A comprehensive histopathological evaluation of the major organs and tissues is generally considered to be the most reliable metric by which adverse toxicological effects are determined. As knowledge of the molecular alterations that underlie the morphological and histochemical changes scored by the anatomic pathologist has increased, molecular techniques have become increasingly used in conjunction with traditional histological evaluation. For example, as the molecular basis of apoptosis has become elucidated, molecular assays that visualize the externalization of membrane proteins (van Engeland et al., 1998) or DNA strand breakage (Thiry, 1992; Wijsman et al., 1993) characteristic of the apoptotic process have been found to be more sensitive and more specific endpoints than traditional evaluation of nuclear and cellular morphological characteristics. Recently, adaptations of the annexin assay have been used to image apoptotic cell populations noninvasively in the human in vivo (Blankenberg et al., 1999; Reutelingsperger et al., 2002).
Application of histochemical and immunohistochemical techniques to visualize (e.g., Bullock and Petrusz, 1986) or quantitatively analyze (Wilson et al., 1990), specific small molecules or proteins in cells and tissues has, of course, long been used to identify specific cellular and extra-cellular constituents associated with normal functions and pathological conditions. In general, these molecular techniques have been used as an adjunct to traditional morphological evaluation using light microscopy. Although it may still be premature to consider a comprehensive reassessment of "standard" regulatory histopathological practice, it is not difficult to envision the development of methodologies suitable for quantitative monitoring of endpoints that are currently evaluated in a qualitative or semi-quantitative manner by visual observation.
Host-defense cell infiltration of damaged tissue is one example of a process that has been characterized to an extent that suggests improved strategies for evaluation. Currently, the degree of infiltration by host-defense cells such as lymphocytes or macrophages is evaluated via a qualitative judgement by the pathologist during visual screening of tissue sections. Objective quantitative approaches that use labeling of well-characterized surface markers of these leukocytic cell populations (Zola, 1992) are easily envisioned, but apparently have not yet been developed or proposed for regulatory testing applications. Likewise, the chemokine and cytokine signals generated in response to tissue damage might serve as biomarkers of cellular responses to tissue damage. Much is now known about the chemokine and cytokine signals that activate and recruit host-defense cells, and immunoassays for leukocyte subclasses are employed routinely in research and clinical testing (Borish and Steinke, 2003; Olson and Ley, 2002).
Figure 2 illustrates the cellular signals and resulting cellular recruitment that are characteristic of tissue damage, and suggests quantitative biomarkers that could be used to characterize the associated pathology. Objective quantitative assays of damage-related host-defense cell signaling and host-defense cell accumulation would be expected to be more sensitive and more objective than the visual screening currently employed (Fig. 2). Additionally, probes for specific cell populations that could be visualized by noninvasive imaging techniques could allow noninvasive monitoring of these responses, as discussed in the section below.