Active oxygen species as factors in multistage carcinogenesis.

Oxygen, a necessary element for the life of a cell, is also the source of active states of oxygen including radicals, which can disrupt cell structure and alter cell function. Increasing evidence indicates that active oxygen species are formed in response to tumor promoters and that the cellular consequences of their actions may play a role in the process of tumor promotion. This report summarizes work from our laboratory that implicates active oxygen species derived in part from phagocytic cells in the tumor promotion process by phorbol esters and other promoters in mouse skin. Work from other laboratories indicates that phorbol ester promoters stimulate the production of active states of oxygen in mouse skin epidermal cells in vivo and in vitro. Oxidative DNA damage in epidermal cells from mice treated topically with the potent promoter phorbol myristate acetate has also been reported. The production of active states of oxygen including free radicals is discussed in relation to the mode of action of complete, first, and second stage promoters in the multistage carcinogenesis model in mouse skin.     

降钙素基因相关肽转基因预防小鼠自身免疫性糖尿病发病及其抗氧化应激机制

有证据表明,活性氧(reactive oxygens pecies,ROS)参与了胰岛β细胞自身免疫损伤,是自身免疫性糖尿病发病的重要原因之一。一氧化氮(NO)和过氧化氢(H2O2)介导了致炎性细胞因子对胰岛B细胞的损害,引起脂质过氧化反应,进而损伤胰岛细胞。降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在心肌细胞中可抑制ROS生成而具有细胞保护作用。通过CGRP裸质粒肌肉注射体细胞电针强化转基因方法,使骨骼肌持续表达CGRP,观察其对小剂量多次注射链脲佐菌素(streptozotocin,STZ)造成的小鼠自身免疫性糖尿病发病的影响,进一步测定胰腺局部活性氧含量以及抗氧化酶的改变,探讨其抗氧化应激机制。结果发现,CGRP裸质粒直接注射入小鼠双后肢胫前肌。继以程控电针刺激导入(体内电穿孔法),可使血浆和骨骼肌组织CGRP表达水平显著增高,且持续4周以上;注射STZ同时给予CGRP转基因治疗可减轻胰岛β细胞损伤,显著降低自身免疫性糖尿病的发病率及血糖水平;CGRP转基因可显著抑制自身免疫性糖尿病小鼠胰腺局部活性氧和丙二醛的生成,增加过氧化氢酶(CAT)及超氧化物歧化酶(superoxide dismutase,SOD)的活性。结果提示,CGRP裸质粒直接注射、电针辅助导人转基因可获得CGRF持续高水平的表达,能够预防小鼠自身免疫性糖尿病的发病,其机制之一可能为CGRP抑制了活性氧对胰岛β细胞的损伤。     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas

Alloxan and streptozotocin are widely used to induce experimental diabetes in animals. The mechanism of their action in B cells of the pancreas has been intensively investigated and now is quite well understood. The cytotoxic action of both these diabetogenic agents is mediated by reactive oxygen species, however, the source of their generation is different in the case of alloxan and streptozotocin. Alloxan and the product of its reduction, dialuric acid, establish a redox cycle with the formation of superoxide radicals. These radicals undergo dismutation to hydrogen peroxide. Thereafter highly reactive hydroxyl radicals are formed by the Fenton reaction. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells. Streptozotocin enters the B cell via a glucose transporter (GLUT2) and causes alkylation of DNA. DNA damage induces activation of poly ADP-ribosylation, a process that is more important for the diabetogenicity of streptozotocin than DNA damage itself. Poly ADP-ribosylation leads to depletion of cellular NAD+ and ATP. Enhanced ATP dephosphorylation after streptozotocin treatment supplies a substrate for xanthine oxidase resulting in the formation of superoxide radicals. Consequently, hydrogen peroxide and hydroxyl radicals are also generated. Furthermore, streptozotocin liberates toxic amounts of nitric oxide that inhibits aconitase activity and participates in DNA damage. As a result of the streptozotocin action, B cells undergo the destruction by necrosis.     

Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell destruction and diabetes development induced by streptozocin

Human type 1 diabetes results from the selective destruction of insulin-producing pancreatic beta cells during islet inflammation. Cytokines and reactive radicals released during this process contribute to beta-cell death. Here we show that mice with a disrupted gene coding for poly (ADP-ribose) polymerase (PARP-/- mice) are completely resistant to the development of diabetes induced by the beta-cell toxin streptozocin. The mice remained normoglycemic and maintained normal levels of total pancreatic insulin content and normal islet ultrastructure. Cultivated PARP-/- islet cells resisted streptozocin-induced lysis and maintained intracellular NAD+ levels. Our results identify NAD+ depletion caused by PARP activation as the dominant metabolic event in islet-cell destruction, and provide information for the development of strategies to prevent the progression or manifestation of the disease in individuals at risk of developing type 1 diabetes.     

HIV-1 Tat-mediated protein transduction of Cu,Zn-superoxide dismutase into pancreatic beta cells in vitro and in vivo

Reactive oxygen species (ROS) are considered an important mediator in pancreatic beta cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, we investigated the HIV-1 Tat protein transduction domain-mediated transduction of Cu,Zn-superoxide dismutase (SOD), which supplies SOD activity exogenously in pancreatic beta cells under oxidative stress. Tat-SOD fusion protein was successfully delivered into insulin-producing RINm5F cells and rat islet cells. The intracellular dismutation activities of SOD were found to increase in line with the amount of protein delivered into the cells. ROS, nitric oxide-induced cell death, lipid peroxidation, and the DNA fragmentation of insulin-producing cells were found to be significantly reduced when the cells were pretreated with Tat-SOD. Next, we examined the in vivo transduction of Tat-SOD into streptozotocin-induced diabetic mice. A single intraperitoneal injection of Tat-SOD resulted in the delivery of this biologically active enzyme to the pancreas. Moreover, increased radical scavenging activity in the pancreas was induced by multiple injections of Tat-SOD, and this enhanced the tolerance of pancreatic beta cells to oxidative stress. These results suggest that the transduction of Tat-SOD offers a new strategy for protecting pancreatic beta cells from destruction by relieving oxidative stress in ROS-implicated diabetes.     

Role of reactive oxygen species in intestinal diseases.

It is well known that reactive oxygen metabolites are generated during several pathologies, and that they are able to disturb many cellular processes and eventually lead to cellular injury. After intestinal ischemia, reactive oxygen species are produced when the ischemic tissue is reperfused. The enzyme xanthine oxidase is thought to play a key role in this process. As a result of this oxygen radical production, the permeability of the endothelium and the mucosa increases, allowing infiltration of inflammatory leukocytes into the ischemic area. Moreover, reactive oxygen species are also indirectly involved in leukocyte activation. In turn, these inflammatory cells respond with the production of oxygen radicals, which play an important role in the development of tissue injury. Thus, intestinal ischemia and reperfusion evokes an inflammatory response. Also during chronic intestinal inflammatory diseases, reactive oxygen metabolites are proposed to play an important role in the pathology. Scavenging of reactive oxygen species will thus be beneficial in these disorders.     

Oxygen radicals in the adult respiratory distress syndrome, in myocardial ischemia and reperfusion injury, and in cerebral vascular damage

Recent work suggests that oxygen radicals may be important mediators of damage in a wide variety of pathologic conditions. In this review we consider the evidence supporting the participation of oxygen radicals in the adult respiratory distress syndrome, in ischemia reperfusion injury in the myocardium, and in cerebral vascular injury in acute hypertension and traumatic brain injury. In the adult respiratory distress syndrome there is active sequestration of polymorphonuclear neutrophils in the pulmonary vascular system. There is evidence that activation of these neutrophils results in the production of oxygen radicals which injure the capillary membrane and increase permeability, leading to progressive hypoxia and decreased lung compliance which are hallmarks of the syndrome. In acute arterial hypertension or experimental brain injury oxygen radicals are important mediators of vascular damage. The metabolism of arachidonic acid is the source of oxygen free radical production in these conditions. In myocardial ischemia and reperfusion injury, the ischemic myocyte is "primed" for free radical production. With reperfusion and reintroduction of molecular oxygen there is a burst of oxygen radical production resulting in extensive tissue destruction. Myocardial ischemia--reperfusion injury shares in common with the other two syndromes activation of the arachidonic acid cascade and acute inflammation. Thus it would appear that the generation of toxic oxygen species may represent a final common pathway of tissue destruction in several pathophysiologic states.     

Glycolate-, Xanthine- and Paraquat-Mediated Inhibition of Nitrate Reductase in Detached Oat Leaves

Leaf segments of 8-day-old oat plants were incubated with 100µMglycolate or xanthine inthe dark or with 100µM paraquatin the light. These treatments were designed to enhance theinternalproduction of activated oxygen species and to allowus to study the possible role of such species in the regulationof the activity of NO₃-induced nitrate reductase (NR) in illuminatedgreen tissues. Each of the treatments inhibited NO₃-induced NR activity. HPMS(an inhibitor of glycolate oxidase) and allopurinol (an inhibitorof xanthine oxidase) protected NR from inactivation by glycolateand by xanthine. Free radical scavengers also protected NR frominactivation by xanthine and paraquat but they had no effecton the inhibition by glycolate. The activities of catalase andsuper oxide dismutase did not increase in response to the varioustreatments. Thus, while the production of active oxygen speciesappears to be stimulated under such conditions, the rates ofthe enzymatic destruction and "scavenging" of these speciesseem to remain unchanged. Hence, the endogenous levels of theseactive oxygen species can be expected to increase in the treatedleaves. We interpret our results as evidence that inhibition by glycolateof NR is caused by the H₂O₂ generated during the oxidation ofglycolate while the effects of xanthine and paraquat dependon the enhanced production of free radicals. It is concluded that activated oxygen species that are formedendogenously may play a role in the regulation of NR in greentissues. (Received August 12, 1991; Accepted February 17, 1992)     

Role of physiological antioxidants in chromium(VI)-induced cellular injury.

Chromium(VI) compounds are well known to be potent toxic and carcinogenic agents. Because chromium(VI) is easily taken up by cells and is subsequently reduced to the trivalent form, the formation of chromium(III) or other intermediate oxidation states such as chromium(V) and (IV) is believed to play a role in the adverse biological effects of chromium(VI) compounds. Recent in vitro studies have shown that this reduction process generates free radical species such as active oxygen radicals. Furthermore, physiological antioxidants are reported to modify the genotoxic and toxic effects of chromate. This article reviewed the recent in vitro and in vivo studies of the effects of antioxidants including active oxygen scavengers; glutathione; vitamins B2, E, and C, on chromate-induced injury such as DNA lesions; lipid peroxidation; enzyme inhibition; cytotoxicity; mutation; and so on. In addition, the mechanism of action of these antioxidants was discussed with respect to the formation of active oxygen radicals and paramagnetic chromium such as chromium(V) and (III). Such studies may help elucidate the mechanism of chromium(VI) toxicity as well as the mechanism of protection.     

Fas is detectable on beta cells in accelerated,but not spontaneous,diabetes in nonobese diabetic mice

Fas (CD95) is a potential mechanism of pancreatic beta cell death in type 1 diabetes. beta cells do not constitutively express Fas but it is induced by cytokines. The hypothesis of this study is that Fas expression should be measurable on beta cells for them to be killed by this mechanism. We have previously reported that up to 5% of beta cells isolated from nonobese diabetic (NOD) mice are positive for Fas expression by flow cytometry using autofluorescence to identify beta cells. We have now found that these are not beta cells but contaminating dendritic cells, macrophages, and B lymphocytes. In contrast beta cells isolated from NODscid mice that are recipients of T lymphocytes from diabetic NOD mice express Fas 18-25 days after adoptive transfer but before development of diabetes. Fas expression on beta cells was also observed in BDC2.5, 8.3, and 4.1 TCR-transgenic models of diabetes in which diabetes occurs more rapidly than in unmodified NOD mice. In conclusion, Fas is observed on beta cells in models of diabetes in which rapid beta cell destruction occurs. Its expression is likely to reflect differences in the intraislet cytokine environment compared with the spontaneous model and may indicate a role for this pathway in beta cell destruction in rapidly progressive models.     

Mammalian metallothioneins: properties and functions

Metallothioneins (MT) are a family of ubiquitous proteins, whose role is still discussed in numerous papers, but their affinity to some metal ions is undisputable. These cysteine-rich proteins are connected with antioxidant activity and protective effects on biomolecules against free radicals, especially reactive oxygen species. In this review, the connection between zinc(II) ions, reactive oxygen species, heavy metal ions and metallothioneins is demonstrated with respect to effect of these proteins on cell proliferation and a possible negative role in resistance to heavy metal-based and non-heavy metal-based drugs.     

Hyperglycemia-induced oxidative stress in diabetic complications

Reactive oxygen species are increased by hyperglycemia. Hyperglycemia, which occurs during diabetes (both type 1 and type 2) and, to a lesser extent, during insulin resistance, causes oxidative stress. Free fatty acids, which may be elevated during inadequate glycemic control, may also be contributory. In this review, we will discuss the role of oxidative stress in diabetic complications. Oxidative stress may be important in diabetes, not just because of its role in the development of complications, but because persistent hyperglycemia, secondary to insulin resistance, may induce oxidative stress and contribute to beta cell destruction in type 2 diabetes. The focus of this review will be on the role of oxidative stress in the etiology of diabetic complications.     

COX-2 inhibition prevents insulin-dependent diabetes in low-dose streptozotocin-treated mice

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease believed to be caused by an inflammatory process in the pancreas leading to selective destruction of the beta cells. Inducible cyclooxygenase (COX-2) is expressed under inflammatory conditions and its product prostaglandin E(2) (PGE(2)) is an important inflammation mediator. We report here that administration of the selective COX-2 inhibitor NS-398 prevents the onset of diabetes in mice brought on by multiple low-doses of streptozotocin (STZ). Histological observations indicated that STZ-mediated destruction of beta cells was prevented by NS-398 treatment. Delayed (day 3) administration of NS-398 was also protective in this model. No protective effect was observed when NS-398 was administered prior to a high, toxic dose of STZ. These results demonstrate the critical importance of COX-2 activity in autoimmune destruction of beta cells, and point to the fact that COX-2 inhibition can potentially develop into a preventive therapy against IDDM.     

Coxsackievirus B4-induced cytokine production in pancreatic cells is mediated through toll-like receptor 4

Coxsackievirus B4 (CBV4), a member of the Picornavirus genus, has long been implicated in the development of insulin-dependent diabetes mellitus (IDDM) caused by virus-induced pancreatic cell damage. The progressive destruction of pancreatic beta cells is responsible for the development of IDDM. It has recently been suggested that CBV4 infection can induce the production of proinflammatory cytokines, and these cytokines seem to be involved in the damage to the insulin-producing cells. In this study we investigated whether toll-like receptors (TLRs) are responsible for triggering the proinflammatory cytokine production in human pancreatic cells in response to CBV4. Here we demonstrate that CBV4 triggers cytokine production through a TLR4-dependent pathway. This interaction seems to be independent of virus attachment and cell entry.     

Detection of tandem CC-->TT mutations induced by oxygen radicals using mutation-specific PCR.

DNA lesions caused by reactive oxygen species (ROS) are considered to be one of the major contributors to DNA damage and mutagenesis. In this study, we developed a modification of allele-specific PCR to detect CC-->TT mutations caused by oxidative damage. These tandem mutations have been previously demonstrated to be indicative of oxygen damage in the absence of UV-irradiation. Using a CC target site in the rat DNA polymerase beta (pol beta) gene and a thermostable restriction enzyme that cuts the wild type sequence but not the TT mutation, we demonstrate that the TT mutation can be preferentially amplified from plasmid DNA damaged by oxygen radicals but not other DNA-damaging agents. We evaluated the potential utility of this assay in screening for mutations in cells and in analyzing those that arise during clonal proliferation in carcinogenesis.     

Inhibition of the nuclear factor-κB pathway prevents beta cell failure and diet induced diabetes in Psammomys obesus

Background

High doses of anti-inflammatory drugs, such as aspirin and salicylates, improve glucose metabolism in insulin resistant and type 2 diabetic patients. It has also been shown that the glucose lowering effect is related to the unspecific ability of these drugs to inhibit inhibitor kinaseβ (IKKβ). In this study we have investigated the effect of a selective IKKβ-inhibitor on beta cell survival and the prevention of diet induced type 2 diabetes in the gerbil Psammomys obesus (P. obesus).

Methodology/Principal Findings

P. obesus were fed a diabetes inducing high energy diet for one month in the absence or presence of the IKKβ-inhibitor. Body mass, blood glucose, HbA_1C, insulin production and pancreatic insulin stores were measured. The effects on beta cell survival were also studied in INS-1 cells and primary islets. The cells were exposed to IL-1β and subsequently reactive oxygen species, insulin release and cell death were measured in the absence or presence of the IKKβ-inhibitor. In primary islets and beta cells, IL-1β induced the production of reactive oxygen species, reduced insulin production and increased beta cell death, which were all reversed by pre-treatment with the IKKβ-inhibitor. In P. obesus the IKKβ-inhibitor prevented the development of hyperglycaemia and hyperinsulinaemia, and maintained pancreatic insulin stores with no effect on body weight.

Conclusions/Significance

Inhibition of IKKβ activity prevents diet-induced diabetes in P. obesus and inhibits IL-1β induced reactive oxygen species, loss of insulin production and beta cell death in vitro.     

Possible role of macrophage-derived soluble mediators in the pathogenesis of encephalomyocarditis virus-induced diabetes in mice.

Pancreatic islets from DBA/2 mice infected with the D variant of encephalomyocarditis (EMC-D) virus revealed lymphocytic infiltration with moderate to severe destruction of pancreatic beta cells. Our previous studies showed that the major population of infiltrating cells at the early stages of infection is macrophages. The inactivation of macrophages prior to viral infection resulted in the prevention of diabetes, whereas activation of macrophages prior to viral infection resulted in the enhancement of beta-cell destruction. This investigation was initiated to determine whether macrophage-produced soluble mediators play a role in the destruction of pancreatic beta cells in mice infected with a low dose of EMC-D virus. When we examined the expression of the soluble mediators interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in the pancreatic islets, we found that these mediators were clearly expressed at an early stage of insulitis and that this expression was evident until the development of diabetes. We confirmed the expression of these mediators by in situ hybridization with digoxigenin-labelled RNA probes or immunohistochemistry in the pancreatic islets. Mice treated with antibody against IL-1beta or TNF-alpha or with the iNOS inhibitor aminoguanidine exhibited a significant decrease in the incidence of diabetes. Mice treated with a combination of anti-IL-1beta antibody, anti-TNF-alpha antibody, and aminoguanidine exhibited a greater decrease in the incidence of disease than did mice treated with one of the antibodies or aminoguanidine. On the basis of these observations, we conclude that macrophage-produced soluble mediators play an important role in the destruction of pancreatic beta cells, resulting in the development of diabetes in mice infected with a low dose of EMC-D virus.     

The emerging role of cardiovascular risk factor-induced mitochondrial dysfunction in atherogenesis

An important role in atherogenesis is played by oxidative stress, which may be induced by common risk factors. Mitochondria are both sources and targets of reactive oxygen species, and there is growing evidence that mitochondrial dysfunction may be a relevant intermediate mechanism by which cardiovascular risk factors lead to the formation of vascular lesions. Mitochondrial DNA is probably the most sensitive cellular target of reactive oxygen species. Damage to mitochondrial DNA correlates with the extent of atherosclerosis. Several cardiovascular risk factors are demonstrated causes of mitochondrial damage. Oxidized low density lipoprotein and hyperglycemia may induce the production of reactive oxygen species in mitochondria of macrophages and endothelial cells. Conversely, reactive oxygen species may favor the development of type 2 diabetes mellitus, mainly through the induction of insulin resistance. Similarly - in addition to being a cause of endothelial dysfunction, reactive oxygen species and subsequent mitochondrial dysfunction - hypertension may develop in the presence of mitochondrial DNA mutations. Finally, other risk factors, such as aging, hyperhomocysteinemia and cigarette smoking, are also associated with mitochondrial damage and an increased production of free radicals. So far clinical studies have been unable to demonstrate that antioxidants have any effect on human atherogenesis. Mitochondrial targeted antioxidants might provide more significant results.