α-particle therapy for synovial sarcoma in the mouse using an astatine-211-labeled antibody against frizzled homolog 10.

Synovial sarcoma (SS) is a rare yet refractory soft-tissue sarcoma that predominantly affects young adults. We show in a mouse model that radioimmunotherapy (RIT) with an α-particle emitting anti-Frizzled homolog 10 (FZD10) antibody, synthesized using the α-emitter radionuclide astatine-211 (211 At-OTSA101), suppresses the growth of SS xenografts more efficiently than the corresponding ß-particle emitting anti-FZD10 antibody conjugated with the ß-emitter yettrium-90 (90 Y-OTSA101). In biodistribution analysis, 211 At was increased in the SS xenografts but decreased in other tissues up to 1 day after injection as time proceeded, albeit with a relatively higher uptake in the stomach. Single 211 At-OTSA101 doses of 25 and 50 µCi significantly suppressed SS tumor growth in vivo, whereas a 50-µCi dose of 90 Y-OTSA101 was needed to achieve this. Importantly, 50 µCi of 211 At-OTSA101 suppressed tumor growth immediately after injection, whereas this effect required several days in the case of 90 Y-OTSA101. Both radiolabeled antibodies at the 50-µCi dosage level significantly prolonged survival. Histopathologically, severe cellular damage accompanied by massive cell death was evident in the SS xenografts at even 1 day after the 211 At-OTSA101 injection, but these effects were relatively milder with 90 Y-OTSA101 at the same timepoint, even though the absorbed doses were comparable (3.3 and 3.0 Gy, respectively). We conclude that α-particle RIT with 211 At-OTSA101 is a potential new therapeutic option for SS.

Targeted cancer cell ablation in mice by an α-particle-emitting astatine-211-labeled antibody against major histocompatibility complex class I chain-related protein A and B.

Major histocompatibility complex class I chain-related protein A and B (MICA/B) are ligands of the immune receptor, natural-killer group 2 member D. MICA/B expression is often found in several types of cancer but is restricted in normal tissues. Here, we show that an α-particle emitting astatine-211 (211At)-labeled antibody targeting MICA/B (211At-anti MICA/B Ab) efficiently ablates cancer cells in vitro and in vivo. We generated 211At-anti MICA/B Ab, an anti-MICA/B antibody conjugated with a highly cytotoxic α-particle emitting radionuclide 211At. 211At-anti MICA/B Ab binds to human osteosarcoma SaOS2 and U2OS cells that exhibit high levels of MICA/B expression and efficiently kills those cells in vitro. Biodistribution analysis using xenograft mouse models of HCT116 p53-/- positive for MICA/B expression, showed increased 211At in the xenografts for up to 22 h after injection as time proceeded. A single dose of 211At-anti MICA/B Ab (1 MBq) showed significant reduction in the tumor growth rate of HCT116 p53-/- xenografts compared to 211At-labeled mouse IgG (1 MBq) at 21 days after injection. No body weight loss and erythrocytopenia was evident in mice that received 211At-anti MICA/B. Leukocytopenia and thrombocytopenia were observed within a week after 211At-anti MICA/B injection, but counts of red blood cells and platelets were recovered to control levels at about 3-4 weeks after injection. Taken together, these data strongly demonstrate that targeted α-particle therapy using 211At-anti-MICA/B Ab emitting highly cytotoxic α-particles is a potential new therapeutic option for several types of cancer.

Locoregional therapy with α-emitting trastuzumab against peritoneal metastasis of human epidermal growth factor receptor 2-positive gastric cancer in mice.

Peritoneal metastasis of gastric cancer (PMGC) is incurable and thus has an extremely poor prognosis. We have found, however, that locoregionally administered trastuzumab armed with astatine-211 (211 At-trastuzumab) is effective against human epidermal growth factor receptor 2 (HER2)-positive PMGC in a xenograft mouse model. We first observed that 211 At-trastuzumab can specifically bind and effectively kill NCI-N87 (N87) cells, which are HER2-positive human metastatic GC cells, both in vitro and in s.c. tumors. We established a PMGC mouse model using N87 xenografts stably expressing luciferase to test α-particle radioimmunotherapy with 211 At-trastuzumab against PMGC. Biodistribution analysis in this PMGC mouse model revealed that the i.p. administration of 211 At-trastuzumab (1 MBq) was a more efficient means of delivery of 211 At into metastatic tumors than i.v. injection; the maximum tumor uptake with i.p. administration was over 60% injected dose per gram of tissue (%ID/g) compared to approximately 18%ID/g with i.v. injection. Surprisingly, a single i.p. injection of 211 At-trastuzumab (1 MBq) was sufficient to completely eradicate intraperitoneally disseminated HER2-positive GC xenografts in two of six treated mice by inducing DNA double-strand breaks, and to drastically reduce the tumor burden in another three mice. No bodyweight loss, leukocytopenia, or significant biochemical changes in liver or kidney function were observed in the treatment group. Accordingly, locoregionally administered 211 At-trastuzumab significantly prolonged the survival time of HER2-positive PMGC mice compared with control treatments. Our results provide a proof-of-concept demonstration that locoregional therapy with 211 At-trastuzumab may offer a new treatment option for HER2-positive PMGC.

Growth retardation and hair loss in transgenic mice overexpressing human H-ferritin gene.

H-ferritin (HF) is a core subunit of the iron storage protein ferritin, and plays a central role in the regulation of cellular iron homeostasis. Recent studies revealed that ferritin and HF are involved in a wide variety of iron-independent functions, including regulating biological processes during physiological and pathological conditions, and can be overexpressed in some human diseases. To investigate the in vivo function of HF, we generated transgenic (tg) mice overexpressing the human HF gene (hHF-tg). We established two independent hHF-tg mouse lines. Although both lines of hHF-tg mice were viable, they showed reduced body size compared to wild-type (WT) mice at 4-12 weeks of age. Serum iron concentration and blood parameters of hHF-tg mice such as hemoglobin and red blood cell counts were comparable to those of WT mice. At 3-5 weeks of age, hHF-tg mice exhibited temporary loss of coat hair on the trunk, but not on the head or face. Histological analyses revealed that although initial hair development was normal, hHF-tg mice had epidermal hyperplasia with hyperkeratosis, dilated hair follicles, bended hair shafts and keratinous debris during the hairless period. In conclusion, we showed that hHF-tg mice exhibited mild growth retardation and temporary hairless phenotype. Our findings highlight the physiological roles of HF and demonstrate that hHF-tg mice are useful for understanding the in vivo functions of HF.

H-ferritin overexpression promotes radiation-induced leukemia/lymphoma in mice.

H-ferritin (HF) is a core subunit of the iron storage protein ferritin and is related to the pathogenesis of malignant diseases. HF overexpression is present in human hematologic malignancies, suggesting that HF overexpression may contribute to the development of hematologic cancers. However, in vivo evidence that HF is directly linked to hematologic tumorigenesis has not yet been shown. In this study, we show that transgenic (tg) mice overexpressing the human HF gene (hHF-tg) developed aggressive radiation-induced thymic lymphoma/leukemia (TL) compared with wild-type (WT) mice, providing evidence that HF overexpression promotes leukemia/lymphomagenesis. Fractionated X-irradiation of hHF-tg mice caused a higher incidence and earlier onset of TL compared with WT mice. Immunological and pathological features of TLs were similar in both groups. However, proliferative activity of hHF-tg lymphoma cells was higher than that of WT lymphoma cells, and microarray analyses revealed that some leukemia/lymphoma-related genes were differentially expressed in hHF-tg TLs compared with WT TLs. To investigate whether cell damage induced by irradiation is related to leukemia/lymphomagenesis, we evaluated apoptotic levels in the thymus and bone marrow (BM) of hHF-tg and WT groups after fractionated X-irradiation. Apoptosis was augmented in the hHF-tg BM, but not in the thymus, compared with the WT BM, suggesting a possible linkage between increased BM apoptosis by HF overexpression and accelerated radiation-induced TL development. Our findings indicate that HF overexpression is closely related to the development of leukemia/lymphoma, which could have implications for the prevention of malignant hematologic diseases.

Molecular imaging of mesothelioma by detection of manganese-superoxide dismutase activity using manganese-enhanced magnetic resonance imaging.

Malignant mesothelioma (MM) is a fatal malignancy with a rapidly increasing incidence in industrialized countries because of the widespread use of asbestos in the past centuries. Early diagnosis of MM is critical for a better prognosis, but this is often difficult because of the lack of disease-specific diagnostic imaging. Here, we report that manganese-enhanced magnetic resonance imaging (MEMRI) represents a promising approach for a more selective mesothelioma imaging by monitoring a high-level expression of manganese-superoxide dismutase (Mn-SOD), which is observed in many MM. We found that most human MM cells overexpressed Mn-SOD protein compared with human mesothelial cells and that NCI-H226 human MM cells highly expressed Mn-SOD and augmented Mn accumulation when loaded with manganese chloride (MnCl(2)). The cells showed marked T(1)-signal enhancement on in vitro MRI after incubation with MnCl(2) because of the T(1) shortening effect of Mn(2+). H226 subcutaneous tumor was preferentially enhanced compared with a lung adenocarcinoma cell tumor and another human MM cell tumor in MnCl(2)-enhanced T(1)-weighted MR image (T(1)WI), correlating with their respective Mn-SOD expression levels. Moreover, in a more clinically relevant setting, H226 xenografted pleural tumor was markedly enhanced and readily detected by MEMRI using manganese dipyridoxyl diphosphate (MnDPDP), a clinically used contrast agent, as well as MnCl(2). Therefore, we propose that MEMRI can be a potentially powerful method for noninvasive detection of MM, with high spatial resolution and marked signal enhancement, by targeting Mn-SOD.

Utility of 211At-Trastuzumab for the Treatment of Metastatic Gastric Cancer in the Liver: Evaluation of a Preclinical α-Radioimmunotherapy Approach in a Clinically Relevant Mouse Model.

A liver metastasis from a primary gastric cancer (LMGC) is relatively common and results in an extremely poor prognosis due to a lack of effective therapeutics. We here demonstrate in a clinically relevant mouse model that an α-particle radioimmunotherapy approach with 211At-labeled trastuzumab has efficacy against LMGCs that are positive for human epidermal growth factor receptor 2 (HER2). Methods:211At was produced in a cyclotron via a 209Bi (α,2n) 211At reaction. 211At-trastuzumab was subsequently generated using a single-step labeling method. NCI-N87 cells (HER2-positive human gastric cancer cells) carrying a luciferase gene were intrasplenically transplanted into severe combined immunodeficiency mice to generate an HER2-positive LMGC model. A biodistribution study was then conducted through the intravenous injection of 211At-trastuzumab (1 MBq) into these LMGC xenograft mice. In parallel with this experimental therapy, phosphate-buffered saline, intact trastuzumab, or 211At-nonspecific human IgG (1 MBq) was injected into control groups. The therapeutic efficacy was evaluated by monitoring tumor changes by chemiluminescence imaging. Body weights, white blood cell counts, and serum markers of tissue damage were monitored at regular intervals. Microdosimetry using a CR-39 plastic detector was also performed. Results: The biodistribution analysis revealed an increased uptake of 211At-trastuzumab in the metastasized tumors that reached approximately 12% of the injected dose per gram of tissue (%ID/g) at 24 h. In contrast, its uptake to the surrounding liver was about 4 %ID/g. The LMGCs in the mouse model reduced dramatically at 1 wk after the single systemic injection of 211At-trastuzumab. No recurrences were observed in 6 of 8 mice treated with this single injection, and their survival time was significantly prolonged compared with the control groups, including the animals treated with 211At-nonspecific antibodies. No severe toxicities or abnormalities in terms of body weight, white blood cell number, liver function, or kidney parameters were observed in the 211At-trastuzumab group. Microdosimetric studies further revealed that 211At-trastuzumab had been delivered at an 11.5-fold higher dose to the LMGC lesions than to the normal liver. Conclusion: α-radioimmunotherapy with 211At-trastuzumab has considerable potential as an effective and safe therapeutic option for LMGC.

Significance of PD-L1 expression in carbon-ion radiotherapy for uterine cervical adeno/adenosquamous carcinoma.

OBJECTIVE: Programmed cell death-ligand 1 (PD-L1) is expressed in tumor cells and has been shown to predict clinical outcomes of several types of malignancies. The aim of this study was to investigate the effects of carbon-ion (C-ion) beam irradiation on PD-L1 expression in human uterine cervical adeno/adenosquamous carcinoma (UCAA) cells and clinical samples and to identify the prognostic factors for outcomes after C-ion radiotherapy (CIRT). METHODS: The effects of C-ion irradiation on PD-L1 expression in human UCAA and cervical squamous cell carcinoma cells were examined by flow cytometry. We examined PD-L1 expression in UCAA biopsy specimens from 33 patients before CIRT started (pre-CIRT) and after 12 Gy (relative biological effectiveness [RBE]) irradiation (post-12Gy-C) in 4 fractions of CIRT to investigate the correlation between PD-L1 status and clinical outcomes. RESULTS: The PD-L1 expression was upregulated by C-ion beam in a dose-dependent manner in HeLa and SiHa cells through phosphorylated Chk1. The overall frequencies of pre-CIRT and post-12Gy-C PD-L1 positivity were 45% (15/33) and 67% (22/33), respectively. The post-12Gy-C PD-L1 expression was significantly elevated compared to the pre-CIRT PD-L1 expression. There was no significant relationship between the pre-CIRT PD-L1 status and clinical outcomes, such as local control (LC), progression-free survival (PFS), and overall survival (OS). However, the post-12Gy-C PD-L1 expression had better correlation with PFS, but not with LC and OS. CONCLUSION: CIRT can induce PD-L1 expression in UCAA and we propose that PD-L1 expression after starting CIRT may become as a predictive prognostic marker in CIRT for UCAA.

131I-IITM and 211At-AITM: Two Novel Small-Molecule Radiopharmaceuticals Targeting Oncoprotein Metabotropic Glutamate Receptor 1.

Targeted radionuclide therapy (TRT) targeting oncoproteins facilitates the delivery of therapeutic radionuclides to tumor tissues with high precision. Herein, we developed 2 new radiopharmaceuticals, 4-131I-iodo- and 4-211At-astato-N-[4-(6-(isopropylamino)pyridine-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (131I-IITM and 211At-AITM), targeting the ectopic metabotropic glutamate receptor 1 (mGluR1) in melanomas for TRT studies. Methods:131I-IITM and 211At-AITM were synthesized by reacting a stannyl precursor with 131I-NaI and 211At in the presence of an oxidizing agent. The therapeutic efficacy and safety of the 2 radiopharmaceuticals were investigated using mGluR1-expressing B16F10 melanoma cells and melanoma-bearing mice. Results:131I-IITM and 211At-AITM were obtained with a radiochemical purity of greater than 99% and radiochemical yields of 42.7% ± 10.4% and 45.7% ± 6.5%, respectively, based on the total radioactivity of used radionuclides. 131I-IITM and 211At-AITM exhibited a maximum uptake of 4.66% ± 0.70 and 7.68% ± 0.71 percentage injected dose per gram (%ID/g) in the targeted melanomas, respectively, and were rapidly cleared from nontarget organs after intravenous injection. Both agents markedly inhibited melanoma growth compared with the controls (61.00% and 95.68%, respectively). In the melanoma model, considerably greater therapeutic efficacy with negligible toxicity was observed using 211At-AITM. Conclusion: The nontoxic radiopharmaceuticals 131I-IITM and 211At-AITM are useful high-precision TRT agents that can be used to target the oncoprotein mGluR1 for melanoma therapy.

Evidence of Local Concentration of α-Particles from 211At-Labeled Antibodies in Liver Metastasis Tissue.

We investigated the local concentration of α-particles from 211At-labeled trastuzumab antibodies against human epidermal growth factor receptor type 2 antigens in liver metastasis tissue of mice. Methods: Mice carrying metastatic cancer in their liver were injected with 211At-agent. After 12 h, the liver was removed and sliced, and 2 tissue samples of liver tissues without lesions and one containing metastatic lesions were mounted on the CR-39 plastic nuclear track detector. Microscope images of the tissues on the CR-39 were acquired. After irradiation for 31 h, the tissues were removed from the CR-39. A microscope image of α-particle tracks on the CR-39 was acquired after chemical etching. The positions of each tissue sample and the emitted α-particle tracks were adjusted to the same coordinates. Results: The positional distribution of α-particle tracks emitted from 211At was consistent within the tissue. The α-particle tracks were mainly allocated in the tumor region of the tissue. The absorbed dose in individual cells segmented by 10-µm intervals was obtained by the spectroscopic analysis of the linear-energy-transfer spectrum. The concentration efficiency-the track density ratio of α-particle tracks in the necrotized tissue, which was the tumor region, to the normal tissue-was found to be 6.0 ± 0.2. In the tumor region, the high-linear-energy-transfer α-particles deposited a large enough dose to cause lethal damage to the cancer cells. Conclusion: The total absorbed dose ranged from 1 to 7 Gy with a peak at around 2 Gy, which would correspond to a 2-3 times higher biologically equivalent dose because of the high relative biological effectiveness of the α-particles emitted from 211At.

Combination Therapy of Intravenously Injected Microglia and Radiation Therapy Prolongs Survival in a Rat Model of Spontaneous Malignant Glioma.

PURPOSE: The aim of this study was to investigate the efficacy of combination therapy with intravenously injected microglia (MI) and radiation therapy (RT) for malignant glioma in rats. METHODS AND MATERIALS: Transgenic rats expressing v-erbB and spontaneously developing malignant glioma were used. The rats were divided into 4 groups: control (n = 19), RT alone (n = 10), MI alone (n = 9), and combination MI and RT (MI + RT) (n = 10). Cranial x-ray irradiation (8 Gy per fraction; once per week) was performed at 50 and 51 weeks of age. Cultured rat microglial cells (5 × 106 cells/rat) were intravenously injected via the tail vein within 30 minutes after RT. RESULTS: No evidence of side effects, including thrombosis or graft-versus-host disease, was noted. Rats treated with RT alone, MI alone, MI + RT, and control survived 60.9, 56.3, 66.0, and 56.1 weeks, respectively. The survival period of MI + RT was significantly longer than that of control (P = .014), MI alone (P = .027), and RT alone (P = .049). Immunohistochemical analysis showed a significantly higher number of tumor-infiltrated MI in the RT alone (P = .041) and MI + RT groups (P = .014) compared with the control. Significantly more CD8-positive lymphocytes were observed in the MI + RT group (P = .049) compared with the control. A positive correlation was found between the number of MI and CD8-positive lymphocytes (R2 = 0.556). A positive correlation was also found between CD8-positive lymphocytes and survival periods (R2 = 0.460). CONCLUSIONS: MI + RT increased infiltrated MI and CD8-positive T cells and prolonged survival in transgenic rats that spontaneously developed malignant glioma. Combined immunocellular therapy and RT may provide a novel treatment strategy for malignant glioma.

Transcriptomic Signatures of Auger Electron Radioimmunotherapy Using Nuclear Targeting (111)In-Trastuzumab for Potential Combination Therapies.

(111)In-labeled trastuzumab modified with nuclear localizing signal (NLS) peptides ((111)In-trastuzumab-NLS) efficiently delivers an Auger electron (AE) emitter (111)In into the cell nucleus and is thus a promising radiopharmaceutical in AE radioimmunotherapy (AE-RIT) for targeted killing of HER2-positive cancer. However, further improvement of its therapeutic efficacy is required. In this study, the authors show a transcriptomic approach to identify potential targets for enhancing the cytotoxic effects of (111)In-trastuzumab-NLS. They generated two types of (111)In-trastuzumab-NLS harboring different numbers of NLS peptides, (111)In-trastuzumab-NLS-S and -L. These radioimmunoconjugates (230 and 460 kBq) showed a significant higher cytotoxicity to SKBR3 human breast cancer cells overexpressing HER2 compared to (111)In-trastuzumab. Microarray analysis revealed that NF-kB-related genes (38 genes) were significantly changed in transcription by (111)In trastuzumab-NLS-L (230 kBq) treatment. Quantitative reverse transcription polymerase chain reaction confirmed the microarray data by showing transcriptional alternation of selected NF-κB target genes in cells treated with (111)In-trastuzumab-NLS-L. Interestingly, bortezomib, a drug known as a NF-κB modulator, significantly enhanced the cytotoxicity of (111)In-trastuzumab-NLS-L in SKBR3 cells. Taken together, the transcriptome data suggest the possibility that the modulation of NF-kB signaling activity is a molecular signature of (111)In-trastuzumab-NLS and coadministration of bortezomib may be efficacious in enhancement of AE-RIT with (111)In-trastuzumab-NLS.

Quantifying initial cellular events of mouse radiation lymphomagenesis and its tumor prevention in vivo by positron emission tomography and magnetic resonance imaging.

Radiation-induced thymic lymphoma (RITL) in mice is induced by fractionated whole-body X-irradiation (FX) and has served as a useful model for studying radiation carcinogenesis. In this model, the initial postirradiation cellular events in the thymus and bone marrow (BM) are critically important for tumorigenesis, and BM transplantation (BMT) prevents RITL. However, direct assessment of these events is so far restricted by the lack of noninvasive monitoring techniques. Here, we have developed positron emission tomography (PET) and magnetic resonance imaging (MRI) methods to quantify the events critical for RITL development and the effects of BMT in living animals. Apparent diffusion coefficients (ADCs) were calculated from diffusion-weighted MRI to evaluate the changes in the BM of mice receiving FX. ADC values dramatically changed in the irradiated BM, corresponding to pathological findings of the irradiated BM, returning to normal levels following BMT sooner than with spontaneous recovery. PET with 4'-[methyl-(11)C]thiothymidine, a novel tracer for cell proliferation, revealed that the irradiated thymus showed significantly higher tracer uptake than the unirradiated thymus 1 week after FX. Interestingly, its increased uptake was completely abolished by BMT, even with very few donor-derived cells in the thymus. Thereafter, the thymus receiving BMT had significantly increased tracer uptake. These findings suggest that BMT first suppresses FX-induced aberrant thymocyte proliferation and then accelerates thymic regeneration. This study demonstrates the feasibility of using PET and MRI for noninvasive monitoring of tumorigenic cellular processes in an animal model of radiation-induced cancer.

Controlled administration of penicillamine reduces radiation exposure in critical organs during 64Cu-ATSM internal radiotherapy: a novel strategy for liver protection.

PURPOSE: (64)Cu-diacetyl-bis (N (4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a promising theranostic agent that targets hypoxic regions in tumors related to malignant characteristics. Its diagnostic usefulness has been recognized in clinical studies. Internal radiotherapy (IRT) with (64)Cu-ATSM is reportedly effective in preclinical studies; however, for clinical applications, improvements to reduce radiation exposure in non-target organs, particularly the liver, are required. We developed a strategy to reduce radiation doses to critical organs while preserving tumor radiation doses by controlled administration of copper chelator penicillamine during (64)Cu-ATSM IRT. METHODS: Biodistribution was evaluated in HT-29 tumor-bearing mice injected with (64)Cu-ATSM (185 kBq) with or without oral penicillamine administration. The appropriate injection interval between (64)Cu-ATSM and penicillamine was determined. Then, the optimal penicillamine administration schedule was selected from single (100, 300, and 500 mg/kg) and fractionated doses (100 mg/kg×3 at 1- or 2-h intervals from 1 h after (64)Cu-ATSM injection). PET imaging was performed to confirm the effect of penicillamine with a therapeutic (64)Cu-ATSM dose (37 MBq). Dosimetry analysis was performed to estimate human absorbed doses. RESULTS: Penicillamine reduced (64)Cu accumulation in the liver and small intestine. Tumor uptake was not affected by penicillamine administration at 1 h after (64)Cu-ATSM injection, when radioactivity was almost cleared from the blood and tumor uptake had plateaued. Of the single doses, 300 mg/kg was most effective. Fractionated administration at 2-h intervals further decreased liver accumulation at later time points. PET indicated that penicillamine acts similarly with the therapeutic (64)Cu-ATSM dose. Dosimetry demonstrated that appropriately scheduled penicillamine administration reduced radiation doses to critical organs (liver, ovaries, and red marrow) below tolerance levels. Laxatives reduced radiation doses to the large intestine. CONCLUSIONS: We developed a novel strategy to reduce radiation exposure in critical organs during (64)Cu-ATSM IRT, thus promoting its clinical applications. This method could be beneficial for other (64)Cu-labeled compounds.

Evaluation of ferritin-overexpressing brain in newly developed transgenic mice.

Aberrant expression of ferritin, a major iron-binding protein, has shown to be involved in neurodegenerative diseases. In this study, we generated transgenic (Tg) mice of human ferritin heavy chain (FTH) gene and investigated the effects of ferritin overexpression in FTH-Tg brain by (1)H-MRI and (1)H-MRS. The mice displayed no apparent neurological symptoms, and no specific morphological and T(2) alterations were found in the brain by MRI, and not even by histological studies. (1)H-MRS, however, revealed that some metabolic markers were significantly altered in FTH-Tg brains compared to wild-type control brains, such as decreases in myo-inositol and glutamine, and an increase in lactate. Our present studies suggested that despite the absence of neurological, morphological, T(2), and histological signatures, brain metabolisms were significantly affected in FTH-Tg mice. This study also highlights the usefulness of (1)H-MRS in the analysis of transgenic mouse models.