Oxidation of di- and polyamines: In vitro effect of amino aldehydes on the vitality of Leishmania promastigotes

Article abstract of DOI:10.1007/s00044-009-9173-0

The aminoaldehydes 4-aminobutanal and 5-aminopentanal, derived from the oxidation of the diamines putrescine and cadaverine, and 1-(3-aminopropyl)-4- aminobutanal and aminodialdehyde, derived from the oxidation of the polyamines spermidine and spermine, were produced utilizing a copper amine oxidase (CAO) from Euphorbia characias latex and tested with in vitro cultivation of Leishmania infantum promastigotes. Whereas the aminoaldehydes derived from the oxidation of the diamines were stimulating factors for growth of Leishmania infantum promastigotes, the aldehydes derived from polyamines oxidation had a drastic inhibitory effect on the vitality and growth of these parasites. Thus, a double scenario arises, showing the use of aldehydes from diamines to obtain a large number of organisms of Leishmania infantum promastigotes to use in serological studies, whereas the aldehydes derived from polyamines could be used as a new strategy for therapeutic treatment against these parasites.

Full text of DOI:10.1007/s00044-009-9173-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:77–83
DOI 10.1007/s00044-009-9173-0
ORIGINAL RESEARCH
Oxidation of di- and polyamines: in vitro effect
of amino aldehydes on the vitality of Leishmania
promastigotes
`
Silvia Massa Æ Delia Spano Æ Francesca Pintus Æ
Rosaria Medda Æ Giovanni Floris
Received: 9 December 2008 / Accepted: 6 February 2009 / Published online: 14 March 2009
¨
Ó Birkhauser Boston 2009
Abstract The aminoaldehydes 4-aminobutanal and 5-aminopentanal, derived
from the oxidation of the diamines putrescine and cadaverine, and 1-(3-aminopro-
pyl)-4-aminobutanal and aminodialdehyde, derived from the oxidation of the
polyamines spermidine and spermine, were produced utilizing a copper amine
oxidase (CAO) from Euphorbia characias latex and tested with in vitro cultivation
of Leishmania infantum promastigotes. Whereas the aminoaldehydes derived from
the oxidation of the diamines were stimulating factors for growth of Leish-
mania infantum promastigotes, the aldehydes derived from polyamines oxidation
had a drastic inhibitory effect on the vitality and growth of these parasites. Thus, a
double scenario arises, showing the use of aldehydes from diamines to obtain a large
number of organisms of Leishmania infantum promastigotes to use in serological
studies, whereas the aldehydes derived from polyamines could be used as a new
strategy for therapeutic treatment against these parasites.
Keywords Leishmania Á Promastigotes Á Aldehydes Á Amine oxidase Á
Diamines Á Polyamines
Introduction
Leishmaniasis is an acute systemic polymorph protozoary disease of skin, mucous
membrane, and internal organs, caused by intracellular obligated parasites
belonging to the Leishmania genus. In clinical medicine leishmaniasis is defined
`
S. Massa Á D. Spano Á F. Pintus Á R. Medda Á G. Floris
Department of Applied Sciences in Biosystems, University of Cagliari, Cagliari, Italy
G. Floris (&)
Dipartimento di Scienze Applicate ai Biosistemi, Citta Universitaria, I–09042 Monserrato, CA, Italy
e-mail: florisg@unica.it
`

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Med Chem Res (2010) 19:77–83
as visceral, cutaneous, and mucocutaneous. Cutaneous and mucocutaneous leish-
maniasis initially appears with a papule, which evolves into an ulcer, and its
cicatrization occurs within an extremely variable range of time. Phlebotomus
perfiliewi, a hematophagous insect belonging to the Phlebotomus genus, is
responsible for cutaneous leishmaniasis transmission, and the etiological agent is
Leishmania infantum. Visceral leishmaniasis manifests with fever, lynphadenopaty,
hepatomegaly, reduced weight, anemia, leukopenia, thrombocytopenia, and infaust
effect if not promptly treated. Phlebotomus perniciosus is mainly responsible
for visceral leishmaniasis transmission, but P. major or P. ariasi can also be
responsible. The etiological agent of visceral leishmaniasis can even be Leishmania
infantum.
Latest studies highlight that intracellular proliferation and differentiation of some
species of Leishmania amastigotes are improved by substances as the polyamines
spermine and spermidine (Cona et al., 1991). Many species of Leishmania are able
to synthesize high levels of putrescine, which seems to play an important role in
proliferation processes and cell differentiation.
The physiological function of aliphatic amines, such as putrescine, cadaverine,
spermidine, and spermine, in living organisms, has yet not been completely
established. Intracellular concentrations of polyamines are highly regulated (Cohen,
1998). If they accumulate excessively within cells, due to either very high
extracellular concentrations or deregulation of the systems that control their
homeostasis, polyamines can cause cytotoxic effects.
Copper amine oxidases (CAOs) [amine: oxygen oxidoreductase (deaminating)
(copper containing); EC 1.4.3.6] are important enzymes in the cellular and
extracellular metabolism of amines. CAOs catalyze the oxidative deamination of
primary amino groups of mono-, di-, and polyamines, extracting two electrons from
amines and transferring them to molecular oxygen, to form the corresponding
aldehyde, ammonia, and hydrogen peroxide (Medda et al., 1995).
Thus CAOs are important enzymes contributing to the regulation of levels of
polyamines, catalyzing their oxidative deamination. Spermidine and spermine may
also become a source of toxic metabolites (Arancia et al., 2004).
The aim of this report is to study the rule played by aldehydes produced from di-
and polyamine on Leishmania promastigotes in vitro and to observe the effects by
monitoring cellular density and vitality.
Materials and methods
Amine oxidase from the latex of Euphorbia characias (ELAO), a perennial
Mediterranean shrub, was purified, following the method described by Padiglia
et al., (1998). Highly purified enzyme preparations (0.5 lM) were incubated in
100 mM Tris-HCl buffer (pH 7.0) at 35°C in presence of 1–5 mM diamine
(putrescine or cadaverine) or 1–5 mM polyamine (spermidine or spermine). After
30 min, when no more di- or polyamine oxidation occurred in the incubation
mixture as observed by high-performance liquid chromatography (HPLC) analysis,
the enzymatic reactions were stopped by heat inactivation (97°C for 5 min).

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The incubates were centrifuged at 18,000 rpm for 15 min and the precipitate was
discarded.
The disappearance of di- and polyamines was monitored using Agilent 1100
series HPLC using the method described by Lozanov et al., (2007).
Leishmania infantum promastigote strain (MHOM-TN-80-IPT1), under cultiva-
tion in the Serology Laboratory of the Zooprofilatic Institute of Cagliari was
`
provided by the Istituto Superiore di Sanita.
Leishmanias, withdrawn from either popliteal and axillary lymph node or rasping
ulcerous lesions of ill dogs, were grown in a specific Tobie medium modified by
Evans, composed of solid (containing beef extract and peptone) and liquid phase
(containing a saline solution and glucose). To this medium gentamycin (0.1 mg /ml)
was added, followed by incubation at 22°C.
After 1 week the cultures were observed using microscopy to verify presence or
absence of parasites. Aliquots of Leishmania infantum promastigotes (106) were
added to five flasks containing culture medium and aminoaldehydes (1–5 mM). As a
control, aliquots of Leishmania infantum promastigotes were inoculated into five
flasks containing culture medium alone or culture medium containing 1–5 mM
putrescine, cadaverine, spermidine or spermine. Analyses of the cultures were done
twice a week, on the fourth and seventh day. Microscopic analyses were carried out by
density estimation determined by Bu¨rker counting chamber. We considered as health
index both the mobility and the form of parasites. On the contrary, in joined cells, low
mobility and globular form were considered a slight illness state of the cultures.
Results
The oxidation of mono-, di-, and polyamines by CAOs is reported in Fig. 1. The
oxidation of putrescine gives hydrogen peroxide and 4-aminobutanal, which yields
1-pyrroline by spontaneous cyclization (Fig. 1a). In similar manner oxidation of
cadaverine gives hydrogen peroxide and 5-aminopentanal, which yields 1-piperei-
dine (Fig. 1b). Oxidation of spermidine gives hydrogen peroxide and 1-(3-
aminopropyl)-4-aminobutanal. The latter spontaneously cyclizes to 1-(3-aminopro-
pyl)pyrrolinium, which undergoes further spontaneous rearrangements to 1,5-
diazobicyclo[4.3.0]nonane (Fig. 1c) (Smith et al., 1986).
The polyamine spermine is oxidized at both the terminal amino groups, giving
two moles of hydrogen peroxide and a dialdehyde that can react with primary amino
groups of free spermine, leading to the formation of aromatic pyrimidinic ring
(Fig. 1d) (Padiglia et al., 1997).
Testing different concentrations of 4-aminobutanal, the aldehyde formed from
putrescine oxidation (1–5 mM), the Leishmania promastigotes showed a negative
drop in density and vitality at the fourth day of treatment (Fig. 2). Unexpectedly, a
considerable increase in both density and vitality of Leishmania promastigotes was
observed at the seventh day of treatment (Fig. 2) for all the tested aldehyde
concentrations. Similar results, if not identical, were obtained using 5-aminopentanal,
the aldehyde formed from the cadaverine oxidation. Neither inhibition nor activation
were seen in the cultures containing 1–5 mM putrescine or cadaverine.

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Fig. 1 Oxidation of di- and polyamines by copper amine oxidase: a putrescine, b cadaverine,
c spermidine, and d spermine
Fig. 2 Density of Leishmania promastigotes grown in the presence of 1–5 mM 4-aminobutanal.
Observation was performed on the fourth ( ) and seventh ( ) day. 4-Aminobutanal is a product formed
from the oxidation of putrescine by ELAO

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Fig. 3 Density of Leishmania promastigotes grown in the presence of 1–5 mM 1-(3-aminopropyl)-4-
aminobutanal. Observation was performed on the fourth ( ) and seventh ( ) day. 1-(3-Aminopropyl)-4-
aminobutanal was derived from spermidine oxidation by ELAO
In the presence of 1-(3-aminopropyl)-4-aminobutanal, the aldehyde derived from
the spermidine oxidation, the tested Leishmania cultures were shown to be sensitive
up to 3 mM aldehyde concentration, for which promastigotes density was halved
(Fig. 3).
Finally, in the presence of the dialdehyde derived from the spermine oxidation,
promastigotes showed a gradual decrease in density from 1 to 3 mM dialdehyde
concentration, followed by a dramatic decrease at 4–5 mM dialdehyde concentra-
tion. Moreover at 5 mM dialdehyde concentration we observed a totally absence of
promastigote vitality with respect to the control cultures (Figs. 4 and 5).
Fig. 4 Density of Leishmania promastigotes grown in the presence of 1–5 mM dialdehyde. Observation
was performed on the fourth ( ) and seventh ( ) day. The dialdehyde was derived from spermine
oxidation by ELAO

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Fig. 5 Microscopic analysis
performed by Bu¨rker counting
chamber. Leishmania
promastigotes growth in the
absence a and presence b of
5 mM dialdehyde derived from
spermine oxidation
Our purpose is to study the rule played by di- and polyamines in the metabolism
of Leishmania infantum, to establish a probable correlation between parasite
proliferation and availability of biogenic amines. Moreover, research on these
protozoans reported to date has not evaluated the activity and vitality of
promastigotes in presence of the aldehyde reaction products of di- and polyamine
oxidation. The results presented herein, in combination with current knowledge of
intracellular proliferation and differentiation of some species of Leishmania, allows
us to establish two main conclusions. First, 4-aminobutanal and 5-aminopentanal
seem to exert a protective effect on growth of promastigotes. This was expected
since many species of Leishmania are able to synthesize high levels of putrescine,
which seems to play an important role in proliferation processes and cell
differentiation. Thus, the aminoaldehydes derived from the oxidation of the
diamines putrescine and cadaverine are stimulating factors on growth of Leishmania

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infantum promastigotes in vitro, and can be used to obtain a large number of
organisms to use in serological studies.
Second, the aldehydes derived from the oxidation of spermidine and spermine
seem to inhibit by the same mechanism, with the dialdehyde showing higher negative
effect on Leishmania promastigote proliferation. It is probably that spermidine and
spermine could become a source of toxic metabolites, or, most intriguingly, that
these aldehydes could interfere with DNA duplication. Thus, the aldehydes derived
from the polyamines spermidine and spermine have a drastic inhibitory effect on
vitality and growth of the Leishmania promastigotes and could be used as a new
strategy for therapeutic treatment against these parasites.
It is difficult to establish a definite role for these aldehydes in the metabolism of
Leishmania promastigotes. Since the normally used therapeutic treatment for
leishmaniasis does not lead to total healing, the discovery of new molecules and
strategies to overcome leishmaniasis remains a target for the future. Thus, we think
that inhibition of Leishmania growth and vitality by aminoaldehydes warrants further
in vivo and ex vivo investigation.
Acknowledgments The authors would like to thank Dott Manuele Liciardi, Dott. Giuseppe Addis,
Marco Cogoni, and Manuela Deidda (Istituto Zooprofilattico Sperimentale della Sardegna) for helpful
collaboration. This study was supported by a grant from Fondazione Banco di Sardegna, Sassari, Italy.
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