RESUMO
The cytochrome P450 1A (CYP1A) mRNA is induced by environmental contaminants such as PAHs, PCBs and dioxins. The present study cloned the CYP1A transcript from the guppy Phalloceros caudimaculatus, which represents a potential fish for toxicological studies in South America. The newly identified CYP1A encodes a protein with 521 amino acids that shared 96-70% identity with other fishes. The characterization of organ- and time-dependent induction of CYP1A using RT-qPCR was evaluated after waterborne exposure to beta-naphthoflavone (BNF; 1µM). The minimum exposure time that elicited significant CYP1A induction was 1h for liver, gill, gut, brain, anal fin and fingerlings; 2h for dorsal fin; and 4h for kidney and tail fin. CYP1A tended to reach peak induction in the first few hours (4h-8h) of experiment in most organs, although levels remained induced until the end of the experiment (96h). Validation of CYP1A use in environmental sample was performed by exposing P. caudimaculatus to elutriate made from sediment of three streams located in adjacent areas of the Patos Lagoon Estuary (RS, Brazil). CYP1A in liver, gills and anal fin was induced by elutriate made from urban (S1) and industrial (S2) sites; and not induced by a reference site located 22 Km from potential contaminant sources, suggesting that environmental contamination plays a role in this induction. The results suggest that fins could be used for CYP1A biomarker analysis and employed in non-lethal biopsy methods for environmental monitoring. The responsiveness of the newly identified CYP1A to BNF and elutriate indicates that the guppy P. caudimaculatus could be used for environmental toxicology investigations in South American environments.
Assuntos
Citocromo P-450 CYP1A1/metabolismo , Poecilia/metabolismo , RNA Mensageiro/metabolismo , Poluentes Químicos da Água/toxicidade , beta-Naftoflavona/toxicidade , Sequência de Aminoácidos , Nadadeiras de Animais/efeitos dos fármacos , Nadadeiras de Animais/metabolismo , Animais , Biomarcadores/metabolismo , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Monitoramento Ambiental , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Dados de Sequência Molecular , Poecilia/crescimento & desenvolvimento , Alinhamento de Sequência , Poluentes Químicos da Água/químicaRESUMO
Alligator gar populations have declined because of overfishing, habitat loss and pollution. Over time, the exposure to different pollutants have affected these fishes as a consequence of their high trophic level, bottom-dwelling habits and long life span. In order to evaluate the physiological effects of pollutants on alligator gar, juveniles (6, 12 and 24 months) were exposed to sub-lethal doses of diazinon, ß-naphthoflavone (BNF) and 17 ß-estradiol (E2) by intraperitoneal injection. After 2 days of exposure, liver samples were taken to determine the activities of acetylcholinesterase, butyrylcholinesterase and carboxylesterase; alkaline and acid phosphatases (ALP and ACP); ethoxyresorufin o-deethylase (EROD); glutathione s-transferase (GST); superoxide dismutase (SOD), and vitellogenin (VTG) concentration. Two additional bioassays consisting on the exposure of compounds through water or food were performed and after 4 and 28 days, respectively, biomarkers were determined. All esterases were inhibited in organisms exposed to diazinon as well as in 6-months gar exposed to E2 and BNF. In contrast, ALP activity increased in gar exposed to diazinon and E2, while ACP activity did not show any variations. No EROD activity was registered after exposure to the different pollutants, despite being one of the most sensitive and common detoxification biomarkers used for fishes. GST activity reduction was detected when gar were exposed to E2 and BNF, while SOD activity increased after exposure to diazinon and E2. Finally, VTG levels were higher in animals exposed to E2 compared to other treatments. Overall, these results suggest that alligator gar juveniles have a low biotransformation metabolism and show that they are especially sensitive to those pollutants affecting the nervous system.
Assuntos
Diazinon/toxicidade , Estradiol/toxicidade , Peixes/metabolismo , Poluentes Químicos da Água/toxicidade , beta-Naftoflavona/toxicidade , Acetilcolinesterase/metabolismo , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Butirilcolinesterase/metabolismo , Carboxilesterase/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Glutationa Transferase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Superóxido Dismutase/metabolismo , Vitelogeninas/metabolismoRESUMO
Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1µM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTq-PCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96h compared to controls (DMSO vehicle; p<0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24h to 96h for gill and liver (â¼15-fold and â¼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4h (â¼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100mgl(-1) MS-222 for 2-5min) in the gill, liver, gonopodium, dorsal or tail fin (p<0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4h, high CYP1A induction was observed in the tail and gonopodium (â¼49-fold and â¼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethalex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America.
Assuntos
Aminobenzoatos/farmacologia , Anestésicos/farmacologia , Citocromo P-450 CYP1A1/biossíntese , Poluentes da Água/toxicidade , beta-Naftoflavona/toxicidade , Nadadeiras de Animais/efeitos dos fármacos , Nadadeiras de Animais/enzimologia , Animais , Ciprinodontiformes/genética , Ciprinodontiformes/metabolismo , Citocromo P-450 CYP1A1/genética , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Cinética , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , RNA Mensageiro/biossíntese , Receptores de Hidrocarboneto Arílico/agonistasRESUMO
South American cyprinodontiform fish are potential candidates to be used as model biomarker species of exposure in environmental toxicology. The aim of this study was to identify molecular and biochemical biomarkers of pollution using Poecilia vivipara (Poecilidae) and Jenynsia multidentata (Anablepidae). Partial nucleotide sequences for cytochrome P-450 1A (CYP1A), a classical biomarker of exposure to organic contaminants in fish, were identified in P. vivipara and J. multidentata (approximately 650 nucleotides) using degenerated primers and polymerase chain reaction (PCR). These sequences shared approximately 90% identity in the predicted amino acid sequence with the corresponding CYP1A region of Fundulus heteroclitus. Real-time quantitative PCR (RT-qPCR) analysis confirmed that CYP1A transcription was markedly induced in the liver and gills of J. multidentata (approximately185-fold and 20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively) 24 h after exposure to 1 µM synthetic CYP1A inducer ß-naphthoflavone (BNF). At 24 h after injection with 1 µg/g environmental carcinogenic contaminant benzo[a]pyrene (BaP), a decreased total antioxidant capacity against peroxyl radicals was observed both in liver of J. multidentata and gills of P. vivipara. BaP injection in both fish did not produce changes in lipid peroxide (thiobarbituric acid-reactive substances, TBARS) levels, suggesting an absence of an oxidative stress condition. The newly identified CYP1A may thus serve as general biomarker of exposure to organic contaminant in future studies using P. vivipara and J. multidentata. Data also indicate the importance of species-specific differences in biomarker responses in these South American cyprinodontiform fish, suggesting distinct resistance/susceptibility properties to polycyclic aromatic hydrocarbons.
Assuntos
Ciprinodontiformes/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Benzo(a)pireno/toxicidade , Biomarcadores , Ciprinodontiformes/classificação , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Brânquias/enzimologia , Brânquias/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Masculino , América do Sul , Especificidade da Espécie , Transcrição Gênica , beta-Naftoflavona/toxicidadeRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme involved in several cellular functions including glycolysis, membrane transport, microtubule assembly, DNA replication and repair, nuclear RNA export, apoptosis, and the detection of nitric oxide stress. Therefore, modifications in the regulatory ability and function of GAPDH may alter cellular homeostasis. We report here that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone, which are well-known ligands for the aryl hydrocarbon receptor (AhR), increase GAPDH mRNA levels in vivo and in vitro, respectively. These compounds fail to induce GAPDH transcription in an AhR-null mouse model, suggesting that the increase in GAPDH level is dependent upon AhR activation. To analyse the consequences of AhR ligands on GAPDH function, mice were treated with TCDD and the level of liver activity of GAPDH was determined. The results showed that TCDD treatment increased GAPDH activity. On the other hand, treatment of Hepa-1 cells with beta-naphthoflavone leads to an increase in microfilament density when compared to untreated cultures. Collectively, these results suggest that AhR ligands, such as polycyclic hydrocarbons, can modify GAPDH expression and, therefore, have the potential to alter the multiple functions of this enzyme.