*2.3. Activities of Xenobiotic-Metabolizing Enzymes and Membrane Transporters*

Table 3 shows the effects of intake of HRW on the activities of xenobiotic-metabolizing enzymes in the liver. No effects were found in the activity of UGT or GST in rats based on intake of HRW for four weeks. In addition, intake of HRW had no significant effect on CYP-mediated reactions.

**Table 3.** Changes in hepatic drug-metabolizing enzyme activities in rats a.


<sup>a</sup> Results are expressed as the mean ± S.D. of eight rats in each group.

The gene expression of CYP enzymes was also not changed (*p* > 0.05) by intake of HRW (Figure 3). Immunoblots of liver membrane transporters are shown in Figure 4. Intake of HRW increased (*p* < 0.05) *p*-glycoprotein and Mrp2 protein expressions in liver. However, HRW had no significant effect on Mrp3 protein expression. These results suggest that intake of HRW may increase the efflux of endogenous substances and exogenous xenobiotics from liver into bile by increasing Mrp2 and *p*-glycoprotein protein expression without affecting the hepatic activity of xenobiotic-metabolizing enzymes in rats.

**Figure 3.** Effects of intake of HRW on the mRNA expression of various CYP enzymes in liver: The results are expressed as the mean ± S.D. of five rats.

**Figure 4.** Effects of intake of HRW on *p*-glycoprotein and Mrp2/3 protein expression in liver of rats (**A**). Protein expression was determined by Western blotting. Densitometric analysis for Mrp2 (**B**), *p*-glycoprotein (**C**) and Mrp3 (**D**) protein levels corrected to each internal control is shown. The results are expressed as the mean <sup>±</sup> S.D. of six rats. Na+/K<sup>+</sup> ATPase acts as an internal control. The protein band was quantified by densitometry, and the level of the control was set at 1. \* Significantly different from the control group at *p* < 0.05.

#### *2.4. Oxidative Stress*

As shown in Table 4, intake of HRW had no significant effects on hepatic GSH or GSSG levels, the GSH/GSSG ratio, or GSH peroxidase activity in rats compared with these animals in the control group. Also, intake of HRW did not change the hepatic TBARS value.


**Table 4.** Effect of intake of HRW on oxidative stress in liver a.

<sup>a</sup> Results are expressed as the mean ± S.D. of eight rats in each group. GSH, reduced glutathione; GSSG, oxidized glutathione; TBARS, thiobarbituric acid reactive substances.

#### **3. Discussion**

Xenobiotic-metabolizing enzymes and membrane transporters are responsible for the detoxification and elimination of xenobiotics from the body. Food components can change xenobiotic metabolism by modifying these enzymes and membrane transporters. This study first showed that intake of HRW had no effect on CYP enzyme activities or antioxidant activity in rat livers. However, intake of HRW increased the efflux pumps of *p*-glycoprotein and Mrp2 in liver. These results indicate that HRW may enhance the excretion of xenobiotics from the liver into bile without altering their metabolism by xenobiotic-metabolizing enzymes. In addition, intake of HRW had no significant effect on oxidative stress in the normal physiologic condition.

CYP-mediated bioactivation and ROS formation may be responsible for chemical or drug toxicity. Increased phase II-conjugated enzymes may facilitate the elimination of xenobiotics [6]. We showed here that intake of HRW did not change the activities of CYP isozymes, UGT, and GST in the liver. Thus, intake of HRW may not change the metabolism of xenobiotics by xenobiotic-metabolizing enzymes in liver.

GSH is the most important biomolecule against ROS-induced tissue injury and can participate in the elimination of xenobiotics through GST [6]. HRW has been shown to be able to scavenge free radicals, especially hydroxyl radical (•OH), and can prevent the progression of various diseases induced by oxidative damage [11,12]. In the present study, the plasma GSH concentration and hepatic GSH, GSH/GSSG, and GSH peroxidase activities were not changed after intake of HRW. In addition, the lipid peroxidation levels in plasma and liver were not affected by HRW. Therefore, in the normal physiologic condition, intake of HRW may not affect antioxidant activity or oxidative stress in liver. These results are consistent with previous results indicating that H2 does not disturb normal cellular metabolic redox reactions [11].

Membrane transporters are effective pumps for elimination of conjugates of xenobiotics from hepatocytes into bile (e.g., Mrp2 and *p*-glycoprotein) [16] and plasma (e.g., Mrp3) [9]. Therefore, increased protein expressions of Mrp2/3 and *p*-glycoprotein also play roles in detoxification processes. These membrane proteins can be induced and can protect normal tissues from endogenous and exogenous toxic substances [16,17]. In the present study, Mrp2 and *p*-glycoprotein increased in the liver whereas Mrp3 did not change significantly after intake of HRW (Figure 4). Therefore, although the mechanism is still unknown, HRW may enhance the excretion of xenobiotics or endogenous toxic substances from the liver into bile and then increase their fecal excretion by increasing the efflux pumps of Mrp2 and *p*-glycoprotein. On the other hand, in this study, a little change on the concentrations of Ca2<sup>+</sup>, Mg2<sup>+</sup>, and SO42<sup>−</sup> between the control water and HRW groups did not have any influence on plasma mineral ions. A higher daily water drinking volume was found in the HRW group compared with the control group, suggesting HRW had better palatability (see descriptions in the Results section). Until now, there is a lack of evidence demonstrated that ions in drinking water may change the xenobiotic-metabolizing enzymes and transporters. Therefore, it is suggested that increased Mrp2 and *p*-glycoprotein expression in the liver after intake of HRW may be attributed to the molecular hydrogen.

Calcium and magnesium are important nutrients in the development and maintenance of human health. Supplementation with magnesium ion (Mg2+) from drinking water may provide substantial contributions to total intakes of Mg2<sup>+</sup> in some populations and may exert beneficial effects on reducing many diseases. A protective effect of Mg2<sup>+</sup> intake from drinking water has been demonstrated on reducing cerebrovascular disease and cardiovascular mortality in humans [18,19], especially in men with lower dietary magnesium intake [20]. A recent study demonstrated that Mg2<sup>+</sup> added to drinking water reduces blood glucose levels by inhibiting the gluconeogenesis pathway in rat liver [21]. In this study, HRW contained a higher Mg2<sup>+</sup> concentration than control water. This may be one of the explanations for the lower plasma glucose level in the HRW group, although H2 is regarded as an effective element to improve glucose intolerance in diabetic mice and some type 2 patients [22,23]. Therefore, HRW manufactured by reacting water with magnesium–carbon hydrogen storage hybrid materials may result in a higher Mg2<sup>+</sup> concentration in drinking water and, thus, may complement daily magnesium, especially in populations with magnesium deficiency.

To date, various commercial apparatuses for HRW production (e.g., manufactured by electrolyzing water or water reacted with magnesium-containing materials) are being developed. These HRW products may have a high dissolved H2 concentration and H2 stability in drinking water. However, the quality and safety of these HRW products should be a concern (e.g., undesirable flavor and/or unknown reaction products).

In summary, intake of HRW for four weeks may not change xenobiotic-metabolizing enzymes or antioxidant activity in liver. Regular consumption of HRW may enhance detoxification process, at least in part, through an increase in the efflux of toxic substances from the liver into bile. In addition to measuring dissolved H2 concentration, the present study also evaluated the water quality of HRW on biological function. Because HRW is becoming more popular worldwide, the results of the present study may provide health and safety information on HRW to consumers.
