Nox4 expression in osteo-progenitors controls bone development in mice during early life.

Tightly regulated and cell-specific NADPH-oxidases (Nox) represent one of the major sources of reactive oxygen species (ROS) signaling molecules that are involved in tissue development and stem cell self-renewal. We have characterized the role of Nox4 in osteo-progenitors during postnatal bone development. Nox4 expression in bone and ROS generation were increased during early osteoblast differentiation and bone development. Stromal osteoblastic cell self-renewal, proliferation and ROS production were significantly lower in samples from whole-body Nox4 knockout mice (Nox4-/-) and conditional knockout (CKO) mice with depletion of Nox4 in the limb bud mesenchyme compared with those from control mice (Nox4fl/fl), but they were reversed after 9 passages. In both sexes, bone volume, trabecular number and bone mineral density were significantly lower in 3-week old CKO and Nox4-/- mice compared with Nox4fl/fl controls. This was reflected in serum levels of bone formation markers alkaline phosphatase (ALP) and procollagen 1 intact N-terminal propeptide (P1NP). However, under-developed bone formation in 3-week old CKO and Nox4-/- mice quickly caught up to levels of control mice by 6-week of age, remained no different at 13-week of age, and was reversed in 32-week old male mice. Osteoclastogenesis showed no differences among groups, however, CTX1 reflecting osteoclast activity was significantly higher in 3-week old male CKO and Nox4-/- mice compared with control mice, and significantly lower in 32-week old Nox4-/- mice compared with control mice. These data suggest that Nox4 expression and ROS signaling in bone and osteoblastic cells coordinately play an important role in osteoblast differentiation, proliferation and maturation.

Soy Formula Is Not Estrogenic and Does Not Result in Reproductive Toxicity in Male Piglets: Results from a Controlled Feeding Study.

Soy infant formula which is fed to over half a million infants per year contains isoflavones such as genistein, which have been shown to be estrogenic at high concentrations. The developing testis is sensitive to estrogens, raising concern that the use of soy formulas may result in male reproductive toxicity. In the current study, male White-Dutch Landrace piglets received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/kg/d) (M + E2) or supplemented with genistein (84 mg/L of diet; (M + G) from postnatal day 2 until day 21. E2 treatment reduced testis weight (p < 0.05) as percentage of body weight, significantly suppressed serum androgen concentrations, increased tubule area, Germ cell and Sertoli cell numbers (p < 0.05) relative to those of Sow or Milk groups. Soy formula had no such effects relative to Sow or Milk groups. mRNAseq revealed 103 differentially expressed genes in the M + E2 group compared to the Milk group related to endocrine/metabolic disorders. However, little overlap was observed between the other treatment groups. These data suggest soy formula is not estrogenic in the male neonatal piglet and that soy formula does not significantly alter male reproductive development.

Binge Ethanol Exposure in Mice Represses Expression of Genes Involved in Osteoblast Function and Induces Expression of Genes Involved in Osteoclast Differentiation Independently of Endogenous Catalase.

Excessive ethanol consumption is a risk factor for osteopenia. Since a previous study showed that transgenic female mice with overexpression of catalase are partially protected from ethanol-mediated trabecular bone loss, we investigated the role of endogenous catalase in skeletal ethanol toxicity comparing catalase knockout to wild-type mice. We hypothesized that catalase depletion would exacerbate ethanol effects. The mice were tested in a newly designed binge ethanol model, in which 12-week-old mice were exposed to 4 consecutive days of gavage with ethanol at 3, 3, 4, and 4.5 g ethanol/kg body weight. Binge ethanol decreased the concentration of serum osteocalcin, a marker of bone formation. The catalase genotype did not affect the osteocalcin levels. RNA sequencing of femoral shaft RNA from males was conducted. Ethanol exposure led to significant downregulation of genes expressed in cells of the osteoblastic lineage with a role in osteoblastic function and collagen synthesis, including the genes encoding major structural bone proteins. Binge ethanol further induced a smaller set of genes with a role in osteoclastic differentiation. Catalase depletion affected genes with expression in erythroblasts and erythrocytes. There was no clear interaction between binge ethanol and the catalase genotype. In an independent experiment, we confirmed that the binge ethanol effects on gene expression were reproducible and occurred throughout the skeleton in males. In conclusion, the binge ethanol exposure, independently of endogenous catalase, reduces expression of genes involved in osteoblastic function and induces expression of genes involved in osteoclast differentiation throughout the skeleton in males.

Antioxidants Associated With Oncogenic Human Papillomavirus Infection in Women.

BACKGROUND: Human papillomavirus (HPV) infection is a major cause of cervical cancer. Studies showed the onset of HPV carcinogenesis may be induced by oxidative stress affecting the host immune system. The association between antioxidants and oncogenic HPV remains unclear. In this study, we aim to identify antioxidants associated with vaginal HPV infection in women. METHODS: The associations between the 15 antioxidants and vaginal HPV infection status (no, low-risk [LR], and high-risk [HR] HPV) were evaluated using 11 070 women who participated in the 2003-2016 National Health and Nutrition Examination Survey (NHANES). RESULTS: We identified serum albumin and 4 dietary antioxidants (vitamin A, B2, E, and folate) inversely associated with HR-HPV infection. Women with a low level of albumin (≤39 g/L) have a significantly higher risk of HR-HPV (odds ratio [OR] = 1.4, P = .009 vs >44 g/L). A Nutritional Antioxidant Score (NAS) was developed based on these 4 dietary antioxidants. The women with the lowest quartile NAS had a higher chance of HR-HPV (OR = 1.3, P = .030) and LR-HPV (OR = 1.4, P = .002) compared with the women with the highest quartile NAS. CONCLUSIONS: We identified 5 antioxidants negatively associated with vaginal HR-HPV infection in women. Our findings provide valuable insights into understanding antioxidants' impact on HPV carcinogenesis.

Nox4 Expression Is Not Required for OVX-Induced Osteoblast Senescence and Bone Loss in Mice.

Estrogen deficiency and aging play critical roles in the pathophysiology of bone as a result of increased oxidative stress. It has been suggested that prevention of NADPH oxidase- (Nox-) dependent accumulation of ROS may be an approach to potentially minimize bone loss caused by these conditions. Using ovariectomized (OVX) and Nox4 gene-deletion mouse models, we investigated the role of Nox4 in OVX-induced bone loss and osteoblast senescence signaling. Six-month-old WT C57Bl6 mice were allocated to a sham control group, OVX, and OVX plus E2 treatment group for 8 weeks. Decreased bone mass including BMD and BMC were found in the OVX group compared with the sham control (p < 0.05); E2 treatment completely reversed OVX-induced bone loss. Interestingly, the prevention of OVX-induced bone loss by E2 was associated with the elimination of increased senescence signaling in bone osteoblastic cells from the OVX group. E2 blunted OVX-induced p53 and p21 overexpression, but not p16 and Nox4 in bone. In addition, 8- and 11-month-old Nox4 KO female mice were OVX for 8 weeks. Significant bone loss and increased bone osteoblastic cell senescence signaling occurred not only in Nox4 KO OVX mice compared with sham-operated animals, but also in 11-month-old Nox4 KO sham mice compared with 8-month-old Nox4 KO sham mice (p < 0.05). These data suggest that Nox4-mediated ROS in bone osteoblastic cells may be dispensable for sex steroid deficiency-induced bone loss and senescence. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

MicroRNA profiles were altered in neonatal piglet mammary glands following postnatal infant formula feeding.

Postnatal dietary modulation of microRNAs (miRNAs) and effects on miRNA-mRNA interactions in tissues remain unknown. This study aimed to investigate whether dietary factors (formula vs. breastfeeding) affect mammary miRNA expression and to determine if these changes are concurrent with developmental alterations of the mammary gland in neonatal piglets. Female Yorkshire/Duroc piglets were fed sow's milk or cow's milk- or soy-based infant formula (from postnatal day 2 to day 21; n=6/group). Differentially expressed miRNAs were determined using mammary miRNA profiling, followed by miRNA and mRNA expressions characterized by quantitative reverse-transcription polymerase chain reaction. Milk and soy formulas reduced expressions of miR-1, -128, -133a, -193b, -206 and -27a; miRNA down-regulation altered mRNA expressions of genes (e.g., Ccnd1, Tgfb3, Igf1r and Tbx3) that were consistent with enhanced cell proliferation and suppressed apoptotic processes in the developing mammary gland. Interestingly, down-regulation of miR-1, -128 and -27a also correlated with increased mRNA genes such as Hmgcs and Hmgcr encoding cholesterol synthesis in the mammary glands in response to lower circulating cholesterol levels. Infant formula feeding affected mammary miRNA profiles in neonatal piglets, concurrent with increased expression of cell proliferation and cholesterol synthesis genes, suggesting early nutritional modulation of miRNAs may contribute to regulation of proliferative status and cholesterol homeostasis of developing mammary glands during infancy.

Potential role of gut microbiota, the proto-oncogene PIKE (Agap2) and cytochrome P450 CYP2W1 in promotion of liver cancer by alcoholic and nonalcoholic fatty liver disease and protection by dietary soy protein.

We have previously demonstrated promotion of diethylnitrosamine (DEN) initiated liver tumorigenesis after feeding diets high in fat or ethanol (EtOH) to male mice. This was accompanied by hepatic induction of the proto-oncogene PIKE (Agap2). Switch of dietary protein from casein to soy protein isolate (SPI) significantly reduced tumor formation in these models. We have linked EtOH consumption in mice to microbial dysbiosis. Adoptive transfer studies demonstrate that microbiota from mice fed ethanol can induce hepatic steatosis in the absence of ethanol suggesting that microbiota or the microbial metabolome play key roles in development of fatty liver disease. Feeding SPI significantly changed gut bacteria in mice increasing alpha diversity (P < 0.05) and levels of Clostidiales spp. Feeding soy formula to piglets also resulted in significant changes in microbiota, the pattern of bile acid metabolites and in inhibition of the intestinal-hepatic FXR/FGF19-SHP pathway which has been linked to both steatosis and hepatocyte proliferation. Moreover, feeding SPI also resulted in induction of hepatic PPARα signaling and inhibition of PIKE mRNA expression coincident with inhibition of steatosis and cancer prevention. Feeding studies in the DEN model with differing dietary fats demonstrated tumor promotion specific to the saturated fat, cocoa butter relative to diets containing olive oil or corn oil associated with microbial dysbiosis including dramatic increases in Lachnospiraceae particularly from the genus Coprococcus. Immunohistochemical analysis demonstrated that tumors from EtOH-fed mice and patients with alcohol-associated HCC also expressed high levels of a novel cytochrome P450 enzyme CYP2W1. Additional adoptive transfer experiments and studies in knockout mice are required to determine the exact relationship between soy effects on the microbiota, expression of PIKE, CYP2W1, PPARα activation and prevention of tumorigenesis.

Chronic Ethanol Feeding in Mice Decreases Expression of Genes for Major Structural Bone Proteins in a Nox4-Independent Manner.

Bone loss in response to alcohol intake has previously been hypothesized to be mediated by excessive production of reactive oxygen species via NADPH oxidase (Nox) enzymes. Nox4 is one of several Nox enzymes expressed in bone. We investigated the role of Nox4 in the chondro-osteoblastic lineage of the long bones in mice during normal chow feeding and during chronic ethanol feeding for 90 days. We generated mice with a genotype (PrxCre +/- Nox4 fl/fl) allowing conditional knockout of Nox4 in the limb bud mesenchyme. Adult mice had 95% knockdown of Nox4 expression in the femoral shafts. For mice on regular chow, only whole-body Nox4 knockout mice had clearly increased cortical thickness and bone mineral density in the tibiae. When chronically fed a liquid diet with and without ethanol, conditional Nox4 knockout mice had slightly reduced dimensions of the cortical and trabecular regions of the tibiae (P < 0.1). The ethanol diet caused a significant reduction in cortical bone area and cortical thickness relative to a control diet without ethanol (P < 0.05). The ethanol diet further reduced gene expression of Frizzled related protein (Frzb), myosin heavy chain 3, and several genes encoding collagen and other major structural bone proteins (P < 0.05), whereas the Nox4 genotype had no effects on these genes. In conclusion, Nox4 expression from both mesenchymal and nonmesenchymal cell lineages appears to exert subtle effects on bone. However, chronic ethanol feeding reduces cortical bone mass and cortical gene expression of major structural bone proteins in a Nox4-independent manner. SIGNIFICANCE STATEMENT: Excessive alcohol intake contributes to osteopenia and osteoporosis, with oxidative stress caused by the activity of NADPH oxidases hypothesized to be a mediator. We tested the role of NADPH oxidase (Nox) 4 in osteoblast precursors in the long bones of mice with a conditional Nox4 knockout model. We found that Nox4 exerted effects independent of alcohol intake, and ethanol effects on bone were Nox4-independent.

Skeletal Toxicity of Coplanar Polychlorinated Biphenyl Congener 126 in the Rat Is Aryl Hydrocarbon Receptor Dependent.

Epidemiological evidence links polychlorinated biphenyls (PCBs) to skeletal toxicity, however mechanisms whereby PCBs affect bone are poorly studied. In this study, coplanar PCB 126 (5 µmol/kg) or corn oil vehicle was administered to N = 5 and 6 male and female, wild type (WT) or AhR -/- rats via intraperitoneal injection. Animals were sacrificed after 4 weeks. Bone length was measured; bone morphology was assessed by microcomputed tomography and dynamic histomorphometry. Reduced bone length was the only genotype-specific effect and only observed in males (p < .05). WT rats exposed to PCB 126 had reduced serum calcium, and smaller bones with reduced tibial length, cortical area, and medullary area relative to vehicle controls (p < .05). Reduced bone formation rate observed in dynamic histomorphometry was consistent with inhibition of endosteal and periosteal bone growth. The effects of PCB 126 were abolished in AhR -/- rats. Gene expression in bone marrow and shaft were assessed by RNA sequencing. Approximately 75% of the PCB-regulated genes appeared AhR dependent with 89 genes significantly (p < .05) regulated by both PCB 126 and knockout of the AhR gene. Novel targets significantly induced by PCB 126 included Indian hedgehog (Ihh) and connective tissue growth factor (Ctgf/Ccn2), which regulate chondrocyte proliferation and differentiation in the bone growth plate and cell-matrix interactions. These data suggest the toxic effects of PCB 126 on bone are mediated by AhR, which has direct effects on the growth plate and indirect actions related to endocrine disruption. These studies clarify important mechanisms underlying skeletal toxicity of dioxin-like PCBs and highlight potential therapeutic targets.

Associations of Liver Disease with Alcohol Use among People Living with HIV and the Role of Hepatitis C: The New Orleans Alcohol Use in HIV Study.

AIM: This cross-sectional analysis of the New Orleans Alcohol Use in HIV (NOAH) study assesses whether current and lifetime alcohol use in people living with HIV (PLWH) are associated with greater liver disease and how hepatitis C-viral (HCV) co-infection (HIV/HCV+) modifies the association. METHODS: Alcohol use was measured by Lifetime Drinking History (LDH), a 30-day Timeline Followback calendar, the Alcohol Use Disorder Identification Test, and phosphatidylethanol. Liver disease was estimated by alanine aminotransferase (ALT), aspartate aminotransferase (AST), AST platelet ratio-index (APRI), fibrosis-4 index (FIB-4) and nonalcoholic fatty liver disease-fibrosis score. Associations between alcohol consumption and liver disease were estimated with multivariable logistic regression. Models were adjusted for age, sex, body-mass index, hepatitis B and HIV viral load. RESULTS: Participants (N = 353) were majority male (69%) and black (84%) with a mean age of 48.3 ± 10 years. LDH was significantly associated with advanced liver fibrosis (FIB-4 aOR = 22.22 [1.22-403.72]) only among HIV/HCV+ participants with an LDH of 100-600 kg. HIV/HCV+ participants had a higher prevalence of intermediate and advanced liver disease markers than HIV/HCV- (P < 0.0001). Advanced markers of liver disease were most strongly associated with hazardous drinking (≥40(women)/60(men) grams/day) (APRI aOR = 15.87 (3.22-78.12); FIB-4 aOR = 6.76 (1.81-7.16)) and PEth ≥400 ng/ml (APRI aOR = 17.52 (2.55-120.54); FIB-4 aOR = 17.75 (3.30-95.630). CONCLUSION: Results indicate a greater association of current alcohol use with liver disease than lifetime alcohol use, which varied by HCV status. These findings stress the importance of reducing alcohol use in PLWH to decrease risk of liver disease and fibrosis.

Skeletal toxicity resulting from exposure of growing male rats to coplanar PCB 126 is associated with disruption of calcium homeostasis and the GH-IGF-1 axis and direct effects on bone formation.

Skeletal toxicity has been reported following exposure to polychlorinated biphenyl (PCB) mixtures. However, molecular mechanisms remain poorly understood. We exposed groups of male 4-5-week-old Sprague-Dawley rats to 3,3', 4, 4', 5-pentachlorobiphenyl (PCB 126), a dioxin-like coplanar PCB congener by a single i.p. injection of 5 µmol/kg in soy oil vehicle or vehicle alone. After 4 weeks, rats were euthanized. PCB exposure resulted in hypocalcemia (P < 0.05) and significant increases in serum PTH without changes in serum phosphorous. Hyperparathyroidism was accompanied by increased expression of mRNAs of vitamin D3 metabolizing cytochrome P450 enzymes CYP27B1 and CYP24 in the kidney (P < 0.05). PCB exposure also reduced body weight, serum IGF-1, and hepatic expression of mRNAs encoding the male-specific GH-pattern-regulated CYP2C11 and CYP3A2 relative to controls (P < 0.05). PCB exposure reduced long bone length, diameter, and surface area, but increased trabecular thickness and volume (P < 0.05). Serum osteocalcin (P < 0.05), a marker and a regulator of bone formation, was reduced, but PCB exposure had no effect on the bone resorption marker RatLaps. Exposure of human intestinal Caco-2 cells to 10-100 nM PCB 126 in the presence of vitamin D3 resulted in inhibition of mRNAs for the calcium transporters TRPV6 and PMCA1b (P < 0.05). In addition, PCB 126 suppressed osteoblastogenesis in primary bone marrow mesenchymal stem cell cultures which was blunted by the AhR antagonist CH-223191. These data provide novel evidence that skeletal toxicity after exposure to PCB 126 is a result of disruption of calcium homeostasis and the GH-IGF-1 axis, and involves direct AhR-mediated effects on bone formation.

Infant Formula Feeding Changes the Proliferative Status in Piglet Neonatal Mammary Glands Independently of Estrogen Signaling.

BACKGROUND: Soy infant formula contains isoflavones, which are able to bind to and activate estrogen receptor (ER) pathways. The mammary gland is sensitive to estrogens, raising concern that the use of soy formulas may promote premature development. OBJECTIVE: We aimed to determine if soy formula feeding increases mammary gland proliferation and differentiation in comparison to other infant postnatal diets. METHODS: White-Dutch Landrace piglets aged 2 d received either sow milk (Sow), or were provided milk formula (Milk), soy formula (Soy), milk formula supplemented with 17-beta-estradiol (2 mg/(kg·d); M + E2), or milk formula supplemented with genistein (84 mg/L of diet; M + G) until day 21. Mammary gland proliferation and differentiation was assessed by histology, and real-time RT-PCR confirmation of differentially expressed genes identified by microarray analysis. RESULTS: Mammary terminal end bud numbers were 19-31% greater in the Milk, Soy, and M + G groups relative to the Sow and M + E2, P <0.05. Microarray analysis identified differentially expressed genes between each formula-fed group relative to the Sow (±1.7-fold, P <0.05). Real-time RT-PCR confirmed 2- to 4-fold increases in mRNA transcripts of genes involved in cell proliferation, insulin-like growth factor 1 (IGF1), fibroblast growth factor 10 (FGF10), and fibroblast growth factor 18 (FGF18), in all groups relative to the Sow, P <0.05. In contrast, genes involved in cell differentiation and ductal morphogenesis, angiotensin II receptor type 2 (AGTR2), microtubule associated protein 1b (MAP1B), and kinesin family member 26b (KIF26B), were significantly upregulated by 2-, 4-, and 13-fold, respectively, in the M + E2 group. Additionally, mRNA expression of ER-specific gene targets, progesterone receptor (PGR), was increased by 12-fold, and amphiregulin (AREG) and Ras-like estrogen regulated growth inhibitor (RERG) expression by 1.5-fold in the M + E2 group, P <0.05. In the soy and M + G groups, mRNA expressions of fatty acid synthesis genes were increased 2- to 4-fold. CONCLUSIONS: Our data indicate soy formula feeding does not promote ER-signaling in the piglet mammary gland. Infant formula feeding (milk- or soy-based) may initiate proliferative pathways independently of estrogenic signaling.

Reduced Serum Osteocalcin in High-Risk Alcohol Using People Living With HIV Does Not Correlate With Systemic Oxidative Stress or Inflammation: Data From the New Orleans Alcohol Use in HIV Study.

BACKGROUND: HIV infection is now largely a chronic condition as a result of the success of antiretroviral therapy. However, several comorbidities have emerged in people living with HIV (PLWH), including alcohol use disorders and musculoskeletal disorders. Alcohol use has been associated with lower bone mineral density, alterations to circulating bone turnover markers, and hypocalcemia. The pathophysiological basis of bone loss in the PLWH population is unclear but has been suggested to be linked to oxidative stress and inflammation. To test the hypothesis that PLWH consuming excessive alcohol have altered markers of bone turnover and/or calcium homeostasis in association with oxidative stress, we correlated measurements of alcohol consumption with markers of oxidative stress and inflammation, serum calcium concentrations, and measurements of bone turnover, including c-terminal telopeptide cross-links (CTX-1) and osteocalcin. METHODS: Data were drawn from cross-sectional baseline data from the ongoing New Orleans Alcohol Use in HIV (NOAH) study, comprised of 365 in care PLWH. Alcohol consumption measures (Alcohol Use Disorders Test, 30-day timeline follow-back calendar, and phosphatidylethanol [PEth]) were measured in a subcohort of 40 subjects selected based on highest and lowest PEth measurements. Multivariate linear regression was performed to test the relationships between alcohol consumption and systemic oxidative stress (4-hydroxynonenal; 4-HNE) and inflammation (c-reactive protein; CRP). RESULTS: Serum calcium and CTX-1 did not differ significantly between the high and low-PEth groups. Individuals in the high-PEth group had significantly lower serum osteocalcin (median low-PEth group: 13.42 ng/ml, inter-quartile range [IQR] 9.26 to 14.99 ng/ml; median high-PEth group 7.39 ng/ml, IQR 5.02 to 11.25 ng/ml; p = 0.0005, Wilcoxon rank-sum test). Osteocalcin negatively correlated with PEth (Spearman r = -0.45, p = 0.05) and self-reported measures after adjusting for covariates. Alcohol consumption showed mild, but significant, positive associations with serum 4-HNE, but not with CRP. Osteocalcin did not correlate with either 4-HNE or CRP. CONCLUSIONS: In this subcohort of PLWH, we detected significant associations between at-risk alcohol use and osteocalcin, and at-risk alcohol use and serum 4-HNE, suggesting suppression of bone formation independent of increased systemic oxidative stress with increasing alcohol consumption.

N-Acetylcysteine prevents the decreases in cardiac collagen I/III ratio and systolic function in neonatal mice with prenatal alcohol exposure.

Prenatal alcohol exposure (PAE) is often associated with congenital heart defects, most commonly septal, valvular, and great vessel defects. However, there have been no known studies on whether PAE affects the resulting fibroblast population after development, and whether this has any consequences in the postnatal period. Our previous study focused on the effects of PAE on the postnatal fibroblast population, which translated into changes in cardiac extracellular matrix (ECM) composition and cardiac function in the neonatal heart. Moreover, our lab has previously demonstrated that alcohol-induced fibrosis is mediated by oxidative stress mechanisms in adult rat hearts following chronic alcohol exposure. Thus, we hypothesize that PAE alters cardiac ECM composition that persists into the postnatal period, leading to cardiac dysfunction, and these effects are prevented by antioxidant treatment. To investigate these effects, pregnant mice were intraperitoneally injected with 2.9 g EtOH/kg body weight on gestation days 6.75 and 7.25. Controls were injected with vehicle saline. Randomly selected dams in both groups were then treated with 100 mg/kg body weight of the antioxidant N-acetylcysteine (NAC) immediately after EtOH or vehicle administration. Left ventricular (LV) chamber dimension and function were assessed in sedated animals on neonatal day 5 using echocardiography. Ejection fraction decreased in the PAE group. NAC treatment prevented this depression of systolic function in PAE neonates. Hearts were analyzed for expression of fibroblast activation markers. Alpha smooth muscle actin (α-SMA) increased in PAE neonatal hearts, and this increase was prevented by NAC treatment. In PAE pups, collagen I decreased, but collagen III expression increased compared to saline animals; the overall collagen I/III ratio significantly decreased. When PAE mice were treated with NAC, collagen I/III ratio did not change. Overall, our data demonstrate that prenatal alcohol exposure produces changes in collagen subtype in neonatal cardiac ECM and a decline in systolic function, and these adverse effects were prevented by NAC treatment.

Improved method for isolating high-quality RNA from mouse bone with RNAlater at room temperature.

Accurate gene expression analysis of bone requires the ability to isolate RNA of good quality. Isolation of intact RNA from frozen bone tissue is problematic since RNA rapidly becomes degraded after thawing. Since we are interested in assessing gene expression from both bone marrow and mineralized bone, we aimed to develop improved simple, robust and statistically validated methods providing high-quality RNA from both mouse femur shaft and femur marrow. RNA integrity was quantified by the RNA Integrity Number (RIN) measured on a TapeStation. While the RNA stabilization reagent RNAlater is not commonly used or recommended for mineralized bone, we found that preservation methods with RNAlater significantly improved the RNA quality with a mean RIN for the femur shaft of 8.0 and a mean RIN for femur marrow of 9.6. With RNAlater, high quality RNA with a mean RIN of 9.3 could also be isolated from lumbar vertebral bone. A further advantage of using RNAlater is that the tissue can be allowed to thaw to room temperature before TRI Reagent lysis without any loss of RNA integrity. A comparison of the TRI Reagent method with a hybrid method combining TRI Reagent lysis with RNeasy column purification showed no difference in RNA integrity. However, the hybrid method seemed to give femur shaft RNA with fewer impurities inhibiting qRT-PCR.

Liver tumorigenesis is promoted by a high saturated fat diet specifically in male mice and is associated with hepatic expression of the proto-oncogene Agap2 and enrichment of the intestinal microbiome with Coprococcus.

Liver cancer results in a high degree of mortality, especially among men. As fatty liver disease is a risk factor for development of hepatocellular carcinoma, we investigated the role of dietary fat type in tumor promotion by high-fat diets in mice after initiation with the chemical carcinogen diethyl nitrosamine. Tumor incidence and multiplicity were significantly greater in males than those in females. In males, fat type had complex effects on tumorigenesis. Preneoplastic foci were most prevalent in mice fed a polyunsaturated fat diet enriched in docosahexaenoic acid, whereas carcinomas and large visible liver tumors were significantly greater in mice fed a saturated fat diet made with cocoa butter relative to mice fed mono- or polyunsaturated fats. Different mechanisms thus seemed involved in early and late tumor promotion. The hepatic transcriptome and gut microbiome were assessed for traits associated with tumorigenesis. Hepatic expression of more than 20% of all genes was affected by sex, whereas fat type affected fewer genes. In males, the saturated fat diet induced expression of the proto-oncogene Agap2 and affected the expression of several cytochrome P450 genes, and genes involved in lipid, bile acid and fatty acid metabolism. The gut microbiome had a higher level of genus Akkermansia and a lower level of Firmicutes in females than in males. Males fed saturated fat had an altered microbiome, including an enrichment of the genus Coprococcus. In conclusion, sex and the dietary fat type affect the gut microbiome, the hepatic transcriptome and ultimately hepatic tumor growth.

Infant Formula Feeding Increases Hepatic Cholesterol 7α Hydroxylase (CYP7A1) Expression and Fecal Bile Acid Loss in Neonatal Piglets.

Background: During the postnatal feeding period, formula-fed infants have higher cholesterol synthesis rates and lower circulating cholesterol concentrations than their breastfed counterparts. Although this disparity has been attributed to the uniformly low dietary cholesterol content of typical infant formulas, little is known of the underlying mechanisms associated with this altered cholesterol metabolism phenotype. Objective: We aimed to determine the molecular etiology of diet-associated changes in early-life cholesterol metabolism with the use of a postnatal piglet feeding model. Methods: Two-day-old male and female White-Dutch Landrace piglets were fed either sow milk (Sow group) or dairy-based (Milk group; Similac Advance powder) or soy-based (Soy group; Emfamil Prosobee Lipil powder) infant formulas until day 21. In addition to measuring serum cholesterol concentrations, hepatic and intestinal genes involved in enterohepatic circulation of cholesterol and bile acids were analyzed by real-time reverse-transcriptase polymerase chain reaction and Western blot. Bile acid concentrations were measured by liquid chromatography-mass spectrometry in serum, liver, and feces. Results: Compared with the Sow group, hepatic cholesterol 7α hydroxylase (CYP7A1) protein expression was 3-fold higher in the Milk group (P < 0.05) and expression was 10-fold higher in the Soy group compared with the Milk group (P < 0.05). Likewise, fecal bile acid concentrations were 3-fold higher in the Soy group compared with the Milk group (P < 0.05). Intestinal mRNA expression of fibroblast factor 19 (Fgf19) was reduced in the Milk and Soy groups, corresponding to 54% and 67% decreases compared with the Sow group. In the Soy group, small heterodimer protein (SHP) protein expression was 30% lower compared with the Sow group (P < 0.05). Conclusions: These results indicate that formula feeding leads to increased CYP7A1 protein expression and fecal bile acid loss in neonatal piglets, and this outcome is linked to reduced efficacy in inhibiting CYP7A1 expression through FGF19 and SHP transcriptional repression mechanisms.

Knockout of the Gsta4 Gene in Male Mice Leads to an Altered Pattern of Hepatic Protein Carbonylation and Enhanced Inflammation Following Chronic Consumption of an Ethanol Diet.

BACKGROUND: Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4-/- mice). METHODS: Adult male WT and GSTA4-/- mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins. RESULTS: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4-/- mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4-/- mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4-/- group. CONCLUSIONS: These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4-/- mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.