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

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

Delayed induction of free radicals in the liver after Cd and Cr exposure.

<p>Liver tissue slices from Cd (A and C) and Cr (B and D) exposed animals were incubated with hydroxy-TEMPO with or without the •OH scavenger DMTU, and examined via ESR. Total free radical levels dose-dependently increased for both metal exposures 1, 3 and 7 days post-injection (A and B). An indication of the contribution of •OH to total ROS was determined by examining the differences in ESR peak height after the addition of DTMU (C and D). Cr exposure dose-dependently increased •OH production at the mid and high doses 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. ^†Significantly different from other days in the same dose.</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

Time-dependent changes in candidate biomarker gene expression.

<p>The figure shows hierarchical clustering of the top 5 up-regulated DEGs on each day for each metal. When the same gene was one of the top genes for a different day of the same metal the next highest up-regulated gene was included for that day. The values shown in the heat map are the log_<b>2</b> ratio of change of the genes compared to the unexposed controls.</p

Activation or inhibition of selected transcriptional regulator activity predicted by IPA.

<p>Predicted activation or inhibition of activity for selected Transcriptional Regulators (TR) predicted by IPA based on the gene response. A number of transcriptional regulators identified are associated with the pathways in Table B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127327#pone.0127327.s002" target="_blank">S2 File</a>. Some regulators may be associated with more than one pathway or stress response, but appear in this table only once.</p><p>*The same TR is identified for both metals.</p><p>**The same TR is differentially affected between Cd and Cr.</p><p>Activation or inhibition of selected transcriptional regulator activity predicted by IPA.</p