Alterations in central nervous system serotonergic and dopaminergic synaptic activity in adulthood after prenatal or neonatal chlorpyrifos exposure.
Exposure to chlorpyrifos (CPF (Control Program Facility) The IBM System/38 operating system that included an integrated relational DBMS. ) alters neuronal development of serotonin (5HT) and dopamine dopamine (dōp`əmēn), one of the intermediate substances in the biosynthesis of epinephrine and norepinephrine. See catecholamine.
One of the catecholamines, widely distributed in the central nervous system. systems, and we recently found long-term alterations in behaviors related to 5HT function. To characterize the synaptic synaptic /syn·ap·tic/ (si-nap´tik)
1. pertaining to or affecting a synapse.
2. pertaining to synapsis.
Of or relating to synapsis or a synapse. mechanisms underlying these effects, we exposed developing rats to CPF regimens below the threshold for systemic toxicity, in three treatment windows: gestational days (GD) 17-20, postnatal postnatal /post·na·tal/ (-na´t'l) occurring after birth, with reference to the newborn.
Of or occurring after birth, especially in the period immediately after birth. days (PN) 1-4, or PN11-14. In early adulthood (PN60), we assessed basal neurotransmitter content and synaptic activity (turnover) in brain regions containing the major 5HT and dopamine projections. CPF exposure on GD17-20 or PN 1-4 evoked long-term increases in 5HT turnover across multiple regions; the effects were not secondary to changes in neurotransmitter content, which was unaffected or even decreased. When the treatment window was shifted to PN11-14, there were no long-term effects. Dopamine turnover also showed significant increases after CPF exposure on GD17-20, but only when the dose was raised above the threshold for overt toxicity; however, hippocampal hip·po·cam·pus
n. pl. hip·po·cam·pi
A ridge in the floor of each lateral ventricle of the brain that consists mainly of gray matter and has a central role in memory processes. dopamine content was profoundly subnormal subnormal /sub·nor·mal/ (-nor´m'l) below normal.
below or less than normal. after exposures below or above the acute, toxic threshold, suggesting outright neurotoxicity neurotoxicity /neu·ro·tox·ic·i·ty/ (noor?o-tok-sis´it-e) the quality of exerting a destructive or poisonous effect upon nerve tissue. . These results indicate that, in a critical developmental period, apparently nontoxic exposures to CPF produce lasting activation of 5HT systems in association with 5HT-associated behavioral anomalies. Key words: brain development, chlorpyrifos, dopamine, organophosphate pesticides, serotonin. doi:10.1289/ehp.7968 available via http://dx.doi.org/[Online 28 April 2005]
The organophosphorus or·gan·o·phos·pho·rus
organ·o·phos pesticide chlorpyrifos (CPF) remains in use throughout the world despite its recent curtailment in the United States [U.S. Environmental Protection Agency Environmental Protection Agency (EPA), independent agency of the U.S. government, with headquarters in Washington, D.C. It was established in 1970 to reduce and control air and water pollution, noise pollution, and radiation and to ensure the safe handling and (EPA EPA eicosapentaenoic acid.
n.pr See acid, eicosapentaenoic.
n. ) 2000, 2002]. The major concern for CPF is its propensity to elicit developmental neurotoxicity at exposure levels below the threshold for systemic toxicity, such that damaging exposures of pregnant women and children may go undetected because of the lack of symptoms (Landrigan 2001; Landrigan et al. 1999; May 2000; Physicians for Social Responsibility 1995; Pope 1999; Slotkin 1999, 2004; Weiss et al. 2004). CPF was originally thought to exert adverse effects on brain development through the same mechanism by which it elicits systemic toxicity, namely, inhibition of cholinesterase cholinesterase /cho·lin·es·ter·ase/ (-es´ter-as) serum cholinesterase, pseudocholinesterase; an enzyme that catalyzes the hydrolytic cleavage of the acyl group from various esters of choline and some related compounds; determination of through its active metabolite, CPF oxon, but it is now evident that other, noncholinergic mechanisms participate in its developmental neurotoxicity (Barone et al. 2000; Clegg and van Gemert 1999a, 1999b; Gupta 2004; Mileson et al. 1998; Pope 1999; Slotkin 1999, 2004). Even at exposures below the threshold for cholinesterase inhibition, CPF itself disrupts the patterns of neural cell replication and differentiation, axonogenesis and synaptogenesis, and the functional development of neurotransmitter and neurotrophin systems, culminating in aberrant behavioral performance (Barone et al. 2000; Casida and Quistad 2004; Gupta 2004; Pope 1999; Qiao et al. 2002, 2003; Yanai et al. 2002). Consequently, CPF-induced damage extends beyond cholinergic cholinergic /cho·lin·er·gic/ (ko?lin-er´jik)
1. parasympathomimetic; stimulated, activated, or transmitted by choline (acetylcholine); said of the sympathetic and parasympathetic nerve fibers that liberate acetylcholine at a pathways to include other neurotransmitter systems, notably the monoamines, norepinephrine norepinephrine (nôr'ĕpīnĕf`rən), a neurotransmitter in the catecholamine family that mediates chemical communication in the sympathetic nervous system, a branch of the autonomic nervous system. , dopamine (DA), and serotonin (5HT), and recent studies show that 5HT systems are especially sensitive (Aldridge et al. 2003, 2004, 2005a, 2005b; Raines et al. 2001; Sachana et al. 2001).
In recent work we found that, during a critical developmental window centered around the immediate perinatal period, CPF elicits immediate and persistent effects on expression of 5HT receptors and the 5HT transporter, biomarkers for 5HT synaptic development and integrity (Aldridge et al. 2003, 2004, 2005a; Raines et al. 2001). Additionally, we found that the same exposures elicited lasting changes in 5HT-related behaviors, resembling animal models of depression (Aldridge et al. 2005a). Notably, these studies were conducted spanning CPF exposures below the threshold for observable toxicity and, more important, could be detected at doses devoid of cholinesterase inhibition in the developing brain (Qiao et al. 2002). In the present study, we assessed 5HT levels and turnover, a measure ofsynaptic activity, in adulthood after prenatal or neonatal CPF exposure in order to provide a mechanistic link between effects on static biomarkers of 5HT synaptic development and altered behavior. Measurements were conducted using the metabolite metabolite, organic compound that is a starting material in, an intermediate in, or an end product of metabolism. Starting materials are substances, usually small and of simple structure, absorbed by the organism as food. ratio method (Slotkin et al. 2000; Xu et al. 2001), assessing the relative amounts of 5HT and its metabolite 5-hydroxyindole-acetic acid (5HIAA HIAA,
n.pr the abbreviation for Health Insurance Association of America. ), with turnover calculated as the 5HIAA:5HT ratio. We examined brain regions with prominent 5HT innervation innervation /in·ner·va·tion/ (in?er-va´shun)
1. the distribution or supply of nerves to a part.
2. the supply of nervous energy or of nerve stimulation sent to a part. that are known to be targets for CPF (Aldridge et al. 2003, 2004, 2005b; Raines et al. 2001). CPF exposure was carried out during the immediate perinatal period, the phase in which the receptor and transporter biomarkers indicate high susceptibility, or at a later postnatal development period in which these effects wane: exposure on gestational days (GD) 17-20 and postnatal days (PN) 1-4 and PN11-14 (Aldridge et al. 2003, 2004, 2005b; Raines et al. 2001). Finally, effects on 5HT systems were contrasted to those on DA levels and turnover, again using the metabolite ratio method (Slotkin et al. 2000; Xu et al. 2001), assessing dihydroxyphenylacetic acid (DOPAC DOPAC 3,4-Dihydroxy-Phenylacetic Acid
DOPAC Disk-Oriented Property and Costing System (Bellcore) ) and homovanillic acid (HVA HvA Hogeschool Van Amsterdam
HVA Hauptverwaltung Aufklärung (East German Foreign Intelligence)
HVA Hazard Vulnerability Analysis
HVA Huron Valley Ambulance (Michigan)
HVA Heating, Ventilation and Air ), calculating the ratio as [DOPAC + HVA]:DA.
Materials and Methods
Animal treatments. All experiments using live animals were carried out in accordance with the Declaration of Helsinki For the political accords, see .
. There is also another Declaration of Helsinki, dealing with the Information Society. Introduction
The Declaration of Helsinki, was developed by the World Medical Association (World Medical Association 2004) and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated prom·ul·gate
tr.v. prom·ul·gat·ed, prom·ul·gat·ing, prom·ul·gates
1. To make known (a decree, for example) by public declaration; announce officially. See Synonyms at announce.
2. by the National Institutes of Health (Institute of Laboratory Animal Resources 1996). Timed-pregnant Sprague-Dawley rats (Zivic Laboratories, Pittsburgh, PA, USA) were housed in breeding cages, with a 12-hr light/dark cycle and with free access to food and water. CPF (Chem Service, West Chester, PA, USA) was dissolved in dimethyl sulfoxide to provide consistent absorption (Whitney et al. 1995) and was injected subcutaneously in a volume of 1 mL/kg body weight; control animals received equivalent dimethyl sulfoxide vehicle injections.
For exposure on GD17-20, dams were injected daily with CPF at 1 and 5 mg/kg body weight, doses that span the threshold for inhibition of fetal brain cholinesterase activity, fetal growth impairment, and reduced maternal weight gain, all of which become evident at [greater than or equal to] 5 mg/kg (Garcia et al. 2003; Qiao et al. 2002). On the day of birth, all pups were randomized ran·dom·ize
tr.v. ran·dom·ized, ran·dom·iz·ing, ran·dom·iz·es
To make random in arrangement, especially in order to control the variables in an experiment. within their respective treatment groups and redistributed to the dams with a litter size of 10 to maintain a standard nutritional status. Randomization randomization (ranˈ·d·m was repeated at intervals of 3 days, and in addition, within each treatment group, dams were rotated among litters to distribute any maternal care-taking differences randomly. Animals were weaned on PN21. On PN60, one male and one female were selected from each litter and were decapitated de·cap·i·tate
tr.v. de·cap·i·tat·ed, de·cap·i·tat·ing, de·cap·i·tates
To cut off the head of; behead.
[Late Latin d . The cerebellum cerebellum (sĕr'əbĕl`əm), portion of the brain that coordinates movements of voluntary (skeletal) muscles. It contains about half of the brain's neurons, but these particular nerve cells are so small that the cerebellum accounts for , including flocculi flocculi (flŏk`yəlī'): see chromosphere. , was removed, and the forebrain forebrain: see brain. was separated from the midbrain midbrain: see brain. and brainstem by a cut made rostral rostral /ros·tral/ (ros´tral)
1. pertaining to or resembling a rostrum; having a rostrum or beak.
2. situated toward a rostrum or toward the beak (oral and nasal region), which may mean superior (in relationships to the thalamus thalamus (thăl`əməs), mass of nerve cells centrally located in the brain just below the cerebrum and resembling a large egg in size and shape. . The forebrain was then further dissected into the regions containing major 5HT projections (cerebral cortex, hippocampus hippocampus
fabulous marine creature; half fish, half horse. [Rom. Myth. and Art: Hall, 154]
See : Monsters , striatum striatum /stri·a·tum/ (stri-a´tum) corpus striatum.stria´tal
n. pl. stri·a·ta ), and the midbrain and brainstem, which contain the preponderance of 5HT cell bodies, were separated from each other. The cerebellum, which is sparse in 5HT neurons or projections, was not evaluated. Tissues were frozen with liquid nitrogen and stored at -45[degrees]C.
For studies of CPF effects in the first few days after birth, animals were given a subcutaneous injection of 1 mg/kg daily on PN1-4; for studies in older animals, which tolerate higher doses (Campbell et al. 1997; Pope and Chakraborti 1992; Pope et al. 1991; Whitney et al. 1995), daily treatment with 5 mg/kg was given on PN11-14. The same randomization procedure was followed as described for prenatal CPF treatment. Neither regimen evoked weight loss or mortality (Campbell et al. 1997; Dam et al. 1998; Johnson et al. 1998; Song et al. 1997). Samples were obtained on PN60 as described above.
Assays. Tissues were thawed and homogenized ho·mog·e·nize
v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es
1. To make homogeneous.
a. To reduce to particles and disperse throughout a fluid.
b. in ice-cold 0.1 M perchloric acid and sedimented for 20 min at 18,000 x g. The supernatant supernatant /su·per·na·tant/ (-na´tant) the liquid lying above a layer of precipitated insoluble material.
the liquid lying above a layer of precipitated insoluble material. solution was collected, and aliquots were used for analysis of 5HT, 5HIAA, DA, HVA, and DOPAC by HPLC HPLC high-performance liquid chromatography.
high performance liquid chromatography.
HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed with electrochemical electrochemical /elec·tro·chem·i·cal/ (-kem´i-k'l) pertaining to interaction or interconversion of chemical and electrical energies.
adj. detection (Slotkin et al. 2000; Xu et al. 2001). Concurrently run standards, containing each of the neurotransmitters and metabolites Metabolites
Substances produced by metabolism or by a metabolic process.
Mentioned in: Interactions (Sigma Chemical Co., St. Louis, MO, USA), were used to calculate the regional content of each neurochemical neu·ro·chem·is·try
The study of the chemical composition and processes of the nervous system and the effects of chemicals on it.
neu . DOPAC and HVA levels in the hippocampus were too low for accurate measurement. Transmitter levels were calculated as nanograms per region, and, because there were no treatment-related differences in brain region weights, the same effects were obtained with calculation as nanograms per gram of tissue or per milligram milligram /mil·li·gram/ (mg) (mil´i-gram) one thousandth (10-3) of a gram.
n. Abbr. mg
A metric unit of mass equal to one thousandth (10-3) of a gram. of protein (data not shown). Similarly, because 5HT and DA turnover were calculated as ratios of metabolites to transmitter, the values are independent of whether region, tissue weight, or protein concentration is used as the denominator term.
Data analysis. Each treatment group comprised six males and six females. We compiled data as means [+ or -] SEs, and treatment-related differences were assessed by analysis of variance (ANOVA anova
see analysis of variance.
ANOVA Analysis of variance, see there ), with data log-transformed because of heterogeneous variance, incorporating factors of treatment (control, CPF), brain region, and sex. Where the initial test identified interactions of CPF treatment with region, we subdivided the effects into the individual brain regions and conducted lower-order tests to examine treatment and sex differences. Similarly, where ANOVA identified interactions of CPF with sex, we subdivided data to examine males and females separately. After subdivisions, we used Fisher's protected least significant difference to evaluate differences between individual groups. However, where CPF treatment did not interact with other variables, we assessed only the main treatment effect. Significance was assumed at the level of p < 0.05 for main effects; however, for interactions at p < 0.1, we examined lower-order main effects after subdivision of the interactive variables (Snedecor and Cochran 1967).
To facilitate comparison of treatment effects across regions with widely differing transmitter levels or turnovers, some results are presented as the percentage change from control values. However, statistical evaluations were always conducted on the original data. Similarly, for convenience, control values for the three different treatment regimens were combined for presentation, but comparisons of CPF effects were conducted only with the appropriately matched control cohort. For reference, control values appear in Table 1.
In brain regions of control rats, levels of 5HT, DA, and their metabolites showed few sex differences (Table 1). 5HIAA was slightly higher in the striatum and midbrain of females, whereas DA levels were lower in midbrain and brainstem. None of the prenatal or postnatal treatment regimens evoked significant deficits in brain region weights (data not shown).
CPF exposure on GD17-20. Across all brain regions, late gestational exposure to CPF elicited a significant net overall reduction (main treatment effect) in 5HT levels (Figure 1A). However, the effect was seen only at the higher dose (5 mg/kg), which elicits maternal weight deficits and significant fetal brain cholinesterase inhibition (Garcia et al. 2003; Qiao et al. 2002). Superimposed su·per·im·pose
tr.v. su·per·im·posed, su·per·im·pos·ing, su·per·im·pos·es
1. To lay or place (something) on or over something else.
2. on the main treatment effect, there were regional differences: most regions showed a decrease in content, but values increased in the striatum. The pattern of effects on 5HT turnover was unrelated to the effects on 5HT content, with a main treatment effect representing a net increase (Figure 1B). In this case, however, effects were statistically significant at both 1 and 5 mg/kg, thus indicating increased 5HT turnover even at CPF exposures below the threshold for maternal toxicity or fetal brain cholinesterase inhibition. Again, some regions (hippocampus, brainstem) showed much larger changes than the others, particularly compared with the striatum.
[FIGURE 1 OMITTED]
The pattern of effects on DA levels and turnover differed substantially from those seen for 5HT. Although DA content was unaffected in most brain regions, there were massive deficits in the hippocampus with either the low or high dose of CPF (Figure 2A). DA turnover was affected only at the higher dose, with significant elevations in cerebral cortex, striatum, and midbrain (Figure 2B); DA turnover could not be assessed in the hippocampus because of the low levels of DA metabolites.
[FIGURE 2 OMITTED]
CPF exposure on PN1-4. With early postnatal treatment, CPF elicited sex-selective alterations in 5HT content (Figure 3A). Males showed no significant change, whereas females showed a small but statistically significant net decrease. Again, the effects on 5HT turnover appeared to be unrelated to changes in 5HT content, displaying a net increase that was not sex selective and with the smallest magnitude of effect in the striatum (Figure 3B).
[FIGURE 3 OMITTED]
Effects on DA levels and turnover after CPF exposure on PN1-4 have appeared previously (Slotkin et al. 2002), exhibiting decreases in cerebrocortical DA content and turnover, increases in both parameters in the striatum, and increased turnover in the midbrain.
CPF exposure on PN11-14. Despite the use of a higher dose (5 mg/kg compared with 1 mg/kg on PN1-4) and the resultant greater degree of cholinesterase inhibition (Qiao et al. 2002; Song et al. 1997), CPF exposure during this later postnatal period had no significant effect on either 5HT content (Figure 4A) or turnover (Figure 4B). Earlier work for DA similarly found no alterations in either levels or turnover (Slotkin et al. 2002).
[FIGURE 4 OMITTED]
In earlier work, we found that CPF exposure during a discrete developmental period in late gestation and early neonatal life elicits lasting up-regulation of the expression of 5HT synaptic proteins, including 5H[T.sub.1A] and 5H[T.sub.2] receptors and the presynaptic presynaptic /pre·syn·ap·tic/ (-si-nap´tik) situated or occurring proximal to a synapse.
Relating to the area on the proximal side of a synaptic gap. , high-affinity 5HT transporter (Aldridge et al. 2004). The present results indicate that these changes in static 5HT biomarkers are associated with functional changes in 5HT levels, and, more important, in 5HT turnover, a dynamic index of synaptic activity. Moreover, the critical period for the alterations of transmitter turnover seen here corresponds precisely to that identified previously for the protein biomarkers, with closure of the window of vulnerability A window of vulnerability or wov is a time frame within which defensive measures are reduced, compromised or lacking.
The term is used with reference to military defences of strategic assets, and also by analogy in computer software to a software vulnerability which is open by the second postnatal week. In both cases, it is of critical importance that the lasting effects on 5HT function are elicited at CPF exposures below the threshold for systemic toxicity and even below that required for inhibition of fetal brain cholinesterase. The main findings here thus reinforce the idea that cholinesterase inhibition per se is an inadequate indicator of thresholds for CPF-induced developmental neurotoxicity.
The increase in 5HT turnover evoked by CPF exposure on either GD17-20 or PN1-4 is indicative of presynaptic hyperactivity, a particularly striking finding in light of the up-regulation of 5HT receptors evoked by the same treatments (Aldridge et al. 2004). Ordinarily, increased 5HT concentrations in the synapse can be expected to evoke receptor down-regulation as a compensation for over-stimulation. As an initial impression, our finding of global presynaptic/postsynaptic up-regulation (receptors, transporter, turnover) might then suggest an overwhelming increase in 5HT function. However, evaluations of 5HT-linked behaviors and cell signaling both indicate quite the opposite, namely, deficiencies in 5HT function (Aldridge et al. 2004, 2005a). Accordingly, "miswiring" of 5HT circuits provides the most likely explanation for the effects of CPF on developing 5HT systems. If 5HT projections do not innervate in·ner·vate
1. To supply an organ or a body part with nerves.
2. To stimulate a nerve, muscle, or body part to action. the appropriate postsynaptic postsynaptic /post·sy·nap·tic/ (-si-nap´tik) distal to or occurring beyond a synapse.
Situated behind or occurring after a synapse. cells, then receptor up-regulation will occur because of the lack of neurotransmitter stimulation, and at the same, presynaptic neurons will be hyperactive as an attempt to compensate for the de facto lack of 5HT effect. With miswiring, these compensations cannot overcome the underlying architectural problem, leading to behavioral deficits resembling 5HT deficiencies as seen in animal models of depression (Aldridge et al. 2005a). Indeed, these types of circuitry defects also occur with prenatal xenobiotic xen·o·bi·ot·ic
Foreign to the body or to living organisms. Used of chemical compounds.
A xenobiotic chemical.
any substance, harmful or not, that is foreign to the animal's biological system. insults that target cholinergic systems. Both phenobarbital phenobarbital /phe·no·bar·bi·tal/ (fe?no-bahr´bi-tal) a long-acting barbiturate, used as the base or sodium salt as a sedative, hypnotic, and anticonvulsant.
n. and heroin disrupt the regional architecture of acetylcholine acetylcholine (əsēt'əlkō`lēn), a small organic molecule liberated at nerve endings as a neurotransmitter. It is particularly important in the stimulation of muscle tissue. projections in the hippocampus, leading to parallel increases in cholinergic receptors, acetylcholine release, and expression of the presynaptic choline choline: see vitamin.
Organic compound related to vitamins in its activity. It is important in metabolism as a component of the lipids that make up cell membranes and of acetylcholine. transporter, accompanied by behavioral and signaling defects that are not rectified by the combined presynaptic/ postsynaptic up-regulation (Steingart et al. 1998; Vatury et al. 2004; Yanai et al. 2004). If this interpretation is correct, deficits of 5HT function elicited by developmental exposure to CPF will also prove to be refractory to drugs that typically restore 5HT function, such as the 5HT reuptake reuptake /re·up·take/ (re-up´tak) reabsorption of a previously secreted substance.
n. inhibitors, resulting in drug-resistant depression. This possibility certainly deserves future examination.
Our findings also support the idea that CPF is frankly neurotoxic neurotoxic
pertaining to or emanating from a neurotoxin.
a case of poisoning by a neurotoxin.
neurotoxic adjective in the developing brain. We found decrements of 5HT levels in both males and females given the higher CPF dose on GD17-20 and also for the PN1-4 regimen in females. Nevertheless, the effects on 5HT turnover occurred at a dose below that required for loss of transmitter, suggesting that the miswiring effect is distinct from outright neurotoxicity. The effects on 5HT levels were also evident in the hippocampus, a region that we found in earlier work to be highly targeted for CPF-induced cellular damage and similar defects in cholinergic projections (Qiao et al. 2003, 2004; Slotkin et al. 2001). In keeping with this, the present results showed prominent loss of hippocampal DA even at 1 mg/kg, a dose devoid of maternal or fetal toxicity or fetal cholinesterase inhibition (Garcia et al. 2003; Qiao et al. 2002). In previous work we found only minor effects on DA levels with CPF treatment on PN1-4, and no such effects with the PN11-14 regimen (Slotkin et al. 2002), indicating early neonatal closing of the window of vulnerability for these particular neurotoxic events. Although hippocampal DA levels are lower than in the other regions, the loss of these projections is likely to play an important role in behavioral alterations. DA projections provide important regulatory inputs to hippocampal cholinergic synapses, with consequent effects on learning and memory (Yanai et al. 1993). Like the effects on 5HT turnover, we also found increased DA turnover after prenatal CPF exposure, with closure of the window of vulnerability in the second postnatal week (Slotkin et al. 2002). The effects on 5HT thus represent one component of a larger spectrum of CPF-induced disruption of synaptic development and function that ultimately contribute to behavioral anomalies.
There are two additional features of the present results that are worth noting. First, with gestational CPF exposure, the loss of 5HT spared the striatum and similarly, whereas the hippocampus showed 40-50% loss of DA, there was no corresponding striatal decrement To subtract a number from another number. Decrementing a counter means to subtract 1 or some other number from its current value. . Effectively, this regional difference de-emphasizes the importance of oxidative stress as a contributory mechanism for the neurotoxic actions of CPF because the striatum is typically more sensitive to such effects than other regions, largely due to its high content of DA, an oxidative neurotransmitter (Bagchi et al. 1995; Crumpton et al. 2000; Gupta 2004; Karen et al. 2001; Qiao et al. 2005). Indeed, we recently evaluated lipid peroxidation with each of the CPF regimens used here and found little or no evidence for oxidative damage with the GD17-20 or PN1-4 regimens, although lipid peroxidation was readily detected after exposure on PN11-14 (Slotkin et al. 2005). Apparently, other, non-cholinesterase-related actions of CPF are more important contributors to neurobehavioral teratogenicity ter·a·to·ge·nic·i·ty
The capability of producing fetal malformation.
teratogenicity, (terˈ· in the critical perinatal period in which 5HT systems are especially vulnerable (Barone et al. 2000; Casida and Quistad 2004; Gupta 2004; Pope 1999; Qiao et al. 2002, 2003; Yanai et al. 2002), although oxidative stress may be relatively more important for later postnatal exposures. The second interesting characteristic is the relative lack of sex selectivity to the effects on 5HT turnover, whereas effects on behavior and 5HT receptor expression show distinct sex differences (Aldridge et al. 2004, 2005a). In fact, many of the functional consequences of CPF exposure represent the elimination of normal sex differences in synaptic function and behavior, partially masculinizing the patterns in females but feminizing males (Aldridge et al. 2004, 2005a). Accordingly, CPF may interfere with sexual differentiation of the brain, which peaks during the vulnerable perinatal period found here (MacLusky and Naftolin 1981; McCarthy 1994); as a consequence, even where CPF effects on males and females might be the same in regard to alterations of 5HT synaptic activity and/or miswiring of 5HT circuits, the outcome nevertheless can be expressed differently for males and females because of CPF's effects on sexual differentiation. Again, future studies should enable us to establish whether this explanation is correct.
In conclusion, the present results reinforce the concept that developmental CPF exposure, at doses below the threshold for maternal or fetal/neonatal toxicity, and below that required for inhibition of fetal brain cholinesterase, nevertheless causes lasting disruption of 5HT synaptic activity when exposure occurs in a critical developmental window centered around the immediate perinatal period. In combination with earlier studies of static synaptic protein biomarkers and 5HT-related behaviors (Aldridge et al. 2004, 2005a), the increase in 5HT turnover seen here suggests strongly that CPF acts through architectural miswiring of 5HT circuits, resulting in functional deficits despite global up-regulation of presynaptic and postsynaptic sensitivity. The developmental neuroteratogenicity of CPF thus encompasses neurotransmitter systems beyond those involving acetylcholine, and the effects seen here for 5HT and DA are likely to contribute to the emergence of behavioral alterations in adolescence and adulthood (Aldridge et al. 2005a; Carret al. 2001; Dam et al. 2000; Icenogle et al. 2004; Levin et al. 2001, 2002; Ricceri et al. 2003).
Aldridge JE, Levin ED, Seidler FJ, Slotkin TA. 2005a. Developmental exposure of rats to chlorpyrifos leads to behavioral alterations in adulthood, involving serotonergic se·ro·to·ner·gic or se·ro·to·ni·ner·gic
Activated by or capable of liberating serotonin, especially in transmitting nerve impulses.
containing or activated by serotonin. mechanisms and resembling animal models of depression. Environ Health Perspect 113:527-531.
Aldridge JE, Meyer A, Seidler FJ, Slotkin TA. 2005b. Developmental exposure to terbutaline terbutaline /ter·bu·ta·line/ (ter-bu´tah-len) a ß agonist; used as the sulfate salt as a bronchodilator and as a tocolytic in the prevention of premature labor. and chlorpyrifos: pharmacotherapy pharmacotherapy /phar·ma·co·ther·a·py/ (-ther´ah-pe) treatment of disease with medicines.
Treatment of disease through the use of drugs. of preterm labor and an environmental neurotoxicant converge on serotenergic systems in neonatal rat brain regions. Toxicol Appl Pharmacol 203:132-144.
Aldridge JE, Seidler FJ, Meyer A, Thillai I, Slotkin TA. 2003. Serotonergic systems targeted by developmental exposure to chlerpyrifos: effects during different critical periods. Environ Health Perspect 111:1736-1743.
Aldridge JE, Seidler FJ, Slotkin TA. 2004. Developmental exposure to chlorpyrifos elicits sex-selective alterations of serotonergic synaptic function in adulthood: critical periods and regional selectivity for effects on the serotonin transporter, receptor subtypes, and cell signaling. Environ Health Perspect 112:148-155.
Bagchi D, Bagchi M, Hassoun EA, Stohs SJ. 1995. In vitro and in five generation of reactive oxygen species reactive oxygen species,
n molecules and ions of oxygen that have an unpaired electron, thus rendering them extremely reactive. Many cellular structures are susceptible to attack by ROS contributing to cancer, heart disease, and cerebrovascular disease. , DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. damage and lactate dehydrogenase leakage by selected pesticides. Toxicology 104:129-140.
Barone S, Das KP, Lassiter TL, White LD. 2000. Vulnerable processes of nervous system development: a review of markers and methods. Neurotoxicology 21:15-36.
Campbell CG, Seidler FJ, Slotkin TA. 1997. Chlorpyrifos interferes with cell development in rat brain regions. Brain Res Bull 43:179-189.
Carr RL, Chambers HW, Guarisco JA, Richardson JR, Tang J, Chambers JE. 2001. Effects of repeated oral postnatal exposure to chlorpyrifos on open-field behavior in juvenile rats. Toxicol Sci 59:260-267.
Casida JE, Quistad 6B. 2004. Organophosphate organophosphate /or·ga·no·phos·phate/ (or?gah-no-fos´fat) an organic ester of phosphoric or thiophosphoric acid; such compounds are powerful acetylcholinesterase inhibitors and are used as insecticides and nerve gases. toxicology: safety aspects of nonacetylcholinesterase secondary targets. Chem Res Toxicol 17:983-998.
Clegg DJ, van Gemert M. 1999a. Determination of the reference dose for chlorpyrifos: proceedings of an expert panel. J Toxicol Environ Health 2:211-255.
Clegg DJ, van Gemert M. 1999b. Expert panel report of human studies on chlorpyrifos and or other organophosphate exposures. J Toxicol Environ Health 2:257-279.
Crumpton TL, Seidler FJ, Slotkin TA. 2000. Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos? Dev Brain Res 121:189-195.
Dam K, Seidler FJ, Slotkin TA. 1998. Developmental neurotoxicity of chlorpyrifos: delayed targeting of DNA synthesis after repeated administration. Dev Brain Res 108:39-45.
Dam K, Soldier FJ, Slotkin TA. 2000. Chlorpyrifos exposure during a critical neonatal period elicits gender-selective deficits in the development of coordination skills and locomotor activity. Dev Brain Res 121:179-187.
Garcia SJ, Seidler FJ, Slotkin TA. 2003. Developmental neurotoxicity elicited by prenatal or postnatal chlorpyrifos exposure: effects on neurospecific proteins indicate changing vulnerabilities. Environ Health Perspect 111:297-303.
Gupta RC. 2004. Brain regional heterogeneity and toxicological mechanisms of organophosphates and carbamates carbamates
effective insecticides which exert their effect by temporarily inhibiting cholinesterase activity. They are also capable of poisoning. Clinical signs are pupillary constriction, muscle tremor, salivation, ataxia and dyspnea. . Toxicol Mech Meth 14:103-143.
Icenogle LM, Christopher C, Blackwelder WP, Caldwell DP, Qiao D, Seidler FJ, et al. 2004. Behavioral alterations in adolescent and adult rats caused by a brief subtoxic exposure to chlorpyrifos during neurulation Neurulation
The process by which the vertebrate neural tube is formed. The primordium of the central nervous system is the neural plate, which arises at the close of gastrulation by inductive action of the chorda-mesoderm on the overlying ectoderm. . Neurotoxicol Teratol 26:95-101.
Institute of Laboratory Animal Resources. 1996. Guide for the Care and Use of Laboratory Animals. 7th ed. Washington, DC:National Academy Press.
Johnson DE, Seidler FJ, Slotkin TA. 1998. Early biochemical detection of delayed neurotoxicity resulting from developmental exposure to chlorpyrifos. Brain Res Bull 45:143-147.
Karen DJ, Li W, Harp PR, Gillette JS, Bloomquist JR. 2001. Striatal dopaminergic pathways as a target for the insecticides permethrin permethrin /per·meth·rin/ (per-meth´rin) a topical insecticide used in the treatment of infestations by Pediculus humanus capitis, Sarcoptes scabiei, or any of various ticks; also applied to objects such as furniture and bedding. and chlorpyrifos. Neurotoxicology 22:811-817.
Landrigan PJ. 2001. Pesticides and polychlorinated biphenyls (PCBs): an analysis of the evidence that they impair children's neurobehavioral development. Mol Genet genet: see civet. Metab 73:11-17.
Landrigan PJ, Claudio L, Markowitz SB, Berkowitz GS, Brenner BL, Romero H, et al. 1999. Pesticides and inner-city children: exposures, risks, and prevention. Environ Health Perspect 107(suppl 3):431-437.
Levin ED, Addy N, Baruah A, Elias A, Christopher NC, Soldier FJ, et al. 2002. Prenatal chlorpyrifos exposure in rats causes persistent behavioral alterations. Neurotoxicol Teratol 24:733-741.
Levin ED, Addy N, Christopher NC, Seidler FJ, Slotkin TA. 2001. Persistent behavioral consequences of neonatal chlorpyrifos exposure in rats. Dev Brain Res 130:83-89.
MacLusky NJ, Naftolin F. 1981. Sexual differentiation of the central nervous system. Science 211:1294-1302.
May M. 2000. Disturbing behavior: neurotoxic effects in children. Environ Health Perspect 108:A262-A267.
McCarthy MM. 1994. Molecular aspects of sexual differentiation of the rodent brain. Psychoneuroendocrinology 19:415-427.
Mileson BE, Chambers JE, Chen WL, Dettbarn W, Ehrich M, Eldefrawi AT, et al. 1998. Common mechanism of toxicity: a case study of organophosphorus pesticides. Toxicol Sci 41:8-20.
Physicians for Social Responsibility. 1995. Pesticides and Children. Washington, DC:Physicians for Social Responsibility.
Pope CN. 1999. Organophosphorus pesticides: do they all have the same mechanism of toxicity? J Toxicol Environ Health 2:161-181.
Pope CN, Chakraborti TK. 1992. Dose-related inhibition of brain and plasma cholinesterase in neonatal and adult rats following sublethal sublethal /sub·le·thal/ (-le´thal) insufficient to cause death.
Not sufficient to cause death. organophosphate exposures. Toxicology 73:35-43.
Pope CN, Chakraborti TK, Chapman ML, Farrar JD, Arthun D. 1991. Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides. Toxicology 68:51-61.
Qiao O, Seidler FJ, Abreu-Villaca Y, Tate CA, Cousins MM, Slotkin TA. 2004. Chlorpyrifos exposure during neurulation: cholinergic synaptic dysfunction and cellular alterations in brain regions at adolescence and adulthood. Dev Brain Res 148:43-52.
Qiao D, Seidler FJ, Padilla S, Slotkin TA. 2002. Developmental neurotoxicity of chlorpyrifos: what is the vulnerable period? Environ Health Perspect 110:1097-1103.
Qiao D, Seidler FJ, Slotkin TA. 2005. Oxidative mechanisms contributing to the developmental neurotoxicity of nicotine and chlorpyrifos. Toxicol Appl Pharmacol 206:17-26.
Qiao D, Seidler FJ, Tate CA, Cousins MM, Slotkin TA. 2003. Fetal chlorpyrifos exposure: adverse effects on brain cell development and cholinergic biomarkers emerge postnatally and continue into adolescence and adulthood. Environ Health Perspect 111:536-544.
Raines KW, Seidler FJ, Slotkin TA. 2001. Alterations in serotonin transporter expression in brain regions of rats exposed neonatally to chlorpyrifos. Dev Brain Res 130:65-72.
Ricceri L, Markina N, Valanzano A, Fortuna S, Cometa MF, Meneguz A, et al. 2003. Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice. Toxicol Appl Pharmacol 191:189-201.
Sachana M, Flaskos J, Nikolaidis E, Hargreaves A, Alexaki-Tzivanidou E. 2001. Inhibition of rat platelet 5-hydroxytryptamine uptake by chlorpyrifos and carbaryl carbaryl (kär`bärəl): see insecticides. . Pharmacol Toxicol 89:195-200.
Slotkin TA. 1999. Developmental cholinotoxicants: nicotine and chlorpyrifos. Environ Health Perspect 107(suppl 1):71-80.
Slotkin TA. 2004. Cholinergic systems in brain development and disruption by neurotoxicants: nicotine, environmental tobacco smoke environmental tobacco smoke (ETS/passive smoke),
n the gaseous by-product of burning tobacco products, including but not limited to commercially manufactured cigarettes and cigars; contains toxic elements harmful to the health of adults and children , organophosphates. Toxicol Appl Pharmacol 198:132-151.
Slotkin TA, Cousins MM, Tare CA, Soldier FJ. 2001. Persistent cholinergic presynaptic deficits after neonatal chlorpyrifos exposure. Brain Res 902:229-243.
Slotkin TA, Oliver CA, Seidler FJ. 2005. Critical periods for the role of oxidative stress in the developmental neurotoxicity of chlorpyrifos and terbutaline, alone or in combination. Dev Brain Res 157:172-180.
Slotkin TA, Seidler FJ, Ali SF. 2000. Cellular determinants of reduced adaptability of the aging brain: neurotransmitter utilization and cell signaling responses after MDMA MDMA 3,4-methylenedioxymethamphetamine.
3,4-Methylenedioxymethamphetamine; a mescaline analog.
MDMA 3,4 methylenedioxy-methamphetamine. See Ecstasy. lesions. Brain Res 879:163-173.
Slotkin TA, Tate CA, Cousins MM, Seidler FJ. 2002. Functional alterations in CNS See Continuous net settlement.
See continuous net settlement (CNS). catecholamine catecholamine (kăt'əkôl`əmēn), any of several compounds occurring naturally in the body that serve as hormones or as neutrotransmitters in the sympathetic nervous system. systems in adolescence and adulthood after neonatal chlorpyrifos exposure. Dev Brain Res 133:163-173.
Snedecor GW, Cochran WG. 1967. Statistical Methods. Ames, IA:Iowa State University Academics
ISU is best known for its degree programs in science, engineering, and agriculture. ISU is also home of the world's first electronic digital computing device, the Atanasoff–Berry Computer. Press.
Song X, Seidler FJ, Saleh JL, Zhang J, Padilla S, Slotkin TA. 1997. Cellular mechanisms for developmental toxicity of chlorpyrifos: targeting the adenylyl cyclase cyclase /cy·clase/ (si´klas) an enzyme that catalyzes the formation of a cyclic phosphodiester.
An enzyme that acts as a catalyst in the cyclization of a compound. signaling cascade. Toxicol Appl Pharmacol 145:158-174.
Steingart RA, Barg J, Maslaton J, Nesher M, Yanai J. 1998. Pre- and postsynaptic alterations in the septohippocampal cholinergic innervations after prenatal exposure to drugs. Brain Res Bull 46:203-209.
U.S. EPA (Environmental Protection Agency). 2000. Administrator's Announcement. Available: http://www.epa.gov/pesticides/ announcement6800.htm [accessed 13 October 2003].
U.S. EPA (Environmental Protection Agency). 2002. Chlorpyrifos: End-Use Products Cancellation Order. Available: http:// www.epa.gov/fedrgstr/EPA-PEST/2002/January/Day-25/ p1764.htm [accessed 6 December 2004].
Vatury O, Barg J, Slotkin TA, Yanai J. 2004. Altered localization Customizing software and documentation for a particular country. It includes the translation of menus and messages into the native spoken language as well as changes in the user interface to accommodate different alphabets and culture. See internationalization and l10n. of choline transporter sites in the mouse hippocampus after prenatal heroin exposure. Brain Res Bull 53:25-32.
Weiss B, Amler S, Amler RW. 2004. Pesticides. Pediatrics 113:1030-1036.
Whitney KD, Seidler FJ, Slotkin TA. 1995. Developmental neurotoxicity of chlorpyrifos: cellular mechanisms. Toxicol Appl Pharmacol 134:53-62.
World Medical Association. 2004. World Medical Association Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects. Available: http://www. wma.net/e/policy/b3.htm [accessed 23 June 2005].
Xu Z, Seidler FJ, Ali SF, Slikker W, Slotkin TA. 2001. Fetal and adolescent nicotine administration: effects on CNS serotonergic systems. Brain Res 914:166-178.
Yanai J, Beer A, Huleihel R, Izrael M, Katz S, Levi Y, et al. 2004. Convergent effects on cell signaling mechanisms mediate the actions of different neurobehavioral teratogens teratogens, (trat´ōjens),
n.pl agents that cause congenital malformations and developmental abnormalities if introduced during gestation. : alterations in cholinergic regulation of PKC PKC Protein Kinase C (biochemistry)
PKC Public Key Cryptography
PKC Public Key Certificate
PKC PaKua Chang (Chinese martial art)
PKC Paroxysmal Kinesigenic Choreoathetosis in chick and avian models. Ann NY Acad Sci 1025:595-601.
Yanai J, Rogel-Fuchs Y, Pick CG, Slotkin T, Soldier FJ, Zahalka EA, et al. 1993. Septohippocampal cholinergic changes after destruction of the A10-septal dopaminergic pathways. Neuropharmaeology 32:113-117.
Yanai J, Vatury O, Slotkin TA. 2002. Cell signaling as a target and underlying mechanism for neurobehavioral teratogenesis teratogenesis /ter·a·to·gen·e·sis/ (ter?ah-to-jen´e-sis) the production of birth defects in embryos and fetuses.teratogenet´ic
n. . Ann NY Acad Sci 965:473-478.
Address correspondence to T.A. Slotkin, Box 3813 DUMC DUMC Duke University Medical Center
DUMC Damascus United Methodist Church (Damascus, MD)
DUMC Demaree United Methodist Church (Illinois) , Duke University Medical Center, Durham, NC 27710 USA. Telephone: (919) 681-8015. Fax: (919) 684-8197. E-mail: email@example.com
This work was supported by National Institutes of Health grants ES10387, ES10356, and ES07031.
The authors declare they have no competing financial interests.
Received 27 January 2005; accepted 28 April 2005.
Justin E. Aldridge, (1) Armando Meyer, (1,2) Frederic J. Seidler, (1) and Theodore A. Slotkin (1)
(1) Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina Durham is a city in the U.S. state of North Carolina. It is the county seat of Durham CountyGR6 and is the fourth-largest city in the state by population. , USA; (2) Centro de Estudos da Saude do Trabalhador e Ecologia Humana, Escola Nacional de Saude Publica, Fundacao Oswaldo Cruz, Rio de Janeiro Rio de Janeiro, city, Brazil
Rio de Janeiro (rē`ō də zhänā`rō, Port. rē` thĭ zhənĕē`r , Brazil
Table 1. Neurotransmitter and metabolite levels (ng/region) in control rat brain regions. Brain region Sex 5HT 5HIAA Cerebral cortex Male 267 [+ or -] 6 186 [+ or -] 5 Female 255 [+ or -] 5 197 [+ or -] 5 Hippocampus Male 113 [+ or -] 3 190 [+ or -] 5 Female 114 [+ or -] 3 202 [+ or -] 5 Striatum Male 165 [+ or -] 4 210 [+ or -] 5 Female 173 [+ or -] 4 231 [+ or -] 5 * Midbrain Male 487 [+ or -] 15 299 [+ or -] 9 Female 461 [+ or -] 13 324 [+ or -] 7 * Brainstem Male 189 [+ or -] 10 91 [+ or -] 6 Female 188 [+ or -] 8 97 [+ or -] 7 Brain region Sex DA DOPAC Cerebral cortex Male 243 [+ or -] 9 59 [+ or -] 2 Female 230 [+ or -] 12 56 [+ or -] 2 Hippocampus Male 1.3 [+ or -] 0.2 BD Female 1.2 [+ or -] 0.2 BD Striatum Male 541 [+ or -] 13 203 [+ or -] 7 Female 470 [+ or -] 13 * 189 [+ or -] 6 Midbrain Male 31 [+ or -] 1 16.1 [+ or -] 0.8 Female 26 [+ or -] 1 * 14.1 [+ or -] 0.5 Brainstem Male 4.0 [+ or -] 0.2 2.2 [+ or -] 0.2 Female 3.4 [+ or -] 0.1 * 2.2 [+ or -] 0.2 Brain region Sex HVA Cerebral cortex Male 42 [+ or -] 2 Female 42 [+ or -] 2 Hippocampus Male BD Female BD Striatum Male 55 [+ or -] 2 Female 50 [+ or -] 2 Midbrain Male BD Female BD Brainstem Male BD Female BD BD, below detection limit. * Significant sex differences.