Chrysin and silibinin sensitize human glioblastoma cells for arsenic trioxide.

Arsenic trioxide (ATO) is highly efficient in treating acute promyelocytic leukemia. Other malignancies, however, are often less sensitive. Searching for compounds sensitizing arsenic resistant tumours for ATO the plant polyphenols, chrysin and silibinin, and the ATP binding cassette (ABC) transporter inhibitor MK-571, respectively, were investigated in human glioblastoma A-172 cells. The sensitivity of A-172 cells to ATO was characterized by a median cytotoxic concentration of 6 µM ATO. Subcytotoxic concentrations of chrysin, silibinin and MK-571, respectively, remarkably increased the sensitivity of the cells to ATO by factors of 4-6. Isobolographic analysis revealed synergistic interaction of the polyphenols and MK-571, respectively, with ATO. Sensitization by chrysin was associated with depletion of cellular glutathione and increased accumulation of arsenic. In contrast, silibinin and also MK-571 increased the accumulation of arsenic more strongly but without affecting the glutathione level. The increase of arsenic accumulation could be attributed to a decreased rate of arsenic export and, additionally, in the case of silibinin and MK-571, to an increasing amount of irreversibly accumulated arsenic. Direct interaction with ABC transporters stimulating export of glutathione and inhibiting export of arsenic, respectively, are discussed as likely mechanisms of the sensitizing activity of chrysin and silibinin.

Incipient cytotoxicity: A time-independent measure of cytotoxic potency in vitro.

Time is an important determinant of toxicity but largely ignored in in vitro toxicity assays where exposure times chosen are rather arbitrary. To investigate the impact of time on the cytotoxic potency of chemicals in vitro, the concentration dependent cytotoxic action of selected chemicals (surfactants, metals, oxidative stressors, a mitochondrial poison) was determined after various exposure times (1-72 h) in cultures of Balb/c 3T3 cells. Time affected the cytotoxic potency as well as the cytotoxic efficacy. The median cytotoxic concentrations, EC50, decreased and in most cases approached an "incipient" value, EC50,∞, within 72 h. Cytotoxicity due to mitochondrial insult occurred after a threshold time which was dependent on the medium glucose concentration. Within the chemicals studied the extent of potency change with time ranged from 3- to >1000-fold and the "time to incipient cytotoxicity", tic, from 4 to >72 h. Hence, also the relative cytotoxic potencies depend on exposure time. Ignoring this may lead to severe bias in toxicological hazard and risk assessment. Therefore it is recommended to determine the incipient cytotoxic potency of chemical compounds, represented by, e.g., the incipient median effect (EC50,∞), no effect (NEC∞) or lowest effect concentrations (LEC∞) instead of measures obtained after arbitrary exposure times. If this is not possible, the 72 h-potency measurements appear to be useful surrogates. These time-independent incipient potency values can be reasonably compared between substances, endpoints, cells and biological test systems and may serve to define points of departure for quantitative in vitro-in vivo extrapolations.

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.

Additivity, antagonism, and synergy in arsenic trioxide-induced growth inhibition of C6 glioma cells: effects of genistein, quercetin and buthionine-sulfoximine.

Arsenic trioxide (ATO) induces clinical remission in acute promyelocytic leukemia and growth inhibition in various cancer cell lines in vitro. Recently, genistein and quercetin were reported to potentiate ATO-provoked apoptosis in leukemia and hepatocellular carcinoma cells. Genistein acted via enhanced ROS generation and quercetin via glutathione depletion. Searching for potential strategies for the treatment of malignant gliomas in this study the capacity of these flavonoids to sensitize rat C6 astroglioma cells for the cytotoxic action of ATO was investigated. ATO inhibited cell growth in a concentration- and time-dependent manner. This effect was accompanied neither by enhanced radical generation nor lipid peroxidation and was not attributed to apoptosis. ATO treatment concentration-dependently increased glutathione levels. Genistein enhanced radical generation. Combined with ATO it inhibited cell growth additively. Additivity was also obtained after cotreatment with ATO and H2O2. Quercetin acted antagonistically on ATO-induced growth inhibition. Quercetin increased glutathione levels. In contrast, buthionine-sulfoximine (BSO) depleted cellular glutathione and acted synergistically with ATO. In conclusion, in C6 cells neither genistein nor quercetin are suited as sensitizing agent, in contrast to BSO. Depletion of cellular glutathione content rather than an increase of ROS generation plays a central role in the enhancement of ATO-toxicity in C6 cells.

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.

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.

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 Ham's nutrient mixture F-10 supplemented with 15% horse serum and 2.5% foetal bovine serum (FBS) or in Dulbecco's Modified Eagle's 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 Ham's 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.

Mechanisms involved in the modulation of astroglial resistance to oxidative stress induced by activated microglia: antioxidative systems, peroxide elimination, radical generation, lipid peroxidation.

Microglia and astrocytes are the cellular key players in many neurological disorders associated with oxidative stress and neuroinflammation. Previously, we have shown that microglia activated by lipopolysaccharides (LPS) induce the expression of antioxidative enzymes in astrocytes and render them more resistant to hydrogen peroxide (H2O2). In this study, we examined the mechanisms involved with respect to the cellular action of different peroxides, the ability to detoxify peroxides, and the status of further antioxidative systems. Astrocytes were treated for 3 days with medium conditioned by purified quiescent (microglia-conditioned medium, MCM[-]) or LPS-activated (MCM[+]) microglia. MCM[+] reduced the cytotoxicity of the organic cumene hydroperoxide in addition to that of H2O2. Increased peroxide resistance was not accompanied by an improved ability of astrocytes to remove H2O2 or an increased expression/activity of peroxide eliminating antioxidative enzymes. Neither peroxide-induced radical generation nor lipid peroxidation were selectively affected in MCM[+] treated astrocytes. The glutathione content of peroxide resistant astrocytes, however, was increased and superoxide dismutase and heme oxygenase were found to be upregulated. These changes are likely to contribute to the higher peroxide resistance of MCM[+] treated astrocytes by improving their ability to detoxify reactive oxygen radicals and oxidation products. For C6 astroglioma cells a protective effect of microglia-derived factors could not be observed, underlining the difference of primary cells and cell lines concerning their mechanisms of oxidative stress resistance. Our results indicate the importance of microglial-astroglial cell interactions during neuroinflammatory processes.

Cytotoxic, cytoprotective and antioxidant activities of resveratrol and analogues in C6 astroglioma cells in vitro.

Resveratrol (3,4',5-trans-trihydroxystilbene) and other hydroxystilbenes exhibit in vitro antioxidant as well as prooxidant effects. The antioxidant properties are assumed to enable these compounds to protect cells from oxidative damage. The prooxidant effects are held likely to be responsible for their cytotoxic, anti-proliferative or pro-apoptotic effects observed in vitro. Regarding antioxidant/prooxidant activities in the past various studies were performed aiming at defining structure-activity relationships for hydroxystilbenes using cell-free systems. In the present study cultured C6 glioma cells were used in order to investigate the relationship between the antioxidant, cytoprotective and cytotoxic activities of resveratrol and selected analogues, e.g., 3,3',4',5-trans-tetrahydroxystilbene (piceatannol), 3,3',5,5'-trans-tetrahydroxystilbene (3,3',5,5'-THS) and 3,3',4',5,5'-trans-pentahydroxystilbene (3,3',4',5,5'-PHS). All these compounds were cytotoxic to growth-arrested C6 cells, with EC50-values between 20 and 85 microM. A higher cytotoxic potency in proliferating cells indicated a specific cytostatic activity of resveratrol and 3,3',4',5,5'-PHS. All hydroxystilbenes studied inhibited cellular radical generation induced by cumene hydroperoxide (CHP). The rank order of antioxidant potency was resveratrol>piceatannol>3,3',5,5'-THS>3,3',4',5,5'-PHS. However, only resveratrol and piceatannol inhibited cellular radical generation at lower than cytotoxic concentrations. At subcytotoxic concentrations only piceatannol was able to protect the cells from damage caused by CHP. Taken together, these results show that neither the cytotoxic or cytostatic activities of hydroxystilbenes nor their cytoprotective and antioxidant activities in living cells can be predicted from their antioxidant and prooxidant activity, respectively, in cell-free systems.

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 (> or =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.

Inhibition of peroxide-induced radical generation by plant polyphenols in C6 astroglioma cells.

Plant polyphenols like flavonoids and hydroxystilbens have been found to possess radical scavenging/antioxidative activity, especially when studied in cell-free systems. A positive effect in such assays, however, does not necessarily indicate a protective activity against deleterious effects of oxidative stress in intact cells. In fact it has been shown that polyphenols can act as anti-oxidants as well as pro-oxidants. The aim of the present study was to investigate whether and with what potency selected polyphenols are able to inhibit cellular radical generation in C6 cells and whether they can induce oxidative stress themselves. Cumene hydroperoxide (CHP) was used as a model to induce radical generation which was measured by means of a fluorometric 2',7'-dichlorodihydro-fluorescein assay. CHP-induced, time and concentration dependent, a manifold increase of DCF fluorescence indicating intracellular radical generation. This process was inhibited by all the flavonoids and the hydroxystilben resveratrol, at low micromolar concentrations. The most potent compounds, luteolin and galangin, already at concentrations of 5 to 10 microM nearly completely abolished the radical generation in the presence of 500 microM CHP. The following ranking of anti-oxidative potency was obtained: luteolingalangin>kaempferol>quercetin>resveratrolgenisteintaxifolin. This ranking is completely different from that obtained by means of a trolox equivalent antioxidant capacity (TEAC) assay in a cell-free system, thus putting the biological relevance of the latter in question. Remarkably, one compound induced oxidative stress itself, namely genistein. This flavonoid inhibited the cellular radical generation in the presence of CHP while it significantly enhanced it in the absence of the peroxide.

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.

The improvement of in vitro cytotoxicity testing for the assessment of acute toxicity in fish.

The use of fish cell line cytotoxicity tests as alternatives to acute lethality tests with fish is hampered by the clearly lower sensitivity of the fish cell line tests. Recently, it has been shown that this is not a unique feature of fish cells. In fact, the sensitivity of mammalian and human cell lines toward the cytotoxic actions of chemicals, in general, is comparable to that of fish cell lines. Reviewing some of our recent investigations, the objective of this paper is to show that the sensitivity of in vitro cytotoxicity testing and the correspondence between in vitro cytotoxic and acute fish toxic concentrations (LC50) can be increased, if: a) inhibition of cell growth instead of cell death is used as the endpoint; and b) the bioavailable free cytotoxic concentration (ECu50) of chemicals in vitro, instead of the nominal cytotoxic concentration (EC50), is used as the measure of cytotoxic potency. Based on these results, a pragmatic in vitro testing strategy for estimating the minimal aquatic toxic potency of chemicals is proposed.

Validation of a prediction model for estimating serum concentrations of chemicals which are equivalent to toxic concentrations in vitro.

The objective of the present study was to evaluate the validity of a recently developed extrapolation model for the prediction of concentrations of chemicals in serum which are equivalent to in vitro effective nominal concentrations. Necessary input data are in vitro toxic concentrations and distribution relevant system and substance specific parameters, e.g. lipid volume fractions and albumin concentrations, octanol/water partition coefficients and specific binding to albumin. It was investigated whether the influence of human and bovine serum, respectively, on nominal cytotoxic potencies (EC(50)-values) of selected chemicals in vitro can be properly predicted using this algorithm. Cytotoxicity was determined as growth inhibition of proliferating Balb/c 3T3 cells after exposure for 72 h. Concentration-effect relationships were measured in the presence of 2% foetal bovine serum (FBS) and, additionally, 18% FBS or human serum (HS), or 1% (w/v) bovine (BSA) or human (HSA) albumin, respectively. Addition of HSA and BSA increased the EC(50)-values of the different chemicals by factors of 2.1 - 22 and 1.7 - 29, respectively. From these measurements values for the specific binding of the test compounds to BSA and HSA were derived. Addition of 18% HS increased the EC(50)-values by factors between 4.2 and 52, while addition of 18% FBS resulted only in 1.5 - 10.4-fold increases. A comparison of experimentally determined and calculated EC(50)-values revealed that the differing influence of human and bovine serum was quite well predicted by the extrapolation model. Deviations did not exceed the factor 3 and were in most cases lower than 2. It is concluded that the extrapolation model is quite well suited to predict equivalent concentrations in serum from in vitro effective concentrations.

Impact of bioavailability on the correlation between in vitro cytotoxic and in vivo acute fish toxic concentrations of chemicals.

The lower sensitivity of in vitro cytotoxicity assays currently restricts their use as alternative to the fish acute toxicity assays for hazard assessment of chemicals in the aquatic environment. In vitro cytotoxic potencies mostly refer to nominal concentrations. The main objective of the present study was to investigate, whether a reduced availability of chemicals in vitro can account for the lower sensitivity of in vitro toxicity test systems. For this purpose, the bioavailable free fractions of the nominal cytotoxic concentrations (EC50) of chemicals determined with a cytotoxicity test system using Balb/c 3T3 cells and the corresponding free cytotoxic concentrations (ECu50) were calculated. The algorithm applied is based on a previously developed simple equilibrium distribution model for chemicals in cell cultures with serum-supplemented culture media. This model considers the distribution of chemicals between water, lipids and serum albumin. The algorithm requires the relative lipid volume of the test system, the octanol-water partition coefficient (K(ow)) and the in vitro albumin-bound fraction of the chemicals. The latter was determined from EC50-measurements in the presence of different albumin concentrations with the Balb/c 3T3 test system. Organic chemicals covering a wide range of cytotoxic potency (EC50: 0.16-527000 microM) and lipophilicity (logK(ow): -5.0-6.96) were selected, for which fish acute toxicity data (LC50-values) from at least one of the three fish species, medaka, rainbow trout and fathead minnow, respectively, were available. The availability of several chemicals was shown to be extensively reduced either by partitioning into lipids or by serum albumin binding, or due to both mechanisms. Reduction of bioavailability became more important with increasing cytotoxic potency. The sensitivity of the Balb/c 3T3 cytotoxicity assay and the correspondence between in vivo and in vitro toxic potencies were increased when the free cytotoxic concentrations instead of the nominal cytotoxic concentrations were used as measure of cytotoxic potency. The few remaining prominent differences between cytotoxic and acute toxic concentrations can be explained by a more specific mechanism of acute toxic action than basal cytotoxicity. It is concluded that the frequently observed low sensitivity of in vitro cytotoxicity test systems, compared to fish acute toxicity assays, at least in part, can be explained by differences in the availability of chemicals in vitro and in vivo. Moreover, neglecting these differences systematically causes a bias of the correlation between in vivo and in vitro toxic potencies of chemicals. Taking them into account, however, increases the predictivity of the in vitro assays.

Comparison of mammalian and fish cell line cytotoxicity: impact of endpoint and exposure duration.

Comparisons of acute toxic concentrations of chemicals to fish in vivo and cytotoxic concentrations to fish cell lines in vitro reveal rather good correlations of the toxic potencies in vitro and in vivo, but a clearly lower sensitivity of the fish cells. To examine whether the low sensitivity is specific for fish cells, cytotoxic potencies of reference chemicals from the Multicenter Evaluation of In Vitro Cytotoxicity program (MEIC) reported for the fish cell lines R1 and RTG-2 were compared with those obtained with the mouse Balb/c 3T3 cell line. Cytotoxic potencies (EC(50) values) for MEIC reference chemicals were determined with exponentially growing Balb/c 3T3 cells using three different test protocols. To assess both endpoints, cell proliferation and cell survival, EC(50) values were measured for the decrease in final cell protein after 24 and 72 h of exposure and for the reduction of cell protein increase during 24 h of exposure. EC(50) values obtained with the fish cell lines R1 and RTG-2 using cell survival as endpoint were taken from the MEIC data base. The comparison of cytotoxic potencies shows that, in general, the fish cell lines and the mammalian cell line are almost equally sensitive towards the cytotoxic action of chemicals. The mammalian cell line assay, however, becomes considerably more sensitive, by factors of 3.4-8.5, than the fish cell line assays, if cell growth instead of cell survival is used as endpoint. It is concluded, that cell proliferation might be a better endpoint than cell survival and that mammalian cell lines might be suited to assess fish acute toxicity.

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.

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.