Centrolobular hepatic necrosis related to covalent binding of metabolites of halogenated aromatic hydrocarbons

EXPERIMENTAL

AND MOLECULAR

PATHOLOGY

18, 80-99 (1973)

Centrolobular Hepatic Necrosis of Metabolites of Halogenated WATSON Laboratory

D.

REID

l~~~G~~~~K~~~~~~

of Chemical Pharmacology, National Institutes of Health, Received

Related to Covalent Binding Aromatic Hydrocarbons

National Bethesda,

June

Heart and Lung Maryland 20014

Institute,

13, 1972

The mechanism of centrolobular hepatic necrosis produced by treating rats or mice with “C-labeled halogenated aromatic hydrocarbons was studied as an animal model of drug-induced tissue lesions. The development of hepatic necrosis was associated with the covalent binding of substantial amounts of radiolabeled material to liver proteins, and autoradiograms revealed that most of the covalently bound material was localized within the necrotic centrolobular hepatocytes. Prior induction of hepatic microsomal enzymes by phenobarbital administration potentiated the covalent binding and necrosis, whereas prior inhibition of hydrocarbon metabolism had the opposite effect, suggesting that the binding and necrosis are caused by toxic metabolites of the hydrocarbons. A positive correlation between the amount of covalent binding and the severity of centrolobular necrosis was obtained after various drug treatments and with several different halogenated benzene derivatives of varying hepatotoxicity. These results suggest that covalent binding of toxic metabohtes may be an important mechanism in the pathogenesis of tissue lesions elicited by a variety of foreign compounds.

Drug-induced tissue lesions such as hepatic or renal necrosis, bone marrow aplasia and certain allergic reactions are a major obstacle in the development of new drugs and represent a clinical problem of considerable importance. An understanding of the biochemical events in the pathogenesis of toxic lesions is prerequisite to rationally designing safe drugs and to eliminating so-called idiosyncratic toxic reactions which occur in the absence of overdosage. It is generally recognized that biochemical and structural damage to tissues produced by relatively inert foreign compounds, such as most lipid-soluble therapeutic agents, might be caused by highly reactive and unstable intermediate products formed during the metabolic conversion of the nontoxic parent compound to harmless polar metabolites. A number of authors have suggested that chemically unreactive carcinogens such as dialkylnitrosamines, azo dyes, N-acetylaminofluorene and polycyclic hydrocarbons may cause tumors by the formation of a covalent linkage between a chemically reactive metabolite and various macromolecules (for reviews see Miller and Miller, 1966; Bergman and Pullman, 1969). Our laboratory has postulated that a wide variety of other drug-induced tissue lesions may be mediated through the covalent binding of active metabolites to macromolecules in target cells (Brodie et al., 1971; Reid, 1972b). 1 Present Prevention,

address: Executive 05ce Washington, D. C. 20506.

of the

President,

80

Copyright All rights

1973 by Academic Press, Inc. oP reproduction in any form reserved.

Special

Action

Office

for

Drug

Abuse

AROMATIC

HYDROCARBONS

AND HEPATIC

NECROSIS

81

Because of the low incidence of drug-induced tissue lesions in laboratory animals, we have investigated the toxic mechanisms of halogenated aromatic industrial solvents known to cause liver necrosis in various animal species (Cameron et al., 1937; Koch-Weser et al., 1953). The first histological change in the liver of rats or mice treated with a single dose of bromobenzene is the depletion of cytoplasmic glycogen in centrolobular hepatocytes at 12-16 hr. Centrolobular necrosis in rats begins at 24 hr, is maximal at 48 hr and resolves by 96 hr ( Koch-Weser et al., 1953; Reid et al., 1971a). Our initial studies have revealed that both the hepatic necrosis and the metabolism of bromobenzene in viva are blocked by prior treatment with piperonyl butoxide or SKF 525-A ( p-diethylaminoethyl diphenylpropyl acetate) and are markedly potentiated by previously inducing hepatic microsomal drug metabolizing enzymes with phenobarbital (Reid et al., 1971a). These findings indicate that the necrosis is caused by a metabolite, presumably bromobenzene epoxide, and not by bromobenzene itself. The present studies demonstrate that after administration of 14C-bromobenzene or other radiolabeled aromatic hydrocarbon to rats and mice a metabolite becomes tightly bound to macromolecules preferentially in the centrolobular region of the liver. Proof of the covalent nature of the binding has been presented (Krishna et al., 1971) and will be published in detail elsewhere. The chemical and autoradiographic studies reported in this communication show the relationship between the covalent binding of radiolabeled aromatic hydrocarbons in viva and the production of centrolobular hepatic necrosis. Some of these results have been published in an abstract (Reid et al., 1971b). MATERIALS

AND METHODS

Experiments were carried out in male Sprague-Dawley rats (160-200 g) and in male C57 Black/6J mice (18-20 g) given food and water ad Zihitum. All compounds to be administered to the animals were dissolved in saline or sesameoil, depending on their solubility in water, and were administered intraperitoneally. Controls received an equal volume (OS-l.0 ml/rat; 0.1-0.2 ml/ mouse) of the appropriate diluent. 14C-Labeled halogenated aromatic hydrocarbons were custom-synthesized by Mallinkrodt Chemical Co. or were purchased from Amersham/Searle. Purity was checked on a gas chromatograph equipped with an electron capture detector (Reid et al., 1971a). Specific activity was adjusted with reagent grade chemicals so that each animal received 10-30 &i of radioactivity. Actual doses of the hepatotoxins are given in the text. Other drug treatments were as follows: To induce microsomal cytochrome P-450 enzymes in the liver, phenobarbital (80 mg/kg) was injected into the animals at 72, 48 and 24 hr prior to the hepatotoxin (Remmer, 1959; Conney and Burns, 1959). SKF 525-A, 75 mg/kg or piperonyl butoxide (400 mg/kg), compounds which inhibit microsomal enzyme activities ( Axelrod et al., 1959; Anders, 1968), were administered 2 hr before the hepatotoxin. The dosage of pyrazole used (375 mg/kg given I hr before bromobenzene) has been shown to alter the activities of hepatic microsomal enzymes as well as that of alcohol dehydrogenase (Lieber et al., 1970). The hepatic concentration of glutathione was lowered by administering diethyl maleate (0.6 mg/kg) 2 hr before bromobenzene ( Boyland and Chasseaud, 1970). In this experiment hepatic glutathione

82

REID

AND

KRISHNA

levels were measured by the method of Cohn and Lyle ( 1966). Aminoacetonitrile (100 mg/kg) was given 72, 48, 24 and 2 hr before bromobenzene in order to block hepatic necrosis (Fiume, 1963). Total urinary excretion of metabolites of 14C-labeled aromatic hydrocarbons was calculated by liquid scintillation counting of an aliquot of urine collected from groups of three rats kept in a metabolism cage for 6 or 24 hr after administration of the hepatotoxin. Extraction of urine with five volumes of heptane: isoamyl alcohol (50: 1) followed by liquid scintillation counting of an aliquot of the organic phase revealed that no unmetabolized hydrocarbon was excreted (Reid et al., 1971a). Animals were killed by cervical fracture at various times after administration of the hydrocarbon and livers were rapidly removed. A 2-mm slice of liver was fixed in buffered formalin and the remainder was frozen on dry ice. Paraffin sections 8 tm thick were prepared by standard techniques (Culling, 1963) and stained with either hematoxylin and eosin or periodic acid Schiff reagent (PAS) and hematoxylin. For autoradiographic studies, paraffin sections mounted on glass slides were coated with Kodak NTB-2 emulsion and developed after 4 wk exposure in a light-free box at -20°C (Baserga and Malamud, 1969). The intrahepatic distribution of 14C-leucine incorporation into proteins was determined autoradiographically by administering 14C-leucine (500 &i/kg; 14C-L-leucine, generally labeled, specific activity 10 mCi/mmole) 0, 6, 24 or 48 hr after a hepatotoxic dose of unlabeled bromobenzene (3.8 mmoles/kg ). In previous studies (Krishna et al., 1971) covalent binding of 14C-bromobenzene to hepatic proteins was demonstrated by exhaustive solvent extraction of trichloroacetic acid precipitates of liver homogenates followed by Sephadex chromatography. Extraction with phenol revealed that virtually no radioactivity was bound to nucleic acid. The proteins were subsequently hydrolyzed with Pronase and assayed in an amino acid analyzer. The 14C-labeled material bound to liver proteins was associated with three or four specific peaks and studies are now in progress to identify the amino acids to which the radioactivity is bound. These studies strongly suggest that the binding of labeled material to liver proteins is covalent, and definitive proof of covalent binding is being sought using mass spectrometry. To handle larger numbers of samples it was necessary to shorten this procedure as follows: Tissues were homogenized in four volumes of water, and 5 ml of the homogenates were extracted three times with 25 ml of heptane:isoamyl alcohol (50: 1 ), which removed virtually all of the unmetabolized 14C-bromobenzene (or other radiolabeled hydrocarbon). A 20-ml aliquot of the first heptane:isoamyl alcohol extraction was washed with 5 ml of sodium hydroxide (1.0 N) to remove phenolic metabolites, and an aliquot of the organic phase was counted to quantitate the amount of unmetabolized 14Cbromobenzene in tissue. Five milliliter of 20% trichloroacetic acid were added to the homogenate after the third heptane:isoamyl alcohol extraction and the mixture was allowed to stand at 4°C for 1 hr. Following centrifugation (2OOOg for 20 mm) and removal of the trichloroacetic acid the precipitate was suspended in methanol at 60°C and shaken vigorously for 10 min. After five extractions with hot methanol significant amounts of radioactivity could no longer be removed from the precipitate by further extractions. The methanol was removed after centrifugation (2OOOg for 10 min) and the precipitate was dissolved in 5 ml of sodium hydroxide ( 1.0 N). Aliquots were taken for liquid scintilla-

AROMATIC

HYDROCARBONS

AND

HEPATIC

83

NECROSIS

tion counting and determination of protein concentration (Gornall et al., 1949), and results were expressed as nmole of radioactive material bound per mg of protein. “Blank” values obtained by adding 1 &i of W-labeled hydrocarbon to 5 ml of normal liver homogenate at 4°C were less than 30 cpm, whereas tissue samples which bound 1 nmole of radioactivity per mg protein gave counts in the range of 2000-3000 cpm, depending on the specific activity. The above method was compared with that described by Nebert and Bausserman (1971) for measuring the covalent binding of aryl hydrocarbons to protein and nucleic acid. Values obtained by the two methods for the amount of covalently bound radiolabeled material in duplicate samples of liver from a ‘C-bromobenzene-treated mouse differed by less than 6%. The data obtained by the method described above also agree with the vaiues for covalent binding obtained by the longer procedure of Krishna et al. (1971) and may therefore be interpreted to indicate covalent binding to liver proteins. RESULTS Distribution of covalently bound 14C-bromobenzene. Twenty-four hours after administration of ‘*C-bromobenzene to mice a considerable amount of radiolabeled material was bound to protein in liver, kidney and, to a lesser extent, in lungs (Table I ) . The amount of binding in the ileum was roughly comparable to that in plasma proteins, whereas in other organs the binding was less than in plasma. It is noteworthy that the distribution of binding correlates well with the activities of microsomal cytochrome P-450 drug-metabolizing enzymes in various tissues (see Conney, 1967). It is not known whether the radioactivity in the plasma was bound to a normal constituent of the plasma or to a protein released from liver or other organs as a result of the toxic action of bromobenzene. Time and dose-response relationships between “C-bromobenzene binding and hepatic necrosis. The time course of accumulation of covalently bound radioactivity in mouse liver after administration of a hepatotoxic dose of 14CTABLE DISTRIBUTION

OF COVALMTLY

Bourw

I ‘IC-BK~MOI~I~:~~ENI,:

Covalent (nmoles/‘mg

Tissue ..-

killed

24 hr

THIS: Rlous~.:a

hinding protein)

--

Liver Kidney Lung Plasma Ileum Spleen Heart Stomach Brain Testis * Mice were (4.8 mmoles/kg).

IN

1.890 1 x38

0.345 0.130 0.113 0.061 0.049 0.049 0.031 0.030 after

administration

of a hepatotoxic

dose

of W-bromobenzene

84

HOURS

REID

AND KRISHNA

AFTER

“C-BROMOEENZENE

ADMINISTRATION

Fro. 1. Covalent binding of “C-bromobenzene to proteins of mouse liver in V&O. Each point represents a mean value of 3-6 mice; bars indicate standard errors. Broken line represents mice which receive a “tracer” dose of ‘“C-bromobenzene (0.41 mmoles/kg, 20 /Xi/ mouse). Solid line indicates animals which received a hepatotoxic dose of l’C-bromobenzene (6.53 mmoles/kg). Livers from animals which received the high dose showed centrolobular glycogen loss at 16 hr and centrolobular necrosis at 24 and 40 hr.

bromobenzene (6.53 mmoles/kg) is shown by the solid line in Fig. 1. There was a progressive increase in the amount of binding until a maximum value was reached at about 12-16 hr, when histopathological changes first began to develop in the centrolobular areas of the liver. After 24 hr the amount of covalently bound material diminished, coincident with the infiltration of necrotic tissue by macrophages, and by 96 hr only minimal amounts of bound radioactivity remained in the liver. In contrast, an order of magnitude less radioactive material was bound after administration of a non-toxic “tracer” dose of l*C-bromobenzene (0.41 mmoles/kg) (Fig. 1). It was subsequently demonstrated (Table II) that after all subnecrogenic doses of 14C-bromobenzene the maximum binding occurred within the first few hours and decreased slowly thereafter, whereas with hepatotoxic doses the binding was greater at 24 hr than at 5 hr. The autoradiographic correlates of the above data are shown in Fig. 2. Twenty-four hours after administration of a “tracer” dose of W-bromobenzene (0.41 mmoles/kg) a low level of radioactivity was evident throughout the liver lobule with a slightly greater accumulation in the centrolobular region than around the portal triad (Figs. 2a and b). Similar autoradiograms were obtained during the first 5 hr after administration of a hepatotoxic dose of 14C-bromoTABLE

II

BROMOBENZENE BOUND TO LIVER PROTEIN AT 5 AND 29 HR AFTER VARIOUS DOSES OF W-BROMOBENZENE Dose (mmoles/kg) 0.24 0.72 1.20

2.15 4.06 8 P < 0.02.

b P < 0.01.

“C-Bromobenzene (nmole/mg 5 hr f 0.040 f 0.045 f 0.052 0.947 f 0.396 1.094 f 0.310 0.126 0.437 0.706

bound to liver prot,ein protein f SE) 24

f f i 1.180 f 2.528 + 0.082 0.184 0.335

Change 7%

Centrolobular necrosis

hr 0.006 0.046" O.OOBb

-36 -58

0.036

+2.5 f131

0.085"

-53

none none none minimal extensive

AROMATIC

HYDROCARBONS

AND

HEPATIC

NECROSIS

85

benzene (4.7 mmoles/kg; Figs. 2c and d). However, by 16 hr after the toxic dose there was a marked accumulation of convalently bound 14C-labeled material in the centrolobular zone (Figs. 2e and f). The concentration of radioactivity in the centrolobular zone was even more striking at 24 and 48 hr, when extensive centrolobular necrosis had developed (Figs. Bg-j). Thus, the progressive increase in covalent binding between 5 and 24 hr after administration of hepatotoxic doses of 14C-bromobenzene occurs mainly in the centrolobular zone. The centrolobular distribution of both the binding and necrosis indicates that these two events are related. Relationship between bromobenzene metabolkm, covalent binding and hepatic necrosis. A previous paper demonstrated that prior treatment with phenobarbital enhances the metabolism of bromobenzene in vivo, accelerates the onset and increases the severity of bromobenzene-induced centrolobular necrosis, whereas pretreatment with SKF 525-A or piperonyl butoxide produces the opposite effects (Reid et al., 1971a). Table III shows that treatment of mice with piperonyl butoxide reduced by 80% the amount of covalently bound radioactive material in the liver 5 hr or 12 hr after administration of 14C-bromobenzene (1.25 mmoles/ kg), suggesting that the binding is due to a metabolite. In contrast, prior administration of phenobarbital produced a twenty-fold increase in covalent binding 5 hr after a hepatotoxic dose of 14C-bromobenzene (4.85 mmoles/kg), a finding consistent with the interpretation that prior induction of hepatic microsomal enzymes enhances the formation of a bromobenzene metabolite capable of alkylating liver proteins. However, with a lower dose of I%-bromobenzene (1.5 mmoles/kg) phenobarbital pretreatment did not significantly alter TABLE EFFI,:CT -~~~

OF PIPERONYL lJC-BROMOUISNZENE

III

BUTOXIDE BINDING

OH PH~NOUAHUITAL IN ItIous~ LIVER

ON

~~~ ~-~

Dose

of

bromobenzene (mmoles li kg! 1.25 1 ..io

Time of killing h)

12 ;5

Covalent binding to liver protein* (Im~oles!mg protein f SE)

Colltrol 0.604 zk O.O.i7 (15)‘)

1.50 4.8.i

.3

r, .5

0.193

Piperonyl 0.124

butoxide

+

(16)

0.011~

0.146

1.054 f

0.1::

Phelwbarbital

Change in covalent binding

+

(1.i)

-86%

o.ooY

-

0.0743 f 0.0096 (12)

0.02’74 zt o.cow

0.794 f 0.144

0.9X9 f o.%‘w (16)

-

(14)

0.443 f 0.084

9.829 f 1.261c i!)i

-

(II)

(121

8 Values indicate means + SE. b Numbers in parentheses indicate number of mice. e P < 0.001. 6 P > 0.1.

(,I01

-62%

86

REID

AND

KRISHNA

FIG. 2. Comparison of centrolobular with periportal areas of mouse liver in paraffin section autoradiograms stained with PAS and hematoxylin. In order to provide maximum resolution of the autoradiograms the focus is on the exposed grains of emulsion, and consequently some of the cellular details of the underlying tissue sections appear somewhat blurred (e.g., Figs. 2C, D, and H ). Animals were killed at various times after bromobenzene-“C: (A) 24 hr after tracer dose (0.47 mmoles/lcg), centrolobular area; (B) periportal area in same section; (C) 5 hr after hepatotoxic dose (4.7 mmoles/kg), centrolobular area; (D) periportal area in same section; (E) 16 hr, hepatotoxic dose, centrolobular area; (F) periportal area in same section; (G) 24 hr, hepatotoxic dose, centrolobular area; (H) periportal area in same section; (I) 48 hr, hepatotoxic dose, centrolobular area; (1) periportal area in same section.

the binding, and after a “tracer” dose of the labeled hydrocarbon (0.13 mmoles/ kg) the amount of binding was actually lower in the phenobarbital-treated animals than in the controls. The latter results are difficult to interpret, but may indicate that in addition to increasing the rate of formation of a toxic metabolite, phenobarbital also enhances the enzymatic inactivation of the toxic intermediate, and that presumably the K, of inactivation is lower than that of formation of the toxic metabolite. In accord with this interpretation, Oesch et al., ( 1971) h ave shown that phenobarbital administration induces epoxide hydrase, a microsomal enzyme which catalyzes the hydrolysis of various aromatic epoxides. Various halogenated benzene derivatives were used to investigate the relationship between metabolism, covalent binding and hepatic necrosis in the rat. We have reported that bromo-, chloro-, iodo- and ortbo-dichlorobenzene elicit hepatic necrosis in rats and that the hepatotoxic action of these comuounds is increased by pretreatment with phenobarbital (Reid et at., 1970; Brodie et al., 1971). In contrast, para-dichlorobenzene is not hepatotoxic to control or phenobarbital-treated rats, and fluorobenzene produces no necrosis in controls and only occasionally causes centrolobular necrosis in phenobarbital-induced animals surviving an LD50 dose. The experiment shown in Table IV compares the binding of bromobenzene, chlorobenzene and iodobenzene with that of fluoro-

AROMATIC

HYDROCARBONS

FIGS.

AND HEPATIC

NECROSIS

87

2C, D, E, and F

benzene at a dosage (1 mmole/kg) where the bromo-, chloro- and iodo- derivatives caused minimal histological changes in the control animals and elicited centrolobular necrosis without mortality in the phenobarbital-treated rats. To circumvent the possible interpretation that the amount of covalent binding was determined soleIy by the presence or absence of necrotic tissue, the binding of ortho- and para-dichlorobenzene was compared at a dose (0.5 mmoles/kg) at which the ortho-derivative was not hepatotoxic even after phenobarbital administration. The covalent binding in liver of control animals was considerably higher after administration of the hepatotoxic compounds (bromo-, chloro-,

88

REID AND KRISHNA

FIGS. 2G, H, I, and J

iodo- and ortho-dichlorobenzenes) than after the nontoxic derivatives (lluoroand para-dichlorobenzenes ) . Furthermore, phenobarbital administration increased the binding of only the hepatotoxic compounds, and (with the exception of ortho-dichlorobenzene) only at the 5 hr time point. The observation that the liver of phenobarbital-treated rats is exposed to a high concentration of a chemically reactive metabolite over a relatively short period of time may explain the accelerated onset and increased severity of the centrolobular necrosis in these animals. Table IV also shows that the increased binding induced by phenobarbital was prevented by giving the animals SKF 525-A 2 hr before

AROMATIC

HYDROCARBONS TABLE

EFFECT

OF PHENOBARBITAL OF HAL~GENATED

Hepatotoxin

AND HEPATIC IV

AND SKF 536-A ADMINISTRATION BENZENE DERIVATIVES TO LIVER

Time of killing

89

NECROSIS

Binding

ON COVALENT

BINDING

PROTEIN IN VIVO

of hepatotoxin

to liver protein8

Control (rimoles/mg protein f SE)

Phenobarbital (nmoles/mg protein f SE)

Phenobarbital + SKF 525-A” (nmoles/mg protein f SE)

Bromobenselle-1°C (1 mmole/kg)

6 hr 24 hl

0.267 f 0.034 0.534 f 0.050

o..YLl f p.031c 0.632 f 0.121

0.36

ChlorobenzeneJ*C (1 mmole/kg)

6 hr 24 hr

0.364 f 0.03 0.604 f 0.044

1.268 f 0.278” 0.564 f 0.04

0.483 i 0.144e

Iodobenzene-14C (1 mmole/kg)

6 hr 24 ht

0.090 f 0.013 0.323 f 0.054

0.545 f 0.129” 0.360 h 0.093

0.666 f 0.304

Fluorobenzene-“C (1 mmole,‘kgj

6 hr 24 hl

0.08.5 Z!Z0.013 0.060 f 0.004

0.054 31 0.008 0.036 f 0.003”

0.052 f 0.005

o-Dichlorobenzene-1% (0.5 mmole/kgj

6 hl 24 hl

0.234 zt 0.015 0.135 f 0.011

0.308 f 0.038 0.207 f 0.008

0.186 zk 0.0140

p-Dichlorobenzene-1% (0.5 mmole/kg)

6 hr 24 hr

0.021 f 0.002 0.010 f 0.002

0.012 f 0.001” 0.012 f 0.001

0.006 + 0.001”

f 0.024d

s Values are the mean of six rats f SE. b SKF 525-A (75 mg/kg ip) was given 1 hr before the hepatotoxin. c P < 0.01 compared with controls. d P < 0.01 compared with phenobarbital alone. 0 P < 0.02 compared with phenobarbital alone. bromo, chloro-, or ortho-dichlorobenzene, although the drug did not reduce the binding nor the hepatic concentration (vide infra) of iodobenzene. The above data are compatible with the interpretation that covalent binding is directly related to the rate of metabolism of the hydrocarbon. This interpretation is supported by Table V which presents the hepatic levels of unmetabolized hydrocarbon and the total urinary excretion of metabolites of the administered hydrocarbon. Tissue levels and urinary metabolites were not measured in Wchlorobenzene-treated rats. Statistical analysis was not attempted on the data in urine because the values represent an average of two pools of three rats each. Phenobarbital treatment accelerated the disappearance from liver of bromo-, iodo- and ortho-dichlorobenzene and increased the urinary excretion of metabolites of these compounds. The effects of phenobarbital were blocked in the case of bromo- and ortho-dichlorobenzene by administration of SKF 525-A. These findings suggest that phenobarbital increased, and SKF 525-A reduced, the rate of hydrocarbon metabolism. The data in Table V also provide an insight into why fluorobenzene and para-dichlorobenzene were not hepatotoxic and did not bind to liver protein (see Table IV). Tissue levels of fluorobenzene were very low, even at 6 hr, while the 24hr urinary excretion of fluorobenzene metabolites was about half that of bromobenzene. It is possible that rapid pulmonary excretion of fluorobenzene reported by Azouz et al. (1952) reduced the amount of hydrocarbon metabolized to a toxic metabolite in the liver. In

90

REID

EFFECTS

Hepatotoxin

AND

KRISHNA

TABLE

V

OF PHENOBARBITAL AND SKF 525-A OF HEPATOTOXINS IN RATS Time of killing

Hepatic

concentration excretion

of hepatotoxin of its metabolites Phenobarbital

Control

Hepatic concen-

Metab-

Hepatic

olites ex-

tration*

ON METABOLISM

(nmolek f

Hepatic

olites ex-

elites

tration

ex-

creted

(nmole/ g (%

of dose)

f

SE)

+

Metab-

concen-

creted

(%

SE)

Phenobarbital SKF 525-A

Metab-

concentration

cretedb

(W

and urinary

bmole/g

of dose)

f 198.3

(%

SE)

of dose)

Bromobenzene-W (1 mmole/kg)

6 24

183.6 i 7.6 f

40.5 0.7

11 49

26.3 f 9.5 i

5.3 1.5

31 75

Iodobenzene-14C (1 mmole/kg)

6 24

154.0 f 7.2 f

18.6 0.9

10 65

47.8 f 12.6 f

29.7” 1.5”

34 73

18.1 f

6.5

22

Fluorobenzene-W (1 mmole/kg)

6 24

1.3 f 0.09 f

0.3 0.04

11 28

0.17 f 0.10 f

0.04 0.02

14 31

2.4 f

1.2

8

o-DichlorobenzenGC (0.5 mmole/kg)

6 24

71.0 f 16.6 f

26.5 2.0

17 42

31.7 f 12.0 f

p-Dichlorobenzene-W (0.5 mmole/kg)

6 24

178.7 f 23.0 f

14.6 2.8

6 29

a Values for b Values for c P < 0.01 d P < 0.01 e P < 0.02 f P < 0.05 g P < 0.05

293.3 20.1

f f

5.0 0.5’

34 74

13.1c 3.5

8 24

hepatic concentrations are the means of six determinations urinary metabolites are the means of two pools of three compared with controls. compared with phenobarbital alone. compared with controls. compared with controls. compared with phenobarbital alone.

rt 31.6d

58.6 290.4

f f

17

8.76 21 62.6

of individual rats.

10

rats.

contrast, para-dichlorobenzene disappeared more slowly from tissues than did the ortho-derivative, and the urinary excretion of para-dichlorobenzene metabolites was less than that of the hepatotoxic hydrocarbons. The apparently slow TABLE EFFECT

OF PYRAZOLE OH SKF OF ~4C-C~Lo~o~~~z~~~~ Number animals

Treat,ment*

525-A

of

VI

ADMINISTRATION ON COVALENT TO LIVER PROTEINS OF MICE Covalent

binding

Significance

(nmoles/mg protein f SE)

--

Saline Pyrazole SKF 52.5-A

a Pyrazole Chlorobenzene

(375 mg/kg) (1 mmole/kg),

-

10 IO 5

iPI

0.364 f 0.053 0.0.53 xk 0.001 0.217 f 0.030

and SKF 525-A (75 mg/kg) were and animals were killed 6 hr later.

BINDING


1 hr before.

14C-

AROMATIC

HYDROCARBONS

AND

TABLE EFFECT

OF PYKAZOLE: BIIVDING

Pretreatment8

(@moles/g 0.750 1.829

* Pyrazole (375 mg/kg, mmoles/kg), and the groups b P < 0.001.

91

NECROSIS

VII

ADMINISTRATION ON W-BROMOBENZENE METABOLISM, AND CENTROLOBULAR NIxROSIS IN RATS

W-Bromobenzene concentration in liver (unbound)

Saline Pyrazole

HEPATIC

f f f

24-hr urinary excretion of W-metabolites

SE)

(pmoles/rat f SE)

0.154 0.08jb

299 f 98 f

Covalent binding in liver

Centrolobular necrosis

(nmoles/mg prot,ein f SE) 13 13b

2.761 1.146

f 0.191 =t 0.045b

ip) or saline was administ.ered 1 hr before of six rats were killed 24 hr later.

Extensive Minimal

14C-bromobenzene

(7.14

rate of para-dichlorobenzene metabolism (Table V) may explain the minimal covalent binding in liver (Table IV) and the lack of hepatotoxicity (KochWeser et al., 1953; Brodie et al., 1971) of this compound. Probably both the rate and pathway of metabolism are important determinants for the amount of covalent binding and tissue necrosis induced by halogenated aromatic hydrocarbons. Further

evidence

for a relationship

between

covalent

binding

and centro-

lobular necrosis. Other drug treatments which blocked 14C-bromobenzeneinduced hepatic necrosis were found to reduce markedly the covalent binding in liver. Treatment with pyrazole inhibited the covalent binding of radiolabeled metabolities in mouse liver 6 hr after 14C-chlorobenzene administration (1 mmole/kg) to a greater extent than did SKF 525-A (Table VI). In rats, prior administration of pyrazole markedly reduced the severity of centrolobular necrosis 24 hr after administration of 14C-bromobenzene (7.14 mmoles/kg, TABLE EFFECT

Species

VIII

OF AMINOACETONITRILE ON COVALENT HEPATOTOXICITY OF W-BROMOBENZFJNE

Pretreatment8

Number of animals

Bromobenzene dose (mmoles/kg)

Mice

Rats

after

6 Animals the last

BINDING

Covalent

binding

Centrolobular necrosis

(nmoles/mg protein f SE)

Saline Aminoacetonitrile Saline

3 4

1.9 1.9

0.817 0.240

f f

1.76 0.18b

Moderate None

9

5.0

2.34

f

0.15

Aminoacetonitrile

9

5.0

1.26

f

0.07”

Moderate to extensive Minimal

were killed 24 hr after administration of ‘4C-bromobenzene dose of saline or aminoacetonitrile as described in Methods.

bP < 0.05. OP < 0.01.

AND

which

was given

2 hr

92

REID

AND

KRISHNA

TABLE EFFECT

IX

OF DIETHYL MALIATF, TREATMENT ON GLUTATHIONE CONCENTRATION, COVALENT BINDINQ OF W-BROMOBENZENE AND CENTROLOBULAR NECROSIS IN MOUSE LIVER*

Measurement

Pretreatment

Hours 0

after

W-bromobenzene 3

Saline Diethyl maleate

4.30 f 0.04b 2.88 f O.llb

2.57 2.50

W-Bromobenzene binding (nmoles/ mg protein)

Saline Diethyl maleate

0 0

0.44 f 0.06

Centrolobular

Saline

None

Diethyl maleate

None

Glutathione tration of liver)

concen(pmoles/g

necrosis

8 Values are the mean of three animals pooled values of three animals treated with

1.11 f

0.3.5

administration

f SE; glutathione W-bromobenzene

28

3.89 2.41

5.03 3.42

0.76 f 0.40 0.82 f 0.18 2.76 f 0.23b 1.93 f 0.70

None

Glycogen loss

12

None

Minimal to moderate

concentrations 1.92 mmoles/kg.

without

Glycogen loss and minimal necrosis

Extensive necrosis SE represent

b P < 0.05.

ip) and inhibited the covalent binding of radioactivity by more than 50yo (Table VII). Pyrazole administration also more than doubled the concentration of unmetabolized 14C-bromobenzene in liver and reduced by two-thirds the 24-hr urinary excretion of l’C-bromobenzene metabolites (Table VII ) . These data suggest that pyrazole blocked bromobenzene-induced necrosis and binding in liver by inhibiting the metabolic conversion of bromobenzene to an active metabolite. Fiume (1963) h as reported that prior treatment with a lathyrogenic compound, aminoacetonitrile, blocks the necrogenic action of bromobenzene. Table VIII confirms that treatment with aminoacetonitrile prevented or markedly reduced the severity of hepatic necrosis induced by bromobenzene (1.9 or 5.0 mmoles/kg) and demonstrates that the lathyrogen inhibited the hepatic binding of *%-bromobenzene in rats and mice. Unlike pyrazole, SKF 525-A and piperonyl butoxide, aminoacetonitrile did not significantly alter either the tissue levels of 14C-bromobenzene or the urinary excretion of metabolites, indicating that the protective effect of the lathyrogen was not related to a decrease in the overall rate of 14C-bromobenzene metabolism. To determine whether hepatic necrosis and covalent binding would be increased by prior depletion of liver glutathione, 14C-bromobenzene ( 1.92 mmoles/ kg) was administered after treatment with diethyl maleate. At the time of 14Cbromobenzene administration hepatic glutathione levels were moderately reduced in animals treated with diethyl maleate (Table IX). Glutathione was reduced to the same concentration in both groups 3 hr after treatment with ‘C-bromobenzene, but the customary rebound increase in glutathione levels 24 hr after bromobenzene administration (Binet and Weller% 1951) was not observed in the diethyl maleate treated mice. Treatment with diethyl maleate

AROMATIC

HYDROCARBONS

AND

HEPATIC

NECROSIS

93

FIG. 3. Parafhn section autoradiograms of Iivers of mice killed 30 min after iv injection of l’C-r.-leucine (500 &i/kg; SA 10 mCi/mmole). (A) Uniform distribution of radioactivity throughout lobule in animal which received no bromobenzene. (B) Six hours after administration of a hepatotoxic dose of unlabeled bromobenzene (3.8 mmoles/kg) there is decreased radioactivity in the centrolobular zone. No necrosis is evident. (C) Forty-eight hours after bromobenzene administration centrolobular necrosis is evident. The concentration of radioactivity is decreased in centrolobular area and increased in periportal area relative to control shown in (A).

not only accelerated the onset and increased the severity of 14C-bromobenzeneinduced centrolobular necrosis, but also more than doubled the hepatic concentration of covalently bound radiolabeled material. These results are com-

94

REID

AND KRISHNA

patible with the interpretation that by reducing the amount of glutathione available to inactivate bromobenzene epoxide, diethyl maleate enhanced the alkylation of liver proteins by the toxic metabolite. To summarize the effects of various unrelated drug treatments on bromobenzene-induced hepatic necrosis and covalent binding, agents which inhibited covalent binding blocked the necrosis, whereas compounds which enhanced the binding potentiated the tissue lesions. These results strongly suggest that covalent binding and centrolobular necrosis are related events. Effects of bromobennene administration on the accumulation of 14C-deucine in mouse Liver. At 6-, 24- or 48-hr intervals after administration of a hepatotoxic dose of unlabeled bromobenzene (3.8 mmoles/kg), 14C-L-leucine (566 &i/kg) was injected intravenously and the animals were killed 30 min later. Autoradiograms of paraffin sections of liver revealed an even distribution of labeled material throughout the liver lobule in control mice not treated with bromobenzene (Fig. 3a). As early as 6 hr after bromobenzene administration there was a striking decrease in 14C-L-leucine accumulation in the centrolobular zones in the absence of any histopathology (Fig. 3b). By 24 hr and 48 hr there was a striking deficit of label in the necrotic centrolobular hepatocytes (Fig. 3c), and at 48 hr there also appeared to be a greater periportal accumulation of 14C-~leucine than in the controls (cf. Figs. 3a and c). These results suggest that bromobenzene blocked, the incorporation of leucine into proteins before histological evidence of centrolobular necrosis developed. DISCUSSION The above studies demonstrate that after administration of 14C-bromobenzene or other hepatotoxic aromatic hydrocarbons a considerable amount of radiolabeled material becomes tightly bound to macromolecules in the liver, The persistence of the binding after extensive solvent extraction strongly suggests, but does not prove, that the binding is covalent. In the autoradiographic studies, no precautions were taken to prevent the extraction of unbound radiolabeled material into the solvents used to prepare the paraffin sections, and it may be assumed that most of the exposed grains of the emulsion indicate radioactive molecules covalently bound to the tissue sections. Proof that a bromobenzene metabolite binds covalently to specific amino acids of liver proteins has been presented by Krishna and co-workers (1971) and will be published in the near future. The close agreement between data obtained by our method of determining binding and the results of Krishna et al. (1971, and unpublished data) validates the assumption that our results indicate covalent binding. Several aspects of the covalent binding of hepatotoxic compounds in the liver deserve comment. The first is the relatively large amount of binding. Radiolabeled material is bound almost exclusively to proteins after administration of a hepatotoxic dose of 14C-bromobenzene (Krishna et al., 1971) . If we assume, that the average protein in liver has a molecular weight of 266,096 we can calculate that at 24 hr after 14C-bromobenzene administration approximately 2 nmoles of radiolabeled material are bound per 5 nmoles of liver protein (Fig. 1). Moreover, the vast majority of the binding occurs in the necrotic centrolobular zone (Figs. 2g and h), which represents only about 25% of the cross-sectional area or approximately +Jsqof the volume of the spherical lobule.

AROMATIC

HYDROCARBONS

AND HEPATIC

NECROSIS

95

Thus, in the centrolobular zone an average of more than one molecule of labeled material is bound to each protein molecule. Secondly, the covalently bound radioactive material is probably a metabolite of 14C-bromobenzene ( or of other hepatotoxins ), since 14C-bromobenzene added to liver homogenates at 4°C did not cause covalent binding, Furthermore, Corsini et al. ( 1971) have demonstrated that 14C-bromobenzene must be metabolized in order to bind to hepatic microsomal proteins in vitro. The data in Tables III, IV and V show that the extent of covalent binding and of centrolobular necrosis produced by 14C-bromobenzene or other hepatotoxic aromatic hydrocarbons depends upon the rate of metaboIism of the hepatotoxins. These results indicate that both the covalent binding and the necrosis are caused by a metabolite. A major pathway in the metabolism of aromatic hydrocarbons is the formation of a complex between their respective epoxides and glutathione. The toxic metabolite of bromobenzene has been postulated to be bromobenzene epoxide (Brodie et al., 1971), the presumed highly reactive precursor of the phenol, catechol and mercapturic acid metabolites of bromobenzene (Booth et al., 1960; Jerina et al., 1968a, b; 1970). In accord with this view, halogenated aromatic hydrocarbons can be converted to intermediates that react covalently with glutathione in vitro (Booth et al., 1961; Brodie et al., 1971) and form bromophenyl mercapturic acid in z;ivo (Baumann and Preusse, 1879; Jaffh, 1879). It is conceivable that bromobenzene epoxide may produce tissue damage by alkylating nucleophilic sites on liver proteins, since in vitro studies by Corsini et al. (1972) have shown that a metabolite of 14C-bromobenzene binds covalently to microsomal protein in the presence of oxygen, liver microsomes, and a NADPH-generating system, and that little binding occurs if the metabolism of 14C-bromobenzene is blocked by SKF 525-A or carbon monoxide. Koch-Weser et al. (1953) sh owed that in contrast to ortho- or meta-disubstituted compounds, para-disubstituted benzene derivatives, presumably because they cannot form sufficiently stable epoxides, are not hepatotoxic and are not excreted as mercapturic acid derivatives. The observation that paradichlorobenzene, which does not form a mercapturic acid, is not hepatotoxic and produces considerably less covalent binding than ortho-dichlorobenzene is compatible with the view that epoxides are responsible for the covalent binding and hepatotoxic effects of aromatic hydrocarbons. Moreover, administration of para-dichlorobenzene, unlike ortho-dichlorobenzene, does not reduce the glutathione concentration in rat liver (Reid, 1972b). The postulate that the liver necrosis is mediated by an active epoxide is strengthened by the finding that phenobarbital administration increases the rates of excretion of p-bromophenyl mercapturic acid (Reid et al., 1971a) and of depletion of hepatic glutathione after bromobenzene administration (D. Jollow and J. R. Gillette, unpublished data). These observations probably reflect an enhanced rate of formation of bromobenzene epoxide. When microsomal enzymes are induced by phenobarbital it is possible that formation of epoxide exceeds the synthesis of glutathione, which may protect tissue sulfhydral groups from alkylation by the epoxide, and as a result more epoxide is free to attack tissue proteins. In accord with this view, prior administration of diethyl maleate, which depletes glutathione in liver (Boyland and Chasseaud, 1970), markedly

96

REID

AND KRISHNA

increases the covalent binding and hepatic necrosis caused by IOW doses of bromobenzene (Table IX). In contrast, prior administration of cysteine, the precursor of glutathione, probably increases the conversion of bromobenzene epoxide to p-bromophenyl mercapturic acid and thereby reduces bromobenzeneinduced hepatotoxicity ( Koch-Weser et al., 1~). Several other experimental observations are compatible with the view that centrolobular necrosis may be caused by the covalent binding of metabolites of hepatotoxins to liver proteins. Both the binding and the necrosis are dose dependent (Table II), and the peak level of binding precedes the development of necrosis by several hours (Fig. 1, and Reid et al., 1971a). Furthermore, hepatotoxic aromatic hydrocarbons exhibit a greater degree of covalent binding than do nontoxic compounds (Table IV), and various unrelated drug treatments exert parallel effects on binding and necrosis (Tables III, IV, VI, IX). In addition to the drug treatments reported in this paper, 3-methylcholanthrene blocks bromobenzene-induced hepatic necrosis and covalent binding in rats (Reid et al., 1971c). Methylcholanthrene increases the activity of epoxide hydrase, the microsomal enzyme which catalyzes the conversion of aromatic epoxides to their corresponding dihydrodiols (Oesch et al., 1971) and presumably exerts its protective effect by enhancing the inactivation of the epoxide (Zampaglione et al., 1971; Reid et al., 1971c). The factors which determine the intrahepatic distribution of covalent binding and drug-induced necrosis are not entirely evident. It has been proposed that tbe intrahepatic localization of the enzymes which produce toxic metabolites may determine the distribution of necrosis (Rees and Tarlow, 1967) and of covalent binding (Reid, 1972a). There is now abundant evidence that many enzymes and even subcellular organelles are preferentially concentrated in either the centrolobular or periportal hepatocytes (see Rappaport, 1963). Ally1 formate and ally1 alcohol elicit periportal necrosis (Piazza, 1915; Eger, 1964) and are metabolized to the reactive aldehyde, acrolein, by a nonmicrosomal enzyme, alcohol dehydrogenase, localized in periportal hepatocytes (Rees and Tarlow, 1967). After administration of 14C-ally1 alcohol, covalent binding of radiolabeled material is concentrated mainly in periportal hepatocytes, and both the binding and periportal necrosis are blocked by prior inhibition of alcohol dehyarogenase by pyrazole (Reid, 1972a). In the case of 14C-bromobenzene-induced hepatic necrosis, the centrolobular distribution of the lesions might result from the enzymatic production in centrolobular hepatocytes of a highly reactive and unstable metabolite which alkylates nearby proteins. The intrahepatic site of bromobenzene metabolism is not known, but histochemical studies reveal a centrolobular distribution for several cytochrome P-450 dependent drug metabolizing enzymes (Wattenberg and Leong, 1962; Koudstaal and Hardonk, 1969, 1970). Furthermore, phenobarbital causeshypertrophy of the smooth endoplasmic reticulum (Remmer and Merker, 1963) mainly in the centrolobular zone (Burger and Herdson, 1966). We have stressed in other publications (Reid et aZ., 1971a, c; 1972) thar halogenated aromatic hydrocarbons are not unique in their conversion to “active” metabolites and that metabolites other than epoxides may react covalently to produce tissue lesions. For example, the quinone of naphthalene has been postulated to produce brown cataracts by binding to proteins in the lens (van Heyningen and Pirie, 1967), and the hepatotoxicity of carbon tetra-

AROMATIC

HYDROCARBONS

AND

HEPATIC

97

NECROSIS

chloride is believed to be mediated by a toxic metabolite (Slater, 1966; Recknagel, 1967; Seawright et al., 1968). We anticipate that metabolism and covalent binding may eventually be imphcated in the pathogenesis of a wide variety of tissue lesions induced by industrial solvents, pesticides, herbicides, chemical pollutants and therapeutic drugs. Our preliminary results suggest that covalent binding of metabolites of chlorobenzene or chloroform in the proximal convoluted tubules leads to renal tubular necrosis in mice (Reid and Ilett, 1972; Reid, 1972c). Metabolites of bromobenzene and naphthalene also bind covalently to the bronchial epithelium where they elicit tissue necrosis (Reid, 1972b, c). Drug metabolism probably plays an important role in the bone marrow aplasia induced by administration of benzene or dimethylbenzanthracene (Ikeda and Ohtsuji, 1971; Suria et al., 1971). Recent experimental studies indicate that liver necrosis caused by at least one therapeutic drug, acetaminophen, is mediated through the covalent binding of a toxic metabolite (Gillette, 1972). Similarly, allergic responses to drugs may be mediated through antigens formed by the reaction of body proteins with trace amounts of reactive drug metabolites (Ledvina, 1969). It is hoped that the accumulation of knowledge concerning the toxic mechanism by which foreign compounds produce tissue lesions will eventually make it possible to design therapeutic agents which are devoid of this type of serious side effect. ACKNOWLEDGMENTS We thank Mr. John George and Miss Cathy Dr. James R. Gillette and Dr. Bernard B. Brodie Greulich and Mrs. Mary Alice Larson for making

Lalush for their expert technical assistance, for their helpful advice, and Dr. Richard C. possible the autoradiographic studies.

REFERENCES I n h’b’t’ ANDERS, M. W. ( 1968). 1 1 ion of microsomal drug metabolism by methylenedioxybenzenes. Biochem. Pharmucol. 17,2367-2370. AXELROD, J., REICHENTHAL, J., and BRODIE, B. B. (1954). Mechanisms of the potentiating action of p-dimethylaminoethyl diphenylpropylacetate. .I. Pharmucol. Exp. Ther. 112, 49-63. AZOUZ, W. M., PARKE, D. V., and WILLIAMS, R. T. ( 1952). Studies in detoxication 42. Fluorobenzene. Biochem. J. 50,702-706. AZOUZ, W. M., PARI(E, D. V., and WILLIAMS, R. T. (1955). Studies in detoxication 62. The metabolism of halogenobenzenes, orthoand para-dichlorobenzenes. Biochem. J. 59, 4% 415. BASERGA, R., and MALAMUD, D. ( 1969). “Modern Methods in Experimental Pathology. Autoradiography. Techniques and Applications,” Chap. 2. Harper and Row, New York. BAUMANN, E., and PREUSSE, C. ( 1879). Ueber bromophenylmercaptursaure. Ber. Deut. Chem. Ges. 12, 806-810. BERGMAN, D. E., and PULLMAN, B. (Eds.) (1969). “Physio-Chemical Mechanisms of Carcinogenesis.” Jerusalem Symposia on Quantum Chemistry and Biochemistry, Vol. 1. The Israel Academy of Sciences and Humanities, Jerusalem. BINET, L., and WELLERS, G. ( 1951). Role du glutathion lors de l’intoxication du rat par le monobromobenzene. Bull. Sot. Chim. Biol. 33,279-285. BOOTH, J,, BOYLAND, E., SATO, T. and Sr~s, P. ( 1960). Metabolism of polycyclic compounds 17. The reaction of 1,2dihydronaphthalene and I,2-epoxy-1,2,3,4-tetrahydronaphthalene with glutathione catalyzed by tissue preparations. Biochem. J. 77, 182-186. BOOTH, J., BOYLAND, E., and SIMS, P. (1961). with glutathione. Biochem. J. 79,5X-524. Bou, E., and CHASSEAUD, L. F. (1970). liver glutathione levels. B&hem. Phnrmucol.

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phenobarbital and 3-methylcholanthrene the rat. Fed. hoc. 30, 448.

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J. R. Influence of of bromobenzene in