249
A Role for a New Vascular Enzyme in the
Metabolism of Xenobiotic Amines
Paul J. Boor, Robert M. Hysmith, and Radhika Sanduja
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Although it has long been thought that environmental toxins may play an underlying role in
vascular diseases such as atherosclerosis, this concept is not supported by any clear-cut
experimental evidence of toxic metabolism by cardiovascular enzymes. In this study, we
demonstrate that allylamine, a selective cardiovascular toxin in vivo, is actively metabolized in
vitro by a purified vascular enzyme (semicarbazide-sensitive amine oxidase), which has been
localized recently to vascular smooth muscle cells. Oxidative deamination of allylamine to a
highly toxic aldehyde, acrolein, was blocked through enzyme inhibition by semicarbazidesensitive amine oxidase inhibitors, which obviate allylamine's in vivo toxic manifestations. This
unique demonstration of metabolism of a known in vivo cardiovascular toxin by semicarbazidesensitive amine oxidase suggests that this vascular enzyme's physiological role may include
metabolism of exogenous amines. (Circulation Research 1990;66:249-252)
T he highly selective cardiovascular toxin allylamine is a highly reactive unsaturated alkylamine that has been used industrially in the
organic synthesis of commercial products including
rubber, pharmaceuticals, and plastics. The extent of
industrial exposure to this chemical is difficult to
ascertain, although several reports suggest that occasional occupational exposure and environmental contamination occur in this and other countries (see
Reference 1 for review). Since the 1930s, there has
been sporadic experimental interest in allylamine
due to this compound's extraordinary and selective
ability to induce progressive acute myocardial necrosis and chronic vascular lesions.2,3
The first suggestion that vascular smooth muscle
was involved in the toxicity of allylamine came in
1974 when Lalich and Paik4 showed that allylamineinduced lesions were markedly diminished in severity
by the concomitant feeding of hydralazine, an antihypertensive agent with direct smooth muscle effects.
We later showed a similar striking protective effect of
other compounds5 and subsequently correlated this
protective in vivo effect with the ability of these
compounds to inhibit the metabolism of allylamine to
a highly toxic aldehyde.6
A common feature of the compounds that protect
against allylamine cardiovascular toxic damage is the
From the Chemical Pathology Division, Department of Pathology, The University of Texas Medical Branch, Galveston, Texas.
Supported by Grant HL-26189 from the National Heart, Lung,
and Blood Institute.
Address for correspondence: Dr. Paul J. Boor, Chemical Pathology Division, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77550.
Received March 10, 1989; accepted July 31, 1989.
inhibition by these compounds of the vascular enzyme
semicarbazide-sensitive amine oxidase (SSAO), also
known as "benzylamine oxidase" or "connective tissue amine oxidase." We hypothesized that this littlestudied vascular enzyme, which is produced by vascular smooth muscle cells, is responsible for the toxic
metabolism of allylamine by vascular tissue.
In this study, using several sources of vascular
SSAO (including purified enzyme), we demonstrate
the oxidative deamination of allylamine to the aldehyde acrolein as a clear example of the capacity of
vascular tissue to metabolize exogenous compounds.
We further demonstrate that SSAO inhibitors that
are capable of obviating in vivo and in vitro lesions
act through enzyme inhibition and not through trapping the distal aldehyde metabolite. One underlying
function of the vascular enzyme SSAO may be deamination of environmental amines.
Materials and Methods
Allylamine was incubated with three sources of
SSAO: 1) homogenates of cultured porcine aortic
smooth muscle cells procured and maintained by
standard methods as previously described,7 2) media
from cultured smooth muscle cells, which contain
high SSAO activity,7 or 3) SSAO purified8 from
smooth muscle cell culture media by established
chromatographic methods. Controls consisted of
blanks and homogenates of porcine aortic endothelial cells that contain no SSAO activity. Incubations
were similar to the standard assay of SSAO activity9
except for the substitution of allylamine for benzylamine as substrate; enzyme source was preincubated
in 0.1 M phosphate buffer in the presence of 0.22 mM
250
Circulation Research Vol 66, No 1, January 1990
TABLE 1. Allylamine Metabolized by Semicarbazide-Sensitive Amine Oxidase
Enzyme source
Smooth muscle
Incubation
Smooth muscle
time (min)
cell homogenate
culture media
Purified SSAO
5
1.4±0.9 (7)
0.3±0.7 (2)
4.5+1.3 (24)
0.8+0.9 (4)
10
4.2+1.3 (22)
6.2+1.1 (33)
30
5.8±1.0 (30)
9.5±0.9 (50)
1.2+1.1 (6)
Values are the mean±SEM of total allylamine (,/g) converted during incubation (recovered as the 2,4dinitrophenylhydrazine adduct of acrolein), n=4. SSAO, semicarbazide-sensitive amine oxidase. Values in parentheses
are conversion percentages.
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deprenyl 20 minutes before addition of allylamine
(1.4 mM final concentration, 210 C, 240 gl total
volume). Reactions were stopped at 5, 10, and 30
minutes by the addition of an equal volume of
saturated solution of 2,4-dinitrophenylhydrazine (2,4DNP) in 2 M sulfuric acid. The product of allylamine
oxidative deamination, acrolein, was measured as the
trapped 2,4-DNP adduct by previously described
high-performance liquid chromatography (HPLC).10
HPLC was performed on an isocratic liquid chromatograph (model 330, Beckman Instruments, Fullerton, California) with an ultrasphere ODS column
(5-,um particle size, 25 cmx 4.6 mm i.d.). Acetonitrilewater (1:1 vol/vol) was used as solvent at a flow rate
of 1 ml/min. The column eluent was monitored at 356
nm. A volume of 40 ,ul was injected in each case.
Comparisons of recovered product were made between groups by analysis of variance.
Experiments were also performed with semicarbazide, which has been shown to protect against
allylamine-induced myocardial lesions in vivo,5 and
phenelzine, which is a known inhibitor of SSAO and
affords in vitro protection against allylamine-induced
injury to vascular smooth muscle cells.1112 The complete set of experiments described above were
repeated except that the 20-minute preincubation
(before addition of allylamine substrate) contained
either semicarbazide or phenelzine (2.2 ,uM).
Results
Acrolein was actively produced by all samples
tested, with brisk, time-dependent production by
smooth muscle cell media and purified SSAO (Table
1). Indeed, during incubation of samples with purified SSAO, the odor of acrolein was evident, suggesting that a significant portion of the highly volatile
aldehyde product (acrolein) was lost. Nevertheless,
on a molar basis, the conversion of allylamine to
acrolein by purified SSAO, based on recovered aldehyde, was high (24% at 5 minutes; 33% at 10
minutes; 50% at 30 minutes). Also, recovery between
the three groups by use of different enzyme sources
was significantly different at 10 and 30 minutes
(p<0.01). Control homogenates of cultured porcine
endothelial cells (which contain no SSAO activity)7
or blanks gave no detectable acrolein. After preincubation with the SSAO inhibitors semicarbazide or
phenelzine (2.2 gM), no acrolein was detected in
incubations of allylamine with the three enzyme
sources (lower limit of acrolein detection, 1 ng).
The possibility exists, however, that these inhibitors
could actually be trapping aldehyde product rather
than inhibiting the SSAO-mediated conversion of allylamine to acrolein. Such a mechanism could also attenuate the in vivo toxicity of allylamine45 by binding the
distal toxic metabolite. Both semicarbazide and
phenelzine readily form adducts with aldehydes, as
does the other chemical with a previously demonstrated
protective effect against allylamine-induced cardiovascular toxicity, hydralazine.5
To test this possibility, authentic standards of
semicarbazide- and phenylethylamine-acrolein
adducts (the latter as a potential product of phenylzine) were synthesized, and their chemical structures were confirmed by mass spectroscopy and
nuclear magnetic resonance. The proton nuclear
magnetic resonance spectra (Figure 1) of acrolein
semicarbazone (I in Figure 1) gave the following (250
MHz, 8, D20): 8.14 ppm (1H, d, J=9.16 Hz, C3-H),
7.14 ppm (1H, m, C2-H), and 6.16 ppm (2H, dd,
J=10.3, 5.2 Hz, Cl-H) (NH2 was exchanged with
D20). The proton nuclear magnetic resonance spectra of the acrolein phenethylamine adduct gave a
mixture of two isomers lla (10%) and lIb (90%) (250
MHz, 8, CDC13): 9.98 ppm (1H, bs, -CHO), 7.54
ppm (5-aromatic protons), 5.4-6.1 ppm (4H, m), 3.6
ppm (2H, dd, J=10.5 and 3.54 Hz), and 3.31 ppm
(2H, dd, J=10.55 and 3.5 Hz).
HPLC methodology for detection of both adducts
was established with a lower detection limit of 16 ng.
No adduct was detected in any of the samples preincubated with semicarbazide or phenelzine. Hence, we
conclude that the action of these compounds in obviating the biotransformation of allylamine to acrolein is
due to direct inhibition of the enzyme SSAO and not
secondary to trapping of the aldehyde.
Discussion
The concept that environmental chemicals, or xenobiotics, may play an underlying toxic role in causing
human atherosclerotic disease is an older idea that,
despite the numerous toxic substances in our environment, has had very little experimental confirmation.'3 Although cardiovascular tissues such as myocardium, vascular smooth muscle, and endothelium
are metabolically active insofar as they possess a
variety of enzymes involved in the metabolism of
Boor et al Vascular Enzyme and Xenobiotic Amine Metabolism
251
0
I
11
C-NH-N=CH-CH=CH,
1
4
3
2
NH2
D20
CH
CH
3
CH
2
a
1
10
.
.
{
10
55
,
.
.
0
.
.
|
.
.
PPM
.
0
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CHi-CHr N=CH-CH=CH,
"ia
CH CH,- NH-CHr-CHr-CHO0
lib
Ar-5H
FIGURE 1. Chemical structures and IHNMR spectra ofadducts e-xpected from reaction of acrolein with semicarbazide-sensitive
amine oxidase inhibitor semicarbazide (I)
and phenylethylamine amine (II). Substances were synthesized and structures were
confirmed by mass spectroscopy and proton
nuclear magnetic resonance (see text for
details); proton nuclear magnetic resonance
spectra of the phenylethylamine-acrolein
adduct gave a mixture of two isomers IIa
(10%) and IIb (90%). High-performance
liquid chromatography of incubated samples in repeated experiments showed that
neither adduct was present (lower detection
limit, 16 ng) after incubation; this absence
confirms that enzyme inhibition is the mechanism through which semicarbazide-sensitive
amine oxidase inhibitors obviate the metabolism of allylamine to acrolein, rather than
"trapping" of the aldehyde. PPM, parts per
million.
CHO
.
I.
10
X
,**
1
9
PPM
.
.
8
.
1
.
7
.
6
5
endogenous substances, no clear example of the
metabolism of a xenobiotic vascular toxin by a cardiovascular enzyme exists. The mode of enzymic
"detoxification" and "toxification" of xenobiotics has
been studied and debated extensively14 in recent
years, primarily with regard to a variety of hepatic
enzyme preparations. A few such enzyme systems
have been identified with relatively low activity in
cardiovascular tissues,15 but studies of their potential
significance have been rare.16
SSAO is an enzyme that is distinct from other
forms of monoamine oxidase (such as MAO A or
MAO B) and has highest activity in blood vessels.17
MAO A and MAO B, which are present in liver,
brain, and heart, have entirely different inhibitor
characteristics from SSAO and apparently are not
capable of metabolizing allylamine in vitro.1'6
Although the metabolic function of SSAO is
unknown, it has widespread distribution in mammalian species,18 and recently the enzyme has been
4
3
localized to the smooth muscle cells of vessels.19 We
have shown that when grown in vitro, porcine vascular smooth muscle cells express SSAO activity and
release enzyme into their culture media7; cultured
endothelial cells or adventitia-derived fibroblasts do
not have SSAO activity; thus, SSAO is further identified as a vascular smooth muscle enzyme.
In this study, we have definitively shown that the
highly selective in vivo cardiovascular toxin allylamine is metabolized by SSAO. Enzymes localized in
vascular tissue, such as SSAO, have been singularly
neglected and are worthy of further study. Very
recently, vascular SSAO has been shown to metabolize methylamineA20 an endogenous amine that may
result as a breakdown product of creatinine, sarcosine, and catecholamines. In that study, the investigators propose that one physiological role of SSAO
may be cellular protection from such endogenous
amines. Our unique demonstration of enzymic
biotransformation of a known in vivo cardiovascular
252
Circulation Research Vol 66, No 1, January 1990
toxin suggests an additional role for this vascular
enzyme, that is, the metabolism of exogenous amines
in our environment with potentially deleterious cardiovascular effects.
References
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KEY WORDS * semicarbazide-sensitive amine oxidase * allylamine
* acrolein * vascular metabolism
A role for a new vascular enzyme in the metabolism of xenobiotic amines.
P J Boor, R M Hysmith and R Sanduja
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Circ Res. 1990;66:249-252
doi: 10.1161/01.RES.66.1.249
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Copyright © 1990 American Heart Association, Inc. All rights reserved.
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