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Apple juice containing high amounts of antioxidative polyphenols might protect the intestine against oxidative cell damage. We investigated the preventive effectiveness of polyphenolic juice extracts of different origins (cider and table apples) in comparison to their major constituents in human colon cell lines (Caco-2, HT29). Parameters studied were (oxidative) DNA damage (Comet assay), glutathione level (photometric kinetic assay), cellular redox status (dichlorofluorescein assay) and antioxidant capacity. The extracts (50-250 microg/mL) modulated DNA damage and redox status in a concentration-dependent manner at 24-h incubation. The pomace extraction technology, applied for juice preparation, and the preferential selection of cider apple varieties influenced the polyphenolic pattern and increased the biological effectiveness of the extracts. The preventive potential of major juice constituents (1-100 microM, 24 h) strongly differed: rutin, epicatechin and caffeic acid clearly reduced (oxidative) DNA damage (Caco-2), chlorogenic acid efficiently decreased cellular reactive oxygen species level (HT29, Caco-2). The aglyca quercetin and phloretin exhibited the highest preventive/antioxidant capacity in all assays. The stability of the compounds inversely correlated with their preventive effectiveness and might contribute to the observed cell specific sensitivities. In conclusion, apple juice extracts distinctly reduce oxidative cell damage in human colon cell lines, an effect, which in part can be accounted for by their major constituents.

The nephrotoxic/carcinogenic mycotoxin ochratoxin A (OTA) occurs as a contaminant in food and feed and may be linked to human endemic Balkan nephropathy. The mechanism of OTA-derived carcinogenicity is still under debate, since reactive metabolites of OTA and DNA adducts have not been unambiguously identified. Oxidative DNA damage, however, has been observed in vitro after incubation of mammalian cells with OTA. In this study, we investigated whether OTA induces oxidative DNA damage in vivo as well. Male F344 rats were dosed with 0, 0.03, 0.1, 0.3 mg/kg bw per day OTA for 4 wk (gavage, 7 days/wk, five animals per dose group). Subsequently, oxidative DNA damage was determined in liver and kidney by the comet assay (single cell gel electrophoresis) with/without use of the repair enzyme formamido-pyrimidine-DNA-glycosylase (FPG). The administration of OTA had no effect on basic DNA damage (determined without FPG); however, OTA-mediated oxidative damage was detected with FPG treatment in kidney and liver DNA of all dose groups. Since the doses were in a range that had caused kidney tumors in a 2-year carcinogenicity study with rats, the oxidative DNA damage induced by OTA may help to explain its mechanism of carcinogenicity. For the selective induction of tumors in the kidney, increased oxidative stress in connection with severe cytotoxicity and increased cell proliferation might represent driving factors.

A promising attempt in the field of tumour therapy is the modulation of intracellular, proliferation-associated signalling pathways. The role of cyclic nucleotide phosphodiesterases (PDEs), key enzymes in cAMP/cGMP signal transduction, was investigated in two human CNS tumour cell lines as well as in the rat glioblastoma cell line C6 in comparison with rat cerebellar astrocytes with the emphasis on target evaluation. We found differential PDE expression patterns in human CNS tumour cell lines as well as in CNS cells of rat origin. In human glioblastoma cells, intracellular cAMP and Ca(2+) levels correlated well with the PDE expression pattern. There were, however, marked differences in PDE expression and Ca(2+) kinetics between the human glioblastoma cell lines. In contrast to human epithelial tumour cells, shown earlier by us to express significantly enhanced cAMP-specific PDE activity, this was not the case in rat glioblastoma cells compared with non-malignant rat astrocytes. Despite different levels of PDE1 and PDE4 expression and activity, cyclic nucleotide and Ca(2+) levels in non-malignant and malignant rat CNS cells were similar. These in vitro data do not support the concept of PDE1C representing a target exploitable for drug treatment of malignant CNS tumours.

Hypoxia inducible factor 1 is regulated by the appearance of the HIF-1alpha subunit. HIF-1alpha is subjected to proteasomal destruction or enhanced protein translation, which requires the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. We investigated how PI3K/Akt and glycogen synthase kinase 3beta (GSK3beta) affect HIF-1alpha in human RKO cells under prolonged periods of severe hypoxia/anoxia. 16- to 32-h lasting incubations attenuated Akt activity and decreased HIF-1alpha protein. This was reproduced by blocking PI3K with LY294002. GSK3beta inhibition by indirubins circumvented the effect of hypoxia/anoxia or LY294002 on HIF-1alpha. Ruling stability regulation of HIF-1alpha protein and/or enhanced transcription of HIF-1alpha mRNA via GSK3beta inhibition out is suggestive for translational modulation of HIF-1alpha under the influence of GSK3beta.

Uremic patients undergoing hemodialysis (HD) are considered to face an elevated risk for atherosclerosis and cancer. This has been attributed in part to an increased oxidative stress. In this pilot study, oxidative cell damage in blood of HD-patients was compared to those of controls: total DNA damage (basic and specific oxidative DNA damage), modulation of glutathione levels (total and oxidized glutathione) and of lipid peroxidation were monitored via the Comet assay (with and without FPG), a kinetic photometric assay and HPLC quantification of plasma malondialdehyde (MDA), respectively. In some samples, leukocytes were analysed for malondialdehyde-deoxyguanosine-adducts (M1dG) with an immunoslot blot technique. HD-patients (n=21) showed a significant increase of total DNA damage (p10(-12)), compared to controls (n=12). In a subset of patients and controls, GSSG levels and M1dG, however, only increased slightly, while tGSH and MDA levels did not differ. The influence of different low flux HD-membranes was tested in a pilot study with nine patients consecutively dialysed on three membrane types for four weeks each. In addition to the individual disposition of the patient, the dialyser membrane had a significant impact on oxidative stress. Total DNA damage was found to be almost identical for polysulfone and vitamin E coated cellulosic membranes, whereas a slight, but significant increase was observed with cellulose-diacetate (p0.001). In patients receiving iron infusion during HD, MDA-formation (n=11) and total DNA damage (n=10) were additionally increased (p0.005). Our results show an increased oxidative damage in HD-patients, compared to healthy volunteers. Significant influences were found for the dialyser membrane type and iron infusion.

We report on the establishment of one transgenic and two endogenous reporter gene assays to determine androgenic/antiandrogenic activity. A transient transactivation assay was developed in COS-7 African green monkey kidney cells. Three plasmids were co-transfected by electroporation: the human androgen receptor expression vector pSG5AR, the reporter gene vector pMamneoLuc, expressing luciferase under the control of the mouse mammary tumor virus (MMTV) promotor containing 4 hormone responsive elements (HREs), and the control plasmid pSVβ . Transcriptional activation was measured by luciferase-mediated chemoluminescence. In T47D human breast cancer cells two endogenous reporter gene systems were established: one based on the androgen-induced expression of prostate-specific antigen (PSA), the other based on the androgen-repressed expression of testosterone repressed message 2 (TRPM-2). PSA protein was measured by enzyme-linked immunosorbent assay (ELISA), TRPM-2 m-RNA by reverse transcriptase polymerase chain reaction (RT-PCR). All three test systems were validated using the physiological androgen dihydrotestosterone (DHT) as agonist and the established antiandrogens hydroxyflutamide and p,p′-dichlorodiphenylethene (p,p′-DDE) as antagonists. The PSA assay was the most sensitive test system with an EC50 of 0.7 nM for DHT-induced response. The transient transactivation assay in COS-7 cells was less sensitive with an EC50 of 9.7 nM DHT. In the PSA assay hydroxyflutamide was a more potent antagonist (IC30 = 0.02 μM) than p,p′-DDE (IC30 = 0.9 μM). Similar results were obtained in the TRPM-2 assay. In the transient transactivation assay in COS-7 cells, both compounds elicited about 30% of the agonistic response induced by 100 nM DHT, thus showing partial agonistic properties. In summary, three highly sensitive and complementary in vitro test systems, together achieving enhanced specificity for detection and assessment of androgenic/antiandrogenic activity have been established and validated.