Chem.-Biol. Interactions, 37 (1981) 165--170 165 © Elsevier/North-Holland Scientific Publishers Ltd.

ACUTE DIELDRIN TOXICITY: EFFECT ON THE UPTAKE OF GLUCOSE AND LEUCINE AND ON BRUSH BORDER ENZYMES IN MONKEY INTESTINE

A. MAHMOOD, N. AGARWAL, S. SANYAL*, P.K. DUDEJA and D. SUBRAHMANYAM**

Departments of Biochemistry and Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012 (India)

(Received March 20th, 1981) (Accepted May 30th, 1981)

SUMMARY

Administration of a single oral dose of dieldrin (20 mg/kg body wt.) to rhesus monkeys considerably elevated the uptake of glucose and the activities of brush border sucrase, lactase, maltase and alkaline phosphatase in intestine compared to control animals. Leucine uptake and leucine amino peptidase activity was significantly depressed in pesticide-treated animals. Kinetic studies with brush border sucrase revealed that augmentation of enzyme activity in pesticide-fed animals was due to an increase in the disaccharidase content .

INTRODUCTION

The widespread use of organochlorine pesticides in agriculture and health care programmes has aroused great concern in recent years on the potential deleterious effects that could occur in man and animals (for review see Refs. 1 and 2). According to WHO surveys, majority of the pesticides enter the human body through an oral route [3]. The intestinal tract thus consti tutes a primary target tissue for interaction before these toxicants reach other organs in the body. In previous reports from this laboratory, it has been demonstra ted that monkeys exposed to DDT exhibit an increased intestinal uptake of nutrients and disaccharidase activities [4,5]. Altera- tions in the chemical composi t ion of the microviUus membrane in pesticide-fed animals has also been described [6]. Whether the stimulatory

*Present address: Eunice Kennedy Shriver Center for Mental Retardation Inc. Waltham, MA 02154 U.S.A. **Present address: Director, CIBA-Giegy Research Center, Goregaon, Bombay.

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action of DDT on gut physiology is specific to this organochlorine insecti. cide or is a general mechanism of action of other members of this class oi pesticides remains to be elucidated. In this paper we describe the results on the effect of acute dieldrin (endo-exo-l,2,3,4,10,10-hexachloro-l,4,5,8- diendo-methylene-6,7-epoxy-l,4,4a,5,8,8a-hexahydro-napthalene) admini- stration on the intestinal uptake of glucose and leucine and on some of the brush border enzymes.

MATERIALS AND METHODS

Animals. Rhesus monkeys weighing 2--3 kg were used in these experi- ments. Animals were adapted to the laboratory environment for a week prior to the commencement of the experiments and were maintained on a stock diet consisting of bread, soaked gram, fruits and green vegetables with free access to water. Animals in the experimental group were fed dieldrin mixed with corn oil (20 mg/kg body wt.) by stomach tube. Control animals received equal amounts of corn oil alone. After 24 h overnight fasted animals were sacrificed under nembutal anaesthesia (30 mg/kg body wt.), the abdomen was opened and jejunal portion of the gut was removed. There were 4 animals in each group.

Intestinal uptake studies. Intestinal uptake of D-glucose and L-leucine was measured in vitro as described elsewhere [7]. Briefly, 120--150 mg of everted intestinal segments were incubated in 5 ml of oxygenated (95% 02 and 5% CO2) Kreb's Ringer buffer (pH 7.4) containing 5 mM D-glucose or L-leucine with trace amounts of D-[U-'4C]glucose or L-[U-'4C]leucine at 37°C. After 10 min, the tissues were removed, gently blotted, weighed and the radioactivity taken up was determined in a scintillation counter. After correcting for the extracellular space [7] the uptake rate was calculated and expressed as pmol /10 min/g wet wt. of tissue.

Enzyme assays. Portions of the intestine were homogenized in 50 mM sodium maleate (pH 6.5) and centrifuged at 1000 Xg for 10 min at 0--4°C. The supernatant was removed and used for the assay of enzymes. Disaccha- ridases were assayed as described previously [8]. Alkaline phosphatase activity was determined by the method of Bergmeyer [9]. Leucine amino peptidase was determined according to Goldbarg and Rutenberg [10]. Protein was estimated by the method of Lowry et al. [11] using bovine serum albumin as the standard.

Kinetic parameters of sucrase activity were determined by measuring the enzyme activity at different substrate concentrations and a double reciprocal plot of the data was made to calculate maximal velocity (Vmax) and the affinity constant (Kin). Statistical analysis of the data was calculated using Student 's t-test.

Reagents. All reagents used were of A.R. quality. Dieldrin was obtained from Fluka (A.G. Switzerland), Radioisotopes were procured from Bhabha Atomic Research Center, Trombay, Bombay. Glucose-oxidase peroxidase kit was from Boehringer-Knoll, F.R.G.

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TABLE I

E F F E C T OF D I E L D R I N ADMINISTRATION ON THE INTESTINAL UPTAKE OF D-GLUCOSE AND L-LEUCINE

Mean ± S.D. o f 8 observat ions f rom 4 animals. Expressed as umol /10 min/g wet tissue.

Group D-glucose L-leucine

Cont ro l 14.7 ± 3.9 12.6 ± 1.8 Dieldrin-fed 92.2 ± 15.2 5.3 ± 1.9 P-value <0 .001 <0 .001

TABLE II

E F F E C T OF D I E L D R I N ADMINISTRATION ON BRUSH B O R D E R ENZYMES IN MONKEY INTESTINE

Values are mean ± S.D. o f 8 de te rmina t ions . One e n z y m e unit is equal to 1 #mol of sub- s t ra te hyd ro lyzed per m inu t e under s tandard assay condi t ions . Expressed as uni ts /g tissue prote in .

E n z y m e Co ntrol Dieldrin-fed P-value

Sucrase 54.9 ± 4.9 78.2 ± 4.2 <0.001 Lactase 5.1 ± 1.7 1016 ± 2.7 <0.001 Maltase 134.0 ± 9.0 168.0 ± 15.2 <0.001 Alkaline phospha ta se 30.5 ± 3.9 47.4 ± 7.1 <0 .001 Leucine-amino pept idase 12.8 -+ 0.5 8.4 ± 0.3 <0.01

40

30

20

I0

i I I I 0.04 0 0.04 0.08 0.12

~ ml~' Fig. 1. Double reciprocal p lo t : sucrase act ivi ty was de t e rmined at subs t ra te concen t ra . t ions in t h e range 8--40 mM in con t ro l (C) and dieldr in-fed (D) mo n k ey s . Each poin t is the mean o f 4 de te rmina t ions . 1/v = (~mol g lucose /min/g p ro te in ) -I.

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R E S U L T S

The results describing the effect of a single oral dose of dieldrin on the intestinal uptake of glucose and leucine are presented in Table I. The uptake of glucose was markedly elevated (6-fold) in insecticide exposed animals compared to the controls. But the uptake of leucine was severely depressed (P < 0.001) under these conditions.

Brush border sucrase, lactase, maltase and alkaline phosphatase activities were also significantly enhanced (25--105%) in the intestine of dieldrin treated animals compared to the control group (Table II). Leucine amino peptidase activity was reduced ( P < 0.01) in pesticide-fed animals. The observed increase in the activity of disaccharidases in dieldrin toxici ty may be due to specific enzyme induction or because of new enzyme formation with an enhanced substrate affinity in response to the pesticide treatment. To examine these possible effects, kinetic characteristics of the brush border sucrase were examined. The Km was calculated from a double reciprocal plot (Fig. 1) for the enzyme in control and experimental animals and gave a value of 40.8 mM. However, the V m a x w a s elevated from 166.7 in the control to 232.6 (pmol glucose/min/g protein) in dieldrin-fed animals. These results tend to suggest that stimulation of sucrase activity afte~ dieldrin exposure is a consequence of net increase in the enzyme content.

DISCUSSION

The results illustrate that dieldrin administration to rhesus monkeys greatly affects the uptake of nutrients and brush border enzymes in the intestinal epithelium. Glucose uptake by intestinal segments and disaccha- ridase and alkaline phosphatase activities were considerably stimulated in pesticide-administered animals. The st imulatory action of dieldrin on sugar uptake and disaccharidase activities is similar to the effects of DDT in monkeys [3,4]. Such observations suggest that chlorinated hydrocarbon pesticides in general act by stimulating the digestion and absorption of sugars. Organochlorine pesticides induce drug metabolizing mixed function oxidase activity [12], gluconeogenic enzymes [13] and ornithine decar- boxylase activity in the liver [14]. Kinetic evidence indicates a close functional link between carrier mediated sugar transport system and sucrase in intestine [15,16]. The fact that both the uptake of glucose and disaccha- ridase activities are similarly affected in dieldrin-fed animals may suggest that the two systems are controlled by a similar or common mechanism(s).

In contrast to the action of dieldrin on the sugar transport system, the uptake of leucine and leucine amino peptidase activity in intestine was drastically impaired in insecticide~exposed monkeys. The inhibitory action of the pesticide is opposite to the st imulatory action of DDT on the amino acid uptake process in intestine [4]. Interestingly, a functional association be tween the amino acid transport system and peptidase activity has also been described in the intestine [17]. The present results showing that

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dieldrin administration induces malabsorption of leucine and reduces leucine amino peptidase activity in monkey intestine, may further provide an indirect evidence for the observed functional association be tween the two systems [17].

In separate experiments (results not shown) it was found that augmenta- tion of the brush border disaccharidases in dieldrin-fed animals is no t a consequence of pesticide interaction at the 'kinetic' level (modification of enzyme protein), since in vitro addition of an alcoholic solution of the pesticide (2 mM) to the enzyme assay mixture did no t influence the enzyme activity. Therefore the observed stimulation of enzyme activities appears to stem from pesticide interactions at the metabolic levels, through enzyme induction. Kinetic studies with brush border sucrase suggested that the increase in the activity of the enzyme is due to enhanced enzyme content . A similar mechanism for the activation of disaccharidases in response to DDT exposure has earlier been postulated in monkeys [4].

Dieldrin feeding to rats has been shown to produce hyperactivi ty of the adrenal glands [14,18]. Recent ly Celano et al. [19] reported that cortisol or 3- iodothyronine administration to rats can elicit precocious appearance of sucrase in the intestine. Thus hormonal imbalances associated with pesticide toxic i ty [14,18] may also be implicated in the observed increase in enzyme levels in dieldrin-fed animals. Organochlorine pesticides are known to produce hyperglycemia and glycosuria [1]. Stimulation of the intestinal functions in these disorders has also been described [20,21] .

ACKNOWLEDGEMENT

These investigations were partly financed by research grants from the Department of Science and Technology and the Council of Scientific and Industrial Research, New Delhi.

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