Excess processing of oxidative damaged bases causes hypersensitivity to oxidative stress and low dose rate irradiation.

Ionizing radiations such as X-ray and γ-ray can directly or indirectly produce clustered or multiple damages in DNA. Previous studies have reported that overexpression of DNA glycosylases in Escherichia coli (E. coli) and human lymphoblast cells caused increased sensitivity to γ-ray and X-ray irradiation. However, the effects and the mechanisms of other radiation, such as low dose rate radiation, heavy-ion beams, or hydrogen peroxide (H2O2), are still poorly understood. In the present study, we constructed a stable HeLaS3 cell line overexpressing human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) protein. We determined the survival of HeLaS3 and HeLaS3/hOGG1 cells exposed to UV, heavy-ion beams, γ-rays, and H2O2. The results showed that HeLaS3 cells overexpressing hOGG1 were more sensitive to γ-rays, OH(•), and H2O2, but not to UV or heavy-ion beams, than control HeLaS3. We further determined the levels of 8-oxoG foci and of chromosomal double-strand breaks (DSBs) by detecting γ-H2AX foci formation in DNA. The results demonstrated that both γ-rays and H2O2 induced 8-oxoguanine (8-oxoG) foci formation in HeLaS3 cells. hOGG1-overexpressing cells had increased amounts of γ-H2AX foci and decreased amounts of 8-oxoG foci compared with HeLaS3 control cells. These results suggest that excess hOGG1 removes the oxidatively damaged 8-oxoG in DNA more efficiently and therefore generates more DSBs. Micronucleus formation also supported this conclusion. Low dose-rate γ-ray effects were also investigated. We first found that overexpression of hOGG1 also caused increased sensitivity to low dose rate γ-ray irradiation. The rate of micronucleus formation supported the notion that low dose rate irradiation increased genome instability.

Prevalence of the main food-borne pathogens in retail food under the national food surveillance system in Japan.

The National Food Surveillance System in Japan was formed in 1998 to monitor the contamination of retail foods with bacterial pathogens. Approximately 2000-3000 samples were tested annually, and the data from food categories that had more than 400 samples collected during 1998-2008 were analysed. With regard to meat, the frequency of positive samples for Salmonella in chicken for raw consumption and ground chicken was 12.7% and 33.5%, respectively. Moreover, Shiga toxin-producing Escherichia coli (STEC) O157 was found in ground meat, organ meat and processed meat, although at a low frequency (0.1%). The prevalence of Campylobacter jejuni/coli was 13.3% and 20.9% in chicken for raw consumption and ground chicken, respectively. In vegetables and fruit, Salmonella was detected in cucumber, lettuce, sprout and tomato samples at a frequency of around 0.1-0.2%. With regard to seafood, Salmonella was found in 0.5% of oysters for raw consumption. Seafood was not contaminated with STEC O157 or Shigella. Serotype Infantis was the most frequently detected serotype of Salmonella in seafood, followed by the serotypes Typhimurium, Schwarzengrund and Manhattan. In ground chicken, 72.2% of the strains were identified as the serotype Infantis. E. coli, as an indicator of food hygiene, was detected in all food categories. The results show the prevalence of the above-mentioned pathogens in the retail food supplied in Japan; further, they indicate that consumption of raw food carries the risk of contracting food-borne infections.

Contamination level and ingestion dose of foodborne pathogens associated with infections.

Intake of a small dose of foodborne pathogens can cause infection. In this study, an estimation of the infectious dose of the pathogens was obtained by conducting microbiological risk assessments. The contamination levels of foodborne pathogens were analysed in 17 outbreaks of Salmonella, Escherichia coli O157, enterotoxigenic E. coli, Vibrio parahaemolyticus, and Campylobacter jejuni occurring in Japan between 2004 and 2006. The infectious dose was estimated in 14 of the 17 outbreaks utilizing existing data. In three outbreaks of Salmonella infection in which the infection rate was 89-100%, the dose of the ingested pathogens was estimated to be 259,000-14,000,000,000 c.f.u. In other outbreaks of Salmonella infection, the infection rate and dose of the ingested pathogens were 10-66·4% and 81-1560 c.f.u. or most probable number (MPN), respectively. The ingested Salmonella dose is likely to be related to the infection rate; however, storage conditions should be taken into account when making this determination. In an outbreak of E. coli O157 infection, the infection rate and ingestion dose were 100% and 2 to <9 c.f.u., respectively, while in an outbreak of enterotoxigenic E. coli infection, they were 93% and 25-1000 c.f.u., respectively. Finally, in an outbreak of C. jejuni infection, the infection rate and ingestion dose were 37·5% and 360 MPN, respectively. These results will be particularly valuable for risk assessment.

Inactivation effects of UV irradiation and ozone treatment on the yeast and the mold in mineral water.

In recent years, bottled mineral water has undergone inactivation by methods other than the traditional heat treatment during the production process; there are fewer reports of the effectiveness of these inactivation methods on yeasts and molds in mineral water than on bacteria and protozoan oocysts. In this study, we evaluated the effects of UV irradiation and ozone treatment compared with heat treatment at 85 degrees C on yeast cells and mold spores inoculated into mineral water. A 5-log reduction occurred at a UV radiation dose of 31,433 microJ/cm2 for Saccharomyces cerevisiae and at 588,285 microJ/cm2 for Penicillium pinophilum. The treatment time for 5-log reduction estimated for UV irradiation was about 0.6 min for S. cerevisiae and about 10.7 min for P. pinophilum; at an ozone concentration of 0.1 ppm, it was 1.75 min for S. cerevisiae and 2.70 min for P. pinophilum, and at a concentration of 0.6 ppm, it was 0.32 min for S. cerevisiae and 0.57 min for P. pinophilum. Comparison of the inactivation effects among the three methods showed that UV irradiation and ozone treatment were less effective than heat treatment at 85 degrees C. Thus, when UV irradiation and ozone treatment are used for inactivation of mineral water, it seems that they need to be combined with heat treatment to achieve a definite effect. Yeast cells are more sensitive to all three inactivation methods than are mold spores, and the sensitivity of yeast cells and mold spores to these inactivation methods may vary among genera.

Nivalenol and the targeting of the female reproductive system as well as haematopoietic and immune systems in rats after 90-day exposure through the diet.

Nivalenol (NIV) is considered to be an important trichothecene mycotoxin produced by Fusarium species because of its frequent contamination in wheat and barley worldwide. The present study examined the subchronic toxicity of NIV in male and female F344 rats fed diets containing 0, 6.25, 25 and 100 mg kg(-1) of the toxin for 90 days. During the experimental period there was a decrease in the white blood cell count at 100 mg kg(-1) in males and at > or =6.25 mg kg(-1) in females. Histopathologically, treatment-related changes were observed in the haematopoietic and immune systems in both sexes and in the female reproductive system at 100 mg kg(-1). Flow cytometric analysis of splenic cells revealed an elevation in the ratio of helper/cytotoxic T-lymphocytes at 100 mg kg(-1). In summary, NIV targets the female reproductive system as well as haematopoietic and immune systems in rats fed NIV for 90 days. Based on a significant decrease in white blood cells in female rats relative to controls, the lowest observable effect level was calculated as 0.4 mg kg(-1) body weight day(-1).

Salmonella prevalence in seafood imported into Japan.

A total of 353 samples of 29 types of seafood were tested for Salmonella prevalence and total microbial population. Salmonella enterica serotype Weltevreden was isolated from 2 of 47 black tiger prawn samples. The contamination levels of Salmonella were in a range of <30 to 40 most probable number per 100 g. In addition, one sample of black tiger prawns and two samples of white shrimp were positive for Salmonella invA gene on PCR assay. Although the mean aerobic bacterial count was greater than 4 log CFU/g in most of the sample types, those in the two Salmonella-isolated samples of black tiger prawn were 7.48 and 5.18 log CFU/g, respectively. These results indicate the possibility that shrimp and prawns contribute to foodborne infections. The improvement of seafood quality is an important issue, and the information on contamination by pathogens should be provided as feedback to the originating country, with the aim of increasing safety.

Detection of Salmonella in spent hens and eggs associated with foodborne infections.

About 16,000 spent hens from 23 farms in the northern area of Japan were purchased in 1996, 1997, 1998, and 1999 to isolate Salmonella in two poultry processing plants. Salmonella was detected in 12 of 23 farms (52.2%). In particular, the serotypes Enteritidis and Infantis were detected in four and three farms, respectively. The prevalence rates in the hens' ceca, immature eggs, and the yolk of mature eggs in oviducts were 14%, 7.2%, and 6.8%, respectively. A total of 23 serotypes were detected. The major serotypes of the strains were Enteritidis, Corvallis, Typhimurium, and Infantis, but most of the strains were untypable. In the same area during 1992 to 1996, Salmonella was detected in eggs associated with four outbreaks of Salmonella Enteritidis infection and one outbreak of Salmonella Infantis infection. The ratio of contamination was approximately 1%, and the level was estimated to be 93 MPN(most probable number)/100 g in one outbreak. In farms that produced the eggs associated with all of the five outbreaks of Salmonella, the serotype Enteritidis or Infantis was isolated from hens. Farms where Salmonella was not detected were not related to any of the outbreaks.

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay.

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.

Real-time PCR method for quantification of Staphylococcus aureus in milk.

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 10(7) to 10(1) CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 10(7) to 10(1) CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63 degrees C for 30 min (low-temperature long-time method) but not at 72 degrees C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

Salmonella prevalence and total microbial and spore populations in spices imported to Japan.

A total of 259 samples of 40 types of spices were tested for Salmonella prevalence and total microbial and spore populations. Salmonella enterica serotypes Weltevreden and Senftenberg were isolated from a black- and red-pepper sample, respectively. Because Salmonella was not detected by the most-probable-number method, it indicated that at least one cell of the microorganism was present in 25 g of sample. The mean aerobic bacterial count was greater than 5.39 log CFU/g in turmeric, garam masala, curry powder, and paprika. The mean bacterial spore counts were greater than 4.33 log CFU/g in turmeric and curry powder. The mean aerobic bacterial count in the two Salmonella-isolated samples was 6.93 log CFU/g. These results indicate that spices can be a source of contamination in the products where they are used as ingredients, and methods to reduce the microbial load in spices should be used.

Migration of formaldehyde and acetaldehyde into mineral water in polyethylene terephthalate (PET) bottles.

The levels of formaldehyde (FA) and acetaldehyde (AA) in polyethylene terephthalate (PET) bottles and in commercial mineral water are reported. All the water samples bottled in Japan contained detectable levels of FA (10.1-27.9 microg l(-1)) and AA (44.3-107.8 microg l(-1)). Of 11 European bottled water samples, eight did not contain either FA or AA, while the remaining three had detectable levels of FA (7.4-13.7 microg l(-1)) and AA (35.9-46.9 microg l(-1)). In three North American bottled water samples, two contained FA (13.6 and 19.5 microg l(-1)) and AA (41.4 and 44.8 microg l(-1)), and one did not. Regardless of the region of origin, all the sterilized water samples contained FA and AA, whilst in contrast, none of the unsterilized water without carbonate contained FA or AA. Of the carbonated water samples, three contained FA and AA, and one did not. When fortified with FA and AA, the commercial water sample without otherwise detectable FA and AA was able to reduce levels, although the commercial water sample containing FA and AA could not. The presence of bacteria in the commercial water samples was investigated using an ATP-based bioluminescent assay and heterotrophic plate count method. The commercial water without FA and AA contained heterotrophic bacteria, whilst the commercial water with FA and AA did not contain detectable bacteria. It is suggested that in this case both FA and AA migrated from PET materials, but were subsequently decomposed by the heterotrophic bacteria in the unsterilized water.

Involvement of eicosanoids and surfactant protein D in extrinsic allergic alveolitis.

The pathophysiology of extrinsic allergic alveolitis (EAA) involves oxidative lung damage as well as interstitial and alveolar inflammation. Macrophages and mast cells are inflammatory components of EAA that produce both leukotrienes (LTs) and prostaglandin D2 (PGD2). In addition, PGD2 is also produced by the free-radical-catalysed peroxidation of arachidonic acid during oxidative stress. Urinary 8-iso prostaglandin F2alpha (8-isoPGF2alpha) and serum surfactant protein D (SP-D) are considered appropriate biomarkers of oxidative stress and interstitial lung disease activity, respectively. The present study aimed to assess the association of these biomarkers with the pathophysiology of EAA. Two cases of acute EAA caused by the inhalation of fungi spores were reported. Eight asthmatic patients and six healthy control subjects were also enrolled in the current study. The serum SP-D and urinary eicosanoid (LTE4, PGD2 metabolite (9alpha,11betaPGF2), 8-isoPGF2alpha) concentrations markedly increased during the acute exacerbation phase. These concentrations decreased following corticosteroid therapy in the EAA patients. There was a significant correlation between serum SP-D and urinary 9alpha,11betaPGF2 concentrations in the EAA patients. In conclusion, although the present study proposes that serum surfactant protein-D and urinary eicosanoids are new biomarkers involved in the various immunological responses in extrinsic allergic alveolitis, further large-scale studies are needed to investigate the role of these compounds, not just as biomarkers, but also as biological potentiators of extrinsic allergic alveolitis.

Increased sensitivity to sparsely ionizing radiation due to excessive base excision in clustered DNA damage sites in Escherichia coli.

PURPOSE: In order to clarify the cellular processing and repair mechanisms for radiation-induced clustered DNA damage, we examined the correlation between the levels of DNA glycosylases and the sensitivity to ionizing radiation in Escherichia coli. MATERIALS AND METHODS: The lethal effects of gamma-rays, X-rays, alpha-particles and H2O2 were determined in E. coli with different levels of DNA glycosylases. The formation of double-strand breaks by post-irradiation treatment with DNA glycosylase was assayed with gamma-irradiated plasmid DNA in vitro. RESULTS: An E. coli mutM nth nei triple mutant was less sensitive to the lethal effect of sparsely ionizing radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of MutM (8-oxoguanine-DNA glycosylase), Nth (endonuclease III) and Nei (endonulease VIII) increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Increased sensitivity to gamma-rays also occurred in E. coli overproducing human 8-oxoguanine-DNA glycosylase (hOgg1). Treatment of gamma-irradiated plasmid DNA with purified MutM converted the covalently closed circular to the linear form of the DNA. On the other hand, overproduction of MutM conferred resistance to H2O2 on the E. coli mutM nth nei mutant. CONCLUSIONS: The levels of DNA glycosylases affect the sensitivity of E. coli to gamma-rays and X-rays. Excessive excision by DNA glycosylases converts nearly opposite base damage in clustered DNA damage to double-strand breaks, which are potentially lethal.

The effect of house design and environment on fungal movement in homes of bronchial asthma patients.

The effect of house building design and environment on the fungal movement in the houses of 41 bronchial asthma (BA) patients has been investigated by examining house dust. The presence and composition of fungi were determined and compared in relation to building structure, house age, size of living room, main flooring material, presence of a living-room rug or air purifier, and frequency of vacuum cleaning. Among these elements, fungal CFU apparently varied only between building structure: wooden-board houses had significantly higher numbers of fungi than reinforced concrete houses (p < 0.01), and wooden mortar or iron-framed prefabricated houses had significantly higher numbers of fungi than reinforced concrete houses (p < 0.05). Classification of the types of fungi present in the house dust of BA patients showed that, regardless of the building designs, there were high levels of osmophilic fungi (group A) and fungi that survive at relatively dry conditions (group B), whereas fungi that survive in very wet conditions (group D) were present at low frequency.

[Fungal allergy - fungal ecology in dwelling environments].

Fungi related to allergies are commonly found in dwelling environments. The predominant fungi Cladosporium, Penicillium, Aspergillus, Alternaria, Wallemia and Rhodotorula live mainly in indoor air, house dust (HD), futons, clothes and contaminated building materials. Fungi in HD are especially important allergens. The fungal CFU and predominant fungi in HD are 10(4) - 10(6)/g and are composed of xerophilic or osmophilic species Aspergillus restrictus, Wallemia and Eurotium but not many yeasts and actinomycetes. Fungal contamination of materials is a serious human health problem because the fungal cells scatter from the materials in the air or HD. The biological activities by fungi also have health implications from the viewpoint of fungal allergens. In this paper, fungal germination, enzyme activities, contaminating cell form and viable or nonviable cells are also discussed.

Fungal flora on board the Mir-Space Station, identification by morphological features and ribosomal DNA sequences.

This report is on the morphological and molecular biological identification, using 18S- and ITS1-rDNA sequences, of the "space fungi" isolated on board the Russian Mir-Space Station as the major constituents of the fungal flora. The six fungal strains were isolated from air by using an air sampler or from condensation. Strains were identified as Penicillium chrysogenum, Aspergillus versicolor, or Penicillium sp. by both methods. The species of space fungi were common saprophytic fungi in our living environment, potential pathogens, and allergens. This study concluded that the environment on board the space station Mir allows the growth of potentially pathogenic fungi as true in residential areas on the earth. Therefore, to prevent infection or other health disorders caused by these fungi, easy and reliable methods should be established to survey the fungal flora in a space station.

Evaluation of 13C-phenylalanine and 13C-tyrosine breath tests for the measurement of hepatocyte functional capacity in patients with liver cirrhosis.

Liver disease is associated with an abnormal elevation of the plasma concentrations of the aromatic amino acids phenylalanine and tyrosine. The liver is the main site of aromatic amino acid metabolism, particularly the hydroxylation of phenylalanine to tyrosine and further tyrosine degradation. In the present study, we have examined the usefulness of the L-[1-13C]phenylalanine breath test (13C-PheBT) and L-[13C]tyrosine breath test (13C-TyrBT) for the detection of hepatic damage in patients with liver cirrhosis. First, the time courses of 13CO2 excretion after the administration of L-[1-13C]phenylalanine and L-[1-13C]tyrosine were compared. The peak times (the time expressed in minutes at which 13CO2 excretion was maximal) were 20 min in both breath tests, but 13C-TyrBT gave a higher peak than 13C-PheBT. Next, the parameters of 13C-PheBT and 13C-TyrBT were compared with biochemical liver function test values. These parameters were well correlated with several liver blood test values conventionally regarded as measures of hepatocyte functional reserve. Therefore, 13C-PheBT and 13C-TyrBT may be useful to assess the degree and progression of hepatic dysfunction.

Identification of principal constituents in enzymatically hydrolyzed coix extract.

The structural elucidation of the main constituents in enzymatically hydrolyzed coix extract, a natural food preservative, was carried out. After peracetylation, five compounds, namely peracetylated forms of glucose, maltose, maltotriose, maltotetraose, and maltopentaose were isolated. The structures were determined by PFG HMQC and HMBC experiments. In addition, by using HPLC with an RI detector, the main components of this coix extract were identified as a mixture of oligosaccharides having one to seven glucose units coupled through alpha-(1-->4) linkages. Since this extract showed no antimicrobial activity, its preservative effect may be caused by its covering of the food surface, thereby blocking contact with air.