Diuron and its metabolites induce mitochondrial dysfunction-mediated cytotoxicity in urothelial cells.

In the environment, or during mammalian metabolism, the diuron herbicide (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is transformed mainly into 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichloroaniline (DCA). Previous research suggests that such substances are toxic to the urothelium of Wistar rats where, under specific exposure conditions, they may induce urothelial cell degeneration, necrosis, hyperplasia, and eventually tumors. However, the intimate mechanisms of action associated with such chemical toxicity are not fully understood. In this context, the purpose of the current in vitro study was to analyze the underlying mechanisms involved in the urothelial toxicity of those chemicals, addressing cell death and the possible role of mitochondrial dysfunction. Thus, human 1T1 urothelial cells were exposed to six different concentrations of diuron, DCA, and DCPMU, ranging from 0.5 to 500 µM. The results showed that tested chemicals induced oxidative stress and mitochondrial damage, cell cycle instability, and cell death, which were more expressive at the higher concentrations of the metabolites. These data corroborate previous studies from this laboratory and, collectively, suggest mitochondrial dysfunction as an initiating event triggering urothelial cell degeneration and death.

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.

Molecular signatures associated with diuron exposure on rat urothelial mitochondria.

Diuron, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, is a worldwide used herbicide whose biotransformation gives rise to the metabolites, 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichloroaniline (DCA). Previous studies indicate that diuron and/or its metabolites are toxic to the bladder urothelium of the Wistar rats where, under certain conditions of exposure, they may induce successively urothelial cell degeneration, necrosis, hyperplasia and eventually tumors. The hypothesis was raised that the molecular initiating event (MIE) of this Adverse Outcome Pathway is the mitochondrial toxicity of those compounds. Therefore, this study aimed to investigate in vitro the metabolic alterations resulting from urothelial mitochondria isolated from male Wistar rats exposure to diuron, DCPMU and DCA at 10 and 100 µM. A non-targeted metabolomic analysis using mass spectrometry showed discriminative clustering among groups and alterations in the intensity abundance of membrane-associated molecules phosphatidylcholine, phosphatidylinositol and phosphatidylserine, in addition to methylhexanoyl-CoA and, particularly for diuron 100 µM, dehydro-L-gulonate, all of them involved in critical mitochondrial metabolism. Collectively, these data indicate the mitochondrial dysfunction as an MIE that triggers cellular damage and death observed in previous studies.

GLP-1 and GIP receptor agonists in the treatment of Parkinson's disease: Translational systematic review and meta-analysis protocol of clinical and preclinical studies.

BACKGROUND: Parkinson's disease (PD) is a progressive multifactorial neurodegenerative condition. Epidemiological studies have shown that patients with type 2 diabetes mellitus (T2DM2) are at increased risk for developing PD, indicating a possible insulin-modulating role in this latter condition. We hypothesized that drugs similar to glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP), used in the treatment of T2DM2, may play a role in PD. OBJECTIVES: The purpose of this study is to systematically review and meta-analyze data of preclinical and clinical studies evaluating the efficacy and safety of GLP-1 and GIP drugs in the treatment of PD. METHODS: Two reviewers will independently evaluate the studies available in the Ovid Medline, Ovid Embase, Web of Science, Cochrane Central Register of Controlled Trials, Cinahl, and Lilacs databases. Preclinical rodent or non-human primate studies and randomized controlled human clinical trials will be included, without language or publication period restrictions. Outcomes of interest in preclinical studies will be primarily locomotor improvements and adverse effects in animal models of PD. For clinical trials, we will evaluate clinical improvements rated by the Movement Disorders Society Unified Parkinson's Disease Rating Scale-parts I, II, III, and IV, and adverse effects. The risk of bias of preclinical studies will be assessed by the SYRCLE tool and CAMARADES checklist and the clinical studies by the Cochrane tool; the certainty of the evidence will be rated by GRADE. DISCUSSION AND CONCLUSION: There is an urge for new PD treatments that may slow the progression of the disease rather than just restoring dopamine levels. This study will comprehensively review and update the state of the art of what is known about incretin hormones and PD and highlight the strengths and limitations of translating preclinical data to the clinic whenever possible. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration number CRD42020223435.

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.

Paraquat and Parkinson's disease: a systematic review and meta-analysis of observational studies.

This investigation aimed to conduct a systematic review of the literature and meta-analysis to determine whether exposure to the herbicide paraquat was associated with the development of Parkinson's disease (PD). Observational studies that enrolled adults exposed to paraquat with PD as the outcome of interest were searched in the PubMed, Embase, LILACS, TOXNET, and Web of Science databases up to May 2019. Two authors independently selected relevant studies, extracted data, and assessed methodological quality. The evidence certainty was assessed by the GRADE approach, which served as basis for a tentative causality assessment, supplemented by the Bradford Hill criteria when necessary. Results from nine case-control studies indicated that PD occurrence was 25% higher in participants exposed to paraquat. The only cohort investigation included demonstrated a non-significant OR of 1.08. Results from subgroup analyses also indicated higher PD frequency in participants that were exposed to paraquat for longer periods or individuals co-exposed with paraquat and any other dithiocarbamate. Data indicate apositive association between exposure to paraquat and PD occurrence, but the weight-of-evidence does not enable one to assume an indisputable cause-effect relationship between these two conditions. Better designed studies are needed to increase confidence in results. Systematic Review Registration: PROSPERO CRD42017069994.

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.

Paraquat and Parkinson's disease: a systematic review protocol according to the OHAT approach for hazard identification.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative condition that has genetic susceptibility, aging, and exposure to certain chemicals as risk factors. In recent decades, epidemiological and experimental studies have investigated the role of pesticides in the development of PD, in particular that of the herbicide paraquat. Here, we, therefore, aim to systematically review the association between paraquat exposure and PD. METHODS: Observational studies (cohort, case-control, and cross-sectional) eligible for this systematic review will enroll any participant who was occupationally and/or environmentally exposed to paraquat. Experimental studies, including in vivo and in vitro assays designed to assess neurotoxicological endpoints or mechanisms of paraquat neurotoxicity, will also be eligible. Outcomes of interest include the following: PD diagnosis; neurobehavioral, biochemical, and/or morphological alterations; and cellular, biochemical, and/or molecular pathways to oxidative stress. Using terms to include all forms of paraquat combined with PD, the following electronic databases will be searched: PubMed, EMBASE, LILACS, Toxnet, and Web of Science, without restrictions as to language, year, or status of publication. A team of reviewers will independently select potential titles and abstracts, extract data, assess risk of bias, and determine the overall quality of evidence for each outcome using the Office of Health Assessment and Translation (OHAT) approach for systematic reviews and evidence integration. Dichotomous data will be summarized as odds ratios, and continuous data will be given as mean differences, both with their respective 95% confidence intervals. DISCUSSION: This is the first time that the OHAT systematic review protocol will be applied to investigate a possible causal association between exposure to paraquat and PD. Results from this study could serve as basis for regulatory agencies to define paraquat levels of concern, supporting its risk assessment process. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42016050861.

QuEChERS-Based Method for Pesticides Analysis in Adipose Tissue Associated with Rat Ovaries.

The concomitant exposure to low doses of various pesticides is one of the most relevant issues in human toxicology today. An experimental toxicology study was developed to evaluate the effects of this type of exposure on the reproductive capacity of females of three species of rats that were exposed to mixtures of dicofol, dieldrin, endosulfan and permethrin at low doses (LOAEL and NOAEL). In this context, we have developed a method for determining pesticides in adipose tissue (0.5 g, 49% lipid) associated with the ovaries, based on the QuEChERS (quick, easy, cheap, effective, rugged and safe) strategy. The method quantification limit (LOQ) was 0.5 mg/kg for dicofol and permethrin, 0.05 mg/kg for endosulfan and dieldrin and 0.2 mg/kg for diclorobenzophenone. Mean recoveries ranged from 75% to 93% with a relative standard deviation <13%. The unspecific selectivity (matrix effect) indicates the mandatory use of analytical curves constructed on the matrix extract. All the analyzed samples (53 adipose tissue associated to ovaries) showed residues of dichlorobenzophenone + dicofol, dieldrin and cis-permethrin while trans-permethrin were detected in 40% of the samples but were below the LOQ. The data indicated the bioaccumulation characteristics of these substances.

A review of the carcinogenic potential of glyphosate by four independent expert panels and comparison to the IARC assessment.

The International Agency for Research on Cancer (IARC) published a monograph in 2015 concluding that glyphosate is "probably carcinogenic to humans" (Group 2A) based on limited evidence in humans and sufficient evidence in experimental animals. It was also concluded that there was strong evidence of genotoxicity and oxidative stress. Four Expert Panels have been convened for the purpose of conducting a detailed critique of the evidence in light of IARC's assessment and to review all relevant information pertaining to glyphosate exposure, animal carcinogenicity, genotoxicity, and epidemiologic studies. Two of the Panels (animal bioassay and genetic toxicology) also provided a critique of the IARC position with respect to conclusions made in these areas. The incidences of neoplasms in the animal bioassays were found not to be associated with glyphosate exposure on the basis that they lacked statistical strength, were inconsistent across studies, lacked dose-response relationships, were not associated with preneoplasia, and/or were not plausible from a mechanistic perspective. The overall weight of evidence from the genetic toxicology data supports a conclusion that glyphosate (including GBFs and AMPA) does not pose a genotoxic hazard and therefore, should not be considered support for the classification of glyphosate as a genotoxic carcinogen. The assessment of the epidemiological data found that the data do not support a causal relationship between glyphosate exposure and non-Hodgkin's lymphoma while the data were judged to be too sparse to assess a potential relationship between glyphosate exposure and multiple myeloma. As a result, following the review of the totality of the evidence, the Panels concluded that the data do not support IARC's conclusion that glyphosate is a "probable human carcinogen" and, consistent with previous regulatory assessments, further concluded that glyphosate is unlikely to pose a carcinogenic risk to humans.

Glyphosate rodent carcinogenicity bioassay expert panel review.

Glyphosate has been rigorously and extensively tested for carcinogenicity by administration to mice (five studies) and to rats (nine studies). Most authorities have concluded that the evidence does not indicate a cancer risk to humans. The International Agency for Research on Cancer (IARC), however, evaluated some of the available data and concluded that glyphosate probably is carcinogenic to humans. The expert panel convened by Intertek assessed the findings used by IARC, as well as the full body of evidence and found the following: (1) the renal neoplastic effects in males of one mouse study are not associated with glyphosate exposure, because they lack statistical significance, strength, consistency, specificity, lack a dose-response pattern, plausibility, and coherence; (2) the strength of association of liver hemangiosarcomas in a different mouse study is absent, lacking consistency, and a dose-response effect and having in high dose males only a significant incidence increase which is within the historical control range; (3) pancreatic islet-cell adenomas (non-significant incidence increase), in two studies of male SD rats did not progress to carcinomas and lacked a dose-response pattern (the highest incidence is in the low dose followed by the high dose); (4) in one of two studies, a non-significant positive trend in the incidence of hepatocellular adenomas in male rats did not lead to progression to carcinomas; (5) in one of two studies, the non-significant positive trend in the incidence of thyroid C-cell adenomas in female rats was not present and there was no progression of adenomas to carcinomas at the end of the study. Application of criteria for causality considerations to the above mentioned tumor types and given the overall weight-of-evidence (WoE), the expert panel concluded that glyphosate is not a carcinogen in laboratory animals.

Alternative Multiorgan Initiation-Promotion Assay for Chemical Carcinogenesis in the Wistar Rat.

The medium-term multiorgan initiation-promotion chemical bioassay (diethylnitrosamine, methyl-nitrosourea, butyl-hydroxybutylnitrosamine, dihydroxypropylnitrosamine, dimethylhydrazine [DMBDD]) with the Fischer 344 rat was proposed as an alternative to the conventional 2-year carcinogenesis bioassay for regulatory purposes. The acronym DMBDD stands for the names of five genotoxic agents used for initiation of multiorgan carcinogenesis. The Brazilian Agency for the Environment officially recognized a variation of this assay (DMBDDb) as a valid method to assess the carcinogenic potential of agrochemicals. Different from the original protocol, this DMBDDb is 30-week long, uses Wistar rats and two positive control groups exposed to carcinogenesis promoters sodium phenobarbital (PB) or 2-acetylaminofluorene (2-AAF). This report presents the experience of an academic laboratory with the DMBDDb assay and contributes to the establishment of this alternative DMBDD bioassay in a different rat strain. Frequent lesions observed in positive groups to evaluate the promoting potential of pesticides and the immunohistochemical expressions of liver cytochrome P450 (CYP) 2B1/2B2 and CYP1A2 enzymes were assessed. Commonly affected organs were liver, kidney, intestines, urinary bladder, and thyroid. PB promoting activity was less evident than that of 2-AAF, especially in males. This study provides a repository of characteristic lesions occurring in positive control animals submitted to a modified alternative 2-stage multiorgan protocol for carcinogenesis in Wistar rat.

Effects of a mixture of pesticides on the adult female reproductive system of Sprague-Dawley, Wistar, and Lewis rats.

The Brazilian federal government Agency for Health Surveillance detected pesticide residues in fresh food available for consumers all over the country. The current study investigated the effects of a mixture of some of those pesticides (dichlorvos, dicofol, dieldrin, endosulfan, and permethrin) on the reproductive system of Sprague-Dawley (SD), Wistar (WT), and Lewis (LEW) rats. Female rats from each strain were randomized into three experimental groups and were fed a control diet or diets added with pesticides mixture at their respective no-observed-effect level (NOEL)/no-observed-adverse-effect level (NOAEL) (low dose) (mg/kg/d): dichlorvos (0.23), dicofol (0.5), dieldrin (0.025), endosulfan (0.7), permethrin (5), or lowest-observed-effect level (LOEL)/lowest-effect level (LEL)/ lowest-observed-adverse-effect level (LOAEL) (toxically effective dose) (mg/kg/d): dichlorvos (2.3), dicofol (2.1), dieldrin (0.05), endosulfan (3.8), and permethrin (25) as reported in the literature. Euthanasia was performed between wk 10 and 12, during the estrous stage. Decreased body weights gain (SD and WT) and increased liver weights (SD, WT, and LEW) were observed in each strain fed the pesticides mixture at the higher levels. At that dose level, rat strains also varied in their responses regarding the estrous cycle, hormonal levels, and number of developing ovarian follicles. The studied mixture of pesticides was found to interfere with the female reproductive system when individual pesticides were mixed above a certain level, indicating a threshold exists for each of the strains studied.

Evaluation of early changes induced by diuron in the rat urinary bladder using different processing methods for scanning electron microscopy.

Diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] is a substituted urea herbicide carcinogenic to the rat urinary bladder at high dietary levels. The suggested non-genotoxic mode of action (MOA) of diuron encompasses cytotoxicity and necrosis followed by regenerative hyperplasia. Prenecrotic swollen cells as observed under scanning electron microscopy (SEM) have been reported as early morphological alterations, putatively related to diuron cytotoxicity. However, these changes were not observed in a previous SEM study conducted in this laboratory. This study evaluated whether these early alterations are actually due to diuron cytotoxicity or artifacts related to different processing methods used for SEM analysis. Male Wistar rats were fed ad libitum with basal diet, 7.1% sodium saccharin (NaS) or 2.500ppm diuron for seven days or 15 weeks. The urinary bladders were processed for histological and labeling indices examinations and for SEM using two different processing methods. The incidence of simple hyperplasia after 15 weeks of exposure to diuron or to NaS was significantly increased. By SEM, the incidences and severity of lesions were significantly increased in the diuron group independently of exposure time. The different SEM processing methods used allowed for visualization of swollen superficial cells after seven days of diuron exposure. Probably the absence these cells in a previous study was due to the use very few animals. Our results support the hypothesis that the swollen cell is an early key event due to diuron-induced cytotoxicity and is the result of a degenerative process involved in the non-genotoxic carcinogenic mode of action of high doses of diuron.

Dose and temporal effects on gene expression profiles of urothelial cells from rats exposed to diuron.

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide that at high dietary levels (2500 ppm) induces rat urinary bladder hyperplasia after 20 weeks of exposure and neoplasia after 2 years. The effects on the urothelium after short-term exposure have not been described. The present 7-day study evaluated the dose-dependency of urothelial alterations in the urinary bladder using light microscopy, scanning electron microscopy, and genome-wide transcriptional profiling. Male Wistar rats were fed 0, 125, 500, 2500 ppm diuron for 7 days. The urinary bladder and isolated urothelial cells of these animals were processed for microscopic examination and gene expression profiling, respectively. No significant treatment-related morphologic effects were observed. The number of differentially expressed genes (DEGs) in the exposed groups increased with diuron levels. Diuron-altered genes involved in cell-to-cell interactions and tissue organization were identified in all treatment groups. After 7 days of diuron exposure, transcriptional responses were observed in the urothelium in the absence of clear morphologic changes. These morphological findings are different from those observed in a previous study in which 20 weeks of diuron exposure was associated with simple hyperplasia secondary to the persistent cytotoxicity and necrosis associated with continuous cellular regeneration. Comparison of the gene expression profiles of rats exposed to the 2500 ppm carcinogenic diuron dose for 7 days versus 20 weeks revealed few similarities between these two time points at the gene or pathway level. Taken together, these data provide insight into the dose- and temporal-dependent morphological and transcriptional changes associated with diuron exposure that may lead to the development of tumors in the rat urinary bladder.

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.

Genetic instability persists in non-neoplastic urothelial cells from patients with a history of urothelial cell carcinoma.

Bladder cancer is one of the most common genitourinary neoplasms in industrialized countries. Multifocality and high recurrence rates are prominent clinical features of this disease and contribute to its high morbidity. Therefore, more sensitive and less invasive techniques could help identify individuals with asymptomatic disease. In this context, we used the micronucleus assay to evaluate whether cytogenetic alterations could be used as biomarkers for monitoring patients with a history of urothelial cell carcinoma (UCC). We determined the frequency of micronucleated urothelial cells (MNC) in exfoliated bladder cells from 105 patients with (n = 52) or without (n = 53) a history of UCC, all of whom tested negative for neoplasia by cytopathological and histopathological analyses. MNC frequencies were increased in patients with a history of UCC (non-smoker and smoker/ex-smoker patients vs non-smoker and smoker/ex-smoker controls; p<0.001), in non-smoker UCC patients (vs non-smoker controls; p<0.01), and in smoker/ex-smoker controls (vs non-smoker controls; p<0.001). Patients with a history of recurrent disease also demonstrated a higher MNC frequency compared to patients with non-recurrent neoplasia. However, logistic regression using smoking habits, age and gender as confounding factors did not confirm MNC frequency as a marker for UCC recurrence. Fluorescent in situ hybridization analysis (using a pan-centromeric probe) showed that micronuclei (MN) arose mainly from clastogenic events regardless of UCC and/or smoking histories. In conclusion, our results confirm previous indications that subjects with a history of UCC harbor genetically unstable cells in the bladder urothelium. Furthermore, these results support using the micronucleus assay as an important tool for monitoring patients with a history of UCC and tumor recurrence.

Diuron metabolites and urothelial cytotoxicity: in vivo, in vitro and molecular approaches.

Diuron is carcinogenic to the rat urinary bladder at high dietary levels. The proposed mode of action (MOA) for diuron is urothelial cytotoxicity and necrosis followed by regenerative urothelial hyperplasia. Diuron-induced urothelial cytotoxicity is not due to urinary solids. Diuron is extensively metabolized, and in rats, N-(3,4-dichlorophenyl)urea (DCPU) and 4,5-dichloro-2-hydroxyphenyl urea (2-OH-DCPU) were the predominant urinary metabolites; lesser metabolites included N-(3,4-dichlorophenyl)-3-methylurea (DCPMU) and trace levels of 3,4-dichloroaniline (DCA). In humans, DCPMU and DCPU have been found in the urine after a case of product abuse. To aid in elucidating the MOA of diuron and to evaluate the metabolites that are responsible for the diuron toxicity in the bladder epithelium, we investigated the urinary concentrations of metabolites in male Wistar rats treated with 2500ppm of diuron, the urothelial cytotoxicity in vitro of the metabolites and their gene expression profiles. DCPU was found in rat urine at concentrations substantially greater than the in vitro IC50 and induced more gene expression alterations than the other metabolites tested. 2-OH-DCPU was present in urine at a concentration approximately half of the in vitro IC50, whereas DCPMU and DCA were present in urine at concentrations well below the IC50. For the diuron-induced MOA for the rat bladder, we suggest that DCPU is the primary metabolite responsible for the urothelial cytotoxicity with some contribution also by 2-OH-DCPU. This study supports a MOA for diuron-induced bladder effects in rats consisting of metabolism to DCPU (and 2-OH-DCPU to a lesser extent), concentration and excretion in urine, urothelial cytotoxicity, and regenerative proliferation.

STEAP1 protein overexpression is an independent marker for biochemical recurrence in prostate carcinoma.

AIMS: To investigate the prognostic value of expression levels of the genes STEAP1 and STEAP2, and of STEAP1 protein, in prostate carcinomas (PCa). METHODS AND RESULTS: STEAP1 and STEAP2 transcript levels were evaluated by RT-qPCR in samples from 35 PCa, 24 adjacent non-neoplastic prostate (AdjP) tissues, five cases of benign prostatic hyperplasia (BPH), and two histologically normal prostates (N). STEAP1 expression was assessed by immunohistochemistry in samples from 198 PCa, 76 AdjP, 22 BPH, and two N. The findings were compared with clinical and pathological parameters and patient outcome. STEAP1 and STEAP2 transcript analysis showed no differences between the groups tested. Although not significant, higher STEAP1 mRNA levels were detected in tumours with high Gleason scores and in patients who presented with biochemical recurrence (BCR). STEAP1 overexpression was detected in PCa, and was significantly associated with high-grade Gleason scores, seminal vesicle invasion, BCR, and worse outcome (metastasis or PCa-specific death). STEAP1 overexpression was significantly associated with shorter BCR-free survival. Multivariate analysis revealed that STEAP1 is an independent marker for BCR. CONCLUSIONS: These findings provide evidence that STEAP1 is a biomarker of worse prognosis in PCa patients.