Sex steroid hormone modulation of NADPH pathways in MCF-7 cells

Hormonal modulation of glucose-6-phosphate dehydrogenase (G6PD) and of utilization pathways of NADPH generated by G6PD was studied in the MCF-7 human breast cancer cell line, using a quantitative cytochemical method. Our results show that G6PD is increased by 17 beta-estradiol (estradiol) and synthetic progestin (promegestone R5020). The synthetic antiestrogen tamoxifen has no effect on G6PD activity. When it is present in the medium with estradiol, tamoxifen can oppose the stimulatory effect of estradiol on G6PD activity. Mifepristone (RU 38486) has no effect on G6PD activity, but it inhibits the R5020 stimulation of G6PD activity. After MCF-7 pretreatment with estradiol, there is a much stronger stimulation of G6PD activity by R5020. When we studied the effect of the steroid on the two utilization pathways of NADPH generated by G6PD activity, we observed that, in the cells treated with estradiol, there is an increase in reducing equivalents generated by G6PD activity which only affects the NADPH2 pathway, and that there is cell growth stimulation. When tamoxifen is present in the medium, we found no effect on the NADPH utilization pathways, nor on cell growth. In the presence of R5020, the NADPH2 pathway activity is increased but, under our experimental conditions, there was no effect on cell growth. On the other hand, even though RU 38486 is without effect on total G6PD activity, it does cause a modification in the distribution of reducing equivalents: the NADPH2 pathway activity is decreased, while the NADPH1 pathway is stimulated.     

Human glucose-6-phosphate dehydrogenase (G6PD) gene transforms NIH 3T3 cells and induces tumors in nude mice

The main physiological function of glucose-6-phosphate dehydrogenase (G6PD) is to produce NADPH and ribose 5-phosphate, which are essential for reductive biosynthesis and nucleic acid synthesis. In normal cells, G6PD expression is tightly controlled; however, in many tumors, regulation of its expression is altered, resulting in a significant increase in G6PD activity. To investigate the potential role of G6PD in tumorigenesis, we transfected NIH 3T3 cells with human G6PD cDNA. Cells overexpressing G6PD showed altered cell morphology and exhibited tumorigenic properties. In contrast to the control cells or cells transfected with mutated G6PD cDNA, G6PD-overexpressing cells were not contact inhibited and exhibited anchorage-independent growth. They divided more quickly and induced rapidly growing, large fibrosarcomas in nude mice. Moreover, the induced tumorigenic properties were positively correlated with the level of G6PD activity. Interestingly, treatment with buthionine SR-sulfoximine (BSO), a glutathione depletion agent, decreased the colony-forming efficiency of G6PD-overexpressing cells in soft agar, which implicates that alteration of the redox balance may be involved in G6PD-induced tumorigenesis. A comparative analysis of the expression level of G6PD in a variety of human cancer cell lines was also performed. Northern- and Western-blot analyses revealed that G6PD was particularly overexpressed in human esophageal cancer cell lines. Our observations indicate that G6PD may act as a potential oncogene, whose overexpression plays a critical role in neoplastic transformation.     

Poor prognosis in nasopharyngeal cancer patients with low glucose-6-phosphate-dehydrogenase activity.

Nasopharyngeal carcinoma (NPC) is endemic among well-defined ethnic groups in several world regions, such as Southeastern China and Taiwan. Glucose-6-phosphate-dehydrogenase (G6PD)- deficiency, a sex-linked disorder, is one of the most common enzymopathies in Taiwan. The major role of G6PD is to generate NADPH to protect cells from oxidative damage, which is a major contributing factor to certain degenerative diseases, such as aging and cancer. In view of the coincidence of epidemic distribution of NPC and G6PD deficiency, as well as the house-keeping function of G6PD in cellular oxidative defense, we investigated the correlation of G6PD activity with NPC. The stage of NPC was classified by AJCC (1997) criteria. G6PD levels were determined in 108 NPC male patients and 75 healthy male individuals. The mean G6PD level of NPC patients was 218.9 U/10(12) RBC or 7.53 U/g hemoglobin (Hb), being much lower than in normal individuals (260.6 U/10(12) erythrocytes (RBC) or 8.92 U / gHb). The level of G6PD activity had no correlation with tumor stage or lymph node or distant metastasis, but was significantly correlated with tumor recurrence (P = 0.004 when using G6PD = 130 U/10(12) RBC as cutoff value). These results indicated that low G6PD activity in patients with NPC is associated with poor prognosis.     

Alterations in Erythrocyte Glutathione Metabolism Associated with Cervical Dysplasias and Carcinoma in Situ

The study was designed to test the hypothesis whether cervical dysplasias of the more severe grades are associated with elevated erythrocyte glutathione levels. Subjects were women who obtained Pap tests and were subsequently found (1) not to have any cervical lesions or (2) to have colposcopically visualized, biopsy-confirmed cervical abnormalities histopathologically diagnosed as mild, moderate, severe dysplasias, or carcinoma in situ (CIS). The erythrocyte levels of reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), and 6-phosphogluconate dehydrogenase (6PGD) were analyzed from coded peripheral venous blood samples. GSH and GR concentrations increased with increasing severity of dysplasia. Exploratory data analysis and multiple pairwise comparisons suggested comparable levels of the glutathione-related variables between these histopathological pairs' (1) mild and moderate dysplasias or (2) severe dysplasia and CIS. We suggest that the changes in erythrocyte glutathione-related indices in conjunction with histopathological diagnosis may have the potential to distinguish between low-and high-grade cervical dysplastic lesions.     

沉默G6PD对卵巢癌细胞顺铂敏感性的影响及其作用机制

目的 探讨葡萄糖-6-磷酸脱氢酶（glucose-6-phosphate dehydrogenase，G6PD）在卵巢癌细胞顺铂耐药中的作用及其潜在的机制。方法 通过低浓度加量持续诱导法构建卵巢癌顺铂耐药细胞株A2780/CDDP，采用Label-free定量蛋白质组学法分析A2780和A2780/CDDP细胞差异蛋白G6PD，Western blot和RT-qPCR检测G6PD表达情况。通过siRNA技术将siRNA-G6PD及其阴性对照（siRNA-NC）转染至A2780/CDDP细胞，然后采用Western blot验证G6PD基因沉默效果，MTT法检测细胞增殖情况，流式细胞术检测细胞凋亡情况，流式细胞仪和酶标仪检测铁死亡相关物质活性氧（ROS）、丙二醛（MDA）、还原型谷胱甘肽（GSH）和脂质过氧化物的表达水平。结果 与A2780细胞相比，A2780/CDDP细胞中有95个上调蛋白和102个下调蛋白，其中上调蛋白中以G6PD表达差异最为显著；这些蛋白大多富集在代谢通路，且其功能主要与细胞氧化应激反应、核糖磷酸代谢途径相关。与A2780细胞相比，A2780/CDDP细胞中G6PD mRNA和蛋白表达水平均显著升高（均P<0.05），铁死亡相关物质ROS、MDA和脂质过氧化物水平均降低（均P<0.05），而GSH水平升高（P=0.001）。沉默G6PD后的A2780/CDDP细胞中顺铂的IC₅₀值降低（P=0.012），细胞凋亡率升高（P=0.006），铁死亡相关物质ROS、MDA和脂质过氧化水平均升高（均P<0.05），而GSH水平降低（P=0.007）。结论 沉默G6PD可能通过诱导卵巢癌顺铂耐药细胞A2780/CDDP中的铁死亡水平而增强顺铂敏感性。     

Alterations of the glutathione cycle enzymes during and after SV40-transformation of human fibroblasts

The activities of several enzymes involved in the antioxidantsystem of the cell were studied in parallel to cytogenetic alterationsat various times after SV40 infection and transformation ofhuman fibroblasts. At early passages after SV40 infection glutathionereductase (GSR), glutathione peroxidase (GPX) glutathione transferase(GST) and glucose-6-phosphate dehydrogenase (G6PD) activitieswere decreased. This associated with the low superoxide dismutase(SOD) and catalase activities previously noticed in these cellssuggested that they are in a highly pro-oxidant status. Althoughchromosomes carrying the genes encoding these enzymes are frequentlyunderrepresented there is no direct relationship between thenumber of chromosomes and enzyme activities. Except for GPXall the activities tend to increase hi established cell linesreaching levels comparable to those of non-transformed fibroblasts.The late increase of G6PD activity may correlate with the frequentduplication of the early replicating X. GSR seems to correlatewith G6PD activity and GPX to SOD total activity. The most strikingalterations affect mitochondrial and peroxisomal enzymes activities:SOD GPX and catalase.     

Poor Prognosis in Nasopharyngeal Cancer Patients with Low Glucose-6-phosphate-dehydrogenase Activity

Nasopharyngeal carcinoma (NPC) is endemic among well-defined ethnic groups in several world regions, such as Southeastern China and Taiwan. Glucose-6-phosphate-dehydrogenase (G6PD)-deficiency, a sex-linked disorder, is one of the most common enzymopathies in Taiwan. The major role of G6PD is to generate NADPH to protect cells from oxidative damage, which is a major contributing factor to certain degenerative diseases, such as aging and cancer. In view of the coincidence of epidemic distribution of NPC and G6PD deficiency, as well as the house-keeping function of G6PD in cellular oxidative defense, we investigated the correlation of G6PD activity with NPC. The stage of NPC was classified by AJCC (1997) criteria. G6PD levels were determined in 108 NPC male patients and 75 healthy male individuals. The mean G6PD level of NPC patients was 218.9 U/10¹²RBC or 7.53 U/g hemoglobin (Hb), being much lower than in normal individuals (260.6 U/10¹²erythrocytes (RBC) or 8.92 U/gHb). The level of G6PD activity had no correlation with tumor stage or lymph node or distant metastasis, but was significantly correlated with tumor recurrence ( P =0.004 when using G6PD=130 U/10¹² RBC as cutoff value). These results indicated that low G6PD activity in patients with NPC is associated with poor prognosis.     

CARD15/NOD2, CD14, and toll-like receptor 4 gene polymorphisms in Greek patients with sarcoidosis

BACKGROUND: Sarcoidosis, similarly to Crohn's disease (CD), is a complex inflammatory disease of unknown etiology. The belief that a genetic susceptibility to the development of sarcoidosis exists was derived from observations of familial clustering of sarcoidosis cases and racial differences in disease prevalence. Taking into account the remarkable similarity in the immunopathophysiology of sarcoidosis and CD, and in further exploring the genetic background of sarcoidosis, we study gene polymorphisms known for their implication in CD. These polymorphisms are in the CARD15/NOD2 gene (R702W, G908R and 3020insC), as well as mutations in the promoter of the CD14 gene (T/C at position -159) and in the TLR4 gene (Asp299Gly and Thr399Ile). METHODS: DNA was obtained from 100 sarcoidosis patients and 150 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis. RESULTS: Although CARD 15/NOD2 mutations were more frequent in cases than in controls, the difference was significant only for the G908R polymorphism (p = 0.024). Interestingly, the same was recorded with reference to the T allele (p = 0.002) and TT genotype (p = 0.017) frequencies of the CD14 promoter. No differences were observed in the 299Gly and 399Ile allele frequencies between patients and controls. Finally, the co-existence of a mutation in the CARD15/NOD2 and the CD14 genes was associated with sarcoidosis at a higher level of significance than any of these mutations separately. CONCLUSION: Our results suggest that the G908R mutation of the CARD15/NOD2 gene, as well as the T allele and TT genotype of the CD14 promoter are associated with increased susceptibility for developing sarcoidosis.     

Analysis of IL6 and IL1A gene polymorphisms in UK and Dutch patients with sarcoidosis

BACKGROUND: Proinflammatory cytokines are a major determinant in the inflammatory events leading to sarcoidosis. Genetic variations in the genes encoding these cytokines might contribute to sarcoidosis susceptibility, disease severity and outcome. METHODS: In the present study we genotyped two clinically well-defined cohorts of Caucasian sarcoidosis patients from different European countries (each with their own controls) for the following polymorphisms using SSP-PCR: IL6 -174(G/C), IL6 intron 4(A/G) and IL1A-889(C/T). In total, 516 individuals were studied (147 UK + 102 Dutch patients, 101 UK + 166 Dutch controls). Disease severity data at presentation included chest radiographic stage, FVC, DL(CO), and extrapulmonary manifestations. Disease progression was evaluated on follow-up chest radiographs and sequential lung function measurements (2, 4 years). RESULTS: No differences in genotype, carriage and allele frequencies of the investigated polymorphisms were found in either of the populations. Analysis of genotype data in relation to disease severity data, however, showed a slightly increased carrier frequency of the rarer-174C allele in patients with Stage IV sarcoidosis (p = 0.03, Pc = 0.09). Pulmonary function progression analysis did not reveal significant associations. CONCLUSIONS: Although the investigated polymorphisms are unlikely to contribute to sarcoidosis susceptibility, the IL6-174C allele might have a role in the genetics underlying sarcoidosis severity or the progression towards pulmonary fibrosis in a particular subgroup.     

BAG3 elevation inhibits cell proliferation via direct interaction with G6PD in hepatocellular carcinomas

Bcl-2 associated athanogene 3 (BAG3) contains multiple protein-binding motifs to mediate potential interactions with chaperons and/or other proteins, which is possibly ascribed to the multifaceted functions assigned to BAG3. The current study demonstrated that BAG3 directly interacted with glucose 6 phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP). BAG3 suppressed the PPP flux, de novo DNA synthesis and cell growth in hepatocellular carcinomas (HCCs). The growth defect of HCCs with forced BAG3 expression can be rescued by enforced G6PD expression. However, BAG3 elevation did not cause a reduction in cellular NADPH concentrations, another main product of G6PD. In addition, supplement of nucleosides alone was sufficient to recover the growth defect mediated by BAG3 elevation. Collectively, the current study established a tumor suppressor-like function of BAG3 via direct interaction with G6PD in HCCs at the cellular level.     

SIRT5通过调控G6PD促进结肠癌细胞的糖代谢及增殖

  目的  探讨SIRT5对结肠癌细胞糖代谢和增殖的作用及其可能的作用机制，以及SIRT5、G6PD在结肠癌组织中的表达情况。  方法  Western blot法检测SIRT5蛋白在结肠癌细胞系HCT116、HT29中的表达水平，构建SIRT5基因过表达及siRNA沉默载体分别转染到SIRT5低表达与SIRT5高表达结肠癌细胞系，葡萄糖氧化酶法、比色法分别检测培养液中葡萄糖、乳酸的含量；CCK-8实验检测细胞活力；克隆形成实验检测细胞增殖能力。RT-PCR与Western blot法分别检测SIRT5对G6PD mRNA及蛋白表达的调控；免疫组织化学法检测结肠癌组织中SIRT5、G6PD的表达情况。  结果  沉默SIRT5后，细胞培养液中葡萄糖的浓度明显升高、乳酸的浓度明显降低，细胞的活性和增殖能力明显减弱；过表达SIRT5后，细胞培养液中葡萄糖的浓度明显降低、乳酸的浓度明显升高，细胞的活性和增殖能力明显增强。SIRT5正向调控G6PD的基因表达与单独转染si-SIRT5相比，共转染si-SIRT5与G6PD质粒恢复了结肠癌细胞的糖代谢及增殖能力；SIRT5、G6PD在结肠癌组织中的表达明显高于癌旁组织。  结论  SIRT5、G6PD在结肠癌组织中高表达，SIRT5通过调控G6PD促进结肠癌细胞糖代谢和增殖。      

Multiple molecular forms of glucose-6-phosphate dehydrogenase in normal, preneoplastic, and neoplastic mammary tissues of mice.

Multiple molecular forms of glucose-6-phosphate dehydrogenase (G6PD) in normal, preneoplastic, and neoplastic mammary tissues were separated by polyacrylamide gel electrophoresis and identified by specific straining for enzyme activity. Mammary tissue from lactating BALB/c mice showed considerable amounts (up to 50%) of a slower-migrating G6PD species, G6PD-III, which was essentially absent from glands of pregnant mice, preneoplastic nodules, and mammary carcinomas. All tissues possessed a faster-migrating species, G6PD-II, which accounted for up to 85% of the total G6PD in the glands of pregnant mice. A third species, G6PD-I, migrating more rapidly than G6PD-II, was found in both abnormal tissues (preneoplastic and neoplastic) and accounted for up to 35% of the total enzymatic activity. G6PD-I was present in moderate amounts (less than 15%) in glands from pregnant mice and was essentially absent from the lactating gland (approximately 5%). The addition of dithiothreitol did not alter the measurable G6PD activity but did increase the relative activity of G6PD-II or G6PD-I, as judged by the intensity of the bands on the gels. Mild oxidation (stirring overnight at 4 degrees in air) resulted in a loss of G6PD activity, but preparations had greater amounts of G6PD-III; presence of dithiothreitol during aeration partially prevented loss of G6PD activity and largely prvented the appearance of G6PD-III. Molecular-weight estimations with preparations from lactating mice yielded a value of 118,000 for G6PD-II and 260,000 for G6PD-III, suggesting a monomer and dimer, respectively. The addition of nicotinamide adenine dinucleotide phosphate stabilized G6PD activity by preventing heat inactivation at 47 degrees; nicotinamide adenine dinucleotide phosphate did not alter the pattern of species present. The data from heat inactivation studies suggest that G6PD-III (dimer) was the more stable species. The addition of nicotinamide adenine dinucleotide phosphate to samples after oxidation in the absence of dithiothreitol (about 70% loss of activity) resulted in no change in patterns and in recovery of full G6PD activity during heating at 47 degrees. A potential relationship between glutathione reductase activity and the pattern of G6PD species observed in the various tissues is noted.     

Benzo(a)pyrene metabolism by lymphocytes from normal individuals and individuals carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase

In vitro growing human lymphocytes (HL) and fibroblasts, isolated from glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects (Mediterranean variant), show a sharp decrease in this enzymatic activity and in NADPH:NADP+ ratio. These cells are less able than controls to hydroxylate benzo(a)pyrene (BaP) when tested in the absence of an exogenous NADPH-generating system. They exhibit great resistance to the toxic effect of BaP. G6PD-deficient fibroblasts are less prone than controls to in vitro transformation by BaP. To investigate whether this depends on a decreased production of active BaP metabolites and BaP:DNA adducts by G6PD-deficient cells, BaP metabolism was studied in G6PD-deficient HL cultured in vitro in the presence of mitogens and treated with BaP for 24 hr. HPLC profiles of organo- and water-soluble metabolites revealed that both types of benzo(a)anthracene (BaA)-induced HL produced: 4,5-, 7,8-, 9,10-diols, 1,3-, 3,6-quinones, 3-, 9-hydroxy and 2 peaks of more polar metabolites. There was a 25-76% decrease of organo- and water-soluble metabolites in the G6PD-deficient cells. When HL were incubated with 7,8-diol, the formation of metabolites mutagenic for Salmonella typhimurium (His-) was very low in G6PD-deficient cells. BaP:deoxyadenosine (dAde) and BaP:deoxyguanosine (dGua) adducts were identified after incubation of both types of HL with BaP. There was a 31-79% fall in adduct formation by G6PD-deficient cells. Our results indicate that G6PD-deficient human lymphocytes are less able to metabolize BaP than normal lymphocytes. We suggest that the NADPH pool is inadequate, in deficient cells, for active BaP metabolism.     

Glucose-6-phosphate dehydrogenase and NADPH oxidase 4 control STAT3 activity in melanoma cells through a pathway involving reactive oxygen species,c-SRC and SHP2

Background: Glucose-6-phosphate dehydrogenase (G6PD) participates in glucose utilization by catalysing the first step of the pentose-phosphate pathway in mammalian cells. Previous studies have shown that changes in G6PD levels can promote tumor cell proliferation or apoptosis via the STAT3/5 pathway in a human melanoma xenograft model. G6PD cooperates with NADPH oxidase 4 (NOX4) in the cellular metabolism of reactive oxygen species (ROS) and in maintaining the intracellular redox state. Methods: In this study, the effect of G6PD or NOX4 silencing in the melanoma line A375 was examined in terms of redox state, proto-oncogene tyrosine-protein kinase Src (c-Src) and the tyrosine-specific protein phosphatase SHP2 expression as well as cell cycle progression. Results: The results demonstrate that: (1) Downregulation of cyclin D1 and CDK4 and up-regulation of p53 and p21 occurred in response to silencing of G6PD and NOX4 thus resulting in G1/S cell cycle arrest and inhibition of A375 cell proliferation. (2) The blockade of cell proliferation is primarily due to a reduced DNA-binding activity of STAT3. (3) The DNA-binding activity of STAT3 was regulated by the upstream factors, c-SRC and SHP2. Silencing of NOX4 in A375 cells inhibited c-SRC and SHP2 regulated STAT3 activity. Conclusion: The data are consistent with a novel G6PD-NOX4-NADPH-ROS-c-SRC/SHP2 pathway controlling STAT3 activity in A375 melanoma cells.     

Glucose-6-phosphate dehydrogenase (G6PD) activity in tumoral tissues of G6PD-deficient subjects affected by larynx carcinoma

The activity of glucose-6-phosphate dehydrogenase (G6PD), the key enzyme of the hexose monophosphate (HMP) shunt pathway, was measured in both normal and tumoral larynx tissues from normal and G6PD deficient subjects. Significant increases of this enzymatic activity were found in tumoral tissues of both normal and G6PD deficient subjects, who were characterized by very low levels of G6PD activity in erythrocytes as well as in larynx tissue.     

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms in sarcoidosis patients

BACKGROUND AND AIM: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a co-stimulatory molecule that is expressed on activated T-cells, and is an important regulator of T-cell activation in T-cell-dependent immune responses. CTLA-4 signals lead to the downregulation of T-cell proliferation and activation. Sarcoidosis is a systemic granulomatous disorder of unknown etiology, which shows abnormal regulation of T cell activation. Polymorphisms of CTLA-4 have been reported to alter CTLA-4 expression, and are associated with many diseases. In the present study we investigated CTLA-4 polymorphisms of promoter -318(C/T) and exon 1 +49(A/G) in sarcoidosis patients and control subjects to determine whether these genetic variations affect sarcoidosis. METHODS: One hundred and six sarcoidosis patients and 100 healthy control subjects were studied. The polymerase chain reaction technique and direct genomic sequencing were used to determine the genotypes of promoter -318(C/T) and exon 1 +49(A/G) in the CTLA-4 gene. RESULTS: We found no difference in the distribution of either genotype between the healthy control subjects and sarcoidosis patients. Among the sarcoidosis patients, the -318(C/T) CC genotype (p = 0.011) and AG or GG genotype at position +49(A/G) (p = 0.004) were increased with ocular involvement compared with those patients without ocular involvement. Furthermore, the +49(A/G) GG genotype was increased in patients with three or more organs affected compared with patients with fewer organs affected (p = 0.019). CONCLUSIONS: The CTLA-4 polymorphisms are not associated with disease susceptibility of sarcoidosis, but these genetic variations significantly influence phenotypes of sarcoidosis.     

Hexose monophosphate shunt enzymes in lung tumors from normal and glucose-6-phosphate-dehydrogenase-deficient subjects

Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate, dehydrogenase the key enzymes of the hexose monophosphate shunt pathway, were measured in both surrounding and tumoral lung tissues from normal and G6PD-deficient subjects. A significant increase of these enzymatic activities in tumoral tissue was found not only in G6PD-normal patients, but also in G6PD-deficient patients with very low or nonmeasurable G6PD activity in both erythrocytes and normal lung tissue.     

Bronchoalveolar glutathione and nitrite/nitrate in idiopathic pulmonary fibrosis and sarcoidosis

BACKGROUND AND AIM OF THE WORK: Bronchoalveolar lavage (BAL) is useful in diagnosis and management of interstitial lung diseases. Glutathione (GSH) represents an important defence molecule against reactive oxygen species produced during inflammation, which underlies both idiopathic pulmonary fibrosis (IPF) and sarcoidosis. Nitric oxide has been suggested as a marker of airway inflammation. The aim of this study was to examine the content of GSH and NO stable metabolites, nitrite/nitrate, in the BAL of a group of patients with IPF and sarcoidosis. METHODS: BAL was performed in 13 patients with newly diagnosed IPF, 13 patients with sarcoidosis and 4 controls. Total GSH and nitrite/nitrate were measured. BAL was repeated, in 5 patients with IPF, after 6 and 12 months and GSH was measured again. RESULTS: IPF patients had significantly lower levels of total GSH compared to sarcoidosis patients or controls (129 +/- 22 vs. 324 +/- 40 and 383 +/- 23 mM; p < 0.001) in the epithelial lining fluid (ELF). In patients with IPF (n = 5), total GSH levels in the ELF increased after 6 and 12 months (T0 = 98 +/- 19; T6 = 219 +/- 22; T12 = 301 +/- 34 microM; p < 0.05) following therapy with immunosuppressive drugs such as corticosteroids, methotrexate, cyclosporin A. Levels of nitrite/nitrate were found significantly elevated in IPF (n = 8) and sarcoidosis (n = 10) patients compared to controls (230 +/- 37 and 216 +/- 36 vs. 86 +/- 11 microM; p < 0.05). CONCLUSION: These observations support the role of inflammation and antioxidant defences in interstitial lung diseases and may provide insights into the pathogenesis of oxidant-induced interstitial disease and their therapy.     

Modifications of the anti-oxidant metabolism during proliferation and differentiation of colon tumor cell lines

The anti-oxidant metabolism was studied at different times after sub-culture in 2 colon cell lines previously characterized for their growth and differentiation properties. The HT29 cell line is mainly composed of proliferative and undifferentiative cells, while the derived 5-fluorouracil (FUra)-adapted cells undergo growth-dependent differentiation, which is complete at post-confluence. In the 2 cell lines, all the anti-oxidant parameters studied appeared to be related to proliferation, with increased activity of superoxide dismutase (SOD) 1 and 2, catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GSR), and glutathione transferase (GST), and decreased glucose-6-phosphate dehydrogenase (G6PD) activity and glutathione content, in parallel with slowing down of proliferation. At post-confluence, these metabolic parameters remained stable, except for GPX activity, which continued to increase, and CAT activity, which decreased. The amounts of SOD1, SOD2 and CAT immunoreactive proteins, estimated by Western blotting, appeared to be correlated to their respective enzymatic activities, SOD1, CAT and GST activity and glutathione content, which remained at similar levels in the 2 cell lines for all times studied, appeared unrelated to the differentiation process. GSR and GPX activity, which was lower in FUraadapted than in parental cells only at post-confluence, could be considered as markers of differentiated cells. The higher SOD2 and lower G6PD activity observed in FUra-resistant cell in comparison with parental cells at all times after sub-culture could be characteristic both of differentiative and of differentiated cells. Interestingly, cytogenetics have previously indicated that deletions of the long arm of chromosome 6, which carry the gene for SOD2, were frequently observed in parental but not in FUra-adapted cells. These results demonstrate that modifications of the anti-oxidant metabolism occur in relation with proliferation and differentiation, and suggest a particular role for SOD2 in these cellular processes.     

Detoxifying activities in alveolar macrophages of rats treated with acetylcysteine, diethyl maleate and/or aroclor

Single or sequential treatment of rats with the thiol N-acetyl-L-cysteine (NAC), the glutathione depletor diethyl maleate (DEM) and the enzyme inducer Aroclor 1254 (AR) produced several significant variations on metabolic activities of pulmonary alveolar macrophages (PAM). Specifically, all three compounds elicited an increase in some oxidoreductase activities, including the two dehydrogenases involved in the hexose monophosphate shunt (G6PD and 6PGD) and NADH- or NADPH-dependent diaphorases. Diaphorase activities were especially increased by sequential treatments with AR and DEM or with DEM and NAC. Both NAC and AR also stimulated other detoxifying mechanisms, such as those related to GSH S-transferase activity and to the NADPH-dependent reduction of hexavalent chromium. Therefore, all the monitored parameters were significantly enhanced not only by the enzyme inducer, but also by the thiol, demonstrating its protective role in the biotransformation of mutagenic/carcinogenic compounds.