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Cytochrome P CYP gene expressions were studied by semi-quantitative polymerase chain reaction PCR to assess oil pollution using liver and gills of juvenile rainbow trout Oncorhynchus mykiss sampled from La Mina stream Bariloche, Argentinaup and downstream of a discharge of petroleum spill from an unproductive wellbore.

Data was complemented with laboratory exposure to water accommodated fraction WAF from the oil spill. Oil exposure induced gill and liver CYP1A transcription 1. CYP2K1 and CYP2M1 are involved in the metabolism of fatty acids and estrogenic hormones, and its down-regulation would indicate metabolic alterations. CYP3A27 levels were only induced in liver of wild trout 1. Hence, patterns of multiple CYP genes expression in rainbow trout gills and liver could be especially valuable for monitoring programs of crude oil pollution and remediation actions on Patagonian freshwater ecosystems.

In vitro effects of endosulfan-based insecticides on mammalian sperm.

CYP, petroleum spill, fish, contaminated stream, Afuatica. The advance of these activities can endanger Patagonian aquatic ecosystems, which are very well preserved until now.

However, in the early 20th century, the black gold fever had already impacted some Patagonian environments in several ways, such ecotoxicologiq the footprint of old unproductive wellbores. One of these old wellbores is located near to Bariloche city, North West Patagonia, and was under production from up to the s, when it was dismantled leaving a permanent heavy oil spill Mancini and Serna Considering the public concern about adverse health effects of persistent oil pollution on highly valued aquatic ecosystems, particularly on water and edible fish, it is necessary to develop tools to assess the impact of this oil spill.

A Oil remains afloat on the surface of stream water. B Oil spill sources running towards the stream.

C Oil extraction well next to the stream in the 30s photograph by Pedro Penna, Parsons Fish have proved to be useful models when it comes to evaluating the health of aquatic ecosystems.

Rainbow trout Oncorhynchus mykiss is a useful model for toxicology studies because of its sensitivity to chemicals Bailey et al.

Since direct measurement of tissue hydrocarbon concentrations generally does not provide a useful indicator of petroleum exposure in fish and could be ecotpxicologia Collier et al. The phase I xenobiotic metabolizing enzymes are represented mainly by cytochrome P CYP multi-enzymatic complex, that catalyzes hydrolysis, oxidation and reduction reactions, by adding or exposing functional groups Danielson Seven CYP families have been reported for the rainbow trout Uno et al.

CYP1A mRNA expression is induced by minuscule amounts of diverse aromatic compounds through the aryl hydrocarbon receptor AhR signaling pathway, being a sensitive biomarker of exposure to aromatic hydrocarbon contaminants Uno et al.

Hence, CYP1A transcript expression serves as reference in biomarkers of crude oil pollution. CYP2K1 is the most abundant constitutively expressed hepatic cytochrome P found in sexually immature rainbow trout and it shows an unusually high sensitivity to carcinogens Bailey et al.

Thus, anomalous expression of both CYP2 could impact on steroid and fatty acid homeostasis in rainbow trout e. Similarly, aromatic hydrocarbons could act as endocrine-disrupting chemicals in O.

The CYP3A27 induction could provide a useful biomarker for the environmental monitoring. Consequently, since several CYPs could be influenced by exposure to polycyclic aromatic hydrocarbons, oil contamination could affect biosynthesis and metabolism of many important endogenous constituents of rainbow trout. In this study we analyzed the suitability of different possible CYPs molecular biomarkers in O.


We focused on gill and liver tissues because the gills are the primary route of hydrocarbon uptake from water, and the liver is the main organ for metabolism of toxic oil constituents Thomas and Rice First, we explored the heavy crude oil effects on these possible biomarkers in wild juvenile rainbow trout that were caught in the La Mina stream, up and downstream of the oil spill.

The spills converge into a unique source point of pollution, with liters of crude oil per day entering the stream in February The spills are the relict of a wellbore, an attempt to have oil production during the early 20th century and then abandoned Mancini and Serna The study was conducted in two m-long reaches: The riparian vegetation of the reaches is well preserved, with dominance of shrubby plants and the herbaceous Festuca gracillima.

The upstream reach is up of a 1. This impairs juvenile fish migrate from the downstream to the upstream reach, but it might not necessarily prevent migration from the upstream to the downstream reach. The stream bottom is composed of boulder cobble substrates and the stream channel has alternated riffle-pool habitats. The climate is cold-temperate, with a mean annual temperature of 8. During the study, the stream reaches were characterized by mean water velocity of 0.

Fish are represented by a single species, the exotic rainbow trout O. Triplicate samples were collected at the upstream reach —, — and — mat the oil spill zero metersat the downstream reach 50, and m and further downstreamand m.

Water samples were collected in polyethylene bottles and filtered through glass-fiber filters: The WAF was prepared according to Singer et al. The TPH was extracted from each with carbon tetrachloride and was estimated by the analysis of the triple peak method EPA at three spectral regions: Wild, juvenile rainbow trout Fish were killed by a blow to the head.

Liver and gills from four fish were aseptically dissected out and pooled into one sample of each organ, obtaining a total of three pooled samples per organ and per reach. For the experiments, juvenile rainbow trout 3.

Fish were kept in 1. Fish were killed and dissected as described above for field samples.

Departamento de Química Biológica

Samples were diluted with ultra-pure water to adjust to a concentration of 2. The number of cycles of denaturation phase was selected to catch the exponential amplification, by previous calibration of each mRNA. The annealing temperatures and primer pairs used during the duplex PCR for each gene are shown in Table I. Bands were analyzed densitometrically using Image J software.

A ecotosicologia ladder Winkler was used as the molecular mass marker. The presence of a single product of the appropriate size, identical to the reference experiments, was considered as a positive result. All statistical analyzes were performed using SPSS We performed post hoc comparisons between ecotoxicologiia and treatments, for both 24 and 96 h, of all pairs of groups using two-sided significance levels with a Bonferroni adjustment.

The TPH was 5. The TPH concentrations in the stream at the oil spill discharge zero meters were TPH concentration in water was 4. TPH concentration in sediments was 0.

CYPs transcriptional expression in wild rainbow trout. The transcript levels of CYP2M1 declined 1. Letters denote significant differences among treatments within each time, 24 h lowercase letters and 96 h capital letters from Bonferroni test Table II. The rainbow trout Oncorhynchus mykiss is widely distributed around the world and is present in most of the freshwater ecosystems of Patagonia.

This, along with the fact that its metabolic pathways related to oil contaminants detoxification are relatively well known, makes this species a good bioindicator Moore et al. We assessed the molecular responses to ecotoxxicologia pollution in juvenile rainbow trout sampled from La Mina stream, 30 km from Bariloche City.

This well-preserved low-order stream is particularly suitable for oil exposure biomarker studies since the control and the impacted areas are consecutive reaches, thus, the studied individuals acuatjca to the same population, and the oil spill is the only cause of water quality change. The field study was combined with laboratory exposure to WAF obtained from the oil source, which affects the same stream. Gene transcript expression of several cytochrome P oxidases was studied by semi-quantitative PCR in both field and laboratory.


These clear and consistent changes reaffirm CYP1A transcript expression as a good semiquantitative biomarker for crude oil exposure. The maximum induction of CYP1A transcript expression has been reported to start eight to sixteen hours after exposure Minura et dcotoxicologia.

This explains the similar response in liver CYP1A transcript levels at 24 and 96 h during our experiments, without dose-dependent effects. In contrast, gill CYP1A transcripts returned to control levels ecotoxicologja 96 h. In the wild trout of this zcuatica, the higher CYP1A induction in liver than in gills could be related to the conserved physiological function of each organ and constitutive expression of each CYP1 forms, as it was previously described in O.

These possible explanations exceed the scope of our research and further studies will be needed to understand gill CYP1A mRNA regulation in rainbow trout. Hence, the response of the last ones to crude oil could be less specific but not less useful indicating some metabolic alterations.

In contrast, more extended time and high PAH concentration ecotoxucologia. Although the results in the WAF experiment are not ecotoxicologix, differences among time response of CYP2 expression could be useful as complementary indicators of pollution by crude oil. We found induced CYP3A27 transcript levels only in liver of impacted wild trout, suggesting an induction at longer time than four days.

However, there are few studies on the nature of PXR ligands in fish Ekins et al. The results obtained in wild rainbow trout could be particularly useful to evaluate other freshwater pollution, since the TPH concentration varied along the stream reach and rainbow trout was not exposed to a constant concentration of the pollution for several hours, unlike the experiments.

The study of mRNA expression patterns of multiple CYP genes in rainbow trout gills and liver would be extended in order to evaluate their use for monitoring programs of crude oil pollution and remediation actions on Patagonian freshwater ecosystems. As far as we know, this is the first ecotoxicological study on the oil spill in La Mina stream, and one of the few related studies in Patagonia. Since oil exploration and extraction activities are expected to be enhanced in this region in acuatca future, further studies and monitoring programs are needed to protect the quality of valuable water ecosystems, fish production in downstream reservoirs and human health.

Also, we wish to thank Dr. Fish models for environmental carcinogenesis: Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish Danio rerio. The application of toxicity test on the specie Atriplex lampa in the risk assessment of oil spills in Patagonian soils.

Rainbow trout cytochrome Ps: Petroleum exposure and associated biochemical effects in subtidal fish after the Exxon Valdez oil spill. Anchorage Alaska, Februarypp. The cytochrome P superfamily: Evolution of pharmacologic specificity in the pregnane X receptor. Petroleum hydrocarbons Spectrophotometric infrared.

Ecotoxicologia acuatica – Jaime Alberto Palacio Baena – Google Books

United States Environmental Protection Agency. Induction patterns of new CYP1 genes in environmentally exposed rainbow trout. The evolution of farnesoid X, vitamin D, and pregnane X receptors: Cloning, tissue distribution, and functional studies of a new cytochrome P 3A subfamily member, CYP3A45, from rainbow trout Oncorhynchus mykiss intestinal ceca.