Progress 08/01/02 to 07/31/07

Outputs
The results of this project showing that the safe, GRAS-listed phenolic food antioxidants significantly reduce aflatoxicosis in turkeys were communicated in a variety of ways. A feature article in was published in the popular science journal "Science News" entitled "Fowl News - Food Additive's Extra Benefit. This article can be viewed at: http://www.sciencenews.org/articles/20050326/food.asp. Several presentations to producer groups were conducted. One recent example was an invited presentation on the topic of reducing the impact of aflatoxin to poultry by chemoprevention and genomics at the National Poultry Breeder's Roundtable May 2007 in St. Louis, MO. Chemoprevention technology developed through this project was presented at a symposium entitled "Neutraceuticals as Double-Edged Swords: Weighing Benefits and Risks of Dietary Chemicals to Human and Animal Health,"at the Society of Toxicology Annual Meeting in Baltimore, MD, March 2004. Communicating results from this project to producer, commodity and other groups in foreign countries also occurred. For example, scientists, representatives of commodity groups, and animal producers were given a presentation entitled "Dietary Cancer Chemoprevention," at the CSIRO Livestock Industries, Australian Animal Health Laboratory, Geelong, Victoria, Australia, August 2003. The P.I. was also invited to present a week-long short course entitled: "Food and Cancer: Molecular mechanisms, biomarkers and prevention," at the Food & Health Research Centre, University of Kuopio, Kuopio Finland, August-September 2005. In attendence were producers, dieticians, and scientists from througout the Euopean Union. The P.I. established collaborations with several European animal health researchers to determine if food-grade antioxidants protect other breeds of poultry and other food animals against AFB1 and other mycotoxins. Furthermore, spin-off projects with European food companies have been intitated to explore other potential chemopreventive agents in the turkey model.

Impacts
The principal outcomes of this project were: 1) the biochemical and molecular reasons for the extreme hypersensitivity of turkeys to the mycotoxin aflatoxin B1 (AFB1) were determined; 2) the turkey as a model for human cancer susceptibility was developed and validated; 3) the demonstration that safe, GRAS-listed phenolic antioxidants prevent aflatoxicosis in poultry resulting in a sigificant improvement in animal health; 4) the mechanism of chemoprevention by antioxidants occurrs through competitive inhibiton of hepatic bioactivation of AFB1; this makes possible the development of other chemopreventives; 5) through support of this project, the first functional proteins from turkeys were sequenced, cloned, expressed and characterized; the amplification technology developed from these studies has been used in various applications in human and veterinary health studies; 6) we determined that phenolic antioxidants, such as butylated hydroxytoluene, are safe to use as chemopreventives, and that residual carry-over into animal products are at least 100-fold less than residue limits set by the U.S. Food and Drug Administration; 7) the results of this project will result in a safer food for consumers because antioxidants also significantly reduce residues of AFB1 in turkey tissues, such as meat, liver, and blood.

Publications

• Guarisco, J.A., Hall, J.O., and Coulombe, R. A. 2007. Butylated hydroxytoluene reduces aflatoxin B1 bioavailability and hepatic adduct formation in turkeys. Poisonous Plants: Global Research and Solutions 197-202.
• Coulombe, R.A., Guarisco, J.A., Klein, P.J. and J.O. Hall. 2005. Chemoprevention of aflatoxicosis in poultry by dietary butylated hydroxytoluene. Animal Feed Science and Technology 121:217-225.
• Klein, P.J., Hall, J.O. and R. A. Coulombe, Jr. 2003. Dietary butulated hydroxytoluene protects against aflatoxicosis in turkeys. In, Poisonous Plants and Related Toxins (T. Acamovic, C.S. Stewart and T.W. Pennycott eds.), pp. 478-48.
• Coulombe, R.A., Klein, P.J. and J.O. Hall. 2003. Butylated hydroxytoluene chemoprotection: Response to Williams. Toxicology and Applied Pharmacology 189:152.
• Klein, P.J. Van Vleet, T.R., Hall, J.O. and R. A. Coulombe, Jr. 2003. Effect of dietary buylated hydroxytoluene on aflatoxin B1-relevant metabolic enzymes in turkeys. Food and Chemical Toxicology 41:671-678.
• Van Vleet, T R, Mace, K and R. A. Coulombe, Jr. 2002. Comparative Aflatoxin B1 Activation and Cytotoxicity in Human Bronchial Cells Expressing Human CYPs 1A2 and 3A4. Cancer Research 62:105-112.
• Van Vleet, T.R., Klein, P.J. and R. A. Coulombe, Jr. 2002. Metabolism and cytotoxicity of aflatoxin B1 in cytochrome P-450-expressing human lung cells. J. Toxicology and Environmental Health, Part A, 65:853-67.

Progress 01/01/06 to 12/31/06

Outputs
In the final phase of this project, we established the safety and the minimum AFB1-chemoprotective concentrations of antioxidants such as butylated hydroxytoluene (BHT). In animals given AFB1 (1 ppm), clinical signs of aflatoxicosis (body weight, serum enzymes, hepatocellular necrosis, and biliary hyperplasia) were brought to control values (no AFB) in the > 100, > 200, > 500, and > 2000 ppm BHT + AFB1 groups, respectively, indicating that BHT chemoprotection is dose-responsive, and that minimal chemprotective concentration for some signs of aflatoxicosis is as little as 100 ppm. We also found that at the highest dietary concentration of BHT tested (4000 ppm), residual concentrations in the lipid fractions of liver and breast meat were < 5 ppm, which is substantially below the FDA standard of 200 ppm, an important prerequisite to FDA approval. Therefore, even at high dietary concentrations, carry-over of BHT into turkey products is negligible. Additionally, ong-term feeding of BHT appears to be safe for turkeys. In a 60 day feeding trial, body and liver weights and overall clinical and serological signs of health were not significantly different from control birds. Finally, we found that BHT must be present continuously in the diet to confer protection against AFB1, and that the period of residual protection against aflatoxicosis is approximately one week. In total, our studies have established the usefulness and safety of BHT and other related phenolic antioxidants as an effective aflatoxin chemopreventive in turkeys.

Impacts
Turkeys are the most susceptible animals to the mycotoxin aflatoxin B1. Our discovery of several GRAS-listed antioxidants that dramatically protect turkeys against dietary AFB1 should help reduce the estimated $100 million annual loss due to this mycotoxin. Antioxidant chemoprotection will also help poultry producers be more productive, and to produce a safer food for the consumer.

Publications

• Reed, K.M., Mendoza, K.M. and R. A. Coulombe, Jr. (2007). Structure and genetic mapping of the Cytochrome P4501A5 gene in the turkey (Meleagris gallopavo). Cytogenetics and Genome Research 116: DOI:10.1159/000097426 (in press).
• Yip, S.S.M. and R.A. Coulombe, Jr. (2006) Molecular cloning and expression of a novel cytochrome P450 from turkey liver with aflatoxin B1 metabolizing activity. Chemical Research in Toxicology 19:30-37.
• Coulombe, R.A. and S.M. Yip. (2006) Cloning, Expression and Partial Characterization of a Novel Cytochrome P450 from Turkey Liver that Catalyzes Epoxidation of Aflatoxin B1. Toxicological Sciences 90 (1): 1832.
• Guarisco, J.A., Hall, J.O., and R.A. Coulombe, Jr. (2006) Butylated hydroxytoluene reduces aflatoxin B1 bioavailability and hepatic adduct formation in turkeys. In Poisonous Plants: Global Research and Solutions (K. Panter, T. Wierenga, J. Phister, eds.) CAB International, London. (in press).

Progress 01/01/05 to 12/31/05

Outputs
We have used turkey cytochrome P4501A5 (P4501A5) which we have recently cloned and expressed (to our knowledge, the first catalytically-active enzyme to be cloned and expressed from turkeys), as a molecular tool to determine the chemoprotective mechanism of the antioxidant butylated hydroxytoluene (BHT) as well as to discover other potential protective agents. We conclusively demonstrated that BHT is a competitive inhibitor of P4501A5-mediated bioactivation of aflatoxin B1 (Ki = 0.98 uM), the probable mechanism of chemoprotection by this antioxidant. In additon to BHT, other FDA-approved antioxidants ethoxyquin (EQ) and butylated hydroxytoluene BHA were potent inhibitors of P4501A5-related enzyme activity with a rank order of EQ >>> BHT > BHA. EQ was approximately 50-fold more potent inhibitor of P4501A5 catalysis activity than either BHT and BHA. Based on these results, we predict that like BHT, EQ and BHA will be potent chemoprotective agents in turkeys.

Impacts
Turkeys are the most susceptible animals to aflatoxin B1. Our discovery of safe chemoprotective antioxidants should help reduce the estimated $100 million annual loss due to this mycotoxin. It will also help poultry producers be more productive, and to produce a safer food for the consumer.

Publications

• Yip, S.S.M. and Coulombe, R.A., Jr. (2005) Molecular cloning and expression of a novel cytochrome P450 from turkey liver with aflatoxin B1 metabolizing activity. Chemical Research in Toxicology. (in press).
• Van Vleet, T.R., Watterson, T.L., Klein, P.J., and R.A. Coulombe, Jr. (2005). Aflatoxin B1 alters the expression of p53 in cytochrome P450-expressing cells. Toxicological Sciences. (in press).

Progress 01/01/04 to 12/31/04

Outputs
Because of the central role of the turkey cytochrome P450 (CYP) 1A homologue in the hypersensitivity of aflatoxin B1 (AFB1) as well as its chemoprevention by antioxidants like butylated hydroxytoluene (BHT), we cloned this isoform from turkey liver polyA+ RNA. Using the 3' and 5' RACE technique with primers designed using multiple sequence alignments for mammalian and avian CYP 1A isoforms, we amplified cDNA for a turkey CYP 1A homologue. A primer set that amplified the entire coding region of turkey CYP 1A produced a 1587 bp cDNA coding for a 528 amino acid protein with amino acid and nucleotide percentage identity 95% homologous to a chicken CYP 1A5. A truncated construct with the 5'-end membrane anchoring signal removed was subcloned into the pCW bicistronic expression vector which contains the redox partner human NADPH-CYP reductase gene immediately downstream of the 1A5 cloning site. Clones of the 28 aa truncated mutant constructs expressed 250 nM/L of spectrally-active CYP, possessed methoxyresorufin O-demethylase (MROD) activity (prototype activity for CYP 1A enzymes. Importantly, the gene product catalyzed the metabolism of AFB1 forming both the activated epoxide AFBO and AFM1, both of which are critical to animal disease caused by this mycotoxin. The role of this homologue in the hypersensitivity of poultry to AFB1 and its chemoprevention will be confirmed in upcoming immuno-inhibition studies in turkey liver microsomes with polyclonal serum made to the expressed gene product. To our knowledge, this is the first CYP cloned from turkey, an important food animal that is hypersensitive to AFB1 and other toxins activated by CYP.

Impacts
Our project has identified safe feed additives that can actually protect animals against the adverse health effects of feed-borne toxins such as aflatoxin. Our research will help reduce losses associated with mycotoxins in poultry feeds thereby helping the poultry industry to be more productive and to produce safer food for consumers.

Publications

• Coulombe, R.A., Guarisco, J.A., Klein, P.J. and J.O. Hall (2004) Chemoprevention of aflatoxicosis in poultry by dietary butylated hydroxytoluene. Animal Feed Science and Technology (in press).
• Coulombe, R.A., Guarisco, J.A., and J.O. Hall (2004). Chemoprevention by dietary butylated hydroxytoluene: inhibtion of AFB1 activation and AFB1-DNA adducts in turkeys. Toxicology and Applied Pharmacology 197, 3 258.

Progress 01/01/03 to 12/31/03

Outputs
Aflatoxin B1 (AFB1) is the most important mycotoxin in occurrence and toxic potency, and poultry, especially turkeys, are the most susceptible food animals. Even trace amounts of AFB1 in poultry feed cause reductions in growth rate, feed efficiency, hatchability and increased susceptibility to diseases. The annual economic impact of aflatoxin-related diseases to the poultry industry is considerable--estimated to exceed $150 million. We previously demonstrated that several GRAS-listed antioxidants, such as butylated hydroxytoluene (BHT), when added to feed, prevent nearly all signs of aflatoxicosis in turkeys. In experiments to determine the mechanism of chemoprevention, we discovered that BHT inhibits cytochrome P-450 (CYP) the hepatic enzyme in turkeys that activates AFB1 to AFB1-epoxide (the DNA-reactive intermediate responsible for the toxic and cancer-causing action of AFB1) by classic Michaelis competitive kinetics (Ki= 5.3 mM). The principal CYP isoform responsible for AFB1 activation is a 1A2 homologue. Livers from BHT turkeys were significantly less active in converting AFB1 to the AFB1-epoxide than control. BHT significantly increased expression of hepatic glutathione S-transferases (GSTs), but not isoforms that detoxify the AFB1-epoxide. Thus, the mechanism of chemoprotection by BHT is likely mediated by inhibition of the activating hepatic CYP in turkey liver, rather than through induction of protective GSTs as we previously thought. Since AFB1 and other mycotoxins are unavoidable in feeds, discovering ways of protecting turkeys through simple dietary intervention with safe chemoprotective agents represents a practical management strategy.

Impacts
Our research will help to significantly reduce the losses associated with mycotoxins in poultry feeds thereby helping the poultry industry to be more productive and to produce safer food for consumers

Publications

• Klein, P.J. Van Vleet, T.R., Hall, J.O. and R. A. Coulombe, Jr. (2003). Effect of dietary buylated hydroxytoluene on aflatoxin B1-relevant metabolic enzymes in turkeys. Food and Chemical Toxicology 41:671-678.
• Coulombe, R.A., Klein, P.J. and J.O. Hall (2003). Butylated hydroxytoluene chemoprotection in poultry. Toxicology and Applied Pharmacology 189:152.
• Klein, P.J., Hall, J.O. and R. A. Coulombe, Jr. (2003). Dietary butulated hydroxytoluene protects against aflatoxicosis in turkeys, in Poisonous Plants and Related Toxins (T. Acamovic, C.S. Stewart and T.W. Pennycott eds.) CAB International, London pp. 478-483.

Progress 01/01/02 to 12/31/02

Outputs
We have recently determined that the molecular mechanisms underlying the extreme susceptibility of poultry to the toxic effects of aflatoxicosis involve a combination of efficient cytochrome P-450 (CYP)-mediated activation and deficient glutathione S-transferase (GST)-mediated detoxification of aflatoxin B1 (AFB1) in turkey liver. We disproved our initial hypothesis that the powerful chemoprotective properties of BHT is through induction of GSTs. While BHT induces several isoforms of alpha and mu-class GST homologues in turkey liver (usually indicative of chemoprotection in mammals), BHT did not induce an isoform with affinity specifically toward microsomally-generated AFB1-8,9-epoxide (the "gold-standard" of AFB1-relevant GST activity). After discovering that GSTs are irrelevant to the mechanism of BHT protection, we abandoned our aim of purifying hepatic GSTs turkeys, and turned our attention to discovering other possible mechanisms. We found that microsomal AFB1 activation in livers from BHT-fed turkeys was significantly lower compared that from controls. BHT also significantly decreased microsomal metabolism of 7-ethoxyresorufin, methoxyresorufin, prototype substrates for CYP 1A and 1A2, respectfully. These findings strongly indicate that BHT protects turkeys by reducing hepatic formation of the toxic AFB1-8,9-epoxide. Preliminary experiments indicate that BHT acts as a competitive inhibitor of CYP- mediated activation of AFB1.

Impacts
Chemoprotection is one of the most promising strategies to reduce cancer incidence in people. Our discovery that BHT protects against nearly all symptoms of aflatoxicosis demonstrates that the time is right to adapt chemoprotection strategies to protect the health of food animals. Our work should identify several treatment strategies that will benefit American agriculture through a) reductions in losses associated with mycotoxins in poultry feeds; b) helping the poultry industry to be more productive; and c) producing a safer food for consumers.

Publications

• No publications reported this period