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Effect of sulfhydryl reagents on peroxidation in microsomes
ARCHIVES
OF
Effect
BIOCFIEYISTRY
AND
of Sulfhydryl
BIOPHYSICS
Reagents
HENRY G. UTLEY, FREDERICK Department
of Physiology
and
X9-32 (1967)
118,
on Peroxidation BERNHEIM,
AND
Pharmacology, Duke University Medical Received April 22, 1966
in Microsomes’ PAUL HOCHSTEIN’ Center, Durham,
North
Carolina
Incubation of mouse liver microsomeswith HgC12,NEM, or PCMB results in the formation of lipid peroxides as measuredby the thiobarbituric acid reaction. Carbon monoxide inhibits peroxidation induced by NEM and the inhibition is reversed by light. Peroxidation induced by HgClz is not greatly affected by EDTA but is increased by ascorbic acid. Microsomes isolated from mice pretreated by intraperitoneal injection of HgClz peroxidized endogenous unsaturated lipid on incubation, and addition of HgClz in vitro further increases the peroxidation. The in vitro stimulation of peroxidation by these SH reagents in liver microsomes increases with age in the rat, and microsomes from male rats are more active than those from female. Pretreatment of mice with phenobarbital for 3 days increases the in vitro effect of HgClz on peroxidation. This stimulation occurs in the smooth-surfaced microsomes. Actinomycin partially inhibits the effect of phenobarbital. Mercuric chloride causes no peroxidation on incubation with shark liver microsomes. Urea causes no peroxidation on incubation with mouse liver microsomes. These results are consistent with the possibility that sulfhydry-reacting agents produce a change in tertiary structure of microsomal Fe, , thereby rendering the prot.ein-bound iron available for catalysis of peroxidation of endogenous lipid.
extracted lipids. This fact, and the general similarity of the lipid composition of microsomes and mitochondria (2), suggested that some factor in the microsomes was involved in the catalytic effect of mercury. Accordingly, an attempt was made to characterize this factor.
Ferrous salts catalyze the peroxidation of unsaturated lipids in mitochondria and microsomes as well as the peroxidation of pure unsaturated lipids (1). Other cations, with the exception of copper, have not been shown t,o catalyze this reaction. In this paper, we report experiments which demonstrate that mercuric ions incubated with liver microsomes may also induce a peroxidation of endogenous lipids. Incubation of mitochondria (fresh, frozen, or previously heated to GO”) with mercuric chloride resulted in no peroxidation, nor did mercuric chloride produce significant peroxidation of lipids extracted from microsomes by a 1:4 ethanolether mixture, although ferrous ions were active in inducing peroxidation of these
MATERIALS AND METHODS Rats or mice were stunned by a blow on the head and decapitated, and the liver was immediately removed and placed in cold sucrose (0.25 M). The tissue was allowed to cool, weighed, and minced with scissors, and microsomes were prepared according to the method of Schneider and Hogeboom (3). Smooth- and rough-surfaced fractions were prepared according to the method of Fouts (4). Microsomes were suspended in isotonic KC1 (1.15 M) to twice the initial tissue weight, and 0.3 ml was added to incubation flasks. Substances to be added in vitro were dissolved in 0.067 RI Naphosphate buffer (pH 7.4) and added to flasks in a volume of 0.2 ml. Flasks were made up to a final volume of 3 ml with buffer and incubated at 37” with shaking.
1 Part, of a thesis submitted by H. G. Utley in partial fulfillment for the requirements of the Ph.D. degree, Duke University. Supported in part by grants GB 1416 from the National Science FoundaGon and AT-(40-l-3329 from the AEC. 2 U.S. Public Health Service Research Career Awardee (KS-GM 4857). 29
30
UTLEY, TABLE
BERNHEIM,
I
AGENTS ON LIPID PEROXIDATION IN MOUSE LIVER MICROSOMES
EFFECT
OF VARIOUS
“Pretreated” mice receive 3 mg/kg/day HgClv for 2 days. CO was added to the appr0priat.e flask as a 1:l mixture with oxygen. Subsequent incubations of flasks containing CO were carried out either in the dark or under a 100 W light bulb. Microsomes were incubated for 90 minutes at 37”. Numbers represent the average of at least three experiments. Additions
and/or treatment
None FeSOl (0.03 mM) + Ascorbate (0.2 mM) + EDTA (0.03 mM) HgClz (0.03 mM) + Ascorbate (0.2 mM) + EDTA (0.03 m&r) + CO-Dark + CO-Light NEM (2 mrvr) + Ascorbate (0.2 mM) + EDTA (0.03 mM) + HgC12 (0.03 mM) PCMB (0.2 mM) + Ascorbate (0.2 mM) + HgClz (0.03 mM) Preheating (54-10”) + HgClz (0.03 mM) HgCla Pretreated + HgClz (0.03 mM) Urea (4 mM)
0 210 339 0 132 201 117 14 69 114 201 105 116 111 219 99 123 147 213 243 0
AND
HOCHSTEIN
various other sulfhydryl reagents on lipid peroxidation in liver microsomes. Maximal peroxidation followed incubation of mouse liver microsomes for 90 minutes with HgClz at 0.03 mM, PCMB at 0.2 mM, or NEM at 2 mM. The effects were not additive. Thus, the addition of both HgC12 and NEM or PCMB caused no greater peroxidation than the addition of each of these singly. However, as in the case with ferrous ions, ascorbate increased the HgClz effect. EDTA caused little inhibition of HgC& or NEMinduced peroxidation. Carbon monoxide inhibited peroxidation induced by NE11 and the inhibition was reversed by light. These experiments suggest the involvement of both iron and sulfhydryl groups in the peroxidation reaction. Urea caused no peroxidation, but preheating of microsomes to 54” for 10 minutes before incubation at 37” had a pronounced effect. Table I also shows that microsomes isolated from mice administered 2 daily doses of HgClz (3 mg per kilogram per day) peroxidize unsaturated lipid to an extent almost as great as that caused by iron added in vitro. Addition of HgC12 in vitro to pretreated animals caused a further increase. Table II shows the effect of HgCl, on peroxidation in microsomes prepared from mice and rats treated in various ways. Mercuric chloride had a greater effect in microsomes from male than female rats, although this difference was not seen in mice and peroxidation was greater in microsomes
Peroxidation was measured by the thiobarbituric acid (TBA) reaction. Aliquots of 0.5 ml of the incubation suspension were removed at appropriate intervals and added to a mixture of 2.0 ml of 2Oy, trichloroacetic acid and 4.0 ml of 0.67yc TBA. The mixture was placed in a boiling water bath for 10 minutes and centrifuged, and the optical density of the supernatant fluid was read at 535 rnp. An extinction coefficient of A&b’& = 1.56 X lo5 was used to calculate the amount of malonyldialdehyde (MA) formed.
F&O*
RESULTS
As reported by others (l), ferrous ions initiated a peroxidation of endogenous lipids as measured by MA formation, which was enhanced by ascorbate and inhibited by EDTA (Table I). This table and Fig. 1 show the effects of treatment with HgClz and
Minutes FIG.
1
103mM)
1
SULFHYDRYL TABLE
EFFECT
REAGENT
II
v&o
EFFECT OF 3 X 10m5M HGCL* ON PEROXIDATION IN LIVER MICROSOMES ISOLATED FROM UNTREATED MICE, MICE PRETREATED WITH PHEN~R~RRIT~L D AT 80 AT 50 MG/KG/D~Y .IND ACTINOMYCIN @/KG/DAY FOR 3 D.4vs SND MICE TREATED WITH PHENOBARBITAL ALONE
The effect of sex and age was tested in rats as indicated below. Microsomes were incubated for 90 minut,es. Numbers represent the amount of MA formed in mpmoles and are the average of at least 3 experiments.
by
urea,
133
324
57
147
represent
the
microsomes
None Phenobarbital None Phenobarbital
Rough Smooth
from
adult
rats
,\licrosomes 3
lipid
days
produced on
with
HgC12.
mals
t’reated
D
produced those found
less
80
at
that
microsomes
produced
with
HgC&.
no
218
young. with
kilogram
per
day
levels
of
in vitro incu-
Microxomes
from
ani-
phenobarbital
pg per
peroxide,
untreated
40
of
highest
both
from
liver
per
subsequent
with
actinomycin
t’hose
,
and
kilogram
but
still
per more
day than
It was also
animals. isolated
from
peroxides
on
shark
incubation
The ner
results
may
consistent
bution
of certain
function
in
steroids.
These
the
the
microsomal
a man-
and enzymes
met’abolism enzymes
in
activity
can
of
drugs
be
altered
distriwhich and in
the
conversion
or iron-containing
enzymes
catalyze
lipid
they
peroxida-
iron were made accessible through in tertiary structure. The relaof insensitivity to inhibition by EDTA
sequent
might
result
from
the
inability
peroxidation
and
this
is
consistent
ESR signal as a result of heat.ing. On the other hand, urea, which converts P-450 to P-420, does not cause peroxidation in microsomes. These various correlations suggest that P-450 is t’he
disappearance
not
of the
involved
in
the
peroxidation
observed and that peroxidation may be induced as a result of an alteration in the structure of microsomal Fe, which makes iron cat’alytically available. This concept is further substantiated by the fact that no peroxides are formed on incubation of HgClz with shark liver microsomes, which are known to be unable to catalyze t,he metabolism of drugs (7) and thus probably lack some component of the microsomal electron transport chain. In further agreement, it’ should be noted that the extent of lipid peroxide formation parallels the activity of these microsomal enzymes (and in particular Fe,) with respect to age and sex. Thus, microsomes from the male rat, which have higher enzyme act’ivity than those of the female (8), form more peroxides those
of
the
female.
However,
micro-
from mouse liver show neither a difference in enzyme activity (S) nor an extent of peroxide formation with respect to sex. Both enzyme activity and extent of peroxide formation also increase wit’h age in the rat. Phenobarbital increases drug hydroxylatsomes
be interpret’ed
with
causes
of EDTA to chelate with protein-bound iron. Preheating of microsomes results in sub-
than
DISCUSSION
micro-
change
probably
pretreated
the
peroxides
bation
in
mice
50 mg
at
7% Inrease
45 G3 64 201
t,han
from
phenobarbital for
mpmoles MA
liver
if t’he
with
Pretreatment
urea
conceivably
Microsomes The numbers ments. hficrosomes
with
heme-
EFFECT OF 3 X 10-j M H&L? ON PEROSIU.~TION IN ROTGH .IND SMOOTH MICROSOMES FROM .~ND MICE TREATED WITH CONTROL AtICE PHENOR~RRITAL :\T 50 MG/KG/DAY FOR 3 D.~YS
90 minutes. of 5 experi-
hemoprotein-like
of cytochrome P-450 to cytochrome P-420. Since both microsomal P-450 and Fe, are
peroxidation
were incubated for represent the average
heat,
For example,
somal component, microsomal Fe, (6). Heating of microsomes also causes the disappearance of the Fe, signal, and incubation of
tive
III
reagents,
cytochrome P-450 is converted another pigment, P-420, by PCMB (5) or urea (6). In addition, PCMB causes the disappearance of the ESR signal thought to
some
TABLE
reacting compounds.
to
tion
42
other
microsomal
either
234
sulfhydryl
and
could
126
3i
ON MICROSOMES
32
UTLEY,
BERNHEIM,
ing activity, as assayed by the oxidative demethylation of aminopyrine, and this activation is accompanied by a parallel increase in TPNH-cytochrome c reductase, cytochrome P-450, and Fe, (6, 9). Proliferation of smooth endoplasmic reticulum accompanies these changes. Simultaneous administration of actinomycin D with phenobarbital greatly inhibits the increases in enzyme activity, but proliferation of endoplasmic reticulum is relatively insensitive to the drug. Results presented here on peroxidation after treatment with these drugs parallel the above-mentioned changes in enzyme activity. Thus, microsomes from animals given both phenobarbital and actinomycin D might peroxidize more than untreated controls as result of an actinomycin-insensitive, phenobarbital - induced, proliferation of smooth endoplasmic reticulum (increased lipid substrate). Microsomes from animals pretreated with phenobarbital alone might peroxidize to an even greater extent a result of both increased lipid substrate and increased availability of catalytically active HgC12altered enzyme. Furthermore, the increased peroxidation after phenobarbital pretreatment occurs mainly in the smooth surfaced microsomes, and this is consistent with the selective in-
AND
HOCHSTEIN
crease in drug metabolizing activity in this fraction after phenobarbital treatment. The maximum peroxidation produced by HgClz is only about a third that produced by the addition of FeS04. The latter presumably peroxidized all or nearly all of the unsaturated lipid in the microsome. The former may act by changing the Fe, component, which, being fixed in the microsome, may only peroxidize the lipids adjacent to it. REFERENCES 1. OTTOLENGHI, A., Arch. Biochem. Biophys. 79, 355 (1959). 2. COLLINS, F. D., AND SHOTLANDER, V. L., Biothem. J. 79, 321 (1961). 3. SCHNEIDER, W. C., AND HOGEBOO~, G. M., Cancer Res. 13, 617 (1953). 4. FOUTS, J. R., Biochem. Biophys. Res. Commun. 6, 373 (1961). 5. COOPER, D. Y., NARASIMHULU, S., ROSENTHAL, O., AND ESTABROOK, R. W., in “Oxidases and Related Redox Systems” (T. King, H. S. Mason, and M. Morrison, eds.), p. 838. Wiley, New York (1965). 6. MASON, H. S., NORTH, J. C., AND VANNESTE, M., Federation Proc. 24, 1172 (1965). 7. BRODIE, B. B., AND MAICKEL, R. P., PTOC. Intern. Pharmacol. Meeting, First, Stockholm. Symposium 6, 229 (1962). 8. CONNEY, A. H., SCHNEIDMAN, K., JACOBSON, M., AND KUNTZMAN, R., Ann. N.Y. Acad. Sci. 133, 98 (1965). 9. ERNSTER, L., AND ORRENIUS, S., Federation Proc. 24, 1190 (1965).
OF
Effect
BIOCFIEYISTRY
AND
of Sulfhydryl
BIOPHYSICS
Reagents
HENRY G. UTLEY, FREDERICK Department
of Physiology
and
X9-32 (1967)
118,
on Peroxidation BERNHEIM,
AND
Pharmacology, Duke University Medical Received April 22, 1966
in Microsomes’ PAUL HOCHSTEIN’ Center, Durham,
North
Carolina
Incubation of mouse liver microsomeswith HgC12,NEM, or PCMB results in the formation of lipid peroxides as measuredby the thiobarbituric acid reaction. Carbon monoxide inhibits peroxidation induced by NEM and the inhibition is reversed by light. Peroxidation induced by HgClz is not greatly affected by EDTA but is increased by ascorbic acid. Microsomes isolated from mice pretreated by intraperitoneal injection of HgClz peroxidized endogenous unsaturated lipid on incubation, and addition of HgClz in vitro further increases the peroxidation. The in vitro stimulation of peroxidation by these SH reagents in liver microsomes increases with age in the rat, and microsomes from male rats are more active than those from female. Pretreatment of mice with phenobarbital for 3 days increases the in vitro effect of HgClz on peroxidation. This stimulation occurs in the smooth-surfaced microsomes. Actinomycin partially inhibits the effect of phenobarbital. Mercuric chloride causes no peroxidation on incubation with shark liver microsomes. Urea causes no peroxidation on incubation with mouse liver microsomes. These results are consistent with the possibility that sulfhydry-reacting agents produce a change in tertiary structure of microsomal Fe, , thereby rendering the prot.ein-bound iron available for catalysis of peroxidation of endogenous lipid.
extracted lipids. This fact, and the general similarity of the lipid composition of microsomes and mitochondria (2), suggested that some factor in the microsomes was involved in the catalytic effect of mercury. Accordingly, an attempt was made to characterize this factor.
Ferrous salts catalyze the peroxidation of unsaturated lipids in mitochondria and microsomes as well as the peroxidation of pure unsaturated lipids (1). Other cations, with the exception of copper, have not been shown t,o catalyze this reaction. In this paper, we report experiments which demonstrate that mercuric ions incubated with liver microsomes may also induce a peroxidation of endogenous lipids. Incubation of mitochondria (fresh, frozen, or previously heated to GO”) with mercuric chloride resulted in no peroxidation, nor did mercuric chloride produce significant peroxidation of lipids extracted from microsomes by a 1:4 ethanolether mixture, although ferrous ions were active in inducing peroxidation of these
MATERIALS AND METHODS Rats or mice were stunned by a blow on the head and decapitated, and the liver was immediately removed and placed in cold sucrose (0.25 M). The tissue was allowed to cool, weighed, and minced with scissors, and microsomes were prepared according to the method of Schneider and Hogeboom (3). Smooth- and rough-surfaced fractions were prepared according to the method of Fouts (4). Microsomes were suspended in isotonic KC1 (1.15 M) to twice the initial tissue weight, and 0.3 ml was added to incubation flasks. Substances to be added in vitro were dissolved in 0.067 RI Naphosphate buffer (pH 7.4) and added to flasks in a volume of 0.2 ml. Flasks were made up to a final volume of 3 ml with buffer and incubated at 37” with shaking.
1 Part, of a thesis submitted by H. G. Utley in partial fulfillment for the requirements of the Ph.D. degree, Duke University. Supported in part by grants GB 1416 from the National Science FoundaGon and AT-(40-l-3329 from the AEC. 2 U.S. Public Health Service Research Career Awardee (KS-GM 4857). 29
30
UTLEY, TABLE
BERNHEIM,
I
AGENTS ON LIPID PEROXIDATION IN MOUSE LIVER MICROSOMES
EFFECT
OF VARIOUS
“Pretreated” mice receive 3 mg/kg/day HgClv for 2 days. CO was added to the appr0priat.e flask as a 1:l mixture with oxygen. Subsequent incubations of flasks containing CO were carried out either in the dark or under a 100 W light bulb. Microsomes were incubated for 90 minutes at 37”. Numbers represent the average of at least three experiments. Additions
and/or treatment
None FeSOl (0.03 mM) + Ascorbate (0.2 mM) + EDTA (0.03 mM) HgClz (0.03 mM) + Ascorbate (0.2 mM) + EDTA (0.03 m&r) + CO-Dark + CO-Light NEM (2 mrvr) + Ascorbate (0.2 mM) + EDTA (0.03 mM) + HgC12 (0.03 mM) PCMB (0.2 mM) + Ascorbate (0.2 mM) + HgClz (0.03 mM) Preheating (54-10”) + HgClz (0.03 mM) HgCla Pretreated + HgClz (0.03 mM) Urea (4 mM)
0 210 339 0 132 201 117 14 69 114 201 105 116 111 219 99 123 147 213 243 0
AND
HOCHSTEIN
various other sulfhydryl reagents on lipid peroxidation in liver microsomes. Maximal peroxidation followed incubation of mouse liver microsomes for 90 minutes with HgClz at 0.03 mM, PCMB at 0.2 mM, or NEM at 2 mM. The effects were not additive. Thus, the addition of both HgC12 and NEM or PCMB caused no greater peroxidation than the addition of each of these singly. However, as in the case with ferrous ions, ascorbate increased the HgClz effect. EDTA caused little inhibition of HgC& or NEMinduced peroxidation. Carbon monoxide inhibited peroxidation induced by NE11 and the inhibition was reversed by light. These experiments suggest the involvement of both iron and sulfhydryl groups in the peroxidation reaction. Urea caused no peroxidation, but preheating of microsomes to 54” for 10 minutes before incubation at 37” had a pronounced effect. Table I also shows that microsomes isolated from mice administered 2 daily doses of HgClz (3 mg per kilogram per day) peroxidize unsaturated lipid to an extent almost as great as that caused by iron added in vitro. Addition of HgC12 in vitro to pretreated animals caused a further increase. Table II shows the effect of HgCl, on peroxidation in microsomes prepared from mice and rats treated in various ways. Mercuric chloride had a greater effect in microsomes from male than female rats, although this difference was not seen in mice and peroxidation was greater in microsomes
Peroxidation was measured by the thiobarbituric acid (TBA) reaction. Aliquots of 0.5 ml of the incubation suspension were removed at appropriate intervals and added to a mixture of 2.0 ml of 2Oy, trichloroacetic acid and 4.0 ml of 0.67yc TBA. The mixture was placed in a boiling water bath for 10 minutes and centrifuged, and the optical density of the supernatant fluid was read at 535 rnp. An extinction coefficient of A&b’& = 1.56 X lo5 was used to calculate the amount of malonyldialdehyde (MA) formed.
F&O*
RESULTS
As reported by others (l), ferrous ions initiated a peroxidation of endogenous lipids as measured by MA formation, which was enhanced by ascorbate and inhibited by EDTA (Table I). This table and Fig. 1 show the effects of treatment with HgClz and
Minutes FIG.
1
103mM)
1
SULFHYDRYL TABLE
EFFECT
REAGENT
II
v&o
EFFECT OF 3 X 10m5M HGCL* ON PEROXIDATION IN LIVER MICROSOMES ISOLATED FROM UNTREATED MICE, MICE PRETREATED WITH PHEN~R~RRIT~L D AT 80 AT 50 MG/KG/D~Y .IND ACTINOMYCIN @/KG/DAY FOR 3 D.4vs SND MICE TREATED WITH PHENOBARBITAL ALONE
The effect of sex and age was tested in rats as indicated below. Microsomes were incubated for 90 minut,es. Numbers represent the amount of MA formed in mpmoles and are the average of at least 3 experiments.
by
urea,
133
324
57
147
represent
the
microsomes
None Phenobarbital None Phenobarbital
Rough Smooth
from
adult
rats
,\licrosomes 3
lipid
days
produced on
with
HgC12.
mals
t’reated
D
produced those found
less
80
at
that
microsomes
produced
with
HgC&.
no
218
young. with
kilogram
per
day
levels
of
in vitro incu-
Microxomes
from
ani-
phenobarbital
pg per
peroxide,
untreated
40
of
highest
both
from
liver
per
subsequent
with
actinomycin
t’hose
,
and
kilogram
but
still
per more
day than
It was also
animals. isolated
from
peroxides
on
shark
incubation
The ner
results
may
consistent
bution
of certain
function
in
steroids.
These
the
the
microsomal
a man-
and enzymes
met’abolism enzymes
in
activity
can
of
drugs
be
altered
distriwhich and in
the
conversion
or iron-containing
enzymes
catalyze
lipid
they
peroxida-
iron were made accessible through in tertiary structure. The relaof insensitivity to inhibition by EDTA
sequent
might
result
from
the
inability
peroxidation
and
this
is
consistent
ESR signal as a result of heat.ing. On the other hand, urea, which converts P-450 to P-420, does not cause peroxidation in microsomes. These various correlations suggest that P-450 is t’he
disappearance
not
of the
involved
in
the
peroxidation
observed and that peroxidation may be induced as a result of an alteration in the structure of microsomal Fe, which makes iron cat’alytically available. This concept is further substantiated by the fact that no peroxides are formed on incubation of HgClz with shark liver microsomes, which are known to be unable to catalyze t,he metabolism of drugs (7) and thus probably lack some component of the microsomal electron transport chain. In further agreement, it’ should be noted that the extent of lipid peroxide formation parallels the activity of these microsomal enzymes (and in particular Fe,) with respect to age and sex. Thus, microsomes from the male rat, which have higher enzyme act’ivity than those of the female (8), form more peroxides those
of
the
female.
However,
micro-
from mouse liver show neither a difference in enzyme activity (S) nor an extent of peroxide formation with respect to sex. Both enzyme activity and extent of peroxide formation also increase wit’h age in the rat. Phenobarbital increases drug hydroxylatsomes
be interpret’ed
with
causes
of EDTA to chelate with protein-bound iron. Preheating of microsomes results in sub-
than
DISCUSSION
micro-
change
probably
pretreated
the
peroxides
bation
in
mice
50 mg
at
7% Inrease
45 G3 64 201
t,han
from
phenobarbital for
mpmoles MA
liver
if t’he
with
Pretreatment
urea
conceivably
Microsomes The numbers ments. hficrosomes
with
heme-
EFFECT OF 3 X 10-j M H&L? ON PEROSIU.~TION IN ROTGH .IND SMOOTH MICROSOMES FROM .~ND MICE TREATED WITH CONTROL AtICE PHENOR~RRITAL :\T 50 MG/KG/DAY FOR 3 D.~YS
90 minutes. of 5 experi-
hemoprotein-like
of cytochrome P-450 to cytochrome P-420. Since both microsomal P-450 and Fe, are
peroxidation
were incubated for represent the average
heat,
For example,
somal component, microsomal Fe, (6). Heating of microsomes also causes the disappearance of the Fe, signal, and incubation of
tive
III
reagents,
cytochrome P-450 is converted another pigment, P-420, by PCMB (5) or urea (6). In addition, PCMB causes the disappearance of the ESR signal thought to
some
TABLE
reacting compounds.
to
tion
42
other
microsomal
either
234
sulfhydryl
and
could
126
3i
ON MICROSOMES
32
UTLEY,
BERNHEIM,
ing activity, as assayed by the oxidative demethylation of aminopyrine, and this activation is accompanied by a parallel increase in TPNH-cytochrome c reductase, cytochrome P-450, and Fe, (6, 9). Proliferation of smooth endoplasmic reticulum accompanies these changes. Simultaneous administration of actinomycin D with phenobarbital greatly inhibits the increases in enzyme activity, but proliferation of endoplasmic reticulum is relatively insensitive to the drug. Results presented here on peroxidation after treatment with these drugs parallel the above-mentioned changes in enzyme activity. Thus, microsomes from animals given both phenobarbital and actinomycin D might peroxidize more than untreated controls as result of an actinomycin-insensitive, phenobarbital - induced, proliferation of smooth endoplasmic reticulum (increased lipid substrate). Microsomes from animals pretreated with phenobarbital alone might peroxidize to an even greater extent a result of both increased lipid substrate and increased availability of catalytically active HgC12altered enzyme. Furthermore, the increased peroxidation after phenobarbital pretreatment occurs mainly in the smooth surfaced microsomes, and this is consistent with the selective in-
AND
HOCHSTEIN
crease in drug metabolizing activity in this fraction after phenobarbital treatment. The maximum peroxidation produced by HgClz is only about a third that produced by the addition of FeS04. The latter presumably peroxidized all or nearly all of the unsaturated lipid in the microsome. The former may act by changing the Fe, component, which, being fixed in the microsome, may only peroxidize the lipids adjacent to it. REFERENCES 1. OTTOLENGHI, A., Arch. Biochem. Biophys. 79, 355 (1959). 2. COLLINS, F. D., AND SHOTLANDER, V. L., Biothem. J. 79, 321 (1961). 3. SCHNEIDER, W. C., AND HOGEBOO~, G. M., Cancer Res. 13, 617 (1953). 4. FOUTS, J. R., Biochem. Biophys. Res. Commun. 6, 373 (1961). 5. COOPER, D. Y., NARASIMHULU, S., ROSENTHAL, O., AND ESTABROOK, R. W., in “Oxidases and Related Redox Systems” (T. King, H. S. Mason, and M. Morrison, eds.), p. 838. Wiley, New York (1965). 6. MASON, H. S., NORTH, J. C., AND VANNESTE, M., Federation Proc. 24, 1172 (1965). 7. BRODIE, B. B., AND MAICKEL, R. P., PTOC. Intern. Pharmacol. Meeting, First, Stockholm. Symposium 6, 229 (1962). 8. CONNEY, A. H., SCHNEIDMAN, K., JACOBSON, M., AND KUNTZMAN, R., Ann. N.Y. Acad. Sci. 133, 98 (1965). 9. ERNSTER, L., AND ORRENIUS, S., Federation Proc. 24, 1190 (1965).