Tomás R. Guilarte

Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes

Synthetic triterpenoids have been developed, which are potent inducers of cytoprotective enzymes and inhibitors of inflammation, greatly improving on the weak activity of naturally occurring triterpenoids. An imidazolide triterpenoid derivative, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im or TP235), has been previously shown to potently protect against hepatic tumorigenesis, acting in part by inducing cytoprotective genes through Keap1-Nrf2-antioxidant response element (ARE) signaling. In these studies, the pharmacodynamic activity of CDDO-Im is characterized in two distinct lines of ARE reporter mice and by measuring increases in Nqo1 transcript levels as a marker of cytoprotective gene induction. Oral administration of CDDO-Im induces ARE-regulated cytoprotective genes in many tissues in the mouse, including liver, lung, kidney, intestines, brain, heart, thymus, and salivary gland. CDDO-Im induces Nqo1 RNA transcripts in some organs at doses as low as 0.3 μmol/kg body weight (orally). A structure activity evaluation of 15 additional triterpenoids (a) confirmed the importance of Michael acceptor groups on both the A and C rings, (b) showed the requirement for a nitrile group at C-2 of the A ring, and (c) indicated that substituents at C-17 dramatically affected pharmacodynamic action in vivo. In addition to CDDO-Im, other triterpenoids, particularly the methyl ester CDDO-Me (TP155) and the dinitrile TP225, are extremely potent inducers of cytoprotective genes in mouse liver, lung, small intestine mucosa, and cerebral cortex. This pharmacodynamic characterization highlights the chemopreventive promise of several synthetic triterpenoids in multiple target organs. [Mol Cancer Ther 2007;6(1):154–62]

Manganese and Parkinson's Disease: A Critical Review and New Findings

Background Excess accumulation of manganese (Mn) in the brain results in a neurological syndrome with cognitive, psychiatric, and movement abnormalities. The highest concentrations of Mn in the brain are achieved in the basal ganglia, which may precipitate a form of parkinsonism with some clinical features that are similar and some that are different to those in Parkinson’s disease (PD). Recently, scientists have debated the possibility that Mn may have an etiological role in PD or that it may accelerate the expression of PD. Objective The goal of this review was to examine whether chronic Mn exposure produces dopamine neuron degeneration and PD or whether it has a distinct neuropathology and clinical presentation. Data source I reviewed available clinical, neuroimaging, and neuropathological studies in humans and nonhuman primates exposed to Mn or other human conditions that result in elevated brain Mn concentrations. Data extraction Human and nonhuman primate literature was examined to compare clinical, neuroimaging, and neuropathological changes associated with Mn-induced parkinsonism. Data synthesis Clinical, neuroimaging, and neuropathological evidence was used to examine whether Mn-induced parkinsonism involves degeneration of the nigrostriatal dopaminergic system as is the case in PD. Conclusions The overwhelming evidence shows that Mn-induced parkinsonism does not involve degeneration of midbrain dopamine neurons and that l-dopa is not an effective therapy. New evidence is presented on a putative mechanism by which Mn may produce movement abnormalities. Confirmation of this hypothesis in humans is essential to make rational decisions about treatment, devise effective therapeutic strategies, and set regulatory guidelines.

Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity.

Abstract: The peripheral benzodiazepine receptor (PBR) is currently used as a marker of inflammation and gliosis following brain injury. Previous reports suggest that elevated PBR levels in injured brain tissue are specific to activated microglia and infiltrating macrophages. We have produced hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the cellular and subcellular nature of the PBR response. Degenerating, argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14 days after TMT exposure. Reactive microglia were also evident at both times with a maximal response observed at 14 days, subsiding by 6 weeks. Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT administration, suggesting that the onset of the astroglia response is delayed, but more persistent, compared with microgliosis. Morphological evidence from [3H]PK11195 microautoradiography and PBR immunohistochemistry indicates that both astrocytes and microglia are capable of expressing high levels of PBR after injury. This was confirmed by double labeling of either Griffonia simplicifolia isolectin B4, a microglial‐specific marker, or glial fibrillary acidic protein, an astrocyte‐specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells.

Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure.

Long‐term deficits in cognitive function are the principal effects of lead (Pb2+) exposure in children and can be modeled in experimental animals. Current therapeutic approaches in the treatment of childhood Pb2+ intoxication are not effective in reversing learning deficits once they have occurred. We report that environmental enrichment reverses long‐term deficits in spatial learning produced by developmental Pb2+ exposure in rats. Enhanced learning performance of Pb2+‐exposed animals reared in an enriched environment was associated with recovery of deficits in N‐methyl‐D‐aspartate receptor subunit 1 (NR1) mRNA and induction of brain‐derived neurotrophic factor (BDNF) mRNA in the hippocampus. The effect of environmental enrichment on NR1 and BDNF gene expression was specific to Pb2+‐exposed animals and was present in the absence of changes in the NR2B subunit of the N‐methyl‐D‐aspartate receptor, GluR1, αCamKII, or PSD‐95 gene expression measured in the same animals. Our findings demonstrate that the learning impairments and NR1 subunit mRNA deficits resulting from developmental Pb2+ exposure are reversible if the animals are provided with an enriched environment even after the exposure has occurred. We propose environmental enrichment as a basis for the treatment of childhood Pb2+ intoxication.

Nigrostriatal dopamine system dysfunction and subtle motor deficits in manganese-exposed non-human primates

We tested the hypothesis that movement abnormalities induced by chronic manganese (Mn) exposure are mediated by dysfunction of the nigrostriatal dopamine system in the non-human primate striatum. Motor function and general activity of animals was monitored in parallel with chronic exposure to Mn and Positron Emission Tomography (PET) studies of in vivo dopamine release, dopamine transporters and dopamine receptors in the striatum. Analysis of metal concentrations in whole blood and brain was obtained and post-mortem analysis of brain tissue was used to confirm the in vivo PET findings. Chronic Mn exposure resulted in subtle motor function deficits that were associated with a marked decrease of in vivo dopamine release in the absence of a change in markers of dopamine (DA) terminal integrity or dopamine receptors in the striatum. These alterations in nigrostriatal DA system function were observed at blood Mn concentrations within the upper range of environmental, medical and occupational exposures in humans. These findings show that Mn-exposed non-human primates that exhibit subtle motor function deficits have an apparently intact but dysfunctional nigrostriatal DA system and provide a novel mechanism of Mn effects on the dopaminergic system.

Cytotoxicity of 3-hydroxykynurenine in a neuronal hybrid cell line

The toxicity of 3-hydroxykynurenine (3HK), an endogenous tryptophan metabolite which is markedly elevated in rat CNS tissue as a result of neonatal vitamin B-6 deficiency, was investigated in a neuronally derived hybrid cell line (N18-RE-105). At concentrations in excess of 100 microM, 3HK was toxic to greater than 85% of cultured cells over the course of 24 h. The time course of 3HK toxicity was studied in cultures exposed to 500 microM 3HK. Cell lysis proceeded linearly to completion in 8-12 h, but the toxic effects of exposure for 2 h were irreversible. 3HK was the most potently toxic among several related kynurenine metabolites tested. The toxic effects of 3HK exposure were markedly attenuated or abolished in the presence of either catalase or glutathione, indicating, a role of oxidative stress in 3HK toxicity.

Manganese neurotoxicity: lessons learned from longitudinal studies in nonhuman primates.

Background Exposure to excess levels of the essential trace element manganese produces cognitive, psychiatric, and motor abnormalities. The understanding of Mn neurotoxicology is heavily governed by pathologic and neurochemical observations derived from rodent studies that often employ acute Mn exposures. The comparatively sparse studies incorporating in vivo neuroimaging in nonhuman primates provide invaluable insights on the effects of Mn on brain chemistry. Objectives The purpose of this review is to discuss important aspects of Mn neurotoxicology and to synthesize recent findings from one of the largest cohorts of nonhuman primates used to study the neurologic effects of chronic Mn exposure. Discussion We reviewed our recent in vivo and ex vivo studies that have significantly advanced the understanding of Mn-induced neurotoxicity. In those studies, we administered weekly doses of 3.3–5.0 (n = 4), 5.0–6.7 (n = 5), or 8.3–10.0 mg Mn/kg (n = 3) for 7–59 weeks to cynomolgus macaque monkeys. Animals expressed subtle deficits in cognition and motor function and decreases in the N-acetylaspartate-to-creatine ratio in the parietal cortex measured by magnetic resonance spectroscopy reflective of neuronal dysfunction. Impaired striatal dopamine release measured by positron emission tomography was observed in the absence of changes in markers of dopamine neuron degeneration. Neuropathology indicated decreased glutamine synthetase expression in the globus pallidus with otherwise normal markers of glutamatergic and GABAergic neurotransmission. Increased amyloid beta (A4) precursor-like protein 1 gene expression with multiple markers of neurodegeneration and glial cell activation was observed in the frontal cortex. Conclusions These findings provide new information on mechanisms by which Mn affects behavior, neurotransmitter function, and neuropathology in nonhuman primates.

Developmental lead exposure causes spatial learning deficits in adult rats

Groups of male rats exposed to lead (Pb) during different developmental periods were tested as adults in a water maze. A highly significant (P < 0.01) impairment in water maze performance was measured in rats exposed to Pb only during gestation and lactation (maternal exposure). At the time of testing (100-106 days old), blood and brain Pb concentrations were at control levels. Significant impairments (P < 0.05) were also present in rats continuously exposed to Pb from conception through adulthood. Post-weaning Pb exposure alone did not result in impaired performance despite significantly elevated blood and brain Pb levels at the time of testing. This study supports the hypothesis that a window of vulnerability to Pb neurotoxicity exists in the developing brain and that Pb exposure can result in long-term cognitive deficits.

Impairment of nigrostriatal dopamine neurotransmission by manganese is mediated by pre-synaptic mechanism(s): Implications to manganese-induced parkinsonism

The long‐term consequences of chronic manganese (Mn) exposure on neurological health is a topic of great concern to occupationally‐exposed workers and in populations exposed to moderate levels of Mn. We have performed a comprehensive assessment of Mn effects on dopamine (DA) synapse markers using positron emission tomography (PET) in the non‐human primate brain. Young male Cynomolgus macaques were given weekly i.v. injections of 3.3–5.0 mg Mn/kg (n = 4), 5.0–6.7 mg Mn/kg (n = 5), or 8.3–10.0 mg Mn/kg (n = 3) for 7–59 weeks and received PET studies of various DA synapse markers before (baseline) and at one or two time points during the course of Mn exposure. We report that amphetamine‐induced DA release measured by PET is markedly impaired in the striatum of Mn‐exposed animals. The effect of Mn on DA release was present in the absence of changes in markers of dopamine terminal integrity determined in post‐mortem brain tissue from the same animals. These findings provide compelling evidence that the effects of Mn on DA synapses in the striatum are mediated by inhibition of DA neurotransmission and are responsible for the motor deficits documented in these animals.

Methamphetamine-induced deficits of brain monoaminergic neuronal markers: Distal axotomy or neuronal plasticity

We examined the effects of methamphetamine (METH) on monoaminergic (i.e. dopamine and serotonin) axonal markers and glial cell activation in the rat brain. Our findings indicate that the loss of dopamine transporters (DAT), serotonin transporters (5-HTT), vesicular monoamine transporter type-2 (VMAT-2) and glial cell activation induced by METH in the striatum and in the central gray are consistent with a degenerative process. Our novel finding of METH effects on monoaminergic neurons in the central gray may have important implications on METH-induced hyperthermia. In other brain regions examined, DAT and 5-HTT deficits after METH administration were present in the absence of lasting changes in VMAT-2 levels or glial cell activation. Brain regions exhibiting protracted deficits in DAT and/or 5-HTT and VMAT-2 levels also expressed increased levels of [(3)H]-R-PK11195 binding to peripheral benzodiazepine receptors, a quantitative marker of glial cell activation. Immunohistochemical assessment of microglia and astrocytes confirmed the PBR results. Microglia activation was more pronounced than astrocytosis in affected regions in most METH-exposed brains with the exception of a small number of rats that were most severely affected by METH based on loss of body weight. In these rats, both microglia and astrocytes were highly activated and expressed a distinct regional pattern suggestive of widespread brain injury. The reason for the pattern of glial cell activation in this group of rats is not currently known but it may be associated with METH-induced hyperthermia. In summary, our findings suggest two neurotoxic endpoints in the brain of METH-exposed animals. Brain regions exhibiting DAT and 5-HTT deficits that co-localize with decreased VMAT-2 levels and glial cell activation may represent monoaminergic terminal degeneration. However, the DAT and 5-HTT deficits in brain regions lacking a deficit in VMAT-2 and glial cell activation may reflect drug-induced modulation of these plasma membrane proteins.