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Abstract

The toxicity of many heavy metals is due to their ability to cause oxidative damage to tissues. Lead is one of the most important metals that pollute the natural environment due to man’s impact The aim of this study is to investigate the potential protective effect of epicatechin alone or combined with lycopene against toxicity of lead in male rats. Five groups of rats were involved in this study; the first was control while the other four injected with lead acetate (100 mg/kg BW) subcutaneous for 2 weeks. On the other hand, the third, fourth and fifth groups were injected with epicatechin, lycopene or epicatechin + lycopene, respectively. Results obtained showed that, the combined treatment (epicatechin + lycopene) exert its effects (100%) against toxic effects against lead by lowering the liver enzymes alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and gamma glutamyle transferase (GGT) activities and decrease lipid peroixdation (MDA) and enhances the superoxide dismutase (SOD) activity. The high-density lipoprotein cholesterol (HDL-c) level was significantly decreased and low-density lipoprotein cholesterol (LDL-c) level was statistically significantly increased in lead-injected rats as compared with control group. The combined treatment with epicatechin and lycopene justify these levels to nearly normal values. The erythrocyte level of total glutathione was decreased in lead-injected rats as compared with control group (p < 0.001). The combined effect is significantly higher than individual treatment lycopene alone or epicatechin. A negative correlation was found between the blood lead and SOD (r = −0.6) and glutathione (r = −0.81) while a positive correlation with MDA level (r = 0.7).

Keywords

lycopene epicatechin lead toxicity

Introduction

Heavy metals cause multiple direct and indirect effects on physiological processes.¹ The toxicity of many heavy metals is due to their ability to cause oxidative damage to tissues and includes enhanced lipid peroxidation, DNA damage and the oxidation of protein sulfhydral groups.² Lead as an environmental and occupational toxicant has been known to damage vital organs and suppress cellular processes.³ The lead-induced toxicity has been explained in different ways. Due to lead competition with essential metals like calcium and zinc, and its high affinity to thiol groups in proteins; the production of reactive oxygen species⁴, lead promote depressing endogenous and enhancing lipid peroxidation.

Lead poisoning causes renal dysfunction, liver cirrhosis, damage to the central nervous system and anemia.⁵ A free radical may be defined as any molecule that has one or more unpaired electrons. Reactive oxygen radicals such as the superoxide anion (O₂ ^_.) the hydroxyl radical (OH^.), the hypochlorite radical (OHCl^.) and hydrogen peroxide (H₂O₂).⁶

Toxic metals increase production of free radicals and decrease availability of antioxidant reserves to respond to the resultant damage. The pathogenesis of lead toxicity is multifactorial, as lead directly interrupts enzyme activation, competitively inhibits trace mineral absorption, binds to sulfhydryl proteins (interrupting structural protein synthesis), alters calcium homeostasis and lowers the level of available sulfhydryl antioxidant reserves in the body.⁷ The etiology of lead toxicity-induced hypertension reveals that the free radical production and lowering of inherent antioxidant reserves resulting from lead toxicity are directly related to vasoconstriction underlying lead-induced hypertension.⁸ The mechanisms of lead-related pathologies, many of which are a direct result of the oxidant effect of lead on tissues and cellular components, may be mitigated by improving the cellular availability of antioxidants.⁹

Organisms have developed many defense mechanisms to protect themselves from injuries by ROS. The small molecule antioxidants, such as vitamin E and vitamin C are able to interact with oxidizing radicals directly.¹⁰

Epicatechin (EC) is one of the most potent antioxidants present in the human diet. Particularly high levels of this compound are found in tea, apples and chocolate. It has been reported that tea extracts and/or its constituents have antibacterial, antiviral, anti oxidative, anti-tumor¹¹ and antimutagenic¹² activities. Also, it was shown that green tea extract can scavenge NO and O₂ ⁻ very effectively.¹³ EC is among the major constituents responsible for the protective and antioxidant biomedical effect exhibited by green tea.

EC did not damage rat liver function after being used for a long term, and it indicated that TPs were a quite safe agent, even at a high dose of 833.3 mg·kg^-1·d^-1, for 6 months.¹⁴ Lycopene, the pigment principally responsible for the characteristic deep-red color of ripe tomato fruits and tomato products, has received much attention in recent years because of its beneficial effect in the treatment of diseases.¹⁵ It was demonstrated that lycopene provided the best protection against singlet oxygen-induced cell damage.^16,17

Epidemiologic studies revealed that, lycopene may increasingly be identified as sharing inverse relationships to cancer with other common carotenoids or as being the only carotenoid to show such an association.¹⁸

Our previous study reported that lycopene in combination with melatonin inhibit the mammary carcinogensis in female rats induced by dimethybenzanthracene.¹⁹ Lycopene in human plasma has a half life of about 2−3 days.¹⁹ Only a few metabolites, such as 5,6-dihydroxy-5,6-dihydro lycopene, have been detected in human plasma.²⁰ Owing to its lipophilic nature, lycopene was found to concentrate in LDL and VLDL fractions and not in HDL fraction of the serum.²¹

Research objective

Aim of the work

The present study aimed to investigate the potential protective role of EC alone or combined with lycopene in reducing the oxidative stress, toxicological and morphological changes in the liver and kidneys of rats injected with lead acetate.

Materials and methods

Animals

Forty adult male Sprague–Dawley rats weighing about 150 g were used in this study. All animals were kept under the same laboratory conditions of temperature (25 ± 2°C) and lighting (12:12 h light: dark cycle) and were given free access to standard laboratory chow and tap water.

Experimental protocol

The rats were divided into five groups (eight rats each).

The first group was served as controls and will receive a subcutaneous injection of physiological saline.

The second group was given subcutaneous injection of lead acetate at a dose of 100 mg/kg body weight.

The third group was given a similar injection of lead acetate, which was preceded, 30 min earlier, by a subcutaneous EC injection with a dose of 10 mg/kg body weight in olive oil.

The fourth group was given a similar injection of lead acetate, which will be preceded, 30 min earlier, by a subcutaneous lycopene injection with a dose of 10 mg/kg in olive oil body weight.

The fifth group was given a similar injection of lead acetate, which will be preceded, 30 min earlier, by a subcutaneous EC and lycopene injection.

Olive oil is commonly employed as a solubilizing agent for lipophilic materials in preclinical studies in rodents.

All of these injections were repeated daily for 14 days. At the end of the treatment period, the rats were sacrificed and blood was collected, divided into two parts, the first for serum separation and the second on heparin for plasma separation. The liver and kidneys were removed and divided into two portions; one part stored at −40°C and the other fixed for histology.

Analysis

Blood Pb was determined by atomic absorption spectrophotometer (Perkin Elmer Zeeman 5100; Perkin Elmer, Norwalk, Connecticut, USA) using an HGA-600 graphite furnace Blood samples were diluted into a ratio of 1:5 with a diluent containing 0.1% Triton X-100 and 0.1% HNO₃ in distilled deionized water.²² The absorption wave length is 284 nm.

Biochemical analysis

Serum total lipids were determined colorimetrically with sulfophosphovanillinic mixture.²³ Serum triglycerides, cholesterol, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), total protein, glucose and the activities of ALT, AST, GGT and ALP were determined automatically using Hitachi auto analyzer 917 (7170) using kits from Biosystem.

Measurement of lipid peroxidation (malondialdhyde)

The level of malondialdhyde (MDA) was determined using thiobarbituric acid and the colored read at 586 nm and 1,1,3,3,-tetramethoxypropane as standard.²⁴

Superoxide dismutase activity

Superoxide dismutase (SOD) activity was estimated based on its ability to inhibit the autooxidation of epinephrine in an alkaline medium (pH 10.2).²⁵

Determination of reduced glutathione

Glutathione was measured using the method of Beutler et al.²⁶ This method is based on the development of a stable yellow color when 2-nitrobenzoic acid is added to sulfhydryl compounds.

Statistical analysis

All the values are presented as means ± SD. One-way analysis of variance (ANOVA) was used, followed by parametric analysis for mean comparisons between each pair, of the JMP statistical package (SPSS version 8).

Results

Table 1 shows that blood lead level and the activities of ALT AST, ALP and GGT enzymes were highly significantly elevated in lead-administrated rats as compared with normal control group (p < 0.001, p < 0.001, p < 0.05, p < 0.001 and p < 0.01, respectively). Administration of EC or lycopene or combined lycopene + EC normalize the parameters to the control level, but the effect of combined action was higher than individual treatment (p < 0.01). There were non-significant changes in the levels of total proteins and glucose level in the studied groups as compared with control group.

Table 1.

Blood lead, serum levels of glucose, total protein and activities of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and gamma glutamyle transferase (GGT) in the studied groups (Mean ± SD)

Parameters	Animal groups
Parameters	(GpI) Control normal	(GpII) Lead	(GpIII) Lead + epicatechin	(GpIV) Lead+ lycopene	(GpV) Lead + epicatechin + lycopene
Lead (μg/dL)
Mean ± SD	0.25 ± 0.01	0.41 ± 0.05	0.36 ± 10	0.31 ± 0.02	0.21 ± 0.01
p Value^a	---	<0.001	<0.01	<0.001	NS
p ^b	--	---	<0.001	<0.001	<0.001
p ^c	---	---	----	---	<0.001
Total protein (mg/dL)
Mean ±SD	5.9 ± 0.9	5.5 ± 0.6	5.1 ± 0.5	6.1 ± 0.9	6.1 ± 0.9
p Value^a	---	NS	NS	NS	NS
p ^b		--	NS	NS	NS
AST (U/L)
Mean ±SD	25 ± 2.3	50 ± 2.0	44 ± 2.5	41 ± 2.6	32 ± 2.6
p value^a	-----	<0.001	<0.001	<0.001	NS
p ^b	-----	-------	<0.001	<0.001	<0.001
p ^c			-----	-----	<0.001
ALT (U/L)
Mean ±SD	22 ± 2.1	46 ± 1.1	39 ± 2.2	32 ± 2.5	28 ± 2.5
p Value^a	---	<0.05	<0.05	<0.01	NS
p ^b		--	<0.05	NS	NS
p ^c		---	----	---	<0. 01
ALP (mg/dL)
Mean ± SD	140 ± 13.2	230 ± 16.8	201 ± 20.0	180 ± 13	160 ± 18
p Value^a		<0.001	<0.01	<0.05	<0.001.
p ^b		--	<0.01	<0.01	<0.01
p ^c		----	---	---	<0.05
GGT(mg/dL)
Mean ± SD	31 ± 3.2	49 ± 1.8	38 ± 2.0	34 ± 3.1	32 ± 3.1
p Value^a		<0.01.	<0.05	NS	NS
p ^b		--	<0.01	NS	NS
Glucose (mg/dL)
Mean ± SD	78 ± 4.5	82 ± 3.3	84 ± 10	79 ± 5.1	80 ± 5.1
p Value^a	---	NS	NS	NS	NS

NS: non-significant.

^a p: compared with control group.

^b p: compared with lead group.

^c p: group V compared with groups III and IV. p < 0.05 was considered as significant.

Serum lipid profiles in (Table 2 ) showed amazing results; total lipids, triglycerides and total cholesterol were non-significantly changed in the studied groups as compared with control one while HDL-c level was significantly decreased and LDL-c level was statistically significantly increased in lead-injected rats as compared with control group. The combined treatment with EC and lycopene justify these levels to nearly normal values.

Table 2.

Serum levels of total, lipids, cholesterol, triacylglycerol, HDL-c and LDL-c in the studied groups (Mean ± SD)

Groups	(GpI) control normal	(GpII) lead	(GpIII) lead + epicatechin	(GpIV) lead + lycopene	(GpV) lead + epicatechin + lycopene
Parameters
T-lipids (mg/dL)
Mean ± SD	185 ± 13.1	179 ± 15	181 ± 17	184 ± 11	181 ± 10
p Value^a	---	NS	NS	NS	NS
p ^b
Triacyglycerol (mg/dL)
Mean ± SD	78 ± 7	73 ± 6	80 ± 4.5	77 ± 5	77 ± 9
p Value^a	---	NS	NS	NS	NS
p ^b
T-cholesterol (mg/dL)
Mean ± SD	62 ± 3.1	59 ± 0.21	56 ± 0.5	68 ± 0.19	112 ± 5.1
p value^a	---	NS	NS	NS	NS
p ^b
LDL-c (mg/dL)
Mean ± SD	28 ± 1.3	37 ± 2.7	27 ± 2.1	27 ± 3.3	23 ± 2.6
p Value^a	---	NS	<0.05	NS	NS
p ^b		--	<0.05	<0.05	NS
p ^c		----	-----	----	<0.05
HDL-c (mg/dL)
Mean ± SD	25 ± 1.1	17 ± 1.9	23 ± 4.3	20 ± 2.2	19 ± 2.5
p Value^a		<0.01	<0.01	<0.05	NS
P ^b			<0.01	<0.01	NS.
p ^c			----	---	<0.01

HDL-c: high-density lipoprotein cholesterol, LDL-c: low-density lipoprotein cholesterol, NS: non-significant.

^a p: compared with control group.

^b p: compared with lead group.

^c p group V compared with groups III and IV; p < 0.05 was considered as significant.

The erythrocyte level of total glutathione in the control and lead-injected rats of different groups are shown in Table 3 . Total glutathione was decreased in lead-injected rats as compared with control group (p < 0.001). The total glutathione decreased by about 40%.

Table 3.

Erythrocytes malondialdhyde and antioxidant enzyme activities; superoxide dismutase (SOD) and glutathione (GSH) level of all studied groups (Mean ± SD)

Groups	(GpI) control normal	(GpII) lead	(GpIII) lead + epicatechin	(GpIV) lead + lycopene	(GpV) Lead + epicatechin + lycopene
Parameters
TBARS (mmol/mL)
Mean ± SD	2.31 ± 0.14	4.1 ± 0.57	3.2 ± 0.27	3.16 ± 0.32	2.16 ± 0.32
p Value^a	-----	<0.001	<0.01	<0.01	NS
p ^b	-----	----	<0.01	<0.01	<0.01
p ^c	------	-----	-----	-----	<0.01
SOD (MU/mg protein)
Mean ± SD	216.8 ± 13.8	105.5 ± 14.0	149.7± 25.8	189.3 ± 13.2	209.3 ± 23.2
p Value^a	-----	<0.001	<0.01	<0.05	NS
p ^b	-----	-----	<0.05	<0.05	<0.05
p ^c	------	-----	-----	----	<0.01
Glutathione (IU/mL)
Mean ± SD	78.4 ± 4.56	44.0 ± 7.86	57.9 ± 7.14	66.6 ± 5.08	70.6 ± 5.08
p Value^a	----	<0.01	<0.01	<0.05	NS
p ^b	----	-----	<0.01	<0.01	<0.01
p ^c	-----	-----	------	-----	<0.01

NS: non-significant.

^a p: compared with control group

^b p compared with Lead group

^c p: Group V compared with groups III and IV. p < 0.05 was considered as significant.

Regarding malondialdhyde (MDA; a marker of lipid peroxidation) level, it was significantly increased (by about 100%) in lead-injected rats as compared with control rats. Lead-injected rats treated with combination of EC and lycopene showed a highly significant reduction of MDA to be the same control more than treatment alone (Table 3).

The effects of lead treatment on the activity of erythrocyte SOD were also assessed. When lead was given to the rats, SOD activity was significantly reduced (80%, as compared with its controls), while it was significantly elevated when combined treatment was given higher than individual treatment.

A positive correlation was recorded between the level of lead in erythrocyte and MDA (r = 0.7) whereas a negative correlation with SOD (r = −0.6).

Discussion

Lead toxicity leads to free radical and cell damage via two separate, pathways: (1) the generation of reactive oxygen species (ROS), including hydroperoxides, singlet oxygen and hydrogen peroxide and (2) the direct depletion of antioxidant reserves.

In this situation, the negative effects on the human system’s ability to deal with increased oxidant stress occur via independent pathways.²⁷

The present study aimed to investigate the potential protective role of EC or lycopene alone or combined together in reducing the oxidative stress, toxicological effect of lead acetate in rats.

The activities of plasma lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) were used as markers for cell membrane damage. It was found that plasma LDH, ALP was significantly elevated in lead-injected rats. However, this elevation returned to a normal level in treatment with epicatechin + lycopene > epicatechin > lycopene, so the combined treatment exerts a higher protection effect against damage induced by lead.

The current study showed that lead acetate ingestion in rats induced a significant elevation of serum ALT, AST and GGT activities as compared with control group. It has reported that serum ALT was elevated significantly more than AST on lead exposure.²⁸ Elevated serum GGT has been observed in chronic hepatobiliary diseases indicating toxic liver damage.²⁹ Similarly, El-Sayed et al.³⁰ and Tandon et al.³¹ reported disturbances in the liver functions after chronic lead exposure. The combined treatment with EC and lycopene showed a protective effect against lead toxicity by ROS scavenging effect released by lead ingestion.

Results obtained showed that, no significant changes in serum total lipid, cholesterol, triglycerides; however, serum LDL-c increased significantly while HDL-c decreased in lead-ingested rats. In contrast with our findings, Skoczynska et al.³² observed a decrease in plasma cholesterol level and lipoproteins in rats poisoned with small doses of lead. These results were also in agreement with the results of Antonowicz and Andrzejak³³ that recorded no disturbances in lipid metabolism in males exposed to lead pollution.

Glutathione is a cysteine-based molecule produced in the interior compartment of the lymphocyte. More than 90% of non-tissue sulfur in the human body is found in the tripeptide glutathione. In addition to acting as an important antioxidant for quenching free radicals, glutathione is a substrate responsible for the metabolism of specific drugs and toxins through glutathione conjugation in the liver.²⁷

The sulfhydryl complex of glutathione also directly binds to toxic metals that have a high affinity for sulfhydryl groups. Mercury, arsenic, and lead effectively inactivate the glutathione molecule so it is unavailable as an antioxidant or as a substrate in liver metabolism. The level of glutathione in the blood was significantly lower in animal of lead exposure than the control group.

Erythrocytes have a high affinity for lead destabilizing effect on cellular membranes, and in red blood cells (RBC) the effect decreases cell membrane fluidity and increases the rate of erythrocyte hemolysis. Hemolysis appears to be the end result of ROS-generated lipid peroxidation in the RBC membrane. Lead can also bind directly to phosphatidylcholine in the RBC membrane, leading to a decrease in phospholipid levels.

The large decrease in glutathione and increase in MDA levels observed in lead-injected rats, as compared with control rats, are in favor of a strong oxidative stress and enhanced ROS formation in rats. It has also been reported that MDA contents decreased in rats treated with epicatechin, lycopene or combined treatment, suggesting decreased lipoperoxidation.

Regarding antioxidant defense, our data showed that the kidney level of glutathione progressively increased in epicatechin + lycopene-treated rats more than individual treatment. Thus, the oxidative stress observed in rats could be related not only to the marked increase in the GSSG/glutathione (GSH) molar ratio but also to the decline in GSH levels. Increased generation of ROS and lipid peroxides has been reported following administration of GSH-depleting agents and after a reduction in liver GSH levels.³⁴

The epicatechin-enhanced antioxidant enzymes may be an attempt by the organism to counterbalance the decreases in the reducing power mediated by GSH.

The lower activity of SOD in lead-injected rats could be a consequence of inhibitory effects due to excess of ROS generation. SOD is inhibited by hydrogen peroxide.³³ Inhibition of the catalytic activity of proteins that express SOD activity could also be a consequence of the stronger lead-induced liver GSH depletion in old animals. It was reported that lead reduces the content of protein sulfhydryl groups and causes protein thiol oxidation in rat liver cells probably due to the lead-induced GSH depletion. Lead might also reduce the protein levels of antioxidant enzymes, because a marked inhibitory effect of the drug on the synthesis of protein in the rat liver has been demonstrated.³⁵The oxidative stress and enhanced thiobarbituric acid reactive substances (TBARS) formation were accompanied by decreased protein levels of SOD. Importantly, the lead-induced decreases in the enzymatic and non-enzymatic antioxidant defense systems in rats makes them more susceptible to oxidative damage, and compromise the antioxidant capacity of the liver to adequately scavenge the ROS generated during lead treatment.³⁶

Conclusion

Daily exposure to lead has damaging effects and generates different diseases. Daily intake of lycopene and epicatechin or its sources prevent the generation of ROS by lead, which has deleterious effects on the cell.

Footnotes

Acknowledgement

The investigators would like deeply to thank the Deanship of Scientific Research, King Abdulaziz University for awarding the grant to undertake studies on our project No.3-11/430 entitled ‘Modulation of lead biohazards using a combination of epicatechin and lycopene in rats.’

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

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