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Biomedical subjects

E Borenfreund

Publications and source records attributed to E Borenfreund.

At least 37 records · Page 2Linked to original sources
PubMed1987

Short-term quantitative in vitro cytotoxicity assay involving an S-9 activating system.

Mouse 3T3 fibroblasts grown in 96-well microtiter plates are used for a test which incorporates an S-9 mixed function oxidase system into a neutral red viability assay for the assessment of the acute cytotoxicity of xenobiotics in vitro. This sensitive, quantitative, semi-automated assay was suitable for the rapid screening of a broad spectrum of substances, including pharmaceuticals, carcinogens, and anti-neoplastic agents. The test was applicable to the analysis of toxic ranges, for the detection of biotransformability of parent compounds and for the evaluation of the cytotoxic effects of chemotherapeutic agents.

Animals↗
PubMed1987

Polycyclic aromatic hydrocarbon in vitro cytotoxicity to bluegill BF-2 cells: mediation by S-9 microsomal fraction and temperature.

The in vitro cytotoxicities of various polycyclic aromatic hydrocarbons (PAHs) to bluegill sunfish BF-2 cells were determined with the neutral red assay, which was modified by the incorporation of an S-9 microsomal fraction. Whereas the PAHs per se were weakly cytotoxic, the presence of the S-9 fraction in the incubation mixture increased the cytotoxicity of many of the PAHs, presumably due to the formation of active metabolites. In addition, the cytotoxicity of the PAHs (whether in the absence or presence of the S-9 microsomal fraction) was potentiated if the 6 h exposure was at 37, rather than at 26 degrees C.

Animals↗
PubMed1987

In vitro cytotoxicity of organic pollutants to bluegill sunfish (BF-2) cells.

BF-2 cells, an established cell line derived from bluegill sunfish, (Lepomis macrochirus), were exposed to 18 organic toxicants, with cytotoxicity being assayed by the neutral red (NR) technique. Based on the concentration of toxicant that reduced lysosomal uptake of neutral red by 50% (NR50), the rank order of cytotoxicity was methyl mercury greater than pentachlorophenol greater than 2,3,5,6-tetrachlorophenol greater than 2,3,5-trichlorophenol greater than 2,3-dinitrotoluene greater than 2,4,6-trichlorophenol greater than 2,4-dichlorophenol greater than 2,4-dichlorotoluene greater than 6-chloro-3-hydroxytoluene greater than o-chlorotoluene greater than 4-chlorophenol greater than 2-chlorophenol, 2,4-dimethylphenol greater than 2,4-dinitrophenol greater than 4-nitrophenol greater than 3-methylphenol greater than phenol greater than toluene. Published in vivo LC50 values were identified for 11 of the 18 test agents and, with the exception of 2,4-dinitrophenol, there was a good correlation between the in vitro cytotoxicity of the test agents and their in vivo waterborne acute toxocity. The potencies of the substituted phenolics and chlorinated toluenes as determined by the in vitro cytotoxicity assay correlated strongly with their log octanol/water partition coefficients (log P). However, the toxicity of 2,3-dinitrotoluene in vitro, and apparently also in vivo, was not a function of its log P value.

Animals↗
PubMed1987

Butyrate-induced cytoarchitectural reorganization of Mallory body-containing rat hepatic tumor cells.

Diethylnitrosamine (CAS: 55-18-5)-transformed 72/22 rat hepatic tumor cells undergo marked cytoarchitectural changes during exposure to sodium butyrate in vitro. Butyrate treatment of this cell line resulted in an increased cell size, volume, and protein content and in structural reorganization within both the intermediate filament and microfilament networks resulting in the generation of a more normal appearing hepatocytic phenotype. Induced changes in the microfilament system involved the accumulation of F-actin at the cellular margins in the form of a peripheral band and in the development of an extensive, predominantly centralized network of thickened cytoplasmic filament bundles. Such butyrate-induced changes in hepatic tumor cellular morphology and microfilament organization were reflected in a 26-51% increase in the amount of cytoskeletal-associated actin in 72/22 cells, as determined by flow cytofluorimetry of permeabilized intact cells or by scanning densitometry of the electrophoretically separated, detergent-resistant cytoskeletal protein fraction, respectively. It is unlikely that this increase in cellular microfilament content was due to a direct effect of butyrate on actin polymerization per se since butyrate (in final concentrations equal to that used in culture) did not alter either actin monomer-polymer transitions or the nucleation reaction in a defined in vitro polymerization assay. The available data suggest that butyrate may regulate the synthesis or modulate the actin-binding capacity of microfilament-associating proteins in cultured cells. Butyrate-induced "normalization" of 72/22 cytoarchitecture was previously shown to be reflected in a reduction or loss in the expression of specific growth traits characteristic of the transformed phenotype. The experimental reversal of defined cytoarchitectural abnormalities and transformed growth characteristics of 72/22 cells by butyrate provided an in vitro model to elucidate both particular cytoskeletal events associated with epithelial cell transformation and the mechanism of action of apparent differentiation-inducing agents, such as sodium butyrate, on responsive tumor cells.

Actins↗
PubMed1986

Alterations in growth rate and cell cycle kinetics of rat liver tumor cells cultured in ethanol-containing medium. In vitro model of proliferative restriction in response to ethanol exposure.

Mechanisms related to the growth suppressive effect of acute ethanol exposure on liver cells were investigated using an established line of ethanol-sensitive rat hepatic tumor cells (32IIIA) and recently developed cytochemical methods for analysis of hepatocyte cell cycle kinetics. Exposure of exponentially growing 32IIIA cells to ethyl alcohol (range 10-100 mM in the growth medium) for a period of 3 days resulted in concentration-dependent decreases (4-25%) in final population density and increases (18-35%) in mean population doubling time compared to untreated cells. Viability was unaffected by ethanol exposure in the concentrations indicated and for the duration period utilized, approximating 94% under all experimental conditions. Multiparametric flow cytometric analysis revealed significant ethanol-associated differences in specific growth parameters and growth state compartments of 32IIIA hepatic tumor cell populations. Most prominent was an ethanol-associated and concentration-dependent (a) increase in the fraction of cells in the G1 phase of the cell cycle, (b) increase in the coefficient of variation in the G1 DNA content measurement, and (c) accumulation (in the G1 phase) of cells with a very low mean RNA content. Increases in each of these cytochemically-defined parameters reflected increasing levels of ethanol in the growth medium. This study indicates that the effects of ethanol on cultured cells of hepatic origin are quite complex. It is concluded that the inhibition of proliferation observed during acute ethanol exposure of liver-derived 32IIIA cells in vitro is due to an accumulation of cells in the G1 compartment.

Animals↗
PubMed1986

In vitro cytotoxicity testing of aquatic pollutants (cadmium, copper, zinc, nickel) using established fish cell lines.

The cytotoxicity of cadmium toward cultured bluegill fry (BF-2) cells was determined using several assay endpoints. The concentrations of cadmium causing a 50% decrease in colony formation, cell replication, uptake of neutral red, population growth (as determined by protein analysis), and uptake of [3H]uridine and 50% detachment of cells (as determined by protein analysis) were 0.03, 0.04, 0.08, 0.09, 0.12, and 0.21 mM cadmium, respectively. The neutral red assay was used to compare the relative sensitivities of bluegill BF-2 cells and RTG-2 cells, derived from the rainbow trout, toward four metals. The concentrations of cadmium, zinc, copper, and nickel causing a 50% reduction in the uptake of neutral red were 0.08, 0.19, 0.55, and 2.0 mM, respectively, with the BF-2 cells and 0.18, 0.64, 1.45, and greater than 10.0 mM, respectively, with the RTG-2 cells. The RTG-2 cells were less sensitive to the metals, in particular to nickel. The less stringent temperature requirements for growth, their greater sensitivity to pollutants, and their markedly shorter doubling time in vitro make the BF-2 cells the preferable cell line for ecotoxicity screening of aquatic pollutants.

Animals↗
PubMed1986

Cytotoxicity of metals, metal-metal and metal-chelator combinations assayed in vitro.

A simple, rapid assay, based on the lysosomal incorporation of neutral red by cells, conveniently carried out in 96-well microtiter plates, was used to evaluate the cytotoxic effect of cationic and anionic metal salts on BALB/c mouse 3T3 fibroblasts. Ranking of the metals according to their decreasing potency was based on spectrophotometrically determined absorbance of the neutral red, extracted from surviving viable cells. The rank order was Cd greater than Hg greater than Ag greater than Zn greater than Mn greater than Cu greater than Co greater than Ni greater than Cr(III) for the cationic metals and Cr2O7 greater than CrO4 greater than AsO2 greater than AsO4 greater than SeO3 greater than SeO4 greater than MnO4 for the anionic metals tested. Cationic metals incubated with cultures in medium containing 1% fetal bovine serum (FBS) were 3-4 times more toxic than in medium with 10% FBS. Cadmium served as a representative metal for the use of this assay not only for concentration, but also for time dependent exposures. Thus a 10% cytotoxic effect after 1 h of incubation with 60 microM cadmium was increased to 90% after 6 h. Examination of the effect of metal-metal interaction on cytotoxicity showed a marked reduction of cadmium toxicity by zinc and to a lesser degree, by nickel. The neutral red assay was also effectively used to investigate the effect of the chelators ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA) and 2,3-dimercaptosuccinic acid (DMSA) on cadmium-induced injury. Cytotoxicity by cadmium was completely inhibited by EDTA, and partially by NTA, but DMSA was ineffective. Reduction of copper toxicity by chelation was less efficient than for cadmium. Use of a chelator as therapy against metal poisoning was only partially effective and limited to administration within 2 h after incubation of cells with cadmium. It is believed that the neutral red assay can be a valuable tool for the screening of cytotoxic and potentially therapeutic agents under controlled in vitro conditions.

Absorption↗
PubMed1986

Incubation of rat hepatic tumor cells with ethanol and acetaldehyde in vitro: effects on growth rate, albumin secretion and cellular protein content.

The in vitro response (defined as changes in growth rate, cellular protein content, and albumin secretion) of liver epithelial cells to the putative hepatotoxins ethanol and acetaldehyde were evaluated using the well-characterized 32IIIA 6/2d rat liver tumor clonal cell line. Exposure of hepatic tumor cells to ethanol (50-100 mM) for a period of 3 days reduced final population density (apparently due to a drug-induced increase in mean cell cycle transit time), reduced secretion of albumin, and increased the mean cellular protein content. Since these ethanol-associated effects were also observed in cells cultured in growth medium containing acetaldehyde (0.1 mM), and were inhibited by simultaneous addition of pyrazole, the changes in the parameters measured in this study appear to be induced by products of ethanol metabolism. These data complement recent in vivo studies implicating acetaldehyde as an inhibitor of hepatocyte secretory function. The 32IIIA 6/2d liver cell system, thus, responds to certain hepatotoxic compounds in a manner analogous to the in vivo organ and may facilitate future analysis of molecular mechanisms underlying alcohol-induced liver disease under defined culture conditions.

Acetaldehyde↗
PubMed1985

A battery of potential alternatives to the Draize test: uridine uptake inhibition, morphological cytotoxicity, macrophage chemotaxis and exfoliative cytology.

Four assays that may serve as components of a battery of alternatives to the conventional Draize test were described. The exfoliative cytology assay is a refinement of the Draize test that may provide a more sensitive and more objective end-point. The macrophage migration assay addresses the inflammatory aspects of the physiological response to irritation. The uridine uptake inhibition assay uses a quantitative, reversible end-point to detect the short-term action of agents on cell membranes and cell phosphorylative potential. Finally, the cytological assay serves as a rapid, easily performed general indicator of cytotoxic action. The two latter assays have been demonstrated to correlate very well with Draize test results and with each other for a wide range of test agents.

Animal Testing Alternatives↗
PubMed1984

In vitro cytotoxicity assays. Potential alternatives to the Draize ocular allergy test.

A short-term cytotoxicity assay carried out in multiwell test plates and a supplementary colony forming assay are both useful for screening and range finding of toxic concentrations of test agents. The highest tolerated dose (HTD), a concentration at which only minimal morphological changes were observed, was designated as endpoint in the assay. Epithelial rabbit cornea cells, murine fibroblasts, Chinese hamster lung cells, human hepatoma cells and mouse macrophage cultures were used as targets. Several of the alcohols tested at HTD in the colony forming assay were found to inhibit colony formation. An ID50 of colony formation was used as a quantitative corroborating test. The ranking of 34 toxicants was found to be virtually the same with all cell types examined. This easily reproducible, rapid in vitro test is cost-effective and can be used for preliminary large scale screening of potential toxicants.

Animal Testing Alternatives↗
PubMed1984

The effect of methadone and naloxone on cultured rat liver cells.

Opioids modify hepatic function in vivo, however, a direct effect of this class of drugs on liver cells has not been demonstrated. The potential effect of opioids on liver parenchymal-type cells, therefore, was studied using cultured albumin-secreting rat hepatoma cells. Liver cells were incubated with methadone or naloxone individually or in combination. Dose-response analysis indicated that concentrations of either or both drugs in excess of 2.5 X 10(-5) M were clearly cytotoxic to cultured hepatoma cells. At a concentration (2.5 X 10(-7) M) at which no adverse effect on hepatoma cell viability or growth rate was observed, naloxone, methadone, or naloxone plus methadone produced a 26, 41, and 63% decrease in trichloroacetic acid-precipitable protein (per 10(6) cells), respectively, when compared to control. The inhibitory effect of naloxone, methadone, or naloxone plus methadone on albumin accumulation in the culture medium was greater than the drug-induced reduction of protein synthesis. These studies demonstrate that opioids have several direct effects on cultured hepatoma cells and, therefore, raise the question of whether clinically significant hepatic dysfunction may be produced by the direct action of opioids on the liver.

Albumins↗
PubMed1981

Alpha-fetoprotein and albumin synthesis by heterotransplanted rat liver tumor cells.

Epithelial cells, isolated from the hepatic tissue of rats bearing carcinogen-induced neoplastic liver nodules, were analyzed by immunological methods for the production of specific liver proteins during alternate in vitro-in vivo passage. Heterotransplantation of several long-term rat liver cell lines into nude mice and subsequent re-establishment in vitro resulted in the derivation of hepatocyte cultures expressing both alpha-fetoprotein and albumin. Prior to passage in vivo these cell lines synthesized by one or neither of these proteins in vitro. Alternate in vitro-in vivo passage thus appears to potentiate tissue-specific protein expression by established liver cell lines.

Albumins↗
PubMed1981

Presence of anti-gamma-FA-reactive antigens in spontaneous and carcinogen-induced malignancies of experimental animals.

Immunochemical analysis of various tumors, representing 12 different tissue sites of origin, revealed a gamma-fetal antigen(gamma-FA)-like component in 17 of 23 malignant neoplasms of the mouse. This antigen was identified in extracts of spontaneous and carcinogen-induced murine tumors as well as in extracts of hepatic tumors initiated in the rat with diethylnitrosamine. The occurrence of gamma-FA in 75% of the malignant tumors examined and its presence in the sera of rodents bearing transplanted and primary cancers suggests an associative relationship of potential diagnostic value. This possibility is supported by the observation that immunoprecipitable gamma-FA appeared in the sera of hepatocarcinogen-treated rats several weeks prior to the detection of alpha-fetoprotein.

Animals↗
PubMed1980

Enhanced albumin production by malignantly transformed hepatocytes during in vitro exposure to dimethylsulfoxide.

The murine BW 1 and rat 32III 6/d tumor cell lines, derived from a spontaneous mouse hepatoma and a carcinogen-induced rat hepatocellular carcinoma, were used to investigate the effect of dimethylsulfoxide (DMSO) on liver cells in vitro. After a 4-day exposure to DMSO in final concentrations of 0.5 and 1.0%, BW 1 cell-associated albumin increased by 41.6 and 94.2%; extracellular albumin levels in these same cultures rose by 131.4 and 214.2%. Exposure of 32III 6/d cells to 2% DMSO produced increases in cell-associated and extra-cellular albumin concentrations of 67.8 and 188.7%, respectively. The lack of inducible gamma-glutamyl transpeptidase in BW 1 cells and its decrease in 32III 6/d cultures following DMSO treatment suggests that the DMSO-mediated enhancement of albumin production is not reflective of a random increase in the expression of cellular genes.

Albumins↗