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Biomedical subjects

R Langenbach

Publications and source records attributed to R Langenbach.

At least 91 records · Page 5Linked to original sources

Promoting effect of feeder cells in maintenance of adult rat hepatocytes.

A procedure is described for maintaining primary cultures of adult rat hepatocytes for prolonged periods of time on layer of irradiated mouse fibroblast cell line (C3H/1OT1/2) and on a secondary lung fibroblasts obtained from Sprague Dawley rats. Morphologically and ultrastructurally the cocultivated hepatocytes retained many characteristics of hepatocytes in vivo. Within 24 hours after seeding, the individual cells were attached on the feeder cell layer and the in vivo polarity of the liver cells reappeared. Electron microscope studies demonstrated the appearance of newly developed bile ducts and junctions between hepatocytes as well as between hepatocytes and feeder cells. Histochemically, these cells were positive for glucose-6-phosphatase and for glycogen. After 14 days in culture the hepatocytes could be reseeded onto fresh C3H1OT1/2 cells. In contrast, hepatocytes maintained on plastic substrate lost their glycogen content and the epithelial character of the liver cells after 5 days in culture, and by day 10 this culture became predominantly fibroblastic. It is suggested that hepatocytes maintained on an irradiated fibroblast feeder layer provide a valuable approach for studying the morphogenesis, cytotoxicity, or the metabolism of different chemicals in vitro.

Animals↗

Mutagenic activities of oxidized derivatives of N-nitrosodipropylamine in the liver cell-mediated and Salmonella typhimurium assays.

The mutagenic activity of N-nitrosobis(2-oxopropyl)amine (BOP), N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP), N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosomethyl-2-oxopropylamine (MOP), and N-nitrosomethyl-2-hydroxypropylamine (MHP) was examined in the Ames liquid incubation assay, using hamster liver homogenate for metabolic activation, and in the hamster liver cell-mediated V79 cell assay. At similar concentrations, the cell-mediated assay showed a greater mutagenic response over background to these nitrosamines than did the bacterial assay. Also, the relative mutagenic potency in the cell-mediated assay (MOP > MHP > BOP > HPOP > BHP) correlated better than that in the Ames assay (HPOP > MHP greater than or equal to BOP = BHP = MOP) with overall carcinogenic potency in the hamster (MOP > BOP > HPOP > BHP). The liver cell-mediated assay may be an important adjunct to the battery of short-term tests for carcinogenicity prescreening.

Animals↗

Carcinogenicity of N-nitrosomethyl(2-oxopropyl)amine in Syrian hamsters.

7-Methylguanine was found in hydrolysates of liver and pancreas DNA from Syrian golden hamsters given a single dose of N-[1-14C]nitrosobis(2-oxopropyl)amine (BOP). This led us to examine the carcinogenicity of a potential methylating metabolite of BOP, N-nitrosomethyl(2-oxopropyl)amine (MOP). MOP was found to be a potent pancreatic carcinogen by either single or weekly s.c. injections. A single MOP treatment (25 mg/kg body weight) induced ductular adenomas and/or adenocarcinomas in 80% of the hamsters. A higher incidence of these neoplasms was found in 93% and 87% of animals receiving, respectively, 3.5 and 1.75 mg MOP per kg body weight weekly for life. However, the lower dose (0.87 mg/kg body weight) was less effective, resulting in a 33% tumor incidence. Compared with the known potent pancreatic carcinogen BOP, MOP seemed to have a greater affinity for the pancreas since considerably lower doses were required to induce similar incidences of equivalent pancreatic tumors. Like BOP, MOP caused tumors of the liver (7 to 100% incidence), kidneys (7 to 80% incidence), and vascular system (7 to 27% incidence). However, in contrast to BOP, which was noncarcinogenic to the upper respiratory tract, MOP-treated animals developed a high incidence of nasal cavity tumors (40% after a single treatment and 27 to 100% after weekly injections). The mutagenesis studies using hamster liver cell-mediated V79 cells confirmed the stronger effect of MOP compared to BOP. The assumption that MOP might be a proximate carcinogenic metabolite of BOP could not be substantiated by our methods for determining the in vivo and in vitro metabolites of BOP.

Adenocarcinoma↗

Culture of adult rat lung cells: benzo(a)pyrene metabolism and mutagenesis.

A method is described for obtaining and culturing large numbers of lung cells from normal adult male rats. The lungs were perfused in situ to remove blood cells and then perfused via the trachea with a trypsin-collagenase solution to initiate tissue digestion. The tissue was further digested in the enzyme solution and approximately 2 X 10(8) viable lung cells were obtained per animal. Primary cultures contained a mixed cell population. Through eight subcultures about 70% of the cell population possessed an epithelial-like morphology, whereas the remaining 30% was fibroblast-like. Three clones of epithelial-like cells were isolated at the fourth subculture. The mass culture lung cells and the epithelial-like clone that was studied retained a normal karyotype and did not grow in soft agar. Both the mass culture cells and the epithelial clone metabolized the lung carcinogen benzo(a)pyrene (BP) to water-soluble products. Furthermore, the mass culture lung cells metabolized BP to intermediate(s) which mutated Chinese hamster V79 cells from ouabain sensitivity to ouabain resistance. These lung cell cultures have potential use in cell transformation, mutation and carcinogen metabolism studies.

Animals↗

Benzo[alpha]pyrene antibody inhibition of benzo[alpha]pyrene-induced mutageneis.

An antibody to benzo[alpha]pyrene (BP) was prepared. The isolated antibody showed a specificity for BP and a low reactivity with another carcinogenic hydrocarbon, 7,12-dimethylbenz[alpha]anthracene (DMBA). The BP-antibody inhibited the in vitro cytotoxic and mutagenic activity of BP in both a rat embryo fibroblast- and a rat lung cell-mediated mutagenesis system. A possible correlation of these in vitro findings to the in vivo carcinogenesis situation is discussed.

9,10-Dimethyl-1,2-benzanthracene↗

Maintenance of adult rat hepatocytes on C3H/10T1/2 cells.

A procedure is described for maintaining primary cultures of adult rat hepatocytes on a layer of irradiated C3H/10T1/2 cells. These hepatocytes were capable of metabolizing the liver carcinogen N-2-acetylaminofluorene to water-soluble products and after 14 days in culture could still metabolize approximately 70% of the Day 1 level. Hepatocytes maintained on the C3H/10T1/2 cells were inducible for the liver-specific enzyme tyrosine aminotransferase, and exhibited approximately a 4-fold induction by hydrocortisone during a 10-day culture period. Morphologically, these hepatocytes retained many characteristics of hepatocytes in vivo. By contrast, hepatocytes maintained on plastic lost both N-2-acetylaminofluorene-metabolizing ability and tyrosine aminotransferase activity by Day 5. This was presumably due to degeneration of the hepatocytes and an overgrowth by fibroblasts. The maintenance of morphologically and biochemically functional hepatocytes in culture on feeder cells may provide a valuable approach for studying drug metabolism and liver cell transformation in vitro.

2-Acetylaminofluorene↗

Stimulation of differentiated functions in human melanoma cells by tumor-promoting agents and dimethyl sulfoxide.

Treatment of cultured human HO melanoma cells with the mouse skin tumor promoter phorbol-12-myristate-13-acetate (PMA) at 5 x 10(-10) to 5 x 10(-7) M resulted in a dose-related inhibition of growth and a stimulation of differentiated functions. These included melanin synthesis and formation of dendrite-like structures. Higher doses of phorbol dibutyrate, a less potent tumor promoter, were required to produce an effect comparable to that of PMA for dendrite induction. Phorbol and two other phorbol esters, which lack tumor-promoting activity, were either inactive or elicited a poor response. In addition to morphological changes, treatment with PMA altered glucosamine incorporation into membrane gangliosides. After PMA treatment, glucosamine incorporation increased 8- to 10-fold in the GM3 ganglioside and decreased 2-fold in the GM1 ganglioside, as compared to phorbol or untreated control. Inhibition of cell growth and stimulation of melanin synthesis were also observed after treatment of the HO cells with dimethyl sulfoxide. Unlike the tumor-promoting agents, dimethyl sulfoxide did not induce the formation of dendrite-like structures in the cells. These findings indicate that HO melanoma cells can be stimulated into terminally differentiated cells after treatment with tumor-promoting agents such as phorbol diesters.

Cell Differentiation↗

Ganglioside and morphological changes in mouse embryo cells with time.

Ganglioside and morphological changes were observed in C3H/10T1/2CL8 cells during a 4-year period of maintenance in the laboratory. The cellular morphology at confluency, as determined by scanning electron microscopy, changed from being polygonal and flattened to a fusiform shape with some piling up of cells. The saturation density increased about three-fold and the level of the simplest ganglioside GM3 (N-acetylneuraminylgalactosylglucosylceramide) increased about two-fold. While the reasons for the alterations are unknown, the data suggest a relationship among ganglioside composition, cellular morphology and regulation of cell growth. The possible implications of such biochemical and biological changes on studies comparing normal and transformed cells are discussed.

Cells, Cultured↗

Liver cell-mediated mutagenesis of mammalian cells by liver carcinogens.

A cell-mediated mutagenesis assay using primary cultures of rat liver cells and V79 Chinese hamster cells has been developed. Liver carcinogens and their structural analogues were studied. Mutations in the V79 cells were characterized by resistance to ouabain. Cocultivation of the liver cells and V79 cells in the presence of the carcinogens N-nitrosodimethylamine, N-nitrosodiethylamine, and aflatoxin B1 caused the induction of ouabain-resistant mutants of V79 cells. In the absence of liver cells, the carcinogens did not induce ouabain resistance. The analogues N-nitrosomethyl-tert-butylamine and aflatoxin G2 were not mutagenic. The carcinogens exhibited a dose-dependent enhancement of mutation frequency. The mutation frequency also increased with increasing numbers of liver cells seeded. It is suggested that such an experimental system may be useful for screening for chemical carcinogens.

Adenosine Triphosphatases↗

Scanning electron microscopy of in vitro chemically transformed mouse embryo cells.

A cloned nontumorigenic control cell line of C3H mouse embryo cells (C3H/1OT1/2CL8) and two cell lines derived from it by treatment in vitro with 7,12-dimethylbenz(a)anthracene (DMBA) or 3-methylcholanthrene (MCA) were studied by scanning electron microscopy. Confluent control cells were polygonal in shape and extensively flattened with smooth surfaces. Both in vitro transformants were pleomorphic to fusiform in shape, thicker than the control cells, and lacked contact inhibition. Microvilli of variable length and small marginal ruffles were characteristic surface alterations of the MCA-transformed cells, while blebs and numerous cytoplasmic strands extending between cells were typical of the DMBA transformant. Inoculation of the DMBA-transformed cells into C3H mice and re-establishment of cells from one of the subsequent fibrosarcomas in culture revealed an increased number of microvilli on the surface of the cells and an alteration in growth pattern. Other surface characteristics remained the same. A possible relationship between surface topography and outer membrane glycolipids is discussed.

9,10-Dimethyl-1,2-benzanthracene↗

[Contemporary animal nutrition and its potential hazards to human health (author's transl)].

The growing challenge to secure wholesome food of animal origin in quantities sufficient to feed the ever increasing world population leads to the compelling need of search for new means to enhance animal production. Such an endeavor often involves the use of pharmacologically active agents. As new substances are continuously introduced into agriculture, the necessity clearly arises to reassess the requirements for the approval of compounds likely to appear in the food of man via the edible tissues of animals. Since a number of animal drugs and feed additives have been recently found to show carcinogenic potency, using examples from their own research, the authors discuss problems encountered while planning animal studies for the safety evaluation of chemicals. Among the most important factors to be reckoned with is the metabolism of the test substance. Most carcinogens require metabolic transformation in order to react with macromolecules and, thus, exert their biological action. Similarly, residues of many drugs in animal tissues appear to be various metabolites rather than the parent compounds themselves. At present, it is not known whether many chemical residues are simply stored in different compartments of the carcass as a result of their physico-chemical properties or whether they are covalently bound to vital macromolecules (e.g., nucleic acids). Hence, their biological significance is not quite clear. The enzyme system which metabolizes numerous drugs, pesticides, as well as other endogenous and exogenous substrates is responsible for both the activation and detoxification of carcinogenic chemicals. The delicate balance between these two processes of opposing toxicological consequence is determined by genetic and environmental factors. Depending upon the metabolic profile of chemicals, certain compounds are carcinogenic in one animal species while not in others. The manipulation of their metabolism by physiological (e.g., stress) or pharmacological (e.g., inducers or inhibitors of microsomal enzymes) means can result in a profound change of various biological actions of chemicals (e.g., cytotoxicity, carcinogenicity, mutagenicity). To ascertain that potential toxicological hazards to human health by animal drugs and feed additives will be recognized during the phase of testing, appropriate test animals have to be selected with great care. It is indispensible that the metabolic break-down of the investigational substance proceeds via similar pathways in both test animals and the target species. This will assure that the same metabolites which, in the form of residues in food, man might be exposed to will have ample opportunity to exert their possible adverse effects to the experimental animals during a life-long feeding of the test substance. Therefore, it can, with a reasonable certainty, be assumed that, in experiments performed under such precautionary measures, toxico-pharmacological properties relevant to human safety evaluation will not remain undetected.

9,10-Dimethyl-1,2-benzanthracene↗

Gangliosides of chemically and virally transformed rat embryo cells.

The gangliosides of control rat embryo cells, 3-methylcholanthrene, Rauscher leukemia virus, and combined 3-methylcholanthrene-Rauscher leukemia virus transformants were examined using [14 C]glucosamine as a tracer. All four cell lines exhibited a complex pattern of gangliosides. While N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosyl-glucosyl-ceramide was the major ganglioside in the control cell line, N-acetylneuraminyl-galactosyl-glucosyl-ceramide was the major ganglioside in the three transformants. The 3-methylcholanthrene transformant possessed a ganglioside pattern different from that of the Rauscher leukemia virus transformant. Hydrolysis of the gangliosides indicated that galactosamine, N-acetyl-and N-glycolylneuraminic acid were the labeled components in all cell lines.

Acetylgalactosamine↗

Cytotoxic and oncogenic activities of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane and metabolites to mouse embryo cells in culture.

The cytotoxic and oncogenic activities of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and its metabolites 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and bis(p-chlorophenyl)acetic acid, were studies at various equimolar concentrations in an in vitro mouse embryo cell culture system that was malignantly transformable. DDD was the most active compound in producing transformation, whereas DDT and DDE showed slight activity. However, none of the transformed foci had the typical malignantly transformed morphology produced by 7,12-dimethylben[a]anthracene and did not produce tumors when inoculated into syngeneic mice.

Animals↗

Nonspecific inhibition of DNA repair by promoting and nonpromoting phorbol esters.

The effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and its nonpromoting structural analogue, 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (Me-TPA), on N-acetoxy-2-acetylaminofluorene-elicited DNA repair and replicative DNA synthesis was measured in normal human fibroblasts. Both esters inhibited DNA repair synthesis, and Me-TPA was nearly as effective as TPA. In addition, TPA inhibited replicative DNA synthesis. These findings showed that inhibition of DNA repair synthesis may not be a major factor in the mechanisms of action of tumor promoters.

Acetoxyacetylaminofluorene↗