ß-ionone inhibits nonalcoholic fatty liver disease and its association with hepatocarcinogenesis in male Wistar rats.

Among the primary neoplasias that affect the liver, hepatocellular carcinoma (HCC) is the most frequent and the third leading cause of death related to cancer. Several risk factors predispose individuals to HCC such as nonalcoholic fatty liver disease (NAFLD), whose incidence has significantly increased worldwide. ß-ionone (ßI) isoprenoid is a known chemopreventive of hepatocarcinogenesis. However, the effects of this compound on NAFLD isolated or in association with hepatocarcinogenesis have not yet been evaluated. A high-fat emulsion administered for 6 weeks resulted in NAFLD in male rats, and oral treatment with ßI during this period significantly attenuated its development. Moreover, the presence of NAFLD potentiated hepatocarcinogenesis induced by the resistant hepatocyte (RH) model in these animals by increasing the number and percentage of the liver section area occupied by placental glutathione S-transferase (GST-P)-positive persistent preneoplastic lesions (pPNLs), that are thought to evolve into HCC. This indicates that this NAFLD/RH protocol is suitable for studies of the influence of NAFLD on the HCC development. Therefore, here we also investigated the chemopreventive effect of ßI under these two associated conditions. In this context, ßI reduced the number and percentage of the liver section area occupied by pPNLs, as well as cell proliferation and the number of oval cells, which are considered potential targets for the development of HCC. Thus, ßI presents not only a promising inhibitory effect on NAFLD isolated but also chemopreventive activity when it is associated with hepatocarcinogenesis.

Antiangiogenic effects of the chemopreventive agent tributyrin, a butyric acid prodrug, during the promotion phase of hepatocarcinogenesis.

Agents that inhibit angiogenic factors may prevent the development of hepatocellular carcinoma (HCC). Thus, the objective of this study was to kinetically evaluate the antiangiogenic activity of tributyrin (TB), a butyric acid prodrug, in the promotion stage of hepatocarcinogenesis. For this purpose, the resistant hepatocyte (RH) model was used for induction of preneoplastic lesions in Wistar rats. During the promotion phase, the animals received TB or maltodextrin (MD) as control daily. The rats were killed at three time-points (P1, P2 and P3). Increased expression of Vegfa and Vegfr2 was observed during promotion phase of hepatocarcinogenesis, which was not reversed by TB treatment. However, TB treatment reduced the expression of cluster of differentiation (CD) 34-positive vessels at P3 and α-smooth muscle actin (α-SMA)-positive vessels at P2 compared with MD. Enhanced levels of hypoxia inducible factor-1α (HIF-1α) and phosphorylated extracellular signal-regulated kinases (pERK) were detected at P3 when compared with P1 and P2 in the MD treatment. TB treatment reduced the levels of HIF-1α and pERK at P3 relative to the MD control. Experiments with human umbilical vein endothelial cells (HUVEC) showed that sodium butyrate (NaBu) inhibited cell migration and tube formation, confirming the antiangiogenic activity of its prodrug TB. In conclusion, antiangiogenic activity of TB is an early event that already occurs in preneoplastic livers, reinforcing its potential chemopreventive effects against HCC.

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.

The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage.

The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200mg/100g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300 mg/100g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40 mg/kg b.w.) twice a week. After 9 weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p<0.05) as well as the ACF with ≥4 crypts (p<0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p<0.05) and reduced DNA damage (p<0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p<0.05). TB administration resulted in increased colonic tissue concentrations of BA (p<0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB.

ß-ionone inhibits persistent preneoplastic lesions during the early promotion phase of rat hepatocarcinogenesis: TGF-α, NF-κB, and p53 as cellular targets.

Dietary isoprenic derivatives such as ß-ionone (ßI) are a promising class of chemopreventive agents. In this study, cellular aspects of ßI protective activities during early hepatocarcinogenesis were evaluated. Male Wistar rats were submitted to "resistant hepatocyte" model and then received daily 16 mg/100 g body weight (b.w.) of ßI (ßI group) or only 0.25 mL/100 g b.w. of corn oil (vehicle, control group [CO]) during 4 wk, specifically during early promotion phase. Compared to controls, ßI inhibited (P < 0.05) the development of persistent preneoplastic lesions (pPNL), considered to be potential hepatocellular carcinoma (HCC) progression sites, and increased remodeling PNL (rPNL) (P < 0.05) that tend to regress to a normal phenotype. Increased ßI hepatic levels (P < 0.05), in the ßI group, were associated with its chemopreventive actions. Compared to control rats, ßI reduced the frequency of both pPNL and rPNL positive for tumor growth factor (TGF)-α (P < 0.05), reduced the frequency of pPNL stained for p65 (nuclear factor-kappaB; NF-κB) (P < 0.05), and reduced the frequency of pPNL positive for cytoplasmic p53 (P < 0.05). Our data demonstrated that ßI targets TGF-α, NF-κB, and p53 in initial phases of hepatocarcinogenesis and specifically inhibits PNL with increased probability to progress to HCC. This isoprenoid may represent a chemopreventive agent of choice for HCC control.

Effects of Ginkgo biloba on chemically-induced mammary tumors in rats receiving tamoxifen.

BACKGROUND: Ginkgo biloba extract (GbE) is used extensively by breast cancer patients undergoing treatment with Tamoxifen (TAM). Thus, the present study investigated the effects of GbE in female Sprague-Dawley (SD) rats bearing chemically-induced mammary tumors and receiving TAM. METHODS: Animals bearing mammary tumors (≥1 cm in diameter) were divided into four groups: TAM [10 mg/kg, intragastrically (i.g.)], TAM plus GbE [50 and 100 mg/kg, intraperitoneally (i.p.)] or an untreated control group. After 4 weeks, the therapeutic efficacy of the different treatments was evaluated by measuring the tumor volume (cm(3)) and the proportions of each tumor that were alive, necrotic or degenerative (mm(2)). In addition, labeling indexes (LI%) were calculated for cell proliferation (PCNA LI%) and apoptosis (cleaved caspase-3 LI%), expression of estrogen receptor-alpha (ER-α) and p63 biomarkers. RESULTS: Overall, the tumor volume and the PCNA LI% within live tumor areas were reduced by 83% and 99%, respectively, in all TAM-treated groups when compared to the untreated control group. GbE treatment (100 mg/kg) reduced the proportions of live (24.8%) and necrotic areas (2.9%) (p = 0.046 and p = 0.038, respectively) and significantly increased the proportion of degenerative areas (72.9%) (p = 0.004) in mammary tumors when compared to the group treated only with TAM. The expression of ER-α, p63 and cleaved caspase-3 in live tumor tissues was not modified by GbE treatment. CONCLUSIONS: Co-treatment with 100 mg/kg GbE presented a slightly beneficial effect on the therapeutic efficacy of TAM in female SD rats bearing mammary tumors.

Effects of swim training on liver carcinogenesis in male Wistar rats fed a low-fat or high-fat diet.

The present study aimed to investigate the beneficial effects of swim training on the promotion-progression stages of rat liver carcinogenesis. Male Wistar rats were submitted to chemically induced liver carcinogenesis and allocated into 4 major groups, according their dietary regimen (16 weeks) and swim training of 5 days per week (8 weeks): 2 groups were fed low-fat diet (LFD, 6% fat) and trained or not trained and 2 groups were fed high-fat diet (HFD, 21% fat) and trained or not trained. At week 20, the animals were killed and liver samples were processed for histological analyses; immunohistochemical detection of persistent or remodeling preneoplastic lesions (pPNL and rPNL) expressing placental glutathione S-transferase (GST-P) enzyme; or proliferating cell nuclear antigen (PCNA), cleaved caspase-3, and bcl-2 protein levels by Western blotting or malonaldehyde (MDA) and total glutathione detection by HPLC. Overall analysis indicated that swim training reduced the body weight and body fat in both LFD and HFD groups, normalized total cholesterol levels in the HFD group while decreased the MDA levels, increased glutathione levels and both number of GST-P-positive pPNL and hepatocellular adenomas in LFD group. Also, a favorable balance in PCNA, cleaved caspase-3, and bcl-2 levels was detected in the liver from the LFD-trained group in relation to LFD-untrained group. The findings of this study indicate that the swim training protocol as a result of exercise postconditioning may attenuate liver carcinogenesis under an adequate dietary regimen with lowered fat intake.

Influência do consumo do café (com e sem cafeína) ou da cafeína isolada sobre a fibrose e a promoção da hepatocarcinogênese química em ratos Wistar machos / Influences the consumption of coffee (with or without caffeine) or caffeine isolated on fibrosis and promotion of chemical hepatocarcinogenesis in male wistar rats

O café e a cafeína são dois potenciais agentes preventivos contra o desenvolvimento ou avanço dos processos de fibrose/cirrose e carcinogênese hepática em humanos, entretanto suas ações são controversas e muitas vezes inconclusivas. Devido a isto, o objetivo deste trabalho foi verificar a ação do café ou da cafeína isolada no fígado de ratos Wistar tratados com tioacetamida (TAA) ou tetracloreto de carbono (CCl4). Para tanto, os dados experimentais foram distribuídos em dois artigos. No primeiro artigo, foram avaliados os efeitos do café convencional, descafeinado e da cafeína isolada na hepatotoxicidade induzida pela TAA em ratos Wistar. Para tanto, 60 os animais foram divididos em 5 grupos experimentais: G1 (controle negativo), G2 (controle positivo tratado com TAA 200 mg/Kg i.p.), G3 (TAA + café convencional), G4 (TAA + café descafeinado) e G5 (TAA + cafeína a 0,1%). Ao final de 8 semanas de tratamento os ratos foram eutanasiados para coleta do sangue (análises séricas) e do fígado (análises histológicas, histoquímicas e moleculares). De maneira geral os animais tratados com café/cafeína (G3-G5) apresentaram níveis da enzima alanina aminotransferase (ALT), área ocupada por colágenos I e III e expressão da proteína TGF-beta1 menores que o grupo controle positivo (G2). Adicionalmente, os grupos G3 e G5 apresentaram menor número de núcleos PCNA positivos em fase S do que o grupo G2. O grupo G3 também apresentou menor número de focos GST-P positivos que o grupo G2. Ademais, os grupos G4 e G5 apresentaram as maiores atividades de MMP-2 ativa. Em conclusão, tanto o café convencional como o descafeinado como a cafeína a 0,1% apresentaram efeitos benéficos, mostrando que os outros componentes do café, mesmo sem a cafeína, ou que somente a cafeína são capazes de reduzir a hepatotoxicidade no fígado de ratos Wistar tratados com TAA..(...)

Consumption of coffee beverages reduces the incidence of liver disease. However, whether these beneficial effects on human health are due to caffeine or other specific components in the beverage remains controversial. There, the present study aimed to study evaluated the protective effects of coffee beverages or caffeine on liver toxicity induced by repeated administration of the hepatotoxicant thioacetamide (TAA) in male Wistar rats. Animals were randomized into five groups: untreated controls (G1) TAA only (G2, 200 mg/Kg b.w. twice a week for 8 weeks, i.p.), TAA+conventional coffee (G3), TAA+decaffeinated coffee (G4) and TAA+caffeine (G5, 0.1% in the drinking water). At the end of 8 weeks, the animals were euthanized and blood and liver samples were collected. Serum ALT levels were lower in animals that received coffee and caffeine (p < 0.001). In addition, liver oxidized glutathione (p < 0.05), fibrosis/inflammation score (p < 0.001) and TGF-beta expression (p ¡Ü 0.001) was reduced in these groups when compared to TAA-only rats. Moreover, conventional coffee and caffeine reduced PCNA S-phase index (p < 0.001) but only conventional coffee reduced cleaved caspase-3 index (p < 0.001) and active metalloproteinase 2 (p ¡Ü 0.004) in the liver from TAA-treated animals.(...)

Maté attenuates DNA damage and carcinogenesis induced by diethylnitrosamine and thermal injury in rat esophagus.

Drinking hot maté has been associated with risk for esophageal cancer in South America. Thus, the aims of this study were to evaluate the modifying effects of maté intake on DNA damage and esophageal carcinogenesis induced by diethylnitrosamine (DEN) and thermal injury (TI) in male Wistar rats. At the initiation phase of carcinogenesis, rats were treated with DEN (8 x 80 mg/kg) and submitted to TI (water at 65 degrees C, 1 ml/rat, instilled into the esophagus). Concomitantly, the animals received maté (2.0%w/v) for 8 weeks. Samples of peripheral blood were collected 4h after the last DEN application for DNA damage analysis. At weeks 8 and 20, samples from esophagus and liver were also collected for histological and immunohistochemical analysis. Maté significantly decreased DNA damage in leukocytes, cell proliferation rates in both esophagus and liver and the number of preneoplastic liver lesions from DEN/TI-treated animals at week 8. A significant lower incidence of esophageal papillomas and liver adenomas and tumor multiplicity was observed in the animals previously treated with maté at week 20. Thus, maté presented protective effects against DNA damage and esophageal and liver carcinogenesis induced by DEN.

Metalloproteinases 2 and 9 activity during promotion and progression stages of rat liver carcinogenesis.

Activity of metalloproteinases 2 and 9 (MMP-2 and 9) during promotion and progression of rat liver carcinogenesis was investigated in a modified resistant hepatocyte model. Development of preneoplastic liver lesions positive for glutathione S-transferase 7-7-(GST-P 7-7-positive PNL) and tumors besides hepatocytes positive for proliferating cell nuclear antigen (PCNA) were quantified and compared to MMP-2 and-9 activity using gelatin zymography. Marked increases in GST-P 7-7-positive PNL development, PCNA labeling indices, MMP-2 (pro, intermediate and active forms) and pro-MMP-9 activity were observed after proliferative stimulus induced by 2-acetylaminofluorene (2-AAF) exposure cycles. After 2-AAF withdrawal, increase in MMP-2 activity was detected only in neoplastic mixed lesions, whereas active MMP-9 was increased in both PLN and neoplastic tissues. Our findings suggest that MMP-2 may be associated with proliferative events induced by 2-AAF rather than with selective growth of PNL and that MMP-9 could be associated with progression of PNL and neoplastic mixed lesions.

Chronic ethanol intake promotes double gluthatione S-transferase/transforming growth factor-alpha-positive hepatocellular lesions in male Wistar rats.

The chronic ethanol intake influence on the gluthatione S-transferase (GST-P) and transforming growth factor alpha (TGF-alpha) expression in remodeling/persistent preneoplastic lesions (PNLs) was evaluated in the resistant hepatocyte model. Male Wistar rats were allocated into five groups: G1, non-treated, fed water and chow ad libitum; G2, non-treated and pair-fed chow (restricted to match that of G3 group) and a maltodextrin (MD) solution in tap water (matched ethanol-derived calories); G3, fed 5% ethanol in drinking water and chow ad libitum; G4, diethylnitrosamine (DEN, 200 mg/kg, body weight) plus 200 parts per million of 2-acetylaminofluorene (2-AAF) for 3 weeks and pair-fed chow (restricted to match that of G5 group) and an MD solution in tap water (matched ethanol-derived calories); G5, DEN/2-AAF treatment, fed ethanol 5% and chow ad libitum. All animals were subjected to 70% partial hepatectomy at week 3 and sacrificed at weeks 12 or 22, respectively. Liver samples were collected for histological analysis or immunohistochemical expression of GST-P, TGF-alpha and proliferating cell nuclear antigen or zymography for matrix metalloproteinases-2 and -9. At the end of ethanol treatment, there was a significant increase in the percentage of liver area occupied by persistent GST-P-positive PNLs, the number of TGF-alpha-positive PNLs and the development of liver tumors in ethanol-fed and DEN/2-AAF-treated groups (G5 versus G4, P < 0.001). In addition, ethanol feeding led to a significant increase in cell proliferation mainly in remodeling and persistent PNLs with immunoreactivity for TGF-alpha at week 22 (P < 0.001). Gelatinase activities were not altered by ethanol treatment. The results demonstrated that ethanol enhances the selective growth of PNL with double expression of TGF-alpha and GST-P markers.