an anaerobic metabolism enhances autoxidation of free haemoglobin, and whether this involves H,O, liberation in blood and haemolymph of the animals.
JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY
Journal of Experimental Marine Biology and Ecology 187 (1995) 63-80
Hypoxia-induced autoxidation of haemoglobin in the benthic invertebrates Arenicola marina (Polychaeta) and Astarte borealis (Bivalvia) and the possible effects of sulphide’ Doris Abele-Oeschger
*, Rolf Oeschger
Universitdt Bremen. Meereszoolgie, FB 2. Biirgemzeister-Smidt-Str. 20, D-27568 Bremerhaven, Germany
Received
21 July 1994; revision
received
3 October
1994; accepted
26 October
1994
Abstract Haemoglobin autoxidation and the simultaneous liberation of H,O, was studied in the blood of the lugworm, Arenicola marina, and the haemolymph of the clam, Astarte borealis, under normoxia, hypoxia, and hypoxia with 200 pmol.ll’ sulphide. In both species Hb autoxidation and hydrogen peroxide liberation into the respiratory fluid increased under hypoxia. Even higher hydrogen peroxide levels were measured under sulphidic hypoxia conditions. Under normoxia and hypoxia, autoxidation of the respiratory pigment seems to be a major source of H,O, in blood and haemolymph. Whereas the body fluids of both species lack the hydrogen peroxide scavenger catalase, they contain considerable amounts of the antioxidant enzymes glutathione reductase (Astarte borealis only) and superoxide-dismutase (SOD). SOD activity in Arenicola marina blood was unaffected by sulphide in vivo and in vitro, while haemolymph SOD activity was significantly reduced during sulphide treatment in Astarte borealis. Catalase activity in the gills of Astarte borealis and the chloragog storage tissue of Arenicola marina were reduced by prolonged hypoxia, while the SOD activities in the tissues were unaffected. A possible involvement of H,O,, partly deriving from autoxidation of respiratory pigments, in H,S oxidation in invertebrate body fluids is discussed. Keywords:
Arenicola
marina;
Astarte
borealis;
Hydrogen
peroxide;
Hypoxia;
Methaemoglobin;
Sulphide
* Corresponding author. Alfred-Wegener-Institut filr Polar- und Meeresforschung, Bremerhaven, Germany. ’ Dedicated to Prof. Hans Theede on the occasion of his 60th birthday. 0022-0981/95/$9.50 0 1995 Elsevier SSDI 0022-0981(94)00172-3
Science B.V. All rights reserved
Columbusstr.,
D-27568
64
D. Ahele-Oeschger,
R. Oeschger
/J. Exp. Mm. Bid. Ed.
187 (1995) 63-80
1. Introduction Autoxidation of haemoglobin (Hb) to methaemoglobin (Hb+ ) involves the producfunctions tion of superoxide anion radicals (0,’ _ ). The ion Fe 2 + in oxy-haemoglobin as electron donor for the univalent reduction of molecular oxygen (Misra & Fridovich, 1972; Elstner, 1990) Hb-Fe2 + + 0, -
Hb-Fe3 + + 0;
-
(1)
According to Winterbourn (1985) evolving oxygen radicals will further react with oxyhaemoglobin yielding oxygen and hydrogen peroxide (H,02): 0;
+ Hb2+-O2
2H’ -
Hb3 + + O2 + H20z
(2)
H,02 formation during autoxidation of vertebrate haemoglobin has been described by Caughey & Watkins (1985) who state that the presence of an effective electron donor, like CN ~, enhances the rate of Hb autoxidation and leads to the direct production of H202
Fe2 + 0, + electron
donor
H’ H,O
Fe3 + .H,O
+ H,O,
+ oxidised donor
(3)
As H20, itself further enhances haemoglobin autoxidation, its uncontrolled presence in blood would lead to a positive feedback on the autoxidative cycle (Misra & Fridovich, 1972). Vertebrate erythrocytes contain high amounts of the H,O, scavenging enzyme catalase to reduce the deleterious effects of this toxic oxygen species. In mammalian erythrocytes catalase and methaemoglobin reductase keep methaemoglobin concentrations well below 1% of total haemoglobin (Elstner, 1990). However, little is known about methaemoglobin formation in invertebrate blood or haemolymph. Misra & Fridovich (1972) state, that haemoglobins from lower animals autoxidise more rapidly than the corresponding mammalian proteins. Generally autoxidation rates increase with elevated temperatures (Elstner, 1990) and with decreasing PO,, and pH (Caughey & Watkins, 1985; Elstner, 1990). The present study deals with two marine benthic infauna species, known for their potential to rapidly adapt to restricted oxygen availability in their habitat. The polychaete Arenicola marina belongs to the intertidal fauna from the German Wadden coast. During summer low tides these animals encounter large and fast variations of abiotic factors like temperature and oxygen availability in the sediment (Toulmond, 1973; Schdttler, 1989). The bivalve Asturte borealis is a sublittoral clam, extraordinarily resistant to anoxia (Theede et al., 1969; Oeschger, 1990). Both species switch to anaerobiosis under insufficient oxygen availability. This involves the accumulation of short chained fatty acids, the end products of anaerobiosis, which cause a decrease in blood pH (Toulmond, 1973; Partner, 1987a,b). Both animals have extracellular haemoglobin in blood (Arenicola marina) or haemolymph (Astute borealis), and it seems likely that haemoglobin autoxidation may occur under hypoxic conditions. Pate1 & Spencer (1963) describe the formation of an oxy-haemoglobin oxidation product, a “brown pigment” in stored crude extracts of Arenicolu marina blood. Their work suggests, that methae-
D. Abele-Oeschger. R. Oeschger 1 J. Exp. Mar. Biol. Ecol. 187 (1995) 63-80
65
moglobin formation results in a change of blood colour from light red to dark red or brown. Interestingly, the amount of brown pigment was positively correlated with sulphide oxidation rates in Arenicola marina blood in vitro. However, Wells & Pankhurst (1980) were not able to detect methaemoglobin absorption at 630 nm in Abarenicola afinis, a related polychaete species, after 22 h of incubation in deoxygenated water in the presence of sulphide. Powell & Arp (1989) state, that methaemoglobin formation in invertebrate blood could be a mere consequence of the poor conditions under which animals are kept in laboratories. Bagarinao & Vetter (1992) were unable to detect methaemoglobin in the blood of the Californian killifish Fzmdulus parvipinnis under normoxic conditions with and without sulphide. The objective of the present study was to clarify whether hypoxia and the onset of an anaerobic metabolism enhances autoxidation of free haemoglobin, and whether this involves H,O, liberation in blood and haemolymph of the animals. Thus methaemoglobin and hydrogen peroxide concentrations were measured in Arenicola marina blood and Astarte borealis haemolymph under normoxic and hypoxic conditions. Hypoxic incubations with sulphide were performed to assess a possible effect of S*- as electron donor, which might further enhance haemoglobin autoxidation and hydrogen peroxide formation. Antioxidant enzyme activities (catalase, superoxide-dismutase, glutathione reductase) were measured in well perfused, metabolically active chloragog (Arenicola marina) and gill (Astarte borealis) tissues, to assess the oxidative stress owing to the internal hydrogen peroxide production. 2. Material and methods 2. I. In vivo hypoxic and sulphide incubations Arenicola marina (L.). For each set of incubations 8-10 worms were collected on intertidal sandflats of the Wadden Sea (Weser Estuary) near Bremerhaven, and kept in natural seawater over night at 10 “C. The incubations were carried out in natural seawater, which was deoxygenated with nitrogen for at least 30 min. The pH was adjusted to 7.8 and controlled every 2 to 5 h. Winkler measurements after the end of the hypoxic incubations yielded oxygen saturations below 2% at 10 “C. Incubations under these extreme hypoxic conditions were conducted over 5 h, to simulate a low tide period, comparable to what the worms encounter in their habitat, and over 2 days to simulate prolonged hypoxia. Incubations under sulphide were carried out for 5 h under hypoxic conditions in a closed system. The concentration of 200 prnol.l-’ sulphide was readjusted every hour, after opening the closed system under argon for photometric determination of the actual sulphide concentrations (Cline, 1969). In this paper the term “sulphide” refers to all forms of dissolved sulphide: H,S, HS -, and S*- . Blood of Arenicola marina (40-100 pi/animal) was sampled from individual animals by opening the body wall and emptying the body cavity of coelomic fluid. Thereafter the large blood vessel passing through the chloragog tissue was opened and the blood sampled into Eppendorf cups. Blood samples were kept on ice under argon, and were immediately analysed for hydrogen peroxide, haemoglobin, and methaemoglobin content, as well as for SOD and catalase activity. The chloragog tissue was analysed for
66
D. Abele-Oeschger,
SOD and catalase Bradford (1976).
R. Oeschger 1 J. Exp. Mar. Bid. Ed.
activity. Protein content
187 (1995) 63-80
of the chloragog tissue was determined
after
Asturte borealis (Schumacher). Clams were collected at Kiel Bight, Western Baltic. Control specimens were kept in natural seawater at 5 “C under normoxia for periods between days and weeks. At one occasion, clams were analysed after keeping them over one night only, to get as close as possible to in situ conditions. These data are referred to as “freshly captured”. All other control animals were acclimated to 10 “C prior to the experiments, i.e. treated like the incubated animals. Incubations were carried out for 2 days and 10 days under hypoxia (pH: 7.8, 10 “C) and for 10 days under hypoxia conditions with 200 pmol.l-’ sulphide. Astarte borealis haemolymph was sampled from individual specimens beneath the mantle with an Eppendorf pipette. Depending on size, an individual yielded between 30 and 80 ~1 of haemolymph. The haemolymph was analysed immediately for the same parameters as the polychaete blood. Catalase, SOD and glutathione reductase activities were measured in the gills and occasionally in the foot. All experiments were carried out between January and April 1994. 2.2. Haemoglobin
and methaemoglobin
measurements
Haemoglobin concentrations were determined in aliquots of blood or haemolymph in 0.9% NaCl at 540 nm. The molar extinction coefficient E,,,,.,-1 at 540 nm = 14 for extracellular oxy-haemoglobin of the polychaete Euzonus mucronata as given by Dangott & Terwilliger (1986) was used for rough determinations of Hb concentrations. Because this approach disregards methaemoglobin (Hb+ ) formation, an exact determination cyan-haemoglobins. was achieved by converting Hb and Hb + to the corresponding After addition of 2.5 mmol.l ml KCN to blood and haemolymph aliquots in 0.9% NaCl, the samples were left at room temperature for 5 min, during which CN - binds only to the methaemoglobin in the sample. Formation of cyanmethaemoglobin resulted in an absorbance increase at 540 nm. At concentrations below 10 mmol.l-‘CNdoes not lead to novel formation of methaemoglobin in a sample, as was shown by Kraus et al. (1992) for Arenicolu cristata. The amount of Hb’ (mmol.ll’) in blood or haemolymph is calculated from the difference of the absorbance at 540 nm before and after the addition of KCN as (A540 k,,-A540)/11, where 11 is E,,,,.,~ 1 at 540 nm given by Assendelft & Zijlstra (1975) for human cyan-methaemoglobin. Subsequently, 2.5 mmol.l-’ K,Fe(CN), were added to convert all oxy-haemoglobin present in the sample to cyan-methaemoglobin. After 5 min reaction time A540 was read again to calculate the total amount of haemoglobin and methaemoglobin in the sample. 2.3. Hydrogen peroxide measurements
in blood and haemolymph
samples
2.3.1. Fluorescence H,O, assay Hydrogen peroxide was analyzed in blood and haemolymph samples using scopoletin as a fluorescent dye. A Kontron SFM 25 fluorimeter was used at 365 nm excita-
D. Abele-Oeschger, R. Oeschger / J. Exp. Mar. Biol. Ecol. 187 (1995) 63-80
67
tion and 490 nm emission wavelengths. The assay is based on the quenching of scopoletin (7-hydroxy-6-methoxy-2H-benzopyran) fluorescence due to oxidation by HzO, in a peroxidase catalysed reaction. To 2.5 ml of phosphate buffer (100 mmol*l-i: 1.36 g KH,PO,+ 1.78 g Na, HP04.2H,0 to 100 ml deionised water, pH 7.0) 20 ~1 of scopoletin solution (Sigma: 0.2 mg in 2 ml phosphate buffer) were added. After stirring, the relative fluorescence was adjusted to 100% reading. Then 20 ~1, equivalent to 14 U of horse-radish peroxidase (EC 1.11.1.7., Serva: 592 U.mg-‘) in phosphate buffer were added, and the subsequent decline of the relative fluorescence, due to the presence of small amounts (< 30 nmol.l-‘) H,O, in the buffer, was recorded on a Kontron Plotter-800. We found that Milli-Q water had higher amounts of up to 50 nmol.l-’ hydrogen peroxide or more, and consequently used deionised water for buffers and standard solutions. After terminating this pre-reaction, 20-50 ~1 of blood or haemolymph were added, and the resulting fluorescence decline recorded. A hydrogen peroxide standard containing 0.4 nmol H,O, in 20 ~1 of deionised water was used to calibrate each sample. Whereas high sensitivity is a clear advantage of the fluorescence assay (detection limit 20 nmol*l-’ H,Oz), a disadvantage is that electron acceptors other than H,O, may interfere. K,Fe(CN), and strong acids spontaneously oxidise scopoletin. Moreover, haemoglobin has peroxidase activity (Halliwell & Gutteridge, 1986), rendering impossible to distinguish scopoletin oxidation by peroxidase from chemical oxidation. In order to confirm, that H,O, was determined, blood proteins were precipitated by adding ~20 mmol*ll’ trichloroacetic acid (TCA) to the sample. Addition of 20 ~1 of the acidified supernatant to the scopoletin assay had no effect on pH in the cuvette. After removal of the blood proteins by centrifugation (10 000 g), the clear supernatant had completely lost its potential to spontaneously oxidise scopoletin. However, when peroxidase and the supernatant were combined, scopoletin oxidation proceeded to the same reading as with haemoglobin. Removal of Astarte borealis haemoglobin by prolonged centrifugation only, yielded the same effect, as removal by acidic precipitation, i.e. loss of peroxidase activity in the supernatant. Peroxidase being required to accomplish scopoletin oxidation in haemoglobin free blood and haemolymph supernatants confirmed the specificity of the assay for H,Oz. 2.3.2.
Polarographic
H,O,
assay
The presence of H,Oz in blood and haemolymph was moreover confirmed by measuring oxygen liberation after the addition of catalase to small volumes (l-l.2 ml) of buffer-sample mixtures in an airtight reaction chamber at 25 “C. Oxygen production was recorded using polarographic oxygen electrodes (Eschweiler, Kiel, Germany) connected to an oximeter (M 200, Eschweiler, Kiel, Germany) and a chart recorder. To 1 ml of previously deoxygenated phosphate buffer (100 mmol.l-‘, pH 7.0), 50-150 ~1 of haemolymph or blood supernatant were added. Precipitation of blood proteins was necessary, because otherwise the liberated oxygen immediately binds to the haem groups, and cannot be detected. Addition of 20-50 ~1 of acidified blood did not cause pH to change in the buffered assay. After equilibration of the initial oxygen saturation in the reaction chamber, 20 ~1 catalase (20 mg.ml-’ phosphate buffer, 2600 U.mg-i) was added with a gas-tight 50 ~1 Hamilton syringe and the resulting oxygen produc-
68
tion recorded. calibration.
D. Abel+Oeschger,
Subsequently,
I?. Oeschger / J. Exp. Mar. Bid. Ed
10 nmol of H20,
187 (I 995) 63-80
in 50 ~1 deionized
water were added for
2.3.3. Enzymatic assays Catalase (EC 1.11.1.6) and superoxide-dismutase (SOD, EC 1.15.1.1) activities were determined in body fluids and tissues. In the tissues, catalase was measured according to Aebi (1985) after extraction into potassium phosphate buffer (50 mmol.ll’, pH 7.0). The body fluids were lysed with water before catalase measurements, and homogenised to break up any kinds of haemocytes, which could have been present. In vitro inhibition of catalase was determined before and after addition of sulphide (final concentration 200 pmol.ll’) to the tissue extract. SOD activity was determined by the inhibition of pyrogallol autoxidation after Marklund & Marklund (1974) at pH 8.2 and 25 ‘C in a pre-aerated tris-succinate buffer (50 mmol.l-I). Glutathione reductase (GR, EC 1.6.4.2.) was determined in the body fluids, the gills and foot tissue of Astarte borealis and the chloragog tissue of Arenicola marina (for detailed descriptions of the SOD- and of the GR-assay see also AbeleOeschger et al., 1994). The total amount of reduced (GSH) and oxidised (GSSG) glutathione was measured spectrophotometrically in respiratory fluids and in tissues using 5,5-dithio-2-nitrobenzoic acid (“Ellmans-reagent”, DTNB). The method is based on the reduction of DTNB by the GSH in the sample. The resulting oxidised GSSG is continuously re-reduced by adding excess GR to the assay. A constant rate of reduction of DTNB is maintained, which can be monitored by an absorbance increase at 405 nm. It is proportional to the amount of GSH + GSSG in the sample, and to the activity of the added GR. A GSSG standard solution (10 pmol.ll’ in water) was used for calibration. For the assay deep-frozen tissue samples were ground with liquid nitrogen and extracted with perchloric acid (1:4 w/v). After centrifugation the extract was neutralised with KOH/KHCO,. 1 ml of NADPH in NaHCO, (8 mg.mll’) and 3 ml DTNB (1 mg. 1 ml-’ NaHCO,) were added to 60 ml of 100 mmol.l-’ phosphate buffer containing 1 mmol.ll’ EDTA (pH 7.0). To 500 ~1 of this “mix”, 50 ~1 of the sample were added, and the reaction was started by addition of 10 ~1 GR (Boehringer, lyophilised, 600 U.mll’). After reading the absorbance increase of the sample (AE,,In+), the reaction was started again with 20 ~1 of the GSSG standard. For a blank, 50 ~1 phosphate buffer were added instead of sample. Glutathione was calculated as pmol GSH + GSSG.g-’ tissue fresh mass and in pmol.ll’ in haemolymph samples. 3. Results 3.1. Hydrogen peroxide concentrations and% methaemoglobin Arenicola marina and Astarte borealis under normoxia
in respiratory fluids
of
Polychaete blood and bivalve haemolymph were compared for haemoglobin content, haemoglobin autoxidation as y0 methaemoglobin, and hydrogen peroxide concentrations under normoxia. The haem concentration was lo-fold higher in Arenicola marina blood (1.8 & 0.7 mmol.ll’, n = 8) as compared to Astarte borealis haemolymph (0.194 + 0.06 mmol.ll’,
D. Abe/e-Oeschger,
loo
0
R. Oeschger / J. Exp. Mar. Bid. Ecol. 187 (1995) 63-80
69
t
0
--__I-
A_-
I
/
’ mmol 1-l hgemoglobin
4
3
Fig. 1. Arenicola marina and Asturte borealis. Linear regression of hydrogen globin (mmol.l-‘) in blood (r = 0.84) and haemolymph (r= 0.76)
peroxide
(~mol~l~‘) vs. haemo-
n = 13). The concentrations of hydrogen peroxide in both respiratory fluids were 154 f 42 ~mol.l11H,02 in Arenicolu marina (n = 8) and 15.3 k 4.6 pmol.ll’ H,Oz in Asturte borealis (n = 13). The relative amount of hydrogen peroxide per mmol Hb was about the same in both species: 85.5 k 17 pmol (Arenicolu marina) and 78.9 f 27 vmol H,02 (Astarte borealis). Haemoglobin autoxidation levels in % methaemoglobin (Hb+ ) of total haemoglobin (Hb + Hb + ) were 4 k 2.8 y0 (Arenicolu marina, n = 8) and 3 + 4.6 y0 (Asturte borealis, n = 13). While the data of Arenicolu marina represent freshly captured animals, i.e. kept under normoxia for less than 24 h, the clams had been kept under normoxia in the laboratory for up to several weeks. Freshly captured Asturte borealis individuals, analysed within 24 h after collection, had less than 0.6 & 1 y0 Hb’ (n = 9). The relatively high standard deviation is due to the fact, that out of 10 freshly captured Asturte borealis individuals, only two clams had detectable amounts of methaemoglobin. In blood and haemolymph, a linear correlation was found between haemoglobin concentrations and hydrogen peroxide levels in individual specimens (Fig. 1). We also found, that under normoxia the percent fractions of methaemoglobin correlated positively with the amount of hydrogen peroxide. 3.1 .I. Hypoxic incubations with and without sulphide The effect of hypoxic and sulphidic incubations on the percentage of methaemoglobin formation, and on the hydrogen peroxide concentrations in the respiratory fluids of
70
D. Abele-Oeschger,
incubated and of control Fig. 3 (Astarte borealis).
R. Oeschger / J. Exp. Mar. Bid. Ed.
animals,
is summarised
187 (1995) 63-80
in Fig. 2 (Arenicolu
marina)
and
Arenicolu
marina.. 5 h hypoxic incubation led to a significant (p< 0.05) increase of haemoglobin autoxidation from 4 + 2.8% Hb’ (n = 7) under normoxia to 10.6 + 5.7% Hb+ . Prolonged hypoxia of 2 days resulted in 12.4 k 7.3% Hb’ (n = 6). 5 h of hypoxic incubation with 200 prnol.l-’ sulphide reduced the percentage of methaemoglobin statistically insignificant to 7.5 f 0.7% compared to hypoxia (n = 6), which was still significantly (p< 0.05) higher, than under normoxia (Fig. 2a). Peroxide concentrations in blood increased steadily from normoxia over short-term (5 h, n = 7) and prolonged (2 days, II = 6) hypoxia, and were even higher after 5 h hypoxic incubation with _ 200 pmol.l-’ sulphide (n = 6, Fig. 2b).
a
A. marina
normox
5h
hypox
2d
hypox
5h
hypox
+
350 ,
8
1
A.
marina
“OrlTlOX
I
5h
hypox
2d
hypox
5h
b
hypox+s
Fig. 2. Arenicola marina. (a) Percent methaemoglobin f SD in blood under normoxia, 5 h hypoxia, 2 day hypoxia and 5 h hypoxic incubation with 200 pmol.1~’ sulphide, n = 5-8. **: significantly different from normoxic control (p c 0.05). (b) pmol.1~ ‘hydrogen peroxide + SD in blood under normoxia, 5 h hypoxia, 2 day hypoxia and 5 h hypoxic incubation with 200 pmol.l-’ sulphide, n = 5-8. ***: significantly different from normoxic control (pcO.01).
D. Abe&Oeschger,
71
R. Oeschger / J. Exp. Mar. Bid. Ecol. 187 (1995) 63-80
20 A.
a
borealis
0 “CWKIOX
2d
hypox
10d
hypox
1Od hypox+a
60
b
A. borealis .s 50 ;: ; cL4o : (II e D 30 r” 320
10 “OVllOX
2d
hypox
10d
hypox
1 Od hypox+s
Fig. 3. Astute borealis. (a) Percent methaemoglobin k SD in haemolymph under normoxia, 2 day hypoxia, 10 day hypoxia and 10 day hypoxic incubation with 200 pmol.l-’ sulphide, n = 8-10. ***: significantly different from normoxic control (p< 0.01). (b) pmol.l-’ hydrogen peroxide k SD in haemolymph under normoxia, 2 day hypoxia, 10 day hypoxia and 10 day hypoxic incubation with 200 prnol.l-’ sulphide, n = 8-10. ***: significantly different from normoxic control (p< 0.01).
Astarte borealis. After 10 days of hypoxic incubation the mean Hb’ content amounted to 11.3 + 4.9% (n = 10) of total haemoglobin in Astarte borealis haemolymph which was significantly higher (PC 0.01) than in the normoxic control group (Fig. 3a). H,O, haemolymph levels in the clams increased statistically insignificant during hypoxic incubations, amounting to 21.3 + 5.3 pmol.l-’ H,O, (n = 10) after 10 days of hypoxia without sulphide (Fig. 3b). 10 days of hypoxic incubation with sulphide (n = 10) yielded significantly (p < 0.0 1) higher hydrogen peroxide levels than mere hypoxia, although methaemoglobin concentrations had decreased in the haemolymph. Under all experimental conditions hydrogen peroxide concentrations were positively correlated with total haemoglobin. Fig. 4 shows linear regressions of the prnol.l-’ hydrogen peroxide concentrations in haemolymph of individual Astarte borealis specimens per total haemoglobin (Hb + Hb + ) under normoxic conditions and under hypoxic
12
D. Abele-Oeschger,
Oo----~~ ’ ~031
R. Oeschger / J. Exp.
Mar. Bid.
Ed.
-
02
187 (1995J
I-
093 094 mmol I -I haemoglobin
63-80
--L
I
095
Fig. 4. Astarte borealis. Hydrogen peroxide (pmol.l-‘) vs. haemoglobin under normoxia 10 day hypoxia (r = 0.95, n = 1 l), and 10 day hypoxia + sulphide (r = 0.95, n = 9)
096
(r = 0.77, n = lo),
conditions with and without sulphide. Like the absolute hydrogen peroxide levels the ratio of hydrogen peroxide:total Hb increased under hypoxia, and further under hypoxia with sulphide. All individuals from the sulphidic incubations had higher hydrogen peroxide haemolymph concentrations than clams kept under normoxia and hypoxia. Hydrogen peroxide levels in Arenicola marina blood were unalfected by experimental hydrogen peroxide exposure. Incubation with 10 pmol.ll’ H,O, did not alter the blood hydrogen peroxide concentrations (Buchner, unpubl. results). Hydrogen peroxide incubations were not performed with the clam, because the sublitloral species Astarte borealis hardly encounters elevated hydrogen peroxide levels in its habitat. 3.1.2. Spectrophotometric scans Arenicola marina and Astarte borealis have characteristic oxy-haemoglobin absorption bands at 540 and 516 nm, and of deoxygenated haemoglobin at 555 nm. When the respiratory fluid of either species was stored in the refrigerator for several days an increase of absorbance at 630 nm showed, that Hb had autoxidised to Hb’. Fig. 5. depicts absorption spectra of Arenicola marina blood, containing zz 30% Hb’ After addition of KCN a decrease at 630 nm and an increase at 540 nm are due to the formation of cyan methaemoglobin. Neither under in vivo nor under in vitro conditions with sulphide, were we able to achieve formation of sullhaemoglobin, which would have been perceived from an absorbance increase at 618 nm (Bagarinao & Vetter, 1992) in Arenicola marina blood. In contrast Astarte borealis haemolymph showed a slight increase at 618 nm 45 min after addition of 375 pmol 1-l sulphide in vitro. Sulfhaemoglobin formation in vivo was never observed in our incubations (200 prnol.1~’ sulphide). Under normoxic conditions formation of sulfmethaemoglobin (SHb+ ) from Hb’ in vitro was observable in
D. Abele-Oeschger, R. Oeschger 1 J. Exp. Mar. Bid. Ed.
187 (199.5) 63-80
13
0.6
z s
0.4 -
13 b $ 0.2 0.0 400
I 500
4 600
680
Fig. 5. Arenicola marina. Spectral scan of whole blood, containing 30% methaemoglobin, before and after addition of KCN. The absorbance increase at 540 nm after KCN addition was used for the determination of methaemoglobin in the sample.
haemolymph which had been completely autoxidised. The SHb+ spectrum compared well with spectral scans given by Bagarinao & Vetter (1992) for SHb’ at a neutral blood pH of 7.8. Characteristic is the absorbance decrease at 630 nm (Hb+ ) and an absorption band at 535 nm with a shoulder at 568 nm. SHb+ formation in vivo was not detected in incubated Astarte borealis specimens after sulphide incubations (Fig. 6.). 3.1.3. Enzymes Table 1 summarises the activities of the antioxidant enzymes catalase, SOD and glutathione reductase in blood and chloragog tissue of Arenicola marina and the haemolymph and the gills of Astarte borealis under normoxia and after hypoxic and sulphidic incubations. For comparison, catalase activities were measured in the body wall of the worm, and in the foot muscle (not referred to in Table 1) of the clam. Arenicola marina. Whereas the chloragog tissue of the polychaetes had high activities of all three antioxidant enzymes, the only enzyme measurable in blood was SOD. Moreover, no glutathione could be detected by the enzymatic assay in polychaete blood, while tissue glutathione levels of 0.14 prnol*g-’ fresh mass were found in the chloragog. SOD activity per ml blood increased statistically insignificant from 0.071 U.ml-’ blood (normoxia, n = 8) to 0.105 U.mll’ under hypoxic (n = 5) and sulphidic conditions (n = 5, Table 1). In contrast to 480 U catalase activity.mg-’ protein in the chloragog storage tissue, 0.4
0.0 Fig. 6. Astarte borealis. Formation of sulfmethaemoglobin in haemolymph after complete autoxidation in formation is achieved by adding 370 pmol.l-’ vitro, yielding a 100% methaemoglobin solution. SHb’ sulphide. Hb + : methaemoglobin, SHb + : sulfmethaemoglobin
74
D. Abele-Oeschger,
R. Oeschger 1 J. Exp. Mar. Bid. Ed.
Table 1 Antioxidant enzyme activities in tissues (n = 8-10) and the bivalve A. borealis Arenicola marina
CAT
blood
15.4 k 5.3 0.07 kO.036 16.1 24.9 0.07 +0.03 16.8 k 5.0 0.105 + 0.04
0.017 +_O.Ol 0.008+0.003* nd
0 nd nd
(n = 8) (n = 5) (n = 5)
18.3 + 5.3 0.105 _t 0.06
0.013 + 0.006
nd
(n=5)
SOD
GR
chloragog
Normoxia Hypoxia (5 h) Hypoxia (2 days) Hypoxia + sulphide (5 h)
480+113 294+ 103* 123 5 18.5**
0 0 0
126+38**
0
Astarte borealis
CAT
Normoxia Hypoxia (2 days) Hypoxia (10 days) Hypoxia + sulfide (10 days)
134.6&44 0 185 253 0 41.1 + 23.5** 0 78.5+27*
0
A. marina
chloragog
blood
haeml
fluids (n = 8) of the polychaete
GR
SOD
chloragog
gills
and respiratory
187 (1995) 63-80
gills
blood
haeml
11.124.1 0.20~0.10 18.8 + 6.5 nd. 18.2k4.1 0.16kO.06
gills
haeml
0.015 2 0.004 0.09 f 0.02* nd
0.058 k 0.024 nd nd
(n = 10) (n = 8) (n = 8)
nd
(n = 8)
16.9 + _ 8.3 0.02 + _ O.Ol** 0.144 f 0.042**
The control group was kept under normoxic conditions for several days (A. borealis) and for 24 h (A. marina). Tissue data in U.mg- ’ protein, means f SD, blood and haemolymph data in U.ml- ‘, means f SD. CAT, catalase; SOD, superoxide-dismutase; GR, glutathione reductase; haeml, haemolymph; nd, not determined. *: significantly different from normoxic controls (p
