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.

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.

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.

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.

Paternal selenium deficiency but not supplementation during preconception alters mammary gland development and 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis in female rat offspring.

Breast cancer is a global public health problem and accumulating evidence indicates early-life exposures as relevant factors in the disease risk determination. Recent studies have shown that paternal nutrition can influence offspring health including breast cancer risk. Selenium is a micronutrient with essential role in central aspects of embryogenesis, male fertility and cancer and that has been extensively studied as a chemopreventive agent in several breast cancer experimental models. Thus, we designed an animal study to evaluate whether paternal selenium deficiency or supplementation during preconception could affect the female offspring mammary gland development and breast cancer susceptibility. Male Sprague-Dawley rats were fed AIN93-G diet containing 0.15 ppm (control diet), 0.05 ppm (deficient diet) or 1 ppm (supplemented diet) of selenium for 9 weeks and mated with control female rats. Mammary carcinogenesis was induced with 7,12-dimethylbenz[a]anthracene (DMBA) in their female offspring. Paternal selenium deficiency increased the number of terminal end buds, epithelial elongation and cell proliferation in the mammary gland of the female rat offspring and these effects were associated with higher susceptibility to DMBA-induced mammary tumors (increased incidence and higher grade tumors). On the other hand, paternal selenium supplementation did not influence any of these parameters. These results highlight the importance of father's nutrition including selenium status as a relevant factor affecting daughter's breast cancer risk and paternal preconception as a potential developmental stage to start disease preventive strategies.

Maternal western style diet increases susceptibility to chemically-induced mammary carcinogenesis in female rats offspring.

The present study investigated whether maternal exposure to western style diet (WD) increases susceptibility to mammary carcinogenesis induced by N-methyl-N-nitrosourea (MNU) in female offspring. Pregnant female Sprague-Dawley rats received WD diet or control diet from gestational day 12 until postnatal day (PND) 21. At PND 21, female offspring received a single dose of MNU (50 mg/kg body weight) and were fed chow diet until PND 110. Mammary gland structures were assessed on whole-mount preparations in the offspring at PND 21, and tumor morphology was examined at PND 110. Immunohistochemical analysis for cell proliferation (PCNA), apoptosis (cleaved caspase-3) and estrogen receptor alpha (ER-α) was performed in mammary terminal end buds (TEBs) at PND 21, and PCNA, ER-α, and p63 analysis in mammary tumors at PND 110. Maternal WD intake induced a significant increase in the number of TEBs (P = 0.024) and in PCNA labeling index (P < 0.020) in the mammary glands at PND 21. Tumor multiplicity, tumor weight, and PCNA labeling indexes were significantly higher in the WD offspring than that of the control offspring (P < 0.05). These findings indicate that maternal western style diet potentially enhanced the development of mammary tumors induced by MNU in female offspring, possibly by affecting the mammary gland differentiation.

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.

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.

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.

Effects of diuron on male rat reproductive organs: a developmental and postnatal study.

This study was performed to determine whether developmental exposure (perinatal and juvenile) to the herbicide diuron exerted adverse effects on adult rat male reproductive system. Pregnant Sprague-Dawley rats received basal diet or diet containing diuron at 500 or 750 ppm from gestational day 12 (GD 12) until the end of lactation period (postnatal day 21, PND 21). After weaning male offspring received basal diet or diet containing diuron until PND 42 (peripubertal age). At PND 90, adult male rats from each experimental group were anesthetized and euthanized for evaluation of body and reproductive organ weights, sperm parameters, plasma testosterone levels, and testicular and epididymal histopathology. Male offspring exposed to diuron at 750 ppm displayed reduced body weight at PND 10, 21, 42, and 90 compared to controls. At PND 90, diuron treatment did not induce significant change in daily sperm production, sperm morphology and motility, and testosterone levels compared to controls. In conclusion, diuron at 750 ppm induced male offspring toxicity but these alterations were not permanent, as evidenced by absence of reproductive-system alterations in adult Sprague Dawley rats.

Developmental exposure to diuron causes splenotoxicity in male Sprague-Dawley rat pups.

This study investigated whether perinatal exposure to diuron [3-(3,4-dichlorophenyl)-1-1-dimethylurea] might exert adverse effects on rat lymphoid organs. Pregnant Sprague-Dawley (SD) rats were exposed to diuron at 500, 750 or 1250 ppm in the diet from gestational days (GD) 12-21 and during lactation. At postnatal day (PND) 42, male pups were euthanized and thymus, spleen, mesenteric lymph node and femur were collected for histopathological analysis. Food consumption and body weight gain were significantly reduced in dams exposed to 1250 ppm during gestation period. Also, Diuron at 750 and 1250 ppm produced: (1) increased relative spleen weight associated histologically with severe congestion in red pulp, (2) enhanced extramedullary hematopoiesis and hemosiderosis as well as (3) depletion of lymphoid follicles in white pulp. Flow cytometric analysis revealed a significant reduction in B lymphocytes (CD45RA+) in male pups but T lymphocytes (CD4+, CD8+ and CD4+/CD8+) were not markedly affected. Thus, data suggest that Diuron-induced maternal toxicity in dams exposed to high dose and perinatal exposure to this herbicide produced spleen toxicity as evidenced by a reduction in B lymphocyte number in male SD pups.

Inhibition of mouse urinary bladder carcinogenesis by açai fruit (Euterpe oleraceae Martius) intake.

Açai, fruit from Euterpe oleraceae Martius, is consumed in natura and in a variety of beverages and food preparations and possesses several potential antioxidant compounds. In a first study for anticarcinogenicity screening, male Swiss mice (n = 20/per group) were chemically-induced to urothelial bladder carcinogenesis for 10 weeks and received a standard diet or a standard diet containing 2.5 and 5 % spray-dried açai pulp (AP) for 10 weeks. At week 20, the incidence of simple and nodular hyperplasia and the incidence and multiplicity of transitional cell carcinoma (TCC) were evaluated. In a second study for antigenotoxicity screening, male Swiss mice (n = 6/per group) were fed standard diet or standard diet containing 5 % AP for three weeks. Urothelial cell suspensions were obtained and challenged with H(2)O(2) for induction of DNA damage and analyzed by comet assay. Overall, dietary 5 % AP reduced TCC incidence and multiplicity (p = 0.019 and p = 0.015, respectively) and tumor cell proliferation and p63 expression (p = 0.02 and p = 0.007, respectively), Furthermore, the group fed the 5 % AP presented a significant reduction (p < 0.01) in DNA damage induced by H(2)O(2), a notable oxidant agent. The results suggest that the spray-dried açai pulp used here inhibits the TCC development in male Swiss mice, probably due to its potential antioxidant action.

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.

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.

Protective effects of lemongrass (Cymbopogon citratus STAPF) essential oil on DNA damage and carcinogenesis in female Balb/C mice.

This study investigated the protective effect of oral treatment with lemongrass (Cymbopogon citratus STAPF) essential oil (LGEO) on leukocyte DNA damage induced by N-methyl-N-nitrosurea (MNU). Also, the anticarcinogenic activity of LGEO was investigated in a multi-organ carcinogenesis bioassay induced by 7,12-dimethylbenz(a)antracene, 1,2-dimethylhydrazine and N-butyl-N-(4-hydroxibuthyl)nitrosamine in Balb/C female Balb/c mice (DDB-initiated mice). In the short-term study, the animals were allocated into three groups: vehicle group (negative control), MNU group (positive control) and LGEO 500 mg kg⁻¹ (five times per week for 5 weeks) plus MNU group (test group). Blood samples were collected to analyze leukocyte DNA damage by comet assay 4 h after each MNU application at the end of weeks 3 and 5. The LGEO 500 mg kg⁻¹ treated group showed significantly lower (P < 0.01) leukocyte DNA damage than its respective positive group exposed to MNU alone at week 3. In the medium-term study, DDB-initiated mice were allocated into three groups: vehicle group (positive control) and LGEO 125 or 500 mg kg⁻¹ (five times per week for 6 weeks; test groups). At week 20, all animals were euthanized and mammary glands, colon and urinary bladder were processed for histopathological analyses for detection of preneoplastic and neoplastic lesions. A slight non-significant effect of treatment with LGEO 500 mg kg⁻¹ in reducing development of alveolar and ductal mammary hyperplasia was found (P = 0.075). Our findings indicate that lemongrass essential oil provided protective action against MNU-induced DNA damage and a potential anticarcinogenic activity against mammary carcinogenesis in DDB-initiated female Balb/C mice.

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.

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.