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Effect of Hexavalent Chromium on Electron Leakage of Respiratory Chain in Mitochondria Isolated from Rat Liver

Background/Aims: In the present study, we explored reactive axygen species (ROS) production in mitochondria, the mechanism of hexavalent chromium (Cr(VI)) hepatotoxicity, and the role of protection by GSH. Methods: Intact mitochondria were isolated from rat liver tissues and mitochondrial basal respiratory rates of NADH and FADH2 respiratory chains were determined. Mitochondria were treated with Cr(VI), GSH and several complex inhibitors. Mitochondria energized by glutamate/malate were separately or jointly treated with Rotenone (Rot), diphenyleneiodonium (DPI) and antimycinA (Ant), while mitochondria energized by succinate were separately or jointly treated with Rot, DPI ‚ thenoyltrifluoroacetone (TTFA) and Ant. Results: Cr(VI) concentration-dependently induced ROS production in the NADH and FADH2 respiratory chain in liver mitochondria. Basal respiratory rate of the mitochondrial FADH2 respiratory chain was significantly higher than that of NADH respiratory chain. Hepatic mitochondrial electron leakage induced by Cr(VI) from NADH respiratory chain were mainly from ubiquinone binding sites of complex I and complex III. Conclusion: Treatment with 50µM Cr(VI) enhances forward movement of electrons through FADH2 respiratory chain and leaking through the ubiquinone binding site of complex III. Moreover, the protective effect of GSH on liver mitochondria electron leakage is through removing excess H2O2 and reducing total ROS.


Introduction
Hexavalent chromium (Cr(VI)) is a well-known highly toxic metal and environmental pollutant, and was categorized as human carcinogen by IARC (International Agency for 2 would be reduced to superoxide anion radical (O 2 -) by electron leaked from mitochondrial respiratory chain, which was further reduced to hydrogen peroxide (H 2 O 2 ) by superoxide dismutase (MnSOD) [4]. Then H 2 O 2 2 -, H 2 O 2 referred as ROS, collectively.
Mitochondria contain several ROS production sites. Determination of the production site is based on using inhibitors of electron transport. For example, if rotenone (complex I inhibitor) could increase ROS production in cells, endogenous ROS production would be from complex I. Moreover, it is well known that GSH in liver mitochondria (mtGSH) plays an important role in clearing away H 2 O 2 and preventing hepatocyte mitochondria from impairment of respiratory function and dysfunction of energy metabolism [6,7]. Mitochondria electron transport chain and the role of GSH in scavenging ROS are illustrated in Figure 1.
Previous studies strongly suggest that ROS produced in mitochondria plays a critical role in Cr(VI)-induced hepatocyte apoptosis. However, electron leakage of mitochondrial respiratory chain induced by Cr(VI) still remains unclear. In the present study, after treating isolated rat mitochondria with Cr(VI), we used glutamate/malate and succinate as substrates and a wide variety of complex inhibitors to explore the ROS production sites in mitochondria, the mechanism of Cr(VI) hepatotoxicity, and the role of GSH protection.

Materials and Methods
Materials L-glutamine, L-Malic acid and sodium succinate were purchased from Sigma (St. Louis, MO, USA).

Mitochondrial isolation
Rats received anesthesia by chloralic hydras after an overnight fast. The rat liver was removed for immediate mitochondrial isolation. Liver mitochondria were isolated by standard differential centrifugation at 4°C PH=7.4). Tissue debris and nuclei were separated from liver homogenate by centrifugation at 1000×g for 10 min. The supernatant solution was collected and centrifuged at 9000×g for 10min. The mitochondria pellet was washed once with isolation buffer and re-suspended in respiratory buffer as mitochondria 2 PO 4 , 2mM MgCl 2 , 10μM EGTA, PH=7.4). Isolated mitochondria were used immediately for measurement of respiration rate and ROS and superoxide formation.

Mitochondrial protein measurements
SF, USA) with bovine serum albumin used as concentration standards. Standards and the samples were measured in duplicates and the mean values were used to calculate the protein concentrations.

Cellular Physiology and Biochemistry
Measurement of mitochondrial purity and functionality measured purity and functionality of mitochondria isolated with our method. We detected mitochondrial purity by detection of succinate dehydrogenase (SDH) activity in liver homogenate, supernatant solution The acid, dehydrated, and embedded with epoxy resin. Thin sections were observed with a Hitachi H-600 transmission electron microscope. Mitochondrial ultra-structure was observed by electron microscope 10,000 and 30000, respectively). Then respiratory function of mitochondria was measured with Clark-type oxygen electrode. One mg of mitochondria protein was incubated in 2 ml of respiratory respiration was initiated by adding 2um ADP, and oxygen consumption was measured as the slope of oxygen consumption curves. The respiratory control ratio (RCR) were calculated as the rate of ADP-stimulated oxygen consumption (State 3 respiratory) divided by the rate of oxygen consumption (state 4 respiratory) when all the ADP has been phosphorylated.

Measurement of mitochondrial basal respiratory rate
2 respiratory chain were measured using substrate-stimulated O 2 consumption recorded with Clark-type oxygen electrode. Mitochondrial Respiration rate was measured by the slope of oxygen consumption curves after substrates were added as nmol O 2 /min/mg prot.

Statistical analysis
All data was expressed as mean±SEM. Statistical comparisons of basal respiratory rates of the mitochondrial respiratory chain were done using t test. Comparisons among multiple treatments were done P<

Mitochondrial purity and functionality
Succinate hydrogenase is tightly bound to the inner mitochondrial membrane and easily precipitated along with mitochondria. The activities of succinate dehydrogenase as that in liver homogenate ( Fig. 2A), which revealed that mitochondria were relatively pure. Mitochondrial ultra-structure observation showed that mitochondria were relatively pure Mitochondrial respiratory control ratios (RCR) were higher than 3, which was indicative of tightly coupled mitochondria (Fig. 2D) [10].

Effect of complex inhibitors on NADH respiratory chain in liver mitochondria treated with Cr(VI)
In glutamate/malate-energized mitochondria, ROS production increased in the Rot group and the Rot+DPI group and decreased in the DPI group. In mitochondria treated with Fig. 6. Effect of complex I inhibitors and complex III inhibitors on FADH 2 respiratory chain in liver mitomitochondrial FADH 2 on ROS production and electron leak rate from mitochondrial FADH 2 (E)(F) Effect of complex III inhibitors (Ant) on ROS production and electron leak rate from mitochondrial FADH 2 # P<

Effect of complex inhibitors on FADH 2 respiratory chain in liver mitochondria treated with Cr(VI)
In succinate-energized liver mitochondria, ROS production and electron leakage 2 respiratory chain. TTFA could increase ROS production and electron leakage in mitochondria The effect of Ant on FADH 2 ROS production and electron leakage in groups treated with Ant were higher than those not treated with Ant (Fig. 6E, 6F, Table 6). Effect of GSH on electron leakage from liver mitochondrial respiratory chain 2 respiratory chains in liver mitochondria were than that in groups treated with Cr(VI)+GSH (P difference in electron leakage was observed between groups treated with Cr (VI) alone and

Discussion
Mitochondria, rich in hepatocytes, provide the main source of cytosolic ATP via the oxidative phosphorylation system. As is well known, Cr(VI) could move into hepatocyte mitochondrial membrane structure and energy metabolism, and further induce cellular apoptosis [11]. Generally, the quantity of ROS generated varies under different sites, substrates and mitochondrial metabolic conditions caused by different chemicals [12]. Many studies reported that critical sites for electron leakage were the ubiquinone binding sites of complex I and complex III, and that of complex II were negligible [13,14]. Some studies also mechanism of how excess ROS is produced in hepatic mitochondria exposed to Cr(VI) has not been elaborated clearly.
Above all, we separated mitochondria from the rat liver by differential centrifugation and ensured mitochondria could maintain integrity and functionality at a suitable level during experiments. Then we used several inhibitors of complexes to characterize the mechanism of enhanced ROS production in hepatic mitochondria treated with Cr(VI). Rot, which is highly lipophilic, could easily penetrate the membrane and selectively block electrons transferring ubiquinone binding site to complex III [19]. Ant, the complex III inhibitor, acts on complex III and inhibit electron driving from cytochrome b (Cyt bH) to ubiquinone, which could increase electron leakage and ROS production [20,21].
ROS production in succinate-energized hepatic mitochondria treated with Cr(VI) were much higher than that in glutamate/malate-energized mitochondria, which may due to Xie/Zhong/Zeng/Guan/Luo: Effect of Hexavalent Chromium on Electron Leakage Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry higher basal respiratory rate of FADH 2 respiratory chain. In glutamate/malate-energized mitochondria, ROS production increased in the Rot group. It is suggested that in mitochondria under normal physiological conditions, Rot inhibit electron driving from iron-sulfur center of electron moving into the reduction center of complex I and then decrease electron leakage.
treated with Cr(VI), the results were different. ROS content and electron leakage increased in the Rot+Cr(VI) group, but not changed in the DPI+Cr(VI) group. The results suggested in mitochondria treated with Cr(VI), which suggests that Ant could inhibit electron moving from Cyt bH to ubiquinone, accumulate semiquinone and increase electron leakage [23,24]. With regard to mitochondrial FADH 2 respiratory chain, it is generally acknowledged that complex II could not release electron by itself and electron could leak from complex I when site of complex III if they move forward along respiratory chain [26]. In succinate-energized mitochondria without Cr(VI) treatment, ROS production and electron leakage decreased in the Rot group and DPI group, and increased in the TTFA group and the Ant group. It is suggested that when electron generated from succinate moves backward from complex II from complex II to ubiquinone, while Ant acts on the ubiquinone binding site of complex III, which leads to electron leakage and excess ROS generation.
As far as succinate-energized mitochondria treated with Cr(VI) are concerned, compared with the Cr(VI) group, ROS content and electron leakage in the Rot+Cr(VI) group mitochondria was irrelevant with electron moving backward from complex II to complex I. Then ROS content and electron leakage increased in the Cr(VI)+TTFA group compared with the Cr(VI) group. It is seemingly found that co-treatment of Cr(VI) and TTFA could promote electron leaked from the ubiquinone binding site of complex II, however, ROS content and electron leakage in the Cr(VI)+Rot+TTFA group and the Cr(VI)+DPI+TTFA group were of electron from complex II to complex I. Furthermore, after Ant inhibits the ubiquinone binding site of complex III in mitochondria exposed to Cr(VI), ROS content and electron leakage from FADH 2 respiratory chain in liver mitochondria through the ubiquinone binding leaking from the ubiquinone binding site of complex II .
GSH, a critical endogenous antioxidant in mitochondria, could transform H 2 O 2 into H 2 O under the effect of glutathione peroxidase and transform itself into oxidized glutathione Excess H 2 O 2 could be cleared during this cycle [27]. Cederbaum et al. found that exogenous S-adenosyl-L-methionine (SAM) supplement, as a precursor of GSH synthesis, could protect 2 respiratory chain of liver mitochondria were similar. GSH could clear over excess ROS production in liver mitochondria induced by 2 be observed on electron leakage. It is indicated that GSH do exhibit protective effects on liver mitochondria by the elimination of excess H 2 O 2 and ROS, but could not decrease electron leakage from mitochondrial respiratory chain.
In summary, our study demonstrated the sites of Cr(VI)-induced ROS production and induced the increase of electron leakage from ubiquinone binding sites of complex I and induced electron leakage from the ubiquinone binding site of complex III when the electron moved forward in the respiratory chain. GSH can alleviate Cr(VI)-induced liver mitochondrial oxidative damage through effective removal of excess H 2 O 2 and total ROS content, rather than the improvement of mitochondrial respiration function to reduce electron leakage.