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Induction of ornithine-urea cycle in a freshwater teleost, Heteropneustes fossilis, exposed to high concentrations of ammonium chloride
~
Comp. Biochem. PhysioL Vol. 108B, No. 3, pp. 315-325, 1994 Copyright © 1994 ElsevierScience Printed in Great Britain. All rights reserved 0305-0491/94 $7.00 + 0.00
Ltd
Pergamon
0305-0491(94)E0023-M
Induction of ornithine-urea cycle in a freshwater teleost, Heteropneustes fossil&, exposed to high concentrations of ammonium chloride N. Saha* and B. K. Rathat *Department of Zoology, North Eastern Hill University, Shillong-793014, India; and tBiochemical Adaptation Laboratory, Centre of Advanced Studies in Zoology, Banaras Hindu University, Varanasi 221005, India An ammoniotelic freshwater teleost, Heteropneustes fossilis, tolerated ambient ammonium chloride concentration up to 75 mM. Ammonia accumulated significantly in all the tissues within 7 days of treatment and the concentration remained high throughout the 4-week period of treatment. The activity of enzymes of the ornithine-urea (o-u) cycle were induced within 7 days, and thereafter remained high in both the liver and kidney of the fish. Urea accumulated significantly in various tissues simultaneous with the induction of o-u cycle enzymes. Accumulated ammonia induced the activity of the enzymes of the o-u cycle for its metabolic conversion to urea. This helped the freshwater fish to avoid toxaemia and to tolerate high concentrations of ammonia in the ambient medium. Key words: Ammonia; Ornithine-urea cycle; Heteropneustes fossilis.
Comp. Biochem. Physiol. 108B, 315-325, 1994.
Introduction Ammonia is produced as a major metabolic end-product during the catabolism of nitrogen containing biomolecules in various animal tissues. Due to its high toxicity, even at very low concentrations in vivo, ammonia is either excreted directly or converted to some less toxic compound such as urea, uric acid or amino acid (Hoar, 1983; Cooper and Plum, 1987). Freshwater teleosts excrete ammonia by diffusion directly to the aquatic medium mainly through the gills (Delaunay, 1931; Baldwin, 1970; Forster and Goldstein, 1969). Ammonia is known Correspondence to: B. K. Ratha, Biochemical Adaptation Laboratory, Centre of Advanced Studies in Zoology, Banaras Hindu University, Varanasi 221005, India. Received 13 October 1993; accepted 7 February 1994.
315
to be a common pollutant in the freshwater system (Alabaster and Lloyd, 1980). Freshwater teleosts are highly sensitive to even very low concentrations of ambient ammonia (Fromm and Gillette, 1968; Olson and Fromm, 1971; Dabrowska and Wlasow, 1986). Interference by higher concentrations of ammonia in the medium in its excretion by diffusion, and absence of a functional ornithine-urea (o-u) cycle (Brown and Cohen, 1960; Huggins et al., 1969; Wilson, 1973) in its conversion to urea in the freshwater teleost, results in ammonia build-up, ultimately causing death. Effective conversion of ammonia to urea through the o-u cycle in vivo has been reported in marine elasmobranchs, marine toad-fish (Opsanus beta), mudskippers, lungfish, a tilapia fish (Oreochromis
316
N. Saha and B. K. Ratha
alcalicus grahami ) living in an alkaline lake, amphibians and mammals (Brown and Cohen, 1960; Read, 1971; Goldstein, 1971; Cohen, 1976; Mommsen and Walsh, 1989; Randall et al., 1989). We reported the presence of a functional o-u cycle with significant activity of all the enzymes in the liver and kidney of three species of freshwater air-breathing teleosts, including Heteropneustesfossilis (Saha and Ratha, 1987, 1989). The physiological level and rate of excretion of urea were also higher compared to other freshwater species (Saha and Ratha, 1989; Saha et al., 1988). However, Heteropneustesfossilis was predominantly ammoniotelic in freshwater, excreting about 85% of its nitrogen as ammonia (Saha et al., 1988). Suppression of excretion of ammonia, and initial suppression, followed by induction of excretion of urea, were reported when Heteropneustes fossilis was exposed to ambient ammonium chloride (Saha and Ratha, 1990). A preliminary report indicating the induction of the o-u cycle under hyper-ammonia stress was presented to the Fourth FAOB Congress (Saha and Ratha, 1986). This paper presents the results of a detailed 4-week study to show induction of the activity of the o-u cycle enzymes by accumulated ammonia as one of the metabolic adaptations favouring a transition from ammoniotelism to ureotelism in a freshwater teleost, Heteropneustes fossilis, in order to tolerate higher ambient ammonia concentrations.
Materials and Methods Animals Heteropneustes fossilis were purchased from commercial sources and maintained in the laboratory in plastic aquaria with bacteria-free filtered tap water at 20 _+ 2°C with a 12 hr: 12 hr light and dark period. Minced pork liver was given as food and water was changed on alternate days. The fish were acclimatized, before being used for experiments, for 4-6 weeks. No sex differentiation was done before setting the experiments.
Experimental set-up Fish of similar sizes (30-35 g) were used for experiments. Food was withheld for
24 hr prior to, and during, the study. The fish were treated in four batches of five each for each concentration in separate plastic buckets containing 41 ammonium chloride solution prepared in bacteria-free filtered tap water. Control fish in four batches of five each were maintained in separate buckets in the medium without ammonium chloride. Streptopenicillin (an antibiotic consisting of streptomycin sulphate 6000 units I.P. equivalent to a 10-mg base, 6000 units of procain penicillin G.I.P. and 2000 units of penicillin G Na) was added (10 mg/1) in the medium to stop microbial growth. The medium was replaced by a fresh solution every 48 hr. All the fish exposed to 100, 150 and 200 mM ammonium chloride died within 5 days and hence, the experiment continued for 4 weeks only with 25, 50 and 75mM ammonium chloride concentrations. Every week, one batch of five fish from each concentration and control were sacrificed for estimations of ammonia and urea in liver, kidney, muscle, brain and blood plasma. The activities of the o-u cycle enzymes were assayed in the liver and kidney of H. fossilis treated with 50 mM ammonium chloride.
Blood sampling Blood sampling and processing for the estimation of ammonia and urea was carried out following the method described earlier (Saha and Ratha, 1989).
Tissue preparation The fish were sacrificed by decapitation immediately after collecting the blood regularly at 12 noon. Tissues such as liver, kidney, muscle and brain were quickly removed, washed in ice-cold saline, blotted dry and kept deep-frozen at - 2 0 ° C until used for estimations. All analyses were completed within 3 days of collection of tissue, during which no change was noticed in any of the parameters studied.
Estimations Total ammonia and urea estimations were made according to the methods described earlier (Saha and Ratha, 1989). Protein was estimated following the method of Lowry et al. (1951) using crystalline bovine serum albumin as the standard.
An ornithine-urea cycle in a freshwater teleost
Enzyme assay
Results
Ten percent homogenate (w/v) of liver and kidney tissue was prepared separately in 0.1% cetyltrimethyl ammonium bromide (CTB) for the assay of activities of the o-u cycle enzymes (Brown and Cohen, 1959). The homogenates were centrifuged at 600 g at 0 __+2°C for 15 min. The supernatants were used for enzyme assays. The enzymes of the o-u cycle, carbamoyl phosphate synthetase (CPS-ammonia dependent; EC 2.7.2.5), ornithine transcarbamylase (OTC; EC 2.1.3.3) and arginase (ARG; EC 3.5.3.1) were assayed in the liver and kidney following the method of Saha and Ratha (1987). It should be noted that the above assay for carbamoyl phosphate synthetase (CPS) does not distinguish between the three different forms of the enzyme, namely CPS I (ammonia-dependent, mitochondrial), CPS II (ammonia-dependent, cytosolic), or CPS III (glutamine-dependent, mitochondrial). Arginino-succinate synthetase (EC 6.3.4.5) and arginino-succinate lyase (EC 4.3.2.1) were assayed together to give the overall reaction as the arginine synthetase system (ASS) (Brown and Cohen, 1959). One unit of enzyme activity was defined as that amount which catalysed the formation of 1 #mole product/hr at 37°C. Activity of the enzymes were expressed as both tissue activity (units/g wet wt of tissue) and specific activity (units/mg protein). Data collected from five replicate fish were statistically analysed and presented as mean ___standard deviation (SD). Comparison of paired mean values was made using Student's t-test (Croxton et al., 1982) and P values greater than 0.05 were taken as non-significant (NS).
Survival in ammonium chloride solution
Chemicals
All the enzymes, co-enzymes, substrates, CTB and bovine serum albumin were obtained from Sigma Chemical Co. (St Louis, MO) and other chemicals used were of analytical grades obtained from indigenous sources. Streptopenicillin was a pharmaceutical preparation from Alembic Chemical Co., Vadodra, India. Deionized and double-glass-distilled ammonia-free water was used in all preparations.
317
H. fossilis exposed to 25 mM ammonium chloride showed minor irritational symptoms which calmed down within hours of treatment. However, the fish exposed to increasing concentrations of ammonium chloride showed increased irritational symptoms. Any disturbance in the experimental bucket resulted in violent disoriented escape attempts. The hyperexcitability calmed down within 48 hr in both 50 and 75 mM ammonium chloride. All the fish exposed to 100, 150 and 200 mM ammonium chloride died within 96, 24 and 12hr, respectively. These concentrations, therefore, could not be used for long-term experimentation. The pH of the medium, which was normally 7.2, decreased to 7.04, 6.94, 6.82, 6.72, 6.60 and 6.48 at 25, 50, 75, 100, 150 and 200 mM ammonium chloride concentrations, respectively. However, the pH and concentration of ammonia in the medium did not decrease during the period of treatment in each bucket due to its replacement every 48 hr. Most of the ammonia (99%) was expected to be in ionic form at the given pH and temperature of the medium and at physiological pH (Emerson et al., 1975). Tissue ammonia and urea levels Ammonia. Ammonia accumulated significantly in different tissues and in blood plasma of fish treated with ammonium chloride (Table 1, Figs 1 and 2). The accumulation increased with increasing concentrations of ambient ammonia. The peak of ammonia accumulation in different tissues which showed dependence on the ambient concentration of ammonium chloride was observed, in general, by the 7th day of treatment. The concentration slowly decreased during the latter part of the experimental period. However, the ammonia concentration in various tissues remained significantly higher than the normal level, even after 4 weeks. Kidney accumulated maximum amounts of ammonia (40.7#mol/g wet wt) followed by liver (34.0), muscle (18.0) and brain (13.7 # mol/g wet wt) of the fish treated with 75 mM ammonium chloride. The maximum rate of accumulation of ammonia was about 10 ×
318
N. Saha and B. K. Ratha
in blood plasma, 3 x in kidney, 2.5 x in liver and brain and 1.5 x in muscle with 75mM ammonium chloride treatment, compared to the control. Urea. Urea concentration increased significantly following the accumulation of ammonia in different tissues of fish exposed to ammonium chloride (Table 2, Figs 1 and 2). The increase continued until the 14th day after which the levels were either maintained (as in plasma and muscle) or slowly decreased (as in brain, liver and kidney). The accumulation of urea was higher at higher concentrations of ambient ammonia. It was maximal in liver (27.8/~mol/g wet wt) followed by kidney (16.8), brain (9.6) and muscle (2.6/~mol/g wet wt) of the fish treated with 75 mM ammonium chloride. In plasma, the urea level increased to 4.8 mM by the 14th day of 75mM ammonium chloride treatment. However, the maximum rate of accumulation of urea was lower than that for ammonia in various tissues. It was
about 6.5 x in plasma followed by 3.5 x in brain, 2.5 x in liver and 2 × in kidney and in muscle with 75 mM ambient ammonium chloride, compared to the control. Activities of the o-u cycle enzymes The enzyme activity of ARG was very high followed by OTC, ASS and CPS in both the liver and kidney tissues of Heteropneustes fossilis. The tissue and specific activity of o-u cycle enzymes, excepting ARG, showed significant induction in both liver and kidney of H. fossilis treated with 50 mM ammonium chloride (Tables 3 and 4; Fig. 1). The activity of the enzymes remained induced throughout the period of study with the highest level usually reached by the 14th day of treatment. The induction of ASS was very high (165-190%) followed by CPS (40-55%) and OTC (20-30%) in the liver. The physiological levels of activity of all the enzymes were lower in kidney compared to those in liver. The pattern and
Table 1. Alterations in the concentration o f a m m o n i a in different tissues (/tmol/g wet wt) and in blood plasma (mmol/1) o f H. fossilis exposed to various ambient concentrations o f a m m o n i u m chloride Days after treatment Tissue
NH¢CI (mM)
Liver (control)
Kidney (control)
Muscle (control)
Brain (control)
Plasma (control)
0
7
0 25 50 75
14.56_+ 1.15
13.33__+ 1.27 y 25.45_+ 1.51" 3 0 . 5 0 ± 1.61 a 33.77_+ 3.04 ~
13.78_+ 21.71 _ 27.99-+ 34.06_+
0.81 y 1.27 a 1.42 a 2.66"
13.98 h- 0.60 y 19.29-+ 1.14" 23.68_+0.89" 32.23 _+ 1.83 ~
13.25 18.76 23.15 30.15
_ 0.78 y __+ 1.12 ~ _+ 0.97 a + 2.68"
0 25 50 75
13.90_+1.22
12.60_+1.29 y 29.44_+2.29 ~ 37.62+0.93" 4 0 . 7 4 ± 1.01"
12.35_+0.43 y 24.67_+ 1.72 ~ 3 6 . 2 7 _ 1.60 a 39.61 -+3.63"
13.02+0.47 y 2 3 . 1 5 + 1.18" 36.65 +2.39" 35.80-+2.22"
13.11 21.75 34.35 32.91
_+ 1.23 y _+ 1.78 a _ 1.00 a __. 2.92"
0 25 50 75
12.43-+ 1.09
10.73 -+ 0.45 y 13.59_+0.86" 16.80___0.36 a 18.14-+0.91"
10.04-+ 0.22 y 13.14_+0.71" 14.78 ± 1.22" 17.17___0.60 a
10.35_ 0.43 y 13.59+0.55 a 14.20+0.99 ~ 16.89___0.45"
10.11 ___0.44 y 12.96 _+ 0.38" 14.02 + 0.74 a 16.25 _+ 0.89"
0 25 50 75
6.74 -+ 0.81
5.53 _+ 0.44 y 8.51 ± 0.47" 9.46 -!-_0 . 4 P 13.68_+2.01 a
5.47 ___0.86 y 9.14 ± 0.56" 9.88 -+ 0.74" 11.82_+0.40"
5.42 + 0.45 y 8.63 _+ 0.44" 9.20 -+ 0.73" 10.45+ 1.23"
5.51 8.24 8.79 10.22
-+ 0.42 y -+ 0.46" _+0.51" ± 1.2P
0 25 50 75
0.47 _+ 0.04
0.50 q- 0.04 y 2.18___0.21" 3.84+0.45" 4.56+0.56 a
0.47 h- 0.07 y 2.42+0.23 a 3.94_+0.36" 4.71 + 0 . 5 P
0.46 + 0.06 y 2.11 ___0.19~ 3.14+0.38 ~ 4.26-+0.47"
0.44 1.98 3.04 4.08
_+ 0.04 r _+ 0.13" ± 0.46 ~ +0.51 a
........
14
Y: P not significant c o m p a r e d to normal control. ~: P significant at <0.001 level c o m p a r e d to fasted control.
21
28
An ornithine-urea cycle in a freshwater teleost
319
200
0 o
I~a~
_ 200
100 0 200
Muscle
-o
4OO
1000 Plasma
Brm
800
-200
6O0
0
400
4o0
200
200
0
I 7
I
14
I
21
I 7
I 28
I 14
I 21
I
28
Days
Fig. 1. Percentage increase in ammonia and urea concentration in different tissues and in blood plasma of H. fossilis exposed to different concentrations of a m m o n i u m chloride. ( O - O Control; C)-C) 25 raM; / k - / k 50 m M and I-1-1-] 75 raM.)
level of induction were also different in the two tissues. The CPS, OTC and ASS were simultaneously induced in kidney during the treatment of ammonium chloride to a comparable level. Normal OTC level in kidney, which was less than 20% of that of liver, showed a maximum level of induction of 221% on the 21st day of treatment. CPS activity increased continuously in kidney with the increasing period of treatment, reaching the highest level (175%) on the 28th day. Maximum ASS activity (136%) in kidney was observed on the 7th day. The specific activity generally showed higher percentage of induction than the tissue activity in all the cases. Discussion
Presence of ammonia in the aquatic medium has been known to be toxic to
ammoniotelic freshwater fish. The 48 hr LCs0 value of ammonia for Cyprinus carpio was 15mg/1 (Dabrowska and Wlasow, 1986). The rates of mortality with 24 hr exposure at 8 and 40 mg/l ammonia were reported to be 50% for trout, Salmo gairdneri (Olson and Fromm, 1971) and 10% for goldfish (Schenone et al., 1982), respectively. Aquatic frog, Xenopus laevis, did not even survive beyond 1 hr at l0 mM ammonium chloride (Janssens, 1972). However, in the present study, H. fossilis tolerated and survived well in a 75 mM ambient ammonium chloride concentration which is very high compared to other freshwater teleosts and aquatic amphibians. This indicated the presence of efficient physiological and biochemical mechanisms for detoxification of excess ammonia in this fish. Ammonia is a neurotoxin. It interferes with energy metabolism, electro-physiologi-
320
N. Saha and B. K. Ratha
cal properties and level of neurotransmitters in the brain (Cooper and Plum, 1987). The fish, on exposure to ammonium chloride in the ambient medium at 25, 50 and 75 mM concentrations, therefore, became immediately hyperexcitable. Acclimatization to the changed environment gradually calmed down the irritational symptoms. A similar response was reported in goldfish (Olson and Fromm, 1971). All the fish exposed to ammonium chloride concentrations above 75 mM died within 4 days. Accumulation of acidic metabolites from glycolysis and Krebs cycle at higher ambient ammonia was suggested to cause anoxia and death due to a decrease in blood pH in coho salmon (Sausa and Meade, 1977). However, Hillaby and Randall (1979) could not observe any decrease in pH when the total ammonia load in the blood of rainbow trout (Salmo gairdneri) was elevated to a toxic level. Therefore, the decreasing pH of the ambient medium from 6.82 to 6.48, with an increasing concentration of
ammonium chloride from 75 to 200 mM, respectively, might not be the main cause of death in the fish. The death could be due to the enhanced accumulation of ammonia in vivo. Ammonia accumulated significantly in all the tissues of H. fossilis studied reaching the highest level within 7 days of exposure to ammonium chloride (Table 1, Figs 1 and 2). Suppression of excretion and absorption of ammonia from the ambient medium by H. fossilis (Saha and Ratha, 1990) probably resulted in this accumulation. The accumulation was tissue-specific. Brain, being highly susceptible to ammonia toxicity (Cooper and Plum, 1987), showed minimum accumulation. Effective enzymatic detoxification of ammonia to glutamate and then to glutamine by the enzymes glutamate dehydrogenase (GDH) and glutamine synthetase (GS), respectively, has been reported in fish brain (Levi et al., 1974; Walton and Cowey, 1977; Iwata et al., 1981; Chakravorty et al., 1989; Das et al.,
O Ammonia • Urea
Liver
[] cPs 50
200
[ ] OTC
[]Ass 40
160
30
120
20
80
0
40
e~
T
o~ 0
40
"
140
30
120
20
80
0
4O
0
7
14
23
.4
28
Days
Fig. 2. Alteration in the percentage Of induction o f the activity (units/g wet wt) o f o u cycle enzymes with relation to the concentration (/zmol/g wet wt) o f a m m o n i a and urea in liver and kidney o f H. :bssilis exposed to 50 m M ambient a m m o n i u m chloride.
An ornithine-urea cycle in a freshwater teleost
321
Table 2. Alterations in the concentration of urea in different tissues (#mol/g wet wt) and in blood plasma (mmol/l) of H. fossilis exposed to various ambient concentrations of ammonium chloride Days after treatment Tissue
NH4C1 (raM)
Liver (control)
Kidney (control)
Muscle (control)
Brain (control)
Plasma (control)
0
7
14
21
28
0 25 50 75
6.23_ 0.61
7.31 +_ 0.78 ~ 10.51 _ 0.60 ~ 12.72-t- 0.82 x 13.21 +_ 1.11~ 9.81+0.87 b 21.86_ 1.50~ 19.45_2.19 b 18.34_ 1.67 b 12.26_ 1.70 b 25.01___3.85 ~ 23.93_2.17 ~ 21.77+2.05 ~ 12.21 + 0.97 a 27.80_ 2.26 a 26.89 +_ 2.45 ~ 25.44 + 2.66 ~
0 25 50 75
5.53 + 0.97
6.48 + 0.81 y 8.22_0.89 d 10.43+0.40 ~ 10.39_ 0.61 a
0 25 50 75
1.31 +__0.32
1.22 _ 1.39 + 1.89 + 1.60 _
0 25 50 75
2.49 _ 0.37
2.87 4.26 4.90 5.05
0 25 50 75
0.76 _ 0.08
0.74 + 2.23 _ 3.16 _ 4.26 +
8.21 _ 0.61 x 12.76_ 1.08 a 15.46_2.29 a 16.74_+ 1.23~
10.82_ 1.33~ 12.30+ 1.26 ' 15.37_2.03 c 15.53_ 1.77 b
11.12 + 0.89 x 12.11 +__1.I0 r 15.87 + 1.70b 16.79 + 1.23~
0.23 y 0.25 f 0.25 c 0.31 f
1.31 0.28 y 1.79 _ 0.39 f 2.24 __-0.22 b 2.16_0.20 b
1.37 _ 0.20 r 1.82 +__0.33 e 2.30 _ 0.38 b 2.41 _0.41 b
1.45 _ 0.27 y 1.78 + 0.36 f 2.46 _ 0.28 c 2.62+0.41 ~
+__0.49 y _ 0.41 b _ 0.31 a _ 0.50 a
2.60 + 0.33 y 6.52+__0.45~ 9.43 + 0.77 a 8.89 + 0.55 a
2.91 7.06 9.66 9.12
4.01 7.11 9.45 8.78
0.74 2.54 3.87 4.82
0.73 _ 2.18 _ 3.54 _ 4.36 _
0.03 y 0.38 a 0.56 a 0.71 a
_
+ 0.04 y +__0.32 a _ 0.48 a _+ 0.79 ~
+ 0.19 y _ 0.25 a _ 0.46 a -t- 0.89 a 0.04 y 0.27 a 0.39 a 0.56 ~
+ 0.38 x +0.30 ~ ___0.39 a + 0.46 a
0.71 + 2.16 + 3.44 + 4.41 _
0.04Y 0.18 a 0.32 a 0.44 a
x and Y: P-values significant at <0.001 and non-significant, respectively, compared to the normal control. a,bc,d,e and r: P-values significant at <0.001, <0.005, <0.01, <0.025, <0.05 and non-significant, respectively, compared to the fasted control.
1991; C h a k r a v o r t y , Das and Ratha, in preparation). Liver and kidney, which are the main organs for ammonia metabolism in fish ( P e q u i n a n d S e r f a t y , 1963; V e l l a s a n d S e r f a t y , 1974; C a s e y et al., 1983), a c c u m u lated high concentrations (35--40/~mol/g t i s s u e ) o f a m m o n i a in H. fossilis. B l o o d being the carrier of metabolites, the concent r a t i o n o f a m m o n i a i n c r e a s e d 3 - 4 t i m e s in the plasma compared to the control animals. The concentration-dependent acc u m u l a t i o n in v a r i o u s t i s s u e s w a s r a t h e r slow at higher concentrations of ambient a m m o n i a a n d g e n e r a l l y s t a b i l i z e d a t a tissue-specific high concentration by the 7th d a y o f e x p o s u r e ( F i g . 2). D i f f e r e n t t i s s u e s probably have different maximum loading capacities for ammonia. Ammonia transport across the membrane takes place either by simple diffusion or by Na+-related carr i e r t r a n s p o r t o f NH~- ( H i l l a b y a n d R a n d a l l , 1979). T h e d i s t r i b u t i o n o f a m m o n i a in
different tissue compartments has been reported to be influenced by both the pH and t h e e l e c t r o c h e m i c a l g r a d i e n t ( W r i g h t et al., 1988). H o w e v e r , t h e m e c h a n i s m o f a m m o n i a t r a n s p o r t i n t h i s fish h a s n o t y e t b e e n studied. It was interesting to note that the m a x i m u m c o n c e n t r a t i o n o f a m m o n i a in t h e plasma did not exceed 5 mM when the a m b i e n t c o n c e n t r a t i o n w a s 75 m M . T h i s was quite a feat which might have cont r i b u t e d t o t h e s u r v i v a l o f t h e fish a t t h a t high ambient concentration of ammonium chloride. T h e c o n t r o l fish, w h i c h w e r e f a s t i n g in fresh water, did not show significant change in t h e a m m o n i a l e v e l s in v a r i o u s t i s s u e s ( T a b l e 1, F i g . 2). H o w e v e r , t h e y s h o w e d gradual reduction in the rate of excretion of ammonia during starvation (Saha and R a t h a , 1990). Metabolic detoxification of ammonia to u r e a c o u l d n o t b e o b s e r v e d in m o s t f r e s h -
322
N. Saha and B. K. Ratha
Table 3. Alterations in the tissue (units/g wet wt) and specific (units/mg protein) activity of o-u cycle enzymes in the liver of H. fossilis exposed to 50 mM ambient ammonium chloride Enzyme activity Days after treatment
CPS
OTC
ASS
ARG
Tissue
Sp. x 102
Tissue
Sp.
Tissue
Sp.
Tissue
Sp.
0 Control 7 P*
4.6 ___0.5
2.9 ___0.3
252 __+23
1.6 __+0.2
28.7 + 5.5
0.21 ___0.04 7700 ___933
50 -+ 7
5.9-+0.8 <0.05
3.8+1.0 <0.05
290___12 <0.025
1.7+0.1 NS
78.1-t-19.8 0.51+0.14 7193+495 <0.005 <0.01 NS
47_+3 NS
14 P
6.4_+0.8 <0.01
4.0+0.4 <0.005
328-t-30 <0.05
2.2__+0.2 82.1__+11.4 0.55+0.08 8298__+455 56-+4 <0.005 <0.001 <0.001 NS NS
21 P
6.0 + 0.7 <0.02
4.4___0.4 <0.001
306 + 27 <0.02
2.2 -+ 0.2 <0.005
75.9 + 15.5 0.53 ___0.01 7909 _+ 873 <0.001 <0.001 NS
28 P
6.4___0.4 <0.05
4.5+0.2 <0.001
293+34 <0.02
2.1_0.3 <0.025
83.7+9.4 <0.001
56 + 6 NS
0.60_+0.06 7507__+357 54___3 <0.001 NS NS
*P value compared to fasted control.
water teleosts due to incomplete o-u cycles (Brown and Cohen, 1960; Huggins et al., 1969; Wilson, 1973). However, the presence of a complete o-u cycle in the liver and kidney of H. fossilis with high activities of all the enzymes, suggested its role in ureogenic ammonia detoxification (Saha and Ratha, 1987). The accumulation of ammonia in vivo was associated with the induction of the activities of the o-u cycle enzymes, except ARG, in both the liver and kidney of H. fossilis within 7 days of treatment with 50 mM ammonium chloride (Tables 3 and 4, Fig. 2). The activity of the induced enzymes remained high throughout the period of the experiment. Even though
arginase activity was assayed at a high unphysiological pH (9.5), its physiological level of activity would also be very high. The high level of activity of all the enzymes of the o-u cycle must have accelerated the rate of ureogenesis (metabolic conversion of ammonia to urea) and thereby controlled the in vivo level of accumulated ammonia. Urea accumulated significantly in all the tissues studied in H. fossilis following the induction of the activities of the enzymes of the o-u cycle (Table 2, Figs 1 and 2). Accumulation of urea was also tissuespecific even though it is a highly permeant solute. Liver accumulated highest concen-
Table 4. Alterations in the tissue (units/g wet wt) and specific (units/mg protein) activity of o-u cycle enzymes in the kidney of H. fossilis exposed to 50 mM ambient ammonium chloride Enzyme activity Days after treatment
CPS
OTC
ASS
ARG
Tissue
Sp. × 102
Tissue
Sp.
Tissue
0 Control 7 P*
3.0+0.3
2.0_+0.3
43+3.3
0.28+0.02
21 +3.6
4.4+0.4 <0.005
3.0+0.2 <0.005
82+2.2 <0.001
0.56___0.05 48___7.6 0.33___0.06 1494+228 10.3_+1.4 <0.001 <0.001 <0.001 NS NS
14 P
5.2+1.0 <0.005
4.0-+0.8 <0.005
102___8 <0.001
21 P
5.1 +0.2 <0.001
4.0___0.2 114__+24 <0.001 <0.001
28 P
7.2___0.4 <0.001
6.0+0.3 <0.001
100+7 <0.001
*P value compared to fasted control.
0.76+0.10 <0.001
43-+7.3 <0.005
0.9__+0.11 4 0 + 4 . 8 <0.001 <0.001 0.77___0.06 41___8 <0.001 <0.005
Sp.
Tissue
Sp.
0.14-+0.03 1586-+ 104 9 . 4 + 0 . 7
0.32-+0.05 1758___222 13.2+ 1.3 <0.001 NS NS 0.31 +0.07 1667+229 <0.001 NS
13+2 <0.02
0.32___0.06 1732___224 12_+1.8 <0.001 NS <0.05
An ornithine-urea cycle in a freshwater teleost
tration of urea (27.8 #mol/g tissue) followed by kidney (16.8#mol/g). Muscle, which constitutes the major bulk of fish tissue, retained a very low concentration (2.6#mol/g) of urea. Either urea was rapidly diffused from the muscle through the skin or the muscle had some special physiological barrier to protect it from excess urea accumulation. Accumulation of urea by about 6 x in blood plasma, 4.5 x in liver, 4 x in brain, 3 x in kidney and 2 x in muscle compared to the control level (Fig. 1) indicated that maximum loading capacity for urea was different in different tissues. The presence of a specific urea transporter protein has been reported in mammalian erythrocytes and kidney tubules (Sands et al., 1992; Gillian and Sands, 1992). Involvement of such urea transporter protein in this fish cannot be ruled out. The excretion of urea, which was suppressed during exposure to higher ambient ammonia (Saha and Ratha, 1990) alone, could not account for the higher level of accumulation of urea in different tissues of Heteropneustes fossilis. Accumulation of ammonia seemed to induce the activity of the o-u cycle enzymes for enhanced conversion of toxic ammonia to less toxic urea. Such ammonia-induced ureogenesis has been reported in ureotelic amphibians (Balinsky et al., 1961; McBean and Goldstein, 1967; Janssens and Cohen, 1968; McBean and Goldstein, 1970; Janssens, 1972). Although the rate of excretion of urea was reported to increase significantly from the 10th day (Saha and Ratha, 1990), the tissue level of both ammonia and urea did not decrease proportionately, and continued at a higher level during exposure to higher ambient ammonia. The control fish, fasting in the ammoniafree medium, also accumulated significant amounts of urea only in the liver and kidney. Increased amino acid catabolism during fasting reported in fish (Johnston, 1981; Walton and Cowey, 1982; Moon, 1983) may have resulted in the production of ammonia and, in turn, urea in the liver and kidney, which had a complete o-u cycle. The results clearly suggest that the exposure of the freshwater air-breathing teleost, Heteropneustes fossilis, to higher
323
ambient ammonium chloride resulted in accumulation in vivo of ammonia and associated induction of the activities of the enzymes of the o-u cycle in liver and kidney which accelerated the rate of ureogenesis. The conversion of excess ammonia to urea and the reported increase in the excretion of urea (Saha and Ratha, 1990) controlled the in vivo concentration of ammonia and urea. Thus, the ammoniotelic fish became ureotelic to survive at that high (75 mM) concentration of ambient ammonium chloride. Acknowledgements--The authors thank the NorthEastern Hill University, Shillong, for facilities, the Council of Scientific and Industrial Research, New Delhi, for awarding an SRF to one of us (N.S.) and the University Grants Commission, New Delhi, for partial financial support.
References Alabaster J. S. and Lloyd R. (1980) In Water Quality Criteria for Freshwater Fish, pp. 297-365. Butterworth, London. Baldwin E. (1970) An Introduction to Comparative Biochemistry, 4th edn, pp. 54-74. Cambridge University Press, London. Balinsky J. B., Cragg M. M. and Baldwin E. (1961) The adaptation of amphibian waste nitrogen excretion to dehydration. Comp. Biochem. Physiol. 3, 236-244. Brown G. W. Jr and Cohen P. P. (1959) Comparative biochemistry of urea synthesis--I. Methods for quantitative assay of urea cycle enzymes in liver. J. biol. Chem. 234, 1769-1774. Brown G. W. Jr and Cohen P. P. (1960) Comparative biochemistry of urea synthesis--Ill. Activities of urea cycle enzymes in various higher and lower vertebrates. Biochem. J. 75, 82-91. Casey A., Perlman D. F. and Campbell J. W. (1983) Hepatic ammoniagenesis in the channel catfish, lctalurus punctatus. Molec. Physiol. 3, 107-126. Chakravorty J., Saha N. and Ratha B. K. (1989) A unique pattern of tissue distribution and sub-cellular localization of glutamine synthetase in a freshwater air-breathing teleost, Heteropneustes fossilis (Bloch). Biochem. Int. 19, 519-527. Cohen P. P. (1976) Evolutionary and comparative aspects of urea biosynthesis. In The Urea Cycle (Edited by Grisolia S., Baguena R. and Mayor F.), pp. 21-28. John Wiley & Sons, New York. Cooper A. J. L. and Plum F. (1987) Biochemistry and physiology of brain ammonia. Physiol. Rev. 67, 440-519. Croxton F. E., Cowden D. J. and Klein S. (1982) Applied General Statistics, 3rd edn. Prentice-Hall of India, New Delhi. Dabrowska H. and Wlasow T. (1986) Sublethal effect of ammonia on certain biochemical and hematological indicators in common carp. Comp. Biochem. Physiol. 83C, 179-184.
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Das J. R., Saha N. and Ratha B. K. (1991) Tissue distribution and subcellular localization of glutamate dehydrogenase in a freshwater air-breathing teleost, Heteropneustes fossilis. Biochem. Syst. Ecol. 19, 207-212. Delaunay H. (1931) L'excretion azot6e des invertebres. Biol. Rev. 6, 265-301. Emerson K., Russo R. C., Lund R. E. and Thurston R. V. (1975) Aqueous ammonia equilibrium calculations. Effect of pH and temperature. J. Fish. Res. Bd Can. 32, 2379-2383. Forster R. P. and Goldstein L. (1969) Formation of excretory products. In Fish Physiology (Edited by Hoar W. S. and Randall D. J.), Vol. 1, pp. 313-350. Academic Press, New York. Fromm P. O. and Gillette J. R. (1968) Effect of ambient ammonia on blood ammonia and nitrogen excretion of rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 12, 489-499. Gillian A. G. and Sands J. M. (1992) Characteristics of osmolarity-stimulated urea transport in rat IMCD. Am. J. Physiol. 262, F1061-F1067. Goldstein L. (1971) Adaptation of urea metabolism in aquatic vertebrates. In Nitrogen Metabolism and the Environment (Edited by Campbell J. W. and Goldstein L.), pp. 55-77. Academic Press, New York. Hillaby B. A. and Randall D. J. (1979) Acute ammonia toxicity and ammonia excretion in rainbow trout (Salmo gairdneri ). J. Fish. Res. Bd Can. 36, 621-629. Hoar W. S. (1983) Excretion. In General and Comparative Physiology, 3rd edn, pp. 585-626. Prentice-Hall, Englewood Cliffs, NJ. Huggins A. K., Skutsch G. and Baldwin E. (1969) Ornithine-urea cycle enzymes in teleostean fish. Comp. Biochem. Physiol. 28, 587-602. Iwata K., Kakuta I., Ikeda M., Kimoto S. and Wada N. (1981) Nitrogen metabolism in mudskipper, Periophthalmus cantonensis: a role of free amino acids in detoxification of ammonia produced during its terrestrial life. Comp. Biochem. Physiol. 68A, 589-596. Janssens P. A. (1972) The influence of ammonia on the transition to ureotelism in Xenopus laevis. J. exp. Zool. 182, 357-366. Janssens P. A. and Cohen P. P. (1968) Biosynthesis of urea in the aestivating African lungfish and in Xenopus laevis under conditions of water shortage. Comp. Biochem. Physiol. 24, 887-898. Johnston I. A. (1981) Quantitative analysis of muscle breakdown during starvation in marine flat fish, Pleuronectes platessa. Cell Tissue Res. 214, 369-386. Levi G., Morisi G., Coletti A. and Catanzaro R. (1974) Free amino acids in fish brain: normal levels and changes upon exposure to high ammonia concentrations in vivo, and upon incubation of brain slices. Comp. Biochem. Physiol. 49A, 623-636. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. McBean R. L. and Goldstein L. (1967) Ornithine-urea cycle activity in Xenopus laevis: adaptation in saline. Science 157, 931-932.
McBean R. L. and Goldstein L. (1970) Accelerated synthesis of urea in Xenopus laevis during osmotic stress. Am. J. Physiol. 219, 1124-1130. Mommsen T. P. and Walsh P. J. (1989) Evolution of urea synthesis in vertebrates: the piscine connection. Science 243, 72-75. Moon T. W. (1983) Metabolic reserves and enzyme activities with food deprivation in immature American eels, Anguilla rostrata. Can. J. Zool. 61, 802-81 I. Olson K. R. and Fromm P. O. (1971) Excretion of urea by two teleosts exposed to different concentrations of ambient ammonia. Comp. Biochem. Physiol. 40A, 999-1007. Pequin L. and Serfaty A. (1963) L'excretion ammoniacale chez un T616ost6en dulcicole Cyprinus carpio L. Comp. Biochem. Physiol. 10, 315-324. Randall D. J., Wood C. M., Perry S. F., Bergman H., Maloiy G. M. O., Mommsen T. P. and Wright P. A. (1989) Urea excretion as a strategy for survival in a very alkaline environment. Nature 337, 165-166. Read L. J. (1971) The presence of high ornithine-urea cycle enzyme activity in the teleost Opsanus tau. Comp. Biochem. Physiol. 39, 409-413. Saha N. and Ratha B. K. (1986) Effect of ammonia stress on ureogenesis in a freshwater air-breathing teleost, Heteropneustes fossilis. In Contemporary Themes in Biochemistry (Edited by Kon O. L., Chung M. C., Hwang P. L. H., Leong S., Loke K. H., Thiyagaraja P. and Wong P. T.), Vol. 6, pp. 342-343. Cambridge University Press, Cambridge. Saha N. and Ratha B. K. (1987) Active ureogenesis in a freshwater air-breathing teleost, Heteropneustes fossilis. J. exp. Zool. 241, 137-141. Saha N., Chakravorty J. and Ratha B. K. (1988) Diurnal variation in renal and extra-renal excretion of ammonia-N and urea-N in a freshwater airbreathing teleost, Heteropneustes fossilis (Bloch). Proc. Ind. Acad. Sci. 97, 529-537. Saha N. and Ratha B. K. (1989) A comparative study of ureogenesis in freshwater air-breathing teleosts. J. exp. Zool. 252, 1-8. Saha N. and Ratha B. K. (1990) Alteration in the excretion pattern of ammonia and urea in a freshwater air-breathing teleost, Heteropneustesfossilis (Bloch) during hyper-ammonia stress. Ind. J. exp. Biol. 28, 597-599. Sands J. M., Gargus J. J., Frolich O., Gunn R. B. and Kakko J. P. (1992) Urinary concentrating ability in patients with Jk (a negative and b negative) blood type who lack carrier-mediated urea transport. J. Am. Soc. Nephrol. 2, 1689-1696. Sausa R. J. and Meade T. L. (1977) The influence of ammonia on the oxygen delivery system of Coho salmon haemoglobin. Comp. Biochem. Physiol. 58A, 23-58. Schenone G., Arillo A., Margiocco C., Melodia F. and Mensi P. (1982) Biochemical bases for environmental adaptation in goldfish (Carassius auratus): resistance to ammonia. Ecotox. Environ. Safety 6, 479-488. Vellas F. and Serfaty A. (1974) L'ammonique et l'ur6e chez un T616ost6en d'eau douce: la carpe (Cyprinus carpio L.). J. Physiol., Paris 68, 591-614.
An ornithine-urea cycle in a freshwater teleost Walton M. J. and Cowey C. B. (1977) Aspects of ammoniogenesis in rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 57B, 143-149. Walton M. J. and Cowey C. B. (1982) Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. 73, 59-79. Wilson R. P. (1973) Nitrogen metabolism in channel catfish, Ictalurus punctatus--II. Evidence for an
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apparent complete ornithine-urea cycle. Comp. Biochem. Physiol. 46B, 625-634. Wright P. A., Randall D. J. and Wood C. M. (1988) The distribution of ammonia and H + between tissue compartments in lemon sole (Parophrys vetulus) at rest, during hypercapnia and following exercise. J. exp. Biol. 136, 149-175.
Comp. Biochem. PhysioL Vol. 108B, No. 3, pp. 315-325, 1994 Copyright © 1994 ElsevierScience Printed in Great Britain. All rights reserved 0305-0491/94 $7.00 + 0.00
Ltd
Pergamon
0305-0491(94)E0023-M
Induction of ornithine-urea cycle in a freshwater teleost, Heteropneustes fossil&, exposed to high concentrations of ammonium chloride N. Saha* and B. K. Rathat *Department of Zoology, North Eastern Hill University, Shillong-793014, India; and tBiochemical Adaptation Laboratory, Centre of Advanced Studies in Zoology, Banaras Hindu University, Varanasi 221005, India An ammoniotelic freshwater teleost, Heteropneustes fossilis, tolerated ambient ammonium chloride concentration up to 75 mM. Ammonia accumulated significantly in all the tissues within 7 days of treatment and the concentration remained high throughout the 4-week period of treatment. The activity of enzymes of the ornithine-urea (o-u) cycle were induced within 7 days, and thereafter remained high in both the liver and kidney of the fish. Urea accumulated significantly in various tissues simultaneous with the induction of o-u cycle enzymes. Accumulated ammonia induced the activity of the enzymes of the o-u cycle for its metabolic conversion to urea. This helped the freshwater fish to avoid toxaemia and to tolerate high concentrations of ammonia in the ambient medium. Key words: Ammonia; Ornithine-urea cycle; Heteropneustes fossilis.
Comp. Biochem. Physiol. 108B, 315-325, 1994.
Introduction Ammonia is produced as a major metabolic end-product during the catabolism of nitrogen containing biomolecules in various animal tissues. Due to its high toxicity, even at very low concentrations in vivo, ammonia is either excreted directly or converted to some less toxic compound such as urea, uric acid or amino acid (Hoar, 1983; Cooper and Plum, 1987). Freshwater teleosts excrete ammonia by diffusion directly to the aquatic medium mainly through the gills (Delaunay, 1931; Baldwin, 1970; Forster and Goldstein, 1969). Ammonia is known Correspondence to: B. K. Ratha, Biochemical Adaptation Laboratory, Centre of Advanced Studies in Zoology, Banaras Hindu University, Varanasi 221005, India. Received 13 October 1993; accepted 7 February 1994.
315
to be a common pollutant in the freshwater system (Alabaster and Lloyd, 1980). Freshwater teleosts are highly sensitive to even very low concentrations of ambient ammonia (Fromm and Gillette, 1968; Olson and Fromm, 1971; Dabrowska and Wlasow, 1986). Interference by higher concentrations of ammonia in the medium in its excretion by diffusion, and absence of a functional ornithine-urea (o-u) cycle (Brown and Cohen, 1960; Huggins et al., 1969; Wilson, 1973) in its conversion to urea in the freshwater teleost, results in ammonia build-up, ultimately causing death. Effective conversion of ammonia to urea through the o-u cycle in vivo has been reported in marine elasmobranchs, marine toad-fish (Opsanus beta), mudskippers, lungfish, a tilapia fish (Oreochromis
316
N. Saha and B. K. Ratha
alcalicus grahami ) living in an alkaline lake, amphibians and mammals (Brown and Cohen, 1960; Read, 1971; Goldstein, 1971; Cohen, 1976; Mommsen and Walsh, 1989; Randall et al., 1989). We reported the presence of a functional o-u cycle with significant activity of all the enzymes in the liver and kidney of three species of freshwater air-breathing teleosts, including Heteropneustesfossilis (Saha and Ratha, 1987, 1989). The physiological level and rate of excretion of urea were also higher compared to other freshwater species (Saha and Ratha, 1989; Saha et al., 1988). However, Heteropneustesfossilis was predominantly ammoniotelic in freshwater, excreting about 85% of its nitrogen as ammonia (Saha et al., 1988). Suppression of excretion of ammonia, and initial suppression, followed by induction of excretion of urea, were reported when Heteropneustes fossilis was exposed to ambient ammonium chloride (Saha and Ratha, 1990). A preliminary report indicating the induction of the o-u cycle under hyper-ammonia stress was presented to the Fourth FAOB Congress (Saha and Ratha, 1986). This paper presents the results of a detailed 4-week study to show induction of the activity of the o-u cycle enzymes by accumulated ammonia as one of the metabolic adaptations favouring a transition from ammoniotelism to ureotelism in a freshwater teleost, Heteropneustes fossilis, in order to tolerate higher ambient ammonia concentrations.
Materials and Methods Animals Heteropneustes fossilis were purchased from commercial sources and maintained in the laboratory in plastic aquaria with bacteria-free filtered tap water at 20 _+ 2°C with a 12 hr: 12 hr light and dark period. Minced pork liver was given as food and water was changed on alternate days. The fish were acclimatized, before being used for experiments, for 4-6 weeks. No sex differentiation was done before setting the experiments.
Experimental set-up Fish of similar sizes (30-35 g) were used for experiments. Food was withheld for
24 hr prior to, and during, the study. The fish were treated in four batches of five each for each concentration in separate plastic buckets containing 41 ammonium chloride solution prepared in bacteria-free filtered tap water. Control fish in four batches of five each were maintained in separate buckets in the medium without ammonium chloride. Streptopenicillin (an antibiotic consisting of streptomycin sulphate 6000 units I.P. equivalent to a 10-mg base, 6000 units of procain penicillin G.I.P. and 2000 units of penicillin G Na) was added (10 mg/1) in the medium to stop microbial growth. The medium was replaced by a fresh solution every 48 hr. All the fish exposed to 100, 150 and 200 mM ammonium chloride died within 5 days and hence, the experiment continued for 4 weeks only with 25, 50 and 75mM ammonium chloride concentrations. Every week, one batch of five fish from each concentration and control were sacrificed for estimations of ammonia and urea in liver, kidney, muscle, brain and blood plasma. The activities of the o-u cycle enzymes were assayed in the liver and kidney of H. fossilis treated with 50 mM ammonium chloride.
Blood sampling Blood sampling and processing for the estimation of ammonia and urea was carried out following the method described earlier (Saha and Ratha, 1989).
Tissue preparation The fish were sacrificed by decapitation immediately after collecting the blood regularly at 12 noon. Tissues such as liver, kidney, muscle and brain were quickly removed, washed in ice-cold saline, blotted dry and kept deep-frozen at - 2 0 ° C until used for estimations. All analyses were completed within 3 days of collection of tissue, during which no change was noticed in any of the parameters studied.
Estimations Total ammonia and urea estimations were made according to the methods described earlier (Saha and Ratha, 1989). Protein was estimated following the method of Lowry et al. (1951) using crystalline bovine serum albumin as the standard.
An ornithine-urea cycle in a freshwater teleost
Enzyme assay
Results
Ten percent homogenate (w/v) of liver and kidney tissue was prepared separately in 0.1% cetyltrimethyl ammonium bromide (CTB) for the assay of activities of the o-u cycle enzymes (Brown and Cohen, 1959). The homogenates were centrifuged at 600 g at 0 __+2°C for 15 min. The supernatants were used for enzyme assays. The enzymes of the o-u cycle, carbamoyl phosphate synthetase (CPS-ammonia dependent; EC 2.7.2.5), ornithine transcarbamylase (OTC; EC 2.1.3.3) and arginase (ARG; EC 3.5.3.1) were assayed in the liver and kidney following the method of Saha and Ratha (1987). It should be noted that the above assay for carbamoyl phosphate synthetase (CPS) does not distinguish between the three different forms of the enzyme, namely CPS I (ammonia-dependent, mitochondrial), CPS II (ammonia-dependent, cytosolic), or CPS III (glutamine-dependent, mitochondrial). Arginino-succinate synthetase (EC 6.3.4.5) and arginino-succinate lyase (EC 4.3.2.1) were assayed together to give the overall reaction as the arginine synthetase system (ASS) (Brown and Cohen, 1959). One unit of enzyme activity was defined as that amount which catalysed the formation of 1 #mole product/hr at 37°C. Activity of the enzymes were expressed as both tissue activity (units/g wet wt of tissue) and specific activity (units/mg protein). Data collected from five replicate fish were statistically analysed and presented as mean ___standard deviation (SD). Comparison of paired mean values was made using Student's t-test (Croxton et al., 1982) and P values greater than 0.05 were taken as non-significant (NS).
Survival in ammonium chloride solution
Chemicals
All the enzymes, co-enzymes, substrates, CTB and bovine serum albumin were obtained from Sigma Chemical Co. (St Louis, MO) and other chemicals used were of analytical grades obtained from indigenous sources. Streptopenicillin was a pharmaceutical preparation from Alembic Chemical Co., Vadodra, India. Deionized and double-glass-distilled ammonia-free water was used in all preparations.
317
H. fossilis exposed to 25 mM ammonium chloride showed minor irritational symptoms which calmed down within hours of treatment. However, the fish exposed to increasing concentrations of ammonium chloride showed increased irritational symptoms. Any disturbance in the experimental bucket resulted in violent disoriented escape attempts. The hyperexcitability calmed down within 48 hr in both 50 and 75 mM ammonium chloride. All the fish exposed to 100, 150 and 200 mM ammonium chloride died within 96, 24 and 12hr, respectively. These concentrations, therefore, could not be used for long-term experimentation. The pH of the medium, which was normally 7.2, decreased to 7.04, 6.94, 6.82, 6.72, 6.60 and 6.48 at 25, 50, 75, 100, 150 and 200 mM ammonium chloride concentrations, respectively. However, the pH and concentration of ammonia in the medium did not decrease during the period of treatment in each bucket due to its replacement every 48 hr. Most of the ammonia (99%) was expected to be in ionic form at the given pH and temperature of the medium and at physiological pH (Emerson et al., 1975). Tissue ammonia and urea levels Ammonia. Ammonia accumulated significantly in different tissues and in blood plasma of fish treated with ammonium chloride (Table 1, Figs 1 and 2). The accumulation increased with increasing concentrations of ambient ammonia. The peak of ammonia accumulation in different tissues which showed dependence on the ambient concentration of ammonium chloride was observed, in general, by the 7th day of treatment. The concentration slowly decreased during the latter part of the experimental period. However, the ammonia concentration in various tissues remained significantly higher than the normal level, even after 4 weeks. Kidney accumulated maximum amounts of ammonia (40.7#mol/g wet wt) followed by liver (34.0), muscle (18.0) and brain (13.7 # mol/g wet wt) of the fish treated with 75 mM ammonium chloride. The maximum rate of accumulation of ammonia was about 10 ×
318
N. Saha and B. K. Ratha
in blood plasma, 3 x in kidney, 2.5 x in liver and brain and 1.5 x in muscle with 75mM ammonium chloride treatment, compared to the control. Urea. Urea concentration increased significantly following the accumulation of ammonia in different tissues of fish exposed to ammonium chloride (Table 2, Figs 1 and 2). The increase continued until the 14th day after which the levels were either maintained (as in plasma and muscle) or slowly decreased (as in brain, liver and kidney). The accumulation of urea was higher at higher concentrations of ambient ammonia. It was maximal in liver (27.8/~mol/g wet wt) followed by kidney (16.8), brain (9.6) and muscle (2.6/~mol/g wet wt) of the fish treated with 75 mM ammonium chloride. In plasma, the urea level increased to 4.8 mM by the 14th day of 75mM ammonium chloride treatment. However, the maximum rate of accumulation of urea was lower than that for ammonia in various tissues. It was
about 6.5 x in plasma followed by 3.5 x in brain, 2.5 x in liver and 2 × in kidney and in muscle with 75 mM ambient ammonium chloride, compared to the control. Activities of the o-u cycle enzymes The enzyme activity of ARG was very high followed by OTC, ASS and CPS in both the liver and kidney tissues of Heteropneustes fossilis. The tissue and specific activity of o-u cycle enzymes, excepting ARG, showed significant induction in both liver and kidney of H. fossilis treated with 50 mM ammonium chloride (Tables 3 and 4; Fig. 1). The activity of the enzymes remained induced throughout the period of study with the highest level usually reached by the 14th day of treatment. The induction of ASS was very high (165-190%) followed by CPS (40-55%) and OTC (20-30%) in the liver. The physiological levels of activity of all the enzymes were lower in kidney compared to those in liver. The pattern and
Table 1. Alterations in the concentration o f a m m o n i a in different tissues (/tmol/g wet wt) and in blood plasma (mmol/1) o f H. fossilis exposed to various ambient concentrations o f a m m o n i u m chloride Days after treatment Tissue
NH¢CI (mM)
Liver (control)
Kidney (control)
Muscle (control)
Brain (control)
Plasma (control)
0
7
0 25 50 75
14.56_+ 1.15
13.33__+ 1.27 y 25.45_+ 1.51" 3 0 . 5 0 ± 1.61 a 33.77_+ 3.04 ~
13.78_+ 21.71 _ 27.99-+ 34.06_+
0.81 y 1.27 a 1.42 a 2.66"
13.98 h- 0.60 y 19.29-+ 1.14" 23.68_+0.89" 32.23 _+ 1.83 ~
13.25 18.76 23.15 30.15
_ 0.78 y __+ 1.12 ~ _+ 0.97 a + 2.68"
0 25 50 75
13.90_+1.22
12.60_+1.29 y 29.44_+2.29 ~ 37.62+0.93" 4 0 . 7 4 ± 1.01"
12.35_+0.43 y 24.67_+ 1.72 ~ 3 6 . 2 7 _ 1.60 a 39.61 -+3.63"
13.02+0.47 y 2 3 . 1 5 + 1.18" 36.65 +2.39" 35.80-+2.22"
13.11 21.75 34.35 32.91
_+ 1.23 y _+ 1.78 a _ 1.00 a __. 2.92"
0 25 50 75
12.43-+ 1.09
10.73 -+ 0.45 y 13.59_+0.86" 16.80___0.36 a 18.14-+0.91"
10.04-+ 0.22 y 13.14_+0.71" 14.78 ± 1.22" 17.17___0.60 a
10.35_ 0.43 y 13.59+0.55 a 14.20+0.99 ~ 16.89___0.45"
10.11 ___0.44 y 12.96 _+ 0.38" 14.02 + 0.74 a 16.25 _+ 0.89"
0 25 50 75
6.74 -+ 0.81
5.53 _+ 0.44 y 8.51 ± 0.47" 9.46 -!-_0 . 4 P 13.68_+2.01 a
5.47 ___0.86 y 9.14 ± 0.56" 9.88 -+ 0.74" 11.82_+0.40"
5.42 + 0.45 y 8.63 _+ 0.44" 9.20 -+ 0.73" 10.45+ 1.23"
5.51 8.24 8.79 10.22
-+ 0.42 y -+ 0.46" _+0.51" ± 1.2P
0 25 50 75
0.47 _+ 0.04
0.50 q- 0.04 y 2.18___0.21" 3.84+0.45" 4.56+0.56 a
0.47 h- 0.07 y 2.42+0.23 a 3.94_+0.36" 4.71 + 0 . 5 P
0.46 + 0.06 y 2.11 ___0.19~ 3.14+0.38 ~ 4.26-+0.47"
0.44 1.98 3.04 4.08
_+ 0.04 r _+ 0.13" ± 0.46 ~ +0.51 a
........
14
Y: P not significant c o m p a r e d to normal control. ~: P significant at <0.001 level c o m p a r e d to fasted control.
21
28
An ornithine-urea cycle in a freshwater teleost
319
200
0 o
I~a~
_ 200
100 0 200
Muscle
-o
4OO
1000 Plasma
Brm
800
-200
6O0
0
400
4o0
200
200
0
I 7
I
14
I
21
I 7
I 28
I 14
I 21
I
28
Days
Fig. 1. Percentage increase in ammonia and urea concentration in different tissues and in blood plasma of H. fossilis exposed to different concentrations of a m m o n i u m chloride. ( O - O Control; C)-C) 25 raM; / k - / k 50 m M and I-1-1-] 75 raM.)
level of induction were also different in the two tissues. The CPS, OTC and ASS were simultaneously induced in kidney during the treatment of ammonium chloride to a comparable level. Normal OTC level in kidney, which was less than 20% of that of liver, showed a maximum level of induction of 221% on the 21st day of treatment. CPS activity increased continuously in kidney with the increasing period of treatment, reaching the highest level (175%) on the 28th day. Maximum ASS activity (136%) in kidney was observed on the 7th day. The specific activity generally showed higher percentage of induction than the tissue activity in all the cases. Discussion
Presence of ammonia in the aquatic medium has been known to be toxic to
ammoniotelic freshwater fish. The 48 hr LCs0 value of ammonia for Cyprinus carpio was 15mg/1 (Dabrowska and Wlasow, 1986). The rates of mortality with 24 hr exposure at 8 and 40 mg/l ammonia were reported to be 50% for trout, Salmo gairdneri (Olson and Fromm, 1971) and 10% for goldfish (Schenone et al., 1982), respectively. Aquatic frog, Xenopus laevis, did not even survive beyond 1 hr at l0 mM ammonium chloride (Janssens, 1972). However, in the present study, H. fossilis tolerated and survived well in a 75 mM ambient ammonium chloride concentration which is very high compared to other freshwater teleosts and aquatic amphibians. This indicated the presence of efficient physiological and biochemical mechanisms for detoxification of excess ammonia in this fish. Ammonia is a neurotoxin. It interferes with energy metabolism, electro-physiologi-
320
N. Saha and B. K. Ratha
cal properties and level of neurotransmitters in the brain (Cooper and Plum, 1987). The fish, on exposure to ammonium chloride in the ambient medium at 25, 50 and 75 mM concentrations, therefore, became immediately hyperexcitable. Acclimatization to the changed environment gradually calmed down the irritational symptoms. A similar response was reported in goldfish (Olson and Fromm, 1971). All the fish exposed to ammonium chloride concentrations above 75 mM died within 4 days. Accumulation of acidic metabolites from glycolysis and Krebs cycle at higher ambient ammonia was suggested to cause anoxia and death due to a decrease in blood pH in coho salmon (Sausa and Meade, 1977). However, Hillaby and Randall (1979) could not observe any decrease in pH when the total ammonia load in the blood of rainbow trout (Salmo gairdneri) was elevated to a toxic level. Therefore, the decreasing pH of the ambient medium from 6.82 to 6.48, with an increasing concentration of
ammonium chloride from 75 to 200 mM, respectively, might not be the main cause of death in the fish. The death could be due to the enhanced accumulation of ammonia in vivo. Ammonia accumulated significantly in all the tissues of H. fossilis studied reaching the highest level within 7 days of exposure to ammonium chloride (Table 1, Figs 1 and 2). Suppression of excretion and absorption of ammonia from the ambient medium by H. fossilis (Saha and Ratha, 1990) probably resulted in this accumulation. The accumulation was tissue-specific. Brain, being highly susceptible to ammonia toxicity (Cooper and Plum, 1987), showed minimum accumulation. Effective enzymatic detoxification of ammonia to glutamate and then to glutamine by the enzymes glutamate dehydrogenase (GDH) and glutamine synthetase (GS), respectively, has been reported in fish brain (Levi et al., 1974; Walton and Cowey, 1977; Iwata et al., 1981; Chakravorty et al., 1989; Das et al.,
O Ammonia • Urea
Liver
[] cPs 50
200
[ ] OTC
[]Ass 40
160
30
120
20
80
0
40
e~
T
o~ 0
40
"
140
30
120
20
80
0
4O
0
7
14
23
.4
28
Days
Fig. 2. Alteration in the percentage Of induction o f the activity (units/g wet wt) o f o u cycle enzymes with relation to the concentration (/zmol/g wet wt) o f a m m o n i a and urea in liver and kidney o f H. :bssilis exposed to 50 m M ambient a m m o n i u m chloride.
An ornithine-urea cycle in a freshwater teleost
321
Table 2. Alterations in the concentration of urea in different tissues (#mol/g wet wt) and in blood plasma (mmol/l) of H. fossilis exposed to various ambient concentrations of ammonium chloride Days after treatment Tissue
NH4C1 (raM)
Liver (control)
Kidney (control)
Muscle (control)
Brain (control)
Plasma (control)
0
7
14
21
28
0 25 50 75
6.23_ 0.61
7.31 +_ 0.78 ~ 10.51 _ 0.60 ~ 12.72-t- 0.82 x 13.21 +_ 1.11~ 9.81+0.87 b 21.86_ 1.50~ 19.45_2.19 b 18.34_ 1.67 b 12.26_ 1.70 b 25.01___3.85 ~ 23.93_2.17 ~ 21.77+2.05 ~ 12.21 + 0.97 a 27.80_ 2.26 a 26.89 +_ 2.45 ~ 25.44 + 2.66 ~
0 25 50 75
5.53 + 0.97
6.48 + 0.81 y 8.22_0.89 d 10.43+0.40 ~ 10.39_ 0.61 a
0 25 50 75
1.31 +__0.32
1.22 _ 1.39 + 1.89 + 1.60 _
0 25 50 75
2.49 _ 0.37
2.87 4.26 4.90 5.05
0 25 50 75
0.76 _ 0.08
0.74 + 2.23 _ 3.16 _ 4.26 +
8.21 _ 0.61 x 12.76_ 1.08 a 15.46_2.29 a 16.74_+ 1.23~
10.82_ 1.33~ 12.30+ 1.26 ' 15.37_2.03 c 15.53_ 1.77 b
11.12 + 0.89 x 12.11 +__1.I0 r 15.87 + 1.70b 16.79 + 1.23~
0.23 y 0.25 f 0.25 c 0.31 f
1.31 0.28 y 1.79 _ 0.39 f 2.24 __-0.22 b 2.16_0.20 b
1.37 _ 0.20 r 1.82 +__0.33 e 2.30 _ 0.38 b 2.41 _0.41 b
1.45 _ 0.27 y 1.78 + 0.36 f 2.46 _ 0.28 c 2.62+0.41 ~
+__0.49 y _ 0.41 b _ 0.31 a _ 0.50 a
2.60 + 0.33 y 6.52+__0.45~ 9.43 + 0.77 a 8.89 + 0.55 a
2.91 7.06 9.66 9.12
4.01 7.11 9.45 8.78
0.74 2.54 3.87 4.82
0.73 _ 2.18 _ 3.54 _ 4.36 _
0.03 y 0.38 a 0.56 a 0.71 a
_
+ 0.04 y +__0.32 a _ 0.48 a _+ 0.79 ~
+ 0.19 y _ 0.25 a _ 0.46 a -t- 0.89 a 0.04 y 0.27 a 0.39 a 0.56 ~
+ 0.38 x +0.30 ~ ___0.39 a + 0.46 a
0.71 + 2.16 + 3.44 + 4.41 _
0.04Y 0.18 a 0.32 a 0.44 a
x and Y: P-values significant at <0.001 and non-significant, respectively, compared to the normal control. a,bc,d,e and r: P-values significant at <0.001, <0.005, <0.01, <0.025, <0.05 and non-significant, respectively, compared to the fasted control.
1991; C h a k r a v o r t y , Das and Ratha, in preparation). Liver and kidney, which are the main organs for ammonia metabolism in fish ( P e q u i n a n d S e r f a t y , 1963; V e l l a s a n d S e r f a t y , 1974; C a s e y et al., 1983), a c c u m u lated high concentrations (35--40/~mol/g t i s s u e ) o f a m m o n i a in H. fossilis. B l o o d being the carrier of metabolites, the concent r a t i o n o f a m m o n i a i n c r e a s e d 3 - 4 t i m e s in the plasma compared to the control animals. The concentration-dependent acc u m u l a t i o n in v a r i o u s t i s s u e s w a s r a t h e r slow at higher concentrations of ambient a m m o n i a a n d g e n e r a l l y s t a b i l i z e d a t a tissue-specific high concentration by the 7th d a y o f e x p o s u r e ( F i g . 2). D i f f e r e n t t i s s u e s probably have different maximum loading capacities for ammonia. Ammonia transport across the membrane takes place either by simple diffusion or by Na+-related carr i e r t r a n s p o r t o f NH~- ( H i l l a b y a n d R a n d a l l , 1979). T h e d i s t r i b u t i o n o f a m m o n i a in
different tissue compartments has been reported to be influenced by both the pH and t h e e l e c t r o c h e m i c a l g r a d i e n t ( W r i g h t et al., 1988). H o w e v e r , t h e m e c h a n i s m o f a m m o n i a t r a n s p o r t i n t h i s fish h a s n o t y e t b e e n studied. It was interesting to note that the m a x i m u m c o n c e n t r a t i o n o f a m m o n i a in t h e plasma did not exceed 5 mM when the a m b i e n t c o n c e n t r a t i o n w a s 75 m M . T h i s was quite a feat which might have cont r i b u t e d t o t h e s u r v i v a l o f t h e fish a t t h a t high ambient concentration of ammonium chloride. T h e c o n t r o l fish, w h i c h w e r e f a s t i n g in fresh water, did not show significant change in t h e a m m o n i a l e v e l s in v a r i o u s t i s s u e s ( T a b l e 1, F i g . 2). H o w e v e r , t h e y s h o w e d gradual reduction in the rate of excretion of ammonia during starvation (Saha and R a t h a , 1990). Metabolic detoxification of ammonia to u r e a c o u l d n o t b e o b s e r v e d in m o s t f r e s h -
322
N. Saha and B. K. Ratha
Table 3. Alterations in the tissue (units/g wet wt) and specific (units/mg protein) activity of o-u cycle enzymes in the liver of H. fossilis exposed to 50 mM ambient ammonium chloride Enzyme activity Days after treatment
CPS
OTC
ASS
ARG
Tissue
Sp. x 102
Tissue
Sp.
Tissue
Sp.
Tissue
Sp.
0 Control 7 P*
4.6 ___0.5
2.9 ___0.3
252 __+23
1.6 __+0.2
28.7 + 5.5
0.21 ___0.04 7700 ___933
50 -+ 7
5.9-+0.8 <0.05
3.8+1.0 <0.05
290___12 <0.025
1.7+0.1 NS
78.1-t-19.8 0.51+0.14 7193+495 <0.005 <0.01 NS
47_+3 NS
14 P
6.4_+0.8 <0.01
4.0+0.4 <0.005
328-t-30 <0.05
2.2__+0.2 82.1__+11.4 0.55+0.08 8298__+455 56-+4 <0.005 <0.001 <0.001 NS NS
21 P
6.0 + 0.7 <0.02
4.4___0.4 <0.001
306 + 27 <0.02
2.2 -+ 0.2 <0.005
75.9 + 15.5 0.53 ___0.01 7909 _+ 873 <0.001 <0.001 NS
28 P
6.4___0.4 <0.05
4.5+0.2 <0.001
293+34 <0.02
2.1_0.3 <0.025
83.7+9.4 <0.001
56 + 6 NS
0.60_+0.06 7507__+357 54___3 <0.001 NS NS
*P value compared to fasted control.
water teleosts due to incomplete o-u cycles (Brown and Cohen, 1960; Huggins et al., 1969; Wilson, 1973). However, the presence of a complete o-u cycle in the liver and kidney of H. fossilis with high activities of all the enzymes, suggested its role in ureogenic ammonia detoxification (Saha and Ratha, 1987). The accumulation of ammonia in vivo was associated with the induction of the activities of the o-u cycle enzymes, except ARG, in both the liver and kidney of H. fossilis within 7 days of treatment with 50 mM ammonium chloride (Tables 3 and 4, Fig. 2). The activity of the induced enzymes remained high throughout the period of the experiment. Even though
arginase activity was assayed at a high unphysiological pH (9.5), its physiological level of activity would also be very high. The high level of activity of all the enzymes of the o-u cycle must have accelerated the rate of ureogenesis (metabolic conversion of ammonia to urea) and thereby controlled the in vivo level of accumulated ammonia. Urea accumulated significantly in all the tissues studied in H. fossilis following the induction of the activities of the enzymes of the o-u cycle (Table 2, Figs 1 and 2). Accumulation of urea was also tissuespecific even though it is a highly permeant solute. Liver accumulated highest concen-
Table 4. Alterations in the tissue (units/g wet wt) and specific (units/mg protein) activity of o-u cycle enzymes in the kidney of H. fossilis exposed to 50 mM ambient ammonium chloride Enzyme activity Days after treatment
CPS
OTC
ASS
ARG
Tissue
Sp. × 102
Tissue
Sp.
Tissue
0 Control 7 P*
3.0+0.3
2.0_+0.3
43+3.3
0.28+0.02
21 +3.6
4.4+0.4 <0.005
3.0+0.2 <0.005
82+2.2 <0.001
0.56___0.05 48___7.6 0.33___0.06 1494+228 10.3_+1.4 <0.001 <0.001 <0.001 NS NS
14 P
5.2+1.0 <0.005
4.0-+0.8 <0.005
102___8 <0.001
21 P
5.1 +0.2 <0.001
4.0___0.2 114__+24 <0.001 <0.001
28 P
7.2___0.4 <0.001
6.0+0.3 <0.001
100+7 <0.001
*P value compared to fasted control.
0.76+0.10 <0.001
43-+7.3 <0.005
0.9__+0.11 4 0 + 4 . 8 <0.001 <0.001 0.77___0.06 41___8 <0.001 <0.005
Sp.
Tissue
Sp.
0.14-+0.03 1586-+ 104 9 . 4 + 0 . 7
0.32-+0.05 1758___222 13.2+ 1.3 <0.001 NS NS 0.31 +0.07 1667+229 <0.001 NS
13+2 <0.02
0.32___0.06 1732___224 12_+1.8 <0.001 NS <0.05
An ornithine-urea cycle in a freshwater teleost
tration of urea (27.8 #mol/g tissue) followed by kidney (16.8#mol/g). Muscle, which constitutes the major bulk of fish tissue, retained a very low concentration (2.6#mol/g) of urea. Either urea was rapidly diffused from the muscle through the skin or the muscle had some special physiological barrier to protect it from excess urea accumulation. Accumulation of urea by about 6 x in blood plasma, 4.5 x in liver, 4 x in brain, 3 x in kidney and 2 x in muscle compared to the control level (Fig. 1) indicated that maximum loading capacity for urea was different in different tissues. The presence of a specific urea transporter protein has been reported in mammalian erythrocytes and kidney tubules (Sands et al., 1992; Gillian and Sands, 1992). Involvement of such urea transporter protein in this fish cannot be ruled out. The excretion of urea, which was suppressed during exposure to higher ambient ammonia (Saha and Ratha, 1990) alone, could not account for the higher level of accumulation of urea in different tissues of Heteropneustes fossilis. Accumulation of ammonia seemed to induce the activity of the o-u cycle enzymes for enhanced conversion of toxic ammonia to less toxic urea. Such ammonia-induced ureogenesis has been reported in ureotelic amphibians (Balinsky et al., 1961; McBean and Goldstein, 1967; Janssens and Cohen, 1968; McBean and Goldstein, 1970; Janssens, 1972). Although the rate of excretion of urea was reported to increase significantly from the 10th day (Saha and Ratha, 1990), the tissue level of both ammonia and urea did not decrease proportionately, and continued at a higher level during exposure to higher ambient ammonia. The control fish, fasting in the ammoniafree medium, also accumulated significant amounts of urea only in the liver and kidney. Increased amino acid catabolism during fasting reported in fish (Johnston, 1981; Walton and Cowey, 1982; Moon, 1983) may have resulted in the production of ammonia and, in turn, urea in the liver and kidney, which had a complete o-u cycle. The results clearly suggest that the exposure of the freshwater air-breathing teleost, Heteropneustes fossilis, to higher
323
ambient ammonium chloride resulted in accumulation in vivo of ammonia and associated induction of the activities of the enzymes of the o-u cycle in liver and kidney which accelerated the rate of ureogenesis. The conversion of excess ammonia to urea and the reported increase in the excretion of urea (Saha and Ratha, 1990) controlled the in vivo concentration of ammonia and urea. Thus, the ammoniotelic fish became ureotelic to survive at that high (75 mM) concentration of ambient ammonium chloride. Acknowledgements--The authors thank the NorthEastern Hill University, Shillong, for facilities, the Council of Scientific and Industrial Research, New Delhi, for awarding an SRF to one of us (N.S.) and the University Grants Commission, New Delhi, for partial financial support.
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