Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

<div><p>Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect <i>in vitro</i>, thus providing targets for focused <i>in vivo</i> studies.</p></div

Enriched Pathways.

<p>Enriched IPA canonical pathways are listed for the transcriptomic, proteomic, and combined data. We considered a pathway to be enriched at a p<.05 and contain more than 2 changing molecules.</p

Modulated Extracellular Transcripts and Proteins.

<p>We identified 26 extracellular proteins and/or genes which encode extracellular protein whose expression was modulated in response to cobalt exposure. We propose them as candidate biomarkers of exposure or effect. We focused on extracellular proteins as they have the best potential to be identified through non-invasive methods.</p

Oral Toxicity Database.

<p>An extensive database was created to access and prioritize the oral hazard of industrial chemicals based on the toxicity, stability, and usage of the chemical. This prioritization was limited to only pure elements and metal compounds.</p

Network of modulated genes and proteins related to HIF-1α.

<p>Many of the differentially expressed genes and/or proteins changing in abundance are regulated by HIF-1α. The arrows show the direction of the relationship, the color indicates the direction of change of the gene or protein with red being an increase and green being a decrease, and the intensity of the color indicating the degree of change.</p

Gel electrophoresis of extracted BALF obtained at 24 hr posttreatment from a control rat (PBS instilled) and a rat exposed intratracheally to 50 mg of DEP/kg body weight

<p><b>Copyright information:</b></p><p>Taken from "Proteomic Analysis of Bronchoalveolar Lavage Fluid: Effect of Acute Exposure to Diesel Exhaust Particles in Rats"</p><p></p><p>Environmental Health Perspectives 2007;115(5):756-763.</p><p>Published online 5 Feb 2007</p><p>PMCID:PMC1867966.</p><p>This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI</p> The four numbered bands in the diesel lane were excised and analyzed by LC/MS

Altered H₂O₂ levels in the liver occur well after Cd or Cr exposure.

<p>H_<b>2</b>O_<b>2</b> concentrations were monitored via a colorimetric assay. All three doses of Cd (A) increased H_<b>2</b>O_<b>2</b> concentrations on day 7 in a dose-dependent manner. Only the high dose of Cr (B) led to increased H_<b>2</b>O_<b>2</b> concentrations on days 3 and 7. Values are mean ±SE, n = 6–7 animals per group. *Significantly different from control on each day, <i>p</i><0.05. ^†<i>p</i> = 0.053.</p

Elevated concentrations of Cd and Cr in the liver 1, 3, and 7 days after a single exposure.

<p>Levels of Cd (A) and Cr (B) were determined via ICP-MS. Both Cd and Cr accumulated in a dose-dependent manner at 24 h. Cd levels remained elevated for all doses on days 3 and 7 post-exposure. Cr levels rapidly declined between days 1 and 3, and this trend continued at the low and medium doses, but in the high dose remained at day 3 levels on day 7. Values are mean ±SE, n = 5–7 animals per group.</p

Cr, but not Cd exposure induced DNA damage in rat liver.

<p>A comet assay was performed to assess DNA strand breaks in hepatocytes from Cd (A) or Cr (B) treated animals. Cd exposure had no effect on Comet tail length. However, Cr induced DNA strand breaks at the medium and high doses, and this was sustained at all days examined. Data are expressed as mean ±SE, n = 2–7 animals per group. ^†Only two comet assays were available for analysis on days 1 and 3. *Significantly different from control on each day, p<0.05.</p

Differentially expressed probe sets and persistent changes in expression following Cd or Cr exposure in the liver.

<p>Venn diagrams of (A) total probe sets from all time points for each metal and (B) probe sets on each day included for enrichment analysis. Probe sets were considered significant with a fold-change ≥ 1.8 or ≤ -1.8 and either an FDR < 0.05 (day 1) or unadjusted <i>p</i>-value < 0.05 (days 3 and 7).</p