Michael Gülden

Cytotoxic potency of H2O2 in cell cultures: Impact of cell concentration and exposure time

Using C6 glioma cells in this study we investigated in detail how exposure time and cell concentration affect the cytotoxic potency of H(2)O(2) in vitro. Median cytotoxic concentrations (EC(50)) decreased from 500 to 30 μM with increasing incubation time from 1 to 24h. Twenty-four hours proved to be sufficient to determine incipient cytotoxic concentrations of H(2)O(2). The incipient EC(50) values were linearly related to the cell concentration. A cell concentration-independent median cytotoxic cell dose (ED(50)) of 430 nmol/mg cell protein or 860 nmol/10(7) cells was derived. Median cytotoxic H(2)O(2) concentrations were completely eliminated from the culture medium at a rate proportional to both the H(2)O(2) and the cell concentrations. In contrast to EC(50) values the corresponding areas under the concentration versus time curve (AUC) were independent of the cell concentration and amounted to 1800 μM×min. With decreasing cell concentration the H(2)O(2) elimination decelerates and, thus, exposure to H(2)O(2) applied as a bolus approaches a continuous exposure to a steady H(2)O(2) concentration. Taken together, our results indicate that the cytotoxic potency of H(2)O(2) administered to cultured cells as a bolus is characterized by the AUC, which depends on its initial concentration, the ability of the cells to eliminate H(2)O(2), and the cell concentration. We recommend expressing the toxic potency of H(2)O(2) in vitro by the incipient toxic cell dose (e.g., nmol H(2)O(2)/mg cell protein or nmol H(2)O(2)/10(7) cells), in particular for comparative purposes.

In vitro-in vivo extrapolation: estimation of human serum concentrations of chemicals equivalent to cytotoxic concentrations in vitro.

In the present study an extrapolation model for estimating serum concentrations of chemicals equivalent to in vitro effective concentrations is developed and applied to median cytotoxic concentrations (EC(50)) determined in vitro. Nominal concentrations of a chemical in serum and in vitro are regarded as equivalent, if they result in the same aqueous concentration of the unbound form. The algorithm used is based on equilibrium distribution and requires albumin binding data, the octanol-water partition coefficient (K(ow)), and the albumin concentrations and lipid volume fractions in vitro and in serum. The chemicals studied cover wide ranges of cytotoxic potency (EC(50): 2.5-530,000 microM) and lipophilicity (logK(ow): -5 to 7). Their albumin binding characteristics have been determined by means of an in vitro cytotoxicity test as described previously. The equivalent serum concentrations of 19 of the 33 compounds investigated, having high protein binding and/or lipophilicity, were substantially higher than the EC(50)-values, by factors of 2.5-58. Prominent deviations between the equivalent nominal concentrations in serum and in vitro were largely restricted to chemicals with higher cytotoxic potency (EC(50)< or =1000 microM). The results suggest that estimates of equivalent serum concentrations based on in vitro data are robust for chemicals with low lipophilicity (logK(ow)< or =2) and low potency (EC(50)>1000 microM). With more potent chemicals or those with higher lipophilicity partitioning into lipids and/or binding to serum proteins have to be taken into account when estimating in vivo serum concentrations equivalent to in vitro effective concentrations.

Peroxide-induced cell death and lipid peroxidation in C6 glioma cells.

Peroxides are often used as models to induce oxidative damage in cells in vitro. The aim of the present study was to elucidate the role of lipid peroxidation in peroxide-induced cell death. To this end (i) the ability to induce lipid peroxidation in C6 rat astroglioma cells of hydrogen peroxide (H2O2), cumene hydroperoxide (CHP) and t-butyl hydroperoxide (t-BuOOH) (ii) the relation between peroxide-induced lipid peroxidation and cell death in terms of time and concentration dependency and (iii) the capability of the lipid peroxidation chain breaking alpha-tocopherol to prevent peroxide-induced lipid peroxidation and/or cell death were investigated. Lipid peroxidation was characterised by measuring thiobarbituric acid reactive substances (TBARS) and, by HPLC, malondialdehyde (MDA), 4-hydroxynonenal (4-HNE) and hexanal. Within 2 h CHP, t-BuOOH and H2O2 induced cell death with EC50 values of 59+/-9 microM, 290+/-30 microM and 12+/-1.1 mM, respectively. CHP and t-BuOOH, but not H2O2 induced lipid peroxidation in C6 cells with EC50 values of 15+/-14 microM and 130+/-33 microM, respectively. The TBARS measured almost exclusively consisted of MDA. 4-HNE was mostly not detectable. The concentration of hexanal slightly increased with increasing concentrations of organic peroxides. Regarding time and concentration dependency lipid peroxidation preceded cell death. Pretreatment with alpha-tocopherol (10 microM, 24 h) prevented both, peroxide-induced lipid peroxidation and cell death. The results strongly indicate a major role of lipid peroxidation in the killing of C6 cells by organic peroxides but also that lipid peroxidation is not involved in H2O2 induced cell death.

Activated Microglia Modulate Astroglial Enzymes Involved in Oxidative and Inflammatory Stress and Increase the Resistance of Astrocytes to Oxidative Stress In Vitro

Neuropathological processes in the central nervous system are commonly accompanied by an activation of microglia and astrocytes. The involvement of both cell populations in the onset and progress of neurological disorders has been widely documented, implicating both beneficial and detrimental influences on the neural tissue. Nevertheless, little is known about the interplay of these glial cell populations, especially under diseased conditions. To examine the effects of activated microglia on astrocytes purified rat astroglial cell cultures were treated with medium conditioned by purified quiescent (MCM[−]) or lipopolysaccharide (LPS)‐activated rat microglia (MCM[+]) and subjected to a comparative proteome analysis based on two‐dimensional gel electrophoresis. No significant down regulation of proteins was observed. The majority of the 19 proteins identified by means of nano HPLC/ESI‐MS/MS in the 12 most prominent protein spots significantly overexpressed (≥2‐fold) in MCM[+] treated astrocytes are involved in inflammatory processes and oxidative stress response: superoxide dismutases (Sod), peroxiredoxins, glutathione S‐transferases (Gst), nucleoside diphosphate kinase B, argininosuccinate synthase (Ass), and cellular retinol‐binding protein I (Rbp1). Sod2, Rbp1, Gstp1, and Ass were also significantly increased on the mRNA level determined by quantitative RT‐PCR. The upregulation of antioxidative enzymes in astrocytes was accompanied by a higher resistance to oxidative stress induced by H2O2. These results show that activated microglia change the expression of antioxidative proteins in astrocytes and protect them against oxidative stress, which might be an effective way to increase the neuroprotective potential of astrocytes under pathological conditions associated with oxidative stress and inflammation.

Cytoprotective activity against peroxide-induced oxidative damage and cytotoxicity of flavonoids in C6 rat glioma cells.

The aim of this study was to investigate the relationship between cytoprotective and cytotoxic activities of selected plant flavonoids in C6 glioma cells. Apigenin, kaempferol, luteolin, and quercetin were cytotoxic at low μM concentrations (LOECs: 5-20 μM), whereas myricetin was less toxic (LOEC>20 μM). Cytotoxicity was not due to H(2)O(2) generation from flavonoids in culture medium. Quercetin, luteolin, and kaempferol protected the cells from peroxide-induced cytotoxicity. Concentration-effect curves for cytoprotection had a biphasic shape. In contrast, apigenin and myricetin did not exhibit any cytoprotective activity. The first three compounds also inhibited cellular lipid peroxidation induced by CHP, while the latter were ineffective. Importantly, concentrations of luteolin and kaempferol protecting cells under oxidative stress were identical to those causing cell damage under normal conditions. Only in case of quercetin there was a narrow range of concentrations protecting cells without being cytotoxic to non-stressed cells. Thus, even for flavonoids with a high antioxidant capacity in cell-free systems the cytoprotective selectivity (LOEC(cytotox)/LOEC(cytoprot)) was very low or even absent. These results should be taken into account when the prophylactic or therapeutic application of flavonoids as antioxidants is discussed.

Serum albumin binding at cytotoxic concentrations of chemicals as determined with a cell proliferation assay.

The aim of the present study was to measure the influence of albumin binding on cytotoxic concentrations of chemicals and to determine binding parameters which can be used for quantitative in vitro-in vivo extrapolations. Protein binding parameters were determined from cytotoxic potencies measured with Balb/c 3T3 cells cultured in the presence of 18 and 600 microM bovine serum albumin (BSA). A subset of 27 chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) project was investigated. At 18 microM BSA the EC(50)-values ranged from 2.54 microM (As(III)) to 527 mM (ethylene glycol). Increasing the BSA concentration either decreased the cytotoxic potency (12 compounds) by factors up to 34 (pentachlorophenol), had no effect (14 compounds), or increased the cytotoxicity (paraquat). Calculated molar ratios of binding ranged from 0.05 (Hg(2+)) to 4.8 moles per mole albumin (acetylic salicylic acid). At 18 microM BSA fractional binding of most of these compounds was low (<25%) but increased up to > or =90% (hexachlorophene, mercuric chloride, thioridazine, pentachlorophenol) at 600 microM BSA. The results obtained in general were compatible with available protein binding data and can be used to calculate equipotent concentrations of chemicals in biological systems containing different albumin concentrations.

Effects of quercetin and quercetin-3-O-glycosides on oxidative damage in rat C6 glioma cells

Flavonoids are reported to be powerful antioxidants in cell free systems. They naturally occur as glycosides rather than as aglycon. In this study the ability of the flavonoid quercetin and its glycosides, quercetin-3-O-rutinoside (rutin), quercetin-3-O-glucoside and quercetin-3-O-(6″-O-acetyl)-glucoside, to protect in vitro rat C6 glioma cells from oxidative damage induced by cumene hydroperoxide was investigated. Cumene hydroperoxide induced cell death and lipid peroxidation. The cytotoxicity of cumene hydroperoxide could be prevented by the radical scavenger dimethyl thiourea and the ferric iron chelator deferoxamine, indicating that its cytotoxic activity is related to the generation of reactive oxygen radicals in the ferrous iron dependent Fenton reaction. Quercetin, in a concentration range of 10-100 μM, but neither rutin nor the other two glycosides, were able to protect C6 cells from cytotoxicity and lipid peroxidation. Furthermore, cytoprotective concentrations of quercetin proved to be cytotoxic itself. These results call in question potential beneficial effects of dietary intake or therapeutic use of naturally occurring flavonoids.

In vitro toxicity testing with microplate cell cultures: Impact of cell binding

In vitro generated data on toxic potencies are generally based on nominal concentrations. However, cellular and extracellular binding and elimination processes may reduce the available free fraction of a compound. Then, nominal effective concentrations do not represent appropriate measures of toxic exposure in vitro and underestimate toxic potencies. In this study it was investigated whether cell binding can affect the availability of chemicals in microplate based toxicity assays. To this end the cytotoxicity of compounds like mercury chloride, digitonin and alcohol ethoxylates, accumulated by cells via different modes, was investigated in 96-well microplate cultures with varying concentrations of Balb/c 3T3 cells. The median effective nominal concentrations of all but one of the tested compounds depended linearly from the cell concentration. Applying a previously developed equilibrium distribution model cell concentration-independent median effective extracellular concentrations and cell burdens, respectively, could be calculated. The compounds were accumulated by the cells with bioconcentration factors, BCF, between 480 and ≥ 25,000. Cell binding of the alcohol ethoxylates was correlated with their lipophilicity. The results show that significant cell binding can occur even at the small cell volume fractions (∼ 1 × 10(-5) to 3 × 10(-3) L/L) encountered in microplate assays. To what extent cell binding affects the bioavailability depends on the BCF and the cell volume fraction. EC50 measurements in the presence of at least two different cell concentrations allow for excluding or detecting significant cell binding and for determining more appropriate measures of toxic exposure in vitro like median effective extracellular (free) concentrations or cell burdens.

Lasting effect of preceding culture conditions on the susceptibility of C6 cells to peroxide-induced oxidative stress

The aim of the present study was to investigate the influence of the maintenance culture conditions on the competence of C6 rat glioma cells to cope with peroxide-induced oxidative stress. C6 cells were maintained either in Hams nutrient mixture F-10 supplemented with 15% horse serum and 2.5% foetal bovine serum (FBS) or in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 5% FBS. The differently cultured cells were exposed under identical conditions to hydrogen peroxide (H₂O₂) and cumene hydroperoxide (CHP) in serum-free DMEM. The cells maintained in high serum Hams F-10 medium (1) were less sensitive towards the cytotoxic action of both peroxides (EC₅₀-values: H₂O₂: 193 ± 23 μM; CHP: 94 ± 16 μM) than the cells maintained in low serum DMEM (EC₅₀-values: H₂O₂: 51 ± 10 μM; CHP: 27 ± 11 μM), (2) eliminated the peroxides (initial concentration: 100 μM) with higher rates (H₂O₂: 56 ± 5.5 vs. 32 ± 2.7, CHP: 32 ± 6 vs. 3.4 ± 0.6 nmol/min mg protein), (3) contained more glutathione (30 ± 2.5 vs. 14 ± 1.1 nmol/mg protein) and (4) owned a higher glutathione peroxidase activity (28 ± 3.4 vs. 9.5 ± 0.8 mU/mg protein). Glutathione reductase and catalase activities were not affected. These results demonstrate that the preceding culture conditions have a lasting effect on the susceptibility of cultured cells to oxidative stressors like peroxides. As cause for these differences a dissimilar supply of the cells with serum born antioxidants like selenium and α-tocopherol is discussed.

In vitro toxicity screening using cultured rat skeletal muscle cells. II. Agents affecting excitable membranes

The screening of chemicals for their potential to interfere with excitable cell membranes should be an important element of in vitro testing for acute toxicity. The suitability for this purpose of a test system using primary cultured rat skeletal muscle cells was evaluated. The test protocol involved the determination of the concentration-dependent effects on three endpoints: (1) spontaneous contractility, (2) membrane integrity and (3) energy metabolism. The chemicals investigated were: NaCl, KCl and CaCl(2); cardiac glycosides (ouabain, digoxin); sodium channel toxins (tetrodotoxin, saxitoxin, veratridine, Anemonia sulcata toxin II, Bolocera tuediae toxin II); an acetylcholine agonist (carbachol); a calcium antagonist (D600) and three membrane-directed insecticides (deltamethrin, DDT, lindane). The response pattern of most of these substances-alteration of contractility at concentrations that neither affected the energy metabolism nor were cytolethal-characterized them as acting either on the excitable membrane or on the excitation-contraction coupling and the contractile apparatus. The results indicate that the test system is suited to assess chemical effects resulting in: (i) changes of resting membrane and threshold potentials, (ii) altered sodium channel function, (iii) opening of endplate channels, (iv) blockade of calcium channels, and (v) inhibition of Na(+)/K(+)-ATPase.