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Pesticides, Food Contaminants, and Agricultural
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Hepatic monooxygenase (CYP1A and CYP3A) and
UDPGT enzymatic activities as biomarkers for long-term
carbofuran exposure in tench (Tinca tinca L)
David Hernández-Moreno a; Francisco Soler-Rodríguez a; M. Prado
Míguez-Santiyán a; Marcos Pérez-López a
a Toxicology Area, Faculty of Veterinary Medicine, Avda de la Universidad, Caceres,Spain To cite this Article: Hernández-Moreno, David, Soler-Rodríguez, Francisco, Míguez-Santiyán, M. Prado and
Pérez-López, Marcos (2008) 'Hepatic monooxygenase (CYP1A and CYP3A) and UDPGT enzymatic activities as
biomarkers for long-term carbofuran exposure in tench (Tinca tinca L)', Journal of Environmental Science and Health,
Part B, 43:5, 395 — 404
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Journal of Environmental Science and Health Part B (2008) 43, 395–404
Copyright C Taylor & Francis Group, LLC
ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/03601230802062117
Hepatic monooxygenase (CYP1A and CYP3A) andUDPGT enzymatic activities as biomarkers for long-termcarbofuran exposure in tench (Tinca tinca L) ANDEZ-MORENO, FRANCISCO SOLER-RODR´IGUEZ, M. PRADO M´IGUEZ-SANTIY ´ Toxicology Area, Faculty of Veterinary Medicine, Avda de la Universidad, Caceres, Spain The effect of a long-term exposure of tenchs to different concentrations (10 and 100 µg/L) of the pesticide carbofuran has beenevaluated. Microsomal hepatic cytochrome P450 subfamily 1A (CYP1A) and 3A (CYP3A) activities, as well as the phase II enzymeuridine diphospho-glucuronosyltransferase (UDPGT) activity were evaluated as adequate biomarkers of fish exposure to environ- Downloaded By: [Pérez-López, Marcos] At: 12:57 25 June 2008 mentally relevant concentrations of the pesticide carbofuran in freshwater ecosystems. A clear time-dependent inhibition of bothCYP1A and UDPGT activities was observed in fish exposed to the highest dose of carbofuran with respect to controls, whereas inthe case of CYP3A activity, values of exposed animals did not show a clear pattern of alteration during the experiment. The results ofthe present study demonstrated that hepatic CYP1A and UDPGT activities from tench could be considered as sensitive biomarkersfor carbamate pesticides in polluted water, thus allowing future and ecologically relevant biomonitoring studies with this species.
Keywords: Biomarker; fish; enzyme; pesticide; carbofuran; tench.
Introduction
organophosphates are extremely toxic and pose a delayedneurotoxicity problem.[7] Among carbamates, the insecti- cide carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl contaminated with toxic chemical substances from methylcarbamate) has been widely used throughout the industrial, agricultural and domestic activities.[1] One world in a variety of registered applications for nematode, of the major families of environmental contaminants is insect or mite control as a result of its efficient translo- constituted by pesticides, widely used substances in current cation by the plant.[8] The action mechanism of carbofu- agricultural practices and farming activities.[2] Indiscrim- ran is primarily through the reversible inhibition of acetyl- inate discharge of pesticides from agricultural run off, cholinesterase enzyme (AChE) and subsequent nervous sys- industrial effluent, and other sources into aquatic media tem disruption. The combination of high solubility and low affects non-target organisms such as fish and prawns, sorption causes carbofuran to be mobile in groundwater, which are very valuable to humans in terms of economical, and its toxic degradation products (3-hydroxy-carbofuran) genetic and recreational resources, and are causing serious are also soluble in water.[9] Carbofuran is being used exten- detrimental effects to ecosystems.[3] Levels of pesticides sively in rice fields to control rice pests, and contamination in superficial waters generally range far below the lethal of water bodies adjacent to rice fields by this carbamate, concentrations for aquatic organisms, but sublethal adverse mainly through run off, is quite possible.[10−11] Moreover, effects may result from exposure of living organisms to carbofuran is very toxic to fish, and generally fish LC50 pesticides at environmentally relevant concentrations.[4−6] values for this carbamate are below 1 mg/L.[12] When considering all classes of pesticides, carbamates Biomarkers can be defined as biochemical and/or physi- are most commonly used, since among the alterna- ological changes in living organisms which can be related to tives, organochlorine has a long residue persistence, and an exposure to, or toxic effect of, an environmental chemi-cal or chemicals.[13] Biomarkers may be used as early warn- Address correspondence to Prof Marcos Perez-Lopez, Toxicology ing indicators, anticipating possible major disturbances at Area, Faculty of Veterinary Medicine (UEX), Avda de la Univer- higher levels of organization and thus they may be of great sidad s/n10071 Caceres, Spain; E-mail: [email protected] value in risk assessment and ecotoxicological studies.[14] In contrast to the simple measurement of contaminants accumulating in body tissues, biomarkers can offer more The central objective of the present study was to monitor complete and biologically more relevant information on hepatic CYP1A and CYP3A enzymatic activities as poten- the potential impact of contaminants on the health of tial biomarkers of fish exposure to the pesticide carbofu- organisms.[15] In fish, however, the physiological values of ran in freshwater ecosystems. In addition, the effects of this many parameters may vary greatly in relation to the species same xenobiotic on the phase II enzyme UDP glucuronosyl of fish, their age and sex as well as seasons of the year.[16] It is, therefore, very important to seek and make use of biomark-ers independent of such physiological fluctuation.[17,18] Regarding biochemical responses in fish to aquatic Material and methods
pollutants, the mixed function oxidase system P450is known to play a major role in the oxidative Animals
metabolism/biotransformation of a wide range of both en-dogenous and exogenous compounds,[19,20] and constitutes Tenchs of both sexes (mean weight 20 ± 11 g) were obtained a part of phase I biotransformation enzymes. Each iso- from a local fish hatchery, “Piscifactor´ıas del Guadiana”, form of the cytochrome P450 participates in the metabolism in Badajoz (Spain). Animals (n = 180) were transported of many different compounds, but one substrate can also in aerated water to the Toxicology unit (Extremadura Uni- be metabolized by several different isoforms.[16] For detec- versity), and randomly divided into six groups of 30 fish.
tion of pollution in aquatic environments, the CYP1 family Each group was placed in an isolated glass aquaria fitted members have been so far proven, at least in the last 20 years, with recirculated, dechlorinated and aerated tap water at a to be the most sensitive indicators.[17,21−23] They respond to temperature of 20 ± 2◦C. All animals were acclimated to water contamination at levels too low to be detected by laboratory conditions for 12 days prior to the initiation of other laboratory methods or at a time when the contami- the experiment in order to reduce differences associated to nant is no longer dissolved in water but persists in the living stress, and they were fed once daily ad libitum with a com- Downloaded By: [Pérez-López, Marcos] At: 12:57 25 June 2008 matter, such as residues of biocidal agents.[16] Another can- mercial dry diet (OVN Dibaq-Diproteg) untill the end of didate for a biomarker is the CYP3A subfamily, the dom- inant CYP form expressed in the digestive and respiratory A continuous flow of dechlorinated tap water of tract of fish[24] and induced in fish by some xenobiotics, such 10 L/hour was provided to produce a replacement of total as pesticides or polychlorinated biphenyls. This family, as volume of 1.5 times each day. Water quality characteris- the CYP1 family, is generally involved in the functional- tics during the whole experiment were determined accord- ization of exogenous and endogenous compounds thereby ing to the American Public Health Association (APHA).[34] creating a more polar and water-soluble compound.[25] Temperature, pH, alkalinity, conductivity and hardness of With respect to phase II biotransformation enzymes, aquatic media averaged 19–23◦C, 7.5 ± 0.4 (Crison pH- such as glutathione S-transferase (GST) or UDPGT, they meter), 3.2 ± 0.8 mmol/L, 0.45–0.93 mOhm and 178 ± are involved in the conjugation of the xenobiotic par- 35 mg/L (as CaCO3), respectively, and dissolved oxygen ent compound or its metabolites with an endogenous content was never situated below 5 mg/L. A 10-hours con- substrate,[25] thus facilitating the excretion of chemicals by the addition of more polar groups to the molecule.[26] PhaseII enzyme’s answer to environmental xenobiotics is less pro- Exposure
nounced than that of phase I.[27] However, even minor al-terations in phase II activity, such as changes in phase II After being acclimated, fish were exposed to a continuous co-substrate levels in response to exposure to xenobiotics, flow system for 90 days: animals were exposed to two spe- can be harmful to aquatic organisms.[15,28−29] As previously cific concentrations of the pesticide carbofuran for 60 days, indicated for phase I-biotransformation enzymes, different and for the last 30 days the constant flow of pesticide was studies have demonstrated that the expression of phase II eliminated, in an attempt to investigate a possible recovery enzymes can be modulated in vivo after exposure to a broad of the considered biochemical parameters.
variety of xenobiotic compounds.[30,31] However, studies on Carbofuran (a 95% product from Faesal, Spain) test so- phase II induction/inhibition responses have been less ex- lutions were prepared in ultra-pure water, by dissolving tensive than those concerning phase I activity.[32,33] the appropriate amount of pesticide standard solution. The The fish, as a bioindicator species, plays an increasingly pesticide was dissolved in water and not in an organic sol- important role in the monitoring of water pollution be- vent since for crop application water is used as a solvent, cause it responds with great sensitivity to changes in the therefore making this a more realistic approach.[35] aquatic environment.[16] The tench, Tinca tinca L, is an ad- A perfusion pump (Gilson, Minipulse 3) was linked to equate test organism in Southern Europe, associated with four experimental tanks, in order to provide constant nom- its widespread occurrence in small streams, reservoirs and inal pesticide concentrations of 10 and 100 µg/L (two ponds, its easy culture in laboratory, and its potential for use aquaria for each assayed concentration) during the 60-day as a regional bioindicator of environmental contamination.
exposure period, the levels of exposure corresponding to the Hepatic monooxygenase and enzymatic UDPGT biomarkers 2% and 20% of the 96 h LC50 value for commercial carbofu- according to the procedure of Thibaut et al.[43] The assay ran in a continuous-flow test with Tilapia nilotica, catfish.[36] consisted of incubating 25 µg of microsomal protein The two remaining tanks were kept with toxicant-free wa- with 200 µM of 7-benzyloxy-4-trifluoromethyl-coumarin ter, and they were considered as controls during the whole (BFC) and 22.5 µM of NADPH (nicotinamide ade- nine dinucleotide phosphate, reduced form) in 100 mM Weekly water samples (250 mL) were taken from each potassium phosphate buffer pH 7.4 (final volume 250 tank for carbofuran analysis in order to assess the effective µL), at 30◦C for 10 min. The reaction was stopped by concentrations of the pesticide, by using an HPLC tech- the addition of 75 µL of 0.5 M Tris-base/acetonitrile nique with acetonitrile and water as mobile phases and a (20:80, v/v), and the fluorescence was directly read in a 200 µL aliquot transferred to a 96-multiwell plate at Exposed and control animals (n = 3 from each aquar- the excitation/emission wavelength pairs of 409 and 530 ium) were removed at the beginning of the experiment and nm, using a Gemini XPS SpectraMax Plus microplate at 10-day intervals. Fish were anesthetized with a solution reader. Quantification was made using the calibration of MS222 (Sigma) at a concentration of 0.5 g/L. Animals curve of the 7-hydroxy-4-trifluofluoromethyl-coumarin were killed, measured, weighed and sexed. Their livers were authentic standard and several blanks containing the immediately dissected, weighed and individually frozen at tested substance were done to remove possible fluorescence −80◦C until subsequent cytochrome P450 monooxygenase emitted by the pesticide.
system and transferase enzyme analysis was carried out.
UDPGT microsomal activity was measured using a mod- ified version of Clarke et al.[44] with p-nitrophenol (pNP) as Biochemical measurements
substrate. Incubation mixtures contained 0.25 mg microso-mal protein (pre-treated for 5 min with 0.2 % Triton X-100 Subcellular fractions were essentially prepared as described on ice), 50 mM Tris/HCl buffer (pH 7.4), 10 mM MgCl2, by F ¨orlin and Andersson[38] and Lavado et al.[39] Livers were 7 mM pNP and 15.5 mM uridine 5 -diphosphoglucuronic Downloaded By: [Pérez-López, Marcos] At: 12:57 25 June 2008 homogenized in cold 100 mM KH2PO4/K2HPO4 buffer acid trisodium salt (UDPGA) in a total volume of 260 mL.
pH 7.4 (1 g every 5 mL), containing 0.1 M KCl, 1 mM The reaction was started at 30◦C by adding the UDPGA dithiothreitol (DTT), 1 mM EDTA, 0.1 mM phenantroline and stopped 30 min later by adding 1.2 mL of 0.2 M and 0.1 mg/mL of trypsine inhibitor. Homogenates were trichloroacetic acid (TCA). After centrifugation, 1.2 mL centrifuged at 1500 g for 15 min, the fatty layer removed, of the supernatant was treated with 10 N KOH and further and the obtained supernatant centrifuged at 12000 g for centrifuged to reduce any particle interference. The remain- 20 min. This last supernatant was further centrifuged at ing pNP was measured spectrophotometrically at 405 nm.
100000 g for 60 min to obtain the cytosolic and microsomal In all cases, reproducibility was based on the relative stan- dard deviation of all triplicate samples.
Microsomal pellets were resuspended in a small vol- ume of 100 mM KH2PO4/K2HPO4 buffer pH 7.4, con-taining 20% w/v glycerol, 1 mM DTT, 1 mM EDTA, Statistical analysis
0.1 mM phenantroline and 0.1 mg/mL of trypsine in-hibitor. Microsomal protein content was measured by the Data were expressed as mean ± standard deviation. The method of Bradford[40] using bovine serum albumin (BSA) differences between the data in the control and the exposed fish were analyzed by means of one-way ANOVA with the Hepatic CYP1A-mediated ethoxyresorufin-O-deethyl- Post hoc Dunnet test, in order to determine statistical dif- ase (EROD) activity was determined in the microsomal ferences among pesticide concentrations for every sampling fraction at 30◦C as described in Burke and Mayer[41]: 10 mL day. A probability level of less than 0.05 was considered of microsomes were incubated for 10 min in a final volume significant (95% confidence interval). The Spearman corre- of 1.0 mL containing 100 mM KH2PO4/K2HPO4 pH 7.4, lation test was individually determined for each established 0.22 mM NADPH, and 3.70 µM 7-ethoxyresorufin. The group (control and exposed) to determine correlations be- reaction was stopped by adding 400 µL of acetonitrile, sam- tween the three biochemical measures. Statistical analysis ples were centrifuged at low speed, and 7-hydroxyresorufin fluorescence determined using a Gemini XPS SpectraMaxPlus microplate reader (Molecular Devices Corporation)at 537/583 nm excitation/emission wavelengths.[42] ERODblanks included microsomal samples plus acetonitrile to Results and discussion
Figures 1, 2 and 3 show the EROD, BFCOD and UDPGT methyl]-coumarin-O-debenzyloxylase (BFCOD) activity microsomal activities, respectively in tench livers dur- was analyzed according to the procedure described by ing the whole experiment. For graphical representation, BD Gentest and optimized for tench liver microsomes, those enzymatic activities were converted in percentage of % of the control
days of assay
Fig. 1. Percentage of the control (100%) (means ± SD) of hepatic ethoxyresorufin-O-deethylase (EROD) activities of tench during the
experience. *: value significantly different from the control (p < 0.05). The vertical dot-line indicates the beginning of the recovery
period.
Downloaded By: [Pérez-López, Marcos] At: 12:57 25 June 2008 days of assay
Fig. 2. Percentage of the control (100%) (means ± SD) of hepatic benzyloxy-4-[trifluoromethyl]-coumarin-O-debenzyloxylase (BF-
COD) activities of tench during the experience. *: value significantly different from the control (p < 0.05). The vertical dot-line
indicates the beginning of the recovery period.
days of assay
Fig. 3. Percentage of the control (100%) (means ± SD) of hepatic uridine diphospho-glucuronosyltransferase (UDPGT) activities of
tench during the experience. *: value significantly different from the control (p < 0.05). The vertical dot-line indicates the beginning
of the recovery period.
Hepatic monooxygenase and enzymatic UDPGT biomarkers inhibition/induction with respect to enzymatic levels deter- min respectively for control and 100 µg/L exposed fish).
mined in control fish, which were assigned at 100%.
Similarly to that previously indicated for EROD activity, Hepatic EROD activities of control fish ranged between inhibition of UDPGT activity in animals exposed to the 39.87 ± 0.055 pmol/mg prot per min (30 days of assay) highest dose of carbofuran were above 20% after 20 days and 55.32 ± 1.431 pmol/mg prot per min (10 days of as- of sampling and until the end of the experiment.
say). Animals exposed to the lowest dose of carbofuran The Spearman correlation test (Table 1) showed a good showed EROD values between 12.41 ± 1.597 and 46.77 ± correlation between individual data of all the investigated 2.278 pmol/mg prot per min, corresponding the maximum biochemical parameters: CYP1A and CYP3A in control difference with respect to controls to day 20 of assay, while fish were significantly correlated, and similarly CYP1A and for animals exposed to the highest dose of pesticide (100 UDPGT in animals exposed to the lowest concentration µg/L) those activities ranged between 7.69 ± 0.846 and (10 µg/L) and between CYP1A and the other two assayed50.68 ± 0.593 pmol/mg prot per min, the maximum dif- biomarkers, CYP3A and UDPGT, in animals exposed to ference with respect to control group corresponding to day the highest carbofuran concentration (100 µg/L), in all 70 of assay (recovery period). In all cases, exposed animals showed decreased levels of EROD activity when compared The CYP1A subfamily plays a major role in xenobi- with those of the control fish, and specifically, inhibition of otic biotransformation through a mechanism mediated EROD activity in animals exposed to the highest dose of by the AhR (aryl hydrocarbon receptor) whose binding carbofuran were above 20% after 20 days of sampling and to a xenobiotic leads to CYP1A induction, protein ex- pression and enzyme activity.[45] The cytochrome P450- When comparing specific EROD levels of the three es- dependent monooxygenase induction/inhibition measured tablished groups on the same sampling days, the differences as EROD activity can be considered as a sensitive indica- with respect to the control group were significant in all cases, tor of exposure to pollutants in fish.[46] In the present study, except on day 50 and 70 of assay for fish exposed to 10 µg/L.
results of EROD analysis exhibit a good temporal and dose- Downloaded By: [Pérez-López, Marcos] At: 12:57 25 June 2008 Those results showed a clear effect of inhibition of CYP1A response relationship with exposure to carbofuran, a pesti- expression caused by carbofuran on exposed animals, and cide which is metabolized by cytochrome P450, leading to indicating that hepatic EROD levels of tench clearly dis- the production of one major ring oxidation metabolite, 3- criminates between both exposed and control group.
hydroxycarbofuran, and two minor metabolites.[47] Never- With respect to the BFCOD activity, mean control val- theless, only a few previous studies have been developed on ues were situated between 9.70 ± 0.509 and 18.85 ± 2.819 fish and CYP1A activity after exposure to carbofuran. One pmol/mg prot per min. Animals exposed to the lowest doseof pesticide ranged between 7.94 ± 0.223 and 22.44 ± 2.899pmol/mg prot per min, and those exposed to the highest Table 1. Correlation study of the considered biomarkers
(CYP1A, CYP3A and UDPGT enzymatic activities) according
dose were situated between 4.33 ± 0.192 and 23.07 ± 1.048 to the three studied groups (control, and carbofuran exposure to pmol/mg prot per min. Maximal difference was observed on sampling day 60, corresponding to a BFCOD activityinduction (60% of induction percentage when compared with the control group), and on sampling day 70 (73% ofinhibition) for animals exposed to 10 and 100 µg/L of pes- ticide respectively. Notwithstanding, activity values of ex- posed animals did not show a clear pattern of alteration, although statistically significant differences with respect to the control group were observed at days 30 and 60 for an- imals exposed to 10 µg/L, and at days 30, 70 and 80 for Similarly to that observed with EROD activity, exposure of tenchs to both doses of carbofuran produced a clear in- hibitory effect on UDPGT microsomal activity during the whole experiment, even during the recovery period. Tenchs exposed to 10 µg/L showed significantly lower UDPGT ac- tivities than controls in 4 of the 10 samplings, whereas for tenchs exposed to 100 µg/L those differences were signifi- cant in 5 of 10 samplings. The highest relative decreases of UDPGT activity of the exposed animals over control fish Correlation is significant at the 0.01 level.
∗Correlation is significant at the 0.05 level.
were observed during the recovery period (70, 80 and 90 CYPIA: Microsomal hepatic cytochrome P450 subfamily 1A.
days of assay) for both concentrations, with a maximum on CYP3A: Microsomal hepatic cytochrome P450 subfamily 3A.
day 80 (0.40 ± 0.012 and 0.25 ± 0.039 nmol/mg prot per UDPGT: uridine diphospho-glucuronosyltransferase.
of them, concerning in vitro toxicity, and working with In- for carbofuran metabolism. Exposure of salmonid species dian catfish, Heteropmeustes fossilis[48] showed that enzyme to other pesticides, for example the organophosphate chlor- induction followed a dose-dependent relationship with this pyrifos, also produced a CYP1A protein level suppression of 30% with respect to control fish.[67] Nevertheless, in gen- Many xenobiotic compounds, such as heavy metals, eral all studies developed on fish species and CYP1A en- dioxin-like compounds, PAHs, β-naphthoflavone or B(a)P zymatic activity have shown induction-processes, for exam- at high concentrations, have shown their effect of inhibition ple towards PAHs and PCBs in livers of feral eel, chan- and/or decrease of liver microsomal cytochromes P4501A nel catfish, mullet, largemouth and common sole;[15,55,63,68] (CYP1A) activity in fish.[32,49−52] The occurrence of enzyme the fungicides propiconazole and ketoconazole in brown inhibition by some compounds coincides with the fact that trout and Atlantic cod;[69−70] β-naphthoflavone, dioxins several CYP1A protein inducers can inhibit EROD activ- and B[a]P in rainbow trout and carps;[71−73] dibenzofurans ity in liver microsomes from fish exposed to high doses in scups[74] or paraquat in Nile tilapia.[75] of xenobiotics, such as some PAHs (polycyclic aromatic With respect to the fish CYP3A enzymatic activity mea- hydrocarbons).[53−55] These protein inducers are also en- sured, only a few works have focused on this aspect. It zyme substrates and appear to be retained in microsomes has been suggested that CYP3A proteins are constitutively and to competitively inhibit the enzyme activity.[55−57] expressed in fish,[76] regulated during sexual maturation The P450-associated enzyme inhibition can also occur by (with males showing higher protein levels than females), mechanism-based inactivation; i.e., the inducer is metabo- and metabolizing endogenous substrates like testosterone lized by P450 into a product that covalently modifies the and progesterone.[64,77,78] Levels of a CYP3A-like protein active site and thereby inactivates the enzyme[50,57], not di- were significantly elevated in Atlantic eel from three mod- rectly suggesting a xenobiotic-induced cytotoxicity[58] but erately polluted sites when compared to control areas, but some kind of enzymatic inhibition. It must also be consid- since this protein was not induced in eel from the most pol- ered that CYP1A catalytic activity varies between differ- luted site its relevance as a biomarker remained unclear.[15] Downloaded By: [Pérez-López, Marcos] At: 12:57 25 June 2008 ent xenobiotics and studies: variables such as species, sex, This conclusion is in accordance with our results, indicat- reproductive stage, compound, route of exposure, time of ing that BFCOD enzymatic activity could be unsuitable exposure, mixed exposure and/or dose may influence the as a specific indicator of inhibition/induction of the cy- tochrome P450 system, due to the fact that during all of A research study developed with Atlantic eel (Anguilla the experiment it was not possible to identify any kind of anguilla) showed a significant decrease in hepatic EROD trend. The mechanism of induction of CYP3A is still un- activity after in vivo exposure to low doses (0.5 mg/L) known. Increased expression of CYP3A-like proteins has of TNT (2,4,6-trinitrotoluene), but curiously no further been observed in liver microsomes of rainbow trout treated decrease was observed when higher doses of this xenobi- with cortisol or pregnenolone-16α-carbonitrile.[76] How- otic were assayed.[60] A similar decrease in EROD activ- ever, dexamethasone, a potent inducer of CYP3A in most ity has been already reported in TNT-exposed rainbow mammals, had no effect on expression of this CYP3A-like trout (Onchorynchus mykiss), allowing us to hypothesize protein in rainbow trout[76] implying variations in CYP3A a potential role of TNT as a competitive inhibitor on regulation between different taxa. Hegelund et al.[79] found EROD enzyme activity.[61] Male sea bass showed a time- only slight induction of CYP3A protein levels in rainbow and concentration-dependent decrease of hepatic EROD trout exposed in vivo to the fungicide ketoconazole, but re- activity after intraperitoneal injection of 17β-estradiol, in lated to a 70% reduction of CYP3A activity at the lowest comparison to control values.[31] In Atlantic salmon (Salmo dose of fungicide tested (12 mg/kg body wt). It must be salar), treatment with nonylphenol (125 ppm) resulted in a signalled that the selectivity of ketoconazole for CYP3A reduction of both CYP1A protein expression and EROD inhibition varies between species, and for example in killi- activity,[62] and inhibition of P450 catalytic activity has been fish the xenobiotic appears to be a more selective inhibitor found after in vivo treatment of gilthead sea bream with towards this activity. Similarly, exposure of Atlantic cod to benzene.[63] In Atlantic salmon, a long-term exposure to different doses of 17β-estradiol showed only a weak inhibi- the endocrine disruptor ethynylestradiol (EE2) also pro- tion of CYP3A activity, whereas exposure to alkylphenols duced a time- and concentration-specific inhibition of hep- resulted in a dose-dependent elevation of this activity.[59] atic microsomal EROD activity,[64] in this case related to Exposure to ketoconazole, ethynylestradiol and nonylphe- the fact that CYP1A catalytic activity has been shown to nol resulted in decreased BFCOD activity in Atlantic cod decrease with increasing cellular estrogen levels in several when compared to control animals, even if no changes on protein levels of CYP3A were quantified.[70,80] On the other hand, Kaur and Sandhir[66] did not measure Lastly, according to UDPGT results, it must be sig- EROD activity, but they observed that cytochrome P450 nalled that phase II enzymes and cofactors in eel liver content increased in rat hepatic microsomes after acute and have been observed as being less susceptible to pollutants chronic carbofuran exposure, and Usmani et al.[47] demon- than phase I enzymes, but the activity of UDP glucuronyl strated that CYP4A4 was the major isoform responsible transferase (UDPGT) in eel could be, however, a useful Hepatic monooxygenase and enzymatic UDPGT biomarkers biomarker.[15] In mammals, UDPGT induction follows an with caution. Piscine organisms are exposed to pollutants Ah-receptor mediated mechanism.[81] This might also be mixtures, and therefore for a correct evaluation of the envi- valid in fish, since significant correlations were observed ronmental quality a battery of biomarkers should always be in treated animals between UDPGT and phase I-enzymes.
used[52] and caution is required when interpreting isolated Because of the abilities of both CYP1A and UDPGT to biomarker data.[22] The baseline data for the biomarker and biotransform the same classes of compounds, the expecta- all factors, both intrinsic and extrinsic, which affect the tion follows that exposure to chemicals that induce CYP1A biomarker, should be known in order to distinguish be- will also trigger UDPGT induction,[28] as the high signifi- tween natural variability (noise) and contaminant-induced cant correlations observed in the present study seem to in- dicate. Gravato and Santos[50] showed similar CYP1A and Moreover, long-term studies must be developed with UDPGT induction patters in sea bass (Dicentrarchus labrax those and other enzymatic activities, in order to evaluate L.) after laboratorial exposure to β-naphthoflavone. Some a broad spectrum of variables which can affect enzyme other laboratory studies have demonstrated the inductive response of UDPGT enzymes to pollutants: rainbow troutexposed to PCBS or bleached kraft mill effluent[82−83]; chan- Acknowledgments
nel fish and flounder exposed to PCBs,[84,85] sea bass ex-posed to β-naphthoflavone[86] or eel exposed to dinitro-o- Authors wish to thank Junta de Extremadura, which finan- cresol.[87] Plaice injected with 3-methylcolanthrene (3-MC) cially supported the present study (2PR04A021).
or Aroclor 1254, hence a prototypical polyaromatic com-pound and a commercial mixture of PCBs, showed, sixdays after treatment, a 1.5–2 fold induction of UDPGT References
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after exposure to β-naphthoflavone.[91] Conversely, a recent [2] Bretaud, S.; Toutant, J.P.; Saglio, P. Effects of carbofuran, diuron, study developed with the antioxidant ethoxyquin and At- and nicosulfuron on acetylcholinesterase activity in goldfish (Caras- lantic salmon, has shown, however, an inhibition of hepatic sius auratus). Ecotoxicol. Environ. Saf. 2000, 47, 117–124.
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