Hong Zhu

Somatic mutations of the mitochondrial genome in human colorectal tumours.

Alterations of oxidative phosphorylation in tumour cells were originally believed to have a causative role in cancerous growth. More recently, mitochondria have again received attention with regards to neoplasia, largely because of their role in apoptosis and other aspects of tumour biology. The mitochondrial genome is particularly susceptible to mutations because of the high level of reactive oxygen species (ROS) generation in this organelle, coupled with a low level of DNA repair. However, no detailed analysis of mitochondrial DNA in human tumours has yet been reported. In this study, we analysed the complete mtDNA genome of ten human colorectal cancer cell lines by sequencing and found mutations in seven (70%). The majority of mutations were transitions at purines, consistent with an ROS-related derivation. The mutations were somatic, and those evaluated occurred in the primary tumour from which the cell line was derived. Most of the mutations were homoplasmic, indicating that the mutant genome was dominant at the intracellular and intercellular levels. We showed that mitochondria can rapidly become homogeneous in colorectal cancer cells using cell fusions. These findings provide the first examples of homoplasmic mutations in the mtDNA of tumour cells and have potential implications for the abnormal metabolic and apoptotic processes in cancer.

Validation of Lucigenin (Bis-N-methylacridinium) as a Chemilumigenic Probe for Detecting Superoxide Anion Radical Production by Enzymatic and Cellular Systems*

Lucigenin is most noted for its wide use as a chemiluminescent detector of superoxide anion radical (O·̄2) production by biological systems. However, its validity as a O·̄2-detecting probe has recently been questioned in view of its ability to undergo redox cycling in several in vitroenzymatic systems, which produce little or no O·̄2. Whether and to what extent lucigenin redox cycling occurs in systems that produce significant amounts of O·̄2 has not been carefully investigated. We examined and correlated three end points, including sensitive measurement of lucigenin-derived chemiluminescence (LDCL), O2 consumption by oxygen polarography, and O·̄2 production by 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin trapping to characterize the potential of lucigenin to undergo redox cycling and as such to act as an additional source of O·̄2 in various enzymatic and cellular systems. Marked LDCL was elicited at lucigenin concentrations ranging from 1 to 5 μm in all of the O·̄2-generating systems examined, including xanthine oxidase (XO)/xanthine, lipoamide dehydrogenase/NADH, isolated mitochondria, mitochondria in intact cells, and phagocytic NADPH oxidase. These concentrations of lucigenin were far below those that stimulated additional O2 consumption or O·̄2 production in the above systems. Moreover, a significant linear correlation between LDCL and superoxide dismutase-inhibitable cytochrome c reduction was observed in the XO/xanthine and phagocytic NADPH oxidase systems. In contrast to the above O·̄2-generating systems, no LDCL was observed at non-redox cycling concentrations of lucigenin in the glucose oxidase/glucose and XO/NADH systems, which do not produce a significant amount of O·̄2. Thus, LDCL still appears to be a valid probe for detecting O·̄2 production by enzymatic and cellular sources.

Detection of mitochondria-derived reactive oxygen species production by the chemilumigenic probes lucigenin and luminol.

Both lucigenin and luminol have widely been used as chemilumigenic probes for detecting reactive oxygen species (ROS) production by various cellular systems. Our laboratory has previously demonstrated that lucigenin localizes to the mitochondria of rat alveolar macrophages and that lucigenin-derived chemiluminescence (CL) appears to reflects superoxide O2(-.) production by mitochondria in the unstimulated macrophages. In this study, we further examined the ability of lucigenin- and luminol-derived CL to assess O2(-.) and H2O2 formation, respectively, by isolated intact mitochondria. Mitochondria were isolated from monocytes/macrophages differentiated from monoblastic ML-1 cells. Incubation of the substrate-supported mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived CL. Luminol-derived CL was also elicited with substrate-supplemented mitochondria in the presence of horseradish peroxidase (HRP). The lucigenin-derived CL was diminished extensively by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6, 6-tetramethylpiperidine-N-oxyl and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. On the other hand, luminol-derived CL was not observed in the absence of HRP and was significantly inhibited by catalase. A spectrum of agents known to specifically affect mitochondrial respiration exhibited corresponding effects on both lucigenin- and luminol-derived CL. Taken together, our results demonstrate that with isolated mitochondria lucigenin-derived CL monitors intramitochondrial O2(-.) production by the mitochondrial electron transport chain, whereas the luminol-derived CL detects H2O2 released from the mitochondria. As such, use of both probes provides a comprehensive and clear assessment of ROS production by mitochondria.

Age-related increase in mitochondrial superoxide generation in the testosterone-producing cells of Brown Norway rat testes: relationship to reduced steroidogenic function?

Aging in Brown Norway rats is accompanied by the reduced production of testosterone by the Leydig cells, the testicular cells responsible for synthesizing and secreting this essential steroid. As yet, the mechanism by which Leydig cell steroidogenesis is reduced is unknown. Herein we assess the production of mitochondrial reactive oxygen species by intact Leydig cells isolated from the testes of young and old rats. To this end, Leydig cells were incubated with lucigenin (bis-N-methylacridinium nitrate), a probe that enters cells, localizes to mitochondria, and yields a significant chemiluminescent response following its reaction with intramitochondrial superoxide. Leydig cells from old rats elicited significantly greater lucigenin-derived chemiluminescence (LDCL) than those from young rats. Electron microscopic stereological analysis revealed that the absolute volume of mitochondria in the old cells was reduced from that in the young. These results, taken together, suggest that there are age-related changes in the production of reactive oxygen species by the mitochondria of Leydig cells, with those of old Leydig cells producing significantly greater levels than those of young Leydig cells. The results are consistent with the proposal that mitochondrial-derived reactive oxygen may play a role in the irreversible decline in the ability of old Leydig cells to produce testosterone.

Differences in xenobiotic detoxifying activities between bone marrow stromal cells from mice and rats: implications for benzene-induced hematotoxicity.

Benzene is a human carcinogen; exposure to benzene can result in aplastic anemia and leukemia. Data from animal models are frequently used in the risk assessment for benzene. In rodent studies, mice have been shown to be more sensitive to benzene-induced hematotoxicity than rats. In this regard, we have observed that bone marrow stromal cells from mice were significantly more susceptible to the cytotoxicity induced by the benzene metabolites hydroquinone (HQ) and benzoquinone (BQ) than cells from rats. Since cellular glutathione (GSH) and quinone reductase (QR) are known to play critical roles in modulating HQ-induced cytotoxicity, we have measured the GSH content and the QR and glutathione S-transferase (GST) activity in stromal cells from both species. In rat cells, the GSH content and the QR specific activity were 2 and 28 times as much as those from mice, respectively. GSH and QR in both mouse and rat stromal cells were inducible by 1,2-dithiole-3-thione (D3T). D3T pretreatment of both mouse and rat stromal cells resulted in a marked protection against HQ-induced toxicity. Pretreatment of both mouse and rat stromal cells with GSH ethyl ester also provided a dramatic protection against HQ-induced toxicity. Conversely, dicoumarol, an inhibitor of QR, enhanced the HQ-induced toxicity in stromal cells from both mice and rats, indicating an important role for QR in modulating HQ-induced stromal toxicity in both species. Buthionine sulfoximine (BSO), which depleted GSH significantly in both species, potentiated the HQ-induced toxicity in mouse but not in rat stromal cells. Surprisingly, incubation of stromal cells with BSO resulted in a significant induction of QR, especially in rats. The failure of BSO to potentiate HQ-induced toxicity in rat stromal cells may be due to the concomitant induction of QR by BSO. Overall, this study demonstrates that the differences in stromal cellular GSH content and QR activity between mice and rats contribute to their respective susceptibility to HQ-induced cytotoxicity in vitro, and may be involved in the greater in vivo sensitivity of mice to benzene-induced hematotoxicity.

A Simple Bioluminescence Imaging Method for Studying Cancer Cell Growth and Metastasis after Subcutaneous Injection of Lewis Lung Carcinoma Cells in Syngeneic C57BL/6 Mice

In vivo imaging of cancer cell growth and invasion is instrumental in studying cancer cell behavior and in developing effective anticancer agents. In this ROS Protocols article, we report the experimental protocol and steps involving the implantation of luciferase-expressing Lewis lung carcinoma (LLC) cells in normal syngeneic C57BL/6 mice. Using the Berthold NightOwl LB981 in vivo imaging system, we observe the time-dependent growth and invasion of the lung cancer cells following subcutaneous injection of luciferase-expressing LLC cells. The three-dimensional image and counts of photon emission of the tumor mass are obtained to estimate the relative size of the tumor. Ex vivo imaging of the isolated lungs supplemented with D-luciferin and adenosine triphosphate (ATP) is obtained to determine lung metastasis of the LLC cells. The LLC cell load in entire mouse lungs is further determined by quantitative bioluminometry with a concurrently run standard curve of the number of LLC cells versus bioluminescence intensity. This in vivo imaging system in live mice, in combination with ex vivo imaging of isolated lungs as well as quantitative bioluminometry of target tissues, may provide important information on the in vivo cancer cell dynamics in immunocompetent syngeneic C57BL/6 mice and offer a valuable tool for studying experimental anticancer agents, including redox-modulating compounds, which are promising anticancer modalities.

Graphene Quantum Dots Protect against Copper Redox-Mediated Free Radical Generation and Cardiac Cell Injury

In this work, we investigated the effects of graphene quantum dots (GQDs) on copper redox-mediated free radical generation and cell injury. Using electron paramagnetic resonance (EPR) spectrometry in conjunction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, we found that GQDs at a concentration as low as 1 μg/ml significantly inhibited Cu(II)/H2O2-mediated hydroxyl radical formation. GQDs also blocked Cu(II)-catalyzed nucleophilic addition of H2O to DMPO to form a DMPO-OH adduct in the absence of H2O2, suggesting a potential for GQDs to inhibit copper redox activity. Indeed, we observed that the presence of GQDs prevented H2O2-mediated reduction of Cu(II) to Cu(I) though GQDs themselves also caused the reduction of Cu(II) to Cu(I). To further investigate the effects of GQDs on copper redox activity, we employed the Cu(II)/hydroquinone system in which copper redox activity plays an essential role in the oxidation of hydroquinone to semiquinone radicals with consequent oxygen consumption. Using oxygen polarography as well as EPR spectrometry, we demonstrated that the presence of GQDs drastically blocked the oxygen consumption and semiquinone radical formation resulting from the reaction of Cu(II) and hydroquinone. These results suggested that GQDs suppressed free radical formation via inhibiting copper redox activity. Lastly, using cultured human cardiomyocytes, we demonstrated that the presence of GQDs also protected against Cu(II)/H2O2-mediated cardiac cell injury as indicated by morphological changes (e.g., cell shrinkage and degeneration). In conclusion, our work shows, for the first time, the potential for using GQDs to counteract copper redox-mediated biological damage.

Assessing underlying mechanisms of quinoid-induced hematopoietic cell toxicity.

Within the bone marrow are a number of cell populations that can serve as potential targets of xenobiotics and/or their metabolites (Trush et al., 1996). These include the hematopoietic and lymphopoietic stem cells, committed progenitors, mature functional blood cells and the cells that comprise the bone marrow stromal microenvironment. The altered function of different populations probably results in the manifestation of different toxicities. Two environmental chemicals that have been linked with toxicity to the bone marrow are benzene and benzo(a)pyrene (BP). There is continued concern about human exposure to benzene arising from a variety of sources including certain occupational settings, hazardous waste sites, automobiles and cigarette smoking. Exposure of humans to benzene is associated with the development of aplastic anemia and leukemia. Polycyclic aromatic hydrocarbons (PAHs), such as BP, have been shown to be myelotoxic in animals (Legraverend et. al., 1983). For example DBA/2 mice, with the Ahd/Ahdgenotype, develop an aplastic anemia-like condition and leukemia in response to orally administered benzo(a)pyrene. In this regard, diet is considered a major route of exposure to PAHs in humans (Buckley and Lioy, 1992). Whether dietary PAHs pose a risk to human bone marrow in poorly investigated from either an experimental or an epidemiologic perspective. The bone marrow toxicity of both benzene and BP has been linked to their biotransformation. Interestingly, both compounds are metabolized to quinone derivatives: benzene to hydroquinone; BP to 1,6-, 3,6-, and 6,12-quinone. Because of the importance of the bone marrow stromal microenvironment, we have studied the toxicity of hydroquinone and BP-derived quinones to stromal cells obtained from DBA/2 mice (Twerdok et al., 1992; Zhu et al., 1995).