INTRODUCTION:

In India, about 75% of the population depends upon agriculture. The 2,4-dichlorophenoxy acetic (2,4-D) acid is one of the most selective herbicides used by Indian farmers in agricultural field to control the growth of broad-leaved weeds^[1]. Its formulations include esters, acids, and several salts, which vary in their chemical properties, environmental behavior and to a lesser extent, toxicity.

Living things are exposed to 2,4-D in many ways: by breathing contaminated air, drinking and bathing in polluted water, contacting contaminated soil, eating contaminated food, ingesting contaminated dust and while working with 2,4-D. Each kind of exposure may involve lesser amounts of 2,4-D but their cumulative effect is a serious concern^[2].

^[3]Wafa et al. and ^[4]Nakbi et al. suggested that 2,4-D exposure can lead to oxidative stress through the unregulated generation of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radical, peroxyl radical and singlet oxygen in varieties of animal species. In mammals, 2,4-D disrupts energy production depleting the body of its primary energy molecule adenosine triphosphate^[5]. The teratogenic, neurotoxic, immunosuppressive, cytotoxic and hepatoxic effects of 2,4-D have been well documented by ^[6]Venkov et al. Recently, this herbicide exerted reproductive effects in rodents leading to loss of reproductive functions^[7,8]. Studies in our laboratory documented that these toxicant induced effects in kidney, testes, and liver of the adult mice^[9,10,11,12].In mice liver necrosis or fatty liver cell changes were observed in two fatal cases following 2,4-D herbicide ingestion^[13]. ^[14]Gorzinski et al. reported necrobiotic changes in the form of hepatocellular, cytoplasmic swelling and homogeneity in rats exposed to 2,4-D daily for 90 days.

Numerous antioxidants are known to mitigate toxicant exerted oxidative stress and other toxic effects in animals. These include hormones, herbal products and –SH containing compounds^[12]. Curcumin (Cur) is known to exhibit antioxidant activities^[15,16]. It interacts with an array of protein targets implicated in various cellular activities including defense antioxidant system, metabolism, carcinogenesis, apoptosis, etc. for protection^{[17, 18]}. The ameliorative effects of curcumin have been demonstrated in various studies and it acted as mitigating agent. These include the induction of nitric oxide synthase and nuclear factor kappa B in gentamicin-induced toxicity^[19], cadmium toxicity on male reproductive system^[20], testicular seminiferous epithelium in metronidazole-treated mice^[21], inhibition of oxidative stress and cytokine activity in endotoxin-induced hepatoxicity^[22] and lead acetate-induced hepatoxicity^[23]. However, very few studies have been conducted on its mitigation with this herbicide; hence this study has been chosen to investigate the role of this compound on liver toxicity in a rodent model.

Fig.1: Chemical Structure of 2,4-D

Fig. 2: Chemical Structure of Curcumin

MATERIALS AND METHODS:

Chemicals:

2, 4-Dichlorophenoxyacetic acid (2,4-D), curcumin and other chemicals used in different assays were procured from Sigma Chemicals (USA ) and Merck and Hi Media (Mumbai).

Animals:

Fifteen weeks old Swiss strain male albino mice (Mus musculus), weighing 30-50 g each, were obtained from Cadila Pharmaceutical, Ahmedabad, Gujarat, India, under the Animal Maintenance Registration No. 167/1999/CPCSEA from the Ministry of Social Justice and Empowerment, Govt. of India.They were kept in stainless steel mesh cages, housed under standard laboratory conditions (26±2º C, 30-70% relative humidity, 12-hour light-dark cycle) with standard animal chow (Pranav Agro Industries, Baroda, India) and drinking water ad libitum. The mice were acclimatized in the animal house for 10 days before use.

Dose and Duration:

The dosage of 2, 4-D (Fig. 1) was used based on LD₅₀ 370 mg/kg in mice followed by ^[24]Gervais et al. The dose of curcumin (Fig. 2) was decided based on earlier work of ^[25]Mach et al.The treatments were given orally (2,4-D, curcumin) to experimental animals. The control animals were provided an only standard diet with distilled water throughout the study. The duration of treatment was for 45 days.

The animals were divided into eight equal groups. The eight groups were mainly: I. Control (untreated), II. Curcumin alone (10mg/kg), III. 2,4-D treated (low dose; 30mg/kg), IV.2,4-D treated (mid dose; 60mg/kg), V. 2,4-D treated (high dose; 90mg/kg), VI. 2,4-D treated + Curcumin (LD+CUR), VII. 2,4-D treated + Curcumin (MD+CUR) and VIII. 2,4-D treated + Curcumin (HD+CUR).

Isolation of hepatic tissues:

The control and treated animals were sacrificed by cervical dislocation at the 46th days of each treatment. The liver was excised carefully, blotted free of blood before weighing and used for carrying out gravimetric, metabolic and oxidative stress indices followed by the histopathological study.

Metabolic Indices:

The metabolic parameters evaluated were acid phosphatase and alkaline phosphatase (ACPase^[26]: EC 3. 1. 3. 2 and ALPase^[26]: EC 3. 1. 3. 1), adenosine triphosphatase (ATPase^[27]: EC 3. 6. 1. 3), succinate dehydrogenase (SDH^[28]: EC 1. 3. 5. 1), total protein (TP^[29]) and glycogen (GLG^[30]) using standard methods.

Oxidative Stress Profile:

The non-enzymatic antioxidant parameters in liver of all groups included glutathione (GSH^[31]), total ascorbic acid (TAA^[32]), total sulfhydryl (Total-SH^[33]) and lipid peroxidation (LPO^[34]) followed by the enzymatic antioxidant enzymes such as superoxide dismutase (SOD^[35]: EC 3. 15. 1. 1), catalase (CAT^[36]: EC 3. 11. 1.), glutathione peroxidase (GPx^[37]: EC 1. 11. 1. 9), glutathione reductase (GR: EC 1. 8. 1. 7^[38]) and glutathione –s-transferase (GST^[39]: EC 2. 5. 1. 18) respectively following standard techniques.

Statistical Analysis:

The data were subjected to statistical analysis such as mean, standard deviation (SD), and analysis of variance (ANOVA), using standard statistical software, the statistical package for social sciences (SPSS; version 16). All values are expressed as mean ± SE of 10 individual samples. A value of P <0.05 was considered significant.

RESULTS:

A significant (P<0.01, P<0.001) reduction was noticed in whole body weight of mice fed with toxicant (Gr. I Vs. IV and V).The same pattern was observed with liver (Gr. I Vs. IV and V). As compared with high, mid and low doses also had indicated significant (P<0.05, P<0.01) changes. Curcumin supplementation at a high dose level of 2,4-D exhibited partial recovery (Gr. I Vs. VIII). But a substantial (P<0.05) mitigation was observed when compared with other doses (Gr. VIII Vs. VII and VI) (Table 1).

In liver tissue, the level of phosphatases, SDH and total proteins showed a significant (P<0.01, P<0.001) reduction. Contrarily a significant (P<0.01, P<0.001) increase in glycogen levels were recorded in high dosed mice. The ATPase levels were (p<0.05) significantly recorvered in curcumin supplementation with 2,4-D high dose treated mice whereas no alterations were observed in ACPase, ALPase, total protein and glycogen levels. A significant (p<0.05) mitigation was seen when compared with the high dose of 2,4-D treated curcumin supplementation group with other groups (Table 2).

Exposure of mice to 2,4-D resulted a marked (P<0.01, P<0.001) reduction in GSH, TAA and -SH levels followed by substantial (P<0.01, P<0.001) rise in LPO levels.The high dose to mice exerted a significant (P<0.05, P<0.01) alterations in GSH, TAA, -SH and LPO level in comparison to other groups. In contrast, except TAA, curcumin partially mitigated the high dose induced toxicity. However, a significant (P<0.05) mitigation was noticed in GSH, -SH and LPO levels by comparing with a high dose of 2,4-D treated mice by curcumin supplementation (Table 3).

Further SOD, CAT, GR and GST enzymes activities of antioxidant system were significantly (P<0.05, P<0.001) decreased in liver of exposed mice. Curcumin was able to alleviate 2,4-D toxicity at a high dose level. Similarly, there were no significant variations observed comparing with low and mid doses. A significant ( P<0.05 ) mitigation was noted in GPx and GR enzyme activities by comparing with control as well as 2,4-D treated curcumin supplementation groups with other groups. But mitigation was partial (P< 0.05) with high dose by curcumin supplementation (Table 4).

Table 1: Body and organ weights of control and experimental groups

PARAMETERS	CON (G.I)	CUR (G.II)	TREATED(2,4-D)			TREATED+CUR
PARAMETERS	CON (G.I)	CUR (G.II)	LD(G.III)	MD(G.IV)	HD(G.V)	LD+ CUR(G.VI)	MD+ CUR (G.VII)	HD +CUR ( VIII)
Total Body Weight gm)	42.63±1.21	41.78±1.37^NS	38.21±1.11^NS,b	32.13±1.6^**,a	29.54±1.43⁺	38.311.43^NS	38.25±1.01^NS	37.21±1.06^*
Liver (mg)	2.78±0.28	2.64±0.13^NS	1.89±0.19^NS,b	2.49±0.16^**,a	0.98±0.14⁺	2.59±0.16^NS,d	2.09±0.11^NS	1.58±0.06^*

Values are Mean ± S.E, NS= Non Significant, (Gr. II, III, VI, VII Vs. Gr. I), (G. VIII Vs G.VII).

*=P<0.05, **=P<0.01, +=P<0.001 (Gr. IV, V, VIII verses Gr. I), a=P<0.05, b=P<0.01 (G.V Vs G.IV and G.III),

d=P<0.05 (G.VIII Vs G.VI).

Table 2: Biochemical indices in the liver of control and experimental groups.

PARAMETERS	CON (G.I)	CUR (G.II)	TREATED(2,4-D)			TREATED+CUR
PARAMETERS	CON (G.I)	CUR (G.II)	LD(G.III)	MD(G.IV)	HD(G.V)	LD +CUR (G.VI)	MD+ CUR (G.VII)	HD+CUR (G.VIII)
Acid phosphatase^m	3.29±0.04	3.09±0.04^NS	3.01±0.02^NS,b	2.01±0.14^**,a	1.67±0.31⁺	2.89±0.18^NS,d	2.82±0.14^NS	2.81±0.13^NS
Alkaline phosphataseⁿ	2.09±0.01	1.64±0.10^NS	1.84±0.11^NS,b	1.56±0.07^**,a	0.98±0.13⁺	1.94±0.11^NS	1.89 ±0.18^NS	1.84±0.17^NS
Adenosine triphosphatase ^o	2.19±0.11	2.13±0.11^NS	2.03±0.01^NS,b	1.41±0.01^**,a	1.09±0.10⁺	2.08±0.01^NS,d	1.68±0.01^NS	1.56±0.18^*
Succinate dehydrogenase ^p	46.18±1.03	42.08±0.51^NS	42.88±0.51^NS,b	35.54±1.21^**,a	28.54±0.11^+,	41.88±0.31^NS,d	40.43±0.51^NS	39.54±1.21^*
Total proteins ^q	16.76±.81	15.13±0.11^NS	13.18±0.24^NS,b	11.58±0.31^**,a	9.58±0.31⁺	14.16±0.13^NS	14.06±0.12^NS	13.14±0.23^NS
Glycogen ^r	1406±43.03	1467±33.81^NS	1647±20.51^NS,b	2051±67.91^**,a	2401±51.1⁺	1617±21.41^NS	1641±67.91^NS	1651±50.19^NS

m,n=µ moles of p-nitro phenol released / mg protein, o= µ moles of inorganic phosphate released / 30 min. / mg protein,

p= µg formazan formed/ 15 min/ mg protein, q=mg/100 mg tissue weight, r=µ moles/ 100 mg tissue weight,

Values are Mean ± S.E, NS= Non Significant, (Gr. II, III, VI, VII Vs. Gr. I), (G. VIII Vs G.VII).*=P<0.05, **=P<0.01, +=P<0.001

(Gr. IV, V, VIII verses Gr. I), a=P<0.05, b=P<0.01 (G.V Vs G.IV and G.III), d=P<0.05 (G. VIII Vs G.VI).

Table 3: Non-enzymatic antioxidants in liver of control and experimental groups

PARAMETERS	CON (G.I)	CUR (CUR)(G.II)	TREATED(2,4-D)			TREATED+CUR
PARAMETERS	CON (G.I)	CUR (CUR)(G.II)	LD(G.III)	MD(G.IV)	HD(G.V)	LD +CUR (G.VI)	MD+ CUR (G.VII)	HD +CUR (G.VIII)
Glutathione^m	68.25±2.04	68.14±2.12^NS	64.21±1.12^NS,b	46.87±2.13^**,a	37.17±1.41⁺	64.11±1.02^NS,d	60.11±1.02^NS	50.11±1.1^*
Total ascorbic acid ⁿ	3.19±0.01	2.84±0.11^NS	2.74±0.21^NS,b	2.26±0.07^**,a	1.98±0.13⁺	2.74±0.11^NS	2.56±0.18^NS	2.36±0.18^NS
Total sulfhydryl ^o	2.19±0.11	2.12±0.11^NS	2.13±0.11^NS,b	1.32±0.11^**,a	2.08±0.10⁺	2.08±0.01^NS,d	1.67±0.11^NS	1.50±0.18^*
Lipid peroxidation ^p	29.76±0.11	32.36±0.51^NS	39.16±0.23^NS,b	45.58±0.31^**,a	47.43±0.31⁺	31.96±0.23^NS,d	39.43±0.21^NS	42.43±0.31^*

m=µ moles/ 100 mg tissue weight, n= mg/ gm tissue weight, o= mg/ 100 mg tissue weight, p= n moles of MDA formed/100mg tissue weight.

Values are Mean ± S.E, NS= Non Significant, (Gr. II, III, VI, VII Vs. Gr. I), (G. VIII Vs G.VII).*=P<0.05, **=P<0.01, +=P<0.001

(Gr. IV, V, VIII verses Gr. I), a=P<0.05, b=P<0.01 (G.V Vs G.IV and G.III), d=P<0.05 (G. VIII Vs G.VI).

Table 4: Enzymatic antioxidant in liver of control and experimental groups.

PARAMETERS	CON (G.I)	CUR (G.II)	TREATED(2,4-D)			TREATED+CUR
PARAMETERS	CON (G.I)	CUR (G.II)	LD(G.III)	MD(G.IV)	HD(G.V)	LD +CUR (G.VI)	MD+ CUR (G.VII)	HD +CUR (G.VIII)
Superoxide dismutase^m	2.15±0.04	2.00±0.01^NS	2.01±0.05^NS,b	1.57±0.03^**,a	1.19±0.01⁺	2.06±0.13^NS,d	1.81±0.03^NS	1.78±0.05^NS
Catalaseⁿ	30.19±0.19	28.71±0.13^NS	28.74±0.07^NS,b	23.23±0.13^**,a	19.58±0.21⁺	28.72±0.06^NS	27.83±0.14^NS	26.78±0.17^NS
Glutathione peroxidase ^o	29.27±0.11	26.61±0.11^NS	25.12±0.01^NS,b	21.41±0.11^*,a	18.17±0.19^**	25.92±0.01^NS,d	25.12±0.01^NS	20.04±0.33^*
Glutathione reductase ^p	34.06±1.03	32.12±1.11^NS	27.77±0.91^NS,b	25.41±1.01^**,a	21.01±1.01⁺	31.13±1.21^NS,d	31.03±1.11^NS	27.20±1.11^*
Glutathine –s-transferase ^q	0.029±0.001	0.033±0.003^NS	0.026±0.001^NS,b	0.017±0.003^**,a	0.012±0.02⁺	0.030±0.001^NS,d	0.034±0.001^NS	0.033±0.001^NS

m= n moles of MDA formed/100mg tissue weight, n= units/ mg protein, o= µ moles of H ₂O ₂consumed/ min/ mg protein,

p= µ moles of GSH consumed/ min/ mg protein, q= n moles of NADPH oxidized/ min/ mg protein. Values are Mean ± S.E,

NS= Non Significant, (Gr. II, III, VI, VII Vs Gr. I) (G. VIII Vs G.VII).*=P<0.05, **=P<0.01, +=P<0.001

(Gr. IV, V, VIII verses Gr. I), a=P<0.05, b=P<0.01 (G.V Vs G.IV and G.III), d=P<0.05 (G. VIII Vs G.VI).

Histopathology:

The liver consists of a network of hepatocytes which are separated by vascular sinusoids. The hepatocytes form organized micro structures which serve as structural and functional units. It also has many lobules, each of which is a hexagonal structure consisting of a central vein surrounded by radiating hepatic cords in control mice (Fig. 1). Treatment of mice with 2,4-D for 45 days produced mild vascular and hepatocellular lesions with necrotic changes and focal areas of necrosis in the liver (Figs 2). Supplementation of curcumin (CUR) (group VIII), few hepatocytes exhibited resumption of the anatomical features similar to that of control mouse (Fig. 3).

(1) (2) (3)

(1) Fig. 1: Normal mouse liver (Gr. 1) section appearances a network of hexagonal shaped cells known as hepatocytes (H), blood vessels BV) followed by sinusoid cells (S). X840.

(2). Fig. 2: This figure revealing necrosis (N) followed by vacuolated hepatocytes (VH) in treated mice (Gr. V) with HD. X840.

(3) Fig. 3: Normal features of hepatic tissue supplemented with curcumin to treated mice (Gr. VIII). X840.

DISCUSSION:

Feeding to serial doses of 2,4-D to mice for 15 weeks significantly decreased body and organ weight due to loss of food intake. This can be explained by the loss of appetite and improper metabolic status. Similar reports were reported by other researchers working with the same toxicant in rodent models^[7,9,10]. Similarly, the deterioration in protein levels in our studies by treatment with 2,4-D was also noticed indicating the loss of body and liver weights and functions in 2,4-D fed mice. Hence, this organ resulted in a loss of energy status affecting SDH and ATPase activities. Further, it is evident that glycogen levels are increased markedly in it altering the level of blood glucose levels affecting carbohydrate metabolism. Moreover, the altered phosphatase levels in the treated liver of mice substantiated pathogenic effects of this tissue in our cohort to support the report of ^[4]Nakai et al. Same results were evident in other tissues like kidney and testes by this treatment in mice and rats^[7,9,10].

Antioxidant profile of tissues is a reflection of defense state of the tissue. The toxicant 2, 4-D is known to be a stress induced agent. In the present study, the decrement level of GSH and total-SH contents might lead to cell injury and death. The reduction of total ascorbate (TAA) level also supported the stress condition imposed by this compound.Thus, this toxicant led to a reduction in non- enzymatic indicators to reflect on the imposition of oxidative stress (OS) in this vital organ.This was still noticed by an increase in lipid peroxidation levels in the liver of intoxicated mice. Other studies, reported by ^[40]Rukkumani et. al. also indicated in an increased oxidation of polyunsaturated fatty acids(PUFAs) leading to loss of membrane stability. Hence, increased lipid peroxides and reduced on enzymatic factors support a definite exerted oxidative stress (OS) that affects the activities of other protective antioxidant enzymatic machinery. Therefore, marked changes in an antioxidant enzyme such as SOD, CAT, GPx, and GR in the liver were observed. These findings were also in agreement with ^[4]Nakbi et al. (2010) who studied oxidative stress in intoxicated rats. Anusuya and Hemlatha^[41] also reported oxidative stress inducted by 2,4-D pesticide/herbicide in fish and found significant reductions in SOD and catalase levels. Further oxidative stress was accepted by the loss of mitochondrial damage in liver cells due to its accumulation to support our data^[42]. These changes in hepatic tissue with respect to toxicant accumulation resulting oxidative stress and metabolic insult leading to pathological consequences in mouse liver is justified.

Curcumin is known to be an excellent antioxidant of plant origin. It shows antioxidant property due to its unique conjugated structure containing two methoxylated phenols and an enol form of di-ketone typically accepting free radical trapping ability^[43]. Further, it also involves in the coupling reaction with lipid and subsequent intramolecular Diels-Alder reaction^[44]. Further, its derivatives des methoxy curcumin and bisdesmethoxy curcumin also have strong antioxidant properties, though curcumin is less soluble in aqueous solution^[45,46]. In our studies, curcumin at its physiological level promoted mitigative effect by inhibiting the toxicant exerted tissue oxidative damage. This is due to maintenance of the defense system indices mice and also assists in the synthesis of enzymes to negate oxidative stress imposed by 2,4-D by curcumin. Hence, most of the enzymatic and non-enzymatic factors exhibited resumption of their levels by supplementation of this product, curcumin. Therefore curcumin has a definite protective effect on this toxicant, herbicide exerted toxicity in mice.

CONFLICT OF INTEREST:

There are no conflicts of interest.

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Received on 08.09.2017 Modified on 18.10.2017

Research J. Pharm. and Tech 2018; 11(2):637-642.

DOI: 10.5958/0974-360X.2018.00119.1