Establishment of an in vitro method of rabbit embryo toxicity with toxicokinetics study.

This report introduces a novel method, rabbit whole embryo culture (WEC) combined with toxicokinetics (TK), for toxicity testing. Rodent WEC has been extensively used for in vitro screening of developmental toxicity. To improve the reliability of in vitro data, it is important to consider TK and species specificity. To test the utility and effectiveness of this method, we investigated the toxic effect of thalidomide on rabbit embryos and its behavior in test systems both in vitro and in vivo under the same experimental condition. The data showed that thalidomide induced embryo malformations such as embryonic brain hypoplasia, short limb buds, and declined embryonic growth both in vitro and in vivo. The toxic effect increased with the increasing exposure of the embryo to thalidomide. In addition, we observed similar toxic effects and exposure-effect relationships in vivo and in vitro. Therefore, we preliminarily conclude that this new method can effectively predict and explain thalidomide toxicity. Furthermore, we investigated the behavior of test compounds in the WEC system for the first time, and this method is expected to be an important technique for in vitro toxicity study after extensive verification.

Inhibitory Effect of Astaxanthin on Testosterone-Induced Benign Prostatic Hyperplasia in Rats.

This study investigates the inhibitory effect of astaxanthin (AST) on testosterone-induced benign prostatic hyperplasia (BPH) in rats. Except for the sham operation, BPH model rats were randomly assigned to five groups: the BPH model control rats, AST-treated BPH model rats (20 mg/kg, 40 mg/kg, and 80 mg/kg), and epristeride (EPR)-treated BPH model rats. After treatment, as compared with the BPH model control rats, the prostate and ventral prostate weights of the AST-treated rats decreased, while there was a marked decline in the 80 mg/kg AST-treated rats. The same effect was also observed in the prostate index and ventral prostate index. The proliferation characteristics of epithelia observed in the BPH model control group were gradually alleviated in the AST-treated rats. As compared with the BPH model control rats, lower epithelial thicknesses of prostates and fewer secretory granules in epithelia were observed in the AST-treated rats. The superoxide dismutase (SOD) activity of prostates increased in all the AST-treated rats with a significant increase in the 40 mg/kg and 80 mg/kg AST-treated rats. The testosterone (T) and dihydrotestosterone (DHT) levels of prostates in the AST-treated groups were lower than those in the BPH model control group, and a significant decline was found in the T level of prostates in the 40 g/kg and 80 mg/kg AST-treated rats and the DHT level of prostates in the 40 mg/kg AST-treated rats. These results indicate that AST might have an inhibitory effect on T-induced BPH in rats, possibly due to SOD activity regulation and T and DHT levels.

A mathematical model for predicting putative association between E2/T ratio and the development of benign prostate hyperplasia in rats.

Benign prostatic hyperplasia (BPH) develops more likely with increasing age and changing serum concentrations of circulating estradiol (E2) and/or testosterone (T). In this study, we explored the relationship between serum E2/T ratio and BPH risk in rats by fitting a mathematical model. A total of 176 rats were randomized to one of the following treatment groups: normal control, castrated control, and 20 more groups of castrated animals treated with increasing dose combinations of T and E2, once daily for 30 days. Serial blood samples were obtained to determine serum T and E2 levels by magnetic bead enzyme-linked immunosorbent assay. Prostate tissue was taken to measure prostate volume. MATLAB software was used to simulate the relationship between prostate/body weight ratio (PBR) and E2/T ratio with a mathematical equation. The values of PBR, E2 and T in the treatment groups were significantly higher than those in the control groups. Stepwise regression showed that PBR was a function of E2 and T. PBR = -0.1782 + 0.0081 E2 + 0.063 T - 0.6 × 10-5 E22 - 0.28 × 10-3 T2. E2/T ratio change may be one of the risk factors for PBR, which is associated with the development of BPH.

Oral exposure to low-dose bisphenol A induces hyperplasia of dorsolateral prostate and upregulates EGFR expression in adult Sprague-Dawley rats.

Prostate is sensitive to endocrine hormone level, and the synergetic effect of estrogen and androgen is critical in prostate growth. The change of signal pathways caused by the imbalance of estrogen and androgen might function in the occurrence of prostate diseases. As a well-known endocrine disruptor compound, bisphenol A (BPA) can disturb the normal function of endocrine hormone and affect prostate development. This study aims to investigate effects of BPA on the dorsolateral prostate (DLP) and the related gene expression of the tissue in adult Sprague-Dawley (SD) rats and to explore the mechanism for the effect of low-dose BPA on DLP hyperplasia. Three-month-old male SD rats were treated with BPA (10.0, 30.0, or 90.0 µg (kg.day)-1, gavage) or vehicle (gavage) for 4 weeks. BPA significantly increased the DLP weight, the DLP organ coefficient, and the prostate epithelium height (p < 0.01) of rats dose-dependently. Microarray analysis and quantitative real-time polymerase chain reaction showed that BPA significantly upregulated the transcriptional levels of some genes, including pituitary tumor transforming gene 1, epidermal growth factor, Sh3kbp1, and Pcna. Furthermore, the expression of PCNA (p < 0.01), androgen receptor (p < 0.01), and EGF receptor (EGFR) (p < 0.001) in DLP was increased significantly by BPA treatment, and the expression of estrogen receptor alpha was also upregulated. The findings evidenced that low-dose BPA could induce DLP hyperplasia in adult rats, and the upregulated EGF/EGFR pathway that was responsive to estrogen and androgen might play an essential role in the DLP hyperplasia induced by low-dose BPA.

Resident fibroblast expansion during cardiac growth and remodeling.

Cardiac fibrosis, denoted by the deposition of extracellular matrix, manifests with a variety of diseases such as hypertension, diabetes, and myocardial infarction. Underlying this pathological extracellular matrix secretion is an expansion of fibroblasts. The mouse is now a common experimental model system for the study of cardiovascular remodeling and elucidation of fibroblast responses to cardiac growth and stress is vital for understanding disease processes. Here, using diverse but fibroblast specific markers, we report murine fibroblast distribution and proliferation in early postnatal, adult, and injured hearts. We find that perinatal fibroblasts and endothelial cells proliferate at similar rates. Furthermore, regardless of the injury model, fibroblast proliferation peaks within the first week after injury, a time window similar to the period of the inflammatory phase. In addition, fibroblast densities remain high weeks after the initial insult. These results provide detailed information regarding fibroblast distribution and proliferation in experimental methods of heart injury.

[Correlation of prostate-specific antigen with the progression and metastasis of human prostate cancer].

Prostate-specific antigen (PSA) is a biomarker for the diagnosis and management of prostate cancer and involved in the development of prostate cancer and/or its progression from the localized to the metastatic stage. This review presents an overview of the roles of PSA in promoting the progression and metastasis of human prostate cancer and its underlying mechanisms, including its serine protease activity, interaction with the cellular membrane receptor, and suppression of specific immune responsiveness, and also points out some of the key problems to be solved.

[Chronic hepatotoxicity evaluation of Chinese medicinal herb Zishen Yutai pill prepared from Polygoni Multiflori Radix preparata in dogs].

To study the chronic hepatotoxicity of Chinese medicine Zishen Yutai pill (ZYP) prepared from Polygonum multiflorum with the recommended dosage in normal Beagle dogs. Low, middle and high doses of ZYP (1.5, 3.0, 6.0 g·kg⁻¹; i.e. 3×, 6× and 12× equivalent doses) were given orally to dogs for 39 consecutive weeks. At the same time, the same volume of deionized water was used as the solvent control group, one time a day. The general condition of the animals was observed every day during the period of administration, and the blood was collected before and 13, 26, 39, 43 weeks after administration to detect the biomarkers related to the hepatotoxicity of the dog serum. 2/7, 3/7 and 2/7 animals were dissected after 13, 39, and 43 weeks of administration to observe the pathological changes of the animal organs, weigh the mass of main organs and conduct pathological examination of the liver. As compared to the solvent control group, 11 liver hepatotoxicity traditional biomarkers such as ALT, AST were found no ZYP-related changes at month 3, 6, 9 of the administration and month 1 in recovery period; There was no significant difference in liver viscera index and liver pathology. Therefore, no obvious hepatotoxicity was shown by ZYP administered up to 6.0 g·kg⁻¹ for 9 months in normal dogs at doses of 1.5, 3.0, and 6.0 g·kg⁻¹.

[Gene regulation of prostaglandin synthase and prostate diseases].

Prostaglandin synthase (PGS) can catalyze the production of various types of prostaglandins and regulate the expression levels of related substances. The regulation mechanisms of the PGS gene are closely related with the occurrence and development of prostate diseases. However, few studies are reported on the regulation mechanisms of PGS in prostatic diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), or on the relationship between PGS gene regulation and prostate diseases. This review aims to analyze their correlation and provide some ideas for the prevention and control of BPH and PCa by intervention of the prostaglandin synthase regulatory pathway.

The TGF-ß pathway mediates doxorubicin effects on cardiac endothelial cells.

Elevated ALK4/5 ligands including TGF-ß and activins have been linked to cardiovascular remodeling and heart failure. Doxorubicin (Dox) is commonly used as a model of cardiomyopathy, a condition that often precedes cardiovascular remodeling and heart failure. In 7-8-week-old C57Bl/6 male mice treated with Dox we found decreased capillary density, increased levels of ALK4/5 ligand and Smad2/3 transcripts, and increased expression of Smad2/3 transcriptional targets. Human cardiac microvascular endothelial cells (HCMVEC) treated with Dox also showed increased levels of ALK4/5 ligands, Smad2/3 transcriptional targets, a decrease in proliferation and suppression of vascular network formation in a HCMVEC and human cardiac fibroblasts co-culture assay. Our hypothesis is that the deleterious effects of Dox on endothelial cells are mediated in part by the activation of the TGF-ß pathway. We used the inhibitor of ALK4/5 kinases SB431542 (SB) in concert with Dox to ascertain the role of TGF-ß pathway activation in doxorubicin induced endothelial cell defects. SB prevented the suppression of HCMVEC proliferation in the presence of TGF-ß2 and activin A, and alleviated the inhibition of HCMVEC proliferation by Dox. SB also prevented the suppression of vascular network formation in co-cultures of HCMVEC and human cardiac fibroblasts treated with Dox. Our results show that the inhibition of the TGF-ß pathway alleviates the detrimental effects of Dox on endothelial cells in vitro.

The comparison of animal models for premature ovarian failure established by several different source of inducers.

The objective of this study was to compare premature ovarian failure animal models established by several different source of inducers. Female ICR mice, KM mice, and SD rats were treated by cyclophosphamide at 120 mg/kg, busulfan at 12 mg/kg, cisplatin at 3 or 4 mg/kg, 4-vinylcyclohexene diepoxide at 160 mg/kg, 35% galactose food pellet, and tripterygium glycosides at 50 mg/kg, respectively. Parameters were analyzed by body weight, serum concentration level of related hormones, ovarian and uterine pathological examination. The results indicated the body weight of mice increased very slowly following single dose of cyclophosphamide (p < 0.05) with damaged ovary; repeated doses of cisplatin could induce body weight significantly decreased (p < 0.01) with a rising trend of serum LH concentration, declining tendency of serum E2 concentration and injured ovary and uterus; 4-vinylcyclohexene diepoxide also hindered the mice growing (p < 0.05) with damaged ovary and uterus; the body weight of mice feed by 35% galactose food pellet increased slowly (p < 0.05) with dramatically higher serum concentration level of galactose, albumin, and total protein (p < 0.001) and injured ovary. Busulfan and tripterygium glycosides did not present obvious evidences. In conclusion, the inducers presented their respective features in such animal models and should be appropriately applied in preventive methods.

Reproductive toxicity of ZishenYutai pill in rats: Perinatal and postnatal development study.

ZishenYutai pill (ZYP) is one of the most commonly used Chinese patent medicines for threatened miscarriage. Although ZYP is widely used, its toxic effects are rarely assessed. We aimed to investigate whether ZYP had reproductive toxicity during perinatal and postnatal period. Pregnant rats (F0) were continuously exposed to 6, 12 and 24 g/kg body weight/d of ZYP by intragastric administration from gestation day15 to post-natal day21. Vehicle and propylthiouracil (PTU, 15 mg/kg) were used as the negative control and positive control, respectively. The mating was done between the treatment (ZYP or PTU) group and negative control group when the F1 pups were born 63-70 days. Body weight, reproductive ability, physical development and neurodevelopment of F0, F1 and F2 pups were observed. The reproductive capacity of F0 and F1 generation decreased significantly after PTU exposure; however, the body weight and reproductive ability of F0, the physical development, weight, feed consumption and reproductive ability of F1, as well as the physical development and body weight of F2 rats were not significantly changed in the ZYP-treated group compared with the negative control group. ZYP exposure had no perinatal toxicity in 3 generations of rats and may be widely used for miscarriage.

Oral administration of low-dose bisphenol A promotes proliferation of ventral prostate and upregulates prostaglandin D2 synthase expression in adult rats.

This study aims to assess the effect of low oral dose of bisphenol A (BPA) on proliferation of ventral prostate (VP) and expression of related genes in adult rats. Three-month-old male Sprague Dawley rats were treated daily with BPA (10, 30, or 90 µg/kg, per os), 17ß-estradiol (E2, 10.0 µg/kg, subcutaneously), or vehicle for 4 weeks. Treatment with 10 µg/kg BPA resulted in increased animal weight and VP epithelial height compared with the controls ( p < 0.01), while such effects were less pronounced in higher BPA doses. Treatment with E2 showed opposite effects, with significantly decreased animal weight and VP epithelial height ( p < 0.01). Interestingly, BPA increased serum E2 and reduced testosterone levels and significantly increased the estrogen to androgen ratio ( p < 0.05). In addition, BPA slightly increased dihydrotestosterone (DHT) levels. Immunohistochemistry data showed that BPA significantly upregulated proliferating cell nuclear antigen expression ( p < 0.01). Furthermore, microarray and reverse transcription polymerase chain reaction analyses showed that BPA induced upregulation of prostaglandin D2 synthase ( Ptgds), Fas, Pbef1, and complement factor B ( Cfb)as well as downregulation of Pttg1 and Fabp4 in the VP. These results indicated that environmental exposure to low doses of BPA may induce proliferation of VP in adult rats by increasing the estrogen to androgen ratio and upregulating expression of Ptgds to promote production of DHT.

[Research progress in mechanism of traditional Chinese medicine treatment of polycystic ovary syndrome].

Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder, which is characterized by hyperandrogenism, insulin resistance and chronic anovulation, and has become a serious threat to the health of adolescents and women of childbearing age.At present,lowering androgen, improving insulin resistance and inducing ovulation are the main methods adopted by doctors to treat the disease, but the adverse reactions of the western medicine and the long-term treatment are hard to be accepted by the patients. PCOS treated by traditional Chinese medicine has achieved a certain effect in recent years.Traditional Chinese medicine is relatively safe and has more effect in many links and targets in improving the symptom of endocrine and metabolic disorder in patients with PCOS. This paper expounds the traditional Chinese medicine pathogenesis of PCOS through clinical and experimental aspects of the literature research：correcting endocrine hormone disorder,the effects of the expression of gene and regulatory factors,improving insulin resistance,correcting lipid metabolic disorder,improving the pregnancy outcome and improving ovarian morphology to summarize the treatment of traditional Chinese medicine in PCOS research results in recent years.

The toxicokinetic profile of curdione in pregnant SD rats and its transference in a placental barrier system detected by LC-MS/MS.

The objective of this study was to determine the toxicokinetic profile of curdione in pregnant SD rats as well as the transference of curdione into the fetus through the placental barrier system using LC-MS/MS. Thirteen pregnant SD rats were treated with 7, 21 and 63 mg/kg curdione once daily from gestational day 6 (GD6) to GD15. Blood samples were collected at different time points on GD6 and GD15. Maternal plasma, placental plasma, placenta tissue, amniotic fluid and fetal tissue were collected for concentration analysis after all the animals were sacrificed following one repeated dose on GD19. The results indicated that Cmax, AUC(0₋t) and AUC(0₋∞) increased in a dose-dependent manner both on GD6 and GD15. At 7 mg/kg group, the total serum clearance value on GD15 was reduced to approximately 16.4% of that on GD6, and the volume of distribution was also significantly decreased (p<0.05). Curdione could be detected in the maternal plasma, placental plasma, placenta tissue, amniotic fluid and fetal tissue, and its concentration in the fetal tissue reached saturation at 21 mg/kg. In conclusion, curdione presents with the risk of accumulation in pregnant SD rats and may affect the fetus via transference through the placental barrier system.

Development and application of an analytical method for curdione quantification in pregnant Sprague-Dawley rats by LC-MS/MS.

The vaginal administration route suffers from relatively low absorption efficiency, which may hinder the identification of the toxicokinetics of curdione in pregnant women. A sensitive analytical method for determining the plasma concentration of curdione was developed and applied in the determination of curdione in pregnant Sprague-Dawley rats as a simulated model. Glimepiride was used as an internal standard and chromatographic separation was achieved on a Capcell Pak C18 MGIII column. A gradient elution profile with 0.5% formic acid (A)-0.5% formic acid-acetonitrile (B) was selected as mobile phase. The selected reaction monitoring mode was used for quantification based on the target fragment ions m/z 237.2 to m/z 135.1 for curdione and m/z 491.3 to m/z 352.1 for the glimepiride. The standard curve was linear over the range of 0.5-500 ng/mL for curdione in rat plasma and yielded a consistent peak pattern, even at the lower limit of quantitation of 0.5 ng/mL. The retention times of curdione and IS were 6.55 and 6.59 min, respectively. The mean recovery of curdione in rat plasma was 95.5-101.1%. The intra-day and inter-day precisions were between 2.35 and 9.08%. This LC-MS/MS method provides a simple and sensitive means for determining the plasma concentration.

Studies on the characteristics and mechanisms of testicular toxicity induced by Hydroxyurea.

OBJECTIVE: Apoptosis plays a dominant role in both spontaneous spermatogenesis and germ cell death. This study was aimed to investigate the functions of related genes in testicular germ cell death induced by Hydroxyurea (HU). METHOD: Wild-type (WT) and FasL transgenic (TG) DBA/C57BL mice were intraperitoneal injected with 400 mg/kg HU. Twelve hours later, testes were collected. Histomorphology of testis was observed by staining with Periodic Acid Schiff (PAS). Apoptosis was assessed by TUNEL assay. mRNA and protein levels of related genes were evaluated by quantitative RT-PCR and Western blot, respectively. RESULTS: The 2 × 2 factorial design comparative experiments between the WT and TG mice showed that the TG mice exhibited a higher basal apoptotic index. The basal mRNA levels of Fas and FasL and protein levels of Fas, FasL, Caspase-3, Caspase-8 and Caspase-9 in the TG mice were also higher than that in the WT mice. Twelve hours after injection of HU, the testicular tubules exhibited no significantly morphological changes but apoptosis index remarkably increased in both the WT and TG mice, with the latter having the higher amplitude. Although, HU up-regulated the mRNA of apoptosis-related genes, such as Fas and FasL, in both the TG and WT mice, the increased amplitude was more obvious in the TG mice. By Western blot analysis, apoptosis-related proteins Fas, FasL Caspase-3, Caspase-8 and Caspase-9 were significantly increased in both the WT and TG mice, with the TG mice exhibiting a greater up-regulation. CONCLUSION: Germ cell apoptosis induced by the HU treatment may be related to the FasL-mediated signal transduction pathway.

[Categories and characteristics of BPH drug evaluation models: a comparative study].

Benign prostatic hyperplasia (BPH) is a worldwide common disease in men over 50 years old, and the exact cause of BPH remains largely unknown. In order to elucidate its pathogenesis and screen effective drugs for the treatment of BPH, many BPH models have been developed at home and abroad. This article presents a comprehensive analysis of the categories and characteristics of BPH drug evaluation models, highlighting the application value of each model, to provide a theoretical basis for the development of BPH drugs.

Development and validation of a stability-indicating high performance liquid chromatographic (HPLC) method for the determination of related substances of micafungin sodium in drug substances.

An isocratic, sensitive and stability-indicating high performance liquid chromatographic (HPLC) method for separation and determination of the related substances of micafungin sodium was developed. The chromatographic separation was achieved on Agilent Zorbax SB-C18 column (250 × 4.6 mm, 5 µm). Forced degradation study confirmed that the newly developed method was specific and selective to the degradation products. The performance of the method was validated according to the present ICH guidelines for specificity, linearity, accuracy, precision and robustness. Regression analysis showed correlation coefficient value greater than 0.999 for micafungin sodium and its six impurities. Limit of detection of impurities was in the range of 0.006%-0.013% indicating the high sensitivity of the newly developed method. Accuracy of the method was established based on the recovery obtained between 98.2% and 102.0% for all impurities. RSD obtained for the repeatability and intermediate precision experiments, was less than 1.0%. The method was successfully applied to quantify related substances of micafungin sodium in bulk drugs.

[Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].

OBJECTIVE: To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader. METHODS: Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data. RESULTS: The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature. CONCLUSION: A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.

Oral exposure to low-dose bisphenol A aggravates testosterone-induced benign hyperplasia prostate in rats.

The declining level of androgen during aging, associated with an inclining level of estrogen, has been hypothesized to be important in the development of benign prostatic hyperplasia (BPH). Within physiologic range, increasing estrogen levels can stimulate prostate to develop and permanently increase prostate size. As an estrogenic endocrine disruptor, bisphenol A (BPA) might be stimulatory to prostate development. We further hypothesized that low dose BPA could induce hyperplasia prostate to proliferate and aggravate the symptom of BPH in male SD rats. BPH was induced by testosterone and then treated with BPA (10, 30, or 90 µg/kg, i.g., daily), 17ß-estradiol (E(2); 50.0 µg/kg, s.c., daily), or vehicle for 4 weeks. We found that weight and volume in rats treated with low dose BPA (10 µg/kg) was higher than that of model control, and BPA significantly increased the relative weight of prostate (p < 0.01). For prostate lobes, BPA 10 µg/kg/day significantly increased relative weight of ventral prostate (VP), weight and relative weight of dorsolateral prostate (DLP) (p < 0.05). And histopathology results showed that height of epithelial cell (HEC) of VP and DLP in BPA group were significantly higher than that of model control (p < 0.01). BPA could also decrease testosterone level and increase prostate-specific antigen level. E(2) treatment also showed an obvious effect on relative weight of VP and DLP, HEC, and hormone levels. We concluded that environment exposure to low dose of BPA may induce prostate to proliferate and aggravate testosterone-induced benign hyperplasia prostate in rats.