abstract: Background Hemodialysis (HD) patients elicit an oxidant-antioxidant imbalance in addition to a selenium deficiency, possibly contributing to cardiovascular disease (CVD) mortality. Objective To evaluate the effect of selenium supplementation on CVD outcomes and antioxidant status in HD patients. Design A randomized controlled intervention trial conducted from October 2012 to January 2013. Participants/setting The study included 27 maintenance HD patients (61.1+17.5y, 14M, 13F) receiving HD in the greater Phoenix, AZ area. Intervention Patients received one of three treatments daily: 2 Brazil nuts, (5g, 181µg/day of selenium as selenomethionine [predicted]), 1 tablet of selenium (200µg/day of selenium as selenomethionine), or control (3 gummy bears). Main outcome measures Antioxidant status outcome measures included total antioxidant capacity, vitamin C, and RBC and plasma glutathione peroxidase (GSH-Px). CVD outcomes measures included brain natriuretic peptide; plasma cholesterol, high density lipoprotein, low density lipoprotein, triglycerides; blood pressure, and thoracic cavity fluid accumulation. Statistical analyses performed Repeated measures ANOVA analyzed changes over time and between groups at months 0 and 2 and months 0 and 3. Results Independent analysis showed the Brazil nuts provided 11µg of selenium/day and the pill provided 266µg of selenium/day. Consequently, the Brazil nut group was combined with the placebo group. 21 patients completed 2 months of the study and 17 patients completed the study in its entirety. Data was analyzed for months 0, 1 and 2. No significant differences were noted for antioxidant status outcome measures with the exception of plasma GSH-Px. Patients receiving the selenium pill had a significant increase in plasma GSH-Px compared to the placebo group (6.0+11 and -4.0+7.6, respectively, p=0.023 for change between month 0 and month 2). No significant differences were seen in total antioxidant capacity or for CVD outcome measures over time or between groups. Conclusions These data indicate that selenium supplementation increased plasma GSH-Px concentration in HD patients; however, oxidative stress was not altered by selenium supplementation. The low vitamin C status of HD patients warrants further research, specifically in conjunction with selenium supplementation. / Dissertation/Thesis / Ph.D. Nutrition 2013

A enzima glutationa peroxidase (GPx-1) possui um importante papel na detoxificação de espécies reativas de oxigênio. Neste estudo foi verificada a influência do polimorfismo Pro198Leu da GPx-1 nos níveis de malondialdeído (MDA) e proteínas carboniladas de 84 voluntários expostos ambientalmente ao chumbo (28 homens e 56mulheres; idade: 18 a 60 anos). Os genótipos para o polimorfismo Pro198Leu da GPx-1 foram determinados por PCR seguido por digestão com enzima de restrição. Chumbo no plasma (Pb_P) e chumbo no sangue total (Pb_S) foram determinados por espectrometria de massas com plasma indutivamente acoplado e espectrometria de absorção atômica com forno de grafite, respectivamente. As freqüências alélicas para os alelos T (que codifica o aminoácido prolina) e C (que codifica o aminoácido leucina) da GPx-1 foram 0,73 e 0,27, respectivamente. Os voluntários foram separados em dois grupos, de acordo com a presença ou ausência do alelo que codifica leucina. Não foi encontrada diferença significativa para as concentrações de Pb_P e Pb_S, bem como para os marcadores de estresse oxidativo entre os grupos. Deste modo, os resultados obtidos sugerem que o polimorfismo Pro198Leu da GPx-1 não torna indivíduos ambientalmente expostos ao chumbo, mais suscetíveis aos danos do estresse oxidativo induzidos pelo metal. / Glutathione peroxidase (GPx-1) plays an important role in scavenger reactive oxygen species (ROS). This study was carried out to assess the effects of GPx-1 gene polymorphism (Pro198Leu) on malondialdehyde (MDA) and carbonyl protein ratio in 84 subjects environmentally exposed to lead. Genotypes for the GPx-1 Pro198Leu polymorphism were determined by PCR and restriction fragment length digestion. Lead in plasma (Pb_P) and lead in total blood (Pb_B) were determined by inductively coupled plasma mass spectrometry and by graphite furnace atomic absorption spectrometry, respectively. The allele frequencies for T (which express the proline aminoacid) and C (which express the leucine aminoacid) were 0.73 e 0.27, respectively. The volunteers were divided in two groups, according to the presence or absence of C allele. No significant differences were found in Pb_P and Pb_B, as well as for the other oxidative biomarkers between the groups. Therefore, the obtained results suggest that the GPx-1 Pro198Leu polymorphism do not affect individuals environmentally exposed to lead greater susceptible to oxidative damage induced for this metal.

O chumbo (Pb) é um metal altamente tóxico no qual os sinais de intoxicação variam bastante ao considerar as diferenças interindividuais. Um dos principais mecanismos de toxicidade do Pb ocorre pela inibição da enzima ácido delta aminolevulínico desidratase (ALAD) no sistema hematopoiético. O Pb também desempenha um importante papel no desbalanço do estado redox, pois sabe-se que ele tem o potencial de aumentar a concentração de espécies reativas de oxigênio (EROS) e inibir enzimas antioxidantes, como por exemplo a glutationa peroxidase (GPx). No entanto, poucos estudos avaliaram estes parâmetros em população ocupacionalmente exposta brasileira. Assim, o presente estudo, objetiva estudar a correlação entre as concentrações de Pb no sangue (Pb-S) de trabalhadores de fábricas de bateria e as atividades das enzimas ALAD e GPx associados com os polimorfismos genéticos da ALAD e GPx. Para tal, foram utilizadas 278 amostras de sangue de trabalhadores expostos ao Pb. As determinações de Pb foram realizadas por espectrometria de massas com plasma acoplado indutivamente (ELAN DRCII Perkin- Elmer). As genotipagens dos polimorfismos genéticos da ALAD e da GPx foram realizadas pela Reação em Cadeia da Polimerase (PCR) em tempo real e as atividades das enzimas ALAD e GPx foram determinadas no sangue por espectrofotometria de UV/VIS. A média da concentração de Pb-S foi de 22,8 ± 14,7 ?g/dL. Foram observadas correlações negativas entre Pb-S e atividade da ALAD (rs -0,24 p<0,01) e Pb-S e atividade de GPx (rs -0,27 p<0,05). Também foi verificada correlação negativa entre a porcentagem de inibição da ALAD e a atividade de GPx (rs -0,21 p<0,01). Em relação aos polimorfismos genéticos, não observamos associação entre os genótipos do ALAD (? -0,19 P>0,05) e GPx (genótipo CT: ? -1,37 P>0,05; genótipo TT: ? -8,37 P>0,05) e a concentração de Pb-S. Não foram observadas associações entre o polimorfismo rs1800668 e a atividade de GPx (genótipo CT: ? -0,016 P>0,05; genótipo TT: ? -0,004 P>0,05). No entanto, foi constatada uma associação entre o genótipo ALAD 1-1 do polimorfismo rs1800435 e a atividade da enzima ALAD (? 3,5 P<0,05). Neste sentido, os resultados deste estudo mostram que o genótipo ALAD 1-1 do polimorfismo rs1800435 do gene ALAD está associado a uma maior atividade da enzima ALAD nos indivíduos expostos ao Pb. Além disso, o polimorfismo rs1800668, localizado no gene que codifica a enzima GPx, não modula a atividade desta enzima nos indivíduos expostos ao metal. Ambos os polimorfismos dos genes ALAD e GPx parecem não influenciar nas concentrações de Pb-S na população estudada. / Lead (Pb) is a highly toxic metal which signs of intoxication vary greatly when considering the interindividual differences. One of the main mechanism of toxicity of Pb is due to inhibition of the enzyme acid delta aminolevulinic dehydratase (ALAD) in the hematopoietic system. Pb also plays an important role in the redox state of unbalance, since it is known that it has the potential of increasing the concentrations of reactive oxygen species (ROS) and inhibit antioxidant enzymes such as glutathione peroxidase (GPx). However, few studies have evaluated these parameters in Brazilian population occupationally exposed. Thus, the present study aims to study the correlation between the concentrations of Pb in the blood (B-Pb) of workers of battery factories and the activities of ALAD and GPx enzymes associated with genetic polymorphisms in ALAD and GPx. Then, blood samples of 278 workers exposed to Pb were collected. Pb was measured by Inductively Coupled Plasma-Mass Spectrometry (Perkin-Elmer ELAN DRCII). The genotyping of genetic polymorphisms of ALAD and GPx was performed by Real-Time Polymerase Chain Reaction (PCR) and activity of ALAD and GPx enzymes in the blood was determined by spectrophotometry UV / VIS. The mean concentration of B-Pb was 22.81 ± 14.73 mg / dL. Negative correlation was found between B-Pb and ALAD activity (rs -0.24 p < 0.01) and B-Pb and GPx activity (rs -0.27 p < 0.05). There was also a negative correlation between the percentage of inhibition of ALAD and GPx activity (rs -0.21 p < 0.01). Considering the genetic polymorphisms, no association between ALAD genotypes (? -0.19 P < 0.05) and GPx (CT genotype: ? -1.37 P > 0.05, TT genotype : ? -8.37 P > 0.05 ) and the concentration of B-Pb. No association between rs1800668 polymorphism and GPx activity (CT genotype: ? -0.016 P > 0.05, TT genotype : ? -0.004 P > 0.05). However an association was found between ALAD 1-1 rs1800435 genotype and ALAD enzyme activity (? 3.5 P < 0.05). Accordingly, the results of this study show that the ALAD 1-1 genotype of rs1800435 polymorphism in ALAD gene is associated with increased activity of ALAD enzyme in individuals exposed to Pb. Furthermore the rs1800668 polymorphism located in a gene encoding the enzyme GPx does not modulate the activity of this enzyme in individuals exposed to the metal. Both polymorphisms of ALAD and GPx genes seem to have no influence in the concentrations of B-Pb in the population studied.

O selênio (Se), mineral traço essencial para o ser humano, exerce sua função no organismo por meio de sua participação nas selenoproteínas, dentre as quais destacam-se as glutationas peroxidase (GPx), importantes no sistema de defesa antioxidante. São conhecidas sete isoformas da GPx, sendo a GPx 1 (citosólica) a mais abundante. Como a atividade dessa enzima encontra-se fortemente correlacionada com a concentração sanguínea de Se, este parâmetro é normalmente utilizado como indicador do balanço metabólico do mineral. A concentração de Se nos alimentos depende do local onde foram cultivados e reflete diretamente o teor do mineral no solo, sendo os alimentos da região Norte e Nordeste mais ricos nesse nutriente quando comparados às demais regiões brasileiras. O consumo inadequado de Se provoca uma diminuição da atividade dessa enzima, o que por sua vez pode afetar a proteção antioxidante. Além disso, estudos vêm mostrando que o polimorfismo Pro198Leu no gene da GPx1 também pode diminuir a atividade da GPx. O presente estudo se propôs a verificar a relação entre esse polimorfismo e o estado nutricional relativo ao selênio de uma população adulta residente no município de Fortaleza/Ceará. A população do estudo foi constituída por 176 indivíduos (79 homens e 97 mulheres), com média de idade de 30,4 ± 8,9 anos. Foram realizadas a avaliação da composição corporal por antropometria e a avaliação do consumo alimentar, por meio de três recordatórios de 24h. Os biomarcadores para avaliação do estado nutricional dos indivíduos quanto ao selênio foram: a concentração de Se plasmático e eritrocitário e a atividade da GPx total no eritrócito. Também foi determinado o polimorfismo Pro198Leu no gene que codifica para a GPx 1 por meio de PCR em Tempo Real. Foram encontradas concentrações médias de 62,6 &#181;g/L para Se plasmático e de 101,5 &#181;g/L para Se eritrocitário, não sendo observada correlação entre os dois. A média de ingestão de selênio foi de 76,88 &#181;g/dia, apresentando correlação positiva com o consumo de energia e proteína. Separando por gênero, encontrou-se que as médias de Se plasmático e de Se ingerido dos homens foram significativamente maiores que as das mulheres (p<0,05). A média da atividade da GPx foi de 38,6 U/g Hb. Para o polimorfismo Pro198Leu no gene que codifica para a GPx 1, foram encontrados 96 (54,5%) indivíduos com genótipo selvagem Pro/Pro, 67 (38,1%) indivíduos Pro/Leu e 13 (7,4%) indivíduos Leu/Leu. A frequência encontrada estava em equilíbrio de Hardy-Weinberg. Não foram encontradas diferenças estatísticas significativas nas concentrações de selênio entre os grupos de acordo com o genótipo, no entanto, para a atividade da GPx, houve diferença entre o grupo Pro/Pro e Pro/Leu (p<0,05). No grupo Pro/Pro, foi encontrada correlação positiva entre a concentração de selênio eritrocitário e a atividade eritrocitária total da GPx (p<0,05). Desta forma, podemos concluir que o estado nutricional da população estudada estava adequado com relação ao selênio e que o polimorfismo Pro198Leu apresentou influência sobre a atividade da GPx, que apresentou-se reduzida nos indivíduos que apresentaram alelo variante em seu genótipo. / Selenium (Se) is an essential trace mineral for humans which exerts its function in the body in selenoproteins, among which we highlight the glutathione peroxidase (GPx), important in the antioxidant defense system. There are seven known GPx isoforms and the GPx 1 (cytosolic) is the most abundant. As the activity of this enzyme is strongly correlated with selenium blood concentration this parameter is usually used as an indicator of the mineral metabolic balance. Selenium concentration in food depends on the location where they were grown and directly reflects the soil mineral content. Food in the North and Northeast are richer in selenium when compared to other Brazilian regions. Inadequate consumption of Se causes a decrease in GPx activity, what affects the antioxidant protection. Furthermore, studies have shown that the Pro198Leu polymorphism at GPx1 gene of can also decrease the activity of GPx. Our study aimed to investigate the relationship between this polymorphism and nutritional selenium status in an adult population living in Fortaleza/Ceará/Brazil. The study population consisted of 176 individuals (79 men and 97 women) with a mean age of 30.4 ± 8.9 years. The body composition was assessed by anthropometry and the food intake assessment was done using three 24-hour records. The selenium Se concentration was measured in plasma and erythrocyte and GPx activity was measured in erythrocytes. The Pro198Leu polymorphism at GPx 1 gene was determined by Real Time PCR. We found concentrations of 62.6 &#181;g/L for Se plasma and 101.5 &#181;g/L for erythrocyte. There was no correlation observed between these two markers. The average selenium intake was 76.88 &#181;g/day and it showed positively related to the energy and protein consumption. Separating by gender, men selenium plasma concentration and selenium consumption were significantly higher than for women (p < 0.05). The GPx activity was 38.6 U/g Hb. The Pro198Leu polymorphism at GPx 1 gene frequency were 96 (54.5%) subjects with Pro/Pro, 67 (38.1%) subjects Pro/Leu and 13 (7.4%) individuals Leu/Leu. The rate found was in Hardy -Weinberg equilibrium. There were no statistically significant differences in selenium concentrations between groups according to the genotype. However, for GPx activity we found difference between the group Pro/Pro and Pro/Leu (p< 0.05). We also found positive correlation between Se erythrocyte concentration and GPx activity in Pro/Pro group (p<0.05). Thus, we conclude that the selenium nutritional status of the population studied was adequate and that Pro198Leu polymorphism showed influence on the activity of GPx, which was lower in individuals with the variant allele in their genotype.

O selênio (Se) é um mineral essencial para o corpo humano, com ação no sistema imunológico, na glândula tireoide, dentre outras funções. É um nutriente importante na proteção das células contra os efeitos dos radicais livres, já que faz parte da enzima glutationa peroxidase (GPX). O consumo alimentar de Se é dependente da concentração desse elemento nos alimentos que varia em função da concentração do mesmo nos solos. Deste modo, este estudo teve como objetivo geral avaliar o estado nutricional relativo ao selênio de indivíduos adultos da cidade de Manaus-AM, por ser considerada região com solo rico nesse nutriente. Casuística: o estudo foi do tipo transversal baseado em amostra não probabilística de conveniência. A população estudada foi composta por 124 voluntários de ambos os gêneros com idade entre 20 a 59 anos. Critérios de inclusão: não ser atleta de elite, não ser etilista, não apresentar doenças crônicas não transmissíveis e não fazer o uso de suplementos vitamínico-minerais e/ou anti-inflamatórios. Não foram incluídos no estudo os participantes que não entregaram os formulários e questionários solicitados (alimentação e/ou ficha cadastral) e/ou que não compareceram para coleta de sangue. A coleta de sangue foi em jejum de 8 a 12 horas para determinação de selênio no plasma e eritrócitos e da atividade da GPX nos eritrócitos, também foram avaliados o consumo alimentar e as medidas antropométricas. Os resultados antropométricos mostraram que 60,0% dos participantes apresentava excesso de peso, a distribuição dos macronutrientes ficou dentro do recomendado pelas DRIs (Dietary Reference Intakes), a ingestão média de selênio ficou acima do recomendado (72±48,0&#181;g/dia), a concentração média para o selênio plasmático foi de 111,4± 37,0 &#181;g/L, de selênio eritrocitário 211± 62,4 &#181;g/L e a atividade da GPX foi de 73± 21,9 U/g Hb, não sendo observada correlação entre as concentrações obtidas para plasma e eritrócito, porém com correlação positiva para Se eritrocitário e atividade da GPX (r= 0,393). Pode-se, portanto concluir do presente estudo que a boa parte dos indivíduos avaliados, habitantes da cidade de Manaus, foi considerada deficiente, apesar de consumirem quantidade desse mineral consideradas adequadas porém, nenhum apresentando parâmetros bioquímicos de deficiência, a maioria dentro da normalidade, entretanto, com um percentual de indivíduos apresentando risco de toxicidade. / Selenium (Se) is an essential mineral for the human body, with function on the immune system, thyroid glands and other ones. It is an important nutrient in protecting cells against the effects of free radicals because it is part of glutathione peroxidase enzyme (GPX). Selenium intake depends on the mineral concentration in aliments which varies based on the mineral content in soils. Thus, this study had as main objective to evaluate the nutritional status related to selenium on adults in Manaus-AM because it is considered as a region of high selenium soil content region. Casuistic: This study was transversal type based in a non-probabilistic convenience sample. The population consisted of 124 volunteers (both genders) aged from 20 to 59 years. Inclusion criteria: To not be an elite athlete or alcoholic, do not have chronic non-communicable diseases and do not use vitamin-mineral supplements and/or anti-inflammatory pills. People who did not submit the requested forms and questionnaires (registration form and/or alimentation) and/or did not attend for blood collection were not included. Blood collection was fasting from 8 to 12 hours for determining selenium in plasma and erythrocytes and the activity of GPX in erythrocytes. Food intake and anthropometric measures were also evaluated. Anthropometric results showed that 60,0% were overweight, macronutrient distribution was within the recommended by DRIs (Dietary Reference Intakes). Average selenium intake was above recommended (72±48,0 &#181;g/day), mean plasmatic selenium concentration was 111,4 ± 37,0 &#181;g/L, for erythrocytes selenium was 211 ± 62,4 &#181;g/L and activity of GPX average was 73 ± 21,9 U/g Hb. No correlation between concentration of Se in plasma and erythrocyte, however, there was positive correlation between GPX activity and Se concentration in erythrocytes (r=0,393). Finally, we conclude that most of the people that were evaluated, citizens of Manaus city, were considered as deficient, although they consumed adequate quantities of this mineral. However, none of them showed biochemical deficiency parameters, most within normality, but with a percentage of them showing toxicity risk.

Free-radical generation is one of the primary causes of myocardial ischemia/reperfusion (I/R) injury. Melatonin is an efficient free-radical scavenger and induces the expression of antioxidant enzymes. We have previously shown that melatonin can prevent free-radical-induced myocardial injury. To date, the mechanism underlying melatonin's cardioprotective effect is not clear. In this study, we assessed the ability of melatonin to protect against I/R injury in mice deficient in glutathione peroxidase 1 (Gpx1). Mice hearts were subjected to 40 min of global ischemia in vitro followed by 45 min of reperfusion. Myocardial I/R injury (expressed as % of recovery of left ventricular developed pressure × heart rate) was exacerbated in mice deficient in Gpx1 (51 ± 3% for Gpx1+/+ mice versus 31 ± 6% for Gpx1-/- mice, P < 0.05). Administration of melatonin for 30 min protected against I/R injury in both Gpx1+/+ mice (72 ± 4.8%) and Gpx1-/- mice (63 ± 4.7%). This protection was accompanied by a significant improvement in left ventricular end-diastolic pressure and a twofold decrease in lactate dehydrogenase (LDH) level released from melatonin-treated hearts. In another set of experiments, mice were subjected to 50 min of ligation of the left descending anterior coronary artery in vivo followed by 4 hr of reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly larger in Gpx1-/- mice than in Gpx1+/+ mice (75 ± 9% versus 54 ± 6%, P < 0.05) and were reduced significantly in melatonin-treated mice (31 ± 3.7% Gpx1-/- mice and 33 ± 6.0% Gpx1+/+ mice). In hearts subjected to 30 min of coronary artery occlusion followed by 3 hr of reperfusion, melatonin-treated hearts had significantly fewer in situ oligo ligation-positive myocytes and less protein nitration. Our results demonstrate that the cardioprotective function of melatonin is independent of Gpx1.

Doxorubicin (DOX)-induced cardiotoxicity is thought to be mediated by the generation of superoxide anion radicals (superoxide) from redox cycling of DOX in cardiomyocyte mitochondria. Reduction of superoxide generates H2O2, which diffuses throughout the cell and potentially contributes to oxidant-mediated cardiac injury. The mitochondrial and cytosolic glutathione peroxidase 1 (Gpx1) primarily functions to eradicate H2O2. In this study, we hypothesize that Gpx1 plays a pivotal role in the clearance of H2O2 generated by DOX. To test this hypothesis, we compared DOX-induced cardiac dysfunction, mitochondrial injury, protein nitration, and apoptosis in Gpx1-deficient and wild type mouse hearts. The Gpx1-deficient hearts showed increased susceptibility to DOX-induced acute functional derangements than wild type hearts, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impaired the mitochondrial function of Gpx1-deficient hearts. Specifically, Gpx1-deficient hearts treated with DOX demonstrated an increased rate of NAD-linked state 4 respiration and a decline in the P/O ratio relative to wild type hearts, suggesting that DOX uncouples the electron transfer chain and oxidative phosphorylation in Gpx1-deficient hearts. Finally, apoptosis and protein nitration were significantly increased in Gpx1-deficient mouse hearts compared to wild type hearts. These studies suggest that Gpx1 plays significant roles in protecting DOX-induced mitochondrial impairment and cardiac dysfunction in the acute phase.

The cardiac toxicity of doxorubicin (DOX), a potent anticancer anthracycline antibiotic, is believed to be mediated through the generation of reactive oxygen species (ROS) in cardiomyocytes. This study aims to determine the function of cellular glutathione peroxidase (Gpx1), which is located in both mitochondria and cytosol, in defense against DOX-induced cardiomyopathy using a line of transgenic mice with cardiac overexpression of Gpx1. The Gpx1-overexpressing hearts were markedly more resistant than nontransgenic hearts to DOX-induced acute functional derangements, including impaired contractility and diastolic properties, decreased coronary flow rate, and reduced heart rate. In addition, DOX treatment impairs mitochondrial function of nontransgenic hearts as evident in a decreased rate of NAD-linked State 3 respiration, presumably a result of inactivation of complex I activity. This is associated with increases in the rates of NAD- and FAD-linked State 4 respiration and declines in P/O ratio, suggesting that the electron transfer and oxidative phosphorylation are uncoupled in these mitochondrial samples. These functional deficits of mitochondria could be largely prevented by Gpx1 overexpression. Taken together, these studies provide new evidence to further support the role of ROS, particularly H2O2 and/or fatty acid hydroperoxides, in causing contractile and mitochondrial dysfunction in mouse hearts acutely exposed to DOX.

Selenium is toxic at high doses, yet metabolically essential in trace amounts, and therefore provides an excellent illustration of the rule of paracelsus that "the dose alone determines the poison". The only mammalian selenoprotein of known function is glutathione peroxidase (GPx). This enzyme is expressed ubiquitously, and is responsible for detoxifying peroxides and hydroperoxides which, if left unchecked, may damage important biomolecules such as DNA and membrane fatty-acids. GPx is a homotetramer; each subunit contains one mole atom of selenium incorporated as a selenocysteine residue in the active site of the enzyme. Using oligonucleotides generated against the known bovine GPx amino-acid sequence, cDNA clones were isolated corresponding to the human GPx mRNA. Sequence analysis indicated that the selenocysteine in the active site of the enzyme was incorporated at an opal terminator (UGA) codon. Therefore the glutathione peroxidase mRNA constitutes the first example of natural suppression of a terminator codon in human cells. Regulation of human GPx gene expression by selenium was examined. Selenium replete HL-60 cells possessed approximately 30-fold more enzymatic GPx activity than selenium deficient cells. However steady-state GPx mRNA levels and rate of transcription of the GPx gene differed by less than 1.5-fold. Cycloheximide abolished the increase in enzymatic activity observed upon selenium replenishment. Cellular immunoreactive GPx protein levels correlated with enzymatic activity, indicating that the human GPx gene is regulated post-transcriptionally by selenium. The mechanism of this post-transcriptional regulation was investigated. A selenium labelled tRNA species was identified which exhibited features in common with a previously characterized tRNAUGA. This data suggested that selenium may be incorporated into GPx via a co-translational mechanism using a selenocysteinyl tRNA intermediate. Selenium did not alter cytoplasmic levels of the tRNAUGA, indicating that accumulation of cytoplasmic suppressor tRNA was not the point of regulation of GPx by selenium. A model is proposed for the co-translational insertion of selenocysteine into GPx mediated by a charged tRNA species present in selenium replete but absent from selenium deficient cells. Models are also proposed to explain the discrimination between the selenocysteine UGA codon and authentic UGA terminator codons. The regulation of the GPx gene was examined during mono-myelocytic differentiation of HL-60 cells in vitro and also during interferon-gamma activation of human peripheral blood macrophages and PMN. During phagocyte cell differentiation or activation, the ability to generate peroxide developed, however the peroxide-destroying capacities of GPx did not increase concomitantly. Complex regulatory patterns involving both transcriptional and translational controls were observed. The association of GPx gene expression with chronic granulomatous disease was explored. No correlation was found with either the autosomal or X-linked forms of the disease, a finding contradictory to previously published material.

No description available.