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ABSTRACTIn Japan, the trend for cervical cancer at younger ages has been increasing. As a countermeasure, the HPV vaccine was introduced as a routine vaccination in April 2013. However, the Ministry of Health, Labour and Welfare (MHLW) announced a “Suspension of its active inoculation recommendation for HPV vaccine” in June 2013. In 2016, 32 months after that suspension, we conducted survey via Internet and compared the results with our previous ones conducted at 9 and 23 months after suspension (in 2014 and 2015, respectively). We examined the ‘time-dependent change’ of the ‘intention of mothers to inoculate their daughters with the HPV vaccine’ in terms of efficacy of external decision-making support. 17.5% of mothers in the first survey replied that they would inoculate their daughters under the current circumstances, 12.1% in the second survey, and 6.7% in the third, showing a consistent decrease in willingness over time (p= 0.03, p< 0.01). If the government recommendation were to be reintroduced, 22.5% of mothers in the first survey replied they would inoculate their daughters, 21.0% in the second survey, which indicated no significant difference (p= 0.65) over the first interval; however, this was significantly decreased to 12.2% in the third survey (p< 0.01). Our study revealed that the intention to inoculate their daughters has been declining among Japanese mothers over time triggered by the suspension.

Modifications in oxygen coordination environments in heterostructures consisting of dissimilar oxides often emerge and lead to unusual properties of the constituent materials. Although lots of attention has been paid to slight modifications in the rigid oxygen octahedra of perovskite-based heterointerfaces, revealing the modification behaviors of the oxygen coordination environments in the heterostructures containing oxides with oxygen vacancies have been challenging. Here, we show that a significant modification in the oxygen coordination environmentsmelting of oxygen vacancy orderis induced at the heterointerface between SrFeO2.5(SFO) and DyScO3(DSO). When an oxygen-deficient perovskite (brownmillerite structure) SrFeO2.5film grows epitaxially on a perovskite DyScO3substrate, both FeO6octahedra and FeO4tetrahedra in the (101)-oriented SrFeO2.5thin film connect to ScO6octahedra in DyScO3. As a consequence of accommodating a structural mismatch, the alternately ordered arrangement of oxygen vacancies is significantly disturbed and reconstructed in the 2 nm thick heterointerface region. The stabilized heterointerface structure consists of Fe3+octahedra with an oxygen vacancy disorder. The melting of the oxygen vacancy order, which in bulk SrFeO2.5occurs at 1103 K, is induced at the present heterointerface at ambient temperatures.

The mitochondrial production of reactive oxygen species (ROS) has been implicated in the anticancer activity of furanonaphthoquinone. However, the mechanism of the activation remains elusive. In the current study, we found that treatment of HeLa cells with 2-methyl-5(or -8)-hydroxy-furanonaphthoquinone (FNQ13) induces mitochondrial swelling, followed by apoptosis. This toxic effect of FNQ13 was reduced by the radical scavengers α-tocopherol and trolox. Cytochemical experiments in isolated mitochondria showed that a combination of FNQ13 and NADH induces the production of H2O2 at the exterior mitochondrial membrane surface. This production of H2O2 was reduced by an antibody to the voltage-dependent anion channel (VDAC). Overexpression of the VDAC by transfection with vdac1 cDNA increased the production of H2O2 by HeLa cells, whereas transfection with a small interfering RNA to VDAC reduced FNQ13-induced H2O2 production and cell death due to an almost complete knockdown of VDAC expression. We also found significant correlations between the expression of VDAC and the induction of H2O2 production and cell death by FNQ13 in 11 human cancer cell lines. These results indicate that the anticancer activity of furanonaphthoquinones depends on the production of reactive oxygen species by mitochondrial permeability transition pores (MPTP) including the VDAC.

Delirium is a frequent symptom of terminally ill cancer patients and can cause serious distress to family members. To clarify the degree of emotional distress of family members concerning terminal delirium, a survey of bereaved families was performed. A questionnaire was mailed to 300 bereaved families with a request to rate the frequency and level of their distress for 12 delirium-related symptoms. A total of 195 responses were analyzed (effective response rate = 65%). Seventy-four percent and 62% of the family members reported that the patients had symptoms of physical restlessness and mood lability, respectively. Psychotic symptoms, such as hallucinations and delusions, were reported by 35%–37%, and somnolence was reported in 92%. The prevalence of cognitive symptoms (e.g., communication difficulty, memory disturbance) ranged between 50% and 72%. More than two-thirds of the bereaved family members perceived all delirium-related symptoms other than somnolence as distressing or very distressing when they occurred “often” or “very often.” For physical restlessness, mood lability, and psychotic symptoms that occurred “sometimes,” 27%–36% of the family members had moderate to high levels of distress. The bereaved family members of terminally ill cancer patients experienced high levels of distress from both the agitation and cognitive symptoms of terminal delirium. Multidisciplinary interventions, including the prevention of agitation and the minimization of cognitive impairment, pharmacological or medical treatments, and supportive and psychoeducational approaches for family members, are needed to alleviate family distress.

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30–40 ng/mL TCDD induced maximal intracellular production of H2O2at 30 minutes and led to severe cell death (0–31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78–97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.

The purpose of this study is to observe dynamic morphological changes induced by ultraviolet (UV) irradiation in a cultured human lens epithelial cell line using electron microscopy, cell viability staining, time-lapsed videography and immunohistochemistry. Human lens epithelial cell line SRA 01-04 was cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% fetal bovine serum. Subconfluent cells were irradiated under a bank of UV lamps, which emitted 275–400nm radiation with a maximum at 310nm. The UV intensity was 20μWcm−2at dosages from 0 to 10mJcm−2. Alterations in the morphology of the living cells were monitored and recorded with phase-contrast microscopy and time-lapsed videography. At different times, the cells were fixed and examined by transmission electron microscopy (TEM), diamidinophenolindole (DAPI) staining, and in situ immunohistochemistry using TdT-mediated dUTP-biotin nick end labeling (TUNEL). Cell viability was also assessed with crystal violet staining. At low doses of UV exposure (2–5mJcm−2), time-lapsed videography revealed definitive cell death that appeared to be primarily apoptotic. The dead cell debris was engulfed and phagocytosed by neighboring living cells. Phase-contrast microscopy and TEM demonstrated that, at UV 10mJcm−2, the cells not only showed typical apoptosis such as nuclear membrane shrinkage, chromatin condensation, and fragmentation into apoptotic bodies, but also necrosis such as swelling of the nucleus and cell body, and disruption of the plasma membrane. In support, DNA staining and in situ immunohistochemical reactions in the UV irradiated cells were both positive. The phagocytotic process was also seen with TEM. UV irradiation thus appears to cause both apoptosis and necrosis in the cultured human lens epithelial cell line. Active migration and phagocytosis of the cells appear to be stimulated by UV-induced damage. These findings may also aid in the understanding of UV injury and repair mechanisms of lens epithelial cells in vivo.

In order to analyze the mutational events and to understand the biological significance of the p53 gene in chemical carcinogenesis, we applied a new yeast-based p53 functional assay to ovarian tumors induced by 7, 12-dimethylbenz[a]anthracene (DMBA), as well as to transitional cell carcinomas of the urinary bladder induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in rats. The assay demonstrated that 15 of 19 DMBA induced tumors harbored clonal p53 mutations, which is consistent with the expectations of the “clonal expansion” hypothesis. The majority of the mutations were purine (AG) to pyrimidine (CT) transversions (12/19) on the non-transcribed (sense) strand (NTS), which is likely to be due to depurination created by DMBA adduct formation on the NTS. In contrast, we found no purine to pyrimidine transversion on the NTS. After cessation of BBN treatment, BBN-induced multifocal lesions in the bladder contained heterogeneous p53 mutations at an early stage. In the later stage, however, clonal p53 mutations were identified in 4 out of 7 bladders analyzed, conforming with the concept of “field cancerization”. The observed base substitutions were G→A (1/6) or C →T transitions (2/6), and mutations at T (3/6) on the NTS in clonal mutations, together with non-clonal mutations, showing a preference of C→T to G→A (17 vs. 0). Thus, preferential repair was found in the transcribed strand of the p53 gene, whether modified by DMBA or by BBN carcinogens. Very similar mutation patterns were observed between clonal and non-clonal mutations in the DMBA- and BBN-induced tumors, indicating that the rat yeast p53 functional assay can be a potential tool for the characterization of in vivo mutation patterns of p53, when modified by chemical carcinogens. Int. J. Cancer 83:700-705, 1999. © 1999 Wiley-Liss, Inc.

We investigated the existence of an NADH-dependent paraquat (PQ) reduction system in rat liver mitochondria (Mt) in respect to the cytotoxic mechanisms of PQ. The outer membrane fractions, free from the contamination of inner membranes but with a few microsomes, catalyzed rotenone-insensitive NADH, but not NADPH, oxidation by menadione or PQ. Anti-NADH–cytochrome b5reductase antibody and its inhibitorp-hydroxymercuribenzonate did not inhibit the NADH–PQ reduction activity. Therefore, the respiratory systems of the inner membranes and microsomal cytochrome P450 systems could not have been responsible for the reaction. Dicoumarol, an inhibitor of NAD(P)H–quinone oxidoreductase (NQO), dose dependently suppressed the NADH oxidation in the outer membrane via PQ as well as menadione, withI50values of 190 (for menadione) and 150 μM (for PQ). Because of a lower sensitivity to NADPH and the higher doses of dicoumarol required for its inhibition, the activity in the outer membrane may be an “NADH–quinone oxidoreductase” which partly differs from the NQO previously reported. This outer membrane enzyme produced superoxide anions in the presence of both NADH and PQ and was too tightly membrane-bound to be extracted by Triton X-100 and deoxycholate. From these results, we concluded that the free radical-producing mitochondrial NADH–quinone oxidoreductase is a novel oxidation–reduction system participating in PQ toxicity. This is in good agreement with our previous results showing that PQ selectively damaged Mtin vivoandin vitro, resulting in cell death (K.-I. Hiraiet al., 1992,Toxicology72, 1–16).

The ultrastructural H2O2-producing site in human polymorphonuclear leukocytes (PMN) stimulated with soluble stimuli was studied using a CeCI3-technique. Cellular aggregation and formation of small vacuoles were observed when PMN were stimulated with 100 µg/ml concanavalin-A, 1 mg/ml Phytohemagglutinin, or 100 µg/ml wheat germ agglutinin for 10 min at 37°C. Electron-dense deposits formed from the reaction of H2O2and CeCI3were observed on the contact surface of the plasma membrane of aggregated PMN stimulated with lectins. Treatment with 5 µg/ml cytochalasin-B before lectin-stimulation induced an enhanced formation of vacuoles, degranulation, rounding of the contour, cellular aggregation, and enhancement of the deposits. Phorbol myristate acetate (PMA; 100 ng/ml) induced strong leukocyte aggregation, the formation of multiple huge vacuoles, degranulation, and H2O2production at almost all of the contact surface between adjoining PMN and between PMN and erythrocytes, mononuclear cells, or thrombocytes. In PMN stimulated with digitonin (8 µg/ml), vacuolar formation, degranulation, multiple projections on the surface, and H2O2production on the whole surface membrane were demonstrated. It is shown that cellular aggregation and cell-to-cell contact have an important role in the induction of O2–production induced by lectins or PMA and that O2–production induced by the detergent is not dependent on leukocyte aggregation.

We tried to demonstrate the electron microscopic histochemical localization of membrane Ca2+-ATPase activity in glutaraldehyde-fixed rat sciatic nerves. Although conventional glutaraldehyde fixatives containing impurities interfered with the reactivity of Ca2+-ATPase, this activity was successfully preserved in the tissues fixed with pure glutaraldehyde as well as in those fixed with paraformaldehyde. In unmyelinated nerve fibers, an ATPase activity depending on 10 mM CaCl2 was detected on the whole external surface of Schwann cell plasma membranes. In myelinated fibers, this activity was localized on the surface of Schwann cell outer loops at the paranodal region of Ranvier nodes and on the axonal membrane at the nodal region. Another activity depending on 0.1 mM CaCl2 was demonstrated on the axolemma of unmyelinated fibers. These results indicated that there may be two types of Ca2+-ATPase activities showing high and low affinity to calcium ions localized in peripheral nerve systems in a different manner between myelinated and unmyelinated fibers.

The cell spreading ability of human lung cancer cells on collagen substrata was examined in comparison with normal human tracheal epithelial cells. Plastic dishes or multiwells were coated with type I, III or IV collagen gel at a concentrate of 200 μg/cm2. Ninety per cent of the normal cells were round on all collagens. Adenocarcinoma RERF-LC-MS and VMRC-LCD cell lines and squamous cell carcinoma VMRC-LCP cell line, which metastasize weakly after intrasplenic transplantation in nude mice, spread relatively poorly. Adenocarcinoma, A549 and SK-LU-1 and squamous cell carcinoma Calu-1 cell lines, which were highly metastatic to liver, spread well. Adenocarcinoma ABC-1 cell line, which is moderately metastatic to liver in nude mice, spread moderately. On type III collagen, three adenocarcinoma cell lines (A549, ABC-1 and VMRC-LCD) gradually started to contract after initial spreading and became round at 24 h. These results suggest that there may be a correlation between the degree of malignancy of human lung cancer cells and their spreading ability on collagen substrata, and that the cell spreading ability may be regulated by type III collagen in some lung cancer cells.

The ultrastructural localization of H2O2production in suspended polymorphonuclear leukocytes (PMN) stimulated with particles was studied using CeCI3technique. PMN stimulated with opsonized zymosan or polystylene latex with or without IgG were incubated in 0.1 MTris-maleate buffer with 1 mMCeCI3and 10 mMaminotriazole. Cells were then fixed and embedded in a resin for electron microscopy. The reaction product of cerium perhydroxide was observed on the phagosomal membranes and on the areas of the plasma membrane engulfing the particles. Catalase or ferricytochrome-c decreased the deposits. p-Benzoquinone (O2scavenger) inhibited the formation of the deposits, but KCN or NaN3enhanced it. Pretreatment with p-diazobenzenesulfonic acid inhibited the reaction. In some PMN pretreated with cytochalasin-B, cellular aggregation was observed. The H2O2production in these cells were observed on the membrane adherent to the particles and on the contact surface of the membrane of adjoining PMN. The plasma membrane was damaged and the electron-dense product was diffused into the cytoplasm. These results clearly show that H2O2production is initiated at the area of the plasma membrane adherent to the particles and that H2O2is released before the completion of phagocytosis.

The peroxidatic activity of rat liver catalase was demonstrated by histochemical staining with 3,3'-diaminobenzidine as hydrogen donor. The activity was so weak that its location was hard to identify in formaldehyde-fixed cells, although high catalatic activity was present, as evidenced by the production of bubbles upon the addition of hydrogen peroxide to the incubation medium. Pretreatment of fixed sections for 60 min at 37°C with formamide, urea or trypsin enhanced the peroxidatic activity significantly. The reaction granules scattered throughout the cytoplasm of the parenchymal cells probably correspond to the peroxisomes.

ABSTRACTAnalogs of furanonaphthoquinone (FNQ) from Tecoma ipeMart had MICs ranging from 1.56 to 25 μg/ml against gram-positive bacteria. FNQ showed significantly lower MICs against methicillin-resistant Staphylococcus aureusthan against methicillin-sensitive S. aureus. FNQ inhibitedHelicobacter pyloriwith an MIC of 0.1 μg/ml. Fungi, including pathogenic species, were sensitive to FNQ with MICs similar to those of amphotericin B.

The effect of the novel anticancer 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) on human osteosarcoma cell lines (HuO9 and HuO9N2) was investigated. The IC50 values of FNQ3 were 5.95 μM for HuO9 and 3.86 μM for HuO9N2, while that for normal fibroblasts (WI-38 cell line) was 35.8 μM. The selectivity in antitumour activity which was estimated from the IC50 ratio of normal fibroblasts to tumour cells was 6.0 and 9.3 fold for HuO9 and HuO9N2, respectively. FNQ3 at 23.6 μM selectively injured mitochondria of HuO9 cells starting at 36 h and HuO9N2 cells at 24 h, whereas WI-38 cells were unaffected even after 72 h. These results demonstrated that FNQ3 was selectively toxic to the mitochondria of osteosarcoma cells similar to carcinoma cells (Pan et al. (1997) J. Electron Microsc. 46: 181), in comparison to normal cells.

The generation of oxygen free radicals was investigated using cytochemistry and its energy-filtering transmission electron microscopy in reference to the toxic mediator for the herbicide paraquat. When isolated intact mitochondria from rat livers were incubated in a medium containing paraquat and NADH, a mitochondrial NADH-quinone oxidoreductase activity generated superoxide anions to cause the destruction of mitochondria which resulted in cell death. The superoxide anions were immediately converted into hydrogen peroxide, which then formed cerium perhydroxide deposits in the presence of cerium ions and precipitated on the outer surface of the mitochondrial outer membrane. This localization was also specifically identified by energy spectralimaging and image-electron energy loss spectral analyses. Precipitation reaction was scavenged by the addition of either cytochrome c or catalase and inhibited by dicoumarol (an inhibitor of NAD(P)H-quinone oxidoreductases). These cytochemical energy-filtering transmission electron microscopic results indicated that paraquat generated free radicals from the outer membrane of mitochondria.

When human polymorphonuclear leukocytes (PMN) were attached to glass coverslips, cells always spread and formed reactive oxygen species prior to any experimental stimulation. To avoid this, a polyvinylidine chloride film was used as an inactive substance to place the cells. Cells engaged in phagocytosis on the film exhibited a specific H2O2-mediated luminol chemiluminescence (LCL) at the cell-particle interface; the cells stimulated with 12-O-tetradecanoylphorbol-13-acetate became aggregated and the LCL was observed at the cell-cell contact. These results corresponded well with those obtained by an electron microscopic H2O2 method.

The intracellular ultrastructural changes induced by the new antitumour agent 2-methylnaphtho [2,3-b]furan-4,9-dione (FNQ3) were investigated in human cervical cancer HeLa cells in comparison with normal cervix cells. The normal cells were isolated from cervixes surgically resected from myoma patients and were keratin positive. FNQ3 at 3–5 μg ml-1 selectively damaged the HeLa cell mitochondria followed by rough-surfaced endoplasmic reticulurn resulting in cell death. In contrast, normal cells remained unaffected at that concentration but were damaged by 20 μg ml-1 FNQ3. The FNQ3-induced tumour cell toxicity was inhibited 52% and 36% by trolox and a water-soluble fraction of the antioxidative substance AOB, respectively. The results indicated that FNQ3 is selectively toxic to HeLa cells at approximately eight times that of normal cells in terms of mitochondrial alteration and free radical formation.

A high incidence of cytoplasmic pore complex similar to Maul's (Exp. Cell Res., 104: 233, 1977) was found in the cultured myeloblastic leukemic cells, Ml line, clone 264-2. The pore complex was composed of one to four pores perforating a single cisterna of the endoplasmic reticulum. When the blastic M1 cells were cultured in the presence of a conditioned medium, they successfully differentiated into macrophages. This resulted in a loss of malignancy, and simultaneously the pore complex changed to the stacked annulate lamellae. The well-developed annulate lamellae were also generally found in the cytoplasm of the cloned macrophages. Mm-1 subline was derived from the M1 line by spontaneous differentiation, and the cells appeared less transplantable to animals. These results indicate that myeloblastic M1 cells possess the simple pore complex, which may develop into the annulate lamellae during cell differentiation into macrophages.

Epoxy resin-embedded metal standards were prepared for accurate quantitative X-ray microanalysis of cerium, nickel, and cobalt. Acetyl acetonate compounds of these metals, which were dissolved in Quetol 651 epoxy resin, up to 204 mM, were used as metal standards. Microanalysis of the standards gave straight calibration lines between X-ray counts and concentrations. These standards are useful for the quantitative microanalysis of cytochemically reacted specimens containing those metal deposits.

Cytochemically demonstrated cytochrome oxidase activities in mitochondria of rat pulmonary alveolar epithelial cells were quantitatively compared. As the standardized procedures, lungs were fixed for 10 mm at 4°C with 2% pure glutaraldehyde, and incubated for different times at 37°C in a medium containing 1.0 mg/ml 3,3'-diaminobenzidine-4HCl, 1 mg/ml cytochrome c, 0.1 mg/ml catalase, 7% sucrose, anb 0.1 M phosphate buffer, pH 7.4. Electron micrographs were then odtained, and the densities of the reaction deposits in the mitochondrial intermembrane-intracristal space were measured in an image analyzer, and were plotted in terms of time in minutes. The initial velocity (maximal rate) of the activity expressed as deposit accumulation rate (% area/60 min) filling the mitochondrial intermembrane-intracristal space of Type I, Type II and Type III cells was 40, 130 and 9.3, respectively. These results indicated that mitochondria are varied in the intensity of cytochrome oxidase activity among 3 types of pulmonary alveolar epithelial cells, and that the image-analyzing measurement of the accumulation rate of reaction product may be useful for quantitative analysis of enzyme activity, in particular, in the cells which are difficult to isolate from complex tissues.

Image analysis quantitation of cytochrome oxidase activity, cytochemicaUy visualized in mitochondria, and the comparison between the activities of pulmonary alveolar Type I and Type II cells were performed. Normal lungs of rats were fixed with 2% glutaraldehyde for 10 min at 4°C and then small blocks were incubated for 60 min at 37°C in a medium containing 1.0 mg/ml (2.8×l0−4M) 3,3′-diamiDobcnzidlnc-tctrahydrochloride, 1 mg/ml cytochrome c, 0.1 mg/ml catalase, 7% sucrose and 0.1 M phosphate buffer at pH 7.4. The intermembrane space, between the outer and inner membranes, and intracristal space of Type II cell mitochondria were filled with deposits whose mean volume was 0.047 μm3. Type I cell mitochondria were smaller in size than those of Type II cells, but because reaction deposits were not fully accumulated within the spaces the resulting net deposit volume was 0.00182 μm3 If the intermembrane spaces of Type I cell mitochondria had been thoroughly filled by the deposits, the mean volume would have been 0.00248 μm3. These results indicate that stereoscopic image analysis of the reaction product is appropriate in the quantitation of cytochemicaUy visualized enzyme activity, and furthermore suggest that Type I cells may be rather deficient in cytochrome oxidase activity.

The cytochrome oxidase activity of rat dorsal root ganglion cells was cytochemically and quantitatively measured. The 100 μm thick tissue slices fixed for 10 min were incubated in a DAB medium for 0, 30, 60, and 90 min at 37°C, and electron-dense deposit areas within the mitochondrial intermembrane-intracristal spaces were measured with a computer-controlled image analyzer. The activity was expressed as deposit accumulation rate filling the mitochondrial space, (1 unit corresponds to the deposit filling 100% of the mitochondrial space per hour). Three different activities of the enzyme in either large pale cells or small dark cells of sensory neurons could be distinguished, based on the quantitative analysis, as large cells with intense (0.83 units), intermediate (0.38), and weak activity (0.22), and small cells with the same degrees of activity (1.04, 0.35, and 0.11, respectively). The results indicate that accumulation rate measurements of reaction product may be useful to quantitatively present the cytochrome oxidase reactivity of mitochondria, and that the degree of enzyme activity may contribute to the identification of functional differences in sensory neurons.

Ultrastructural and cytochemical changes in rat livers after paraquat administration were Investigated. A single intraperitoneal injection of 150 mg/ kg paraquat dichioride caused primary toxicological changes in the hepatocyte mitochondria., such as decrease in the electron density of the matrices, disintegration of crlstae, disappearance of intramitocliondrial granules, and irreversible swelling. Ca-ATPase activity in those mitochondria disappeared. In vitro experiments, the activity of fixed normal livers was significantly inhibited by 10 mM paraquat. No significant effect on the plasma membrane Ca-ATPase activity was observed. These results suggest that the mitochondrial damage caused by paraquat may be related to the possible inhibition of their Ca-ATPase activity, resulting in cell injury.

The ultrastructural localization of uterine peroxidase activity in the human endometrium was studied. Very weak peroxidase activity was present in a few surface nonciliated cells with very weak activity in the early proliferative phase; the activity was enhanced and distributed throughout the nonciliated cells during the late proliferative to secretory phase. The activity was observed in the endoplasmic reticulum and nuclear envelope but not in the secretory granules and Golgi apparatus. The surface ciliated cells and all glandular cells were entirely devoid of the activity throughout the menstrual cycle. Nucleolar channel system was transiently formed in the surface and glandular nonciliated cells in the postovulatory phase. The channels of the system in the surface cells showed intense peroxidase activity, but those in the glandular cells were negative. These results indicate that the human uterine peroxidase activity appears only in the superficial nonciliated cells and changes in its intensity depending on the menstrual cycle, and that the nucleolar channel system is closely related with the nuclear envelope showing peroxidase activity.

The platinum-diaminobenzidine (Pt-DAB) reaction, which yields a black, electron-dense, insoluble, Pt-containing reaction product at the active site of cytochrome oxidase without postosmification, has been developed. This reaction permits us to quantitate the oxidase activity in an individual mitochondrion or in its constituent part by the energy dispersive X-ray analysis (EDAX), based on the platinum atoms incorporated in the product.

Although the intracellular toxic sites of paraquat, a herbicide toxic to human bodies, have remained unclear for a long time, we recently demonstrated paraquat-induced mitochondrial injury in rat lung and liver in vivo. In the present study, cultured human lung cells (A549 adenocarcinoma cell line) which received 0.15 mM paraquat (equivalent to 40 mg/kg i.v.) showed selective mitochondrial breakage at 6–24 hr and died at 36–48 hr. These results suggest that mitochondria are the initial toxic site of paraquat in vitro as well as in vivo in contrast to the previously proposed microsome theory.