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Esophageal squamous cell carcinoma (ESCC) is among the ten most frequent and deadly cancers, without effective therapies for most patients. More recently, drugs targeting deregulated growth factor signaling receptors have been developed, such as HGF-MET targeted therapy. We assessed MET and HGF genetic alterations and gene and protein expression profiles in ESCC patients from the Brazilian National Cancer Institute and publicly available datasets, as well as the intratumor heterogeneity of the alterations found. Our analyses showed that HGF and MET genetic alterations, both copy number and mutations, are not common in ESCC, affecting 5 and 6% of the cases, respectively. HGF showed a variable mRNA expression profile between datasets, with no alterations (GSE20347), downregulation (GSE45670), and upregulation in ESCC (our dataset and GSE75241). On the other hand, MET was found consistently upregulated in ESCC compared to non-tumor surrounding tissue, with median fold-changes of 5.96 (GSE20347), 3.83 (GSE45670), 6.02 (GSE75241), and 5.0 (our dataset). Among our patients, 84% of the tumors showed at least a two-fold increase in MET expression. This observation was corroborated by protein levels, with 55% of cases exhibiting positivity in 100% of the tumor cells. Intratumor heterogeneity was evaluated in at least four tumor biopsies from five patients and two cases showed a consistent increase in MET expression (at least two-fold) in all tumor samples. Our data suggested that HGF-MET signaling pathway was likely to be overactivated in ESCC, representing a potential therapeutic target, but eligibility for this therapy should consider intratumor heterogeneity.

Hot beverage consumption is a risk factor for esophageal squamous cell carcinoma, but the underlying mechanisms are still unknown. We developed an experimental mouse model to understand the mechanism of thermal lesion to esophageal carcinogenesis. Female BALB/c mice were treated by gavage with water at different temperatures three times a week and nitrosamines in the drinking water. Water at 70 °C, but not at lower temperatures, initially induced an esophageal necrosis that healed and became resistant to necrosis after further administrations. However, when 70 °C water was associated with N-nitrosodiethylamine at doses above 1 ppm, there was interference in epithelial regeneration, allowing recurrent thermal injury and inflammation. Recurrent thermal injury resulted in hyper proliferative premalignant lesions being induced earlier (at 4 weeks) and at a higher frequency (4-fold increase at 16 weeks) when compared to mice treated with NDEA only. Ki-67 immunostaining revealed that recurrent thermal injury induced basal cell proliferation resulting in the expansion of epithelial basal cells, confirmed by the increase in cytokeratin 14 positive cells with concomitant reduction of differentiated cytokeratin 5 positive cells. We conclude that recurrent thermal lesion may act as a tumor promoter though a strong proliferation stimulus of esophageal epithelial basal cells.

The International Agency for Research on Cancer (IARC) and the US National Cancer Institute (NCI) have initiated a series of cancer-focused seminars [Scelo G, Hofmann JN, Banks RE et al. International cancer seminars: a focus on kidney cancer. Ann Oncol 2016; 27(8): 1382-1385]. In this, the second seminar, IARC and NCI convened a workshop in order to examine the state of the current science on esophageal squamous cell carcinoma etiology, genetics, early detection, treatment, and palliation, was reviewed to identify the most critical open research questions. The results of these discussions were summarized by formulating a series of 'difficult questions', which should inform and prioritize future research efforts.

Early nutrition plays a long-term role in the predisposition to chronic diseases and influences the metabolism of several drugs. This may happen through cytochromes P450 (CYPs) regulation, which are the main enzymes responsible for the metabolism of xenobiotics. Here, we analyzed the effects of maternal protein restriction (MPR) on the expression and activity of hepatic offspring’s CYPs during 90 days after birth, using Wistar rats as a mammal model. Hepatic CYP1A1, CYP1A2, CYP2B1, CYP2B2 and CYP2E1 mRNA and protein expression, and associated catalytic activities (ECOD, EROD, MROD, BROD, PROD and PNPH) were evaluated in 15-, 30-, 60-, and 90-day-old offspring from dams fed with either a 0% protein (MPR groups) or a standard diet (C groups) during the 10 first days of lactation. Results showed that most CYP genes were induced in 60- and 90-day-old MPR offspring. The inductions detected in MPR60 and MPR90 were of 5.0- and 2.0-fold (CYP1A2), 3.7- and 2.0-fold (CYP2B2) and 9.8- and 5.8– fold (CYP2E1), respectively, and a 3.8-fold increase of CYP2B1 in MPR90. No major alterations were detected in CYP protein expression. The most relevant CYP catalytic activities’ alterations were observed in EROD, BROD and PNPH. Nevertheless, they did not follow the same pattern observed for mRNA expression, except for an induction of EROD in MPR90 (3.5-fold) and of PNPH in MPR60 (2.2-fold). Together, these results suggest that MPR during lactation was capable of altering the expression and activity of the hepatic CYP enzymes evaluated in the offspring along development.

Yerba mate extract, made from leaves of Ilex paraguariensis, is a tea-like beverage consumed in South America. Considering reports that I. paraguariensis leaves have been associated with antioxidant and anticancer activity, the aim of the present work was to investigate the effect of yerba mate beverages available in the Brazilian market on DNA damage induced by known mutagens. Salmonella/microsome assay was performed with strains TA97, TA98, TA100, and TA102, with and without metabolic activation. For the antimutagenic analysis, co-, pre- and post-treatments were performed. No significant mutagenicity was induced by the yerba mate samples for any of the strains tested. A significant antimutagenic effect against 4-nitroquinoline-1-oxide was detected for each sample using TA97 in the presence of S9 mix. The results suggest that the tested samples of yerba mate have the potential to protect DNA against oxidative damage induced by 4-nitroquinoline-1-oxide, independent of their processing or packaging.

Introduction Esophageal cancer (EC) is the eighth most frequent cancer and the sixth leading cause of cancer-related deaths worldwide, with squamous cell carcinoma (ESCC) corresponding to 80% of the cases. SPRR3 is a differentiation-associated gene that belongs to SPRR (small proline rich) family that showed a gradual loss of expression in malignant transformation of the healthy oesophagus into ESCC. However, studies of the molecular mechanisms involved in SPRR3 silencing are limited. Thus, the aim of this study is to examine DNA methylation as a regulatory mechanism of SPRR3 expression in ESCC. Material and methods Three CpG sites of SPRR3 (A, B for promotor region and C for 5´UTR region) were analysed by pyrosequencing in esophageal cancer cell lines (TE-1, TE-13 and OE21) treated with the demethylating agent decitabine, and in tumour and matched normal surrounding mucosa from patients with ESCC. RT-qPCR was performed to evaluated SPRR3 , DNMT1, DNMT3A and DNMT3B expression in the same samples. Results and discussions Decitabine treatment was able to induce SPRR3 demethylation and reestablished esophagin expression in the three cell lines tested. The methylation of all CpG sites analysed was significantly higher in tumours in comparison with the adjacent tissues (p SPRR3 methylation to distinguish the adjacent mucosa from tumour samples using ROC curve analyses was significant for each of the CpG sites examined (A, sensitivity 93.10% and specificity=86.21%, p SPRR3 mRNA expression for all CpG sites evaluated (A and B p SPRR3 hypermethylation, we evaluated DNMTs expression (higher expression in tumour in comparision with the normal surrounding mucosa, p DNMT1 , p=0.0005 DNMT3A and p DNMT3B ) which are inversely correlated with SPRR3 expression ( DNMT1 p=0.0013, DNMT3A p=0.0108 and DNMT3B p SPRR3 methylation and DNMTs mRNA expression for all CpG sites evaluated exceptd for DNMT3A and Site C. Conclusion Thus, our data provide evidences that DNA methylation induced by DNMTs is a possible mechanism involved for SPRR3 silencing in ESCC.

Introduction Laryngeal squamous cell carcinoma (LSCC) is one of the most incidence tumours in the world, especially in developing countries, such as Brazil. The main risk factors for LSCC are tobacco and alcohol consumption and it usually occurs in patients older than 60 years. Similarly to other head and neck tumours, LSCC is a major health problem because of poor prognosis and slight improvement in the five-year survival during the past four decades. Therefore, our objective was to better understand the LSCC transcriptome, identifying possible prognosis biomarkers and novel therapy targets. Material and methods Was carried out a gene expression profile analysis using the Human Exon 1.0 ST microarray chip. To this end, the expression profile of 14 tumour tissues were compared with 12­matched non­malignant mucosa. Results and discussion Transcriptome analysis pointed out 817 differentially expressed genes (DEG), 315 of which with overexpression in tumour and 502 underexpressed when compared with matched non­malignant mucosa. Performing a survival analysis across all overexpressed DEG, 24 genes were related to patients survival ( ACOX1, ACVR1, ADH7, AGFG2, ALCAM, BTBD11, C12orf75, CDK14, CYP2C19, GBP6, GLTO, GNG4, LOX, LYPD6B, ME1, NPEPPS, ODC1, PMM1, PTGR1, SERPINA3, ST3GAL4, TDP52L1, ZDHHC13 and ZNF75 0). The prognostic values of these 24 genes were, then, validated in an independent LSCC data set of The Cancer Genome Atlas (TCGA) and the expression data, patient’s clinic and pathological features were analysed in Log-rank Test. Perineural invasion, commitment of surgical margins and expression of LOX, ALCAM, BTBD11 and LYPD68 showed p ALCAM expression were classified as independent prognostic factors to LSCC patients. LSCC-patients displaying ALCAM high expression presented 4.5-fold increased death risk. ALCAM gene encodes the activated leukocyte cell adhesion molecule protein, also known as CD166, which is a member of a subfamily of immunoglobulin receptors. This protein binds to T-cell differentiation antigen CD6, and is implicated in the processes of cell adhesion and migration. Conclusion ALCAM expression might be an independent prognosis biomarker to LSCC patients. Furthermore, in vitro, in vivo and pre-clinical analyses must be performed to reinforce this hypothesis.

Introduction The AID/APOBEC family consists of proteins that participate in a process known as DNA editing. APOBEC signatures are present in many tumour genomes as C-to-T and as C-to-G in TpC context. In esophageal squamous cell carcinoma (ESCC), an APOBEC-mediated mutational signature in 50% of tumour samples suggests that APOBEC-catalysed deamination provides a source of DNA damage. Head and neck squamous cell carcinoma (HNSCC) also shows high APOBEC3B mRNA levels and display the putative APOBEC mutational signature. HNSCC and esophageal cancer are the 5th and 6th most frequent types of cancer, respectively, in Brazil among men. HNSCC and ESCC are tumours with similarities in morphology and etiologic factors. Based on these findings, this study was designed to evaluate the contribution of APOBECs to the mutational signature of HNSCC and ESCC. Material and methods Mutational and gene expression profile were evaluated by RNA-seq in samples from INCA. Further validation was performed by RT-qPCR. Furthermore, data regarding exome sequencing and RNA-seq were also obtained from TCGA database. Bioinformatics analyses were performed using R software. All differences were considered statistically significant when p Results and discussions Through the exome sequencing data from TCGA of HNSCC and ESCC samples, we observed that the predominant mutational signature these tumours was of APOBEC signature followed by tobacco signature, except for larynx cancer (LSCC). So, we focused on ESCC and LSCC analyses. Using RNAseq, APOBEC3A , APOBEC3B , APOBEC3D , APOBEC3F , APOBEC3G and APOBEC3H were found overexpressed in tumour when compared with matched surrounding non-tumour mucosa of 14 ESCC patients. Furthermore, in 8 LSCC patients, the same APOBECs, aside from APOBEC3H , were found overexpressed in tumour when compared with matched surrounding non-tumour mucosa. Besides, the mutation fractions of C-to-G and C-to-T in TpC context were lower in LSCC than in ESCC samples. We also observed a positive correlation between APOBEC3H expression with total APOBEC mutations (r 2 : 0.347, p value: 0.027); with C-to-T APOBEC mutations (r 2 : 0.380, p value: 0.019); and between APOBEC3D expression and total APOBEC mutations (r 2 : 0.326, p value: 0.033); with C-to-T APOBEC mutations (r 2 : 0.298, p value: 0.043) and with C-to-G APOBEC mutations (r 2 : 0.294, p value: 0.045) in ESCC patients. Conclusion The deregulation of the APOBEC family of genes is a common feature in ESCC and LSCC, but the APOBEC signature seems to be able to distinguish these two tumours.

Esophageal cancer is among the most common and fatal tumors in the world. Eighty percent of esophageal tumors are esophageal squamous cell carcinoma (ESCC). Brazil is one of the high incidence areas in the West, where tobacco and alcohol consumption have been associated with ESCC. However, polymorphisms in xenobiotic metabolizing genes may also contribute to the risk. Therefore, in this study, we analyzed the risk of ESCC associated with tobacco and alcohol consumption and with polymorphisms of CYP2A6 (CYP2A6*2), CYP2E1 (CYP2E1*5B, CYP2E1*6), GSTP1 (Ile105Val), GSTM1 and GSTT1 null genotypes in 126 cases and 252 age- and gender-matched controls. Data on the amount, length and type of tobacco and alcohol consumed were collected, and DNA was extracted from blood lymphocytes from all individuals. Polymorphisms were analyzed by polymerase chain reaction (PCR)-multiplex (GSTM1 and T1), PCR-Restriction Fragment Length Polymorphism (CYP2E1*5B and *6 and GSTP1 Ile105Val) or allele-specific PCR amplification (CYP2A6*2). Risks were evaluated by multivariate conditional regression analysis. As expected, tobacco [odds ratio (OR) = 6.71, 95% confidence interval (95% CI) 3.08-14.63] and alcohol (OR = 16.98, CI 7.8-36.98) consumption, independently or together (OR = 26.91, CI 13.39-54.05) were risk factors. GSTP1 Ile105Val polymorphism was an independent risk factor (OR = 2.12, CI 1.37-3.29), whereas GSTT1 wild-type was an independent protective factor for ESCC (OR = 0.37, CI 0.16-0.79). There was approximately 80% statistical power to detect both results. There was no risk associated with CYP2A6, CYP2E1 and GSTM1 polymorphisms. In conclusion, this study suggests an opposite role of GSTP1 and GSTT1 polymorphisms for the risk for ESCC.

Cytochrome P450 (CYP) is a superfamily of enzymes involved in the metabolism of endogenous compounds and xenobiotics. CYP2A6 catalyzes the oxidation of nicotine and the activation of carcinogens such as aflatoxin B1 and nitrosamines. CYP2E1 metabolizes ethanol and other low-molecular weight compounds and can also activate nitrosamines. The CYP2A6 and CYP2E1 genes are polymorphic, altering their catalytic activities and susceptibility to cancer and other diseases. A number of polymorphisms described are ethnic-dependent. In the present study, we determined the genotype and allele frequencies of the main CYP2A6 and CYP2E1 polymorphisms in a group of 289 volunteers recruited at the Central Laboratory of Hospital Universitario Pedro Ernesto. They had been residing in the city of Rio de Janeiro for at least 6 months and were divided into two groups according to skin color (white and non-white). The alleles were determined by allele specific PCR (CYP2A6) or by PCR-RFLP (CYP2E1). The frequencies of the CYP2A6*1B and CYP2A6*2 alleles were 0.29 and 0.02 for white individuals and 0.24 and 0.01 for non-white individuals, respectively. The CYP2A6*5 allele was not found in the population studied. Regarding the CYP2E1*5B allele, we found a frequency of 0.07 in white individuals, which was statistically different (P < 0.05) from that present in non-white individuals (0.03). CYP2E1*6 allele frequency was the same (0.08) in both groups. The frequencies of CYP2A6*1B, CYP2A6*2 and CYP2E1*6 alleles in Brazilians are similar to those found in Caucasians and African-Americans, but the frequency of the CYP2E1*5B allele is higher in Brazilians.

The importance of fish-farming using economic indigenous fish in agriculturalareas has been steadily increasing in Brazil. Despite the usage of organophosphatepesticides (OP) to protect crops in these lands, there is a lack of informationconcerning the biochemistry of OP intoxication of freshwater Brazilian fish.Inhibition of brain acetylcholinesterase (AChE) activity has been considered themain event responsible for the toxicity of organophosphorus compounds tovertebrates (Saunders and Harper 1994). Among some non-Brazilian fish species,the correlation between reduced activity levels of brain ACE and mortality havebeen established as being variable (Gibson et al. 1969; Coppage 1972;Kozlovskaya and Mayer 1984; Chambers and Carr 1995). As a result, the use ofbrain AChE inhibition to evaluate fish intoxication by anticholinesterasecompounds is controversial. Such variability may be due to assays of brain AChEperformed without proper kinetic adaptations. Aside from that, acetylcholine maynot be the only substrate hydrolyzed in the brain of a fish; propionylcholine andbutyrylcholine might also have their ester bonds split. It is also reasonable tosuppose that before reaching the brain, a considerable amount of pesticide shouldbe found in serum of fish, producing inhibition of serum propionyl, butyryl andacetylcholinesterases, as well. Consequently, sensitivity of brain and serumcholinesterases to inhibition by OP should be biochemically characterized in agiven fish species, in order to make it easier to evaluate how much intoxicated thefish is after OP absorption.The present investigation was designed to learn about the kinetic properties ofcholinesterases from pacu, Piaractus mesopotamicus, an economically importantBrazilian fish species. Our main point was to look for the response of serum andbrain cholinesterase activities of pacu to paraoxon, a potent anticholinesteraseorganophosphate pesticide which results from parathion bioactivation (Neal 1967;Cunha Bastos et al. 1992). Also, this work was intended to study whether serumand brain cholinesterases activities could be equally used for biodetectingsublethal exposure of pacu specimens to parathion.

Cytochrome P450 (CYP) 2A enzymes are involved in the metabolism of numerous drugs and hormones and activate different carcinogens. Human CYP2A6, mouse CYP2A5 and rat CYP2A3 are orthologous enzymes that present high similarity in their amino acid sequence and share substrate specificities. However, different from the human and mouse enzyme, CYP2A3 is not expressed in the rat liver. There are limited data about expression of CYP2A3 in extrahepatic tissues and its regulation by typical CYP inducers. Therefore, the objective of the present study was to analyze CYP2A3 mRNA expression in different rat tissues by RT-PCR, and to study the influence of 3-methylcholanthrene, pyrazole and ß-ionone treatment on its expression. Male Wistar rats were divided into four groups of 5 rats each, and were treated ip for 4 days with 3-methylcholanthrene (25 mg/kg body weight), pyrazole (150 mg/kg body weight), ß-ionone (1 g/kg body weight), or vehicle. Total RNA was extracted from tissues and CYP2A3 mRNA levels were analyzed by semiquantitative RT-PCR. CYP2A3 mRNA was constitutively expressed in the esophagus, lung and nasal epithelium, but not along the intestine, liver, or kidney. CYP2A3 mRNA levels were increased in the esophagus by treatment with 3-methylcholanthrene and pyrazole (17- and 7-fold, respectively), in lung by pyrazole and ß-ionone (3- and 4-fold, respectively, although not statistically significant), in the distal part of the intestine and kidney by 3-methylcholanthrene and pyrazole, and in the proximal part of the intestine by pyrazole. CYP2A3 mRNA was not induced in nasal epithelium, liver or in the middle part of the intestine. These data show that, in the rat, CYP2A3 is constitutively expressed in several extrahepatic tissues and its regulation occurs through a complex mechanism that is essentially tissue specific.

The association between alcohol consumption and esophageal cancer is particularly marked in Northern France where an apple brandy, Calvados, is drunk. Calvados contains branched chain alcohols such as isoamyl alcohol (IAA) and it has been suggested that these play a particular role in the etiology of the cancer. In this study the effect of IAA, alone or with ethanol, on the ethylation of DNA by N-nitrosodiethylamine was evaluated in living rats, and the effect of IAA on the metabolism of N-nitrosodiethylamine by rat liver microsomes or esophageal epithelium was measured. As previously reported co-administration of ethanol increases the ethylation of esophageal DNA by the nitrosamine by almost 2-fold, but IAA did not alter the proportion of DNA ethylation between liver and esophagus. When both alcohols were given together with the nitrosamine, the alteration on DNA ethylation produced by ethanol remained unchanged. Different from ethanol, IAA inhibited NDEA metabolism catalyzed not only by rat liver microsomes (K(i)=0.42 mM), but also by esophageal epithelium (K(i)=0.52 mM), thus possessing an affinity also for the esophageal NDEA metabolizing P450.