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TUE
JWRN.%I.
OF
B~o~oa~ca~.
CHEMISTRY
0
1994
by
The
American Society
for
Biochemistry and Molecular Biology, Inc
Vol.
269,
No.
11.
Issue
of
March
18,
pp.
8022-8028,
1994
Printed
in
U.S.A.
Interactions
of
Cyclic Hydrocarbons with Biological Membranes*
(Received for publication, June
14,
1993, and in revised form, November 29, 1993)
Jan
SikkematB, Jan
A.
M.
de Bontt, and Bert Poolmann
From the $Diuision
of
Industrial Microbiology, Department
of
Food Science, Wageningen Agricultural University,
P
0.
Box
8129,
6700
EV
Wageningen and the IDepartment
of
Microbiology, University
of
Groningen,
P
0.
Box
14,
9750
AA
Haren,
The
Netherlands
Many cyclic hydrocarbons,
e.g.
aromatics, cycloal-
kanes, and terpenes, are toxic to microorganisms. The
primary site of the toxic action is probably the cytoplas-
mic membrane, but the mechanism of the toxicity is still
poorly understood. The effects of cyclic hydrocarbons
were studied in liposomes prepared from
Escherichia
coli
phospholipids. The membrane-buffer partition coef-
ficients of the cyclic hydrocarbons revealed that these
lipophilic compounds preferentially reside in the mem-
brane. The partition coefficients closely correlated with
the partition coefficients of these compounds in a stan-
dard octanol-water system. The accumulation of hydro-
carbon molecules resulted in swelling of the membrane
bilayer, as assessed by the release of fluorescence self-
quenching of fluorescent fatty acid and phospholipid
analogs. Parallel to the expansion of the membrane, an
increase in membrane fluidity was observed. These ef-
fects
on
the integrity of the membrane caused an in-
creased passive flux of protons and carboxyfluorescein.
In cytochrome
c
oxidase containing proteoliposomes,
both components of the proton motive force, the pH gra-
dient and the electrical potential, were dissipated with
increasing concentrations of cyclic hydrocarbons. The
dissipating effect was primarily the result of an
in-
creased permeability of the membrane for protons
(ions). At higher concentrations, cytochrome
c
oxidase
was also inactivated. The effective concentrations of the
different cyclic hydrocarbons correlated with their par-
tition coefficients between the membrane and aqueous
phase. The impairment of microbial activity by the cy-
clic hydrocarbons most likely results from hydrophobic
interaction with the membrane, which affects the func-
tioning of the membrane and membrane-embedded pro-
teins.
Cyclic hydrocarbons, such
as
aromatics, alicyclics, and
ter-
penes, interact with biological membranes (de Smet et
al.,
1978; Sikkema et
al.,
1992; Uribe et al., 1985, 1990). These
interactions lead to changes in structure and function of the
membranes, which in turn, may impair growth and activity of
the cells (Sikkema et
al.,
1992). The widespread use of cyclic
hydrocarbons (e.g. fuels, solvents, starting compounds for or-
ganic synthesis) and their release in the environment makes
knowledge of their metabolism and toxicity of eminent impor-
tance. The toxicity of cyclic hydrocarbons
has
been well noted
(Smith, 19931, but knowledge about their mode of interaction
Industrial Biotechnology. The costs
of
publication of this article were
:*
This
work
was
supported
by
the Dutch Programme Committee
on
defrayed in part by the payment
of
page charges. This article must
therefore
be
hereby marked “aduertisernent” in accordance with 18
U.S.C. Section 1734
solely
to indicate this fact.
9:
To whom correspondence should
be
addressed: Snow Brand
Euro-
pean Research Laboratories BV, Zernikepark
6,
9747
AN,
Groningen,
The Netherlands. Fax: +31-50.745766.
with cells and the cause of toxicity is scarce. Uribe and co-
workers studied the toxicity of p-pinene (Uribe et
al.,
1985) and
cyclohexane (Uribe
et
al.,
1990) on intact yeast cells and iso-
lated mitochondria. Both compounds exerted their action
at
the
level of the membrane and membrane-embedded enzymes. Re-
cently, we have reported
the
effects of the aromatic hydrocar-
bon tetralin on the structure and function of both bacterial and
liposomal membranes (Sikkema et
al.,
1992). Our data showed
that
tetralin accumulated in the membrane (partition coeffi-
cient approximately 1,100), causing “expansion” of the mem-
brane surface area, inhibition of primary ion pumps, and in-
crease in proton permeability.
As
a
result the electrical
potential and pH gradient were dissipated, which may have
been the primary cause of inhibition of ceilular growth. Further
experiments with other aromatic and alicyclic hydrocarbons
indicated
that
the observed effects were not specific for tetralin
and
that
a direct relationship can be found between the parti-
tioning of
a
particular compound in the membrane and its
effect on the structural integrity and functional properties of
the membrane (this paper). Effects of polar and non-polar com-
pounds on biological membranes have been reported for fatty
acids (Rottenberg, 19901, ethanol in yeast (Cartwright
et
al.,
1986; Lei50 and van Uden, 1984), and anesthetics in erythro-
cytes (Seeman, 1972). The explanation most given for the ob-
served toxicity of these compounds is disruption of membrane
structure by hydrophobic interaction with the lipid bilayer due
to their lipophilicity.
In this investigation, the toxic effects of different cyclic hy-
drocarbons were studied and related to their hydrophobicity
and partitioning into the membrane. The results show
that
effects of cyclic hydrocarbons on structural and functional prop-
erties
of
membranes are closely related to their accumulation
in the membrane. The data give
a
rationale for
the
frequently
observed correlation between the toxicity of lipophilic com-
pounds to microorganisms and the partition coefficients of such
compounds in
a
standard octanol-water system (log
P
or
kow;
Leo et al., 1971).
MATERIALS AND METHODS
Preparation
of
Liposomes-Escherichia coli phospholipids, obtained
from Sigma, were washed with acetone/ether (Kagawa and Racker,
1971). The commercially obtained
E.
coli
lipids contained phosphati-
dylethanolamine
(72
mol
%),
lyso-phosphatidylethanolamine
(5.2
mol
%),
and cardiolipin (20.5 mol
%)
(In ’t Veld
et
al.,
1992). Lipids dissolved
in CHC1,NeOH
(9:1,
viv)
were mixed in appropriate quantities and
dried under
a
stream
of
N,
gas.
Traces
of
solvent were then removed
under vacuum for
1
h.
Dried
lipid
was
suspended in
50
mM potassium
phosphate (pH 7.0)
at
a
concentration
of
20 mg lipidiml and dispersed
by
ultrasonic irradiation using
a
bath sonicator (Sonicor, Sonicor In-
struments,
New
York). Single membrane liposomes (Chapman, 1984;
Elferink et
al.,
1992) were obtained by sonication (probe type sonicator,
MSE, West Sussex, United Kingdom) for 300
s
at maximal amplitude,
using intervals of 15-s sonication and 45-s rest, at
4
“C
under
a
constant
stream of N2 gas.
Partitioning
of
Lipophilic Cornpounds-Partitioning of lipophilic
compounds over membrane
and
buffer phases was determined in
a
E.
8022
This is an Open Access article under the CC BY license.
Membrane Interactions of Cyclic Hydrocarbons 8023
coli phospholipid liposomeipotassium phosphate buffer system (De
Young and Dill, 1988; Katz and Diamond, 1974). Increasing amounts of
the radiolabeled compounds were added to 50 rnM potassium phosphate
(pH 7.0) containing liposomes (5.0 mg of phospholipid/ml; final volume
0.5 ml). After equilibration (30 mini, the liposomes were spun down in
an Airfuge (Beckman Instruments) for 30 min at 135,000 x g. By this
method all liposomes were pelleted, as was assessed by phosphate
analyses of control incubations performed in MOPS’ buffer (50 nm, pH
71 (Rouser et al., 1970). The supernatant was removed with a Pasteur
pipette, and two portions of 100 pl were pipetted in a scintillation vial.
The pellet was resuspended in scintillation fluid. Both the pellet and the
supernatant fractions were analyzed radiometrically in a scintillation
counter. The results presented are the mean and the standard deviation
of six independent measurements. Control experiments without lipo-
somes were performed in parallel to account for losses of solvent due to
possible evaporation and/or attachment to tubes and pipettes. The in-
ternal volume of the liposomes (3 pVmg phospholipid) was taken into
account. Partition coefficients of the non-radioactive lipophilic com-
pounds carvone and tetralin were determined by gas chromatography,
as described previously (Sikkema et al., 19921.
Membrane Expansion and Extraction ofPhospholipids-The expan-
sion of liposomal membranes and extraction of phospholipids from the
liposomes due to the addition of lipophilic compounds was monitored in
liposomes labeled with the fluorescent fatty acid, octadecyl rhodamine-
a-chloride CR,,; Molecular Probes Inc., Junction City, OR1 or the fluo-
rescent phospholipid analog, N-(lissamine rhodamine+sulfonyl)phos-
phatidylethanolamine (N-Rh-PE; Avanti Polar Lipids Inc., Alabaster,
AL). The method is based on the relief of fluorescence self-quenching
(Hoekstra et al., 1984) of rhodamine+-chloride as a result of expansion
of the membrane and/or extraction of the probe from the membrane.
The fatty acid probe was incorporated into liposomal membranes at a
concentration of 4 mol Q phospholipid phosphorous. Maximum rhoda-
mine fluorescence was determined upon the addition of 1%’ (v/v) Triton
X-100. Fluorescent changes were measured in a spectrofluorometer
(Perkin-Elmer Cetus) using the excitation-emission pair 560 and 590
nm. In order to discriminate between fluorescence increases due to
expansion of the membrane and extraction of membrane constituents,
incubation mixtures with different concentrations of lipophilic com-
pounds were centrifuged at 135,000 x g (Beckman Airfuge, 30 min).
Subsequently, the fluorescence of the supernatant was determined rela-
tive to the supernatant of an incubation without hydrocarbon added.
Additionally, supernatants of incubations containing E. coli phospho-
lipid liposomes in MOPS (50 m&f, pH 7.0~ and varying amounts of cyclic
hydrocarbons were assayed for released phospholipids by phosphate
analysis (Rouser et al., 19701.
Membrane Fluidity Measurements IFluorescence Polarization
Measurements-DPH (1,6-diphenyl-1,3,5hexatrienel and TMADPH
~l-[4-~trimethylamino)phenyl~-6-phenylhexa-l,3,5-triene) steady-state
polarization measurements were carried out as described (In ‘t Veld et
al., 19911. Membrane fluidity is used as a qualitative measure and is
defined as the inverse of microviscosity. Microviscosity can be deduced
from the steady-state fluorescence polarization of (TMA-)DPH probes
(Shinitzky and Barenholz, 1978).
The degree of fluorescence polarization was calculated from Equation
1 (Lentz, 1989; Shinitzky and Barenholz, 1978):
(Eq. 1)
r,,, steady-state fluorescence polarization; I,, , fluorescence intensity at
430 nm, measured parallel to the emitted light; I, : fluorescence inten-
sity at 430 nm, measured perpendicular to the emitted light.
Reconstitution of Cytochrome c Oxidase into Proteoliposomes-Ace-
ton-ether-washed E. coli lipid (40 mg) and n-octyl+-n-glucopyranoside
(18 mgl in 2 ml of 50 mM potassium phosphate (pH 7.0) was cosonicated
until clarity under a constant stream of N, gas at 4 “C using a probe
sonicator. Cytochrome c oxidase (9 nmol of heme a was added, and the
suspension was dialyzed at 4 “C for 4 h against a 500-fold volume of 50
rnM potassium phosphate (pH 7.01. Dialysis was repeated for another 4
h and continued overnight at 4 “C (Hinkle et al., 1972).
Internal pH of Cytochrome c Onidase Containing Proteoliposomes-
Internal pH changes were measured by following the fluorescence of
’ The abbreviations used are: MOPS, 4-morpholinepropanesulfonic
acid; DPH, l&diphenyl-1,3,5-hexatriene; TMA-DPH, 1-14.(trimethyl-
aminolphenyll-6-phenylhexa-1,3,5triene; CF, carboxyfluorescein;
DMF, N-dimethylformamide; TMPD; N,N,N’,N’-tetramethyl-p-phe-
nylenediamine.
entrapped pyranine (Eastman Kodak Co.) (Clement and Gould, 1981).
To incorporate pyranine into proteoliposomes (20 mg phospholipid/ml),
100 nmol of pyranine was added to 0.5 ml of proteoliposomes and rap-
idly mixed. The suspensions were rapidly frozen in liquid nitrogen and
subsequently thawed slowly (approximately 30 min) at room tempera-
ture. The suspension was sonicated for 8 s using a probe type sonicator
at an amplitude of 4. To remove external pyranine, the proteoliposomes
were washed in 10 ml of 50 rnM potassium phosphate (pH 7.01 and
centrifuged for 45 min at 280,000 x g in a Beckman type Ti-75 rotor at
4 “C. Fluorescent changes were measured at excitation and emission
wavelengths of 460 and 508 nm, respectively. Calibration was per-
formed by titration with acid or base upon addition of nigericin to a tinal
concentration of 20 nM. At pH 7.0, the ApH generated by the cytochrome
c oxidase-containing proteoliposomes was 0.8-0.9.
Electrxal Potential across Membranes of Proteoliposomes-The
transmembrane electrical potential C&/J) of cytochrome c oxidase con-
taining liposomes was determined by monitoring the distribution of
tetraphenylphosphonium (TPP’) across the membrane with a TPP+-
sensitive electrode as previously described (Lolkema et al., 1982).
Proton Fluxes through Liposomal Membranes-A$-Induced proton
fluxes were estimated in liposomes in the presence of varying concen-
trations of a particular hydrocarbon compound in a well stirred ther-
mostated 2-ml cuvette, using phenol red (20 pg/ml, final concentration)
as external pH indicator. Absorbance changes (AsGO - AslO) were con-
verted into H+ fluxes by calibrating with known amounts of oxalic acid
or KOH (de Vrij
et
al., 1988). Valinomycin-induced potassium diffusion
potentials were imposed across the liposomal membrane by loo-fold
dilution of the liposomes (20 mg of phospholipid/ml) into the same
medium, in which sodium ions were substituted for potassium ions and
supplemented with phenol red (20 ug/ml). Generation of the electrical
potential was initiated by adding valinomycin (2 pi, final concentra-
tion).
Determination of Cytochrome c Oxidase Activity-Cytochrome c oxi-
dase activity was measured spectrophotometrically by monitoring the
decrease in the absorbance of the alpha peak of reduced cytochrome c,
using an extinction coefficient (reduced minus oxidized) of •‘,s,)-~.,~~ =
19.5 mM-’ cm-’ (de Vrij et al., 1988).
Measurement of Carboxyfluorescein
Efflur-Release of carboxyfluo-
rescein (CF) from liposomes, resulting in relief of its fluorescence self-
quenching, was determined in a Perkin Elmer spectrofluorometer
equipped with a thermostated cell holder, using the excitation-emission
pair 430 and 520 nm, respectively. Encapsulation of 5,6carboxyfluores-
cein (CF, Eastman Kodak Chemical Co.) was achieved by preparing
liposomes in 50 rn.21 CF following the protocol for the formation of pyra-
nine containing liposomes (see above).
Chemicals-All hydrocarbons used were of the highest available
commercial grade. Radiochemicals were obtained from the Radiochemi-
cal Centre, Amersham, United Kingdom (1 ‘%lphenylalanine, l’%ltolu-
enesulfonic acid, I “‘Clbenzoic acid, [“%I4-chlorobenzoic acid) and
Sigma
(l’%ltoluene, l’%lnaphthalene, and I ‘%lphenanthrene.
l”HlTPP+, used for the determination of the partition coefficient, was
obtained from Amersham.
Addition of Cyclic Hydrocarbons-The
hydrocarbons were prepared
as solutions inN-dimethylformamide (DMF). In all
cases,
the amount of
DMF was 2% (v/v) of the total volume. In this concentration DMF had
no effect on any parameter studied except for the binding of TPP+ to
membranes (binding of TPP’ was less in the presence of DMF); AI/I
values were corrected accordingly (Lolkema
et
al., 1982).
RESULTS
Partitioning
of
Lipophilic Compounds-In order to gain in-
sight in the effects of lipophilic compounds, at subsaturating
concentrations in the aqueous phase, on biological membranes
it is essential to know the partitioning behavior of such com-
pounds in a membrane-buffer system. As a model system for a
biological membrane, liposomes were used prepared from
E.
coli
phospholipids.
Membrane-buffer partition coefficients in the
E. coli
phos-
pholipid liposomes/potassium phosphate buffer were deter-
mined for compounds varying in hydrophobicity/lipophilicity,
i.e.
phenylalanine, benzoic acid, 4-chlorobenzoic acid, 4-tolu-
enesulfonic acid, TPP’, carvone, toluene, naphthalene, tetralin,
and phenanthrene. The choice of most of these molecules stems
from their availability in radioactive form. As a measure for the
hydrophobicity of the compounds, the octanol-water partition
8024
Membrane
partition coefficient membrane/buffer
10000
1000
100
10
1
Interactions
of
Cyclic Hydrocarbons
0.l”l
1
10
100
1000
10000
portition coefflcient octanol/woter
FIG.
1.
Relationship between the partition coefficients in an
E.
coli
phospholipid membrane-potassium phosphate buffer (pH
7.0;
50
mM) system and the partition coefficients in the standard
n-octanol-water system of 4-toluenesulfonic acid
(1
),
4-chloro-
benzoic acid
(2),
benzoic acid
(3),
phenylalanine
(4),
TPP’
(5),
carvone
(6),
toluene
(7),
naphthalene
(8),
tetralin
(9),
and phen-
anthrene
(10).
The
experimental points represent the mean and stan-
dard deviation
of
six independent measurements.
coefficients were taken (Leo et al., 1971). The distribution of the
lipophilic compounds over the aqueous and the lipid phase was
determined at several solvent to lipid ratios. When the meas-
ured aqueous concentration was plotted against the lipid
to
solvent ratio, a saturation curve was obtained (Sikkema et aZ.,
1992). The partition coefficient was calculated, from the linear
part of this curve (below maximum aqueous solubility). The
membrane-buffer partition Coefficients were plotted as
a
func-
tion of the octanol-water partition Coefficients (Fig. 1). Despite
differences in structural features of the molecules, a good cor-
relation between the partitioning in a membrane-buffer system
and
a
standard octanol-water system was observed. The corre-
lation line for lipophilic compounds with logP values between
approximately
1
and 4.5, is described by Equation
2:
log
P,,
=
0.97
X
log
Po,,
~
0.64
(Eq.
2)
The correlation coefficient for the four aromatic hydrocar-
bons toluene, naphthalene, tetralin, and phenanthrene is
0.9967. With this equation, the membrane-buffer partition co-
efficients of 20 cyclic hydrocarbons were calculated from their
octanol-water partition coefficients. In Table I the membrane-
buffer partition coefficients of these cyclic hydrocarbons to-
gether with other physical and chemical data of these com-
pounds are given.
Expansion
of
the Membrane-Due to the accumulation of
lipophilic compounds in the lipid bilayer, changes in the mem-
brane structure and even swelling
of
the membrane can be
expected. The effect of accumulation in the membrane surface
area was monitored by using liposomes prepared from
E.
coli
phospholipids that were labeled with Rl8 or N-Rh-PE. The ra-
tionale of this method is that expansion of the membrane leads
to dilution
of
the probe in the membrane which can be meas-
ured as a relief in fluorescence self-quenching. Since the fluo-
rescence signal
is
related
to
the lipid concentration (Hoekstra et
al., 19841, a change in fluorescence will be proportional to a
change in surface area.
An
increase in fluorescence could, how-
ever, also be due to extraction of the fluorescent probe from the
membrane by the hydrocarbon. Ultracentrifugation of lipo-
somes equilibrated with varying amounts of toluene, cyclohex-
ane, and tetralin showed that
at
the most 16.3,
11,
and 9.4% of
the fluorescence increase with 150 pmol of toluene, 15 pmol of
cyclohexane, and
5
pmol of tetralidmg phospholipid, respec-
tively, could be attributed to probe extraction from the mem-
brane. In addition, supernatants of incubations containing li-
posomes and varying concentrations of hydrocarbon were
checked for the presence of free phospholipids. The highest
concentrations of each hydrocarbon applied in the experiments
with R,*-labeled liposomes (see Fig. 2) did not result in extrac-
tion of more than 10% of the phospholipid content. The data for
the different compounds were: decalin, 8.6% of total phospho-
lipid phosphate solubilized at 3 pmoVmg PL; anthracene,
8.4%
at
1
pmoVmg PL; biphenyl, 9.0%
at
2.5 pmoVmg PL; a-pinene,
9.3%
at
2.5 pmoVmg PL; tetralin, 6.2% at
5
pmoVmg PL; naph-
thalene, 7.8% at 6 pmoVmg PL; cyclohexane, 9.9% at 15
FrnoVmg PL; o-xylene, 9.6%
at
60 pmoVmg PL; ethylbenzene,
8.7%
at
70
pmoVmg PL; toluene, 9.4% at 150 pmoVmg PL;
benzene, 9.1%
at
250 pmoVmg PL. At higher concentrations
solubilization of the liposomes did occur, which was not only
detected by a rapid increase of free phospholipids in the super-
natant but also by the increase of turbidity
of
the suspension in
the cuvette. In a set of control experiments it was shown that
the hydrocarbon solvent had no direct effect on fluorescence
intensity, which could have occurred as
a
result of modification
of the microenvironment of the probe. In these experiments the
same concentrations of solvents (Fig.
2)
were mixed with lipo-
somes labeled with non-self-quenching concentrations of the
fluorescent probes. Taken together, these results indicate that
the observed increase in Rl8 fluorescence was primarily due to
swelling of the membrane. Different solvents exhibit different
concentration dependencies and extents of apparent membrane
expansion (Fig. 2). For instance, in the presence of decalin the
increase in rhodamine fluorescence not only occurred at a much
lower concentration than with benzene, but the extent of fluo-
rescence increase was also higher. The difference in effective
concentration at which the rhodamine fluorescence increased
parallels the change in hydrophobicity of the compounds and
the partitioning into the membrane. The differences in the
extent of the fluorescence increase could be due to differences in
maximum solubility of the hydrocarbon in the membrane but
may also reflect differences in location in the membrane. Re-
sults similar
to
those presented in Fig. 2 were obtained with
N-Rh-PE-labeled liposomes (data not shown).
Changes in Membrane Fluidity
as
a
Result
of
Interaction
with Hydrocarbons-The fluidity of a membrane bilayer can be
assessed by determining the fluorescence polarization
of
DPH
or TMA-DPH. Although the precise location of DPH in the
membrane is still not clear, this probe most likely resides near
the center of the bilayer (Lentz, 1989). Less ambiguities exist
about the location of TMA-DPH since its hydrophilic group
anchors the molecule at the headgroup region of the bilayer
thereby aligning the DPH moiety with the phospholipid acyl
chains. All hydrocarbons except biphenyl decreased the polar-
ization of DPH whereas TMA-DPH polarization was not sig-
nificantly affected (Fig. 3). The different locations in the mem-
brane of DPH and TMA-DPH and the different effects
of
tetralin, cyclohexane, naphthalene, and toluene on the fluores-
cence polarization of DPH and TMA-DPH suggest that the
hydrocarbons perturb the bilayer structure primarily by accu-
mulating into the interior rather than into the peripheral re-
gions of the membrane.
Effects
of
Hydrocarbons on the Proton Motive Force-The
accumulation of hydrocarbons in the lipid bilayer, and the con-
sequent change in membrane-structure due
to
membrane-ex-
pansion, change in membrane fluidity, and/or disruption of
lipid-protein interactions could have
a
strong effect on the func-
tioning of the membrane as a selective barrier for ions and
hydrophilic molecules. The permeability for protons and other
ions is especially of importance since ion leaks directly affect
the energy transducing properties of the membrane. To analyze
the effect of hydrocarbons on the generation of the transmem-
brane pH gradient (ApH) and electrical potential
(A$)
in arti-
ficial membranes, beef heart mitochondrial cytochrome c oxi-
Membrane Interactions
of
Cyclic
Hydrocarbons
8025
TABLE
I
Physical data offhe cyclic hydrocarbons used in this study
~~
Compound
Formula
M,"
Solubilityh logP
Benzene
Cyclohexane
Toluene
Ethylbenzene
o-Xylene
Naphthalene
Tetralin
o-di-Ethylbenzene
a-Pinene
13-Pinene
y-Terpinene
Limonene
Decalin
Biphenyl
Anthracene
Phenanthrene
78.11
84.16
106.17
92.14
106.17
128.17
132.21
134.22
136.24
136.24
136.24
136.24
138.25
154.21
178.23
178.23
mmolll,
25
"C
22.9
0.683
6.28
1.27
2.02
0.797
0.125'
h
-
0.101'
0.126
0.040
0.025
-
-
2.13b
3.44b.d
2.6gb
3.15b
3.12
3.37h
3.86d
4.10f
4.46''
4.46"
4.46f
4.46''
4.83f
4.04'
4.45p
4.46R
POnV
PMmexP
Pl,r,Hcalch'
135 27
2,754 498
490 59
2
8.5 93
1,413 260
1,318 243
2,344 527
2
38 426
7,244 1,100
2
56 1,271
12,590 2,173
28,840 4,855
28,840 4,855
28,840 4,855
28,840 4,855
67,608 11,094
10,965 1,900
28,184 4,748
28,840 4,937
-c
86 4,855
Data obtained from Handbook of Chemistry and Physics.
Eastcott
et
al.,
1988.
Rekker,
1977.
Sikkema and de Bont,
1991.
Tomlinson and Haflcenscheid,
1986.
Data not available.
Riddick
et
al..
1986.
<'
Calculated from
Po,
data found in literature and applied
to
Equation
2.
"Calculated via fragmental constants (Rekker and de Kort,
1979).
rhodamine ffuorescence
(%)
30<
0.01
01
1
10
100
hydrocorbon
odded
l~mol/mg
PL)
FIG.
2.
Effect of cyclic hydrocarbons on the relief of fluores-
ture contained liposomes
(0.2
mg of
E.
coli
phospholipid/ml) labeled
cence self-quenching of Rls-labeled liposomes.
The reaction mix-
with R,,
(4
mol
%)
in potassium phosphate
(50
mM, pH
7.0).
The
changes in fluorescence were monitored using the excitation-emission
pair
560
and
590
nm. The temperature of the solution was kept at
30
"C.
A,
benzene;
A,
toluene;
V,
ethylbenzene;
V,
o-xylene;
0,
cyclohexane;
0,
naphthalene;
0,
tetralin;
0,
biphenyl;
+,
a-pinene;
e,
decalin.
dase was reconstituted into liposomes as proton motive force
generating mechanism. At an external pH of
7.0,
and in the
presence of the electron donor system ascorbate-TMPD-cyto-
chrome
c,
cytochrome
e
oxidase containing proteoliposomes
generated
a
-ZApH
and
A$
of
-54
and
-60
mV, respectively.
The results show that all hydrocarbons tested dissipated the
ApH (Fig.
4)
and that the inhibitory concentrations directly
correlated with the partitioning of the compound into the mem-
brane as well
as
with the increase in rhodamine fluorescence
and DPH polarization measurements. The
A$
was found to
decrease in
a
similar way as the ApH (data not shown).
Site(s)
of
Action
of
Hydrocarbons-The observed decrease in
ApH and
A$
could be the result of an increase in passive proton
or
ion fluxes, and/or inhibition
of
the energy transducing activ-
ity of the cytochrome
c
oxidase. Incubation
of
cytochrome
e
oxidase containing liposomes with different concentrations of
benzene, cyclohexane, tetralin, decalin, and biphenyl showed
A
DPH
polarization
0.19
0.01
O.?
1
10
100
hydrocarbon added
Ipmolhg
PL)
TMA-DPH
polorizotion
0'30'
O.7
O'*I
0.26
0.251
0.01
0.1
1
10
100
hydrocarbon odded l~rnol/mg
PL)
FIG.
3.
Polarization of DPH and TMA-DPH
as
a result of the
interaction
of
different cyclic hydrocarbons with
E.
coli
phos-
pholipid liposomes.
The measurements were performed in a spec-
trofluorometer at an excitation wavelength
of
360
nm; the emission was
recorded at
430
nm. The cuvette, thermostated at
30
"C, contained
0.2
mg/ml of either TMA-DPH-
or
DPH-labeled liposomes (probe
to
lipid
molar ratio in both instances
1-250)
in
50
mM potassium phosphate (pH
7.0).
0,
naphthalene;
0,
cyclohexane;
0,
tetralin;
0,
biphenyl;
+,
a-pi-
nene.
A,
effect of hydrocarbons on the polarization of DPH.
B,
effect
of
hydrocarbons on the polarization of TMA-DPH.
8026
Membrane Interactions
of
Cyclic Hydrocarbons
ApH
(%I
50
-
0
0.01
01
1
10
100
hydrocarbon odded
itunol/mg
PL)
FIG.
4.
Effect
of
cyclic hydrocarbons
on
the
ApH
generated
by
cytochrome
c
oxidase
containing
proteoliposomes. Energization
of
cytochrome
c
oxidase containing proteoliposomes was achieved in the
presence
of
cytochrome
c
(20
PM),
TMPD
(200
p~),
and ascorbate
(10
mM). The assay was performed in
50
mM potassium phosphate (pH
7.01,
in a cuvette thermostated at
30
"C.
A,
benzene;
A,
toluene;
V,
ethylben-
zene;
V,
o-xylene;
0,
naphthalene;
0,
cyclohexane;
*,
o-di-ethylbenzene;
0,
tetralin;
0,
biphenyl;
+,
a-pinene;
+,
decalin.
that indeed inhibition of the enzyme activity occurred. Com-
parison of the sensitivity of cytochrome
e
oxidase reconstituted
in liposomes with the enzyme in Triton X-100 solution indi-
cated that the membrane embedded enzyme was more affected
by hydrocarbons (Fig.
51,
as could be expected from the accu-
mulation of the molecules in the membrane. Since the enzyme
in solution is associated with detergent micelles it is difficult to
compare the inhibitory effects on the reconstituted and "free"
enzyme quantitatively.
Dissipation of the ApH
as
a result of an increased proton
permeability of the membrane was assessed by determining
the passive proton influx across the liposomal membrane.
Po-
tassium-loaded liposomes were diluted into potassium-free me-
dium in the presence of valinomycin, and the initial rates of
H'
influx in the absence and presence of different amounts of
hydrocarbon were determined (Fig.
6).
Increasing amounts of
hydrocarbon were needed
to
increase the proton permeability
of the membrane going from anthracene, decalin, tetralin, cy-
clohexane, and toluene to benzene. The concentrations of hy-
drocarbons that affected the proton permeability were in the
same range as those that inhibited cytochrome
c
oxidase.
Permeability
of
Liposomal Membranes
for
CF-To assess the
effect of cyclic hydrocarbons on the permeability of the mem-
brane for low molecular weight molecules, the efflux of the
fluorescent dye CF was examined. In the presence of various
hydrocarbons an increased leakage of CF
(M,.
=
376) was ob-
served that paralleled the increase in permeability of the mem-
brane to protons. The concentration
at
which leakage of car-
boxyfluorescein was observed was only slightly higher than the
hydrocarbon concentrations needed to increase the proton per-
meability (data not shown).
DISCUSSION
Due to the hydrophobic character of hydrocarbons, the pri-
mary site of their toxicity is the membrane. Hydrocarbons ac-
cumulate in the lipid bilayer according
to
a
partition coeficient
that is specific for the compound applied. Since partitioning of
a compound between
a
membrane and an aqueous phase
is
difficult
to
determine, and may vary with the composition of the
membrane, attempts have been made
to
find
a
parameter for
partitioning. The octanol-water system, which has been applied
for
many years in anesthesiology and environmental biology
(Leo
et al.,
1971; Verschueren, 1983), proved to be the most
suitable model system (Lieb and Stein, 1986). For the
E.
coli
hydrocorbon odded
(prnol/rnl)
400
01
1
10
100
hydrocarbon odded
(Nrnol/mg
PL)
FIG.
5.
Activity
of
cytochrome
c
oxidase
as
determined by
monitoring
the
oxidation
of
reduced
cytochrome
c
(A.M)-A640).
The reaction mixture contained either proteoliposomes
(solid lines)
or
Triton
X-100-
(0.5%,
v/v) solubilized proteoliposomes
(dotted lines),
in
50
mM potassium phosphate (pH
7.0);
the assay was performed at
30
"C.
Ap was decoupled by the addition
of
valinomycin
(2
VM,
final concen-
tration), and nigericin
(0.1
p~,
final concentration). Activity
of
the cy-
tochrome
c
oxidase is expressed as mole cytochrome
c
per mole enzyme/
second. The maximum activity
of
the cytochrome
c
oxidase in uncoupled
liposomes was
425
s-l,
and
of
the enzyme in solubilized liposomes was
370
5-l.
A,
benzene;
0,
cyclohexane;
0,
tetralin;
0,
biphenyl;
+,
decalin.
zoo*
JH+
(nmol/rnm.
mg
PL)
o!
'
"""I
'
'
"""I
'
'
"""I
'
'
0.1
1
10
100
hydrocarbon added
(Nrnol/rng
PLI
ability
of
E.
coli
phospholipid
membranes. Liposomes
(0.2
mg
of
FIG.
6.
The
effect
of
cyclic
hydrocarbons
on
the
proton
perme-
phospholipidiml) were washed and resuspended in a medium in which
sodium ions were substituted
for
potassium ions and to which phenol
red
(20
pg/ml) was added. To initiate the potassium diffusion potential,
valinomycin
(2
PM,
final concentration) was added. Subsequently, ab-
sorbance changes were measured atA,,,Aelo to determine the external
pH changes caused by proton influx as a compensatory effect
on
the
imposed diffusion potential.
A,
benzene;
0,
naphthalene;
0,
tetralin;
0,
biphenyl;
+,
decalin;
H,
anthracene.
phospholipid liposomal membrane-potassium phosphate buffer
system, the octanol-water partition coefficient
of
a wide variety
of compounds showed good correlation with the membrane-
buffer partition coefficient (Fig.
1).
The ratio between these
partition coefficients, however, may differ significantly depend-
ing on the type of membrane (Antunes-Madeira and Madeira,
1984, 1985, 1986, 1987;
Katz
and Diamond, 1974; Seeman,
1972). Therefore, each membrane system should be tested be-
fore quantitative estimations
of
the partition coefficients can be
made.
The cyclic hydrocarbons were dissolved in DMF in order to
increase the dissolution rate of the hydrocarbons. The use of a
cosolvent is especially relevant for solid hydrocarbons, such as
naphthalene, biphenyl, phenanthrene, and anthracene.
By
US-
ing a cosolvent the distribution
of
the hydrocarbons in the
Membrane Interactions
of
Cyclic Hydrocarbons
8027
rhodamine fluorescence
1%)
.,”
I I
20
I
0
0
00
0.50
1
00 1.50
2.00
2
so
actual membrane concentration
(Hmol/rng
PL)
FIG.
7.
Increase in
Rls
fluorescence as a function of the con-
centration of cyclic hydrocarbons in the membrane as calcu-
lated from the membrane-buffer partition coefficient (Table
I).
A,
benzene;
A,
toluene;
V,
ethylbenzene;
V,
o-xylene;
5,
cyclohexane;
0,
naphthalene;
0,
tetralin;
0,
biphenyl.
aqueous phase and the membrane phase will come to equilib-
rium rapidly.
Accumulation of compounds in the membrane may lead to
alteration of the membrane structure and function. An impor-
tant change is the apparent increase in surface
area
of the
membrane, due to swelling of the membrane upon accumula-
tion of lipophilic compounds (Machleidt
et
al.,
1972; Seeman,
1972). The expansion observed with hydrocarbons was more
than two times higher than the expansion by alcohols (Seeman
et
al.,
1971). This variation is probably due to differences in
the
type of hydrophobic interaction and part of the membrane
where lipophilic compounds reside (see also below). Differences
in the methods applied to determine the increase in surface
area
were of less importance, since experiments with n-alcohols
(butanol to decanol) in the
E. coli
phospholipidlRl, system gave
results that did not significantly differ from
the
data reported
by Seeman and co-workers.2 The hydrocarbon concentrations
that
are
present in the membrane can be calculated from the
estimated membrane-buffer partition coeficients (Table
I).
When the
RI8
fluorescence data from Fig. 2 are plotted against
the membrane concentrations of the hydrocarbons
a
concentra-
tion range
at
which “swelling” occurs can be seen (Fig. 7). Up to
a
concentration, in the membrane, of approximately
0.5
pmoVmg phospholipid
(el
hydrocarbon molecule/2 phospholip-
ids)
an
increase in membrane surface area is observed, after
which an apparent maximum is reached. The extent of
R1,
fluorescence increase
at
an
actual membrane concentration
higher than
0.5
pmoYmg phospholipid (Fig. 7) was highest for
the compounds with the highest Pm,
i.e.
biphenyl and
tetra-
lin, naphthalene and cyclohexane were intermediate, whereas
o-xylene, ethylbenzene, toluene, and benzene were lowest. The
cause of this phenomenon is not readily understood although
the
extent of
the
R,,
fluorescence increase parallels the molar
volumes of the molecules (Table
I).
The increase in membrane fluidity
as
estimated from DPH
polarization measurements (Fig.
3)
is already apparent
at
slightly lower cyclic hydrocarbon concentrations than the in-
crease in membrane surface
area
(Fig. 2). This is most clear for
a-pinene and tetralin, although for cyclohexane and naphtha-
lene this effect can also be seen.
No
significant effect of the
hydrocarbons on the polarization of TMA-DPH was observed.
These results suggest
that
the
hydrocarbons partition to the
central part of the membrane, which directly affects
the
polar-
ization of the DPH.
In
principle one could envisage an effect of
J.
Sikkema,
W.
van den Heuvel, and
J.
A.
M.
de Bont, unpublished
results.
hydrocarbons on the distribution of DPH and TMA-DPH in the
membrane. However, if for instance DPH would become inter-
calated among acyl chains, one would expect
a
decrease in
polarization not only with DPH but also with TMA-DPH.
As
a
result of accumulation of hydrocarbons in the mem-
brane the activity of cytochrome
c
oxidase is lowered and the
proton (ion) permeability increases. Both effects act synergi-
cally on
the
magnitude of the ApH and
A+
generated by cyto-
chrome
c
oxidase. Since
a 50%
reduction of cytochrome
c
oxi-
dase activity only causes
a
small drop in the ApH and
A+
(Sikkema
et
al., 1992), the drop in the components of the proton
motive force will primarily be caused by the increased proton
permeability. To our knowledge the effects of hydrocarbons on
the generation and maintenance of the proton motive force
have neither quantitatively nor qualitatively been analyzed
so
far. Uribe and co-workers (Uribe
et
al.,
1985, 1990) reported
results which are in accordance with ours and support the view
that an important part of the toxicity of hydrocarbons is ex-
erted by effects on the proton motive force.
The action
of
general anesthetics on cell functioning, which is
similar to the effects observed for cyclic hydrocarbons, is often
ascribed to interaction of the anesthetic compounds with the
membrane (Overton, 1896; Seeman, 1972). This hypothesis,
which ascribes the inhibitory action of anesthetics fully to
changes in membrane integrity, is named the lipid theory of
anesthesia.
The
competing theory is the protein-interaction
theory, which states that anesthesia is
a
result of interaction of
anesthetic molecules with various enzymes involved in cellular
metabolism (Franks and Lieb, 1987). Our studies clearly indi-
cate
that
the
effects of hydrocarbons on the functioning of bio-
logical membranes involves both effects on the permeability
(protons (ions) but also larger molecules,
e.g.
CF) and the ac-
tivity of membrane enzymes (cytochrome
c
oxidase). The effects
on enzyme activity can be due to altered protein-lipid interac-
tions (hydrogen bonding and others), membrane thickness,
flu-
idity, and/or phospholipid headgroup hydration (Yeagle, 1989).
Therefore, it is remarkable
that
the obvious combination of the
lipid theory of anesthesia (Overton, 1896) and the protein-in-
teraction theory advocated by Franks and Lieb ((1987); La-
Bella, 1981) has not gained more attention
so
far.
A remarkable outcome of
our
studies is the observation that
the effect of cyclic hydrocarbons on the structural and func-
tional properties of biological membranes ((proteo)liposomes) is
directly related to accumulation in these membranes;
the
effect
is independent of the structural features of the molecules. The
accumulation of cyclic hydrocarbons in the membranes is pro-
portional to the concentration in
the
aqueous phase and the
membrane-aqueous phase partition coefficient. This latter pa-
rameter relates directly to the partitioning of these cyclic hy-
drocarbons in
a
standard octanol-water system, which allows
predictions to be made about the toxicity of other lipophilic
compounds on basis of their logP values. Since bacteria highly
differ in their sensitivity toward cyclic hydrocarbons it will be
important to establish how the membrane bilayers of these
organisms differ and how the
PwB
is affected by the phospho-
lipid composition of the membrane. Future studies are aimed
at
addressing these questions.
Acknowledgments-We
thank Prof.
Wil
Konings for critical reading
of the manuscript. Marian Vermue is gratefully acknowledged for cal-
culating logP values.
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