Leonard I. Sweet

Xenobiotic-induced apoptosis: significance and potential application as a general biomarker of response

The process of apoptosis, often coined programmed cell death, involves cell injury induced by a variety of stimuli including xenobiotics and is morphologically, biochemically, and physiologically distinct from necrosis. Apoptotic death is characterized by cellular changes such as cytoplasm shrinkage, chromatin condensation, and plasma membrane asymmetry. This form of cell suicide is appealing as a general biomarker of response in that it is expressed in multiple cell systems (e.g. immune, neuronal, hepatal, intestinal, dermal, reproductive), is conserved phylogenetically (e.g. fish, rodents, birds, sheep, amphibians, roundworms, plants, humans), is modulated by environmentally relevant levels of chemical contaminants, and indicates a state of stress of the organism. Further, apoptosis is useful as a biomarker as it serves as a molecular control point and hence may provide mechanistic information on xenobiotic stress. Studies reviewed here suggest that apoptosis is a sensitive and early indicator of acute ...The process of apoptosis, often coined programmed cell death, involves cell injury induced by a variety of stimuli including xenobiotics and is morphologically, biochemically, and physiologically distinct from necrosis. Apoptotic death is characterized by cellular changes such as cytoplasm shrinkage, chromatin condensation, and plasma membrane asymmetry. This form of cell suicide is appealing as a general biomarker of response in that it is expressed in multiple cell systems (e.g. immune, neuronal, hepatal, intestinal, dermal, reproductive), is conserved phylogenetically (e.g. fish, rodents, birds, sheep, amphibians, roundworms, plants, humans), is modulated by environmentally relevant levels of chemical contaminants, and indicates a state of stress of the organism. Further, apoptosis is useful as a biomarker as it serves as a molecular control point and hence may provide mechanistic information on xenobiotic stress. Studies reviewed here suggest that apoptosis is a sensitive and early indicator of acute and chronic chemical stress, loss of cellular function and structure, and organismal health. Examples are provided of the application of this methodology in studies of health of lake trout (Salvelinus namaycush) in the Laurentian Great Lakes.

Effects of selected polybrominated diphenyl ether flame retardants on lake trout (Salvelinus namaycush) thymocyte viability, apoptosis, and necrosis

Polybrominated diphenyl ether (PBDE) flame-retardants have been identified as an emergent contaminants issue in many parts of the world. In vitro analyses were conducted to test the hypothesis that selected PBDEs congeners affect viability, apoptosis, and necrosis of thymocytes from laboratory-reared lake trout (Salvelinus namaycush). At current environmental levels (< 1 mg/L), effects of the tested PBDEs on thymocytes were negligible. However, at 100 mg/L, major effects were seen for congener brominated diphenyl ether 47 (BDE-47) and minor effects were seen for congener BDE-99.

Age-related changes in mature CD4+ T cells: cell cycle analysis.

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.

Acute and chronic life cycle toxicity of acenaphthene and 2,4,6-trichlorophenol to the midge Paratanytarsus parthenogeneticus (Diptera: Chironomidae).

The acute and life cycle toxicities of acenaphthene and 2,4,6-trichlorophenol (2,4,6-TCP) were evaluated using the parthenogenic dipteran larva, Paratanytarsus parthenogeneticus, of the family Chironomidae. Static 48 h acute toxicity tests employed third instar larvae, and the flow-through 20-day life cycle tests were initiated with eggs. No acute lethal effect was observed in the saturated concentration (2.1 mg/l) of acenaphthene in water, and the median lethal concentration of 2,4,6-TCP was approximately 40 mg/l. In the life cycle toxicity test with acenaphthene, hatchability of the midge was affected by 50% at an exposure concentration of approximately 0.17 mg/l. The median effective concentration for larval, pupal and adult development was found between 0.06 and 0.07 mg/l, respectively, and these were significantly lower than the effective concentration for hatching success. With regard to 2,4,6-TCP toxicity, the median effective concentration for midge hatchability was 4.23 mg/l. The larval development, pupal formation and adult emergence, however were impeded at 1.60, 1.46 and 1.33 mg/l, respectively. Each level was noticeably lower than the median effective concentration for hatchability. The concentrations for each compound that interfered with the development to fourth instar, pupa and adult were not significantly different. These results indicate that larval developments after hatching were the most sensitive stages and affected the success of midge growth.

Effects of polychlorinated biphenyls, hexachlorocyclohexanes, and mercury on human neutrophil apoptosis, actin cytoskelton, and oxidative state.

Apoptosis, or programmed cell death, has been proposed as a biomarker for environmental contaminant effects. In this work, we test the hypothesis that in vitro assays of apoptosis are sensitive indicators of immunological effects of polychlorinated biphenyls, hexachlorocyclohexanes, and mercury on human neutrophils. Apoptosis, necrosis, and viability as well as the related indicators F-actin levels, and active thiol state were measured in purified human neutrophils after treatment with contaminants. Effective concentrations observed were 0.3μM (60μg/L) mercury, 750μg/L Aroclor 1254, and 50μM (14,500μg/L) hexachlorocylcohexanes. Concentrations of contaminants that induced apoptosis also decreased cellular F-actin levels. Active thiols were altered by mercury, but not organochlorines. Comparison of these data with levels of contaminants reported to be threats to human health indicate neutrophil apoptosis is a sensitive indicator of mercury toxicity.

The Role of Wiskott-Aldrich Syndrome Protein in Signaling Cytoskeletal Remodeling |[bull]| 793

Analysis of Wiskott-Aldrich Syndrome Protein (WASP) has revealed several domains, including domains homologous to actin regulating proteins. It has also been shown that expression of WASP in cells induces actin polymerization and that this is dependent upon WASP binding CDC42, a small GTP-binding protein involved in cytoskeletal signaling. These observations have led to the hypothesis that WASP plays a role in signaling to the actin cytoskeleton and that an underlying defect in this pathway due to mutations in WASP leads to the clinical manifestations associated with the classic Wiskott-Aldrich Syndrome (WAS) or the milder form, X-linked thrombocytopenia (XLT). To test this theory we have examined the kinetics of actin polymerization in neutrophils (PMNs), monocytes, lymphocytes and platelets in response to a variety of stimuli in WAS and XLT patients. In addition, we obtained scanning electron microscopic (SEM) images of platelets during actin remodeling in order to examine the ability of these cells to produce cytoarchitectural features associated with stimulation (microspikes, filopodia). PMNs were stimulated through signal transduction pathways important to phagocytosis (3G8 antibody), chemotaxis (fMLP), and adhesion (IB4 antibody). Stimulation of these pathways in PMNs isolated from WAS and XLT patients resulted in an actin polymerization response that was kinetically and quantitatively indistinguishable from control cells. Stimulation of monocytes from both WAS and XLT patients with fMLP also resulted in a normal actin polymerization response. As platelets and lymphocytes are thought to be the most severely affected cells in this disorder, these cells were examined for their ability to dynamically remodel their actin cytoskeleton. Lymphocytes isolated from WAS as well as XLT patients were able to produce a normal actin remodeling response to stimulation with phorbol ester and Bryostatin. SEM analysis of platelets isolated from WAS and XLT patients showed that they were able to produce robust microspikes and filopodia upon activation with PGF2α. These cells also produced an actin polymerization response to ADP and PGF2α that was indistinguishable from control cells. These observations present comprehensive evidence that signaling and functioning of the actin cytoskeleton is normal in peripheral blood cells isolated from WAS as well as XLT patients. These data call into question the hypothesis that WASP plays a critical role in signaling to the actin cytoskeleton and that an underlying defect in this pathway leads to the clinical manifestations seen in WAS.