PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 15, 143-148 (1981) Effect of a Single Oral Dose of DDT on Intestinal Uptake of Nutrients and Brush Border Enzymes in Protein -Calorie-Malnourished Monkeys A. MAHMOOD, N. AGARWAL, P. K. DUDEJA, S. SANYAL,’ R. MAHMOOD, AND D. SUBRAHMANYAM~ Depurtments of Biochemistry und Gastroenterology. Postgraduute Institute of Medical Educution und Reseurch, Chundigarh-160012. Indiu Received December 23, 1980: accepted February 9. 1981 The effect of a single oral dose of DDT (150 mg/kg body wt) has been investigated on the intestinal uptake of certain nutrients and on brush border enzymes in protein-calorie-malnour- ished monkeys. In contrast to the stimulatory action of DDT on intestinal functions in normal-fed monkeys (A. Mahmood, N. Agarwal, S. Sanyal. P. K. Dudeja, and D. Subrahmanyam, Pest. Biochem. Physiol. 12, 141 (1979) ) pesticide feeding to malnourished animals inhibited the uptake of alanine and phenylalanine and depressed sucrase, lactase, and leucine amino-peptidase activities. Glucose and leucine uptake was also reduced, albeit insignificantly and alkaline phosphatase activ- ity was unaffected under these conditions. Protein-energy malnutrition per se considerably ele- vated the uptake of nutrients and disaccharidase activities. Analysis of the chemical composition of microvillus membrane in underfed and pesticide-exposed malnourished animals revealed alter- ations in protein, sialic acid, and phospholipid fractions of the brush borders. Membrane choles- terol and triglyceride contents remained unaltered in protein-energy-deficient and DDT-fed mon- keys. Exposure to organochlorine pesticides is well documented to induce metabolic and pathological derangements in experimental animals and in humans (for review see (1, 2) ). Further it has also been demonstrated that toxicity of these pesticides is aggra- vated in malnutrition (3, 4). A marked re- duction in vitamin A level in liver was ob- served in animals maintained on low- protein diets, after exposure to DDT (5, 6). Dieldrin administration to vitamin C- deficient guinea pigs also failed to induce the mixed-function oxidase activity in hepatocytes (7). In previous reports from this laboratory we demonstrated that DDT administration considerably stimulated the absorption of glucose and amino acids and elevated disaccharidase activities in mon- key intestine (8, 9). In view of these obser- ’ Present address: Department of Biochemistry. Eunice Kennedy Shriver Center for Mental Retarda- tion, Waltham, Mass. 02154. ” Present address: Director, Ciba-Geigy Research Centre. Goregaon East. Bombay-400 063. India. vations, present studies were undertaken to examine the effect of DDT feeding on the digestive and absorptive functions of the intestinal epithelium in protein-calorie malnutrition (PCM). These results suggest that acute DDT treatment to protein- calorie-deficient monkeys severely impairs the absorption of glucose and amino acids and inhibits brush border enzymes. Chem- ical composition of the microvillus mem- brane was also affected in malnutrition and in DDT toxicity compared to the control animals. MATERIALS AND METHODS Animals. Rhesus monkeys (Macacu mulatta) approximately 2 kg body wt were used in these experiments. Animals were acclimatized to laboratory conditions and fed on laboratory stock diet consisting of wheat bread, soaked Bengal gram, fruits, and vegetables, and had free access to water. After stabilization, animals were di- vided into two groups: I. Control group. 143 0048.3575/81/020143-06$02.00/O Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
Transcript
PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 15, 143-148 (1981)
Effect of a Single Oral Dose of DDT on Intestinal Uptake of Nutrients and Brush Border Enzymes in Protein -Calorie-Malnourished Monkeys
A. MAHMOOD, N. AGARWAL, P. K. DUDEJA, S. SANYAL,’ R. MAHMOOD, AND D. SUBRAHMANYAM~
Depurtments of Biochemistry und Gastroenterology. Postgraduute Institute of Medical Educution und Reseurch, Chundigarh-160012. Indiu
Received December 23, 1980: accepted February 9. 1981
The effect of a single oral dose of DDT (150 mg/kg body wt) has been investigated on the intestinal uptake of certain nutrients and on brush border enzymes in protein-calorie-malnour- ished monkeys. In contrast to the stimulatory action of DDT on intestinal functions in normal-fed monkeys (A. Mahmood, N. Agarwal, S. Sanyal. P. K. Dudeja, and D. Subrahmanyam, Pest. Biochem. Physiol. 12, 141 (1979) ) pesticide feeding to malnourished animals inhibited the uptake of alanine and phenylalanine and depressed sucrase, lactase, and leucine amino-peptidase activities. Glucose and leucine uptake was also reduced, albeit insignificantly and alkaline phosphatase activ- ity was unaffected under these conditions. Protein-energy malnutrition per se considerably ele- vated the uptake of nutrients and disaccharidase activities. Analysis of the chemical composition of microvillus membrane in underfed and pesticide-exposed malnourished animals revealed alter- ations in protein, sialic acid, and phospholipid fractions of the brush borders. Membrane choles- terol and triglyceride contents remained unaltered in protein-energy-deficient and DDT-fed mon- keys.
Exposure to organochlorine pesticides is well documented to induce metabolic and pathological derangements in experimental animals and in humans (for review see (1, 2) ). Further it has also been demonstrated that toxicity of these pesticides is aggra- vated in malnutrition (3, 4). A marked re- duction in vitamin A level in liver was ob- served in animals maintained on low- protein diets, after exposure to DDT (5, 6). Dieldrin administration to vitamin C- deficient guinea pigs also failed to induce the mixed-function oxidase activity in hepatocytes (7). In previous reports from this laboratory we demonstrated that DDT administration considerably stimulated the absorption of glucose and amino acids and elevated disaccharidase activities in mon- key intestine (8, 9). In view of these obser-
’ Present address: Department of Biochemistry. Eunice Kennedy Shriver Center for Mental Retarda- tion, Waltham, Mass. 02154.
” Present address: Director, Ciba-Geigy Research Centre. Goregaon East. Bombay-400 063. India.
vations, present studies were undertaken to examine the effect of DDT feeding on the digestive and absorptive functions of the intestinal epithelium in protein-calorie malnutrition (PCM). These results suggest that acute DDT treatment to protein- calorie-deficient monkeys severely impairs the absorption of glucose and amino acids and inhibits brush border enzymes. Chem- ical composition of the microvillus mem- brane was also affected in malnutrition and in DDT toxicity compared to the control animals.
MATERIALS AND METHODS
Animals. Rhesus monkeys (Macacu mulatta) approximately 2 kg body wt were used in these experiments. Animals were acclimatized to laboratory conditions and fed on laboratory stock diet consisting of wheat bread, soaked Bengal gram, fruits, and vegetables, and had free access to water. After stabilization, animals were di- vided into two groups: I. Control group.
143 0048.3575/81/020143-06$02.00/O Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved
144 MAHMOOD ET AL.
animals were fed a synthetic diet (27.0 g/kg body wt/day) as described in Table 1. II. PCM group, animals were given 13.5 g/kg body wtiday of the synthetic diet. It was earlier established that normal protein re- quirement of these animals is 4.84 g/kg body wt/day and 110 KcaYkg body wt/day (see legend to Table 1). This nutritional reg- imen was continued up to 12 weeks and thereafter animals in PCM group were di- vided into two groups. Half of the animals were administered DDT dissolved in corn oil (150 mg/kg body wt) orally using Ryle’s tube. The other half of PCM and control animals (group I) received the same amount of corn oil alone. After being fasted 48 hr overnight, animals were sacrificed under nembutol anesthesia (30 mg/kg body wt). Blood was directly drained from the heart for determination of total serum proteins. Starting from the ligament of Treitz, a 25-cm portion of the jejunum was removed, immediately flushed with ice-cold saline, thoroughly cleaned, and everted. There were three animals in each group.
Determination of intestinal uptake of glucose and amino acids. Intestinal uptake of D-glucose, L-alanine, L-leucine, and L- phenylalanine was measured using radio- labeled substrates as previously described (9). Uptake rates were corrected for the extracellular space, measured separately using [3H]inulin (10).
Preparation of brush border membranes. Procedure of Schmitz et al. (11) was employed to prepare the brush borders. Membrane fragments were suspended in 50 mi!4 sodium maleate, pH 6.5, and exhibited 20-fold enrichment of brush border sucrase (marker enzyme) activity over the crude homogenate with nearly 75% recovery.
Enzyme assays and chemical determina- tions. Mucosa was scrapped from portions of the jejunum using glass slide, a 5% (wt/v) homogenate was prepared in 50 mM sodium maleate pH 6.5 and centrifuged at 1OOOg for 10 min at 0-4°C. Supernatant was removed and used as such for enzyme assays. Brush border sucrase (BBS) and lactase (BBL) were assayed as described earlier (12). Al- kaline phosphatase (AP) activity was de- termined by the method of Bergmeyer (13). Leucine amino-peptidase (LAP) was as- sayed as described (14). Protein estimation was done according to Lowry et al. (15). Extraction, separation, and quantification of the membrane lipids was done as previ- ously reported (9). Sialic acid was deter- mined by the method of Warren (16). All enzyme activities were expressed as Units/g protein (1 Unit of enzyme activity is equivalent to 1 pmol substrate hydrolyzed min under standard assay conditions). Statistical analysis of the data was done using Student’s t test.
Reagents. All reagents used were of an-
TABLE 1 Composition of the Synthetic Diet Fed to Monkeys for I2 Weeks
Note. A multivitamin tablet (Bayer Ltd., India) was provided to each animal daily with tap water ud libitum. Control animals received 27.0 g of this diet/kg body wt/day, which yields 4.84g protein and 110 kcakkg body wt/ day. Animals in the PCM group received 13.5 g/kg body wtiday of the diet, which yields 2.42 g protein and 55 kcakkg body wtlday.
n The composition of the salt mixture was that described by Hubbel et al. (25).
DDT TOXICITY AND MALNUTRITION 145
alytical grade. DDT (Hindustan Insec- ticides Ltd., New Delhi, India) was purified prior to use as described previously (17). D-[U-lJC]Glucose, L-[U-14C]alanine, L-[U- 14C]leucine, and L-[U-14C]phenylalanine were obtained from Bhabha Atomic Re- search Center, Bombay, India. [3H]Inulin was procured from the Radiochemical Centre, Amersham, England. Glucose- oxidase-peroxidase containing kit was from Boehringer, Knoll, West Germany.
RESULTS
Animals in the PCM group exhibited se- vere signs of malnutrition such as loss of hair, edema on the face, and sluggish be- havior after 3 weeks of feeding on protein-calorie-deficient diet; body weight did not show appreciable change until the end of 4 weeks, when a 12% reduction in the body weight was observed. At the end of 12 weeks, PCM animals reflected 25% reduction in body weight, whereas animals in the control group grew at a mean rate of 26.4 g/day during the experimental period. At the time of sacrificing, water logging in the brain tissue was noticed and serum protein level of protein-malnourished ani- mals was 7.56% compared to 8.6% in the control group (P < 0.05).
The results describing the effect of DDT administration on the intestinal uptake of different nutrients are shown in Table 2. A considerable increase (40- 126%) in the uptake of glucose, alanine, leucine, and
phenylalanine in the intestine of PCM ani- mals was observed compared to control group. However, DDT administration to protein-energy-deficient animals depressed the uptake of alanine and phenylalanine (P < 0.05) compared to the oil-fed PCM group. The uptake of glucose and leucine in the PCM + DDT group was also re- duced, but this change was not significant compared to the PCM group.
The activities of various brush border en- zymes in protein-calorie deficiency and after pesticide treatment were also studied and these results are presented in Table 3. BBS and BBL activities were elevated two- to fourfold in PCM intestine compared to control animals; levels of AP and LAP in PCM, however, did not show any appreci- able alteration from control values. DDT feeding to undernourished monkeys strong- ly inhibited the BBS, BBL, and LAP (P < 0.01) activities compared to oil-fed PCM animals. AP activity was stimulated under these conditions, but the increase was not statistically significant.
Microvillus membrane lining the surface of the enterocytes is the primary site re- sponsible for the terminal stages of diges- tion and absorption in the gut. As described above DDT feeding to malnourished mon- keys greatly influences the uptake capacity of the intestine and the enzyme activities associated with the brush borders, there- fore we analyzed the chemical composition of the brush border membranes to see
TABLE 2 Effect of PCM und DDT Exposure on the Intestinul Uptuke of Nutrients in Rhesus Monkeys
pmol/lO min/g wet tissue”
Substrate Control PCM PCM + DDT
D-GhCOSe (II)* 5.66 zk 0.28 11.14 t 1.05* 9.39 t 1.29 L- Alanine (10) 4.07 t 0.63 9.22 k 0.76* 5.46** i 0.75 L-Leucine (10) 9.93 + 0.81 14.99 2 1.47’ 12.47 t 0.63 t.-Phenylalanine (10) 6.41 + 0.38 9.02 e 0.32* 7.02** i 0.63
” Values are mean + SE. ’ Figures in parentheses indicate the number of determinations.
*P < 0.001 compared to controls. ** P < 0.05 compared to PCM group.
146 MAHMOOD ET AL.
TABLE 3 Effect of DDT Administration on Brush Border Enzymes in Monkeys Exposed to
’ Values are mean ? SE of three animals run in duplicate. * P < 0.001 compared to control group.
** P < 0.01 compared to PCM group.
whether membrane constituents are also affected under these situations. These re- sults are depicted in Table 4. Membrane protein and sialic acid contents were in- creased although the change in protein content was insignificant but phospholipids were decreased in the PCM group com- pared to the controls. Pesticide administra- tion to malnourished monkeys reduced the sialic acid content of membranes but aug- mented the phospholipids. Other fractions, viz. cholesterol and triglycerides, did not reveal any change in the membrane frag- ments isolated from control and experi- mental animals.
DISCUSSION
We have previously described that both acute and chronic DDT administrations produce hyperactivities of the intestine in monkeys (8, 9). Present results, however, indicate that acute DDT toxicity in PCM impairs the uptake of sugar and amino acids and diminishes the BBS, BBL, and LAP activities of the enterocytes.
Intestinal uptake of glucose, alanine, leucine, and phenylalanine and brush bor- der disaccharidase activities were greatly elevated in PCM groups compared to con- trol animals. These observations, in gen- eral, are in agreement with studies with lower laboratory animals that feeding of
protein-deficient diet or fasting result in en- hanced nutrient absorption and elevated disaccharidase levels in the intestine (18, 19). Recently, it has also been demon- strated that undernutrition imposed during weaning also stimulates the digestive and absorptive functions of the intestinal epithelium in rats (20).
Susceptibility of malnourished animals to intestinal infections because of reduced immunity has been described (21). The present observations indicate that pro- tein-energy-deprived animals become susceptible to the toxic effects of DDT, as it refers to intestinal functions. An impair- ment in the activities of hepatic drug- metabolizing enzymes in response to diel- drin toxicity in protein deficiency has been observed (22). Depletion of vitamin A stores in protein-deficient rats exposed to DDT has also been described (3, 5, 6). Wagstaff and Street (7) demonstrated that vitamin C deficiency interferes in the in- duction of mixed-function oxidase activity by dieldrin in the liver. Inhibition of stimu- lation of microsomal enzyme activities in phenobarbitone-treated protein deficient animals has also been observed by Marshall and McLean (23).
We do not know the exact mechanism leading to altered mode of action of DDT with regard to intestinal functions in mon- keys with protein-energy-malnutrition.
DDT TOXICITY AND MALNUTRITION 147
TABLE 4 Effect of DDT Administration on the Chemical Composition oj
Microvillus Men&runes in Rhesus Monkeys
Membrane mgilO0 mg dry membranes”
- fraction Control PCM PCM + DDT
-- Protein 71.4 f 3.2 82.2 2 4.1 75.2 2 4.4 Sialic acid” 0.67 + 0.02 0.88 + 0.03* 0.65 t 0.04** Phospholipids 14.7 + 0.7 12.6 -t 0.4* 15.9 2 0.6*’ Free cholesterol 5.7 t 0.2 5.5 + 0.2 5.7 k 0.1 Esterified
” Values are mean t- SE of three observations done in duplicate. ’ fimoU100 mg dry membranes.
* P < 0.05 compared to control group. ** P < 0.01 compared to PCM group.
But it is likely that in absence of sufficient protein nutrition, metabolic alterations in the epithelial cells occur and the aberrated tissues become susceptible to inhibitory actions of the pesticide. Also as shown above, PCM itself markedly augmented the uptake of nutrients and disaccharidase ac- tivities in intestine. Since these systems in undernutrition already exist in a state of “stimulation,” further enhancement in these activities in response to DDT admin- istration may not be possible owing to lim- ited energy supply. Young et al. (6) have shown that rats fed low-protein diets retain significantly larger concentrations of DDT in the liver, because of low levels of mixed-function oxidase activity. The ob- served intestinal derangements in DDT toxicity in PCM may also be attributed to this phenomenon. DDT administration to PCM animals produced only mild changes in the chemical composition of the brush borders (Table 4). These results are quite distinct from the effects of pesticide in normal-fed animals, where striking altera- tions in the membrane make-up were ob- served (24).
Nevertheless, the present results that DDT exposure of malnourished animals in- duces malfunctions of the gut are of some serious considerations in view of the exten- sive use of these toxicants in health care
programs and in agriculture in the devel- oping countries, where malnutrition is an equally prevalent disorder. It is apparent that nutritional status of the animals has a strong bearing on the metabolic interactions of the pesticides. Further studies on the long-term exposure of the insecticides in various nutritional disorders are in progress in this laboratory, to elucidate the etio- pathogenesis of intestinal derangements in pesticide toxicity.
ACKNOWLEDGMENTS
These studies were financed in part by research grants from the Department of Science and Technol- ogy, Government of India and the Council of Scientific and Industrial Research, New Delhi. N.A. was recipi- ent of a Senior Research Fellowship from the Indian Council of Medical Research, New Delhi.
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