Analysis of in vitro chemoprevention of genotoxic damage by phytochemicals, as single agents or as combinations.

Cancer chemoprevention with low-dose combinations of bioactive phytochemicals instead of single agents has been suggested to induce less toxicity and improve efficacy. In this study, we selected four plant food-based phytochemicals, viz. chlorogenic acid (CLA), pelargonidin (PEL), resveratrol (RES) and epigallocatechin gallate (EGCG) to evaluate the in vitro chemoprevention of genotoxic damage in HL-60 cells. These agents were tested either individually or as a combination at two concentrations (with a 10-fold difference) against the genotoxins mitomycin C (MMC), diepoxybutane (DEB) and patulin (PAT). Our preliminary ferric reducing antioxidant power (FRAP) assay demonstrated additive effects when PEL, CLA, RES and EGCG were combined. Results of the cytokinesis-block micronucleus test showed significant protection against genotoxic damage induced by PAT, DEB and MMC when CLA, PEL, RES and EGCG were tested individually. This protective effect of the phytochemicals was not concentration-related. Both low- and high-concentration combinations of CLA, PEL, RES and EGCG showed significant reducing effects on the frequencies of micronuclei induced by PAT, DEB and MMC. However, the micronucleus test did not provide indications of additive or synergistic effects with this combination of phytochemicals. In conclusion, the chemo-preventive effects of PEL, CLA, RES and EGCG against genotoxic damage induced by MMC, DEB and PAT are indicative of a 'saturation effect' when higher concentrations and combinations of these phytochemicals are used.

Differential gene expression involved in oxidative stress response caused by triethylene glycol dimethacrylate.

Triethylene glycol dimethacrylate (TEGDMA) is a comonomer that is released from dental resin-based materials into hydrophilic solvents. The compound reduces cell vitality, and causes genotoxicity in mammalian cells in vitro. Here, we used gene expression profiling, combined with pathway analysis tools, to identify the molecular events associated with TEGDMA cytotoxicity in human fibroblasts using Affymetrix HG-U133A 2.0 GeneChip arrays. Increased ROS production and a cell cycle delay caused by 3mm TEGDMA after a 6h exposure were related to a cell response at the transcriptional level. The predominant biological processes associated with the genes that were differentially expressed in untreated and treated cell cultures included oxidative stress, cellular growth, proliferation and morphology, cell death, gene expression as well as DNA replication and repair. The most significantly upregulated genes were GEM (17-fold), KLHL24, DDIT4, TGIF, DUSP5 and ATF3, which are all related to the regulation of the cell structure, stress response, and cell proliferation. TXNIP was the most downregulated transcript (five-fold), whose gene product regulates the cellular redox balance. The downregulation of NRG1, ASPM, FBXO5, and PLK2 is linked to the regulation of cell proliferation and cell structure. The underlying mechanisms of the up- and downregulation of genes seem to be activated by the production of ROS, and the related regulation of the cellular redox balance disturbed in the presence of TEGDMA appears to be of the utmost importance. The coordinated induction of genes coding for oxidative stress response and antioxidant proteins is a critical mechanism of protection against TEGDMA-induced cell damage.

Application of single-cell cultures of mouse splenocytes as an assay system to analyze the immunomodulatory properties of bacterial components.

Recently, various bacterial components have been suggested as initiating and modulating immune activation, thereby substantially affecting the complex and dynamic host/pathogen interactions. Herein, we present a valuable and simple methodology for determining the capacity of bacteria as well as defined bacterial structures to stimulate cellular effectors of the innate and cognate immune system. This assay format is based on the exposure of freshly prepared single-cell cultures of splenic cells derived from naive mice with the immunogen of interest. Herein, the determination of exclusive panels of cytokines by the ELISA, ELISpot, and FACS technology will serve as an indicator for the activation of defined arms of the immune system. An increased knowledge about microbial components with immunomodulatory properties will substantially contribute to a more detailed understanding of the dynamic interplay between the host and potential pathogens and, based on this knowledge, to the development of novel substances for the prevention and therapy of microbial infections.

Influence of TEGDMA on the mammalian cell cycle in comparison with chemotherapeutic agents.

OBJECTIVES: The dental resin monomer triethylene glycol dimethacrylate (TEGDMA) caused a cell cycle arrest in response to DNA damage. However, the underlying mechanisms are unclear. Therefore, the influence of TEGDMA on the cell cycle was analyzed in comparison with the chemotherapeutic agents adriamycin and mitomycin C (MMC), which arrest the cell cycle through different mechanisms. METHODS: RAW264.7 mouse macrophages were exposed to TEGDMA, adriamycin, or MMC, and flow cytometry (FACS) was used for cell cycle analyses. In addition, the number of surviving cells was determined by a crystal violet assay, and viability in treated cultures was determined by FACS after staining of cells with trypan blue. Morphological changes in cells were interpreted using forward and side scatter (FSC/SSC) cell physical criteria. RESULTS: The exposure of cells to 1mM TEGDMA resulted in a delay of the cell cycle in G1 phase since 85.3% of the cells were found in G1 compared with 47.4% in untreated controls. Adriamycin also increased the number of cells (72.1%) in G1 compared to controls. Caffeine, an inhibitor of the checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), had no effect on the TEGDMA and adriamycin-induced cell cycle arrest. In contrast, MMC delayed the cell cycle in G2 since cell numbers increased to 22.1% compared to 10.7% in controls. The effect of MMC on G2 was even increased by low caffeine concentrations (100-400muM), but 1000muM caffeine inhibited MMC activity. SIGNIFICANCE: Our results suggest that the mechanism of a TEGDMA-induced arrest of the cell cycle is different from the effect of the direct-acting interstrand crosslinking agent MMC. Since TEGDMA produced oxidative stress, it probably acts indirectly on the cell cycle through reactive oxygen species, unless TEGDMA-DNA adducts are shown experimentally.

The influence of Ni(II) on surface antigen expression in murine macrophages.

Biomedical alloys may release nickel ions during corrosion phenomena and, in addition to their interaction with oral tissues, these ions may also influence characteristic properties of the immune system cells. The aim of this study was to evaluate the effect of nickel chloride on the expression of functionally distinct surface antigens in murine RAW macrophages. The expression of the surface antigens CD14, CD40, MHC class I, MHC class II, CD80, CD86, CD54 was analyzed by flow cytometry. The bacterial endotoxin lipopolysaccharide (LPS) was used as a positive control to induce antigen expression. Cells were stimulated with NiCl(2) (0.1 and 0.5mm) in the presence and absence of LPS (0.1 or 25 microg/ml). After exposure periods of 6, 24 and 48 h, LPS caused a time- and dose-dependent increase in the expression of all surface antigens. CD14 expression was up-regulated by 0.1 microg/ml LPS by about 10-fold after 24h and 100-fold after 48 h. After 48 h, NiCl(2) alone up-regulated the expression of all surface antigens between 2- and 4-fold, while in cells stimulated by LPS, 0.1mm NiCl(2) was effective only on CD14, CD40 and MHC class I. Moreover, 0.5mm NiCl(2) even inhibited the LPS-induced expression of all surface antigens, except for CD54, which was still significantly up-regulated. These results show that nickel chloride is able to induce an up-regulation of surface antigen expression, but a high concentration may impair essential functions of macrophages stimulated by LPS.

TEGDMA-induced oxidative DNA damage and activation of ATM and MAP kinases.

The development of strategies for the protection of oral tissues against the adverse effects of resin monomers is primarily based on the elucidation of underlying molecular mechanisms. The generation of reactive oxygen species beyond the capacity of a balanced redox regulation in cells is probably a cause of cell damage. This study was designed to investigate oxidative DNA damage, the activation of ATM, a reporter of DNA damage, and redox-sensitive signal transduction through mitogen-activated protein kinases (MAPKs) by the monomer triethylene glycol dimethacrylate (TEGDMA). TEGDMA concentrations as high as 3-5 mM decreased THP-1 cell viability after a 24h and 48h exposure, and levels of 8-oxoguanine (8-oxoG) increased about 3- to 5-fold. The cells were partially protected from toxicity in the presence of N-acetylcysteine (NAC). TEGDMA also induced a delay in the cell cycle. The number of THP-1 cells increased about 2-fold in G1 phase and 5-fold in G2 phase in cultures treated with 3-5 mM TEGDMA. ATM was activated in THP-1 cells by TEGDMA. Likewise, the amounts of phospho-p38 were increased about 3-fold by 3 mM TEGDMA compared to untreated controls after a 24h and 48h exposure period, and phospho-ERK1/2 was induced in a very similar way. The activation of both MAPKs was inhibited by NAC. Our findings suggest that the activation of various signal transduction pathways is related to oxidative stress caused by a resin monomer. Signaling through ATM indicates oxidative DNA damage and the activation of MAPK pathways indicates oxidative stress-induced regulation of cell survival and apoptosis.

Inhibition of cytokine and surface antigen expression in LPS-stimulated murine macrophages by triethylene glycol dimethacrylate.

Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) cause a shift in the cellular redox balance which influences redox-sensitive signaling pathways. The immediate response of the innate immune system to inflammatory challenges is controlled by related pathways. Therefore, the influence of TEGDMA on the expression of the pro- and anti-inflammatory cytokines TNF-alpha, IL-6, and IL-10 and surface antigens (CD14, CD40, CD80, CD86, CD54, MHC class I and II) was analyzed in RAW264.7 macrophages. No significant change in cytokine production or surface antigen expression was detected after the macrophages were treated with increasing TEGDMA concentrations for 6, 24, and 48h. However, co-stimulation with the bacterial endotoxin lipopolysaccharide (LPS) and TEGDMA resulted in a concentration-dependent inhibition of LPS-induced release of TNF-alpha, IL-6, and IL-10 by about 90% as detected by ELISA. Flow-cytometric analyses indicated an LPS-stimulated expression of all surface antigens. The LPS-induced expression of CD14 was inhibited by high TEGDMA concentrations. CD40 and CD80 expressions were down-regulated by TEGDMA in LPS-stimulated cells, and CD86 as well as MHC class I expression was inhibited to a lesser extent. The LPS-stimulated expression of CD54 (ICAM-1) was increased about twofold by increasing TEGDMA concentrations after a 24 and 48h exposure. Thus, the ability of macrophages to induce an appropriate immune response is inhibited by TEGDMA which reduces cytokine production and expression of surface antigens.

Characterization of the Helicobacter pylori cysteine-rich protein A as a T-helper cell type 1 polarizing agent.

Predominant T-helper 1 (Th1) responses with increased gamma interferon (IFN-gamma) levels have been proposed to play an important role in Helicobacter pylori-induced gastritis and peptic ulceration. However, bacterial factors contributing to the initiation of Th1 polarization of H. pylori-specific immune responses have not been characterized in detail thus far. We report here on the identification of Helicobacter cysteine-rich protein A (HcpA) as a novel proinflammatory and Th1-promoting protein. The capacity of HcpA to induce immune activation was studied in splenocyte cultures of naive H. pylori-negative mice. HcpA stimulated the release of high concentrations of the proinflammatory and Th1-promoting cytokines interleukin-6 (IL-6) and IFN-gamma, in addition to significant levels of IL-12, tumor necrosis factor alpha, and IL-10. The observed cytokine profile was comparable to that induced by lipopolysaccharide but differed in the kinetics and maximum levels of cytokine production. In addition, HcpA-induced cytokine release resembled that observed upon incubation with H. pylori except for IL-10, which was only moderately released upon HcpA stimulation. Both HcpA- and H. pylori-mediated IFN-gamma production was drastically reduced by a neutralizing antibody against IL-12 but not by an anti-IL-2 antibody. Thus, HcpA seems to represent a novel bacterial virulence factor triggering the release of a concerted set of cytokines to instruct the adaptive immune system for the initiation of proinflammatory and Th1-biased immunity.

Induction of maturation and cytokine release of human dendritic cells by Helicobacter pylori.

Helicobacter pylori causes a persistent infection in the human stomach, which can result in chronic gastritis and peptic ulcer disease. Despite an intensive proinflammatory response, the immune system is not able to clear the organism. However, the immune escape mechanisms of this common bacterium are not well understood. We investigated the interaction between H. pylori and human dendritic cells. Dendritic cells (DCs) are potent antigen-presenting cells and important mediators between the innate and acquired immune system. Stimulation of DCs with different concentrations of H. pylori for 8, 24, 48, and 72 h resulted in dose-dependent interleukin-6 (IL-6), IL-8, IL-10 and IL-12 production. Lipopolysaccharide (LPS) from Escherichia coli, a known DC maturation agent, was used as a positive control. The cytokine release after stimulation with LPS was comparable to that induced by H. pylori except for IL-12. After LPS stimulation IL-12 was only moderately released compared to the large amounts of IL-12 induced by H. pylori. We further investigated the potential of H. pylori to induce maturation of DCs. Fluorescence-activated cell sorting analysis of cell surface expression of maturation marker molecules such as CD80, CD83, CD86, and HLA-DR revealed equal upregulation after stimulation with H. pylori or LPS. We found no significant differences between H. pylori seropositive and seronegative donors of DCs with regard to cytokine release and upregulation of surface molecules. These data clearly demonstrate that H. pylori induces a strong activation and maturation of human immature DCs.