Exposure to Diazinon Alters In Vitro Retinogenesis: Retinospheroid Mor
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《毒物学科学杂志》
Department of Developmental Biology and Neurogenetics, University of Technology Darmstadt, Institute of Zoology, Schnittspahnstrasse 3, D-64287, Darmstadt, Germany
To whom correspondence should be addressed at TU Darmstadt, Institute of Zoology, Department of Developmental Biology and Neurogenetics, Schnittspahnstrasse 3, 64287, Darmstadt, Germany. Fax: 0049–6151–166548. E-mail: paraoanu@bio.tu-darmstadt.de.
ABSTRACT
Developing embryos are more vulnerable than adults to acute cholinergic intoxication by anticholinesterases, including organophosphorus pesticides. These agents affect the process of neural development itself, leading to permanent deficits in the architecture of the nervous system. Recent evidence on direct roles of acetylcholinesterase (AChE) on neuronal differentiation provides additional grounds for investigating the developmental toxicity of anticholinesterases. Therefore, the effect of the organophosphate diazinon on the development of chick retinal differentiation was studied by an in vitro reaggregate approach. Reaggregated spheres from dissociated retinal cells of the E6 chick embryo were produced in rotation culture. During the whole culture period of 10 days, experimental cultures were supplemented with different concentrations of the pesticide, from 20 to 120 μM diazinon. The pesticide-treated spheres were reduced in size, and their outer surface was irregular. More importantly, inner structural distortions could be easily traced because the structure of control spheroids can be well characterized by a histotypical arrangement of laminar parts homologous to the normal retina. Acetylcholinesterase activity in diazinon-treated spheres was reduced when compared with controls. As a dramatic effect of exposure to the pesticide, inner plexiform layer (IPL)-like areas in spheroids were not distinguishable anymore. Similarly, photoreceptor rosettes and Müller radial glia were strongly decreased, whereas apoptosis was stimulated. The expression of transcripts for choline-acetyltransferase and muscarinic receptors was affected, revealing an effect of diazinon on the cholinergic system. This further proves the significance of cholinesterases and the cholinergic system for proper nervous system development and shows that further studies of debilitating diazinon actions on development are necessary.
INTRODUCTION
Diazinon is a broad-spectrum pesticide widely used in agricultural practice throughout the world. Toxicity of this pesticide has been correlated with its inhibitory effect on acetylcholinesterase (AChE) activity. In the classical view, AChE regulates cholinergic neurotransmission by hydrolyzing synaptic acetylcholine (ACh), whereas butyrylcholinesterase (BChE) is supposed to represent a less important enzyme with an uncertain role in metabolizing xenobiotic esters. Acetylcholinesterase predominates in neurons and muscle cells wherever cholinergic synapses are found. Butyrylcholinesterase, on the other hand, occurs primarily in non-neural or non-synaptic sites like liver, lung, plasma, and glia. But the complete view is more complex, because AChE also occurs in non-neural and embryonic tissues like red blood cells, megakaryocytes, and migrating neural crest cells (Lapidot-Lifson et al., 1989; Lev-Lehman et al., 1997; Miki et al., 1983). Similarly, BChE appears in limited groups of neurons (Darvesh and Hopkins, 2003), although its function at such sites is still not described.
The distributions of AChE and BChE have led to the hypothesis that cholinesterases function in ways unrelated to cholinergic neurotransmission (for review see Layer and Willbold, 1995; Massoulie et al., 1993; Soreq and Siedman, 2001). These functions were called "non-classical" and range from cell adhesion (Paraoanu and Layer, 2004; Sharma and Bigbee, 1998) to cell migration (Layer and Kaulich, 1991) and hematopoesis (Soreq et al., 1994). It has also been suggested that AChE facilitates axonal outgrowth and synapse formation—in other words, that it serves as a "morphogenic molecule" in neurons (Sharma et al., 2001). Considering all these functions of cholinesterases, we must ask whether anticholinesterase pesticides might harm immature (developing) organisms by hindering the architectural development of their nervous systems. This would be rather speculative, especially when it involves judging the risks of low-level environmental exposures. Diazinon remains a commonly used insecticide in the United States, although restrictions for residential use of diazinon have been recently applied (Whitmore et al., 2003). Moreover, there is evidence of widespread exposure to diazinon in the general population (Whyatt et al., 2002).
Might anticholinesterases even disturb the structure of emerging nervous systems by affecting development itself To address this question we used an in vitro system of aggregating retinal cells of the chicken embryo to investigate the influence of the organophosphorus pesticide diazinon on the proliferation and differentiation of retinal neurons and glia. Reaggregated retinal cells of the 6-day-old chick embryo develop into three-dimensional retina-like spheres. These retinospheroids include cellular and fibrous areas that resemble an outer and inner nuclear layer and an outer and inner plexiform layer of the normal retina (Layer and Willbold, 1993). During the first 3 days in vitro many cells still proliferate; after day 4 the system represents a differentiating neuronal network. We found effects of diazinon on AChE and BChE activity, retinospheroid morphology, size, differentiation of cell types, and muscarinic receptor expression.
MATERIALS AND METHODS
Cell preparation, rotary culture, pesticide treatment.
The detailed procedure of retina cell preparation and generation of retinospheroids in vitro has been described elsewhere (Willbold and Layer, 1992). In brief, retinae of embryonic day 6 chicken embryos (White Leghorn) were isolated and dissociated by tryptic digestion and mechanical trituration (15 strokes). To raise rosetted retinospheroids, 2 ml suspensions of cells were reagregated in bacteriological 35-mm dishes (about two eyes/dish) in aggregation medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum, 2% chicken serum, 1% glutamine, 0,1% penicillin/streptomycin, 20 μg/ml gentamycin) on a gyratory shaker (70 rpm, 37°C, 5% CO2) in the dark. Diazinon (O,O-diethyl-O-[2-isopropyl-4-methyl-6-pyrimidyl] phosphorothioate), purity 99%, was purchased from Sigma, Germany, solubilized in 75% methanol and added at concentrations of 20, 40, 80, and 120 μM (0.00625 mg/ml, 0.0125 mg/ml, 0.025 mg/ml, and 0.0375 mg/ml). Concentrations lower than 20 μM diazinon showed no effect and are not presented here. Besides the untreated control, a series of methanol-treated retinospheroids were analyzed. Vehicle-treated retinospheroids (up to a 1% concentration) developed completely normally up to the end of the observation period and showed no differences in size and cell type differentiation when compared to the untreated spheroids; the final concentrations of vehicle were 0.125%, 0.25%, 0.5%, and 0.75% corresponding to 20, 40, 80, and 120 μM diazinon, respectively. However, concentrations of 1% methanol affected the retinospheroid morphology; this limited our study to a maximal diazinon concentration of 120 μM. All comparisons show diazinon-exposed retinospheroids and the control group (not vehicle treated). Medium was changed every 2 days; the culture period was as indicated. All chemicals and media for cell culture were purchased from Gibco (Eggenstein, Germany).
Preparation of retinospheroid extracts.
Spheroids were cultured at 37°C in rotary culture in Dulbecco's modified Eagle's medium (DMEM) aggregation medium. The aggregates (about 200) were collected and washed twice with phosphate-buffered saline (PBS), 5 min at room temperature. They were then homogenized in 100 μl of ice-cold homogenization buffer (1 mM NaHCO3, 0.2 mM MgCl2X6H2O, 0.2 mM CaCl2X2H2O, 1 mM spermidine, pH 8.0) at 4°C. The cell debris was separated by centrifugation at 12,000 x g, 4°C, 10 min, and the supernatant was analyzed.
Protein determination, Ellman assay.
Protein concentrations were routinely determined according to the method of Bradford (1976). Bovine serum albumin (BSA) was used as a standard. Cholinesterase activities were determined from whole cell extracts by the Ellman assay (Ellman, 1961). Acetylcholinesterase and BChE activities were measured using acetylthiocholin (ATCh) as substrate at a final concentration of 3 mM ATCh, plus 0.1 M Ellman buffer pH 8.0, and 0.6 mM DTNB in 500μl final volume at 412 nm and 25°C. Acetylcholinesterase activity was measured by incubating the cell extract 3 min in presence of 10–3 M BChE-specific inhibitor iso-OMPA in the same reaction mixture. Butyrylcholinesterase activity was measured by incubating the cell extract 3 min in presence of 10–3 M of the specific AChE inhibitor BW284c51. All assays were carried out in triplicate. Possible interference of the lysis buffer and substrate autolysis was eliminated by use of different combinations of blank measurements. One unit of cholinesterase activity is the amount of enzyme that catalyzes the formation of 1 μmol product per min under the conditions mentioned above.
RNA, DNA isolation, and RT-PCR studies.
Total RNA from retinospheroids was isolated with the RNeasy kit (Qiagen, Hilden, Germany) and used in reverse-transcriptase polymerase chain reaction (RT-PCR) studies. RNA concentration and purity were determined by measuring the absorbance at 260 nm. For eachs ample, 2 μg RNA was used to generate cDNA with the reverse reaction using AMV-RT from Promega (Reverse Transcription Systems, Promega, Mannheim). The primers used to amplify were the following: chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CCT CTC TGG CAA AGT CCA AG-3', 5'-TGG CTG TCA CCA TTG AAG TC-3'; chicken choline-acetyltransferase (ChAT), 5'-CTC CGA GCC GAT TCA GTA AG-3', 5'-TGC TTT CAC AAA AGC AAA CG-3'; chicken muscarinic receptor M2, 5'-AGC CAG CAA GAG TCG GAT AA-3', 5'-TGC ATG TCA GCT TGG AGT TC-3'; chicken muscarinic receptor M3, 5'-GCA GGA CTA CAG GCT TCA GG-3', 5'-AGG GCA ACT TTG TGG AAT TG-3'. The primers were synthesized by Carl Roth (Karlsruhe, Germany) on the basis of available sequences.
Cycle parameters for GAPDH, ChAT, M2, and M3 included denaturation for 1 min at 95°C, followed by primer annealing at 55°C for 1 min and extension at 72°C for 1 min. In each experiment, the last cycle was followed by a 5-min elongation step at 72°C. The PCR products (GAPDH, 800 bp; ChAT, 345 bp; M2, 431 bp; M3, 358 bp) were visualized on 0.8% ethidium bromide–stained agarose gels. Quantification of the PCR products was performed by separating PCR products in agarose gels containing ethidium bromide, and the labeling intensity of the PCR product, which is linearly related to the amount of DNA, was quantified using a Bio-Rad imaging system and analyzed with the accompanying software program (Bio-Rad Multianalyst). All RT-PCR measurements aimed at determining the relative abundance of ChAT and muscarinic receptor mRNAs in control and treated retinospheroids were performed during the linear phase of amplification. The results were normalized using as internal standard GAPDH. The cycle numbers were typically 30. The RT-PCR conditions (primer concentrations, input RNA, choice of RT primer, cycling conditions) were initially optimized and were identical for all samples. Appropriate precautions were taken to avoid contamination and RNA degradation.
Genomic DNA was isolated using the Wizard SV Genomic DNA purification system (Promega, Mannheim, Germany) according to the manufacturer's instructions.
Histological procedures.
For preparation of frozen sections, the retinospheroids were fixed in 4% formaldehyde (Merck, Darmstadt, Germany) for 45 min at 25°C. After fixation, the aggregates were washed twice in PBS for 10 min at 25°C and then transferred into a solution of 25% sucrose (Merck, Darmstadt, Germany) in PBS at 4°C; they were then sectioned at 10–16 μm on a cryostat (Microm, Heidelberg, Germany).
Immunostainings.
Frozen sections were incubated for 30 min in PBS/0.3% BSA and 0.1% Triton X-100. For antibody staining, they were then incubated for 2–3 h in a 1/100 dilution in PBS/0.1% Triton X-100 monoclonal antibodies directed against calretinin (Abcam, Biozol), glutamine synthetase (Transduction Laboratoires), Pax6 (Developmental Studies Hybridoma Bank, The University of Iowa) or, alternatively polyclonal antibody against chicken rhodopsin CERN 901 (a generous gift of Dr. W. J. de Grip, Nijmegen, the Netherlands). The sections were washed twice for 10 min in PBS, followed by an incubation for 1 h with a Cy3-labeled goat anti-rabbit IgG or Cy3-labeled rabbit anti-mouse (Dianova, Hamburg, Germany) at a 1/1000 dilution in PBS. Finally, the sections were washed three times in PBS and the cell nuclei were stained with DAPI (0.1 μg/ml 4',6-diamidine-2-phenylindol-dihydrochloride in PBS) for 1 min at room temperature. Apoptosis was investigated using the DeadEnd colorimetric TUNEL system (Promega, Mannheim, Germany) on retinospheroid sections according to the manufacturer's protocol.
Karnovsky and Roots staining.
The staining procedure of Karnovsky and Roots (1964) is widely used for studies of cholinesterase expression patterns. We used AChE and BChE histochemistry to follow up the cholinesterase inhibition at the cellular level. The retinospheroid sections were incubated for 10 min in 0.1 M Tris-maleate buffer, pH 6. After the equilibration step, the sections were incubated for up to 2 h (AChE) and up to 5 h (BChE) in 0.1% ATCh, 0.1 M C6H5Na3O7X2H2O, 30 mM CuSO4, 5 mM K3Fe(CN)6 in Tris-maleate buffer.
Microscopy and photography.
Whole retinospheroids and stained sections were documented using a Zeiss Axiophot microscope with DIC (Nomarski) and fluorescence optics. Photomicrographs were taken using an Intas camera guided by a computer program (Diskus 1280, CH Hilgers, Knigswinter). The figures were produced using Adobe Photoshop 7.
Statistical analysis.
Statistical analyses were performed with the aid of two software packages (GraphPad Software, and Statistica 5, Statsoft). Values are presented as means ± standard error (SE) of triplicate experiments. Statistical analyses for all experiments were performed by one-way and factorial analysis of variance (ANOVA), followed by Tukey HSD tests. Values of p < 0.05 were considered statistically significant.
RESULTS
The Size, Cell Number, Protein, and DNA Content of Diazinon-Treated Retinospheroids Is Affected
Treatment with diazinon affected strongly the growth and morphogenesis of retinospheroids (Figs. 1–2). This treatment during culture resulted in the formation of smaller spheroids with average surface of only 30–90% that of control retinospheroids (Figs. 1–2). The effects were concentration dependent; the most affected spheroids were the ones treated with 80 μM diazinon (Fig. 2D). The most dramatic decrease was seen after 10 days in culture (ANOVA; F3, 16 = 42.888, p = 0.000001). Significant were also the differences at 6 days in culture (ANOVA; F3, 16 = 4.7956, p = 0.014). In parallel, spheroid sections were also stained with DAPI in order to analyze the general organization (Fig. 2D). The decrease in size is associated with a reduced cell number.
The outer surface of diazinon-treated spheroids was rough; they presented fewer inner rosettes, and fusion processes between two or more spheroids were observed (Fig. 2D). Protein concentration was measured; in general, it tended to decrease as the diazinon concentrations increased (Fig. 2A). The results were analysed with ANOVA and found to be marginally significant (p < 0.1). A corresponding result was found at the genomic DNA level. The samples treated with 80 μM diazinon had a clearly reduced amount of DNA (Fig. 2B).
To further analyze the cause of size reduction, a TUNEL assay was used for detecting apoptosis (Fig. 3). Interestingly, intense apoptosis was observed at the border of the structure, indicating that these cells undergo apoptosis and afterwards are lost by the retinospheroids (Fig. 3B and E).
The AChE and BChE Activities Are Inhibited
The expression of both AChE and BChE was analyzed in cell extracts of retinospheroids at different culture stages with the Ellman test. The AChE activity in control retinospheroids increased up to a maximum at day 6 in culture, followed by a decrease up to day 10 in culture (Fig. 4A). The diazinon treatment resulted in a decrease of AChE activity (Fig. 4A) that was concentration dependent. The strongest effect was observed at day 6 in culture where the activity of control was much higher than of the treated samples (ANOVA; F3,9 = 33.219, p = 0.00003). The sample treated with 80 μM diazinon was always highly significantly different when compared with controls, indicating that this concentration is the most effective. Interestingly, after 10 days in culture the spheroids treated with this concentration of pesticide show the same AChE activity as the controls, suggesting a mechanism of "overproduction" of enzyme under long-term treatment with diazinon.
The decrease in AChE expression can be also followed at the histological level (see Fig. 5). In particular, strongly stained areas that represent AChE-positive "inner plexiform like" (IPL) areas are decreased in number and also in areal size (Fig. 5, left panels). The AChE-positive cells in diazinon-treated spheroids are less organized and tend to localize at the border.
The activity of BChE was also investigated using the Ellman assay and the Karnovsky-Roots histochemistry. The retinospheroids show a lower activity when compared with AChE. Interestingly, an inhibition of BChE was observed at low diazinon concentrations (Fig. 4B), but an upregulation occurred at higher diazinon concentrations. At the histological level, the BChE activity is organized in IPL-like specific areas (Fig. 6, left panels). The size of stained areas and the intensity of the staining are decreased at diazinon concentrations of 20 μM, suggesting a strong effect of the pesticide on BChE.
Effect of Diazinon on Retinal Neurons (Amacrine Cells, Ganglion Cells, and Photoreceptors) and Müller Glia Cells
To characterize whether the treatment with diazinon was accompanied with changes in the development of retinal cells, different antibody stainings were used to follow the differentiation of retinal cell types over a period of 10 culture days. The retinospheroids were collected at different stages, paraformaldehyde-fixed, and cryosectioned. Figure 7 shows retinospheroid sections at different culture stages stained with an anti-calretinin antibody. Calretinin is a calcium-binding protein that labels subpopulations of amacrine and ganglion cells in the chicken retina (Rogers, 1989). In control retinospheroids, calretinin-positive cells concentrate around IPL-like areas and extend processes into the cell-free spaces (Fig. 7, left panels). In diazinon-treated retinospheroids the calretinin-positive cells are not organized and their processes are not stained by the antibody. It appears that at concentrations of 80 μM diazinon, neurite growth into neuropil areas (IPL) was strongly inhibited (Fig. 7, right panels).
Amacrine and ganglion cells represent the only cell populations of the chicken retina that express AChE activity (Layer, 1983; Ramirez et al., 1989). Therefore, it would be expected that these cells would be most affected by the diazinon treatment. A further marker, Pax6, was used to label amacrine and ganglion cells (Belecky-Adams et al., 1997). Pax6-positive cells were found at high numbers surrounding the IPL-like areas (Fig. 8A). Interestingly, the processes of Pax6-positive cells become intensively stained with increasing diazinon concentration. At a concentration of 80 μM diazinon, distinct organization was no longer visible, but the processes of Pax6-positive cells formed a thick inner neuropil network. The formation of this network could not be observed with the calretinin antibody.
The differentiation of rod photoreceptors was also investigated. Photoreceptors organize in so-called rosettes (see Fig. 9A) or appear in unorganized regions of retinospheroids (Rothermel et al., 1997). Cryosections were immunostained with the rod-specific antiserum CERN 901. Diazinon affected the number and the organization of rods (Fig. 9B, C, and D). No distinct rosettes could be observed, indicating that the pesticide also affected the organization of the cells that do not express AChE.
Müller glia cells are astrocyte-like radial glia cells that are formed exclusively in the retina and are crucially involved in the development of the retina's architecture and circuitry (Willbold and Layer, 1998). Glutamine synthetase represents a Müller glia cell marker in the retina. Diazinon led to a drastic decrease in the number and organization of Müller cells (Fig. 10B, C, and D). This documents that diazinon treatment leads to a degeneration of Müller glia cells.
Other Components of the Cholinergic System Are Affected
It is already known that organophosphates not only affect the cholinesterases; they also act on the muscarinic acetylcholine receptors (Lein and Fryer, 2005). Semi-quantitative RT-PCR revealed that the diazinon-treated spheres did not show drastic changes; nevertheless slight increases in the number of transcripts for ChAT, M2, and M3 was observed with increasing diazinon concentration until culture day 8 (see Fig. 11). These slight increases were followed on day 10 by a decrease in the ChAT transcripts.
DISCUSSION
Diazinon was examined in three-dimensional retinal cell cultures to determine whether cholinesterase inhibition led to abnormal cellular differentiation and outgrowth of axons. The results show that diazinon was deleterious to the growth and histogenesis of reaggregated spheres from chicken embryonic retinae. More specifically, the size of reaggregates was reduced, the differentiation and organization of retinal cell types was affected, and the expression of other cholinergic markers was changed.
The first and most dramatic effect of diazinon treatment was the size reduction. All concentrations used were effective and the effects were dose-related. This is also supported by studies in humans, where diazinon exposures led to an impaired fetal growth (Whyatt et al., 2004), and in chick embryo, where teratogenic effects of diazinon were observed (Misawa et al., 1982). These effects were due mainly to growth retardation and alteration of differentiation at early stages of development. Studies with other organophosphates could show that developmental exposure decreased brain DNA and RNA synthesis (Dam et al., 1998).
A decrease in size can be associated with increased cell death or/and less proliferation within the retinospheroids. TUNEL assay showed that the cells treated with diazinon undergo apoptotic cell death. The most affected cells were at the border, indicating that the first layer of cells exposed to the pesticide is also the one most affected. These cells respond to the pesticide by producing higher amounts of AChE (see Fig. 5) in order to overcome enzymatic inhibition and undergo apoptosis. Therefore decrease in size can be due to apoptosis, followed by loss of cells from the retinospheroid. It seems that the cells that undergo apoptosis also express high levels of AChE. Interestingly, Jin and collaborators (2004) could show that AChE promotes apoptosis, first by inhibiting cell growth and then by impeding subsequent transfer to the cell nucleus, where it promotes cell death by an unknown mechanism.
We could show that the histogenesis in retinal reaggregates was stongly affected by concentrations of 40 μM diazinon, starting with cell aggregation processes. This might be explained by the adhesive properties of AChE (Johnson and Moore, 2003; Paraoanu and Layer, 2004). Diazinon-treated cells show an instability of the cell-cell contacts. This was also observed earlier (Layer et al., 1992), where a specific AChE-inhibitor led to similar effects.
Given our results, one has to consider a dual aspect of this pesticide's actions on retinal development, comprising both cholinergic and non-cholinergic components. All organophosphorus compounds have a mechanism of toxicity in common, that is, phosphorylation of AChE causing accumulation of acetylcholine, overstimulation of cholinergic receptors, and consequently, signs of cholinergic toxicity. On the other hand, organophosphate exposures have also been shown to downregulate muscarinic receptors, inhibit the adenylate cyclase signaling cascade, decrease brain DNA and RNA synthesis, and suppress neurite outgrowth and cell adhesion (Anderson and Key, 1999; Bigbee et al., 1999; Dam et al., 1998; Lein and Fryer, 2005; Li and Casida, 1998; Song et al., 1997). Therefore, developmental exposures may be more dangerous than previously thought, both because acetylcholinesterase may have a direct role on neuronal differentiation, and moreover, the organophosphorus compounds also act on other components of the cholinergic system.
In the retina, two populations of cells express AChE: amacrine cells and ganglion cells. We used specific antibodies to study the development of these two cell populations under diazinon treatment. The two cell types should normally be most affected by diazinon treatment. It is described that AChE typically appears during neural development while axons are growing and before synaptic connections form (for review see Layer, 1991). This transient AChE expression is no accident of development, but is correlated with an AChE role of promoting axonal outgrowth or synaptic connection (Anderson and Key, 1999; Bigbee et al., 1999, 2000). The present study established that both amacrine cells and ganglion cells were affected by diazinon in their numbers and distribution. Although the reduction in number of cells can be correlated with a size reduction of the retinospheroids, the effect of diazinon on their distribution remains to be elucidated. In control retinospheroids the AChE-positive amacrine and ganglion cells organize around so called IPL-areas and extend their processes into these areas. In treated spheroids such an organization is no longer distinguishable; at high concentrations the AChE-positive cells migrate to the outer surface of the structure, and no IPL-like areas are seen. The migration of AChE-positive cells to the border of the retinospheroid could be interpreted as a defense mechanism by which the three-dimensional structure tries to overcome the exposure by increasing AChE production at its margin.
Our results show that not only the AChE-positive cells were affected but also other cell types, e.g., rod photoreceptors and Müller glia cells. These represent cells with no cholinergic innervation, and which also do not express cholinesterases. Interestingly, similar "non-cholinergic" effects were observed for another organophosphate, chlorpyrifos (for review see Slotkin, 1999). Some of the non-cholinergic effects of chlorpyrifos involve changes in the protein G–mediated signaling and adenylyl cyclase activity (Song et al., 1997). Cyclic AMP is universally involved in the control of cell replication and differentiation in virtually all prokaryotic and eukaryotic cells (Hultgardh-Nilsson et al., 1994), so that perturbation of this pathway during development would be expected to have a significant impact on brain cell development. A similar scenario can happen for diazinon too.
Other components of the cholinergic system, e.g., ChAT, and the muscarinic receptors were analyzed at the transcript level. Slight changes were observed for the muscarinic receptors 2 and 3, consisting of an upregulation of the transcripts. This can be easily explained as a reaction to the increased amount of acetylcholine available.
In conclusion, this study illustrates that retinospheroids represent a promising model system for the evaluation of developmental neurotoxicity of organophosphate pesticides and indicate also the need for additional research on possible deleterious effects of pesticides usage (both singly and in combination) on the developing organisms.
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To whom correspondence should be addressed at TU Darmstadt, Institute of Zoology, Department of Developmental Biology and Neurogenetics, Schnittspahnstrasse 3, 64287, Darmstadt, Germany. Fax: 0049–6151–166548. E-mail: paraoanu@bio.tu-darmstadt.de.
ABSTRACT
Developing embryos are more vulnerable than adults to acute cholinergic intoxication by anticholinesterases, including organophosphorus pesticides. These agents affect the process of neural development itself, leading to permanent deficits in the architecture of the nervous system. Recent evidence on direct roles of acetylcholinesterase (AChE) on neuronal differentiation provides additional grounds for investigating the developmental toxicity of anticholinesterases. Therefore, the effect of the organophosphate diazinon on the development of chick retinal differentiation was studied by an in vitro reaggregate approach. Reaggregated spheres from dissociated retinal cells of the E6 chick embryo were produced in rotation culture. During the whole culture period of 10 days, experimental cultures were supplemented with different concentrations of the pesticide, from 20 to 120 μM diazinon. The pesticide-treated spheres were reduced in size, and their outer surface was irregular. More importantly, inner structural distortions could be easily traced because the structure of control spheroids can be well characterized by a histotypical arrangement of laminar parts homologous to the normal retina. Acetylcholinesterase activity in diazinon-treated spheres was reduced when compared with controls. As a dramatic effect of exposure to the pesticide, inner plexiform layer (IPL)-like areas in spheroids were not distinguishable anymore. Similarly, photoreceptor rosettes and Müller radial glia were strongly decreased, whereas apoptosis was stimulated. The expression of transcripts for choline-acetyltransferase and muscarinic receptors was affected, revealing an effect of diazinon on the cholinergic system. This further proves the significance of cholinesterases and the cholinergic system for proper nervous system development and shows that further studies of debilitating diazinon actions on development are necessary.
INTRODUCTION
Diazinon is a broad-spectrum pesticide widely used in agricultural practice throughout the world. Toxicity of this pesticide has been correlated with its inhibitory effect on acetylcholinesterase (AChE) activity. In the classical view, AChE regulates cholinergic neurotransmission by hydrolyzing synaptic acetylcholine (ACh), whereas butyrylcholinesterase (BChE) is supposed to represent a less important enzyme with an uncertain role in metabolizing xenobiotic esters. Acetylcholinesterase predominates in neurons and muscle cells wherever cholinergic synapses are found. Butyrylcholinesterase, on the other hand, occurs primarily in non-neural or non-synaptic sites like liver, lung, plasma, and glia. But the complete view is more complex, because AChE also occurs in non-neural and embryonic tissues like red blood cells, megakaryocytes, and migrating neural crest cells (Lapidot-Lifson et al., 1989; Lev-Lehman et al., 1997; Miki et al., 1983). Similarly, BChE appears in limited groups of neurons (Darvesh and Hopkins, 2003), although its function at such sites is still not described.
The distributions of AChE and BChE have led to the hypothesis that cholinesterases function in ways unrelated to cholinergic neurotransmission (for review see Layer and Willbold, 1995; Massoulie et al., 1993; Soreq and Siedman, 2001). These functions were called "non-classical" and range from cell adhesion (Paraoanu and Layer, 2004; Sharma and Bigbee, 1998) to cell migration (Layer and Kaulich, 1991) and hematopoesis (Soreq et al., 1994). It has also been suggested that AChE facilitates axonal outgrowth and synapse formation—in other words, that it serves as a "morphogenic molecule" in neurons (Sharma et al., 2001). Considering all these functions of cholinesterases, we must ask whether anticholinesterase pesticides might harm immature (developing) organisms by hindering the architectural development of their nervous systems. This would be rather speculative, especially when it involves judging the risks of low-level environmental exposures. Diazinon remains a commonly used insecticide in the United States, although restrictions for residential use of diazinon have been recently applied (Whitmore et al., 2003). Moreover, there is evidence of widespread exposure to diazinon in the general population (Whyatt et al., 2002).
Might anticholinesterases even disturb the structure of emerging nervous systems by affecting development itself To address this question we used an in vitro system of aggregating retinal cells of the chicken embryo to investigate the influence of the organophosphorus pesticide diazinon on the proliferation and differentiation of retinal neurons and glia. Reaggregated retinal cells of the 6-day-old chick embryo develop into three-dimensional retina-like spheres. These retinospheroids include cellular and fibrous areas that resemble an outer and inner nuclear layer and an outer and inner plexiform layer of the normal retina (Layer and Willbold, 1993). During the first 3 days in vitro many cells still proliferate; after day 4 the system represents a differentiating neuronal network. We found effects of diazinon on AChE and BChE activity, retinospheroid morphology, size, differentiation of cell types, and muscarinic receptor expression.
MATERIALS AND METHODS
Cell preparation, rotary culture, pesticide treatment.
The detailed procedure of retina cell preparation and generation of retinospheroids in vitro has been described elsewhere (Willbold and Layer, 1992). In brief, retinae of embryonic day 6 chicken embryos (White Leghorn) were isolated and dissociated by tryptic digestion and mechanical trituration (15 strokes). To raise rosetted retinospheroids, 2 ml suspensions of cells were reagregated in bacteriological 35-mm dishes (about two eyes/dish) in aggregation medium (Dulbecco's modified Eagle's medium, 10% fetal calf serum, 2% chicken serum, 1% glutamine, 0,1% penicillin/streptomycin, 20 μg/ml gentamycin) on a gyratory shaker (70 rpm, 37°C, 5% CO2) in the dark. Diazinon (O,O-diethyl-O-[2-isopropyl-4-methyl-6-pyrimidyl] phosphorothioate), purity 99%, was purchased from Sigma, Germany, solubilized in 75% methanol and added at concentrations of 20, 40, 80, and 120 μM (0.00625 mg/ml, 0.0125 mg/ml, 0.025 mg/ml, and 0.0375 mg/ml). Concentrations lower than 20 μM diazinon showed no effect and are not presented here. Besides the untreated control, a series of methanol-treated retinospheroids were analyzed. Vehicle-treated retinospheroids (up to a 1% concentration) developed completely normally up to the end of the observation period and showed no differences in size and cell type differentiation when compared to the untreated spheroids; the final concentrations of vehicle were 0.125%, 0.25%, 0.5%, and 0.75% corresponding to 20, 40, 80, and 120 μM diazinon, respectively. However, concentrations of 1% methanol affected the retinospheroid morphology; this limited our study to a maximal diazinon concentration of 120 μM. All comparisons show diazinon-exposed retinospheroids and the control group (not vehicle treated). Medium was changed every 2 days; the culture period was as indicated. All chemicals and media for cell culture were purchased from Gibco (Eggenstein, Germany).
Preparation of retinospheroid extracts.
Spheroids were cultured at 37°C in rotary culture in Dulbecco's modified Eagle's medium (DMEM) aggregation medium. The aggregates (about 200) were collected and washed twice with phosphate-buffered saline (PBS), 5 min at room temperature. They were then homogenized in 100 μl of ice-cold homogenization buffer (1 mM NaHCO3, 0.2 mM MgCl2X6H2O, 0.2 mM CaCl2X2H2O, 1 mM spermidine, pH 8.0) at 4°C. The cell debris was separated by centrifugation at 12,000 x g, 4°C, 10 min, and the supernatant was analyzed.
Protein determination, Ellman assay.
Protein concentrations were routinely determined according to the method of Bradford (1976). Bovine serum albumin (BSA) was used as a standard. Cholinesterase activities were determined from whole cell extracts by the Ellman assay (Ellman, 1961). Acetylcholinesterase and BChE activities were measured using acetylthiocholin (ATCh) as substrate at a final concentration of 3 mM ATCh, plus 0.1 M Ellman buffer pH 8.0, and 0.6 mM DTNB in 500μl final volume at 412 nm and 25°C. Acetylcholinesterase activity was measured by incubating the cell extract 3 min in presence of 10–3 M BChE-specific inhibitor iso-OMPA in the same reaction mixture. Butyrylcholinesterase activity was measured by incubating the cell extract 3 min in presence of 10–3 M of the specific AChE inhibitor BW284c51. All assays were carried out in triplicate. Possible interference of the lysis buffer and substrate autolysis was eliminated by use of different combinations of blank measurements. One unit of cholinesterase activity is the amount of enzyme that catalyzes the formation of 1 μmol product per min under the conditions mentioned above.
RNA, DNA isolation, and RT-PCR studies.
Total RNA from retinospheroids was isolated with the RNeasy kit (Qiagen, Hilden, Germany) and used in reverse-transcriptase polymerase chain reaction (RT-PCR) studies. RNA concentration and purity were determined by measuring the absorbance at 260 nm. For eachs ample, 2 μg RNA was used to generate cDNA with the reverse reaction using AMV-RT from Promega (Reverse Transcription Systems, Promega, Mannheim). The primers used to amplify were the following: chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CCT CTC TGG CAA AGT CCA AG-3', 5'-TGG CTG TCA CCA TTG AAG TC-3'; chicken choline-acetyltransferase (ChAT), 5'-CTC CGA GCC GAT TCA GTA AG-3', 5'-TGC TTT CAC AAA AGC AAA CG-3'; chicken muscarinic receptor M2, 5'-AGC CAG CAA GAG TCG GAT AA-3', 5'-TGC ATG TCA GCT TGG AGT TC-3'; chicken muscarinic receptor M3, 5'-GCA GGA CTA CAG GCT TCA GG-3', 5'-AGG GCA ACT TTG TGG AAT TG-3'. The primers were synthesized by Carl Roth (Karlsruhe, Germany) on the basis of available sequences.
Cycle parameters for GAPDH, ChAT, M2, and M3 included denaturation for 1 min at 95°C, followed by primer annealing at 55°C for 1 min and extension at 72°C for 1 min. In each experiment, the last cycle was followed by a 5-min elongation step at 72°C. The PCR products (GAPDH, 800 bp; ChAT, 345 bp; M2, 431 bp; M3, 358 bp) were visualized on 0.8% ethidium bromide–stained agarose gels. Quantification of the PCR products was performed by separating PCR products in agarose gels containing ethidium bromide, and the labeling intensity of the PCR product, which is linearly related to the amount of DNA, was quantified using a Bio-Rad imaging system and analyzed with the accompanying software program (Bio-Rad Multianalyst). All RT-PCR measurements aimed at determining the relative abundance of ChAT and muscarinic receptor mRNAs in control and treated retinospheroids were performed during the linear phase of amplification. The results were normalized using as internal standard GAPDH. The cycle numbers were typically 30. The RT-PCR conditions (primer concentrations, input RNA, choice of RT primer, cycling conditions) were initially optimized and were identical for all samples. Appropriate precautions were taken to avoid contamination and RNA degradation.
Genomic DNA was isolated using the Wizard SV Genomic DNA purification system (Promega, Mannheim, Germany) according to the manufacturer's instructions.
Histological procedures.
For preparation of frozen sections, the retinospheroids were fixed in 4% formaldehyde (Merck, Darmstadt, Germany) for 45 min at 25°C. After fixation, the aggregates were washed twice in PBS for 10 min at 25°C and then transferred into a solution of 25% sucrose (Merck, Darmstadt, Germany) in PBS at 4°C; they were then sectioned at 10–16 μm on a cryostat (Microm, Heidelberg, Germany).
Immunostainings.
Frozen sections were incubated for 30 min in PBS/0.3% BSA and 0.1% Triton X-100. For antibody staining, they were then incubated for 2–3 h in a 1/100 dilution in PBS/0.1% Triton X-100 monoclonal antibodies directed against calretinin (Abcam, Biozol), glutamine synthetase (Transduction Laboratoires), Pax6 (Developmental Studies Hybridoma Bank, The University of Iowa) or, alternatively polyclonal antibody against chicken rhodopsin CERN 901 (a generous gift of Dr. W. J. de Grip, Nijmegen, the Netherlands). The sections were washed twice for 10 min in PBS, followed by an incubation for 1 h with a Cy3-labeled goat anti-rabbit IgG or Cy3-labeled rabbit anti-mouse (Dianova, Hamburg, Germany) at a 1/1000 dilution in PBS. Finally, the sections were washed three times in PBS and the cell nuclei were stained with DAPI (0.1 μg/ml 4',6-diamidine-2-phenylindol-dihydrochloride in PBS) for 1 min at room temperature. Apoptosis was investigated using the DeadEnd colorimetric TUNEL system (Promega, Mannheim, Germany) on retinospheroid sections according to the manufacturer's protocol.
Karnovsky and Roots staining.
The staining procedure of Karnovsky and Roots (1964) is widely used for studies of cholinesterase expression patterns. We used AChE and BChE histochemistry to follow up the cholinesterase inhibition at the cellular level. The retinospheroid sections were incubated for 10 min in 0.1 M Tris-maleate buffer, pH 6. After the equilibration step, the sections were incubated for up to 2 h (AChE) and up to 5 h (BChE) in 0.1% ATCh, 0.1 M C6H5Na3O7X2H2O, 30 mM CuSO4, 5 mM K3Fe(CN)6 in Tris-maleate buffer.
Microscopy and photography.
Whole retinospheroids and stained sections were documented using a Zeiss Axiophot microscope with DIC (Nomarski) and fluorescence optics. Photomicrographs were taken using an Intas camera guided by a computer program (Diskus 1280, CH Hilgers, Knigswinter). The figures were produced using Adobe Photoshop 7.
Statistical analysis.
Statistical analyses were performed with the aid of two software packages (GraphPad Software, and Statistica 5, Statsoft). Values are presented as means ± standard error (SE) of triplicate experiments. Statistical analyses for all experiments were performed by one-way and factorial analysis of variance (ANOVA), followed by Tukey HSD tests. Values of p < 0.05 were considered statistically significant.
RESULTS
The Size, Cell Number, Protein, and DNA Content of Diazinon-Treated Retinospheroids Is Affected
Treatment with diazinon affected strongly the growth and morphogenesis of retinospheroids (Figs. 1–2). This treatment during culture resulted in the formation of smaller spheroids with average surface of only 30–90% that of control retinospheroids (Figs. 1–2). The effects were concentration dependent; the most affected spheroids were the ones treated with 80 μM diazinon (Fig. 2D). The most dramatic decrease was seen after 10 days in culture (ANOVA; F3, 16 = 42.888, p = 0.000001). Significant were also the differences at 6 days in culture (ANOVA; F3, 16 = 4.7956, p = 0.014). In parallel, spheroid sections were also stained with DAPI in order to analyze the general organization (Fig. 2D). The decrease in size is associated with a reduced cell number.
The outer surface of diazinon-treated spheroids was rough; they presented fewer inner rosettes, and fusion processes between two or more spheroids were observed (Fig. 2D). Protein concentration was measured; in general, it tended to decrease as the diazinon concentrations increased (Fig. 2A). The results were analysed with ANOVA and found to be marginally significant (p < 0.1). A corresponding result was found at the genomic DNA level. The samples treated with 80 μM diazinon had a clearly reduced amount of DNA (Fig. 2B).
To further analyze the cause of size reduction, a TUNEL assay was used for detecting apoptosis (Fig. 3). Interestingly, intense apoptosis was observed at the border of the structure, indicating that these cells undergo apoptosis and afterwards are lost by the retinospheroids (Fig. 3B and E).
The AChE and BChE Activities Are Inhibited
The expression of both AChE and BChE was analyzed in cell extracts of retinospheroids at different culture stages with the Ellman test. The AChE activity in control retinospheroids increased up to a maximum at day 6 in culture, followed by a decrease up to day 10 in culture (Fig. 4A). The diazinon treatment resulted in a decrease of AChE activity (Fig. 4A) that was concentration dependent. The strongest effect was observed at day 6 in culture where the activity of control was much higher than of the treated samples (ANOVA; F3,9 = 33.219, p = 0.00003). The sample treated with 80 μM diazinon was always highly significantly different when compared with controls, indicating that this concentration is the most effective. Interestingly, after 10 days in culture the spheroids treated with this concentration of pesticide show the same AChE activity as the controls, suggesting a mechanism of "overproduction" of enzyme under long-term treatment with diazinon.
The decrease in AChE expression can be also followed at the histological level (see Fig. 5). In particular, strongly stained areas that represent AChE-positive "inner plexiform like" (IPL) areas are decreased in number and also in areal size (Fig. 5, left panels). The AChE-positive cells in diazinon-treated spheroids are less organized and tend to localize at the border.
The activity of BChE was also investigated using the Ellman assay and the Karnovsky-Roots histochemistry. The retinospheroids show a lower activity when compared with AChE. Interestingly, an inhibition of BChE was observed at low diazinon concentrations (Fig. 4B), but an upregulation occurred at higher diazinon concentrations. At the histological level, the BChE activity is organized in IPL-like specific areas (Fig. 6, left panels). The size of stained areas and the intensity of the staining are decreased at diazinon concentrations of 20 μM, suggesting a strong effect of the pesticide on BChE.
Effect of Diazinon on Retinal Neurons (Amacrine Cells, Ganglion Cells, and Photoreceptors) and Müller Glia Cells
To characterize whether the treatment with diazinon was accompanied with changes in the development of retinal cells, different antibody stainings were used to follow the differentiation of retinal cell types over a period of 10 culture days. The retinospheroids were collected at different stages, paraformaldehyde-fixed, and cryosectioned. Figure 7 shows retinospheroid sections at different culture stages stained with an anti-calretinin antibody. Calretinin is a calcium-binding protein that labels subpopulations of amacrine and ganglion cells in the chicken retina (Rogers, 1989). In control retinospheroids, calretinin-positive cells concentrate around IPL-like areas and extend processes into the cell-free spaces (Fig. 7, left panels). In diazinon-treated retinospheroids the calretinin-positive cells are not organized and their processes are not stained by the antibody. It appears that at concentrations of 80 μM diazinon, neurite growth into neuropil areas (IPL) was strongly inhibited (Fig. 7, right panels).
Amacrine and ganglion cells represent the only cell populations of the chicken retina that express AChE activity (Layer, 1983; Ramirez et al., 1989). Therefore, it would be expected that these cells would be most affected by the diazinon treatment. A further marker, Pax6, was used to label amacrine and ganglion cells (Belecky-Adams et al., 1997). Pax6-positive cells were found at high numbers surrounding the IPL-like areas (Fig. 8A). Interestingly, the processes of Pax6-positive cells become intensively stained with increasing diazinon concentration. At a concentration of 80 μM diazinon, distinct organization was no longer visible, but the processes of Pax6-positive cells formed a thick inner neuropil network. The formation of this network could not be observed with the calretinin antibody.
The differentiation of rod photoreceptors was also investigated. Photoreceptors organize in so-called rosettes (see Fig. 9A) or appear in unorganized regions of retinospheroids (Rothermel et al., 1997). Cryosections were immunostained with the rod-specific antiserum CERN 901. Diazinon affected the number and the organization of rods (Fig. 9B, C, and D). No distinct rosettes could be observed, indicating that the pesticide also affected the organization of the cells that do not express AChE.
Müller glia cells are astrocyte-like radial glia cells that are formed exclusively in the retina and are crucially involved in the development of the retina's architecture and circuitry (Willbold and Layer, 1998). Glutamine synthetase represents a Müller glia cell marker in the retina. Diazinon led to a drastic decrease in the number and organization of Müller cells (Fig. 10B, C, and D). This documents that diazinon treatment leads to a degeneration of Müller glia cells.
Other Components of the Cholinergic System Are Affected
It is already known that organophosphates not only affect the cholinesterases; they also act on the muscarinic acetylcholine receptors (Lein and Fryer, 2005). Semi-quantitative RT-PCR revealed that the diazinon-treated spheres did not show drastic changes; nevertheless slight increases in the number of transcripts for ChAT, M2, and M3 was observed with increasing diazinon concentration until culture day 8 (see Fig. 11). These slight increases were followed on day 10 by a decrease in the ChAT transcripts.
DISCUSSION
Diazinon was examined in three-dimensional retinal cell cultures to determine whether cholinesterase inhibition led to abnormal cellular differentiation and outgrowth of axons. The results show that diazinon was deleterious to the growth and histogenesis of reaggregated spheres from chicken embryonic retinae. More specifically, the size of reaggregates was reduced, the differentiation and organization of retinal cell types was affected, and the expression of other cholinergic markers was changed.
The first and most dramatic effect of diazinon treatment was the size reduction. All concentrations used were effective and the effects were dose-related. This is also supported by studies in humans, where diazinon exposures led to an impaired fetal growth (Whyatt et al., 2004), and in chick embryo, where teratogenic effects of diazinon were observed (Misawa et al., 1982). These effects were due mainly to growth retardation and alteration of differentiation at early stages of development. Studies with other organophosphates could show that developmental exposure decreased brain DNA and RNA synthesis (Dam et al., 1998).
A decrease in size can be associated with increased cell death or/and less proliferation within the retinospheroids. TUNEL assay showed that the cells treated with diazinon undergo apoptotic cell death. The most affected cells were at the border, indicating that the first layer of cells exposed to the pesticide is also the one most affected. These cells respond to the pesticide by producing higher amounts of AChE (see Fig. 5) in order to overcome enzymatic inhibition and undergo apoptosis. Therefore decrease in size can be due to apoptosis, followed by loss of cells from the retinospheroid. It seems that the cells that undergo apoptosis also express high levels of AChE. Interestingly, Jin and collaborators (2004) could show that AChE promotes apoptosis, first by inhibiting cell growth and then by impeding subsequent transfer to the cell nucleus, where it promotes cell death by an unknown mechanism.
We could show that the histogenesis in retinal reaggregates was stongly affected by concentrations of 40 μM diazinon, starting with cell aggregation processes. This might be explained by the adhesive properties of AChE (Johnson and Moore, 2003; Paraoanu and Layer, 2004). Diazinon-treated cells show an instability of the cell-cell contacts. This was also observed earlier (Layer et al., 1992), where a specific AChE-inhibitor led to similar effects.
Given our results, one has to consider a dual aspect of this pesticide's actions on retinal development, comprising both cholinergic and non-cholinergic components. All organophosphorus compounds have a mechanism of toxicity in common, that is, phosphorylation of AChE causing accumulation of acetylcholine, overstimulation of cholinergic receptors, and consequently, signs of cholinergic toxicity. On the other hand, organophosphate exposures have also been shown to downregulate muscarinic receptors, inhibit the adenylate cyclase signaling cascade, decrease brain DNA and RNA synthesis, and suppress neurite outgrowth and cell adhesion (Anderson and Key, 1999; Bigbee et al., 1999; Dam et al., 1998; Lein and Fryer, 2005; Li and Casida, 1998; Song et al., 1997). Therefore, developmental exposures may be more dangerous than previously thought, both because acetylcholinesterase may have a direct role on neuronal differentiation, and moreover, the organophosphorus compounds also act on other components of the cholinergic system.
In the retina, two populations of cells express AChE: amacrine cells and ganglion cells. We used specific antibodies to study the development of these two cell populations under diazinon treatment. The two cell types should normally be most affected by diazinon treatment. It is described that AChE typically appears during neural development while axons are growing and before synaptic connections form (for review see Layer, 1991). This transient AChE expression is no accident of development, but is correlated with an AChE role of promoting axonal outgrowth or synaptic connection (Anderson and Key, 1999; Bigbee et al., 1999, 2000). The present study established that both amacrine cells and ganglion cells were affected by diazinon in their numbers and distribution. Although the reduction in number of cells can be correlated with a size reduction of the retinospheroids, the effect of diazinon on their distribution remains to be elucidated. In control retinospheroids the AChE-positive amacrine and ganglion cells organize around so called IPL-areas and extend their processes into these areas. In treated spheroids such an organization is no longer distinguishable; at high concentrations the AChE-positive cells migrate to the outer surface of the structure, and no IPL-like areas are seen. The migration of AChE-positive cells to the border of the retinospheroid could be interpreted as a defense mechanism by which the three-dimensional structure tries to overcome the exposure by increasing AChE production at its margin.
Our results show that not only the AChE-positive cells were affected but also other cell types, e.g., rod photoreceptors and Müller glia cells. These represent cells with no cholinergic innervation, and which also do not express cholinesterases. Interestingly, similar "non-cholinergic" effects were observed for another organophosphate, chlorpyrifos (for review see Slotkin, 1999). Some of the non-cholinergic effects of chlorpyrifos involve changes in the protein G–mediated signaling and adenylyl cyclase activity (Song et al., 1997). Cyclic AMP is universally involved in the control of cell replication and differentiation in virtually all prokaryotic and eukaryotic cells (Hultgardh-Nilsson et al., 1994), so that perturbation of this pathway during development would be expected to have a significant impact on brain cell development. A similar scenario can happen for diazinon too.
Other components of the cholinergic system, e.g., ChAT, and the muscarinic receptors were analyzed at the transcript level. Slight changes were observed for the muscarinic receptors 2 and 3, consisting of an upregulation of the transcripts. This can be easily explained as a reaction to the increased amount of acetylcholine available.
In conclusion, this study illustrates that retinospheroids represent a promising model system for the evaluation of developmental neurotoxicity of organophosphate pesticides and indicate also the need for additional research on possible deleterious effects of pesticides usage (both singly and in combination) on the developing organisms.
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