Phosgene: A metabolite of chloroform

BIOCHEMICAL

Vol. 79, No. 3, 1977

PHOSGENE: Lance

R. Pohl,

A METABOLITE

B. Bhooshan, Laboratory

National

Institute

National Received

Noel

F. Wnittaker*

Lung,

and Gopal

Krishna

Pharmacology,

and Blood

Institute

and the Laboratory

of Chemistry

of Arthritis,

Institutes

October

OF CHLOROFORM

of Chemical

Heart, *

National

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Metabolism,

of Health,

and Digestive

Bethesda,

Maryland

Diseases 20014

20,1977

Summary Cysteine inhibited the --in vitro covalent binding of [ 14 C] chloroform, (CHCl ) to microsomal protein and concomitantly trapped a reactive metabolite,3piesumably phosgene (COCl > as 2-oxothiazolidine-4-carboxylic acid. When the incubation was conductzd'in an atmosphere of [lgo] 0 the trapped COC12 contained [180]. These findings suggest that the C-H b%d of CHCl is oxidized by a cytochrome P-450 monooxygenase to produce trichlorometh&ol, which spontaneously dehydrochlorinates to yield the toxic agent phosgene. INTRODUCTION Chloroform purposes

until

it

in mice

(1)

animals

(4-7).

active

metabolite

when rats necrosis

that

of CHCl are

with

treated

inhibited

a reactive

metabolite

for

[ 14C]

is

Copyright 0 1977 by Academic Press, Inc. AI1 rights of reproducfion in any form reserved.

produced

with

the inhibitor decreases

phenobarbital

(6,7).

of hepatic

irreversibly

of liver

The results

a re-

For example,

the extent

inducer

with

that

toxicity.

bound

tumors

and experimental

are potentiated

CHC13 administration

liver

suggest

its

CHC13,

of [ 14 C] label

pretreated

produces

in man (2,3)

by pretreatment

in rats

and industrial

investigations

a known

that

solvent

toxicity

and binding

phenobarbital,

medicinal

responsible with

necrosis

The finding

glutathione

is

3

for this

of several

the amount Both

used that

and renal

The results

or mice

and are

butoxide. liver

and hepatic

(5-7).

of animals

was widely

was established

parallels

protein

lism,

(CHC13)

to liver

by pretreatment microsomal

metabo-

piperonyl the level further

of

suggests

of --in vitro

684 ISSN

0006-291

X

BIOCHEMICAL

Vol. 79, No. 3, 1977

studies

with

rat

and mouse liver

vations

by establishing

metabolite

which

binds

metabolic

process

is

cytochrome

P-450,

In this activated treated

oxygen is

investigation,

rats.

formed

14

support

the --in vivo

Cl CHC13 is metabolized to microsomal

dependent inducible we report

(5,6,8,9).

by phenobarbital

(5,8,9).

that

microsomes

of an [laO]

by an oxidative

protein

to be mediated

evidence

02 experiment dechlorination

obser-

to a reactive

and appears

COC12, by liver

The results is

[

microsomes

covalently

which

to phosgene,

metabolite

that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This by a

CHC13 is metabolically of phenobarbital establish

prethat

this

mechanism.

EXPERIMENTAL Synthesis: 2-Oxothiazolidine-4-carboxylic acid was synthesized from cysteine and COCl, following the procedure of Kaneko et al. (10)._ This acid was methylated with diazomethane or deutero-diazomethane, which were prepared from Diazald (Aldrich) following the directions supplied with the reagents. The products were then analyzed by gas chromatography-chemical ionization mass spectroscopy. Preparation of microsomes: Male Sprague-Dawley rats (180-200 g) were obtained from Hormone Assay Laboratories (Chicago, Ill.). The animals were allowed free access to water and food (Purina Lab Rat Chow) and were pretreated with phenobarbital (80 mg/kg, in saline, i.p.) 72, 48 and 24 hrs before the experiment. Liver microsomes were prepared as previously described (8). Effect of cysteine on in vitro covalent binding to microsomal protein: Each 1 ml incubation mixture contained 1 mg microsomal protein, 0.10 mM NADH, 0.20 mM NADP, 2.00 mM glucose-6-phosphate, 1 unit-of glucose-6-phosphate dehydrogenase, 20 mM Tris (pH 7.4), 153 mM KCl, 1.00 mM [14C] CHC13 (New England Nuclear) (1 mCi/mmole, added in 10 ~1 of dimethylformamide) with or without 2.0 mM cysteine. The incubation was conducted in a sealed vial at 37'C for 10 min. The reaction was stopped by the addition of methanol (4 ml) and the covalent binding of the [14C] label to microsomal protein was determined as previously described (8). A 45 ml incubation Trapping the reactive metabolite with cysteine: mixture containing cysteine was incubated in a sealed flask under the conditions outlined above for the 1 ml mixture. After 10 min the reaction was stopped by the addition of methanol (180 ml). The mixture was then centrifuged and the supernatant phase was evaporated under vacuum. The resulting residue was dissolved in water (2 ml, pH 6) and washed with ethyl acetate (3 ml, 3x). The aqueous solution was then acidified with concentrated hydrochloric acid to pH 1 and extracted four times with 3.0 ml of ethyl acetate. The combined ethyl acetate extracts of the acidic aqueous solution was then evaporated (N ) to dryness. The residue was dissolved in methanol (100 ~1) and methylate 2 with diazomethane, which was generated from Diazald (Aldrich) following the directions given on the reagent bottle. The derivatized product was then analyzed by gas chromatography-chemical ionization mass spectrometry (CC-CWS).

685

BIOCHEMICAL

Vol. 79, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Trapping the metabolite with cysteine in an atmosphere of [ 1801 0 : The large scale reaction was repeated in an atmosphere of [isO] 0 (Stzhler Isotope Chemicals, 99%). The actual enrichment of 11803 0 in thg reaction flask was determined by simultaneously conducting the para2hydroxylation of acetanilide (1 mM) with liver microsomes in another flask (11). The experiment was performed by interconnecting the two reaction flasks and a sealed vial of ]180] 02 (100 ~1) with vacuum tubing. The system was then evacuated with a vacuum pump and purged with nitrogen. This process was repeated nine more times. After a final evacuation, the seal to the 11801 O2 vial was broken and the [180] 02 atmosphere was allowed to distribute into the two reaction flasks. Nitrogen gas was then added to bring the pressure up to approximately 1 atmosphere. The atmospheric concentration of two reaction flasks were calculated to be approximately 20% [ lBases 0] O2 inandthe80% The incubations were stopped after 10 min by the addition of MeOH. The N2' trapped phosgene was isolated and analyzed as described above, while phydroxyacetanilide was isolated from the acetanilide reaction by following the procedure described by Hinson et al. (11). This product was then further purified and characterized by GC-CIMS. Gas chromatography chemical ionization mass spectrometry (GC-CIMS): The GC-CIMS were performed on a Finnigan 1015 D instrument which was equipped with a chemical ionization source, a Finnigan 9500 gas chromatograph and a Model 6000 data system. The samples were injected onto a 150 cm glass column (2 mm id) which was packed with 3% OV-225 on Gas Chrom Q, lOO120 mesh. Helium was employed as the carrier gas at a flow rate of 25 mllmin. The column temperature was 175' for the CHC13 studies and 230°C for the acetanilide study. The mass spectrometer was operated at a source pressure of 1 Torr, a source temperature of 15O"C, and an electron energy of 100 eV. Methane or ammonia were used as the reagent gas. RESULTS When cysteine liver

microsomes

hibited

from

the binding

determine active

cysteine

metabolite

incubation Since

it

CHC13, cysteine

of the metabolite

Moreover,

was anticipated

oxothiazolidine-4-carboxylic with

rats,

did

this

not that

have reacted acid an authentic

when cysteine

contain

cysteine.

with (10).

this

686

that

of CHCl 3 in one major

was added

was not present

intermediate isotope

To

a re-

of

to produce dilution

of 2-oxothiazolidine-4-

to

in the

COC12, was a metabolite

Reverse

standard

1).

by trapping

indicated

metabolite

in-

(Table

the metabolism

this

phosgene,

significantly

protein

inhibition

study

was produced

[ 14 C] CHC13 and

of it

to microsomal

we investigated

product

that

should

pretreated

A preliminary

mixture. mixtures

mixture

[ 14 C] label

of chloroform,

radiolabeled

incubation

incubation

had produced

of cysteine.

nonvolatile

to the

phenobarbital

of the

whether

the presence

the

was added

2-

analysis

BIOCHEMICAL

Vol. 79, No. 3, 1977

Table 1 binding

Effect [14C]

of

Incubation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of cysteine chloroform

on the --in vitro irreversible to liver microsomal proteina

conditions

Irreversible bindingb pmolelmg protein/l0 min

No cysteine

2178 + 163

Cysteine a

b

carboxylic

14 [ C] chloroform from phenobarbital as described in

acid

a large

this

scale

idea.

1B) were

ated

molecular

time

sponded chain

to the loss (MH+-60)

performing

of this

and methane

identical.

(Fig.

the

of carbon

fragment

monoxide

respectively.

These

the GC-CIMS of deuterium

carboxylate,

which

was prepared

to obtain

study

are

structural enough

presented

1A

standard

of

metabolite

at m/e 162 represents ions

the proton-

at m/e 134 and 102 correand the carbomethoxy

fragmentation methyl

the authentic

product

in Figs.

1A) and the derivatized

(MH+-28)

labeled

from

this

CIMS of the methylated

The ion


error

to confirm

was conducted

acid

nearly ion

In order

incubation

2-oxothiazolidine-4-carboxylic (Fig.

as the mean + standard

The results

The retention

80

(1 mM) was incubated with liver microsomes pretreated rats in an atmosphere of air the Experimental Section.

supported

GC-CIMS analysis.

and 1B.

1032 ?

The results are expressed of 5 incubations.

assignment, for

(2 mM)

pathways

were

side

confirmed

by

2-oxothiazolidine-4carboxylic

acid

and

deutero-diazomethane. To establish scale time

reaction

that

CHC13 was oxidatively

was repeated

of methylated

standard

1D) were

again

standard

and the metabolite


virtually

an ammonia

addition

metabolized

in an atmosphere (Fig. identical.

02.

1C) and the derivatized The ammonia

contain ion

of (180]

to COC12, the large

a protonated

at m/e 179 (M+lB)

687

The retention metabolite

(Fig.

CIMS of the authentic molecular

ion

and a fragment

at m/e 162 ion

at m/e

Vol. 79, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Gas chromatography - Chemical ionization mass spectra (GC-CIMS). A. The methane CIMS of authentic methyl 2-oxothiazolidine-4-carboxylate. As described in the Experimental Section, this product was synthesized from 2-oxothiazolidine-4-carboxylic acid and diazomethane and analyzed by GC-CIMS, retention time 5.34 min. B. the methane CIMS of the methylated metabolite of CHCl . As described in the Experimental Section, this metabolite was isolate a from the --in vitro incubation mixture of CHC13 and cysteine with liver microsomes from phenobarbital-pretreated rats. The metabolite was derivatized with diazomethane and analyzed by GC-CIMS, retention time 5.34 min. C. The ammonia CIMS of authentic methyl 2-oxothiazolidine-4-carboxylate, retention time, 4.92 min. D. The ammonia CIMS of the methylated metabolite of CHCl from the [~SO] 0 incubation. As described in the Experimental Section: this metabolite fs isolated from the in vitro incubation of CHCl and cysteine which was performed in an atmosphere OfSO] 02. The metabo 4.ite was derivatized with diazomethane and analyzed by GC-CIMS, retention time, 4.92 min.

688

BIOCHEMICAL

Vol. 79, No. 3, 1977

102 @I+-60).

In addition,

164,

181 and 104 (Fig.

into

the 2-0~0

cent

incorporation

which

contained

revealed

that

O2'

Since

into

the para

it our

it

appears

this

into

represent

the metabolite

doublets product

also

contained

position

of acetanilide

approximately

ions

at m/e

the incorporation

of

acid.

[180]

The per-

was calculated

to be 76%.

metabolite

nearly

at least

of acetanilide,

76% incorporation

96% of the oxygen

is derived

[ 16 0] Oz.

or from

the presence

incorporated oxygen

in the atmosphere

This

of

of [ 180]

from molecular

20% of the O2 present

was in the form of

degassing

contains

at m/e 152 and 154 @HI+) and 109 and 111 (M-42),

shown that

incubations

incomplete

[I801

has been

that

ions

CIMS of the parahydroxylated

ion this

These

metabolite

of 2-oxothiazolidine-4-carboxylic

of

The GC-ammonia

the methylated

1D).

position

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

could

[160]

(ll), during

have resulted

in the

[180]

from

O2 used

in

experiment.

DISCUSSION The results

of this

activated

by liver

mechanism

(Fig.

investigation

microsomes 2).

of rat

The first

of CHC13 to trichloromethanol monooxygenase

(5,8,9).

the hydroxylation However,

spontaneously COC12 could

cysteine

bound inhibits

and concomitantly

process.

would

to form carbon (16,17)

(5,6,8,9

and Table

of [14C]

reacts

a metabolite

with

acid

(Fig. the

cytochrome

is an anticipated

dioxide, or with 1).

process

to be unstable (13).

since (12).

and

The electrophilic

a known metabolite microsomes

to yield

The observations

the O2 is

existence incorporated

of a

that

protein

of CHC13 to produce

[180]

D-450

reaction

documented

phosgene

suggests that

the hydroxylation

inducible

CHC13 to microsomal

lA,B),

finding

dechlorination

involves

be expected

to produce

the binding

Moreover,

process

is a well

and --in vivo

product

zolidine-4-carboxylic this

carbons

water

(14,15)

step

CHC13 is metabolically

an oxidative

of this

metabolic

dehydrochlorinate

CHC13 &vitro covalently

This

with

that

by a phenobarbital

case the product

react

through

step

of aliphatic

in this

suggest

(Table

1)

2-oxothiaof COC12 in into

the

BIOCHEMJCAL

Vol. 79, No. 3, 1977

a-c-a

AND BIOPHYSICAL RESEARCH COMMUNlCATlONS

? I cl

*ncl I

Fig. 2. Proposed oxidative dechlorination mechanism vation of CHCl by liver microsomes of rat. 3

2-0~0

position

incubation that

is

Although

the mechanism

microsomes finding

that

pretreatment

CHC13 (7)

suggests

for

(1,2,4,5)

Indeed,

chloroamphenicol (Fig. as well

2) is

the depletion tumors

of activation

the

results

(18,19)

protects

important

(1)

that

that

the

also

responsible

for

as other

halogenated

hydrocarbons.

the

oxidative

the metabolic

the

relevance finding

This (6,7)

of

process

by this occur

with

chlorinated

of this

compound. other antibiotic mechanism compound

ACKNOWLEDGMENTS We wish to thank Dr. James R. Gillette for critically reviewing this manuscript and Christine McDaniels for her valuable technical assistance.

690

in

may

and the renal

dehalogenation

activation

2).

hepatotoxicity

--in vivo.

may also with

(Fig.

the previous

are produced

of investigations

the proposal

of CHC13 by rat

glutathione

here

acti-

when the

toxicologic

against

of liver

suggested

suggest

the

However,

is

lC,D)

mechanism

activation

understood,

mechanism

and liver

The mechanism drugs.

the metabolic

cysteine

this

(Fig.

dechlorination

to be elucidated. with

that

be responsible

toxicity

for

the metabolic

18 [ 0] O2 confirms

of

by an oxidative

is now more clearly remains

acid

in an atmosphere

produced

of this

also

conducted

COC12 is

liver

rats

of 2-oxothiazolidine-4-carboxylic

for

Vol. 79, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Eschenbrenner, A.B. (1944) .I. Natl. Cancer Inst. 5, 251-255. von Oettingen, W.F. (1964) The Halogenated Hydrocarbons of Industrial and Toxicological Importance, pp. 77-108, Elsevier Publ. Co. Amsterdam. Conlon, M.F. (1963) Brit. Med. J. 2, 1177-1178. Klaassen, C.D., and Plaa, G.L (1966) Toxicol. Appl. Pharmacol. 9, 139-151. Ilett, K.F., Reid, W.D., Sipes, I.G., and Krishna, G. (1973) Exp. Mol. Pathol. 19, 215-229. Brown, B.R., Sipes, I.G., and Sagalyn, A.M. (1974) Anesthesiology 41, 554-561. Docks, E.L., and Krishna, G. (1976) Exp. Mol. Pathol. 24, 13-22. Sipes, I.G., Krishna, G., and Gillette, J.R. (1977) Life Sci. 20, 1541-1548. Uehleke, H., and Werner, Th. (1975) Arch. Toxicol. 34, 289-308. Koneko, T., Shimokobe, T., Ota, Y., Toyokawa, E., Inui, T., and Shiba, T. (1964) Bull. Chem. Sot. Jap. 37, 242-244. Hinson, J.A., Nelson, S.D., and Mitchell, J.R. (1977) Mol. Pharmacol. 13, 625-633. Testa, B., and Jenner, P. (1976) Drug Metabolism - Chemical and Biological Aspects, pp. 10-25, Dekker Inc., New York. Seppelt, K. (1977) Angew. Chem. Int. Ed. Engl. 16, 322-323. Paul, B.B., and Rubinstein, D. (1963) J. Pharmacol. Exp. Therap. 141, 141-148. Rubinstein, D., and Kaniss, L. (1964) Canad. J. Biochem. 42, 1577-1585. Fry, J., Taylor, T., and Hathway, D.E. (1972) Arch. Int. Pharmacodyn. 196, 98-111. Brown, D.M., Langley, P.F., Smith, D., and Taylor, D.C. (1974), Xenobiotica 4, 151-163. Pohl, L.R., and Krishna, G. (1977) Biochem. Pharmacol., in press. Pohl, L.R., Nelson, S.D., and Krishna, G. (1977) Biochem. Pharmacol., in press.

691