DETERMINATION OF SOME ANABOLIC HORMONE RESIDUES IN CATTLE MEAT CONSUMPTION IN VAN, TURKEY.

The aim of this study was to determine some anabolic hormone residues (zeranol, trenbolone, DES, and testosterone) in cattle meat offered in the markets of the Van province. Cattle meat samples used in the study were collected from supermarkets, butchers, and slaughterhouses in Van Province between September 2013 and September 2014. A total of 80 samples consisting of approximately 50-150 g fat-free portions of cattle meat were collected and transported to the laboratory in the shortest time possible, after they were stored at 20° C until the analysis. Residual amounts of zeranol, In this study, analyzes of anabolic materials were performed by ELISA technique. It is known that zeranol analysis in biological materials can shift towards positive direction (Cooper et al ., 2003). In zeranol analyzes made on the meat samples, zeranol can be detected in low amounts even if the samples contain no zeranol. Positivity values can be much higher than normal when extraction is not performed appropriately. For these reasons, the results can be misinterpreted if they are not evaluated carefully.

The aim of this study was to determine some anabolic hormone residues (zeranol, trenbolone, DES, and testosterone) in cattle meat offered in the markets of the Van province. Cattle meat samples used in the study were collected from supermarkets, butchers, and slaughterhouses in Van Province between September 2013 and September 2014. A total o f 80 samp les consisting of approximately 50-150 g fat-free portions of cattle meat were collected and transported to the laboratory in the shortest time possible, after they were stored at -20° C until the analysis. Residual amounts of zeranol, trenbolone, DES and testosterone in the samples were determined by competitive enzy me immunoassay (ELISA ) technique. Preparative kits (RIDESCREEN® Test Kits, R-Biopharm A G, Darmstadt, Germany) were used for the extraction and residue detection procedures, as recommended by the manufacturer company. Of all the samples in the study, the number of positive samples corresponding to each of the inspected residues were as follows: 37 (46.25%) t renbolone, 34 (42.5%) zeranol, 22 (27.5%) testosterone, and 11 (13.75%) DES. The highest residual values were determined as 230.7 ng/kg for the trenbolone in August, 1810.0 ng/kg fo r zeranol in September, 2004.49 ng/kg for testosterone in October, and 171.83 ng/kg for DES in November. In th is study, trenbolone, zeranol, testosterone and DES residues were detected in the cattle meat offered in Van province markets. Residue quantities determined in cattle meat samp les do not exceed acceptable residue limits. However, p rohibited except for cases of anabolic veterinary treat ment applicat ions. The results obtained in this study show that different anabolic materials are being used for increased meat production, even though they are forbidden. For this reason, effective monitoring and inspections must be carried out at all stages from production to consumption with regard to the use of anabolic agents in livestock. It is important to remember that ELISA provides good results and rapid screening, but incorrect results may be obtained due to cross reactions.
At least one of the three of the protein consumed daily by a sufficiently balanced diet must be derived fro m sources of animal orig in (Uran, 2013). Meat and meat products are impo rtant food types as high-quality protein sources with high biological values. For this reason, demand for quality meat and meat products is steadily increasing. In developing countries, the consumption of food orig inating fro m animal sources has recently approached the le vels of developed countries. The production of animal origin foods, especially meat and meat products, has substantially increased throughout the world to meet the increased demand (Bender 1992;Bo land et al., 2013;Wolk, 2017).
Meat and meat products, which are increasingly consumed all over the world, are types of food that are open to various manipulations with the purpose of by passing, legal regulations. The reasons for the cheating attempts on meat products are the high prices, the difficulties of their production, and the inability of the current production capabilit ies to meet the demand adequately (Barai et al., 1992;Cawthorn et al., 2013). In order to increase the production of meat and meat products, and to close the an imal protein barrier, some fertilizers were used all over the world. Antibiotics, chemotherapeutics, ionophores, hormones and hormone -like anabolic substances were used for this purpose. While these hormones and hormone-like anabolic substances increase the weight-gain performance and meat yield of the animals, when used unconsciously in overdoses, they accumulate in various organs and tissues and leave residues, which can lead to various health problems (Akıllı, 1996;Saraç et al., 1999;Şanlı, 1989). In recent years there is growing suspicions and evidence about a connection between the consumption of high amounts of red meat, especially processed meat products, with some important and common diseases such as diabetes, coronary heart disease, heart failure , stroke and cancer (Wolk, 2017). With these worries, the European Union Food Safety Agency (EFSA) has reported that all six types of hormones can cause endocrine, developmental, immunological, neurobiological, immunoto xic, genotoxic, and carcinogenic effe cts for the susceptible risk groups, and has forbidden the import of meat obtained from countries which do not regulate their meat productions accordingly (Donovan, 2015).
Hormones play an extremely important role in maintaining the normal life functions of the body. This hormone balance is necessary to obtain maturation, sexual development, regeneration, and the replacement of many physiological functions. Disruption of hormone balance can cause malfunctioning of certain organs or disorders that affect the whole body. The hormone levels in children and adults can be affected by hormone consumption. The consumption of meat and meat products derived fro m hormone-applied animals can cause consumers to be exposed to various levels of hormone residues. There is a concern that hormones, which are very important for the human body, can cause health risks if they are consumed, even in residual quantities found in foods of animal origin. The amount of exposure to hormone residues varies individually depending on the consumer's eating habits, the amount of hormone residues in the animal's flesh, and the product's concentration (Anonymous, 2000). The presence of androgenic hormone residues in the meat (such as testosterone, trenbolone acetate), may affect v irilizat ion in wo men (menstruation) and cause menstrual cycle disorders; while estrogenic hormone residues have been reported to cause femin izing impotence, and infertility in men. In addit ion to these general hormonal activit ies of hormone residues, they may also act as carcinogens, and cause nervous system co mplications and arteriosclerosis (Hero, 2017).

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Veterinary drugs with anabolic effects, even some used for therapeutic and prophylactic purposes, also may be used to enhance the ability of the animal to benefit fro m the feed. That being said, many of them are prohibited in the European Union and are only allowed to be used in specific situations under strict control measures (Reig and Toldra, 2008). Growth regulating substances acting like hormones are called "anabolics".
Sex hormones such as estrogens (estradiol), androgens (testosterone) and progestogens (progesteron) are sometimes to provide more live weight gain in animals, to increase utilizat ion from the feed, to develop better carcass and meat quality. Synthetic non-steroidal estrogens such as diethylstilbestrol (DES), hexoestrol, dienestrol and zeranol, and synthetic steroids such as trenbolone acetate (TBA), testosterone propionate, melengestrol acetate, methyltestosterone are used as hormonal effect anabolic agents (Hero, 2017). Estrogenic and androgenic agents were used in the meat industry for about 50 years to increase meat production and profitability. These agents can be used alone, or in co mbination with some other agents. Such estrogenic preparatio ns are effective for the growth of the dendritic systems, wh ile androgenic preparations are especially potent in the growth of the ovary (Dunshea et al., 2005). Weight gain in butchered cattle using anabolic hormone imp lants is increased by 10-20% co mpared to untreated animals. In addition, 8% less fat was detected in slaughtered animals using imp lants compared to animals not using the same implant (Dikeman, 2007). Because of these positive effects, the uncontrolled and widespread use of anabolic agents has increased. These drugs are now prohibited and the productions of some of them were stopped (Toldra and Reig, 2006). In particu lar, the use of 6 of the growth -promoting hormones from anabolics is risky in terms of hu man health. These are; 17β estradiol, testosterone, zeranol, progesterone, trenbolone acetate, and melengestrol acetate (Anonymous, 1999).
This study was designed to contribute to the identificat ion of trace levels of these banned hormones and to statutory auditing and monitoring activities, thereby contributing to the minimizat ion of the negative consequences of the uncontrolled hormone use. The situation is particularly of importance these days: as the date of the greater aid approaches, more breeders are tempted to use these illegal hormone med icines in order to increase the weight gain of the animals in a very short period of time.

Materials And Methods:
Meat samples were collected fro m large markets, butchers, and slaughterhouses. 80 samp les of 50-150 g fat-free cattle meat were collected between September 2013 and September 2014 and were stored at -20 °C until analysis. Analyzes were performed using the ELISA test (Bio-tek Instruments ELX 800 UV Reader, Bio-tek Instruments In the evaluation of the data obtained from the samples in the study were used the numbers of the samples, the corresponding average amounts of these numbers, the standard errors, the min imu m and maximu m values.

Results:
The amounts of hormones detected in analyzed samp les and the distributions according to the months are given in table 1, table 2, table 3 and table 4.

Discussion:
Anabolic agents are substances that increase growth in cattle, sheep and even in pet animals, and they cause substantial weight gain. These effects are induced by increasing protein synthesis and reducing fat tissue (Lone 1997;Turner et al., 1995). A lthough the use of hormones for weight increase purposes contributes to productivity, the opinion among European countries and the United States towards their use is vastly different. The European Union forbids the use of anabolic hormones as a growth promoter in slaughterhouse animal breeding. The FDA, on the other hand, has allowed the use of some naturally occurring hormones (estradiol and testosterone) in animal breeding (Sawaya et al., 1998b).
According to the Communiqué of the Turkis h Food Codex in our country, the follo wing hormones and substances are prohibited for use on animals with food value, or may only be used under some specific conditions; stilbenes, stilbene derivatives, anti-thyroidal substances, steroids suitable for anabolic purposes, resorcylic acid lactones including zeranol and β-agonist substances (Anonymous, 2003).
There are two basic problems encountered during an inspection of hormone residues in animal products: to distinguish the animals own naturally occurring hormone levels fro m that of the residues, and the fact that each animal species has different hormone levels (Şener, 1994). Generally, the use of growth promoting substances should be inspected from three perspectives: the producer, the animal, and the consumer. It would be ideal to use a growth accelerator substance that is economic fro m the producer's perspective, does not adversely affect the biological functions from the animal perspective, and does not adversely affect human health from consumer's perspective. The use of a material that does not meet all of these three conditions is strictly prohibited (Alpan, 1989). Despite the fact that the use of anabolic substances is prohibited in our country, these residual hormones were detected in meat and meat products through various researches, some of which reported very amounts of residual anabolics. In terms of consumer health and safe food consumption, it is necessary to carry out hormone analyzes on animal products more frequently, to increase the number of controls, and to initiate legal proceedings on those who were determined to have used them.
There are various studies that show the presence of residual hormones and similar substances with anabolic effects in animal-derived foods. In a study conducted by Kadim et al. (2010) in order to determine the residual levels of various antibiotics and hormones in chicken meat, it was reported that the residual levels of 17β -estradiol and testosterone were above the acceptable limits.
Sadek et al. (1998) investigated the presence of anabolic agent residues in meat and poultry samples collected from supermarkets and small-scale butcher shops and found that supermarket pickles contained higher levels of TBA residues than the samples fro m the butcher's shops. Similarly, they detected DES and estradiol residues in cattle meat and liver samples, albeit at low levels. They also detected that none of the samples contained zeranol.
In the study conducted by Nazlı et al. (2005a), none of the samples were found to contain DES and clenbuterol residues, whereas zeranol and DES residue levels were above the limit values in all 30 o f the samples. Fro m these determinations, it is suggested that hormone residues in meat and meat products may pose a risk for hu man health and that the control mechanis ms need to work better to prevent mass -scale health problems. In another study (2005b), the same researchers reported that they found zeranol in all 60 meat and meat products samples, TBA in 48 samples, and DES residues in 21 samples, whereas no clenbuterol was identified in any of the samp les.
135 Yılmaz et al. (2007) reported that they have determined zeranol in 18 samples, while they detected 6 samples of testosterone, 7 samples of TBA, 1 sample of DES and 22 of 17β-estradiol amongst 155 cattle meat in Tekirdağ. In another study (Akkaya et al., 2004), 300 chicken meat samples were analyzed for the presence of DES, zeranol, estradiol, testosterone, progesterone, andclenbuterol, and no anabolic hormone residues were found in any of the samples.
In the study conducted by Oruç et al. (2007) in Bursa, 2 of the 81 cattle meat samp les were found to contain zeranol, 11 o f the 80 samp les were found to contain DES, wh ile 3 o f the 29 samples were found to contain testosterone. In addition, 72 of the samples were analyzed for the presence of clenbuterol and 29 for 17β-estradiol residues and it was determined that the meat samp les did not contain the residues of the corresponding hormones. Zeranol (α -zeranol) is a non-steroidal resorcylic acid lactone formed o f zearalenone, a mycoto xin, and has estrogenic effects (Aksoy and Dagoglu 1998;Durmaz 1997;Daxenberger et al 2000). A joint committee between the World Health Organizat ion and World Agriculture Organizat ion has determined a tolerance level of 2 μg/kg zeranol for ed ible t issues of animals. In this study, it was found that of the 80 samples, 9 had 500 ng/kg (11.25%) zeranol, wh ile 6 had 400-500 ng/kg (7.5%), and 9 had 300-400 ng/kg 300 ng/kg (11.25%) of zeranol. When inspected on a per month basis, the highest values were found to be that of September and October. Even these values, however, do not exceed the limit of 2μg / kg (2000 ng/kg). Nazlı et al.

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In this study, analyzes of anabolic materials were performed by ELISA technique. It is known that zeranol analysis in biological materials can shift towards positive direction (Cooper et al., 2003). In zeranol analyzes made on the meat samp les, zeranol can be detected in low amounts even if the samples contain no zeranol. Positiv ity values can be much higher than normal when extraction is not performed appropriately. For these reasons, the results can be misinterpreted if they are not evaluated carefully.
Testosterone, although allowed to be used under certain circu mstances in some countries such as the US, it is prohibited in Turkey and the EU countries. Testosterone-implanted animals were reported to have a residual level of 0.031-0.360 μg/kg of testosterone, and animals with no hormones were reported to have a level of 0.006-0.029 μg/kg (Jeong et al., 2010). In this study, over 500 ng/kg testosterone was found in 15 of the 80 samples (19%): 400-500 ng/kg in 2 samp les, 300-400 ng/kg in 1 samp le, 200-300 ng/kg in 2 samples and 100-200 ng/kg in 1 sample. When evaluated in a per-month basis, testosterone was not detected in samples belonging to January, April, May, August, and September. It was also determined that the highest testosterone level in meat samples occurred in October. Oruç et al. (2007) found testosterone levels at a mean value of 329.96 ng/kg in three out of 29 samples (7%). The results obtained in our study are higher compared to the results found by Oruç et al.
Diethylstilbestrol is a synthetic estrogenic compound with a potent accelerating effect on physical develop ment. DES is not allowed at any levels in anima l tissues since it has a carcinogenic effect and can't be metabolized by the organism (Durmaz 1997 When compared to the other months, the highest TBA level in meat samp les was found in August, the highest zeranol value of the meat samples was detected for September and October samples, the highest testosterone value was in October, and the highest value of DES was found in November. Considering that the great eid of 2013 was in October, this may indicate that some breeders took the advantage of approaching great aid by using illegal hormone med icines. Even though the sales and use of hormonal agents are forbidden in Turkey, it's possible that some contraband may be arriving fro m Iran.

Conclusions:
As a result; this study concluded that hormones or similar substances were actually being used in animals and while the zeranol and TBA levels detected in the samples did not exceed the tolerance limits effective control mechanisms still have to be developed to prevent the use of anabolic agents. In light of the findings obtained from the study, it was concluded that anabolic agents such as zeranol, DES and testosterone were being used in certain amounts even though they are forbidden in Turkey. The results indicate that consumption of the meat offere d in the market may pose a risk for public health as some samples contain the residues of these substances. As a matter of fact, developed countries have implemented many regulations to protect consumer health fro m the threat of anabolic substances in animal breeding, and to control their presence in foodstuffs. Information on whether or not anabolic is used in animal breeding in our country is not enough. The use of anabolic substances that are regarded a sharm less in terms of consumer health should be allowed. Those allo wed should be effectively inspected. Such agents should be licensed by the relevant ministry sold by veterinary prescriptions, and should be monitored under veterinary surveillance during their use, and if necessary after theiruse.
Considering ELISA tests may return false or higher-than-normal positive results due to cross -reactions for many of the substances, verificat ion of the levels of hormones determined in this study by further verificat ion tests is recommended. Many researchers have emphasized the necessity of confirming the hormone and drug residue values 137 obtained by ELISA method for meat products using more advanced devices and techniques simu ltaneously (Seo et al., 2005;Fuh et al., 2004;Sun et al., 2010;Chaoyang et al., 2008) .