Cyto-Genotoxicity of Tritiated Stainless Steel and Cement Particles in Human Lung Cell Models.

During the decommissioning of nuclear facilities, the tritiated materials must be removed. These operations generate tritiated steel and cement particles that could be accidentally inhaled by workers. Thus, the consequences of human exposure by inhalation to these particles in terms of radiotoxicology were investigated. Their cyto-genotoxicity was studied using two human lung models: the BEAS-2B cell line and the 3D MucilAirTM model. Exposures of the BEAS-2B cell line to particles (2 and 24 h) did not induce significant cytotoxicity. Nevertheless, DNA damage occurred upon exposure to tritiated and non-tritiated particles, as observed by alkaline comet assay. Tritiated particles only induced cytostasis; however, both induced a significant increase in centromere negative micronuclei. Particles were also assessed for their effects on epithelial integrity and metabolic activity using the MucilAirTM model in a 14-day kinetic mode. No effect was noted. Tritium transfer through the epithelium was observed without intracellular accumulation. Overall, tritiated and non-tritiated stainless steel and cement particles were associated with moderate toxicity. However, these particles induce DNA lesions and chromosome breakage to which tritium seems to contribute. These data should help in a better management of the risk related to the inhalation of these types of particles.

Marine Bacteria under Low-Intensity Radioactive Exposure: Model Experiments.

Radioactive contaminants create problems all over world, involving marine ecosystems, with their ecological importance increasing in the future. The review focuses on bioeffects of a series of alpha and beta emitting radioisotopes (americium-241, uranium-(235 + 238), thorium-232, and tritium) and gamma radiation. Low-intensity exposures are under special consideration. Great attention has been paid to luminous marine bacteria as representatives of marine microorganisms and a conventional bioassay system. This bioassay uses bacterial bioluminescence intensity as the main testing physiological parameter; currently, it is widely applied due to its simplicity and sensitivity. Dependences of the bacterial luminescence response on the exposure time and irradiation intensity were reviewed, and applicability of hormetic or threshold models was discussed. A number of aspects of molecular intracellular processes under exposure to low-intensity radiation were analyzed: (a) changes in the rates of enzymatic processes in bacteria with the bioluminescent system of coupled enzymatic reactions of NADH:FMN-oxidoreductase and bacterial luciferase taken as an example; (b) consumption of an intracellular reducer, NADH; (c) active role of reactive oxygen species; (d) repairing of the DNA damage. The results presented confirm the function of humic substances as natural radioprotectors.

Comparative Study of the γH2AX Foci Forming in Human Lung Fibroblasts Incubated in Media Containing Tritium-Labeled Thymidine or Amino Acids.

We compared the formation of γH2AX foci (marker of DNA double-strand breaks) in human lung fibroblasts (MRC-5 line) during their 24-h incubation in a medium containing 3H-labeled thymidine or amino acids (glycine, alanine, and proline) with specific radioactivity from 100 to 400 MBq/liter. A linear dependence of changes in the number of γH2AX foci on the specific radioactivity of the medium was revealed. The quantitative yield of DNA double-strand breaks under the influence of 3H-thymidine was more than 2-fold higher than under the influence of 3H-labeled amino acids. Comparative analysis of the yields of DNA double-strand breaks during cell incubation with 3H-labeled amino acids showed that 3H-alanine produced more pronounced effect that 3H-proline, which is consistent with the data on the content of their non-radioactive analogs in chromatin proteins.

Histamine H2 receptor radioligands: triumphs and challenges.

Since the discovery of the histamine H2 receptor (H2R), radioligands were among the most powerful tools to investigate its role and function. Initially, radiolabeling was used to investigate human and rodent tissues regarding their receptor expression. Later, radioligands gained increasing significance as pharmacological tools in in vitro assays. Although tritium-labeling was mainly used for this purpose, labeling with carbon-14 is preferred for metabolic studies of drug candidates. After the more-or-less successful application of numerous labeled H2R antagonists, the recent development of the G protein-biased radioligand [3H]UR-KAT479 represents another step forward to elucidate the widely unknown role of the H2R in the central nervous system through future studies.

Radiolabeled Amino Acid Uptake Assays in Primary Bone Cells and Bone Explants.

Radiolabeled amino acid uptake assays are a highly sensitive method used to characterize the uptake of amino acids by cells or tissues in culture. This method is an excellent tool to quantify changes in amino acid consumption that are associated with states of cellular differentiation and/or disease. The methods presented here can be adapted to measure the transport of all amino acids and can be applied to cultured cells and bone explants.

Enzymatic Responses to Low-Intensity Radiation of Tritium.

The present study considers a possible role of enzymatic reactions in the adaptive response of cells to the beta-emitting radionuclide tritium under conditions of low-dose exposures. Effects of tritiated water (HTO) on the reactions of bacterial luciferase and NAD(P)H:FMN-oxidoreductase, as well as a coupled system of these two reactions, were studied at radioactivity concentrations ≤ 200 MBq/L. Additionally, one of the simplest enzymatic reactions, photobiochemical proton transfer in Coelenteramide-containing Fluorescent Protein (CLM-FP), was also investigated. We found that HTO increased the activity of NAD(P)H:FMN-oxidoreductase at the initial stage of its reaction (by up to 230%); however, a rise of luciferase activity was moderate (<20%). The CLM-FP samples did not show any increase in the rate of the photobiochemical proton transfer under the exposure to HTO. The responses of the enzyme systems were compared to the 'hormetic' response of luminous marine bacterial cells studied earlier. We conclude that (1) the oxidoreductase reaction contributes significantly to the activation of the coupled enzyme system and bacterial cells by tritium, and (2) an increase in the organization level of biological systems promotes the hormesis phenomenon.

An Agonist Radioligand for the Proinflammatory Lipid-Activated G Protein-Coupled Receptor GPR84 Providing Structural Insights.

The orphan G protein-coupled receptor (GPCR) GPR84 is expressed on immune cells mediating proinflammatory and immunostimulatory effects. In this study, we prepared the fully efficacious, nonbiased GPR84 agonist 6-hexylamino-2,4(1H,3H)-pyrimidinedione (6) in tritium-labeled form ([3H]PSB-1584) by hydrogenation of a hexenyl-substituted precursor with tritium gas. The radioligand was characterized by kinetic, saturation, and competition assays using membranes of Chinese hamster ovary cells recombinantly expressing the human GPR84. [3H]6 reversibly labeled the receptor with high affinity (KD 2.08 nM). Structurally diverse orthosteric and allosteric ligands, including newly designed and synthesized compounds, were studied in competition binding assays. A homology model of GPR84 was generated to perform docking studies rationalizing the experimental data. The radioligand was additionally used for labeling GPR84 in native cells and tissues. [3H]6 constitutes the first GPR84 agonist radioligand representing a powerful tool for this poorly investigated GPCR, which has potential as a future drug target.

Comparative Analysis of the Formation of γH2AX Foci in Human Mesenchymal Stem Cells Exposed to 3H-Thymidine, Tritium Oxide, and X-Rays Irradiation.

We performed a comparative study of the formation of γН2АХ foci (a marker of DNA doublestrand breaks) in human bone marrow mesenchymal stem cells after 24-h incubation with 3Н-thimidin and tritium oxide with low specific activities (50-800 MBq/liter). The dependence of the number of γH2AX foci on specific activity of 3H-thymidine was described by a linear equation y=2.21+43.45x (R2=0.96), where y is the number of γH2AX foci per nucleus and x is specific activity in 1000 MBq/liter. For tritium oxide, the relationship was described by a linear equation y=2.52+6.70x (R2=0.97). Thus, the yield of DNA double-strand breaks after exposure to 3H-thymidine was 6.5-fold higher than after exposure to tritium oxide. Comparison of the effects of tritium oxide and X-ray radiation on the yield of DNA double-strand breaks showed that the relative biological efficiency of tritium oxide in a dose range of 3.78-60.26 mGy was 1.6-fold higher than that of X-ray radiation. Improvement of the methods of analysis of DNA double-strand breaks repair foci is highly promising in the context of creation of highly sensitive biodosimetry technologies for tritium compounds in humans.

Tritiated water exposure disrupts myofibril structure and induces mis-regulation of eye opacity and DNA repair genes in zebrafish early life stages.

Tritium (3H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H2O2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae.

Measurement of Rates of Cholesterol and Fatty Acid Synthesis In Vivo Using Tritiated Water.

Every organ in the body is capable of synthesizing cholesterol de novo but at rates that vary with a constellation of factors. A significant proportion of the hydrogen atoms present in cholesterol that is synthesized in the body are derived from water. Thus, although water ordinarily makes up the bulk of body mass, the acute enrichment of the body water pool with a sufficiently large amount of tritiated water over a short interval of time (usually 1 h) yields measurable rates of incorporation of the labeled water into newly generated cholesterol and also fatty acids. Such data can provide a quantitative measure of how specific genetic, dietary, and pharmacological manipulations impact not just the rate of cholesterol synthesis in particular organs but also rates of whole-body cholesterol production and turnover.

The binding orientations of structurally-related ligands can differ; A cautionary note.

Crystal structures can identify ligand-receptor interactions and assist the development of novel therapeutics, but experimental challenges sometimes necessitate the use of homologous proteins. Tropisetron is an orthosteric ligand at both 5-HT3 and α7 nACh receptors and its binding orientation has been determined in the structural homologue AChBP (pdbid: 2WNC). Co-crystallisation with a structurally-related ligand, granisetron, reveals an almost identical orientation (pdbid; 2YME). However, there is a >1000-fold difference in the affinity of tropisetron at 5-HT3 versus α7 nACh receptors, and α7 nACh receptors do not bind granisetron. These striking pharmacological differences prompt questions about which receptor the crystal structures most closely represent and whether the ligand orientations are correct. Here we probe the binding orientation of tropisetron and granisetron at 5-HT3 receptors by in silico modelling and docking, radioligand binding on cysteine-substituted 5-HT3 receptor mutants transiently expressed in HEK 293 cells, and synthetic modification of the ligands. For 15 of the 23 cysteine substitutions, the effects on tropisetron and granisetron were different. Structure-activity relationships on synthesised derivatives of both ligands were also consistent with different orientations, revealing that contrary to the crystallographic evidence from AChBP, the two ligands adopt different orientations in the 5-HT3 receptor binding site. Our results show that even quite structurally similar molecules can adopt different orientations in the same binding site, and that caution may be needed when using homologous proteins to predict ligand binding.

On the mechanism of biological activation by tritium.

The mechanism of biological activation by beta-emitting radionuclide tritium was studied. Luminous marine bacteria were used as a bioassay to monitor the biological effect of tritium with luminescence intensity as the physiological parameter tested. Two different types of tritium sources were used: HTO molecules distributed regularly in the surrounding aqueous medium, and a solid source with tritium atoms fixed on its surface (tritium-labeled films, 0.11, 0.28, 0.91, and 2.36 MBq/cm(2)). When using the tritium-labeled films, tritium penetration into the cells was prevented. The both types of tritium sources revealed similar changes in the bacterial luminescence kinetics: a delay period followed by bioluminescence activation. No monotonic dependences of bioluminescence activation efficiency on specific radioactivities of the films were found. A 15-day exposure to tritiated water (100 MBq/L) did not reveal mutations in bacterial DNA. The results obtained give preference to a "non-genomic" mechanism of bioluminescence activation by tritium. An activation of the intracellular bioluminescence process develops without penetration of tritium atoms into the cells and can be caused by intensification of trans-membrane cellular processes stimulated by ionization and radiolysis of aqueous media.

A novel radioligand for the ATP-gated ion channel P2X7: [3H] JNJ-54232334.

The ATP-gated ion channel P2X7 has emerged as a potential central nervous system (CNS) drug target based on the hypotheses that pro-inflammatory cytokines such as IL-1ß that are released by microglia, may contribute to the etiology of various disorders of the CNS including depression. In this study, we identified two closely related P2X7 antagonists, JNJ-54232334 and JNJ-54140515, and then tritium labeled the former to produce a new radioligand for P2X7. JNJ-54232334 is a high affinity ligand for the rat P2X7 with a pKi of 9.3±0.1. In rat cortical membranes, [3H] JNJ-54232334 reached saturable binding with equilibrium dissociation (Kd) constant of 4.9±1.3 nM. The compound displayed monophasic association and dissociation kinetics with fast on and off rates. In rat brain sections, specific binding of [3H] JNJ-54232334 was markedly improved compared to the previously described P2X7 radioligand, [3H] A-804598. In P2X7 knockout mouse brain sections, [3H] A-804598 bound to non-P2X7 binding sites in contrast to [3H] JNJ-54232334. In rat or wild type mouse brain sections [3H] JNJ-54232334 bound in a more homogenous and region independent manner. The ubiquitous expression of P2X7 receptors was confirmed with immunohistochemistry in rat brain sections. The partial displacement of [3H] A-804598 binding resulted in the underestimation of the level of ex vivo P2X7 occupancy for JNJ-54140515. Higher levels of P2X7 ex vivo occupancy were measured using [3H] JNJ-54232334 due to less non-specific binding. In summary, we describe [3H] JNJ-54232334 as a novel P2X7 radioligand, with improved properties over [3H] A-804598.

The influence of different cellular environments on PET radioligand binding: an application to D2/3-dopamine receptor imaging.

Various D2/3 receptor PET radioligands are sensitive to endogenous dopamine release in vivo. The Occupancy Model is generally used to interpret changes in binding observed in in vivo competition binding studies; an Internalisation Hypothesis may also contribute to these changes in signal. Extension of in vivo competition imaging to other receptor systems has been relatively unsuccessful. A greater understanding of the cellular processes underlying signal changes following endogenous neurotransmitter release may help translate this imaging paradigm to other receptor systems. To investigate the Internalisation Hypothesis we assessed the effects of different cellular environments, representative of those experienced by a receptor following agonist-induced internalisation, on the binding of three D2/3 PET ligands with previously reported sensitivities to endogenous dopamine in vivo, namely [3H]spiperone, [3H]raclopride and [3H]PhNO. Furthermore, we determined the contribution of each cellular compartment to total striatal binding for these D2/3 ligands. These studies suggest that sensitivity to endogenous dopamine release in vivo is related to a decrease in affinity in the endosomal environment compared with those found at the cell surface. In agreement with these findings we also demonstrate that ∼25% of total striatal binding for [3H]spiperone originates from sub-cellular, microsomal receptors, whereas for [3H]raclopride and [3H]PhNO, this fraction is lower, representing ∼14% and 17%, respectively. This pharmacological approach is fully translatable to other receptor systems. Assessment of affinity shifts in different cellular compartments may play a crucial role for understanding if a radioligand is sensitive to endogenous release in vivo, for not just the D2/3, but other receptor systems.

Effect of tritium on luminous marine bacteria and enzyme reactions.

The paper studies chronic effect of tritiated water, HTO, (0.0002-200 MBq/L) on bioluminescent assay systems: marine bacteria Photobacterium phosphoreum (intact and lyophilized) and coupled enzyme reactions. Bioluminescence intensity serves as a marker of physiological activity. Linear dependencies of bioluminescent intensity on exposure time or radioactivity were not revealed. Three successive stages in bacterial bioluminescence response to HTO were found: (1) absence of the effect, (2) activation, and (3) inhibition. They were interpreted in terms of reaction of organisms to stress-factor i.e. stress recognition, adaptive response/syndrome, and suppression of physiological function. In enzyme system, in contrast, the kinetic stages mentioned above were not revealed, but the dependence of bioluminescence intensity on HTO specific radioactivity was found. Damage of bacteria cells in HTO (100 MBq/L) was visualized by electron microscopy. Time of bioluminescence inhibition is suggested as a parameter to evaluate the bacterial sensitivity to ionizing radiation.

Tritium contamination of hematopoietic stem cells alters long-term hematopoietic reconstitution.

PURPOSE: In vivo effects of tritium contamination are poorly documented. Here, we study the effects of tritiated Thymidine ([(3)H] Thymidine) or tritiated water (HTO) contamination on the biological properties of hematopoietic stem cells (HSC). MATERIALS AND METHODS: Mouse HSC were contaminated with concentrations of [(3)H] Thymidine ranging from 0.37-37.03 kBq/ml or of HTO ranging from 5-50 kBq/ml. The biological properties of contaminated HSC were studied in vitro after HTO contamination and in vitro and in vivo after [(3)H] Thymidine contamination. RESULTS: Proliferation, viability and double-strand breaks were dependent on [(3)H] Thymidine or HTO concentrations used for contamination but in vitro myeloid differentiation of HSC was not affected by [(3)H] Thymidine contamination. [(3)H] Thymidine contaminated HSC showed a compromised long-term capacity of hematopoietic reconstitution and competition experiments showed an up to two-fold decreased capacity of contaminated HSC to reconstitute hematopoiesis. These defects were not due to impaired homing in bone marrow but to an initial decreased proliferation rate of HSC. CONCLUSION: These results indicate that contaminations of HSC with doses of tritium that do not result in cell death, induce short-term effects on proliferation and cell cycle and long-term effects on hematopoietic reconstitution capacity of contaminated HSC.

Lysosomal trapping of a radiolabeled substrate of P-glycoprotein as a mechanism for signal amplification in PET.

The radiotracer [(11)C]N-desmethyl-loperamide (dLop) images the in vivo function of P-glycoprotein (P-gp), a transporter that blocks the entry of drugs that are substrates into brain. When P-gp is inhibited, [(11)C]dLop, a potent opiate agonist, enters and becomes trapped in the brain. This trapping is beneficial from an imaging perspective, because it amplifies the PET signal, essentially by accumulating radioactivity over time. As we previously demonstrated that this trapping was not caused by binding to opiate receptors, we examined whether [(11)C]dLop, a weak base, is ionically trapped in acidic lysosomes. To test this hypothesis, we measured [(3)H]dLop accumulation in human cells by using lysosomotropics. Because the in vivo trapping of dLop was seen after P-gp inhibition, we also measured [(3)H]dLop uptake in P-gp-expressing cells treated with the P-gp inhibitor tariquidar. All lysosomotropics decreased [(3)H]dLop accumulation by at least 50%. In P-gp-expressing cells, tariquidar (and another P-gp inhibitor) surprisingly decreased [(3)H]dLop uptake. Consequently, we measured [(11)C]dLop uptake before and after tariquidar preadministration in lysosome-rich organs of P-gp KO mice and humans. After tariquidar pretreatment in both species, radioactivity uptake in these organs decreased by 35% to 40%. Our results indicate that dLop is trapped in lysosomes and that tariquidar competes with dLop for lysosomal accumulation in vitro and in vivo. Although tariquidar and dLop compete for lysosomal trapping in the periphery, such competition does not occur in brain because tariquidar has negligible entry into brain. In summary, tariquidar and [(11)C]dLop can be used in combination to selectively measure the function of P-gp at the blood-brain barrier.

EdU incorporation is an alternative non-radioactive assay to [(3)H]thymidine uptake for in vitro measurement of mice T-cell proliferations.

RATIONALE: T lymphocyte proliferations can be measured by [(3)H]thymidine incorporation. However, many labs avoid this technique because of the need to use radioactive substrates. In addition, [(3)H]thymidine incorporation method does not permit simultaneous characterization of the proliferating cells. We developed the 5-ethynyl-2'-deoxyuridine (EdU) and Cu(I)-catalyzed cycloaddition "click" reaction assay to measure T-cell responses by flow cytometry. METHODS: Spleen cells from normal, immune-deficient purine nucleoside phosphorylase (PNP) defective (PNP-/-) mice or PNP-/- mice with partial immune reconstitution were stimulated with anti-CD3 antibodies. The correlation (r) between [(3)H]thymidine and EdU incorporations into stimulated T cells was measured and the stimulation index (SI), the ratio between stimulated and non-stimulated cells, was calculated. Flow cytometry was used to characterize the proliferating cells. RESULTS: EdU and [(3)H]thymidine incorporation into normal spleen cells were strongly correlated (r=0.89). Following stimulation, EdU incorporation into spleen cells from normal and immune-reconstituted PNP-/- mice was significantly increased compared to PNP-/- immune-deficient mice. Immune-deficient PNP-/- mice had increased [(3)H]thymidine and EdU incorporation into non-stimulated spleen cells, indicative of spontaneous proliferation. Analysis of EdU incorporation showed that the increased proliferation was due primarily to cells expressing CD3, CD4 and IgM. CONCLUSION: EdU-Click technology accurately measures proliferation of murine T lymphocyte and can be used as an alternative to [(3)H]thymidine assays. The EdU-Click technology also allows identification of proliferating cells.

Metabotropic glutamate receptor II in the brains of Parkinsonian patients.

Modulation of basal ganglia group II metabotropic glutamate receptors (mGluR2/3) is a potential therapeutic alternative to levodopa in Parkinson disease (PD). We used receptor-binding autoradiography of the mGluR2/3-selective radioligand [H]LY341495 in postmortem brain specimens from PD patients (n = 14) and controls (n=11) to investigate possible contributions of changes in ligand binding of this receptor to levodopa-associated motor complications experienced premortem in PD patients. The PD patients included those with and without histories of dyskinesias and those with and without "wearing off," which is defined as a reduced period of benefit from levodopa. Specific binding of [H]LY341495 to mGluR2/3 in the basal ganglia was higher in the caudate nucleus than the putamen and lower by approximately half in the external and internal globus pallidus (GPi) in controls. [H]LY341495-specific binding was reduced in the caudate and GPi in patients without wearing-off (-22% caudate, -30% GPi), compared with controls and with patients who had experienced wearing-off; there were no differences among PD patients with or without dyskinesias. These data suggest that an adaptive downregulation of mGluR2/3 in PD patients without wearing-off may compensate for increased glutamate. They indicate a key role for mGluR2/3 in control of movement and the potential for mGluR2/3-targeted drugs in the management of wearing-off fluctuations in PD.

Asenapine exerts distinctive regional effects on ionotropic glutamate receptor subtypes in rat brain.

Asenapine, a new pyschopharmacologic agent being developed for the treatment of schizophrenia and bipolar disorder, has a unique human receptor binding signature with strong affinity for dopaminergic, alpha-adrenergic, and, in particular, serotonergic receptors raising the possibility of interactions with glutamatergic receptors. Changes in ionotropic glutamate (Glu) N-methyl-D-aspartic acid (NMDA) receptors and 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) receptors in rat forebrain regions were quantified after repeated administration of multiple doses of asenapine (0.03, 0.1, or 0.3 mg/kg, subcutaneous, twice/day) or vehicle for 4 weeks. Brain sections were collected from the medial prefrontal cortex (mPFC), dorsolateral frontal cortex, caudate putamen (CPu), nucleus accumbens (NAc), and hippocampus (HIP), and processed for in vitro receptor autoradiography. Four weeks of treatment with 0.03, 0.1, or 0.3 mg/kg of asenapine significantly (P < 0.01) decreased binding of [3H]MK-801 to NMDA/MK-801 modulatory sites in NAc (by 27%, 29%, and 26%, respectively), medial CPu (by 25%, 28%, and 24%), and lateral CPu (by 24%, 31%, and 26%). In contrast, the same doses of asenapine did not alter binding of [3H]glycine to NMDA/glycine modulatory sites in any of the brain regions examined. [3H]AMPA binding to AMPA receptors was selectively and significantly (P < 0.001) elevated in hippocampal CA(1) (41%) and CA(3) (40%) regions but only at the highest dose tested. These results indicate that chronic treatment with asenapine has region-specific and dose-dependent effects on ionotropic Glu-receptor subtypes in rat forebrain, which might contribute to the unique psychopharmacologic properties of asenapine.