The LEARn model: an epigenetic explanation for idiopathic neurobiological diseases.

Neurobiological disorders have diverse manifestations and symptomology. Neurodegenerative disorders, such as Alzheimer's disease, manifest late in life and are characterized by, among other symptoms, progressive loss of synaptic markers. Developmental disorders, such as autism spectrum, appear in childhood. Neuropsychiatric and affective disorders, such as schizophrenia and major depressive disorder, respectively, have broad ranges of age of onset and symptoms. However, all share uncertain etiologies, with opaque relationships between genes and environment. We propose a 'Latent Early-life Associated Regulation' (LEARn) model, positing latent changes in expression of specific genes initially primed at the developmental stage of life. In this model, environmental agents epigenetically disturb gene regulation in a long-term manner, beginning at early developmental stages, but these perturbations might not have pathological results until significantly later in life. The LEARn model operates through the regulatory region (promoter) of the gene, specifically through changes in methylation and oxidation status within the promoter of specific genes. The LEARn model combines genetic and environmental risk factors in an epigenetic pathway to explain the etiology of the most common, that is, sporadic, forms of neurobiological disorders.

Importance of tau in cognitive decline as revealed by developmental exposure to lead.

Previous reports by us have determined that developmental exposure to the heavy metal lead (Pb) resulted in cognitive impairment in aging wildtype mice, and a latent induction in biomarkers associated with both the tau and amyloid pathways. However, the relationship between these two pathways and their correlation to cognitive performance needs to be scrutinized. Here, we investigated the impact of developmental Pb (0.2%) exposure on the amyloid and tau pathways in a transgenic mouse model lacking the tau gene. Cognitive function, and levels of intermediates in the amyloid and tau pathways following postnatal Pb exposure were assessed on young adult and mature transgenic mice. No significant difference in behavioral performance, amyloid precursor protein (APP), or amyloid beta (Aß) levels was observed in transgenic mice exposed to Pb. Regulators of the tau pathway were impacted by the absence of tau, but no additional change was imparted by Pb exposure. These results revealed that developmental Pb exposure does not cause cognitive decline or change the expression of the amyloid pathway in the absence of tau. The essentiality of tau to mediate cognitive decline by environmental perturbations needs further investigation.

Lead (Pb) exposure and its effect on APP proteolysis and Abeta aggregation.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with clinical manifestations appearing in old age, however, the initial stages of this disease may begin early in life. AD is characterized by the presence of excessive deposits of aggregated beta-amyloid (Abeta) peptides, which are derived from the beta-amyloid precursor protein (APP) following processing by beta-secretase and gamma-secretase. Recently, we have reported that developmental exposure of rats to Pb resulted in latent elevation of APP mRNA, APP, and Abeta in old age. Here we examined whether latent up-regulation in APP expression and Abeta levels is exacerbated by concurrent disturbances in APP processing or Abeta aggregation. Among the environmental metals tested, only Abeta solutions containing Pb promoted the formation of Abeta aggregates at nanomolar concentrations. The lifetime profiles of alpha-, beta-, and gamma-secretases remained constant in adult and aging animals, and developmental exposure to Pb did not alter them. Furthermore, the addition of various concentrations of Pb (0.1 to 50 microM) to cerebral cortical extracts derived from control animals also did not affect the proteolytic activities of these enzymes. Therefore, we propose that amyloidogenesis is promoted by a latent response to developmental reprogramming of the expression of the APP gene by early exposure to Pb, as well as enhancement of Abeta aggregation in old age. In rodents, these events occur without Pb-induced disturbances to the enzymatic processing of APP. The aforementioned results provide further evidence for the developmental basis of amyloidogenesis and late-life disturbances in AD-associated proteins by environmental agents.

Developmental exposure to lead (Pb) alters the expression of the human tau gene and its products in a transgenic animal model.

Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregation of the tau protein in the human brain. The best known of these illnesses is Alzheimer's disease (AD); a disease where the microtubule associated protein tau (MAPT) becomes hyperphosphorylated (lowering its binding affinity to microtubules) and aggregates within neurons in the form of neurofibrillary tangles (NFTs). In this paper we examine whether environmental factors play a significant role in tau pathogenesis. Our studies were conducted in a double mutant mouse model that expressed the human tau gene and lacked the gene for murine tau. The human tau mouse model was tested for the transgene's ability to respond to an environmental toxicant. Pups were developmentally exposed to lead (Pb) from postnatal day (PND) 1-20 with 0.2% Pb acetate. Mice were then sacrificed at PND 20, 30, 40 and 60. Protein and mRNA levels for tau and CDK5 as well as tau phosphorylation at Ser396 were determined. In addition, the potential role of miRNA in tau expression was investigated by measuring levels of miR-34c, a miRNA that targets the mRNA for human tau, at PND20 and 50. The expression of the human tau transgene was altered by developmental exposure to Pb. This exposure also altered the expression of miR-34c. Our findings are the first of their kind to test the responsiveness of the human tau gene to an environmental toxicant and to examine an epigenetic mechanism that may be involved in the regulation of this gene's expression.

Differential effects of difluoromethylornithine on basal and induced activity of cerebral ornithine decarboxylase and mRNA.

The induction of the activity of cerebral ornithine decarboxylase (EC 4.1.1.17) and mRNA by electrical stimulation exhibits regional differences. The effects of the enzyme inhibitor difluoromethylornithine on these regional variations was examined. Administration of this inhibitor resulted in pronounced depression of both basal and induced activity of ornithine decarboxylase in the hippocampus. Basal activity of the enzyme in the neocortex and the cerebellum appeared to be resistant to difluoromethylornithine but the induced enzyme activity was sensitive to the effects of this inhibitor. Susceptibility to difluoromethylornithine may be directly correlated with a slower turnover rate for ornithine decarboxylase. These results suggest that ornithine decarboxylase in the hippocampus may possess a longer half-life than its counterparts in other regions of the brain. Pretreatment with difluoromethylornithine had no effect on the induced ornithine decarboxylase mRNA in the neocortex. Thus, elevated activity of ornithine decarboxylase enzyme, due to electrical stimulation, appears to not have any effect on either the transcription or the decay rate of the induced ornithine decarboxylase mRNA. These findings support the concept of region-specific regulation of cerebral ornithine decarboxylase.

Electrically stimulated rapid gene expression in the brain: ornithine decarboxylase and c-fos.

A single electroconvulsive shock (ECS) resulted in a major induction of cerebral ornithine decarboxylase (ODC) mRNA and a rapid and transient elevation of ODC enzyme activity. The proto-oncogene c-fos was also transiently induced under the same conditions. Following a rapid rise in mRNA levels, the messages for these proteins take different courses. The c-fos mRNA fell to below control levels by 1 h, while the ODC mRNA remained elevated beyond 24 h. The ECS-induced elevation of ODC enzyme activity was not abolished by adrenalectomy but was attenuated significantly by the anti-convulsant MK-801. These results imply that the induction of cerebral ODC may be neuronal activity dependent, and suggest that the ODC/polyamine system may be linked to the proposed third messenger cascade, involving c-fos, which couples cell stimulation to gene expression, resulting in long-term adaptive responses.

c-fos and ornithine decarboxylase gene expression in brain as early markers of neurotoxicity.

An increase of proto-oncogene c-fos expression in cerebral cortex of rats treated with subconvulsant doses of the pesticide organochlorine lindane (gamma-hexachlorocyclohexane) has been detected using Northern blots. Immunohistochemical studies show that Fos protein was already increased in neuronal nuclei 3 h after treatment. The administration of the benzodiazepine diazepam prior to lindane totally blocked the activation of this proto-oncogene expression. Parallel to this increased expression of c-fos an activation of the ornithine decarboxylase (ODC) gene and enzyme was also observed. High levels of ODC mRNA and increased enzyme activity in cortex were found in rats following lindane treatment. These changes were attenuated by prior treatment of animals with diazepam. The co-induction of c-fos and ODC suggests a potential link between the ODC/polyamine system and the short-acting proto-oncogenes in stimulus-transcription coupling events.

Lead exposure alters Egr-1 DNA-binding in the neonatal rat brain.

Zinc finger proteins (ZFP) contain a structural motif (Cys-2 His-2) found in a large family of eukaryotic transcriptional regulatory proteins, such as Sp1. Previous studies have shown that Sp1 DNA-binding was disrupted by exposure to lead (Pb), due to action on its zinc finger domain. In this paper, we discuss the results of studies with another ZFP, Egr-1, an early growth response gene, which is functionally involved in cell proliferation and differentiation. Egr-1 DNA-binding was studied by gel shift mobility assays in several brain regions of developing rat pups. We observed a distinct developmental profile of Egr-1 DNA-binding with a gradual increase from the early to late postnatal days in all the brain regions examined. Lactational exposure to Pb resulted in a modulation of Egr-1 DNA binding manifested by premature peaks in DNA-binding reminiscent of the in vivo changes previously reported for Sp1. These data are consistent with earlier findings that exposure to Pb both in vivo and in vitro causes a modulation in the DNA-binding of ZFP such as Sp1, Egr-1 and TFIIIA. The commonality by which Pb exposure alters the DNA-binding patterns of ZFP suggests that divalent Pb may be interacting directly with the Zn moiety of these proteins.

Exposure to lead-acetate modulates the developmental expression of myelin genes in the rat frontal lobe.

Postnatal exposure to high levels (4%) of lead (Pb) have been shown to disrupt myelin formation and result in abnormal conduction of nerve impulses, components necessary for information processing in the CNS. To investigate whether the pathological changes in myelin, due to Pb exposure, might be partially mediated by modulations of the expression of genes involved in CNS myelin, we have examined the developmental profiles of the proteolipid protein (PLP) and myelin basic protein (MBP), two major structural constituents of CNS myelin and 2',3'-cyclic nucleotide 3' phosphodiesterase (CNP), a non-structural enzyme associated with myelin formation. Rat pups were postnatally exposed, from birth to weaning, to moderate amounts of Pb (0.2%), in the drinking water of the dam, and their frontal cortices were assayed for changes in the expression profile of the above genes by Northern Analysis. On PND 20, Pb resulted in a dramatic stimulation of the mRNA levels of PLP and a small increase in MBP mRNA levels, but had no effect on the CNP message. These data suggest that moderate levels of Pb selectively interfere with the gene expression of structural proteins of CNS myelin and may thus influence the composition of myelin in this way.

Correlation between lead-induced changes in cerebral ornithine decarboxylase and protein kinase C activities during development and in cultured PC 12 cells.

Exposure to lead (Pb) interferes with neurodevelopment and disturbs ornithine decarboxylase (ODC) activity. ODC the key regulatory enzyme of the polyamine pathway, is a potential substrate for protein kinase C (PKC). Therefore, we examined developmental changes in PKC activity and its relationship to ODC activity. Male rats were lactationally exposed to 0.2% Pb-acetate from birth to weaning. PKC and ODC activity were measured on postnatal days (PND) 3, 5, 10, 20 and 30. We found that the basal patterns of ODC and PKC activities resembled each other in both the neocortex and cerebellum and Pb-exposure attenuated both enzymes in a similar manner. To determine whether any link existed between these enzymes, ODC and PKC activities were induced to increase using nerve growth factor (NGF) in the presence and/or absence of ODC (difluoromethylornithine, DFMO) and PKC (staurosporine) inhibitors, in control and Pb-exposed Pheochromocytoma (PC-12) cells. Staurosporine decreased both ODC activity and PKC activity, while DFMO had no effect on PKC activity. These data suggest that ODC may be regulated by PKC and that Pb-induced developmental alterations in ODC activity may be secondary to changes in the integrity of PKC.

Developmental profiles of ornithine decarboxylase activity in the hippocampus, neocortex and cerebellum: modulation following lead exposure.

Ornithine decarboxylase (ODC) is a growth-associated enzyme which is critical for cell growth and transformation. ODC activity follows a specific ontogenetic pattern of activity in distinct brain regions according to their developmental stage. Perturbations in the pattern of ODC activity have been associated with brain damage including arrested cerebral growth. Modulations in the pattern of ODC activity were examined in the hippocampus, neocortex and cerebellum of neonatal rats (PND 3, 6, 9, 15) exposed via the dam to 0.2% lead-acetate (Pb2+ prenatally (gestational day 13 to birth), postnatally (PND 1-15) or perinatally (gestational day 13 to PND 15). Prenatal exposure to Pb2+ perturbed the profile of ODC activity in all three brain regions examined, while postnatal exposure to Pb2+ resulted in prolonged stimulations of ODC activity in the cerebellum. Following prenatal exposure, these effects were manifested as a stimulation of ODC activity in the hippocampus, a repression of activity in the neocortex and a combination of these effects in the cerebellum. Perinatal exposure to Pb2+ transiently modulated the pattern of ODC activity similarly in all three brain regions, in a characteristic manner irrespective of their developmental stage. These Pb(2+)-induced modulations of ODC activity suggest that polyamine-dependent processes may play a significant role in the manifestation of Pb(2+)-induced neurotoxicity dependent upon developmental factors at specific exposure periods.

Intracellular signaling pathways involved in mediating the effects of lead on the transcription factor Sp1.

It has been well established that exposure to Pb during critical periods of brain development results in both cognitive and behavioral deficits. Although the mechanism by which Pb induces developmental neurotoxicity is unknown, it may involve alterations in transcription of genes that are essential for growth and differentiation. Recent studies reveal that Pb interferes with growth and differentiation by acting on the transcription factor Sp1. Pb-induced changes in the activity of Sp1 may be consequent to alterations in intermediates in signal transduction pathways. This study examines both in vivo and in vitro the role of signaling factors in mediating the effects of Pb on Sp1 DNA-binding. Hippocampal developmental profiles of Sp1 DNA-binding, PKC, and MAPK protein levels were monitored in Pb-exposed rats. Pb exposure resulted in an induction of Sp1 DNA-binding during PND 5-10 followed by a subsequent decline on PND 15-20. The protein expression profiles for PKCalpha and MAPK followed a relatively similar pattern. To examine the interdependence between Sp1 DNA-binding, PKCalpha, and MAPK, PC12 cells were exposed to Pb and/or NGF. Pb or NGF exposure increased Sp1 DNA-binding. Addition of the PKC inhibitor (staurosporine) diminished NGF and Pb-induced Sp1 DNA-binding, while the MAPK inhibitor (PD 98059), completely abolished both basal and induced Sp1 DNA-binding. These findings demonstrate that Sp1 DNA-binding is regulated by PKC and MAPK, which may serve as mediators through which Pb may indirectly modulate Sp1 DNA-binding.

Lead-induced developmental perturbations in hippocampal Sp1 DNA-binding are prevented by zinc supplementation: in vivo evidence for Pb and Zn competition.

Zinc finger protein (ZFP) transcription factors are essential for regulation of gene expression in the developing brain. We previously reported that Pb exposure perturbed the DNA-binding of ZFP such as Sp1 and Egr-1 in the cerebellum, which play critical role in CNS development. In this study, we focused on hippocampal Sp1 DNA-binding and mRNA expression in neonatal Pb-exposed animals. The expression pattern of an Sp1 target (NMDAR1) gene was also monitored. To study in vivo and in vitro competition between Pb and Zn, we supplemented animals with Zn, and examined the effects of both metals on hippocampal Sp1 DNA-binding and the DNA-binding of a recombinant Sp1 protein (rhSp1). Tissue metal analysis revealed that only the disposition of Pb in the brain but not its distribution in the blood was influenced by the presence of Zn. The developmental profile of Sp1 DNA-binding exhibited a peak on PND 15 which subsequently declined to adult levels. Consistent with earlier studies, Pb exposure produced premature peaks of Sp1 DNA-binding on PND 5 which later returned to adult levels. The basal and Pb-induced developmental patterns of Sp1 mRNA departed from its DNA-binding profiles. However, the expression patterns of the NMDAR1 gene were relative to Sp1 DNA-binding. Supplementation with zinc provided a protective effect on Pb-induced changes in Sp1 DNA-binding. Moreover, Pb and Zn directly interfered with the DNA-binding of rhSp1 in vitro. These data suggest that Pb and Zn can compete both in vivo and in vitro at the zinc finger domain of Sp1 with a consequential effect on Sp1 DNA-binding, subsequent gene expression and brain development.

Correlations between developmental ornithine decarboxylase gene expression and enzyme activity in the rat brain.

The rise and decline in cerebral ODC activity during specific stages of development has been attributed to cytoplasmic intermediates which regulate ornithine decarboxylase activity. Here we examine whether transcriptional regulation contributes to the production of the developmental profiles of ODC activity. Postnatal cerebellar and neocortical tissue were obtained from Long-Evans hooded rats at postnatal days (PND) 5, 10, 15, 20, 25, 30, 90 and probed for ODC and actin gene expression, by Northern analysis. Our results indicate that ODC gene expression in the cerebellum was elevated at PND 5 and 10 followed by a gradual drop to the adult low levels by PND 20. By contrast, high levels of ODC gene expression in the neocortex were seen at PND 5 with an abrupt decrease at day 10 to low adult levels. The expression of the ODC gene in the neocortex follows closely the pattern for the ODC enzyme activity; however, it tends to remain elevated longer in the cerebellum. The levels of actin gene expression exhibited a distinct developmental profile in the postnatally developing cerebellum. However, actin mRNA levels remained unchanged in the neocortex, consistent with the prenatal development of this region. Our findings suggest that ODC gene expression may play an important role in the production of the ontogenetic patterns of ODC activity.

Sp1 as a target site for metal-induced perturbations of transcriptional regulation of developmental brain gene expression.

Differential gene expression is partially regulated by zinc finger proteins (ZFP) such as Sp1, which may be potential targets for perturbations by environmental metals. In this paper, we discuss the selective effects of lead (Pb) and other heavy metals on the in vitro and in vivo DNA-binding of Sp1, and the developmental expression of its target genes. We have found that the presence of Pb, Zn and Cd in a DNA-binding assay differentially modulated the binding of Sp1 to its specific DNA sequence, while Ca, Mg and Ba, did not. In PC12 cells, cultured in the presence of low concentrations of Pb, a premature enhancement of Sp1 DNA-binding was observed. Similarly, Sp1 DNA-binding in the cerebellum of Pb-exposed animals was shifted to the first week after birth, while the developmental profile of a non-ZFP, NFkB, was not. Furthermore, selective premature peaks of myelin basic protein and proteolipid protein mRNA expression were observed to occur in a manner relative to the changes in Sp1 DNA-binding. Since these genes are high targets for Sp1, these data suggest that exposure to heavy metals may alter developmental gene expression and brain development through selective modulation of the transcriptional activity of Sp1.

Regional variations in spermine levels in the developing rat brain following exposure to lead.

Polyamines are ubiquitous compounds involved in growth and differentiation, and omithine decarboxylase (ODC), is their rate-limiting enzyme. In an effort to examine whether changes in ODC enzyme activity are reflected in alterations in the availability of polyamines, levels of the more stable end-product spermine, were examined following exposure to lead (Pb). Rats were lactationally exposed to 0.2% Pb-acetate from birth to weaning. At postnatal days 3, 5, 10, 15, and 20, pups were sacrificed, and spermine was extracted from their brain tissue and the extracts were analyzed by high-performance liquid chromatography coupled with a fluorescence detector. Basal spermine levels in the cerebellum were found to be higher than those in the neocortex, and exhibited a characteristic developmental profile. In Pb-exposed animals, spermine levels were attenuated in both brain regions, however, there was a rebound in cerebellar levels during the third week after birth. While basal spermine levels are in concert with reported patterns of ODC activity, they depart from each other following a toxic challenge to the cerebellum. This study suggest that developmental polyamine levels are not necessarily a direct translation of ODC activity and that modulations in their biosynthesis depend on regional stages of growth.

Lead exposure in pheochromocytoma (PC12) cells alters neural differentiation and Sp1 DNA-binding.

Previous studies have revealed that lead modulates the DNA-binding profile of the transcription factor Sp1 both in vivo and in vitro (Dev Brain Res 1998;107:291). Sp1 is a zinc finger protein, that is selectively up-regulated in certain developing cell types and plays a regulatory role during development and differentiation (Mol Cell Biol 1991;11:2189). In NGF-stimulated PC12 cells, Sp1 DNA-binding activity was induced within 48 h of exposure of NGF naïve cells. Exposure of undifferentiated PC12 cells to lead alone (0.1 microM) also produced a similar increase in Sp1 DNA-binding. Since lead altered the DNA-binding profile of Sp1 in newly differentiating cells, neurite outgrowth was assessed as a morphological marker of differentiation to determine whether or not the effects of lead on differentiation were restricted to the initiation phase (unprimed) or the elaboration phase of this process (NGF-primed). NGF-primed and unprimed PC12 cells were prepared for bioassay following exposure to various concentrations of NGF and/or lead. Neurite outgrowth was measured at 48 and 72 h during early stages of NGF-induced differentiation and at 14 h in NGF primed/replated cells. In the absence of NGF, exposure to lead alone (0.025, 0.05, 0.1 microM) promoted measurable neurite outgrowth in unprimed PC12 cells at 48 and 72 h. A similar phenomenon was also observed in primed/replated PC12 cells at 14 h. However, this effect was two to five times greater than unprimed control cells. In the presence of NGF, a similar trend was apparent at lower concentrations, although the magnitude and temporal nature was different from lead alone. In most cases, the administration of higher lead concentrations (1 and 10 microM), in both the absence or presence of NGF, was less effective than the lower concentrations in potentiating neurite outgrowth. These results suggest that lead alone at low doses may initiate premature stimulation of morphological differentiation that may be related to lead-induced alterations in Sp1 binding to DNA.

Disruption of the zinc finger domain: a common target that underlies many of the effects of lead.

The health risks associated with exposure to heavy metals such as lead (Pb) remain a major public health concern. The zinc finger is a major structural motif involved in protein-nucleic acid interactions and is present in the largest superfamily of transcription factors. Zinc (Zn) ions coordinate this finger-like structure through bonds created with cysteine and histidine residues. Little information exists on the effects of heavy metals on proteins that contain structural repeats of this kind. Studies by us in the nervous system have shown that factors containing such motifs could be potential targets for perturbation by Pb. We have observed that metals such as Pb interfered with the DNA-binding properties of Sp1 and Egr-1, both in vivo and in vitro. Pb could also directly interfere with the DNA-binding of a recombinant human Sp1 protein. More recently, the effects of Pb on the DNA-binding of the zinc finger protein transcription factor IIIA (TFIIIA) have been demonstrated. Analysis on the effects of Pb on Sp1 revealed that alterations in its DNA-binding were commensurate with changes in the expression of its target genes. The action of Pb on Sp1, Egr-1, and TFIIIA suggests that it can also target other cellular proteins that contain the zinc finger motif and reveals this protein domain as a potential mediator for Pb-induced alterations in protein function. Thus by specifically targeting zinc finger proteins (ZFP), Pb is able to produce multiple responses through its action on a common site that is present in enzymes, channels and receptors.

Modulation of developmental cerebellar ornithine decarboxylase activity by lead-acetate.

The developing brain is particularly susceptible to the neurotoxic effects of lead exposure. The ontological profile of ornithine decarboxylase (ODC) activity in the cerebellum was examined following lactational exposure of rats to 0.2% lead acetate (Pb). Relative to controls, Pb-exposure induced ODC activity levels in a transient manner with a 50% increase at postnatal day (PND) 6, a 20% increase at PND 9, returning to control basal levels by PND 15. These effects were seen at exposure levels of Pb that did not alter the normal growth and body weight of either the lactating dam or the developing pups. Basal cerebellar ODC activity in homogenates was increased with addition of low concentrations of Pb acetate (0.01 microM and 0.1 microM), while concentrations of 1 microM or greater were inhibitory. The effects of Pb acetate on tissue ODC activity in vitro were not mimicked by the addition of calcium chloride. Unlike tissue ODC activity, incubation of these metals with a pure ODC protein preparation exhibited fluctuations in ODC activity possibly due to the ionic interactions of Pb or calcium chloride. Calcium homeostatic mechanisms appeared to be unchanged with Pb exposure, at this dose, in that neither 45Ca-uptake (both mitochondrial and microsomal) nor synaptosomal Ca(2+)-ATPase activity was altered. These data suggest that alterations in ODC activity may be indicative of subtle toxicant induced perturbations during early development. Although the precise mechanism by which Pb may induce ODC activity in developing tissue is unknown, our results suggest that Pb may specifically alter ODC activity via cytosolic interactions.

Intranigral iron infusion in the rat. Acute elevations in nigral lipid peroxidation and striatal dopaminergic markers with ensuing nigral degeneration.

Iron is known to induce lipid peroxidation and recent evidence indicates that both iron and lipid peroxidation are elevated in the substantia nigra in Parkinson's disease (PD). To test whether excess intranigral iron induces lipid peroxidation, we infused an iron citrate solution (0.63 nmol in 0.25 microL) into the rat substantia nigra and measured nigral thiobarbituric acid reactive products at 1-h, 1-d, 1-wk, and 1-mo postinfusion. In a separate group of iron-infused animals, histologic analysis within the substantia nigra through 1-mo postinfusion was accomplished by thionine- and iron-staining, with concurrent assessment of striatal neurochemical markers. Concentrations of nigral thiobarbituric acid reactive products were significantly elevated at 1 h and 1 d in iron-infused animals compared to vehicle-infused and unoperated animals, with a return to control values by 1 wk. Similarly, striatal dopamine turnover was acutely elevated, suggesting damage to dopaminergic neurons, which was confirmed histologically. Although iron-staining within the iron diffusionary area was increased through the postinfusion month, there was an apparent progression of the cellular character of staining from predominantly neuronal to reactive glial and finally to oligodendroglial by 1 mo postinfusion. This progression of cellular iron-staining may indicate a shifting of infused iron to a more bound unreactive form, thus explaining only an acute elevation in lipid peroxidation through 1 d following intranigral iron infusion. The data indicate that damage to nigral neurons induced by iron infusion is transciently associated with a marker of oxidative damage and supports the possibility that iron-induced oxidative stress contributes to the pathogenesis of PD.