* Toxicology Program in the Department of Environmental and Occupational Health Sciences, and The Department of Pharmacology, University of Washington, Seattle, Washington 98195-7234
3 To whom correspondence should be addressed at Department of Environmental and Occupational Health Sciences, Box 357243, 1705 Pacific Street, University of Washington, HSB, Room F561C, Seattle, WA 98195-7234. Fax: (206) 685-3990. E-mail: zxia{at}u.washington.edu.
Received August 22, 2003; accepted November 5, 2003
ABSTRACT
Chlorpyrifos, an acetylcholinesterase (AChE) inhibitor, is a widely used organophosphate pesticide. Recent concern has focused on its neurotoxicity that is not attributable to AChE inhibition. Here, we report that chlorpyrifos and chlorpyrifos-oxon, but not 3,5,6-trichloro-2-pyridinol (TCP; the breakdown product of chlorpyrifos and chlorpyrifos-oxon), induce apoptosis in primary cortical neurons cultured from embryonic day 17 or newborn rats. It is generally agreed that chlorpyrifos-oxon is approximately three orders of magnitude more potent than chlorpyrifos in inhibition of brain acetylcholinesterase activity. However, our data demonstrate that chlorpyrifos-oxon is only slightly more potent than chlorpyrifos in inducing apoptosis. This indicates that chlorpyrifos-induced apoptosis may occur independently of AChE inhibition, although AChE activity was not measured in this study. Furthermore, chlorpyrifos activates the ERK1/2 and p38 MAP kinases. Surprisingly, blocking ERK1/2 activation by the MEK inhibitor SL327 caused a small but statistically significant inhibition of apoptosis, while blocking p38 with SB202190 significantly accelerated apoptosis induced by chlorpyrifos. This suggests a pro- and anti-apoptotic role for ERK1/2 and p38, respectively. Although chlorpyrifos did not stimulate total JNK activity, it caused a sustained activation of a sub-pool of JNK in the nucleus and stimulated phosphorylation of c-Jun, a downstream target of JNK. Transient expression of a dominant negative c-Jun mutant inhibited chlorpyrifos-induced apoptosis, suggesting a role for JNK and JNK-mediated transcription in this cell death. Together, our data suggest apoptosis as a novel toxic endpoint of chlorpyrifos neurotoxicity in the brain that may be independent of AChE inhibition. Furthermore, activation of the ERK1/2 and JNK MAP kinases contributes to, while activation of the p38 MAP kinase counteracts chlorpyrifos-induced apoptosis in cortical neurons.
Key Words: chlorpyrifos; apoptosis; cortical neuron; CNS; MAP kinase; p38; c-Jun; ERK1/2; JNK; SAPK; SL327; SB202190.
In multicellular organisms, normal development and homeostasis in adults require a carefully regulated balance between pathways that stimulate cell proliferation and cell death. Apoptosis is a form of programmed cell death and inappropriate apoptosis may contribute to various neurodegenerative conditions including Parkinson's disease and Alzheimer's disease. Recent studies suggest that environmental toxicants including pesticides may contribute to the development of several neurodegenerative disorders including Parkinson's disease and amyotrophic lateral sclerosis (Nelson, 1995). Epidemiological studies have established an association between increased risk for Parkinson's disease and exposure to pesticides including the chlorpyrifos-family organophosphates (Checkoway and Nelson, 1999
; Di Monte et al., 2002
; Le Couteur et al., 1999
). Furthermore, pesticide exposure impairs learning and memory in children (Guillette et al., 1998
) and affects hippocampus-dependent spatial memory in rodents (Levin et al., 2001
, 2002
; Terry et al., 2003
). In the developing brain, changes in the amount of apoptosis after exposure to environmental toxicants could cause structural changes that in turn lead to developmental deficits in brain function, including learning and memory. Hence, mechanistic studies of pesticide-induced neuronal apoptosis may provide valuable new information concerning the molecular basis of pesticide-induced apoptosis in neurons and the role of environmental toxicants in neurodegeneration. Furthermore, these studies may also provide insight regarding the impact of pesticide exposure on children's health and central nervous system (CNS) development, and suggest treatments to counteract the neurotoxicity of pesticides.
Chlorpyrifos (O, O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate), an organophosphate pesticide, is widely used throughout the world. Prior to, and possibly even since, the recent agreement between the major manufacturer, Dow AgroSciences, and the EPA limiting the use of chlorpyrifos, it was the most widely used pesticide in the United States. Its primary target of toxicity is the CNS. Because of the large number of humans that are exposed to chlorpyrifos, we used chlorpyrifos as a model pesticide to investigate the role of pesticide exposure in the induction of neuronal apoptosis and to elucidate the underlying molecular mechanisms.
Chlorpyrifos is a well-known acetylcholinesterase (AChE) inhibitor. However, recent concern has focused on its neurotoxicity independent of AChE inhibition (Auman et al., 2000; Crumpton et al., 2000b
). In this study, we investigated whether chlorpyrifos induces apoptosis in primary cultured CNS neurons. We chose primary cultured cortical neurons from newborn rats as our in vitro model system because the neocortex is essential to cognition as well as learning and memory. Furthermore, neurons in this region are frequently damaged during neurodegeneration.
Several signaling pathways have been implicated in the regulation of neuronal apoptosis (Hetman and Xia, 2000). These include the extracellular signal-regulated protein kinase (ERK) 1/2, the c-Jun NH2-terminal protein kinase (JNK), and the p38 MAP kinase. ERK1/2 are preferentially activated by growth factors and neurotrophic factors, while JNK and p38 are preferentially activated by cell stress-inducing signals, such as oxidative stress, environmental stress, and toxic chemical insults (Davis, 2000
). Using nerve growth factor (NGF)-withdrawal induced PC12 cell apoptosis as a model, it was discovered that activation of the JNK and p38 MAP kinases induce apoptosis while the ERK1/2 protect against this apoptosis (Xia et al., 1995
). It has been hypothesized that the balance between growth factor activated survival pathways and stress-activated death pathways determines whether a cell survives or dies (Xia et al., 1995
). Although transient activation of JNK or p38 has been implicated in proliferation and even cellular survival, many subsequent studies have confirmed an anti-apoptotic role for ERK1/2 and a pro-apoptotic role for sustained activation of p38 and JNK (Figueroa-Masot et al., 2001
; Hetman and Xia, 2000
; Ip and Davis, 1998
; Kawasaki et al., 1997
; Namgung and Xia, 2000
; Yang et al., 1997
).
In the present study, an in vitro primary neuron culture system was used to assess whether exposure to chlorpyrifos or its metabolites could induce apoptosis and to elucidate the underlying signaling mechanisms. Our data indicate that chlorpyrifos and chlorpyrifos-oxon, but not TCP, induce neuronal apoptosis. Furthermore, we implicate several MAP kinase signaling pathways in chlorpyrifos-induced neurotoxicity.
MATERIALS AND METHODS
Primary cortical neuron cultures.
Cortical neurons were prepared from newborn (P0) or embryonic day 17/18 (E17/18) Sprague-Dawley rats (B&K, Kent, WA) as described (Xia et al., 1996) with modifications. Briefly, dissociated cortical neurons were plated at a density of 2 x 106 cells per 35 mm plate, and cultured in Neurobasal A (Gibco BRL, Rockville, MD) medium supplemented with 2% B27 (Gibco BRL), 25 mM glutamate, 18 mM glucose, 0.5 mM L-glutamine, 50 U/ml penicillin, and 0.05 mg/ml streptomycin in a humidified incubator with 5% CO2 at 37°C. Half of the culture medium was changed every 2-3 days with fresh medium excluding glutamate. Plates and glass coverslips (Bellco, Vineland, NJ) were coated with poly-D-lysine and laminin (Collaborative Biomedical Product, Bedford, MA).
Drug treatment.
Cortical neurons were cultured for 5-7 days before chlorpyrifos, chlorpyrifos-oxon, or TCP exposure. Chlorpyrifos (99.2% pure) and chlorpyrifos-oxon (96% pure; ChemService, Inc., West Chester, PA) were dissolved in ethanol and freshly prepared for each experiment. TCP (98% pure; ChemService, Inc.) was dissolved in ethanol to a stock solution of 100 mM. Aliquots of TCP were then diluted in media and pH was adjusted with 1N NaOH before treating cells. All treatments were one-time, single-dose exposures. All compounds were handled in a chemical hood. SL327 (DuPont Pharmaceuticals Company) and SB202190 (CalBiochem, La Jolla, CA) were added to the culture 1 h before chlorpyrifos exposure.
MTT metabolism assay.
This was done as described (Hetman et al., 1999).
Quantitation of apoptosis by nuclear morphological changes.
To visualize nuclear morphology, cells were fixed in 4% paraformaldehyde and stained with 2.5 µg/ml of the DNA dye Hoechst 33258 (bis-benzimide; Sigma, St. Louis, MO). Apoptosis was quantitated by scoring the percentage of apoptotic cells in the adherent cell population. Uniformly stained nuclei were scored as healthy, viable neurons. Condensed or fragmented nuclei were scored as apoptotic. In order to obtain unbiased counting, slides were coded and cells were scored blind without knowledge of their prior treatment.
Transient transfection of cortical neurons, detection of transfected cells, and quantitation of apoptosis in transfected cells.
Cortical neurons were transiently transfected on the third day in vitro (DIV 3) using a calcium-phosphate co-precipitation protocol as described (Hetman et al., 1999; Namgung and Xia, 2000
). Neuron cultures were always cotransfected with an expression vector encoding enhanced green fluorescent protein (eGFP) as a marker for transfected cells (Hetman et al., 1999
; Xia et al., 1995
). Neuron cultures were treated with chlorpyrifos at one day after transfection for 24 h and cells were harvested by fixation in a solution containing 4% paraformaldehyde and 4% sucrose. Transfected cells were identified by eGFP fluorescence. Apoptosis in transfected cells was assayed by nuclei fragmentation and condensation after staining with Hoechst 33258 (2.5 µg/ml; Hetman et al., 1999
; Xia et al., 1995
). Transfected cells were scored blind for apoptosis under a fluorescence microscope at the single neuron level. The percentage of apoptotic cells in the total transfected cell population was quantitated.
Western analysis.
This was done as described (Figueroa-Masot et al., 2001). Anti-ERK2 antibody and anti-phospho specific antibodies for ERK1/2, JNK and p38 were purchased from Promega, anti-phospho c-Jun antibody from Cell Signaling Technology (Beverly, MA), anti-JNK1 from Pharmingen, anti-p38 from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-ß-actin from Sigma.
Immunohistochemistry for endogenous phospho-JNK, phospho-c-Jun, and MAP-2 proteins in neurons.
This was done as described (Figueroa-Masot et al., 2001).
Statistical analysis.
Data are presented as means ± SE. Results are from three or more independent experiments. We employed one-way ANOVA for statistical analysis of the data. Data with statistical values of p < 0.05 are generally accepted as statistically significant (Zar, 1996).
RESULTS
Chlorpyrifos Disrupts Mitochondria Function and Induces Apoptosis in Postnatal Cortical Neurons
To examine the neurotoxic effects of chlorpyrifos, cortical neurons prepared from newborn rats (P0) were treated for 24, 48, and 72 h to various concentrations of chlorpyrifos or to vehicle control ethanol (0.015% final concentration; Fig. 1). The neurotoxic effect of chlorpyrifos was first measured using the MTT metabolism assay (Hetman et al., 1999). The MTT assay is based on the colorimetric conversion of the yellow, water-soluble tetrazolium, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the blue, water-insoluble formazan (Mosmann, 1983
). This conversion is catalyzed by cellular mitochondrial dehydrogenases. Because the rate of this reaction is proportional to the number of surviving cells and functional mitochondria, the MTT assay is widely used to quantify viable cells and to assess mitochondrial function (Mosmann, 1983
). Chlorpyrifos reduced MTT metabolism in a dose- and time-dependent manner (Fig. 1), suggesting that chlorpyrifos decreases cortical neuron viability and/or interferes with mitochondrial function.
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Chlorpyrifos-Oxon Induces Apoptosis in Postnatal Cortical Neurons
Chlorpyrifos-oxon is the active metabolite of chlorpyrifos. Because many of the toxic effects of chlorpyrifos have been attributed to chlorpyrifos-oxon inhibition of the brain AChE activity (Ecobichon, 2001), we examined the effect of chlorpyrifos-oxon on P0 cortical neuron apoptosis. P0 cortical neurons were treated with 0 (vehicle control), 20, or 50 µM chlorpyrifos-oxon, and its effect on MTT metabolism or apoptosis examined at 24, 48, and 72 h following exposure. Like chlorpyrifos, chlorpyrifos-oxon reduced MTT metabolism (Fig. 3A) and induced apoptosis (Fig. 3B) in cortical neurons. The dose response and kinetics of MTT metabolism were very similar in neurons treated with chlorpyrifos and chlorpyrifos-oxon. At 50 µM concentrations, chlorpyrifos-oxon did not induce more apoptosis than chlorpyrifos. Although 30 µM chlorpyrifos did not induce an appreciable level of apoptosis, 20 µM chlorpyrifos-oxon was able to induce apoptosis in these neurons from basal level of 29 to 51% at 72 h post treatment. This suggests that at lower concentrations, these neurons are more sensitive to chlorpyrifos-oxon than to the parent compound. Chlorpyrifos-oxon is about three orders of magnitude more potent than chlorpyrifos in inhibition of brain AChE activity (Das and Barone, 1999
; Huff et al., 1994
). Thus, if chlorpyrifos-induced apoptosis is mediated by AChE inhibition, the oxon form should induce apoptosis at concentrations two to three orders of magnitude lower than chlorpyrifos. Because chlorpyrifos-oxon was only slightly more potent than chlorpyrifos in inducing apoptosis, our data suggest that chlorpyrifos-induced apoptosis is unlikely due to AChE inhibition.
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ERK1/2 is Pro-apoptotic in Chlorpyrifos-Induced Cortical Neuron Apoptosis
To determine if various MAP kinase signaling pathways are involved in the regulation of chlorpyrifos-induced apoptosis, we first determined whether they are activated by chlorpyrifos. ERK1/2 activation was assayed by Western analysis using an anti-phospho-ERK1/2 antibody that specifically recognizes activated ERK1/2. Chlorpyrifos (50 µM) induced ERK1/2 phosphorylation (Fig. 5A), indicative of its activation. This activation persisted for 4 h. The importance of ERK1/2 activation for induction of apoptosis was investigated by inhibition of ERK1/2 using SL327. SL327 is a water soluble, structural homologue of the specific MKK1/2 inhibitor U0126 and has been widely used to study neuronal signaling mechanisms (Atkins et al., 1998; Hetman et al., 2002
). MKK1/2 are upstream kinases that phosphorylate and activate ERK1/2 (Lewis et al., 1998
). Treatment with 50 µM SL327 caused a partial but statistically significant (p < 0.005) reduction in chlorpyrifos-induced apoptosis (Fig. 5B). These data suggest that activation of the ERK1/2 signaling pathway contributes to the induction of cortical neuron apoptosis after chlorpyrifos treatment.
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The objectives of this study were to determine if chlorpyrifos induces apoptosis in primary cultured CNS neurons and to elucidate the underlying mechanisms of chlorpyrifos neurotoxicity. Our data indicate that chlorpyrifos and chlorpyrifos-oxon, but not TCP, induce apoptosis in primary cortical neurons. They also reduced MTT metabolism. Furthermore, chlorpyrifos activated ERK1/2, p38, and JNK signaling pathways. Blocking ERK1/2 or JNK signaling inhibited chlorpyrifos-induced apoptosis, suggesting that ERK1/2 and JNK act as pro-apoptotic pathways to mediate chlorpyrifos-induced apoptosis. In contrast, blocking p38 signaling potentiated chlorpyrifos-induced apoptosis, indicating that p38 is activated as a compensatory survival mechanism to counteract chlorpyrifos neurotoxicity.
Much of chlorpyrifos neurotoxicity has been attributed to AChE inhibition via its active metabolite chlorpyrifos-oxon. However, recent studies suggest that additional mechanisms may also be at play. For example, there is evidence to suggest that the effects of chlorpyrifos on the developing brain could be quantitatively and/or qualitatively different from that on adult brain (Atterberry et al., 1997; Won et al., 2001
). Immature animals, although able to recover from AChE inhibition much more rapidly than their adult counterparts, are more sensitive to chlorpyrifos than to chlorpyrifos-oxon (Pope et al., 1991
; Song et al., 1997
; Whitney et al., 1995
). Consequently, recent research has focused on identifying chlorpyrifos toxicity independent of its effect on AChE inhibition (Auman et al., 2000
; Crumpton et al., 2000b
). For example, chlorpyrifos inhibits macromolecule and protein synthesis in PC12 cells that cannot be blocked by muscarinic or nicotinic antagonists (Song et al., 1998
). Our data show that both chlorpyrifos and chlorpyrifos-oxon induce apoptosis in cortical neurons. These results are consistent with a previous report that chlorpyrifos causes mitotic abnormalities and apoptosis in neuroepithelium of cultured rat embryos (Roy et al., 1998
). Chlorpyrifos-oxon is about two to three orders of magnitude more potent than chlorpyrifos in inhibition of brain AChE activity. However, chlorpyrifos-oxon was only slightly more potent than chlorpyrifos in inducing cortical neuron apoptosis. Thus chlorpyrifos-induced apoptosis in primary cortical neurons may occur independently of AChE, although AChE activity was not measured in this study. It will be interesting to investigate if there is any correlation between AChE inhibition and chlorpyrifos-induced apoptosis.
TCP is the major breakdown product of the detoxification of chlorpyrifos and chlorpyrifos-oxon. It is generally thought to be nontoxic. Our results showed that TCP does not induce cortical neuron apoptosis at concentrations up to 130 µM, consistent with reports in the literature that TCP is a relatively nontoxic metabolite. However, because TCP may selectively accumulate in fetal brain (Hunter et al., 1999), our data do not exclude a role for TCP in mediating the developmental neurotoxicity of chlorpyrifos.
Interestingly, chlorpyrifos treatment decreased MTT metabolism before it caused nuclear morphological changes characteristic of apoptosis. Many apoptotic insults cause changes in nuclear morphology before they have a detectable effect on MTT metabolism because early apoptotic cells maintain mitochondria function and MTT metabolism (Hetman et al., 1999; Namgung and Xia, 2000
; Thompson, 1995
). However, for some other insults, disruption of mitochondrial potential is an early event and precedes nuclear changes (Dumont et al., 1999
; Kroemer et al., 1997
; Stridh et al., 1998
; Zamzami et al., 1996
). Our data suggest that mitochondrial inhibition resulting from chlorpyrifos may be an early biomarker of pesticide exposure, perhaps shifting the balance towards apoptosis.
Chlorpyrifos reduced MTT metabolism in cortical neurons at low concentrations that did not induce apoptosis assayed by nuclear fragmentation and condensation. For example, although low concentrations of chlorpyrifos (< 30 µM) reduced MTT metabolism as early as 24 h, they did not induce apoptosis even after 72 h treatment. Therefore, chlorpyrifos may interfere with mitochondrial function at low concentrations without causing apoptosis, and MTT metabolism may be an early biomarker for chlorpyrifos toxicity. Although MTT metabolism is generally used to assay for cell viability, our data open the possibility that MTT metabolism may not always correlate with cell viability because a reduction in MTT metabolism may occur without an ultimate decrease in cell viability. For instance, mitochondrial function may be repaired without losing cell viability. This is exemplified in glutamate treated cells in which neurons surviving the early necrotic phase recover from mitochondrial potential and energy levels (Ankarcrona et al., 1995). Alternatively, chlorpyrifos may induce an early phase of necrosis, thus the decrease in MTT metabolism in a subpopulation of the neurons. Those neurons surviving the necrotic phase may die by apoptosis at higher concentration. This latter case is less likely because we did not observe morphological changes associated with necrosis when cells were treated with chlorpyrifos at concentrations lower than 30 µM. Regardless, apoptosis induced by chlorpyrifos treatment is one of the major and novel manifestations of chlorpyrifos toxicity.
There are few or no data regarding the relative distribution or concentration of chlorpyrifos in human brain after exposure. In the literature, 50-150 µM chlorpyrifos has been routinely used to study chlorpyrifos toxicity (Crumpton et al., 2000a; Dam et al., 1999
; Jett and Navoa, 2000
; Roy et al., 1998
). In this study, we used 30-80 µM (10.5-28 µg/ml) chlorpyrifos, concentrations consistent with most of the previous studies examining chlorpyrifos toxicity. Interestingly, co-exposure of chlorpyrifos with rotenone synergistically induced neuronal apoptosis in a neuronal cell line (Wang and Xia, unpublished observation), suggesting that chlorpyrifos may synergize with other pesticides to induce neuronal apoptosis.
Our data showed that chlorpyrifos and its oxon metabolite induce apoptosis in cortical neurons. Because chlorpyrifos is a widely used pesticide and apoptosis plays an important role in the pathology of many forms of neurodegeneration, our results support the hypothesis that pesticide exposure may increase the risk of neurodegeneration. Because the brains of newborn rats are developmentally equivalent to the human brain in the third trimester, and E17 neurons are even more sensitive than P0 neurons to chlorpyrifos-induced apoptosis, fetuses whose mothers are slow metabolizers of chlorpyrifos may be at an increased risk of toxicity from chlorpyrifos-induced apoptosis. Furthermore, changes in the amount of apoptosis in the developing brain could cause structural changes that in turn could lead to developmental deficits in many areas of the brain function, including learning and memory. Thus our data may have implications on early developmental exposure of pesticides on children's learning and memory.
It is interesting that chlorpyrifos exposure caused sustained activation of three families of MAP kinases, the ERK1/2, the p38, and the JNK. Activation of ERK1/2 usually protects neurons from apoptosis, while sustained activation of p38 and JNK by stress stimuli often causes neuron cell death (Figueroa-Masot et al., 2001; Hetman et al., 1999
; Hetman and Xia, 2000
; Ip and Davis, 1998
; Kawasaki et al., 1997
; Namgung and Xia, 2000
; Xia et al., 1995
; Yang et al., 1997
). Surprisingly, here we report that blocking ERK1/2 activation protected neurons from, while blocking p38 accelerated apoptosis induced by chlorpyrifos. These data suggest that ERK1/2 contribute to chlorpyrifos-induced apoptosis. Furthermore, to our knowledge, this is the first evidence that p38 is activated by an insult as a compensatory mechanism.
Although chlorpyrifos did not induce JNK phosphorylation when total cell lysates were used for Western analysis, it increased nuclear JNK phosphorylation, indicative of nuclear JNK activation. Moreover, chlorpyrifos stimulated c-Jun phosphorylation, providing additional evidence for nuclear JNK activation. Expression of a dominant negative c-Jun inhibited chlorpyrifos-induced apoptosis. These data suggest that nuclear JNK activation and JNK stimulated, c-Jun-mediated transcription play a critical role in chlorpyrifos-induced cortical neuron apoptosis. The fact that a distinct pool of JNK is activated in the nucleus by chlorpyrifos is reminiscent to nuclear JNK activation by taxol (Figueroa-Masot et al., 2001). These results suggest that specific nuclear JNK activation may be a general mechanism of insult-induced apoptosis in CNS neurons.
In summary, we discovered that chlorpyrifos induces apoptosis and reduces mitochondrial function of cortical neurons. This apoptosis may be independent of AChE inhibition and is regulated by a complex of MAP kinases including ERK1/2, JNK, and p38. Activation of these MAP kinases may also underlie other aspects of chlorpyrifos neurotoxicity in addition to apoptosis. Taken together, our results indicate apoptosis and perturbation of mitochondrial function as novel toxic endpoints of chlorpyrifos-induced neurotoxicity, which may have implications for pesticide exposure in humans both during development and in neurodegeneration in adults.
ACKNOWLEDGMENTS
We thank DuPont Pharmaceuticals Company for providing us with SL327. This work was supported by APP#3010 from Burroughs Wellcome Fund for New Investigator Award in Toxicology (Z.X.), the UW NIEHS sponsored Center for Ecogenetics and Environmental Health (NIEHS P30ES07033), grants NS44069 and EPA-R826886/ES09601.
NOTES
1 These authors contributed equally to this work.
2 Present address: Department of Oriental Medicine, College of Oriental Medicine, Daejeon University, South Korea.
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