Prenylation enhances cytotoxicity of apigenin and liquiritigenin in rat H4IIE hepatoma and C6 glioma cells.

Antioxidative as well as cytotoxic effects of the prenylated flavonoids licoflavone C (8-prenylapigenin) and isobavachin (8-prenylliquiritigenin) were investigated in comparison to the corresponding non-prenylated flavonoids (apigenin, liquiritigenin) and vitexin (apigenin-C8-glucoside) using metabolically active H4IIE hepatoma and metabolically poorly active C6 glioma cells. None of the substances showed radical scavenging activities in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-assay nor were they effective in protection against H2O2-induced intracellular 2',7'-dichlorodihydrofluorescein (H2DCF) oxidation (fluorescent probe for oxidative stress) in H4IIE and C6 cells. When the intrinsic effects of the substances were investigated, licoflavone C and isobavachin exerted a pronounced toxicity in both H4IIE (IC50 values of 42+/-5 and 96+/-19 micromol/L) and C6 cells (IC50 values of 37+/-6 and 69+/-3 micromol/L) while the non-prenylated analogues as well as the glycosylated derivate vitexin showed almost no cytotoxic effect up to 250 micromol/L. In H4IIE cells the induction of apoptotic cell death by licoflavone C and icobavachin was detected as an activation of caspase 3/7 (6- and 3.3-fold, respectively). Based on these experiments we suggest that C8-prenylation of a flavonoid enhances the cytotoxicity inducing an apoptotic cell death in H4IIE cells without affecting antioxidative properties.

Resveratrol induces apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6 glioma cells.

Resveratrol (trans-3,5,4',-trihydroxystilbene) is assumed to possess cancer-preventive and cancer-therapeutic properties. The aim of this project was to analyze cellular effects of resveratrol in metabolically active H4IIE rat hepatoma cells in comparison to metabolically poorly active C6 rat glioma cells. Resveratrol is rapidly taken up by both cell types and acts as a potent intracellular antioxidant. On the other hand, resveratrol in higher concentrations is relatively toxic to both cell lines as measured by the neutral red accumulation assay. In H4IIE cells, resveratrol concentrations rapidly decline to very low levels during the first hours of incubation due to formation of resveratrol glucuronides. The first resveratrol effect found at 3h after the start of resveratrol treatment was the induction of mild DNA damage as detected by the comet assay. Cell death was caused via induction of apoptosis as detected by caspase activation, oligonucleosomal DNA fragmentation and formation of apoptotic nuclei. Following DNA damage, resveratrol led to an activation of caspases 2 and 8/10 at 6h and consequently of caspase 3 at 12h, but failed to activate caspase 9. In contrast to H4IIE cells, resveratrol is not metabolised in C6 glioma cells and accumulates to concentrations which are assumed to drive the cell into necrosis. This suggests that the mode of cell death caused by resveratrol and the usefulness of resveratrol for cancer prevention and treatment critically depends on the metabolic capacity of the tumor cell to be eradicated.

Pro-apoptotic effects of the flavonoid luteolin in rat H4IIE cells.

Polyphenols are ubiquitous substances in the diet. Their anti-oxidative, anti-inflammatory and anti-viral effects are of interest for human health, and polyphenols such as luteolin are used at high concentrations in food supplements. The aim of this project was to determine the intrinsic effects of luteolin in H4IIE rat hepatoma cells. Luteolin is relatively toxic, cell death was caused via induction of apoptosis as detected by DNA-ladder formation, by nuclear fragmentation and activation of apoptotic enzymes (caspase-2, -3/7, -9 and -8/10). Luteolin (250 microM, 24 h) increased the caspase-3/7 activity four-fold and the caspase-9 activity six-fold. In a time course experiment caspase-9 is activated after 6h, while caspase-2 and -3/7 are activated after 12 h. After 24 h, caspase-8/10 also displays activation. We found a concentration-dependent increase in malondialdehyde release suggesting a prooxidative effect of luteolin. Furthermore, we analysed DNA strand break formation by luteolin and found a distinct increase of DNA strand breaks after incubation for 3h with 100 microM luteolin, a concentration which induces oligonucleosomal DNA cleavage at 24h. In conclusion, the sequence of events is compatible with the assumption that luteolin triggers the mitochondrial pathway of apoptosis, probably by inducing DNA damage.

Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes.

We are developing strategies to use naive T lymphocytes in cancer therapy. For this purpose, we are deriving T cells with specificity of recognition for defined tumor cells. To direct effector lymphocytes toward tumor cells, we have manipulated the recognition specificity of naive rat and mouse T lymphocytes and a mouse T-cell line. The cells were stably transduced with a chimeric T-cell receptor (TCR) component. The zeta chain of the TCR consists of a single transmembrane protein with a short extracellular domain and an intracellular domain for TCR signaling. We provided an extracellular tumor cell recognition domain to the zeta chain. Human heregulin beta1 (ligand to the erbB-3 and erbB-4 receptors) and three different single-chain antibodies specific for the human and rat Neu/erbB-2 receptors were used. One single-chain antibody (C11) is directed against the rat Neu protein, and one single-chain antibody (FRP5) is directed against the human erbB-2 receptor. The single-chain antibody (R-AK) directed against the Mr 14,000 fusion protein of orthopox viruses served as a control. An efficient procedure was devised to introduce the chimeric genes into primary rat and mouse T lymphocytes. Retrovirus-producing packaging cell lines were cocultured with the T cells activated by phytohemagglutinin and interleukin 2. T-cell lines were transduced by exposure to retrovirus-containing supernatants from helper cell lines. Expression of the fusion genes was determined by fluorescence-activated cell sorting analysis. More than 80% of the naive rat and mouse T cells and 85-100% of the cells from the established T-cell lines expressed the fusion genes within 48 h after infection. The expression of the fusion genes was maintained for at least 10 days after infection. Target cells expressing Neu/erbB-2, erbB-3, or erbB-4 were lysed in vitro with high specificity by T cells expressing the corresponding recognition proteins. No selection of a marker gene is necessary to confer a predetermined recognition specificity. The described experiments are important for a gene therapy approach to cancer treatment with autologous T cells.

Alterations of antioxidant enzyme expression in response to hydrogen peroxide.

The effect of hydrogen peroxide (H2O2) on the expression of different antioxidant enzymes was investigated in primary rat hepatocytes and the rat hepatoma H4IIE cell line. Catalase mRNA expression and enzyme activity decreased during rat hepatocyte culture. Exposure of hepatocytes to H2O2 prevented this decrease in catalase mRNA expression, catalase expression was induced 2-fold. MnSOD message levels showed a peak after 12 h of culture and MnSOD enzyme activity increased similarly. MnSOD mRNA expression was also induced after exposure to H2O2. Cu/ZnSOD mRNA expression remained constant during culturing and was not affected by H2O2 treatment. In confluent hepatoma H4IIE cells catalase mRNA expression was lower than in early hepatocyte cultures and could be induced 2-fold upon treatment with H2O2. Actinomycin D alone caused the same amount of induction of catalase mRNA in rat hepatocytes as in combination with H2O2. Exposure of hepatocytes to cycloheximide did not influence the induction of catalase mRNA by H2O2. In rat hepatoma H4IIE cells the induction of catalase mRNA by H2O2 was prevented by the addition of actinomycin D or cycloheximide. Although induction of catalase mRNA by H2O2 was found in rat hepatocytes and H4IIE cells, gene expression of catalase does not appear to be regulated in both cell types in the same manner.

Enhancement of epoxide hydratase activity in rat lung, kidney and liver by dietary antioxidants.

Enhancement of pulmonary epoxide hydratase activity in the rat was obtained by feeding a diet which contained 0.5% ethoxyquin (EQ) or 3,5-di-tert-butyl-4-hydroxytoluene (BHT). The enhancement was less marked (1.6-fold) than in the liver, where a 2-4-fold enhancement of enzyme activity was found after feeding 0.5% antioxidant. A minimal enhancing concentration of 0.1% EQ or BHT in food was established for hepatic epoxide hydratase. In kidney microsomes, elevation of enzyme activity was obtained with 0.5% EQ (1.8-fold), but not with 0.5% BHT. No concomitant increase of cytochrome P-450 content and of aryl hydrocarbon hydroxylase activity was found in the tissues studied.

Modulation of benzo[a]pyrene-induced morphological transformation of Syrian hamster embryo cells by butylated hydroxytoluene and butylated hydroxyanisole.

The Syrian hamster embryo cell transformation assay has been used to investigate the effect of two synthetic antioxidants on morphological transformation induced by the initiator benzo[a]pyrene (BP). A two-stage protocol was employed with an initiation phase of 2 days and a subsequent promotion phase of 5 days. When 10 microM butylated hydroxytoluene (BHT) were present in the promotion phase instead of the solvent the transformation frequency at 0.1 micrograms BP/ml increased from 0.27% to 0.55%; at 100 microM of BHT the transformation frequency was 0.77%. Butylated hydroxyanisole (BHA) also enhanced the percentage of transformed colonies from 0.40% (10 microM) to 0.49% (100 microM), respectively. No significant initiating activity was detected for both antioxidants when tested in the initiation phase instead of BP; when the antioxidants were applied simultaneously with BP (1 microgram/ml) during the initiation phase the transformation frequency was decreased from 0.64% to 0.15% (100 microM BHT) and to 0.17% (100 microM BHA), respectively. These results show that the dual action of phenolic antioxidants on chemical carcinogenesis, which depends on the administration schedule, can be imitated in an in vitro test system. In addition to their anti-initiation effect, BHT and BHA, while devoid of intrinsic initiator potency, exert a moderate promotional activity on hamster embryo cell cultures. Their ability to enhance tumorigenesis by various carcinogens in vivo is likely to be at least partially related to such promotion-like effects on cell growth and morphology.

Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS.

OBJECTIVE: Measurement of hydrogen peroxide concentrations in breath condensate of mechanically ventilated patients with ARDS and with risk factors for developing ARDS. DESIGN: Open study in intensive care patients. SETTING: Intensive care units of the Clinics of the University of Göttingen, a primary care center. PATIENTS: 10 post-operatively ventilated patients as a control group and 26 patients with acute respiratory failure, 7 of them with ARDS, 12 with polytrauma, 4 with pneumonia, 3 with cardiogenic or nephrogenic pulmonary edema. INTERVENTIONS: None. MEASUREMENTS: Breath condensate was collected by a special cold trap and was analysed for H2O2 by a chemiluminescence method. Daily measurements were performed for 4.2 +/- 2.6 days (mean +/- SD) as soon as possible after manifestation of respiratory failure. RESULTS: Patients with acute respiratory failure exhibited higher H2O2 concentrations than control patients (median 95 nmol/l, range 76-144 nmol/l), with the highest median value found in the ARDS group (552 nmol/l, range 154-893). After clinical improvement, H2O2 concentrations decreased to the range of the control group. CONCLUSION: Since high concentrations of H2O2 in breath condensate were only found in patients with ARDS or with risk factors for ARDS, the results add to the existing evidence that reactive oxygen species are associated with some acute lung diseases.

The influence of oxidative stress on catalase and MnSOD gene transcription in astrocytes.

The brain is particularly vulnerable to oxygen free radicals, which have been implicated in the pathology of several neurological disorders. The antioxidant enzyme (AOE) system of the brain may play an important role in the protection against such oxidative stress. We investigated the influence of oxidative stress on the transcription of catalase and MnSOD mRNA. Primary rat astroglial cell cultures were treated either with hydrogen peroxide (H2O2), as a direct mediator of oxidative stress, or with the redox cycling compound paraquat. Both substances led to an increase of catalase and MnSOD mRNA levels. To further elucidate the mechanisms residing behind this increase, transfection experiments were performed. Transient transfection of primary astroglial cells with a reporter plasmid containing the upstream region of the catalase gene showed a decrease in reporter gene activity after exposure of transfected cells to either H2O2 or paraquat. In contrast, transfection experiments done with reporter plasmids for the MnSOD upstream region resulted in an increase of reporter gene activity after H2O2 as well as after paraquat treatment of transfected cells. These results indicate transcriptional regulation of MnSOD and post-transcriptional regulation of catalase gene expression after oxidative stress in primary rat astrocytes.

[False diagnosis of space occupying lesions as multiple sclerosis (author's transl)]. / Verkennung spinaler Raumforderungen als multiple Sklerose

Two cases are described in which severe neurological deficits arose because of the wrong diagnosis of Multiple Sclerosis over many years. In both cases the correct diagnosis of a space occupying lesion was possible by clinical signs, particularly by the X-ray examination.

Induction of rat hepatic UDP-glucuronosyltransferases by dietary ethoxyquin.

Dietary administration of 0.5% ethoxyquin markedly enhanced rat hepatic UDP-glucuronosyltransferase activities. Both 3-methylcholanthrene- and phenobarbital-inducible glucuronidation reactions were stimulated by the antioxidant. In contrast, phenobarbital-inducible bilirubin glucuronidation was not affected by ethoxyquin.

Binding of benzo(a)pyrene metabolites to cellular DNA in perfused rat lungs.

The influence of pretreatment with monooxygenase inducers on total irreversible binding of metabolically activated [3H]-benzo(a)pyrene to cellular DNA and the formation of benzo(a)pyrene metabolite-deoxyribonucleoside adducts after cytochrome P-448 induction was studied in perfused rat lungs. Pretreatment with the cytochrome P-448 inducer beta-naphthoflavone increasing binding by a factor of 23. In lungs of induced animals, 0.45 pmoles of benzo(a)pyrene equivalents were bound per mg DNA. Binding to RNA and to protein was also considerably induced by beta-naphthoflavone. Phenobarbital treatment did not significantly increase binding to cellular macromolecules of rat lung. Analysis of hydrolyzed DNA of lungs from beta-naphthoflavone-treated rats by Sephadex LH 20 chromatography revealed the formation of at least two nucleoside adducts with metabolically activated benzo(a)pyrene one of which is probably due to modification of the DNA with a benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide and the other to modification of DNA with secondary metabolites of benzo(a)pyrene phenols.

Differential inhibition of biphenyl hydroxylation in perfused rat liver.

A differential inhibition of biphenyl hydroxylation by alpha-naphthoflavone and metyrapone was observed in isolated perfused rat liver. alpha-Naphthoflavone inhibited 2- and 4-hydroxylation in livers from beta-naphthoflavone-pretreated animals but had no effect on both reactions in livers from phenobarbital-pretreated animals. Metyrapone inhibited 2- and 4-hydroxylation in phenobarbital-stimulated livers, but only insignificant inhibition of 2-hydroxylation and a slight enhancement of 4-hydroxylation by metyrapone was observed in beta-naphthoflavone-stimulated livers. Conjugation of 2-hydroxybiphenyl and 4-hydroxybiphenyl by isolated perfused livers was also studied. 4-Hydroxybiphenyl preferentially formed sulphates in livers from untreated animals but after induction glucuronidation was as effective as sulphation or even exceeded sulphation. Only glucuronic acid conjugates of 2-hydroxybiphenyl were detected.

Synthetic antioxidants: biochemical actions and interference with radiation, toxic compounds, chemical mutagens and chemical carcinogens.

Biological actions of 4 commonly used synthetic antioxidants--butylated hydroxyanisole, butylated hydroxytoluene, ethoxyquin and propyl gallate--on the molecular, cellular and organ level are complied. Such actions may be divided into modulation of growth, macromolecule synthesis and differentiation, modulation of immune response, interference with oxygen activation and miscellaneous. Moreover, an overview of beneficial and adverse interactions of these antioxidants with exogenous noxae is given. Beneficial interactions include radioprotection, protection against acute toxicity of chemicals, antimutagenic activity and antitumorigenic action. Possible mechanisms of the antitumorigenic action of antioxidants are discussed. This discussion is centered around antioxidant properties which may contribute to a modulation of initiation-related events, especially their ability to interfere with carcinogen metabolism. The beneficial interactions of antioxidants with physical and chemical noxae are contrasted to those leading to unfavorable effects. These include radiosensitization, increased toxicity of other chemicals, increased mutagen activity and increased tumor yield from chemical carcinogens. At present, the latter one can most adequately be characterized as tumor promotion at least in the case of butylated hydroxytoluene. It is concluded that current information is insufficient to promote expectations as to the use of antioxidants in the prevention of human cancer.

Production of reactive oxygen species due to metabolic activation of butylated hydroxyanisole.

The synthetic antioxidant butylated hydroxyanisole (BHA) stimulates superoxide formation in rat liver microsomes up to 10-fold. This stimulation is prevented by the monooxygenase inhibitor metyrapone and does not occur when NADH is consumed instead of NADPH indicating that metabolic activation is required for superoxide production. The BHA metabolite tert-butylhydroquinone (TBHQ) is much more active than BHA in stimulating superoxide production, and the amounts of TBHQ and formaldehyde formed from BHA in microsomes are sufficient to explain the effect of BHA. In buffer and in a xanthine oxidase system, superoxide production by TBHQ also takes place. TBHQ autoxidizes to tert-butylquinone (TBQ) and TBQ exceeds TBHQ by far in its capacity for superoxide production in microsomes. Thus, a 30-fold increase of basal superoxide production is induced by 5 microM TBQ. In rat forestomach, the target organ of BHA carcinogenicity in rodents, stimulation of superoxide production by BHA and more markedly by TBHQ and TBQ is also observed. Excess production of superoxide in microsomes by TBHQ is accompanied by excess production of hydrogen peroxide and of hydroxyl radicals. It is concluded that TBQ undergoes redox cycling leading to an oxidative burst in the presence of enzymes capable of one electron reduction of TBQ and that the BHA metabolite TBHQ enters the redox cycle by autoxidation. No oxygen activating properties can be ascribed to BHA itself.

Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-alpha from rat hepatocyte cultures.

Western blot analysis of conditioned media from hepatocytes exposed to H2O2 revealed that a 28 kDa protein was released dose-dependently in response to 1-10 mM H2O2. The 28 kDa protein was present in freshly isolated hepatocytes and exhibited cross-reactivity towards an antibody against CINC/gro. The intracellular amount of the protein decreased in parallel to the H2O2-induced release into the medium. The CINC-related protein was absent in media harvested after 1 h of treatment. The delivery of CINC-related protein correlated with the extent of cell damage as judged from lactate dehydrogenase leakage. Likewise, exposure of hepatocytes to 10-50 mM acetaminophen resulted in a dose-dependent release of the CINC-related protein after 24 h of culture. In contrast, monomeric CINC (molecular weight approximately 6.5 kDa) but not the 28 kDa CINC-related protein was released by lipopolysaccharide (LPS)-stimulated Kupffer cells. The amount of monomeric CINC liberated by Kupffer cells was diminished upon acetaminophen-treatment. Also, the release of tumor necrosis factor-alpha by hepatocytes was reduced after exposure to high acetaminophen doses (40-50 mM). In contrast to this finding, TNF-alpha release from hepatocyte cultures was not affected after H2O2 treatment. These data suggest that damaged hepatocytes release proinflammatory cytokines which may aggravate liver injury through activation of neutrophils and monocytes. The results indicate that the appearance of the CINC-related protein is due to impairment of plasma membrane integrity as the consequence of massive cell damage. In addition, APAP inhibited the release of monomeric CINC from LPS-activated Kupffer cells and of TNF-alpha from hepatocytes even at concentrations that were not sufficient to affect cell viability.

Effect of dietary antioxidants on benzo[a]pyrene metabolism in rat liver microsomes.

Feeding of rats with 1% ethoxyquin (EQ) and butylated hydroxytoluene (BHT) but not butylated hydroxyanisole (BHA) increases the formation rate of benzo[a]pyrene (BP)-4,5-dihydrodiol from BP in hepatic microsomes. The production of other BP-dihydrodiols and of BP phenols is decreased after treatment with EQ, BHT and BHA. EQ and BHT are more effective than BHA in inducing epoxide hydrolase (EH) activity towards styrene oxide as the substrate.

Ortho-phenanthroline modulates enzymes of cellular energy metabolism.

The lipophilic iron chelator 1,10-phenanthroline has been used in mechanistic studies on intracellular oxidant damage because iron is assumed to play a role in the endogenous formation of highly reactive oxygen species. This study shows that 1,10-phenanthroline has enzyme-modulatory properties in addition to its antioxidant activity. In rat hepatocytes, 1,10-phenanthroline caused inhibition of respiration and enhancement of cellular ATP content, pyruvate release and CO2 formation from glycerol resulting from a modulatory action of 1,10-phenanthroline on various enzymes involved in cellular energy metabolism. In intact mitochondria and in submitochondrial particles, oxygen consumption, complex I activity, and ATPase degradation are inhibited by 1,10-phenanthroline. In submitochondrial particles, complex II activity can also be suppressed by 1,10-phenanthroline. The purified cytosolic enzymes lactate dehydrogenase and glycerol-3-phosphate dehydrogenase are inhibited while purified glyceraldehyde-3-phosphate dehydrogenase is activated by 1,10-phenanthroline. The results suggest that 1,10-phenanthroline modulates various enzyme activities linked to cellular energy metabolism and that this property must be taken into account when using 1,10-phenanthroline as a tool in experiments on oxidant effects in cells.

Formation of the semiquinone anion radical from tert-butylquinone and from tert-butylhydroquinone in rat liver microsomes.

The tert-butylsemiquinone anion radical is formed from tert-butylhydroquinone and from tert-butylquinone in rat liver microsomes. In the presence of oxygen, the quinone and, less extensively, the hydroquinone induce excess production of superoxide in microsomes. It is concluded that autoxidation of the semiquinone formed from the quinone by microsomal enzyme activity is responsible for superoxide formation and that the hydroquinone enters the redox cycle via autoxidation. tert-Butylquinone induces injury of the hepatocyte plasma membrane. tert-Butylhydroquinone and tert-butylquinone are metabolites of the antioxidant butylated hydroxyanisole. The semiquinone-dependent superoxide formation may contribute to the toxic actions of butylated hydroxyanisole.