Effect of Vitamins A, E and C on Liver Enzyme Activity in Rats Exposed to
Organophosphate Pesticide Diazinon
Sakineh Sadat Hosseini Payam
Diazinon, a commonly used organophosphorus pesticide, has been widely used throughout the world in agriculture and horticulture to control insects that feed on crops, ornamentals, lawns, fruits, vegetables and other food products. The toxicity of the DZN causes adverse effects on many organs. The purpose of this study was to examine the protective effect of vitamins A, E and C on liver enzymes alanine transaminase (ALT), Aspartate aminotransferase (AST) and Lactate Dehydrogenase (LDH) in rats exposed to diazinon. In this study, male wistar rats were randomly divided into 10 different groups. The groups were administered normal saline, soybean oil (as the solvent for diazinon and fat-soluble vitamins), diazinon, (30 mg kg-1), vitamins E, C and A (100, 500 mg kg-1 and 400 IU kg-1, respectively) and a combination of diazinon with the same dose of each vitamin intraperitoneally i.p.daily for 14 days. Seven days after the final injection, the animals were anesthetized and blood samples were taken. The photometric method was used to measure the activity of the enzymes. The activities of ALT and AST in the diazinon group were significantly higher than that observed in the control group; however, the diazinon/vitamin E, A, C group displayed significant reduction in ALT and AST activities compared to the diazinon group. The lowest level of LDH enzyme activity was observed in the dazinon/vitamin C group and this was statistically lower than the diazinon group. The results of this study revealed that vitamin E, A and C have a potent protective effect against diazinon-induced hepatotoxicity in rats, which may be due to the scavenging of free radicals and increased antioxidant status.
to cite this article:
Mohammad Shokrzadeh, Sepideh Shobi, Hossein Attar, Sahel Shayegan, Sakineh Sadat Hosseini Payam and Faezeh Ghorbani, 2012. Effect of Vitamins A, E and C on Liver Enzyme Activity in Rats Exposed to
Organophosphate Pesticide Diazinon. Pakistan Journal of Biological Sciences, 15: 936-941.
Received: December 16, 2012;
Accepted: January 18, 2013;
Published: March 04, 2013
Organophosphate pesticides are the most common cause of poisoning in the world.
This group of pesticides is not only used in agricultural fields, gardens and
residential areas but is also used for pest control and the protection of public
health, as well as in industry and veterinary medicine (Fattahi
et al., 2009; Helferich and Winter, 2000).
Diazinon is one of the most important organophosphate insecticides and is usually
used as an emulsion of 0.1 to 0.2% in farm fields and citrus groves to eliminate
pests, particularly the stem borer (Esmaili et al.,
1992; Dutta and Maxwell, 2003). The mechanism of
action is via the inhibition of the cholinesterase enzyme (Oliveira-Silva
et al., 2001; Wu et al., 1996). Typically,
diazinon is dissolved in alcohol and acetone and creates an emulsion in water
(Coppage and Mattews, 1974). Exposed individuals such
as farm workers have reported headaches, neurological complications, skin complications,
liver and kidney problems, seizures and even death (Vittozzi
et al., 2001). Most organophosphate compounds are converted to toxic
metabolites in the liver by the cytochrome P450 system through oxidative
dephosphorylation (IPCS, WHO and ILO, 1995). Organophosphates
work by inhibiting the enzyme acetylcholinesterase in sympathetic synaptic and
parasympathetic synaptic terminals, resulting in the accumulation of the neurotransmitter
acetylcholine in the nerve synapses and a consequent excessive stimulation of
nicotinic and cholinergic receptors (Civen et al.,
1977). The destructive effects of organophosphate compounds are not restricted
to enzyme inhibition; non-cholinergic effects, including damage to cell membranes,
free radical production and impaired antioxidant activity have also been observed
(IPCS, WHO and ILO, 1995). One mechanism that has received
much consideration is the production of free radicals by these compounds and
subsequent changes in the cell antioxidant system and peroxidation membrane
lipids (Sarabia et al., 2009). Many organophosphate
insecticides cause oxidative stress and generate Reactive Oxygen Species (ROS)
(Pokorny, 2001). ROS, such as superoxide anion and hydrogen
peroxide and hydroxyl radicals, are very active and can damage lipids, DNA,
nucleic acids and proteins, which may cause genetic mutations. Vitamins E, C
and A are known to be antioxidant agents and some studies have shown that these
vitamins can inhibit free radicals and mitigate their toxic effects (Kalender
et al., 2007). Vitamin E, which belongs to the fat-soluble vitamin
family, protects the cell membrane and lipoproteins against peroxidation (Jurczuk
et al., 2007; Kalender et al., 2005).
Several studies have shown that vitamin E can also act effectively to prevent
peroxidation in biological systems via the inhibition of free radicals (Ballantyne
et al., 1995). Vitamin C is a low-weight molecular antioxidant and
is effective in the aqueous phase in protecting different parts of cells against
free radicals of oxygen and nitrogen dissolved in water (IPCS,
WHO and ILO, 1995). The combination of vitamins C and E reduces lipid peroxidation
and regenerates antioxidant enzyme activity (Akturk et
al., 2006). There is some evidence that the major role of vitamin A
as an antioxidant is to eliminate oxygen radicals and prevent the formation
of lipid hydroperoxidase. Liver enzymes are commonly found in liver cells and
when the liver is damaged, liver cells release their enzymes into the blood
stream; thus, increased levels of these enzymes are a symptom of liver damage.
The first step in the diagnosis of liver damage is a simple blood test that
shows the presence of certain liver enzymes in the blood. Aminotransferases
are the most sensitive liver enzyme (Dixon and Webb, 1964).
Aspartate amino transferase (AST) is in the cytoplasm and mitochondria of heart,
liver and muscle cells; if these tissues are damaged, there is a corresponding
increase in AST activity in the serum. Alanine transaminase (ALT), like AST,
is distributed in most tissues, but at lower levels than AST. This enzyme is
primarily used in the diagnosis of liver disease. In evaluating the performance
of liver enzymes, ALT is more specific than AST and is known to reach higher
levels earlier in the disease process (Pratt and Kaplan,
2005). Lactate dehydrogenase (LDH) is a cytoplasmic enzyme that is used
clinically to diagnose cell injury; as such, it is a useful marker for toxic
chemical exposure and cell lysis. The activity of this enzyme has a direct relationship
with cell mortality. In complete liver failure, blood lactate levels are significantly
higher than the reference range (Kalender et al.,
2007). Far-reaching effects have been reported for organophosphate compounds.
One study showed that under the lethal density of organophosphate pesticides
(quinal Foss), the level of liver-specific LDH was reduced but the level of
kidney-specific LDH was increased (Das and Mukherjee, 2000).
In another study, the hepatotoxic role of diazinon and the preventive role of
vitamin E on biochemical markers in the serum (ALT, AST) was explored and vitamin
E was shown to reduce hepatotoxicity but not prevent it completely (Kalender
et al., 2005). The effects of diazinon on the antioxidation and peroxidation
systems of rat kidney lipids was studied and diazinon was found not to produce
significant changes compared to a control (Abbasnejad et
The purpose of this study was to examine the effects of the chronic administration of diazinon on the activities of liver enzymes in the presence and absence of antioxidant vitamins.
MATERIALS AND METHODS
Chemicals and systems: Chemical materials include diazinon (technical form: 96% purity; Supelco, United States) which was prepared by the Golsam company in Golestan and dissolved in soybean oil. The diazinon was injected intraperitoneally in a non-lethal dose (1/2 LD50). Vitamins A, E and C were obtained from Sigma company in appropriate doses suspended in soybean oil (as a solvent for vitamins A and E) and were injected intraperitoneally daily to the study groups (after toxin injection with diazinon). Other chemicals that were required in high purities were purchased from Merck or Sigma. This study was performed at the year of 2012.
Animal study: In this study, male Wistar rats weighing 180±10 g at the age of 8 weeks were purchased from the Pasteur Institute of Iran (Amol). Rats were kept in good condition at the university animal section under 12 h light and 12 h darkness and food and water were given.
Animals care: For experimental study, animals were randomly divided into 10 separate groups. Animals were comprised of the following groups:
||Group received normal saline
||Group received soybean oil (as the solvent for diazinon and
||Group received diazinon/vitamin E
||Group received diazinon/vitamins E and C (at the abovementioned
Each of abovementioned treatments was administered intraperitoneally for 14 days. Seven days after the final injection, the animals were euthanized using a 3:1 mixture of ketamine and xylazine. Prior to the surgery on the abdomen of mice, through a subcutaneous incision, 1.5-2 cc of blood was drawn straight from the heart of mice and transferred to a centrifuge tube, incubated for 30 m in in vitro and then centrifuged for 15 min with 1500 rpm. Approximately 1 cc of serum was obtained.
Measurement of enzymes level: The Pars Azmon kit was used to photometrically measure the activity levels of all three enzymes (AST, ALT and LDH). These enzymes results were reported in units of IU L-1.
Data analysis: Data analysis was performed using the Prism software and one way ANOVA and Tukey tests were performed with a 5% significant level (Mean±SD).
RESULTS AND DISCUSSION
In this study, the protective effect of vitamins C, E and A on rat liver enzyme
activity (ALT, AST, LDH) in the presence of diazinon exposure was studied. The
results are shown in Table 1. The activity of ALT in the diazinon
group was significantly higher (66.5±11.35 IU L-1) than that
observed in the control group (52.66±8.144 IU L-1), however,
the diazinon/vitamin E, A, C group displayed significant reduction to the diazinon
group. The highest ALT activity was observed in the diazinon group (66.5±11.35
IU L-1), while the lowest was observed in the diazinon/vitamin C+E
group (48.25±3.2 IU L-1) (p<0.001). A statistically significant
AST activity difference was observed in the diazinon group (122.25±17.34
IU L-1) relative to the normal saline treated group (105.5±5.06
IU L-1). The diazinon/vitamin A, C and C+E group displayed significant
relative to the diazinon group. The highest levels of AST enzyme activity were
unexpectedly observed in the diazinon/vitamin E group (172.33±7.23 IU
L-1). Significant reduction in LDH enzyme activity were observed
in the diazinon group (359.75±66.82 IU L-1) compared to the
control group (542±169.91 IU L-1) and overall, significant
changes in the vitamin groups were observed compared to the diazinon group.
A Tukey test showed that LDH enzyme activity in the dazinon/vitamin A group
(521±259.08 IU L-1) was significantly increased compared to
the diazinon group. The lowest level of LDH enzyme activity was observed in
the dazinon/vitamin C (272±47.08 IU L-1) group and this was
statistically lower than the diazinon group.
This study investigated the activity of liver enzymes in male rats chronically
exposed to diazinon and the role of vitamins A, E and C in the reduction of
diazinon toxicity. The results showed a significant difference in ALT enzyme
activity in the diazinon group compared with the normal control group. The current
study was conducted under chronic contact for over 14 days. It is possible that
glutathione (GSH) acts as an antioxidant against free radicals at first, but
considering the lapse, rats adapted to the various toxin levels, as GSH increased
to reduce toxicity; in the diazinon/vitamin groups, diazinon may have promoted
the regeneration of GSH, thus, reducing toxin levels and ALT activity.
|| Comparison of the effects of diazinon and vitamins A, E and
C on rat liver enzymes in the studied groups
|ALT: Alanine transaminase, AST: Aspartate aminotransferase,
LDH: Lactate dehydrogenase, aValues are the Mean±SD for
each group of 5 mice, ap<0.001 compared to the normal control,
bp<0.001 compared with the diazinon treated group, cp<0.05
compared with the diazinon treated group
Vitamin E/Diazinon significantly increased AST enzyme activity compared with
Diazinon (p <0.01). One explanation for this is that when toxic compounds
reach the body, the bodys natural antioxidants (GSH) try to clear the
poison and repair enzyme activity. It is possible that GSH was unable to completely
neutralize diazinon-related toxicity and vitamin E as an antioxidant acted synergistically
with GSH, but the toxicities resulting from chronic diazinon contact caused
AST levels to increase in the serum even in the presence of vitamin E. The LDH
levels observed in the diazinon group were not significantly increased compared
to the control group. This may be because chronic contact to diazinon may cause
reproduce glutathione, decrease toxin and fix LDH activity. In the diazinon/vitamin
A group, LDH activity increases significantly more than that of diazinon group
(p<0.001). This may be because Vitamin A decreases diazinon liver damage
and because freed liver enzyme in blood stream and liver enzyme produced by
gene, too. The results of this study are consistent with the results of other
studies (Karakilcik et al., 2004; Yousef
et al., 2003; El-Shenawy et al., 2009).
However, a few studies have found contradictory results (Gokcimen
et al., 2007; Salehi and Jafary, 2010; Kalender
et al., 2005; Etim et al., 2006).
One study showed the effect of different doses of diazinon on changes in LDH
and AST levels (Gokcimen et al., 2007). Another
study found that LDH activity was reduced 24 h after the injection of diazinon
and paraoxon due to a failure of the antioxidant defense system to protect against
free radicals and tissue oxidative damage. (Salehi and
Jafary, 2010). Increased serum concentrations of liver enzymes and changes
in antioxidant levels were reported to result from chlorpyrifos and sipermetrien
in mice (Khan et al., 2005). Lambda-cyhalothrin
(LTC) significantly decreased LDH activity in rat brains (Fetoui
et al., 2008). After Lindane and diazinon intoxication, LDH levels
were shown to increase (Kalender et al., 2005;
Etim et al., 2006). Klervos organophosphates
created different changes in plasma LDH levels depending on dose and time (Petroianu
et al., 2006). Significant plasma AST reductions in carp following
acute diazinon toxicity have been reported (Luskova et
al., 2002; Sastry and Sharma, 1980). In addition,
it has been found that vitamin E partially counteracted the toxic effect of
DZN and repaired tissue damage in the liver and kidney (El-Shenawy
et al., 2009). The presence of ascorbic acid (vitamin C) may also
diminish the adverse effects of Aflatoxin B1 (AFB1) on
most hematological and biochemical values and enzymatic activities in rabbits
(Yousef et al., 2003). Another study found that
AFB1 affected some liver enzymes and other biochemical parameters,
but that vitamins C, E and C+E partially prevented an increase in these liver
enzymes and biochemical parameters that were induced by AFB1 (Karakilcik
et al., 2004).
The liver is one of the main tissues affected by the poison. With regard to vitamin levels, changes in the related enzyme levels were observed. The activities of ALT and AST in the diazinon group were significantly higher than that observed in the control group; however, the diazinon/vitamin E, A, C group displayed significant reduction in ALT and AST activities compared to the diazinon group. LDH enzyme activity decreased significantly in the diazinon/vitamin C group compared to the diazinon-exposed group. Differences in chemical composition, the species studied and dose and exposure time vary across the studies. Variations in the chemical structure of organophosphates and their differential effects on various tissues necessitate additional studies to understand the mechanisms of action of this compound.
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