TOXICOLOGY AND APPLIED PHARMACOLOGY 36, 11 l-120 (1976)

Influence of Starvation and Hepatic Microsomal Enzyme Induction on the Mobilization of DDT Residues in Rats’,’

GERARD LAMBERT AND JULES BRODEUR~

Department of Pharmacology, Faculte de MPdecine, Universite’ de Montrtial, MontrPal, Canada

Received August 27, 1975; accepted December I,1975

Influence of Starvation and Hepatic Microsomal Enzyme Induction on the Mobilization of DDT Residues in Rats. LAMBERT, G. AND BRODEUR, J. (1976). Toxicol. Appl. Pharmacol. 36, 111-120. Adult female Sprague- Dawley rats were treated with DDT (10 mg/kg po daily for 10 days) and were then treated with hepatic microsomal enzyme inducers and/or starved for various periods of time in order to study depletion of pesticide residues from the blood, brain, and abdominal fat tissue. The enzyme inducers used, and the daily doses administered over a period of 14 days, were: pheno- barbital sodium 50 mg/kg, pregnene-16a-carbonitrile (PCN) 30 mg/kg, Bcr-fluoro-1 l/Y, 17-dihydroxy-3-oxo-4-androstene-17a-propionic acid (CS-1) 40 mg/kg, and dexamethasone 4 mg/kg for 7 days and 2 mg/kg for 7 days. Starvation consisted in withholding solid food, but not water, for periods of 3 or 5 consecutive days, or 3 or 5 nonconsecutive days. At the end of the treatment period, the animals were sacrificed for determination of&-DDT, p,p’-DDD, and&-DDE. Phenobarbital and CS-1 reduced the concentra- tion of the total residues in all three tissues examined, but phenobarbital was superior to the steroid. On the other hand, starvation for 5 consecutive days was more effective than any other period of starvation for reducing the concentration of the residues. Combination of a phenobarbital pre- treatment with starvation for 5 consecutive days led to additive tissue depletion of the residues. The degree of effectiveness of the combined treatment was better when the animals were starved early during the period of phenobarbital administration. These results provide good evidence that the combination of enzyme induction and starvation is a superior means of clearing contaminated tissues of DDT than either treatment alone. However, maxima1 efficacy is obtained only when both treatments are interrelated optimally with respect to the time and the duration of admis- tration.

Various attempts have been made to eliminate pesticide residues from contaminated organisms (Street, 1969; Liska and Stadelman, 1969). Adipose tissue is an important site of storage for organochlorine pesticides. Therefore, in various species, starvation results in the mobilization of the residues, their redistribution to other tissues including blood, brain, and liver, and their partial elimination by way of urinary and fecal

1 This research was supported by a group grant in drug toxicology from the Medical Research Council of Canada.

2 Presented in part at the Twelfth Annual Meeting, Society of Toxicology, March 18-22, 1973, New York, N.Y.

3 Member, Medical Research Council Group in Drug Toxicology. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

111 Printed in Great Britain

112 LAMBER?‘ AND BRODEUR

excretion (Dale et nl., 1962; Donaldson et al.. 1968; Ecobichon and Saschenbrecker 1969; Brown, 1970: Smith et a/.. 1970). However, this approach is not without risk, since extensive mobilization of the residues from the adipose tissue can result in high circulating concentrations of pesticides and, possibly, in the accumulation of toxic concentrations in target organs (Fitzhugh and Nelson, 1947; Ecobichon and Saschen- brecker, 1969: Davidson et al., 1971).

Another approach takes advantage of the fact that at least part of the biotrans- formation of DDT to inactive metabolites is carried out under the catalytic action of the liver drug-metabolizing microsomal enzymes (Moreilo, 1965; Sanchez, 1967). It has been shown that the treatment of contaminated animals with various microsomal enzyme inducers has generally been followed by a reduction in the concentration of pesticide residues in the adipose tissue and an increase in the quantity of the pesticide metabolites excreted in the urine and feces (Street, 1964; Koransky et a/., 1964: Street et al., 1966a: Street et al., 1966b; Cueto and Hayes, 1967; Alary et al., 1968; Mayer et a/., 1970; Alary et al., 1971). Stimulation of microsomal drug metabolizing enzymes has been proposed as the most likely mechanism for this effect.

The present investigation was undertaken in rats previously treated with DDT to determine whether starvation or microsomal enzyme induction produces maximal depletion of pesticide residues from various tissues. An attempt was also made to study whether or not the combination of starvation and microsomal enzyme induction could have any advantage over either treatment alone in mobilizing the residues of DDT. The data obtained strongly suggest that the experimental conditions of starvation and microsomal enzyme induction can be optimized in such a way as to justify the combina- tion of both treatments as a means of accelerating decontamination.

METHODS

Animals. Adult female Sprague-Dawley rats weighing between 180 and 200 g were used for this investigation. The animals were kept in a room maintained at 22°C and, unless otherwise stated, they were fed Purina laboratory chow and water ad libitum.

Chemicals. The organochlorine insecticide used was p,p’-DDT{l,l-bis-(p-chloro- phenyl)-2,2,2-trichloroethane4)}. The claimed purity of the sample was 99 y/,. p,p’-DDT was dissolved in corn oil (5 mg/ml).

Various drugs were used as microsomai enzyme inducers as follows: Phenobarbital sodium5 was dissolved in a water-l y; -Tween-80 solution to yield a final concentration of 25 mg/ml. PCN6 (3fi-hydroxy-20-0x0-5 pregnene- 16c+carbonitrile), CS-I6 (potassium salt of 9cc-fluoro- 11 p, 17-dihydroxy-3-oxo-4-androstene- 17a-propionic acid), and dexamethasone6 (16a-methyl-9a-fluoroprednisolone) were all suspended in a water- 1 yi-Tween-80 solution so that final concentrations were 15, 20, and 2 mg/ml, respec- tively. The latter three compounds are catatoxic steroids known to be potent enzyme inducers (Selye, 197 1).

Experimental protocols: Influence ofpretreatment with various enzyme inducers on the concentration of tissue residues in rats treated with DDT. p,p’-DDT was given by gastric

4 Purchased from Aldrich Chemical Co., Inc., Milwaukee, Wise. 5 Purchased from B.D.H. Chemicals, Toronto, Canada. 6 Gift from Dr. Hans Selye, Institut de mkdecine et chirurgie exp&imentales, Universite de Montrtal,

Mont&al, Canada.

MOBILIZATION OF DDT RESIDUES 113

gavage at a daily dose of 10 mg/kg body weight for 10 consecutive days. The adminis- tration of the inducers was started 4 days later for a period of 14 consecutive days. The chemicals were given by gavage at a daily dose of 50 mg/kg for phenobarbital, 30 mg/kg for PCN, 40 mg/kg for CS-1, and 4 mg/kg during the first 7 days and 2 mg/kg during the next 7 days for dexamethasone. A control group was given an equivalent volume of a solution of water-l ‘A-Tween-80. Twenty-four hours after the last dose of the inducers the animals were sacrificed by decapitation. Samples of blood, brain, and abdominal fat tissue were then analyzed for p,p’-DDT, p,p’-DDD’, and p,p’DDEs.

Influence qf various periods of starcation on the concentration qf tissue residues in rats contaminated with DDT. All animals were givenp,p’-DDT as described above. During the period of 14 days beginning 4 days after the end of contamination, they were sub- mitted to various modalities of starvation, A first group was deprived of solid food, but not water, for a period of 3 consecutive days and was then allowed to feed ad libitum for the remaining 11 days. A second group was starved on 3 alternate days and then had normal access to food for 9 days. The third and fourth groups were starved during periods of 5 consecutive and 5 alternate days, respectively, and were then allowed to resume normal feeding until the end of the 1Cday treatment period. A fifth group served as control and was not submitted to any form of food deprivation. On Day 15 after the beginning of treatment (starvation) period, the animals were sacrificed for residue analysis as described above.

Influence of various combinations of an enzyme inducer and difSerent starvation periods on the concentration of tissue residues in rats contamined with DDT. Again, all animals were treated with p,p’-DDT. During the 14day experimental period (Days 1 to 14) beginning 4 days after treatment with DDT, one group was given phenobarbital sodium in saline at a daily dose of 50 mg/kg during the entire period of treatment: a second group was starved for 5 consecutive days (Days 1 to 5); a third group was starved for 5 different consecutive days (Days 4 to 8); the fourth and fifth groups received pheno- barbital for 14 days and were also exposed to starvation according to the same procedure used for the second and third groups, respectively. A saline group served as control. The animals were sacrificed and their tissues analyzed as described above.

Analysis ofthe residues. p,p’-DDT,p,p’-DDD, andp,p’-DDE were extracted from the whole brain and abdominal fat tissue (5 g) according to the solvent extraction procedure of Saschenbrecker and Ecobichon (1967). Residues from blood were extracted directly with benzene. Equal volumes of blood and benzene were thoroughly mixed for 1 min. The organic layers recovered after three consecutive extractions were combined and evaporated to dryness. The dry residue was dissolved in a small volume of benzene for GLC determination. Only “pesticide grade” solvents were used during extraction procedures.

The analysis of the residues was carried out on an Aerograph model 2100 gas chro- matograph using a tritium electron capture detector. A U-shaped Pyrex glass column (1.8 x 0.64 cm) containing a 5% DC-200 silicone on acid-washed chromosorb W (60/80 mesh) was used. Optimal temperatures (“C) were 230 (injector), 200 (column), and 215 (detector). The optimal nitrogen flow rate was 60 ml/min. Retention times were found to be 5 min for p,p’-DDT, 4 min for p,p’-DDD, and 3 min for p,p’-DDE. The

7 2,2-bis-(p-chlorophenyl)-l,l-dichloroethane. ’ 2,2-bis-(p-chlorophenyl)-l,l-dichloroethylene.

114 LAMBERT AND BRODEUR

concentration of residues was expressed in terms of micrograms per gram of wet tissue for brain and adipose tissue, and nanograms per milliliter of whole blood.

Statistical analysis. A unidimensional analysis of variance was used to analyze the results presented in Tables I and 2. For those presented in Tables 3 and 4, the influence of either treatment (phenobarbital or starvation) on the tissue concentration of the various residues was assessed by an analysis of variance for a 2 x 2 factorial experi- mental design; the latter also allowed for analysis of possible interactions between both factors. The level of significance was set at p < 0.05.

RESULTS

Injuence of pretreatments M*ith Llarious enzyme inducers on the concentration of tissue residues in rats treated with DDT. The residues of DDT found in the blood, brain, and abdominal fat tissue following pretreatment with various enzyme inducers have been tabulated in Table 1. The data are presented as total residues which represent the sum

TABLE 1 INFLUENCE OF VARIOUS HEPATIC MICROSOMAL ENZYME INDUCERS ON THE

CONCENTRATION OF TOTAL DDT RESIDUES IN VARIOUS TISSUES OF RATS PREVIOUSLY CONTAMINATED WITH DDT

Total residues”

Treatment Whole blood Brain

hg/ml) ~ccg/d Adipose tissue

tid!)

Water-l %-Tween-80 Phenobarbital PCN cs-1 Dexamethosone SEd

263b 2.15 342 112” 0.92” 222’ 179’ I .80’ 306 122’ 1.55” 252” 231 I .73c 265’ 27 0.10 24

a Total residues represent the sum of p,p’-DDT, p,p’-DDD, and p&-DDE residues.

b Mean of determinations made on six animals. c Significantly different from the water-l %-Tween-30 group as calculated by the

Dunnettt’s test (Winer, 1971). d Standard error of the mean of any group, calculated as follows: (Mean square

(error)/n)“‘, where n is the number of animals per treatment group (Snedecor and Co&ran, 1967).

ofp,p’-DDT, p,p’-DDD, and p,p’-DDE residues. During this investigation, the relative importance of each type of residue was found to be 67 to 83 % forp,p’-DDT, 4 to 117: for p,p’-DDD, and 13 to 24 % for p,p’-DDE. It can be seen that the concentration of residues was significantly decreased in all samples by phenobarbital and CS-1, pheno- barbital being more effective than the steroid. PCN significantly reduced the residues in the blood and brain only, whereas dexamethasone was effective in decreasing the residues in the brain and adipose tissue.

Phenobarbital and PCN pretreatments did not interfere with normal growth of the animals. At the beginning of the treatment period, the body weight of the control,

MOBILIZATION OF DDT RESIDUES 115

phenobarbital, and PCN groups were 216.3 + 3.0, 215.0 + 2.0, and 211.7 f 3.1 g (mean + SE), respectively, compared to 242.3 +_ 3.2, 239.0 + 4.8, and 241.0 of: 5.4 g for the same groups at the end of the treatment. On the other hand, CS-1 administration resulted in a slight stimulation of growth as evidenced by an increase in body weight from 220.0 + 3.0 g at the beginning to 272.3 5 3.8 g at the end of the treatment period. Finally, considerable loss in body weight was observed during Week 1 of treatment with dexamethasone 4 mg/kg (from 215.0 & 4.4 g at the beginning to 190.3 + 5.8 g on Day 4). Consequently, the dosage was reduced to 2 mg/kg during Week 2, thus prevent- ing a further marked decrease in body weight. By the end of the treatment period, the body weight of the dexamethasone group was 171.3 ? 2.7 g.

Influence of various periods of starvation on the concentration of tissue residues in rats contaminated with DDT. The residues of DDT found in the blood, brain, and adipose tissue following various periods and sequences of starvation are presented in Table 2.

TABLE 2

INFLUENCE OF VARIOUS PERIODS OF STARVATION ON THE CONCENTRATION OF

TOTAL DDT RESIDUES IN VARIOUS TISSUES OF RATS PREVIOUSLY CONTAMIN- ATED WITH DDT

Total residues”

Duration of starvation Whole blood (days) (nglml)

Brain Wd

Adipose tissue Wg)

0 3 consecutive 5 consecutive 3 nonconsecutive 5 nonconsecutive SEd

152b 1.83 349 111 1.47 265 80’ 0.88” 157”

138 1.43 249’ 154 1.31 278 15 0.18 30

‘-’ See Table 1.

A significant reduction in the concentration of residues in all tissues was observed only after 5 consecutive days of starvation. A moderate, but significant, reduction in residues was also found in adipose tissue after 3 nonconsecutive days of starvation.

Food deprivation during 3 or 5 consecutive days resulted in body weight losses of 14% (216.3 + 1.2 g in controls vs 186.3 + 2.0 g after 3 days of starvation) and 19% (221.7 + 1.7 g in controls vs 180.0 rf: 5.2 g after 5 days of starvation), respectively. Three days after resuming normal feeding, the body weight of previously starved rats com- pared with that of the controls (225.7 + 1.6 g in controls vs 221.0 f 2.8 g in animals starved during 3 days; 221.3 f 4.5 g in controls vs 223.3 f 7.0 g in those starved during 5 days). Similarly, when the animals were starved on alternate days, daily body weight losses amounted to nearly 10 %, but were rapidly corrected upon resumption of normal feeding. The following figures provide a typical example of body weight fluctuations under such experimental conditions: 217.7 + 1.9 g before starvation, 199.0 + 1.7 g after a l-day period of starvation and 215.2 + 2.7 g after a l-day period of normal feeding.

116 LAMBERT AND BRODEUR

InfIuence of various conlbinatiorls of phenobarbital and dQ,Grent starvation periods ou the concentration of tissue residues in rats contaminated with DDT. The previous series of experiments have shown quite clearly that among the four microsomal enzyme inducers used, phenobarbital was the most effective in reducing the concentration of DDT residues in certain tissues. In addition, the results have demonstrated that submitting the animals to starvation for 5 consecutive days resulted in a greater deple- tion of tissue residues than did any other treatment investigated. Tables 3 and 4 show the influence of combining phenobarbital and starvation on the ultimate mobilization of the residues.

TABLE 3

INFLUENCE OF PHENOBARBITAL COMBINED WITH STARVATION (DAYS 1 TO 5) ON THE CONCENTRATION OF TOTAL DDT RESIDUES IN VARIOUS TISSUES OF RATS PREVIOUSLY

CONTAMINATED WITH DDT

Total residues”

Phenobarbital*

Whole blood Brain (m/ml> hdd

- + - +

Adipose tissue cw/g>

- +

Starvationb - 146’ 74 1.55 0.94 293 207

Sid 71 42 11 0.68 0.10 0.45 195 112 27

RESULTS OFTHEANALYSIS OF VARIANCEWAISJEOF F)

Sources of variation DF’ Whole blood Brain Adipose tissue

Phenobarbital (P) I 20.99f 16.35f 9.92f Starvation (S) 1 23.41f 43.26f 1 2.32f Interaction (P x S) 1 3.60 3.34 0.00

n Total residues represent the sum of p,p’-DDT, p,p’-DDD, and p&-DDE residues. * Phenobarbital and starvation were used as treatments according to a 2 x 2 factorial experi-

mental design, each factor thus being used at two levels, e.g., absence (-1, or presence (+> of the factor. The results presented in this table are arranged in a bidimensional manner for each tissue.

’ Mean of determinations made on six animals. d Standard error of the mean of any group, calculated as indicated in Table 1. ’ DF, degrees of freedom. f Denotes significant effect of the treatment.

Starvation on Days I ro 5 (Table 3). The total residues were significantly decreased in all samples examined by both phenobarbital and starvation. The lack of a significant interaction for these residues indicates that the effects of each treatment were additive.

Starvation on Days 4 to 8 (Table 4). Phenobarbital and starvation significantly lowered the residues in the blood and brain only. Again, the statistical analysis revealed a lack of a significant interaction between both treatments.

MOBILIZATION OF DDT RESIDUES 117

TABLE 4

INFLUENCE OF PHENOBARBITAL COMBINED WITH STARVATION (DAYS 4 TO 8) ON THE CONCENTRATION OF TOTAL DDT RESIDUES IN VARIOUS TISSUES OF RATS PREVIOUSLY

CONTAMINATED WITH DDT

Total residues”

PhenobarbitaP

Whole blood Brain Wml) WP)

- f - +

Adipose tissue hm

- +

Starvationb - + SEd

146” 14 1.55 0.94 293 207 109 54 1.19 0.62 251 255

11 0.09 33

RESULTS OF THE ANALYSIS OF VARIANCE (VALUE OF F)

Sources of variation DF’ Whole blood Brain Adipose tissue

Phenobarbital (P) 1 36.15f 40.92f 1.54 Starvation (S) 1 7.39-f 13.34f 0.01 Interaction (P x S) 1 0.60 0.04 1.90

O-l See Table 3.

The results of the latter series of experiments, therefore, provide good evidence that for all tissues examined, the combination of phenobarbital and starvation does lead to additive tissue depletion of DDT-type residues. This is true whether the starvation period occurs early during the phenobarbital treatment or at a later time. However, the degree of effectiveness of the combined treatment is far better when starvation occurs early.

DISCUSSION

The results obtained during this investigation confirm the efficacy of enzyme induc- tion and starvation as a means of mobilizing pesticide residues from the tissues of previously contaminated organisms. However, for maximal efficacy, each treatment has to be administered under carefully selected experimental conditions. When com- bined, enzyme induction and starvation lead to no more than additive effects, Maximal efficacy is obtained when the mobilization of the residues by starvation, on the one hand, and the accelerated biotransformation due to enzyme induction, on the other hand, are interrelated optimally with respect to the time and the duration of the respec- tive treatments.

During starvation, highly lipid soluble chemicals are mobilized from adipose tissue stores and redistributed throughout the body. High concentrations of these materials can therefore be found in various tissues. However, this study demonstrates that the concentration of the residues falls well below the pretreatment values only a few days after resumption of feeding due to the partial elimination of the mobilized residues

118 LAMBERT AND BRODEUR

and to the redistribution of the latter as a consequence of the rapid correction of body weight and repletion of fat stores upon refeeding. Above all, the pattern of starvation appears to infhtence the magnitude of residue mobilization, the best results being obtained when the animals are deprived of food for a few consecutive days. Thus, the degree of completeness of fat depletion seems to bear some relationship with the degree of residue mobilization.

Amongst the potent enzyme inducers used during this study, phenobarbital rated as the most effective in decreasing the concentration of total residues in whole blood, brain, and adipose tissue, this being paralleled quite closely by decreases in the con- centration of&-DDT itself. Although it was not the purpose of this investigation to provide direct evidence for the mechanism of action of phenobarbital, it can reasonably be assumed that the barbiturate acted through hepatic microsomal enzyme induction. Thus, it has previously been shown by Alary et al. (1971) that repeated administration of phenobarbital to rats results in accelerated degradation of DDT to DDD by liver homogenates, and higher urinary elimination of the water-soluble metabolite DDA” under in viro conditions. In addition, other studies conducted under experimental conditions similar to that of the present investigation have shown that a phenobarbital pretreatment is able to bring about a significant increase in the concentration of p,p’-DDD in the liver (unpublished observations).

The failure to show more than additive effects with combinations of both treatments might appear to be surprising for the following reasons. First, the evidence points to different mechanisms of action for phenobarbital and starvation with respect to the elimination of residues, phenobarbital stimulating the biotransformation and starva- tion resulting mostly in the redistribution of the residues. Since both aspects of residue disposition were enhanced by the respective treatments, one might logically expect more than an additive response. Second, it has been shown that the combination of enzyme induction and starvation leads to definite potentiation of the in rifro hepatic microsomal enzyme activity (Greim, 1971; Brodeur and Lambert, 1973). This too would suggest the possibility of obtaining more than an additive response.

However, there might be an explanation for the data obtained in this study. Measure- ment of tissue concentrations of a residue is obviously not the most appropriate parameter for estimating its elimination from the body. This is especially true when a challenge, such as starvation, is known to influence some aspects of drug translocation in tissues (Brodeur et al., 1974). Indeed, determination ofurinaryand fecal metabolite(s) is usually the most reliable index of drug elimination. It can therefore be reasoned that, from a mechanistic point of view, the measurement of DDA would have been more informative.

In conclusion, these data show that when rats treated with DDT are submitted to a treatment combining enzyme induction and starvation, the end-results are additive when estimated in terms of tissue depletion of pesticide residues. It is still possible, however, that the mechanisms leading to the elimination of the residues might act in more than an additive manner. This is an interesting observation, since it is now evident that the combination of enzyme induction and starvation is a superior means of clearing contaminated tissues of certain pesticides than either treatment alone.

9 2,2-bis-(p-chlorophenyl)acetic acid.

MOBILIZATION OF DDT RESIDUES 119

ACKNOWLEDGMENTS

We are indebted to Dr. Robert Elie for his help in the statistical analysis of the data. The expert technical assistance of Mrs. Madeleine Langlois and Mrs. Sanae Yamaguchi is grate- fully acknowledged.

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