Anthocyanidins and their glucosides contain multiple bindingsites for unpaired free radical electrons 158,160,192 which conferpotent antioxidant activity 159, . Anthocyanins are stable atstomach pH and, following their passage into the intestinal tract,most are absorbed into the bloodstream, although with low efficiency. Punicalagins and ellagitannins are largepolyphenols (Figure 6) that are stable at stomach pH and pass intothe small intestine unchanged 142,146, although a small fraction arehydrolyzed within the neutral pH environment of the human smallintestine, forming combinations of ellagic and gallic acids 139, . After mixing, 0.2 ml of 42-mmol l−1 thiobarbituric acid solution was added to a final concentration of 7 mmol l−1 and then placed in a 95 °C dry bath for 1 h. An aliquot of 50 µl of each sample was pipetted into a test tube containing 0.6 ml of 0.44 mol l−1 phosphoric acid. Following the addition of 10 μmol l−1 H2DCFDA (final concentration), the appearance of DCF fluorescence was typically followed for 1 h, using excitation at 486 nmol l−1 and emission at 530 nmol l−1. After cells were 90% confluent, they were washed three times with Hank's balanced salt solution. Cells were plated on 48-well plates at an initial density of 2×104 treated cells per well and grown for 3 days. Glyphosate has been demonstrated to elevate oxidative levels (12), and exposure to glyphosate in the environment has been shown to reduce levels of sex hormones, including testosterone (13). Vitamin C, acting as an antioxidant, can reverse the decreased testosterone levels induced by lead exposure (9). For example, the oxidative properties of foods and medications encountered in daily life can significantly influence testosterone levels. Previous studies on the effect of diet and lifestyle on testosterone levels have shortcomings or limitations, which may prevent people (men) from doing a good job of intervention according to diet and lifestyle. The oxidative balance score (OBS) and testosterone levels were derived from interview data and laboratory measurements. Briefly, approximately 10 µl of a cell suspension in phosphate-buffered saline was mixed with 40 µl of trypan blue, and the numbers of stained (dead cells) and unstained cells (live cells) were counted using a hemocytometer. TM3 cells were plated in 12-well plates at a density of (2×105–5×105 cells/well) and grown for 24 h. The TM3 cell line (ATCC no. CRL-1714; ATCC, Manassas, VA, USA) was selected as the cell model in this study. Potential risks include erythrocytosis, edema, gynecomastia, prostate stimulation and suppression of sperm production. Following the chemical induction of hypoxia, intracellular HIF-1α stabilization and activation increased to 132%±3% (P2 or testosterone treatment). To test whether the cytoprotective effect is specific for testosterone, we further investigated the effect of an inert steroid, epitestosterone, on cell viability. After chemical induction of hypoxia with 100 µmol l−1 CoCl2, whole-cell lysates were taken directly from samples in extract buffer (50 mmol l−1 Tris (pH 7.4), 150 mmol l−1 NaCl, 5 mmol l−1 EDTA, 0.1% sodium dodecylsulfate, 1 mmol l−1 phenazine methosulfate and complete protease inhibitor). Testosterone-treated cells were lysed by adding 2 ml of a guanidium thiocyanate phenol RNAzol B solution (Biotex, Houston, TX, USA). In order to demonstrate the effect of testosterone on intracellular steroidogenesis, alterations in StAR messenger RNA (mRNA) and AR mRNA were detected. The eluent was monitored with a Jasco fluorescence detector (with excitation at 525 nmol l−1 and emission at 550 nmol l−1). Sodium fluoride, a substance widely present in drinking water and food, can influence testosterone production by altering oxidative stress levels in the testes (8). This study underscores the importance of comprehensive antioxidant approaches, particularly lifestyle OBS, in male testosterone deficiency. Moreover, red blood cell resistance to free radicals is also positively correlated with survival in this species (Alonso-Alvarez et al. 2006), suggesting that the fitness cost of testosterone-induced increase in oxidative stress might be substantial. Cotinine, a metabolite of cigarette smoke, exhibits a non-linear relationship with testosterone levels. Many lifestyle habits can also significantly influence testosterone levels. Surprisingly, a high-fat diet in parental generations can lead to the occurrence of testosterone deficiency in offspring (32). The highest OBS group exhibited a 38% reduction in the risk of testosterone deficiency compared to the lowest OBS group. In this study, we experimentally tested the hypothesis that high testosterone levels necessary for the expression of secondary sexual traits can generate a cost in terms of oxidative stress. In agreement with the prediction, we found that red blood cell resistance to a free radical attack was the highest in males implanted with flutamide and the lowest in males implanted with testosterone. These observationssupport the hypothesis that ROS play an important role in age-relatedreductions in Leydig cell testosterone production.
