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Quantitation of aberrant cell nuclei and mitotic figures in Feulgen’s stained sections of mouse duodenum via image analysis.

Evolving Roles of the Toxicologic Pathologist in Multi-disciplinary Research

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The role of the toxicologic pathologist is continuously evolving and becoming ever more complex, as novel tools are being developed to identify structural and biochemical changes that result from exposure to toxic substances, and elucidate mechanisms of action by which toxins may potentially impact animal and human health.  Recently, EPL has been working with ToxStrategies to investigate morphologic effects of the environmental contaminant hexavalent chromium [Cr(VI)] on the small intestines of mice.  Thus far, this work has involved conventional histopathological examinations, plus image analysis techniques that have been used to quantitate compartmental changes in duodenal architecture (Fig. 1), and markers of DNA damage such as the presence of aberrant mucosal epithelial nuclei (Fig. 2) and the abundance of gamma-H2AX immunoreactivity (Fig. 3).  The combined results of these efforts have indicated that intestinal damage caused by oral exposure of mice to Cr(VI) appears to occur primarily in the villus compartment as opposed to the crypts.

To better understand the pathogenesis of Cr(VI) toxicity, a method known as X-ray fluorescence spectromicroscopy (XFSM) was employed, in collaboration with ToxStrategies and the U.S. Army Corps of Engineers, to determine the precise anatomic site of Cr(VI) localization in the mouse intestine following 13 weeks of oral exposure.  This technique, which utilizes a particular type of particle accelerator called a Synchrotron, allows for high-resolution mapping of various chemical elements in specially prepared, unstained histologic sections (Fig. 4).  When XFSM was applied to duodenal sections of mice that had received toxicologically relevant concentrations of Cr(VI) via the oral route, it became evident that while constituent elements such as calcium and sulfur were diffusely distributed throughout the intestinal mucosa, chromium tended to concentrate within the lamina propria of the villus tips, and was almost entirely absent from the crypt region (Fig. 5).  This same pattern was consistently present among different Cr(VI)-treated mice, and was also found in Cr(VI)-treated rats.  These results provide additional evidence that the villus is the primary target of Cr(VI)-induced small intestinal damage.

The preceding example further demonstrates the value of the contemporary toxicologic pathologist as part of multi-disciplinary expert team, and the level of scientific advancement that can be achieved through collaborative research efforts.

Figure 1
Quantitation of aberrant cell nuclei and mitotic figures in Feulgen’s stained sections of mouse duodenum via image analysis.

 

Figure 2
Image analysis-based measurements of mucosal and villus areas in whole slides scans of mouse duodenum.

 

Figure 3
Immunostaining of gamma-H2AX, a marker for DNA damage, in the duodenal crypts of untreated and treated mice.

 

Figure 4
Schematic of Synchrotron-based X-ray fluorescence spectromicroscopy (XFSM) technique.

 

Figure 5
XFSM used to demonstrate that chromium (Cr) becomes concentrated in the villus tips of the mouse duodenum following 13 weeks of oral Cr(VI) dosing, but is relatively absent from crypts (asterisks). Conversely, elements such as calcium (Ca) and sulfur (S) are distributed diffusely throughout the duodenal mucosa. Lighter colors = higher concentrations.

 

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