**2. Biomarkers for oxidative stress in human disease**

Oxidative/nitrosative stress, such as alcohol-induced increase of reactive oxygen/nitrogen species (ROS/NOS), is now recognized to be a common cause or a prominent feature of many acute and chronic inflammatory diseases(Dalle-Donne et al., 2006). However, up to date, there are not ideal biomarkers and/or methods available to assess oxidative stress status in human diseases. Thus, we are exploring and searching for new biomarkers of oxidative stress, such as miRNA, to objectively measure and evaluate the role of miRNA in the alcohol-induced oxidative stress.

Biomarkers may provide information on three progressive levels of disease outcome(Dalle-Donne et al., 2006): (*a*) as measurable endpoints of damage of biomolecules such as lipids and proteins; (*b*) as functional markers of, for example, cognitive function; and (*c*) as endpoints related to specific disease. In many cases, oxidative stress is an early and common pathphysiological process. Thus, it is very important to find a series of biomarkers to early detect the alcohol-induced oxidative stress. Studying the association between a biomarker and alcohol-induced oxidative stress could benefit for early detection and, therefore, prevention of diseases associated with oxidative stress.

commencement of translation and expeditive restoration of cells back to their normal state; (2) since miRNAs regulate numerous targets, they have the capacity to powerfully and efficiently coordinate a stress response involving numerous genes; (3) owing to their small size and high stability, miRNAs may be less susceptible to certain types of stress, like genotoxic insults. Hence, their ability to modulate stress response would be less likely to be compromised under oxidative-stress. Given the recent development in the field of miRNA

To test our hypothesis, we chose to study Alcoholic Liver Disease (ALD) as an oxidative stress model because: (1) it has high morbidity and mortality with no satisfactory therapy; (2) we showed that oxidative damage is the major mechanism for ALD; (3) we have established and validate that ALD model in rats is a good model for studies of oxidative stress. Our studies demonstrated that: (1). Nitric oxide-induced oxidative stress is required for alcohol-induced gut leakiness and liver damage in this model; (2) The miRNA expression profile was identified by miRNA microarray analysis. The miRNAs signatures were validated by TaqMan real time PCR assay. Our research results demonstrated that the differentially expressed miRNAs are the sensitive and specific biomarkers for alcoholinduced oxidative stress. (3) We showed that oats supplementation, a diet with strong antioxidative effect that is widely used in diets to prevent many diseases associated with oxidative damage, prevents ALD in rats by preventing alcohol-induced oxidative tissue

Numerous markers of oxidative stress and antioxidant status have been evaluated, but there has been little systematic effort to validate sensitive and specific biomarkers for oxidative damage in animal models. The application of miRNA as new biomarkers will lead to: a) identification susceptible individuals who are at risk for oxidative stress and would thus benefit from interventions that provide antioxidants; b) novel strategies to prevent and treat

Oxidative/nitrosative stress, such as alcohol-induced increase of reactive oxygen/nitrogen species (ROS/NOS), is now recognized to be a common cause or a prominent feature of many acute and chronic inflammatory diseases(Dalle-Donne et al., 2006). However, up to date, there are not ideal biomarkers and/or methods available to assess oxidative stress status in human diseases. Thus, we are exploring and searching for new biomarkers of oxidative stress, such as miRNA, to objectively measure and evaluate the role of miRNA in

Biomarkers may provide information on three progressive levels of disease outcome(Dalle-Donne et al., 2006): (*a*) as measurable endpoints of damage of biomolecules such as lipids and proteins; (*b*) as functional markers of, for example, cognitive function; and (*c*) as endpoints related to specific disease. In many cases, oxidative stress is an early and common pathphysiological process. Thus, it is very important to find a series of biomarkers to early detect the alcohol-induced oxidative stress. Studying the association between a biomarker and alcohol-induced oxidative stress could benefit for early detection and, therefore,

**2. Biomarkers for oxidative stress in human disease** 

prevention of diseases associated with oxidative stress.

research, we predict that miRNA will be promised biomarkers for oxidative stress.

damage.

oxidative injury.

the alcohol-induced oxidative stress.

The most intuitive goals for a biomarker are to help diagnose symptomatic and presymptomatic disease and to provide surrogate endpoints to demonstrate clinical efficacy of new treatments(Ogino and Wang, 2007). The usefulness of the ideal biomarker of oxidative damage lies in its ability to provide early indication of disease and/or its progression (Fig. 1).

Fig. 1. Biomarkers for oxidative stress

When investigating the status of alcohol-induced oxidative stress, it is unclear what the most appropriate biomarker is and how to measure them. At present, the biomarkers of oxidative stress/damage and the methods used to measure them are different among different study groups. Thus, it is difficult to compare the study findings in different groups to determine which one is the best biomarker to evaluate an individual's oxidative status in relation to alcohol consumption.

In addition, the validity of many biomarkers remains to be established. The biomarkers that have been developed and currently been used to evaluate the oxidative stress have several shortcomings(Dalle-Donne et al., 2006), such as: (*a*) the limited specificity of the assay itself for the product of oxidative/nitrosative damage being measured; (*b*) the fact that the analyte being measured is not a specific product of a specific ROS/RNS; (*c*) the lack of sufficient sensitivity to detect concentrations of the product being measured in healthy individuals, thus not allowing the definition of a reference interval; (*d*) concentrations of the product being measured being influenced by external factors such as the lipid content of the diet; or (*e*) the assay being too invasive for in vivo investigations in humans.

So far, several oxidative stress biomarkers have been used in clinics to assess patient's reaction to oxidative stress, their accuracy, sensitivity, or specificity need to be improved

Using miRNA as Biomarkers to Evaluate the Alcohol-Induced Oxidative Stress 323

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

Oats, like many other plant materials, contain numerous constituents vitamins, minerals, essential fatty acids, -glucan (fermentable fibers), and phytochemicals, including several phenolic compounds. These constituents have been found to possess many types of bioactivity, including antioxidant, antiproliferaton, anti-inflammatory, and detoxification

We hypothesized that oats supplementation protects through its effects on oxidative pathways. We had two primary rationales for our hypothesis. First, it has been generally accepted that oats are of benefit to human health and normal gut growth and function not only because of their nutrient and fiber values, but also, because of their antioxidant and anti-inflammatory activities. Second, several studies have demonstrated the importance of oxidative stress and upregulated iNOS in alcohol-induced tissue injury and organ dysfunction. More specifically, several reports demonstrated the pivotal role of the upregulation of iNOS and oxidative stress in alcohol-induced gut leakiness. For example, our in vitro studies showed that preventing the upregulation of iNOS that is induced by alcohol, using both iNOS inhibitors and dominant negative mutant for iNOS, prevented

Furthermore, we recently showed that inhibition of iNOS by L-NIL reduces EtOH-induced NO overproduction, oxidative tissue injury and gut leakiness in alcohol-treated rats. Our current study, which uses immunohistochemical staining, provides direct evidence that EtOH induces iNOS activation in colonic epithelium and that oats prevent this effect and

We confirmed our in vitro findings in an animal model of alcoholic steatohepatitis (ASH). We showed that chronic, daily alcohol administration to rats caused gut leakiness. More importantly, we showed that EtOH-induced gut leakiness in rats was associated with

effects, which may contribute to the promotion of good health (Chen et al., 2007).

alcohol-induced disruption of the barrier integrity of intestinal cell monolayers.

prevent alcohol-induced intestinal mucosal oxidative stress.

EtOH-induced oxidative injury in blood, intestinal epithelium, or liver.

**5. Oats supplementation is an antioxidant**

(Dalle-Donne et al., 2006; Ogino and Wang, 2007). The identification of novel biomarkers is urgently needed. miRNA may hold great promise as a biomarker for oxidative stress.
