Effects of hydrolyzed chicken liver on digestibility, fecal and urinary characteristics, and fecal metabolites of adult dogs.

High-protein low-carbohydrate diets have been widely used for adult maintenance dogs, as well as in specific weight loss diets and maintenance programs. However, increasing dietary protein may increase undigested protein in the hindgut, modifying intestinal fermentation and fecal metabolite concentrations. The aim of this study was to evaluate the effects of protein source and concentration on apparent total tract digestibility (ATTD) of nutrients, metabolizable energy (ME), fecal and urinary characteristics, and fecal metabolites of dogs. Twelve healthy adult dogs were distributed into six treatments (n = 6 per diet) in a balanced incomplete Latin square design consisting of three periods of 30 days each. Six diets, varying in protein source [poultry byproduct meal (PBPM) and hydrolyzed chicken liver powder (HCLP)] and concentration [24, 32, and 40% crude protein (CP) on dry matter (DM) basis] were tested: PBPM24 (PBPM based diet with 24% CP); PBPM32 (PBPM with 32% CP); PBPM40 (PBPM with 40% CP); HCLP24 (HCLP based diet with 24% CP); HCLP32 (HCLP with 32% CP); HCLP40 (HCLP with 40% CP). The ATTD of CP was greater in dogs-fed HCLP and higher protein concentrations diets (P < 0.05). However, dogs-fed HCLP diets had lower ATTD of fat and carbohydrates, and ME (P < 0.05). Similarly, high-protein diets reduced the ATTD of DM, OM, fat, carbohydrates, and energy (P < 0.05). High-protein diets increased the daily fecal output and moisture (P = 0.004 and P < 0.05, respectively), as well as the fecal score (P < 0.0001), verified as soft, moist stools, but still within the ideal range. Fecal valerate concentration was greater in dogs-fed PBPM at 32% CP (P = 0.007). Fecal isobutyrate tended to increase in dogs-fed PBPM and high-protein diets (P < 0.10). Also, dogs-fed PBPM and high-protein diets had greater fecal concentrations of isovalerate, branched-chain fatty acids (BCFA), and ammonia (P < 0.05). Finally, the fecal lactate concentration increased in dogs-fed HCLP and high-protein diets (P < 0.05). The HCLP increased the ATTD of CP, being a highly digestible protein. Although the inclusion of HCLP slightly increased fecal score and moisture, it decreased the amount of fecal metabolites of protein fermentation ammonia and BCFA, both of which are associated with proteolytic fermentation in the colon.

Feeding companion animals with high-protein diets has been a demand of the market and pet owners. However, the protein quality and quantity consumed can interfere with the amount of undigested protein that reaches the hindgut and be fermented. Intestinal fermentation can be desired when well controlled. This study tested two protein sources (hydrolyzed chicken liver and poultry byproduct meal) combined at three dietary protein concentrations (24, 32, and 40% crude protein on dry matter basis) and their effects on the apparent total tract digestibility (ATTD), fecal and urinary characteristics, and fecal metabolites of healthy adult dogs. In summary, diets containing higher inclusion of hydrolyzed chicken liver had improved protein ATTD. However, the same diets impaired the ATTD of fat and carbohydrates and decreased metabolizable energy. High-protein diets retained more water in the feces and increased the fecal output. Fecal consistency was affected, scored as soft and moist stools, but remained within an acceptable score. Dogs-fed poultry byproduct meal diets had greater concentrations of fecal protein fermentation metabolites, such as ammonia and branched-chain fatty acids, possibly related to a greater amount of undigested protein that reached the hindgut and was fermented.

Effects of Diets Based on Hydrolyzed Chicken Liver and Different Protein Concentrations on the Formation and Deamination of Biogenic Amines and Total Antioxidant Capacity of Dogs.

Biogenic amines are synthesized through the bacterial decarboxylation of amino acids, commonly found in high levels in animal by-product meals due to spoilage. Furthermore, biogenic amines and other metabolites can be produced by the fermentation of proteins in the hindgut according to the protein source and concentration of crude protein (CP) in the diet. Thus, this study aimed to evaluate two protein sources (poultry by-product meal (PBPM) and hydrolyzed chicken liver powder (HCLP)) and three CP concentrations (24, 32, and 40%) and their effects on the consumption and fecal excretion of biogenic amines, plasma monoamine oxidase (MAO) and diamine oxidase (DAO) activities, and total antioxidant capacity (TAC) of healthy adult dogs after 30 days of feeding the experimental diets. Twelve dogs were randomly distributed into six treatments (n = 6/treatment): PBPM24 (PBPM with 24% CP); PBPM32 (PBPM with 32% CP); PBPM40 (PBPM with 40% CP); HCLP24 (HCLP with 24% CP); HCLP32 (HCLP with 32% CP); HCLP40 (HCLP with 40% CP). The PBPM and PBPM-based diets had higher concentrations of putrescine, cadaverine, tyramine, histamine, agmatine, and total biogenic amines. In contrast, HCLP and HCLP-based diets contained higher concentrations of spermidine, phenylethylamine, and spermine. The PBPM and PBPM-diets had higher biogenic amine index (BAI) indicating lower quality due to the high content of putrescine, cadaverine and tyramine. Dogs fed diets with PBPM and higher protein concentrations consumed more putrescine, cadaverine, tyramine, agmatine, and total amines (p < 0.0001), while dogs fed with HCLP consumed more spermidine, phenylethylamine, and spermine (p < 0.0001). Fecal excretion of phenylethylamine was greater in dogs fed HCLP32 and HCLP40 diets (p = 0.045). Dogs fed with HCLP tended to excrete more spermidine and tryptamine via feces, while higher protein concentrations tended to increase fecal excretion of cadaverine (p < 0.10). Plasma MAO activity was higher in dogs fed HCLP24 and PBPM32 diets (p = 0.024). The plasma activities of DAO and TAC were not different between diets (p > 0.05). Although we did not evaluate the intestinal activities of MAO and DAO, our results suggest that healthy adult dogs have an efficient deamination process on the gut epithelium.

Bisphenol A analogues induce a feed-forward estrogenic response in zebrafish.

Because exposure to bisphenol A (BPA) has been linked to health problems in humans and wildlife, BPA analogues have been synthesized to be considered as replacement molecules. We here have examined estrogenic activity of BPA and five of its analogues, BPAF, BPE, BPC, BPC-Cl, and BPS by a combination of zebrafish-based in vivo and in vitro assays. We used transgenic estrogen reporter (5xERE:GFP) fish to study agonistic effects of bisphenols. Exposures to BPA, BPAF, BPE, and BPC, induced GFP expression in estrogen reporter fish at low exposure concentrations in the heart valves and at higher concentrations in the liver, whereas BPC-Cl activated GFP expression mainly in the liver, and BPS faintly in the heart only. The in vivo response was compared to in vitro estrogenicity of bisphenol exposure using reporter cells that express the zebrafish estrogen receptors driving expression of an estrogen response element (ERE)-luciferase reporter. In these cells, BPA, BPAF, BPC, BPE and BPS preferentially activated Esr1, whereas BPC-Cl preferentially activated Esr2a. By quantitative PCR we found that exposure to BPAF induced expression of the classical estrogen target genes vtg1, esr1, and cyp19a1b in a concentration response manner, but the most responsive target gene was f13a1a. Exposure to BPC-Cl resulted in a different expression pattern of vtg1 and f13a1a with an activation at low concentrations, followed by a declining expression at higher concentrations. Because expression of f13a1a was strongly activated by all compounds tested, we suggest including this mRNA as a biomarker for estrogenicity in larval fish. We further showed that exposure to BPAF and BPC-Cl increased E2 levels in zebrafish larvae, indicating that bisphenol exposures result in a feed-forward response that can further augment the estrogenic activity of these compounds.

Hydrolyzed chicken liver used as single source of animal protein in diet and its effect on cytokines, immunoglobulins, and fecal microbiota profile of adult dogs.

Dogs with food allergies and enteropathies may require hydrolyzed diets to prevent or reduce clinical signs, therefore the protein sources used in these diets must be previously characterized and evaluated in healthy dogs. This study aimed to investigate the effects of a hydrolyzed chicken liver powder-based diet (HCLP) versus a poultry by-product meal and bovine meat and bone meal-based diet (Control), on complete blood count (CBC), cytokine, immunoglobulins responses (assessed on days 0, 15, 30 and 45), and fecal microbiota (assessed on day 45) in healthy adult dogs. The CBC did not differ between diets (P>0.05), remaining within reference range. Total plasma IL-4 concentrations were decreased over time independent of the dietary treatment (P<0.001). Total plasma IgA decreased on day 30 compared to days 0 and 45 in dogs fed the control diet (P<0.001). Total plasma IgE concentrations were reduced on days 30 and 45 in dogs fed the control diet, and on days 15 vs 30 and 15 vs 45 in dogs fed HCLP diet (P = 0.001). The 16S rRNA gene sequencing showed similar species richness and abundances of phyla and genera between diets (P>0.05). ß-diversity principal coordinate analysis plots demonstrated that HCLP group had a higher similarity than control. Based on our results, healthy adult dogs fed a HCLP based diet maintained normal values for hematological and immunological characteristics, and fecal microbiota after 45 days of feeding.

Adesão de mães de bebês prematuros com indicadores clínicos de saúde emocional à grupos psicoeducativos / Adherence of mothers of premature babies with clinical indicators of emotional health to psychoeducational groups / Adhesión de madres de prematuros con indicadores clínicos de salud emocional a grupos psicoeducativos

Diversos fatores podem interferir na saúde emocional materna e, dentre eles, o nascimento prematuro do bebê. Este estudo pretendeu: a) descrever e comparar a saúde emocional de mães de bebês prematuros que aderiram ou não a grupos psicoeducativos, com indicadores clínicos de ansiedade, estresse e depressão pós-parto e, b) identificar quais variáveis sociodemográficas influenciaram a saúde emocional das participantes de cada um dos grupos. A amostra foi composta por 42 mães de bebês prematuros que apresentaram indicadores clínicos de saúde emocional, que foi avaliada por meio de inventários de estresse, ansiedade e depressão. Elas compuseram dois grupos: 22 mães que participaram dos grupos e 20 que não participaram. Não foram observadas diferenças significativas entre as variáveis sociodemográficas e nem entre os indicadores de saúde emocional entre os dois grupos. Todavia, é importante identificar os indicadores emocionais clínicos de mães de bebês e oferecer intervenções para minimizá-los, enquanto medidas preventivas de saúde mental da população feminina e, consequentemente, infantil.

Several factors can interfere in maternal emotional health and, among them, the premature birth of the baby. This study aimed: a) to describe and compare the emotional health of mothers of premature babies who joined or not to psychoeducational groups, with clinical indicators of anxiety, stress and postpartum depression and, b) identify which sociodemographic variables influenced the emotional health of participants in each of the groups. The sample consisted of 42 mothers who presented clinical indicators of emotional health, which was assessed through inventories of stress, anxiety and depression. They composed two groups: 22 mothers who participated in the groups and 20 who did not. No significant differences were observed between sociodemographic variables or between emotional health indicators between the two groups. However, it is important to identify the clinical emotional indicators of mothers of premature babies and offer interventions to minimize them, as preventive measures for mental health in the female population and, consequently, in children.

Varios factores pueden interferir con la salud emocional materna y, entre ellos, el nacimiento prematuro del bebé. Este estudio tuvo como objetivo: a) describir y comparar la salud emocional de madres de bebés prematuros que ingresaron o no a grupos psicoeducativos, con indicadores clínicos de ansiedad, estrés y depresión posparto y, b) identificar qué variables sociodemográficas influyeron en la salud emocional de las madres en cada uno de los grupos. La muestra estuvo conformada por 42 madres de bebés prematuros que presentaron indicadores clínicos de salud emocional, la cual fue evaluada a través de inventarios de estrés, ansiedad y depresión. Compusieron dos grupos: 22 madres que participaron en los grupos y 20 que no. No se observaron diferencias significativas entre variables sociodemográficas ni entre indicadores de salud emocional entre los dos grupos. Sin embargo, es importante identificar los indicadores clínicos emocionales de las madres de bebés prematuros y ofrecer intervenciones para minimizarlos, como medidas preventivas para la salud mental de la población femenina y, en consecuencia, de los niños.

Lactose-free Dulce de leche: compositional characterization, browning and texture profile.

Our objective was to elaborate lactose-free Dulce de leche (DL) and evaluate the influence of the hydrolysis of this sugar on the attributes of the products. Fluid milk used was divided into two portions and, in one of them, enzymatic hydrolysis of lactose was carried through. Next, the homogenization of milk was performed at 20 MPa. Four different treatments were studied. The final products were evaluated in relation to their composition and physico-chemical characteristics. The main results show that the homogenized lactose-free DL obtained a higher concentration of free 5-Hydroxymethylfurfural (HMF) (133.77 ± 3.42 µmol l-1). Consequently, browning was more intense due to Maillard Reaction. Texture parameters were higher (1611.00 ± 598.78 g hardness and 19.52 ± 2.46 g gumminess) when compared to the homogenized traditional product (28.45 ± 1.16 µmol l-1 free HMF, 437.17 ± 279.3 g hardness, and 406.20 ± 311.69 g gumminess). Lactose-free products are in high demand by consumers; however, the results of this work highlight the challenges to properly control the browning and the texture parameters of DL.

Partial Substitution of Maize for Sorghum With or Without Supplemental Hydrolysable Tannins on Digestibility and Postprandial Glycemia in Adult Dogs.

The effect of partial substitution of maize for sorghum, containing condensed tannins (CT), with or without the addition of a purified hydrolysable tannin extract (HT), on dog apparent digestibility and glycemic response were evaluated. The trial was conducted with eight adult dogs distributed in four treatments: (M) 50% maize; (MS) 25% maize + 25% sorghum; (MHT) 50% maize + 0.10% HT; (MSHT) 25% maize + 25% sorghum + 0.10% HT; in a balanced incomplete Latin square design in three periods, with two dogs per diet, per period. Data were analyzed by ANOVA procedure and glycemic response by repeated measures ANOVA over time (P < 0.05). The phenolic compounds analyzed were not detected after extrusion process, with a reduction mainly in diets containing sorghum. There were no differences in the digestibility coefficients of nutrients and energy between the dietary treatments (P > 0.05). Fecal and urinary characteristics were not changed by the addition of sorghum and HT (P > 0.05). The fecal score remained within the ideal classification as hard, dry, firm stools. A moderate increase in fecal pH was observed on dogs fed diets containing sorghum (P = 0.0948). Additionally, the partial replacement of maize for sorghum associated or not with HT do not alter the glycemic aspects evaluated among dietary treatments (P > 0.05). Availability of nutrients from maize and sorghum were similar. Tannins did not interfere in the nutritional capacity of the ingredients.

Untargeted GC/MS-based approach for identification of anti-inflammatory alkaloids from Hippeastrum elegans (Amaryllidaceae) using a human neutrophil model.

Hippeastrum elegans is an Amaryllidaceae species producing alkaloids with pharmaceutical potential including lycorine and galanthamine. Herein, we developed a non-targeted metabolomic study associated to chemometrics and biological evaluations to identify the H. elegans constituents that were able to reduce the human neutrophils proinflammatory mechanisms. The alkaloid fractions were extracted from bulbs cultivated for 15 months (m) and harvested in six harvest periods (5, 7, 9, 11, 13, and 15 m). The GC-MS analysis allowed the detection of 41 alkaloids being 31 identified. All alkaloid components varied over the cultivation and most of them were lycorine-type skeletons. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) distinguished three groups according to the chemical profile (group I: 5, 7, and 9 m; group II: 11 m and group III: 13 and 15 m). Therefore, the biological assays were only performed with one of the representative samples of each group: 7 m, 11 m and 15 m. None of them was toxic to human neutrophils by LDH activity and MTT test. The 7 m and 15 m-alkaloid fractions showed anti-inflammatory effects by reducing human neutrophil degranulation. However, the former one was more effective in inhibiting the cell activation based on the reduction of myeloperoxidase (MPO) release and reactive oxygen species (ROS) production. Afterwards, Partial Least Squares analysis (PLS) indicated lycorine and 11,12-dehydro-2-methoxy-assoanine as the compounds responsible for the anti-inflammatory activity of the bioactive fraction. Thus, the 7 m-alkaloid fraction of H. elegans seems to be a promising anti-inflammatory drug that deserves additional research.

Human milk contamination by nine organochlorine pesticide residues (OCPs).

Organochlorine pesticides (OCPs) are widely used around the world as insecticides, herbicides, fungicides, nematicides, and rodenticides. Despite banned in Brazil, the usage remains occurring in many countries. The persistence and extreme mobility of OCPs contribute to the contamination of the environment and the human body. The OCPs bioaccumulation in adipose tissue triggers the excretion into human milk during breastfeeding. Hence, the present study determined eighteen OCPs residues in the breast milk of mothers from the Western Region of Bahia State, Brazil. Nine different residue species were found, including beta-Hexachlorocyclohexane (9.24 ± 0.00 ng g-1 fat), delta- Hexachlorocyclohexane (22.15 ± 10.48 ng g-1 fat), Heptachlor (58.08 ± 74.13 ng g-1 fat), Aldrin (142.65 ± 50.65 ng g-1 fat), Dieldrin (774.62 ± 472.68 ng g-1 fat), Endosulfan I (408.44 ± 245.51 ng g-1 fat), Dichloro-diphenyl-dichloro-ethylene (29.17 ± 22.42 ng g-1 fat), Dichloro-diphenyl-trichloro-ethane (28.87 ± 0.00 ng g-1 fat) and Methoxychlor (1699.67 ± 797.43 ng g-1 fat). The Methoxychlor presence in all samples may reveal a recent exposure, while Dieldrin and Endosulfan I analyses can point to distant past exposure.

Evaluation of teeth injuries in Beagle dogs caused by autoclaved beef bones used as a chewing item to remove dental calculus.

Dental calculus (DC) is the most widespread oral problem in domestic dogs. Chewing items are used to remove DC from the tooth surface; they also favor oral health and animal welfare. Raw beef bone mastication also shortly reduces DC in adult dogs. However, it can cause oral lesions and hence is not popular. This study evaluated the impact of bone mastication on the dental roots, enamel, and gingiva of dogs. Twelve adult Beagle dogs were randomly divided into 2 treatment groups in a completely randomized block design: cortical bone (CB) or spongy bone (SB). Intraoral radiographs were obtained on days 0 and 14, and calculus assessment was performed using images captured on days 0, 3, 6, 9, 12, and 14; an integration program was used to measure the proportion between the area covered by calculus and the total area of teeth. DC was completely removed from the first and second premolars and molars from both the arcades in less than 3 days of supplementation, indicating that these teeth were frequently used for chewing (P < 0.10). Bones were highly effective for DC removal and gingival inflammation reduction. Despite the hardness of bones, no lesions or teeth root and enamel fracture, or esophageal or intestinal obstructions-complications related to bone ingestion-were noted. However, SB showed some gingival lesions (n = 4) and bone remnants between teeth (n = 2). Gingival lesions were caused by the daily and continuous supply of new pieces of bone for 13 days. Specific pieces of bone should be used for oral home care programs because they shortly remove almost 90% of DC, allowing longer intervals between periodontal cleaning procedures. Long-term studies are required to evaluate the use of bones and evaluate their impact on teeth and periodontium after prolonged supplementation.

High-throughput screening tools facilitate calculation of a combined exposure-bioactivity index for chemicals with endocrine activity.

High-throughput and computational tools provide a new opportunity to calculate combined bioactivity of exposure to diverse chemicals acting through a common mechanism. We used high throughput in vitro bioactivity data and exposure predictions from the U.S. EPA's Toxicity and Exposure Forecaster (ToxCast and ExpoCast) to estimate combined estrogen receptor (ER) agonist activity of non-pharmaceutical chemical exposures for the general U.S. population. High-throughput toxicokinetic (HTTK) data provide conversion factors that relate bioactive concentrations measured in vitro (µM), to predicted population geometric mean exposure rates (mg/kg/day). These data were available for 22 chemicals with ER agonist activity and were estimated for other ER bioactive chemicals based on the geometric mean of HTTK values across chemicals. For each chemical, ER bioactivity across ToxCast assays was compared to predicted population geometric mean exposure at different levels of in vitro potency and model certainty. Dose additivity was assumed in calculating a Combined Exposure-Bioactivity Index (CEBI), the sum of exposure/bioactivity ratios. Combined estrogen bioactivity was also calculated in terms of the percent maximum bioactivity of chemical mixtures in human plasma using a concentration-addition model. Estimated CEBIs vary greatly depending on assumptions used for exposure and bioactivity. In general, CEBI values were <1 when using median of the estimated general population chemical intake rates, while CEBI were ≥1 when using the upper 95th confidence bound for those same intake rates for all chemicals. Concentration-addition model predictions of mixture bioactivity yield comparable results. Based on current in vitro bioactivity data, HTTK methods, and exposure models, combined exposure scenarios sufficient to influence estrogen bioactivity in the general population cannot be ruled out. Future improvements in screening methods and computational models could reduce uncertainty and better inform the potential combined effects of estrogenic chemicals.

Differential activity of BPA, BPAF and BPC on zebrafish estrogen receptors in vitro and in vivo.

The high volume production compound bisphenol A (BPA) is of environmental concern largely because of its estrogenic activity. Consequently, BPA analogues have been synthesized to be considered as replacement molecules for BPA. These analogues need to be thoroughly evaluated for their estrogenic activity. Here, we combined mechanism zebrafish-based assays to examine estrogenic and anti-estrogenic activities of BPA and two of its analogues, bisphenol AF (BPAF) and bisphenol C (BPC) in vitro and in vivo. In vitro reporter cell lines were used to investigate agonistic and antagonistic effects of the three bisphenols on the three zebrafish estrogen receptors. The transgenic Tg(5 × ERE:GFP) and Cyp19a1b-GFP zebrafish lines were then used to analyze estrogenic and anti-estrogenic responses of the three bisphenols in vivo. BPA, BPAF and BPC were agonists with different potencies for the three zebrafish estrogen receptors in vitro. The potent zfERα-mediated activity of BPA and BPAF in vitro resulted in vivo by activation of GFP expression in zebrafish larvae in the heart (zfERα-dependent) at lower concentrations, and in the liver (zfERß-dependent) at higher concentrations. BPC induced zfERß-mediated luciferase expression in vitro, and the zfERß agonism led to activation of GFP expression in the liver and the brain in vivo. In addition, BPC acted as a full antagonist on zfERα, and completely inhibited estrogen-induced GFP expression in the heart of the zebrafish larvae. To summarize, applying a combination of zebrafish-based in vitro and in vivo methods to evaluate bisphenol analogues for estrogenic activity will facilitate the prioritization of these chemicals for further analysis in higher vertebrates as well as the risk assessment in humans.

An Approach for Using In Vitro and In Silico Data to Identify Pharmaceuticals with Potential (Anti-)Estrogenic Activity in Aquatic Vertebrates at Environmentally Relevant Concentrations.

Endocrine-active pharmaceuticals can cause adverse reproductive and developmental effects in nontarget organisms. Aquatic vertebrates may be susceptible to the effects of such pharmaceuticals given that the structure of hormone receptors and the physiology of the endocrine system are highly conserved across vertebrates. To aid in the regulatory review of the environmental impact of drugs, we demonstrate an approach to screen and support the prioritization of pharmaceuticals based on their ability to interact with estrogen receptors (ERs) at environmentally relevant concentrations. Tox21 in vitro results from ER agonist and antagonist assays were retrieved for 1123 pharmaceuticals. In silico predictions from the Collaborative Estrogen Receptor Activity Prediction Project (CERAPP) models were used to estimate ER agonist and antagonist activity for an additional 170 pharmaceuticals not tested in the Tox21 assay platform. The estrogenic effect ratio (EER) and anti-estrogenic effect ratio (AEER) were calculated by comparing the activity concentration at half-maximal response (AC50) for ER agonism and antagonism, respectively, with estimated pharmaceutical concentrations in fish tissue based on estimates of environmental exposures. A total of 73 and 127 pharmaceuticals were identified as ER agonists and antagonists, respectively. As expected, 17ß-estradiol and 17α-ethinylestradiol displayed EERs > 1, and raloxifene and bazedoxifene acetate displayed AEERs > 1, thus indicating that these pharmaceuticals have the potential to reach fish tissue levels that exceed concentrations estimated to interact with ERs. Four pharmaceuticals displayed EERs between 0.1 and 1, and 6 displayed AEERs between 0.1 and 1. This approach may help determine the need for submission of environmental assessment data for new drug applications and support prioritization of pharmaceuticals with the potential to disrupt endocrine signaling in vertebrates. Environ Toxicol Chem 2019;38:2154-2168. © 2019 SETAC.

Effect of partial substitution of rice with sorghum and inclusion of hydrolyzable tannins on digestibility and postprandial glycemia in adult dogs.

Sorghum is used as a substitute for rice in dog food, owing to its nutritional similarity and low cost. However, its use has been associated with negative effects, like a reduction in palatability, digestibility, and enzyme activity, which can decrease nutrient absorption. The presence of condensed tannins (CT) in sorghum may cause these effects. Another tannin group, the hydrolysable tannins (HT), is known for its antioxidant properties. Research has shown the nutritional effects of sorghum on dogs, but the effect of HT on dogs remains unknown. We evaluated the effects of substituting rice with sorghum containing CT and inclusion of commercial extract of HT on digestibility, fecal and urinary characteristics, and postprandial blood glucose levels in adult dogs. Eight adult Beagle were randomly subjected to 4 treatments: (R) 50% rice; (RS) 25% rice + 25% sorghum; (RHT) 50% rice + 0.10% HT; (RSHT) 25% rice + 25% sorghum + 0,10% HT. Tannins did not affect food intake. The digestibility of dry matter, organic matter, crude protein (CP), acid hydrolyzed fat, gross energy, and metabolizable energy (ME) decreased with sorghum inclusion (P < 0.05). Greater fecal dry matter was observed with the RHT diet. HT associated with sorghum, RSHT diet, reduced ME (P < 0.05). Sorghum inclusion enhanced fecal output, without altering fecal score (P > 0.05). No alterations in urinary characteristics were observed. Sorghum and HT did not affect the postprandial blood glucose response measured by the area under the curve (P > 0.05). The substitution of rice by sorghum decreased CP digestibility and ME of the diets. Sorghum can be considered as a source of carbohydrates with lower digestibility of protein and energy than rice. HT may potentiate the effect of CT, but more research is needed to evaluate its potential use in dog nutrition.

Identification of candidate reference chemicals for in vitro steroidogenesis assays.

The Endocrine Disruptor Screening Program (EDSP) is transitioning from traditional testing methods to integrating ToxCast/Tox21 in vitro high-throughput screening assays for identifying chemicals with endocrine bioactivity. The ToxCast high-throughput H295R steroidogenesis assay may potentially replace the low-throughput assays currently used in the EDSP Tier 1 battery to detect chemicals that alter the synthesis of androgens and estrogens. Herein, we describe an approach for identifying in vitro candidate reference chemicals that affect the production of androgens and estrogens in models of steroidogenesis. Candidate reference chemicals were identified from a review of H295R and gonad-derived in vitro assays used in methods validation and published in the scientific literature. A total of 29 chemicals affecting androgen and estrogen levels satisfied all criteria for positive reference chemicals, while an additional set of 21 and 15 chemicals partially fulfilled criteria for positive reference chemicals for androgens and estrogens, respectively. The identified chemicals included pesticides, pharmaceuticals, industrial and naturally-occurring chemicals with the capability to increase or decrease the levels of the sex hormones in vitro. Additionally, 14 and 15 compounds were identified as potential negative reference chemicals for effects on androgens and estrogens, respectively. These candidate reference chemicals will be informative for performance-based validation of in vitro steroidogenesis models.

Prediction of Estrogenic Bioactivity of Environmental Chemical Metabolites.

The US Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program (EDSP) is using in vitro data generated from ToxCast/Tox21 high-throughput screening assays to assess the endocrine activity of environmental chemicals. Considering that in vitro assays may have limited metabolic capacity, inactive chemicals that are biotransformed into metabolites with endocrine bioactivity may be missed for further screening and testing. Therefore, there is a value in developing novel approaches to account for metabolism and endocrine activity of both parent chemicals and their associated metabolites. We used commercially available software to predict metabolites of 50 parent compounds, out of which 38 chemicals are known to have estrogenic metabolites, and 12 compounds and their metabolites are negative for estrogenic activity. Three ER QSAR models were used to determine potential estrogen bioactivity of the parent compounds and predicted metabolites, the outputs of the models were averaged, and the chemicals were then ranked based on the total estrogenicity of the parent chemical and metabolites. The metabolite prediction software correctly identified known estrogenic metabolites for 26 out of 27 parent chemicals with associated metabolite data, and 39 out of 46 estrogenic metabolites were predicted as potential biotransformation products derived from the parent chemical. The QSAR models estimated stronger estrogenic activity for the majority of the known estrogenic metabolites compared to their parent chemicals. Finally, the three models identified a similar set of parent compounds as top ranked chemicals based on the estrogenicity of putative metabolites. This proposed in silico approach is an inexpensive and rapid strategy for the detection of chemicals with estrogenic metabolites and may reduce potential false negative results from in vitro assays.

Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model.

In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative dose-response information on estrogenic activity of chemical compounds.

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development.

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development.

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors.

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERß), whereas the zebrafish genome encodes three ERs, zfERα and two zfERßs (zfERß1 and zfERß2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28°C as compared to 37°C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERß selective agonists displayed greater potency for zfERα as compared to zfERßs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology.

Halogenated bisphenol-A analogs act as obesogens in zebrafish larvae (Danio rerio).

Obesity has increased dramatically over the past decades, reaching epidemic proportions. The reasons are likely multifactorial. One of the suggested causes is the accelerated exposure to obesity-inducing chemicals (obesogens). However, out of the tens of thousands of industrial chemicals humans are exposed to, very few have been tested for their obesogenic potential, mostly due to the limited availability of appropriate in vivo screening models. In this study, we investigated whether two commonly used flame retardants, the halogenated bisphenol-A (BPA) analogs tetrabromobisphenol-A (TBBPA) and tetrachlorobisphenol-A (TCBPA), could act as obesogens using zebrafish larvae as an in vivo animal model. The effect of embryonic exposure to these chemicals on lipid accumulation was analyzed by Oil Red-O staining, and correlated to their capacity to activate human and zebrafish peroxisome proliferator-activated receptor gamma (PPARγ) in zebrafish and in reporter cell lines. Then, the metabolic fate of TBBPA and TCBPA in zebrafish larvae was analyzed by high-performance liquid chromatography (HPLC) . TBBPA and TCBPA were readily taken up by the fish embryo and both compounds were biotransformed to sulfate-conjugated metabolites. Both halogenated-BPAs, as well as TBBPA-sulfate induced lipid accumulation in zebrafish larvae. TBBPA and TCBPA also induced late-onset weight gain in juvenile zebrafish. These effects correlated to their capacity to act as zebrafish PPARγ agonists. Screening of chemicals for inherent obesogenic capacities through the zebrafish lipid accumulation model could facilitate prioritizing chemicals for further investigations in rodents, and ultimately, help protect humans from exposure to environmental obesogens.