Accumulation, elimination, sequestration, and genetic variation of lead (Pb2+) loads within and between generations of Drosophila melanogaster.

We examined accumulation, sequestration, elimination, and genetic variation for lead (Pb) loads within and between generations of Drosophila melanogaster. Flies were reared in control or leaded medium at various doses and tested for their Pb loads at different stages of development (larvae, eclosion, newly-eclosed adults, and mature adults). Pb loads were tested using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). We found that D. melanogaster readily accumulated Pb throughout their lifespan and the levels of accumulation increased with Pb exposure in the medium. Wandering third-instar larvae accumulated more Pb than mature adults; this phenomenon may be due to elimination of Pb in the pupal cases during eclosion and/or depuration in adults post-eclosion. The accumulated Pb in mature adults was not transferred to F1 mature adult offspring. Using a set of recombinant inbred strains, we identified a quantitative trait locus for adult Pb loads and found that genetic variation accounted for 34% of the variance in Pb load. We concluded that D. melanogaster is a useful model organism for evaluating changes in Pb loads during development, as well as between generations. Furthermore, we found that genetic factors can influence Pb loads; this provides an essential foundation for evaluating phenotypic variation induced by the toxic effects of Pb.

Asymmetrical positive assortative mating induced by developmental lead (Pb2+) exposure in a model system, Drosophila melanogaster.

Anthropogenic pollutants have the potential to disrupt reproductive strategies. Little is known about how lead (Pb2+) exposure disrupts individual-level responses in reproductive behaviors, which are important for fitness. Drosophila melanogaster was used as a model system to determine the effects of: 1) developmental lead exposure on pre-mating reproductive behaviors (i.e., mate preference), and 2) lead exposure and mating preferences on fitness in the F0 parental generation and F1 un-exposed offspring. Wild-type strains of D. melanogaster were reared from egg stage to adulthood in control or leaded medium (250 µM PbAc) and tested for differences in: mate preference, male song performance, sex pheromone expression, fecundity, mortality, and body weight. F0 leaded females preferentially mated with leaded males (i.e., asymmetrical positive assortative mating) in 2-choice tests. This positive assortative mating was mediated by the females (and not the males) and was dependent upon context and developmental exposure to Pb. Neither the courtship song nor the sex pheromone profile expressed by control and leaded males mediated the positive assortative mating in leaded females. Leaded females did not incur a fitness cost in terms of reduced fecundity, increased mortality, or decreased body weight by mating with leaded males. These results suggest that sublethal exposure to lead during development can alter mate preferences in adults, but not fitness measures once lead exposure has been removed. We suggest that changes in mate preference may induce fitness costs, as well as long-term population and multi-generational implications, if pollution is persistent in the environment.

Epigenetics of early-life lead exposure and effects on brain development.

The epigenetic machinery plays a pivotal role in the control of many of the body's key cellular functions. It modulates an array of pliable mechanisms that are readily and durably modified by intracellular or extracellular factors. In the fast-moving field of neuroepigenetics, it is emerging that faulty epigenetic gene regulation can have dramatic consequences on the developing CNS that can last a lifetime and perhaps even affect future generations. Mounting evidence suggests that environmental factors can impact the developing brain through these epigenetic mechanisms and this report reviews and examines the epigenetic effects of one of the most common neurotoxic pollutants of our environment, which is believed to have no safe level of exposure during human development: lead.

Drosophila melanogaster as a model for lead neurotoxicology and toxicogenomics research.

Drosophila melanogaster is an excellent model animal for studying the neurotoxicology of lead. It has been known since ancient Roman times that long-term exposure to low levels of lead results in behavioral abnormalities, such as what is now known as attention deficit hyperactivity disorder (ADHD). Because lead alters mechanisms that underlie developmental neuronal plasticity, chronic exposure of children, even at blood lead levels below the current CDC community action level (10 µg/dl), can result in reduced cognitive ability, increased likelihood of delinquency, behaviors associated with ADHD, changes in activity level, altered sensory function, delayed onset of sexual maturity in girls, and changes in immune function. In order to better understand how lead affects neuronal plasticity, we will describe recent findings from a Drosophila behavioral genetics laboratory, a Drosophila neurophysiology laboratory, and a Drosophila quantitative genetics laboratory who have joined forces to study the effects of lead on the Drosophila nervous system. Studying the effects of lead on Drosophila nervous system development will give us a better understanding of the mechanisms of Pb neurotoxicity in the developing human nervous system.

Pb2+: an endocrine disruptor in Drosophila?

Environmental exposure to Pb(2+) affects hormone-mediated responses in vertebrates. To help establish the fruit fly, Drosophila melanogaster, as a model system for studying such disruption, we describe effects of Pb(2+) on hormonally regulated traits. These include duration of development, longevity, females' willingness to mate, fecundity and adult locomotor activity. Developmental Pb(2+) exposure has been shown to affect gene expression in a specific region of the Drosophila genome (approximately 122 genes) involved in lead-induced changes in adult locomotion and to affect regulation of intracellular calcium levels associated with neuronal activity at identified synapses in the larval neuromuscular junction. We suggest ways in which Drosophila could become a new model system for the study of endocrine disruptors at genetic, neural and behavioral levels of analysis, particularly by use of genomic methods. This will facilitate efforts to distinguish between behavioral effects of Pb(2+) caused by direct action on neural mechanisms versus effects of Pb(+2) on behavior mediated through endocrine disruption.

Genetical toxicogenomics in Drosophila identifies master-modulatory loci that are regulated by developmental exposure to lead.

The genetics of gene expression in recombinant inbred lines (RILs) can be mapped as expression quantitative trait loci (eQTLs). So-called "genetical genomics" studies have identified locally acting eQTLs (cis-eQTLs) for genes that show differences in steady-state RNA levels. These studies have also identified distantly acting master-modulatory trans-eQTLs that regulate tens or hundreds of transcripts (hotspots or transbands). We expand on these studies by performing genetical genomics experiments in two environments in order to identify trans-eQTL that might be regulated by developmental exposure to the neurotoxin lead. Flies from each of 75 RIL were raised from eggs to adults on either control food (made with 250 microM sodium acetate), or lead-treated food (made with 250 microM lead acetate, PbAc). RNA expression analyses of whole adult male flies (5-10 days old) were performed with Affymetrix DrosII whole genome arrays (18,952 probesets). Among the 1389 genes with cis-eQTL, there were 405 genes unique to control flies and 544 genes unique to lead-treated ones (440 genes had the same cis-eQTLs in both samples). There are 2396 genes with trans-eQTL which mapped to 12 major transbands with greater than 95 genes. Permutation analyses of the strain labels but not the expression data suggests that the total number of eQTL and the number of transbands are more important criteria for validation than the size of the transband. Two transbands, one located on the 2nd chromosome and one on the 3rd chromosome, co-regulate 33 lead-induced genes, many of which are involved in neurodevelopmental processes. For these 33 genes, rather than allelic variation at one locus exerting differential effects in two environments, we found that variation at two different loci are required for optimal effects on lead-induced expression.

Genetic aspects of behavioral neurotoxicology.

Considerable progress has been made over the past couple of decades concerning the molecular bases of neurobehavioral function and dysfunction. The field of neurobehavioral genetics is becoming mature. Genetic factors contributing to neurologic diseases such as Alzheimer's disease have been found and evidence for genetic factors contributing to other diseases such as schizophrenia and autism are likely. This genetic approach can also benefit the field of behavioral neurotoxicology. It is clear that there is substantial heterogeneity of response with behavioral impairments resulting from neurotoxicants. Many factors contribute to differential sensitivity, but it is likely that genetic variability plays a prominent role. Important discoveries concerning genetics and behavioral neurotoxicity are being made on a broad front from work with invertebrate and piscine mutant models to classic mouse knockout models and human epidemiologic studies of polymorphisms. Discovering genetic factors of susceptibility to neurobehavioral toxicity not only helps identify those at special risk, it also advances our understanding of the mechanisms by which toxicants impair neurobehavioral function in the larger population. This symposium organized by Edward Levin and Annette Kirshner, brought together researchers from the laboratories of Michael Aschner, Douglas Ruden, Ulrike Heberlein, Edward Levin and Kathleen Welsh-Bohmer conducting studies with Caenorhabditis elegans, Drosophila, fish, rodents and humans studies to determine the role of genetic factors in susceptibility to behavioral impairment from neurotoxic exposure.

Variations at a quantitative trait locus (QTL) affect development of behavior in lead-exposed Drosophila melanogaster.

We developed Drosophila melanogaster as a model to study correlated behavioral, neuronal and genetic effects of the neurotoxin lead, known to affect cognitive and behavioral development in children. We showed that, as in vertebrates, lead affects both synaptic development and complex behaviors (courtship, fecundity, locomotor activity) in Drosophila. By assessing differential behavioral responses to developmental lead exposure among recombinant inbred Drosophila lines (RI), derived from parental lines Oregon R and Russian 2b, we have now identified a genotype by environment interaction (GEI) for a behavioral trait affected by lead. Drosophila Activity Monitors (TriKinetics, Waltham, MA), which measure activity by counting the number of times a single fly in a small glass tube walks through an infrared beam aimed at the middle of the tube, were used to measure activity of flies, reared from eggs to 4 days of adult age on either control or lead-contaminated medium, from each of 75 RI lines. We observed a significant statistical association between the effect of lead on Average Daytime Activity (ADA) across lines and one marker locus, 30AB, on chromosome 2; we define this as a Quantitative Trait Locus (QTL) associated with behavioral effects of developmental lead exposure. When 30AB was from Russian 2b, lead significantly increased locomotor activity, whereas, when 30AB was from Oregon R, lead decreased it. 30AB contains about 125 genes among which are likely "candidate genes" for the observed lead-dependent behavioral changes. Drosophila are thus a useful, underutilized model for studying behavioral, synaptic and genetic changes following chronic exposure to lead or other neurotoxins during development.

Behavioral effects of chronic exposure to low levels of lead in Drosophila melanogaster.

Through human activity lead has become a serious environmental neurotoxin, known to affect activity levels, attention and both sensory and cognitive function in children. Study of lead would be facilitated by having a model system that could be manipulated easily and quickly. We find Drosophila melanogaster ideal as such, and we have been studying effects of lead on courtship, fecundity and locomotor activity. We raised Canton-S flies from eggs to adult day 6-7 on medium made with lead acetate solution (2-100 microgram/g), or with distilled water, and we measured adult body lead burdens by means of Inductively Coupled Plasma Mass Spectrometry (ICP-MS). To measure courtship effectiveness, five virgin females and five virgin males were transferred into an empty vial and the number of females that mated within 20min was recorded. To measure fecundity, all adult offspring from eggs produced by one female within 12 days of mating were counted. To measure locomotor activity, individual flies were transferred to a grid-labeled petri dish and the number of lines crossed in 30s was counted. The number of females mating within 20min was increased significantly by exposure to 2 or 8 microgram/g lead, and was decreased significantly by exposure to 20 or 50 microgram/g lead. Fecundity was increased significantly by exposure to 2 microgram/g lead, but was unaffected by exposure to 20 microgram/g lead. Locomotor activity was consistently higher for males than for females, and was significantly reduced only by exposure to 50 microgram/g lead, and then only for males. We thus defined for Drosophila a lowest observable effect level (LOEL) of 2 microgram/g lead, which is considerably lower than the doses shown previously to affect this animal. The dose-response curve was biphasic for the number of females mating within 20min, an example of hormesis, a non-linear response that has been reported for low levels of stressors as diverse as pollutants and radiation. We hope from further studies with Drosophila to understand better how lead affects the developing nervous system, and thus ultimately its effects on children.

Effects of chronic lead exposure on the neuromuscular junction in Drosophila larvae.

Long term or chronic exposure to lead is associated with cognitive and other deficits in humans, which may reflect lead-induced changes in synaptic development and function. We believe that Drosophila has great potential as a model system for studying such changes. To test this, we compared the structure of single, identified synapses between identified axons (axons 1 and 2) and muscle fibers (fibers 6 and 7) in untreated 3rd instar larvae, and in larvae reared on medium made with 100 microM lead acetate in distilled water. We used three approaches to examine the motor terminals on muscle fibers 6 and 7 in segment 2: (1) all terminals were stained with an antibody to HRP; (2) only the terminals of axon 1 were stained by injecting biotinylated Lucifer yellow into it; and (3) the regions of the terminal containing synaptic vesicles were stained with an antibody to synaptotagmin, which provides an estimate of "synaptic" terminal area. Lead burdens were determined by inductively coupled plasma mass spectrometry; hemolymph lead levels at the neuromuscular junction were likely to be micromolar. We observed that lead exposure did not significantly affect the average terminal area or the average muscle fiber area, but did significantly affect the uniformity of the matching between muscle area and motor terminal size that normally occurs during development. There was a significant positive correlation between motor terminal size and muscle area in control, but not in lead-exposed larvae. The sensitivity of Drosophila larval synaptic development to lead opens the way to using the powerful genetic and molecular tools available for this system to study the underlying mechanisms of this sensitivity. We would hope that from such an understanding may come strategies for dealing with lead-induced deficits in children.