In vivo spin-trapping of trichloromethyl radicals formed from CCl4

0006.2952/79/0715-2231

Biochemical Pharmacology, Vol. 28, pp. 2231 - 2235. 0 Pergamon Press Ltd. 1979. Printed in Great Britain

IN VIVO SPIN-TRAPPING --

OF TRICHLOROMETHYL

Research

Laboratory,

Oklahoma

The spin-trapping brief to obtain resonance radical, through

spectrum

this technique

system

1979; accepted

This technique

simulation

definitively

(PBN) is lipid-soluble

in the liver -in situ, we have investigated

plished -in vivo. romethyl

adduct formed

from the microsomal

The results

described

radical

membrane

by electron

direct evidence

by the reaction

rats (250-300 g body wt),fed

a commercial

are formed

can be accomof the trichlo-

rat ration, were used

NY.

Organic

Rochester,

from the Fisher Scientific E-9 electron

agent,

spin resonance

TX.

spectrometer

quality

grade) were obtained

CHC13 and methanol

equipped

with an X-band

at a dosage of 120 pl/lOO

either

To whom inquiries

requests

The

in 0.02 M with 1.0 ml of

tube after the rats had been fasted for 20 hr.

the spin-trapping

and reprint

(E-101-15)

g body weight.

CC1 4 or the spin trap, or both, were homogenized by stomach

were

All e.s.r. spectra were recorded

PBN, was given at a dosage of 1 ml of a 0.14 M solution

buffer, pH 7.4.

received

Reagent

Co., Houston,

Ccl4 was administered

corn oil and administered

*

of

AND METHODS

Chemicals,

trol animals

long half-

Since formation

for the formation

from Eastman

phosphate

of the -Ccl3

that spin-trapping

and Ccl4 (spectrophotometric

spin-trapping

from Ccl4 by

of using the spin-trapping

Phenyl-t-butylnitrone

bridge.

employed

that *Ccl3 radicals

in all experiments.

microwave

radical

from Ccl4 by the liver in the intact animal.

Male, Sprague-Dawley

with a Varian

spin

We have recently

of the -Ccl3 radical

mean

too

a more stable derivative

with chloroform.

the feasibility

MATERIALS

purchased

have a half-life

and has a relatively

herein indicate

The results also provide

directly

procedure.

the production

The radical

which

the nature of the primary

in an -in vitro system does not necessarily

in vivo. --

radicals

not only produces

for determining

and a computer

(1).

Foundation

9 May 1979)

levels which can be detected

to demonstrate

Research

City, OK 73104, U.S.A.

the opportunity

life, and can be extracted

technique

Medical

has been used to detect

radical with phenyl-t-butylnitrone

this radical

Oklahoma

technique

analysis

a liver microsomal

and Kuo-Lan Fong

16 April

spectroscopy.

but provides

Noguchi

(Received

concentration

(e.s.r.)

FORMED FROM CC14

Toshikazu

Edward K. Lai, Paul B. McCay,* Biomembrane

RADICALS

102.00/O

Con-

agent mixed with corn oil, or Ccl4 in corn

should 223 1

be sent.

2232

Preliminary

Communications

0.5 ml samples of the lipid extracts measurement

of electron

spin resonance

signal of liver extracted (B) cc+

Electron

by Poyer s

&

(1).

signals.

in the analysis (A)

Electron

from rats given Ccl4 and PBN orally

spin resonance

NADPH and PBN.

were placed

signal obtained

The incubation (C) Control

tubes for

spin resonance in corn oil.

in vitro with liver microsomes,

system was the same as that described

study showing absence

of e.s.r.

signal

in the liver lipid extract when fresh liver, Ccl4 and PBN were added simultaneously

to chloroform-methanol

study showing absence

of e.s.r.

extracting

signal

in extracted

rats were given PBN in corn oil without showing absence

of e.s.r.

signal

given Ccl4 in corn oil without

medium.

CC14.

(E)

(D) Control

liver lipid when Control

study

in liver lipid extract when rats were

PBN.

In the present study, the signal was not observed in the lipid extract obtained when fresh liver tissue, methanol,

and the extraction

administration extraction

procedure

carried

to rats of the spin-trapping

of liver lipids, yielded

(Figure 1D). absence

CC14, and the spin-trapping agent

out as above

radical

that produced is not formed

to chloroform-

(Figure 1C).

agent in corn oil without

an extract

Hence, the spin-trapped

were added simultaneously

In addition,

CC14, followed

no measurable

e.s.r. signal

in the rat liver -in vivo in the

of Ccl4 administration.

Fig. 2. isolated

Electron

spin resonance

Methods.

studies on lipid extracts

of fractions

from liver of rats given Ccl4 and PBN in corn oil orally.

(5.0 g) was homogenized The protein

and fractionated

content

by

as described

Liver

under Materials

per gram of liver of the fractions

and

obtained

Preliminary

oil, or corn oil only. The treated

in chloroform-methanol

of liver by the method extracting centrated

procedure

and the solvent

rats has been described power,

25 mW; modulation

membrane previously

removed

scan time, 30 min or 2 hr.

removed,

rapidly weighed,

(3).

from 5.0 g

The CHC13 layer was recovered

at the end of the

and evaporated

gas.

immediately

fraction,

amplitude,

in all studies.

(2:l). Total lipids were then extracted

under nitrogen

in a Varian

or was stored at -20" for later study.

(nuclear-plasma

was constant

Livers were immediately

of Folch -et al. (2).

volume was either analyzed

spectrometer, fractions

The amount of corn oil administered

rats were killed after 2 hr.

and homogenized

2233

Communications

E-9 electron

The preparation

mitochondria

1 G; time constraint,

spin resonance

of liver subcellular

and microsomes)

The e.s.r. spectrometer

The con-

from the treated

settings

were microwave

10 set; scan range, 100 G; and

The spectra were taken at room temperature,

24".

RESULTS Figure

1A shows the e.s.r.

liver of a rat which agent,

PBN.

had been given Ccl4 orally

The signal

liver microsomes,

is identical

from a chloroform-methanol in corn oil together

to that which

NADPH, and the spin-trapping

to be the spin-trapped

Fig. 1.

signal obtained

agent

adduct of *Ccl3 by computer

Studies

on the spin-trapping

vitro and in vivo.

Where indicated,

orally

under Materials

as described

lipids were extracted

is obtained

of

with the spin-trapping

when Ccl4 is incubated

with

(Figure lB), and which was demonstrated

simulation

(1).

of the trichloromethyl rats were administered

and Methods.

from 5.0 g of liver.

(2:l) extract

After

radical -in the compounds

1 hr., the total

After removal

of the solvent,

2234

Preliminary Communications

was

as follows:

microsomes,

plasma membrane-nuclei,

16 mg.

Total lipids were extracted

and, after solvent removal, an electron

spin resonance

Mitochondrial (D)

lipids.

Supernatant

20 mg; mitochondria,

from each fraction

lipid samples of 0.5 ml were analyzed spectrometer.

(C)

fraction

Nuclei-plasma

(A)

Microsomal

membrane

membrane,

and fractionated

(b) mitochondria,

and (c) microsomes,

was extracted

analysis

of each lipid extract

which contains

by the same procedure

fraction

(Figure 2).

subcellular

(B)

lipids.

The livers were then removed, (a) nuclei-plasma

The total lipid from each subcellular

as described

above.

shows that the microsomal

the spin adduct

any of the extracted

lipids.

at 4" into three groups of organelles:

fraction

in

lipids.

Rats were treated with the spin trap and CC14 in vivo. homogenized,

39 mg;

Electron

fraction

spin resonance

is the only organelle

tlo signal was detected

in the aqueous

phase of

fractions. DISCUSSION

There are two significant support diate

for the concept

in the metabolism

that the formation

aspects

that the trichlor~ethyl

of the radical appears

at a detectable

vitro.

fraction

There

is very little

likelihood

solvent

and liver tissue from untreated

There

resulted

result

against

that the radical

speculation

of CC14 by liver microsomes

addition

(2:1), followed

for initiating

of the

of CCl4, PBN,

by immediate

action of CC14 (7-9).

the events

of liver microsomes

lie have not determined

signal. such as

that ultimately scavengers

Other investigators

of the labeled carbon

of damage to the liver caused by CC14.

free radical,

free radical

s

after administra-

the h~ogeni~ation

that a highly reactive

and would explain why lipid-soluble

(11).

with the lipid-

a lipid extract which did not give an e.s.r.

binding of l4 CC14 to lipid and conversion

the extent

(4-6), and

since the spin-trapped

adduct observed

rats to chloroform-methanol

the hepatotoxic

by antioxidants

interme-

but not with other membranous

is formed -in vitro during

that both 14C and 35 Cl bind to lipid and to protein

inhibited

function

is very stable, and is associated

radical, may be responsible

in liver damage,

protection

to be a microsomal

in view of the fact that simultaneous

in obtaining

has been considerable

the trichloromethyl

is , in fact, a metabolic

just as in the case of the metabolism

liver in the extracting

strong

level.

tion of CC14 and PBN to intact animals

h~ogenization,

radical

with that organelle,

The PBN adduct of the *CC13 radical extractable

First, the data provide

of CC14 in the rat liver -in vivo as has been postulated

radical was found to be associated fractions

of these results.

afford

have shown

(lo), but that

in 14CC14 to 14C02 are not

if PBN administration

itself reduces

Preliminary

The second significant using a spin-trapping

aspect of this study is that it establishes

agent to investigate

are that this type of approach formation

of highly reactive

To this end, we are currently to be damaged

-We

Wanda R. Honeycutt supported

free radical

may be useful

free radicals applying

by CC14 for evidence

Acknowledgements

2235

Communications

phenomena

in obtaining

produced

the technique

of formation

information

the metabolism

described

in the preparation

in part by NIH Grants AMO6978-17

of other compounds. known

radical -___ in vivo.

thank Mr. Larry J. Olson for his expert technical

for her assistance

on the

above to other tissues

of the trichloromethyl

of

The implications

in vivo. --

more direct

during

the feasibility

assistance

of this manuscript.

and Mrs.

This work was

and AMO8397-18.

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