Dietary cellulose has no effect on the regeneration of hemoglobin in growing rats with iron deficiency anemia

The objective of the present study was to determine the effect of cellulose on intestinal iron absorption in rats during recovery from iron deficiency anemia. Twenty-one-day-old male Wistar-EPM rats were fed an iron-free ration for two weeks to induce anemia. At 5 weeks of age, the rats were divided into two groups (both groups receiving 35 mg of elemental iron per kg diet): cellulose group (N = 12), receiving a diet containing 100 g of cellulose/kg and control (N = 12), receiving a diet containing no cellulose. The fresh weight of the feces collected over a 3-day period between the 15th and 18th day of dietary treatment was 10.7 ± 3.5 g in the group receiving cellulose and 1.9 ± 1.2 g in the control group (P<0.001). Total food intake was higher in the cellulose group (343.4 ± 22.0 g) than in the control (322.1 ± 13.1 g, P = 0.009) during the 3 weeks of dietary treatment. No significant difference was observed in weight gain (cellulose group = 132.8 ± 19.2, control = 128.0 ± 16.3 g), hemoglobin increment (cellulose group = 8.0 ± 0.8, control = 8.0 ± 1.0 g/dl), hemoglobin level (cellulose group = 12.3 ± 1.2, control = 12.1 ± 1.3 g/dl) or in hepatic iron levels (cellulose group = 333.6 ± 112.4, control = 398.4 ± 168.0 µg/g dry tissue). We conclude that cellulose does not adversely affect the regeneration of hemoglobin, hepatic iron level or the growth of rats during recovery from iron deficiency anemia.

Lack of evidence for the pathogenic role of iron and HFE gene mutations in Brazilian patients with nonalcoholic steatohepatitis

The hypothesis of the role of iron overload associated with HFE gene mutations in the pathogenesis of nonalcoholic steatohepatitis (NASH) has been raised in recent years. In the present study, biochemical and histopathological evidence of iron overload and HFE mutations was investigated in NASH patients. Thirty-two NASH patients, 19 females (59%), average 49.2 years, 72% Caucasians, 12% Mulattoes and 12% Asians, were submitted to serum aminotransferase and iron profile determinations. Liver biopsies were analyzed for necroinflammatory activity, architectural damage and iron deposition. In 31 of the patients, C282Y and H63D mutations were tested by PCR-RFLP. Alanine aminotransferase levels were increased in 30 patients, 2.42 ± 1.12 times the upper normal limit on average. Serum iron concentration, transferrin saturation and ferritin averages were 99.4 ± 31.3 g/dl, 33.1 ± 12.7% and 219.8 ± 163.8 µg/dl, respectively, corresponding to normal values in 93.5, 68.7 and 78.1% of the patients. Hepatic siderosis was observed in three patients and was not associated with architectural damage (P = 0.53) or with necroinflammatory activity (P = 0.27). The allelic frequencies (N = 31) found were 1.6 and 14.1% for C282Y and H63D, respectively, which were compatible with those described for the local population. In conclusion, no evidence of an association of hepatic iron overload and HFE mutations with NASH was found. Brazilian NASH patients comprise a heterogeneous group with many associated conditions such as hyperinsulinism, environmental hepatotoxin exposure and drugs, but not hepatic iron overload, and their disease susceptibility could be related to genetic and environmental features other than HFE mutations.

Morphological and functional alterations of the intestine of rats with iron-deficiency anemia

The present study was designed to assess the intestinal absorption of D-xylose and jejunal morphometry in rats with iron-deficiency anemia. Male Wistar rats were randomly divided into a control group (diet containing 50 mg Fe/kg, N = 12) and an anemic group (diet containing <5 mg Fe/kg, N = 12). The animals were housed in individual metabolic cages and deionized water and diet were provided ad libitum for 6 weeks. Hemoglobin and hematocrit were determined at 0, 2, 4, and 6 weeks. At the end of the study the rats were submitted to a D-xylose absorption test (50 mg/100 g body weight) and sacrificed and a jejunal specimen was obtained for morphometric study. At the end of the study the hemoglobin and hematocrit of the anemic rats (8.7 ± 0.9 g/dl and 34.1 ± 2.9%, respectively) were significantly (P < 0.05) lower than those of the controls (13.9 ± 1.4 g/dl and 47.1 ± 1.5%, respectively). There was no statistical difference in D-xylose absorption between the anemic (46.5 ± 7.4%) and control (43.4 ± 9.0%) groups. The anemic animals presented statistically greater villus height (445.3 ± 36.8 µm), mucosal thickness (614.3 ± 56.3 µm) and epithelial surface (5063.0 ± 658.6 µm) than control (371.8 ± 34.3, 526.7 ± 62.3 and 4401.2 ± 704.4 µm, respectively; P < 0.05). The increase in jejunum villus height, mucosal thickness and epithelial surface in rats with iron-deficiency anemia suggests a compensatory intestinal mechanism to increase intestinal iron absorption.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

Baroreflex function in conscious rats submitted to iron overload

Our hypothesis is that iron accumulated in tissue, rather than in serum, may compromise cardiovascular control. Male Fischer 344 rats weighing 180 to 220 g were divided into 2 groups. In the serum iron overload group (SIO, N = 12), 20 mg elemental iron was injected ip daily for 7 days. In the tissue iron overload group (TIO, N = 19), a smaller amount of elemental iron was injected (10 mg, daily) for 5 days followed by a resting period of 7 days. Reflex heart rate responses were elicited by iv injections of either phenylephrine (0.5 to 5.0 µg/kg) or sodium nitroprusside (1.0 to 10.0 µg/kg). Baroreflex curves were determined and fitted to sigmoidal equations and the baroreflex gain coefficient was evaluated. To evaluate the role of other than a direct effect of iron on tissue, acute treatment with the iron chelator deferoxamine (20 mg/kg, iv) was performed on the TIO group and the baroreflex was re-evaluated. At the end of the experiments, evaluation of iron levels in serum confirmed a pronounced overload for the SIO group (30-fold), in contrast to the TIO group (2-fold). Tissue levels of iron, however, were higher in the TIO group. The SIO protocol did not produce significant alterations in the baroreflex curve response, while the TIO protocol produced a nearly 2-fold increase in baroreflex gain (-4.34 ± 0.74 and -7.93 ± 1.08 bpm/mmHg, respectively). The TIO protocol animals treated with deferoxamine returned to sham levels of baroreflex gain (-3.7 ± 0.3 sham vs -3.6 ± 0.2 bpm/mmHg) 30 min after the injection. Our results indicate an effect of tissue iron overload on the enhancement of baroreflex sensitivity.

APOA1/C3/A4 gene cluster variability and lipid levels in Brazilian children

Genetic studies have suggested that polymorphisms of genes coding for apolipoproteins are significant determinants of serum lipoprotein and lipid levels in adults. However, only a few studies have investigated the association of these polymorphisms in children. Therefore, in the present investigation we studied the distribution of APOA1 -75 G>A, +83 C>T, APOC3 -482 C>T, -455 T>C and 3238 C>G, and APOA4 Q360H and T347S polymorphisms and their influence on plasma lipoprotein levels in children from a Brazilian northeastern admixed population. The seven polymorphic sites were genotyped in 414 children aged 5 to 15 years (mean 8.9 ± 2.9). The genotypes of the seven polymorphic sites were assessed by PCR-RFLP methods. The frequencies of the less common alleles were, in general, intermediate among parental populations, as expected. Strong linkage disequilibrium was detected between polymorphisms at the APOA1, APOC3 and APOA4 loci in this admixed population sample. Overall the genotype effects seen in adults were weaker or absent in children. The APOC3/-455 and APOA4 T347S variants showed significant effects on HDL cholesterol in girls (P = 0.033 and P = 0.016, respectively). Significantly higher plasma total (P = 0.003) and LDL cholesterol (P = 0.004) levels were observed in boys who were carriers of the 3238G allele at the APOC3/3238 C>G site. These results disclosed an overall absence of associations between these polymorphisms and lipids in children. This finding is not unexpected because expression of the effect of these polymorphisms might depend on the interaction with environmental variables both internal and external to the individual.

Effect of iron overload on the severity of liver histologic alterations and on the response to interferon and ribavirin therapy of patients with hepatitis C infection

The objective of the present study was to determine the presence of hepatic iron overload in patients with chronic HCV infection and to correlate it with histologic alterations, HCV genotype and response to therapy. Liver tissue samples from 95 patients with chronic hepatitis C were divided into two groups: group I, presence of iron overload in hepatic tissue (Perls' staining) and group II, no iron overload. Hepatic iron overload was detected in 30 (31.6%) of 95 patients. Of the 69 patients tested by genotyping, 49 (71.01%) were genotype 1 and 20 (28.99%) genotype non-1. Iron overload was detected in 14 (28.6%) patients with genotype 1 and in 6 (30%) with genotype non-1 (P = 0.906). There was a significant difference in fibrosis stage between groups (P = 0.005). In group I (N = 30), one patient had stage F0/F1 of fibrosis, while in group II (N = 65), 22 (33.8%) patients had minimal or no fibrosis. Fibrosis stage F2/F3 was observed in 70% of group I patients compared to 46.2% of group II. Eighty-five patients were treated with a combination of interferon and ribavirin; 29 of them (34.1%) had a sustained virologic response and 8 (27.6%) of them had hepatic iron overload. Iron overload was detected in 18 (32.1%) of the 56 non-responders (P = 0.73). Hepatic iron overload was frequent among patients with chronic hepatitis C and was associated with a more severe stage of liver fibrosis. There was no association between iron overload and HCV genotype and response to interferon and ribavirin therapy.

Lycopene and ß-carotene protect in vivo iron-induced oxidative stress damage in rat prostate

It has been suggested that iron overload may be carcinogenic. In the present study, we evaluated the effect of plasma and prostate carotenoid concentration on oxidative DNA damage in 12-week-old Wistar rats treated with intraperitoneal (ip) ferric nitrilotriacetate (Fe-NTA) (10 mg Fe/kg). Plasma ß-carotene and lycopene concentrations were measured as a function of time after ip injection of carotenoids (10 mg kg-1 day-1 ß-carotene or lycopene) in rats. The highest total plasma concentration was reached 3 and 6 h after ip injection of lycopene or ß-carotene, respectively. After 5 days of carotenoid treatment, lycopene and ß-carotene were present in the 0.10-0.51 nmol/g wet tissue range in the prostate. Using a sensitive method to detected 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) by HPLC/EC, the level of 8-oxodGuo in rat prostate DNA was significantly higher (6.3 ± 0.6 residues/10(6) dGuo) 3 h after Fe-NTA injection compared with control rats (1.7 ± 0.3 residues/10(6) dGuo). Rats supplemented with lycopene or ß-carotene for 5 days prior to Fe-NTA treatment showed a reduction of about 70% in 8-oxodGuo levels to almost control levels. Compared with control rats, the prostate of Fe-NTA-treated animals showed a 78% increase in malondialdehyde accumulation. Lycopene or ß-carotene pre-treatment almost completely prevented lipid damage. Epidemiological studies have suggested a lower risk of prostate cancer in men reporting a higher consumption of tomato products. However, before associating this effect with tomato sauce constituents, more information is required. The results described here may contribute to the understanding of the protective effects of carotenoids against iron-induced oxidative stress.

Iron(III) as Lewis acid catalyst in organosilicon and carbonyl chemistry

Iron is one of the most common elements in the earth’s crust and thus its availability and economic viability far exceed that of metals commonly used in catalysis. Also the toxicity of iron is miniscule, compared to the likes of platinum and nickel, making it very desirable as a catalyst. Despite this, prior to the 21st century, the applicability of iron in catalysis was not thoroughly investigated, as it was considered to be inefficient and unselective in desired transformations. In this doctoral thesis, the application of iron catalysis in combination with organosilicon reagents for transformations of carbonyl compounds has been investigated together with insights into iron catalyzed chlorination of silanes and silanols. In the first part of the thesis, the synthetic application of iron(III)-catalyzed chlorination of silanes (Si-H) and the monochlorination of silanes (SiH2) using acetyl chloride as the chlorine source is described. The reactions proceed under ambient conditions, although some compounds need to be protected from excess moisture. In addition, the mechanism and kinetics of the chlorination reaction are briefly adressed. In the second part of this thesis a versatile methodology for transformation of carbonyl compounds into three different compound classes by changing the conditions and amounts of reagents is discussed. One pot reductive benzylation, reductive halogenation and reductive etherification of ketones and aldehydes using silanes as the reducing agent, halide source or cocatalyst, were investigated. Also the reaction kinetics and mechanism of the reductive halogenation of acetophenone are briefly discussed.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Hydrogen peroxide induces a specific DNA base change profile in the presence of the iron chelator 2,2’ dipyridyl in Escherichia coli

Pretreatment of Escherichia coli cultures with the iron chelator 2,2’-dipyridyl (1 mM) protects against the lethal effects of low concentrations of hydrogen peroxide (<15 mM). However, at H2O2 concentrations equal to or greater than 15 mM, dipyridyl pretreatment increases lethality and mutagenesis, which is attributed to the formation of different types of DNA lesions. We show here that pretreatment with dipyridyl (1 mM) prior to challenge with high H2O2 concentrations (≥15 mM) induced mainly G:C→A:T transitions (more than 100X with 15 mM and more than 250X with 20 mM over the spontaneous mutagenesis rate) in E. coli. In contrast, high H2O2 concentrations in the absence of dipyridyl preferentially induced A:T→T:A transversions (more than 1800X and more than 300X over spontaneous mutagenesis for 15 and 20 mM, respectively). We also show that in the fpg nth double mutant, the rpoB gene mutation (RifS-RifR) induced by 20 mM H2O2 alone (20X higher) was increased in 20 mM H2O2 and dipyridyl-treated cultures (110X higher), suggesting additional and/or different lesions in cells treated with H2O2 under iron deprivation. It is suggested that, upon iron deprivation, cytosine may be the main damaged base and the origin of the pre-mutagenic lesions induced by H2O2.

HFE gene mutations and iron status of Brazilian blood donors

Mutations of the HFE and TFR2 genes have been associated with iron overload. HFE and TFR2 mutations were assessed in blood donors, and the relationship with iron status was evaluated. Subjects (N = 542) were recruited at the Hemocentro da Santa Casa de São Paulo, São Paulo, Brazil. Iron status was not influenced by HFE mutations in women and was independent of blood donation frequency. In contrast, men carrying the HFE 282CY genotype had lower total iron-binding capacity (TIBC) than HFE 282CC genotype carriers. Men who donated blood for the first time and were carriers of the HFE 282CY genotype had higher transferrin saturation values and lower TIBC concentrations than those with the homozygous wild genotype for the HFE C282Y mutation. Moreover, in this group of blood donors, carriers of HFE 63DD plus 63HD genotypes had higher serum ferritin values than those with the homozygous wild genotype for HFE H63D mutation. Multiple linear regression analysis showed that HFE 282CY leads to a 17.21% increase (P = 0.018) and a 83.65% decrease (P = 0.007) in transferrin saturation and TIBC, respectively. In addition, serum ferritin is influenced by age (3.91%, P = 0.001) and the HFE 63HD plus DD genotype (55.84%, P = 0.021). In conclusion, the HFE 282Y and 65C alleles were rare, while the HFE 63D allele was frequent in Brazilian blood donors. The HFE C282Y and H63D mutations were associated with alterations in iron status in blood donors in a gender-dependent manner.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

The miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation of mouse embryonic cardiomyocytes

MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3’ untranslated regions (3’UTR) of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3’UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3’UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.

Association of sulfur dioxide exposure with circulatory system deaths in a medium-sized city in Brazil

There is a demonstrable association between exposure to air pollutants and deaths due to cardiovascular diseases. The objective of this study was to estimate the effects of exposure to sulfur dioxide on mortality due to circulatory diseases in individuals 50 years of age or older residing in São José dos Campos, SP. This was a time-series ecological study for the years 2003 to 2007 using information on deaths due to circulatory disease obtained from Datasus reports. Data on daily levels of pollutants, particulate matter, sulfur dioxide (SO2), ozone, temperature, and humidity were obtained from the São Paulo State Environmental Agency. Moving average models for 2 to 7 days were calculated by Poisson regression using the R software. Exposure to SO2 was analyzed using a unipollutant, bipollutant or multipollutant model adjusted for mean temperature and humidity. The relative risks with 95%CI were obtained and the percent decrease in risk was calculated. There were 1928 deaths with a daily mean (± SD) of 1.05 ± 1.03 (range: 0-6). Exposure to SO2 was significantly associated with mortality due to circulatory disease: RR = 1.04 (95%CI = 1.01 to 1.06) in the 7-day moving average, after adjusting for ozone. There was an 8.5% decrease in risk in the multipollutant model, proportional to a decrease of SO2 concentrations. The results of this study suggest that residents of medium-sized Brazilian cities with characteristics similar to those of São José dos Campos probably have health problems due to exposure to air pollutants.