Biomedical subjects

R Storeng

Publications and source records attributed to R Storeng.

30 records · Page 2Linked to original sources

PubMed1990

New colorimetric cytotoxicity assay for anticancer-drug screening.

We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

Adenocarcinoma↗

PubMed1989

Altered regulation of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines.

The cytochrome P450 (CYP) systems catalyze the metabolic transformation of a wide variety of xenobiotics including procarcinogens present in cigarette smoke condensate as well as atmospheric pollutants. The CYP1A1 isoenzyme is of particular interest because it has been implicated as a risk factor in the etiology of lung cancer in heavy cigarette smokers. The identification and expression of the structural CYP1A1 gene in either normal human lung or lung cancer cells has not been reported. Because of its potential significance in human lung cancer, we investigated the expression of the CYP1A1 structural gene in 24 established human lung cancer cell lines including 15 non-small cell (eight adenocarcinomas, three large cell undifferentiated carcinomas, two bronchioloalveolar cell carcinomas, and two squamous cell carcinomas) and nine small cell lung carcinomas. CYP1A1 mRNA was detected in 14 of 15 (93%) of the non-small cell lung carcinoma cell lines examined following 24-hour treatment with benz[a]anthracene (BA) and in nine of 15 (60%) of the non-small cell lines cultured without an inducer in the medium. When the small cell lung cancer lines were evaluated for CYP1A1 gene expression, two of nine (22%) expressed detectable CYP1A1 mRNA in both BA-induced cell cultures and constitutive (control) cultures. A positive correlation was noted between BA-induced CYP1A1 mRNA levels and the corresponding aryl hydrocarbon hydroxylase activity expressed as absolute BA-induced enzyme activity (r = 0.74; P less than .01; n = 24), which further demonstrated that CYP1A1 mRNA expression reflects CYP1A1 enzyme activity in the individual cell lines. These observations represent the first known demonstration of constitutive (non-induced) CYP1A1 gene expression in human cells and suggest altered regulation of the CYP1A1 gene in selected lung cancer cell lines. These human pulmonary carcinoma cell lines, which have documented regulatory defects, could be useful for further identification of the mechanisms associated with CYP1A1 gene regulation.

Adenocarcinoma↗

PubMed1989

A human tumor lung metastasis model in athymic nude rats.

Experimental lung metastases regularly developed in athymic Han:rnu/rnu Rowett rats after i.v. injection of LOX human malignant melanoma cells. When 5 x 10(5) tumor cells were injected into 4-week-old rats, 89% of the animals died of lung tumors, with a mean survival time of 18 days. With 5- and 6-week-old rats, however, the fraction of animals that died decreased to 80 and 46%, with mean survival times of 35 and 38 days, respectively. The number of detectable lung colonies in each animal was about 35 in 5- and 6-week-old animals, compared to nearly 300 in 4-week-old rats. In the latter, a correlation was found between the number of tumor cells injected and the number of detectable lung colonies. The capacity of the LOX tumor to grow s.c. and to form experimental lung metastases was, by and large, similar in young nude rats and in nude mice, and no significant difference in morphology between the different tumors in the two species was seen. A high-resolution radiographic method was used to visualize lung colonies in the nude rats, and single tumors with diameters as small as 2-4 mm could be detected. By this method, for the first time, the effect of chemotherapy on a human tumor growing in a visceral organ of a rodent host could be followed by repeat X-ray examinations, mimicking a situation commonly faced in the clinic. This procedure may prove particularly useful for experimental chemotherapy studies, and may be extended to other human tumors that frequently metastasize to the lungs. Indications were obtained that some host-specific differences in tissue-preferenced growth might exist, a possibility that will be further explored.

Animals↗

PubMed1987

Effects of 3H-thymidine on preimplantation mouse embryos in vitro.

The effect of 3H-thymidine on in vitro development of preimplantation mouse embryos was studied. Two-cell and 4-8-cell embryos from B6CBA/F1 mice were continuously exposed to 3H-thymidine in medium containing 3H-thymidine in concentrations ranging from 10-500 nCi/ml. The effect of the radioactive precursor on embryo development to the blastocyst stage was studied by morphological observation, counting the blastocyst cell number and measuring 3H-thymidine incorporation. The continuous presence of 3H-thymidine significantly inhibited development of 2-cell and 4-8-cell embryos to the blastocyst stage. Embryos cultured from the 2-cell stage were more sensitive to 3H-thymidine than those exposed from the 4-8-cell stage. Even in morphologically normal blastocysts the cell number was significantly reduced. A 2 hr pulse of 100 nCi/ml 3H-thymidine at the blastocyst stage, did not affect the blastocyst formation or the blastocyst cell number and the amount of incorporated 3H-thymidine was sufficient to provide a reliable quantitation of DNA synthesis during the culture of preimplantation embryos in vitro. Continuous incubation with 3H-thymidine in order to measure DNA synthesis of preimplantation mouse embryos should be avoided when DNA synthesis is used as a means of evaluating toxic effect of an agent. Adverse radiation effects by 3H-thymidine on preimplantation mouse embryos during toxicity testing can be avoided by pulse labelling.

Animals↗

PubMed1987

Toxic effects of lipopolysaccharide from Bacteroides intermedius and Escherichia coli assessed in the pre-implantation mouse embryo culture system.

A comparative study on the toxic effects of endotoxin (lipopolysaccharide, LPS) from the strictly anaerobic Bacteroides intermedius BM1 and from Escherichia coli 055 was performed. Pre-implantation mouse embryos at the 2-cell stage from LPS responder (C57BL/6J) and low responder (C3H/Hej) mouse strains were exposed to the endotoxins, and development was observed during 72 h in vitro. Toxic effects of both endotoxins on embryos from both mouse strains were demonstrated. B. intermedius LPS was less toxic than E. coli LPS. The C3H/Hej embryos were more susceptible to endotoxins than the C57BL/6J embryos. A biphasic dose-response relationship was observed, as 100 micrograms/ml LPS was less embryotoxic than 1, 10 or 500 micrograms/ml, suggesting more complex mechanisms of effect than non-specific cell toxicity.

Animals↗

PubMed1986

Effect of Shigella toxin on preimplantation mouse embryos in vitro.

Preimplantation mouse embryos at the 4-cell to 8-cell stage were exposed to Shigella dysenteriae toxin at concentrations of 0.001-100 pg/ml in vitro. The effect of the toxin was studied by morphological observation of the embryos to the blastocyst stage, by assessing protein synthesis with 14C-leucine incorporation, and by measuring embryonic adenosine triphosphate (ATP) content. Preimplantation mouse embryos were highly sensitive to the toxin. All variables investigated were adversely influenced by the toxin. After a lag period of 24 hr, 0.01 pg/ml toxin inhibited development to the blastocyst stage and protein synthesis. Toxin concentrations of 1.0 pg/ml resulted in a significant decrease in ATP content.

Adenosine Triphosphate↗

PubMed1985

Sensitivity of preimplantation mouse embryos to abrin.

The effect of the plant toxin abrin on preimplantation mouse embryos in vitro was studied. Two-cell embryos from C57BL/6J or B6CBA/F1/Bom female X B6CBA/F1/Bom male mice were exposed to abrin for 72 hrs in vitro, in medium containing abrin in concentrations ranging from 0.1 to 10,000 pg/ml. Two-cell mouse embryos were also exposed to corresponding concentrations of ricin in vitro. The effect of abrin was evaluated by daily morphological observation of the embryos up to the blastocyst stage and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. The effect of ricin was evaluated by morphological observation of the embryos up to the blastocyst stage. The results showed that 2-cell mouse embryos were highly sensitive to abrin, 1 pg/ml being the lowest concentration of the toxin that significantly inhibited the development of 2-cell embryos to the blastocyst stage. With ricin statistically significant inhibition of blastocyst formation was obtained at a concentration of 10 pg/ml. The ID50 for abrin was calculated to be 24 pg/ml and for ricin 47 pg/ml. A dose-dependent lag period was observed before the effect of abrin or ricin on the formation of blastocysts became evident. Incorporation of 3H-thymidine, 3H-uridine and 14C-leucine into DNA, RNA and protein, respectively, was also inhibited by abrin after different time intervals in culture.

Abrin↗

PubMed1984

Recovery of mouse embryos after short-term in vitro exposure to toxic nickel chloride.

The development of preimplantation mouse embryos in vitro was adversely affected by the addition of nickel chloride (NiCl2 X 6H2O) to the culture medium. For day 3 (4-8 cell) embryos developmental cessation occurred after 48 h in culture, in NiCl2 X 6H2O-containing medium. However, transfer to NiCl2 X 6H2O-free medium after 5 min, 1 h, and 3 h exposure, resulted in regaining of the developmental capacity for a proportion of the exposed embryos. The in vivo development, in pseudopregnant recipients, of in vitro nickel-exposed embryos was not significantly different from that in control embryos. The results indicated that the effect of NiCl2 X 6H2O on the development of day 3 mouse embryos in vitro was reversible after a short exposure period.

Animals↗

PubMed1983

Freezing of mouse embryos in the oviduct.

The survival of 2-cell and 4-8-cell mouse embryos after freezing and storage in liquid nitrogen, -196 degrees C, was examined. Both isolated embryos and embryo-containing oviducts were frozen to -40 degrees C at a cooling rate of 0.3 degrees C/min, and then transferred to -196 degrees C for one week. They were rapidly thawed in a 37 degrees C water bath at a rate of approximately 275 degrees C/min. Survival was assayed by in vitro cultivation of thawed embryos. The results indicated that freezing 8-cell mouse embryos directly or in the oviducts, is a reliable procedure for storing 8-cell embryos. By the procedure used, survival rates of 64% for the directly frozen embryos and 66% for the embryos frozen in the oviducts were obtained.

Animals↗

PubMed1981

Nickel toxicity in early embryogenesis in mice.

The development of mouse embryos was studied after intraperitoneal injection of nickel chloride in the preimplantation period. A single intraperitoneal injection of NiCl2 . 6H2O in 0.154 M NaCl corresponding to 20 mg/kg body wt was given to groups of female mice on days 1, 2, 3, 4, 5 or 6 of gestation. Control groups were injected with 0.154 M NaCl. Caesarean section was performed on day 19 of gestation and the following parameters were recorded: implantation frequency, frequency of early and late resorptions, frequency of liver normal fetuses, abnormal fetuses and stillborns, and the weight of each fetus. The implantation frequency of females treated with nickel chloride on the first day of gestation was significantly lower than that of the controls. The size of the litters in the control groups was larger than that of the nickel treated dams, significant difference being observed on days 1, 3 and 5. NiCl2 . 6H2O injection also resulted in diminished body weights of fetuses on day 19 of gestation. The groups of nickel treated mice had a larger frequency of both early and late resorptions and the frequency of stillborn and abnormal fetuses exceeded that of the control groups. This study shows that, by the procedure used, nickel chloride may influence mouse embryos during the passage through the oviduct with subsequent effect on the development after implantation.U

Abnormalities, Drug-Induced↗

PubMed1980

Effect of nickel chloride and cadmium acetate on the development of preimplantation mouse embryos in vitro.

Preimplantation mouse embryos were used to investigate the toxic effect of nickel chloride and cadmium acetate on early embryo development in vitro. Embryos at the 2- and 4-8 cell stage were cultured in approximately 0.05 ml of mouse embryo culture medium (No. 16), overlaid with paraffin oil and incubated in a humidified atmosphere of 5% CO2 in air for 48 h. NiCl2 . 6H2O was added to the culture medium at concentrations of 10-1000 microM, Cd(CH3COO)2 . 2H2O at concentrations of 10-50 microM. Morphological criteria were used to check embryonic development. Ten micromolars of nickel chloride affected adversely the development of Day 2 embryos (2-cell stage), whereas 300 microM was needed to affect Day 3 embryos (8-cell stage). Toxic effect of cadmium acetate on Day 2 embryos was observed at a concentration of 10 microM.

Animals↗

PubMedDate not supplied

Inhibitory effect of ricin on the development of preimplantation mouse embryos.

Preimplantation mouse embryos were exposed to ricin, a plant toxin, in vitro and in vivo. The effect was evaluated by morphological observations of the exposed embryos as well as by means of protein synthesis. Ricin was highly toxic to the preimplantation mouse embryo in vitro, the effect being greater on the 2-cell than on the 4-8 cell stage. Intraperitoneal injection of ricin induced a small number of fetuses with exencephaly.

Abnormalities, Drug-Induced↗