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Biological defense system against xenobiotics in meat-producing animals

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Title: Biological defense system against xenobiotics in meat-producing animals
Other Titles: 外来性の化学物質に対する食肉用動物の生体防御システム
Authors: Abdelrahem Abdallah Darwish, Wageh Sobhy1 Browse this author
Authors(alt): アブドエルラヒム アブダラハ ダルウィッシュ, ワギ ソブヒ1
Issue Date: 24-Sep-2010
Publisher: Hokkaido University
Abstract: Meat-producing animals are frequently exposed during their lifetime to a lot of xenobiotics which affect on their biological systems, growth, disease response and lead to changes on the carcass quality. These changes may have some public health impact if people consumed such contaminated meat or meat products. Meat-producing animals have developed enzyme systems which help them to metabolize such xenobiotics. Studying of the profile of the different enzymes used in xenobiotics metabolism may be a good tool to reflect the pre-slaughter exposure to xenobiotics in the meat-producing animals. As many of these enzymes tend to increase upon exposure to xenobiotics, so these enzymes are considered as biomarkers for xenobiotics exposure.Cytochrome P450 superfamily and other phase II enzymes are considered as major enzyme systems that play important roles in xenobiotics metabolism. Thus, in this thesis, I studied the biological response to xenobiotics in meat-producing animals by studying the characterization of different phase I and II enzymes, mainly CYP1A, UGT1A1 and GSTA1 subfamilies, in these animals.In chapter I, I investigated the tissue-specific mRNA expression of different cytochrome P450 (CYP) isoforms, UDP glucuronosyl transferase 1A1 (UGT1A1) and glutathione-S-transferase (GSTA1) in the different tissues of cattle using quantitative real-time polymerase chain reaction (qPCR). CYP1A1-like mRNA was expressed in all of the tissues examined including liver, with the highest expression level in kidney. CYP1A2-, 2E1- and 3A4-like mRNAs were only expressed hepatically. Interestingly, significant expression of CYP2B6-like mRNA was recorded in lung tissue, while CYP2C9-like mRNA was expressed in liver and kidney tissues of the examined cattle. UGT1A1- and GSTA1-like mRNA were expressed in all of the examined tissues, except the mammary glands, and the highest expression levels were recorded in kidney. The high expression of UGT1A1 in lung tissue and GSTA1 in liver tissue was unique to cattle, this has not been reported for rats or mice. The findings of this chapter strongly suggest that the liver, kidneys and lungs of cattle are the major organs contributing to xenobiotic metabolism. Moreover, induction of CYP1A1, UGT1A1 and GSTA1 are considered as good biological biomarkers for pre-slaughter exposure to xenobiotics. In chapter II, I extended my study to include other growing sources for meat production such as deer and horses. Thus, I investigated and characterized the metabolic activities of CYP1A in deer, cattle and horses in comparison to those of rats using ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-demethylation (MROD) assays. Also, I performed an inhibition study for these activities using anti-rat CYP1A1 antibody and identified that these activities were due to the CYP1A subfamily. Interspecies differences in the CYP1A-dependent activities were highly observed in this chapter. In particular, I found that the horse had the highest EROD and MROD activities among the examined animal species. In the kinetic analysis, the horses showed the highest Vmax and catalytic efficiency (Vmax/Km), followed by the cattle, deer and rats. In chapter III, I compared the mutagenic activation activity of hepatic microsomes from the three meat-producing animals (cattle, deer and horses) with those of rats as a reference species. In the Ames Salmonella typhimurium TA98 assay, the liver microsomes of all examined animals mutagenically activated benzo[a]pyrene, an ideal promutagens, in terms of production of histidine-independent revertant colonies. The microsomes of horses had the highest ability to produce revertant colonies of the examined animals under both low and high substrate concentrations. Inhibition of this mutagenic activity using α-naphthoflavone, anti-rat CYP1A1, anti-rat CYP3A2 and anti-rat CYP2E1 antibodies suggested that this activity was mainly because of CYP1A1 in these animals as well as in rats. The addition of co-factors for two phase II enzymes, microsomal UDP glucuronosyl transferase (UGT) and cytosolic glutathione-S-transferase (GST), reduced the production of the revertant colonies in a concentration-dependent manner. Interestingly, horses had the highest reduction rate among the examined animals, suggesting that phase II enzymes play a great role in producing a state of balance between the bioactivation and detoxification of xenobiotics in these meat-producing animals. In chapter IV, I elucidated that accumulation of carotenoids is a possible cause for inter-species difference in CYP1A-dependent activity in this group of animals. The relationship between inter-species differences in CYP1A-dependent activity and the accumulated carotenoids and retinoids as candidates of dietary CYP1A inducers in ungulate species was clarified. Interestingly, there were positive correlations between the accumulated carotenoids, such as β-carotene, with both EROD activity and CYP1A protein expression. These correlations were negative with the accumulated retinoids, such as retinol. The β-carotene was major component of carotenoids in ungulates, and known as an inducer of CYP1A. On the other hand, the retinol is reported as the reducer of CYP1A. Other factors which affect CYP1A1 expression, such as polycyclic aromatic hydrocarbons, were also analyzed. To cancel the effects of inter-species difference in CYP1A induction signal cascade among these animals, the rat cell line (H4-II-cells) was treated with the extracted carotenoids from the examined animals. CYP1A expression and dependent activities in the treated cells had confirmed that the carotenoid accumulation is, at least in part, a regulator for the inter-species differences in CYP1A expression and activities.In chapter V, I determined a partial sequence of CYP1A1 in the camel and its phylogenetic position. The deduced amino acid sequence of camel CYP1A1 showed the highest identity 94% with those of sheep and cattle CYP1A1. In a phylogenetic analysis, the camel CYP1A1 isoform was located beside sheep and cattle CYP1A1. When I studied the distribution of camel CYP1A1 mRNA in different tissues, I found that this isoform was expressed in all tissues except the hump. Interestingly, the lungs of all the camels and tongues of two of the three animals showed high expressions of CYP1A1 mRNA, and this may indicate exposure to ligands of aryl hydrocarbon receptor such as environmental pollutants or flavonoids.In conclusion, in this thesis, I clarified the biological defense systems to xenobiotics in the meat-producing animals. I confirmed the inter-species differences in CYP1A expression and dependent activities. Subsequently, I explained the mechanism of the protection of these animals against the mutagenic activation of promutagens and procarcinogens. Also, I declared a possible cause for the inter-species differences in CYP1A dependent activities and expression. Moreover, I characterized cytochrome P450s and phase II enzymes in some ungulates such as camel, cattle, deer, horses and deer in comparison to rats.
Conffering University: 北海道大学
Degree Report Number: 甲第9708号
Degree Level: 博士
Degree Discipline: 獣医学
Type: theses (doctoral)
Appears in Collections:学位論文 (Theses) > 博士 (獣医学)

Submitter: 石塚 真由美

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