The JmjC-domain-containing histone demethylases (JHDMs) can remove histone lysine-methylation and thereby regulate gene expression. The JmjC-domain uses iron Fe (II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl-lysine. We hypothesize that reactive oxygen species will oxidize Fe (II) to Fe (III), thereby attenuating the activity of JmjC-domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3 hours) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H2O2) for 3 hours, exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; pre-incubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2′, 7′-dichlorofluorescein (DCF), GSH/GSSG ratio and protein carbonyl content. A cell free system indicated H2O2 inhibited histone demethylase activity where increased Fe (II) rescued this inhibition. TET protein also showed a decreased activity under oxidative stress. Cells exposed to a low dose and long term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.

The rapid development of high-volume horizontal hydraulic fracturing for mining natural gas from shale has posed potential impacts on human health and biodiversity. The produced flow back waters after hydraulic stimulation is known to carry high levels of saline and total dissolved solids. To understand the toxicity and potential carcinogenic effects of these waste waters, flow back water from five Marcellus hydraulic fracturing oil and gas wells were analyzed. The physicochemical nature of these samples was analyzed by inductively coupled plasma mass spectrometry and scanning electron microscopy / energy dispersive X-ray spectroscopy. A cytotoxicity study using colony formation as the endpoint was carried out to define the LC50 values of test samples using human bronchial epithelial cells (BEAS-2B). The BEAS-2B cell transformation assay was employed to assess the carcinogenic potential of the samples. Barium and strontium were among the most abundant metals in these samples and the same metals were found elevated in BEAS-2B cells after long-term treatment. BEAS-2B cells treated for 6 weeks with flow back waters produced colony formation in soft agar that was concentration dependant. In addition, flow back water-transformed BEAS-2B cells show a better migration capability when compared to control cells. This study provides information needed to assess the potential health impact of post-hydraulic fracturing flow back waters from Marcellus Shale natural gas mining.

No abstract