Reduced hepatic global hydroxymethylation in mice treated with non-genotoxic carcinogens is transiently reversible with a methyl supplemented diet.

Non-genotoxic carcinogens (NGCs) are known to cause perturbations in DNA methylation, which can be an early event leading to changes in gene expression and the onset of carcinogenicity. Phenobarbital (PB) has been shown to alter liver DNA methylation and hydroxymethylation patterns in mice in a time dependent manner. The goals of this study were to assess if clofibrate (CFB), a well-studied rodent NGC, would produce epigenetic changes in mice similar to PB, and if a methyl donor supplementation (MDS) would modulate epigenetic and gene expression changes induced by phenobarbital. CByB6F1 mice were treated with 0.5% clofibrate or 0.14% phenobarbital for 7 and 28 days. A subgroup of PB treated and control mice were also fed MDS diet. Liquid Chromatography-Ionization Mass Spectrometry (LC-MS) was used to quantify global liver 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels. Gene expression analysis was conducted using Affymetrix microarrays. A decrease in liver 5hmC but not 5mC levels was observed upon treatment with both CFB and PB with varying time of onset. We observed moderate increases in 5hmC levels in PB-treated mice when exposed to MDS diet and lower expression levels of several phenobarbital induced genes involved in cell proliferation, growth, and invasion, suggesting an early modulating effect of methyl donor supplementation. Overall, epigenetic profiling can aid in identifying early mechanism-based biomarkers of non-genotoxic carcinogenicity and increases the quality of cancer risk assessment for candidate drugs. Global DNA methylation assessment by LC-MS is an informative first step toward understanding the risk of carcinogenicity.

Evaluation of miR-122 as a Serum Biomarker for Hepatotoxicity in Investigative Rat Toxicology Studies.

MicroRNAs are short noncoding RNAs involved in regulation of gene expression. Certain microRNAs, including miR-122, seem to have ideal properties as biomarkers due to good stability, high tissue specificity, and ease of detection across multiple species. Recent reports have indicated that miR-122 is a highly liver-specific marker detectable in serum after liver injury. The purpose of the current study was to assess the performance of miR-122 as a serum biomarker for hepatotoxicity in short-term (5-28 days) repeat-dose rat toxicology studies when benchmarked against routine clinical chemistry and histopathology. A total of 23 studies with multiple dose levels of experimental compounds were examined, and they included animals with or without liver injury and with various hepatic histopathologic changes. Serum miR-122 levels were quantified by reverse transcription quantitative polymerase chain reaction. Increases in circulating miR-122 levels highly correlated with serum elevations of liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH). Statistical analysis showed that miR-122 outperformed ALT as a biomarker for histopathologically confirmed liver toxicity and was equivalent in performance to AST and GLDH. Additionally, an increase of 4% in predictive accuracy was obtained using a multiparameter approach incorporating miR-122 with ALT, AST, and GLDH. In conclusion, serum miR-122 levels can be utilized as a biomarker of hepatotoxicity in acute and subacute rat toxicology studies, and its performance can rival or exceed those of standard enzyme biomarkers such as the liver transaminases.

Recombinant human interleukin-6 enhances the immunoglobulin secretion of a rabbit-rabbit hybridoma.

The generation of stable rabbit-rabbit hybridomas is now possible by the recent development of a rabbit fusion partner. The ability to generate rabbit monoclonal antibodies (MAbs) can be advantageous because these rabbit immunoglobulins tend to exhibit higher affinity than murine MAbs. Furthermore, it has been observed that, in general, rabbits will elicit an immune response to antigens of limited immunogenicity in mice. Unfortunately, these rabbit-rabbit hybridomas secrete only 200 ng/mL to 5 microg/mL of immunoglobulin, which may limit larger scale production of rabbit antibodies. This study sought to determine if interleukin 6 (IL-6), which has been reported to have proliferative and secretory stimulating effects on some murine hybridomas, had any effect on a rabbit cell line that secretes a monoclonal IgG specific for estradiol. The results demonstrated that recombinant human IL-6 had a dose-dependent enhancing effect on the IgG secretion of the rabbit-rabbit hybridoma. The enhancing effect was consistent when the cells were continuously passed in the presence of IL-6. However, IL-6 did not affect the growth of the hybridoma. In contrast, no discernible effect was accomplished with recombinant mouse IL-6. Furthermore, no basal IL-6 activity was detected in the rabbit hybridoma extracellular medium. The IL-6 enhancement effect observed in this study may help to increase the immunoglobulin yield of rabbit hybridomas and to assist in the understanding of the mechanism(s) behind the lowered secretion level.