Mycotoxins are secondary metabolites of various fungi that have toxic effects on humans and animals and its effects are called "mycotoxicosis". The severity of mycotoxicosis also varies depending on factors such as age, gender, nutritional status in organisms exposed to this toxin
29. Among the studies on mycotoxins, aflatoxins take the first place which are known to have very strong hepatotoxic effects. They are mostly synthesized by A. flavus, A. nomius, A. parasiticus, A. bombycis and A. pseudotamarii
2-4. Mean and standard deviation (mean±standard deviation) AFM1 levels in goat milk according to country is 14.5±8.4 ng/L in Italy
30, 19±13.8 ng/L in Syria
31, in Pakistan 2.0±5.0 ng/L
32, 31.8±13.7 ng/L in Iran
33, 7.6±8.94 ng/L in Croatia
34. Ozdemir et al.
35 reported that the mean and minimum-maximum (mean (min-max)) AFM1 concentration in goat milk was 19.23 (5.16-116.78) ng/L in March and April in goat breeding enterprises in Kilis province. Karadal et al.
36 determined that mean (min-max) AFM1 concentration in goat milk was 3.07 (0.33-11.79) ng/L in goat breeding enterprises in Niğde province.
In our study, the AFM1 concentration was found to 31.21±5.00 ng/L in the Group 1, and 24.66±4.76 ng/L in the Group 2. In also in our study, samples were taken in (April). Age-related changes were taken into account, therefore, healthy goats in lactation were selected in the close age range. Therefore, the changes that would occur due to these factors have been eliminated. Considering the nutritional status, the both groups of animals are given ground barley and then grazed in the bush. While the animals in Group 1 are grazed in the bush area close to the district waste disposal area, the animals in Group 2 are grazed in the normal bush area. Considering this information; it was observed that the AFM1 concentration in the milk of the goats in Group 1 was significantly higher (P<0.001) compared to the goats in Group 2. This situation believed to be associated with the consumption of mycotoxins produced by molds growing on the discarded food wastes. There are six types of aflatoxins: B1, B2, G1, G2, M1 and M2 37. and they are listed as AFB1 > AFG1 > AFB2 > AFG2 in terms of toxic effect. AFM1 and AFM2 are toxic metabolites of AFB1 and AFB2, respectively. Excretion occurs during lactation in animals fed with aflatoxins contaminated feed. This excretion occurs in the form of AFM1 and AFM2. In the studies, a linear relationship was found between the rate of AFB1 consumed by the animal and the rate of AFM1 excreted from its milk 38,39. It varies according to the breed of animal, milking period, milking interval and time. It has been reported that approximately 0.3-6.2% of AFB1 in milk is metabolized to AFM1 40. Feeding the offspring with milk and dairy products containing AFM1 can cause serious problems 14,41,42. In the present study, AFM1 levels were determined especially in milk due to the above concerns. Aflatoxin has been found in the ruminants milk and poultry eggs which were fed with aflatoxin-contaminated feeds 43. When feeds containing aflatoxin is taken into the gastrointestinal tract, it is converted to partially water-soluble conjugation products by the rumen microflora in the digestive tract of ruminants. They are easily absorbed and transported with a significant amount of serum albumin. It is distributed to soft tissues, mainly to liver. Aflatoxins in the circulating blood are highly retained in the liver. While some of the aflatoxins in the liver combine to large molecules such as hepatocytes, DNA, the binding surfaces of endoplasmic steroids and enzymes, another part is converted into oil- and water-soluble aflatoxins P1, Q1, B2a, aflatoxicol M1 and M2 in forms and rates that differ according to species. The shaped metabolism products are mostly excreted with bile 44. Aflatoxins are also metabolized by the urinary system. In most of the animal species, 50% of the amount excreted is with biliary secretion, while the amount removed by urinary elimination is about 15-25% 45. In the present study, low level of AFM1 was also observed in the animals grazed on the normal pasture and given the barley crumb, compared to the animals grazing on the edge of the waste, which indicates that aflatoxin can be seen in normal pasture as well. In addition, it was thought that the source of this low aflatoxin might be caused by cracking barley. Mortality rates from aflatoxicosis are reported to vary between 39% and 50%. Aflatoxicosis can be defined as acute or chronic due to aflatoxin’s dose and exposure time 46-48. The main toxic effects that occur in acute overdose exposure are hepatotoxicity, nephrotoxicity and sometimes death. In chronic exposure, genotoxicity, carcinogenicity and reproductive disorders are encountered 49. Animals such as ducks, sheep, turkeys, dogs, pigs and rats are most susceptible to AFB1. Monkeys, chickens, mice and ruminants are more resistant to AFB1 50.
Regarding the immunostimulatory effects, there is an increasing evidence that aflatoxins elicit a biphasic immune response with a stimulating effect in the first phase and a suppressive effect in the second phase 51. According to Valtchev et al. 52, short-term exposure to low-dose aflatoxins stimulates the immune system, while prolonged exposure to high doses shows immunosuppressive effects. For example, upregulation of the expression of toll-like receptors has been observed in different immune cells in different organs when exposed to very low levels of aflatoxin. Cytokines are stimulated in response to aflatoxin exposure 50. With the stimulation of cytokinin, an acute phase response develops and leads to the formation of acute phase proteins. Hp is one of these acute phase proteins. In inflammatory conditions, stress, tissue damage, and some non-tissue damage-related conditions are also increased 23,53,54.
In studies on Hp in goats, Hp values vary according to the measurements made with different ELISA kits. 19,55-58 It is also noteworthy that the heights in the standard deviation values of Hp values 55,56. Gonzalez et al. 55 reported that the mean and standard deviation values in healthy goats were 89±105 μg/mL. As it can be seen, the standard deviation value of the Hp value is higher than the mean and shows serious variability. In addition, in the same study, it was noted that the Hp value increased up to 4 times due to the disease 55. For this reason, comparison between groups would be more appropriate instead of comparison with reference values in goats. In the light of this information in our study, the Hp value was found to be 188.62±137.75 μg/mL in the group with high AFM1 level, and 24.66±4.76 μg/mL in the group with low AFM1 level. In addition, a moderately significant positive correlation was found between AFM1 and Hp values (r=0.374; P=0.017). It can be seen from the results that, as AFM1 increases, acute phase response develops, and Hp value increases.
Free Radicals are reactive molecules containing one or more unpaired electrons in their outermost orbitals. Radicals containing oxygen in their main skeleton are called free oxygen radicals 59. Free radicals are produced in physiological amounts in all living cells. When they are overproduced, they cause cell and tissue damage. These effects of free radicals are eliminated by some enzymes and molecules called antioxidants. Oxidative stress is occur due to an imbalance between the production of free oxygen radicals and their elimination by antioxidants. Free radical reactions cause oxidation of lipids, proteins and polysaccharides and DNA damage, which has significant toxic biological effects 7,60,61.
TOS can be used as an indicator for oxidants produced by the organism and taken up by environmental factors 62. It is reported that TOS levels will increase in inflammatory diseases 10. OSI is a key factor in determining oxidative stress 41. Various studies have been conducted to evaluate OSI in the determination of oxidative stress in farm animals. As a result of these studies, it has been reported that OSI values increase significantly in diseases where oxidative stress occurs 63.
Antioxidants serve to protect cells from the destructive effects of oxidative stress. Considering the abundance of antioxidant substances and pathways; in the prevention of oxidative stress and in determining the overall antioxidant capacity, determining the quantitative antioxidant power or TAS level in biological samples is a serious point. Antioxidative status evaluation can also be done by measuring TAS levels 7. Antioxidants neutralize free radicals and protect the body against oxidative stress. The level of antioxidants decreases during oxidative stress 7,10,63.
TAS value in healthy goats ranges from 1.18 to 1.79 mmol/L 64. In our study, the TAS value was found to be 1.94±0.36 mmol/L in the group with high AFM1 level, and as 1.45±0.09 mmol/L in the group with low AFM1 level. In addition, a moderately significant positive correlation was found between AFM1 and TAS values (r= 0.320; P= 0.044).
The TOS value in healthy goats ranges from 1.05 to 1.36 mmol/L 64. In our study, the TOS value was found to be 2.02±1.31 mmol/L in the group with high AFM1 level, and 3.42±1.11 mmol/L in the group with low AFM1 level. In addition, a moderately significant negative correlation was found between AFM1 and TOS values (r= -0.388; P= 0.013).
In our study, the OSI value was found to be 0.10±0.07 in the group with high AFM1 level, and 0.23±0.080 in the group with low AFM1 level. In addition, a moderately significant negative correlation was found between AFM1 and OSI values (r= - 0.419; P= 0.07).
Radical damage that frequently occurs in the organism is lipid peroxidation. Fat radicals are formed as a result of the separation of a hydrogen from fatty acids in the cell membrane. As a result, aldehydes, which are cytotoxic products, form hydrocarbon gases such as pentane. Of these toxic products, malonaldehyde is the last step of aldehydes. MDA is used to determine lipid peroxidation. MDA is an indirect indicator of injury caused by reactive oxygen species 3. It is known that there is a positive relationship between milk AFM1 levels and blood malondialdehyde (MDA) levels 3,65-67. Therefore, the positive relationship between MDA and AFM1 is an indication that AFM1 leads to a significant increase in lipid peroxidation.
However, its effect on other free radicals is not clear. Because AFM1, which is known to increase MDA, caused not an increase but even a decrease in TOS, which is an indicator of oxidative stress. It caused an increase in TAS. This has been attributed to the absence of an imbalance between the production of free oxygen radicals and their elimination by antioxidants.
Another possible reason is although AFM1 increases MDA by increasing lipid peroxidation; in our study, it was observed that the oxidative stress was lower in the group with high AFM1 and the oxidative stress was higher in the group with low AFM1. However, in the findings of antioxidant capacity; it was observed that the antioxidant system was higher in the group with high AFM1 and the antioxidant system was lower in the group with low AFM1. This situation has been associated with the moderately high AFM1 level stimulating the immune system by stimulating antioxidant systems and suppressing oxidative stress.
In conclusion, this study showed that AFM1 levels were found to be high in milk of goats grazing on the edge of the dump. It was observed that AFM1 caused an increase in TAS and Hp values, and a decrease in TOS and OSI values. It was observed that while AFM1 increased TAS and Hp values increased, it caused a decrease in TOS and OSI values It is thought that the moderately high AFM1 level can stimulate the immune system by suppressing oxidative stress by stimulating the antioxidant systems. In future studies, AFM1 level, oxidant and antioxidant capacity and acute phase response should be examined in more detail together with the immune system. In addition, the edges of waste disposal centers should be surrounded with wire to prevent animals from reaching toxic and moldy products.