Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying noncholinesterase targets. Here we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography–mass spectrometry (LC–MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-<i>N</i>-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues. The MS data have been deposited to the ProteomeXchange with identifier PXD001107

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

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