**4. Animal model of Alcoholic Liver Disease (ALD) is a good model for studying the alcohol-induced oxidative damage**

ALD is one of the most common and serious complications of heavy drinking. It is a major health problem in the US, consuming 15% of total health care dollars, and associated with 20% mortality(Maher, 2002). However, the mechanisms linking Ethanol (EtOH) consumption to ALD are not completely understood.

Our in vitro, in vivo animal, and ex-vivo human studies provided compelling evidence for the central involvement of iNOS activation in EtOH-induced gut leakiness. Indeed,

(Dalle-Donne et al., 2006; Ogino and Wang, 2007). The identification of novel biomarkers is

miRNAs are highly conserved regulatory molecules expressed in eukaryotic cells. They are short noncoding RNAs that regulate gene expression by binding to target mRNAs, which leads to reduced protein synthesis and sometimes decreased steady-state mRNA levels(Ambros, 2004; Carthew, 2006; Carthew and Sontheimer, 2009; Li and Carthew, 2005). Although hundreds of miRNAs have been identified, much less is known about their biological function. There is evidence that miRNAs affect pathways fundamental to metabolic control in higher organisms such as adipocyte and skeletal muscle differentiation. Also, some miRNAs are implicated in lipid, amino acid, and glucose homeostasis. Thus miRNA abnormalities may contribute to common metabolic diseases and there may be novel therapeutic opportunities based on miRNA targeting. Indeed, the expression of certain genes can depend more on levels of regulatory miRNAs than on levels of mRNAs. miRNAs act through a mechanism similar to that of short interfering RNAs (siRNA). The expression of miRNA target genes can be fine-tuned in animals by altering the

There are many evidences indicated that miRNAs are able to modulate the cellular response to oxidative stress both in vitro and in vivo(Babar et al., 2008). We hypothesize that miRNAs may be biomarkers for oxidative stress due to following several reasons. First, since miRNA are post-transcriptional gene regulators, they may be able to function as 'quick responders' to oxidative stress. For example, upon exposure to stress, miRNA may rapidly localize to P-bodies or stress granules to regulate key genes involved in the oxidative stress response. After the stress is mitigated, miRNA inhibition may be promptly abated, allowing commencement of translation and expeditive restoration of cells back to their normal state. Second, since miRNA regulate numerous targets, they have the capacity to powerfully and efficiently coordinate a stress response involving numerous genes. Third, owing to their small size and high stability, miRNA may be less susceptible to certain types of stress, such as genotoxic insults. Hence, their ability to modulate stress response would be less likely to be compromised under high-stress condition. Given the recent development in the field of miRNA research, we predict that

urgently needed. miRNA may hold great promise as a biomarker for oxidative stress.

**3. miRNA may be biomarkers for oxidative stress** 

concentrations or identities of miRNAs within cells.

miRNA will be promised biomarkers for oxidative stress.

**studying the alcohol-induced oxidative damage** 

consumption to ALD are not completely understood.

**4. Animal model of Alcoholic Liver Disease (ALD) is a good model for** 

ALD is one of the most common and serious complications of heavy drinking. It is a major health problem in the US, consuming 15% of total health care dollars, and associated with 20% mortality(Maher, 2002). However, the mechanisms linking Ethanol (EtOH)

Our in vitro, in vivo animal, and ex-vivo human studies provided compelling evidence for the central involvement of iNOS activation in EtOH-induced gut leakiness. Indeed, several of our studies (Banan et al., 2000a; Banan et al., 2000b; Banan et al., 2007; Keshavarzian et al., 2001; Keshavarzian and Fields, 2000; Keshavarzian and Fields, 2003; Keshavarzian et al., 1999; Tang et al., 2009a; Tang et al., 2009c) have shown that iNOS activation is required for EtOH-induced gut leakiness. We reported that: **1)** EtOH increases iNOS activity and NO levels in intestinal monolayers and increases monolayer permeability. A specific iNOS inhibitor (L-NIL) prevented EtOH-induced monolayer leakiness; **2)** EtOH no longer can cause leakiness in monolayers incapable of upregulating iNOS (i.e., transfected with dominant negative iNOS antisense); **3)** iNOS is increased in intestinal mucosa of alcoholics with ALD and in alcohol-treated rats with gut leakiness and endotoxemia; **4)** Daily gavage of the specific iNOS inhibitor L-NIL prevented iNOS upregulation and oxidative stress in the intestinal mucosa of alcohol-fed rats and also prevented alcohol-induced gut leakiness; **5)** Daily gavage of Lactobacillus GG or supplementation of the diet with oats prevented nitration of intestinal mucosal proteins, oxidative stress, and gut leakiness in alcohol-fed rats; **6)** A daily, alcohol-containing (Nanji) diet for 4 weeks caused gut leakiness in wild type mice but NOT in iNOS knockout mice. The unanswered question is whether miRNAs are the biomarkers for EtOH-induced oxidative injury in blood, intestinal epithelium, or liver.
