Biomedical subjects

I M Shapiro

Publications and source records attributed to I M Shapiro.

At least 19 recordsLinked to original sources

PubMed2001

Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism.

In a previous investigation we showed that phosphate ions (Pi) induced apoptosis of terminally differentiated hypertrophic chondrocytes. To explore the mechanism by which Pi induces cell death, we asked the following two questions. First, can we prevent Pi-induced apoptosis by inhibiting plasma membrane Na-Pi cotransport? Second, which specific Na-Pi transporters are expressed in chondrocytes and are they developmentally regulated? Terminally differentiated hypertrophic chondrocytes were isolated from chick tibial cartilage and cell death was measured in the presence of 3-7 mmol/L Pi. To ascertain whether apoptosis was linked to a rise in cellular Pi loading, we examined the effect of phosphonoformic acid (PFA), a competitive inhibitor of Na-Pi cotransport on Pi-induced apoptosis in chondrocytes. We found that 1 mmol/L PFA blocked anion-induced cell death and prevented an increase in the cell Pi content. In a parallel study, we determined that the bisphosphonate, alendronate, also protected chondrocytes from death, albeit at a lower concentration than PFA. Using a DNA end-labeling procedure, we showed that the Pi-treated cells were apoptotic and, as might be predicted, the presence of PFA blocked induction of the death sequence. Next, we examined the expression of two Pi transporters in relation to chondrocyte maturation and anion treatment. We noted that there was expression of the constitutive transporter, Glvr-1, and a type II cotransporter in chick growth plate cells. Although these transport systems are active in terminally differentiated cells, it is probable that the initiation of apoptosis may require the induction of other Pi-transport systems. It is concluded that, at the mineralization front, cell death is linked directly to the elevation in environmental anion concentration and the concomitant rise in intracellular Pi levels.

Alendronate↗

PubMed2000

Effect of varying physical properties of porous, surface modified bioactive glass 45S5 on osteoblast proliferation and maturation.

The objective of this study was to determine the effect of porous bioactive glass (45S5) substrate characteristics on the expression and maintenance of the osteoblastic phenotype. We cultured ROS 17/2. 8 cells on substrates with different pore size and porosity for periods up to 14 days and analyzed the characteristics of the cells and extracellular matrix. Results of the study show that the glass substrates supported the proliferation and growth of osteoblast-like cells. Although the morphologies of the cells differed on the various substrates, their shape and the extent of membrane ruffling suggested that they maintained high levels of metabolic activity. Cells on all substrates expressed high levels of alkaline phosphatase activity and produced extracellular matrices that mineralized to form nonstoichiometric, carbonated, calcium-deficient apatites. An important finding was that at a given porosity of 44%, the pore size neither directed nor modulated the in vitro expression of the osteoblastic phenotype. In contrast, porosity did affect cellular function. We noted that at an average pore size of 92 microm, as the porosity increased from 35 to 59%, osteoblast activity was reduced. As designed in this experiment, an increase in the porosity led to a corresponding increase in total surface area of the specimens. With increasing porosity and surface area, glass reactions in the media may persist for longer durations at higher intensities, thereby affecting local media composition. As such, we suggest that extensive conditioning treatments before cell seeding can reduce this effect. Our results also revealed that the expression of the osteoblastic phenotype is enhanced by the ongoing glass dissolution. The reaction pathway at the origin of this effect still needs to be elucidated. Taken together, the findings support the overall hypothesis that in vitro cell activity can be controlled by a careful selection of substrate properties.

Alkaline Phosphatase↗

PubMed2000

Bioactive glass serves as a substrate for maintenance of phenotype of nucleus pulposus cells of the intervertebral disc.

The objective of the study was to investigate the capability of bioactive glass 45S5 to serve as a substrate for nucleus pulposus cells in vitro. Nucleus pulposus cells were isolated from adult rabbit discs and seeded onto bioactive glass. At selected time intervals, the cells and glass were evaluated. We found that the cells rapidly attached to the substrate, colonizing it within 12 h. By 21 days, they had formed a lawn of cells over the glass substrate. DNA measurements showed a progressive increase in cell number with time. The phenotype was maintained; the cells expressed aggrecan, and collagen type II and I, but were negative for collagen type X. CD44, a cell-surface glycoprotein that binds hyaluronate, was also expressed by these cells. Electron dispersive X-ray analysis and Fourier transform infrared spectroscopy revealed calcium phosphate-rich layer formation on the substrate surface. The results of this study suggest that nucleus pulposus cell proliferation may be an anchorage dependent event, and that the cells use the calcium phosphate-rich layer to facilitate cell adhesion, and subsequent proliferation. These findings point to the importance of the conditioned bioactive glass as a substrate for nucleus pulposus cells.

Aggrecans↗

PubMed2000

Mercury-induced apoptosis in human lymphoid cells: evidence that the apoptotic pathway is mercurial species dependent.

There is growing evidence that heavy metals, in general, and mercurial compounds, in particular, are toxic to the human immune system. In this regard, we have previously shown that both inorganic and organic mercurials are potent human T-cell apoptogens; moreover, mitochondria appear to be a target organelle for the induction of cell death. To ascertain whether both mercury species utilize the same molecular pathway to trigger the apoptotic cascade, cells were treated with MeHgCl or HgCl2 and mitochondrial activity was examined. We show that both mercury species affect mitochondrial activity by inducing the development of a membrane permeability transition. This state is characterized by a decline in both the transmembrane potential and the intracellular pH, as well as the generation of reactive oxygen species. We also determined that mercury exposure results in a decline in the T-cell GSH content. Since mitochondrial dysfunction and the development of a permeability transition may result in the release of cytochrome c, a factor that promotes apoptosis, we assessed the abilities of both species of mercury to induce the translocation of cytochrome c from mitochondria to the cytosol. We noted that MeHgCl caused a significant increase in cytosolic cytochrome c. Surprisingly, however, HgCl2 did not alter the level of cytosolic cytochrome c. We next determined whether the mercurials could alter the level of the anti-apoptotic protein Bcl-2. Our results demonstrate that HgCl2 induces a significant elevation in the Bcl-2 content of T-cells; in contrast, T-cells treated with MeHgCl did not exhibit altered levels of this anti-apoptotic protein. Regardless of whether cytochrome c is released from the mitochondria, both mercurial species were capable of activating the caspase cascade, as evident by cleavage of poly (ADP-ribose) polymerase. Thus, our study shows that, whereas each of the mercury species shares common features in the apoptotic process, profound differences exist in a number of key steps in the pathway. The significance of these differences is discussed.

Adult↗

PubMed2000

Inorganic phosphate induces apoptosis of osteoblast-like cells in culture.

The major goal of this investigation was to test the hypothesis that one of the major products of bone resorption, inorganic phosphate (Pi), activates osteoblast apoptosis. Osteoblast-like cells were isolated from explants of human bone. In monolayer culture, these cells showed an osteogenic phenotype. Thus, the cells exhibited raised alkaline phosphatase activity, expressed osteogenic messenger RNA transcripts, and formed biological mineral. When these cells were treated with 1-7 mmol/L Pi there was a dose- and time-dependent decrease in cell viability. Accordingly, after 48 h, 5 mmol/L Pi reduced the number of viable osteoblast-like cells by 25%; 7 mmol/L Pi reduced the number of cells by 60%. By 96 h, following treatment with 5 mmol/L Pi, the percentage of viable cells was 30%, whereas 7 mmol/L Pi caused an almost complete loss of osteoblast viability. Osteoblast death was blocked by treating the cells with phosphonoformic acid, an inhibitor of the plasma-membrane Na-Pi transporter. Using morphological and end-labeling procedures, we confirmed that cell death was through apoptosis. To probe the mechanism of cell death, osteoblast-like cells were probed with rhodamine 123, a dye that is responsive to the membrane potential. We noted that Pi-treated cells displayed a profound loss of mitochondrial membrane potential, suggesting that the anion activated the death program through the induction of a mitochondrial membrane permeability transition. We conclude that high levels of osteoblast apoptosis observed at sites of bone resorption may be linked to release of Pi from bone mineral.

Apoptosis↗

PubMed2000

Evaluation of osteoblast response to porous bioactive glass (45S5) substrates by RT-PCR analysis.

Previous studies have shown that neonatal rat calvaria osteoblasts elaborate substantial amounts of extracellular material with bone-like characteristics when cultured on porous bioactive glass substrates in vitro. However, the osteoblastic response to this material has not been fully characterized. The objective of this study was to characterize osteoblast response to porous bioactive glass substrates following the expression of the classical markers for osteoblast differentiation. In this study we synthesized porous bioactive glass substrates, seeded them with osteoblast-like cells (ROS 17/2.8) and followed the temporal expression of alkaline phosphatase (AP) activity, as well as the expression of mRNA for collagen type I (Coll-1), osteonectin (OSN), osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP). The data confirm that porous bioactive glass substrates are capable of supporting the in vitro growth and maturation of osteoblast-like cells. At a porosity of 42% and an average pore size of 80 microm, the substrates promote the expression and maintenance of the osteoblastic phenotype. The results additionally suggest that there is both a solution-mediated and a surface-controlled effect on cell activity.

Alkaline Phosphatase↗

PubMed1999

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

1-Carboxyglutamic Acid↗

PubMed1999

Induction of apoptosis in human T-cells by methyl mercury: temporal relationship between mitochondrial dysfunction and loss of reductive reserve.

The objective of our study was to define the mechanism by which MeHgCl induces human T-cell apoptosis. We asked the question: does mercury disrupt the Deltapsim and induce a mitochondrial permeability transition state? Using two fluorescent reagents, JC-1 and DiOC6(3), we demonstrated that MeHgCl exposure resulted in a decrease in the Deltapsim. Since a decline in Deltapsim can disturb the pHi, we employed SNARF-1 to assess pHi; results indicate that mercury treatment reduced the pHi from 7.0 to 6.5. Consistent with these observations, we noted that uncoupled electron transfer reactions generated ROS, while cardiolipin, a mitochondrial phospholipid, was oxidized. In concert with the biochemical changes, there was a decrease in overall dimension of the mitochondria of mercury-treated cells and a loss in cristae architecture. The toxicant also depleted the thiol reserves of the cell and promoted translocation of cytochrome c from the mitochondria to the cytosol. Furthermore, when T cells were thiol-depleted, there was increased susceptibility to MeHgCl-induced apoptosis. Finally, we established a temporal relationship between the decline in Deltapsim, generation of ROS, and depletion of thiol reserves. The earliest detectable event was at the level of the mitochondrion; in the presence of MeHgCl there was a profound reduction in mitochondrial Deltapsim and a decline in GSH levels within 1 h. Subsequently, a further decrease in thiol reserves was linked to the generation of ROS. We propose that the target organelle for MeHgCl is the mitochondrion and that induction of oxidative stress leads to activation of death-signaling pathways.

Adult↗

PubMed1999

Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes.

Epiphyseal chondrocytes end their life cycle through apoptosis. While this event provides a mechanism for the removal of terminally differentiated cells from cartilage, agents that promote this physiological process have not been defined. To address this issue, using a cell culture technique that models events that take place in the growth plate, we asked the following questions: Can agents that promote chondrocyte maturation and cartilage mineralization serve as specific triggers for cell death? Are chondrocytes susceptible to apoptogens at a singular developmental stage? Treatment of embryonic tibial chondrocytes with inorganic phosphate (Pi) induced death in a dose- and time-dependent manner. Within 48 hr, 3 mM Pi increased chondrocyte death by 30%; lower concentrations of Pi induced death after 48 hr. To ascertain if death was due to apoptosis, we evaluated Pi-induced death by a number of different methods and compared the results to those induced by the apoptogen, staurosporine. Analysis of the death process indicated that cartilage cells shared many of the common biological features of the apoptotic process. Thus, there was DNA fragmentation, terminal deoxynucleotidyl transferase (TUNEL) labeling, an increase in cells in the sub-G1 fraction of the cell cycle, and morphological evidence of apoptosis. To explore the specificity of the Pi effect, the experiment was repeated using embryonic sternal cephalic and caudal chondrocytes, cells that are at an earlier developmental stage than the terminally differentiated tibial cells. We noted that these cells remained vital despite a major increase in the medium Pi content. Results of this study suggest that Pi is a stage-specific inducer of apoptosis in maturing chondrocytes and that this role may be linked to chondrocyte maturation and mineralization of the extracellular matrix.

Animals↗

PubMed1999

Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate.

In the companion article, we reported that the local phosphate (Pi) concentration triggers apoptosis in epiphyseal chondrocytes. The goal of the current investigation was to evaluate the apoptotic process in relationship to the energy status of cells in the growth plate. For these studies, we used sections of the adolescent growth plate, as well as cells isolated from the tissue. We found that there was a maturation-dependent loss of mitochondrial function in growth plate chondrocytes and these cells generated energy by glycolysis. Since treatment with the uncoupler 2,4-dinitrophenol as well as the site-specific inhibitors antimycin A and rotenone failed to elicit a further increase in the activity of the glycolytic pathway, we concluded that oxidative metabolism was minimum in these cells. Flow cytometric studies of growth plate cells and confocal microscopy of growth plate sections using the mitochondrial probes Rh123 and DiOC6(3) provided unequivocal evidence that there was loss of mitochondrial membrane potential in hypertrophic cells. Furthermore, the intrinsic fluorescence of the flavoprotein lipoamide dehydrogenase complex of the electron transport chain revealed that the mitochondria were in an oxidized state. Finally, we assessed Bcl-2 expression in these cells. Although immunohistochemical and Western blot analysis showed that the chick cells contained a low level of the anti-apoptotic protein Bcl-2, reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that transcripts were present in chondrocytes. Based on these observations, we suggest that terminally differentiated chondrocytes undergo a maturation-dependent loss of mitochondrial function. In concert with the low expression of Bcl-2, they become sensitive to signals for programmed cell death. We hypothesize that Pi triggers apoptosis in these energy-compromised cells by promoting a mitochondrial membrane transition, thereby inducing the death process.

Adenosine Triphosphate↗

PubMed1999

Effect of serum proteins on osteoblast adhesion to surface-modified bioactive glass and hydroxyapatite.

Previous studies indicate that modification of the surface of porous bioactive glass promotes osteoblast function. We hypothesize that bone formation on treated bioactive glass is due to the selective adsorption of serum attachment proteins. To test this hypothesis, we examined the profile of proteins adsorbed to treated bioactive glass and compared these proteins with those adsorbed to untreated bioactive glass and porous hydroxyapatite. Porous bioactive glass was treated with Tris-buffered electrolyte solution to generate a calcium phosphate-rich surface layer and then immersed in tissue-culture medium containing 10% serum. Proteins adsorbed to the ceramic surfaces were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Porous hydroxyapatite bound a higher amount of total protein than did the other substrates. However, surface-modified porous bioactive glass adsorbed more fibronectin than did hydroxyapatite. The effect of serum-protein adsorption on osteoblast adhesion to bioactive glass and hydroxyapatite was also evaluated. Cell adhesion to porous bioactive glass that was surface-modified and serum-treated was significantly greater than to porous bioactive glass that was either surface-modified or serum-treated. Furthermore, cell adhesion to porous bioactive glass treated to form the dual layer of calcium phosphate and serum protein was significantly higher than adhesion to porous hydroxyapatite with adsorbed serum protein. Results of the study strongly suggest that adsorption of serum fibronectin to the surface of modified porous bioactive glass coated with calcium phosphate may be responsible for enhanced osteoblast adhesion.

Absorption↗

PubMed1999

Discovery: Osf2/Cbfa1, a master gene of bone formation.

This report reviews the current research that has impacted on our understanding of osteogenesis. Recent studies indicate that the transcription factor Osf2 (osteoblast specific transcription factor 2)/Cbfa1 (core binding factor activity 1) serves as a Master Gene regulating osteoblast-specific gene expression. The gene is expressed in cells of the osteoblast lineage only, and this expression is regulated by calciotropic agents. Moreover, when expressed in non-skeletal cells, the cells assume many of the characteristics of an osteoblast. In knockout experiments designed to assess the importance of the gene in osteogenesis, no evidence of bone formation could be observed in animals that are homozygous for the deletion. Studies of the heterozygote indicate that osteoblast function is compromised: there is a severe reduction in the number of bone cells, the tissue is deficient in bone proteins, and the activity of the enzyme alkaline phosphatase is low. It was noted that the heterozygote displays abnormalities that are remarkably similar to those exhibited by cleidocranial dysplastics. Indeed, Osf2 mapped close to a chromosomal locus on chromosome 6p21, long suspected of being involved with the disease. A search conducted for Osf2 mutations in kindreds with cleidocranial dysplasia revealed deletions, insertions, and missense mutations; these mutations are found to segregate with patients who are defined clinically as cleidocranial dysplastic. Aside from providing a new insight into a disease state that has so far avoided molecular analysis, results of the studies emphasize that the loss of a Master Gene drastically alters the development and maintenance of the appendicular skeleton and the craniofacial complex.

Animals↗

PubMed1999

Developmental expression of creatine kinase isoenzymes in chicken growth cartilage.

We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid-treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2-7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.

Animals↗

PubMed1998

Si-Ca-P xerogels and bone morphogenetic protein act synergistically on rat stromal marrow cell differentiation in vitro.

This study describes a novel bioactive xerogel glass as a carrier for bone morphogenetic protein (BMP) and the value of this carrier in terms of stimulating osteogenic activity of rat stromal marrow cells in vitro. These cells were seeded onto the surface of xerogel glass disks with BMP either incorporated in the glass, adsorbed to the surface of the glass, or added to the culture media and then compared to cells on glass with no added BMP or to cells on tissue culture plastic (TCP) with and without BMP. Cells were cultured for 6 and 10 days and examined for total DNA, alkaline phosphatase activity, and osteocalcin and total protein production. Stromal cell differentiation, as measured by alkaline phosphatase activity and osteocalcin synthesis was most increased when the BMP was incorporated or adsorbed onto the xerogel glass. Cells on xerogel glass without BMP were more differentiated than cells grown on plastic with BMP, thereby demonstrating the additive effect of a bioactive substrate and BMP on osteoblastic cell differentiation. These data indicate that xerogel glass effects differentiation of cells with osteogenic potential and that it can serve as a delivery vehicle for BMP.

Alkaline Phosphatase↗

PubMed1998

Low-level methylmercury exposure causes human T-cells to undergo apoptosis: evidence of mitochondrial dysfunction.

There is growing evidence that heavy metals, in general, and mercurial compounds, in particular, are immunotoxic to the human immune system. The major focus of our study is to demonstrate that methylmercuric chloride (MeHgCl) kills human lymphocytes by inducing apoptosis. T-cells exposed to 0.6-5 microM MeHgCl for 24 h were analyzed by flow cytometry. Methylmercury-treated cells exhibited increased Hoechst 33258 fluorescence while maintaining their ability to exclude the vital stain 7-aminoactinomycin. Furthermore, T-cells exposed to methylmercury exhibited changes in light scatter patterns that included decreased forward light scatter and increased side light scatter. The light scatter and fluorescent changes were consistent with morphological alterations displayed by cells during apoptosis. Cell death was further evaluated by assessing annexin V binding to the plasma membrane. Methylmercury-treated cells exhibited increased annexin V binding indicative of phosphatidylserine translocation to the outer leaflet of the plasma membrane. Using the fluorescent probe DiOC6(3), we noted that methylmercury exposure resulted in a decrease in mitochondrial transmembrane potential (Psim). Since a low Psim is associated with altered mitochondrial function, we also determined if exposure to methylmercury potentiated reactive oxygen species (ROS) generation. We noted that treated cells generated ROS, as evidenced by oxidation of hydroethidine and the generation of the fluorescent product, ethidium. Finally, we evaluated the effect of methylmercury on T-cell GSH content utilizing the fluorescent probe monochlorobimane; in the presence of MeHgCl, there is a marked loss in reduced cell thiols. The results of the study indicate that a key event in the induction of T-cell apoptosis by mercuric compounds is depletion in the thiol reserve which predisposes cells to ROS damage and at the same time activates death signaling pathways.

Apoptosis↗

PubMed1998

Inhibition of poly(ADP-ribose) polymerase rescues human T lymphocytes from methylmercury-induced apoptosis.

The objective of this investigation was to determine the role of poly(ADP-ribose) polymerase (PARP) in methylmercuric chloride (MeHgCl)-induced T-cell apoptosis. Following exposure of human T-cells to 2.5 microM MeHgCl, we observed PARP activation within 45 min. Maximal activation was observed at 90 min after MeHgCl treatment; thereafter, PARP activity declined. The loss in enzyme activity was coincidental with the cleavage of 116-kDa intact PARP protein to an 85-kDa fragment. To address the relationship between PARP activation and induction of apoptosis, we first examined the redox status of T cells treated with MeHgCl. We found that exposure of T cells to low concentrations of this toxicant resulted in decreased levels of reduced pyridine nucleotides and an increase in the relative amounts of oxidized flavoproteins. Thus, the possibility exists that activation of PARP leads to NAD+ depletion and thereby alters mitochondrial redox status. To determine if PARP activation is indeed part of the proapoptotic (destructive) response or a component of the antiapoptotic (protective) response, we employed two inhibitors: 3-aminobenzamide and nicotinamide. Pretreatment of T cells with these inhibitors protected cells from MeHgCl-induced apoptosis; this was seen as a reduction in the uptake of Hoechst 33258 and DNA fragmentation. Moreover, these inhibitors blocked MeHgCl-induced oxidative stress as evidenced by a reduction in reactive oxygen species (ROS) generation. These agents, however, failed to block MeHgCl-dependent decline in mitochondrial transmembrane potential (delta psi m). We conclude that PARP activation leads to proapoptotic events that contribute to MeHgCl-induced cell death.

Apoptosis↗

PubMed1998

Mercuric chloride induces apoptosis in human T lymphocytes: evidence of mitochondrial dysfunction.

The major objective of our study was to define the mechanism by which mercuric chloride (HgCl2) induces human T-cell death. Human peripheral blood T-cells were exposed to 0-40 microm HgCl2 and then analyzed for biochemical and molecular features of T-cell apoptosis. HgCl2-treated cells exhibited increased Hoechst 33258 fluorescence while maintaining their ability to exclude the vital stain 7-aminoactinomycin D. To further evaluate cell death and distinguish between apoptosis and necrosis, translocation of phosphatidylserine to the outer layer of the plasma membrane (annexin V binding), DNA fragmentation (TUNEL assay), and cleavage of poly (ADP-ribose) polymerase (PARP) were assessed. In the presence of 20-40 microm HgCl2, T-cells exhibited increased annexin V binding (28%) and DNA fragmentation (31%). HgCl2-dependent PARP cleavage was also observed by Western blot analysis. Because degradative changes associated with apoptosis are often preceded by disruption of mitochondrial function, HgCl2-treated cells were assessed for disruption of the mitochondrial transmembrane potential (DeltaPsim) and development of the mitochondrial permeability transition state. Using DiOC6(3), we demonstrated that HgCl2 exposure resulted in a decrease in the DeltaPsim. Because a decline in DeltaPsim can disturb the intracellular pH (pHi), we used the fluorescent probe, SNARF-1, to assess intracellular acidification. Treatment of T-cells with HgCl2 resulted in reduced pHi from 7.0 to 6.7. Concomitant with these observations, the fluorescent probe, hydroethidine, was utilized to demonstrate that uncoupled mitochondrial electron transport resulted in increased reactive oxygen species (ROS) generation. Interestingly, in spite of these alterations to mitochondrial function, translocation of cytochrome c to the cytosol was not detected; this correlated with enhanced bcl-2 levels in HgCl2-treated cells. In conclusion, HgCl2 exposure results in oxidative stress and activation of death signaling pathways leading to apoptosis. Collectively, our studies indicate that individual mercurial species are capable of inducing T-cell death by activating specific apoptotic cascades.

Apoptosis↗

PubMed1997

Mercuric compounds inhibit human monocyte function by inducing apoptosis: evidence for formation of reactive oxygen species, development of mitochondrial membrane permeability transition and loss of reductive reserve.

The focus of this investigation was to examine the effects of low concentrations of organic mercuric compounds on human monocyte function and to relate these effects to apoptosis. Following exposure of monocytes to 0-5 microM MeHgCl, phagocytic function and capacity to generate a respiratory burst, following PMA activation, were determined. We found that the mercury-treated cells exhibited reduced phagocytic activity. Exposure to the same mercury concentration range, also caused a marked increase in cell death. To ascertain if monocyte death was due to apoptosis, a number of flow cytometric studies were performed. Mercury-treated cells exhibited increased Hoechst 33258 fluorescence, while maintaining their ability to exclude the vital dye 7-aminoactinomycin D. Furthermore, monocytes exhibited changes in light scatter patterns that were consistent with apoptosis; these included decreased forward light scatter and increased side scatter. The percentage of cells undergoing apoptosis was dependent upon the mercury content of the medium, regardless of whether the metal was present as methyl, ethyl or phenyl mercury. Mercury-treated cells also exhibited changes in lipid organization within the plasma membrane as evidenced by increased uptake of the fluorescent probe, merocyanine 540, and by elevated annexin V binding to phosphatidylserine. Using the fluorescent probes DiOC6(3) and rhodamine 123 we noted that within 1 h of exposure to mercury, monocytes exhibited a decrease in mitochondrial transmembrane potential (psi m). Since a decreased psi m is associated with altered mitochondrial function, the hypothesis that mercury potentiated reactive oxygen species (ROS) generation and that these species promoted apoptosis was tested. We noted that treated cells generated ROS, as evidenced by oxidation of hydroethidine and the generation of the fluorescent product, ethidium. Finally, since ROS would also lower monocyte reductive reserve, we also measured GSH levels in mercury-treated cells. Chemical measurement of GSH indicated that there was thiol depletion. We suggest that the low thiol reserve predisposes cells to ROS damage and at the same time activates death-signaling pathways.

Apoptosis↗