Stimulatory effects of chlordane on hepatic microsomal drug metabolism in the rat

TOXICOLOGY

AND APPLIED

Stimulatory Microsomal

PHARMACOLOGY

Effects

5, 3 7l-386 ( 1963)

of

Drug

Chlordanel

Metabolism

LARRY G. HART, ROBERT W. SHULTICE, Department

of Pharmacology,

September

Hepatic

in the

Rat”

AND JAMES R. FOUTS

College of Medicine, Iowa City, Iowa

Received

on

State

University

of

Iowa,

7, 1962

Various chlorinated hydrocarbons have been widely usedas insecticides. The toxicity of such compounds has been studied in detail (Negherbon, 1959; von Oettingen, 1955; U.S. Department of Health, Education, and Welfare, 1956). In this paper we are reporting a stimulatory effect of one such insecticide on hepatic microsomal drug metabolizing enzymes. This effect came to our attention accidentally. A group of adult male rats had been starved in order to study the effects of starvation on drug metabolism. Normally, starvation depresses hepatic microsomal drug metabolism (Dixon et aZ., 1960). In this group of starved animals, microsomal enzyme activity was higher than from any previous group of rats we had studied. One possible reason for the increased rate of drug metabolism was the exposure of these rats to a chlordane (1,2,4,5,6,7,8,8-octachloro-4,7-methane-3a,4,7,7a-tetrahydroindane) spray. This insecticide was used to eradicate Cimex Eectularius (bed bugs) in the room housing the experimental animals. The rats were exposed to the chlordane about 1 week before they were used to study effects of starvation on drug metabolism. Several compounds, including phenobarbital, 3-methylcholanthrene, 3,4benzpyrene, aminopyrine, and zoxazolamine, none of which bear any structural relationship to chlordane, have been shown by Conney et al. (1960) to stimulate microsomal drug metabolizing enzyme activity in laboratory animals. 1 Chlordane, 1,2,4,5,6,7,8,8-octachloro-4,7-methane-3a,4,7,7a-tetrahydroindane, known as Velsicol 1068, CD-68, Toxichlor, and Octa-klor. 2 Supported by United States Public Health Service Grant GM-06034. 371

is also

372

LARRY

G.

HART

ET

AL.

METHODS

Animals

and Materials

Both adult (200-250 g) and weanling (SO-80 g) male Holtzman rats were used. The animals were maintained on water and lab chow ad libitum. Rats were killed by a blow on the head. Their livers were immediately excised and placed in a vessel immersed in ice. Livers were homogenized with a Potter homogenizer (plastic pestle) in the cold. All homogenates were prepared such that each gram of liver was suspendedin 2 ml of cold isotonic ( 1.15%)) KCl. The supernatant fraction (the supernatant obtained by centrifugation for 15 minutes at 9000 g), containing microsomal and soluble enzymes, was prepared from the homogenate with a high speed angle centrifuge. Technical chlordane, a mixture containing 60-7.5% of the ‘
Design

Adult rats were divided into three different dose groups; one group receiving 10 mg/kg, a second group receiving 25 mg/kg, and the third group receiving 100 mg/kg of chlordane. Two other groups were given 3 consecutive daily dosesof chlordane, 10 mg/kg and 25 mg/kg being the dosesused. All doseswere given by intraperitoneal injection. The LDBo of chlordane given by intraperitoneal injection to rats is 200 mg/kg (Stohlman et al., 1950).

CHLORDANE

ON

DRUG

METABOLISM

IN

RATS

373

Chlordane was administered in single doses of 10 mg/kg and 30 mg/kg to 2 different groups of rats at the same time that another group of rats was injected with the first of 3 daily doses of 10 mg/kg. Enzyme activity for hexobarbital and aminopyrine was determined on livers from all 3 groups 1 day after the third injection of 10 mg/kg of chlordane or 3 days after the single injection of 10 mg/kg or 30 mg/kg of chlordane. One group of weanling rats was injected with a single dose of 25 mg/kg, and another group received 3 consecutive daily doses of 10 mg/kg of chlordane. To test the effects of y-chlordane, one group of adult rats received a single dose of 10 mg/kg, a second group received 3 consecutive daily doses of 10 mg/kg, and a third group received 3 consecutive daily doses of 100 mg/kg of y-chlordane. Control animals in all cases were injected with an equal volume of corn oil. m-Ethionine, an amino acid antagonist, was administered concurrently with chlordane to a group of weanling rats. This was done to determine whether the increases in enzyme activity caused by chlordane could be prevented. Ethionine, at doses of 210mg/kg and 280 mg/kg, was given intraperitoneally, 15 minutes prior to an intraperitoneal injection of 10 mg/kg of chlordane. This treatment with both ethionine and chlordane was continued for 3 days. Chlordane was added to the incubation mixtures to determine the direct effect of this material on enzyme activity. Final concentrations of chlordane added to the incubate were 1.7 X lo-” M, 3.4 X 10-“&f, 1.7 X 1OP’ M, 3.4 X lo-‘iM, 8.5 X lo-” d4, and 1.7 X lo-” M. The concentration of chlordane in ZI~VO,assuming complete absorption from the intraperitoneal site of injection and uniform distribution in body water, would be 3.4 X 1O-5IM at lOmg/kg, the lowest dose used, and 3.4 X lo-” M at 100 mg/kg, the highest dose used. Determination

of Enzyme Activity

Metabolic pathways studied were the side chain oxidation of hexobarbital, the N-dealkylation of aminopyrine, and oxidation of the ring sulfur of chlorpromazine. The disappearance of the substrate was measured to follow the metabolism of hexobarbital and chlorpromazine. Appearance of the metabolite was determined in measuring the metabolism of aminopyrine. Hexobarbital was assayed by the method of Cooper and Brodie

374

LARRY

G.

HART

ET

AL.

(1955). Chlorpromazine was determined by the method of Salzman and Brodie ( 1956). The metabolite of aminopyrine, 4-aminoantipyrine, was determined by the method of Brodie and Axelrod (1950). The conditions of incubation, cofactors used, and concentrations thereof were the same as those reported by McLuen and Fouts ( 1961). Statistical methods used are described by Snedecor (1956). For our experiments, we chose the level of significance as P 5 0.05. RESULTS

Effects of Single and Multiple Metabolizing Enzyme Activity

D,oses of Chlordane on Drug in Adult Rats

Enzyme activity was determined at 1, 8, 15, 22, and in some cases 29 days after the last injection of chlordane. With one dose, enzyme activity increased slightly at 1 day post injection, but with one exception (chlorpromazine metabolism) was not significantly different from control (Table 1). At the lowest dose used, 10 mg/kg, maximal stimulation was noted at 8 days for all three pathways of drug metabolism with a decrease in enzyme activity thereafter. At the intermediate dose of 25 mg/kg, a maximal effect was reached at 15 days with the exception of the rate of metabolism of chlorpromazine. For chlorpromazine, increased enzyme activity was present at 1 day and was sustained through 15 days. At the highest dose of chlordane, 100 mg/kg, peak effects were seen at 8 days and maintained for 2 weeks, enzyme activity being significantly greater than control at 29 days for the metabolisms of aminopyrine and chlorpromazine. When a single dose of chlordane was compared with effects of 3 consecutive daily doses of 10 mg/kg and 25 mg/kg, stimulation after 3 doses occurred earlier (1 day after the third injection) and declined earlier, activity in most cases having decreased considerably by 15 days after injection (Table 1 vs. Table 2). When a single dose of 10 mg or 30 mg/kg of chlordane was given at the same time as the first of 3 doses of 10 mg/kg there was no difference in the rate of metabolism of hexobarbital and aminopyrine among the 3 groups (Table 3). Enzyme activity for hexobarbital and aminopyrine was also elevated significantly over control levels. These data contrast with those mentioned in the previous paragraph, which show no effect when the single dose was given at the same time as the third of 3 doses.

ard

b P 2

deviation

Q Values

Chlorpromazine

Aminopyrine

0.05.

in

this

For

per

all

gram

table

indicate

other

liver

100 w/k Control

Control

10 w/k Control 25 mg/kg

Control

(wet

values

supernatant

in

P > 0.05.

weight)

metabolism

by All

expressed

& 0.10

1.09

f 0.46 ?I 0.71

1.43 1.17

2 hours.

? 0.17 k o.16b

fraction groups

had

and

0.35

f r k

0.26 0.47 0.41

4 control

animals.

drug

0.08

& O.lOb k

0.91 0.57

& 0.27 ‘- 0.16

-c 0.11

0.56 1.03 0.84

s 0.19

c 0.14b & 0.08

0.11 0.15

0.05

0.58

0.61 0.29

I? 0.00

0.24

0.46

2 k

RAT+

metabolized

o.41b

& 1.15

-c 0.68 do 0.79

k

22

IN ADULT

3.04 1.58

1.80

2.44 1.46

1.76

micromoles

injection

METABOLISM

in average

31 0.06

2 0.13 zk O.lSb

I? 0.14”

4 experimental

0.50

0.72 1.40

1.03

1.11 1.58

0.05"

-c

1.55

& o.lob * 0.17

1.61 1.22

2 0.13 -c 0.12

k 0.06

1.02 0.86

rt 0.05

-

& 0.05 k 0.13”

0.46 0.74

-

2 0.05 _t 0.08b

0.21

& 0.10

2 0.19’) & 0.42

-c 0.47

0.43 0.80

zk o/W’ I-c 0.14

0.24

0.52

last

AZ 0.59 k 0.41b

k

15

the

DRUG

0.52

6.59 3.70

” o.oiP -c 0.10

4.51

3.29 5.33

4.23

i o.W ‘-c 0.35

0.65 0.56

0.411,

after

zk 0.70

2 2

k

Days

2.88 0.73

f 0.12 2 0.10

0.65 0.31

mdkg Control 100 mg/kg

25

0.50 0.35

6.35 3.78

1.13

2.77 2.59

3.76

8

1 MICROSOMAL

_t 0.14 ‘-r- 0.21

2 0.06

0.81

TABLE ON HEPATIC

0.75 0.45

?I 0.15 k 0.08

0.55 0.56

1.15 0.59

1.12

0.74 0.49

1.04

10 m&g Control

i e

k

5.90

Control

100 mg/kg Control

4.57 5.45

t f

5.36 6.17

25

Control mdkg

k

1

OF CHLORDANE

4.64

10

Hexobarbital

DOSE

w/kg

of chlordane

Dose

OF A SINGLE

Drug substrate

EFFECTS

0.23

0.68

0.44 0.26

2.61 3.13

29

c

r

2

-

-

-

r ”

-

-

-

stand-

0.16

mob

0.06

o.11"

k 1.17 ” 0.97

-

-

-

-

1.70 c o.lob 1.04 * 0.17

25 w/k

Control

--

0.39 k 0.11

Control

10 w/kg Control

0.84 k o.oSb 0.40 -t 0.13 1.49 2 0.28b

10 w/kg Control 25 w/kg

1.67 I+ 0.89

Control

1

OF CHLORDANE

4.68 k 0.32b 2.93 -t 0.80 3.24 r!z 0.2Sb

DOSES

10 w/kg Control 25 w/k

dose of chlordane

Daily

OF THREE

TABLE

1.31 -c 0.11” 0.90 t 0.13 -

0.72 k 0.20

0.88 e 0.18" 0.40 k 0.05 1.69 C 0.26"

5.54 & 0.47

2

Days 15

-t 0.16 -t 0.31

zk 0.05 c 0.10

0.35 A 0.08

0.34 & 0.08 0.31 t 0.05 0.76 t o.llb

3.22 I+ I.15

0.65 0.54 1.14 0.86

METABOLISM

the last injection

DRUG

2.72 4 0.29 1.69 k 0.35 3.74 t 1.11

after

MICROSOMAL

5.51 & 0.48b 3.72 t 0.46 6.89 k 0.22b

8

ON HEPATIC

0.44

0.89 k 0.20" 0.52 2 0.10

-

0.25 ? 0.09

0.38 21 0.10” 0.24 I+ 0.05 0.39 -e 0.07

3.02 t

3.30 k 0.66 3.09 _' 0.28 3.71 & 2.00

22

IN ADULT

Q Values in the table indicate metabolism by supernatant fraction expressed in average micromoles drug ard deviation per gram liver (wet weight) in 2 hours. All groups had 4 experimental and 4 control animals. h P 2 0.05. For all other values P > 0.05.

Chlorpromazine

Aminopyrine

Hexobarbital

Drug substrate

EFFECTS

29

metabolized

-+- stand-

0.63 2 0.31 0.81 k 0.19

--

0.32 c 0.06

0.44 -t 0.10 0.41 iI 0.08 0.46 k 0.12

3.00 T!Y 0.58

~- 3.86 k 1.07 3.43 _' 1.08 3.74 -c 1.48

RAT@

F

i?

? x 2 H

F F *

GIVEN

WHEN

TABLE

c

” O.ZlC

0.34a

METABOLISM

i

0.3OC

6.37 k o.47b 1.07 _c o.zlb 1.61

IN ADULT DOSE+

10 w/kg for 3 days

CONSECUTIVE

of Chlordane

OF THREE

DRUG

RATS

1.40

z!I 0.15

4.78 -+ 0.53 0.43 " 0.04

Control

metabolism by supernatant fraction expressed in average micromoles drug metabolized & stand(wet weight) in 2 hours. Singly dosed animals were injected at the same time that the first of 3 dosed animals. Enzyme activity for all 3 groups was determined the day following the third inanimals. All groups had 6 animals. with control, but P > 0.05 when compared with each other (with the exception of hexobarbital, days, P = O.OS-0.02). with control or with each other.

0.33C

Dose(s)

6.83 k 0.60b

3Ow/k

1.55

t

3 MICROSOMAL

AS THE FIRST

1.55

TEVIE

ON HEPATIC

1.07

-e 0.39*

AT THE SAME

OF CHLORDANE

0.89 r+ o.17b

7.01

10 w/k

DOSES

OF SINGLE

a Values in the table indicate ard deviation per gram liver doses was given to the multiply jection to the multiply dosed 0 P 2 0.05 when compared 10 mg/kg vs. 10 mg/kg for 3 c P > 0.05 when compared

Hexobarbital Aminopyrine Chlorpromazine

Drug substrate

EFFECTS

g 2

z

+I $ 8 i

B

:: 2

E

$ m

E

E s

378

LARRY

G.

HART

ET

AL.

Effects of Single and Multiple Doses of Chlordune Metabolizing Enzyme Activity in Wean&g Rats

on Drug

Peak effects of chlordane on enzyme activity in young rats were quite similar to those obtained with adults. A single dose of 25 mg/kg produced no effect at 1 day, but maximal enhancement of activity was seen by 8 days after the injection. Three doses of 10 mg/kg caused stimulation the day following the last injection (Table 4). Effects of y-Chlordane in Adult Rats

on Drug Metabolizing

Enzyme Activity

The y-isomer of chlordane when given for 3 days, at both a low dose of 10 mg/kg and a high dose of 100 mg/kg, caused a stimulation of drug enzyme activity the day following the last injection of the insecticide. At the larger dose, this increased activity was maintained through 22 days for the metabolism of aminopyrine and through 29 days for the metabolism of hexobarbital (Table 5). In those animals treated with the lower dose, enzyme activity remained elevated for at least 15 days following the last injection (Table 5). Activity was not measured after 15 days. This enhanced enzyme activity was comparable at both doses to that seen in rats pretreated with the technical chlordane for 3 days (Table 2 vs. Table 5). After a single dose of 10 mg/kg of y-chlordane a stimulatory effect was not seen until 15 days, and then only for the metabolism of chlorpromazine (Table 5). This is in contrast to the same dose of technical chlordane where enzyme activity was significantly greater than control at 8 days (Table 1 vs. Table 5). Reversal by m-Ethimine Wean&g Rats

of Stimulatory

Ejects

of Chlordane in

nn-Ethionine has been shown to inhibit increases in microsomal drug metabolizing enzyme activity caused by various compounds (Conney et al., 1956, 1957, 1960; Hart et al., 1962). Increases in the rate of in vitro metabolism of hexobarbital and aminopyrine caused by chlordane in weanling rats were completely blocked by ethionine. At both doses of ethionine used (210 mg/kg and 280 mg/kg), enzyme activity was maintained at levels not significantly different from those in control animals (Table 6).

TABLE

0.34 -c 0.05 0.34 & 0.06

25 w/kg

after

DRUG

0.29 dr 0.08 0.25 2 0.08

I .55 -c o.25b 0.40 I? 0.02

10 mg/kg Control

Aminopyrine

0.28 f 0.17 r

0.020 0.04

2.91 ? 0.56 2.02 c 0.51

0.60 k 0.25 0.30 -c 0.02

metabolized

IN WEANLING

2.21 k 0.53b 1.27 -c 0.26

15

the last injection

METABOLISM

a Values in the table indicate metabolism by supernatant fraction expressed in average micromoles drug ard deviation per gram liver (wet weight) in 2 hours. All groups had 4 experimental and 4 control animals. h P 9 0.05. For all other values P > 0.05.

2.94 I? 0.92 1.99 & 0.41

5.65 k 0.58b 2.12 * 0.30

1.43 -c 0.31b 0.53 k 0.16

5.01 c 0.37b 2.92 -c 0.52

8

Days

MICROSOMAL

10 mg/k Control

Three doses of chlordane

Control

4

Hexobarbital

Aminopyrine

Control

2.84 k 0.28 2.66 2 0.62

1

ON HEPATIC

25 n-&kg

OF CHLORDANE

Hexobarbital

Single

DOSES

of chlordane

THREE

dose

AND

Drug substrate

EFFECTS OF ONE

22

& stand-

0.45 ” 0.06 0.32 31 0.10

3.71 Ik 0.40 2.91 I!I 1.41

0.36 2 0.09 0.29 & 0.06

3.84 k 0.67 3.04 -c 0.61

RAW

$ 2

2

E

g ~ g 0 E z + gs

8 ZJ ;5

AND

10 mg/kg Control

Chlorpromazine

10 w/kg Control

Chlorpromazine

1.21 -t- O.lSh 0.87 -c 0.07

1 .lO 2 0.06’7 0.51 & 0.04 -

8

0.28” 0.08 0.14” 0.04

1.34 2 o.15b 0.98 2 0.19

2 2 2 e

3.56 I+ 0.31 5.24 -r 0.487’ 3.27 IL 0.10

5.96 2 1.22”

0.97 -c 0.14 0.98 IL 0.19

0.41 -r- 0.14 0.35 k 0.08

4.14 -c 0.75 3.56 21 0.31

0.78 0.35 0.82 0.43

5

15

o.22b

0.09” 0.06 O.Ogh 0.11 1.19 * 0.13b 0.80 ?I 0.2 1

-c iz 31 2

5.56 k 0.34 5.40 -c 0.59” 3.40 k 0.87

6.93 31

1.13 k 0.13” 0.80 1+ 0.21

0.62 & 0.07 0.57 k 0.06

0.88 0.57 0.64 0.34

DRUG

-

0.65 2 0.09” 0.35 k 0.13

4.64 21 0.37” 2.73 e 0.62

-

-

-

-

22

METABOLISM

the last injection

4.59 k 0.23 5.56 k 0.34

after

MICROSOMAL

~-

Days

ON HEPATIC

TABLE

u Values in this table indicate metabolism by supernatant fraction expressed in average micromoles drug ard deviation per gram liver (wet weight) in 2 hours. All groups had 4 experimental and 4 control animals. b P 2 0.05. For all other values P > 0.05.

10 mg/kg Control 100 mdkg Control

6.62 k 0.321,

0.79 k 0.12 0.87 t 0.07

0.50 t 0.06 0.51 & 0.04

5.30 t 0.55 5.33 ?I 0.5Ob 2.41 k 0.41

-

1

OF Y-CHLORDANE

4.46 C 0.17 5.30 zk 0.55

DOSES

10 w/k

THREE

Control 100 w/k Control

Aminopyrine

Hexobarbital

10 mg/kg Control

Aminopyrine

Three doses of y-chlordane

10 w/kg Control

Single dose of v-chlordane

OF ONE

Hexobarbital

Drug substrate

EFFECTS

-

-

-

-

29

RAT+

2 stand-

-

0.43 t 0.03 0.34 k 0.06

-

5.09 t 0.172, 2.66 k 0.62

metabolized

IN ADULT

F

z

5

?4

r % z 0

2 10 mg/kg 280 mg/kg

Aminopyrine

BY

6

0.14c

(3)

METABOLISM

(3) (3)

0.30 e o.osc 0.24 5 O.lOc

k

+ ethionine

Chlordane

DRUG

1.59 -c 0.321’ (5) 0.79 c o.16b (5)

1.77

MICROSOMAL

1.98 2~ 0.63C (3)

10 mg/kg

OF HEPATIC

TABLE

4.55 c 1.02b (5) 4.37 -c 0.79 (5)

Chlordane,

CIILORDANE

IN

RATS~

0.42 k 0.12 0.30 k 0.03

(4)

(5)

2.38 in 0.70 (5) 2.70 L 0.52 (4)

Control

WEANLINC

a Values in the table indicate metabolism by supernatant fraction expressed in average micromoles drug metabolized -+ standard deviation per gram liver (wet weight) in 2 hours. Numbers in parentheses represent animals used for each experiment. Animals were treated with ethionine and chlordane for 3 days, enzyme activity being determined the following day. rj P 2 0.05. P indicates the probability that there is no difference between the means of the animals treated with chlordane alone versus animals treated with chlordane plus ethionine or control animals other than that caused by random error in measurement. c P > 0.05. P indicates the probability that there is no difference between the means of the animals treated with chlordane plus ethionine and the control animals other than that caused by random error in measurement.

2 10 mg/kg 280 mp/kg

STIMULATION

Hexobarbital

ON

Dose of ethionine

OF DL-ETHIONINE

Drug substrate

EFFECTS

382 Addition

LARRY

of

G. HART

ET

AL.

ChEordane in Vitro

Chlordane added to the incubation mixtures had no significant effect on the rate of hexobarbital and aminopyrine metabolism when compared with control incubation mixtures where chlordane was not added. Histologic changes from normal were not observed in the liver samples from rats treated with chlordane even at the highest dose used (100 w&4. DISCUSSION

Chlordane, a chlorinated hydrocarbon insecticide, administered acutely by injection of sublethal doses to adult and weanling rats, stimulated hepatic microsomal enzyme activity for the metabolism of certain drugs. There are many drugs and chemicals which have this stimulatory property. However, the duration of stimulatory effects by most of these compounds is relatively short. In most cases, we have found that the enhancement of drug metabolizing enzyme activity is present for only a few days after discontinuing administration of the stimulatory substance. Contrasted to these agents, chlordane, when given as a single dose to either adult or weanling rats, had essentially no immediate effects on enzyme activity, but at 8 or more days post injection there was an increased microsomal drug enzyme activity which was significantly greater than control. No obvious dose-response relationships were found either with single or multiple doses of chlordane. Three consecutive daily doses of chlordane gave an immediate enhancement of enzyme activity (one day after the third injection). This response was usually followed by a more rapid loss of enzyme activity (returning to control levels by 15 days) as compared with response of singly dosed rats. The apparent lag period in effects on the rate of drug metabolism might be explained by the metabolism and/or the physical properties of chlordane. Chlordane and its postulated metabolites are quite insoluble in water and polar solvents, but miscible with aliphatic and aromatic hydrocarbon solvents. Conceivably, these materials may be taken up by adipose tissue and then released slowly into the circulation and thereby exert their effects. Davidow et al. (1951), Davidow and Radomski (1953), and others (U.S. Department of Health, Education, and Welfare, 1956) have shown that chlordane and its metabolites, primarily the epoxide, are

CHLORDANE

ON

DRUG

METABOLISM

IN

RATS

383

stored in the fat and that such storage follows either a single large dose or repeated small doses. These compounds are then eliminated only gradually from the fat stores. Stohlman et al. (1950) have shown that after the administration of chlordane to rabbits the organic chlorides in the urine are increased to a significant extent during the first 24 hours, and that after a single dose of 10 mg/kg the peak is reached on the third or fourth day, small amounts being excreted for several days thereafter. A dose of 100 mg/kg of chlordane would probably be released over a longer time than a dose of 100 mg/kg. This concept would be consistent with the more prolonged stimulation of drug metabolizing enzyme activity by the higher doses of chlordane. With multiple doses, the first dose might be taken up as postulated. By the time the third dose had been administered sufficient amounts of chlordane and/or metabolites from the first dose would have been released from the fat into the circulation and carried presumably to the liver to produce the early stimulatory effect seen one day after the last injection (Table 3). Our results indicate that this hypothesis is consistent with the urinary excretion studies cited previously (Stohlman et al., 1950). When the same time period elapsed before measurement of drug enzyme activity after injection of a single dose or the first of 3 doses of chlordane, there was no difference in the rate of metabolism of hexobarbital and aminopyrine between these singly and multiply dosed animals. Thus it would appear that it is the first dose regardless of its size (10 mg vs. 30 mg) which is causing most of the stimulatory effect seen 3 days after the first injection and that a lag period is required for chlordane or its metabolites to be taken up by the fat and then released. It has been shown by Davidow and Radomski (1953) that heptachlor, one of the components of the technical chlordane mixture, is rapidly metabolized to an epoxide. This metabolite is quite stable and because of its lipid solubility is stored in the fat. When technical chlordane was fed to rats, analysis of the fat showed a colored reaction product whose absorption spectrum was different from that of the technical chlordane (Davidow, 1950). Furthermore, if the individual components of technical chlordane, including y- and l3-chlordane and heptachlor, were fed to rats, the infrared absorption spectra of all the metabolites found in the fat appeared identical (Davidow et al., 1951). Thus if the stimulatory effect of chlordane is produced by its metabolite(s) , the time required for metabolism, storage in the fat and release from the fat of the metabo-

384

LARRY

G. HART

ET

AL.

lite(s) would be a further explanation for the lag period between administration of the compound and stimulation of drug enzyme activity. The mechanism by which chlordane acts to produce its stimulatory effect is not understood. An amino acid antagonist, Dr.-ethionine, can block the increases in enzyme activity caused by chlordane. Enzyme activity in the chlordane treated animal is depressed by concurrent administration of ethionine to a level which is not significantly different from control values. Thus it would appear that ethionine exerts its inhibitory effects predominantly on the chlordane-induced enzyme activity. Ethionine is thought to act by interfering with synthesis of a peptide or protein. This protein could be an enzyme activator, a part of the system synthesizing the drug enzymes, a part of a system antagonizing the degradation or inhibition of the drug enzymes, or the drug enzymes themselves. Chlordane or its metabolites may act by directly or indirectly stimulating synthesis of this peptide or protein. However, there is little evidence available at this time to allow a choice between these alternatives. It is known that chlordane has no effect on drug enzyme activity when added in vitro. To produce increases in these enzyme activities, the chlordane must be administered to the animal. Other routes of administration of chlordane and related compounds might be studied for their effects on the rate of drug metabolism. When higher animals and man are exposed to insecticides most of that absorbed is either by inhalation or through the skin. We used the intraperitoneal route primarily because a more rigid control of dosage could be maintained by this route. The pure y-isomer of chlordane was shown to be active in stimulating drug enzyme activity. We have no information at this time whether all the stimulation by technical chlordane is due only to this y-isomer. It is possible that some of the stimulatory effects may be caused by other components such as heptachlor. Further investigation is planned in this area. SUMMARY Technical chlordane, a halogenated hydrocarbon insecticide, isomers, y-chlordane, were shown to have stimulatory effects enzyme activity for the metabolism of certain drugs.

and one on hepatic

of its pure microsomal

Adult and weanling rats were injected intraperitoneally with both a single dose and three consecutive daily doses of chlordane at dose levels of 10, 25, and lOOmg/kg. Drug enzyme activity was determined on livers from these rats at 1, 8, 15, 22, and 29 days after the last injection.

CHLORDANE

m-Ethionine control levels.

inhibited

ON

the

DRUG

increases

METABOLISM

in

enzyme

It was postulated that the latent and prolonged its metabolites were due to its storage in body

IN

activity,

keeping

stimulatory effects fat and subsequent

Chlordane is stimulatory to drug enzyme activity animal. Addition of chlordane to liver homogenates in vitro does not affect drug enzyme activity.

385

RATS

only when or particulate

this

activity

at

of chlordane or gradual release.

administered to the fractions incubated

ACKNOWLEDGMENTS We wish to acknowledge the help of Dr. J. W. Smith, Department of Pathology, State University of Iowa, in histologic examinations of hepatic tissue; and Mrs. Donna Goodwin and Mrs. Anne Trankle, Department of Pharmacology, State University of Iowa, in various assays. We also wish to thank the Velsicol Chemical Corporation for generously contributing the y-chlordane used in our studies. REFERENCES B. B., and AXELROD, J. (1950). The fate of aminopyrine (Pyramidon) in man and methods for the estimation of aminopyrine and its metabolites in biological materials. J. Pharmacol. Exptl. Therap. 99, 171-184. CONNEY, A. H., MILLER, E. C., and MILLER, J. A. (1956). The metabolism of methylated aminoazo dyes. V. Evidence for induction of enzyme synthesis in the rat by 3-methylcholanthrene. Cancer Res. 16, 450-459. CONNEY, A. H., MILLER, E. C., and MILLER, J. A. (1957). Substrate-induced synthesis and other properties of benzpyrene hydroxylase in rat liver. J. Biol. Chem. 223, 753-766. CONNEY, A. H., DAVISON, C., GASTEL, R., and BURNS, J. J. (1960). Adaptive increases in drug-metabolizing enzymes induced by phenobarbital and other drugs. J. Pharmacol. Exptl. Therap. 130, 1-8. B. B. (1955). The enzymatic metabolism of hexoCOOPER, J. R., and BRODIE, barbital (Evipal). J. Pharmacol. Exptl. Therap. 114, 409-417. DAVIDOW, B. (1950). A spectrophotometric method for the quantitative estimation of technical chlordane. J. Assoc. Ofic. Agr. Chemists 39, 886-894. DAVIDOW, B., and RADOMSKI, J. L. (1953). The metabolite of heptachlor, its estimation, storage, and toxicity. J. Pharmacol. Exptl. Therap. 107, 266-272. DAVIDOW, B., HAGAN, E. C., and RADOMSKI, J. L. (1951). A metabolite of chlordane in tissues of animals. Federation Proc. 10, 291. DIXON, R. L., SCHULTICE, R. W., and FOUTS, J. R. (1960). Factors affecting drug metabolism by liver microsomes. IV. Starvation. Proc. Sot. Exptl. Biol. Med. 103, 333-335. HART, L. G., ADAMSON, R. H., DIXON, R. L., and FOUTS, J. R. (1962). Stimulation of hepatic microsomal drug metabolism in the newborn and fetal rabbit. J. Pharmacol. Exptl. Thevap. 137, 103-106. MCLUEN, E. F., and FOUTS, J. R. (1961). The effect of obstructive jaundice on drug metabolism in rabbits. 1. Pharmacol. Exptl. Therap. 131, 7-11. BRODIE,

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W. 0. (1959). Handbook of Toxicology, Vol. III, Insecticides, A Compendium. Wright Air Development Center Technical Report 55-16, Wright-Patterson Air Force Base, Ohio. SALZMAN, N. P., and BRODIE, B. B. (1956). Physiological disposition and fate of chlorpromazine and a method for its estimation in biological material. J. Pharmacol. Exptl. Therap. 118, 46-54. SNEDECOR, G. W. (1956). Statistical Methods, 5th ed. Iowa State College Press, Ames, Iowa. STOHLMAN, E. F., THORP, W. T. S., and SMITH, M. I. (1950). Toxic action of chlordan. Arch. Ind. Hyg. Occup. Med. 1, 13-19. U.S. Department of Health, Education, and Welfare. (1956). Clinical Memoranda on Economic Poisons, Vol. V. U.S. Government Printing Office, Washington, DC. VON OETTINGEN, W. F. (1955). The halogenated hydrocarbons, their toxicity and potential dangers. Public Health Serv. Publ. No. 414. NEGHERBON,