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Hematological responses of the Neotropical teleost matrinxã (Brycon cephalus) to environmental nitrite
Comparative Biochemistry and Physiology, Part C 139 (2004) 135 – 139 www.elsevier.com/locate/cbpc
Hematological responses of the Neotropical teleost matrinxa˜ (Brycon cephalus) to environmental nitrite Ive M. Avilez, Alexandre E. Altran, Lu´cia H. Aguiar, Gilberto Moraes* Departament of Genetics and Evolution, Federal University of Sa˜o Carlos, Rod Washington Luı´s, km 235, Sa˜o Carlos, SP CEP 13565-905, Brazil Received 21 May 2004; received in revised form 1 October 2004; accepted 3 October 2004
Abstract Environmental increase in nitrite impairs the function of several aquatic species, including fishes. Nitrite reacts with hemoglobin yielding the non-functional methemoglobin (metHb), and many physiological disturbances can arise. The physiological mechanisms to cope with nitrite are still unclear in fish. Hematological parameters, the role of NADH–methemoglobin reductase system and the electrolytic balance were studied in the freshwater teleost Brycon cephalus (matrinxa˜) exposed to 0.2, 0.4 and 0.6 mg/L of nitrite N–NO2 for 24 and 96 h. Hematocrit, total hemoglobin and the red blood cell (RBC) number decreased. Methemoglobin content increased from 1% to 69% for 24 h of exposure and drastically from 5–6% to 90% for 96 h. The activity of NADH–methemoglobin reductase system displayed a tendency of increase in response to nitrite concentration or time of exposure. In the plasma, nitrite was accumulated to values 30-fold higher than the environmental concentration. The plasma K+ concentration increased only in fish exposed to NO2 for 24 h. No changes in plasma protein and Na+ were observed during nitrite exposure but Cl-presented a punctual increase at 0.2 mg/L N–NO2–96 h. The hematological data suggest that nitrite caused functional and hemolytic anemia. Furthermore, the electrolytic balance was relatively undisturbed, and the nitrite clearance in matrinxa˜ is likely depending on other factors than NADH–methemoglobin reductase system. D 2004 Elsevier Inc. All rights reserved. Keywords: Brycon cephalus; Fish; Functional and hemolytic anemia; Hematology; Matrinxa˜; Methemoglobin; NADH–methemoglobin reductase system; Nitrite
1. Introduction Water pollution and industrial waste usually elevate the nitrite concentrations (Nikinmaa, 1992; Heckman et al., 1997) due to ammonia oxidation. This nitrification process depends on the bacterial activity and the oxygen level. High ammonia concentrations can be observed in aquaculture systems as a consequence of high stocking densities (Hargreaves, 1998; Hagopian and Riley, 1998). Nitrite from such conditions causes many physiological problems for reared fish. Fish has been proposed as a good animal model for disclosing the physiology that underlies the toxicology of nitrite (Jensen, 2003). Environmental nitrite crosses the gill * Corresponding author. Tel.: +55 16 260 8376; fax: +55 16 260 8377. E-mail address: [email protected] (G. Moraes). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.10.001
barrier by competing with chloride for chloride uptake sites (Gaino et al., 1984; Williams and Eddy, 1986) and is accumulated in the plasma (Shechter et al., 1972; Bath and Eddy, 1980). Thus, plasma nitrite concentrations are usually higher than environmental ones (Eddy and Willians, 1987); from the plasma, nitrite subsequently enters tissue cells (Arillo et al., 1984), and within the red blood cells (RBCs) it oxidizes hemoglobin Fe2+ to Fe3+ yielding methemoglobin (metHb), which is unable to transport oxygen (functional anemia) (Scarano and Saroglia, 1984). This effect is supposed to result in tissue hypoxia (Cameron, 1971; Bath and Eddy, 1980; Doblander and Lackner, 1996; Vedel et al., 1998) even in the presence of environmental oxygen (functional hypoxia). Methemoglobin formation in RBCs is easier as hemoglobin is in the non-oxygenated form (Jensen, 1990, 1992; Willians et al., 1993) and at low pH values (Jensen, 2003). Its content varies among fish and
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depends on factors such as the external nitrite concentration and the time of exposure. Nitrite causes other blood disturbances such as decrease of total hemoglobin, hematocrit and red cell counts. This phenomenon causes hemolytic anemia (Scarano and Saroglia, 1984). Several osmoregulatory responses including hyponatremia, hypochloremia (Jensen et al., 1987), branchial chloride cells failure (Gaino et al., 1984) and inhibition of chloride uptake (Williams and Eddy, 1986) are observed in freshwater teleosts exposed to nitrite. Recovery from effects of nitrite has been reported in nitrite-free water (Schoore et al., 1995) and a couple of ways involved in such processes are proposed. The first one involves the NADH–methemoglobin reductase system (Diaphorase I), which reduces hemoglobin Fe3+ to Fe2+ (Huey and Beitinger, 1981; Freeman et al., 1983; Scott and Harrington, 1985; Woo and Chiu, 1997). Nitrate synthesis is another way proposed as mechanism to detoxify nitrite (Doblander and Lackner, 1997). Catalase and cytochrome oxidase-aa3 are proposed to take role in that process (Doblander and Lackner, 1996); however, both mechanisms are still unclear. In the present study, hematological changes and osmoregulatory responses, in the freshwater Neotropical teleost Brycon cephalus (Gqnther, 1869) (matrinxa˜) exposed to environmental nitrite, were investigated. Few data concerning the effect of nitrite exposure on the induction of NADH– methemoglobin reductase system are now available. The most are just regarded to its presence in fish (Huey and Beitinger, 1981; Freeman et al., 1983; Scott and Harrington, 1985). Matrinxa˜ is widely reared in South America and is a low-tolerant species to environmental nitrite (LC50 0.86 mg/L N–NO2–96 h; Avilez et al., 2004). Therefore, the methemoglobin formation and the role of NADH– methemoglobin reductase system were also examined.
(n=6 in each tank). The fish were allowed to rest for 24 h, and 0.2, 0.4 or 0.6 mg/L (sublethal concentrations) of sodium nitrite N–NO2 were added to experimental tanks except the control. The nitrite exposure remained for 24 h. At the same time, another experiment was carried out under the same conditions for 96 h. Both experiments were carried out in semistatic system. After the exposure to nitrite, the fish were collected, anesthetized with MS 222 and a blood sample of 2 ml was drawn from the caudal vein into a heparinized syringe. 2.3. Hematological parameters Hematocrit (Hct) was determined on blood samples centrifuged at 12,000g for 3 min in capillary tubes. Total hemoglobin (Total Hb) was determined spectrophotometrically on 10 AL of blood in 2.0 mL of Drabkin solution at 540 nm. Methemoglobin (metHb) was colorimetrically quantified on 6 AL of whole blood at 563 nm as described by Matsuoka (1997). Red blood cell (RBC) (NRBC, ml 1) was counted under a light microscope with a Neubauer chamber. Mean corpuscular volume (MCV) was calculated from Hctd 10/NRBC, the mean corpuscular hemoglobin (MCH) content from Total Hbd 10/NRBC and the mean corpuscular hemoglobin concentration (MCHC) from Total Hbd 10/Hct. A blood aliquot was centrifuged at 12,000g for 3 min. The plasma was used for flame photometric determinations of Na+ and K+ on a Digimed DM-61, and optical determinations of Cl at 480 nm (APHA, 1980) and NO2 at 520 nm (Shechter et al., 1972). Total protein was determined by a UV method at 215 and 225 nm (Vilella et al., 1972). 2.4. NADH–methemoglobin reductase system activity
Juveniles of B. cephalus had a mean weight of 90F5 g ´ guas Claras, (FS.D.) and were obtained from the fish farm A Moco´ca, SP, Brazil. The fish were brought to the lab aquaculture facilities, acclimated for 2 months, fed commercial food and exposed to natural photoperiod, controlled temperature (25F1 8C) and aerated water. The water quality parameters in the experiment were: oxygen content, 7.5 mg/ L; pH, 6.8F0.2; temperature, 24F1 8C; conductivity, 74.3 AS cm 1; total alkalinity, 37 mg/L as CaCO3; hardness, 28 mg/L as CaCO3, ammonia concentration, 0.01 mg/L; chloride concentration, 0.294 mg/L; and nitrite concentration, 0 mg/L.
Blood samples were centrifuged and the packed red cells were washed at least three times with saline solution (0.9%) by centrifugation at 1000g for 10 min. White cells were removed by suction of the upper layer. Red cells were resuspended into 0.04 mL of mercaptoethanol–EDTA solution and the erythrocytes were lysed by thermal shock in a liquid nitrogen bath (Beutler, 1984). The resulting hemolysate was used as enzyme source. The enzyme assay was performed in a 0.2 M Tris–HCl pH 7.5 buffer solution containing 1.2 mM 2,6-dichlorophenol indophenol, 6 mM NADH, and a suitable enzyme aliquot. The substrate consumption was optically followed at 600 nm, and one enzyme activity unit equals a decrease in 1.0 absorbance unity per min, at 25 8C (Worthington Biochemical, 1972). The specific activity isexpressed in U (units) per mg of hemoglobin (U/mg total Hb).
2.2. Experimental design
2.5. Statistics
Twenty-four fish, starved for 1 day, were equally distributed in four tanks with 250 L of nitrite-free water
STATISTICA 5.5 was used to compare data. Normality of the data was evaluated by the Shapiro–Will W test with a 95%
2. Materials and methods 2.1. Fish
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Fig. 1. Methemoglobin concentration in B. cephalus exposed to environmental nitrite for 24 and 96 h. The values are expressed as mean (FS.E.M.); (*) significantly different from control group at Pb0.05.
confidence limit. The parametric test ANOVA was used to compare the groups, the Post-Test Duncan for multiple comparisons was applied, and the two-way ANOVAwas used to compare NADH MetHb reductase between 24 and 96 h. Pb0.05 was considered statistically significant.
3. Results The blood parameters of matrinxa˜ were affected by every nitrite level tested. The methemoglobin content increased markedly (Fig. 1), and the hematocrit, total hemoglobin and red blood cell number decreased (Table 1) except the group exposed to 0.2 mg/L for 24 h. The MCHC was elevated in fish exposed to 0.4 mg/L for 24 h, but decreased in fish exposed to nitrite for 96 h. No significant changes were observed in the MCV and MCH (Table 1). The NADH
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Fig. 2. Plasma nitrite concentration in B. cephalus exposed to environmental nitrite for 24 and 96 h. The values are expressed as mean (FS.E.M.); (*) significantly different from control group at Pb0.05.
methemoglobin reductase system was detected in the red blood cells of matrinxa˜, and the activity was affected by external nitrite concentrations or time of exposure (two-way ANOVA; Table 1). Plasma nitrite concentration increased as function of external nitrite (Fig. 2). The maximal value reached was 2.1 mM, 30-fold higher than the environmental concentration. Plasma potassium was elevated in the 24 h at all nitrite levels treatment, but was as in controls after 96 h. Plasma chloride increased in fish exposed to 0.2 mg/L for 96 h, whereas no changes were observed in the plasma protein or Na+ (Table 2).
4. Discussion The most prominent effects in fish exposed to nitrite are the increases in methemoglobin content and the plasma nitrite
Table 1 Hematological parameters of Brycon cephalus exposed to nitrite for 24 and 96 h Time
Parameters
Nitrite concentration (mg/L)
Total blood
0
0.2
0.4
0.6
24 h
Hct (%) Total Hb (g dL 1) RBC (106 cells mm3) MCV (fl) MCH (pg) MCHC (g dL 1) NADH–MetHb reductasea Hct (%) Total Hb (g dL 1) RBC (106 cells mm 3) MCV (fl) MCH (pg) MCHC (g dL 1) NADH–MetHb reductasea
33.5F0.85 9.54F0.33 2.45F0.31 150.7F23.1 42.68F6.28 2.85F0.11 0.09F0.0071 34.6F1.33 10.27F0.56 3.17F0.37 115.0F10.1 33.94F2.88 2.95F0.06 0.22F0.0271
28.0F0.73* 7.03F0.36* 2.26F0.15 127.3F9.6 31.37F1.14 2.51F0.15 0.10F0.0161 28.6F0.66* 7.60F0.43* 2.39F0.13* 120.9F4.9 31.93F1.72 2.64F0.11* 0.33F0.0632
22.9F0.76* 8.2F0.16* 1.51F0.13* 158.6F18.0 56.31F4.81 3.61F0.12* 0.08F0.0101 25.3F0.76* 6.58F0.21* 1.85F0.10* 137.9F5.6 35.86F1.56 2.60F0.05* 0.34F0.0342
23.5F1.23* 7.15F0.59* 1.64F0.22* 152.3F14.5 46.14F5.32 3.02F0.14 0.14F0.0241 23.1F1.28* 5.94F0.51* 1.79F0.11* 130.0F6.5 33.49F3.08 2.55F0.12* 0.31F0.0282
96 h
Values are expressed as means (FS.E.M.). The numbers 1 and 2 at the upright side means statistically different at Pb0.05 (ANOVA two-way). a Indicates U mg Hb 1. * Means significantly different from control at Pb0.05 (ANOVA).
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Table 2 Plasma parameters of Brycon cephalus exposed to nitrite for 24 and 96 h Time
Parameter
Nitrite concentration (mg/L)
Plasma
0
0.2
0.4
0.6
24 h
Sodium (meq L 1) Potassium (meq L 1) Chloride (meq L 1) Protein (mg mL 1) Sodium (meq L 1) Potassium (meq L 1) Chloride (meq L 1) Protein (mg mL 1)
126.1F5.03 2.28F0.15 125.4F4.8 0.49F0.02 127.3F4.66 2.80F0.18 114.1F3.3 0.48F0.02
159.8F12.3* 3.91F0.33* 143.5F15.5 0.52F0.03 134.5F4.03 2.70F0.15 127.1F3.3* 0.50F0.01
151.6F12.3* 3.98F0.58* 138.0F9.2 0.47F0.05 128.8F3.09 2.68F0.20 111.5F2.8 0.45F0.01
138.0F7.27* 3.85F0.36* 144.3F8.4 0.54F0.03 127.0F2.00 2.56F0.14 121.7F2.4 0.41F0.01
96 h
Values are expressed as means (FS.E.M.). * Significantly different from control at Pb0.05.
concentrations (Cameron, 1971; Shechter et al., 1972). Methemoglobin formation is supposed to result in tissue hypoxia which causes significant stress (Arillo et al., 1984; Hilmy et al., 1987; Woo and Chiu, 1997). A common response to cope with hypoxia is an increase in hematocrit. This could be caused by increases in red cell counts and the content of hemoglobin to maintain the oxygen delivery (Swift, 1981; Peterson, 1990). However, several species do not present it, and the opposite effect of nitrite has been reported (Hilmy et al., 1987; Tucker et al., 1989). In matrinxa˜, the decreases in hematocrit, hemoglobin content and red cells counts, in addiction to the constant MCV can be attributed to blood cell lyses as suggested in carp (Jensen, 1990; Knudsen and Jensen, 1997). This effect would reduce the methemoglobin content; however, the total hemoglobin available would consequently also being reduced. Thus, functional anemia is a consequence of hemolytic anemia (Scarano and Saroglia, 1984). Hemolytic anemia is a result of the increase in energetic demand of red blood cells due to the methemoglobin reduction process by NADH–methemoglobin reductase. Consequently, the time the red blood cells spend in circulation is reduced as well they are destroyed by the spleen and kidney (Scarano et al., 1984). The decreases in Hct, NRBC and total Hb observed in matrinxa˜ are indicative of both functional and hemolytic anemia. The reduction of methemoglobin can be mediated by the NADH–methemoglobin reductase system. The enzyme is presumed to be present in several species, including fish (Huey and Beitinger, 1981; Freeman et al., 1983; Scott and Harrington, 1985; Woo and Chiu, 1997), because basal levels of methemoglobin were reached whenever the fish were allowed to recover in nitrite-free water. This recovery has been attributed to the NADH–methemoglobin reductase system in the channel catfish, in spite of that no enzyme assay was done (Schoore et al., 1995). This enzyme system is unresponsive in Lates calcarifer exposed to nitrite (Woo and Chiu, 1997); however, our data are suggestive of this enzyme in inducible by nitrite concentration or time of exposure. However, in spite of being non-inductive, the role of the NADH–methemoglobin reductase system has been proposed to be prevention of high levels of methemoglobin (Woo and Chiu, 1997). The control levels of plasma nitrite
and methemoglobin in matrinxa˜ are very low, and the raised level of both followed different patterns. The nitrite increase was exponential in the course of the exposures, while the methemoglobin reached a plateau. Plasma nitrite increased 30-fold compared to environmental concentration. This suggests a non-passive transport through the gills and consequent plasma accumulation. These responses have been reported previously (Huey et al., 1980), and it was suggested that the hemoglobin oxidation reached a steadystate equilibrium due to opposing effects of nitrite and the NADH–methemoglobin reductase activity. Changes in plasma electrolytes are observed in some fish exposed to nitrite (Jensen et al., 1987; Jensen, 1990; Woo and Chiu, 1997). Plasma potassium concentration is proposed to be associated to nitrite uptake probably due to loss of intracellular potassium from RBCs (Jensen, 1990, 1992) and skeletal muscle (Knudsen and Jensen, 1997) in Cyprinus carpio. However, high levels of plasma potassium in matrinxa˜ occur only at the early stages of exposure to nitrite. An increase in plasma sodium levels has been reported in seawater L. calcarifer exposed to nitrite (Woo and Chiu, 1997), and this effect is associated with environmental concentration of nitrite. Plasma chloride does not change in most freshwater fish. The observed chloride cell hypertrophy seems to prevent a decrease in plasma Cl concentration despite the competition for uptake site by nitrite (Gaino et al., 1984). In accordance with that no changes of the ions Cl , except in fish exposed to 0.2 mg/L for 96 h, and Na+ were observed in matrinxa˜. The plasma protein concentration can be reduced in fish exposed to nitrite (Hilmy et al., 1987), but in matrinxa˜ it remained constant. This ionic profile suggests that non-significant disturbances were observed in the electrolytic balance of matrinxa˜ exposed to sublethal nitrite concentrations.
5. Conclusions The present study reports the presence of NADH– methemoglobin reductase system in the freshwater fish matrinxa˜ exposed to environmental nitrite. This enzyme seems to be inductive which suggests its role in nitrite
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detoxification processes. Hemolytic anemia and a marked increase in methemoglobin content suggest that the nitrite exposed fish experience functional anemia. The high sensitivity of the species to toxic effects of nitrite is more likely due to metabolic failure than electrolytic unbalance.
Acknowledgements We thank Antoˆnio Donizete Aparecido da Silva and colleagues from the lab for technical support and suggestions. This research was sponsored by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP).
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Huey, D.W., Simco, B.A., Criswell, D.W., 1980. Nitrite-induced methemoglobin formation in channel catfish. Trans. Am. Fish. Soc. 109, 558 – 562. Jensen, F.B., 1990. Nitrite and red cell function in carp: control factors for nitrite entry, membrane potassium ion permeation, oxygen affinity and methemoglobin formation. J. Exp. Biol. 152, 149 – 166. Jensen, F.B., 1992. Influence of hemoglobin conformation, nitrite and eicosanoids on K+ transport across the carp red blood cell membrane. J. Exp. Biol. 171, 349 – 371. Jensen, F.B., 2003. Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol., A 135, 9 – 24. Jensen, F.B., Andersen, N.A., Heisler, N., 1987. Effects of nitrite exposure on blood respiratory proprieties, acid–base and electrolyte regulation in carp (Cyprinus carpio). J. Comp. Physiol. 157B, 533 – 542. Knudsen, P.K., Jensen, F.B., 1997. Recovery from nitrite-induced methemoglobinaemia and potassium balance disturbances in carp. Fish Physiol. Biochem. 16, 1 – 10. Matsuoka, T., 1997. Determination of methemoglobin and carboxyhemoglobin in blood by rapid colorimetry. Biol. Pharm. Bull. 20 (11), 1208 – 1211. Nikimaa, M., 1992. How does environmental pollution affect red cell function in fish? Aquat. Toxicol. 22, 227 – 238. Peterson, M.S., 1990. Hypoxia-induced physiological changes in two mangrove swamp fishes: sheepshead minnow, Cyprinodon variegatus Lacepede and sailfin molly, Poecilia latipinna (Lesueur). Comp. Biochem. Physiol., A 97 (1), 17 – 21. Scarano, G., Saroglia, M.G., 1984. Recovery of fish from functional and hemolytic anemia after brief exposure to a lethal concentration of nitrite. Aquaculture 43, 421 – 426. Scarano, G., Saroglia, M.G., Gray, R.H., Tibaldi, E., 1984. Hematological responses of Sea bass Dicentrarchus labrax to sublethal nitrite exposures. Trans. Am. Fish. Soc. 113, 360 – 364. Schoore, J.E., Simco, B.A., Davis, K.B., 1995. Responses of blue catfish and channel catfish to environmental nitrite. J. Aquat. Anim. Health 7, 304 – 311. Scott, E.M., Harrington, J.P., 1985. Methemoglobin reductase activity in fish erytrocytes. Comp. Biochem. Physiol., B 82 (3), 511 – 513. Shechter, H., Gruener, N., Shuval, I., 1972. A micromethod for the determination of nitrite in blood. Anal. Chim. Acta 60, 93 – 99. Swift, D.J., 1981. Changes in selected blood component concentratios of raibow trout, Salmo Gardneri, exposed to hypoxia or sublethal concentration of phenol or ammonia. J. Fish Biol. 19, 45 – 61. Tucker, C.S., Francis-Floyd, R., Beleau, M.H., 1989. Nitrite-induced anemia in channel catfish, Ictalurus punctatus rafinesque. Bull. Environ. Contam. Toxicol. 43, 295 – 301. Vedel, N.E., Korsgaard, B., Jensen, F.B., 1998. Isolated and combined exposure to ammonia and nitrite in rainbow trout (Oncorhynchus mykiss): effects on electrolyte status, blood respiratory properties and brain glutamine/glutamate concentrations. Aquat. Toxicol. 41, 325 – 342. Vilella, G.G., Bacila, M., Tasdaldi, H., 1972. Te´cnicas e experimentos de bioquı´mica, 1st ed. Guanabara-Koogan, Rio de Janeiro, RJ. 552 pp. Williams, E.M., Eddy, F.B., 1986. Chloride uptake in freshwater teleosts and its relationship to nitrite uptake and toxicity. J. Comp. Physiol. 156 B, 867 – 872. Willians, E.M., Glass, M.L., Heisler, N., 1993. Effects of nitrite-induced methemoglobinaemia on oxygen affinity of carp blood. Environ. Biol. Fishes 37, 407 – 413. Woo, N.Y.S., Chiu, S.F., 1997. Metabolic and osmoregulatory responses of the sea bass Lates calcarifer to nitrite exposure. Environ. Toxicol. Water Qual. 12 (3), 257 – 264. Worthington Biochemical Corporation, 1972. Wortihington Enzyme Manual—Enzymes, Enzymes Reagents and Related Biochemicals. Freehold, New Jersey, USA.
Hematological responses of the Neotropical teleost matrinxa˜ (Brycon cephalus) to environmental nitrite Ive M. Avilez, Alexandre E. Altran, Lu´cia H. Aguiar, Gilberto Moraes* Departament of Genetics and Evolution, Federal University of Sa˜o Carlos, Rod Washington Luı´s, km 235, Sa˜o Carlos, SP CEP 13565-905, Brazil Received 21 May 2004; received in revised form 1 October 2004; accepted 3 October 2004
Abstract Environmental increase in nitrite impairs the function of several aquatic species, including fishes. Nitrite reacts with hemoglobin yielding the non-functional methemoglobin (metHb), and many physiological disturbances can arise. The physiological mechanisms to cope with nitrite are still unclear in fish. Hematological parameters, the role of NADH–methemoglobin reductase system and the electrolytic balance were studied in the freshwater teleost Brycon cephalus (matrinxa˜) exposed to 0.2, 0.4 and 0.6 mg/L of nitrite N–NO2 for 24 and 96 h. Hematocrit, total hemoglobin and the red blood cell (RBC) number decreased. Methemoglobin content increased from 1% to 69% for 24 h of exposure and drastically from 5–6% to 90% for 96 h. The activity of NADH–methemoglobin reductase system displayed a tendency of increase in response to nitrite concentration or time of exposure. In the plasma, nitrite was accumulated to values 30-fold higher than the environmental concentration. The plasma K+ concentration increased only in fish exposed to NO2 for 24 h. No changes in plasma protein and Na+ were observed during nitrite exposure but Cl-presented a punctual increase at 0.2 mg/L N–NO2–96 h. The hematological data suggest that nitrite caused functional and hemolytic anemia. Furthermore, the electrolytic balance was relatively undisturbed, and the nitrite clearance in matrinxa˜ is likely depending on other factors than NADH–methemoglobin reductase system. D 2004 Elsevier Inc. All rights reserved. Keywords: Brycon cephalus; Fish; Functional and hemolytic anemia; Hematology; Matrinxa˜; Methemoglobin; NADH–methemoglobin reductase system; Nitrite
1. Introduction Water pollution and industrial waste usually elevate the nitrite concentrations (Nikinmaa, 1992; Heckman et al., 1997) due to ammonia oxidation. This nitrification process depends on the bacterial activity and the oxygen level. High ammonia concentrations can be observed in aquaculture systems as a consequence of high stocking densities (Hargreaves, 1998; Hagopian and Riley, 1998). Nitrite from such conditions causes many physiological problems for reared fish. Fish has been proposed as a good animal model for disclosing the physiology that underlies the toxicology of nitrite (Jensen, 2003). Environmental nitrite crosses the gill * Corresponding author. Tel.: +55 16 260 8376; fax: +55 16 260 8377. E-mail address: [email protected] (G. Moraes). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.10.001
barrier by competing with chloride for chloride uptake sites (Gaino et al., 1984; Williams and Eddy, 1986) and is accumulated in the plasma (Shechter et al., 1972; Bath and Eddy, 1980). Thus, plasma nitrite concentrations are usually higher than environmental ones (Eddy and Willians, 1987); from the plasma, nitrite subsequently enters tissue cells (Arillo et al., 1984), and within the red blood cells (RBCs) it oxidizes hemoglobin Fe2+ to Fe3+ yielding methemoglobin (metHb), which is unable to transport oxygen (functional anemia) (Scarano and Saroglia, 1984). This effect is supposed to result in tissue hypoxia (Cameron, 1971; Bath and Eddy, 1980; Doblander and Lackner, 1996; Vedel et al., 1998) even in the presence of environmental oxygen (functional hypoxia). Methemoglobin formation in RBCs is easier as hemoglobin is in the non-oxygenated form (Jensen, 1990, 1992; Willians et al., 1993) and at low pH values (Jensen, 2003). Its content varies among fish and
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depends on factors such as the external nitrite concentration and the time of exposure. Nitrite causes other blood disturbances such as decrease of total hemoglobin, hematocrit and red cell counts. This phenomenon causes hemolytic anemia (Scarano and Saroglia, 1984). Several osmoregulatory responses including hyponatremia, hypochloremia (Jensen et al., 1987), branchial chloride cells failure (Gaino et al., 1984) and inhibition of chloride uptake (Williams and Eddy, 1986) are observed in freshwater teleosts exposed to nitrite. Recovery from effects of nitrite has been reported in nitrite-free water (Schoore et al., 1995) and a couple of ways involved in such processes are proposed. The first one involves the NADH–methemoglobin reductase system (Diaphorase I), which reduces hemoglobin Fe3+ to Fe2+ (Huey and Beitinger, 1981; Freeman et al., 1983; Scott and Harrington, 1985; Woo and Chiu, 1997). Nitrate synthesis is another way proposed as mechanism to detoxify nitrite (Doblander and Lackner, 1997). Catalase and cytochrome oxidase-aa3 are proposed to take role in that process (Doblander and Lackner, 1996); however, both mechanisms are still unclear. In the present study, hematological changes and osmoregulatory responses, in the freshwater Neotropical teleost Brycon cephalus (Gqnther, 1869) (matrinxa˜) exposed to environmental nitrite, were investigated. Few data concerning the effect of nitrite exposure on the induction of NADH– methemoglobin reductase system are now available. The most are just regarded to its presence in fish (Huey and Beitinger, 1981; Freeman et al., 1983; Scott and Harrington, 1985). Matrinxa˜ is widely reared in South America and is a low-tolerant species to environmental nitrite (LC50 0.86 mg/L N–NO2–96 h; Avilez et al., 2004). Therefore, the methemoglobin formation and the role of NADH– methemoglobin reductase system were also examined.
(n=6 in each tank). The fish were allowed to rest for 24 h, and 0.2, 0.4 or 0.6 mg/L (sublethal concentrations) of sodium nitrite N–NO2 were added to experimental tanks except the control. The nitrite exposure remained for 24 h. At the same time, another experiment was carried out under the same conditions for 96 h. Both experiments were carried out in semistatic system. After the exposure to nitrite, the fish were collected, anesthetized with MS 222 and a blood sample of 2 ml was drawn from the caudal vein into a heparinized syringe. 2.3. Hematological parameters Hematocrit (Hct) was determined on blood samples centrifuged at 12,000g for 3 min in capillary tubes. Total hemoglobin (Total Hb) was determined spectrophotometrically on 10 AL of blood in 2.0 mL of Drabkin solution at 540 nm. Methemoglobin (metHb) was colorimetrically quantified on 6 AL of whole blood at 563 nm as described by Matsuoka (1997). Red blood cell (RBC) (NRBC, ml 1) was counted under a light microscope with a Neubauer chamber. Mean corpuscular volume (MCV) was calculated from Hctd 10/NRBC, the mean corpuscular hemoglobin (MCH) content from Total Hbd 10/NRBC and the mean corpuscular hemoglobin concentration (MCHC) from Total Hbd 10/Hct. A blood aliquot was centrifuged at 12,000g for 3 min. The plasma was used for flame photometric determinations of Na+ and K+ on a Digimed DM-61, and optical determinations of Cl at 480 nm (APHA, 1980) and NO2 at 520 nm (Shechter et al., 1972). Total protein was determined by a UV method at 215 and 225 nm (Vilella et al., 1972). 2.4. NADH–methemoglobin reductase system activity
Juveniles of B. cephalus had a mean weight of 90F5 g ´ guas Claras, (FS.D.) and were obtained from the fish farm A Moco´ca, SP, Brazil. The fish were brought to the lab aquaculture facilities, acclimated for 2 months, fed commercial food and exposed to natural photoperiod, controlled temperature (25F1 8C) and aerated water. The water quality parameters in the experiment were: oxygen content, 7.5 mg/ L; pH, 6.8F0.2; temperature, 24F1 8C; conductivity, 74.3 AS cm 1; total alkalinity, 37 mg/L as CaCO3; hardness, 28 mg/L as CaCO3, ammonia concentration, 0.01 mg/L; chloride concentration, 0.294 mg/L; and nitrite concentration, 0 mg/L.
Blood samples were centrifuged and the packed red cells were washed at least three times with saline solution (0.9%) by centrifugation at 1000g for 10 min. White cells were removed by suction of the upper layer. Red cells were resuspended into 0.04 mL of mercaptoethanol–EDTA solution and the erythrocytes were lysed by thermal shock in a liquid nitrogen bath (Beutler, 1984). The resulting hemolysate was used as enzyme source. The enzyme assay was performed in a 0.2 M Tris–HCl pH 7.5 buffer solution containing 1.2 mM 2,6-dichlorophenol indophenol, 6 mM NADH, and a suitable enzyme aliquot. The substrate consumption was optically followed at 600 nm, and one enzyme activity unit equals a decrease in 1.0 absorbance unity per min, at 25 8C (Worthington Biochemical, 1972). The specific activity isexpressed in U (units) per mg of hemoglobin (U/mg total Hb).
2.2. Experimental design
2.5. Statistics
Twenty-four fish, starved for 1 day, were equally distributed in four tanks with 250 L of nitrite-free water
STATISTICA 5.5 was used to compare data. Normality of the data was evaluated by the Shapiro–Will W test with a 95%
2. Materials and methods 2.1. Fish
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Fig. 1. Methemoglobin concentration in B. cephalus exposed to environmental nitrite for 24 and 96 h. The values are expressed as mean (FS.E.M.); (*) significantly different from control group at Pb0.05.
confidence limit. The parametric test ANOVA was used to compare the groups, the Post-Test Duncan for multiple comparisons was applied, and the two-way ANOVAwas used to compare NADH MetHb reductase between 24 and 96 h. Pb0.05 was considered statistically significant.
3. Results The blood parameters of matrinxa˜ were affected by every nitrite level tested. The methemoglobin content increased markedly (Fig. 1), and the hematocrit, total hemoglobin and red blood cell number decreased (Table 1) except the group exposed to 0.2 mg/L for 24 h. The MCHC was elevated in fish exposed to 0.4 mg/L for 24 h, but decreased in fish exposed to nitrite for 96 h. No significant changes were observed in the MCV and MCH (Table 1). The NADH
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Fig. 2. Plasma nitrite concentration in B. cephalus exposed to environmental nitrite for 24 and 96 h. The values are expressed as mean (FS.E.M.); (*) significantly different from control group at Pb0.05.
methemoglobin reductase system was detected in the red blood cells of matrinxa˜, and the activity was affected by external nitrite concentrations or time of exposure (two-way ANOVA; Table 1). Plasma nitrite concentration increased as function of external nitrite (Fig. 2). The maximal value reached was 2.1 mM, 30-fold higher than the environmental concentration. Plasma potassium was elevated in the 24 h at all nitrite levels treatment, but was as in controls after 96 h. Plasma chloride increased in fish exposed to 0.2 mg/L for 96 h, whereas no changes were observed in the plasma protein or Na+ (Table 2).
4. Discussion The most prominent effects in fish exposed to nitrite are the increases in methemoglobin content and the plasma nitrite
Table 1 Hematological parameters of Brycon cephalus exposed to nitrite for 24 and 96 h Time
Parameters
Nitrite concentration (mg/L)
Total blood
0
0.2
0.4
0.6
24 h
Hct (%) Total Hb (g dL 1) RBC (106 cells mm3) MCV (fl) MCH (pg) MCHC (g dL 1) NADH–MetHb reductasea Hct (%) Total Hb (g dL 1) RBC (106 cells mm 3) MCV (fl) MCH (pg) MCHC (g dL 1) NADH–MetHb reductasea
33.5F0.85 9.54F0.33 2.45F0.31 150.7F23.1 42.68F6.28 2.85F0.11 0.09F0.0071 34.6F1.33 10.27F0.56 3.17F0.37 115.0F10.1 33.94F2.88 2.95F0.06 0.22F0.0271
28.0F0.73* 7.03F0.36* 2.26F0.15 127.3F9.6 31.37F1.14 2.51F0.15 0.10F0.0161 28.6F0.66* 7.60F0.43* 2.39F0.13* 120.9F4.9 31.93F1.72 2.64F0.11* 0.33F0.0632
22.9F0.76* 8.2F0.16* 1.51F0.13* 158.6F18.0 56.31F4.81 3.61F0.12* 0.08F0.0101 25.3F0.76* 6.58F0.21* 1.85F0.10* 137.9F5.6 35.86F1.56 2.60F0.05* 0.34F0.0342
23.5F1.23* 7.15F0.59* 1.64F0.22* 152.3F14.5 46.14F5.32 3.02F0.14 0.14F0.0241 23.1F1.28* 5.94F0.51* 1.79F0.11* 130.0F6.5 33.49F3.08 2.55F0.12* 0.31F0.0282
96 h
Values are expressed as means (FS.E.M.). The numbers 1 and 2 at the upright side means statistically different at Pb0.05 (ANOVA two-way). a Indicates U mg Hb 1. * Means significantly different from control at Pb0.05 (ANOVA).
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Table 2 Plasma parameters of Brycon cephalus exposed to nitrite for 24 and 96 h Time
Parameter
Nitrite concentration (mg/L)
Plasma
0
0.2
0.4
0.6
24 h
Sodium (meq L 1) Potassium (meq L 1) Chloride (meq L 1) Protein (mg mL 1) Sodium (meq L 1) Potassium (meq L 1) Chloride (meq L 1) Protein (mg mL 1)
126.1F5.03 2.28F0.15 125.4F4.8 0.49F0.02 127.3F4.66 2.80F0.18 114.1F3.3 0.48F0.02
159.8F12.3* 3.91F0.33* 143.5F15.5 0.52F0.03 134.5F4.03 2.70F0.15 127.1F3.3* 0.50F0.01
151.6F12.3* 3.98F0.58* 138.0F9.2 0.47F0.05 128.8F3.09 2.68F0.20 111.5F2.8 0.45F0.01
138.0F7.27* 3.85F0.36* 144.3F8.4 0.54F0.03 127.0F2.00 2.56F0.14 121.7F2.4 0.41F0.01
96 h
Values are expressed as means (FS.E.M.). * Significantly different from control at Pb0.05.
concentrations (Cameron, 1971; Shechter et al., 1972). Methemoglobin formation is supposed to result in tissue hypoxia which causes significant stress (Arillo et al., 1984; Hilmy et al., 1987; Woo and Chiu, 1997). A common response to cope with hypoxia is an increase in hematocrit. This could be caused by increases in red cell counts and the content of hemoglobin to maintain the oxygen delivery (Swift, 1981; Peterson, 1990). However, several species do not present it, and the opposite effect of nitrite has been reported (Hilmy et al., 1987; Tucker et al., 1989). In matrinxa˜, the decreases in hematocrit, hemoglobin content and red cells counts, in addiction to the constant MCV can be attributed to blood cell lyses as suggested in carp (Jensen, 1990; Knudsen and Jensen, 1997). This effect would reduce the methemoglobin content; however, the total hemoglobin available would consequently also being reduced. Thus, functional anemia is a consequence of hemolytic anemia (Scarano and Saroglia, 1984). Hemolytic anemia is a result of the increase in energetic demand of red blood cells due to the methemoglobin reduction process by NADH–methemoglobin reductase. Consequently, the time the red blood cells spend in circulation is reduced as well they are destroyed by the spleen and kidney (Scarano et al., 1984). The decreases in Hct, NRBC and total Hb observed in matrinxa˜ are indicative of both functional and hemolytic anemia. The reduction of methemoglobin can be mediated by the NADH–methemoglobin reductase system. The enzyme is presumed to be present in several species, including fish (Huey and Beitinger, 1981; Freeman et al., 1983; Scott and Harrington, 1985; Woo and Chiu, 1997), because basal levels of methemoglobin were reached whenever the fish were allowed to recover in nitrite-free water. This recovery has been attributed to the NADH–methemoglobin reductase system in the channel catfish, in spite of that no enzyme assay was done (Schoore et al., 1995). This enzyme system is unresponsive in Lates calcarifer exposed to nitrite (Woo and Chiu, 1997); however, our data are suggestive of this enzyme in inducible by nitrite concentration or time of exposure. However, in spite of being non-inductive, the role of the NADH–methemoglobin reductase system has been proposed to be prevention of high levels of methemoglobin (Woo and Chiu, 1997). The control levels of plasma nitrite
and methemoglobin in matrinxa˜ are very low, and the raised level of both followed different patterns. The nitrite increase was exponential in the course of the exposures, while the methemoglobin reached a plateau. Plasma nitrite increased 30-fold compared to environmental concentration. This suggests a non-passive transport through the gills and consequent plasma accumulation. These responses have been reported previously (Huey et al., 1980), and it was suggested that the hemoglobin oxidation reached a steadystate equilibrium due to opposing effects of nitrite and the NADH–methemoglobin reductase activity. Changes in plasma electrolytes are observed in some fish exposed to nitrite (Jensen et al., 1987; Jensen, 1990; Woo and Chiu, 1997). Plasma potassium concentration is proposed to be associated to nitrite uptake probably due to loss of intracellular potassium from RBCs (Jensen, 1990, 1992) and skeletal muscle (Knudsen and Jensen, 1997) in Cyprinus carpio. However, high levels of plasma potassium in matrinxa˜ occur only at the early stages of exposure to nitrite. An increase in plasma sodium levels has been reported in seawater L. calcarifer exposed to nitrite (Woo and Chiu, 1997), and this effect is associated with environmental concentration of nitrite. Plasma chloride does not change in most freshwater fish. The observed chloride cell hypertrophy seems to prevent a decrease in plasma Cl concentration despite the competition for uptake site by nitrite (Gaino et al., 1984). In accordance with that no changes of the ions Cl , except in fish exposed to 0.2 mg/L for 96 h, and Na+ were observed in matrinxa˜. The plasma protein concentration can be reduced in fish exposed to nitrite (Hilmy et al., 1987), but in matrinxa˜ it remained constant. This ionic profile suggests that non-significant disturbances were observed in the electrolytic balance of matrinxa˜ exposed to sublethal nitrite concentrations.
5. Conclusions The present study reports the presence of NADH– methemoglobin reductase system in the freshwater fish matrinxa˜ exposed to environmental nitrite. This enzyme seems to be inductive which suggests its role in nitrite
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detoxification processes. Hemolytic anemia and a marked increase in methemoglobin content suggest that the nitrite exposed fish experience functional anemia. The high sensitivity of the species to toxic effects of nitrite is more likely due to metabolic failure than electrolytic unbalance.
Acknowledgements We thank Antoˆnio Donizete Aparecido da Silva and colleagues from the lab for technical support and suggestions. This research was sponsored by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP).
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