The Effects of Biopesticide on the Antioxidant Enzyme Activities of Lemna minor
Muhammed Atamanalp1, Gonca Alak1*, Özden Fakıoğlu2, Arzu Uçar1 and Veysel Parlak2
1Department of Aquaculture, Fisheries Faculty, Atatürk University, Turkey
2Department of Basic Science, Fisheries Faculty, Atatürk University, Turkey
Submission: February 07, 2019; Published: February 22, 2019
*Correspondence author: Gonca Alak, Department of Aquaculture, Atatürk University, Turkey
How to cite this article:Muhammed Atamanalp, Gonca Alak, Özden Fakıoğlu, Arzu Uçar, Veysel Parlak. The Effects of Biopesticide on the Antioxidant
Enzyme Activities of Lemna Minor. Oceanogr Fish Open Access J. 2019; 9(3): 555761. DOI: 10.19080/OFOAJ.2019.09.555761
In this study, Lemna minor which were identified as suitable plant material for ecotoxicological investigations in recent years, was exposed to different levels of bio pesticides (40-80-120 μl 100 ml), for 21 days. Treatment and control groups’ plants antioxidant enzyme activities glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT), glucose-6-phosphate dehydrogenase (G6PD) glutathione reductase (GR) glutathione-S-transferase (GST)] and malondialdehyde level (MDA) were analysed weekly (0., 7., 14, and 21. day) at during the research. The obtained data indicated that the administration doses caused changes on the antioxidant enzyme activities and even the induction effect for some enzymes (p <0.05). The inducing effects of concentrations on antioxidant enzyme activity differed from group to group. It has also been observed with low MDA levels in the subject groups that do not cause oxidative damage
It has become inevitable to use pesticides in order to get more efficiency from the unit area in agricultural activities all over the world. Initially, the use of these chemicals was welcomed and later revealed that these compounds had many negative effects on the environment and human health. Pesticides can stay around for years without degradation. Pesticides that are once transmitted to the ecosystem, cause accumulation through the aquatic nutrient chain and this accumulation is increasing in the upper levels of the food chain . The use of pesticides causes serious problems on human health and environment. Pesticides effect the organism by blocking cellular processes and causing changes in the cell. Some pesticides’ residual effect lasts for days, weeks or months. These effects of the pesticides vary according to the chemical properties of the compound, the amount of concentration, temperature, humidity, pH and microorganism activities.
Increased concern about the environment and health, high consciousness on the harms of synthetic chemicals has led to intensification of studies on natural control methods of harmful livings. Biopesticides are groups of pesticides that have reduced risks compared to synthetic pesticides. Effecting in narrow area, slow moving ability and special effect shapes are the advantages of these. They are preferred since they do not make residue,
remove the harmful effect without destroying the harmful living, and affect only the target harmful, limited resistant, economic, and friendly to environment and health.
Among these pesticides, azadiracthin is a natural insecticide widely used today which obtained by drying and powdering Azadirachta indica tree leaves . Duckweed (Lemna minor) has been identified as a suitable plant material for ecotoxicological investigations in recent years [3-7]. Lemna absorbs its nutrients, ammonium and phosphate forms, from roots. Ammonium is an important source for duckweed. Duckweed is preferred because of its rapid growth rate, low fiber and high protein content. It is also used to increase water quality in different water reserves. Obermeier et al.  reported that Lemna sp. might be used for phytoremediation of low-level contamination with metals and organic xenobiotics. But some authors recommend a more detailed analysis of the development of the oxidative burst following copper exposure and of the enzymatic metabolism of pethoxamide in order to elucidate the extent of its removal from water. Under abiotic stress conditions, highly toxic and reactive molecules called reactive oxygen species (ROS) are formed in plants. These molecules disrupt the structure of proteins, lipid carbohydrates and DNA, leading to the formation of oxidative stress. The plants have antioxidant defense systems to prevent this damage, this study was planned and conducted to investigate the effects of azadirachtin on antioxidant defense
systems of biopesticides on duckweed (L. minor).
The duckweeds are found intensive in the basins of the Pulur,
Karasu and Tortum streams in the Erzurum city borders. The
plants are collected fresh from these areas and transferred to
the Atatürk University Faculty of Fisheries. Plant adaptation was
provided in suitable aquariums at Algae Unit of faculty.
The adapted for 4 weeks duckweeds were divided into four
groups, a control group and 3 different doses of biopesticide.
Three different concentrations of 40-80-120 μl of stock solution
were applied to 100 ml test medium. Weekly (7, 14. and 21.
days) samples were made during the 21-day trial
The samples were placed in a porcelain mortar and
thoroughly crushed with a hammer, and a small amount of liquid
nitrogen was added and obtained fine powder. Then KH2PO4
buffer solution was added to the samples, the homogenates were
centrifuged at 15.000g at 15 minute 4 ° C and removed from the
centrifuge tube . After centrifugation, the supernatant fraction
was obtained, and enzyme activities were directly measured via
Nicotinamidadenin dinucleotidephosphate (NADP+) is
reduced by glucose 6-phosphate dehydrogenase in the presence
of glucose 6-phosphate. The formation rate of NADPH is
dependent on glucose 6-phosphate dehydrogenase activity and
can be measured by an increase in absorbance at 340 nm .
The absorbance reductions at 340 nm of the NADPH
concentration, which is active at the conversion of GSH-Pxcatalyzed
GSSG to glutathione reductase (GSSG-Rd) catalyzed
GSH conversion, are monitored for 2 minutes .
This enzyme catalyzes the reaction of the glutathione group
with CDNB. It gives absorbance at 340 nm and is calculated by
optical density change . Protein levels of each sample were
determined bovine serum albumin (BSA) as the standard .
The data obtained from the enzyme activity measurements
are given as mean ± standard deviation. (n=9). The findings
were subjected to analysis of variance (ANOVA) and averages
were compared using Duncan’s multiple comparison test. The
importance level is taken as 0.05
a, b: There is no statistical difference between the averages, indicated by the same letter in the same column. *p<0.05, NS: Not Significant, Specific
enzyme activities EU/mg protein, MDA is calculated as nmol/ml
Antioxidant enzyme activities and MDA levels of treatment
groups at the end of chronic treatment are given in Table 1. In
the present study, different values were determined for all the
treatment groups in terms of enzyme activity compared to the
control. Different concentration applications showed an inducing
effect on antioxidant enzyme activity and not causing oxidative
damage was surveyed with low MDA levels in the corresponding
groups. In terms of all the parameters examined, the statistical
difference between control and treatment groups was found
to be significant (p<0.05). Antioxidant enzymes have a vital
prescription in the regulation of cell balance. Inductions are a
consequence of the response to contaminants, and antioxidant
enzyme activities and lipid peroxidation are important indicators
of cell damage in toxicological studies [2,16-19].
The presence of an oxygen-rich atmosphere ensured
the development of an endogenous antioxidant system that
counteracts reactive oxygen species (ROS) and reactive nitrogen
species (RNS) (20). This reduction of O2 metabolism products is
controlled by the enzymatic (SOD), (CAT) and (GSH-Px) cellular
defense mechanisms [21-23]. Biopesticide application generally
resulted in an increase in duckweed CAT-specific activity and
higher values were obtained than the control. There was a
decrease in CAT activity in the 40 μL concentrations group. It
can be interpreted that these changes may occur in cases where
adaptation cannot be achieved and with high H2O2. It can also be
assumed that the decline in CAT activity is due to the suppression
of protein synthesis by free radicals .
Some enzymes are defined as “catalytically perfect”
or “kinetically excellent”. Examples of such enzymes
include triosephosphate isomerase, carbonic anhydrase,
acetylcholinesterase, catalase, b-lactamase and superoxide
dismutase. When the production of O2
- anion is high in the cells,
the induction of the SOD enzyme takes place and the O2
is converted to H2O2. That’s why; the increase in SOD activity
is a result of increased O2
- production. Because of the catalytic
activity of many enzymes, such as dehydrogenases and oxidases,
and the oxidation of many biomolecules in thiols that take place
in aerobic environments, O2
- anion is formed . Glutathione
peroxidase transfers the electrons of GSH used as a substrate in
the presence of H2O2 to H2O2 and catalyzes the oxidation of GSH
to GSSG. GPx activity increased in treatment groups compared to
control. With this increase, we are thinking that oxidative stress
may be induced by increasing GSSG amount, GSH / GSSG ratio
and GPx activity. Again, in order to increase the oxidative toxicity
resistance, it can be said that the increase in the enzyme activity
It is thought that in the Lemna minor affected by biopesticide,
decreasing GSH level in the first few days with GSH / GSSG
increasing in the next few days may be associated with newly
established GSH balance or other detoxification mechanisms. It
is estimated that increases in GSH levels in all time are due to an
adaptive response to oxidative stress. In addition, the inability to
observe significant changes in GR activity in all groups may be
due to extracellular transport of GSSG in place of GSH to inhibit its
cytotoxic effects. The increases in GSH level under the pollutant
effect are explained by the regulation and activations that the
enzymes involved in GSH synthesis can carry out to replace the
GSH level [26,27]. In this study, it is thought that the decrease of
GST in the pesticide-treated groups, comparison with the control
is due to the increase of O2 . High dose administration has
severely reduced GST activity. It is known that this may be due
to changes in antioxidant enzymes according to different species
and tissues in response to oxidative stress .
GSH antioxidant defense mechanism and NADP synthesized
in the pentose phosphate metabolic pathway in which G6PD
and 6PGD coexist are used to produce the enzymes in this
mechanism. The importance of G6PD and 6PGD in metabolism
has been known for many years. GSH antioxidant defense mechanism and NADP synthesized in the pentose phosphate
metabolic pathway in which G6PD and 6PGD coexist are used to
produce the enzymes in this mechanism. Therefore, G6PD and
6PGD are thought to be antioxidant enzymes G6PD and 6PGD
enzymes have been found to decrease significantly in fish which
effected by pollution. This is since these enzymes are the first
enzymes of the pentose phosphate pathway and that the increase
or decrease in these enzymes is not only due to exposure to
pollutants but also because these enzymes are potential targets
of toxic chemicals .
In this study, different concentrations of azadiracthin
resulted in a decrease in G6PD activity. In this process,
biopesticides cause O2 production and inhibition in enzyme
activity . When lipid hydroperoxides resulting from lipid
peroxidation break down, most biologically active aldehydes
form. These compounds are either metabolized at the cell level
or diffuse from the baseline domains and radiate damage to
other parts of the cell. Malondialdehyde (MDA), measurable with
thiobarbutyric acid, is used to determine these damages. MDA
is not a specific or quantitative indicator of fatty acid oxidation
but correlates well with the degree of lipid peroxidation [2,18].
It has reported that the enzyme GSH-Px inhibits lipid
peroxidation primarily by protecting the damaged cell, peroxypolyanionic
fatty acids, short chain fatty acids, and GSH-Px
enzymes using GSH instead of MDA production into hydroxyl
fatty acids . Based on the results obtained from the study,
it has become necessary to regularly monitor the pollution in
natural water environments, to determine the effects on livings,
to establish a database with these studies, and to determine the
impact levels of pollutants harmful for aquaculture.
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