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Xanthohumol (XN) is a prenylated chalcone with antimutagenic and anticancer activity from hops. A nonaqueous reverse polarity capillary electrophoretic method for the determination of XN in hop extract was developed and validated. The optimal parameters were a 64.5 cm long fused-silica capillary with 50 microm id at 25 degrees C; 30 kV negative voltage (anode at detector side of the capillary); nonaqueous buffer with 75 mM NaOH and 50 mM boric acid in methanol; hydrodynamical injection with 10 mbar for 40 s; and detection at 440 nm. XN, isoxanthohumol (IX), colupulone, adlupulone, and n-lupulone were well resolved on the electropherogram. The LOD for XN was 0.05 mg/L and RSD for peak area was below 3%. The amount of XN in different samples of hop pellets varied from 0.14 to 0.42%.

Background. Microcystins (MCs) are cyclic heptapeptides that are considered to be liver specific toxins. They are potent tumour promoters and recent studies indicate that they are also genotoxic. In this study we measured DNA damage in lymphocytes, liver, kidney (cortex and medulla), lung, spleen and brain cells of male Fisher F344 rats that were exposed to sublethal dose (every second day 10 μg/kg b.w.; i.p) of micro-cystin-YR (MCYR) for one month. Methods. At the end of exposure the animals were sacrificed, the lymphocytes were isolated from blood taken from jugular vein, liver cells were obtained by perfusion with collagenase A and the cells from other organs were isolated by incubating small tissue pieces with collagenase A. The DNA damage in isolated cells was measured with the single cells gel electrophoresis (SCGE) also called the comet assay. Results. A significant increase of the % tail DNA in MCYR-exposed animals compared to the nonexposed control ones was observed in brain (2.5 fold), liver (2.1 fold), kidney medulla (1.9 fold), kidney cortex (1.8 fold) and lung (1.7 fold) cells, while the DNA from lymphocytes and spleen cells was not affected. Conclusion. This study demonstrated that subchronic exposure to sublethal doses of MCs can induce systemic genotoxicity in mammals, and it affects not only the liver but also other vital organs.

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma-glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 microM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl(2) (1, 10 microM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.

Microcystin-LR (MCLR) is a liver-specific toxin known as a tumour promoter in experimental animals. Its mechanisms of hepatotoxicity have been well documented; however, the mechanisms of other effects, in particular those related to its genotoxicity, are not well understood. In our previous studies, we showed that MCLR-induced DNA strand breaks are transiently present and that the damage is mediated by reactive oxygen species (ROS). In this study, we show that exposure of HepG2 cells to non-cytotoxic doses of MCLR-induced time-dependent alterations in the level of intracellular reduced glutathione (GSH). These comprised a rapid initial decrease followed by a gradual increase, reaching a maximum after 6h of exposure, before returning to the control level after 8h. During the first 4h, expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, increased, indicating an increased rate of de novo synthesis of GSH. The most important observation of this study, combined with the results of our previous studies is the correlation between the time course of alterations of intracellular GSH content and the formation and disappearance of MCLR-induced DNA damage. When the intracellular GSH level was reduced, MCLR-induced DNA damage was observed to increase. Later, when the level of intracellular GSH was normal or elevated, new DNA damage was not induced and existing damage was repaired. To confirm the role of GSH system in MCLR-induced genotoxicity, the intracellular GSH level was moderated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor. Pre-treatment with BSO dramatically increased the susceptibility of HepG2 cells to MCLR-induced DNA damage, while pre-treatment with NAC almost completely prevented MCLR-induced DNA damage. Thus, intracellular GSH is shown to play a critical role in the cellular defence against MCLR-induced DNA damage in HepG2 cells.

Microcystins are cyclic heptapeptides produced by different freshwater cyanobacterial species such as Microcystis aeruginosa. They have been shown to induce DNA damage in vitro and in vivo, however, the mechanisms of their genotoxic activity remain unclear. With the comet assay we demonstrate that, in human hepatoma HepG2 cells, microcystin-LR (MCLR) induced DNA strand breaks which were transiently present and probably produced during the cellular repair of MCLR-induced DNA damage. Digestion of DNA from MCLR-treated HepG2 cells with purified formamidopyrimidine-DNA glycosylase (Fpg), which recognizes specific oxidized purines, displayed a greater extent of DNA strand breaks than non-digested DNA, providing evidence that MCLR induced oxidation of purines. The number of DNA strand breaks detected after digestion with Fpg increased with time of exposure of the cells to MCLR, indicating that oxidized purines were not repaired. Using the 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluoroprobe we showed that MCLR, at non-cytotoxic concentrations, induced a time and dose dependent increase of intracellular reactive oxygen species (ROS) formation in HepG2 cells. The role of ROS in MCLR-induced DNA damage was further confirmed by exposing the cells to MCLR in the presence of different ROS scavengers. The formation of DNA strand breaks and oxidized purines was completely prevented by a superoxide dismutase mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), an iron chelator, deferoxamine (DFO), a precursor of glutathione (GSH) and intracellular ROS scavenger, N-acetyl-L-cysteine (NAC), and partly by hydroxyl radical scavengers dimethylsulphoxide (DMSO) and 1,3-dimethyl-2-thiourea (DMTU). The results provide evidence that the genotoxicity of MCLR is mediated by ROS.

Arsenic is a well-established human carcinogen and is ubiquitous in the environment. For decades, arsenic has been considered to be a nongenotoxic carcinogen because it is only weakly active or, more often, completely inactive in bacterial and mammalian cell mutation assays. In this review, evidence is presented that when assayed using model systems in which both intragenic and multilocus mutations can readily be detected, arsenic is, indeed, found to be a strong, dose-dependent mutagen which induces mostly multilocus deletions. Furthermore, the roles of reactive oxygen and reactive nitrogen species in mediating the genotoxic response are presented in a systematic and logical fashion in support of a working model. The data suggest that antioxidants may be a useful interventional treatment in reducing the deleterious effects of arsenic.

In 1976, Sporn has defined chemoprevention as “the use of pharmacologic or natural agents that inhibit the development of invasive breast cancer either by blocking the DNA damage that initiates carcinogenesis, or by arresting or reversing the progression of premalignant cells in which such damage has already occurred.” Although the precise mechanism or mechanisms that promote a breast cancer are not completely established, the success of several recent clinical trials in preventive settings in selected high-risk populations suggests that chemoprevention is a rational and an appealing strategy. Breast cancer chemoprevention has focused heavily on endocrine intervention using selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs). Achieving much success in this particular setting and new approaches as low-dose administration are actually under investigations in several topics. Unfortunately, these drugs are active in prevention of endocrine responsive lesions only and have no effect in reducing the risk of estrogen-negative breast cancer. Thus, recently new pathways, biomarkers, and agents likely are to be effective in this subgroup of cancers and were put under investigation. Moreover, the identification of new potential molecular targets and the development of agents aimed at these targets within cancer have already had a significant impact on advanced cancer therapy and provide a wealth of opportunities for chemoprevention. This paper will highlight current clinical research in both ER-positive and ER-negative breast cancer chemoprevention, explaining the biologic effect of the various agents on carcinogenesis and precancerous lesions, and finally presenting an excursus on the state-of-the-art about new molecular targets under investigations in breast cancer settings.

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in antiinvasive and antimetastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, classspecific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca 074Me, inhibited invasion from 20 40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.

Cadmium is an important heavy metal environmental toxicant, which is classified as a human carcinogen. The comet assay was used to evaluate the levels of DNA damage in a metabolically competent HepG2 cell line after treatment with low, non-cytotoxic and physiologically relevant concentrations of cadmium, alone and in combination with the dietary mutagen 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and with the environmental mutagen benzo[a]pyrene (B(a)P). After exposure of the cells to 10, 100 and 1000 nM CdCl(2), a dose- and time-dependent increase of DNA damage was detected. Maximal damage was found after 12 h of treatment, but declined with further incubation with CdCl(2). The increased synthesis of metallothioneins on exposure to CdCl(2) up to 12 h suggests that they are responsible for the adaptation of HepG2 cells to the DNA damaging effects of CdCl(2). Co-treatment of the cells with CdCl(2) (10-1000 nM) and IQ (300 microM) induced a dose-dependent increase of DNA damage compared to cells treated with IQ alone. Co-genotoxic activity was also observed by increased formation of micronuclei in cells exposed to IQ and 1000 nM CdCl(2); at this concentration, CdCl(2) alone also induced micronuclei in HepG2 cells. Our results support the hypothesis that direct and indirect mechanisms are involved in cadmium-induced DNA damage.

Corrigendum to ‘‘Protective effects of xanthohumol against the genotoxicity of heterocyclic aromatic amines MeIQx and PhIP in bacteria and in human hepatoma (HepG2) cells’’ [Food and Chemical Toxicology 50 (2012) 949–955] Olga Viegas , Bojana Žegura , Marko Pezdric , Matjaž Novak , Isabel M.P.L.V.O. Ferreira , Olivia Pinho , Metka Filipic c,⇑ a Faculdade de Ciencias da Nutricao e Alimentacao da Universidade do Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal REQUIMTE, Laboratorio de Bromatologia e Hidrologia, Departamento de Ciencias Quimicas, Faculdade de Farmacia da Universidade do Porto, Rua Anibal Cunha 164, 4099-030 Porto, Portugal Department for Genetic Toxicology and Cancer Biology, National Institute of Biology, Vecna pot 111, SI-1000 Ljubljana, Slovenia

A qualitative risk assessment identified Salmonella spp., Yersinia enterocolitica, Toxoplasma gondii and Trichinella spp. as the most relevant biological hazards in the context of meat inspection of swine. A comprehensive pork carcass safety assurance is the only way to ensure their effective control. This requires setting targets to be achieved in/on chilled carcasses, which also informs what has to be achieved earlier in the food chain. Improved Food Chain Information (FCI) enables risk-differentiation of pig batches (hazard-related) and abattoirs (process hygiene-related). Risk reduction measures at abattoir level are focused on prevention of microbial contamination through technology- and process hygiene-based measures (GMP/GHP- and HACCP-based), including omitting palpation/incision during post-mortem inspection in routine slaughter, as well as hazard reduction/inactivation meat treatments if necessary. At farm level, risk reduction measures are based on herd health programmes, closed breeding pyramids and GHP/GFP. Chemical substances listed in Council Directive 96/23/EC were ranked into four categories. Dioxins, dioxin-like polychlorinated biphenyls and chloramphenicol were ranked as being of high potential concern. However, chemical substances in pork are unlikely to pose an immediate or short term health risk for consumers. Opportunities for risk-based inspection strategies by means of differentiated sampling plans taking into account FCI were identified. Regular update of sampling programmes and inclusion of inspection criteria for the identification of illicit use of substances were also recommended. Meat inspection is a key component of the overall surveillance system for pig health and welfare but information is currently under-utilised. The changes proposed to the pig meat inspection system will lead to some reduction in the detection probability of diseases and welfare conditions. The difference is likely to be minimal for diseases/conditions that affect several organs. To mitigate the reduced detection probability, palpation and/or incision should be conducted as a follow-up to visual inspection whenever abnormalities are seen

We investigated the effects of two types of TiO2 nanoparticles (

Magdalena Stevanović1, Branimir Kovačević2, Jana Petković3, Metka Filipič3, Dragan Uskoković11Institute of Technical Sciences of Serbian Academy of Sciences and Arts, 2Institute of General and Physical Chemistry, Belgrade, Serbia; 3Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, SloveniaAbstract: Highly stable dispersions of nanosized silver particles were synthesized using a straightforward, cost-effective, and ecofriendly method. Nontoxic glucose was utilized as a reducing agent and poly- α, γ, L-glutamic acid (PGA), a naturally occurring anionic polymer, was used as a capping agent to protect the silver nanoparticles from agglomeration and render them biocompatible. Use of ammonia during synthesis was avoided. Our study clearly demonstrates how the concentration of the capping agent plays a major role in determining the dimensions, morphology, and stability, as well as toxicity of a silver colloidal solution. Hence, proper optimization is necessary to develop silver colloids of narrow size distribution. The samples were characterized by Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, field-emission scanning electron microscopy, transmission electron microscopy, and zeta potential measurement. MTT assay results indicated good biocompatibility of the PGA-capped silver nanoparticles. Formation of intracellular reactive oxygen species was measured spectrophotometrically using 2,7-dichlorofluorescein diacetate as a fluorescent probe, and it was shown that the PGA-capped silver nanoparticles did not induce intracellular formation of reactive oxygen species.Keywords: silver nanoparticles, poly-α, γ, L-glutamic, green synthesis, morphology, cytotoxicity

LGMDs are a heterogeneous group of genetic disorders characterized by progressive muscle wasting and weakness with onset in the proximal muscles. These diseases present a large clinical variability regarding age of onset, rate of progression, pattern of skeletal muscle involvement, heart damage and mode of inheritance, with both autosomal recessive and dominant forms. The most common clinical forms are autosomal recessive classified as LGMD 2 (A-J). In general, they have more severe course compared to dominant forms so-called LGMD1 (A-F). All of them are incurable and often life-threatening disorders of children and young adults. Therefore they represent a considerable health and economic burden, not only on the patients and their families, but also on the whole community. Our inability to treat these affections effectively makes preventing any recurrence very important. For this reason it is necessary to know their exact genetic cause to provide adequate genetic counseling, to predict risks for the patient such as the development of cardiomyopathy, and to be able to take advantage of specific treatment when they become available. Difficulties in classification are often caused by the relatively common sporadic occurrence of autosomal recessive forms as well as by interfamilial clinical variability. Molecular genetic studies have demonstrated different causative mutations in the genes encoding a disparate collection of proteins involved in all aspects of muscle cell biology. These novel genes encode highly diverse proteins with different localization within or at the surface of the skeletal muscle fiber, such as the sarcolemma, the sarcomere, the muscle cytosol, the nucleus and the glycosilation pathway enzyme. Epidemiological data vary especially in LGMD2.