Elevated 5-hydroxymethylcytosine in the Engrailed-2 (EN-2) promoter is associated with increased gene expression and decreased MeCP2 binding in autism cerebellum.

Epigenetic mechanisms regulate programmed gene expression during prenatal neurogenesis and serve as a mediator between genetics and environment in postnatal life. The recent discovery of 5-hydroxymethylcytosine (5-hmC), with highest concentration in the brain, has added a new dimension to epigenetic regulation of neurogenesis and the development of complex behavior disorders. Here, we take a candidate gene approach to define the role 5-hmC in Engrailed-2 (EN-2) gene expression in the autism cerebellum. The EN-2 homeobox transcription factor, previously implicated in autism, is essential for normal cerebellar patterning and development. We previously reported EN-2 overexpression associated with promoter DNA hypermethylation in the autism cerebellum but because traditional DNA methylation methodology cannot distinguish 5-methylcytosine (5-mC) from 5-hmC, we now extend our investigation by quantifying global and gene-specific 5-mC and 5-hmC. Globally, 5-hmC was significantly increased in the autism cerebellum and accompanied by increases in the expression of de novo methyltransferases DNMT3A and DNMT3B, ten-eleven translocase genes TET1 and TET3, and in 8-oxo-deoxyguanosine (8-oxo-dG) content, a marker of oxidative DNA damage. Within the EN-2 promoter, there was a significant positive correlation between 5-hmC content and EN-2 gene expression. Based on reports of reduced MeCP2 affinity for 5-hmC, MeCP2 binding studies in the EN-2 promoter revealed a significant decrease in repressive MeCP2 binding that may contribute to the aberrant overexpression of EN-2. Because normal cerebellar development depends on perinatal EN-2 downregulation, the sustained postnatal overexpression suggests that a critical window of cerebellar development may have been missed in some individuals with autism with downstream developmental consequences. Epigenetic regulation of the programmed on-off switches in gene expression that occur at birth and during early brain development warrants further investigation.

Inter-laboratory comparison of turkey in ovo carcinogenicity assessment (IOCA) of hepatocarcinogens.

In three independent laboratories carcinogens (diethylnitrosamine, DEN, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and non-carcinogens (N-nitrosoproline, nicotine) were evaluated in turkey eggs for in ovo carcinogenicity assessment (IOCA). Compounds were injected into aseptic fertilized eggs. After incubation for 24 days, foci of altered hepatocytes (FAH), some with a pseudoglandular structure and/or signs of compression of the surrounding tissue were observed in the fetal liver. All laboratories were able to distinguish unequivocally the hepatocarcinogen-exposed groups from those exposed to non-carcinogens or the vehicle controls, based on the pre-specified evaluation parameters: tumor-like lesions, pseudoglandular areas and FAH. In addition to focal changes, only the carcinogens induced hepatocellular karyomegaly. Lower doses of the carcinogens, which did not induce FAH, were sufficient to induce hepatocellular karyomegaly. After exposure to 4 mg DEN, gall bladder agenesis was observed in all fetuses. The IOCA may be a valuable tool for early investigative studies on carcinogenicity and since it does not use rodents may complement chronic rat or mouse bioassays. Test substances that are positive in both rodents and fertilized turkey eggs are most probably trans-species carcinogens with particular significance for humans. The good concordance observed among the three laboratories demonstrates that the IOCA is a reliable and robust method.

Gold nanoparticles synthesis and biological activity estimation in vitro and in vivo.

UNLABELLED: The aim of the work was the synthesis of gold nanoparticles (GNP) of different sizes and the estimation of their biological activity in vitro and in vivo. MATERIALS AND METHODS: Water dispersions of gold nanoparticles of different sizes have been synthesized by Davis method and characterized by laser-correlation spectroscopy and transmission electron microscopy methods. The GNP interaction with tumor cells has been visualized by confocal microscopy method. The enzyme activity was determined by standard biochemical methods. GNP distribution and content in organs and tissues have been determined via atomic-absorption spectrometry method; genotoxic influence has been estimated by "Comet-assay" method. RESULTS: The GNP size-dependent accumulation in cultured U937 tumor cells and their ability to modulate U937 cell membrane Na(+),K(+)-АТР-ase activity value has been revealed in vitro. Using in vivo model of Guerin carcinoma it has been shown that GNP possess high affinity to tumor cells. CONCLUSIONS: Our results indicate the perspectives of use of the synthesized GNP water dispersions for cancer diagnostics and treatment. It's necessary to take into account a size-dependent biosafety level of nanoparticles.

The use of nanoferromagnetics to increase the cytotoxic effect of antitumor drugs.

AIM: To investigate the influence of ferromagnetic nanoparticles on antitumor effect of doxorubicin and mitochondria oxidative phosphorylation. METHODS: The study was carried out on the mice-hybrids (C57Bl/6xDBA/2) with intraperitoneally (i/p) transplantated Ehrlich ascitic carcinoma. Single i/p injection of doxorubicin (Dox), stabilized ferromagnetic nanoparticles (Fe3O4; 20-40 nm; FM) or their combination were performed 7 days after tumor transplantation. The cytotoxic effect of agents, morphology and cell cycle of tumor cells were studied 24, 48 and 72 h after Dox administration. RESULTS: The investigations showed that ferromagnetic nanoparticles increased the cytotoxic effect of doxorubicin on Ehrlich ascsmall i, Ukrainiantic carcinoma mainly 48 h after agents' administration. The largest number of apoptotic cells was observed in group of animals in which doxorubicin was administered before ferromagnetic nanoparticles. Moreover, the ferromagnetic nanoparticles at concentration 1.45 microg Fe/ml and, particularly, 7.25 microg Fe/ml decreased mitochondria oxygen consumption in phosphorylation state that may negatively influence their living capability. CONCLUSIONS: Obtained data point out the perspective of use of certain sized FM nanoparticles to increase the cytotoixc effect of antitumor drugs.