A mixture of glyphosate and 2,4-D herbicides enhances the deleterious reproductive outcomes induced by Western diet in obese male mice.

The consumption of Western diet (WD) - enriched in fats and sugars - is associated with overweight, obesity and male reproductive disorders. In addition to WD intake, crops and dairy products display residues of herbicides, including glyphosate and 2,4-D that are widely applied worldwide. The concomitant exposure to WD and herbicides - mimicking contemporary scenarios - is not fully investigated. Thus, we evaluated the effects of glyphosate and 2,4-D, alone or in mixture, on WD-induced alterations in the male genital system. Male C57BL6J mice were submitted to WD (chow containing 20% lard, 0.2% cholesterol, 20% sucrose, and high sugar solution with 23.1 and 18.9 g/L of D-fructose and D-glucose) for 6 months. Concomitantly to WD, the animals received glyphosate (0.05, 5, or 50 mg/kg/day), 2,4-D (0.02, 2 or 20 mg/kg/day) or their mixture (0, 05 + 0.02, 5 + 2, or 50 + 20 mg/kg/day) by intragastrical administration (5×/week). Doses were based on Acceptable Daily Intake (ADIs) or No Observed Adverse Effect Level (NOAEL) values. Herbicide exposure did not alter the WD-induced obesity, hypercholesterolemia and hyperglycemia. WD induced sperm cell abnormalities, reduced the number, volume and area of Leydig cells, enhanced the frequency of epididymal abnormalities, decreased the proliferation in both germinal and epididymal epithelia, and reduced the number of androgen receptor (AR) positive epididymal cells. Remarkably, the herbicide mixtures promoted such WD-induced effects: increased the frequency of sperm cell and epididymal abnormalities (absence of sperm, cytoplasmic vacuoles, and clear cell hypertrophy) (5 + 2 and 50 + 20 doses); decreased Leydig cell nuclei volume and area (5 + 2 and 50 + 20 doses), reduced epididymal cell proliferation (all mixtures), and AR expression (50 + 20 dose). In addition, herbicide mixtures reduced serum testosterone levels (5 + 2 and 50 + 20 doses). Our findings indicate that the mixture of glyphosate and 2,4-D herbicides, mimicking environmentally relevant scenarios, promotes WD-induced changes in the male genital system.

Panx1 knockout promotes preneoplastic aberrant crypt foci development in a chemically induced model of mouse colon carcinogenesis.

Colorectal cancer, which is the third leading cause of cancer-related deaths worldwide, is a multistep disease, featuring preneoplastic aberrant crypt foci (ACF) as the early morphological manifestation. The roles of hemichannel-forming transmembrane Pannexin 1 (Panx1) protein have not been investigated in the context of colon carcinogenesis yet, although it has contrasting roles in other cancer types. Thus, this study was conducted to examine the effects of Panx1 knockout (Panx1-/- ) on the early events of chemically induced colon carcinogenesis in mouse. Wild type (WT) and Panx1-/- female C57BL6J mice were submitted to a chemically induced model of colon carcinogenesis by receiving six intraperitoneal administrations of 1,2-dimethylhydrazine (DMH) carcinogen. Animals were euthanized 8 h (week 7) or 30 weeks (week 37) after the last DMH administration in order to evaluate sub-acute colon toxicity outcomes or the burden of ACF, respectively. At week 7, Panx1 genetic ablation increased DMH-induced genotoxicity in peripheral blood cells, malondialdehyde levels in the colon, and apoptosis (cleaved caspase-3) in colonic crypts. Of note, at week 37, Panx1-/- animals showed an increase in aberrant crypts (AC), ACF mean number, and ACF multiplicity (AC per ACF) by 56%, 57% and 20%, respectively. In essence, our findings indicate that Panx1 genetic ablation promotes preneoplastic ACF development during chemically induced mouse colon carcinogenesis, and a protective role of Panx1 is postulated.

Arthrospira (Spirulina) platensis feeding reduces the early stage of chemically induced rat colon carcinogenesis.

Colorectal cancer is the third most diagnosed cancer worldwide and linked to dietary/lifestyle factors. Arthrospira (Spirulina) platensis (AP) contains bioactive compounds with beneficial effects in vivo/in vitro. We evaluated the effects of AP feeding against 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Male Sprague Dawley rats were given subcutaneous injections of DMH (4 × 40 mg/kg body weight) (G1-G3) or vehicle (G4-G5) twice a week (weeks 3-4). During weeks 1-4, animals were fed a diet containing 1 % (G2) or 2 % (G3-G4) AP powder (w/w). After this period, all groups received a balanced diet until week 12. Some animals were euthanised after the last DMH injection (week 4) for histological, immunohistochemical (Ki-67, γ-H2AX and caspase-3) and molecular analyses (real time-PCR for 91 genes), while other animals were euthanised at week 12 for preneoplastic aberrant crypt foci (ACF) analysis. Both AP treatments (G2-G3) significantly decreased the DMH-induced increase in γ-H2AX (DNA damage) and caspase 3 (DNA damage-induced cell death) in colonic crypts at week 4. In addition, Cyp2e1 (Drug metabolism), Notch1, Notch2 and Jag1 genes (Notch pathway) and Atm, Wee1, Chek2, Mgmt, Ogg1 and Xrcc6 genes (DNA repair) were also down-regulated by 2 % AP feeding (G3) at week 4. A significant reduction in ACF development was observed in both AP-treated groups (G2-G3) at week 12. In conclusion, findings indicate that AP feeding reduced acute colonic damage after DMH, resulting in fewer preneoplastic lesions. Our study provided mechanistic insights on dietary AP-preventive effects against early colon carcinogenesis.

Modifying effects of 2,4-D and Glyphosate exposures on gut-liver-adipose tissue axis of diet-induced non-alcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD), which is linked to western diet (WD) intake, affects 30% of the world's population and involves the crosstalk of liver steatosis, hypertrophy/inflammation of adipose tissue and deregulation of gut microbiome. Glyphosate and 2,4-D are some of the most applied herbicides worldwide, and their roles in NAFLD have not been investigated. Thus, the present study evaluated whether glyphosate and 2,4-D, in single or mixed exposure, alter WD-induced NAFLD in a mouse model. Male C57Bl/6 mice (n = 10/group) received a fat (30% lard, 0.02% cholesterol), and sucrose-rich diet (20%) and high sugar solution (23.1 and 18.9 g/L of fructose and glucose) for 6 months. Simultaneously, animals received glyphosate (0.05 or 5 mg/kg/day), 2,4-D (0.02 or 2 mg/kg/day), or their combination (0.05 +0.02 or 5 +2 mg/kg/day) by intragastrical administration (5 ×/week). Doses were based on the Acceptable Daily Intake (ADIs) or No Observed Adverse Effect Level (NOAEL) levels. Herbicide exposures featured differential responses. WD-induced obesity, hypercholesterolemia, and hyperglycemia remained unaltered. Compared to the group receiving only WD, only the concomitant exposure to WD and 2,4-D (2 mg) enhanced the percentage of mice with moderate/severe hepatic inflammation, CD68 macrophage infiltration, and malondialdehyde levels in the liver. In line, this herbicide modulated immune response- (including Cd4, C8b, Cd28, Cxcr3, Cxcr6) and oxidative stress-related (such as Gsta1, Gsta2, Gsta4, Gstm1, Gstm2, Gstm3, Gstm4, Nqo1, Gpx2) genes in the hepatic transcriptome analysis. This exposure also enriched pro-inflammatory Deferribacteres phylum in fecal microbiome. In general, the herbicide mixtures did not feature the same effects attributed to 2,4-D isolated exposure. Our findings indicate that 2,4-D, at a dose within the toxicological limits, was able to induce disturbances in mainly at the liver and gut axes involved in NAFLD development in male mice.

Protective Effects of Omega-3 Supplementation against Doxorubicin-Induced Deleterious Effects on the Liver and Kidneys of Rats.

Anthracycline doxorubicin (DOX) is still widely used as a chemotherapeutic drug for some solid tumors. Although DOX is highly effective, its side effects are limiting factors, such as cardio, nephro and hepatotoxicity. As such, approaches used to mitigate these adverse effects are highly encouraged. Omega 3 (ω-3), which is a class of long-chain polyunsaturated fatty acids, has been shown to have anti-inflammatory and antioxidant effects in preclinical bioassays. Thus, we evaluated the protective effects of ω-3 supplementation on hepatotoxicity and nephrotoxicity induced by multiple DOX administrations in rodents. Male Wistar rats (10 rats/group) were treated daily with ω-3 (400 mg/kg/day) by gavage for six weeks. Two weeks after the first ω-3 administration, the rats received DOX (3.5 mg/kg, intraperitoneal, 1×/week) for four weeks. DOX treatment reduced body weight gain increased systemic genotoxicity and caused liver-related (increase in serum ALT levels, thickness of the Glisson's capsule, compensatory proliferation and p65 levels) and kidney-related (increase in serum urea and creatinine levels, and incidence of tubular dilatation) deleterious outcomes. In contrast, ω-3 supplementation was safe and abrogated the DOX-related enhancement of systemic genotoxicity, serum urea and creatinine levels. Furthermore, ω-3 intervention reduced by 50% the incidence of kidney histological lesions while reducing by 40-50% the p65 protein level, and the proliferative response in the liver induced by DOX. Our findings indicate that ω-3 intervention attenuated the DOX-induced deleterious effects in the liver and kidney. Therefore, our findings may inspire future mechanistical investigations and clinical interventions with ω-3 on the reported outcomes.

Dietary Capsaicin Reduces Chemically Induced Rat Urinary Bladder Carcinogenesis.

Capsaicin (CAP) is the compound responsible for pungency in chili peppers, presenting several biological properties. But its general safety and effectiveness in the context of carcinogenesis has not been fully clarified. Thus, the present study evaluated whether dietary CAP modifies the development of urothelial lesions induced by the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in male Sprague-Dawley rats. Animals were randomly allocated into 6 groups: G1 - treated with 0.05% BBN in drinking water (weeks 1-12) and received a balanced diet (weeks 1-20); G2 and G3-treated with BBN (weeks 1-12) and received a balanced diet with 0.01 or 0.02% CAP (weeks 1-20), respectively; G4 and G5- only received a balanced diet with 0.01 or 0.02% CAP (weeks 1-20), respectively; G6 - only received a balanced diet (weeks 1-20). At the end of week 20, the incidence and types of urothelial lesions, proliferating cell nuclear antigen (PCNA) labeling index, and matrix metalloproteinases (MMP) 2 and 9 activities were analyzed. A significant reduction was observed in the incidence and multiplicity of simple (p = 0.020 and p = 0.011) and nodular/papillary (p = 0.030 and p = 0.003) hyperplasias and papillomas/carcinomas (p = 0.023 and p = 0.020), epithelial proliferation (p = 0.007) and in the activity of the intermediate form of MMP-2 (p < 0.001) and pro-MMP-9 activities (p < 0.002), in BBN + 0.02% CAP (G3) group in comparison to BBN (G1) group. Capsaicin intake per se did not alter body weight, liver and kidney weights, urothelial histology or serum biochemical parameters. Thus, dietary CAP was safe and showed a protective effect against rat BBN-induced urothelial carcinogenesis.

Caffeine and Chlorogenic Acid Combination Attenuate Early-Stage Chemically Induced Colon Carcinogenesis in Mice: Involvement of oncomiR miR-21a-5p.

Colorectal cancer (CRC) is one of most common cancers worldwide, with high rates of mortality. Epidemiological findings demonstrate that coffee consumption reduces the risk of developing CRC by ~13%. In general, in vivo and in vitro findings demonstrate the antiproliferative, antioxidant and proapoptotic effects of brewed coffee or major bioavailable coffee compounds. Thus, it was assessed whether caffeine (CAF) and/or chlorogenic acid (CGA) attenuates the early-stage of chemically induced mouse colon carcinogenesis. Male Swiss mice were submitted to a 1,2-dimethylhydrazine/deoxycholic acid (DMH/DCA)-induced colon carcinogenesis model. These animals received CAF (50 mg/kg), CGA (25 mg/kg) or CAF+CGA (50 + 25 mg/kg) intragastrically for five times/week for ten weeks. CAF+CGA had the most pronounced effects on decreasing epithelial cell proliferation (Ki-67) and increasing apoptosis (cleaved caspase-3) in colonic crypts. This treatment also decreased the levels of proinflammatory cytokines IL-6, IL-17 and TNF-α, and downregulated the oncomiR miR-21a-5p in the colon. Accordingly, the analysis of miR-21a-5p targets demonstrated the genes involved in the negative regulation of proliferation and inflammation, and the positive regulation of apoptosis. Ultimately, CAF+CGA attenuated preneoplastic aberrant crypt foci (ACF) development. Our findings suggest that a combination of coffee compounds reduces early-stage colon carcinogenesis by the modulation of miR-21a-5p expression, highlighting the importance of coffee intake to prevent CRC.

Protective Effects of Dietary Capsaicin on the Initiation Step of a Two-Stage Hepatocarcinogenesis Rat Model.

Capsaicin (CPS), an ingredient of Capsicum plants, has anti-inflammatory, antioxidant and antitumoral properties. The mechanisms of CPS on hepatocarcinogenesis preclinical bioassays are not described. Thus, the protective effects CPS were evaluated in the early stages of chemically-induced hepatocarcinogenesis. Male Wistar rats received diet containing 0.01% or 0.02% CPS for 3 weeks. Afterwards, animals received a dose of hepatocarcinogen diethylnitrosamine (DEN, 100 mg/kg body weight). From weeks 4-12, groups had their diet replaced by a 0.05% phenobarbital supplemented one to promote DEN-induced preneoplastic lesions. Animals were euthanized 24 h after DEN administration (n = 5/group) or at week 12 (n = 9/group). The estimated CPS intake in rats resembled human consumption. At the end of week 3, dietary 0.02% CPS attenuated DEN-induced oxidative damage and, consequently, hepatocyte necrosis by reducing serum alanine aminotransferase levels, liver CD68-positive macrophages, lipid peroxidation, while increasing antioxidant glutathione system. Additionally, 0.02% CPS upregulated vanilloid Trpv1 receptor and anti-inflammatory epoxygenase Cyp2j4 genes in the liver. Ultimately, previous 0.02% CPS intake decreased the number of GST-P-positive preneoplastic lesions at week 12. Thus, CPS attenuated preneoplastic lesion development, primarily by diminishing DEN-induced oxidative liver injury. Findings indicate that CPS is a promising chemopreventive agent when administered after and during the early stages of hepatocarcinogenesis.

Modifying effects of menthol against benzo(a)pyrene-induced forestomach carcinogenesis in female Swiss mice.

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon widespread in the environment and closely associated to tobacco use, which is an important risk factor for highly incident stomach cancer. Menthol, a monoterpene extracted from Mentha genus species, has multiple biological properties, including anti-inflammatory and gastroprotective properties, but its effects on carcinogenesis are still to be fully understood. Thus, we evaluated the modifying effects of Ment against BaP-induced forestomach carcinogenesis. Female Swiss mice received BaP by intragastrical (i.g.) administration (50 mg/kg of body weight [b wt], 2×/week), from weeks 1-5 weeks. Concomitantly, mice received Menthol at 25 (Ment25) or 50 (Ment50) mg/kg b wt (i.g, 3×/week). Animals were euthanized at weeks 5 (n = 5 mice/group) or 30 (n = 10 mice/group). At week 5, both Ment doses reduced peripheral leukocyte blood genotoxicity 4 h after the last BaP administration, but only Ment50 attenuated this biomarker 8 h after the last BaP administration. In accordance to these findings, both Ment interventions attenuated BaP-induced increase in the percentage of H2A.X-positive forestomach epithelial cells. Moreover, Ment50 reduced cell proliferation and apoptosis (i.e., Ki-67 and caspase-3, respectively) in forestomach epithelium but exerted no significant effects on NFκB, and Nrf2 protein levels. At week 30, Ment50 reduced by ~55% the incidence of BaP-induced forestomach diffuse hyperplasia and multiplicity of forestomach tumors (squamous cell papillomas and carcinomas). Our findings indicate that Ment50, administered during initiation phase, attenuates forestomach carcinogenesis by reducing early genotoxicity, cell proliferation, and apoptosis induced by BaP.

Sorafenib reduces steatosis-induced fibrogenesis in a human 3D co-culture model of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) affects around 25% of the worldwide population. Non-alcoholic steatohepatitis (NASH), the more progressive variant of NAFLD, is characterized by steatosis, cellular ballooning, lobular inflammation, and may culminate on hepatic stellate cell (HSC) activation, thus increasing the risk for fibrosis, cirrhosis, and HCC development. Conversely, the antifibrotic effects of sorafenib, an FDA-approved drug for HCC treatment, have been demonstrated in 2D cell cultures and animal models, but its mechanisms in a NAFLD-related microenvironment in vitro requires further investigation. Thus, a human 3D co-culture model of fatty hepatocytes and HSC was established by culturing hepatoma C3A cells, pre-treated with 1.32 mM oleic acid, with HSC LX-2 cells. The fatty C3A/LX-2 spheroids showed morphological and molecular hallmarks of altered lipid metabolism and steatosis-induced fibrogenesis, similarly to the human disease. Sorafenib (15 µM) for 72 hours reduced fatty spheroid viability, and upregulated the expression of lipid oxidation- and hydrolysis-related genes, CPT1 and LIPC, respectively. Sorafenib also inhibited steatosis-induced fibrogenesis by downregulating COL1A1, TGFB1, PDGF, and TIMP1 and by decreasing protein levels of IL-6, TGF-ß1, and TNF-α in fatty spheroids. The demonstration of the antifibrotic properties of sorafenib on steatosis-induced fibrogenesis in a 3D in vitro model of NAFLD supports its clinical use as a therapeutic agent for the treatment of NAFLD/NASH patients.

Capsaicin reduces genotoxicity, colonic cell proliferation and preneoplastic lesions induced by 1,2-dimethylhydrazine in rats.

Capsaicin (8-Methyl-N-vanillyl-(trans)-6-nonenamide) is the major pungent ingredient found in chili peppers consumed worldwide. Most reports on capsaicin potential carcinogenicity have yielded inconsistent findings. Some studies have shown that capsaicin exerts anti-proliferative and pro-apoptotic effects on different cancer cell lines, while others have reported an association between capsaicin at high doses with mutagenicity and carcinogenicity. Thus, this study aimed at assessing the effects of capsaicin administration on 1,2-dimethyl-hydrazine (DMH)-induced colon carcinogenesis in male Wistar rats. Our results show that capsaicin administration, before and during carcinogen exposure, modified DMH-induced cytotoxicity and genotoxicity, promoting anti-proliferative and pro-apoptotic responses through the expression of the genes involved in apoptosis, cell cycle suppression and cell/tissue differentiation. Furthermore, capsaicin reduced aberrant crypt foci (ACF) number and multiplicity, although there were no differences in tumor incidence and multiplicity among the groups. Taken together, the results suggest that capsaicin may have a preventive effect against DMH-induced colorectal carcinogenesis.

ß-ionone modulates the expression of miRNAs and genes involved in the metastatic phenotype of microdissected persistent preneoplastic lesions in rats submitted to hepatocarcinogenesis.

MicroRNAs (miRNAs) are post-transcriptional gene expression regulators which expression is frequently altered in hepatocellular carcinoma (HCC). ß-ionone (ßI) is noted for its ability to inhibit persistent preneoplastic lesions (pPNLs) in liver rats. We evaluated the expression of miRNAs involved in carcinogenesis and possible targets modulated by ßI, in pPNLs and surrounding of microdissected tissues. Rats subjected to resistant hepatocyte model were treated during promotion stage with ßI (16 mg/100 g body weight) or corn oil (CO; 0.25 mL/100 g body weight; controls). Five animals receive no treatment (NT). In CO group, 38 and 29 miRNAs showed reduced expression relative to NT (P < 0.05) in pPNLs and surrounding, respectively. No miRNAs showed increased expression in surrounding of the CO compared to NT group; however, 30 miRNAs showed increased expression (P ≤ 0.05) in pPNLs of the CO group. There was no difference between ßI and CO groups (P > 0.05) in the expression of miRNAs in surrounding. In pPNLs ßI increased expression of miR-122 and miR-34a (P ≤ 0.05) and reduced of Igf2 (P ≤ 0.05), target of the latter, compared to CO. Additionally, ßI decreased the expression of miR-181c and its target Gdf2 (P ≤ 0.05). ßI reduced the expression of miR-181b and miR-708 (P ≤ 0.05) and increased the expression of their respective target mRNAs Timp3 and Mtss1 (P ≤ 0.05), relative to CO group. Modulation of miRNAs target genes by ßI was confirmed in vitro. ßI is a promising chemopreventive agent in the initial stages of hepatocarcinogenesis, as it modulates the expression of the miRNAs and target genes that can alter the metastatic phenotype of HCC. © 2016 Wiley Periodicals, Inc.

Impact of maternal and postnatal zinc dietary status on the prostate of pubescent and adult rats.

Zinc is important for cell physiology and alteration of its levels during development can modulate a series of biological events. The aim of this study was to investigate whether dietary zinc deficiency or supplementation during morphogenesis and early postnatal development could interfere in prostate maturation. Pregnant rats were exposed to a standard diet (NZ:35 mg Zn/kg chow), low-zinc diet (LZ:3 mg of Zn/kg chow) and zinc-supplemented diet (HZ:180 mg/Kg chow) from gestational day 10 (GD10) through postnatal day 21 (PND21). After weaning, male offspring were divided into three groups that were submitted to the same food conditions as their mothers until PND53. The animals were euthanized at PND53 and PND115. The ventral prostate was removed, weighed and its fragments were subjected to histological, western blot and zymography analysis. PND53: body and prostate weight were lower in LZ compared to NZ; the epithelial compartment was reduced while the stromal compartment was increased in LZ compared to NZ; there was an increase in the amount of collagen and reduction in AR and SIRT1 expression in LZ compared to NZ. PND115: body weight was lower in LZ compared to NZ and prostate weight was similar among the groups; peripheral physiological hyperplasia was observed, as well as an increased epithelial proliferation index and reduced PAR4 expression in LZ and HZ compared to NZ. Zinc deficiency during prostate morphogenesis and differentiation is potentially harmful to its morphology, however, by restoring the standard dietary environment, the gland responds to the new microenvironment independent of the previous dietary condition.

Early Life and Postnatal Western Diet Feeding and Susceptibility to Chemically Induced Colonic Aberrant Crypt Foci in Male Rats Offspring.

The modifying effects of a Western diet (WD) during early life on the susceptibility to colon carcinogenesis induced by dimethylhydrazine (DMH) were examined in male rats as later adults. Three groups were studied: a lifetime control diet-fed group, a test group fed WD since pregnancy from dams until postnatal day (PND) 42, and a group fed WD at adulthood. At PND 70, all groups received the carcinogen DMH and were euthanized 10 wk later. Colonic aberrant crypt foci (ACF) were scored (number and crypt multiplicity) and the altered pattern of ß-catenin expression was evaluated in the colonic lesions. ACF multiplicity (≥4 crypts) was significantly higher in the group fed WD at early life than in the group fed the control diet. ACF number, crypt multiplicity, and the number of high-grade dysplastic lesions were significantly higher in the group fed WD at adulthood than in the groupfed the control diet. The number of lesions with altered ß-catenin expression was higher in the groups receiving WD at early life or at adulthood than in the lifetime control-diet-fed group. These findings indicate that WD exposure at early life increased the susceptibility to colon carcinogenesis at adulthood.

Apoptotic index for prediction of postmolar gestational trophoblastic neoplasia.

BACKGROUND: Although 85% of patients with a complete hydatidiform mole achieve spontaneous remission after a few months, 15% of them will experience gestational trophoblastic neoplasia, which requires chemotherapy. To date, there is no biomarker to predict post-molar gestational trophoblastic neoplasia before the initiation of human chorionic gonadotropin surveillance. OBJECTIVE: The purpose of this study was to assess the relationship between the expression of apoptosis markers in the molar villous trophoblasts and the subsequent development of gestational trophoblastic neoplasia after the evacuation of a complete hydatidiform mole. STUDY DESIGN: This was a retrospective cohort study of patients with complete hydatidiform mole who were diagnosed, treated, and followed at the Center of Trophoblastic Diseases (Botucatu/São Paulo State and Rio de Janeiro/Rio de Janeiro State, Brazil) from 1995-2014. Patients were divided temporally into derivation (1995-2004) and validation (2005-2014) cohorts. Immunohistochemistry was used to examine tissue expression of the apoptosis inhibitor survivin or the pro-apoptotic enzyme caspase-3. Survivin stains for cytoplasmic and nuclear expression were evaluated independently. Caspase-3 expression was measured as an apoptotic index of positive staining cells over negative staining cells multiplied by 100. Receiver operating characteristic curves were then constructed, and the area under the curve was calculated to test the performance characteristics of the staining to predict the subsequent development of gestational trophoblastic neoplasia. RESULTS: The final study population comprised 780 patients, with 390 patients in each temporal cohort: 590 patients entered spontaneous remission, and 190 patients experienced post-molar gestational trophoblastic neoplasia. Neither nuclear nor cytoplasmic survivin expression performed well as a predictor of subsequent gestational trophoblastic neoplasia. The caspase-3 apoptotic index was a strong risk factor for subsequent gestational trophoblastic neoplasia development. When the apoptotic index was <4%, the risk of gestational trophoblastic neoplasia had an odds ratio of 35.55 (95% confidence interval, 14.02-90.14; P < .0001) in the derivation cohort and an odds ratio of 25.71 (95% confidence interval, 10.13-65.29; P < .0001) in the validation cohort. However, in both cohorts, the positive predictive value for gestational trophoblastic neoplasia of an apoptotic index <4.0% was modest (49% in the derivation cohort and 41% in the validation cohort); the negative predictive value for gestational trophoblastic neoplasia of an apoptotic index ≥4.0% was high (97% in both cohorts). CONCLUSION: The subsequent development of gestational trophoblastic neoplasia after evacuation of complete hydatidiform mole is tied closely to the apoptotic index, which may be a useful biomarker for future prospective studies.

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.

An overview of chemically induced rodent models for sporadic colorectal cancer: Histopathological and translational perspectives.

Globally, colorectal cancer (CRC) is one of the most frequently diagnosed human gastrointestinal neoplasia and the second leading cause of cancer-related death in both men and women. Despite considerable efforts currently devoted to the study of the biology and treatment of CRC, patient prognosis and survival are still poor. Sporadic CRC is a complex multistep disease and usually emerges in the setting of lifestyle and dietary changes mainly observed in industrialized countries with high human development index (HDI) (westernized style). The molecular pathogenesis of sporadic CRC presents genetic heterogeneity with APC, RAS, PIK3CA, TGFBR, SMAD4, and TP53 mutations usually detected during the progression of this malignancy. The establishment of sporadic CRC models has become essential for both basic and translational research to improve our understanding of the pathophysiology, unravel new molecular drivers, and preventive/therapeutic improvement of this malignancy. Chemically induced rodent models of sporadic CRC recapitulate most key morphological and genetic/epigenetic events observed during the promotion and progression of this malignancy, establishing effective diagnostic and prevention strategies to be translated into clinical practice. The present review gathers the main features of the state-of-the-art evidence on chemically induced rodent models, widely applied for translational modelling of sporadic CRC with a specific focus on histopathology and prevention perspectives. Our narrative review reinforces the persistent value of these bioassays and encourages the use of multimodel strategies for further investigations.

Exploring in vitro modeling in hepatocarcinogenesis research: morphological and molecular features and similarities to the corresponding human disease.

The hepatocellular carcinoma (HCC) features a remarkable epidemiological burden, ranking as the third most lethal cancer worldwide. As the HCC-related molecular and cellular complexity unfolds as the disease progresses, the use of a myriad of in vitro models available is mandatory in translational preclinical research setups. In this review paper, we will compile cutting-edge information on the in vitro bioassays for HCC research, (A) emphasizing their morphological and molecular parallels with human HCC; (B) delineating the advantages and limitations of their application; and (C) offering perspectives on their prospective applications. While bidimensional (2D) (co) culture setups provide a rapid low-cost strategy for metabolism and drug screening investigations, tridimensional (3D) (co) culture bioassays - including patient-derived protocols as organoids and precision cut slices - surpass some of the 2D strategies limitations, mimicking the complex microarchitecture and cellular and non-cellular microenvironment observed in human HCC. 3D models have become invaluable tools to unveil HCC pathophysiology and targeted therapy. In both setups, the recapitulation of HCC in different etiologies/backgrounds (i.e., viral, fibrosis, and fatty liver) may be considered as a fundamental guide for obtaining translational findings. Therefore, a "multimodel" approach - encompassing the advantages of different in vitro bioassays - is encouraged to circumvent "model-biased" outcomes in preclinical HCC research.

Maternal protein restriction combined with postnatal sugar consumption alters liver proteomic profile and metabolic pathways in adult male offspring rats.

This study investigated the impact of maternal protein restriction (MPR) and early postnatal sugar consumption (SUG) on the liver health of adult male descendant rats. Male offspring of mothers fed a normal protein diet (NPD) or a low protein diet (LPD) were divided into four groups: Control (CTR), Sugar Control (CTR + SUG), LPD during gestation and lactation (GLLP), and LPD with sugar (GLLP + SUG). Sugar consumption (10% glucose diluted in water) began after weaning on day 21 (PND 21), and at 90 days (PND 90), rats were sacrificed for analysis. Sugar intake reduced food intake and increased water consumption in CTR + SUG and GLLP + SUG compared to CTR and GLLP. GLLP and GLLP + SUG groups showed lower body weight and total and retroperitoneal fat compared to CTR and CTR + SUG. CTR + SUG and GLLP + SUG groups exhibited hepatocyte vacuolization associated with increased hepatic glycogen content compared to CTR and GLLP. Hepatic catalase activity increased in GLLP compared to CTR. Proteomic analysis identified 223 differentially expressed proteins (DEPs) among experimental groups. While in the GLLP group, the DEPs enriched molecular pathways related to cellular stress, glycogen metabolic pathways were enriched in the GLLP + SUG and CTR + SUG groups. The association of sugar consumption amplifies the effects of MPR, deregulating molecular mechanisms related to metabolism and the antioxidant system.

Lack of protective effects of zinc gluconate against rat colon carcinogenesis.

Zinc has been proposed as a promising chemopreventive candidate against colon cancer. However, few studies on the potential beneficial effects of this trace element on cancer chemoprevention are available. The present study was designed to investigate the potential modifying influence of zinc gluconate (ZnGly) on the initiation step of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH). Male Wistar rats received orally ZnGly (15 mg elemental zinc/kg, 3 times per wk) 2 wk before and during DMH treatment (3 × 40 mg/kg, once a wk). The animals were euthanized at the end of 4th and 16th wk. Colons were analyzed for aberrant crypt foci (ACF) and tumor development. Blood and colon zinc levels, cell proliferation, and apoptosis indexes in colonic crypts were analyzed 24 h after the last DMH administration. Oral treatment with ZnGly did neither alter the number of ACF nor the indexes of cell proliferation and apoptosis in the colonic mucosa. The incidence and multiplicity of colon tumors induced by DMH and their histopathological patterns were not modified by previous treatment with ZnGly. These findings indicate a lack of chemopreventive action of zinc gluconate supplementation on the initiation step of rat colon carcinogenesis induced by DMH.