Tao Lin

Dynamic Effect of Di-2-(Ethylhexyl) Phthalate on Testicular Toxicity: Epigenetic Changes and Their Impact on Gene Expression

This study investigated epigenetic (specifically, DNA methylation) changes and their impact on gene expression in testes induced by maternal exposure to Di-2-(ethylhexyl) phthalate (DEHP) in mice. Testicular dysgenesis syndrome was induced in fetuses and pups by maternal exposure to DEHP at 500 mg/kg/d, and testes were excised for analysis on gestation day (GD) 19 and postnatal days (PNDs) 3, 21, 56, and 90. High-performance liquid chromatography (HPLC) was performed to analyze DNA methylation status, and expression levels of the DNA methyltransferases were examined by quantitative real-time polymerase chain reaction (qPCR). Testis-specific gene, insulin-like hormone 3 (Insl3), and testosterone production were also detected. DEHP significantly increased DNA methylation levels on GD 19 and PND 3 (P < .05 and P < .05) but not on PNDs 21, 56, and 90. DEHP also significantly increased the expression of DNA methyltransferases. For DNA methyltransferase 1, the difference was not significant on PND 21, and DNA methyltransferase 3a and 3b returned to normal levels on PND 56. Fetal testes were a main target for DEHP as evidenced by a reduction in Insl3 expression and testosterone production. Effects of DEHP on Insl3 expression continued until PND 21. The DEHP-induced suppression of testosterone had not recovered on PND 56. Changes in DNA methylation may play an important role in abnormal testicular function caused by environmental factors such as maternal exposure to DEHP, which may be a mechanism of DEHP-mediated testicular toxicity.

Di(2-Ethylhexyl) Phthalate (DEHP) Increases Transforming Growth Factor-β1 Expression in Fetal Mouse Genital Tubercles

Phthalates are known to elicit marked effects on the developing male reproductive tract as evidenced by hypospadias. Recently, transforming growth factor-β1 (TGF-β1) was postulated to play an essential role in the development of genital tubercles (GT), and TGF- β1 was found to act as a phthalates-responsive gene. In this study, the effects of di(2-ethylhexyl) phthalate (DEHP) were examined on the expression of TGF-β1 in fetal mice, as GT development is dependent upon this factor. Pregnant C57BL/6 mice were exposed to corn oil or DEHP (100, 200, or 500 mg/kg/d orally) from embryonic day 12 (ED12) to ED17. Data showed a significant inhibition of male fetal GT development following DEHP treatment. Hypospadic-like urethral orifice and abnormal urethra were evaluated macroscopically and by histology at ED19. By using reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, the expression of TGF-β1 was upregulated in DEHP-treated mice. These results suggest that hypospadias may be induced by DEHP exposure involving modification of TGF-β1 levels.

Fine particulate matter leads to reproductive impairment in male rats by overexpressing phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway

Maintenance of male reproductive function depends on normal sperm generation during which process Sertoli cells play a vital role. Studies found that fine particulate matter (PM) causes decreased male sperm quality, mechanism of which unestablished. We aim to investigate the definite mechanism of PM impairment on male reproduction. Male Sprague-Dawley rats were daily exposed to normal saline (NS) or PM2.5 with the doses of 9 mg/kg.b.w and 24 mg/kg.b.w. via intratracheal instillation for seven weeks. Reproductive function was tested by mating test and semen analysis after last exposure. Testes were collected to assess changes in histomorphology, and biomarkers including connexin 43 (Cx43), superoxide dismutase (SOD), phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (p-Akt). Male rats exposed to PM2.5 showed noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate and severe testicular damage in histomorphology. After PM2.5 exposure, the levels of Cx43 was significantly downregulated, and SOD was upregulated and downregulated significantly with different dose, respectively. Protein expression of PI3K and p-Akt dramatically enhanced, and the later one being located in Sertoli cells, the upward or declining trend was in dose dependent. PM2.5 exposure leads to oxidative stress impairment via PI3K/Akt signaling pathway on male reproduction in rats.

The Mechanism of Environmental Endocrine Disruptors (DEHP) Induces Epigenetic Transgenerational Inheritance of Cryptorchidism

Discussion on the role of DEHP in the critical period of gonadal development in pregnant rats (F0), studied the evolution of F1-F4 generation of inter-generational inheritance of cryptorchidism and the alteration of DNA methylation levels in testis. Pregnant SD rats were randomly divided into two groups: normal control group and DEHP experimental group. From pregnancy 7d to 19d, experimental group was sustained to gavage DEHP 750mg/kg bw/day, observed the incidence of cryptorchidism in offspring and examined the pregnancy rate of female rats through mating experiments. Continuous recording the rat’s weight and AGD value, after maturation (PND80) recording testis and epididymis’ size and weight, detected the sperm number and quality. Subsequently, we examined the evolution morphological changes of testicular tissue for 4 generation rats by HE staining and Western Blot. Completed the MeDIP-sequencing analysis of 6 samples (F1 generation, F4 generation and Control). DEHP successfully induced cryptorchidism occurrence in offspring during pregnancy. The incidence of cryptorchidism in F1 was 30%, in F2 was 12.5%, and there was no cryptorchidism coming up in F3 and F4. Mating experiment shows conception rate 50% in F1, F2 generation was 75%, the F3 and F4 generation were 100%. HE staining showed that the seminiferous epithelium of F1 generation was atrophy and with a few spermatogenic cell, F2 generation had improved, F3 and F4 generation were tend to be normal. The DNA methyltransferase expression was up-regulated with the increase of generations by Real Time-PCR, immunohistochemistry and Western Blot. MeDIP-seq Data Analysis Results show many differentially methylated DNA sequences between F1 and F4. DEHP damage male reproductive function in rats, affect expression of DNA methyltransferase enzyme, which in turn leads to genomic imprinting methylation pattern changes and passed on to the next generation, so that the offspring of male reproductive system critical role in the development of imprinted genes imbalances, and eventually lead to producing offspring cryptorchidism. This may be an important mechanism of reproductive system damage.

Ultrasound microbubble-mediated delivery of the siRNAs targeting MDR1 reduces drug resistance of yolk sac carcinoma L2 cells

BackgroundMDR1 gene encoding P-glycoprotein is an ATP-dependent drug efflux transporter and related to drug resistance of yolk sac carcinoma. Ultrasound microbubble-mediated delivery has been used as a novel and effective gene delivery method. We hypothesize that small interfering RNA (siRNA) targeting MDR1 gene (siMDR1) delivery with microbubble and ultrasound can down-regulate MDR1 expression and improve responsiveness to chemotherapeutic drugs for yolk sac carcinoma in vitro.MethodsRetroviral knockdown vector pSEB-siMDR1s containing specific siRNA sites targeting rat MDR1 coding region were constructed and sequence verified. The resultant pSEB-siMDR1 plasmids DNA were encapsulated with lipid microbubble and the DNA release were triggered by ultrasound when added to culture cells. GFP positive cells were counted by flow cytometry to determine transfection efficiency. Quantitative real-time PCR and western blot were performed to determine the mRNA and protein expression of MDR1. P-glycoprotein function and drug sensitivity were analyzed by Daunorubicin accumulation and MTT assays.ResultsTransfection efficiency of pSEB-siMDR1 DNA was significantly increased by ultrasound microbubble-mediated delivery in rat yolk sac carcinoma L2 (L2-RYC) cells. Ultrasound microbubble-mediated siMDR1s delivery effectively inhibited MDR1 expression at both mRNA and protein levels and decreased P-glycoprotein function. Silencing MDR1 led to decreased cell viability and IC50 of Vincristine and Dactinomycin.ConclusionsOur results demonstrated that ultrasound microbubble-mediated delivery of MDR1 siRNA was safe and effective in L2-RYC cells. MDR1 silencing led to decreased P-glycoprotein activity and drug resistance of L2-RYC cells, which may be explored as a novel approach of combined gene and chemotherapy for yolk sac carcinoma.

Potential spermatogenesis recovery with bone marrow mesenchymal stem cells in an azoospermic rat model.

Non-obstructive azoospermia is the most challenging type of male infertility. Stem cell based therapy provides the potential to enhance the recovery of spermatogenesis following cancer therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the potential to differentiate or trans-differentiate into multi-lineage cells, secrete paracrine factors to recruit the resident stem cells to participate in tissue regeneration, or fuse with the local cells in the affected region. In this study, we tested whether spermatogenically-induced BMSCs can restore spermatogenesis after administration of an anticancer drug. Allogeneic BMSCs were co-cultured in conditioned media derived from cultured testicular Sertoli cells in vitro, and then induced stem cells were transplanted into the seminiferous tubules of a busulfan-induced azoospermatic rat model for 8 weeks. The in vitro induced BMSCs exhibited specific spermatogonic gene and protein markers, and after implantation the donor cells survived and located at the basement membranes of the recipient seminiferous tubules, in accordance with what are considered the unique biological characteristics of spermatogenic stem cells. Molecular markers of spermatogonial stem cells and spermatogonia (Vasa, Stella, SMAD1, Dazl, GCNF, HSP90α, integrinβ1, and c-kit) were expressed in the recipient testis tissue. No tumor mass, immune response, or inflammatory reaction developed. In conclusion, BMSCs might provide the potential to trans-differentiate into spermatogenic-like-cells, enhancing endogenous fertility recovery. The present study indicates that BMSCs might offer alternative treatment for the patients with azoospermatic infertility after cancer chemotherapy.

Role of Nitric Oxide Synthase in Bladder Pathologic Remodeling and Dysfunction Resulting From Partial Outlet Obstruction

OBJECTIVES To investigate the relationship between nitric oxide synthase (NOS) and oxidative stress and pathologic remodeling in the partial obstructed bladder of a rat model. METHODS We surgically established partial bladder outlet obstruction (PBOO) in 2 groups of rats and allowed it to persist for 3-6 weeks. Normal and sham-operated rats served as the controls. Each group contained 6 rats for a total of 24 rats. Cystometry was used to evaluate the bladder function. The bladders were removed, and histopathologic measurements were performed to evaluate bladder hypertrophy and fibrosis and NOS immunolocalization. Biochemical measurements were used to evaluated NOS mRNA and activity and the oxidative stress level. RESULTS The obstructed rats experienced significant increases in bladder weight, muscle hypertrophy, and deposits of collagen fibers compared with the normal and sham-operated groups. PBOO debilitated bladder contractibility, increased the residual urine volume and voiding interval, decreased the voiding volume, and caused poor bladder emptying, with an increased residual urine volume and decompensated bladder in the PBOO rats at 6 weeks. The elevation in malondialdehyde and reduction in superoxide dismutase activity in the PBOO rats suggested that oxidative stress injury occurred in the obstructed bladder. Lower inducible NOS and endothelial NOS (eNOS) mRNA expression was demonstrated through quantitative polymerase chain reaction. In particular, eNOS was significantly downregulated in the PBOO rats compared with the normal and sham-operated rats. The normal and PBOO bladder tissues did not express detectable levels of neuronal NOS mRNA or exhibit neuronal NOS immunoreactivity. The total NOS activity had decreased progressively in the PBOO groups in conjunction with the significantly decreased eNOS activity compared with that in the normal and sham-operated groups. CONCLUSIONS These findings suggest that decreases in NOS activity and expression (mainly of eNOS) concomitant with increases in reactive oxygen species generation represent the underlying pathogenic mechanism of obstructed bladder remodeling and dysfunction.

Laparoscopic Orchiopexy for Treating Inguinal Canalicular Palpable Undescended Testis

BACKGROUND AND PURPOSE This study investigated the safety and efficacy of using the minimally invasive operation of laparoscopic orchiopexy to treat palpable inguinal canalicular undescended testes. PATIENTS AND METHODS Laparoscopic orchiopexy was performed on 90 Chinese patients (103 testes) with inguinal canalicular palpable undescended testes. Patients with testes that were nonpalpable, ectopic, retractable, or palpable but distal to the external ring were excluded. Patients in the study ranged from 8 months to 6 years old (mean age: 17 months). Of the 103 testes undergoing operation, 26.7% were on the left side of the body and 58.9% were on the right; 14.4% of the patients had bilateral undescended testes. Laparoscopic techniques were used by the same surgeon to move the spermatic vessels and transfer the testis into the scrotum. RESULTS All 103 testes were successfully descended by laparoscopy. In the first 46 unilateral cases, the operation took significantly longer for the first 15 patients than the next 31, which were completed in 32.7 +/- 5.2 min. Of all 90 patients, a complication was encountered in only one, and this occurred at the beginning of the surgeons learning curve. Of 77 unilateral undescended testes, a patent processes vaginalis was found in 90.3% of the cases on the ipsilateral side and 15.6% on the contralateral side. All of the testes maintained an adequate size and intrascrotal position with no atrophy or recurrent inguinal hernia during a follow-up of 6 to 12 months. CONCLUSIONS The laparoscopic approach to orchiopexy is a safe way to descend the inguinal canalicular palpable testis. It offers several advantages of a minimally invasive technique and yield effective results.

Urban fine particulate matter exposure causes male reproductive injury through destroying blood-testis barrier (BTB) integrity.

Blood-testis barrier (BTB) provides a suitable microenvironment for germ cells that is required for spermatogenesis. Exposure to particulate matter (PM) is recognized to occasion male reproductive impairment, but the mechanism of which remains unclear. Male Sprague-Dawley (SD) rats were used to establish animal models with PM2.5 exposure concentration of 0, 10, and 20mg/kg.b.w. once a day for four weeks. Success rate of mating, sperm quality, epididymal morphology, expressions of spermatogenesis markers, superoxide dismutases (SOD) activity and expression in testicular tissues, and expressions of BTB junction proteins were detected. In addition, in vitro experiments were also performed. After PM2.5 treatment, reactive oxygen species (ROS) production and apoptosis of Sertoli cells were analyzed. Our results indicated that after PM2.5 exposure male rats presented inferior uberty and sperm quality, with decreased expressions of spermatogenesis markers, escalated SOD activity and expression levels, and reduced expressions of tight junction, adherens junction, and gap junction proteins in testicular tissues. Meantime, PM2.5-treated Sertoli cells displayed increased SOD production and apoptosis. PM2.5 exposure engenders male reproductive function injury through breaking BTB integrity.

Incidence and risk of hypertension associated with vascular endothelial growth factor receptor tyrosine kinase inhibitors in cancer patients: a comprehensive network meta-analysis of 72 randomized controlled trials involving 30013 patients

Background Tyrosine kinase inhibitors (TKIs) have been developed during the last decade that target the vascular endothelial growth factor receptor (VEGFR) are currently being evaluated as treatments for malignant tumors. The increased application of VEGFR-TKIs means that the probability of hypertension is a serious concern. However, the reported incidence varies markedly between clinical trials. Here, we undertook an up-to-date, comprehensive meta-analysis on clinical works to build the incidence of hypertension along with VEGFR-TKIs. The goal was to understand better of the overall venture of cancer patients’ hypertension treated with these drugs. Methods Databases (EMBASE, PubMed, and Cochrane library) and the abstracts of the American Society of Clinical Oncology annual meeting and European Society of Medical Oncology were searched to identify related studies. 95% confidence intervals (CIs), summary incidences, and relative risk (RR) were calculated utilizing either fixed-effects models on the basis of the heterogeneity of the included studies or random-effects. Results Seventy-two randomized controlled trials (including 30013 patients) were involved. The total incidence of high-grade and all-grade hypertensive events along with VEGFR-TKIs was 23.0% (95% CI, 20.1–26.0%) and 4.4% (95% CI, 3.7–5.0%), respectively. The use of VEGFR-TKIs remarkably enhanced the venture of developing high-grade (RR, 4.60; 95% CI, 3.92–5.40; P < 0.001) and all-grade (RR, 3.85; 95% CI, 3.37–4.40; P < 0.001) hypertensive events. Subgroup analyses revealed that the risk of a hypertensive event varied significantly in accordance with tumor type, VEGFR-TKI, trial phase, VEGFR-TKIs-based regimen, control therapy, and chemotherapy regimen. Conclusions Patients with cancer that receive VEGFR-TKIs are at a remarkable venture of developing hypertension. Therefore, suitable treatment and monitoring should be introduced to avoid cardiovascular complications.