Skin sensitization in silico protocol.

The assessment of skin sensitization has evolved over the past few years to include in vitro assessments of key events along the adverse outcome pathway and opportunistically capitalize on the strengths of in silico methods to support a weight of evidence assessment without conducting a test in animals. While in silico methods vary greatly in their purpose and format; there is a need to standardize the underlying principles on which such models are developed and to make transparent the implications for the uncertainty in the overall assessment. In this contribution, the relationship between skin sensitization relevant effects, mechanisms, and endpoints are built into a hazard assessment framework. Based on the relevance of the mechanisms and effects as well as the strengths and limitations of the experimental systems used to identify them, rules and principles are defined for deriving skin sensitization in silico assessments. Further, the assignments of reliability and confidence scores that reflect the overall strength of the assessment are discussed. This skin sensitization protocol supports the implementation and acceptance of in silico approaches for the prediction of skin sensitization.

Evaluation of the potential anti-cancer activity of the antidepressant sertraline in human colon cancer cell lines and in colorectal cancer-xenografted mice.

Evidence has been provided of the anti-proliferative activity of certain antidepressants, mainly the selective serotonin reuptake inhibitors (SSRIs). We tested the effect of different antidepressants on cell viability and proliferation of human colorectal carcinoma cell lines HT29 and the multi-drug resistant (MDR) LS1034. The SSRIs, paroxetine and sertraline, induced a dose-dependent inhibition of cell viability and proliferation in the two cell lines (IC50 8-15 micro M). When compared to cytotoxic agents e.g. doxorubicin, vincristine and 5-fluorouracil, the SSRIs showed comparable activity (HT29) or a superior effect (LS1034). Using flow cytometry analysis, we found that the two SSRIs arrested cells at the G0/G1 stage and stimulated DNA fragmentation in a dose-dependent manner. The SSRIs (10 and 20 microM) increased caspase-3 activation. Western blot analysis showed an increase after 24 h in c-Jun and a decrease in the expression of the anti-apoptotic protein Bcl-2. The results suggest a proapoptotic activity for the active SSRIs accompanied by mitogen-activated protein kinase cascade activation and Bcl-2 inhibition. In vivo, we used CD1 nude mice xenografted subcutaneously with HT29 cells. On day 8, after cell inoculation sertraline or paroxetine (15 mg/kg x3/week i.p.) were administered to animals (6/group), which were monitored weekly (for 5 weeks) for tumor volume and body weight. At 5 weeks, the animals survived, with no significant difference in body weight. Sertraline, though not paroxetine, significantly inhibited tumor growth. Collectively, our results suggest that the widely-used antidepressant, sertraline, possesses a potential anti-tumor activity, which circumvents the MDR mechanism. Since SSRI therapy is frequently indicated in cancer patients, the use of sertraline in colon cancer patients with co-morbidity of depression seems attractive.

Evidence for an inhibitory immunomodulatory effect of selected antidepressants on rat splenocytes: possible relevance to depression and hyperactive-immune disorders.

Antidepressants have been found to possess antiproliferative effect. In the immune system depression may activate pro-inflammatory cytokines. Therefore, the aim of this study was to assess the immunomodulatory activity of antidepressants in naïve rat. Rat splenocytes were activated with con A and treated with paroxetine, sertraline or clomipramine ex vivo. We found that the antidepressants inhibit cell viability and proliferation at IC50 of 5-8 microM of mitogen-stimulated rat splenocytes. This inhibitory effect was accompanied by cell cycle arrest and increase in apoptotic events as assayed by FACS. Moreover, antidepressants decrease the secretion of the TH1 factor--TNFalpha. In addition, the antidepressants reduced the expression of the enzyme cyclooxygenase2 which is involved in inflammation. On the cellular level we show the up-regulation of MAPK death signaling pathway and suppression of the anti-apoptotic factor--Bcl-2. These findings reveal the immunomodulatory effect of the selected antidepressants. These data suggest a novel use of antidepressants or their derivatives.

Immunomodulatory effect of selective serotonin reuptake inhibitors (SSRIs) on human T lymphocyte function and gene expression.

Antidepressants have an antiproliferative effect in some cell lines. Depression may be associated with activation of some pro-inflammatory cytokines. Therefore, we evaluated the ex-vivo immunomodulatory effect of selective serotonin reuptake inhibitors (SSRIs) in T cells. We found that the SSRIs, paroxetine and sertraline decreased T-cell viability with IC50 around 10 microM. The inhibition obtained with exposure to the SSRIs was more pronounced than that achieved with dexamethasone. Moreover, these SSRIs inhibit the secretion of the TH1 factor-tumor necrosis factor(TNF)alpha from the cells. On the molecular level, the SSRIs suppressed signal transducer and activator of transcription 3 (Stat3) and cyclooxygenase(Cox)2 protein expression. The inhibitory effects were accompanied by alterations in gene expression as assessed in the gene array. These findings reveal an immunomodulatory effect of the SSRIs paroxetine and sertraline in human T cells. The clinical implications of our findings merit further investigation.

Differential attenuation of oxidative/nitrosative injuries in early prostatic neoplastic lesions in TRAMP mice by dietary antioxidants.

BACKGROUND: Dietary antioxidants with yet unproven efficacies in averting prostate cancer (PCa) are widely used in the United States as preventives. Experimental evidence establishing a causal relationship between oxidative and nitrosative stress (OS/NS) and PCa development and showing its modulation by dietary antioxidants would help justify their usage. METHODS: The TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) mouse model was used to demonstrate the OS/NS-associated damage, as evident by 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxynonenal (4-HNE)-protein-adducts and nitrotyrosine (Ntyr), in prostatic premalignant lesions, and to evaluate the antioxidant efficacy of various dietary supplements [natural antioxidant (NAO) from spinach extracts, (-) epigallocatechin-3-gallate (EGCG), or N-acetylcystein (NAC)] during the early PCa development. RESULT: We show, for the first time, that oxidative/nitrosative damages of genomic DNA and cellular proteins were discretely localized in premalignant lesions, but not in adjacent morphologically normal epithelia, of TRAMP prostates; these injuries were absent in age-matched nontransgenic littermates. The extent of OS/NS-related injuries correlated well with the tempo of development and prevalence of premalignant lesions in various prostatic lobes and exhibited a clear trend of increase from 12 to 20 weeks of age. Treatment of TRAMP mice with various antioxidants as dietary supplements resulted in differential alleviation of OS/NS-related prostatic injuries. The antioxidant potencies of the dietary supplements did not fully correlate with their documented antiPCa actions, suggest that they may exert additional "nonantioxidant," antitumor effects in this model. CONCLUSIONS: Our data indicate that in TRAMP mice, OS/NS injuries are likely involved in early prostatic tumorigenesis and can be modulated by various antioxidants.

Halofuginone inhibits serum-stimulated pericardial tissue retraction in vitro.

We developed an in vitro model of tissue contraction in which living pericardium, in response to serum, contracted and the cells in situ expressed proliferating cell nuclear antigen (PCNA) and synthesized collagen. Here we evaluated the effects of halofuginone on these serum-stimulated pericardial tissue responses. Parietal pericardium was incubated with media containing increasing doses of halofuginone and evaluated for tissue contraction, evident by tissue curling. Proliferation was measured by MTS metabolism and PCNA expression. Furthermore, collagen synthesis was compared between samples incubated with halofuginone, cytochalasin B, cytochalasin D, aphidicolin, or cytosine arabinoside (AraC), using Masson's trichrome and the monoclonal antibody to sheep type I procollagen, SP1. D8. Halofuginone inhibited tissue contraction, cellular proliferation, and collagen synthesis in a dose-dependent manner. In contrast, cytochalasin B, cytochalasin D, aphidicolin, and AraC, shown previously to prevent cellular proliferation, did not prevent type I collagen synthesis. Halofuginone has been implicated as an agent in the prevention of wound-healing fibrosis. This study suggests that halofuginone may have an added benefit in the inhibition of pericardial tissue contraction, which appeared to be related to the synthesis of type I procollagen.

Composition, efficacy, and safety of spinach extracts.

Spinach leaves, containing several active components, including flavonoids, exhibit antioxidative, antiproliferative, and antiinflammatory properties in biological systems. Spinach extracts have been demonstrated to exert numerous beneficial effects, such as chemo- and central nervous system protection and anticancer and antiaging functions. In this review article, we present a compilation of data generated in our laboratories and those of other investigators describing the chemical composition of spinach, its beneficial effects, relative safety information, and its recommended inclusion in the human diet. A powerful, water-soluble, natural antioxidant mixture (NAO), which specifically inhibits the lipoxygenase enzyme, was isolated from spinach leaves. The antioxidative activity of NAO has been compared to that of other known antioxidants and found to be superior in vitro and in vivo to that of green tea, N-acetylcysteine (NAC), butylated hydroxytoluene (BHT), and vitamin E. NAO has been tested for safety and is well tolerated in several species, such as mouse, rat, and rabbit. NAO has been found to be nonmutagenic and has shown promising anticarcinogenic effects in a few experimental models, such as skin and prostate cancer; it has not shown any target-organ toxicity or side effects. The current review provides epidemiological and preclinical data supporting the efficacy of extracts of spinach and the safety of its consumption.

A natural antioxidant mixture from spinach does not have estrogenic or antiestrogenic activity in immature CD-1 mice.

The use of natural antioxidants and flavonoids in nutritional and pharmaceutical applications is increasing. Because some phytochemicals such as genistein, found in soy products, have estrogenic activity, we investigated the estrogenic potential of a natural antioxidant mixture (NAO) isolated from spinach leaves, using an in vivo uterotrophic bioassay and an in vitro transcriptional activation assay for the estrogen receptor (ER). Outbred female CD-1 mice (17 d old) were given subcutaneous injections of 17beta-estradiol or genistein [500 and 500,000 microg /(kg x d), respectively] as positive controls or NAO [1000 to 1,000,000 microg/(kg x d)] for 3 d. Uterine wet weight/body weight ratios were determined. Both 17beta-estradiol and genistein significantly increased uterine wet weight ratios compared with untreated controls, but NAO did not. Histological examination of the uterus showed that 17beta-estradiol and genistein increased epithelial cell height, number and gland development, but NAO did not. Estrogenic activity of NAO was investigated in vitro using the ER transcriptional activation assay. BG1Luc4E2 cells expressing ER were stably transfected with a luciferase reporter gene responsive to estrogens. 17beta-estradiol dose dependently increased luciferase activity; NAO had no effect. When NAO was tested for antiestrogenic activity, it did not lessen the effects of 17beta-estradiol. These data suggest that NAO does not have estrogenic or antiestrogenic activity. Thus, an antioxidant mixture has been identified that does not have potentially adverse estrogenic activity.

Forestomach tumor induction by 2,4-hexadienal in F344N rats and B6C3F1 mice.

2,4-Hexadienal (2,4-Hx) was studied for its toxicity and carcinogenicity because of its alpha, beta-unsaturated aldehyde structure and potential link between exposure to lipid peroxidation products in the diet and human malignancies. Male and female F344N rats and B6C3F1 mice received 2,4-Hx in corn oil by gavage for 16 days, 14 weeks, or 2 years. In the 16-day studies 2,4-Hx induced forestomach necrosis and ulceration at 240 mg/kg and forestomach epithelial hyperplasia at 80 mg/kg in rats and mice. In the 14-week studies the chemical induced forestomach hyperplasia and nasal olfactory atrophy or necrosis at 120 mg/kg in rats and mice. In the 2-year studies 2,4-Hx induced squamous cell papilloma and carcinoma of the forestomach in male and female rats at 45 and 90 mg/kg and in male and female mice at 120 mg/kg. Two male mice in the 120 mg/kg group had uncommon squamous cell carcinoma of the oral cavity (tongue). Mechanistic studies indicated that the forestomach carcinogenesis in rats and mice may be due to depletion of glutathione as a result of oxidative stress induced by 2,4-Hx.

Slowing tumorigenic progression in TRAMP mice and prostatic carcinoma cell lines using natural anti-oxidant from spinach, NAO--a comparative study of three anti-oxidants.

The TRAMP model and human prostatic cancer (PCA) cell lines DU145 and PC3 are useful forchemopreventive studies. We compared the efficacy of 3 anti-oxidants [a water-soluble natural anti-oxidant. NAO (200 mg/kg). found in spinach leaves; epigallocatechin-3 gallate, EGCG (200 mg/kg), a major green tea polyphenol; and N-acetylcysteine, NAC (125 mg/kg)] plus vehicle in slowing spontaneous tumorigenic progression in TRAMP and wild-type male mice. Sacrifices occurred on weeks 5, 9, and 13. Prostatic histopathology and oxidative-stress blood markers were evaluated. Hyperplasias were ranked by a combination of severity grade and distribution (focal, multifocal, and diffuse). The effectivity of each tested compound in reducing the severity/focalness of hyperplasia varied from lobe to lobe. NAO exerted a significant effect on the dorsal and lateral lobes; NAC, on the anterior and ventral lobes, and EGCG, on the ventral lobe. When the most severe hyperplasia in all 4 lobes of TRAMPs was evaluated, only NAO reduced hyperplasia at weeks 9 and 13. Plasma peroxide levels in TRAMPs were reduced following oral administration of NAO or NAC for 13 weeks; EGCG only slightly reduced these levels. In NAO-treated DU 145 and PC3 PCA cells, inhibition of cellular proliferation occurred in a dose-dependent manner, increasing numbers of G1 cells and reducing ROS levels. The anti-oxidative and antiproliferative properties of NAO may explain its efficacy in slowing the spontaneous prostatic carcinogenic process in the TRAMP and its effects in the cell lines.