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Recently, we induced memory-like T cells from human naïve T cells in a hypoxic culture. In this study, we attempted induction of both memory B cells and plasma cells from human naïve B cells in hypoxic cultures. Although the hypoxic culture (1% O2) did not support the survival of naïve B cells, it supported the survival of B cells that were activated by interleukin 4 (IL-4) and lipopolysaccharides (LPS). On activation culture Day 7, there was no survivor in 20% O2, but 29.5% of the activated B cells survived in 1% O2. Next, we withdrew both IL-4 and LPS from the B-cell culture preactivated for 7 days in 1% O2, and the culture was continued for another 7 days. As a result, 12.4% of the B cells survived. These memory-like B cells decreased in body size and number of cytoplasmic granules. Moreover, we tried to induce plasma-like cells. Human naïve B cells were activated by two-step culture for 9 days, after which all activators were withdrawn from the medium, and the cells were cultured for an additional 6 days in 1% O2. The viability was 0.2%, but over 92% of the survivors showed a CD20-/CD27+/CD38+ plasma cell phenotype.

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here, we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits, both purified naive CD8+ T cells and purified naive CD4+ T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O2 atmosphere) for several more days; during this period, most of the cells died, but some survived in a quiescent state for a month. The survivors had small round cell bodies, expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia, if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats., Pre-activated human naïve CD4+ or CD8+ T cells were cultured in activator-free medium to induce cell death for 5 to 10 days. Although no cell survived in normal oxygen condition (20% O2), 2 to 30% of the activated T cells could survive in hypoxia (1% O2) and became memory-like T cells. We named this simple procedure as ‘the Death Assay’., Graphical Abstract Graphical Abstract

Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Lanthanum carbonate is a popular chemical which is administered for patients with end-stage kidney disease to reduce the absorption of phosphate, and lanthanum deposition in the gastroduodenal mucosa has recently been reported. The aim of this study was to assess whether any histologic changes of the gastric mucosa are related to the deposition of lanthanum. Twenty-four patients who revealed the histology of lanthanum deposition on gastroduodenal biopsy between 2011 and 2014 were included in the study, and their clinical records and gastroduodenal biopsies obtained from 2011 to 2015 were reviewed, adding the review of gastroduodenal biopsies before 2011 if possible. Analysis of the deposited materials by scanning electron microscopy-energy dispersive x-ray spectroscopy was performed for a representative gastric biopsy. All patients were diagnosed as having renal insufficiency due to chronic kidney disease and treated with dialysis for more than 5 years, with confirmation of lanthanum carbonate use for 22 patients. Of 121 gastric biopsies and 10 duodenal ones between 2011 and 2015, 86 gastric biopsies (71.1%) and 3 duodenal biopsies (30%), respectively, revealed histology consistent with lanthanum deposition, which was confirmed by scanning electron microscopy-energy dispersive x-ray spectroscopy analysis for a representative case. The deposition tended to occur in the gastric mucosa with regenerative change, intestinal metaplasia, or foveolar hyperplasia (P

The significant increase in chromosomal instability with aging is well known, but the underlying mechanism is not fully understood. Our earlier studies showed a high frequency of abnormal mitosis, such as mitotic slippage or incomplete mitosis in near-senescent human fibroblasts. This study examined the centrosome aberrations in mitotic and interphase cells from different passages of several strains of human fibroblasts. Analysis by laser scanning cytometry showed increased frequencies of abnormal mitotic cells with supernumerary (>2/cell) centrosomes and misaligned chromosomes in later passages in all strains examined. Numerical centrosome aberrations were prominent in a polyploid subpopulation. In metaphase cells, numerical centrosome aberrations were correlated significantly with chromosome misalignment. Fluorescent in situ hybridization analysis using a centromere-specific probe revealed a correlation between chromosome aneusomy and centrosome over-duplication. These results suggest that abnormal duplication of centrosomes in association with cellular aging may be responsible for the increase in chromosomal instability with aging.

Intestinal alkaline phosphatase (IAP) is a brush-border membrane ectoenzyme (BBM-IAP) that is released into the lumen (L-IAP) after a high-fat diet. We examined the effects of oil feeding and the addition of mixed-lipid micelles on the formation of L-IAP in oil-fed rat intestine, Caco-2 cell monolayers, and mouse intestinal loops. We localized IAP in the duodenum of rats fed corn oil using fluorescence microscopy with enzyme-labeled fluorescence-97 as substrate. Four hours after oil feeding, L-IAP increased ∼10-fold accompanied by the loss of BBM-IAP, consistent with BBM-IAP release. Rat IAP isozyme mRNAs progressively increased 4–6 h after oil feeding, followed by the increase of IAP activity in the subapical location at 6 h, consistent with the restoration of IAP protein. Postprandial lipid-micelle components, sodium taurocholate with or without oleic acid, mono-oleylglycerol, cholesterol, or lysophosphatidylcholine (lysoPC) were applied singly or as mixed-lipid micelles to the apical surface of polarized Caco-2 cell monolayers. LysoPC increased L-IAP >10-fold over basal release. LysoPC released IAP into the apical medium more than other intestinal brush-border enzymes, 5′-nucleotidase, sucrase, aminopeptidase N, and lactase, without comparable lactate dehydrogenase release or cell injury. LysoPC increased human IAP mRNA levels by 1.5-fold in Caco-2 cells. Luminally applied lysoPC also increased release of IAP preferentially in mouse intestinal loops. These data show that lysoPC accelerates the formation of L-IAP from BBM-IAP, followed by enhanced IAP synthesis, suggesting the role that lysoPC might play in the turnover of brush-border proteins.

Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc.

Background/Aims: We previously reported that endothelin (ET)-1 may be involved in the contraction of hepatic sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently cell morphology. To clarify the role of ET receptors [endothelin A receptor (ETAR) and endothelin B receptor (ETBR)] in ET-1-induced defenestration, we studied the size of hepatic SEF under various experimental conditions. Methods: Liver sinusoidal endothelial cells (LSECs) isolated from rat livers by collagenase perfusion were cultured and divided into four groups: control, ET-1 (10−6–10−10 M)-treated, ET-1+selective ETAR antagonist (BQ610)-treated and ET-1+ETBR antagonist (BQ788)-treated groups. SEF morphology was observed by scanning electron microscopy. Protein expressions of ETAR and ETBR, Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. F-actin stress fiber formation was observed by confocal microscopy. Active Rho was measured by Ren's modification. Intracellular free Ca2+ concentration ([Ca2+]i) was measured by fluorescence digital imaging using fura-2 AM by Aqua cosmos. Results: ET-1 induced a reduction in the number and size of SEF. ETAR antagonist pretreatment inhibited defenestration induced by low ET-1 concentrations (10−8–10−10 M), whereas ETBR antagonist pretreatment did not block defenestration at low to high ET-1 concentrations (10−6–10−10 M). F-actin stress fibers, Rho A levels and phosphorylated myosin light-chain kinase levels remained the same in various treatments. Active Rho was not detected in control and various treatments. ET-1 did not increase [Ca2+]i. Western blot showed prominent ETBR but scarce ETAR protein expression in LSECs. Conclusions: The present findings demonstrated that ETBR- and ETAR-induced contractile mechanisms are not involved in ET-1-induced defenestration, and that Rho is also not activated. Therefore, ET-1 induces hepatic defenestration by mechanisms other than receptor-mediated contraction.

To study the role of cell death in the aging process, cell death during spontaneous cellular senescence in vitro was examined with normal human fibroblasts. A small subset of the senescent cells showed aberrant morphology such as remarkable nuclear fragmentation or multiple micronuclei, and such cells often showed positive reactions with antibody to phosphorylated pRb. Cells showing caspase activation and binding of Annexin V, which indicate apoptotic change, increased in the senescent phase in flow cytometry analysis. Propidium iodide-positive cells, however, also increased with passaging. The results suggest that both apoptosis and necrosis are involved in cell death of senescent human fibroblasts.

Nickel compounds are known to be carcinogenic to humans and show genotoxicity, including the ability to induce chromosome aberrations and neoplastic transformation in vitro. The mutagenicity of nickel compounds is, however, equivocal and the mechanisms of carcinogenesis are still not clear. In this study, the possibility that nickel compounds induce genetic or chromosomal instability was examined, because recent studies in cancer research show that these conditions are critically involved in carcinogenesis. V79 Chinese hamster cells were treated with 320 microM nickel sulfate for 24 h at low cell density (100 cells/100 mm diameter dish) and clones derived from single cells surviving Ni treatment were isolated. When cells grew up to 23-25 population doublings post-treatment, mutation frequency at the HPRT locus and the chromosome aberration frequency of each clone were examined. Five out of 37 clones (13.5%) derived from Ni-treated cells showed a remarkably increased frequency of HPRT mutations (>or=1 x 10(-4)), while only one out of 37 control clones (2.7%) showed this high mutation rate. In addition, 17 out of 37 clones (45.9%) from Ni-treated cells showed structural chromosomal aberrations in 10% or more of cells (up to 45.5%), while only three out of 31 control clones (9.7%) showed this high aberration rate. Out of 37 clones derived from Ni-treated cells, eight (21.6%) and 11 (29.7%) clones showed an increased frequency (>or=5%) of aneuploid and polyploid cells, respectively, while only a few control clones showed such an increase in aneuploid and polyploid cells. These results indicate that nickel sulfate can induce genetic and chromosomal instability in V79 cells.

Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.

CYP1A1 is involved in the metabolism of benzopyrene, a suspected lung carcinogen; it is therefore conceivable that genetically determined variations in its activity modify individual susceptibility to lung cancer. The role of the CYP1A1 MspI polymorphism in lung cancer has been widely studied but has not been fully clarified. We have included 2,451 cases and 3,358 controls in a pooled analysis of 22 case-control studies on CYP1A1 and lung cancer risk. We found a clear association between the CYP1A1 homozygous MspI restriction fragment length polymorphism (RFLP) and lung cancer risk in Caucasians (age- and gender-adjusted odds ratio = 2.36; 95% confidence interval 1.16-4.81); other associations were weaker or not statistically significant. The association with the homozygous variant was equally strong for squamous cell carcinomas and adenocarcinomas among Caucasians. We analyzed the risk by duration of smoking: for Caucasian subjects with the MspI RFLP combined variants (homozygotes plus heterozygotes), the increase in the risk of lung cancer was steeper than among the individuals with the homozygous reference allele. Our analysis suggests that Caucasians with homozygous variant CYP1A1 polymorphism have a higher risk of lung cancer. The data were more consistent among Caucasians, with a strong association between the homozygous variant in both squamous cell carcinomas and adenocarcinomas, and a stronger association in men than in women. The analyses were more inconsistent and failed to reach statistical significance in Asians. This observation might be due to design specificities or unknown effect modifiers in the Asian studies.

Effects of uranium ore dust on cell proliferation, lipid peroxidation and micronuclei formation were compared with silica (DQ12) and titanium oxide in normal human distal airway epithelial cells (NHDE), human lung cancer cells (A549) and human lung fibroblast cells. Cell proliferation was significantly inhibited with uranium ore dust and silica but not with titanium oxide. Lipid peroxidation was significantly enhanced only with uranium ore dust. Micronuclei formation was significantly stimulated with uranium ore dust in A549 and NHDE cells, but not in fibroblast cells. Silica stimulated micronuclei formation only in A549 cells. The results showed the outstanding effect of uranium ore dust on lipid peroxidation and micronuclei formation in human lung cells compared to silica and titanium dioxide.

Point mutations at codon 12 of the c-Ki-ras gene were investigated in 103 biopsy tissues from 38 patients with gastric adenoma. DNA from minute specimens of formalin-fixed paraffin-embedded tissue was amplified by nested polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP) method was used to detect mutations of the c-Ki-ras gene. Mutations were detected in 4 of the 38 gastric adenomas (10.5%), but were not detected in intestinal metaplasias and gastric adenocarcinomas. Of the 4 mutation-positive gastric adenomas, the GGT sequence in codon 12 changed to GAT in 2 cases, to GTT in 1 case and to TGT in 1 case. We classified gastric adenomas into three degrees of atypia; mutations were detected only in biopsy tissues with moderate and severe atypia but were not detected in biopsy tissues with mild atypia. When different biopsy tissues from a mutation-positive gastric adenoma were examined, the mutation was not always detected in every tissue sample examined. This suggests that mutations are not homogeneously distributed, even in the same adenoma. It is possible to follow up the mutation of biopsy materials of gastric adenoma by this method.

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4 days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2 weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3 days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.

Centrosome overduplication or amplification has been observed in many human cancers and in premalignant tissue, but the mechanisms leading to such centrosome aberrations are not fully understood. We previously showed that abnormal mitotic cells with supernumerary centrosomes increase with replicative senescence in human fibroblasts, especially in a polyploid subpopulation. This study examines localization of p53 protein at centrosomes in mitotic cells, which is often observed in association with DNA damage response, to investigate a possible association between p53 localization and numerical centrosome aberrations induced by cellular senescence. Cultures at later passages or the 4th day after exposure to H2O2showed increased frequencies of mitotic cells with supernumerary centrosomes, especially in a polyploid subpopulation. Immunohistochemical analysis frequently showed p53-positive foci in mitotic cells, and some were localized at centrosomes. The number of p53-positive foci in mitotic cells and its localization to centrosomes increased with replicative and premature senescence. Supernumerary centrosomes showed higher frequencies of p53 localization compared to normally duplicated centrosomes. Centrosome-associated p53 protein was phosphorylated at Ser15. These data suggest a possible association between localization of p53 protein and numerical centrosome aberrations in replicatively or prematurely senescent cells.

Aberrant nuclear morphology, such as nuclei with irregular shapes or fragmented nuclei, is often observed in senescent cells, but its biological significance is not fully understood. My previous study showed that aberrant nuclear morphology in senescent human fibroblasts is attributable to abnormal mitosis in later passages. In this study, the production of abnormal nuclei in association with premature senescence was investigated. Premature senescence was induced by brief exposure of human fibroblasts to hydrogen peroxide (H(2)O(2)), and mitosis was observed by time-lapse microscopy. In addition, cell cycle and nuclear morphology after exposure to H(2)O(2) were also analyzed using a laser scanning cytometer. Time-lapse analysis revealed that the induction of premature senescence caused abnormal mitoses, such as mitotic slippage or incomplete mitosis, especially in later days after H(2)O(2) exposure and often resulted in abnormal nuclear morphology. Analysis by laser scanning cytometer showed significantly higher frequency of abnormal cells with deformed nuclei and abnormal mitotic cells with misaligned chromosomes in a hypertetraploid subpopulation. These results suggest that unstable hypertetraploid cells, formed in association with H(2)O(2)-induced premature senescence, cause abnormal mitosis that leads to aberrant nuclear morphology.

In order to investigate anthracosis of the human lung, especially its causal relationship with atmospheric pollution and the occurrence of lung cancer, intrapulmonary particulate pollutants (IPP) from autopsy cases and patients lobectomized because of lung cancer were separated by alkali digestion of the lung tissue, and their elemental constitution was analyzed by a wavelength-dispersive X-ray fluorescence spectrometer. Silicon was the most abundant mineral constituent of non-carbonaceous fraction of IPP, followed by calcium, magnesium, iron, aluminum and other trace elements. The levels of silicon and aluminum in IPP were significantly higher in individuals treated at Saitama Medical School Hospital than in those from Tokyo. Farmers showed higher levels of silicon and aluminum than other occupational categories, whereas male blue-collar workers showed higher levels of calcium and lead than farmers. The level of iron in IPP of male smokers tended to be higher than in non-smokers. In cases of lung cancer, especially of the hilar type, the levels of iron, calcium, copper, lead, chromium and nickel in IPP tended to be higher than in non-lung cancer cases, whereas the levels of silicon and aluminum were lower than in non-lung cancer cases. On the basis of these results, pulmonary anthracosis was considered to be etiologically related to the occurrence of lung cancer.

We have developed an in vitro model for studying vascular injury. After 7-10 days in a three-dimensional collagen gel culture, capillary-like tubes were formed in the collagen gels. We injured these capillary-like tubes with a laser microdissection system or a scrape method with razors and then examined the collagen gel culture by phase contrast and electron microscopy. After laser injury, profuse necrotic cells were observed around the injured capillary-like tubes and within the tubular lumen compared to the razor injury. We then isolated total RNA from these cultures and prepared cDNA for investigations by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real time RT-PCR revealed the up-regulation of transcription factor early growth response-1 (Egr-1) after both laser and razor injury, accompanied by the up-regulation of fibroblast growth factor-2 (FGF-2), a proangiogenic factor downstream of Egr-1. The effective laser energy is concentrated on the minute focal spot only. These methods provide a useful in vitro model for studying vascular injury.

Although apoptosis has been shown in vivo to be involved in the aging process, in vitro studies of age-dependent apoptosis are limited. In this study, apoptosis was examined in normal human fibroblasts exposed to H(2)O(2) to induce premature senescence and in spontaneously senescent human fibroblasts. Although apoptosis was not observed for several days after exposure to H(2)O(2), morphological changes indicating apoptosis were evident in about 5% of cells 7 days after exposure to 80microM H(2)O(2), concomitantly with expression of senescent phenotype. The apoptotic changes were preceded by caspase activation in majority of the exposed cells. As well as H(2)O(2)-induced senescent cells, spontaneously senescent human fibroblasts showed apoptotic changes in about 2% of cells and majority of the senescent cells also showed activation of caspases. The results indicate that the apoptosis pathway is activated in H(2)O(2)-induced and spontaneously senescent human fibroblasts in vitro.

The ability of nickel sulfate (NiSO 4 ) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100–400 μM NiSO 4 for 24 h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay. Aneuploid cells were induced in a significant fraction of the cell population 24–48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO 4 . The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose. These results indicate that NiSO 4 has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.

Drug-induced fever due to histamine H2-receptor blockers was experienced by a 55-year-old man. The patient became febrile 5 days after receiving cimetidine, and continued to be febrile until the drug was stopped. His maximum body temperature was above 40 degrees C. Challenge tests with cimetidine and ranitidine showed that the fever was caused by the H2-blocker. The patient's serum IgE concentration increased markedly to 2590 IU/ml 10 days after admission, and skin tests for cimetidine and ranitidine were positive. Lymphocyte stimulation tests were positive for both drugs (stimulation indices: 193% for cimetidine and 325% for ranitidine). Cimetidine-induced fever has generally been thought to be due to a direct effect on the thermoregulatory centre in the hypothalamus, on the basis of experimental studies of the injection of cimetidine to the cerebral ventricles. However, clinical evidence has not excluded an allergic involvement in this type of drug-induced fever. This patient's fever was proven to be due to administration of the H2-blocker, and the mechanism of action was IgE-mediated.

DNA of minute specimens taken from formalin-fixed and paraffin-embedded tissue was used as a template for the polymerase chain reaction (PCR) to examine whether the c-Ki-ras gene is activated in atypical bronchioloalveolar cell hyperplasia (ABH) of human lung. The c-Ki-ras gene was successfully amplified on 131 samples from 29 cases of lung adenocarcinoma (87.3% of all samples used as templates) by the nested PCR method. Point mutations at codon 12 of the c-Ki-ras gene could be detected in carcinoma tissue of 6 cases (20.7% of all cases), also detected in ABHs showing moderate to severe atypia of 2 cases. PCR amplification is a useful technique for studying pin-point pathological lesions in a routine paraffin section at the molecular level.

Concentrations of nine metals (Fe, Ca, Mg, Zn, Cu, Co, Ni, Pb and Cr) concentrations in lung tissues from 224 lung cancer cases were compared with those in other cases to achieve an understanding of their contribution to the development of lung cancer and the varieties after the development of cancer. Comparisons of metal concentrations in each cell type of lung cancer were also performed. All cases were collected from routine autopsies in Tokyo and Saitama, Japan. The copper concentration in tissue from lung cancers was significantly higher than that in other specimens, although calcium, magnesium, zinc and cobalt concentrations in lung cancers were significantly lower than those in other cases. There were no significant differences in the 99% intervals (excluding extremely high values for occupationally exposed cases) for chromium, nickel and lead concentrations between lung cancers and other cases, although these values were lower in lung cancers. However, in comparisons of men only, the chromium concentration, the degree of lung contamination and the severity of pulmonary emphysema in lung cancer cases were significantly higher than those in other specimens. Moreover, percentages of lung cancer in men at each degree of contamination and each severity of emphysema increased with increasing grades. Thus, this finding could be evidence that the exposure to contaminants other than chromium and nickel in the air had affected the development of lung cancer, except for occupationally exposed individuals. Therefore, almost all chromium and nickel in lung tissue might not deposit in carcinogenic forms such as hexavalent chromium or nickel subsulfide. Comparison of the characteristics in each cell type of lung cancer revealed that calcium and chromium concentrations in lung tissue (not including tumor) or squamous cell carcinomas were higher than those in the other cell types. In small cell carcinomas, calcium magnesium and zinc concentrations were low.

Repeated oral administration of an amine-curing agent for epoxy resin, bis(4-amino-3-methylcyclohexyl)methane, gave rise to severe damage in the choroid plexus of rat brain. The damaged epithelium presented varying degrees of swelling and hydropic vacuolation on light microscopy, and varying numbers of vacuoles and inclusion bodies, frequently with lamellar structure, on transmission electron microscopy. Scanning electron microscopy of the choroid plexus disclosed some irregularity in the size of epithelial cells and occasional loss of microvilli. These changes in the choroid plexus were closely correlated with the dosage of the agent administered and the period of administration. In spite of the severe changes in the choroid plexus, no neurological abnormalities were observed in the animals during the experimental period.

Repeated oral administration of bis(4-amino-3-methylcyclohexyl)methane to rats induced unusual ultrastructural changes in Clara cells of the bronchiolar epithelium, involving marked accumulation of electron-dense inclusion bodies with a lamellar structure in the cytoplasm. The appearance of the inclusion bodies was speculated to result from accumulation of complexes formed between the agent and phospholipids within lysosomes, a condition known as drug-induced lipidosis. This finding appeared noteworthy, since it might reflect high potential for metabolism of xenobiotic compounds in Clara cells.