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Nephrotoxic and haematological effects of mercuric chloride in the plaice (Pleuronectes platessa L.)
Aquatic Toxicology, 8 (1986) 77-84
77
Elsevier
AQT
00188
NEPHROTOXIC AND I-IAEMATOLOGICAL CHLORIDE
IN THE
PLAICE
EFFECTS
(PLEURONECTES
OF MERCURIC
P L A T E S S A L.)
T.C. FLETCHER and A. WHITE
N.E.R.C. Institute of Marine Biochemistry, St. Fittick's Road, Aberdeen ABI 3RA, U.K. (Received 5 June 1985; revised version received 20 December 1985; accepted 20 January 1986)
Exposure of plaice (Pleuronectesplatessa L.), a marine teleost, to 0.3 ppm Hg (as HgCI2) resulted in a reduction in blood haematocrit values over seven days due to erythrocyte lysis. The decrease was significant within four days of exposure and by seven days there was a 72% reduction in prebleed haematocrit values. There was also a steady decline in serum lysozyme but not in serum protein concentration. The spleen is actively phagocytic for effete erythrocytes and although not erythropoietic, splenomegaly occurred by day 6. Urine analysis showed significant proteinuria by day 3, but, although kidney proximal tubule damage was severe, the onset of lysozymuria was not observed until the seventh day of exposure, presumably only occurring when tubular damage extended to the absorptive area of the first proximal segment. Lysozymuria, as in mammals, may be a relatively specific indication of renal tubular damage in fish. Key words: kidney; mercuric chloride; lysozyme; urine; erythrocytes; plaice
INTRODUCTION
Teleosts show extensive renal accumulation of heavy metals following both acute and chronic exposure. This is inevitable given the role of the kidney in the elimination of potentially toxic chemicals and must in some instances be associated with renal damage, although the causative mechanisms are difficult to establish (Pritchard and Renfro, 1984). The plaice, Pleuronectesplatessa, is no exception and the kidney, together with the spleen and blood cells, exhibits high rates of inorganic Hg uptake from sea water (Pentreath, 1976). Trump, Jones and Sahaphong (1975) have attempted to detail the cellular changes in teleost kidneys exposed to HgCIE and observed selective necrosis of the second and probably first proximal segments of the tubules. Injection of rats with HgCI2 also results in localized tubular damage with the appearance of the enzyme lysozyme in the urine (Prockop and Davidson, 1964). They found, however, that little lysozymuria occurred when specific glomerular damage was induced by injection of an antiserum to rat kidney. Urinary excretion of lysozyme can therefore be indicative of tubular dysfunction. The ubiquitous lysozyme whose biological role is still not known (Joll~s and Joll~s, 1984) has been 0166-445X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
78
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characterized in the plaice (Fletcher and White, 1976) although its concentration in urine was not examined. The current work was therefore undertaken to establish whether urinary lysozyme levels could be used as a marker of Hg-induced nephrotoxicity in the plaice. MATERIALS AND METHODS
Plaice (250-350 g), caught by seine net o f f the Aberdeen coast, were transferred to the aquarium for an acclimation period of 2 wk in tanks of re-cycled sea water maintained at 10-12°C. The fish were then transferred to individual plastic tanks, 48 x 28 x 25 cm deep containing 6 1 aerated sea water with or without 0.3 p p m Hg (as HgC12). These exposure solutions were changed every 24 h to maintain Hg concentrations and reduce the accumulation of waste products. The plaice were not fed during the exposure period. For some experiments, blood samples ( < 0.1 ml) were collected daily f r o m the caudal vein of individual fish into heparinized microhaematocrit tubes and the percentage of cells measured in relation to the plasma, after centrifuging for 5 min at 12000 × g. The haematocrit values were measured for all fish at the start of an experiment. At the end of the experimental period, blood was collected f r o m the caudal vein, the plaice killed by a blow on the head and the body weighed. Final haematocrits were determined and blood smears were made and stained with Giemsa. Serum was collected from the blood after clotting at 5°C and either tested directly or stored at - 2 0 ° C . The body cavity of the fish was opened and if the bladder was visibly distended, the urine collected into a syringe with a 21-G needle. Spleens and kidneys were removed and weighed and the spleen index calculated as spleen weight (mg) per weight of fish (g). The kidneys were homogenized with a Polytron ® Homogenizer (Northern Media, Brough, York, U.K.) in a six-fold volume of phosphate buffer (0.06 M, p H 6.0 containing 0.09°7o NaC1), centrifuged at 3000 x g at 4°C and the supernatant fraction analyzed for lysozyme and protein concentrations. Total protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. Lysozyme activity was assayed as described by Fletcher and White (1976). Three times crystallized hen egg white lysozyme (Worthington) was used as a standard and and results expressed as/~g lysozyme per ml of serum or urine and/~g per mg protein for kidney. Statistical treatment of the data was performed by Student's t-test. Significant differences compared with controls were established at __<0.01. RESULTS
From the results presented in Table 1, the most marked effect of exposure to
80 HgCI2 is the r e d u c t i o n in h a e m a t o c r i t value f o r each g r o u p . This r e d u c t i o n is signific a n t on d a y s 4, 6 a n d 7 (all at P < 0.001) a n d on d a y 5 ( P < 0 . 0 1 ) . T h e m e a n h a e m a t o c r i t value (_+ SE) for the 62 plaice n o t e x p o s e d to H g ( T a b l e I) was 18.03 +_ 0.67°70. T h e p r e b l e e d values for the six e x p o s e d g r o u p s were similar to the c o n t r o l
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Fig. 1. Plaice blood smears stained with Giemsa. (a) Before exposure to HgCI2. (b) 7 days after exposure to 0.3 ppm HgCI2.
81
value, but were reduced by 72°7o after 7 days. This reduction was due to lysis of the red cells, with 30070 of the blood samples showing haemolysis by day 2 of exposure, increasing to 70°7o by day 3. Examination of blood smears made on day 7 of Hgexposure showed a dramatic reduction in the number of erythrocytes. The few remaining cells were in the process of losing their discrete outline and cytoplasm, with deposited nuclei visible in the smear (Fig. 1). Haematocrit values from fish bled daily also showed a steady decline (Fig. 2) and when compared with daily-bled controls the difference was significant on days 1 and 2 ( P < 0 . 0 1 ) and on day 7 (P<0.001). The serum lysozyme values (Table I) all declined after exposure to Hg from day 3 onwards but the difference was only significant on day 4 ( P < 0.01). There was no significant change in the serum protein concentration over the experimental period: 16 plaice with a mean prebleed value of 27.3 _+ 1.2 mg protein/ml had a value of 28.0 _+ 1.5 after the 7 days o f exposure. The bladder was not catheterized and urine samples were only collected immediately after the plaice were killed and could not therefore be collected as easily as blood samples for routine monitoring of the effects of Hg. The urine samples however showed significant proteinuria within 3 days o f exposure ( P < 0.001) when compared with the day 0 control group values. The statistical difference was maintained at P < 0.01 on day 5 and P < 0.001 on day 7. Unlike the protein values, the lysozyme values for the same urine samples were only significantly elevated above the controls on day 7 ( P < 0.001). Similar results were found for the lysozyme content of the whole kidney which, although expressed on a protein basis, were also only significantly high on day 7 ( P < 0.001). The spleen indices (Table I) were significantly higher than the control group after 6 days ( P < 0.001) and seven days ( P < 0.01) o f Hg-exposure.
100
=
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60
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g
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c
o
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I
I
I
I
I
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0
1
2
3
4
5
6
7
Days
Fig. 2. Percentage change in daily haematocrit values of plaice exposed to 0.3 ppm HgCI2 (#) and controls (e). Values represent mean and SE of 6 fish.
82 DISCUSSION The most probable cause of Hg toxicity is the affinity for SH-groups. Lysozyme is an unusual protein in that it lacks sulphydryl groups although Ting and Schrier (1977) reported some binding of Hg to hen egg white lysozyme with carboxyl and amide groups participating. Balazs and Roepke (1966) found, however, that urine from rats dosed with HgClz in no way interfered with the assay of lysozyme and no problems were experienced with assays of plaice tissues. Preston (1960) reported considerable variation in the packed cell volume of plaice blood, in relation to weight and season, but the anaemia characteristic of metal toxicity (Dawson, 1979) was apparent in our Hg-exposed plaice with a rapid and significant decrease in haematocrit values and visible haemolysis. Significant decreases in haematocrit values have been reported for another member of the family Pleuronectidae, the winter flounder, Pseudopleuronectes americanus (Dawson, 1979) and also for striped bass, Morone saxatilis (Dawson, 1982), after exposure to 10 ppb HgCI2 for 60 days. Within 48 h of exposure to 0.181 ppm HgCiz, degenerative changes in erythrocyte morphology have been observed in Barbus conchonius (Gill and Pant, 1985). Conflicting results can, however, appear with the length of exposure of different species because of their different LCs0 for HgCI2. Juneja and Mahajan (1983) found increased haematocrit values over 7 days in the freshwater Channa punctatus exposed to 0.136 ppm HgClz, although this could be due to erythropoiesis initially over-compensating in order to meet the higher oxygen requirements caused by erythrocyte loss. Damage to erythrocytes would, however, be consistent with known membrane effects of Hg, Arbuthnott (1962) having observed the haemolytic action of HgCI2 on mammalian cells, while the uptake of Hg by plaice blood cells (Pentreath, 1976) provides the conditions for direct cell damage. Although the affinity of Hg for SH-groups in membrane proteins would affect membrane conformation and permeability, Ribarov and Benov (1981) have suggested that the peroxidation of membrane iipids is also a possible mechanism of damage to the erythrocyte membrane in metal-induced haemolysis. The significant increase in spleen index by day 6 coincides with low haematocrit values. The spleen is an important phagocytic organ in the plaice, with avidity for erythrocytes (MacArthur et al., 1983) and there is the possibility that effete erythrocytes could account for some increase in splenic weight. Stimulation of erythropoietic tissue has been observed in the spleen and kidney of C. punctatus over the first 7 days of exposure to HgCI2 at 0.136 ppm (Juneja and Mahajan, 1983). This is unlikely to account for the splenomegaly observed in the plaice, where erythropoiesis appears to be a minor activity of the spleen (Ellis et al., 1976). Mercuric chloride has, however, been reported to be mitogenic for human peripheral lymphocytes (Caron et al., 1970). Mercury is concentrated in the plaice spleen (Pentreath, 1976) but whether it directly stimulates proliferation of the splenic lymphoid tissue is not known.
83 The lysozyme present in m a m m a l i a n serum, as a low molecular weight protein, is filtered by the kidney glomerulus and reabsorbed by the proximal tubular cells and is not normally present at concentrations greater than 4 # g / m l urine (Prockop and Davidson, 1964). Lysozyme concentrations in the range 0-9.8/~g/ml urine have been reported in P. americanus (Maack and Kinter, 1969) and are consistent with the values found in the plaice. Maack and Kinter (1969) injected winter flounder with egg white lysozyme and found filtration and reabsorption as in mammals. The plaice exposed to Hg showed reduced serum lysozyme levels which could be due to increased excretion or decreased enzyme production. Our results show increased enzyme excretion, although lysozymuria was only significant after seven days exposure. Presumably there was no concomitant increase in enzyme release into the serum. At day 7 there was also a significant increase in the lysozyme content of the whole kidney. Although the lysozyme values for the kidney were expressed on a protein basis, there was little change in the protein content of comparable weights of kidney and the results represent a real increase in total lysozyme. The plaice kidney, as with rats (Litwack, 1955), normally contains more lysozyme activity than other tissues (Fletcher and White, 1973). This is due in the plaice to the abundance of leucocytes in the kidney stroma which are probably the source of the endogenous lysozyme (Murray and Fletcher, 1976) and to the kidney being a site of lysozyme catabolism (Ottosen, 1978), as in mammals. The kidney is the main haemopoietic tissue of the plaice (Ellis et al., 1976) and because of the lytic effects of the Hg on circulating blood cells, it is possible that stimulation of this tissue includes an increase in lysozyme producing leucocytes. It is interesting that none of this extra kidney lysozyme can boost the serum levels. Lysozymuria is indicative of kidney proximal tubule damage in the plaice, but it appears only when the damage is extensive, while significant proteinuria occurs earlier in Hg exposure. The collection of urine from the plaice is, however, more difficult than the collection of blood and the easily performed haematocrit determination gives an early warning of anaemia. Only when the latter occurs might it then be useful to collect and analyze urine. ACKNOWLEDGEMENTS We thank Mr. C.K. Murray for sectioning and staining kidneys from the plaice and Ms. B.E. Stroud for photography. REFERENCES Arbuthnott, J.P., 1962. Haemolytic action of mercurials. Nature (London) 196, 277-278. Balazs, T. and R.R. Roepke, 1966. Lysozymuria induced in rats by nephrotoxic agents. Proc. Soc. Exp. Biol. Med. 123, 380-385. Caron, G.A., S. Poutala and T.T. Provost, 1970. Lymphocyte transformation induced by inorganic and organic mercury. Int. Arch. Allergy Appl. Immunol. 37, 76-87.
84 Dawson, M.A., 1979. Hematological effects of long-term mercury exposure and subsequent periods of recovery on the winter flounder, Pseudopleuronectes americanus. In: Marine pollution: functional responses, edited by W.B. Vernberg, A. Calabrese, F.P. Thurberg and F.J. Vernberg, Academic Press, New York, pp. 171-182. Dawson, M.A., 1982. Effects of long-term mercury exposure on hematology of striped bass, Morone saxatilis. Fish. Bull. 80, 389-392. Ellis, A.E., A.L.S. Munroe and R.J. Roberts, 1976. Defence mechanisms in fish 1. J. Fish Biol. 8, 67-78. Fletcher, T.C. and A. White, 1973. Lysozyme activity in the plaice (Pleuronectes platessa L.). Experientia 29, 1283-1285. Fletcher, T.C. and A. White, 1976. The lysozyme of the plaice Pleuronectesplatessa L. Comp. Biochem. Physiol. 55B, 207-210. Gill, T.S. and J.C. Pant, 1985. Mercury-induced blood anomalies in the freshwater teleost Barbus conchonius Ham. Water Air Soil Pollut. 24, 165-171. Joll~s, P. and J. Joll~s, 1984. What's new in lysozyme research? Mol. Cell. Biochem. 63, 165-189. Juneja, C.J. and C.L. Mahajan, 1983. Haematological and haemopoietic changes in fish Channa punctatus due to mercury pollution in water. Indian J. Anim. Res. 17, 63-71. Litwack, G., 1955. Photometric determination of lysozyme activity. Proc. Soc. Exp. Biol. Med. 89, 401-403. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Maack, T. and W.B. Kinter, 1969. Transport of protein by flounder kidney tubules during long-term incubation. Am. J. Physiol. 216, 1034-1043. MacArthur, J.l., T.C. Fletcher and A.W. Thomson, 1983. Distribution of radiolabeled erythrocytes and the effect of temperature on clearance in the plaice (Pleuronectes platessa L.). J. Reticuloendothel. Soc. 34, 13-21. Murray, C.K. and T.C. Fletcher, 1976. The immunohistochemical localization of lysozyme in plaice (Pleuronectes platessa L.) tissues. J. Fish Biol. 9, 329-334. Ottosen, P.D., 1978. Reversible peritubular binding of a cationic protein (lysozyme) to flounder kidney tubules. Cell. Tiss. Res. 194, 207-218. Pentreath, R.J., 1976. The accumulation of inorganic mercury from sea water by the plaice, Pleuronectes platessa L. J. Exp. Mar. Biol. Ecol. 24, 103-119. Preston, A., 1960. Red blood values in the plaice (Pleuronectes platessa L.). J. Mar. Biol. Assoc. U.K. 39, 681-687. Pritchard, J.B. and J.L. Renfro, 1984. Interactions of xenobiotics with teleost renal function. In: Aquatic toxicology, Vol. 2, edited by L.J. Weber, Raven Press, New York, pp. 51-106. Prockop, D.J. and W.D. Davidson, 1964. A study of urinary and serum lysozyme in patients with renal disease. N. Engl. J. Med. 270, 269-274. Ribarov, S.R. and L.C. Benov, 1981. Relationship between the hemolytic action of heavy metals and lipid peroxidation. Biochim. Biophys. Acta 640, 721-726. Ting, D.C.Y. and E.E. Schrier, 1977. Binding of mercury (II) ion to hen egg white lysozyme and bovine pancreatic ribonuclease A. J. Agric. Food Chem. 25, 158-162. Trump, B.F., R.T. Jones and S. Sahaphong, 1975. Cellular effects of mercury on fish kidney tubules. In: The pathology of fishes, edited by W.E. Ribelin and G. Migaki, The University of Wisconsin Press, Madison, pp. 585-612.
77
Elsevier
AQT
00188
NEPHROTOXIC AND I-IAEMATOLOGICAL CHLORIDE
IN THE
PLAICE
EFFECTS
(PLEURONECTES
OF MERCURIC
P L A T E S S A L.)
T.C. FLETCHER and A. WHITE
N.E.R.C. Institute of Marine Biochemistry, St. Fittick's Road, Aberdeen ABI 3RA, U.K. (Received 5 June 1985; revised version received 20 December 1985; accepted 20 January 1986)
Exposure of plaice (Pleuronectesplatessa L.), a marine teleost, to 0.3 ppm Hg (as HgCI2) resulted in a reduction in blood haematocrit values over seven days due to erythrocyte lysis. The decrease was significant within four days of exposure and by seven days there was a 72% reduction in prebleed haematocrit values. There was also a steady decline in serum lysozyme but not in serum protein concentration. The spleen is actively phagocytic for effete erythrocytes and although not erythropoietic, splenomegaly occurred by day 6. Urine analysis showed significant proteinuria by day 3, but, although kidney proximal tubule damage was severe, the onset of lysozymuria was not observed until the seventh day of exposure, presumably only occurring when tubular damage extended to the absorptive area of the first proximal segment. Lysozymuria, as in mammals, may be a relatively specific indication of renal tubular damage in fish. Key words: kidney; mercuric chloride; lysozyme; urine; erythrocytes; plaice
INTRODUCTION
Teleosts show extensive renal accumulation of heavy metals following both acute and chronic exposure. This is inevitable given the role of the kidney in the elimination of potentially toxic chemicals and must in some instances be associated with renal damage, although the causative mechanisms are difficult to establish (Pritchard and Renfro, 1984). The plaice, Pleuronectesplatessa, is no exception and the kidney, together with the spleen and blood cells, exhibits high rates of inorganic Hg uptake from sea water (Pentreath, 1976). Trump, Jones and Sahaphong (1975) have attempted to detail the cellular changes in teleost kidneys exposed to HgCIE and observed selective necrosis of the second and probably first proximal segments of the tubules. Injection of rats with HgCI2 also results in localized tubular damage with the appearance of the enzyme lysozyme in the urine (Prockop and Davidson, 1964). They found, however, that little lysozymuria occurred when specific glomerular damage was induced by injection of an antiserum to rat kidney. Urinary excretion of lysozyme can therefore be indicative of tubular dysfunction. The ubiquitous lysozyme whose biological role is still not known (Joll~s and Joll~s, 1984) has been 0166-445X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
78
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79
characterized in the plaice (Fletcher and White, 1976) although its concentration in urine was not examined. The current work was therefore undertaken to establish whether urinary lysozyme levels could be used as a marker of Hg-induced nephrotoxicity in the plaice. MATERIALS AND METHODS
Plaice (250-350 g), caught by seine net o f f the Aberdeen coast, were transferred to the aquarium for an acclimation period of 2 wk in tanks of re-cycled sea water maintained at 10-12°C. The fish were then transferred to individual plastic tanks, 48 x 28 x 25 cm deep containing 6 1 aerated sea water with or without 0.3 p p m Hg (as HgC12). These exposure solutions were changed every 24 h to maintain Hg concentrations and reduce the accumulation of waste products. The plaice were not fed during the exposure period. For some experiments, blood samples ( < 0.1 ml) were collected daily f r o m the caudal vein of individual fish into heparinized microhaematocrit tubes and the percentage of cells measured in relation to the plasma, after centrifuging for 5 min at 12000 × g. The haematocrit values were measured for all fish at the start of an experiment. At the end of the experimental period, blood was collected f r o m the caudal vein, the plaice killed by a blow on the head and the body weighed. Final haematocrits were determined and blood smears were made and stained with Giemsa. Serum was collected from the blood after clotting at 5°C and either tested directly or stored at - 2 0 ° C . The body cavity of the fish was opened and if the bladder was visibly distended, the urine collected into a syringe with a 21-G needle. Spleens and kidneys were removed and weighed and the spleen index calculated as spleen weight (mg) per weight of fish (g). The kidneys were homogenized with a Polytron ® Homogenizer (Northern Media, Brough, York, U.K.) in a six-fold volume of phosphate buffer (0.06 M, p H 6.0 containing 0.09°7o NaC1), centrifuged at 3000 x g at 4°C and the supernatant fraction analyzed for lysozyme and protein concentrations. Total protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as standard. Lysozyme activity was assayed as described by Fletcher and White (1976). Three times crystallized hen egg white lysozyme (Worthington) was used as a standard and and results expressed as/~g lysozyme per ml of serum or urine and/~g per mg protein for kidney. Statistical treatment of the data was performed by Student's t-test. Significant differences compared with controls were established at __<0.01. RESULTS
From the results presented in Table 1, the most marked effect of exposure to
80 HgCI2 is the r e d u c t i o n in h a e m a t o c r i t value f o r each g r o u p . This r e d u c t i o n is signific a n t on d a y s 4, 6 a n d 7 (all at P < 0.001) a n d on d a y 5 ( P < 0 . 0 1 ) . T h e m e a n h a e m a t o c r i t value (_+ SE) for the 62 plaice n o t e x p o s e d to H g ( T a b l e I) was 18.03 +_ 0.67°70. T h e p r e b l e e d values for the six e x p o s e d g r o u p s were similar to the c o n t r o l
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Fig. 1. Plaice blood smears stained with Giemsa. (a) Before exposure to HgCI2. (b) 7 days after exposure to 0.3 ppm HgCI2.
81
value, but were reduced by 72°7o after 7 days. This reduction was due to lysis of the red cells, with 30070 of the blood samples showing haemolysis by day 2 of exposure, increasing to 70°7o by day 3. Examination of blood smears made on day 7 of Hgexposure showed a dramatic reduction in the number of erythrocytes. The few remaining cells were in the process of losing their discrete outline and cytoplasm, with deposited nuclei visible in the smear (Fig. 1). Haematocrit values from fish bled daily also showed a steady decline (Fig. 2) and when compared with daily-bled controls the difference was significant on days 1 and 2 ( P < 0 . 0 1 ) and on day 7 (P<0.001). The serum lysozyme values (Table I) all declined after exposure to Hg from day 3 onwards but the difference was only significant on day 4 ( P < 0.01). There was no significant change in the serum protein concentration over the experimental period: 16 plaice with a mean prebleed value of 27.3 _+ 1.2 mg protein/ml had a value of 28.0 _+ 1.5 after the 7 days o f exposure. The bladder was not catheterized and urine samples were only collected immediately after the plaice were killed and could not therefore be collected as easily as blood samples for routine monitoring of the effects of Hg. The urine samples however showed significant proteinuria within 3 days o f exposure ( P < 0.001) when compared with the day 0 control group values. The statistical difference was maintained at P < 0.01 on day 5 and P < 0.001 on day 7. Unlike the protein values, the lysozyme values for the same urine samples were only significantly elevated above the controls on day 7 ( P < 0.001). Similar results were found for the lysozyme content of the whole kidney which, although expressed on a protein basis, were also only significantly high on day 7 ( P < 0.001). The spleen indices (Table I) were significantly higher than the control group after 6 days ( P < 0.001) and seven days ( P < 0.01) o f Hg-exposure.
100
=
80
u
60
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g
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o
20
I
I
I
I
I
I
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0
1
2
3
4
5
6
7
Days
Fig. 2. Percentage change in daily haematocrit values of plaice exposed to 0.3 ppm HgCI2 (#) and controls (e). Values represent mean and SE of 6 fish.
82 DISCUSSION The most probable cause of Hg toxicity is the affinity for SH-groups. Lysozyme is an unusual protein in that it lacks sulphydryl groups although Ting and Schrier (1977) reported some binding of Hg to hen egg white lysozyme with carboxyl and amide groups participating. Balazs and Roepke (1966) found, however, that urine from rats dosed with HgClz in no way interfered with the assay of lysozyme and no problems were experienced with assays of plaice tissues. Preston (1960) reported considerable variation in the packed cell volume of plaice blood, in relation to weight and season, but the anaemia characteristic of metal toxicity (Dawson, 1979) was apparent in our Hg-exposed plaice with a rapid and significant decrease in haematocrit values and visible haemolysis. Significant decreases in haematocrit values have been reported for another member of the family Pleuronectidae, the winter flounder, Pseudopleuronectes americanus (Dawson, 1979) and also for striped bass, Morone saxatilis (Dawson, 1982), after exposure to 10 ppb HgCI2 for 60 days. Within 48 h of exposure to 0.181 ppm HgCiz, degenerative changes in erythrocyte morphology have been observed in Barbus conchonius (Gill and Pant, 1985). Conflicting results can, however, appear with the length of exposure of different species because of their different LCs0 for HgCI2. Juneja and Mahajan (1983) found increased haematocrit values over 7 days in the freshwater Channa punctatus exposed to 0.136 ppm HgClz, although this could be due to erythropoiesis initially over-compensating in order to meet the higher oxygen requirements caused by erythrocyte loss. Damage to erythrocytes would, however, be consistent with known membrane effects of Hg, Arbuthnott (1962) having observed the haemolytic action of HgCI2 on mammalian cells, while the uptake of Hg by plaice blood cells (Pentreath, 1976) provides the conditions for direct cell damage. Although the affinity of Hg for SH-groups in membrane proteins would affect membrane conformation and permeability, Ribarov and Benov (1981) have suggested that the peroxidation of membrane iipids is also a possible mechanism of damage to the erythrocyte membrane in metal-induced haemolysis. The significant increase in spleen index by day 6 coincides with low haematocrit values. The spleen is an important phagocytic organ in the plaice, with avidity for erythrocytes (MacArthur et al., 1983) and there is the possibility that effete erythrocytes could account for some increase in splenic weight. Stimulation of erythropoietic tissue has been observed in the spleen and kidney of C. punctatus over the first 7 days of exposure to HgCI2 at 0.136 ppm (Juneja and Mahajan, 1983). This is unlikely to account for the splenomegaly observed in the plaice, where erythropoiesis appears to be a minor activity of the spleen (Ellis et al., 1976). Mercuric chloride has, however, been reported to be mitogenic for human peripheral lymphocytes (Caron et al., 1970). Mercury is concentrated in the plaice spleen (Pentreath, 1976) but whether it directly stimulates proliferation of the splenic lymphoid tissue is not known.
83 The lysozyme present in m a m m a l i a n serum, as a low molecular weight protein, is filtered by the kidney glomerulus and reabsorbed by the proximal tubular cells and is not normally present at concentrations greater than 4 # g / m l urine (Prockop and Davidson, 1964). Lysozyme concentrations in the range 0-9.8/~g/ml urine have been reported in P. americanus (Maack and Kinter, 1969) and are consistent with the values found in the plaice. Maack and Kinter (1969) injected winter flounder with egg white lysozyme and found filtration and reabsorption as in mammals. The plaice exposed to Hg showed reduced serum lysozyme levels which could be due to increased excretion or decreased enzyme production. Our results show increased enzyme excretion, although lysozymuria was only significant after seven days exposure. Presumably there was no concomitant increase in enzyme release into the serum. At day 7 there was also a significant increase in the lysozyme content of the whole kidney. Although the lysozyme values for the kidney were expressed on a protein basis, there was little change in the protein content of comparable weights of kidney and the results represent a real increase in total lysozyme. The plaice kidney, as with rats (Litwack, 1955), normally contains more lysozyme activity than other tissues (Fletcher and White, 1973). This is due in the plaice to the abundance of leucocytes in the kidney stroma which are probably the source of the endogenous lysozyme (Murray and Fletcher, 1976) and to the kidney being a site of lysozyme catabolism (Ottosen, 1978), as in mammals. The kidney is the main haemopoietic tissue of the plaice (Ellis et al., 1976) and because of the lytic effects of the Hg on circulating blood cells, it is possible that stimulation of this tissue includes an increase in lysozyme producing leucocytes. It is interesting that none of this extra kidney lysozyme can boost the serum levels. Lysozymuria is indicative of kidney proximal tubule damage in the plaice, but it appears only when the damage is extensive, while significant proteinuria occurs earlier in Hg exposure. The collection of urine from the plaice is, however, more difficult than the collection of blood and the easily performed haematocrit determination gives an early warning of anaemia. Only when the latter occurs might it then be useful to collect and analyze urine. ACKNOWLEDGEMENTS We thank Mr. C.K. Murray for sectioning and staining kidneys from the plaice and Ms. B.E. Stroud for photography. REFERENCES Arbuthnott, J.P., 1962. Haemolytic action of mercurials. Nature (London) 196, 277-278. Balazs, T. and R.R. Roepke, 1966. Lysozymuria induced in rats by nephrotoxic agents. Proc. Soc. Exp. Biol. Med. 123, 380-385. Caron, G.A., S. Poutala and T.T. Provost, 1970. Lymphocyte transformation induced by inorganic and organic mercury. Int. Arch. Allergy Appl. Immunol. 37, 76-87.
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