Sanae Kanno

Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells

It has been suggested that oxidative stress plays a major role in various forms of cell death, including necrosis and apoptosis. We have previously reported that fluoride (NaF) induces apoptosis in HL-60 cells by caspase-3 activation. The main focus of this investigation was to arrive at a possible pathway of the apoptosis induced by NaF upstream of caspase-3, because the mechanism is still unknown. The present study showed that after exposure to NaF, there was an increase in MDA and 4-HNE and a loss of mitochondrial membrane potential (deltaPsi(m)) was also observed in NaF-treated cells. There was a significant increase in cytosolic cytochrome c, which is released from the mitochondria. We have reported a downregulation of Bcl-2 protein in NaF-treated cells. The antioxidants N-acetyl cysteine (NAC), glutathione (GSH) protected the cells from loss of deltaPsi(m), and there was no cytochrome c exit or Bcl-2 downregulation, and we suggest that these antioxidants prevent apoptosis induced by NaF. These results suggested that perhaps NaF induced apoptosis by oxidative stress-induced lipid peroxidation, causing loss of deltaPsi(m), and thereby releasing cytochrome c into the cytosol and further triggering the caspase cascade leading to apoptotic cell death in HL-60 cells.

Multi-walled carbon nanotubes injure the plasma membrane of macrophages

Carbon nanotubes (CNTs) are emerging nanotechnology materials which are likely to be mass-produced in the near future. However, prior to mass-production, certain health-related concerns should first be addressed. For example, when inhaled, the thin-fibrous shape and the biopersistent characteristics of CNTs may cause pulmonary diseases, in a manner similar to asbestos. In the present study, mouse macrophages (J774.1) were exposed to highly-purified multi-walled CNTs (MWCNTs, 67 nm) or to UICC crocidolite in order to evaluate the toxicity of these nano-size fibers. The cytotoxicity of MWCNTs was found to be higher than that of crocidolite. The toxic effect of MWCNTs was not affected by N-acetylcysteine, an antioxidant, or buthionine sulfoximine, a glutathione synthesis inhibitor. cDNA microarray analyses suggested that the cytotoxicity of MWCNTs could not be explained satisfactorily by either an increase or decrease of gene expression, although mRNA levels of some cytokines were slightly increased by MWCNTs. Moreover, MWCNTs did not significantly activate either MAP kinases such as ERK, JNK and p38, nor common apoptosis pathways such as caspase 3 and PARP. Electron microscopic studies indicated that MWCNTs associate with the plasma membrane of macrophages and disrupt the integrity of the membrane. Several proteins were found to adsorb onto MWCNTs when MWCNT-exposed macrophages were gently lysed. One of these proteins was macrophage receptor with collagenous structure (MARCO). MARCO-transfected CHO-K1 cells associated with MWCNTs more rapidly than mock-transfected cells. These results indicate that MWCNTs probably trigger cytotoxic effects in phagocytotic cells by reacting with MARCO on the plasma membrane and rupturing the plasma membrane.

Extrapulmonary translocation of intratracheally instilled fine and ultrafine particles via direct and alveolar macrophage-associated routes

Translocation of inhaled ultrafine particles from the lungs into the blood may impair cardiovascular function. We administered ultrafine (20-nm) and fine (200-nm) gold colloid or fluorescein-labeled polystyrene particles to mice intratracheally and examined their localization in the lung and extrapulmonary organs. Fifteen minutes after instillation, dispersed and agglomerated 20-nm gold colloid particles were observed on the surface of endothelial cells, on the alveolar surface, in endocytotic vesicles of alveolar epithelial cells, and in the basement membrane of the lung. A small but noteworthy amount of gold was detected in the liver, kidney, spleen, and heart by inductively coupled plasma-mass spectrometry. After administration of 20- or 200-nm fluorescent particles, free particles were detected infrequently in blood vessels, on the endocardial surface, and in the kidney and liver only in the mice that received 20-nm particles, whereas phagocytes containing 20- or 200-nm particles were found in the extrapulmonary tissues. Fluorescent particle-laden alveolar macrophages administered intratracheally translocated from alveoli to extrapulmonary organs via the blood circulation. Thus, small amounts of ultrafine particles are transported across the alveolar wall into the blood circulation via endocytotic pathways, but particle-laden alveolar macrophages translocate both ultrafine and fine particles from the lungs to the extrapulmonary organs.

Subchronic Exposure to Arsenic Through Drinking Water Alters Expression of Cancer-Related Genes in Rat Liver

Although arsenic exposure causes liver disease and/or hepatoma, little is known about molecular mechanisms of arsenic-induced liver toxicity or carcinogenesis. We investigated the effects of arsenic on expression of cancer-related genes in a rat liver following subchronic exposure to sodium arsenate (1, 10, 100 ppm in drinking water), by using real-time quantitative RT-PCR and immunohistochemical analyses. Arsenic accumulated in the rat liver dose-dependently and caused hepatic histopathological changes, such as disruption of hepatic cords, sinusoidal dilation, and fatty infiltration. A 1-month exposure to arsenic significantly increased hepatic mRNA levels of cyclin D1 (10 ppm), ILK (1 ppm), and p27Kip1 (10 ppm), whereas it reduced mRNA levels of PTEN (1 ppm) and β-catenin (100 ppm). In contrast, a 4-month arsenic exposure showed increased mRNA expression of cyclin D1 (100 ppm), ILK (1 ppm), and p27Kip1 (1 and 10 ppm), and decreased expression of both PTEN and β-catenin at all 3 doses. An immunohistochemical study revealed that each protein expression accords closely with each gene expression of mRNA level. In conclusion, subchronic exposure to inorganic arsenate caused pathological changes and altered expression of cyclin D1, p27Kip1, ILK, PTEN, and β-catenin in the liver. This implies that arsenic liver toxicity involves disturbances of some cancer-related molecules.

RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation.

RGD motif-containing peptides have been used in various studies of cell adhesion and growth. We report that RGD triggered apoptosis at a concentration of 1 mmol/L, whereas RAD-containing peptides failed to induce apoptosis in HL-60 cells. RGD-treated cells revealed internucleosomal DNA fragmentation. Western blot reveals caspase-3 activation in RGD peptide-treated cells. A caspase-3 inhibitor z-VAD-FMK completely blocked the apoptosis, but a caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-2 inhibitor (z-VDVAD-FMK) did not block the apoptosis, suggesting that caspase-3 might have a critical role in the execution process of apoptosis induced by RGD. RGD peptides have been used extensively to inhibit tumor metastasis. Our results should help in further understanding the RGD peptide-induced apoptosis, which is important since RGD peptides have a potential role in therapies of the future.

Selective removal of copper bound to metallothionein in the liver of LEC rats by tetrathiomolybdate.

LEC rats (Long-Evans with a cinnamon-like coat color) have a genetical defect in Cu metabolism. Male LEC rats aged 10 weeks were injected ip with TTM at a dose of 5 or 10 mg/kg body weight for 8 consecutive days and killed one day after the last injection. Cu that had accumulated in the liver at a concentration of 251 micrograms/g liver was decreased to 82.7 or 74.3 micrograms/g liver respectively, by the treatment. Although most of Cu was bound to MT as a soluble form before TTM treatment, the metal remaining in the liver after the treatment was present almost exclusively in the non-soluble fraction. Zinc (Zn) present, bound to MT before the treatment, was also partly removed from the liver by TTM, and the Zn remaining in the liver after the treatment was revealed to be bound to MT (Zn-MT) by high performance liquid chromatography-atomic absorption spectrophotometry. Iron (Fe) in the liver was not affected by TTM treatment. Cu in the kidneys and spleen increased by TTM treatment, while Zn and iron were not affected. Treatment of LEC rats with severe jaundice effectively cured the animals from otherwise lethal signs by only two ip injections of TTM at a dose of 10 mg/kg body weight.

Localization of zinc after in vitro mineralization in osteoblastic cells

The present study was designed to investigate the incorporation of zinc (Zn) into cultured UMR-106 osteoblasts in response to mineralization caused by the addition of β-glycerophosphate. As a result of the induced mineralization, the contents of calcium (Ca), phosphorus (P), and Zn in the monolayer increased, whereas the magnesium (Mg) content did not change. The activity of alkaline phosphatase (ALP) also increased during the process.The zinc distribution in the cell monolayer was studied using Zinquin, a fluorescent zinc ion chelator. The double fluorescent labeling with Zinquin and calcein revealed that zinc was localized both as intracellular vesicles and extracellular clusters, whereas calcium was colocalized with extracellular zinc. These results suggest that zinc is involved in the mineralization process of UMR-106 cells.

Mechanisms for removal of copper from metallothionein by tetrathiomolybdate

Mechanisms for removal of copper (Cu) from metallothionein (MT) by tetrathiomolybdate (TTM) were examined in vivo and in vitro using the LEC rat, which accumulates Cu as MT owing to the hereditary disorder of this strain. In our previous experiment, repeated intraperitoneal injections of TTM were shown to remove approximately two-thirds of the Cu from the liver, and the Cu remaining in the liver changed from soluble MT-bound forms to nonsoluble unidentified forms. The present single intravenous injection of TTM changed only part of the distribution in the soluble fraction, and dimeric MT was assumed to be formed. The liver supernatant was treated in vitro with high and low doses of TTM. The former treatment removed all Cu bound to MT and the Cu distributed to high molecular weight proteins, while the latter treatment produced dimeric MT. The results indicate that Cu accumulated as MT can be removed differently by TTM according to its relative dose both in vivo and in vitro. Excess TTM removes Cu completely from MT, leaving apothionein, while lesser TTM than Cu removes Cu incompletely, leaving MT with unoccupied sulfhydryl groups which coordinate with Cu intermolecularly to form dimeric and polymeric MT through the -S-Cu-S- bridge.

Enhanced synthesis of metallothionein as a possible cause of abnormal copper accumulation in LEC rats

Long-Evans rats with a cinnamon-like coat color (LEC) is an inbred strain accumulating copper (Cu) in the liver abnormally and showing spontaneous hepatitis and hepatoma. The present study was intended to clarify how Cu accumulates in the LEC rat liver. For this purpose, the distribution profiles of Cu and zinc (Zn) and the inducibility of metallothionein (MT) synthesis were examined in the liver between Cu-loaded Long Evans agouti (LEA, the original strain of LEC) rats and were compared with those in control LEC rats. LEA rats (female, five weeks old) were injected subcutaneously with CuCl2 daily at a dose of 3 mg Cu/kg body weight for 2, 4, 6, and 9 days. The concentration of Cu (124 micrograms/g) accumulated in the LEA rat liver after four injections was comparable to that in control LEC rats. Only 20% of Cu in the liver of LEA rats was recovered in the supernatant fraction in the form of MT, while Cu in the LEC rat liver (113 micrograms/g) was recovered mostly in the supernatant fraction, and was bound to MT. Although the increased concentration of Cu in the LEA rat liver was further elevated after additional injections of Cu, the amount of MT did not increase further. The MT mRNA content in the LEA rat liver remained lower than that of LEC rats even after further injections of Cu. Therefore, the present results suggest that LEC rats can accumulate Cu at a high concentration in the liver because of their extremely high inducibility of MT.

Inflammatory responses of rat alveolar macrophages following exposure to fluoride

Abstract Inhalation exposure to fluoride compounds has been associated with respiratory failure. We have addressed effects of fluoride on alveolar macrophages and lung responses to intratracheally (i.t.) instilled fluoride in rats. I.t. instillation of fluoride at doses of 200 and 400 μg F/rat caused significant polymorphonuclear leukocyte (PMN) infiltration in the rat lung at 20 h post-administration, while 100 μg fluoride did not recruit a significant number of PMNs in the alveolar space. Total RNA was extracted from the lung lavage cells obtained from 5 h post i.t. instillation and mRNA levels of chemokines and proinflammatory cytokines were semi-quantitatively evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). I.t. instillation of fluoride significantly enhanced mRNA expression of cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory proteins-1α and -2. Fluoride-induced augmentation in IL-1β mRNA expression was also examined by Northern hybridization following in vitro exposure of alveolar macrophages to fluoride. However, the enhancement of IL-1β mRNA expression following in vitro exposure to fluoride was observed only at 500 μM, a dose higher than the 50% lethal concentration (LC50). Non-specific adhesion of alveolar macrophages to the plastic dish was significantly increased following in vitro exposure to fluoride. The fluoride-induced non-specific adhesion was significantly reduced by anti-CD18, suggesting that β2 integrin played a role in the increase of adherence. Those results suggest that fluoride activates alveolar macrophages, enhances the production of chemokines and proinflammatory cytokines, and causes PMN infiltration in the lung.