Indole-3-carbinol and chlorogenic acid combination modulates gut microbiome and attenuates nonalcoholic steatohepatitis in a murine model.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, affecting almost 32% of the population and ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Recent findings indicate that the fast-growing prevalence of NAFLD might be linked to adherence to a Westernized diet (WD), mostly composed of fat/sugar-enriched foods. The WD has been reportedly targeted as a potential driver of gut-liver axis unbalance, suggesting a major role in NASH. On the other hand, bioactive food compounds feature as a potential chemopreventive strategy against NASH, due to their beneficial effects (i.e, anti-inflammatory/oxidant activity and modulation of gut microbiome). Brassicaceae vegetables are known for their high amount of isothiocyanates and polyphenols, as indole-3-carbinol (I3C) and chlorogenic acid (CGA). Thus, we sought to assess the effects of human relevant doses of I3C and CGA isolated or in combination (5/125 mg/Kg of body weight, respectively) on a diet/chemical-induced murine model of NASH. I3C + CGA oral treatment diminished NAFLD activity score (NAS) (p < 0.0001), as well as alleviated the hepatic lipid (p = 0.0011) accumulation, prevented hepatic stellate cell (HSC) activation (p < 0.0001), and subsequent fibrosis (p < 0.0001). The combination also reduced the number of both hepatic CD68-positive macrophages (p < 0.0001) and cleaved caspase-3 hepatocytes (p < 0.0001) and diminished the malondialdehyde levels (p = 0.0155). Additionally, the combination of I3C + CGA restored the relative abundance of Alistipes (p = 0.0299), Allobaculum (p = 0.0014), Bacteroides (p = 0.0046), and Odoribacter (p = 0.0030) bacteria genera on the gut microbiome. Taken together, these findings show that the combination of I3C + CGA at populational-relevant ingestion, rather than the I3C or CGA alone, was able to modulate gut microbiome and attenuate NASH in this hybrid model mouse.

Maternal dietary zinc status alters offspring female mammary gland development and response to acute 7,12-dimethylbenzanthracene insult.

We evaluated the effects of prenatal and postnatal dietary zinc (Zn) deficiency or supplementation on mammary gland morphology and on acute response to 7,12-dimethylbenzanthracene (DMBA) in pubertal female rats. On gestational day 10 (GD 10), rat dams were allocated randomly into three experimental groups of 10: a Zn-adequate diet group (ZnA) fed 35 mg Zn/kg chow, a Zn-deficient diet group (ZnD) fed 3 mg ZN/kg chow and a Zn-supplemented diet group (ZnS) fed 180 mg Zn/kg chow. After weaning, female offspring were fed the same diet as their dams until postnatal day 53 (PND 53). All animals received a single 50 mg/kg dose of DMBA on PND 51 and were euthanized on PND 53. Female ZnD offspring exhibited significantly less weight gain compared to the ZnA group and reduced mammary gland development compared to the ZnD and ZnA groups. By PND 53, the Ki-67 labeling index in mammary gland epithelial cells was significantly greater for the ZnS group than for the ZnA and ZnD groups. Apoptosis and ER-α indices did not differ among groups. The ZnD group exhibited significantly increased lipid hydroperoxide (LOOH) levels and decreased catalase and glutathione peroxidase (GSH-Px) activity compared to the ZnA and ZnS groups. The ZnS group exhibited significantly reduced superoxide dismutase (SOD) activity compared to the ZnA and ZnS groups. We observed atypical ductal hyperplasia in the mammary gland of female ZnS group offspring compared to the ZnA and ZnD groups and decreased expression of the Api5 and Ercc1 genes related to apoptosis inhibition and DNA damage repair, respectively. Both the Zn-deficient and Zn-supplemented diet exerted adverse effects on offspring mammary gland morphology and acute response to DMBA.

Unraveling Hepatic Metabolomic Profiles and Morphological Outcomes in a Hybrid Model of NASH in Different Mouse Strains.

Nonalcoholic fatty liver disease (NAFLD) encompasses nonalcoholic steatohepatitis (NASH) and affects 25% of the global population. Although a plethora of experimental models for studying NASH have been proposed, still scarce findings regarding the hepatic metabolomic/molecular profile. In the present study, we sought to unravel the hepatic metabolomic profile of mice subjected to a hybrid model of NASH, by combining a Western diet and carbon tetrachloride administration, for 8 weeks, in male C57BL/6J and BALB/c mice. In both mouse strains, the main traits of NASH-metabolic (glucose intolerance profile), morphologic (extensive microvesicular steatosis and fibrosis, lobular inflammation, and adipose tissue-related inflammation/hypertrophy), and molecular (impaired Nrf2/NF-κB pathway dynamics and altered metabolomic profile)-were observed. The hepatic metabolomic profile revealed that the hybrid protocol impaired, in both strains, the abundance of branched chain-aromatic amino acids, carboxylic acids, and glycosyl compounds, that might be linked to the Nrf2 pathway activation. Moreover, we observed a strain-dependent hepatic metabolomic signature, in which the tricarboxylic acid metabolites and pyruvate metabolism were dissimilarly modulated in C57BL/6J and BALB/c mice. Thus, we provide evidence that the strain-dependent hepatic metabolomic profile might be linked to the distinct underlying mechanisms of NASH, also prospecting potential mechanistic insights into the corresponding disease.

Biodistribution Profile of Magnetic Nanoparticles in Cirrhosis-Associated Hepatocarcinogenesis in Rats by AC Biosusceptometry.

Since magnetic nanoparticles (MNPs) have been used as multifunctional probes to diagnose and treat liver diseases in recent years, this study aimed to assess how the condition of cirrhosis-associated hepatocarcinogenesis alters the biodistribution of hepatic MNPs. Using a real-time image acquisition approach, the distribution profile of MNPs after intravenous administration was monitored using an AC biosusceptometry (ACB) assay. We assessed the biodistribution profile based on the ACB images obtained through selected regions of interest (ROIs) in the heart and liver position according to the anatomical references previously selected. The signals obtained allowed for the quantification of pharmacokinetic parameters, indicating that the uptake of hepatic MNPs is compromised during liver cirrhosis, since scar tissue reduces blood flow through the liver and slows its processing function. Since liver monocytes/macrophages remained constant during the cirrhotic stage, the increased intrahepatic vascular resistance associated with impaired hepatic sinusoidal circulation was considered the potential reason for the change in the distribution of MNPs.

Multigenerational Effects of an Environmentally Relevant Phthalate Mixture on Reproductive Parameters and Ovarian miRNA Expression in Female Rats.

Phthalates are endocrine-disrupting chemicals used in many consumer products. Our laboratory previously developed an environmentally relevant phthalate mixture consisting of 6 phthalates and found that it disrupted female fertility in mice. However, it was unknown if maternal exposure to the mixture affects reproductive parameters and ovarian post-transcription in the F1 and F2 generation of female rats. Thus, we tested the hypothesis that maternal exposure to the phthalate mixture affects folliculogenesis, steroidogenesis, and ovarian microRNA (miRNA) in the F1 and F2 generations of female rats. Pregnant female rats were divided into 4 groups and orally dosed daily from gestational day 10 to postnatal day 21 with corn oil (control group), 20 µg/kg/day, 200 µg/kg/day, or 200 mg/kg/day of the phthalate mixture. Maternal exposure to the phthalate mixture impaired folliculogenesis in the F1 and F2 generations of female rats and affected steroidogenesis in the F1 generation of female rats compared to control. Further, the phthalate mixture altered ovarian expression of some genes related to the cell cycle and steroidogenesis compared to control in the F1 and F2 generations of female rats. The mixture also increased ovarian expression of rno-mir-184 that is involved with the oocyte maturation process. Collectively, our data show that maternal exposure to the phthalate mixture affects folliculogenesis and steroidogenesis in the F1 and F2 generations of female rats and alters ovarian miRNA expression in the F1 generation of female rats.

Vitamin D3 supplementation alleviates chemically-induced cirrhosis-associated hepatocarcinogenesis.

Vitamin D3 (VD3) deficiency has been associated with increased risk for cirrhosis and hepatocellular carcinoma, a highly incident malignant neoplasia worldwide. On the other hand, VD3 supplementation has shown some beneficial effects in clinical studies and rodent models of chronic liver disease. However, preventive effects of dietary VD3 supplementation in cirrhosis-associated hepatocarcinogenesis is still unknow. To investigate this purpose, male Wistar rats submitted to a combined diethylnitrosamine- and thioacetamide-induced model were concomitantly supplemented with VD3 (5,000 and 10,000 IU/kg diet) for 25 weeks. Liver samples were collected for histological, biochemical and molecular analysis. Serum samples were used to measure 25-hydroxyvitamin D [25(OH)D] and alanine aminotransferase levels. Both VD3 interventions decreased hepatic collagen deposition and pro-inflammatory p65 protein levels, while increased hepatic antioxidant catalase and glutathione peroxidase activities and serum 25(OH)D, without a clear dose-response effect. Nonetheless, only the highest concentration of VD3 increased hepatic protein levels of VD receptor, while decreased the number of large preneoplastic glutathione-S-transferase- (>0.5 mm²) and keratin 8/18-positive lesions, as well the multiplicity of hepatocellular adenomas. Moreover, this intervention increased hepatic antioxidant Nrf2 protein levels and glutathione-S-transferase activity. In summary, dietary VD3 supplementation - in special the highest intervention - showed antifibrotic and antineoplastic properties in chemically-induced cirrhosis-associated hepatocarcinogenesis. The positive modulation of Nrf2 antioxidant axis may be mechanistically involved with these beneficial effects, and may guide future clinical studies.

Prenatal exposure to a mixture of different phthalates increases the risk of mammary carcinogenesis in F1 female offspring.

Phthalates metabolites have been detected in the urine of pregnant and breastfeeding women. Thus, this study evaluated the adverse effects of maternal exposure to a mixture of six phthalates (Pth mix) on the mammary gland development and carcinogenesis in F1 female offspring. Pregnant female Sprague-Dawley rats were exposed daily to vehicle or Pth mix (35.22% diethyl-phthalate, 21.03% di-(2-ethylhexyl)-phthalate, 14.91% dibutyl-phthalate, 15.10% diisononyl-phthalate, 8.61% diisobutyl-phthalate, and 5.13% benzylbutyl-phthalate) by gavage at 20 µg/kg, 200 µg/kg or 200 mg/kg during gestational day 10 (GD 10) to postnatal day 21 (PND 21). After weaning (PND 22), some female offspring were euthanized for mammary gland analyses while other females received a single dose of N-methyl-N-nitrosourea (MNU, 50 mg/kg) or vehicle and then tumor incidence and multiplicity were recorded until PND 180. Maternal Pth mix exposure increased the number of Ki-67 and progesterone receptor-positive epithelial cells in the mammary gland from Pth mix 200 at µg/kg and 200 mg/kg groups. In addition, tumor incidence and mean number were higher only in Pth mix at 200 mg/kg when compared to the vehicle-treated group, and percentage of tumor-free animals was lower in Pth mix at 200 µg/kg and 200 mg/kg groups. The findings indicate that perinatal Pth mixture exposure increased susceptibility to MNU-induced mammary carcinogenesis in adult F1 female offspring.

Synbiotic sheep milk ice cream reduces chemically induced mouse colon carcinogenesis.

Sheep dairy products containing prebiotic and probiotic ingredients may have health-promoting properties. Thus, this study evaluated the effects of sheep milk ice cream [conventional full-fat (CONV), full-fat enriched with probiotic (PROB, 100 mg % wt/wt of Lacticaseibacillus casei 01), or nonfat synbiotic (SYNB, Lacticaseibacillus casei 01 and inulin, 10% wt/wt)] on carcinogen-induced colonic crypt cytotoxicity and premalignant lesion development. Male Swiss mice received 2 doses of colon carcinogen azoxymethane (AOM, 15 mg/kg of body weight) at wk 3 and 4. Two weeks before and during AOM administrations (4 wk) mice were treated with CONV, PROB, or SYNB by gavage (10 mL/kg). Mice were euthanized at wk 4 or 25 (n = 5 or 10 mice/group, respectively). At wk 4, a significant reduction in micronucleated colonocytes was observed in PROB and SYNB groups, and a significant decrease in both p53 expression and apoptosis indexes in colonic crypts was observed in SYNB group. At wk 25, both PROB and SYNB interventions reduced the mean number of colonic premalignant lesions. However, only SYNB group showed lower incidence and number of high-grade premalignant lesions in the colonic mucosa. These findings indicate that PROB or SYNB sheep milk ice cream, especially SYNB intervention, can reduce chemically induced mouse colon carcinogenesis.

Maternal Low-Protein Diet Deregulates DNA Repair and DNA Replication Pathways in Female Offspring Mammary Gland Leading to Increased Chemically Induced Rat Carcinogenesis in Adulthood.

Studies have shown that maternal malnutrition, especially a low-protein diet (LPD), plays a key role in the developmental mechanisms underlying mammary cancer programming in female offspring. However, the molecular pathways associated with this higher susceptibility are still poorly understood. Thus, this study investigated the adverse effects of gestational and lactational low protein intake on gene expression of key pathways involved in mammary tumor initiation after a single dose of N-methyl-N-nitrosourea (MNU) in female offspring rats. Pregnant Sprague-Dawley rats were fed a normal-protein diet (NPD) (17% protein) or LPD (6% protein) from gestational day 1 to postnatal day (PND) 21. After weaning (PND 21), female offspring (n = 5, each diet) were euthanized for histological analysis or received NPD (n = 56 each diet). At PND 28 or 35, female offspring received a single dose of MNU (25 mg/kg body weight) (n = 28 each diet/timepoint). After 24 h, some females (n = 10 each diet/timepoint) were euthanized for histological, immunohistochemical, and molecular analyses at PDN 29 or 36. The remaining animals (n = 18 each diet/timepoint) were euthanized when tumors reached ≥2 cm or at PND 250. Besides the mammary gland development delay observed in LPD 21 and 28 groups, the gene expression profile demonstrated that maternal LPD deregulated 21 genes related to DNA repair and DNA replication pathways in the mammary gland of LPD 35 group after MNU. We further confirmed an increased γ-H2AX (DNA damage biomarker) and in ER-α immunoreactivity in mammary epithelial cells in the LPD group at PND 36. Furthermore, these early postnatal events were followed by significantly higher mammary carcinogenesis susceptibility in offspring at adulthood. Thus, the results indicate that maternal LPD influenced the programming of chemically induced mammary carcinogenesis in female offspring through increase in DNA damage and deregulation of DNA repair and DNA replication pathways. Also, Cidea upregulation gene in the LPD 35 group may suggest that maternal LPD could deregulate genes possibly leading to increased risk of mammary cancer development and/or poor prognosis. These findings increase the body of evidence of early-transcriptional mammary gland changes influenced by maternal LPD, resulting in differential response to breast tumor initiation and susceptibility and may raise discussions about lifelong prevention of breast cancer risk.

Burdock (Arctium lappa L.) root attenuates preneoplastic lesion development in a diet and thioacetamide-induced model of steatohepatitis-associated hepatocarcinogenesis.

Nonalcoholic steatohepatitis (NASH) is considered growing risk factor for hepatocellular carcinoma development in high-income countries. Diet- and chemically induced rodent models have been applied for the translational study of NASH-associated hepatocarcinogenesis due to their morphological and molecular similarities to the corresponding human disease. Arctium lappa L. (burdock) root tea has been extensively consumed in Traditional Chinese Medicine due to its potential therapeutic properties. Indeed, the bioactive compounds of A. lappa root, as the polyphenols, have already showed antioxidant and anti-inflammatory properties in different in vivo and in vitro bioassays. In this study, we investigated whether burdock root ethanolic extract (BRE) administration attenuates NASH-associated hepatocarcinogenesis. Eight-week-old male Wistar rats received choline-deficient high-fat diet for 8 weeks and multiple thioacetamide doses for 4 weeks in order to induce NASH and preneoplastic glutathione-S-transferase pi (GST-P)+ preneoplastic foci. Subsequently, rats were treated with BRE (100 or 200 mg/kg body weight) or vehicle by oral gavage for 2 weeks. BRE displayed high levels of chlorogenic and caffeic acids and BRE administration reduced total fatty acid and lipid hydroperoxide levels, while increasing the activities of antioxidant superoxide dismutase and catalase enzymes in the liver. Furthermore, burdock intervention diminished the size of GST-P+ remodeling preneoplastic lesions (PNLs) and displayed a trend on reducing hepatocyte proliferation (Ki-67) inside them. These findings suggest that short-term exposure to BRE alleviated remodeling PNL development in NASH-associated hepatocarcinogenesis.

Dietary hemin promotes colonic preneoplastic lesions and DNA damage but not tumor development in a medium-term model of colon carcinogenesis in rats.

Red and processed meat consumption has been strongly related to increase the risk of colorectal cancer (CRC), although its impact is largely unknown. Hemin, an iron-containing porphyrin, is acknowledged as a putative factor of red and processed meat pro-carcinogenic effects. The aim of this study was to investigate the effects of high dietary hemin on the promotion/progression stages of 1,2-dimethylhydrazine (1,2-DMH)-induced colon carcinogenesis. Twenty-four Wistar male rats were given four subcutaneous 1,2-DMH injections and received either balanced diet or balanced diet supplemented with hemin 0.5 mmol/kg for 23 weeks. Colon specimens were analyzed for aberrant crypt foci (ACF) and tumor development. Dietary hemin significantly increased ACF number and fecal water cytotoxicity/genotoxicity in Caco-2 cells when compared to 1,2-DMH control group. However, tumor incidence, multiplicity and cell proliferation did not differ between 1,2-DMH + hemin and 1,2-DMH control group. Gene expression analysis of 91 target-genes revealed that only three genes (Figf, Pik3r5 and Tgfbr2) were down-regulated in the tumors from hemin-fed rats compared to those from 1,2-DMH control group. Therefore, the findings of this study show that high hemin intake promotes mainly DNA damage and ACF development and but does not change the number nor incidence of colon tumors induced by 1,2-DMH in male rats.

Impact of gestational low protein diet and postnatal bisphenol A exposure on chemically induced mammary carcinogenesis in female offspring rats.

This study evaluated the effect of gestational low protein diet (LPD) and/or postnatal bisphenol A (BPA) exposure on mammary gland development and carcinogenesis in female offspring. Pregnant Sprague-Dawley rats were fed a normal protein diet (NPD, 17% protein) or LPD (6% protein). At weaning, female offspring were distributed in four groups (NPD, LPD, NPD + BPA, and LPD + BPA) and received vehicle or BPA in drinking water (0.1%), during postnatal day (PND) 21 to 51. On PND 51, some female offspring were euthanized or received a single dose of 7,12-dimethylbenzoanthracene (DMBA, 30 mg/kg, i.g.) and were euthanized on PND 250. On PND 51, neither gestational LPD nor postnatal BPA exposure, individually or in combination, significantly altered the development of mammary gland tree, mean number of terminal structures or estrogen receptor beta (ER-ß), proliferating cell nuclear antigen (PCNA) or caspase-3 protein expression in the mammary tissue. A significant reduction in mammary epithelial area (%) was observed in both LPD groups and a significant increase in ER-α protein expression was detected only in LPD group. In LPD + BPA group was observed a significant increase in both fat pad area (%) and in mean number of mammary epithelial cells positive for progesterone receptor (PR). On PND 250, the groups that received BPA presented lower latency and higher tumor incidence and tumor multiplicity and LPD + BPA group more aggressive tumors. These findings suggest that postnatal BPA exposure associated with gestational LPD is able to induce morphological changes in the mammary gland and increase susceptibility to mammary carcinogenesis.

Exposure to an Environmentally Relevant Phthalate Mixture During Prostate Development Induces MicroRNA Upregulation and Transcriptome Modulation in Rats.

Environmental exposure to phthalates during intrauterine development might increase susceptibility to neoplasms in reproductive organs such as the prostate. Although studies have suggested an increase in prostatic lesions in adult animals submitted to perinatal exposure to phthalates, the molecular pathways underlying these alterations remain unclear. Genome-wide levels of mRNAs and miRNAs were monitored with RNA-seq to determine if perinatal exposure to a phthalate mixture in pregnant rats is capable of modifying gene expression during prostate development of the filial generation. The mixture contains diethyl-phthalate, di-(2-ethylhexyl)-phthalate, dibutyl-phthalate, di-isononyl-phthalate, di-isobutyl-phthalate, and benzylbutyl-phthalate. Pregnant females were divided into 4 groups and orally dosed daily from GD10 to PND21 with corn oil (Control: C) or the phthalate mixture at 3 doses (20 µg/kg/day: T1; 200 µg/kg/day: T2; 200 mg/kg/day: T3). The phthalate mixture decreased anogenital distance, prostate weight, and decreased testosterone level at the lowest exposure dose at PND22. The mixture also increased inflammatory foci and focal hyperplasia incidence at PND120. miR-184 was upregulated in all treated groups in relation to control and miR-141-3p was only upregulated at the lowest dose. In addition, 120 genes were deregulated at the lowest dose with several of these genes related to developmental, differentiation, and oncogenesis. The data indicate that phthalate exposure at lower doses can cause greater gene expression modulation as well as other downstream phenotypes than exposure at higher doses. A significant fraction of the downregulated genes were predicted to be targets of miR-141-3p and miR-184, both of which were induced at the lower exposure doses.

Maternal western-style diet enhances the effects of chemically-induced mammary tumors in female rat offspring through transcriptome changes.

Previous studies have shown that early life intake of high-fat diet or western-style diet (WD) enhances the development of mammary tumors in adult female rats. Thus, we hypothesized that maternal WD throughout pregnancy and the lactation period could speed up the development of MNU-induced mammary tumors and alter their gene expression. For this, the present study investigated the gene expression profile of chemically-induced mammary tumors in female rat offspring from dams fed a WD or a control diet. Pregnant female Sprague-Dawley rats received a WD (high-fat, low-fiber and oligoelements) or a control diet from gestational day 12 until post-natal day (PND) 21. At PND 21, female offspring received a single dose of N-Methyl-N-Nitrosourea (MNU, 50 mg/kg body weight) and were fed a control diet for 13 weeks. Tumor incidence, multiplicity, and latency were recorded and mammary gland samples were collected for histopathology and gene expression analysis. Tumor multiplicity and histological grade were significantly higher and tumor latency was lower in WD offspring compared to control offspring. Transcriptome profiling identified 57 differentially expressed genes in tumors from WD offspring as compared to control offspring. There was also an increase in mRNA expression of genes such as Emp3, Ccl7, Ets1, Abcc5, and Cyr61, indicative of more aggressive disease detected in tumors from WD offspring. Thus, maternal WD diet increased MNU-induced mammary carcinogenesis in adult female offspring through transcriptome changes that resulted in a more aggressive disease.

Lyophilized açaí pulp (Euterpe oleracea Mart) attenuates colitis-associated colon carcinogenesis while its main anthocyanin has the potential to affect the motility of colon cancer cells.

This study evaluated the possible protective effects of lyophilized açaí pulp (AP) in a colitis-associated carcinogenesis (CAC) rat model and the modifying effect of cyanidin 3-rutinoside (C3R) on the motility of RKO colon adenocarcinoma cells, using the wound healing assay. Male Wistar rats were induced to develop CAC using 1,2-dimethylhydrazine (DMH) and 2,4,6-trinitrobenzene acid (TNBS). Animals were randomly assigned to different groups that received basal diet or basal diet supplemented with 5.0% or 7.5% lyophilized AP. The findings indicate: 1) C3R (25 µM) has the potential to reduce RKO cell motility in vitro; 2) ingestion of lyophilized AP reduces the total number of aberrant crypt foci (ACF), ACF multiplicity, tumor cell proliferation and incidence of tumors with high grade dysplasia; 3) AP increases the gene expression of negative regulators of cell proliferation such as Dlc1 and Akt3, as well as inflammation (Ppara). Thus, lyophilized AP could exert a potential antitumor activity.

Genetic ablation of pannexin1 counteracts liver fibrosis in a chemical, but not in a surgical mouse model.

Liver fibrosis is the final common pathway for almost all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components mainly due to transdifferentiation of quiescent hepatic stellate cell into myofibroblasts-like cells, which in turn is driven by cell death and inflammation. In the last few years, paracrine signaling through pannexin1 channels has emerged as a key player in the latter processes. The current study was set up to investigate the role of pannexin1 signaling in liver fibrosis. Wild-type and whole body pannexin1 knock-out mice were treated with carbon tetrachloride or subjected to bile duct ligation. Evaluation of the effects of pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, oxidative stress, inflammation and regenerative capacity. In parallel, to elucidate the molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. While pannexin1 knock-out mice treated with carbon tetrachloride displayed reduced collagen content, hepatic stellate cell activation, inflammation and hepatic regeneration, bile duct ligated counterparts showed increased hepatocellular injury and antioxidant enzyme activity with a predominant immune response. Gene expression profiling revealed a downregulation of fibrotic and immune responses in pannexin1 knock-out mice treated with carbon tetrachloride, whereas bile duct ligated pannexin1-deficient animals showed a pronounced inflammatory profile. This study shows for the first time an etiology-dependent role for pannexin1 signaling in experimental liver fibrosis.

TAT-Gap19 and Carbenoxolone Alleviate Liver Fibrosis in Mice.

Although a plethora of signaling pathways are known to drive the activation of hepatic stellate cells in liver fibrosis, the involvement of connexin-based communication in this process remains elusive. Connexin43 expression is enhanced in activated hepatic stellate cells and constitutes the molecular building stone of hemichannels and gap junctions. While gap junctions support intercellular communication, and hence the maintenance of liver homeostasis, hemichannels provide a circuit for extracellular communication and are typically opened by pathological stimuli, such as oxidative stress and inflammation. The present study was set up to investigate the effects of inhibition of connexin43-based hemichannels and gap junctions on liver fibrosis in mice. Liver fibrosis was induced by administration of thioacetamide to Balb/c mice for eight weeks. Thereafter, mice were treated for two weeks with TAT-Gap19, a specific connexin43 hemichannel inhibitor, or carbenoxolone, a general hemichannel and gap junction inhibitor. Subsequently, histopathological analysis was performed and markers of hepatic damage and functionality, oxidative stress, hepatic stellate cell activation and inflammation were evaluated. Connexin43 hemichannel specificity of TAT-Gap19 was confirmed in vitro by fluorescence recovery after photobleaching analysis and the measurement of extracellular release of adenosine-5'-triphosphate. Upon administration to animals, both TAT-Gap19 and carbenoxolone lowered the degree of liver fibrosis accompanied by superoxide dismutase overactivation and reduced production of inflammatory proteins, respectively. These results support a role of connexin-based signaling in the resolution of liver fibrosis, and simultaneously demonstrate the therapeutic potential of TAT-Gap19 and carbenoxolone in the treatment of this type of chronic liver disease.

Enhancement of colon carcinogenesis by the combination of indole-3 carbinol and synbiotics in hemin-fed rats.

The risk of developing colorectal cancer (CRC) could be associated with red and processed meat intake. Experimental data supports that hemin iron, found abundantly in red meat, promotes CRC in mice and rats, while indole-3 carbinol (I3C) and synbiotics (syn) exert anti-carcinogenic activities in most studies of colon carcinogenesis. This study aimed to investigate the modifying effects of I3C and syn (inulin + Bifidobacterium lactis), given separately or together, on dimethylhidrazine (DMH)-induced colon carcinogenesis in hemin-fed rats. All animals were given four subcutaneous DMH injections and then, two weeks after carcinogen exposure, they began a basal diet containing hemin, hemin + I3C, hemin + syn, or hemin + I3C + syn for 23 weeks. The combination of I3C + syn significantly increased fecal water genotoxicity, tumor volume and invasiveness when compared to the hemin-fed control group. The groups fed I3C or syn alone had a significant reduction in the number of preneoplastic aberrant crypt foci (ACF) lesions compared to the hemin-fed group. Dietary I3C also reduced fecal water genotoxicity. Gene expression analysis of colorectal tumors demonstrated that the combination of dietary I3C + syn increased transcript levels for Raf1 and decreased tumor progression and invasiveness related to the genes Cdh1 and Appl1. This analysis also revealed that the Tnf and Cdh1 genes were significantly up- and down-regulated, respectively, in tumors of rats that received I3C, in comparison with the hemin-fed group. These findings reveal that the joint administration of I3C and syn enhanced the development of colon tumors induced by DMH in hemin-fed rats, while they potentially reduced ACF development when given alone.

Maternal Resveratrol Treatment Reduces the Risk of Mammary Carcinogenesis in Female Offspring Prenatally Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) presents adverse effects on breast development/carcinogenesis. This study aimed to identify the ability of resveratrol (Res) to modify the adverse effects of TCDD in a female offspring. Pregnant female Wistar rats were allocated into four groups: TCDD, TCDD + Res, Res, and control. TCDD (1 µg/kg) was orally administered as a single dose on gestational day (GD) 15, and Res was orally administered during GD10-21 and lactation at a dose of 20 mg/kg/day. Female offsprings were euthanized on a specific postnatal day (PND) for hormonal analysis (PND 22, 48-51), vaginal opening (PND 30-48), and mammary gland morphology (PND 22). Other females received two doses of N-nitroso-N-methylurea (MNU, 50 mg/kg) on PNDs 22 and 51 and were euthanized on PND 24 (Ki-67, ER-α and apoptosis indexes or molecular analysis) or PND 180 (tumor assay). TCDD exposure altered the development of the mammary structure while these alterations were partially improved by maternal Res. Two days after first MNU administration, some genes associated with apoptosis were altered in the mammary tissue from the TCDD group (Bax and Caspase 3 down- and Bcl-2 upregulated) but were also partially reestablished by maternal Res. Mammary gland bcl-2 and bcl-xl proteins expression was increased while the apoptosis index was reduced by TCDD exposure but restored by maternal Res. An increase in number of mammary tumors was observed in female offspring from the TCDD group compared to the other groups. The results indicate that most mammary changes induced in female offspring through TCDD exposure or after MNU administrations were reduced by maternal resveratrol treatment.

Vitamin D3 supplementation attenuates the early stage of mouse hepatocarcinogenesis promoted by hexachlorobenzene fungicide.

Hexachlorobezene (HCB), a fungicide widely distributed in the environment, promotes the development of hepatocellular preneoplastic lesions (PNL) and tumors in rodents. In contrast, vitamin D3 (VD3) supplementation presents a potential role for the prevention/treatment of chronic liver diseases. Thus, we investigated whether VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis. Female Balb/C mice were injected a single dose of diethylnitrosamine (DEN, 50 mg/kg) at postnatal day 15. From day 40 onwards, mice were fed with a standard diet containing 0.02% HCB alone or supplemented with VD3 (10,000 or 20,000 IU/Kg diet) for 20 weeks. Untreated mice were fed just standard diet. After this period, mice were euthanized and liver and serum samples were collected. Compared to the untreated group, DEN/HCB treatment decreased total hepatic glutathione levels and glutathione peroxidase (GSH-Px) activity while increased lipid peroxidation, p65 protein expression, cell proliferation/apoptosis and the PNL development. In contrast, dietary VD3 supplementation enhanced vitamin D receptor (VDR) protein expression, total glutathione levels and GSH-Px activity while diminished lipid hydroperoxide levels. Also, VD3 supplementation decreased p65 protein expression, hepatocyte proliferation, the size and the liver area occupied by PNL. Therefore, our findings indicate that VD3 supplementation attenuates the early stage of HCB-promoted hepatocarcinogenesis.