Japanese quail acute exposure to methamidophos: Experimental design, lethal, sub-lethal effects and cholinesterase biochemical and histochemical expression

Science of the Total Environment 450–451 (2013) 334–347

Contents lists available at SciVerse ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Japanese quail acute exposure to methamidophos: Experimental design, lethal, sub-lethal effects and cholinesterase biochemical and histochemical expression Manousos Foudoulakis a,⁎, 1, Christos Balaskas b,⁎⁎, 1, Attila Csato c, Csaba Szentes c, Gerassimos Arapis a a b c

Laboratory of Ecology and Environmental Sciences, Agricultural University of Athens, Iera Odos 75, Athens 11854, Greece Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, Iera Odos 75, Athens 11854, Greece Ecotoxicological Laboratory, 7136 Fácánkert Puszta 1, Hungary

H I G H L I G H T S ► ► ► ► ►

We examined the effects of methamidophos to Japanese quail in an acute oral test. We estimated ChE activity in various tissues using various substrate-inhibitors. Changes concerning the activity of plasma, brain and liver ChEs, were reversible. Cyto-architectural and histochemical changes were persistent. We assessed enteric neuronal function in a major absorption site, the duodenum.

a r t i c l e

i n f o

Article history: Received 22 January 2012 Received in revised form 14 October 2012 Accepted 15 October 2012 Available online 9 November 2012 Keywords: Oral acute test Methamidophos Quail Cholinesterase Histochemistry

a b s t r a c t We exposed the Japanese quail (Coturnix coturnix japonica) to the organophosphate methamidophos using acute oral test. Mortality and sub-lethal effects were recorded in accordance to internationally accepted protocols. In addition cholinesterases were biochemically estimated in tissues of the quail: brain, liver and plasma. Furthermore, brain, liver and duodenum cryostat sections were processed for cholinesterase histochemistry using various substrates and inhibitors. Mortalities occurred mainly in the first 1–2 h following application. Sub-lethal effects, such as ataxia, ruffled feathers, tremor, salivation and reduced or no reaction to external stimuli were observed. Biochemical analysis in the brain, liver and plasma indicates a strong cholinesterase dependent inhibition with respect to mortality and sub-lethal effects of the quail. The histochemical staining also indicated a strong cholinesterase inhibition in the organs examined and the analysis of the stained sections allowed for an estimation and interpretation of the intoxication effects of methamidophos, in combination with tissue morphology visible by Haematoxylin and Eosin staining. We conclude that the use of biochemistry and histochemistry for the biomarker cholinesterase, may constitute a significantly novel approach for understanding the results obtained by the acute oral test employed in order to assess the effects of methamidophos and other chemicals known to inhibit this very important nervous system enzyme. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Various studies carried out in both terrestrial and aquatic organisms have demonstrated (WHO, 1993) that exposure to organophosphorus pesticides (OPs), known for their anti-cholinesterase (ChE) properties, poses a significant occupational hazard in agriculture, and in addition exerts toxic effects to non-target organisms. The activity of ChEs, inclusive of both acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, also called pseudo-cholinesterase or non-specific cholinesterase,

⁎ Corresponding author. Tel.: +30 210 5745224; fax: +30 210 529 4464x5. ⁎⁎ Corresponding author. Tel.: +30 210 5294389; fax: +30 210 5294388. E-mail addresses: [email protected] (M. Foudoulakis), [email protected] (C. Balaskas). 1 Equal contribution to the work. 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.10.066

EC 3.1.1.8), has been commonly used as a biomarker for pollution evaluation and risk assessment (Fossi et al., 1992; Thompson and Walker, 1994; Soler-Rodriguez et al., 1998; Storm et al., 2000). BuChE is mainly found in plasma but is also present in brain, liver, muscle, and other tissues (Geula et al., 1995; Monteiro et al., 2005). Being found in both the developing and mature brain, it suggests that BuChE may play important roles in neurogenesis, neuronal development and cell proliferation (Geula and Nagykery, 2006; Mack and Robitzki, 2000). Moreover, its physiological role seems also related to detoxification processes and lipid metabolism (Mack and Robitzki, 2000; Darvesh et al., 2003). The mechanism of toxic action of OPs insecticides, or their metabolites, is based on the irreversible inhibition of the enzyme AChE, which hydrolyzes the neurotransmitter acetylcholine (ACh) to end the cholinergic neural transmission in both the central and peripheral nervous

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

systems of vertebrates (Carr and Chambers, 2001). Accumulation of ACh in synapses, due to AChE inhibition, results in overstimulation followed by depression or paralysis and eventual death (Abou-Donia, 1992). OPs are also capable to induce apoptosis by multifunctional pathways in tissues such as brain (Caughlan et al., 2004). In addition to cellular changes, the use of OPs is known to result in physiological changes in various organs (liver, kidney) and systems (nervous, immune and reproductive system) (Aly and El-Gend, 2000; Gomes et al., 1999; Nagymajtenyi et al., 1998; Rawlings et al., 1998), inclusive of behavioral and/or even psychological dysfunction (Beauvais et al., 2000). Signs of sub-lethal ChE inhibition in birds include nausea, lethargy, nutation, wing-drop, loss of righting-reflex, paralysis, opisthotonos and coma (Somers et al., 1991). In the end, death, secondary to the acute cholinergic syndrome by OPs, is usually due to a combination of excessive bronchial secretion, bronchospasm and paralysis of the respiratory center (Klaassen, 2008). Japanese quail (Coturnix coturnix japonica) is a well established bio-indicator in accordance with the OECD guidelines and the European legislation for the risk assessment of pesticides, biocides and chemicals (OECD 223, 2002; OECD 205, 1984; OECD 206, 1984). Methamidophos (O,S-Dimethyl phosphoramidothioate) was selected as a model compound given its well-established AChE inhibitory action (Brimijoin, 2005; Sheets et al., 1997) and one of the most widely used OPs in agriculture (Suresh et al., 2007; Tacal and Lockridge, 2010). The aim of this study was to evaluate acute lethal and sub-lethal effects of methamidophos, by means of ChE biochemistry and histochemistry in a variety of organs (brain, liver and duodenum), as well as plasma of Japanese quail. 2. Materials and methods 2.1. Chemicals Methamidophos technical (purity 98.2%) was supplied by Bayer CropScience [batch no. 2377 (M01585)].

335

Given the fact that for the middle and high doses high mortality was observed soon after dosing, we re-dosed 5 new birds for each of the above-mentioned treatments in order to take samples 30 min and 1 h after. 2.3. Biological observations and measurements The birds were monitored for sub-lethal effects of toxicity every 2 h during daylight for the 14-day duration of the study. In particular, only for day 0 the birds were also monitored at 30 min, 1, 2 and 3 h after the administration of the gavage doses. Body weights were determined at initiation of the study (day 0) and on days 3, 7 and 14. Feed consumption was estimated for each treatment and control group every day. 2.4. Sampling and preparation of tissues Birds were euthanized by decapitation. Tissue sampling for both biochemical and histochemical analysis was performed on day 0/1, and on days 3/4 and 14; evidently dead and alive quails were used. Blood was collected into heparinized tubes for plasma preparation using a syringe inserted in the heart, immediately after decapitation, and whole brain, liver and duodenum samples were removed and immediately placed at − 80 °C until the time of the biochemical assay (not for duodenum), and for histological evaluation. Notably, no control quails' were sampled at 30 min and 1 h after the administration of the gavage dose. This did not pose a statistical problem given that the biochemical and histochemical observations for the control did not differ between samplings, as logically expected for the whole duration of the trial (Tables 2–7). 2.4.1. Preparation for biochemical analysis 2.4.1.1. Plasma. For the enzyme assay blood was collected from sacrificed animals. Plasma was separated by centrifugation at 4000 g for 5 min, aspirated and stored in aliquots at − 80 °C.

2.2. Animals This study was designed to assess the acute oral toxicity (LD50) of methamidophos to the Japanese quail (Coturnix coturnix japonica). The study design was in compliance with the OECD guidelines draft 223 (2002), and the principles of the US EPA (1996). The study described in this report was conducted in compliance with Good Laboratory Practice standards. Japanese quails (3.5 months old) were bred in the Ecotoxicological Laboratory, Fácáncert, Hungary. The birds were weighed individually shortly before the beginning of the test and were allocated to the test groups by a randomization plan on the basis of their body weights (bw). This was done with the aim, all treatment groups to have similar mean bw and bw distribution at the beginning of the trial. The birds were marked individually by numbered leg bands. The quails were offered a commercial poultry diet ad libitum throughout maintenance before the study and during the test with the exception of a fasting period of about 16 h prior to dosing. Relative humidity was 30–75%, temperature 18–22 °C and illumination on an 8:16 LD cycle under natural intensity. Before intoxication, and one week before the beginning of the test, to allow acclimatization, the birds were assigned to individual cages (103 × 51 × 37 cm) with a floor area for 10 birds (in the control 8 birds) which contained an automatic drinker and food hopper. The birds received a single dose of the test material in an aqueous solution by oral intubation using a disposable syringe and a catheter. Birds were randomly assigned to a control group (not treated) or groups exposed to methamidophos at nominal concentrations of 1, 2.2, 5, 11.2 and 25 mg/kg bw. The observation period lasted 14 days.

2.4.1.2. Brain and liver. For the enzyme assay the left hemisphere of the brain inclusive of the left part of the cerebellum and liver samples were weighed and homogenized in 0.05 M Tris–HCl (pH 7.6, T-3253 Sigma). To solubilize membrane bound AChE, Triton X-100 (3% v/v in distilled water, T8787 Sigma) was also added. Homogenates were then decanted into an Eppendorf tube and centrifuged at 9000 g for 30 min at 4 °C and the supernatant stored in aliquots at − 80 °C. 2.4.2. Preparation for histochemical analysis 2.4.2.1. Fixation. Pieces of the removed tissues, the right hemisphere of the brain and the right part of the cerebellum, liver and duodenum were immersed in 4% paraformaldeyde (P6148 Sigma) in 0.1 M phosphate-buffered saline (PBS, pH 7.4, 79,382 Fluka), at a volume of at least 20 times the volume of the tissue, for 1 h at room temperature. Tissues were washed two times for 30 min in PBS before storage overnight at 4 °C in PBS containing 7% sucrose and 0.1% sodium azide. 2.4.2.2. Cryostat sections. The fixed tissue was attached to a piece of cork (1×1 cm) and embedded in cryoprotection medium (Tissue-Tek, JUNG), and rapidly frozen in liquid-nitrogen-cooled isopentane (AC126470010 Acros). Isopentane acts as a cryoprotective agent against the liquid nitrogen. 10–30 μm thick transverse sections were cut using Leica CM1500 cryostat. A number of frozen sections (4 to 5) from different layers of the brain, liver and duodenum were thaw-mounted on poly-L-lysinecoated glass slides and finally stored at −4 °C.

336

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

2.4.2.3. Microscopy slides poly-L-lysine coating. Untreated slides were washed in acid alcohol solution (70% ethanol and 1% HCl 1 N) for 90 min followed by running tap water for additional 90 min. After the washing, slides were dipped into poly-L-lysine solution (P8920 Sigma) for 10 s. Finally they were drained in the oven (37 °C) for 24 h.

253949.1610) for 2 min, rinsed with tap water and then dipped in Eosin Y solution (Merck Certistain 1.15935.0025) for 1 min followed by rinsing again in tap water. The slides were then dehydrated in ascending ethanol solutions (2× 50%, 70%, 90%, 2 × 100%) followed by 2 × xylene, air dried and finally mounted with Depex (18,243 Serva) and coverslips.

2.5. Enzyme assays Plasma and supernatants from brain and liver preparations were assayed for ChE activity in vitro after the exposure of varying concentrations of methamidophos. All assays were performed according to Ellman et al. (1961) adapted to microplate and incubations were performed at 25–30 °C using the appropriate volume of homogenate or plasma, various alkylthiocholine substrates selective for ChEs, AChE and BuChE. AChE is highly selective for acetyl esters as substrates and BuChE preferentially hydrolyze butyryl and propionyl esters, although it also hydrolyzes a wider range of esters, including ACh. In a typical assay, 0.250 ml of the reaction solution [30 ml of phosphate buffer (0.1 M, pΗ 7.6)], 1 ml of DTNB (D8130 Sigma) 10 mM as chromagen reagent [5,5′ dithio-bis 2-(nitrobenzoic acid)] and 0.2 ml of various alkylthiocholine substrates 0.075 M [acetylthiocholine iodide (ASCh, A5751 Sigma) or S-butyrylthiocholine iodide (BuSCh, B3253 Sigma) or propionylthiocholine iodide (PrSCh, P2880 Sigma)] were added to each well while the volume of the samples for the homoganates (brain or liver) and plasma was 10 and 5 μl, respectively. The change of absorbance per min at 405 nm (ΔA) during 5 min was measured at 25–30 °C using a microplate microreader (Model M2E Molecular Devices). The reaction solution (buffer+DTNB+substrate) was used as blank. The concentration of protein in the tissue samples was determined by the Bradford method (Bradford, 1976), adapted to microplate, at 595 nm. Commercial bovine serum albumin (BSA, ICN 160069 Sigma), was used as standard. ChEs were classified as AChE or BuChE with the aid of diagnostic substrates and inhibitors given that they both lack complete selectivity (Fairbrother et al., 1991). In fact enzymatic activity was studied in the presence of the various alkylthiocholine substrates separately and with selective inhibitors. In vertebrates, eserine (E8375 Sigma), 1,5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51, A9013 Sigma) and tetraisopropyl pyrophosphoramide, (iso-OMPA, T1505 Sigma) are selective for ChE, AChE and BuChE, respectively. The expression of ChE justifies its classification as AChE in all respects when it hydrolyzes ASCh, but not BuSCh, is sensitive to BW284C51 but not to iso-OMPA. Respectively, the expression of ChE justifies its classification as BuChE in all respects when it hydrolyzes ASCh and is sensitive to iso-OMPA, or it hydrolyzes BuSCh. As regards the use of the substrate PrSCh, the expression of ChE justifies its classification both as AChE and BuChE. The tissue homogenates were pre-incubated with the inhibitors (10 −4 M, final concentration each) for 3 min before addition of the substrate. AChE was assayed with ASCh as the substrate after removal of BuChE activity by incubation of enzyme extracts with iso-OMPA. ChE activity of the treatments was expressed as μmol substrate hydrolyzed × min −1 × mg protein −1 and as the percentage of the average activity compared to the controls.

2.6.2. ChE staining of cryostat sections The histochemical reaction was carried out using a variation of the Karnovsky and Roots (1964) method proposed by Baker et al. (1986) with various alkylthiocholine substrates, ASCh, BuSCh and PrSCh. ChEs are classified as AChE or BuChE with the aid of diagnostic substrates and inhibitors as mentioned previously in the biochemical enzyme assays. For ASCh histochemistry, sections were incubated in a hyaluronidase solution (0.33 mg per 100 ml, H 3506 Sigma) with a 10−4 M solution of iso-OMPA. Hyaluronidase is used to increase the permeability of the tissue (Baker et al., 1986). For BuSCh and PrSCh histochemistry sections were incubated in the hyaluronidase solution alone. Furthermore, as a positive control for PrSCh histochemistry, 10 − 4 M BW284C51 and 10 − 4 M iso-OMPA solutions were used, in addition to hyaluronidase. The reaction medium consists of two parts (A and B) which, when mixed, form the working solution; the medium was kept at 4 °C, where it is stable for hours. Part A consists of 0.1 M acetate buffer pH 5 with 1.5% Triton X-100 (65 ml), 0.15 mM sodium citrate (10 ml), 30 mM copper sulfate (10 ml) and 5 mM potassium ferricyanide [K3Fe(CN)6] (10 ml). Part B consists of various substances depending on the ChEs to be evaluated, and in particular: For AChE: 10 −4 M iso-OMPA and ASCh (50 mg). For BuChE: 10 −4 M BuSCh (50 mg). For ChEs that hydrolyze PrSCh: 10 −4 M PrSCh (50 mg). For positive control of ChEs that hydrolyze PrSCh: 10 −4 M iso-OMPA and 10 −4 M BW284C51 and PrSCh (50 mg). The tissues were washed in distilled water for 1 h, fixed in 4% paraformaldehyde in PBS (0.1 M, pH 7.4) for 30 min, and mounted with 70% glycerol in PBS.

2.6.3. Microscopic examination For digital microphotography and observation of H&E and ChEs stained sections, Olympus 1011 light microscope and digital camera (OLYMPUS BX50) were used combined with image analysis software (Image-Pro Plus Version 3.1. for Windows 95, MEDIA CYBERNETICS, USA). In order to express a qualitative account of ChE histochemistry each sectional profile was examined, by the same person, under the light microscope and was assigned 3 different marks given by this person. The marks referred to i) staining intensity [from light (0.5) to very dark (5), with a step of 0.5 between marks], ii) the staining density [from scarce (0.5) to very dense (5), with a step of 0.5 between marks] and iii) the staining extent [from low (0.1) to extensive staining area (3)]. Their product value, which served as an indicator of the staining pattern is presented in Tables 8–10; the assigned marks are not therefore displayed in the relevant tables.

2.6. Histochemistry 2.6.1. Haematoxylin and eosin (H&E) staining of cryostat sections This staining technique was performed according to a modification of the original method of Harris (1900); slides mounted with the cryostat sections were dipped in paraformaldehyde solution (0.1 M, pH 7.4) for 3 min at room temperature before staining. Subsequently, the slides were dipped in freshly filtered Harris haematoxylin (Pancreac DC

2.7. Statistical analysis The LDx values were calculated following the probit model. The statistical evaluation was performed using the SPSS software version 13.0. Results are presented as mean ± SD. Statistical analysis is not presented because of the high variance observed.

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347 Table 1 Sub-lethal effects of Japanese quail, following the administration of several doses of methamidophos, in an avian acute oral test. Methamidophos (mg/kg bw)

No. of birds with sub-lethal effects, type of sub-lethal effects and severity Time of sampling and number of quails per treatment 30 min

1h

2h

3h

4.5 h

>4.5 h

1d

2d

14 d

0 1 2.2 5

0 0 0 0

0 0 0 10Aa 10Ra 10Sa 10Ta

0 0 0 10Ac 10Rc 10Sc 10Tc

0 0 2Aa 5Ac 5Rc 5Sc 5Tc 1D

0 0 2Aa 5Ac 5Rc 5Sc 5Tc

0 0 1Fa 2Fa

0 0 0 0

0 0 0 0

11.2

10Aa 10Ta

10Ac 10Tc 10Lc 10Wc

2Ac 2Tc 2Lc 2Wc

2D

x

x

25

10Aa 10La

10Nc

2Ac 2Tc 2Lc 2Wc 8D 10D

0 0 2Aa 6Ac 6Rc 6Sc 6Tc 4D 2Ac 2Tc 2Lc 2Wc

x

x

x

x

x

337

The NOEL was determined to be 1 mg/kg bw, based on sub-lethal effects at 2.2 mg/kg bw (LOEL). 3.1.3. Body weight and feed consumption At day 7, bw gain was 18.7, 5.8, 8.2 and 7.7%, while for day 14 the bw gain was 25.2, 5.5, 7.5 and 11.2% for the control and the dose rates of 1, 2.2 and 5 mg/kg bw, respectively. As regards the rate of feed consumption, this was similar between the control and the dose treatments during the first 7 days, while for the remainder 7 days feed consumption for the dose treatments was approximately 25% lower from the control. 3.2. Biochemical results for cholinesterases

Key to the type of sub-lethal effects: F = ruffled feathers, W = can't use wings, S = salivation, A = difficulties in walking (ataxia), L = leg weakness, R = reduce reaction to external stimuli, T = tremor, N = no reaction. Severity of sub-lethal effect symptoms: a = light or for a short period, b = moderate, c = severe. D = Dead birds. x= All the individuals were dead.

3. Results 3.1. Biological results 3.1.1. Mortality Mortality occurred mainly within 1–2 h after the administration of the gavage dose (except for 2 birds in the dose of 11.2 mg/kg bw which died on day 1). Mortality during the study is presented in detail in Table 1. The LD50 of methamidophos to Japanese quail is 2.2 b LD50 b 11.2 mg/kg bw. Most of the quails of the 25 mg/kg bw group displayed gross hemorrhagic features over the surface of their brain, as revealed during sampling.

3.1.2. Sub-lethal effects All quails in the control group were in good health throughout the trial. In the methamidophos groups light, moderate or severe toxic signs were observed. These signs included ataxia, ruffled feathers, leg weakness, tremor, salivation and reduced or no reaction to external stimuli (Table 1). The survivors in all dose rates had completely recovered from the mid of day 1 onwards.

3.2.1. Plasma ChEs present in plasma showed a high activity when ASCh, BuSCh or PrSCh were used as substrate. The results obtained using ASCh or BuSCh as a substrate, with BW284C51, iso-OMPA and eserine as inhibitors suggested that the enzyme is only BuChE given the insensitivity induced by BW284C51, the 98% inhibition induced by iso-OMPA and 99% inhibition induced by eserine. The results obtained using PrSCh as a substrate suggested that BuChE is able to hydrolyze PrSCh as well, as inhibition with BW284C51, isoOMPA and eserine displayed similar results as with ASCh or BuSCh (insensitivity induced by BW284C51, 99% inhibition induced by iso-OMPA and 100% inhibition induced by eserine). In the control BuChE activities, with BuSCh or PrSCh were similar as presented in Tables 2 and 3. The BuChE activity with ASCh was similar to BuSCh (not presented in Tables). BuChE activity was significantly inhibited 18% to 100% from 0.5 h up to day 1. This inhibition did not persist and from day 3 onwards, BuChE activity was significantly increased from 31 to 785% and remained increased until the end of the trial (14th day; 65–206%) (Table 2). 3.2.2. Brain ChEs present in brain showed a high activity when ASCh or PrSCh was used as substrate while when BuSCh was used as substrate a very low activity was observed. The results obtained using ASCh as a substrate, with BW284C51, iso-OMPA and eserine as inhibitors suggested that the enzymes are AChE and traces of BuChE given the 86% inhibition induced by BW284C51, the 8% inhibition induced by iso-OMPA and 94% inhibition induced by eserine. When using BuSCh as a substrate, with BW284C51, iso-OMPA and eserine as inhibitors and due to the very low activity suggested that the enzyme is traces of BuChE given the insensitivity induced by BW284C51, the 39% inhibition induced by iso-OMPA and 62% inhibition induced by eserine.

Table 2 ChE activity (using BuSCh as a substrate) in plasma from Japanese quail, following the administration of several doses of methamidophos, in an avian acute oral test. Methamidophos (mg/kg bw) 0 1 2.2 5 11.2 25

Plasma ChE activity using BuSCh as a substrate (μmol × min − 1 × mg protein − 1). 30 min

0.2 0.7

1h

−1.2 ± 3.1 (100a) 0.1 ± 0.1 (100) 0.5 ± 0.3 (99)

Day 1

Day 3

Day 14

35.6 ± 2.5 (0) 15.9 ± 5.1(55) 29.0 ± 8.3 (18)

42.7 ± 32.8 (0) 55.9 ± 29.5 (−31) 89.1 ± 0.9 (−109) 377.6 ± 81.2 (−785) x x

36.8 ± 8.5 (0) 60.9 ± 21.4 (−65) 112.7 ± 32.1 (−206) 65.7 ± 12.4 (−79) x x

0.4 (99) x

Data are expressed as mean ± SD. Inhibition as a percentage to the control birds is indicated in ( ). x = All the individuals were dead. a No control quails were sampled at 30 min and 1 h after the administration of the gavage dose. Inhibition is expressed as a percentage to the control birds on day 1.

338

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

Table 3 ChE activity (using PrSCh as a substrate) in plasma from Japanese quail, following the administration of several doses of methamidophos in an avian acute oral test. Plasma ChE activity using PrSCh as a substrate (μmol × min − 1 × mg protein − 1)

Methamidophos (mg/kg bw)

30 min 0 1 2.2 5 11.2 25

1h

−0.4 ± 1.4 (100a) 0.7 ± 0.7 (100) 1.5 ± 1.8 (100)

−0.1 −0.7

Day 1

Day 3

Day 14

33.2 ± 1.7 (0) 17.6 ± 03.5 (47) 25.9 ± 10 1 (22)

41.9 ± 31.8 59.7 ± 18.5 (-42) 81.1 ± 11.6 (−93) 357.0 ± 111.2 (−751) x x

34.8 ± 11.4 (0) 55.0 ± 7.3 (−58) 60.2 ± 29.7 (−142) 60.2 ± 22.8 (−73) x x

1.3 (96) x

Data are expressed as mean ± SD. Inhibition as a percentage to the control birds is indicated in ( ). x = All the individuals were dead. a No control quails were sampled at 30 min and 1 h after the administration of the gavage dose. Inhibition is expressed as a percentage to the control birds on day 1.

Finally, the results obtained when using PrSCh as a substrate suggested, in contrast with results from the plasma, that the enzyme is primarily AChE. AChE is able to hydrolyze PrSCh in the brain as inhibition with BW284C51, iso-OMPA and eserine was 82%, 9% and 96%, respectively. In the control the highest ΑChE activity was found with ASCh or PrSCh as presented in Tables 4 and 5. Less significant activity was found when the substrate used was BuSCh (29.0 ± 13.18 SD μmol × min −1 × mg protein −1; data not presented in tables). For all doses, methamidophos produced 53–66% inhibition in brain AChE activity of living birds up to the 1st day after the exposure. AChE activity recovered from day 1 onward, for the living birds (Table 4). Notably, PrSCh as a substrate revealed much longer effects as ChE activity did not recover on day 14 (Table 5).

AChE given the 56% inhibition induced by iso-OMPA and the 63% inhibition induced by eserine. BuSCh and PrSCh as substrates suggested that the enzyme is BuChE. BW284C51 as inhibitor displayed similar results with the plasma given the insensitivity induced by BW284C51. In the control low ChE activity was found with BuSCh and PrSCh as presented in Tables 6 and 7. The ChE activity with ASCh was similar (not presented in Tables). For all doses, methamidophos produced 34–71% inhibition in liver BuChE activity up to the 1st day after the exposure. From day 3 onwards, BuChE activity was increased compared to the control, with the exception of the medium dose (5 mg/kg bw) (Table 6). Similar results were obtained for ChEs activity, when PrSCh was used as a substrate (Table 7).

3.2.3. Liver ChEs present in liver showed a low activity when ASCh, BuSCh or PrSCh was used as substrates. Similar results, which indicate low ChE activity, were obtained when using ASCh as a substrate with iso-OMPA and eserine as inhibitors. These results suggested that the enzyme is BuChE and traces of

3.3. Histochemical results 3.3.1. Brain 3.3.1.1. H&E stained sections. H&E staining in the cryostat sections of the brain revealed its structure in the control group and allowed for

Table 4 ChE activity (using ASCh without iso-OMPA as a substrate) in brain from Japanese quail, following the administration of several doses of methamidophos, in an avian acute oral test. Brain ChE using ASCh without iso-OMPA as a substrate (μmol × min − 1 × mg protein − 1)

Methamidophos (mg/kg bw)

30 min 0 1 2.2 5 11.2 25

1h

170.2 ± 42.2 (54 [62]a) 126.2 ± 11.3 (66 [72]) 167.3 ± 58.2 (55 [90])

137.8 55.2

Day 1

Day 3

Day 14

374.1 ± 108.9 (0) 177.6 ± 52.2 (53) 197.1 ± 25.9 (47)

373.4 ± 126.3 (0) 209.3 ± 13.4 (44) 193.9 ± 14.8 (48) 229.3 ± 7.5 (39) x x

266.2 ± 53.4 (0) 263.5 ± 29.7 (1) 223 ± 20.2 (16) 197.5 ± 91.0 (26) x x

126.3(66) x

Data are expressed as mean ± SD. Inhibition as a percentage to the control birds is indicated in ( ) for live and in [ ] for dead quails. x = All the individuals were dead. a No control quails were sampled at 30 min and 1 h after the administration of the gavage dose. Inhibition is expressed as a percentage to the control birds on day 1.

Table 5 ChE activity (using PrSCh as a substrate) in brain from Japanese quail, following the administration of several doses of methamidophos in an avian acute oral test. Methamidophos (mg/kg bw) 0 1 2.2 5 11.2 25

Brain ChE using PrSCh as a substrate (μmol × min−1 × mg protein−1) 30 min

62.2 52.2

1h

145.3 ± 23.0 (65[73]a) 106.9 ± 44.1 (74[67]) 154.4 ± 69.3 (63[90])

Day 1

Day 3

Day 14

415.3 ± 204.9 (0) 221.3 ± 149.3 (47) 154.7 ± 40.4 (63)

373.2 ± 246.0 (0) 166.0 ± 6.0 (56) 159.4 ± 54.7 (57) 129.4 ± 79.3 (65) x x

313.6 ± 70.5 199.8 ± 53.0 221.1 ± 95.4 167.2 ± 37.4 x x

86.9 (79) x

Data are expressed as mean ± SD. Inhibition as a percentage to the control birds is indicated in ( ) for live and in [ ] for dead quails. x = All the individuals were dead. a No control quails were sampled at 30 min and 1 h after the administration of the gavage dose. Inhibition is expressed as a percentage to the control birds on day 1.

(0) (36) (30) (47)

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

339

Table 6 ChE activity (using BuSCh as a substrate) in liver from Japanese quail, following the administration of several doses of methamidophos, in an avian acute oral test. Methamidophos (mg/kg bw)

Liver ChE activity using BuSCh as a substrate (μmol × min−1 × mg protein−1) 30 min

0 1 2.2 5 11.2 25

11.4 7.4

1h

11.3 ± 2.6 (59a) 11.0 ± 5.2 (60) 9.2 ± 1.3 (67)

Day 1

Day 3

Day 14

27.6 ± 22.9 (0) 18.2 ± 10.5 (34) 17.1 ± 7.5 (38)

25.7 ± 18.7 (0) 38.2 ± 1.7 (49) 26.9 ± 15.8 (−5) 27.9 ± 10.4 (−9) x x

26.4 ± 28.0 (0) 42.3 ± 29.4 (−60) 42.2 ± 27.6 (−59) 23.7 ± 7.0 (10) x x

7.9 (71) x

Data are expressed as mean ± SD. Inhibition as a percentage to the control birds is indicated in ( ). x = All the individuals were dead. a No control quails were sampled at 30 min and 1 h after the administration of the gavage dose. Inhibition is expressed as a percentage to the control birds on day 1.

comparison between treatments. The inclusion of methamidophos in the treated groups resulted in dose-dependent changes in the cyto-architecture of the brain of the quails used for over a period of 14 days. In particular, these changes include decreased affinity for H&E staining in the cytoplasm of neuronal cells, revealed as lighter staining in areas of the brain tissue sections. In addition, increased cytoplasmic vacuoles and nuclei showing signs of condensation in the nucleoplasm were observed. These findings of both cytoplasmic and nuclear degeneration, often observed together in the same cells were evident for the lower doses (1 and 2.2 mg/kg bw) (Fig. 1a). Notably, the outer cell layers of the brain sections were less affected when compared with the inner layers. Spongiform morphology was more discernible as time from the treatment passed on, up to a point when tissue restoration appeared to compensate for the damage caused by methamidophos. In the middle dose (5 mg/kg bw) the histopathological changes observed were more severe compared to those observed for the lower doses. These changes included caryomegaly or pyknotic nuclei often in groups of cells. Spongiform morphology was also observed and coincided with decreased affinity for H&E staining in the examined sections and was more prominent in the center of the tissue sectional profile. As regards the two higher doses (11.2 and 25 mg/kg bw) which resulted in the eventual death of all the quails within the treatment the sections represent post-mortem histology. As such, extreme vacuolization within the cells, H&E differential staining in the section profile and extreme spongiform morphology were evident, but less severe in the periphery of the sectional profile (Fig. 1b). The process of the tissue decay was apparent in some cases. 3.3.1.2. AChE stained sections. Histochemical staining for AChE results in the visualization of neurocyte perikarya (the nuclei do not stain) and nerve bundles (groups of individual neuronal axons). The combined use of ASCh as a substrate with iso-OMPA as an inhibitor resulted in intensely positive AChE neuronal cells, with histochemical reaction product residing within the cytoplasm, often closely associated with the cytoplasmic membranes, displaying a

granular pattern. The cell and nucleus membranes of these cells are also stained. Furthermore, the proximal dendrites of these neurons and in addition neuronal axon and nerve sectional profiles are evidently stained. A comparison between the treatments (doses and time after exposure) revealed a lower staining as it is presented in Table 8 (Fig. 2a). In the sectional profiles of brains which received lower or middle doses and in particular 14 days following exposure (none of the quails received higher doses lived that long); a broader network of individual neuronal axons or nerves stained for AChE was evident (Fig. 2b). ΑChE positive cells were evident throughout the sectional profiles. 3.3.1.3. BuChE stained sections. The use of BuSCh resulted in extremely faint BuChE granular staining closely associated with the neuronal cytoplasmic membranes. The stain is more evident mainly at the periphery of the sectional profiles, often in the nuclei while no axon or dendrite-like processes were visibly stained. The overall histochemical reaction was estimated at 8.7% of the reaction observed for AChE. The stained BuChE sectional profiles for the lower and the middle doses displayed large variations between individuals within the groups (Table 8). Most BuChE positive cells resided in the cortex of the brain of the control quails (Fig. 2c). The distribution of the BuChE positive cells in the sectional profiles differed in the lower and middle doses for the quails which survived the treatment. In particular for the lower doses (after day 3) and for the middle dose, the BuChE positive cells were evident throughout the sectional profile (Fig. 2d). 3.3.1.4. ChEs that hydrolyze PrSCh stained sections. The use of PrSCh resulted in extensive staining which was evidently not restricted to neurons but in addition stained red blood cells within vascular sectional profiles. In the case of neuronal staining, this was similar to that observed for BuChE, but evidently not as granular. In red blood cells both the cytoplasm and the cell membrane were stained. The overall histochemical reaction was estimated at 77% of the reaction observed for AChE.

Table 7 ChE activity (using PrSCh as a substrate) in liver from Japanese quail, following the administration of several doses of methamidophos, in an avian acute oral test. Methamidophos (mg/kg bw)

Liver ChE activity using PrSCh as a substrate (μmol × min − 1 × mg protein − 1) 30 min

0 1 2.2 5 11.2 25

14.9 13.5

1h

20.4 ± 11.4 (18a) 14.0 ± 3.0 (43) 13.6 ± 2.4 (45)

Day 1

Day 3

Day 14

24.8 ± 18.8 (0) 33.4 ± 19.8 (−35) 18.4 ± 6.0 (26)

29.8 ± 19.1 (0) 45.7 ± 0.5 (−54) 30.7 ± 13.6 (−3) 47.1 ± 6.2 (−58) x x

40.8 ± 22.1 69.9 ± 50.4 40.5 ± 21.6 34.6 ± 11.8 x x

21.9 (12) x

Data are expressed as mean ± SD. Inhibition as a percentage to the control birds is indicated in ( ). x = All the individuals were dead. a No control quails were sampled at 30 min and 1 h after the administration of the gavage dose. Inhibition is expressed as a percentage to the control birds on day 1.

(0) (−72) (1) (15)

340

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

The staining pattern for all doses was more prominent as it is presented in Table 8. When both inhibitors (iso-OMPA and BW284C51) were used as a positive control the results still displayed a similar staining pattern, with consistently lesser background staining. In addition, it appears that fewer sectional profiles associated with blood cells were stained. ChEs that hydrolyze PrSCh positive cells were evident throughout the sectional profiles (Fig. 2e). 3.3.2. Liver 3.3.2.1. H&E stained sections. For the lower doses, our observations include restricted differential Η&Ε staining within the sectional profiles of the liver lobules, caryomegaly and in some cases apoptotic liver cells, when compared to the controls (Fig. 1c). In the middle dose (5 mg/kg bw) differential Η&Ε staining within the sectional profiles of the liver lobules was observed. In addition, more extensive (when compared to the lower doses) lesions of the cyto-architecture, often a spongiform morphology visibly not attributed to vasculature, was evident in the sectional profiles. Furthermore, the regeneration of the tissue appeared to lag behind in some cases.

Table 8 ChE histochemistry in Japanese quail brain evaluation, following the administration of several doses of methamidophos, in an avian acute oral test. Methamidophos (mg/kg bw)

AChE stained sections

BuChE stained sections

ChEs that hydrolyze PrSCh stained section

0 1 2.2 5 11.2 25

60. 0 ± 7.5 44.8 ± 7.6 (−25.3) 39.9 ± 18.2 (−33.5) 49.1 ± 24.8 (−18.1) 60.6 ± 11.2 (1) 42.9 ± 21.7 (−28.5)

5.2 ± 2.6 5.9 ± 4.1 4.8 ± 7.7 4.0 ± 4.2 1.3 ± 2.4 1.3 ± 1.7

46.2 ± 11.6 55.8 ± 16.5 (20.7) 50.2 ± 12.7 (8.6) 54.9 ± 9.5 (18.8) 55.4 ± 10.7 (19.9) 55.3 ± 8.9 (19.7)

() () () () ()

Data are expressed as mean from all the sampling dates ± SD. Staining differentiation expressed as a percentage to the control birds is shown in ( ). As the staining pattern for BuChE was significantly less evident no percentage expression is reported.

As for the higher doses, the most impressive observation was that of liver cell cytoplasm shrinkage, often not completely associated with cell death, but evidently responsible for the disruption of the cyto-architecture (Fig. 1d). Spongiform morphology, abnormal, often apoptotic cells were evident.

Fig. 1. H&E staining of cryostat sections from brain, liver and duodenum of quails treated with various doses of methamidophos. a. Brain, 2.2 mg/kg bw, day 14: Lighter vs. darker (differential) cellular staining intensity is evident (arrows). b. Brain, 25 mg/kg bw, day 0: extreme cellular vacuolization and differential staining intensity is evident (arrows). c. Liver, 2.2 mg/kg bw, day 3: scattered apoptotic cellular profiles (arrowhead) are visible. Cell hypertrophy combined with karyomegaly (arrow) is also detected. d. Liver, 25 mg/kg bw, day 0: acute disruption of the cyto-architecture is visible in the liver lobule vein (asterisk) surrounding parenchyma. e. Duodenum, 2.2 mg/kg bw, day 1: the morphology of the duodenal wall shows no signs of disruption. f. Duodenum, 11.2 mg/kg bw, day 0: the mucosal surface displays variable degree of cellular architecture disruption; the epithelial cell lining is not continuous (arrows). In addition, the profiles of enteric ganglia in both myenteric and submucosal plexuses are visibly affected; the enteric ganglion cells are not discernible (arrowheads).

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

341

Fig. 2. ChEs histochemistry on cryostat sections from brain of quails treated with various doses of methamidophos. a. AChE staining, 11.2 mg/kg bw, day 1: extensive staining in cellular profiles in the cortex is evident. b. AChE staining, 5 mg/kg bw, day 14: note the presence of a broader network of neurites and nerve bundles. c. BuChE staining, 0 mg/kg bw, day 3: BuChE positive cells are visible in the cortex. d. BuChE staining, 1 mg/kg bw, day 14: in contrast to figure c, BuChE stained cells are not confined in the cortex, but appear scattered both in the cortex and underlying structures of the brain. e. ChEs that hydrolyze PrSCh staining, 25 mg/kg bw, day 0: ChEs that hydrolyze PrSCh stained cells are scattered in various structures of the brain.

3.3.2.2. AChE stained sections. The reaction product for AChE staining was restricted to scarce cellular profiles within all treatments, displaying a granular pattern more closely associated with the cell membrane of hepatocytes (Fig. 3a). 3.3.2.3. BuChE stained sections. The reaction product for BuChE was evident mainly in the cytoplasmic membranes and to a much lesser extent in the cytoplasm of cellular profiles, often associated with the vasculature, without exempting liver cells. The BuChE staining during the study is presented in Table 9. BuChE positive cells were evident throughout the sectional profiles (Fig. 3b). 3.3.2.4. ChEs that hydrolyze PrSCh stained sections. The reaction product for ChEs that hydrolyze PrSCh was evident mainly in the cytoplasmic membranes and to a much lesser extent in the cytoplasm of cellular profiles, often associated with the vasculature, without exempting liver cells. The staining was less granular when compared to BuChE. The ChEs that hydrolyze PrSCh staining during the study is presented in Table 9.

When both inhibitors (iso-OMPA and BW284C51) were used as a positive control the staining pattern was no longer evident. ChEs that hydrolyze PrSCh positive cells were evident throughout the sectional profiles (Fig. 3c). 3.3.3. Duodenum 3.3.3.1. H&E stained sections. There were no significant cyto-architectural changes observed for the lower doses (1 and 2.2 mg/kg bw) and over the period of 14 days in the duodenal wall (Fig. 1e). As regards the middle dose (of 5 mg/kg bw) some lesions were evidently restricted in the mucosal surface and involved epithelial cells. The examination of the duodenum of quails which received the higher doses (11.2 and 25 mg/kg bw) revealed extensive damage – when disintegration has not occurred – to the mucosal surface (in some cases the epithelial cells were necrotic or even absent) and to the remainder of the duodenal wall (muscle layers, enteric ganglia) (Fig. 1f). 3.3.3.2. AChE stained sections. Histochemical staining for AChE results in the visualization of enteric neuronal perikarya (the nuclei do not stain) and nerve bundles (groups or individual neuronal axons). The

342

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

Fig. 4. ChEs histochemistry on cryostat sections from duodenum of quails treated with various doses of methamidophos. a. AChE staining, 11 mg/kg bw, day 0: AChE staining is visible in both myenteric (arrow) and submucosal (arrowhead) ganglia as well as in intramural nerves; a staining profile similar to that observed in controls. b. ChEs that hydrolyze PrSCh staining, 2.2 mg/kg bw, day 3: the distribution of ChEs that hydrolyze PrSCh staining is significantly more extensive than AChE staining (a), closely resembling that of BuChE staining (not shown). Positively stained cells are visible especially in the mucosa, residing in various cell types. In addition, stained ganglion cells (arrow) are detected; the latter are negative for BuChE (not shown).

Fig. 3. ChEs histochemistry on cryostat sections from liver of quails treated with various doses of methamidophos. a. AChE staining, 1 mg/kg bw, day 14: AChE stained cells are visible in the liver parenchyma. b. BuChE staining, 0 mg/kg bw, day 3: the distribution of BuChE staining is significantly more extensive than AChE staining (a). c. ChEs that hydrolyze PrSCh staining, 1 mg/kg bw, day 14: the distribution of ChEs that hydrolyze PrSCh staining is significantly more extensive than AChE staining (a), closely resembling that of BuChE staining (b).

stained enteric neurons resided in both myenteric and submucosal ganglia while axons and nerves were distributed throughout the duodenal wall sectional profile (Fig. 4a). Stained cells were scarcely seen within the sectional profiles and therefore the overall value is quite low (the histochemical reaction Table 9 ChE histochemistry in Japanese quail liver evaluation, following the administration of several doses of methamidophos, in an avian acute oral test. Methamidophos (mg/kg bw) 0 1 2.2 5 11.2 25

AChE stained sections

∅

BuChE stained sections

ChEs that hydrolyze PrSCh stained section

62.6 ± 21.4 61.2 ± 15.1 (−2.2) 51.0 ± 9.8 (−18.5) 52.5 ± 15.0 (−16.1) 49.1 ± 18.4 (−21.5) 38.8 ± 15.3 (−38.1)

50.6 ± 23.8 44.4 ± 10.4 39.5 ± 10.2 38.4 ± 25.6 41.8 ± 18.6 39.4 ± 17.2

(−12.3) (−22.0) (−24.2) (−17.3) (−22.1)

Data are expressed as mean from all the sampling dates ± SD. Staining differentiation expressed as a percentage to the control birds is shown in ( ). ∅ As the reaction product for AChE staining was restricted to scarce cellular profiles within all treatments, no data are reported.

for AChE was estimated at 2.6–3.1% of the reaction observed for the ChEs that hydrolyze PrSCh and BuChE, of the control stained sections, respectively). The AChE staining during the study is presented in Table 10. 3.3.3.3. BuChE stained sections. The use BuSCh resulted in extensive staining in the duodenal wall sectional profile, with the exception of ganglia, nerves and the tunica muscularis. The stain reaction product resided in the cytoplasm and cell membranes of the various constituent tissues of the remainder duodenal wall. The BuChE staining during the study is presented in Table 10. 3.3.3.4. ChEs that hydrolyze PrSCh stained sections. The use of PrSCh resulted in extensive staining of the cell membranes and the cytoplasm of all cellular type profiles, inclusive of enteric neurons and

Table 10 ChE histochemistry in Japanese quail duodenum evaluation, following the administration of several doses of methamidophos, in an avian acute oral test. Methamidophos (mg/kg bw)

AChE stained sections

BuChE stained sections

ChEs that hydrolyze PrSCh stained section

0 1 2.2 5 11.2 25

1.5 ± 2.2 0.05 ± 0.05 ( ) 0.3 ± 0.7 ( ) 0.4 ± 0.8 ( ) 1.2 ± 1.9 ( ) 2.9 ± 2.4( )

48.0 ± 6.36 49.5 ± 28.3 (3.1) 46.6 ± 17.6 (−2.9) 36.3 ± 25.8 (−24.3) 22.3 ± 20.1 (−53.5) 11.1 ± 10.7(−76.9)

57.5 ± 8.4 54.5 ± 15.1 (−5.2) 61.9 ± 8.1 (7.6) 40.3 ± 19.9 (−29.9) 25.7 ± 12.9 (−55.3) 12.0 ± 9.1 (−79.1)

Data are expressed as mean from all the sampling dates ± SD. Staining differentiation expressed as a percentage to the control birds is shown in ( ). As the staining pattern for AChE was markedly less evident no percentage expression is reported.

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

axons within the duodenal wall with the exception of the tunica muscularis. Often an array of cells localized below the mucosal basement membrane is visibly stained, outlining the sectional profile of the villi; no staining is evident in the epithelial cells of the mucosa (Fig. 4b). The ChEs that hydrolyze PrSCh staining during the study are presented in Table 10. When both inhibitors (iso-OMPA and BW284C51) were used as a positive control the staining pattern was no longer evident. 4. Discussion 4.1. A novel multidisciplinary methodological approach The acute toxic trial involves a variety of parameters that need to be evaluated in order to conclude as regards the appearance of lethal and sub-lethal effects of an OP pesticide, methamidophos, in adult quails following 14 days acute exposure. In this study, we attempted to combine observation on mortality and behavioral alterations with biochemical analysis of specific tissue (brain, liver and plasma) ChEs, using various substrates and inhibitors in order to determine the OP effect on each ChE type. H&E staining of tissue sections was used to examine the morphology of the same tissues. The brain and the liver, both well documented previously as targets of OP effect, and the duodenum were also processed ChE histochemistry to assess the toxic effects on the same types of ChEs, using the same, as for biochemistry, substrates and inhibitors. Some, such as PrSCh, alone or in conjunction with inhibitors, have never been used before or have never been tested at all in the organs selected (ChEs in the liver). Inhibition of brain ChEs activity has been the most widely used biomarker of OP poisoning on birds in terms of inhibition of brain AChE (Parker and Goldstein, 2000; Soler-Rodriguez et al., 1998; Vyas et al., 1996). The liver is the primary organ involved in xenobiotic metabolism and is a major target organ for chemicals and drugs. Hepatotoxicity is therefore an important endpoint in the evaluation of the effect of a particular xenobiotic (Sayim, 2007; Yehia et al., 2007). In most cases of OP poisoning, the principal risk, at least for terrestrial vertebrates is considered to arise through ingestion. Τhe avian duodenum is part of the gastrointestinal tract (GIT), is anatomically well-defined, plays a major role in digestion and furthermore contains an extensive network of enteric plexuses, where ChEs are known to be expressed (Larini, 1979). 4.2. Macroscopic observations In our trials the estimated toxicological end points for methamidophos were: 2.2b LD50 b 11.2 mg/kg bw and NOEL=2.2 mg/kg bw. According to Johnson et al. (1991) the oral LD50 of racemic methamidophos for hens is 25 mg/kg. Other reports on mammals, indicate LD50 = 13– 30 mg methamidophos/kg (Pesticide Management Education Program (PMEP) (2008)), while Burruel et al. (2000) reported no mortality in rats up to 7.5 mg methamidophos/kg bw. High acute toxicity to quails has also been indicated by other OPs, such as chlorpyrifos (LD50 = 13.3–32 mg/kg bw) (Hudson et al., 1984; Johnson et al., 2001). The symptoms of bird poisoning with anti-ChE insecticides vary from light to severe even 1 h after the administration of the toxicant, which can eventually cause death. This short response time is to no surprise given that methamidophos is a direct inhibitor of AChE [i.e., does not require metabolic activation; Pope (1999)]. It has been documented, for other anti-ChE pesticides, that the onset and peak of behavioral effects matched quite closely the onset and peak of ChE inhibition in brain (Moser and Padilla, 1998; McDaniel and Moser, 2004). Other reports demonstrated that the behavioral toxicity of OPs is not necessarily a direct reflection of brain ChE inhibition, and that other mechanisms of

343

action may be involved in the neurotoxicity of certain OPs (McDaniel and Moser, 2004). The survivors in all dose rates had completely recovered from the mid of day 1 onwards. According to Brunet et al. (1997) the visual symptoms of intoxication lasted from 100 to 750 min after intoxication with increasing doses of dimethoate from 1.75 to 15.00 mg/kg bw. Food consumption for all the dose rates for the last 7 days was lower compared to the control. In direct analogy to the feed consumption, bw in birds receiving 1, 2.2 and 5 mg/kg bw methamidophos increased 5.5%–11.2%, mainly during the first 7 days, compared to the measured 25.2% for the control which kept growing normally for the duration of the trial. Similar observations were reported for three granivorous species, with dimethoate (Brunet et al., 1997; Brunet and Cyr, 1992). Post mortem examination revealed brain hemorrhage in quails which received the highest dose of 25 mg/kg bw of methamidophos. According to Kubo et al. (2000), stimulation of the paraventricular nucleus of the hypothalamus causes an increase in arterial pressure by means of a cholinergic input from the lateral parabrachial nucleus to the rostral ventrolateral medulla (RVLM). Furthermore, the angiotensin system in the anterior hypothalamus is overactive in spontaneously hypertensive rats, which leads to raised levels of ACh release in the RVLM (Kubo et al., 2002). This chain leads to hypertension. In conclusion, our data indicates that even though the visual signs of intoxication disappeared within hours after ingestion of the pesticide, the physiological impact of a single sub-lethal dose of methamidophos on non-target avian species may last several days after the pesticide was sprayed on a field. These results are in agreement with those of Hudson et al. (1984), who reported that animals are symptom free 24 h after exposure to sub-lethal doses of OPs. However, the birds were still affected by the pesticide several days after intoxication, regardless of showing increased or decreased activity levels. The effects on the activity level of birds, persisted about 6–15 days after ingestion of about 5.5 mg dimethoate/kg bw (Brunet and Cyr, 1992) or 12 days of a dose of 6.69 mg dimethoate/kg bw (Brunet et al., 1997). 4.3. Biochemical analysis of cholinesterases' activities Τhe inhibition of enzyme activity seen in tissue preparations following in vivo exposure provides a net reflection of the sensitivity of the organism in terms of uptake, biotransformation and detoxification patterns. The present study shows that methamidophos administered in various doses for 14 days led, within 1 day after the administration of the gavage doses, to a considerable decrease in ChE activity in brain, plasma and liver. This pattern indicates also that even at very low levels of exposure, ChE activity may be considerably reduced. ChE activity slightly hydrolyzed BuSCh; an indication of a low activity of BuChE as reported for various parts of rat brain (Gupta, 2004) and in different ages (Lassiter et al., 1998). To determine whether other non-specific esterases that are able to metabolize the substrate ASCh might be present in our crude homogenate preparations, we used the non-selective ChE inhibitor eserine. The enzyme activity was then inhibited almost 100%, confirming that activity measured in our experimental conditions was due to ChE and not to other types of esterases (Thompson and Walker, 1994). In biomarker studies, such knowledge is very important since tissues may contain significant amounts of non-specific esterases, which contribute to the measured activity and may be more or less sensitive than ChE to anti-ChE chemicals (Garcia et al., 2000). The data indicates that AChE is the predominant form present in the brain. The percentage of brain ChE inhibition found in this study ranged from a mean of about 60% from 1 h after the exposure to a mean of about 15% after 14 days. Notably, for the highest doses (11.2 and 25 mg/kg bw), methamidophos produced significant higher inhibition,

344

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

85 and 87%, in brain ChE activity 30 min after the administration of the gavage doses (Table 4). It is noted that PrSCh as a substrate reveals much longer effects as ChE activity did not recover on day 14 in contrast with ASCh, where the mean brain ChE inhibition remained high, approximately 38%. The lack of recovery compared to ASCh is to be explained upon. It may reflect differences in the composition of AChE and BuChE molecular forms and their relative sensitivity to substrates. AChE and BuChE exist in most tissues in both globular (free) and membrane bound forms. The degree of free ChEs activity varies from entirely free ChEs activity in the plasma and heart to almost entirely membrane bound (i.e. insoluble in a buffer without a detergent or salt) as in the brain tissue. There is, however, a variety of different molecular forms ranging from globular monomers (G1), dimers (G2), and tetramers (G4) to asymmetric collagen-tailed forms (A4, A8 and A12) in which catalytic tetramers are associated with anchoring proteins (Massoulié et al., 1999). ChE activity (using PrSCh as a substrate) did not recover compared with the ones using ASCh, probably due to the different proportion of the membrane bound and soluble ChEs' forms in the brain during recovery. Maximum inhibitions of brain AChE in birds generally occur in the first few hours after OP exposure, as shown in Japanese quail 4 and 8 h after treatment with dicrotophos (Fleming and Grue, 1981) and chlorpyriphos (Soler-Rodriguez et al., 1998), respectively. Similar results were seen in zebra finches 1–3 h after exposure to fenitrothion (Holmes and Boag, 1990) and in starlings 6 h after treatment with triazophos (Thompson et al., 1991). Interestingly, brain AChE activity did not correlate with dose. The lack of correlation suggests that the effects may have been the result of peripheral stimulation (Fryday et al., 1995). Τhe liver showed the lowest ChE activity with ASCh, compared to all the tissues examined. For liver and plasma, all substrates were hydrolyzed almost at the same rate. Plasma ChEs are generally more sensitive than brain AChE to inhibition by these compounds (Thompson et al., 1991) and have a rapid recovery rate, in terms of a few hours (Holmes and Boag, 1990; Ludke et al., 1975). For all tissues and plasma a dose–response inhibition of all cholinesterases cannot be established. Plasma ChE was inhibited 100% within 30 min, but recovered rapidly, and in fact 3 days later, ChE values were significantly higher than those of controls and remained higher even 14 days after the exposure. A similar trend was also obtained for the various doses in the liver. Thompson et al. (1995) reported high correlation in the sensitivity to inhibition of liver and serum BuChE, after the exposure to various OPs to four avian species and supports the theory that the liver is the site of production and release for serum esterases. According to Kacham et al. (2006) carboxylesterase inhibition in plasma and liver of chlorpyrifos and parathion in neonatal rats was similar among the treatment groups at 4, 8, and 24 h. In addition the rate of activation of organophosphate pesticides was far higher in the liver than in the brain (Kacham et al., 2006; Thompson et al., 1995). Henderson et al. (1994) showed that when pigeons were treated with diazinon via gavage at 1.0 mg/kg bw, plasma ChE activity decreased to 20% of control within 2 h of treatment and subsequently increased to 60% of control within 4 h and 75% of control within 24 h. Plasma ChE levels reached those of the control within 120 h of treatment. The inhibition of serum esterases has been proposed as a non-destructive biomarker for biomonitoring the exposure of wild fauna to OPs and carbamates (Fairbrother et al., 1989; Fossi et al., 1992; Soler-Rodriguez et al., 1998; Thompson et al., 1991; Thompson and Walker, 1994). The rapid recovery rates together with large variations between individuals are the main disadvantages of serum B esterases as non-destructive biomarkers in birds (Fairbrother et al., 1989). Despite behavioral changes observed in open-field tests at various oral doses of chlorpyrifos (2 and 4 mg/kg), repeated daily oral dosing

of chicks with chlorpyrifos for 7 days at 2 mg/kg was not associated with significant ChE inhibition in the brain, plasma and liver. The high dose of chlorpyrifos (4 mg/kg) when given repeatedly caused significant ChE inhibition in the brain and liver with behavioral changes mainly similar to those induced by the lower dose. This finding suggests lack of significant association between the extent of ChE inhibition and the behavioral changes induced by chlorpyrifos. Several authors indicate that ChE inhibition cannot be associated all the time with the occurrence and severity of signs of OP poisoning, and this is especially true at low doses of the OPs (Wilson, 1998; Worek et al., 2005). The recovery of brain AChE activity was slower than that of plasma. It reached up to 48% inhibition of that observed in controls 3 days after methamidophos administration. Two weeks after the exposure in our study, the level of the enzyme in the brain ranged from almost similar to the control up to 26% of inhibition in the experimental groups. In liver the inhibition and recovery of the ChE activity was relevant to plasma. Although enzyme assays indicate that the birds excrete OPs within hours in the droppings (O'Brien and Yamamoto, 1970), the slow recovery in activity level does not necessarily parallel the gradual disappearance of methamidophos in the blood. In fact, the activity level was shown to be proportional to brain AChE activity in birds (Bakre and Rajasekaran, 1989). Fleming and Grue (1981) reported that ChE levels take much more time to return to normal levels than do OPs to be eliminated. Brain ChE activities of mallard ducks, bobwhite quails, barn owls, starlings and common grackles were shown to take in average 26 days to recover after being depressed by 55–64% with oral doses of dicrotophos (Fleming and Grue, 1981). ChE activity is species-specific (Hill, 1988; Thompson, Walker and Hardy, 1991) and the magnitude of ChE depression and the time required for recovery has been demonstrated to depend on the toxicant and the dose used, as it correlates strongly with the degree of initial ChE depression (Fleming and Grue, 1981). Several experiments were conducted in an attempt to establish a relationship between intoxication symptoms and AChE activity (Fairbrother et al., 1991). Several authors have pointed out the need for further analyses to diagnose whether death is truly caused by the OPs (Holmes and Boag, 1990). Thus, the behavioral signs of intoxication cannot provide an estimate of brain AChE inhibition. Therefore, the activity level should constitute a more sensitive indicator of changes in brain AChE activity than intoxication symptoms. In our study, an inhibition of brain AChE more than 62%, 1 h after the administration of the dose was adequate to cause death in the greatest majority of cases; while a few quails survived even at inhibition levels up to 66%. Brain ChE inhibition of about two standard deviations below normal (control) has been generally accepted as indicative of anti-ChE exposure and ChE activity less than 50% of the normal has been used to assert the cause of death. However, birds may survive with brain ChE activity levels exceeding 50% depression (Ludke et al., 1975). Hill (1988) reported that avian mortality from OP insecticide exposure is usually associated with brain ChE inhibition of more than 80% in the field. Ludke et al. (1975) established the criterion that brain AChE inhibition exceeding 20% or 2 standard deviations below normal activity in birds is an index of stress, and an inhibition greater than 50% causes death attributable to the OP. Subtle effects may, however, occur at lower levels of inhibition (Greig-Smith et al., 1992). According to Wilson (1998) and Worek et al. (2005), plasma ChE inhibition by 20–30% usually indicates exposure to OP, whereas 50% inhibition or more is associated with serious poisoning and adverse effects. 4.4. Histochemical observations The staining of neurons containing acetylcholine has relied on histochemical methods which detect enzymes implicated in the metabolism

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

of the molecule, such as AChE (degradation) (Ali and McLelland, 1978; Smith et al., 1977). The histochemistry showed significant variation depending on the potency of ChEs in the brain, liver and duodenum of the adult quail. The correlation between ChE activity detected by histochemical and biochemical methods in brain and liver from untreated animals was consistent to some extent and in some cases and served as a basis for the analysis of the treated quails. We may therefore, assess quantitatively by biochemistry the OP effects and in addition define the exact anatomical position of this expression. The latter is very important when the exact mechanism of this effect is to be defined as it offers a glimpse of the functional anatomy and pathology. Our results indicate that methamidophos induces histochemical alterations in the brain of the exposed quails, visible by H&E staining. These alterations range from atypical cellular morphology (caryomegaly, pyknotic nuclei and increased cytoplasmic vacuoles) to tissue cytoarchitecture disruption such as spongiform morphology. The gravity and the extent of these alterations appear, in most but not in all cases, to be dose dependent. It is upon first glance rather surprising that a clear relation between dose and effect is not visible, but organ-tissue specific traits, such as the blood–brain barrier, and/or individual variations within the species—demonstrated previously by biochemistry, may account for these discrepancies. Experimental studies in mammals have reported neuronal necrosis in hippocampus, enthorinal and frontal cortex, amygdaloid complex, caudate nuclei, and thalamus in acute OP intoxication (Britt et al., 2000; Lemercier et al., 1983). Pelegrino et al. (2006) reported morphological changes in specific regions of the brain of rats which received 2.5 or 5.0 mg/kg methamidophos. As the molecular layer is composed basically by dendritic and axonal ramifications of neurons, it is believed that at low repeated doses, methamidophos would induce loss or atrophy of neuronal ramifications (Pelegrino et al., 2006). The brain lesion is a feature of the neuropathies and toxic states in which the primary injuries are found in neuronal cell bodies (Jortner, 2000, 2011). The distinct brain temporal and spatial histochemical distribution patterns of AChE (with ASCh as a substrate and iso-OMPA as an inhibitor) and BuChE (with BuSCh as a substrate) clearly demonstrate the presence of two discernible enzymes. In the rat brain, both AChE (Lassiter et al., 1998) and BuChE (Geula and Nagykery, 2006; Lassiter et al., 1998) histochemical staining have been documented. AChE staining values were lower than the control, irrelevant of the sampling periods. The histochemical staining pattern observed after the exposure to methamidophos was not altered, in a fashion similar to the biochemical results. The effect of methamidophos inhibition on AChE appears to be the same irrespective of the dosage as visible in Table 8. This may be due to the fact that even in the smallest dose the OP is able to exert an inhibitory action on the enzyme, which remains unchanged even when this dosage is increased; suggesting that, the brain is the primary target organ, and thus confirming all previous reports (references). Bajgar et al. (2007) reported good correlation between biochemical and histochemical results detecting AChE activity in different rat brain areas following intoxication with three nerve agents. It is worth to point out that the sectional profiles of brains subjected to various doses, and in particular 14 days following exposure, displayed a broader network of individual neuronal axons and nerves stained for AChE. BuChE histochemical staining was limited to few cell bodies, significantly fewer than AChE, and never stained nerves in the sections examined. These observations are in general agreement with other descriptions of the distribution of BuChE-positive structures in the adult rat brain (Darvesh et al., 1992; Lassiter et al., 1998; Tago et al., 1992). PuSCh histochemical staining was evidently not restricted to neurons but in addition resided in red blood cells and often labeled vascular sectional profiles. The use of PrSCh as a substrate and with iso-OMPA and BW284C51 as inhibitors displayed the same staining pattern

345

while the overall value for ChEs that hydrolyze PrSCh was higher than the control for all the dose rates. The lack of qualitative difference compared to PrSCh alone is to be explained upon. It may reflect tissuedependent differences in the composition of AChE molecular forms and their relative sensitivity to inhibitors. AChE and BuChE exist in most tissues in both globular (free) and membrane bound forms (Massoulié et al., 1999). When animals were treated with the AChE inhibitor chlorpyrifos (Ricceri et al., 2003), or soman (Lintern et al., 1998), the G4 form in the brain was preferentially inhibited. PrSCh staining values were lower than the ones for AChE for the control, probably due to the high proportion of the membrane bound ChEs' forms in the brain. PrSCh staining values for the various doses were increased compared to the control, in contrast with AChE, irrelevant of the sampling periods. This is not in agreement with the biochemical results where the mean brain ChEs activity remained inhibited up to the end of the trial. The majority of the insecticides are bio-transformed in metabolites by the liver, through enzymes from the soluble fractions of mitochondria and microsomes. Thus, the liver is the organ that contains the major concentration of OP residues and it suffers variable levels of damage as a consequence of this process (Sayim, 2007; Yehia et al., 2007). The effects on quail metabolism are great, especially immediately following exposure and for a period of time afterwards. One single exposure to such a pollutant has been documented to result in withholding of the bile secreted by the hepatocytes within the cells from which it is actually never released, in addition to effects in protein, carbohydrate, and lipid metabolism. Our results indicate that methamidophos induces histochemical alterations in the liver of exposed quails. These alterations range from changes in the cellular morphology and cell death to severe apoptosis and spongiform morphology in the higher doses. In fact, in the higher doses, hepatocyte cytoplasm shrinkage was very evidently observed. The histochemical observations described above demonstrate clearly that the liver is the major detoxification organ in the quail and it suffered greatly following exposure to methamidophos. This liver damage is consistent with functional failure which may irreversibly result to death. The liver capacity to adopt and regenerate is enormous and this was also well documented in our study, as areas of cell renewal and liver re-building were also observed in quail given medium doses, when regeneration was observed. Hepatocyte proliferation, vacuolated cytoplasm and focal necrosis proceeding to hepatomegaly in rat receiving DDT have been correlated to a regenerative liver response to pesticides (Sayim, 2007). In liver, AChE staining was restricted to scarce cellular profiles within all treatments, displaying a granular pattern more closely associated with the cell membrane of hepatocytes. AChE staining values were very low compare BuChE and ChEs that hydrolyze PrSCh in order to evaluate them. The BuChE staining was evident mainly in the cytoplasmic membranes and to a much lesser extent to the cytoplasm of cellular profiles, often associated with the vasculature, without exempting liver cells. BuChE staining values were dose-dependent lower than the control, except the lowest dose, irrelevant of the sampling periods. This is not in agreement with the biochemical results where the mean liver BuChE inhibition remained increased for the lower doses. PuSCh staining resided mainly in the cytoplasmic membranes and to a much lesser extent to the cytoplasm of cellular profiles, often associated with the vasculature, without exempting liver cells. The use of PrSCh as a substrate and with iso-OMPA and BW284C51 as inhibitors resulted in complete elimination of the staining. It appears that iso-OMPA totally inhibited the staining of BuChE. PrSCh staining values were relevant to BuSCh in duodenum with lower values of approximately 19%. The distribution area of the staining pattern for BuChE and ChEs that hydrolyze PrSCh was evident everywhere irrelevant of the dose rates and the time past after exposure.

346

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347

It is well established that the GIT easily absorbs chemicals if the no ionized form is soluble in lipids. The intestine is another site for biotransformation of insecticides (Larini, 1979), and therefore, local intoxication of the enterocytes can damage their structure. Our results indicate that methamidophos induces dose-dependent histochemical alterations in the duodenum of exposed quails. These alterations range from lesions in the mucosal surface (in the middle dose) to extensive damage of the mucosa and the duodenal wall (in the higher doses). In duodenum, AChE staining resided in the enteric neurons in both myenteric and submucosal ganglia while axons and nerves were distributed throughout the duodenal wall sectional profile. According to Chanda et al. (2010) the AChE inhibition at different regions of the GIT following inhalation exposure to nerve agent sarin was significantly for groups both 4 h and 24 h post-sarin exposure; among the seven chosen regions of the guinea pig GIT, duodenum showed the highest AChE activity in control animals. AChE staining values in our studies were too low compared to BuChE and ChEs that hydrolyze PrSCh in order to evaluate the results. Histochemistry revealed intense and extensive BuChE staining in the sectional profile of the duodenal wall, with the exception of ganglia, intramural nerves and tunica muscularis. In previous studies BuChE staining was observed in epithelial cells of the rat intestine, mainly in the crypts and the villi, suggesting that BuChE may be involved in lipid metabolism or cell regeneration (Hermite et al., 1996). BuChE staining values in our study were lower than the control for the middle and higher doses, irrelevant of the sampling periods. PuSCh staining revealed extensive labeling of cell membranes in all cellular profiles in the duodenal wall, inclusive of enteric neurons. The use of PrSCh as a substrate and with iso-OMPA and BW284C51 as inhibitors resulted in complete elimination of the staining. It appears that iso-OMPA totally inhibited the staining of BuChE. PrSCh staining values in our study were relevant to BuSCh in duodenum with higher values of approximately 20%. The present study shows that changes concerning the biochemical activity of plasma, brain and liver ChEs, are reversible, as reported for other vertebrates treated with OPs (Jortner, 2000; Yehia et al., 2007), whereas the histopathological changes persist longer as reported also for other vertebrates treated with OP (Britt et al., 2000; Jortner, 2000; Yehia et al., 2007). 5. Conclusions Following an acute oral test of the Japanese quail with five methamidophos doses, we observed dose-dependent lethal and sub lethal effects, relevant to biochemical alterations in both AChE and BuChE activity in brain, liver and plasma. We also observed morphological changes, evident by H&E histochemistry, as well as variations in both AChE and BuChE staining patterns in the brain, liver and duodenum. The use of various substrates and inhibitors allowed for a concise interpretation of our observations. We therefore recommend the use of biochemistry and histochemical staining of the biomarker ChE, in combination with tissue morphology visible by H&E staining as a novel methodological approach, in order to significantly improve the laboratory interpretation of the data obtained by the acute oral test employed to assess the effects of methamidophos and other chemicals known to inhibit this very important nervous system enzyme. Acknowledgments We thank Bayer CropScience for providing us methamidophos technical. We would like to thank Prof. Maria Chrysagi for providing us the microplate microreader in order to measure the activity of the ChEs, and Dimitrios Apostolopoulos for assisting the histochemical staining. This work was a part of a project entitled “ENVIRONMENT – PYTHAGORAS II – funding of research groups in Agricultural University

of Athens”. The project is co-funded by European social fund and national resources — Operational Program for Education and Initial Vocational Training “EDUCATION” II (EPEAEK ΙΙ). References Abou-Donia MB. Triphenyl phosphate: a type II organophosphorus compound-induced delayed neurotoxic agent. In: Champers JE, Levi PE, editors. Organophosphates: chemistry, fate and effects. San Diego: Academic Press; 1992. p. 327–51. Ali HA, McLelland J. Avian enteric nerve plexuses. Cell Tissue Res 1978;189:537–48. Aly NM, El-Gend KS. Effect of dimethoate on the immune system of female mice. J Environ Sci Health 2000;35:77–86. Bajgar J, Hajek P, Slizova D, Krs O, Fusek J, Kuca K, et al. Changes of acetylcholinesterase activity in different rat brain areas following intoxication with nerve agents: biochemical and histochemical study. Chem Biol Interact 2007;165(1):14–21. Baker DG, McDonald DM, Basbaum CB, Mitchell RA. The architecture of nerves and ganglia of the ferret trachea as revealed by acetyl-cholinesterase histochemistry. J Comp Neurol 1986;246:513–26. Bakre P, Rajasekaran M. Acute toxicity of certain pesticides to house sparrow (Passer domesticus). Geobios 1989;16:249–51. Beauvais SL, Jones SB, Brewer SK, Little EE. Physiological measures of neurotoxicity of diazinon and malathion to larval rainbow trout (Onchorhynchus mykiss) and their correlation with behavioural measures. Environ Toxicol Chem 2000;19:1875–80. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dyebinding. Anal Biochem 1976;72: 248–54. Brimijoin S. Can cholinesterase inhibitors affect neural development? Environ Toxicol Pharmacol 2005;19(3):429–32. Britt Jr JO, Martin JL, Okerberg CV, Dick EJ. Histopathologic changes in the brain, heart and skeletal muscle of rhesus macaques, ten days after exposure to soman (an organophosphorus nerve agent). Comp Med 2000;50:133–9. Brunet R, Cyr A. The impact of dimethoate on rhythms of three granivorous bird species. Agric Ecosys Environ 1992;41:327–36. Brunet R, Girard C, Cyr A. Comparative study of the signs of intoxication and changes in activity level of red-winged blackbirds (Agelaius phoeniceus) exposed to dimethoate. Agric Ecosys Environ 1997;64:201–9. Burruel RV, Raabe GO, Overstreet WJ, Wilson WB, Wiley ML. Paternal effects from methamidophos administration in mice. Toxicol Appl Pharmacol 2000;165: 148–57. Carr RL, Chambers JE. Toxic responses of the nervous system. In: Schlenk DM, Benson WH, editors. Target organ toxicity in marine and freshwater teleosts, vol. 2. London: Taylor and Francis; 2001. p. 27–95. Caughlan A, Newhouse K, Namgung U, Zhengui X. Chlorpyrifos induces apoptosis in rat cortical neurons that is regulated by a balance between p38 and ERK/JNK MAP kinases. Toxicol Sci 2004;78:125–34. Chanda S, Song J, Rezk P, Sabnekar P, Doctor PB, Sciuto MA, et al. Gastrointestinal acetylcholinesterase activity following endotracheal microinstillation inhalation exposure to sarin in guinea pigs. Chem Biol Interact 2010;187:309–11. Darvesh S, Smereczynsky A, Hopkins DA. Distribution of butyrylcholinesterase in the rat brain. Abst Soc Neurosci 1992;18:1505. Darvesh S, Hopkins DA, Geula C. Neurobiology of butyrylcholinesterase. Nat Rev Neurosci 2003;4:131–8. Ellman L, Courtey KD, Andreas Jr V, Featherstone RM. A new rapid colorimetric determination of cholinesterase activity. Biochem Pharmacol 1961;7:88–95. Fairbrother A, Bennett RS, Bennett JK. Sequential sampling of plasma cholinesterase in mallards (Anas platyrhynchos) as indicator of exposure to cholinesterase inhibitors. Environ Toxicol Chem 1989;8:117–22. Fairbrother A, Marden BT, Bennett JK, Hooper MJ. Methods used in determination of cholinesterase activity. In: Mineau P, editor. Cholinesterase inhibiting insecticides: their impact on wildlife and the environment. Chemicals in AgricultureNew York: Elsevier; 1991. p. 57–8. Fleming WJ, Grue CE. Recovery of cholinesterase activity in five avian species exposed to dicrotophos, an organophosphorus pesticide. Pestic Biochem Physiol 1981;16: 129–35. Fossi MC, Leonziom CI, Massi A, Lari L, Casini S. Serum esterase inhibition in birds: a non-destructive biomarker to assess organophosphorus and carbamate contamination. Arch Environ Contam Toxicol 1992;23:99-104. Fryday S, Hart A, Marczylo T. Effects of sublethal exposure to an organophosphate on the flying performance of captive starlings. Bull Environ Contam Toxicol 1995;55:366–73. Garcia ML, Castro B, Ribeiro R, Guilhermino L. Characterization of cholinesterase from guppy (Poecilia reticulata) muscle and its in vitro inhibition by environmental contaminants. Biomarkers 2000;5:274–85. Geula C, Nagykery N. Butyrylcholinesterase activity in the rat forebrain and upper brainstem: postnatal development and adult distribution. Exp Neurol 2006;204: 640–57. Geula C, Mesulam MM, Kuo CC, Tokuno H. Postnatal development of cortical acetylcholinesterase-rich neurons in the rat brain: permanent and transient patterns. Exp Neurol 1995;134:157–78. Gomes J, Dawodu AH, Lloyd O, Revitt DM, Anilal SV. Hepatic injury and disturbed amino acid metabolism in mice following prolonged exposure to organophosphorus pesticides. Hum Exp Toxicol 1999;18:33–7. Greig-Smith PW, Walker CH, Thompson HM. Ecotoxicological consequences of interactions between avian esterases and organophosphorus compounds. In: Ballantyne

M. Foudoulakis et al. / Science of the Total Environment 450–451 (2013) 334–347 B, Marrs TC, editors. Clinical and experimental toxicology of organophosphates and carhamates. Oxford: Butterworth-Heinemann Ltd.; 1992. p. 295–303. Gupta RC. Brain regional heterogeneity and toxicological mechanisms of organophosphates and carbamates. Toxicol Mech Methods 2004;14:103–43. Harris HR. On the rapid conversion of haematoxylin into haematein in staining reactions. J Appl Microsc 1900;3:377–80. Henderson J, Yamamoto J, Fry D, Seiber J, Wilson B. Oral and dermal toxicity of organophosphate pesticides in the domestic pigeon (Columba livia). Bull Environ Contam Toxicol 1994;52:633–40. Hermite A, Sine JP, Colas B. Ultrastructural localization of butyrylcholinesterase in epithelial cells of rat intestine. Eur J Histochem 1996;40:299–304. Hill EF. Brain cholinesterase activity of apparently normal wild birds. J Wildl Dis 1988;24:51–61. Holmes SB, Boag PT. Inhibition of brain and plasma cholinesterase activity in zebra finches orally dosed with fenitrothion. Environ Toxicol Chem 1990;9:323–34. Hudson RH, Tucker RK, Haegele MA. Handbook of Toxicity of Pesticides to Wildlife., 153. US Dept. of Interior, Fish and Wildlife Serv, Resource Publ; 1984. p. 90. Johnson MK, Vilanova E, Read DJ. Anomalous biochemical responses in tests of the delayed neuropathic potential of methamidophos (O, S-dimethyl phosphorothioamidate), its resolved isomers and of some higher O-alkyl homologues. Arch Toxicol 1991;65(8): 618–24. Johnson BL, Calabrese EJ, Callahan BG, Pastorok RA, Chapman PM. Chlorpyrifos: ecotoxicological risk assessment for birds and mammals in corn agroecosystems. Hum Ecol Risk Assess 2001;7(3):473–637. Jortner BS. Mechanisms of toxic injury in the peripheral nervous system: neuropathologic considerations. Toxicol Pathol 2000;28:54–69. Jortner BS. Preparation and analysis of the peripheral nervous system. Toxicol Pathol 2011;39(1):66–72. Jan. Kacham R, Karanth S, Baireddy P, Liu J, Pope C. Interactive toxicity of chlorpyrifos and parathion in neonatal rats: role of esterases in exposure sequence-dependent toxicity. Toxicol Appl Pharmacol 2006;210:142–9. Karnovsky MJ, Roots L. A direct-coloring thiocholine method for cholinesterases. J Histochem Cytochem 1964;12:219–21. Klaassen CD, editor. Casarett and Doull's Toxicology, the Basic Science of Poisons. 7th ed. New York: McGraw-Hill; 2008. Kubo T, Hagiwara Y, Sekiya D, Chiba S, Fukumori R. Cholinergic inputs to rostral ventrolateral medulla pressor neurons from hypothalamus. Brain Res Bull 2000;53: 275–82. Kubo T, Hagiwara Y, Endo S, Fukumori R. Activation of hypothalamic angiotensin receptors produces pressor responses via cholinergic inputs to the rostral ventrolateral medulla in normotensive and hypertensive rats. Brain Res 2002;953:232–45. Larini L. Toxicologia dos inseticidas. São Paulo: Sarvier; 1979. Lassiter ΤL, Barone Jr S, Padilla S. Ontogenetic differences in the regional and cellular acetylcholinesterase and butyrylcholinesterase activity in the rat brain. Dev Brain Res 1998;105(1):109–23. Lemercier G, Carpentier H, Sentenac-Roumanou P, Morlis P. Histological and histochemical changes in the central nervous system of the rat poisoned by an irreversible anticholinesterase organophosphorus compound. Acta Neuropathol 1983;61: 123–9. Lintern MC, Wetherell JR, Smith ME. Differential recovery of acetylcholinesterase in guinea pig muscle and brain regions after soman treatment. Hum Exp Toxicol 1998;17:157–62. Ludke JL, Hill EF, Dieter MP. Cholinesterase (ChE) response and related mortality among birds fed ChE inhibitors. Arch Environ Contam Toxicol 1975;3:1-21. Mack A, Robitzki A. The key role of butyrylcholinesterase during neurogenesis and neural disorders: an antisense-5′butyrylcholinesterase-DNA study. Prog Neurobiol 2000;60(6):607–28. Massoulié J, Anselmet A, Bon S, Krejci E, Legay C, Morel N, et al. The polymorphism of acetylcholinesterase: post-translational processing, quaternary associations and localization. Chem Biol Interact 1999;119–120:29–42. McDaniel LK, Moser CV. Differential profiles of cholinesterase inhibition and neurobehavioral effects in rats exposed to fenamiphos or profenofos. Neurotoxicol Teratol 2004;26(3):407–15. Monteiro M, Quintaneiro C, Morgado F, Soares AMVM, Guilhermino L. Characterization of the cholinesterases present in head tissues of the estuarine fish Pomatoschistus microps: application to biomonitoring. Ecotoxicol Environ Saf 2005;62:341–7. Moser VC, Padilla S. Age- and gender-related differences in the time course of behavioral and biochemical effects produced by oral chlorpyrifos in rats. Toxicol Appl Pharmacol 1998;149(1):107–19. Nagymajtenyi L, Schulz H, Pappa A, Desi I. Developmental neurotoxicological effects of lead and dimethoate in animal experiments. Neurotoxicology 1998;19:617–22. O'Brien RD, Yamamoto I. Biochemical toxicology of insecticides. New York: Academic Press; 1970. p. 218.

347

OECD 205. Avian dietary toxicity test. Paris: Organisation for Economic Co-operation and Development; 1984. OECD 223. Avian Acute Oral Toxicity test. Draft guidelines for testing of chemicals. Paris: Organisation for Economic Co-operation and Development; 2002. Parker ML, Goldstein MI. Differential toxicities of organophosphate and carbamate insecticides in the nestling European starling (Sturnus vulgaris). Arch Environ Contam Toxicol 2000;39:233–42. Pelegrino JR, Calore EE, Saldiva PHN, Almeida VF, Peres NM, Vilela-de-Almeida L. Morphometric studies of specific brain regions of rats chronically intoxicated with the organophosphate methamidophos. Ecotoxicol Environ Saf 2006;64:251–5. Pesticide Management Education Program (PMEP). Methamidophos (Monitor) Chemical Profile, 4/95; 2008). Pope CN. Organophosphorus pesticides: do they all have the same mechanism of toxicity? J Toxicol Environ Health B Crit Rev 1999;2:161–81. Rawlings NC, Cook SJ, Waldbiling D. Effects of the pesticides carbofuran, chlorpyrifos, dimethoate, lindane, triallate, trifluralin, 2,4-D and pentachlorophenol on the metabolic endocrine and reproductive endocrine system in ewes. J Toxicol Environ Health 1998;54:21–36. Ricceri L, Markina N, Valanzano A, Fortuna S, Cometa MF, Meneguz A, et al. Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice. Toxicol Appl Pharmacol 2003;191(3):189–201. Sayim F. Dimethoate-induced biochemical and histopathological changes in the liver of rats. Exp Toxicol Pathol 2007;59(3–4):237–43. Sheets LP, Hamilton BF, Sangha GK, Thyssen JH. Subchronic neurotoxicity screening studies with six organophosphate insecticides: an assessment of behavior and morphology relative to cholinesterase inhibition. Fundam Appl Toxicol 1997;35: 101–19. Smith J, Cochard P, Le Douarin NM. Development of choline acetyltransferase and cholinesterase activities in enteric ganglia from presumptive adrenergic and cholinergic levels of the neural crest. Cell Diff 1977;6:199–216. Soler-Rodriguez F, Miguez-Santiyan MP, Reja-Sanchez A, Roncero-Cordero V, GarciaCambero JP. Recovery of brain acetylcholinesterase and plasma cholinesterase activities in quail (coturnix coturnix) after chlorpyriphos administration and effect of pralidoxime treatment. Environ Toxicol Chem 1998;17:1835. Somers JD, Barrett MW, Khan AA. Simulated field ingestion of carbofuran-contaminated feedstuffs by pheasants. Bull Environ Contam Toxicol 1991;47:521–8. Storm EJ, Rozman KK, Doull J. Occupational exposure limits for 30 organophosphate pesticides based on inhibition of red blood cell acetylcholinesterase. Toxicology 2000;150:1-29. Suresh BP, Purushotham G, Aleem R, Rajasekhar B, Mahesh Y, Mike M, et al. Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2-hydroxylase in a gram-negative Serratia sp. strain DS001. Environ Biotechnol 2007;73(6):1452–62. Tacal O, Lockridge O. Methamidophos, dichlorvos, O-methoate and diazinon pesticides used in Turkey make a covalent bond with butyrylcholinesterase detected by mass spectrometry. J Appl Toxicol 2010;30(5):469–75. Jul. Tago H, Maeda T, McGeer PL, Kimura H. Butyrylcholinesterase-rich neurons in rat brain demonstrated by a sensitive histochemical method. J Comp Neurol 1992;325: 301–12. Thompson HM, Walker CH. Blood esterases as indicators of exposure to organophosphorus and carbamate insecticides. In: Fossi MC, Leonzio C, editors. Nondestructive biomarkers in vertebrates. Boca Raton,FL: Lewis Publishers; 1994. p. 37–62. Thompson HM, Walker CH, Hardy AR. Changes in activity of avian serum esterases following exposure to organophosphorus insecticides. Arch Environ Contam Toxicol 1991;20:514–8. Thompson HM, Langton SD, Hart ADM. Prediction of inter-species differences in the toxicity of organophosphorus pesticides to wildlife—a biochemical approach. Camp Biorhem Physiol 1995;IIIC(1):1-12. US EPA. United States Environmental Protection Agency Ecological Effects Guidelines, OPPTS 850.2100. Avian acute oral toxicity test; 1996. Vyas BN, Kuenzel JW, Hill FE, Romo AG, Komaragiri VSM. Regional cholinesterase activity in white-throated sparrow brain is differentially affected by acephate (orthene). Camp Biochem Physiol 1996;113C(No. 3):381–6. WHO. Methyl parathion (Environmental Health Criteria-EHC 145, 1992). International Programme on Chemical Safety. Environ Health CritGeneva: World Health Organization; 1993. p. 1-135. Wilson WB. Cholinesterase inhibition. In: Wexler P, editor. Encyclopedia of toxicology, vol. 1. CA, San Diego: Academic Press; 1998. p. 326–40. Worek F, Koller M, Thiermenn H, Szinicz L. Diagnostic aspects of organophosphate poisoning. Toxicology 2005;214:182–9. Yehia AHM, El-Banna GS, Okab BA. Diazinon toxicity affects histophysiological and biochemical parameters in rabbits. Exp Toxicol Pathol 2007;59(3–4):215–25.