Complete elimination of established neuroblastoma by synergistic action of gamma-irradiation and DCs treated with rSeV expressing interferon-beta gene.

Dendritic cell (DC)-based immunotherapy has been investigated as a new therapeutic approach to intractable neuroblastomas; however, only limited clinical effect has been reported. To overcome the relatively low sensitivity of neuroblastomas against immunotherapy, we undertook a preclinical efficacy study to examine murine models to assess the combined effects of gamma-irradiation pretreatment and recombinant Sendai virus (ts-rSeV/dF)-mediated murine interferon-beta (mIFN-beta) gene transfer to DCs using established c1300 neuroblastomas. Similar to intractable neuroblastomas in the clinic, established c1300 tumors were highly resistant to monotherapy with either gamma-irradiation or DCs activated by ts-rSeV/dF without transgene (ts-rSeV/dF-null) that has been shown to be effective against other murine tumors, including B16F10 melanoma. In contrast, immunotherapy using DCs expressing mIFN-beta through ts-rSeV/dF (ts-rSeV/dF-mIFNbeta-DCs) effectively reduced tumor size, and its combination with gamma-irradiation pretreatment dramatically enhanced its antitumor effect, resulting frequently in the complete elimination of established c1300 tumors 7-9 mm in diameter, in a high survival rate among mice, and in the development of protective immunity in the mice against rechallenge by the tumor cells. These results indicate that the combination of ts-rSeV/dF-mIFNbeta-DCs with gamma-irradiation is a hopeful strategy for the treatment of intractable neuroblastomas, warranting further investigation in the clinical setting.

Impact of deletion of envelope-related genes of recombinant Sendai viruses on immune responses following pulmonary gene transfer of neonatal mice.

We demonstrated previously that the additive-type recombinant Sendai virus (rSeV) is highly efficient for use in pulmonary gene transfer; however, rSeV exhibits inflammatory responses. To overcome this problem, we tested newly developed non-transmissible constructs, namely, temperature-sensitive F-deleted vector, rSeV/dF (ts-rSeV/dF) and a rSeV with all the envelope-related genes deleted (rSeV/dFdMdHN), for pulmonary gene transfer in neonatal mice, by assessing their toxicity and immune responses. The gene expression in the lungs of neonatal ICR mice peaked on day 2, then gradually decreased until almost disappearing at 14 days after infection in all constructs. Loss of body weight and mortality rate, however, were dramatically improved in mice treated with SeV/dFdMdHN (mortality=0%, n=41) and ts-rSeV/dF (24.2%, n=33) compared with additive rSeV (70.7%, n=58). Although the deletion of envelope-related genes of SeV had a small impact on the production of antibody and cytotoxic T-lymphocyte activity in both adults and neonates, a dramatic reduction was found in the events related to innate responses, including the production of proinflammatory cytokines, particularly in the case of neonates. These results indicate that pulmonary gene transfer using SeV/dFdMdHN warrants further investigation for its possible use in developing safer therapeutics for neonatal lung diseases, including cystic fibrosis.

Insights into infant neuroblastomas based on an analysis of neuroblastomas detected by mass screening at 6 months of age in Japan.

BACKGROUND/PURPOSE: Mass screening (MS) for neuroblastoma (NB) at 6 months of age in Japan was discontinued in 2004. We have previously reported that the majority of NB detected by MS showed a good prognosis, with only a few cases demonstrating an unfavorable outcome (J Pediatr Surg 2002, Cancer 2001). This study aims to provide insights into infant NB by assessing the details of the clinical courses in patients treated with a standard regimen and the biological features of such cases using highly sensitive methods at one institution in Japan. METHODS: In 76 NB detected through MS treated at Kyushu University Hospital, the clinical features and MYCN amplification, 1p deletion, 17q gain, the expression level of TRKA using FISH and the quantitative PCR were analyzed. RESULTS: Of these 76 persons with NB treated at one institution, 97 % are still alive, while 2 cases died from other diseases. Three patients experienced a recurrence after complete remission (CR), and 2 patients demonstrated refractory disease since the initial diagnosis. Two of the 3 NB patients with recurrence have demonstrated a 2nd CR, while one case still has multiple active diseases. Regarding the findings of highly sensitive biological analyses, 5/74 (7 %) showed MYCN amplification, 2/24 (8 %) cases had a 1p deletion, 3/33 (9 %) cases had a 17q gain, 5/50 (10 %) cases had diploidy, 1/25 (4 %) cases had a low expression of TRKA, and 2/76 (3 %) cases had an unfavorable histology. Of the 76 NB, 13 tumors (17 %) had one or more unfavorable factors (UF). Of the 5 refractory NB, 1 case had 3 UF, 1 case had 2 UF, 1 case had 1 UF, and 2 cases had no UF. As a result, 60 % of the refractory NB had one or more UF. CONCLUSIONS: Of the NB detected by MS at one institution in Japan, 17 % had one or more unfavorable factors (UF) and might have a higher risk of recurrence than the patients with no UF, although the unfavorable biology of several refractory cases is still unclear even after highly sensitive analyses. At least one-fifth of the NB cases detected by MS are anticipated cases. In infantile neuroblastomas, it may therefore be most important to analyze biologically prognostic factors using highly sensitive methods followed by immediate surgical intervention. Since the MS program has been discontinued in Japan, it will be necessary in future to assess the mortality and characteristics of NB detected clinically.

Prenatal ultrasonographic and magnetic resonance imaging findings of congenital cloacal anomalies associated with meconium peritonitis.

BACKGROUND/PURPOSE: Cloacal malformations are rare anomalies, occurring in females, and in which they demonstrate a single perineal orifice for urethra, vagina, and rectum. Prenatal ultrasonograms (US) of cloacal malformations sometimes show ascites, hydrocolpos, and hydronephrosis. We herein describe the characteristic prenatal US and magnetic resonance imaging (MRI) findings of the cloacal malformations associated with meconium peritonitis. METHODS: The pre- and postnatal records of 11 newborn patients with cloacal malformations, treated in our hospital from 1988 to 2004, were reviewed. All fetuses underwent prenatal US by experienced obstetricians, whereas in addition, fetal MRI was performed in 1 patient. RESULTS: The prenatal US and/or MRI findings showed fetal ascites, a multicystic pelvic mass, oligohydramnios, and bilateral hydronephrosis in 5 of 11 patients with cloacal malformations. In these 5 cases, postnatal examinations showed associated hydrocolpos, hydrometrocolpos, and bilateral hydronephrosis; furthermore, 4 of these 5 cases also showed meconium peritonitis at laparotomy. CONCLUSIONS: The prenatal US and MRI findings, showing fetal ascites, multicystic pelvic mass, bilateral hydronephrosis, and oligohydraminios are highly suggestive of the cloacal malformations associated with meconium peritonitis.

A case of an inflammatory myofibroblastic tumor in the lung which expressed TPM3-ALK gene fusion.

We herein describe a 4-year-old boy who after being treated for pneumonia showed an abnormal shadow at the hilus of the right lung on chest X-rays with continued inflammatory findings in his laboratory data. CT and MR investigations suggested the existence of a neoplasm at that site. An open biopsy was thus performed for a definite diagnosis. The histological findings and the expression of TPM3-ALK fusion gene confirmed a diagnosis of an inflammatory myofibroblastic tumor. A right upper and middle lobectomy including the tumor was thus performed for a complete resection. In addition to the histological diagnosis, the detection of the tumor specific fusion gene provided objective evidence in making a diagnosis.

The effect of a valine-rich diet on intestinal adaptation to massive small bowel resection in the rat.

BACKGROUND/PURPOSE: Recently, valine, which is one of the branched chain amino acids, has been reported to enhance liver regeneration after hepatectomy in the rat. The aim of this study was to investigate the effect of enteral valine supplementation on intestinal adaptation. MATERIALS/METHODS: Seven-week-old male Lewis rats underwent a 90% small bowel resection. The rats were randomly divided into two groups: group V (valine-rich diet) and group S (standard rat chow), according to the diet. The rats were sacrificed at the operation day and on postoperative days (POD) 7, 14, 30, and 60. The metrics were body weight (BW), blood amino acids, urine organic acids, and morphology of the residual small intestine. RESULTS: The BW and the intestinal wet weight, jejunal crypt depth, and proliferating cell nuclear antigen-positive cells in group V at POD 7 were significantly higher than those values in group S, while those in group V at POD 30 and 60 were smaller than those in group S. The urine methylmalonic acid (MMA) level in group V at POD 30 and 60 was much higher than in group S. CONCLUSION: Valine enhanced intestinal adaptation after massive small bowel resection in the acute phase. However, the long-term supplementation disturbed intestinal adaptation, which might be due to the high production of MMA.

Relationship between real-time monitoring of the graft motility and mucosal histology in swine intestinal transplantation.

We studied the correlation between the motility and the mucosal histology of the small bowel seeking to detect rejection in an early stage by real-time monitoring using a swine model. Intestinal transplantation (ITx) was performed orthotopically using FK506 immunosuppression. The distal about 20 cm segment of the allograft was exteriorized as a Thiry-Vella stoma for biopsies. Strain gauge (SG) force transducers were attached to the graft for real-time monitoring of graft motility. Pigs without ITx were used as controls (group 1). Rejection was classified into four groups by histologic findings: nonrejection (group 2), mild rejection (group 3), moderate rejection (group 4), and severe rejection (group 5). Migrating motor complex (MMC) phase III was analyzed for the following parameters: duration, amplitude, interval, motility index, velocity, and frequency of propagation. In group 2, all parameters were almost the same as those for group 1. In contrast, groups 4 and 5 showed most parameters significantly lower than those in group 1. In group 3, the contractility of the MMC was not significantly altered, but the frequency of the propagation was decreased significantly. In conclusion, graft motility detected by a real-time SG method correlated with the grade of mucosal histology. This method is useful to detect rejection at an early stage by examining the frequency of MMC propagation.

MYCN gene amplification is a powerful prognostic factor even in infantile neuroblastoma detected by mass screening.

MYCN is the most powerful prognostic factor in cases of older children. However, how MYCN is related to the prognosis of infantile cases is not clear. A mass screening program was carried out by measuring urinary catecholamine metabolites (VMA and HVA) from 6-month-old infants. Of 2084 cases detected by the screening program, MYCN amplification (MNA) was examined by Southern blot analyses in 1533 cases from 1987 to 2000. Of the 1533 cases examined, 1500 (97.8%) showed no MNA, 20 cases (1.3%) showed MNA from three to nine copies, and 13 (0.8%) cases showed more than 10 copies. The 4-year overall survival rates of these three groups (99, 89 and 53%, respectively) were significantly different (P<0.001), indicating that MYCN copy number correlates with the prognosis. Cases with MNA more than 10 copies were more advanced than those without amplification (stage III, IV vs I, II, IVs; P<0.001). Patients with MNA more than 10 copies had significantly higher serum levels of neuron-specific-enolase (NSE) and ferritin than non-amplified patients (P=0.049, P=0.025, respectively). MYCN amplification was strongly correlated with a poor prognosis in infantile neuroblastoma cases. Therefore, for the selection of appropriate treatment, an accurate determination of MNA is indispensable.

The distribution of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the lungs of congenital diaphragmatic hernia patients and age-matched controls.

AIMS: In congenital diaphragmatic hernia (CDH), the pathogenesis of abnormal pulmonary morphology is still incompletely understood. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are known to play an important role in the turnover of the extracellular matrix (ECM) during development and in remodelling of tissue. The aim of this study was to investigate differences in the expression of MMPs and TIMPs between CDH lungs and controls, against the background of the abnormal pulmonary vasculature in CDH. METHODS: We studied 12 lungs of term CDH patients who died < 24 h after birth and 11 normal age-matched control lungs, by immunohistochemistry with antibodies against human MMP-1, -2, -9, TIMP-1 and -2. RESULTS: There was a clear increase in the number of MMP-1-reactive capillaries and fibroblasts in CDH lungs compared with controls. In contrast, TIMP-2 reactivity in these structures was decreased in CDH lungs. The arterial endothelium and medial smooth muscle expressed MMP-2, -9 and TIMP-2 in both CDH and control lungs. In small arteries (< 100 microm in diameter), the positive surface area of MMP-2, -9 and TIMP-2 was significantly larger in CDH lungs than in controls. There was no difference in the distribution and expression of TIMP-1 between CDH lungs and normal controls. CONCLUSION: The differences in staining pattern of MMPs and TIMPs between normal and CDH lungs suggest that these enzymes might play a role in the abnormal remodelling of the interstitium and the pulmonary arteries in CDH lungs. This could contribute to our understanding of the abnormal lung morphology and the occurrence of pulmonary hypertension, which forms one of the major obstacles to the successful treatment of these patients.

Esophageal atresia with double tracheoesophageal fistula--a case report and review of the literature.

Esophageal atresia with double tracheoesophageal fistula (TEF) is a very rare anomaly, and the accurate preoperative diagnosis of proximal TEF is very difficult. This paper describes a baby girl who presented with esophageal atresia with double, proximal, and distal TEF. The distal TEF was diagnosed before operation, whereas the proximal TEF was found intraoperatively. Overlooking the presence of proximal TEF can lead to increased morbidity and mortality due to severe respiratory infection and the necessity of a second operation. Great care must therefore be taken to not overlook the presence of proximal TEF in patients with this anomaly.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases during normal human pulmonary development.

AIMS: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in lung development because they play an important role in the turnover of the extracellular matrix. Although limited data on MMP and TIMP expression are available from animal studies during prenatal pulmonary development, little is known about their expression during human fetal lung development. The aim of this study was to investigate the expression of MMP-1, -2, -9, TIMP-1, -2 and -3 in human fetal lungs from 9 to 42 weeks of gestation. METHODS AND RESULTS: Forty-five normal human fetal lung samples were analysed by immunohistochemistry. MMP-1, -9, TIMP-1, -2 and -3, but not MMP-2, were expressed in the epithelium at all gestational ages. The endothelium of all vessels and the arterial smooth muscle cells expressed MMP-1, -2, -9, TIMP-2 and -3, but not TIMP-1, at all developmental stages. CONCLUSION: The extensive distribution of MMPs and TIMPs throughout all stages of human lung development suggests that they play a significant role in the remodelling that occurs in the interstitium and epithelial basement membrane during lung development and in pulmonary vascular development. These data will serve as a base line for comparison with neonatal lung pathology, including pulmonary hypertension.

An abnormal distribution of C-kit positive cells in the normoganglionic segment can predict a poor clinical outcome in patients with Hirschsprung's disease.

PURPOSE: The loss or decrease of interstitial cells of Cajal (ICCs) has been implicated in several disorders of human intestinal motility. We have encountered a few cases suffering from severe constipation or enterocolitis resulting in patient death after a definitive operation for HD, even though the normoganglionic intestine had been successfully pulled through. We investigated the distribution of ICCs using c-kit immunostaining in the normoganglionic segment and compared these findings with the clinical outcome after a definitive operation in each case. PATIENTS AND METHODS: The distributions of ICCs were investigated by using c-kit immunostaining in the normoganglionic segment in the resected bowel in 15 cases with HD. The distributions of protein gene product 9.5 (PGP 9.5) as a general neuronal marker and those of NADPH-diaphorase (NADPH-d) as a marker of nitric-oxide neurons were also examined. The numbers of ICCs and neurons were evaluated quantitatively. The histopathological results were compared with the clinical outcome after definitive operation in each case. RESULTS: C-kit immunoreactive cells showed a normal distribution in the normoganglionic segment in 13 cases, while they were markedly (less than 50% compared with the other cases) decreased in 2 cases. The distributions of PGP 9.5 and NADPH-d were almost the same in all cases. The bowel movements of 13 cases showing normal c-kit distribution were satisfactory. In contrast, the bowel movements were impaired in 2 cases with a decreased number of c-kit positive cells. One infant suffered from severe persistent constipation and thus had to undergo a resection of a dilated colon. The other infant died of sepsis due to postoperative enterocolitis and showed a markedly dilated colon. CONCLUSION: A decreased number of c-kit positive cells in the normoganglionic segment can thus allow us to predict a poor clinical outcome after definitive surgery, probably due to poor intestinal motility. Therefore examining the c-kit distribution in a resected bowel specimen in patients with HD should be mandatory in order to select the optimal postoperative treatment regimen for each case.

Endoscopic diverticulotomy for symptomatic pediatric esophageal diverticula.

There have so far been few reports on esophageal diverticulum in children. We experienced two symptomatic pediatric cases with esophageal diverticulum. Our cases manifested high fever and dysphagia with chest pain during swallowing. The patients underwent endoscopic diverticulotomy. The septum between the diverticulum and the esophagus was cut using the argon plasma coagulation (APC 3000) system. We recommend an endoscopic diverticulotomy as an effective treatment modality for such symptomatic cases.

Clinical characteristics and the prognosis of rhabdomyosarcoma - a report from the Study Group for Pediatric Solid Malignant Tumors in the Kyushu Area, Japan.

AIM: There have been no nationwide group studies for patients with rhabdomyosarcoma in Japan. This study aims to assess the actual state of treatments and their outcome. PATIENTS AND METHODS: From 1982 to 1996, 79 rhabdomyosarcomas were registered by the Study Group for Pediatric Solid Malignant Tumors in the Kyushu Area. The prognostic factors and treatments were assessed based on the 5-year survival rate. The staging was done according to the Intergroup Rhabdomyosarcoma Study (IRS) Clinical Grouping Classification. RESULTS: The 5-year survival rate for all patients was 39.1 %. The survival rates for each factor were as follows, according to 1) group; 77.8 % for Group I, 51.9 % for Group II, 33.7 % for Group III, and 20.2 % for Group IV; 2) primary site: 56.3 % for the head and neck, 43.8 % for the parameningeal region, 12.5 % for the extremity, 58.3 % for the genitourinary region, and 30.5 % for the others; 3) histology: 35.8 % for the embryonal type, 36.8 % for the alveolar type. CONCLUSIONS: Altogether, the outcome of this study was poor. To improve outcomes, a new nationwide group study for rhabdomyosarcoma, which we belong to, has just started in Japan.

High expression of telomerase is an independent prognostic indicator of poor outcome in hepatoblastoma.

Telomerase, an enzyme related with cellular immortality, has been extensively studied in many kinds of malignant tumours for clinical diagnostic or prognostic utilities. Telomerase activity is mainly regulated by the expression of hTERT (human telomerase reverse transcriptase), which is a catalytic component of human telomerase. To evaluate whether the levels of hTERT mRNA provides a molecular marker of hepatoblastoma malignancy, we examined hTERT mRNA expression levels in the primary hepatoblastoma tissues by fluorescent RT-PCR using LightCycler technology and followed up the clinical outcomes in 63 patients listed in the Japanese Study Group of Pediatric Liver Tumor between 1991 and 2002. The hTERT mRNA expression was detected in 61 (96.8%) specimens and their expression levels ranged between 0.1/1000 and 745.1/1000 copies of PBGD gene that was used as an internal control. Among these cases, frozen 39 tumour samples and 14 adjacent noncancerous liver tissues were analysed for semiquantitative telomerase assay. In the 39 tumour samples, the levels of telomerase activity ranged between 0.11 and 2709 TPG and 12 (30.7%) had high telomerase activity (>100 TPG), whereas only nine of 14 noncancerous liver tissue samples showed telomerase activity which was less than 1.0 TPG. The levels of telomerase activity were significantly correlated with the levels of hTERT mRNA expression (P<0.001). The frequency of high hTERT mRNA expression and/or high telomerase activity did not significantly associate with the clinicopathological factors except for stage of disease. The prognosis of the patients with high hTERT mRNA expression was significantly worse than that of others (P<0.01), as was the patients with high telomerase activity (P<0.01). Multivariate analysis indicated that high levels of hTERT mRNA expression as well as telomerase activity are independent prognosis-predicting factors in patients with hepatoblastoma.

Immunosuppressive effect of nucleoside-nucleotide-free diet in rat allogeneic small intestinal transplantation.

BACKGROUND: We evaluated the effects of nucleosides (NS) and nucleotides (NT) on the rejection of rat allogeneic small intestinal transplants. METHODS: A 2-cm segment of jejunum from fetal Fischer rats (RT-1(lvl)) was transplanted at day 19 of gestation into the abdominal wall of 7-week-old Lewis rats (RT-1(l)) by a nonvascular technique. Two weeks before transplantation, recipient rats were separated into an NS-NT-free group and an NS-NT-supplemented group. At 2 days after transplantation, histologic study of the grafts was performed with hematoxylin-eosin staining and interleukin-2 (IL-2) production estimated in recipient blood using an ELISA method. The morphologic findings were graded in a blind fashion on a scale of 0 to 4, with 0 indicating an intact intestinal structure. RESULTS: Mean plasma IL-2 levels of the NS-NT-free group were significantly lower than those of the NS-NT-supplemented group. The mean rejection score of the NS-NT-free group was also significantly lower than that of the NS-NT-supplemented group. CONCLUSIONS: Administration of an NS-NT-free diet reduces acute rejection in rat small intestinal transplantations.

Real-time monitoring for detecting rejection using strain gauge force transducers in porcine small bowel transplantation.

PURPOSE: The clinical results of small bowel transplantation (SBT) have not been satisfactory mainly because of the immunological barrier. It is important to detect the presence of and to perform adequate treatment of rejection as early as possible to improve graft survival. Therefore, we have established a pig model to monitor graft motility as a means to detect rejection in real time. METHODS: Orthotropic SBT was performed in 25 pigs using FK-506 (0.05 to 0.1 mg/kg/d) immunosuppression. The interdigestive motor patterns were evaluated using strain gauge force transducers (SG). Seven pigs without SBT were treated as controls (C). Animals that displayed migrating motor complex (MMC) activity as evidenced by duration, amplitude, and interval in the graft were alive more than 10 days with adequate oral feeding: the functional graft (FG) group. In contrast the rejection (R) group did not show these activities on data recorded within 10 days before death due to rejection. RESULTS: The FG group showed MMC propagated throughout the graft with all parameters almost the same as the control group except for the duration. In contrast, all parameters in the group R were significantly lower than those in group FG, suggesting that group R motility was obviously impaired by rejection. CONCLUSIONS: The SG method may afford real-time monitoring of transplanted bowel motility that could be useful to detect rejection after SBT.

Improved survival outcome for hepatoblastoma based on an optimal chemotherapeutic regimen--a report from the study group for pediatric solid malignant tumors in the Kyushu area.

BACKGROUND/PURPOSE: The survival outcome for patients with hepatoblastoma normally depends on the resectability of the tumor. In Japan, the pre and/or postoperative chemotherapy protocol using a combination of cisplatin (CDDP) and tetrahydropyranyl-Adriamycin (THP-ADR) has been the standard treatment since 1991. This study aims to assess exactly what influence the establishment of this chemotherapy protocol has had on both the tumor resectability and the outcome of patients with hepatoblastoma. METHODS: From 1982 to 1997, 60 patients with hepatoblatoma were treated in the Kyushu area, Japan. Based on the pretreatment extent of disease (PRETEXT), the outcome and tumor resectability were compared between group A (1982 to 1990, n = 27, PRETEXT I:5, II:8, III:6, IV:8) and group B (1991 to 1997, n = 33, PRETEXT I:9, II:9, III:5, IV:10). RESULTS: The 5-year survival rates (group A and group B) were 33% and 73% for all cases (P <.01), 100% and 89% for PRETEXT I, 38% and 89% for II (P <.05), 17% and 80% for III (P <.01), and 0% and 40% for IV (P <.01), respectively. The 5-year survival rates for patients with metastases were 0% for group A (n = 5) and 57% for group B (n = 7; P <.01). The rates of a complete resection of primary tumor were 48% for group A and 67% for group B. In particular, a significant difference was found regarding the complete resection rate between groups A and B in the patients with PRETEXT III (17% for group A and 80% for group B; P <.01). In the patients with an incomplete tumor resection (14 for group A, 11 for group B), the 5-year survival rates were 0% for group A and 45% for group B (P <.01). CONCLUSIONS: The optimal chemotherapeutic regimen of CDDP and THP-ADR was thus found to greatly contribute to the improved survival rate of hepatoblastoma patients. Preoperative chemotherapy resulted in an increased resectability of the tumor, whereas postoperative chemotherapy played an important role in the increased cure rate of cases with either an incomplete tumor resection or metastasis. However, refractory cases with PRETEXT IV or metastasis may still require the development of an even more effective treatment modality, including the use of blood stem cell transplantation.

The effect of a prenatal androgen disruptor, vinclozolin, on gubernacular migration and testicular descent in rats.

BACKGROUND/PURPOSE: Androgen has been shown to regulate inguinoscrotal testicular descent. This study aims to clarify the effect of one of the major endocrine disrupters, vinclozolin (V), on both gubernacular migration and inguinoscrotal testicular descent in rats. METHODS: Time-pregnant rats were segregated into 2 groups. In group I, the rats were administered 200 mg/kg/d of V by gavage on days 15 to 18 of gestation. In group II, the rats were administered the same volume of solvent and were used as controls. At birth, the anogenital distance was measured in pups, and gubernacular migration was examined at 10 days of age in some of male offspring. Next, the incidence of testicular descent and the growth of external genitalia were investigated in the remaining male offspring at 60 days of age. The chi2 test was used for statistical analysis of the results. RESULTS: At birth, the anogenital distance (AGD) index decreased significantly more in group I than in group II in male offspring. However, there was no significant difference in the AGD index between the 2 groups in the female offspring. At 10 days of age, an aberrant migration of the gubernaculum was found in the 51.5% of V-treated rats in group I. At 60 days of age, the incidence of cryptorchidism was 57.7% in group I and 0% in group II (P <.05). In addition, hypospadias with cleft phallus and pseudo vagina with a blind pouch also were observed in some of the V-treated rats. CONCLUSIONS: Prenatal administration with V thus caused intrauterine defects, which resulted in testicular maldescent caused by the induction of an aberrant migration of the gubernaculum associated with an abnormal extension of the processus vaginalis, and this may have been caused by the antiandrogenic effect of V in utero.

Short-time exposure to vinclozolin in utero induces testicular maldescent associated with a spinal nucleus alteration of the genitofemoral nerve in rats.

BACKGROUND/PURPOSE: Vinclozolin (V), a known antiandrogen, has been used widely to protect fruits, vegetables, and turf from fungus damage. The aim of this study was to clarify the effect of V on both the development of the spinal cord nucleus and testicular descent in rats. METHODS: Pregnant rats were administered 200 mg/kg/d of V from day 16 to 18 of gestation. At 5 days of age, the genitofemoral nerve (GFN) of male pups was identified on the psoas muscle, and diamidinophenyl indole was applied to the proximal cut end of the GFN. Forty-eight hours later, the T11 to L4 level of the spinal cord was removed, and 30-microm frozen serial sections were made. Next, the spinal nuclei labeled in a retrograde fashion by diamidinophenyl indole (DAPI) were examined with a fluorescence microscope. Additional male pups survived until 60 days of age to evaluate the position of the testes. RESULTS: The size of the DAPI-labeled spinal nuclei were smaller in the V-treated rats than in the control rats. The average number of the DAPI-labeled spinal nuclei decreased significantly more in the V-treated rats (176+/-33) than in the controls (247+/- 21; P <.05) during the newborn period. At 60 days of age, 15 of the 26 male rats showed either unilateral or bilateral undescended testes in the V-treated rats. The incidence of cryptorchidism was also significantly higher in the V-treated rats (57.7%) than in the controls (0%; P <.05). CONCLUSIONS: The antiandrogenic effect of the prenatal administration of V inhibited the development of the GFN nucleus in the spinal cord and induced testicular maldescent in rats. These results support the hypothesis that androgens regulate the descent of the testis through GFN development.