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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.Many bacterial pathogens that infect mammals have developed specific traits to avoid the innate and specific immune defenses of the host (3,6,12,17). A characteristic common to several invasive enteric pathogens (e.g., Shigella, Salmonella, and Yersinia species) is the ability to induce macrophage apoptosis via a type III protein secretion system (8,9,16,21). Macrophage apoptosis in response to Shigella and Salmonella infections triggers severe inflammation via the action of proinflammatory cytokines (22). Yersiniae induce apoptosis in macrophages by suppressing the signaling pathway that leads to the production of proinflammatory cytokines (1, 10). Macrophage cell death may lead to either the induction or the inhibition of an inflammatory response. In studying the interaction between phagocytes and Vibrio vulnificus, most efforts have focused on the capsule (7,11,13,18). Encapsulated isolates of V. vulnificus are more resistant to phagocytosis by human polymorphonuclear leukocytes and murine peritoneal macrophages than are unencapsulated isolates (4,5,15). However, the cytotoxic effects on phagocytes have not been clearly demonstrated. In this study, we examined the apoptotic effects of nine isolates of V. vulnificus on macrophages.First, we examined each isolate of V. vulnificus for apoptotic activity toward a macrophage-like cell line, J774, by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis. Five clinical isolates of V. vulnificus, the K series of strains, were isolated from the blood of individual septicemic patients at Kurashiki Central Hospital in Japan between 1985 and 1999. Two environmental strains, E4 and E10, were isolated from seafood in Florida. Strain R41 was isolated from plankton in Okayama Prefecture in Japan. Strain G83 was isolated from seafood in the Republic of Korea. Bacteria in the logarithmic growth phase were obtained by cultivation with Luria-Bertani broth at 37°C. The desired bacterial concentration was checked by plating serial dilutions of the samples on agar and counting CFU after incubation. J774 cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL Life Technologies, Rockville, Md.) supplemented with 2 mM glutamine, 2 mM sodium pyruvate, and 20% heattreated fetal calf serum. Cells were seeded in 24-well tissue culture plates at 10 5 cells/well. Each isolate of V. vulnificus was inoculated into the wells at a multiplicity of infection of approximately 1.0. After incubation at 37°C for 150 min, the Mebstain Apoptosis Kit (Immunotech, Marseilles, France) was used to label the free 3Ј-OH ends of DNA fragments with fluorescein as recommended by the manufacturer. The wellkno...

Pretreatment of primary cultures of rat hepatocytes with alpha-tocopherol succinate (vitamin E) for 20 h prior to exposure to K2Cr2O7 resulted in a marked decrease of chromium (VI)-induced cytotoxicity, as evaluated by the leakage of lactate dehydrogenase, without affecting cellular uptake and the subcellular distribution of chromium. The levels of chromium (VI)-induced lipid peroxidation, as monitored by malondialdehyde formation, were also inhibited by pretreatment with the vitamin. Pretreatment with vitamin E normalized the levels of nonenzymatic antioxidants such as glutathione and vitamin C suppressed by dichromate, and caused a distinct accumulation of vitamin E in hepatocytes. However, vitamin E pretreatment did not affect the activities of enzymatic antioxidants including glutathione reductase, superoxide dismutase, and catalase suppressed by dichromate. These results indicate that the protective effect of vitamin E against chromium (VI)-induced cytotoxicity as well as lipid peroxidation, may be associated more with the level of nonenzymatic antioxidants than the activity of enzymatic antioxidants.

According to published information, the lung is the only clear target organ for tumors when mice are exposed to cigarette smoke. Liver, skin, and upper digestive tract are target organs when orally or dermally exposed to cigarette smoke condensate, but not kidney, brain, or bone marrow. We tested the genotoxicity of cigarette smoke in the known target organ (lung), possible target organs (stomach and liver), and non-target organs (kidney, brain, and bone marrow) of the mouse using the alkaline single-cell gel electrophoresis (SCG, or comet) assay, as modified by us. We also tested the effect of free radical scavengers on the genotoxicity of the smoke. Male ICR mice were exposed to cigarette smoke. DNA single-strand breaks (SSB) were measured by the SCG assay 15, 30, 60, 120, and 240 min after the exposure. Fifteen min after the animals were exposed for 1 min to a 6-fold dilution of smoke, SSB appeared in the lungs, stomach, and liver; the damage in the lungs and liver returned to almost control levels by 60 min, and that of the stomach by 120 min. Kidney, brain, and bone marrow DNA were not damaged. Exposure to more dilute smoke (12- or 24-fold dilution) did not cause DNA damage. Single oral pretreatment (100 mg/kg) of either ascorbic acid (VC) or alpha-tocopherol acetate (VE) 1 h before smoke inhalation prevented SSB in the stomach and liver, while VE but not VC significantly reduced SSB in the lung. Five consecutive days of either VC or VE (100 mg/kg/day) pretreatment completely prevented SSB in the lung, stomach, and liver. Thus, the SCG assay detected DNA SSB, induced by cigarette smoke, in the known target organ, two possible target organs, and none of the non-target organs. Antioxidants could prevent those effects, suggesting that free radicals may have been a source of the damage. Our results suggest the importance of the SCG assay as a tool in the study of genotoxicity and carcinogenicity.

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 Gramnegative 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.