Correlation between hydrophobicity of short-chain aliphatic alcohols and their ability to alter plasma membrane integrity

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FUNDAMENTAL AND APPLIED TOXICOLOGY 36, 6 2 - 7 0 (1997)
ARTICLE NO. FA962252
Correlation between Hydrophobicity of Short-Chain Aliphatic Alcohols
and Their Ability to Alter Plasma Membrane Integrity
SUSAN C. MCKARNS,* CORWIN HANSCH,! WILLIAM S. CALDWELL,* WALTER T. MORGAN,*
SARAH K. MOORE,* AND DAVID J. DOOLITTLE*
*Research and Development, R. J. Reynolds Tobacco Company, Winston-Salem, North Carolina;
and ^Department of Chemistry, Pomona College, Claremont, California
Received December 12, 1995; accepted September 10, 1996
ical effects of other, as yet untested, aliphatic alcohols and aliphatic alcohol-like compounds (e.g., anesthetics) on the plasma
Correlation between Hydrophobicity of Short-Chain Aliphatic
Alcohols and Their Ability to Alter Plasma Membrane Integrity.
membrane,
o 1997 society of Toxkoiogr.
MCKARNS, S. C , HANSCH, C, CALDWELL, W. S., MORGAN, W. T.,
MOORE, S. K., AND DOOLITTLE, D. J. (1997). Fundam. AppL Tox-
icol. 36, 62-70.
The quantitative relationship between chemical structure and
biological activity has received considerable attention in the fields
of pharmacology and drug development More recently, quantitative structure-activity relationships (QSARs) have been used for
predicting chemical toxicity. It has been proposed that alcohols
may elicit their toxic effects through hydrophobic interactions with
the cellular membrane. The objective of this study was to evaluate
the role of hydrophobicity in the loss of membrane integrity following acute exposure to short-chain aliphatic alcohols in rat liver
epithelial cells in vitro. The series of alcohols studied included
methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol,
1-heptanol, 1-octanol, 2-butanol, 2-methyl-l-propanol, and 2methyl-2-propanol. The lactate dehydrogenase (LDH) assay was
used to quantify membrane integrity. The logarithm of the octanol/
water partition coefficient (log P) was used to quantify hydrophobicity. LDH50 values, representing alcohol concentrations yielding
a 50% increase in LDH release relative to untreated controls (i.e.,
mild disruption of membrane integrity), and EC50 values, representing alcohol concentrations yielding 50% of the maximal release
of LDH (i.e., moderate disruption of LDH release), were experimentally determined for each alcohol. The LDH50 and EC50 values were then used to derive the QSAR relationship. The aqueous
alcohol concentrations yielding LDH50 or EC50 values ranged
from 8.9 X 10~4 M (LDH50 for octanol) to 3.5 M (EC50 for methanol), and the log P of the alcohols ranged from -0.77 (methanol)
to 3.00 (octanol). From these data, we have derived two QSAR
equations describing the role of hydrophobicity in the release of
LDH from rat liver epithelial cells following a 1-hr alcohol exposure. The QSAR equation for LDH50 values, log (1/LDH50) =
0.896 log P + 0.117 (n = 11, SD = 0.131), was nearly identical to
the QSAR equation for EC50 values, log (1/EC50) = 0.893 log P
+ 0.101 (n = 11, SD = 0.133], suggesting that similar structureactivity relationships exist at both mild and moderate levels of
membrane disruption. Our data indicate that an increase in LDH
release was positively and linearly correlated with the hydrophobicity (r = 0.993). These data may help predict the potential biolog-
0272-0590/97 $25.00
Copyright © 1997 by the Society of Toxicology.
All rights of reproduction in any form reserved.
62
The pathologies associated with ethanol toxicity, including neurological impairments, liver disease, and certain cancers, are relatively well defined (Lieber, 1994,
review; Mufti, 1991; Mufti and Sipes, 1991). However,
the mechanisms which underlie these cytotoxic events are
not clearly understood. Altered membrane integrity, increased cell membrane fluidity, lipid peroxidation, oxidative stress, impaired protein secretion, mitochondrial injury, acetaldehyde interaction with cellular protein and
membrane lipids, and cellular hypoxia are all proposed
potential mechanisms of ethanol toxicity (for review, see
Zakin and Boyer, 1990). The importance of each of these
potential mechanisms of action in the cascade of ethanol
induced toxicity is currently under investigation.
A large body of evidence suggests that the primary site
of action of ethanol in the liver is the cell membrane (for
reviews, see Lieber, 1994; Hunt, 1985; Hunt, 1975). Interestingly, an apparent adaptive response to this increased membrane fluidity effect exists and reportedly involves an increase in cell membrane rigidity (Lyon and Goldstein, 1982).
Recent studies suggest that biochemical changes (e.g., protein alteration, lipid peroxidation, and oxidative stress) may
also be associated with ethanol-induced hepatic cellular injury (Ties and Nagy, 1995; Shaw et al., 1995; Sergent et al,
1995; Chiarpotto et al, 1995; Devi et al., 1993). Likewise,
specific biochemical actions involving GAB A, receptor-coupled chloride channels and glutamate receptors (Hoffman
and Tabakoff, 1993; Dietrich et al., 1989, review) have been
postulated to mediate the effects of ethanol within the central
nervous system. Thus, it appears that the potential biological
effects of ethanol may be mediated by biochemical (e.g.,
receptor-mediated) and/or biophysical (e.g., physical disruption of the membrane) processes, and these processes may
be cell-type-, concentration-, and time-dependent.
HYDROPHOBICITY OF SHORT-CHAIN ALIPHATIC ALCOHOLS
63
TABLE 1 Short-Chain Aliphatic Alcohols Studied
Alcohol
Structure
Methanol
Common applications*
Antifreeze, octane booster for gasoline, fuel for model airplanes and picnic stoves, paint removers,
adhesives, inks, alternative motor fuel, solvent for manufacture of Pharmaceuticals
OH
Ethanol
Alcoholic beverages, lotion, tonics, colognes, perfumes, octane booster for gasoline, topical
antiseptic, solvent for pharmaceutical preparations
OH
1-Propanol
Industrial solvent for resins and cellulose esters, laboratory solvent
OH
1-Butanol
Industrial solvent for fats, waxes, resins, shellac and varnish, manufacture of lacquers, rayon and
detergents, laboratory solvent
OH
2-Butanol
Industrial cleaners, paint removers, solvent for the manufacture of flavors, perfumes,
and dyestuffs
OH
2-Methy 1-1 -propanol
Paint removers, varnish removers, manufacture of esters for fruit flavor essences
CH,
2-Methyl-2-propanol
1-Pentanol
H3C
Denaturant for ethanol, octane booster for gasoline, manufacture of flavors and perfumes
OH
Laboratory solvent, solvent for organic synthesis
OH
1-Hexanol
Manufacture of Pharmaceuticals such as antiseptics amd hypnotics
1 -Heptanol
Laboratory solvent
1-Octanol
OH
Manufacture if perfumes and plasticizers
' Budavari « a/. (1989).
Although ethanol has been the most widely studied, many
short-chain aliphatic alcohols are toxic as well (Nelson et al.,
1990). As a class, short-chain aliphatic alcohols are relatively
small molecules consisting of a polar, hydrophilic hydroxyl
group and a nonpolar, hydrophobic hydrocarbon chain that
has a high affinity for membranes (Hunt, 1975). Therefore,
these compounds have the potential to act by physical insertion into the target membrane, disrupting the structural organization of the lipid bilayer, altering plasma membrane integrity, and subsequently disrupting normal membrane function
(Lyon et al., 1981). It has been hypothesized that aliphatic
alcohols dissolve in hydrophobic regions of the membrane
and increase membrane fluidity (Goldstein et al., 1981;
McCreery and Hunt, 1978; Chin and Goldstein, 1977; Roth
and Seeman, 1972). According to this proposed model, the
potency of an alcohol to alter membrane integrity should
correlate with the hydrophobicity of the alcohol. The more
hydrophobic the alcohol, the more readily it should disrupt
membrane integrity.
The hydrophobicity of an alcohol can be determined by
measuring its ability to partition between hydrophobic and
hydrophilic solvents. The most widely used measure of
hydrophobicity is the logarithm of the octanol/water partition coefficient, log P (Lindenburg, 1951). Log P can be
determined by first allowing the alcohol to equilibrate between water and octanol phases, and then measuring its
concentration in each phase. The magnitude of log P is
dependent upon a number of variables including the size
and molecular structure of the compound (Lindenberg,
1951). For instance, the magnitude of log P of aliphatic
alcohols increases with the number of carbons (Paterson
et al., 1972). The magnitude of log P for structural isomers
of aliphatic alcohols is straight-chain primary > branchedchain primary > secondary > tertiary (Lyon et al., 1981).
The depressant, hypnotic, and membrane-disruptive potencies of short-chain aliphatic alcohols have been highly
correlated with hydrophobicity in neuronal membranes
(Lyon et al., 1981). However, the role that hydrophobicity
plays on the toxicity of these compounds in liver cells has
not yet been extensively characterized.
The objective of this study was to develop a quantitative
structure-activity relationship (QSAR) to characterize the
64
McKARNS ET AL.
B. Ediancl
150-
125/
100-
i
75-
50-
M-
1
4
5
*
*
0.0
0.5
*
1.0
1.5
1.0
0.0
0.1
CUJ
0,»
0.4
0.5
0 . 1-S<rtano<
i
I
T —-"
i
0.00
0.01
0.02
0.04
0.04
0.05
O.M
0.000
0.005
0.010
0.015
CO
X
Q
0000
0.002
0.004
O.OOt
0.00*
0.010
0.00000
0.0002J
O.OOOSO
0.00075
0.00100
J. 2-H«0iy<- 1-prop«>ol
2S0-
200-
I
150-
I
100-
j
50-
0-
* -
—
0.0
0.1
O.J
O.J
0.4
0.5
0.0
0 1
0.2
O.I
Alcohol (M)
0.4
0 5
0.0
0.1
0.2
0.1
0.4
0.6
65
HYDROPHOBICITY OF SHORT-CHAIN ALIPHATIC ALCOHOLS
TABLE 2
Experimentally Determined LDH50 and EC50 Values
Alcohol"
R group"
Methanol
Ethanol
1 -Propanol
2-Methyl-2-propanol
2-Butanol
2-Methyl-1 -propanol
1-Butanol
1-Pentanol
1-Hexanol
1-Heptanol
1-Octanol
CH 3
C2H5
CjH,
C(CH 3 ) 3
CH(CH3)C2H5
CH 2 CH(CH 3 ),
CjH,
C 3 H,,
C«H13
C 7 H, 5
C,H 17
LDH50*
EC5(T
(M)
(M)
3.4
1.7
0.47
0.36
0.19
0.16
0.16
0.047
0.012
0.0045
0.00089
3.5
1.6
0.46
0.32
0.16
0.15
0.16
0.047
0.011
0.0042
0.00090
" The short-chain aliphatic alcohols tested in this study.
b
The hydrophobic R groups of the alcohols tested in this study.
c
Values derived from the concentration—response curves. Data expressed
in molar units.
correlation between an alcohol's hydrophobicity and its
potential to disrupt plasma membrane integrity in rat liver
epithelial cells following acute exposure in vitro. The release of endogenous lactate dehydrogenase (LDH) was
used to quantify disruption of membrane integrity. From
these data, we have developed a QSAR describing a positive and linear (r = 0.993 and p < 0.001) correlation
between the disruption of plasma membrane integrity and
hydrophobicity for short-chain, aliphatic alcohols in rat
liver cells following acute exposure in vitro.
MATERIALS AND METHODS
Cells and cell culture. The WB rat hepatic epithelial cell line (Tsao et
al., 1984) was used in this study. Cells were grown and maintained in
Ham's F12-K nutrient mixture (Kaighn's modified) supplemented with 5%
(v/v) heat-inactivated fetal bovine serum (FBS), 1% L-glutamine, and 50
/ig/ml gentamycin. The cell cultures were grown and maintained at 37°C
in a humidified incubator containing 5% CO 2 . Stock cultures were maintained in Corning 75-cm 2 plastic tissue culture flasks. F12-K growth medium, FBS, and Hank's balanced salt solution (HBSS) were purchased from
JRH Biosciences (Lenexa, KS). Gentamycin was purchased from Sigma
Chemical Co. (St. Louis, MO).
Chemicals.
1-Butanol (99.5%), 99.5% 2-butanol, 99.5+% ethanol, 98%
1-heptanol, 98% 1-hexanol, 99.5+% 2-methyl-l-propanol, 99.5+% 2methyl-2-propanol, 99+% 1-pentanol, 9 9 + % 1-propanol, and 99+% 1octanol, were purchased from Aldrich Chemical Company (Milwaukee,
WI). Methanol (100%) was purchased from Sigma.
Alcohol treatment
Two x 103 cells suspended in 2 ml culture medium,
supplemented with 5% FBS, were pipetted into Corning 35-mm-diameter
plastic tissue culture plates. Medium was aspirated from each plate and 2
ml of fresh growth medium (without FBS) containing the test compound
was added to confluent monolayers for 1 hr at 37°C. Alcohol dosing solutions were sonicated in autoclaved Pyrex glass flasks at 37°C during preparation, maintained in covered vials in order to minimize evaporation, and
used within 4 hr of preparation.
Lactate dehydrogenase release. Quantification of lactate dehydrogenase (LDH) in cell culture medium following a 1-hr chemical exposure was
used to quantify plasma membrane integrity. An increase of LDH in the
medium after exposure to a xenobiotic is correlated with the breakdown of
the plasma membrane integrity (Danpure, 1984). It should be noted that an
increase of LDH release is not always associated with irreversible cell
death, and may, in some instances, represent reversible graded increases in
plasma membrane permeability (Danpure, 1984). In our assays, LDH was
quantified using a commercially available method based on previously published procedures (Azuma et al, 1986). LDH was measured in cell culture
supematants after a 1-hr incubation at 37°C in serum-free growth medium
with or without the test compound. Immediately following the 1-hr treatment, a 100-/il aliquot of culture medium from each plate was analyzed
for LDH activity using an optimized LDH test kit (DG1340-K; Sigma).
This assay is based on the reduction of NAD to NADH by LDH. The
reaction was carried out at room temperature and absorbance was monitored
at 340 nm using a Beckman DU-70 spectrophotometer. An increase in
LDH activity in the extracellular medium is interpreted as a loss of plasma
membrane integrity. A minimum of nine readings (three plates, three readings per plate) were collected for each data point.
OctanoUwater partition coefficient (P). Published octanol/water partition coefficients for the alcohols were obtained from Hansch et al. (1995).
Derivation of the LDH50 and EC50 Values. LDH50 and EC50 endpoints were used to develop the QSAR of the alcohols. The LDH50 values
are defined as the concentrations which elicited a 50% increase of LDH
release relative to the untreated control. The EC50 values are defined as
the concentrations which elicited 50% of the maximal cellular LDH release.
The two measures were obtained to compare very subtle changes of membrane integrity (LDH50) with marked cytotoxic effects (EC50).
To determine LDH50, the experimental data were fit using a Boltzmann
sigmoidal model (Eq. 1).
y = (A, - A2y(\
+ (exp((AT - x^dx))
+
(1)
In this model, A, and A2 are values corresponding to the lower and upper
limits on the dose-response curve, x is the concentration, Xo is the dose
corresponding to the midpoint (50%) between A, and A2, and dx denotes
the slope of the dose-response curve. A,, A2, xc, and dx were obtained
using the computer-generated Boltzmann model.
Equation (1) was rearranged and solved for x to yield Equation (2).
LDH50 = *50 = dx*\a((A,
-
- 1) +
(2)
In this model, _y50 is the amount of LDH release corresponding to a 50%
increase from the untreated control (v50 = ].5*A,). A,,A2,xo,dx,
and >>50
were determined from Eq. (1).
The EC50 values were obtained by determining rhe alcohol concentrations which yielded 50% of the maximum LDH release. The concentrationresponse data for each alcohol were fit to a Boltzmann sigmoidal model
FIG. 1. Concentration-response curves generated for each alcohol following a 1-hr in vitro exposure in WB-344 rat liver epithelial cells. Each data
point represents a minimum of nine readings (three plates, three readings per plate). The data are expressed as the mean ± standard deviation.
66
McKARNS ET AL.
TABLE 3
Parameters Used To Derive the Quantitative Structure-Activity Relationships (QSARs)
Alcohol*
f
Log/*
Observed log
(1/LDH50)1'
Predicted log
(1/LDH50)'
A Log
(1/LDH50/
Observed log
(l/EC50)»
Predicted log
(1/EC50)*
A Log
(1/EC50)'
Methanol
Ethanol
1-Propanol
2-Methyl-2-propanol
2-Butanol
2-Methyl-l-propanol
1-Butanol
1-Pentanol
1-Hexanol
1-Heptanol
1-Octanol
0.17
0.49
1.8
2.2
4.1
5.8
7.9
36
110
520
1,000
-0.77
-0.31
0.25
0.35
0.61
0.76
0.88
1.6
2.0
2.7
3.0
-0.53
-0.22
0.33
0.45
0.73
0.81
0.81
1.3
1.9
2.3
3.1
-0.59
-0.18
0.33
0.41
0.65
0.78
0.89
1.5
1.9
2.5
2.8
0.06
-0.04
0.01
0.04
0.08
0.03
-0.08
-0.2
0
-0.2
0.3
-0.54
-0.20
0.34
0.50
0.79
0.81
0.81
1.3
2.0
2.4
3.1
-0.57
-0.16
0.34
0.43
0.66
0.80
0.91
1.5
1.9
2.6
2.8
-0.03
-0.04
0
0.07
0.13
0.01
-0.1
-0.2
0.1
-0.2
0.3
* The short-chain aliphatic alcohols tested in this study.
* P values represent the octanol/water partition coefficient for each alcohol. These published values were obtained from Hansch et al. (1995).
c
Log P values represent the logarithm of the octanol/water partition coefficient for each alcohol.
' T h e observed log (1/LDH50) values represent the experimentally determined values. These values were derived from the concentration-response
curves (Fig. 1).
' The predicted log (1/LDH50) values represent the predicted values. These values were computed using Eq. (3) (text).
/r
These values represent [observed log (1/LDH50) - predicted log (1/LDH50)].
' The observed log (1/EC50) values represent the experimentally determined values. These values were derived from the concentration-response curve
(Fig- 1).
* The calculated log (1/EC50) values represent the predicted values. These values were computed using Eq. (4) (text).
' These values represent [observed log (1/EC50) - predicted log (1/EC50)].
using Origin version 3.5 software (Microcal Inc.). The EC50 values were
computer-generated using the Origin software.
RESULTS
among the alcohols studied (Table 3). These P values were
obtained from published work by Hansch et al. (1995).
LDH50 and EC50 values were transformed to the logarithms
of (1/LDH50) and (1/EC50) (Table 3). Log (1/LDH50) and
log (1/EC50) were plotted against log P to develop the
QSARs describing the relationship of hydrophobicity to loss
of membrane integrity (Fig. 2). These QSARs are described
by Eqs. (3) and (4).
A series of short-chain aliphatic alcohols (i.e., methanol,
ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1heptanol, 1-octanol, 2-butanol, 2-methyl-1-propanol, and 2methyl-2-propanol) were studied (Table 1). Each alcohol
tested increased LDH release in a concentration-dependent
log 1/LDH50 = 0.896 log P + 0.117
manner (Fig. 1).
„ = n , r = 0.993, SD = 0.131
(3)
LDH50 and EC50 values were determined to quantify and
compare the potency of each alcohol (Table 2). The LDH50
log 1/EC50 = 0.895 log P + 0.101
and EC50 values were calculated from the concentration n= 11, r = 0.993, SD = 0.133,
(4)
response curves (Fig. 1). These LDH50 and EC50 values
were then used to formulate QSARs to describe mild versus
moderate disruption of membrane integrity (Eqs. 3 and 4). where n is the number of data points, r is the correlation
The LDH50 and EC50 values were nearly identical for each coefficient, and SD is the standard deviation.
alcohol, indicative of the steep concentration-response
Both log (1/LDH50) and log (1/EC50) (r = 0.993) were
curves. The aqueous alcohol concentrations resulting in the positively and linearly correlated with hydrophobicity (Figs.
LDH50 and EC50 endpoints varied approximately 4000-fold 2a and 2b, respectively). The slopes (0.896 and 0.895, reamong the alcohols and ranged from 8.9 X 10"4 M (octanol) spectively) and y intercepts (0.117 and 0.101, respectively)
to 3.5 M (methanol), indicative of the wide range of toxicity differed little between the LDH50 and EC50 data, suggesting
of the series of alcohols tested.
a possible similarity in the mechanism(s) of biological action
A 6000-fold range, from 0.17 (methanol) to 1000 (octa- between these two portions of the concentration-response
nol), in octanol/water partition coefficients (/>) was present curve. The potency to disrupt membrane integrity (expressed
67
HYDROPHOBICITY OF SHORT-CHAIN ALIPHATIC ALCOHOLS
a
l-octanol %
3-
s'^
2-
t3
l-heptanol
l-hcxanol I*'
2-methyl-1-propanol
A
t-l
s'
1 ~
y ^ * 1-pentanol
2-butanol A # # i -butanol
2-methy1-2-propanol %
y/^\ -propanol
0-
/•m«nan < | t h a n 0 1
-1 0993,n-ll,SD-0.131
p<00001
-2-
1
'
1
'
1
'
1
1
-1
1
'
LogP
b
l-octanol^
3-
s^
y / ^ 1-heptanol
1-hexanol ^ /
22-methyl-1-propanol
Log
1-
0-
-1 -
-2-
2 butanol
Lf
,
//^
1-pentanol
tanol
2-methyl-2-propanol
J}
y/^ 1 -propanol
/""ethano!
^—
^^methanol
l
'
l
y-0.895 log P +0 101
r-O993,n-ll,SD-O133
P< 0.0001
'
1
1
|
1
|
1
-1
LogP
FIG. 2. The correlation of (a) mild, i.e., LDH50, and (b) moderate, i.e., EC50, disruption of membrane integrity with octanol:water partition
coefficients. Note die log scales and the correlation (r = 0.993) over a wide range of lipid solubilities and alcohol potencies.
68
McKARNS ET AL.
1-octanol
3-
^ /
•
/ • 1-heptanol
2-
^n-hexanol
^*
1 -
2-butanol
l-propanol
- /
1-pentanol
y
' w 1 -butanol, 2-methy 1-1 -propanol
™2-methyl-2-propanol
0J*ethanol
jnethanol
y-0.518logP-l 257
r = 0990.n-ll,SD 0.159
P-<0.0001
-1 •
0
1
l
i
I
•
I
2
i
1
3
'
4
1
'
5
1
'
1
6
'
7
1
8
'
1
9
Total Number of Carbons in Alcohol
FIG. 3. The correlation of disruption of membrane integrity with the number of carbons in the alcohol. A linear regression of alcohol potency
expressed as the logarithm of (1/LDH50) and the number of carbons (r = 0.990).
as 1/LDH50) increased logarithmically with the number carbon atoms in the molecule (slope = 0.52, r = 0.99, and p
< 0.0001) (Fig. 3). A plot (not shown) of the EC50 versus
the number of carbon atoms resulted in a similar pattern
with the same slope (i.e., 0.52).
The relative contribution of the molecular shape of the
alcohols to alter LDH release is unclear from these data.
The order of potency for these compounds in neuronal membranes has been reported to be straight-chain primary >
branched-chain primary > secondary > tertiary (Lyon et
ai, 1981). Figure 3 suggests a trend toward straight-chain
> tertiary (1-butanol > 2-methyl-2-propanol); however, we
are unable to differentiate any difference between isoprimary
and secondary alcohols (2-methyl-l-propanol = 2-butanol).
The log P for each alcohol was inserted in Eqs. (3) and
(4) to generate the predicted LDH50 and EC50 values for
disruption of membrane integrity. The difference between
the predicted and experimentally determined LDH50 values
(ALDH50) and the difference between the calculated and
experimentally determined EC50 values (AEC50) values are
listed in Table 3. The negligible difference between the experimentally determined and predicted values is suggestive
of the accuracy of these QSAR equations to predict the
potential of alcohols, ranging in log P from 0.17 to 3.0, to
disrupt membrane integrity under the conditions studied.
DISCUSSION
It is well established that the hydrophobicity of a xenobiotic is often associated with biological action (Hansch and
Dunn, 1972). A number of models for assessing hydrophobic
interactions have been reported. A particularly simple yet
effective model is the disaggregation of silanized glass beads
(Cecil, 1967). Silanized glass beads have a tendency to cluster together when placed in water and these clusters can be
broken up by the addition of hydrophobic compounds like
alcohols. The equation describing this purely physical effect
of alcohols on silanized glass bead aggregation is similar to
equations 3 and 4 and other equations describing alcoholmediated membrane damage (Hansch et ai, 1989). A direct
correlation between membrane fluidity and aliphatic alcohol
hydrophobicity has been documented for neuronal membranes (Lyon et al, 1981). The objective of this study was
to develop a quantitative structure-activity relationship to
describe the relationship between hydrophobicity and disruption of nonneuronal membrane integrity for a series of shortchain aliphatic alcohols following acute exposure in vitro.
Our results correlating alcohol potency with hydrophobicity (slope = 0.895) and the number of carbons in the
alcohol chain (slope = 0.520) nearly parallel similar studies
in neuronal membranes (Lyons et ai, 1981), and are in close
HYDROPHOBICITY OF SHORT-CHAIN ALIPHATIC ALCOHOLS
agreement with other studies suggesting that alcohol potency
on membrane damage is mediated via biophysical processes
(Hunt, 1985, review; McCreery and Hunt, 1978; and Hansch
and Glave, 1971).
Overall molecular shape does not appear to be a major
contributor to the potency to disrupt membrane integrity in
our proposed model. The behavior of the three branchedchain alcohols (2-butanol, 2-methyl-l-propanol and 2methyl-2-propanol) appears to be described by QSAR Eqs.
(3) and (4), albeit the behavior of 2-methyl-2-propanol (Fig.
3) suggests that the steric effects may require some additional investigation. Our results are not in agreement with
studies suggesting that steric hindrance is important in the
ability of aliphatic alcohols to alter membrane fluidity in
neuronal membranes (Lyon et al., 1981). Although cell type
may be an important factor in this discrepancy, changes in
enzyme activity and cell differentiation during cell culture
should not be overlooked as possible contributing factors as
well ales and Nagy, 1995).
In contrast to the toxic effect examined in the present
study, several studies report a beneficial or protective effect
of alcohols (Seeman, 1972; Roth and Seeman, 1972, 1971).
The protective effects of alcohols have been reported to
occur at lower concentrations than needed to cause cell lysis,
to be concentration-dependent, and to increase with an increase in the numbers of carbons in the alcohol. These reports are consistent with our data which suggest a possible
membrane stabilizing effect (i.e., a concentration-dependent
inhibition of LDH release) of 1-octanol (Fig. 1).
In conclusion, under the conditions tested, an alcohol's
potential to increase the release of LDH is positively and
linearly correlated with its hydrophobicity in rat liver cells
(r = 0.993). Our results are consistent with numerous other
investigations suggesting that biophysical processes are a
major contributor to the biological effects of alcohols on
membranes. One could speculate that while biophysical processes may be largely responsible for the toxicity of hydrophobic alcohols (e.g., heptanol and octanol), biochemical
mechanisms may play a more essential role in mediating the
toxicity of alcohols with lower hydrophobicity (e.g., ethanol). Clarification of the mechanisms of actions of individual
alcohols is necessary to characterize fully the relevance of
hydrophobicity in the toxicity of these compounds.
The QSAR developed from this study may help predict the
potential biological effects of other, as yet untested, aliphatic
alcohols and aliphatic alcohol-like compounds (e.g., anesthetics). Furthermore, these data may help direct future efforts in
understanding the mechanisms underlying changes in plasma
membrane integrity following acute alcohol exposure.
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