The effect of age and long-term low-level DDT exposure on the response to enzyme induction in the rat

TOXICOLOGY AND APPLIED PHARMACOLOGY31,469-480

The Exposure

(1975)

Effect of Age and Long-Term on the Response to Enzyme

Low-Level Induction

DDT in the

Rat1

R. W. CHADWICK, R. S. LINKO, J. J. FREAL, AND A. L. ROBBINS” Environmental Biochemistry

Protection Agency, Pesticides and Toxic Substances E&is Laboratory, and Physiology Branch, Research Triangle Park, North Carolina 27711 Received

June IS, 1974;

accepted

September

13, 1974

The Effect of Age and Long-Term Low-Level DDT Exposure on the Response to Enzymeinduction in the Rat. CHADWICK, R. W., LINKO, R. S., FREAL, J. J. AND ROBBINS, A. L. (1975). Toxicol. Appl. Pharmacol. 31, 469-480. Some investigationssuggestthere is an age-dependentsusceptibility to enzyme induction in animals,while other work indicatesthat the apparentlack of responsein older animalsmay bethe result of an induction lag period. The existenceof suchan induction lag in rats aswell asthe effect of long-term low-level DDT exposureon the responseto enzymeinduction by dieldrin were investigatedin this study. Adult and weanling rats were sacrificedat 24,48, 72, 96, and 120hr after ip administration of 2.5 mg/kg of dieldrin. Twenty-four hours before the rats weresacrificed,10 mg/kg of lindane in peanut oil was administered po. Within 24 hr after the administration of dieldrin, there were significant increasesin the in vitro activity of various hepatic microsomalenzymesfrom the weanling rats. Dieldrin did not producea consistentincreasein the enzymeactivity of the adults and increasesthat did occur were much smallerthan those in the weanlings.In general,throughout the study, the weanlingshad the highest enzyme activity, the DDT-fed adults were intermediate and the control adults had the lswest activity. Weanlings metabolize more lindane to chlorophenolsby 72 hr after receivingdieldrin and storelesslindaneby 96 hi after dieldrin administrationthan either aduli group of rats. Data from this experimentdoesnot support the existenceof an induction time lag in adult rats. The exposure of man and animalsto foreign substancessuchasdrugs and environmental chemicals has increased tremendously since World War II. Fortunately, animals have the capacity for an increased metabolism of these xenobiotics through enzyme induction. It follows therefore, that knowledge of the factors which influence this adaptive responseare important to public welfare. It has been known for sometime that drug-metabolism is relatively deficient in both old (Kato et al., 1954)and newborn animals (Jondorf et cl., 1958; Fouts and Adamson, 1959). However, there are conflicting reports regarding the effect of age on enzyme induction. Thus. whereas Kato and Takanaka (1968) found that drug-metabolizing enzymes in young rats responded much more markedly to phenobarbital than the ’ Presented at the 12th Annual Meeting of the Society of Toxicology, New York, March 1973. ’ Current address: Environmental Protection Agency, Pesticides and Toxic Substances Effects Laboratory, Field Studies Section, P.O. Box 219, Wenatchee, Wash. 98801. Copvright 7“ 1975 by Academic Prers, Inc. 469 All rights of re?roduction PI-inicd nn Great Britain

in any form

reserved.

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ET AL.

enzymes of old rats, Gillett (1969) observed little difference in the absolute amount of DDT-induced enzyme activity due to age. Furthermore, work by Adelman (1971) indicated that lower enzyme activity in older animals may just be due to a lag in their responseto enzyme inducing agents.In every agegroup of rats tested, the induced value characteristic of the young rats was attained in no more than four to five days after the administration of phenobarbital. This study was designedto compare drug-metabolism in weanling and old adult rats at 24 hr intervals up to and including five days after the administration of a single dose of the insecticide 1,2,3,4,10,10-hexachloro-exo-6,7-epoxy-l ,4,4a,5,6,7,8,8a-octahydro1,Cendo, exo-5,Cdimethanonaphthalene (dieldrin).3 In addition, the effect of exposure, for one year, to five ppm l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT)3 upon basal enzyme activity and upon the response to stimulation by dieldrin was also examined. METHODS Animals used in this study were either one month or fifteen months old female Sprague-Dawley rats. Half of the adult animals had received 5 ppm DDT in their feed since they were 3 months old. On day 0, eight weanlings, four control adults and four DDT-fed adults were sacrificed. Twenty hours previous to this, these animals were

FIG. 1. Sacrifice schedule for the adult and weanling rats. Time (in days) after the administration of dieldrin is represented by the shaded area (t&4)adjacent to each group. Time interval (in days) at which lindane was administered is represented by the solid black rectangular (m) sections adjacent to each group. All rats except group 1 received dieldrin and lindane.

given ip injections of dimethyl sulfoxide (DMSO) and po injections of peanut oil. An additional 40 weanlings, 20 control adults and 20 DDT-fed adults were given injections of 2.5 mg/kg of dieldrin in DMSO on day 0. Eight weanlings, four control adults, and four DDT-fed adults were sacrificed at 1, 2, 3. 4, and 5 days after the administration of dieldrin. Each group except those sacrificed on day0, received 10mg/kg of y-hexachlorocyclohexane (lindane)3 in peanut oil po 24 hr before they were sacrificed. The dosage schedule is illustrated in Fig. 1. 3 The pesticides were obtained from the Perrine Laboratory

Repository.

AGE,

DDT,

AND

ENZYME

INDUCTION

471

When the animals were sacrificed, liver samples were homogenized and analyzed for the enzyme activity involved in the 0-demethylation of p-nitroanisole (Kinoshita et al., 1966), the oxidative hydrolysis of O-ethyl 0-p-nitrophenyl phenylphosphonothioic acid (EPN)3 (Kinoshita et al., 1966), the reduction of the azo group of sodium p-dimethylaminoazobenzenesulfonate (methyl-orange) (Fouts et al., 19.57), the glucuronidation of p-nitrophenol (Chadwick et al., 1971), the desulfuration of 0, O-diethyl 0-p-nitrophenyl phosphorothioate (parathion)3 (Davis et al., 1973). and the dearylation of parathion3 (Davis et al., 1973). Individual 0.2 g samplesof both adipose tissueand liver were minced three times in tissue grinders using 3 ml portions of benzene each time. The combined extracts were then diluted to an appropriate volume and analyzed on a Tracer MT-220 gas chromatograph equipped with both an electron capture detector and a Coulson electrolytic conductivity detector (operated in the oxidative mode). The electron capture detector was usedto analyze pesticide concentrations in the liver and adiposetissue.The column usedconsisted of a 6 ft x 0.25 in. glassU-tube packed with 4-6 % SE-30/QF-1 on SO/l00 meshgaschrom Q,4 isothermally maintained at 195°C. The flow of the nitrogen carrier gas was regulated at 72 cm3/min. On the day each group of animals were sacrificed, individual 24 hr urine sampleswere collected under toluene for analysis of lindane metabolites. The urines were adjusted to pH 7 and extracted twice with equal volumes of benzeneto recover free chlorophenols. The residual urine was then acidified and hydrolyzed in an autoclave for 15 min under 15 lb pressureand extracted twice with equal volumes of benzenefor recovery of the conjugated chlorophenols. The benzene extracts were partitioned with a volume of 0.15 N NaOH equivalent to 25 “/; of the volume of the organic phase. The 0.15 N NaOH extract was then acidified and extracted with a 2 ml volume of benzene. This extract was analyzed on a Tracer MT-220 gaschromatograph equipped with a Coulson electrolytic conductivity detector operated in the oxidative mode. The column usedfor the analysis consisted of a 6 ft x 0.25 in. glassU-tube packed with 5 % DEGS5 on SO/100meshGas chrom Q,’ isothermally maintained at 180°C. The flow of nitrogen carrier gas was regulated at 90 cm3/min. Duncan’s Multiple Range Test was used as an aid in the interpretation of the data from this study (Duncan, 1955). Comparisons were considered significantly different at 17< 0.05 unlessotherwise stated.

RESULTS 0- Denzethylation

of p-Nitroanisole

Initially on day zero, before the administration of dieldrin or lindane, the DDTtreated adults have significantly greater O-demethylase activity than either the weanlings or the control adults (Fig. 2). Within 24 hr after the administration of dieldrin, the 0-demethylase activity of the weanlings becomessignificantly greater than the control adults. The weanlingsexhibit peak enzyme activity three days after dieldrin administration and then decline to that of the adults on the last day. The 0-demethylase activity of either group of adults does not change significantly with time during the study. 4 The packing material was obtained from Applied Science Laboratories, Inc., State College, Pa. 5 Diethyleneglycosuccinate was obtained from Analabs Inc. Hamden, Conn.

472

CHADWICK

ET AL.

T

0

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ADMINISTRATION

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TIME AFTER DIELDRIN

FIG.

5

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(days)

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Oxidative Hydrolysis

DDT,

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ENZYME

INDUCTION

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of EPN

On day zero both adult groups show significantly higher activity in the oxidative hydrolysis of EPN than the weanlings (Fig. 2). However, within 24 hr after the administration of dieldrin, the weanlings exhibit significantly higher enzyme activity than the adults. Again the weanlings exhibit maximal activity 48-72 hr after receiving dieldrin and then decline. The control adults peak on the fifth day after receiving dieldrin while the DDT-treated adults show no significant change in enzyme activity with elapsed time. Azo-Reductase Again the adults show significantly higher enzyme activity than the weanlings prior to the dieldrin treatment (Fig. 2). However, 24 hr after dieldrin administration the azoreductase levels in the weanlings are significantly higher than that of either adult group. Moreover, even though the azo-reductase activity of the weanlings peaks on the first day after dieldrin, it remains significantly higher than that of the adults throughout the rest of the study, except day three when both weanlings and the DDT-treated adults are significantly higher than the control adults. Though both adult groups exhibit maximal enzyme activity 3 days after receiving dieldrin, their enzyme activity at this point is still significantly less than that of the weanlings at 1 day after the administration of dieldrin. Ghcuronoyl

Transferase

Twenty-four hours after dieldrin administration, the weanlings and the DDTtreated adults have significantly greater glucuronoyl transferase activity than that of the control adults (Fig. 3). However, at 0, 2, 3, and 4 days after dieldrin, there are no significant differences in activity. Finally on day five, the weanlings exhibit significantly greater activity than both groups of adults. The enzyme activity of the DDT-treated adults falls off with time from the initial peak on day zero. Desulfk-ation

qf Parathion

Initially and except for the first and fifth days after the administration of dieldrin, the conversion of parathion to paraoxon by the weanlings and the DDT-treated adults was significantly greater than that of the control adults (Fig. 3). However, it will be noted that in contrast to the weanlings who show a prolonged increase in enzyme activity after receiving dieldrin, the adults exhibit a decline. after a transitory initial increase.

FIG. 2. (Upper Panel) Effect of age and long-term, low-level DDT exposure on O-demethyiase (bg of p-nitroanisole 20 mg liver’hr). Each point represents the mean of eight animals k SE for the weanlings and four animals k SE for the adult rats. (Middle Panel) Effect of age and long-term, low-level DDT exposure on the oxidative hydrolysis of EPN (fig of EPNjZO mg liver:hr). Each point represents the mean of eight animals + SE for the weanlings and four animals + SE for the adults. (Lower Panel) Effect of age and long-term, low-level DDT exposure on azoreductase (pg of methyl orange/IO mg liver/ 30 min). Each point represents the mean of eight animals f SE for the weanlings and four animals i SE for the adult rats.

474

ET AL.

CHADWICK

TIME AFTER DIELDRIN

ADMINISTRATION

[days)

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05-

04-

2 =

03-

02 t 0 I

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FIG.

ADMINISTRATION

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ldoysl

AGE,

Dearyfation

DDT,

AND

ENZYME

475

INDUCTION

of Parathion

On day zero the DDT-treated adults show significantly greater activity in the dearylation of parathion to diethylphosphorothioate than either the control adults or weanlings (Fig. 3). However, by day two after receiving dieldrin, the weanlings have significantly higher enzyme activity than the adults and remain the most active group throughout the rest of the study. Total Chlorophenol Excretion Figure 4 shows the excretion of total chlorophenols from the metabolism of lindane by the control adults, the DDT-treated adults and the weanlings at various times after receiving a single dose of dieldrin.

c DW 1

cow 2 TIME AFTER DlELDRlN

CDW 3

CDW d

ADMINISTRATION

CDW 5 (dqr]

FIG. 4. Effect of age and long-term low-level DDT exposure on the urinary excretion of lindanederived chlorophenols (7: of administered findane! hr urine sample). The lower section of each bar represents the conjugated chlorophenols while the upper portion represents the free chlorophenols excreted. Each section is the mean of eight animals i SE for the weanlings and four animals & SE for the adult rats. The bars are labeled C, D, and W at each time interval to represent the control adults, DDT-treated adults and weanlings respectively.

Both of the DDT-treated adults and the weanlings excrete significantly more chlorophenols than the controi adults throughout the study. While conjugated chlorophenols constitute the major part of this increasedexcretion by DDT-treated adults at 24 hr after dieldrin, the increasedexcretion by weanlings is due to significantly higher levels of free

FIG. 3. (Upper Panel) El?ect cf age and long-term, low-level DDT exposure on glucuronyl transferase (pg p-nitrophenol]?O mg liver/30 min). Each point represents the mean of eight animals + SE for the wean!ings and four animals 2 SE, for ihe adult rats. (Middle Panel) Effect of age and long-term lowlevel DDT exposure on the desulfuration cf parathion (pg parathionj55.6 mg liver/30 min). Each point represents the mean of eight animals + SE for the weanlings and 4 animals f SE for the adult rats. (Lower Panel) Effect of age and long-term, low-level DDT exposure on the dearylation of parathion (/lg of parathionj55.6 mg liver/30 min). Each point represents ihe mean of eight animals i SE for the weanlings and four animals + SE for the adult rats.

476

CHADWICK ET AL.

I

;

I

;

TIME AFTER DIELDRIN

4

ADMINISTRATION

; (days)

FIG. 5. Effect of age and long-term low-level DDT exposure on the distribution chlorophenol metabolites individual excreted chlorophenol mean x 100 1 ( total excreted chlorophenol mean

of lindane-derived

The individual trichlorophenol means are each represented as rectangular sections of the lower bars, while the upper bars represent the mean tetrachlorophenols excreted. Each section or bar represents the mean of eight animals for the weanlings and four animals for the adults. The three bars at each time interval represent from left to right, the control adults, the DDT-treated adults, and the weanlings respectively. The SE for the total tetrachlorophenols and total trichlorophenols of each group are represented by vertical lines. 2or

C’ 2

I

I 2 TIME AFTER DIELDRIN

I 3 ADMINISTRATION

4

5 tdayil

FIG. 6. Effect of age and long-term low-level DDT exposure on hepatic lindane content (ppm lindane in liver tissue). Each point represents the mean of eight animals k SE for the weanlings and four animals f SE for the adult rats. Weanling rats (&--); adult DDT-treated rats (--); adult control rats (----).

AGE,

DDT,

AND

ENZYME

477

INDUCTION

chlorophenols. However, the weanlings excrete significantly more conjugated chlorophenols than the control adults by 48 hr after receiving dieldrin and significantly more than both adult groups 72 hr after dieldrin treatment. Tri- and Tetrachlorophenol

Excretion

Figure 5 indicates the relative proportion of each chlorophenol excreted by the control adults, the DDT-treated adults and the weanlings. The bars in the top part of the figure represent the tetrachlorophenols and it was in the excretion of these metabolites that the greatest differences were noted. At first the DDT-treated adults excrete a significantly greater proportion of tetrachlorophenols than the other rats, but from three days after the administration of dieldrin until the end of the study, both the weanlings and the DDT-treated adults excrete a significantly greater proportion of tetrachlorophenols than the control adults. Hepatic Lindane Content Within 24 hr after the administration of dieldrin, the concentrationof lindane in the livers of the weanlings and DDT-treated adults was significantly lower than that of the control adults (Fig. 6). Significantly lower lindane content is indicative of increased mobilization of the pesticide from the liver and both the weanlings and DDT-treated adults maintain significantly lower levels than the control adults throughout the study except for day two. Fat Storage of Lindane Three days after dieldrin administration the storage of lindane in the fat is significantly less in the weanlings than in the control adults (Fig. 7), and by 4 days the fat content of lindane in weanlings was significantly lower than either adult group.

Y 2

70

-

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-

50

-

AO-

f I e

30-

20

-

1

2 T,ME

AFTER

1 5

3 DiELDRlN

ADMINISTRATI9N

Idays!

Fc. 7. Effect of age and long-term low-level DDT exposure on lindane in adipose tissue (ppm lindane in adipose tissue). Each point represents the mean of eight animals + SE for the weanlings and four animals k SE for the adult rats. Weanling rats (---): adult DDT-treated rats (--); adult control rats (----).

478

CHADWICK

ET AL.

Hepatic Dieldrin Content From 24 hr after the administration of dieldrin until the final day of the study, the weanlingshave a significantly higher concentration of dieldrin in the liver than the adults (Fig. 8). The control adults have lessthan detectable amounts until day three. The relative availability of dieldrin to the hepatic enzyme induction site(s) may account for some of the observed differences between the weanlings and adults with respect to enzyme activity and lindane metabolism.

L

1 1

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3

4

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TIME AFTER DIELDRIN

ADMINISTRATION

[days)

FIG. 8. Effect of age and long-term low-level DDT exposure on hepatic dieldrin content (ppm dieldrin in liver tissue). Each point represents the mean of eight animals + SE for the weanlings and four animals f SE for the adult rats. Weanling rats (---); adult DDT-treated rats (--); adult control rats (----).

DISCUSSION The effect of long-term, low level DDT-exposure on enzyme induction and the existence of an age-dependent time lag for enzyme induction in adult rats were investigated. In general, after the administration of dieldrin, the weanlings attain the highest enzyme activities, the DDT-treated adults are intermediate, and the control adults are lowest. Rats with long-term low-level DDT exposure, maintain a higher basal enzyme level than control adults. However, responseto enzyme induction, from a single doseof dieldrin, doesnot appear to be altered significantly by long-term DDT exposure over that observed in the control adults. Moreover, neither adult group reachesthe peak dieldrin-induced enzyme activity exhibited by the weanlings during the time intervals studied. The comparative responsepatterns of various in vitro enzyme systemsto dieldrin stimulation differ markedly from each other over the 5 day test period. Whereas the weanlings reach peak O-demethylase activity 3 days after dieldrin administration and then decline to adult levels, their azo-reductaseactivity remainssignificantly higher than that of the adults from day one to the end of the study. Furthermore, while the adults

AGE,

DDT,

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ENZYME

INDUCTION

479

exhibit no change in O-demethylase activity with time and a peak azo-reductase activity 3 days after receiving dieldrin, they actually show a significant decline in desulfurase activity. Figure 9 summarizes the point in time at which the experimental animals reach a significant maximum for each of eight parameters; O-demethylase, total chlorophenol excretion, glucuronyl transferase, dearylation, per cent tetrachlorophenol excretion, oxidative hydrolysis, oxidative desulfuration and azo-reductase. Dieldrin increased each of the above responses in weanling rats. On the other hand, dieldrin administration did not appear to influence maximum activity of the adult controls in four of the eight responses. Similarly dieldrin had no effect on the maximum activity of the DDTtreated adults in five of the eight responses.

FIG. 9. Effect of age and long-term, low-level DDT exposure on the time at which peak metabolic activity occurs. The horizontal bars labeled C, D, or W represent the time at which maximum activity occurred for the control adults, the DDT-treated adults or the weanlings respectively. Absence of a bar indicates that there was no significant change with time.

Enzyme stimulation in the rat is dependent on such diverse factors as environmental contamination, age of the animal, elapsedtime after exposure to an inducing agent, and the type of enzyme systemexamined. If the variation in elapsedtime between exposure to an inducer and maximum enzyme activity (noted in Fig. 9) indicates the existence of different half lives for different induction processes,then this fact could be of importance in toxicology. For example it has recently been reported that chemically reactive metabolites mediate many different kinds of serious toxicity including carcinogenesis, mutagenesis,cellular necrosis, hypersensitivity reactions, methemoglobinemia. hemolytic anemia, blood dyscrasias and fetotoxicities (Gillette et al., 1974). When toxic metabolites are formed along minor pathways, toxicity would not be observed until theseroutes becamesubsequentlyactivated. Thus anything which can potentially change the pattern of metabolism of a xenobiotic such asdose, age, exposure to other environmental contaminants, etc., might be expected to significantly alter not only its toxicity but the onset of toxicity as well. The observed dissimilarities in responseto enzyme induction by the weanling and the adult groups of rats may simply be due to the higher concentration of dieldrin in the livers of the weanlings.

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Wright et a/. (1972) found that regression of dieldrin-induced drug-metabolizing enzyme activity could be correlated with the rate of decay of dieldrin from the liver. It is possible that the decreasedconcentration of dieldrin found in the livers of the adult rats, compared to weanlings, reflects increased plasmabinding of the insecticide by the older animals. Many toxic materials are bound onto plasmaproteins, particularly serum albumin (Kasik, 1971)and the albumin synthesisof old rats is 50 “/, higher than that of young rats (Ove et al., 1972; Chen et al., 1973). Data from this experiment do not support the existence of an induction time lag in adult rats and further suggestthat observed age-dependent susceptibility to enzyme induction may be due to a decreasedavailability of the inducing agent to the hepatic enzyme induction site(s) in older animals.

REFERENCES

R. C. (1971).Age-dependenteffectson enzymeinduction. A biochemicalexpression of aging. Exp. Gerorztol. 6, 75-87. CHADWICK, R. W., CRANMER, M. F. AND PEOPLES, A. J. (1971).Comparative stimulation of yHCH metabolismby pretreatmentof rats with yHCH, DDT, and DDT + yHCH. Toxicol. ADELMAN,

Appl. Pharmacol. 18, 685-695. CHEN, J. C., OVE, P. AND LANSING, A. I. (1973) Ifz vitro synthesis of microsomal protein and albumin in young and old rats. Biochim. Biophys. Acta 312, 598-607. DAVIS, J. E., CRANMER, M. F. AND PEOPLES, A. J. (1973). Gas-liquid chromatography of parathion metabolites as a sensitive index of induction of liver microsomal mixed-function oxidases. Anal. Biochem. 53, 522-530. DUNCAN, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, 142. FOLJTS, J. R., KAMM, J. J. AND BRODIE, B. B.(l957). Enzymaticreductionofprontosilandother azo dyes. J. Pharmacol. Exp. Ther. 120, 291-300. FOUTS, J. R. AND ADAMSON, R. H. (1959).Drug metabolismin the newborn rabbit. Science 129, 897-898. GILLETT, J. (1969). Microsomal epoxidation: Effect of age and duration of exposure to dietary DDT on induction. Bull. Environ. Contam. Toxico/. 4, 159-168. GILLETTE,

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MITCHELL,

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B. B. (I 974).Biochemicalmechanisms of drug

toxicity. In Annual Review of Pharmacology (H. W. Elliot, Ed.), Vol. 14, pp. 271-288. Annual Reviews Inc. Palo Alto, CA. JONDORF, W. R., MAICKEL, R. P. AND BRODIE, B. B. (1958). Inability of newborn mice and guinea pigs tometabolizedrugs. Biochem. Phamacol. 1,352-354. KASIK, J. E. (1971). Absorption, metabolism and excretion of toxic substances. /. Occup. Med. 13, 8-13. KATO, R., VASSANELLI, P., FRONTINO, G. AND CHIESARA, E. (1964). Variation in the activity of liver microsomal drug metabolizing enzymes in rats in relation to the age. Biochenz. Pharmace/. 13, 1037-1951. K~~0,R.~~~T~~~~~~~,A.(l968).Effectofphenobarbitalonelectrontransportsystemoxidation and reduction of drugs in liver microsomes of rats of different age. J. Biochem. 63,406408. KINOSHITA, F. K., FRAWLEY, J. P. AND DUBOIS, K. P. (1966). Quantitative measurements of induction of hepatic microsomal enzymes by various dietary levels of DDT and toxaphene in rats. Toxicol. Appl. Pharmacol. 9, 505-513. OVE, P., OBENRADER, M. AND LANSING, A. (1972). Synthesis and degradation of liver proteins in young and old rats. Biochitn. Biophys. Acta 277, 21 l-221. WRIGHT, A. S., POTTER. D., WOODER, M. F. AND DONNINCER, C. (1972). The effects of dieldrin on the subcellular structure and function of mammalian liver cells. Fd. Comet. Toxicol. 10, 31 l-332.