الفهرس 
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Abstract
Vegetable oils contain high-energy dietary unsaturated fatty acids that are not only essential part of human nutrition but also contribute significantly to transportation of nutrients in the body. When vegetable oils are heated for a long time in the open air at high temperature, various chemical reactions (hydrolysis, oxidation and polymerization) occur. As a result of reactions, cooking oil produce volatile components which result into degradation the oil. Sunflower and soybean oil are the most used vegetable oils in the household and industry for deep frying purposes. Both sunflower and soybean oils are rich in tocopherols.
Reusing cooking oil in food preparation, especially during deep-frying, is a common practice to save costs. Repeated heating of the oil accelerates oxidative degradation of lipids, forming hazardous reactive oxygen species and depleting the natural antioxidant contents of the cooking oil. Long-term ingestion of foods prepared using reheated oil could severely compromise one’s antioxidant defense network, leading to pathologies such as liver disease, hypertension, diabetes and vascular inflammation.
One of the chemical byproducts formed during cooking at very high temperatures is a group of complex heterogeneous compounds known as advanced glycation end products (AGEs) which are a family of compounds that are the products of nonenzymatic reactions between reducing sugars and proteins, lipids, or nucleic acids. Roasting and broiling food at high temperatures is a common practice in cooking.
Advanced glycation end products can cause browning, fluorescence, and protein cross-linking, as well as the formation of flavor and aroma compounds. In addition, AGEs can occur endogenously in the body as well as exogenously in food. Although the formation of AGEs is a part of normal metabolism, it can become pathogenic if AGEs in tissues and circulation reach extremely high levels.
In addition, AGEs are important in clinical science because they are associated with oxidative stress (OS) and inflammation, processes that eventually cause most chronic diseases, including cardiovascular disease, diabetes, chronic kidney disease, and neurodegenerative diseases. Of note, AGEs cause OS but OS also leads to AGE formation. The liver is the main exposure organ of absorbed dietary AGEs and specialized liver cells, in particular endothelial and Kupffer cells, play an important role in clearing AGEs from the circulation.
This study aimed to evaluate the effect of consumption of repeated heated oils (RHOs) and advanced glycation end products (AGEs) on genetic and metabolic changes in male rats.
Mixed vegetable oil (sunflower and soybean) was heated for 10 minutes at 180°C in a stainless-steel wok, then left to cool at room temperature for five hours, this sample of oil was called one-time heated oil (1-THO). The heating and cooling process of oils were repeated five and ten times to obtain (5-THO) and (10-THO) respectively.
The experimental diets were prepared weekly using fresh and heated oils. The fat composition in the standard diet was replaced by mixture oil in fresh form, heated once, five times or ten times as follows:
Diet A: Standard balanced diet was prepared using fresh corn oil.
Diet B: Standard balanced diet was prepared using fresh mixed vegetable oil (Fresh MVO)
Diet C: Standard balanced diet was prepared using single- time heated mixed vegetable oil (1-THO).
Diet D: Standard balanced diet was prepared using five-time heated mixed vegetable oil (5-THO)
Diet E: Standard balanced diet was prepared using ten-time heated mixed vegetable oil (10-THO).
The diets (A, B, C, D, and E) were autoclaved for 30 minutes at 125∘C to obtain the high AGEs diet.
One hundred rats (Sprague –Dawley strain) weighing (120±10g) were divided into ten groups: 10 rats in each group. The experiment was divided into two parts as follows:
Part (1): Study the effects of consumption of repeated heated oils. It contained five groups as follow:
group 1: Healthy control group, rats fed on diet contained fresh corn oil.
group 2: Rats fed on diet which contained fresh MVO.
group 3: Rats fed on diet which contained 1-THO.
group 4: Rats fed on diet which contained 5-THO.
group 5: Rats fed on diet which contained 10-THO.
Part (2): Study the effects of consumption of repeated heated oils and advanced glycation end products (AGEs). It contained six groups as follow:
group 1: Healthy control group. As in part one, it’s the negative control group (joint group between part 1 and part 2)
group 1\: Rats fed on diet contained fresh corn oil after autoclaved (positive control).
group 2\: Rats fed on diet which contained fresh MVO after autoclaved.
group 3\: Rats fed on diet which contained 1-THO after autoclaved.
group 4\: Rats fed on diet which contained 5-THO after autoclaved.
group 5\: Rats fed on diet which contained 10-THO after autoclaved.
Food intake was recorded daily. The animals were weighted weekly to monitor body weight change and FER. At the end of experimental period (8 weeks) rats were scarified under Isoflurane anesthesia after 12 hours fasting with water ad libitum. Blood samples were collected from the eye. The liver and thyroid gland were separated immediately, and weighted, then part of the organ’s specimens was taken and fixed in formalin 10% for microscopic examination.
The following physical, nutritional, biological, and biochemical parameters were measured:
Peroxide value and fatty acid composition of oils samples.
Food intake, body weight change, and feed efficiency ratio.
Relative liver and thyroid gland weight.
Serum liver enzymes activity (ALT, AST. ALP)
Serum lipids profile (TC, TAG, HDL-C, LDL-C, and VLDLC) levels and atherogenic index.
Serum fasting glucose, insulin, and HOMA-IR levels.
Serum oxidative stress levels (AGEs, MDA and NO).
Antioxidant status (blood GSH level, serum TAC level and serum GPX activity).
Serum inflammatory level (hs-CRP and TNF-α).
Serum anti-inflammatory level (IL-10).
Evaluation of cell metabolic changes biomarkers in liver tissues (ATP production level, Acyl-COA oxidase, MRCC-I and MRCC-II, HMG-COA reductase activities).
Serum thyroid gland function tests (F-T3, F-T4, TSH and thyroidal iodide transporter) levels.
Relative gene expression of p53 in liver and thyroid gland tissues.
Serum behavioral changes markers (serotonin, dopamine, and norepinephrine) levels.
In addition, a microscopic examination of liver and thyroid gland was performed.
The results of the current study are summarized as follow:
1.Peroxide Value and Fatty Acid Composition of Oil Samples
Peroxide values of oils samples were (2.5, 11.1, 15.1, 24.9, and 23.9) meq/kg in fresh corn oil, fresh MVO, 1-THO, 5-THO, and 10-THO respectively.
Data showed that peroxide value of fresh MVO was higher than peroxide value (PV) of fresh corn oil. In addition, the PV increased with the increase in the number of heating cycles until 5 heating cycles then the value slightly decreased as shown in 10 heating cycles. Therefore, an increased level of PV of oil samples until 5-THO sample may be due to the high concentration of hydroperoxides formed, followed by a decreased PV level as in 10-THO sample may be due to presence of secondary oxidation products.
The present results showed that there were substantial differences in the fatty acid composition of the investigated oils. It was observed that corn oil contained the highest levels of unsaturated fatty acids while had the lowest levels of saturated fatty acids. from the current results it could be concluded that as the cycle of heating process increases the percentage of saturated and trans fatty acids increases as well as the percentage of unsaturated fatty acids decreases.
2. Food Intake, Body Weight Change, Feed Efficiency Ratio and Relative Organs Weight
Repeated heated oil caused significant reduction in food intake, body weight change, FER, and relative thyroid weight, while caused significant increase in relative liver weight as compared to healthy control, fresh MVO, and 1-THO rat groups. Generally, the worst effect on biological measurements was shown in 10-THO group followed by 5-THO group.
While consumption of AGEs diet containing fresh corn and MVO oils causes significant increments in food intake and body weight change as compared to healthy control group. On the other hand, consumption of AGEs diet containing repeated heated oils (5-THO and 10-THO) showed the worst effect which manifested by significant reduction in food intake, body weight change, relative thyroid weight while caused significant elevation in relative liver weight as compared to all experimental rat groups.
Long term consumption of oxidized oils and fats has been reported to cause growth retardation, thrombosis, fatty livers, essential fatty acid deficiency, nucleic acid deficiency and micronutrient malnutrition leading to deactivation of key metabolic enzymes. The present results reported the ability of AGEs to increase body weight by increasing energy intake. While, after consumption of AGEs diet containing heated oils for 5 and 10 times caused reduction in body weight and food intake. This may be due to highly toxic products accumulated from repeated heated oil and Millard reaction. These toxic products reduce nutritional value of diet and cause off- odor that decreases food intake. These compounds also cause enlargement of liver and reduction in thyroid gland weight.
3. Liver Function Assessment
All heated oils and heated diets rat groups showed liver function enzymes disturbance that was manifested by marked elevation in serum ALT, AST and ALP activities as compared to healthy control group. Generally, the worst effect was shown in rat groups consumed 10-THO followed by 5-THO either before or after diet autoclaved.
The elevation in serum AST, ALT and ALP activities may be resulted from the increase permeability of hepatocyte cell membrane which may occur due to the toxic effect of thermal oxidized oils products and dAGEs which cause cellular damage and in turn leakage of the enzymes outside the cellular structure to extracellular fluid.
4. Lipids Profile Measurement
Disturbance in serum lipids profile was illustrated in all heated oil and heated diets groups which detected by marked elevation in TC, TAGs, LDL-C, VLDL-C levels and atherogenic index as well as marked reduction in serum HDL-C level as compared to healthy control. It could be concluded that the worst hyperlipidemic effect was shown in heated diets containing repeated heated oils.
These results may be due to formation of highly toxic compounds during heating process and oxidation of polyunsaturated fatty acids (PUFAs) and increase concentration of saturated fatty acid which lead to lipid peroxidation and oxidative stress.
5. Serum Glucose Hemostasis
It was noticed that there was significant elevation in serum fasting glucose, insulin and HOMA-IR levels in all heated oils and heated diets rat groups compared to healthy control group. In addition, the worst effect was shown in heated diets containing repeated heated oil.
The mechanism of hyperinsulinemia caused by oxidative heated oil and AGEs consumption may involve impaired insulin secretion due to impaired function of pancreatic beta cells or increased liver extraction of insulin from the portal blood. It is known that oxidative heated oil feeding induces higher oxidative stress, which may result in oxidative damage to the pancreas.
6. Oxidative Stress and Antioxidant Biomarkers:
Rats consumed repeated heated oils and AGEs diets showed imbalance between oxidative stress and antioxidant status that can be inferred by elevation in serum AGEs, MDA and NO levels and significant reduction in serum TAC level, blood GSH level and GPX activity as compared to healthy control rat groups.
Thermally oxidized oils induce oxidation of PUFAs and increase oxidative stress. Glycation and oxidative stress are closely linked, and both phenomena are referred to as ‘‘glycoxidation’. All steps of glycoxidation generate oxygen-free radical production, some of them being common with lipidic peroxidation pathways. In addition, heating of oils and diets decrease its antioxidant properties.
7. Inflammatory and Anti-inflammatory Markers
The present study showed that all heated oils and heated diets rat groups showed significant elevation in serum TNF-α, hs-CRP levels, and showed significant reduction in serum IL-10 level as compared to healthy control group. These results occur due to excessive production of free radicals produced during lipid peroxidation and glycoxidation may lead to oxidative stress, subsequently activating several molecular pathways that generate inflammatory molecules.
8. Cell Metabolic Changes Biomarkers in Liver Tissues
Consumption of repeated heated oils and AGEs showed disturbance in cell metabolic function which manifested by significant elevation in hepatic Acyl COA oxidase and HMG-COA reductase activities as well as significant reduction in hepatic level of ATP production and MRCC-I and MRCC-II activities as compared to healthy control group. This may be due to the formation of toxic products, carbonyl compounds and ROS that increase oxidative stress that alter mitochondrial function and ATP production.
9. Thyroid Gland Function Tests
Thermally oxidative oils and AGEs consumption showed bad effect on thyroid gland function which cleared by significant reduction in serum free T3, T4 and thyroidal iodide transporter levels and significant elevation in serum TSH level as compared to healthy control group.
During the heating process of oils and diets, many chemical processes occur, like the oxidation of different fatty acids and triacylglycerols, polymer or cyclic compound formation, loss of volatile compounds, AGEs and the formation of polycyclic aromatic hydrocarbons that could downregulate the expressions and activity of the key molecules of thyroid hormone synthesis.
10. Hepatic and Thyroidal P53 Gene Expression
The data showed that there was significant elevation in hepatic and thyroidal P53 levels in all heated oils and diets rat groups as compared to healthy control rat groups. It could be concluded that the worst effect on hepatic and thyroidal P53 levels was shown in rat groups consumed heated diets containing repeated heated oils (5-THO and 10-THO). This mutagenic effect may be due to the formation of oxidative compounds of lipid peroxide or heterocyclic aromatic amines (HAAs) that induce cell apoptosis, and make the cells stagnate in G0/G1phase.
11. Behavioral Changes Markers Level
The present result showed that there was significant depletion in serum levels of dopamine and serotonin as well as significant elevation in serum norepinephrine in all repeated heated oils and heated diets groups as compared to healthy control group. Advanced glycation end products neurotoxicity is associated with the enhancement of lipid peroxidation and the reduction of antioxidative capacity in nerve tissue which is mostly caused by a primary depletion of antioxidant.
12. Microscopic Examination of Liver and Thyroid Gland
Liver sections from repeated heated oils and heated diets showed dilatation in the central vein, while the parenchyma showed focal area of degenerated and sever fatty changes hepatocytes with few inflammatory cell’s infiltration in between. In addition, thyroid sections from repeated heated oils and heated diets showed that the follicles showing empty and narrow obliterated lumen.