Metabolic responses of channel cat fi sh ( Ictalurus punctatus ) exposed to phenol and post-exposure recovery

Metabolic adjustments were studied in channel catfi sh Ictalurus punctatus exposed to 1.5 mg L of phenol (10% LC50) for four days and recovered for seven days. Lower triacylglycerol (TGA) stores and increased muscle fat free acids (FFA) suggest fat catabolism in muscle. Remarkable liver FFA decrease (-31%) suggests liver fat catabolism as well. Increased muscular ammonia levels and ASAT (aspartate aminotransferase) and decreased plasma aminoacids suggest higher muscular amino acid uptake. Constant levels of glucose and increased liver glycogen stores, associated with lower amino acids in plasma, indicate gluconeogenesis from amino acids. This is supported by higher hepatic ALAT and ASAT. Higher hepatic LDH followed by lower plasma lactate may indicate that plasma lactate was also used as gluconeogenic substrate. Biochemical alterations were exacerbated during the post-exposure recovery period. Reduction in muscle and plasma protein content indicate proteolysis. A higher rate of liver fat catabolism was resulted from a remarkable decrease in hepatic TGA (-58%). Catabolic preference for lipids was observed in order to supply such elevated energy demand. This study is the fi rst insight about the metabolic profi le of I. punctatus to cope with phenol plus its ability to recover, bringing attention to the biological consequences of environmental contamination.


INTRODUCTION
Phenols and their derivatives are aromatic, organic compounds present in a wide variety of biotic and abiotic factors, and under numerous circumstances, they can be considered pollutants.Domestic and industrial effl uents are the major source of such pollutants in aquatic environments, causing damages at several levels of biological organization (Moens et al. 2007).Despite the maximum limit for the concentration of phenol in treated effl uents (0.5 mg L -1 ) and freshwater (0.003 -1 mg L -1 ) be restrictive in Brazil (Brasil 2005, Cetesb 2014a), phenol contamination in water basins is often from either industrial wastewaters or accidental phenol discharges.Total phenols concentrations are above the allowed limit in some Brazilian freshwater and coastal water points (Cetesb 2014b).Moreover, phenols concentrations raging from 0.37 to 3.92 mg L -1 are reported in the Brazilian rivers (Possa FERNANDA D. MORAES et al. et al. 2008, Watthier et al. 2008, Porto and Ethur 2009).When toxic substances reach the fi sh, it can be absorbed through the skin, gills, intestine and mucous membranes.Into bloodstream, it spreads out to body compartments and causes several physiological disturbances as hematological and metabolic disorders (Hori et al. 2006, Avilez et al. 2008, Moraes et al. 2015).Depending on the environmental concentration, phenol can lead to death to fi shes (Gupta et al. 1982, Saha et al. 1999, Moraes et al. 2015).Multiple mechanism of action of phenol or some phenoli c compounds have been reported, including: drug antagonists (Roche and Bogé 2000), induction of genotoxic ity (Bolognesi et al. 2006), carcinogenesis and mutagenesis (Tsutsui et al. 1997, Yin et al. 2006), endocrine disruption (Kumar and Mukherjee 1988), and metabolic disruption (Hori et al. 2006).
The evaluation of metabolic parameters has currently been used as biomarkers of environmental contamination by phenol (Hori et al. 2006, Abdel-Hameid 2007, Avilez et al. 2008, Varadarajan et al. 2014), pesticides (Venkateswara Rao 2006, Aguiar et al. 2004, Sancho et al. 2009, Venturini et al. 2015) and metals (Pretto et al. 2014).Evaluation of metabolic profi le can help to elucidate how metabolic disruption caused by xenobiotics may affect fi sh physiology and behavioral state.(Oost et al. 2003, Scott and Sloman 2004, Schlenk et al. 2008).The metabolism of protein in fi shes can be altered by phenol.Adjustments in the activities of alanine (ALAT) and aspartate (ASAT) aminotransferases and changes in tissular protein concentration are reported in fi sh exposed to phenol (Barse et al. 2006, Dangé 1986b, Gupta et al. 1983, Hori et al. 2006, Sannadurgappa et al. 2007).Phenolic compounds affect the lactate dehydrogenase (LDH) activity, altering the carbohydrate metabolism through the inter-conversion of lactatepyruvate (Barse et al. 2006, Dangé 1986a, Gupta et al. 1983, Hori et al. 2006, Sannadurgappa et al. 2007).Adjustments in the lipid metabolism are also observed in Oreochromis mossambicus exposed to phenol ( Sannadurgappa et al. 2007).
Biological effects of phenol are relevant, particularly when several derivatives of it should present similar effects.This is particularly relevant to fi sh, which is able to uptake and retain different xenobiotic dissolved in water via active or passive processes (Fernández-Vega et al. 2015).Channel catfi sh Ictalurus punctactus is a freshwater fi sh responsive to phenol contamination (Moraes et al. 2015).At the present days, it is distributed in several countries, including Brazil (FAO 2015), and it is easily maintained in the lab conditions.Besides, I. punctatus have large amount of fat, which is subject to phenols bioaccumulation, as reported to the catfi sh Clarias gariepinus, and Carassius carassius exposed to phenol and effl uents, respectively (Ibrahem 2012, Zheng et al. 2015).
Even at low levels, environmental contamination may result in no perceptible, immediate or acute consequences.Sublethal concentrations (as 10% of LC50) can be explored through biochemical biomarkers to predict impairment at the high levels of organization.It is assumed that risks can be mitigated when contaminants are applied to aquatic environments at concentrations 10 -50% of LC50 (Touart 1995).In addition, studies on the post-exposure recovery period (Aguiar et al. 2004, Begum 2004, Sancho et al. 2009, Pretto et al. 2014, Venturini et al. 2015) can elucidate the ability of fi sh to reestablish the homeostasis, when the stressor is discontinued.
Once a toxicant enters an organism, several biochemical and physiological responses occur which may be adaptive or may lead to toxicity (Begum 2004).The biochemical responses of organisms are the most sensitive and relatively early events of pollutant damages and can be accessed into few hours; besides, biochemical alterations could be part of mitigating effects from toxicant presence (Pickering and Pottinger 1995, Begum 2004, Schlenk et al. 2008).Among the many biochemical responses, those observed at metabolic level are particularly relevant since energy is partially and insidiously shifted to attend the detoxifi cation processes.No data are available concerning the effects of phenol on metabolic activity of I. punctatus.Therefore, the evaluation of intermediary metabolism of I. punctatus exposed to phenol plus its ability to recover was the fi rst insight into the metabolic adaptability of the species to cope with this kind of stressor, allowing us to infer on the magnitude of the metabolic expenditures and losses.

CHEMICALS
Phenol was purchased from Sigma-Aldrich and it had previously been dried into a vacuum desiccator at room temperature in the dark.All chemicals and reagents were purchased from Sigma-Aldrich, Merck or Mallinckrodt.

FISH MAINTENANCE AND EXPERIMENTAL DESIGN
The fi sh I. punctatus were obtained from a local commercial fi sh farm and, acclimated for 15 days in a water fl ow-through system of 2000 L fi ber tanks at folowing: temperature 25-27 o C; pH 7.0-7.2;[NH 3 -NH 4 + ] 0.4-0.6 mg L -1 ; and dissolved oxygen 5.0-6.0 mg L -1 .The fi sh samples (37-48 g; 15.3-17.9cm) were fed twice a day with commercial pellets until satiety.After acclimation, 48 fi sh were transferred to the experimental system wherein they equally distributed into eight 250L-tanks (n=6 fi sh/ tank) with the same water quality of a new fl owthrough system and fed the same regimen.The fi sh were kept undisturbed in this new condition for seven days and the feeding was discontinued 24 hours before starting the phenol exposure to prevent fecal debris deposition over the assay.The experiment was carried out in duplicates in a semi-static system.Afterward, the water fl ow was ceased and phenol was added to four tanks to fi nal concentration of 1.5 mg L -1 (10% of 96 hour LC50 for I. punctatus, Moraes et al. 2015).The four tanks remaining were kept with clean water and assigned as control.Over the exposure period, water was renewed each 24 hours, the phenol concentration was readjusted, and the water quality was preserved.This procedure was done in nearly 20 minutes and the whole semi-static exposure lasted four days.At the fourth day, four tanks (two exposures and two controls) were randomly pointed and the fi sh were netted to sample tissues and blood.In the other four tanks the water was kindly renewed to keep the fi sh undisturbed and the tanks returned to the same initial fl ow-through system for seven more days (post-exposure recovery).Ended this period, the fi sh were equally netted for blood and tissue sampling.

BLOOD AND TISSUE SAMPLING
At the ending of each experimental spans (four days of exposure or seven days of recovery), the fi sh were dived into 40 mg L -1 eugenol solution for one minute (Inoue et al. 2003), blood was withdrawn from the caudal vein with heparinized sy ringes and t hen the fi sh were immediately killed by transecting the spinal cord.Liver and white muscle samples were quickly excised over a chilly glass plate, rinsed with cold saline solution and quickly plunged in liquid nitrogen.Total blood was centrifuged for three minutes at 13,400 x g and the pellets were discarded.Plasma and tissues were kept at -80 °C until analyses.

ENZYME ASSAYS
White muscle and liver samples were homogenized at 4 o C in a buffer containing 50% 10 mM phosphate buffer pH 7.0 plus 50% glycerol at a ratio 1:10 (w/w).For enzyzme extraction, a rotative highspeed homogenizer was used for tissue disruption.To cool the sample down to 4 o C tubes were place in ice bath.The tissue homogenates were fi rstly centrifuged at 600  g for three minutes at 4 °C FERNANDA D. MORAES et al.
and secondly the supernatants were centrifuged at 6,000  g for eigth minutes at 4 °C.The protein concentrations were determined in the supernatants which were used as crude enzyme source.White muscle and liver ALAT (alanine aminotransferase) and ASAT (aspartate aminotransferase) were assayed kinetically (Bergmeyer et al. 1978); LDH (lactate dehydrogenase) and MDH (malate dehydrogenase) were used as auxiliary enzyme for ALAT and ASAT assays, respectively.The fi nal concentration of the reaction mixture was 500 mM alanine (ALAT) or 220 mM aspartate (ASAT), 10 mM 2-ketoglutaric acid and liver/muscle homogenate adjusted to 0.04/0.40mg of protein to a fi nal volume of 1.0 mL with 100 mM Tris buffer, pH 7.5.All the reagents were previously diluted into 100 mM Tris buffer, pH 7.5.The reaction was followed by NADH + H + extinction, read at 340nm and expressed in mIU per mg of protein.One IU corresponded to 1.0 μmol of NADH oxidized per minute (ε o = 6.22 mM -1 .cm - ).Enzyme activity of LDH (lactate dehydrogenase) was assayed according to Hochachka et al. (1978).The tissue homogenates were prepared as described above.The LDH reaction was carried out in a medium containing 5.0mM pyruvic acid, 0.1mM NADH, 42.5 mM TRIS buffer pH 7.5, and liver/muscle homogenate adjusted to 0.02/0.10mg of protein to a final volume of 1.0 mL.The reaction was followed by NADH + H + extinction, read at 340nm and expressed in mIU per mg of protein.One IU corresponded to 1.0 μmol of NADH hydrolyzed per minute (ε o = 6.22 mM -1 .cm - ).Liver and white muscle activities of ACP (acid phosphatase) and ALP (alkaline phosphatase) were assayed by endpoint method read at 405nm (Bergmeyer 1986).The reaction was based in hydrolyses of 120 mM p-nitrophenyl phosphate, in 50mM sodium citrate buffer pH 5.0 (ACP) or 50mM glycine buffer pH 8.5 plus 10mM MgCl 2 (ALP) and liver/muscle homogenate adjusted to 0.04/0.40mg of protein to a fi nal volume of 1.0 mL.The reaction was stopped by 2M NaOH addition and the p-nitrophenolate was optically determined at 405nm and expressed in mIU per mg of protein.One IU corresponded to 1.0 μmol of hydrolyzed p-nitrophenyl phosphate hydrolyzed per minute (ε o = 18,200 mM -1 .cm - ) METABOLITES Metabolites were quantified in liver and white muscle acidic homogenates prepared in 20% trichloroacetic acid (TCA) or neutral homogenates made in distilled water.Plasma was only diluted in 20% TCA or water before using.Homogenates were performed in a rotative high-speed tissue disruptor with three strokes of 20 seconds under ice bath.The homogenates were centrifuged for three minutes at 21,000 x g and the supernatant extracts were used.Ammonia (Gentzkow and Masen 1942), lactate (Harrower and Brown 1972) and pyruvate (Lu 1939) were photometrically quantifi ed in acid extracts.Free amino acids (Copley 1941), glucose (Bergmeyer 1986), free fatty acids (Novák 1965) and triacylglycerol (Stein and Myers 1995) were photometrically determined in extracts at neutral pH.Hepatic and muscular glycogen content was determined by the method of Bidinotto et al. (1997).The tissues were solubilized with 6.0N KOH in a boiling water bath for fi ve minutes, and the glycogen was precipitated by ethanol and K 2 SO 4 saturated solution.After centrifugation at 2000 x g for three minutes, the pellets were resuspended in distilled water and the glycogen was quantifi ed as glucosyl-glucose by phenol-sulfuric acid (Dubois et al. 1956).

PROTEIN
The homogenate and plasma protein concentrations were determined with Bradford reagent against bovine albumin solution as standard (Kruger 1994).

STATISTICAL APPROACH
The biochemical parameters from each group are presented as mean ± SE.Groups (including controls) were tested for homogeneity concerning the respective replicates.The data were analyzed concerning the groups normality by Kolmogorov-Smirnov test and the differences among means were evaluated by Student's t-test at the level of P < 0.05.Each treatment (exposed to phenol and post-exposure recovery) was evaluated against the respective control group.

ETHICS
The conditions and experimental procedures of this study were approved by Ethic Committee for Animal Research of Federal University of São Carlos (CEEA n o 039).

RESULTS
I. punctatus exposed to phenol did not exhibit any evident signs of poisoning.The metabolic profi le of I. punctatus depicted relevant tissular and plasmatic adjustments (Table I).All differences described in this section were statistical signifi cant.The liver glycogen stores enhanced after exposure, however, in white muscle it was not mobilized.The exposure to phenol caused a sinking of plasmatic concentrations of amino acids, protein, free fatty acids (FFA), triacylglycerol (TAG) and lactate.After the post-exposure recovery period, plasma lactate concentration increased, while protein concentration decreased.The levels of glucose and TAG were fallen in the muscle after exposure, and pyruvate and protein just after the post-exposure recovery period.However, the levels of muscle ammonia and FFA were heightened, and this one was kept high even after post-exposure recovery period.The relevant changes in the liver were observed after the post-exposure recovery period.Except by decrease of the liver FFA levels after exposure, those of glucose and ammonia were increased after post-exposure recovery while the TAG level was dropped.The activities of the enzymes assayed depicted changes either after the exposure or the post-exposure recovery period (Table II).In the liver, the enzymatic activity of ALAT, ASAT, LDH and ALP were increased; ACP activity increased only after post-exposure recovery period.In white muscle, ACP activity was reduced after exposure.The activity of the ALAT was increased in white muscle after post-exposure recovery.μ) or mg (mg) per g of tissue or per mL of plasma.C-4 control of exposure for 4 days; P-4 exposed for 4 days; C-7 control of recovery; P-7 recovered for 7days; L = liver; Wm = white muscle; P = plasma; FFA = free-fatty acids; TAG = triacylglycerol; Values are followed by mean ± SE (*) difference between the exposed group and the corresponding control is signifi cant at P<0.05 (n=12).The specific activities of: ALAT (alanine aminotransferase), ASAT (aspartate aminotransferase), ACP (acid phosphatase), ALP (alkaline phosphatase) and LDH (lactate dehydrogenase) are expressed in mIU (mg of protein) -1 .L = liver; Wm = white muscle.C-4 control of exposure for 4 days; P-4 exposed for 4 days; C-7 control of recovery; P-7 recovered for 7days.The values are followed by mean ± SE; (*) difference between the exposed group and the corresponding control is signifi cant at P<0.05 (n=12).

DISCUSSION
The intermediary metabolism of I. punctatus was altered due to phenol exposure.A switch from anabolic to catabolic processes is commonly observed in stressed fi sh.It involves major changes in intermediary metabolism and may have important consequences for somatic growth (Pickering and Pottinger 1995).The energy demanded to cope with poisoning was clearly observed in white muscle of I. punctatus.Increase in the glycolytic activity in white muscle of I. punctatus could be inferred by the presence of less glucose.This seems to be a general response of fi sh exposed to phenol (Hori et al. 2006, Abdel-Hameid 2007), and such decrease was to attend energetic expenditures caused by phenol.In this particular, a glycogen synthesis, to justify the glucose decrease, was not carried out since no glycogen increase was observed in the white muscle.It is expected over the muscular fermentative processes an increase of lactate concentration and LDH activity.Then, invariable activity of LDH enzyme and steady concentrations of lactate suggest that glucose oxidation has been the metabolic preference of I. punctatus.Although glycogen can provide the immediate energy requirements of fi sh under stress (Fernández-Vega et al. 2015), it was observed the maintenance of the glycogen bulks in the muscle of I. punctatus, as also reported to O. mossambicus (Dangé 1986a) andB. amazonicus (Hori et al. 2006) exposed to phenol.This metabolic scenario points out an exogenous source of glucose to the white muscle of I. punctatus.Therefore, the metabolic preference of white muscle in I. punctatus seemed to be oxidative, in which glucose was certainly fundamental to the mitochondrial metabolic pathways, and lipid catabolism.
High level of fat catabolism have decreased the triacylglycerol stores (17%) followed by an increase of FFA in white muscle of I. punctatus.Lipids are the major energy reserves in fi sh and their mobilization suggest high energy demands state (Fernández-Vega et al. 2015).Catabolism of such amount of lipid in I. punctatus refl ects a higher demand for ATP as a consequence of elevated stress and detoxifi cation process.Lipid catabolism is also observed in O. mossambicus exposed to phenol for 30 days (Sannadurgappa et al. 2007).However, not only glucose and lipids were required to cope with phenol poisoning.The increase in the white muscle ammonia in I. punctatus was a consequence of an increase in the amino acids catabolism.The activity increase of white muscle ASAT corroborates such assumption since this enzyme provides oxaloacetate to the oxidative metabolism.In spite of the amino acids steadystate, which is usual among tissues (Houlihan et al. 1995), the plasma amino acids decreased in I. punctatus.Decrease of plasma amino acids and increase of ammonia in the muscle indicate amino acids catabolism in the muscle.Alterations on muscle protein metabolism is clearly reported in catfi sh Rhamdia quelen exposed to cadmium for 7 days, whereas protein concentration decreased, and amino acids and ammonia concentrations increased (Pretto et al. 2014).
The exposure to phenol was likely effective in expending metabolic energy to detoxifi cation processes in the liver.The increase of liver glycogen in fi sh exposed to phenol demands glucose input.Considering that fi sh were kept starved the only source of glucose backbones should be amino acids.In I. punctatus, constant levels of liver glucose and decrease of plasma amino acids are suggestive that gluconeogenesis has occurred from free amino acids supplied by peripheral tissues.This assumption is strengthened by the increase of hepatic ALAT and ASAT.Such increases of ALAT and ASAT are usual biomarkers in fi sh exposed to xenobiotics (Gupta et al. 1983, Dangé 1986b, Begum 2004, Sancho et al. 2009).Enhanced activity of aminotransferases can be providing the oxaloacetate, pyruvate, α-ketoglutarate and glutaric FERNANDA D. MORAES et al. acid due to increase of energy demand imposed by stressing conditions (Begum 2004).The oxaloacetic acid and α-ketoglutarate might have been channeled into the Krebs's cycle, as proposed to C. batrachus exposed to carbofuran, which presents ALAT and ASAT induction in the liver (Begum 2004).The increase of hepatic LDH of I. punctatus would also support gluconeogenesis from lactate in the liver cells, as reported in Brycon amazonicus and Oreochromis aureus exposed to phenol (Abdel-Hameid 2007, Hori et al 2006).Increased hepatic LDH and ALAT activity are also reported to O. mossambicus exposed to 10% LC50 of phenol (Varadarajan et al. 2014).The rise in muscular ALAT activity of Cyprinus carpio exposed to 4-tert-butylphenol suggests amino acid catabolism to support the hepatic gluconeogenesis (Barse et al. 2006).Therefore, glucose synthesized de novo in the liver of I. punctatus should be responsible for maintaining the ob served normoglycemia, and th is profile was enhanced in the post-exposure recovery period.Normoglycemia accompanied by hepatic gluconeogenesis is also reported in silver catfi sh Rhamdia quelen exposed to cadmium for 7 days (Pretto et al. 2014).Significant addition of metabolic energy was required to I. punctatus carried out phenol detoxification processes, which resorted to lipid catabolism in the liver to supply such demand.This explains the remarkable decrease (31%) of the liver content of FFA.
In the course of the post-exposure recovery period, the biochemical alterations were exacerbated.The teleost Piaractus mesopotamicus exposed to 10% LC50 of trichlorfon also presented metabolic alterations over the post-exposure recovery period (Venturini et al. 2015).In I. punctatus, reduction in the muscle protein content was observed, indicating that proteolysis was necessary in the recovery period.R. quelen recovered for 7 days to cadmium exposure also presented muscle proteolysis (Pretto et al. 2014).Decrease of pyruvate concentration and increase of ALAT activity in the white muscle are suggestive that alanine was synthesized and likely exported to the liver, performing the alanine cycle.The increased glucose concentration in the liver of I. punctatus indicates that rate of liver gluconeogenesis was maintained, and this fact was provided by amino acids backbones.Increased liver ammonia of I. punctatus indicates catabolism of amino acids, as also reported to the catfi shes Clarias batrachus and R. quelen after post-exposure recovery of carbofuran and cadmium, respectively (Begum 2004, Pretto et al. 2014).High rates of fat catabolism were active in liver of I. punctatus and it is resulted from the remarkable decrease (58%) of liver TGA.Thus, depletion of energy stores, such as muscular proteolysis and hepatic lipolysis, was observed at the end of the recovery post-exposure period in the I. punctatus, indicating metabolic high cost of compensatory responses of stressed fi sh, as early proposed to Anguilla anguilla exposed to propanil (Fernández-Vega et al. 2015).
Proteolytic activities can be resultant from catabolic processes induced by higher metabolic demand and cleaning of dead cells or components of the poisoned cells.Consequently, increase of hydrolytic enzymes from lysosomes, such as ACP, or bounded to cell membrane, such as ALP, can be observed in plasma.Structural damages followed by leaking of cell components reduce the content of enzymes in poisoned cells.Hepatocytes of rainbow trout Oncorhynchus mykiss exposed to alkyphenols for 96 hours lost the cell membrane integrity (Tollefsen et al. 2007).The increase of plasmatic ALP and ACP reported in the African catfi sh Clarias gariepinus exposed to the phenyl derivative λ-cyhalohtrin and in the O. mossambicus exposed to phenol is attributed to a leakage of hepatic of enzymes into the blood (Okechukwu andAuta 2007, Varadarajan et al. 2014).The profi le of phosphatases activity observed in I. punctatus was likely due a leakage of muscle ACP in consequence of intoxication.The levels of hepatic ALP were raised likely to provide phosphate radicals to metabolic processes of detoxifi cation, as already reported to C. carpio exposed to terc-buthylphenol (Barse et al. 2006).
In summary, the results indicate that I. punctatus is adversely affected by sublethal concentration of phenol.The increased rates of carbohydrates, proteins and fat oxidation were to at tend the metabolic demand, resultant from stress and detoxification processes.This fact is usually accepted in other fi shes exposed to a number of xenobiotics (Scott andSloman 2004, Fernández-Vega et al. 2015).Since a long time, the preference for other fuels rather than carbohydrates is widely accepted in fi shes (Thi llart and Raaij 1995).I. punctatus presented a catabolic preference for lipids to cope with phenol exposure in the liver.Proteins were hydrolyzed in white muscle as a secondary metabolic source of energy.The energy level expended by I. punctatus exposed to sublethal concentration of phenol was signifi cant enough to bring attention to the resultant biological consequences.Even after post-exposure recovery period, metabolic alterations were maintained, indicating that compensation or adaptation was not achieved by I. punctatus.The injuries caused by sublethal concentration of phenol could have ecological implications.The metabolic losses due to phenol contamination can be refl ected in impairments on growth, disease resistance and reproduction.