Murine acetaminophen hepatotoxicity: Temporal interanimal variability in plasma glutamic-pyruvic transaminase profiles and relation to in vivo chemical covalent binding

FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

7, 17-25 (1986)

Murine Acetaminophen Hepatotoxicity: Temporal Interanimal Variability in Plasma Glutamic-Pyruvic Transaminase Profiles and Relation to in Vivo Chemical Covalent Binding’ PETER G. WELLS*

Facultyof Pharmacy,

University

of Toronto,

AND ESTHER C. A. To3 19 Russell

Street,

Toronto,

Ontario,

Canada

M5S

IA1

Murine Acetaminophen Hepatotoxicity: Temporal Intcranimal Variability in Plasma GlutamicPyruvic Transaminase Profiles and Relation to in Vivo Chemical Covalent Binding. WELLS, P. G., AND TO, E. C. A. (1986). Fundam. Appl. Toxicol. 7, 17-25. The hepatotoxicity of acetaminophen is thought to be dependent upon its enzymatic bioactivation to a reactive intermediary metabolite which binds covalently to essential cellular macromolecules, thereby causing cellular death. Traditional in vivo methods using smaller mammals are mechanistically restrictive in that measures of hepatotoxicity, such as plasma glutamic-pyruvic transaminase (GPT), and chemical covalent binding to hepatocellular protein are performed at different times in separate groups of animals. We developed a microanalytical technique which allowed repetitive plasma GPT sampling from individual mice, followed by delayed determination of covalent binding in the same mouse 36 hr after acetaminophen administration. Diethyl ether anesthesia was used to enhance acetaminophen hepatotoxicity. The repetitive sampling technique permitted an accurate determination of the peak GPT concentration, which exhibited a marked interanimal variability in the time of occurrence. Individual peak GPT concentrations correlated with the respective covalent binding of acetaminophen in each mouse (r = 0.82, p < 0.05), while the traditional method using a fixed sampling time (24 hr) failed to correlate (r = 0.50, p > 0.05). Ether produced a 39-fold enhancement in the severity of acetaminophen hepatotoxicity; however, a single, fixed-time sample taken at either 12 or 24 hr produced a substantial and inconsistent over- or underestimate of this toxicologic enhancement. This study shows that chemical hepatotoxicity as reflected by plasma GPT concentration cannot be quantified accurately by a single blood sample obtained from a given animal, regardless of the chosen sampling time. An accurate determination of the peak plasma GPT concentration in any single animal requires repetitive blood sampling, preferably in the absence of general anesthesia. 0 1986 Society of Toxicology.

Acetaminophen (Tylenol) is a widely usedanalgesic/antipyretic drug. When taken in large doses,acetaminophen causeshepatic necrosis in humans (Davidson and Eastman, 1966; Proudfoot and Wright, 1970) and in animals (Boyd and Bereczky, 1966; Mitchell et al.,

1973a), presumably via the covalent binding of its electrophilic, reactive intermediary metabolite to nucleophilic sites on essential hepatocellular macromolecules (Jollow et al., 1973; Potter et al., 1973). Saturable, enzymatically catalyzed conjugations of acetaminophen with glucuronic acid and sulfate prevent the formation of the toxic reactive intermediate (Fig. 1) and account respectively for about 60 and 30% of acetaminophen elimination. A small fraction (5- 10%) of acetaminophen is bioactivated by hepatic cytochromes P-450 to the toxic reactive intermediate (Potter et al., 1973), which normally is detoxified by

’ A preliminary report of this work was presented at the annual meeting of the Canadian Federation of Biological Societies (Proc. Canad. Fed. Biol. Sot. 28, 228, 1985). Supported by grants from the Atkinson Foundation, the Banting Research Foundation, the Connaught Fund, and the Medical Research Council of Canada. 2 To whom correspondence should be addressed. 3 Recipient of an Ontario Graduate Scholarship.

17

0272-0590186 $3.00 Copyright 0 1986 by the Society of Toxicology All rights of reproduction in any form reserved.

18

hlCUROlllOE

WELLS AND TO

Tra”SwraSB bH ACETAMl3OPHEW

LULFITE

REACTIVE IITERMEUIATE CELLULAR WACROMOLECULES

-i

If \HCCH,

MACROMOLECULE OH

CELLULAR NECROSIS

FIG. 1. Postulated relation of acetaminophen biotransformation to hepatotoxicity. Over 60% of acetaminophen is eliminated via enzymatic conjugation with glucuronic acid, producing a nontoxic, water-soluble metabolite. A small amount (5-10%) is bioactivated by the hepatic cytochromes P-450 to a reactive intermediary metabolite which, if not detoxified by conjugation with glutathione, can bind covalently to essential hepatocellular macromolecules, thereby initiating a process leading to cellular necrosis (see text).

enzymatically mediated conjugation with hepatic reduced glutathione (GSH) (Mitchell et al.. 1973b). While much is known about the toxicology of acetaminophen, its use as a biochemical probe for in vivo toxicological studies raises questions, particularly when small animals such as the mouse are used. In the direct assessment of hepatocellular alterations and necrosis produced by acetaminophen, histological examination employing light and electron microscopy requires sacrificing the animal. Thus, time-response studies inevitably must involve separate groups of animals sacrificed at different times. One indirect assessment of

hepatotoxicity involves quantification of the amount of intracellular glutamic-pyruvic transaminase enzyme (GPT, alanine aminotransferase) released into the blood by dying hepatocytes. In smaller rodents such as the mouse, blood sampling for GPT often involves sacrificing the animal and taking the entire available blood volume via cardiac puncture to obtain volumes adequate for standard GPT assays. As with histological techniques, for time-response studies this method requires either sacrificing groups of animals at different times or assuming that a single time sample can accurately approximate the peak GPT concentration for all treatment groups. The former method increases both the variability of concurrent measurements for chemical disposition and tissue response and the number of animals required; more importantly, this method precludes an integrated study of longitudinally related parameters in the same animal. The assumption in the latter method of a constant timing for the occurrence of peak GPT concentrations may not always be correct, as shown in the study reported herein. In larger rodents such as the rat, milliliter volumes of blood can be obtained via cardiac puncture under general anesthesia without killing the animal. While not commonly employed for GPT determinations, this method would permit repetitive toxicologic assessment in larger rodents; however, the use of anesthesia in in vivo studies creates its own interpretive problems, as shown in this study. Mechanistic studies involving the hepatotoxicity of acetaminophen generally include quantification of its covalent binding to hepatocellular proteins. Traditionally, such measurements are made within 6 hr of administering radiolabeled acetaminophen, when covalent binding is highest. However, toxicological tissue response generally is more delayed, and plasma GPT concentrations in mice may not peak until 24 hr after acetaminophen administration (Wells et al., 1980). This means that covalent binding and GPT concentrations must be measured in different groups of animals, thereby precluding direct

GPT RELATION

TO COVALENT

examination of the relation between tissue response and the covalent binding of acetaminophen in the same animal. The objectives of this study were to develop a method for the accurate determination of the peak plasma concentration of GPT in individual mice and to examine the relation of this index of acetaminophen hepatotoxicity to the covalent binding of acetaminophen to hepatocellular protein in the same animal. This involved the development of a microanalytical technique to measure plasma GPT concentrations repetitively in a single mouse, combined with a delay in measuring the covalent binding of acetaminophen in each animal until after the peak plasma GPT concentration was achieved. METHODS Animak Male CD-I mice4 weighing 20-25 g were housed in plastic cages using ground corn cob bedding5 with a 12-hr dark cycle automatically maintained. The mice were allowed to acclimatize for 1 week prior to treatment. Food6 and tap water were provided ad libitum. Treatments. To enhance the hepatotoxicity of acetaminophen, diethyl ether (ether)’ was administered for 5 min as a general anesthetic to mice housed in an inhalation chamber, with anesthetic efficacy monitored by abolition of the righting reflex. Following recovery from anesthesia, animals were transferred to their original cages and treated 6 hr later with acetaminophen (N-acetyl-paminophenol), 300 mg/kg, ip. The 6-hr time interval between administration of ether and acetaminophen had been shown previously to produce the maximal toxicological enhancement (Wells et al., 1986). Solutions were prepared immediately prior to use by dissolution of acetaminophen in normal saline adjusted to pH 9.5 with 5 N NaOH. The solutions were prepared to deliver the appropriate dose in a volume of 0.0 1 ml/g body wt. Toxicology. Acetaminopben-induced hepatotoxicity was assessedby measuring the amount of intracellular glutamic-pyruvic transaminase enzyme released into the blood by injured liver cells. Plasma concentrations of GPT are a Charles River Canada Inc., St. Constant, Quebec, Canada. s Northeastern Products Corp., Warrensburg, N.Y. 6 Purina Mouse Chow, Woodlyn Laboratories Ltd., Guelph, Ontario, Canada. ’ Baker Chemical Co., Phillipsburg, NJ. * Sigma Chemical Co., St. Louis, MO.

BINDING

19

both, in general, a sensitive and discriminating indicator of hepatocellular injury (Adolph and Lorenz, 1982) and, in particular, a hallmark and quantitative index of acetaminophen hepatotoxicity (Rumack and Matthew, 1975; Ameer and Greenblatt, 1977; Black, 1980, 1984), with concentrations over 1000 [U/liter being predictive of severe hepatic damage in humans (Prescott et al., 1976). Using heparinized microcapillary tubes,g 50-~1 blood samples were obtained repetitively from the tail vein of each mouse at 6, 12, 24, and 36 hr following administration of acetaminophen. Plasma was separated by centrifugation at 1OOOgfor 15 min at 4”C, stored at 4”C, and analyzed within 24 hr. Under these conditions, GPf activity declines less than 5% (Adolph and Lorenz, 1982). GPf concentrations in plasma were determined with a double-beam spectrophotometer” by a calorimetric method (Reitman and Frankel, 1957) using a standardized assaykit.’ Quantities of reagents were reduced in accordance with the decreased volume of plasma samples. Standard GPT curves generated for each assay were virtually superimposable, and analysis of Sigma controls for normal and elevated GPT concentrations were similarly accurate and reproducible. Preliminary experiments were conducted to assure an equivalence of our microanalytical GPT technique with the standard method employing larger plasma volumes obtained via cardiac puncture. In these studies only, 15 animals were treated ip with between 200 and 700 mg/kg of acetaminophen, and a 50-~1 blood sample was obtained 24 hr later via the tail vein. Immediately thereafter, animals were killed by cervical dislocation and a blood sample was obtained via cardiac puncture. The first study was to assess whether a SO-~1 volume of blood obtained via cardiac puncture gave the same plasma GPT value as a 200~~1 volume (recommended volume for Sigma kit) obtained from the same sample. The second study assessedwhether a 50-~1 blood sample obtained from the tail vein gave the same plasma GPT concentration as an equal volume of blood obtained via cardiac puncture at the same time. The repetitive microsampling technique was employed in all subsequent studies. In the first study, the plasma GPT profile following acetaminophen administration was characterized as described above, with and without ether pretreatment, using six animals per group. In the second study, the same design was employed with the addition of radiolabeled acetaminophen to permit the measurement of covalently bound drug. An initial study was conducted using animals treated with ether and acetaminophen (n = 4) followed by a complete study of animals treated with acetaminophen alone or in combination with ether, using eight animals per group. Covalent binding. To allow simultaneous quantification of covalent binding, radiolabeled acetaminophen (p[G, 9 Microcaps, Drummond Scientific Co., Bloomall, Pa. lo Mcdel Lambda 3, Perkin-Elmer Canada Ltd., Toronto, Ontario, Canada.

20

WELLS AND TO

‘Hlhydroxyacetanilide, sp act = 1.8 Ci/mmol),” 2 &i/g body wt, was administered in solution with the unlabeled acetaminophen 300 mg/kg, ip. Animals were sacrificed by cervical dislocation 36 hr following the administration of acetaminophen, after obtaining a final blood sample. Covalent binding to hepatocellular protein was determined using exhaustive washing with hot methanol as previously ‘described (Wells et al.,1980). Data are expressed as the binding of radiolabeled acetaminophen resulting from a radiolabeled acetaminophen dose of 0.117 mg/kg. To obtain the equivalent values for total binding (radiolabeled plus unlabeled acetaminophen), the radiolabeled binding (data presented in Table 1 and Fig. 5 should be multiplied 3y a factor of 2565. Statistical analysis. Statistical comparisons of differIencesand correlations between groups were determined Ising a standard, computerized statistical program’* modlfied for microcomputers (SPSS-PC). Multiple comparisons among groups were determined by analysis of variance ;ollowed by a range test, while paired data were analyzed by the Student t test.

RESULTS There was no difference in plasma GPT concentrations derived from 50- and 200-p] blood volumes obtained via cardiac puncture (data not shown). Similarly, plasma GPT measurements derived from a 50-p] blood sample obtained via the tail vein were identical to those obtained via cardiac puncture in the same animal at the same time (Fig. 2). The temporal plasma GPT profiles for the individual mice demonstrated qualitatively a l.oxicologic enhancement for animals treated with ether plus acetaminophen compared with t.hose receiving acetaminophen alone (Fig. 3). In untreated control animals, the plasma GPT concentration was 41 + 6 IU/liter (X + SD), and this concentration was not increased at any time after treatment with ether alone. A number of important observations are evident. First, the peak plasma GPT concentrations were shifted from 6 or 12 hr in the acetaminophen control group to 12 or 24 hr in the group receiving both ether and acetaminophen (intergroup variability) (Fig. 3). Second, it was particularly evident in the group receiving I1 New England Nuclear, Lachine. Quebec, Canada. ‘* SPSS Inc., Chicago, Ill.

100

1000

10.000

1 0,000

TAIL VEIN

Iblasma 6PT,lUiLl FIG. 2. Relation of plasma glutamic-pyruvic transaminase (GPT) concentrations obtained via tail vein sampling and via cardiac puncture. A 50-J blood sample from male CD-l mice treated with acetaminophen, 200 to 700 mg/ kg, ip, was obtained via each route in the same animal in the absence of general anesthesia (see Methods). All blood samples were obtained 24 hr after acetaminophen administration. The solid line represents equivalence.

combined treatment that there was a substantial discrepancy among the different animals in the time of occurrence of peak plasma GPT concentrations (intragroup variability). For example, for one animal, there was a twofold decrease in GPT going from 12 to 24 hr, while for another animal, there was a twofold increase (Fig. 3). Third, for the marginally toxic dose of acetaminophen alone, GPT concentrations peaked at 6 or 12 hr and approached normal levels by 24 hr, while with enhancement of acetaminophen hepatotoxicity by ether, the GPT profile was much less predictable, and generally was elevated in a more prolonged fashion. The mean data from the individual profiles in Fig. 3 allow a quantitative assessment of the enchancement of acetaminophen hepatotoxicity by ether (Fig. 4). The importance of repetitive sampling from each individual animal can be seen by comparing mean data by time (Fig. 4A) with the mean of peak GPT concentrations for each individual animal, regardless of their time of occurrence (Fig. 4B). While the timing for occurrence of mean peak

GPT RELATION

TO COVALENT

21

BINDING THER & ACETAMlNOPHEN

I

*

0

1

12

1

I

24

.

*

I

36

TIME ( hr 1

FIG. 3. Temporal plasma GPT profiles in individual male CD-1 mice treated ip with a 300 mg/kg dose of acetaminophen alone or 6 hr following 5 min general anesthesia with diethyl ether (ether). Ether atone had no effect on plasma GPT concentrations.

GPT concentrations in the combined treatment group compared to the respective control group did coincide in this experiment (Fig. 4A), it did not coincide in a subsequent experiment which included covalent binding studies (Table 1). In the latter experiment, for the group given ether plus acetaminophen, the true mean peak GPT concentration was underestimated by 30% (p = 0.059) using the 24hr sample (Table l), and the respective 24-hr GPT value for animals given acetaminophen 2 10.000

6 2 E 3 !: s

A

1000:

2

*

10.000

+

T

i l

B

1000 :

Et & APAP

“‘\,‘“”

100 :

Li 0 i

l

100 :

1oc

lo0

12

24

36

APAP

Et & APAP

TIME ( hr 1

FIG. 4. (A) Temporal profile of mean plasma GPT concentrations for mate CD- 1 mice calculated from data given in Fig. 3. Animals were treated with acetaminophen (APAP) alone or 6 hr after ether (Et), as described in Fig. 3. (B) Mean values of the individual peak plasma GPT concentrations irrespective of temporal occurrence. Data points and bars represent the mean GPT value & SE for 6 animals. Asterisks indicate a significant difference from the acetaminophen control (p < 0.05).

alone was similarly inaccurate. Therefore, depending upon the relative inaccuracies of GPT values for groups receiving combined treatment versus acetaminophen alone sampled at the same time, a study design choosing a single sampling time of either 12 or 24 hr could make a substantial under- or overestimate of the toxicologic enhancement. For example, if the peak plasma GPT concentration was chosen for each individual animal (Fig. 4B), the mean of these data demonstrated an accurate and significant 36-fold enhancement of acetaminophen hepatotoxicity by ether. In a replicate experiment, the accurate enhancement as determined by individual peak plasma GPT data was 39-fold (Table 1); however, the 24-hr sample produced an underestimated 32-fold enhancement, and the 12-hr sample produced a remarkably overestimated enhancement of 89-fold. A similar observation was evident with regard to the dependence of plasma GPT concentration on the covalent binding of acetaminophen to hepatocellular protein remaining at 36 hr after drug administration. When the peak plasma GPT concentration for each animal was chosen (Table l), there was a significant correlation between covalent binding and plasma GPT data (r = 0.82, p < 0.05). A similar correlation was obtained in a replicate experiment (r = 0.91, n = 4), and the com-

22

WELLS AND TO TABLE 1 Emc-r OFSAMPLINGTIMEONTHERELATIONOFPLASMAGPT CONCENTRATION TOCOVALENTBINDINGOFRADIOLABELEDACETAMINOPHENIN VIVO” Plasma GPT concentration (IU/liter) Treatment b

l2-hr sample

Acetaminophen

24hr sample

21 27 9 33

6 30 57 36

XT SE Ether plus acetaminophen

27~~

16

Correlation’

2411 t- 1811*

2.43 2.32 1.72 1.21 2.19

111

111 142

2.36

28

76

2550

r = 0.79, p < 0.05

r = 0.50, p > 0.05

38

1.97 + 0.42 3.64 5.51 4.56 3.64 9.8 1 6.69 10.70 12.86

2250 2400 2400 2850 3150 6300

1350 1350 k 46F

2

1.71

1950 2100

1950 2100 2250 2400 2400

2044

1.75

40 45 48 54 60 105

64%

1500

Covalent binding“ (Wmg protein)

39 45 48 54 60 105 48

240 1950 1350 1950 2850 3150 6300

X f SE

Individual peak’

2925

z!z 1418*+

7.18

+ 3.51*

r = 0.82, p < 0.05

’ Plasma glutamic-pyruvic transaminase (GPT) concentration was obtained repetitively from the tail vein of CD-l male mice from 0 to 36 hr after acetaminophen administration. b Acetaminophen, unlabeled 300 mg/kg, ip, plus radiolabeled 2 &i/g, was administered alone or 6 hr after 5 min general anesthesia with diethyl ether. ’ The individual peak was the maximal plasma GPT concentration obtained in each animal irrespective of its time of occurrence. d Covalent binding of radiolabeled acetaminophen was determined 36 hr after acetaminophen administration. The value for total binding (radiolabeled plus unlabeled acetaminophen) can be calculated as described under Methods. ’ Difference between the 24-hr mean GPI concentration and the true mean peak GPT value (p = 0.059). /Correlation of GPT concentration with covalent binding of acetaminophen to hepatocellular protein in the same animal. The correlations with covalent binding are calculated for the GPT samples at 12 and 24 hr and at the individual peaks, irrespective oftime ofoccurrence. Included for each group are both the acetaminophen controls and the animals pretreated with ether. * Mean values which are significantly different from the respective acetaminophen controls (p -c 0.05).

bined data from these two studies are shown in Fig. 5. However, if GPT data from the traditional24-hr sampling time was chosen (Table 1), there was no such correlation (r = 0.50, p > 0.05). The 39-fold increase in acetaminophen hepatotoxicity produced by ether pretreatment was associated with a 3-fold increase

in the amount of acetaminophen covalently bound to hepatocellular protein (Table 1). DISCUSSION The microanalytical technique for determination of murine plasma GPT concentra-

GPT RELATION 1

TO COVALENT

BINDING

23

tions. Depending upon the sampling time chosen, erroneous under- or overestimates of the enhancement of acetaminophen hepatotoxicity by ether were produced, and these re4000. sults were highly variable in replicate experiments. One would expect to find false-positive 2000. and false-negative results by this method, particularly when toxicologic enhancements are oc more subtle than with the effects of ether re0 4 8 12 16 ported herein. While the traditional method COVALENT BINDING l pg / m g of protean ) of sacrificing a series of different groups of animals at each time point can detect major difFIG. 5. Relation in the same animal of peak plasma GPT concentration with respect to the covalent binding ferences in GPT concentrations between of radiolabeled acetaminophen to hepatocellular protein treatment groups (Wells et al., 1980), this apremaining at 36 hr after acetaminophen administration. pears to be a relatively inaccurate and potenThis figure combines individual peak GPT data from Table tially insensitive method requiring unwar1 with those from a replicate experiment. ranted numbers of animals. The rather dramatic effect of ether on acetaminophen hepatotoxicity also underscores an inherent tions in 50-~1 blood samples obtained from the tail vein gave equivalent results to those methodological danger in employing general obtained via cardiac puncture without ether anesthesia, however briefly, in repetitive samanesthesia. This was an important question, pling studies. since sampling from central and peripheral Accordingly, an accurate and reproducible sites has been reported to result in different measure of maximal hepatotoxicity required estimates of pharmacokinetic parameters for the determination of the peak GPT concentration for each animal, irrespective of the time drugs (Johannessen et al., 1982), and a similar discrepancy could have been true for GPT es- of occurrence (Fig. 4B, Table 1). This obsertimates. vation was internally consistent with the sigThe method of repetitive blood sampling in nificant correlation of individual peak GPT our studies demonstrated that there was a sig- concentration and acetaminophen covalent nificant interanimal variability in the temporal binding in the same animal. The most accurate occurrence of peak plasma GPT concentraanalysis of repetitive GPT data would involve tions secondary to acetaminophen hepatotoxquantification of the total area under the icity. This variability was evident with both plasma GPT time curve rather than peak data, intra- and intergroup comparisons. The intrain a manner analogous to the quantification group variability for animals receiving com- of plasma drug disposition. This would be true bined treatment was reflected in as much as particularly in cases where one treatment twofold errors in the estimate of individual group demonstrates a more prolonged release peak GPT concentrations if any single sam- of GPT into the blood compared with another pling time point was chosen rather than the group, indicative of ongoing hepatic necrosis. individual peak GPT concentrations irrespec- However, while area data could be more retive of timing. Substantial intergroup vari- vealing than peak data, particularly in cases ability was similarly evident in this study, and of chronic low-level hepatic necrosis, such an appears to be potentially greater in studies with analysis would not be readily interpretable in the context of the general toxicological litermore pronounced toxicologic enhancement (Wells et al., 1980) particularly with regard ature which reports single GPT concentrato the 12-hr sample. Thus, single time point tions. Area analysis therefore could be reserved GPT determinations can represent an inac- for those comparisons which would be intercurate measure of the peak GPT concentrapreted incorrectly on the basis of peak data. 6000.

24

WELLS AND TO

Delayed measurement of covalent binding of acetaminophen to hepatocellular protein 36 hr following acetaminophen administration correlated well with the peak plasma GPT concentration from the same animals, suggesting that this persistent binding in a given animal was an accurate quantitative reflection of earlier biochemical determinants of individual hepatotoxicity. While the covalent binding of acetaminophen at 36 hr represented only one-tenth of the binding observed in other studies at 2 hr, the threefold magnitude of enhancement of acetaminophen binding by ether observed at 36 hr was identical (To and Wells, 1986). Structural identification of the covalent adducts would be necessary to examine whether persistent binding might represent specific, toxicological binding to essential cellular macromolecules, since the possibility of identifying an irreparable lesion could be obscured by the competing loss of critical protein adducts via hepatocellular necrosis. However, the quantitative relationship of persistent binding to early binding and ultimate hepatotoxic response raises the possibility that such persistent binding may in this case provide a useful biological separation of toxicologically critical binding from nonspecific binding, the latter representing a nonenzymatic pathway for detoxification of the reactive intermediate of acetaminophen. Further molecular studies will be necessary to test this hypothesis. Another advantage with the method of delayed determination of covalent binding is that it permits simultaneous measures of in vivo acetaminophen biotransformation, covalent binding, and toxicological tissue response (reflected indirectly by GPT concentrations), all in the same animal. This would reduce considerably the interanimal variability resulting from the use of different groups of animals for determination of these parameters, while permitting a direct measure of the dependence of tissue response upon chemical factors. In the present case, our data would support the hypothesis that ether potentiates the hepatotoxicity of acetaminophen by increasing hepatocellular exposure to its reactive intermediate, as reflected by increased covalent binding of

acetaminophen. Data reported elsewhere suggest that this increased exposure may result from an imbalance caused by ether in the eliminating, bioactivating, and detoxifying pathways of acetaminophen biotransformation (To and Wells, 1986). Traditional methods where covalent binding is determined prior to the attainment of peak plasma GPT concentrations preclude the accurate testing of such hypotheses, since the two measurements cannot be performed in the same animal. It appears in retrospect that such studies in some casescould be conducted as early as 24 hr after administration of acetaminophen, since in the present study no peak GPT concentrations occurred at 36 hr. In general, our studies indicate that for in vivo studies of acetaminophen hepatotoxicity, methods permitting identification of both peak plasma GPT concentration for each animal and persistent covalent binding of acetaminophen in the same animal may provide more useful information while requiring fewer animals. ACKNOWLEDGMENTS The assistance of Grazyna M. Kalabis and Nurjehan A. Vassanji in the performance of these studies is gratefully acknowledged.

REFERENCES ADOLPH, A., AND LORENZ, R. (1982). Enzyme

Diagnosis

Karger, New York. AMEER, B., AND GREENBLATT, D. J. (1977). Acetaminophen. Ann. Intern. Med. 87, 202-209. BLACK, M. (1980). Acetaminophen hepatotoxicity. Gasin Diseases

troenterology

of the Heart,

Liver

and Pancreas.

78, 382-392.

BLACK, M. (1984). Acetaminophen hepatotoxicity. Annu. Rev. Med.

35, 577-593.

BOYD, E. M., ANDBERECZKY,G. M. (1966). Liver necrosis from paracetamol. Brit. J. Pharmacol. 26,606-6 14. DAVIDSON,D. G. D., AND EASTMAN, W. N. (1966). Acute liver necrosis following overdose of paracetamol. Brit. Med.

J. 2,497-499.

JOHANNESSEN,W. M., TYSSEBOTN, I. M., AND AARBAKKE, J. (1982). Antipyrine and acetaminophen kinetics in the rat: Comparison of data based on blood samples from the cut tail and a cannulated femoral artery. J. Pharm. Sci. 71, 1352-1356.

GF’T RELATION

TO COVALENT

JOLLOW, D. J., MITCHELL, J. R., POTTER, W. Z., DAVIS, D. C., GILLETTE, J. R., AND BRODIE, B. B. (1973). Acetaminophen-induced hepatic necrosis. II. Role of covalent binding in vivo. J. Pharmacol. Exp. Ther. 187, 195-202. MITCHELL, J. R., JOLLOW, D. J., POTTER, W. Z., DAVIS, D. C., GILLETTE, J. R., AND BRODIE, B. B. (1973a). Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism. J. Pharmacol. Exp. Thu. 187, 185 194. MITCHELL, J. R., JOLLOW, D. J., POTTER, W. Z., GILLETTE, J. R., AND BRODIE, B. B. (1973b). Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione. J. Pharmacol. Exp. Ther. 187, 211-217. POTTER, W. Z., DAVIS, D. C., MITCHELL, J. R., JOLLOW, D. J., GILLETTE, J. R., AND BRODIE, B. B. (1973). Acetaminophen-induced hepatic necrosis. III. Cytochrome P-450-mediated covalent binding in vitro. J. Pharmacol. Exp. Ther. 187,203-2 10. PRESCOTT, L. F., PARK, J., SUTHERLAND, G. R., SMITH, I. J., AND PROVDFOOT, A. T. (1976). Cysteamine, methionine, and penicillamine in the treatment of paracetamol poisoning. Lancet 2, 109-l 13.

25

BINDING

PROVDFOOT, A. T., AND WRIGHT, N. (1970). Acute paracetamol poisoning. Brit. Med. J. 3, 557-558. REITMAN, S., AND FRANKEL, S. (1957). A calorimetric method for the determination of serum ghttamic-oxaloacetic and glutamic-pyruvic transaminases. Amer. J. Clin. Pathol.

28, 56-63.

RVMACK, B. H., AND MATTHEW, H. (1975). Acetaminophen poisoning and toxicity. Pediatrics 55, 87 l-876. TO, E. C. A., AND WELLS, P. G. (1986). Biochemical changes associated with the potentiation of acetaminophen hepatotoxicity by brief anesthesia with diethyl ether. Biochem. Pharmacol., in press. WELLS, P. G., BOERTH, R. C., OATES, J. A., AND HARBISON, R. D. (1980). Toxicologic enhancement by a combination of drugs which deplete hepatic glutathione: Acetaminophen and doxorubicin (Adriamycin). Toxicol. Appl. Pharmacol. 54, 197-209. WELLS, P. G., RAMJI, P., AND Ku, M. S. W. (1986). Delayed enhancement of acetaminophen hepatotoxicity by general anesthesia using diethyl ether or halothane. Fundam.

Appl.

Toxicol.

6,299-306.