METHODS Dogs
Mice
Antidotes
Read before the Aspirin and Acetaminophen Symposium,
New York, November 4-5, 1977.
ADDRESS FOR REPRINTS: (E.P.) Toxicology Research
Unit, McNeil Laboratories, Inc., 500 OffIce Center Drive,
Fort Washington, PA 10034.
Pathophysiology
of Acetam
inophen
Overdosage
Toxicity:
Implications
for Management
Elliot Piperno, M.S., D.V.M., A. Harris Mosher, V.M.D., Ph.D., Daniel A. Berssenbruegge, James D. Winkler, and Roger B. Smith, Ph.D.
From time Toxicology Research Unit and flu’ Pathology Section, 1(’.’ei1 Laboratories, Inc., tort %i ashington,
Pen nsylrimnui
ABSTRACT. Acute acetaminophen intoxication was studied
in the dog to characterize pathogenesis and in the mouse as a
model for antidotal research. In the dog, overt toxicity was
manifested principally by cyanosis, facial and paw edema,
gastrointestinal disturbance, and coma. Typical laboratory
findings were metheinoglobinemia, hemoconcentration,
let,-kocytosis, and hepatic centrolobular necrosis. In the mouse,
physical signs of acetaminophen overdose appeared to be
central in origin; sequelae included anemia, leukopenia,
thrombocvtopenia, and hepatic centrolobular necrosis.
The antidotal profile of acetylcysteine in mice was
char-acterized. When acetylcysteine therapy was instituted early
(one hour after acetaminophen overdose), it conferred
dose-related protection from lethality coupled with
hepatoprotec-tion, as judged from transaminase activity. When
acetylcys-teine therapy was instituted relatively late (4#{189}hours after
acetaminophen overdose), its beneficial effect on survival
persisted but was unaccompanied by distinct
hepatoprotec-tion, indicating that SGPT activity was an unreliable
prog-nostic indicator. Acetlcysteine was well tolerated in mice
even when administered in the presence of preexisting
acetaminophen-induced liver damage. Pediatrics
62(suppl):88O-889, 1978, acetaminophen, overdose,
acetyl-cysteine.
The objectives of these studies were to find a safe, effective antidote for acetaminophen poison-ing and to characterize its antidotal profile. The dog was selected as a model for background pathogenesis work because the clinical course following overdosage is delayed as it is in man, much historical control pathology data were available in this species, and serial blood samples could be taken with relative ease. Systematic screening of potential antidotes was done in mice because this animal model was already estab-lished as a reliable predictor of antidotal efficacy in acetaminophen overdose.2
Purebred female beagles, weighing 7.0 to 9.4 kg each, were gavaged with a single 600-mg/kg dose of acetaminophen (100 mg/nil) after an overnight fast. Parameters examined included physical signs, hematology profile, clinical chemistry profile, and urinalysis. Twelve dogs made up the test group and six dogs served as vehicle-treated controls. Blood collection (via the jugular vein) was performed in the pretest phase and at approx-imately two and 24 hours or six and 48 hours after acetaminophen overdose. Complete necropsies and histopathological evaluations were likewise performed during these intervals on overdosed dogs.
Female CF-i mice (non-Swiss albino), 8 to 14 weeks of age and weighing approximately 20 to 30 gni each, were used. All mice were fasted five hours prior to being overdosed with acetamino-phen. Specific details of experiments are included in table footnotes and figure legends.
TABLE I
PHysIcAl. SICN INCII)EN(E FoLLowING ADMINISTRATION OF ACETAMINOPHEN, 600 MG/K(; ORALLY, IN TIlE Doc
Allifll(1lS Ct/a-00515
Re-
(‘0111-beney Lae- nina-tion
Tre-iiiors
Ptyal-ism
J’acial
Pruritis
Orbi-tal
Ede-111(1
Periorb-ital
Edema
Paw
Ede-111(1
tae-ial
File-111(1
Enu’-
Di,sp-sis rica
f(’l-(‘?l(Z
Coma Death
Approximate onset, hr 2 2 2-4 4 1-4 4 4-24 24 24 24 2-48 24 48 30 31
Necropsied at 2 hr
1318 - - -
-1319 + - - -
-1320 + - - -
-Necropsied at 6 hr
1324 + + - - - + + - - - + - - -
-1325 + - + - + - + - - -
-1326 - - + - - -
-Necropsied at 24 hr
1321 + - - -
-1322 + - - - - + + + - - -
-1323 + + + - - - + - - -
-Necropsied at 48 hr
1327 + - + - - - + + - + - - - -
-1328 + - + - + - + + + + +#{176} + - + +
1329 + + + + - - + + + - +#{176} - + -
-#{176}Frankblood present.
experiment. Methionine (L isomer) was adminis-tered at a concentration of 25 nig/ml.
RESULTS
Dog Pathogenesis Study
Physical Signs’ (Table I). Physical signs of acetaminophen overdose (600 mg/kg orally) were characterized principally by cyanosis, edema of the facial adnexa and paws, emesis, and, in one instance, coma followed by death. Cyanosis, the earliest sign noted (approximately two hours after acetaminophen overdose), was confirmed to be a reflection of methemoglobinemia, which in-creased in severity between the second and sixth hours and subsided by the 24th hour (Table II). This pattern held true for the single case that progressed to coma and death (dog 1328) as well as for survivors. Facial and paw edema were relatively common findings, occasionally pre-ceded or accompanied by lacrimation and appar-ent pruritis. Gastrointestinal signs included delayed, bloody emesis and melena. Excluding the case of coma, none of the dogs exhibited overt impairment of the sensorium.
Periodic urinalysis (Chemstrip 8) indicated the occasional appearance of blood and/or bilirubin
and the more frequent, unrelated appearance of a reddish-brown discoloration. Pilot studies had indicated that the intensity of the discoloration may be related to the presence of total urinary conjugates of acetaminophen.
Clinical Laboratory Paranieters. The clinical laboratory profiles of the three most severely affected dogs are presented in Table II. Hemato-logic aberrations distinctly related to overdose included methemoglobinemia, hemoconcentra-tion, leukocytosis, and, terminally, a degenerative left shift.
Evaluation of blood coagulation and chemistry parameters disclosed an early trend toward alka-losis and hypokalemia followed by distinct eleva-tions in SGPT level, bilirubin value (principally unconjugated), prothrombin time, ammonia con-centration, and creatinine phosphokinase level. No remarkable alterations occurred in BUN, creatinine, glucose, cholesterol, and amylase concentrations where tested.
Dog 1322
+24
hr
Normal’
Dog 1328
A
Pretest + 6
hr
+30
hrf
Dog 1329
.
--.--Voruial Pretest +2
hr
Normal Pretest +hr +45 hr
White blood ‘cIls (XID/cu 12.7 ± 2.6 13.2 10.5 12.7± 2.6 12.0 8.6 :3.0 12.7 ± 2.6 14.0 14.0 38.9
Nesitrophils, segmented (%) 55’75 . . . 84 91 55-75 . . . (15 5 55-75 . . . 80 81
Neutrophils, hand (%) 0- . . . I 3 0-6 . . . 16 38 0-6 . . . 9 8
Lymphoeytes (%) 20.35 . . . 8 4 20-35 . . . 15 4 20-3.5 . . . I 1 9
Monocvtes (%) 2-6 . . . 5 2 2-6 . . 3 11 2-6 . . . 0 3
Iosinophils (%) I:) . . . 2 0 1-3 ... I 0 1-3 . . . 0 0
Basophils (%) 0-I . . . 0 0 0.1 . . . (1 t) (I-I . . . 0 0
NucleatedRHCs/IOOWBCs O’I ... ... ... (Ii ... 4 -- 2 0 (1
Red blood cells (X IO’/ei* mm) 6$R ± 0.86 5.65 6.46 5.72 6.95 ± 0.86 5.40 8.98 9.76 6.95 ± 0.86 7.23 7.36 8.71
11CI1OgI()I)ifl (gsss/dI) 16.1 ± 1.8 13.7 16.0 14.9 16.1 ± 1.8 13.8 21.5 22.0 1.1 ± 1.8 16.1 17.1 18.9
Ilernatocrit (%) 47.5 ± 5.9 40.0 42.8 41.5 47.5 ± 5.9 38.2 55.9 64 47.5 ± 5.9 47.7 43.2 52.1
Methemoglobin (%) 0661.9 . . . 48.3 9.2 0.66-1.9 . . . 53.5 1.97 0.66-1.9 . . . 68.1 10.3
5CPT(,nU/mI) 31±7 ... 32 ... 31±7 30 54 9,900 31#{247}7 27 42 29,500
Bmlinihin (mg/dI)
Total 0141.2 . . . 0.2 . . . 0.1.0.2 0.1 0.1 . . . 0.1-0.2 0.1 0.2 6.1
Direct 0 . . . 0 . . . 0 0 0 . . . 0 0 0 1.6
Indirect 0.1-0.2 . . . 0.2 . . . 0.1-0.2 0.1 0.1 . . . 0.1-0.2 0.1 0.2 4.5
Prothromhiis tune (see) 6.5 ± 0.3 6.5 8.2 8.0 6.5 ± 0.3 6.5 6.5 14.0 6.5 ± (1.3 6(1 5.5 21.5
(isieoce(rng/dI) 83± 11 90 104 94 83± 11 93 83 ... 83± 11 103 106 85
Ammonia (ig/dI) 65’119 . . . 114 109 65-119 . . . 78 237 85-119 . . . 191 409
Cholesterol (sug/di) 159 -t 26 187 204 167 159 ± 26 137 151 116 159 ± 26 146 124 149
Sodium (mEq/Iitcr) 145 ± 2 144 142 . . . 145 ± 2 146 145 . . . 145 #{247}2 145 41 138
Potassi,im (,isEq/Iiter) 4.8 ± 0.3 4.8 4.2 . . . 4.8 ± 0.3 4.3 4.0 . . . 4.8 ± 0.3 4.8 4.0 3.5
Chlorides (rnEq/Iiter) 108 ± 3 108 1 12 . . . 108 ± 3 109 1 I 1 . . . 108 t 0.3 108 1 12 III)
Carlxndioxideeapacity 24±1 2.3 18 ... 24± 1 25 17 ... 24 I 22 18 20
(rnFq/liter)
.kxiiyla.ce (ItT/liter) 1,746 ± 702 . . . 1,571 . . . 1.746 ± 702 1,820 1,250 . . . 1,746 ± 702 2,22(1 2,360 :3,502
Creatinine phosphokinase 49 ± 19 . . . 49 ± 19 83 . . . 424 49 19 46 . . . 170
(mU/mI)
BUN (mg/ 100 ml) 15.8 ± 3.5 12 12 11 15.8 ± 3.5 1 1 15 33 15.8 ± 3.5 I I 16 50
Creatinine (mg/I00 nil) 0.84 ± 0.13 0.9 1.1 0.6 0.8.4 ± 0.13 0.7 0.9 0.9 0.84 #{247}0.13 0.9 0.9 1.0
‘Group pretest mean ± SD or established normal range. tComato.
TABLE II
HEMATOLOCY AND BLOOD CHEMISTRY PROFILES FOLLOWING ADSI INISTRATION OF ACETAM I NOPII EN, 600 MC /KG ORALLY
even though one of the dogs was clearly icteric and several of the dogs were known to have marked elevations in SGPT level.
Histologic Lesions Related to Treatment (Table IV). No definitive histologic lesions were present in dogs killed two to three hours after treatment. One dog (1319) had trace-sized foci of vacuolar degeneration in the liver and another (1318) had a trace amount of necrosis in a mesenteric lymph node, both possibly related to treatment.
Lesions considered to be related to treatment were present in two of the three dogs killed approximately six hours after treatment. One dog
(
1324) had a severe edema with hemorrhage and eosinophilic infiltrates in the palpebral conjuncti-va and subcutis of the eyelid. This dog also had a trace amount of necrosis in a mesenteric lymph node and a small number of foamy macrophages in Peyer’s patches of the small intestine, also considered possibly related to treatment. The other dog (1325) had a marked amount ofpal-pebral edema and a trace amount of centrolobu-lar vacuolar degeneration in the liver. This dog also had a trace amount of necrosis in a mesent-eric lymph node and foamy macrophages present in Peyer’s patches of the small intestine.
All dogs killed approximately 24 hours after treatment had moderate to marked amounts of palpebral edema with eosinophils present. In one dog, a moderate amount of hemorrhage was also present as well as edema in the feet. Two dogs killed during this time period had moderate and marked amounts of centrolobular hepatic necrosis with associated large eosinophilic cytoplasmic inclusions. Two dogs in this group also had a moderate amount of renal tubular dilatation that was considered to be possibly related to treat-ment.
TABLE III
CROSS PATHOLOGIC FINnIN;s ATTRIBUTED TO
ACETAMINOPHEN ADMINISTRATION
Animals
All
Negative findings
Liver, ileocecal valve, large
intestine, kidney, lungs,
spleen, adrenal gland,
mammary gland, heart
Positive findings
Eyelid: edema 1321, 1322, 132.3,
1324, 1327, 1328,
1329
1322, 1323
1318, 1319, 1320
1329 1329 1329 Mouse 1325 1328
The focal gastric mucosal congestion was also possibly related to treatment.
Both dogs killed approximately 48 hours after treatment had marked centrolobular hepatic necrosis with varying degrees of a peripheral zone of vacuolar degeneration. Palpebral edema had subsided to a small amount in these dogs and was associated with hemorrhage. A small conjunctival ulcer with cellular infiltrate was present in one dog. Hemorrhage was present in the urinary bladder of both dogs and was associated with small acute thrombi in one dog. One dog had a moderate amount of congestion, hemorrhage, and erythrophagia in a mesenteric lymph node. Lesions considered to be possibly related to treatment were restricted to one dog (i329) and consisted of renal tubular dilatation with asso-ciated hemorrhage and cellular infiltrate, gastric hemorrhage and edema, and mucosal hemorrhage in the small intestine.
Other lesions, summarized in Table IV, were not considered to be related to treatment because of duration (chronic appearance), distribution between groups (frequently in these controls or others), or other known etiology.
General Toxicity and Pathology. The clinical course of acetaminophen overdosage (1,500 mg/ kg orally) in the CF-i mouse is rapid. Debilitation occurs within one hour, and death, when it ensues, usually within 24 hours. Physical signs are characterized by decreased spontaneous activity, tremors, hyperreactivity to noise, and loss of righting reflex. Unlike that of the dog, the senso-rium of the mouse appears to be markedly affected.
The most consistent findings noted in the hematologic profile of acetaminophen-overdosed
mice were anemia, leukopenia, and thrombocyto-penia. In the small population studied, the ane-mia appeared to correlate with subsequent death (Fig. i) and was largely prevented by therapy with the antidote acetylcysteine (Fig. 2).
Liver histopathologic studies disclosed minimal periportal hepatocellular vacuolization by one-half hour, moderate to marked centrolobular congestion by three hours, centrolobular hemor-rhage by 4#{189}hours, the presence of many pyknot-ic nuclei by six hours, and varying degrees of centrolobular necrosis characterized by pyknotic nuclei and eosinophilic intracytoplasmic globules by nine hours.
Antidotal Research. After systematically screening a variety of both sulfhydryl- and non-sulfhydryl-containing potential antidotes,
acetyl-Feet: subcutaneous edema
Blood: brown discoloration
Gastric mucosa: dark discoloration
Small intestine: red contents
Urinary bladder: subserosal
hemorrhage
Gallbladder: edematous
swelling of wall
Fat: icterus
cysteine emerged as the most promising agent as judged by survival and intrinsic safety. Tested side by side with mercaptamine (cysteamine) as a reference compound, acetylcysteine was more efficacious when administered relatively late and distinctly safer.3
Dose-Response (Survival) Studies. The results of seven-day survival studies, using single doses of acetylcysteine administered one or 4#{189}hours after acetaminophen overdose, are represented in Fig. 3. Methionine was evaluated in parallel for comparison purposes. Protection associated with the 4#{189}-hour treatment period has special signifi-cance because acetaminophen-induced liver in-volvement is known to occur by this time.
As noted in Fig. 3, early acetylcysteine therapy afforded dose-dependent protection. Relatively late therapy (4#{189}hours after acetaminophen ingestion) likewise afforded protection, but the dose-response profile tended to flatten, with disproportionately higher efficacy noted at the lower dosage levels. Methionine, on the other hand, tended to produce a bell-shaped response when administered early that was further exag-gerated when administered relatively late (4#{189} hours after acetaminophen overdose).
TABLE IV
HISTOLOGIC FINDINGS IN ACETAMINOPHEN-OVERDOSED Docs AND CorrrisoLs#{176}
Acetaminophen-Overdased Dogs Controls
,i-;-8 1.320 1321 1322 1323 1324 1325 1326 1327 132Sf 1329 1330 1331 1332 1333 1.334 1335
Posttreatment
survival (hr:min) 2:10 2:47 3:27 24:09 24:35 25:01 6:10 6:37 657 48:11 3050 48:28 49:25 495370:0070:33 71:09 71:21
Liver N N N N N
Hematopoiesls 1 ±
Hydropic degeneration 1
vaIar degeneration ±I ± 1 3t 3*
Necrosis 31 2* 3* It SI
Cytoplasmtc Inclusions II 1
Cellularlnfiltrate ±* 1 ±
Bladder N N N N N N N N N
Congestion 1 3 1 1 1 1
Hemorrhage 1 3 1
ThrombosIs 21
Cellular infiltrate 1
Eyelid N
Edema 2* 31 3* 41 31 31 11 21 II
---Hemorrh&ge 21 21 1* 11 -I
Ukeatio 11
-Cellular Inffltrate 21 3* 2* 3 3 1 1
Kidney N N N N
Hyperplasia 1
MineralizatIon I ± ± ± ± ± ± ± ± ±
Hemorrhage 1
Tubular dilatation 21 2* 2*
--.--Cellularinfiltrate ± 1 1 1 I
Mesenteric lymph lode N N N N N N N N N NNN
Congestion 2*
Hemorrhage 2*
Erythrophagi* 21
Necroia ±1 ± ±
Cellular Infi1trte 1 1
Gallbladder N N N N N N N N N N N N N N N N
Cysts - X
Edema 3*
Hemorrhage 1*
Lungs N N N N N N N N N
Hair x
Focal fibissls ±
Hemorrhage ±
Cellular infiltrate ± 2 ± I ± 1 ± ±
Stomach N N N N N N N N N N N N N
Congestion 1
Hemorrhage
11
Edema 21 1
Cellular infiltrate 1 1
Small intt1ne N N N N N N N N N N N
Ectopic gastric mucoa X
I
Dilated crypta --- 1 1
Hemorrhage 11 __
-Cellular infiltrate fl ± 1 1 1
Colon N N N N N N N N N NN N N N
Congestion 1 1
Hemorrhage 1 1
Cellular infiltrate I
Ileocolic valve N
Congestion 1 1 1 1
Hemorrhage 1 -. -.__________
Feet N -_______
Edema 1*
F,s____ N N N N N N N N N N N N N N N N N
Trachea - Cellular infiltrate
Heart
Arteritis
Adrenal gland
Aretansinsphen’Overdo.,ed Dogs Control.,
1318 131.9 1320 1321 1322 1.323 1.324 132.5 1.326 1327 132Sf 1329 1330 1331 1.532 1.3.3.3 1.3:14 133.5
N N N N N N N N N N N N N N N N
N N N N N N N N N N N N N N N N
3 2
N N N N N N N N N N N N N N N N
- Hvperpla.sia 1 1
Tongue - N N N N N N N N N N N N N N N N N
(:ellsttar infiltrate I
Parathyroid gland N N N N N N N N N N N N N N N N N
Cyst x
Pancreas N N N N N N N N N N N N N N N N N
Cellular infiltrate ±
Pituitary gland N N N N N N N N
Cyst X X X X X X X X X X
Utents N N N N N N N N N N
Immature X X X X X X X X
Thvroidgland N N N N N N N N N N N N N
llvperplasia ± ± 2
Cellular infiltrate ±
Cyst x
Mammary gland N N N N N N N N N N N N N N
(;tgmtion I
Hemorrhage ± 1
Ilylserl)lasia
TABLE IV (CONTINUED)
Thyinsis N N N N N N N N N N N N N N
Cyst X
Involution I ± I 1
Splet.n N N N N N N N N N N
Congestioii 1 I 1 1
Sidene.is I I 1 1 1
Necrosis I
Salivarvgland N N N N N N N N N N N N N N N N N
(:akILs -- __________________-
__
CeII,ilar i,fiItrate 1
Ovaries N N N N N N N N N N N N N N N N N N
Skin N N N N N N N N N N N N N N N N N N
Eyes N N N N N N N N N N N N N N N N N N
Costodsondraljiinetion N N N N N N N N N N N N N N N N N N
Central nermnts systetn N N N N N N N N N N N N N N N N N N
‘N indicates normal; X, present; ±,trace or minimal; 1, small or slight; 2. moderate; 3, marked; 4, severe. tI)ied.
Relatmt to treatment. §Pos.sihly related to treatment.
that neither drug exacerbated acetaminophen-induced mortality (unpublished data).
Dose-response studies conducted with intrave-nously and orally administered acetylcysteine indicated that potency (milligrams per kilogram) was comparable for both routes.4
Transaminase Profiles. Hepatoprotection, in-ferred from transaminase activity, was marked when acetylcysteine was orally administered one hour after acetaminophen overdose (Table V) but minimal or nil when administered 4#{189}hours after overdose (Table VI). Methionine likewise af-forded no distinct hepatoprotection when admin-istered 4#{189}hours after acetaminophen overdose (Table VI).
Acetylcysteine MechanLsm Studies. The plasma half-life of parent acetaminophen (1,200 mg/kg orally) in fasted CF-i mice, determined to be approximately one hour, was unaffected by early acetylcysteine therapy (1,200 mg/kg orally). Likewise, urinary clearance of parent compound plus metabolites was not significantly altered by acetylcysteine therapy.
)DEAD AT 61/2 HR.
DEAD AT 6 HR.
.-...o 40’
30’
20
10’
.-. CONTROLS
o--o APAP 1500mg/kg
I I I I I I I I
‘2 2 3 4 5 6 7 8 9
HOURS POST APAP
FIG. 1. Effect of acetaminophen (APAP) overdose on hematocrit profile in mouse. Each group
comprised three serially bled mice.
APAP+ NAC
UNTREATED CONTROLS
ALONE 50’
40’
0 0
Lu I
30’
20
FIG. 2. Effect ofacetylcysteine (NAC) treatment on
acetamin-ophen (APAP)-induced anemia in the mouse. Each point
represents mean of three serially bled mice. Acetaminophen
was administered at dose of 1,500 mg/kg orally.
Acetylcys-teine was administered one hour later at dose of 1,200 mg/kg orally.
50
I.-C)
0
‘Ii
x
other conjugates, most notably mercapturate. Curiously, this profile was similar to that observed in mice made tolerant to a lethal dose of acetaminophen (1,200 mg/kg orally) by pretreat-ment with sublethal doses of the drug (250 mg/kg orally twice daily for four days).5
0 2 4 6
HOURS P0 ST APAP
The degree to which acetaminophen metabo-lite covalently binds to liver tissue after various acetylcysteine treatment times is shown in Fig. 4. Covalent binding was significantly reduced (P < .05) by approximately 38% when acetylcys-teine was coadministered with acetaminophen but not when it was administered two to four hours later.
DISCUSSION AND CONCLUSIONS
The mouse is generally accepted as the most reliable animal model for predicting the clinical efficacy of acetaminophen antidotes. It is espe-cially susceptible to glutathione depletion’ and experiences an accelerated pathogenesis follow-ing acetaminophen overdosage. Acetaminophen antidotal research in the mouse model was prin-cipally responsible for the clinical introduction of
both cysteamine”2 and acetylcysteinet as
antidotes. The clinical use of methionine in this indication,’0 however, was based on original investigations in the rat,” with the subsequent support of dog’2 and mouse studies.” Currently, acetylcysteine and methionine appear to be the most useful clinical antidotes from the standpoint of efficacy, safety, and availability.
cou-50
87
73 73
53
0000O
0 0 O
0 0 O
Cs se
mg/kg mg/kg
NO NAC 1 HR. NAC 4HRS. METH 1 HR. METH 4’/HRS.
THERAPY POST APAP POST APAP POST APAP POST APAP
mg/kg mg/kg
FIG. 3. Effect of acetylcysteine (NAG) and methionine on survival following acetaminophen
(APAP) overdosage in the mouse. Fifteen mice constituted each dosage group. Acetaminophen
was administered at dose of 1,500 mg/kg orally.
300
200
100
Treatment Time IHr.J
Time of Sacrifice (Hr.J
0 2 4 0 2 4
2 4 6 24 2424
HOURS POST APAP ADMINISTRATION
100
93
-J
. > >
U)
>-4
0
pled with facial edema. Both of these effects have been reported in the cat after acetaminophen overdosage,’ and it is hypothesized that the two phenomena are related.’ There is evidence to indicate that methemoglobinemia is a conse-quence of the formation of free amines.’ Compared to man, the cat and the dog are both reported to produce relatively higher levels of
0. <LU
LU
E
LU0
<C
> 0 0
free amines after acetaminophen administra-tion.’T We have not been able to demonstrate niethemoglobinemia in the mouse after acetamin-ophen overdosage.
Both the dog and mouse experience severe hepatic centrolobular necrosis after acute acet-aminophen intoxication despite their differing physical and hematologic responses.
Interesting-FI;. 4. Effect of acetylcysteine (NAC) treatment (750 Ing/kg orally) on covalent binding of
acetaminophen (APAP) metaholite to liver tissue. Carbon 14-labeled acetaininophen was
administered at dose of 750 mg/kg orally. Four mice constituted each group. Asterisk indicates
TABLE V
EFcr OF EARLY ACETYLCYSTEINE THERAPY ON ACETAMINOPHEN-INDUCED ELEVATIONS OF
PLASMA GPT LEVEL IN THE M0uSE#{176}
Median Plasma GPT Leeel (JU/Lite
hr After Acetaminophen Ingestion
r),
0.5 1 2 .3 .3.5 4.5 6 9 24
Negative control 32 41 50 39 . . . 31 30 26
Acetaminophen plus 29 31 28 23 1 14 672 836 4,026 1,660
vehicle
Acetaminophen plus 37 31 20 13 34 39 23 47 29
acetylcysteine
#{176}Acetylcysteine was administered orally at a dose of 1,200 mg/kg one hour after acetaminophen
overdose of 1,200 mg/kg orally. Six different mice constituted each treatment group per time period.
ly, the most severely affected dog (1328), the only one that died, had only minimal hepatic necrosis although its SGPT level was 9,900 mU/ml approximately one hour prior to death. Dissocia-tion between the intensity of hepatic necrosis and subsequent death has likewise been reported in man’8 and in the rat.’9 Therefore, liver necrosis per se may not be the cause of death but is merely a frequently appearing morphological manifesta-lion of a more critical functional lesion(s). The dissociation is further highlighted by our finding
TABLE VI
that when acetylcysteine therapy is initiated relatively late in mice, it largely prevents lethali-ty but offers little or no hepatoprotection, as judged from SGPT activity.
Because acetylcysteine chemically constitutes the central portion of the glutathione molecule, and the latter is known to be depleted in acetamin-ophen overdosage,’ the protective effect of acetylcysteine may be related to its ability to replenish glutathione or mimic its function. This hypothesis, however, stands to be proven. The
EFFECT OF MODERATELY LATE ORAL ANTIDOTAL THERAPY ON ACETAMINOPHEN-INDUCED
ELEVATED SGPT LEVELS IN THE MOUSE#{176}
SGPT Level (lU/Liter)
9 hr After 24 hr After
Acetaminophen Acetaniinophen
Ingestion lngestion
.4edian Range N Median Range V
Negative control 29 14-56 10 34 14-153 10
Positive control 8,390 1,705-> 13,000 16 6,040 2,480-10,980 10
Acetylcysteine (mg/kg)
200 11,990 164->13,000 10 5,070 128-11,700 10
40#{243} 3,827 452-12,260 10 2,008 302-6,260 109
800 6,610 96-12,720 10 3,540 2-10,740
Methionine (mg/kg)
200 4,460 20-> 13,000 9 1,660 27-> 13,000 10
9
400 6,320 250-> 13,000 10 3,280 2,450- > 13,000
800 6,840 1,050-> 13,000 9 7,540 586-> 13,000 7
#{176}Crouppopulation at antidotal therapy 4#{189}hours after acetaminophen ingestion was 18 for
positive controls and 10 in all other groups. Changes in population size reflect mortality with the
exception of the nine-hour low-dose methionine group figure, which represents a single missed
TABLE VII
EFFECT OF ACETYLCYSTEINE THERAPY ON RELATIVE DIsTRIBuTIoN OF ACETAM INOPHEN URINARY
% of Total (.lean ± SD)
Acetylcysteine No
Therapy Therapy
Parent compound 20.8 ± 4.0 22.8 ± 3.4
Glucuronide 42.5 ± 4.2 40.0 ± 4.7
Sulfate 11.5 ± 3.3 17.5 ± 1.3
Cysteine 18.8 ± 2.2 16.8 ± 4.8
Mercapturate 6.3 ± 1.0 3.0 ± 0
#{176}Acetylcysteine was administered at a dose of 1,000 mg/kg
orally one hour after acetaminophen overdose of .1,000
mg/kg orally. Values represent four urine samples (combined
urine from six mice per sample) collected during the first 4#{189}
hours after acetaminophen ingestion.
replenishment argument is supported by the find-ing that the analogue cysteine has been shown to replenish hepatic glutathione in mice overdosed With acetaminophen.2#{176}
In conclusion, preclinical studies in mice mdi-cate that acetylcysteine is both well tolerated and
highly efficacious when administered relatively
late after acute acetaminophen overdosage. Its protective effect does not appear to be associated with facilitated plasma or urinary clearance of drug, major alterations in the urinary metabolite profile, or removal of covalently bound metabo-lite from the liver. Early administration of acetyl-cysteine did prevent acetaminophen-induced
anemia in mice. However, since it did so at a time when it was known to be hepatoprotective, the site of this action (RBCs? liver?) and its impor-tance is unclear but certainly worthy of further pursuit.
REFERENCES
1. Mitchell JR. Thorgeirsson SS, Potter WZ, et al:
Acet-aminophen-induced hepatic injury: Protective role
of glutathione in man and rationale for therapy.
Cliii Pharniacol flier 16:674, 1974.
2. Prescott LF, Newton RW, Swainson CP, et al:
Success-ful treatment of severe paracetamol overdosage
with cysteamine. Lancet 1:588, 1974.
3. Piperno E, Berssenbruegge DA: Reversal of
experimen-tal paracetamol toxicosis with N-acetylcysteine.
L(ltICet 2:738, 1976.
4. Piperno E, Berssenbruegge DA: Toxicological Research
Report No. 495 (761012). Fort Washington, Pa,
McNeil Laboratories mc, 1976.
5. Piperno E, Winkler JD, Berssenbruegge DA: Antidotal
profile of N-acetylcysteine in the acetaminophen
overdosed mouse. Read before the Second French,
American, and Canadian International Congress of
Clinical and Analytical Toxicology, St Adele,
Quebec, August 1977.
6. Lyons L, Studdiford JS, Somniaripa AM: Treatment of
acetaminophen overdosage with N-acetylcysteine.
N Engl I Med 296: 174, 1977.
7. Peterson RG, Rumack BH: Treating acute
acetamino-phen poisoning with acetylcysteine. JAMA
237:2406, 1977.
8. Peterson RG, Rumack BH: Treatment of acetaminophen
poisoning with acetylcysteine. Read before the
Second French, American, and Canadian
Interna-tional Congress of Clinical and Analytical
Toxicol-ogy, St Adele, Quebec, August 1977.
9. Prescott LF, Ballantyrie A, Park J, et al: Treatment of
paracetamol (acetaminophen) poisoning with
N-acetylcysteine. Lancet 2:432, 1977.
10. Crome P, Vale JA, Volans GN, et al: Oral methionine in
the treatment of severe paracetamol
(acetamino-phen) overdose. Lancet 2:829, 1976.
11. McLean AEM: Prevention of paracetamol poisoning.
Lancet 1:729, 1974.
12. Maxwell LF, Cotty VF, Marcus AD, et al: Prevention of
acetaminophen (paracetamol) poisoning. Lancet
2:610, 1975..
13. Legros J: Animal studies-a theoretical basis for
treat-ment (paracetamol toxicity). J Int Med Res
4(suppl):46, 1976.
14. Finco DR, Duncan JR, Schall WD, et al:
Acetamino-phen toxicosis in the cat. J Am Vet Med Assoc
166:469, 1975.
15. Leyland A: Probable paracetamol toxicity in a cat. Vet
Rec 94:104, 1974.
16. Brodie BB, Axlerod J: The fate of acetophenetidin in
man. J Pharmacol Exp flier 97:58, 1949.
17. Welch RM, Conney AH, Burns JJ: The metabolism of
acetophenetidin and N-acetyl-p-aminophenol in the
cat. Biochenz Pharmacol 15:521, 1966.
18. Dixon MF: Paracetamol hepatotoxicity. Lancet 1:35,
1976.
19. Boyd EM, Bereczky GM: Liver necrosis from
paraceta-mol. Br I Pharmacol 26:606, 1966.
20. Strubelt 0, Siegers CP, Schutt A: The curative effects of
cysteamine, cysteine, and dithiocarb in
experimen-tal paracetamol poisoning. Arch Toxicol 33:55,
1974.
ACKNOWLEDGMENT
We gratefully acknowledge the groundwork studies of
Robert K. Dix, the literature review support of Bernice
Friedmann, and the technical assistance of Edith R.
Williams, James McMunn, Peter Taggart, and Mary
Wolferth.
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