Neutrophil pyroptosis mediates pathology of P. aeruginosa lung infection in the absence of the NADPH oxidase NOX2.

Nod-like receptor family, CARD domain-containing 4 (NLRC4) inflammasome activation is required for efficient clearance of intracellular pathogens through caspsase-1-dependent pyroptosis in macrophages. Although neutrophils have a critical role in protection from Pseudomonas aeruginosa infection, the mechanisms regulating inflammasome-mediated pyroptosis in neutrophils and its physiological role are largely unknown. We sought to determine the specific mechanisms regulating neutrophil pyroptosis in P. aeruginosa strain PAO1 (PAO1) lung infection and to identify the pathological role of this process. Nox2-/- models with reduced neutrophil antibacterial activity exhibited increased neutrophil pyroptosis, which was mediated by flagellin, a pathogenic PAO1 component. We also demonstrate that PAO1-induced pyroptosis depended on NLRC4 and Toll-like receptor 5 (TLR5) in neutrophils generated from Nlrc4-/- or Tlr5-/- mice. Our study reveals previously unknown mechanisms and physiological role of neutrophil pyroptosis during P. aeruginosa lung infection. Furthermore, our findings regarding neutrophil pyroptosis in the context of neutrophil dysfunction may explain the causes of acute and/or chronic infectious diseases discovered in immune-compromised patients.

Octanal-induced inflammatory responses in cells relevant for lung toxicity: expression and release of cytokines in A549 human alveolar cells.

Inhalation is an important route of aldehyde exposure, and lung is one of the main targets of aldehyde toxicity. Octanal is distributed ubiquitously in the environment and is a component of indoor air pollutants. We investigated whether octanal exposure enhances the inflammatory response in the human respiratory system by increasing the expression and release of cytokines and chemokines. The effect of octanal in transcriptomic modulation was assessed in the human alveolar epithelial cell line A549 using oligonucleotide arrays. We identified a set of genes differentially expressed upon octanal exposure that may be useful for monitoring octanal pulmonary toxicity. These genes were classified according to the Gene Ontology functional category and Kyoto Encyclopedia of Genes and Genomes analysis to explore the biological processes related to octanal-induced pulmonary toxicity. The results show that octanal affects the expression of several chemokines and inflammatory cytokines and increases the levels of interleukin 6 (IL-6) and IL-8 released. In conclusion, octanal exposure modulates the expression of cytokines and chemokines important in the development of lung injury and disease. This suggests that inflammation contributes to octanal-induced lung damage and that the inflammatory genes expressed should be studied in detail, thereby laying the groundwork for future biomonitoring studies.

Low frequency mutation of the Ephrin receptor A3 gene in hepatocellular carcinoma.

EphA3 is a component of the Eph/ephrin tyrosine kinase system, which participates in vasculature development. This receptor/ligand system is associated with various signaling pathways related to cell growth and viability, cytoskeletal organization, cell migration, and anti-apoptosis. Accumulated evidence suggests that aberrant regulation of EphA3 and its genetic alterations are implicated in the development and progression of various cancers. However, despite a high incidence of EphA3 over-expression, no such investigation has been performed in hepatocellular carcinoma. Thus, we investigated genetic alterations of the EphA3 gene in 73 cases of hepatocellular carcinoma by single-strand conformational polymorphism and sequencing. One novel D219V missense mutation was found in the extracellular domain of EphA3, and two genetic alterations in the intracellular sterile-alpha-motif (SAM) domain of EphA3 appeared to be polymorphisms. Although the functional assessments of this mutant are incomplete, it is believed that this novel EphA3 mutation may contribute to the development of hepatocellular carcinoma.

Mutational analysis of JAK1 gene in human hepatocellular carcinoma.

UNLABELLED: The Janus kinase 1 (JAK1) gene encodes a cytoplasmic tyrosine kinase that is noncovalently associated with a variety of cytokine receptors and plays a nonredundant role in cell proliferation, survival, and differentiation. The mutated forms of JAK1 often altered the activation of JAK1 and then changed the activation of JAK1/STAT pathways, and this may contribute to cancer development and progression. Thus, to investigate whether genetic mutations of JAK1 gene are associated in hepatocellular carcinoma (HCC) progression, we analyzed genetic alterations of JAK1 gene in 84 human HCCs by single-strand conformational polymorphism (SSCP) and direct sequencing. Of 24 exons of JAK1 gene, 12 exons were previously reported to have mutations, we searched genetic alteration of JAK1 in these exons. Overall, one missense mutation (1.2%) was found. In addition, 12 cases (14%) were found to have single nucleotide polymorphism (14%) in exon 14. Taken together, we found one novel missense mutation of JAK1 gene in hepatocellular carcinomas with some polymorphisms. Although the functional assessment of this novel mutant remains to be completed, JAK1 mutation may contribute to the tumor development in liver cancer. KEYWORDS: JAK1 gene, hepatocellular carcinoma, mutation.

The effect of methyl methanesulfonate (MMS)-induced excision repair on p53-dependent apoptosis in human lymphoid cells.

The tumor suppressor p53 product has been shown to play an important role in preventing carcinogenesis by at least two different mechanisms, by evoking cell cycle arrest and eliciting DNA repair on one hand, or by eliminating damaged cells by induction of apoptosis on the other hand. As a first step toward understanding the relationship between protective responses and apoptosis after genotoxic stress, we examined the effect of DNA strand breaks generated from repair processes in respect to acute cellular responses against DNA damage, and on p53-dependent apoptosis in human lymphoid cells. We used two isogenic cell lines, TK6 harboring wild-type p53, and WI-L2-NS, which carries a mutant p53. A significant difference in sensitivity was observed at 50 microg/ml methyl methane-sulfonate (MMS) between the two cell lines used. In addition, a clear p53-mediated contribution to apoptosis in MMS-induced cell death was observed. However, we did not observe any differences in repair of MMS-lesions, as determined by comet assay, between the two cell lines. These data suggest that the differences in apoptosis induction in the two lines are not a reflection of differences in strand-break frequency or repair capacity.

Salsolinol, a naturally occurring tetrahydroisoquinoline alkaloid, induces DNA damage and chromosomal aberrations in cultured Chinese hamster lung fibroblast cells.

Salsolinol (SAL) is a tetrahydroisoquinoline neurotoxin that has been speculated to contribute to pathophysiology of Parkinson's disease and chronic alcoholism. The compound is also found in certain beverages and food stuffs, including soy sauce, beer and bananas. Despite potential human exposure to SAL and its endogenous formation, little is known about the genotoxic or carcinogenic potential of this substance. In the present investigation, SAL induced DNA damage in cultured Chinese hamster lung (CHL) fibroblasts as assessed by single cell gel electrophoresis (Comet). CHL cells treated with SAL also exhibited higher frequencies of chromosomal aberrations than did vehicle-treated controls. Our recent study has revealed that SAL in combination with Cu(II) causes the strand scission in phiX174 supercoiled DNA [Neurosci. Lett. 238 (1997) 95]. In line with this notion, addition of cupric ion potentiated the DNA damaging and clastogenic activity of SAL. Antioxidant vitamins, such as Vitamin C and Vitamin E, and reduced glutathione inhibited clastogenicity of SAL, suggesting the involvement of reactive oxygen species (ROS) in SAL-induced DNA damage and genotoxicity in CHL cells.

Identification of propentofylline metabolites in rats by gas chromatography/mass spectrometry.

Propentofylline (PPF, 3-methyl-1-(5-oxohexyl)-7-propylxanthine) has been reported to be a compound for treatment of both vascular dementia and dementia of the Alzheimer type. The short half-life (about 15 min) of PPF at the terminal elimination phase and poor bioavailability after oral administration of PPF to rabbits (Kim et al., 1992) suggest in part that this drug takes the extensive first-pass metabolism in the liver. In addition, the metabolic pathway for PPF remains unclear. The objective of this experiment is to identify urinary metabolites of PPF in rats. For the identification of the metabolites, rat urine was collected after oral administration of 100 mg/kg PPF. PPF metabolite, 3-methyl-1-(5-hydroxyhexyl)-7-propylxanthine, was synthesized and confirmed by gas chromatography/mass spectroscopy (GC/MS) and 1H nuclear magnetic resonance spectroscopy. The urinary metabolites of PPF were extracted with diethyl ether and identified by electron impact and chemical ionization GC/MS. One urinary metabolite was confirmed to be 3-methyl-1-(5-hydroxyhexyl)-7-propylxanthine by synthesized authentic compound. Several metabolites of monohydroxy- and dihydroxy-PPF were identified based on mass fragmentation of both intact and trimethylsilylated derivatives of PPF metabolites and the novel structure of these metabolites is suggested based on mass spectra.

Isolation of csm1 encoding a class V chitin synthase with a myosin motor-like domain from the rice blast fungus, Pyricularia oryzae.

A gene encoding a chitin synthase with a myosin motor-like domain (csm1) was isolated from Pyricularia oryzae using a PCR fragment amplified from a fungal chitin synthase conserved region. The deduced amino acid sequence of csm1 is homologous to that of CsmA of Aspergillus nidulans (65% identity). The putative gene product of csm1 is consisted of the myosin motor-like domain and a chitin synthase domain as in A. nidulans csmA. The chitin synthase domain of its C-terminus was also homologous to Aspergillus fumigatus ChsE (61.4% identity) and Ustilago maydis Chs6 (48.6% identity) that encode class V chitin synthases. Northern analysis demonstrated that the csm1 was expressed throughout the mycelial growth of P. oryzae. This is the first report on the isolation of the gene encoding a class V chitin synthase with the myosin motor-like domain from P. oryzae.

Mild hyperthermia-induced apoptosis is dependent on p53 in human lymphoid cells.

Mild hyperthermia is known to enhance apoptosis. The p53 tumor-suppressor gene product has been shown to function in apoptosis in response to genotoxic stress. However, there is little information regarding the mechanism of p53-dependent apoptosis induced by heat stress. In present study, a p53 contribution in mild hyperthermia-induced apoptosis was investigated in human lymphoid system. After 30-minute exposure at 44 degrees C, the accumulation of p53 protein was clearly observed in TK6 and ML-1 cells. Using comet assay, the more significant sensitivity to hyperthermic apoptosis was found in TK6 (wild-type p53) than in WI-L2-NS (mutated in p53). Furthermore, the significantly rapid shifting from early apoptotic phase to late apoptotic was observed in heat-induced p53 TK6 cells. These findings suggest that p53-dependent apoptosis is efficaciously induced by mild hyperthermia as non-genotoxic stress in human lymphoid system.

Effect of p53 tumor suppressor on nucleotide excision repair in human colon carcinoma cells treated with 4-nitroquinoline 1-oxide.

In probing the mechanism of nucleotide excision repair (NER) in response to 4-nitroquinoline 1-oxide (4NQO)-induced DNA damage, the effect of p53 tumor suppressor was investigated. The effect of p53 protein on the repair of damaged DNA was examined by comet assay. Expression of p53 and p21(Waf1/Cip1) proteins was measured by the Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry, respectively. Compared to RKO cells having the wild-type p53 gene, increased cytotoxicity by 4NQO was observed in RKOmp53 cells with a mutation in p53 protein. DNA single strand breaks (SSB), indicative of the DNA repair, were considerably increased in 4NQO-treated RKO cells. Also, the expression of p53 and p21 proteins was significantly increased in 4NQO-treated RKO cells. In RKOmp53 cells, no effect of 4NQO on p21 expression was observed. Our findings suggest that 4NQO-induced NER is p53-dependent and involves up-regulation of its downstream regulator, p21(Waf1/Cip1) proteins.

Pharmacokinetics of propentofylline and the quantitation of its metabolite hydroxypropentofylline in human volunteers.

Propentofylline (PPF, 3-methyl-1-(5-oxohexyl)-7-propylxanthine) has been reported to be effective for the treatment of both vascular dementia and dementia of the Alzheimer type. The pharmacological effects of PPF may be exerted via the stimulation of nerve growth factor, increased cerebral blood flow, and inhibition of adenosine uptake. The objectives of this experiment are to determine the kinetic behavior of PPF, to identify, and to quantify its metabolite in human. Blood samples were obtained from human volunteers following oral administration of 200 mg of PPF tablets. For the identification and quantification of the metabolite, 3-methyl-1-(5-hydroxyhexyl)-7-propylxanthine (PPFOH), PPFOH was synthesized and identified by gas chromatography/mass spectroscopy (GC/MS) and 1H-nuclear magnetic resonance spectroscopy. The molecular weight of synthesized metabolite is 308 dalton. The PPF and PPFOH in plasma were extracted with diethyl ether and identified by electron impact GC/MS. The plasma concentrations of PPF and PPFOH were determined by gas chromatography/nitrogen phosphorus detector in plasma and their pharmacokinetic parameters were determined. The mean half-life of PPF was 0.74 hr. The areas under the curve (AUCs) of PPF and PPFOH were 508 and 460 ng.hr/ml, respectively. Cmax of PPF was about 828.4 ng/ml and the peak concentration was achieved at about 2.2 hr (Tmax). These results indicate that PPF is rapidly disappeared from blood due to extensive metabolism into PPFOH.

Polymorphism of angiotensin converting enzyme gene is associated with circulating levels of plasminogen activator inhibitor-1.

The deletion (D) allele of the insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene is strongly associated with an increased level of circulating ACE. The ACE gene polymorphism may influence the production of angiotensin II (Ang II). It has been shown that Ang II modulates fibrinolysis, that is, Ang II increases plasminogen activator inhibitor-1 (PAI-1) mRNA and plasma PAI-1 levels in vitro and in vivo. Considered together, we tested the hypothesis that the deletion allele of the ACE gene might be associated with increased levels of PAI-1. We related the ACE genotype to PAI-1 antigen levels in 603 men and 221 women attending a routine health screening. As a whole, the plasma PAI-1 level was not strongly associated with ACE genotype. Since the PAI-1 level was significantly influenced by well-known risk factors for coronary artery disease (CAD), we further analyzed the data after excluding subjects with major cardiovascular risk factors. In low-risk male subjects, the DD genotype had significantly higher levels of plasma PAI-1 (DD: 20.3 +/- 2.2; DI: 13.9 +/- 1.1; II: 13.6 +/- 1.3 ng/mL, P = .010 by ANOVA). In low-risk female subjects, the DD genotype showed a tendency to a high level of plasma PAI-1 without statistical significance. When analysis was restricted to postmenopausal women (age > or = 55 or FSH > or = 35 ng/mL), the DD genotype showed a significantly higher level of PAI-1 than subjects with the DI and II genotypes (27.7 +/- 6.2 versus 15.6 +/- 1.8 ng/mL, P = .028). The DD polymorphism of the ACE gene is associated with high PAI-1 levels in male and possibly in postmenopausal female subjects who have lower conventional cardiovascular risk factors. These results suggest that the increased ACE activity caused by DD polymorphism may play an important role in elevating the level of plasma PAI-1. Our data support the notion that the genetic variation of ACE contributes to the balance of the fibrinolytic pathway.

Cardiac tamponade due to a rupture of the coronary arteriovenous aneurysm--a case report.

We experienced an unusual case of cardiac tamponde caused by a rupture of the coronary arteriovenous aneurysm in a 54-year-old woman. The patient was suffered from sudden chest pain and syncope, and was initially managed by pericardiocentesis following an echocardiogram which revealed a massive pericardial effusion with signs of cardiac tamponade. She was referred to our hospital under the impression of aortic dissection with cardiac tamponade. She underwent an emergency operation and was found to have a 2 x 2 cm sized bleeding cystic mass protruding from the proximal anterior descending coronary artery. The aneurysm was excised and the openings connected with the coronary artery and right ventricular outflow tract were closed with sutures from the inside of aneurysm. Subsequent coronary arteriography supported the diagnosis.

Survey of natural occurrence of trichothecene mycotoxins and zearalenone in Korean cereals harvested in 1992 using gas chromatography/mass spectrometry.

For the survey of the natural occurrence of trichothecene mycotoxins, produced by species of fungi imperfecti such as Fusarium and Trichothecium, a sensitive analytical method was developed for the simultaneous detection and quantitation of the major trichothecene mycotoxins, viz. T-2 toxin (T-2), HT-2 toxin (HT-2), nivalenol (NIV), fusarenon-X (F-X), deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-Ac DON), and zearalenone (ZEN), using gas chromatography/mass spectrometry-selected ion monitoring (GC/MS-SIM) mode after trimethyl silyl derivatization. The incidence of NIV and DON in 30 barley samples were 93% and 67%, respectively; the average contents of NIV and DON in positive samples were 390 ng/g (range 40-2038) and 106 ng/g (range 5-361) respectively. In 15 maize samples, the incidences of NIV and DON were 53% and 93% respectively and the average contents were 168 ng/g and 145 ng/g, respectively. These results suggest that NIV and DON were the major contaminating trichothecene mycotoxins in Korean barley and maize samples harvested in 1992.

Fate and distribution of [14C]S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine, an antimutagenic mixed disulfide from disulfiram, in rats.

S-(N,N-Diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC) is a mixed disulfide from disulfiram and N-acetyl-L-cysteine, which possesses putative anticarcinogenic and antimutagenic properties. The present study describes the absorption, distribution, metabolism and excretion of 14C-labeled AC-DDTC in rats. AC-DDTC was well absorbed after oral administration. Based on the excretion of radioactivity in urine, the minimum absorption was about 73%. The rate of absorption was very rapid, with the peak level of radioactivity in plasma after 15 min of administration. Mean Cmax value for N,N-diethyldithiocarbamate (DDTC) after oral dose of AC-DDTC (20 mg/kg) was 3.8 +/- 0.2 nmol/ml at 15 min and the mean residence time was 47.1 +/- 2.8 min. After oral administration of [14C]AC-DDTC, radioactivity was distributed relatively rapidly. Maximum concentrations were observed in the liver (0.443% dose/g), kidneys (0.496% dose/g), oesophagus (0.313% dose/g) and in the adrenals (0.364% dose/g) at 30 min to 1 h after dosing. Liver was the only organ which contains a considerable amount of radioactivity (0.091% dose/g) 24 h after dosing. Two metabolites of AC-DDTC following oral administration were identified in the plasma and liver by GC and HPLC using extractive alkylation technique, namely DDTC and its methyl ester. Urinary excretion was a major route of elimination of radioactivity derived from [14C]AC-DDTC, in that about 73% of the dose was recovered in urine whereas only 14% was found in feces over 7 days.

Metabolism and pharmacokinetics of S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine in rats.

The metabolism and pharmacokinetics of a mixed disulfide S-(N,N-diethyldithiocarbamoyl)-N-acetyl-L-cysteine (AC-DDTC) were studied in rats. Two metabolites of AC-DDTC following i.v. and p.o. administration were identified in plasma and liver by HPLC and GC, namely N,N-diethyldithiocarbamate (DDTC) and the methyl ester of DDTC (Me-DDTC). AC-DDTC was very unstable in vivo and could not be detected neither in plasma nor in urine. Pharmacokinetic parameters of DDTC following intravenous administration of AC-DDTC (20 mg/kg) were calculated. DDTC has a low affinity to rat tissue and the total body clearance was 9.0 +/- 3.4 ml/min/kg. The mean residence time (MRT) was 111.5 +/- 16.3 min. After oral administration of 20 mg/kg AC-DDTC, maximal plasma concentration (Cmax) was 3.8 +/- 0.2 nmol/ml and the bioavailability was 7.04%. Cmax for DDTC at a dose of 120 mg/kg AC-DDTC was 40.1 +/- 2.2 nmol/ml. MRT was 47.1 +/- 2.8 min at a dose of 20 mg/kg and 110.5 +/- 6.0 min at 120 mg/kg.

Chronic toxicity of nivalenol in female mice: a 2-year feeding study with Fusarium nivale Fn 2B-moulded rice.

Groups of 42 7-wk-old female C57BL/6CrSlc SPF mice were fed diets containing 0, 6, 12 and 30 ppm nivalenol (NIV) for 2 years. Body-weight gain was reduced in all treated groups of animals and feed efficiency was reduced, significantly so, in the high-dose group. The absolute weights of the liver in the 30-ppm group, and of the kidneys in the 12- and 30-ppm groups were significantly reduced, compared with those of the controls. When expressed relative to brain weight there was a reduction in the kidney weight of the 12-ppm NIV group only. Some leucopenia was seen in the treated mice, particularly in the 30-ppm group, although this was not statistically significant, and there were dose-dependent increases in the serum concentrations of alkaline phosphatase and non-esterified fatty acids. No tumours attributable to NIV were found in any of the experimental groups. Naturally occurring tumours, mostly lymphomas, were of similar incidence in all groups, but developed later and appeared to grow more slowly in the mice of the 30-ppm group than in those of other groups. The incidence of amyloidosis, particularly in the small intestine, was low in the two higher dose groups compared with that in the control group. The mortality rate of the 30-ppm NIV group was lower than that of the control group and this may be partly due to the lower tumour incidence in the earlier period and partly due to the lower incidence of amyloidosis.

The acute and chronic toxicities of nivalenol in mice.

In an attempt to ascertain precisely the toxic effects of nivalenol (NIV), we conducted the determination of LD50 values, and interim kills during the carcinogenic study in mice. LD50 values (mg/kg) of NIV in 6-week-old male ddY mice were determined as 38.9 (po), 7.4 (ip), 7.2 (sc), and 7.3 (iv). Seven-week-old female C57BL/6CrSlc SPF mice were fed diets containing 0, 6, 12, and 30 ppm (mg/kg) NIV over 1 year, and were assessed for effects on body weight gain, feed efficiency, terminal organ weights, hematology, and histopathology. The rates of body weight gain and feed efficiency showed a good dose-dependent correlation in all experimental periods. Gross and histopathological evaluation of the liver, thymus, spleen, kidneys, stomach, adrenal glands, pituitary gland, ovaries, sternum, bone marrow, lymph node, brain, and small intestines with or without Peyer's patch portion from control and all NIV-exposed mice revealed that these tissues were normal in appearance and in histopathological structure. Also, no changes were observed in the ultrastructural studies on the bone marrow. Dietary NIV did, however, cause dose-dependent decreases of absolute organ weights (mg) and increases of relative organ weights (mg/g body weight) in the terminal organ weights recorded. A significant leukopenia was observed in the 30 ppm group at 6 months and in all NIV-treated groups at 1 year. No marked changes were observed in the other hematological parameters. These results indicated that 6 ppm or more of dietary NIV for 1 year showed a characteristic toxic effect of trichothecene mycotoxins in mice.