Dose- and time-dependent effects of doxorubicin on cytotoxicity, cell cycle and apoptotic cell death in human colon cancer cells.

The cytostatic drug doxorubicin is a well-known chemotherapeutic agent which is used in treatment of a wide variety of cancers. A key factor in the response of cancer cells to chemotherapeutic drugs is the activation of the apoptotic pathway, a pathway that is often impaired in chemoresistant colon cancer cells. The aim of the present study was to investigate the effects of doxorubicin in Hct-116 human colon carcinoma cells in order to clarify if a time/concentration range for optimal doxorubicin-induced apoptosis exists. We compared a treatment schedule were cells were bolus incubated for 3h with doxorubicin followed by 24h in drug-free medium, with a continuous doxorubicin treatment schedule for 24h. Bolus incubation was carried out to determine effects of doxorubicin accumulated during the first 3h, whereas continuous incubation allowed further (continuous) exposure to doxorubicin. We found that bolus (3h) treatment with doxorubicin resulted in a dose-dependent decrease of viable cells and concomitant increase of apoptosis. Additionally, bolus (3h) doxorubicin incubation led to phosphorylation of p53 at serine 392, induction of p21, G2 arrest and increase of proapoptotic protein Bax. In contrast, continuous (24h) treatment with doxorubicin reduced the number of living cells with no parallel raise in the amount of dead cells. Continuous (24h) treatment with 5 microM doxorubicin resulted in cell cycle arrest in G0/G1 phase that was neither accompanied by phosphorylation and activation of p53 nor enhanced expression of p21. These results suggest that doxorubicin is able to induce cell death by apoptosis only at particular dose and treatment conditions and imply a completely different cellular response following bolus or continuous exposure to doxorubicin.

The use of Fluoro-Jade in primary neuronal cell cultures.

Fluoro-Jade (FJ) and its derivatives are widely used for histological staining of neurons undergoing neurodegeneration. With this dye, the entire structure of these neurons can be stained in a fast and reliable way in histopathological slices of the brain, with results comparable to those obtained with other methods such as the Nissle technique or silver staining. The question arose as to whether this method might be useful for in vitro neuronal cell cultures. Primary cortical neuronal cell cultures have been used as a sensitive and reliable system to detect compounds which induce neurodegenerative lesions (Schmuck et al. 2000). Additionally, various biochemical endpoints in this system allow the mode of action of these compounds to be identified. The target mechanism of FJ staining is unknown, and it may therefore be useful to compare FJ staining with one of the central endpoints in compound-induced neurodegeneration, interaction with the cytoskeleton as demonstrated by accumulations of neurofilaments (200 kD). Cortical neuronal cells were cultivated under standardized serum-free conditions. Once they had developed a stable network, the cells were treated with acrylamide, mipafox, diethyldithiocarbamate, glutamate, paraquat, paraoxon, and IDPN (ss,ss-imino dipropionitrile) for 7 days in the concentration range of 0.1-50 microg/ml. One half of the cell culture samples were tested directly after 7 days, the others were allowed to recover during a 7-day treatment-free period. Subsequently viability testing and quantification for FJ staining were performed. All compounds except paraoxon increased FJ staining after 7 days, and this signal increased slightly during the recovery period with glutamate and acrylamide. With mipafox and IDPN the signal decreased slightly. Paraoxon increased FJ staining only after the recovery period. The intensity of FJ staining did not always correlate with neurofilament destruction or cytotoxicity. It can therefore be assumed that FJ targets a different cellular endpoint. Interestingly, paraoxon, a compound which does not induce neurodegeneration, increased FJ staining only in the recovery phase; this pointed to a neurotoxic mechanism which sets it apart from the other model compounds.

The forkhead transcription factor FOXO4 sensitizes cancer cells to doxorubicin-mediated cytotoxicity.

The forkhead superfamily of transcription factors, which play major roles in control of cellular proliferation, oxidative stress and apoptosis, are becoming more and more considered as crucial therapeutic targets in cancer. In this study, we addressed the contribution of class O of forkhead box transcription factor (FOXO) 4 transcription factor, a forkhead superfamily member, to cytotoxicity mediated by the anthracyclic drug doxorubicin. FOXO4 can be phosphorylated by phosphatidylinositol-3-kinase/AKT signaling resulting in its inactivation and nuclear exclusion. Under stress conditions, FOXO4 can be phosphorylated via jun N-terminal kinase (JNK) leading to increased transcriptional activation of the transcription factor. Our results show that doxorubicin incubation led to phosphorylation of AKT and concomitantly to AKT-dependent inactivation and nuclear exclusion of the tumor suppressor FOXO4 in Hct-116 cells. We found that inhibition of FOXO4 nuclear exclusion by blockage of AKT phosphorylation following overexpression of dominant-negative AKT enhanced doxorubicin-mediated cytotoxicity. Overexpression of wild-type FOXO4 led to an increase in doxorubicin-mediated cytotoxicity, which was further exacerbated by overexpression of a solely nuclear-localized FOXO4 mutant. In contrast, though doxorubicin resulted in JNK activation, modulation of JNK-dependent regulation of FOXO4 was of no effect to doxorubicin cytotoxicity. These results show for the first time that in Hct-116 cells sustained nuclear localization of FOXO4 seems to be one crucial point enhancing doxorubicin-induced cytotoxicity and apoptosis. Targeting FOXO4 or AKT may lead to new chances in sensitizing cancer cells to cytostatic drugs thereby allowing use of lower drug concentrations and minimizing drug-induced adverse effects in patients.

Increased expression of catalase in human hepatoma cells by the soy isoflavone, daidzein.

The reduced incidence of cancer that has been observed in Asian population traditionally consuming soy-based food has been linked to the antioxidant potential of soy isoflavones, in particular daidzein and genistein. The present study was undertaken in order to test the antioxidative potential of daidzein and to examine the effect of daidzein treatment on the expression of the antioxidant enzyme catalase in the human hepatoma cell lines Huh-7 and HepG2. Daidzein itself did not display radical scavenging activity but it significantly increased the activity of the antioxidant enzyme catalase. Huh-7 cells were much more susceptible to daidzein cytotoxicity than HepG2 cells and showed much lower basal activity in luciferase reporter gene assays with the 3.2 kb fragment of the human catalase promoter. However, treatment with daidzein at a non-toxic concentration resulted in a similar induction of promoter activity in both cell lines. Reporter gene studies with different promoter constructs in HepG2 cells restrict the potential localization of the main regulatory elements for basal and inducible activity of the catalase promoter to a region approximately 120 bp to 300 bp upstream of the start codon of the catalase gene. From our results, we conclude that in human hepatoma cells daidzein at a non-toxic concentration increases the activity of human catalase and induces the transcription of the catalase gene via interaction with the proximal part of the promoter.

Catalase overexpression impairs TNF-alpha induced NF-kappaB activation and sensitizes MCF-7 cells against TNF-alpha.

The pleiotropic cytokine tumor necrosis factor alpha (TNF-alpha) can induce apoptosis but also supports cell survival pathways. Among the possible anti-apoptotic mechanisms of TNF-alpha is the activation of the transcription factor NF-kappaB. Since reactive oxygen species (ROS) are assumed to contribute to TNF-alpha mediated cytotoxicity but can also facilitate NF-kappaB activation this study investigates the relationship between TNF-alpha treatment, NF-kappaB activation and the expression of the anti-oxidative enzyme catalase. TNF-alpha treatment caused downregulation of catalase expression in MCF-7, Caco-2 and Hct-116 cancer cell lines. Overexpression of catalase in MCF-7 cells, resulting in lower intracellular ROS levels upon challenge with H(2)O(2), caused a transient nuclear p65 translocation upon TNF-alpha treatment as compared to the sustained NF-kappaB activation in wild type cells. This was due to a lack of sufficient H(2)O(2) to co-stimulate NF-kappaB activation as demonstrated by the observation that addition of exogenous H(2)O(2) led to a second increase of NF-kappaB activity. The rapid decline of nuclear translocation of NF-kappaB in the catalase overexpressing cells resulted in a slower increase of NF-kappaB mediated reporter gene expression. These results indicate that TNF-alpha mediated downregulation of catalase expression and accordingly sufficient H(2)O(2) is required for appropriate function of the NF-kappaB dependent survival pathway.

Effects of the flavonoids kaempferol and fisetin on thermotolerance, oxidative stress and FoxO transcription factor DAF-16 in the model organism Caenorhabditis elegans.

Flavonoids present in many herbal edibles possess a remarkable spectrum of biochemical and pharmacological actions and they are assumed to exert beneficial effects to human health. Although the precise biological mechanisms of their action has not been elucidated yet many of the protective properties of flavonoids are attributed to their antioxidative activity since oxidative stress is regarded as a main factor in the pathophysiology of various diseases and ageing. Oxidative stress results from excessive generation of reactive oxygen species (ROS) or diminished antioxidative defence and thus antioxidants are able to counteract such situations. We used the multicellular model organism Caenorhabditis elegans that is conserved in molecular and cellular pathways to mammals to examine the effects of the flavonoids kaempferol and fisetin with respect to their protective action in individual living worms. Both flavonoids increased the survival of C. elegans, reduced the intracellular ROS accumulation at lethal thermal stress, and diminished the extent of induced oxidative stress with kaempferol having a stronger impact. Kaempferol but not fisetin attenuated the accumulation of the ageing marker lipofuscin suggesting a life prolonging activity of this flavonoid. In addition to these effects that may be attributed to their antioxidative potential kaempferol and fisetin caused a translocation of the C. elegans FoxO transcription factor DAF-16 from the cytosol to the nucleus indicating a modulatory influence of both flavonoids on signalling cascade(s).

Investigations of protective effects of the flavonoids quercetin and rutin on stress resistance in the model organism Caenorhabditis elegans.

Oxidative stress as a result of excessive generation of reactive oxygen species (ROS) or diminished antioxidative defence is regarded as a main factor in the pathophysiology of various diseases and ageing. Many flavonoids that are present in herbal edibles have antioxidative properties and possess a remarkable spectrum of biochemical and pharmacological actions. They are assumed to exert beneficial effects but the precise biological mechanism of their action is unknown. In this project, we studied effects of the flavonoids quercetin and rutin in the multicellular model organism Caenorhabditis elegans that exhibits a strong conservation in molecular and cellular pathways to mammals. Both flavonoids reduced the ROS accumulation at thermal stress and the extent of induced oxidative stress with quercetin having a stronger impact. The higher antioxidative activity of quercetin may explain the protection against lethal thermal stress and the reduction in accumulation of the ageing marker lipofuscin exerted by quercetin but not by rutin. The subcellular distribution of the FoxO transcription factor DAF-16 was only affected by quercetin indicating a modulatory effect of quercetin on signalling cascade(s). These results suggest that quercetin may act as an antioxidant as well as a modulator of cellular signalling processes to exert its protective properties.

Downregulation of NF-kappaB activation in a H4IIE transfectant insensitive to doxorubicin-induced apoptosis.

Cytostatic drugs are administered to cancer patients in order to drive the tumor cells into apoptosis by DNA damage signalling pathway(s). DNA damage also leads to NF-kappaB activation, and it is controversial whether this is exclusively part of a survival process, thus enabling drug resistance, or whether it can also lead to a pro-apoptotic response, thus supporting the therapeutic purpose of drug administration. In the present work, the pathway and outcome of NF-kappaB activation was compared in the doxorubicin sensitive H4IIE rat hepatoma cell and the H4IIE-derived transfectant Yv2-12 which is insensitive to doxorubicin induced apoptosis. In the wild type H4IIE cell, doxorubicin induces serine 536 phosphorylation and nuclear translocation of p65 which however results in reduced rather than increased expression of the anti-apoptotic protein XIAP. Apoptosis in H4IIE cells is accompanied by rapid production of intracellular reactive oxygen species, caspase activation and increased expression of the pro-apoptotic protein Bax. The doxorubicin-insensitive Yv2-12 transfectant differs from its wild type counterpart by the complete failure to activate NF-kappaB in response to doxorubicin. In contrast, serine 536 phosphorylation and nuclear translocation of p65 are even reduced by doxorubicin treatment while the expression of XIAP and Bax remain virtually unchanged. These results show that NF-kappaB activation by doxorubicin in our experimental system proceeds by an atypical pathway resulting in a pro-apoptotic effect and that insensitivity to doxorubicin-induced apoptosis was accompanied by a loss of NF-kappaB activation.

Protective and detrimental effects of kaempferol in rat H4IIE cells: implication of oxidative stress and apoptosis.

Flavonoids are ubiquitous substances in fruits and vegetables. Among them, the flavonol kaempferol contributes up to 30% of total dietary flavonoid intake. Flavonoids are assumed to exert beneficial effects on human health, e.g., anticancer properties. For this reason, they are used in food supplements at high doses. The aim of this project was to determine the effects of kaempferol on oxidative stress and apoptosis in H4IIE rat hepatoma cells over a broad concentration range. Kaempferol is rapidly taken up and glucuronidated by H4IIE cells. The results demonstrate that kaempferol protects against H2O2-induced cellular damage at concentrations which lead to cell death and DNA strand breaks in the absence of H2O2-mediated oxidative stress. Preincubation with 50 microM kaempferol exerts protection against the loss of cell viability induced by 500 microM H2O2 (2 h) while the same concentration of kaempferol reduces cell viability by 50% in the absence of H2O2 (24 h). Preincubation with 50 microM kaempferol ameliorates the strong DNA damage induced by 500 microM H2O2 while 50 microM kaempferol leads to a significant increase of DNA breakage in the absence of H2O2. Preincubation with 50 microM kaempferol reduces H2O2-mediated caspase-3 activity by 40% (4 h) while the same concentration of kaempferol leads to the formation of a DNA ladder in the absence of H2O2 (24 h). It is concluded that the intake of high dose kaempferol in food supplements may not be advisable because in our cellular model protective kaempferol concentrations can also induce DNA damage and apoptosis by themselves.

Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis.

Dietary flavonoids possess a wide spectrum of biochemical and pharmacological actions and are assumed to protect human health. These actions, however, can be antagonistic, and some health claims are mutually exclusive. The antiapoptotic actions of flavonoids may protect against neurodegenerative diseases, whereas their proapoptotic actions could be used for cancer chemotherapy. This study was undertaken to determine whether a cytoprotective dose range of flavonoids could be differentiated from a cytotoxic dose range. Seven structurally related flavonoids were tested for their ability to protect H4IIE rat hepatoma cells against H(2)O(2)-induced damage on the one hand and to induce cellular damage on their own on the other hand. All flavonoids proved to be good antioxidants in a cell-free assay. However, their pharmacologic activity did not correlate with in vitro antioxidant potential but rather with cellular uptake. For quercetin and fisetin, which were readily taken up into the cells, protective effects against H(2)O(2)-induced cytotoxicity, DNA strand breaks, and apoptosis were detected at concentrations as low as 10-25 micromol/L. On the other hand, these flavonoids induced cytotoxicity, DNA strand breaks, oligonucleosomal DNA fragmentation, and caspase activation at concentrations between 50 and 250 micromol/L. Published data on quercetin pharmacokinetics in humans suggest that a dietary supplement of 1-2 g of quercetin may result in plasma concentrations between 10 and 50 micromol/L. Our data suggest that cytoprotective concentrations of some flavonoids are lower by a factor of 5-10 than their DNA-damaging and proapoptotic concentrations.

Polyphenols from plants used in traditional Indonesian medicine (Jamu): uptake and antioxidative effects in rat H4IIE hepatoma cells.

Phytochemical investigation of plants used in traditional Indonesian medicine (Jamu) yielded lignans (pinoresinol, 9 alpha-hydroxypinoresinol and salicifoliol), flavonoids (3-O-beta-(D)-glucopyranosyl-(1-->6)-beta-(D)-glucopyranosylkaempferol, luteolin and apigenin) and coumarins (coumarin, 8-hydroxycoumarin and 5-hydroxycoumarin). The beneficial effects of the respective plants for human health are thought to be associated with antioxidative activity. In the present study, the antioxidative capacity of the isolated compounds was determined in an in-vitro assay. Luteolin and kaempferol (cleavage product of 3-O-beta-(D)-glucopyranosyl-(1-->6)-beta-(D)-glucopyranosylkaempferol, which is thought to be formed in the intestine) showed strong antioxidant activity; pinoresinol and 9 alpha-hydroxypinoresinol showed only minor antioxidative effects. The coumarins, as well as apigenin and 3-O-beta-(D)-glucopyranosyl-(1-->6)-beta-(D)-glucopyranosylkaempferol were inactive. The antioxidative effects of luteolin, kaempferol and pinoresinol were further investigated in H4IIE rat hepatoma cells. A strong protective effect of kaempferol and luteolin was found against H2O2-mediated intracellular reactive oxygen species formation measured using the dichlorofluorescein assay and H2O2-mediated DNA strand breaks. Pinoresinol did not have a protective effect against H2O2-mediated DNA-damage, but in the dichlorofluorescein assay, an antioxidative effect was detectable. During studies with H4IIE cells, kaempferol, luteolin and pinoresinol were taken up by the cells within 60 min. The flavonoids were found to be relatively toxic at higher concentrations, while pinoresinol was less cytotoxic. In conclusion, kaempferol and luteolin, at low concentrations (< or = 50 microM), protect H4IIE cells against oxidative stress but are cytotoxic at higher concentrations; the biological effects of pinoresinol are less prominent in comparison. These results are important for the identification of pharmacologically active substances from traditional Indonesian medicinal plants.

Antioxidant enzymes and apoptosis.

The role of antioxidant enzymes can be interpreted in terms of fine tuning of the concentration of reactive oxygen species which are required in the redox regulation of the cell cycle and of programmed cell death. This review summarizes findings from papers published in the last few years which deal with the relation between apoptosis and the two antioxidant enzymes, manganous superoxide dismutase (MnSOD) and catalase. With respect to MnSOD, the literature is much in favor of an inhibitory action in apoptosis. Increased MnSOD activity has been shown to prevent cell death via the receptor-mediated apoptotic pathway as well as cell death via the mitochondrial pathway. The literature on the influence of catalase activity on apoptosis is less consistent. Evidence for both an antiapoptotic and a proapoptotic role of catalase can be found. From the results reviewed here, two schemes for the involvement of MnSOD and catalase in the regulation of apoptosis can be extracted: 1) Both MnSOD and catalase inhibit apoptosis by removing superoxide anion radicals or H2O2, respectively, because these reactive oxygen species are mediators required for the apoptotic program or inhibit a survival pathway. 2) An increase in H2O2 by downregulation or inhibition of catalase activity and/or upregulation of MnSOD activity inhibits apoptosis while a decrease in H2O2 by upregulation of catalase activity and/or downregulation of MnSOD activity supports apoptosis, possibly because of a supportive role of H2O2 in a survival pathway. The data reported so far do not allow for an explanation why some cell models appear to fit the first scheme while the second scheme appears to correctly describe other cell models. The present state of the literature reveals that antioxidant enzymes play a more intricate role in cell physiology than previously assumed.

Genotoxicity of the isoflavones genistein, daidzein and equol in V79 cells.

Hormonally active chemicals in the human diet, such as man-made estrogenic chemicals or plant-derived compounds (phytoestrogens), have become a matter of public concern. A significant part of human exposure to phytoestrogens is attributable to soy isoflavones. Besides their estrogenic properties, soy isoflavones also exert genotoxic actions. In this paper, the micronucleus (MN) assay in V79 cells was used to study chromosomal genotoxicity. Genistein caused a clear dose-related induction of MN within the range of 5-25 microM; MN rates were declining at higher genistein concentrations. This was probably due to cytotoxicity of genistein since reduced neutral red uptake and MTT formation with an IC(50) of about 75 microM occurred. Daidzein induced a comparatively shallow increase in the number of MN between 25 and 100 microM. In contrast, the daidzein metabolite equol caused an increase in the number of MN up to 25 microM with no further increase at higher concentrations. Additional staining with anti-kinetochore (CREST) antibodies served to determine if the micronuclei contain whole chromosomes or acentric fragments. Genistein induced mostly CREST(-) micronuclei, i.e. MN with chromosomal fragments, thus indicative of a clastogenic mode of action. MN induced by high concentrations of daidzein were partly CREST(+) and CREST(-), whilst equol induced mostly CREST(+) micronuclei indicative of an aneugenic action. These results point to a differential genotoxicity of phytoestrogens.

Antioxidants protect primary rat hepatocyte cultures against acetaminophen-induced DNA strand breaks but not against acetaminophen-induced cytotoxicity.

Acetaminophen, a safe analgesic when dosed properly but hepatotoxic at overdoses, has been reported to induce DNA strand breaks but it is unclear whether this event preceeds hepatocyte toxicity or is only obvious in case of overt cytotoxicity. Moreover, it is not known whether the formation of reactive oxygen species (ROS) is involved in the formation of the DNA strand breaks. In the present study, the dose-response curves for cytotoxicity and DNA strand breaks and the response to antioxidant protection have been compared. In primary hepatocytes from untreated male rats, cytotoxicity as measured by the MTT test and by Neutral Red accumulation was obvious at 10 mM acetaminophen but DNA strand breaks as measured by the comet assay were only found at 25-30 mM acetaminophen. Non-cytotoxic concentrations of three compounds with antioxidant activity, the glutathione precursor N-acetylcysteine (100 micro M), the plant polyphenol silibin (25 micro M) and the antioxidant vitamin alpha-tocopherol (50 micro M), were not able to inhibit acetaminophen toxicity at any acetaminophen concentration, while they completely prevented the formation of DNA strand breaks at 25-30 mM acetaminophen. The occurrence of oxidative stress in our experiments was indicated by a slight increase of malondialdehyde formation at 40 mM acetaminophen and by an adaptive increase in catalase mRNA concentration. We conclude that in acetaminophen-treated hepatocytes ROS-independent cell death and ROS-dependent DNA strand breaks occur which appear not to be causally related as judged from their dose dependency and their response to antioxidants.

Germany: toxicology information on the World Wide Web.

Following a brief introduction to the history of toxicology in Germany, this article deals with institutions concerned with toxicological information and research in Germany. The web addresses, responsibilities and main topics of federal authorities, federal state authorities, institutions for statutory accident insurance and prevention, non-university and university research institutes, funding institutions, scientific societies, industry representatives and poison information centers are described and links are given to databases provided on the web sites of these institutions.

Resistance to tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in rat hepatoma cells expressing TNF-alpha is linked to low antioxidant enzyme expression.

In order to study the mechanisms of resistance to tumor necrosis factor-alpha (TNF-alpha), we have constructed two stable transfectants producing TNF-alpha (Yv12-2 and Yv13-44) from the rat hepatoma H4IIE cell, which does not produce TNF-alpha. H4IIE cells were highly sensitive to apoptosis induced by TNF-alpha, whereas Yv2-12 and Yv13-44 cells were resistant. Manganous superoxide dismutase was not up-regulated in Yv2-12 and Yv13-44 cells and was unresponsive to induction by exogenous TNF-alpha and by H2O2 in H4IIE cells and in the transfectants. Catalase expression and activity were lower in Yv2-12 and Yv13-44 cells than in H4IIE cells; furthermore, the transfectants were more susceptible to H2O2. Treatment with exogenous TNF-alpha down-regulated catalase in H4IIE cells but not in Yv2-12 and Yv13-44 cells. Treatment of H4IIE cells with the catalase inhibitor 3-amino-1,2,4-triazole rendered them resistant to exogenous TNF-alpha. These data suggest a causal relationship between resistance to TNF-alpha and low catalase activity. Expression of copper and zinc containing superoxide dismutase was also decreased, whereas expression of glutathione peroxidase-1 was unchanged in Yv2-12 and Yv13-44 cells. Data from a microarray point to a down-regulation of genes in the resistant clones that code for antioxidative proteins and proteins involved in glutathione synthesis and function. We assume that a prooxidant signal linked to the down-regulation of antioxidant defense may be associated with resistance to apoptosis induced by TNF-alpha.

The effect of quercetin on the mRNA expression of different antioxidant enzymes in hepatoma cells.

The flavonol quercetin shows a wide range of effects in biological systems. We investigated whether quercetin exerts its proposed antioxidant properties via the antioxidant enzyme system. Quercetin in a concentration range from 5 to 100 microM decreased manganese superoxide dismutase, glutathione peroxidase, and copper zinc superoxide dismutase mRNA expression levels each by 30-40% in rat hepatoma H4IIE cells. Catalase mRNA expression levels increased about 30% but only with the cytotoxic concentration of 100 microM. Despite the down-regulation of antioxidant enzyme mRNA expression quercetin treatment of cells induced only a mild oxidative stress. Pretreatment of H4IIE cells with quercetin even protected against an oxidative stress resulting from hydrogen peroxide exposure. In conclusion, the antioxidant capacity of quercetin was shown not to be due to the antioxidant enzyme system.

The phytoestrogen daidzein affects the antioxidant enzyme system of rat hepatoma H4IIE cells.

Phytoestrogens such as the soy isoflavonoid daidzein have potential health benefits. The antioxidant properties of phytoestrogens are considered to be responsible in part for their protective effects. The antioxidant enzyme (AOE) system plays an important role in the defense of cells against oxidative insults. To determine whether flavonoids can exert antioxidative effects not only directly but also indirectly by modulating the AOE system, we investigated the influence of the flavonoid daidzein on the expression of different AOE. Daidzein treatment of hepatoma H4IIE cells increased catalase mRNA expression two- to threefold. Expression levels of copper zinc superoxide dismutase (CuZnSOD) were not affected by exposure to daidzein. Manganese superoxide dismutase (MnSOD) mRNA expression levels decreased slightly and glutathione peroxidase (GPx) levels increased slightly after daidzein exposure. Changes in AOE mRNA expression levels were significant at 300 micromol/L daidzein. To elucidate the mechanisms underlying the strong increase in catalase mRNA, transfection experiments were performed. Transient transfection of hepatoma cells with reporter plasmids containing different parts of the upstream region of the catalase gene showed a significant one- to threefold increase in reporter gene activity after daidzein exposure. This indicates that daidzein can directly activate the rat catalase promoter region. Despite the increase in catalase mRNA, daidzein pretreatment of cells did not protect against oxidative stress resulting from H(2)O(2) exposure. On the contrary, daidzein itself exerted a mild oxidative stress. In conclusion, the changes in the AOE system provoked by daidzein affected the oxidant rather than the antioxidant properties of daidzein.

Neurotoxic mode of action of artemisinin.

We recently described a screening system designed to detect neurotoxicity of artemisinin derivatives based on primary neuronal brain stem cell cultures (G. Schmuck and R. K. Haynes, Neurotoxicity Res. 2:37-49, 2000). Here, we probe possible mechanisms of this brain stem-specific neurodegeneration, in which artemisinin-sensitive neuronal brain stem cell cultures are compared with nonsensitive cultures (cortical neurons, astrocytes). Effects on the cytoskeleton of brain stem cell cultures, but not that of cortical cell cultures, were visible after 7 days. However, after a recovery period of 7 days, this effect also became visible in cortical cells and more severe in brain stem cell cultures. Neurodegeneration appears to be induced by effects on intracellular targets such as the cytoskeleton, modulation of the energy status by mitochondrial or metabolic defects, oxidative stress or excitotoxic events. Artemisinin reduces intracellular ATP levels and the potential of the inner mitochondrial membrane below the cytotoxic concentration range in all three cell cultures, with these effects being most dominant in the brain stem cultures. Surprisingly, there were substantial effects on cortical neurons after 7 days and on astrocytes after 1 day. Artemisinin additionally induces oxidative stress, as observed as an increase of reactive oxygen species and of lipid peroxidation in both neuronal cell types. Interestingly, an induction of expression of AOE was only seen in astrocytes. Here, manganese superoxide dismutase (MnSOD) expression was increased more than 3-fold and catalase expression was increased more than 1.5-fold. In brain stem neurons, MnSOD expression was dose dependently decreased. Copper-zinc superoxide dismutase and glutathione peroxidase, two other antioxidant enzymes that were investigated, did not show any changes in their mRNA expression in all three cell types after exposure to artemisinin.