**1. Introduction**

Xenobiotics, such as drugs or toxic chemicals, can be metabolized and eliminated by xenobioticmetabolizing enzymes and membrane transporters. The liver is the major tissue responsible for detoxification of xenobiotics. The xenobiotic-metabolizing enzymes include phase I and phase II enzyme systems. The cytochrome P450 (CYP) enzymes are the major phase I enzymes responsible for the metabolism of endogenous molecules (e.g., sterols and fatty acids) and exogenous xenobiotics (e.g., drugs and toxic chemicals), resulting in the formation of more water-soluble and less toxic metabolites [1]. However, some CYP enzymes, such as CYP1A1, 3A, and 2E1, are involved in the bioactivation of chemicals, such as benzo[a]pyrene, aflatoxin B1, and acetaminophen [2–4], and produce electrophile intermediates that may covalently bind to proteins, lipids, and DNA. These enzyme reactions may therefore produce more reactive oxygen species (ROS) and increase oxidative damage to tissues [5]. Uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT) and glutathione S-transferase (GST) are two important phase II detoxifying enzymes that catalyze the conjugation reactions, resulting

in the formation of water-soluble glucuronate and glutathione conjugates to facilitate the excretion of xenobiotics. Induction of phase II detoxifying enzymes and reduction of ROS is most pronounced in the prevention of chemical-induced tissue injuries and carcinogenesis [6]. Phase III membrane transporters include *p*-glycoprotein and multidrug resistance-associated proteins, such as Mrp2/3, which may function to shuttle xenobiotics or their metabolites across cellular membranes and to facilitate the excretion of these compounds from the liver into bile (e.g., *p*-glycoprotein and Mrp2) and blood (e.g., Mrp3) [7–9].

Molecular hydrogen (H2) acts as an antioxidant by selectively reducing particularly strong oxidants such as the hydroxyl radical (•OH), which can exhibit relatively stronger oxidative activities than other ROS in cells [10]. It is noteworthy that H2 has no cytotoxicity even at high concentrations. Consumption of hydrogen water (H2 dissolved in water) is a convenient, easily administered, and safe way to ingest H2. Hydrogen water can be made by several methods: infusing H2 gas into water under high pressure, electrolyzing water to producing H2, and reacting magnesium metal or its hydride with water [11]. Despite the low solubility of H2 gas in water, which can be only up to 1.6 ppm under 1 atmospheric pressure at room temperature, consumption of hydrogen-rich water (HRW) has been shown to be effective for ameliorating various diseases caused by oxidative stress in animal and clinical studies [11,12]. These findings suggest that H2 may be a versatile element with therapeutic activity.

The quality of drinking water is an important health and safety information [13]. It is known that dissolved H2 in drinking water will gradually decrease over time. Until now, in addition to the H2 concentration, the quality of HRW has not been adequately described. In addition, the effects of intake of HRW on the activity of xenobiotic-metabolizing enzymes and on the membrane transporters involved in the metabolism of various drugs or chemicals are still unknown. In this study, therefore, we first assessed quality parameters of HRW, such as the dissolved H2 concentration, total dissolved solids (TDS), ions, and water cluster. Then, the effects of intake of HRW on xenobiotic-metabolizing enzymes, membrane transporters, and antioxidant activity in rat liver were investigated.

#### **2. Results**
