Krüppel-like Factor 9 (KLF9) Suppresses Hepatocellular Carcinoma (HCC)-Promoting Oxidative Stress and Inflammation in Mice Fed High-Fat Diet.

Obesity, oxidative stress, and inflammation are risk factors for hepatocellular carcinoma (HCC). We examined, in mice, the effects of Krüppel-like factor 9 (KLF9) knockout on: adiposity, hepatic and systemic oxidative stress, and hepatic expression of pro-inflammatory and NOX/DUOX family genes, in a high-fat diet (HFD) context. Male and female Klf9+/+ (wild type, WT) and Klf9-/- (knockout, KO) mice were fed HFD (beginning at age 35 days) for 12 weeks, after which liver and adipose tissues were obtained, and serum adiponectin and leptin levels, liver fat content, and markers of oxidative stress evaluated. Klf9-/- mice of either sex did not exhibit significant alterations in weight gain, adipocyte size, adipokine levels, or liver fat content when compared to WT counterparts. However, Klf9-/- mice of both sexes had increased liver weight/size (hepatomegaly). This was accompanied by increased hepatic oxidative stress as indicated by decreased GSH/GSSG ratio and increased homocysteine, 3-nitrotyrosine, 3-chlorotyrosine, and 4HNE content. Decreased GSH to GSSG ratio and a trend toward increased homocysteine levels were observed in the corresponding Klf9-/- mouse serum. Gene expression analysis showed a heightened pro-inflammatory state in livers from Klf9-/- mice. KLF9 suppresses hepatic oxidative stress and inflammation, thus identifying potential mechanisms for KLF9 suppression of HCC and perhaps cancers of other tissues.

Lesion Genotype Modifies High-Fat Diet Effects on Endometriosis Development in Mice.

Endometriosis is a chronic, estrogen-dependent gynecologic disorder that affects reproductive-aged women and to a lesser extent, post-menopausal women on hormone therapy. The condition is associated with systemic and local immune dysfunctions. While its underlying mechanisms remain poorly understood, endometriosis has a genetic component and propensity for the disease is subject to environmental, nutritional, and lifestyle influences. Previously, we showed that high-fat diet (HFD) increased ectopic lesion numbers, concurrent with systemic and peritoneal changes in inflammatory and oxidative stress status, in immunocompetent recipient mice ip administered with endometrial fragments null for Krüppel-like factor 9 gene. Herein, we determined whether HFD modifies lesion parameters, when recipient peritoneal environment is challenged with ectopic wild-type (WT) endometrial fragments, the latter simulating retrograde menstruation common in women during the menstrual period. WT endometrium-recipient mice fed HFD (45% kcal from fat) showed reduced lesion incidence, numbers, and volumes, in the absence of changes in systemic ovarian steroid hormone and insulin levels, relative to those fed the control diet (CD, 17% kcal from fat). Lesions from HFD- and CD-fed recipients demonstrated comparable gene expression for steroid hormone receptors (Esr and Pgr) and cytokines (Il-6, Il-8, and CxCL4) and similar levels of DNA oxidative biomarkers. HFD moderately altered serum (3-nitrotyrosine and methionine/homocysteine) and peritoneal (reduced glutathione/oxidized glutathione) pro-oxidative status but had no effect on peritoneal inflammatory (tumor necrosis factor α and tumor necrosis factor receptor 1) mediators. Results indicate that lesion genotype modifies dietary effects on disease establishment and/or progression and if translated, could be important for provision of nutritional guidelines to women with predisposition to, or affected by endometriosis.

Expression Changes in Epigenetic Gene Pathways Associated With One-Carbon Nutritional Metabolites in Maternal Blood From Pregnancies Resulting in Autism and Non-Typical Neurodevelopment.

The prenatal period is a critical window for the development of autism spectrum disorder (ASD). The relationship between prenatal nutrients and gestational gene expression in mothers of children later diagnosed with ASD or non-typical development (Non-TD) is poorly understood. Maternal blood collected prospectively during pregnancy provides insights into the effects of nutrition, particularly one-carbon metabolites, on gene pathways and neurodevelopment. Genome-wide transcriptomes were measured with microarrays in 300 maternal blood samples in Markers of Autism Risk in Babies-Learning Early Signs. Sixteen different one-carbon metabolites, including folic acid, betaine, 5'-methyltretrahydrofolate (5-MeTHF), and dimethylglycine (DMG) were measured. Differential expression analysis and weighted gene correlation network analysis (WGCNA) were used to compare gene expression between children later diagnosed as typical development (TD), Non-TD and ASD, and to one-carbon metabolites. Using differential gene expression analysis, six transcripts (TGR-AS1, SQSTM1, HLA-C, and RFESD) were associated with child outcomes (ASD, Non-TD, and TD) with genome-wide significance. Genes nominally differentially expressed between ASD and TD significantly overlapped with seven high confidence ASD genes. WGCNA identified co-expressed gene modules significantly correlated with 5-MeTHF, folic acid, DMG, and betaine. A module enriched in DNA methylation functions showed a suggestive protective association with folic acid/5-MeTHF concentrations and ASD risk. Maternal plasma betaine and DMG concentrations were associated with a block of co-expressed genes enriched for adaptive immune, histone modification, and RNA processing functions. These results suggest that the prenatal maternal blood transcriptome is a sensitive indicator of gestational one-carbon metabolite status and changes relevant to children's later neurodevelopmental outcomes. LAY SUMMARY: Pregnancy is a time when maternal nutrition could interact with genetic risk for autism spectrum disorder. Blood samples collected during pregnancy from mothers who had a prior child with autism were examined for gene expression and nutrient metabolites, then compared to the diagnosis of the child at age three. Expression differences in gene pathways related to the immune system and gene regulation were observed for pregnancies of children with autism and non-typical neurodevelopment and were associated with maternal nutrients.

Sex-Specific Effects of Dietary Methionine Restriction on the Intestinal Microbiome.

Dietary methionine restriction is associated with improved health outcomes and an increase in lifespan in animal models. We have previously shown that an increase in dietary methionine induces alteration in the intestinal microbiome. The composition of the intestinal microbiota is a determinant of health and we, therefore, hypothesized that dietary methionine restriction would also induce changes in the murine microbiome. After one month on a methionine-restricted diet, five-month-old male and female C57BL/6 mice had decreased levels of serum methionine, without changes in body weight. We identified a decrease in the hepatic methylation status of animals fed a methionine-restricted diet compared to controls. This decrease was not associated with changes in DNA or protein methylation in the liver. In males, we saw an increase in families Bacteroidaceae and Verrucoccaceae (mostly A. mucinophila) and a decrease in Rumminococcaceae in animals fed a methionine-restricted diet compared to controls. In females, Bacteroidales family S24-7 was increased two-fold, while families Bacteroidaceae, Verrucoccaceae, Rumminococcaceae, and Rikenellaceae were decreased compared to controls. In summary, feeding a methionine-restricted diet for one month was associated with significant and sex-specific changes in the intestinal microbiome.

Complex epigenetic patterns in cerebellum generated after developmental exposure to trichloroethylene and/or high fat diet in autoimmune-prone mice.

Trichloroethylene (TCE) is an environmental contaminant associated with immune-mediated inflammatory disorders and neurotoxicity. Based on known negative effects of developmental overnutrition on neurodevelopment, we hypothesized that developmental exposure to high fat diet (HFD) consisting of 40% kcal fat would enhance neurotoxicity of low-level (6 µg per kg per day) TCE exposure in offspring over either stressor alone. Male offspring were evaluated at ∼6 weeks of age after exposure beginning 4 weeks preconception in the dams until weaning. TCE, whether used as a single exposure or together with HFD, appeared to be more robust than HFD alone in altering one-carbon metabolites involved in glutathione redox homeostasis and methylation capacity. In contrast, opposing effects of expression of key enzymes related to DNA methylation related to HFD and TCE exposure were observed. The mice generated unique patterns of anti-brain antibodies detected by western blotting attributable to both TCE and HFD. Taken together, developmental exposure to TCE and/or HFD appear to act in complex ways to alter brain biomarkers in offspring.

C/EBPδ protects from radiation-induced intestinal injury and sepsis by suppression of inflammatory and nitrosative stress.

Ionizing radiation (IR)-induced intestinal damage is characterized by a loss of intestinal crypt cells, intestinal barrier disruption and translocation of intestinal microflora resulting in sepsis-mediated lethality. We have shown that mice lacking C/EBPδ display IR-induced intestinal and hematopoietic injury and lethality. The purpose of this study was to investigate whether increased IR-induced inflammatory, oxidative and nitrosative stress promote intestinal injury and sepsis-mediated lethality in Cebpd-/- mice. We found that irradiated Cebpd-/- mice show decreased villous height, crypt depth, crypt to villi ratio and expression of the proliferation marker, proliferating cell nuclear antigen, indicative of intestinal injury. Cebpd-/- mice show increased expression of the pro-inflammatory cytokines (Il-6, Tnf-α) and chemokines (Cxcl1, Mcp-1, Mif-1α) and Nos2 in the intestinal tissues compared to Cebpd+/+ mice after exposure to TBI. Cebpd-/- mice show decreased GSH/GSSG ratio, increased S-nitrosoglutathione and 3-nitrotyrosine in the intestine indicative of basal oxidative and nitrosative stress, which was exacerbated by IR. Irradiated Cebpd-deficient mice showed upregulation of Claudin-2 that correlated with increased intestinal permeability, presence of plasma endotoxin and bacterial translocation to the liver. Overall these results uncover a novel role for C/EBPδ in protection against IR-induced intestinal injury by suppressing inflammation and nitrosative stress and underlying sepsis-induced lethality.

Modulation of dietary methionine intake elicits potent, yet distinct, anticancer effects on primary versus metastatic tumors.

Methionine dependency describes the characteristic rapid in vitro death of most tumor cells in the absence of methionine. Combining chemotherapy with dietary methionine deprivation [methionine-deficient diet (MDD)] at tolerable levels has vast potential in tumor treatment; however, it is limited by MDD-induced toxicity during extended deprivation. Recent advances in imaging and irradiation delivery have created the field of stereotactic body radiotherapy (SBRT), where fewer large-dose fractions delivered in less time result in increased local-tumor control, which could be maximally synergistic with an MDD short course. Identification of the lowest effective methionine dietary intake not associated with toxicity will further enhance the cancer therapy potential. In this study, we investigated the effects of MDD and methionine-restricted diet (MRD) in primary and metastatic melanoma models in combination with radiotherapy (RT). In vitro, MDD dose-dependently sensitized mouse and human melanoma cell lines to RT. In vivo in mice, MDD substantially potentiated the effects of RT by a significant delay in tumor growth, in comparison with administering MDD or RT alone. The antitumor effects of an MDD/RT approach were due to effects on one-carbon metabolism, resulting in impaired methionine biotransformation via downregulation of Mat2a, which encodes methionine adenosyltransferase 2A. Furthermore, and probably most importantly, MDD and MRD substantially diminished metastatic potential; the antitumor MRD effects were not associated with toxicity to normal tissue. Our findings suggest that modulation of methionine intake holds substantial promise for use with short-course SBRT for cancer treatment.

Cebpd Is Essential for Gamma-Tocotrienol Mediated Protection against Radiation-Induced Hematopoietic and Intestinal Injury.

Gamma-tocotrienol (GT3) confers protection against ionizing radiation (IR)-induced injury. However, the molecular targets that underlie the protective functions of GT3 are not yet known. We have reported that mice lacking CCAAT enhancer binding protein delta (Cebpd-/-) display increased mortality to IR due to injury to the hematopoietic and intestinal tissues and that Cebpd protects from IR-induced oxidative stress and cell death. The purpose of this study was to investigate whether Cebpd mediates the radio protective functions of GT3. We found that GT3-treated Cebpd-/- mice showed partial recovery of white blood cells compared to GT3-treated Cebpd⁺/+ mice at 2 weeks post-IR. GT3-treated Cebpd-/- mice showed an increased loss of intestinal crypt colonies, which correlated with increased expression of inflammatory cytokines and chemokines, increased levels of oxidized glutathione (GSSG), S-nitrosoglutathione (GSNO) and 3-nitrotyrosine (3-NT) after exposure to IR compared to GT3-treated Cebpd+/+ mice. Cebpd is induced by IR as well as a combination of IR and GT3 in the intestine. Studies have shown that granulocyte-colony stimulating factor (G-CSF), mediates the radioprotective functions of GT3. Interestingly, we found that IR alone as well as the combination of IR and GT3 caused robust augmentation of plasma G-CSF in both Cebpd⁺/+ and Cebpd-/- mice. These results identify a novel role for Cebpd in GT3-mediated protection against IR-induced injury, in part via modulation of IR-induced inflammation and oxidative/nitrosative stress, which is independent of G-CSF.

Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes.

The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.

Inflammatory and oxidative stress-related effects associated with neurotoxicity are maintained after exclusively prenatal trichloroethylene exposure.

Trichloroethylene (TCE) is a widespread environmental toxicant with immunotoxic and neurotoxic potential. Previous studies have shown that continuous developmental exposure to TCE encompassing gestation and early life as well as postnatal only exposure in the drinking water of MRL+/+ mice promoted CD4+ T cell immunotoxicity, glutathione depletion and oxidative stress in the cerebellum, as well increased locomotor activity in male offspring. The purpose of this study was to characterize the effects of exclusively prenatal exposure on these parameters. Another goal was to investigate potential plasma oxidative stress/inflammatory biomarkers to possibly be used as predictors of TCE-mediated neurotoxicity. In the current study, 6 week old male offspring of dams exposed gestationally to 0, 0.01, and 0.1mg/ml TCE in the drinking water were evaluated. Our results confirmed that the oxidized phenotype in plasma and cerebellum was maintained after exclusively prenatal exposure. A Phenotypic analysis by flow cytometry revealed that TCE exposure expanded the effector/memory subset of peripheral CD4+ T cells in association with increased production of pro-inflammatory cytokines IFN-γ and IL-17. Serum biomarkers of oxidative stress and inflammation were also elevated in plasma suggesting that systemic effects are important and may be used to predict neurotoxicity in our model. These results suggested that the prenatal period is a critical stage of life by which the developing CNS and immune system are susceptible to long-lasting changes mediated by TCE.

Loss of C/EBPδ enhances IR-induced cell death by promoting oxidative stress and mitochondrial dysfunction.

Exposure of cells to ionizing radiation (IR) generates reactive oxygen species (ROS). This results in increased oxidative stress and DNA double strand breaks (DSBs) which are the two underlying mechanisms by which IR causes cell/tissue injury. Cells that are deficient or impaired in the cellular antioxidant response are susceptible to IR-induced apoptosis. The transcription factor CCAAT enhancer binding protein delta (Cebpd, C/EBPδ) has been implicated in the regulation of oxidative stress, DNA damage response, genomic stability and inflammation. We previously reported that Cebpd-deficient mice are sensitive to IR and display intestinal and hematopoietic injury, however the underlying mechanism is not known. In this study, we investigated whether an impaired ability to detoxify IR-induced ROS was the underlying cause of the increased radiosensitivity of Cebpd-deficient cells. We found that Cebpd-knockout (KO) mouse embryonic fibroblasts (MEFs) expressed elevated levels of ROS, both at basal levels and after exposure to gamma radiation which correlated with increased apoptosis, and decreased clonogenic survival. Pre-treatment of wild type (WT) and KO MEFs with polyethylene glycol-conjugated Cu-Zn superoxide dismutase (PEG-SOD) and catalase (PEG-CAT) combination prior to irradiation showed a partial rescue of clonogenic survival, thus demonstrating a role for increased intracellular oxidants in promoting IR-induced cell death. Analysis of mitochondrial bioenergetics revealed that irradiated KO MEFs showed significant reductions in basal, adenosine triphosphate (ATP)-linked, maximal respiration and reserved respiratory capacity and decrease in intracellular ATP levels compared to WT MEFs indicating they display mitochondrial dysfunction. KO MEFs expressed significantly lower levels of the cellular antioxidant glutathione (GSH) and its precursor- cysteine as well as methionine. In addition to its antioxidant function, GSH plays an important role in detoxification of lipid peroxidation products such as 4-hydroxynonenal (4-HNE). The reduced GSH levels observed in KO MEFs correlated with elevated levels of 4-HNE protein adducts in irradiated KO MEFs compared to respective WT MEFs. We further showed that pre-treatment with the GSH precursor, N-acetyl L-cysteine (NAC) prior to irradiation showed a significant reduction of IR-induced cell death and increases in GSH levels, which contributed to the overall increase in clonogenic survival of KO MEFs. In contrast, pre-treatment with the GSH synthesis inhibitor- buthionine sulfoximine (BSO) further reduced the clonogenic survival of irradiated KO MEFs. This study demonstrates a novel role for C/EBPδ in protection from basal as well as IR-induced oxidative stress and mitochondrial dysfunction thus promoting post-radiation survival.

Metabolic history impacts mammary tumor epithelial hierarchy and early drug response in mice.

The emerging links between breast cancer and metabolic dysfunctions brought forth by the obesity pandemic predict a disproportionate early disease onset in successive generations. Moreover, sensitivity to chemotherapeutic agents may be influenced by the patient's metabolic status that affects the disease outcome. Maternal metabolic stress as a determinant of drug response in progeny is not well defined. Here, we evaluated mammary tumor response to doxorubicin in female mouse mammary tumor virus-Wnt1 transgenic offspring exposed to a metabolically compromised environment imposed by maternal high-fat diet. Control progeny were from dams consuming diets with regular fat content. Maternal high-fat diet exposure increased tumor incidence and reduced tumor latency but did not affect tumor volume response to doxorubicin, compared with control diet exposure. However, doxorubicin-treated tumors from high-fat-diet-exposed offspring demonstrated higher proliferation status (Ki-67), mammary stem cell-associated gene expression (Notch1, Aldh1) and basal stem cell-like (CD29(hi)CD24(+)) epithelial subpopulation frequencies, than tumors from control diet progeny. Notably, all epithelial subpopulations (CD29(hi)CD24(+), CD29(lo)CD24(+), CD29(hi)CD24(+)Thy1(+)) in tumors from high-fat-diet-exposed offspring were refractory to doxorubicin. Further, sera from high-fat-diet-exposed offspring promoted sphere formation of mouse mammary tumor epithelial cells and of human MCF7 cells. Untargeted metabolomics analyses identified higher levels of kynurenine and 2-hydroxyglutarate in plasma of high-fat diet than control diet offspring. Kynurenine/doxorubicin co-treatment of MCF7 cells enhanced the ability to form mammosphere and decreased apoptosis, relative to doxorubicin-only-treated cells. Maternal metabolic dysfunctions during pregnancy and lactation may be targeted to reduce breast cancer risk and improve early drug response in progeny, and may inform clinical management of disease.

High-Fat Diet Promotion of Endometriosis in an Immunocompetent Mouse Model is Associated With Altered Peripheral and Ectopic Lesion Redox and Inflammatory Status.

Endometriosis is a benign gynecological condition that causes considerable morbidity due to associated infertility, debilitating pelvic pain and inflammatory dysfunctions. Diet is a highly modifiable risk factor for many chronic diseases, but its contribution to endometriosis has not been extensively investigated, due partly to the paradoxical inverse association between obesity and disease incidence. Nevertheless, chronic exposure to dietary high-fat intake has been linked to greater systemic inflammation and oxidative stress, both features of women with endometriosis. Here, we evaluated the effects of a high-fat diet (HFD) (45% fat kcal) on endometriosis progression using an immunocompetent mouse model where ectopic lesion incidence was induced in wild-type recipients by ip administration of endometrial fragments from transcription factor Krüppel-like factor 9-null donor mice. We show that HFD significantly increased ectopic lesion numbers in recipient mice with no significant weight gain and modifications in systemic ovarian steroid hormone and insulin levels, relative to control diet-fed (17% fat kcal) mice. HFD promotion of lesion establishment was associated with reductions in stromal estrogen receptor 1 isoform and progesterone receptor expression, increased F4/80-positive macrophage infiltration, higher stromal but not glandular epithelial proliferation, and enhanced expression of proinflammatory and prooxidative stress pathway genes. Lesion-bearing HFD-fed mice also displayed higher peritoneal fluid TNFα and elevated local and systemic redox status than control diet-fed counterparts. Our results suggest that HFD intake exacerbates endometriosis outcome in the absence of ovarian dysfunction and insulin resistance in mice and warrants further consideration with respect to clinical management of endometriosis progression and recurrence in nonobese patients.

Effects of local irradiation combined with sunitinib on early remodeling, mitochondria, and oxidative stress in the rat heart.

BACKGROUND AND PURPOSE: Thoracic (chemo)radiation therapy is increasingly administered with tyrosine kinase inhibitors (TKI). While TKI have adverse effects on the heart, it is unknown whether combination with other cancer therapies causes enhanced toxicity. We used an animal model to investigate whether radiation and sunitinib interact in their effects on the heart. MATERIAL AND METHODS: Male Sprague-Dawley rats received local heart irradiation (9Gy per day, 5days). Oral sunitinib (8 or 15mg/kg bodyweight per day) started on day 1 of irradiation and continued for 2weeks. Cardiac function was examined with echocardiography. Cardiac remodeling, cell death, left ventricular (LV) oxidative stress markers, mitochondrial morphology and mitochondrial permeability transition pore (mPTP) opening were assessed. RESULTS: Cardiac diameter, stroke volume, and LV volume, mass and anterior wall thickness increased in time, but only in the vehicle group. Sunitinib reduced LV inner diameter and volume in systole, which were counteracted by radiation. Sunitinib and radiation showed enhanced effects on mitochondrial morphology and mPTP opening, but not on cardiac troponin I, mast cell numbers or markers of oxidative stress. CONCLUSIONS: This study found no early enhanced effects of radiation and sunitinib on cardiac function or structure. Long-term effects remain to be determined.

Radiation-induced changes in DNA methylation of repetitive elements in the mouse heart.

DNA methylation is a key epigenetic mechanism, needed for proper control over the expression of genetic information and silencing of repetitive elements. Exposure to ionizing radiation, aside from its strong genotoxic potential, may also affect the methylation of DNA, within the repetitive elements, in particular. In this study, we exposed C57BL/6J male mice to low absorbed mean doses of two types of space radiation-proton (0.1 Gy, 150 MeV, dose rate 0.53 ± 0.08 Gy/min), and heavy iron ions ((56)Fe) (0.5 Gy, 600 MeV/n, dose rate 0.38 ± 0.06 Gy/min). Radiation-induced changes in cardiac DNA methylation associated with repetitive elements were detected. Specifically, modest hypomethylation of retrotransposon LINE-1 was observed at day 7 after irradiation with either protons or (56)Fe. This was followed by LINE-1, and other retrotransposons, ERV2 and SINE B1, as well as major satellite DNA hypermethylation at day 90 after irradiation with (56)Fe. These changes in DNA methylation were accompanied by alterations in the expression of DNA methylation machinery and affected the one-carbon metabolism pathway. Furthermore, loss of transposable elements expression was detected in the cardiac tissue at the 90-day time-point, paralleled by substantial accumulation of mRNA transcripts, associated with major satellites. Given that the one-carbon metabolism pathway can be modulated by dietary modifications, these findings suggest a potential strategy for the mitigation and, possibly, prevention of the negative effects exerted by ionizing radiation on the cardiovascular system. Additionally, we show that the methylation status and expression of repetitive elements may serve as early biomarkers of exposure to space radiation.

The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

The Contribution of Peroxisome Proliferator-Activated Receptor Alpha to the Relationship Between Toxicokinetics and Toxicodynamics of Trichloroethylene.

Exposure to the ubiquitous environmental contaminant trichloroethylene (TCE) is associated with cancer and non-cancer toxicity in both humans and rodents. Peroxisome proliferator-activated receptor-alpha (PPARα) is thought to be playing a role in liver toxicity in rodents through activation of the receptor by the TCE metabolite trichloroacetic acid (TCA). However, most studies using genetically altered mice have not assessed the potential for PPARα to alter TCE toxicokinetics, which may lead to differences in TCA internal doses and hence confound inferences as to the role of PPARα in TCE toxicity. To address this gap, male and female wild type (129S1/SvImJ), Pparα-null, and humanized PPARα (hPPARα) mice were exposed intragastrically to 400 mg/kg TCE in single-dose (2, 5 and 12 h) and repeat-dose (5 days/week, 4 weeks) studies. Interestingly, following either a single- or repeat-dose exposure to TCE, levels of TCA in liver and kidney were lower in Pparα-null and hPPARα mice as compared with those in wild type mice. Levels of trichloroethanol (TCOH) were similar in all strains. TCE-exposed male mice consistently had higher levels of TCA and TCOH in all tissues compared with females. Additionally, in both single- and repeat-dose studies, a similar degree of induction of PPARα-responsive genes was observed in liver and kidney of hPPARα and wild type mice, despite the difference in hepatic and renal TCA levels. Additional sex- and strain-dependent effects were observed in the liver, including hepatocyte proliferation and oxidative stress, which were not dependent on TCA or TCOH levels. These data demonstrate that PPARα status affects the levels of the putative PPARα agonist TCA following TCE exposure. Therefore, interpretations of studies using Pparα-null and hPPARα mice need to consider the potential contribution of genotype-dependent toxicokinetics to observed differences in toxicity, rather than attributing such differences only to receptor-mediated toxicodynamic effects.

A tocotrienol-enriched formulation protects against radiation-induced changes in cardiac mitochondria without modifying late cardiac function or structure.

Radiation-induced heart disease (RIHD) is a common and sometimes severe late side effect of radiation therapy for intrathoracic and chest wall tumors. We have previously shown that local heart irradiation in a rat model caused prolonged changes in mitochondrial respiration and increased susceptibility to mitochondrial permeability transition pore (mPTP) opening. Because tocotrienols are known to protect against oxidative stress-induced mitochondrial dysfunction, in this study, we examined the effects of tocotrienols on radiation-induced alterations in mitochondria, and structural and functional manifestations of RIHD. Male Sprague-Dawley rats received image-guided localized X irradiation to the heart to a total dose of 21 Gy. Twenty-four hours before irradiation, rats received a tocotrienol-enriched formulation or vehicle by oral gavage. Mitochondrial function and mitochondrial membrane parameters were studied at 2 weeks and 28 weeks after irradiation. In addition, cardiac function and histology were examined at 28 weeks. A single oral dose of the tocotrienol-enriched formulation preserved Bax/Bcl2 ratios and prevented mPTP opening and radiation-induced alterations in succinate-driven mitochondrial respiration. Nevertheless, the late effects of local heart irradiation pertaining to myocardial function and structure were not modified. Our studies suggest that a single dose of tocotrienols protects against radiation-induced mitochondrial changes, but these effects are not sufficient against long-term alterations in cardiac function or remodeling.

Maternal metabolic perturbations elicited by high-fat diet promote Wnt-1-induced mammary tumor risk in adult female offspring via long-term effects on mammary and systemic phenotypes.

Many adult chronic diseases are thought to be influenced during early life by maternal nutrition; however, the underlying mechanisms remain largely unknown. Obesity-related diseases may be due partly to high fat consumption. Herein, we evaluated mammary tumor risk in female mouse mammary tumor virus-Wnt-1 transgenic (Tg) offspring exposed to high-fat diet (HFD) or control diet (CD) (45% and 17% kcal from fat, respectively) during gestation and lactation, with CD provided to progeny at weaning. In Tg offspring, maternal HFD exposure increased mammary tumor incidence and decreased tumor latency without affecting tumor volume. Tumor risk was associated with higher tumor necrosis factor-α and insulin and altered oxidative stress biomarkers in sera and with early changes in mammary expression of genes linked to tumor promotion [interleukin 6 (Il6)] or inhibition [phosphatase and tensin homolog deleted on chromosome 10 (Pten), B-cell lymphoma 2 (Bcl2)]. Corresponding wild-type progeny exposed to maternal HFD displayed accelerated mammary development, higher mammary adiposity, increased insulin resistance and early changes in Pten, Bcl2 and Il6, than CD-exposed offspring. Dams-fed HFD showed higher serum glucose and oxidative stress biomarkers but comparable adiposity compared with CD-fed counterparts. In human breast cancer MCF-7 cells, sera from maternal HFD-exposed Tg offspring elicited changes in PTEN, BCL2 and IL6 gene expression, mimicking in vivo exposure; increased cell viability and mammosphere formation and induced measures [insulin receptor substrate-1 (IRS-1), IRS-2] of insulin sensitivity. Serum effects on IRS-1 were recapitulated by exogenous insulin and the PTEN-specific inhibitor SF1670. Hyperinsulinemia and PTEN loss-of-function may thus, couple maternal HFD exposure to enhanced insulin sensitivity via increased mammary IRS-1 expression in progeny, to promote breast cancer risk.