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Arch Toxicol
DOI 10.1007/s00204-017-1998-6
MOLECULAR TOXICOLOGY
On the mechanisms of melatonin in protection of aluminum
phosphide cardiotoxicity
Mohammad Hossein Asghari1,2 · Milad Moloudizargari3 · Maryam Baeeri4 · Amir Baghaei5 · Mahban Rahimifard4 ·
Reza Solgi6 · Abbas Jafari7 · Hamed Haghi Aminjan1 · Shokoufeh Hassani4 · Ali Akbar Moghadamnia2 ·
Seyed Nasser Ostad1 · Mohammad Abdollahi1,4
Received: 1 February 2017 / Accepted: 22 May 2017
© Springer-Verlag Berlin Heidelberg 2017
device was used to record the electrocardiographic (ECG)
parameters. Heart tissues were studied in terms of oxida-
tive stress biomarkers, mitochondrial complexes activities,
ADP/ATP ratio and apoptosis. Abnormal ECG records as
well as declined heart rate and blood pressure were found
to be related to AlP administration. Based on the results,
melatonin was highly effective in controlling AlP-induced
changes in the study groups. Significant improvements
were observed in the activities of mitochondrial complexes,
oxidative stress biomarkers, the activities of caspases 3 and
9, and ADP/ATP ratio following treatment with melatonin
at doses of 40 and 50 mg/kg. Our results indicate that mela-
tonin can counteract the AlP-induced oxidative damage in
the heart. This is mainly done by maintaining the normal
balance of intracellular ATP as well as the prevention of
oxidative damage. Further research is warranted to evalu-
ate the possibility of using melatonin as an antidote in AlP
poisoning.
Keywords Aluminum phosphide · Melatonin · Apoptosis ·
Oxidative stress · Mitochondrial dysfunction
Introduction
Aluminum phosphide (AlP), a lethal solid pesticide, is
frequently used to protect stored food products and dur-
ing food transformation processes (Bumbrah et al. 2012).
This pesticide has no effects on seed viability and leaves
minimal residues on food products. Moreover, its usage
is cost-effective and has shown high efficacy against all
life stages of insects (Anand et al. 2011; Bumbrah et al.
2012). Although this pesticide is a high-risk agent for
non-target species including humans, the advantages out-
weigh the risks of usage and due to the aforementioned
Abstract Aluminum phosphide (AlP), one of the most
commonly used pesticides worldwide, has been the lead-
ing cause of self-poisoning mortalities among many Asian
countries. The heart is the main organ affected in AlP poi-
soning. Melatonin has been previously shown to be ben-
eficial in reversing toxic changes in the heart. The present
study reveals evidence on the probable protective effects of
melatonin on AlP-induced cardiotoxicity in rats. The study
groups included a control (almond oil only), ethanol 5%
(solvent), sole melatonin (50 mg/kg), AlP (16.7 mg/kg), and
4 AlP + melatonin groups which received 20, 30, 40 and
50 mg/kg of melatonin by intraperitoneal injections follow-
ing AlP treatment. An electronic cardiovascular monitoring
* Mohammad Abdollahi
Mohammad@TUMS.Ac.Ir;
Mohammad.Abdollahi@UToronto.Ca
1 Department of Toxicology and Pharmacology, Faculty
of Pharmacy, Tehran University of Medical Sciences, Tehran,
Iran
2 Department of Pharmacology, Faculty of Medicine, Babol
University of Medical Sciences, Babol, Iran
3 Student Research Committee, Department of Immunology,
School of Medicine, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
4 Toxicology and Diseases Group, Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences,
Tehran 1417614411, Iran
5 Department of Toxicology and Pharmacology, Faculty
of Pharmacy, Alborz University of Medical Sciences, Karaj,
Iran
6 Legal Medicine Research Center, Legal Medicine
Organization of Iran, Hamedan, Iran
7 Department of Occupational Health, School of Public Health,
Urmia University of Medical Sciences, Urmia, Iran
Arch Toxicol
1 3
advantages, it is still widely used by farmers (Mostafalou
et al. 2013). The availability of this chemical in Asian
pesticide markets has made it a favorable agent for those
who intend to commit suicide. It is the most commonly
used suicidal poison in India. An increasing number of
self-poisonings with AlP are being reported among Ira-
nians (Mehrpour et al. 2012; Moghadamnia 2012). More
than 70% of individuals exposed to AlP die from its
toxic effects in different body organs. Cardiotoxicity is
the primary cause of death in AlP-poisoned cases. Dys-
rhythmias, congestive heart failure (CHF) and refractory
hypotension have been shown to be the most important
cardiovascular disturbances induced by AlP (Bogle et al.
2006).
AlP reacts to the presence of hydrochloric acid or water
in the stomach by releasing the fatal phosphine gas. The
precise mechanisms of AlP toxicity are not yet known;
however, studies on animals show that it possibly works via
inhibiting cytochrome oxidase in the mitochondria (Anand
et al. 2011; Moghadamnia 2012).
Unfortunately, there is no specific antidote or effective
drug to manage the cardiotoxic effects of AlP. The protec-
tive effects of some drugs such as triiodothyronine (Abdol-
ghaffari et al. 2015), vasopressin (Jafari et al. 2015), iron
sucrose (Solgi et al. 2015), Mg nanoparticle (Baeeri et al.
2013) and acetyl-l-carnitine (Baghaei et al. 2016) have
been previously investigated; however, the results were not
promising enough due to various reasons such as low safety
profiles. Melatonin, as an amphiphilic molecule, crosses all
morphophysiological barriers and can be particularly found
in mitochondria, potentially protecting it against oxida-
tive stress (Govender et al. 2014; Yang et al. 2014). This
indoleamine is a free radical scavenger with good solubil-
ity in both aqueous and organic phases which maintaining
a high capacity to modulate homeostasis mitochondrial
(Paradies et al. 2015). Additionally, melatonin is shown
to be advantageous to classic antioxidants, such as vita-
min E, and vitamin C (Korkmaz et al. 2009). A huge body
of evidence has been studied on the toxicity of pesticides
and the correlation of exposure to these compounds with
many disorders (Mostafalou and Abdollahi 2017). The pro-
tective actions of melatonin have been previously reported
against various pesticide and metal toxicities (Asghari et al.
2017b; Romero et al. 2014). The results of studies on the
antioxidant, anti-apoptotic and antiarrhythmic properties
of melatonin give rise to the opinion that it can be highly
potential in counteracting the underlying mechanisms of
AlP-induced cardiotoxicity. Also, it may probably improve
the clinical manifestations following AlP exposure, due to
its pain relieving and anxiolytic properties (Asghari et al.
2017a). On such a basis, we decided to examine the possi-
ble cardioprotective effects of melatonin and the underling
mechanisms in AlP-poisoned rats.
Materials and methods
Chemicals
ELISA kits for the evaluation of oxidative stress biomark-
ers and mitochondria isolation kit were obtained from
Cayman Chemical Co. (USA) and BioChain Inc. (USA),
respectively. AlP was obtained from Samiran Pesticide For-
mulating Co. (Iran). All the other chemicals were obtained
from Sigma-Aldrich (GmbH, Munich, Germany).
Animals
Guidelines for the ethical use of animals were followed and
a certification code was deposited by the institute commit-
tee of ethics (IR.TUMS.REC.1394.1432), approving all the
animal experiments conducted in this study. For the animal
studies, 200–250 g male Wistar rats were acclimatized in a
room with 50–55% humidity, temperature of 20–25 °C and
the light/dark cycle of 12 h, during which standard rat diet
was available ad libitum.
Protocols
Determination of AlP LD100
In the previous studies, the LD50 and LD100 of AlP
were reported to be 12.5 (Jafari et al. 2015) and 20 mg/
kg (Anand et al. 2012) of rat body weight, respectively.
We also tried to determine the LD100 of AlP for each
experiment of our study. To do this, different doses of AlP
between 12 and 20 mg/kg were selected. Almond oil was
used as the solvent and the selected doses were orally given
to the animals. The control group was solely administered
with the same amount of almond oil used as the solvent.
In each group, four rats were placed. The mortality was
recorded 24 h post-treatment. Ultimately, the LD100 of AlP
was calculated as 16.7 mg/kg, using the probit test. Since
most doses of AlP that are deliberately ingested by indi-
viduals for self-harm intentions are supra-lethal, a single
LD100 was considered. The present study was conducted
to obtain data on the biochemical changes at the time of
death following AlP poisoning. This could thus mimic a
real case of human AlP poisoning.
Experimental design
Considering the hemodynamic parameters, four differ-
ent doses of melatonin (20, 30, 40, 50 mg/kg) were cho-
sen. After the determinations of LD100 of AlP (16.7 mg/
kg) and doses of melatonin (MLT), the rats were randomly
Arch Toxicol
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allotted into eight groups, including Control, Ethanol 5%,
AlP, MLT 50, AlP + MLT 20, AlP + MLT 30, AlP + MLT
40, and AlP + MLT 50, each in which six rats were placed.
Almond oil was used as the solvent for AlP which was
given orally (through gavage). Melatonin was dissolved in
ethanol 5% and administered intraperitoneally. The animals
in the groups 1 and 2 were only treated with appropriate
volumes of almond oil and 5% ethanol, respectively. To
measure the hemodynamic parameters, general anesthesia
was induced and maintained by injecting 60.6 mg/kg of
ketamine/xylazin and three repetitive injections (30.3 mg/
kg), 45, 90 and 150 min after AlP treatment (LD100 dose).
After anesthesia was induced, a PowerLab system (Pow-
erLab 4/35 Data Acquisition Systems, AD Instruments,
Australia) was used to noninvasively record the parameters
including heart rate (HR), electrocardiogram (ECG) and
blood pressure (BP). The animals received intraperitoneal
injections of melatonin 30 min after treatment with AlP. In
order for the biochemical studies, the heart was surgically
isolated and the blood was washed out by rinsing in ice-
cold saline and was then instantly freezed at −80 °C for
biochemical evaluation. The flowchart of the experimental
study design is shown in Fig. 1.
Determination of electrocardiogram (ECG) parameters
The anesthetized rats were immobilized and the ECG was
monitored after attaching the electrodes to both hands and
paws of the animals. PowerLab system software was used
for data analysis and QRS complexes as well as the seg-
ments of QTc, and ST were measured. The tail cuff of Pow-
erLab was also placed on the external part of the tail, where
the pulse can be detected, to record the systolic BP and HR.
Tissue sampling and mitochondrial isolation
The AlP and the other treatment groups were observed till
death, following the administrations. The animals were
killed, the heart tissues were isolated, washed with ice-cold
saline and were then cut into several sections. All the sec-
tions were stored at −80 °C various biochemical studies
except for one small section (100 mg) which was used for
mitochondrial complex assays. The mitochondria isolation
kit protocol was then followed.
Activity assessment of complex I
NADH consumption is the principal of this assay, which
is characterized by the translocation of electrons initially
too complex I and then to an electron acceptor, i.e., syn-
thetic ubiquinone. The method was followed to measure
the activity of complex I in the heart homogenate. Briefly,
an approximate amount of total mitochondrial protein
(100–200 µg) was mixed with the reaction media and the
changes in the absorbance of NADH were determined dur-
ing 3 min at 340 nm. Rotenone was then added to meas-
ure the rotenone-unaffected activity of NADH–cytochrome
b oxidase. The overall net activity was finally determined
Fig. 1 The methodology of the
experimental study is illustrated
Arch Toxicol
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by subtracting the rotenone-unaffected activity from the
entire activity. The unit, nmol/min/mg protein, was used to
express the results (Sherwood and Hirst 2006).
Activity assessment of complex II
The oxidation of 2,6-dichlorophenolindophenol (DCPIP)
by complex II (succinate dehydrogenase) decreases the
absorbance at 600 nm which can be used as an indicator of
its activity. In brief, after the addition of a specific amount
of total mitochondrial protein (10–50 µg), the baseline
and the ubiquinone-initiated activity of the complex were
both determined for 3 and 3–5 min, respectively. A stand-
ard curve of DCPIP was then used to calculate the final net
activity of complex II which was then expressed as nmol
DCIP/min/mg of mitochondrial protein (Karami-Mohajeri
et al. 2014).
Activity assessment of complex IV
Shortly, after the addition of mitochondrial protein the
reaction media, the decline in the absorbance at 540 nm
was measured during 3–6 min as the reduced form of
cytochrome c was being converted to the oxidized form by
cytochrome-c oxidase. The unit, K/min/mg mitochondrial
protein was used to express the results (Cooperstein and
Lazarow 1951).
Assessment of ADP/ATP ratio in the heart
The ratio was determined based on a study by Hosseini
et al. (Hosseini et al. 2010). The frozen samples were
thawed and immediately homogenized (4 °C). The homog-
enized sample was centrifuged at 2000g for 10 min, the
supernatant was gathered and was then applied to an HPLC
system (Waters Chromatography Division, Milford, MA,
USA) at a flow rate of 1 ml/min to do the detection. After
the quantification of ATP and ADP levels, the ADP/ATP
ratio was calculated using a standard curve.
Activity assessment of glutathione peroxidase (GPX)
After homogenizing the heart tissue based on the kit pro-
tocol, GPX activity was measured in the heart tissue and
the serum samples. A Cayman’s kit (Cayman Chemical
Co., Ann Arbor, MI, USA) was used to assess the activity
of GPX, based on a glutathione reductase-coupled proce-
dure. The oxidized form of glutathione is formed when the
hydroperoxide molecule is reduced by GPx.
Activity assessment of superoxide dismutase (SOD)
To assess the activity of SOD, the method of a study by
Pourkhalili et al. in which the production of a red formazon
dye acts as an indicator of the activity of this enzyme, was
followed (Pourkhalili et al. 2011).
Lipid peroxidation (LPO) assessment
The principle for the assessment of LPO is based on a
method in which the amount of produced malondialdehyde
(MDA) as an end product of LPO corresponds to the extent
of peroxidation. This is determined using a spectrophotom-
eter. To do this, the method of Ranjbar et al. was followed
(Ranjbar et al. 2010).
Reactive oxygen species (ROS) assessment
DCF-DA was used to measure the amount of peroxides.
Five micrometres of DCF-DA was added to the supernatant
and incubated at 37 °C for 30 min. The amount of DCF, the
fluorescent end product, was then determined at excitation
and emission wavelengths of 488 and 525 nm, respectively
(Momtaz et al. 2010).
Activity assessment of caspases 3 and 9
The method of Hosseini et al. was followed to assess the
activities of caspases 3 and 9. Shortly, the heart samples
were rinsed with ice-cold saline after weighing. Homogen-
ates of the tissues were prepared using lysis buffer contain-
ing MgCl2 (2 mM), KCl (50 mM), EDTA (2 mM), Triton
X100 (%1), HEPES (50 mM; pH 7.4) and were centrifuged
at 12,000g for 20 min. The substrates of caspases 3 and 9
were added to specific amounts of the supernatant and were
incubated at 37 °C for 4 h. Absorbance was determined
at a wavelength of 405 nm and the results were shown as
nanomoles per hour milligrams of protein (Hosseini et al.
2013).
Determination of CK‑MB activity and troponin I level
in heart tissue
The activity of creatinine kinase-MB (CK-MB) and tro-
ponin-I levels, as cardiac markers, was determined in fro-
zen homogenates of the heart tissue using commercial kits
from ZellBio GmbH, Germany. The absorbance of samples
was checked by an enzyme linked immunosorbent assay
(ELISA) reader.
Arch Toxicol
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Statistical analysis
Results are expressed as mean ± standard deviation (SD).
One-way ANOVA tests were used to perform the statistical
analysis. Tukey was used as the post hoc test. p values less
than 0.05 were considered significant.
Results
No significant differences were observed between the etha-
nol 5% and the control group (results not shown).
Survival time
We started to monitor the ECG abnormalities, 15 min after
AlP treatment. Death was the final outcome in all the ani-
mals in the AlP and the treatment groups. The median sur-
vival time of the AlP group was 55 min [95% confidence
interval (CI) 36.99–73]. In the groups receiving melatonin
following AlP administration, the median survival times
were 90 min (95% CI 69.59–110.4), 85 min (95% CI
66.99–103), 180 min (95% CI 122.38–237.61) and 320 min
(95% CI 266.1–373.9) at doses of 20, 30, 40 and 50 mg/kg,
respectively.
ECG, HR and BP
AlP-poisoned rats treated with melatonin showed
improved hemodynamic parameters. The BP and HR
started to decrease significantly 30 min after AlP treat-
ment (Tables 1, 2). Interestingly, melatonin mitigated the
progressive decrease of BP in the AlP group; however, BP
did not change significantly in the sole melatonin group.
ECG abnormalities including widened QRS, elevated ST
and prolonged QTc were also observed in the AlP group
(Table 3). Melatonin administration vanished the aforemen-
tioned ECG abnormalities.
Activity of mitochondrial respiratory complexes
The activities of mitochondrial complexes were sepa-
rately evaluated to analyze the cardiac mitochondrial
function. None of the treatment groups showed sig-
nificant changes in the activity of complex II. How-
ever, the activities of complexes I and IV decreased
significantly in the AlP group compared to the con-
trol group (p < 0.05). Melatonin at doses of 40 and
50 mg/kg was able to drastically increase the activi-
ties of these two complexes in comparison to the AlP
group (Table 4).
ADP/ATP ratio assessment as an indicator of cardiac
energy
A significant increase in the ADP/ATP ratio was observed
following AlP treatment (p < 0.05). The administration of
50 mg/kg melatonin could decrease this ratio considerably
(Table 4).
The activities of GPX and SOD
Cardiac SOD and GPX activities in the AlP group
showed a significant decrease compared to the control
Table 1 Changes in blood
pressure in various groups
Data are mean + SD of six animals in each group. The control group received almond oil alone; AlP
group received only aluminum phosphide (LD100); MLT 50 group received only melatonin (50 mg/kg);
AlP + MLT 20 group received AlP + melatonin (20 mg/kg); AlP + MLT 30 group received AlP + mela-
tonin (30 mg/kg); AlP + MLT 40 group received AlP + melatonin (40 mg/kg); AlP + MLT 50 received
AlP + melatonin (50 mg/kg)
D died
a Significantly different from control groups at p < 0.05
b Significantly different from AlP group at p < 0.05
Time (min)
0–30 30–60 60–90 90–120 120–150 150–180
Control 99 ± 4 97 ± 5 97 ± 4 97 ± 6 96 ± 5 96 ± 4
MLT 100 ± 7 99 ± 6 97 ± 8 97 ± 6 100 ± 7 100 ± 6
AlP 99 ± 8 70 ± 8a48 ± 4aD D D
AlP +MLT 20 104 ± 7 95 ± 6 83 ± 6 74 ± 7a67 ± 4aD
AlP + MLT 30 102 ± 7 91 ± 5 80 ± 3a71 ± 2a67 ± 4aD
AlP + MLT 40 100 ± 7 93 ± 7 87 ± 6 79 ± 3 73 ± 3 74 ± 4
AlP + MLT 50 98 ± 5 92 ± 6b83 ± 5b77 ± 4 73 ± 2 71 ± 1a
Arch Toxicol
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group (p < 0.05). Nevertheless, melatonin administra-
tion at doses of 40 and 50 mg/kg enhanced the activity
of SOD. It also increased the activity of GPX at a dose
of 50 mg/kg (Fig. 2).
Results of oxidative stress assessment
LPO levels in the AlP group were higher than those of the
control group (p < 0.05). However, rats treated with 40
and 50 mg/kg melatonin showed lower MDA levels. ROS
Table 2 Changes in heart rate
in various groups
Data are mean + SD of six animals in each group. The control group received almond oil alone; AlP
group received only aluminum phosphide (LD100); MLT 50 group received only melatonin (50 mg/kg);
AlP + MLT 20 group received AlP + melatonin (20 mg/kg); AlP + MLT 30 group received AlP + mela-
tonin (30 mg/kg); AlP + MLT 40 group received AlP + melatonin (40 mg/kg); AlP + MLT 50 received
AlP + melatonin (50 mg/kg)
D died
a Significantly different from control groups at p < 0.05
b Significantly different from AlP group at p < 0.05
Time (min)
0–30 30–60 60–90 90–120 120–150 150–180
Control 279 ± 25 276 ± 21 272 ± 23 272 ± 34 270 ± 36 257 ± 30
MLT 278 ± 19 291 ± 34 289 ± 26 296 ± 16 280 ± 31 279 ± 16
AlP 253 ± 12 139 ± 36a46 ± 11aDDD
AlP + MLT 20 254 ± 11 194 ± 15 134 ± 13a80 ± 9a47 ± 12aD
AlP + MLT 30 268 ± 27 197 ± 93 166 ± 22ab 93 ± 21a49 ± 15aD
AlP + MLT 40 258 ± 10 202 ± 7 155 ± 14a110 ± 15 73 ± 6 72 ± 6a
AlP + MLT 50 292 ± 28 225 ± 25b173 ± 20ab 111 ± 19 65 ± 7 75 ± 8a
Table 3 Changes in ECG parameters of various groups
Data are mean + SD of six animals in each group. The control group received almond oil alone; AlP group received only aluminum phosphide
(LD100); MLT 50 group received only melatonin (50 mg/kg); AlP + MLT 20 group received AlP + melatonin (20 mg/kg); AlP + MLT 30
group received AlP + melatonin (30 mg/kg); AlP + MLT 40 group received AlP + melatonin (40 mg/kg); AlP + MLT 50 received AlP + mela-
tonin (50 mg/kg)
D died
a Significantly different from control groups at p < 0.05
b Significantly different from AlP group at p < 0.05
Time (min) Variable Control MLT AlP AlP + MLT 20 AlP + MLT 30 AlP + MLT 40 AlP + MLT 50
0–30 QRS (ms) 11.64 ± 0.32 12.67 ± 0.23 23.88 ± 0.73a21.41 ± 0.90a21.12 ± 0.55a,b 17.43 ± 0.48a,b 13.17 ± 0.41b
QTc (ms) 96.73 ± 2.49 89.70 ± 1.97 155.44 ± 7.02a103.50 ± 1.93 99.51 ± 2.37b114.70 ± 3.27a,b 101.56 ± 2.53b
ST (µv) 35.89 ± 2.04 39.92 ± 0.77 80.59 ± 2.05a69.25 ± 1.26a,b 67.57 ± 1.04a73.80 ± 2.63a,b 43.58 ± 2.22a,b
30–60 QRS (ms) 14.47 ± 1.08 14.44 ± 0.35 26.96 ± 1.47a23.12 ± 1.83a24.08 ± 1.75a20.97 ± 1.47 15.54 ± 0.49b
QTc (ms) 81.84 ± 1.30 80.18 ± 1.70 203.82 ± 4.33a189.88 ± 1.78a148.09 ± 1.73a,b 142.79 ± 2.00a,b 88.77 ± 2.81a,b
ST (µv) 31.25 ± 0.91 30.22 ± 0.99 138.42 ± 2.42a121.48 ± 2.01a138.57 ± 3.51a92.36 ± 2.75a,b 38.64 ± 2.32b
60–90 QRS (ms) 14.53 ± 0.41 16.56 ± 0.45 59.90 ± 1.11a45.93 ± 2.70a,b 38.79 ± 0.69a,b 21.54 ± 1.77b15.03 ± 0.65b
QTc (ms) 95.17 ± 2.72 103.91 ± 4.17 239.27 ± 2.53a183.86 ± 3.01a,b 166.53 ± 2.38a,b 146.65 ± 2.77a,b 110.85 ± 6.28b
ST (µv) 39.44 ± 1.32 39.50 ± 0.78 171.20 ± 1.33a137.60 ± 2.04a,b 122.13 ± 0.80a,b 70.74 ± 2.91a,b 43.21 ± 1.17b
90–120 QRS (ms) 12.37 ± 0.84 12.49 ± 0.97 D 48.27 ± 3.16a30.00 ± 0.79a19.18 ± 1.14 15.25 ± 0.58
QTc (ms) 103.01 ± 3.59 101.86 ± 1.75 D 192.88 ± 3.34a174.00 ± 2.17a138.55 ± 4.47a113.77 ± 4.11
ST (µv) 34.79 ± 1.26 31.07 ± 0.72 D 83.24 ± 2.28a85.15 ± 1.45a50.11 ± 2.06a35.77 ± 1.47
120–150 QRS (ms) 15.77 ± 0.77 15.41 ± 0.65 D 33.60 ± 1.97a31.02 ± 1.27a17.81 ± 0.65 15.89 ± 0.60
QTc (ms) 98.02 ± 2.07 97.90 ± 1.15 D 208.70 ± 1.48a216.10 ± 3.16a142.19 ± 7.30a109.19 ± 8.27
ST (µv) 20.99 ± 0.89 23.54 ± 0.92 D 72.92 ± 2.26a74.05 ± 1.67a48.18 ± 3.57a26.56 ± 3.87
150–180 QRS (ms) 16.54 ± 0.58 16.32 ± 0.38 D D D 20.07 ± 1.12 20.54 ± 1.86
QTc (ms) 88.92 ± 0.81 93.73 ± 3.11 D D D 133.66 ± 4.52a96.68 ± 2.98
ST (µv) 29.34 ± 1.09 27.56 ± 1.35 D D D 43.54 ± 2.38a33.21 ± 2.62
Arch Toxicol
1 3
production in the AlP group was significantly higher than
its production in the control group (p < 0.05). Melatonin
administration at doses of 40 and 50 mg/kg decreased the
amount of ROS significantly (Fig. 2).
Caspase‑3 and 9 activities
Exposure to AlP increased the activities of caspases 3 and
9 in heart tissue compared to control group (p < 0.05).
Table 4 Effects of various treatments on the activity of mitochondrial complexes and ADP/ATP ratio in heart tissue
Data are mean + SD of six animals in each group. The control group received almond oil alone; AlP group received only aluminum phosphide
(LD100); MLT 50 group received only melatonin (50 mg/kg); AlP + MLT 20 group received AlP + melatonin (20 mg/kg); AlP + MLT 30
group received AlP + melatonin (30 mg/kg); AlP + MLT 40 group received AlP + melatonin (40 mg/kg); AlP + MLT 50 received AlP + mela-
tonin (50 mg/kg)
a Significantly different from control groups at p < 0.05
b Significantly different from AlP group at p < 0.05
Control MLT AlP AlP + MLT20 AlP + MLT30 AlP + MLT40 AlP + MLT50
Complex I
(nmol/min/mg)
230 ± 2.83 227 ± 5.32 148 ± 17.52a155 ± 8.43a168 ± 7.43a218 ± 9.38b223 ± 9.27b
Complex II
(nmol/min/mg)
85.21 ± 2.14 82.35 ± 2.48 83.74 ± 5.29 78.98 ± 2.37 79.36 ± 3.71 79.63 ± 3.91 81.93 ± 6.37
Complex IV (K/min/mg) 515.1 ± 17.97 516.7 ± 18.02 335.9 ± 35.87a375.7 ± 11.55a385.8 ± 10.94a424.9 ± 13.20a, b 493.9 ± 7.07b
ADP/ATP ratio 1.25 1.54 2.79a2.57a2.93a2.25a1.71b
Fig. 2 Effects of treatments on oxidative stress parameters in rat
heart tissue. Data are mean + SD of six animals in each group. The
control group received almond oil alone; AlP group received only
aluminum phosphide (LD100); MLT 50 group received only mela-
tonin (50 mg/kg); AlP + MLT 20 group received AlP + melatonin
(20 mg/kg); AlP + MLT 30 group received AlP + melatonin (30 mg/
kg); AlP + MLT 40 group received AlP + melatonin (40 mg/kg);
AlP + MLT 50 received AlP + melatonin (50 mg/kg). a Significantly
different from control groups at p < 0.05. b Significantly different
from AlP group at p < 0.05
Arch Toxicol
1 3
Administration of melatonin at doses of 30 and 40 mg/kg
significantly reduced the activity of caspase 3, while caspase
9 was inhibited at the doses of 40 and 50 mg/kg (Fig. 3).
Cardiac biomarkers (cTnI and CK‑MB)
The animals in the AlP group showed an increase in
CK-MB activity, compared to control group (p < 0.05).
Melatonin treatment at doses of 30, 40 and 50 mg/kg
diminished the increased activity of this enzyme. Moreo-
ver, the levels of troponin I increased in the AlP group and
were then declined following the administration of mela-
tonin at doses of 30, 40 and 50 mg/kg (Fig. 4).
Discussion
The present study was carried out to examine the cardio-
protective effects of melatonin in acute AlP poisoning and
the underlying mechanisms. The potential of melatonin
prevents oxidative damage and apoptosis and to restore
the activities of mitochondrial enzymes and cellular ATP
storage makes it a potential candidate to reverse the toxic
effects induced by AlP in the heart. Research has shown
that AlP-induced alterations in the functions of cardiovas-
cular system such as severe hypotension and decreased
HR account for the high rates of mortality in AlP-poisoned
patients (Moghadamnia 2012). A key finding of the present
study was that melatonin can halt the progressive drop in
the BP of AlP-poisoned rats without exerting any signifi-
cant changes in the BP of healthy controls.
In a previous study on the effects of melatonin on noc-
turnal BP, the administration of melatonin versus placebo
did not induce any significant changes (Laudon and Zis-
apel 2011). It has been shown that the decreased BP as
a result of melatonin administration is associated with a
vasodilatation induced by melatonin in a receptor-inde-
pendent manner. Controversial results have been reported
in many of the studies conducted so far indicating that the
activation of melatonin receptors causes reduced cAMP
levels and induces phosphatidylinositol-4,5-bisphosphate
hydrolysis which consequently leads to vasoconstric-
tion (Paulis and Simko 2007). However, it is also shown
that the activation of endothelial MT2 receptors increases
intracellular Ca2+ in these cells (Pogan et al. 2002).
Fig. 3 Effects of treatments on caspase-3 and -9 activities in rat
heart tissue. Data are mean + SD of six animals in each group. The
control group received almond oil alone; AlP group received only
aluminum phosphide (LD100); MLT 50 group received only mela-
tonin (50 mg/kg); AlP + MLT 20 group received AlP + melatonin
(20 mg/kg); AlP + MLT 30 group received AlP + melatonin (30 mg/
kg); AlP + MLT 40 group received AlP + melatonin (40 mg/kg);
AlP + MLT 50 received AlP + melatonin (50 mg/kg). a Significantly
different from control groups at p < 0.05. b Significantly different
from AlP group at p < 0.05
Fig. 4 Effects of treatments on cardiac biomarkers. Data are
mean + SD of six animals in each group. The control group
received almond oil alone; AlP group received only aluminum phos-
phide (LD100); MLT 50 group received only melatonin (50 mg/
kg); AlP + MLT 20 group received AlP + melatonin (20 mg/
kg); AlP + MLT 30 group received AlP + melatonin (30 mg/
kg); AlP + MLT 40 group received AlP + melatonin (40 mg/kg);
AlP + MLT 50 received AlP + melatonin (50 mg/kg). a Significantly
different from control groups at p < 0.05. b Significantly different
from AlP group at p < 0.05
Arch Toxicol
1 3
Treatment of the rats with 50 mg/kg of melatonin
induced no significant changes in their BP. These results
indicate that melatonin has no effect on BP at its basal
levels during healthy state. This is while previous stud-
ies have shown that melatonin decreases BP of humans
and animal models at hypertensive state (Simko and Pau-
lis 2007). It can be inferred that this substance regulates
BP via a modulatory mechanism rather than producing
a constant hypotensive effect. Our findings confirm the
modulatory role of melatonin in BP regulation since mel-
atonin administration to the AlP-treated rats prevented
the severe drop in their BP. Moreover, melatonin pre-
vented AlP-induced bradycardia and improved the func-
tion of cardiomyocytes through its antioxidant and antiar-
rhythmic actions.
As mentioned earlier, AlP induces several ECG abnormal-
ities such as prolongation of QTc, ST alterations, and QRS
widening (Baghaei et al. 2014). ST-segment represents the
end of ventricular depolarization and the initiation of repo-
larization and is shown to be either elevated or depressed
by AlP administration, signifying myocardial and pericar-
dial damage (Shah et al. 2009; Soltaninejad et al. 2012). ST
elevation is correlated closely with mortality rate of acute
AlP poisoning and its improvement can be considered as
an indicator of therapeutic success in ameliorating AlP poi-
soning (Karami-Mohajeri et al. 2013). In the present study,
fAlP poisoning was followed by ST elevation which showed
a significant reduction in response to melatonin treatment.
Melatonin also alleviated the QRS widening observed as a
result of AlP toxicity. Prolonged QTc was another noticeable
change in the ECG of AlP-treated rats which was mitigated
by melatonin administration as well. Melatonin might have
exerted its protective roles at least partially via the modula-
tion of exaggerated production of oxidative and nitrosative
stress markers and alleviating the electrophysiologic dys-
function in the heart (Bertuglia and Reiter 2007; Sahna et al.
2002). Melatonin has been shown to decrease vasoconstric-
tion and the permeability of vessels and leukocyte adhesion.
This is while it also has the ability to enhance capillary per-
fusion in ischemia/reperfusion models. Reducing the inci-
dence of ventricular tachycardia as well as complete removal
of ventricular fibrillation is among other effects of melatonin
in the heart (Bertuglia and Reiter 2007).
It is shown that AlP can cause considerable alterations
in oxidative stress biomarkers. An assessment of MDA
content in different human and animal samples showed
that AlP significantly increases levels of LPO. Moreover,
AlP is shown to increase ROS levels via disruption of the
electron transfer chain (ETC) which leads to the overpro-
duction of free radicals along with some alterations in the
antioxidant mechanisms (Anand et al. 2013; Kariman et al.
2012; Tehrani et al. 2013). One study reported the efficacy
of melatonin in protecting against LPO in many animal
tissues studied under numerous oxidizing conditions; how-
ever, its exact mechanism is not yet clear (García et al.
2014). The results of studies on phospholipid-containing
vesicles demonstrate that melatonin is found near the polar
heads of these molecules in membrane lipid layers (Reiter
et al. 2014). Such specific positioning of melatonin gives
this molecule a higher chance of protecting phospholip-
ids against free radicals. Melatonin can easily penetrate
into subcellular compartments because of its small size
and amphiphilic properties. Among subcellular organelles,
endogenous levels of melatonin are higher in membranes
and the mitochondria even at low blood levels of this
indoleamine (Venegas et al. 2012). It is also found that
melatonin is a potent lipid peroxyl radical (LOO·) scaven-
ger. In the recent years, the melatonin metabolites, c3OHM
and AMK, have been introduced to be highly effective
LOO· scavengers (Marchetti et al. 2011; Mekhloufi et al.
2007). Our results showed that melatonin treatment (40
and 50 mg/kg) can decrease LPO and ROS levels in AlP-
poisoned rats.
Although there is no general agreement on the rela-
tionship between AlP and SOD activity, there are contro-
versial reports indicating that AlP affects SOD activity, an
effect which is responsible for the dismutation of super-
oxide radicals into H2O2. Some studies have reported that
phosphine induces cellular toxicity through the inhibition
of SOD activity and via influencing cellular antioxidant
defense (Ayobola 2012; Mehrpour et al. 2012). However,
others claim that phosphine increases H2O2 levels through
enhancing SOD activity (Anand et al. 2011; Gurjar et al.
2011). Thus, H2O2 overload leads to protein denatura-
tion, lipid peroxidation in cell membranes and increased
MDA levels (Anand et al. 2011). It has been also shown
that melatonin contributes to Glutathione (GSH) recycling
and maintaining high GSH/GSSG ratio through facilitating
gene expression of GPX, SOD and catalase, and promoting
de novo synthesis of GSH through stimulating the activity
of γ-glutamyl-cysteine synthase (Escames et al. 2010). Our
findings showed that melatonin administration at doses of
40 and 50 mg/kg increases the activities of GPX and SOD.
Studies show that complex IV (cytochrome c oxidase) is
the primary site where phosphine interferes with ETC and
dysregulates the ATP levels and energy requirements of the
cell (Dua et al. 2010; Nath et al. 2011; Singh et al. 2006).
Phosphine seems to interact with any enzyme and mac-
romolecule containing heme groups and is a nonspecific
cytochrome inhibitor. Through decreasing heme structure
in hemoglobin, it induces methemoglobinemia (Anand
et al. 2012; Lall et al. 2000; Shadnia et al. 2011). Despite
the results of some previous studies, our finding did not
show any significant reduction in the activity of complex
II. However, complex I and IV activities were significantly
reduced in heart tissue of AlP-poisoned rats. Interestingly,
Arch Toxicol
1 3
such a decrease in the activities of complexes I and IV was
absent in the rats treated with melatonin.
Thanks to its specific molecular characteristics, mela-
tonin is a highly lipophilic molecule and easily crosses cell
membranes to accumulate in subcellular compartments,
especially in the mitochondria. It affects mitochondrial
homeostasis through several mechanisms, finally leading
to improved ETC activity (Leon et al. 2005). In one study,
the protective role of melatonin on mitochondrial and oxi-
dative damage induced by ruthenium red was evaluated.
Melatonin could increase the activities of complexes I and
IV, whereas the activities of complexes II and III remained
unaffected (Martin et al. 2000). Reports have shown that
the antioxidant action of melatonin is not the only respon-
sible mechanism through which melatonin regulates the
activities of the complexes I and IV. A unique feature of
melatonin is that it can enhance electron flow in the ETC
complexes via functioning as an electron donor as well
as an electron acceptor due to its high redox potential
(Paradies et al. 2015).
The apoptotic and necrotic effects of phosphine have
been evaluated in several studies (Anand et al. 2012; Shah
et al. 2009). Electron microscopy of some tissues showed
that AlP exposure induces mitochondrial swelling due to
the release of pro-apoptotic factors such as cytochrome c
from the intermembraneous space which gives rise to the
activation of caspase-3 and 9 (Heusch et al. 2010). Activity
assessment of caspases 3 and 9 in the present study showed
an increase in the activities of these two enzymes, which
was more pronounced in case of caspase 3. This implies
that mechanisms other than mitochondrial involvement are
engaged in AlP toxicity. Our result also showed that the
activity of caspase-9 at doses of 40 and 50 mg/kg and that
of caspase-3 at doses of 30, 40 and 50 mg/kg was reduced
by melatonin. Melatonin also acts as an anti-apoptotic
agent via inhibiting the dimerization/activation of Bax, a
pro-apoptotic protein. It also antagonizes the effect of Bax
at the mitochondrial level via inducing significant re-local-
ization of Bcl-2 (Radogna et al. 2008, 2015). Melatonin,
due to its structural features, inhibits mitochondrial perme-
ability transition pores and is accumulated in the mitochon-
dria (Andrabi et al. 2004).
There are a number of diagnostic markers which are
released into the blood stream following myocardial infarc-
tion (Patel et al. 2010). Enzymes are among the best of
these markers which are desirable indicators of heart tis-
sue damage mainly due to their hight tissue specific-
ity. CK-MB is a specific enzyme abundantly found in the
myocardium and is widely used as a diagnostic marker.
Its usage as an early indicator of myocardial damage has
made this enzyme an important diagnostic marker (Farvin
et al. 2004). CK-MB levels were dramatically increased
in the AlP-treated rats, while melatonin administration
significantly decreased the activity of this enzyme. It was
concluded that melatonin might have inhibited the leakage
of CK-MB via maintaining the integrity of cellular plasma
membranes.
Although it is claimed that troponin I (cTnI) is highly
sensitive and a specific marker in myocardial cell injury
detection (Acikel et al. 2003), there are scant data confirm-
ing the relationship between the observed increases in cTnI
and histological findings in myocardial tissue following
AlP-induced cardiotoxicity in rats. According to the find-
ings of the current study, it was revealed that AlP exposure
increased troponin I levels in the heart tissue. Also, cardiac
troponin I levels significantly decreased as a result of mela-
tonin administration.
Based on the valuable properties of melatonin, it can be
potentially used for the management of AlP-induced clini-
cal manifestations via reversing the molecular mechanisms
of its toxicity. Commonly, AlP-poisoned patients are pre-
sented with anxiety and agitation. AlP intake by humans
can cause epigastric pain (Moghadamnia 2012). It has been
revealed that melatonin can play an antinociceptive role in
a variety of experimental animal models and in humans;
therefore, it is concluded that melatonin and its analogs
may be also applied in pain management. Despite the lack
of evidence on the effectiveness of melatonin as a premedi-
cation, it has been used as an alternative to midazolam in
adults and children (Marseglia et al. 2015). According to
the available data, there is a possibility of using melatonin
and its analogs as anesthetic agents or as adjuvants to com-
mon anaesthetics. It is worth mentioning that these proper-
ties of melatonin have been exploited in diagnostic situa-
tions requiring sedation. In another study, using melatonin
as a premedication prevented postoperative early agitation
in children after sevoflurane anesthesia (Özcengiz et al.
2011).
Although melatonin administration resulted in pro-
longed survival of the treated animals in comparison to
the untreated controls, death was the ultimate outcome
in all groups at various time points following melatonin
administration. One reason that might justify this is the
single administration of melatonin which might interfere
with the acute cardiotoxicity of AlP but not with its more
long-lasting effects due to its limited half-life. It is also
possible that the toxicity of AlP in other organs such as
the kidney and liver is the cause of death, since the acute
toxicity of AlP in the heart had been already blocked by
melatonin administration. Further research is warranted
in which multiple doses of melatonin, based on its half-
life, may at least partially clarify the ambiguities herein.
The administration of melatonin via systemic infusion
may also aid in sustaining a constant baseline concentra-
tion of this substance, possibly resulting in longer sur-
vival times. Moreover, the use of other protective agents
Arch Toxicol
1 3
which have the potential to counteract the AlP-induced
toxicity in other organs, in combination with melatonin,
may clarify the hypothesis that toxicity in other organs
might have been the cause of eventual death in the
animals.
Conclusion
The protective role of melatonin in various ailments has
been previously shown. The present study was carried
out to evaluate its probable protective effects against alu-
minum phosphide-induced cardiotoxicity and the under-
lying mechanisms. Based on the results, the average sur-
vival time increased following melatonin administration
in AlP-poisoned rats which might be attributable to the
protective effects of melatonin against various mecha-
nisms underlying cardiotoxicity of this agent. Electro-
cardiographic abnormalities and mitochondrial dys-
function including oxidative stress, ATP depletion and
apoptosis were observed following AlP poisoning. Mela-
tonin proved helpful in ameliorating these changes and
showed therapeutic effects by inhibiting mainstream
pathways of oxidative stress and cell injury. Our results,
along with the safe history of melatonin administration,
invite for further assessment of its therapeutic effects in
clinical settings of AlP poisoning.
Acknowledgements This study was in part supported by a Grant
from TUMS coded 94-04-33-30892. The authors wish to thank INSF.
Author contributions MA gave the idea, AAM, and SNO were
advisors; MHA did the study and participated in the literature search
and drafted the article; MM participated in drafting and editing the
article. MB, AJ, MR, HH, SH AB, and RS helped in performing the
experimental part of the study. All authors were involved in data anal-
ysis and interpretation. MA supervised whole study. All authors read
and approved the final version.
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts of
interest
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