The effect of beclobric acid and clofibric acid on peroxisomal beta-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes.

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey (Macaca fascicularis) and human liver. Determination of peroxisomal fatty acid beta-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal beta-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 microM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations greater than 300 microM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid beta-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisome-proliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.

Monitoring of organ formation in rat embryos after in vitro exposure to azathioprine, mercaptopurine, methotrexate or cyclosporin A.

Rat embryos in the organ formation phase (days 9.5-11.5 post coitum) were cultivated in pure rat serum in the presence of an Aroclor 1254 pretreated liver microsomal preparation (S9-mix). Various concentrations of the immunosuppressive drugs azathioprine (AZ), 6-mercaptopurine (MP), methotrexate (MTX) or cyclosporin A (CS-A) were added at the beginning of the culture period. Forty-eight hours later, malformations were observed in the AZ, MP and MTX treated embryos at concentrations as low as 1 microgram/ml, 1.8 micrograms/ml and 0.05 microgram/ml, respectively. This indicates that these drugs have a direct effect on embryonic development. They selectively affected the rhombencephalic and telencephalic brain regions. Other malformations were seen in the caudal trunk, the heart and forelimb regions, and in the vesicular structures. It is suggested that the similarity of the pharmacological action of these drugs, that is, the DNA de novo synthesis inhibition, was the cause of the comparable types of malformations observed. Higher AZ, MP and MTX concentrations caused concentration-dependent increases in the types and incidences of malformations, as well as inhibited overall growth and differentiation. CS-A, a new type of immunosuppressant agent, had no effect on the morphogenetic events at the concentrations tested. These results are generally in agreement with the literature data, indicating that AZ, MP and MTX induce malformations in whole-animal systems, whereas CY-A does not. When AZ and MTX were assayed in the rat species in vivo, on the other hand, embryolethalities and retardations, but few malformations, were observed. The possibility of controlled exposure in vitro may, therefore, offer the advantage that clearer distinctions between embryolethal and teratogenic effects can be made.

Teratogenicity induced in cultured rat embryos by the serum of procarbazine treated rats.

Male rats were injected with single intraperitoneal doses of 50-200 mg/kg of procarbazine hydrochloride (P). Their undiluted serum was then used as the culture medium for 9.5-day-old embryos to evaluate its teratogenic and toxic potential in vitro. A 48-h exposure to this medium resulted in a variety of dysmorphogenic effects. Somite, limb bud and otic vesicle deformities were seen at all dose levels, and neural tube and optic vesicle abnormalities were frequently observed in the 150 and 200 mg/kg groups. Embryonic growth and differentiation were only moderately affected at any dose levels. In a second set of experiments, embryos were directly exposed to 100-150 micrograms/ml P combined with a liver enzymatic activating system. No abnormalities were detected but toxicity occurred within narrow concentration ranges; 125 micrograms/ml affected growth and differentiation slightly, and 150 micrograms/ml suppressed the embryonic development severely. Exposure to 125 micrograms/ml P without the liver enzymatic activating system had no effect on embryonic growth and differentiation, suggesting that the metabolites responsible for the toxic effect were generated by the liver enzymatic preparation. Our results indicate that stable metabolites of P, responsible for its teratogenic action, are formed only within body compartments, whereas the formation of these metabolites does not take place in vitro in the presence of hepatic enzyme preparations.

Assessment of the teratogenic potential of acrolein and cyclophosphamide in a rat embryo culture system.

Rat conceptuses were explanted from the uterus on day 10.5 of gestation. The embryos within the yolk-sacs were then transferred to culture bottles containing pure male rat serum either with or without liver microsomes and NADPH. Acrolein (AC), present worldwide in the environment and one of the intermediate metabolites of cyclophosphamide (CPA), was added to these culture mediums. The conceptuses were grown for a period of 48 h after which the morphological features and their degree of differentiation were examined and the DNA and protein contents determined. The effects produced by AC were compared with those obtained by CPA treatment, using the same culture conditions. AC treated embryos and yolk-sacs showed slight but statistically significant inhibition of growth at concentrations of 100 microM and 150 microM. Higher dose levels (200 microM and 250 microM) resulted in a drastic inhibition of growth and differentiation. However, no gross structural defects were observed at the dose-levels used. In contrast, conceptuses cultivated in the presence of CPA (350 microM), liver microsomes and NADPH showed characteristic morphologic lesions. Our findings indicate that AC is lethal to embryos within a narrow dose-range, but has no teratogenic potential. Therefore, AC is not the metabolite which is responsible for the teratogenic effects observed after CPA treatment in vivo. The results also demonstrate that the postimplantation embryo culture system can discriminate between embryolethal and teratogenic effects and that whole embryos in culture can respond to teratogens in a manner similar to embryos exposed in vivo.

Embryonic and fetal development: fundamental research.

Much progress has been made over the past decades in the development of in vitro techniques for the assessment of chemically induced effects in embryonic and fetal development. In vitro assays have originally been developed to provide information on the mechanism of action of normal development, and have hence more adequately been used in fundamental research. These assays had to undergo extensive modification to be used in developmental toxicity testing. The present paper focuses on the rat whole embryo culture system, but also reviews modifications that were undertaken for the in vitro chick embryo system and the aggregate cultures of fetal rat brain cells. Today these tests cannot replace the existing in vivo developmental toxicity tests. They can, however, be used to screen chemicals for further development or further testing. In addition, these in vitro tests provide valuable information on the mechanisms of developmental toxicity and help to understand the relevancy of findings for humans. In vitro systems, combined with selected in vivo testing and pharmacokinetic investigations in animals and humans, can thus provide essential information for human risk assessment.

Interlaboratory evaluation of embryotoxicity in the postimplantation rat embryo culture.

The embryotoxicity of eight xenobiotic compounds in rat postimplantation whole embryo culture was blindly tested in four laboratories according to a standard protocol. The results show that the four nonteratogens amaranth, penicillin, isoniazid, and saccharin did not affect embryogenesis apart from general toxicity at very high concentrations in culture for amaranth and isoniazid. There was good concordance of results across the laboratories. The four teratogens (retinoic acid, 6-aminonicotinamide, acetylsalicylic acid, and vincristine) induced a variety of specific embryotoxic effects, which were in most cases similar in all laboratories. These results indicate that the definition for specific embryotoxicity used, as well as the culture duration and embryonic age are crucial for concordant scoring. Other methodologic differences did not significantly influence scoring of embryotoxicity. Therefore, within the limits of the end points and embryonic stage represented in the method, embryo culture appears as a useful method for embryotoxicity screening, which can be reproducibly applied in different laboratories.

Interlaboratory evaluation of three culture media for postimplantation rodent embryos.

The first aim of the study was to compare the ability of rat serum, human serum, and a mixture of human and rat serum (4:1) to support in vitro development of rodent postimplantation embryos. The comparison was made in three laboratories using rat embryos and in one laboratory using mouse embryos. Batches of sera, initial developmental stage, duration of culture, and endpoints were identical in the laboratories. The second aim of the study was to evaluate if other variables that could not be standardized would significantly influence the results of the laboratories. No reproducible difference was observed among the culture media or among the laboratories except that growth and differentiation were slower in the laboratory using mouse embryos. Further experiments are needed to exclude small differences in performance of the media.

Teratogenicity in vitro-A comparative study of four antimycotic drugs using the whole-embryo culture system.

The embryotoxic effects caused in vitro by the four antimycotic compounds griseofulvin, ketoconazole, naftifine and terbinafine were compared. Rat embryos (9.5 days old) were cultivated in rat serum for 48 hr both in the presence and in the absence of the individual test compounds. A rat liver microsomal preparation and NADPH were added to the cultures. Effects on growth and differentiation were evaluated and dysmorphogenic effects on various embryonic features were recorded. The compounds could be ranked as follows: in terms of the concentrations that affected growth in vitro, griseofulvin = naftifine = ketoconazole < terbinafine, and, with regard to retarded differentation, griseofulvin = ketoconazole = naftifine < terbinafine, while for morphological findings, ketoconazole < griseofulvin = naftifine < terbinafine. The ratio between the concentration affecting rat embryonic morphology in vitro and the human peak plasma levels after multiple drug application was approximately 0.2 for ketoconazole, 3 for griseofulvin, 20 for terbinafine and 2000 for naftifine. The results of this study verified that this in vitro system can be used to classify test compounds according to their embryotoxic effects. The findings reported here were in agreement with those of in vivo studies. In both systems, ketoconazole and griseofulvin had relatively high teratogenic potential and terbinafine and naftifine had none.

Application of the post-implantation rat embryo culture system to in vitro teratogenicity testing.

Originally developed for studying basic mechanisms in developmental biology, the post-implantation embryo culture system has been extended and applied to the testing of chemicals in the field of prenatal toxicology. The endpoints used in a procedure that has been developed for the routine short-term assessment of teratogenicity in vitro are growth (crown-rump length), differentiation (somite number) and morphology (together with measurement of yolk-sac growth and vascularization) in rat embryos explanted on day 9.5 of gestation and cultured for 48 hr in 100% homologous rat serum containing the test compound. Studies involving exposure to trichlorophenoxyacetic acid and to acetylsalicylic acid demonstrate the ability of this system to distinguish between non-specific toxicity and specific effects on development.

Rodent whole-embryo culture as a teratogen screening method.

Postimplantation embryo techniques can sensitively detect compounds with adverse biological activity. The versatility of the in vitro embryo toxicity/teratogenicity screening assay has been augmented by the addition of adult hepatic metabolic activation systems, which influence the results for some compounds in the embryo culture system. In this assay, cyclophosphamide produces negative results without metabolic activation and positive results with activation; metabolic activation changes the results for cytochalasin D from positive to negative. Another improvement has been the evaluation of the embryo toxicity/teratogenicity of various water-immiscible solvents. As these studies revealed that sonicated corn oil/serum preparations were nontoxic at concentrations of up to 10% of the culture medium, these solvents can now be used to expose cultured embryos to water-insoluble compounds (e.g., chlorinated pesticides). This in vitro rodent whole-embryo culture makes it possible to investigate extremely rapid cell division and specific time-related morphogenic events. Because in vitro development so closely parallels that occurring in vivo, the in vitro embryo culture system appears to be particularly relevant for the detection of teratogenic and embryotoxic compounds. A variety of compounds have been assayed in vitro via a combination of metabolic activation system and/or water-insoluble chemical delivery systems. Correlation between the published results of the in vitro screening assay and the published results of in vivo embryo toxicology and teratology studies has been very good. However, most of the compounds used in in vitro testing have been strongly teratogenic, and little information is available on compounds that are not teratogenic in in vivo tests. There is also a good correlation between the concentrations of teratogenic agents that cause adverse effects in vitro and the in vivo teratogenic doses of these compounds.

Xenobiotic influences on embryonic differentiation, growth and morphology in vitro.

Direct in vitro exposure of post-implantation rat embryos to 18 known teratogens induced typical malformations in all cases. Of 21 non-teratogens in vivo, 20 induced, in vitro either no malformations at all, even at high concentrations, or abnormal development could only be observed at concentrations which affected growth and differentiation significantly. Responses of chemically induced exposed embryos in vitro occurred within wide concentration ranges. Actinomycin D, for example, affected embryonic development at a concentration as low as 3 X 10(-4) micrograms/ml, whereas other substances had no effect at concentrations up to 9 X 10(2) micrograms/ml.

DNA-damaging potential of diethylstilbestrol evaluated in the germ cell unscheduled DNA synthesis assay.

Despite the fact that the nonsteroidal estrogen diethylstilbestrol (DES) exerts its toxic effects primarily on the reproductive system, little is known about the possible interference of this compound with germ cell DNA. The measurement of unscheduled DNA synthesis (UDS) in spermatocytes and early spermatids of mice germ cells is a valid indicator for the DNA-damaging potential of a compound. UDS occurrence was thus determined after IP administration of 10, 30, 60 or 180 mg/kg DES to male mice. Tritiated thymidine ( [3H]dThd) was then injected into the testes, the spermatozoa were serially collected, the sperm heads isolated, and UDS determined by the amount of [3H]dThd incorporation. [3H]dThd measurements in germ cells of mice which were treated with 10 mg/kg DES were comparable to those of the controls. Higher incorporation of [3H]dThd, indicating UDS, was measured in sperm cells which had been spermatocytes at the time of treatment with 30 and 60 mg/kg DES; this increase was statistically significant at 60 mg/kg. Administration of 180 mg/kg DES caused [3H]dThd incorporation which was comparable to that of the controls, suggesting that DES interfered with repair mechanisms or delayed spermatogenic cycles at high dose levels. General toxicity was manifested in a dose-dependent decrease of the sperm cell numbers in the spermatogenic stages investigated. This study provides evidence that DES, or its metabolite(s), reached the germ cells of adult mice in sufficient amounts to produce DNA damage. The levels of radioactivity measured were comparable to those measured after cyclophosphamide treatment, but [3H]dThd incorporation was about 10 times less than in methylmethane sulfonate-treated animals.

Postimplantation embryo culture for the assessment of the teratogenic potential and potency of compounds.

Whole rat embryos cultured during the early stages of organogenesis were subjected to a panel of selected chemicals. Of seventeen known in vivo teratogens, seventeen also induced specific malformations in embryos grown in culture. Of ten chemicals which were reported to be negative in in vivo rat teratogenicity studies, eight also did not provoke dysmorphogenic effects in vitro. Of five additionally tested retinoids, all induced multiple malformations. However, concentrations used to induce these effects varied considerably, isotretinoin inducing malformations at 10(-5) M and arotinoid at 10(-11) M. The results indicate qualitatively as well as quantitatively a high predictability of this in vitro system and suggest that the postimplantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

Post-implantation embryo culture: validation with selected compounds for teratogenicity testing.

1. Some chemical compounds selected by experts for the validation of in vitro teratogenicity testing were investigated in whole rat embryos cultured during the early stages of organogenesis. All sixteen known in vivo teratogens tested also induced specific malformations in embryos grown in culture. 2. Of the nine compounds which were negative in in vivo rat teratogenicity studies, none provoked dysmorphogenic effects in cultured embryos. Abnormal development of the embryos was only observed with these compounds at concentrations also high enough to affect significantly overall growth and/or differentiation. 3. The results showed a high predictability of this system for the compounds tested and suggest that the post-implantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.

Evidence for reopening of the cranial neural tube in mouse embryos treated with cadmium chloride.

Use of the whole-embryo culture technique resulted in experimental evidence that the pathogenesis of exencephaly in mouse embryos after cadmium chloride treatment results from reopening of the cranial neural tube.

Effects of cyproheptadine on the rat yolk sac membrane and embryonic development in vitro.

Electron-microscopic examinations of rat embryonic yolk sacs treated in vitro with 1.5 X 10(-5) M cyproheptadine showed proliferation of the lysosomal structures; other organelles remained unaffected, and also overall yolk-sac growth and vascularization were comparable to non-treated samples. Radioactive measurements with 125I-labelled albumin showed that yolk sacs and embryos of the cyproheptadine-treated group incorporated less radioactivity than the controls. Embryos inside the yolk sacs, treated either for 24 or 48 h, were severely retarded in growth and differentiation (approximately 50% of the controls). It is suggested that the specific action of cyproheptadine on yolk-sac lysosomal structures, combined with reduced macromolecular transport, is the cause of inhibited embryonic development.

In vitro exposure of male and female mice gametes to cadmium chloride during the fertilization process, and its effects on pregnancy outcome.

The effect of cadmium chloride (Cd) on gamete fusion in vitro was evaluated, with further observations of the embryonic development and assessment of the pregnancy outcome of the in vitro fertilized mice. Oocytes were recovered from superovulated B6C3F1 females. Of 1210 control oocytes, 53.2% cleaved into two-cell stage embryos. Of these, 46.6% developed into blastocyst stage embryos which were then surgically transferred to pseudopregnant female CD-1 mice. Of a total of 63 implanted embryos, 8 (12.7%) developed in utero to live fetuses. Teratological examinations of these "test-tube" mice revealed no signs of abnormalities caused by in vitro culture. Male and female gametes were exposed to 0.4, 0.8, or 1.6 microM of Cd and a decrease in sperm motility was noted in the 1.6 microM group. Nevertheless, even in the highest concentration used, 56.4% of the ova cleaved into the two-cell stage, thus indicating no effect of Cd on initial gamete interaction. Gametes that had been treated with 0.4 and 0.8 microM Cd developed to blastocysts at rates comparable to that of the controls. In the 1.6 microM group, however, only one (3.2%) of the two-cell embryos developed to the blastocyst stage. Blastocysts from 0.4 microM Cd-treated gametes were then transferred to surrogate dams. Statistically significant blastocyst losses were recorded during the implantation period, whereas the pregnancy rate and the numbers of resorbed and live fetuses, were comparable to those of the controls. The offspring exhibited no malformations, and their body weights remained within the control values.