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// Isidro Aleman-Avila 1, 2 , Mayra Jimenez-Morales 1 , Olga Beltran-Ramirez 1 , Rosa Elda Barbosa-Cobos 3 , Silvia Jimenez-Morales 4 , Fausto Sanchez-Munoz 5 , Guillermo Valencia-Pacheco 6 , Luis M. Amezcua-Guerra 5 , Yaneli Juarez-Vicuna 5 , Dulce Milagro Razo-Blanco Hernandez 7 , Maria Concepcion Aguilera-Cartas 8 , Ricardo F. Lopez-Villanueva 9 , Oscar Peralta-Zaragoza 10 , Carlos Tovilla-Zarate 11 and Julian Ramirez-Bello 1 1 Endocrine and Metabolic Disease Unit Research, Hospital Juarez of Mexico, Mexico City, Mexico 2 Superior School of Medicine Postgraduate Program, National Polytechnic Institute, Mexico City, Mexico 3 Rheumatology Department, Hospital Juarez of Mexico, Mexico City, Mexico 4 Laboratory of Cancer Genomics, National Institute of Genomic Medicine, Mexico City, Mexico 5 Immunology Department, National Institute of Cardiology, Mexico City, Mexico 6 Hematology Laboratory, Regional Research Center, Autonomous University of Yucatan, Yucatan, Mexico 7 Research Direction, Hospital Juarez of Mexico, Mexico City, Mexico 8 Endocrinology Department, Hospital Juarez of Mexico, Mexico City, Mexico 9 Rheumatology Department, Regional Hospital General (ISSSTE), Health Service Yucatan, Yucatan, Mexico 10 Direction of Chronic Infections and Cancer, Research Center in Infection Diseases, National Institute of Public Health, Cuernavaca, Mexico 11 Multidisciplinary Academic Division of Comalcalco, Juarez Autonomous University of Tabasco, Comalcalco, Mexico Correspondence to: Julian Ramirez-Bello, email: dr.julian.ramirez.hjm@gmail.com Keywords: systemic lupus erythematosus, rheumatoid arthritis, Graves’ disease, microRNA gene, susceptibility Received: December 21, 2016 Accepted: June 30, 2017 Published: July 27, 2017 ABSTRACT Recently, different microRNA (miRNA) gene polymorphisms have been evaluated in patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Graves’ disease (GD). In the present study, we examined three single-nucleotide polymorphisms (SNPs) located in the pre-miR-146a (rs2910164G/C), pre-miR-196a-2 (rs11614913C/T), and pre-miR-499 (rs3746444A/G) genes. Our study population included 900 Mexican patients with RA, SLE, or GD, as well as 486 healthy control individuals with no family history of inflammatory or autoimmune diseases. Genotyping was performed using TaqMan probes and a 5′ exonuclease assay. None of the investigated SNPs were associated with RA or GD susceptibility under any genetic model (co-dominant, recessive, or dominant). Genotype and allele frequencies of the miR-196a-2 rs11614913C/T polymorphism were similar between SLE cases and controls. In contrast, the miR-146a rs2910164G/C and miR-499 rs3746444A/G polymorphisms were associated with SLE susceptibility. These SNPs were not associated with lupus nephritis (LN). Our results suggest that polymorphisms in miR-146a, miR-196a-2 , and miR-499 are not associated with RA or GD susceptibility. This is the first report documenting that the miR-146a rs2910164G/C and miR-499 rs3746444 polymorphisms are associated with SLE susceptibility but not with LN.

Different strategies have been adopted aiming to improve either performance, water quality and health on intensive shrimp farming, including the manipulation of the bacterial community in biofloc technology (BFT) systems. The experimental design was completely randomized, with three treatments in triplicate developed in BFT without water exchange; CONT (control, only BFT without external addition of bacteria); NIT (addition of nitrifying bacteria in BFT water); LACT (BFT with addition of Lactobacillus rahamnosus in the pelletized feed). The experiment was carried out under controlled conditions, with juveniles of white shrimp Litopenaeus vannamei (0.56 g / ind as initial weight). During the bioassay (35 days), the basic variables of water and nitrogenous compounds (TAN, NO2-N and NO3-N) were monitored. At the end of the experiment, the abundance of ammonium oxidizing bacteria AOB), heterotrophic bacteria and Vibrio-like bacteria, the proximal composition of the biofloc and the shrimp tissue were analyzed. There were significant differences in the levels of DO, pH, TSS and alkalinity. In the NIT treatment, the minimum amount of TAN and NO2-N was recorded, and the nitrification curves occurred in less time. In terms of bacteria profile, the highest abundance of heterotrophic bacteria in water was recorded in the LACT treatment (36.8 × 106 CFU / mL) and the highest amount of OAB and Vibrio-like bacteria (yellow colonies) occurred in the NIT treatment (7.5 × 106 and 10.6 × 103 CFU / mL, respectively). The proximal composition of biofloc was different, the protein varied from 20.04 to 25.59%, the highest corresponded to CONT. Similarly, the proximal composition of shrimp was different, the protein varied from 69.61 to 71.42%, the maximum was recorded in LACT. The best productive parameters were registered in the LACT and NIT treatments, (specific growth 5.31, 5.18% / day; survival 99.6, 99.0% and FCR: 0.94, 1.00, respectively). In our experimental conditions, the addition of external bacteria (nitrifying and Lactobacillus rhamnosus) had a significant positive impact on the quality of the water, proximal composition and productive parameters of the shrimp, as well as on the abundance of bacteria.

Establishing a transcriptomic profile of human hepatocellular liver cancer (HCC) progression is a complex undertaking. A rat model of HCC was employed to develop a transcriptomic profile. Using three interventions, preneoplastic lesions appeared after 30 days and they progressed to HCC by 9 months. Preneoplastic and cancer lesions were characterized for transcriptomic analysis, and RNA from total liver homogenates was obtained at 1, 7, 11 and 16 days after the initiation treatment. RNA from dissected persistent preneoplastic lesions, adjacent tissue or cancer tissue was used for 30 days, and 5, 9, 12 and 18 months. The GeneChip® Rat Exon 1.0 ST arrays, Partek software and an Affymetrix console were employed for these analyses. LGALS3BP was differentially expressed at each time point, from the initial period, through the preneoplastic evolution period and until the end of cancer progression period. Twelve differentially expressed genes common to the preneoplastic evolution and to the cancer progression period were detected, which included ABCC3. Validation of the microarrays was confirmed by reverse transcription-quantitative polymerase chain reaction of six genes, including LGALS3BP and ABCC3. Of note, the proteins of these two genes are associated with the multidrug response complex, and evasion of immune surveillance and negative regulation of T cell proliferation. This model is useful for identifying candidate genes, and to validate them with regards to determining their relevance in rat HCC progression.

The functional PTPN22 R620W polymorphism (rs2476601) is clearly associated with susceptibility to several autoimmune diseases (ADs). However, the PTPN22 R263Q polymorphism (rs33996649) has been scarcely explored in different ADs. Here we aimed to examine the associations of the PTPN22 R620W and R263Q polymorphisms with susceptibility to or protection against rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and Graves' disease (GD) among Mexican patients.We conducted a case-control study including 876 patients (405 with SLE, 388 with RA, and 83 with GD) and 336 healthy control individuals. PTPN22 genotypes were determined using the TaqMan 5' allele discrimination assay.PTPN22 R620W was associated with GD susceptibility (OR 4.3, p = 0.004), but was not associated with SLE (OR 1.8, p = 0.19). We previously demonstrated that this polymorphism is associated with RA susceptibility (OR 4.17, p = 0.00036). Moreover, PTPN22 R263Q was associated with protection against SLE (OR 0.09, p = 004) and RA (OR 0.28, p = 0.045), but was not associated with GD.Our data provide the first demonstration that PTPN22 R620W confers GD susceptibility among Latin-American patients. Moreover, this is the second report documenting the association of PTPN22 R263Q with protection against SLE and RA.

S-adenosylmethionine (SAM), N-acetylcysteine (NAC) and quercetin exhibit a chemoprotective effect. Likely this effect is mediated by counteracting, oxidative stress and NF-kB activation. To test this hypothesis F344 rats were subjected to hepatocarcinogenesis with or without antioxidants. NAC decreased foci in number and area, SAM and quercetin decreased area. Lipid-peroxidation was decreased by antioxidants, but only SAM increased glutathione. SAM, in its regulation from IKK downwards, abolished the NF-kB activation. NAC decreased IKK and IkB-a phosphorylation, and Rel-A/p65 and NF-kB binding, though the last two were affected with less intensity compared to the NF-kB inhibitor. Quercetin decreased Rel-A/p65, without modifying upstream signalling. Although all antioxidants inhibited oxidative stress as shown by reduction of lipid peroxidation, not all exerted the same effect on NF-kB signalling pathway and only SAM increased GSH. The mechanisms exerted by SAM in the reduction of foci makes this compound a potential liver cancer therapeutic agent.

We present a study of the chemoprotective effects of two caffeic acid phenethyl ester (CAPE)-related structures: LQM717 and LQM706. The modified resistant hepatocyte model in rats was used to study the chemoprevention of these CAPE analogues, which are inexpensive and easily obtained. In the liver cancer model used, we detected extensive necrosis and lipid peroxidation after 24 h, many altered hepatic foci, putatively preneoplastic lesions with γ-glutamyl transpeptidase staining after 30 days, and liver tumors at 12 months. We tested the effect of the CAPE analogues on necrosis, lipid peroxidation, proliferation, p65 activation, altered hepatic foci, and tumors. Both compounds exerted protective effects on lipid peroxidation, necrosis, cell proliferation, p65 activation, and preneoplastic lesions. Rats under a carcinogenic protocol showed a 52, 71.74, and 51.6% decrease in the number of preneoplastic nodules when pretreated with CAPE, LQM706, and LQM717, respectively. At 12 months after carcinogenic treatment, eight of eight rats developed liver cancer, whereas in the group of rats that received pretreatment with CAPE, LQM706, or LQM717, 62.5, 83.3, or 42.85%, respectively, had tumors. In conclusion, LQM717 has the potential to enhance chemoprotection activity much better than CAPE by markedly reducing the formation of liver cancers in this model, and this is a compound that is easy to obtain.

Studies of cancer chemoprevention with caffeic acid phenethyl ester (CAPE) in the resistant hepatocyte model of hepatocarcinogenesis have shown the participation of CYP drug metabolizing enzymes. To prevent neoplastic and preneoplasic lesions, we must specifically identify which CYP activities are modified in the mechanism of action of CAPE. Male Fischer-344 rats were pretreated with CAPE twelve hours before administration of diethylnitrosamine (DEN) and were sacrificed twelve hours after CAPE and twelve hours, twenty-four hours, twenty-four days, and twelve months after DEN. Other rats were treated with the CYP inhibitors α-naphthoflavone or SKF525A and sacrificed twenty-four hours and twenty-four days after DEN. Microsomes were obtained from livers to quantify protein using Western blot. Diethylnitrosamine metabolism was measured based on nitrite formation and liver histology using GGT histochemistry. Caffeic acid phenethyl ester diminished the protein levels of CYP1A2 and CYP2B1/2. The inhibition of CYP2B1/2 prevented the appearance of preneoplastic lesions. Microsomal assays demonstrated that CAPE interfered with DEN activation diminishing nitrites similar to SKF525A and probably mediated by CYP2B1/2 inhibition. A single dose of CAPE before DEN treatment reduced the appearance of tumors by 43%. These results confirmed that CAPE is a promising agent to confer chemoprotection in liver cancer and should be considered for human therapies.

Cancer is a disease characterized by uncontrolled growth and division of genetically altered cells and its emergence requires several elements, including self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, tissue invasion and metastasis, and sustained angiogenesis (Hanahan and Weinberg, 2011). Cancer is thought to evolve along a multi-step process, cancer cells are the descendants of a normal cell in which some kind of internal or external stress causes a change in its genetic code. This event is said to “initiate” the cell to a precancerous state. In a second stage, this precancerous cell divides in response to a promoting agent to produce daughter cells, and these daughter cells divide to produce more daughter cells, and so on. The genetic instabilities passed down through the generations finally result in one cell that no longer requires the promoting agent to stimulate its proliferation, and a cancer cell is born with the ability to make proteins such as growth factors that stimulate proliferation. Finally in the third stage of carcinogenesis, progression, this cancer cells divides to produce daughter cells, these cells also divide, and soon there is a population of cancer cells with the ability to invade and metastasize (Vincent & Gatenby, 2008).

We have previously evaluated the chemopreventive effect of celecoxib on preneoplastic lesions in rat liver. However, though the effects of celecoxib have been tested in a variety of carcinomas, there has not been a study on the modulation of gene expression in response to this drug. Here, we evaluated the effect of celecoxib on the gene expression profile associated with hepatocarcinogenesis. Male Sprague-Dawley rats underwent the modified resistant hepatocyte model and were fed a diet containing 1500 ppm of celecoxib. Gene expression profiles were evaluated using DNA microarrays and further validations were performed using quantitative PCR, western blotting and immunohistochemical staining. Celecoxib modulated the expression of 46 genes, and those regulated by growth hormone were selected for further analysis. Celecoxib significantly upregulated the expression of the Cyp2b1/2, Cyp3a1, and alpha2-urinary globulin (alpha2uG) genes and restored the expression of Cyp2b3 to normal. The protein expression of Cyp2b1/2 was increased, but the expressions of Cyp3a1 and alpha2uG were only restored to normal levels. The increased Cyp2b1/2 expression in response to celecoxib was mainly confined to preneoplastic lesions. A search for the upstream mediator of these genetic alterations found that carcinogenesis inactivated by 87% the signal transducer and activator of transcription 5 (Stat5), a transcription factor that is activated by growth hormone signaling, but celecoxib treatment restored its activation. In conclusion, these results suggest that celecoxib exerts anticancer effects on altered hepatic cells by restoring mRNA and the protein expression levels of specific genes, in part through the reactivation of Stat5.

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, shows anticarcinogenic properties in the modified resistant hepatocyte model when administered before initiation or promotion of hepatocarcinogenesis process; however, information about the mechanism underlying this chemoprotection is limited. The aim of this work was to characterize the effect of CAPE on cytochrome P450 (CYP), which is involved in diethylnitrosamine (DEN) metabolism during the initiation stage of chemical hepatocarcinogenesis. Male Fischer-344 rats were treated as in the modified resistant hepatocyte model. Liver samples were obtained at four different times: at 12 h after pretreatment with CAPE and at 12 and 24 h and 25 days after DEN administration. Liver damage was determined by histology with hematoxylin and eosin, measurement of total CYP levels and enzyme activity, and gamma-glutamyl transpeptidase-positive (GGT+) staining of hepatocyte foci. CAPE administration prevented DEN-induced necrosis at 24 h. It also decreased O-dealkylation of 7-ethoxy-resorufin (EROD), O-dealkylation of 7-methoxyresorufin (MROD), and 7-pentoxy-resorufin activities at 12 h after its administration and EROD and MROD activities at 12 h after administration of DEN. CAPE treatment decreased GGT+ foci by 59% on day 25. Our results suggest that CAPE modifies the enzymatic activity of CYP isoforms involved in the activation of DEN, such as CYP1A1/2 and CYP2B1/2. These findings describe an alternative mechanism for understanding the ability of CAPE to protect against chemical hepatocarcinogenesis.

AIM: To study the effect of celecoxib (CXB) on diethylnitrosamine activation through the regulation of cytochrome P450 in a hepatocarcinogenesis model. METHODS: Six-week-old male Sprague-Dawley rats were randomly divided into five groups, a non-treated group (NT), a diethylnitrosamine-treated group (DEN), a DEN+CXB-treated group (DEN+CXB), and CXB 8 d-treated and CXB 32 d-treated groups. The effects of celecoxib on the enzymatic activities of CYP1A1, 2A, 2B1/2, and 2E1 were assessed in hepatic microsomes 24 h after DEN administration. Changes in CYP1A1 and CYP2B1/2 protein expression were also evaluated. The rate of DEN metabolism was measured by the production of the deethylation metabolite acetaldehyde, and the denitrosation metabolite nitrite. RESULTS: DEN+CXB administration produced a significant increase in the enzymatic activities of CYP2B1/2 and 1A1, whereas it did not change the activities of CYP2A and 2E1, compared to that of the DEN group. CXB treatment for eight days did not produce a significant effect on enzymatic activity when compared to the NT group; however, when it was administered for prolonged times (CXB 32 d group), the enzymatic activities were increased in a similar pattern to those in the DEN+CXB group. The observed increase in the enzymatic activities in the DEN+CXB group was accompanied by an increase in the CYP2B1/2 protein levels; no changes were observed in the levels of CYP1A1. In vitro, CXB increased the denitrosation of DEN, a pathway of metabolic detoxification. The addition of SKF-525A, a preferential inhibitor of CYP2B, abrogated the denitrosation of DEN. CONCLUSION: These results suggest that the mechanism of action of CXB involves enhancement of the detoxification of DEN by an increasing denitrosation via CYP2B1/2.