Keith Bailey

Interpreting Stress Responses during Routine Toxicity Studies: A Review of the Biology, Impact, and Assessment

Stress often occurs during toxicity studies. The perception of sensory stimuli as stressful primarily results in catecholamine release and activation of the hypothalamic–pituitary–adrenal (HPA) axis to increase serum glucocorticoid concentrations. Downstream effects of these neuroendocrine signals may include decreased total body weights or body weight gain; food consumption and activity; altered organ weights (e.g., thymus, spleen, adrenal); lymphocyte depletion in thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophils with decreased lymphocytes and eosinophils); and altered reproductive functions. Typically, only some of these findings occur in a given study. Stress responses should be interpreted as secondary (indirect) rather than primary (direct) test article–related findings. Determining whether effects are the result of stress requires a weight-of-evidence approach. The evaluation and interpretation of routinely collected data (standard in-life, clinical pathology, and anatomic pathology endpoints) are appropriate and generally sufficient to assess whether or not changes are secondary to stress. The impact of possible stress-induced effects on data interpretation can partially be mitigated by toxicity study designs that use appropriate control groups (e.g., cohorts treated with vehicle and subjected to the same procedures as those dosed with test article), housing that minimizes isolation and offers environmental enrichment, and experimental procedures that minimize stress and sampling and analytical bias. This article is a comprehensive overview of the biological aspects of the stress response, beginning with a Summary (Section 1) and an Introduction (Section 2) that describes the historical and conventional methods used to characterize acute and chronic stress responses. These sections are followed by reviews of the primary systems and parameters that regulate and/or are influenced by stress, with an emphasis on parameters evaluated in toxicity studies: In-life Procedures (Section 3), Nervous System (Section 4), Endocrine System (Section 5), Reproductive System (Section 6), Clinical Pathology (Section 7), and Immune System (Section 8). The paper concludes (Section 9) with a brief discussion on Minimizing Stress-Related Effects (9.1.), and a final section explaining why Parameters routinely measured are appropriate for assessing the role of stress in toxicology studies (9.2.).

N-Methyl-N-Nitrosourea (MNU): A Positive Control Chemical for p53+/− Mouse Carcinogenicity Studies

This study evaluated the effects of a single intraperitoneal injection of N-methyl-N-nitrosourea (MNU) in citrate buffer (pH 4.5) at a dose of 75 mg/kg in thirty male and thirty female p53+/− mice followed by a six-month observation period. Fifteen control mice per sex received a single intraperitoneal injection of citrate buffer. Fifty-six of sixty mice treated with MNU died or were sacrificed before the end of the observation period. Twenty-four males and twenty-seven females treated with MNU developed malignant lymphoma of the thymus; of these, twenty-three males and twenty-seven females had corresponding enlargement or masses in the thymus at necropsy. Lymphoblasts in thymic lymphomas stained positively for mouse CD3 antigen, indicating a T-cell lineage. One control female mouse had malignant lymphoma of the spleen that did not involve the thymus. Nine males and five females treated with MNU had adenomas or adenocarcinomas of the small intestine, whereas no intestinal neoplasms were observed in control mice. These findings support the use of a single dose of MNU as a positive control chemical in six-month p53+/− mouse carcinogenicity studies and suggest that examination of the thymus alone is sufficient to evaluate the validity of the model system.

Unexpected thrombocytopenia and anemia in cynomolgus monkeys induced by a therapeutic human monoclonal antibody.

Cynomolgus monkeys dosed with a therapeutic monoclonal antibody (mAbY.1) at ≥50 mg/kg had unexpected acute thrombocytopenia (nadir ∼3,000 platelets/µl), sometimes with decreases in red cell mass. Increased activated macrophages, mitotic figures, and erythrophagocytosis were observed in the spleen. Binding of mAbY.1 to cynomolgus peripheral blood cells could not be detected in vitro. mAbY.1 induced phagocytosis of platelets by peripheral blood monocytes from cynomolgus monkeys, but not from humans. mAbs sharing the same constant domain (Fc) sequences, but differing from mAbY.1 in their variable domains, bound competitively to and had similar biological activity against the intended target. None of these antibodies had hematologic liabilities in vitro or in vivo. Neither the F(ab’)2 portion of mAbY.1 nor the F(ab’)2 portion on an aglycosylated Fc (IgG1) framework caused phagocytosis of platelets in vitro. These data suggest that the hematologic effects of mAbY.1 in cynomolgus monkeys likely occurred through an off-target mechanism, shown to be driven by 1 to 3 amino acid differences in the light chain. The hematologic effects made mAbY.1 an unsuitable candidate for further development as a therapeutic agent. This example demonstrates that nonclinical safety studies may be essential for understanding off-target effects of mAbs prior to clinical trials.

Pulsed terahertz imaging of breast cancer in freshly excised murine tumors

Abstract. This paper investigates terahertz (THz) imaging and classification of freshly excised murine xenograft breast cancer tumors. These tumors are grown via injection of E0771 breast adenocarcinoma cells into the flank of mice maintained on high-fat diet. Within 1 h of excision, the tumor and adjacent tissues are imaged using a pulsed THz system in the reflection mode. The THz images are classified using a statistical Bayesian mixture model with unsupervised and supervised approaches. Correlation with digitized pathology images is conducted using classification images assigned by a modal class decision rule. The corresponding receiver operating characteristic curves are obtained based on the classification results. A total of 13 tumor samples obtained from 9 tumors are investigated. The results show good correlation of THz images with pathology results in all samples of cancer and fat tissues. For tumor samples of cancer, fat, and muscle tissues, THz images show reasonable correlation with pathology where the primary challenge lies in the overlapping dielectric properties of cancer and muscle tissues. The use of a supervised regression approach shows improvement in the classification images although not consistently in all tissue regions. Advancing THz imaging of breast tumors from mice and the development of accurate statistical models will ultimately progress the technique for the assessment of human breast tumor margins.

Assessment of Terahertz Imaging for Excised Breast Cancer Tumors with Image Morphing

This paper presents an image morphing algorithm for quantitative evaluation methodology of terahertz (THz) images of excised breast cancer tumors. Most current studies on the assessment of THz imaging rely on qualitative evaluation, and there is no established benchmark or procedure to quantify the THz imaging performance. The proposed morphing algorithm provides a tool to quantitatively align the THz image with the histopathology image. Freshly excised xenograft murine breast cancer tumors are imaged using the pulsed THz imaging and spectroscopy system in the reflection mode. Upon fixing the tumor tissue in formalin and embedding in paraffin, a formalin-fixed paraffin-embedded (FFPE) tissue block is produced. A thin slice of the block is prepared for the pathology image while another THz reflection image is produced directly from the block. We developed an algorithm of mesh morphing using homography mapping of the histopathology image to adjust the alignment, shape, and resolution to match the external contour of the tissue in the THz image. Unlike conventional image morphing algorithms that rely on internal features of the source and target images, only the external contour of the tissue is used to avoid bias. Unsupervised Bayesian learning algorithm is applied to THz images to classify the tissue regions of cancer, fat, and muscles present in xenograft breast tumors. The results demonstrate that the proposed mesh morphing algorithm can provide more effective and accurate evaluation of THz imaging compared with existing algorithms. The results also showed that while THz images of FFPE tissue are highly in agreement with pathology images, challenges remain in assessing THz imaging of fresh tissue.