Vol.3, No.4B, 1-7 (2013) Open Jo urnal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34A2001
Action of protoporphyrin-IX (PP-IX) in the lifespan of
Drosophila melanogaster deficient in endogenous
antioxidants, Sod and Cat
Emilio Pimentel1*, Luz M. Vidal1, Martha Patricia Cruces1, Mariusz Krzysztof Janczur2
1Departamento de Biología, Instituto Nacional de Investigaciones Nucleares (ININ), Ocoyoacac, México;
*Corresponding Author:
2Facultad de Ciencias, Universidad Autónoma del Estado de México, Toluca, México
Received 12 August 2013; revised 21 September 2013; accepted 2 October 2013
Copyright © 2013 Emilio Pimentel et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Protoporphyirin-IX (PP-IX) is a precursor of the
biosynthesis of the hemo group, most of the
cytochromes and the chlorophylls. The PP-IX is
used for medical purposes, and recently a report
indicated that it exhibits a dual action since it
can decrease or increase the genetic damage
caused by N-nitroso-N-ethylurea (ENU) in so-
matic cells of Drosophila. PP-IX is known to be
able to act as an anti- or pro-oxidant agent. The
aim of the presen t research was to study the role
of PP-IX on the lifespan of Drosophila melano-
gaster, taking into account the fact that in-
creasing levels of ROS can accelerate the aging
process. The Canton-S strain (CS) was used as
well as Sod and Cat which are deficient in the
endogenous enzymes, superoxide dismutase
and catalase, respectively. Groups of females
and males were treated separately with 5 mg/ml
of PP-IX solution. The comparison of survival
curves indicates that this pigment extended the
lifespan of CS. In contrast, Sod strain showed
that the opposite effect and had no effect in Cat
strain. The fact that PP-IX reduces the mean
lifespan in Sod deficient strain might suggest a
pro-oxidant action of PP-IX, and consequently
the cumulating of ROS as a superoxide could
have a mutagenic effect as was shown recently.
The result s presen ted evidence o f the dual effect
of PP-IX.
Keywords: Protoporphyrin IX (PP-IX); Lifespan;
Sod; Cat; Reactive Oxygen Species; Longevity;
Drosophila melanogaster
1. INTRODUCTION
Aerobic metabolism and exposure to physical or che-
mical agents of anthropogenic origin generate reactive
oxygen species (ROS) that are characterized by an un-
paired electron. The excess of ROS in the cell causes
oxidative stress which may damage DNA, RNA, proteins
and lipids producing degenerative diseases, and may
accelerate the normal process of aging [1]. Although
many ROS are continuously generated during metabo-
lism, neither cells nor their components are damaged due
to the action of the physiological defense enzymes that
include the following: superoxide dismutase (Sod), cata-
lase (Cat) and glutathione peroxidase [2].
In addition, the cells can prevent oxidative stress
through the action of compounds known as antioxidants
that are defined as any substance which, when it is pre-
sent in concentrations lower than those of an oxidizable
substrate, delays or inhibits oxidation [3]. Since many
antioxidants are found in food, their levels can increase.
The compounds that have been more extensively studied
for their antioxidant activity are ascorbic acid, α-toco-
pherol and β-carotene. These compounds have a similar
structure which in most cases is responsible for their ac-
tivity, and which includes at least an aromatic ring and
one or more hydroxyl groups that can act as electron do-
nors [4].
Studies in vitro as well as in vivo have shown that
chlorophyll and its semi-synthetic derivative, sodium
copper chlorophilin (SCC), are able to decrease or com-
pletely inhibit DNA damage induced by both physical [5]
or chemical agents [6,7]. From the results obtained with
different test systems, it was established that SCC works
primarily through three mechanisms: a) as an antioxidant,
inactivating free radicals, b) forming complexes with
mutagens/carcinogens or their precursors, and c) inhibit-

E. Pimentel et al. / Open Journal of Anima l Sciences 3 (2013) 1-7
2
ing the enzymes involved in the activation of carcino-
gens [8]. However, although most of the studies have
provided evidence that SCC is an excellent antimutagen
and/or anticarcinogen, some others have shown that this
compound can increase DNA damage, either spontane-
ously or induced by other agents [9-11].
Previous studies demonstrated that SCC may act as
promoter and inhibitor of the mutagenesis in Drosophila.
In our study 48 h-old larvae were pretreated for 24 h with
SCC or sucrose and then treated with chromium (VI) ox-
ide (CrO3), gamma rays, N,N-Dimethylhydrazine (DMH)
or ENU immediately following completion of the pre-
treatment period (0-day delay) or delayed 1, 2 or 3 days.
After delays of 0 and 1 day, clear evidence was found of
a protective effect of SCC. Contrarily, after delays of 2
and 3 days the results showed a reversal effect, in other
words that SCC-related genetic damage appeared more
frequently than the events in the sucrose control sug-
gesting a promoting effect. This dual effect of SCC with
different agents suggested that it does not depend on the
mechanism of action of the agent [10]. At present, evi-
dence indicates that both the lower concentrations and
the SCC metabolites such as PP-IX are involved in the
mutagenic effect, and that copper could be responsible
for antimutagenic activity [10].
Protoporphyrin-IX is a precursor of the hemo group,
the chlorophylls and most of the cytochromes [12]. Some
studies have provided evidence that the PP-IX can act as
either pro-oxidant or antioxidant; for example, in the
presence of light, PP-IX stimulates lipid peroxidation by
Fe+2 and ascorbate [13] whereas lipid peroxidation is
inhibited in darkness. Increased levels of Sod were also
found in mice treated with PP-IX, suggesting that it in-
duces the formation of superoxide radicals [14]. In rat
strain CF1, Sod was rapidly induced after a single dose
of PP-IX suggesting that superoxide radicals had been
generated [14].
The most accepted hypothesis of aging was proposed
by Gerschman [15] and Harman [1] who suggest that
aging results from the accumulation of damage caused
by free radicals; this is supported with evidence found by
different systems [16-21]. The use of strains deficient in
the expression of endogenous antioxidants has been
practicable in testing the effect of dietary antioxidants in
lifespan. Drosophila melanogaster has been extensively
used to test the effect of dietary exogenous antioxidants
in lifespan because it offers many advantages: its lifespan
is short, 80 - 90 days (in laboratory conditions) and no
mitotic divisions take place in the adult [22]. In addition,
different mutant strains in genes involved in antioxidant
defense such as catalase and superoxide dismutase en-
zymes could be used [16,20]. It is known that through
the lifespan of Drosophila the activity of Cat decreases
[18,23-25] however, the increased expression of the Cat
gene does not extend the lifespan of flies, but rather pro-
tects against oxidative stress [26].
Some studies have shown that in both, mice [27] and
Drosophila [28] a deficiency of the Mn-Sod gene (Sod2)
severely reduces lifespan and increases the degree of
oxidative stress which in turn, increases the levels of
DNA damage and tumor incidence [29]. Unlike Cat, the
over expression of Sod-Cu/Zn in Drosophila increases
the lifespan [30,31] proportionally to the activity of the
enzyme. Moreover, the simultaneous expression of Mn-
Sod and Sod-Cu/Zn has an additive effect [32], whereas
the simultaneous expression of the enzyme Mn-Sod and
Cat does not provide additional benefits [33].
Since free radicals accelerate the aging process, the
consumption of antioxidants has been promoted due to
its ability to inactivate free radicals. Reports have shown
that a concentration of 20 µg/mL of vitamin E, added in
the culture medium of Drosophila, increased lifespan by
16% compared with the control group. However, adverse
effects were also detected because its higher concentra-
tions (200 μg/mL) reduced the lifespan significantly [34].
One of the best documented mechanisms of action of
SCC is its activity as an antioxidant; it is known to be
capable of preventing lipid peroxidation [35,36]. How-
ever, as mentioned earlier this compound, as well as
PP-IX are capable of inducing genetic damage probably
through generation of ROS [10,14]. To evaluate this pos-
sibility, and based on the fact that increased ROS levels
accelerate the aging process, we defined our aim in this
research as the assessment of the role of PP-IX in the
lifespan of D. melanogaster strains deficient in CuZn-
Sod and Cat.
2. MATERIALS AND METHODS
2.1. Drosophila melanogaster Strains
Canton-S wild type, Sod and Cat strains were used.
The last two were obtained from the Bloomington Dro-
sophila Stock Center. Sod and Cat enzymes constitute an
evolutionary conserved ROS defense system against su-
peroxide; Sod converts superoxide anions to H2O2, and
Cat prevents free hydroxyl radical formation by breaking
down H2O2 into oxygen and water.
Sod [n1] red [1]/TM3, Sb [1] Ser [1]: In Drosophila,
deficiency of cytoplasmic CuZn-Sod (Sod 1) in Sod1-null
mutants imparts reduced lifespan, neurodegeneration,
infertility, and hypersensitivity to further oxidative stress
[37-39]. The gene encoding the enzyme is located in
chromosome 3 [40]. Homozygotes have a significantly
shorter mean and maximum lifespan than normal and are
sensitive to paraquat, ionizing radiation and hyperoxia
compared to control flies.
Cat [n1]/TM3, Sb [1] Ser [1]: This strain is deficient in
the enzyme Cat, the gene encoding the enzyme is also

E. Pimentel et al. / Open Journal of Anima l Sciences 3 (2013) 1-7 3
found in chromosome 3, in the region 44.3 [40]. Cat en-
zyme is involved in the decomposition of H2O2 generated
during cellular metabolism [41]. Only about 10% of ho-
mozygous Cat [n1] flies eclose from their pupal cases.
Homozygous Cat [n1] mutants also show a reduced life-
span, living half as long as wild type flies [42].
Three groups of 25 males and virgin females 16 h old
were placed separately for 24 h in homeopathic vials
(10.5 × 2.4 cm) containing 0.7 mg of Drosophila instant
medium (Formula 4 - 24 Carolina Biological Supply Co.)
with 2.5 mL of tap water or 5 mg/mL solution of PP-IX.
In all, 100 to 225 females or males were tested. Three
experiments were performed for each group (0 and PP-
IX). Twice a week dead flies were counted, and live in-
dividuals were transferred to vials with freshly corre-
sponding treatment. The PP-IX disodium salt (CAS
50865-01-5) was purchased from Sigma Chemical Com-
pany (St. Louis, MO).
2.2. Statistical Analysis
The Kaplan-Meier test was used to obtain the cumula-
tive survival curves for each experimental group, and for
each sex. Data for survival were plotted and curves were
compared using the Wilcoxon test (XLSTAT software)
[43]. The General Linear Model (GLM) was used to de-
termine any interactions between sex, strains or treat-
ment.
3. RESULTS
Based on the toxicity test in which three concentra-
tions for PP-IX were tested (0.5, 5 and 50 mg/mL.), we
found that the 5 mg/mL concentration was appropriate
for chronic treatment in adults. The mean lifespan (MLS)
and maximum lifespan (ML) for each sex and treatment
were evaluated. MLS is the time where half of the treated
individuals died and ML is the total lifespan of individu-
als.
Ta b l e 1 shows the values of both traits of each tested
strain. The analysis of variance (ANOVA) at 95% confi-
dence showed significant differences among strains with
a relation: Sod < Cat < CS (see Figure 1.). The treatment
with PP-IX significantly prolonged the MLS of females
(by 17.4 days) and males (by 9.7 days.) in CS as well as
Cat (by 1.6 and 3.2 days for females and males, respect-
tively.); however, in the latter strain no noteworthy dif-
ferences were found. In contrast, PP-IX shortened the
MLS significantly in both sexes of Sod strain, where the
females exhibited a shorter MLS (12 days.) than males (7
days.). Regarding the ML, PP-IX reduced it significantly
only in the Sod strain; the reduction of ML was different
between females (25 days less.) and males (11 days less.)
with respect to the control. No significant differences
were found between sex in CS and Cat strains.
Figure 2 shows the survival curves for each strain
separately by sex after chronic treatment with PP-IX;
data corresponding to females are given in the left panel
and for males in the right one. Taking together data for
females and males, the results reflect that PP-IX in-
creases the MLS by 13.5 days (26%) in CS, by 2 days
(5%) in Cat but reduces it in Sod by 10 days (26%) with
respect to the control.
4. DISCUSSION
Longevity is a quantitative trait, with continuous phe-
notypic variation attributable to the joint segregation of
Table 1. Effect of chronic treatment with 5 mg/ml of PP-IX on
the lifespan of CS, Sod and Cat strains.
Strain Sex
Chronic
Tr eat m en t
PP-IX
(5 mg/mL)
n MLS
(days) ± SE
ML
[days]
P value
(0 vs.
PP-IX)
CS ♀ 0 22550.7 ± 2.4 84
PP-IX 15068.1 ± 3.0 84 <0.0001
♂ 0 22553.3 ± 2.4 84
PP-IX 15063.0 ± 0.1 84 <0.0001
Sod ♀ 0 20041.8 ± 2.6 70
PP-IX 12529.6 ± 3.3 45 <0.0001
♂ 0 20036.2 ± 2.6 49
PP-IX 12529.0 ± 3.3 38 <0.0001
Cat ♀ 0 10044.9 ± 3.7 59
PP-IX 15046.5 ± 3.0 63 n.s.
♂ 0 12543.2 ± 3.3 63
PP-IX 15046.4 ± 3.0 66 n.s.
n= Number of individuals tested, MLS = Mean lifespan and ML = Maxi-
mum lifespan. The MLS were calculated from the general linear model
(least square means). P values were obtained from comparisons between the
survival proportion curves. The confidence intervals of MLS were obtained
from the Kaplan-Meir analysis.
M LS (Days ± SE)
F
igure 1. Mean lifespan (MLS) relationship between strains.

E. Pimentel et al. / Open Journal of Anima l Sciences 3 (2013) 1-7
4
Figure 2. Represents the survival for females (a), (b), (c) and for males (d), (e), (f) Green-
wood confidence intervals were employed. Data for survival were plotted and curves were
compared using the Wilcoxon test (XLSTAT software) (Addinsoft 2011). Log-rank, Wil-
coxon and Tarone-Ware analysis showed a significant difference (p < 0.0001) when treat-
ments PP-IX and control were compared for each sex of strain. Probability level was: alpha
= 0.05.
multiple interacting quantitative trait loci and with ef-
fects that are highly sensitive to the environment. D.
melanogaster has been a historically important system
for investigating the genetic basis of longevity, which
relies on two sources: its powerful genetic tool as a mo-
del system, and a natural ecology that provides substan-
tial genetic variation across significant environmental
heterogeneity.
In a previous study PP-IX was shown to be able to act
as an antimutagen immediately after being administrated
to the flies and as a mutagen a few days later [10]. One
possible explanation of this effect is that PP-IX provokes
damage through generation of ROS. The present study
showed that in Sod -deficient strain, chronic treatment
with PP-IX reduced the MLS by 12 days for females and
by 7 days for males. The ML was shorter by 25 days for
females and by 11 days for males. Nevertheless, neither
MLS nor ML was affected in Cat-deficient individuals.
These results are in accordance with those reported by
Lebovitz et al. [37] in mice and by Duttaroy [28] in
Drosophila who showed that the deficiency in the mito-
chondrial Mn-Sod gene significantly reduces life expec-
tancy due to the increase of the superoxide radical. The
same was observed in the absence of the gene encoding
the cytosolic CuZn-Sod, but the effect was not as severe
as when a deficiency of mitochondrial Sod occurs. It is
OPEN A CCESS

E. Pimentel et al. / Open Journal of Anima l Sciences 3 (2013) 1-7 5
likely that under those conditions PP-IX produces no
action on the superoxide generated by the radiation, but
probably acts as a pro-oxidant generating superoxide as
demonstrated by Afonso et al. [14].
Another example that supports this hypothesis is that
PP-IX has been used for several years in photodynamic
therapy [14,44-46]. This is so due to its ability to induce
superoxide radicals that react with molecular oxygen
producing peroxide radicals which cause lipid peroxide-
tion, and leading to different cell damages such as struc-
tural changes of the cell membrane, damage to proteins,
inactivation of receptors, enzymes and ion channels, all
of which can lead to cell death [47].
The protective activity of PP-IX observed in the CS
strain in this study could be the result of the elimination
of free radicals, particularly H2O2. Afonso et al. [14]
found that Cat activity increased 30% in rats of the strain
CF1, 2 h after the injection of PP-IX, with the decrease
in H2O2 levels. Although the results obtained with Cat
strain showed a minimum increase in the MLS and ML,
in the wild type strain its effect was more evident; PP-IX
increased the MLS by 26% compared to the control.
However, a report indicated that the over expression of
Cat by 80% did not increase the lifespan of Drosophila,
but protected it against oxidative stress [48]. Furthermore,
Griswold et al. [49] molecularly characterized six alleles
and found that Cat [n2], Cat [n3], Cat [n5] and Cat [n6]
reduced the Cat activity, viability and normal longevity,
but the alleles Cat [n1] and Cat [n4] showed no activity
of Cat, yet longevity and viability were reduced. Mackay
et al. [50] showed that Cat null allele caused a significant
increase in the frequency of spontaneous mutations. The
increase in MLS in CS and Cat could be attributable to
the antimutagenic effect reported by other authors [5,
51-53], who suggest that the protective effect of both
PP-IX and SCC, (porphyrin bound to Cu+2.) is due to
their ability to arrest free radicals generated by radiation
and to increase the expression of endogenous antioxi-
dants [54].
Although the PP-IX treatment reduced the MLS in the
Sod-deficient strain (Probably because of its pro-oxidant
action inducing the formation of O2− radicals, the H2O2
produced by this increase in superoxide.), the antioxidant
action of PP-IX could be added to the action of glu-
tathione peroxidase enzyme which can also reduce H2O2.
According to the effect of PP-IX on both the wild type
CS and the Cat strain, we suggest three possible mecha-
nisms of action: a) as inductor of the antioxidant en-
zymes to achieve normal levels of activity; b) as an anti-
oxidant, reducing the levels of peroxide; and c) both ac-
tions occurring simultaneously in the organism pro-
voking an increase in the lifespan of the treated organ-
isms. The CS strain is the best evidence of the c) mecha-
nism, since in this strain PP-IX delayed death and strongly
increased MLS at birth in both sexes by 13.5 days.
5. ACKNOWLEDGEMENTS
This research was supported by a grant from Consejo Nacional de
Ciencia y Tecnología (CONACyT), México, Grant Number 167461 and
involved in part research carried out by L.M., Vidal to obtain the Mas-
ter Degree in Environmental Sciences at Universidad Autónoma del
Estado de México (UAEM). The authors wish to acknowledge the
splendid technical assistance of Hugo Suarez Contreras and Alicia
Hernández Arenas.
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