Safety assessment for nail cosmetics: Framework for the estimation of systemic exposure through the nail plate.

All cosmetics products, including nail care products, must be evaluated for their safety. The assessment of systemic exposure is a key component of the safety assessment. However, data on the exposure, especially via ungual route (nail plate) are limited. Based on the physicochemical properties of human nails and permeability data of topical onychomycosis drugs, the nail plate is considered a good barrier to chemicals. We examine factors impacting penetration of nail care ingredients through the nail plate, including properties of the nails of the ingredients and formulations. The molecular weight, vapor pressure, logP, water solubility, and keratin binding, as well as formulations properties e.g., polymerization of acrylate monomers are considered important factors affecting penetration. To estimate systemic exposure of nail care ingredients through the nail plate, a standardized framework is applied that quantifies the impacts of these properties on penetration with an adjustment factor for each of these influencing properties. All the adjustment factors are then consolidated to derive an integrated adjustment factor which can be used for calculation of the systemic exposure dose for the ingredient. Several case studies are presented to reflect how this framework can be used in the exposure assessment for nail cosmetic products.

Raw single-wall carbon nanotubes induce oxidative stress and activate MAPKs, AP-1, NF-kappaB, and Akt in normal and malignant human mesothelial cells.

BACKGROUND: Single-wall carbon nanotubes (SWCNTs), with their unique physicochemical and mechanical properties, have many potential new applications in medicine and industry. There has been great concern subsequent to preliminary investigations of the toxicity, biopersistence, pathogenicity, and ability of SWCNTs to translocate to subpleural areas. These results compel studies of potential interactions of SWCNTs with mesothelial cells. OBJECTIVE: Exposure to asbestos is the primary cause of malignant mesothelioma in 80-90% of individuals who develop the disease. Because the mesothelial cells are the primary target cells of asbestos-induced molecular changes mediated through an oxidant-linked mechanism, we used normal mesothelial and malignant mesothelial cells to investigate alterations in molecular signaling in response to a commercially manufactured SWCNT. METHODS: In the present study, we exposed mesothelial cells to SWCNTs and investigated reactive oxygen species (ROS) generation, cell viability, DNA damage, histone H2AX phosphorylation, activation of poly(ADP-ribose) polymerase 1 (PARP-1), stimulation of extracellular signal-regulated kinase (ERKs), Jun N-terminal kinases (JNKs), protein p38, and activation of activator protein-1 (AP-1), nuclear factor kappaB (NF-kappaB), and protein serine-threonine kinase (Akt). RESULTS: Exposure to SWCNTs induced ROS generation, increased cell death, enhanced DNA damage and H2AX phosphorylation, and activated PARP, AP-1, NF-kappaB, p38, and Akt in a dose-dependent manner. These events recapitulate some of the key molecular events involved in mesothelioma development associated with asbestos exposure. CONCLUSIONS: The cellular and molecular findings reported here do suggest that SWCNTs can cause potentially adverse cellular responses in mesothelial cells through activation of molecular signaling associated with oxidative stress, which is of sufficient significance to warrant in vivo animal exposure studies.

Requirements of phosphatidylinositol-3 kinase and mammalian target of rapamycin for estrogen-induced proliferation in uterine leiomyoma- and myometrium-derived cell lines.

OBJECTIVE: This study was undertaken to investigate the effects of 17beta-estradiol (E2) on G1 cell cycle progression and proliferation in uterine fibroid and myometrial cells and the roles of phosphatidylinositol-3 kinase and mammalian target of rapamycin in mediating these estrogen effects. STUDY DESIGN: The human uterine smooth muscle-derived cells (UtSM) and uterine leiomyoma-derived cells (UtLM) were treated with varying doses of E2 with or without pretreatment with LY294002, a phosphatidylinositol-3 kinase inhibitor, or rapamycin, a mammalian target of rapamycin inhibitor. The effects of E2 on cell cycle progression and proliferation and the roles of phosphatidylinositol-3 kinase and mammalian target of rapamycin in E2-induced effects were studied. RESULTS: Compared with controls, E2 significantly induced G1 cell cycle progression and proliferation in uterine smooth muscle-derived cells and uterine leiomyoma-derived cells. These effects, however, were significantly blocked when LY294002 or rapamycin was used. CONCLUSION: E2 significantly induces G1 cell cycle progression and cell proliferation in uterine smooth muscle-derived cells and uterine leiomyoma-derived cells, in which phosphatidylinositol-3 kinase and mammalian target of rapamycin are essentially required.

Role of mitochondria in silica-induced apoptosis of alveolar macrophages: inhibition of apoptosis by rhodamine 6G and N-acetyl-L-cysteine.

Induction of apoptosis by silica in alveolar macrophages (AM) may be a critical step in silica-induced lung injury and pulmonary fibrosis. This study investigated the mechanism(s) through which silica induces apoptosis in AM and their production of proinflammatory cytokines. Using N-acetyl-L-cysteine (NAC) for glutathione (GSH) synthesis and removal of reactive oxygen species (ROS), and rhodamine 6G (R6G) to inhibit the mitochondrial-dependent function, this study found that silica-induced apoptosis of rat AM in primary culture is mitochondria dependent and exhibits a mechanism involving ROS generation, increased mitochondrial release of cytochrome c, and the activation of caspase 9, but not caspase 8, activity. Silica-induced apoptosis was accompanied by a lowering of intracellular and mitochondrial GSH (mGSH) and was blocked by pretreatment of cells with NAC or R6G. When cells were exposed to silica and then treated with either NAC or R6G, silica-induced apoptosis was not affected by the blocking agent. In addition, R6G, which inhibited cellular ATP production and mitochondrial ROS generation, had no effect on apoptosis induced by exogenous hydrogen peroxide or superoxide. Pretreatment of cells with NAC or R6G also inhibited silica-induced production of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha, but the inhibition of these cytokines with agents known to block their secretion did not protect cells from silica-induced apoptosis. Data indicate that silica-induced apoptosis is mediated through mitochondrial generation of ROS, which may be inhibited by pretreatment of cells with R6G that prevents ROS generation, or with NAC that maintains a high level of mGSH. The secretion of IL-1beta and TNF-alpha by silica-exposed AM was markedly inhibited by NAC and R6G, suggesting that the production of these cytokines is also ROS dependent.

Suppression of phagocytic and bactericidal functions of rat alveolar macrophages by the organic component of diesel exhaust particles.

Exposure to diesel exhaust particles (DEP) was shown to increase the susceptibility of the lung to bacterial infection in rats. In this study, the effects of DEP on alveolar macrophage (AM) phagocytic and bactericidal functions and cytokine secretion by AM and lymphocytes in response to Listeria monocytogenes infection were investigated in vitro and the roles of different DEP components in these processes were compared. Exposure to DEP or the organic extracts of DEP (eDEP) significantly decreased the phagocytosis and killing of L. monocytogenes by AM obtained from normal rats. Washed DEP (wDEP) also decreased AM phagocytosis and bacterial killing to a lesser extent, whereas carbon black (CB) reduced AM phagocytosis but had no significant effect on AM bactericidal activity. DEP or eDEP concentration-dependently suppressed L. monocytogenes-induced secretion of tumor necrosis factor-alpha, interleukin (IL)-1beta, and IL-12 by AM and of IL-2 and interferon-gamma by lymphocytes obtained from L. monocytogenes-infected rats, but augmented the AM secretion of IL-10. wDEP or CB, however, exerted little or no effect on these L. monocytogenes-induced cytokines. These results provide direct evidence that DEP, through the actions of organic components, suppresses AM phagocytic and bactericidal functions in vitro. Inhibition of AM phagocytic function and alterations of AM and lymphocyte cytokine secretion by DEP and DEP organic compounds may be implicated in the diminished AM bactericidal activity and the lymphatic arm of the host immune system, thus resulting in an suppressed pulmonary clearance of L. monocytogenes and an increased susceptibility of the lung to bacterial infection.

Silica-induced apoptosis in alveolar macrophages: evidence of in vivo thiol depletion and the activation of mitochondrial pathway.

Studies have shown that silica induces apoptosis through mechanisms that also regulate the inflammatory responses of lung cells to silica exposure. Although implicated in cell culture studies, the major in vivo pathway through which silica induces apoptosis has not been characterized. The present study is to study the role of mitochondria in silica-induced oxidative stress and apoptosis in vivo. Rats were intratracheally instilled with saline or silica (20 mg/kg) and sacrificed at 3 days post-exposure unless otherwise specified. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage and measured for apoptosis and secretion of inflammatory mediators in the presence or absence of appropriate inhibitors. Concurrent studies were carried out to determine the presence of intracellular reactive oxygen species (ROS) via confocal microscopy, mitochondrial trans-membrane potential by flow cytometry, mitochondrial release of cytochrome c, and the activation of caspase activities in AM by Western blot analysis. Silica was shown to induce elevated levels of intracellular ROS, resulting in a marked decrease in intracellular glutathione (GSH) and cysteine and a sustained presence of apoptotic AM in silica-exposed rats up to two weeks post-exposure. The apoptotic AM were characterized by decreased mitochondrial trans-membrane potential, increased mitochondrial release of cytochrome c, activated caspase 9 (but not caspase 8) and caspase 3 activities, and PARP degradation, comparing to cells from the saline control. Silica induced AM production of IL-1 and TNF-alpha, which may be inhibited by ex vivo treatment of cells with N-acetylcysteine (NAC) or microtubule modifiers such as tetrandrine and taxol. NAC was shown to prevent intracellular GSH depletion and silica-induced production of IL-1beta and TNF-alpha but not apoptosis in AM from silica-exposed rats. These results show that silica-induced apoptosis is mediated through the mitochondrial pathway but not through cellular production of inflammatory cytokines, ROS generation, however, induces both apoptosis and cellular secretion of inflammatory mediators.

Sustained effect of inhaled diesel exhaust particles on T-lymphocyte-mediated immune responses against Listeria monocytogenes.

Studies have shown that exposure to diesel exhaust particles (DEP) suppresses pulmonary host defense against bacterial infection. The present study was carried out to characterize whether DEP exposure exerts a sustained effect in which inhaled DEP increase the susceptibility of the lung to bacterial infection occurring at a later time. Brown Norway rats were exposed to filtered air or DEP by inhalation at a dose of 21.2 +/- 2.3 mg/m3, 4 h/day for 5 days, and intratracheally instilled with saline or 100,000 Listeria monocytogenes (Listeria) 7 days after the final DEP exposure. Bacterial growth and cellular responses to DEP and Listeria exposures were examined at 3 and 7 days post-infection. The results showed that inhaled DEP prolonged the growth of bacteria, administered 7 days post DEP exposure, in the lung as compared to the air-exposed controls. Pulmonary responses to Listeria infection were characterized by increased production of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12, and IL-10 by alveolar macrophages (AM) and increased presence of T lymphocytes and their CD4+ and CD8+ subsets in lung draining lymph nodes that secreted elevated levels of IL-2, IL-6, IL-10, and interferon (IFN)-gamma. Diesel exhaust particles were found to inhibit Listeria-induced production of IL-1beta and TNF-alpha, which are responsible for the innate immunity, and IL-12, which initiates the development of T helper (Th)1 responses, but enhance Listeria-induced AM production of IL-10, which prolongs Listeria survival in these phagocytes. The dual action of DEP on AM production of IL-12 and IL-10 correlated with an inhibition of the development of bacteria-specific T lymphocytes by DEP. Cytokine production by lymphocytes from DEP- and Listeria-exposed rats showed a marked decrease in the production of IL-2, IL-10, and IFN-gamma compared to Listeria infection alone, suggesting either that DEP inhibit the production of cytokines by lymphocytes or that these lymphocytes contained T-cell subsets that are different from those of Listeria infection alone and less effective in mediating Th1 immune responses. This study demonstrates that inhaled DEP, after a 7-day resting period, increase the susceptibility of the lung to bacterial infection occurring at a later time by inhibiting macrophage immune function and suppressing the development of T-cell-mediated immune responses. The results support the epidemiological observations that exposure to DEP may be responsible for the pulmonary health effects on humans.

Effect of diesel exhaust particles on allergic reactions and airway responsiveness in ovalbumin-sensitized brown Norway rats.

We have previously demonstrated that exposure to diesel exhaust particles (DEP) prior to ovalbumin (OVA) sensitization in rats reduced OVA-induced airway inflammation. In the present study, Brown Norway rats were first sensitized to OVA (42.3 +/- 5.7 mg/m3) for 30 min on days 1, 8, and 15, then exposed to filtered air or DEP (22.7 +/- 2.5 mg/m3) for 4 h/day on days 24-28, and challenged with OVA on day 29. Airway responsiveness was examined on day 30, and animals were sacrificed on day 31. Ovalbumin sensitization and challenge resulted in a significant infiltration of neutrophils, lymphocytes, and eosinophils into the lung, elevated presence of CD4+ and CD8+ T lymphocytes in lung draining lymph nodes, and increased production of serum OVA-specific immunoglobulin (Ig)E and IgG. Diesel exhaust particles pre-exposure augmented OVA-induced production of allergen-specific IgE and IgG and pulmonary inflammation characterized by marked increases in T lymphocytes and infiltration of eosinophils after OVA challenge, whereas DEP alone did not have these effects. Although OVA-sensitized rats showed modest response to methacholine challenge, it was the combined DEP and OVA exposure that produced significant airway hyperresponsiveness in this animal model. The effect of DEP pre-exposure on OVA-induced immune responses correlated with an interactive effect of DEP with OVA on increased production of reactive oxygen species (ROS) and nitric oxide (NO) by alveolar macrophages (AM) and alveolar type II (ATII) cells, NO levels in bronchoalveolar lavage fluid, the induction of inducible NO synthase expression in AM and ATII cells, and a depletion of total intracellular glutathione (GSH) in AM and lymphocytes. These results show that DEP pre-exposure exacerbates the allergic responses to the subsequent challenge with OVA in OVA-sensitized rats. This DEP effect may be, at least partially, attributed to the elevated generation of ROS in AM and ATII cells, a depletion of GSH in AM and lymphocytes, and an increase in AM and ATII cell production of NO.

Exposure of brown Norway rats to diesel exhaust particles prior to ovalbumin (OVA) sensitization elicits IgE adjuvant activity but attenuates OVA-induced airway inflammation.

Exposure to diesel exhaust particles (DEP) during the sensitization process has been shown to increase antigen-specific IgE production and aggravate allergic airway inflammation in human and animal models. In this study, we evaluated the effect of short-term DEP exposure on ovalbumin (OVA)-mediated responses using a post-sensitization model. Brown Norway rats were first exposed to filtered air or DEP (20.6 +/- 2.7 mg/m3) for 4 h/day for five consecutive days. One day after the final air or DEP exposure (day 1), rats were sensitized with aerosolized OVA (40.5 +/- 6.3 mg/m3), and then again on days 8 and 15, challenged with OVA on day 29, and sacrificed on days 9 or 30, 24 h after the second OVA exposure or the final OVA challenge, respectively. Control animals received aerosolized saline instead of OVA. DEP were shown to elicit an adjuvant effect on the production of antigen-specific IgE and IgG on day 30. At both time points, no significant airway inflammatory responses and lung injury were found for DEP exposure alone. However, the OVA-induced inflammatory cell infiltration, acellular lactate dehydrogenase activity and albumin content in bronchoalveolar lavage (BAL) fluid, and numbers of T cells and their CD4+ and CD8+ subsets in lung-draining lymph nodes were markedly reduced by DEP on day 30 compared with the air-plus-OVA exposure group. The OVA-induced nitric oxide (NO) in the BAL fluid and production of NO, interleukin (IL)-10, and IL-12 by alveolar macrophages (AM) were also significantly lowered by DEP on day 30 as well as day 9. DEP or OVA alone decreased intracellular glutathione (GSH) in AM and lymphocytes on days 9 and 30. The combined DEP and OVA exposure resulted in further depletion of GSH in both cell types. These results show that short-term DEP exposure prior to sensitization had a delayed effect on enhancement of the sensitization in terms of allergen-specific IgE and IgG production, but caused an attenuation of the allergen-induced airway inflammatory responses.

Roles of reactive oxygen species and heme oxygenase-1 in modulation of alveolar macrophage-mediated pulmonary immune responses to Listeria monocytogenes by diesel exhaust particles.

Diesel exhaust particles (DEP) have been shown to suppress alveolar macrophage (AM)-mediated pulmonary immune responses to Listeria monocytogenes in vivo. In this study, effects of DEP-derived reactive oxygen species (ROS) and heme oxygenase (HO)-1 on AM-mediated immune responses to L. monocytogenes were investigated. Brown Norway rats were intratracheally inoculated with 100,000 L. monocytogenes, and AM were isolated at 7 days post-infection. Exposure to DEP or their organic extract (eDEP), but not the washed DEP (wDEP) or carbon black, increased intracellular ROS and HO-1 expression in AM. Induction of ROS and HO-1 by eDEP was partially reversed by alpha-naphthoflavone, a cytochrome P450 1A1 inhibitor, and totally blocked by N-acetylcysteine. In addition, exposure to eDEP, but not wDEP, inhibited lipopolysacchride-stimulated secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-12 (IL-12), but augmented production of IL-10 by AM. Kinetic studies showed that modulation of cytokines by eDEP was preceded by ROS and HO-1 induction. Furthermore, pretreatment of AM with superoxide dismutase (SOD) or zinc protoporphrin IX (Znpp), which attenuated eDEP-induced HO-1 expression/activity, substantially inhibited eDEP effect on IL-10. Finally, direct stimulation with pyrogallol (PYR), a superoxide donor, upregulated HO-1 and IL-10 but decreased secretion of IL-12 in L. monocytogenes-infected AM. These results show that DEP, through eDEP-mediated ROS, induce HO-1 expression and IL-10 production and at the same time inhibit AM production of TNF-alpha and IL-12 to dampen the host immune responses. The results also suggest that HO-1 may play an important role in regulating production of IL-10 by DEP-exposed and L. monocytogenes-infected AM.

Suppression of cell-mediated immune responses to listeria infection by repeated exposure to diesel exhaust particles in brown Norway rats.

Diesel exhaust particles (DEP) have been shown to alter pulmonary immune responses to bacterial infection. Exposure of rats to 100 mg/m(3) DEP for 4 h was found to aggravate Listeria monocytogenes(Listeria) infection at 3 days postinfection, but the bacteria were largely cleared at 7 days postinfection due to the development of a strong T cell-mediated immunity. In the present study, we examined the effects of repeated DEP exposure at lower doses on pulmonary responses to bacterial infection. Brown Norway rats were exposed to DEP by inhalation at 20.62 +/- 1.31 mg/m 3 for 4 h/day for 5 days, followed by intratracheal inoculation with 100,000 Listeria at 2 h after the last DEP exposure. DEP-exposed rats showed a significant increase in lung bacterial load at both 3 and 7 days postinfection. The repeated DEP exposure was shown to suppress both the innate, orchestrated by alveolar macrophages (AM), and T cell-mediated responses to Listeria. DEP inhibited AM production of interleukin- (IL-) 1beta, tumor necrosis factor- (TNF-) alpha, and IL-12 but enhanced Listeria-induced AM production of IL-10, which has been shown to prolong the survival of intracellular pathogens such as Listeria. DEP exposure also suppressed the development of bacteria-specific lymphocytes from lung-draining lymph nodes, as indicated by the decreased numbers of T lymphocytes and their CD4(+) and CD8(+) subsets. Furthermore, the DEP exposure markedly inhibited the Listeria-induced lymphocyte secretion of IL-2 at day 7, IL-10 at days 3 and 7, and interferon- (IFN-) gamma at days 3 to 10 postinfection when compared to air-exposed controls. These results show a sustained pattern of downregulation of T cell-mediated immune responses by repeated low-dose DEP exposure, which is different from the results of a single high-dose exposure where the acute effect of DEP aggravated bacteria infection but triggered a strong T cell-mediated immunity.