Fish Physiol Biochem (2019) 45:71–82
https://doi.org/10.1007/s10695-018-0535-8
Antioxidant activities of Moringa oleifera leaf extract
against pendimethalin-induced oxidative stress
and genotoxicity in Nile tilapia, Oreochromis niloticus (L.)
Heba S. Hamed & Yasser S. El-Sayed
Received: 4 April 2018 / Accepted: 27 June 2018 / Published online: 7 July 2018
# Springer Nature B.V. 2018
Abstract To assess the ameliorative effects of Moringa
oleifera (MO) leaf extract on haematological and bio-
chemical changes, liver DNA damage and oxidative
stress biomarkers in Nile tilapia (Oreochromis niloticus)
exposed to a sublethal concentration (0.52 mg/l) of
pendimethalin (PM). Tilapia fish were allocated into
four equal groups in tri-replicates as follows: first group
was the control group, second group was treated with
MO (20 ml/30 l water), third group was exposed to
0.52 mg PM/l and fourth group was exposed to
0.52 mg PM/l and treated with MO leaf extract (20 ml/
30 l water) for 28 days. At the end of this period, blood
and liver tissue samples were collected and haematolog-
ical and biochemical changes, hepatic DNA fragmenta-
tion and oxidative stress biomarkers were analysed.
Pendimethalin caused significant reduction in haemato-
logical profile [White blood cells (WBCs) and red blood
cells (RBCs) counts, haemoglobin (Hb) concentration
and haematocrit (Ht) level]; meanwhile, serum aspartate
aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP), creatinine,
uric acid, glucose, cortisol, cholesterol and lactate
H. S. Hamed (*)
Department of Zoology, Faculty of Women for Arts, Science and
Education, Ain Shams University, Cairo 11757, Egypt
e-mail: heba.salah84@yahoo.com
e-mail: hebasalah84@women.asu.edu.eg
Y. S. El-Sayed
Department of Veterinary Forensic Medicine and Toxicology,
Faculty of Veterinary Medicine, Damanhour University,
Damanhour 22511, Egypt
dehydrogenase (LDH) were significantly increased.
On the other hand, serum total protein, albumin, globu-
lin and acetylcholinesterase (AChE) were decreased.
Significant reduction in hepatic superoxide dismutase
(SOD), catalase (CAT), total antioxidant capacity (TAC)
and glutathione peroxidase (GSH-Px) levels and marked
increments of hepatic malondialdehyde (MDA) and
DNA fragmentation were observed in PM-exposed fish
compared to the control group. The addition of Moringa
oleifera leaf extract into the water could overcome the
negative impacts of pendimethalin and normalise the
examined parameters nearly to the control values.
Moringa oleifera was used for the first time to protect
tilapia fish against PM-induced toxicity. The present
study revealed that Moringa oleifera has potent antiox-
idant and antigenotoxic actions against pendimethalin
toxicity.
Keywords Moringa oleifera . Pendimethalin .
Oreochromis niloticus . Haematology . Biochemistry .
DNA fragmentation . Oxidative stress biomarkers
Introduction
The heavy use of pesticides in areas adjacent to fish
residing in waterways may lead to fish health conse-
quences in these areas (Shelley et al. 2012).
Pendimethalin (PM) (N-(1-ethylpropyl)-3,4-dimethyl-
2,6-dinitrobenzenamine; PD) is a dinitroaniline herbi-
cide which prevents chromosomal separation and
72
formation of cell wall in plants. It had been categorised
as a persistent bioaccumulative toxic (PBT) herbicide
and a group C carcinogen (possible human carcinogen),
contaminating the soil and the aquatic system due to
runoff and leaching (USEPA 1997a, b; Tabassum et al.
2015). However, studies on the effects of pendimethalin
on fish are limited. It may cause neurotoxicity like other
herbicides (Rahman and Thomas 2014). Consequently,
fish must activate several responses to resist these toxic
effects, and these responses alter the fish metabolism,
such as survival skills, growth, reproduction and immu-
nity (Danion et al. 2012). Pendimethalin may also cause
oxidative stress in many fish species, inducing liver
injury to critical cellular molecules, such as lipids and
proteins, and DNA damage (Dai et al. 2012; Tabassum
et al. 2015). In the past few years, interest in medicinal
plants has been increased worldwide (Marques and
Farah 2009) and their active constituents were used
against toxicity induced by diverse chemicals, drugs
and xenobiotics (Abdou and Abdel-Daim 2014).
Moringa oleifera (MO) belongs to the family of
Moringaceae, and it is widely distributed throughout
the world, especially in Asian and African countries
(Moyo et al. 2011; Fakurazi et al. 2012). Roots, fruits,
leaves and flowers of MO are used as vegetables
(Siddhuraju and Becker 2003). Phytochemical studies
have shown that its leaves are mainly rich in potassium,
calcium, phosphorous, iron, vitamins A and D, β-caro-
tene, flavonoids, vitamin C, vitamin E, polyphenol ox-
idase, ascorbic acid, oxidase and catalase (Yang and
Frenkel 2002; Khatun et al. 2003; Aslam et al. 2005;
Manguro and Lemmen 2007; Amaglo et al. 2010).
Medicinally, leaves of MO are used for the treatment
of many diseases, thus coined as “the miracle tree”.
Many reports documented that MO has antibiotic,
antitrypanosomal, hypotensive, antispasmodic, antiul-
cer, anti-inflammatory, hypocholesterolemic and hy-
poglycemic activities (Anwar et al. 2007; Coppin
et al. 2013). Recent investigations indicated that
MO has hepatoprotective effects (Das et al. 2012;
Sharifudin et al. 2013).
Nile tilapia, Oreochromis niloticus, is one of the most
common freshwater fishes used in toxicological studies
(Garcia-Santos et al. 2006), because of its high growth
rates, easy adaption to commercial diets and resistance
to diseases and injuries (Figueiredo-Fernandes et al.
2006). There are many attempts to use leaf extractions
of medicinal plants to help fish which live in polluted
ecosystems for detoxification and for antioxidant
Fish Physiol Biochem (2019) 45:71–82
protection (Sayed and Hamed 2017), to improve its
growth and health (Hamed and Abdel-Tawwab 2017),
but their use to improve fish resistance against PM
toxicity is limited. Hence, the present investigation
was conducted to assess PM toxic effects on haemato-
logical and biochemical profiles, oxidative stress bio-
markers and hepatic DNA damage of Nile tilapia and to
evaluate the potential activity of Moringa oleifera leaf
extract as an antioxidant and antigenotoxic agent against
PM toxicity on Nile tilapia as a fish model.
Materials and methods
Chemicals
PM used in this study was commercially available in
the product (Stomp® 50% EC, BASF PLC). It is an
orange-yellow emulsive herbicide of dinitroaniline
family, whose active ingredient is pendimethalin
( U S E PA 1 9 9 7 a , b ) . T h e c h e m i c a l n a m e o f
pendimethalin is n-(1-ethylpropyl)-3,4-dimethyl-2,6-
dinitrobenzene amine with empirical formula
C13H19N3O4; water solubility is 0.3 mg/l at 25 °C
and aqueous photolysis of 60 days (USEPA 1997a,
b). Biochemical kits for aspartate aminotransferase
(AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP), creatinine, uric acid, glucose,
cholesterol, lactate dehydrogenase (LDH), total pro-
tein, albumin, superoxide dismutase (SOD), catalase
(CAT), total antioxidant capacity (TAC), glutathione
peroxidase (GSH-Px) and malondialdehyde (MDA)
were purchased from Bio-Diagnostic Co. (Cairo,
Egypt). Cortisol and acetylcholinesterase (AChE) kits
were obtained from Gamma Trade Co. (Cairo, Egypt).
Preparation of MO ethanolic leaf extract
The fresh leaves of MO plant were picked from trees
grown on sandy soil in El-Sharkia Governorate, Egypt.
The collected leaves were purified, washed with dis-
tilled water and dried at room temperature for 3 weeks,
after which the leaves were powdered into a coarse
form. About 1000 g of the coarse form was macerated
in absolute ethanol and was left to stand for 48 h. The
resulting ethanol extract was filtered through muslin
cloth on a plug of glass wool in a glass column, con-
centrated and evaporated to dryness at 45 °C using a
rotary evaporator to avoid denaturation of the active
Fish Physiol Biochem (2019) 45:71–82
ingredients. After that, the concentrated extract was
diluted to 1000 ml using a polysaccharide as a carrier
and stored in the refrigerator (Okechukwu UPC 2013).
Gas chromatography–mass spectrometry analysis
The analysis of the chemical composition of ethanolic
extract of MO leaves was performed using a Trace GC
Ultra-ISQ mass spectrometer (Thermo Scientific, Wal-
tham, MA) with a direct capillary column TG-5MS
(30 m × 0.25 mm × 0.25 mm film thickness). The
column oven temperature was initially held at 40 °C
and then increased by 5 °C/min to 280 °C. The injector
and detector (MS transfer line) temperatures were kept
at 250 °C. Helium was used as a carrier gas at a constant
flow rate of 1 ml/min. Extract derivatisation was done
using BSTFA/TMCS (80/20, v/v) for 1 h at 70 °C, after
evaporation to dryness of dichloromethane/methanol
mixture. The resulting solution was dried and then dis-
solved in hexane. The solvent delay was 2 min, and
diluted samples of 1 ml were injected automatically
using Autosampler AS3000 (Thermo Scientific, Wal-
tham, MA) coupled with GC in the splitless mode. EI
mass spectra were collected at 70 eV ionisation voltages
over the range of m/z 50–650 in full scan mode. The ion
source and quadrupole temperatures were set at 200 and
150 °C, respectively. The components were identified
by comparison of their retention times (RTs) and mass
spectra with those of WILEY 09 (Flavor & Fragrance
Natural & Synthetic Compounds) and NIST 11 (Nation-
al Institute of Standards and Technology, Gaithersburg,
MD) mass spectral databases.
Fish maintenance
A total number of 260 O. niloticus were obtained from
the Central Laboratory of Abbassia Fish Farm, Egypt,
with an average body weight of 60 ± 10 g. Fish were
kept in 80-l glass aquaria containing dechlorinated tap
water under laboratory circumstances (conductivity
2000 l/cm; pH 6.5; oxygen 88–95% saturation; water
temperature 25–26 °C; total hardness 150 mg/l as
CaCO3; photoperiod 12:12 light/dark) for 2 weeks
for acclimatisation. Fish were fed a commercial pellet
diet (3% of body weight per day) and transferred to a
fresh volume of water daily to reduce impurities from
metabolic wastes.
73
Determination of 96-h LC50 of pendimethalin
The 96-h LC50 of pendimethalin-based herbicide
Stomp® 50% EC (PM) in O. niloticus was determined
using the method of Litchfield and Wilcoxon (1949).
Briefly, five groups of eight fish per each group were
exposed to different PM concentrations (0.0, 1.23, 2.46,
4.92, 9.84 and 19.68 mg/l), and it was 5.15 mg/l.
Experimental setup and sampling
Fish were divided into equal four groups, and each
group consisted of three replicates of 13 fish per
aquarium and fed on a commercial pellet diet (3% of
body weight) which was given to fish up to satiation
twice a day at 08:00 and 13:00 for 28 days. The first
group was the control group. The second group was
treated with MO leaf extract (20 ml/30 l water). The
third group was exposed to 0.52 mg PM/l. The fourth
group was exposed to 0.52 mg PM/l and treated with
MO leaf extract (20 ml/30 l water) for 28 days. Every
day, the water of the aquaria was changed with clean
water containing the same PM and MO concentrations
during the experiment period.
Blood sampling
At the end of the experiment, six fish per each group
were collected and anaesthetised with 0.02% benzo-
caine solution. Blood samples were collected from the
caudal vessels of fish. Blood samples were divided
into two parts: part I was performed on the same day
using sodium citrate as an anticoagulant (Falkner and
Houston 1966) for determination of haematological
parameters, and in part II, the collected blood samples
were allowed to clot in clean dry centrifuge tubes at
room temperature and then centrifuged at 3000 rpm at
4 °C for 15 min. Sera were collected for determination
of biochemical parameters.
Haematological examination
WBCs and RBCs were counted by a haemocytometer
according to the method of Kanaeu (1985). Haemoglobin
(Hb) concentrations and haematocrit (Ht) values were
estimated by using the cyanmethaemoglobin method de-
scribed by van Kampen and Zijlstra (1961) and the
microhaematocrit method of Jain (2000), respectively.
74
Biochemical analysis
Serum AST and ALT were estimated according to the
method described by Reitman and Frankel (1957). ALP
was determined using an enzymatic colorimetric meth-
od according to Tietz et al. (1983). Creatinine was
measured according to the method of Larsen (1972).
Uric acid was estimated according to the method de-
scribed by Whitehead et al. (1991). Serum glucose and
cortisol were assessed using the methods described by
Trinder (1969) and Foster and Dunn (1974), respective-
ly. Cholesterol was detected using the method of Allain
et al. (1974). LDH was determined according to the
method described by Vassault (1983). Serum total
protein and albumin were measured using the methods
described by Lowry et al. (1951) and Doumas et al.
(1971), respectively. Serum globulin was determined
by subtracting the albumin value from the total protein
value of the same sample. AChE was determined ac-
cording to Knedel and Böttger (1967).
Liver MDA and antioxidant biomarkers
Samples from liver tissues were homogenised in cold
phosphate buffer saline (0.1 M, pH 7.4) using a Potter-
Elvehjem glass/Teflon homogeniser. After filtration, the
homogenate was centrifuged at 1600 rpm at 4 °C for
10 min. Supernatants were stored at − 20 °C until anal-
ysis. SOD activity was determined using the supernatant
(20%) according to the method described by Nishikimi
et al. (1972). CAT enzyme was estimated according to
the method of Aebi (1984). TAC and GSH-Px levels
were detected using the method of Koracevic et al.
(2001) and Paglia and Valentine (1967), respectively.
MDA levels were determined according to Mihara and
Uchiyama (1978).
Liver DNA fragmentation measurement
DNA fragmentation of hepatic specimens of tilapia
fish was determined according to the method de-
scribed by Kurita-Ochiai et al. (1999) using a spec-
trophotometer (Micro-lab 200; Vital Scientific,
Dieren, The Netherlands) at 575 or 600 nm against
reagent blank. The percentage of fragmented DNA
was assessed by the following formula: % of
fragmented DNA = fragmented DNA / (fragmented
DNA + intact DNA) × 100.
Fish Physiol Biochem (2019) 45:71–82
Statistical analyses
The results were presented as mean ± SE of three repli-
cates. Prior to statistical analysis, all data were tested for
normality of distribution using the Kolmogorov–
Smirnov test. The homogeneity of variances among
different treatments was tested using Bartlett’s test.
Then, data were subjected to two-way ANOVA to eval-
uate effects of PM toxicity and MO leaf extract. Differ-
ences between means were tested at the 5% probability
level using Duncan’s test. All the statistical analyses
were done using the SPSS program (version 20; SPSS,
Richmond, VA, USA) as described by Dytham (1999).
Results
Chemical composition of MO
The gas chromatography–mass spectrometry (GC-MS)
analysis of MO showed that it contains steroids, pheno-
lics, flavonoids, alkaloids, tannins and esters. The iden-
tified bioactive components of MO are listed in Table 1,
with their respective RT and percent composition (area
%), where the most important substances are oleic acid,
3-(octadecyloxy) propyl ester, docosatetraenoic acid,
ethyl iso-allocholate, androst-5,7-dien-3-ol-17-one, vi-
tamin A aldehyde, octadecadiynoic acid and pyrimidin-
2-one. The major identified compound was the oleic
acid, 3-(octadecyloxy) propyl ester (23.10%), while
from total ion chromatogram (TIC), we noted that the
steroid androst-5,7-dien-3-ol-17-one has appeared at
multiple RTs with total percent composition of
17.51%. The chromatogram of the number of peaks of
the compounds detected is shown in Fig. 1.
Haematological parameters
Tilapia fish treated with 0.52 mg PM/l showed a marked
(P < 0.05) decrease in the WBC and RBC count, Hb
concentration and Ht level compared to the control
group (Table 2). Combined treatment with PM and
MO leaf extract (20 ml/30 l water) resulted in a signif-
icant improvement in blood parameters to be near the
normal values (Table 2). Nile tilapia administrated MO
leaf extract exhibited no significant difference in hae-
matological parameters compared to the control.
Fish Physiol Biochem (2019) 45:71–82 75

Table 1 Bioactive components of ethanolic extract of Moringa oleifera (MO) leaves as determined by using gas chromatography–mass
spectrometry (GC-MS)

Number Retention time Compound name MW Molecular formula Area %

1 4.11 1,3,5-Cycloheptatriene 92 C7H8 1.93

2 5.59 p-Xylene 106 C8H10 6.52
3 6.93 Pyrimidin-2-one, 325 C13H19N5O5 1.21
4-[N-methylureido]-1-[4-methylaminocarbonyloxymethyl
4 7.06 Octadecadiynoic acid, methyl ester 290 C19H30O2 2.71
5 7.46 Vitamin A aldehyde 284 C20H28O 0.58
6 24.50 Androst-5,7-dien-3-ol-17-one 286 C19H26O2 17.51
7 24.73 Ethyl iso-allocholate 436 C26H44O5 30.17
8 28.79 6,9,12,15-Docosatetraenoic acid, methyl ester 346 C23H38O2 6.49
9 29.25 Oleic acid, 3-(octadecyloxy) propyl ester 592 C39H76O3 23.10

Biochemical profile
Nile tilapia exposed to PM showed marked increments
in levels of serum liver enzymes (AST, ALT, ALP),
serum renal products, creatinine and uric acid compared
to the control fish (Table 3). Serum glucose, cortisol,
cholesterol and LDH levels were also increased
(P < 0.05) significantly in PM-exposed fish. On the
other hand, significant (P < 0.05) reductions in serum
total protein, albumin, globulin and AChE levels were
observed compared to the control tilapia (Table 3). In
contrary, the addition of MO leaf extract (20 ml/30 l
water) into the water along with PM-exposed fish nor-
malised levels of all the above-mentioned parameters to
be closer to the levels of the control group (Table 3).
Fish of the control group (first group) and those treated
with MO leaf extract (second group) have nearly the
same values of the tested biochemical parameters.
Liver MDA and antioxidant biomarkers
Exposure of tilapia fish to PM caused marked (P < 0.05)
reductions in hepatic SOD, CAT, TAC and GSH-Px
activities, as presented in Table 4, and a significant
(P < 0.05) rise in the level of MDA of the same tissue
compared to the control group (Table 4). Co-
administration of tilapia fish with MO leaf extract (sec-
ond group) showed no significant difference in liver
antioxidant enzyme activities compared to the control
group (first group). Treatment of PM-exposed fish with

Fig. 1 Gas chromatography–

mass spectrometry profile for
Moringa oleifera (MO) ethanolic
leaf extract showing that MO
extract contains oleic acid, 3-
(octadecyloxy) propyl ester
(23.10%), docosatetraenoic acid
(6.49%), ethyl iso-allocholate
(30.17%), androst-5,7-dien-3-ol-
17-one (17.51%), vitamin A
aldehyde (0.58%),
octadecadiynoic acid (2.71%) and
pyrimidin-2-one (1.21%)
76 Fish Physiol Biochem (2019) 45:71–82

Table 2 Changes in the haematological parameters (mean ± SD) of Nile tilapia (O. niloticus) exposed to (0.52 mg/l) PM with and without
MO leaf extract administration for 28 days

Parameters Experimental groups

1st group 2nd group 3rd group 4th group

WBCs (103/mm3) 8.08 ± 0.38b 8.50 ± 0.40a 5.14 ± 0.23c 7.70 ± 0.20b
6 3 a a c
RBCs (10 /mm ) 2.30 ± 0.21 2.50 ± 0.22 1.38 ± 0.19 1.98 ± 0.20b
Hb (g/dl) 7.16 ± 0.15a 7.01 ± 0.30a 4.26 ± 0.07b 6.92 ± 0.13a
b a c
Ht (%) 34.48 ± 0.53 35.70 ± 0.67 22.63 ± 0.79 33.79 ± 0.91b

Means with different superscript letters in the same row for each parameter are significantly different (P < 0.05)

MO leaf extract resulted in a significant improvement in Discussion

liver oxidative stress biomarkers.
Liver DNA fragmentation
Data presented in Fig. 2 revealed that PM-exposed
fish experienced a significant (P < 0.05) increase in
the percentage of liver DNA fragmentation com-
pared to the controls. In contrast, combined treat-
ment with PM and MO leaf extract (20 ml/30 l
water) provided a remarkable decrease in the per-
centage of hepatic DNA fragmentation compared to
the PM-intoxicated group (Fig. 2).
The aquatic environment is continuously being con-
taminated with different pesticides from agriculture
activities (Kadry et al. 2012). Rezende et al. (2018)
postulated that the alterations in routine metabolism of
Nile tilapia exposed to titanium dioxide nanoparticles
(TiO2 NPs) may be related to stress which are useful
for biomonitoring the toxic effects of xenobiotics. The
96-h LC50 value of PM for O. niloticus was 5.15 mg/l.
The results are verified by El-Sayed et al. (2015) who
recorded that the 96-h LC50 for monosex Nile tilapia
was 4.92 mg/l. In contrast to the aforementioned
values, the 96-h LC50 values of PM for rainbow trout,

Table 3 Changes in the biochemical parameters (mean ± SD) of Nile tilapia (O. niloticus) exposed to (0.52 mg/l) PM with and without MO
leaf extract administration for 28 days

Parameters Experimental groups

1st group 2nd group 3rd group 4th group

AST (U/l) 51.71 ± 0.44c 52.03 ± 1.02bc 64.25 ± 1.09a 53.09 ± 0.43b
b b a
ALT (U/l) 46.28 ± 0.31 46.87 ± 0.66 58.73 ± 0.80 44.94 ± 0.37c
c b a
ALP (U/l) 7.23 ± 0.17 7.58 ± 0.28 11.90 ± 0.34 7.03 ± 0.09c
c c a
Creatinine (mg/dl) 0.56 ± 0.06 0.60 ± 0.05 1.17 ± 0.02 0.71 ± 0.01b
Uric acid (mg/dl) 11.14 ± 0.43b 10.96 ± 0.62bc 14.91 ± 0.06a 10.58 ± 0.23c
c c a
Glucose (mg/dl) 48.56 ± 0.31 49.34 ± 0.76 78.04 ± 1.54 51.81 ± 1.78b
c c a
Cortisol (μg/dl) 4.23 ± 0.31 4.18 ± 0.06 10.06 ± 0.13 6.28 ± 0.12b
b b a
Cholesterol (mg/dl) 146.89 ± .3.94 145.86 ± 2.77 180.60 ± 1.48 140.01 ± 1.18c
b b a
LDH (U/l) 26.19 ± 1.48 26.12 ± 0.89 39.20 ± 0.91 24.09 ± 0.62c
Total protein (g/dl) 5.92 ± 0.08ab 6.10 ± 0.14a 4.30 ± 0.14b 5.76 ± 0.05ab
a a c
Albumin (g/dl) 3.60 ± 0.02 3.52 ± 0.04 2.37 ± 0.05 3.27 ± 0.11b
b a c
Globulin (g/dl) 2.33 ± 0.09 2.57 ± 0.12 1.93 ± 0.11 2.50 ± 0.15a
a a b
AChE (U/l) 447.60 ± 4.16 450.20 ± 1.92 387.20 ± 4.76 449.80 ± 5.81a

Means with different superscript letters in the same row for each parameter are significantly different (P < 0.05)
Fish Physiol Biochem (2019) 45:71–82 77

Table 4 Changes in liver antioxidant enzymes and malondialdehyde (MDA) levels (mean ± SD) of Nile tilapia (O. niloticus) exposed to
(0.52 mg/l) PM with and without MO leaf extract administration for 28 days

Parameters Experimental groups

1st group 2nd group 3rd group 4th group

SOD (μg/mg protein) 35.68 ± 0.42b 37.18 ± 0.33a 28.14 ± 0.47c 35.32 ± 0.51b
b a c
CAT (μg/mg protein) 22.39 ± 0.31 23.19 ± 0.60 15.99 ± 0.34 22.30 ± 0.54b
TAC (μmol/mg protein) 41.91 ± 0.17b 42.30 ± 0.30b 31.04 ± 0.27c 48.30 ± 1.01a
a a c
GSH-Px (nmol/g protein) 16.06 ± 0.53 16.52 ± 0.80 10.83 ± 0.13 14.93 ± 0.80b
b b a
MDA (nmol/g protein) 19.75 ± 0.40 20.10 ± 0.14 32.66 ± 0.71 18.93 ± 0.25c

Means with different superscript letters in the same row for each parameter are significantly different (P < 0.05)

bluegill sunfish and channel catfish were 0.52, 0.92
and 1.9 mg/l, respectively, as documented by Munn
et al. (2006) and USEPA (1997a, b). The difference in
PM LC50 values among different fish species may be
attributed to the difference in the absorption of PM
herbicide and its accumulation, biotransformation and
excretion (Omitoyin 2006). Blood is a pathophysio-
logical reflector of the whole body, and therefore,
blood parameters are important in diagnosing the
structural and functional status of fish exposed to
toxicants (Adhikari et al. 2004; El-Sayed et al. 2007;
El-Sayed and Saad 2008). Leucocytes play an impor-
tant role in the non-specific or innate immunity, which
indicate the health status of fishes (Secombes 1996;
Ural 2013). The present study indicates that the leu-
cocyte count was significantly decreased in PM-
exposed fish. This finding agrees with the previous
investigations of Marzouk et al. (2012) and Velisek
et al. (2010) who observed significant reduction in
leucocytes of female African catfish and common carp
exposed to different herbicides, respectively. The
reduction in white blood cell count indicates the
malfunctioning of the haematopoietic system of fish
caused by herbicide toxicity (Ellis 1981; Marzouk
et al. 2012). RBC count, Hb concentrations and Ht
values were also reduced significantly in PM-exposed
fish. Bosisio et al. (2017) observed a marked decrease
in erythrocyte count and haemoglobin concentrations
of Nile tilapia submitted to different salinities.
Corroborating this study, Marzouk et al. (2012) re-
corded significant reductions in RBC count, Hb con-
centration and Ht levels of female African catfish
exposed to atrazine, which could be attributed to the
inhibition of erythropoiesis, haemosynthesis or in-
creasing the rate of erythrocyte destruction in the
haematopoietic organ, which gives an indicator of
anaemia (Vani et al. 2011). Treatment with MO
(20 ml/30 l water) enhanced the haematological pa-
rameters of PM-exposed fish. Hence, leaves of MO
exhibited a positive relationship in the improvement
of anaemia (Eshak and Osman 2013). Moreover, the
phenolic compounds in leaves of MO can bind to

Fig. 2 Changes in the percentage 35

of liver DNA fragmentation of
Nile tilapia (O. niloticus) exposed 30
to (0.52 mg/l) PM with and
without MO leaf extract 25
administration for 28 days
1st group
20
2nd group
15 3rd group
4th group
10

0
DNA %
78
the erythrocyte membranes, giving these mem-
branes protection against the damage resulting
from PM toxicity. Our findings suggest that MO
has an antihaemolytic effect in tilapia fish towards
PM exposure.
The studied biochemical parameters of fish are
sensitive indicators of early changes due to the haz-
ardous exposure to pesticides (Bakhshwan et al.
2009). Liver ALT, AST and ALP enzymes are com-
monly used in the diagnosis of fish diseases caused by
environmental pollution (Firat et al. 2011). Therefore,
they are considered as relevant stress indicators. The
activity of liver enzymes showed significant incre-
ments in PM-exposed fish compared to the controls.
Similar results were verified in Nile tilapia (El-
Sharkawy et al. 2011) and African catfish (Hamed
2016) exposed to PM and deltamethrin, respectively.
These increases in the bloodstream are usually due to
the hepatic potency of pesticide toxicity resulting in
cellular damage and necrosis in the liver tissues
(Hamed and Osman 2017; Harvey et al. 1994). Inter-
estingly, using MO leaf extract against PM toxicity
attenuated serum liver activities, which indicated that
MO possesses hepatoprotective properties. These
findings are consistent with Hamza (2010) who stated
that MO extract can act against CCl4-induced liver
damage. Likewise, the increased values of creatinine
and uric acid in the experimental fish group exposed to
PM may be due to kidney dysfunction, which leads to
reduced renal blood flow with a reduction in glomer-
ular filtration rate and a decrease in creatinine and uric
acid excretion resulting in azotemia (Chang 1996).
Results are in conformity with those reported by
Hamed and Osman (2017) who recorded a marked
increase in serum creatinine and uric acid levels, sug-
gesting nephrotoxicity of African catfish exposed to
deltamethrin. Moringa oleifera administration to PM-
exposed fish (fourth group) for 28 days normalises the
levels of serum renal biomarkers.
Glucose and cortisol are commonly used as indica-
tors of stress in fish due to the pollution and the physical
factors (Authman et al. 2013; Hamed and Osman 2017;
Harabawy and Ibrahim 2014). In the current study,
levels of glucose and cortisol were significantly in-
creased in the blood of PM-exposed fish to enhance
gluconeogenesis response of stressed fish as an attempt
to satisfy its energy demands (Winkaler et al. 2007;
Hamed and Osman 2017). This finding is corroborated
by the previous reports of El-Sayed et al. (2015), El-
Fish Physiol Biochem (2019) 45:71–82
Sharkawy et al. (2011), Hamed (2015a, b) and Marzouk
et al. (2012). Simultaneous treatment with MO (20 ml/
30 l water) resulted in a significant decrease in glucose
and cortisol levels.
At the same time, cholesterol level increased
(P < 0.05) significantly in PM-exposed fish. The ob-
served hypercholesteraemia could be explained by the
changes in the permeability of hepatic cells and the
disruption of lipid metabolism because of the accumu-
lation of pesticide in the liver (Yousef et al. 2003).
Increased cholesterol levels have been reported in Nile
tilapia exposed to cypermethrin (El-Sharkawy et al.
2011) and African catfish exposed to 4-nonyl phenol
(Sayed and Hamed 2017). The addition of MO re-
stored cholesterol to about normal level. This result
is in agreement with those of Mansour et al. (2014)
who reported that the leaf extract of MO significantly
lowered serum cholesterol level in hypercholesterol-
aemic rats. Proteins are considered a good tool for
monitoring the negative effects of environmental
stressors and aquatic pollution on fishes as well as
physiological homeostasis (Saravanan et al. 2011;
Tayel et al. 2007). Tilapia fish treated with PM
showed significant depletion in total protein
(hypoproteinaemia), albumin and globulin levels
compared to the control group. The reduction in
serum total protein may be related to adjustment of
fish to its new environmental circumstances as a
stress response (Hamed 2016). The decrease in the
albumin level may reflect the reduction of the blood
viscosity (Sharaf-Eldeen 2002). Also, the decline in
globulin concentration in the blood of PM-exposed
fish (third group) may be an indication of reduced
immunity in the body since the liver became unable
to synthesise enough globulins for immunological
activities (Sunmonu and Oloyede 2007). The current
study revealed that MO activated hepatoprotective
effects in tilapia fish against the decrease in liver
synthetic function by upregulating serum total pro-
tein and albumin levels as compared to the PM-
exposed fish group.
In the present investigation, the exposure of tilapia
fish to 0.52 mg PM/l exhibited a significant decline in
the activity of AChE enzyme. The results are in ac-
cordance with those observed in common carp ex-
posed to carbofuran (Clasen et al. 2014) and Nile
tilapia exposed to malathion (Hamed 2015b). The
inhibition of AChE enzyme could be attributed to
oxidative stress generated by pesticide exposure
Fish Physiol Biochem (2019) 45:71–82
(Salbego et al. 2010). On the other side, treatment of
PM-exposed fish with MO (20 ml/30 l water) resulted
in a significant enhancement in AChE level.
The antioxidant profile of PM-exposed fish changed
through a decrease in hepatic SOD, CAT, TAC and
GSH-Px activities of PM-exposed fish as compared to
that in control fish. Similar results were verified in the
tilapia fish liver (Abdelkhalek et al. 2015) and African
catfish liver (Hamed 2016) after exposure to deltameth-
rin, respectively. Depletion in hepatic antioxidant en-
zymes could be attributed to the excess of free radicals
produced through the detoxification process of pollut-
ants (Monteiro et al. 2006) which make the fish cells
more vulnerable to oxidative damage (Banaee et al.
2013). Moreover, the current study indicated a signifi-
cant increment in the hepatic MDA level of PM-
exposed fish. A similar pattern of increases in the
MDA levels was also recorded in the hepatic tissue of
African catfish exposed to atrazine (Kadry et al. 2012)
and tilapia fish exposed to pendimethalin (El-Sayed
et al. 2015). The increment in MDA level could be
attributed to excess production of ROS which attack
the cell membrane, as a result of the impairment in
antioxidant enzymes (Choi et al. 2014; Hamed 2015b).
In this respect, the administration of MO leaf extract
(20 ml/30 l water) along with PM-exposed fish amelio-
rated the reductions in hepatic SOD, CAT, TAC and
GSH-Px activities and restored the MDA level to nor-
mal levels. Hence, this investigation indicates the anti-
oxidant property of MO which could be possibly the
primary mechanism of protection against PM toxicity.
The antioxidant property of MO may be due to the
presence of antioxidant compounds that was confirmed
in this study, such as vitamin A, which quench ROS,
chelate metal ions and regenerate membrane-bound an-
tioxidants. These findings are matched with previous
reports of Arabshahi et al. (2007), Ashok Kumar and
Pari (2003) and Hamza (2010). The present study ex-
hibited that PM toxicity resulted in a significant increase
in the percentage of hepatic DNA fragmentation and
indicated that PM generates free radicals and enhances
lipid peroxidation in the liver tissues, which linked to
the genotoxicity expressed by DNA adduct formation
and to the disturbance of calcium homeostasis due to an
impairment of the endoplasmic reticulum membrane
(Abdel-Wahhab et al. 2016; Pfohl-Leszkowicz et al.
1998). The current work confirms that oxidative stress
is an important factor in genotoxicity (Abdel-Wahhab
et al. 2016; Sayed and Hamed 2017). Interestingly, the
79
biological mechanism of MO to reduce genotoxicity of
PM-exposed fish is mainly attributed to the antioxidants
existing in its leaves, suggesting that MO has an
antigenotoxic effect.
Conclusion
The findings of this experiment showed that Moringa
oleifera leaf extract alleviates the harmful impacts of
PM on Nile tilapia. Also, this investigation confirmed
the antioxidant role and the antigenotoxic role of
Moringa oleifera to reinstate the haematological and
biochemical variables, antioxidant imbalance and liver
DNA damage induced by PM toxicity.
Acknowledgements The authors wish to thank Dr. Mamoun
Abd El-Kareem of the Atomic and Molecular Physics Unit, De-
partment of Experimental Nuclear Physics, Nuclear Research
Centre, Egyptian Atomic Energy Authority, Inshas, Cairo, Egypt,
for his kind helpful for the GC-MS analysis and interpretations.
Compliance with ethical standards
Conflict of interest The authors declare that they have no con-
flict of interest.
Ethical statement Experimental design and fish handling were
approved by the Research Ethical Committee of the Faculty of
Women for Arts, Science and Education, Ain Shams University,
Cairo, Egypt.
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