"2. Materials and methods

2.1. Chemicals

TPP (CAS No.:115-86-6; purity: >99%) and TCEP (CAS No.:115- 98-8; purity: >97%) were purchased from Sigma-Aldrich. Both of the chemicals used as received. The kits for determining superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities, glutathione (GSH) and MDAcontents were purchased fromthe Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). The BCA protein assay kit from Beyotime (Nantong, China). TRIzol reagent for RNA isolation was purchased from Takara Biochemicals (Dalian, China). The reverse transcriptase kit wasprovidedby Toyobo (Tokyo,Japan). Themouse testosterone (T) ELISA kits from RapidBio Lab (RapidBio, USA).

2.2. Animals and experimental design

Totally 35 four-week old adolescent male ICR mice (Mus musculus) were purchased from the China National Laboratory Animal ResourceCenter (Shanghai,China).Allthemice were keptinnormal facilities (light-on at eight o’clock, light-off at twenty o’clock; illumination with strip lights, 200 lx at cage level; 22 ± 1 ◦C).Water and food were available ad libitum. After one week, all the mice were divided into five groups randomly (seven mice each group). Different concentrations of TPP and TCEP were dissolved in amount of anhydrous ethanol and uniformly mixed into feed completely.After anhydrous ethanol volatilized entirely, the male mice were orally administered TPP and TCEP (0, 100 and 300 mg/kg/bodyweight, respectively) contained diet daily for 35 days. During the treated period, water was available ad libitum, while food was available only at night. At day 36, all of 35 mice were sacrificed. The mice were anesthetized by ether before sacrificing. Livers and testes were quickly removed and weighed, after that they were immediately frozen in liquid nitrogen and kept at −80 ◦C until use. In each group, two mice were randomly selected and their right testes were quickly removed and stored in 4% paraformaldehyde for histopathological analysis. All experiments were performed in accordance with the Guiding Principles in the Use of Animals in Toxicology.

2.3. Determination of SOD, CAT, GPX and GST activities, GSH and MDA contents in the liver

Livers were defrosted and homogenized with 10 volumes of icecold saline. The homogenate was centrifuged at 5000 × g at 4 ◦C for 15 min to obtain the supernatant for the enzyme activity assays. The enzyme activities of SOD, CAT, GPX, and GST as well as the GSH and MDA content in the liver were determined according to the manufacturer’s instructions (Jin et al., 2010, 2011, 2014). Briefly, lipid peroxidation products (measured as MDA) were quantified with the thiobarbituric acid (TBARS) method, and the MDA concentration was expressed as nmol/mg protein. The GSH content was measured by the dithiobisnitrobenzoic acid (DTNB) method and expressed as mg/g protein. The SOD activity assay was based on water-soluble four azole nitrogen (WST) method. SOD could inhibitthe oxidation reaction offour azole nitrogen with O2 −• produced by the xanthine oxidase system. The formazan dye produced by the oxidation reaction had absorbance at 450 nm. One unit of SOD activity was defined as the amount of enzyme required to inhibit the oxidation reaction by 50% and expressed as U/mg protein. The CAT activity was determined through ammonium molybdate (AHM) method. One unit of CAT activity was defined as 1 s the amount of enzyme required to consume 1 mol H2O2 and expressed as U/mg protein. The GPX activity was quantified through measuring the oxidation rate of reduced GSH to oxidized glutathione (GSSG) by using the H2O2 catalyzed by GPX. One unit of GPX was defined as 1 min the amountthat reduced 1 mol/L of GSH and expressed as U/mg protein. The GST activity was measured by 2,4-dinitrobenzene (CDNB) method. One unit of GST was defined as 1 min the amount that reduced 1 mol/L of GSH and expressed as U/mg protein. Protein concentration was analyzed by bicinchoninic acid (BCA) as a detection reagent for Cu+ following the reduction of Cu2+ by protein in an alkaline environment. All the measurements were made on a microplate reader (Power wave XS, Bio-TEK, USA) according to the manufacturer’s instructions. 2.4. Histopathological analysis

The testes chosen for histopathological analysis were processed sequentially in ethanol, xylene and paraffin. Then testes were embedded in paraffin wax, sectioned (6 m) using a Leica RM2235 (Germany) and mounted on slides. The sections were observed by a microscope (Olympus, Japan) after staining with hematoxylin and eosin (Jin et al., 2012). The average numbers of the seminiferous tubules in 10 same square disparate viewing areas were counted at 100× magnification.

2.5. Gene expression analysis

The total RNA in the livers and testes were isolated by TRIzol reagent according to the manufacturer’s specification. After denaturalization, RNA was used to synthesize cDNA by a reverse transcriptase kit. A 1-L portion of each RT product was used directly for real-time quantitative polymerase chain reaction (RTqPCR) and performed on an Eppendorf MasterCycler® ep RealPlex4 (Wesseling-Berzdorf, Germany). Oligonucleotide primers were used to detect the expression of house-keeping gene GAPDH, antioxidant relatedgenes Sod1, Sod2,Gpx1,Gpx2, Cat andGsta1, lowdensity lipoprotein receptor (LDL-R), scavenger receptor class B type 1 (SR-B1), peripheral benzodiazepine receptor (PBR), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), cytochrome P450 17˛-hydroxysteroid dehydrogenase (P450-17˛), 3ˇ-hydroxysteroid dehydrogenase (3ˇHSD) and 17ˇ-hydroxysteroid dehydrogenase (17ˇ-HSD) genes using the SYBR Green system. Primers were designed and synthesized, and the detail information was shown in Table S1. The transcript of GAPDH was used to standardize the results by eliminating variations in mRNA and cDNA quantity and quality. The PCR protocol is 1 min at 95 ◦C for denaturation, followed by 40 cycles of 15 sat 95 ◦C and 1 min at 60 ◦C. The 2−Ct method was used to analyze the relative quantification of gene expression among the treatment groups (Livak and Schmittgen, 2001).

2.6. Determination of testicular T concentration

Testes were defrosted and homogenized with 10 volumes of ice-cold saline. The homogenate was centrifuged at 5000 rpm at 4 ◦C for 15 min to obtain the supernatant for further use. The level of T in 10 L supernatant of each sample was determined by the corresponding mouse ELISA kits according to the manufacturer’s specification. The ELISA assays were measured on a microplate reader by using 96-well micro well plate. The total protein level in each sample was determined as described above.

2.7. Data analysis

Experimental data is expressed as mean ± SEM and p < 0.05 is considered significant. Data is evaluated by one-way analysis of variance (ANOVA) using Fisher’s protected least significant difference test(StatView 5.0 forWindows;Abacus Concepts, Berkeley, CA)."

Male ICR Mice