Vibrio vulnificus detected in the spleen leads to fatal outcome in a mouse oral infection model.

Vibrio vulnificus causes rapid disseminating septicemia by oral infection in infected individuals who have an underlying disease, especially chronic liver diseases. Although the elucidation of specific risk factors for V. vulnificus infection in patients with liver diseases is of urgent importance, no appropriate experimental animal model that mimics the liver diseases in this bacterial infection has been available so far. To discover these risk factors, we generated a liver disordered mouse by performing bile duct ligation (BDL). Hepatitis developed in the BDL mice; however, this did not affect mortality in mice after orogastric administration of V. vulnificus, suggesting that the liver disorders caused by the BDL were not risk factors for V. vulnificus septicemia. When the dead and surviving mice were compared, V. vulnificus could be detected from the spleen only in the dead group. Furthermore, significantly higher numbers of V. vulnificus were detected from the intestines in the dead group than in the surviving group ( P < 0.001). These findings suggested that proliferation of the challenge inoculum in the intestine was needed for the oral infection with V. vulnificus, and that the elimination of V. vulnificus in the liver and/or spleen plays a critical role in survival of the host.

Inhibition of binding of Vibrio vulnificus hemolysin (VVH) by MßCD.

Vibrio vulnificus secrets a pore-forming toxin called Vibrio vulnificus hemolysin (VVH). In this study, we showed that methyl-beta-cyclodextrin (MßCD), an oligosaccharide, decreased binding of VVH to Chinese hamster ovary (CHO) cells, resulting in inhibition of its cytotoxicity. When the VVH was incubated with MßCD, cytotoxicity of the toxin was inhibited from 100.3 ± 7.2% to 19.6 ± 5.3%. Binding analysis showed that the amount of VVH on the cells was decreased from 101.4 ± 9.2% to 18.1 ± 8.0% only when MßCD was present in the culture media. Our results indicate that the inhibition of cytotoxicity of VVH by MßCD was due to a decrease in the amount of toxin binding to CHO cells.

Relationship between localization on cellular membranes and cytotoxicity of Vibrio vulnificus hemolysin.

Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis in target cells. A detergent resistant membrane domain (DRM) fraction of the cells after sucrose gradient centrifugation includes cholesterol-rich membrane microdomains which have been called "lipid rafts". It was reported that some pore-forming toxins require association with DRM and/or lipid rafts to exert their cytotoxicity. It has also been thought that cellular cholesterol is involved in VVH cytotoxicity because VVH cytotoxicity was inhibited by pre-incubation with cholesterol. However, both cellular localization and mode of action of VVH cytotoxicity remain unclear. In this study, we investigated the relationship between VVH localization on the cellular membrane and its cytotoxicity. Oligomers of VVH were detected from DRM fractions by sucrose gradient ultracentrifugation but all of these oligomers shifted from DRM fractions to non-DRM fractions after treatment with methyl-beta-cyclodextrin (MßCD), a cholesterol sequestering agent. On the other hand, immunofluorescence analysis showed that VVH did not co-localize with major lipid raft markers on cellular membrane of CHO cells. These data suggested that VVH localized at membrane regions which are relatively abundant in cholesterol but which are not identical with lipid rafts. To determine the linkage between localization and cytotoxicity of VVH, cytotoxicity was evaluated in MßCD-treated CHO cells. The cytotoxicity of VVH was not decreased by the MßCD treatment. In addition, the amount of VVH oligomer did not decrease in MßCD-treated CHO cells. Thus, we found that the amount of oligomer on cellular membrane is important for induction of cytotoxicity, whereas localization to lipid rafts on the cellular membrane was not essential to cytotoxicity.

Smoking induces bimodal DNA damage in mouse lung.

To clarify the relationship between DNA damage and free radical generation caused by smoking in vivo, DNA damage was investigated in the mouse lung by single-cell gel electrophoresis assay after exposure to cigarette smoke (CS) or gas phase cigarette smoke (GPCS). Although GPCS did not induce DNA lesions, bimodal peaks of DNA damage were detected in mouse lung exposed to CS, one immediately after exposure and another 15 min later. Pretreatment with a specific hydroxyl radical (•OH) scavenger completely prevented both types of DNA damage induced by CS. Electron spin resonance (ESR) study of the kinetics of free radical generation in CS or GPCS revealed that •OH could be detected immediately after the spin trapping of CS without chelators (first •OH generation), whereas •OH was also generated gradually with a time lag when the spin trapping was performed with chelators (second •OH generation). Our ESR study also indicated that the first •OH peak was probably generated from H(2)O(2) via a metal-independent pathway, whereas the second •OH peak might have been generated from H(2)O(2) and other sources via at least two different metal-masked pathways. The bimodal DNA damage induced in lung by smoking appears to be the result of a time lag between the first •OH generation and second •OH generation after exposure to the tar in CS.

The aromatic ring of phenylalanine 334 is essential for oligomerization of Vibrio vulnificus hemolysin.

Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.

Oligomerization is essential for apoptotic activity of Vibrio vulnificus hemolysin.

Vibrio vulnificus hemolysin (VVH), a pore forming toxin, is thought to be a virulence factor of this bacterium. It is well known that VVH induces apoptosis as well as cell lysis in susceptible target cells. Although pore formation is an essential step in cell lysis, it is unknown whether this step is necessary for VVH-induced apoptosis. In this study, Chinese hamster ovary (CHO) cells were exposed to non-oligomerized mutant F334I, in which phenylalanine 334 was replaced by isoleucine. The rate of apoptosis caused by the wild type VVH (VVH wt) was 41.5 +/- 6.4 %, whereas that caused by F334I was 0.4 +/- 0.8% at the same concentration. Our results clearly showed that oligomerization is essential for the cell lytic activity as well as apoptotic activity of this toxin.

Reduction in peripheral lymphocytes and thymus atrophy induced by organotin compounds in vivo.

To clarify the involvement of apoptosis in the immunotoxicity of organotin compounds, we examined the induction of apoptosis in the peripheral lymphocytes and thymus of mice treated with triphenyltin (TPT), tributyltin (TBT) or dexamethasone (Dex). Application of TPT or TBT and Dex resulted in a transient reduction in peripheral lymphocytes at 3 to 6 hr, and thymus atrophy was observed at 6 and 24 hr after administration. Lymphocyte subpopulation analysis showed that TPT and TBT induced a greater reduction in B cells than in T cells. The maximum levels of organotin in the blood were about 450 ng TPT/ml in the TPT-treated mice, and 170 ng TBT/ml in the TBT-treated mice. When the isolated peripheral lymphocytes were incubated with the organotins at 500 ng/ml, TPT and TBT induced necrosis in over 70% of cells, while both organotins caused lower percentages of apoptosis as well as necrosis after 3 hr at 100 ng/ml. In the thymus, although in vivo treatment of mice with Dex caused apoptosis, neither apoptotic nor necrotic thymocytes were observed in the TPT- and TBT-treated mice, indicating that the thymus atrophy might be caused by the antiproliferative effects of these organotin compounds. Thus, our results did not support the idea that apoptosis played a decisive part in the immunotoxicity of the organotin compounds in vivo.

Improvement of Experimentally Induced Hepatic and Renal Disorders in Rats using Lactic Acid Bacteria-fermented Soybean Extract (BiofermenticsTM).

The effects of lactic acid bacteria-fermented soybean extract (Biofermentics; BF) on experimental models of hepatic and renal disorders were investigated in vivo and in vitro. In rat, hepatitis induced by feeding of deoxycholic acid (DCA, 0.5 wt/wt, n = 6) or intraperitoneal injection of d-galactosamine (GMN, 500 mg/body wt, n = 6), the increase in serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) levels were inhibited significantly (P < 0.05) by feeding a diet containing 5% dried BF. Moreover, the BF-administered rat group showed lower concentrations of blood urea nitrogen and a larger amount of urine as compared with values in the control group. Pretreatment of primary cell cultures of rat hepatic and renal cells with BF prior to exposure to dichromate (K(2)Cr(2)O(7)) resulted in a marked decrease of dichromate-induced cytotoxicity as evaluated by the leakage of lactate dehydrogenase The levels of dichromate-induced lipid peroxidation, as monitored by malondialdehyde formation, were also reduced by pretreatment of hepatocytes with BF. These results suggest that BF may play a role in hepatic and renal disorders, and may be useful for maintaining health in humans as well.

Estimation of hydroxyl radical generation by salicylate hydroxylation method in kidney of mice exposed to ferric nitrilotriacetate and potassium bromate.

Hydroxyl radical (*OH) generation in the kidney of mice treated with ferric nitrilotriacetate (Fe-NTA) or potassium bromate (KBrO3) in vivo was estimated by the salicylate hydroxylation method, using the optimal experimental conditions we recently reported. Induction of DNA lesions and lipid peroxidation in the kidney by these nephrotoxic compounds was also examined. The salicylate hydroxylation method revealed significant increases in the *OH generation after injection of Fe-NTA or KBrO3 in the kidneys. A significant increase in 8-hydroxy-2'-deoxyguanosine in nuclei of the kidney was detected only in the KBrO3 treated mice, while the comet assay showed that the Fe-NTA and KBrO3 treatments both resulted in significant increases in DNA breakage in the kidney. With respect to lipid peroxidation, the Fe-NTA treatment enhanced lipid peroxidation and ESR signals of the alkylperoxy radical adduct. These DNA breaks and lipid peroxidation mediated by *OH were diminished by pre-treatment with salicylate in vivo. These results clearly demonstrated the usefulness of the salicylate hydroxylation method as well as the comet assay in estimating the involvement of *OH generation in cellular injury induced by chemicals in vivo.

Assessment of DNA damage in multiple organs of mice after whole body X-irradiation using the comet assay.

The comet assay was performed to elucidate the linearity of calibration curves and detection limits for DNA damage in multiple organs of whole body X-irradiated mice, and rates of reduction in DNA damage by DNA repair during the irradiation period were estimated in the respective organs by comparing the rates of increase in DNA damage at different absorbed dose rates of X-rays. Of the assay parameters, tail length and the percentage DNA in the tail showed a higher sensitivity to DNA damage in most organs than Olive tail moment. Data at the higher absorbed dose rates (2.22 or 1.44 Gy/min) showed good correlations between absorbed doses and these two parameters, with correlation coefficients of more than 0.7 in many organs. However, this assay had difficulty detecting DNA damage at the lower absorption dose rate (0.72 Gy/min). The estimated rates of increase in DNA damage and those of DNA repair during the irradiation period in the respective organs suggested differences in the radiosensitivity of nuclear DNA and DNA repair capacity among organs. Our results indicated that absorbed dose rates of 1.0-1.3 Gy/min or greater were needed to induce detectable DNA damages by the comet assay in many organs.

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation.

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.

Depletion of lymphocytes, but not neutrophils, via apoptosis in a murine model of Vibrio vulnificus infection.

Vibrio vulnificus causes severe sepsis in humans. There are several reports about the relationship between host immunity and bacterial growth in V. vulnificus infection. However, the effect on leukocytes of V. vulnificus infection in vivo has not been elucidated. A murine model of V. vulnificus infection was used to investigate its effects on leukocytes in this study. Bacteria were recovered from the blood of mice 3 h after subcutaneous injection in the right lower flank. They were detected in 87.5 % (n = 7/8) of mice at 6 h, but this value decreased to 12.5 % (n = 1/8) at 12 h. In contrast, the number of lymphocytes in peripheral blood had already started to decrease at 3 h, and reached a minimum at 6-9 h post-inoculation. Typical DNA laddering, a hallmark of apoptosis, was also detected in thymocytes and splenocytes at 6 and 9 h, and showed a tendency to disappear by 12 h. Although the number of lymphocytes decreased in the model, the numbers of neutrophils did not. These results suggested that V. vulnificus has selective cytotoxicity for lymphocytes in peripheral blood in vivo, and the lymphocyte depletion was probably associated with apoptosis in vivo.

Effects of butyltin compounds on mitochondrial respiration and its relation to hepatotoxicity in mice and Guinea pigs.

Because the mechanisms responsible for the difference in toxicity between different experimental animal species remain unclear, the effects of tributyltin chloride (TBTC) and dibutyltin dichloride (DBTC) on mitochondrial respiration were compared among the livers of mice and guinea pigs in vitro and in vivo. Further, the levels of these butyltin compounds and their derivatives in the mitochondrial fractions of the hepatocytes were investigated in these animal species. Administration of TBTC and DBTC to mice resulted in the obvious elevation of serum enzymatic activities, as well as the inhibition of succinate-linked State 3 respiration in hepatic mitochondria at 24 h after administration. On the other hand, these metal compounds failed to induce such hepatotoxicity or to inhibit mitochondrial respiration in guinea pigs. There was no significant difference between mice and guinea pigs in the IC50 (metal concentration observed in 50% inhibition of mitochondrial respiration) of TBTC and DBTC against the succinate-linked State 3 respiration of hepatic mitochondria in vitro, although the mitochondrial respiration of succinate-linked State 3 was inhibited in the liver of mice treated with the metals in vivo. The levels of total butyltin compounds in the mitochondrial fractions of hepatocytes were higher in the mice than in the guinea pigs, and the main butyltin compound in the mitochondrial fractions was DBTC in both species at 24 h after TBTC or DBTC administration. The amount of sulfhydryl groups, which were capable of binding with DBTC, in mice hepatic mitochondria was twice as large as that in guinea pigs, and the affinity of DBTC for the isolated hepatic mitochondria was higher in mice than in guinea pigs in vitro. These results suggested that the induction of hepatotoxicity by TBTC and DBTC in vivo was closely associated with the depression of mitochondrial respiration and that the difference in susceptibility to the metal-induced mitochondrial damages between mice and guinea pigs might result from the high affinity of butyltin compounds, in particular DBTC, for hepatic mitochondria in mice containing higher levels of sulfhydryl groups, compared with guinea pigs.

Comparison of hepatotoxicity and metabolism of butyltin compounds in the liver of mice, rats and guinea pigs.

The hepatotoxicity of tributyltin chloride (TBTC) and dibutyltin dichloride (DBTC) was compared among mice, rats and guinea pigs in vivo. Further, the metabolism of these butyltin compounds in the liver was also investigated in these species. The oral administration of TBTC and DBTC to mice induced obvious liver injury, as demonstrated by both serodiagnosis and histopathological diagnosis. The concentrations of TBTC and DBTC that induced hepatotoxicity in mice at 24 h after oral administration were 180 and 60 micro mol/kg, respectively. In the case of rats, the liver injury induced by TBTC and DBTC was detected at 24 h by the serodiagnosis, but not by histopathological diagnosis. On the other hand, in guinea pigs, TBTC and DBTC administration did not produce any clear liver injury at 24 h, as evaluated by these two diagnostic methods. Thus, the following ranking was obtained with regard to increasing order of sensitivity to liver injury caused by TBTC and DBTC: mice, rats and guinea pigs. The total butyltin contents in the liver of mice were equivalent at 3 h and 24 h after the administration of TBTC or DBTC; however, the contents in the liver of rats and guinea pigs were relatively lower at 3 h and higher at 24 h than those of mice, although there were no differences between rats and guinea pigs in the total liver butyltin content. Concerning the liver metabolism of these butyltin compounds, the main form of butyltin compounds in these animals treated with TBTC was DBTC within 3 h after oral administration, while the main metabolites at 24 h were different in each species, indicating that the liver metabolism of TBTC might vary by animal type. When the animals were treated with DBTC orally, DBTC was hardly metabolized in the livers of these animals even at 24 h, and the liver levels of DBTC were two times greater in mice and guinea pigs than in rats at 3 h and were lower in mice at 24 h than in rats and guinea pigs. The analysis of cellular distributions of DBTC in the liver at 3 h after the administration showed that the levels of DBTC in the nuclear, microsomal and mitochondrial fractions of mice hepatocytes were relatively higher than in those of rats, which were greater than in those of guinea pigs. These results suggest differences in the sensitivity of mice, rats and guinea pigs to hepatotoxicity caused by butyltin compounds and demonstrate that the difference in the sensitivity of these animals to the hepatotoxicity induced by TBTC and DBTC may be partly due to differences in hepatic metabolism of TBTC and in the distribution of DBTC within cell organelles, respectively.

Vibrio vulnificus induces macrophage apoptosis in vitro and in vivo.

In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of V. vulnificus.