Mechanism of protection against carbon tetrachloride by prior carbon tetrachloride administration

EXPERIMENTAL

AND

Mechanism

MOLECULAR

16, 281-285 (1972)

of Protection Against Prior Carbon Tetrachloride

GIANCARLO Department

PATHOLOGY

Carbon Tetrachloride Administration’

CGAZIO,~ ROBERT R. KOCH, AND RICHARD

of Physiology,

School of Medicine, Cleveland, Ohio

Case &lOS

Received

14, 1971

October

Western

by

0. RECKNAGEL Reserve

University,

A small dose of carbon tetrachloride, equal to $0 of an LDss, administered intragastrically to rats, confers a remarkable protection against subsequently administered and ordinarily lethal doses of the same liver poison. Protection sets in after 12 hours. It is fully developed by 24 hours and lasts for 3 days, after which it gradually fades. The development of tolerance is due to the capacity of the small initial dose of carbon tetrachloride to depress the liver microsomal drug-metabolizing system. Forty-eight hours posttreatment with a small, protective dose of carbon tetrachloride, hexobarbital sleeping time is increased more than 3-fold, and conversion of WCl, to exhaled “CO2 is depressed to half the control rate.

Within 24 hours postadministration of small doses of carbon tetrachloride, mice were completely resistant to double the Lng5 of phalloidin (Floersheim, ‘1966). Phalloidin is a toxic principle from the deadly white mushroom, Amanita phalloides. The protection phenomenon is not limited to phalloidin. Experimental work reported here shows that administration of a small dose of carbon tetrachloride protects rats from large and ordinarily lethal doses of this haloalkane. While our work was in progress, another study appeared (Dambrauskas and Cornish, 1970) which reported that after exposure of rats to carbon tetrachloride there was marked increase in resistance to a subsequently administered dose of this toxic agent. Results of in viva experiments presented here indicate that protection is due to suppression of liver microsomal mixed-function oxidase activity. A preliminary report of this work has been published (Ugazio et al., ‘1971). MATERIALS

AND METHODS

Male rats of the Sprague-Dawley strain were used. The small initial dose of carbon tetrachloride, referred to as the protective dose, was 25 ~1 CC14/100 gm rat body weight. The protective dose was administered intragastrically in a volume of mineral oil equivalent to 0.5 ml/100 gm body weight. Rats serving as 1 This work was supported by Grant AM-01489 from the National Institute of Arthritis and Metabolic Diseases, and by Training Grant 5 TO1 GM 00899 from the National Institutes of Health, USPHS. a Present address: Istituto di Patologia Generale, Facolta di Scienze. Universita di Sassari, Viale Mancini 5,07100, Sassari, Italy. 281 Copyright

@ 1972 by Academic

Press,

Inc.

282

UGAZIO,

KOCH,

AND

RECKNAGEL

controls received only mineral oil. At various times after administration of either mineral oil or the protective dose of carbon tetrachloride, the animals were challenged with a much larger dose of carbon tetrachloride. The challenging dose was again administered intragastrically in mineral oil. In all cases, the animals were under light ether anesthesia for intragastric intubation. Animals were fasted overnight before receiving the protective dose of carbon tetrachloride, or pure mineral oil, as the situation required. Animals were maintained in the fasting condition when the time interval between administration of the protective and challenging doseswas 12 hours or less. For longer time intervals, food was offered 6 hours postadministration of the protective dose, but was again withdrawn for at least 6 hours before administration of the challenging dose. In all cases, food was offered ad libitum 6 hours postadministration of the challenging dose; water was available ad Zibitum throughout the experiment. For measurement of hexobarbital sleeping time, the rats were given an ip injection of 12 mg hexobarbital sodium in saline/100 gm body weight. After onset of anesthesia, the rats were placed in the supine position. Sleeping time was taken as the time interval from the moment of administration of the hexobarbital until the moment of awakening. The latter was recorded as the moment when the rat assumedthe prone position. Measurement of conversion of 14CC14to l*COz in expired air was carried out as follows. A rat was given an intragastric dose of 500 ~1 14CC14/100 gm body weight. Radioactivity administered was 6.503 X 10” dpm/lOO gm body weight. The rat was placed in a sealed metabolism container improvised from a large glass desiccator. Filtered air was passedthrough a water tower, and then into the metabolism container, under a pressure head of 5 cm water. Air was withdrawn through a tube situated in the bottom of the metabolism container. Air leaving the system was first passedthrough a toluene tower; the latter was immersed in an ice-bath. This step removes expired 14CC14. From the toluene tower the air was passed through 2 N NaOH in a small glass vial suitable for radioactivity determination. This step traps expired 14C02. The 14C03 was precipitated as Baz 14C03, washed twice with 5 ml methyl alcohol, and finally suspended in 1 ml methyl alcohol. The scintillation vial was filled with thixotropic gel powder (Packard, Cab-0-Sil), after which was added 10 ml of a toluene scintillant mixture. Each liter of the toluene scintillant mixture contained 4 gm of 2,5-diphenyloxazole (Packard, PPO) and 50 mg 1,4-b&2- (5-phenyloxazolyl) -benzene (Packard, POPOP) . Radioactivity was determined in a Packard Scintillation Spectrometer (Series 2000, Model 2211). RESULTS For the particular conditions used in these experiments, 400 ~1 CC14/100 gm ‘body weight is an LD?~; 500 ~1 is an LDQ~ (Table 1). Up to 6 hours postadministration of the protective dose of carbon tetrachloride, there is no protection against an LD?~; by 12 hours there is significant protection. Protection against an Lng5is complete by 24 hours and remains so for 3 days, after which the protection gradually wears off (Table 1).

Ccl,

PROTECTION

AGAINST

233

CClr

Hexobarbital sleeping time was greatly prolonged 48 hours postadministration of a protective dose of carbon tetrachloride (Table 2). Three rats were each given a protective dose of 25 ~1 CCla/lOO gm body weight. After 48 hours, these three rats were given 500 ~1 of “Ccl,, intragastritally, per 100 gm body weight. Conversion of 1%C14 to exhaled 14C@, was followed for 6 hours, and compared to the rate of conversion in three rats not given the protective dose. Administration of the protective dose markedly diminished the capacity of the rats to convert l”CC14 to 14C02 (Table 3). DISCUSSION The experimental work reported in this communication confirms and extends the report of Dambrauskas and Cornish (1970). The latter showed that after exposure of rats to carbon tetrachloride vapors, the animals became resistant to a TABLE EFFECT

OF -4 SM.~LL

DOSE OF CC& CHALLENGING

Challenging dose of CC14 (&‘lOO gm body weight)

Time intervala (hours or days) 1 3 6 12 1 1 2 3 5 7 7 No No

protective protective

I

ON SURVIVAL OF RATS DOSB OF CC14

AFTER

Number Number

400 400 400 400 400 500 500 500 500 500 400 400* 500*

hour hour hour hour day day days days days days days dose given dose given

A SECOND,

surviving challenged O/IO l/10 o/10 6/10 16/16 g/g lO/lO g/g 7/g 4/9 415 8/26 3/46

a The time interval is the elapsed time after the protective dose when challenging dose was given. 5 For unprotected rats, under these condit.ions, 400 ~1 of Ccl4 is about LD~o; 500 pl is an LDgg. TABLE HEXOBARBITAL

SLEEPING

TIME

PROTECTIVE Treatment Mineral

oil only

25 pl CC14/100

II

OF RATS 48 HOURS DOSE OF CC14 Number of rats 10

gm body weight

10

AFTER

-4 SMALL

Sleeping time (minutes f SE) 50.3

f

175 f

5.3 15.0

the an

284

UGAZIO,

KOCH,

AND

TABLE

RECKNAGEL III

CO2 48 HOURS POSTADMINISTRATION TO RATS OF A SMALL PROTECTIVE DOSE OF Ccl&

CONVERSION

OF CCll

Time postadministration of 500 pl of XC14 (hours) 1 2 3 4 6

TO EXHALED

Unprotected rats n=3 (pg CC$cc.;Verted 0 73.2 158.9 213.2 250.1 277.1

f f f f f

12.3 25.3 30.8 33.4 33.9

Protected rats 12=3 (pg Ccl4 converted to CO?) 30.0 69.6 106.8 128.9 148.9

f 7.3 f 14.9 f 20.7 xt 26.2 f 27.2

normally lethal exposure to carbon tetrachloride inhalation. Dambrauskas and Cornish (1970) found that an oral dose of 200 ~1 CCl&OO gm body weight also resulted in development of tolerance when the animals were tested after 48 hours. The data of our Table 1 show that tolerance to a large dose of carbon tetrachloride can be developed with a very small, intragastric dose of the same haloalkane. There is no protection for the first 6 hours; protection is partial by 12 hours and fully manifested by 24 hours. It is complete for 3 days, after which susceptibility gradually reappears. One significant aspect of our study is that the time course of development of protection is more clearly etched when the protective dose of carbon tetrachloride is given as a single intragastric dose rather than via inhalation for 6 hours. Dambrauskas and Cornish (1970) found that in rats protected by prior exposure to carbon tetraehloride vapors, conversion to CHC& of subsequently administered Ccl4 was significantly less than in otherwise normal rats. They regarded the above finding as consistent with a large body of evidence (Recknagel, 1967; Recknagel and Glende, in press; Slater, 1966) which indicates that carbon tetrachloride toxicity depends in some way on carbon tetrachloride metabolism. The data of our Table 3 show that 48 hours after receiving a small dose of carbon tetrachloride, conversion of 14CC14to 14C02in expired air was reduced to half the rate of conversion as measured in unprotected rats. These data indicate that an effective level of carbon tetrachloride metabolism is intimately related to carbon tetrachloride lethality. In a series of papers (Alpers and Isselbacher, 1967; Alpers and Isselbacher, 1968; Alpers et al., 1968) data were presented which led the authors to suggest that some of the toxic effects of carbon tetrachloride may not be related to carbon tetrachloride metabolism or attendant lipid peroxidation. A critical analysis of this work has been made (Recknagel and Glende, in press). The study reported here has an important bearing on this question. From the data presented in Table 1 it is clear that a normally lethal dose of carbon tetrachloride has been rendered nonlethal by the simple expedient of administration of l/20 of an ~~~~ 24 hours previously. If a solvent action, or some other action not involving carbon tetrachloride metabolism were a significant feature of carbon tetrachloride lethality, then one might expect that an LDQ~ of carbon tetrachloride would kill at least some of the rats. In actual fact, the rats are completely resistant.

CClr

PROTECTION

AGAINST

285

CC14

This argues strongly against any significant role for a nonchemical action of carbon tetrachloride in the mechanism of carbon tetrachloride lethality. The greatly prolonged hexobarbital sleeping time (Table 2) 48 hours p&administration of the protective dose reveals the mechanism of the pro:ection. Hexobarbital is metabolized by the liver microsomal mixed-function oxidase system. Prolongation of the hexobarbital sleeping time indicates that ackivity of this drug-metabolizing system has been depressed. The liver microsomal mixedfunction oxidase system is also the locus of the enzymic apparatus involved in the metabolism of carbon tetrachloride (Rubinstein and Panics, 1964) and in its in vitro prooxidant action (Glende and Recknagel, 1969). Clearly, loss of the power of carbon tetrachloride to kill the rat is coincident with depression of the enzymic system necessary for carbon tetrachloride metabolism. Direct in 2,&o assay for components of the liver drug-metabolizing enzyme system indicates (Glende, unpublished) that the onset of carbon tetrachloride tolerance is coextensive in time with depression of liver microsomal drug-metabolizing activity. As liver mixed-function oxidase activity is regained, the rats again become susceptible to the lethal effects of this liver poison. The protection phenomenon as reported here is significant for another reason ; it provides a simple and highly useful model system for further investigative work. The protective dose of carbon tetrachloride, although only l/20 of an LDg5, appears to have a remarkably localized and large depressant effect on liver microsomal drug-metabolizing activity. Rats given the protective dose show no overt signs of toxicity. These rats should provide new opportunities for study of the cellular dynamics of toxic injury and repair (Recknagel et al., in press). REFERENCES ALPERS,

liver

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Ii.

J. AC~U

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The

effect

of

carbon

tetrachloride

on

rat

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