Armando Vega-López

Relations of oxidative stress in freshwater phytoplankton with heavy metals and polycyclic aromatic hydrocarbons

The phytoplankton is the basis of food webs and also, may bioconcentrate different toxic substances. This phenomenon is well documented, but there are few studies on the toxic effects on the phytoplankton community. In the current study the relation of oxidative stress (TBARS, ROOH, RC=O) and antioxidant defenses (activities of SOD, CAT, GPx and GST) of two phytoplankton communities with BCF of heavy metals (Cu, Fe, Mn, Pb, Zn) and of PAHs (naphthalene, pyrene, indenol, benzo[a]pyrene, benzo[a]anthracene, benzo[b]fluoranthene) was tested. Three sampling surveys were conducted bimonthly on the surface and bottom at different sampling points in the lakes Menor and Mayor of the 2nd section of Chapultepec Park. Also negative and positive controls obtained in the laboratory were included. Toxicity relationships were analyzed using the integrated biomarker response (IBR). Both green algae and cyanobacteria dominated. The contents of ROOH and protein oxidation assessed as RC=O were higher in the Lago Menor, a water body that contains ancient sediments. Through the IBR it was demonstrated that these damages were influenced by Pb, indenol and benzo[b]fluoranthene. In contrast, TBARS content was higher in Lago Mayor, which has sediment in formation. Through IBR it was estimated that Cu, Fe, Mn, Pb, indenol and benzo[b]fluoranthene were related to this damage. However, oxidative stress was accompanied by an induction of CAT and SOD, in contrast, GPx and GST had low or null activity. The field data were similar to the positive controls. We demonstrated for the first time that, although the phytoplankton suffers oxidative stress elicited by metals and PAHs, this community is able to counter this damage through antioxidant defenses. The effects of organic or inorganic toxics in phytoplankton depend on their bioavailability that is modulated by the sediment and also by its physicochemical properties and the characteristics of the abiotic medium.

Differential effects of esculetin and daphnetin on in vitro cell proliferation and in vivo estrogenicity

Esculetin (6,7-dihydroxycoumarin) and daphnetin (7,8-dihydroxycoumarin) are secondary metabolites of plants used in folk medicine. These compounds have showed great antiproliferative activity in several tumor cell lines and have been proposed as potential anticancer agents. However, the estrogenic potential of these two compounds has to date not been reported. The present study compared esculetin and daphnetin on the inhibition of cell proliferation and cell cycle progression of the MCF-7 estrogen-responsive human carcinoma cell line. In vivo and in vitro estrogenic activity for both compounds was also evaluated. Esculetin inhibited cell proliferation after 72 h exposure (IC50=193 ± 6.6 μM), while daphnetin evidenced inhibiting effects starting at 24-h exposure (72 h, IC50=73 ± 4.1 μM). Both effects showed changes in cyclin D1 gene expression. In non-estrogenic conditions (E-screening assay), esculetin produced biphasic response on proliferation of the MCF-7 cells; at 10(-8)-10(-6)M, concentrations induced proliferative effects as EC50=4.07 × 10(-9)M (E(2)=2.91 × 10(-12)M); at higher concentrations (10(-5)-10(-4)M), cell proliferation was inhibited. Relative proliferative effect at E(2) was 52% (E(2)=100), relative proliferative potency was 0.072 (E(2)=100). Additionally, esculetin tested in vivo showed estrogenic effects at 50-100mg/kg doses; relative uterotrophic effect at E(2) was 37%, with relative uterotrophic potency registered at 0.003. In contrast, daphnetin did not induce estrogenic effects in vitro or with in vivo models. The low estrogenic activity of esculetin could prove useful in postmenopausal therapy but not as a safe antitumor agent in estrogen-dependent tumors. Daphnetin-based antiproliferative selectivity with MCF-7 cells showed that daphnetin is a promising antitumoral agent also acting on estrogen dependent tumors.

Implications of cytochrome 450 isoenzymes, aryl-esterase and oxonase activity in the inhibition of the acetylcholinesterase of Chirostoma jordani treated with phosphorothionate pesticides

Organophosphate pesticides must be metabolized by cytochrome-P450 isoenzymes such CYP 2C19 as CYP 3A4 to induce neurotoxicity, but damage apparently depends on the activity of aryl esterases of the oxonase type that are involved in detoxication of these compounds. However, information on this subject is not available in fish. Chirostoma jordani has sustained significant population reductions, probably due to changes in land-use as well as pesticide impact; nevertheless, no specific studies demonstrating this are available. This study shows for the first time that the activity of cytochrome-P450 isoenzymes (CYP 2B6, CYP 2C19, CYP 3A4) in C. jordani is involved in diazinon and chlorpyrifos bioactivation. However, higher toxicity of chlorpyrifos cannot be explained solely because its bioactivation. Differences in toxicity between both pesticides are due to the activity of aryl esterases and oxonases that are responsible for oxon detoxication. Both hepatic enzymes metabolize diazoxon more efficiently than chlorpyrifos oxon. At lethal concentrations, detoxication is particularly important since mortality was lower with diazinon (LC50=1.5 μg/L) than with chlorpyrifos (LC50=0.17 μg/L). At sublethal levels, maximum acetylcholinesterase inhibition took place at 4h in both brain and muscle and was of lower magnitude in diazinon-treated fish. This is due to the higher affinity of both aryl esterases for diazoxon, which allows higher detoxication rates and therefore greater recovery of acetylcholinesterase activity.

Fatty acid metabolism in fish species as a biomarker for environmental monitoring

Pollution by Organic Contaminants (OC) in aquatic environments is a relevant issue at the global scale. Lipids comprised of Fatty Acids (FA) play many important roles in the physiology and life history of fishes. Toxic effects of OC are partly dependent on its bioaccumulation in the lipids of aquatic organisms due its physicochemical properties. Therefore, there is an increasing interest to investigate the gene expression as well as the presence and activity of proteins involved in FA metabolism. The attention on Peroxisome Proliferation Activate Receptors (PPARs) also prevails in fish species exposed to OC and in the transport, biosynthesis and β-oxidation of FA. Several studies have been conducted under controlled conditions to evaluate these biological aspects of fish species exposed to OC, as fibrates, endocrine disrupting compounds, perfluoroalkyl acids, flame retardants, metals and mixtures of organic compounds associated with a polluted area. However, only fibrates, which are agonists of PPARs, induce biological responses suitable to be considered as biomarkers of exposure to these pollutants. According to the documented findings on this topic, it is unlikely that these physiological aspects are suitable to be employed as biomarkers with some noticeable exceptions, which depend on experimental design. This emphasises the need to investigate the responses in fish treated with mixtures of OC and in wild fish species from polluted areas to validate or refute the suitability of these biomarkers for environmental or fish health monitoring.

Evidence of Bioactivation of Halomethanes and its Relation to Oxidative Stress Response in Chirostoma riojai, an Endangered Fish from a Polluted Lake in Mexico

Halomethanes (HMs) are produced autochthonously in water bodies through the action of ultraviolet light in the presence of HM precursors, such as dissolved organic carbon and halogens. In mammals, toxic effects induced by HMs are diverse and include oxidative stress, which is also induced by divalent and polyvalent metals; however, in fish little information is available on HM metabolism and its possible consequences at the population level. In the present study, high CYP 2E1 and GST theta-like activities were found in viscera of the Toluca silverside Chirostoma riojai from Lake Zumpango (LZ; central Mexico). Formaldehyde, one of the HM metabolites, was correlated with CYP 2E1 activity and also induced lipid peroxidation in viscera. Hepatic CYP 2E1 activity was correlated with GST theta-like activity, suggesting the coupling of both pathways of HM bioactivation and its consequent oxidative damage. Sediment metals, among others, were also responsible for oxidative stress, particularly iron, lead, arsenic and manganese. However, under normal environmental conditions, the antioxidant enzymes of this species sustain catalysis adapted to oxidative stress. Findings suggest that this fish species apparently has mechanisms of adaptation and recovery that enable it to confront toxic agents of natural origin, such as metals and other substances formed through natural processes, e.g., HMs. This has allowed C. riojai to colonize LZ despite the high sensitivity of this species to xenobiotics of anthropogenic origin.

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.

Pro-oxidant and antioxidant response elicited by CH2Cl2, CHCl3 and BrCHCl2 in Goodea gracilis using non-invasive methods.

The development of non-invasive methods aimed to evaluate the effects of many toxicants is required. Although there are some studies conducted in successful ways, a lack of information prevails especially for those substances that could be formed autochthonously in the water bodies, such as halomethanes (HMs). In this study, induction of pro-oxidant forces (CH2O, O2, H2O2), oxidative stress (TBARS, RCO) and antioxidant defenses (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in the skin mucus layer regarding to the liver of Goodea gracilis exposed to CH2Cl2, CHCl3 and BrCHCl2 were evaluated, in addition to the hepatic cytochrome P450 (CYP 2E1) and glutathione S-transferase theta (GSTT) activities. Regardless of the implicit toxicity involved in the bioactivation of the HMs, carried out by the CYP 2E1 and GST, it was noticeable that this process induces oxidative stress. The usefulness of the mucus layer for the evaluation of the oxidative stress response was demonstrated, despite some peculiar characteristics concerning induction of oxidative stress in liver and skin mucous layer. However, for the understanding of the induction of reactive oxygen species in both targets it is essential to evaluate the activity of antioxidant defenses; otherwise the interpretation of toxic effects elicited by HMs would be erroneous. In the skin mucus layer, lower activities of the enzymes involved in antioxidant defense than in liver were observed. The evaluation of the biomarkers in the skin mucus layer involved in the oxidative stress is useful due the consistent response regarding to concentration of the HMs.

Estrogenic effects of polychlorinated biphenyls and relation to cytochrome P4501A activity in the endangered goodeid fish Ameca splendens

The present study examines the relationships between cytochrome P4501A (CYP1A) activity and vitellogenin (VTG) induction in Ameca splendens elicited by a polychlorinated biphenyl (PCB) mixture. Ethoxyresorufin-O-deethylase (EROD) activity, mRNA levels of VTG, and VTG induction were evaluated in male and female fish exposed for 1, 2, 4, 8, and 16 d to a commercial PCB mixture. Polychlorinated biphenyls induced higher EROD in both sexes and this induction was higher in females than in males. Maximum EROD and VTG induction occurred on day 1 in females, while in males these maxima occurred on days 8 and 16. A correlation between EROD and VTG induction was found only in males (p<0.001), and VTG induction was also higher in males than in females (p<0.01). Exposure to PCBs elicited increases in VTG expression and induction over time in males, while in females these decreased at the end of the exposure period. Deficiencies in the feedback mechanisms of male A. splendens exposed in the wild to xenoestrogens such as PCBs have probably contributed to alter the sex ratio of wild populations of this species.

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 Morelets 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 Morelets crocodile that is subject to special protection in Mexican guidelines.

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-β-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.