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.

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.

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.