Unequivocal demonstration that malondialdehyde is a mutagen
Ashis K.Basu and Lawrence J.Marnettl
Department of Chemistry, Wayne State University, Detroit,
MI 48202, USA.
(Received on I December 1982; accepted on 28 December
1982)
Abstract
Malondialdehyde (MDA), a product of lipid peroxidation
and prostaglandin biosynthesis, has been mported to be
mutagenic and carcinogenic. Recent evidence suggests,
however, that strongly mutagenic impurities are generated
during the preparation of MDA that may contribute to the
observed biological activity. Since MDA is widely produced
in animal tissue it is important to establish whether it is actually mutagenic and carcinogenic. We have utilized three
complementary methods for the preparation of highly
purified MDA for biological testing. These are chromatographic purification of the sodium salt of MDA, sublimation
of the free acid of MDA, and basic hydrolysis of Pa-nitrophenoxy)acrolein. The latter is a uniqw method that we have
developed specifically to generate MDA under non-acidic
conditions wherr it is stable. MDA prepared by each method
induced -5 revertants/pmol in Salmonella typhimunum his
D 3052. This unequivocally demonstrates that MDA is a
weak mutagen.
Introduction
Malondialdehyde (MDA)* is a three carbon dialdehyde
which is widely produced in mammalian organisms as a side
product of prostaglandin biosynthesis and an end product of
polyunsaturated lipid peroxidation (1,2,3). Mukai and Goldstein (4) and Shamberger et al. (5) reported that MDA is
mutagenic in certain strains of Salmone//atyphimurium and
carcinogenic in Swiss mice (6). Studies by Yau indicate that
MDA is cytotoxic and mutagenic when administered to a
murine L5178 Y lymphoma cell line cultured in vitro (7). The
MDA used for these studies was prepared by the aeidcatalyzed hydrolysis of tetraalkoxypropanes. Marnett and
Tuttle showed that this procedure generates side products
which are highly mutagenic (8). P-Ethoxyacrolein and 0methoxyacrolein, formed from tetraethoxypropane and tetramethoxypropane, respectively, are 25 and 40 times more
mutagenic than MDA (8). Some of the biological effects attributed to MDA may have been due to these impurities and
the mutagenicity as well as carcinogenicity of MDA is uncertain. Although highly purified NaMDA (>99% pure by
n.m.r. spectroscopy) was weakly mutagenic in S. typhimurium his D 3052, it is difficult to rule out the possibility that
the observed mutagenicity was due to a trace impurity which
was highly mutagenic (8). Because of the potential irnportance of MDA as a mediator of spontaneous carcinogenesis,
an unequivocal evaluation of its biological activities is essenT o whom reprint requests should be addressed.
'Abbreviations: MDA, rnalondialdehyde in its conjugate base form;
H-MDA, free acid of malondialdehyde in its enolic tautomer form; NaMDA,
sodium malondialdehydate; TMP, tetramethoxypropane; PNPA, @-(pnitre
phenoxy)acrolein; d, doublet; dd, doublet of doublet; s, single.
O IRL Press Ltd., Oxford, England
tial. We have, therefore, employed alternative methods of
purification of MDA and have developed a new procedure
for its synthesis to provide very highly purified material by
three complementary routes. The different batches of MDA
were tested for mutagenicity in Salmonella. We report here
that MDA is, indeed, a weak mutagen.
Materials and Methods
Tebamethoxypropan: W P ) , pniuophenol and propargyl alcohol were
purchased from Aldrich Chemical Co. Dowex 50 W-X4 was purchased from
J.T.Baker Chemical Co. Sephadex LH-20 was purchased from Sigma
Chemical Co. Water was distilled twice in glass.
NaMDA prepmtion and puri/kntion by Sephadev LH-20
Marnett and Tuttle's procedure was used (8). The lyophilized NaMDA was
kept under vacuum for 12 h at room temperature and then stored at - 20°C.
NaMDA pur~pcafionby svblimafion of H-MDA
An adaptation of a procedure by Maschel and Leonard developed for the
purification of a-substituted malondialdehydes was employed (9). Highly
purified NaMDA (470 mg, 5 mmol) made by the above procedure was
suspended in 25 ml dry ether and 4 mmd I N HCl in dry ether was slowly added with vigorous stirring at ambient temperature. The suspension was allowed to stir for another 3 min, precipitated NaCl and excess NaMDA were
filtered off, and the ether was evaporated under vacuum. The white solid o b
tained was immediately stored at -85°C until further ura. No appreciable
decomposition or polymerization was observed at this temperature w i h n
24 h. Sublimation of the H-MDA at -5 mTorr for 1 h at room temperature
yielded 15-20% H-MDA. The H-MDA adhering to the cold finger of the
sublimation apparatus was dissolved rapidly in a minimum quantity of 1 N
NaOH which was timated to pH 7.0 with 2 N HCl. The resultant NaMDA
solution was then lyophilited and stored at -20°C.
Following Spiinig and Weickmann (I I), 1 g propynal(l0) in 10 g absolute
ethanol was p h c d in a round bottom flask to which 2.48 g pnimophenol in
5.4 g ethanol was added with vigorous stirring. After I5 min, the clear reaction mixture turned to a curdy yellow mass. The yellow precipitate was filtered
and the mother liquor was concentrated to give another crop of yellow
precipitate. The combined yellow filter cake was recrystallized twioe from a b
solute ethanol. Off-white crystals of PNPA (m.p. 1 17- 118°C) were obtained
which gave acceptable combustion analysis. The material exhibited a single
peak on reversed-phase h.p.1.c. N.m.r. (CDCIJ; 6 6.04 dd, J = 8, 13 Hz
(1 H); 7.27 multiplet, (2 H); 7.65 d, J = 13 Hz (1 H); 8.33 d. J = 10 Hz
(2 H); 9.59 d, J = 8 Hz (I H).
Rate meanrrement of the hydrolysk of PNPA
The hydrolysis of PNPA was followed by pnitrophenolate release at
400 nm in a Cary 210 Specmophotometer following the addition of NaOH.
AU experiments were carried out with a large excess of NaOH and followed
pseudo-first order kinetics. Second order rate constants were obtained by
dividing each observed first order rate constant by theconcentration of NaOH
according to the equation: k = '"Z where [B], was the concentration of
-
1%
IBI,
NaOH used. A value of 3 I/mol/s was obtained. Using this value, we can
calculate the time needed for hydrobk to proceed to any extent desired.
Prepration of NaMDA from PNPA
PNPA (150 mg, 0.78 mmoD was dissolved in minimum quantity of ethanol
and meated with 1.6 mmol of aqueous NaOH. The total volume of the reaction mixture was increased to 5 ml by addition of distilled water and the mixture was stirred for 5 min (calculated and actual extent of hydrolysis
>99.999%).The reaction mixture was applied to a 50 g Sephadex LH-20 column and eluted with water. The absorption profile of the fractions at both
267 nm and 400 nm indicated clearcut separation of NaMDA and sod~um
pnitrophenolate. No detectable absorption of pnitrophenol (330 nm) was
observed in any of these fractions. The NaMDA fraction was lyophilized and
aored at -20'C.
NaMDA was dissolved in distilled water and was a e M by passage
through 0.2 am Acrodiscs (Gelman Instrument Co.). The concenwtions of
A.K.Basu and LJ.Maraea
NaMDA for all studies were determined from the absorbance at 267 nm (t =
34 000) (12).
S. typhimuriwn strains were kindly provided by Professor Bruce Ames,
University of California, Berkeley. Mutagenicity assays were carried out by
the standard plate incorporation method: to 0. I ml of a sterile NaMDA solution
2 ml of molten top agar at 45'C and 0.1 ml of an overnight nutrient broth
cultureof hk D3052 were added. The contents weremixed by vonexing at low
speed and the r e s u l k mixture was poured onto a minimal agar plate. After
incubation at 37'C for 48 h macroscopic colonies were counted (13). The
spontaneous revenion rate, determined in parallel incubation was subtracted
from each experimental value. Triplicate plates were run for each data point.
Survivors were determined on nutrient agar plates after appropriate dilution.
The effect of metabolic activation was assessed using S 9 prepared from
Aroclor 1254-treated Long-Evans rats (13).
Statktical analysis
The mutagenicity data were analyzed by the use of the F-test (14).
Results and Discussion
We adopted three different approaches for the preparation
of NaMDA. These are as follows: The T K D hydrolysate
generated under acidic conditions is titrated to pH 7.0 with
aqueous NaOH and the isolated Nah4DA is recrystallized
twice from acetone. The NaMDA obtained is subjected to
Sephadex LH-20 chromatography which effectively separates
all organic impurity. The material is >99% pure by n.m.r.
spectroscopy and g.c. analysis indicates that the principal
mutagenic impurities present in the hydrolysate, 0-methoxyacrolein and 3,3-dimethoxypropionaldehyde are undetectable.
We introduced a further purification step utilizing the
volatile character of the free acid of MDA (H-MDA).
Basically the procedure involves acidification of highly
purified NaMDA obtained by the above method to form
H-MDA and the purification of H-MDA by vacuum
sublimation. This approach has been used by other investigators to prepare H-MDA for spectroscopic studies
(15 - 18). A direct mutagenic study of H-MDA is not possible
due to its acidity (pKa = 4.46), and, therefore, the purified
H-MDA is converted to NaMDA, the form which occurs at
physiological pH ( -99.9%). This method separates any nonvolatile impurity which may still be present after the
Sephadex LH-20 purification.
We considered the possibility that since the H-MDA is
derived by the acid hydrolysis of TMP,some mutagenic impurities that might be present in the NaMDA preparations
might also be present in the H-MDA. For mutagenic impurities to be carried along with the H-MDA, they must be
volatile. We have already shown that the volatile mutagens 0alkoxyacroleins and dialkoxypropionaldehydes can be
removed effectively by solvent extraction and Sephadex
LH-20 chromatography and the NaMDA we used as our starting material had already been purified by that method. It is
unlikely that other volatile compounds are present in a TMP
hydrolysate since the other potential impurities are polymers
of MDA which are not volatile (8). Nevertheless, we cannot
absolutely rule out the possibility that volatile mutagenic impurities might be formed during acidic hydrolysis of TMP
and are somehow carried through both purification procedures. For this reason, we developed a new method of synthesis of Nah4DA under basic conditions. This approach involves alkaline hydrolysis of PNPA. PNPA is prepared by
the addition of pnitrophenol to propynal (Equation 1).
It is a stable, crystalline solid which can be obtained in a
highly pure state. It offers the advantage that its hydrolysis
can be accurately and conveniently followed by monitoring
the absorbance of the pnitrophenolate ion as well as the con-
Equation 1
Equation 2
Flg. 1. Reversion of S. typhimuriwn hisD3052 with MDA obtained by three
different methods of preparation: A-A,
TMP hydrolysis; 0- - -5,
sublimation of H-MDA, 0.. . . O , PNPA hydrolysis.
jugate base of MDA (Equation 2).
In addition, since it is prepared from propynal rather than
tetraalkoxypropanes, it cannot contain the same impurities
which might be formed during the acidic hydrolysis of tetraalkoxypropanes.
The mutagenicity of Nah4DA obtained by the three different approaches was simultaneously measured in hk
D 3052, the strain with the highest reported sensitivity to
MDA mutagenicity (8). A metabolic activation system was
omitted since it had no effect on the mutagenicity. In each experiment, the number of revertants at a particular concentration of Nah4DA was very similar despite the difference in the
method of preparation. The results from a typical experiment
are shown in Figure 1. When all the data from the experiment
are averaged a straight line results with a correlation coefficient of 0.98. No method of preparation consistently gives
higher or lower number of revenants from experiment to experiment. When the data from three such experiments are
compared, all nine lines have statistically the same slope,
although different batches of material were used every time.
The combined data from these three experiments, a total of
117 points are shown in Figure 2. The linear regression
analysis of these points generates a straight line (Figure 2) that
fits the equation: e x ) = 4.44 x + 5.40. The correlation coefficient (0.96) for this line is lower than each of the individual
lines (0.99) or that of a single experiment (0.98), but considering the small but definite difference in experimental condi-
MUTAGENICITY Of MDA - COMBWJED DATA
m).
L.J.M. is a recipient of a Faculty Research Award of the American
C a n c a Society (FRA 243).
Referenoes
Fig. 2. Combined data from three different mutagenicity experiments with
MDA made by different methods: a total of 117 data points were introduced.
tions from day to day, and the number of total data points introduced, this result is not surprising. The non-zero intercept
is an artifact of the curve-fitting. The slight deviation from
linearity is not a consequence of toxicity since NaMDA is not
toxic at 20 pmol/plate.
These results unequivocally demonstrate that highly
purified NaMDA prepared by different and complementary
methods is mutagenic in S. typhimurium. It is unreasonable
to presume that all these methods generate the same
mutagenic impurity in exactly the same proportion. Furthermore, we routinely assayed the purity of each batch of
NaMDA by n.m.r, and g.c. Since PNPA itself is highly
mutagenic (A.K.Basu and L.J.Marnett, manuscript in
preparation), we considered the possibility that it contributes
to the observed mutagenicity of the batches made by its
hydrolysis. The amount of p-nitrophenolate generated by the
basic hydrolysis of PNPA places an upper limit of 0.001 % on
the percentage of PNPA which can be present in the NaMDA
before purification. If one assumes that all the PNPA remains in the NaMDA after purification (an unreasonable
assumption), one can calculate that unreacted PNPA could
contribute a maximum of 0.5 revertant/pmol NaMDA.
These calculations taken with the observation that NaMDA
prepared by three different methods exhibits identical
mutagenicity, suggests that PNPA makes no contribution.
The unequivocal demonstration of the mutagenicity of
MDA is important because of widespread distribution of this
compound in animal tissue. Although the specific mutagenicity is low, the strains that detect it have not been maximized
for sensitivity in the detection of mutagens of this structure.
Therefore, it is conceivable that it exhibits more potent effects
in mammalian cells. An accurate assessment of the carcinogenicity of MDA will require complete testing in appropriate
animal models.
Acknowledgements
We are grateful to Robert Sachs for his help in statistical analysis. This work
was supported by a research grant from the National Institutes of Health (CA
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