Hepatic microsomal metabolism and covalent binding of 2,4-dinitrotoluene

TOXICOLOGY

AND

APPLIED

Hepatic

GARY Chemical

Industry

62, 325-334 (1982)

PHARMACOLOGY

Microsomal

Metabolism and Covalent 2,4-Dinitrotoluene

M. DECAD,’ M. ELIZABETH Institute

of Toxicology,

P.O. Box

GRAICHEN, 12137,

Received July 27. 1981; accepted Hepatic Microsomal G. M., GRAICHEN,

Metabolism

M. E., AND DENT,

Research

Binding

AND JOHN G. DENT’ Triangle

September

Park,

North

Toxicol.

Carolina

27709

16, 1981

and Covalent Binding of 2,4-Dinitrotoluene. J. G. (1982)

of

Appl.

Pharmacol.

DECAD,

62, 325-334.

The effects of 2,4-dinitrotoluene (2,4-DNT) on xenobiotic metabolizing enzymes and the hepatic metabolism and covalent binding of this compound to microsomal proteins in vitro were studied. Male Fischer-344 rats received po doses of DNT daily for 5 days at 14, 35, and 70 mg/kg/day. Hepatic oxygen-insensitive cytosolic azoreductase activity was increased and microsomal nitroreductase was decreased by DNT treatments. A small but significant increase in liver/body weight ratio and in hepatic cytochromes P-450 and b, occurred in the absence of changes in microsomal biphenyl hydroxylase or aryl hydrocarbon hydroxylase activities. The patterns of in vitro microsomal metabolism of DNT were dependent on oxygen tension: under aerobic conditions, 2,4-dinitrobenzyl alcohol (DNBAlc) was the major metabolite whereas under anaerobic conditions no DNBAlc was detected; 2-amino-4-nitrotoluene (2A4NT) and 4-amino-2-nitrotoluene (4A2NT) were the major metabolites. Pretreatment of rats with phenobarbital or Aroclor 1254 increased the metabolism of 2,4-DNT to DNBAlc by six- to sevenfold. Metabolism to the alcohol was inhibited by SKF-525A. These data suggested that oxidative metabolism of 2,4-DNT to DNBAlc was mediated by cytochrome P-450-dependent mixed-function oxidases. Covalent binding studies showed that a maximum of only 7 pmol of 2,4-DNT-derived radioactivity was bound per milligram of microsomal protein per hour; this binding was increased to 1.0 nmol bound/mg protein/hr in microsomes from phenobarbital of Aroclor 1254-pretreated rats. It is concluded that 2,4-DNT treatment had little effect on the activity of some hepatic xenobiotic metabolizing enzymes and was readily metabolized by liver preparations in vitro. The pathways of in vitro metabolism were dependent on oxygen tension. This in vitro metabolism produced mostly polar metabolites which did not bind appreciably to microsomal macromolecules.

Technical grade dinitrotoluene a mixture of dinitrotoluene

(DNT)3 is isomers. Of

these, 2,4-dinitrotoluene (2,4-DNT) represents about 15% of the total; 2,6-dinitrotoluene, 20%; and the remainder consists of other dinitrotoluene isomers. Dinitrotoluenes are used in the production of trinitrotoluene and of toluene diisocyanate which is used in the production of polyurethane foams, coatings, and elastomers. Methemoglobinemia, cyanosis, anemia, and jaundice were reported in man as a result of DNT exposure in the workplace (McGee et al., 1942). Acute exposure also led to methemoglobinemia and cyanosis, and

’ Present Address: Dr. G. M. Decad, IBM Corporation, Building 28, 5600 Cottle Road, San Jose, Calif. 95193. ’ To whom correspondence should be addressed. 3 Abbreviations used: DNT, technical grade dinitrotoluene; 2,4-DNT, 2,4-dinitrotoluene; 4A2NT, 4amino-2nitrotoluene; 2A4NT. 2-amino-4-nitrotoluene; 2Ac4NT, 2-(N-acetyl)amino-4-nitrotoluene; DNBAlc, 2,4-dinitrobenzylalcohol; DAT, 2,4-diaminotoluene; SKF-525A, @-diethylaminoethyldiphenylpropylacetate; HPLC, high-performance liqid chromatography; PB, phenobarbital; AR, Aroclor 1254. 325

0041-008X/82/020325-10$02.00/0 Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

326

DECAD,

GRAICHEN,

in some animal species, atrophy of the testes with aspermatogeneis (Ellis et al., 1979). Rats chronically exposed to DNT in the diet for 1 to 2 years developed hepatocellular carcinomas (Ellis et al., 1979; CIIT, 1979). Nitroaromatic compounds are metabolized to nitroso- and hydroxylamino intermediates (Bueding and Jolliffe, 1946; Poirier and Weisburger, 1974) and reactive nitro free radicals (Mason, 1979). The reduction of the nitro groups is catalyzed by a cytosolic

and/or microsomal nitroreductase (Fouts and Brodie, 1957). Different nitroreductases are thought to be present in the cytosol and microsomal fractions of the liver (Kato et al., 1969). Both liver microsomal and cytosolic enzymes reduce 2,4-dinitrophenol under aerobic conditions (Eiseman et al., 1972). Urine is the major route of excretion for metabolites of 2,4-DNT in the rat (Rickert and Long, 198 1). 2,4-Dinitrobenzylalcoholglucuronide, 2,4-dinitrobenzoic acid, 2amino-4-nitrobenzoic acid, and 4-(N-acetyl)-amino-2-nitrobenzoic acid account for 85% of the urinary metabolites. Since 2,4-DNT is hepatocarcinogenic in the rat, the role of hepatic drug-metabolizing enzymes in the activation and/or detoxification of this compound was investigated. The effect of 2,4-DNT treatment in vivo on various hepatic enzyme activities measured in vitro was examined. In addition, the in vitro metabolism and covalent binding of 2,4-DNT to liver subcellular fractions were investigated. METHODS Animals and treatments. Adult male Fischer-344 (CD F/CrlBR) inbred albino rats obtained from Charles River Breeding Laboratories, Inc. (Wilmington, Mass.) were housed four per cage in hanging stainless-steel cages over Deotized Animal Cage Board (UpJohn Co., Kalamazoo, Mich.). All animals had free access to food (Wayne Certified Lab Blox, Allied Mills, Inc., Chicago, Ill.) and water. 2,CDinitrotoluene (99.97% pure by gas chromatography-mass spectroscopy) was a gift from Air Products, Inc., and was administered by gavage to rats (180 to 200 g body wt) between 0800 and 0830 hr for 5 consecutive days. The treatments (four rats per

AND

DENT

dose) were 70, 35, and 14 mg/kg body wt; 2,4-DNT was dissolved in corn oil at concentrations such that each rat received 2.0 ml/kg body wt and controls received an equivalent volume of corn oil alone. Sodium phenobarbital was administered ip at a dose of 80 mg/kg/day for 3 days and rats were killed 24 hr after the last dose; Aroclor 1254, a gift from Monsanto Company (St. Louis, MO.), 500 mg/ml in corn oil, was given in a single ip injection (500 mg/kg) 5 days prior to death. Control rats received ip doses of saline or corn oil. Chemicals. Two sources of ring-labeled [U-‘4C]2,4DNT were used. A preparation having a specific activity of 5.7 mCi/mmol (>99% radiochemical purity), purchased from New England Nuclear Corporation (Boston, Mass.), was used for all experiments except those in which covalent binding of 2,4-DNT in microsomes from control and 2,4-DNT-treated animals was measured. For these experiments, a preparation with a specific activity of 12.0 mCi/mmol(>99% radiochemically pure) obtained from Midwest Research Institute (Kansas City, MO.), was used. Radiochemical purity was confirmed by high-performance liquid chromatography (HPLC). A sample of SKF-525A was a gift of Smith, Kline and French Company (Philadelphia, Pa.). All chemicals and solvents were the best grades commercially available. The scintillation medium was ACS (Amersham Corp., Arlington Heights, Ill.). 2,4-DNT, 2,4-dinitrobenzoic acid (DNBA), and 2,4-diaminotoluene (DAT) were obtained from Aldrich Chemical Company (Milwaukee, Wise.). 2-Amino-4-nitrobenzoic acid (2A4NBA), 2-amino-4-nitrotoluene (2A4NT), and 4-amino-2-nitrotolune (4A2NT) were obtained from ICN Pharmaceuticals, Inc. (Plainview, N.Y.). Purity of the commercially available standards was >99% as assessed by HPLC. 2,4-Dinitrobenzyl alcohol (DNBalc) and 2-amino-4nitrobenzyl alcohol (2A4NBalc) were gifts from the U.S. Army (Environmental Protection Research Division, U.S. Army Medical Research and Development Command, Washington, D.C.) and found to be >99% pure as assessed by HPLC. 4-(NAcetyl)Amino-2-nitrotoluene (4Ac2NT) and 2-(N-acetyl)amino-4nitrotoluene (2Ac4NT) were synthesized as described by Bond and Rickert (1981). Preparation of liver fractions. Rats were killed by cervical dislocation. The livers were quickly removed and washed in ice-cold 1.15% KC], 20 mM Tris-HCl, pH 7.4. Livers were homogenized and microsomes were prepared by differential centrifugation (Dent et al., 1976). Microsomes were resuspended to a final concentration of I5 to 25 mg protein/ml in 20 mM Tris-HCl buffer containing 250 mM sucrose and 5.4 mM EDTA. Protein was determined by the biuret method with an American Monitors Total Protein Kit (American Monitors Co., Indianapolis, Ind.). Enzyme activities. All enzyme activities were measured under conditions which were within the linear range of the assay with respect to time and protein con-

IN

VITRO

METABOLISM

centration. The following assays were performed on freshly prepared microsomes: biphenyl 4-hydroxylase (Creaven et al., 1965) NADPH cytochrome c reductase (Pederson et al., 1973), aryl hydrocarbon hydroxylase (total polar metabolites formed from benzo[ alpyrene) (van Cantford et al., 1977), and pnitrobenzoic acid reductase (Fouts and Brodie, 1957). Methyl red azoreductase was measured in freshly prepared cytosol by the method of Huang et al. (1978). For anaerobic incubations the buffers were degassed under vacuum and then equilibrated with nitrogen; reaction mixtures were placed in test tubes, stoppercd with rubber septums, and placed in a shaking water bath at 37°C. A nitrogen stream was continuously introduced into the head space of incubations through the septum; in addition, the buffer contained an oxygen scavenger system consisting of glucose (10 mM), glucose oxidase (12.5 units/ml), and catalase (150 units/ml). Cytochrome P-450 concentrations (measured from the reduced CO difference spectra), cytochrome b,, and total microsomal heme concentrations (Omura and Sato, 1964) were determined within 48 hr, during which time samples were stored at -80°C. Metabolism of 2,4-DNT. Microsomes or cytosol from control or pretreated rats were incubated in 2.0 ml 66 mM Tris-HCl, pH 7.4, 4 pmol glucose 6-phosphate, 2 units glucose-6-phosphate dehydrogenase, 10 rmol MgC&, 2 rmol NADP, and 200 nmol (1.5 &i) [‘%]2,4-DNT added in DMSO (final concentration, 0.1% DMSO); the final protein concentration was 2 mg/ ml. All incubations were performed in triplicate. Anaerobic incubations were as described above. Other incubations were performed in room air (test tubes open to room atmosphere) or pure oxygen (oxygen introduced into the head space through the rubber septum). All incubations were for 1 hr at 37°C in a shaking water bath. Following incubation, each sample was flash frozen in dry ice-acetone and stored at -8O”C, or clarified through a Whatman aqueous filtration unit (0.22-pm

TABLE EFFECT OF 2,4-DINITR~TOLUENE

Treatment”

h/W Control 14 35 70

Microsomal azoreductas@ 9.13 8.45 8.47 8.94

2 k k k

G 5 days, po. Controls received ’ Methyl red oxygen-insensitive ‘Oxygen-sensitive pnitrobenzoic

’ Biphenyl

4-hydroxylase;

0.89 0.29 0.33 0.19

filter). One hundred microliters of the filtrate was injected onto HPLC column(s). Separations were performed with a Waters Associates high-performance liquid chromatography system at room temperature utilizing a linear gradient of 15 to 70% methanol in 0.01 M NaZHP04, pH 7.4, over 40 min, at a flow rate of 2 ml/min. Two Lichrosorb RP- 18 reverse-phase columns, supplied by E. M. Merck & Co. (Darmstadt, West Germany) connected in series were employed. Radioactivity in the column eluates was detected with a Berthold BP5026 radioactivity monitor equipped with a 200~1 flow-through cell and quantitated by liquid scintillation counting of collected fractions. Retention volumes of metabolites were compared to those of authentic nonradiolabeled standards. Determination of bound radioactivity. Radioactivity bound to microsomal protein was assessed by exhaustive extraction (Sun and Dent, 1980). Liquid scintillation counting. Liquid scintillation counting was performed in a Tracer Mark IV liquid scintillation spectrometer. The automatic external standard channels ratio method allowed conversion of counts per minute to disintergrations per minute. Statistical anal&. Data were analyzed by analysis of variance, completely random design. Where signiticant F values were obtained, tests for significance were performed by the least significant difference test (Steel and Torrie, 1960). The level of significance chosen was p < 0.05.

RESULTS Effect of 2,4-Dinitrotoluene Liver Enzymes

Nitroreductase’

10.45 12.38 14.92 17.23

1.244 0.998 0.990 0.984

1.04 0.89 2.29/ 0.48’

biphenyl

produced metabolites

mg protein-’ produced

AND NITRO-I-REDUCTASES

BP40H“

f 0.088 k 0.067’ f 0.094’ 5~ 0.038’

corn oil only. n = 4, values are X + SEM. azoreductase; nmol 4-(iV,N-dimethylamino)aniline acid nitroreductase; nmol paminobenzoic

nmol 4-OH

on

1

Cytosolic azoreductaseb

’ Aryl hydrocarbon hydroxylase; nmol total polar ‘Significantly different from control at p < 0.05.

Treatment

Treatment with 2,4-DNT resulted in a significant decrease in microsomal anaerobic

PRETREATMENT ON HEPATIC AzoAND MIXED-FUNCTION OXIDASES

+ f + ?

327

OF 2,4-DINITROTOLUENE

acid

1.69 1.60 1.59 1.42

mg protein-’

f 0.11

min-‘.

481 516 543 440

k 0.17 f 0.19 f 0.13

produced mg protein-’ produced mg protein-’

min-‘.

AHH’

miii’. min-I.

+ 2 f t

32 65 60 28

DECAD, GRAICHEN,

AND DENT

nitroreductase activity at all dose levels, but not in a dose-related manner (Table 1). A dose-related increase in liver cytosolic aerobic azoreductase activity was observed with significant elevation at the two higher doses (Table 1). Following a 70 mg/kg dose of 2,4-DNT, a small but statistically significant increase in relative liver weight, cytochrome P-450, and cytochrome b5 (Table 2) was observed. Cytosolic and microsomal protein, total microsomal heme, cytochrome c reductase, microsomal azoreductase, 4-hydroxylation of biphenyl and arylhydrocarbon hydroxylase were not significantly affected by treatment. Effect of 2,4-Dinitrotoluene Pretreatment on in Vitro Metabolism and Covalent Binding of 2,4-Dinitrotoluene The metabolism of 2,4-DNT was examined with hepatic cytosol and microsomes from control and 2,CDNT-pretreated rats (Table 3). Under a nitrogen atmosphere, reduced metabolites predominated in hepatic cytosol, with greater amounts of 4A2NT produced than 2A4NT. Treatment of rats with 2,4-DNT resulted in a slight decrease in the metabolism of 2,4-DNT in cytosol, especially reduction of the 4-nitro group. Small amounts of a metabolite with a retention volume of 44 ml, and a compound with cochromatographed with 2-(Nacetyl)amino-4-nitrotoluene, were detected. Under aerobic conditions, no metabolism of 2,4-DNT was detected in cytosol preparations (data not shown). In a pure oxygen atmosphere, liver microsomes metabolized 2,4-DNT primarily to DNBAlc (Table 3). Under anaerobic conditions the major microsomal metabolites were 2A4NT and 4A2NT in addition to a metabolite with a relative retention volume of 0.58. At normal oxygen concentrations (air atmosphere) the extent of both reductive and oxidative metabolism was diminished, 4A2NT and DNBAlc being the only metabolites detected. Treatment of rats with 2,4-

IN VITRO

METABOLISM

329

OF 2,4-DINITROTOLUENE

TABLE

3

METABOL~SMOFZ,~-DINITROTOLUENEINCYTOSOLANDMICROSOMESFROMCONTROL AND&&DNT-TREATEDRATS

nmol product/mg Incubation conditions Cytosol Controlb DNTd Microsomes Control

DNT

Unk”

2Ac4NT

2,4-DNBAlc

Nitrogen Nitrogen

1.94 +_ 0.08 1.42 f 0.07

ND ND

Oxygen Air Nitrogen Oxygen Air Nitrogen

ND ND 7.93 _+ 1.18 ND ND 10.15 + 1.18

14.71 + 1.85 + ND 12.63 + 1.84 + ND

3.67 + 1.06 1.02 f 0.21 0.14 0.22 1.05 0.33

’ Standards (retention volume in ml): Unk, Unknown (42); 2,4-DNBAlc, tyl)amino-4-nitrotoluene (55); 4A2NT, 4-amino-2-nitrotoluene (58); 2A4NT, volume of 76 ml in this system. b Controls received five daily doses of corn oil. r Not detectable. d Treatment five daily doses of 70 mg/kg 2,4-DNT.

DNT did not appreciably alter the pattern of 2,4-DNT metabolism in hepatic microsomes or cytosol (Table 3) but did cause a slight increase in the rate of reductive metabolism under anaerobic conditions. Effects of Enzyme Inducers on Metabolism of 2,4-DNT

In an oxygen or air atmosphere, DNBAlc was the major metabolite produced by liver microsomes from phenobarbital (PB) or Aroclor 1254 (AR)-treated rats (Table 4). The amount of DNBAlc formed decreased with decreasing oxygen tensions and the production of this metabolite was almost completely suppressed (>90%) by addition of SKF-525A to PB microsomes. However, DNBAlc formation was much less sensitive to SKF-525A, being inhibited by about 50% with microsomes from Aroclor 1254-treated animals. Only small amounts of 4A2NT and 2A4NT (~2 nmol/mg protein/hr) were detected under aerobic incubations. In a nitrogen atmosphere, reduction of DNT to 4A2NT and 2A4NT represented the major

proteinlhr

ND ND ND ND ND ND

4A2NT

2A4NT

42.63 + 2.35 28.60 f 2.50

22.88 k 0.73 18.93 k 0.85

2.05 1.45 6.05 1.63 1.63 11.19

ND ND 16.49 + 6.03 ND ND 27.09 f 1.79

2,4-dinitrobcnzylalchol 2-amino-4-nitrotoluene

f f f + f f

0.28 0.06 1.54 0.10 0.24 3.76

(50); 2Ac4NT, 2-(N-ace(61). DNT had a retention

route of metabolism. Alcohol formation was almost totally eliminated. SKF-525A inhibited the reductive metabolism of DNT but to a lesser extent than it inhibited the oxidative metabolism. The production of 4A2NT was suppressed by SKF-525A and the production of 2A4NT was stimulated. When incubations were performed under nitrogen for 30 min followed by oxygen for 30 min, reduced metabolites (principally 2A4NT and 2N4AT) predominated. No detectable metabolism of 2,4-DNT occurred in liver microsomes from phenobarbital or Aroclor 1254-treated rats in the absence of NADPH and a regenerating system or with boiled microsomes (not shown). In Vitro Covalent Binding

Very little covalent binding (pmol/mg protein/hr) was detected in microsomes prepared from control or DNT-treated animals (Table 5). Phenobarbital or AR, which increased in vitro metabolism of DNT, also markedly increased covalent binding. In microsomes from control and DNT-treated

microsomes

1.83 f 0.09 1.18 f 0.12 ND

2.47 + 0.21

ND ND ND ND

3.66 f 0.47 1.33 f 0.18

ND ND ND ND

3.46 + 0.44

11.05 ? 2.80

f 6.16 -t f f k

7.73 0.27 0.04 0.07

++ f +

2.06 1.47 2.53 2.24

1.66 + 0.02

0.93 f 0.05 1.39 f 0.09

36.00 60.13 56.40 33.63

1.82 + 0.43

44.69 2.98 0.98 2.45

4.43 f 0.72

54.52

2,4-DNBalc

nmol

4

1.95 k 0.08

1.46 + 0.13 1.46 f 0.16

ND ND ND ND

2.00 f 0.36

ND ND 1.36 f 0.16 1.92 + 1.19

ND

ND

2Ac4NT

product/mg

protein/hr

f 6.23

17.32

f 3.17

7.39 + 1.83 2.07 f 0.20

0.48 ND+ 0.06 0.60 f 0.03 0.42 + 0.04

31.48

2.02 f 0.76 ND 9.99 f 4.16 3.79 f 0.96

0.78 f 0.03

0.62 f 0.06

4A2NT

f 0.41 + 1.20

0.03 f+ 0.04 + 0.05 ND

f 7.05

14.13 f 0.41

15.93 24.39

0.44 0.44 0.41

31.14

1.39 -t 0.76 ND 14.73 + 2.90 24.97 f 3.34

ND

ND

2A4NT

k 0.50 + 0.60 4.69 zk 0.24

15.42 14.29

ND ND ND ND

6.13 + 1.14

ND ND 10.82 k 1.7 18.16 + 3.6

ND

ND

Unk

’ Incubations were performed under atmospheres of: oxygen, 100% 02; air, room air; nitrogen, 100% N2; nitrogen plus oxygen, 30 min in 100% N2 then flushed with 100% O2 and incubated for a further 30 min. When SKF-525A was used it was added to a final concentration of 0.1 mM and reaction mixtures were preincubated for 5 min prior to addition of substrate. ’ Standards (retention volume, ml): Unk, unknown (42, 47, and 73); 2,4-DNBAlc, 2,4-dinitrobenzylalcohol (50); 2Ac4NT, 2-(N-acetyl)amino-4-nitrotoluene (55); 4A2NT, 4-amino-2nitrotoluene (58); 2A4NT, 2-amino-4-nitrotoluene (61). DNT had a retention volume of 76 ml in this system. ’ Not detected.

Nitrogen tSKF-525A Nitrogen plus Oxygen

tSKF-525A Oxygen Air +SKF-525A

Aroclor

+SKF-525A Nitrogen tSFK-525A Nitrogen plus Oxygen

ND ND 2.43 + 0.68 1.86 + 0.34

ND

0.79 + 0.12

Unk

2.61 k 1.45 ND 2.84 f 1.01 0.70 f 0.20

ND

Air

ND’

Unk

tSKF-525A

Standard?

PB microsomes Oxygen

Incubation conditions”

TABLE

Fi

J

w

k

“F

K

T

2 “Q o

IN

VITRO

METABOLISM

TABLE COVALENT

BINDING

OF RADIOACTIVITY

331

OF 2,4-DINITROTOLUENE 5

TO LIVER MICROSOMES

INCUBATED

WITH 2,4-[ “C]D~~~~~~~~~~~~~

Microsomes from rats treated Incubation conditions” Oxygen +SKF-525A Air +SKF-525A Nitrogen +SFK-525A Nitrogen plus oxygen

Saline 2.2 + o.2b 3.9 + 0.3 20.8 f 6.0

DNT

Phenobarbital

2.2 + 0.1 7.7 f 0.3 10.4 + 3.0 -

1160 170 960 240 1170 1180

Aroclor

1254

+ 60 + 20 + 70 -t 30 * 90 ?z 90

1060 f 170 790 t 260 2090 + 110 630 + 120 1030+ 60 960 z!z270

500 + 40

740 t- 70

n Incubations were performed for 60 min in 100% O,, oxygen; room air, air; 100% nitrogen, nitrogen; 30 min 100% NZ followed by 30 min 100% 02, nitrogen plus oxygen. Incubation contained 100 PM [14C]DNT and where SKF-525A was used it was added to a final concentration of 100 FM and preincubated for 5 min prior to addition of substrate. b Values represent X f SEM, n = 3 or 4. Data are shown after subtraction of blank (binding in the absence of regenerating system and NADPH), expressed as pmol of 2,4-DNT hound/mg protein/hr. rats, the amount of covalent binding increased 5 to IO-fold as the oxygen concentration of the incubations decreased from 2 1 to 0%. A similar trend was not evident with

microsomes

from PB- or AR-treated rats. incubations SKF-525A reduced the extent of covalent binding. By contrast, in anaerobic incubations, SKF-525A did not affect the extent of binding. In incubations performed for 30 min in nitrogen followed by 30 min in oxygen, less binding occurred than in either aerobic or anaerobic incubations alone. In the aerobic

DISCUSSION The objectives of this investigation were ( 1) to determine the effect of 2,4-DNT pretreatment of rats on hepatic xenobiotic metabolizing enzymes measured in vitro and (2) to investigate the in vitro hepatic metabolism and covalent binding of 2,4-DNT to better evaluate the role of metabolism in the hepatotoxicity of this compound. Pretreatment of rats with 2,4-DNT altered only slightly the activity of some hepatic enzymes which may play a role in the metabolism of

2,4-DNT. Microsomal nitroreductase activity was depressed by five consecutive doses of 2,4-DNT, confirming the results of Kozuka et al. (1979) who observed a decrease in nitroreductase activity in rats fed a diet containing 0.5% 2,4-DNT for 4 weeks. Hepatic microsomal mixed-function oxidase activity was not markedly altered in rats after five daily po doses of 2,4-DNT. Cytosolic oxygen-insensitive azoreductase activity was increased by treatment of rats with 2,4-DNT. This enzyme is identical to DTdiaphorase (Huang et al., 1979). Treatment of rats with 2,4-DNT had no significant effect on in vitro microsomal metabolism of 2,4-DNT, whereas treatment with phenobarbital or Aroclor 1254 markedly stimulated both oxidative and reductive microsomal metabolism. Under aerobic conditions, the primary metabolic reaction involved oxidation at the methyl group whereas anaerobically, the nitro groups were reduced. SKF-525A effectively inhibited aerobic microsomal metabolism of 2,4-DNT, suggesting that the cytochrome P-450 containing mixed-function oxidases (MFOs) play a role in 2,4-DNT hepatic metabolism. Hepatic cytosol metabolized 2,4-DNT

332

DECAD, GRAICHEN,

only under anaerobic conditions in contrast to the results observed with microsomes. In cytosol preparations the 4-nitro group was more rapidly reduced than the 2-nitro. The converse was observed using microsomes. The oxygen sensitivity of the cytosolic reduction of 2,4-DNT is in contrast with the findings of Eiseman et al. (1972) who demonstrated that 2-amino-4-nitrophenol was the major metabolite produced from 2,4-dinitrophenol by both rat liver microsomes and cytosol. The reductive metabolism of 2,4dinitrophenol in both microsomes and cytosol was only partially inhibited by oxygen, whereas, reduction of 2,4-DNT was almost totally inhibited by oxygen. It seem likely that these differences in both the positional specificity and oxygen sensitivity of the metabolism of 2,4-DNT and 2,4-dinitrophenol relate to the methyl and hydroxyl groups. While both hydroxyl and methyl groups are electron donating and would therefore increase the electronegativity at both the ortho and para positions, the hydroxyl group is a considerably stronger electron-donating group than the methyl group. The hydroxyl group in 2,4-dinitrophenol presumably activates both the ortho- and para-nitro groups toward reduction to such an extent that the reduction can occur even under aerobic conditions. Studies of the covalent binding of 2,4DNT derived radioactivity in microsomes from control and 2,4-DNT pretreated animals revealed that very little binding occurred in control microsomes incubated under aerobic conditions. Between 2 and 7 pmol-eq of 2,4-DNT were bound per milligram protein per hour representing approximately 1000 times less binding than has been observed with 2,4-diaminotoluene and control microsomes (Aune et al., 1979). Under the conditions used, the amount of covalent binding detected was small relative to the binding observed in vitro with other carcinogens, such as atlatoxin B, and 2-acetylaminofluorene, where binding to protein, RNA, and DNA was 1 to 100 pmol of car-

AND DENT

cinogen per mole of amino acid or nucleotide residue (Farber, 1968). Treatment with enzyme inducers caused a disproportionately large increase in binding relative to the stimulation of metabolism. The results demonstrate that hepatic microsomes metabolize 2,4-DNT aerobically to principally DNBAlc and anaerobically to aminonitrotoluenes. Both oxidative and reductive metabolism is stimulated by inducers of hepatic MFO activity and the oxidative pathways are sensitive to inhibition by SKF525A. Furthermore, substantial anaerobic reductive metabolic activity resides in the cytosol. The small amount of covalent binding detected in control microsomal incubations suggests that the microsomes are not the primary site of production of reactive intermediates. Evidence is accumulating to implicate both the gut flora and enterohepatic recirculation in the hepatotoxicity of 2,4-DNT. The reductive capacity of the gut flora exceeds that of the liver by a factor of 1000 (Dent et al., 198 1). Thus, the gut represents a much more important site for the production of reduced metabolites of DNT than liver, although the reduced metabolites produced by the gut flora are similar to those produced in the liver. In axenic rats, hepatic covalent binding of radioactivity following an oral dose of [14C]2,4-DNT is much less than observed in conventional rats and the pattern of DNT metabolites is different in axenic animals (Rickert et al., 1981). Furthermore, Medinsky and Dent (1981) demonstrated that the pattern of urinary metabolites of 2,4-DNT of rats with cannulated bile ducts is very similar to that found in the urine of axenic animals. Preliminary results (Mirsalis et al., 1981) indicate that intestinal bacterial are required for the induction of DNA repair by DNT. These observations combined with the finding that DNBAlcglucuronide is the major biliary metabolite of 2,4-DNT (Bond et al., 1981; Medinsky and Dent, 1981) indicate that there is an interrelationship between liver and bacterial

IN

VITRO

METABOLISM

metabolism of 2,4-DNT and suggest that this relationship may be critical in the toxicity of DNT. We have demonstrated in this study that DNT treatment of rats has little or no effect on in vitro metabolism of DNT, supporting our previous observations that feeding DNT to rats at a dose of 35 mgjkglday did not affect the pattern of urinary metabolites (Rickert et al., 1981). The oxidative metabolism of DNT occurring at the methyl group is catalyzed by microsomal enzymes and is increased by inducers of MFO activity and inhibited by SKF-525A. Interestingly microsomes do not catalyze the further oxidation of DNBAlc to 2,4DNBAcid. Isolated hepatocytes produce small quantities of DNBAcid and 2-amino-4-nitrobenzoic acid (Bond and Rickert, 198 1 ), and isolated perfused livers produce small quantities of DNBAcid, 2A4NBAcid, and 4Ac2NBAcid (Bond et al., 198 1). The capacity to further oxidize the alcohol probably resides in the cytosol and may be catalyzed by alcohol and aldehyde dehydrogenase. Gillette (1959) has reported that p-nitrobenzylalcohol was further oxidized by liver alcohol and aldehyde dehydrogenase. The low capacity of liver microsomes to reductively metabolize DNT under aerobic conditions suggests that the gut flora is the primary site of reductive metabolism. This observation coupled with the low level of covalent binding detected in microsomes suggests that an interaction between metabolism of DNT in gut flora and the liver is involved in its toxicity.

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