Proliferating Cell Nuclear Antigen Labeling Index Research Articles

Based polymer nanoparticles from bee pollen attenuate non-small lung cancer through enhancement of apoptosis and cell cycle arrest in vivo

Bee pollen extract (BPE)-based polymer nanoparticles (BPENP) were fabricated using bovine serum albumin (BSA) and targeted with folic acid and were further characterized. Mice groups are: Group 1 received saline, whereas Groups 2, 3, 4, 5, and 6 received a single dose of urethane, followed by weekly injections of butylated hydroxy-toluene (BHT). After the BHT injection, the mice in Groups 3, 4, 5, and 6 received BPE, Avastin, BPENP, and BPENP + Avastin, respectively. The number and size of tumors were decreased in Group 6 compared to those in the other groups. The ratios of early and late apoptotic cells in Groups 3, 4, 5, and 6 (42.8%, 41.4%, 26.2%, and 45.4%, respectively) were higher than that in the untreated group. The PCNA-labeling indexes (LI)% in tissues and lesions from Group 6 were lower than those in the other groups; on the other hand, the Caspase-3 LI (%) was higher than those in the other groups. No significant differences in HRAS and MAPK levels were observed between Group 6 and the other groups. However, the level of Bax was significantly increased, whereas those of Bcl2 and P21 were decreased in Group 6 compared to those in Groups 4 and 5. According to the results of the current study's in vivo lung cancer mouse model, adjuvant chemotherapy given in conjunction with a polyphenolic substance derived from bee pollen significantly activates the apoptotic pathways as measured by flow cytometry, immunohistochemistry, and apoptotic genes. It also significantly reduces tumor volume and growth as measured by histopathology.Graphical

Comparative Analysis of Cluster of Differentiation 57 and Proliferating Cell Nuclear Antigen Expression in Different Grades of Oral Squamous Cell Carcinoma: An Immunohistochemical Study.

The immune defense against tumor cells is mainly mediated by the natural killer (NK) cells. Cluster of differentiation 57 (CD57) is a 110-kd glycoprotein, typically expressed by the NK cells, attacks the cancer cells and inhibits tumor development. Proliferating cell nuclear antigen (PCNA) is a 36-kd auxiliary protein for DNA polymerase delta that correlates with cell proliferation and DNA synthesis. It is an essential component of DNA replication, DNA recombination, and DNA repair. The uncoordinated proliferation of PCNA protein characterizes the biological behavior of malignant lesions. The aim of the present study is to compare and correlate the expression of CD57 and PCNAin different grades of oral squamous cell carcinoma (OSCC) by immunohistochemistry. This retrospective analysis comprises 30 samples of various grades of OSCCsand10 samples of healthy mucosa.Sections of 4-5 µm thickness were done and stained with monoclonal anti-PCNA and anti-CD57 antibodies.The statistical package for social science (SPSS) version 16.0 software (IBM Corp., Armonk, NY) was used to analyze the data in this study. The expression of CD57 and PCNA was compared and correlated between the groups using analysis of variance (ANOVA) post hoc, Dunnet t-test, and Pearson's correlation coefficient test.For statistical significance, a p-value of 0.05 or less was used. A significant decrease in CD57 labeling index was seen from well-differentiated squamous cell carcinoma (16.63 ± 2.33) to poorly differentiated squamous cell carcinoma (5.53 ± 1.20) whereas the significant increase in PCNA labeling index was noted from well-differentiatedsquamous cell carcinoma (45.88 ± 2.20), followed by moderately differentiated and poorly differentiated squamous cell carcinoma (72.77 ± 4.35). The combination of CD57 and PCNA biomarkers appears to be good indicators of the immune status of the patient and the aggressiveness of the lesion.

Citrinin inhibits the function of Leydig cells in male rats in prepuberty

Citrinin, a mycotoxin existing in fruits, has nephrotoxicity, hepatotoxicity and embryotoxicity. The effects of citrinin on Leydig cell development in prepuberty remains unclear. Male Sprague-Dawley rats were gavaged with 0, 1, 2.5, and 5 mg/kg citrinin from postnatal days 21–28. Citrinin at 5 mg/kg significantly decreased serum testosterone levels, while increasing serum LH and FSH levels. Citrinin at 1–5 mg/kg markedly downregulated Hsd17b3 and HSD17B3 expression, while upregulating Srd5a1 (SRD5A1) and Akr1c14 (AKR1C14) expression at 2.5 and/or 5 mg/kg. Citrinin at 5 mg/kg also significantly increased PCNA-labeling index in Leydig cells. Citrinin at 5 mg/kg significantly raised testicular MDA amount, whiling at 2.5 and 5 mg/kg downregulating SOD1 and SOD2 expression. Citrinin at 5 mg/kg markedly decreased the ratio of Bcl2 to Bax, in consistent with the increased apoptosis in Leydig cells judged by TUNEL assay. Enzymatic assay revealed that citrinin inhibited rat testicular HSD3B1 activity at 100 µM and HSD17B3 activity at 10–100 μM. Citrinin at 50 μM and higher also induced reactive oxygen species (ROS) and apoptosis of R2C cell line. In conclusion, citrinin inhibits Leydig cell development at multiple levels via different mechanisms and oxidative stress partially plays a role.

18. REFLUX OF DUODENAL CONTENTS INDUCES ESOPHAGEAL CARCINOGENESIS (SCC, ADC)

Abstract The incidence of esophageal cancer patients who have undergone distal gastrectomy is increasing recently. Distal gastrectomy is a good model for studying the clinical effects of duodenal content reflux. It is known that reflux of duodenal contents can induce mucosal injury, stimulate cell proliferation, and promote tumorigenesis. We examined the expression of COX2 and P53 in rat esophageal lesions induced by duodenal contents reflux after total gasytrectomy. Thirty 8 week old male wistar rats were exposed to duodenal content esophageal reflux. All animal underwent an esophagoduodenal anastomosis(EDA) with total gastrectomy in order to produce chronic esophagitis. Ten rats were the sham (Control). They were sacrified at the 40th week. Their esophagi were examined for HE, COX2,P53 and Proliferating cell nuclear antigen (PCNA). After 40 weeks of reflux, dysplasia(100%), squamous cell carcinoma(SCC)(40%) and adenocarcinoma (ADC) (30%)were found. PCNA Labeling index was higher in dysplastic and cancer tissue than that of normal. Overexpression of COX2 were shown in ADC and SCC. Wild type p53 accumulation were found in ADC, not in SCC. Moreover, SCC developed in places distant from the anastomosis compared to ADC. As compared with ADC, SCC is less frequently inflammatory. This means that histological features may depend on the volume of reflux contents; small amounts of reflux causes SCC and a large volume of reflux causes ADC. Reflux of duodenal contents into the esophagus led to ADC and SCC in rat. Small amounts of reflux causes SCC and a large volume of reflux causes ADC. COX2 may play an important role in esophageal cancer by duodenal content reflux. Our present results suggest an association between wild type P53 accumulation and COX2 expression in ADC, with no such relation seen in SCC.

Immunohistochemical expression of FAP and PCNA in neoplastic epithelial colonic lesions

Background Colorectal carcinoma is one of the leading causes of morbidity and mortality worldwide. Fibroblast-activation protein is expressed in cancer-associated fibroblasts and its expression is upregulated during the transformation of benign colorectal tissue into cancer. Proliferating-cell nuclear antigen expression was used as a marker of cell proliferation and is considered as a marker able to identify aggressive adenomas that may in turn become malignant. Aim To study the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in neoplastic epithelial colonic lesions and their role in adenoma–carcinoma sequence. Patients and methods This retrospective study aimed to evaluate the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in 80 cases of neoplastic epithelial colonic lesions. Results In malignant colorectal epithelial tumors, high fibroblast-activation protein expression was detected with an increase in histological grade, depth of invasion, vascular invasion, and lymph-node metastasis. No fibroblast-activation protein expression was detected in all benign cases. In included benign cases, there was a significant relation between proliferating-cell nuclear antigen labeling index and both histological type and grade of dysplasia. In the included malignant cases, there was a significant relation between proliferating-cell nuclear antigen labeling index with grade of tumor and the depth of invasion. Conclusions Both fibroblast-activation protein and proliferating-cell nuclear antigen labeling index suggested to have a role in early detection of malignant transformation and had a prognostic role in malignant cases.

Immunohistochemical analysis of proliferating cell nuclear antigen and minichromosome maintenance complex component 7 in benign and malignant salivary gland tumors.

Background:Proliferation markers have been used to determine the behavior and prognosis of benign and malignant tumors; this study was aimed to compare the immunohistochemical (IHC) expression of proliferating cell nuclear antigen (PCNA) and novel marker minichromosome maintenance complex component 7 (MCM7) in common salivary gland tumors including pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (AdCC), to find a possible significant correlation between benign and malignant tumors.Materials and Methods:In this cross-sectional study, a total of 90 cases, including 30 PAs, 30 MECs, and 30 AdCCs, were collected. The IHC expressions of PCNA and MCM7 were evaluated. Their expressions were compared with each other and between benign and malignant tumors. Statistical analysis was performed by Chi-square and Tukey's test. P value was considered 0.05.Results:Out of 30 cases of PA, 28 cases (93.3%) were PCNA positive and 28 cases (93.3%) were MCM7 positive. In the AdCC cases, 29 cases (96.6%) were PCNA positive and 29 cases (96.6%) were MCM7 positive. In the MEC cases, all cases (100%) were PCNA positive and 23 cases (76.6%) were MCM7 positive. The labeling index (LI) of MCM7 and PCNA was evaluated, and this index was lower in MCM7 LI than PCNA in all tumors. The MCM7 and PCNA expression showed a significant difference in PA and MEC (P < 0.001).Conclusion:PCNA expression was higher than MCM7 expression in salivary gland tumors. However, more studies are needed to evaluate the malignant activity of these tumors with group of markers such as MCM family members.

Nuclear Expression of Pygo2 Correlates with Poorly Differentiated State Involving c-Myc, PCNA and Bcl9 in Myanmar Hepatocellular Carcinoma.

In Myanmar, hepatocellular carcinoma (HCC) is commonly seen in young adult and associated with poor prognosis, while the molecular mechanisms that characterize HCC in Myanmar are unknown. As co-activation of Wnt/β-catenin signaling and c-Myc (Myc) are reported to associate with malignancy of HCC, we immunohistochemically investigated the expression of Pygo2 and Bcl9, the co-activators of the Wnt/β-catenin signaling, Myc and PCNA in 60 cases of Myanmar HCC. Pygo2 expression was confirmed by in situ hybridization. The signal intensity was measured by image analyzer and then statistically analyzed. As a result, the expression of Pygo2 was significantly higher in HCC compared to normal liver tissue and the nuclear signal was the most intense in poorly differentiated HCC. Cytoplasmic Bcl9 was expressed in the normal liver tissue but decreased in HCC with the progression of histopathological grade. Myc was significantly higher in poorly differentiated HCC, whereas PCNA labeling index increased with the progression of histopathological grade. Nuclear Pygo2 showed strong correlation with nuclear Myc (P < 0.01) and PCNA (P < 0.001), and inversely correlated with cytoplasmic Bcl9 (P < 0.01). Our results suggested Wnt/β-catenin and Myc signaling is commonly activated in Myanmar HCC and that the correlative upregulation of nuclear Pygo2 and Myc characterizes the malignant features of HCC in Myanmar.

Cisplatin induces differentiation in teratomas derived from pluripotent stem cells.

IntroductionCurrently, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be induced to differentiate at the cellular level but not to form mature tissues or organs suitable for transplantation. ESCs/iPSCs form immature teratomas after injection into immunodeficient mice. In humans, immature teratomas often transform into fully differentiated mature teratomas after administration of anticancer agents.MethodsWe first investigated the ability of cisplatin to induce changes in mouse ESCs/iPSCs in vitro. Next, we designed experiments to analyze ESC/iPSC-derived immature teratoma tissue in vivo after treatment of cisplatin. Groups of six mice carrying ESC- or iPSC-derived teratomas were given either low or high dose intraperitoneal injection of cisplatin, while the control group received saline for 4 weeks.ResultsTreatment of ESC/iPSC cultures with cisplatin for 3 days caused a dose-related decrease in cell numbers without inducing any morphological changes to the cells. ESC/iPSC-derived teratomas showed lower growth rates with a significantly higher mature components ratio in a concentration dependent manner after cisplatin treatment (P < 0.05); however, immunohistochemical analyses demonstrated a significantly reduced PCNA labelling index and an increase in an apoptosis marker on immature neural components (P < 0.05) along with emergence of h-Caldesmon+ mature smooth muscle cells in treated mice. Moreover, newly differentiated components not found in the control group, such as mature adipose tissue, cartilage, and pancreas, as well as striated muscle, salivary glands, gastric mucosa with fundic glands, and hair follicles emerged. The identities of these components were confirmed by immunostaining for specific markers.ConclusionsCisplatin has the ability to reduce immature components in ESC/iPSC-derived teratomas, presumably through apoptosis, and also to induce them to differentiate.

Platelets Stimulate Liver Regeneration in a Rat Model of Partial Liver Transplantation.

Living donor liver transplantation (LDLT) is sometimes associated with impaired regeneration and severe ischemia/reperfusion injury (IRI) in the graft, resulting in small-for-size syndrome (SFSS). Platelets were previously reported to stimulate liver regeneration in models of hepatectomy, but the evidence in partial liver transplantation (LT) is lacking. In this study, a rat model of partial LT was used, and the impact of thrombopoietin (TPO)-induced perioperative thrombocytosis on graft regeneration, IRI, and survival was investigated. In experiment 1, a 30% partial LT was performed. Under thrombocytosis, SFSS was attenuated, as shown by decreased levels of serum aminotransferases, bilirubin, and ascites. Serum hepatocyte regeneration-related cytokines, including insulin-like growth factor-1, hepatocyte growth factor, interleukin 6 (IL6), and tumor necrosis factor α (TNF-α), were elevated. In addition, the proliferative signaling pathways, Ki-67-labeling index, proliferating cell nuclear antigen (PCNA)-labeling index, mitotic index, and liver/body weight ratio were increased under thrombocytosis. The platelet-induced regeneration was independent of TPO because increases in the Ki-67-labeling and PCNA-labeling indexes were eliminated after reducing platelet counts by antiplatelet serum in rats administered with TPO. For IRI, thrombocytosis did not aggravate oxidative stress or downstream signaling pathways, necrosis, or apoptosis in the graft. After Kupffer cell (KC) depletion, the platelet-induced attenuation of serum aminotransferases, increased serum levels of IL6 and TNF-α, and proliferation-related signaling pathways were eliminated. Moreover, platelet accumulation in the graft decreased substantially. In experiment 2, a 20% partial LT was performed, and thrombocytosis improved postoperative survival. In conclusion, our results suggested that thrombocytosis stimulated graft regeneration and prolonged survival without aggregating IRI after partial LT, and KCs vitally contributed to platelet-derived regeneration. Platelet therapies to increase perioperative platelet counts may improve the outcomes after LDLT.

Two-Stage Portal Vein Ligation Facilitates Liver Regeneration Safely in Rats with Liver Cirrhosis

Background Portal vein (PV) embolization is performed prior to extended hepatectomy for the damaged liver to increase future remnant liver volume and prevent postoperative liver failure. This study examined whether two-stage PV ligation (PVL) increased regeneration and hypertrophy of the future remnant liver compared to conventional PVL, and whether two-stage PVL was safe for damaged liver. Method We produced a cirrhotic liver rat model with perioperatively maintained fibrosis. Rats were divided into: Group A (70%PVL), ligation of left branch of PV; Group B (90%PVL), ligation of right and left branches of PV; and Group C (two-stage 90%PVL), two-stage PVL with left branch ligation of PV followed by right branch ligation 7 days later. To evaluate liver regeneration, liver weight ratios, proliferating cell nuclear antigen (PCNA) labeling index (LI), mitotic index (MI), and TdT-mediated dUTP-biotin nick end labeling (TUNEL) LI in the non-ligated caudate lobe were measured. Results Fourteen-day survival rate was 20% in Group B but 100% in Group C. TUNEL LI differed significantly between Groups A and B at 2 and 7 days postoperatively. Weight ratios were significantly higher in Group C than in Groups A and B at 14 days postoperatively. PCNA LI and MI in the non-ligated caudate lobe decreased to preoperative levels by 7 days postoperatively in Groups A and B, but remained elevated until 14 days postoperatively in Group C. Conclusion In cirrhotic liver rats, two-stage PVL avoided the lethal liver failure seen with one-stage PVL, and significantly facilitated liver regeneration more than one-stage PVL.

Long-Term Nonclinical Pulmonary Safety Assessment of Afrezza, a Novel Insulin Inhalation Powder.

Afrezza delivers inhaled insulin using the Gen2 inhaler for the treatment of patients with type 1 and type 2 Diabetes. Afrezza was evaluated in long-term nonclinical pulmonary safety studies in 2 toxicology species. Chronic inhalation toxicology studies in rat (26 weeks) and dog (39 weeks) and an inhalation carcinogenicity study in rats were conducted with Technosphere insulin (Afrezza) and with Technosphere alone as a vehicle control. Respiratory tract tissues were evaluated by histopathology and cells expressing proliferating cell nuclear antigen (PCNA) were quantified in lungs of rats. Microscopic findings in rats exposed to Afrezza were attributed to the Technosphere particle component, were confined to nasal epithelia, and consisted of eosinophilic globules and nasal epithelial degeneration. There were no Afrezza-related changes in pulmonary PCNA labeling indices in alveoli, large bronchioles, or terminal bronchioles. Microscopic findings in rats exposed to Technosphere particles included eosinophilic globules, mucus cell hyperplasia, and epithelial degeneration in the nasal cavities. PCNA labeling indices were increased in large bronchioles and terminal bronchioles but not in alveoli. There were no Technosphere particle-related findings in the dog study. Afrezza did not exhibit carcinogenic potential in the 2-year study in rats. These nonclinical inhalation studies support the use of Afrezza in humans over extended periods.

Paraquat exposure delays late-stage Leydig cell differentiation in rats during puberty

Paraquat is a fast and non-selective herbicide that is widely used in crop cultivation and conservation tillage systems. Animal experiments have shown that paraquat decreases sperm quality and testicular organ coefficient, but its effects on the development of Leydig cells remain unclear. The objective of the current study was to investigate the effects of paraquat exposure on the Leydig cell development in rats during puberty. Twenty-eight male 35-day-old Sprague-Dawley rats were divided into 4 groups: 0, 0.5, 2.0, and 8 mg kg−1 d−1 paraquat. Paraquat was gavaged for 10 d. Adult Leydig cells were isolated and treated with paraquat for 24 h. Paraquat in vivo significantly decreased body and testis weights at 8 mg kg−1 and lowered serum testosterone levels at 2 and 8 mg kg−1 without affecting the levels of serum luteinizing hormone and follicle-stimulating hormone. Paraquat did not alter Leydig cell number and PCNA labeling index. Real-time PCR showed that paraquat down-regulated the expression of Lhcgr, Scarb1, Cyp11a1, Cyp17a1, and Hsd17b3 genes and their proteins at 2 or 8 mg kg−1, while it up-regulated the expression of Srd5a1 at 8 mg kg−1. Paraquat increased ROS and decreased testosterone production by Leydig cells at 1 and 10 μM after in vitro 24-h exposure. Vitamin E (40 μg/ml) reversed paraquat-induced ROS and suppression of testosterone synthesis in vitro. In conclusion, paraquat directly delays Leydig cell differentiation to block testosterone synthesis via down-regulating the expression of critical testosterone synthesis-related genes and up-regulating the expression of testosterone metabolic enzyme (Srd5a1) gene and possibly via increasing ROS production.

Partial Epithelial-Mesenchymal Transition Was Observed Under p63 Expression in Acquired Middle Ear Cholesteatoma and Congenital Cholesteatoma.

Partial epithelial-mesenchymal transition (p-EMT) is a process by which epithelial cells partially lose their intercellular adhesion and change to obtain migration ability. The transcription factor p63 regulates the expression of cadherin family and induces epithelial cell proliferation. In this study, we hypothesized that p-EMT under p63 expression may be a key factor in epithelial cell growth in middle ear cholesteatoma. Specimens were surgically excised from patients with congenital cholesteatoma (CC) (n = 48), acquired middle ear cholesteatoma (AC) (n = 120), and normal skin tissue (n = 34). We analyzed immunohistochemically for the EMT marker (N-cadherin), adherence junction marker (E-cadherin), and tight junction marker (claudin-1, claudin-4, occludin). We also examined the labeling index (LI) of p63 and Proliferating cell nuclear antigen (PCNA) (late S phase marker), and Snail expression as a mobility marker. The expression of p63 (CC 51.0 ± 7.4%, AC 50.0 ± 5.9%) was significantly higher in the thickened epithelium of CC and AC compared with normal skin tissue (p < 0.0001). The loss of E-cadherin was observed (CC 50.0%, AC 55.8%) but the expression patterns in the tight junction were almost normal. N-cadherin was partially detected in the basal and upper layer of epithelium in CC and AC. In contrast to that of normal skin tissue, the LI of PCNA was significantly higher in AC (p < 0.0001). The positive rate of Snail was significantly higher in CC (p < 0.0001). This study indicates that p-EMT via the p63 signaling pathway might plays an essential role in epithelial growth in AC and CC formation, although tight junction formation and terminal differentiation were not affected in those processes.

The Effect of Estrogen on Hepatic Fat Accumulation during Early Phase of Liver Regeneration after Partial Hepatectomy in Rats.

Fatty liver is common in men and post-menopausal women, suggesting that estrogen may be involved in liver lipid metabolism. The aim of this study is to be clear the role of estrogen and estrogen receptor alpha (ERα) in fat accumulation during liver regeneration using the 70% partial hepatectomy (PHX) model in male, female, ovariectomized (OVX) and E2-treated OVX (OVX-E2) rats. Liver tissues were sampled at 0–48 hr after PHX and fat accumulation, fatty acid translocase (FAT/CD36), sterol regulatory element-binding protein (SREBP1c), peroxisome proliferator-activated receptor α (PPARα), proliferative cell nuclear antigen (PCNA) and ERα were examined by Oil Red O, qRT-PCR and immunohistochemistry, respectively. Hepatic fat accumulation was abundant in female and OVX-E2 compared to male and OVX rats. FAT/CD36 expression was observed in female, OVX and OVX-E2 at 0–12 hr after PHX, but not in male rats. At 0 hr, SREBP1c and PPARα were elevated in female and male rats, respectively, but were decreased after PHX in all rats. The PCNA labeling index reached a maximum at 36 hr and 48 hr in OVX-E2 and OVX rats, respectively. ERα expression in OVX-E2 was higher than OVX at 0–36 hr after PHX. In conclusion, these results indicated that estrogen and ERα might play an important role in fat accumulation related to FAT/CD36 during early phase of rat liver regeneration.

Stilbene compound trans-3,4,5,4´-tetramethoxystilbene, a potential anticancer drug, regulates constitutive androstane receptor (Car) target genes, but does not possess proliferative activity in mouse liver

The constitutive androstane receptor(CAR) activation is connected with mitogenic effects leading to liver hyperplasia and tumorigenesis in rodents. CAR activators, including phenobarbital, are considered rodent non-genotoxic carcinogens. Recently, trans-3,4,5,4´-tetramethoxystilbene(TMS), a potential anticancer drug (DMU-212), have been shown to alleviate N-nitrosodiethylamine/phenobarbital-induced liver carcinogenesis. We studied whether TMS inhibits mouse Car to protect from the PB-induced tumorigenesis. Unexpectedly, we identified TMS as a murine CAR agonist in reporter gene experiments, in mouse hepatocytes, and in C57BL/6 mice in vivo. TMS up-regulated Car target genes Cyp2b10, Cyp2c29 and Cyp2c55 mRNAs, but down-regulated expression of genes involved in gluconeogenesis and lipogenesis. TMS did not change or down-regulate genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, or Mdm2, which were up-regulated by another Car ligand TCPOBOP. TMS did not increase liver weight and had no significant effect on Ki67 and Pcna labeling indices in mouse liver in vivo. In murine hepatic AML12 cells, we confirmed a Car-independent proapoptotic effect of TMS. We conclude that TMS is a Car ligand with limited effects on hepatocyte proliferation, likely due to promoting apoptosis in mouse hepatic cells, while controlling Car target genes involved in xenobiotic and endobiotic metabolism.

Paraquat exposure delays stem/progenitor Leydig cell regeneration in the adult rat testis

Paraquat, a widely used nonselective herbicide, is a serious hazard to human health. However, the effects of paraquat on the male reproductive system remain unclear. In this study, adult male Sprague Dawley rats were intraperitoneally injected ethane dimethane sulfonate (EDS, 75 mg/kg) to initiate a regeneration of Leydig cells. EDS-treated rats were orally exposed to paraquat (0.5, 2, 8 mg/kg/day) from post-EDS day 17 to day 28 and effects of paraquat on Leydig and Sertoli cell functions on post-EDS day 35 and day 56 were investigated. Paraquat significantly decreased serum testosterone levels at 2 and 8 mg/kg. Paraquat lowered Leydig cell Hsd17b3, Srd5a1, and Hsd11b1 mRNA levels but increased Hsd3b1 on post-EDS day 35. Paraquat lowered Cyp11a1, Cyp17a1, and Hsd11b1 but increased Srd5a1 on post-EDS day 56. However, paraquat did not alter Leydig cell number and PCNA labeling index. Epididymal staining showed that few sperms were observed in paraquat-treated rats. Primary culture of adult Leydig cells showed that paraquat diminished testosterone output and induced reactive oxygen species generation at 1 and 10 μM and apoptosis rate at 10 μM. In conclusion, a short-term exposure to paraquat delays Leydig cell regeneration from stem/progenitor Leydig cells, causing low production of testosterone and an arrest of spermatogenesis.

Stem Leydig cell regeneration in the adult rat testis is inhibited after a short-term triphenyltin exposure

Triphenyltin (TPT) is an organotin compound and may be an endocrine disruptor, impairing the male reproductive system. However, the effect of short-term TPT exposure on stem Leydig cell regeneration later on remains unknown. Here, we show that TPT affects stem Leydig cell regeneration in the adult rat testis. Adult male Sprague Dawley rats were gavaged with TPT (0, 0.5, 1.0, 2.0 mg/kg body weight/day) for 10 days, followed by a single intraperitoneal injection of ethane dimethane sulfonate (EDS, 75 mg/kg body weight) to eliminate Leydig cells. Testis parameters and hormone levels were investigated on post-EDS days 21, 35, and 56. TPT significantly reduced serum testosterone levels, decreased Leydig cell number and cell size, and down-regulated its specific gene and protein expression at 1.0 and 2.0 mg/kg even 56 days after cession of treatment. TPT lowered PCNA-labeling index of progenitor Leydig cells on post-EDS day 21. TPT also lowered AKT1 and AKT2, and ERK1/2 phosphorylation on post-EDS day 56. This study reveals that a short-term exposure to TPT blocks stem Leydig cell regeneration in the long term thus delaying spermatogenesis.

Pulmonary and pleural toxicity of potassium octatitanate fibers, rutile titanium dioxide nanoparticles, and MWCNT-7 in male Fischer 344 rats

Potassium octatitanate (K2O·8TiO2, POT) fibers are used as an alternative to asbestos. Their shape and biopersistence suggest that they are possibly carcinogenic. However, inhalation studies have shown that respired POT fibers have little carcinogenic potential. We conducted a short-term study in which we administered POT fibers, and anatase and rutile titanium dioxide nanoparticles (a-nTiO2, r-nTiO2) to rats using intra-tracheal intra-pulmonary spraying (TIPS). We found that similarly to other materials, POT fibers were more toxic than non-fibrous nanoparticles of the same chemical composition, indicating that the titanium dioxide composition of POT fibers does not appear to account for their lack of carcinogenicity. The present report describes the results of the 3-week and 52-week interim killing of our current 2-year study of POT fibers, with MWCNT-7 as a positive control and r-nTiO2 as a non-fibrous titanium dioxide control. Male F344 rats were administered 0.5 ml vehicle, 62.5 µg/ml and 125 µg/ml r-nTiO2 and POT fibers, and 125 µg/ml MWCNT-7 by TIPS every other day for 2 weeks (eight doses: total doses of 0.25 and 0.50 mg/rat). At 1 year, POT and MWCNT-7 fibers induced significant increases in alveolar macrophage number, granulation tissue in the lung, bronchiolo-alveolar cell hyperplasia and thickening of the alveolar wall, and pulmonary 8-OHdG levels. The 0.5 mg POT- and the MWCNT-7-treated groups also had increased visceral and parietal pleura thickness, increased mesothelial cell PCNA labeling indices, and a few areas of visceral mesothelial cell hyperplasia. In contrast, in the r-nTiO2-treated groups, none of the measured parameters were different from the controls.

Inhibitory effects of pentoxifylline on inflammation-related tumorigenesis in rat colon

Chronic inflammation in the colorectum increases the risk of colorectal cancer development. Pentoxifylline, a medicine used for improving the circulation, has been reported to inhibit TNF-α production and to ameliorate inflammatory bowel disease and non-alcoholic steatohepatitis. In this study, we investigated the effects of pentoxifylline on inflammation-related colon tumorigenesis in a rodent model using Kyoto APC delta rats, which have APC mutation and are susceptible to colon carcinogenesis. Male Kyoto APC delta rats were treated with azoxymethane and dextran sodium sulfate, and were subsequently administered water, with or without pentoxifylline. At the end of the experiment, the development of colorectal tumor was significantly inhibited in the pentoxifylline group. The pentoxifylline treatment also lowered the levels of oxidative stress markers and mRNAs of pro-inflammatory cytokines, including TNF-α and IL-6, in the colon mucosa. The PCNA labeling index and the inflammation score were also decreased in the colon of rats in the pentoxifylline -treated group. We also used an endoscopy to observe the tumor progression and inflammation in the colon of rats, revealing that inflammation grade was significantly lower in pentoxifylline-treated group at several points during the experiment. These findings suggest that pentoxifylline treatment might be useful for chemoprevention of inflammation-related colon cancer.

Immunohistochemical Study of the Ameliorative Effect of Vitamin E on Liver Regeneration after Different Periods of Partial Hepatectomy

The liver is almost unique in its capacity for regeneration after hepatectomy but the exact mechanisms are not yet fully clarified. Antioxidants have been shown to promote liver regeneration after major hepatectomy. The present study evaluated the ameliorative effect of vitamin E administration on the liver regeneration after different periods of partial hepatectomy (PH) in rats. Fifty-six adult male albino rats were divided into three groups: Control sham operated group; partially hepatectomized group which were divided into three subgroups sacrificed at 1day, 3 days and 7days after the operation respectively; Partially Hepatectomized group with vitamin E pretreatment before PH where the rats were given a daily oral dose of vitamin E until the time of sacrifice of the rats. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and labeling index were demonstrated. After PH, the PCNA positive hepatocytes and the PCNA labeling indices were significantly high after the 1st day and then much decreased after the 3rd day, to be followed by a slight increase at the 7th day. Vitamin E pretreatment in PH rats resulted in a decrease in PCNA positive cells and its labeling indices in the 1st day with a gradual increase in the 3rd and 7th days. Vitamin E has an inhibitory effect in the first 24 hours on liver regeneration followed by stimulatory effect at the third and seventh days after PH. These data indicated that vitamin E pretreatment has an important role in regulation and enhancement of liver regeneration after PH.