锰超氧化物歧化酶在食管鳞癌组织中的表达及其临床意义

目的 探讨锰超氧化物歧化酶(MnSOD)在食管鳞癌组织中的表达及其与食管鳞癌临床病理特征及生物学行为的关系.方法 采用免疫组织化学SP法和逆转录聚合酶链反应(RT-PCR),检测45例食管鳞癌组织及距肿瘤组织边缘＞5 cm的正常组织中MnSOD蛋白和mRNA的表达水平,并分析其与食管癌临床病理特征及生物学行为之间的关系.结果 MnSOD蛋白在45例食管鳞癌组织中的阳性表达率为31.1%,明显低于正常食管组织中的阳性表达率(86.7%,P＜0.05).MnSOD mRNA在45例食管鳞癌组织的相对表达量为0.310±0.036,亦明显低于其在正常食管组织中的相对表达量(0.482±0.053,P＜0.05).MnSOD蛋白和mRNA的表达与食管鳞癌的病变长度、浸润深度和组织学分级有关,即病变长度越长、浸润深度越深、组织学分级越高者,MnSOD表达水平越低(均P＜0.05).MnSOD蛋白和mRNA的表达与食管鳞癌的淋巴结转移状况、病变部位和病变类型均无关(均P＞0.05).结论 食管鳞癌组织中,MnSOD蛋白和mRNA的表达水平均明显降低,检测MnSOD的表达对食管癌的临床治疗和预后判断有一定的参考价值.     

Colocalization of MnSOD expression in response to oxidative stress

The loss of manganese superoxide dismutase function has been associated with increased incidence of Barrett's esophagus and esophageal adenocarcinoma. In previous studies, we have demonstrated that loss of MnSOD resulted in severe esophageal damage by both endogenous and exogenous bile. However, the alterative manner of MnSOD in esophageal epithelium is largely unknown. In this study, we investigated the expression and localization of MnSOD in response to the exposure to bile salts in an esophageal epithelial cell line. Het‐1A cells were seeded at 5 × 10⁵ and 10⁷ and incubated with taurocholate, cholate, glycochlate, deoxycholate, and the mixture of these bile salts. Mitochondria and cytoplasma were separated, and the expression and localization of MnSOD was determined by Western blot and immunocytochemical assay. Proliferation rates were strongly inhibited in the groups with taurocholate and bile salts mixture at 4 h, with 0.367 ± 0.042 and 0.396 ± 0.046, respectively, compared to 0.684 ± 0.054 in untreated groups (P < 0.05). An increased apoptotic rate compared to untreated group (3.65 ± 0.59) were significantly increased in taurocholate group and in bile salts mixture group were 33.62 ± 10.25 and 31.52 ± 8.97 at 4 h, respectively (P < 0.05). The protein level of MnSOD in mitochodria was increased at 4 h, but with a decreased enzymatic activity after bile salts treatment. Cytoplasmic MnSOD was detected in the cells with bile salts treatment. Immunocytochemical staining demonstrated that esophageal epithelial cell underwent morphological alteration and MnSOD relocalization after bile salts treatment. This is the first study to demonstrate cellular cytosolic MnSOD expression and that this relocalization to the cytosol is a cause for decreased MnSOD enzymatic activity. This suggests that bile salts may contribute to the dysfunction of mitochondria, by enzymatically inhibiting of MnSOD localization and thus activation in the mitochondria. © 2009 Wiley‐Liss, Inc.     

Plasma antioxidant concentration,not superoxide dismutase polymorphism,is associated with breast cancer risk in Korean women

Disturbances in redox regulation are suggested to be involved in the development of breast cancer. We conducted a hospital-based case-control study to examine the hypothesis that lower plasma antioxidant concentration is related to higher risk of breast cancer and that genetic polymorphism of manganese superoxide dismutase (SOD2) modifies the relationship between breast cancer risk and plasma antioxidant. Genotyping for SOD2 Val16Ala polymorphism was performed by a 5′ exonuclease assay, and plasma concentrations of retinol, carotenoids, and tocopherols were determined by high-performance liquid chromatography. Unconditional logistic regression models were used to estimate crude and multivariate odds ratios with a 95% confidence interval. The variant allele frequencies of SOD2 Val16Ala (TC or CC type) were 26% for the control subjects and 23% for the breast cancer patients, and the variant genotype was not a risk factor for breast cancer. Higher plasma retinol concentration was associated with a lower risk, whereas higher plasma β-carotene, α-tocopherol, or γ-tocopherol concentrations were associated with a higher risk of breast cancer. SOD2 CT or CC genotype was associated with lower risk (odds ratio, 0.53; 95% confidence interval, 0.30-0.93; P for interaction = .025) in subjects with low plasma γ-tocopherol concentration. Our findings suggest that the SOD2 Val16Ala variant is not related to the risk of breast cancer in Korean women; however, it may affect the association between plasma γ-tocopherol levels and the risk of breast cancer.     

Tumor necrosis factor alpha-mediated nitric oxide production enhances manganese superoxide dismutase nitration and mitochondrial dysfunction in primary neurons: an insight into the role of glial cells

Tumor necrosis factor-alpha (TNF-alpha), a ubiquitous pro-inflammatory cytokine, is an important mediator in the immune-neuroendocrine system that affects the CNS. The present study demonstrates that treatment with TNF-alpha activates microglia to increase TNF-alpha production in primary cultures of glial cells isolated from wild-type (WT) mice and mice deficient in the inducible form of nitric oxide synthase (iNOSKO). However, mitochondrial dysfunction in WT neurons occurs at lower concentrations of TNF-alpha when neurons are directly treated with TNF-alpha or co-cultured with TNF-alpha-treated microglia than iNOSKO neurons similarly treated. Immunofluorescent staining of primary neurons co-cultured with TNF-alpha-treated microglia reveals that the antioxidant enzyme in mitochondria, manganese superoxide dismutase (MnSOD), is co-localized with nitrotyrosine in WT but not in iNOSKO primary neuronal cells. Importantly, the percentage of surviving neurons is significantly reduced in WT neurons compared with iNOSKO neurons under identical treatment conditions. Together, the results suggest that TNF-alpha activates microglia to produce high levels of TNF-alpha and that production of nitric oxide (NO) in neurons is an important factor affecting MnSOD nitration and subsequent mitochondrial dysfunction.     

锰超氧化物歧化酶基因的多态性与食管癌发生和发展的关系

目的 探讨锰超氧化物歧化酶(MnSOD)基因单核苷酸多态性与食管癌的发生及病变进展的关系.方法 采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法,分析103例食管癌患者(食管癌组)和195例正常对照人群(对照组)的MnSOD基因启动子区上游第9位点单核苷酸多态性,比较不同基因型与食管癌发病风险以及病变部位、病变长度、最大直径和临床分期之间的关系.结果 在食管癌组中,MnSOD基因启动子区上游第9位点变异基因型(TC+CC)分布的频率为28.2%,明显高于对照组(17.4%;X~2=4.645,P＜0.05);与携带TT基因型者相比,携带C等位基因(TC+CC)的个体患食管癌的风险增加了1.889倍(95%CI为1.052～3.391).食管癌组中,病变长度≤5 cm者,变异基因型分布的频率为16.3%;病变长度＞5 cm者,变异基因型分布的频率为36.7%,差异有统计学意义(X~2=5.147,P＜0.05).MnSOD 9 T→C变异与食管癌的病变长度呈正相关,但与食管癌的病变部位、最大直径以及临床分期之间无明显的相关性.结论 MnSOD 9 T→C变异增加了食管癌的发病风险,其可以作为食管癌遗传易感性的标志物,用于易感个体的预警,并且该基因的多态性可能与食管癌病变的纵向进展相关.     

前列腺癌与增生组织中MnSOD基因片段的克隆分析

目的:探讨前列腺癌(Pca)及良性前列腺增生(BPH)组织中MnSOD基因的突变情况。方法:用RT-PCR的方法分别检测30例Pca组织中和20例BPH组织中SOD2基因片段的突变情况。结果:在6例前列腺癌组织标本和4例良性前列腺增生(BPH)组织中发现SOD2基因的点突变位点,其中前列腺癌组织标本(P3)中发现了SOD2基因的两个相连的点突变位点:372nt G---T颠换和373nt T---G颠换。结论:MnSOD基因的突变在前列腺癌的发生和发展过程中可能具有重要意义。     

MnSOD 9Ala/Val基因多态性与冠心病的关系

目的探讨锰超氧化物歧化酶9 Ala/Val(MnSOD9 Ala/Val)基因多态性与冠心病、总超氧化物歧化酶(T-SOD)和锰超氧化物歧化酶(MnSOD)活性以及丙二醛(MDA)浓度的关系。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测262例冠心病患者和100例对照组的MnSOD9 Ala/Val基因多态性的基因型,采用比色法测定血浆T-SOD、MnSOD活性以及MDA浓度。结果与对照组比较,冠心病患者的血浆T-SOD和MnSOD活性[(23.61±16.51)U/ml与(44.01±22.68)U/ml,P<0.001和(21.56±13.11)U/ml与(28.79±8.65)U/ml,P<0.001]明显降低,MDA浓度显著增高[(2.47±0.73),(2.15±0.55)nmol/ml,P<0.01];冠心病患者的MnSOD 9 AA基因型和A等位基因频率较对照组明显增多(64.2%与43.0%,P=0.001和80.0%与67.0%,P=0.014);MnSODAA基因型的MnSOD活牲较VV基因型明显降低[(22.87±13.47)与(32.04±9.19)U/ml,P<0.01)];MnSOD与MDA负相关(r=-0.15,P<0.01)。结论冠心病患者的血浆T-SOD和MnSOD活性明显降低,MDA浓度显著增高;MnSOD 9 Ala/Val基因多态性AA基因型的血浆MnSOD活性降低。     

Bilobalide protects astrocytes from oxygen and glucose deprivation‐induced oxidative injury by upregulating manganese superoxide dismutase

Bilobalide (BB), a constituent of the Ginkgo biloba extract, is a neuroprotective agent with multiple mechanisms of action. To further explore the potential therapeutic effects of BB in stroke, we investigated its effects on primary astrocytes using the oxygen and glucose deprivation‐reoxygenation (OGD‐R) model. Cell viability was measured by lactate dehydrogenase release assay and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. Cell death was measured by annexin 5 conjgated with fluorescein isothiocyanate (V‐FITC) assay, and reactive oxygen species (ROS) production was measured by 2′,7′‐Dichlorodihydrofluorescein Diacetate (DCFH‐DA) probe. Manganese superoxide dismutase (MnSOD) expression was measured by western blot and immunofluorescence. Mitochondrial membrane potential was monitored using JC‐1 staining. Our results show that OGD‐R downregulated MnSOD and impaired mitochondrial function, which further enhanced ROS production in primary astrocytes. As a result, cell viability was compromised, and cell death increased. BB treatment protected astrocytes from those injuries mainly by restoring MnSOD level as MnSOD inhibitor abolished the effects of BB. In conclusion, we demonstrated that OGD‐R induced astrocytic injury, but BB increased the expression of MnSOD, the ROS scavenger, to reverse the exacerbated astrocytic injury.     

Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells

Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24 h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.     

In silico assessment of the potential allergenicity of transgenes used for the development of GM food crops

Genetically modified (GM) crops require allergenicity and toxicity assessment of the novel protein(s) to ensure complete safety to the consumers. These assessments are performed in accordance with the guidelines proposed by Codex (2003) and ICMR (2008). The guidelines recommend sequence homology analysis as a preliminary step towards allergenicity prediction, later in vitro experiments may be performed to confirm allergenicity. In the present study, an in silico approach is employed to evaluate the allergenic potential of six transgenes routinely used for the development of GM food crops. Among the genes studied, manganese superoxide dismutase (MnSOD) and osmotin shares greater than 90% identity with Hev b 10 and Cap a 1w, respectively. Chitinase shares greater than 70% identity with allergens namely Pers a 1 and Hev b 11, and fungal chitinase showed significant IgE binding with 7 of 75 patients’ sera positive to different food extracts. Glucanases (alfalfa, wheat) and glycine betaine aldehyde dehydrogenase gene share 50% homology with allergens like – Ole e 9, Cla h 10 and Alt a 10. The results demonstrate the allergenic potential of six genes and can serve as a guide for selection of transgenes to develop GM crops.