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Alterations in the intestinal brush border membrane structure and function in chronic DDT-exposed monkeys
PESTICIDE
BIOCHEMISTRY
Alterations
AND
12, 141- 146 (19791
PHYSIOLOGY
in the Intestinal Brush Border Membrane Structure Function in Chronic DDT-Exposed Monkeys
A. MAHMOOD. N. AGARWAL, S. SANYAL, Departments
of Gastroc~nterology
and
P. K. DUDEJA,
Biochrmistry.
Rc.srurcA.
Postgraduate
Chandigarh
160012.
and
AND D. SUBRAHMANYAM
Institute
of Medical
Education
and
India
Received April 3, 1979: accepted June 21, 1979 Intestinal uptake of o-glucose. t-leucine, and glycine is considerably enhanced in monkeys exposed to subacute doses of DDT for 100 days. The brush border sucrase and lactase activities were also significantly elevated but there was no change in alkaline phosphatase. lactate dehydrogenase, and Mg”-ATPase activities in the pesticide-administered animals compared to controls. Kinetic evidence revealed that augmentation of disaccharidases in the pesticide-treated animals is because of an increase in the enzyme content. Cholesterol and triglyceride fractions of the brush border membrane were also elevated but sialic acid content was reduced in the insecticide-treated animals compared to control group. These results suggest that observed changes in the functional activity of the enterocytes in DDT toxicity are possibly related to the alterations in the brush border membrane structural organization. INTRODUCTION
The organochlorine pesticides have been widely used in agriculture and health care programs in various parts of the world. But the extensive use of these drugs has aroused a great concern in recent years, because of the potent deleterious effects of these compounds to the experimental animals and to humans (l), which include fatty infiltration of the liver (2). endocrine imbalances (3, 4). induction of the “mixed function oxidase” activity in the liver (5), and nervous disorders (6). Stimulation of the sugar absorption and disaccharidases in the intestine of monkeys given a single oral dose of DDT has also been described (7). According to WHO surveys, chronic exposure to pesticides is a frequent problem and 90% of these toxicants consumed by human subjects enter the body through an oral route (8). Therefore the present studies were undertaken to investigate the effect of long-term exposure of DDT on the intestinal digestive and absorptive functions in monkeys, an animal species phylogenetitally nearer to man. In addition, the effect of DDT administration on the chemical composition of the brush border membrane has also been studied.
MATERIALS
AND METHODS
Animals. Male Rhesus monkeys weighing 3-4 kg body wt were used in these experiments. Before commencement of the experiment, animals were adapted tc the laboratory conditions and were maintained on a laboratory stock diet consisting of soaked Bengal gram, wheat bread, green vegetables, and fruits with free access to water. Animals were divided into two groups. The animals in the experimental group were given DDT mixed with corn oil by stomach tube in the dose of 10 mg/kg body wt and the control animals received the same amount of corn oil alone. After 100 days, the animals were sacrificed by injecting nembutol (30 mg/kg body wt) iv and the intestines were removed quickly, flushed with cold saline, and everted. MeasuretnerIt of intestinal uptake. The intestinal uptake of sugar and amino acids was determined using tissue accumulation method as described previously (9). Intestinal segments (120- 150 mg) were incubated in Kreb’s Ringer buffer, pH 7.4, containing 5 mM o-glucose or L-leucine or glycine with a trace of the radioactive substrates for 10 min at 37°C. At the end of incubation tissues were removed, blotted, weighed, and 141 0048.3575/79/080141-06$02.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
142
MAHMOOD
radioactivity was determined as described by Robinson and Alvarado (10). After correcting for the extracellular space as described earlier (9), absorption was calculated and expressed as ~molihrig tissue wet weight. Prepumtion
of brush border membtxms.
Brush border membranes were isolated from the jejunal portions of the gut following the method of Schmitz et al. (11). The membrane preparation was identical to the Pg fraction of these workers and was essentially free from other subcellular contaminations such as mitochondria, microsomes, basolateral membranes, and DNA. The membrane fragments were suspended in 50 mM sodium maleate, pH 6.5, and the final membrane preparation exhibited a 22- to 25fold increase in the brush border sucrase activity over the crude homogenate with 75-80s recovery. Enzyme ussays arld ciwnrical dcternzirzations. Tissues were homogenized in 50 mM
sodium maleate, pH 6.5 (10% w/v) and centrifuged at 1OOOgfor 10 min at 4°C. The pellet was discarded and the supernatant was used as such for enzyme determinations. Brush border sucrase (BBS) and brush border lactase (BBL) were assayed by measuring D-glucose formation with glucose oxidase and peroxidase systems (12, 13). Alkaline phosphatase (AP) was assayed by the method of Eichholz (14). Lactate dehydrogenase (LDH) and Mg”+-adenosinetriphosphatase (Mg’+-ATPase) activities were measured as described (15, 16). All enzyme activities were calculated in enzyme units (1 enzyme unit = pmoles of product formed per minute under the standard conditions). Membrane lipids were extracted in chloroform:methanol (2: 1) following the procedure of Folch et (I/. (17). Various lipid fractions were separated on silica gel G thinlayer plates using petroleum ether:diethylether:acetic acid (9O:lO:l) as the solvent system and identified by simultaneously running the authentic standards. Phospholipid phosphorous was determined by
ET
AL.
the method of Bartlett (18). Cholesterol was estimated by the method of Zak (19). Triglycerides were determined according to Van Handel and Zilversmit (20). Sialic acid was estimated by the method of Warren (21). Protein estimation was done according to Lowry et a/. (22). Reagents. All chemicals were of analytical grade. Glucostat was obtained from Worthington Biochemical Corporation, New Jersey. DDT was from Wilson Brothers Laboratories. D-[U-‘C]glucose (74 mCi/mmol), L-[U-“Clleucine (45.3 mCi/mmol), and [U-L”C]glycine (17.5 mCi/mmol) were procured from Bhabha Atomic Research Center, Bombay. RESULTS
The effect of chronic DDT administration on the monkeys was examined on the intestinal permeability of D-glucose, Lleucine, and glycine using everted intestinal segments. These results are presented in Table 1. There occurred a considerable increase in the absorption of these substrates in the pesticide-treated animals compared to controls. The observed increase in the uptake of these nutrients in DDT-fed animals is most likely due to an increase in the number of transport proteins (induction) rather than the effect of DDT at the kinetic level (modification of the carrier molecule), since in previous studies it was demonstrated that in \litro addition of the insecticide to the incubation medium containing tissues from control animals did not exert any influence on the sugar uptake process (7). To elucidate whether chronic DDT treatment also affects the digestive activities associated with the mucosal membrane, the BBS, BBL, and AP activities were analyzed in the control and experimental group of animals. As shown in Table 2, BBS and BBL activities exhibited a 35-40s increase in the pesticide-fed monkeys over the controls; however, there was no change in the AP activity. Also LDH and Mg’+-ATPase activities remained unaltered in the pesticide-treated animals.
DDT
AND
INTESTINAL TABLE
143
FUNCTIONS 1
Substrate
II
Control” (pmolihrlg tissue)
DDT-Fed” (~molihrig tissue)
o-Glucose r-Leucine Glycine
IO 6 6
4.95 2 1.31 2.62 2 0.91 5.12 -+ 1.30
7.10 2 1.55 5.58 t 1.90 8.12 t 1.89
” Mean
P co.01 co.05 co.05
? S.D
The observed increase in the activities of the disaccharidases in insecticide-exposed monkeys may be due to an increase in the enzyme concentration (induction) or it may result because of new enzyme formation with high substrate affinity in response to DDT administration. In order to examine these possibilities, we studied the kinetics of BBS in control and experimental groups of animals. The enzyme activity was determined at various substrate concentrations (12-40 mM) and the double reciprocal plot made for the data is depicted in Fig. 1. These results demonstrate that there is no change in the affinity constant of the enzyme W,,, = 27 mM in both the control and experimental animals) but the maximal velocity (Vmax) increased from 0.16 in control to 0.23 @moles glucoseiminlmg protein) in the pesticide-treated animals. These observations suggest that the apparent enhancement in the sucrase activity in DDT toxicity is due to an increase in the enzyme
Control” (Units/g protein)
Enzyme Sucrase Lactase Alkaline phosphatase Lactate dehydrogenase Mg”-ATPase ” II = 8- 10; N.S., h Mean -t S.D.
84.9 i 4.5 i
4.8 1.1
49.7 +
7.2
171.0 2 27.0 60.2 + 4.4 not significant.
protein. Similar results were obtained for BBL (data not shown). From the results described above, it is inferred that pesticide feeding to monkeys primarily influences the functional activities associated with brush border membrane, since marked changes occur in the permeability of nutrients and disaccharidases in enterocytes following DDT administration to monkeys. Therefore the effect of DDT feeding to monkeys was also investigated on the chemical composition of the brush borders to examine whether the observed changes in the functional activities of the microvillus membrane are related to alterations in the membrane structure. These results are represented in Table 3. There was no significant change in the membrane protein and phospholipid constituents in the pesticide-treated animals. However, the sialic acid content was reduced (P < 0.05), whereas the free cholesterol and triglyceride fractions of the
DDT-Fed” (Units/g protein) 112.1 i- 16.2 6.3 k 0.9 42.7 i-
P :0.01 -‘O.Ol
4.5
N.S.
206.1 -+ 21.8 68.7 2 7.4
N.S. N.S.
144
MAHMOOD
ET AL.
duces considerable changes in the functional and structural organization of the brush border membrane in monkey intestine. Qualitatively, the changes observed in the uptake of o-glucose, L-leucine, and glycine and the disaccharidase activities are similar to the alterations observed in acute DDT-fed animals (7), but there are certain quantitative variations in these parameters under the two experimental conditions. As shown in Table 2, BBS and BBL activities were enhanced by 3540% after chronic pesticide exposure, but administration of a single dose of the insecticide stimulated these activities to the extent of 2- to 4-fold. Such differences in the pesticide potency to induce these changes obviously relate to different experimental conditions. BBS and BBL levels were appreciably augmented in DDT-fed animals, but there was no change in the brush border AP activity, which is known to be localized in the microvillus membrane (14). Also pesticide administration did not influence the LDH and Mg”+-ATPase activities of the epithelial tissue. These results suggest that changes observed in the enterocytes in response to DDT are specific to disaccharidases and the transport systems. An interesting conclusion that emerges from these studies is the fact that pesticide feeding to monkeys primarily influences the structural and functional integrity of the mucosal surface. Such a facilitative action of DDT may be
FIG. 1. Double reciprocal plot: brush border suerase from control and DDT-fed monkeys. Enzyme activity was determined in the Pz fraction at sucrose cow centrations (S) ranging betn,een 12 and 40 mM. Reac,tion rate (V) is expressed as voles glucoselminimg protein. Each point is the mean of at least four determinations.
membrane were significantly (P < 0.01) elevated in the drug-administered animals compared to control group. Thus the observed perturbations in the permeability characteristics of the enterocytes and BBS and BBL activities in DDT toxicity are possibly related to some modifications in the structural organization of the microvillus membrane. The histological examination of the epithelial tissue, however, did not reveal any marked alterations in the morphology of the tissue (unpublished data). DISCUSSION
The results presented in this paper suggest that chronic exposure to DDT inTABLE Effect
of Chronic
Membrane fraction
Control” (mg/lOO
Protein Sialic acid Total phospholipids Free cholesterol Esterified cholesterol Triglycerides ” n = 6-8: N.S., ’ Mean + S.D.
DDT Administration Border Membrane
16.20 2 0.88 2 15.92 ?r 6.80 i
6.72 0.16 0.82 0.16
1.56 -+ 0.12 2.64 + 0.26 not significant.
3
on the Chemical Composition in Monkey Intestine”
mg dry
membrane
DDT-Fed weight)
81.80 0.64 16.88 7.56
t zt -+ t
of Brash
’ P
4.60 0.20 0.92 0.08
N.S. =:o.os N.S.
1.88 2 0.16 3.74 t 0.58
co.01 -co.01
DDT
AND
INTESTINAL
due to its high lipid solubility and lipids constitute an integral component of the biological membranes. There is much experimental evidence which emphasizes a close functional link between BBS and the carrier-mediated sugar transport system in the intestine (23, 24). It is interesting that DDT toxicity induces similar changes in the sugar absorption and BBS in this tissue. The exact mechanism by which DDT treatment enhances the absorption of nutrients and the brush border disaccharidases is not clear at present. The kinetic evidence indicates that enhancement of disaccharidase activities is, however, due to an increase in the enzyme content. The stimulating action of DDT on the enterocyte functions may be due to specific induction of the proteins responsible for these functions or indirectly it may arise because of the hormonal imbalances associated with pesticide toxicity (2). Chronic DDT feeding is known to produce hypoinsulinism (4) and the hyperactivities of the adrenal glands (3). Such hormonal disorders are well known to enhance the digestive and absorptive activities of the intestine (25. 26). Analysis of the chemical composition of the brush border membranes in control and DDT-exposed animals revealed that sialic acid content was reduced, whereas the cholesterol and triglyceride fractions were highly elevated in insecticide-fed monkeys compared to controls. Thus the observed changes in the brush border membrane structure and function in DDT-fed monkeys for a long period are of considerable interest in view of the fact that the majority of the pesticides consumed by living populations enter the body through an oral route (8). ACKNOWLEDGMENT
These investigations were financed by the Department of Science and Technology of the Government of India, New Delhi. REFERENCES
1. T. S. S. Dikshith, DDT-The and hazards, J. Sci. Ind.
problems of residue Res. 37, 316 (1978).
145
FUNCTIONS
2. K. K. Kohli. S. C. Sharma. S. C. Bhatia, and T. A. V. Subramanian, Biochemical effects of chlorinated insecticides: DDT and dieldrin, J. Sci. Ind. Res. 34, 462 (1975). 3. D. Kupfer and W. H. Bulger, Interactions of chlorinated hydrocarbons with steroid hormones, Fed. Proc. Amer. Sot. E.rp, Biol. 35, 2603 (1976). 4. E. T. Yau and J. H. Mennear, The inhibitory effect of DDT on insulin secretion in mice, To.ricol. Appl. Pharm. 39, 81 (1971). 5. L. G. Hart and J. R. Fouts, Effect of acute and chronic DDT administration on hepatic microsomal drug metabolism in the rat, Proc,. SOCK. E.up. Biol. Med. 114, 388 (1963). 6. P. D. Hrdina and R. L. Singhal. Neurotoxic effects of DDT: Protection by cycloheximide, J. Phurm. Pharmucol. 24. 167 (1972). 7. A. Mahmood, N. Agarwal, S. Sanyal, and D. Subrahmanyam. Effects of DDT (chlorophenotane) administration on glucose uptake and brush border enzymes in monkey intestine. Ac,ttr Pharrnatwl. Toricol. 43, 99 (1978). 8. World Health Organization, “Report on Health Hazards of the Human Environment.” p. 205, WHO, Geneva. 1972. 9. F. Alvarado and A. Mahmood, Cotransport of organic solutes and sodium ions in the small intestine: A general model amino acid transport. Biochc~mi.sfq 13, 2882 (1974). 10. J. W. L. Robinson and F. Alvarado, Interactions between sugar and amino acid transport systems at the brush border: A comparative study, F’jlw,qers Arch. 326, 48 (1971). 11. J. C. Schmitz, H. Preiser, D. Maestracci. B. K. Ghosh. J. J. Cerdo, and R. K. Crane, Purification of human intestinal brush border membrane. Biochem. Biophxs. Ac.ftr 322. 98 ( 1973). 12. A. Dahlqvist. Method for the assay of intestinal disaccharidases. AnuI. Biwhrm. 7. 18 (1964). 13. A. Mahmood and F. Alvarado. The activation of intestinal brush border sucrase by alkali metal ions: An allosteric mechanism similar to that for the Na activation of non-electrolytes transport systems in intestine, Arch. Biochrm. Bioplys. 168,
585
(1975).
14. A. Eichholz, Structural and functional organization of the brush border of intestinal epithelium. III. Enzymatic activities and chemical composition of various fractions of tris disrupted brush borders. Bicw.hr,m. Bioplly\. Ac,tcl 135, 475 (1967). 15. 1. D. P Wootton, “Microanalysis in Medical Biochemistry.” 4th ed., p. 114, Churchill. London. 1964. 16. A. Mahmood, K. Singh, and A. K. Ahuja, Distribution of Na. K-ATPase in rat intestine. fndim J. Biochem. Biophys. 9, 279 (1972). 17. J. Folch, M. Lees, and G. H. Sloane Stanley, A
146
MAHMOOD simple method of total lipids Chum.
226,
497
ET
for the isolation and purification from animal tissues, .1. Biol. (1957).
G. R. Bartlett. Phosphorus assay in column chromatography, J. Biol. Cl7rn7. 234, 466 (1959). 19. B. Zak, Simple rapid microtechnique for total cholesterol, Amer. .I. C/in. Prrthol. 27, 583
18.
(A. Hottinger Karger, Basal,
and H. 1968.
Berger,
Eds.).
p. 32,
24.
D. Ramaswamy, P. Malathi, and R. K. Crane, Demonstration of hydrolase related glucose transport in brush border membrane vescicles prepared from guinea pig intestine, Bi0che~7. Bi0ph.v~. Rrs. Commtrn. 68, 162 (1976).
25.
A. Mahmood and S. D. Varma. Uber die wirkung des alooxan diabetes von insulin and diabetogenen hormonen (thyroxin. hydrocortison and oxytocin) auf den intestinalen transport von glycin, Z. Gustvo~nfrro/. 9. 425 (1971).
(1957).
E. Van Handel and D. B. Zilversmit. Micromethod for direct determination of serum triglycerides,.I. Ltrb. C/in. Med. 50, 152 (1957). 21. L. Warren, The thiobarbituric acid assay of sialic acids, J. Biol. Cherrr. 234, 1971 (1959). 22. 0. H. Lowry, N. J. Rosebrough. A. L. Farr, and R. L. Randall, Protein measurement with Folin phenol reagent, J. B&l. Chew. 193, 265 (1951). 23. G. Semenza, in “Modem Problems in Pediatrics” 20.
AL.
26. A. Mahmood, R. M. Pathak. and N. Agarwal, fect of chronic alloxan diabetes and insulin ministration on intestinal brush border zymes. Exprrienfiu 34, 741 (1978).
Efaden-
BIOCHEMISTRY
Alterations
AND
12, 141- 146 (19791
PHYSIOLOGY
in the Intestinal Brush Border Membrane Structure Function in Chronic DDT-Exposed Monkeys
A. MAHMOOD. N. AGARWAL, S. SANYAL, Departments
of Gastroc~nterology
and
P. K. DUDEJA,
Biochrmistry.
Rc.srurcA.
Postgraduate
Chandigarh
160012.
and
AND D. SUBRAHMANYAM
Institute
of Medical
Education
and
India
Received April 3, 1979: accepted June 21, 1979 Intestinal uptake of o-glucose. t-leucine, and glycine is considerably enhanced in monkeys exposed to subacute doses of DDT for 100 days. The brush border sucrase and lactase activities were also significantly elevated but there was no change in alkaline phosphatase. lactate dehydrogenase, and Mg”-ATPase activities in the pesticide-administered animals compared to controls. Kinetic evidence revealed that augmentation of disaccharidases in the pesticide-treated animals is because of an increase in the enzyme content. Cholesterol and triglyceride fractions of the brush border membrane were also elevated but sialic acid content was reduced in the insecticide-treated animals compared to control group. These results suggest that observed changes in the functional activity of the enterocytes in DDT toxicity are possibly related to the alterations in the brush border membrane structural organization. INTRODUCTION
The organochlorine pesticides have been widely used in agriculture and health care programs in various parts of the world. But the extensive use of these drugs has aroused a great concern in recent years, because of the potent deleterious effects of these compounds to the experimental animals and to humans (l), which include fatty infiltration of the liver (2). endocrine imbalances (3, 4). induction of the “mixed function oxidase” activity in the liver (5), and nervous disorders (6). Stimulation of the sugar absorption and disaccharidases in the intestine of monkeys given a single oral dose of DDT has also been described (7). According to WHO surveys, chronic exposure to pesticides is a frequent problem and 90% of these toxicants consumed by human subjects enter the body through an oral route (8). Therefore the present studies were undertaken to investigate the effect of long-term exposure of DDT on the intestinal digestive and absorptive functions in monkeys, an animal species phylogenetitally nearer to man. In addition, the effect of DDT administration on the chemical composition of the brush border membrane has also been studied.
MATERIALS
AND METHODS
Animals. Male Rhesus monkeys weighing 3-4 kg body wt were used in these experiments. Before commencement of the experiment, animals were adapted tc the laboratory conditions and were maintained on a laboratory stock diet consisting of soaked Bengal gram, wheat bread, green vegetables, and fruits with free access to water. Animals were divided into two groups. The animals in the experimental group were given DDT mixed with corn oil by stomach tube in the dose of 10 mg/kg body wt and the control animals received the same amount of corn oil alone. After 100 days, the animals were sacrificed by injecting nembutol (30 mg/kg body wt) iv and the intestines were removed quickly, flushed with cold saline, and everted. MeasuretnerIt of intestinal uptake. The intestinal uptake of sugar and amino acids was determined using tissue accumulation method as described previously (9). Intestinal segments (120- 150 mg) were incubated in Kreb’s Ringer buffer, pH 7.4, containing 5 mM o-glucose or L-leucine or glycine with a trace of the radioactive substrates for 10 min at 37°C. At the end of incubation tissues were removed, blotted, weighed, and 141 0048.3575/79/080141-06$02.00/O Copyright All rights
0 1979 by Academic Press. Inc. of reproduction in any form reserved.
142
MAHMOOD
radioactivity was determined as described by Robinson and Alvarado (10). After correcting for the extracellular space as described earlier (9), absorption was calculated and expressed as ~molihrig tissue wet weight. Prepumtion
of brush border membtxms.
Brush border membranes were isolated from the jejunal portions of the gut following the method of Schmitz et al. (11). The membrane preparation was identical to the Pg fraction of these workers and was essentially free from other subcellular contaminations such as mitochondria, microsomes, basolateral membranes, and DNA. The membrane fragments were suspended in 50 mM sodium maleate, pH 6.5, and the final membrane preparation exhibited a 22- to 25fold increase in the brush border sucrase activity over the crude homogenate with 75-80s recovery. Enzyme ussays arld ciwnrical dcternzirzations. Tissues were homogenized in 50 mM
sodium maleate, pH 6.5 (10% w/v) and centrifuged at 1OOOgfor 10 min at 4°C. The pellet was discarded and the supernatant was used as such for enzyme determinations. Brush border sucrase (BBS) and brush border lactase (BBL) were assayed by measuring D-glucose formation with glucose oxidase and peroxidase systems (12, 13). Alkaline phosphatase (AP) was assayed by the method of Eichholz (14). Lactate dehydrogenase (LDH) and Mg”+-adenosinetriphosphatase (Mg’+-ATPase) activities were measured as described (15, 16). All enzyme activities were calculated in enzyme units (1 enzyme unit = pmoles of product formed per minute under the standard conditions). Membrane lipids were extracted in chloroform:methanol (2: 1) following the procedure of Folch et (I/. (17). Various lipid fractions were separated on silica gel G thinlayer plates using petroleum ether:diethylether:acetic acid (9O:lO:l) as the solvent system and identified by simultaneously running the authentic standards. Phospholipid phosphorous was determined by
ET
AL.
the method of Bartlett (18). Cholesterol was estimated by the method of Zak (19). Triglycerides were determined according to Van Handel and Zilversmit (20). Sialic acid was estimated by the method of Warren (21). Protein estimation was done according to Lowry et a/. (22). Reagents. All chemicals were of analytical grade. Glucostat was obtained from Worthington Biochemical Corporation, New Jersey. DDT was from Wilson Brothers Laboratories. D-[U-‘C]glucose (74 mCi/mmol), L-[U-“Clleucine (45.3 mCi/mmol), and [U-L”C]glycine (17.5 mCi/mmol) were procured from Bhabha Atomic Research Center, Bombay. RESULTS
The effect of chronic DDT administration on the monkeys was examined on the intestinal permeability of D-glucose, Lleucine, and glycine using everted intestinal segments. These results are presented in Table 1. There occurred a considerable increase in the absorption of these substrates in the pesticide-treated animals compared to controls. The observed increase in the uptake of these nutrients in DDT-fed animals is most likely due to an increase in the number of transport proteins (induction) rather than the effect of DDT at the kinetic level (modification of the carrier molecule), since in previous studies it was demonstrated that in \litro addition of the insecticide to the incubation medium containing tissues from control animals did not exert any influence on the sugar uptake process (7). To elucidate whether chronic DDT treatment also affects the digestive activities associated with the mucosal membrane, the BBS, BBL, and AP activities were analyzed in the control and experimental group of animals. As shown in Table 2, BBS and BBL activities exhibited a 35-40s increase in the pesticide-fed monkeys over the controls; however, there was no change in the AP activity. Also LDH and Mg’+-ATPase activities remained unaltered in the pesticide-treated animals.
DDT
AND
INTESTINAL TABLE
143
FUNCTIONS 1
Substrate
II
Control” (pmolihrlg tissue)
DDT-Fed” (~molihrig tissue)
o-Glucose r-Leucine Glycine
IO 6 6
4.95 2 1.31 2.62 2 0.91 5.12 -+ 1.30
7.10 2 1.55 5.58 t 1.90 8.12 t 1.89
” Mean
P co.01 co.05 co.05
? S.D
The observed increase in the activities of the disaccharidases in insecticide-exposed monkeys may be due to an increase in the enzyme concentration (induction) or it may result because of new enzyme formation with high substrate affinity in response to DDT administration. In order to examine these possibilities, we studied the kinetics of BBS in control and experimental groups of animals. The enzyme activity was determined at various substrate concentrations (12-40 mM) and the double reciprocal plot made for the data is depicted in Fig. 1. These results demonstrate that there is no change in the affinity constant of the enzyme W,,, = 27 mM in both the control and experimental animals) but the maximal velocity (Vmax) increased from 0.16 in control to 0.23 @moles glucoseiminlmg protein) in the pesticide-treated animals. These observations suggest that the apparent enhancement in the sucrase activity in DDT toxicity is due to an increase in the enzyme
Control” (Units/g protein)
Enzyme Sucrase Lactase Alkaline phosphatase Lactate dehydrogenase Mg”-ATPase ” II = 8- 10; N.S., h Mean -t S.D.
84.9 i 4.5 i
4.8 1.1
49.7 +
7.2
171.0 2 27.0 60.2 + 4.4 not significant.
protein. Similar results were obtained for BBL (data not shown). From the results described above, it is inferred that pesticide feeding to monkeys primarily influences the functional activities associated with brush border membrane, since marked changes occur in the permeability of nutrients and disaccharidases in enterocytes following DDT administration to monkeys. Therefore the effect of DDT feeding to monkeys was also investigated on the chemical composition of the brush borders to examine whether the observed changes in the functional activities of the microvillus membrane are related to alterations in the membrane structure. These results are represented in Table 3. There was no significant change in the membrane protein and phospholipid constituents in the pesticide-treated animals. However, the sialic acid content was reduced (P < 0.05), whereas the free cholesterol and triglyceride fractions of the
DDT-Fed” (Units/g protein) 112.1 i- 16.2 6.3 k 0.9 42.7 i-
P :0.01 -‘O.Ol
4.5
N.S.
206.1 -+ 21.8 68.7 2 7.4
N.S. N.S.
144
MAHMOOD
ET AL.
duces considerable changes in the functional and structural organization of the brush border membrane in monkey intestine. Qualitatively, the changes observed in the uptake of o-glucose, L-leucine, and glycine and the disaccharidase activities are similar to the alterations observed in acute DDT-fed animals (7), but there are certain quantitative variations in these parameters under the two experimental conditions. As shown in Table 2, BBS and BBL activities were enhanced by 3540% after chronic pesticide exposure, but administration of a single dose of the insecticide stimulated these activities to the extent of 2- to 4-fold. Such differences in the pesticide potency to induce these changes obviously relate to different experimental conditions. BBS and BBL levels were appreciably augmented in DDT-fed animals, but there was no change in the brush border AP activity, which is known to be localized in the microvillus membrane (14). Also pesticide administration did not influence the LDH and Mg”+-ATPase activities of the epithelial tissue. These results suggest that changes observed in the enterocytes in response to DDT are specific to disaccharidases and the transport systems. An interesting conclusion that emerges from these studies is the fact that pesticide feeding to monkeys primarily influences the structural and functional integrity of the mucosal surface. Such a facilitative action of DDT may be
FIG. 1. Double reciprocal plot: brush border suerase from control and DDT-fed monkeys. Enzyme activity was determined in the Pz fraction at sucrose cow centrations (S) ranging betn,een 12 and 40 mM. Reac,tion rate (V) is expressed as voles glucoselminimg protein. Each point is the mean of at least four determinations.
membrane were significantly (P < 0.01) elevated in the drug-administered animals compared to control group. Thus the observed perturbations in the permeability characteristics of the enterocytes and BBS and BBL activities in DDT toxicity are possibly related to some modifications in the structural organization of the microvillus membrane. The histological examination of the epithelial tissue, however, did not reveal any marked alterations in the morphology of the tissue (unpublished data). DISCUSSION
The results presented in this paper suggest that chronic exposure to DDT inTABLE Effect
of Chronic
Membrane fraction
Control” (mg/lOO
Protein Sialic acid Total phospholipids Free cholesterol Esterified cholesterol Triglycerides ” n = 6-8: N.S., ’ Mean + S.D.
DDT Administration Border Membrane
16.20 2 0.88 2 15.92 ?r 6.80 i
6.72 0.16 0.82 0.16
1.56 -+ 0.12 2.64 + 0.26 not significant.
3
on the Chemical Composition in Monkey Intestine”
mg dry
membrane
DDT-Fed weight)
81.80 0.64 16.88 7.56
t zt -+ t
of Brash
’ P
4.60 0.20 0.92 0.08
N.S. =:o.os N.S.
1.88 2 0.16 3.74 t 0.58
co.01 -co.01
DDT
AND
INTESTINAL
due to its high lipid solubility and lipids constitute an integral component of the biological membranes. There is much experimental evidence which emphasizes a close functional link between BBS and the carrier-mediated sugar transport system in the intestine (23, 24). It is interesting that DDT toxicity induces similar changes in the sugar absorption and BBS in this tissue. The exact mechanism by which DDT treatment enhances the absorption of nutrients and the brush border disaccharidases is not clear at present. The kinetic evidence indicates that enhancement of disaccharidase activities is, however, due to an increase in the enzyme content. The stimulating action of DDT on the enterocyte functions may be due to specific induction of the proteins responsible for these functions or indirectly it may arise because of the hormonal imbalances associated with pesticide toxicity (2). Chronic DDT feeding is known to produce hypoinsulinism (4) and the hyperactivities of the adrenal glands (3). Such hormonal disorders are well known to enhance the digestive and absorptive activities of the intestine (25. 26). Analysis of the chemical composition of the brush border membranes in control and DDT-exposed animals revealed that sialic acid content was reduced, whereas the cholesterol and triglyceride fractions were highly elevated in insecticide-fed monkeys compared to controls. Thus the observed changes in the brush border membrane structure and function in DDT-fed monkeys for a long period are of considerable interest in view of the fact that the majority of the pesticides consumed by living populations enter the body through an oral route (8). ACKNOWLEDGMENT
These investigations were financed by the Department of Science and Technology of the Government of India, New Delhi. REFERENCES
1. T. S. S. Dikshith, DDT-The and hazards, J. Sci. Ind.
problems of residue Res. 37, 316 (1978).
145
FUNCTIONS
2. K. K. Kohli. S. C. Sharma. S. C. Bhatia, and T. A. V. Subramanian, Biochemical effects of chlorinated insecticides: DDT and dieldrin, J. Sci. Ind. Res. 34, 462 (1975). 3. D. Kupfer and W. H. Bulger, Interactions of chlorinated hydrocarbons with steroid hormones, Fed. Proc. Amer. Sot. E.rp, Biol. 35, 2603 (1976). 4. E. T. Yau and J. H. Mennear, The inhibitory effect of DDT on insulin secretion in mice, To.ricol. Appl. Pharm. 39, 81 (1971). 5. L. G. Hart and J. R. Fouts, Effect of acute and chronic DDT administration on hepatic microsomal drug metabolism in the rat, Proc,. SOCK. E.up. Biol. Med. 114, 388 (1963). 6. P. D. Hrdina and R. L. Singhal. Neurotoxic effects of DDT: Protection by cycloheximide, J. Phurm. Pharmucol. 24. 167 (1972). 7. A. Mahmood, N. Agarwal, S. Sanyal, and D. Subrahmanyam. Effects of DDT (chlorophenotane) administration on glucose uptake and brush border enzymes in monkey intestine. Ac,ttr Pharrnatwl. Toricol. 43, 99 (1978). 8. World Health Organization, “Report on Health Hazards of the Human Environment.” p. 205, WHO, Geneva. 1972. 9. F. Alvarado and A. Mahmood, Cotransport of organic solutes and sodium ions in the small intestine: A general model amino acid transport. Biochc~mi.sfq 13, 2882 (1974). 10. J. W. L. Robinson and F. Alvarado, Interactions between sugar and amino acid transport systems at the brush border: A comparative study, F’jlw,qers Arch. 326, 48 (1971). 11. J. C. Schmitz, H. Preiser, D. Maestracci. B. K. Ghosh. J. J. Cerdo, and R. K. Crane, Purification of human intestinal brush border membrane. Biochem. Biophxs. Ac.ftr 322. 98 ( 1973). 12. A. Dahlqvist. Method for the assay of intestinal disaccharidases. AnuI. Biwhrm. 7. 18 (1964). 13. A. Mahmood and F. Alvarado. The activation of intestinal brush border sucrase by alkali metal ions: An allosteric mechanism similar to that for the Na activation of non-electrolytes transport systems in intestine, Arch. Biochrm. Bioplys. 168,
585
(1975).
14. A. Eichholz, Structural and functional organization of the brush border of intestinal epithelium. III. Enzymatic activities and chemical composition of various fractions of tris disrupted brush borders. Bicw.hr,m. Bioplly\. Ac,tcl 135, 475 (1967). 15. 1. D. P Wootton, “Microanalysis in Medical Biochemistry.” 4th ed., p. 114, Churchill. London. 1964. 16. A. Mahmood, K. Singh, and A. K. Ahuja, Distribution of Na. K-ATPase in rat intestine. fndim J. Biochem. Biophys. 9, 279 (1972). 17. J. Folch, M. Lees, and G. H. Sloane Stanley, A
146
MAHMOOD simple method of total lipids Chum.
226,
497
ET
for the isolation and purification from animal tissues, .1. Biol. (1957).
G. R. Bartlett. Phosphorus assay in column chromatography, J. Biol. Cl7rn7. 234, 466 (1959). 19. B. Zak, Simple rapid microtechnique for total cholesterol, Amer. .I. C/in. Prrthol. 27, 583
18.
(A. Hottinger Karger, Basal,
and H. 1968.
Berger,
Eds.).
p. 32,
24.
D. Ramaswamy, P. Malathi, and R. K. Crane, Demonstration of hydrolase related glucose transport in brush border membrane vescicles prepared from guinea pig intestine, Bi0che~7. Bi0ph.v~. Rrs. Commtrn. 68, 162 (1976).
25.
A. Mahmood and S. D. Varma. Uber die wirkung des alooxan diabetes von insulin and diabetogenen hormonen (thyroxin. hydrocortison and oxytocin) auf den intestinalen transport von glycin, Z. Gustvo~nfrro/. 9. 425 (1971).
(1957).
E. Van Handel and D. B. Zilversmit. Micromethod for direct determination of serum triglycerides,.I. Ltrb. C/in. Med. 50, 152 (1957). 21. L. Warren, The thiobarbituric acid assay of sialic acids, J. Biol. Cherrr. 234, 1971 (1959). 22. 0. H. Lowry, N. J. Rosebrough. A. L. Farr, and R. L. Randall, Protein measurement with Folin phenol reagent, J. B&l. Chew. 193, 265 (1951). 23. G. Semenza, in “Modem Problems in Pediatrics” 20.
AL.
26. A. Mahmood, R. M. Pathak. and N. Agarwal, fect of chronic alloxan diabetes and insulin ministration on intestinal brush border zymes. Exprrienfiu 34, 741 (1978).
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