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Biomedical subjects

D Neubert

Publications and source records attributed to D Neubert.

At least 145 records · Page 8Linked to original sources
PubMed1983

Interference of acetoxyalkyl-nitrosamines with limb bud differentiation in organ culture.

alpha-acetoxynitrosamines have been used as model compounds to study biological activity of N-nitrosamines. After hydrolytic cleavage they yield an "active intermediate" (the hydroxynitrosamine) which presumably also arises as metabolite of N-nitrosamines. We tested eight N-nitrosamines mono-substituted at the alpha-carbon with an acetoxy group for their teratogenic potential in a mouse limb bud culture system. The following results were obtained: 1. The methyl compound (MOAc-MNA) - a derivative of dimethynitrosamine - proved to be the strongest teratogen in this group of chemicals. 2. The tertiary-butyl derivative - releasing a carbonium ion of low chemical reactivity - possessed the lowest activity in our test system. 3. The primary alpha-acetates with unbranched side chains (POAc-MNA or BOAc-MNA) were more active than the corresponding derivatives with branched side chains. 4. The secondary alpha-acetate (EOAc-ENA) was clearly less active than the primary alpha-acetate (EOAc-MNA). 5. A teratogenic potential could also be demonstrated in the organ culture system with the cyclic derivative: N-nitroso-alpha-acetoxy-pyrrolidine (NAPYR). 6. With the use of limb buds from 12-day-old mouse embryos the explants showed the highest susceptibility to the teratogens on the first day of culture. No effect could be produced if the substances were added to the culture medium at the third day of culture or later. 7. When initiating the culture with limb buds from 11-day-old mouse embryos the concentrations needed to induce typical effects were lower than those tested with 12-day-old explants. 8. Typical and pronounced impairment with morphogenetic differentiation could be induced by MOAc-MNA if the substance was present in the medium for less than 60 min - the shortest period tested was 15 min. 9. A different abnormality pattern could be induced with the various substances tested. This may partly be explained by a quite different stability of the compounds in the test system. 10. N-Nitrosamines must be expected to be highly teratogenic if they can be "activated" in embryonic tissues. Such an activation into potent electrophilic agents does not occur in rodents under normal conditions. It will be interesting to study if such activity can be "induced" in embryonic tissues of rodents or if it is already present in embryonic tissues of primates.

Animals↗
PubMed1981

Manifestation of carcinogenesis as a stochastic process on the basis of an altered mitochondrial genome.

Computer calculations are used to show the feasibility of a concept which explains the manifestation of a pathological cell function from a latent state by the phenomenon of extrachromosomal inheritance (through the mitochondrial genome) in mammalian cells. A hypothesis is submitted in which this principle is applied to the process of carcinogenesis. According to this concept, the manifestation of a tumor cell--after the initiation stage--entirely depends on stochastic events, i.e., random distribution of mitochondria during cell divisions, with an accumulation of the lesion in a few out of many cells. We feel that this concept comprises a better explanation of many characteristics and peculiarities of the phenomenon of carcinogenesis than do attempts which explain tumor formation as a phenomenon caused by mutation in a nuclear genome. A consideration of the principles presented automatically leads to a number of specific consequences with regard to carcinogenesis. Some of these consequences are discussed. They include: 1. the process of malignant transformation should not be irreversible for all the cells of a progeny; 2. the number of mitochondria in a cell type should be inversely correlated to tumor frequency; 3. the latent period should mainly be determined by the cell division rate and the "extent" of the initiating event; 4. susceptibility to carcinogenesis may be substantially higher if the number of mitochondria per cell line is increasing or decreasing, i.e., during the embryonic and fetal periods; 5. heterogeneous types of cells may arise from a single "initiated" cell, and 6. the process of malignant transformation should not necessarily be confined to one generation of the species. In addition, experimental approaches to support the submitted concept are suggested.

Animals↗
PubMed1980

Estimation of creatine phosphokinase and hydroxyproline in the developing limb: its use in evaluating the effect of teratogens on myogenesis and chondrogenesis.

Forelimbs of mouse fetuses were examined for tissue-specific, drug-induced alterations in their biochemical composition. The activity of the enzyme creatine phospholinase (CPK; to estimate myogenesis) and the content of hydroxyproline (HP; to estimate chondrogenesis) were compared in homogenates of control and treated mouse-fetus forelimbs on day 14 of gestation. In addition, the content of DNA, RNA, and protein was also measured. Injection of 6-aminonicotinamide (6-AN) (15 mg/kg) on day 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a signific 10 resulted in an overall growth retardation in day 14 fetuses and all biochemical parameters tested were reduced. The ratio of PH:CPK was unaffected by 6-AN treatment. Retinoic acid (vitamin A acid; 100 mg/kg), administered to pregnant female mice on day 10, produced severe forelimb defects and resulted in a significant reduction in day 14 forelimb HP and RNA content, without altering CPK, DNA, or protein; thus, the HP:CPK ratio was decreased. These results indicated that 1) 6-AN nonspecifically retards growth and cyto-differentiation in limbs; 2) retinoic acid inhibits synthesis of collagen and RNA; 3) retinoic acid has a differential effect upon chondrogenic and myogenic tissues of the limb, and 4) the comparison of HP content and CPK activity in tissue homogenates is an acceptable method of evaluating teratogenic compounds for selective effects.

6-Aminonicotinamide↗
PubMed1980

Testing for embryotoxicity.

No animal species exists with which the situation in man can be completely mimicked. This also holds true for non-human primates. With animal experiments only certain aspects of the whole complex situation can be analyzed. In order to achieve this successfully, animal species and experimental set-ups have to be chosen carefully to represent the situation existing in humans in as suitable a model as possible. The more the model deviates from the situation existing in humans, the less will be the predictability. It has to be decided, and this is mainly a political decision, what risk is acceptable. Both terms "risk" and "acceptable" have to be specified. Animal studies beyond routine procedures will have to include the use of more and other species (e.g., primates), and in vitro systems adjusted to the special problem to be solved. Today more information is needed on the pharmacokinetic and "toxico" dynamic properties of old and new chemicals. This will supply data on the embryotoxic/teratogenic doses of a substance or on their non-embryotoxic/teratogenic doses relevant to man. A drastic reduction in the number of chemicals pregnant women are exposed to will greatly enhance the chance to perform experiments on the remaining substances successfully and the chance to obtain valid data.

Animals↗
PubMed1980

Significance of organ culture techniques for evaluation of prenatal toxicity.

A discussion of the applicability of in vitro techniques now available for research in prenatal toxicology is presented. Advantages and disadvantages of the various in vitro methods (such as cultivation of preimplantation embryos, whole embryo culture, and organ culture) as applied to various problems of experimental research are described. As a typical example, the experience gained in our laboratory with the organ culture of mammalian limb buds is detailed. Various aspects of the research with this type of organ culture - e.g., different techniques of culturing, extent of differentiation achieved in culture, induction of abnormalities in culture, supplementing the system with drug-metabolizing capacities and means for quantification of the data - are discussed. It is concluded that certain in vitro techniques using mammalian embryonic tissues are very suitable tools for elucidating the mode of action of teratogenic agents, and that they may serve as a "model" for several basic processes also for the situation probably existing in humans. Such organ culture, and other in vitro methods, provide little, if any advantage over in vivo experiments if a "mass screening" of a possible teratogenic potential of chemicals (hazards for the human population) is attempted.

Abnormalities, Drug-Induced↗
PubMed1976

Circadian fluctuations in the activity of DNA-dependent RNA polymerases I and II in the nuclei isolated from mouse tissues and the significance for pharmacological and toxicological studies on transcription.

For pharmacological and toxicological studies on the effect of drugs on transcriptional processes the basic fluctuation in activity during a 24-h period must be known. We have measured the activity of nuclear and mitochondrial DNA-dependent RNA polymerases in cell fractions from mice kept under defined dark/light and feeding schedules. The following results have been obtained: 1. Circadian variations in the activity of RNA polymerases are especially pronounced in the nuclei of liver tissue. These changes depend predominantly on feeding habits and they are comparatively independent on the dark/light schedule. A maximum in the activity of nuclear DNA dependent RNA polymerases can be observed shortely after the beginning of the feeding period. 2. The nuclei isolated from other tissues, such as brain, kidney or embryos during the late stage of embryogenesis shown considerably smaller, if any, fluctuations in connection with the feeding habits. These tissues seem to be especially suitable for pharmacological or toxicological studies on transcriptional processes. 3. While both nuclear DNA-dependent RNA polymerases shown a strong dependence on the feeding habits no obvious circadian viriations can be observed in the activity of the DNA-dependent RNA polymerase localized in the mitochondrial fractions of the same tissue. The regulation of these polymerases in different cell compartments apparently occurs quite independently. The conclusions for the design of experiments on induction processes occurring in liver tissues, drawn from the results presented, are discussed.

Animals↗
PubMed1976

In vitro system for toxicological studies on the development of mammalian limb buds in a chemically defined medium.

In organ culture systems using the Trowell setup, morphogenetic differentiation (which largely mimics the development reached in vivo within 2--3 days) can be obtained in limb buds of mouse embryos during a culture period of 6 days. We succeeded in improving the technique and in achieving good differentiation of the limb buds in a chemically defined culture medium from which all additions of the heterologous serum used in previous studies were omitted. With this technique of using a chemically defined medium, the following results were obtained: (1) A high degree of reproducibility can be obtained in the grade of differentiation if the experimental conditions are standardized and limb buds of the same developmental stage (somite stage) are used. (2) The technique is applicable to species other than mice, such as rats and rabbits, however, the results obtained so far are not so satisfactory as those acquired with mouse limb buds. (3) In an attempt to offer some colloid osmotic pressure, macromolecules like polyvinylpyrrolidone (Periston), polydextrans (Macrodex) or polypeptides (Haemaccel) were added to the culture medium. None of these macromolecules had a beneficial effect on the differentiation of the limb buds in vitro; in the case of Macrodex the differentiation was even impaired. (4) In comparison with the development in a serum-containing medium, the results attained with a chemically defined medium are just as good when judged from microscopical and some biochemical studies (total content of DNA, RNA, protein, and protein-bound hydroxyproline in the explants). The applicability of the test system for the evaluation of embryotoxic effects is discussed.

Animals↗
PubMed1976

Activity of nuclear DNA-dependent RNA polymerases in mouse limb buds differentiating in vivo or in organ culture.

1) A method was developed to measure the activity of DNA dependent RNA polymerase reactions I (alpha-amanitin-insensitive) and II (alpha-amanitin-sensitive) in homogenates of very small amounts of tissue. This technique was used to study the activity of these polymerase reactions in limb buds from mouse embryos developing in vivo or in an organ culture system. This culture system allows the morphogenetic differentiation of limb buds in early stages of embryonic development (day 10-11 of gestation greater than or equal to 37 - 45 pairs of somites) from a blastema stage to well recognizable cartilaginous bone analgen. 2) An increase in the activities of both types of RNA polymerases was found to occur in vivo in limb buds on day 12 of gestation when compared with the activity measured on day 11 of gestation. A similar increase with a maximum about 24-48h after initiation of the cultures could be observed in the explants differentiating in organ culture. To our knowledge this is the first time that induction processes proceeding in mammalian embryonic tissues could be correlated with an increased activity of RNA polymerases. 3)A rough estimate shows that the rate of transcription due to RNA polymerase I measured in homogenates or in isolated nuclei (measured with substrate concentrations giving about 80% of the maximal rate) would account for the assumed rate of rRNA synthesis in vivo. The measurement of the RNA polymerase activity in isolated nuclei, thus, may give some information about the transcriptional processes occurring under "physiological" conditions. 4) The mammalian organ culture system presented provides a suitable model for studying embryonic differentiation processes.

Amanitins↗
PubMed1975

Symmetries of genetic code-doublets.

The fact that 64 base triplets code only about 20 essential amino acids implies a strong degeneracy of certain base doublets. It is shown that the set of degenerate base doublets and the set of non-degenerate base doublets are highlly structured. A mathematical formalism is introduced which allows a systematic description of the consequences of an exchange of bases in a doublet. By this formalism it is shown that the two mentioned set have in fact the same structure.

Biological Evolution↗