By G. Boge, D. Bussiere, G. Peres
Water Research, Volume 22, Issue 4, April 1988, Pages 441-447, ISSN 0043-1354, DOI: 10.1016/0043-1354(88)90038-3.

Abstract: Resume
Ce travail a concerne la recherche des effects du chrome (sous la forme de bichromate de potassium) sur l'activite de deux enzymes de la bordure en brosse intestinale; la phosphatase alcaline et la maltase, ainsi que sur l'absorption d'un acide amine: la glycine.
Une technique originale a ete utilisee pour mesurer l'activite des deux enzymes sur des poissons vivants. Elle consiste a perfuser les substrats (glucose-1-phosphate pour la phosphatase et maltose pour la maltase) dans la lumiere de l'intestin puis a suivre l'apparition du glucose produit.
Le chrome perfuse entraine une diminution de l'activite de la phosphatase alcaline dont l'intensite depend de sa concentration et de la duree de l'intoxication. Une inhibition voisine de 50% a ete enregistree apres 30 min de perfusion par une concentration de bichromate de 1,36 mM et apres 90 min pour une concentration 10 fois plus faible.
A la concentration de 0,136 mM une stimulation transitoire de l'activite de l'enzyme a ete observee au cours des 60 premieres minutes de perfusion.
L'activite de la maltase n'est en revanche pas influencee par le chrome, meme a une concentration tres elevee: 136 mM.
L'etude de l'absorption de la glycine a ete egalement effectuee chez des truites vivantes au moyen de la meme technique de perfusion, L'absorption est tres sensible au chrome, mais seulement pour des concentrations de toxiques tres elevees: 136 mM, et par consequent peu significatives d'un point de vue ecotoxicologique.
Ce travail demontre la particuliere sensibilite de l'activite de la phosphatase alcaline de l'intestin de la truite vis vis du chrome hexavalent et presente une nouvelle methode pour etudier chez des poissons vivants les activitees de la bordure en brosse en relation avec une intoxication par la voie intraluminale.
A new procedure has been developed to study the effects of hexavalent chromium (potassium dichromate) on brush border membrane functions of the intestines of living trout (alkaline phosphatase and maltase activities, glycine absorption).
Manifold recylced perfusions were carried out with saline solutions containing the specific substrate, the hydrolysis of which liberates glucose (Bussiere et al., 1987). At the end of each period, glucose concentrations were determined using the glucose oxydase method (Hugget and Nixon, 1957).
For the determination of alkaline phosphatase activity, glucose-1-phosphate (20 mM) was used as substrate rather than classic para-nitrophenyl phosphate because the product of its hydroloysis; para-nitrophenol, is rapidly absorbed by the intestine (Bussiere et al., 1987). Phloridzin (10-4 M) and KCl (150 mM), were added to the perfusates to prevent the glucose absorption, pH was adjusted to 10 with CHES (20 mM).
For the determination of maltase activity, maltose (28 mM) was used as substrate in KCl (150 mM) solutions containing phloridzin (10-4 M). pH was adjusted to 6.5 with MES (20 mM).
The absorption of glycine by the intestine of trout was also determined in vivo using a perfusion technique (Peres et al., 1973). The intestinal lumen was perfused with a saline solution containing glycine 14C (0.5 mM) and buffered at pH 7.4 with HEPES 20 mM. The perfusate was recycled in the intestine during 30 min periods and non absorbed glycine was collected and measured.
Effects of hexavalent chromium on enzyme activities
On each trout, a perfusion was first carried out in the absence of toxicant. Chromium was then added to the solutions and succeeding 30 min perfusions were carried out. After the chromium perfusions, the intestines were rinsed and perfused with a medium devoid of chromium. At the end of each period, alkaline phosphatase and maltase activities were measured.
Two different dichromate concentrations were used, 1.36 and 0.136 mM. The highest concentration is generally lethal for trout after 24 h exposure in the aquatic environment.
Chromium leads to a severe decrease of the alkaline phosphatase activity which increases with the chromium concentration and with time.
An approximative 50% inhibition of this activity was observed after 30 min with 1.36 mM dichromate. Such an inhibition is obtained after 90 min with the lowest chromium concentration.
During the early perfusion with 1.36 mM dichromate, a transient stimulation of the alkaline phosphatase activity has been observed which is progressively replaced by a characteristic inhibition.
After chromium removal from the perfusion medium, the enzyme activity remains at a lower level than in the first control experience. This means that no recovery of the initial activity exists.
No effect of chromium on maltase activity has been observed.
Effect of hexavalent chromium on glycine absorption
The intestinal absorption of amino acids in fish requires, as in mammals, the presence of sodium in the intraluminal medium, and can be highly depressed when sodium is replaced by potassium (Boge et al., 1980a). To avoid prejudicial effects of potassium on amino acid absorption, potassium dichromate was replaced by sodium dichromate and sodium was present in the perfusate at a final concentration of 272 mM.
Chromium effects on each trout were studied after 60 min perfusions following control determinations.
The results obtained revealed that chromium is a potent inhibitor of glycine absorption in trout but only when the concentration is very high: a 90% inhibition was obtained at 136 mM dichromate. This effect disappears when this concentration is ten times lower.
These results point out the particular interest in ecotoxicology of the alkaline phosphatase activity which is a sensible target for chromium in trout intestine. A new method was presented which permits the measurement of this activity on living fishes, in relation to the presence of a toxic molecule.
Keywords: chrome hexavalent; phosphatase alcaline; maltase; absorption glycine; intestin truite

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