Special Issue on the Pathobiology of Laboratory Nonhuman Primates: A Review of Species, Substrain, Geographical Origin, Age, and Modality-Related Factors.

Nonhuman primates (NHPs) are utilized in nonclinical safety testing due to their phylogenetic proximity to humans and similarity in physiology and anatomy. However, ethical considerations and the increased demand for NHPs, coupled with the current shortage in their supply, have increased the calls to minimize their use. In addition, the increased demand and supply shortage of NHPs have increased the use of animals sourced from different geographical origins, and animals of different ages, which can complicate the interpretation of study results. Coupled with the relative uniqueness of findings induced by novel therapeutic modalities, there is an increasing need for a deeper understanding of the systemic pathobiology of NHPs. Here we provide a brief preview of the two main themes discussed in this special issue, which include the influence of geographical origin, age, and sex on background pathology, clinical pathology reference values, other relevant toxicology endpoints, and organ system pathology.

Morphological characterization of canine retrobulbar lymphoma.

This report characterized seven cases of canine retrobulbar lymphoma that have been diagnosed during 2008 to 2014 by immunophenotyping of CD3 and Pax5. Classification of lymphoma were performed according to the revised WHO guidelines. Four retrobulbar lymphomas were of T-cell origin, while the others were of B-cell. Out of 7 cases, four subtypes were diagnosed in this study; T-cell-rich large B-cell lymphoma (3/7), T-cell lymphoblastic lymphoma (2/7), peripheral T-cell lymphoma (1/7), and cutaneous nonepitheliotropic lymphoma (1/7). T-cell-rich large B-cell lymphoma was found to be the most frequent subtype found.

Evaluation of insulin-like growth factor acid-labile subunit as a potential biomarker of effect for deoxynivalenol-induced proinflammatory cytokine expression.

Consumption of the trichothecene deoxynivalenol (DON) suppresses growth in experimental animals - an adverse effect that was used to establish the tolerable daily intake for this toxin. DON ingestion has been recently found to suppress plasma insulin-like growth factor acid-labile subunit (IGFALS), a protein essential for growth. Studies were conducted to explore the feasibility of using plasma IGFALS as a biomarker of effect for DON. In the first study, weanling mice were fed 0, 1, 2.5, 5 and 10 ppm DON and weight and plasma IGFALS determined at intervals over 9 wk. Reduced body weight gains were detectable beginning at wk 5 in the 10 ppm dose and wk 7 at the 5 ppm dose. Plasma IGFALS was significantly depressed at wk 5 in the 5 and 10 ppm groups at wk 9 in the 10 ppm group. Depressed IGFALS significantly correlated with reduced body weight at wk 5 and 9. Benchmark dose modeling revealed the BMDL and BMD for plasma IGFALS reduction were 1.1 and 3.0 ppm DON and for weight reduction were 2.1 and 4.5 ppm DON. In the second study, it was demonstrated that mice fed 15 ppm DON diet had significantly less plasma IGFALS than mice fed identical amounts of control diet. Thus DON's influence on IGFALS likely reflects the combined effects of reduced food intake as well as its physiological action involving suppressors of cytokine signaling. Taken together, these findings suggest that plasma IGFALS might be a useful biomarker for DON's adverse effects on growth.

Body composition and hormonal effects following exposure to mycotoxin deoxynivalenol in the high-fat diet-induced obese mouse.

SCOPE: To characterize the effects of ingesting the common foodborne mycotoxin deoxynivalenol (DON) on body weight and composition in the high-fat (HF) diet-induced obese mice, a model of human obesity. METHODS AND RESULTS: Female B6C3F1 mice were initially fed HF diets containing 45% kcal (HF45) or 60% kcal (HF60) as fat for 94 days to induce obesity. Half of each group was either continued on unamended HF diets or fed HF diets containing 10 mg/kg DON (DON-HF45 or DON-HF60) for another 54 days. Additional control mice were fed a low-fat (LF) diet containing 10% kcal as fat for the entire 148-day period. DON induced rapid decreases in body weights and fat mass, which stabilized to those of the LF control within 11 days. These effects corresponded closely to a robust transient decrease in food consumption. While lean body mass did not decline in DON-fed groups, further increases were suppressed. DON exposure reduced plasma insulin, leptin, insulin-like growth factor 1, and insulin-like growth factor acid labile subunit as well as increased hypothalamic mRNA level of the orexigenic agouti-related protein. CONCLUSION: DON-mediated effects on body weight, fat mass, food intake, and hormonal levels in obese mice were consistent with a state of chronic energy restriction.

Kinetics of satratoxin g tissue distribution and excretion following intranasal exposure in the mouse.

Intranasal exposure of mice to satratoxin G (SG), a macrocyclic trichothecene produced by the indoor air mold Stachybotrys chartarum, selectively induces apoptosis in olfactory sensory neurons (OSNs) of the nose and brain. The purpose of this study was to measure the kinetics of distribution and clearance of SG in the mouse. Following intranasal instillation of female C57B16 mice with SG (500 microg/kg bw), the toxin was detectable from 5 to 60 min in blood and plasma, with the highest concentrations, 30 and 19 ng/ml, respectively, being observed at 5 min. SG clearance from plasma was rapid and followed single-compartment kinetics (t(1/2) = 20 min) and differed markedly from that of other tissues. SG concentrations were maximal at 15-30 min in nasal turbinates (480 ng/g), kidney (280 ng/g), lung (250 ng/g), spleen (200 ng/g), liver (140 ng/g), thymus (90 ng/g), heart (70 ng/g), olfactory bulb (14 ng/g), and brain (3 ng/g). The half-lives of SG in the nasal turbinate and thymus were 7.6 and 10.1 h, respectively, whereas in other organs, these ranged from 2.3 to 4.4 h. SG was detectable in feces and urine, but cumulative excretion over 5 days via these routes accounted for less than 0.3% of the total dose administered. Taken together, SG was rapidly taken up from the nose, distributed to tissues involved in respiratory, immune, and neuronal function, and subsequently cleared. However, a significant amount of the toxin was retained in the nasal turbinate, which might contribute to SG's capacity to evoke OSN death.

Pulmonary responses to Stachybotrys chartarum and its toxins: mouse strain affects clearance and macrophage cytotoxicity.

We investigated differences in the pulmonary and systemic clearance of Stachybotrys chartarum spores in two strains of mice, BALB/c and C57BL/6J. To evaluate clearance, mice were intratracheally instilled with a suspension of radiolabeled S. chartarum spores or with unlabeled spores. The lungs of C57BL/6J mice showed more rapid spore clearance than the lungs of BALB/c mice, which correlated with increased levels of spore-associated radioactivity in the GI tracts of C57BL/6J as compared with BALB/c mice. To identify mechanisms responsible for mouse strain differences in spore clearance and previously described lung inflammatory responses, we exposed alveolar macrophages (AMs) lavaged from BALB/c and C57BL/6J mice to S. chartarum spores, S. chartarum spore toxin (SST), and satratoxin G (SG) in vitro. The S. chartarum spores were found to be highly toxic with most cells from either mouse strain being killed within 24 h when exposed to a spore:cell ratio of 1:75. The spores were more lethal to AMs from C57BL/6J than those from BALB/c mice. In mice, the SST elicited many of the same inflammatory responses as the spores in vivo, including AM recruitment, pulmonary hemorrhage, and cytokine production. Our data suggest that differences in pulmonary spore clearance may contribute to the differences in pulmonary responses to S. chartarum between BALB/c and C57BL/6J mice. Enhanced AM survival and subsequent macrophage-mediated inflammation may also contribute to the higher susceptibility of BALB/c mice to S. chartarum pulmonary effects. Analogous genetic differences among humans may contribute to reported variable sensitivity to S. chartarum.

Bacterial- and viral-induced inflammation increases sensitivity to acetaminophen hepatotoxicity.

Acetaminophen (APAP)-induced hepatotoxicity accounts for nearly half of acute liver failure cases in the United States. The doses that produce hepatotoxicity vary considerably and many risk factors have been proposed, including liver inflammation from viral hepatitis. Interestingly, inflammatory stress from another stimulus, bacterial endotoxin (lipopolysaccharide, LPS), renders the liver more sensitive to hepatotoxicity from numerous xenobiotic agents. The purpose of these studies was to test the hypothesis that inflammation induced by LPS or infection with reovirus increases sensitivity to APAP-induced liver injury. For LPS-induced inflammation, C57BL/6J mice were treated with either saline or LPS (44 x 10(6) EU/kg, ip) 2 h before treatment with APAP (100-400 mg/kg, ip) or saline. No elevation in serum alanine aminotransferase (ALT) activity was observed in mice that received vehicle or LPS alone. LPS co-treatment produced a leftward shift of the dose-response curve for APAP-induced hepatotoxicity and led to significantly greater tumor necrosis factor-alpha (TNF) production than APAP alone. Reovirus serotype 1 (10(8) PFU, iv) induced inflammation in Balb/c mice as evidenced by increases in hepatic mRNAs for macrophage inhibitory protein-2, interleukin-6, and TNF. Co-administration of reovirus and APAP at doses of 450 and 700 mg/kg (2 h after reovirus) led to increases in serum ALT activity, whereas neither reovirus nor APAP alone produced liver injury. Consistent with the increases in serum ALT activity, histopathologic examination revealed centrilobular necrosis with marked neutrophilic accumulation only in livers of mice treated with LPS/APAP or with reovirus/APAP. The results suggest that normally noninjurious doses of APAP are rendered hepatotoxic by modest inflammation, whether bacterial or viral in origin.

Suppression of insulin-like growth factor acid-labile subunit expression--a novel mechanism for deoxynivalenol-induced growth retardation.

Consumption of deoxynivalenol (DON), a trichothecene mycotoxin commonly detected in cereal-based foods, causes impaired growth in many animal species. While growth retardation is used as a basis for regulating DON levels in human food, the underlying mechanisms remain poorly understood. Oral exposure of mice to DON rapidly induces multiorgan expression of proinflammatory cytokines, and this is followed by upregulation of several suppressors of cytokine signaling (SOCS), some of which are capable of impairing growth hormone (GH) signaling. The purpose of this study was to test the hypothesis that impairment of the GH axis precedes DON-induced growth retardation in the mouse. Subchronic dietary exposure of young (4-week old) mice to DON (20 ppm) over a period of 2-8 weeks was found to (1) impair weight gain, (2) result in a steady-state plasma DON concentration (40-60 ng/ml), (3) downregulate hepatic insulin-like growth factor acid-labile subunit (IGFALS) mRNA expression, and (4) reduce circulating insulin-like growth factor 1 (IGF1) and IGFALS levels. Acute oral exposure to DON at 0.5-12.5 mg/kg body weight (bw) markedly suppressed hepatic IGFALS mRNA levels within 2 h in a dose-dependent fashion, whereas 0.1 mg/kg bw was without effect. DON-induced IGFALS mRNA upregulation occurred both with and without exogenous GH treatment. These latter effects co-occurred with robust hepatic suppressors of cytokine signaling 3 upregulation. Taken together, these data suggest that oral DON exposure perturbs GH axis by suppressing two clinically relevant growth-related proteins, IGFALS and IGF1. Both have potential to serve as biomarkers of effect in populations exposed to this common foodborne mycotoxin.

Induction of suppressors of cytokine signaling by the trichothecene deoxynivalenol in the mouse.

Deoxynivalenol (DON), a trichothecene mycotoxin found in grains and cereal-based foods worldwide, impairs weight gain in experimental animals but the underlying mechanisms remain undetermined. Oral exposure to DON induces rapid and transient upregulation of proinflammatory cytokine expression in the mouse. The latter are known to induce several suppressors of cytokine signaling (SOCS), some of which impair growth hormone (GH) signaling. We hypothesized that oral exposure to DON will induce SOCS expression in the mouse. Real-time PCR and cytokine bead array revealed that oral gavage with DON rapidly (1 h) induced tumor necrosis factor-alpha and interleukin-6 mRNA and protein expression in several organs and plasma, respectively. Upregulation of mRNAs for four well-characterized SOCS (CIS [cytokine-inducible SH2 domain protein], SOCS1, SOCS2, and SOCS3) was either concurrent with (1 h) or subsequent to cytokine upregulation (2 h). Notably, DON-induced SOCS3 mRNAs in muscle, spleen and liver, with CIS1, SOCS1, and SOCS2 occurring to a lesser extent. Hepatic SOCS3 mRNA was a very sensitive indicator of DON exposure with SOCS3 protein being detectable in the liver well after the onset of cytokine decline (5 h). Furthermore, hepatic SOCS upregulation was associated with about 75% suppression of GH-inducible insulin-like growth factor acid labile subunit. Taken together, DON-induced cytokine upregulation corresponded to increased expression of several SOCS, and was associated with suppression of GH-inducible gene expression in the liver.

Tissue distribution and proinflammatory cytokine gene expression following acute oral exposure to deoxynivalenol: comparison of weanling and adult mice.

The frequent presence of deoxynivalenol (DON) in cereal-based foods and the high intake of these foods by children raises particular concerns about the relative susceptibility of this subpopulation to adverse effects evoked by this mycotoxin. We tested the hypothesis that both toxicokinetics and proinflammatory cytokine gene expression following a oral DON exposure at 5mg/kg bw differ between weanling (3-4 wk) and young adult (8-10 wk) female mice. DON was rapidly taken up with maximum plasma concentrations reaching 1.0 microg/ml in adult mice at 15 min, whereas DON levels were approximately twice as much in weanling mice at these times. DON was rapidly cleared in both weanling and adult mice with concentrations being reduced by 78% and 81% of the peak levels, respectively, after 2h. DON accumulation and clearance in spleen, liver, lung and kidney followed similar kinetics to that of plasma with tissue burdens also reaching twice that of adult mice. When TNF-alpha, IL-1beta and IL-6 mRNAs in spleens (a primary source of systemic proinflammatory cytokines) were used as biomarkers of the DON's effects, expression of these mRNAs was two to three times greater in weanling than adult mouse. However, differences in proinflammatory cytokine expression were less robust or not apparent in the liver or lung. Taken together, these data suggest that young mice are modestly more susceptible than adult mice to the adverse effects of DON and that this might result from a greater toxin tissue burden.

Immunochemical assessment of deoxynivalenol tissue distribution following oral exposure in the mouse.

Deoxynivalenol (DON or vomitoxin) is a trichothecene mycotoxin commonly found in cereal grains that adversely affects growth and immune function in experimental animals. A competitive enzyme-linked immunosorbent assay (ELISA) was used to monitor the kinetics of distribution and clearance of DON in tissues of young adult B6C3F1 male mice that were orally administered 25mg/kg bw of the toxin. DON was detectable from 5 min to 24h in plasma, liver, spleen and brain and from 5 min to 8h in heart and kidney. The highest DON plasma concentrations were observed within 5-15 min (12 microg/mL) after dosing. There was rapid clearance following two-compartment kinetics (t(1/2)alpha=20.4 min, t 1/2 beta=11.8h) with 5% and 2% maximum plasma DON concentrations remaining after 8 and 24h, respectively. DON distribution and clearance kinetics in other tissues were similar to that of plasma. At 5 min, DON concentrations in mug/g were 19.5+/-1.9 in liver, 7.6+/-0.5 in kidney, 7.3+/-0.8 in spleen, 6.8+/-0.9 in heart and 0.8+/-0.1 in the brain. DON recoveries in tissues by ELISA were comparable to a previous study that employed (3)H-DON and 25mg/kg bw DON dose. The ELISA was further applicable to the detection of DON in plasma of mice exposed to the toxin via diet. This approach provides a simple strategy that can be used to answer relevant questions in rodents of how dose, species, age, gender, genetic background and route/duration of exposure impact DON uptake and clearance.

Tissue distribution and proinflammatory cytokine induction by the trichothecene deoxynivalenol in the mouse: comparison of nasal vs. oral exposure.

Oral exposure to the trichothecene deoxynivalenol (DON), a common cereal grain contaminant, adversely affects growth and immune function in experimental animals. Besides foodborne exposure, the potential exists for DON to become airborne during the harvest and handling of grains and therefore pose a risk to agricultural workers. The purpose of this study was to compare the effects of oral and intranasal exposure to DON (5mg/kg bw) on tissue distribution and proinflammatory cytokine induction in the adult female mouse. Competitive direct ELISA revealed that, regardless of exposure route, DON concentrations in plasma, spleen, liver, lung and kidney were maximal within 15-30 min and declined by 75-90% after 120 min. However, plasma and tissue DON concentrations were 1.5-3 times higher following intranasal exposure as compared to oral exposure. The functional significance of elevated DON tissue concentrations was assessed by measuring IL-1beta, IL-6, and TNF-alpha mRNA responses in spleen, liver and lung. Oral exposure to DON-induced robust proinflammatory cytokine gene expression after 60 and 120 min. In contrast, inductions of IL-1beta, IL-6 and TNF-alpha mRNAs in nasally exposed mice were 2-10, 2-5 and 2-4 times greater, respectively, than those in the tissues of orally exposed mice. Taken together, these data suggest that DON was more toxic to the mouse when nasally exposed than when orally exposed, and that this might relate to greater tissue burden of the toxin.

Neurotoxicity and inflammation in the nasal airways of mice exposed to the macrocyclic trichothecene mycotoxin roridin a: kinetics and potentiation by bacterial lipopolysaccharide coexposure.

Macrocyclic trichothecene mycotoxins produced by indoor air molds potentially contribute to symptoms associated with damp building illnesses. The purpose of this investigation was to determine (1) the kinetics of nasal inflammation and neurotoxicity after a single intranasal instillation of roridin A (RA), a representative macrocyclic trichothecene; and (2) the capacity of lipopolysaccharide (LPS) to modulate RA's effects. C57Bl/6 female mice were intranasally instilled once with 50 mul of RA (500 mug/kg body weight [bw]) in saline or saline only and then nose and brain tissues were collected over 72 h and processed for histopathologic and messenger RNA (mRNA) analysis. RA-induced apoptosis specifically in olfactory sensory neurons (OSNs) after 24 h postinstillation (PI) causing marked atrophy of olfactory epithelium (OE) that was maximal at 72 h PI. Concurrently, there was marked bilateral atrophy of olfactory nerve layer of the olfactory bulbs (OBs) of the brain. In the ethmoid turbinates, upregulated messenger RNA (mRNA) expression of the proapoptotic gene FAS and the proinflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)-6, IL-1, and macrophage inhibitory protein-2 was observed from 6 to 24 h PI, whereas expression of several other proapoptotic genes (PKR, p53, Bax, and caspase-activated DNAse) was detectable only at 24 h PI. Simultaneous exposure to LPS (500 ng/kg bw) and a lower dose of RA (250 mug/kg bw) magnified RA-induced proinflammatory gene expression, apoptosis, and inflammation in the nasal tract. Taken together, the results suggest that RA markedly induced FAS and proinflammatory cytokine expression prior to evoking OSN apoptosis and OE atrophy and that RA's effects were augmented by LPS.