Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: Transcriptome analysis of the remaining turkey liver mRNA samples from the chemoprevention trial were processed and assembled with VELVET software resulting in assemblies of contigs averaging just over 230 bp. Queries with this sequence set have identified over 13,000 unique hits to the chicken Reference mRNA sequence gene set. Partitioning of the dataset has identified unique sets of genes corresponding to the treatment group. Preliminary analysis of the liver transcriptome of AFB1-challenged birds indicates up regulation of key MHC and other genes indicative of the inflammatory response. We are completing this analysis using the turkey reference gene set and are developing a turkey-specific analysis pipeline to support additional transcriptome studies. PARTICIPANTS: Moroni Feed, Inc. Jeff Hall, USU; Bill Helferich, University of Illinois; Dave Williams, Oregon State University; Len Bjeldanes, University of California; Tiffany Weir, Colorado State University, Marie-Louise Ricketts, University of Nevada, Reno; Pratibha Nerurkar, University of Hawaii; Jim Pestka, Michigan State University; Mike Denison, University of California; Ron Riley, USDA-ARS Athens, GA; Hani El-Nezami, University of Hong Kong, Paul Turner, University of Leeds; Rami Dalloul, Virginia Tech; Valio LTD, Helsinki, Finland. TARGET AUDIENCES: Consumers, poultry producers, food processors, food safety specialists, veterinary health professionals, government regulators, food companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
"1. Our findings will bring the promise of sustainable, safe food supplements with cancer fighting capabilities nearer to reality. Chemopreventive dietary factors such as antioxidants, and probiotic Lactobacillus have already achieved a high degree of consumer acceptance, and its use as a safe cancer preventive shows great potential. 2. Other possible impacts resulting from this research will be an improved understanding of the mechanisms responsible for the beneficial effects of cancer chemopreventives such as probiotics. This knowledge will help form the basis for an informed evolution of dietary habits to improve health. Because probiotic Lactobacillus can be incorporated into dairy fermented dairy products such as yogurt, another potential outcome of this work will be to stimulate the small farm economy and that of dairy producers by increasing markets for their products."
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The combined presence of 1A5 and 3A37, which obey different kinetic models, both of which metabolize AFB1 to the exo-AFBO and to detoxification products aflatoxin M1 (AFM1) and aflatoxin Q1 (AFQ1), respectively, complicates the kinetic analysis of AFB1 in turkey liver microsomes (TLMs). Antisera directed against 1A5 and 3A37, thereby individually removing the catalytic contribution of these enzymes, were used to identify the P450 responsible for epoxidating AFB1in TLMs. In control TLMs, AFB1 was converted to exo-AFBO in addition to AFM1 and AFQ1 confirming the presence of functional 1A5 and 3A37. Pretreatment with anti-1A5 inhibited exo-AFBO formation, especially at low, submicromolar (~0.1μM), while anti-3A37, resulted in inhibition of exo-AFBO formation, but at higher (>50μM) AFB1 concentrations. Metabolism in immunoinhibited TLMs resembled that of individual enzymes: 1A5 produced exo-AFBO and AFM1, conforming to Michaelis-Menten, while 3A37 produced exo-AFBO and AFQ1 following the kinetic Hill equation. At 0.1μM AFB1, close to concentrations in livers of exposed animals, 1A5 contributed to 98% of the total exo-AFBO formation. At this concentration, 1A5 accounted for a higher activation:detoxification (50:1, exo-AFBO: AFM1) compared to 3A37 (0.15: 1, exo-AFBO: AFQ1), suggesting that 1A5 is high, while 3A4 is the low affinity enzyme in turkey liver. The data support the conclusion that P450 1A5 is the dominant enzyme responsible for AFB1 bioactivation and metabolism at environmentally-relevant AFB1 concentrations in turkey liver. PARTICIPANTS: Moroni Feed, Inc. Jeff Hall, USU; Bill Helferich, University of Illinois; Dave Williams, Oregon State University; Len Bjeldanes, University of California; Tiffany Weir, Colorado State University, Marie-Louise Ricketts, University of Nevada, Reno; Pratibha Nerurkar, University of Hawaii; Jim Pestka, Michigan State University; Mike Denison, University of California; Ron Riley, USDA-ARS Athens, GA; Hani El-Nezami, University of Hong Kong, Paul Turner, University of Leeds; Rami Dalloul, Virginia Tech; Valio LTD, Helsinki, Finland. TARGET AUDIENCES: Consumers, poultry producers, food processors, food safety specialists, veterinary health professionals, government regulators, food companies. PROJECT MODIFICATIONS: Due to the accelerated destruction of poultry facility located at the N. Logan veterinary science farm, and delay of construction of replacement facilities, chemoprevention and intervention trials were put on hold this year.
Impacts
1. Cloning and functionally characterizing genes that dictate sensitivity to dietary carcinogens is an important step in the development of our animal model for human cancer sensitivity, which may help shed light on the molecular basis of cancer susceptibility in people. 2. Results of this work will not only define the liver transcriptome of the turkey but aid in the annotation of genes in the turkey whole genome sequence. 3. We filed for a provisional patent on our technology to develop a diagnostic SNP-based molecular marker kit to identify disease resistance in poultry
Publications
- UTAO+476 Baldwin, T. T., Riley, R., Zitomer, N. C., Voss, K., Coulombe, R. A., Pestka, J., Williams, D., & Glenn, A. E., (2011). The current state of mycotoxin biomarker development in humans and animals and the potential for application to plant systems: World Mycotoxin Forum Journal, 4(3): 257-270. (Published).
- UTAO+476 Rawal, S., & Coulombe, R. A., (2011). Metabolism of Aflatoxin B1 in Turkey Liver Microsomes: The Relative Roles of Cytochromes P450 1A5 and 3A34: Toxicology and Applied Pharmacology, 254(3): 349-354. (Published).
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: We conducted a trail to see whether probiotic Lactobacillus (LGG) would protect against symptoms of aflatoxicosis in turkeys in a 28-day protocol. In this study, 10-day old male Nicholas strain turkeys were randomized into one of four groups: LGG only, LGG+AFB1, PBS control, and AFB1 only. Birds were pretreated with LGG (5 X 10 CFU/0.5 ml PBS) or PBS by oral gavage for the first 10 days, before starting a 10-day dietary AFB1 treatment (1 ppm). Chemoprevention by LGG was most evident when body weights were considered. The LGG + AFB1-treated group showed significantly higher body and liver weights than the AFB1 only group. We are using RNA-Seq approaches to characterize the transcriptome level changes in the liver of birds exposed to AFB1 with and without probiotics (Lactobacilli) to investigate the response to aflatoxin exposure. To date we have sequenced RNA pools from four treatment groups (AFB1-treated, Lactobacillus-treated, AFB1+Lactobacillus and control) using the Illumina GAIIx sequencer at the Mayo Clinic. Gene expression changes are being quantified in response to aflatoxin, the probiotic and to both together in order to isolate the compensatory effects of the probiotic on aflatoxin exposure. Preliminary results indicate an immune system response as part of AFB1 exposure. Given this observed role of inflammation in this process, we have expanded our study to include the RNA-seq analysis of the spleen transcriptome. To date we have constructed 12 libraries from 3 individuals each from the four treatment groups described above. Libraries were pooled and sequenced on the GAIIx system at the University of Minnesota. Since each library was individually bar coded, sequences can be sorted by individual allowing for statistical analysis of the immune response. Sequencing has been completed and is awaiting analysis. PARTICIPANTS: Moroni Feed, Inc. Jeff Hall, USU; Bill Helferich, University of Illinois; George Bailey, Oregon State University; Len Bjeldanes, University of California; Pratibha Nerurkar, University of Hawaii; Jim Pestka, Michigan State University; Mike Denison, University of California; Ron Riley, USDA-ARS Athens, GA; Hani El-Nezami, University of Hong Kong, Paul Turner, University of Leeds; Rami Dalloul, Virginia Tech; Valio LTD, Helsinki, Finland. TARGET AUDIENCES: Consumers, poultry producers, food processors, food safety specialists, veterinary health professionals, government regulators, food companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. Our results showing a positive and profound protective effect of Lactobacillus representas an important confirmation that this intervention is successful in turkeys, the most susceptible animal known to the toxic effects of AFB1. These results have lead the way to the planning of clinical trials. 2. Our findings will bring the promise of sustainable, safe food supplements with cancer fighting capabilities nearer to reality. Lactobacillus has already achieved a high degree of consumer acceptance, and its use as a safe cancer preventive shows great potential. 3. Other possible impacts that will directly result from this research will be an improved understanding of the mechanisms responsible for the beneficial effects of cancer chemopreventives such as probiotics. This knowledge will help form the basis for an informed evolution of dietary habits to improve health. Because probiotic Lactobacillus can be incorporated into dairy fermented dairy products such as yogurt, another potential outcome of this work will be to stimulate the small farm economy and that of dairy producers by increasing markets for their products. 4. Results of this work will not only define the liver transcriptome of the turkey but aid in the annotation of genes in the turkey whole genome sequence.
Publications
- Dalloul, R., & Coulombe, R. A., 2010. Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo) genome assembly and analysis. PLoS Biology, 8(9): 1-21.
- Rawal, S., Kim, J., & Coulombe, R. A., 2010. Aflatoxin in poultry: metabolism, toxicity, and prevention: Research in Veterinary Science, 89: 325-331.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: In rodent models and in three field trials, probiotic bacteria such as Lactobacillus, the type used in fermented foods such as yogurt, has been shown to prevent the cancer-causing ability of several food-borne carcinogens. Combinations of different probiotic bacteria are often used by the food industry as biopreservatives, making them a suitable candidate to study their chemopreventive action. The presumed mechanism of action is non-specific carcinogen binding of cell wall proteins which reduces intestinal absorption and hence cancer risk. In collaboration with investigators at the University of Kuopio Finland, the University of Leeds, and the University of Hong Kong, we explored whether probiotic Lactobacillus (LGG) would protect against symptoms of aflatoxicosis in turkeys in our 28-day protocol which we have used to identify the chemopreventive properties of phenolic antioxidants. In this study, 10-day old male Nicholas strain turkeys were randomized into one of four groups: LGG only, LGG+AFB1, PBS control, and AFB1 only. Birds were pretreated with LGG (5 X 10 CFU/0.5 ml PBS) or PBS by oral gavage for the first 10 days, before starting a 10-day dietary AFB1 treatment (1 ppm). Chemoprevention by LGG was most evident when body weights were considered. The LGG + AFB1-treated group showed significantly higher body and liver weights than the AFB1 only group. Serum AFB1-lysine adducts were reduced in the LGG + AFB1 group compared to the AFB1 only group, but this was not statistically significant. Similarly, a serum panel reflecting hepatotoxicity (ALP, GGT, total protein, total albumin) and incidence of hepatic histopathologic effects (biliary hyperplasia and hepatic necrosis) showed no protective trend of LGG. The results of a full transcriptome analysis from birds in this study is pending. PARTICIPANTS: Moroni Feed, Inc.; Jeff Hall, USU; Bill Helferich, University of Illinois; George Bailey, Oregon State University; Len Bjeldanes, University of California; Pratibha Nerurkar, University of Hawaii; Jim Pestka, Michigan State University; Mike Denison, University of California; Ron Riley, USDA-ARS Athens, GA; Hani El-Nezami, University of Hong Kong, Paul Turner, University of Leeds; Rami Dalloul, Virginia Tech; Valio LTD, Helsinki, Finland. TARGET AUDIENCES: Consumers, poultry producers, food processors, food safety specialists, veterinary health professionals, government regulators, food companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Although the US food supply continues to be reasonably safe, diet is now known to be an important factor in our most prevalent chronic diseases. Estimates suggest that diet is responsible for between 35-80% of human cancer incidence. Furthermore, genetics appear to play a smaller role in human cancer than once thought. Thus, identifying potential beneficial dietary components holds great promise in reducing human suffering and the great financial burden of associated health care, and represents the foremost potential impact of this research. Other possible impacts that will directly result from this research will be an improved understanding of the mechanisms responsible for the beneficial effects of cancer chemopreventives such as probiotics. This knowledge will help form the basis for an informed evolution of dietary habits to improve health. Because probiotic Lactobacillus can be incorporated into dairy fermented dairy products such as yogurt, another potential outcome of this work will be to stimulate the small farm economy and that of dairy producers by increasing markets for their products. While body weights are an important endpoint for aflatoxicosis, our data seems to indicate that LGG was unable to protect against the high dietary AFB1 concentration in our research protocol.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Estimates suggest that diet is responsible for between 35-80% of human cancer incidence. Consequently, the overall goal of this research is to improve food safety, and reduce the incidence of diet-related cancer by discovering natural and synthetic chemopreventives in foods, and to determine their mechanism of action. Another output is to adapt and validate models to study the cancer process in people. PARTICIPANTS: Moroni Feed, Inc. Jeff Hall, USU; Bill Helferich, University of Illinois; George Bailey, Oregon State University; Len Bjeldanes, University of California; Pratibha Nerurkar, University of Hawaii; Jim Pestka, Michigan State University; Mike Denison, University of California; Ron Riley, USDA-ARS Athens, GA. TARGET AUDIENCES: Consumers, poultry producers, food processors, food safety specialists, veterinary health professionals, government regulators, food companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have shown that butylated hydroxytoluene (BHT), at 1000 and 4000 ppm, protects against aflatoxicosis in turkeys through mechanisms that include competitive inhibition of P450-mediated AFB1 bioactivation. We wished to determine whether the observed chemoprotection is dose-related, and also determine the minimum chemopreventive concentration of dietary BHT. In this study, the diets of 10-day-old male turkey were supplemented with BHT (100, 200, 500, 1000, 2000, and 4000 ppm) for 10 days, then AFB1 (1 ppm) was added to the diets and continued for another 10 days, then the birds were sampled. The AFB1-induced reduction of liver weight and liver/body weight ratio was reversed in ameliorated in the > 1000 ppm BHT groups, while 2000 and 4000 ppm BHT protected against AFB1-induced increases in serum enzymes aspartate aminotransferase (AST), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH). Biliary hyperplasia was reduced in > 500 ppm groups, and there was also a decreasing incidence of hepatocellular necrosis at concentrations > 500 ppm. The data indicate that BHT chemoprevention in turkeys is dose-responsive, and that the minimal measurable concentration for protection against some signs of aflatoxicosis is as low as 100 ppm.
Publications
- Coulombe, R. 2007. GenBank Accession DQ450083. Cytochrome P450 3A37 from Melaegris gallopavo.
- Carlisle, N. and Rosetta, L. 2007. Meth cops swear they can sweat off toxins. Salt Lake Tribune, Salt Lake City, UT.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Estimates suggest that diet is responsible for between 35-80% of human cancer incidence. Consequently, the overall goal of this research is to improve food safety, and reduce the incidence of diet-related cancer by discovering natural and synthetic chemopreventives in foods, and to determine their mechanism of action. Another goal is to adapt and validate models to study the cancer process in people. We determined the mechanism by which dietary antioxidants, specifically butylated hydroxytoluene, profoundly prevents aflatoxicosis in turkeys. affects excretion and residual aflatoxin B1 in various tissues. Turkeys were pre-treated with dietary BHT (4000 ppm, 10 days), then given radiolabeled AFB1 by oral gavage, and sampled at intervals up to 24 hr thereafter. As a requirement for eventual FDA approval of cancer chemopreventives, we also conduced a long-term safety trail in which turkeys were fed BHT for up to 40 days.
PARTICIPANTS: Moroni Feed, Inc. Jeff Hall, USU; Bill Helferich, University of Illinois; George Bailey, Oregon State University; Len Bjeldanes, University of California; Pratibha Nerurkar, University of Hawaii; Jim Pestka, Michigan State University; Mike Denison, University of California; Ron Riley, USDA-ARS Athens, GA.
TARGET AUDIENCES: Consumers, poultry producers, food processors, food safety specialists, veterinary health professionals, government regulators, food companies.
Impacts
Dietary BHT inhibits the metabolic conversion of AFB1 to the reactive intermediate AFB1-8,-9-epoxide (AFBO), exhibiting Michaelis-Menton competitive inhibition kinetics (Ki = 0.81 uM). Likewise, livers from BHT-fed turkeys were significantly less active in AFBO formation compared to those from control birds. When turkeys were fed BHT for up to 40 days, residual BHT was present in liver, breast meat, thigh meat, and abdominal fat in concentrations substantially below U.S. FDA guidelines for this antioxidant, but in concentrations greater than the Ki, likely sufficient to inhibit bioactivation of AFB1 in vivo. BHT-induced hydropic degeneration in the livers of BHT fed animals was significantly greater in birds that remained on BHT treatment for up to 30 days, but this lesion diminished in animals fed for 40 days or when returned to a control diet. The data indicate that the observed chemopreventive properties of BHT in turkeys may be due, at least in part, to its ability
to inhibit hepatic AFB1 epoxidation, and also that the BHT-induced hydropic degeneration is reversible, and does not appear to cause long-term effects. These data will be useful when FDA approval for antioxidants as disease a preventive is sought. Other potential impacts include improved animal health, a safer food product for consumers, and incresed profitability to the producer.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Poultry, especially turkeys, are extremely sensitive to the mycotoxin aflatoxin B1 (AFB1). Turkeys are hypersensitive to AFB1 because of a combination of extremely efficient hepatic bioactivation, and deficient detoxification of AFB1. In turkeys, AFB1 is enzymatically bioactivated by cytochrome P450 (CYP) 1A5, and to a minor degree, by CYP3A homologues of unknown identity. Using 3', 5' RACE, we cloned and sequenced two CYP3A homologues from turkey liver, which we strongly suspect from previous studies will have AFB1 bioactivation activity. CYP3A37 (Genbank DQ450083) has an open reading frame (ORF) of 1512 bp and is predicted to be 504 amino acids with 76% sequence identity to human 3A4 and 97% identity to chicken CYP3A37. A second gene CYP3A80 (Genbank DQ450082) is slightly larger with an ORF of 1530 bp coding for a protein predicted to be 510 amino acids, also with an amino acid similarity of 76% to human 3A4 and 78% sequence identity to chicken CYP3A80. We are in
the process of expressing these genes in E. coli to determine their AFB1 bioactivation capabilities. Determining the genes responsible for the hypersensitivity of turkeys to AFB1 is the first step in devising genetic and/or management strategies for health improvement.
Impacts
Identifying genes associated with disease susceptibility is the first step in devising genomic markers for mycotoxin disease resistance in susceptible domestic food animals such as poultry. This research will help American agriculture by improving animal health.
Publications
- Coulombe, R.A. and S.M. Yip. (2006). Heterologous expression of a cytochrome P450 from turkey liver that activates aflatoxin B1. In Poisonous Plants: Global Research and Solutions (K. Panter, T. Wierenga, J. Phister, eds.) CAB International, London. (in press).
- Guarisco, J.A., Hall, J.O., and R.A. Coulombe, Jr. (2006). Butylated hydroxytoluene chemoprevention of aflatoxicosis in turkeys is dose-related. Toxicological Sciences 90 (1): 1991.
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Progress 01/01/05 to 12/31/05
Outputs
We determined the effect of dietary antioxidants on the residual concentrations of aflatoxin B1 (AFB1) as well as on the presence of related biomarkers of toxicity in turkeys, the most susceptible animal species to this mycotoxin. Ten-day old turkeys were pretreated for ten days with dietary butylated hydroxytoluene (BHT; 4000 ppm) then given a single oral dose of [3H]AFB1. Turkeys in the BHT group had significantly reduced AFB1 concentrations in the serum, liver, breast meat and fat compared to controls at several time points up to 24 hr. Formation of hepatic AFB1-DNA adducts (an important AFB1 biomarker) was also significantly reduced in BHT-fed birds. This data is supported by our findings that BHT competitively inhibits AFB1 bioactivation in turkey. In total, our data demonstrates that BHT significantly reduces AFB1 bioavailability, as well as AFB1 residues in turkey products.
Impacts
This is the first demonstration that dietary antioxidants actually reduce the residual concentrations of a naturally-occurring food-borne carcinogen aflatoxin B1 (AFB1) in turkeys. Because AFB1 is a known human carcinogen, this research will help improve the safety of our food supply by reducing human exposure to natural cancer-causing chemicals.
Publications
- Coulombe, R.A. and S.M. Yip (2006) Heterologous expression of a cytochrome P450 from turkey liver that activates aflatoxin B1. Natural Toxins (in press).
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Progress 01/01/04 to 12/31/04
Outputs
Our earlier studies demonstrated that the extreme sensitivity of poultry to AFB1 is associated with a combination of efficient hepatic cytochrome P-450 (CYP)-mediated activation and deficient glutathione S-transferase (GST)-mediated detoxification of AFB1. We then discovered that dietary butylated hydroxytoluene (BHT) protects against nearly all clinical signs of aflatoxicosis in turkeys, an effect mediated primarily by competitive inhibition of AFB1 activation, rather than by induction of protective GSTs. We now show that 10-day pretreatment with BHT (4000 ppm, in feed) significantly reduced AFB1 bioavailability as well as hepatic AFB1-DNA adduct formation compared to control turkeys at each time point 2-24 hrs after oral administration of [3H]-AFB1. Aflatoxin concentrations in whole liver were decreased in BHT-fed animals compared to controls. Despite induction of hepatic GST indicator activities, we found no specific AFB1 detoxification by GST in the livers of
birds from any group. These data support our hypothesis that the observed in vivo chemoprevention by BHT is due to inhibition of hepatic CYP-mediated AFB1 activation to reactive toxic intermediate(s).
Impacts
Our discovery that several GRAS-listed food-grade antioxidants protect turkeys against feed-borne toxins such as aflatoxin B1 as well as reduce the content of aflatoxin B1 in turkey products is an important step to significantly reduce the losses associated with mycotoxins in poultry feeds. Our research will also help the poultry industry to be more productive and to produce safer food for consumers.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Some epidemiological evidence suggests a link between the inhalation of aflatoxin B1 (AFB1)-contaminated grain dusts and increased lung cancer risk. To determine possible risk, we compared AFB1 action in human bronchial epithelial cells (BEAS-2B) transfored to stably express human CYP 1A2 (B1A2) and 3A4 (B3A4), the principal CYPs thought to activate this mycotoxin in human liver. All three cell types retained catalytically-active glutathione S-transferase (GST). B1A2 and B3A4 cells expressed MROD and nifedipine oxidase activities, respectively, and were 3,000- and 70-fold more susceptible, respectively, to the cytotoxic effects of AFB1 than BEAS-2B. When cultured with a range of low, environmentally relevant AFB1 concentrations (0.02-1.5 uM), control cells formed barely detectable AFB1-DNA adducts, while B1A2 cells formed significantly more adducts than B3A4 cells. Formation of AFB1-DNA adducts in B1A2 was inhibited by the CYP 1A2 inhibitor 7,8 benzoflavone, while
those in B3A4 cells were inhibited by the CYP 3A4 inhibitor 17b-ethynylestradiol. Only the CYP-transfected cell lines expressed CYP mRNA. When adjusted for CYP mRNA expression, B1A2 cells formed more cytotoxic and DNA-alkylating species at low AFB1 concentrations, while B3A4 cells were more efficient at high concentrations. Untreated B1A2, and B3A4 cells constitutively expressed p53. AFB1 caused a concentration-dependent decline in p53 expression in B1A2 cells, and to a lesser extent, in B3A4 cells, which continued for at least 12 hr after exposure. There was a concomitant concentration-dependent increase in expression of the 76 kDa MDM2 isoform in B1A2 cells. Our results affirm the hypothesis that, as in human liver microsomes, CYP 1A2 in human lung cells appears to have a more important role than CYP 3A4 in the bioactivation of low AFB1 concentrations associated with many human exposures. It is possible that under conditions where appropriate CYPs are expressed in lung, inhalation
of AFB1 may result in increased risk to lung cancer in certain agricultural settings where such exposure occurs.
Impacts
Relatively little is known about the potential risk posed by inhalation of AFB1-contaminated grain dusts in agricultural settings. This work may help determine that risk, and may be useful in setting safety guidelines and possible regulation to protect those exposed. This work will help determine risk posed by inhaled grains contaminated with AFB1 in certain agricultural settings
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
Aflatoxin B1 (AFB1) is a food-borne mycotoxin and one of the most potent carcinogens known. It is universally present in corn, peanuts and other agricultural commodities, and is not affected by conventional food processing. AFB1 causes significant losses to agriculture, and is likely responsible for human liver cancer in many countries. AFB1 must first be metabolically activated to the aflatoxin B1-8,9-epoxide (AFBO) before it is toxic and carcinogenic. Once metabolized to AFBO, it reacts rapidly (half life = 0.5 sec) with cellular nucleophiles; thereafter it is non-toxic, and therefore safe for consumption. The purpose of this research project is to determine if plants can be genetically modified to activate AFB1 in situ prior to human or animal consumption. We are inserting cloned DNA (cDNA) from rainbow trout cytochrome P-450 (CYP) 2K1 gene into plants via Agrobacterium tumefaciens. With this gene it is hypothesized the plant will activate and neutralize the AFB1
before it reaches the point of human or animal consumption. When this project was undertaken, no suitable DNA plasmid containing a maize promotor that would work with Agrobacterium existed. We have completed the plasmid construction phase of this project. We have prepared a set of plasmids containing CYP 2K1 that will be transferred into Nicotiana tobaccum via A. tumefaciens. The plasmids are driven by the cauliflower mosaic virus 35s, a strong dicot promotor. The plasmids also have been introduced into Agrobacterium. We are currently awaiting for sufficient growth of tobacco seedlings to transfer the gene into the plant. We have also developed plasmids containing potentially beneficial mutations that will allow expression in the test plant Nicotiana tobaccum. If successful, we will then transform Zea maize. We are also investigating a method of purifying the 2K1 gene product. This will not only allow beneficial study of the protein, but may lead to methods of removing AFB1
contamination from food and the environment.
Impacts
To our knowledge, this project represents the first application of biotechnology to specifically produce a safer food supply, and to protect consumers from a known cancer-causing chemical in their food. Accordingly, a "cancer-fighting" biotechnologically-derived product may engender a more positive public impression of the potential of agricultural biotechnology than previous products.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Aflatoxin B1 (AFB1) is a food-borne mycotoxin and one of the most potent carcinogens known. It is universally present in corn, peanuts and other agricultural commodities, and is not affected by conventional food processing. AFB1 causes significant losses to agriculture, and is likely responsible for human liver cancer in many countries. AFB1 must first be metabolically activated to the aflatoxin B1-8,9-epoxide (AFBO) before it is toxic and carcinogenic. Once metabolized to AFBO, it reacts rapidly (half life = 0.5 sec) with cellular nucleophiles; thereafter it is non-toxic, and therefore safe for consumption. The purpose of this research project is to determine if plants can be genetically modified to activate AFB1 in situ prior to human or animal consumption. We are inserting cDNA from rainbow trout cytochrome P-450 (CYP) 2K1 gene into plants via the well-established method of transformation by Agrobacterium tumfaciens. With this gene it is hypothesized the plant will
activate and neutralize the AFB1 before it reaches the point of human or animal consumption. When this project was undertaken, no suitable DNA plasmid containing a maize promotor that would work with Agrobacterium existed. We synthesized one using the maize ubiquitin promotor. We have also developed plasmids containing potentially beneficial mutations that will allow expression in the test plant Nicotiana tobaccum. If successful, we will then transform maize. We are also investigating a method of purifying the 2K1 gene product. This will not only allow beneficial study of the protein, but may lead to methods of removing AFB1 contamination from food and the environment.
Impacts
To our knowledge, this project represents the first application of biotechnology to specifically produce a safer food supply, and to protect consumers from a known cancer-causing chemical in their food. Accordingly, a "cancer-fighting" biotechnologically-derived product may engender a more positive public impression of the potential of agricultural biotechnology than previous products.
Publications
- Van Vleet, T R, Mace, K and R. A. Coulombe, Jr. (2001). Comparative Aflatoxin B1 Activation and Cytotoxicity in Human Bronchial Cells Expressing Human CYPs 1A2 and 3A4. Cancer Research (in press).
- Van Vleet, T.R., Klein, P.J. and R. A. Coulombe, Jr. (2001). Metabolism and Cytotoxicity of aflatoxin B1 in cytochrome P-450-expressing human lung cells. J. Toxicology and Environmental Health (in press).
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Progress 01/01/00 to 12/31/00
Outputs
Inhaled aflatoxin B1 (AFB1) is thought to be a lung cancer risk in certain agricultural settings where people are exposed to respirable grain dust contaminated with this mycotoxin. To examine the possible occupational risk, we characterized the metabolism of AFB1 in normal human bronchial epithelial cells (NHBE), the cell type thought to be the progenitor of most, if not all, human lung cancers. Our data indicated that these cells metabolize AFB1 poorly compared to human liver cells. However, when induced with 3-methylcholanthrene, a cytochrome P450 (CYP) inducer, NHBE cells metabolize AFB1 to its putative active intermediate, the AFB1-8,9-epoxide. Increased AFB1 activation was reflected in western immunoblots showing that 3-MC pretreatment significantly increased the expression of CYP 1A2 compared to control cells. We also utilized SV40-transformed normal human bronchial epithelial cells (BEAS-2B) that stably express CYPs 1A2 and 3A4, the most important isoforms that
activate AFB1 in human liver. These cells retain most if not all of the biochemical characteristics of NHBE cells, including glutathione S-transferase activity. CYP 1A2-expressing cells were approximately 70-fold more susceptible to the cytotoxic action of AFB1 compared to the 3A4-expressing cells. The CYP1A2-expressing cells also activate AFB1 to a DNA binding species. Expression of tumor suppressor p53 protein (p21) in BEAS-2B cells is up-regulated in control cells. AFB1 treatment acts to decrease expression of this protein. AFB1 treatment decreases expression of MDM2, a protein which represses expression of p53. Our data indicates that as in human liver, CYP 1A2 appears to be an important isoform in AFB1 activation in lung cells. Uninduced human lung cells do not activate AFB1 to a significant extent. However, there may increased risk to inhaled AFB1 in smokers in whom expression of this isoform may be permanently up-regulated.
Impacts
Determining the effects of AFB1 in human lung cells will help to assess the risk posed to people occupationally exposed to airborne mycotoxins in food industries. Our data may help government and industry set safe standards for airborne AFB1 in the agricultural workplace.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
Aflatoxin B1 (AFB1) is a naturally-occuring carcinogen present, in varying amounts, in foods such as corn and peanuts. In collaboration with investigators at Oregon State University, we are experimenting with introducing an efficient AFB1 metabolizing enzyme into plant tissue culture as a way of increasing in situ detoxification of this toxin in the field. We are transforming maize tissue culture with trout hepatic cytochrome P-450 (CYP) 2K1. Collaborators have shown that this CYP isozyme has a low Km and high Vmax toward the AFB1-8,9-epoxide. This CYP isform appears to be a "pure epoxidator" in that it forms no other metabolites except the epoxide. We have amplified the cDNA from the 2K1 gene received from OSU, and are now in the process of transformation various plant target species using the Agrobacterium tranfection system.
Impacts
Transforming maize with a detoxifying enzyme is an application of biotechnology to specifically produce a safer food supply, and to protect consumers from a known cancer-causing chemical in their food. Accordingly, a "cancer-fighting" biotechnologically-derived product may engender a more positive public impression of the potential of agricultural biotechnology than previous products.
Publications
- Coulombe, R.A., Jr. (1999) Natural Toxins and Chemopreventives in Plants, In B. Helferich and C. Winter (eds.) Food Toxicology. CRC Press, Inc., Boca Raton, Fl. (in press).
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Progress 01/01/98 to 12/31/98
Outputs
In collaboration with investigators at Oregon, and Texas, we are experimenting with introducing an efficient aflatoxin B1 (AFB1) metabolising enzyme into corn as a way of increasing in situ detoxification of this toxin in the field. We are transforming maize tissue culture with cytochrome P450 (CYP) 2K1, an enzyme isolated from rainbow trout. This CYP has the lowest known Km and the highest Vmax toward the AFB1, and produces purely the activated AFB1 metabolite, the electrophilic AFB1-8,9-epoxide. No other metabolites are formed from AFB1. The 1859 bp 2K1 cDNA clone was inserted into pSPORT, and amplified. We are now in the process of transformation using the Agrobacterium tranfection system.
Impacts
(N/A)
Publications
- VanVleet, T.R., K. Mace, and R.A. Coulombe, Jr. 1998. Metabolism and cytotoxicity of aflatoxin B1 in genetically engineered human lung cells. Toxicological Sciences 42:1970
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Progress 01/01/97 to 12/31/97
Outputs
Working with investigators at Colorado State University, we have assessed the toxicity and potential medicinal value of plants important in hispanic and native american folk and ceremonial medicine. Analysis of plant extracts that were significantly cytotoxic (Brickellia californica and Cryptantha jamesii, for example) showed both to contain toxic unsaturated pyrrolizidine alkaloids (PAs). We have shown that these PAs are genotoxic by cross-linking with cellular DNA. Using a variety of mammalian cell and viral inhibition assay systems, we discovered that a component of Indian parsley (Ligusticum porteri), the most widely-used herbal product in the American Southwest, has potent antiviral activity against several types of viruses that infect people. Ligustilide, a major component of this plant has modest antiviral activity. A synthetic phenylphthalate derivative of ligustilide had striking antiviral activity against several viruses. At this time, we have not determined
the mechanism of antiviral action of these compounds.
Impacts
(N/A)
Publications
- Drew, G.L, Stermitz, F.R. and R.A. Coulombe, Jr. Molecular interactions of pyrrolizidine alkaloids with critical cellular targets. Fifth International Symposium on Poisonous Plants, San Angelo, TX, May, 1997.
- Drew, G.L., Stermitz, F.R. and R.A. Coulombe, Jr. Activated pyrroles cross-link DNA with actin in mammalian cells. Presented at the annual meeting of the American Association for Cancer Research, San Diego, April, 1997.
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Progress 01/01/96 to 12/30/96
Outputs
Activated pyrrolizidine alkaloid pyrroles cross-link actin to DNA. This was confirmed by Western immunoblot analysis of DNA-protein complexes purified from two mammalian systems exposed to dehydrosenecionine and dehydromonocrotaline. A polyclonal anti-actin antibody was used. Actin is an important protein involved in cell division and cell structure, and is involved in gene regulation as well. It is likely that the anti-mitotic action of PAs is mediated, at least in part, by its reaction with actin. We have also demonstrated for the first time that a related pyrrolic anticarcinogen, mitomycin C, also cross-links actin with DNA in mammalian cells. We have further determined that the presence of a free sulfhydryl group is critical to the PA-induced DNA-protein cross-link. Thus, glutathione and cysteine are preferable alternate nucelophiles compared to DNA bases. Methionine, which does not have a free sulfhydryl group, does not form DNA-protein cross-links with pyrrolic
PAs. Thus, it is possible that sulfhydryl-containing compounds may be useful antidotes for pyrrolizidine alkaloid poisoning.
Impacts
(N/A)
Publications
- COULOMBE, R.A. JR. AND G. DREW. 1996. Molecular interactions of pyrrolizidine alkaloids with DNA. Presented at the Annual meeting of the Western Regional FoodSafety Project. October, 1996. Lake Tahoe, CA.
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Progress 01/01/95 to 12/30/95
Outputs
In collaboration with investigators at Colorado State University, we have been screening a number of plants native to Utah and the Rocky Mountain region that are used by Native Americans as part of traditional herbal medicine. Two plants that are commonly used by Native Americans, Cryptantha jamesii, and Brickellia grandiflora were found to contain high levels of the toxic pyrrolizidine alkaloid (PA) lycopsamine as a major component. This is of concern as small children might be especially at risk by consumption of products derived from these plants. Lycopsamine is a toxic, DNA cross-linking compound, and we will test its toxicity and DNA cross-linking potency compared to other PAs.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
The nature of DNA-protein interactions induced by Pas was investigated because the large portion of PA-induced cross-links was protein associated. The cellular DNA-protein cross-linking activity of the pyrrolic metabolites was dehydrosenecionine>dehydromonocrotaline>dehydroseneciphylline >dehydroriddelline >dehydroretronecine. The protein-associated fraction accounted for approximately 50% of the total cellular DNA cross-links. However, dehydroretronecine induced few DNA-protein cross-links; none were detected following treatment with indicine N-oxide. Thus, the macrocyclic diester pyrroles are potent cellular DNA-protein cross-links.Because a large portion of PA-induced cellular DNA cross-links involved proteins, the DNA cross-linked proteins were characterized in cell cultures by isolating and separating the cross-linked proteins using SDS-PAGE and 2-D gel electrophoresis. In cells treated with the macrocyclic alpha,beta-unsaturated diesters (senecionine,
seneciphylline, riddelline, retrorsine), the macrocyclic alpha,beta-saturated diester (monocrotaline) and open diester (heliosupine), proteins from 40-60 kD cross-linked with DNA.
Impacts
(N/A)
Publications
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Progress 01/01/93 to 12/30/93
Outputs
The DNA cross-link activity of a series of pyrrolizidine alkaloids (PA) in pBR322 and lambda-phage DNA target systmes has been investigated. A retardation of the electrophoretic migration of DNA occurs when it is cross-linked. There are inherent differences between cross-link potency within PA structural groups, and that differences in potency are similar to that seen in cellular systems when parent PAs are used. For example, the alpha, beta-unsaturated macrocyclic diester pyrroles dehydrosenecionine and dehydroriddelliine are more potent cross-linkers than the saturated macrocyclin pyrrole dehydromonocrotaline. The simple necine base pyrrole dehydroretroncecine was the least potent of this group. Indicine N-oxide did not produce detectable cross-links. In plasmid pBR322, the cross-linking was dose-dependent in the DNA:PA range of 1:05 to 1:2. In PA-exposed cells, protein-associated cross-links were isolated and analyzed by 2-D gel electrophoresis. The major
DNA-binding proteins were found in the basic portion of the gel, and the staining pattern was similar to that of the known DNA cross-link agent cis-platinum. PAs cross-link DNA in a pattern similar to that of other potent bifunctional alkylating agents used as antitumor agents.
Impacts
(N/A)
Publications
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Progress 01/01/91 to 12/30/91
Outputs
Hamsters that were exposed to long-term pulmonary aflatoxin B1 (AFB1) (bi-weeklydoses) developed bronchogenic carcinomas beginning at 18 months following cessation of dosing. Persistent airway atypia, reduced airway atypia, reduced airway clearance, and increased airway cellular mitosis were among other observations. Preneoplastic and neoplastic tumors in the liver were also found. Studies were continued to determine the effects of AFB1 on the hypothalamus-pituitary-adrenal gland axis. Mice (C57Bl/6) were treated orally with 30, 150, or 750 mg/kg AFB1 daily for four weeks. A much higher dose of AFB1 was needed to produce the immunosuppressive effects on blastogenic response, IL-2 production, and primary antibody production of splenic cells, compared to previous studies in other mice strains. The amount of circulating anti-SRBC antibody was reduced. Decreases were observed in the helper-T cell and B cell populations in phenotyping splenic lymphocytes. No significant
changes were observed in natural killer cell activity, mixed lymphocyte response, hypothalamic biogenic amine concentrations and corticotropin releasing factor, and of ACTH and coricosterone in plasma.
Impacts
(N/A)
Publications
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Progress 01/01/90 to 12/30/90
Outputs
T-2 mycotoxin is an amphipathic molecule known to interact with biomembranes. Ofthe many trichothecene mycotoxins, T-2 toxin, HT-2 toxin, acetyl T-2 toxin, neosolaniol, and T-2 tetraol were selected for the cytotoxicity test on normal human fibroblasts and mutant I-cell human fibroblasts (which lack most of their lysosomal hydrolases), using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cleavage and cell count. The cytotoxicity of the 5 trichothecene mycotoxins was similar in both cell lines, indicating that lysosomal enzymes are not involved in the cytotoxicity of these toxins. The order of cytotoxicity is T-2 toxin > HT-2 toxin = neosolaniol > acetyl T-2 toxin > T-2 tetraol in both cell lines. Cells pretreated with E-64, a specific inhibitor of cysteine proteases, showed no significant difference in susceptibility to the T-2 toxin. This supports the earlier observation that lysosomal membranes or their hydrolytic enzymes might not be
responsible for mediating the toxicity of T-2 toxin or its congeners.
Impacts
(N/A)
Publications
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Progress 01/01/89 to 12/30/89
Outputs
We previously reported that primary fetal bovine Kidney (PFBK) cells were consistently more sensitive to the cytotoxic effects of fusarium T-2 toxin than Madin-Darby bovine kidney (MDBK) cells in culture. T-2 toxin inhibited incorporation of labeled thymidine, uridine, and leucine in both culture types; at lower concentrations of the toxin, PFBK cells were affected to a greater extent than MDBK cells. T-2 toxin inhibited both the transport of thymidine as well as thymidine incorporation into macromolecules in MDBK cells during initial periods, but did not affect uridine incorporation. The cellular enzymes, K+-dependent phosphatase and succinic dehydrogenase were inhibited in MDBK but not in PFBK cultures; acid phosphatase was not influenced in either culture types. In a cell-free system none of the above enzymes were affected by T-2 until the toxin concentration exceeded 10-5 M. The immunotoxic potential of N-ethyl-N-nitrosourea (ENU), a potent and transplacental
neurocarcinogen, was evaluated in male CD-1 mice. The animals received i.p. injections of ENU-0, 2, 8 or 32 mg/kg body weight, twice weekly for three weeks. Splenic lymphocytes were cultured in the presence of mitogens, lipopolysaccharide, pokeweed mitogen, concanavalin A and phytohaemagglutinin. Mixed lymphocyte cultures in the presence of allogeneic cells were also tested. Blastogenic response decresed in a dose-dependent manner, as measured by the 3H-thymidine uptake by splenocytes.
Impacts
(N/A)
Publications
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Progress 01/01/88 to 12/30/88
Outputs
Young adult male CD-1 mice were treated orally twice weekly for three weeks with0, 0.05, 0.15 or 0.65 mg/kg of aflatoxin B1 (AFB1) in corn oil. Two days after the last dose, the mice were killed by decapitation and the concentrations of the brain catecholamines, norepinephrine (NE) and dopamine (DA), and their metabolites, 3-methoxy-4-hydroxymandelic (VMA), homavanillic acid (HVA) and dihydroxyphenyl acetic acid (DOPAC) and the indoleamine serotonin (5-HT) and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were determined by high pressure liquid chromatography in six discrete brain regions. Major effects of AFB1 were found in the concentrations of NE in most brain areas. Endogenous concentrations of DA were increased in the striatum and hypothalamus. The VMA level in the hypothalamus and striatum were decreased by the treatment. The activity of tyrosine hydroxylase, tryptophan hydroxylase, amino acid decarboxylase and monoamine oxidase (the enzymes important in
synthetic and degradation pathways of biogenic amine) were often consistent with the changes observed in metabolizing enzymes. There was an increase noted in tryptophan hydroxylase activity. Activities of amino acid decarboxylase and monoamine oxidase were increased although the changes were not consistent in all regions or at all dose levels of AFB1. These results suggest that dietary exposure to AFB1 diets may cause alterations in various biogenic amine concentrations and related metabolizing enzymes.
Impacts
(N/A)
Publications
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Progress 01/01/87 to 12/30/87
Outputs
The potential DNA-interstrand (DD) and DNA-protein (DP) cross-linking activity of seven pyrrolizidine alkaloids (PA) in cultured Madin-Darby bovine kidney epithelial cells (MDBK) using gravity-flow alkaline elution was investigated. Cells were cultured two hr with the PA (100,300 and 500 uM) and rat liver S9 containing a NADPH-generating system. The alkaloids which produced either DD or DP cross-links (ranked from highest to lowest) were: seniciphylline (DP > DD), riddelline (DP > DD), retrorsine (DP > DD), senecionine (DP > DD), monocrotaline (DD > DP), heliosupine (DP only), retronecine (neither DP nor DD). None produced DNA single-strand breaks. The teratogenicity of trienoic acid (Rol3-6307) and of ethyl-(E)-7-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)-3-methyl-2,4 ,6-octatrienoate (Rol3-2389) was evaluated in hamsters. Both retinoids induced malformations similar to those induced by all-trans-retinoic acid (RA). On a molar basis these retinoids were
18 times more potent teratogens than all-trans-RA.
Impacts
(N/A)
Publications
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Progress 01/01/86 to 12/30/86
Outputs
The disposition of intratracheally (i/t) and orally administered (3H)AFB1 was studied in male Sprague-Dawley rats. Blood-concentration data from both groups approximated a two-compartment open model. The time to speak for the i/t group (hr) was less than that for the oral group (3 hr), but the disappearance of label from the blood followed a nearly identical course in both groups. Rats were treated po twice weekly for 3 weeks with a low (32.8 ug/kg) and high dose (327.9 ug/kg) of aflatoxin B1 (AFB1) in corn oil. The major effects were found in striatal dopamine and serotonin concentrations, with decreases of 37 and 29%, respectively. A corresponding decline was observed in the dopamine metabolites, homovanillic acid (44%) and dihydroxyphenylacetic acid (30%). Cytotoxicity of citrinin, a fungal metabolite and a common food contaminant, was evaluated in an established cell line, Madin-Darby bovine kidney (MDBK) cells and primary fetal bovine kidney (PKBK) cells. Citrinin
produced a low order of cytotoxicity in cultured renal cells. MDBK cells were more sensitive than the PFBK cells. Groups of pregnant hamsters were fed diets consisting of cassava meal:laboratory chow (80:20) during days 3-14 of gestation. Reduced fetal body weight and reduced ossification were associated with cassava diets. High cyanide cassava diets caused a significant increase in the numbers of runts.
Impacts
(N/A)
Publications
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Progress 01/01/85 to 12/30/85
Outputs
Studies have continued on the assessment of cassava in inducing congential malformations in hamsters. Golden Syrian hamsters given 80% cassava in diets produced marginally significant effects but a similar observation was also notice when animals were fed a diet poor in protein quality, the one comparable to cassava diets. The rate of absorption of amygdalin (a potent teratogenic cyano-glycoside) was compared to linamarin (the glycoside in cassava). The liberation and absorption of cyanide was greater in the case of linamarin than amygdalin, and this may account for the higher maternal toxicity of the former glycoside. Investigations were carried out on the structure-activity relationship of various retinoic acid derivatives. The results available to date suggest that a hydroxyl residue at carbon 3 reduced teratogenic effects compared to its methoxy congener. Replacement of the ring in all-trans-retinoic acid by a cyclo-pentenyl structure failed to alter its
teratogenic potential. The research efforts involving mycotoxins in food have been continued. The potential of fusarium T-2 toxin, a trichothecene mycotoxin, as an immunomodulatory chemical was investigated in mice. Following an oral treatment, an increased mitotic activity within spleen was observed. Another mycotoxin, verrucarin A, was also found to be immunopotentiatory. The experiments involving the suitability of in vitro cell-culture methods to assess toxicity are in progress.
Impacts
(N/A)
Publications
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Progress 01/01/84 to 12/30/84
Outputs
Lymphatic cells, prepared from syngeneic male NFS mice, were cultured with either lipopolysaccharide (LPS), pokeweed mitogen (PWM), phytohaemagglutinin (PHA), or concanavalin A (Con A) for 48 hours. T-2 toxin (10 -2 - 10 -7M) was added at 0 or 24 hr. Exposure to T-2 toxin (10 -11 - 10 -10M) after 24 hr. caused an increase in 3Hl-thymidine uptake splenic cells. PWM stimulation increased in this system, the response to LPS was increased to a lesser extent. However, T cell responses to PHA and Con A decreased. Forty-eight hr exposure to T-2 toxin (10 -11 - 10 -12M) decreased both the PWM and LPS responses. Thymic cells (immature T cells) were even more sensitive to T-2 toxin; Con A responses decreased at 10 -12M. The pharmacokinetic disposition of intratracheally (i.t.) and orally (p.o.) administered 3H!AFB1 in male S-D rats was investigated. Blood, urine and feces were collected at selected intervals following a single dose of AFB1 (600 ug-kg) in saline. Blood
concentration data from both groups approximated a two-compartment open model. Although the time-to-peak of the i.t. group (1 hr) was slightly less than that from the p.o. group (3 hr), disappearance of label from blood followed a nearly identical course in both groups. The plasma half-lives (t 1-2) were 87.7 and 19.8 hr for i.t. and p.o. groups, respectively.
Impacts
(N/A)
Publications
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Progress 01/01/83 to 12/30/83
Outputs
Selected drugs are being utilized as models of putative colon carcinogens in a study of the influence of major types of dietary fiber upon drug pharmacokinetics. Adult, male Wistar rats were pretreated with standardized, isocaloric hydrated gelatin diets containing no fiber or 15% (w/w) cellulose, lignin, hemicellulose (Metamucil), or pectin for 30 days. The pharmacokinetics of acetaminophen, FD & C Red No. 2 and mirex were examined following oral administration in three separate experiments. There was no effect of fiber type on the rate of acetaminophen elimination as determined by the interpretation of the plasma data using the computer programs. Minimal quantities of Red No. 2 were absorbed from the rat's intestinal tract, but its microbial metabolite, naphthionic acid, was readily taken up. Pectin produced a 5-fold higher peak plasma concentration of naphthionic acid than control animals on fiber free diet. Cellulose feeding lowered peak plasma concentration
of naphthionic acid compared to the fiber control animals. Red No. 2 decreased intestinal transit times in all diet groups, including controls. Hemicellulose and pectin feeding lowered peak plasma concentrations of mirex compared to control and cellulose fed animals. Lignin, however, produced higher peak plasma concentrations of mirex and a 4-fold higher rate of mirex elimination.
Impacts
(N/A)
Publications
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Progress 01/01/82 to 12/30/82
Outputs
Interaction of ionic cadmium with copper metabolism was studied in mice togetherwith observatons of its influence on iron and zinc. Metal accumulations in tissues and sub-cellular distributions were correlated with the physiological changes from the interaction. BHA was shown to depress the immune responses of mice; activation was shown to occur during its metabolism with binding to DNA. A cell culture system using embryonic chick retinal cells was developed to score neurotoxicity, and verified with a number of neurotoxic chemicals; the protective influence of vit. E and Se in that sytem was demonstrated. Neurotoxic chemicals (e.g., hexanedione, Leptophos, TOTP, methylmercury) were compared for effects on levels of brain biogenic amines and their metabolites, against behavioral changes in the treated animals. Studies of toxicant effects in the pulmonary system were initiated using intratracheal instillation to evaluate effects of CR + 6 on alveolar macrophage
function and inhalation (nose only) to study systemmic effects of formaldehyde exposure upon the immune system as a component of its general toxicity.
Impacts
(N/A)
Publications
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Progress 01/01/81 to 12/30/81
Outputs
Progress included study of the influence of dietary protein (level) upon tehe invivo metabolism of BHA in the rat, including the potential interaction of polyoxyethylene sorbitan, as measured by BHA pharmacokinetics. The effect of BHA upon immune responsiveness in the mouse was investigated in terms of the inducibility of splenic lymphocyte transformations, immunoglobulin levels in plasma and other indices. an animal model to evaluate hypotheses for chemical-induced neurotoxicity was tested with acrylamide, 2,5-hexanedione, methyl mercury, TOCP, and leptophos. Behavioral confirmation of central neuropathy was made in the treated rats; brain catecholamines and serotonin levels were then measured in animals similarly treated. Differential effects of chemicals producing demyelinating vs non-demyelinating neurotoxicities were observed, but no consistent pattern of CNS transmitter concentration was established with respect to the type of chemical studied. Similar
neurotoxic chemicals wer tested in cells in culture (chick retinoblastoma and rat synaptosomes) and cytotoxic indices and activities of membrane enzymes were correlated with the type and degree of neurotoxic action of the chemical. Toxic effects of vanadium and toxic interactions between cadmium and copper, or between cadmium and zinc, lead, ad mercury, were evaluated in lab animals.
Impacts
(N/A)
Publications
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Progress 01/01/80 to 12/30/80
Outputs
A number of heavy metal ions and organic toxicants were used in efforts to develop in vitro test systems for specific toxic effects: chick ganglia cultures for cytotoxicity, uptake of monoamines and K +-dependent phosphatase inhibitions by brain synaptosomes as indices of neurotoxicity; and aggregation of embryonic retinal cells as an index of inhibited organogenesis. Accumulation kinetics of V, Cd, Pd in animal tissues has been under continuous investigation, including definition of dosage limits in the Cu/Cd interaction, as well as of specific environmental movements of metals leading to animal exposure. Natural food toxicants investigations included study of patulin/rubratoxin B interactiuons, and of flatulance-causing potentials of verbascose, stachyose and raffinose. Immunotoxicity investigations included evaluation in animals or in lymphocyte culture of BHT, tri-o-tolylphosphate, and vanadate for immune suppression or stimulation, and for hypersensitivity toward
pesticides, 2,4-D and malathion. A number of chemicals, primarily neurotoxic pesticides, were tested for effects on serotonin and norepinephrine turnover in brain in efforts to define the neurochemsitry of such toxicity.
Impacts
(N/A)
Publications
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Progress 01/01/79 to 12/30/79
Outputs
Neural cell culture investigations of postulated processes inducing delayed neurotoxicity syndromes are being conducted with toxic metals and selected organic compounds, including neurotoxic solvents (e.g., n-hexane) and organophosphates. Direct effects upon cell membrane enzyme functions are being correlated with similar effects in brain tissue produced upon in vivo exposure to these toxicants and, for comparison, to non-neurotoxic analogues or similar chemicals. Industrial air pollution studies are being continued with emphasis upon a large coal-fired electrical generating unit now under construction. Pre-operation flora and fauna samples are being taken to establish the background of heavy metal, fluoride, selenium, arsenic and other potential coal-derived toxicants. Long term monitoring is planned to observe the trends of these toxicants in representative organisms during operation of the generating unit. Continued support of research on toxic secondary
metabolites of potato and other food commodities is provided to collaborators in the W-122 regional project on natural toxicants in foods.
Impacts
(N/A)
Publications
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Progress 01/01/78 to 12/30/78
Outputs
Project activities included development of preliminary data supporting grant proposals for study of delayed neurotoxicity (of pesticides, haloalkanes, and metal ions), of natural toxicants or co-toxicants in foods, and of relationships between dietary fiber, toxicant metabolism, and gastro-intestinal cancer. Analyses of aflatoxin and other suspected mycotoxins of cottonseed and silage were conducted in connection with an episode of cattle abortions with concomitant used of contaminated cottonseed. Study of Cadmium - metallothionein relationships in domestic levestock species were completed; hepatic and renal MT from cattle, swine, and chickens were compared after cadmium dosing.
Impacts
(N/A)
Publications
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Progress 01/01/77 to 12/30/77
Outputs
The distribution of cadmium from the diet into various animal tissues and milk or eggs was determined in feeding trials lasting up to 3 months (cows) 6 months (growing pigs) or 12 weeks (hens). Cadmium levels in meat, milk and eggs were not increased by feeding up to 10 ppm added cadmium; liver and kidney accumulated large quantities of cadmium which was directly associated with the formulation of the Cd-binding protein, metallathioneine (MT). Lead administration on contrast, did not affect MT content of liver or kidney. Tissue MT levels continued to increase for several weeks after terminating Cd administration and the Cd levels in liver and kidney remained elevated without sign of depleting. The project data indicated that only the organ meats (e.g., liver and kidney and bone (Pb only) are liable to convey significant residuals Ph and Cd from animal feeds to the human diet. Neurotoxic (TOTP, DFP) and non-neurotoxic (Bidrin) organophosphates were compared for
effects on protein synthesis in a never cell culture by measuring 14-C-leucine uptake vs glucose incorporation. All compounds were somewhat cytotoxic, however, Bidrin did not inhibit leucine uptake at concentrations where neurotoxic compounds did. Further screening may reveal selective changes in protein synthesis corresponding to effects of neurotoxic organophosphates.
Impacts
(N/A)
Publications
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Progress 01/01/76 to 12/30/76
Outputs
Research during 1976 was continued upon the following topics: Organophosphates and neurotoxicity; model studies were carried out in chick dorsal root cell culture. Cytotoxicity and ATPase activities were used as indices of effect. Heavy metal (Pb and Cd) toxicology; metal distribution from feed into animal products, Cd metal-lothionein occurance in organs of livestock species, and metal ion effects on immune mechanisms were studied. Dietary protein effect on detoxifying enzyme activity; a model accounting for the dietary methionine regulation of hepatic microsomal enzyme activity was developed based upon control of phosphatidyl choline content of microsomes. Supporting data from that model were developed. Study of sulfur dioxide and bisulfite interaction with plasma and tissue proteins was initiated. A comparative study of dieldrin effects on brain monoamine oxidase activity and 5-hydroxytrptamine and 5-indoleacetic acid concentrations was conducted in several
species.
Impacts
(N/A)
Publications
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Progress 01/01/75 to 12/30/75
Outputs
Pharmacodynamics of lead administered orally as lead acetate is being investigated in lactating dairy cows, growing-finishing hogs, and laying hens. Biochemical indices of lead exposure have been quantified and lead levels in milk, meat, eggs, bone and selected organs determined. Similar investigation with dairy cows and swine is now in progress employing orally administered cadmium chloride. Laying hens will be added to that study in the near future. Effects of dietary CdCly on the development of tumor cells transplanted into treated, receptive mice were studied. It is hoped to use this technique as the basis for quantitative screening of environmental chemicals for their effects upon cellular immunity. A study of the effects of dietary protein, ascorbate, and calcium levels upon fluoride toxicology in the guinea pig is nearing completion. Physical properties of bone were shown to be affected differentiallyby F under the different variables, particularly the calcium
level. A large series of organophosphate and carbamates has been evaluated as agents producing direct cytotoxic effects on nerve cells in tissue culture. Correlation with the propensity of the chemical to produce delayed neurotoxicity in exposed mammals and birds is being sought. Regional research contributed by this project involves a study of the alkaloid formation in potatoes under various conditions of storage and fungal infection; the toxicology of products isolated from infected potatoes is under investigation.
Impacts
(N/A)
Publications
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Progress 01/01/74 to 12/30/74
Outputs
A survey of several toxic metals and fluoride in soils, vegetation and animal tissues from 16 selected sites about the Magna copper smelter has been completedand interpreted in terms of health hazards. An intensive study of lead pharmacodynamics including levels in milk, and biochemical parameters indicativeof lead exposure (such as blood and urine ALA levels, free porphyrins in blood and tissue ALAD activity) is being conducted with dairy cows and growing pigs. Protein quality in rat diets is being investigated as a variable affecting the activity of hepatic microsomal SAM-dependent methyltransferase. In turn, that enzyme forms the critical microsomal phospholipid needed for drug metabolizing oxidase activity. Used shortening from deep-fat frying as dietary fat did not yield sufficient non-urea adducting fatty acid products to affect liver microsomal adaptive enzymes. Nerve cell culture study with TOTP showed direct cytotoxic effects (degeneration) which may
related to the delayed neurotoxicity of certain organophosphorus compounds. The hypothesis of an auto-immune basis for delayed toxicity was not supported by any of several lines of study. Chicken spinal cord synaptosomal ATPase was found to be slowly inhibited by TOTPand Mevinphos in vitro suggesting its involvement in the delayed toxicity. Dietary protein level is being studied as a factor in F toxicity with primary attention directed toward adaptations in osseous tissue.
Impacts
(N/A)
Publications
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Progress 01/01/73 to 12/30/73
Outputs
FO-I(in drinking water) effects on guinea pig growth and serum FO-I levels, FO-Iaccumulation in bone and bone physical properties have been studied in relation to duration of exposure. Survey of pathological effects on animals living in the vicinity of FO-I emitting industries were continued. Analyses of animal tissue sections have been processed for histopatholigic examination. Studies ofthe effect of neurotoxic organophosphates (primarily TOTP) on immune responses in chickens were completed. Histologic evidence of immune stimulation was obtained, but attempts to obtain protection against the delayed paralysis syndrome by immune suppressants were not successful. An embryonic chick ganglianerve culture system was developed to evaluate the specific cytotoxicity of neurotoxic pesticides. Thermally oxidized corn oil and a non-urea-adducting fraction of fatty acids prepared from oxidized corn oil were shown to cause extreme liver enlargement, increased hepatic
microsomal protein and microsomal enzyme activities when fed to female rats in a standard purified diet. Evidencethat the microsomal mixed-function oxidase system is regulated by methyl transferase activity was obtained. Selected halogenated phenols are being tested as thyroid antagonists by evaluating uncoupling of oxidative phosphorylation and altering thyroxine-induced mitochondrial swelling.
Impacts
(N/A)
Publications
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Progress 01/01/72 to 12/30/72
Outputs
An investigation of the effect of dietary protein levels on chronic fluoride toxicity is in progress with primary attention directed toward adaptations in osseous tissue. Delayed neurotoxicity (paralytic), a problem with some organophosphorus insecticides, is being investigated in chickens using TOTP (tri-orthotolyl phosphate) as a model toxicant. The persistence in tissues and metabolism of DTyP TOTP has been studied in considering autoimmune and other possible mechanisms for the effect. Effects of protein quality and fat level inthe diet on the liver adaptation to organochlorine toxicants are being investigated with emphasis on microsomal phospholipid alterations as governing the enzyme responses observed. Levels of lead, arsenic, cadmium, molybdenum, fluoride, zinc, and copper in soils, vegetation, and animal tissues from 16 selected sites neighboring the Magna smelter have been determined during two years of sampling. These are being interpreted in terms of
general health hazards, specifically with respect to the animals at those locations.
Impacts
(N/A)
Publications
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