Chemosphere 90 (2013) 2115–2122

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Chemosphere

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Effects of the neurotoxic thionophosphate pesticide chlorpyrifoson differentiating alternative models

Andrea Amaroli a,⇑, Maria Grazia Aluigi b, Carla Falugi b, Maria Giovanna Chessa a

a Laboratorio di Protozoologia, Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università degli Studi di Genova, Genova, Italyb Laboratorio di Biologia dello Sviluppo, Dipartimento di Scienze della Terra, dell’Ambiente e della Vita (DISTAV), Università degli Studi di Genova, Genova, Italy

h i g h l i g h t s

" There are controversial opinions about the effects, at low doses, of Chlorpyrifos on neurodevelopment." We exposed to a wide range of CPF concentrations three models compatible with the 3Rs Strategy." We evaluated the effect of CPF on cholinesterase activity, growth and differentiation." We revealed that developing organisms are sensitive to CPF also at the doses found in food for children.

a r t i c l e i n f o

Article history:Received 30 May 2012Received in revised form 23 October 2012Accepted 1 November 2012Available online 1 December 2012

Keywords:ProtozoaSea urchinStem cellCholinesteraseNeurotoxic drugsChlorpyrifos

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.chemosphere.2012.11.005

⇑ Corresponding author. Fax: +39 0103538209.E-mail address: amaroli@dipteris.unige.it (A. Amar

a b s t r a c t

Studies by researchers worldwide have revealed that, even in industrialised nations, people, infants andthe aged in particular, are even more exposed to neurotoxic drugs as a consequence of the increasedquantity of pesticide residues in food. This phenomenon, as underlined by The Worldwatch Institute(2006), is linked to the exponential increase in the use of these toxic compounds over the last 40 years,up from 0.49 kg per hectare in 1961 to 2 kg in 2004, with the result that these substances are found in thedaily diet.

Many studies have demonstrated how the assumption of pesticides in the neonatal period and earlyinfancy can alter the development and function of the nervous, immune, endocrine and reproductiveapparatuses. Moreover, the unequivocal relationship between brain tumours, infant leukemia and pesti-cides are well recognised.

On the basis of the above information, the effects of the neurotoxic thionophosphate pesticide chlor-pyrifos (CPF) have been tested, considering biomarkers of toxicity and toxicity endpoint, on the biologicalmodels Dictyostelium discoideum, Paracentrotus lividus, and NTera2 Cells, as they are compatible with the3Rs strategy (Reduction, Replacement, and Refinement in animal experiments). Our results have revealedthat developing organisms are particularly sensitive to the toxic effects of CPF.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

For several years, there has been a growing awareness of thedevelopmental role of molecules related to the cholinergic system.In particular, acetylcholinesterase (AChE, EC 3.1.1.7), the lyticenzyme that removes ACh from its receptors, has been demon-strated to be involved in regulating a number of events related toembryonic development, from fertilisation, to cell proliferation,gastrulation movements, neurogenesis and growth. In theseevents, AChE is involved in the modulation of cell proliferation(Angelini et al., 2004), cell-substrate interaction, messages

ll rights reserved.

oli).

mediated by ion changes (Aluigi et al., 2005), and apoptosis (Aluigiet al., 2010b). For these reasons, there has been an increase inattention to the possible negative effects of pollutants on choliner-gic molecules and therefore on human and, particularly, childhealth (Eskenazi et al., 1999). The reported outcomes shed a newlight on the mechanisms of damage exerted by neurotoxic pesti-cides, i.e. cholinesterase inhibitors (namely organophosphorusand carbamate compounds) (Aardema et al., 2008) present in theenvironment and food from agricultural sites (Abdel Rasoul et al.,2008). Many studies have demonstrated how the assumption ofpesticides in the neonatal period and early infancy can alter thedevelopment and function of the nervous, immune, endocrineand reproductive apparatuses (Glynn et al., 2008; Grandjeanet al., 2008; Borchers et al., 2010), and how children exposed tochlorpyrifos while in the womb have an increased risk of delays

2116 A. Amaroli et al. / Chemosphere 90 (2013) 2115–2122

in mental and motor development and an increased occurrence ofpervasive developmental disorders such as Attention DeficitHyperactivity Disorder (ADHD) (Rauh et al., 2006). Overexposureof the infant to pesticides can also increase the risk of developingallergic pathologies (Proskocil et al., 2008). Finally, the unequivocalrelationship between brain tumours, infant leukemia and pesti-cides are well recognised (Proskocil et al., 2008).

Infants are in contact with pesticides during early developmentthrough the maternal blood, and this pre-load is increased bydirect exposure once they are born, of varying degrees, dependingon the family lifestyle and location (urban or agricultural sites)(Berry, 1997; Akland et al., 2000; Curl et al., 2003; Rauh et al.,2006; Abdel Rasoul et al., 2008). According to the European FoodSafety Authority (EFSA) (Tucker, 2008) it is now clear that thereis a need to adopt a tiered approach to the toxicological evaluationand intake estimation of these pollutants. A harmonisedconsumption survey has also been identified as an importantoutstanding task. In addition EFSA and the EU Member Statesare continuing to cooperate to develop new methods to meetthe challenges of cumulative risk assessment; for example in theimplementation of more representative residue surveillanceschemes.

In this frame, the need for new models for toxicity testsemerges, in order to establish a correlation between benchmarkdoses and effects which should be cost effective, bioethically com-patible, high throughput and at different degrees of complexity, toidentify either the action mechanisms of old and new pesticides, orthe general effects at a systemic level (EFSA Scientific Committee,2009).

In order to be able to establish reference doses (BMD doses), asrecommended by EFSA for genotoxic and carcinogenetic sub-stances, we propose using chlorpyrifos (CPF), a neurotoxic pesti-cide widely used all over the world, whose toxicity is well-known. In fact, the carcinogenetic activity of CPF (Blair et al.,2011) may be due to the depression of AChE activity and mutationon AChE gene (Perry and Soreq, 2004) exerted by organophosphatecompounds. In addition, there are controversial opinions about theeffects, at low doses, of CFP on neurodevelopment (Eaton et al.,2008).

To carry out this task, we will use the following biologicalmodels chosen on the basis of their compatibility with the 3RsStrategy (Replace, Reduce and Refine animal testing) (Russell andBurch, 1959), which has been adopted by ECVAM as the basis forthe development of new toxicity tests (Atterwil et al., 1994).

Dictyostelium discoideum (Protozoa) included in the eightbioassay alternatives to vertebrate models for the study of humandisease by the U.S. National Institute of Health (Williams et al.,2006). Paracentrotus lividus (Echinodermata), included in the fivebioassay alternatives to mammalian models, for neurotoxicitystudies in the nervous system’s embryogenesis by the EuropeanCentre for the Validation of Alternative Methods (ECVAM). NTera2cells-clone D1 (NT2), pluripotent cells able to develop in choliner-gic nervous cells and generally deemed an ethical substitute forgerminal neurogenic stem cells.

In previous works we detected the presence and the role ofa pseudocholinesterase, named propionylcholinesterase (PrChE),in the cell to cell interactions of D. discoideum, (Falugi et al.,2002; Amaroli et al., 2003), and how this enzyme can reactto environmental stress in the same way as the molecules ofmacroinvertebrate and vertebrate models (Delmonte Corradoet al., 2005, 2006; Amaroli, 2011). Moreover, our previous re-sults showed how pesticides have serious effects on the acetyl-cholinesterase activity of P. lividus (Pesando et al., 2003; Aluigiet al., 2010a) and how the exposure to diazinon affects thebalance between cell viability/apoptosis in NT2 cells (Aluigiet al., 2010b).

2. Material and methods

2.1. Chlorpyriphos (CPF)

The pesticide CPF is a crystalline thionophosphate insecticidewhich inhibits AChE activity and is used to control insect pests.

This pesticide was chosen for the experiments because it is oneof the most active anti-cholinesterase (ChE) agents (Aluigi et al.,2005), and its metabolites were found in very high concentrationsin the blood and urine of young children fed with non-organic fruitand vegetables (Lu et al., 2008). In addition, the CPF is an organo-phosphate insecticide which as a lipophilic molecule, can easilypass through the cell membrane into the cytoplasm (Uzun et al.,2010). Purified CPF was obtained from PESTANAL, through Sigmapurchase.

The stock solution of CPF was obtained by a dilution of the pes-ticide in dimethyl sulfoxide (DMSO). The toxicity of this solventwas assessed by exposing the cells and the organisms to DMSOat the maximum final concentration employed in the experiments[<10�3] (Sciarrino and Matranga, 1995). DMSO at that concentra-tion had no effect on the cells and the organisms tested in thiswork.

2.2. D. discoideum growth and differentiation

The life cycle of D. discoideum includes two phases: the repro-ductive and the developmental phase. The reproductive phase con-sists of growth and multiplication by binary fission of single-cellamoebae feeding on bacteria. Starvation triggers the developmen-tal phase (the streaming, mound, first finger, and mexican hatstages), and results in the formation of the fruiting body anchoredto the substratum.

The reproduction phase, used in this work, was axenically in-duced by inoculating the fruiting bodies in Falcon flasks containingAX-2 axenic medium as described in Amaroli et al. (2006). Thedevelopmental phase was induced by transferring some drops ofthe AX-2 culture, onto a B2 Escherichia coli monolayer growingon a nutrient agar-N plate (Amaroli et al., 2003). The plates wereincubated in a moist chamber for 3 d at 25 �C (Swan et al., 1977)to allow the cells to exhaust the supply of bacteria (reach starvingconditions) and migrate and aggregate. When the fruiting bodieshad developed, the plates were kept at 4 �C.

2.3. P. lividus growth and differentiation

Embryos and larvae of the sea urchin P. lividus were reared fromfertilisation in ultra-filtered, pasteurised pelagic sea water,1 larva mL�1 sea water, in tanks of the same size (100 mL), shape(cylindrical) and material (borosilicate glass). The pelagic sea waterwas collected from the water column and maintained for at leastone month in a glass tank of 100 L at 20 �C. The salinity of thiswater was 3.7%, and the pH 8.00. The sea urchin spermiotoxicitytest (ISO 2007) showed that the quality of our pelagic sea waterwas higher (15% ± 2.3) than artificial sea water. The pelagic seawater was collected far enough off the coast that it was not af-fected by agricultural pesticides, and kept in a glass tank for1 month to allow the decantation of heavy metals. Before our rear-ing experiments, the water was ultra-filtered through a filter with0.2 lm pores.

The larvae were fed with Cricosphaera elongata microalgae fromthe end of the vitellophagic phase (48 h) according to appropriateprotocols (Fenaux et al., 1994), and maintained at a temperature of18 �C. Metamorphosis was obtained according to standard proce-dures, by exposing 20-day-old larvae to the presence of stonesfreshly taken from clean sea sites.

A. Amaroli et al. / Chemosphere 90 (2013) 2115–2122 2117

2.4. NTera2-D1 cell growth and differentiation

Human NT2 cells were obtained from American Type CultureCollection (ATCC) and cultured in flasks in a humidified incubatorat 37 �C with a 5% CO2 atmosphere. The culture medium consistedof high glucose Dulbecco’s modified eagle’s medium (DMEM)supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin. Neurogenic commitment was performed byexposure to 10�6 M retinoic acid (RA) (Andrews, 1984; Llaneset al., 1995).

2.5. D. discoideum experimental samples

The samples for the spectrophotometric evaluation of the ChEactivities were obtained by centrifugation of cell cultures at a con-centration of 106 cells mL�1. The cell pellet thus obtained was sub-divided into samples of 10 lL. The control samples were exposedto commercial mineral water, buffered to pH 6.7, for 10 min at atemperature of 20 �C, and the experimental samples were exposedto different concentrations of CPF (10�4 M, 10�5 M, 10�6 M,10�7 M, 10�8 M, 10�10 M) for 10 min at a temperature of 20 �C.

For the evaluation of the effects of CPF on cell density, 140 mL ofthe cell culture at a concentration of 104 cells mL�1, was subdi-vided into six samples of 20 mL each. Five of the six samples wereexposed to CPF at concentrations of 10�4 M, 10�5 M, 10�6 M,10�7 M, 10�8 M, and the sixth sample was left untreated as control.All the experiments were carried out for 72 h and the cell densitywas determined at the time 0 h and every 24 h, by counting thenumber of cells using a Neubauer chamber.

For the evaluation of the effects of CPF on differentiation, agarplates were prepared by melting 3 gr of agar Difco in 140 mL of10 mM phosphate buffer (PB), pH 6. After 20 min in autoclave at120 �C, 20 mL of the agar was transferred onto a 9-cm Petri dishand considered as control. The remaining 120 mL of the agar wassubdivided into six samples with the addition of CPF at concentra-tions of 10�4 M, 10�5 M, 10�6 M, 10�7 M, 10�8 M, 10�10 M andtransferred to 9-cm Petri dishes. Subsequently, amoeba culturesat a concentration of 106 cells mL�1 were collected by centrifuga-tion at 600 rpm at 20 �C for 10 min and the pellet obtained dilutedin 50 mL of 10 mM PB, pH 6 at 6 �C. After an incubation period of6 h at 6 �C, 4 mL of the starving amoebae were transferred to thePetri dishes described above. After an incubation of 10 min at20 �C the excess liquid was removed and the Petri dishes withthe amoebae were incubated in a moist chamber for 48 h at20 �C. All the samples were monitored and photographed at thetime 0 h and every 24 h.

2.6. Electrophorus electricus experimental samples

As commercial acetylcholinesterase (AChE) from E. electricus isknown to have high sensitivity to neurotoxic pesticides it was uti-lised as the standard for comparing the ChE of D. discoideum. 1 lLof 500U AChE was diluted in 1 mL of 0.1 M PB, pH 8.0, and exposedto CPF at concentrations of 10�4 M, 10�5 M, 10�6 M, 10�7 M,10�8 M, 10�10 M. The samples were processed as described in thesection ‘‘Spectrophotometric evaluation of ChE activities’’.

2.7. P. lividus experimental samples

The mating, gastrulation and larval stages of P. lividus, obtainedas previously described, were exposed to CPF at final concentra-tions of 10�4 M, 10�5 M, 10�6 M, 10�7 M, 10�8 M. The developmentof the exposed samples was periodically monitored and photo-graphed to evaluate any anomalies with respect to the controlsnot exposed to CPF.

2.8. NT2 experimental samples

Native and RA-exposed NT2 cells were cultured in the presenceof CPF at concentrations of 10�4 M, 10�5 M, 10�6 M, 10�7 M,10�8 M.

For the evaluation of the effects of CPF on cell density, the NT2cells exposed and not exposed to CPF were observed for 72 h andthe cell density of the living cells was determined at the time 0 hand every 24 h by an MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Mosmann, 1983) as describedby Aluigi et al. (2007).

For the evaluation of the effects of CPF on AChE activity the cellswere processed as described in the section ‘‘Spectrophotometricevaluation of ChE activities’’.

2.9. Spectrophotometric evaluation of ChE activities

The samples of both the D. discoideum and NT2 cells were col-lected by centrifugation and the proteins extracted in a solution of0.1% Triton X-100 in 0.1 M PB, pH 8.0 (Falugi et al., 2002). The ChEactivities of both these samples and of the AChE of E. electricus wererecorded at k = 412 nm with a Unikon 930 spectrophotometer (Kon-tron Instruments) for the first 10 min after exposure to propionylthiocholine iodide (PrTChI) or acetyl thiocholine iodide (AcTChI),both used as substrate, according to Ellman’s method (Ellmanet al., 1961) modified (Falugi et al., 2002; Aluigi et al., 2010a).

The total protein content was evaluated spectrophotometricallyusing the BioRad protein assay, based on the methods of Bradford,according to the manufacturer’s instructions.

2.10. Cytochemical localisation of ChE activity

The NT2 cells were fixed in a solution of 2% paraformaldehydeand 0.5% glutaraldehyde. The samples were then tested accordingto the method of Karnovsky and Roots (1964) and Karnovsky andRoots (1964) using AcTChI as substrate. The controls were testedwithout the substrate.

2.11. Statistical analysis

Each experiment was carried out at least three times. For statis-tical analysis a two-way ANOVA test was used, followed by theStudent-Newman-Kelus multicomparison test.

3. Results

3.1. The effect of CPF on the PrChE activity of D. discoideum

The spectrophotometric analysis (Fig. 1), carried out with themethod of Ellman et al. (1961), on sample cells of D. discoideum ex-posed to the CPF pesticide at the following concentrations 10�4 M,10�5 M, 10�6 M, 10�7 M, 10�8 M, 10�10 M and exposed to the sub-strate PrTChl demonstrated that PrChE activity was inhibited in adose-dependent way by concentrations from 10�4 M to 10�8 M.On the contrary, cells exposed to the pesticide at a concentrationof 10�10 M did not show PrChE activity significantly different fromthe controls.

3.2. The effect of CPF on the cell density of D. discoideum

After 24 h the cells exposed to CPF concentrations of 10�4 Mand 10�5 M had a lower cell density than the controls (Fig. 2).The samples exposed to concentrations from 10�6 M to 10�8 Mwere not significantly different from the controls.

CPF [M]

PrChE D. discoideum

AChE E. electricus

AChE NT2 cells

Fig. 1. PrChE activity (%) of D. discoideum and AChE activity (%) of E. electricus andNT2 cells revealed spectrophotometrically after treatment with CPF [M]. (�)Significant statistical differences from the controls, P < 0.05; Student–Newman–Keuls multicomparison test.

hours [h]

Cel

l den

sity

x 1

04

10−4M

10-5M

10-6M

10-8M

Fig. 2. Effect of exposure to CPF, for 24 h, 48 h and 72 h, on the cell density of D.discoideum.

2118 A. Amaroli et al. / Chemosphere 90 (2013) 2115–2122

Forty-eight h after exposure to concentrations from 10�4 M to10�6 M of CPF the cell density of the samples was lower than thecontrols but that of those exposed to concentration 10�8 M wasnot significantly different. A similar result was also recorded 72 hafter exposure.

3.3. The effect of CPF on the cellular differentiation of D. discoideum

The differentiation of samples of D. discoideum raised in an agardish containing the pesticide was different from that of the con-trols (Fig. 3). In fact, while the starving control cells reached theslug stage after 24 h, the samples exposed to CPF concentrationsfrom 10�4 M to 10�8 M had a reduced aggregation capacity, onlyforming a tentative aggregation that did not develop into any ofthe typical stages of the life cycle of D. discoideum. This phenome-non was even more obvious 48 h after exposure, when fruitingbodies were evident among the controls but the exposed samplesstill remained at the tentative aggregation stage. The cells exposedto a CPF concentration of 10�10 M in the agar dish did not show anydifferences from the controls (data not shown).

3.4. The effect of CPF on the AChE activity of E. electricus

The exposure of the AChE of E. electricus to the same pesticideconcentrations as used in our experiment revealed an AChEinhibition trend similar to that observed for the PrChE of D. discoid-eum (Fig. 1).

3.5. The effects of CPF on the life cycle of P. lividus

Samples of P. lividus were exposed to CPF concentrations of10�4 M, 10�5 M, 10�6 M during the mating stage and showed irre-versible damage from the beginning of gastrulation (data notshown). That damage caused death in the embryonic stage, makingit impossible to observe the gastrula, larval and immature stages ofsea urchins. On the contrary, the samples exposed to CPF concen-trations of 10�7 M and 10�8 M had similar development to the con-trols (data not shown).

The samples exposed to concentrations of 10�4 M, 10�5 M,10�6 M, 10�7 M, 10�8 M at the beginning of the gastrulation stageshowed signs of dose-dependent damage. While a CPF concentra-tion of 10�4 M caused the death of all the gastrulas, exposure toconcentrations of 10�5 M and 10�6 M left an increasing numberof larvae to metamorphose. However, 35 h after fertilisation, manycontrol gastrulas had an evident embryonic celoma, with primarymesenchymal cells formed from large micromeres, and secondarymesenchymal cells, made up of of small micromeres (Fig. 4A),while the gastrulas exposed to CPF, at concentrations of 10�5 Mand 10�6, had anomalies such as the absence of a celomatic cavityand the loss of endodermic cell cohesion (Fig. 4A0 and A00). CPF con-centrations of 10�7 M and 10�8 M did not cause anomalies and thegastrulas appeared similar to the controls (data not shown).

Some of the gastrulas resistant to CPF concentrations of 10�5 Mand 10�6 M continued to develop, and 50 h after fertilisationformed larvae with dimensions similar to the controls (Fig. 4B),but with shorter than normal perioral arms (Fig. 4B0 and B00) andgenerally unable to metamorphose.

The samples exposed to CPF concentrations of 10�5 M and10�6 M during the larval stage metamorphosed into immaturesea urchins faster than the controls (Fig. 4C). However, they lackedcertain skeletal components and spines and, sometimes, some ped-icles (Fig. 4C0 and C00). The samples exposed to concentrations of10�5 M died within a few days, while many of those exposed to10�6 M developed but with the formation of elongated spines orother skeletal components, (data not shown). Exposure to CPF con-centrations of 10�7 M and 10�8 M did not cause anomalies in thelarval and immature stages, which had similar development tothe controls, (data not shown).

3.6. Effect of CPF on the cell growth of NT2

Cells exposed to CPF concentrations of 10�4 M, 10�5 M, 10�6 M,10�7 M, and 10�8 M, for 24, 48 and 72 h, showed variations in cellgrowth from the controls after 72 h. Exposure to a concentration of10�4 M caused the death of 18%, 40% and 63% of the initial popula-tion after 24 h, 48 h and 72 h respectively. Instead, the cells ex-posed to concentrations of 10�5 M and 10�6 M had similardevelopment to the controls, as did those exposed to concentra-tions of 10�7 M and 10�8 M (Fig. 5).

3.7. Effect of CPF on the AChE activity of NT2 cells

Cells exposed to CPF concentrations of 10�4 M, 10�5 M, 10�6 Mfor 24 h, and analysed with the spectrophotometric method of Ell-man et al. (1961), had a dose-dependent inhibition to AChE activity(Fig. 1). Cells exposed to CPF concentrations of 10�7 M and 10�8 M,for 24 h, had AChE activity similar to that of the controls. Cells ex-posed to an RA concentration of 10�6 M and CPF concentrations of10�5 M and 10�6 M, and analysed with the cytochemical method ofKarnovsky and Roots (1964), showed significant differences fromthe controls (Fig. 6). In fact, while the differentiated confluent cellsof the controls showed AChE activity localised at the cell mem-brane, and particularly in the cell–cell adhesion zone (Fig. 6A),the differentiated confluent cells exposed to CPF showed localised

A’ A A’'

B B’ B’'

C C’ C’'Fig. 4. Effect of CPF on the developmental cycle of P. lividus. Control gastrula (A), gastrula exposed to CPF 10�5 M (A0) and CPF 10�6 M (A00) for 24 h; control larva (B), larvaexposed to CPF 10�5 M (B0) and CPF 10�6 M (B00) for 24 h; control immature sea urchin (C), immature sea urchin exposed to CPF 10�5 M (C0) and CPF 10�6 M (C00) for 24 h.Bars = 25 lm.

control 24hr

control 48hr

CPF 10-4M 24hr CPF 10-5M 24hr CPF 10-6M 24hr CPF 10-8M 24hr

CPF 10-4M 48hr CPF 10-5M 48hr CPF 10-6M 48hr CPF 10-8M 48hr

Fig. 3. Effect of exposure to CPF, for 24 h and 48 h, on the developmental cycle of D. discoideum.

A. Amaroli et al. / Chemosphere 90 (2013) 2115–2122 2119

cytochemical staining similar to but less than that of the controls(Fig. 6E). The differentiated confluent cells exposed to CPF concen-trations of 10�7 M and 10�8 M, showed localised AChE activity andcytochemical staining similar to that of the controls (data notshown).

The undifferentiated non-confluent control cells had AChEactivity localised at the perinuclear level (Fig. 6B), as did the cellsexposed to CPF concentrations of 10�7 M and 10�8 M (data notshown), while the undifferentiated non-confluent cells exposed

to RA (Fig. 6D) and CPF concentrations of 10�5 M and 10�6 M(Fig. 6F) had a more obvious cytochemical colouration than thecontrols, localised at the perinuclear, cytoplasmic and membranelevels.

4. Discussion

The results of this work demonstrate that developing organismsare particularly sensitive to the toxic effects of CPF. This substance

Perc

enta

ge o

f vi

able

cel

ls

M

10-5M

10-6M

10−4

Fig. 5. Effect of exposure to CPF, for 24 h, 48 h, and 72 h, on the NT2 cell density.

2120 A. Amaroli et al. / Chemosphere 90 (2013) 2115–2122

can enter the organism via various pathways, but particularlythrough food, and can play an important role in the health of in-fants. Several studies have highlighted that infants raised on bio-logical products have pesticide levels six times lower, if notinexistent, than infants whose nutrition comes from intensivefarming, suggesting that baby food is the primary source of in-gested pesticides for infants in an urban and suburban environ-ment (Melnyk et al., 1997; Fenske et al., 2000; Lu et al., 2001;Williams and Hammit, 2001).

Although several nations have instituted programmes to deter-mine the level of pesticides in food (FDA, 1996; USDA, 1997), norelevant action has yet been taken to curb this problem.

In our study the CPF was tested at various concentrations,including those in the bibliography (Latif et al., 2011; Nougadèreet al., 2012), on biomarkers of toxicity and toxicity endpoints.

As is clearly indicated in our results in NT2 and Dictyosteliumcells the CPF have serious effects on its specific biomarker, thecholinesterase activity. Moreover, in our previous works weshowed how the AChE of P. lividus is inhibited by exposition toconcentration from 10�7 M up to 10�3 M of basudin and diazinon(Pesando et al., 2003) and to concentrations from 10�6 M up to

A

DFig. 6. AChE activity in NT2 cells revealed with the cytochemical method of Karnovskydifferentiated cells exposed to retinoic acid (RA) 10�6 M (C), undifferentiated cells exposcells exposed to 10�6 M and (F). The white arrows show the proper histochemical activ

10�4 M of CPF (Aluigi et al., 2010a). Our data suggest how thePrChE activity of D. discoideum is more sensitive (10�8 M) thanthe AChE of NT2 cells and P. lividus (10�6 M CPF).

High doses of CPF (10�4 M) induce the death of NT2 cells andgastrulas of P. lividus suggesting a cytotoxic effect of this pesticides.Recently, a strict correlation was found between AChE expressionand apoptosis (Zhang et al., 2002; Aluigi et al., 2010b). In fact, astrong inhibition of AChE induces a block of ACh receptors whoseexcitation is known to prevent apoptosis (Resende and Adhikari,2009).

Low doses of CPF do not induce cells death in our models.Nevertheless, at these concentrations the cell proliferation and dif-ferentiation of D. discoideum are altered and, moreover, the devel-opment of NT2 cells and P. lividus larvae are affected, suggesting, inagreement with the data in literature (Falugi et al., 2002; Angeliniet al., 2004; Aluigi et al., 2005; Aardema et al., 2008; Aluigi et al.,2010b; Amaroli, 2011), a correlation between cholinesterase inhi-bition and their alteration. In particular, the anomalies observedat the level of the perioral arms of the larva of P. lividus are a signnot only of morphological but also nerve damage, as the nervesthat synchronise the ciliary movement of the larva run along thosearms, which are also where the principal nervous ganglion, the api-cal ganglion, is situated (Marois and Carew, 1997). Furthermore,the malformation of the perioral arms, supported by skeletalstructures that form from inorganic crystallisation centres onglycoprotein deposited by mesenchymal cells, such as humanbone, suggests that CPF may play a role in skeletal anomalies in in-fants (Wilt, 1999). That hypothesis is supported by a study thatdemonstrated the effects of CPF on the differentiation of the oste-ogenic stem cells (Hoogduijn et al., 2006).

Concentrations of 10�5 M and 10�6 M inhibited AChE activity indifferentiated NT2 cells and induced, in undifferentiated NT2 cells,the development of cell morphology towards the neuronal pheno-type, underlined by a variation in the localisation of the AChE thatpasses from the perinuclear level of the undifferentiated cell tothat of the membrane characterising the excitable cell. Such a sit-uation, which can be positive for the aged or someone affected by aneurodegenerative disease, is negative in the case of a developing

F

B C

Eand Roots (1964). Differentiated control cells (A), undifferentiated control cells (B),ed to RA 10�6 M (D), differentiated cells exposed to CPF 10�6 M (E), undifferentiatedity of AChE. Bars = 10 lm.

A. Amaroli et al. / Chemosphere 90 (2013) 2115–2122 2121

embryo, as every developmental stage occurs at a precise momentand development outside of this moment could cause redundancyand disorder in the formation of the synapses.

The concentrations studied inhibited the activity of the PrChEenzyme in D. discoideum in a dose-dependent way and affectedthe developmental cycle, reducing cell growth in the 72 h afterexposure and impeding aggregation and differentiation. This effectcan be linked to the role of ChE in cell migration during the gastru-lation of vertebrates and the decidedly non-cholinergic activity ofChE to bind extracellular matrix molecules such as laminin andfibronectin (Drews, 1975).

5. Conclusion

In conclusion, on the basis of our results on the effect of CPF onthe organisms considered here, it would seem reasonable to claimthat the levels of pesticides present in food (Latif et al., 2011;Nougadère et al., 2012) can provoke problems in infant growthvia cholinesterase inhibition. The results appear even more inter-esting when taking into consideration the fact that all the modelsused meet the criteria of recent European Union initiatives thatrecommend the use of non-vertebrate alternative models (3RsStrategy) for tests and meet the ever increasing requests forRegistration, Evaluation, Authorisation and Restriction of ChemicalSubstances (REACH) checks on chemical substances that are poten-tially dangerous for human health and the environment.

Furthermore, the ECVAM regulations for good practice intoxicity tests require that laboratory models be bioethically com-patible, readily available, qualitatively congruous with the cost ofthe experiments, usable for more than one test, and suitable forcomparing results (Atterwil et al., 1994).

In view of these requirements, the high sensibility of D.discoideum to CPF appears particularly interesting. In fact, althoughP. lividus and NT2 cells are excellent models that provide reliableresults, they present respectively problems of seasonal difficultiesin obtaining gametes and high costs and long preparation times.On the contrary, the fast and cheap standardised growth procedureof D. discoideum meets all the requirements of the internationalcommunity.

Acknowledgments

This research has been partly funded by the Fondazione CARIGE(Genoa, Italy), 2009–2010.

Disclaimers/competing interest declaration: the authors haveno competing financial interests.

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