Victoria A. Wong

Reactive Oxygen Species as a Signal in Glucose-Stimulated Insulin Secretion

One of the unique features of β-cells is their relatively low expression of many antioxidant enzymes. This could render β-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H2O2. In both models, insulin secretion could be stimulated by provision of either exogenous H2O2 or diethyl maleate, which raises intracellular H2O2 levels. Provision of exogenous H2O2 scavengers, including cell permeable catalase and N-acetyl-l-cysteine, inhibited glucose-stimulated H2O2 accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H2O2, had no effect on GSIS. Because oxidative stress is an important risk factor for β-cell dysfunction in diabetes, the relationship between glucose-induced H2O2 generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H2O2 derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function.

Low-Level Arsenic Impairs Glucose-Stimulated Insulin Secretion in Pancreatic Beta Cells: Involvement of Cellular Adaptive Response to Oxidative Stress

Background Chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with incidence of type 2 diabetes (T2D). A key driver in the pathogenesis of T2D is impairment of pancreatic β-cell function, with the hallmark of β-cell function being glucose-stimulated insulin secretion (GSIS). Reactive oxygen species (ROS) derived from glucose metabolism serve as one of the metabolic signals for GSIS. Nuclear factor-erythroid 2–related factor 2 (Nrf2) is a central transcription factor regulating cellular adaptive response to oxidative stress. Objectives We tested the hypothesis that activation of Nrf2 and induction of antioxidant enzymes in response to arsenic exposure impedes glucose-triggered ROS signaling and thus GSIS. Methods and results Exposure of INS-1(832/13) cells to low levels of arsenite led to decreased GSIS in a dose- and time-dependent fashion. Consistent with our hypothesis, a significantly enhanced Nrf2 activity, determined by its nuclear accumulation and induction of its target genes, was observed in arsenite-exposed cells. In keeping with the activation of Nrf2-mediated antioxidant response, intracellular glutathione and intracellular hydrogen peroxide–scavenging activity was dose dependently increased by arsenite exposure. Although the basal cellular peroxide level was significantly enhanced, the net percentage increase in glucose-stimulated intracellular peroxide production was markedly inhibited in arsenite-exposed cells. In contrast, insulin synthesis and the consensus GSIS pathway, including glucose transport and metabolism, were not significantly reduced by arsenite exposure. Conclusions Our studies suggest that low levels of arsenic provoke a cellular adaptive oxidative stress response that increases antioxidant levels, dampens ROS signaling involved in GSIS, and thus disturbs β-cell function.

When Nanoparticles Get in the Way: Impact of Projected Area on In Vivo and In Vitro Macrophage Function

Previous reports by others establish that particle surface area is related to a change in macrophage function as measured by the ability to clear particles from the alveolar spaces. However, for nanoparticles the relation may not be strictly due to surface chemistry: The cumulative projected area of the particles may reflect the degree to which the inner or outer surface of the macrophage is shielded from other objects or molecules. We apply this alternative interpretation to in vitro measurements of macrophage uptake of 26-nm-diameter fluorescent beads and to in vivo data presented in a classic inhalation toxicology paper on nano-sized TiO2 particles. In their paper, Oberdörster et al. (Environ. Health Perspect. 102[suppl. 5]:173–179, 1994) reported that following inhalation exposure to 20-nm or 250-nm TiO2 particles, the half-times for alveolar clearance of polystyrene test particles were proportional to square centimeters of TiO2 particle surface per million macrophages; macrophage toxicity from TiO2 particle surface was assumed to be the cause of the decrease in the clearance rate of polystyrene test particles. When TiO2 particle projected area was incorporated into the in vivo macrophage dosimetry calculations, particle projected areas ranged in value from covering only a fraction (0.1) of the macrophage surface to covering the cell surface 4 times over. The observed decrease in macrophage mediated alveolar clearance of polystyrene test particles was directly related to the potential for TiO2 particles to mask the surface of the macrophage—a possibility that was visualized in vitro with confocal laser scanning microscopy.

Benzene-Induced Hematotoxicity and Bone Marrow Compensation in B6C3F1 Mice

Long-term inhalation exposure of benzene has been shown to cause hematotoxicity and an increased incidence of acute myelogenous leukemia in humans. The progression of benzene-induced hematotoxicity and the features of the toxicity that may play a major role in the leukemogenesis are not known. We report the hematological consequences of benzene inhalation in B6C3F1 mice exposed to 1, 5, 10, 100, and 200 ppm benzene for 6 hr/day, 5 days/week for 1, 2, 4, or 8 weeks and a recovery group. There were no significant effects on hematopoietic parameters from exposure to 10 ppm benzene or less. Exposure of mice to 100 and 200 ppm benzene reduced the number of total bone marrow cells, progenitor cells, differentiating hematopoietic cells, and most blood parameters. Replication of primitive progenitor cells in the bone marrow was increased during the exposure period as a compensation for the cytotoxicity induced by 100 and 200 ppm benzene. In mice exposed to 200 ppm benzene, the primitive progenitor cells maintained an increased percentage of cells in S-phase through 25 days of recovery compared with controls. The increased replication of primitive progenitor cells in concert with the reported genotoxicity induced by benzene provides the components necessary for producing an increased incidence of lymphoma in mice. Furthermore, we propose this mode of action as a biologically plausible mechanism for benzene-induced leukemia in humans exposed to high concentrations of benzene.

Immunotexicological effects of benzene inhalation in male Sprague-Dawley rats

The inhalation of benzene is toxic to various components of the immunologie system in rodents. Spleen and thymus weights, total spleen and femur marrow cell counts, enumeration of spleen B- and T-lymphocytes, and an assessment of humoral immunocompetence, were used to evaluate the immunotoxicity of benzene in male Sprague-Dawley rats. Rats were exposed to 0, 30, 200 or 400 ppm benzene for 6 h/day, 5 days/week for 2 or 4 weeks. An early indicator of immunotoxicity was a reduction in the number of B-lymphocytes after 2 weeks of 400 ppm. After 4 weeks of 400 ppm, there was a reduction in thymus weight and spleen B-, CD4+ /CD5+ and CD5+ T-lymphocytes. Rats exposed to 30, 200 or 400 ppm benzene for 2 or 4 weeks and challenged with sheep red blood cells developed a humoral response comparable to that of the control (0 ppm) animals. Enumeration of spleen T- and B-lymphocytes in rats exposed to benzene and challenged with SRBC showed only a transient reduction in spleen B-lymphocytes after 2 weeks of exposure to 400 ppm. These data suggest that there are no immunotoxicological effects of exposure to 200 ppm benzene or less, in rats exposed for 6 h/day, 5 days/week for 2 or 4 weeks.

Exposure of hematopoietic stem cells to benzene or 1,4-benzoquinone induces gender-specific gene expression.

Chronic exposure to benzene results in progressive decline of hematopoietic function and may lead to the onset of various disorders, including aplastic anemia, myelodysplastic syndrome, and leukemia. Damage to macromolecules resulting from benzene metabolites and misrepair of DNA lesions may lead to changes in hematopoietic stem cells (HSCs) that give rise to leukemic clones. We have shown previously that male mice exposed to benzene by inhalation were significantly more susceptible to benzene‐induced toxicities than females. Because HSCs are targets for benzene‐induced cytotoxicity and genotoxicity, we investigated DNA damage responses in HSC from both genders of 129/SvJ mice after exposure to 1,4‐benzoquinone (BQ) in vitro or benzene in vivo. 1,4‐BQ is a highly reactive metabolite of benzene that can cause cellular damage by forming protein and DNA adducts and producing reactive oxygen species. HSCs cultured in the presence of 1,4‐BQ for 24 hours showed a gender‐independent, dose‐dependent cytotoxic response. RNA isolated from 1,4‐BQ–treated HSCs and HSCs from mice exposed to 100 ppm benzene by inhalation showed altered expression of apoptosis, DNA repair, cell cycle, and growth control genes compared with unexposed HSCs. Rad51, xpc, and mdm‐2 transcript levels were increased in male but not female HSCs exposed to 1,4‐BQ. Males exposed to benzene exhibited higher mRNA levels for xpc, ku80, ccng, and wig1. These gene expression differences may partially explain the gender disparity in benzene susceptibility. HSC culture systems such as the one used here will be useful for testing the hematotoxicity of various substances, including other benzene metabolites.

Effects of benzene on splenic, thymic, and femoral lymphocytes in mice

Chronic exposure to high concentrations of benzene, primarily by inhalation, can affect the function of the human immune system. Limited data are available on the immunotoxic effects of low concentrations of benzene. This study evaluated the effects of 1, 5, 10, 100, and 200 ppm benzene on lymphocytes in mice exposed by inhalation for up to 8 weeks. Exposure to 100 or 200 ppm benzene induced rapid and persistent reductions in femoral B-, splenic T- and B-, and thymic T-lymphocytes. The percentage of femoral B-lymphocytes and thymic T-lymphocytes in apoptosis was increased 6- to 15-fold by 200 ppm benzene compared to controls. Replication of femoral B-lymphocytes was increased during the exposure period in the bone marrow as a compensation for the lymphocyte loss induced by 100 and 200 ppm benzene. Exposure of mice to 10 ppm benzene or less did not have a statistically significant effect on numbers or replication of the lymphocyte populations evaluated. A reduced number of splenic B-lymphocytes after 2 weeks of exposure to benzene appeared to be the most sensitive end point and time point for evaluating benzene cytotoxicity in this study.

Nasal uptake of inhaled acrolein in rats.

An improved understanding of the relationship between inspired concentration of the potent nasal toxicant acrolein and delivered dose is needed to support quantitative risk assessments. The uptake efficiency (UE) of 0.6, 1.8, or 3.6 ppm acrolein was measured in the isolated upper respiratory tract (URT) of anesthetized naive rats under constant-velocity unidirectional inspiratory flow rates of 100 or 300 ml/min for up to 80 min. An additional group of animals was exposed to 0.6 or 1.8 ppm acrolein, 6 h/day, 5 days/wk, for 14 days prior to performing nasal uptake studies (with 1.8 or 3.6 ppm acrolein) at a 100 ml/min airflow rate. Olfactory and respiratory glutathione (GSH) concentrations were also evaluated in naive and acrolein-preexposed rats. Acrolein UE in naive animals was dependent on the concentration of inspired acrolein, airflow rate, and duration of exposure, with increased UE occurring with lower acrolein exposure concentrations. A statistically significant decline in UE occurred during the exposures. Exposure to acrolein vapor resulted in reduced respiratory epithelial GSH concentrations. In acrolein-preexposed animals, URT acrolein UE was also dependent on the acrolein concentration used prior to the uptake exposure, with preexposed rats having higher UE than their naive counterparts. Despite having increased acrolein UE, GSH concentrations in the respiratory epithelium of acrolein preexposed rats were higher at the end of the 80 min acrolein uptake experiment than their in naive rat counterparts, suggesting that an adaptive response in GSH metabolism occurred following acrolein preexposure.

Genotoxicity and cytotoxicity in male B6C3F1 mice following exposure to mixtures of 1,3-butadiene and styrene

1,3‐Butadiene and styrene are oxidized, in part, by cytochrome P450 2E1 and have been shown to metabolically interact in rodents exposed by inhalation to mixtures of both compounds. Because the reactive metabolites of butadiene and styrene are thought to be responsible for the toxicity of each compound, metabolic interactions may alter the response in animals exposed to mixtures of butadiene and styrene compared with the response in animals exposed to butadiene alone or styrene alone. The purpose of this study was to quantitate alterations in genotoxicity and cytotoxicity in male B6C3F1 mice exposed to mixtures of butadiene and styrene. Male B6C3F1 mice were exposed to 6.25, 62.5, 200, or 625 ppm butadiene alone, 50 ppm styrene alone, or mixtures of 6.25, 62.5, 200, or 625 ppm butadiene and 50 ppm styrene. Genotoxicity was assessed by quantitating the frequency of micronucleated polychromatic erythrocytes in bone marrow. Cytotoxicity was assessed by counting total spleen and thymus cells and by quantitating the frequency of polychromatic erythrocytes in the peripheral blood. Butadiene and mixtures of butadiene and styrene were genotoxic in mice, as shown by a significant increase in the frequency of micronucleated polychromatic erythrocytes. The increased frequency following exposure to mixtures of butadiene and styrene was not significantly different compared with the frequency following exposure to butadiene alone. Styrene and mixtures of butadiene and styrene were cytotoxic in mice, as shown by significantly decreased number of spleen cells. Exposure to mixtures of butadiene and styrene with butadiene concentrations of 62.5 or 625 ppm significantly reduced the number of thymus cells. Exposure to 200 ppm or 625 ppm butadiene alone, or to mixtures of 200 ppm or 625 ppm butadiene and 50 ppm styrene, significantly reduced the frequency of polychromatic erythrocytes in the peripheral blood. The results of the study demonstrate that exposure to mixtures of butadiene and styrene does not reduce the respective genotoxicity of butadiene or cytotoxicity of styrene. Environ. Mol. Mutagen. 29: 335–345, 1997.

Changes in intracellular pH play a secondary role in hydrogen sulfide-induced nasal cytotoxicity.

Hydrogen sulfide (H2S) is a naturally occurring gas that is also associated with several industries. The potential for widespread human inhalation exposure to this toxic gas is recognized as a public health concern. The nasal epithelium is particularly susceptible to H2S-induced pathology. Cytochrome oxidase inhibition is postulated as one mechanism of H2S toxicity. Another mechanism by which the weak acid H2S could cause nasal injury is intracellular acidification and cytotoxicity. To further understand the mechanism by which H2S damages the nasal epithelium, nasal respiratory and olfactory epithelial cell isolates and explants from naive rats were loaded with the pH-sensitive intracellular chromophore SNARF-1 and exposed to air or 10, 80, 200, or 400 ppm H2S for 90 min. Intracellular pH was measured using flow cytometry or confocal microscopy. Cell lysates were used to quantify total protein and cytochrome oxidase activity. A modest but statistically significant decrease in intracellular pH occurred following exposure of respiratory and olfactory epithelium to 400 ppm H2S. Decreased cytochrome oxidase activity was observed following exposure to >10 ppm H2S in both respiratory and olfactory epithelia. None of the treatments resulted in cytotoxicity. The intracellular acidification of nasal epithelial cells by high-dose H2S exposure and the inhibition of cytochrome oxidase at much lower H2S concentrations suggest that changes in intracellular pH play a secondary role in H2S-induced nasal injury.