Nat2, Xrcc1 And Hogg1 Polymorphisms, Cigarette Smoking, Alcohol Consumption And Risk Of Upper Aerodigestive Tract Cancer.

To evaluate associations between polymorphisms of the N-acetyltransferase 2 (NAT2), human 8-oxoguanine glycosylase 1 (hOGG1) and X-ray repair cross-complementing protein 1 (XRCC1) genes and risk of upper aerodigestive tract (UADT) cancer. A case-control study involving 117 cases and 224 controls was undertaken. The NAT2 gene polymorphisms were genotyped by automated sequencing and XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms were determined by Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Slow metabolization phenotype was significantly associated as a risk factor for the development of UADT cancer (p=0.038). Furthermore, haplotype of slow metabolization was also associated with UADT cancer (p=0.014). The hOGG1 Ser326Cys polymorphism (CG or GG vs. CC genotypes) was shown as a protective factor against UADT cancer in moderate smokers (p=0.031). The XRCC1 Arg399Gln polymorphism (GA or AA vs. GG genotypes), in turn, was a protective factor against UADT cancer only among never-drinkers (p=0.048). Interactions involving NAT2, XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms may modulate the risk of UADT cancer in this population.

Changes In Liver Cell Dna Methylation Status In Diabetic Mice Affect Its Ft-ir Characteristics.

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas -CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of -CH3 groups. Other spectral differences were found at 1700-1500 cm(-1) and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

Gut Bacteria Products Prevent Aki Induced By Ischemia-reperfusion.

Short-chain fatty acids (SCFAs) are fermentation end products produced by the intestinal microbiota and have anti-inflammatory and histone deacetylase-inhibiting properties. Recently, a dual relationship between the intestine and kidneys has been unraveled. Therefore, we evaluated the role of SCFA in an AKI model in which the inflammatory process has a detrimental role. We observed that therapy with the three main SCFAs (acetate, propionate, and butyrate) improved renal dysfunction caused by injury. This protection was associated with low levels of local and systemic inflammation, oxidative cellular stress, cell infiltration/activation, and apoptosis. However, it was also associated with an increase in autophagy. Moreover, SCFAs inhibited histone deacetylase activity and modulated the expression levels of enzymes involved in chromatin modification. In vitro analyses showed that SCFAs modulated the inflammatory process, decreasing the maturation of dendritic cells and inhibiting the capacity of these cells to induce CD4(+) and CD8(+) T cell proliferation. Furthermore, SCFAs ameliorated the effects of hypoxia in kidney epithelial cells by improving mitochondrial biogenesis. Notably, mice treated with acetate-producing bacteria also had better outcomes after AKI. Thus, we demonstrate that SCFAs improve organ function and viability after an injury through modulation of the inflammatory process, most likely via epigenetic modification.

Evaluation of muscle strength and motor abilities in children with type II and III spinal muscle atrophy treated with valproic acid

Background: Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects the motoneurons of the spinal anterior horn, resulting in hypotonia and muscle weakness. The disease is caused by deletion or mutation in the telomeric copy of SMN gene (SMN1) and clinical severity is in part determined by the copy number of the centromeric copy of the SMN gene (SMN2). The SMN2 mRNA lacks exon 7, resulting in a production of lower amounts of the full-length SMN protein. Knowledge of the molecular mechanism of diseases has led to the discovery of drugs capable of increasing SMN protein level through activation of SMN2 gene. One of these drugs is the valproic acid (VPA), a histone deacetylase inhibitor. Methods: Twenty-two patients with type II and III SMA, aged between 2 and 18 years, were treated with VPA and were evaluated five times during a one-year period using the Manual Muscle Test (Medical Research Council scale-MRC), the Hammersmith Functional Motor Scale (HFMS), and the Barthel Index. Results: After 12 months of therapy, the patients did not gain muscle strength. The group of children with SMA type II presented a significant gain in HFMS scores during the treatment. This improvement was not observed in the group of type III patients. The analysis of the HFMS scores during the treatment period in the groups of patients younger and older than 6 years of age did not show any significant result. There was an improvement of the daily activities at the end of the VPA treatment period. Conclusion: Treatment of SMA patients with VPA may be a potential alternative to alleviate the progression of the disease.

Effects of protein deprivation and re-feeding on P2X(2) receptors in enteric neurons

AIM: To investigate the effects of malnutrition and refeeding on the P2X(2) receptor, nitric oxide synthase (NOS), calretinin, calbindin and choline acetyltransferase (ChAT) in neurons of the rat ileum. METHODS: We analyzed the co-localization, numbers and sizes of P2X(2)-expressing neurons in relation to NOS-IR (immunoreactive), calbindin-IR, ChAT-IR, and calretinin-IR neurons of the myenteric and submucosal plexus. The experimental groups consisted of: (1) rats maintained on normal feed throughout pregnancy until 42 d post-parturition (N); (2) rats deprived of protein throughout pregnancy and 42 d post-parturition (D); and (3) rats undernourished for 21 d post-parturition and then given a protein diet from days 22 to 42 (DR). The myenteric and submucosal plexuses were evaluated by double labeling by immunohistochemical methods for P2X(2) receptor, NOS, ChAT, calbindin and calretinin. RESULTS: We found similar P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of myenteric and submucosal neurons from the N, D and DR groups. Double labeling of the myenteric plexus demonstrated that approximately 100% of NOS-IR, calbindin-IR, calretinin-IR and ChAT-IR neurons in all groups also expressed the P2X(2) receptor. In the submucosal plexus, the calretinin-IR, ChAT-IR and calbindinIR neurons were nearly all immunoreactive for the P2X(2) receptor. In the myenteric plexus, there was a 19% increase in numbers per cm(2) for P2X(2) receptor-IR neurons, 64% for NOS-IR, 84% for calretinin-IR and 26% for ChAT-IR neurons in the D group. The spatial density of calbindin-IR neurons, however, did not differ among the three groups. The submucosal neuronal density increased for calbindin-IR, calretinin-IR and ChAT-IR neurons. The average size of neurons in the myenteric plexus neurons in the D group was less than that in the controls and, in the re-fed rats; there was a 34% reduction in size only for the calretinin-IR neurons. CONCLUSION: This work demonstrates that expression of the P2X(2) receptor is present in inhibitory, intrinsic primary afferent, cholinergic secretomotor and vasomotor neurons. Undernutrition affected P2X(2) receptor expression in the submucosal plexus, and neuronal and size. These changes were rescued in the re-fed rats. (C) 2010 Baishideng. All rights reserved.

Antimicrobial compounds produced by Lactobacillus sakei subsp sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product

Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.

The azo dye Disperse Orange 1 induces DNA damage and cytotoxic effects but does not cause ecotoxic effects in Daphnia similis and Vibrio fischeri

Azo dyes constitute the largest group of colorants used in industry and can pass through municipal waste water plants nearly unchanged due to their resistance to aerobic treatment, which potentially exposes humans and local biota to adverse effects. Unfortunately, little is known about their environmental fate. Under anaerobic conditions, some azo dyes are cleaved by microorganisms forming potentially carcinogenic aromatic amines. In the present study, the azo dye Disperse Orange 1, widely used in textile dyeing, was tested using the comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and Microtox (R) assays. The human hepatoma cell line (HepG2) was used in the comet assay and for cell viability. In the mutagenicity assay. Salmonella typhimurium strains with different levels of nitroreductase and o-acetyltransferase were used. The dye showed genotoxic effects with respect to HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0 mu g/mL. In the mutagenicity assay, greater responses were obtained with the strains TA98 and YG1041, suggesting that this compound mainly induces frameshift mutations. Moreover, the mutagenicity was greatly enhanced with the strains overproducing nitroreductase and o-acetyltransferase, showing the importance of these enzymes in the mutagenicity of this dye. In addition, the compound induced apoptosis after 72 h in contact with the HepG2 cells. No toxic effects were observed for either D. similis or Vibrio fischeri. (C) 2011 Elsevier B.V. All rights reserved.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

Localization of N-acetyltransferases NAT1 and NAT2 in human tissues

Human acetyl coenzyme A-dependent N-acetyltransferase (EC 2.3.1.5) (NAT) catalyzes the biotransformation of a number of arylamine and hydrazine compounds. NAT isozymes are encoded at 2 loci; one encodes NAT1, formerly known as the monomorphic form of the enzyme, while the other encodes the polymorphic NAT2, which is responsible for individual differences in the ability to acetylate certain compounds. Human epidemiological studies have suggested an association between the acetylator phenotype and particular cancers such as those of the bladder and colon. In the present study, NAT1- and NAT2-specific riboprobes were used in hybridization histochemistry studies to localize NAT1 and NAT2 mRNA sequences in formalin-fixed, paraffin-embedded human tissue sections. Expression of both NAT1 and NAT2 mRNA was observed in liver, gastrointestinal tract tissues (esophagus, stomach, small intestine, and colon), ureter, bladder, and lung. In extrahepatic tissues, NAT1 and NAT2 mRNA expression was localized to intestinal epithelial cells, urothelial cells, and the epithelial cells of the respiratory bronchioles. The observed heterogeneity of NAT1 and NAT2 mRNA expression between human tissue types may be of significance in assessing their contribution to known organ-specific toxicities of various arylamine drugs and carcinogens.

Role of neurotrophin receptor p75NTR in mediating neuronal cell death following injury

1. The neurotrophin receptor p75NTR has been shown to mediate neuronal cell death after nerve injury. 2. Down-regulation of p75NTR by antisense oligonucleotides is able to inhibit both sensory and motor neuron death and this treatment is more effective than treatment with growth factors. 3. p75NTR induces cell death by a unique death signalling pathway involving transcription factors (nuclear factor kappa B and c-jun), Bcl-2 family members and caspases.

An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes

Arylamine N-acetyltransferase (NAT) was first identified as the inactivator of the anti-tubercular drug isoniazid, The enzyme was shown to catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of the hydrazine drug. The rate of inactivation of isoniazid was polymorphically distributed in the population and was one of the first examples of pharmacogenetic variation, NAT was identified recently in Mycobacterium tuberculosis and is a candidate for; modulating the response to isoniazid, Genome sequences have revealed many homologous members of this unique family of enzymes. The first three-dimensional structure of a member of the NAT family identifies a catalytic triad consisting of aspartate, histidine and cysteine proposed to form the activation mechanism. So far, all procaryotic NATs resemble the human enzyme which acetylates isoniazid (NAT2), Human NAT2 is characteristic of drug-metabolizing enzymes: it is found in liver and intestine, In humans and other mammals, there are up to three different isoenzymes. If only one isoenzyme is present, it is like human NAT1. Human NAT1 and its murine equivalent specifically acetylate the folate catabolite p-amino-benzoylglutamate. NAT1 and its murine homologue each have a ubiquitous tissue distribution and are expressed early in development at the blastocyst stage, During murine embryonic development, NAT is expressed in the developing neural tube. The proposed endogenous role of NAT in folate metabolism, and its multi-allelic nature, indicate that its role in development should be assessed further.

Differential Toxicity of Disperse Red 1 and Disperse Red 13 in the Ames Test, HepG2 Cytotoxicity Assay, and Daphnia Acute Toxicity Test

Azo dyes are of environmental concern due to their degradation products, widespread use, and low-removal rate during conventional treatment. Their toxic properties are related to the nature and position of the substituents with respect to the aromatic rings and amino nitrogen atom. The dyes Disperse Red 1 and Disperse Red 13 were tested for Salmonella mutagenicity, cell viability by annexin V, and propidium iodide in HepG2 and by aquatic toxicity assays using daphnids. Both dyes tested positive in the Salmonella assay, and the suggestion was made that these compounds induce mainly frame-shift mutations and that the enzymes nitroreductase and O-acetyltransferase play an important role in the observed effect. In addition, it was shown that the presence of the chlorine substituent in Disperse Red 13 decreased the mutagenicity about 14 times when compared with Disperse Red 1, which shows the same structure as Disperse Red 13, but without the chlorine substituent. The presence of this substituent did not cause cytotoxicity in HepG2 cells, but toxicity to the water flea Daphnia similis increased in the presence of the chlorine substituent. These data suggest that the insertion of a chlorine substituent could be an alternative in the design of dyes with low-mutagenic potency, although the ecotoxicity should be carefully evaluated. (C) 2010 Wiley Periodicals, Inc. Environ Toxicol 26: 489-497, 2011.

Metabolic activation of aromatic amines by human pancreas

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas, Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens, Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethylamine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/ 4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [H-3]N-hydroxy-ABP) of pancreatic cytosols was high, about two-thirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using P-32-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.

The role of xenobiotic metabolizing enzymes in arylamine toxicity and carcinogenesis: Functional and localization studies

In both animal models and humans, the first and obligatory step in the activation of arylamines is N-hydroxylation. This pathway is primarily mediated by the phase-I enzymes CYP1A1, CYP1A2 and CYP4B1. In the presence of flavonoids such as alpha-naphthoflavone and flavone, both CYP3A4 and CYP3A5 have also been shown to play a minor role in the activation of food-derived heterocyclic amines. The further activation of N-hydroxyarylamines by phase-II metabolism can involve both N,O-acetylation and N,O-sulfonation catalyzed by N-acetyltransferases (NAT1 and NAT2) and sulfotransferases, respectively. Using an array of techniques, we have been unable to detect constitutive CYP1A expression in any segments of the human gastrointestinal tract. This is in contrast to the rabbit where CYP1A1 protein was readily detectable on immunoblots in microsomes prepared from the small intestine. In humans, CYP3A3/3A4 expression was detectable in the esophagus and all segments of the small intestine. Northern blot analysis of eleven human colons showed considerable heterogeneity in CYP3A mRNA between individuals, with the presence of two mRNA species in same subjects. Employing the technique of hybridization histochemistry (also known as in situ hybridization), CYP4B1 expression was observed in some human colons but not in the liver or the small intestine. Hybridization histochemistry studies have also demonstrated variable NAT1 and NAT2 expression in the human gastrointestinal tract. NAT1 and NAT2 mRNA expression was detected in the human liver, small intestine, colon, esophagus, bladder, ureter, stomach and lung. Using a general aryl sulfotransferase riboprobe (HAST1), we have demonstrated marked sulfotransferase expression in the human colon, small intestine, lung, stomach and liver. These studies demonstrate that considerable variability exists in the expression of enzymes involved in the activation of aromatic amines in human tissues. The significance of these results in relation to a role for heterocyclic amines in colon cancer is discussed.

The Genetic Landscape of the Childhood Cancer Medulloblastoma

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.