Time-course of oral toxicity to contaminated groundwater in male Sprague Dawley rats.

Assessing toxicity of complex mixtures of contaminants from industrial sites with historic and ongoing contamination remains a challenge for risk assessors. Groundwater from a pesticide packaging site in Canada containing a complex mixture of known and unknown contaminants was examined in male rats to determine the target organ toxicity. This study determined the time-course of toxicity (7, 14, 28, and 60 days) following ad libitum oral exposure to 0.05% v/v contaminated groundwater compared to tap water (control) in male Sprague Dawley rats (n=5 /group/time). Exposure to groundwater resulted in inflammation, indicated by a statistically significant increase in plasma lymphocyte and neutrophil counts on days 7 and 60, respectively, but a reduction in the plasma alpha 2 macroglobulin levels by day 60. Gonadotoxicity was indicated by a reduced Johnsen score (grading spermatogenesis) in all exposed groups at all time points, while seminiferous epithelial height was reduced on days 7, 14, and 28 compared to controls. Plasma testosterone was reduced in exposed groups on days 7 and 28, accompanied by elevated testicular lipid peroxidation at all time points compared to control. In contrast, lipid peroxidation in the lungs from exposed rats was elevated on days 7, 14, and 28. Plasma symmetric dimethylarginine was elevated on day 14 in the exposed group indicating renal impairment. Taken together, these results indicate that testes, kidney, immune and lung are target organs for the contaminated groundwater from this industrial site. The current study highlights the challenge in hazard assessment for complex mixtures and highlights the need for effects-directed analysis and the continued, albeit limited, use of animal models in toxicity testing.

Determining the effects of Candida utilis-fermented pea starch vs. unfermented pea starch, alone or in whole diets, on palatability and glycemic response in dogs and cats.

Current research suggests yeast fermentation has the potential to improve palatability of pea-based diets for both cats and dogs. However, to be useful, fermentation should not compromise other healthy attributes of peas such as a low glycemic response. Fermentation of uncooked pea starch with Candida utilis (ATCC 9950) appeared to increase crude protein, crude fiber content, inorganic compounds (phosphorus and iron) and phenols. Whole diets were designed with fermented and unfermented pea starch to assess palatability, food intake, and glycemic responses in unacclimated, mixed sex Beagle dogs and mixed breed cats (n = 8 and n = 7, respectively). For palatability testing, a control diet was formulated with 30% corn starch as well as test diets with 30% inclusion of fermented or unfermented pea starch (all lab-made), then compared to a commercial diet containing pea starch (Legacy/Horizon). Fermentation had little effect on rapidly digestible starch either in uncooked starch form or when incorporated into whole diets, but did decrease resistant starch by 15% and increase slowly digestible starch by 20%. Palatability tests using either two choices or four choices at a time revealed a significant preference for the fermented pea starch diet (p < 0.01) in both species. For the glycemic responses, a total of four different pea products were included: unfermented pea starch, fermented pea starch, and 30% inclusion of unfermented and fermented pea starch in whole formulated diets. There were no significant changes in glycemic responses with the fermented pea diet compared to the unfermented diet, demonstrating that healthful low glycemic properties of pea starch were retained after C. utilis fermentation. Overall, C. utilis-fermentation technique was successfully adapted to pea starch where it resulted in increased palatability and food intake in dogs and cats, with potential to positively contribute to overall health benefits for both species.

Hazard assessment of complex legacy-contaminated groundwater mixtures using a novel approach method in adult fathead minnows.

Traditional risk assessment methods face challenges in the determination of drivers of toxicity for complex mixtures such as those present at legacy-contaminated sites. Bioassay-driven analysis across several levels of biological organization represents an approach to address these obstacles. This study aimed to apply a novel transcriptomics tool, the EcoToxChip, to characterize the effects of complex mixtures of contaminants in adult fathead minnows (FHMs) and to compare molecular response patterns to higher-level biological responses. Adult FHMs were exposed for 4 and 21 days to groundwater mixtures collected from a legacy-contaminated site. Adult FHM showed significant induction of micronuclei in erythrocytes, decrease in reproductive capacities, and some abnormal appearance of liver histology. Parallel EcoToxChip analyses showed a high proportion of upregulated genes and a few downregulated genes characteristic of compensatory responses. The three most enriched pathways included thyroid endocrine processes, transcription and translation cellular processes, and xenobiotics and reactive oxygen species metabolism. Several of the most differentially regulated genes involved in these biological pathways could be linked to the apical outcomes observed in FHMs. We concluded that molecular responses as determined by EcoToxChip analysis show promise for informing of apical outcomes and could support risk assessments of complex contaminated sites.

Characterization of molecular and apical effects of legacy-contaminated groundwater on early life stages of fathead minnows.

Mechanistic toxicology approaches represent a promising alternative to traditional live animal testing; however, the often-noted uncertainties concerning the linkages between effects observed at molecular and apical levels curtails the adoption of such approaches. The objective of this study was to apply a novel transcriptomics tool, EcoToxChips, to characterize the effects of complex mixtures of contaminants in fish and to compare molecular response patterns to higher-level biological responses including swimming behavior, deformities, and mortality. Fathead minnow (FHM) embryos were exposed for seven days to increasing concentrations of groundwater collected from moderate (MIAZ) and high (HIAZ) industrial activity zones of a legacy contaminated site. There was a concentration-dependent disruption of photo-dependent swimming responses associated with avoidance behavior patterns and spinal deformities (HIAZ and MIAZ), and an induction of pericardial edema and mortality (HIAZ-10%). Parallel EcoToxChip analyses showed a shift from a majority of upregulated genes at lower concentrations to a majority of downregulated genes at higher concentrations for both treatment conditions. Many of the significantly differentially regulated genes were involved in biological pathways including induction of oxidative stress, activating of several metabolic processes and growth, cell death, and inhibition of signal transduction signaling processes. Several contaminants present in the groundwater mixtures could have contributed to an exceedance of antioxidant system capacities that possibly led to the deformities, altered swimming behaviours, and mortality observed in FHMs. Therefore, molecular response patterns could be linked to apical outcomes observed in this study. Overall, the results observed in this study demonstrate that transcriptomics approaches such as the EcoToxChip system could be supportive of risk assessment of complex contaminated sites.

Low birth weight and reduced postnatal nutrition lead to cardiac dysfunction in piglets.

Heart disease is the leading cause of death in humans and evidence suggests early life growth-restriction increases heart disease risk in adulthood. Therefore, this study sought to investigate the effects of low birth weight (LBW) and postnatal restricted nutrition (RN) on cardiac function in neonatal pigs. We hypothesized that LBW and RN would reduce cardiac function in pigs but this effect would be reversed with refeeding. To investigate this hypothesis, pigs born weighing <1.5 kg were assigned LBW, and pigs born >1.5 kg were assigned normal birth weight (NBW). Half the LBW and NBW pigs underwent ~25% total nutrient restriction via intermittent suckling (assigned RN) for the first 4 wk post-farrowing. The other half of piglets were allowed unrestricted suckling access to the sow (assigned NN). At 28 d of age (weaning), pigs were weaned and provided ad libitum access to a standard diet. Echocardiographic, vascular ultrasound, and blood pressure (BP) measurements were performed on day 28 and again on day 56 to assess cardiovascular structure and function. A full factorial three-way ANOVA (NN vs. RN, LBW vs. NBW, male vs. female) was performed. Key findings include reduced diastolic BP (P = 0.0401) and passive ventricular filling (P = 0.0062) in RN pigs at 28 d but this was reversed after refeeding. LBW piglets have reduced cardiac output index (P = 0.0037) and diastolic and systolic wall thickness (P = 0.0293 and P = 0.0472) at 56 d. Therefore, cardiac dysfunction from RN is recovered with adequate refeeding while LBW programs irreversible cardiac dysfunction despite proper refeeding in neonatal pigs.

Heart disease is the leading cause of death in humans, and in addition to the known modifiable risk factors, evidence suggests early life undernutrition increases heart disease risk in adulthood. Specifically, low birth weight (LBW) has been linked to poor infant cardiac development which could be made worse by an inadequate postnatal diet. Globally, 160 million children under the age of five experience a poor nutritive environment leading to growth-restriction highlighting the need for continued research. Using a pig model, the present investigation examined the effects of LBW and a restricted diet during postnatal life on cardiac structure and function in preweaning and post-weaning piglets. The most important findings were (1) nutrient-restricted piglets had reduced cardiac function at 28 d old but refeeding reversed cardiac dysfunction at 56 d, indicating that nutrient-induced cardiac dysfunction can be reversed, and (2) LBW pigs presented with cardiac dysfunction at 56 d regardless of feeding level, suggesting potential for an increased risk of heart disease in adulthood with LBW.

Examining the subchronic (28-day) effects of aqueous Cd-BaP co-exposure on detoxification capacity and cardiac function in adult zebrafish (Danio rerio).

The present study aimed to examine the effects of environmentally relevant concentrations of cadmium (Cd) and Benzo[a]Pyrene (BaP) in the adult zebrafish (Danio rerio). To this end, fish were exposed to either 1 or 10 µg/L Cd or 0.1 or 1 µg/L BaP in isolation, or a co-exposure containing a mixture of the two toxicants. Our results showed extensive modulation of the expression of key antioxidant genes (GPx, SOD1, catalase), detoxifying genes (MT1, MT2, CYP1A1) and a stress biomarker (HSP70) differing between control, single toxicant groups and co-exposure groups. We additionally carried out histopathological analysis of the gills, liver, and hearts of exposed animals, noting no differences in tissue necrosis or apoptosis. Finally, we carried out ultrasonographic analysis of cardiac function, noting a significant decrease of E-wave peak velocity and end diastolic volume in exposed fish. This in turn was accompanied by a decrease in stroke volume and ejection fraction, but not cardiac output in co-exposed fish. The present study is the first to demonstrate that a subchronic aqueous exposure to a Cd-BaP mixture can extensively modulate detoxification capacity and cardiac function in adult zebrafish in a tissue-specific manner.

Oligosaccharides and diet-related dilated cardiomyopathy in beagles.

Introduction: In 2018 the US Food and Drug Administration reported a potential link between grain-free, legume-containing dog foods and the development of canine dilated cardiomyopathy in atypical breeds. One hypothesis was that high oligosaccharide content in legumes reduced bioavailability of taurine, an amino acid with some previous links to canine dilated cardiomyopathy. Methods: To address this hypothesis, in the present study, 8 Beagle dogs consumed four diets: a husbandry commercial dental diet, and three test diets formulated with either 30% rice (control), 30% pea (grain-free) or 30% rice with the addition of 1% raffinose (the predominant oligosaccharide found in peas). The study was conducted in a randomized, crossover design with 5 week feeding periods. Measurement of basic health parameters (weight, body condition score, complete blood cell count, chemistry panel), plasma amino acids, cardiac biomarkers (plasma N-terminal pro-brain natriuretic peptide (NT-proBNP) and cardiac-specific troponin I), fecal bile acids and echocardiographic exams were completed pre-study after feeding the husbandry diet as well as after each test feeding period. Results: Echocardiography showed 9-11% reduction in ejection fraction and 17- 20% greater left ventricular end systolic volume with the husbandry diet compared to both grain-containing test diets. Concentrations of plasma NT-proBNP were 1.3-2 times greater after the husbandry diet compared to the grain-based diet, with the oligosaccharide and pea-based diets showing intermediate levels. Plasma taurine levels were unchanged across diets, while plasma methionine levels were highest and cysteine/cystine levels were lowest after dogs ate the husbandry diet. Discussion: Results indicate that raffinose in the diet is sufficient, but not required to see an increase NT-proBNP, but did not induce any changes in cardiac function after 5 weeks of feeding. Whether this could progress to reduction in cardiac function with longer term feeding is uncertain. A reduced cardiac function along with the greatest increase in NT-proBNP was observed after feeding the husbandry diet that contained the highest amount of insoluble fiber but did not contain legumes or oligosaccharide. Further research into the impact of insoluble fiber in the dental diet is needed to support these novel observations.

Apical and mechanistic effects of 6PPD-quinone on different life-stages of the fathead minnow (Pimephales promelas).

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone) is an emerging contaminant of concern that is generated through the environmental oxidation of the rubber tire anti-degradant 6PPD. Since the initial report of 6PPD-quinone being the cause of urban runoff mortality syndrome of Coho salmon, numerous species have been identified as either sensitive or insensitive to acute lethality caused by 6PPD-quinone. In sensitive species, acute lethality might be caused by uncoupling of mitochondrial respiration in gills. However, little is known about effects of 6PPD-quinone on insensitive species. Here we demonstrate that embryos of fathead minnows (Pimephales promelas) are insensitive to exposure to concentrations as great as 39.97 µg/L for 168 h, and adult fathead minnows are insensitive to exposure to concentrations as great as 9.4 µg/L for 96 h. A multi-omics approach using a targeted transcriptomics array, (EcoToxChips), and proton nuclear magnetic resonance (1H NMR) was used to assess responses of the transcriptomes and metabolomes of gills and livers from adult fathead minnows exposed to 6PPD-quinone for 96 h to begin to identify sublethal effects of 6PPD-quinone. There was little agreement between results of the EcoToxChip and metabolomics analyses, likely because genes present on the EcoToxChip were not representative of pathways suggested to be perturbed by metabolomic analysis. Changes in abundances of transcripts and metabolites in livers and gills suggest that disruption of one­carbon metabolism and induction of oxidative stress might be occurring in gills and livers, but that tissues differ in their sensitivity or responsiveness to 6PPD-quinone. Overall, several pathways impacted by 6PPD-quinone were identified as candidates for future studies of potential sublethal effects of this chemical.

Effects of a 28-day feeding trial of grain-containing versus pulse-based diets on cardiac function, taurine levels and digestibility in domestic dogs.

In 2018, the US Food and Drug Administration reported a link between canine dilated cardiomyopathy (DCM) and grain-free diets. Evidence to support a link has emerged, but the specific ingredients responsible and the role of taurine or other causative factors remain unclear. We hypothesized dogs fed pulse-based, grain-free diets for 28 days will show decreased macronutrient digestibility, increased fecal bile acid excretion, and reduced plasma cystine, cysteine, methionine and taurine, causing sub-clinical cardiac or blood changes indicative of early DCM. Three diets were formulated using white rice flour (grain), whole lentil (grain-free), or wrinkled pea (grain-free) and compared to the pre-trial phase on a commercial grain-based diet. After 28 days of feeding each diet, the wrinkled pea diet impaired stroke volume and cardiac output, increased end-systolic ventricular diameter and increased plasma N-Terminal Pro-B-type Natriuretic Peptide (NT-ProBNP), albeit in a sub-clinical manner. Digestibility of some macronutrients and sulphur-containing amino acids, excluding taurine, also decreased with pulse-based compared to grain-based diets, likely due to higher fiber levels. Plasma taurine levels were unchanged; however, plasma methionine was significantly lower after feeding all test diets compared to the commercial diet. Overall, DCM-like changes observed with the wrinkled pea diet, but not lentil diet, after only 4 weeks in a breed not known to be susceptible support a link between pea-based diets and canine nutritionally-mediated DCM.

Solidified saturated fats coating subunit vaccines greatly extended vaccine booster release and contributed to a Th1/Th2 mixed immune response in mice.

Delayed release of vaccine coupled with a soluble vaccine acts as a primer and a booster with only a single administration, which would be very beneficial to livestock producers. We developed a subdermal pellet consisting of solid-phase pure stearic acid (SA) or palmitic acid (PA) that was used to encapsulate a small volume liquid vaccine consisting of fluorescently labeled *Ovalbumin (Cy5-*OVA) formulated with Emulsigen-D +/- Poly I:C (EMP) adjuvants. Mice were also immunized via the subcutaneous route with Cy5-*OVA-EMP (soluble liquid). The vaccine leached out of the pellet with very little dissolution of the fat itself resulting in the sustained subdermal delivery of antigens and adjuvants. Cy5-*OVA was still visible 60 days post administration in mice immunized with stearic acid-coated or palmitic acid-coated pellets. In these mice, persistently high IgG1 and IgG2a antibody titres were detected as well as significant IFNγ production at least 60 days post-injection. These responses were significantly higher than those observed after a single subcutaneous injection of the vaccine. A repeat trial with the pellets alone +/- the soluble vaccine showed comparable immune responses after surgical implantation of the pellet, suggesting that pellet alone may be sufficient. The PA-coated vaccines led to dermal inflammation in the mice that would limit usefulness of this vehicle, but this was largely absent when SA was used to coat the pellets. These data suggest that the SA-coated adjuvanted vaccine prolonged the release of the vaccine and triggered a comparable immune response to the mice that received the two liquid injections, and a single pellet vaccine should be tested as a novel immunization method for livestock.

Modulation of Cd and BaP uptake rate during acute aqueous co-exposure in adult zebrafish (Danio rerio).

Cadmium and Benzo[a]pyrene are two toxicants of great environmental importance given their frequency and ability to cause extensive toxicity in aquatic organisms including fish. There is evidence that fish can modulate their respective uptake rate during simultaneous exposures, albeit the mechanism behind this is poorly understood. The present study aimed to examine this interaction by exposing adult zebrafish to either 89.3 nM Cd, 4.25 nM BaP or a combination of the two for 72 hrs prior to examining the uptake rate of either toxicant via short-term exposures (3-6 hrs) to radiotracers (109Cd and 14C-BaP). Our results showed that Cd uptake rate increased significantly in the gills when animals were pre-exposed to both toxicants simultaneously, resulting in an increased maximum uptake rate (Jmax). The increased Cd uptake rate did not correspond to increased expression of gill Cd transporters such as the epithelium calcium channel (ECaC) or the divalent metal transporter 1 (DMT1). Furthermore, BaP uptake rate increased significantly at the whole-body level when animals were exposed to both 5.03 nM 14C-BaP and 89.3 nM Cd concurrently. Additionally, we ran a time-course and observed BaP uptake rate is highest in the 6-12 hrs following the beginning of the exposure. Our results provide evidence that the increased bioaccumulation of Cd and BaP observed during co-exposures is at least in part due to an increase in uptake rate and is driven by separate mechanisms.

Bleeding Complications in Patients With Severe Frostbite Injury.

Frostbite is caused by exposure to cold temperatures and can lead to severe injury resulting in amputations. Tissue plasminogen activator (tPA) is a thrombolytic agent that has demonstrated efficacy preventing amputation in frostbite patients. The goal of frostbite management with tPA is to salvage tissue without causing clinically significant bleeding complication. The purpose of this study was to characterize bleeding complications in severe frostbite patients managed with and without tPA. Retrospective chart review of severe frostbite patients admitted to a single ABA verified burn center. Bleeding events were grouped: category 0: no bleed; category 1: bleed not resulting in change or intervention; category 2: bleed resulting in change of management; and category 3: bleed resulting in change of management and intervention. Over a 7-year period, 188 patients were included in the study. Most patients had no documentation suggesting a bleeding complication: 69.7% category 0, 19.1% category 1, 4.8% category 2, and 6.4% category 3. There was no significant difference in category 2 or 3 bleeding complications between patients treated with or without tPA. Overall, 9 of the 143 patients (6.3%) treated with tPA had a category 2 or 3 bleeding complication within 12 hours of tPA completion and 12 of 143 (8.4%) within 24 hours of tPA completion. Based on the low risk of severe bleeding and significant benefit relative to limb or digit salvage demonstrated in this study, we conclude that tPA is safe and effective for the treatment of frostbite in appropriately selected patients.

Assessing non-protein nitrogen sources in commercial dry dog foods.

Protein is a macronutrient required by dogs for growth and maintenance metabolism. However, a portion of the crude protein listed on pet foods may actually arise from non-digestible organic nitrogen or potentially toxic inorganic non-protein nitrogen sources. Neither non-protein source is retained or used by the animal. However, these compounds may result in adverse effects such as methemoglobin formation and increased oxidative stress or potentially beneficial effects such as improved vascular distensibility and decreased inflammation. To analyze nitrogen retention and screen for non-protein nitrogen, four commercial, dry kibble dog foods and one laboratory-made diet were evaluated and then fed to beagles during two separate feeding trials. During the first trial, dogs were randomly assigned each diet (n = 4 dogs/diet) and fed chromium oxide-coated diets for 48 h, followed by total urine and marked fecal collection, as well as plasma collection for total nitrogen, nitrate, ammonia, and urea determination. The amount of nitrogen retained (93%-96%) did not differ among commercial diets. Protein total tract apparent digestibility (TTAD) ranged from 69% to 84%, with the high protein diets significantly higher than the laboratory-made and mid-ranged diets (1-way ANOVA: P < 0.05). The high protein diet also contained the highest concentration of nitrate with subsequent elevations in plasma nitrotyrosine levels (indicator of oxidative stress). During the second trial, eight dogs (n = 8) were fed the same diets for 6 d, after which echocardiography was completed with blood, urine, and feces collected. For health end-points, methemoblobin, plasma nitrotyrosine, and C-reactive protein (CRP; indicator of inflammation) levels were measured. Methemoglobin levels were significantly lower in the high protein diet (P > 0.05), possible due to the stimulation of methemoglobin reductase while nitrotyrosine was unchanged and CRP was undetectable. Furthermore, there was a positive relationship between crude protein, crude fat (simple linear regression: P = 0.02, r 2 > 0.6), price (P = 0.08, r 2 > 0.6), and caloric density (P = 0.11, r 2 > 0.6). There were no significant cardiovascular differences among any of the diets (P > 0.05). Ultimately, this study shows that in commercial diets, price does reflect protein content but that feeding dogs high protein diets for a long period of time may provide an excess in calories without a change in cardiovascular function or detectable increases in inflammation.

Altered cerebrovascular regulation in low birthweight swine.

Full-term low birthweight (LBW) offspring exhibit peripheral vascular dysfunction in the postnatal period; however, whether such impairments extend to the cerebrovasculature remains to be elucidated. We used a swine model to test the hypothesis that LBW offspring would exhibit cerebrovascular dysfunction at later stages of life. Offspring from 14 sows were identified as normal birthweight (NBW) or LBW and were assessed at 28 (similar to end of infancy) and 56 (similar to childhood) days of age. LBW swine had lower absolute brain mass, but demonstrated evidence of brain sparing (increased brain mass scaled to body mass) at 56 days of age. The cerebral pulsatility index, based on transcranial Doppler, was increased in LBW swine. Moreover, arterial myography of isolated cerebral arteries revealed impaired vasoreactivity to bradykinin and reduced contribution of nitric oxide (NO) to vasorelaxation in the LBW swine. Immunoblotting demonstrated a lower ratio of phosphorylated-to-total endothelial NO synthase in LBW offspring. This impairment in NO signaling was greater at 28 vs. 56 days of age. Vasomotor responses to sodium nitroprusside (NO-donor) were unaltered, while Leu31, Pro34 neuropeptide Y-induced vasoconstriction was enhanced in LBW swine. Increases in total Y1 receptor protein content in the LBW group were not significant. In summary, LBW offspring displayed signs of cerebrovascular dysfunction at 28 and 56 days of age, evidenced by altered cerebral hemodynamics (reflective of increased impedance) coupled with endothelial dysfunction and altered vasomotor control. Overall, the data reveal that normal variance in birthweight of full-term offspring can influence cerebrovascular function later in life.

The Effects of Fermentation of Low or High Tannin Fava Bean-Based Diets on Glucose Response, Cardiovascular Function, and Fecal Bile Acid Excretion during a 28-Day Feeding Period in Dogs: Comparison with Commercial Diets with Normal vs. High Protein.

We have shown that feeding dogs fava bean (FB)-based diets for 7 days is safe and FB flour fermentation with Candida utilis has the potential to decrease FB anti-nutritional factors. In the present study, the effects of 28-day feeding of 4 different FB-based test dog foods containing moderate protein (~27% dry matter (DM)) were compared with two commercial diets with normal protein (NP, grain-containing, ~31% DM protein) or high protein (HP, grain-free, ~41% DM protein). Health parameters were investigated in beagles fed the NP or HP diets or using a randomized, crossover, 2 × 2 Latin square design of the FB diets: unfermented high-tannin (UF-HT), fermented high-tannin (FM-HT), unfermented low-tannin (UF-LT), and fermented low-tannin (FM-LT). The results showed that fermentation increased glucose tolerance, increased red blood cell numbers and increased systolic blood pressure, but decreased flow-mediated vasodilation. Taken together, the overall effect of fermentation appears to be beneficial and improved FB nutritional value. Most interesting, even though the HP diet was grain-free, the diet did contain added taurine, and no adverse effects on cardiac function were observed, while glucose tolerance was impaired compared to NP-fed dogs. In summary, this study did not find evidence of adverse cardiac effects of pulses in 'grain-free' diets, at least not in the relatively resistant beagle breed over a 28-day period. More importantly, fermentation with C. utilis shows promise to enhance health benefits of pulses such as FB in dog food.

Nesfatin-1-like peptide is a negative regulator of cardiovascular functions in zebrafish and goldfish.

Nucleobindins (NUCB1 and NUCB2) were originally identified as calcium and DNA binding proteins. Nesfatin-1 (NEFA/nucleobindin-2-Encoded Satiety and Fat-Influencing proteiN-1) is an 82 amino acid anorexigenic peptide encoded in the N-terminal region of NUCB2. We have shown that nesfatin-1 is a cardiosuppressor in zebrafish. Both NUCB1 and NUCB2 possess a -very highly conserved bioactive core. It was found that a nesfatin-1-like peptide (NLP) encoded in NUCB1 suppresses food intake in fish. In this research, we investigated whether NLP has nesfatin-1-like effects on cardiovascular functions. NUCB1/NLP-like immunoreactivity was found in the atrium and ventricle of the heart and skeletal muscle of zebrafish. Intraperitoneal injection (IP) of either zebrafish NLP or rat NLP suppressed cardiac functions in both zebrafish and goldfish. Irisin and RyR1b mRNA expression was downregulated by NLP in zebrafish cardiac and skeletal muscles. However, cardiac ATP2a2 mRNA expression was elevated after NLP injection. Administration of scrambled NLP did not affect irisin, RyR1b or ATP2a2 mRNA expression in zebrafish. Together, these results implicate NLP as a suppressor of cardiovascular physiology in zebrafish and goldfish.

Toxicokinetic Models for Bioconcentration of Organic Contaminants in Two Life Stages of White Sturgeon (Acipenser transmontanus).

The white sturgeon (Acipenser transmontanus) is an endangered ancient fish species that is known to be particularly sensitive to certain environmental contaminants, partly because of the uptake and subsequent toxicity of lipophilic pollutants prone to bioconcentration as a result of their high lipid content. To better understand the bioconcentration of organic contaminants in this species, toxicokinetic (TK) models were developed for the embryo-larval and subadult life stages. The embryo-larval model was designed as a one-compartment model and validated using whole-body measurements of benzo[a]pyrene (B[a]P) metabolites from a waterborne exposure to B[a]P. A physiologically based TK (PBTK) model was used for the subadult model. The predictive power of the subadult model was validated with an experimental data set of four chemicals. Results showed that the TK models could accurately predict the bioconcentration of organic contaminants for both life stages of white sturgeon within 1 order of magnitude of measured values. These models provide a tool to better understand the impact of environmental contaminants on the health and the survival of endangered white sturgeon populations.

The sympathetic/beta-adrenergic pathway mediates irisin regulation of cardiac functions in zebrafish.

Irisin is a 23 kDa myokine encoded in its precursor, fibronectin type III domain containing 5 (FNDC5). The exercise-induced increase in the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) promotes FNDC5 mRNA, followed by the proteolytic cleavage of FNDC5 to release irisin from the skeletal or cardiac muscle into the blood. Irisin is abundantly expressed in skeletal and cardiac muscle and plays an important role in feeding, modulates appetite regulatory peptides, and regulates cardiovascular functions in zebrafish. In order to determine the potential mechanisms of acute irisin effects, in this research, we explored whether adrenergic or muscarinic pathways mediate the cardiovascular effects of irisin. Propranolol (100 ng/g B·W) alone modulated cardiac functions, and when injected in combination with irisin (0.1 ng/g B·W) attenuated the effects of irisin in regulating cardiovascular functions in zebrafish at 15 min post-injection. Atropine (100 ng/g B·W) modulated cardiovascular physiology in the absence of irisin, while it was ineffective in influencing irisin-induced effects on cardiovascular functions in zebrafish. At 1 h post-injection, irisin downregulated PGC-1 alpha mRNA, myostatin-a and myostatin-b mRNA expression in zebrafish heart and skeletal muscle. Propranolol alone had no effect on the expression of these mRNAs in zebrafish and did not alter the irisin-induced changes in expression. At 1 h post-injection, irisin siRNA downregulated PGC-1 alpha, troponin C and troponin T2D mRNA expression, while upregulating myostatin a and b mRNA expression in zebrafish heart and skeletal muscle. Atropine alone had no effects on mRNA expression, and was unable to alter effects on mRNA expression of siRNA. Overall, this research identified a role for the sympathetic/beta-adrenergic pathway in regulating irisin effects on cardiovascular physiology and cardiac gene expression in zebrafish.

A Novel Multispecies Toxicokinetic Modeling Approach in Support of Chemical Risk Assessment.

Standardized laboratory tests with a limited number of model species are a key component of chemical risk assessments. These surrogate species cannot represent the entire diversity of native species, but there are practical and ethical objections against testing chemicals in a large variety of species. In previous research, we have developed a multispecies toxicokinetic model to extrapolate chemical bioconcentration across species by combining single-species physiologically based toxicokinetic (PBTK) models. This "top-down" approach was limited, however, by the availability of fully parameterized single-species models. Here, we present a "bottom-up" multispecies PBTK model based on available data from 69 freshwater fishes found in Canada. Monte Carlo-like simulations were performed using statistical distributions of model parameters derived from these data to predict steady-state bioconcentration factors (BCFs) for a set of well-studied chemicals. The distributions of predicted BCFs for 1,4-dichlorobenzene and dichlorodiphenyltrichloroethane largely overlapped those of empirical data, although a tendency existed toward overestimation of measured values. When expressed as means, predicted BCFs for 26 of 34 chemicals (82%) deviated by less than 10-fold from measured data, indicating an accuracy similar to that of previously published single-species models. This new model potentially enables more environmentally relevant predictions of bioconcentration in support of chemical risk assessments.

The Effect of Fermentation of High- or Low-Tannin Fava Bean on Glucose Tolerance, Body Weight, Cardiovascular Function, and Blood Parameters in Dogs After 7 Days of Feeding: Comparison With Commercial Diets With Normal vs. High Protein.

Fava bean, which is available in high- and low-tannin varieties, is not an approved pet food ingredient and was not included in the "assumed to be safe" category based on its ability to cause favism and hemolytic anemia in susceptible humans. The effects of 7-day feeding of test canine diets containing moderate protein (~27%) were compared with two control commercial diets with normal (NP, grain-containing, ~25% protein) or high protein (HP, grain-free, ~41% protein). Fava bean diets were formulated either with or without Candida utilis fermentation processing to reduce antinutritional factors. Glucose tolerance, body weight, cardiovascular function, and blood parameters were investigated in beagles fed the NP or HP diets or a randomized, crossover, 2 × 2 Latin square design of the fava bean diets: unfermented high-tannin (UF-HT), fermented high-tannin (FM-HT), unfermented low-tannin (UF-LT), and fermented low-tannin (FM-LT). After 7 days, HP decreased red blood cells (RBC) (P < 0.05) compared with NP, while FM increased RBC compared with UF. HP increased blood bicarbonate, calcium, phosphorus, urea, cholesterol, and albumin:globulin ratio while decreasing bilirubin, liver enzymes, and total protein. Sodium:potassium ratio was increased in UF-HT, decreased in FM-HT, and intermediate in LT regardless of fermentation. Blood phosphorus was increased in HT. Blood amylase was increased in FM-HT and decreased in FM-LT, being intermediate in UF regardless of fava bean variety. Blood direct bilirubin was decreased in HT regardless of fermentation. Of note, left ventricular end-systolic volume and cardiac output were increased in NP compared with HP-fed dogs, but were normal and had no significant differences among the fava bean diets. As expected, plasma taurine, cystine, and cysteine levels were increased in HP- compared with NP-fed dogs. Plasma cysteine levels were increased in HT- compared with LT-fed dogs and in FM- compared with UF-fed dogs. Taken together, these results show that fava bean appears to be safe as a dog food ingredient at least in the short term, and its nutritional value appears improved by fermentation. Moreover, blood chemistry parameters and cardiovascular function were impacted by protein content which merits further investigation with longer term feeding trials.