Comparison of species, virulence genes and clones of Aeromonas isolates from clinical specimens and well water in Okinawa Prefecture, Japan.

AIMS: To reveal the sources of Aeromonas infection in Okinawa Prefecture of Japan, the species, virulence genes and clones of strains isolated from clinical specimens and well water were compared. METHODS AND RESULTS: The properties of both isolates were investigated by sequencing of rpoD, detection of 10 virulence genes using PCR and genotyping with pulsed-field gel electrophoresis. In all, 68 clinical and 146 well water strains of Aeromonas were isolated and the main species were A. caviae, A. dhakensis, A. hydrophila and A. veronii biovar sobria. Aeromonas dhakensis possessed various virulence genes; however, A. caviae possessed only fla. The same or similar clones were distributed in certain areas of Okinawa and one clone had survived several months in the biliary system of two patients, respectively. CONCLUSION: Although the same Aeromonas clone was not isolated from clinical and well water samples, our study revealed the detected patterns of virulence genes in both isolates, the distribution of identical/similar clones in the Okinawan environment and long-time survival in patient's organs. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated the association between Aeromonas patients and well water exposure. This study provides the properties of species, virulence genes and clones of Aeromonas isolated from samples of these origins.

Selective 8-hydroxyguanine formation in pancreatic DNA due to a single intravenous administration of 4-hydroxyaminoquinoline 1-oxide in rats.

8-Hydroxyguanine (8-OHG) formation, a possible initiating event, was determined in pancreatic and liver DNA and compared with the genesis of acinar cell and hepatocyte necrosis in male Wistar rats given a single intravenous administration of 4-hydroxyaminoquinoline 1-oxide (4-HAQO). At the non-necrotic but tumorigenic dose of 7.0 mg/kg body weight, 8-OHG was selectively generated in pancreatic DNA, in the absence of acinar cell necrosis, at the 6 and 24 h time points and repaired by the 48 h time point. When rats were exposed to 4-HAQO at a necrotic dose of 14.0 mg/kg body weight, 8-OHG was also selectively formed in pancreatic DNA with the same time-dependence of generation and repair, while acinar cell necrosis became evident at the 24 h time point and progressed thereafter. Whereas no hepatocyte necrosis was detected in any rats, 8-OHG values for liver DNA merely expressed slight increases only at the 24 and 48 h time points in rats given 14.0 mg/kg body weight of 4-HAQO. The present data suggest that formation of oxidative DNA damage, assayed by 8-OHG, in pancreatic DNA is independent from toxicity and may be involved, along with quinoline adducts, in mutational events underlying 4-HAQO-induced rat acinar cell carcinogenesis.

Different roles of 8-hydroxyguanine formation and 2-thiobarbituric acid-reacting substance generation in the early phase of liver carcinogenesis induced by a choline-deficient, L-amino acid-defined diet in rats.

The present study was performed to assess the roles of hepatocellular oxidative damage to DNA and constituents other than DNA in rat liver carcinogenesis caused by a choline-deficient, L-amino acid-defined (CDAA) diet by examining the effects of the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD). The parameters used for cellular oxidative damage were the level of 8-hydroxy-guanine (8-OHGua) for DNA and that of 2-thiobarbituric acid-reacting substance (TBARS) for constituents other than DNA. A total of 40 male Fischer 344 rats, 6 weeks old, were fed the CDAA diet for 12 weeks with or without DPPD (0.05, 0.10 or 0.20%) or butylated hydroxytoluene (BHT, 0.25%). In the livers of the rats, the numbers and sizes of glutathione S-transferase (EC 2.5.1.18) placental form (GSTP)- and/or gamma-glutamyltransferase (GGT, EC 2.3.2.2)-positive lesions and levels of 8-OHGua and TBARS were determined. The GSTP-positive lesions of 0.08 mm2 or larger were all stained positively for GGT as well in cross-sectional area, whereas the smaller lesions were generally negative for GGT. DPPD and BHT reduced the size of the GSTP-positive lesions without affecting their total numbers. At the same time, they reduced TBARS generation without affecting 8-OHGua formation in DNA. The present results indicate that oxidative DNA damage (represented by 8-OHGua formation) and damage to constituents other than DNA (represented by TBARS generation) may play different roles in rat liver carcinogenesis caused by the CDAA diet; the former appears to be involved in the induction of phenotypically altered hepatocyte populations while the latter may be related to the growth of such populations.

Inhibitory effects of 2-O-octadecylascorbic acid and other vitamin C and E derivatives on the induction of enzyme-altered putative preneoplastic lesions in the livers of rats fed a choline-deficient, L-amino acid-defined diet.

Effects of a lipophilic derivative of vitamin C, 2-O-octadecylascorbic acid (CV-3611), as well as its parent L-ascorbic acid (AscA), DL-alpha-tocopherol (alpha-T) and its hydrophilic derivative, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), on the number and size of gamma-glutamyltransferase (GGT)-positive putative preneoplastic lesions were examined and compared with their influences on 8-hydroxyguanine formation in DNA and 2-thiobarbituric acid-reacting substance generation in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet for 12 weeks. A total of 90 male Fischer 344 rats, 6 weeks old, were divided into 18 groups each consisting of five rats. Group 1 received the CDAA diet alone; Groups 2, 3 and 4 received the CDAA diet containing respectively 0.01, 0.05 and 0.10% CV-3611; Groups 5-7, 8-10 and 11-13 similarly received the CDAA diet containing AscA, alpha-T and Trolox, respectively, at these same low, middle and high concentrations; Group 14 received a choline-supplemented, L-amino acid-defined (CSAA) diet alone; Groups 15-18 were given the CSAA diet containing CV-3611, AscA, alpha-T and Trolox, respectively, all at the 0.10% level. While all four vitamin derivatives exerted inhibitory effects on all four parameters, in each case dose-dependently, CV-3611 demonstrated the most pronounced effects. The present results indicated that lipophilic vitamin C derivatives may be particularly effective chemopreventive agents against CDAA diet-associated, oxidative stress-related hepatocarcinogenesis via its superior antioxidative properties.

Enhancing effect of oxymetholone, an anabolic steroid, on development of liver cell foci in rats initiated with N-diethylnitrosamine.

The promoting potential of oxymetholone (OXM) administration on development of liver cell foci was investigated in male F344 rats previously treated with N-diethylnitrosamine (DEN). One week after a single injection of DEN (100 mg/kg, i.p.), rats were given OXM at a dietary level of 0.2% for the first 4 weeks and then at a concentration of 0.1% for an additional 35 weeks. All rats were killed at week 40 for histopathological and immunohistopathological examination of liver tissue. The numbers and areas of both clear cell and glutathione S-transferase placental form (GST-P) positive foci were significantly increased in the group treated with DEN and OXM as compared with the respective values for the DEN alone group. The results thus suggested that OXM possesses promoting potential for rat liver carcinogenesis.

In situ freezing of the rat urinary bladder: DNA adduct formation in the bladder epithelium demonstrated by 32P-postlabeling assay.

In situ freezing of the urinary bladder has been demonstrated to exert tumor-initiating potential in two-stage urinary bladder carcinogenesis in the rat. In the present experiment, DNA modification was examined after in situ freezing of the whole urinary bladder performed by pinching with frozen forceps at -15 degrees C or -30 degrees C for 2 s. The 32P-postlabeling analysis revealed at least 2 DNA adducts in the epithelial cells of the urinary bladder collected 3 days after freezing. Single-strand breaks of DNA were also found by means of the alkaline elution assay in the bladder epithelium collected 10 min after freezing. Thus, the previously demonstrated tumor-initiating activity of in situ freezing in urinary bladder carcinogenesis was revealed to be associated with substantial DNA damage and adduct formation.

In situ freezing of the urinary bladder: a trigger of rapid development of sodium o-phenylphenate-induced urinary bladder tumors in the rat.

The effect of in situ freezing of the urinary bladder on sodium o-phenylphenate (OPP-Na)-induced urinary bladder tumor development was investigated in male F344 rats. Freezing was performed at the start of the experiment by touching the serosal surface of the bladder with a frozen steel rod. As a result, three out of 27 rats (11%) developed bladder tumors within 78 weeks when 0.5% OPP-Na feeding was started 2 weeks after freezing and one out of 27 rats (4%) when the feeding was started 12 weeks after freezing. 0.5% OPP-Na alone did not induce any bladder lesions. In a second experiment, 19 out of 25 rats (76%) developed bladder tumors (carcinomas in 12 rats and papillomas in seven rats) when 2% OPP-Na was administered from 6 weeks after freezing, whereas only one rat (5%) demonstrated a bladder carcinoma in the group given 2% OPP-Na without prior freezing. In neither experiment were tumors induced by freezing alone. Enzyme histochemistry revealed no remarkable changes in enzyme activities of regenerative hyperplasia induced by freezing. The results indicate that in situ freezing of the urinary bladder acts as a trigger of rapid development of OPP-Na-induced rat urinary bladder tumors.

[Twenty-eight day repeated dose toxicity testing of diphenylamine in F344 rats].

A twenty-eight day repeated dose toxicity test of diphenylamine (DPA) was carried out in male and female F344 rats at dose levels of 1000, 333, 111 or 0 mg/kg/day. Thirty-six animals of both sexes were divided into 6 groups of equal number, 4 groups being used for the 28 days dosing study and the remainder for investigation of recovery. Inhibition of body weight gain, increase of liver, spleen and kidney weights, and anemia were observed in the highest dose groups in both sexes. The same groups demonstrated mucosal hyperplasia in the forestomach, dilatation, degeneration or necrosis of renal tubules in the corticomedullary junction, and hyperplasia in the bone marrow histopathologically. Slight increase of spleen, liver and kidney weights as well as slight degeneration of renal tubules were evident in several animals receiving the dose level of 333 mg/kg/day. Repair of histopathological lesions and anemia occurred within 14-day resting period. Based on these findings, under the present experimental conditions, the no observable effect level of DPA was 111 mg/kg/day.