Guo-Xin Hu

Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero.

Exposures to di-(2-ethylhexyl) phthalate (DEHP) have been shown to be associated with decreased adult testosterone (T) levels and increased Leydig cell numbers. As yet, little is known about DEHP effects in utero on fetal Leydig cells (FLC). The present study investigated effects of DEHP on FLC function. Pregnant Long–Evans female rats received vehicle (corn oil) or DEHP at 10, 100, or 750 mg/kg by oral gavage from gestational day (GD)2–20. At GD21, T production, FLC numbers and distribution, and testicular gene expression were examined. The percentage of FLC clusters containing 6–30 cells increased in all treatment groups, with 29 ± 2% in control vs. 37 ± 3, 35 ± 3, and 56 ± 4% in rats receiving 10, 100, and 750 mg/kg DEHP, respectively. In contrast, FLC numbers were 33% and 39% lower than control after exposures to 100 and 750 mg/kg DEHP, respectively. At these doses, mRNA levels of leukemia inhibitory factor (LIF) increased. LIF was found to induce cell aggregation in FLCs in vitro, consistent with the hypothesis that DEHP induced FLC aggregation. Testicular T levels were doubled by the 10 mg/kg dose and halved at 750 mg/kg. The mRNA levels of IGF-1 and c-Kit ligand (KITL) were induced by 10 mg/kg DEHP. These results, taken together, indicate that fetal exposures to DEHP have effects on FLC number, distribution, and most importantly, steroidogenic capacity and suggest that abnormal expressions of IGF1, KITL, and LIF genes may contribute to the reproductive toxicity of phthalates.

Phthalate-induced testicular dysgenesis syndrome: Leydig cell influence

Phthalates, the most abundantly produced plasticizers, leach out from polyvinyl chloride plastics and disrupt androgen action. Male rats that are exposed to phthalates in utero develop symptoms characteristic of the human condition referred to as testicular dysgenesis syndrome (TDS). Environmental influences have been suspected to contribute to the increasing incidence of TDS in humans (i.e. cryptorchidism and hypospadias in newborn boys and testicular cancer and reduced sperm quality in adult males). In this review, we discuss the recent findings that prenatal exposure to phthalates affects Leydig cell function in the postnatal testis. This review also focuses on the recent progress in our understanding of how Leydig cell factors contribute to phthalate-mediated TDS.

Rapid mechanisms of glucocorticoid signaling in the Leydig cell

Stress-mediated elevations in circulating glucocorticoid levels lead to corresponding rapid declines in testosterone production by Leydig cells in the testis. In previous studies we have established that glucocorticoids act on Leydig cells directly, through the classic glucocorticoid receptor (GR), and that access to the GR is controlled prior to the GR by a metabolizing pathway mediated by the type 1 isoform of 11beta-hydroxysteroid dehydrogenase (11betaHSD1). This enzyme is bidirectional (with both oxidase and reductase activities) and in the rat testis is exclusively localized in Leydig cells where it is abundantly expressed and may catalyze the oxidative inactivation of glucocorticoids. The predominant reductase direction of 11betaHSD1 activity in liver cells is determined by an enzyme, hexose-6-phosphate dehydrogenase (H6PDH), on the luminal side of the smooth endoplasmic reticulum (SER). Generation of the pyridine nucleotide cofactor NADPH by H6PDH stimulates the reductase direction of 11betaHSD1 resulting in increased levels of active glucocorticoids in liver cells. Unlike liver cells, steroidogenic enzymes including 17beta-hydroxysteroid dehydrogenase 3 (17betaHSD3) forms the coupling with 11betaHSD1. Thus the physiological concentrations of androstenedione serve as a substrate for 17betaHSD3 utilizing NADPH to generate NADP+, which drives 11betaHSD1 in Leydig cells primarily as an oxidase; thus eliminating the adverse effects of glucocorticoids on testosterone production. At the same time 11betaHSD1 generates NADPH which promotes testosterone biosynthesis by stimulating 17betaHSD3 in a cooperative cycle. This enzymatic coupling constitutes a rapid mechanism for modulating glucocorticoid control of testosterone biosynthesis. Under stress conditions, glucocorticoids also have rapid actions to suppress cAMP formation thus to lower testosterone production.

Activation of the AMP activated protein kinase by short-chain fatty acids is the main mechanism underlying the beneficial effect of a high fiber diet on the metabolic syndrome

The metabolic syndrome, a cluster of conditions including abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure, is a natural consequence of over nutrition and a sedentary lifestyle. The prevalence of the metabolic syndrome has increased to epidemic proportions in the world. The exact pathogenesis of the metabolic syndrome remains unclear, but it is known to be a complex interaction between genetic, metabolic, and environmental factors. Promotion of physical activity and dietary management are still the main methods for the prevention and management of the metabolic syndrome. Numerous experimental and clinical studies have demonstrated the beneficial effects of high fiber diet on the metabolic syndrome. The principal beneficial effects of a fiber-rich diet in these patients are: prevention of obesity, improved glucose levels, and control of the profile of blood lipids. Dietary fiber may also favor the control of arterial blood pressure. How dietary fiber exerts its beneficial effects on the metabolic syndrome is not well understood. AMP activated protein kinase (AMPK) functions as a major cellular fuel gauge and a master regulator of metabolic homeostasis. Several lines of evidence suggest that AMPK can be activated by short-chain fatty acids (SCFA) either directly or indirectly. It is our hypothesis that the main mechanism underlying the beneficial effect of a high fiber diet on the metabolic syndrome is the increased SCFA production in the colon leading to a higher concentration of SCFA in the portal vein, which activates the AMPK in the liver.

Genetic variations of human CYP2D6 in the Chinese Han population

AIM The purpose of this study was to determine the genetic polymorphisms of the CYP2D6 gene and to elucidate the allele distribution pattern in the Chinese Han population. MATERIALS & METHODS We used PCR and bidirectional sequencing methods to analyze all nine exons of the CYP2D6 gene in 2129 unrelated, healthy Chinese Han subjects from two geographical locations in China: the northern and southern regions. RESULTS In total, 165 mutated sites were detected in 2129 participants, of which 67 sites were reported for the first time. Among these novel mutation sites, 22 were nonsynonymous and 12 were named as novel alleles (*87-*93, *94A, *94B and *95-*98) by the Human CYP Allele Nomenclature Committee. In addition, 29 previously reported alleles and 84 genotypes were also detected in 1954 volunteers. Functional prediction of novel variants revealed that eight variants might have a deleterious effect on CYP2D6. Linkage disequilibrium analysis and tagSNP selection were performed separately. By using these methods, distinct differences were found between the two regions. CONCLUSION This study provides the most comprehensive data concerning CYP2D6 polymorphisms in the Chinese Han population to date and increases the number of known alleles; these findings may greatly contribute to the development of personalized medicine for the Chinese Han population.

In Utero and Lactational Exposures to Diethylhexyl-Phthalate Affect Two Populations of Leydig Cells in Male Long-Evans Rats

Abstract Diethylhexylphthalate (DEHP) has been classified as an antiandrogen. However, whether in utero and lactational exposures of DEHP affect Leydig cells has not been well established. In the present study, the effects of DEHP exposures on fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) were assessed. Pregnant dams of Long-Evans rats were treated with 0, 10, and 750 mg/kg body weight DEHP from Gestational Day 12.5 to Postnatal Day (PND) 21.5. Fetal Leydig cell clustering and FLC-specific gene expression were examined. Anogenital distances (AGDs) of male pups were assessed at PND 2. Serum testosterone levels of male pups and mRNA levels of ALC-specific genes were measured at PNDs 21 and 49. The AGDs of male pups were significantly shorter in the group treated with 750 mg/kg DEHP (mean ± SEM, 3.68 ± 0.16 mm) compared with control (4.62 ± 0.13 mm). The FLCs were aggregated after 10 and 750 mg/kg DEHP exposures. Several FLC-specific genes, including luteinizing hormone receptor (Lhcgr) and steroidogenic enzyme genes, were downregulated at both doses. Serum testosterone levels were significantly lower compared with control at PND 21 after treatment of 10 or 750 mg/kg DEHP, and continued to be lower even up to 49 days postpartum at the higher dose. The mRNA levels for Lhcgr and steroidogenic enzyme genes were significantly lower at both doses of DEHP at PND 21, whereas there were no significant differences for these genes at PND 49. In conclusion, in utero and continued lactational exposures to DEHP exert long-term disruption of steroidogenesis of ALCs.

Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

The objective of the present study was to investigate the effects of genistein and equol on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) in human and rat testis microsomes. These enzymes (3beta-HSD and 17beta-HSD3), along with two others (cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17alpha-hydroxylase/17-20 lyase), catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3beta-HSD activity (0.2 micromol L(-1) pregnenolone) with half-maximal inhibition or a half-maximal inhibitory concentration (IC(50)) of 87 +/- 15 (human) and 636 +/- 155 nmol L(-1) (rat). Genisteins mode of action on 3beta-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD(+). There was no difference in genisteins potency of 3beta-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3beta-HSD, genistein had lesser effects on human and rat 17beta-HSD3 (0.1 micromol L(-1) androstenedione), with an IC(50) >or= 100 micromol L(-1). On the other hand, equol only inhibited human 3beta-HSD by 42%, and had no effect on 3beta-HSD and 17beta-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3beta-HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.

Age-Dependent Accumulation of 8-Oxoguanine in the DNA and RNA in Various Rat Tissues

The relationship between the oxidative damage of nucleic acids and aging of animals was investigated by analyzing the nucleic acids derived from various tissue specimens of naturally aged Sprague-Dawley (SD) rats. For this purpose, we established an accurate and sensitive isotope-diluted LC-MS/MS method to determine the levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGsn) in DNA and 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) in RNA. An age-dependent increase in oxidative DNA and RNA damage was observed in the various organs examined, including the brain, liver, kidneys, and testes. Similar increases in the 8-oxo-dGsn and 8-oxo-Gsn contents were observed in three parts of the brain, the hippocampus, cerebral cortex, and cerebellum, among which, the values for the hippocampus were always the highest. When the oxidized guanosine metabolites were quantified with urine, a similar age-dependent increase was observed for both 8-oxo-dGsn and 8-oxo-Gsn. However, unlike the results of nucleic acid samples derived from the tissues, the amount of 8-oxo-Gsn was significantly higher compared to that of 8-oxo-dGsn, probably reflecting the fact that RNA degradation occurs more frequently than DNA degradation. Our finding indicates that the amount of urinary 8-oxo-Gsn could be considered as a biomarker for the sensitive measurement of oxidative stress and aging.

Adjudin Targeting Rabbit Germ Cell Adhesion as a Male Contraceptive : A Pharmacokinetics Study

Adjudin (1-(2,4-dichlorobenzyl)-1H-indazole-3-carbohydrazide; formerly called AF-2364) has been shown to inhibit spermatogenesis by disrupting anchoring junctions at the Sertoligerm cell interface. This, in turn, leads to germ cell loss from the seminiferous epithelium, and transient infertility. Adjudins efficacyin inhibiting spermatogenesis, the recovery of spermatogenesis after cessation of the drug, and side effects were examined in adult male Japanese rabbits. The pharmacokinetics profiles of adjudin in rabbits after oral administration and after intravenous injection were compared. Rabbits received 25 mg/kg adjudin once weekly for 4 consecutive weeks either by intravenous injection or by gavage. Vehicle-treated rabbits were used as controls. At 1, 2, 3, 4, and 8 weeks after treatment, testes were removed for microscopic examination to assess the status of spermatogenesis. Four weeks after intravenous cessation of adjudin, the recovery of spermatogenesis also was monitored. Blood was withdrawn after first administration to measure plasma concentrations of adjudin by high-performance liquid chromatography. Four weeks after intravenous treatment, examination of testis sections showed rapid exfoliation of elongated/elongating spermatids and the presence of large multinucleated cells; more than 95% of germ cells were absent from the seminiferous epithelium. Intravenous treatment showed a more severe disturbance of spermatogenesis compared with gavage treatment, which was correlated with bioavailability of the drug. The areas under the curve for intravenous injection and gavage were 20.11 +/- 1.90 and 2.23 +/- 0.45 mg x h x L(-1), respectively. These results illustrate the potential of adjudin as a male contraceptive, and the efficacy is associated with the bioavailability of the drug.

Inutero exposure to diisononyl phthalate caused testicular dysgenesis of rat fetal testis

Diisononyl phthalate (DINP) is a synthetic material that has been widely used as a substitute for other plasticizers prohibited due to reproductive toxicity in consumer products. Some phthalates have been associated with testicular dysgenesis syndrome in male fetus when female pregnant dams were exposed to them. The present study investigated effects of DINP on fetal Leydig cell function and testis development. Female pregnant Sprague Dawley rats received control vehicle (corn oil) or DINP (10, 100, 500, and 1000 mg/kg) by oral gavage from gestational day (GD) 12 to 21. At GD 21.5, testicular testosterone production, fetal Leydig cell numbers and distribution, testicular gene and protein expression levels were examined. DINP showed dose-dependent increase of fetal Leydig cell aggregation with the low observed adverse-effect level (LOAEL) of 10 mg/kg and multinucleated gonocyte with LOAEL of 100 mg/kg. At 10 mg/kg, DINP also significantly increased fetal Leydig cell size, but inhibited insulin-like 3 and 3β-hydroxysteroid dehydrogenase gene expression and protein levels. DINP inhibited testicular testosterone levels at 1000 mg/kg. The results indicate that in utero exposure to DINP affects the expression levels of some fetal Leydig cell steroidogenic genes, gonocyte multinucleation and Leydig cell aggregation.