Pentoxifylline and Norcantharidin Modify p62 Expression in 2D and 3D Cultures of B16F1 Cells.

Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.

Integrated evaluation of the biological response of the earthworm Eisenia fetida using two glyphosate exposure strategies: soil enriched and soils collected from crops in Southeastern Mexico.

Under laboratory conditions, the toxicological effects of pesticides tend to be less variable and realistic than under field conditions, limiting their usefulness in environmental risk assessment. In the current study, the earthworm Eisenia fetida was selected as a bioindicator for assessing glyphosate toxic effects in two different trials to solve this dilemma. In Trial 1, the worms were exposed for 7 and 14 days to concentrations of a commercial glyphosate formulation (1 to 500 mg a.i. kg-1) currently used in the field. In Trial 2, the worms were kept in nine soils collected from different plots with crops for 14 days of exposure. In both experiments, glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and acetylcholinesterase (AChE) activities and contents of lipid peroxidation (LPO) were evaluated. In T1, the glyphosate formulation produced a 40% inhibition of AChE activity and a significant increase in GST, SOD, CAT, and GPx activities and LPO contents in E. fetida on day 7. In T2, higher concentrations of glyphosate were detected in the soils of soybean, papaya, and corn (0.92, 0.87, and 0.85 mg kg-1), which induced a positive correlation between the levels of glyphosate residues with GST, SOD, CAT, GPx, and LPO and a negative correlation with AChE. These findings indicate that crop soils polluted with glyphosate elicited higher oxidative stress than under laboratory conditions, confirmed by IBRv2, PCA, and AHC analyses.

Tumor-Infiltrating iNKT Cells Activated through c-Kit/Sca-1 Are Induced by Pentoxifylline, Norcantharidin, and Their Mixtures for Killing Murine Melanoma Cells.

The involvement of NK and other cytotoxic cells is considered the first defense line against cancer. However, a significant lack of information prevails on the possible roles played by factors considered characteristic of primitive cells, such as c-kit and Sca-1, in activating these cells, particularly in melanoma models subjected to treatments with substances under investigation, such as the case of norcantharidin. In this study, B16F1 murine melanoma cells were used to induce tumors in DBA/2 mice, estimating the proportions of NK and iNKT cells; the presence of activation (CD107a+) and primitive/activation (c-kit+/Lya6A+) markers and some tumor parameters, such as the presence of mitotic bodies, nuclear factor area, NK and iNKT cell infiltration in the tumor, infiltrated tumor area, and infiltrating lymphocyte count at 10x and 40x in specimens treated with pentoxifylline, norcantharidin, and the combination of both drugs. Possible correlations were estimated with Pearson's correlation analysis. It should be noted that, despite having demonstrated multiple correlations, immaturity/activation markers were related to these cells' activation. At the tumor site, iNKT cells are the ones that exert the cytotoxic potential on tumor cells, but they are confined to specific sites in the tumor. Due to the higher number of interactions of natural killer cells with tumor cells, it is concluded that the most effective treatment was PTX at 60 mg/kg + NCTD at 0.75 mg/kg.

Effectiveness of radiotherapy and targeted radionuclide therapy for melanoma in preclinical mouse models: A combination treatments overview.

Cutaneous melanoma is an aggressive and highly metastatic skin cancer. In recent years, immunotherapy and targeted small-molecule inhibitors have improved the overall survival of patients. Unfortunately, most patients in advanced stages of disease exhibit either intrinsically resistant or rapidly acquire resistance to these approved treatments. However, combination treatments have emerged to overcome resistance, and novel treatments based on radiotherapy (RT) and targeted radionuclide therapy (TRT) have been developed to treat melanoma in the preclinical mouse model, raising the question of whether synergy in combination therapies may motivate and increase their use as primary treatments for melanoma. To help clarify this question, we reviewed the studies in preclinical mouse models where they evaluated RT and TRT in combination with other approved and unapproved therapies from 2016 onwards, focusing on the type of melanoma model used (primary tumor and or metastatic model). PubMed® was the database in which the search was performed using mesh search algorithms resulting in 41 studies that comply with the inclusion rules of screening. Studies reviewed showed that synergy with RT or TRT had strong antitumor effects, such as tumor growth inhibition and fewer metastases, also exhibiting systemic protection. In addition, most studies were carried out on antitumor response for the implanted primary tumor, demonstrating that more studies are needed to evaluate these combined treatments in metastatic models on long-term protocols.

Combinational photodynamic and photothermal - based therapies for melanoma in mouse models.

BACKGROUND: Melanoma is a highly metastatic skin cancer with limited response to current therapies in advanced patients. To overcome resistance, novel treatments based on photodynamic and photothermal therapies (PDT and PTT, respectively) have been developed to treat melanoma in preclinical murine models. Despite success inhibiting implanted tumors' growth, there has been limited evaluation of their long-term effectiveness in preventing metastasis, recurrence, or improving survival rates. METHODS: Combined and multidrug therapies based on PDT and/or PTT to treat cutaneous malignant melanoma in the preclinical mouse model were reviewed from 2016 onwards. PubMed® was the database in which the search was performed using mesh search algorithms resulting in fifty-one studies that comply with strict inclusion rules of screening. RESULTS: B16 melanoma-bearing C57BLACK6 mice model was the most used to evaluate immunotherapies, chemotherapies, and targeted therapies in combination with PDT and/or PTT. Combined therapies demonstrated a synergistic effect, resulting in intense antitumor activity. The most extensively studied protocol for developing metastatic models involved the intravenous administration of malignant cells, with some combined therapies being tested. Furthermore, the review presents the composition of the nanostructures utilized for delivering the drugs and light-responsive agents and the treatment plans for each combined approach. CONCLUSIONS: The identified mechanisms to simulate metastatic melanoma models and the therapeutic combinations may aid in evaluating the systemic protection of combined PDT and PTT-based therapies, particularly in conducting short-term preclinical experiments. Such simulations could have relevance to clinical studies.

Norcantharidin Nanoemulsion Development, Characterization, and In Vitro Antiproliferation Effect on B16F1 Melanoma Cells.

Melanoma is a highly lethal type of cancer that has had an increase in incidence in the last decades. Nevertheless, current therapies lack effectiveness and have highly disabling side effects, which calls for new therapeutic strategies. Norcantharidin (NCTD) is an acid derivative with potential antitumor activity isolated from natural blister beetles. However, its solubility limitations restrict its use. To address this issue, we developed an oil-in-water nanoemulsion using commonly available cosmetic ingredients, which increased NCTD solubility 10-fold compared to water. The developed nanoemulsion showed a good droplet size and homogeneity, with adequate pH and viscosity for skin application. In vitro drug release studies showed a sustained release profile, ideal for prolonged therapeutic effects. Accelerated stability studies proved that the formulation was reasonably stable under stress conditions, with particle separation fingerprints, instability index, particle size, and sedimentation velocity analyses being conducted. To assess the therapeutic potential of the developed formulation, in vitro studies were conducted on melanoma B16F1 cells; results showed an IC50 of 1.026 +/- 0.370 mg/kg, and the cells' metabolic activity decreased after exposure to the NCTD nanoemulsion. Hence, a new "easy-to-make" nanoformulation with therapeutic potential on melanoma cells was developed, as a possible adjuvant for future melanoma treatment.

Norcantharidin toxicity profile: an in vivo murine study.

Norcantharidin (NCTD) is the demethylated analog of cantharidin, with allegedly reduced toxicity. However, there is still limited information regarding its posology and potential risk in its use in cancer treatment. Healthy BDF1 mice were intraperitoneally administered with norcantharidin (0, 3, 6, 12, and 25 mg/kg) every 24 h for 6 days. Survivor mice were euthanized, and the brain, lungs, kidneys, spleen, and liver were procured for enzymatic and histopathological analysis in the liver and kidney. DL50 were 8.86 mg/kg for females and 11.77 mg/kg for males. The treatments with 3.0 mg/kg and 6.0 mg/kg significantly modified the phosphorylase, alanine transaminase, and γ-glutamyl transferase activities; however, an organ-specific response was detected. A significant dose-dependent decrease was observed in the kidney for ROS, while the liver had the opposite effect. Histopathological analysis revealed a significant elevation in hepatocytes' nuclei average size and total area (3 mg/kg), as well as centrilobular vein and adjacent sinusoidal capillaries showed a significant difference. The portal triad presented a significant difference in veins and capillarity count in 6 mg/kg. Renal samples showed cortex convoluted tubules' average size significantly augmented in both doses' groups, and tubule count was found augmented in 6 mg/kg. These physiological effects of NCTD can be exploited as treatment strategies if able to operate in an established posology and proper testing.

Phenotype of Peripheral NK Cells in Latent, Active, and Meningeal Tuberculosis.

The mechanisms underlying the immunopathology of tuberculous meningitis (TBM), the most severe clinical form of extrapulmonary tuberculosis (TB), are not understood. It is currently believed that the spread of Mycobacterium tuberculosis (Mtb) from the lung is an early event that occurs before the establishment of adaptive immunity. Hence, several innate immune mechanisms may participate in the containment of Mtb infection and prevent extrapulmonary disease manifestations. Natural killer (NK) cells participate in defensive processes that distinguish latent TB infection (LTBI) from active pulmonary TB (PTB). However, their role in TBM is unknown. Here, we performed a cross-sectional analysis of circulating NK cellCID="C008" value="s" phenotype in a prospective cohort of TBM patients (n = 10) using flow cytometry. Also, we addressed the responses of memory-like NK cell subpopulations to the contact with Mtb antigens in vitro. Finally, we determined plasma levels of soluble NKG2D receptor ligands in our cohort of TBM patients by enzyme-linked immunosorbent assay (ELISA). Our comparative groups consisted of individuals with LTBI (n = 11) and PTB (n = 27) patients. We found that NK cells from TBM patients showed lower absolute frequencies, higher CD69 expression, and poor expansion of the CD45RO+ memory-like subpopulation upon Mtb exposure in vitro compared to LTBI individuals. In addition, a reduction in the frequency of CD56brightCD16- NK cells characterized TBM patients but not LTBI or PTB subjects. Our study expands on earlier reports about the role of NK cells in TBM showing a reduced frequency of cytokine-producing cells compared to LTBI and PTB.

In vitro anti-proliferative effect and in vivo antitumor action of daphnetin in different tumor cells.

ANTECEDENTES: Los efectos antiinflamatorios de la dafnetina (7,8-dihidroxicumarina) han sido bien documentados, pero su potencial como agente anticanceroso es controversial y no se ha explorado suficientemente. MATERIAL Y MÉTODOS: En este trabajo se evalúa el efecto antiproliferativo in vitro de la dafnetina en tres líneas celulares mediante ensayos de MTT, así como su efecto antitumoral in vivo en cuatro diferentes tipos de tumores en ratones. RESULTADOS: Con una correlación entre los resultados in vitro e in vivo, los tipos de células probadas tienen diferente sensibilidad al compuesto. Las siguientes líneas celulares están ordenadas de acuerdo con la potencia antiproliferativa in vitro de la dafnetina: células de melanoma B16 (IC50 = 54 ± 2.8 µM) > células de adenocarcinoma de mama MXT (IC50 = 74 ± 6.4 µM) > células de carcinoma de colon C26 (IC50 = 108 ± 7.3 µM). In vivo, la dosis antitumoral óptima de dafnetina fue de 40 mg/kg, y las magnitudes de inhibición fueron las siguientes: tumor B16 (48%) > tumor MXT (40%) > tumor fibrosarcoma S180 (30%) > tumor C26 (20%). CONCLUSIÓN: Los resultados indican que la dafnetina podría tener un impacto como adyuvante para mejorar la efectividad de la quimioterapia convencional.

BACKGROUND: The anti-inflammatory effects of daphnetin (7,8-dihidroxicoumarin) have been well-documented, but the potential of daphnetin as an anticancer agent is controversial and remains insufficiently explored. MATERIAL AND METHODS: In this work, we evaluated the in vitro anti-proliferative effect of daphnetin in three cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, as well as its in vivo antitumor effect in four different types of mouse tumor. RESULTS: With a correlation between in vitro and in vivo results, the tested cell types have different sensitivity to the compound. The following cell lines are arranged according to the in vitro anti-proliferative potency of daphnetin: B16 melanoma cells (inhibitory concentrations 50 [IC50] = 54 ± 2.8 µM) > mitoxantrone (MXT) breast adenocarcinoma cells (IC50 = 74 ± 6.4 µM) > C26 colon carcinoma cells (IC50 = 108 ± 7.3 µM). In vivo, the optimal antitumor dose of daphnetin was 40 mg/kg and the magnitudes of inhibition were the following: B16 tumor (48%) > MXT tumor (40%) > S180 fibrosarcoma tumor (30%) > C26 tumor (20%). CONCLUSION: Our results indicate that daphnetin might have an impact as adjuvant to improve the effectiveness of conventional chemotherapy.

Distribution of heavy metals in crop soils from an agricultural region of the Yucatan Peninsula and biochemical changes in earthworm Eisenia foetida exposed experimentally.

Heavy metals (HM) are natural components of agricultural soils. However, excessive use of agrochemicals (fertilizers and pesticides) can increase the concentration of these elements, making them harmful to crops and soil biota. Therefore, this study aimed to evaluate the concentrations of 6 HM (Cr, Mn, Cu, Zn, Al, and Fe) in soils collected from a typical agricultural region of the Yucatan Peninsula, Mexico, and relate their effects on weight, biotransformation (EROD, GST), and oxidative stress (SOD, CAT, LPO, and MT) in the earthworm Eisenia foetida. The results showed different concentrations of HM accumulation in soils, in the following order, Fe (598.74 ± 223.81 µg g-1), Al (145.30 ± 33.54 µg g-1), Cr (88.38 ± 36.23 µg g-1), Mn (69.53 ± 22.48 µg g-1), Zn (54.10 ± 12.04 µg g-1), and Cu (24.91 ± 12.40 µg g-1), the soils with maize, soybean, and chihua squash crops being the ones with the highest distribution and availability of these elements. These concentrations of metals did not produce mortality. However, Cr, Fe, and Al induced a significant biomass loss of E. foetida. The integrated biomarker response version 2 revealed that soil with maize cultivation was affected the most, with the earthworms exposed to this soil showing greater variations in the dominant biological responses including EROD, TBARS, SOD, and MT (IBRv2 = 9.73). PCA analysis indicated a strong positive relationship between these oxidative stress biomarkers and Cu and Zn concentrations. These findings provide a reference guide that should be part of the monitoring and management programs for different agricultural types in the region under study.

Combination of pentoxifylline and α-galactosylceramide with radiotherapy promotes necro-apoptosis and leukocyte infiltration and reduces the mitosis rate in murine melanoma.

Despite the success for the treatment of melanoma such as targeted molecular therapy, the use of such treatments are expensive For this reason, this study was carried out to explore the anti-cancer properties of available drugs that are able to modify the melanoma prognosis. The study was conducted in two phases: Evaluation of pharmacological effects of pentoxifylline (PTX) administered above (60 mg/kg) which is the therapeutic dose that is aimed at reducing the side-effect of radiotherapy, and of α- galactosylceramide (GalCer) administered at 100 µg/kg, as well as their combination using a murine model (BDF1 mice) of melanoma cell line (B16-F1, ATCC). For the radiotherapy phase, 9 Gy was applied in the tumor area, before (3 days), during (30 min) and after (3 days) the PTX + GalCer treatment. In both study phases, the mitosis rate, leukocyte infiltration and necro-apoptosis were assessed using histological and immunohistochemical approach and tumor volume evaluation as biomarkers. All treatments showed good prognosis results estimated as reduction of mitosis rate (PTX + GalCer after radiotherapy and GalCer), increased leukocyte infiltrate (PTX + GalCer after radiotherapy and GalCer) and necro-apoptosis augmentation (PTX + GalCer after radiotherapy and radiotherapy control). Nevertheless, a lower development of tumor volume was found in GalCer treatment. In this way, it is possible to suggest that the integrated treatment with immuno-stimulators such as GalCer, plus drug used for peripheral vascular disease (PTX) after radiotherapy is probably an alternative for controlling aggressive melanoma in murine model.

Proteomic analysis of oxidized proteins in the brain and liver of the Nile tilapia (Oreochromis niloticus) exposed to a water-accommodated fraction of Maya crude oil.

Crude oil (CO) is a super mixture of chemical compounds whose toxic effects are reported in fish species according to international guidelines. In the current study a proteomic analysis of oxidized proteins (ox) was performed on the brain and liver of Nile tilapia exposed to WAF obtained from relevant environmental loads (0.01, 0.1 and 1.0 g/L) of Maya CO. Results have shown that oxidation of specific proteins was a newly discovered organ-dependent process able to disrupt key functions in Nile tilapia. In control fish, enzymes involved on aerobic metabolism (liver aldehyde dehydrogenase and brain dihydrofolate reductase) and liver tryptophan--tRNA ligase were oxidized. In WAF-treated liver specimens, fructose-bisphosphate aldolase (FBA), ß-galactosidase (ß-GAL) and dipeptidyl peptidase 9 (DPP-9) were detected in oxidized form. oxDPP-9 could be favorable by reducing the risk associated with altered glucose metabolism, the opposite effects elicited by oxFBA and oxß-GAL. oxTrypsin showed a clear adverse effect by reducing probably the hepatocyte capacity to achieve proteolysis of oxidized proteins as well as for performing the proper digestive function. Additionally, enzyme implicated in purine metabolism adenosine (deaminase) was oxidized. Cerebral enzymes of mitochondrial respiratory chain complex (COX IV, COX5B), of glycosphingolipid biosynthesis (ß-N-acetylhexosaminidase), involved in catecholamines degradation (catechol O-methyltransferase), and microtubule cytoskeleton (stathmin) were oxidized in WAF-treated specimens. This response suggests, in the brain, an adverse scenario for the mitochondrial respiration process and for ATP provision as for ischemia/reoxygenation challenges. Proteomic analysis of oxidized proteins is a promising tool for monitoring environmental quality influenced by hydrocarbons dissolved in water.

Lipid metabolism and pro-oxidant/antioxidant balance of Halamphora oceanica from the Gulf of Mexico exposed to water accommodated fraction of Maya crude oil.

Diatoms play key roles in primary production and carbon fixation at a global scale and in some cases these species live on marine ecosystems impacted by crude oil (CO) spills. Halamphora oceanica, a new diatom species from the Southwest of the Gulf of Mexico was isolated and cultured in the laboratory and was exposed to water accommodated fraction (WAF) of different Maya CO loads at 0.01, 0.1, 1 and 10g/L by 96h. A battery of biomarkers involved in oxidative stress (O2•, H2O2, TBARS, ROOH, RC=O, SOD, CAT, GPx), biotransformation and conjugation (total CYP450 activity and GST) moreover fatty acid (FA) metabolism (FA levels, fatty-acid synthase and acyl-CoA oxidase) were measured. Obtained results suggest that increases of PAHs in the medium (below to EC50) acts as external forces able to turn-on regulatory mechanisms on H. oceanica involved in both, on the PAHs uptake and changing its aerobic metabolism to anaerobic metabolism. However, the growth of this microalgae species evaluated as chlorophyll "a" and pheophytin levels increased as the WAF concentration indicating that PAHs and other hydrosoluble hydrocarbons were used as carbon and energy sources by unidentified enzymes not evaluated in the current study. Our hypothesis was also corroborated by IBRv2. In the current study, we suppose the change from aerobic to anaerobic metabolism as a strategy for Halamphora oceanica survival exposed to petroleum hydrocarbons.

Oxidative stress response in the skin mucus layer of Goodea gracilis (Hubbs and Turner, 1939) exposed to crude oil: A non-invasive approach.

The skin of the fish is the foremost target of oxidative stress due to the generation of Reactive Oxygen Species (ROS) originated in the environment and in the skin itself. In this study, a non-destructive assay was developed to evaluate the effects of crude oil (0.0001-0.1mg/L, 96h) on oxidative stress response in the Skin Mucus Layer (SML) of the dusky splitfin goodeid (Goodea gracilis). The response in the SML was compared with recognized target organs through the Integrated Biomarker Response (IBRv2) and a slight addition to the method was proposed. Crude oil was extremely toxic and elicited a clear induction of ROS in the SML, as in the brain, liver and muscle. By the exposure to crude, a significant change in the activities of Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx) as well as on lipid peroxidation (TBARS) and carbonyl protein (RCO) levels was detected. Also, increases in the activity of EROD were found. The general IBRv2 proposed in this study (gIBRv2) showed that oil causes the higher oxidative response in the SML (60.049) under different concentrations of petroleum, which was greater in the brain (56.749), muscle (56.561) and liver (55.775). The results of the study revealed an organ-specific antioxidant defense response that was dependent on the load of petroleum. These results contributed to the understanding of the complexity of oxidative stress response in fish exposed to crude oil using the Skin Mucus Layer as a target for environmental monitoring studies.

Usefulness of oxidative stress biomarkers evaluated in the snout scraping, serum and Peripheral Blood Cells of Crocodylus moreletii from Southeast Campeche for assessment of the toxic impact of PAHs, metals and total phenols.

In this study, we assessed the effects of inorganic and organic pollutants [As, Cu, Fe, Mn, Pb, Zn, PAHs (11 compounds) and total phenols] from a panel of biomarkers [O2, H2O2, thiobarbituric acid reactive substances (TBARS), carbonyl proteins (RCO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total cytochrome P450 activities] evaluated in the Snout Scraping (SS), Serum (S) and Peripheral Blood Cells (PBC) of the Morelet's crocodile (Crocodylus moreletii) inhabiting the reference locality (Lake Mocu) and polluted locality (Champoton River) using Principal Component Analysis (PCA). In male crocodiles from the reference site, only H2O2 in PBC was related to levels of fluoranthene on the Keel of Caudal Scales (KCS), but, in females, no association was detected. In contrast, a sex-linked response was detected in specimens from the polluted locality. Levels of benzo[a]pyrene, benzo[a]anthracene, chrysene, pyrene, phenanthrene, acenaphthene, Zn, Cu, and Pb in KCS of the female crocodil were related to the oxidative stress biomarkers on PBC, incluing the total CYP450 activity and levels of O2, H2O2 in serum. However, in male crocodiles, the oxidative stress in SS and in the serum (TBARS, RCO, CAT, GPx), and SOD in PBC was related to As, Pb, Cu, Fe, and benzo[a]pyrene water concentrations and to the burdens of As, Fe, Mn, indeno[1,2,3cd]pyrene in KCS. These results confirm the usefulness of minimal or non-invasive methods of evaluating the oxidative stress response for the environmental monitoring program on the wild Morelet's crocodile that is subject to special protection in Mexican guidelines.

Fat-associated lymphoid cluster in Cyprinus carpio: Characterisation and its relation with peritoneal haemangiosarcoma.

FALC cells are natural helper cells producing Th2-type cytokines, which express c-kit, Sca-1, IL7R and CD45 in mouse and human. These cells are involved in allergic responses and contribute to the inflammatory reactions of adipose tissue; however, a lack of information prevails about the presence of these cells in other species. The aim of the study was to identify and characterise FALC cells in the common carp (Cyprinus carpio) using immunohistochemistry and molecular biology techniques as well as to explore their relationships with their microenvironment. Histological description of the FALC was performed using H&E and polyclonal antibodies were used against cell-surface markers such as c-kit, Sca-1 and CD45. Furthermore, gene expression of c-kit, Sca-1 and IL7R was assessed. C. carpio FALC cells express the same surface markers reported in FALC of the mouse at both the pre- and post-transcriptional level. By exposure to the soluble fraction of helminths, FALC cells produce abundant Th2 cytokines (IL-5, IL-6 and IL-13) but do not synthesise IL-1α. Additionally, FALC cells probably participate in vascular remodelling of the intestine vessels, inducing tumours because a malignant haemangiosarcoma in the peritoneal cavity was found. In this tumour, abundant FALC with their characteristic cell-surface markers were detected. The findings of this study suggest the involvement of some proto-oncogenes such as c-kit and Sca-1, and the deregulation of Src kinases modulated by CD45 present in C. carpio FALC with the ontogeny of peritoneal haemangiosarcoma in this fish species.

Relationship between biomarkers and endocrine-disrupting compounds in wild Girardnichthys viviparus from two lakes with different degrees of pollution.

Despite great efforts worldwide to evaluate the effects of endocrine-disrupting compounds (EDCs) in fish, there is little information available about the interactions of EDCs with the disruption of the sexual endocrine axis in fish species with matrotrophic viviparity and intraluminal gestation. To understand these interactions, six sampling campaigns were performed within a period of 1 year in two lakes with different degrees of pollution. A battery of biomarkers of the oestrogenic response was assessed in the liver [vitellogenin, CYP 1A1, epoxide hydrolase activity, and metallothioneins (MT)] and MT in the head of Girardinichthys viviparus. Linear correlation analysis and canonical correspondence analysis were performed to explore the relationship between the oestrogenic response with EDCs and with metals. The biomarker responses were assessed using the water content of EDCs (oestrone, 17-ß-oestradiol, oestriol, 17-α-ethinyl oestradiol, total phenols, bisphenol A, nonyl phenol, octyl phenol), as well as the PAHs indene[1,2,3-c,d]pyrene, naphthalene, pyrene, benzo[a]anthracene, benzo[k]fluoranthene and benzo[a]pyrene) and metals (Cu, Fe, Mn, Pb and Zn). Greater disruption of the sexual endocrine axis occurred in fish of both sexes inhabiting the polluted lake whose effects were apparently influenced by CYP 1A1 activity and by 17-α-ethinyl oestradiol. In addition, non-estrogenic mechanisms in the hypothalamus and pituitary glands in male fish were observed, elicited by endogenous levels and the water concentration of Pb. In contrast, in females from the less polluted lake, VTG induction was related to exogenous oestrogens. The disruption of the hypothalamic-pituitary-gonadal axis is a complex process influenced by both endogenous and exogenous factors and contributes to male feminisation by exposure to EDCs.

The relationship between the bioactivation and detoxification of diazinon and chlorpyrifos, and the inhibition of acetylcholinesterase activity in Chirostoma jordani from three lakes with low to high organophosphate pesticides contamination.

In fish, a number of studies have linked acetylcholinesterase (AChE) inhibition with exposure to organophosphate pesticides (OPs); however, evidence suggests the need to study aspects related to the bioactivation and detoxification of OPs, since their neurotoxicity is dependent on these processes. Thus, the study aim was to examine the relations between chlorpyrifos (CPF) and diazinon (DZN) bioactivation by hepatic CYP450 izoenzymes (CYP 2B6, CYP 2C19, CYP 3A4) and detoxification by aryl esterases and oxonases with brain and muscle AChE activity in Chirostoma jordani from three lakes with low to high OPs contamination in water and sediments. We found two patterns of bioactivation in vitro: (i) in fish from a lake with high CPF pollution, the main isoenzymes involved in this process were CYP 2C19>CYP 2B6>CYP 3A4, and (ii) in fish captured in a lake with a high concentration of DZN, the isoenzymes were CYP 3A4>CYP 2C19>CYP 2B6. Bioactivation is shown in this study to be fundamental in brain and muscle AChE inhibition in vivo. The rate of bioactivation of CPF was lower than for DZN. CPF bioactivation was accompanied by reduced detoxification and higher neurotoxicity, which was inversely dependent on the environmental contamination of CPF. Detoxification was also inversely correlated with environmental contamination by CPF, and was higher with diazoxon than chlorpyrifos-oxon. Oxonases were the most relevant enzymes involved in detoxification. The current findings suggest a series of strategies between the bioactivation and detoxification of OPs that allowed the survival of C. jordani despite of OPs pollution levels.

Measurement of suppressor activity of T CD4⁺CD25⁺ T reg cells using bromodeoxyuridine incorporation assay.

The suppressor effect of T regulatory lymphocytes in co-cultures with T effector cells obtained by magnetic columns from healthy donors and activated by CD3/CD28 was measured by a proliferation assay using BrdU incorporation and an ELISA test. Tritiated thymidine incorporation was used as a reference since it is the gold standard for proliferation assays. Both methods were used simultaneously in the same samples in order to compare them. Correlation between them was statistically significant (p < 0.001). The purification using magnetic columns was very efficient since CD4⁺CD25⁺ cells were also FOXP3⁺ therefore; they were identified as suppressor T cells. The use of BrdU incorporation in suppression assays is an excellent method that avoids the use of radioactive contaminating materials.

Pro-oxidant and antioxidant responses in the liver and kidney of wild Goodea gracilis and their relation with halomethanes bioactivation.

In mammals, it has been shown that halomethanes (HM) are bioactivated by enzymes such as CYP 2E1 and the theta isoform of GST to produce reactive metabolites. However, in fish, little information is available, although HM can form autochthonously in aquatic environments. This study assessed the effect of HM in dusky splitfin (Goodea gracilis) from three lakes of the Valley of Mexico by analysing specific HM biomarkers as well as a broad range of biomarkers. The concentration of HM was a function of its half-life (higher in deep waters), while its precursors and solar radiation are secondary factors that determine its concentration. The kidney showed higher basal metabolism than the liver, probably because of its function as a haematopoietic and filtration organ. Using integrated biological response version 2 (IBRv2), it was found that the hepatic and renal O2· content is a pro-oxidant force capable of inducing oxidative stress (ROOH, TBARS and RC=O). Early damage was found to be dependent on low concentrations of HM in Major Lake, whereas late damage was observed in fish exposed to higher concentrations of HM in Zumpango Lake and Ancient Lake. The activities of enzymes involved in antioxidant defence seemed to be inefficient. The quantitative assessment of biomarkers (ANOVA) and the estimate of parameter A obtained from IBRv2 provided different information. However, the data support the greater predictive power of IBRv2, but it requires a series of interrelated biomarkers to infer these possibilities. G. gracilis presents marked patterns of adaptation, which are dependant on the HM concentrations in environmental mixtures, although the response is complex and many toxicants could induce similar responses.