Deletion of NADPH oxidase 2 in chondrocytes exacerbates ethanol-mediated growth plate disruption in mice without major effects on bone architecture or gene expression.

BACKGROUND: Excess reactive oxygen species generated by NADPH oxidase 2 (Nox2) in response to ethanol exposure mediate aspects of skeletal toxicity including increased osteoclast differentiation and activity. Because perturbation of chondrocyte differentiation in the growth plate by ethanol could be prevented by dietary antioxidants, we hypothesized that Nox2 in the growth plate was involved in ethanol-associated reductions in longitudinal bone growth. METHODS: Nox2 conditional knockout mice were generated, where the essential catalytic subunit of Nox2, cytochrome B-245 beta chain (Cybb), is deleted in chondrocytes using a Cre-Lox model with Cre expressed from the collagen 2a1 promoter (Col2a1-Cre). Wild-type and Cre-Lox mice were fed an ethanol Lieber-DeCarli-based diet or pair-fed a control diet for 8 weeks. RESULTS: Ethanol treatment significantly reduced the number of proliferating chondrocytes in the growth plate, enhanced bone marrow adiposity, shortened femurs, reduced body length, reduced cortical bone volume, and decreased mRNA levels of a number of osteoblast and chondrocyte genes. Conditional knockout of Nox2 enzymatic activity in chondrocytes did not consistently prevent any ethanol effects. Rather, knockout mice had fewer proliferating chondrocytes than wild-type mice in both the ethanol- and control-fed animals. Additional analysis of tibia samples from Nox4 knockout mice showed that loss of Nox4 activity also reduced the number of proliferating chondrocytes and altered chondrocyte size in the growth plate. CONCLUSIONS: Although Nox enzymatic activity regulates growth plate development, ethanol-associated disruption of the growth plate morphology is independent of ethanol-mediated increases in Nox2 activity.

Diet Supplementation with Soy Protein Isolate, but Not the Isoflavone Genistein, Protects Against Alcohol-Induced Tumor Progression in DEN-Treated Male Mice.

Diethylnitrosamine-treated male mice were assigned to 4 groups: a casein-based 35% high fat ethanol liquid diet (EtOH), an EtOH diet made with soy protein isolate protein (EtOH/SOY), an EtOH liquid diet supplemented with genistein (EtOH/GEN) and a chow group. EtOH feeding, final concentration 5% (v/v), continued for 16 wks. EtOH increased incidence and multiplicity of basophilic lesions and adenomas compared to the chow group, (p < 0.05). The EtOH/SOY group had reduced adenoma progression when compared to the EtOH and EtOH/GEN group, (p < 0.05). Genistein supplementation had no protective effect. Soy feeding significantly reduced serum ALT concentrations (p < 0.05), decreased hepatic TNFα and CD-14 expression and decreased nuclear accumulation of NFκB protein in EtOH/SOY-treated mice compared to the EtOH group (p < 0.05). With respect to ceramides, high resolution MALDI-FTICR Imaging mass spectrometry revealed changes in the accumulation of long acyl chain ceramide species, in particular C18, in the EtOH group when compared to the EtOH/SOY group. Additionally, expression of acid ceramidase and sphingosine kinase 1 which degrade ceramide into sphingosine and convert sphingosine to sphingosine-1-phosphate (S1P) respectively and expression of S1P receptors S1PR2 and S1PR3 were all upregulated by EtOH and suppressed in the EtOH/SOY group, p < 0.05. EtOH feeding also increased hepatocyte proliferation and mRNA expression of ß-catenin targets, including cyclin D1, MMP7 and glutamine synthase, which were reduced in the EtOH/SOY group, p < 0.05. These findings suggest that soy prevents tumorigenesis by reducing inflammation and by reducing hepatocyte proliferation through inhibition of EtOH-mediated ß-catenin signaling. These mechanisms may involve blockade of sphingolipid signaling.

Alcohol consumption, Wnt/ß-catenin signaling, and hepatocarcinogenesis.

Alcohol is a well-established risk factor for hepatocellular carcinoma, and the mechanisms by which alcohol liver cancer is complex. It has been suggested that ethanol (EtOH) metabolism may enhance tumor progression by increasing hepatocyte proliferation. To test this hypothesis, ethanol (EtOH) feeding of male mice began 7 weeks post-injection of the chemical carcinogen diethylnitrosamine (DEN), and continued for 16 weeks, with a final EtOH concentration of 28% of total calories. As expected, EtOH increased the total number of cancerous foci and liver tumors identified in situ fixed livers from the EtOH+DEN group compared to corresponding pair-fed (PF)+DEN and chow+DEN control groups. In the EtOH+DEN group, tumor multiplicity corresponded to a 3- to 4-fold increase in proliferation and immunohistochemical staining of ß-catenin in non-tumorigenic hepatocytes when compared to the PF+DEN and chow+DEN groups, p<0.05. Analysis of EtOH-treated livers from a previously published rat model of chronic liver disease revealed increases in hepatocyte proliferation accompanied by a hepatic depletion of retinol and retinoic acid stores (p<0.05), nuclear accumulation of ß-catenin (p<0.05), increased cytosolic expression p-GSK3ß (p<0.05), significant upregulation of soluble Wnts, Wnt2, and Wnt7a, and increased expression of several ß-catenin targets involved in tumor promotion and progression, cyclin D1, c-myc, WISP1, and MMP7 (p<0.05). These data suggest that chronic EtOH consumption activates the Wnt/ß-catenin signaling pathway, which increases hepatocyte proliferation thus promoting tumorigenesis following an initiating insult in the liver.

Dietary fat source alters hepatic gene expression profile and determines the type of liver pathology in rats overfed via total enteral nutrition.

To determine if dietary fat composition affects the progression of nonalcoholic fatty liver disease (NAFLD), we overfed male Sprague-Dawley rats low (5%) or high (70%) fat diets with different fat sources: olive oil (OO), corn oil (CO), or echium oil (EO), with total enteral nutrition (TEN) for 21 days. Overfeeding of the 5% CO or 5% EO diets resulted in less steatosis than 5% OO (P < 0.05). Affymetrix array analysis revealed significant differences in hepatic gene expression signatures associated with greater fatty acid synthesis, ChREBP, and SREBP-1c signaling and increased fatty acid transport (P < 0.05) in the 5% OO compared with 5% CO group. The OO groups had macrosteatosis, but no evidence of oxidative stress or necrosis. The 70% CO and 70% EO groups had a mixture of micro- and macrosteatosis or only microsteatosis, respectively; increased oxidative stress; and increased necrotic injury relative to their respective 5% groups (P < 0.05). Oxidative stress and necrosis correlated with increasing peroxidizability of the accumulated triglycerides. Affymetrix array analysis comparing the 70% OO and 70% CO groups revealed increased antioxidant pathways and lower expression of genes linked to inflammation and fibrosis in the 70% OO group. A second study in which 70% OO diet was overfed for 50 days produced no evidence of progression of injury beyond simple steatosis. These data suggest that dietary fat type strongly influences the progression of NAFLD and that a Mediterranean diet high in olive oil may reduce the risk of NAFLD progressing to nonalcoholic steatohepatitis.

Soy protein isolate reduces hepatosteatosis in yellow Avy/a mice without altering coat color phenotype.

Agouti (A(vy)/a) mice fed an AIN-93G diet containing the soy isoflavone genistein (GEN) prior to and during pregnancy were reported to shift coat color and body composition phenotypes from obese-yellow towards lean pseudoagouti, suggesting epigenetic programming. Human consumption of purified GEN is rare and soy protein is the primary source of GEN. Virgin a/a female and A(vy)/a male mice were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) (the same approximate GEN levels as in the above mentioned study) for 2 wks prior to mating. A(vy)/a offspring were weaned to the same diets and studied at age 75 d. Coat color distribution did not differ among diets, but SPI-fed, obese A(vy)/a offspring had lower hepatosteatosis (P < 0.05) and increased (P < 0.05) expression of CYP4a 14, a PPARalpha-regulated gene compared to CAS controls. Similarly, weanling male Sprague-Dawley (SD) rats fed SPI had elevated hepatic Acyl Co-A Oxidase (ACO) mRNA levels and increased in vitro binding of PPARalpha to the PPRE promoter response element. In another hepatosteatosis model, adult SD rats fed a high fat/cholesterol diet, SPI reduced (P < 0.05) steatosis. Thus, 1) consumption of diets made with SPI partially protected against hepatosteatosis in yellow mice and in SD rats, and this may involve induction of PPARalpha-regulated genes; and 2) the lifetime (in utero, neonatal and adult) exposure to dietary soy protein did not result in a shift in coat color phenotype of A(vy)/a mice. These findings, when compared with those of previously published studies of A(vy)/a mice, lead us to conclude that: 1) the effects of purified GEN differ from those of SPI when GEN equivalents are closely matched; 2) SPI does not epigenetically regulate the agouti locus to shift the coat color phenotype in the same fashion as GEN alone; and 3) SPI may be beneficial in management of non-alcoholic fatty liver disease.

Effects of chronic ethanol on hepatic and renal CYP2C11 in the male rat: interactions with the Janus-kinase 2-signal transducer and activators of transcription proteins 5b pathway.

Chronic alcohol intake in male rats results in: 1) demasculinization of the GH pulse pattern; 2) reduced serum testosterone concentrations; and 3) decreased expression hepatic CYP2C11. Hepatic CYP2C11 expression is regulated by the male pattern of GH through the Janus-kinase/signal transducer and activators of transcription proteins (JAK/STAT) signal transduction pathway in the male rat. Renal CYP2C11 is regulated by testosterone, not GH. The involvement of the JAK/STAT5b signal transduction pathway in renal CYP2C11 signaling has not been studied. We tested the hypothesis that ethanol reduces CYP2C11 levels by interfering with the JAK/STAT5b pathway. Using a total enteral nutrition (TEN) model to feed rats a well-balanced diet, we have studied the effects of chronic ethanol intake (21 d) on hepatic and renal JAK/STAT pathway of adult male rats (8-10/group). We found decreased hepatic and renal expression of CYP2C11 in ethanol-fed rats with concomitant decreases in STAT5b and phospho-STAT5b, decreased in vitro hepatic STAT5b binding to a CYP2C11 promoter element and no effects on hepatic GHR levels. Ethanol caused tissue specific effects in phospho-JAK2 and JAK2, with increased levels in the liver, but decreased JAK2 expression in the kidney. We conclude that ethanol suppression of CYP2C11 expression is clearly associated with reductions in STAT5b levels, but not necessarily in reductions of JAK2 levels. The mechanisms underlying ethanol-induced suppression of STAT5b is yet to be determined, as is the question of whether this is secondary to hormonal effects or a direct ethanol effect.