Malignant human cell transformation of Marcellus Shale gas drilling flow back water

Abstract

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 are known to carry high levels of saline and total dissolved solids. To understand the toxicity and potential carcinogenic effects of these wastewaters, flow back waters 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 to be elevated in BEAS-2B cells after long-term treatment. BEAS-2B cells treated for 6weeks with flow back waters produced colony formation in soft agar that was concentration dependent. In addition, flow back water-transformed BEAS-2B cells show 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.

Keywords: Barium; Cytotoxicity; Epigenetics; Marcellus Shale; Mutagenesis; Strontium.

Figure 1

SEM/EDX analysis of Marcellus flow back water. Representative SEM images reveal the morphology of crystals and particles in flow back samples. The rectangles indicate the regions that were analyzed by the EDX for the composition of the particulates. Samples were prepared at high (A) and low densities (B, C). At low density conditions, individual particles can be isolated and EDX can be performed on each particle.

Figure 2

Cytotoxicity of Marcellus flow back, soluble barium and strontium. The colony formation curve was generated by culturing replated BEAS-2B cells for 16 days after exposed to various concentrations of flow back (A), soluble barium (B) and strontium (C)for 3 weeks. (B) and then the number of surviving colonies was determined.

Figure 3

Anchorage free growth of BEAS-2B cells exposed to Marcellus flow back. BEAS-2B cells were exposed to various concentrations of flow back for 6 weeks, and assessed for anchorage free growth using a soft agar assay. 3w later, cell colonies were stained with INT/BCIP and photographed. Panel A shows representative plates in soft agar assay, control: distilled water, SF: filtered pristine lake water from Sterling Forest. Numbers of colonies formed by BEAS-2B cells exposed to flow back well 1 and flow back well 3 (B) were counted and presented as the mean ± SD (n = 3), dd: distilled water, SF: filtered pristine lake water from Sterling Forest. Panel C shows representative image and the actual number of tumor formation in nude mice after subcutaneously injection of control clones (ctrl clone 1) and flow back water well 1-transformed cells (Well1 clone 1) and flow back water well 3-transformed cells (Well3 clone 1).

Figure 4

Analysis of transformed cells derived from soft agar. (A) representative image of normal BEAS-2B cells derived from spontaneously derived colonies of untreated cells (ctrl clone), flow back well 1 transformed cells (well 1 clone) or flow back well 3 transformed cells (well 3 clone) grown in low density. (B) control and flow back transformed cells were seeded at 2500 cells/well in 24-well plates. Cells were trypsinized and counted at the indicated time point. Results were represented as mean ± SD (n=3), DD: distilled water, SF: filtered pristine lake water from Sterling Forest.

Figure 5

Enhanced cell migration of transformed BEAS-2B. (A) Representation (top) and quantification (bottom) of matrigel invasion assay showing the in vitro migration of transformed BEAS-2B cells. *p<0.05 (B) flow back transformed clones healed the wound faster than the control clones. All images were captured at 100 X magnification, and the same field of view was captured at each time point with guidance from the grid on the cell culture plates.

Figure 6

RNA-Seq analysis of transformed BEAS-2B cells. (A) Hierarchical clustering dendrogram shows 1298 genes resulting from a multiple hypothesis test, FDR<0.05, for all 8 clones (6 flow back water-transformed clones, 2 control clones). Genes that were increased in expression compared to their control clones are shown in red while under-expressed genes are depicted in blue. (B) A Multidimensional scaling plot shows that clones derived from individual flow back water wells treated cells generally cluster together.(C and D) GO analyses showing the major canonical pathways associated with the genes (C)upregulated and (D)downregulated in flow back well water sample transformed BEAS-2B cells. (E) Identification of the overrepresented transcription factor binding motifs at the promoters of genes upregulated in flow back well water sample transformed BEAS-2B cells.