Testicular alterations in cryptorchid/orchiopexic rats chronically exposed to acrylamide or di-butyl-phthalate.

Exposure of Sprague-Dawley (SD) rats to acrylamide (AA) or di-butyl-phthalate (DBP) from the 12th gestational day to the 16th postnatal week (PNW) has been shown to reduce the effectiveness of orchiopexy in recovering the testicular alterations associated with experimental cryptorchidism established at weaning. Herein, we provide information about the long-term effects of AA or DBP on the testes of cryptorchid/orchiopexic rats. Male offspring exposed in utero to 10 mg/kg/day AA or 500 mg/kg/day DBP underwent bilateral surgical cryptorchidism at the 3rd PNW and orchiopexy at the 6th week, with continuous exposure to the chemicals through diet until the 58th week. Regardless of the test chemical, there were severe qualitative/quantitative alterations in the seminiferous tubules and increased numbers of Leydig cells. There was an increase and decrease in the number of tubules with c-Kit- and placental alkaline phosphatase-labeled germ cells, respectively, as compared to those in the control group, suggesting an imbalance between apoptosis and cell proliferation processes. The histological scores of the testicular lesions at the end of this one-year study were higher than those in the previous 16-week study, indicating that exposure of rats to the toxicants AA or DBP enhanced the testicular alterations induced by the chemicals beginning at the intra-uterine life, and impaired the effectiveness of orchiopexy in restoring the testes to normal morphology. Although the present experimental protocol does not completely replicate the natural human undescended testes, our findings may contribute to understanding the alterations occurring in cryptorchid/orchiopexic testes potentially exposed to exogenous chemicals for extended periods.

Time response of rat testicular alterations induced by cryptorchidism and orchiopexy.

Cryptorchidism is one of the main risk factors for infertility and testicular cancer. Orchiopexy surgery corrects cryptorchidism effects. Different models of cryptorchidism developed in the rat include surgery. We assessed testicular alterations in rats submitted to surgical cryptorchidism and examined their potential for reversibility at different time points in order to verify time dependency effect(s) on the recovery of the undescended testes. Cryptorchidism was induced in 3-week-old rats. Animals were euthanized 3, 6 or 11 weeks after surgery to evaluate the morphological progression of cryptorchidism-induced germinative epithelial alterations. Other groups underwent orchiopexy 3, 5 or 9 weeks after surgical cryptorchidism, before or after puberty. Animals were euthanized 3 or 8 weeks after orchiopexy. Controls underwent sham surgery at the same time points as the surgical groups. Cryptorchid testes showed decreased weight, germinative epithelial degeneration, apoptosis and vacuolation, corresponding to impairment of spermatogenesis and of Sertoli cells. Some tubules has a Sertoli cell-only pattern and atrophy. The intensity of damage was related to the duration of cryptorchidism. After orchiopexy, spermatogenesis completely recovered only when testicular relocation occurred before puberty and the interval for recovery was extended. These results indicate that age, sexual maturity and extension of germ cell damage were relevant for producing germ cell restoration and normal spermatogenesis. We provide original observations on the time dependency of testicular alterations induced by cryptorchidism and their restoration using morphologic, morphometric and immunohistochemical approaches. It may be useful to study germ cell impairment, progression and recovery in different experimental settings, including exposure to exogenous chemicals.

Preliminary preclinical study of Chol-DsiRNA polyplexes formed with PLL[30]-PEG[5K] for the RNAi-based therapy of breast cancer.

RNA interference molecules have tremendous potential for cancer therapy but are limited by insufficient potency after i.v. administration. We previously found that Chol-DsiRNA polyplexes formed between cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase the activity of Chol-DsiRNA against a stably expressed reporter mRNA in primary murine syngeneic breast tumors after daily i.v. dosing. Here, we provide a more thorough preliminary preclinical study of Chol-DsiRNA polyplexes against the therapeutically relevant target protein, STAT3. We found that Chol-DsiSTAT3 polyplexes greatly increase plasma exposure, distribution, potency, and therapeutic activity of Chol-DsiSTAT3 in primary murine syngeneic 4T1 breast tumors after i.v. administration. Furthermore, inactive Chol-DsiCTRL polyplexes are well tolerated by healthy female BALB/c mice after chronic i.v. administration at 50 mg Chol-DsiCTRL/kg over 28 days. Thus, Chol-DsiRNA polyplexes may be a good candidate for Phase I clinical trials to improve the treatment of breast cancer and other solid tumors.

Urinary crystal formation and urothelial effects of pyroxasulfone administered to male rats.

Pyroxasulfone induced a low incidence of urinary bladder tumors in male rats in a 2-year bioassay at 1000 and 2000 ppm, with occasional urinary calculi. No increased incidence of tumors of any tissue occurred in female rats or in mice of either gender. We performed three short-term studies to evaluate early development of pyroxasulfone-induced urinary crystals and urothelial cytotoxicity with consequent regenerative proliferation. First, male rats were treated with dietary 50, 1000 or 2000 ppm pyroxasulfone for 1, 3 or 7 days. The urothelium was examined by light and scanning electron microscopy (LM, SEM) and bromodeoxyuridine labeling index (BrdU LI). In two other studies, male rats were treated with dietary 20 000 ppm pyroxasulfone for 1 week. Urine collected at various times of day was examined by SEM and energy dispersive spectroscopy (EDS) or by LM, SEM, EDS, and infrared spectroscopy (IFS). Urinary crystals were present at various time points. EDS and IFS showed some contained calcium; others contained organic matter. Cytotoxicity was detected by SEM as cellular swelling, craters, and necrosis and by LM as cellular hypertrophy. Increased cell proliferation was detected by LM (hyperplasia), SEM (piling up of round cells), and by increased BrdU LI. There was no evidence of increased apoptosis. These findings support a mode of action for pyroxasulfone-associated bladder tumors in male rats involving formation of urinary crystals leading to urothelial cytotoxicity and regenerative proliferation. This is a high dose phenomenon, therefore, pyroxasulfone is not likely to be carcinogenic to humans at exposure levels that do not cause crystals with subsequent calculi formation in the urinary tract.

Histopathology re-examination of the NTP toxicity/carcinogenicity studies of tert-butyl alcohol to identify renal tumor and toxicity modes of action.

Tert-butyl alcohol (TBA) targets the rat kidney following repeated exposures, including renal tubule tumors. The mode of action (MOA) of these tumors, concluded by a pathology working group, involves both alpha2u-globulin nephropathy (α2u-gN) and exacerbated chronic progressive nephropathy (CPN), but has been disputed and an undefined MOA proposed. This study further reviews the histology slides of male and female rat kidneys from the NTP drinking water 13-week toxicity and 2-year carcinogenicity studies, including the 15-month interim sacrifice group. The papillary epithelial lining alteration formerly referred to as "transitional cell hyperplasia" develops as part of advanced CPN and does not represent a separate toxicity. No changes were observed in the kidney pelvis urothelium. The only alterations in subchronic male rats involved α2u-gN and CPN, without test article-related alterations in females. Focused examination of areas of parenchyma unaffected by CPN in TBA-treated male and female rats of the chronic studies revealed no renal tubule abnormalities other than from the effects of α2u-gN and CPN. Unrelated to toxicity were spontaneous amphophilic or vacuolar tubule proliferative lesions. All observed TBA-associated non-neoplastic and neoplastic histopathological changes in the kidney can be explained by α2u-gN or enhanced CPN, neither of which are relevant to humans.

Isolation and molecular characterization of spermatogonia from male Sprague-Dawley rats exposed in utero and postnatally to dibutyl phthalate or acrylamide.

The increased incidence of testicular disorders in young men and the possible influence of environmental chemicals, such as dibutyl phthalate (DBP) and acrylamide (AA), requires experimental models for identifying modes of action. Most published reproductive toxicologic studies use RNA samples from the total testis to evaluate testicular gene expression; however, analyses of isolated cell types could provide a more specific tool. Among testicular germ cells, spermatogonia are critical since they represent the onset of spermatogenesis. This study aimed, (1) to establish a technique for spermatogonia isolation; (2) to apply this isolation technique to verify possible gene expression alterations (Pou5f1, Kitlg, Mki-67, Bak1 and Spry4) in prepubertal post-natal day, (PND24) and pubertal (PND45) testes after in utero and postnatal exposure to DBP or AA. The technique was efficient for isolation of a majority of spermatogonia. In utero DBP exposure led to reduced litter body weight at birth, reduced anogenital distance of male pups on PND4, and increased frequency of male nipple retention on PND14 compared to controls. DBP-exposed relative testes weights were reduced only at PND24 compared to control but they did not differ at PND45. DBP-exposed animals showed reduced expression levels of Pou5f1 and Mki67 on PND24, and reduced expression of Pou5f1 and Spry4 on PND45. AA exposure reduced expression of Pou5f1, Mki67, and Spry4 at PND45 although not significantly. Our results suggest that DBP acts by reducing cell proliferation and impairing differentiation in prepubertal and pubertal testes.

Epithelium Lining Rat Renal Papilla: Nomenclature and Association with Chronic Progressive Nephropathy (CPN).

Chronic progressive nephropathy (CPN) occurs commonly in rats, more frequently and severely in males than females. High-grade CPN is characterized by increased layers of the renal papilla lining, designated as urothelial hyperplasia in the International Harmonization of Nomenclature and Diagnostic Criteria classification. However, urothelium lining the pelvis is not equivalent to the epithelium lining the papilla. To evaluate whether the epithelium lining the renal papilla is actually urothelial in nature and whether CPN-associated multicellularity represents proliferation, kidney tissues from aged rats with CPN, from rats with multicellularity of the renal papilla epithelium of either low-grade or marked severity, and from young rats with normal kidneys were analyzed and compared. Immunohistochemical staining for uroplakins (urothelial specific proteins) was negative in the papilla epithelium in all rats with multicellularity or not, indicating these cells are not urothelial. Mitotic figures were rarely observed in this epithelium, even with multicellularity. Immunohistochemical staining for Ki-67 was negative. Papilla lining cells and true urothelium differed by scanning electron microscopy. Based on these findings, we recommend that the epithelium lining the papilla not be classified as urothelial, and the CPN-associated lesion be designated as vesicular alteration of renal papilla instead of hyperplasia and distinguished in diagnostic systems from kidney pelvis urothelial hyperplasia.

Identification of Methylated Dithioarsenicals in the Urine of Rats Fed with Sodium Arsenite.

Biotransformation of inorganic arsenic results in the formation of methylarsenicals of both oxygen and sulfur analogues. Aiming to improve our understanding of metabolism of inorganic arsenic in animals, we conducted an animal feeding study with an emphasis on identifying new arsenic metabolites. Female F344 rats were given 0, 1, 10, 25, 50, and 100 µg/g of arsenite (iAs(III)) in the diet. Arsenic species in rat urine were determined using high performance liquid chromatography (HPLC) separation and inductive coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI MS/MS) detection. Nine arsenic species were detected in the urine of the iAs(III)-dosed rats. Seven of these arsenic species were consistent with previous reports, including iAs(III), arsenate, monomethyarsonic acid, dimethylarsinic acid, trimethylarsine oxide, monomethylmonothioarsonic acid, and dimethylmonothioarsinic acid. Two new methyldithioarsencals, monomethyldithioarsonic acid (MMDTA(V)) and dimethyldithioarsinic acid (DMDTA(V)), were identified for the first time in the urine of rats treated with iAs(III). The concentrations of both MMDTA(V) and DMDTA(V) in rat urine were dependent on the dosage of iAs(III) in diet. The concentration of DMDTA(V) was approximately 5 times higher than that of MMDTA(V). MMDTA(V) has not been identified in any biological samples of animals, and DMDTA(V) has not been reported as a metabolite of inorganic arsenic in the rats. The identification of novel methylated dithioarsenicals as metabolites of inorganic arsenic in the rat urine provided further insights into the understanding of the metabolism of arsenic.

Critical role of toxicologic pathology in a short-term screen for carcinogenicity.

Carcinogenic potential of chemicals is currently evaluated using a two year bioassay in rodents. Numerous difficulties are known for this assay, most notably, the lack of information regarding detailed dose response and human relevance of any positive findings. A screen for carcinogenic activity has been proposed based on a 90 day screening assay. Chemicals are first evaluated for proliferative activity in various tissues. If negative, lack of carcinogenic activity can be concluded. If positive, additional evaluation for DNA reactivity, immunosuppression, and estrogenic activity are evaluated. If these are negative, additional efforts are made to determine specific modes of action in the animal model, with a detailed evaluation of the potential relevance to humans. Applications of this approach are presented for liver and urinary bladder. Toxicologic pathology is critical for all of these evaluations, including a detailed histopathologic evaluation of the 90 day assay, immunohistochemical analyses for labeling index, and involvement in a detailed mode of action analysis. Additionally, the toxicologic pathologist needs to be involved with molecular evaluations and evaluations of new molecularly developed animal models. The toxicologic pathologist is uniquely qualified to provide the expertise needed for these evaluations.

Inorganic arsenic: A non-genotoxic carcinogen.

Inorganic arsenic induces a variety of toxicities including cancer. The mode of action for cancer and non-cancer effects involves the metabolic generation of trivalent arsenicals and their reaction with sulfhydryl groups within critical proteins in various cell types which leads to the biological response. In epithelial cells, the response is cell death with consequent regenerative proliferation. If this continues for a long period of time, it can result in an increased risk of cancer. Arsenicals do not react with DNA. There is evidence for indirect genotoxicity in various in vitro and in vivo systems, but these involve exposures at cytotoxic concentrations and are not the basis for cancer development. The resulting markers of genotoxicity could readily be due to the cytotoxicity rather than an effect on the DNA itself. Evidence for genotoxicity in humans has involved detection of chromosomal aberrations, sister chromatid exchanges in lymphocytes and micronucleus formation in lymphocytes, buccal mucosal cells, and exfoliated urothelial cells in the urine. Numerous difficulties have been identified in the interpretation of such results, including inadequate assessment of exposure to arsenic, measurement of micronuclei, and potential confounding factors such as tobacco exposure, folate deficiency, and others. Overall, the data strongly supports a non-linear dose response for the effects of inorganic arsenic. In various in vitro and in vivo models and in human epidemiology studies there appears to be a threshold for biological responses, including cancer.

Diuron-induced rat urinary bladder carcinogenesis: mode of action and human relevance evaluations using the International Programme on Chemical Safety framework.

Diuron, a high volume substituted urea herbicide, induced high incidences of urinary bladder carcinomas and low incidences of kidney pelvis papillomas and carcinomas in rats exposed to high doses (2500 ppm) in a 2-year bioassay. Diuron is registered for both occupational and residential uses and is used worldwide for more than 30 different crops. The proposed rat urothelial mode of action (MOA) for this herbicide consists of metabolic activation to metabolites that are excreted and concentrated in the urine, leading to cytotoxicity, urothelial cell necrosis and exfoliation, regenerative hyperplasia, and eventually tumors. We show evidence for this MOA for diuron using the International Programme on Chemical Safety (IPCS) conceptual framework for evaluating an MOA for chemical carcinogens, and the United States Environmental Protection Agency (USEPA) and IPCS framework for assessing human relevance.

Time course of urothelial changes in rats and mice orally administered arsenite.

Inorganic arsenic (arsenite and arsenate) at high exposures is a known human carcinogen, inducing tumors of the urinary bladder, skin, and lungs. In two experiments, we examined the urothelial proliferative effects of treatment with 173 ppm sodium arsenite (100 ppm arsenic) in the drinking water for 6 and 24 hr, and 3, 7, and 14 days in female F344 rats and 43.3 ppm sodium arsenite (25 ppm arsenic) in female C57BL/6 wild-type and arsenic (+3 oxidation state) methyltransferase knockout (As3mt KO) mice that are unable to methylate arsenicals. In the rat and both mouse genotypes, scanning electron microscopy showed cytotoxic urothelial changes as early as 6 hr after the start of arsenic exposure. The severity of As(III)-induced cytotoxic urothelial changes increased over time in the rat and in the As3mt KO mouse. Light microscopy showed an increase in urothelial hyperplasia in the rat. No significant increases in bromodeoxyuridine-labeling index were observed. The data support the hypothesis that the sequence of events in the mode of action for urothelial effects of orally administered inorganic arsenic in the rat and mouse involves superficial cytotoxicity with consequent regenerative increased cell proliferation similar to the findings associated with the administration of dimethylarsinic acid (DMA(V)) in rats.

Arsenic speciation in the blood of arsenite-treated F344 rats.

Arsenic speciation in blood can improve understanding of the metabolism and toxicity of arsenic. In this study, arsenic species in the plasma and red blood cells (RBCs) of arsenite-treated female F344 rats were characterized using anion exchange and size exclusion chromatography separation with inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI MS/MS) detection. Arsenite (iAs(III)), arsenate (iAs(V)), monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), trimethylarsine oxide (TMAO(V)), monomethylmonothioarsonic acid (MMMTA(V)), and dimethylmonothioarsinic acid (DMMTA(V)) were detected in the plasma, with DMA(V) being the predominant metabolite. Upon oxidative pretreatment with 5% hydrogen peroxide (H2O2), plasma proteins released bound arsenic in the form of DMA(V) as the major species and MMA(V) as the minor species. The ratio of protein-bound arsenic to total arsenic decreased with increasing dosage of iAs(III) administered to the rats, suggesting a possible saturation of the binding capacity of the plasma proteins. The proportion of the protein-bound arsenic in the plasma varied among rats. In the H2O2-treated lysates of red blood cells of rats, DMA(V) was consistently found as the predominant arsenic species, probably reflecting the preferential binding of dimethylarsinous acid (DMA(III)) to rat hemoglobin. iAs(V), MMA(V), and trimethylarsine oxide (TMAO(V)) were also detected in the hydrogen peroxide-treated lysates of red blood cells. Importantly, DMMTA(V) and MMMTA(V) have not been reported in rat blood, and the present finding of DMMTA(V) and MMMTA(V) in the rat plasma is toxicologically relevant because these pentavalent thioarsenicals are more toxic than their counterparts DMA(V) and MMA(V). Identifying novel thiolated arsenicals and determining protein-bound arsenicals in the blood provide useful insights into the metabolism and toxicity of arsenic in animals.

Evaluation of the carcinogenicity of inorganic arsenic.

Inorganic arsenic (iAs) at high exposures is a human carcinogen, affecting mainly the urinary bladder, lung and skin. We present an assessment of the mode of action (MOA) of iAs's carcinogenicity based on the United States Environmental Protection Agency/International Programme on Chemical Safety (USEPA/IPCS) framework, focusing primarily on bladder cancer. Evidence is presented for a MOA involving formation of reactive trivalent metabolites interacting with critical cellular sulfhydryl groups, leading to cytotoxicity and regenerative cell proliferation. Metabolism, kinetics, cell transport, and reaction with specific proteins play a critical role in producing the effects at the cellular level, regardless of cell type, whether urothelium, lung epithelium or epidermis. The cytotoxicity induced by iAs results in non-cancer toxicities, and the regenerative cell proliferation enhances development of epithelial cancers. In other tissues, such as vascular endothelium, different toxicities develop, not cancer. Evidence supporting this MOA comes from in vitro investigations on animal and human cells, from animal models, and from epidemiological studies. This MOA implies a non-linear, threshold dose-response relationship for both non-cancer and cancer end points. The no effect levels in animal models (approximately 1 ppm of water or diet) and in vitro (>0.1 µM trivalent arsenicals) are strikingly consistent. Cancer effects of iAs in humans generally are not observed below exposures of 100-150 ppb in drinking water: below these exposures, human urine concentrations of trivalent metabolites are generally below 0.1 µM, a concentration not associated with bladder cell cytotoxicity in in vitro or animal models. Environmental exposures to iAs in most of the United States do not approach this threshold.

Evaluation of expression profiles of hematopoietic stem cell, endothelial cell, and myeloid cell antigens in spontaneous and chemically induced hemangiosarcomas and hemangiomas in mice.

It is unclear whether the process of spontaneous and chemically induced hemangiosarcoma and hemangioma formation in mice involves the transformation of differentiated endothelial cells (ECs) or recruitment of multipotential bone marrow-derived hematopoietic stem cells or endothelial progenitor cells (EPCs), which show some degree of endothelial differentiation. In the present study, immunohistochemical staining for hematopoietic stem cell markers (CD45 and CD34), EC markers (vascular endothelial growth factor receptor 2 [VEGFR2], CD31, and factor VIII-related antigen), and a myeloid lineage marker (CD14) was employed to better define the origin of hemangiosarcomas and hemangiomas in mice. Staining was negative for CD45, factor VIII-related antigen, and CD14 and positive for CD34, VEGFR2, and CD31, indicating that mouse hemangiosarcomas and hemangiomas are composed of cells derived from EPCs expressing CD34, VEGFR2, and CD31 but not factor VIII-related antigen. The lack of CD45 expression suggests that mouse vascular tumors may arise from EPCs that are at a stage later than hematopoietic stem cells. Since factor VIII-related antigen expression is known to occur later than CD31 expression in EPCs, our observations may indicate that these tumor cells are arrested at a stage prior to complete differentiation.  In addition, myeloid lineage cells do not appear to contribute to hemangiosarcoma and hemangioma formation in mice.

Evaluation of direct and indirect effects of the PPARγ agonist troglitazone on mouse endothelial cell proliferation.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists are used in the treatment of type 2 diabetes mellitus and hyperlipidemias. In carcinogenicity studies, some of these agonists induced hemangiomas/hemangiosarcomas in mice, but not in rats. We hypothesized that increased endothelial cell (EC) proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice. We previously showed that the sarcomagenic PPARγ agonist troglitazone (TG) increased EC proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses (400 and 800 mg/kg) after four weeks of treatment. In vitro, TG had a mitogenic effect on mouse microvascular mouse ECs by increasing cell proliferation and survival. The current studies showed that treatment of mouse ECs in vitro induced alterations in proliferation pathway gene expression, especially the expression of insulin-like growth factor-1, but had no effect on mouse oxidative stress pathways. In vivo, treatment with vitamin E did not inhibit TG-induced EC proliferation in liver and adipose tissue. In addition, no hypoxic effect was detected in adipose tissue of TG-treated mice; however, TG had a minor effect on hepatocellular hypoxia. These results provide additional evidence supporting a direct mitogenic effect in the mode of action of TG-induced hemangiosarcomas in mice.

Cell proliferation of rat bladder urothelium induced by nicotine is suppressed by the NADPH oxidase inhibitor, apocynin.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis in a two-stage carcinogenesis model. Nicotine also induced cytotoxicity in the bladder urothelium in a short-term study. In the present study, male rats were treated with nicotine (40 ppm) in drinking water co-administered with the NADPH oxidase inhibitor, apocynin (0, 250 or 750 mg/kg) in diet for 4 weeks. The apocynin treatment induced no clinical toxic effects. Reduction of reactive oxygen species (ROS) by apocynin was confirmed by immunohistochemistry of 8-OHdG in the bladder urothelium. Incidences of simple hyperplasia, cell proliferation and apoptosis were reduced by apocynin treatment in the bladder urothelium. However, despite reduction of cell proliferation (labeling index), apocynin did not affect the incidence of simple hyperplasia, apoptosis, or ROS generation in the kidney pelvis urothelium, in addition to 8-OHdG positivity induced by nicotine being lower. In vitro, apocynin (500 µM) reduced ROS generation, but induced cell proliferation in bladder cancer cell lines (T24 and UMUC3 cells). These data suggest that oxidative stress may play a role in the cell proliferation of the bladder urothelium induced by nicotine.