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.

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.

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.

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.

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.

Cotinine, a major nicotine metabolite, induces cell proliferation on urothelium in vitro and in vivo.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. In a previous study, nicotine enhanced rat urinary bladder carcinogenesis using a rat urinary bladder two-stage carcinogenesis model. In the present study, nicotine metabolites (cotinine, trans-3'-hydroxy cotinine and N'-nitrosonornicotine) were evaluated in a cell proliferation assay using urinary bladder urothelial cell lines. Cotinine (0.1 to 1 mM) induced the highest cell proliferation compared to the others, including nicotine, in three bladder cancer cell lines (RT4, T24 and UMUC3 cells). By Western blot, cotinine induced phosphorylation of Stat3 and expression of cyclin D1 in UMUC3 cells. The cell proliferation induced by cotinine was blocked by inhibitors of nicotinic receptors (10 nM SR16584 or 10 µM methyllycaconitine citrate) and Stat3 (100 nM stattic). In an in vivo study, cotinine (13, 40 and 120 ppm) in drinking water also induced cell proliferation and simple hyperplasia in urinary bladder and renal pelvis urothelium of rats, but to a lesser degree compared to nicotine (40 ppm). Cytotoxicity detected by scanning electron microscopy and apoptosis in the bladder urothelium were induced by nicotine but not cotinine. These data suggest that cotinine is able to induce urothelial cell proliferation both in vitro and in vivo, and high urinary concentrations may enhance urothelial carcinogenesis.

Mitochondrial lipid droplet formation as a detoxification mechanism to sequester and degrade excessive urothelial membranes.

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due to its coverage by urothelial plaques consisting of 2D crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Because mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism.

Evaluation of immunologic and intestinal effects in rats administered an E 171-containing diet, a food grade titanium dioxide (TiO2).

The toxicity of dietary E 171, a food grade titanium dioxide was evaluated. A recent study reported rats receiving E 171 in water developed inflammation and aberrant crypt foci (ACF) in the gastrointestinal tract. Here, rats received food containing E 171 (7 or 100 days). The 100-day study included feeding E 171 after dimethylhydrazine (DMH) or vehicle only pretreatment. Food consumption was similar between treatment groups with maximum total cumulative E 171 exposure being 2617 mg/kg in 7 days and 29,400 mg/kg in 100 days. No differences were observed due to E 171 in the percentage of dendritic, CD4+ T or Treg cells within Peyer's patches or the periphery, or in cytokine production in plasma, sections of jejunum, and colon in 7- or 100-day E 171 alone fed rats. Differences were observed for IL-17A in colon (400 ppm E 171 + DMH) and IL-12p70 in plasma (40 ppm E 171 + DMH). E 171 had no effect on histopathologic evaluations of small and large intestines, liver, spleen, lungs, or testes, and no effects on ACF, goblet cell numbers, or colonic gland length. Dietary E 171 administration (7- or 100-day), even at high doses, produced no effect on the immune parameters or tissue morphology.

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.

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.

Orally administered nicotine effects on rat urinary bladder proliferation and carcinogenesis.

Tobacco smoking is a major risk factor for human cancers including urinary bladder carcinoma. Cigarette smoke inhalation in mice and orally administered nicotine in rats and mice increased urothelial cell proliferation. Nicotine, a major component of smoke, induced cell proliferation in multiple cell types in vitro. In the present study, the enhancing effects of nicotine on F344 rat bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Nicotine administered in drinking water for 32 weeks following 4 weeks of BBN treatment significantly increased the incidence and number of urothelial carcinomas dose-dependently. Ki67 and pSTAT3 labeling indices and expression of nicotinic acetylcholine receptor alpha 7 (nAChRα7) in non-tumor bladder urothelial lesions were significantly increased by nicotine, but the TUNEL assay for apoptosis showed no increase. In a 4 week study, inhibitors of nicotinic acetylcholine receptor decreased nicotine-induced urothelial simple hyperplasia and Ki67 labeling index in the bladder and kidney pelvis at a single cytotoxic dose of nicotine (40 ppm). Urothelial cytotoxicity with regenerative proliferation was observed by light and scanning electron microscopy. In vitro, nicotine was not cytotoxic to rat or human immortalized urothelial cells (do not express nicotine receptors) below millimolar concentrations, nor in human RT4, T24 or UMUC3 urothelial carcinoma cells (express nicotine receptors). However, nicotine slightly, but statistically significantly, increased cell proliferation at micromolar concentrations in human urothelial carcinoma cells. These data suggest that nicotine enhances urinary bladder carcinogenesis by inducing cytotoxicity with regenerative proliferation. The possible role of direct mitogenesis, involving nAChR and STAT3 signaling and of nicotine receptors requires further investigation at non-cytotoxic doses of nicotine.

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.

Effect of polyhexamethylene biguanide on rat liver.

Polyhexamethylene biguanide (PHMB), an amphiphilic polymeric biocide, increased liver tumor incidence in male and female rats at 1000 and 1500 mg/L in drinking water, but not at 500 mg/L in previous studies. In another study, PHMB administered in diet at 4000 mg/kg was negative for hepatocellular tumors. The present studies evaluated bioavailability and distribution of PHMB administered in drinking water and diet and possible modes of action (MOA). PHMB in drinking water was unpalatable during the first 3 days, resulting in markedly decreased food consumption and decreased body weight. Ki-67 labeling index was increased in hepatocytes and endothelial cells dose responsively with PHMB administered in drinking water but not diet. Vitamin E had no effect on this. There was no cytotoxicity by histopathology or serum enzymes, and no increase in cytokines TNFα, IL-1α or NF-κB. Focal iron deposition in sinusoidal lining cells was detected. Microarray analyses were non-contributory. No effect on CAR or PPARα activation was detected. 14C-PHMB administered at 500, 1000, or 1500 mg/L in the drinking water or 4000 mg/kg in the diet was nearly completely absorbed and excreted in urine, with some fecal excretion. The hypothesized MOA for liver tumors induced by PHMB in drinking water is: 1) severe dehydration and starvation because of unpalatability, followed by ingestion with rapid absorption and urinary excretion; 2) increased hepatocyte proliferation; and 3) induction of hepatocellular foci and tumors. The PHMB-induced rat hepatocellular tumors are unlikely to pose a human cancer risk. However, the actual MOA has not been determined.

Low-dose dose-response for reduced cell viability after exposure of human keratinocyte (HEK001) cells to arsenite.

The in vitro arsenite (AsIII) cytotoxicity dose-response (DR) of human keratinocytes (HEK001) was examined at greater statistical resolution than ever previously reported using the MTT assay to determine cell viability. Fifty-four 96-well plates were treated with AsIII concentrations of 0.25, 0.5, 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, or 30 µM. Because of unexpected variation in viability response patterns, a two-stage DR analysis was used in which data on plate-specific viability (%), estimated as 100% times the ratio of measured viability in exposed to unexposed cells, were fit initially to a generalized lognormal response function positing that HEK001 cells studied consisted of: a proportion P of relatively highly sensitive (HS) cells, a proportion Po of relatively resistant cells, and a remaining (1-P-Po) fraction of typical-sensitivity (TS) cells exhibiting the intermediate level of AsIII sensitivity characteristic of most cells in each assay. The estimated fractions P and Po were used to adjust data from all 54 plates (and from the 28 plates yielding the best fits) to reflect the condition that P = Po = 0 to provide detailed DR analysis specifically for TS cells. Four DR models fit to the combined adjusted data were each very predictive (R2 > 0.97) overall but were inconsistent with at least one of the data set examined (p < 10-5). Adjusted mean responses at ≤3 µM were approximately equal (p > 0.30) and exceeded 100% significance (p ≤ 10-6). A low-dose hormetic model provided the best fit to the combined adjusted data for TS cells (R2 = 0.995). Marked variability in estimates of P (the proportion of apparent HS cells) was unexpected, not readily explained, and warrants further study using additional cell lines and assay methods, and in vivo.

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.

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.

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.