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

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

Metal contents in the edible tissues of camel and sheep: human dietary intake and risk assessment in Saudi Arabia

Camel and sheep meats are considered major sources of animal-derived protein, vitamins and minerals in certain parts of the world such as Arabian Peninsula, India and Middle East countries. Despite these health benefits, the safety of sheep and camel meat may be compromised by the presence of heavy metals. The monitoring of metal concentrations is therefore important to ensure compliance with food safety regulations and consequent consumer protection. This study was undertaken to estimate the toxic metals (lead, cadmium and arsenic) and the essential trace elements (copper, zinc and iron) contents in the edible tissues (muscle, livers and kidneys) of both camel and sheep slaughtered at Al-Ahsa, Saudi Arabia. The dietary intake and human health risk assessment of the examined metals due to consumption of these tissues by Saudi population were estimated. The results indicate that the tested metals were detected in all the tissue samples with variable concentrations. Residual concentrations of all examined metals were within the maximum permissible limits set by European Union, Food and Agricultural Organization except for the arsenic. Human health risk assessment revealed that the detected concentrations of cadmium and lead might not possess health hazards. Nevertheless, care is imperative regarding As especially in Saudi Arabia because of the excessive meat consumption

Cytochrome P450 3A mRNA expression along goat and rat gastrointestinal tracts

The cytochrome P450 (CYP) 3A family is involved in the elimination processes of almost 50% of commonly used drugs. CYP3A mRNA expressions in goat and rat gastrointestinal tracts in comparison to the liver were investigated using real-time PCR. In goats, the expression of CYP3A-like mRNAs was comparatively higher in the liver than in the gastrointestinal tract. The intestinal expression of CYP3A-like mRNA showed a gradual decrease from the duodenum to the ileum. In rats, the highest CYP3A62 mRNA expression was found in the duodenum followed by the liver. This study provides insights into the contribution of CYP3A enzymes to xenobiotic metabolism, especially in small ruminants such as goats

Estimation and Human Health Risk Assessment of Organochlorine Pesticides in Raw Milk Marketed in Zagazig City, Egypt

Milk is nearly a perfect natural food and is widely used by all segments of our population especially for infants and the elderly. Organochlorine pesticides (OCPs) have been used worldwide, particularly in many African countries as in Egypt for the control of pests. OCPs are characterized by their bioaccumulation in the environment, especially in the food chain, where they find their way into the human body. The objectives of this study were initially to estimate the residual concentrations of different OCPs in three kinds of fresh and raw milk from different animals (cattle, buffalo, and goat) marketed in Egypt. Additionally, human dietary intake and risk assessment of OCPs were calculated. The tested OCPs included pp-DDT and its metabolites pp-DDD and pp-DDE; hexachlorohexanes (HCHs) including α HCH and γ HCH; heptachlor and heptachlor epoxide; aldrin and endrin; chlordane, methoxychlor, and hexachloride benzene. The recorded results revealed that goat and buffalo milk samples had the highest incidence of OCPs’ contamination (75% for each), while this percentage was 50% in cow’s milk. The mean values of ΣOCPs were 317.83 ± 34.11, 605 ± 50.54, and 1210.57 ± 99.55 (ppb/ww) in the examined cattle, buffalo, and goat milk samples, respectively. All examined OCPs were within the maximum permissible limits (MPLs) set by World Health Organization with only 10% of goat milk samples exceeding this MRL. The estimated daily intake, noncancer, and cancer health risk assessment of the tested OCPs revealed the potential cancer risk especially among children consuming goat’s milk. The public health importance of such OCPs was discussed

beta-carotene and retinol reduce benzo[a]pyrene-induced mutagenicity and oxidative stress via transcriptional modulation of xenobiotic metabolizing enzymes in human HepG2 cell line

Benzo[a]pyrene (B[a]P) is one of the polycyclic aromatic hydrocarbons which is formed due to smoking of foods, incomplete combustion of woods, vehicle exhausts, and cigarettes smokes. B[a]P gets entry into human and animal bodies mainly through their diets. Metabolic activation of B[a]P is required to induce mutagenesis and carcinogenesis in animal and human studies. Carotenoids and retinoids are phytochemicals that if ingested have multiple physiological interferences in the human and animal bodies. In this study, we firstly investigated the protective effects of beta-carotene, beta-apo-8-carotenal, retinol, and retinoic acid against B[a]P-induced mutagenicity and oxidative stress in human HepG2 cells. Secondly, we tested the hypothesis of modulating xenobiotic metabolizing enzymes (XMEs) by carotenoids and retinoids as a possible mechanism of protection by these micronutrients against B[a]P adverse effects. The obtained results declared that beta-carotene and retinol significantly reduced B[a]P-induced mutagenicity and oxidative stress. Tested carotenoids and retinoids reduced B[a]P-induced phase I XMEs and induced B[a]P reduced phase II and III XMEs. Thus, the protective effects of these micronutrients are probably due to their ability of induction of phase II and III enzymes and interference with the induction of phase I enzymes by the promutagen, B[a]P. It is highly recommended to consume foods rich in these micronutrients in the areas of high PAH pollution

Hygienic status of meat served at hospitals and its improvement after HACCP implementation

Examination of the microbial quality of meat served to patients at hospitals received little attention. Therefore, this study investigated the microbial status of meat served at hospitals in Zagazig city, Egypt. Furthermore, the effects of the implementation of hazard analysis and critical control point (HACCP) on the microbial status of meat were examined. Microbiological examination in this study included general microbial indicators (total aerobic plate count and most probable number of coliforms), isolation and identification of specific food-poisoning microorganisms including Escherichia coli, Salmonella spp., and Staphylococcus aureus. Due to the lack of the available information about the virulence of the isolated pathogens and their multidrug resistance profile in Egypt, multiplex PCR was used to detect the virulence-associated genes of Escherichia coli including shiga toxin, shiga toxin 2 and intimin in addition to invasive and hyper-invasive locus genes of Salmonella spp. Furthermore, Staphylococcus aureus enterotoxin (SE) coding-genes including SEA, SEB, SEC and SED were also investigated. Finally, antibiograms of the isolated food poisoning organisms were tested. The achieved results revealed inadequate hygienic measures performed at hospital kitchens, in terms of the high microbial load of meat either raw or cooked. Such meat was subjected to contamination by different types of microorganisms. The isolated strains showed variable degrees of virulence and multidrug resistance for the commonly used antibiotics in Egypt, which may therefore cause sever adverse outcomes to patients and stuff if such contaminated meat is served. Implementation of HACCP parameters significantly improved the microbiological quality of meat