Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method for Analyzing Blueberry Leaf Metabolites That Inhibit Adult T-cell Leukemia Proliferation.

Metabolic profiling is often used to identify possible correlations between a compound's metabolic profile and biological activity. Direct-injection electron ionization-mass spectrometry "fingerprinting" is useful for characterizing biological materials. We demonstrate the utility of direct-injection electron ionization-mass spectrometry for metabolic profiling using 100 different extracts of leaves from 20 blueberry cultivars collected at 5 time points from April to December 2008. A qualitative direct-injection electron ionization-mass spectrometry method was used to profile the major and/or minor constituents in the blueberry leaf extracts. Blueberry leaf extracts could be distinguished by principal component analysis based on the absolute intensity of characteristic fragment ions. Twenty cultivars were categorized into four species, and the most appropriate discriminative marker m/z value for identifying each cultivar was selected statistically. Correlated m/z values indicating the collection month were determined in the same analysis, and air temperature variance factors were extracted from score plots by principal component analysis. We previously reported that blueberry extracts inhibit the proliferation of adult T-cell leukemia cells. Leaves of Vaccinium virgatum collected in December of 2008 exhibited significantly greater inhibition of adult T-cell leukemia cell proliferation than other species. Highly bioactive cultivars or species were identified by direct-injection electron ionization-mass spectrometry metabolomics analysis of blueberry leaf extracts. The components extracted based on our direct-injection electron ionization-mass spectrometry analyses could be used to construct a model to predict anti-adult T-cell leukemia bioactivity. This is the first study to report a relationship between seasonal variation and bioactivity of natural products using a direct-injection electron ionization-mass spectrometry metabolomics method.

Quantification of three triterpenic acids in dried rosemary using HPLC-fluorescence detection and 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride derivatization.

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) and detected using HPLC-fluorescence (FL). Dried rosemary (50 mg) was ground, added to 3 ml of ethanol, sonicated for 40 min, then the sample solution was added to a mixture of 1% trimethylamine and 1 mM DIB-Cl in acetonitrile. The mixture was settled for 5 min at room temperature, then the DIB-triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250 × 4.6 mm, 6 µm) eluted with 25 mM acetate buffer (pH 4.5)/methanol/acetonitrile (= 8:10:82 v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λex ) and 490 nm (λem ) and the maximum retention time of the derivatives was 30 min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r ≥ 0.997) in the range 2.5-100 ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5 ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.

Establishment of a Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics Method and Its Application to Lichen Profiling.

Direct-injection electron ionization-mass spectrometry (DI-EI-MS) is a multivariate analysis method useful for characterizing biological materials. We demonstrated the use of DI-EI-MS for metabolic profiling using several closely related lichen species: Cladonia krempelhuberi, C. gracilis, C. pseudogymnopoda, and C. ramulosa. The methodology involves conversion of total ion chromatograms to integrated chromatograms and assessment of reproducibility. The qualitative DI-EI-MS method was used to profile the major and/or minor constituents in extracts of lichen samples. It was possible to distinguish each lichen sample by altering the electron energy in DI-EI-MS and examining the resulting data using one-way analysis of variance. Previously undetectable peaks, which are easy to fragment could be revealed by varying the electron energy. Our results suggest that metabolic profiling using DI-EI-MS would be useful for discriminating between subgroups within the same species. This is the first study to report the use of DI-EI-MS in a metabolomics application.

Activation of Cellular Immunity in Herpes Simplex Virus Type 1-Infected Mice by the Oral Administration of Aqueous Extract of Moringa oleifera Lam. Leaves.

Moringa oleifera Lam. is used as a nutritive vegetable and spice. Its ethanol extract has been previously shown to be significantly effective in alleviating herpetic skin lesions in mice. In this study, we evaluated the alleviation by the aqueous extract (AqMOL) and assessed the mode of its anti-herpetic action in a murine cutaneous herpes simplex virus type 1 (HSV-1) infection model. AqMOL (300 mg/kg) was administered orally to HSV-1-infected mice three times daily on days 0 to 5 after infection. AqMOL significantly limited the development of herpetic skin lesions and reduced virus titers in the brain on day 4 without toxicity. Delayed-type hypersensitivity (DTH) reaction to inactivated HSV-1 antigen was significantly stronger in infected mice administered AqMOL and AqMOL augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice at 4 days post-infection. AqMOL administration was effective in elevating the ratio of CD11b(+) and CD49b(+) subpopulations of splenocytes in infected mice. As DTH is a major host defense mechanism for intradermal HSV infection, augmentation of the DTH response by AqMOL may contribute to their efficacies against HSV-1 infection. These results provided an important insights into the mechanism by which AqMOL activates cellular immunity. Copyright © 2016 John Wiley & Sons, Ltd.

Multimetal-Substituted Epsilon-Iron Oxide ϵ-Ga0.31 Ti0.05 Co0.05 Fe1.59 O3 for Next-Generation Magnetic Recording Tape in the Big-Data Era.

From the viewpoints of large capacity, long-term guarantee, and low cost, interest in magnetic recording tapes has undergone a revival as an archive storage media for big data. Herein, we prepared a new series of metal-substituted ϵ-Fe2 O3 , ϵ-Ga(III) 0.31 Ti(IV) 0.05 Co(II) 0.05 Fe(III) 1.59 O3 , nanoparticles with an average size of 18 nm. Ga, Ti, and Co cations tune the magnetic properties of ϵ-Fe2 O3 to the specifications demanded for a magnetic recording tape. The coercive field was tuned to 2.7 kOe by introduction of single-ion anisotropy on Co(II) (S=3/2) along the c-axis. The saturation magnetization was increased by 44 % with Ga(III) (S=0) and Ti(IV) (S=0) substitution through the enhancement of positive sublattice magnetizations. The magnetic tape media was fabricated using an actual production line and showed a very sharp signal response and a remarkably high signal-to-noise ratio compared to the currently used magnetic tape.

Prediction of the Adult T-Cell Leukemia Inhibitory Activity of Blueberry Leaves/Stems Using Direct-Injection Electron Ionization-Mass Spectrometry Metabolomics.

Although Vaccinium virgatum Aiton leaves and stems inhibit adult T-cell leukemia (ATL) cells, leaves and stems can differ between individual plants and by time and location. In this study, leaf and stem components were profiled in the same individual plant using direct-injection electron ionization-mass spectrometry (DI-EI-MS) metabolomics, with the aims of analyzing the anti-ATL activity, and quantifying proanthocyanidins (PACs). Leaves, stems, and leaf/stem mixtures showed distinct and characteristic spectra. Anti-ATL activity was stronger in stems than leaves, and the PAC content was higher in stems than leaves. These data were subjected to bivariate analysis to identify the factor (m/z) responsible for the inhibitory effect of ATL based on the highest coefficient of determination (R2). The results of this DI-EI-MS metabolomics analysis suggest that among PACs contained in V. virgatum stems and leaves, the fragment ion at m/z 149 contributes significantly to anti-ATL activity.

Efficacy of Brazilian Propolis against Herpes Simplex Virus Type 1 Infection in Mice and Their Modes of Antiherpetic Efficacies.

Ethanol extracts (AF-06, 07, and 08, 10 mg/kg) of Brazilian propolis were administered orally to cutaneously herpes simplex virus type 1 (HSV-1)-infected mice three times daily on days 0 to 6 after infection to evaluate their efficacies against HSV-1 infection and significantly limited development of herpetic skin lesions. AF-07 and 08 significantly reduced virus titers in brain and/or skin on day 4 without toxicity, but AF-08 had no anti-HSV-1 activity in vitro. AF-06 and 08 significantly enhanced delayed-type hypersensitivity (DTH) to inactivated HSV-1 antigen in infected mice. Oral AF-08-administration significantly augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice, while direct exposure of splenocytes of infected mice to AF-06 significantly elevated IFN-γ production in vitro. Thus, AF-08 might have components that are active in vivo even after oral administration and those of AF-06 might be active only in vitro. Because DTH is a major host defense for intradermal HSV-1 infection, augmentation of DTH response by AF-06 or 08, directly or indirectly, respectively, may contribute to their efficacies against HSV-1 infection. In addition, AF-06 and 07 possibly contain anti-HSV-1 components contributing to their efficacies. Such biological activities of Brazilian propolis may be useful to analyze its pharmacological actions.

Increased glutathione level is not involved in enhanced bleomycin sensitivity in cisplatin-resistant 2780CP cells.

The cytotoxic activity of bleomycin (BLM) was evaluated in cisplatin (CDDP)-sensitive (A2780) and -resistant (2780CP) human ovarian cancer cells, and the mechanism of increased antitumor activity of BLM in the 2780CP cells was investigated. Compared with the A2780 cells, the 2780CP cells exhibited a 4.5-fold increase in resistance to CDDP, but were 4.0-fold more sensitive to BLM. The cellular glutathione (GSH) levels in the 2780CP cells were significantly higher than those in the A2780 cells, however, GSH depletion in the 2780CP cells below the levels in the A2780 cells by using buthionine-[S,R]-sulfoximine (BSO) did not affect the sensitivity to BLM. BLM decreased 5-bromo-2'-deoxyuridine (BrdU) incorporation after 24-h exposure by 27.5%-90% compared to that of the untreated control at BLM doses of 25-500 ng/ml in the 2780CP cells, but only by 1.5% -45.8% in the A2780 cells. Furthermore, in the 2780CP cells, the percentage of S-phase cells markedly decreased, with an increase in G2/M-phase cells as determined by flow cytometry after exposure to BLM. The enhanced cytotoxity of BLM in CDDP-resistant 2780CP cells could be attributed to BLM-induced G2/M accumulation and significantly inhibited DNA synthesis, not to increased cellular GSH levels.

8-hydroxydeoxyguanosine generated in the earthworm Eisenia fetida grown in metal-containing soil.

Heavy metal pollution of soil causes biological problems, such as mutagenicity to living organisms, including human beings. However, few methods have been developed to assess metal mutagenicity in soil. To avoid metal mutagenicity, an adequate bio-monitoring method is required. In the present study, to determine if the analysis of oxidative DNA damage generated in the earthworm is a useful bio-monitoring method for soil mutagenicity, the accumulation of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, in Eisenia fetida (Savigny, 1826) treated with cadmium chloride (CdCl2) or nickel chloride (NiCl2) was analyzed. E. fetida was treated with Cd (10 or 200 microg/g soil) or Ni (10 or 200 microg/g soil) for 1, 2, and 3 weeks or 3 months. After metal exposure, the metal concentration in E. fetida was analyzed by atomic absorption spectrometry and the 8-OH-dG accumulated in E. fetida was analyzed by HPLC analyses and immunohistochemistry. Atomic absorption spectrometry revealed that Cd, but not Ni, accumulated within E. fetida. The 8-OH-dG levels in the DNA of E. fetida treated with Cd for 3 months were significantly higher than those in control E. fetida. Moreover, immunohistochemical analyses revealed that positive signals for 8-OH-dG accumulation in seminal vesicles were detected only in E. fetida treated with 10 microg of Cd for 3 months. Although some points remain unresolved, a bio-monitoring system analyzing the DNA damage generated in the earthworm might be useful for the assessment of the mutagenicity of soil contaminated with various heavy metals, such as Cd.

Effect of copper and role of the copper transporters ATP7A and CTR1 in intracellular accumulation of cisplatin.

An investigation was carried out as to whether copper affected the intracellular accumulation of cisplatin (cis-diamminedichloroplatinum(II); CDDP) and whether changes in the expression of ATP7A and CTR1 were related to acquired resistance using CDDP-sensitive (KB) and -resistant (KBR/0.8, KBR/1.2) cells. Intracellular platinum accumulation and platinum-DNA adducts were significantly lower in the CDDP-resistant sublines compared with KB cells. Treatment with 750 microM CuSO4 increased the amount of intracellular platinum 1.8-, 3.2-, and 3.9-fold in KB, KBR/0.8, and KBR/1.2 cells respectively, and increased the platinum-DNA adducts by 1.9-fold in KB cells and by 3.2-fold in KBR/1.2 cells. The level of ATP7A was greatly reduced and CTR1 expression slightly decreased in KBR/1.2 cells compared with KB cells. ATP7A expression was markedly increased by exposure to CDDP with or without copper in KB cells but not in KBR/1.2 cells. CDDP and copper did not increase the level of CTR1 in KB or KBR/1.2 cells. These results indicate that a high concentration of copper causes a significant increase in the cellular accumulation of CDDP and binding of platinum to DNA independently of CTR1 expression in KB cells and CDDP-resistant sublines thereof and that the acquisition of CDDP resistance is associated with a greatly reduced level of ATP7A and a marginally lower expression of CTR1.

Effects of lifestyle on urinary 1-hydroxypyrene concentration.

This study aimed to clarify the variation of urinary excretion of 1-hydroxypyrene, which is a major metabolite of pyrene, in relation to lifestyle, including factors such as diet and smoking. The study subjects were 251 workers (male: 196, female: 55, mean age: 44.3) who were not occupationally exposed to PAHs. Urine specimens were collected from 8:00 a.m. to 11:00 a.m. and their 1-hydroxypyrene concentrations were determined by HPLC. A questionnaire was distributed in order to learn gross aspects of the subjects' lifestyles, i.e., smoking, alcohol consumption, coffee/black tea intake, and dietary habits. Multiple linear regression analysis revealed that cigarette consumption most strongly affected the 1-hydroxypyrene level in urine, followed by dietary balance. The urinary 1-hydroxypyrene concentrations of smokers were about 2 times higher than those of non-smokers. Subjects who ate more meat and/or fish excreted 1.5-2 times more 1-hydroxypyrene in urine than those who ate more vegetables.

Application of Mixture Analysis to Crude Materials from Natural Resources (V) [1]: Discrimination of Glycyrrhiza uralensis and G. glabra by El mass spectrometry.

The roots and stolons of some Glycyrrhiza species are used worldwide for traditional folk medicines and commercial pharmaceuticals. Phenolic constituents such as flavonoids and coumarins are medicinal and vary according to species. Therefore, species identification is important for quality analysis. In order to identify Glycyrrhiza species by chemical fingerprinting, methanol extracts of the root bark of Glycyrrhiza uralensis Fischer and G. glabra Linn6 were analyzed using EI-MS. Differences in kinds and quantity of components are reflected in complex EI-MS data and determining characteristic peaks for each species is straightforward.. The chaiacteristic peaks were determined statistically by volcano plot, a multivariate analysis method. EI-MS data of G. uralensis and G. glabra showed differential patterns, and the notable peaks in each pattern were identified. Peaks at m/z 153 and 221 are signature peaks of G. uralensis, and at 11/z 173, 309, and 324 are those of G. glabra. In conclusion, we found species-specific patterns by EI-MS that distinguish G. uralensis and G. glabra. This method based on chemical constituent patterns can be applied to identify other Glycyrrhiza species and similar natural products.

Detection of 4-oxo-2-hexenal, a novel mutagenic product of lipid peroxidation, in human diet and cooking vapor.

Since the diet plays an important role in the development of human cancer, it is important to identify mutagens in foods. We have detected a novel mutagenic product, 4-oxo-2-hexenal (4-OHE), in a model lipid peroxidation reaction mixture [H. Kasai, M. Maekawa, K. Kawai, K. Hachisuka, Y. Takahashi, H. Nakamura, R. Sawa, S. Matsui, T. Matsuda, 4-Oxo-2-hexenal, a mutagen formed by omega-3 fat peroxidation, causes DNA adduct formation in mouse organs, Ind. Health 43 (2005) 699-701]. In the present study, the contents of 4-OHE in various food samples were determined by a GC/MS method. Commercial perilla oil (derived from the seed of Perilla frutescens var. frutescens), which is rich in linolenic acid triglyceride (TG), the edible part of broiled fish, and various fried foods contained 4-OHE in the range of 1-70 microg/g. Furthermore, from the ethyl acetate trap (extracts) of the smoke released during the broiling of fish, 4-OHE was also detected by GC/MS. These results provide a warning to humans, who may be exposed to this mutagen. The 4-OHE may be produced from omega-3 polyunsaturated fats, such as alpha-linolenic acid-, docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-TG, which are more easily oxidized than omega-6 fats, such as linoleic acid-TG.

Electron Ionization Mass Spectrometry-based Metabolomics Studies of Sophoraflavescens can Identify the Geographical Origin of Root Samples.

An electron ionization mass spectrometry (EI-MS)-based metabolomic approach was applied to Sophora flavescens to identify the geographical origin of each sample. The score plot from principal component analysis using the EI-MS data showed that Japanese S. flavescens samples tended to cluster away from Chinese S. flavescens samples. Statistical techniques showed that ions arising from kurarinol and kushenol H, which we previously identified as marker molecules for Japanese S. flavescens, were characteristic of Japanese S. flavescens. Therefore, metabolomics based on EI-MS data is a valuable tool for confirming the geographical origins of S. flavescens samples. The results suggest that EI-MS-based metabolomics is suitable for the quality control of traditional medicines containing many components.

Aldehyde dehydrogenase 2 activity affects symptoms produced by an intraperitoneal acetaldehyde injection, but not acetaldehyde lethality.

Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde. Approximately 45% of Chinese and Japanese individuals are inactive ALDH2 phenotype; acute acetaldehyde toxicity has not been evaluated in these populations. We compared the acute acetaldehyde toxicity between wild-type (Aldh2+/+) and Aldh2-inactive transgenic (Aldh2-/-) mice who were administered an intraperitoneal (ip) injection of a single dose of acetaldehyde. This comparison was made based on the LD(50) values of acetaldehyde and the symptoms following the ip injection. Blood acetaldehyde level was measured in the 400 mg/kg dose group. Immediately after administration of acetaldehyde, the mice exhibited hypoactivity and staggering gait. Subsequently, symptoms such as pale skin, prone position, coma, and abnormal deep respiration were observed. In cases of death, dyspnea, wheezing, and hypothermia were observed from 15 to 30 min after the administration. In cases of survival, crouching, bradypnea, flushing and piloerection were observed. Significant latency of symptom recovery was found in the Aldh2+/- mice as compared with the Aldh2+/+ mice; however, no statistical difference was observed in the acetaldehyde LD(50) values. This might be attributable to the absence of a significant difference in the blood acetaldehyde concentrations in both mice during the first 0-15 min following administration; however, acetaldehyde elimination delay was observed in the Aldh2-/- mice as compared with the Aldh2+/+ mice. Acetaldehyde toxicity difference was observed between the Aldh2+/+ and Aldh2-/- mice; however, no difference in acetaldehyde lethality was observed by administration of a single dose of an ip acetaldehyde injection.

Paired acute inhalation test reveals that acetaldehyde toxicity is higher in aldehyde dehydrogenase 2 knockout mice than in wild-type mice.

Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme that oxidizes acetaldehyde. Approximately 45% of Chinese and Japanese individuals have the inactive ALDH2 genotypes (ALDH2*2/*2 and ALDH2*1/*2); acute inhalation toxicity of acetaldehyde has not been evaluated in these populations. We compared the toxicity between wild-type (Aldh2+/+) and Aldh2-inactive transgenic (Aldh2-/-) mice by using the paired acute inhalation test modified from the acute toxic class method (OECD TG433). Blood acetaldehyde level was measured 4 hr after the inhalation. A pair of Aldh2+/+ and Aldh2-/- mice was put into a chamber and was exposed to 5000 ppm of acetaldehyde. At the start of the inhalation, the mice exhibited hypoactivity and closing of the eyes. Subsequently, symptoms such as crouching, bradypnea, and piloerection were observed. Flushing was observed only in the Aldh2+/+ mice. Symptoms such as tears, straggling gait, prone position, pale skin, abnormal deep respiration, dyspnea, and one case of death were observed only in the Aldh2-/- mice. The symptoms did not change 1 hr after inhalation in the Aldh2+/+ mice. In contrast, in the Aldh2-/- mice, the symptoms became more severe until the end of the inhalation. The blood acetaldehyde level in the Aldh2-/- mice was approximately twice that in the Aldh2+/+ mice 4 hr after inhalation. The Aldh2-/- mice evidently showed more severe toxicity as compared with the Aldh2+/+ mice due to acute inhalation of acetaldehyde at a concentration of 5000 ppm. Acetaldehyde toxicity in Aldh2+/+ and Aldh2-/- mice was estimated and classified one class different. Based on this study, acetaldehyde inhalations were inferred to pose a higher risk to ALDH2-inactive human individuals.

Diminished alcohol preference in transgenic mice lacking aldehyde dehydrogenase activity.

Aldehyde dehydrogenase (ALDH) 2 plays a major role in the detoxification of aldehyde and is known to be responsible for alcohol preference. A diminished enzyme activity due to mutation of the Aldh2 gene is associated with high alcohol sensitivity and a low alcohol tolerance in humans. The genomic background distinguishing an alcohol preference and avoidance in various inbred mouse strains is not clear. We created Aldh2-negative mice by transgenic knockout of the Aldh2 gene into the high alcohol preference C57BL/6 background. The Aldh2 gene targeting (Aldh-/-) mice exhibited an alcohol avoidance characteristic. After free-choice ethanol and water drinking, brain and liver acetaldehyde concentrations of Aldh2-/- mice were almost equal to those of wild-type (Aldh2+/+) mice although the Aldh2-/- mice drank less ethanol than the Aldh2+/+ mice. This result indicates that a direct effect of the Aldh2 genotype plays an important role on alcohol preference and acetaldehyde concentration in the brain is correlated with alcohol avoidance. This highlights the potential benefits of alcoholism and alcohol-related disease research in the animal model of ALDH2 alleles.

A case-case study comparing the usefulness of serum trace elements (Cu, Zn and Se) and tumor markers (CEA, SCC and SLX) in non-small cell lung cancer patients.

UNLABELLED: Serum copper (Cu), zinc (Zn) the Cu/Zn ratio (Cu/Zn) and selenium (Se) were evaluated in 84 patients with non-small cell lung cancer (NSCLC) before surgery. Serum carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC) and sialyl Lewis X-i antigen (SLX) levels were also determined in the same cases. The cut-off values of Cu, Zn and Se were established to create two categories with equal numbers of patients. We investigated the clinical and prognostic usefulness of assays of serum trace elements (Cu, Zn, Cu/Zn and Se) and compared levels of serum trace elements and serum tumor markers (CEA, SCC and SLX) in NSCLC patients. Furthermore, we evaluated the usefulness of serum trace elements, when compared with tumor markers, in assessing prognosis for NSCLC. IN CONCLUSION: (1) a preoperative increase in Cu/Zn level predicted tumor progression more effectively than changes in Cu or Zn levels; (2) Se levels seemed to vary with age, but there was no relationship between Se level and disease stage; (3) the measurement of Cu/Zn was useful for assessing both prognosis and extent of the disease in NSCLC patients, similar to the measurement of serum tumor markers such as CEA; and (4) the measurement of the Cu/Zn had prognostic significance, but was inferior to disease stage in predicting outcome. Cu and Zn in serum are storable and the determination of Cu/Zn level is so simple and inexpensive that it can be helpful in determining clinical stages and predicting the prognoses of NSCLC patients.

Comparison of Cultivars and Seasonal Variation in Blueberry (Vaccinium Species) Leaf Extract on Adult T-Cell Leukemia Cell Line Growth Suppression.

The inhibitory effects of blueberry leaves on the proliferation of adult T-cell leukemia (ATL) cell lines have previously been reported. A comparison of blueberry leaf extracts from different cultivars and seasonal variation were investigated regarding their effects on ATL cell line proliferation. The inhibitory effects of 80% ethanol leaf extracts from different blueberry cultivars collected from April to December in 2006 or 2008 were evaluated using two ATL cell lines. The bioactivities of leaf extracts of rabbit-eye blueberry (Vaccinium virgatum Aiton; RB species), southern highbush blueberry (V. spp.; SB species), northern highbush blueberry (V. corymbosum L.; NB species), and wild blueberry (V. bracteatum Thunb.; WB species) were compared. Of these, leaves of the RB species collected in December showed a significantly stronger inhibitory effect in both cell lines than the SB, NB, or WB species. These results suggest elevated biosynthesis of ATL-preventative bioactive compounds in the leaves of the RB species before the defoliation season.