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Abstract

Because of the widespread use of dichlorvos (DDVP) for domestic applications, evaluation of their toxic effects is of major concern to public health. Lycopene may lower oxidative stress by a mechanism that is not fully elucidated. The present study was undertaken to evaluate the protective efficacy of lycopene in terms of normalization of altered biochemical parameters following DDVP treatment in rats. Animals were divided into four groups. The first group was used as control, while groups 2, 3, and 4 were orally treated with lycopene (10 mg kg⁻¹ body weight (b.w.)), DDVP (1.6 mg kg⁻¹ b.w.), and DDVP plus lycopene, respectively. Results showed that oral administration of DDVP for 30 days increased the levels of lipid peroxidation markers such as malondialdehyde, 4-hydroxynonanal, and protein carbonyl content in liver. Also, a decrease in levels of vitamin C, vitamin E, and reduced glutathione was detected due to DDVP administration. These were accompanied by a decrease in the activities of antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase in the liver tissue. Moreover, DDVP increased the activities of serum transaminases, alkaline phosphatase, lactate dehydrogenase, and lipoxygenase, and the levels of bilirubin, total cholesterol, low-density lipoprotein cholesterol, triglyceride and DNA–protein crosslinks, and 8-hydroxy-2-deoxyguanosine, while decreased the level of high-density lipoprotein cholesterol. Our results provide new insights into the biochemical studies of relation between DDVP hepatotoxicity and lycopene treatment. Administration of lycopene to DDVP-treated rats reverted the status of hepatic markers to near-normal levels. These data suggest that lycopene can protect against the liver damage induced by DDVP.

Keywords

Dichlorvos lycopene liver oxidative stress antioxidants

Introduction

Organophosphate (OP) pesticides are among the most widely used synthetic chemicals for agricultural and domestic pest control. Nowadays, the extensive use of OP insecticides in agriculture and public health results in environmental pollution and a large number of acute and chronic poisoning events. For this reason, there is growing public concern about the accumulation of these insecticides in food products and water supply.¹ Dichlorvos (2,2-dichlorovinyl dimethyl phosphate; DDVP), an OP pesticide, is used throughout the world for the protection of stored products and crops and as an insecticide for public health.^2,3 DDVP is readily absorbed through all routes of exposure.⁴ DDVP exposure has been linked to substantial adverse health effects on several organ systems, including the respiratory system⁵ and reproductive system.⁶ Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants, the excessive accumulation of the reactive oxygen species (ROS), or both. Despite the potential danger of the ROS, cells have a variety of defense mechanisms to neutralize the harmful effects of free radicals.⁷

The carotenoids are a family of fat-soluble pigments that are prevalent in numerous fruits and vegetables. Lycopene, an aliphatic hydrocarbon, is one of the 600 known naturally occurring carotenoids. It is of particular interest, as it might have high potential to prevent DNA damage caused by ROS. Lycopene is an antioxidant found in tomato, tomato products, and in other red fruits and vegetables.⁸ The known protective effect of tomatoes against some types of pathologies mediated by oxidative processes has been attributed to lycopene.⁹ It is the pigment principally responsible for the characteristic deep red color of ripe tomato fruits and tomato products and has attracted attention because of its biological and physicochemical properties, especially related to its effects as a natural antioxidant.^10,11 Although it has no provitamin A activity, lycopene exhibits a physical quenching rate constant with singlet oxygen almost twice as high as that of β-carotene and 10 times as high as that of α-tocopherol.¹² This compound acts as a powerful antioxidant and is an effective agent against chronic inflammatory-related diseases, including cardiovascular diseases and some types of cancer, which involve radical formation and cytokine production, being one of the main carotenoids in numerous medicinal plants and fruits.^13,14 Lycopene has received particular attention in recent years as a result of studies, indicating that it is a highly efficient antioxidant and has a singlet oxygen and free radical scavenging capacity.^15,16 Lycopene may contribute to the prevention or amelioration of oxidative damage to cells and tissues both in vivo and in vitro.^17,18 Moreover, lycopene also exerts its effects via other mechanisms that include gene function regulation, hormone and immune modulation, carcinogen metabolism, and metabolic pathways involving phase II drug-metabolizing enzymes.¹⁹ Early research suggested the protective role of lycopene against cardiovascular disease, cancer, diabetes, osteoporosis, and male infertility. Recent scientific and clinical research has been devoted to a possible correlation between lycopene consumption and general health.¹⁵ In addition to anticarcinogenic effects, lycopene has been found to have ameliorative properties against several toxic agents, such as ionizing radiation or potent oxidants like hypochlorous acid or mercuric chloride. Treatment with lycopene attenuated induced oxidative stress returning the lipid peroxidation level, the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, and the reduced glutathione (GSH) concentration to values near to the controls. However, the mechanisms through which lycopene exerts its antioxidant properties have not yet been elucidated.^15,20

To our knowledge, there are no reports available on the effect of lycopene on DDVP-induced toxicity in experimental animals. In light of the above information, the present study has been undertaken to evaluate the ameliorating effect of lycopene on DDVP-induced biochemical alterations in the liver of rats.

Materials and methods

Chemicals

DDVP (purity 98%) was obtained from Merck (Germany). Lycopene (95%) was obtained from Sigma-Aldrich (St Louis, Missouri, USA). All the other chemicals and reagents used in our biochemical assays were of analytical reagent grade, purchased from commercial sources.

Animals

Healthy male albino Wistar rats weighing 200–220 g, bred in Central Animal House, Prince Mohamed Bin Fahd Center for Research and Consultation Studies, Dammam University, Dammam, Kingdom Saudi Arabia (KSA), were used in this study. The animals were housed eight per polypropylene cage and were maintained in accordance with the guidelines of the Council of Medical Research, Dammam University, KSA. The animals were kept at 25 ± 3°C and relative humidity (50–55%) in a 12-h light/12-h dark cycle with ad libitum access to standard rodent diet and water. The rats were allowed to acclimatize to the animal facility for at least 7 days before the start of the experiment.

Experimental design

The animals were randomly divided into four groups with eight rats in each group.

Group 1: Rats that were given corn oil at a dose of 1 mL kg⁻¹ body weight (b.w.) via oral gavage once per day for 30 days were negative control. Group 2: Rats orally treated with a daily dose of lycopene (10 mg kg⁻¹ b.w.) for 30 days according to the procedure followed by Turk et al.²¹ Lycopene dissolved in corn oil was prepared freshly each day. Group 3: Rats received DDVP (1.6 mg kg⁻¹ b.w.), which is 1/50 median lethal dose (LD₅₀) each day in corn oil and given via oral gavage to rats once per day for 30 days.²² Group 4: Rats were orally treated with DDVP as in group 3 plus a daily dose of lycopene (10 mg kg⁻¹ b.w.) as in group 2 after 2 h for 30 days.

At the end of the treatment, animals were killed by cervical decapitation under light ether anesthesia during morning hours. Trunk blood samples were collected from the killed animals and centrifuged for serum separation. The liver tissue was minced and homogenized (10%, w/v) in 0.15 M Tris–hydrochloric acid (HCl) buffer (pH 7.4) and centrifuged (3000g for 10 min).

Biochemical estimations

MDA, 4HNE, and total PCC

Malondialdehyde (MDA) and 4-hydroxynonanal (4HNE) were estimated by the method described by Jacobson et al.²³ Briefly, 200 µL aliquot of tissue homogenates (10% w/v in Tris-HCl buffer, 20 mM, pH 7.4) were transferred to 650 µL of 10.3 mM 1-methyl-2-phenylindole in acetonitrile and vortex mixed. To assay MDA + 4HNE, 150 µL of 15.4 M methanesulfonic acid was added, vortexed, and incubated at 45°C for 40 min. To assay MDA alone, 150 µL of 37% HCl was added instead of methanesulfonic acid, vortexed, and incubated at 45°C for 60 min. After incubation, samples were kept on ice, centrifuged at 9500g for 5 min and absorbance was measured at 586 nm. The levels of MDA and 4HNE are expressed as nanomole per gram tissue using extinction coefficient 1.1 × 10⁵ M⁻¹ cm⁻¹.

Total protein carbonyl content (PCC) was determined in the liver by a spectrophotometric method described by Levine et al.²⁴ Briefly, tissue homogenate was centrifuged at 10,000g for 20 min to separate cytosol, and then 0.5 mL of cytosolic fraction and 0.5 mL of trichloroacetic acid (TCA) were added. Later, 0.5 mL of 2,4-dinitrophenylhydrazine (DNPH) was added and kept for 1 h at room temperature. Pellet was washed thrice with 1 mL of ethanol–ethyl acetate mixture, and the pellet was dissolved in 1 mL of guanidine hydrochloride, and the color developed was read at 366 nm. The level of PCC was expressed as nanomole per milligram protein.

Determination of nonenzymatic antioxidants

Vitamin C concentration was measured as described by Omaye et al.²⁵ To 0.5 mL of liver homogenate, 1.5 mL of 6% TCA was added and centrifuged (10,000g, 20 min). To 0.5 mL of supernatant, 0.5 mL of DNPH reagent (2% DNPH and 4% thiourea in 9 N sulfuric acid) was added and incubated for 3 h at room temperature. After incubation, 2.5 mL of 85% sulfuric acid was added, and the color developed was read at 530 nm after 30 min.

Vitamin E was estimated by the method of Desai.²⁶ Vitamin E was extracted from liver tissue with the addition of 1.6 mL ethanol and 2.0 mL petroleum ether and centrifuged. The supernatant was separated and evaporated in air atmosphere. To the residue, 0.2 mL of 0.2% 2,2′-dipyridyl and 0.2 mL of 0.5% ferric chloride were added and kept in the dark for 5 min. An intense red-colored layer obtained on addition of 4 mL butanol was read at 520 nm.

GSH was determined by the method of Ellman.²⁷ One milliliter of supernatant was treated with 0.5 mL of Ellman’s reagent (19.8 mg of 5,5′-dithiobisnitrobenzoic acid (DTNB) in 100 mL of 0.1% sodium citrate) and 3.0 mL of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm in a spectrophotometer. To prevent the autoxidation of GSH, the samples were reduced with potassium borohydride prior to analysis.

Assay of antioxidant enzymes

SOD activity was determined by the method of Kakkar et al.²⁸ Tissue homogenate (0.5 mL) was diluted with 1 mL of water. In this mixture, 2.5 mL of ethanol and 1.5 mL of chloroform (all reagents chilled) were added and shaken for 1 min at 4°C and then centrifuged. The enzyme activity in the supernatant was determined. The assay mixture contained 1.2 mL of sodium pyrophosphate buffer (0.025 M, pH 8.3), 0.1 mL of 186 µM phenazine methosulfate, 0.3 mL of 30 µM nitroblue tetrazolium (NBT), 0.2 mL of 750 µM nicotinamide adenine dinucleotide (NADH), appropriately diluted enzyme preparation and water in a total volume of 3 mL. Reaction was started by the addition of NADH. After incubation at 30°C for 90 min, the reaction was stopped by the addition of 1 mL glacial acetic acid. The reaction mixture was stirred vigorously and shaken with 4 mL of n-butanol. The intensity of the chromogen in the butanol layer was measured at 560 nm against butanol blank. A system devoid of enzyme served as control. One unit of the enzyme activity was defined as the enzyme reaction, which gave 50% inhibition of NBT reduction in 1 min under the assay conditions.

The activity of CAT was determined by the method followed by Sinha.²⁹ The reaction mixture (1.5 mL) contained 1.0 mL of 0.01 M phosphate buffer (pH 7.0), 0.1 mL of tissue homogenate, and 0.4 mL of 2 M hydrogen peroxide (H₂O₂). The reaction was stopped by the addition of 2.0 mL of dichromate–acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio). Then, the absorbance was read at 620 nm: CAT activity was expressed as micromoles of H₂O₂ consumed per minute milligram protein.

GSH-Px activity was estimated by the method of Rotruck et al.³⁰ Briefly, the reaction mixture contained 0.2 mL of 0.4 M phosphate buffer (pH 7.0), 0.1 mL of 10 mM sodium azide, 0.2 mL tissue homogenized in 0.4 M phosphate buffer (pH 7.0), 0.2 mL glutathione, and 0.1 mL of 0.2 mM H₂O₂. The contents were incubated for 10 min at 37°C, 0.4 mL 10% TCA was added to stop the reaction, and centrifuged at 3200g for 20 min. The supernatant was assayed for GSH content using Ellman’s reagent (19.8 mg, DTNB in 100 mL 0.1% sodium nitrate). The activity was expressed as microgram of GSH consumed per minute milligram protein.

The glutathione-S-transferase (GST) activity was determined spectrophotometrically by the method of Habig et al.³¹ The reaction mixture contained 1.0 mL 100 mM phosphate buffer (pH 6.5), 0.1 mL 30 mM 1-chloro-2,4-dinitrobenzene (CDNB), and 0.7 mL double-distilled water. After preincubating the reaction mixture for 5 min at 37°C, the reaction was started by the addition of 0.1 mL tissue homogenate and 0.1 mL of GSH as substrate. After 5 min, the absorbance was read at 340 nm. Reaction mixture without the enzyme was used as a blank. The activity of GST was expressed as micromoles of GSH-CDNB conjugate formed per minute per milligram protein using an extinction coefficient of 9.6 mM⁻¹ cm⁻¹.

Determination of liver function parameters

The activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and total bilirubin were assayed spectrophotometrically according to the standard procedures using commercially available diagnostic kits (Sigma diagnostics (I) Pvt. Ltd, Baroda, Gujarat, India). Gamma glutamyl transferase (GGT) activity was determined by the method of Rosalki et al.³² using γ-glutamyl-p-nitroanilide as substrate. Total cholesterol (Tc), low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), and triglyceride (Tg) were assessed in serum using a commercially available spectrophotometric enzymatic kit (Thermo Trace-BECGMAN, Germany) and analyzed by an autoanalyzer (Bayer ope-RA).

DPC determination

DNA-protein cross-links (DPCs) were measured using a previously described procedure.³³ Briefly, DNA was released from suspended cells after exposure to sodium dodecyl sulfate and sheared into fragments of relatively uniform length by repeated aspiration using a pipette. DNA wash buffer (0.1 M potassium chloride, 0.1 mM ethylenediamine tetraacetic acid, 20 mM Tris-HCl, pH 7.4) was added to the mixture, and then centrifuged at 100,000g for 10 min, and protein-bound DNA precipitated. Then, 1 mL supernatant was washed and precipitated again. After washing thrice, the final pellet was resuspended and incubated with proteinase K at 50°C for 3 h to digest protein and leave the protein-bound DNA intact. Thus, 3 mL of free DNA and 1 mL of protein-bound DNA was separated and collected. Fluorescence measurement is at an excitation wavelength of 350 nm and an emission wavelength of 460 nm. Finally, a DPC coefficient was expressed as the percentage of protein-DNA to total DNA (free DNA + protein-bound DNA).

8-OHdG determination

Levels of 8-hydroxy-2-deoxyguanosine (8-OH-dG) in liver supernatants were measured using enzyme-linked immunosorbent assay kit.³⁴ All procedures were conducted according to manufacturer’s instructions. Concentrations were determined in duplicate for each sample.

Measurement of LOX activity

Hepatic lipoxygenase (LOX) activity was determined by measuring the increase in absorbance at 234 nm produced by the synthesis of hydroperoxy derivatives from arachidonic acid.³⁵

Protein determination

Protein in the tissue samples was precipitated by 10% TCA and dissolved in 0.1 N sodium hydroxide for determination. Protein was determined by the method of Lowry et al.³⁶ using bovine serum as standard.

Statistical analysis

Data were analyzed by one-way analysis of variance, followed by Duncan’s multiple range test (DMRT) using a statistically software package (SPSS for Windows, V.13.0, Chicago, USA). Results were presented as mean ± SD, p < 0.05 and were considered as statistically significant.³⁷

Results

Effect on MDA, 4HNE, and PCC levels

Exposure of animals to DDVP alone caused induction of oxidative stress in liver as evidenced by significant (p < 0.001) increase in the levels of MDA, 4HNE, and PCC, the major end products of lipid peroxidation as compared to negative control group (Table 1). The observed increase was markedly high in 4HNE (+225.88%) and PCC (+221.76%) when compared with increase in MDA (+78.19%) levels. Lycopene treatment alone caused no significant changes of these parameters when compared with the negative control (p > 0.05). In addition, the treatment of lycopene with DDVP reduced the increases in liver MDA (−18.98%), 4HNE (−42.21%), and PCC (−44.60%) levels as compared to DDVP-intoxicated group; however, they were not at control levels.

Table 1.

Effects of lycopene treatment on MDA, 4HNE, and total PCC levels in liver of DDVP intoxicated rats.^a

Groups	Control	Lycopene (10 mg kg⁻¹)	DDVP (1.6 mg kg⁻¹)	DDVP (1.6 mg kg⁻¹) + lycopene (10 mg kg⁻¹)
MDA (nmol g⁻¹)	9.22 ± 0.53^b	10.02 ± 0.62^b	16.43 ± 0.64^c	13.31 ± 0.51^d
4HNE (nmol g⁻¹)	2.82 ± 0.21^b	3.22 ± 0.10^b	9.19 ± 0.42^c	5.31 ± 0.54^d
PCC (nmol mg⁻¹)	1.47 ± 0.12^b	1.21 ± 0.12^b	4.73 ± 0.41^c	2.62 ± 0.17^d

MDA: malondialdehyde; 4HNE: 4-hydroxynonanal; PCC: protein carbonyl content; DDVP: 2,2-dichlorovinyl dimethyl phosphate; DMRT: Duncan’s multiple range test.

^aValues are given as mean ± SD from eight rats in each group. Values not sharing a common superscript letters (b, c, and d) differ significantly at p < 0.001 (DMRT).

Effect on nonenzymatic antioxidants

The levels of vitamin C, vitamin E, and GSH were significantly decreased in DDVP-treated rats by −50%, −47.61%, and −41.94%, respectively, in comparison with the control group (p < 0.01; Table 2). Administration of lycopene along with DDVP significantly (p < 0.01) increased the levels of nonenzymatic antioxidants by +41.86%, +45.45%, and +37%, respectively, in the liver, as compared to positive control group, without reaching the control level.

Table 2.

Effects of lycopene treatment on the levels of nonenzymatic antioxidant status in liver of DDVP-intoxicated rats.^a

Groups	Control	Lycopene (10 mg kg⁻¹)	DDVP (1.6 mg kg⁻¹)	DDVP (1.6 mg kg⁻¹) + lycopene (10 mg kg⁻¹)
Vitamin C (µmol mg⁻¹ tissue)	0.86 ± 0.05^b	0.92 ± 0.07^b	0.43 ± 0.05^c	0.61 ± 0.06^d
Vitamin E (µmol mg⁻¹ tissue)	0.42 ± 0.03^b	0.48 ± 0.05^b	0.22 ± 0.02^c	0.32 ± 0.03^d
GSH (mg g⁻¹ tissue)	3.91 ± 0.11^b	4.22 ± 0.43^b	2.27 ± 0.26^c	3.11 ± 0.26^d

DDVP: 2,2-dichlorovinyl dimethyl phosphate; DMRT: Duncan’s multiple range test; GSH: reduced glutathione.

^aValues are given as mean ± SD from eight rats in each group. Values are not sharing a common superscript letters (b, c, and d) differ significantly at p < 0.01 (DMRT).

Effect on antioxidant enzymes

As shown in Table 3, administration of DDVP alone caused a significant (p < 0.05) decrease in the activities of SOD (−44.85%), CAT (−48.63%), GPx (−28.36%), and GST (−38.60%) in liver tissue in comparison to negative control group. Administration of lycopene alone caused insignificant change in the activities of these antioxidant enzymes compared to control rats. In addition, a significant recovery relating to these parameters was observed (+70.81%, +80.65%, +50.42% and +66.32%) for SOD, CAT, GPx, and GST, respectively, in response to the presence of lycopene with DDVP as compared to positive control group.

Table 3.

Effects of lycopene treatment on hepatic SOD, CAT, GPx, and GST activities in DDVP-intoxicated rats.^a

Groups	Control	Lycopene (10 mg kg ⁻¹ )	DDVP (1.6 mg kg⁻¹)	DDVP (1.6 mg kg⁻¹) + lycopene (10 mg kg⁻¹)
SOD (units per milligram protein)	11.37 ± 0.62^b	10.73 ± 0.25^b	6.27 ± 0.21^c	10.71 ± 0.47^b
CAT (µmol min⁻¹ mg⁻¹ protein)	67.62 ± 13.66^b	71.57 ± 12.24^b	34.73 ± 5.22^c	62.74 ± 9.71^b
GPx (µg min⁻¹ mg⁻¹ protein)	16.36 ± 3.15^b	17.72 ± 3.21^b	11.72 ± 1.11^c	17.63 ± 2.36^b
GST (µmol min⁻¹ mg⁻¹ protein)	14.22 ± 1.25^b	15.16 ±2.11^b	8.73 ± 1.36^c	14.52 ± 1.47^b

SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GST: glutathione-S-transferase; DDVP: 2,2-dichlorovinyl dimethyl phosphate; DMRT: Duncan’s multiple range test.

^aValues are given as mean ± SD from eight rats in each group. Values not sharing a common superscript letters (b, c, and d) differ significantly at p < 0.05 (DMRT).

Effect on liver functional markers

DDVP treatment caused abnormal liver function in all rats. Activities of serum hepatospecific enzymes such as AST, ALT, ALP, LDH, and GGT, and the level of bilirubin were significantly (p < 0.05) increased in DDVP-treated rats by +54.52%, +81.47%, +36.62%, +43.12%, +58.44%, and +39.70%, respectively, as compared to negative control. Treatment with lycopene alone did not cause any significant changes in the activity of these enzymes (p > 0.05). Treatment of lycopene (10 mg kg⁻¹) with DDVP exhibited a significant recovery as it decreased the activities of AST (−19.72%), ALP (−19.93), and bilirubin (−17.89%) to near the control values, although it had no effect on LDH activity (−6.85%) when compared to DDVP-treated group (Table 4).

Table 4.

Effects of lycopene treatment on the activities of serum hepatic markers in DDVP intoxicated rats.^a

Groups	Control	Lycopene (10 mg kg ⁻¹ )	DDVP (1.6 mg kg⁻¹)	DDVP (1.6 mg kg⁻¹) + lycopene (10 mg kg⁻¹)
AST (U L⁻¹)	42.22 ± 3.24^b	45.61 ± 3.15^b	65.24 ± 4.31^c	52.37 ± 3.27^b
ALT (U L⁻¹)	26.24 ± 2.33^b	27.36 ± 1.71^b	47.62 ± 4.15^c	31.52 ± 2.26^d
ALP (U L⁻¹)	83.52 ± 6.38^b	85.37 ± 6.72^b	114.11 ± 6.44^c	91.36 ± 5.88^b
LDH (U L⁻¹)	113.63 ± 8.52^b	118.39 ± 9.33^b	162.63 ± 11.35^c	151.48 ± 10.71^c
GGT (U L⁻¹)	0.77 ± 0.09^b	0.75 ± 0.07^b	1.22 ± 0.09^c	0.92 ± 0.06^d
Bilirubin (mg dL⁻¹)	0.68 ± 0.05^b	0.71 ± 0.03^b	0.95 ± 0.07^c	0.78 ± 0.04^b

DDVP: 2,2-dichlorovinyl dimethyl phosphate; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase, LDH: lactate dehydrogenase; GGT: gamma glutamyl transferase; DMRT: Duncan’s multiple range test.

^aValues are given as mean ± SD from eight rats in each group. Values not sharing a common superscript letters (b, c, and d) differ significantly at p < 0.05 (DMRT).

Effect on lipid profiles

DDVP treatment caused significant increase in serum levels of Tc (+42.57%; p < 0.01), LDL-c (+90.39%; p < 0.001), and Tg (+63.80%; p < 0.001), and significant decrease in HDL–c (−18.40%; p < 0.05) as compared to negative control (Figure 1). Results revealed that there are no significant changes in lipid profile of rats treated with lycopene alone, while the presence of lycopene with DDVP alleviated the adverse effects of DDVP as it restored the altered lipid profiles to the normal values.

Figure 1.

Effects of lycopene treatment on serum Tc, LDL-c, HDL-c, and Tg levels in DDVP-intoxicated rats. Values are given as mean ± SD of eight rats in each group. Values not sharing a common superscript letters (a, b, and c) show statistically significant differences in Tc (p < 0.01), LDL-c (p < 0.001), HDL-c (p < 0.05), and Tg (p < 0.001; DMRT). Tc: total cholesterol; LDL-c: low-density lipoprotein-cholesterol; HDL-c: high-density lipoprotein-cholesterol; Tg: triglyceride; DDVP: 2,2-dichlorovinyl dimethyl phosphate; DMRT: Duncan’s multiple range test.

Effect on DNA damage

DNA damage was evaluated to assess the genotoxicity of DDVP. The DPC coefficient can be increased in oxidative stress. The covalent crosslinking of proteins to DNA presents a major physical challenge to the metabolic machinery of DNA. A significant (p < 0.01) increase in the DPC coefficient (+56.25%) and 8-OHdG level (+62.36), indicators of DNA damage, in liver tissue was observed in DDVP-treated group compared to negative control (Figures 2 and 3). Lycopene treatment with DDVP significantly decreased levels of DPC (−20%) and 8-OH-dG (−14.67) when compared to those seen in the DDVP-treated group (p < 0.01).

Figure 2.

Effects of lycopene treatment on DPCs coefficient of liver homogenates of DDVP-intoxicated rats. Values are given as mean ± SD of eight rats in each group. Values not sharing a common superscript letters (a, b, and c) differ significantly at p < 0.01 (DMRT). DPC: DNA-protein crosslink; DDVP: 2,2-dichlorovinyl dimethyl phosphate; DMRT: Duncan’s multiple range test.

Figure 3.

Effects of lycopene treatment on 8-OH-dG level of liver homogenates of DDVP-intoxicated rats. Values are given as mean ± SD of eight rats in each group. Values not sharing a common superscript letters (a, b, and c) differ significantly at p < 0.01 (DMRT). 8-OH-Dg: 8-hydroxy-2-deoxyguanosine; DDVP: 2,2-dichlorovinyl dimethyl phosphate; DMRT: Duncan’s multiple range test.

Effect on the enzymatic activity of LOX

Our data (Figure 4) showed a significant (p < 0.001) increase in LOX activity (+71.55) in DDVP-treated rats when compared with the controls. However, lycopene treatment with DDVP minimized the DDVP-induced alteration of enzyme activity by 37.3% as compared to the positive control group.

Figure 4.

Effects of lycopene treatment on hepatic LOX activity of DDVP intoxicated rats. Values are given as mean ± SD from eight rats in each group. Values not sharing a common superscript letters (a, b, and c) differ significantly at p < 0.001 (DMRT). LOX: lipoxygenase; DDVP: 2,2-dichlorovinyl dimethyl phosphate; DMRT: Duncan’s multiple range test.

Discussion

Results of the present study showed that DDVP treatment generated oxidative stress in rat liver tissue as evidenced by significant elevation of lipid peroxidation products MDA, 4HNE, and PCC. OP pesticides induced peroxidative damage of membranes and accumulation of lipid peroxidation products has been reported in cells, tissues, and serum of rats.^15,38 The impact of lipid peroxidation on membrane lipids, membrane receptors, and membrane-bound enzymes can alter the function, structure, and fluidity of membranes and may result in altered ion flux.² Increased lipid peroxidation products, such as 4HNE, contribute to conditions associated with oxidative stress.²³ Increased oxidative stress represents an imbalance between intracellular production of free radicals and the cellular defense mechanisms. Extensive research demonstrated that OP pesticides cause oxidative stress in a dose- and time-dependent manner and increase levels of MDA.³⁸ The enhanced production of liver lipid peroxides observed in our study is in agreement with other studies.⁸ Lycopene may protect against the in vivo oxidation of lipids and proteins.³⁹ The data show that oral coadministration of lycopene decreased the MDA, 4HNE, and PCC levels although their values still were more than that of the baseline. However, this decrease in lipid peroxidation products may be due to the free radical scavenging properties of lycopene.

Exposure to OP is often followed by impairment of the antioxidant defense and characterized by the depletion of tissue and circulating nonenzymatic antioxidants, including GSH, vitamin C, and vitamin E.⁴⁰ OPs interact with sulfhydryl groups of GSH leading to its inactivation.¹ In this study, the decreased level of GSH caused by DDVP toxicity increased the susceptibility of the liver to free radical damage. The central role of GSH in antioxidative defense may be attributed to its ability to regenerate another water-soluble antioxidant, ascorbic acid, via the ascorbate–glutathione cycle.⁴¹ Hence, depletion of intracellular GSH is usually regarded a measure of oxidative stress. Vitamin C and vitamin E are the other major nonenzymatic antioxidants having synergetic action in scavenging oxygen derived free radicals, and these vitamins are likely to be most susceptible to free radical oxidation. Our study showed that the levels of these vitamins decreased significantly in the liver of DDVP-treated rats. It might contribute to the development of pesticide-induced hepatic damage.³⁸

Oral administration of DDVP (1/50 LD₅₀) for 30 days decreased SOD, CAT, GPX, and GST activities in rat liver, consistent with the study of Ogutcu et al.⁴² In the DDVP group, low levels of these enzymatic antioxidants might be related to the consumption of these enzymes due to increased oxidative stress in the liver, therefore increased usage in scavenging free radicals induced by the pesticide, thus causing irreversible inhibition in their activities.⁴³ The levels of these antioxidants might provide a clear indication on the extent of cytotoxic damage.¹⁵ Diminished in the activities of these antioxidants upon DDVP exposure may lead to increased oxidative modifications of cellular membrane and intracellular molecules. The basis of OP toxicity in the production of oxidative stress may be due to either their “redox cycling” activity, where they readily accept an electron to form free radicals and then transfer them to oxygen to generate superoxide anions and then H₂O₂ through dismutation reactions or to ROS generation via changes in normal antioxidant homeostasis resulting in antioxidant depletion if the requirement of continuous antioxidants is not maintained.³⁹ Previous studies have already demonstrated oxidative damage following chronic administration of DDVP,⁴⁴ supporting this observation. However, treatment of lycopene with DDVP increased the enzymatic antioxidant activities and the levels of nonenzymatic antioxidants in the liver of the DDVP-treated rats. The present results showed that lycopene enhanced antioxidant capacity, thus protecting the liver against the DDVP-induced damages, as shown by the maintenance of the enzymatic activities. Yonar and Sakin⁴⁵ suggested that the administration of lycopene might alleviate deltamethrin-induced oxidative stress. It may also contribute to the prevention or amelioration of oxidative damage to cells and tissues both in vivo and in vitro.^17,18 Moreover, studies have suggested that the anticancer effects of lycopene are related to their effectiveness as antioxidants.⁴⁶ Treatment with lycopene may have increased the GSH levels via increased GSH biosynthesis or by increasing the levels of other antioxidants. In addition, Bose and Agrawal⁴⁷ reported that lycopene elevated the concentrations of GSH, which plays a major role in maintaining high GPx and GST activities.

Liver injury following OP exposure is well established,⁴² and the hepatic dysfunction is followed by the elevated levels of serum marker enzymes indicating cellular leakage and loss of functional integrity of hepatic membrane.⁴⁸ This correlates with our results, which showed increased activities of AST, ALT, and ALP in the serum of DDVP-treated rats. Serum LDH, a cytoplasmic marker enzyme, and GGT are other well-known indicators of cell and tissue damage by toxic substances and their levels were also substantially increased in DDVP intoxicated rats. The observed elevation in the concentration of serum bilirubin in DDVP-treated rats is also consistent with the presence of hepatic damage. In contrast, the administration of lycopene along with DDVP treatment lowered the serum activities of these enzymatic markers. However, lycopene treatment with DDVP showed insignificant difference in LDH activity when compared with those seen in the DDVP-treated group. The elevation in serum LDH activity after lycopene treatment may be due to the isoforms secreted by different organs such as kidney, thymus, and adrenal, gland but not for the isoform that is secreted by liver cells.^49,50 OP insecticides generally cause an increase in Tc and total lipid levels.^51,52 In this study, DDVP caused an increase in the Tc, LDL-cholesterol, and Tg levels. The increase in the level of serum Tc may be attributed to the effects of the pesticide on the permeability of liver cell membranes, the blockage of the liver bile ducts, causing a reduction or cessation of cholesterol secretion into the duodenum.⁵³ The present study supports the results of other investigations; an increase in the serum cholesterol level may be a sign of liver damage. Some pesticides cause a decrease in the HDL-c and increase in Tg levels.⁵² Our data suggested the possibility that lycopene provides protection against DDVP-induced liver injury and lipidemic profiles in rats by altering liver function, resulting in a change in lipid synthesis and lipid metabolism, as a consequence, exerts an in vivo hepatoprotective effect.

In this study, the level of 8-OHdG was increased markedly in the liver of DDVP-treated rats, which correlated with the level of ROS, suggesting that DNA is a common target of ROS induced by DDVP in liver. This oxidized DNA product is important because it is relatively easily formed and is mutagenic and carcinogenic. It is a good biomarker of oxidative stress of an organism and a potential biomarker of carcinogenesis.⁴¹ Proteins can bind to DNA to form DPC, which is associated with DNA damage. DNA damage can result in the arrest or induction of transcription, induction of signal transduction pathways, replication errors, and genomic instability.^54
–56 The present study showed DNA damage following DDVP treatment as indicated by increase in DPC coefficient. Our results are consistent with several studies showing that OP increases levels of the indicators of DNA damage or leads to oxidative damage to various tissues or cells.⁵² Excessive levels of ROS and oxidative stress in cells and tissues induce damage to DNA molecules, which can lead to tissue lesions.⁵⁷ Lycopene treatment for 30 days along with DDVP provided better protection against the DNA damage of liver cells versus DDVP-treated group. However DNA damage detected was the result of oxidative stress. Lycopene may protect in vivo DNA against oxidation.^10,18 Kong et al.⁵⁸ reported that lycopene is a suitable agent for preventing chemically induced DNA and chromosome damage. It also significantly reduced the genotoxicity induced by H₂O₂ in vitro.

It is known that 5-, 12-, and 15-LOX is overexpressed in experimental liver disease⁵⁹ and that LOX inhibitors show a partial hepatoprotective effect in several toxicants that induced hepatopathy.⁶⁰ Our data clearly show an increase in LOX activity in DDVP-intoxicated rats when compared with the controls. To our knowledge, it is the first record on the LOX activity as an indicator of DDVP hepatotoxicity. When rats were cotreated with lycopene, LOX activity was significantly reduced. In previous in vitro studies, it has been shown that LOX interacts directly with natural antioxidants, such as resveratrol, quercetin, and phenolic compounds.^15,61 The results obtained in this work reveal that lycopene contributes to hepatic protection by inhibiting the LOX pathway, as it is a feasible interaction partner with LOX.

In conclusion, the present data recorded that DDVP intoxication enhanced lipid peroxidation in liver of rats. This was associated with decreased levels of GSH, vitamin C, and vitamin E, and activities of antioxidant enzymes (SOD, CAT, GPx, and GST) in the liver. Also, it is capable of inducing marked alterations in some biochemical parameters as well as DNA damage. However, the coadministration of lycopene with DDVP attenuated the observed harmful effects of DDVP on the tested biochemical and antioxidant parameters. On the basis of this study, lycopene cotreatment contributes favorably to maintaining the equilibrium between lipid peroxidation induction and antioxidant enzymatic activities. Accordingly, care must be taken to avoid mammalian and human exposure to DDVP, and attention should be paid to sources of its residues in foods as well as occupational sources. Also, it should be taken into consideration that supplementation of natural antioxidant such as lycopene may act as a protective agent against the toxicity of DDVP.

Footnotes

Acknowledgments

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no. RGP-VPP 297.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

Deanship of Scientific Research at King Saud University, research group no. RGP-VPP 297.

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Lycopene attenuates dichlorvos-induced oxidative damage and hepatotoxicity in rats

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Animals

Experimental design

Biochemical estimations

MDA, 4HNE, and total PCC

Determination of nonenzymatic antioxidants

Assay of antioxidant enzymes

Determination of liver function parameters

DPC determination

8-OHdG determination

Measurement of LOX activity

Protein determination

Statistical analysis

Results

Effect on MDA, 4HNE, and PCC levels

Effect on nonenzymatic antioxidants

Effect on antioxidant enzymes

Effect on liver functional markers

Effect on lipid profiles

Effect on DNA damage

Effect on the enzymatic activity of LOX

Discussion

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

References