Investigating the Mechanistic Basis for Epigenetic Modifications Induced by Tungsten

Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten, remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. This work demonstrates tungsten’s ability to induce carcinogenic-related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (BEAS-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. In order to examine the epigenetic mechanisms that mediate tungsten’s tumorigenicity, we investigated if tungsten alters the levels of global histone methylation and if these changes are due to tungsten influencing the histone demethylases. We found that cells acutely treated with tungsten displayed significantly increased numbers of H34me3 and H3K9me2 histone marks on a global scale. This increase was due to down-regulation in the protein levels of the histone demethylases JMJD1A and JARID1A. The increase in global histone methylation remained when cellular SAM levels were depleted. The decrease in histone demethylase proteins was found to be due to a reduction in their gene expression. Epigenetic alterations induced by tungsten in the histone demethylase genes caused the repression. We also evaluated insoluble tungsten, tungsten oxide (WO3). WO3 is an occupational exposure hazard. The primary route of WO 3 exposure is inhalation and WO3 is known as a pulmonary irritant. WO3 exposure led to stochastic results, which were likely due to the random effects of the particles. Given the carcinogenic potential of other metals, it is likely that tungsten will exert carcinogenic outcomes. This study evaluates cancer-associated endpoints induced by tungsten exposure in both in vitro and in vivo models. To evaluate the mechanisms that underlie tungsten-induced carcinogenesis, alterations to the epigenome are assessed. Arsenic, cadmium, nickel, and chromium (VI) are poor mutagens; however, they exert their carcinogenic potential via epigenetic mechanisms. The literature is currently void of investigations examining the epigenetic effects of tungsten. Given the evidence characterizing metals as epimutagens, it is likely that tungsten toxicity and carcinogenesis is mediated via epigenetic mechanisms

Nickel and cadmium-induced SLBP depletion: A potential pathway to metal mediated cellular transformation.

Both nickel and cadmium compounds have been established as group I carcinogens for several decades. Despite over-whelming evidence of these compounds' carcinogenicity in humans, the specific underlying molecular mechanisms that govern metal induced cellular transformation remain unclear. In this study, we found that there were slightly different effects on decreased SLBP mRNA and protein as well as increased polyA H3.1 in our nickel exposed cells. This suggested that nickel and arsenic have similar effects on canonical histone mRNA transcription and translation. We also saw that the depletion of SLBP protein was reversed by inhibiting the proteosome. Finally, we showed that inhibiting the SLBP mRNA and protein levels were rescued by epigenetic modifiers suggesting that nickel's effects on SLBP may be mediated via epigenetic mechanisms. Taken together these results suggest a similar mechanism by which both arsenic and nickel may exert their carcinogenic effects

Tungsten or wolfram: Friend or foe?

Tungsten or wolfram was regarded for many years as an enemy within the tin smelting and mining industry, because it conferred impurity or dirtiness in tin mining. However, later it was considered an amazing metal for its strength and flexibility, together with its diamond like hardness and its melting point which is the highest of any metal. It was first believed to be relatively inert and an only slightly toxic metal. Since early 2000, the risk exerted by tungsten alloys, its dusts and particulates to induce cancer and several other adverse effects in animals as well as humans has been highlighted from in vitro and in vivo experiments. Thus, it becomes necessary to take a careful look at all the most recent data reported in the scientific literature, covering the years 2001-2016. In fact, the findings indicate that much more attention should be devoted to thoroughly investigate the toxic effects of tungsten and the involved mechanisms of tungsten metal or tungsten metal ions

Nickel exposure depletes SLBP protein in Beas2B and BL41 cells.

<p>Whole cell lysates from BL41 (A) and Beas2B (C) cells exposed to the Ni Cl₂ for 48 hours show marked decreases in SLBP in representative western blots. Relative protein amounts for were quantified and shown in charts (B) and (D).</p

Epigenetic modifiers increase/rescue SLBP protein and mRNA levels in Nickel exposed Beas2B cells.

<p>Nickel exposed Beas2B cells were co-treated with Ni Cl₂ and sodium butyrate (Na–Buty). SDS-PAGE gel (A). Quantification of the western blot indicated that SLBP protein levels are rescued in cells exposed to nickel and sub-cultured with or without Na–Buty (C). Total RNA was collected from cells co-treated with Na–Buty, 5 AzC, or nothing and converted to cDNA. RT-qPCR analysis indicated that SLBP mRNA levels were increased by both epigenetic modifiers (B).</p

Both nickel and cadmium significantly decrease SLBP mRNA in exposed cells.

<p>Total RNA was collected from nickel and cadmium exposed cells and converted to cDNA. RT-qPCR analysis showed clear decreases in SLBP mRNA in Nickel treated BL41(A) and Beas2B cells (B). Cadmium exposed BL41 cells exhibit a similar pattern (C).</p

Cadmium induces heritable depletion of SLBP protein in BL41 cells.

<p>BL41 cells were exposed to 0μM, 1μM, 2.5μM, or 5μM Ni Cl₂ for 48 hours. After removing the media and washing in PBS, whole cell lysates were collected and run on a 12% SDS PAGE gel after 4 days (A) and 6 days (C). While a decrease in SLBP remains after 4 days, by day 6 SLBP levels are restored to above baseline. The relative protein amounts are quantified for 4 days (B) and 6 days (D).</p

Increase of total H3.1 mRNA in nickel and cadmium exposed cells.

<p>Total RNA was isolated from Beas2B and BL41 cells exposed to 0μM or 250μM Ni Cl₂ and 0μM or 5μM Cd Cl₂. RT-qPCR was preformed and revealed an increase in the amount of H3.1 mRNA in nickel treated BL41 (A) and Beas2B (B). BL41 (C) and Beas2B (D) showed increased total H3.1 after Cd Cl₂ also.</p

Increased proteosomal degradation plays a role in nickel mediated SLBP protein depletion.

<p>Nickel exposed Beas2B cells were co-treated with 10 μM MG-132 for 1 hour before the end of a 48-hour nickel treatment. Whole cell lysates were collected and run on a 4–15% SDS-PAGE gel (A) Densitometry (B) indicated that inhibited proteosomal degradation rescues SLBP protein levels.</p

Nickel induces heritable depletion of SLBP protein in Beas2B cells.

<p>Beas2B cells were exposed to 0μM, 250μM, or 400μM Ni Cl₂ for 48 hours. Cells were then washed with PBS and sub-cultured for 3 and 7 days post-exposure. Whole cell lysates were collected and run on a 4–15% gradient SDS PAGE gel. After 3 days (A) and 7 days (B) the decrease in SLBP is still apparent. The relative protein amounts across several experimental replicates are also quantified for both 3 days (C) and 7days (D) post nickel exposure.</p