Environment, endocrinology, and biochemistry influence expression of stress proteins in bottlenose dolphins.

Natural and anthropogenic stressors have been reported to impact the health of marine mammals. Therefore, investigation of quantifiable biomarkers in response to stressors is required. We hypothesized that stress protein expression would be associated with biological and health variables in wild and managed-care bottlenose dolphins (Tursiops truncatus). To test this hypothesis, our study objectives were to (1) determine if stress proteins in skin, white blood cells (WBCs), and plasma could be measured with an antibody-based microarray, (2) measure stress-protein expression relative to biological data (location, sex, age, environment), and (3) determine if stress-protein expression was associated with endocrine, hematological, biochemical and serological variables and gene expression in bottlenose dolphins. Samples were collected from two wild groups (n = 28) and two managed-care groups (n = 17). Proteins involved in the HPA axis, apoptosis, proteotoxicity, and inflammation were identified as stress proteins. The expression of 3 out of 33 proteins was significantly (P < 0.05) greater in skin than plasma and WBCs. Male dolphins had significantly greater expression levels for 10 proteins in skin compared to females. The greatest number of stress-associated proteins varied by the dolphins' environment; nine were greater in managed-care dolphins and 15 were greater in wild dolphins, which may be related to wild dolphin disease status. Protein expression in skin and WBCs showed many positive relationships with measures of plasma endocrinology and biochemistry. This study provides further understanding of the underlying mechanisms of the stress response in bottlenose dolphins and application of a combination of novel methods to measure stress in wildlife.

Nanoformulation of Talazoparib Increases Maximum Tolerated Doses in Combination With Temozolomide for Treatment of Ewing Sarcoma.

The Pediatric Preclinical Testing Program previously identified the PARP inhibitor talazoparib (TLZ) as a means to potentiate temozolomide (TMZ) activity for the treatment of Ewing sarcoma. However, the combination of TLZ and TMZ has been toxic in both preclinical and clinical testing, necessitating TMZ dose reduction to ~15% of the single agent maximum tolerated dose. We have synthesized a nanoparticle formulation of talazoparib (NanoTLZ) to be administered intravenously in an effort to modulate the toxicity profile of this combination treatment. Results in Ewing sarcoma xenograft models are presented to demonstrate the utility of this delivery method both alone and in combination with TMZ. NanoTLZ reduced gross toxicity and had a higher maximum tolerated dose than oral TLZ. The dose of TMZ did not have to be reduced when combined with NanoTLZ as was required when combined with oral TLZ. This indicated the NanoTLZ delivery system may be advantageous in decreasing the systemic toxicity associated with the combination of oral TLZ and TMZ.

Development and application of an antibody-based protein microarray to assess physiological stress in grizzly bears (Ursus arctos).

A novel antibody-based protein microarray was developed that simultaneously determines expression of 31 stress-associated proteins in skin samples collected from free-ranging grizzly bears (Ursus arctos) in Alberta, Canada. The microarray determines proteins belonging to four broad functional categories associated with stress physiology: hypothalamic-pituitary-adrenal axis proteins, apoptosis/cell cycle proteins, cellular stress/proteotoxicity proteins and oxidative stress/inflammation proteins. Small skin samples (50-100 mg) were collected from captured bears using biopsy punches. Proteins were isolated and labelled with fluorescent dyes, with labelled protein homogenates loaded onto microarrays to hybridize with antibodies. Relative protein expression was determined by comparison with a pooled standard skin sample. The assay was sensitive, requiring 80 µg of protein per sample to be run in triplicate on the microarray. Intra-array and inter-array coefficients of variation for individual proteins were generally <10 and <15%, respectively. With one exception, there were no significant differences in protein expression among skin samples collected from the neck, forelimb, hindlimb and ear in a subsample of n = 4 bears. This suggests that remotely delivered biopsy darts could be used in future sampling. Using generalized linear mixed models, certain proteins within each functional category demonstrated altered expression with respect to differences in year, season, geographical sampling location within Alberta and bear biological parameters, suggesting that these general variables may influence expression of specific proteins in the microarray. Our goal is to apply the protein microarray as a conservation physiology tool that can detect, evaluate and monitor physiological stress in grizzly bears and other species at risk over time in response to environmental change.

Ecotoxicological risks associated with land treatment of petrochemical wastes. I. Residual soil contamination and bioaccumulation by cotton rats (Sigmodon hispidus).

Petrochemical waste contains both organic and inorganic contaminants that can pollute soil and may pose significant ecological risks to wildlife. Petrochemical waste typically is disposed of in land treatment units, which are widespread throughout Oklahoma and the United States. Few studies have been conducted evaluating possible toxicity risks to terrestrial organisms residing on these units. In this study, the extent of soil contamination with fluoride (F), metals, and organic hydrocarbons, the bioaccumulation of F and metals in cotton rats (Sigmodon hispidus), the relationship between contaminants in soil and in tissues of cotton rats, and the level of potentially toxic polycyclic aromatic hydrocarbons (PAHs) in soil were determined on land treatment units. Over a 2-yr period, cotton rats and soils were collected and analyzed from 5 land treatment and matched reference units. The number of land treatment units with soil metal contamination (in parentheses) included: Cr, Cu, Pb (5). Al, As, Ni, Sr, Zn (4). Ba (3). and Cd, V (2). The number of land treatment units with soil PAH contamination (in parentheses) were naphthalene, phenanthrene, benzo[g,h,i]perylene (3). acenaphthene, anthracene, pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[a]pyrene, indeno[1,2,3-c,d]pyrene, dibenz[a,h]anthracene (2). and acenaphthylene, fluorene, fluoranthene, benzo[k]fluoranthene (1). Total PAH and total petroleum hydrocarbons (TPH) were elevated at all five land treatment units. Mean sums of benzo[a]pyrene (BaP) equivalents (BaPequiv ) were not affected on

Ecotoxicological risks associated with land treatment of petrochemical wastes. II. Effects on hepatic phase I and phase II detoxification enzymes in cotton rats.

The purpose of this study was to evaluate possible exposure and resultant hepatic effects of petrochemical waste on cotton rats (Sigmodon hispidus) living on landfarmed sites. Male and female cotton rats were collected in summer, fall, and winter from four landfarm sites and four ecologically similar reference sites. Hepatic methoxyresorufin O-deethylase (MROD) activity was significantly induced in male and female rats collected from landfarms compared to rats collected from reference sites. In contrast, changes in ethoxyresorufin O-deethylase (EROD) activity were inconsistent due to season, sex, and treatment variation. A significant decrease in EROD and MROD activity was found in cotton rats held for 48 h prior to sacrifice compared to rats euthanized on the day of capture. These results indicate that when using hepatic EROD and MROD activities as biochemical markers of exposure to aryl hydrocarbon receptor agonists, animals should be euthanized as quickly as possible after capture. The cotton rats collected from one landfarm unit exhibited a pattern of consistent elevation of EROD, MROD, and pent-oxyresorufin O-deethylase (PROD) activity. This unit also had a pattern of elevated CYP1A2 protein expression determined by Western blotting. There were no consistent alterations from contaminant exposure on hepatic glutathione S-transferase (GST) activity, glutathione levels, or CYP1A1 protein. Hepatic EROD and MROD activities varied considerably between seasons and sex of rats. In conclusion, consistent induction of hepatic EROD and MROD activities in cotton rats was found in three out of four sampled landfarm sites compared to the rats collected from the reference sites, indicating exposure to contaminants-likely polyaromatic hydrocarbons.

Ecotoxicological risks associated with land treatment of petrochemical wastes. III. Immune function and hematology of cotton rats.

Landfarming is a widely used method of treating petrochemical waste through microbial bio-degradation. The effects of residual petrochemical contamination on wildlife, especially terrestrial mammals, are poorly understood. The effects of contaminants on the immune system and hematology of cotton rats (Sigmodon hispidus) living on five abandoned petrochemical landfarms (units 1-5) in Oklahoma were studied. Cotton rats were sampled seasonally (summer and winter) from each landfarm and from five ecologically matched reference sites for 2 yr (1998-2000) and returned to the laboratory for immunological and hematological assays. Overall analysis indicated that rats inhabiting landfarms exhibited decreased relative spleen size compared to rats collected from reference sites, with the landfarm at unit 1 showing the greatest reduction. Cotton rats collected from landfarms also had increased hemoglobin, hematocrit, and platelet levels and decreased blood leukocytes during summer. During winter, an increase in the number of popliteal node white blood cells was observed from rats collected on landfarms. No marked difference was detected for lymphocyte proliferation in response to concanavalin A, pokeweed, or interleukin-2. Lymphokine-activated killer cell lytic ability showed a seasonal pattern, but no treatment differences. No differences between landfarm and reference sites were detected in the hypersensitivity reaction of rats given an intradermal injection of phytohemagluttinin (PHA-P). Comparisons within individual sites indicated that two sites (units 1 and 3) had the greatest effects on immune function and hematology of cotton rats. The results of this study suggest that residual petrochemical waste affects the immune system and hematology of cotton rats living on abandoned landfarms during summer and is complicated by variation in the contaminants found on individual petroleum sites.