Neonatal exposure to parathion alters lipid metabolism in adulthood: Interactions with dietary fat intake and implications for neurodevelopmental deficits.

Organophosphates are developmental neurotoxicants but recent evidence also points to metabolic dysfunction. We determined whether neonatal parathion exposure in rats has long-term effects on regulation of adipokines and lipid peroxidation. We also assessed the interaction of these effects with increased fat intake. Rats were given parathion on postnatal days 1-4 using doses (0.1 or 0.2mg/kg/day) that straddle the threshold for barely detectable cholinesterase inhibition and the first signs of systemic toxicity. In adulthood, animals were either maintained on standard chow or switched to a high-fat diet for 7 weeks. We assessed serum leptin and adiponectin, tumor necrosis factor-alpha (TNFalpha) in adipose tissues, and thiobarbituric acid reactive species (TBARS) in peripheral tissues and brain regions. Neonatal parathion exposure uncoupled serum leptin levels from their dependence on body weight, suppressed adiponectin and elevated TNFalpha in white adipose tissue. Some of the effects were offset by a high-fat diet. Parathion reduced TBARS in the adipose tissues, skeletal muscle and temporal/occipital cortex but not in heart, liver, kidney or frontal/parietal cortex; it elevated TBARS in the cerebellum; the high-fat diet again reversed many of the effects. Neonatal parathion exposure disrupts the regulation of adipokines that communicate metabolic status between adipose tissues and the brain, while also evoking an inflammatory adipose response. Our results are consistent with impaired fat utilization and prediabetes, as well as exposing a potential relationship between effects on fat metabolism and on synaptic function in the brain.

Neonatal parathion exposure disrupts serotonin and dopamine synaptic function in rat brain regions: modulation by a high-fat diet in adulthood.

The consequences of exposure to developmental neurotoxicants are influenced by environmental factors. In the present study, we examined the role of dietary fat intake. We administered parathion to neonatal rats and then evaluated whether a high-fat diet begun in adulthood could modulate the persistent effects on 5HT and DA systems. Neonatal rats received parathion on postnatal days 1-4 at 0.1 or 0.2 mg/kg/day, straddling the cholinesterase inhibition threshold. In adulthood, half the animals in each exposure group were given a high-fat diet for 8 weeks. We assessed 5HT and DA concentrations and turnover in brain regions containing their respective cell bodies and projections. In addition, we monitored 5HT1A and 5HT2 receptor binding and the concentration of 5HT presynaptic transporters. Neonatal parathion exposure evoked widespread increases in neurotransmitter turnover, indicative of presynaptic hyperactivity, further augmented by 5HT receptor upregulation. In control rats, consumption of a high-fat diet recapitulated many of the changes seen with neonatal parathion exposure; the effects represented convergent mechanisms, since the high-fat diet often obtunded further increases caused by parathion. Neonatal parathion exposure causes lasting hyperactivity of 5HT and DA systems accompanied by 5HT receptor upregulation, consistent with "miswiring" of neuronal projections. A high-fat diet obtunds the effect of parathion, in part by eliciting similar changes itself. Thus, dietary factors may produce similar synaptic changes as do developmental neurotoxicants, potentially contributing to the increasing incidence of neurodevelopmental disorders.

Consumption of a high-fat diet in adulthood ameliorates the effects of neonatal parathion exposure on acetylcholine systems in rat brain regions.

BACKGROUND: Developmental exposure to a wide variety of developmental neurotoxicants, including organophosphate pesticides, evokes late-emerging and persistent abnormalities in acetylcholine (ACh) systems. We are seeking interventions that can ameliorate or reverse the effects later in life. OBJECTIVES: We administered parathion to neonatal rats and then evaluated whether a high-fat diet begun in adulthood could reverse the effects on ACh systems. METHODS: Neonatal rats received parathion on postnatal days 1-4 at 0.1 or 0.2 mg/kg/day, straddling the cholinesterase inhibition threshold. In adulthood, half the animals were switched to a high-fat diet for 8 weeks. We assessed three indices of ACh synaptic function: nicotinic ACh receptor binding, choline acetyltransferase activity, and hemicholinium-3 binding. Determinations were performed in brain regions comprising all the major ACh projections and cell bodies. RESULTS: Neonatal parathion exposure evoked widespread abnormalities in ACh synaptic markers, encompassing effects in brain regions possessing ACh projections and ACh cell bodies. In general, males were affected more than females. Of 17 regional ACh marker abnormalities (10 male, 7 female), 15 were reversed by the high-fat diet. CONCLUSIONS: A high-fat diet reverses neurodevelopmental effects of neonatal parathion exposure on ACh systems. This points to the potential for nonpharmacologic interventions to offset the effects of developmental neurotoxicants. Further, cryptic neurodevelopmental deficits evoked by environmental exposures may thus engender a later preference for a high-fat diet to maintain normal ACh function, ultimately contributing to obesity.

Selective and irreversible inhibitors of aphid acetylcholinesterases: steps toward human-safe insecticides.

Aphids, among the most destructive insects to world agriculture, are mainly controlled by organophosphate insecticides that disable the catalytic serine residue of acetylcholinesterase (AChE). Because these agents also affect vertebrate AChEs, they are toxic to non-target species including humans and birds. We previously reported that a cysteine residue (Cys), found at the AChE active site in aphids and other insects but not mammals, might serve as a target for insect-selective pesticides. However, aphids have two different AChEs (termed AP and AO), and only AP-AChE carries the unique Cys. The absence of the active-site Cys in AO-AChE might raise concerns about the utility of targeting that residue. Herein we report the development of a methanethiosulfonate-containing small molecule that, at 6.0 microM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum) without any measurable inhibition of the human AChE. Reactivation studies using beta-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in aphids, thus offering new insight into the relative functional importance of the two insect AChEs. More importantly, by demonstrating that the Cys-targeting inhibitor can abolish AChE activity in aphids, we can conclude that the unique Cys may be a viable target for species-selective agents to control aphids without causing human toxicity and resistance problems.

Is fipronil safer than chlorpyrifos? Comparative developmental neurotoxicity modeled in PC12 cells.

Fipronil, a GABA(A) receptor antagonist, is replacing many insecticide uses formerly fulfilled by organophosphates like chlorpyrifos. Few studies have addressed the potential for fipronil to produce developmental neurotoxicity. We compared the neurotoxicity of fipronil and chlorpyrifos in undifferentiated and differentiating neuronotypic PC12 cells, evaluating indices of cell replication, cell number, differentiation, and viability for short- and long-term exposures. Fipronil inhibited DNA and protein synthesis in undifferentiated PC12 cells and evoked oxidative stress to a greater extent than did chlorpyrifos, resulting in reduced cell numbers even though cell viability was maintained. In differentiating cells, fipronil displayed an even lower threshold for disruption of development, reducing cell numbers without impairing cell growth, and promoting emergence of neurotransmitter phenotypes; superimposed on this effect, the phenotypic balance was shifted in favor of dopamine as opposed to acetylcholine. Differentiation also enhanced the susceptibility to fipronil-induced oxidative stress, although antioxidant administration failed to provide protection from cell loss. At low concentrations maintained for prolonged periods, fipronil had a biphasic effect on cell numbers, increasing them slightly at low concentrations, implying interference with apoptosis, while nevertheless reducing cell numbers at higher concentrations. Our results suggest that fipronil is inherently a more potent disruptor of neuronal cell development than is chlorpyrifos. The neurodevelopmental effects are not predicated on GABA(A) antagonist properties, since PC12 cells lack the GABA(A) receptor. If fipronil is intended to provide greater safety than chlorpyrifos, then this will have to entail advantages from factors that are yet unexamined: exposure, persistence, pharmacokinetics.

Exposure of neonatal rats to parathion elicits sex-selective reprogramming of metabolism and alters the response to a high-fat diet in adulthood.

BACKGROUND: Developmental exposures to organophosphate pesticides are virtually ubiquitous. These agents are neurotoxicants, but recent evidence also points to lasting effects on metabolism. OBJECTIVES: We administered parathion to neonatal rats. In adulthood, we assessed the impact on weight gain, food consumption, and glucose and lipid homeostasis, as well as the interaction with the effects of a high-fat diet. METHODS: Neonatal rats were given parathion on postnatal days 1-4 using doses (0.1 or 0.2 mg/kg/day) that straddle the threshold for barely detectable cholinesterase inhibition and the first signs of systemic toxicity. In adulthood, animals were either maintained on standard lab chow or switched to a high-fat diet for 7 weeks. RESULTS: In male rats on a normal diet, the low-dose parathion exposure caused increased weight gain but also evoked signs of a prediabetic state, with elevated fasting serum glucose and impaired fat metabolism. The higher dose of parathion reversed the weight gain and caused further metabolic defects. Females showed greater sensitivity to metabolic disruption, with weight loss at either parathion dose, and greater imbalances in glucose and lipid metabolism. At 0.1 mg/kg/day parathion, females showed enhanced weight gain on the high-fat diet; This effect was reversed in the 0.2-mg/kg/day parathion group, and was accompanied by even greater deficits in glucose and fat metabolism. CONCLUSIONS: Neonatal low-dose parathion exposure disrupts glucose and fat homeostasis in a persistent and sex-selective manner. Early-life toxicant exposure to organophosphates or other environmental chemicals may play a role in the increased incidence of obesity and diabetes.

Rats gain excess weight after developmental exposure to the organophosphorothionate pesticide, chlorpyrifos.

Pesticides that target molecules with critical roles in brain function deserve careful scrutiny for potential developmental neurotoxicity. In this study, time-pregnant rats were dosed daily by gavage with chlorpyrifos (2.5 mg/kg) from gestational day 7 through the end of lactation on postnatal day 21 (PND 21), and offspring were weighed regularly from birth until brain harvest at PND 22 or young adulthood (PND 95-101). The chlorpyrifos exposure caused excess weight gain in males beginning at PND 45 and reaching levels 10.5% above control by PND 72, while volumetric measurements showed that the exposed males were also 12% larger than controls. The body weight response showed an inverted U-shaped relation to chlorpyrifos dose. These data suggest delayed disturbances in body weight and density as previously unsuspected adverse consequences of developmental exposure to an environmental pesticide. Although we do not regard our findings as definitive evidence that chlorpyrifos exposure is a risk factor for obesity, the potential implications nonetheless deserve serious consideration.

A method to determine precise benchmark doses for carbamate anticholinesterases.

In determining benchmark doses for risk assessment and regulation of carbamate anticholinesterase pesticides like formetanate, oxamyl, and methomyl, one needs to quantitate low levels of cholinesterase inhibition. For improved accuracy while using fewer subjects, we developed an assay based on the recognized ability of carbamates to protect cholinesterase from irreversible inactivation. This assay measures enzyme that survives diisopropylfluorophosphate exposure in vitro and then reactivates by decarbamylation after small molecules are removed with size-exclusion centrifugation. The 99% silencing of unprotected cholinesterase yields a low background. Comparisons of recovered activity with initial activity (representing carbamate-free enzyme) use each sample as its own control. As a result, carbamate-protection assays can demonstrate a statistically significant 2-3% inhibition of brain cholinesterase in a single experimental group of modest size. When applied to brain samples from formetanate-treated rats, such an assay predicted a benchmark dose of 0.19 mg/kg for 10% inhibition (BMD10), with a lower 95% confidence limit of 0.15 mg/kg (BMDL10). Protection assays should enable precise determinations of benchmark doses for other carbamates, as well as accurate assessment of in vivo inhibition half-lives under low-dose scenarios.

Automated measurement of acetylcholinesterase activity in rat peripheral tissues.

The accepted mechanism of toxicity of many organophosphorous and carbamate insecticides is inhibition of acetylcholinesterase activity. In mammals, part of the toxicity assessment usually includes monitoring blood and/or brain acetylcholinesterase inhibition. Other tissues, however, contain cholinesterase activity (i.e. acetyl- and butyryl-cholinesterase), and the inhibition of that activity may be informative for a full appraisal of the toxicity profile. The present group of studies first optimized the variables for extraction and solubilization of cholinesterase activity from various rat tissues and then refined an existing automated method, in order to differentially assess acetyl and butyrylcholinesterase activity in those tissues. All these studies were conducted using tissues from untreated, Long-Evans, adult rats. The first studies determined the effect of Triton X-100 or salt (NaCl) on the extraction and solubilization of cholinesterase activity from retina, brain, striated muscle, diaphragm, and heart: phosphate buffer plus detergent (1% Triton X-100) yielded the highest activity for most tissues. For striated muscle, however, slightly more activity was extracted if the phosphate buffer contained both 1% Triton X-100 and 0.5 M NaCl. It was also noted that the degree of homogenization of some tissues (e.g. striated muscle) must be increased for maximal solubilization of all cholinesterase activity. Subsequent studies developed a method for assessing the level of acetylcholinesterase, butyrylcholinesterase and total cholinesterase activity in these tissues using an automated analyzer. In conclusion, automated assay of acetylcholinesterase activity in cholinergically innervated tissues in the rat (other than brain) is achievable and relatively convenient.