Physiological responses to sulfide toxicity by the air-breathing catfish, Hoplosternum littorale (Siluriformes, Callichthyidae)

Comparative Biochemistry and Physiology, Part C 139 (2004) 251 – 257 www.elsevier.com/locate/cbpc

Physiological responses to sulfide toxicity by the air-breathing catfish, Hoplosternum littorale (Siluriformes, Callichthyidae) E.G. Affonsoa,*, V.L.P. Polezb, C.F. Correˆab, A.F. Mazonc, M.R.R. Arau´joc, G. Moraesb, F.T. Rantinc a

Department of Aquaculture, National Research Institute of Amazon, Av. Andre´ Arau´jo, 2936, P.O. Box 478, 69083-000-Manaus, AM, Brazil b Department of Genetics and Evolution, Federal University of Sao Carlos, Via Washington Luı´s, Km 235, Sao Carlos, Sao Paulo, Brazil c Department of Physiological Sciences, Federal University of Sao Carlos, Via Washington Luı´s, Km 235, Sao Carlos, Sao Paulo, Brazil Received 9 August 2004; received in revised form 19 November 2004; accepted 20 November 2004

Abstract Hemolytic anemia accompanied by changes in the immunology system is one of the sulfide intoxication harmful effects on Hoplosternum littorale. Hematological parameters are considered as effective indicators of stress caused by this hydrogen sulfide. During sulfide exposure, H. littorale neither alters the methemoglobin concentration nor forms sulfhemoglobin in the presence of high levels of dissolved sulfide in the water. Cytochrome c oxidase shows little activity in the gills and blood of H. littorale when exposed to sulfide. Alternative metabolic routes are suggested through which the accumulation of pyruvate leads to the formation of an end product other than lactate. D 2004 Elsevier Inc. All rights reserved. Keywords: Cytochrome c oxidase; Hematology; Hoplosternum littorale; Metabolism; Methemoglobin; Sulfhemoglobin; Sulfide

1. Introduction Hydrogen sulfide (H2S) is highly toxic to aerobic organisms due to its binding to the cytochrome c oxidase, the last enzyme of the electron transport system. This compound is naturally produced by anaerobic decomposition of organic matter and sulfate reduction during high water season in the Central Amazon (Affonso, 2000). Sulfide has been reported as meaningful environmental factor for Amazonian fish species. Their tolerance limits and physiological strategies to cope with environmental sulfide have been investigated (Affonso, 2000; Affonso et al., 2002). The armored catfish tamoata (Hoplosternum littorale), a facultative air-breather, inhabits shallow, stagnant waters, poor in oxygen and rich in sulfide. This freshwater fish is considered one of the most tolerant species to sulfide (96 h, LC50 37 AM H2S; Affonso, 2000). Besides keeping the proper oxygen

* Corresponding author. Tel.: +55 92 642 6088; fax: +55 92 642 1384. E-mail address: [email protected] (E.G. Affonso). 1532-0456/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2004.11.007

content in the blood, the increase of the air-breathing frequency seems to help this species to withstand the sulfide toxicity (Brauner et al., 1995; Affonso, 2000). This may be a general adaptive response of H. littorale to cope with different stressful situations (Duncan, 1998; Brauner et al., 1999). However, the air breathing by itself cannot hold the respiratory homeostasis for long periods of exposure to sulfide (Duncan, 1998; Brauner et al., 1999). The high tolerance to the sulfide toxic effects on the fish respiratory processes is mainly due to the metabolic adjustments instead of the ability to detoxicate it (Bagarinao, 1992; Affonso et al., 2002). However, these adjustments are not exclusive for the toxic effects of sulfide since most of these factors also occur under hypoxic conditions (Affonso et al., 2002). In the present work the blood and metabolic changes were investigated in tamoata continuously exposed to sulfide for 96 h. The in vivo formation of sulfhemoglobin (SHb), which is a green hemoglobin derivative with lower O2 affinity, and the activity of cytochrome c oxidase were also assayed in a static short-term exposure for 6 h.

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2. Materials and methods 2.1. Fish collection and maintenance The fish, ranged from 60 to 180 g, were collected in the Piracicaba River, SP, Brazil and maintained in tanks with aerated flow-through water. The water quality parameters were 28F1 8C, pH 7.2–7.8, PO2 120–140 mm Hg. Fish were fed with fish pellets for 3 weeks and the feeding was discontinued 24 h before the experiments.

monitored with methylene blue (Cline, 1969), through flow injection analysis (FIA). The water was maintained normoxic (PwO2N130 mm Hg) at constant temperature of 28F1 8C and pH 7.2–7.8. Six fish of both groups were sampled at 12, 24, 48 and 96 h. Blood samples were taken from the caudal vein into heparinized syringes. Fish were previously anesthetized by immersion into a benzocaine solution (1 g 10 L 1) and killed by severing the spinal cord. Samples of liver, white muscle and heart were taken and kept at –20 8C for posterior biochemical analysis. The entire process was carried out in about 10 min.

2.2. Continuous fish exposure to sulfide 2.3. Blood and tissue analysis Two groups of tamoatas (n=24) were transferred to a couple of 200-L aquaria (control and test) with the same water quality, and kept undisturbed for 24 h. Into the test aquarium a sulfide stock solution was continuously pumped with a peristaltic pump at a rate of 3 mL min 1 to keep a final concentration of 13–19 AM for 96 h. The sulfide stock solution was 250 mM Na2S, prepared with washed and dried crystals dissolved into deoxygenated distilled water. The water was renewed at 500 mL min 1. The total water sulfide concentration, here meaning H2S, HS and S , was

Blood samples were divided into three aliquots and kept in an ice bath throughout the analyses (2 h). One blood aliquot was used for hematology. Hematocrit (Ht) was determined by microhematocrit technique, red blood cells (RBC) were counted in a Neubauer chamber and the hemoglobin concentration ([Hb]) was determined with Drabkin’s reagent at 540 nm. The mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated from

Fig. 1. Hematocrit (Ht), red blood cell count (RBC), hemoglobin concentration ([Hb]), mean cell volume (MCV), cell hemoglobin (MCH), and cell hemoglobin concentration (MCHC) of Hoplosternum littorale exposed to sulfide for 96 h, relative to the control. Points are meanFS.E.M.; n=6. Asterisks indicate significant differences ( Pb0.05) in relation to the control values.

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the Ht, [Hb] and RBC. Blood smears were stained with May Grqnwald-Giemsa for identification and quantification of leukocytes. Methemoglobin concentration was quantified in hemolysates done with 37 mM phosphate buffer at pH 7.3, and read at 540, 560, 576 and 630 nm according to Benesch et al. (1973). The second blood aliquot was assayed for lactate and pyruvate determination. The samples were deproteinized with 8% perchloric acid (PCA), homogenized, centrifuged at 5000g for 10 min at 8 8C, and lactate and pyruvate were determined enzymatically. A third aliquot was centrifuged, and the plasma analyzed for glucose by a glucose oxidase method. Lactate, pyruvate and glucose of the tissue were determined in white muscle, liver and heart (200 mg). The samples were prepared as detailed above for lactate and pyruvate blood analyses and determined enzymatically on a MRX microplate reader (Dynex Technologies). 2.4. Static fish exposure to sulfide Eighteen fish were distributed into three 100 L aquaria and left undisturbed for 24 h. One aquarium was free of sulfide and was considered as the control. The two others contained 44 and 72 AM sulfide. The pH values were kept at 7.1–7.5 in all experimental aquaria. Sulfide concentrations were predetermined using the data obtained by Affonso (2000) for this species. After 6 h of sulfide exposure the animals were sampled and the blood was collected as described above to evaluate SHb formation in vivo. The concentration of SHb was determined according to the method described by Bagarinao and Vetter (1992). The fish exposed to 72 AM sulfide and the control were killed and tissue (muscle, gill, spleen, kidney, heart, brain, liver and blood) samples were collected to determine the cytochrome c oxidase (EC 1.9.3.1) activity, following the method of Yonetani and Ray (1965) and Hand and Somero (1983). Tissue samples were homogenized into 9 volumes of 10 mM Tris, pH 7.2, and centrifuged at 2000g for 10 min at 5 8C. One aliquot (25 AL) of the each tissue homogenate was Table 1 Differential leukocyte counts of Hoplosternum littorale exposed to 13–19 AM sulfide for 96 h Time 12 h Control Sulfide 24 h Control Sulfide 48 h Control Sulfide 96 h Control Sulfide

Lymphocytes

Neutrophils

Monocytes

Eosinophils

76.6F3.3 43.0F7.2*

10.6F2.4 22.3F3.4*

7.7F2.1 14.4F5.0

0.7F0.3 0.6F0.2

73.8F1.5 59.7F2.8*

12.8F3.0 24.4F2.1*

11.0F1.7 24F8.9

0.17F0.17 0.18F0.18

67.7F5.4 57.5F7.8

16.6F3.3 23.4F4.8

15.2F4.3 21.1F5.5

0.24F0.17 1.4F0.7

77.7F4.1 76.6F2.4

11.0F2.4 15.1F4.5

11.1F2.6 8.2F2.9

0.0F0.0 0.0F0.0

Averages are expressed in percentages. Asterisks indicate significant differences ( Pb0.05) in relation to the control values. MeanFS.E.M.; n=6.

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Fig. 2. Methemoglobin concentrations in Hoplosternum littorale exposed to sulfide and on control for 96 h. MeanFS.E.M.; n=6.

added to 2.0 ml of a reaction mixture containing to a final concentration: 100 mM KH2PO4 at pH 6.0, 0.5 mM EDTA and 50 AM of reduced cytochrome c. The cytochrome c (horse heart, type III, Sigma) was reduced by using sodium dithionite and subsequently removed by gel filtration into Sephadex G-25. Enzyme activities were calculated from the linear rate of absorbance decrease at 550 nm, using the cytochrome c extinction coefficient 18.5 mM 1 cm 1, and were expressed in Amol cytochrome c min 1 g tissue 1. 2.5. Statistics Experimental fish mean blood parameter and metabolite level values were statistically analyzed by two-way ANOVA. The differences were considered to be significant when Pb0.05 using the Tukey test.

3. Results 3.1. Hematological observations The values of Ht, RBC and Hb were lower than control in the course of continuous sulfide exposure. However, increase of hematocrit and RBC were observed at 96 h (Fig. 1). Lymphocytes and neutrophils exhibited significant differences in fish exposed for 12 and 24 h (Table 1).

Fig. 3. Sulfhemoglobin concentrations in Hoplosternum littorale exposed to 44 AM and 72 AM for 6 h to sulfide. Values are expressed as ratio A 618/ A 576. Bars show meanFS.E.M.; n=6.

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Table 2 Cytochrome c oxidase of Hoplosternum littorale assayed in tissues after 72 AM sulfide exposure for 6 h Tissues

3.4. Glucose Acute hypoglycemia was observed in the fish exposed for 48 h to sulfide (Table 3). The white muscle glucose increased slowly after 48 h of exposure. No significant differences were observed in the other tissues.

Cytochrome c oxidase activity (Amol cyt c min 1 g fresh weight 1)

Muscle Gill Spleen Kidney Heart Brain Liver Blood

Control

Fish exposed to 72 AM sulfide

6.11F2.3 4.5F0.95* 4.8F1.4 11.6F2.7 11.7F3.0 14.3F2.3 13.9F1.3 0.47F0.02*

6.4F0.22 1.3F0.27 6.8F0.74 13.1F0.45 12.6F1.5 18.9F1.5 12.4F0.97 0.17F0.09

3.5. Lactate Lactate concentrations decreased in white muscle, liver, and heart only at the first 12 h of exposure to sulfide (Table 3). In the liver, the lactate concentrations increased after 24 h.

Asterisks indicate significant differences ( Pb0.05) in relation to the control values. MeanFS.E.M.; n=6.

3.6. Pyruvate High concentrations of pyruvate were detected in the white muscle, liver and heart of tamoata in the first 12 h of exposure to sulfide (Table 3). In the liver, pyruvate increased at 24 and 96 h of exposure to sulfide.

3.2. Methemoglobin and sulfhemoglobin in vivo Methemoglobin did not change in the fish continuously exposed to sulfide (Fig. 2) and SHb concentrations were the same in animals exposed to 44 and 72 AM of sulfide (Fig. 3).

4. Discussion 3.3. Cytochrome c oxidase activity 4.1. Hematological parameters The cytochrome c oxidase activity in the gills and red blood cells of fish exposed to sulfide was high compared to the control (Table 2). However, the blood enzyme was very low compared to other tissues. This enzyme activity was unchanged in muscle, kidney, heart, and liver. In spite of being non-responsive to sulfide, spleen and brain displayed high enzyme activity.

Increases of hematocrit, red blood cells and hemoglobin, have been detected in H. littorale exposed to hypoxia for 24 h (Moura et al., 1997). According to Val (1993), some bimodal breathing fish like H. littorale tend to increase the hematocrit and hemoglobin concentration during hypoxia, raising the oxygen content in the blood.

Table 3 Glucose, lactate and pyruvate levels in the different tissues of Hoplosternum littorale exposed to 13–19 AM sulfide for 96 h, relative to those of the unexposed control Duration of exposure 12 h

24 h

48 h

96 h

Control

Sulfide

Control

Sulfide

Control

Glucose Plasma Muscle Liver Heart

4.39F0.3 0.27F0.04 7.8F0.7 1.2F0.1

3.73F0.28 0.46F0.1 7.1F1.9 1.2F0.3

4.95F0.71 0.43F0.05 5.4F0.3 1.1F0.1

5.16F0.51 0.73F0.18 9.0F1.4 1.3F0.2

5.13F0.75 0.32F0.01 6.1F0.5 1.6F0.3

1.7F0.23* 1.1F0.31* 6.3F0.8 1.5F0.8

Lactate Blood Muscle Liver Heart

0.64F0.04 26.0F2.1 1.8F0.3 7.8F1

0.78F0.2 5.0F1.3* 0.99F0.1* 3.2F0.8*

0.78F0.08 16.0F2.0 1.45F0.25 5.2F0.6

0.8F0.25 18.5F3.5 2.05F0.15 5.9F0.9

0.9F0.2 19.5F2.5 1.4F0.2 4.5F0.7

1.7F0.25* 15.5F3.5 2.9F0.6* 6.8F1.3

0.5F0.1 17.0F2.0 1.2F0.1 3.2F0.7

0.3F0.07 16.0F2.0 1.6F0.1* 5.5F0.9

Pyruvate Blood Muscle Liver Heart

0.24F0.02 0.22F0.03 0.27F0.02 0.48F0.03

0.21F0.02 0.63F0.1* 1.8F0.38* 0.98F0.5

0.21F0.1 0.44F0.09 0.58F0.1 0.8F0.04

0.22F0.02 0.11F0.01 1.2F0.13* 0.5F0.02

0.20F0.08 0.28F0.05 0.61F0.1 0.7F0.03

0.27F0.06 0.23F0.07 0.64F0.16 1.5F0.5

0.07F0.01 0.28F0.05 0.57F0.09 1.0F0.03

0.27F0.03 0.18F0.04 1.8F0.29* 0.06F0.02

The values are expressed in Amol mL 1 (blood or plasma) and Amol g relation to the control values. MeanFS.E.M.; n=6.

1

Sulfide

Control 6.6F0.91 0.4F0.05 4.5F0.2 1.7F0.2

Sulfide 1.7F0.11* 1.0F0.06* 8.2F1.5 2.1F0.2

(muscle, liver and heart). Asterisks indicate significant differences ( Pb0.05) in

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Sulfide, which permeates easily into the gill epithelium, avoids the oxygen binding by the hemoglobin. The result is tissular hypoxia, similar to that caused by cyanide and reduction of oxygen. Usually, studies on the effects of pollutants that cause internal hypoxia report an increase of hematological parameters (Hilmy et al., 1987; Areechon and Plumb, 1990). However, this was not observed in H. littorale continuously exposed to sulfide in normoxic conditions for 96 h. Conversely, our results show that sulfide reduced the three main hematological variables. These data agree with those previously reported by Affonso (2001) since the presence of sulfide in a Central Amazon floodplain lake reduced the Ht and Hb concentration in H. littorale. Similar reduction of the hematological parameters is also observed in Colossoma macropomum (Affonso et al., 2002). However, this is not a general pattern for fish. The exposure of Fundulus parvipinnis to sulfide for 2 h caused increase in such blood parameters (Bagarinao and Vetter, 1993). The immediate release of red blood cells from the spleen is usually a short-term compensatory response. If H. littorale increases the number of cells in its blood stream before being exposed to sulfide for 12 h, this could be an immediate response for improving the capacity of oxygen transport. However, 12 h after exposure to sulfide the red blood cells of H littorale seem to be affected. There are several causes for the anemia of fish exposed to pollutants (Heath, 1995). The inhibition of enzyme precursors for hemoglobin synthesis may be considered one of these causes (Tephly et al., 1978). The binding of Hb to a pollutant can inhibit the linkage with oxygen (Massano, 1974), induces methemoglobin formation (Costa et al., 2004), results in malformation of erythrocytes (Houston et al., 1993) and may cause lipid peroxidation, which results in hemolysis (Palace et al., 1993). Methemoglobin and sulfhemoglobin formation in H. littorale should not be considered the cause for the decreases of hematological parameters, since methemoglobin concentrations remained unchanged and practically there was no SHb formation. Blood cell morphology also suggested that there were no deformities. Hemolysis is the most plausible reason due to the fragility of the erythrocyte membrane. The hematimetric indexes indicate that red cell volume and Hb cellular concentration were unvaried. In addition to the action on erythrocytes, pollutants may also alter the fish leukocyte differential count (Guilhermino et al., 1998; Kumar et al., 1999; Rosenberg et al., 2003). Our results show that the leukocytes of H. littorale were affected by sulfide stressing action. Lymphopenia was observed in the first two periods of exposure to sulfide. The main role of the lymphocytes in the organism is the production of antibodies. This is related to about 50–80% of the of leukocyte population in fish (Fange, 1992). Decreases of these cells were also observed in fish exposed to cadmium (Newman and Maclean, 1974), high temperatures

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(Moura et al., 1994), and hypoxia (Moura et al., 1997). H. littorale exposed to hypoxia, but allowed to access the surface, presented an opposite lymphocyte responses. This was attributed to the air-breathing behavior, which facilitates infections and stimulates immune responses (Moura et al., 1997). However, the exposure to sulfide also increases the air-breathing frequency in H. littorale (Affonso, 2000). Therefore, the lymphocytosis should not be a consequence of infections resulting from the air-breathing behavior, as proposed by Moura et al. (1997). Probably, the stress caused by sulfide impairs the production of lymphocytes. Different results were also obtained on the neutrophil counting in H. littorale when compared to hypoxia (Moura et al., 1997). These authors reported neutrophilia in 12 and 24 h of hypoxia exposure. Neutrophils, as well as monocytes, are responsible for phagocytic processes in the organism. Neutrophilia should mean increased phagocytic activity bringing about both infectious and aseptic processes. Hemolytic anemia brought about by sulfide might be most likely reason for the occurrence of neutrophilia in H. littorale. 4.2. Methemoglobin and sulfohemoglobin formation In spite of the toxic effects of sulfide, adaptive mechanisms have also been reported in fish (Bagarinao and Vetter, 1989, 1992, 1993; Vismann, 1991). The most feasible mechanism is the sulfide oxidation to a less toxic form. Some heme compounds can be responsible for such activity, and the methemoglobin (mtHb) is one amongst them. MtHb binds to sulfide as well as to other cytochrome c oxidase inhibitors such as cyanide and azide. Therefore, it protects vertebrates against H2S toxicity (Smith and Gosselin, 1964, 1966; Smith et al., 1977) through a complex tightly bound to the molecule. Despite the mtHb concentration for the sulfide exposure in mammals (Smith and Gosselin, 1966) and fish (Torrans and Clemens, 1982), its synthesis is not a general process to prevent toxicity, even in highly tolerant species living in sulfide-rich environments (Bagarinao and Vetter, 1992). For example, C. macropomum does not produce mtHb at the same rate of sulfide exposure (Affonso et al., 2002). Similar response was observed in the present study. Therefore, mtHb synthesis seems not to be the only defense in H. littorale to cope with environmental sulfide. On the other hand, the oxygen transport is not impaired by SHb formation in this species, since its concentration was negligible. Sulfhemoglobin is a compound that impairs oxygen transport (Carrico et al., 1978). However, the lack of SHb synthesis is common in sulfide intoxicated mammals and fishes. Therefore SHb formation seems to be not a significant fact in sulfide poisoning (Parkel and Nagel, 1984; Curry and Gerkin, 1987; Bagarinao and Vetter, 1992; Affonso et al., 2002). Sulfhemoglobin formation in fishes has been observed only in in vitro experiments (Bagarinao and Vetter, 1992; Vo¨lkel and Berenbrink, 2000; Affonso et al., 2002).

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4.3. Cytochrome c oxidase activity Cytochrome c oxidase is a multi-subunit protein that catalyses electron transfer from ferrocytochrome c to molecular oxygen and the concomitant translocation of protons across the inner mitochondrial membrane. The activity of cytochrome c oxidase in the tissues of tamoata exposed to high sulfide concentrations (72 AM) showed that this enzyme is only inhibited in the gills and blood. This occurs probably because these tissues are primarily in contact with the surrounding water. Similar results were observed in some marine species (Bagarinao and Vetter, 1989, 1993). The effects of sulfide on cytochrome c oxidase in isolated mitochondria of F. parvipinnis suggest that mitochondria is able to oxidize sulfide before it reaches inhibitory concentrations (Bagarinao and Vetter, 1993). This should be one of the reasons for the high tolerance of species such as tamoata to sulfide-rich environments (61 AM total sulfide; as found in a floodplain lake according to Affonso, 2000). Further data from in vitro experiments are needed in order to elucidate the role of mitochondria and cytochrome c oxidase in the sulfide detoxification process. 4.4. Metabolic changes Some aquatic organisms perform anaerobiosis under high sulfide concentrations, even in the presence of high oxygen levels (Torrans and Clemens, 1982; Powell and Somero, 1986; Bagarinao and Vetter, 1989, 1993; Johns et al., 1997; Affonso et al., 2002). For example, C. macropomum is predominantly anaerobic for up to 24 h of exposure to sulfide. After this period, aerobic metabolism is reestablished preventing acidosis and metabolic depression (Affonso et al., 2002). Differently of that species, the airbreather H. littorale does not accumulate lactate in the course of the first 24 h of exposure to sulfide. The lactate decrease and pyruvate increase in muscle, liver, and heart, in the first 12 h of exposure to sulfide indicate lactate consume (Table 3). Similar responses are reported for H. littorale in the presence of crude oil in the water (Duncan, 1998). This species increases pyruvate in the plasma with low levels of lactate. In addition, no changes in the lactate dehydrogenase (LDH) level were observed when fish is exposed to nitrite for 24 h (Duncan et al., 1998). These authors proposed that there is an alternative metabolic pathway, which produces a compound other than lactate. This seems to be the case when the species is exposed to sulfide and other reducing agents such as nitrite and petroleum oil. The fate of glucose from the tissues, and glycogen from liver and muscle, suggests that carbohydrates are not the only substrate to cope with such stressors. Therefore, it is proposed that H littorale uses other substrates, such as amino acids and lipids, as energy sources. The increase of lactate in the liver at 48 h suggests that glycolysis breaks down glucose in that tissue. Duncan (1998) reported high hepatic glycogen levels in H littorale after 72 h of exposure to crude oil. However,

lactate and pyruvate in the muscle and heart of H. littorale, at 12 and 96 h of exposure to sulfide, suggest depression of the metabolism in those tissues. Those results coincided with the air-breathing frequency observed in H. littorale under sulfide exposure, which practically suppressed the air breathing after 24 h. Therefore, metabolic responses of H. littorale may be distinct for each tissue. In summary, the blood parameters of H. littorale, red and white blood cells, are important indexes of contamination caused by H2S. H. littorale does not form SHb in vivo, which eliminate this compound as the likely cause for poisoning by sulfide. Except in the gills and blood, cytochrome c oxidase was not changed by high sulfide concentrations. An alternative metabolic pathway, in addition to glycolysis, is probably the way to supply the energetic demands under sulfide exposure in H. littorale. This seems to be a general response in fish to cope with compounds that cause tissue hypoxia. Acknowledgements This work was supported by CAPES, INPA and UFSCar. The authors are thankful to Nelson S.A. Matos (in memoriam) for his dedicated work in the field. References Affonso, E.G., 2000. O ga´s sulfı´drico e a respirac¸a˜o de duas espe´cies de peixes teleo´steos, Hoplosternum littorale e Colossoma macropomum: Distribuic¸a˜o, toleraˆncia e adaptac¸a˜o. PhD thesis. Federal University of Sa˜o Carlos. 107 pp. Affonso, E.G., 2001. Respiratory characteristics of Hoplosternum littorale (Siluriformes, Callichthyidae). Acta Amazoˆn. 31, 249 – 262. Affonso, E.G., Polez, V.L.P., Correˆa, C.F., Mazon, A.F., Arau´jo, M.R.R., Moraes, G., Rantin, F.T., 2002. Blood parameters and metabolites in the teleost fish Colossoma macropomum exposed to sulfide or hypoxia. Comp. Biochem. Physiol., C 133, 375 – 382. Areechon, N., Plumb, J., 1990. Sublethal effects of malathion on channel catfish, Ictalurus punctatus. Bull. Environ. Contam. Toxicol. 44, 435. Bagarinao, T., 1992. Sulfide as an environmental factor and toxicant: tolerance and adaptations in aquatic organisms. Aquat. Toxicol. 24, 21 – 62. Bagarinao, T., Vetter, R.D., 1989. Sulfide tolerance and detoxification in shallow-water marine fishes. Mar. Biol. 103, 291 – 302. Bagarinao, T., Vetter, R.D., 1992. Sulfide–hemoglobin interactions in the sulfide tolerance salt marsh resident, the California killifish Fundulus parvipinnis. J. Comp. Physiol. 162B, 614 – 624. Bagarinao, T., Vetter, R.D., 1993. Sulfide tolerance and adaptation in California killifish, Fundulus parvipinnis, a salt marsh resident. J. Fish Biol. 42, 729 – 748. Benesch, R.E., Benesch, R., Yung, S., 1973. Equation for the spectrophotometric analysis of hemoglobin mixtures. Anal. Biochem. 55, 215 – 218. Brauner, C.J., Ballantyne, C.L., Randall, D.J., Val, A.L., 1995. Air breathing in the armoured catfish (Hoplosternum littorale) as an adaptation to hypoxic, acidic, and hydrogen sulphide rich water. Can. J. Zool. 73, 739 – 744. Brauner, C.J., Ballantyne, C.L., Vijayan, M.M., Val, A.L., 1999. Crude oil exposure affects air-breathing frequency, blood phosphate levels and ion regulation in an air-breathing teleost fish, Hoplosternum littorale. Comp. Biochem. Physiol., C 123, 127 – 134.

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