Evaluation of Drug Sorption on Laboratory Materials with Abraham Solvation Parameters of Drugs and its Prevention.

PURPOSE: Undesired drug sorption on laboratory material surfaces reduces the performance of analytical methods and results in the generation of unreliable data. Hence, we characterized the sorption of drugs and evaluated the sorption extent using a linear free energy relationship (LFER) model with Abraham solvation parameters of drugs. Furthermore, to prevent sorption, the effects of additives, such as organic solvents and salts, were evaluated. METHODS: The sorption of fifteen model drugs (concentration: 2 µM), with various physicochemical properties, on materials in 0.2% dimethyl sulfoxide aqueous solutions was evaluated. Drug sorption extent on the materials was determined using high-performance liquid chromatography. The obtained results were analyzed using an LFER model with Abraham solvation parameters of the drugs. The effect of additives on the sorption of itraconazole, one of the most hydrophobic drugs among those tested in this study, was investigated. RESULTS: Sorption was dependent on the physicochemical properties of drugs, rather than the type of materials used, and additives altered the rate of drug sorption. Equations were developed to evaluate the sorption extent (nmol) of drugs to glass and polypropylene using the Abraham solvation parameters of the drugs. CONCLUSIONS: LFER modeling with Abraham solvation parameters of drugs enabled us to evaluate drug sorption on materials. All the additives altered the rate of drug sorption, and some organic solvents effectively prevented sorption. The developed LFER model would be useful for assessment of the sorption properties of compounds in in vitro evaluations in drug discovery research and various other biochemical fields.

Hmga2 regulation of tooth formation and association with Sox2 and Nanog expression.

Tooth formation is accomplished under strict genetic programs. Although patients with chromosome 12q14 aberration shows tooth phenotype including the size and eruption timing with bone growth anomaly, its etiology is uncertain. Here, we examined expression of Hmga2, which is encoded at chromosome 12q14, in mouse tooth germs and analyzed the involvement in lower first molar (M1) and mandibular bone development. Hmga2 expression was immunohistochemically detected at enamel organ and the surrounding mesenchyme of the M1 germs. The expression was dynamically changed with gestation and rapidly decreased in postnatal mice. In Hmga2-/- mice, the M1 germs and crowns were diminished in size, and formation and eruption of molars were delayed with mandibular bone growth retardation. Hmga2 cDNA or siRNA transfection showed that Hmga2 transcriptionally up-regulates expression of stem cell factors, Sox2 and Nanog. They were co-localized with Hmga2 in the germs, but differentially distributed at enamel organ and mesenchyme in Hmga2-/- mice. These results demonstrate that Hmga2 expressed in tooth germs regulates the growth, sizing and eruption and stem cell factor expression in different compartment of the germ and associates with mandibular bone growth. Although future studies are needed, the present study demonstrates HMGA2 regulation of tooth genesis with skeletal development.

Isolation and characterization of embryonic ameloblast lineage cells derived from tooth buds of fetal miniature swine.

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

Establishment and characterization of a cell line designated Nur-1 derived from human endometrioid adenocarcinoma of uterine corps.

A new cell line designated Nur-1 has been established from human endometrioid adenocarcinoma, Grade 1, pT1a, PN1 (3/24), Stage IIIc (International Federation of Gynecology and Obstetrics/Union for International Cancer Control (FIGO/UICC TNM Classification of Malignant Tumours, 7th ed.). Cytological findings of Nur-1 cells reveal anaplastic and pleomorphic features such as anisonucleosis, nucleolar pleomorphism, and piling-up tendency in cellular arrangement. Distribution of the chromosome number is found at the hyperploid range, and the apparent marker chromosome has not been identified. The original tumor and graft of the Nur-1 cell line had a large amount of estrogen receptors and progesterone receptors, as revealed by immunohistochemistry. The cytokeratin pattern of the tumor was positive for cytokeratin-7 and negative for cytokeratin-20. However, a few cells were positive for cytokeratin-20 in the original tumor. Nur-1 cells express mRNA of estrogen receptors and progesterone receptors, cytokeratin-7, and cytokeratin-20 at 105 passages. These findings are consistent with the cytokeratin pattern of endometrial glandular cells. The cells make contact with each other via interdigitation and desmosomes. They possess bundles of microtubules and tonofilaments and many free ribosomes. Some cells have various sizes of phagosomes. The Nur-1 cell line exceeded 102 passages in 5 years, and multiplication of the cells is stable. The modal number of the Nur-1 cell line is 91-92 (56 %). The Nur-1 cells develop well-differentiated adenocarcinoma in tumors sustained in nude mice that resemble the original tumors.

Novel management of acute or secondary biliary liver conditions using hepatically differentiated human dental pulp cells.

The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine.

Postnatal mandibular cheek tooth development in the miniature pig based on two-dimensional and three-dimensional X-ray analyses.

The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time-course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X-ray analyses using soft X-ray for two-dimensional observations and micro-CT for three-dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X-ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth.

Isolation and characterization of epithelial and myogenic cells by "fishing" for the morphologically distinct cell types in rat primary periodontal ligament cultures.

The periodontal ligament (PDL) contains various cell populations and plays a central role in the maintenance, repair, and regeneration of the periodontium, i.e., tooth-supporting structures. Because primary cells isolated from PDL tissue are heterogeneous, the establishment of an effective isolation method for cells of interest is desired. In the present study, two morphologically distinct cell types were identified in confluent primary cultures derived from rat PDL. To isolate these cell populations, a small piece of filter paper soaked with trypsin-EDTA was placed directly onto the target cell population, enabling the cells to detach from the culture dish. The filter papers were then transferred into fresh culture dishes to establish outgrowth cultures; these two steps constitute the "cell fishing" method. The "fished" cell types were propagated and subcultured for further analyses. In morphological evaluation, immunocytochemical analyses, and reverse transcription-polymerase chain reaction, the isolated cells exhibited a polygonal appearance or a mono- or multinucleated appearance, with a high cytoplasm-to-nucleus ratio, leading to their being characterized as epithelial or myogenic cell populations, respectively. Surprisingly, a notable proportion of the multinuclear cells in the primary and subsequent isolated cultures demonstrated dramatic, spontaneous contractions, a feature typical of skeletal muscle cells. Finally, the isolated cell populations maintained a normal karyotype with a diploid chromosomal number. These results demonstrated that physiological epithelial and skeletal muscle cells can be obtained from primary PDL cultures without artificial induction using growth factors or chemicals, and can be propagated as individual lineage-committed cell populations; the populations consisted of differentiated and progenitor cells that maintained chromosomal stability. This simple, classical culture procedure provides new insights into the biological properties of PDL cells, which are potentially important for the differentiation of tissue or somatic stem cells and for the development of future cell-based therapies for dental and muscular diseases.

Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

Establishment and characterization of the rhabdomyosarcoma cell line designated NUTOS derived from the human tongue sarcoma: Special reference to the susceptibility of anti-cancer drugs.

Primary alveolar type of rhabdomyosarcoma (RMS) tumor tissue was collected from the tongue of a 17-year-old Japanese woman and used to successfully establish a rhabdomyosarcoma cell line, which has been designated NUTOS. The chromosomal distribution revealed that the NUTOS cell line was hyper-tetraploid with chromosomal translocation. The cells were grown in Dulbecco's modified eagle medium/F12 supplemented with 15% fetal bovine serum, 0.1% non-essential amino acids solution (NEAA), 50 microg of streptomycin, 50 U/mL of penicillin and 0.25 microg /mL of Fungizone. The NUTOS shapes included small spindles, large spindles and long, thick multinucleated cells. All three cell types were immunostained with anti-desmin antibody, which is a marker protein for middle sized myofilaments. Furthermore, immunocytochemical staining revealed that the cells were positively immunostained with anti-MyoD, myogenin, alpha-sarcomeric actin, myosin and troponin T. Mitotic figures were only observed in the small spindle cells. These cells were coadunated with each other at the lateral portion of the apex of the cells. Subsequently, these cells grew into large multinucleated cells. Autonomic contractions (approximately 20 times/min) were observed in both the large spindle cells and the large multinucleated cells. NUTOS cells incorporated serotonin from the serum in the growth medium. Histopathological observations of the NUTOS cell grafts in the subcutis of nude mice exhibited characteristics similar to those seen for the primary rhabdomyosarcoma of the tongue. Susceptibility tests for the anti-cancer drugs revealed that NUTOS cells were susceptive to cisplatin, paclitaxel, and docetaxel, but not to adriacin.

Novel amelanotic and melanotic cell lines NM78-AM and NM78-MM derived from a human oral malignant melanoma.

Abstract Novel cell lines, designated NM78-AM and NM78-MM, have been established from a malignant melanoma of the cheek oral mucosa. NM78-AM cells were spherical, grew in suspension as clusters, and produced no melanin. In contrast, NM78-MM cells were adherent and produced melanin granules. Initially, NM78-AM cells were grown on fibroblast feeder cells or in growth media supplemented with 10% conditioned medium from fibroblasts, but eventually grew in standard growth media alone. NM78-AM cells had interdigitating microvilli and formed cell clusters. They had large nucleoli, desmosomes, lipid droplets, and well-developed Golgi apparatuses. In contrast, NM78-MM cells grew as adherent neuron-like cells. They had large prominent nucleoli, irregular nuclear membranes, a number of mitochondria, well-developed Golgi apparatuses, melanosomes at various stages of development in the cytoplasm, and the cells secreted melanin granules. Projections from these melanotic cells formed anastomoses with each other. NM78-MM cells stained immunofluorescently for internexin, neuron specific enolase, NF-200, and glial fibrillary acidic protein. These cells were severely aneuploid, approximating to triploidy, and had many marker chromosomes. We used a real-time monitoring system to evaluate oxygen concentrations in culture medium to investigate the susceptibility of both cell lines to various anti-cancer drugs. NM78-AM cells were slightly sensitive to actinomycin D, but not to cisplatin, irinotecan, the irinotecan metabolite SN-38, taxol, taxotere, bleomycin and methotrexate; NM78-MM cells were sensitive to cisplatin, and not to taxol, taxotere, carboplatin, and irinotecan. These new cell lines, NM78-AM and NM78-MM, will be very important for the development of new chemotherapeutics for oral malignant melanoma.

Association of lifestyle factors, polymorphisms in adiponectin, perilipin and hormone sensitive lipase, and clinical markers in Japanese males.

According to recent genome-wide association studies, a number of single nucleotide polymorphisms is reported to be associated with diseases or several clinical markers. Among them, adiponectin (ADIPOQ) and perilipin (PLIN) polymorphisms are major factors of obesity. However, the association between lifestyle factor, these polymorphisms and obesity-related clinical markers in Japanese is not well researched. Therefore, the aim of present study is to investigate the association between lifestyle factor, polymorphisms of lipid metabolic genes, and clinical markers in 148 middle-aged Japanese males. The study revealed that ADIPOQ 45 T>G and ADIPOQ 276 G>T genotypes were significantly associated with triglyceride, total cholesterol, hemoglobin A1c (HbA1c) in blood and body mass index (BMI). PLIN4 11482 G>A and hormone sensitive lipase (LIPE)-60 C>G genotypes were respectively associated with BMI and serum triglyceride. Not only genetic factors but also lifestyle factors influence several clinical markers. The BMI of subjects who like sweets and have the GG allele in ADIPOQ 276 G>T was higher than that of subjects who don't like sweets. The habit of eating fruits and fish affected low-density lipoprotein-cholesterol of the GT allele and HbA1c of the TT allele in ADIPOQ 276 G>T. Those findings indicate improvement and conservation of lifestyle depending on genetic predisposition in ADIPOQ, PLIN and LIPE should be encouraged.

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs.

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 degrees C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.

Phytochelatins do not correlate with the level of Cd accumulation in Chlamydomonas spp.

Chlamydomonas acidophila KT-1 and Chlamydomonas acidophila DVB238 exhibit a strong heavy metal tolerance, but C. acidophila DVB238 can accumulate a much higher amount of Cadmium (Cd) than C. acidophila KT-1. Phytochelatins (PCs) are known to play an important role in the detoxification of several toxic heavy metals, but the relationship between PCs and Cd accumulation is not clear. PC metabolism and Cd accumulation were investigated by using three Chlamydomonas strains including Chlamydomonas reinhardtii C-9 as a standard alga. The results showed that the PC content did not correlate closely with the level of Cd accumulation, maintenance of a high GSH level seeming to be more important for Cd accumulation. The ultrastructure of C. acidophila KT-1 was extremely disrupted by a great increase in starch granules, which resulted in a moribund state, but hyper-accumulator C. acidophila DVB238 did not exhibit an increase in starch granules in its cells, in spite of Cd accumulation in its chloroplasts, cytosol and vacuoles. These results indicated that C. acidophila DVB238 probably has a developed detoxification system preventing such as destruction of the cells due to Cd toxicity.

Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism.

Ultrastructural changes induced by heavy metals (cadmium, zinc, and copper) and polyphosphate metabolism were studied in Chlamydomonas acidophila. Transmission electron microscopy indicated that cadmium led to the most drastic morphometric changes. An increase in number and volume of starch grains and vacuoles as well as the presence of electron dense deposits in vacuole and membrane whorls were observed. Energy-dispersive X-ray analysis revealed that vacuolar deposits inside cells treated with cadmium contained phosphate and cadmium. These ultrastructural changes were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo (31)P-nuclear magnetic resonance. It was also observed that cadmium treatment caused polyphosphate degradation and increased vacuolar short-chains and orthophosphates.