Low level exposure to inorganic mercury interferes with B cell receptor signaling in transitional type 1 B cells.

Mercury (Hg) has been implicated as a factor contributing to autoimmune disease in animal models and humans. However the mechanism by which this occurs has remained elusive. Since the discovery of B cells it has been appreciated by immunologists that during the normal course of B cell development, some immature B cells must be generated that produce immunoglobulin reactive to self-antigens (auto-antibodies). However in the course of normal development, the vast majority of immature auto-reactive B cells are prevented from maturing by processes collectively known as tolerance. Autoimmune disease arises when these mechanisms of tolerance are disrupted. In the B cell compartment, it is firmly established that tolerance depends in part upon negative selection of self-reactive immature (transitional type 1) B cells. In these cells negative selection depends upon signals generated by the B Cell Receptor (BCR), in the sense that those T1 B cells who's BCRs most strongly bind to, and so generate the strongest signals to self-antigens are neutralized. In this report we have utilized multicolor phosphoflow cytometry to show that in immature T1 B cells Hg attenuates signal generation by the BCR through mechanisms that may involve Lyn, a key tyrosine kinase in the BCR signal transduction pathway. We suggest that exposure to low, environmentally relevant levels of Hg, disrupts tolerance by interfering with BCR signaling in immature B cells, potentially leading to the appearance of mature auto-reactive B cells which have the ability to contribute to auto-immune disease.

Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells.

Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualize a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercury's neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of mercury exposure, this novel finding for the first time suggests a testable molecular mechanism explaining the overall anti-inflammatory properties of n-3 PUFAs.

Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

STUDY QUESTION: Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? SUMMARY ANSWER: Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. WHAT IS KNOWN ALREADY: Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. STUDY DESIGN, SIZE, DURATION: BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), ß-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and ß-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. MAIN RESULTS AND THE ROLE OF CHANCE: Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, ß-catenin and ß-actin did not differ, compared with control. LIMITATIONS, REASONS FOR CAUTION: Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. WIDER IMPLICATIONS OF THE FINDINGS: These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. STUDY FUNDING/COMPETING INTERESTS: National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. TRIAL REGISTRATION NUMBER: Not applicable.

Novel Lethal Form of Congenital Hypopituitarism Associated With the First Recessive LHX4 Mutation.

BACKGROUND: LHX4 encodes a member of the LIM-homeodomain family of transcription factors that is required for normal development of the pituitary gland. To date, only incompletely penetrant heterozygous mutations in LHX4 have been described in patients with variable combined pituitary hormone deficiencies. OBJECTIVE/HYPOTHESIS: To report a unique family with a novel recessive variant in LHX4 associated with a lethal form of congenital hypopituitarism that was identified through screening a total of 97 patients. METHOD: We screened 97 unrelated patients with combined pituitary hormone deficiency, including 65% with an ectopic posterior pituitary, for variants in the LHX4 gene using Sanger sequencing. Control databases (1000 Genomes, dbSNP, Exome Variant Server, ExAC Browser) were consulted upon identification of variants. RESULTS: We identified the first novel homozygous missense variant (c.377C>T, p.T126M) in two deceased male patients of Pakistani origin with severe panhypopituitarism associated with anterior pituitary aplasia and posterior pituitary ectopia. Both were born small for gestational age with a small phallus, undescended testes, and mid-facial hypoplasia. The parents' first-born child was a female with mid-facial hypoplasia (DNA was unavailable). Despite rapid commencement of hydrocortisone and T4 in the brothers, all three children died within the first week of life. The LHX4(p.T126M) variant is located within the LIM2 domain, in a highly conserved location. The absence of homozygosity for the variant in over 65 000 controls suggests that it is likely to be responsible for the phenotype. CONCLUSION: We report, for the first time to our knowledge, a novel homozygous mutation in LHX4 associated with a lethal phenotype, implying that recessive mutations in LHX4 may be incompatible with life.

The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

INTRODUCTION: The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. OBJECTIVES/METHODS: We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). RESULTS: Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. CONCLUSIONS: Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.

Low level lead exposure in vitro stimulates the proliferation and expansion of alloantigen-reactive CD4(high) T cells.

T cells are believed to be critical functional targets of Pb immunotoxicity. In this study, low concentrations of lead (i.e., as low as 0.1 microM approximately 2 microg/dl) were found to markedly enhance the allogeneic mixed lymphocyte reaction-an assay of CD4(+) T cell responsiveness. Cell cycle analysis of cells recovered from allogeneic mixed lymphocyte cultures revealed that Pb stimulated a substantial increase in the proportion of cycling alloreactive CD4(+) T cells. The enhanced alloproliferative response was characterized by an increased population of lymphoblasts expressing heightened cell surface expression of CD4 (i.e., CD4(high) cells). Successive rounds of cell division were monitored using the cell division dye 5- (and 6)-carboxyfluorecein diacetate succinimyl ester and it was determined that the CD4(high) subpopulation comprised the expanding alloreactive T cells, which ultimately took on the phenotype of memory/effector T cells (i.e., CD44(high), CD45RB(low), CD69(high), and CD162(high)). Enhancement of T cell proliferation by lead was selective for responsiveness to alloantigen, as lead had no effect on T cell proliferation induced by mitogens or superantigen, processes that unlike alloreactivity are not dependent on antigen presentation. Collectively, these data suggest that Pb enhances alloantigen-specific T cell proliferation through an indirect mechanism involving altered antigen processing/presentation, resulting in marked clonal expansion or repertoire expansion of alloreactive T cell clones. Consistent with this suggestion was the finding that a single exposure to Pb during alloantigen priming elicited a population of CD4(+) T cells that was hyperresponsive to further alloantigen stimulation and neither lead dependent nor lead responsive.

Low concentrations of inorganic mercury inhibit Ras activation during T cell receptor-mediated signal transduction.

Mercury is widespread in the environment and consequently there are large populations that are currently exposed to low levels of mercury as a result of ubiquitous environmental factors. Whether these environmental levels of mercury are harmful is a matter of current debate, with epidemiological and animal studies suggesting detrimental effects on the immune and nervous systems. However, specific cellular effects of low concentrations of mercury have been hard to characterize. We now demonstrate that subtoxic concentrations of HgCl(2) can potently (maximal at 1 microM) increase Ras.GTP levels in Jurkat, a human T cell line. Remarkably, this activation of Ras occurs without a concomitant increase in MAP kinase activation, suggesting that mercury may direct Ras into a nonproductive state. In addition to its direct effect on Ras, concentrations of HgCl(2) as low as 0.6 microM inhibited the ability of the T cell receptor to activate Ras and MAP kinase. The inhibitory effect of mercury is selective, as activation of MAP kinase by phorbol diesters remain intact. Since the Ras/MAP kinase pathway is both highly conserved and central to signal transduction processes mediated by a myriad of diverse membrane receptor systems in a variety of cell types, these results suggest a mechanism for adverse health effects resulting from exposure to low levels of mercury. They also support a model for regulation of the Ras/MAP kinase pathway, whereby partial but unproductive activation of Ras can diminish signaling from cell surface receptors.

Sensitivity of myelomonocytic leukemia cells to arsenite-induced cell cycle disruption, apoptosis, and enhanced differentiation is dependent on the inter-relationship between arsenic concentration, duration of treatment, and cell cycle phase.

Arsenite treatment has been found to induce clinical remission in patients with acute promyelocytic leukemia. Although the potential therapeutic value of arsenite may lie in triggering apoptosis, it has not been established that cytotoxicity is the sole mechanism of action. We have used a myelomonocytic leukemia cell line (U937) to characterize the concentration-dependent effects of arsenite on cell growth, viability, apoptosis, and differentiation. Arsenite has multiple effects on U937 cells. Low concentrations of arsenite (i.e., < or = 1 microM) potentiate vitamin-D(3)-induced differentiation. Two markers of monocyte differentiation, Mac-1 expression and nitroblue tetrazolium reduction, are increased in arsenite-exposed, D(3)-costimulated cells. Concentrations of arsenite >10 microM rapidly induce the death of cells irrespective of cell cycle phase. Intermediate concentrations of arsenite (i.e., 5 to 10 microM) are cytostatic initially. Cell cycle analysis using elutriated, synchronous cell populations revealed that intermediate concentrations of arsenite delay both G(1) and G(2) transit. G(2) cells appear to be most sensitive to arsenite, in that transit through G(2)/M is more delayed than transit through G(1), and apoptosis is induced in these cells as they emerge from an aberrant G(2)/M. Arsenite-induced apoptosis was caspase-3 dependent. Arsenite-mediated cytotoxicity was reduced in the presence of the broad caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone; however, caspase inhibition did not reverse arsenite-induced cytostasis. Thus, arsenite has multiple effects on U937 cells that are dependent on concentration and cell cycle phase. Specifically, cell cycle transit and differentiation are more sensitive to arsenite than is the induction of apoptosis.

Mercuric chloride damages cellular DNA by a non-apoptotic mechanism.

Mercury is a xenobiotic metal that is well known to adversely affect the immune system, however, little is known as to the molecular mechanism. Recently, it has been suggested that mercury may induce immune dysfunction by triggering apoptosis in immune cells. Here, we studied the effects of Hg(2+) (HgCl(2)) on U-937 cells, a human cell line with monocytic characteristics. We found that these cells continued to proliferate when exposed to low doses of mercury between 1 and 5 microM. Using the single cell gel electrophoresis (SCGE) or 'comet' assay, we found that mercury damaged DNA at these levels. Between 1 and 50 microM Hg(2+), comet formation was concentration-dependent with the greatest number of comets formed at 5 microM mercury. However, the appearance of mercury-induced comets was qualitatively different from those of control cells treated with anti-fas antibody, suggesting that although mercury might damage DNA, apoptosis was not involved. This was confirmed by the finding that cells treated with 5 microM mercury were negative for annexin-V binding, an independent assay for apoptosis. These data support the notion that DNA damage in surviving cells is a more sensitive indicator of environmental insult than is apoptosis, and suggests that low-concentrations of ionic mercury may be mutagenic.

Meso-2,3-dimercaptosuccinic acid induces calcium transients in cultured rhesus monkey kidney cells.

The maintenance of intracellular Ca2+ homeostasis is critical to many cellular functions that rely on the calcium ion as a messenger. While attempting to characterize the effects of lead on intracellular calcium levels ([Ca2+]i) in LLC-MK2 Rhesus Monkey kidney cells, we observed that treatment with the metal chelating drug, meso-2,3-dimer-captosuccinic acid (DMSA) evoked transient increases in [Ca2+]i. Changes in [Ca2+]i were monitored using the Ca2+ indicator dye Fura-2 and a dual wavelength fluorescence imaging system. In the presence of 2 mM extracellular Ca2+, DMSA treatment caused a concentration-dependent (15-500 microM) transient increase in [Ca2+]i returning to baseline levels within 30-60 s. Pharmacologic concentrations of DMSA (30 microM) stimulated a three-fold increase in [Ca2+]i, which was spatiotemporally comparable to Ca2+ transients induced by other calcium agonists. Depletion of inositol trisphosphate (IP3)-sensitive [Ca2+]i stores with the smooth endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin did not prevent DMSA-elicited increases in [Ca2+]i, suggesting that Ca2+ mobilized by DMSA was either extracellular or from an non-IP3 releasable Ca2+ pool. Treatment with glutathione, cysteine, or 2-mercaptoethanol caused similar but not identical calcium transients. Adenosine-5'-trisphosphate (ATP) also elicited transient increases in [Ca2+]i similar to those of DMSA. No transient increases in [Ca2+]i were elicited by DMSA or ATP in the absence of extracellular calcium. These data indicate that DMSA and other sulfhydryl compounds trigger an influx of extracellular calcium, suggesting a previously unobserved and unanticipated interaction between DMSA and the Ca2+ messenger system.

Low and nontoxic levels of ionic mercury interfere with the regulation of cell growth in the WEHI-231 B-cell lymphoma.

WEHI-231 is a mouse B-cell line, which is a well-established model for studying signal transduction in B lymphocytes, normally responding to cross-linking of the B-cell receptor (BCR) complex by the rapid upregulation of protein tyrosine kinase activity, followed by increased intracellular calcium and activation of protein kinase C. In WEHI-231, activation of protein kinase C is functionally associated with downregulation of DNA synthesis, followed by the induction of apoptosis. We have found in WEHI-231, that at low and environmentally relevant exposure levels (0.1 microM) mercury is not toxic, but still interferes with signal transduction in that it attenuates the growth inhibitory effects of BCR cross-linking. The molecular target for mercury resulting in attenuation of the BCR-mediated growth inhibitory signal is likely proximal to activation of the BCR complex, as HgCl2 had no effect on the negative growth signal generated downstream by direct activation of protein kinase C with phorbol 12-myristate 13-acetate. Treatment of WEHI-231 cells with high and toxic concentrations of Hg results in a marked increase in protein tyrosine phosphorylation in a great many proteins; whereas treatment of WEHI-231 cells with 0.1 microM mercury is not toxic. Under these conditions mercury selectively perturbed BCR-mediated protein tyrosine phosphorylation of a 75 kDa protein, without grossly affecting tyrosine phosphorylation levels of most other proteins. These data suggest that low levels of mercury, which are not toxic, may still contribute to immune dysfunction by interfering with antigen-receptor-mediated and protein-kinase-dependent signal transduction in lymphocytes.

Lead inhibits meso-2,3-dimercaptosuccinic acid induced calcium transients in cultured rhesus monkey kidney cells.

Previously we have shown that meso-2,3-dimercaptosuccinic acid (DMSA, 15-500 microM) elicits concentration-dependent increases in intracellular calcium levels ([Ca2+]i) in untreated rhesus monkey kidney cells (LLC-MK2) (Pokorski et al., 1997, unpublished results). Little is known about the restorative effects of the chelating agent 2,3-dimercaptosuccinic acid on intracellular calcium homeostasis in the presence of lead. Lead interacts at numerous sites in Ca2+ homeostasis and may mimic Ca2+ to interfere with Ca2+-mediated intracellular signaling. To examine the effects of lead on [Ca2+]i and DMSA-induced calcium transients, LLC-MK2 were plated on 35 mm coverslip dishes (10(4) cells/dish) and pre-treated with non-cytotoxic concentrations of lead (0-100 microM) for 24 h. Cells were washed, loaded with the calcium-sensitive probe Fura-2/AM, rinsed again, and examined in loading buffer in the absence of any additional lead. Intracellular calcium was measured using a dual-wavelength calcium imaging system. Basal [Ca2+]i levels did not change between Pb-exposed (0-50 microM, 24 h) and non-lead exposed cells. In cells treated with > or = 10 microM lead for 24 h, the ability of DMSA to elicit a calcium response was blocked. These results provide evidence that pre-exposure to lead blocks the entry of extracellular calcium into LLC-MK2 cells when stimulated by specific calcium mobilizing agents.

Protection against CD95-mediated apoptosis by inorganic mercury in Jurkat T cells.

Dysregulation of CD95/Fas-mediated apoptosis has been implicated as a contributing factor in autoimmune disorders. Animal studies clearly have established a connection between mercury exposure and autoimmune disease in rodents, while case reports have suggested a link between accidental mercury contamination and autoimmune disease in humans. The mechanism(s) for these associations are poorly understood. Using the Jurkat cell model, we have found that low levels (

Lead intoxication impairs the generation of a delayed type hypersensitivity response.

The effect of oral administration of lead, as Pb-acetate, via the drinking water on the murine delayed type hypersensitivity (DTH) response was investigated. The DTH response of BALB/c mice sensitized intravenously with sheep red blood cells (SRBC) was found to be suppressed markedly in lead-intoxicated mice. Suppression of the DTH correlated with increasing blood Pb concentration. Suppression of the DTH response by Pb intoxication depended on the route of administration of the sensitizing antigen, as Pb intoxication did not impair the DTH reaction when mice were sensitized to SRBC via intraperitoneal injection. Since DTH reactions are regulated in large part by Th1 cells, these data establish an in vivo model system based on a rational route of Pb exposure in which to study further the modulation of Th1-mediated immune effector function by Pb.

Low levels of ionic mercury modulate protein tyrosine phosphorylation in lymphocytes.

The ability of ionic mercury to induce protein tyrosine phosphorylation in mouse spleen cells and in the mouse WEHI-231 B-cell lymphoma was investigated. We have confirmed previous studies which showed that exposure to high levels (several hundred microM) of mercury lead to very large increases in the level of protein tyrosine phosphorylation in these cell systems. However we have also demonstrated that low levels (in the order of 0.1 to 1.0 microM) of mercury also significantly upregulate protein tyrosine phosphorylation. Mercury induced protein tyrosine phosphorylation is inhibited by the mercury chelator penicillamine and by pretreating treating target cells with the sulfhydryl blocking reagent N-hydroxymaleimide. These results suggest that exposure to low levels of mercury could potentially interfere with lymphocyte signal transduction and so offer a possible explanation as to how mercury exposure could lead to immune cellular dysfunction. On a molecular level, the results suggest that the site(s) of action with respect to mercury dependent induction of protein tyrosine phosphorylation is likely a free disulfide group or groups located on the outer leaflet of the plasma membrane.

Ethical issues in pain management.

The modern hospice movement has played a significant role in the development of palliative care. Effective palliation is of crucial importance in achieving quality of life and a dignified death for the terminally ill. While the inherent risk in palliative care, respiratory depression, remains an open medical question, an understanding of the ethical and moral principle of double effect demonstrates the prudential nature of palliative care and how it is an application of the ethical and moral norm, respect for patient autonomy.

Lack of Ca2+ involvement in thymocyte apoptosis induced by chelation of intracellular Zn2+.

BACKGROUND: Chelation of intracellular Zn2+ with N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) triggers apoptosis predominantly in mature thymocytes. A rise in intracellular-free Ca2+ concentration ([Ca2+]i) has been associated with induction of thymocyte apoptosis by a variety of agents. Zn2+ can affect intracellular Ca2+ homeostasis, so the aim of this study was to investigate whether TPEN-induced apoptosis is mediated by Ca2+ signalling. EXPERIMENTAL DESIGN: The possible role of Ca2+ in TPEN-induced apoptosis was investigated. Apoptotic markers used were DNA cleavage into oligonucleosomal fragments and formation of apoptotic nuclei. The change in [Ca2+]i in thymocytes after TPEN treatment was monitored using the fluorescence Ca2+ indicator dye, fura-2. The requirement of an increase in [Ca2+]i for TPEN-induced apoptosis was examined in thymocytes preloaded with the intracellular Ca2+ buffer, bis-(o-aminophenoxy)-ethane-N, N,N',N'-tetraacetic acid, or incubated in nominally Ca(2+)-free medium supplemented with EGTA. The effect of an increase in [Ca2+]i on TPEN-induced DNA fragmentation was studied by using thapsigargin or ionomycin to elevate [Ca2+]i in thymocytes. RESULTS: No increase in [Ca2+]i could be detected before DNA fragmentation in thymocytes during TPEN treatment. Buffering intracellular Ca2+ with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid or incubating cells in nominally Ca(2+)-free medium with EGTA had little effect on TPEN-induced DNA fragmentation and formation of apoptotic nuclei. Increasing thymocyte [Ca2+]i with thapsigargin or ionomycin administration during TPEN treatment resulted in an additive effect on TPEN-induced DNA fragmentation in thymocytes. CONCLUSIONS: Our study shows that TPEN induces apoptosis in thymocytes by Ca(2+)-independent mechanisms and that apoptosis triggered by Zn2+ chelation and [Ca2+]i elevation affects distinct thymocyte subpopulations.

Chelation of intracellular zinc triggers apoptosis in mature thymocytes.

BACKGROUND: Thymocyte apoptosis has been shown to be regulated by intracellular levels of cations. Elevation of [Ca2+]i can induce cell death by apoptosis, whereas, Zn2+ prevents it. EXPERIMENTAL DESIGN: A membrane permeable metal ion chelator, N,N,N'N',-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN), was used to examine the role of intracellular zinc in thymocyte (rat and human) apoptosis. Characteristics of apoptosis that were assessed included: chromatin degradation into oligosomal-sized fragments and nuclear condensation. The necessity for protein synthesis in TPEN-induced apoptosis was ruled out using the inhibitors, cycloheximide and emetine. Metal ion specificity for TPEN was established by competition with exogenous cations. FACS analysis was employed to determine the phenotype of the TPEN-sensitive thymocyte populations. RESULTS: TPEN induced DNA fragmentation within 4 hours of exposure before the onset of cell death (6 hours). Addition of equimolar exogenous Zn2+ or Cu2+, but not Mn2+ or Fe2+, at the initiation of culture prevented TPEN-induced apoptosis. A membrane impermeable chelator, diethylenetriaminepentaacetic acid, did not induce thymocyte apoptosis indicating that chelation of intracellular Zn2+ was required to trigger DNA fragmentation. The identity of the critical intracellular Zn(2+)-binding site(s) is currently unknown, but it appears that increased thymocyte [Ca2+]i may displace Zn2+ from these intracellular sites. TPEN treatment resulted in the depletion of thymocytes having a mature phenotype with respect to CD3, CD4, and CD8. Moreover, lymph node cells were more sensitive to TPEN than thymocytes. CONCLUSIONS: These experiments show that Zn2+ chelation has disparate effects on immature and mature T cells, and suggest that zinc availability controls the cell death or selection pathway during thymopoiesis.

Genistein induces apoptosis in immature human thymocytes by inhibiting topoisomerase-II.

The toxicity of genistein, an inhibitor of tyrosine kinases and topoisomerase-II, on human thymocytes was investigated. Genistein induced marked chromatin fragmentation indicative of apoptosis in human thymocyte cultures. Genistein-induced thymocyte apoptosis is unlikely due to an inhibition of basal tyrosine kinase activity, since another tyrosine kinase inhibitor, herbimycin A, does not induce thymocyte apoptosis, whereas other topoisomerase-II inhibitors do. The thymocyte subpopulation most sensitive to genistein-induced apoptosis exhibited a CD3-CD4+CD8+ phenotype. This subpopulation of thymocytes is also sensitive to glucocorticoid-induced apoptosis; however, differences between genistein- and glucocorticoid-induced apoptosis were noted. In particular, unlike glucocorticoid-induced apoptosis, genistein-induced apoptosis does not involve changes in [Ca2+]i and cannot be blocked by activation of protein kinase C.