High levels of reactive oxygen metabolites in colon cancer tissue: analysis by chemiluminescence probe.

Reactive oxygen metabolites (ROM) have been postulated to contribute to the development of various carcinomas, including colon cancer. Indeed, the effects of ROM scavengers are being tested for chemoprevention of adenocarcinoma of the colon. However, there has been no evidence to indicate that high levels of ROM are indeed present in cancerous tissue. In this study, we used a chemiluminescence probe to estimate ROM levels in cancerous and neighboring noncancerous colonic tissues from seven patients with colon cancer. Cancerous tissues contained significantly (p less than 0.05) more luminol-enhanced chemiluminescence (4,808 +/- 2,282 counts/min/mg protein) than neighboring noncancerous tissues (2,175 +/- 1,111). The addition of an ROM scavenger, catalase (2, 4, and 8 micrograms/ml), to the tissue suspension inhibited chemiluminescence produced by both noncancerous (-74%, -85%, and -71%) and cancerous (-11%, -61%, and -53%) tissues. This study shows that colonic cancerous tissue contains high levels of ROM, which may play an important role in the pathogenesis of colon cancer.     

化学发光法检测胃窦粘膜氧自由基及分类

目的探讨幽门螺杆菌对胃粘膜氧自由基的影响及变化。方法用化学发光法对１４２例ＨＰ（＋）和７２例ＨＰ（－）胃炎及溃疡病患者的胃窦粘膜进行氧自由基的测定。并分别用叠氮钠、超氧歧化酶、硫脲和过氧化氢酶对氧自由基进行分类测定。结果ＨＰ（＋）组胃窦粘膜发光值中位数为４２．３（９５％ＣＩ：８．７～２５６．２）ｃｏｕｎｔｓ·ｍｇ－１·ｍｉｎ－１,ＨＰ（－）组为１．６（９５％ＣＩ：－２．１～１１．３）ｃｏｕｎｔｓ·ｍｇ－１·ｍｉｎ－１,ＨＰ（＋）组明显高于ＨＰ（－）组,Ｐ＜０．０１。按炎症程度２１例中度炎症ＨＰ（＋）发光值中位数为５６．７（９５％ＣＩ：１４．２～１５７．３）ｃｏｕｎｔｓ·ｍｇ－１·ｍｉｎ－１,１８例ＨＰ（－）为２．３（９５％ＣＩ：－２．１～１１．８）ｃｏｕｎｔｓ·ｍｇ－１·ｍｉｎ－１。抑制试验显示叠氮钠、超氧歧化酶、硫脲和过氧化氢酶均能显著抑制氧自由基（Ｐ＜０．０５）,其中以叠氮钠最为明显。结论幽门螺杆菌感染时引起胃粘膜氧自由基增多。用化学发光法测定微量活组织中氧自由基比较敏感。抑制试验可以检测到不同的氧自由基。     

Relationship between obesity and serum reactive oxygen metabolites in adolescents

Objectives

Various cross-sectional studies have revealed a significant positive relationship between systemic oxidative stress and obesity-related indices such as body mass index (BMI, kg/m²). However, little is known of the role of oxidative stress during adolescence. The aim of this study was to determine the association between obesity and serum reactive oxygen metabolites (ROM) in adolescents.

Method

A total of 595 healthy junior high school students from northern Japan were enrolled in the study. Oxidative stress was evaluated by measuring serum levels of ROM. Obesity indices included BMI and percentage body fat (PBF). The analyses were stratified by sex and controlled for age and menarche. Partial correlation coefficients and analysis of covariance were also analyzed.

Results

In female students, ROM levels increased with increasing BMI and PBF. Therefore, ROM levels were significantly higher in the underweight group than in the BMI-classified overweight–obese (P < 0.001) and normal weight groups (P < 0.05). ROM levels were significantly higher in the high PBF group than in the underweight (P < 0.05) and normal groups (P < 0.001).

Conclusion

The results of this study show that, regardless of menarche, obesity indicators such as BMI and PBF are correlated with the level of oxidative stress in female adolescents.     

Characteristics of reactive oxygen metabolites in serum of early teenagers in Japan

Objectives

The relationship between the incidence of cardiovascular disease and the state of oxidative stress in blood has been studied to some extent. Several lines of evidence underscore the importance of primary prevention of cardiovascular disease beginning in childhood. However, little is known about the current state of oxidative stress in childhood. This study was carried out to determine the current state of the level of reactive oxygen metabolites (ROM) in serum of early teenagers.

Methods

This study enrolled 595 healthy junior high school students from the town of Nanbu located in northern Japan. Oxidative stress was evaluated by measuring the serum level of ROM, and antioxidant capacity was evaluated by measuring the serum level of biological antioxidant potential (BAP).

Results

Although the ROM level in female students [308.6 ± 63.1 Carratelli units (U.CARR)] was slightly higher than that in males (299.9 ± 55.2 U.CARR), the difference was not statistically significant. The BAP level in males was significantly higher than that in females. The levels of ROM and BAP detected in males in the first grade were higher than those in the other grades. In females, only first-graders’ BAP was higher than that in other grades.

Conclusions

The current study found that the ROM level in males was negatively correlated with grade. These results suggest the presence of factor(s) that increase oxidative stress in Japanese puberty.     

稽留流产绒毛中线粒体膜电位和活性氧的变化

目的:通过对稽留流产患者绒毛滋养叶细胞中线粒体膜电位(mitochondrial membrane potential MMP)、活性氧物质(reactive oxygen species ROS)的测定,探讨MMP、ROS与稽留流产的关系。方法:稽留流产患者20例作为研究组,正常早孕要求流产妇女20例作为对照组。研究组20例胚胎稽留宫内时间<4周。应用荧光分光光度计和流式细胞仪检测研究组及对照组绒毛滋养叶细胞中线粒体膜电位(MMP)、活性氧物质(ROS)的水平。结果:与正常早孕妇女相比较,稽留流产患者绒毛滋养叶细胞中MMP水平显著降低(P<0.05),ROS水平显著增高(P<0.05)。结论:绒毛滋养叶细胞中线粒体膜电位下降、活性氧物质增多可能是稽留流产发生的信号。     

Effects of combined radiofrequency radiation exposure on levels of reactive oxygen species in neuronal cells

The objective of this study was to investigate the effects of the combined RF radiation (837 MHz CDMA plus 1950 MHz WCDMA) signal on levels of intracellular reactive oxygen species (ROS) in neuronal cells. Exposure of the combined RF signal was conducted at specific absorption rate values of 2 W/kg of CDMA plus 2 W/kg of WCDMA for 2 h. Co-exposure to combined RF radiation with either H₂O₂ or menadione was also performed. The experimental exposure groups were incubator control, sham-exposed, combined RF radiation-exposed with or without either H₂O₂ or menadione groups. The intracellular ROS level was measured by flow cytometry using the fluorescent probe dichlorofluorescein diacetate. Intracellular ROS levels were not consistently affected by combined RF radiation exposure alone in a time-dependent manner in U87, PC12 or SH-SY5Y cells. In neuronal cells exposed to combined RF radiation with either H₂O₂ or menadione, intracellular ROS levels showed no statically significant alteration compared with exposure to menadione or H₂O₂ alone. These findings indicate that neither combined RF radiation alone nor combined RF radiation with menadione or H₂O₂ influences the intracellular ROS level in neuronal cells such as U87, PC12 or SH-SY5Y.     

Production and consumption of reactive oxygen species by fullerenes

Reactive oxygen species (ROS) are one of the most important intermediates in chemical, photochemical, and biological processes. To understand the environmental exposure and toxicity of fullerenes better, the production and consumption of ROS (singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals) by Buckminster fullerene (C(60) ) and fullerenol were investigated in aqueous systems. Fullerenol exhibits higher photoproduction efficiency of singlet oxygen and superoxide than aqueous suspensions of C(60) aggregates (aqu/nC(60) ), and this higher efficiency results in higher steady-state concentrations of these two ROS. Transmission electron microscopy indicates that the C(60) molecules in aqu/nC(60) are much more closely packed than the C(60) cages in fullerenol. These observations provide additional evidence that the lower ROS production efficiency of aqu/nC(60) is attributable primarily to efficient self-quenching of C(60) triplet states. Production of singlet oxygen by aqu/nC(60) is accelerated by increasing oxygen concentration and in part is sensitized by fluorescent photoproducts that accumulate during irradiation. The fullerenes react slowly with singlet oxygen (second-order rate constant <4?×?10(5) M(-1) s(-1) ), but react rapidly with hydroxyl radicals (second-order rate constants of 5.4?×?10(9) and 4?×?10(8) M(-1) s(-1) for aqu/nC(60) and fullerenol, respectively). These results show that environmental conditions, including light exposure and oxygen concentration, have the potential to impact the generation of toxic ROS by fullerenes.     

胃癌患者胃组织和血清中葡萄糖代谢产物的变化

目的 探讨胃癌患者胃组织和血清中糖代谢相关产物的变化及其意义。方法 采用气相色谱联合质谱分析技术对40例胃癌患者手术切除的胃癌组织和血清中糖代谢相关产物进行定量分析,并与配对的胃癌患者癌旁正常胃黏膜组织和健康体检者血清进行对照,对胃癌组织和血清的代谢物进行相关性分析。结果 胃组织中发现5种代谢物,分别为琥珀酸、延胡索酸、L-(+)-乳酸、α-酮戊二酸和D-葡萄糖,其中延胡索酸、L-(+)-乳酸和α-酮戊二酸在胃癌和正常胃黏膜组织中分别为（0.332±0.136）%和（0.276±0.095）%、（0.606±0.254）%和（0.408±0.184）%、（0.856±0.583）%和（0.534±0.421）%,3种代谢物在胃癌组织中高于正常胃黏膜组织,差异均有统计学意义(P值分别为0.043、0.033、0.022);琥珀酸和D-葡萄糖在两种组织中差异均无统计学意义(P均＞0.05)。血清中发现6种代谢物参与糖代谢,分别为L-(+)-乳酸、延胡索酸、2-氧-甲磺酰基阿拉伯糖、D-吡喃葡萄糖苷、D-吡喃果糖、D-葡萄糖,其中延胡索酸和2-氧-甲磺酰基阿拉伯糖在胃癌组和健康对照组血清中分别为（0.165±0.329）%和（0.307±0.205）%、（0.152±0.172）%和（0.250±0.217）%,胃癌组低于健康对照组,差异均有统计学意义(P值分别为0.030、0.049);其余4种代谢物在两组间差异均无统计学意义(P均＞0.05)。在胃癌患者的组织和血清中均检测到L-(+)-乳酸、延胡索酸和D-葡萄糖,相关性分析显示胃癌组织和血清中L-(+)-乳酸(r=-0.095,P=0.617)、延胡索酸（r=-0.056,P=0.768）和D-葡萄糖（r=-0.088,P=0.725)均无相关性。结论 胃癌组织和血清的糖代谢均发生了不同程度的变化,但两者无相关性。     

Lrig1在结肠癌组织和细胞株中表达意义

目的 探讨富含亮氨酸重复序列免疫球蛋白样蛋白1（Lrig1）在结肠癌组织和细胞株中的表达,并比较其与正常结肠组织和正常结肠上皮细胞表达的差异性。方法 采用免疫组化、逆转录聚合酶链式反应（RT-PCR）和蛋白印迹（WB）方法检测结肠癌组织和癌旁正常组织以及各细胞株中的表达水平,并比较其差异性。结果 免疫组化结果显示,结肠癌组织中Lrig1的表达水平（31.43%,22/70）明显低于癌旁正常结肠组织（86.11%,31/36）。Lrig1在结肠癌组织中的表达与分化、分期及有无淋巴结转移有关（P<0.05）,与性别、年龄和肿瘤大小无关（P>0.05）。RT-PCR结果显示,与正常肠上皮细胞株NCM460（0.95±0.12）比较,Lrig1 mRNA在结肠癌细胞株SW480和HCT116中相对表达量降低,分别为（0.65±0.09）和（0.47±0.06）,组间比较具有统计学意义（P<0.05）。WB的结果显示,与NCM460（0.98±0.16）比较,Lrig1蛋白在SW480和HCT116细胞中的相对表达量降低,分别为（0.71±0.06）和（0.54±0.05）,组间比较差异具有统计学意义（P<0.05）。结论 Lrig1在结肠癌的发生和发展中可能起着抑癌基因的作用。     

鸡肉组织中二甲硝咪唑残留物测定

目的 研究染毒后鸡肉组织中二甲硝眯唑（DMz）的残留情况，可能存在的原型药物及其自由代谢产物、结合残留物。方法 利用生物酶解技术、化学技术对样品进行处理，得到游离态及结合态残留物组分，进一步采用高效液相色谱-电喷雾离子化-质谱（HPLC-ESI-MS）法检测其组成结构。结果 在染毒动物鸡体12h内肌肉组织样品中发现有原型药物DMZ（m／z 141），较多的游离羟基化代谢物2-羟基二甲硝咪唑（DMZO）（m／z 157）及少量的去甲基化游离代谢物2-甲基-5硝基咪唑（m／z 127）的存在，未发现明显的葡萄糖苷酸化及磺化代谢物存在。结论 DMZOH为DMZ的主要代谢物，未发现明显的结合态残留物存在。     

Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis

The aim of the present study was to evaluate the effect of different vitrification protocols on reactive oxygen species (ROS) and apoptosis in human ovarian tissue. Human ovarian tissue pieces were exposed to different vitrification solutions. The intracellular redox state level was measured using the fluorescent dye dichlorodihydrofluorescein diacetate. Imaging of apoptotic cells was monitored by anti-caspase-3 immunolabelling after vitrification and warming. Following equilibration in either 40% ethylene glycol (EG) (v/v), 0.35 M sucrose + 10% egg yolk extract (v/v) or 40% EG (v/v), 18% Ficoll-70 (w/v) + 0.35 M sucrose for 6 min, ovarian pieces were cooled to -196 degrees C using four different protocols. Tissue that was cooled very rapidly (plunged directly into liquid nitrogen in straws or on grids or plunged directly into metal filings precooled to -196 degrees C) showed no statistically significant increase in either tissue ROS levels or the number of apoptotic cells after warming. In contrast, cooling using a less rapid method (nitrogen vapour at -120 degrees C) resulted in significantly elevated ROS levels and apoptosis after warming. There were no significant differences between the two vitrification solutions. This indicates that human ovarian tissue pieces should be vitrified using very rapid cooling rates.     

Sequence-specific DNA damage by reactive oxygen species: Implications for carcinogenesis and aging

Reactive oxygen species (ROS) generated by environmental chemicals can cause sequence-specific DNA damage, which may lead to carcinogenesis and aging. We investigated the mechanism of DNA damage by environmental chemicals (catechol, propyl gallate and bisphenol-A), homocysteine and UVA radiation using human cultured cell lines and³²P-labeled DNA fragments. Carcinogenic catechol induced piperidine-labile sites frequently at thymine residues in the presence of Cu(II) and NADH. Furthermore, catechol increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, a hydrogen peroxide (H₂O₂)-resistant cell line derived from HL-60. Thus, it is concluded that oxidative DNA damage through generation of H₂O₂ plays an important role in the carcinogenic process of catechol. In addition, an environmental factor, bisphenol-A, and a dietary factor, propyl, gallate, also induced sequence-specific DNA damage via ROS generation. UVA, as well as UVB, contributes to photoaging. In humans, telomere shortening is believed to be associated with cell senescence. In this study, we investigated the shortening rate of telomeres in human WI-38 fibroblasts exposed to UVA irradiation. The telomere length (as measured by terminal restriction fragment length) in WI-38 fibroblasts irradiated with UVA decreased with increasing the irradiation dose. UVA irradiation with riboflavin caused damage specifically at the GGG sequence in the DNA fragments containing telomere sequence (TTAGGG)₄. We concluded that the GGG-specific damage in telomere sequence induced by UVA irradiation participates in the increase of the telomere shortening rate. In this report, we show our experimental results and discuss the mechanisms of sequence-specific DNA damage in relation to carcinogenesis and aging. This article is based upon the research that was given Encouragement Award at the 74th Annual Meeting of the Japanese Society for Hygiene held in Tokyo, Japan on March 24–27, 2004.     

Punicalagin reduces H2O2-induced cytotoxicity and apoptosis in PC12 cells by modulating the levels of reactive oxygen species

Background: Oxidative stress has long been linked to neuronal cell death in many neurodegenerative diseases. Antioxidant conventional supplements are poorly effective in preventing neuronal damage caused by oxidative stress due to their inability to cross the blood brain barrier. Hence the use of molecules extracted from plants and fruits such as phenolics, flavonoids, and terpenoids compounds constitute a new wave of antioxidant therapies to defend against free radicals.

Objective: In this study we examined the effects of punicalagin, a ellagitannin isolated from the pomegranate juice, on a rat adrenal pheochromocytoma cell line, treated with hydrogen peroxide, evaluating the viability, oxidation potential, mitochondrial function, and eventual apoptosis.

Methods: This study was performed on PC12 cells pretreated with punicalagin (0.5, 1, 5, 10 e 20?µM) 24 hours before of the damage by hydrogen peroxide (H₂O₂). H₂O₂ concentration (300?µM) used in our study was determined by preliminary experiments of time course. The cell viability and ROS production were evaluated by MTS assay and cytofluorometry assays, respectively. Subsequently, the number of apoptotic-positive cells and mitochondrial transmembrane potential, were measured by flow cytometry, in the same experimental paradigm. Finally, the expression of Bax and enzymatic activity of Caspase 3, some of the principle actors of programmed cell death, were investigated by semiquantitative PCR and utilizing a colorimetric assay kit, respectively.

Results: We found that pretreatment with punicalagin protected the cells from H₂O₂-induced damage. In particular, the protective effect seemed to be correlated with a control both in radical oxygen species production and in mitochondrial functions. In fact the cells treated with H₂O₂ showed an altered mitochondrial membrane integrity while the pretreatment with punicalagin retained both the cellular viability and the mitochondrial membrane potential similar to the control. Furthermore, the punicalagin, modulated the apoptotic cascade triggered reducing Bax gene expression and Caspase 3 activity.

Discussion: Results of the present study demonstrated a neuroprotective effect of punicalagin on H₂O₂-induced PC12 cell death, including mitochondria damage and expression of apoptotic gene Bax; therefore we hypothesize a possible prevent role for this molecule in neurodegenerative diseases related to oxidative stress.     

Carcinoma of the colon in asbestos-exposed workers: analysis of asbestos content in colon tissue.

Epidemiological studies have indicated an increased incidence of carcinoma of the colon in asbestos workers. The present study evaluated the colon tissue asbestos burden, by light and electron microscopic analytic techniques, in patients with a history of occupational asbestos exposure and colon cancer. Asbestos fibers and/or asbestos bodies were present in colon tissue from 14 of 44 (31.8%) asbestos workers with colon carcinoma (range 142,199 to 15,231, 543 fibers/g/wet weight, mean 2,517,823). Chrysotile was identified in 9 patients and amosite in 3 patients. Both amosite and chrysotile were found in the colonic wall in one individual. Other forms of asbestos (e.g., crocidolite, tremolite, or anthophyllite) were not found. Asbestos fibers and asbestos bodies were not found in colon tissue from 20 control patients (colon carcinoma and no asbestos exposure). Asbestos fibers frequently enter and reside in the wall of the colon and are often intimately associated with tumor tissue at the site of colon carcinoma in workers with asbestos exposure and colon carcinoma.     

The influence of dietary levels of vitamin a and fat on colon cancer

Rats fed diets high (24%) or low (5%) in fat were given dietary levels of vitamin A (retinyl acetate) ranging from 0.3 to 30 μg/food. The lowest tumor incidence was in the group fed diets high in vitamin A and low in fat. When the diet was high in fat and low in vitamin A, tumor incidence and frequency were significantly increased over that in rats fed the high‐fat diet with normal levels of vitamin A (10 μg/g feed). However, even with a high level of fat in the diet, raising the level of vitamin A above 10 μg/g feed had no further beneficial effect. Thus, although there was a significant interaction between vitamin A and fat, it is the latter that appears to require the most attention, once the vitamin A intake is adequate. These data support the view that we should set as a goal an adequate, diversified diet that is low in fat but that an excessive intake of vitamins such as vitamin A that are toxic should be avoided.     

The influence of dietary levels of vitamin A and fat on colon cancer

Rats fed diets high (24%) or low (5%) in fat were given dietary levels of vitamin A (retinyl acetate) ranging from 0.3 to 30 micrograms/g food. The lowest tumor incidence was in the group fed diets high in vitamin A and low in fat. When the diet was high in fat and low in vitamin A, tumor incidence and frequency were significantly increased over that in rats fed the high-fat diet with normal levels of vitamin A (10 micrograms/g feed). However, even with a high level of fat in the diet, raising the level of vitamin A above 10 micrograms/g feed had no further beneficial effect. Thus, although there was a significant interaction between vitamin A and fat, it is the latter that appears to require the most attention, once the vitamin A intake is adequate. These data support the view that we should set as a goal an adequate, diversified diet that is low in fat but that an excessive intake of vitamins such as vitamin A that are toxic should be avoided.     

Chemotherapy diminishes lipid storage capacity of adipose tissue in a preclinical model of colon cancer

Background

Accelerated loss of adipose tissue in cancer is associated with shorter survival, and reduced quality of life. Evidence is emerging suggesting tumour association with alterations in adipose tissue, but much less is known about drug-related mechanisms contributing to adipose atrophy. Identification of mechanisms by which tumour and cancer treatments, such as chemotherapy, affect adipose tissue are required to develop appropriate therapeutic interventions to prevent fat depletion in cancer. This pre-clinical study aimed to assess alterations in adipose tissue during the clinical course of cancer.

Methods

Fischer 344 rats bearing the Ward colorectal tumour were euthanized before chemotherapy, after 1- cycle, or 2-cycles of a combination chemotherapy consisting of Irinotecan (CPT-11) combined with 5-fluorouracil (5-FU), which recapitulates first line treatment for human colorectal cancer. Periuterine adipose tissue was isolated. Healthy rats served as a reference group. Histological analysis (hematoxylin and eosin), Real-time PCR (TaqMan) and proteomic analysis (LC-MS/MS) were performed.

Results

Larger adipocytes (3993.7?±?52.6 μm²) in tumour-bearing animals compared to the reference group (3227.7?±?36.7 μm²; p?<?0.001) was associated with reduced expression of proteins involved in mitochondrial fatty acid oxidation. The presence of a tumour has a significant effect on phospholipid but not triglyceride fatty acid composition. There were greater proportions of saturated fatty acids concurrent with lower monounsaturated fatty acids within the PL fraction of adipocytes in tumour-bearing animals. Chemotherapy treatment decreased the size of adipocytes (2243.9?±?30.4 μm²; p?<?0.001) and led to depletion of n-3 polyunsaturated fatty acids in adipose tissue triglyceride. Evaluation of the proteome profile revealed decreased expression of proteins involved in ATP generation, β-oxidation, and lipogenesis. Overall, adipose tissue may not be able to efficiently oxidize fatty acids to provide energy to maintain energy demanding pathways like lipogenesis inside the tissue.

Conclusions

In conclusion, metabolic adaptations to mitochondrial impairment may contribute to diminished lipid storage capacity of adipose tissue following chemotherapy delivery.

     

Identifying prognostic structural features in tissue sections of colon cancer patients using point pattern analysis

Diagnosis and prognosis of cancer are informed by the architecture inherent in cancer patient tissue sections. This architecture is typically identified by pathologists, yet advances in computational image analysis facilitate quantitative assessment of this structure. In this article, we develop a spatial point process approach to describe patterns in cell distribution within tissue samples taken from colorectal cancer (CRC) patients. In particular, our approach is centered on the Palm intensity function. This leads to taking an approximate-likelihood technique in fitting point processes models. We consider two Neyman-Scott point processes and a void process, fitting these point process models to the CRC patient data. We find that the parameter estimates of these models may be used to quantify the spatial arrangement of cells. Importantly, we observe characteristic differences in the spatial arrangement of cells between patients who died from CRC and those alive at follow up.