Cellular mechanisms for developmental toxicity of chlorpyrifos: targeting the adenylyl cyclase signaling cascade

Developmental neurotoxicity caused by chlorpyrifos exposure is generally thought to target cholinesterase but chlorpyrifos may also act on cellular intermediates, such as adenylyl cyclase, that serve global functions in the coordination of cell development. In the current study, neonatal rats were exposed to apparently subtoxic doses of chlorpyrifos (no weight loss, no mortality) either on Postnatal Days 1-4 or on Postnatal Days 11-14, and the effects on components of the adenylyl cyclase cascade were evaluated in brain regions that are enriched (forebrain) or sparse (cerebellum) in cholinergic innervation, as well as in a nonneural tissue (heart). In all three, chlorpyrifos evoked deficits in multiple components of the adenylyl cyclase cascade: expression and activity of adenylyl cyclase itself, functioning of G-proteins that link neurotransmitter and hormone receptors to cyclase activity, and expression of neurotransmitter receptors that act through this cascade. Disruption of signaling function was not restricted to transduction of cholinergic signals but rather extended to adrenergic signals as well. In most cases, the adverse effects were not evident during the immediate period of chlorpyrifos administration, but appeared after a delay of several days. These results suggest that chlorpyrifos can affect cell development by altering the activity and reactivity of the adenylyl cyclase signaling cascade, a major control point for trophic regulation of cell differentiation. The effects are not restricted to cholinergic targets, nor even to the central nervous system. Hence, disruption of cell development by chlorpyrifos is likely to be more widespread than previously thought.     

Modeling the developmental neurotoxicity of chlorpyrifos in vitro: macromolecule synthesis in PC12 cells

Exposure to apparently subtoxic doses of chlorpyrifos during late stages of brain development affects cell acquisition through a mixture of cholinergic and noncholinergic mechanisms. In the current study, we modeled these effects in vitro using rat pheochromocytoma (PC12), a cell line that, upon nerve-growth factor (NGF)-induced differentiation, develops the appearance and function of cholinergic target neurons, including the expression of cholinergic receptors. In the undifferentiated state (no NGF), chlorpyrifos evoked an immediate (1 h), robust, concentration-dependent inhibition of DNA synthesis as evaluated by [3H]thymidine incorporation, with a threshold of 0.5-1.5 microg/ml. Continuous exposure for up to 24 h maintained the same degree of inhibition. The effects were selective for DNA synthesis, as much smaller inhibitions were found for synthesis of RNA or protein. In contrast, direct cholinergic stimulation of the cells by 100 microM nicotine had much smaller effects on DNA synthesis. Moreover, the effects of chlorpyrifos on DNA synthesis could not be blocked by nicotinic or muscarinic antagonists, confirming that the effects were not mediated primarily through cholinergic hyperstimulation consequent to cholinesterase inhibition or to direct receptor-mediated effects. When PC12 cells underwent NGF-induced differentiation, the rate of cell replication fell dramatically and neurite extension was evident both from morphological examination and from biochemical markers (increased protein:DNA ratio). After introduction of NGF, chlorpyrifos maintained its ability to inhibit DNA synthesis acutely. However, the ability to inhibit RNA and protein synthesis initially intensified and then disappeared, indicating a shift in macromolecular targets as differentiation proceeded. We also tested the effects of long-term exposure to chlorpyrifos during the process of NGF-induced differentiation. Continuous chlorpyrifos exposure resulted in severe reductions in macromolecule synthesis and a deficit in the total number of cells, effects similar to those seen with chlorpyrifos treatment in vivo. At the highest concentrations, neurite extension was also inhibited. Our results suggest that chlorpyrifos can interact directly with developing neural cells to inhibit replication and neuritic outgrowth.     

Serotonin transporter expression in rat brain regions and blood platelets: aging and glucocorticoid effects

Hyperactivity of the hypothalamus-pituitary-adrenal axis is more common in elderly depression than in younger cohorts and glucocorticoids are known to influence serotonergic systems. The current study explores the interaction of glucocorticoids with aging on serotonin transporter expression and function. Continuous infusions of dexamethasone (26 days) reduced transporter expression in the aged brain but the ability of imipramine to inhibit synaptosomal [3H]serotonin uptake was unimpaired. These effects were unique to aged animals, as prior work with young adults found no effects of dexamethasone on transporter expression. In contrast to the effects in the brain, there were no differences in platelet transporter expression between young and old rats nor did dexamethasone treatment affect the values in the aged group: thus, the platelet may not reliably model these aspects of CNS function. The results suggest that there are basic biologic differences in the effects of glucocorticoids in aged vs. young brain that could contribute to lowered effectiveness to antidepressants in elderly depression; if transport capacity is already reduced by the effects of increased glucocorticoids, further inhibition of transport by antidepressants would have proportionally less impact on synaptic serotonin concentrations.     

Young adolescent and maternal depression: Assessment, interrelations, and family predictors.

The present study examined (a) the relation between self-report and behavioral ratings of depression for young adolescents and their mothers; (b) the relation between adolescent and maternal depression; and (c) family correlates and predictors of adolescent and maternal depression. Sixty-nine nonclinic adolescents and their mothers completed self-report measures and participated in two behavioral observations 1 year apart. Self-report and behavioral-rating measures of depression were related for mothers but not for adolescents, and maternal depression and adolescent depression were not related to one another. In addition, marital conflict predicted maternal depression as measured by both self-reports and behavioral ratings, whereas parent–adolescent conflict predicted only self-reported adolescent depression. The differences found between maternal and adolescent depression are discussed, and the findings are contrasted with those reported for clinically depressed mothers and children. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Ontogeny of beta-adrenoceptor/adenylyl cyclase desensitization mechanisms: the role of neonatal innervation

The ability of adrenergic stimulation to elicit desensitization of the beta-receptor/adenylyl cyclase signaling cascade is not an inherent property of cells but rather is acquired during the period in which sympathetic innervation develops. This study examines whether innervation provides the signal that enables target cardiac and hepatic cells to learn to desensitize their responses. Neonatal rats were sympathectomized with 6-OHDA on postnatal day 1 and were treated at various ages with a regimen of isoproterenol known to elicit desensitization in adults. In control rats, desensitization first appeared between days 6 and 15. Desensitization was heterologous, involving changes in the efficiency of G-protein coupling, as there were parallel decreases in isoproterenol-stimulated adenylyl cyclase activity, basal activity and fluoride-stimulated activity (maximal G-protein activation) without changes in forskolin-Mn2+-stimulated activity (total cyclase catalytic activity). The lesioned animals showed a delay in the onset of desensitization as isoproterenol did not evoke decreased responsiveness until day 25 in the heart; the liver did not display agonist-induced desensitization even at day 25. The effects of lesioning on development of desensitization were entirely separable from those on regulation of beta-receptors themselves: agonist-induced decreases in receptor binding appeared by day 15 in both control and lesioned animals. Uniquely in the youngest animals (6 days old), isoproterenol treatment produced heterologous sensitization of adenylyl cyclase responses rather than desensitization, with a parallel increase in basal, isoproterenol-, fluoride- and forskolin-Mn2+-stimulated activity; the latter indicates induction of total catalytic activity as the primary mechanism of sensitization. The lesioned neonates did not show sensitization, despite the fact that during this period, sympathetic pathways are not functionally competent. Our results indicate that innervation provides a timing signal for the onset of desensitization capabilities of sympathetic target cells, but is not absolutely required for the cells to learn how to desensitize. Prior to the onset of desensitization, agonists induce sensitization that may be important in preserving physiological responsiveness during ontogenetic surges of adrenergic activity. The absence of sensitization in lesioned animals implies that, before physiological function is completely established, early pioneer synapses provide a trophic signal that enables cells to increase their sensitivity to stimulation during the perinatal transition period.     

Acute effects of cocaine on ornithine decarboxylase activity in fetal and neonatal rat heart: evidence for cardiotoxicity

Fetal exposure to cocaine is associated with increased perinatal cardiac risk. In the current study, we examined the effects of acute cocaine administration on ornithine decarboxylase (ODC) activity in fetal and neonatal rat heart. ODC is a key regulatory enzyme in the control of cell differentiation and growth, and rapid changes in ODC are associated with the response to cell injury. Administration of 30 mg/kg s.c. of cocaine to pregnant rats on the 20th day of gestation caused acute elevation of fetal cardiac ODC that persisted throughout the ensuing 24 h. In contrast, the same dose given directly to neonatal rats the day after birth evoked only a short-term (1-h) stimulation of ODC that was reversed by 4 h after treatment. By 4 days of age and subsequently, cocaine was unable to elicit acute stimulation of heart ODC and only evoked inhibition of enzyme activity. Elevated progesterone levels during pregnancy have been shown to sensitize the maternal myocardium to cocaine-induced catecholaminergic effects; the greater sensitivity of fetal heart ODC to cocaine, as compared to neonatal heart, supports the hypothesis that similar enhancement of fetal cardiac irritability can contribute to cocaine-induced cell damage.     

Chlorpyrifos elicits mitotic abnormalities and apoptosis in neuroepithelium of cultured rat embryos

Case records of 27 dogs with medically managed congenital portosystemic shunts were reviewed. Fourteen were followed up by telephone questionnaires to the owners. Age, breed, sex, clinical signs and blood results were similar to previous studies. Weight and quality of life were stable or improved on treatment in all cases. Total serum protein concentration and alanine aminotransferase and alkaline phosphatase activities fell significantly during treatment. Fourteen dogs were euthanased, four were lost to follow-up and nine remained alive. Mean survival time for the dogs euthanased was 9.9 months. Mean follow-up period for the dogs still alive was 56.9 months and all had survived more than 36 months from diagnosis. Surviving dogs with intrahepatic shunts had a significantly shorter follow-up period than dogs with extrahepatic shunts. Two prognostic indicators were identified, age at initial signs and blood urea concentration on presentation, both correlating with survival time. It was demonstrated that a significant proportion of dogs with portosystemic shunts managed medically have a good prognosis.