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Biomedical subjects

T Tokiwa

Publications and source records attributed to T Tokiwa.

At least 55 records · Page 3Linked to original sources
PubMed1988

[Culture methods of human liver cells].

Attempts to isolate and cultivate human liver cells have been described. Many viable liver cells have been obtained when dissociated with collagenase followed by dispase. The morphology and differentiated functions have been maintained for more than 3 weeks when human fetal liver cells were cultured not only in medium containing 10(-3) M hydrocortisone, but also on collagen gel substrates with 5 x 10(-7) M hydrocortisone. The colony-forming capacity of primary cultured fetal human livers has also been described in the presence of conditioned medium.

Cell Differentiation↗
PubMed1987

Production of albumin and alpha-fetoprotein in primary culture of fetal human liver cells on collagenous substrata in the presence of hydrocortisone.

When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, alpha-fetoprotein (AFP), of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin and AFP is facilitated by synergies between HC and collagenous substrata.

Albumins↗
PubMed1987

Immunochemically detected nuclear envelope-associated cytochrome P-450 component(s) in rat hepatocyte culture lines.

The presence and location of cytochrome P-450 in Donryu rat hepatocyte culture lines, Ac2F cells and 3 other cell lines were assessed by indirect immunofluorescence examination using anti-cytochrome P-450 monoclonal antibodies. Ac2F cells and other hepatocyte cell lines were selectively stained at their nuclear envelope, but not the cytoplasm, with a monoclonal antibody selective to a high-spin form of cytochrome P-448 (P-448H), although this monoclonal antibody stained primary cultured normal rat hepatocytes at both cellular components and did not stain hepatoma cells of 2 transplantation lines. The results of unscheduled DNA synthesis assay with Ac2F cells using several carcinogenic aromatic amines (4-aminoazobenzene derivatives and amino acid pyrolysis products) suggested that this nuclear envelope-associated cytochrome P-450 activates a restricted portion of these aromatic amines, i.e., a tryptophan pyrolysis component and a glutamic acid pyrolysis component. These results indicate that rat hepatocyte culture lines lack (or contain a reduced amount of) the cytoplasmic cytochrome P-450 but maintain a characteristic type of cytochrome P-450, probably a kind of cytochrome P-448H in their nuclear envelope, and this may be involved in oxidative metabolism of a restricted portion of aromatic amines.

Animals↗
PubMed1986

The effect of conditioned medium on fetal human liver cells in primary culture.

A medium conditioned by rat embryo cultures (RCM) promoted the adhesion of liver cells from human fetuses to plastic dish. Colonies were also formed in primary cultures of the same cells in the presence of RCM. The majority of the colonies formed were composed of large polygonal cells with a few colonies composed of both clear epithelial-like cells and fibroblast-like cells. RCM was superior to the rat embryo feeder layer for promotion of colony formation of cells. A number of colonies were formed from fetal human livers when a conditioned medium from human hepatoma cells (HCM) was used, but most of the colonies formed were composed of fibroblast-like cells. The cells derived from the polygonal cell colonies, which were formed in the presence of RCM, have been passaged four times and they are still growing with albumin-producing capacity. The effect of RCM was reduced by various physico-chemical treatments.

Animals↗
PubMed1986

Primary cultures of human livers and their albumin-producing capacity.

Primary cultures of surgically obtained noncancerous portions of human liver tissues were made. Liver tissues were poorly dissociated with collagenase, but well dissociated with dispase. The yield and viability of cells were improved somewhat when dissociated with collagenase followed by dispase. The mean cell yield was 1.1 X 10(6) cells/g liver. The epithelial-like morphology of the dissociated liver cells was maintained for about one week, but thereafter degenerative alteration of cells was observed. In liver explant culture, an active outgrowth of cells was observed for more than one month. Albumin production in culture fluids from dissociated livers was detectable for about 2 weeks, but later became undetectable, while that from explant culture was detectable for at least one month. These data demonstrate that adult human hepatocytes can be isolated from noncancerous portions of livers with relatively high yield, and that albumin production of the dissociated cells is detectable for several days.

Adult↗
PubMed1986

An attempt to eliminate fibroblast-like cells from primary cultures of fetal human livers.

The elimination of fibroblast-like cells from primary cultures of fetal human livers was studied. A fibroblast-like cell line (HuF), which was obtained by subculturing fetal human liver cells 4 or more times, was briefly treated with hydrocortisone (HC) or putrescine (PUT). The growth of HuF cells was inhibited by HC at a concentration of 10(-2) M and by PUT at a concentration higher than 10(-3) M. Long-term treatment of HuF cells with 10(-3) M HC inhibited the growth of the cells. Primary cultures of fetal human livers were made in medium containing HC or PUT, and morphological and functional examinations were made. The cultures were predominantly composed of epithelial-like cells, with few fibroblast-like cells, when the HC concentration was 10(-5)M to 10(-3) M. A high amount of albumin was secreted at these concentrations of HC. On the other hand, at 10(-3) M PUT, many epithelial-like cells were seen, but albumin was undetectable. The present results indicate that albumin-producing epithelial-like cells can be selectively maintained in medium containing HC, in primary cultures of fetal human livers.

Albumins↗
PubMed1986

Malignant transformation of rat liver cells by N-benzoyloxy-3'-methyl-4-methylaminoazobenzene and its related compound.

N-Benzoyloxy-3'-methyl-4-methylaminoazobenzene-(N-benzoyloxy-3'-Me-MAB) was highly toxic to rat liver epithelial cells (Ac2F) and induced many more chromosome aberrations and unscheduled DNA synthesis than did 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). N-Benzoyloxy-3'-Me-MAB and its related compound, N-acetoxy-4-methylaminoazobenzene, efficiently induced malignant transformation of the cells, but 3'-Me-DAB was not so effective. The cells transformed by N-acyloxy-4-methylaminoazobenzene (N-acyloxy-MAB) derivatives showed significant increases in plating efficiency in liquid medium and in the size of aggregates in rotation culture. Such increases were not seen in the 3'-Me-DAB-transformed cells. The results indicate that the N-acyloxy form of the 4-dimethylaminobenzene derivatives seems very likely to be the ultimate metabolite.

Animals↗
PubMed1985

Asbestos-associated chromosomal changes in human mesothelial cells.

Replicative cultures of human pleural mesothelial cells were established from noncancerous adult donors. The cells exhibited normal mesothelial cell characteristics including keratin, hyaluronic acid mucin, and long branched microvilli, and they retained the normal human karyotype until senescence. The mesothelial cells were 10 and 100 times more sensitive to the cytotoxic effects of asbestos fibers than normal human bronchial epithelial or fibroblastic cells, respectively. In addition, cultures of mesothelial cells that survived two cytotoxic exposures of amosite fibers were aneuploid with consistent specific chromosomal losses indicative of clonal origin. These aneuploid cells exhibit both altered growth control properties and a population doubling potential of greater than 50 divisions beyond the culture life span (30 doublings) of the control cells.

Asbestos↗
PubMed1984

Effects of nickel sulfate on growth and differentiation of normal human bronchial epithelial cells.

Epidemiological studies have shown that inhalation of nickel compounds enhances the risk of human respiratory cancer. Cultures of normal human bronchial epithelial cells were continuously exposed to a dose (5-20 micrograms/ml) of NiSO4 that reduced their colony forming efficiency by 30-80%. After 40 days of incubation, the cultures consisted of large, squamous cells; mitotic cells were not observed. The cells were then maintained in medium without NiSO4. After 40-75 total days of incubation, colonies of mitotic cells appeared at a rate of 1 colony per 100 000 cells originally at risk; no colonies appeared in control cultures or in cultures exposed to less than 5 micrograms NiSO4/ml for 90 days. Twelve NiSO4-altered cell cultures isolated from five experiments have been expanded into mass cultures. Most of the cell lines have an increased population doubling potential (greater than 50 divisions). Some exhibit aberrations in the squamous (terminal) differentiation process whereas others have lost the requirement for epidermal growth factor for clonal growth. Aneuploidy and marker chromosomes have also been noted. However, none of these NiSO4-altered cell cultures are anchorage independent nor do they produce tumors upon injection into athymic nude mice.

Bronchi↗
PubMed1983

High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells.

A protoplast fusion method was developed to stably transfect human cells with pSV2-derived plasmids at frequencies greater than 10(-3). This procedure made it possible to test the biological effect of a hepatitis B virus (HBV) gene independent of the viral structures required for infection. A pSV2gpt+ plasmid constructed to carry a subgenomic fragment of HBV that contained the core antigen gene (HBc gene) was transfected into human cells. A human epithelial cell line was stably transfected with the HBc+ gene by selecting recipient cells for expression of guanine phosphoribosyl transferase expression. With this gpt+/HBc+ cell line it was shown that growth in serum-free medium or treatment with 5'-azacytidine stimulates the production of the HBV core antigen. A hepatocellular carcinoma carrying the entire HBV genome was stimulated to produce the HBc gene product in response to the same factors that stimulated HBcAg production in the gpt+/HBc+ cell line constructed by transfection. The temporal relation between the cytopathologic response and HBc gene expression was similar for both cell types, indicating a primary role for HBc gene expression in the cytopathology of HBV-infected human liver.

Azacitidine↗
PubMed1983

Effect of 3'-methyl-N,N-dimethyl-4-aminoazobenzene in the presence or absence of liver microsomes on the liver cells having low tumor-producing capacity in culture.

A cell strain having low tumor-producing capacity was exposed in culture to 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) in the presence or absence of liver microsomes, and whether or not the cells will progress to those having high tumor-producing capacity was examined. When transplanted into rats, the cells treated with 3'-Me-DAB four (Exp-I) or thirteen times (Exp-II) formed larger tumors than untreated control cells, the latter treatment being more efficient in this regard. Furthermore, the tumors formed by the cells treated with 3'-Me-DAB in the presence of liver microsomes were considerably larger than those formed by the cells treated with 3'-Me-DAB alone. The subcutaneous tumors produced by the cells treated with 3'-Me-DAB with S-15 Mix showed poorly differentiated histology compared with those produced by control and 3'-Me-DAB-treated cells. The frequency of lung metastasis tended to increase by 3'-Me-DAB with S-15 Mix. The cells treated with 3'-Me-DAB in the presence or absence of liver microsomes differed from untreated control cells in vitro in some properties, including the size of aggregates in rotation culture, plating efficiency in liquid medium and morphology. These observations suggest that cell malignancy was promoted by 3'-Me-DAB alone as well as by 3'-Me-DAB in the presence of liver microsomes.

Animals↗
PubMed1982

Effect of 3'-methyl-4-dimethylaminoazobenzene in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations in a diploid clone derived from normal rat liver cells in culture.

The effect of 3' methyl-4-dimethylaminoazobenzene (3'-Me-DAB) in the induction of malignant transformation and of 8-azaguanine-resistant mutations and chromosomal aberrations was studied in a diploid strain derived from normal rat liver cells. The cells were malignantly transformed by treatment with 3'-Me-DAB 1.7 micrograms/ml for 130 to 221 d or 1.7 micrograms/ml for 53 d followed by 24.9 micrograms/ml for 27 to 77 d. The untreated control cells did not transform spontaneously until the 232nd d in culture. Some properties of the 3'-Me-DAB-treated cells were compared to those of untreated control cells but no reliable marker for predicting the tumorigenic potential of the cells was found. The single addition of 3'-Me-DAB caused little induction of 8-azaguanine-resistant mutations and chromosomal aberrations to the cells. However, mutations and chromosomal aberrations were significantly induced by N-acetoxy-4-methylaminoazobenzene, an active metabolite of 4-dimethylaminoazobenzene or 3'-Me-DAB in the presence of liver microsomes.

Animals↗
PubMed1982

Effects of steroid sex hormones and adriamycin on human bladder cancer cells in culture.

The effects of steroid sex hormones on the established cell lines derived from human urinary bladder cancer, T24, and from human transitional cell cancer of the urinary tract, 253J, were examined using the colony formation method. Of the seven kinds of steroid hormones tested, estradiol-17 beta was intensively cytotoxic for both cells. The cytotoxic effect was depended on the dose and time of treatment. The combined effect of Adriamycin and estradiol-17 beta on T24 cells could be recognized at low concentrations of Adriamycin (less than or equal to 10(-3) micrograms/ml) after exposure for 24 h.

Aged↗
PubMed1982

[Absorption, distribution, metabolism and excretion of netilmicin in rats (I). Metabolic fate of netilmicin after a single dose (author's transl)].

The absorption, distribution and excretion of netilmicin (NTL) in rats were examined by the microbiological assay method. Plasma level and urinary excretion of NTL in rats after intramuscular administration were compared with those of 5 other aminoglycoside antibiotics, gentamicin, sisomicin, dibekacin, amikacin and tobramycin. 1. After intravenous administration, plasma level of NTL declined rapidly with a half-life of about 18 minutes. In the cases of intramuscular, intraperitoneal and subcutaneous administrations, plasma levels reached maximum at 15 minutes and declined with similar half-life as intravenous administration. 2. When 20 mg/kg of NTL was administered intramuscularly, NTL was highly distributed into the kidney, while poorly into the brain. The disappearance rates of NTL from the tissues except the kidney were almost same as that from the plasma. Concentration of NTL in kidney was about 3 times higher than that in plasma at 15 minutes after administration and declined with a half-life of about 7 days. 3. NTL excreted in the bile within 24 hours was only 0.2% of the dose administered (20 mg/kg, i.m.). An average concentration of bile during the first 2 hours was 3 mcg/ml. 4. Irrespective of the route of administration (i.v., i.m., i.p. and s.c.), NTL was excreted rapidly in the urine and 70.0 approximately 81.0% of the dose was recovered within 6 hours. 5. Plasma level and urinary excretion of NTL after intramuscular administration (20 mg/kg) were almost similar to those of 5 other aminoglycoside antibiotics. 6. There was no difference between NTL and GM in plasma level after high dose (100 mg/kg, i.m).

Animals↗
PubMed1982

[Absorption, distribution, metabolism and excretion of netilmicin in rats (II). Accumulation study of netilmicin after repeated administration].

Repeated administration of netilmicin (NTL), a new aminoglycoside antibiotic, to rats at daily intramuscular dose of 10 or 20 mg/kg for 22 days did not affect the plasma level and the plasma half-life of the drug. The concentrations of NTL in the kidney increased markedly after repeated administration, and reached peak level after 8 and 15 doses for 10 and 20 mg/kg, respectively. Cumulative effect of NTL after repeated dose was also observed in the liver, spleen and lung, although the peak concentrations of NTL in these organs were below 1/50 of that in the kidney. Blood and tissue levels of NTL in rats were determined after a single intramuscular administration of 14C-NTL at a dose of 20 mg/kg following 21 repeated intramuscular administrations of NTL at daily dose of 20 mg/kg. The repeated dose of NTL had no effect on the blood level-time curve of radioactivity. The concentration of NTL in the kidney determined by radioassay was about 1/3 of that determined by bioassay, whereas the half-lives in the tissue levels determined by these two assays were nearly identical with each other. The half-life in the lung determined by bioassay was almost identical to that determined by radioassay, whereas the former was rather shorter than the latter in the liver and spleen.

Animals↗
PubMed1982

[Absorption, distribution, metabolism and excretion of netilmicin in rats (III). Metabolic fate of 14C-netilmicin after intramuscular administration (author's transl)].

The metabolic fate of 14C-netilmicin (14C-NTL) was studied in rats after intramuscular administration (20 mg/kg). 1. Binding ratio of 14C-NTL to rat plasma protein, determined by the ultracentrifugal method, was 15 approximately 20% during the first 1 hour after intramuscular administration. 2. Binding ratio of 14C-NTL to HSA, determined by the equilibrium dialysis method, was 15 approximately 23%. Binding constant (K) and maximum binding number (n) were calculated as 1.48 X 10(4) and 3.3 mole/mole, respectively. Half of 14C-NTL bound to HSA was dissociated from HSA by gel-filtration. 3. The radioactivity in tail vein blood reached peak level at 10 minutes after intramuscular administration and declined rapidly. 4. Since the distribution rate of 14C-NTL into the blood cells was low during the first 2 hours, the plasma level at that time was about 1.5 times higher than whole blood level. 5. The recovery of radioactivity in the bile (0 approximately 24 hours) was only 0.13% of the dose administered. An average concentration during 2 hours after intramuscular injection was 2.5 mcg/ml. 6. Within 2 hours after administration, approximately 40% of the dose was recovered in the urine. Within 24 hours, 87.8% of the dose was excreted in the urine, 2.6% in the feces, 0.3% in the cage washing and 8.8% in the carcass. Total recovery ratio was 99.5% of the dose. 7. The radioactivity was widely distributed in the tissues, especially high in the kidney and bone, low in the brain. A high concentration of radioactivity was found in the renal cortex at 24 hours after administration. 8. Netilmicin was excreted into the urine unchanged and no metabolites were detected by TLC at all.

Animals↗