Null activity of selenium and vitamin e as cancer chemopreventive agents in the rat prostate.

To evaluate the potential efficacy of selenium and vitamin E as inhibitors of prostate carcinogenesis, four chemoprevention studies using a common protocol were done in a rat model of androgen-dependent prostate cancer. After stimulation of prostate epithelial cell proliferation by a sequential regimen of cyproterone acetate followed by testosterone propionate, male Wistar-Unilever rats received a single i.v. injection of N-methyl-N-nitrosourea (MNU) followed by chronic androgen stimulation via subcutaneous implantation of testosterone pellets. At 1 week post-MNU, groups of carcinogen-treated rats (39-44/group) were fed either a basal diet or a basal diet supplemented with l-selenomethionine (3 or 1.5 mg/kg diet; study 1), dl-alpha-tocopherol (vitamin E, 4,000 or 2,000 mg/kg diet; study 2), l-selenomethionine + vitamin E (3 + 2,000 mg/kg diet or 3 + 500 mg/kg diet; study 3), or selenized yeast (target selenium levels of 9 or 3 mg/kg diet; study 4). Each chemoprevention study was terminated at 13 months post-MNU, and prostate cancer incidence was determined by histopathologic evaluation. No statistically significant reductions in prostate cancer incidence were identified in any group receiving dietary supplementation with selenium and/or vitamin E. These data do not support the hypotheses that selenium and vitamin E are potent cancer chemopreventive agents in the prostate, and when considered with the recent clinical data reported in the Selenium and Vitamin E Cancer Prevention Trial (SELECT), show the predictive nature of this animal model for human prostate cancer chemoprevention.

Ascorbic acid increases healing of excision wounds of mice whole body exposed to different doses of gamma-radiation.

Because of the practical importance of acute radiation exposure associated with combined injuries, it is imperative to investigate the efficacy of cost-effective nutritional factors in the reconstruction of irradiated wounds. Therefore, effect of pretreatment of ascorbic acid was studied on the healing of excised wounds in mice exposed to 2, 4, 6 and 8 Gy whole body gamma-radiation. A full-thickness wound was created on the dorsum of the irradiated mice and the progression of wound contraction was monitored by capturing video images of the wound at various varying days after irradiation. Irradiation caused a dose dependent delay in wound contraction and wound healing time, while ascorbic acid pretreatment resulted in a significant acceleration in the rate of wound contraction and a decrease in the mean wound healing time. To understand the mechanism of healing, collagen, hexosamine, DNA, nitrite and nitrate contents were measured in the granulation tissue of wounded mice treated with ascorbic acid before exposure to 6 Gy gamma-radiation. Ascorbic acid treatment prior to irradiation enhanced the synthesis of collagen, hexosamine, DNA, nitrite and nitrate contents. The histological assessment of wound biopsy revealed an improved collagen deposition, and increase in fibroblast and vascular densities. The present study demonstrates that ascorbic acid pretreatment has a beneficial effect on the irradiated wound and could be a substantial therapeutic strategy to accelerate wound repair in irradiated wounds and in the cases of combined injury situations.

Chemoprevention of rat prostate carcinogenesis by dietary 16alpha-fluoro-5-androsten-17-one (fluasterone), a minimally androgenic analog of dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) is a potent inhibitor of prostate carcinogenesis in rats. However, concerns related to the possible androgenicity of DHEA may preclude its use for chemoprevention of human prostate cancer. Studies were performed to compare the androgenicity of DHEA and a fluorinated DHEA analog, 16alpha-fluoro-5-androsten-17-one (fluasterone), and to determine the chemopreventive activity of fluasterone in the rat prostate. Comparisons of accessory sex gland weight and histology in gonadectomized male rats demonstrated that fluasterone is less androgenic than is DHEA. Fluasterone conferred significant protection against prostate carcinogenesis induced in Wistar-Unilever rats by a sequential regimen of N-methyl-N-nitrosourea+testosterone. Chronic administration of fluasterone at levels of 2000 and 1000 mg/kg diet reduced the incidence of adenocarcinoma in the dorsolateral/anterior prostate from 64% in dietary controls to 28 and 31%, respectively. Other than a dose-related suppression of body weight gain, chronic exposure to fluasterone induced no clinical evidence of toxicity; suppression of body weight gain may be either a pharmacological effect or a minimally toxic effect of the compound. These data demonstrate that a minimally androgenic analog of DHEA protects against prostate carcinogenesis induced in rats by a chemical carcinogen + androgen. The reduced androgenicity of fluasterone may obviate toxicities associated with the androgenicity of the parent compound. On this basis, fluasterone merits consideration for evaluation in clinical trials for prostate cancer prevention. The chemopreventive activity of a non-androgenic DHEA analog suggests that at least a portion of the chemopreventive activity of DHEA in the rat prostate is unrelated to hormonal effects.

Augmentation of wound healing by ascorbic acid treatment in mice exposed to gamma-radiation.

PURPOSE: Because of the crucial practical importance of acute radiation exposure associated with combined injuries, the study was undertaken to investigate the effect of various doses of ascorbic acid on the survival and healing of wounds in mice exposed to whole-body gamma-radiation. MATERIALS AND METHODS: Animals were given double-distilled water or different doses of ascorbic acid by intraperitoneal injection before exposure to 0 or 10 Gy whole-body gamma-radiation to evaluate the effect of ascorbic acid on radiation-induced mortality. The animals were monitored daily for the symptoms of radiation sickness and mortality. In a separate experiment, animals were administered with either double-distilled water or different doses of ascorbic acid before exposure to 0 or 6 Gy whole-body gamma-radiation to investigate the effect of ascorbic acid on the irradiated wound. A full-thickness skin wound was created on the dorsum of the irradiated mice and the progression of wound contraction was monitored by capturing video images of the wound at various post-irradiation periods. RESULT: Treatment of mice with various doses of ascorbic acid elevated survival of mice and a highest number of survivors (67 and 33% for 10 and 30 days post-irradiation) was observed for 250 mg kg(-1) (p<0.002 and<0.02 for 10- and 30-day survival, respectively). Ascorbic acid treatment caused a dose-dependent elevation in the wound contraction and highest contraction was observed for 250 mg kg(-1). The wound contraction was significantly greater at 3 (p<0.005), 6 (<0.05) and 9 (<0.05) days post-irradiation with 250 mg kg(-1) ascorbic acid. The complete healing of the wound was effected by day 22.8 post-irradiation in the ascorbic acid-treated irradiation group. CONCLUSION: Administration of ascorbic acid protected mice against radiation-induced sickness, mortality and improved healing of wounds after exposure to whole-body gamma-radiation. Additional studies will be directed toward analysing the role of successive administration of ascorbic acid to protect non-target tissues during radiotherapy and in initiating and supporting the cascade of tissue repair processes in radiotherapy delayed wounds.

The evaluation of the acute toxicity and long term safety of hydroalcoholic extract of Sapthaparna (Alstonia scholaris) in mice and rats.

The acute and sub-acute toxic effects of various doses of hydroalcoholic extract of Alstonia scholaris (ASE) was studied in mice and rats. The acute toxicity in mice depended on the season of collection of plant. The highest acute toxicity was observed in the ASE prepared from the summer collection followed by winter. The least toxicity was observed in the extract prepared from the bark of A. scholaris collected in the monsoon season. The administration of different doses of ASE showed a dose dependent increase in the toxicity in all species of mice. The Swiss albino mice were found to be the most sensitive followed by the DBA and C(57)BL. The crossbred mice were resistant when compared to the pure inbred strains. The oral administration of ASE was non-toxic up to a dose of 2000 mg/kg b. wt., while maximum number of animals succumbed to death after administration of 1100 mg/kg ASE by intraperitoneal route. The rats were more sensitive than the mice as the LD(50) dose of ASE was lesser for the former than the latter. The sub-acute toxicity in the rats was carried out with 120 and 240 mg/kg b. wt. ASE (1/10th and 1/5th of the LD(50) dose of ASE). The 240 mg was observed to be more toxic than 120 mg/kg ASE since it caused mortality and deformity in various organs of the recipient animals. The various biochemical parameters like AST, ALT, ACP, ALP, CK, LDH, creatinine, urea, ammonia, glucose and LPx were higher at 240 mg/kg ASE when compared with the 120 mg and the non-drug treated animals. In contrast, the total protein, albumin, DNA, RNA, cholesterol, glucose, glutathione, total thiols declined in the 240 mg/kg ASE treated animals when compared with non-drug treated controls. The hematological analysis showed a dose dependent decrease in the RBC, WBC, hemoglobin, neutrophils and monocytes, while a significant increase in the lymphocytes, eosinophils and basophils was observed. The observed toxic effect of ASE may be due to the presence of echitamine. Our studies shows that at high doses, A. scholaris exhibited marked damage to all the major organs of the body.

Improved sensitivity for solid-support invasive cleavage reactions with flow cytometry analysis.

A new configuration of the solid-support invasive cleavage reaction provides a small reaction-volume format for high-sensitivity discrimination of nucleic acid targets with single nucleotide differences. With target concentrations as low as 2 amol/assay, the solid-support invasive cleavage reaction clearly distinguishes single base mutations. Two oligonucleotides tethered to the solid support hybridize to the target nucleic acid, forming a tripartite substrate that can be recognized and cleaved by Cleavase, a structure-specific 5'-nuclease. Each cleavage event yields fluorescence signal on the surface. When microspheres serve as the solid-support surface, analysis by fluorometer imparts real-time information about change in the reaction signal over time. Flow cytometry provides an alternative detection technology that collects endpoint information about the reaction signal on individual microspheres. A reaction volume of 10 microL with as few as 3000 microspheres is sufficient to distinguish single nucleotide differences at target concentrations less than 200 fM. This sensitivity level is within the range required for analysis of SNPs in genomic DNA. In addition, the flow cytometry format has multiplexing potential, making the microsphere-based invasive cleavage assay attractive for high-throughput genomic applications.

Molecular characterization of a calcium binding translationally controlled tumor protein homologue from the filarial parasites Brugia malayi and Wuchereria bancrofti.

We have cloned homologues of the mammalian translationally controlled tumor protein (TCTP) from the human filarial parasites Wuchereria bancrofti and Brugia malayi. TCTP genes from B. malayi and W. bancrofti were expressed in a T7 promoter vector as histidine tagged fusion proteins. Both the recombinant B. malayi TCTP (rBm-TCTP) and recombinant W. bancrofti TCTP (rWb-TCTP) have a molecular mass of approximately 28 kDa with the histidine tag. Sequence analyses showed that there is a 98% similarity between the two filarial TCTPs at amino acid levels and are immunologically cross-reactive. Analysis of soluble proteins from various lifecycle stages of B. malayi suggested that the expression of Bm-TCTP might be differentially regulated and occurs in multimeric form. Recombinant TCTP were found to form multimers in solution under non-reducing conditions. The tendency for filarial TCTPs to become multimers was predicted by the presence of the Lupas coiled coil structure in their sequence. Despite the absence of a signal sequence, Bm-TCTP is present abundantly in the excretory/secretions (ES) of microfilariae. Characterization studies showed that both Bm- and Wb-TCTPs are calcium-binding proteins and have histamine-releasing function in vitro. When injected intraperitoneally both the filarial TCTPs induced inflammatory infiltration of eosinophils into the peritoneal cavity of mice suggesting that the filarial TCTPs may have a role in the allergic inflammatory responses associated with filarial infections.

Suppression of cutaneous inflammation by intradermal gene delivery.

Biological effects of in vivo transfection of a potential anti-inflammatory gene, designated Sm16, cloned from the human parasite Schistosoma mansoni were analyzed in these studies. A single intradermal injection of a full-length cDNA of Sm16 resulted in the expression of Sm16 in the epidermis, dermis, skin migratory cells and skin-draining lymph nodes of mice for up to 7 days. Subsequently the anti-inflammatory effect of this gene expression was evaluated by inducing an inflammatory response in the skin of mice. These studies showed that Sm16 gene delivery resulted in a significant suppression of cutaneous inflammation as shown by a reduction in cutaneous edema, decrease in neutrophil infiltration, suppression of pro-inflammatory cytokine expression and down-regulation of ICAM-1 expression in the skin inflammatory site. Cells collected from the skin-draining lymph nodes showed reduced proliferation to mitogen. Multiple intradermal injection of Sm16 cDNA failed to induce any antibody response in mice for up to 8 weeks after initial injection. These findings suggest a potential for developing Sm16 gene delivery as a therapeutic agent for treating inflammatory skin disorders.