Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [¹⁴C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercaptopurine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effects of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.     

Magnesium (and trace substance) deficiencies in the pathogenesis of cancer

Except for a few experimental models of magnesium (Mg)-deficiency-induced neoplasms, less attention has been paid in the past quarter century in the Western world to this macromineral than to the trace elements; e.g., selenium (Se) and zinc (Zn), and to vitamins, deficiencies of which are each considered probable factors in oncogenesis. Although early epidemiologic studies showed an inverse correlation between the amount of Mg in soil and water and the incidence of (gastric) cancer, and several animal studies supported the premise that Mg has a prophylactic effect against induction of cancer, other studies showed that Mg supplementation increased the growth of established experimental tumors. Thus, enthusiasm for this approach subsided. The early epidemiologic findings have since been confirmed, and there have been studies demonstrating the importance of Mg in maintaining immunocompetence, and others indicating that immunodeficiencies increase susceptibility to the development of cancer. Evidence has now accrued that indicates that Mg deficiency increases susceptibility to chemical oncogens. The abnormal metabolism of tryptophan (yielding a carcinogenic metabolite) that indicates functional or absolute pyridoxine deficiency is an indirect clue to Mg deficiency. Vitamin B₆-activated enzymes require Mg as a cofactor. However, the early warnings against the use of Mg as part of an antineoplastic program against established cancer were justified, since rapidly metabolizing cells (such as cancers) are dependent on Mg. There are similarities between experiences with Mg and with Se and Zn. All three are required for normal metabolism; Se also protects against free radicals in the environment. Mg and Zn have increased established tumor growth, and their depletion has been applied to antineoplastic programs, with risks comparable to those of using antimetabolic agents.     

Evaluating long-term cellular effects of the arsenic species thio-DMAV: qPCR-based gene expression as screening tool

Thio-dimethylarsinic acid (thio-DMA^V) is a human urinary metabolite of the class 1 human carcinogen inorganic arsenic as well as of arsenosugars. Thio-DMA^V exerts strong cellular toxicity, whereas its toxic modes of action are not fully understood. For the first time, this study characterises the impact of a long-term (21 days) in vitro incubation of thio-DMA^V on the expression of selected genes related to cell death, stress response, epigenetics and DNA repair. The observed upregulation of DNMT1 might be a cellular compensation to counterregulate the in a very recent study observed massive global DNA hypomethylation after chronic thio-DMA^V incubation. Moreover, our data suggest that chronic exposure towards subcytotoxic, pico- to nanomolar concentrations of thio-DMA^V causes a stress response in human urothelial cells. The upregulation of genes encoding for proteins of DNA repair (Apex1, Lig1, XRCC1, DDB2, XPG, ATR) as well as damage response (GADD45A, GADD45G, Trp53) indicate a potential genotoxic risk emanating from thio-DMA^V after long-term incubation.     

Microcystins: measuring human exposure and the impact on human health

Abstract

Context: Freshwater cyanobacterial toxins, microcystins, may be a contributing factor to the development of hepatocellular cancer and colorectal cancer.

Objectives: This review summarizes the toxicity data, exposure routes and the methodologies available to determine exposure to elucidate the relationship to liver and colorectal cancer.

Methods: Literature searches were conducted using Medline, PubMed and Web of Science.

Results: There is evidence of human poisonings resulting from exposure to microcystins, however current methods rely on targeted approaches only suitable for acute exposure. No methods exist for the determination of chronic exposure to microcystins.

Conclusions: With the growing evidence of exposure to microcystins and the possible links to cancer, methods to measure medium to long-term human exposure are needed. The identification and validation of candidate biomarkers are key to undertaking urgently required epidemiological studies.     

Listeria monocytogenes induces host DNA damage and delays the host cell cycle to promote infection

Listeria monocytogenes (Lm) is a human intracellular pathogen widely used to uncover the mechanisms evolved by pathogens to establish infection. However, its capacity to perturb the host cell cycle was never reported. We show that Lm infection affects the host cell cycle progression, increasing its overall duration but allowing consecutive rounds of division. A complete Lm infectious cycle induces a S-phase delay accompanied by a slower rate of DNA synthesis and increased levels of host DNA strand breaks. Additionally, DNA damage/replication checkpoint responses are triggered in an Lm dose-dependent manner through the phosphorylation of DNA-PK, H2A.X, and CDC25A and independently from ATM/ATR. While host DNA damage induced exogenously favors Lm dissemination, the override of checkpoint pathways limits infection. We propose that host DNA replication disturbed by Lm infection culminates in DNA strand breaks, triggering DNA damage/replication responses, and ensuring a cell cycle delay that favors Lm propagation.     

Microfluidic method reduces osmotic stress injury to oocytes during cryoprotectant addition and removal processes in porcine oocytes

Oocyte cryopreservation is an important technology in assisted reproduction and fertility preservation. However, the developmental potential of cryopreserved oocyte remains poor. Osmotic stress injury (OSI) during cryoprotectants (CPAs) loading and unloading steps has critical impact on successful cryopreservation. In order to minimize OSI to oocytes, a microfluidic device was designed and fabricated to achieve continuous CPA concentration change. MII porcine oocytes were loaded and unloaded CPAs with step-wise and microfluidic methods, oocyte volume changes were recorded and compared, loading and unloading duration of microfluidic methods were optimized. The survival and developmental rate of treated oocytes in step-wise and microfluidic linear methods were also evaluated. The results showed that oocyte volume changes with microfluidic method were obviously less than step-wise method, and the survival, cleavage and blastocyst rate of oocytes were 95.3%, 64.4%, and 19.4%, respectively, which were significantly higher than the traditional step-wise method (79.4%, 43.6%, and 9.7%) (p < 0.05). In conclusion, microfluidic device can effectively reduce the osmotic damage to oocytes and improve the survival rate and developmental rate of oocytes, which may provide a new path for oocyte cryopreservation.     

Discovery of novel chalcone-dithiocarbamates as ROS-mediated apoptosis inducers by inhibiting catalase

Novel chalcone-dithiocarbamate hybrids were designed, synthesized and evaluated for antiproliferative activity against selected cancer cell lines (MGC803, MCF7, and PC3). Among these analogues, (E)-2-oxo-2-((4-(3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)amino)ethyl-4-(2-hydroxyethyl)piperazine-1-carbodithioate (12d) showed the best inhibitory activity against PC3 cells (IC₅₀ = 1.05 μM). Cellular mechanism studies elucidated 12d could inhibit colony formation, arrest cell cycle at G2/M phase and induce DNA damage against PC3 cells. Compound 12d also induced mitochondrial apoptosis by caspase activation, MMP decrease, ROS production and catalase (CAT) inhibition. Importantly, 12d inhibited epithelial-mesenchymal transition (EMT) process by regulating EMT-related proteins (E-cadherin, N-cadherin, Vimentin, MMP2, MMP9). These results indicated that 12d is a promising lead compound and deserves further investigation for prevention and treatment of human prostate cancer.     

Quantitative Measurement of the Diastereoisomers of CIS Thymidine Glycol in Gamma-Irradiated DNA

A technique for determing the relative content of each of the diastereoisomers of cis thymidine glycol (dTG) in DNA exposed to ionizing radiation has been developed. [³H]thymidine DNA was gamma-irradiated, digested to 2′-deoxyribonucleosides, authentic [¹⁴C] (+, -) cis dTG added to the digestate and the mixture resolved by HPLC. ³H fractions coeluting with [¹⁴C] (+, -) dTG were collected and acetylated.

The acetoxy derivatives.of (+) and (-) cis dTG were easily resolved by a second HPLC analysis and their absolute configuration determined by NMR amd mass spectroscopies. We have constructed a dose-response curve for formation of each isomer in gamma-irradiated DNA and shown that they are formed in equal amounts. This technique may be used to determine the relative formation of cis dTG isomers in DNA resulting from other oxidative stresses and whether repair of these is influenced by their configuration.