Beneficial effect of pine honey on trichlorfon induced some biochemical alterations in mice

ARTICLE IN PRESS Ecotoxicology and Environmental Safety 73 (2010) 1084–1091

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Beneficial effect of pine honey on trichlorfon induced some biochemical alterations in mice ¨ ¨ Gokhan Eraslan a,n, Murat Kanbur a, Sibel Silici b, Mursel Karabacak b a b

University of Erciyes, Faculty of Veterinary Medicine, Department of Pharmacology and Toxicology, Kayseri, Turkey University of Erciyes, S. C - ıkrıkc- ıoglu Vocational Collage, Department of Animal Science, Kayseri, Turkey ˘

a r t i c l e in f o

a b s t r a c t

Article history: Received 3 October 2008 Received in revised form 29 September 2009 Accepted 22 February 2010 Available online 29 March 2010

Forty-eight male BALB/c mice, weighing 30–35 g, were used in the study, and were divided into groups of 12 each. The four groups established in the study included one control group and three experimental groups. The first group served as the control group, while Groups 2, 3 and 4 were administered 1 g/ kg bw/day pine honey, 180 mg/kg bw/day trichlorfon (  1/5LD50) and 1 g/kg bw/day pine honey plus 180 mg/kg bw/day trichlorfon, respectively, by the oral route using a catheter for 21 days. At the end of 21 days post-administration, blood and tissue (liver, kidney, brain and heart) samples were collected. Serum levels/activities of total protein, albumin, glucose, cholesterol, triglyceride, BUN, creatine, uric acid, magnesium, sodium, potassium, chloride, total bilirubin, GGT, LDH, AST, ALT and ALP were determined. Furthermore, tissue MDA levels and CAT, SOD and GSH-Px activities were analyzed. According to the data obtained, when administered at the indicated dose and for the indicated time period, trichlorfon was determined to lead to negative alterations in most of the biochemical parameters investigated. The administration of pine honey was determined to alleviate this effect. & 2010 Elsevier Inc. All rights reserved.

Keywords: Beneficial effect Trichlorfon Pine honey Oxidative stress Biochemical alterations Mice

1. Introduction Trichlorfon (dimethyl(2,2,2-trichloro-1-hydroxyethyl)phosphonate), an organophosphate insecticide, is used to control a variety of pests, including fish parasites existent in aquatic environment, and also ectoparasites and endoparasites of domestic animals. Although highly toxic to target organisms, its effects on organisms in the surrounding environment are either very limited or absent. It degrades rapidly in soil. For this reason, several formulations, including such as emulsifiable concentrate, powder, dust, granules, a solution and ultra-low volume concentrates have been developed, which have extensive use in the environment (EPA, 1985; Gallo and Lawryk, 1991; Thomaz et al., 2009). The nonenzymatic metabolite of this pesticide is dichlorvos, which is more toxic than the main compound. Dichlorvos is also used as a pesticide. Furthermore, trichlorfon is used in the treatment of Alzheimer’s disease in humans, under the name metrifonate. Introduced into both human and veterinary medicine, dichlorvos has toxic potential to both humans and animals. Severe symptoms of toxicity may develop upon accidental or intentional exposure to high doses of trichlorfon in veterinary or agricultural preparations. In case of exposure by oral and dermal routes,

n ¨ Corresponding author. Erciyes Universitesi Veteriner Fakultesi, Farmakoloji ve ¨ Toksikoloji Anabilim Dalı, Sumer Mah. Barıs- Manco Cad, Kayseri, Turkey. E-mail address: [email protected] (G. Eraslan).

0147-6513/$ - see front matter & 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2010.02.017

trichlorfon is a moderately toxic insecticide, and decreases the cholinesterase activity required for normal nervous system functions (Ringman and Cummings, 1999; Kaya, 2002; Karademir-Catalgol et al., 2007; Feng et al., 2008). Clinical symptoms observed in case of acute exposure to trichlorfon include headache, giddiness, nervousness, blurred vision, weakness, nausea, cramps, loss of muscle control or reflexes, convulsion, or coma. The oral intake of high doses of trichlorfon may result in the development of polyneuropathy (EPA, 1985; Gallo and Lawryk, 1991). The oral median lethal dose (LD50) of trichlorfon is 800 mg/ kg in mice (Haley et al., 1975). The potential to generate free radicals is reported among the mechanisms of action of organophosphate insecticides, including trichlorfon. Free radicals (hydrogen peroxide, superoxide anion, hydroxyl radicals and others) peroxidize cellular structures due to their high content of unsaturated fatty acids, oxidize proteins and cause DNA damage by binding to sensitive regions of DNA. These effects trigger toxicity and result in reversible and irreversible damage of tissues and organs (Rui et al., 2004; Zhou et al., 2004; Valavanidis et al., 2006; Karademir-Catalgol et al., 2007; Pe´rez et al., 2007; Soltaninejad and Abdollahi, 2009; Thomaz et al., 2009). Pine honey is obtained upon the collection of the honeydew secreted by the insect Marchalina hellenica (Gennadius), and the sap of pine trees pertaining to the species Pinus brutia Ten and Pinus halepensis Mill, by honeybees (Apis mellifera L.). Pine honey has no incisive taste or aroma, a very low tendency to crystallize and a thick consistency, and is resistant. Chemically, honey

ARTICLE IN PRESS G. Eraslan et al. / Ecotoxicology and Environmental Safety 73 (2010) 1084–1091

contains sugar (70–80%), water (10–20%) and minor compounds (1%) such as organic acids, minerals, proteins, phenolic compounds and amino acids. However, the percentage of these compounds is greater in pine honey, compared to other types of honey. Pine honey is produced from the sap of pine trees, and is known to have high mineral content. Therefore, pine honey is classified as high quality honey. Turkey and Greece are the major pine honey producing countries in the world. In Turkey, pine honey is generally produced in Marmaris and its vicinity. Pine honey is dark brown and intensely flavored. Also black pine honey is even richer, thicker and darker (Valant et al., 2000; Tsigouri et al., 2004; Tananaki et al., 2007; Akbulut et al., 2008). Various types of honey are known to have antioxidant properties. The correlation between the antioxidant capacity and composition of honey has been investigated in a number of studies. Data obtained from these studies have demonstrated such a correlation to exist particularly for phenolic compounds. However, not only phenolic compounds but also the amino acid composition of honey is reported to play a major role in antiradical effect (Pe´rez et al., 2007; Baltruˇsaityte_ et al., 2007; Bertoncelj et al., 2007; Blasa et al., 2007; Viuda-Martos et al., 2008; Rasmussen et al., 2008). Amongst studies conducted on the composition of pine honey produced in Turkey, according to Akbulut et al. (2008), pine honey, collected from Western Anatolia, has high phenolic content (323.8 mg gallic acid/ 100 g) and antiradical activity (35.32 DPPH, IC50), as well as calcium (2.665 mg/kg), potassium (3.802 mg/kg) and phosphorus (903.7 mg/ kg) levels. According to Louveaux et al. (1978), honeydew index lower than 3 what may show the floral origin of the pine honey. The present study was firstly aimed at the determination of the possible effects of trichlorfon and its oxidative stress potential. The second objective was to determine if pine honey would alleviate the possible adverse effects of trichlorfon.

2. Materials and methods 2.1. Animal material Forty-eight male BALB/c mice, weighing 30–35 g, were used in the study. The animals were divided into four groups, including one control group and three

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experimental groups. The animals were exposed to a 12-h light/12-h dark cycle, housed at a fixed temperature of 22 7 1 1C, and provided ad libitum feed and water. The animals were given solid pellet feed containing 23% crude protein, 7% crude cellulose and 2600 kcal/kg metabolic energy. The protocol of this study was approved by the Ethics Committee of Erciyes University Faculty of Veterinary Medicine.

2.2. Preparation and analysis of honey samples Pine honey (Pinus brutia Ten) samples were obtained from the MuglaMarmaris (southwest part) of Turkey. The uniformity of honey was ensured by means of melissopalynological analysis. Qualitative analysis of honey was performed as described by Louveaux et al. (1978) with minor modifications. A honey sample of 20 g was dissolved in 100 mL of distilled water and centrifuged at 1500g (Pendleton, 2006). The pollen sediment was mounted in glycerin-gelatin and sealed with paraffin. 500 pollen grains were counted, to determine relative frequency (percentage of each pollen type in the pollen content of a sample). Quantitative pollen analysis included the addition of tablets of Lycopodium clavatum L. spores (Moar, 1985). 10 g of honey was dissolved in 40 mL of distilled water, and two tablets of L. clavatum spores dissolved in 5 mL of 5% hydrochloric acid, were added. The resulting sediment was concentrated by centrifugation at 1000g for 10 min. Centrifugation was performed until all sediments were pooled in a single tube. The sediment was mounted in glycerin-gelatin, and sealed with paraffin without any chemical treatment. Until 500 pollen grains were reached, spores, pollen and honeydew elements were counted. The pollen concentration (number of pollen grains in 10 g of honey: NPG) was calculated according to this formula: (pollen counted/L. clavatum spores counted  spores added. The honeydew index (HDE/P): ratio of honeydew elements) (HDE) to pollen grains of nectariferous plants (P) was evaluated (Louveaux et al., 1978). The analyses of pine honey for amino acids, and phenolic and others compounds were performed in compliance with the modified from methods of Ozcan and Senyuva (2006) and Bankova et al. (2002), respectively. The analysis of total phenolic compounds was performed as described by Singleton and Rossi (1965).

2.3. Administration of trichlorfon and pine honey The first group served as the control group. The second group was administered 1 g/kg bw/day pine honey. The third group was administered 180 mg/kg bw/day (  1/5LD50) trichlorfon (Haley et al., 1975). Finally, the fourth group received 180 mg/kg bw/day trichlorfon plus 1 g/kg bw/day pine honey. All applications were performed orally using a catheter for 21 days. In all trial periods, pine honey was administered 8 h after trichlorfon was given. Pine honey was given in deionised water while trichlorfon was administered in sunflower oil. Both honey and trichlorfon were given in a volume of 0.5 mL and at the previously indicated doses. The control group was given sunflower oil alone.

Table 1 Chemical components of typical pine honey obtained by GC–MS. Compounds Fatty and aromatic acids Hexanoic acid Benzoic acid 1H-pyrrole-3.4-diacetic acid Phosphoric acid Nonanoic acid 2-Butenedioic acid 2,4-Hexadienoic acid Pentanoic acid Propanoic acid Hexadecanoic acid Dodecanoic acid Benzeneacetic acid Thiocyanic acid 1,2 Benzenedicarboxylic acid Butyric acid Alcohol and ketones Phenethyl alcohol Isoborneol 1-Decanol 2-Cyclooctanone Azepino pyrimidin-11-one 2H-Benzimidazol-2-one Ethanone

%TIC

RT (min)

11.79 17.83 17.92 18.82 13.62 18.93 19.73 19.79 20.11 20.19 20.3 20.93 21.71 23.65 23.79

0.31 0.21 1.33 0.18 0.92 5.27 0.17 0.26 6.94 0.26 0.25 1.28 0.24 0.24 32.23

11.57 11.96 12.86 14.18 15.66 17.05 18.21

1.69 1.15 0.12 0.21 0.13 0.19 0.52

Compounds

%TIC

Benzoxazole-2-thiol Benzophenone 2-Nonadecanone Phenolic compounds 2,6-Bis(1,1dimethylethyl)-phenol Coumarin Esters Methyl dihydrojasmonate Isopropyl myristinate Thiocyanic acid carbazole-3,6-diyl ester

19.41 20.49 28.71

0.48 0.59 0.22

21.19 23.26

0.26 0.40

20.61 21.85 23.95

0.30 0.36 0.47

Others Cyclotetrasiloxane Isothiazole Dimethyl anthranilate Pyrazine Benzene Butyl butyrate 2,4,6-Trimethyl-2-2H-pyran Cyclohexasiloxane Benzaldehyde Trans-2-hexanal Hexasiloxane n-Decanal

13.62 14.13 14.78 15.1 15.66 16.29 17.19 19.2 19.5 21.1 24.95 13.43

0.17 0.37 0.23 0.11 0.13 8.02 11.31 0.52 0.36 0.60 2.68 1.46

RT: retention time, TIC: the total ion current generated depends on the characteristics of the compound concerned and it is not a true quantification.

RT (min)

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2.4. Collection and preparation for analysis of tissues

a, double-beam UV/VIS).

Following the collection of blood samples, the liver, kidneys, brain and heart were extracted in all animals. Subsequently, the organs were washed in cold distilled water. Following the removal of fat and connective tissue, the tissues were homogenized with 1/5 buffer adjusted to a pH value of 7.2 with 5 N NaOH (140 mM KCl, 10 mM NaHCO3, 3 mM KH2PO4 and 2 mM K2HPO4/L), using a homogenizer (Heidolph, SilentCrusher M). Subsequently, the homogenized tissues were centrifuged at 20,000 rpm for 1 h (Sigma, 3 K30) and the supernatant was transferred to Eppendorf tubes.

2.7. Analysis of serum biochemical parameters The measurement of serum total protein, albumin, glucose, cholesterol, triglyceride, blood urea nitrogen (BUN), protein, uric acid, creatine, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma glutamyl transpeptidase (GGT), potassium, magnesium, sodium and chloride levels/activities were performed using a Konelab 60i model auto-analyzer and a kit of the same brand.

2.5. Collection of blood samples 2.8. Statistical analysis At the end of 21 days, blood samples were collected into dry tubes from all animals under light ether anesthesia. Each tube was centrifuged at 3000 rpm for 10 min (Heraeus, Labofuge 200) for the separation of serum. 2.6. Analysis of oxidative stress markers The method described by Ohkawa et al. (1978) was used for the determination of tissue MDA levels in all tissues. The results were expressed as nmol/mg protein. The protein levels in the tissues were determined in accordance with the method described by Lowry et al. (1951) and modified by Miller (1959). The results were expressed as mg-protein/mL homogenate. Measurements were performed in accordance with the method described by Sun et al. (1988) for the detection of tissue SOD activity. Catalase activity measurements were performed in accordance with the method described by Luck (1965). GSH-Px activity measurements were performed in compliance with the method described by Paglia and Valentine (1967). Enzyme analysis results were given in U/mg-protein. All oxidative stress

Table 2 Free amino acid values (mg/100 g honey) of pine honey by LC–MS. Free amino acid

Amounts

Aspartic acid Serine Glycine Lysine Cysteine Glutamic acid Threonine Alanine Proline Valine Methionine Tyrosine Tryptophan Histidine Arginine Cystine Phenylalanine Hydroxyproline Leucine–isoleucine Glutamine

9.78 0.67 1.63 15.85 3.42 4.07 11.39 4.23 36.33 4.73 2.67 3.37 0.59 3.73 16.85 3.42 10.59 1.03 1.54 2.53

3. Results Analyses demonstrated the HDE/P ratio, which may confirm the presence of pine honey indicators in the studied honey sample, to be greater than 1 but lower than 3. The level of total phenolic compounds was determined as 56.70 mg gallic acid equivalents (GAE)/g honey. Some other compounds determined to exist in the composition of pine honey are given in Tables 1 and 2. Compared to the controls, no statistically significant alteration was determined in any of the parameters investigated (oxidative stress markers and biochemical parameters except for total bilirubin) in the group which was administered pine honey alone (Figs. 1–13). All tissue MDA levels were determined to have increased significantly in the group which received trichlorfon alone, in comparison to the control group. When compared to the control group, amongst the antioxidant enzymes studied, SOD activity was determined to have decreased significantly in all tissues, CAT activity was ascertained to have decreased significantly in liver and to have increased significantly in kidney and heart tissues, while GSH-Px activity was determined to have decreased significantly in liver, kidney and brain tissues and to have increased significantly in heart tissue (Figs. 1–4). Furthermore, compared to the controls, in the group which was administered trichlorfon alone, amongst the serum biochemical parameters investigated, total protein level, and LDH and ALT activities were found to have decreased significantly, whereas glucose, triglyceride, cholesterol, BUN, potassium, sodium and chloride levels, and ALP activities were determined to have increased significantly (Figs. 5–13). In the group which received trichlorfon plus pine honey, the values of oxidative stress markers were ascertained to have Group 1

MDA

8 7

Group 2

*

6 nmol/mg-prot

The SPSS 13.0 statistical software package for Windows was used for statistical calculations. Data was given in the form of arithmetical mean values and 7standard deviations. Differences between the experimental groups and the controls were evaluated by the Mann–Whitney U-test according to P o0.05.

*

Group 3 Group 4

**

5 4

*

3 2 1 0 Liver

Kidney

Brain

Hearth

Fig. 1. Tissue MDA levels treated with pine honey and trichlorfon (significant difference from the control group at nPo 0.05, honey; Group 3, trichlorfon; Group 4, trichlorfon plus pine honey.

nn

P o0.01). Group 1, control; Group 2, pine

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Group 1

SOD

9

Group 2

8

Group 3

7 U/mg-prot

1087

Group 4

6

***

5

**

4 3

*** ***

2 1 0

Kidney

Liver

Brain

Hearth

Fig. 2. Tissue SOD activities treated with pine honey and trichlorfon (significant difference from the control group at

Po 0.001).

Group 2 Group 3

1

Group 4 U/mg-prot

nnn

Group 1

CAT

1.2

P o 0.01,

nn

0.8

**

**

0.6 0.4 *

0.2 0 Kidney

Liver

Brain

Hearth

Fig. 3. Tissue CAT activities treated with pine honey and trichlorfon (significant difference from the control group at nPo 0.05,

nn

Po 0.01).

GSH-Px

U/mg-prot

4 3.5

Group 1

3

Group 2 Group 3

**

2.5

Group 4

2 1.5 1

***

***

***

***

***

0.5 0 Liver

Kidney

Brain

Hearth

Fig. 4. Tissue GSH-Px activities treated with pine honey and trichlorfon (significant difference from the control group at

drawn closer to those of the control group, while compared to the controls, statistically significant differences were determined only in liver and heart GSH-Px activities (Figs. 1–4). With respect to serum biochemical parameters, compared to the control group, only BUN levels were determined to be significantly different in the group that was administered pine honey plus trichlorfon (Figs. 5–13).

4. Discussion A wealth of studies exist on the oxidative stress potential and adverse effects of several organophosphate pesticides and their

P o 0.01,

nn

nnn

Po 0.001).

detoxification (Kalender et al., 2005; Durak et al., 2009; Celik and Suzek, 2008, 2009). Very recently, pesticide intoxications and the beneficial effects of bee products have started to be investigated (Eraslan et al., 2008, 2009a, 2009b; Kanbur et al., 2009). In the present study, the effects of pine honey, which is widely produced and consumed in Turkey, in possible cases of trichlorfon intoxication have been investigated. The study bears originality in pioneering future studies to be conducted in this field. Some compounds, which were analyzed in the pine honey used in this study, have antioxidant effect. Phenolic compounds show antiradical effect, and radical scavenging potential has been determined by some researchers in some honey types (Pietta, 2000; Baltruˇsaityte_ et al., 2007; Bertoncelj et al., 2007; Blasa et al.,

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200

Group 1

**

Group 2

180

Group 3

160

Group 4

mg/dL

140 120 100 80 60 40 20 0 Glucose

Fig. 5. Serum glucose levels treated with pine honey and trichlorfon (significant difference from the control group at

Group 1

90

*

80

**

Group 2 Group 3

70

Group 4

60 mg/dL

P o0.01).

nn

50 40 30

**

20

**

10 0 Triglyceride

T-Cholesterol

BUN

Fig. 6. Serum triglyceride, T-cholesterol and BUN levels treated with pine honey and trichlorfon (significant difference from the control group at nPo 0.05,

Group 1

9

Group 2

8

Group 3

7

Group 4

mg/dL

6 5

P o 0.01).

nn

*

4 3 2 1 0 T-Protein

Uric Acid

Albumin

Fig. 7. Serum T-protein, uric acid and albumin levels treated with pine honey and trichlorfon (significant difference from the control group at nP o 0.05).

2007; Viuda-Martos et al., 2008; Rasmussen et al., 2008). Also, similar to phenolic compounds, the amino acid composition of honey also exhibits antiradical effect (Pe´rez et al., 2007). When compared to the control group, in the group administered trichlorfon alone, the increase in MDA levels, which is one of the most important indicators of lipid peroxidation (Del Rio et al., 2005; Michel et al., 2008), demonstrated the generation of a high level of free radicals by trichlorfon. Free radicals led to the increase of the level of MDA, a final product of peroxidation, by

peroxidizing sensitive structures. In addition, among the other parameters investigated for the evaluation of oxidative stress (Lemineur et al., 2006; Katayama, 2006; Michel et al., 2008), the alterations observed either in the form of the inhibition or stimulation of the activity of antioxidant enzymes, point out to the formation of a high level of free radicals in tissues, and also prove that antioxidant enzymes play an active role in the conversion of these harmful compounds into less harmful or harmless components. Apart from free radicals, trichlorfon itself

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Group 1

0.6

Group 2 0.5

Group 3

*

Group 4

mg/dL

0.4 0.3 0.2 0.1 0 T-Bilirubin

Creatine

Fig. 8. Serum T-bilirubin and Creatine levels treated with pine honey and trichlorfon (significant difference from the control group at nPo 0.05).

Group 1

300

Group 2 250

Group 3 Group 4

U/L

200 150 100

* **

50 0 AST

ALT

ALP

Fig. 9. Serum AST, ALT and ALP activities treated with pine honey and trichlorfon (significant difference from the control group at nPo 0.05,

P o 0.01).

nn

Group 1

3000

Group 2

2500

Group 3 Group 4

U/L

2000

**

1500 1000 500 0 LDH

Fig. 10. Serum LDH activities treated with pine honey and trichlorfon (significant difference from the control group at

may also have an effect on the mentioned antioxidant enzymes, directly. Amongst previously conducted studies on the oxidative stress potential of trichlorfon, in a study carried out by Karademir-Catalgol et al. (2007), upon the exposure of human erythrocytes to different concentrations of trichlorfon for different time periods, erythrocyte MDA levels and SOD and CAT activities were determined to increase, while GSH-Px activity was reported to decrease. Thomaz et al. (2009) investigated the effects of trichlorfon exposure on fish tissues (liver, gills, heart) in the species Nile tilapida, and determined increase in LPO levels in heart tissues and both increase and decrease in SOD and CAT

nn

Po 0.01).

activities in certain tissues. In another study (Rui et al., 2004), they reported blood LPO levels to have increased and SOD activity to have decreased in trichlorfon intoxication in humans. In a similar study (Zhou et al., 2004), increased erythrocyte LPO levels and decreased SOD, CAT and GSH-Px activities were reported in trichlorfon intoxication. The results of the present study are in agreement with previous studies with respect to alterations in oxidative stress markers. Alterations in certain biochemical parameters, most probably related to the generation of free radicals via pesticide exposure, demonstrate tissue damage (Banerjee et al., 2001; Bajgar, 2004).

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4

Group 1

3.5

Group 2 Group 3

3

Group 4

U/L

2.5 2 1.5 1 0.5 0 GGT Fig. 11. Serum GGT activities treated with pine honey and trichlorfon.

Group 1

14

Group 2 **

12

Group 3 Group 4

mmol/L

10 8 6 4 2 0 Potassium

Magnesium

Fig. 12. Serum potassium and magnesium levels treated with pine honey and trichlorfon (significant difference from the control group at

Group 1

180 **

160

Group 2 Group 3

140

mmol/L

P o 0.01).

nn

*

120

Group 4

100 80 60 40 20 0 Sodium

Chloride

Fig. 13. Serum sodium and chloride levels treated with pine honey and trichlorfon (significant difference from the control group at nP o 0.05,

In the present study, significant alterations determined in ALP, LDH and ALT activities and glucose, cholesterol, triglyceride and total protein levels in mice which were administered trichlorfon alone particularly may suggest functional disorder of the liver. One or more of the significant alterations in BUN and some electrolyte (sodium, chloride and potassium) levels may demonstrate in particular disorders of the kidneys and heart. Furthermore, most of the parameters having drawn closer to values of the control group, in the group which received trichlorfon plus pine honey, demonstrate pine honey to have antiradical effect. This effect is thought to be related to the

nn

Po 0.01).

phenolic and proteinaceous compounds found in the composition of honey. The radical scavenging effect of these compounds, mentioned in detail above, is a common fact. No previous study exists on the investigation of the effects of trichlorfon and pine honey on biochemical parameters. Neither does a previous study exist in which other bee products have been investigated together with trichlorfon. For this reason, the comparison of the data obtained in the present study was not able to be made. However, there are studies in which other pesticides have been compared to bee products (Eraslan et al., 2008, 2009a, 2009b; Kanbur et al., 2009), and in these studies, based on oxidative stress markers and

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other biochemical parameters, it has been determined that the adverse effects of pesticides were alleviated in groups which were administered both the investigated pesticide and the bee product together.

5. Conclusion In the present study, the administration of trichlorfon to mice at a dose of 180 mg/kg bw/day for a period of 21 days caused significant alterations in certain biochemical parameters. One of the main underlying reasons for this is the generation of a very high level of free radicals by trichlorfon, which cannot be compensated physiologically. For alterations determined in the oxidative stress markers further support this. The administration of pine honey alleviated the adverse affects of trichlorfon, based on the same parameters. Pine honey is considered to display antiradical effect due to its composition and in particular its content of phenolic compounds and amino acids. Therefore, in order to minimize the risk of foodborne trichlorfon intoxication and to alleviate any adverse effects, it is concluded that pine honey may be safely included in the daily diet of livings, such that it is either directly consumed or used as a food additive. References Akbulut, M., Ozcan, M.M., Coklar, H., 2008. Evaluation of antioxidant activity, phenolic, mineral contents and some physicochemical properties of several pine honeys collected from Western Anatolia. Int. J. Food Sci. Nutr. 23, 1–13. Bajgar, J., 2004. Organophosphates/nerve agent poisoning: mechanism of action, diagnosis, prophylaxis, and treatment. Adv. Clin. Chem. 38, 151–216. ˇ Baltruˇsaityte_ , V., Venskutonis, P.R., Ceksteryt e_ , V., 2007. Radical scavenging activity of different floral origin honey and beebread phenolic extracts. Food Chem. 101, 502–514. Banerjee, B.D., Seth, V., Ahmed, R.S., 2001. Pesticide-induced oxidative stress: perspectives and trends. Rev. Environ. Health 16, 1–40. Bankova, V., Popova, M., Bogdanov, S., Sabatini, A.G., 2002. Chemical composition of European propolis: expected and unexpected results. Z. Naturforsch. [C] 57, 530–533. Bertoncelj, J., Doberˇsek, U., Jamnik, M., Golob, T., 2007. Evaluation of the phenolic content, antioxidant activity and colour of Slovenian honey. Food Chem. 105, 822–828. Blasa, M., Candiracci, M., Accorsi, A., Piacentini, M.P., Piatti, E., 2007. Honey flavonoids as protection agents against oxidative damage to human red blood cells. Food Chem. 104, 1635–1640. Celik, I., Suzek, H., 2008. Subacute effects of methyl parathion on antioxidant defense systems and lipid peroxidation in rats. Food Chem. Toxicol. 46, 2796–2801. Celik, I., Suzek, H., 2009. Effects of subacute exposure of dichlorvos at sublethal dosages on erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats. Ecotoxicol. Environ. Saf. 72, 905–908. Del Rio, D., Stewart, A.J., Pellegrini, N.A., 2005. Review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr. Metab. Cardiovasc. Dis. 15, 316–328. Durak, D., Uzun, F.G., Kalender, S., Ogutcu, A., Uzunhisarcikli, M., Kalender, Y., 2009. Malathion-induced oxidative stress in human erythrocytes and the protective effect of vitamins C and E in vitro. Environ. Toxicol. 24, 235–242. E.P.A, 1985. Chemical Profile. Trichlorophon, Washington, DC, pp. 5–107. Eraslan, G., Kanbur, M., Silici, S., 2009a. Effect of carbaryl on some biochemical changes in rats: the ameliorative effect of bee pollen. Food Chem. Toxicol. 47, 86–91. Eraslan, G., Kanbur, M., Silici, S., Altinordulu, S., Karabacak, M., 2008. Effects of cypermethrin on some biochemical changes in rats: the protective role of propolis. Exp. Anim. 57, 453–460. Eraslan, G., Kanbur, M., Silici, S., Liman, B.C., Altinordulu, S., Soyer Sarica, Z., 2009b. Evaluation of protective effect of bee pollen against propoxur toxicity in rat. Ecotoxicol. Environ. Saf. 72, 931–937. Feng, T., Li, Z.B., Guo, X.Q., Guo, J.P., 2008. Effects of trichlorfon and sodium dodecyl sulphate on antioxidant defense system and acetylcholinesterase of Tilapia nilotica in vitro. Pestic. Biochem. Phys. 92, 107–113. Gallo, M.A., Lawryk, N.J., 1991. Organic phosphorus pesticides. In: Hayes Jr., W.J., Laws Jr., E.R. (Eds.), Handbook of Pesticide Toxicology. Academic Press, New York, NY. Haley, T.J., Farmer, J.H., Harmon, J.R., Dooley, K.L., 1975. Estimation of the LD1 and extrapolation of the LD0.1 for five organophosphate pesticides. Arch. Toxicol. 34, 103–109.

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