Modulation of human glutathione s-transferases by polyphenon e intervention.

PURPOSE: Green tea consumption has been associated with decreased risk of certain types of cancers in humans. Induction of detoxification enzymes has been suggested as one of the biochemical mechanisms responsible for the cancer-preventive effect of green tea. We conducted this clinical study to determine the effect of repeated green tea polyphenol administration on a major group of detoxification enzymes, glutathione S-transferases (GST). METHODS: A total of 42 healthy volunteers underwent a 4-week washout period by refraining from tea or tea-related products. At the end of the washout period, a fasting blood sample was collected, and plasma and lymphocytes were isolated for assessment of GST activity and level. Following the baseline evaluation, study participants underwent 4 weeks of green tea polyphenol intervention in the form of a standardized Polyphenon E preparation at a dose that contains 800 mg epigallocatechin gallate (EGCG) once a day. Polyphenon E was taken on an empty stomach to optimize the oral bioavailability of EGCG. Upon completion of the intervention, samples were collected for postintervention GST assessment. RESULTS: Four weeks of Polyphenon E intervention enhanced the GST activity in blood lymphocytes from 30.7 +/- 12.2 to 35.1 +/- 14.3 nmol/min/mg protein, P = 0.058. Analysis based on baseline activity showed that a statistically significant increase (80%, P = 0.004) in GST activity was observed in individuals with baseline activity in the lowest tertile, whereas a statistically significant decrease (20%, P = 0.02) in GST activity was observed in the highest tertile. In addition, Polyphenon E intervention significantly increased the GST-pi level in blood lymphocytes from 2,252.9 +/- 734.2 to 2,634.4 +/- 1,138.3 ng/mg protein, P = 0.035. Analysis based on baseline level showed that this increase was only significant (P = 0.003) in individuals with baseline level in the lowest tertile, with a mean increase of 80%. Repeated Polyphenon E administration had minimal effects on lymphocyte GST-mu and plasma GST-alpha levels. There was a small but statistically significant decrease (8%, P = 0.003) in plasma GST-alpha levels in the highest tertile. CONCLUSIONS: We conclude that 4 weeks of Polyphenon E administration resulted in differential effects on GST activity and level based on baseline enzyme activity/level, with GST activity and GST-pi level increased significantly in individuals with low baseline enzyme activity/level. This suggests that green tea polyphenol intervention may enhance the detoxification of carcinogens in individuals with low baseline detoxification capacity.     

Rheology of hemoglobin S gels: possible correlation with impaired microvascular circulation

The sequence of pathogenic events in sickle cell disease begins with the genetic abnormality and proceeds through molecular and red cell abnormalities to clinical events of vascular obstruction, hemolysis, and crisis. The least studied event, central in the sequence, is altered viscosity and rheology of the gelled deoxyhemoglobin S. In this work, shear is shown not only to measure the formation of gels, but to alter the progress of gelation. Thus, intraerythrocytic shear may be an important factor in pathogenesis. Increasing shear decreases the delay time for gelation as measured directly and by experiments in which shear rate is altered during the delay period. After the delay time, during the growth stage, characterized by a large increase in viscosity, shearing increases the rate of viscosity increase. On the other hand, as previously shown, shearing breaks down solid-like gels. These two effects of shear, one detrimental and the other possibly beneficial, may contribute to the variations known to exist in the clinical picture of sickle cell disease. The growth stage progress curve of gelation is here shown to be exponential in shape. This suggests that fiber breakage occurs under shear and/or that new fibers nucleate on the surface of existing fibers (i.e. heterogeneous nucleation). Finally, the progress curve is shown to be composed of plastic (i.e. solid-like) as well as viscous components early in gel development.     

The interaction of 2,3-diphosphoglycerate with various human hemoglobins

Oxygen equilibria were measured on a number of human hemoglobins, which had been "stripped" of organic phosphates and isolated by column chromatography. In the presence of 2 x 10(-4) M 2,3-diphosphoglycerate (2,3-DPG), the P(50) of hemoglobins A, A(2), S, and C increased about twofold, signifying a substantial and equal decrease in oxygen affinity. Furthermore, hemoglobins Chesapeake and M(Milwaukee-1) which have intrinsically high and low oxygen affinities, respectively, also showed a twofold increase in P(50) in the presence of 2 x 10(-4) M 2,3-DPG. In comparison to these, hemoglobins A(IC) and F were less reactive with 2,3-DPG while hemoglobin F(I) showed virtually no reactivity. The N-terminal amino of each beta-chain of hemoglobin A(IC) is linked to a hexose. In hemoglobin F(I) the N-terminal amino of each gamma-chain is acetylated. These results suggest that the N-terminal amino groups of the non-alpha-chains are involved in the binding of 2,3-DPG to hemoglobin.     

Evaluation of a new enzyme immunoassay system for free thyroxine (Enzymun-Test FT4)

Free thyroxine (FT4) represents the metabolically active fraction of the circulating thyroid hormone thyroxine (T4). In this paper the results of the evaluation of a newly developed FT4 assay are reported. This assay system is based on an enzyme-labelled one-step immunoassay. The within-run imprecision, checked using serum pools and several commercial reference materials, showed a coefficient of variation (CV) of between 0.8 and 9.8%, depending on the reference material used. The between-run imprecision showed a CV of between 1.0 and 13.2%. Accuracy experiments yielded values between 80 and 116%. When the new FT4 was compared with the calculation of an index for free thyroxine (FT4I; derived from either the ratio of T4/thyroxine binding coefficient of from T4/thyroxine binding globulin) in a number of samples in the hypo-, eu- and hyperthyroid range, a good correlation was obtained. The same was true when the new FT4 assay was compared with a widely used two-step radioimmunoassay (y = -0.146 + 0.943 x). In euthyroid subjects the measured FT4 concentration was 10.3-25.8 pmol/l. No effect was evident when the influence of EDTA and citrate was investigated, whereas addition of heparin led to an increase in FT4 concentration of about 12 to 15%. Investigation of the possible influence of a large number of drugs showed that probenezid, carbamazepine and furosemide led to an increase in the measured FT4 concentration. Dialysis increased the FT4 concentration, as measured in patients before and after haemodialysis. No effect of alteration in protein concentration and/or protein distribution of FT4 concentration could be detected. In pregnancy, FT4 values were within the normal range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of catalase activity in the malignant phenotype plays a functional role in an in vitro model for tumor progression

We have developed an in vitro model to study the molecular mechanisms of tumor progression. Using repeated treatments with ionizing radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), we caused malignant progression of a papilloma producing mouse keratinocyte cell line, 308 cells. In a previous study we have shown that the malignant variants of 308 cells have elevated reactive oxygen species (ROS) levels, and have established a functional role for the pro-oxidant state in the progressed phenotype (Carcinogenesis 20 (1999) 2063). In this study, we have evaluated the status of intracellular defense mechanisms for ROS scavenging in the progressed phenotype to identify sources that contribute to their pro-oxidant state. Our results demonstrate that a reduction in several anti-oxidant defense mechanisms, including catalase and glutathione S-transferase mu, correlates with the emergence of the malignant phenotype. We provide evidence that attenuation of catalase activity may play a functional role in the malignant progression of mouse keratinocytes.     

Genetic polymorphisms in oxidative stress‐related genes are associated with outcomes following treatment for aggressive B‐cell non‐Hodgkin lymphoma

Variable survival outcomes are seen following treatment for aggressive non‐Hodgkin lymphoma (NHL). This study examined whether outcomes for aggressive B‐cell NHL are associated with single nucleotide polymorphisms (SNPs) in oxidative stress‐related genes, which can alter drug metabolism and immune responses. Genotypes for 53 SNPs in 29 genes were determined for 337 patients given anthracycline‐based therapies. Their associations with progression‐free survival (PFS) and overall survival (OS) were estimated by Cox proportional hazard regression; associations with hematologic toxicity were estimated by logistic regression. To validate the findings, the top three SNPs were tested in an independent cohort of 572 DLBCL patients. The top SNPs associated with PFS in the discovery cohort were the rare homozygotes for MPO rs2243828 (hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.14–3.06, P = 0.013), AKR1C3 rs10508293 (HR = 2.09, 95% CI = 1.28–3.41, P = 0.0032) and NCF4 rs1883112 (HR = 0.66, 95% CI = 0.43–1.02, P = 0.06). The association of the NCF4 SNP with PFS was replicated in the validation dataset (HR = 0.66, 95% CI = 0.44–1.01, P = 0.05) and the meta‐analysis was significant (HR = 0.66, 95% CI = 0.49–0.89, P < 0.01). The association of the MPO SNP was attenuated in the validation dataset, while the meta‐analysis remained significant (HR = 1.64, 95% CI = 1.12–2.41). These two SNPs showed similar trends with OS in the meta‐analysis (for NCF4, HR = 0.72, 95% CI = 0.51–1.02, P = 0.07 and for MPO, HR = 2.06, 95% CI = 1.36–3.12, P < 0.01). In addition, patients with the rare homozygote of the NCF4 SNP had an increased risk of hematologic toxicity. We concluded that genetic variations in NCF4 may contribute to treatment outcomes for patients with aggressive NHL. Am. J. Hematol. 89:639–645, 2014. © 2014 Wiley Periodicals, Inc.     

Fragility and structure of hemoglobin S fibers and gels and their consequences for gelation kinetics and rheology

Pathogenesis in sickle cell disease depends on whether red blood cells can pass the microvasculature during the delay time before hemoglobin S gelation and cell rigidification occur. Here we observe individual hemoglobin S fibers by differential interference contrast (DIC) microscopy and show that hemoglobin S gels and fibers are fragile and easily broken by mechanical perturbation, and that breakage results in vast acceleration of gelation kinetics due to the creation of new, growing fiber-ends. Hence, in vivo this may be an important factor, in addition to hemoglobin concentration and degree of deoxygenation, that governs delay time and pathogenesis. Pathogenesis also depends on gel rheology and cell rigidification, which depend on fiber cross-linking. We show different mechanisms by which X-shaped, Y-shaped, and "zippering" cross-links form. Finally, we estimate the "on" rate constant for fiber growth to be about 200 mmol/(L.s) and obtain a value for the heterogeneous nucleation rate at 13.5 mmol/L heme.     

Kinetics of hemoglobin S polymerization and gelation under shear: I. Shape of the viscosity progress curve and dependence of delay time and reaction rate on shear rate and temperature

Polymerization and gelation of deoxyhemoglobin S makes red blood cells (RBCs) rigid and is the immediate basis of pathogenesis in sickle cell disease. Hence, characterization of hemoglobin S viscosity and its time- dependent development as RBCs pass through the microvasculature is important in understanding pathogenesis. Because RBCs and the intraerythrocytic milieu in vivo are subject to shear, the shear dependence of polymerization kinetics is also important. In steady- state cone-plate viscometry we find: (1) gelation under shear progresses exponentially with time; (2) shear markedly increases exponential rate and (3) shortens delay time independent of when in the delay time it is applied; (4) shear greatly decreases the temperature dependence of the exponential rate and delay time; (5) simultaneous with its acceleratory effect on polymerization, shear breaks down gel structure. We conclude that shear acts to accelerate gelation by breaking fibers and creating new growing ends, a process that occurs in addition to the homogeneous and heterogeneous nucleation of new fibers that occurs in the absence of shear. Fibers that break are part of a gel network rather than in free solution. The shear dependence of gelation rates means that the critical clinical issue, whether the delay time is long enough and gelation slow enough to permit deoxygenated cells to pass through the microvasculature before they rigidify, depends on in vivo shear rates as well as on degree of unsaturation and hemoglobin concentration.