ASSESSMENT OF MITOCHONDRIAL STRUCTURE, FUNCTION AND ANTIOXIDANT DEFENSE MECHANISMS OF INFLAMMATION-INDUCED OXIDATIVE STRESS IN POULTRY FED ALLOPURINOL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004970
• Project Start Date: Nov 5, 2014
• Project End Date: Nov 4, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
WEST VIRGINIA UNIVERSITY
886 CHESTNUT RIDGE RD RM 202
MORGANTOWN,WV 26505-2742

Performing Department
Animal & Veterinary Science

Non Technical Summary
This proposal will strengthen the collective knowledge of mitochondrial structure and function in avian species, particularly poultry. At this time, very little research has been completed in chickens concerning mitochondria and the relationship to health status. Subpopulations of cardiac mitochondria have not yet been determined in birds, therefore, this will be the first attempt to understand cardiac function at a cellular level of an aging animal throughout its production cycle. If this project is sucessful, it will be posible to contribute to an understanding of how cellular function and immune response effect the overall health status in normal production settings, but additionally providing a deeper understanding of pathogen response. Uric acid is suggested to be the most important factor in the amelioration of oxidative burden in birds. Results from numerous studies have shown a correlation between the increase in plasma uric acid concentrations and the reduction in oxidative stress. In view of this observation, further research is critical for understanding the regulation of uric acid production in health and disease with the view to reduce the oxidative burden.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3299	1020	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1020 - Physiology;

Keywords

oxidative stress

inflammation

uric acid

poultry

production cycles

Goals / Objectives
The Project Personnel propose to perform studies that will determine mitochondrial structure and function during normal and induced oxidative stress states in broiler chickens and laying hens throughout their individual production cycles. The primary objective of our research is to link plasma and tissue uric acid concentrations in poultry undergoing oxidative stress, and to identify relative differences in mitochondrial function and inflammation in selected tissues under conditions of oxidative stress and uric acid supplementation. The long-term goal of this research proposal is to elucidate the efficacy of uric acid as a protector against oxidative stress. Oxidative stress plays an important role in inflammation. It is in the purpose of these studies to determine to what extent inflammation is contributing to their production cycles.

Project Methods
Research Design and MethodsBroiler StudiesStudy 1AThis study will a preliminary understanding of the effect of allopurinol on mitochondrial function. The purpose of this study is to ascertain if there is an effect of acute treatment with allopurinol on mitochondrial function in heart and liver tissues. Broilers (4 wks of age) will be separated into two treatments: control (n=12) which will be fed a commercial grower diet and treatment(n=12) which will be fed an addition of allopurinol (30mg/kg body weight) for no less than one week. At this time half of the birds will be euthanized. Heart and liver tissues will be taken for analysis of State III and State IV mitochondrial respiration using polarography. The remaining birds will be administered allopurinol for an additional week before euthanasia and the same analysis procedures. This will allow a better understanding of the effect of chronic uric acid depression on mitochondrial function in these tissues. Allopurinol inhibits xanthine oxidase thereby inhibiting uric acid production and increasing oxidative stress in chickens as evidenced by several studies. Birds will be given allopurinol (30mg/kg body weight) in the feed to induce oxidative stress. Plasma uric acid will be measured to validate the effect of allopurinol. Heart and liver tissue will be collected from birds immediately after euthanasia. Total mitochondria will be isolated from fresh tissue using a commercial kit (Biovision mitochondrial/cytosolic fractionation kit). State III (active) and State IV (resting) respiration will be measured using polarography. This measurement will help to determine the function of the mitochondria. The cytosolic fraction will be analyzed for purine markers (xanthine, hypoxanthine, and uric acid) using high performance liquid chromatography (HPLC). Weight gain, feed intake, and feed conversion will be used to assess performance of the birds in all studies.Study 2This study will focus on inflammation in liver tissue. Liver mitochondria from Study 1 show a significant reduction in State III (p= 0.01) and State IV (p= 0.007) respiration as illustrated in Figure 1, when the birds are fed allopurinol which suggests dysfunction of the mitocondria. There was no difference in respiration of cardiac mitochondria between the control and treated groups. The question remains as to why we are seeing this difference. It is hypothesized that the liver is under oxidative stress due to inflammation. It is established in the literature that mitochondria will become dysfunctional in inflammatory responses and in oxidative stress situations. Broiler chickens (5wks) will be fed allopurinol for two weeks in this study at a dose of 35mg/kg BW as in previous studies. Liver will be removed after two weeks of treatment and analyzed, using real time RT-PCR, for oxidative stress and inflammatory cytokines (IL-6, IL-12-P35, IL-1β, and IL-8). Inducible nitric oxide synthase (iNOS) will also be measured in this study to establish inflammation and immune response. Uric acid, SOD, catalase, and GPX activity or presence should be measured in this tissue or plasma as well to establish a reduction in antioxidant defense. Total antioxidant capacity will be measured. Isolated mitochondria from study 2A from heart and liver tissue will be assessed for superoxide radical production and possible hydroxyl radical production. More experimentation will be needed here to ascertain if this measurement can be done using electron spin resonance (ESR). Preliminary results from study 1b suggest that heart tissue can be analyzed in this manner, but further work is needed.Study 2b:This study will look at the parameters described above over a period of time which simulates chronic oxidative stress . Broilers (n=40) will be placed on a dose of allopurinol at 35mg/kg BW beginning at 1 week of age and ending at 6 weeks of age or on a comercial control diet. At 2, 4, and 6 weeks of age, a selected group of birds will be euthanized and the liver will be removed for analysis of inflammation, total antioxidant activity and tissue pentodine. This will provide an understanding of chronic oxidative stress over time and how this effects the liver.Study 2c:This study will focus on the alleviation of oxidative stress by feeding inosine, a precursor to uric acid in the purine degradation pathway. It has been shown that a dose of 161g/kg feed will increase uric acid concentrations in both plasma and tissues (Settle et al.,2012). When allopurinol and inosine are fed in a mixture, it has been seen that while there is an increase in plasma uric acid, it is still significantly decreased when compared to an inosine fed animal (Table 1). This suggests that there is still oxidative damage to tissues. With this model, inflammation, iNOS, nitrites, and antioxidant activity will be assessed in a group of 20 broilers. The birds (n=5) will be placed on one of four treatments: comercial control diet, inosine only, inosine in a mixture with allopurinol (35mg/kg BW), and a group that is pair-fed with the inosine birds to confirm an effect of inosine and not feed intake. The diets will be fed for one week. All birds will be euthanized and tissues will be removed for analysis.Laying Hen StudiesMitochondrial function, to our knowledge, has not been determined for laying hens at any stage of production. Hens (n = 5/group) will be randomly selected from a flock of 375 birds every 12 weeks beginning at 8 weeks of age and ending at 104 weeks. It is hypothesized that mitochondrial function will different at the different stages of production of the bird due to ageing and stresses from production cycles. Performance of the hens will be assessed in terms of growth, feed intake, and feed conversion before egg laying and once they begin to lay eggs. This will give an overview of mitochondrial function throughout the lifespan of a laying hen undergoing several dietary and management changes from pullet to a hen at the end of her production. Blood samples will be obtained at each time point for determination of oxidative stress by measuring leukocyte oxidative activity and plasma and tissue concentrations of UA, nitrite content, and total antioxidant capacity as well as selected mitochondrial antioxidants (SOD, catalase, and GPX). Immediately afterwards the birds will be killed and samples of liver, kidney, intestine and heart removed. Total mitochondria and mitochondrial subpopulations will be isolated from fresh tissue. State III and State IV respiration will be assessed. Structure will be assessed using for the cardiac subpopulations using flow cytometry. These studies will be through years 1-2 and will finish analysis during year 2 and 3

Progress 11/05/14 to 11/04/19

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Diminished levels of urate have been linked to oxidative stress in avians and mammals. Urate, a major antioxidant that lowers reactive oxygen/nitrogen species (ROS/RNS), is thought to ameliorate the effects. Research studiesexamined the effect of CLA and berberine supplementation on markers of oxidative-stress in poultry. The sphingolipid ceramide has alsobeen linked to oxidative stress as markers in mammals; however, the relationship between ceramide supply and oxidative stress in broiler chickens is not understood. Therefore, we employed a lipidomic approach to investigate the changes in circulating ceramides in context of oxidative stress in birds. Overall, total and saturated ceramides were significantly decreased overtime in the healthy growing chicks. In treatments effects, total and saturated ceramides decreased with allopurinol supplementation. Notably, plasma ceramides C16:0 and 18:0 did not follow the overall trend. The use of allopurinol to induce this state might have exacerbated the liver, primary organ for lipid synthesis in birds, hence affecting the results. Results also showed that there was a 10-fold increase in the relative mRNA expression of superoxide dismutase 2 and 3 as well as glutathione peroxidase 1 and 3 in CLA+ALLO and BRB+ALLO treatment groups. Notably, CLA increased the expression of uncoupling protein 19-fold compared to control while the addition of allopurinol blocked these changes. In contrast, there was a slight increase in the uncoupling protein in the BRB+ALLO treatment. Despite the increase in mRNA expression of the antioxidants genes, these results suggest that at the dosages administered, CLA and berberine were not effective in reversing the oxidative stress induced by allopurinol. The relationship between reduced urate, the immune system and the pathogenesis of the intestine, the liver or the brain is not well characterized in avians. Moreover, recent studies have proposed that an increased permeability in the intestine due to some insult can induce inflammation in peripheral organs such as the brain. The results of our studies showed that urate was reduced in all treatment groups regardless of time, treatment groups exhibited reduced amounts of bile acids throughout time and female treatment groups exhibited increased amounts of LDH and AST over time.

Publications

• Type: Journal Articles Status: Other Year Published: 2020 Citation: Plasma ceramides as potential markers of oxidative stress in broiler chickens. Vincent Dartigue, Zach Phipps, Eduardo Rico, Joseph McFadden, Hillar Klandorf. The effects of conjugated linoleic acid and berberine supplementation on markers of allopurinol activated oxidative stress in broiler chickens Vincent Dartigue, Knox Van Dyke, Hillar Klandorf. Allopurinol alters the expression of cytokines and mediators of immune cells in the systemic tissue that are associated with the inflammasome by decreasing plasma urate. Lundrim Marku, Natalie Green, Brooke Zedar, Melissa Trani, Jianbo Yao*, Ashok Bidwai, Hillar Klandorf

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Uric acid has crucial roles in orchestrating Th2 (T-helper cell 2) responses, which mediate reactive oxygen and nitrogen species, and are associated with acute and chronic inflammation. Allopurinol, a purine analogue, serves as a xanthine oxidase inhibitor which when introducted intothe diet generates an inflamed state that is characterized by decreased plasma uric acid along with increased in nitrotyrosylated cellular proteins. Although we understand the involvement of uric acid in the balance of oxidants and antioxidants, much less is known about theinnate immunes system and Th2 response in uricoteles. Our current invesigation is measuring the expression profiles of gut, liver and cerebral structural and restorative genes, specifically, in Gallus gallus domesticus. Liver panel blood chemistries, quantitative PCR, immunohistochemistry and fluorescence microscopy are being used to determine the effects of allopurinol on systemic tissue, the innate immune system, and the Th2 response. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student training How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Data is currnetnly being collected and analyzed

Impacts
What was accomplished under these goals? Liver panel blood chemistries, quantitative PCR, immunohistochemistry and fluorescence microscopy are being analyzed to visualize and measure the effects of allopurinol on systemic tissue, the innate immune system, and the Th2 response.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Reactive oxidative species (ROS) are oxygen base molecules that are leaked from the electron transport chain during mitochondrial cellular respiration. They are kept in balance by endogenous and exogenous antioxidants, which may include conjugated linoleic acid (CLA) and a plant root extract called berberine. A redox shift towards oxidants causes a state called oxidative stress, which is detrimental if prolonged. A ten week study examined the effect CLA and berberine supplementation on markers of oxidative stress in broiler birds. Broilers (n=60) were divided into six groups; the diet of group four to six included allopurinol (25mg/kg body weight) which induced an oxidative stress state. Pen 1(P1) served as the control, P2 CLA oil (50% substitution), P3 berberine supplementation (20g/kg feed), P4 allopurinol alone, P5 CLA oil + allopurinol and P6 berberine + allopurinol. Body weight, plasma uric acid as a marker of oxidative stress, blood glucose, lipid peroxidation via TBARs assay of serum, and relative gene expression of five endogenous liver antioxidants were measured during the course of the study. The addition of allopurinol to the diet induced an oxidative stress state when compared to the baseline groups as observed by a significant reduction in plasma uric acid. In P5 and P6 treatment, we observed on average a 10 fold increase in the relative mRNA expression of superoxide dismutase 2 and 3 as well as glutathione 3 and 7. Notably, CLA increased the expression of uncoupling protein 34 fold compared to control while the addition of allopurinol blocked these changes; in contrast there was a slight increase in the berberine+allopurinol treatment. Despite the increase in mRNA expression of the antioxidants genes, the result of the uric acids assay suggest that at the dosages administered CLA and berberine were not effective in reversing the oxidative stress induced by allopurinol.

Publications

• Type: Other Status: Published Year Published: 2018 Citation: The Effects of Conjugated Linoleic Acid and Berberine Supplementation on Markers of Oxidative Stress in Broiler Chickens. Exptl. Biol. (2018)

Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Reactive oxidative species (ROS) are oxygen base molecules that are leaked from the electron transport chain during mitochondrial cellular respiration. They are kept in balance by endogenous and exogenous antioxidants, which may include conjugated linoleic acid (CLA) and a plant root extract called berberine. A redox shift towards oxidants causes a state called oxidative stress, which is detrimental if prolonged. A ten week study examined the effect CLA and berberine supplementation on markers of oxidative stress in broiler birds. Broilers (n=60) were divided into six groups; the diet of group four to six included allopurinol (25mg/kg body weight) which induced an oxidative stress state. Pen 1(P1) served as the control, P2 CLA oil (50% substitution), P3 berberine supplementation (20g/kg feed), P4 allopurinol alone, P5 CLA oil + allopurinol and P6 berberine + allopurinol. Body weight, plasma uric acid as a marker of oxidative stress, blood glucose, lipid peroxidation via TBARs assay of serum, and relative gene expression of five endogenous liver antioxidants were measured during the course of the study. The addition of allopurinol to the diet induced an oxidative stress state when compared to the baseline groups as observed by a significant reduction in plasma uric acid. In P5 and P6 treatment, we observed on average a 10 fold increase in the relative mRNA expression of superoxide dismutase 2 and 3 as well as glutathione 3 and 7. Notably, CLA increased the expression of uncoupling protein 34 fold compared to control while the addition of allopurinol blocked these changes; in contrast there was a slight increase in the berberine+allopurinol treatment. Despite the increase in mRNA expression of the antioxidants genes, the result of the uric acids assay suggest that at the dosages administered CLA and berberine were not effective in reversing the oxidative stress induced by allopurinol.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Vincent Dartigue, Hillar Klandorf*, Elizabeth Falkenstein, Knox Van Dyke. The Effects of Conjugated Linoleic Acid and Berberine Supplementation on Markers of Oxidative Stress in Broiler Chickens.

Progress 11/05/14 to 09/30/15

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Tabatha Settle,my PhD student,obtained a research position at NIOSH in Morgantown, WV. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Birds have a remarkable longevity for their body size despite an increased body temperature, higher metabolic rate, and increased blood glucose concentrations compared to most mammals. As the end-product of purine degradation, uric acid (UA) is generated in the xanthine/hypoxanthine reactions catalyzed by xanthine oxidoreductase (XOR). In the first study, Cobb × Cobb broilers (n = 12; 4 weeks old) were separated into 2 treatments (n = 6); control (CON) and allopurinol (AL) 35 mg/kg BW (ALLO). The purpose of this study was to assess mitochondrial function in broiler chickens in response to potential oxidative stress generated from the administration of AL for 1 wk. There was a significant reduction in state 3 respiration (P = 0.01) and state 4 respiration (P = 0.007) in AL-treated birds compared to the controls. The purpose of the second study was to assess the effect of AL on gene expression of inflammatory cytokines interferon-γ (IFN)-γ, IL-1β, IL-6, and IL-12p35, as well as inducible nitric oxide synthase and XOR in liver tissue. Cobb × Cobb broilers were separated into two groups at 4 wk age (n = 10); CON and ALLO. After 1 wk AL treatment, half of the birds in each group (CON 1 and ALLO 1) were euthanized while the remaining birds continued on AL treatment for an additional week (CON 2 and ALLO 2). A significant increase in gene expression of XOR, IFN-γ, IL-1β, and IL-12p35 in ALLO 2 birds as compared to birds in CON 2 was detected. Liver UA content was significantly decreased in both ALLO 1(P = 0.003) and ALLO 2 (P = 0.012) birds when compared to CON 1 and CON 2, respectively. The AL reduced liver UA concentrations and increased expression of inflammatory cytokines. Additional studies are needed to determine if AL causes a direct effect on mitochondria or if mitochondrial dysfunction observed in liver mitochondria was due indirectly through increased oxidative stress or increased inflammation.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Settle, T. Falkenstein, E, and Klandorf, H. Effect of allopurinol and inosine administration on xanthine oxidoreductase gene expression in selected tissues of broiler chickens. Int. J. Poult. Sci. 2015;14:37-43.