Lethal and sublethal responses of an aquatic insect Culex quinquefasciatus (Diptera: Culicidae) challenged with individual and joint exposure to dissolved sodium selenate and methylmercury chloride

Pollutants rarely occur alone in the natural environment, and few studies have focused on the potential interactions between metals or metalloids. In this study an aquatic insect, the southern house mosquito (Culex quinquefasciatus: Diptera), was used to test the individual and joint effects of dissolved sodium selenate (Se) and methyl mercury chloride (MeHg). We conducted ovipositional preference tests and 14-day chronic toxicity studies to determine lethal and sublethal responses of C. quinquefasciatus to a range of Se and MeHg concentrations and mixtures. No evidence was found for female ovipositional preference in field trials using artificial ponds. Larvae were more sensitive to MeHg than Se, with LC(50) values of 30 microg/L (CI = 28-31 microg/L) and 11 mg/L (CI = 10-12 mg/L) respectively. In addition, larval survival was significantly reduced at concentrations as low as 25 microg/L of MeHg and 8 mg/L of Se. A synergistic interaction was observed in the toxicity of the Se-MeHg mixtures to C. quinquefasciatus larvae. Larval mosquito survival was significantly reduced at 7.5 microg/L MeHg + 2.75 mg/L Se and an LC(50) value of 9 microg/L MeHg + 3.4 mg/L Se was determined for a fixed ratio mixture. The rate of growth of the larvae was analyzed using a Growth Index that provided a sensitive measure of the developmental effects of toxicant exposure. Sodium selenate at concentrations as low as 2 mg/L caused a significant decrease in growth between larvae in treatment versus control solutions after only 4 days. Similarly, MeHg at concentrations as low as 25 microg/L and a Se-MeHg mixture of 3 microg/L MeHg plus 1.1 mg/L Se caused significant growth reductions after only 2 and 3 days, respectively. These are the first reported survival and developmental data for an aquatic insect exposed to MeHg and Se-MeHg mixtures.     

Prenatal methylmercury exposure hampers glutathione antioxidant system ontogenesis and causes long-lasting oxidative stress in the mouse brain

During the perinatal period, the central nervous system (CNS) is extremely sensitive to metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-induced developmental neurotoxicity remains obscure, several studies point to the glutathione (GSH) antioxidant system as an important molecular target for this toxicant. To extend our recent findings of MeHg-induced GSH dyshomeostasis, the present study was designed to assess the developmental profile of the GSH antioxidant system in the mouse brain during the early postnatal period after in utero exposure to MeHg. Pregnant mice were exposed to different doses of MeHg (1, 3 and 10 mg/l, diluted in drinking water, ad libitum) during the gestational period. After delivery, pups were killed at different time points - postnatal days (PND) 1, 11 and 21 - and the whole brain was used for determining biochemical parameters related to the antioxidant GSH system, as well as mercury content and the levels of F(2)-isoprostane. In control animals, cerebral GSH levels significantly increased over time during the early postnatal period; gestational exposure to MeHg caused a dose-dependent inhibition of this developmental event. Cerebral glutathione peroxidase (GPx) and glutathione reductase (GR) activities significantly increased over time during the early postnatal period in control animals; gestational MeHg exposure induced a dose-dependent inhibitory effect on both developmental phenomena. These adverse effects of prenatal MeHg exposure were corroborated by marked increases in cerebral F(2)-isoprostanes levels at all time points. Significant negative correlations were found between F(2)-isoprostanes and GSH, as well as between F(2)-isoprostanes and GPx activity, suggesting that MeHg-induced disruption of the GSH system maturation is related to MeHg-induced increased lipid peroxidation in the pup brain. In utero MeHg exposure also caused a dose-dependent increase in the cerebral levels of mercury at birth. Even though the cerebral mercury concentration decreased to nearly basal levels at postnatal day 21, GSH levels, GPx and GR activities remained decreased in MeHg-exposed mice, indicating that prenatal exposure to MeHg affects the cerebral GSH antioxidant systems by inducing biochemical alterations that endure even when mercury tissue levels decrease and become indistinguishable from those noted in pups born to control dams. This study is the first to show that prenatal exposure to MeHg disrupts the postnatal development of the glutathione antioxidant system in the mouse brain, pointing to an additional molecular mechanism by which MeHg induces pro-oxidative damage in the developing CNS. Moreover, our experimental observation corroborates previous reports on the permanent functional deficits observed after prenatal MeHg exposure.     

Effects of methyl mercury on the microtubule system of mouse lymphocytes

The effects of in vivo and in vitro methyl mercury (MeHg) treatments on the microtubule system of murine splenic lymphocytes were examined by immunofluorescence microscopy. In vitro exposures to 1 to 10 microM MeHg resulted in time- and concentration-dependent microtubule disassembly. Lymphocytes isolated from mice receiving a single 10 mg/kg injection displayed microtubule damage when examined 2 and 5 days post-treatment. The capacity of in vivo and in vitro treated lymphocytes to respond to the mitogen concanavalin A (Con A) was generally inhibited by MeHg. There was a good correlation between the degree of microtubule disassembly and the inhibition of mitogen responsiveness. In vivo and in vitro treatments that resulted in extensive microtubule damage suppressed the ConA response and blocked lymphocytes early in the stimulation sequence. In vitro MeHg treatment late in mitogenesis caused a rapid, concentration-dependent inhibition of [3H]thymidine incorporation. These results suggest that damage to the microtubule system can serve as an indicator of MeHg toxicity and may underlie the toxicant's effects on lymphocyte functions.     

Delayed effects of embryonic exposure of zebrafish (Danio rerio) to methylmercury (MeHg)

Since previous short-term bioassays of methylmercury (MeHg) indicated no morphological effects in zebrafish (Danio rerio) after embryonic exposures below 20 microg/l MeHg, studies were done to determine whether embryonic exposure to MeHg at lower concentrations would induce behavioral effects. Newly fertilized embryos were exposed to 0, 5, 10 or 15 microg MeHg/l for selected exposure durations: single day, multiple day or continuous exposure from fertilization through hatching. Larvae were maintained in an essential salt solution after hatching. Spontaneous swimming performance and prey capture experiments were conducted. Continuous embryonic exposure to 15 microg/l caused delayed mortality syndrome (DMS). These larvae hatched normally and appeared normal, but beginning at Day 3 post-hatch (ph), general activity was severely reduced and by Day 5 ph, larvae were completely moribund; many had faint heartbeats, severely enlarged body cavities and upward flexures of the spinal cord. Most of these larvae were dead by Day 6 ph. Multi- and single-day embryonic exposures to 15 microg/l caused reduced swimming activity and prey capture ability, and by Day 4 ph, these larvae also began to show signs of DMS. Continuous embryonic exposure to 10 microg/l significantly reduced spontaneous swimming activity, which did not improve after 5 days in clean water. Similar results were seen in larvae exposed during the last 24 h of embryonic development. Prey capture ability was also impaired in larvae exposed continuously to 10 microg/l, even after 4 days in clean water. Single-day exposures to 10 microg/l did not affect prey capture ability. Larvae from the 5-microg/l exposures were not significantly different from controls for either parameter. This study reinforces the idea that functional impairment is a more subtle response to developmental toxicants than mortality or the production of morphological defects.     

Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure

Methylmercury (MeHg) is an environmental neurotoxicant which induces neuropathological changes in both the central nervous and peripheral sensory nervous systems. Our recent study demonstrated that down-regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which is known to promote neuritic extension, preceded MeHg-induced damage in cultured cortical neurons, suggesting that MeHg-mediated axonal degeneration is due to the disturbance of neuritic extension. Therefore we hypothesized that MeHg-induced axonal degeneration might be caused by neuritic extension/retraction incoordination. This idea brought our attention to the Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) pathway because it has been known to be associated with the development of axon and apoptotic neuronal cell death. Here we show that inhibition of the Rho/ROCK pathway prevents MeHg-intoxication both in vitro and in vivo. A Rho inhibitor, C3 toxin, and 2 ROCK inhibitors, Fasudil and Y-27632, significantly protected against MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured cortical neuronal cells exposed to 100 nM MeHg for 3 days. Furthermore, Fasudil partially prevented the loss of large pale neurons in dorsal root ganglia, axonal degeneration in dorsal spinal root nerves, and vacuolar degeneration in the dorsal columns of the spinal cord in MeHg-intoxicated model rats (20 ppm MeHg in drinking water for 28 days). Hind limb crossing sign, a characteristic MeHg-intoxicated sign, was significantly suppressed in this model. The results suggest that inhibition of the Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction incoordination and is effective for the prevention of MeHg-induced axonal degeneration and apoptotic neuronal cell death.     

Low level postnatal methylmercury exposure in vivo alters developmental forms of short-term synaptic plasticity in the visual cortex of rat

Methylmercury (MeHg) has been previously shown to affect neurotransmitter release. Short-term synaptic plasticity (STP) is primarily related to changes in the probability of neurotransmitter release. To determine if MeHg affects STP development, we examined STP forms in the visual cortex of rat following in vivo MeHg exposure. Neonatal rats received 0 (0.9% NaCl), 0.75 or 1.5 mg/kg/day MeHg subcutaneously for 15 or 30 days beginning on postnatal day 5, after which visual cortical slices were prepared for field potential recordings. In slices prepared from rats treated with vehicle, field excitatory postsynaptic potentials (fEPSPs) evoked by paired-pulse stimulation at 20–200 ms inter-stimulus intervals showed a depression (PPD) of the second fEPSP (fEPSP2). PPD was also seen in slices prepared from rats after 15 day treatment with 0.75 or 1.5 mg/kg/day MeHg. However, longer duration treatment (30 days) with either dose of MeHg resulted in paired-pulse facilitation (PPF) of fEPSP2 in the majority of slices examined. PPF remained observable in slices prepared from animals in which MeHg exposure had been terminated for 30 days after completion of the initial 30 day MeHg treatment, whereas slices from control animals still showed PPD. MeHg did not cause any frequency- or region-preferential effect on STP. Manipulations of [Ca²⁺]_e or application of the GABA_A receptor antagonist bicuculline could alter the strength and polarity of MeHg-induced changes in STP. Thus, these data suggest that low level postnatal MeHg exposure interferes with the developmental transformation of STP in the visual cortex, which is a long-lasting effect.     

Non-dioxin-like polychlorinated biphenyls interfere with neuronal differentiation of embryonic neural stem cells

Developmental exposure to food contaminants, such as polychlorinated biphenyls (PCBs), has been considered as a possible cause of neurodevelopmental disorders. We have investigated the effects of noncytotoxic concentrations of PCBs 153 and 180 on spontaneous differentiation of rat embryonic neural stem cells (NSCs). Upon removal of basic fibroblast growth factor to induce spontaneous differentiation, cells were exposed to 100 nM of the selected PCBs for 48 h and analyzed after 5 days. Both PCBs 153 and 180 induced a significant increase in the number of neurite-bearing Tuj1-positive cells with a concomitant decrease in proliferating cells, as detected by FUCCI transfection and EdU staining. Measurements of spontaneous Ca2? oscillations showed a decreased number of cells with Ca2? activity after PCB exposure, further confirming the increase in neuronal cells. Conversely, exposure to methylmercury (MeHg), which we evaluated in parallel, led to an increased number of cells with Ca2? activity, in agreement with the previously observed inhibition of neuronal differentiation. Analysis with quantitative PCR of the Notch pathway revealed that PCBs have a repressive action on Notch signaling, whereas MeHg activates it. Altogether, the data indicate that nanomolar concentrations of the selected non-dioxin-like PCBs and MeHg interfere in opposite directions with neuronal spontaneous differentiation of NSCs through Notch signaling. Combined exposures to PCBs and MeHg resulted in an induction of apoptosis and an antagonistic interaction on spontaneous neuronal differentiation. NSCs are further proven to be a valuable in vitro model to identify potential developmental neurotoxicants.     

In vivo methylmercury exposure induced long-lasting epileptiform activity in layer II/III neurons in cortical slices from the rat

Prenatal and postnatal methylmercury (MeHg) exposure has been shown to increase neuronal excitability and seizure susceptibility. To determine if early postnatal MeHg exposure causes a similar effect, we examined changes in field potentials in layer II/III neurons in cortical slices of rat following in vivo MeHg treatment. Rats received 0 (0.9% NaCl), 0.75 mg/kg/day or 1.5 mg/kg/day MeHg subcutaneously for 15 or 30 days beginning on postnatal day 5, after which cortical slices were prepared for field potential recordings. In slices from rats treated with vehicle, single pulse stimulation of layer IV of cortical slices induced a typical field excitatory postsynaptic potential (fEPSP) with a single spike. This type of fEPSPs was also seen in slices from rats with 15 day treatment with 0.75 mg/kg/day or 1.5 mg/kg/day MeHg. However, 30-day treatment with either MeHg dose resulted in fEPSPs with multiple spikes (epileptiform activity) in 40% of animals examined. This epileptiform activity remained observable in 50–60% animals in which MeHg exposure had been terminated for 30 days. However, slices from control animals still showed fEPSPs with single spike. Thus, these data suggest that postnatal MeHg exposure in vivo altered neuronal excitability and induced a long-lasting hyperexcitability in cortical neurons.     

Differing effects of toxicants (methylmercury, inorganic mercury, lead, amyloid β, and rotenone) on cultured rat cerebrocortical neurons: differential expression of rho proteins associated with neurotoxicity

Methylmercury (MeHg), inorganic mercury (IHg), lead (Pb), amyloid-β peptide (Aβ), and rotenone (RTN) are well-known toxicants. Here, we demonstrate that these five toxicants exhibit differing effects on cerebrocortical neurons. The concentration responsible for 30% loss of viability (EC30) values 3 days after exposure was approximately 100nM for MeHg, IHg, and RTN and 10μM for Aβ. Neuritic degeneration and subsequent apoptotic cell death were observed in these toxicant-treated cells. In contrast, the EC30 value 3 days after exposure to Pb was > 10μM. We clarified the differential expression of Ras homolog gene (Rho) family proteins (Ras-related C3 botulinum toxin substrate 1 [Rac1], cell division cycle 42, and Ras homolog gene family, member A [RhoA]) upon exposure to these five toxicants. Exposure to 100nM MeHg, IHg, or RTN downregulated the expression of Rac1, related to neuritic extension, but did not affect RhoA, related to retraction. At a higher concentration (1μM), IHg and RTN also acted through the suppression of Rac1, whereas increased MeHg toxicity was not associated with the expression of Rho family proteins. On the other hand, Pb and Aβ showed no effects on the expression of Rho proteins. Modification of the balance of neuritic extension and retraction by the suppression of Rho A rescued the neurotoxicity of 100nM MeHg, IHg, and RTN. The results indicate that the imbalance of neuritic extension and retraction by the suppression of Rac1 by 100nM MeHg, IHg, and RTN causes cerebrocortical neuron axonal degeneration and cell death. By contrast, the neurotoxicities of Pb, Aβ, and MeHg (at higher concentrations) are conferred by other toxic mechanisms.     

p21(WAF1/CIP1) inhibits cell cycle progression but not G2/M-phase transition following methylmercury exposure

Methylmercury (MeHg) is an environmentally prevalent organometal that is particularly toxic to the developing central nervous system (CNS). Prenatal MeHg exposure is associated with reduced brain size and weight and a reduced number of neurons, which have been associated with impaired cell proliferation. We evaluate the role of p21, a cell cycle protein involved in the G1- and G2-phase checkpoint control, in the cell cycle inhibition induced by MeHg. Primary mouse embryonic fibroblasts (MEFs) of different p21 genotypes (wild-type, heterozygous, and null) were isolated at day 14 of gestation and treated at passages 4-6 with either 0, 2, 4, or 6 microM MeHg or 50 nM colchicine for 24 h. Changes in cell cycle distribution after continuous toxicant treatment were analyzed by DNA content-based flow cytometry using DAPI. MeHg induced an increase in the proportion of cells in G2/M at 2 and 4 microM MeHg (p < or = 0.05) irrespective of p21 genotype. Effects of MeHg on cell cycle progression were subsequently evaluated using BrdU-Hoechst flow cytometric analysis. Inhibition of cell cycle progression was observed in all p21 genotypes after continuous exposure to MeHg for 24 and 48 h. p21 null (-/-) cells reached the second-round G1 at a higher fraction compared to the wild type (+/+) and heterozygous (+/-) cells (p < or = 0.05). These data support previous observations that MeHg inhibits cell cycle progression through delayed G2/M transition. Whereas the G2/M accumulation induced by MeHg was independent of p21 status, a greater proportion of p21(-/-) cells were able to complete one round of cell division in the presence of MeHg compared to p21(+/-) or p21(+/+) cells. These data suggest a role for p21 in retarding cell cycle progression, but not mitotic inhibition, following exposure to MeHg.     

Long-term effects of developmental exposure to low doses of PCB 126 and methylmercury

Methylmercury (MeHg) and polychlorinated biphenyls (PCBs) are food contaminants often found in fish. Experimental and epidemiological studies indicate that both PCBs and MeHg are developmental neurotoxicants, and some reports suggest that they may cause additive and/or synergistic neurotoxicity. We had previously investigated the effects of exposure to low doses of MeHg (0.5 mg/kg/day in drinking water) and PCB 126 (100 ng/kg/day in food) alone or in combination, from gestational day 7 to post-partum day 21, on neurobehavioral development in Wistar rats. The main finding was hyperactivity in male rats exposed to PCB 126, and in female animals exposed to PCB 126 + MeHg at 4 months of age (Vitalone et al., 2008). Since effects caused by developmental exposure may be exacerbated as the animal ages, aim of the present study was to investigate behavioral effects of the same developmental exposure to PCB 126 and/or MeHg up to the age of 20 months. Results indicate that aging did not enhance the behavioral effects of early exposures; however, behavioral alterations found in the first months of life in male rats exposed to PCB 126, or in female rats exposed to PCB 126 + MeHg, were persistent. Furthermore, an additional effect (increased body weight) was unmasked in adulthood in male rats exposed to PCB 126. These results indicate that developmental exposure to a low, environmentally relevant dose of PCB 126 causes long-lasting hyperactivity in male rats, and a significant increase in body weight.     

Methylmercury effects on reproduction and offspring size at birth

This article describes a study of the toxic, reproductive, and developmental effects of chronic methylmercury (MeHg) exposure in Macaca fascicularis monkeys. Adult and infant monkeys were studied using procedures to assess maternal and newborn blood Hg concentrations, menstrual cyclicity, conception rate, reproductive outcome, maternal toxicity, and offspring size at birth. Maternal intakes of 0, 50, 70, or 90 μg/kg/d MeHg hydroxide were studied. Maternal blood Hg concentrations reached equilibrium by 10 weeks of exposure. The half-life of blood Hg for adult females ranged from 15 to 40 days ( ) and did not vary with dose. Maternal MeHg exposure did not affect the length of the menstrual cycle or the conception rate. Maternal MeHg exposure did significantly reduce the number of viable deliveries at blood Hg concentrations above 1.5 ppm. Maternal blood Hg concentrations at delivery were significantly lower than newborn concentrations. No effect of maternal MeHg exposure on offspring size at birth was observed. Maternal toxicity was related to blood Hg concentrations above 2.0 ppm following approximately one year of exposure. Results indicate that MeHg exposure can affect reproductive outcome at levels that do not overt toxicity.     

Methylmercury effects on reproduction and offspring size at birth

This article describes a study of the toxic, reproductive, and developmental effects of chronic methylmercury (MeHg) exposure in Macaca fascicularis monkeys. Adult and infant monkeys were studied using procedures to assess maternal and newborn blood Hg concentrations, menstrual cyclicity, conception rate, reproductive outcome, maternal toxicity, and offspring size at birth. Maternal intakes of 0, 50, 70, or 90 micrograms/kg/d MeHg hydroxide were studied. Maternal blood Hg concentrations reached equilibrium by 10 weeks of exposure. The half-life of blood Hg for adult females ranged from 15 to 40 days (mean = d) and did not vary with dose. Maternal MeHg exposure did not affect the length of the menstrual cycle or the conception rate. Maternal MeHg exposure did significantly reduce the number of viable deliveries at blood Hg concentrations above 1.5 ppm. Maternal blood Hg concentrations at delivery were significantly lower than newborn concentrations. No effect of maternal MeHg exposure on offspring size at birth was observed. Maternal toxicity was related to blood Hg concentrations above 2.0 ppm following approximately one year of exposure. Results indicate that MeHg exposure can affect reproductive outcome at levels that do not cause overt toxicity.     

The effects of methylmercury on motor activity are sex- and age-dependent, and modulated by genetic deletion of adenosine receptors and caffeine administration

Adenosine and its receptors are, as part of the brain stress response, potential targets for neuroprotective drugs. We have investigated if the adenosine receptor system affects the developmental neurotoxicity caused by the fish pollutant methylmercury (MeHg). Behavioral outcomes of low dose perinatal MeHg exposure were studied in mice where the A(1) and A(2A) adenosine receptors were either partially blocked by caffeine treatment or eliminated by genetic modification (A(1)R and A(2A)R knock-out mice). From gestational day 7 to day 7 of lactation dams were administered doses that mimic human intake via normal diet, i.e. 1microM MeHg and/or 0.3g/l caffeine in the drinking water. This exposure to MeHg resulted in a doubling of brain Hg levels in wild type females and males at postnatal day 21 (PND21). Open field analysis was performed at PND21 and 2 months of age. MeHg caused time-dependent behavioral alterations preferentially in male mice. A decreased response to amphetamine in 2-month-old males pointed to disturbances in dopaminergic functions. Maternal caffeine intake induced long-lasting changes in the offspring evidenced by an increased motor activity and a modified response to psychostimulants in adult age, irrespectively of sex. Similar alterations were observed in A(1)R knock-out mice, suggesting that adenosine A(1) receptors are involved in the alterations triggered by caffeine exposure during development. Perinatal caffeine treatment and, to some extent, genetic elimination of adenosine A(1) receptors, attenuated the behavioral consequences of MeHg in males. Importantly, also deletion of the A(2A) adenosine receptor reduced the vulnerability to MeHg, consistent with the neuroprotective effects of adenosine A(2A) receptor inactivation observed in hypoxia and Parkinson's disease. Thus, the consequences of MeHg toxicity during gestation and lactation can be reduced by adenosine A(1) and A(2A) receptor inactivation, either via their genetic deletion or by treatment with their antagonist caffeine.     

Protective properties of quercetin against DNA damage and oxidative stress induced by methylmercury in rats

Aim of the study was to find out whether consumption of quercetin (QC), an abundant flavonoid in the human diet, protects against DNA damage caused by exposure to organic mercury. Therefore, rats were treated orally with methylmercury (MeHg) and the flavonoid with doses that reflect the human exposure. The animals received MeHg (30???g/kg/bw/day), QC (0.5?C50?mg/kg/bw/day), or combinations of both over 45?days. Subsequently, the glutathione levels (GSH) and the activities of glutathione peroxidase (GPx) and catalase (CAT) were determined, and DNA damage was measured in hepatocytes and peripheral leukocytes in single cell gel electrophoresis assays. MeHg decreased the concentration of GSH and the activity of GPx by 17 and 12%, respectively and caused DNA damage to liver and blood cells, while with QC no such effects were seen. When the flavonoid was given in combination with MeHg, the intermediate and the highest concentrations (5.0 and 50.0?mg/kg/bw/day) were found to cause DNA protection; DNA migration was reduced by 54 and 65% in the hepatocytes and by 27 and 36% in the leukocytes; furthermore, the reduction in GSH and GPx levels caused by MeHg treatment was restored. In summary, our results indicate that consumption of QC-rich foods may protect Hg-exposed humans against the adverse health effects of the metal.     

Accumulation of methylmercury or polychlorinated biphenyls in in vitro models of rat neuronal tissue

In vivo exposure levels for neurotoxicants are often reported in parts per million (ppm) concentration in tissue, whereas exposure levels in experiments utilizing in vitro models are most commonly reported in micromolar (muM) concentration in the exposure solution. The present experiments sought to determine whether or not in vitro solution concentration was an appropriate dose-metric for comparison to in vivo tissue levels for lipophilic compounds. To do so, the accumulation of the polychlorinated biphenyl (PCB) mixture Aroclor 1254 (A1254) or methylmercury (MeHg) was examined in three commonly utilized in vitro neuronal tissue models: nerve growth factor differentiated pheochromocytoma (PC12) cells, primary cultures of rat neocortical cells, and adult rat hippocampal slices. Tissues were exposed to A1254 (0.65 ppm) or to MeHg (0.0033-0.33 ppm) in serum-free media for 1 or 24 h. Total PCB or mercury accumulation was measured by dual column gas chromatography with electron capture detection or by cold vapor atomic absorption, respectively. PC12 cells accumulated 66.7 and 103.8 ppm PCBs after 1 and 24 h exposure to A1254. Neocortical neurons also accumulated significant concentrations of PCBs, but less so than PC12 cells. After 1 h exposure to 0.65 ppm A1254, slices contained 3.46 and 0.81 ppm PCBs when exposed in a static and perfused system, respectively. After 1 h exposure to 0.0033, 0.033, and 0.33 ppm MeHg, PC12 cells contained 0.3, 2.2, and 17.7 ppm mercury, respectively; after 24 h, PC12 cells contained 0.4, 2.8, and 21.9 ppm. Hippocampal slices accumulated 1.7 and 4.8 ppm mercury after 1 and 3 h exposure to 0.33 ppm MeHg. For comparison, mercury accumulation in rat fetal and pup brain tissue after maternal exposure [0, 0.1, 1.0, or 2.0 mg/kg/day MeHg from gestational day (GD) 6-15] ranged from 0.05 to 7.89 ppm in 0.1 mg/kg dose animals on postnatal day 10 and 2.0 mg/kg dose animals on GD16, respectively. These results demonstrate that accumulation of PCBs and MeHg in vitro is tissue-, time-, and concentration-dependent and indicates that tissue levels rather than exposure concentrations are a more appropriate metric for comparison of in vitro to in vivo effects.     

Butenenitriles have low axonopathic potential in the rat

IDPN (3,3'-iminodipropionitrile) causes a neurofilamentous proximal axonopathy. This study addressed the hypothesis that the butenenitriles (allylnitrile, cis-crotononitrile and trans-crotononitrile) have an IDPN-like axonopathic potential. First, male adult rats were exposed (i.p.) to IDPN, allylnitrile, cis-crotononitrile or trans-crotononitrile at 3.25 mmol/kg/day, 0.89 mmol/kg/day, 1.79 mmol/kg/day, or 3.75 mmol/kg/day for 3 consecutive days, respectively; lumbar dorsal root ganglia were examined for axonal swelling eight days after dosing. IDPN caused axonal swelling, a few swollen axons were recorded in one trans-crotononitrile animal, and no axonal abnormalities were observed following cis-crotononitrile or allylnitrile. To further evaluate trans-crotononitrile, additional rats were given this nitrile through a 10-day i.p. dosing schedule (2.5 mmol/kg/day, 2.75 mmol/kg/day, 3.0 mmol/kg/day or 3.25 mmol/kg/day) or a 9-week drinking water exposure (12.3, 24.6 and 49.1mM, three weeks each), and examined by light and electron microscopy. Semithin sections revealed no overt swelling in axons from several locations of the nervous system after trans-crotononitrile; quantitative analysis in the L5 dorsal root ganglion showed no increase in proximal axon diameter in comparison to control animals. At the transmission electron microscopy level, pathological effects were mild; they were mostly found in the animals submitted to the 10-day dosing regimen, and did not include evidence of significant axonal swelling. Although an axonopathic potential for the three unsaturated 4-carbon nitriles cannot be excluded, the present data indicated that this potential is significantly lower than that of IDPN.     

Chlorpyrifos exerts opposing effects on axonal and dendritic growth in primary neuronal cultures

Evidence that children are widely exposed to organophosphorus pesticides (OPs) and that OPs cause developmental neurotoxicity in animal models raises significant concerns about the risks these compounds pose to the developing human nervous system. Critical to assessing this risk is identifying specific neurodevelopmental events targeted by OPs. Observations that OPs alter brain morphometry in developing rodents and inhibit neurite outgrowth in neural cell lines suggest that OPs perturb neuronal morphogenesis. However, an important question yet to be answered is whether the dysmorphogenic effect of OPs reflects perturbation of axonal or dendritic growth. We addressed this question by quantifying axonal and dendritic growth in primary cultures of embryonic rat sympathetic neurons derived from superior cervical ganglia (SCG) following in vitro exposure to chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and trichloropyridinol (TCP). Axon outgrowth was significantly inhibited by CPF or CPFO, but not TCP, at concentrations > or =0.001 microM or 0.001 nM, respectively. In contrast, all three compounds enhanced BMP-induced dendritic growth. Acetylcholinesterase was inhibited only by the highest concentrations of CPF (> or =1 microM) and CPFO (> or =1 nM); TCP had no effect on this parameter. In summary, these compounds perturb neuronal morphogenesis via opposing effects on axonal and dendritic growth, and both effects are independent of acetylcholinesterase inhibition. These findings have important implications for current risk assessment practices of using acetylcholinesterase inhibition as a biomarker of OP neurotoxicity and suggest that OPs may disrupt normal patterns of neuronal connectivity in the developing nervous system.     

Early acute necrosis and delayed apoptosis induced by methyl mercury in murine peritoneal neutrophils

There is growing evidence that heavy metals in general, and mercurial compounds in particular, are immunotoxic. The purpose of this study was to explore the mechanism of MeHg in inducing cell death of mouse peritoneal neutrophils. In this paper we demonstrate that MeHg induces apoptosis and necrosis depending on MeHg concentration. In vitro exposure of mouse peritoneal neutrophils to MeHg resulted in a time- and concentration-dependent cell death. MeHg (15 microM) induced neutrophil necrosis in 13 min. The type of cell death was attributed to necrosis based on cells permeable to the fluorescent dye, propidium iodide and DNA appeared as a smear. With fura-2 microfluorimetric technique, we found that the entry of external Ca2+ into the cytosol played a crucial role in inducing cell necrosis by 15 microM MeHg. However, at lower concentrations, MeHg (10 microM)-induced apoptosis is confirmed by the observation of morphological features characterised by apoptotic bodies and fragmented DNA ladder. MeHg (10 microM) caused an immediate fall in pHi as revealed by the pH-sensitive fluorescent probe 2'7'-bis (carboxyethyl)-5(6)-carboxyfluorescein. We have found that MeHg induced cellular acidification prior to DNA fragmentation so as the other two apoptosis-inducing agents (ZnCl(2) and EGTA). Furthermore, acid-activated endonuclease was increased by MeHg in neutrophils, which we considered to play a possible role in chromatin digestion leading to apoptosis. Taken together, these findings indicate that MeHg induces necrosis at higher concentrations by a rapid increase of [Ca2+]i and apoptosis at lower concentrations by acid activation of endonuclease.     

Influence of cypermethrin toxicity on ionic regulation and gill Na(+)/K(+)-ATPase activity of a freshwater teleost fish Cyprinus carpio

The effects of acute and sublethal toxicity of cypermethrin, a synthetic pyrethroid insecticide on plasma electrolytes (Na(+), K(+) and Cl(-)) levels and gill Na(+)/K(+)-ATPase activity in a common carp Cyprinus carpio were examined. The 24h LC(50) value of cypermethrin for C. carpio was 1.86ppm. During acute exposure, cypermethrin caused adverse effects in the form of hyponatreima, hypokalemia and hypochloremia and inhibition of gill Na(+)/K(+)-ATPase activity. In sublethal exposure to cypermethrin (0.186ppm for 35 days), plasma Na(+) was decreased throughout the exposure period except 7th day whereas plasma K(+) level was increased up to 28th day, then declined. However, plasma Cl(-) level was decreased. Likewise, gill Na(+)/K(+)-ATPase activity was decreased as the exposure period extended. The present study indicates that cypermethrin was highly toxic to freshwater fish and ion levels in blood as measured by specific ion concentrations (Na(+), K(+) and Cl(-)) and changes in gill Na(+)/K(+)-ATPase activity may represent a sensitive and useful nonspecific biomarkers of chemical exposure and effects.