Protection of Liver Injury by Vitamin C, E and GSH …Jyotsna A. Patil1, Arun J. Patil1, Sanjay P. Govindwar2, Ajit V. Sontakke1 1Department of Biochemistry, Krishna Institute of Medical

506

J Pharm Chem Biol Sci, December 2015-February 2016; 3(4):506-517

Journal of Pharmaceutical, Chemical and Biological

Sciences ISSN: 2348-7658

Impact Factor (GIF): 0.615 Impact Factor (SJIF): 2.092

December 2015-February 2016; 3(4): 506-517

Protection of Liver Injury by Vitamin C, E and GSH after Methomyl Toxicity in Rat

Jyotsna A. Patil1, Arun J. Patil1, Sanjay P. Govindwar2, Ajit V. Sontakke1

1Department of Biochemistry, Krishna Institute of Medical Sciences University, Karad- 415539, Maharashtra, India 2 Department of Biochemistry, Shivaji University, Kolhapur – 416008, Maharashtra, India *Corresponding Author: Dr. Arun J. Patil, Professor in Biochemistry, Krishna Institute of Medical Sciences University, Karad- 415539, Dist. Satara, Maharashtra, India Received: 19 December 2015 Revised: 26 December 2015 Accepted: 29 December 2015

Original Research Article

ABSTRACT

Methomyl (Lannate), a carbamate pesticide induces liver injury by increasing lipid peroxide,

superoxide dismutase and inhibiting microsomal cytochrome P450 which is prevented by supplementation of vitamins. Aims and objectives: To study effect of supplementation of vitamins C, E and Glutathione (GSH) on lipid peroxide and superoxide dismutase, mixed function oxidase in methomyl treated rats. Materials and Methods: Adult male rats (weighing 200-230 g) were divided into 4 groups each of 6 animals. Animals from group 2, 3 and 4 were given a dose of 1, 2 and 4 mg methomyl/kg body weight (bw) intra peritoneal, respectively for 3 consecutive days. Second set of experiment adult male rats were divided into 4 groups. Animals from group 2, 3, 4 were injected selected dose methomyl (4mg/kg bw) for 1, 3 and 5 successive days. Third set of experiment adult male rats were divided into 5 groups. Animals from group 2 were injected methomyl 4mg/kg bw. Animals from group 3, 4, and 5 were injected methomyl (4mg/kg bw) along with Vitamin C, E and GSH (100 mg/kg bw each) respectively. Group-1 of each category received an equivalent amount of saline as control. Results: Increased dose and duration of methomyl treatment to rats increases the microsomal LP and SOD. Supplementation of vitamin C, E and GSH (100mg/kg bw each) to methomyl pretreated groups received in saline water separately and observed that microsomal LP and SOD was significantly increased in methomyl (P<0.01and P<0.05) treated rats and decreases in supplementation of vitamin C, E and GSH to methomyl-pretreated rats. Group-1 injected 0.9% saline and served as control in all the experiment. alteration in microsomal mixed function oxidases observed in Methomyl toxicity. Protection of liver is observed after supplementation of vitamin C, E and GSH on mixed function oxidases.

Keyword: Methomyl; vitamin C; Vitamin E; Glutathione (GSH); Oxidative Stress; Lipid Peroxidation; Mixed Function Oxidase

Jyotsna et al 507


INTRODUCTION

Lannate is widely used for the control of a large

variety of insects (leafhoppers and thrips), on a

wide range of crops viz. fruits, vines, hops,

vegetables, grains soybeans, cotton and

ornamentals throughout the world [1]. The

active ingredient in lannate is a methomyl, (S-

methyl-N-(methylcabomyl)-thioacetimidate), a

compound of the oxime carbamate group.

Methomyl has been classified as a pesticide of

category-I toxicity [2]. It is an insecticide of low

chronic, but high acute toxicity. This acts by

direct contact or following ingestion through

the stomach.

Metabolic pathway for methomyl in the rat

includes the displacement of the S-methyl

moiety by glutathione and enzymatic

transformation to produce a mercapturic acid

derivative. Another pathway involves hydrolysis

to give S-methyl-N-hydroxy thioacetimidate,

which is rapidly broken down to carbon dioxide

[1]. Methomyl treated rats showed

histopathologic changes in kidney and spleen of

male and female rats. Similarly, enzymatic

alterations of acetyl cholinesterase and liver

glucose-6-phosphate dehydrogenase were also

observed [3].

Microsomal cytochrome P450 (CYP450) consists

of multigene family that plays important role in

the metabolism of a wide variety of

endogenous compounds and xenobiotics

including drugs, carcinogens, toxic chemicals,

steroids and fatty acids [4]. Methomyl is known

as a potent cholinesterase inhibitor. A decrease

in the level of cytochrome P450 and the activity

of drug metabolizing enzymes at higher dose

level in male rats indicates destruction of

cytochrome P450. Decreased hemoglobin

content indicates that methomyl may involve in

heme synthesis. A significant increase in liver

enzymes (ALT, AST and ALP) indicates liver

damage [5]. It is established that many

pesticides, in common use, can produce some

toxic and adverse effects on the liver, kidney

and other biological systems, when tested on

various types of experimental animals through

their mode of action or by production of free

radicals that damage all cell components [6].

Pesticide chemicals can induce oxidative stress

by generating free radicals and altering

antioxidant levels of the free radical scavenging

enzyme activity [7]. Exposure to endosulfan and

chlorpyrifos can differentially modify

endogenous antioxidants like SOD, GPX and

GSH, which can lead to the development of

oxidative stress in some tissues [8]. Chlorpyifos

intoxication causes a significant decrease in the

reduced Glutathione (GSH), Catalase (CAT) and

Glutathione–S-Transferase (GST) activities [9].

A major contributor to non-enzymatic

protection against lipid peroxidation is vitamin

C and E, well known free radical scavengers

[10].Vitamins E, C and GSH are antioxidants

which exert their effect by curbing the excess

free radicals formation and thereby controlling

MDA levels which indicate lipid peroxidation.

The genesis of present study is to see whether

the antioxidants supplementation help to

alleviate the oxidative stress in pesticide toxicity

in experimental animals which may have

implications in managing human who exposed

to pesticide during spraying on grape gardens.

MATERIAL AND METHODS

Chemicals and Reagents

Methomyl (Lannate) was obtained from Du

point, USA; 2-thiobarbituric acid (2, 6-

dihydroxypyrimidine-2-thio;TBA) from Merck.

Reduced Nicotinamide Adenine Dinucleotide

Phosphate (NADPH), Oxidized Nicotinamide

Adenine Dinucleotide Phosphate (NADP),

Cytochrome c, Glucose 6- Phosphate, Glucose

6- Phosphate Dehydrogenase, Aminopyrine,

Aniline Hydrochloride, Malondialdehyde (MDA),

Pyrogallol, Glutathione (reduced) were

obtained from Sigma Chemical Co. (St. Louis

MO), Sucrose, Phenol, Trichloroacetic Acid,

Sodium Chloride, Potassium Chloride, Calcium

Chloride and other chemicals were of analytical

Jyotsna et al 508


grade, obtained from Qualigens Chemical

(Bombay). Vitamins E and C are from local

medical stores.

Animals

Three month old male (weighing 200-230 g, 80-

100days) Wistar rats were obtained from

Haffkine Institute, Bombay, India. The animals

were housed in standard cages and were given

an appropriate standard laboratory diet

(Hindustan Lever Ltd, Bombay) and tap water

ad libitum. The animals were used after

clearance from the Institutional Animal Ethics

committee.

Experimental Groups

Adult male rats (weighing 200-230 g) were

divided into 4 groups each of 6 animals. Animals

from group 2, 3 and 4 were given a dose of 1, 2

and 4 mg methomyl/kg body weight intra

peritoneal, respectively for 3 consecutive days.

In the second set of experiment adult male rats

were divided into 4 groups. Animals from group

2, 3, 4 were injected methomyl (4mg/kg bw) for

1, 3 and 5 successive days. In the third set of

experiment adult male rats were divided into 5

groups. Animals from group 2 were injected

methomyl 4mg/kg body weight. Animals from

group 3, 4, and 5 were injected methomyl

(4mg/kg bw) along with Vitamin C, E and GSH

(100mg/kg bw each) respectively. Group-1 of

each category received an equivalent amount

of saline as control. The volume injected into

rats of body weight 200 g was 1 ml. between 8:

00 am and 9:00 am.

Preparation of Microsomes

The rats used in this study were killed 24 hr

after the last dose by cervical dislocation. Their

livers were perfused in situ with ice cold 1.15 %

KCl containing 0.05 mM EDTA, rapidly excised,

blotted dry, weighed, minced and homogenized

with 2 volumes of ice cold 0.25 M sucrose

solution, in a Potter–Elvehjem type

homogenizer. The homogenate was centrifuged

at 10,000 x g for 10 min in a refrigerated

centrifuge (REMI C-24). The microsomes were

isolated by the procedure of Cinti et al [11]. The

microsomal pellet was suspended in phosphate

buffer (0.1 M, pH 7.4) and the suspension was

used for microsomal enzyme assays.

Microsomal protein content was measured by

the Biuret method using bovine serum albumin

as standard [12].

Enzyme assays

The levels of microsomal electron transport

components, cytochrome P450 and cytochrome

b5 were determined using Hitachi UV-visible

recording spectrophotometer by the procedure

of Omura and Sato [13, 14]. Cytochrome c

reductase activity was determined by the

method of Masters et. al [15]. Aminopyrine N–

demethylase activity was assayed according to

the procedure of Schenkman et. al [16].

Formaldehyde liberated during N-

demethylation was estimated by the procedure

of Nash [17]. Aniline hydroxylase assay was

performed using the procedure reported by

Govindwar and Dalvi [18].

Biochemical Assay

The biochemical measurements in microsomes

were performed according to method given

below in brief.

Assay of Superoxide Dismutase Activity

The measurement of superoxide dismutase

(SOD; EC1.15.1.1) is based on the principle in

which superoxide anion is involved in auto-

oxidation of pyrogallol at alkaline pH 8.5. The

SOD inhibits the autoxidation of pyrogallol,

which can be determined as an increase in the

absorbance per 2 min at 420 nm (1988). SOD

activity was expressed in units / mg protein

[19].

Jyotsna et al 509


Estimation of Hepatic Microsomal Lipid

Peroxide

Kohn and Liver sedge described the colorimetric

reaction of thiobarbituric acid (TBA) with an

unknown substance forming during the aerobic

incubation of tissue homogenates. Patton and

Kurtz later identified this as malondialdehyde

(MDA), a secondary product of lipid

peroxidation. The reaction of lipid peroxides

with TBA has been widely adopted as a

sensitive assay method for lipid peroxidation in

animal tissues. Yagi reported an assay method

for lipid peroxides in blood plasma by TBA

reaction under optimum conditions, the

reaction conditions especially the pH of the

reaction mixture must be carefully determined

while measuring lipid peroxide level in animal

tissues. Optical density of organic layer

measured at 532nm on spectrophotometer

against blank. The concentration of MDA

calculated by using standard graph [20].

Spectrophotometric measurements

The spectrophotometric measurements were

performed by using a Hitachi uv-visible recorder

(Japan).

Statistics

Statistical analysis was done by using one-way

analysis of variance and Tuckey krammer post

test. The level of significance was set at 0.05.

RESULTS

Administration of methomyl at different dose

for three days resulted in significant increase in

microsomal lipid peroxidation (LP) and

superoxide dismutase (SOD) level by 26.66%,

and 16.71% at 1 mg/kg, 41.11%, and 23.58% at

2 mg/kg, 48.33%, and 41.12% at 4 mg/kg body

weight (Table 1, fig. 1).

Selected dose of methomyl (4mg/kg bw)

injected for different days resulted in significant

increase in lipid peroxidation and SOD by

44.51% and 14.29% at 1 day, 63.94% and 32%

at 3 days, 114 % and 34% at 5 days of adult rats

as compared to control rats (Table 2, Fig. 2).

Microsomal LP and SOD was significantly

increased by 36.22%, and 22.89% in methomyl,

and antioxidants supplementation showed that

increase LP and SOD by 18.87%, and 7.39% in

methomyl + vitamin E, 14.03%, and 9.96% in

methomyl + vitamin C, 23.46%, and 11.44% in

methomyl + GSH treated rats, respectively as

compared to control rats. Microsomal proteins

was significantly decreased by 37.25% at

methomyl treated, 35% at methomyl + vitamin

E, 16.47% at methomyl + GSH and 14.29% at

methomyl + vitamin C, treated rats as

compared to control (Table 3, 4). These results

indicate that the microsomal protein decreased

more in methomyl treated rats as compared to

vitamin C and GSH supplementation to

methomyl-pretreated rats. Microsomal

cytochrome b5, and cytochrome P450, levels

were significantly inhibited by 43.28%, and -

33.33% due to methomyl, 27.27% and 23.23%

by methomyl + vitamin E, 12.12 % and 27.27%

by methomyl + GSH, 24.24% and 23.23% by

methomyl + vitamin C, , treated rats

respectively as compared control rats (Table

3,4). Whereas, no significant change was

observed in case of cytochrome c reductase in

all the three groups except methomyl treated

rats increased by 25.91% as compared to

control rats. Drug metabolizing enzymes i.e.

aminopyrine N-demethylase, and aniline

hydroxylase were significantly decreased by

43.7% and 29.31% due to methomyl, 30.14

%and 24.13% by methomyl + vitamin E, 21.37%

and 18.96% by methomyl + GSH, and 28.07%

and 21.12%, by methomyl + vitamin C, treated

rats respectively as compared to control rats

(Table 3,4).

Jyotsna et al 510


Table 1: Effect of Different Doses of Methomyl on Microsomal Lipid Peroxide and Superoxide

Dismutase of Adult Rats. [Each Group Consist Six Rats]

Parameter

Control group

Dose of Methomyl

1 mg/kg 2 mg/kg 4 mg/kg

LP a 3.6 ± 0.28 4.56 ± 0.25* * 5.08 ± 0.68* * * 5.34 ± 0.45* * *

SOD b 29.1 ± 4.33 34 ± 2.75* * 36 ± 2.36* * * 41.11 ± 1.7* * *

Values are means of three experiments ± SD * * P < 0.01, * * * P < 0.001, Lipid peroxide (LP),

Superoxide dismutase (SOD). a nmol/mg of microsomal protein, b Unit/mg of microsomal proteins.

26.66

41.11

48.33

16.71

23.58

41.12

0

10

20

30

40

50

60

LP, SOD

Pe

rce

nta

ge

ch

an

ge

Fig. 1: Percentage Change of Effect of Different Doses of Methomyl on Microsomal Lipid Peroxide

and Super Oxide Dismutase of Adult Rats. [Reference Table - 1]

1 mg/ kg, 2 mg/ kg, 4 mg/kg Lipid peroxide (LP), Superoxide dismutase (SOD)

Table 2: Effect of Different Duration of Methomyl Doses on Microsomal Lipid Peroxide and

Superoxide Dismutase of Adult Rats.

Parameter

Control group

Duration of methomyl doses in days

1 day 3 days 5 days

LP a 3.19 ± 0.42 4.61 ± 0.42* * 5.23 ± 0.24* * * 6.84 ± 0.40* * *

SOD b 29.38 ± 2.01 33.58 ± 1.10* 38.81 ± 1.97* * 39.63 ± 1.93* * *

Values are means of three experiments ± SD * P < 0.05, * * P < 0.01, * * * P < 0.001, Lipid peroxide

(LP), Superoxide dismutase (SOD). a nmol / mg of microsomal protein, b Unit /mg of microsomal

proteins.

Jyotsna et al 511


Fig. 2: Percentage Change of Effect of Duration of Methomyl Dose on Microsomal Lipid Peroxide

and Super Oxide Dismutase of Adult Rats. [Reference Table – 2]

1 day, 3 days 5 day Lipid peroxide (LP), Superoxide dismutase (SOD).

Table 3: Alteration in Male Rats Liver Microsomal Protein, Electron Transport Components and

Drug Metabolizing Enzymes, Lipid Peroxide, Superoxide Dismutase Due to Methomyl (4 mg/Kg),

Vitamin E (100 mg/kg) + Methomyl (4 mg/kg),Vitamin C (100 mg/kg)+ Methomyl (4 mg/Kg) and

GSH (100 mg/kg) + Methomyl (4 mg/kg) of Three Days Treatments

Parameters Control Methomyl Vit E+Met GSH+Met Vit C+ Met

MP a 14.2±2.73

(10 - 17.9)

8.91 ± 0.62* *

(8.2 - 9.9)

9.23 ± 0.47* *

(8.5 - 9.7)

11.86 ±1.39•

(9.9 - 14.10)

12.17 ± 1.51•

(10.22-13.30)

Cyt b5 b 0.33 ± 0.056

(0.26 – 0.40)

0.19 ±0.04* * *

(0.14 – 0.24)

0.24 ±0.07* * *

(0.14 – 0.34)

0.29 ± 0.072•

0.21 – 0.38)

0.25±0.023* *

(0.22 – 0.28)

Cyt P450c 0.49 ± 0.054

(0.42 – 0.57)

0.33± 0.056* *

( 0.28 – 0.40)

0.38 ± 0.034*

(0.32 – 0.42)

0.36 ± 0.05* *

(0.26 – 0.45)

0.38 ± 0.064*

(0.27 – 0.49)

Cyt C red d 22.50 ± 2.73

(19 - 26)

28.33 ± 2.58* *

( 25 – 32)

24.80 ± 2.67 •

(20.80 - 29)

23.83 ± 2.71 •

(19 - 27)

25.16 ± 3.25 •

(20 – 29)

AND e 6.27 ±1.64

(4.18 – 8.40)

3.53 ± 1.30* *

(1.90 – 5.4)

4.38 ± 0.577*

(3.80 – 5.40)

4.93 ± 1.17•

(2.90 – 6.50)

4.51 ± 1.10*

(2.70 – 5.60)

AH f 2.32 ± 0.589

(1.38 – 2.90)

1.64 ± 0.498*

(1.0 – 2.30)

1.76 ± 0.44•

(1.20 - 2.30)

1.88 ± 0.343*

(1.40 – 2.40)

1.83 ± 0.541•

(1.40 – 2.6)

LP g 3.92 ± 0.76

(2.96 – 4.89)

5.34 ± 0.37* *

(4.9 – 5.90)

4.66 ± 0.62*

(3.96 – 5.40)

4.84 ± 0.69*

( 3.90 – 5.80)

4.47 ± 0.53*

(3.74 – 5.20)

SOD h 45.16 ± 9.94

(35 – 60)

55.50 ± 10.5*

(39 – 67)

48.50 ± 5.82•

(38 – 55)

50.33 ± 7.96•

(36 – 59)

49.66 ± 8.45•

(34 – 59)

Values are means of three experiments ± SD; six animals in each group.a mg of protein/g liver, b

nmole/mg of microsomal protein, c nmole cytochrome c reduced/min/mg of microsomal protein, d

nmole formaldehyde liberated/min/mg of microsomal protein, e nmole p-aminophenol

formed/min/mg of microsomal protein,g nmol/mg of microsomal protein, h Unit//mg of microsomal

proteins.

Jyotsna et al 512


Microsomal proteins (MP), Cytochrome b5 (Cyt b5), Cytochrome P450 (Cyt. P450) Cytochrome c

reductase ( Cyt C red), Aminopyrine N-demethylase (AND), Aniline hydroxylase (AH), Lipid peroxide

(LP), Superoxide dismutase (SOD). * P < 0.05, * * P < 0.01, * * * P < 0.001, • Non significant with compared to control.

Table 4: Percentage Change of Alteration in Rats Liver Microsomal Protein, Electron Transport

Components and Drug Metabolizing Enzymes, Lipid Peroxide, Superoxide Dismutase due to

Methomyl (4 Mg /Kg), Vitamin E (100 Mg/Kg) + Methomyl (4 Mg/Kg), Vitamin C (100 Mg/Kg) +

Methomyl (4 Mg/Kg) And GSH (100 Mg/ Kg) + Methomyl (4 Mg/Kg) of Three Days Treatments

With Respect to Control Group Rats.

Parameters Treatments

Methomyl Vit E + Met GSH + Met Vit C + Met

MP a -37.25 -35.0 -16.47 -14.29

Cyt b5b -43.28 -27.27 -12.12 -24.24

Cyt P450c -33.33 -23.23 -27.27 -23.23

Cyt C red d 25.91 10.22 5.91 11.82

AND e -43.70 -30.14 -21.37 -28.07

AH f -29.31 -24.13 -18.96 -21.12

LP g 36.22 18.87 23.46 14.03

SOD h 22.89 7.39 11.44 9.96 a mg of protein/g liver, b nmole/mg of microsomal protein. c nmole cytochrome c reduced/min/mg of

microsomal protein. d nmole formaldehyde liberated/min/mg of microsomal protein. e nmole p-

aminophenol formed/min/mg of microsomal protein.g nmol/mg of microsomal protein, h Unit/mg

of microsomal proteins [Ref. table 3].

DISCUSSION

In the present study methomyl treatment

resulted in increased oxidative stress in the rat

as evidenced by enhanced levels of

thiobarbituric acid reactive substancse (TBARS),

accompanied by concomitant increase in the

levels of superoxide scavenging enzymes SOD,

in liver. The increase levels are dose and

duration dependent.

Increase formation of reactive oxygen and

nitrogen species resulting an increase in the

lipid peroxidation in several tissues mainly

brain, skeletal muscle, RBC, etc. A deplete

antioxidant status was reported in several

studies of various pesticide exposed population

[21- 25]. However, in case of short-term

pesticides exposure, the antioxidants enzymes

are increased due to more generation of free

radicals, and in case of long-term pesticides

exposure, the antioxidants enzyme levels are

depleted due to continuous utilization for

scavenging the reactive oxygen species. We

observed increased SOD level may be short-

term methomyl exposure.

Pesticides may increase oxidative damage

because they are more active to oxygen free

radical that re-oxidizes to make superoxide or

the pesticides may itself be free radicals or they

may deplete antioxidants defences. Paraquat is

reduced free radical that reoxidises to make a

superoxide and regenerate paraquat, which

accumulates selectively. The overall effect of

pesticides is the production of more free

radicals [26]. Pesticides may irritate lung

macrophages, encouraging them to generate

Jyotsna et al 513


the superoxide radical. The organisms use

antioxidant enzymes as natural endogenous

protection against the generation of oxygen

species [27].

Superoxide dismutase is an antioxidant enzyme

that catalyses the dis-mutation of the highly

reactive superoxide anion to O2 and the less

reactive species H2O2. Peroxide can be

destroying by catalase or Glutathione Peroxide

reactions [28]. In humans, there are three forms

of SOD, 1) Cytosolic: Cu/Zn SOD 2)

Mitochondrial: Mn-SOD, and 3) Extra cellular

SOD [29]. If there is an increase in the

production of free radicals in the body, extra

antioxidants produced. However, if large

numbers of extra free radicals produced results

cell damage and death, due to imbalance

between free radicals and antioxidants [26].

Selected dose of methomyl treatment at

4mg/kg bw for 3 days decreases in cytochrome

P450 content and the activity of drug

metabolising enzymes at dose and duration

dependent manner in male rats indicate

destruction of cytochrome P450, this coincides

with our earlier report. Many pesticides

including organochlorine and

organophosphorous compounds have been

reported to inhibit the activity and alteration in

the expression of various cytochrom P450

isoforms during its oxidative biotransformation.

These changes may increase the sensitivity of

cell against reactive endogenous metabolites or

other xenobiotics [30].

Methomyl treatment to rats increased

microsomal LP due to more generation of free

radicals and to scavenge these radicals,

microsomal SOD activity increased. The

increase microsomal LP and SOD decreases by

supplementation of vitamin E, C, and GSH may

be due to antioxidants properties of these

compounds. Vitamin E acts as a chain breaking

antioxidants for lipids in biological membranes

[31, 32]. The GSH is an endogenous thiol

antioxidant that has a multifaceted role in

xenobiotic metabolism and is a first line of

defence against oxidant-mediated cell injury

[33].

Present result indicate that the microsomal

protein decreased more in methomyl treated

rats as compared to vitamin C and GSH

supplementation to methomyl pre-treated rats

(Table 4). From this, it is clear that the vitamin C

and GSH may prevent the adverse effects of

methomyl on protein biosynthesis, or it may

have some role in protein biosynthesis. Several

studies reported that the methomyl inhibits the

protein biosynthesis [34, 35]. and induces

oxidative stress. In oxidative stress, decreased

protein biosynthesis has well documented in

literature. In this study, vitamin C and GSH may

be involved in scavenging the free radicals

generated by methomyl and decreases the

oxidative stress and indirectly preventing the

inhibition of protein biosynthesis by methomyl

in rats.

Vitamin C enhances the synthesis of

immunoglobulin (antibodies) and increase the

phagocytic action of leucocytes and peptide

hormone synthesis. Many peptide hormones

contain carboxyl terminal amide, which is

derived from terminal glycine. Vitamin C is

required for hydroxylation of glycine carried by

peptidylglycine hydroxylase. The vitamin C is

also involved in collagen, ferritin, serotonin and

carnitine synthesis [31, 32]. From these vitamin

C functions, it clearly understood that the

vitamin C might enhance the microsomal

proteins biosynthesis.

Glutathione supplementation to methomyl-pre-

treated rats increases the microsomal proteins

as compared to methomyl treatment. It could

be explain by the role of glutathione in “γ -

glutamyl cycle” for absorption of amino acids

from gut [33, 34]. Glutathione enhance amino

acid absorption across the gut, which may be

indirectly accelerating the microsomal protein

biosynthesis.

Ascorbic acid seems to take part in electron

transport system of mammalian ‘microsomes’,

due to its easy oxidation with reversible

Jyotsna et al 514


reduction of ascorbic acid. Enzyme like ascorbic

acid oxidase, cytochrome oxidase, flavine

transhydrogenase participate in the electron

transport system, where ascorbic acid takes

part between NADH and cytochrome b5. The

detail mechanism of the role of vitamin C is not

known, but suggested that the coupled reaction

with hydroxylation [31]. Therefore, vitamin C

supplementation may be restored the

cytochrome b5 activity in case of methomyl pre-

treated rats. Oxidative modification of

microsomal protein produced by NADPH-450

reductase/P450/O2system is exclusively

prevented by ascorbic acid this may be

explained by the consideration that perferryl

radical, P450. Fe3+O2 is reduced and inactivated

by ascorbic acid and not by GSH, alpha

tocopherol (Vit E) and other antioxidants used

[35].

Microsomal cytochrome P450 enzyme activity

has reduced in methomyl treated rats may be

due to inhibition of various cytochrome P450

isoforms or it may be due to inhibition of heme

biosynthesis, resulting the decrease activity of

various cytochromes. Vitamin C

supplementation to methomyl pre-treated rats

increased the microsomal cytochrome b5 and

cytochrome P450 activity may be the enhancing

the heme biosynthesis. Ascorbic acid enhances

iron absorption by keeping it in the ferrous

form due to its reducing property. It also helps

in the formation of ferritin (storage form of

iron) and mobilization of iron from ferritin,

which is required for heme biosynthesis [31,

32]. Lead arsenate (di plumbic hydrogen

arsenate) used as insecticide, and fungicide.

The arsenic trioxide depletes ascorbic acid [36].

Deficiency of ascorbic acid inhibits cytochrome

content, and microsomal mixed function

oxidases reported in literature [37].

In earlier discussion, we have seen that vitamin

C increasing the hepatic microsomal protein

biosynthesis. Moreover, our results are

consistent with others report in literature.

Glutathione, a thiol tripeptide, is the most

important endogenous antioxidant that is found

in mM concentrations in tissues and is

responsible for maintaining the cellular redox

state with smaller thiols such as thioredoxin,

glutaredoxin and peroxiredoxin [38].

Methomyl mainly detoxifying by conjugation

with GSH forming mercapturic acid that excrete

in urine. Increased microsomal protein,

cytochrome b5 and cytochrome P450,

aminopyrine N-demethylase, aniline

hydroxylase enzymes activity after

supplementation of GSH to methomyl pre-

treated rats may be due to immediate

conjugation of methomyl by GSH, reduces its

adverse effects on mixed function oxidase and

drug metabolizing enzymes along with decrease

oxidative stress by improving antioxidant

status.

Increased cytochrome b5 and cytochrome P450,

aminopyrine N-demethylase, aniline

hydroxylase enzymes activity after

supplementation of Vit E to methomyl

pretreated rats may due to the role of vitamin E

as an antioxidant. Vit E allows free radicals to

abstract a hydrogen atom from the antioxidant

molecule rather than from polyunsaturated

fatty acids, thus breaking the chain of free

radical reactions, the resulting antioxidant

radicals being a relatively unreactive species

[39].Thus vit E inhibiting the formation of free

radical, and decreases the methomyl toxicity.

The basis of pesticide toxicity in the production

of reactive oxygen species may be due to:

1) Their “redox-cycling” activity-they readily

accept an electron to form free radicals and

then transfer them to oxygen to generate

superoxide anions and hence hydrogen

peroxide through dismutation reaction.

2) Generation of free radicals probably because

of the alteration in the normal hemostasis of

the body resulting in oxidative stress, if the

requirement of continueous antioxidants is not

maintained [40].

Jyotsna et al 515


The efforts of the endogenous antioxidant

enzymes to remove the continueously

generated free radicals initially increase due to

an induction but later enzyme depletion results,

resulting in oxidative cell damage [41].

In case of short-term pesticides exposure, the

antioxidants enzymes are increased due to

more generation of free radicals, and in case of

long-term pesticides exposure, the antioxidants

enzyme levels are depleted due to continuous

utilization for scavenging the reactive oxygen

species.

Present study can conclude that

supplementation of Vitamin C, E and

glutathione decreases oxidative stress and

improves antioxidant SOD in methomyl exposed

rats. Antioxidant role of these may be one of

the key mechanisms of its protective effect.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no

competing interests.

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Cite this article as: Jyotsna A. Patil, Arun J. Patil, Sanjay P. Govindwar, Ajit V. Sontakke. Protection of Liver

Injury by Vitamin C, E and GSH after Methomyl Toxicity in Rat. J Pharm Chem Biol Sci 2015;

3(4): 506-517

Documents

Protection of Liver Injury by Vitamin C, E and GSH …Jyotsna A. Patil1, Arun J. Patil1, Sanjay P. Govindwar2, Ajit V. Sontakke1 1Department of Biochemistry, Krishna Institute of Medical