investigations show for the first time that pharmacokinetic studies are essential for the interpretation of in vitro experiments.

Prenatal toxicity of acyclovir in rats

Pregnant rats were treated during organogenesis with s.c. injections of acyclovir and the embryos were evaluated on day 11.5 of gestation (crown-rump length, somites, protein content, score, abnormalities, histological examination). After eight injections of 50 mg/kg body wt on days 9, 10, and 11 of pregnancy a reduction of the crownrump length was noticed. After 100 mg/kg this effect was more pronounced. With two or three applications of this dose on day 10 specific embryonic abnormalities were visible: the shape of the head was abnormal, the width of the skull had decreased resembling a beak-like visceral cranium. With a single administration of 200 mg/kg on day 10 we found a similar but slightly more pronounced outcome. A drastic change of all variables was obtained after eight injections of 100 mg/kg on days 9, 10, and 11. Comparatively we measured maternal plasma concentrations of acyclovir 1 h after the administration of 50, 100 or 200 mg/kg body wt. After an injection of 50 mg/kg on days 9, 10, and 11 of gestation (three injections/day) the plasma levels ranged from 19.1 to 40.0 mg/l (1 mg/l = 4.44 μM). No cumulation was observed. In contrast, a cumulative effect was detected following a dose of 100 mg/kg. After the first injection of this dose a mean value (± SD) of 60.3±14.7 mg/l (n = 16) was obtained. In this case a third injection increased the mean plasma level to 124.6±16.6 mg/l (n = 5). Further injections, however, led to decreasing levels. One hour after administration of 200 mg/kg body wt acyclovir levels ranged from 120.0 to 163.9 mg/l. We conclude that acyclovir, at doses leading to plasma concentrations well above the therapeutic level in the dam, interferes with the embryonic development in the rat. Acyclovir induces typical gross structural abnormalities which have been first observed using a whole embryo culture system.

Effect of acyclovir on mammalian embryonic development in culture.

Abstract1.Acyclovir [9-(2-hydroxyethoxymethyl)guanine] interfered with embryonic development in vitro when assessed with the “whole-embryo” culture technique. The “no-observed-effect level” was at 10 μM acyclovir; 2. Minor impairment of embryonic development (retarded development of ear anlagen) was observed in vitro at 25 μM acyclovir in the culture medium. At high concentrations (100 or 200 μM) development of the ear anlagen was largely inhibited. At concentrations of 50 μM acyclovir or higher, additional disturbances of embryonic differentiation in vitro became obvious, resulting in gross structural abnormalities, especially of the brain (telencephalon); 3. Histological examinations confirmed and extended these observations: at 100 μM acyclovir alterations of the neuroepithelium of the ventricles were pronounced, the telencephalon had developed poorly or was almost completely absent, and necroses were seen in the ear anlagen, the maxillar branch and within the somites; 4. In a limb bud culture (mouse embryos, starting with day 11 of gestation) acyclovir interfered with the differentiation of cartilaginous bone anlagen at concentrations of 200 μM and more in the culture medium. A concentration of 100 μM induced no significant effect. Thus, this organ culture system is less sensitive to the action of acyclovir when compared with whole-embryo culture; 5. Contrary to the results achieved with acyclovir, physiological nucleosides (2′-deoxyguanosine and 2′-deoxyadenosine) did not interfere with embryonic development in vitro even at the highest concentration tested (500 μM).

All-trans retinoic acid and 13-cis-retinoic acid in the rat whole-embryo culture: abnormal development due to the all-trans isomer

Abstract9.5-day-old rat embryos were exposed to 3 μg 13-cis retinoic acid (13-cis-RA)/ml culture medium or 1 μg all-trans retinoic acid (all-trans-RA)/ml culture medium for different time intervals of the culture period (test substance-exposure periods alternated with test substance-free periods). These studies were performed to confirm the hypothesis (Klug et al. 1989) that the effect of 13-cis-RA on embryonic development in vitro is predominantly caused by its isomerisation to all-trans-RA. A 12 h exposure of the rat embryos to 13-cis-RA during different periods of culture did not interfere with normal development. However, a 12 h exposure of the embryos to all-trans-RA in the first three quarters of the culture period significantly interfered with normal development and caused clear-cut and typical abnormalities. Embryonic exposure to 13-cis-RA, for periods of more than 12 h, caused severe interference with normal development and led to branchial effects very similar to those observed following to a 12 h exposure to alltrans-RA.

Effects of valproic acid, some of its metabolites and analogues on prenatal development of rats in vitro and comparison with effects in vivo

Using a whole-embryo culture system valproic acid (VPA) and some of its metabolites (2-en-VPA, 4-en-VPA, 4,4′-dien-VPA) and analogues (ethyl-propyl-acetic acid, propyl-butyl-acetic acid, di-butyl-acetic acid, 2-methyl-2-ethyl-hexanoic acid, 1-methyl-1-cyclohexanoic acid) were tested for their potential to induce abnormal development. With regard to embryonic growth, development and abnormality rate, the tested compounds showed a wide range of “teratogenic potency” in vitro. In order to verify some of the in vitro results, in vivo experiments were performed. Pregnant rats were treated subcutaneously on day 10 of gestation with 2×330 mg VPA/kg, or 2×400 mg 2-en-VPA/kg, respectively. Evaluation of the embryos was performed on day 11.5 of gestation, corresponding to the in vitro experiments. VPA showed a high potential to induce abnormal development in vivo as well as in vitro, whereas 2-en-VPA was inactive under our experimental conditions. Problems connected with the evaluation of the predictive value of an in vitro test system for the detection of embryotoxic effects, such as “validation” and significance of pharmacokinetic data, are discussed.

In vitro and in vivo studies on the prenatal toxicity of five virustatic nucleoside analogues in comparison to aciclovir

Several virustatic agents are known to be teratogenic in laboratory animals. Since routinely performed in vivo studies do not always offer the best conditions to detect the teratogenic potential of a drug, we used a combined in vivo/in vitro approach for comparative studies on the prenatal toxicity of five nucleoside analogues. Rat embryos were exposed for 48 h to various concentrations of vidarabine-phosphate (VAP), ganciclovir (GCV), 2′,3′-dideoxyadenosine (ddA), 2′,3′-dideoxycytidine (ddC) and zidovudine (= azidothymidine, AZT) in a whole-embryo culture system. The steepness of the concentration-response curves as well as the induced abnormality pattern (head, neural tube, shape) were similar for these compounds. However, a wide range in embryotoxic potency was observed: VAP was the most potent compound (100% abnormal embryos at 25 μM) in this in vitro system, while AZT showed the lowest potency to interfere with normal embryonic development (40% abnormal embryos at 3000 μM). In addition to these experiments we treated rats on day 10 of gestation with three s.c. injections (8 a.m.; 12 a.m.; 4 p.m.) of 200 mg of each drug/kg body wt. The embryos were evaluated on day 11.5 of gestation, i.e. at a time of development corresponding to the developmental stage at the end of the whole-embryo culture. The same criteria were used as during the in vitro studies for the evaluation of these in vivo exposed embryos. With VAP and GCV we obtained similar results with both exposure routes (in vitro and in vivo), while no abnormalities were detectable with the other compounds after exposure in utero. When the results from the in vitro and in vivo studies are compared with data of similar experiments conducted in our laboratory with the nucleoside analogue aciclovir (ACV) under identical conditions (Klug et al. 1985 a; Stahlmann et al. 1988), the following conclusions can be drawn: under in vitro conditions VAP showed the highest potential of the virustatics to interfere with embryonic development, the toxic potential of AZT was surprisingly low. Under our experimental in vivo conditions ACV reveals the highest teratogenic potential, whereas ddC, ddA, and AZT exhibited an obviously lower toxicity.

Pharmacokinetic aspects of drug effects in vitro (II) placental transfer to the embryo and activity of some carboxylic acids structurally related to valproic acid in whole embryos in culture.

The transfer of a group of short/medium chain-length carboxylic acids, related to the antiepileptic drug valproic acid (VPA), to the whole rat embryo in vitro was investigated. The protein binding of the drugs in the culture medium determined the placental transfer in vitro: at comparable total concentrations, the substances that bound to a lesser degree (VPA and its metabolite 2-propyl-4-pentenoic acid; 4-en-VPA) reached higher embryonic levels than the more highly bound substances, octanoic acid (OA), 2-methyl-2-ethylcaproic acid (MEC), and the VPA metabolite, 2-propyl-2-pentenoic acid (2-en-VPA). Consequently, the amount of drug added to the culture did not correlate with the transfer to the embryo, but the concentration of the free drug in the culture medium correlated highly with embryonic exposure. The concentration of the drugs in the cultured embryos, the embryonic membranes and the subembryonic fluid were higher than the corresponding free concentrations in the medium. The difference in the teratogenic potency of the substances tested was clearly related to their intrinsic activity, since even high embryonic concentrations of 2-en-VPA, MEC and OA did not produce adverse effects in contrast to VPA and 4-en-VPA which were effective at levels several-fold lower. The effective concentrations of VPA in the cultured embryos were almost ten times lower than those in embryos in vivo. The factors responsible for the high vulnerability of the cultured embryos to the action of VPA are as yet unknown. Our results indicate that the determination of drug concentrations in cultured embryonic tissue is imperative for a rational interpretation of experimental data obtained from in vitro studies; a full validation of in vitro systems must incorporate pharmacokinetic studies.