Lead acetate was purchased from Sigma Chemicals (St. Louis, MO, USA). APG (Cat. No. 520-36-5) was purchased from (Matrix Scientific, Columbia, SC). Levels of total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein cholesterol (HDL-C) were determined by standard kits (BIOMED Diagnostics, Germany). the units were expressed as mg/dL.Malondialdehyde level, as an indicator for lipid peroxidation, Superoxide dismutase, Catalase and glutathione peroxidase activities were determined using commercial kits (catalog # MD2528, SD2520, CA2516, GP2524 respectively) obtained from Diagnostic, Giza, Egypt. TNF α was determined using Human ELISA Kit (catalog # EA100365) OriGene Technologies Inc., Rockville, MD, IL-6 was determined using Rat Interleukin 6 (IL-6) ELISA Kit (catalog # MBS726707), IL-4 was determined using Rat Interleukin 4 (IL-4) ELISA Kit (catalog #MBS162452) , Interleukin 10 (IL-10) was determined using Interleukin 10 (IL-10) ELISA Kit ELISA Kit (catalog #MBS764911) MyBioSource, Inc., San Diego, USA. Determination of caspase-3 was done using Biotechne kit (catalog # BF3100) (USA). T4 ELISA kit (Cat. No. 60863) was purchased from Kamiya Biomedical Company, WA, USA. T3 solid-phase competitive ELISA kit (Cat. No. T3043T-100) was obtained from CalbioTech Inc., Spring Valley, Canada. TSH ELISA kit (Cat. No. CSBE05115r) was obtained from CUSABIO Biotech Co., LTD, Wuhan, China. Kit for determination of T was purchased from K-assay, WA, USA. LH and FSH kits were obtained from Biovender, Tokyo, Japan. Determination of caspase-3 was done using Biotechne kit (catalog # BF3100) (USA).
Twenty-four adult male rats (Rattus norvegicus) weighing 180-200gm were purchased from the animal house of Faculty of Science king Faisal University, Saudi Arabia. All experimental procedures were done according to the research ethics at King Faisal University. The rats were housed in plastic cages (6 per cage), floored with soft a wood shaving that was changed three times per week. The animals were acclimatized for 2 weeks prior the study and were maintained under a 12 h light/dark cycle at (25 °C ± 2 °C), with free access to water and rat chow.
Preparation of treatment materials:
- Lead acetate dose: dosage of 20mg/kg body weight (b.w.) was dissolved in normal saline and administrated intraperitoneally (i.p.) (El-Neweshy and El-Sayed, 2011).
- Apigenin dose: Apigenin (20mg/kg b.w.) was dissolved in normal saline and administrated (i.p.) (Venigalla et al., 2015, Yang et al., 2017).
Animals were randomly divided into four groups of six animals each as follows:
Group I: served as the control group. Rats were daily injected (i.p.) with normal saline (0.9% Na cl) as vehicle.
Group II: Served as lead acetate – treated group. Rats were daily injected (i.p) with lead acetate dissolved in normal saline at a dose of 20mg/kg b.w.
Group III: Served as lead acetate and Apigenin-treated group. Rats were daily injected (i.p) with lead acetate dissolved in normal saline at a dose of 20mg/kg (b.w.) followed by injection (i.p) with Apigenin dissolved in normal saline at dose of 20mg/kg b.w.
Group IV: Served as Apigenin-treated group. Rats were daily injected (i.p) with Apigenin dissolved in normal saline at dose of 20mg/kg b.w.
At the end of the 4-week experimental period, the animals were sacrificed and samples of blood and testis tissues were collected for analysis.
Estimation of studied parameters:
a) Evaluation of lead level:
The lead concertation was determined in blood and cerebellum tissue using Atomic Absorption Spectroscopy.
b) Biochemical parameters:
I-Determination of serum lipid profile parameters
Levels of total cholesterol (TC), triglycerides (TGs), and high-density lipoprotein cholesterol (HDL-C) were determined by standard kits according to the instructions of the supplier. The levels of LDL-cholesterol (LDL-C) was calculated using Friedewal's formula, the units were expressed as mg/dL.
Preparation of tissue homogenate
Chosen tissues were immediately removed and washed using chilled saline solution. These tissues were minced and separately homogenized (10 % w/v) in ice-cold sodium-potassium phosphate buffer (0.01 M, pH 7.4) containing 1.15 % KCl using a homogenizer (Potter–Elvehjem). The homogenate was centrifuged at 10,000 xg for 20 min at 4°C and the resultant supernatant was used for the assay of the enzyme activities, the level of MDA and the protein. The protein content of the tissues was determined by the method of Lowry et al. (1951).
II-Oxidative stress markers
- Activities of catalase, superoxide dismutase, glutathione peroxidase were determined in chosen tissues using commercial kits. The enzymes activities were expressed as U/mg protein. The level of MDA, as an index of lipid peroxidation, was determined through its reaction with thiobarbituric acid (TBA) using the lipid peroxidate kit according to the instruction of the supplier. TBARS values were calculated as nmol MDA per g tissue.
III- Inflammatory markers
The sera TNF-µ , IL-6, IL-4 and IL-10 levels were measured using standard kits according to the manufacturer's protocol.
IV- Apoptosis marker
The levels of caspase-3 enzyme in cerebellum tissue were measured according to the manufacturer's protocol.
V-Estimation of serum hormonal concentrations
Thyroid hormones were determined by measuring the serum levels of thyroxine (T4), triiodothyronine (T3) and thyroid-stimulating hormone (TSH). T4 was determined following the protocol described in T4 ELISA kit (Cat. No. 60863), Kamiya Biomedical Company, WA, USA. The data of T4 were measured as ng/ml. T3 was estimated using a solid-phase competitive ELISA kit (Cat. No. T3043T-100) obtained from CalbioTech Inc., Spring Valley, Canada. The T3 values were expressed as pg/dl. TSH was estimated using ELISA kit (Cat. No. CSBE05115r) obtained from CUSABIO Biotech Co., LTD, Wuhan, China. The records of TSH were given in μIU/ml. Serum levels of testosterone, LH, and FSH were detected according to Sakuma (2009), Shioya and Wakabayashi (1998), and Teerds et al. (1989), respectively. The values of testosterone were expressed as pg/ml whereas LH and FSH values were expressed as ng/ml.
VI-Assessment of sperm concentration, motility, and abnormality
The left testis of each rat was harvested, then minced in pre-warmed saline (37 °C), and the resulted suspension was used in semen analysis. To analyze sperm motility, 1 drop of sperm suspension was placed on a glass slide to analyze 200 motile sperm in 4 different fields. The motility of the sperm was evaluated microscopically within 2– 4 min of their isolation from the testis, and data were expressed as percentage motility (Morrissey et al. 1988). The sperms were counted using a hemocytometer following the method of Freud and Carol (1964). The technique of Evans and Maxwell (1987) was adopted for sperm abnormality study. Briefly, smears were made by placing a drop from the sperm suspension and one or two drops of the previously warmed (37 °C) eosin–nigrosin stain. The smears were allowed to dry in the air and then examined under the microscope using a high power (100×) oil immersion objective. The normal sperm cells were counted and the percentage was calculated.
C. Microscopic examination
For preparation of samples used in the examination by light microscope, specimens of testis were collected from all experimental groups and fixed in 10% neutral buffered formalin and routinely processed for stained with hematoxylin and eosin stain (H& E) (Bancroft and Camble, 2002), then sections were examined using light microscope.
D- Statistical analysis
All variables were compared using one-way analysis of variance (ANOVA) followed by LSD multiple range test. Differences at P<0.05 were considered as statically significant.