The intertwining between lead and ethanol in the model organism Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) is a model organism widely used to evaluate the mechanistic aspects of toxicants with the potential to predict responses comparable to those of mammals. We report here the consequences of developmental lead (Pb) exposure on behavioral responses to ethanol (EtOH) in C. elegans. In addition, we present data on morphological alterations in the dopamine (DA) synapse and DA-dependent behaviors aimed to dissect the neurobiological mechanisms that underlie the relationship between these neurotoxicants. Finally, the escalation to superior animals that parallels the observed effects in both experimental models with references to EtOH metabolism and oxidative stress is also discussed. Overall, the literature revised here underpins the usefulness of C. elegans to evidence behavioral responses to a combination of neurotoxicants in mechanistic-orientated studies.

Blood lead levels and enzymatic biomarkers of environmental lead exposure in children in Cordoba, Argentina, after the ban of leaded gasoline.

Lead (Pb) is a developmental neurotoxicant found in industrial activities, many of them already prohibited worldwide. This study aimed to evaluate current blood Pb (PbB) levels in children in Cordoba, Argentina, and to compare these with similar studies performed before Pb was banned in gasoline in 1996. We also sought to identify mechanistically relevant biomarkers by measuring δ-aminolevulinic acid dehydratase (δ-ALAD), superoxide dismutase (SOD), and catalase (CAT) activities. We finally aimed to determine whether sociodemographic characteristics are associated with Pb toxicity. Blood samples collected from 161 healthy children between September 2009 and February 2010 revealed mean PbB levels of 2.58 ± 0.30 µg/dl. Enzymatic δ-ALAD, CAT, and SOD activities showed no significant variations when plotted against PbB levels. Finally, children living in the suburbs have higher PbB levels than their city counterparts, while low socioeconomic status increased δ-ALAD inhibition compared with that of middle-income children. Overall, these results evidenced a substantial reduction in exposure to Pb in this pediatric population over a decade after Pb was restricted in gasoline and reveal the importance of pursuing novel biomarkers of toxicity along with the sociodemographic profile to complement Pb diagnosis.

Enhanced stimulus sequence-dependent repeated learning in male offspring after prenatal stress alone or in conjunction with lead exposure.

Both lead (Pb) exposure and prenatal stress (PS) can produce cognitive deficits, and in a prior study we demonstrated enhanced cognitive deficits in repeated learning of female rats exposed to both of these developmental insults (Cory-Slechta et al., 2010). However, PS can also lead to improved cognitive outcomes that are both gender- and context-dependent. Thus, the current study examined whether Pb ± PS likewise produced repeated learning deficits in males, either after maternal or lifetime Pb exposure. Repeated learning was evaluated using a multiple schedule of repeated learning and performance that required learning 3-response sequences in male offspring that had been subjected to either maternal Pb (0 or 150 ppm) or lifetime Pb exposure (0 or 50 ppm) beginning two months prior to dam breeding, to prenatal immobilization restraint stress (gestational days 16-17), or to both Pb and PS. Blood Pb, corticosterone, hippocampal glucocorticoid receptor density and brain monoamines were also measured. In contrast to outcomes in females, sequence-specific enhancements of repeated learning accuracy were produced by PS, particularly when combined with Pb, results that appeared to be more robust in combination with lifetime than maternal Pb exposure. A common behavioral mechanism of these improvements appears to be an increased reinforcement density associated with increased response rates and shorter session times seen with PS ± Pb that could shorten time to reinforcement. Trends toward lower levels of nucleus accumbens dopamine activity seen after both maternal Pb and lifetime Pb combined with PS suggest a possible role for this region/neurotransmitter in enhanced accuracy, whereas PS ± Pb-induced corticosterone changes did not exhibit an obvious systematic relationship to accuracy enhancements. While PS ± Pb-based increases in accuracy appear to be an improved outcome, the benefits of increased response rate are by no means universal, but highly context-dependent and can lead to adverse behavioral effects in other conditions.

Reduction in hospital admissions for acute coronary syndrome after the successful implementation of 100% smoke-free legislation in Argentina: a comparison with partial smoking restrictions.

BACKGROUND: Several studies have shown a decrease in acute coronary syndrome (ACS) admissions after the implementation of 100% smoke-free legislation. However, no studies have been conducted in developing countries. METHODS: We conducted a time series analysis of ACS hospital admissions in Santa Fe province and Buenos Aires city, Argentina. In 2006, Santa Fe implemented a 100% smoke-free law and Buenos Aires implemented a partial law with designated smoking areas and exceptions. Age-standardised ACS admissions rates were compared before and after the implementation of the laws in each district. Smoking prevalence, compliance with legislation and exposure to secondhand smoke (SHS) was also assessed in both districts. RESULTS: In Santa Fe an immediate decrease in ACS admissions was observed after implementation (-2.5 admissions per 100,000, p=0.03; 13% reduction), compared with no change in Buenos Aires city (rate ratio Santa Fe vs Buenos Aires: 0.74, 95% CI 0.63 to 0.86, p≤0.001). In Santa Fe, the immediate effect was followed by a persistent decrease in admissions due to ACS (-0.26 admissions per 100,000 per month). Smoking prevalence did not change significantly in either district during the same period. In both districts, there was a reduction in self-reported SHS exposure, with a trend towards lower exposure in Santa Fe province. No other comprehensive tobacco control interventions were implemented during the study period. CONCLUSIONS: A 100% smoke-free law was more effective than a partial restriction law in reducing ACS admissions. An immediate effect was followed by a sustained decrease in ACS admissions. Smoke-free initiatives can be also effective in decreasing acute coronary events in developing countries.

Interactions of lifetime lead exposure and stress: behavioral, neurochemical and HPA axis effects.

Lead (Pb) and stress co-occur as risk factors, share biological substrates and produce common adverse effects. We previously found that prenatal restraint stress (PS) or offspring stress (OS) could enhance maternal Pb-induced behavioral, brain neurotransmitter level and HPA axis changes. The current study examined how lifetime Pb exposure, consistent with human environmental exposure, interacts with stress. Dams were exposed to Pb beginning 2 mos prior to breeding (0, 50 or 150ppm in drinking water), PS on gestational days 16 and 17, or the combination. Offspring continued on the same Pb exposure as the dam. A subset of Pb+PS offspring also received 3 additional stress challenges (OS), yielding 9 exposure groups/gender: 0-NS, 0-PS, 0-OS, 50-NS, 50-PS, 50-OS, 150-NS, 150-PS and 150-OS. As with maternal Pb (Virgolini et al., 2008a), lifetime Pb and stress influenced Fixed Interval (FI) behavior primarily in females. Relative to 0-NS control, reductions in postreinforcement pause (PRP) times were seen only with combined Pb+PS (50-PS, 50-OS, 150-PS). Stress increased FI response rates when Pb alone was without effect (150-PS, 150-OS), but gradually mitigated rate increases produced by Pb alone (50-PS, 50-OS), effects that appear to be due primarily to PS, as they were of comparable magnitude in PS and OS groups. Individual subject data suggest that enhanced Pb and PS effects reflect increasing numbers of subjects shifting to the high end of the normal range of FI performance values, consistent with a dose-response type of Pb+stress additivity. Consistent with reports of cortico-striatal mediation of both interval timing (PRP) and FI rates, principal component analyses suggested potential mediation via altered frontal cortex norepinephrine, reduced nucleus accumbens dopaminergic control and enhanced striatal monoamine control. Altered FI performance, whether occurring through changes in response rate, PRP, or both, represent behavioral inefficiency and potentially sub-optimal or even dysfunctional resource/energy use.

Experimental manipulations blunt time-induced changes in brain monoamine levels and completely reverse stress, but not Pb+/-stress-related modifications to these trajectories.

This study sought to further understand how environmental conditions influence the outcomes of early developmental insults. It compared changes in monoamine levels in frontal cortex, nucleus accumbens and striatum of male and female Long-Evans rat offspring subjected to maternal Pb exposure (0, 50 or 150ppm in drinking water from 2 months pre-breeding until pup weaning)+/-prenatal (PS) (restraint on GD16-17) or PS+offspring stress (OS; three variable stress challenges to young adults) determined at 2 months of age and at 6 months of age in littermates subsequently exposed either to experimental manipulations (EM: daily handling and performance on an operant fixed interval (FI) schedule of food reward), or to no experience (NEM; time alone). Time alone (NEM conditions), even in normal (control) animals, modified the trajectory of neurochemical changes between 2 and 6 months across brain regions and monoamines. EM significantly modified the NEM trajectories, and except NE and striatal DA, which increased, blunted the changes in monoamine levels that occurred over time alone. Pb+/-stress modified the trajectory of monoamine changes in both EM and NEM conditions, but these predominated under NEM conditions. Stress-associated modifications, occurring mainly with NEM OS groups, were fully reversed by EM procedures, while reversals of Pb+/-stress-associated modifications occurred primarily in nucleus accumbens, a region critical to mediation of FI response rates. These results extend the known environmental conditions that modify developmental Pb+/-stress insults, which is critical to ultimately understanding whether early insults lead to adaptive or maladaptive behavior and to devising behavioral therapeutic strategies. That time alone and a set of EM conditions typically used as outcome measures in intervention studies can themselves invoke neurochemical changes, moreover, has significant implications for experimental design of such studies.

Alterations in glucocorticoid negative feedback following maternal Pb, prenatal stress and the combination: a potential biological unifying mechanism for their corresponding disease profiles.

Combined exposures to maternal lead (Pb) and prenatal stress (PS) can act synergistically to enhance behavioral and neurochemical toxicity in offspring. Maternal Pb itself causes permanent dysfunction of the body's major stress system, the hypothalamic pituitary adrenal (HPA) axis. The current study sought to determine the potential involvement of altered negative glucocorticoid feedback as a mechanistic basis of the effects in rats of maternal Pb (0, 50 or 150 ppm in drinking water beginning 2 mo prior to breeding), prenatal stress (PS; restraint on gestational days 16-17) and combined maternal Pb+PS in 8 mo old male and female offspring. Corticosterone changes were measured over 24 h following an i.p. injection stress containing vehicle or 100 or 300 microg/kg (females) or 100 or 150 microg/kg (males) dexamethasone (DEX). Both Pb and PS prolonged the time course of corticosterone reduction following vehicle injection stress. Pb effects were non-monotonic, with a greater impact at 50 vs. 150 ppm, particularly in males, where further enhancement occurred with PS. In accord with these findings, the efficacy of DEX in suppressing corticosterone was reduced by Pb and Pb+PS in both genders, with Pb efficacy enhanced by PS in females, over the first 6 h post-administration. A marked prolongation of DEX effects was found in males. Thus, Pb, PS and Pb+PS, sometimes additively, produced hypercortisolism in both genders, followed by hypocortisolism in males, consistent with HPA axis dysfunction. These findings may provide a plausible unifying biological mechanism for the reported links between Pb exposure and stress-associated diseases and disorders mediated via the HPA axis, including obesity, hypertension, diabetes, anxiety, schizophrenia and depression. They also suggest broadening of Pb screening programs to pregnant women in high stress environments.

Influence of low level maternal Pb exposure and prenatal stress on offspring stress challenge responsivity.

We previously demonstrated potentiated effects of maternal Pb exposure producing blood Pb(PbB) levels averaging 39microg/dl combined with prenatal restraint stress (PS) on stress challenge responsivity of female offspring as adults. The present study sought to determine if: (1) such interactions occurred at lower PbBs, (2) exhibited gender specificity, and (3) corticosterone and neurochemical changes contributed to behavioral outcomes. Rat dams were exposed to 0, 50 or 150ppm Pb acetate drinking water solutions from 2 mos prior to breeding through lactation (pup exposure ended at weaning; mean PbBs of dams at weaning were <1, 11 and 31microg/dl, respectively); a subset in each Pb group underwent prenatal restraint stress (PS) on gestational days 16-17. The effects of variable intermittent stress challenge (restraint, cold, novelty) on Fixed Interval (FI) schedule controlled behavior and corticosterone were examined in offspring when they were adults. Corticosterone changes were also measured in non-behaviorally tested (NFI) littermates. PS alone was associated with FI rate suppression in females and FI rate enhancement in males; Pb exposure blunted these effects in both genders, particularly following restraint stress. PS alone produced modest corticosterone elevation following restraint stress in adult females, but robust enhancements in males following all challenges. Pb exposure blunted these corticosterone changes in females, but further enhanced levels in males. Pb-associated changes showed linear concentration dependence in females, but non-linearity in males, with stronger or selective changes at 50ppm. Statistically, FI performance was associated with corticosterone changes in females, but with frontal cortical dopaminergic and serotonergic changes in males. Corticosterone changes differed markedly in FI vs. NFI groups in both genders, demonstrating a critical role for behavioral history and raising caution about extrapolating biochemical markers across such conditions. These findings demonstrate that maternal Pb interacts with prenatal stress to further modify both behavioral and corticosterone responses to stress challenge, thereby suggesting that studies of Pb in isolation from other disease risk factors will not reveal the extent of its adverse effects. These findings also underscore the critical need to extend screening programs for elevated Pb exposure, now restricted to young children, to pregnant, at risk, women.

CNS effects of developmental Pb exposure are enhanced by combined maternal and offspring stress.

Lead (Pb) exposure and elevated stress are co-occurring risk factors. Both impact brain mesolimbic dopamine/glutamate systems involved in cognitive functions. We previously found that maternal stress can potentiate Pb-related adverse effects in offspring at blood Pb levels averaging approximately 40 microg/dl. The current study of combined Pb exposure and stress sought to extend those results to lower levels of Pb exposure, and to examine relationships among consequences in offspring for fixed interval (FI) schedule-controlled behavior, neurochemistry and corticosterone levels. Dams were exposed to maternal Pb beginning 2 months prior to breeding (0, 50 or 150 ppm in drinking water), maternal restraint stress on gestational days 16 and 17 (MS), or the combination. In addition, a subset of offspring from each resultant treatment group was also exposed intermittently to variable stressors as adults (MS+OS). Marked "Pb-stress"-related increases in response rates on a fixed interval schedule, a behavioral performance with demonstrated sensitivity to Pb, occurred preferentially in female offspring even at mean blood Pb levels of 11 microg/dl when 50 ppm Pb was combined with maternal and offspring stress. Greater sensitivity of females to frontal cortex catecholamine changes may contribute to the elevated FI response rates as mesocorticolimbic systems are critical to the mediation of this behavior. Basal and final corticosterone levels of offspring used to evaluate FI performance differed significantly from those of non-behaviorally tested (NFI) littermates, demonstrating that purported mechanisms of Pb, stress or Pb/stress effects determined in non-behaviorally trained animals cannot necessarily be generalized to animals with behavioral histories. Finally, the persistent and permanent consequences of Pb, stress and Pb+stress in offspring of both genders suggest that Pb screening programs should include pregnant women at risk for elevated Pb exposure, and that stress should be considered as an additional risk factor. Pb+stress effects observed in the absence of either risk factor alone (i.e., potentiated effects) raise questions about the capacity of current hazard identification approaches to adequately identify human health risks posed by neurotoxicants.

New and evolving concepts in the neurotoxicology of lead.

Lead (Pb) is a xenobiotic metal with no known essential function in cellular growth, proliferation, or signaling. Decades of research characterizing the toxicology of Pb have shown it to be a potent neurotoxicant, especially during nervous system development. New concepts in the neurotoxicology of Pb include advances in understanding the mechanisms and cellular specificity of Pb. Experimental studies have shown that stress can significantly alter the effects of Pb, effects that could potentially be mediated through alterations in the interactions of glucocorticoids with the mesocorticolimbic dopamine system of the brain. Elevated stress, with corresponding elevated glucocorticoid levels, has been postulated to contribute to the increased levels of many diseases and dysfunctions in low socioeconomic status populations. Cellular models of learning and memory have been utilized to investigate the potential mechanisms of Pb-induced cognitive deficits. Examination of long-term potentiation in the rodent hippocampus has revealed Pb-induced increases in threshold, decreases in magnitude, and shorter retention times of synaptic plasticity. Structural plasticity in the form of adult neurogenesis in the hippocampus is also impacted by Pb exposure. The action of Pb on glutamate release, NMDA receptor function, or structural plasticity may underlie perturbations in synaptic plasticity and contribute to learning impairments. In addition to providing insight into potential mechanisms of Pb-induced cognitive deficits, cellular models offer an opportunity to investigate direct effects of Pb on isolated biological substrates. A target of interest is the 78-kDa molecular chaperone glucose-regulated protein (GRP78). GRP78 chaperones the secretion of the cytokine interleukin-6 (IL-6) by astrocytes. In vitro evidence shows that Pb strongly binds to GRP78, induces GRP78 aggregation, and blocks IL-6 secretion in astroglial cells. These findings provide evidence for a significant chaperone deficiency in Pb-exposed astrocytes in culture. In the long term, chaperone deficiency could underlie protein conformational diseases such as Alzheimer's Disease (AD). Lead exposure in early life has been implicated in subsequent progression of amyloidogenesis in rodents during old age. This exposure resulted in an increase in proteins associated with AD pathology viz., beta-amyloid precursor protein (beta-APP), and beta-amyloid (Abeta). These four new lines of research comprise compelling evidence that exposures to Pb have adverse effects on the nervous system, that environmental factors increase nervous system susceptibility to Pb, and that exposures in early life may cause neurodegeneration in later life.

Permanent alterations in stress responsivity in female offspring subjected to combined maternal lead exposure and/or stress.

Elevated lead (Pb) exposures preferentially impact low socioeconomic status (SES) populations, the same groups thought to sustain the highest levels of environmental stress. As co-occurring risk factors, therefore, Pb and stress could interact, a possibility further supported by the fact that both act on mesocorticolimbic dopamine systems of the brain. We recently demonstrated in rats that maternal Pb exposure could permanently increase basal corticosterone levels of offspring consistent with altered hypothalamic pituitary adrenal (HPA) axis function. The current study was thus designed to test the hypothesis that stress responsivity of offspring should likewise be altered, with the outcome differing in response to Pb, stress or Pb+stress. The impact of intermittent variable stress challenges (restraint, novelty, cold) on behavior sensitive to Pb exposure (fixed interval (FI) schedule-controlled responding) and on stress-induced corticosterone changes were evaluated in adult female offspring of dams that had been exposed to Pb (150 ppm) in drinking water from 2 months prior to breeding through lactation with or without maternal restraint stress on days 16 and 17 of gestation. This design yielded four treatment groups: (NS/0, no maternal Pb, no maternal stress; S/0, no maternal Pb, maternal stress; NS/150, maternal Pb, no maternal stress; and S/150, maternal Pb exposure and maternal stress). While maternal Pb alone and stress alone each altered components of stress responsivity, the greatest number of effects was seen in response to Pb + stress. This included alterations in FI performance following both restraint and cold stress and in the corticosterone response to cold stress. Collectively, these studies reveal that maternal Pb exposure alone can permanently alter stress responsivity and that the profile of effects produced by maternal Pb differ from those produced by maternal Pb in conjunction with stress, findings which have both mechanistic and risk assessment significance.

Glutamate and dopamine in nucleus accumbens core and shell: sequence learning versus performance.

This study sought to determine whether neurochemical changes associated with chronic postweaning lead (Pb) exposure, namely, enhanced dopamine (DA) activity and/or blockade of NMDA function in nucleus accumbens (NAC), underlie the learning impairments also associated with this Pb regimen, and whether core or shell subregions of nucleus accumbens would be more important to such effects. If so, then mimicking these neurochemical changes in normal (control) rats should reproduce these Pb-induced learning impairments. For this purpose, the effects of DA (20-80 microg), the non-competitive NMDA antagonist MK-801 (1.0-2.5 microg) or DA+MK-801 (40+1.0, 80+2.5 microg) were infused in core or shell of nucleus accumbens in normal rats and effects on a multiple schedule of repeated learning (RL) and performance (P) evaluated. In core, MK-801 mimicked the effects of Pb exposure, selectively reducing RL accuracy with no corresponding changes in P accuracy, an effect derived from an increased frequency of perseverative errors. DA produced non-specific changes, reducing accuracy levels in RL and P components. Accuracy and rate effects of DA could be reversed by concurrent administration of the higher MK-801 dose. In shell, MK-801, primarily the lower dose, reduced accuracy in both the RL and P components, while DA did not produce any systematic effects. Collectively, these results point to a greater importance of core as compared to shell in the mediation of learning of spatial sequences, and suggest that inhibition of glutamatergic NMDA function may play a critical role in the selective learning impairments associated with chronic low level Pb exposure.

Behavioral responses to ethanol in rats perinatally exposed to low lead levels.

Wistar rats were exposed to 220 ppm of lead (Pb) in the drinking water from conception to the end of the nursing period (postnatal day 25). Maternal blood Pb levels at this time were 25 microg/dl. Male offspring were tested at the age of 35 or 70 days. We studied the anxiolytic response to 0.5-2.0 g/kg ethanol in an elevated plus maze test and preference for increasing ethanol solutions (2%, 4%, and 6%, v/v) in a free-choice paradigm; we also determined basal blood levels of corticosterone. Results demonstrated that, at 35 days of age, experimental rats were hypersensitive to the anxiolytic effect of ethanol and showed greater voluntary intake of this drug. In addition, 35-day-old Pb-treated rats exhibited higher basal levels of corticosterone as compared with those of controls. These differences disappeared at 70 days. Our findings are discussed in terms of either Pb-induced alterations in the development of the CNS or higher levels of corticosterone in experimental animals. Possible Pb-ethanol effects interactions are also considered.

Spatial learning in rats exposed to acute ethanol intoxication on gestational day 8.

Pregnant Wistar rats were treated on gestational day 8 (GD 8) with two IP injections of either ethanol (2.9 g/kg in 24% v/v saline solution) or saline. Offspring were tested in the water-maze task at 45 or 90 days of age. The escape latencies of rats trained with a submerged escape platform at a fixed location were similar between control and experimental rats. Analyses of responses on a probe trial carried out 10 days after the training period, revealed that 90-day-old females prenatally exposed to alcohol were less likely to swim in the target region. No differences were observed in this free-swim trial in 45- and 90-day-old male, and 45-day-old female animals. Binding studies of low-affinity GABAA sites in the hippocampus showed an increase in affinity of [3H]GABAA for their binding sites in 90-day-old female offspring prenatally intoxicated with ethanol. Our results demonstrate that acute intoxication with ethanol on GD 8 did not modify acquisition but impaired the retention of spatial learning only in adult female rats. It is possible that the impaired retention will be consequence of higher GABAA receptor affinity.

Effects of acute ethanol intoxication during pregnancy on central dopaminergic system in male rats.

Pregnant albino rats received 2 IP injections, spaced by 4 h, of either ethanol (2.9 g/kg in 24% v/v saline solution) or saline, on gestational day (GD) 8. During adulthood, male rats prenatally exposed to alcohol exhibited an increased stereotyped behavioral response to 12 mg/kg of amphetamine (AMPH) or 1 mg/kg of apomorphine (APO), whereas the stereotypy induced by 6 mg/kg of AMPH showed no difference between control and experimental animals. Also, the hypoactivity response elicited by small doses of APO was not significantly affected by the prenatal treatment with alcohol. Analysis of dopaminergic function in the striatum and nucleus accumbens demonstrated no change on dopamine (DA) levels in both structures in alcohol pre-exposed 55- and 180-day-old rats. A reduction in striatum 3-4 dihydroxyphenylacetic acid levels was observed at both ages. These results indicate that an acute intoxication with alcohol on GD 8 induces a long-lasting decrease in striatal but not in nucleus accumbens DA metabolism. As a consequence, a lower striatal DA release might produce a compensatory supersensitivity of postsynaptic DA sites. This interpretation is consistent and correlates with behavioral results.