INTRODUCTION

Bifenthrin (BIF) is a pyrethroid (PYR) insecticide. BIF is acyclopropanecarboxylate ester of alcohol. It is characterizedwith strong insecticidal properties due to its ability to alterfunctioning of nerves by modifying the kinetics of voltage-sensitive sodium channels in insects (1, 2). BIF and othersynthetic PYRs are considered to be safe for mammals becausethey are readily cleaved at the central ester bond to relativelynon-toxic metabolites in the liver, which are passed with urine(3-5). The relative resistance of mammalians to PYRs isattributed to their higher body temperature than of insects, fastermetabolism in the liver and lower sensitivity of sodium channels(6). However, there were numerous cases of PYR poisoning innon-target organisms described (7-9). Acute poisoning with BIFalters gait and other motor functions in rodents (1, 10, 11).

For long time it was believed that PYRs acted only via fastdisregulation of the nervous system, without any significantcytotoxic effect. However, there is evidence, that exposure toPYRs may also produce neuron death in adult animals (12),inhibition of nervous system development in rodent newborns(13) hepatic toxicity, changes in blood morphology, disrupt the

endocrine system (14, 15), produce reproductive toxicity (16), aswell as oxidative stress (OS) (17-19).

PYRs are used in medicine and veterinary medicine againstvectors (ticks and mosquitoes) and ectoparasites (scabies), aswell as in agriculture for crop control, and to fight domesticpests. Due to the global climate change significant changes inglobal disease patterns are expected (20). The incidence ofmalaria, Lyme disease and tick-borne encephalitis are toincrease. Therefore residents and tourist to the endemic areas areexpected to be chronically exposed to PYRs. Farmers andgreenhouse workers can be chronically exposed to theinsecticides at workplace in their efforts to increase crops (21,22). The use of PYRs in agriculture increases as they replaceorganophosphorous insecticides. In Poland current annual use ofPYRs is estimated to be over 80,000 kg of active ingredient (23),and one of them is BIF. Members of the public are exposed totraces of PYRs via food of plant origin, water, indoor air andtextiles (24, 25). Therefore a new questions arise: whether PYRsact in a dose-additive fashion in the course of subacutepoisoning, are there other target organs for BIF apart from brainand if there is one common mechanism of its’ toxic action indifferent organs.

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2015, 66, 1, 129-137www.jpp.krakow.pl

B. NIERADKO-IWANICKA, A. BORZECKI, B. JODLOWSKA-JEDRYCH

EFFECT OF SUBACUTE POISONING WITH BIFENTHRIN ON LOCOMOTOR ACTIVITY,MEMORY RETENTION, HAEMATOLOGICAL, BIOCHEMICAL

AND HISTOPATHOLOGICAL PARAMETERS IN MICE

1Chair and Department of Hygiene, Medical University of Lublin, Lublin, Poland; 2Chair and Department of Histology andEmbryology with Experimental Cytology Unit, Medical University of Lublin, Lublin, Poland

Bifenthrin (BIF) is a pyrethroid (PYR) insecticide. The target point for PYR’s toxic action are voltage sensitive sodiumchannels in the central nervous system (CNS). Intoxication with PYRs results in motor activity impairment and death ininsects. Although PYRs are considered to be safe for mammals, there were numerous cases of pyrethroid poisoning inhumans, animals and pets described. The general population is chronically exposed to PYRs via grain products, dust andindoor air. Therefore new questions arise: whether PYRs act in a dose-additive fashion in the course of subacutepoisoning, are there other target organs (but brain) for BIF and if there is one common mechanism of its’ toxic action indifferent organs. The objective of this work was to characterize the effect of BIF at the doses of 4 or 8 mg/kg injectedintraperitoneally (i.p.) daily for 28 consecutive days on memory and motor activity, hematological, biochemical andhistopathological parameters in mice. BIF at the doses of 8 mg/kg or 4 mg/kg of body mass was administered i.p. dailyto the mice for 28 consecutive days. Motor function was measured on day 1, 7, 14 and 28 and memory retention wastested in a passive avoidance task on day 2, 7, 14 and 28. BIF significantly impaired memory retention on day 2. BIFdecreased locomotor activity at every stage of the experiment in a single dose depending manner. No behavioralcumulative effect was observed. Subacute poisoning with the higher dose of BIF caused anaemia, elevated white bloodcell count (WBC), elevated alanine transaminase (ALT), superoxide dismuthase (SOD), and decreased glutathioneperoxidase (GPx) activity. Lymphocyte infiltrates were visualized in the livers. In conclusion: subacute poisoning withBIF decreases locomotor activity in a single dose proportionate manner. BIF damages also the liver and alters bloodmorphology. The possible common mechanism of these effects can be oxidative stress.

K e y w o r d s : bifenthrin, pyrethroid, locomotor activity, memory, oxidative stress, liver function, blood morphology

The objective of this work was to characterize the effect ofBIF at the doses of 4 or 8 mg/kg injected intraperitoneally (i.p.)daily for 28 consecutive days on memory and motor activity,hematological, biochemical and histopathological parametersin mice.

MATERIALS AND METHODS

Experimental animals

Non-gravid female albino Swiss mice weighing 18–24 gapproximately 6 weeks of age purchased from a licensed breeder(T. Gorzkowski, Warsaw, Poland) were used in the study. Allanimals were given a 7-day acclimation period and maintainedon a 12 h light/dark cycle. Food and tap water were provided adlibitum. Temperature was maintained at 21 ± 2°C. The mice wererandomly divided into groups of 8 animals each. A total of 48animals were used in the experiment.

All the experimental procedures were conducted withrespect for the law regulations of the European Community andPoland. They were conducted at the Chair and Department ofHygiene, Medical University of Lublin, Poland. The LocalEthics Committee for Animal Experiments in Lublin hadapproved the experiment (Opinion No. 4/2009, dated: Jan. 9th

2009). The experiments were performed between 8:00 a.m. and6:00 p.m.

Chemicals

BIF [2-methylbiphenyl-3-ylmethyl (Z)-(1RS)-cis-3-(2-chloro-3,3,3-trifluorprop-1-enyl)-2,2-dimethyl cyclopropanecarboxylate]99% purity was purchased from the manufacturer (Institute ofIndustrial Organic Chemistry, Annopol, Warsaw, Poland). Tween60 (poloxyethylene sorbitan monostearate) purchased fromLaboratorium Reagenzien, Germany was used to preparesolutions in bidistilled water.

Experimental procedures

Dosing

On the first day of the experiment 16 mice injected i.p. withBIF at the dose of 8 mg/kg bw. BIF at the dose of 8 mg wasdissolved in 9.9 ml of saline with 0.1 ml of Tween as pure PYRspoorly dissolve in water. The volume of 10 ml of solution wasprepared per 1,000 g of mice body mass. A mouse of body mass20 g received 0.2 ml solution i.p. in one injection. The injectionswere repeated once daily for 28 subsequent days. Body mass(b.w.) of each animal was recorded daily before BIFadministration. The same number of animals was given BIF at thedose of 4 mg/kg b.w. for 28 consecutive days. Controls were 16animals injected with respective volume of saline i.p. for 28 days.

Locomotor activity

To evaluate the influence of BIF on locomotor activity, 8animals from each group (control, BIF 8 mg, BIF 4 mg) weretested in Opto-Varimex 4 Activity Meter (Columbus, OH, USA).Each monitoring instrument (Opto-Varimex) consisted of aPlexiglass cage (44.5 × 44.5 cm) covered with perforatedPlexiglass cover connected to the Auto-Track interface andintercrossed by 16 pairs of horizontal infrared beams equallyspaced at 1 cm above the floor. Interruption of a beam generatedan electrical impulse, which was subsequently processed and sentto a computer linked to the Auto-Track interface. The Auto-Tracksystem detected behavioral parameters including locomotor

activity. Interrupting five or more beams was recorded aslocomotor movement activity. Testing was conducted at the timeof peak effects of BIF. Locomotor activity monitoring started 30min after the injection and was continued for 60 min.

Passive avoidance task

The remaining animals (8 from each group: control, BIF 8mg, BIF 4 mg) were tested in a step-through passive avoidancetask (PA). PA is regarded as a good measure of long-termmemory retention (26). The task relies on the natural preferenceof rodents for dark, enclosed spaces. PA is regarded as a goodmeasure of long-term memory retention. Thirty minutes after BIFinjection each animal was placed in a well lit box (15 × 12 × 15cm) adjacent to a darkened one. The dark box had an electric gridfloor. Thirty seconds after placing an animal in the centre of theilluminated box, a passage joining the two boxes was opened.After entering the dark box, the mouse was affected with anelectric foot shock (2 mA for 2 s). Twenty four hours after thetraining, memory retention test was conducted. The latency toenter the darkened box was recorded. The test ended when themouse entered the darkened box or when 180 s elapsed.

Training was repeated on day 6,13, 27. PA test was done onday 2, 7, 14 and 28.

Laboratory tests

On the 29 day all the animals were decapitated and theirblood samples were collected to clot in order to measure alaninetransaminase (ALT) activity in the blood sera. Blood sampleswith EDTA were also collected in order to measure bloodmorphology. The livers of 50% animals from each group wereused for measurement of superoxide dismutase (SOD) andglutathione peroxidase (GPx) activity. The livers of theremaining animals were used for histopathological examination.

Blood samples were centrifugated for 4 min at the speed of3,400 rpm. Cobas Integra 800 apparatus manufactured byRoche, France, was used to measure ALT activity. Sysmex XT2000i automatic hematologic analysator was used to measureblood morphology.

The SOD activity was measured with use of RANSOD kitmanufactured by Randox Laboratories Ltd., with aspectrophotometric method (27). The livers were homogenizedwith a mechanic blender MPW- 120 in a 0.1 M buffer of Tris-HCl, 7.4 pH (0.5 g of tissue per 5 ml buffer). The homogenateswere centrifugated for 15 min twice at the speed of 5,000 rpm.The supernatants were collected for SOD activity measurement.The activity of GPx was measured with use of RANSEL kitmanufactured by Randox Laboratories Ltd., with thespectrophotometric method (28). The supernatants for GPxdetermination were prepared in the same way as for SODactivity measurement.

Livers from the remaining animals were used formicroscopic examination. The livers were assessed with a lightmicroscope. The livers were buffered in 10% formalin,dehydrated in alcohol-acetone series, subjected to xylene,embedded in paraffin blocks, cut into sections and stained withhematoxillin and eosin (H + E). They were assessed in lightmicroscopy, magnification ×40.

Statistical analysis

The PA results were shown as medians ± 25th and 75th

percentile and results obtained in the remaining tests as means ±S.E.M., and evaluated by one-way analysis of variance ANOVAfollowed by Dunnett’s test. The P value <0.05 was consideredstatistically significant.

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RESULTS

Locomotor activity was significantly reduced in a singledose proportionate manner in animals exposed to BIF on day 1

(Fig. 1), 7 (Fig. 2), 14 (Fig. 3) and 28 (Fig. 4) in comparisonwith control group. No cumulative effect of BIF was observed.The results obtained in the step-through passive avoidance taskare shown in Fig. 5. BIF significantly impaired memory

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0 10 20 30 40 50 60 700

1000

2000ControlBIF 4 mg/kgBIF 8 mg/kg

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dist

ance

trav

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Fig. 1. Effect of exposure toa single dose of BIF onlocomotor activity in mice(mean ± S.E.M.). N = 8, *P <0.05 vs. control, Anova,Dunnett’s test.

0 10 20 30 40 50 60 700

500

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1500ControlBIF 4 mg/kgBIF 8 mg/kg

*****

time [min]

dist

ance

trav

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d [c

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Fig. 2. Effect of a 7-dayexposure to BIF onlocomotor activity in mice(mean ± S.E.M.). N = 8, *P <0.05 vs. control, Anova,Dunnett’s test.

0 10 20 30 40 50 60 700

1000

2000ControlBIF 4 mg/kgBIF 8 mg/kg

* * ** *

time [min]

dist

ance

trav

elle

d [c

m]

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Fig. 3. Effect of a 14-dayexposure to BIF onlocomotor activity in mice(mean ± S.E.M.). N = 8, *P <0.05 vs. control, Anova,Dunnett’s test.

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0 10 20 30 40 50 60 700

1000

2000ControlBIF 4 mg/kgBIF 8 mg/kg

* * **

time [min]

dist

ance

trav

elle

d [c

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Fig. 4. Effect of a 28-dayexposure to BIF onlocomotor activity in mice(mean ± S.E.M.). N = 8, *P <0.05 vs. control, Anova,Dunnett’s test.

0

100

200

300ControlBIF 4 mg/kgBIF 8 mg/kg

**

2 7 14 28

day

Late

ncy

[s]

Fig. 5. The effect of BIFintoxication on memoryretention in the step-throughpassive avoidance task(median ± 25th and 75th

percentile). N = 8. *P <0.05vs. control, Anova, Dunnett’stest.

Fig. 6. Light microscopyexamination of the liver of amouse exposed to BIF at thedose of 8 mg/kg for 28 days.Lymphocyte infiltratesaround blood vessels andbiliary ducts (arrow).Haematoxillin and eosinstaining. Magnification ×40.

retention on day 2. The results of blood morphology, ALTactivities in the blood sera, SOD and GPx activities in the liversare shown in Table 1. Subacute poisoning with the higher doseof BIF caused anaemia, elevated white blood cell count (WBC),and lymphocyte percentage (LYM), elevated alaninetransaminase (ALT), superoxide dismuthase (SOD), anddecreased glutathione peroxidase (GPx) activity. In the lightmicroscopy examination in the livers of mice receiving BIF atthe dose of 8 mg/kg for 28 days lymphocyte infiltrates aroundblood vessels and biliary ducts were seen (Fig. 6). Lessinfiltrates and widened blood vessels were visualized in thelivers of mice receiving the lower dose of BIF. Body mass gainwas recorded daily (Fig. 7). Animals treated with BIF gainedweight at a slower rate that controls.

DISCUSSION

Pyrethroids are neurotoxins. The primary target sitesunderlying PYR’s toxicity are voltage-gated sodium channels.PYRs enhance sodium channel activity by shifting activation tomore negative membrane potentials and by inhibiting channelinactivation. The altered sodium channels cause repetitive firingand depolarizing block in neurons (1). Type II PYRs typicallyproduce longer delays in channel inactivation than do type Icompounds (29). In target organisms acute poisoning withPYRs results in motor activity impairment. In insects moderatedoses of PYRs produce hyperexcitability, an increase in motoractivity with altered mode of flying or walking (30). Higherdoses cause immobility of walking insects and falling down

among flying insects. The highest doses cause immediatehindlimb paralysis, movement incoordination and eventuallyprostration and death (31).

BIF is classified as producing T syndrome (aggressivesparring, sensitivity to external stimuli, tremor progressing toprostration) or type I pyrethroid (32, 33). BIF is considered to beof moderate toxicity in vivo (10) and low efficacy on the sodiumchannels in vitro (34).

In the present study we have recorded reduced locomotoractivity in mice exposed to BIF for 28 subsequent days. Theseresults come in line with results published by other authors,however majority of articles show description of acute BIFneurotoxicity. In rats acute oral exposure to BIF at the dose of 75mg/kg causes whole-body tremors, twitching, staggered gait,uncoordinated movement/ataxia, splayed hindlimbs, abnormalposture, clonic convulsions, and abdominogenital staining (1).Wolansky (10) wrote that the threshold dose of BIF of 1.28mg/kg was enough to reduce motor activity in rats. Wolanskyreported that BIF’s LD50 was 55 mg/kg. In his experiments thePYR was administerd to rats in corn oil by gavage. Ther authorindicated the following potencies of PYRs in vivo: esfenvalerate> lambda-cyhalothrion > beta-cyfluthrin > tefluthrin >deltamethrin > BIF > fenpropathrin > cypermethrin > permethrin> s-bioallethrin > resmethrin. In our experiment BIF wasadministered i.p. to mice at the dose of 4 or 8 mg/kg. The i.p.LD50 for BIF calculated at our department (the HygieneDepartment of the Medical University in Lublin) was 16.1mg/kg(35). In our former study BIF at the dose of 1.61 mg/kg wasadministered to mice in a single injection and the dose was highenough to impair memory tested in a Y-maze and impair

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0 10 20 300

10

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30

40ControlBIF 4 mg/kgBIF 8 mg/kg

* ** * * **

* * ** * * ** * ** *

day

body

mas

s [g

]

Fig. 7. Effect of BIF on bodymass gain. N = 16,*P <0.05vs. control, Anova, Dunnett’stest.

Control BIF 4 mg/kg BIF 8 mg/kg RBC [million/mm3] 9.49 ± 0.1 (N = 16) 8.4 ± 0.1 (N = 16) 8.1 ± 0.4* (N = 16) WBC [/mm3] 8,071 ± 1,108 (N = 16) 12,103 ± 2,226* (N = 16) 12,805 ± 2,675*(N = 16) LYM [%] 60 ± 4 (N = 16) 68 ± 11 (N = 16) 75 ± 8* (N = 16) PLT [thousand/mm3] 640 ± 149 (N = 16) 789 ± 134 (N = 16) 613 ± 210 (N = 16) HGB [g/dl] 14.08 ± 0.2 (N = 16) 13.9 ± 0.4 (N = 16) 11.1 ± 0.8* (N = 16) ALT [U/l] 50 ± 11 (N = 16) 78.4 ± 10.3 (N = 16) 133.4 ± 38.2* (N = 16) SOD-liver [U/g of tissue] 1,420 ± 85 (N = 8) 1,312 ± 180 (N = 8) 1,648 ± 307 (N = 8) GPx-liver [U/g of tissue] 63.6 ± 11.6 (N = 8) 49.9 ± 13.6 (N = 8) 28.1 ± 15* (N = 8)

Table 1. Derived hematological and biochemical parameters in mice exposed to BIF for 28 days, mean ± S.E.M., N = 16, *P <0.05 vs.control. Anova, Dunnett’s test.

movement co-ordination but there was no significant effect onlocomotor activity (35). Therefore this time we’ve made thedecision to use a higher dose of the PYR. Scollon et al. (36)demonstrated that BIF reduced motor activity in rats. The dosesused in their experiment were even higher than used in ourpresent study. In Scollon’s experiment motor activity wasmeasured 4 and 7 hours after exposure to BIF at the dose of 0–16mg/kg or 0–9 mg/kg. And they have found out that therelationship between motor activity and brain concentration ofBIF was not significantly different between the two time points.To sum up majority of studies confirm that PYRs affect motoractivity in rodents. Another question remains open: if this is dueto sodium channel activation.

Cao et al. (34) described his experiments carried on primarycultures of cerebrocortical neurons. He showed that BIFproduced just a modest increase in sodium ions influx. Therelative potencies of different PYRs in his in vitro study were notidentical to those reported by Wolansky et al. (10). According toCao et al. (34) the rank order of in vitro potency wasdeltamethrin > s-bioallethrin > beta-cyfluthrin > lambda-cyhalothrion > esfenvalerate > tefluthrin > fenpropathrin >cypermethrin > BIF, while permethrin and resmethrin wereinactive. However the in vitro system lacks significant metaboliccapability and consists of a single tissue, so the results obtainedthis way not always are able to explain the results obtained invivo. In vivo the xenobiotic can interfere with multiple neuronalpathways, act of wider variety of ion channels and can affectneurotransmitter release.

It is known that PYRs also decrease chloride currentsthrough voltage-dependent chloride channels (1). At highconcentrations PYRs can also act on GABA-gated chloridechannels inhibiting chloride influx (37-39), which is responsiblefor neurological effects: the seizures seen in severe acutepoisoning and the altered motor activity. Exposure to PYRs canaffect cholinergic/dopaminergic neurochemistry (40). However,the early studies on PYRs’ structure-toxicity relationships, thatgave basis for the current classification of these pesticides intotype I and II, were carried after an acute mice poisoning withhigh doses of intracerebrally injected PYRs (33). Such dosinghad little relevance to the possible environmental exposure ofhumans and other non-target organisms to PYRs.

A newly studied target points for PYRs are voltage-gatedcalcium channels. Cao et al. studied the activation of voltage-gated calcium channels by PYRs (41). Thery have demonstratedthat PYRs stimulated Ca 2+ entry into neurons subsequent to theiraction on the voltage-gated sodium channels. However BIF (atconcentrations 10 µM, 30 µM, 50 µM and 100 µM) producedvery little Ca 2+ influx response in neocortical neurons. In thatstudy of Cao et al. the rank order of PYRs’ efficacy was:tefluthrin = deltamethrin > lambda-cyhalothrion > beta-cyfluthrin > esfenvalerate > fenpropathrin> cypermethrin > BIF> s-bioallethrin. In a recent study of Cao et al. (42) the authorsinvestigated whether nanomolar BIF (EC50 = 58 nM) alteredsynchronous Ca 2+ oscillations necessary for activity-dependentdendritic development. Acute exposure to BIF increased thefrequency of synchronous Ca 2+ oscillations and decreased theiramplitude. These changes were independent on voltage-gatedsodium channels since 100 nM BIF had no effect on the sodiumcurrent and did not influence neuronal resting membranepotential. The metabotropic glutamate receptor antagonist wasdemonstrated to normalize the BIF-triggered increase insynchronous Ca 2+ oscillations frequency without changing thebaseline synchronous Ca 2+ oscillations. The authors concludedthat BIF amplified metabotropic glutamate receptor signalingindependent of sodium channel modification. This is thepossible mechanism of reducing motor activity in mammalsafter exposure to pyrethroids. However, further studies are

needed to check if the effects demonstrated in vitro can beconfirmed in vivo.

As the bifenthrin’s action on the neuronal firing is irreversiblethe mechanism of amplifying the metabotropic glutamatereceptor signaling may explain why BIF demonstrates long termtoxicity although it is quickly metabolized and cleaned.

The data about the influence of BIF on memory processes inmammals are scarce. Our former experiment demonstrated, thata single administration of BIF at the dose of 1.6 mg/kgadministered i.p. caused movement incoordination and impairedmemory retention in PA in mice after brain oligemia (43). In thepresent study only during the first test in PA memory retentionwas impaired. It is possible, that in the course of subacutepoisoning the brain structures responsible for memory retention‘acclimatize’ to BIF. Hornychowa et al. also detected memoryimpairment in rodents after subacute poisoning with PYRs (44).Similar memory deficits were deteceted in experiments withdeltamethrin (45). Such behavioral changes confirm that thecentral nervous system is the main neuotoxicity target for BIFand other PYRs. Subacute poisoning with PYR causesneurodegeneration and neuroinflammation (46). PYRs appliedfor 4 weeks to rats cause nerve cell loss in layer III of frontalcortex, dentate gyrus, and hippocampus, cause dopamine anddopamine transporter decreased in hippocampus and striatum(40). The neurodegenerative effect of PYRs in the nigrostriatalpathway resembles the changes occurring in human brains in thecourse of Parkinson’s disease (PD). Another similarity betweentoxic effects of PYRS and PD is that deltamethrin reduces thelevel of dopamine (DA) in rat brain (47).

Our results show not only that subacute poisoning with BIFreduces locomotor activity at every stage of testing in a dose-proportionate manner, but also that there is no dose-additivity.The lack of cumulative effect is probably due to fast pyrethroidmetabolism in the liver. Pyrethroids are metabolized mainly bythe liver esterases and cytochrome P450 (3).

The OS is an imbalance between reactive oxygen species(ROS) and antioxidants. The ROS can damage DNA, proteinsand lipids, change cell’s metabolism, affect gene expression andposttranslational modifications of proteins, which acceleratesageing, neurodegeneration and development of atherosclerosis,hypertension, type II diabetes as well as cancer (48). There isalso data that ROS can cause cytoplasmic calcium influx byphospholipase C activation and phosphorylation of 1,4,5-inositoltriphosphate-sensitive calcium channels, potentially affectingneurotransmitter release, memory processes and motor activity(49). Some antidepressant drugs together with chromiumtrichloride at the dose of 32 mg/kg were shown to increase motoractivity in rodents via interaction with monoaminergic(noradrenergic, dopaminegic and serotonin) systems in rodents(50). Moreover, ROS induce activity of cyclooxygenases leadingto inflammation and vasculitis (49). Vascular pathology oftenleads to significant impairment of learning and memory (51).

The SOD and GPx are antioxidant enzymes ubiquitous inliving organisms acting as an endogenous defense against ROS(52, 53). Dar et al. showed in their study that repeated oraladministration of BIF at the dose of 5.8 mg/kg daily for 20 or 30days led to an increase in lipid peroxidation, and a decrease inSOD activity (17). Dubey et al. reported significant hepatic OSand hepatic damage in rats exposed to deltamethrin, which isalso a pyrethroid, administered with fluoride (18).

Krzepilko (54) studied the effect of exposure of yeast cellextracts to BIF on their oxidative-antioxidative system. Theydemonstrated that the investigated PYR insecticide reducedconcentration of thiol groups in the yeast cell extracts. It confirmsthat PYRs cause OS. The results obtained in our experiment showthat repeated exposure to BIF may cause hepatic OS and affectliver structure. In the present study significant changes in animals’

134

blood morphology were recorded and these results come in linewith data reported by other authors (19, 46, 55-57). The OS stressis also very likely to be responsible for bone marrow damage andchanges in blood cells of mammals in the course of intoxicationwith (19, 56). Elevation of neutrophil and lymphocyte counts after15-day exposure of mice to deltamethrin and neutropenia in longduration (30-day) study were recoreded by Tewari and Gill (57).The leucocytes play an essential role in ROS mediated injurythrough extracellular release of superoxide free radical, which iscytotoxic. The lymphocyte infiltrates were found in liver samplesof mice examined in our study, and the lymphocytes visualizedmay be responsible for release of free radicals. It indicates that theliver is another target point for BIF’s toxicity, possibly viametabolites as its’ metabolism occurs mainly in the liver. Theelevated ALT activities in the blood sera of experimental animalsexposed to the highest dose of BIF in our study are in agreementwith results obtained by other authors (57) and seem to confirmthe notion that BIF shows significant liver-toxicity.

The brain functioning, haematopoiesis and BIF’smetabolism in the liver require energy. The oxidative stresstriggered by pesticides seems to be one common mechanism ofdamaging all theses organs, as pesticides cause depletion ofmitochondrial energy. They inhibit Na, K-ATPases and causesinduction of proteolytic enzymes. This leads to DNAfragmentation and apoptosis (58).

Many authors provided evidence that vitamin C and E,selenium as well as SOD supplementation or nimesulide havethe ability to ameliorate the overwhelming OS (8, 52, 56, 58-61).

In our experiment animals exposed to BIF gained weight atsignificantly slower rate than controls. This is in agreement withresults of other experiments (62). The xenobiotic apparently alteredmice metabolism and energy production, that in spite of free accessto food and water they couldn’t grow normally. It is possible thatacting as endocrine disruptors, PYRs affect also proper functioningof the hypothalamus-pituitary-ovarian axis (63, 64).

In conclusion, we state that subacute poisoning with BIF leadsto neurotoxic effects: a decreases in locomotor activity in a singledose proportionate manner and transient memory impairment. BIFdamages also the liver and alters blood morphology. The possiblecommon mechanism of these effects can be OS.

Conflict of interests; None declared.

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R e c e i v e d : October 17, 2014A c c e p t e d : January 20, 2015

Author’a address: Assoc. Prof. Barbara Nieradko-Iwanicka,Chair and Department of Hygiene, Medical University ofLublin, 11 Radziwillowska Street, 20-080 Lublin, Poland.E-mail: bnieradkoiwanicka@wp.pl

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