Hugo F. Olivares-Rubio

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.

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.

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.

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.

DISRUPTION OF HYPOTHALAMUS-PITUITARY-LIVER-GONADS AXIS IN THE ENDANGERED Girardinichthys viviparus EXPOSED TO ENVIRONMENTALLY RELEVANT CONCENTRATIONS OF A MIXTURE OF METALS AND WITH 17α-ETHYNIL ESTRADIOL

Girardinichthys viviparus is an endemic and endangered Mexican fish species with matrotrophic viviparity that only inhabits in some polluted water bodies in the Valley of Mexico. In the current study, G. viviparus of both sexes were exposed for 21 days to a mixture of metals with relevant environmental concentrations (T1) and to the same mixture spiked with 25 ng of 17α-ethynil estradiol (EE 2 )/L (T2). Some biomarkers involved in endocrine disruption of the hypothalamus-pituitary-liver-gonads axis such as gonadotropins I and II (GtH I and GtH II, respectively), and estradiol (E 2 ) concentrations in the head and gonads were measured. Vitellogenin (VTG) in the liver and gonads, and metallothionein (MT) in the head, liver and gonads were assessed. Increases of GtH I and decreases in GtH II, and alterations of E 2 in the head and gonads were found in fish treated with T1 and T2. Higher content of hepatic and gonadal VTG only in fish treated with T2 was detected. MT was notably induced by T2; however, a time-dependent MT reduction was observed. In both treatments, the hypothalamic-pituitary control point was most affected and their alterations were documented by gonadal and head content of E 2 . In female fish, it is most likely that endogenous levels of E 2 diminished the alterations elicited by EE 2 on this control point of the axis in contrast with male fish. The endocrine disruption of this fish species is a dynamic and complex process.