Olive oil protects against 2,4-dichlorophenoxyacetic acid-induced oxidative renal dysfunction in...

Research Article

Olive oil protects against 2,4-dichlorophenoxyaceticacid-induced oxidative renal dysfunction in adult rats

Amel Nakbi1, Wafa Tayeb1, Samia Dabbou1, Issam Chargui2, Manel Issaoui1, Ameur Ferih1,

Zohra Ben Ali2, Mohammed A. Alsaif3 and Mohamed Hammami1,3

1 Biochemistry Laboratory, UR03ES08 ‘Human Nutrition & Metabolic Disorders’, Faculty of Medicine,

University of Monastir, Monastir, Tunisia2 Laboratory of Histology and Cytogenetic, Faculty of Medicine of Monastir, Monastir, Tunisia3 College of Applied Medical Sciences, VPP Unit, King Saud University, Riyadh, Saudi Arabia

This study was aimed to evaluate the protective effect of extra virgin olive oil (EVOO) and its fractions

(hydrophilic (HF) and lipophilic (LF)) treatment on 2,4-dichlorophenoxiacetic acid (2,4-D)-induced

nephrotoxicity in rats. The Wistar rats (n ¼ 80, 210–230 g) were divided into eight groups having ten

animals, i.e., control group (C), (2,4-D) group that received 2,4-D (5 mg/kg b.w.), (2,4-D/EVOO)

group was treated with 2,4-D plus EVOO, (2,4-D/olive oil hydrophilic fraction, OOHF) group that

received 2,4-D plus HF, (2,4-D/olive oil lipophilic fraction, OOLF) group was treated with 2,4-D plus

LF, (EVOO) group that received only EVOO, (OOHF) group was given HF and (OOLF) group was

treated with the LF. These components were administered daily by gavage for 4 wk. 2,4-D administration

affected plasma urea and creatinine levels, which increased while uric acid significantly decreased. A

marked increase of malondialdehyde level and with a significant decrease in enzymatic antioxidant’s

activities, were also observed in 2,4-D treated rats. The co-administration of EVOO and its fractions

along with 2,4-D resulted in a reversal of 2,4-D induced biochemical changes in kidney accompanied by a

significant decrease in lipid peroxidation and an increase in the level of the antioxidant defence system.

Light microscopy investigations revealed that 2,4-D exposure induced numerous histopathological

alterations in the kidney. EVOO administration with 2,4-D severely reduced the toxicity of 2,4-D

and preserved the normal histological architecture of the renal tissue. However, a milder

histopathological improvement was observed in animals co-treated with HF or LF plus 2,4-D. In

conclusion, the present study suggest that the nephroprotective potential of EVOO against 2,4-D

toxicity might be due to the synergic effect of the two fractions, which could be useful for achieving

optimum effects in 2,4-D-induced renal damage.

Keywords: 2,4-D / Kidney / Olive oil / Oxidative stress / Rat

Received: June 20, 2011 / Revised: October 13, 2011 / Accepted: November 8, 2011

DOI: 10.1002/ejlt.201100213

1 Introduction

2,4-Dichlorophenoxyacetic acid (2,4-D) is a selective herbi-

cide of the phenoxyacetic acid group, with weak aromatic acid

properties [1]. Its herbicidal activity is mediated by an auxin

like capacity to alter normal protein synthesis and cell division

in plant meristems and leaves [2, 3]. Although it has recently

been suggested that its herbicidal activity may also be due to

an increase in the production of reactive oxygen species

(ROS) leading to the generation of oxidative stress in the

weed [4, 5]. Exposure of humans and animals occurs through

Additional corresponding author: Amel Nakbi, nakbia@yahoo.fr

Correspondence: Professor Mohamed Hammami, Laboratory of

Biochemistry, Faculty of Medicine, UR03/ES-08, ’Human Nutrition and

Metabolic Disorders’, Monastir 5019, Tunisia

E-mail: mohamed.hammami@fmm.rnu.tn

Fax: þ216 73 460733

Abbreviations: CAT, catalase; DC, conjugated dienes; 2,4-D, 2,4-

dichlorophenoxiacetic acid; EVOO, extra virgin olive oil; GPx,

glutathione peroxidase; GR, glutathione reductase; LDL, low density

lipoprotein; MDA, malondialdehyde; OOHF, olive oil hydrophilic fraction;

OOLF, olive oil lipophilic fraction; ROS, reactive oxygen species; SFA,

saturated fatty acid; SOD, superoxide dismutase; UFA, unsaturated fatty

acid

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contaminated air, drinking water, soil, and foodstuff or

during production of the herbicide. It has been reported that

2,4-D may cause a health-risk [1, 6]. Dose-dependent toxic

effects include damage to the eye, thyroid, kidney, adrenals,

and ovaries or testes. In addition, researchers have observed

neurotoxicity, reproductive toxicity and developmental

toxicity [7, 8]. The kidney is the critical target organ of

chronic 2,4-D toxicity [6, 9] and is well documented by a

number of studies in experimental animals. In fact, subacute

2,4-D administration induces dose-dependent histopatho-

logical degenerative effects in the rat kidney cortex [10].

Several lines of evidence indicate that oxidative stress and

ROS formed in the presence of 2,4-D could be responsible for

its toxic effects in many settings in vitro [11, 12] and in vivo

[13, 14]. Consequently, increased tissue oxidative stress can

lead to cell damage.

Most works have focused on the beneficial effect of plant

foods on oxidative stress. Constituents of some dietary

plant products, such as unsaturated fatty acids (UFAs)

and phenolics, endowed with antioxidant and anti-

inflammatory properties, which may play a protective role

in several human pathologies [15–17]. Most of the studies

comparing the effects of a MUFA-rich diet with PUFA-rich

diet on low density lipoprotein (LDL) oxidation parameters has

found a higher resistance of LDL particles to oxidation after the

consumption of MUFA-rich diet [15, 16]. Furthermore, in

vitro studies suggest that PUFAs are more pro-inflammatory

than MUFAs and saturated fatty acids (SFAs) [18].

Phenolics are also powerful scavengers of superoxide

anions and hydrogen peroxide and are capable of preventing

the generation of ROS by intact leukocytes as it has been

demonstrated by other authors [19, 20]. Numerous in vitro

studies have shown these phenolics to possess strong radical

scavenging activity at least equal in potency with other

important dietary antioxidants, such as ascorbic acid and

a-tocopherol [21, 22]. Phenolic compounds are also potent

in vitro inhibitors of LDL oxidation and are capable of break-

ing the peroxidative chain reaction [23, 24].

One from the Mediterranean diet, olive oil appears to be a

functional food with various components such as MUFAs that

may have health benefits and is also a good source of phyto-

chemicals, including polyphenolic compounds. Thus, it is likely

that extra virgin olive oil (EVOO) may be useful to alleviate or

protect against oxidant-induced various disease conditions,

including nephrotoxicity caused by 2,4-D. The present study

was established to examine the possible beneficial effect of

EVOO and its fractions on 2,4-D-induced renal functional

and structural abnormalities in experimental animals.

2 Materials and methods

2.1 Chemicals

2,4-D commercial formulation (Desormone Lourd) consist-

ing of 600 g/L 2,4-D ester butylglycol, register number

H.96064 was used in the experiments. 2-Thiobarbituric

acid (TBA) was obtained from Sigma Chemicals Co

(Taufkirchen, Germany). 1,1,3,3-Tetramethoxypropane

were purchased from Sigma Chemical Co. (St. Louis,

MO). Folin-Ciocalteu phenol reagent was purchased from

Fluka Biochemika (Buchs, Switzerland). All other chemicals

used were of analytical grade and were obtained from Sigma

Chemicals Co or Merck (Darmstadt, Germany).

2.2 Oil sample analysis

The EVOO used was harvested from the North of Tunisia.

The olive oil hydrophilic fraction (OOHF) was extracted

from EVOO by the method of Montedoro et al. [25]. The

olive oil lipophilic fraction (OOLF) was obtained from

EVOO as follows: the EVOO was homogenized for 1 min

with water (1:1, v/v), and the oil was separated by centrifu-

gation; this procedure was repeated six times. Then, the oil

fraction (OOLF) was filtered through a cellulose acetate

membrane [26, 27].

Extra virgin olive oil and its fractions analysis were per-

formed as reported in our previous paper [27]. In fact, the

determination of fatty acids was accomplished through the

quantification of their methyl esters (FAMEs) by gas chroma-

tography using a model 5890 series II instruments (Hewelett-

Packard Ca Palo Alto, California, USA) as described in

ref. 28. Carotenoids and chlorophylls (mg/kg oil) were deter-

mined spectrophotometrically according to Minguez-

Mosquera’s method [29]. Phenolic compounds were

extracted, estimated colorimetrically and expressed as

hydroxytyrosol equivalents as reported by Montedoro et al.

[25]. a-Tocopherol was evaluated according to Gimeno et al.

[30] on a Hewlett-Packard system (Waldbronn, Germany) as

described in ref. [31].

2.3 Animal treatment

This study was carried out in healthy, male adult Wistar rats,

weighing 200–230 g. The animals were housed under stand-

ard laboratory conditions of light (12-h light-dark periods),

temperature (22 � 38C), and relative humidity of 40%. The

animals were given standard diet (SICO, Sfax Tunisia) and

tap water ad libitum. According to our previous papers [26, 27],

the animals were randomly divided into eight experimental

groups: C (control), D (5 mg/kg b.w. of 2,4-D), D/EVOO

(5 mg/kg b.w. of 2,4-D þ 300 mL of EVOO), D/OOHF

(5 mg/kg b.w. of 2,4-D þ 1 mL of OOHF), D/OOLF

(5 mg/kg b.w. of 2,4-D þ 300 mL of OOLF), EVOO

(300 mL of EVOO), OOHF (1 mL of OOHF, the same

amount of phenols that we found in 300 mL of

oil ¼ 0.16 mg), and OOLF (300 mL of OOLF), each group

consisted of ten rats.

At the end of the experimental period of 4 wk, the animals

were sacrificed under diethyl ether anaesthesia; plasma

samples were collected and stored at �808C for analysis.

470 A. Nakbi et al. Eur. J. Lipid Sci. Technol. 2012, 114, 469–478

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Kidney tissues were excised from sacrificed animals,

cleaned and individually weighted. Some samples were

minced and homogenized (10%, w/v) in an appropriate buf-

fer (pH 7.4) and centrifuged. The resulting supernatants

were collected and stored at �808C until used for enzyme

assays. The others were immediately fixed in Bouin liquid for

histological studies.

2.4 Kidney biomarkers analysis

The levels of urea, uric acid, creatinine, and albumin in

plasma were estimated spectrophotometrically using the

available diagnostic kits supplied by Randox Laboratories

(Ardmore, Northern Ireland, UK). The protein content

was determined according to Bradford [32].

2.5 Kidney lipoperoxidation

The level of lipid peroxidation products was measured as

thiobarbituric acid reactive metabolites (TBA-rm) according

to Yagi [33]. One hundred and twenty-five microliters of

plasma or supernatants were homogenized by sonication with

50 mL of TBS, 125 mL of TCA-BHT in order to precipitate

proteins and then centrifuged (1000g, 10 min, 48C). Two

hundred microliters of the supernatant were mixed with

40 mL of HCl (0.6 M) and 160 mL of TBA dissolved in

Tris, and the mixture was heated at 808C for 10 min. The

absorbance of the resultant supernatant was read at 530 nm.

The TBA-rm amount was calculated using a 156 mM/cm

extinction coefficient.

2.6 Kidney activities of antioxidant enzymes

Antioxidant enzyme activities were analyzed using a BioRad

UV–Visible spectrophotometer with a ‘‘kinetics’’ program

(BioRad, Mares la Coquette, France). The measurement

of superoxide dismutase (SOD), glutathione peroxidase

(GSH-Px), and glutathione reductase (GR) activities in

supernatants were performed by the commercially available

diagnostic kits supplied by Randox Laboratories. Catalase

(CAT) activity was measured at 258C according to Aebi’s

method [34] by measuring H2O2 concentration decrease at

240 nm.

2.7 Histopathological studies

For qualitative analysis of kidney histology, the tissue samples

were fixed for 48 h in Bouin liquid. The specimens were

washed and dehydrated through a graded series of ethanol

and were sequentially embedded in paraffin wax blocks.

Blocks were made and sectioned at 5 mm thickness using a

rotary microtome. Sections were rehydrated in distilled water

and stained with hematoxylin–eosin (H–E) then examined

under light microscopy. The images were obtained by a

digital camera system attached to the microscope. A mini-

mum of three fields for each kidney slide were examined and

scored semiquantitatively for severity of changes by a path-

ologist unaware of the type of treatment. The scoring was

done as none (�), mild (þ), moderate (þþ), and severe

(þþþ).

2.8 Statistical analysis

The data obtained were expressed as means (�SD) and

analyzed using the Statistical Package for the Social

Sciences (SPSS) program, release 11.0 for Windows

(SPSS, Chicago, IL, USA). Tukey’s test was used to deter-

mine any significant differences between analytical

parameters means of supplemented diet (one way analysis

of variance, ANOVA; 95% confidence interval). Student’s

t-test was used when comparison between two groups was

required. Statistical significance was considered at p<0.05.

3 Results

3.1 Analytical parameters of extra virgin olive oiland its fractions

Figure 1 shows that EVOO and OOLF presented approxi-

mately the same fatty acid composition with 17% of SFA,

65% of MUFA, and 15% of PUFA, whereas the standard diet

was more abundant in PUFA (55%) [26, 27]. The minor

compounds of EVOO and its fractions are present in Fig. 2.

Data showed that EVOO and OOHF were rich in phenols

(580 and 384 mg/kg of olive oil, respectively) while the

OOLF was deprived from phenols but preserved the higher

content of a-tocopherol (490 mg/kg of olive oil) and other

minor compounds as pigments (Fig. 2).

Figure 1. Fatty acids composition (%) of extra virgin olive oil

(EVOO), lipophilic fractions (OOLF), and standard diet fed to rats.

Data are expressed as mean values (WSD) of three independent

experiments. SFA, saturated fatty acids; MUFA; PUFA. Values fol-

lowed by same letters are not significantly different (Tukey’s test,

p<0.05).

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3.2 Body and kidney weights

The body weights of the studied groups of rats up to 28 days

are presented in Fig. 3. At the end of the experimental diet, no

statistical difference in body weight was observed among

treated and control groups. Mean absolute and relative kid-

ney weights of rats in the 2,4-D exposure groups were sig-

nificantly less than the control group at the sacrifice

(p<0.05). EVOO and its fraction treatments alone produced

a near equal absolute and relative kidney weight with the

kidney of control rats. The 2,4-D þ EVOO and its fraction

groups restored the effect of 2,4-D by increasing the kidney

weights at near a control level (Table 1).

3.3 Kidney biochemical parameters

Results in Table 2 showed biochemical parameters indicators

of kidney function. This table shows a significant decrease in

plasma levels of uric acid (28.3%) and albumin (24.8%),

associated with an increase of urea (5.7%) and creatinine

(42.5%) levels in 2,4-D-treated rats when compared with the

control group. 2,4-D exposure significantly decreased total

protein in the kidney tissue by 23% of the control level. The

EVOO and its extracts treatment alone had no significant

effect on levels of urea, uric acid, creatinine, and protein

compared to the control group. The administration of

EVOO or its extracts along with 2,4-D significantly restored

the levels of renal function markers when compared to 2,4-D

treated rats. A significant, but incomplete, improvement in

creatinine level was observed in the 2,4-D/OOLF-treated

rats.

3.4 Antioxidant enzymes activities and lipidperoxidation

Data in Table 3 indicate that a single daily dose of 2,4-D

(5 mg kg�1 day�1) for 28 days significantly decreased anti-

oxidant enzymes (CAT, SOD, and GPx) as compared to the

control group. This enzyme inhibition was markedly pre-

vented upon concomitant administration of EVOO and its

extras in the 2,4-D-treated animals (Table 3). This improve-

ment, however, was incomplete as the GPx activity in the

OOLF group plus or minus 2,4-D remained low when com-

pared to the control group. The MDA level in kidney hom-

ogenates was significantly augmented by 63% in the 2,4-D-

treated group when compared to the control group. A sig-

nificant improvement in MDA levels was observed in the

Figure 2. Minor compounds (mg/kg of olive oil) of EVOO and its

lipophilic (OOLF) and hydrophilic fractions (OOHF). Data are

expressed as mean values (WSD) of three independent

experiments.

Figure 3. Growth curves of rats from different experimental groups

during 28 days.

Table 1. Extra virgin olive oil and its extract effects on absolute and

relative kidney weight in 2,4-D treated rats

Absolute kidney

weight (g)

Relative kidney

weight (g/100 g b.w.)

C 2.15 � 0.29 0.83 � 0.01

2,4-D 1.85 � 0.10a) 0.70 � 0.04c)

2,4-D/EVOO 2.03 � 0.22b) 0.78 � 0.03a),d)

2,4-D/OOHF 2.09 � 0.27b) 0.78 � 0.04a),d)

2,4-D/OOLF 2.16 � 0.32b) 0.80 � 0.11b)

EVOO 2.14 � 0.29 0.83 � 0.08

OOHF 2.12 � 0.33 0.81 � 0.07

OOLF 2.06 � 0.39 0.79 � 0.09

Data are expressed as means � SD (n ¼ 10 rats per group). C,

controls group; 2,4-D, 2,4-D treated group; 2,4-D/EVOO, 2,4-D

plus extra virgin olive oil; 2,4-D/OOHF, 2,4-D plus hydrophilic

fraction; 2,4-D/OOLF, 2,4-D plus lipophilic fraction; EVOO, extra

virgin olive oil treated group alone; OOHF, group treated with

hydrophilic fraction of olive oil alone; OOLF, group treated with

lipophilic fraction of olive oil alone. Comparison between groups was

made using unpaired Student’s t-test.a) p<0.05; c) p<0.01 (compared to control group (C)).b) p<0.05; d) p<0.01 (compared to 2,4-D group).

472 A. Nakbi et al. Eur. J. Lipid Sci. Technol. 2012, 114, 469–478

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EVOO or its extracts administered along with 2,4-D-treated

rats (Table 3).

3.5 Kidney histological studies

Histopathological studies showed that control rats pre-

sented normal rat kidney with normal tubular brush-bor-

ders and intact glomeruli (Fig. 4A). Contrarily, histology of

the 2,4-D treated groups revealed renal corpuscular

degeneration, which was marked by glomeruli atrophy,

epithelial cell degeneration of distal convoluted tubules

(DCTs), and dilatation with dislocation of brush-borders

of proximal convoluted tubules. A congestion, necrosis,

and vascular/tissular inflammation were observed in this

group (Fig. 4B and Table 4). However, the pathological

lesions induced by 2,4-D were remarkably reduced by the

administration of EVOO and its extracts (Fig. 4C–E).

A higher improvement was observed in the group supple-

mented with EVOO along with 2,4-D with a moderate

amelioration for the 2,4-D/OOLF and 2,4-D/OOHF

groups.

The histopathological changes were in agreement with

biochemical parameters. There were no histological altera-

tions in the kidney of EVOO, OOHF, and OOLF groups

showing the same histological structure of kidney (Fig. 4A

and Table 4) when compared to control.

Table 2. The effect of extra virgin olive oil and its fractions on 2,4-D induced changes in renal functional markers

Urea

(mmol/L)

Uric acid

(mmol/L)

Creatinine

(mmol/L)

Albumin

(g/dL)

Total protein

(g/dL)

C 6.97 � 0.62 202.22 � 49.06 105.85 � 16.26 4.44 � 0.44 1.56 � 0.11

2,4-D 7.37 � 0.69 145.00 � 29.89b) 150.87 � 5.51c) 3.34 � 0.36c) 1.19 � 0.13c)

2,4-D/EVOO 6.95 � 0.67 193.00 � 47.08a) 117.33 � 24.54d) 4.32 � 0.48d) 1.71 � 0.19d)

2,4-D/OOHF 6.71 � 1.07 199.66 � 48.90a) 116.75 � 18.39d) 4.20 � 0.47d) 1.59 � 0.29a)

2,4-D/OOLF 6.45 � 0.83a) 204.66 � 43.69a) 136.00 � 3.67c),d) 4.67 � 0.42d) 1.57 � 0.24a)

EVOO 6.43 � 0.83 210.85 � 53.61 106.00 � 19.07 4.11 � 0.42 1.48 � 0.10

OOHF 6.58 � 1.15 184.33 � 53.81 103.00 � 17.01 4.03 � 0.61 1.48 � 0.17

OOLF 6.18 � 0.76 205.20 � 55.71 109.28 � 16.69 4.22 � 1.21 1.73 � 0.20

Data are expressed as means � SD (n ¼ 10 rats per group). C, controls group; 2,4-D, 2,4-D treated group; 2,4-D/EVOO, 2,4-D plus extra

virgin olive oil; 2,4-D/OOHF, 2,4-D plus hydrophilic fraction; 2,4-D/OOLF, 2,4-D plus lipophilic fraction; EVOO, extra virgin olive oil

treated group alone; OOHF, group treated with hydrophilic fraction of olive oil alone; OOLF, group treated with lipophilic fraction of olive oil

alone. Comparison between groups was made using unpaired Student’s t-test.a) p<0.05; c) p<0.01 (compared to control group (C)).b) p<0.05; d) p<0.01 (compared to 2,4-D group).

Table 3. The effect of extra virgin olive oil and its fractions on kidney antioxidant enzyme activities and MDA levels of 2,4-D treated rats

SOD

(U/mg protein)

GPx

(U/mg protein)

CAT

(mmol/min mg protein)

MDA

(mM/g protein)

C 5.28 � 0.79 0.86 � 0.18 19.92 � 1.82 1.00 � 0.14

2,4-D 3.80 � 0.53a) 0.69 � 0.17 12.56 � 0.91c) 1.63 � 0.11c)

2,4-D/EVOO 6.50 � 2.42b) 0.89 � 0.20 17.00 � 2.54b) 0.99 � 0.10d)

2,4-D/OOHF 6.49 � 2.21b) 0.75 � 0.17 17.84 � 0.35b) 1.02 � 0.19d)

2,4-D/OOLF 4.29 � 1.93 0.51 � 0.14 17.39 � 1.42 0.93 � 0.27d)

EVOO 5.33 � 1.19 0.87 � 0.18 20.66 � 2.54 0.93 � 0.27

OOHF 5.41 � 1.88 0.72 � 0.22 18.22 � 3.78 1.07 � 0.15

OOLF 6.11 � 2.26 0.52 � 0.07 18.63 � 2.01 0.95 � 0.15

Data are expressed as means � SD (n ¼ 10 rats per group). C, controls group; 2,4-D, 2,4-D treated group; 2,4-D/EVOO, 2,4-D plus extra

virgin olive oil; 2,4-D/OOHF, 2,4-D plus hydrophilic fraction; 2,4-D/OOLF, 2,4-D plus lipophilic fraction; EVOO, extra virgin olive oil

treated group alone; OOHF, group treated with hydrophilic fraction of olive oil alone; OOLF, group treated with lipophilic fraction of olive oil

alone. Comparison between groups was made using unpaired Student’s t-test.a) p<0.05; c) p<0.01 (compared to control group (C)).b) p<0.05; d) p<0.01 (compared to 2,4-D group).

Eur. J. Lipid Sci. Technol. 2012, 114, 469–478 Olive oil protects against oxidative renal dysfunction 473

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Figure 4. Histopathology of rat kidney cortex

from control and experimental groups. Kidney

sections were stained using the hematoxylin–

eosin method (H&E 32T). (A) Kidney section,

from control, EVOO, OOHF, and OOLF groups,

showing a normal appearance of glomeruli (G)

and an intact Bowman’s capsule (BC) with adja-

cent proximal (PCT) and distal convoluted

tubules (DCT). (B) 2,4-D (5 mg/kg) treated rat

revealed renal corpuscular degeneration show-

ing a glomeruli atrophy (G At), tubular dilatation

(TD) with an expansion (exp) of the space

between glomeruli and the Bowman’s capsule.

(C) 2,4-D/EVOO group showing a higher

improvement of the histological alteration of

the kidneys induced by 2,4-D. (D and E) 2,4-

D/OOHF and 2,4-D/OOLF groups showing a

moderate improvement with manifestation of

the congestion (Cg).

Table 4. Histopathological findings in the kidney of the control group and experimental groups

Glomerular

atrophy

Tubular

dilatation

Tissular and

vascular congestion

Tubular

degeneration Necrosis

C S S S S S2,4-D þþþþ þþþþ þþþþ þþþþ þþ2,4-D/EVOO ### ### ## ### ###2,4-D/OOLF # ## # ## #2,4-D/OOHF # ## # ## #EVOO S S S S SOOHF � � � � �OOLF � � � � �

�, none; þþ, moderate; þþþ, severe; #, mild improvement; ##, moderate improvement; ###, severe improvement.

Data were carried from six rats per group.

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4 Discussion

There are various chemical products and drugs, which cause

damage to renal tissues by ROS production [35]. The phe-

noxyacetic acid herbicides have previously been shown to

accumulate at high levels in liver and kidney via the organic

acid transport system, and this accumulation decreases the

oxygen uptake by the renal cortical slices and results in

uncoupling of renal mitochondria raising the possibility of

organ-specific toxicity secondary to transport [10]. Seeing the

deleterious effects of chlorophenoxy compounds on kidney,

the current study was investigated on the reno-protective

effect of EVOO and its fractions against 2,4-D-induced oxi-

dative stress in rats.

Among the chlorophenoxy herbicides used for pest con-

trol with their higher pesticidals activity and considerably

lower mammalian toxicity compared to other pesticides

[10], 2,4-D is the most used in Tunisia. The precise mech-

anism of its acute toxicity is not elucidated but can comprise

the disruption of plasma and intracellular membranes or

uncoupling of oxidative phosphorylation [36]. This last

mechanism may be involved in the generation of oxidative

stress and subsequent induction of cytotoxic and genotoxic

effects, which may lead, among others to reproductive and

developmental toxicity [5]. In the current study, 2,4-D

induced a kidney dysfunction which was demonstrated by

a decrease of kidney weight, alterations of biochemical

parameters, and histological damages. This kidney injury

was recovered by treatment of EVOO and its fractions.

The presence of high levels of plasma urea and creatinine

and lower level of uric acid and albumin are possible

indicators of kidney injuries induced through 2,4-D treat-

ment. Moreover, 2,4-D exposure caused a significant

reduction in kidney total protein level, which indicated the

disorders in protein synthesis and metabolism [3].

Furthermore, the decreases in the plasma total protein and

albumin values may be due to hepatic injury, including

degenerative and necrotic changes.

The results of the present study have demonstrated that sub-

acute treatment with 2,4-D could affect the antioxidant defense

systems of animals. In fact, it has been observed that 2,4-D

induced a decrease in SOD, CAT, and GPx activities and

enhanced MDA level in kidney. These data confirmed the

findings of Celik et al. [14] indicating that 2,4-D at 50 and

100 ppm produced substantial systemic organ toxicity the eryth-

rocyte, liver, brain, heart, and kidney, during the period of a

25-day subacute exposure. In fact, the results indicated that

2,4-D might affect antioxidant potential enzymes and lipid per-

oxidation in the kidney. Moreover, it has been suggested that a

decrease in the activities of primary antioxidants, CAT and SOD,

may be due to accumulation of ROS induced by 2,4-D. An

observation that strengthens this hypothesis is that SOD activity

can be inhibited by hydrogen peroxide treatment [35, 37].

The current study revealed that supplementation of

EVOO and its fractions significantly restored the level of

biochemical markers of kidney function as well as the renal

antioxidant defence system. In fact, the biochemical

parameters such as total protein, albumin, and uric acid were

increased after administration of EVOO and its fractions,

where the better improvement was observed in 2,4-D/

EVOO group for total protein. However, urea and creatinine

levels in plasma were decreased in 2,4-D treated rats supple-

mented with EVOO or its fractions compared to 2,4-D

group. These data were in agreement with previous reports,

which proved that the beneficial effects of oils were evidenced

by reduced plasma urea and creatinine concentrations in the

group receiving oils compared to the non-oil treatment

animals receiving gentamicin only [38]. While the present

results showed that the creatinine level remained significantly

higher in the 2,4-D/OOLF compared to controls which can

be due to the absence of phenolic compounds in the LF

fraction.

Supplementation of EVOO, in the present study, signifi-

cantly improved the tissue level of enzymatic antioxidants

compared to 2,4-D-treated rats. These effects have been

attributed to its high MUFA content, mainly the oleic acid.

Due to its high MUFA content, olive oil seems to have

protective properties with regard to LDL oxidation.

Indeed, several investigators have compared the influence

of dietary MUFA and PUFA on LDL oxidation. It could

first be shown in rabbits that oleate-rich LDL particles are

remarkably resistant to oxidative modification [39, 40].

Moreover, dietary fatty acids can influence the susceptibility

of cells to oxidative stress, probably by changing cell mem-

brane fatty acid composition. In fact, cells enriched with

MUFA have been shown to be less susceptible to oxidative

damage, whereas n-6 PUFA increased the susceptibility to

oxidative damage. Additionally, dietary MUFA from EVOO

has been shown to protect mitochondrial membranes from

rat heart cells against ageing-related per oxidative damage

[41]. In addition, in rats, low levels of 5% lipid-enriched diet

(canola oil, fish oil, and butter) in a chronic nutritional

regimen can modify the age-related changes in renal function

and that the impact of different types of lipid-supplemented

diets on renal function depends on the kind of lipid present in

the diet [42, 43]. However, OOLF did not show any improve-

ment in the SOD and GPx antioxidant enzyme activities.

This might indicate the usefulness of EVOO, as an excellent

source of antioxidants, in modulating 2,4-D-induced neph-

rotoxicity since EVOO has other components with antioxi-

dant properties, mainly polyphenol [44]. The antioxidant

efficiency of olive phenols has been assessed in various tests

such as the inhibition of LDL oxidation [45]. Phenolic com-

pounds have been shown to possess free radical-scavenging

and metal-chelating activities in addition to the reported

anticarcinogenic properties [46, 47]. As a very rich source

of polyphenol compounds, the strong antioxidant and oxygen

radicals scavenging effects of tea have been documented

[48–50]. Hydroxytyrosol, the main phenolic compound in

olives and their by-products, is a phenolic compound which

Eur. J. Lipid Sci. Technol. 2012, 114, 469–478 Olive oil protects against oxidative renal dysfunction 475

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

has been shown to possess diverse healing properties for its

antioxidant and anti-inflammatory activities [51, 52].

Moreover, low rate in free radicals and important activity

in antioxidant enzymes such as SOD, CAT, and GPX in liver

and kidney tissues were observed in diabetic rats [53].

Additionally, the hypoglycemic and antioxidant activities of

hydroxytyrosol prevent oxidative stress; in fact administration

of hydroxytyrosol to diabetic rats prevented kidney toxicity

observed by low rates in creatinine and urea in plasma, which

increased in diabetic rats [53].

2,4-D administration mediated lipid peroxidation of lipid

structures of renal tissues, resulting in subcellular damages as

observed in the histopathological examination. In this study,

The optic microscopic findings have shown characteristic

morphological alterations such as glomeruli atrophy, epithelial

cell degeneration of DCTs, and dilatation with dislocation of

brush-borders of proximal convoluted tubules in kidneys of

2,4-D treated rats. A congestion, necrosis, and vascular/tissu-

lar inflammation were also observed. Similar histopathological

changes were observed by Uyanikgil et al. [10] in renal of rats

treated with 2,4-D. The severe changes were not observed in

the groups treated with EVOO and its fractions suggesting the

protective effects of EVOO in attenuating 2,4-D-induced

morphological changes. In fact, previous researches have

shown that usual glomerulosclerosis, glomerular enlargement,

and glomeruli loss in spontaneously hypertensive rats have

been prevented (fish, canola, and palm oils) or attenuated

(olive and soybean oils) by this oil long-term administration

[43, 54]. However, cyclosporine-dependent changes in renal

histology were only partially reversed by the administration of

the hydroxytyrosol in rats [55]. In the current study, a higher

improvement was observed in the 2,4-D/EVOO group with a

moderate amelioration for the 2,4-D/OOLF and 2,4-D/

OOHF groups. The histopathological changes were in agree-

ment with biochemical parameters. There were no histological

alterations in the kidney of EVOO, OOHF, and OOLF groups

showing the same histological structure of kidney when

compared to control.

5 Conclusions

This study substantiated the scientific evidence in favor of the

pharmacological use of olive oil in renal disorders in folk

medicine. According to the data obtained in the present

study, the administration of 2,4-D to rats at a dose of

5 mg/kg b.w./day for a period of 28 days caused significant

changes in oxidative stress markers and lipid peroxidation in

kidney rats. These changes were observed in the form of the

improvement of values in the groups, which were adminis-

tered EVOO and its fractions. However, the LF and HF

fractions supplemented separately showed a moderate

improvement in the histology kidney toxicity induced by

2,4-D. While EVOO supplemented to rats showed better

improvement. These data should be explained in terms of

cooperation between the two fractions.

This research was supported by a grant from the ‘‘Ministere de

l’Enseignement Superieur et de la Recherche Scientifique’’ Tunisia

(UR03/ES-08 ‘‘Nutrition Humaine et Desordres Metaboliques’’

et USCR-Spectrometrie de Masse, University of Monastir).

The authors have declared no conflict of interest.

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