No effect of acidification or freezing on urinary metanephrine levels.

BACKGROUND: Urinary metanephrine is a reliable method to estimate catecholamine secretion. Traditionally, urinary metanephrines are collected into chilled containers containing hydrochloric acid (HCl) and most laboratories freeze urinary samples before analysis. It is uncertain if these pre-analytic procedures alter metanephrine values. AIM: To evaluate if acidifying and freezing urine samples affect the accuracy of urinary metanephrine measurements. METHODS: Random urine samples from healthy individuals were collected. Urine samples were distributed into two containers: with HCl 50% homogenized with urine to obtain pH < 2, and without HCl. Each container was divided again into aliquots for immediate measurement or freezing. One aliquot with acid (group 1) and another without acid (group 2) were sent immediately to the laboratory for testing (HPLC), while the other two aliquots, one with acid (group 3) and another without it (group 4) were frozen for 3 months at - 20 °C. Bland-Altman's test was used to analyze inter-assay agreement between measurements. RESULTS: A total of 15 individuals were included (mean age 27.5 ± 5.9 years, 8 male and 14 white). No difference was observed on mean urinary metanephrine/creatinine ratio between groups: group 1: 0.23 ± 0.11, group 2: 0.22 ± 0.07, group 3: 0.25 ± 0.13, group 4: 0.25 ± 0.15 mg/g creatinine; P > 0.05 for all the comparisons). Bland-Altman's analysis showed agreement between the standard method (group 1) and the experimental method (group 4). CONCLUSION: Measurement of urinary metanephrines by HPLC method is not influenced by sample acidification nor freezing at - 20 °C for 3 months.

Persistent effects of pre-Columbian plant domestication on Amazonian forest composition.

The extent to which pre-Columbian societies altered Amazonian landscapes is hotly debated. We performed a basin-wide analysis of pre-Columbian impacts on Amazonian forests by overlaying known archaeological sites in Amazonia with the distributions and abundances of 85 woody species domesticated by pre-Columbian peoples. Domesticated species are five times more likely than nondomesticated species to be hyperdominant. Across the basin, the relative abundance and richness of domesticated species increase in forests on and around archaeological sites. In southwestern and eastern Amazonia, distance to archaeological sites strongly influences the relative abundance and richness of domesticated species. Our analyses indicate that modern tree communities in Amazonia are structured to an important extent by a long history of plant domestication by Amazonian peoples.

Long-term decline of the Amazon carbon sink.

Atmospheric carbon dioxide records indicate that the land surface has acted as a strong global carbon sink over recent decades, with a substantial fraction of this sink probably located in the tropics, particularly in the Amazon. Nevertheless, it is unclear how the terrestrial carbon sink will evolve as climate and atmospheric composition continue to change. Here we analyse the historical evolution of the biomass dynamics of the Amazon rainforest over three decades using a distributed network of 321 plots. While this analysis confirms that Amazon forests have acted as a long-term net biomass sink, we find a long-term decreasing trend of carbon accumulation. Rates of net increase in above-ground biomass declined by one-third during the past decade compared to the 1990s. This is a consequence of growth rate increases levelling off recently, while biomass mortality persistently increased throughout, leading to a shortening of carbon residence times. Potential drivers for the mortality increase include greater climate variability, and feedbacks of faster growth on mortality, resulting in shortened tree longevity. The observed decline of the Amazon sink diverges markedly from the recent increase in terrestrial carbon uptake at the global scale, and is contrary to expectations based on models.

HbA(1c) measurement for the diagnosis of diabetes: is it enough?

AIM: To analyse the performance of HbA(1c) in diagnosing Type 2 diabetes based on fasting plasma glucose and/or 2-h plasma glucose measurements after a 75-g oral glucose tolerance test. METHODS: This is a study of diagnostic test accuracy in individuals referred to the Clinical Pathology Department for oral glucose tolerance testing. After fasting overnight, HbA(1c), fasting plasma glucose and 2-h plasma glucose were measured. The receiver operating characteristic curve was used to evaluate the diagnostic performance of HbA(1c). RESULTS: Four hundred and ninety-eight subjects (195 male, mean age 56 years) were enrolled and 115 (23.1%) were diagnosed with diabetes according to glucose-based methods and only 56 (11.2%) individuals were identified by HbA(1c) ≥ 6.5% (48 mmol/mol) (sensitivity 20.9%, specificity 95.3%). There is poor agreement between the newly recommended criterion and the current glucose-based diagnostic criteria (κ = 0.217; P < 0.001), probably because the diagnostic methods identify different populations of patients. Adding a glucose-based method into an algorithm, as proposed by the UK Department of Health, improved HbA(1c) performance. CONCLUSIONS: HbA(1c) ≥ 6.5% (48 mmol/mol) showed limited sensitivity to diabetes diagnosis, although with high specificity. The results suggest that this cut-off point would not be enough to diagnose diabetes. Its use as the sole diabetes diagnostic test should be interpreted with caution to assure the correct classification of diabetic individuals.

Impact of metabolic syndrome and C-reactive protein on outcome after coronary stenting.

Metabolic syndrome (MS) identifies cardiovascular risk; however, there is little information regarding the evolution of patients with MS after stent implantation. The aim of this single-center study is to evaluate the possible association between MS and clinical restenosis, after adjustment for highsensitivity C-reactive protein (hs-CRP) and angiographic predictors of restenosis. In a longitudinal study, 159 patients (89 with and 70 without MS) were studied. Criteria for MS were: elevated blood pressure (systolic >or=130 mmHg, diastolic >or=85 mmHg or drug treatment for hypertension; elevated fasting glucose (>100 mg/dl) or drug treatment for elevated glucose; reduced HDL-cholesterol (<40 mg/dl in men and <50 mg/dl in women) or drug treatment for reduced HDL-cholesterol; elevated triglycerides (>or=150 mg/dl) or drug treatment for elevated triglycerides; and obesity (body mass index >28.8 kg/m2). The primary end point was the rate of major adverse clinical events (MACE): cardiovascular death, myocardial infarction, or target lesion revascularization (TLR) during the 12-month follow-up period. The secondary end point was the rate of TLR. MS was neither identified as predictor of MACE [hazard ratio (HR): 0.844; 95% CI: 0.41-1.74; p=0.648], nor TLR (HR: 1.05; 95% CI: 0.44-2.50; p=0.91), even when controlled for hs-CRP levels and angiographic predictors of restenosis. Also, no significant interaction between MS and hs-CRP was found (p=0.135 and p=0.194, for MACE and TLR, respectively). This study shows that patients with MS do not have an additional risk of MACE, even when controlled for angiographic predictors of restenosis and hs-CRP.

HLA-G polymorphism and transitional cell carcinoma of the bladder in a Brazilian population.

The morphologic appearance and clinical behavior of the human urinary bladder papillary transitional cell carcinoma (TCC) probably result from a complex interaction between carcinogenic insults and host resistance during the patient's life. While the main recognized risk factors are of environmental origin (e.g. smoking), relatively little information exists about the susceptibility to TCC development. The human leukocyte antigen G (HLA-G) molecule plays an important role in immune response regulation and has been implicated in the inhibition of the cytolytic function of natural killer and cytotoxic T cells. Several lines of evidence indicate that HLA-G polymorphisms influence the expression level and production of different HLA-G isoforms. The aim of this study was to explore a possible influence of the HLA-G polymorphism on the susceptibility to urinary bladder TCC development and progression in smokers and nonsmokers Brazilian subjects. The HLA-G locus was found to be associated with susceptibility to TCC development and progression. The G*0104 allelic group (specially the G*010404 allele) and the G*0103 allele were associated with a tobacco-dependent influence on TCC development. The G*0104 group was associated with progression to high-grade tumors, irrespective of smoking habit, while the G*0103 allele was associated to high-grade tumor only in smoking patients. Our results are an evidence that the HLA-G locus itself, or as part of an extended haplotype encompassing this chromosome region (particularly the HLA-A given the high linkage disequilibrium observed between them in this data series), may be associated with TCC susceptibility and tumor progression, suggesting a tobacco-dependent influence of these polymorphisms.

Lack of chemopreventive effects of ginger on colon carcinogenesis induced by 1,2-dimethylhydrazine in rats.

Ginger (Zingiber officinale Roscoe) has been proposed as a promising candidate for cancer prevention. Its modifying potential on the process of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) was investigated in male Wistar rats using the aberrant crypt foci (ACF) assay. Five groups were studied: Groups 1-3 were given four s.c. injections of DMH (40 mg/kg b.w.) twice a week, during two weeks, whereas Groups 4 and 5 received similar injections of EDTA solution (DMH vehicle). After DMH-initiation, the animals were fed a ginger extract mixed in the basal diet at 0.5% (Group 2) and 1.0% (Groups 3 and 4) for 10 weeks. All rats were killed after 12 weeks and the colons were analyzed for ACF formation and crypt multiplicity. The rates of cell proliferation and apoptosis were also evaluated in epithelial colonic crypt cells. Dietary consumption of ginger at both dose levels did not induce any toxicity in the rats, but ginger meal at 1% decreased significantly serum cholesterol levels (p<0.038). Treatment with ginger did not suppress ACF formation or the number of crypts per ACF in the DMH-treated group. Dietary ginger did not significantly change the proliferative or apoptosis indexes of the colonic crypt cells induced by DMH. Thus, the present results did not confirm a chemopreventive activity of ginger on colon carcinogenesis as analyzed by the ACF bioassay and by the growth kinetics of the colonic mucosa.

Typing class I HLA-A gene using a nested PCR-RFLP procedure.

In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A, One Lambda Inc.) showed an agreement higher than 95%. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations.

Typing class I HLA-A gene using a nested PCR-RFLP procedure

In order to detect several new HLA-A class I alleles that have been described since 1998, the original PCR-RFLP method developed to identify the 78 alleles recognized at that time at high resolution level was adapted by us for low and medium resolution levels using a nested PCR-RFLP approach. The results obtained from blood samples of 23 subjects using both the PCR-RFLP method and a commercial kit (MicroSSP1A®, One Lambda Inc.) showed an agreement higher than 95 percent. The PCR-RFLP adapted method was effective in low and medium resolution histocompatibility evaluations.

Conditions associated with very low values of glycohaemoglobin measured by an HPLC method.

AIMS: To identify the causes of very low glycohaemoglobin (GHb) values in a sample of patients with diabetes in southern Brazil using high performance liquid chromatography. METHODS: Between August 1996 and December 2001 all samples from patients with diabetes at a university hospital with GHb values below the reference range (4.7-6.0% HbA(1c)) were submitted to cellulose acetate electrophoresis. Medical records were reviewed to identify conditions that might be associated with these low values. RESULTS: Among 29 657 samples analysed, 130 patients had GHb < 4.7%. Seventy three patients (56%) were heterozygous for HbS, HbC, or HbD (19 black, two mulatto, and 52 white patients). The other 57 patients (44%) without Hb variants had low haematocrit and haemoglobin values (42 patients) or other conditions such as pregnancy, lipaemia, malignancy, cirrhosis, acetylsalicylic acid use, and absence of diabetes (15 patients). CONCLUSIONS: The presence of an Hb variant may falsely lower GHb measurements. However, anaemia is also a source of negative interference. The haematological status should be considered for the correct interpretation of GHb results.

Chemoprevention of preneoplastic liver foci development by dietary mushroom Agaricus blazei Murrill in the rat.

The chemopreventive potential of an Agaricus blazei (Ab) Murrill mushroom meal was investigated in a medium-term rat liver carcinogenesis assay. Male Wistar rats initiated for hepatocarcinogenesis with diethylnitrosamine (DEN, 200 mg/kg i.p.) were fed during a 6-week period with the dry powdered mushroom strains Ab 29 or 26, each one with opened (OB) or closed basidiocarp (CB), mixed at 10% level in a basal diet. All experimental animals and controls were subjected to partial hepatectomy at week 3 and killed at week 8. Chemopreventive activity of the mushroom meal was observed for the Ab 29 (OB and CB) and Ab 26 (CB) strains in terms of the number of putative preneoplastic altered foci of hepatocytes which express either the enzyme glutathione S-transferase, placental form (GST-P+) or the transforming growth factor-alpha, and for the Ab 29 (OB) and Ab 26 (CB) strains on the size of GST-P+ foci. This was associated with inhibition of foci cell proliferation in the animals fed the Ab 29 (OB) and Ab 26 (CB) strains. The results suggest that the protective influence of the Ab meal against the DEN potential for rat liver carcinogenicity depends on both the strain and period of mushroom harvest.

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9 NaCl. All animals were subjected to 70 partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9 NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci

Aberrant crypt foci and colon cancer: comparison between a short- and medium-term bioassay for colon carcinogenesis using dimethylhydrazine in Wistar rats.

Aberrant crypt foci (ACF) in the colon of carcinogen-treated rodents are considered to be the earliest hallmark of colon carcinogenesis. In the present study the relationship between a short-term (4 weeks) and medium-term (30 weeks) assay was assessed in a model of colon carcinogenesis induced by dimethylhydrazine (DMH) in the rat. Six-week-old male Wistar rats were given subcutaneous injections of DMH (40 mg/kg) twice a week for 2 weeks and killed at the end of the 4th or 30th week. ACF were scored for number, distribution pattern along the colon and crypt multiplicity in 0.1% methylene-blue whole-mount preparations. ACF were distinguished from normal crypts by their larger size and elliptical shape. The incidence, distribution and morphology of colon tumors were recorded. The majority of ACF were present in the middle and distal colon of DMH-treated rats and their number increased with time. By the 4th week, 91.5% ACF were composed of one or two crypts and 8.5% had three or more crypts, while by the 30th week 46.9% ACF had three or more crypts. Thus, a progression of ACF consisting of multiple crypts was observed from the 4th to the 30th week. Nine well-differentiated adenocarcinomas were found in 10 rats by the 30th week. Seven tumors were located in the distal colon and two in the middle colon. No tumor was found in the proximal colon. The present data indicate that induction of ACF by DMH in the short-term (4 weeks) assay was correlated with development of well-differentiated adenocarcinomas in the medium-term (30 weeks) assay.

Aberrant crypt foci and colon cancer: comparison between a short- and medium-term bioassay for colon carcinogenesis using dimethylhydrazine in Wistar rats

Aberrant crypt foci (ACF) in the colon of carcinogen-treated rodents are considered to be the earliest hallmark of colon carcinogenesis. In the present study the relationship between a short-term (4 weeks) and medium-term (30 weeks) assay was assessed in a model of colon carcinogenesis induced by dimethylhydrazine (DMH) in the rat. Six-week-old male Wistar rats were given subcutaneous injections of DMH (40 mg/kg) twice a week for 2 weeks and killed at the end of the 4th or 30th week. ACF were scored for number, distribution pattern along the colon and crypt multiplicity in 0.1 percent methylene-blue whole-mount preparations. ACF were distinguished from normal crypts by their larger size and elliptical shape. The incidence, distribution and morphology of colon tumors were recorded. The majority of ACF were present in the middle and distal colon of DMH-treated rats and their number increased with time. By the 4th week, 91.5 percent ACF were composed of one or two crypts and 8.5 percent had three or more crypts, while by the 30th week 46.9 percent ACF had three or more crypts. Thus, a progression of ACF consisting of multiple crypts was observed from the 4th to the 30th week. Nine well-differentiated adenocarcinomas were found in 10 rats by the 30th week. Seven tumors were located in the distal colon and two in the middle colon. No tumor was found in the proximal colon. The present data indicate that induction of ACF by DMH in the short-term (4 weeks) assay was correlated with development of well-differentiated adenocarcinomas in the medium-term (30 weeks) assay

Single-cell gel (comet) assay detects primary DNA damage in nonneoplastic urothelial cells of smokers and ex-smokers.

A protocol for DNA damage assessment by the single-cell gel (SCG)/comet assay in human urinary bladder washing cells was established. Modifications of the standard alkaline protocol included an increase to 2% of sodium sarcosinate in the lysis solution, a reduction in the glass-slide area for comet analysis, and a cutoff value for comet head diameter of at least 30 microm, to exclude contaminating leukocytes. Distinguishing cell populations is crucial, because significant differential migration was demonstrated for transitional and nontransitional cells, phenomena that may confound the results. When applying the modified protocol to urinary bladder cells from smokers without urinary bladder neoplasia, it was possible to detect a significant (P = 0.03) increase in DNA damage as depicted by the tail moment (6.39 +/- 3.23; mean +/- 95% confidence interval; n = 18) when compared with nonsmokers (1.94 +/- 1.41; n = 12). No significant differences were observed between ex-smokers and current smokers regarding comet parameters. Inflammation was not a confounding factor, but DNA migration increased significantly with age in nonsmokers (r = 0.68; P = 0.014). Thus, age matching should be a concern when transitional cells are analyzed in the SCG assay. As it is well known, DNA damage may trigger genomic instability, a crucial step in carcinogenesis. Therefore, the present data directly support the classification of individuals with smoking history as patients at high risk for urinary bladder cancer.

Dose- and sex-related carcinogenesis by N-bis(2-hydroxypropyl)nitrosamine in Wistar rats.

An initiation-promotion medium-term bioassay for detection of chemical carcinogens, developed in the male F344 rat, uses 0.1% N-bis(2-hydroxypropyl)nitrosamine (DHPN) among five genotoxic chemicals for the initiation of carcinogenesis in multiple organs. To establish this bioassay in the Wistar strain, the effects of two dose levels of DHPN were evaluated on the main DHPN rat target organs: lung, thyroid gland, kidneys and liver. Four groups of male and female animals were studied: Control -- untreated group; Multi-organ initiated group (also referred to as DMBDD, based on the initials of the five initiators) -- treated sequentially with N-diethylnitrosamine (DEN, i.p.), N-methyl-N-nitrosourea (MNU, i.p.), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN, drinking water), N, N'-dimethylhydrazine (DMH, s.c.) and DHPN (drinking water) for 4 weeks; a third group treated with 0.1% DHPN in drinking water for 2 weeks and the last group treated with 0.2% DHPN in drinking water for 4 weeks. The animals were sacrificed after 30 weeks. DHPN at 0. 2% induced preneoplasia in the liver and kidneys of rats of both sexes, the number and area of the putative preneoplastic liver glutathione S-transferase-positive hepatocyte foci being significantly increased in these animals. It also induced benign and malignant tumors in female and in male rats. However, there was no relationship between the increased incidence of preneoplastic lesions and tumor development in the 0.2% DHPN-exposed groups of both sexes. DHPN at 0.1% induced only a few preneoplastic lesions in the liver and kidney and no tumors in both male and female rats. A clear dose and sex-related carcinogenic activity of DHPN was registered, although Wistar rats of both sexes showed a relative resistance to the carcinogenic activity of this compound.

Lymphoproliferative response and T lymphocyte subsets in a medium-term multi-organ bioassay for carcinogenesis in Wistar rats.

The lymphoproliferative response and T lymphocyte subsets were evaluated at different stages of carcinogenesis in male Wistar rats sequentially initiated with N-diethylnitrosamine (DEN), N-butyl-N-4(hydroxybutyl)nitrosamine (BBN), N-methyl-N-nitrosourea (MNU), dihydroxy-di-N-propylnitrosamine (DHPN) and N, N'-dimethylhydrazine (DMH) (DMBDD initiation). One group was evaluated at the 4th week and other initiated group at the 30th week. Two initiated groups were also exposed through diet to 2-acetylaminofluorene (2-AAF) or phenobarbital (PB), from the 6th until the 30th week. Two groups received only 2-AAF or PB until the 30th week. Five groups were studied to evaluate the effects of each initiator. The lymphoproliferative response was induced in vitro by concanavalin A and the percentage of T lymphocyte subsets was determined by flow cytometry. All groups submitted to initiation only, initiation plus promotion, or promotion only, developed significantly more preneoplastic lesions than the untreated control group. The main target organs for tumor development were the liver, colon, urinary bladder, kidneys and Zymbal glands, mainly in the group treated with DMBDD+2-AAF. There were no alterations of the lymphoproliferative response and of the T lymphocyte subsets percentage in the DMBDD-treated group at the 4th and 30th weeks. At the 30th week, the T lymphocyte subsets percentage was also not affected in the initiated groups after treatments with 2-AAF or PB. The lymphoproliferative response, however, was decreased in the DMBDD+2-AAF group and in the groups treated only with 2-AAF or PB. The present results indicate that the initiating chemicals used in the DMBDD initiation protocol do not exert any influence on the immune system. The alteration of lymphoproliferative response induced at the advanced stage of carcinogenesis without alteration of T lymphocyte subsets may indicate that the influence of 2-AAF and PB on the immune system is functional and not toxic.

Promotion of hepatocarcinogenesis by hexachlorobenzene in energy-restricted rats.

The interaction between dietary energy restriction and low dose of the fungicide hexachlorobenzene (HCB) was evaluated in a rat liver medium-term bioassay for carcinogenesis. Male Wistar rats were fed a control or a 50% energy-restricted diet, both added or not with 50 ppm HCB, for 6 weeks. HCB exposure or energy restriction separately did not exert any influence on the development of glutathione S-transferase placental form (GST-P(+)) foci of hepatocytes. Simultaneous HCB exposure and energy restriction induced a significant increase in liver centrilobular hypertrophy and GST-P(+) foci development. Our findings suggest that energy restriction increases liver response to low dose of HCB, unmasking the promoting potential of this fungicide.

Effects of uracil calculi on cell growth and apoptosis in the BBN-initiated Wistar rat urinary bladder mucosa.

The different potential of initiated and non-initiated urinary bladder mucosa (UBM) to develop neoplasia was quantitatively evaluated in the male Wistar rat. Initiation of carcinogenesis was accomplished with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Stimuli for cell proliferation and apoptosis were obtained by exposure followed by withdrawal of 3% Uracil in the diet. The proliferation index (PI) was estimated in UBM immunostained for the proliferating nuclear cell antigen (PCNA). The apoptotic index (AI) and the density of papillary/nodular hyperplasia (PNH) were estimated in hematoxilin-eosin stained sections. PNH was the main proliferative response to the mechanical irritation by uracil, irrespective of previous initiation with BBN. Uracil exposure induced higher PI and PNH density in the initiated rats. After uracil withdrawal, there was a significant increase of the AI in both uracil-treated groups, which correlated well to the respective PNH density. However, at the end of the experiment, PNH incidence and density were significantly higher in the BBN-initiated mucosa, which also presented 18% incidence of papillomas and 27% of carcinomas. Therefore, under prolonged uracil calculi trauma, the UBM of BBN-initiated Wistar rats gives rise to epithelial proliferative lesions that progress to neoplasia through acquired resistance to apoptosis.