2.1. Study Subjects
High Fructose Corn Syrup 55 (HFCS-55)
HFCS-55%, was kindly provided and certified by Zar Fructose Co., Ltd., Tehran, Iran in March 2020, under the code number of ZFQS04 with confirmed analytical sheet. According to analytical sheet, 100 ml of HFCS-55 provides 71.72 g carbohydrate (Fructose and Glucose), also the estimated energy of 100 ml of HFCS-55 is 286.9 Kcal.
Sucrose Syrup 75%
Purified and crystallized sucrose (saccharose) with over 99.7 ºZ polarization was provided and certified by Merck Millipore Co., Ltd., Germany under the CAS number of 57-50-1 with accessible analysis sheet in March 2020.Sucrose is made up of one molecule of glucose and one molecule of fructose joined together. It is a disaccharide, a molecule composed of two monosaccharaides: glucose and fructose. Sucrose produces naturally in plants, from which, table sugar refines. Sucrose is the most abundant disaccharide and the major product of photosynthesis. Sucrose syrup 75% was prepared by dissolving 75 g Sucrose with properties mentioned above in distilled water and adjusting up to 100 ml. We prepared each 100 ml of sucrose syrup 75% which provides about 280-290 Kcal energy.
Experimental animals and housing conditions
This study was carried out in a group of 15 mature regularly cycling female Wistar rats, aged 5-6 weeks, with initial body weights of 165-180 g which were purchased from Pasture Institute of Iran. Each 5 female rats were housed together in one standard cage in 12-h light/dark cycle (07:00-19:00), 23 ± 2°C temperature, relative humidity of 30-70%,8-15 times/h air change with access to tap water and standard diet ad libitum. The cage cleaning schedule, air filtration and recirculation, health checks and facility maintenance were carried out following to one-week adaptation and acclimation period.
This study was approved by the ethics committee of Islamic Azad University (IR. IAU. TMU REC.1399.216), and the animal care protocol was consistent with the committee’s guidelines for the care of animals accordance to the Standard Operating Procedures of Ministry of Health and Medical Education of Iran for the Care and Use of Laboratory Animals and Canadian Council of Animal Care (CCAC) Guidelines for Care and Use of Experimental Animals.
2.2. Repeated dose oral toxicity study
The practical model of the experiment was conducted based on OECD 408 toxicology guideline.
After 10 days adaptation period, healthy female rats were randomly divided into 3 groups (5 rats/group) and labeled as Fo (HFCS-55), So (Sucrose 75%), and Co (control) groups. They were given free access to HFCS–water (HFCS group), Sucrose-water (Sucrose group) or deionized water (control group). Water and food consumption, general behavior and body weights were daily measured and recorded. Daily calorie intakes were calculated based on the calories levels in Table 1 and adjusted to the same levels based on the calorie levels obtained from ingested fructose or sucrose and standard rodents chow (3.34 kcal/g).
Clinical variables were considered as drinking and eating patterns, quality of response to environmental stimuli, surface of body reactions, body hair changes, drowsiness, any change in stool and urine colors, bringing up the tails, abnormal or ataxic gaits, any changes in eyes, salivation, changes in tear and total weight. At day 90 overnight fasted rats were sacrificed after blood collections by heart puncture under light carbon dioxide anesthesia and whole bloods were drawn for further biochemical analysis.
2.3 Biochemical Analysis
The collected blood samples were centrifuged at 1500 g for 15 min at 4o C to obtain serums, and then the whole plasma were isolated and kept at -80 C until further analysis. The levels of glycemic factors were measured using an auto analyzer (HITACHI 917 / OLYMPUS AU640 & COBAS INTEGRA). Other than above factors. ,lipid profile of animals was evaluated using serum concentration of total cholesterol, HDL, LDL and ratios using spectrophotometric enzyme assay kits (Amara Siri SS, 2020) in Bahar Toxicology Laboratory in Tehran.
2.4. Hormonal Assay
In order to determine the variations in serum concentrations of FSH, LH, Testosterone, Estradiol and progesterone , tail blood samples were collected according to standard protocol at day 15, 30,45,60, 75 and 90 of study and the serum was separated by cold centrifuge and collected in microtubes. Hormone Levels were compared with control by Chemiluminescence Immunoassays (CLIA) method using Cobas E411 from Roche Company with serial number of 15D3-16.
2.5. Recovery studies
A commercial rat serum pool (catalog no. M5905, Sigma Chemical Co., St. Louis, MO) was spiked with various hormone concentrations, and percent recovery and parallelism to the assay standard curve were determined. For each assay, the serum pool was spiked either with hormones across the assay range or vehicle to determine endogenous hormonal levels in the pool. Samples were run in duplicate, and each assay was repeated to confirm results. Hormonal recovery from each spiked sample was determined by subtracting hormone values in vehicle-spiked controls from hormone spiked samples.
2.6. Necropsy and Histopathological studies
During necropsy study, reproductive organs including uterus and ovaries were dissected out. Intact organs were rinsed and weighed with physiological serum to remove substances that may interfere with later stages and weighted. Organs were fixed in 10% formalin solution at the next step. To remove the water, the desired tissue was dehydrated with degrees of alcohol (30, 50, 70, 80, 90 and absolute alcohol). To strengthen the fixed tissues, they were placed in a paraffin blocks and finally 5 micron thin sections were prepared with a microtome. Multiple sections from each block were subsequently prepared at 5-micrometer diameters and stained with hematoxylin and eosin (H&E) for microscopically evaluations. The sections were examined under the light microscope (Olympus BX-51, Olympus, Tokyo, Japan) by expert animal pathologist and scored.
2.7 Immunohistochemcial Assay of sex hormone receptors
As previously described (Bidgoli SA ), (Bidgoli SA, 2011), dewaxed and rehydrated tissue sections were subjected to antigen retrieval using microwave oven and boiling citrate buffer (pH=6.0). Endogenous peroxidase activity and nonspecific binding sites were blocked by incubating sections by 0.3% hydrogen peroxide in methanol for30 min and 3% BSA for 60 min, respectively. Sections were then incubated 30 min at Room Temperature with AR (Clone AR441, Dakocytomation) that recognize the nuclear expression of rat proteins in reproductive tissues. The results were visualized using Opti View DAB detection kit based on the manufacturer's instruction with necessary modifications. Sections were also counterstained with Meyer's haematoxyline. In each series, a section in which incubation with the primary antibody was omitted used as negative control. The ideal staining conditions were established in our preliminary experiments. Staining was considered negative only after careful examination of the entire tissue section. All samples were assessed and scored by two independent pathologists. Histoscores of androgen receptor was assessed according to Intensity score and Allred score (P+I). In each case, a histoscore with a potential range of 0–300 was calculated as follows:
No immunoreactive cell: negative (0)
≤ 1% of cells are immunoreactive: Weak positive (1+)
1–10% of cells are immunoreactive: Intermediate positive (2+)
11–33% of cells are immunoreactive: Strong positive (3+)
>34% of cells are immunoreactive: Very strong positive (4+)
2.7. Statistical analysis
Study groups compared with one-way analysis of variance (ANOVA) and Post-hoc test. By student’s t-test the difference between two sample means compared. Parametric values were expressed as mean ± standard deviation (SD) and the level of significance was set at p<0.05 in SPSS Statistics Software (version 21).