Dose-response Curves For Induction Research Articles

Assessment of MCF-7 cells as an in vitro model system for evaluation of chemical oxidative stressors

Studies have been carried out to establish an experimental in vitro model system for routine testing of oxidative stress inducers through biochemical analysis using human breast carcinoma (MCF-7) cell line. Hydrogen peroxide (H 2 O 2 ) has been chosen as a test chemical oxidant to assess the level of induced glutathione (GSH), lipid peroxidation (LPO), superoxide dismutase (SOD), catalase, lactate dehydrogenase (LDH) release and cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays in MCF-7 cells. Cells were exposed with H 2 O 2 in the range of 0.1 to 1.6 mM in MEM culture medium up to 24 h. The sensitivity of the system was examined by determining the dose response curve for induction of mitochondrial activity and growth inhibition. The concentrations of H 2 O 2 above 0.5 mM were found to be cytotoxic, whereas, lower concentrations did not cause any significant decrease in cell viability. Results of the study showed a decrease in GSH level at 12 and 24 h (39 and 44% of control) and maximum increase (60% of control) in LPO at 24 h. In case of catalase and SOD, a concentration of 0.5 mM of H 2 O 2 was found instantly effective and caused reduction in activity within 2 h, with which decreases significantly up to 24 h. The results indicate that the H 2 O 2 induced oxidative stress mediated cytotoxicity in MCF-7 cells and usefulness of these cell types as sensitive biological system for routine testing of chemical oxidative stressors. Key words: Hydrogen peroxide (H2O2), MCF-7 cells, Cytotoxicity oxidative stress.

Inhibition of normal rat kidney cell growth by transforming growth factor-β is mediated by collagen

Abstract We have investigated the mechanism of inhibition of the serum-free monolayer growth of normal rat kidney (NRK) cells by transforming growth factor-beta (TGF-beta). NRK cells grown on fibronectin-coated dishes exhibited a biphasic response to TGF-beta. Monolayer growth was slightly stimulated by subpicomolar concentrations, while picomolar concentrations of TGF-beta inhibited NRK cell growth in the presence or absence of epidermal growth factor. NRK cells exhibited a similar biphasic growth response to exogenous type I collagen. TGF-beta induced a 3-5-fold increase in the deposition of type I collagen-like proteins into the extracellular matrix of NRK cells during serum-free growth. Type I collagen-like proteins were identified by their sensitivity to degradation by purified bacterial collagenase and by Western blot analysis. The TGF-beta dose-response curves for induction of extracellular matrix-localized collagen and inhibition of NRK cell growth were similar. Finally, the inclusion of a purified bacterial collagenase, which did not degrade TGF-beta or TGF-beta receptors, or alter control NRK growth, prevented exogenous collagen or TGF-beta from inhibiting the serum-free growth of NRK cells. Our results demonstrate that an increase in collagen secretion plays an important role in the inhibition of the growth of NRK cells by TGF-beta.

The binding of 1-(2-hydroxyethyl)-1-nitrosourea to DNA in vitro and to DNA of thymus and marrow in C57BL mice in vivo

To investigate the hypothesis that the similarity of dose-response curves for induction of thymic lymphoma in C57BL mice was due to similar DNA alkylation profiles for 1-ethyl-1-nitrosourea (ENU) and 1-(2-hydroxyethyl)-1-nitrosourea (HNU), we measured the reaction of the two agents with DNA in vitro and in target tissues in vivo. At equimolar doses, alkylation of DNA by HNU was about 20% greater than that by ENU in vitro. As a percentage of total DNA-bound alkyl groups, relative reaction at a minor groove site (3 of adenine) was similar for the two agents, but HNU caused greater relative alkylation at the major groove sites, O 6 and N-7 of guanine. At equi-oncogenic doses, alkylation at the O 6 of guanine in liver and thymus was similar for both agents, but O 6-alkylguanine formation in bone marrow by HNU was almost twice that by ENU. Because alkylation at O 6 of guanine has previously been shown to be a key procarcinogenic lesion in this system, these findings suggest the thymus, rather than the marrow as a primary target for tumor induction by these agents, although involvement of marrow alkylation cannot be ruled out.

Induction Dose???Response Curves for Midazolam and Ketamine in Premedicated ASA Class III and IV Patients

Using probit analysis, dose-response curves for induction of anesthesia with midazolam or ketamine were constructed in ASA class III and IV patients premedicated with morphine, 0.1 mg/kg, and glycopyrrolate, 4 micrograms/kg. For ketamine, ED50 values for abolition of the response to verbal commands, eyelash stimulation, and painful stimulation were 0.9, 1.3, and 1.3 mg/kg, respectively; corresponding ED95 values were 1.6, 2.3, and 4.3 mg/kg, which are within the range of clinically recommended doses. For midazolam, ED50 values for verbal commands, eyelash stimulation, and painful stimulation were 0.19, 0.24, and 0.36 mg/kg, significantly greater than those previously reported for unpremedicated ASA class I and II patients. The corresponding ED95 values, 0.35, 0.43, and 1.04 mg/kg exceed previously reported values and are appreciably greater than the doses used in most previous studies of midazolam induction. Midazolam decreased systolic blood pressure slightly but significantly (from 138 +/- 4 to 128 +/- 4 mm Hg, mean +/- SEM, P less than 0.005), while diastolic blood pressure and heart rate remained unchanged. In contrast, ketamine increased systolic blood pressure (from 141 +/- 4 to 164 +/- 5 mm Hg, P less than 0.005), diastolic blood pressure (from 71 +/- 3 to 88 +/- 4 mm Hg, P less than 0.005), and heart rate (from 84 +/- 2 to 102 +/- 4 beats/min, P less than 0.005). On the basis of these data, we conclude that in ASA class III and IV patients, midazolam induction allows for hemodynamic stability and avoids the significant tachycardia and hypertension associated with equipotent doses of ketamine.

Baclofen and muscimol: behavioural and neurochemical sequelae of unilateral intranigral administration and effects on 3H-GABA receptor binding.

Log dose-response curves for induction of contralateral rotational behaviour in the rat by unilateral intranigral injections of the GABA agonist muscimol and the GABA analogue baclofen have been compared. Baclofen, 5--1000 ng, produced a maximal rotational response that was only 40% of that produced by 0.25--100 ng muscimol, and log dose-response curves failed to show parallelism. The behavioural effects of both drugs were only weakly antagonised by haloperidol and were not antagonised by 6-hydroxydopamine lesions of ipsilateral dopamine (DA) neurons, indicating that these responses were independent of DAergic mechanisms. The effects of baclofen were weakly antagonised by picrotoxin. Intranigral muscimol and baclofen substantially elevated striatal DA concentrations. While muscimol also substantially elevated striatal dihydroxyphenylacetic acid (DOPAC) but not homovanillic acid (HVA), bactofen did not significantly effect either DOPAC or HVA. Baclofein, GABA and muscimol displaced specific 3H-GABA binding in vitro with IC50's of 40 micron, 400 nM and 40 nM respectively. These results indicate that muscimol and baclofen do not act via a unitary GABAergic mechanism, but suggest that baclofen may be a partial GABA agonist, at least at nigral GABA receptors.

Effect of N-nitroso-N-methylurea on viability and mutagenic response of repair-deficient strains of Escher1chia coli

Various E. coli mutants, deficient in DNA repair, differed in their response to increasing concentrations of N -nitroso- N -methylurea (NMU). Loss of viability due to exposure to NMU was greatest in those strains with a reduced capacity for repair of single-strand breaks. Viability of wild-type and uvrA − strains was not affected by NMU concentrations up to 3.0 m M . Some loss of viability occurred, at the higher NMU concentrations, in both strains carrying exrA − while strains carrying uvrA − polA − or recA − were the most sensitive. The results support the hypothesis that the lethal effect of NMU on repair-deficient E. coli was due to its ability to induce single-strand breaks. Induction of mutations by NMU was observed in all the strains used and the results suggested that NMU damage per se was the major mutational event. The dose response curve for induction of revertants by NMU was, however, influenced by the repair system(s) present. The number of revertants scored at the higher NMU concentrations was greater in those strains lacking the recA and polA dependent repair functions than in the wild-type strain. However, at NMU concentrations below 2.0 m M the numbers of revertants induced in exrA − carrying strains, prossessing accurate rec -dependent repair, were lower than the comparable wild-type values. The evidence suggests that the uvrA gene product also acts on some, possibly non-mutagenic, types of NMU damage and that error-prone repair of these lesions increases the number of potential revertants.

Effect of N-nitroso- N-methylurea on viability and mutagenic response of repair-deficient strains of Escherichia coli

Various E. coli mutants, deficient in DNA repair, differed in their response to increasing concentrations of N-nitroso- N-methylurea (NMU). Loss of viability due to exposure to NMU was greatest in those strains with a reduced capacity for repair of single-strand breaks. Viability of wild-type and uvrA − strains was not affected by NMU concentrations up to 3.0 m M. Some loss of viability occurred, at the higher NMU concentrations, in both strains carrying exrA − while strains carrying uvrA −polA − or recA − were the most sensitive. The results support the hypothesis that the lethal effect of NMU on repair-deficient E. coli was due to its ability to induce single-strand breaks. Induction of mutations by NMU was observed in all the strains used and the results suggested that NMU damage per se was the major mutational event. The dose response curve for induction of revertants by NMU was, however, influenced by the repair system(s) present. The number of revertants scored at the higher NMU concentrations was greater in those strains lacking the recA and polA dependent repair functions than in the wild-type strain. However, at NMU concentrations below 2.0 m M the numbers of revertants induced in exrA − carrying strains, prossessing accurate rec-dependent repair, were lower than the comparable wild-type values. The evidence suggests that the uvrA gene product also acts on some, possibly non-mutagenic, types of NMU damage and that error-prone repair of these lesions increases the number of potential revertants.

Mitogen-induced lymphocyte-mediated cytotoxicity in vitro: effect of mitogens selectively activating T or B cells.

AbstractConcanavalin A (Con A) and phytohemagglutinin (PHA) are selective T cell mitogens, whereas lipopolysaccharide (LPS) and purified protein derivative of tuberculin (PPD) are selective B cell mitogens. Con A and PHA induced mouse lymphocyte‐mediated cytotoxicity in vitro, which was equally expressed on autologous, allogeneic and heterologous target fibroblasts, as measured by thymidine release from prelabeled targets. The dose response curves of Con A‐induced lymphocyte‐mediated cytotoxicity and DNA synthesis were parallel, 5 μg/ml being optimal, both higher and lower concentrations being less effective. Con A pretreated mouse lymphocytes failed to express cytotoxicity even though they were irreversibly activated with regard to DNA synthesis and were morphologically transformed. However, when Con A pretreated lymphocytes were added to target fibroblasts in the presence of soluble Con A, the cytotoxic effect markedly exceeded that induced by Con A in non‐pretreated lymphocytes. It is suggested that cytotoxicity not only requires close contact between target and aggressor lymphocyte, but actual binding of the lymphocyte to the target, in this case effectuated by Con A.Con A and PHA failed to induce lymphocyte‐mediated cytotoxicity in B lymphocytes. LPS and PPD did not induce cytotoxicity in normal or in B lymphccytes. Even LPS or PPD pretreated B lymphocytes failed to exert cytotoxicity when aggregated to the targets by Con A. It is suggested that the lymphocytes responsive to LPS and PPD are not the non‐T cells responsible for cell‐mediated cytotoxicity on antibody‐coated target cells.Con A and PHA, but not LPS and PPD, induced lymphocyte‐mediated cytotoxicity in human lymphocytes on allogeneic and heterologous target fibroblasts. The dose response curves for induction of cytotoxicity and DNA synthesis by Con A were not always identical in this case, cytotoxicity persisting at low Con A concentrations, which failed to activate DNA synthesis.