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Effect of cadmium and zinc on intestinal absorption of xylose and tryptophan in the fresh water teleost fish, Heteropneustes fossilis
Chemosphere, Vol.13, No.8, pD 889 - 898, 1984 Printed in Great Britain
0045-6535/84 $3.00 + .00 ©1984 Perqamon Press Ltd.
EFFECT OF CADFJ.IU~< /~q[] ZINC ON INTESTINAL ABSORPTION OF XYLOSE ~ J D TRYPTOPHA~ IN THE FRESH UATER TELEOST F I S H , HETEROPNEUSTES F O S S I L I S
K.V. SASTRY* and S. SUBHADRA Environmental Toxicology Laboratory, Department of Zoology, College, Muzaffarnagar - 251 001, India.
D.A.V.
(P.G.)
SU~ARY The effect of cadmium and of zinc on the rate of uptake of a pentose sugar xylose and an aminoacid tryptophan by the intestine of a teleost fish, Heterooneustes fossilis was studied under two experimental conditions. In the first, four concentrations of cadmium or zinc (1.O mM, O.1 mM, O.O1 mM and O.OO1 ~¢i) mixed with the nutrient solution were filled in the intestinal sacs, and the rate of absorption was recorded after 1 h at 23°C. In the second experiment fish were exposed by bath to a sublethal concentration of cadmium (0.26 mg/1) or zinc (4 mg/l) for 15 and 30 days and the rate of absorption of the two nutrients was measured. The activity of intestinal Na +, K + activated adenosine triphosphamase was also assayed. The two heavy metals at all the four concentrations decreased the rate of intestinal transport of nutrients. Increase in the concentration of each of the heavy metals decreased the uptake of nutrients, but the decreases were not linear. The rate of intestinal absorotion of the two nutrients was also reduced by exposure of fish to the heavy metals in vivo. The activity of Na +, K + ATPase decreased in vitro with all four concentrations of cadmium and zinc and was diminished in fish exposed for 15 and 30 days. Of the two heavy metals, cadmium was more effective in reducing the rate of transport of xylose and tryptophan. INTRODUCTION Heavy meta]s discharged from industries are a major source of pollution and may threaten the life of aquatic organisms, especially fishes. Kumar (I) reported 0.22 ]Jg/l - 2.29 ng/l of cadmium and 5.4 ~g/l - 6.5 ~g/l of zinc in unpolluted water and 1.8 zg/l - 11.5 pg/l of cadmium and I0 pg/l of zinc in polluted water of the Jamuna river near the Union Territory of Delhi. Cadmium and zinc occur simultaneously in the environment (2,3) and are toxic to fish. Muramoto (4) studied the effect of cadmium on fish after 47, 58, 73, 85 and 95 days and observed that different concentrations of cadmium in water ranging from O.O1 to O.I ppm produced vertebral damage. Merlini (5) noted hepatic storage alterations of vitamin BI2 in the fresh water fish, Leoomis qibbosus exposed to 0.04 ppm of cadmium for 2 weeks. Bengtsson (6) reported minnows (Pnoxinus phoxinus) exposed to 0.20 and 2.4 mg/l of zinc developed hemorrhages, lesions and vertebral damaqe in tests lasting up to 9 months. Although most toxicity tests with fish have used short-term acute exposures, attempts are being made to determine the most sensitive effects of toxicants through chronic exposures of fish over their complete life cycle (7). Heavy metals present in water can enter the digestive system of fishes through consumption of food chain organisms (8) and can adversely affect the processes of digestion of food material (9~ and absorption of nutrients by t~e intestine (iO). However, very little information is avail* Present address:
Post Graduate Department of Zoology, Rohtak (Haryana), India.
889
M.D. University,
890
able on the effect of heavy metals on the absorption of nutrients by the intestine of teleost fishes. This aspect is important, as m a l a b s o r o t i o n of nutrients by the intestine can disturb the m e t a b o l i s m and nutritive value of fishes. Earlier reports from this laboratory (ll,12) have shown that m e r c u r y and cadmium decrease the rate of transport of glucose and fructose significantly. The present work Sas been undertaken to examine if heavy metals can adversely affect the uptake of nutrients by the intestine oi fish and to probe the possible mechanism. In the first experiment, nutrients solutions mixed with heavy metals were directly instilled into the intestinal sacs of the fish, and in the second experiment fish were exposed to a sublethal concentration of cadmium or zinc for 15 or 30 days by bath and the rate of absorption of the two nutrients were measured. Experiments were also conducted to observe if cadmium or zinc exert any effect on the intestinal brush border Na +, K + activated adenosine triphosphatase. MATERIALS
AND METHODS
Living specimens of ~[eter0pneustes fossilis were collected from local fresh water sources and maintained in laboratory glass aquaria. Prior to experimentation, the fish were allowed to acclimate to laboratory conditions for a week. During this period the fish were fed witn commercial fisin food twice a day. Feeding was stopped 24 h before the experiments to clear any food material out of the alimentary canal. The water characteristics were pH 7.6; temperature 20+3°C; hardness 160 ppm (as CaCO3); alkalinity 165 ppm (as CaCO3) and dissolved oxygen c o n c e n t r a t i o n 7.2 ppm. Intestinal sacs were prepared accordino to the method of Musacchia (13). ~our concentrations of cadmium sulphate or zinc sulphate (I.O m~, O.l mM, O.Ol mM and O.OO1 mM) containing 30 mM of xylose or 30 mH of tryotophan were prepared in saline solution. The fish were placed on a dissecting board and cotton plugs soaked in water were placed in the mouth, gills and over the skin of the fish to keep the surface moist. A midline abdominal incision was made in order to expose the viscera. The intestine was cut at the p v l o r i c - d u o d e n a l junction and infront of the rectum. Into the duodena] end of the intestine, a syringe tube attachment was inserted and the intestinal contents were flushed out with saline solution. A ligature was applied to the rectal end of the intestine and another loose li@ature was held in p o s i t i o n towards the duodenal end, to be tied after filling of the intestinal sacs with appropriate solutions. The intestinal sacs were separately filled with 0.5 ml of nutrient saline solution containing each concentration of the heavy metal solution. In control fish equal volume of nutrient saline solution without cadmium or zinc was used. The luminal fluids of both control and experimental fish were collected after i h at 23°C and their velume was recorded. The concentration of xylose and tryotophan in the recovered solutions were determined according to the colorimetric methods of Park and Johnson (14) and Graham et al. (15). The drv weight of the intestines was recorded by dehydrating th~ t ~ s u e in an oven'at iOO C until the w e i g h t became c o n s t a n t . In another experiment, 240 fish were divided into eight e q u a l orouos. The first group was exposed under static conditions to a sublethal concentration (0.26 m9/l) of cadmium for 15 days and the second qrouo for 30 days. The third group was exposed to a sublethal c o n c e n t r a t i o n (A mg/!) of zinc for 15 days and the fourth group for 30 days. The remaining four groups served as controls. W a t e r in the aquaria was renewed after every 2e hr and fresh cadmium or zinc solutions were added to bring the concentration of the heavy metals to the requisite level. On !6th and 31st days, the rate
891
of intestinal a b s o r p t i o n was d e t e r m i n e d in both control and heavy metal e x p o s e d fish by instilling xylose and t r y p t o o h a n in saline solution seoararely as d e s c r i b e d above. The effect of c a d m i u m and zinc on the intestinal brush border adenosine triphospF~atase a c t i v i t y (ATPase) was studied under three e x p e r i m e n t a l cor~ditions. In the first, the intestinal sacs of e x p e r i m e n t a l fish were fiJied s e p a r a t e l y witi~ saline solutions containing the four c o n c e n t r a t i o n s of cadmium and zinc. In control fish, saline s o l u t i o n without cadmium and zinc was used. After i h at 23°C the intestinal sacs from both control and e x o e r i m e n t a l fish were c o l l e c t e d and the luminal contents were discarded. Ti',e intestinal mucosa was scraped and weighed to the n e a r e s t millicram. The p r o c e d u r e d e s c r i b e d by Jampol and Epstein (16) was adopted for the p r e p a r a t i o n of enzyme extract and d e t e r m i n a t i o n of the enzyme activity. In the second experiment, the intestines from 40 normal fish were c o l l e c t e d an(] ~ o m o g e n i z e d as above, i n c u b a t i o n m e d i u m containing the subsirate and four c o n c e n t r a t i o n s of c a d m i u m or zinc (l.O m},<, O.l m~A, 0.01 mM ant 0 . 0 0 1 ~A) were prepared. In controls, heavy metals were excluded from the incubation medium. Enzyme extracts were added to these two types of solutions and the activity of ATPase was determined at 23°C. In the third experiment, fish were exposed to sublethal c o n c e n t r a t i o n s of c a d m i u m or zinc as d e s c r i b e d above and the activity of intestinal Na +, K + d e p e n d e n t AYPase was determined. 7he first and second e x p e r i m e n t s are treated as in vitro and the third as in vivo.
The s i g n i f i c a n c e o f d i f f e r e n c e s b e tw een c o n t r o l and e x p e r i m e n t a l was c a l c u l a t e d by t h e S t u d e n t ' s ' t ' t e s t d e s c r i b e d by F i s h e r ( 1 7 ) .
means
RESULTS No marked d i f f e r e n c e was observed in the rates of intestinal a b s o r p t i o n of xylose and t r y p t o p h a n in the four groups of control fish and b e t w e e n the two sugars (Table 1 to 4). Inclusion of cadmium or zinc in the instilled m e d i u m or exposure of fish to either of the two heavy metals in the ambient w a t e r d e c r e a s e d the rate of t r a n s p o r t of xylose and tryptophan. Increase in the c o n c e n t r a t i o n of each of the two heavy metals in the instilled s o l u t i o n resulted in a p r o g r e s s i v e decrease in the rate of a b s o r p t i o n of xy]ose and tryptophan, but the decrease was not linear. At the hiqhest c o n c e n t r a t i o n of the two metals used (1.O ~4) d e c r e a s e in t h ~ r a t e of t r a n s p o r t of t r y p t o p h a n was g r e a t e r than that of xylose. The n e r c e n t a g e of d e c r e a s e after 30 days of exposure was almost equal to that of O.1 m~Z in the first experiment. Yhe a c t i v i t y of }~a+,K + ATPase was reduced in all the three experimental conditions. Increase in the c o n c e n t r a t i o n of each of the two heavy metals d e c r e a s e d enzyme activity. I n h i b i t i o n was maximal at the hiohest c o n c e n t r a t i o n of cadmium or zinc (1.O mZ). Inclusion of cadmium or zinc in the saline s o l u t i o n instilled in the intestinal sacs p r o d u c e d a creater r e d u c t i o n in enzyme activity than was seen in the intestinal h o m o g e n a t e s or in vivo. Intestinal brush border AYPase a c t i v i t y was affected by chronic exposure of H e t e r o p n e u s t e s f o s s i l i s to c a d m i u m or zinc, and 30 days of e x p o s u r e p r o d u c e d greater r e d u c t i o n in enzyme a c t i v i t y than did exposure fol 15 days.
892
D I SCUS S ION The results obtained in the present study reveal that the presence of cadmium or zinc in the intestine or in the ambient v,ater can decrease the rate of transport of xylose and tryptophan and the activity of intestinal brush border Na+,K + ATPase. The rate of absorption of eaci~ of the two nutrients is inversel]~ related to tt~e concentration of each of the two heavy metals in the instilled solution and to the period of exposure of test fish to the heavy metals. Hioher concentration of both the heavy metals decreased nutrient absorption-rates to a greater extent than do lower concentrations in vitro. The decreases were most markeo betv,~een O.O1 and O.OO1 m~.] of cadmium or zinc. Thirty days exposure to eacli heavy metal in vivo produced a creater decrease in the rate of absorption of the two nutrients than did ~xposure for 15 days. Thus, the p o s s i b i l i t y that exposure for periods lonoer than SO days may produce an even greater reduction in the rate of transport of nutrients cannot be r~!led out. ,qlvarado (18) reported that suoar and amino acid transport by the intestine is carrier-mediated. Heavy metals like m~rcury, lead, cadmium and zinc, due to their strong affinity for ligands like phosphate cysteinyl and histidyl side chains of proteins, can bind to the carrier protein molecules, resultinQ in inhibition of suqar and amino acid transport. L{vidence for reduct{on of absorotion of nu{rients by heavy metals comes from the observations of Wapnir e__~ta]. (19), who reported inhibition of glucose transport in rats rece]v~nq 20 mq/Kg body w e i g h t of lead acetate. Heavy metals may also bind to the proteins of the brush border membrane of the mucosal cells and alter their permeability. This view lacks e x p e r i m e n t a l v a l i d a t i o n and needs further study. Keiic~i et al. (9<~,) reported the binding of cadmium to the supernatant components in rat intestinal mucosa. Shears and Hletc~ier (21) also observed binding of zinc to t~e soluble proteins of intestinal mucosa in ti~e winter flounder (gseud_~_p_lu_r_onectes" americanus). Inhibition of the activity of enzymes involved in t,~e active transport of nutrients may be one of the factors responsible for the observed decrease in the rate of absorption of x~/]ose and tryptophan. Schultz et al. (22) and Crane (23) have established a direct correlation between Na + concentration and sugar and amino acid transport. Decrease in the activity of Na +, K+ ATPase may lead to a decrease in the transport rate of Na + that in turn may reduce the uptoke of suqars and amino acids by the intestine of fish. Wembrane bound ![a+, K + AT~Sase is believed to play an important role in th~ active transport of nutrients. ATPase breaks down ATP, releasing energy required for t~e t r a n s p o r t process. Inhibition in the activity of this enzyme may thus decrease the rate of transport of nutrients. In the present sL~dv, Li~e activity of ATPase was reduced in all the three experim~nta! conditions. Addition of each of the two heavy metals to the saline solution instilled or to the intestinal mucosal extracts decreased the activity of ATPase to a greater tnan in the intestinal homogenate. This difference may be due to the direct a c c e s s i b i l i t y of the heavy metal ions to t~e enzyme molecules in the former preparation. Inhibition of intestinal brush border ATPase and alkaline phosphatase activities in rats exposed to cadmium has been reported by Sugav lara and Suga,wara (24) bo%n in v~vo and in vitro.
893
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Table 2. Effect of cadmium in vivo and in vitro on the activity of Na +, K + ATPase in t-K[ intestine of Heteropneu~ites fossilis. Concentration of cadmium/duration of exposure
~R~ inorganic phosphate/mg of p r o te in_Zh Control
Experimental
% Alter
In vitro ist experimental condition 1.0 m~1 (112.41 rag/l) 0.I m~1 (ll.2a mg/l) 0.01 ~4(i.12 mg/l) 0.001 mh1(O.ll rag/l)
3 3 3 3
9.59+--0.36 9.59_+0.36 9.59+0.36 9.59_+0.36
2.70_+0.19 *~ 3.11_+0.16-* 3.85__+0.12~ 7. la_--eO.30-
71.85 67.57 59.85 2,%.55
9.27+0.33 9.72T0.33 9.98~0.43 9.98±0.43
4.86+0.28** 5.62~).21-* 6.41~0.308.09p0.27-
=~ j~.O0 42.18 35.77 18.94
7.86_+0.25* 6.10+0.22"*
16.03 33.33
2nd experi91ental condition i.o ~A
(n~.4±
rag/l)
0.I m~,~ (11.24 mg/l) 0.01 m~A (1.12 rag/l) 0.001 m}~(O.ll mg/l)
s 3
3 3
In vivo 0.26 mg/l for 15 days 0.26 m9/l for 30 days
3 3
9.36_+0.31 9.15~0.30
Values are mean + S.E. Va!,les are statistically significant from control value at *P<0.05; **P
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Table
4.
Effect o f zinc in v i v o and in v i t r o on t h e acti,~~tv o{ Na + K + A T P a s e in the i n t e s t i n e of H e t e r o p n e u s t e : ~:o ~-~ , l
Concentration zinc/duration exposure
ubl
of of
inorcanic phosohate/mc: of orotein/_~ . . . . . . . . . . . . . . . . . . . . . . Exoerimen%a]
Control
~ &]t~.
In v i t r o 1st
condition
experimental
1 . 0 m~,4 ( 6 5 . 3 7 m g / 1 ) 0.i m~' ( 6 . 5 3 m q / l ) O . O l m.~! ( 0 . 6 [ ) rag/!) 0 . 0 0 1 mI'4(O 0 7 m g / ! )
2nd exoerimental
3 3 3 3
9.72p.42 9.72_+0.42 9.72+0.42
2.98+0 T7~ 2.59+(0.25"*
,.
7.02+0.34*
~-.t<, 4","
,~q "'" 71! .3') f v. 7 '
condition 3 3 3 3
] .0 ~wt (6,5.37 mg/l 0.i mbl ( 6 . 5 3 mg/l 0.0] mbl (0.65 mg/l 0 . 0 0 1 rp~,'~(O.07 m9/l
o .72+0.42 9.72+0.42 9.29_+(9.40 9.29=0.4 0
~. ~" 69-@: 3 6 * °
5.08,~.35, 6 ]~T:O 5o* 8.7D---'0.37( ) _, -
~~
.
,","
o-, ~c '~: ~' ,....... '~
In v i vo a mg/l f o r L5 days 4 rag/! for 30 days Values
are
mean
3 3
7.90+0.28:' 6.34+7.26*
1,~ % :F'.&.':
+ S.E.
Values are statistically *P < 0 . 0 % ; **P< 0.0!.
(-) S t a t i s t i c a l l y n : number
9.49q0.33 o . z~ l ~ r, ~ -~. 39
signific~nt
insienificant.
of e x p e r i m e n t .
from
control
w~iue
~1
897
Any one o f t~lese factors or a combination o f all tr:ese factors may be responsible for the observed decrease in transnort rate of xylose or tryotophan :~C ~9~()",'/L EDG EME NT
One of the authors (KVS) is grateful to the University Grants Commission, New Del~i for a 'Career Award' and to the autr~orities of D.A.V. College, Muzaffarnagar for facilities. The J.R.F. to Subhadra by CSIR, New Deli~i is gratefully acknowledge. i:
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V. I
2.
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14.
g.r . Park and ~.3. Johnson, Colorimetr}c ana]ys~s of suqars, Cited from L:etnods in Enzymology, Vol. !iI, edited by S.P. Colowick and H.O. Kaplan, Academic Press, ]'jew York, 86 (1963).
15.
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P.K. Crane,
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in USA 1 June
1984)
Bull. Environ. Contam.
Toxicol., i~,
0045-6535/84 $3.00 + .00 ©1984 Perqamon Press Ltd.
EFFECT OF CADFJ.IU~< /~q[] ZINC ON INTESTINAL ABSORPTION OF XYLOSE ~ J D TRYPTOPHA~ IN THE FRESH UATER TELEOST F I S H , HETEROPNEUSTES F O S S I L I S
K.V. SASTRY* and S. SUBHADRA Environmental Toxicology Laboratory, Department of Zoology, College, Muzaffarnagar - 251 001, India.
D.A.V.
(P.G.)
SU~ARY The effect of cadmium and of zinc on the rate of uptake of a pentose sugar xylose and an aminoacid tryptophan by the intestine of a teleost fish, Heterooneustes fossilis was studied under two experimental conditions. In the first, four concentrations of cadmium or zinc (1.O mM, O.1 mM, O.O1 mM and O.OO1 ~¢i) mixed with the nutrient solution were filled in the intestinal sacs, and the rate of absorption was recorded after 1 h at 23°C. In the second experiment fish were exposed by bath to a sublethal concentration of cadmium (0.26 mg/1) or zinc (4 mg/l) for 15 and 30 days and the rate of absorption of the two nutrients was measured. The activity of intestinal Na +, K + activated adenosine triphosphamase was also assayed. The two heavy metals at all the four concentrations decreased the rate of intestinal transport of nutrients. Increase in the concentration of each of the heavy metals decreased the uptake of nutrients, but the decreases were not linear. The rate of intestinal absorotion of the two nutrients was also reduced by exposure of fish to the heavy metals in vivo. The activity of Na +, K + ATPase decreased in vitro with all four concentrations of cadmium and zinc and was diminished in fish exposed for 15 and 30 days. Of the two heavy metals, cadmium was more effective in reducing the rate of transport of xylose and tryptophan. INTRODUCTION Heavy meta]s discharged from industries are a major source of pollution and may threaten the life of aquatic organisms, especially fishes. Kumar (I) reported 0.22 ]Jg/l - 2.29 ng/l of cadmium and 5.4 ~g/l - 6.5 ~g/l of zinc in unpolluted water and 1.8 zg/l - 11.5 pg/l of cadmium and I0 pg/l of zinc in polluted water of the Jamuna river near the Union Territory of Delhi. Cadmium and zinc occur simultaneously in the environment (2,3) and are toxic to fish. Muramoto (4) studied the effect of cadmium on fish after 47, 58, 73, 85 and 95 days and observed that different concentrations of cadmium in water ranging from O.O1 to O.I ppm produced vertebral damage. Merlini (5) noted hepatic storage alterations of vitamin BI2 in the fresh water fish, Leoomis qibbosus exposed to 0.04 ppm of cadmium for 2 weeks. Bengtsson (6) reported minnows (Pnoxinus phoxinus) exposed to 0.20 and 2.4 mg/l of zinc developed hemorrhages, lesions and vertebral damaqe in tests lasting up to 9 months. Although most toxicity tests with fish have used short-term acute exposures, attempts are being made to determine the most sensitive effects of toxicants through chronic exposures of fish over their complete life cycle (7). Heavy metals present in water can enter the digestive system of fishes through consumption of food chain organisms (8) and can adversely affect the processes of digestion of food material (9~ and absorption of nutrients by t~e intestine (iO). However, very little information is avail* Present address:
Post Graduate Department of Zoology, Rohtak (Haryana), India.
889
M.D. University,
890
able on the effect of heavy metals on the absorption of nutrients by the intestine of teleost fishes. This aspect is important, as m a l a b s o r o t i o n of nutrients by the intestine can disturb the m e t a b o l i s m and nutritive value of fishes. Earlier reports from this laboratory (ll,12) have shown that m e r c u r y and cadmium decrease the rate of transport of glucose and fructose significantly. The present work Sas been undertaken to examine if heavy metals can adversely affect the uptake of nutrients by the intestine oi fish and to probe the possible mechanism. In the first experiment, nutrients solutions mixed with heavy metals were directly instilled into the intestinal sacs of the fish, and in the second experiment fish were exposed to a sublethal concentration of cadmium or zinc for 15 or 30 days by bath and the rate of absorption of the two nutrients were measured. Experiments were also conducted to observe if cadmium or zinc exert any effect on the intestinal brush border Na +, K + activated adenosine triphosphatase. MATERIALS
AND METHODS
Living specimens of ~[eter0pneustes fossilis were collected from local fresh water sources and maintained in laboratory glass aquaria. Prior to experimentation, the fish were allowed to acclimate to laboratory conditions for a week. During this period the fish were fed witn commercial fisin food twice a day. Feeding was stopped 24 h before the experiments to clear any food material out of the alimentary canal. The water characteristics were pH 7.6; temperature 20+3°C; hardness 160 ppm (as CaCO3); alkalinity 165 ppm (as CaCO3) and dissolved oxygen c o n c e n t r a t i o n 7.2 ppm. Intestinal sacs were prepared accordino to the method of Musacchia (13). ~our concentrations of cadmium sulphate or zinc sulphate (I.O m~, O.l mM, O.Ol mM and O.OO1 mM) containing 30 mM of xylose or 30 mH of tryotophan were prepared in saline solution. The fish were placed on a dissecting board and cotton plugs soaked in water were placed in the mouth, gills and over the skin of the fish to keep the surface moist. A midline abdominal incision was made in order to expose the viscera. The intestine was cut at the p v l o r i c - d u o d e n a l junction and infront of the rectum. Into the duodena] end of the intestine, a syringe tube attachment was inserted and the intestinal contents were flushed out with saline solution. A ligature was applied to the rectal end of the intestine and another loose li@ature was held in p o s i t i o n towards the duodenal end, to be tied after filling of the intestinal sacs with appropriate solutions. The intestinal sacs were separately filled with 0.5 ml of nutrient saline solution containing each concentration of the heavy metal solution. In control fish equal volume of nutrient saline solution without cadmium or zinc was used. The luminal fluids of both control and experimental fish were collected after i h at 23°C and their velume was recorded. The concentration of xylose and tryotophan in the recovered solutions were determined according to the colorimetric methods of Park and Johnson (14) and Graham et al. (15). The drv weight of the intestines was recorded by dehydrating th~ t ~ s u e in an oven'at iOO C until the w e i g h t became c o n s t a n t . In another experiment, 240 fish were divided into eight e q u a l orouos. The first group was exposed under static conditions to a sublethal concentration (0.26 m9/l) of cadmium for 15 days and the second qrouo for 30 days. The third group was exposed to a sublethal c o n c e n t r a t i o n (A mg/!) of zinc for 15 days and the fourth group for 30 days. The remaining four groups served as controls. W a t e r in the aquaria was renewed after every 2e hr and fresh cadmium or zinc solutions were added to bring the concentration of the heavy metals to the requisite level. On !6th and 31st days, the rate
891
of intestinal a b s o r p t i o n was d e t e r m i n e d in both control and heavy metal e x p o s e d fish by instilling xylose and t r y p t o o h a n in saline solution seoararely as d e s c r i b e d above. The effect of c a d m i u m and zinc on the intestinal brush border adenosine triphospF~atase a c t i v i t y (ATPase) was studied under three e x p e r i m e n t a l cor~ditions. In the first, the intestinal sacs of e x p e r i m e n t a l fish were fiJied s e p a r a t e l y witi~ saline solutions containing the four c o n c e n t r a t i o n s of cadmium and zinc. In control fish, saline s o l u t i o n without cadmium and zinc was used. After i h at 23°C the intestinal sacs from both control and e x o e r i m e n t a l fish were c o l l e c t e d and the luminal contents were discarded. Ti',e intestinal mucosa was scraped and weighed to the n e a r e s t millicram. The p r o c e d u r e d e s c r i b e d by Jampol and Epstein (16) was adopted for the p r e p a r a t i o n of enzyme extract and d e t e r m i n a t i o n of the enzyme activity. In the second experiment, the intestines from 40 normal fish were c o l l e c t e d an(] ~ o m o g e n i z e d as above, i n c u b a t i o n m e d i u m containing the subsirate and four c o n c e n t r a t i o n s of c a d m i u m or zinc (l.O m},<, O.l m~A, 0.01 mM ant 0 . 0 0 1 ~A) were prepared. In controls, heavy metals were excluded from the incubation medium. Enzyme extracts were added to these two types of solutions and the activity of ATPase was determined at 23°C. In the third experiment, fish were exposed to sublethal c o n c e n t r a t i o n s of c a d m i u m or zinc as d e s c r i b e d above and the activity of intestinal Na +, K + d e p e n d e n t AYPase was determined. 7he first and second e x p e r i m e n t s are treated as in vitro and the third as in vivo.
The s i g n i f i c a n c e o f d i f f e r e n c e s b e tw een c o n t r o l and e x p e r i m e n t a l was c a l c u l a t e d by t h e S t u d e n t ' s ' t ' t e s t d e s c r i b e d by F i s h e r ( 1 7 ) .
means
RESULTS No marked d i f f e r e n c e was observed in the rates of intestinal a b s o r p t i o n of xylose and t r y p t o p h a n in the four groups of control fish and b e t w e e n the two sugars (Table 1 to 4). Inclusion of cadmium or zinc in the instilled m e d i u m or exposure of fish to either of the two heavy metals in the ambient w a t e r d e c r e a s e d the rate of t r a n s p o r t of xylose and tryptophan. Increase in the c o n c e n t r a t i o n of each of the two heavy metals in the instilled s o l u t i o n resulted in a p r o g r e s s i v e decrease in the rate of a b s o r p t i o n of xy]ose and tryptophan, but the decrease was not linear. At the hiqhest c o n c e n t r a t i o n of the two metals used (1.O ~4) d e c r e a s e in t h ~ r a t e of t r a n s p o r t of t r y p t o p h a n was g r e a t e r than that of xylose. The n e r c e n t a g e of d e c r e a s e after 30 days of exposure was almost equal to that of O.1 m~Z in the first experiment. Yhe a c t i v i t y of }~a+,K + ATPase was reduced in all the three experimental conditions. Increase in the c o n c e n t r a t i o n of each of the two heavy metals d e c r e a s e d enzyme activity. I n h i b i t i o n was maximal at the hiohest c o n c e n t r a t i o n of cadmium or zinc (1.O mZ). Inclusion of cadmium or zinc in the saline s o l u t i o n instilled in the intestinal sacs p r o d u c e d a creater r e d u c t i o n in enzyme activity than was seen in the intestinal h o m o g e n a t e s or in vivo. Intestinal brush border AYPase a c t i v i t y was affected by chronic exposure of H e t e r o p n e u s t e s f o s s i l i s to c a d m i u m or zinc, and 30 days of e x p o s u r e p r o d u c e d greater r e d u c t i o n in enzyme a c t i v i t y than did exposure fol 15 days.
892
D I SCUS S ION The results obtained in the present study reveal that the presence of cadmium or zinc in the intestine or in the ambient v,ater can decrease the rate of transport of xylose and tryptophan and the activity of intestinal brush border Na+,K + ATPase. The rate of absorption of eaci~ of the two nutrients is inversel]~ related to tt~e concentration of each of the two heavy metals in the instilled solution and to the period of exposure of test fish to the heavy metals. Hioher concentration of both the heavy metals decreased nutrient absorption-rates to a greater extent than do lower concentrations in vitro. The decreases were most markeo betv,~een O.O1 and O.OO1 m~.] of cadmium or zinc. Thirty days exposure to eacli heavy metal in vivo produced a creater decrease in the rate of absorption of the two nutrients than did ~xposure for 15 days. Thus, the p o s s i b i l i t y that exposure for periods lonoer than SO days may produce an even greater reduction in the rate of transport of nutrients cannot be r~!led out. ,qlvarado (18) reported that suoar and amino acid transport by the intestine is carrier-mediated. Heavy metals like m~rcury, lead, cadmium and zinc, due to their strong affinity for ligands like phosphate cysteinyl and histidyl side chains of proteins, can bind to the carrier protein molecules, resultinQ in inhibition of suqar and amino acid transport. L{vidence for reduct{on of absorotion of nu{rients by heavy metals comes from the observations of Wapnir e__~ta]. (19), who reported inhibition of glucose transport in rats rece]v~nq 20 mq/Kg body w e i g h t of lead acetate. Heavy metals may also bind to the proteins of the brush border membrane of the mucosal cells and alter their permeability. This view lacks e x p e r i m e n t a l v a l i d a t i o n and needs further study. Keiic~i et al. (9<~,) reported the binding of cadmium to the supernatant components in rat intestinal mucosa. Shears and Hletc~ier (21) also observed binding of zinc to t~e soluble proteins of intestinal mucosa in ti~e winter flounder (gseud_~_p_lu_r_onectes" americanus). Inhibition of the activity of enzymes involved in t,~e active transport of nutrients may be one of the factors responsible for the observed decrease in the rate of absorption of x~/]ose and tryptophan. Schultz et al. (22) and Crane (23) have established a direct correlation between Na + concentration and sugar and amino acid transport. Decrease in the activity of Na +, K+ ATPase may lead to a decrease in the transport rate of Na + that in turn may reduce the uptoke of suqars and amino acids by the intestine of fish. Wembrane bound ![a+, K + AT~Sase is believed to play an important role in th~ active transport of nutrients. ATPase breaks down ATP, releasing energy required for t~e t r a n s p o r t process. Inhibition in the activity of this enzyme may thus decrease the rate of transport of nutrients. In the present sL~dv, Li~e activity of ATPase was reduced in all the three experim~nta! conditions. Addition of each of the two heavy metals to the saline solution instilled or to the intestinal mucosal extracts decreased the activity of ATPase to a greater tnan in the intestinal homogenate. This difference may be due to the direct a c c e s s i b i l i t y of the heavy metal ions to t~e enzyme molecules in the former preparation. Inhibition of intestinal brush border ATPase and alkaline phosphatase activities in rats exposed to cadmium has been reported by Sugav lara and Suga,wara (24) bo%n in v~vo and in vitro.
893
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Table 2. Effect of cadmium in vivo and in vitro on the activity of Na +, K + ATPase in t-K[ intestine of Heteropneu~ites fossilis. Concentration of cadmium/duration of exposure
~R~ inorganic phosphate/mg of p r o te in_Zh Control
Experimental
% Alter
In vitro ist experimental condition 1.0 m~1 (112.41 rag/l) 0.I m~1 (ll.2a mg/l) 0.01 ~4(i.12 mg/l) 0.001 mh1(O.ll rag/l)
3 3 3 3
9.59+--0.36 9.59_+0.36 9.59+0.36 9.59_+0.36
2.70_+0.19 *~ 3.11_+0.16-* 3.85__+0.12~ 7. la_--eO.30-
71.85 67.57 59.85 2,%.55
9.27+0.33 9.72T0.33 9.98~0.43 9.98±0.43
4.86+0.28** 5.62~).21-* 6.41~0.308.09p0.27-
=~ j~.O0 42.18 35.77 18.94
7.86_+0.25* 6.10+0.22"*
16.03 33.33
2nd experi91ental condition i.o ~A
(n~.4±
rag/l)
0.I m~,~ (11.24 mg/l) 0.01 m~A (1.12 rag/l) 0.001 m}~(O.ll mg/l)
s 3
3 3
In vivo 0.26 mg/l for 15 days 0.26 m9/l for 30 days
3 3
9.36_+0.31 9.15~0.30
Values are mean + S.E. Va!,les are statistically significant from control value at *P<0.05; **P
895
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Table
4.
Effect o f zinc in v i v o and in v i t r o on t h e acti,~~tv o{ Na + K + A T P a s e in the i n t e s t i n e of H e t e r o p n e u s t e : ~:o ~-~ , l
Concentration zinc/duration exposure
ubl
of of
inorcanic phosohate/mc: of orotein/_~ . . . . . . . . . . . . . . . . . . . . . . Exoerimen%a]
Control
~ &]t~.
In v i t r o 1st
condition
experimental
1 . 0 m~,4 ( 6 5 . 3 7 m g / 1 ) 0.i m~' ( 6 . 5 3 m q / l ) O . O l m.~! ( 0 . 6 [ ) rag/!) 0 . 0 0 1 mI'4(O 0 7 m g / ! )
2nd exoerimental
3 3 3 3
9.72p.42 9.72_+0.42 9.72+0.42
2.98+0 T7~ 2.59+(0.25"*
,.
7.02+0.34*
~-.t<, 4","
,~q "'" 71! .3') f v. 7 '
condition 3 3 3 3
] .0 ~wt (6,5.37 mg/l 0.i mbl ( 6 . 5 3 mg/l 0.0] mbl (0.65 mg/l 0 . 0 0 1 rp~,'~(O.07 m9/l
o .72+0.42 9.72+0.42 9.29_+(9.40 9.29=0.4 0
~. ~" 69-@: 3 6 * °
5.08,~.35, 6 ]~T:O 5o* 8.7D---'0.37( ) _, -
~~
.
,","
o-, ~c '~: ~' ,....... '~
In v i vo a mg/l f o r L5 days 4 rag/! for 30 days Values
are
mean
3 3
7.90+0.28:' 6.34+7.26*
1,~ % :F'.&.':
+ S.E.
Values are statistically *P < 0 . 0 % ; **P< 0.0!.
(-) S t a t i s t i c a l l y n : number
9.49q0.33 o . z~ l ~ r, ~ -~. 39
signific~nt
insienificant.
of e x p e r i m e n t .
from
control
w~iue
~1
897
Any one o f t~lese factors or a combination o f all tr:ese factors may be responsible for the observed decrease in transnort rate of xylose or tryotophan :~C ~9~()",'/L EDG EME NT
One of the authors (KVS) is grateful to the University Grants Commission, New Del~i for a 'Career Award' and to the autr~orities of D.A.V. College, Muzaffarnagar for facilities. The J.R.F. to Subhadra by CSIR, New Deli~i is gratefully acknowledge. i:
I.
V. I
2.
J.J. Lingane, Analytical Chemistry of Selected f'etal]ic Elements, Reinhold Publ. Co., New York, N.Y., 143 (1966).
3.
II.A. Schroeder, A.P. Nason, Dis., 2__OO, 179 (1967).
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6.
B.E.
7.
D.A. Benoit, E.N. Leonard, G.M. Christensen and J.T. Fiandt, Trans. Am. Fish. Soc., i__O_~5,550 (1976).
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Environ. Safety, ~, 47 (197a).
K.V. Sastry and P.K. Gupta, Environ. Res., 1__9_9 , 221 (1979). K.V
Sas%ry and Kin. Subhadra, V{ater Soil and Aim Pollut.
9~
0o3
(igs3). ii.
K.V. Sastry and D.R. Rao, V~ater, Soil and Air Poilu%., ( 1982 ).
19, la~3
12.
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13.
X.J. h'1~sacchia, Intestinal absorption of olucose in vivo, Cited from ExDeriments in Physiology and Biochemistry, V-~l__f~ edited by G.A. Kerkut, Academic Press, London, 379 (1967).
14.
g.r . Park and ~.3. Johnson, Colorimetr}c ana]ys~s of suqars, Cited from L:etnods in Enzymology, Vol. !iI, edited by S.P. Colowick and H.O. Kaplan, Academic Press, ]'jew York, 86 (1963).
15.
C.F. Graham, E.P. Smith, S.~,~. Hier and D. Klein, Cited from f?e%hods in Enzymology, Vol. Ill, edited by S.P. Colowick and N.O. Kap!an, Academic Press, New York, 613 (1963).
898
16.
L.M. Jampol and F.?q. Eostein, Am. J. Physiol., 218, 607 (1970).
17.
R.A. Fisher, Statistical Methods for Research Workers, Oliver and Boyd, London (1950).
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F. Alvarado, Science, 151, iO11 (1966).
19.
R. Wapnir, S. Moak, F. Lieschitz and S. Teicnberg, J. Lab. Clin. Med., 94, 144 (1979).
20.
T. keiichi, N. Kaori and O. Kunio, Eisei KaQaku , 19, 202 (1973).
21.
M.A. Shears and G.L. Fletcher, Gomp. Biochem. Physiol., 6~r R, 297 (1979).
22.
S.G. Schultz, P.F. Curran, R.A. Chez and R.E. Fuisz, J. Can. Physiol., 5_0_, 1241 (1967).
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P.K. Crane,
24.
N. SuQawara and Co Sugawara, 653
(Received
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l!%h ed.
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in USA 1 June
1984)
Bull. Environ. Contam.
Toxicol., i~,