Differential Neuroprotective Effects of N-Acetylcysteine against Dithianon Toxicity in Glutamatergic, Dopaminergic, and GABAergic Neurons: Assessment Using Zebrafish.

Despite the widespread agricultural use of dithianon as an antifungal agent, its neurotoxic implications for humans and wildlife have not been comprehensively explored. Using zebrafish embryonic development as our model, we found that dithianon treatment induced behavioral alterations in zebrafish larvae that appeared normal. Detailed quantitative analyses showed that dithianon at ≥0.0001 µgmL-1 induced cytoplasmic and mitochondrial antioxidant responses sequentially, followed by the disruption of mitochondrial and cellular homeostasis. Additionally, dithianon at 0.01 and 0.1 µgmL-1 downregulated the expressions of glutamatergic (slc17a6b), GABAergic (gad1b), and dopaminergic (th) neuronal markers. Contrarily, dithianon upregulated the expression of the oligodendrocyte marker (olig2) at concentrations of 0.001 and 0.01 µgmL-1, concurrently suppressing the gene expression of the glucose transporter slc2a1a/glut1. Particularly, dithianon-induced increase in reactive oxygen species (ROS) production was reduced by both N-acetylcysteine (NAC) and betaine; however, only NAC prevented dithianon-induced mortality of zebrafish embryos. Moreover, NAC specifically prevented dithianon-induced alterations in glutamatergic and dopaminergic neurons while leaving GABAergic neurons unaffected, demonstrating that the major neurotransmission systems in the central nervous system differentially respond to the protective effects. Our findings contribute to a better understanding of the neurotoxic potential of dithianon and to developing preventive strategies.

The Korean undiagnosed diseases program phase I: expansion of the nationwide network and the development of long-term infrastructure.

BACKGROUND: Phase I of the Korean Undiagnosed Diseases Program (KUDP), performed for 3 years, has been completed. The Phase I program aimed to solve the problem of undiagnosed patients throughout the country and develop infrastructure, including a data management system and functional core laboratory, for long-term translational research. Herein, we share the clinical experiences of the Phase I program and introduce the activities of the functional core laboratory and data management system. RESULTS: During the program (2018-2020), 458 patients were enrolled and classified into 3 groups according to the following criteria: (I) those with a specific clinical assessment which can be verified by direct testing (32 patients); (II) those with a disease group with genetic and phenotypic heterogeneity (353 patients); and (III) those with atypical presentations or diseases unknown to date (73 patients). All patients underwent individualized diagnostic processes based on the decision of an expert consortium. Confirmative diagnoses were obtained for 242 patients (52.8%). The diagnostic yield was different for each group: 81.3% for Group I, 53.3% for Group II, and 38.4% for Group III. Diagnoses were made by next-generation sequencing for 204 patients (84.3%) and other genetic testing for 35 patients (14.5%). Three patients (1.2%) were diagnosed with nongenetic disorders. The KUDP functional core laboratory, with a group of experts, organized a streamlined research pipeline covering various resources, including animal models, stem cells, structural modeling and metabolic and biochemical approaches. Regular data review was performed to screen for candidate genes among undiagnosed patients, and six different genes were identified for functional research. We also developed a web-based database system that supports clinical cohort management and provides a matchmaker exchange protocol based on a matchbox, likely to reinforce the nationwide clinical network and further international collaboration. CONCLUSIONS: The KUDP evaluated the unmet needs of undiagnosed patients and established infrastructure for a data-sharing system and future functional research. The advancement of the KUDP may lead to sustainable bench-to-bedside research in Korea and contribute to ongoing international collaboration.

TNNT1 myopathy with novel compound heterozygous mutations.

Nemaline myopathies are clinically and genetically heterogeneous disorders caused by several different genes. One of them is TNNT1, which was initially described in Amish families and has not been reported in Asian populations. Although most TNNT1 myopathies are caused by loss-of-function mutations, several recent studies have shown that missense mutations can also be pathogenic. A 16-year-old Korean boy with progressive muscle weakness visited the Seoul National University Hospital. He showed generalized myopathy, which was predominant in the paraspinal and neck muscles. Moreover, nemaline rods were observed in a muscle biopsy. Whole-exome sequencing of DNA samples of the patient and his younger brother, who had a similar phenotype, revealed novel compound heterozygous mutations in TNNT1 (c.724G>C (p.Ala242Pro) and c.611+1G>A). Sanger sequencing of cDNA extracted from muscle samples of the patient confirmed partial or total skipping of exon 11 in the splicing variant. The impact of the missense variant on muscle integrity and locomotor activity was verified using a zebrafish loss-of-function model. Here, we reported novel familial cases of TNNT1 myopathy with intermediate clinical presentations caused by compound heterozygous mutations and demonstrated their functional defects using an animal model.

Ipconazole Disrupts Mitochondrial Homeostasis and Alters GABAergic Neuronal Development in Zebrafish.

Ipconazole, a demethylation inhibitor of fungal ergosterol biosynthesis, is widely used in modern agriculture for foliar and seed treatment, and is authorized for use in livestock feed. Waste from ipconazole treatment enters rivers and groundwater through disposal and rain, posing potential toxicity to humans and other organisms. Its metabolites remain stable under standard hydrolysis conditions; however, their neurodevelopmental toxicity is unknown. We investigated the potential neurodevelopmental toxicity of ipconazole pesticides in zebrafish (Danio rerio). Our behavioral monitoring demonstrated that the locomotive activity of ipconazole-exposed zebrafish larvae was reduced during early development, even when morphological abnormalities were undetected. Molecular profiling demonstrated that the mitochondrial-specific antioxidants, superoxide dismutases 1 and 2, and the genes essential for mitochondrial genome maintenance and functions were specifically reduced in ipconazole-treated (0.02 µg/mL) embryos, suggesting underlying ipconazole-driven oxidative stress. Consistently, ipconazole treatment substantially reduced hsp70 expression and increased ERK1/2 phosphorylation in a dose-dependent manner. Interrupted gad1b expression confirmed that GABAergic inhibitory neurons were dysregulated at 0.02 µg/mL ipconazole, whereas glutamatergic excitatory and dopaminergic neurons remained unaffected, resulting in an uncoordinated neural network. Additionally, ipconazole-treated (2 µg/mL) embryos exhibited caspase-independent cell death. This suggests that ipconazole has the potential to alter neurodevelopment by dysregulating mitochondrial homeostasis.

IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish.

Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.

Transgenic fluorescent zebrafish lines that have revolutionized biomedical research.

Since its debut in the biomedical research fields in 1981, zebrafish have been used as a vertebrate model organism in more than 40,000 biomedical research studies. Especially useful are zebrafish lines expressing fluorescent proteins in a molecule, intracellular organelle, cell or tissue specific manner because they allow the visualization and tracking of molecules, intracellular organelles, cells or tissues of interest in real time and in vivo. In this review, we summarize representative transgenic fluorescent zebrafish lines that have revolutionized biomedical research on signal transduction, the craniofacial skeletal system, the hematopoietic system, the nervous system, the urogenital system, the digestive system and intracellular organelles.

Histopathological assessment of laterality defects in zebrafish development.

Laterality defects during embryonic development underlie the aetiology of various clinical symptoms of neuropathological and cardiovascular disorders; however, experimental approaches to understand the underlying mechanisms are limited due to the complex organ systems of vertebrate models. Zebrafish have the ability to survive even when the heart stops beating for a while during early embryonic development and those adults with cardiac abnormalities. Therefore, we induced laterality defects and investigated the occurrence of situs solitus, situs inversus, and situs ambiguus in zebrafish development. Histopathological analysis revealed heterotaxy in both embryos and juvenile fish. Additionally, randomization of left-right asymmetry of the brain and heart in individual zebrafish embryos under artificial experimental pressure further demonstrated the advantage of transparent zebrafish embryos as an experimental tool to select or reduce the embryos with laterality defects during early embryonic development for long-term studies, including behavioural and cognitive neuroscience investigations.

Eif2b3 mutants recapitulate phenotypes of vanishing white matter disease and validate novel disease alleles in zebrafish.

Leukodystrophy with vanishing white matter (VWM), also called Childhood Ataxia with Central Nervous System Hypomyelination, is caused by mutations in the subunits of the eukaryotic translation initiation factor, EIF2B1, EIF2B2, EIF2B3, EIF2B4 or EIF2B5. However, little is known regarding the underlying pathogenetic mechanisms, and there is no curative treatment for VWM. In this study, we established the first EIF2B3 animal model for VWM disease in vertebrates by CRISPR mutagenesis of the highly conserved zebrafish ortholog eif2b3. Using CRISPR, we generated two mutant alleles in zebrafish eif2b3, 10- and 16-bp deletions, respectively. The eif2b3 mutants showed defects in myelin development and glial cell differentiation, and increased expression of genes in the induced stress response pathway. Interestingly, we also found ectopic angiogenesis and increased VEGF expression. Ectopic angiogenesis in the eif2b3 mutants was reduced by the administration of VEGF receptor inhibitor SU5416. Using the eif2b3 mutant zebrafish model together with in silico protein modeling analysis, we demonstrated the pathogenicity of 18 reported mutations in EIF2B3, as well as of a novel variant identified in a 19-month-old female patient: c.503 T > C (p.Leu168Pro). In summary, our zebrafish mutant model of eif2b3 provides novel insights into VWM pathogenesis and offers rapid functional analysis of human EIF2B3 gene variants.

Velutin, an Aglycone Extracted from Korean Mistletoe, with Improved Inhibitory Activity against Melanin Biosynthesis.

The abnormal regulation of melanin synthesis leads to a wide range of pigmentary disorders. Although various melanin biosynthesis inhibitors have been developed, their efficacy and long-term safety needs to be further improved, and thus the goal of this study is to develop promising natural compound inhibitors of melanin biosynthesis. Here, we obtained aglycone flavonoid extract through the microwave-assisted hydrolysis of glycoside extract from Korean mistletoe in acidic condition. The aglycone extract inhibited tyrosinase activity more efficiently with better antioxidant activity than glycoside extract in vitro. The microwave-assisted aglycone extract of mistletoe was further analyzed for in vivo activity, and the results showed the aglycone extract inhibited both early melanocyte development and melanin synthesis more efficiently in zebrafish embryo in a dose-dependent manner. Our in vivo toxicity assay quantitatively measured cell death in zebrafish embryos and showed that the microwave-assisted aglycone extract of mistletoe had no significant effect on cell death (p < 0.001), indicating that aglycone extract is more biocompatible than glycoside extract. Furthermore, our in vitro and in vivo analyses successfully identified and characterized velutin, an aglycone of a homoflavoyadorinin B glycoside, as a major inhibitory component in the microwave-assisted mistletoe extract. Ultimately, this study showed that the novel natural compound inhibitor velutin, which was generated through microwave-assisted extraction from mistletoe, improved the efficacy of melanin biosynthesis inhibition with little toxicity.

3D light-sheet assay assessing novel valproate-associated cardiotoxicity and folic acid relief in zebrafish embryogenesis.

Precise in vivo toxicological assays to determine the cardiotoxicity of pharmaceuticals and their waste products are essential in order to evaluate their risks to humans and the environment following industrial release. In the present study, we aimed to develop the sensitive imaging-based cardiotoxicity assay and combined 3D light-sheet microscopy with a zebrafish model to identify hidden cardiovascular anomalies induced by valproic acid (VPA) exposure. The zebrafish model is advantageous for this assessment because its embryos remain transparent. The 3D spatial localization of fluorescence-labeled cardiac cells in and around the heart using light-sheet technology revealed dislocalization of the heart from the outflow tract in two-day-old zebrafish embryos treated with 50 µM and 100 µM VPA (P < 0.01) and those embryos exposed to 20 µM VPA presented hypoplastic distal ventricles (P < 0.01). These two observed phenotypes are second heart field-derived cardiac defects. Quantitative analysis of the light-sheet imaging demonstrated that folic acid (FA) supplementation significantly increased the numbers of endocardial and myocardial cells (P < 0.05) and the accretion of second heart field-derived cardiomyocytes to the arterial pole of the outflow tract. The heart rate increased in response to the cellular changes occurring in embryonic heart development (P < 0.05). The present study disclosed the cellular mechanism underlying the role of FA in spontaneous cellular changes in cardiogenesis and in VPA-associated cardiotoxicity. The 3D light-sheet assay may be the next-generation test to evaluate the risks of previously undetected pharmaceutical and environmental cardiotoxicities in both humans and animals.

Enzymatic construction of shRNA library from oligonucleotide library.

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. OBJECTIVE: We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. METHODS: The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. RESULTS: The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. CONCLUSION: Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.

Promoter cross-talk affects the inducible expression of intronic shRNAs from the tetracycline response element.

BACKGROUND: RNA interference (RNAi), defined as double-stranded, RNA-mediated gene silencing, is a useful tool for functional genomic studies. Along with increasing information about genomic sequences due to the innovative development of genome-sequencing technologies, functional genomic technologies are needed to annotate the genome and determine the processes by which each gene is regulated. Lentiviral vectors have been used to efficiently deliver reagents, such as small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), into cells and tissues for functional genomic analyses. OBJECTIVE: We developed a lentiviral vector that efficiently expresses intronic shRNA from the tetracycline regulatory element (TRE) promoter in a doxycycline-dependent manner. METHODS: We developed a lentiviral vector system that contains reverse tetracycline-controlled transactivator 3 (rtTA3) and the TRE promoter, which are necessary for the doxycycline-inducible expression of shRNAs that are expressed as intronic miR-30a precursors. We then measured the cross-talk between the cytomegalovirus (CMV) and TRE promoters in the vector. RESULTS: We found that nearby promoters influence each other and that the TRE promoter should be located far from other promoters, such as the CMV promoter, in a vector. The orientation of a promoter with respect to other promoters also influences its transcriptional activity. A head-to-head orientation of the CMV and TRE promoters maintains the lowest level of transcription from TRE in the absence of doxycycline, compared to the tail-to-tail and head-to-tail orientations. CONCLUSION: Based on these findings, we were able to construct a lentiviral vector that faithfully expresses intronic miR-30a shRNA precursors in a doxycycline-inducible manner.

Enhancement of gene knockdown efficiency by CNNC motifs in the intronic shRNA precursor.

BACKGROUND: Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Of several methods to express shRNA, a method of transcribing shRNAs embedded in microRNA precursors has been more widely used than the one that directly expresses shRNA from RNA polymerase III promoters because the microRNA precursor form of shRNA is known to cause lower levels of cytotoxicity and off-target effects than the overexpressed shRNAs from the RNA polymerase III promoters. OBJECTIVE: We study the primary sequence features of microRNA precursors, which enhance their processing into mature form, helps design more potent shRNA precursors embedded in microRNA precursors. METHODS: We measure the enhancement of gene knockdown efficiency by adding CNNC motifs in the 3' flanking region of shRNA precursor embedded in the human miR-30a microRNA precursor. RESULTS: By systemically adding three CNNC motifs in the 3' flanking region of shRNA precursor, we found that addition of two CNNC motifs saturates their enhanced knockdown ability of shRNA and that the CNNC motif in the + 17 to + 20 from the drosha cleavage site is most important for the shRNA-mediated target gene knock down. We also did see little knockdown of target gene expression by the shRNA precursor lacking CNNC motif. CONCLUSION: Since genetic studies generally require techniques that could reduce gene expression at different degrees, the findings in this study will allow us to use RNAi for genetic studies of reducing gene expression at different degrees.

Improved dynamic monitoring of transcriptional activity during longitudinal analysis in the mouse brain.

Bioluminescence imaging has proven to be a highly sensitive technique for assessing in vitro transcriptional activity toward understanding gene regulation patterns; however, application of this technique is limited for brain research. In particular, the poor spatiotemporal resolution is a major hurdle for monitoring the dynamic changes of transcriptional activity in specific regions of the brain during longitudinal analysis of living animals. To overcome this limitation, in this study, we modified a lentivirus-based luciferase glucocorticoid receptor (GR) reporter by inserting destabilizing sequence genes, and then the reporter was stereotaxically injected in the mouse infralimbic prefrontal cortex (IL-PFC). Using this strategy, we could successfully pin-point and monitor the dynamic changes in GR activity in IL-PFC during normal stress adaptation. The modified reporter showed a 1.5-fold increase in temporal resolution for monitoring GR activity compared to the control, with respect to the intra-individual coefficients of variation. This novel in vivo method has broad applications, as it is readily adaptable to different types of transcription factor arrays as well spanning wide target regions of the brain to other organs and tissues.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy.

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Extent of vertical cementing as a predictive factor for radiolucency in revision total knee arthroplasty.

PURPOSE: Limited information is available regarding the effects of cementing extent on implant stability in patients who have undergone revision total knee arthroplasty (TKA). As such, the goals of this study were: (1) to determine the correlation between the extent of vertical cementing and implant loosening; (2) to determine whether the extent of cementing is a potential predictive factor for radiolucency; and (3) to evaluate the minimal amount of cement needed for a stable implant during revision TKA using a hybrid technique. METHODS: One hundred nine stemmed/revision TKAs with a mean follow-up period of 5 years were retrospectively analysed. In each case, a single varus-valgus constrained implant was used and fixed with a hybrid technique. Implant stability was evaluated according to the modified Knee Society radiographic scoring system. The extent of vertical cementing was defined as the longitudinal length from the implant base to the end of the radiopaque line around the stem on radiograph. Its correlation with implant stability was analysed, and the minimal value for a stable implant was evaluated with a receiver operating characteristic (ROC) analysis. RESULTS: The mean extent of vertical cementing was longer in stable implants (femur: p = 0.001, tibia: p = 0.004) and significantly correlated with implant stability (femur: p < 0.001, tibia: p = 0.001). A logistic regression analysis revealed that the risk of loosening was 8.7-16.1 times higher when the extent of cementing was <40 mm, which was located at the stem-implant junction of the modular implant. The minimal extent of vertical cementing was estimated to be 60 mm for a stable femoral implant and 50 mm for a tibial implant. CONCLUSIONS: The hybrid fixation technique with a cementing extent >60 mm for the femur and 50 mm for the tibia was durable at a mean follow-up period of 5 years. Vertical cementing 10-20 mm above the stem-implant junction is recommended when performing revision TKA using this technique. LEVEL OF EVIDENCE: IV.

3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.

Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins.

Targeted protein degradation is a powerful tool in determining the function of specific proteins or protein complexes. We fused nanobodies to SPOP, an adaptor protein of the Cullin-RING E3 ubiquitin ligase complex, resulting in rapid ubiquitination and subsequent proteasome-dependent degradation of specific nuclear proteins in mammalian cells and zebrafish embryos. This approach is easily modifiable, as substrate specificity is conferred by an antibody domain that can be adapted to target virtually any protein.

Comparison of patellar resurfacing versus preservation in high flexion total knee arthroplasty.

PURPOSE: Recently, high flexion design total knee arthroplasty (TKA) has been introduced to improve clinical outcomes. The purpose of this study is to compare the midterm outcomes between patellar resurfacing (PR) and patellar preservation (PP) in high flexion TKA. METHODS: A total of 373 knees of primary TKAs were performed using high flexion design, 339 knees involved PR group and 34 knees involved PP group. After applying exclusion criteria, 1:3 matching was performed by the matching criteria. After matching, 69 knees in PR group and 23 knees in PP group remained. Radiographic outcomes, clinical outcomes, patients' satisfaction, ability and pain related to the high flexion activities were also evaluated. RESULTS: There was no significant difference in radiograph measurements, KS function score and WOMAC score (n.s). However, PR group showed better outcomes in KS knee score (P = 0.001) and HSS score (P = 0.03). There was no significant difference in postoperative satisfaction and ability of high flexion activities between the groups, but the pain at the high flexion activities in PP group was worse than that in PR group. CONCLUSION: In high flexion design of TKA, PR resulted in better midterm outcomes in regard to KS knee score, HSS score and knee pain related to the high flexion activities. The selective PR is recommended when performing primary TKA with high flexion design. LEVEL OF EVIDENCE: IV.

Benzyl isothiocyanate suppresses high-fat diet-stimulated mammary tumor progression via the alteration of tumor microenvironments in obesity-resistant BALB/c mice.

We previously reported that a high-fat diet (HFD) and M2-macrophages induce changes in tumor microenvironments and stimulate tumor growth and metastasis of 4T1 mammary cancer cells in BALB/c mice. In this study, we attempted to determine whether benzyl isothiocyanate (BITC) inhibits HFD-induced changes in tumor progression and in tumor microenvironments. Four groups of female BALB/c mice (4-week-old) were fed on a control diet (CD, 10 kcal% fat) and HFD (60 kcal% fat) containing BITC (0, 25, or 100 mg/kg diet) for 20 weeks. Following 16 weeks of feeding, 4T1 cells (5×10(4) cells) were injected into the mammary fat pads, and animals were killed 30 d after the injection. HFD feeding increased solid tumor growth and the number of tumor nodules in the lung and liver, as compared to the CD group, and these increases were inhibited by BITC supplementation. The number of lipid vacuoles, CD45+ leukocytes and CD206+ M2-macrophages, expression of Ki67, levels of cytokines/chemokines, including macrophage-colony stimulating factor (M-CSF) and monocyte chemoattractant protein-1, and mRNA levels of F4/80, CD86, Ym1, CD163, CCR2, and M-CSF receptor were increased in the tumor tissues of HFD-fed mice, and these increases were inhibited by BITC supplementation. In vitro culture results demonstrated that BITC inhibited macrophage migration as well as lipid droplet accumulation in 3T3-L1 cells. These results suggest that suppression of lipid accumulation and macrophage infiltration in tumor tissues may be one of the mechanisms by which BITC suppresses tumor progression in HFD-fed mice.