Neonatal chlorpyrifos exposure targets multiple proteins governing the hepatic adenylyl cyclase signaling cascade: implications for neurotoxicity

Developmental Brain Research 121 (2000) 19–27 www.elsevier.com / locate / bres

Research report

Neonatal chlorpyrifos exposure targets multiple proteins governing the hepatic adenylyl cyclase signaling cascade: implications for neurotoxicity q J.T. Auman, F.J. Seidler, T.A. Slotkin* Department of Pharmacology & Cancer Biology, Duke University Medical Center, Box 3813 DUMC, Durham, NC 27710, USA Accepted 15 February 2000

Abstract Chlorpyrifos has been hypothesized to interact with receptors and transduction proteins involved in the production of cyclic AMP, contributing to adverse effects on cell replication and differentiation. We studied the effects of neonatal chlorpyrifos exposure on hepatic adenylyl cyclase (AC) activity, as the liver accumulates the highest concentrations of chlorpyrifos and is the site for generation of its active metabolite, chlorpyrifos oxon. Newborn rats were given 1 mg / kg of chlorpyrifos s.c. on PN1-4. On PN5, 24 h after the last dose, AC catalytic activity was induced as assessed by the response to the direct AC stimulant, Mn 21 . In contrast, AC activation dependent upon interaction of the enzyme with G-proteins (forskolin) did not show any enhancement, suggesting impairment of G-protein function. This conclusion was confirmed by impaired responsiveness to fluoride, which directly activates G-proteins. In addition, the response of AC to hormonal signals was altered in a receptor-selective manner, with an enhanced response to glucagon but not to the b-adrenoceptor agonist, isoproterenol. The effects of chlorpyrifos on AC signaling displayed a critical developmental period of vulnerability, as treatment of older rats (PN11-14) failed to cause substantial induction of AC or interference with G-protein signaling, although it did still enhance the glucagon response. In all cases, the effects of chlorpyrifos disappeared within a few days of discontinuing treatment. These results stand in contrast to the delayed deterioration of AC signaling seen in the brain after the same chlorpyrifos treatment. The temporal and organ selectivity of chlorpyrifos’ effects on the AC cascade suggest that disruption of membrane signaling occurs consequent to selective effects on cell development, rather than representing a direct interaction between chlorpyrifos and signaling proteins.  2000 Elsevier Science B.V. All rights reserved. Keywords: Adenylyl cyclase; b-Adrenoceptor; Chlorpyrifos; Development; Glucagon receptor; Liver; Organophosphate insecticides

1. Introduction Chlorpyrifos has become one of the most commonly used insecticides in agriculture and in domestic ‘‘crack and crevice’’ treatment, largely due to its persistence and its safety relative to many other organophosphate insecticides. Chlorpyrifos has only a limited capability to elicit delayed peripheral neuropathies [28], generally requiring exposures well above the threshold for acute systemic toxicity [39,50]. Nevertheless, there has been considerable recent concern over chlorpyrifos exposure of pregnant women, Abbreviations: AC, adenylyl cyclase; ANOVA, analysis of variance; PN, postnatal days q Supported by USPHS ES-10387, ES-07031 and ES-10356 *Corresponding author. Tel: 11-919-681-8015; fax: 1-919-684-8197. E-mail address: [email protected] (T.A. Slotkin)

infants and children [32,46], especially in light of the possibility of exposures above the No Observed Adverse Effect Level after routine home application [16,18,37]. It is also increasingly evident that polymorphisms of the genes encoding enzymes responsible for the catabolism of organophosphates may produce especially sensitive human subpopulations that show adverse effects at exposure levels thought generally to be safe [10,19]. Studies in developing rats indicate that chlorpyrifos is far more toxic to the immature organism [47,48,61], despite the fact that cholinesterase activity recovers quicker in the fetus and neonate [33,41,42]. Accordingly, using just cholinesterase inhibition, the standard biomarker for organophosphate exposure, may substantially underestimate the degree and biological impact of chlorpyrifos exposure [25]. This conclusion is reinforced by recent evidence that chlorpyrifos targets brain cell development

0165-3806 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 00 )00021-3

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by mechanisms either independent of, or poorly related to, cholinesterase inhibition [46,56]. Studies conducted both in vivo and in vitro have implicated chlorpyrifos in inhibition of DNA synthesis [13,58,61], impaired cell acquisition [5,52], altered levels of specific macromolecules [5,27], impaired axonogenesis [35,58], increased release of neurotransmitters that play a trophic role in brain development [12] and impaired function of signaling cascades involved in neurotrophic activity [57]. Cyclic AMP provides one of the most basic trophic signals in differentiation of all cells [9,17,23,59], and it is thus important to note that in vitro studies suggest that chlorpyrifos and / or its active metabolite, chlorpyrifos oxon, can bind to G-protein-coupled membrane receptors and may also interact with G-proteins or AC itself [20,21,60]. Administration of chlorpyrifos to neonatal rats in vivo also leads to impaired signal transduction through the AC pathway [57]. Nevertheless, there are some logical inconsistencies in ascribing these effects to a direct interaction of chlorpyrifos or chlorpyrifos oxon with specific elements of AC signaling. First, direct, in vitro effects on receptors, Gproteins and AC require extremely high concentrations, well above those experienced with in vivo exposures. Second, developmental exposure to chlorpyrifos targets brain development at levels apparently devoid of systemic toxicity or growth impairment, whereas direct effects on cyclic AMP generation would be expected to exhibit little or no tissue selectivity and should render chlorpyrifos generally teratogenic. Although there are individual case reports of chlorpyrifos-induced terata [55], controlled animal studies have failed to find significant dysmorphogenesis [4], and high doses are needed to produce outright embryotoxicity [43]. Third, even within the brain, many of the effects of in vivo chlorpyrifos administration on AC signaling appear after a delay of several days rather than during the period of chlorpyrifos exposure [57]. These results suggest that disruption of AC signaling, although important for the adverse effects of chlorpyrifos on brain development, is likely to occur consequently to other cellular targets such as cell replication, macromolecule synthesis, reactive oxygen generation or gene transcription [2,3,11,56]. To address this issue, we examined the effects of chlorpyrifos administration to neonatal rats on hepatic AC signaling. The liver accumulates far more chlorpyrifos than does the brain and is also the primary site for generation of chlorpyrifos oxon [6,24,42,45]. Accordingly, if chlorpyrifos or chlorpyrifos oxon interact directly with elements of the AC signaling cascade after relevant in vivo exposures, the liver should show much greater and persistent effects than those seen in the brain. We evaluated the AC pathway at four functional levels. First, we examined the constitutive activity of AC itself using the direct enzymatic stimulant, Mn 21 , which replaces Mg 21 at metal binding sites [26] and acts without the requirement for participation of G-proteins [36]. Second, we studied AC stimulation by forskolin, which acts

directly on the enzyme by binding to the catalytic core [26], and which does require association of AC with G-proteins for maximal effect [54]. Third, we evaluated the AC response to G-protein activation by fluoride. Fourth, we assessed the responses to stimulation of badrenoceptors and glucagon receptors, the two G s -linked membrane proteins that regulate gluconeogenesis and glycogenolysis, the major physiological functions of hepatic AC signaling.

2. Methods

2.1. Animals and treatments All experiments using live animals were carried out in accordance with the declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Timed-pregnant rats (Zivic Miller Laboratories, Allison Park, PA) were housed with a 12-h dark–light cycle in breeding cages with free access to food and water. Pups from all litters were randomized on the day after birth and redistributed to the dams with litter sizes of 10–12 pups to ensure standardized nutrition and maternal care. In addition, for all experiments, equal numbers of animals from a given litter were assigned to each of the treatment groups and dams were reassigned randomly to the litters on a daily basis so as to distribute any differences in maternal caretaking equally across all animals. Chlorpyrifos was dissolved in dimethylsulfoxide to ensure rapid and complete absorption [61] and was injected subcutaneously in a volume of 1 ml / kg body weight so as to deliver 1 or 5 mg / kg; control rats received equivalent volumes of dimethylsulfoxide. For studies of effects of chlorpyrifos in the first few days after birth, animals were given 1 mg / kg daily on PN1-4 and studies were conducted on PN5 and 10. For studies in older animals, pups were randomized on PN11, given daily treatment with 5 mg / kg of chlorpyrifos on PN11-14, and studies were conducted on days 15 and 20. Older animals tolerate higher doses [47,48,61], so that the two regimens at the different ages are toxicologically equivalent in that they lie just below the threshold for evidence of systemic toxicity (reduced weight gain, mortality) [5,61]. Nevertheless, these doses elicit adverse effects on brain cell development [5,12,13,27,56,57,61]. For each experiment, rats were decapitated and the livers rapidly removed. Tissues were frozen immediately in liquid nitrogen and maintained at 2458C until assayed; preliminary studies showed no degradation of AC after freezing.

2.2. AC activity Tissues were thawed and homogenized (Polytron, Brink-

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mann Instruments, Westbury NY) in 39 volumes of icecold buffer containing 145 mM NaCl, 2 mM MgCl 2 , and 20 mM Tris (pH 7.5), strained through several layers of cheesecloth to remove connective tissue, and the homogenates were sedimented at 40 000 g for 15 min. The pellets were washed twice by resuspension (Polytron) in homogenization buffer followed by resedimentation, and were then dispersed with a homogenizer (smooth glass fitted with a Teflon pestle) to achieve a final protein concentration of 0.5–1 mg / ml in a buffer consisting of 250 mM sucrose, 20 mM MgCl 2 , and 50 mM Tris (pH 7.5). The membrane preparations were diluted 10-fold with 250 mM sucrose, 1 mM EGTA and 10 mM Tris (pH 7.4) prior to the assay. Aliquots of membrane preparation containing 25–40 mg protein were incubated for 30 min at 308C with final concentrations of 100 mM Tris–HCl (pH 7.4), 10 mM theophylline, 1 mM adenosine 59-triphosphate, 2 mM MgCl 2 , 1 mg bovine serum albumin, and a creatine phosphokinase-ATP-regenerating system consisting of 10 mM sodium phosphocreatine and 8 IU phosphocreatine kinase, and 10 mM GTP in a total volume of 250 ml. The enzymatic reaction was stopped by placing the samples in a 90–1008C water bath for 5 min, followed by sedimentation at 3000 g for 15 min, and the supernatant solution was assayed for cyclic AMP using radioimmunoassay kits. Preliminary experiments showed that the enzymatic reaction was linear well beyond the assay time period and was linear with membrane protein concentration; concentrations of cofactors were optimal and, in particular, the addition of higher concentrations of GTP produced no further augmentation of activity. The contributions of G-protein-linked processes to AC were evaluated in several ways. First, we compared activity of the AC moiety itself with direct stimulation by 100 mM forskolin or 10 mM MnCl 2 [7]. Forskolin-stimulated activity is enhanced by G s -AC association, whereas Mn 21 -stimulation is not [36]. Second, the net G-proteinlinked response of AC activity to maximal activation of all G-proteins was evaluated by addition of 10 mM NaF [7]. Finally, the AC response to stimulation of cell surface receptors linked to G s were evaluated with 100 mM isoproterenol, a b-adrenoceptor stimulant, and 3 mM glucagon, which operates through a different receptor coupled to G s . The concentrations of all the agents used here have been found previously to be optimal for effects on AC and / or were confirmed in preliminary experiments [7,62–64].

2.2.1. Data analysis Data are presented as means and standard errors. Prior to conducting tests of specific treatments and ages, a global ANOVA was conducted for the entire data set, with factors of treatment, treatment period, age, sex and type of AC measurement (basal, forskolin, Mn 21 , isoproterenol, glucagon); data were log-transformed because of heterogeneous variance, and the type of AC measurement was

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considered a repeated measure for each membrane preparation. This initial test did not reveal any significant interactions of sex3treatment or sex3treatment3other variables (data not shown), so results of males and females were combined for subsequent analyses). The subsequent tests first examined the effects of chlorpyrifos on all AC measurements (repeated measure) across all ages, followed by evaluation of effects for each treatment subset (PN1-4, PN11-14). Data were then analyzed separately for each age (ANOVA for treatment3AC measurement) and finally for treatment effects on each individual type of AC measurement using Fisher’s Protected Least Significant Difference. For all tests, significance was assumed at P,0.05. For convenience, some data are presented as the percent change from control values but statistical significance was always assessed on the unmanipulated data.

2.2.2. Materials Chlorpyrifos was purchased from Chem Service Inc. (West Chester, PA). Radioimmunoassay kits for cyclic AMP determinations were obtained from Amersham Corp. (Chicago, IL). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

3. Results In control rats, basal AC activity, measured in the presence of GTP so as to involve G-proteins, displayed a substantial ontogenetic decline between PN5 and PN20 (Fig. 1). A different developmental pattern was obtained when AC was stimulated by Mn 21 , which bypasses the involvement of G-proteins: Mn 21 -stimulated AC activity rose between PN5 and PN10 and then declined to a much smaller extent than was seen for basal AC. These differences suggest that participation of G-proteins changes during development, superimposed on the catalytic activity of AC itself as evaluated with Mn 21 . Accordingly, we also evaluated the development of the AC response to forskolin, which, although it acts on AC directly, is dependent on the association of G-proteins with AC. Forskolin-stimulated activity showed a pattern midway between those of basal AC and Mn 21 -stimulated AC, with a small increment on PN10 and a subsequent decline proportionally less than that of basal AC but more than that of Mn 21 -stimulated AC (age3AC measure: P,0.0001 for forskolin vs. basal, P,0.0001 for forskolin vs. Mn 21 ). Furthermore, the development of AC responses to fluoride, isoproterenol and glucagon all differed significantly from either the pattern for Mn 21 or for forskolin (age3AC measure, P,0.0001 for each), indicating superimposition of ontogenetic shifts in G-protein expression or function (fluoride), and in b-adrenoceptor and glucagon receptor expression or function. Indeed, the responses to the two G s linked receptor agonists also showed distinct developmental differences from each other (age3AC measure, P,

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Fig. 1. Hepatic AC activity in control animals. Data represent means and standard errors obtained from 14 to 18 animals at each age. ANOVA across all ages and AC measurements appears at the top of the panel; in addition, every type of measurement shows significant changes with age (P,0.0001 for each). Asterisks denote ages at which AC activity differs from the PN5 value. Abbreviations: Mn 21 5manganese; fsk5forskolin; F 2 5fluoride; iso5isoproterenol, glu5glucagon. Note different ordinate for forskolin (scale on the right) as compared to other measures (scale on the left).

0.0001 for isoproterenol vs. glucagon). The response to isoproterenol declined monotonically with age, whereas the response to glucagon increased on PN10 before declining to a much smaller extent than did the b-adrenergic response. Isoproterenol evoked approximately a 2.5fold increase over basal activity at all ages; whereas glucagon produced a response equivalent to that of isoproterenol on PN5, it became twice as effective as isoproterenol by PN20. In light of the involvement of ontogenetic changes in AC catalytic activity, involvement of G-proteins, and differences in receptor-mediated activities, we evaluated the effects of chlorpyrifos on all these processes. Global statistical analysis across all treatments, ages and AC measures (Table 1) indicated that the effects of chlorpyrifos differed depending upon the type of AC measurement (interaction of treatment3AC measure), implying that the alterations were dependent on different sets of

signaling proteins mediating the AC response (direct effects on AC, effects on G-proteins, effects on receptor function). Furthermore, the differential effects were dependent on the age at which measurements were made (interaction of treatment3age3AC measure). Accordingly, we subdivided the global statistical analyses into the two separate treatment cohorts (PN1-4, PN11-14). In both these cases, differential effects on the AC response to stimulants were still apparent (interaction of treatment3 AC measure); however, the two treatment paradigms did not elicit equivalent effects (treatment3treatment period, P,0.0007; treatment3treatment period3AC measure, P, 0.05). Accordingly, data are presented separately for the two types of treatment and for each age point within the treatment groups. We also found that the effects of chlorpyrifos differed significantly between the two direct AC stimulants, Mn 21 and forskolin (interactions of treatment3AC measure and treatment3age3AC measure),

TABLE 1 Chlorpyrifos effects on AC activity–statistical analyses Comparison

Treatment Main effect

Treatment 3 Age

Treatment 3 AC measure

Treatment3Age 3 AC measure

All ages, all measures Chlorpyrifos PN1-4 Chlorpyrifos PN11-14 Mn 21 vs. Forskolin Forskolin vs. F 2 Isoproterenol vs. glucagon

NS NS NS P,0.03 NS P,0.02

NS NS NS NS P,0.04 P,0.007

P,0.0001 P,0.001 P,0.0001 P,0.0007 NS P,0.006

P,0.0001 P,0.0001 NS P,0.003 NS P,0.02

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indicating separable targeting of both AC and of G-proteins. In contrast, the effects on forskolin- and fluoridestimulated AC activity, which both share a requirement for G-proteins, were not separable from each other (no interaction of treatment3AC measure). The two different receptor agonists, isoproterenol and glucagon, showed marked disparities in the effects of chlorpyrifos on their AC response (interactions of treatment3AC measure and treatment3age3AC measure). There were no significant effects of either chlorpyrifos regimen on body or liver weight, nor did chlorpyrifos evoke any mortality or other overt signs of systemic toxicity (data not shown). Twenty-four hours after chlorpyrifos treatment on PN14 (Fig. 2, top panel), basal AC activity was unchanged despite the fact that the Mn 21 -stimulated activity was significantly augmented. As this disparity implies that alterations extended to G-protein /AC interactions, we next examined the effects on the forskolin and fluoride responses. Chlorpyrifos failed to increase the response to forskolin and actually decreased the response to fluoride. The AC response to b-adrenoceptor stimulation by isoproterenol, which comprises elements of both AC catalytic activity and G s function, was unaffected by chlorpyrifos exposure. In contrast, the response to glucagon was enhanced. In an additional study using a higher dose (5 mg / kg daily), we obtained an even larger augmentation of the AC response to glucagon, 50612% increase above control levels (n514, P,0.0006). In keeping with earlier results [5,61], 5 mg / kg given on PN1-4 was associated with significant mortality and impairment of weight gain in survivors, so detailed studies were not performed at that dose. By PN10, six days after the last chlorpyrifos injection, most of the effects of the 1 mg / kg dose regimen on AC signaling were much less notable (Fig. 2, bottom panel). Although forskolin-stimulated activity was significantly elevated, the effect was not statistically distinguishable from the residual effect on Mn 21 -stimulated activity, which by itself was not significant from control values. The inhibitory effect on the AC response to fluoride also disappeared by PN10 and the enhanced response to glucagon was completely reversed. Animals receiving 5 mg / kg of chlorpyrifos on PN11-14 also displayed significant alterations in hepatic AC activity. However, despite the higher dose, the effects on all but one of the AC measures was less than had been seen in younger animals treated with the lower dose. On PN15, 24 h after the last dose of chlorpyrifos, there was a statistically significant increase in Mn 21 -stimulated AC activity but the magnitude of effect was quite small (Fig. 3, top panel). Furthermore, there were no differential effects on forskolin-stimulated activity as compared to Mn 21 -stimulated activity, nor was there any defect in the response to fluoride. Again, although there were no changes in the ability of isoproterenol to stimulate AC activity, the response to glucagon was enhanced substantially in the

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Fig. 2. Effects of 1 mg / kg of chlorpyrifos (PN1-4) on hepatic AC activity measured on PN5 and PN10, presented as the percentage change from control values (see Fig. 1). Data represent means and standard errors obtained from 12 to 18 animals at each age. Abbreviations: Mn 21 5 manganese; fsk5forskolin; F 2 5fluoride; iso5isoproterenol, glu5 glucagon. ANOVA across all measures appears at the top of each panel and asterisks denote individual values that differ from the corresponding control.

chlorpyrifos group. By PN20, six days after the last chlorpyrifos injection, all parameters had returned to normal (Fig. 3, bottom panel).

4. Discussion In keeping with earlier results, the catalytic activity of hepatic AC in control rats showed an early postnatal peak and subsequent decline to lower levels by the third postnatal week [29,30,44]. However, superimposed on this pattern, there were distinct differences in response to

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Fig. 3. Effects of 5 mg / kg of chlorpyrifos (PN11-14) on hepatic AC activity measured on PN15 and PN20, presented as the percentage change from control values (see Fig. 1). Data represent means and standard errors obtained from 18 animals at each age. Abbreviations: Mn 21 5manganese; fsk5forskolin; F 2 5fluoride; iso5isoproterenol, glu5glucagon. ANOVA across all measures appears at the top of each panel and asterisks denote individual values that differ from the corresponding control.

stimulants of the signaling proteins that regulate AC function. First, AC activity stimulated by forskolin was much higher in general than that stimulated by Mn 21 , and the two also showed significantly different ontogenetic profiles, despite the fact that both act directly on AC itself. Forskolin and Mn 21 differ in two ways: they bind to different epitopes of the AC molecule [26], and the effects of forskolin, but not Mn 21 , are enhanced by the association of AC with G s [36,54]. Differential responses of AC to these stimulants thus implies either that the conformation of the AC molecule, and / or the number or function of G-proteins, undergo significant ontogenetic changes. In previous work with other tissues, developmental shifts in

the expression of specific AC subtypes have been identified [8,49,53], as have those for expression and function of G s and G i [15,51,62], and we have identified attendant alterations in the relative responses to Mn 21 and forskolin [64]. In the current study, we confirmed the pivotal role played by G-proteins by comparing the response to Mn 21 with that to fluoride and receptor agonists that operate through G s . Unlike the response to Mn 21 , which showed an ontogenetic peak of activity, the response to fluoride showed a monotonic decline from PN5 to PN20, and the magnitude of the decline (.50%) was significantly greater than that seen for Mn 21 (25%). Furthermore, the AC response to two different membrane receptors linked to G s displayed disparate ontogenetic patterns from that of Mn 21 , fluoride or each other, indicating that each receptormediated response undergoes a separable developmental course that is distinct from that of AC or the G-proteins. The concentration of b-adrenoceptors falls substantially over the first few postnatal weeks [22,38], likely contributing to the larger decline that we saw in the response to isoproterenol as compared to glucagon. Administration of chlorpyrifos to neonatal rats on PN1-4 affected signaling through the AC pathway at every level of organization. First, catalytic activity was induced as evidenced by an increase in the response to Mn 21 . If that were the only effect on AC, then all other measures should simply reflect the basic change in catalytic activity of the AC moiety. However, the response to forskolin was unchanged, suggesting either that chlorpyrifos elicited a shift in the AC isoform being expressed to one with an enhanced response to Mn 21 vs. forskolin, or alternatively, that G-protein linkages to AC were impaired, since forskolin requires association of G s with AC to produce its maximal effect. The results obtained with fluoride, which directly activates G-proteins, confirmed that chlorpyrifos has an adverse effect on G-protein function. Consequently, isoproterenol, which operates through the G s -linked badrenoceptor, showed an effect summating those on Mn 21 and fluoride: increases in AC catalytic activity were offset by adverse effects on G s -mediated activity. In contrast, chlorpyrifos enhanced the AC response to glucagon, which also shares G s as its coupling protein; accordingly, chlorpyrifos treatment must either induce glucagon receptors or enhance their ability to elicit a response, since otherwise, it should have produced no change, as was seen for isoproterenol. Despite the fact that receptor and G-protein activities, and the activity of AC itself are altered by neonatal chlorpyrifos exposure, it is still unclear whether these effects represent direct actions of chlorpyrifos on the activity or expression of these proteins. Certainly, the initial effects of chlorpyrifos on hepatic AC signaling on PN5 are greater than those seen in the brain after the same treatment [57], in keeping with the fact that the liver is exposed to higher concentrations of chlorpyrifos and chlorpyrifos oxon [6,24,42,45]. Additionally, the fact that

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hepatic AC signaling regresses toward normal within a few days of the termination of exposure would seem to support a direct effect. However, at the same time, there are features that cannot be explained by a simple relationship. First, one would then expect to see the same effects of chlorpyrifos on AC signaling regardless of the age of exposure. Instead, when chlorpyrifos was given on PN1114, the only robust effect was the augmented response to glucagon. AC induction, assessed with Mn 21 , was much smaller than had been seen with PN1-4 chlorpyrifos treatment, and there were no effects on G-protein signaling as assessed with fluoride; and this was despite the much higher dose given to the older animals. The existence of a critical period for effects on AC signaling argues in favor of changes in signaling consequent to alterations in cell development, rather than the other way around. Similarly, direct targeting of signaling proteins would be expected to produce uniform effects in different tissues sharing the same proteins. Instead, earlier work on AC signaling in the brain indicates a marked deterioration in the function of multiple signaling proteins in the period from PN5 to PN10 [57], effects which were absent in the liver despite its receiving a higher effective concentration of chlorpyrifos and chlorpyrifos oxon. This supports the idea that chlorpyrifos is a selective developmental neurotoxin, exerting effects on the function of brain cells that are not shared by other tissues [56]. The only AC signaling component showing a similar effect on PN5 and PN15 was the enhanced glucagon response. Although it is not possible to tell from these results alone whether chlorpyrifos acts directly on glucagon receptor expression or function, or elicits secondary changes that affect glucagon signaling, there are potential ramifications to this effect. The perinatal transition period is one of intense metabolic activity, requiring the rapid mobilization of stored glycogen as well as gluconeogenesis, effects which are keyed to the activity of AC-linked receptors for catecholamines and glucagon [1,14,31,34,38,40]. Hyperactivity of glucagon receptors may lead to premature depletion of hepatic energy stores, impairing the ability to withstand metabolic stress. Future studies should address the potential physiologic implications of the developmental toxicity of chlorpyrifos outside the central nervous system.

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