Ortho-substituted but not coplanar PCBs rapidly kill cerebellar granule cells.

Several PCB congeners were assessed for their cytotoxicity on cerebellar granule cells in an attempt to compare their structure-activity relationship as potential neurotoxicants and to assess the mechanisms associated with their toxicity. Flow cytometry was used to monitor the changes of a number of biochemical endpoints: membrane integrity, intracellular free calcium concentration ([Ca(2+)](i)), reactive oxygen species (ROS) production, mitochondrial membrane potential (Delta psi(m)), and cell size. The non-coplanar, ortho-substituted congeners, PCB 8 (2,4'-dichlorobiphenyl), PCB 28 (2,4,4'-trichlorobiphenyl), PCB 47 (2,4,2',4'-tetrachlorobiphenyl), and PCB 52 (2,5,2',5'-tetrachlorobiphenyl) (10 microM) killed neurons to different degrees within 30 min. Loss of viability was accompanied by increased [Ca(2+)](i) and decreased Delta psi(m). No significant changes of ROS level were observed during exposure. The coplanar congeners, PCB 77 (3,4,3',4'-tetrachlorobiphenyl), PCB 80 (3,5,3',5'-tetrachlorobiphenyl), and PCB 81 (3,4,5,4'-tetrachlorobiphenyl) (10 microM), had no effects on membrane integrity, [Ca(2+)](i) or Delta psi(m) in this time period of exposure. In Ca(2+)-free Tyrode's medium, there was no [Ca(2+)](i) increase after exposure to the ortho-substituted congeners, but also no reduction in loss of membrane integrity, suggesting Ca(2+) influx was not the cause of viability loss. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (1-2 microM), caused a large decrease of Delta psi(m), but only a slight loss of viability, which suggested that Delta psi(m) is not the primary cause of PCB 52-induced cell death. These studies show that ortho-substituted PCBs are toxic to cerebellar granule cells; however, their toxic action is not secondary to elevation of intracellular calcium, a change in mitochondrial membrane potential, or free radical generation.     

Ortho-substituted PCBs kill thymocytes.

The effects of exposure of acutely dissociated rat thymocytes to various polychlorinated biphenyl (PCB) congeners were examined using flow cytometry. Non-planar, ortho-substituted congeners caused a rapid cell death at low micromolar concentrations, while coplanar, dioxin-like congeners at the same concentration were without significant effect. The most potent of the congeners studied was PCB 52 (2,2',5,5'-tetrachlorobiphenyl), which had an IC50 of 3.96 microM at 20 min. Prior to loss of viability there was a decrease in mitochondrial membrane potential Delta Psi m, an accumulation of intracellular calcium, and a progressive leakiness of the plasma membrane. Application of PCB 52 in calcium-free medium reduced the calcium accumulation, but did not reduce cell death. Agents that depolarized mitochondria also did not induce the same degree of cell death caused by PCB 52. Cyclosporin A, which prevents opening of the mitochondria permeability transition channel, protected against cell death but did not protect against mitochondrial depolarization or calcium accumulation. Rapamycin and FK 506 at high concentration provided partial protection against cell death. These observations indicate that the ortho-substituted PCB 52 disrupts plasma, mitochondrial and endoplasmic reticulum membranes. We hypothesize that PCB 52 incorporates into lipid bilayers and with its bulky, three-dimensional ortho-substituted congener structure disrupts membrane function to a greater degree than coplanar congeners.     

Tea catechins protect against lead-induced cytotoxicity, lipid peroxidation, and membrane fluidity in HepG2 cells.

Recent studies have shown that lead causes oxidative stress by inducing the generation of reactive oxygen species (ROS) and reducing the antioxidant defense system of cells. This suggests that antioxidants may play an important role in the treatment of lead poisoning as a kind of excellent scavenger of free radicals and chelator of heavy metal. Whether tea catechins have protective effects against oxidative stress after lead treatment in cell systems remains unclear. The present study was designed to elucidate if tea catechins have any protective effects on lipid peroxidation damage in lead-exposed HepG2 cells. Exposure of HepG2 cells to Pb(++) decreased cell viability and stimulated lipid peroxidation of cell membranes as measured by the thioburbituric acid reaction. Electron spin resonance (ESR) spin-labeling studies indicated that lead exposure could decrease the fluidity in the polar surface of cell membranes. Tea catechin treatment significantly increased cell viability, decreased lipid peroxidation levels, and protected cell membrane fluidity in lead-exposed HepG2 cells in a concentration-dependent manner. The galloylated catechins showed a stronger effect than nongalloylated catechins. Cotreatment with (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG) showed a synergistically protective effect. The results suggest that tea catechin supplementation may have a role to play in modulating oxidative stress in lead-exposed HepG2 cells.     

Correction by pentoxifylline of the abnormal fluorescence polarization of erythrocyte membranes from diabetic patients

Summary Erythrocytes from diabetic patients show abnormal rheology. Pentoxifylline, a methylxanthine, improves the abnormal deformability of diabetic erythrocytes, but its mechanism of action remains unclear. We have studied the effect of pentoxifylline on the lipid order of erythrocyte membranes from controls and patients with Type I diabetes. We studied the structural organization of membrane lipids in individual erythrocyte ghosts by fluorescence polarization using a cell sorter. Fluorescence polarization values (P) for 17 controls (P=0.244) and 20 diabetic patients (P=0.215) were significantly different. Pentoxifylline added in vitro had no effect on normal membranes, but significantly increased at 10^–5 mol·l^–1 (P=0.233), and normalized at 10^–4 mol·l^–1 (P=0.243), the P value of membrane ghosts from diabetics.     

异丙酚对PC12细胞和脂质体膜流动性的影响

目的：研究异丙酚对PC12细胞和脂质体膜流动性的影响，探讨其全麻作用机理，方法：以PC12细胞作为神经细胞模型，以脂质体作为生物膜模型，利用荧光偏振技术离体动态地检测30min内异丙酚对PC12细胞各向异性r、荧光偏振度P和微粘度η及脂质体微度η的影响，分析细胞膜流动性的改变。结果：各浓度的异丙酚均可引起PC12细胞膜的各向异性r、荧光偏振度P和微粘度η的显性降低，尤其在5min内r,p,η值下降迅速，5min后各指标下降的趋势渐渐平缓。与PC12细胞相类似，除了1mg.L^-1组外，10、100mg.L^-1的异丙酚均显降低脂质体微粘度η值。结论：异丙酚能显增加PC12细胞和脂质体的膜流动性，提示其全麻作用原理可能是通过改变神经细胞的功能或结构产生。     

Effect of glutamate receptor ligands on mitochondrial membrane potential in rat dissociated cerebellar cells

The effect of three different glutamate receptor ligands on mitochondrial membrane potential has been studied in rat pup dissociated cerebellar cells by measuring rhodamine 123 fluorescence. L-glutamate, NMDA (N-methyl-D-aspartate) and kainate (from 10^–8 to 10^–3 M) decreased in a concentration-dependent manner the mitochondrial membrane potential with EC₅₀ values of 6.7±1.7, 3.8±0.5, and 37.4±14 M, respectively.Dizocilpine ((+)MK 801) was able to inhibit the NMDA- and L-glutamate-induced decrease in rhodamine 123 fluorescence, while kainate-induced fluorescence-decreases were unaffected. However, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) totally prevented the effect of kainate on mitochondrial membrane potential, but failed to block the L-glutamate effect. It is concluded that, in our cell preparation, L-glutamate exerts its action mainly through NMDA-subtype receptors, and that Ca²⁺ and Na⁺ entry through ionotropic glutamate receptors could be responsible for an impairment of mitochondrial membrane potential.     

异丙酚对PC12细胞膜流动性及[Ca~(2+)]_i的影响

目的　研究异丙酚对PC12细胞膜流动性及 [Ca2 + ]i的影响。方法　以PC12细胞作为神经细胞模型 ,加入不同浓度的异丙酚 ,用荧光偏振法测定细胞膜微粘度 η的动态变化 ,用激光扫描共聚焦显微镜检测 [Ca2 + ]i 随时间变化曲线。结果　①异丙酚各剂量组的 η值均降低 ,并呈现剂量依赖性 ;② 30、10 0mg·L-1的异丙酚在加药后 2 0～ 30s使[Ca2 + ]i 短暂升高 ,[Ca2 + ]i的峰值分别比加药前增加了119%和 14 0 % ,但在 5 0s内均恢复到加药前水平。结论　异丙酚的全麻作用机制可能与细胞膜流动性增高有关     

Mechanisms of the apoptotic and necrotic actions of trimethyltin in cerebellar granule cells.

In evaluating mechanisms of trimethyltin (TMT)-initiated neuronal damage, the present study focused on involvement of reactive oxygen species, protein kinase C (PKC), and glutamate receptors. Exposure of cerebellar granule cells to TMT (0.01-0.1 microM) produced primarily apoptosis, but higher concentrations were associated with cellular lactate dehydrogenase efflux and necrosis. TMT increased generation of cellular reactive oxygen species, which was inhibited by either L-NAME (inhibitor of nitric oxide synthase, NOS) or catalase, indicating that both NO and H(2)O(2) are formed on TMT exposure. Since chelerythrine (selective PKC inhibitor) also inhibited oxidative species generation, PKC appears to play a significant role in TMT-induced oxidative stress. The metabotropic glutamate receptor antagonist, MCPG, (but not MK-801) prevented oxidative species generation, indicating significant involvement of metabotropic receptors (but not NMDA receptors) in TMT-induced oxidative stress. NOS involvement in the action of TMT was confirmed through measurement of nitrite, which increased concentration dependently. Nitrite accumulation was blocked by L-NAME, chelerythrine, or MCPG, showing that NO is generated by TMT and that associated changes in NOS are regulated by a PKC-mediated mechanism. Oxidative damage by TMT was demonstrated by detection of elevated malondialdehyde levels. It was concluded that low concentrations of TMT (0.01-0.1 microM) cause apoptotic cell death in which oxidative signaling is an important event. Higher concentrations of TMT initiate necrotic death, which involves both an oxidative and a non-oxidative component. TMT-induced necrosis but not apoptosis in granule cells is mediated by glutamate receptors.     

In vitro toxicity of tetrabromobisphenol-A on cerebellar granule cells: cell death, free radical formation, calcium influx and extracellular glutamate.

Tetrabromobisphenol-A (TBBPA) is one of the worlds most widely used brominated flame retardant. The present study reports effects of TBBPA on primary cultures of cerebellar granule cells (CGC). Using the trypan blue exclusion assay, we show that TBBPA induces death of CGC at low micro molar concentrations. Cell death was reduced by the NMDA receptor antagonist MK-801 (3 microM), the antioxidant vitamin E (50 microM), and in calcium-free buffer. We further demonstrate that TBBPA's toxicity was accompanied by apoptosis-like nuclear shrinkage, chromatin condensation, and DNA fragmentation. Other hallmarks of apoptosis such as caspase activity were, however, absent, indicating an atypical form of apoptosis. TBBPA increased intracellular free calcium in a concentration-dependent manner. TBBPA also induced an increase in extracellular glutamate in a time-dependent manner. TBBPA gave a concentration-dependent increase information reactive oxygen species (ROS) of measured with 2,7-dichlorofluorescein diacetate. The ROS formation was inhibited by the extracellular signal-regulated protein kinase (ERK) inhibitor U0126 (10 microM), the tyrosine kinase inhibitor erbstatin-A (25 microM), eliminating calcium from the buffer and by the superoxide dismutase inhibitor diethyldithio-carbamic acid (DDC, 100 microM). Further analysis with Western blot confirmed phosphorylation of ERK1/2 after exposure to TBBPA. We found that TBBPA induces ROS formation, increases intracellular calcium, extracellular glutamate, and death of CGC in vitro at concentrations comparable to those of polychlorinated biphenyl. These findings implicate TBBPA as a predicted environmental toxin and bring out the importance of awareness of its hazardous effects.     

Relationship Between Drug Absorption Enhancing Activity and Membrane Perturbing Effects of Acylcarnitines

Acylcarnitines with chain lengths of 2 to 18 carbon atoms were tested for their effects on rat intestinal brush border membrane order (S) by fluorescence polarization of 1,6-diphenyl-l ,3,5-hexatriene (DPH). These results were compared to the previously reported effectiveness of the acylcarnitines as absorption enhancers of the poorly absorbed antibiotic cefoxitin. Acylcarnitines with fatty acids less than 12 carbon units in length were ineffective in increasing drug absorption and perturbing brush border membrane order. Long-chain acylcarnitines (12–18 carbons) significantly increased the bioavailability of cefoxitin and decreased the lipid order of brush border membranes. The results suggest that, in order to promote drug absorption, the acylcarnitines must surpass a critical chain length (10 carbon units) to partition effectively into the membrane and, in addition, must perturb the lipid order beyond a threshold value (15–20%). Membrane perturbing capacity may serve as an indicator of the absorption enhancing potential of other aliphatic-type compounds.     

温度对南极红酵母Rhodotorula sp.NJ298抗氧化系统及质膜流动性的影响

目的研究在不同温度下南极红酵母Rhodotorula sp. NJ298抗氧化酶活性变化,丙二醛(MDA)、类胡萝卜素含量变化,及细胞质膜流动性的变化。方法采用分光光度法研究了-3,0,8℃3种温度下培养的南极红酵母Rhodotorula sp. NJ298超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性,及丙二醛(MDA)和类胡萝卜素含量,同时测定了对H2O2的抗性,并应用荧光探针法测定了质膜流动性。结果在-3℃低温时,NJ298 SOD酶活性下降,CAT和POD酶活性升高,MDA含量没有明显变化,类胡萝卜素含量增加,对H2O2的抗性增加,质膜维持正常流动性。结论在低温时,NJ298类胡萝卜素积累,在积累过程中,虽然SOD酶活力下降,但类胡萝卜素替代SOD酶行使主要的抗氧化功能,并使NJ298抗氧化能力增强,保护膜系统,维持质膜正常流动性,这在NJ298低温适应性中具有重要作用。     

Measurement of GABAA receptor function in rat cultured cerebellar granule cells by the Cytosensor microphysiometer

1. γ-Aminobutyric acid (GABA), acting via the GABA_A receptor, increased the extracellular acidification rate of rat primary cultured cerebellar granule cells, measured by the Cytosensor microphysiometer.
2. The optimal conditions for the measurement of GABA_A receptor function in cerebellar granule cells by microphysiometry were: cells seeded at 9–12×10⁵ cells/transwell cup and maintained in vitro for 8 days, GABA stimulation performed at 25°C, with a stimulation time of 33 s.
3. GABA stimulated a concentration-dependent increase in the extracellular acidification rate with an EC₅₀ of 2.0±0.2 μM (mean±s.e.mean, n=7 experiments) and maximal increase (E_max) over basal response of 15.4±1.2%.
4. The sub-maximal GABA-stimulated increase in acidification rate could be potentiated by the 1,4-benzodiazepine, flunitrazepam (100 nM). The 10 nM GABA response showed the maximal benzodiazepine facilitation (GABA alone, 1.4 μV s⁻¹, GABA+flunitrazepam, 3.8 μV s⁻¹, mean increment over basal, n=7).
5. The GABA-stimulated increase in acidification rate was inhibited by the GABA_A antagonist, bicuculline (100 μM) (90% inhibition at 1 mM GABA).
6. The results of this study show that activation of GABA_A receptors in rat cerebellar granule cells caused an increase in the extracellular acidification rate; an effect which was potentiated by benzodiazepines and inhibited by a GABA_A receptor antagonist. This paper defines the conditions and confirms the feasibility of using microphysiometry to investigate GABA_A receptor function in primary cultured CNS neurones. The microphysiometer provides a rapid and sensitive technique to investigate the regulation of the GABA_A receptor in populations of neurones.

     

Non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95) amplifies ionotropic glutamate receptor signaling in embryonic cerebellar granule neurons by a mechanism involving ryanodine receptors.

The mechanisms by which non-coplanar 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and rapamycin interact with ryanodine receptor (RyR) complexes to alter Ca2+ signaling, were explored in intact cerebellar granule neurons. PCB 95 (10 microM, 20 min) significantly increased the number of neurons responding to caffeine. PCB 95 sensitization of RyR-mediated responses was further supported by the observations that ryanodine pretreatment blocked response to caffeine and coplanar 2,4,4',5-tetrachlorobiphenyl (PCB 66), which lacks RyR activity, failed to sensitize neurons. PCB 95 did not significantly alter levels of resting cytosolic Ca2+ nor thapsigargin-sensitive Ca2+ stores, suggesting a more complex mechanism than sensitization from increased cytosolic Ca2+ or an increased endoplasmic reticulum/cytosolic Ca2+ gradient. The immunosuppressant, rapamycin, sensitized neurons to caffeine in a manner similar to PCB 95, suggesting a common mechanism. PCB 95 or rapamycin significantly enhanced Ca2+ responses following N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl4-isoxasolepropiate (AMPA) receptor activation. Store depletion or direct block of RyR with ryanodine enhanced responses to NMDA. PCB 95 further enhanced these responses to NMDA. These results suggest that PCB 95 and rapamycin enhance NMDA- and AMPA-mediated Ca2+ signals by modifying a functional association of the FKBP12/RyR complex that results in amplification of glutamate signaling in cultured cerebellar granule neurons in culture.     

Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells.

Rotenone inhibits spontaneously and chemically induced hepatic tumorigenesis in rodents through the induction of apoptosis. However, the mechanism for the induction of apoptosis by rotenone has not been defined. Mitochondrial dysfunction, in particular the induction of the mitochondrial membrane permeability transition (MPT), has been implicated in the cascade of events involved in the induction of apoptosis. Inhibition of the mitochondrial electron-transport chain reduces the mitochondrial transmembrane potential (delta(psi)m), which may induce the formation of the mitochondrial permeability transition pore and the subsequent MPT. Fluorescent microscopy of Hoechst 33258-stained WB-F344 cells, a rat-liver cell line, was utilized to examine the effect of the mitochondrial respiratory chain inhibitor, rotenone (0.5-5 microM), atractyloside (5-10 microM), and cyclosporin A (2.5-10 microM) on apoptosis. A time- and concentration-dependent increase in liver cell apoptosis was observed following treatment with rotenone and atractyloside (11.7- and 7.7-fold, respectively, over solvent control). Cotreatment with 7.5- and 10 microM-cyclosporin A for 12 h inhibited the apoptogenicity of 5-microM rotenone treatment. A similar effect was observed following cyclosporin A cotreatment with atractyloside. Rotenone induced a rapid increase in apoptosis (within 20 min of treatment). By 2 h of treatment, the morphological appearance of apoptosis was similar to that observed in cultures treated continuously with rotenone for 12 h. Inhibition studies demonstrated that cyclosporin A prevented apoptosis if the exposure to it occurred prior to the 20-min threshold necessary to induce apoptosis by rotenone. Mitochondrial function was examined by staining with the mitochondrial membrane potential (delta(psi)m)-sensitive fluorochrome, MitoTracker Red (CMXRos) and confirmed utilizing cytofluorometric analysis of DiOC6(3)-stained cells. Rotenone (5.0-microM) and atractyloside (5.0-microM) reduced the percent of CMXRos or DiOC6(3)-positive (delta(psi)m-positive) liver cells within 15 min and throughout the duration of the study (6 h) to approximately 65-80% and 50-80% of control. However, cotreatment with concentrations of cyclosporin A that inhibited the apoptogenicity of rotenone and atractyloside prevented the rotenone- and atractyloside-induced reduction of the delta(psi)m. Therefore, the apoptogenic effect of rotenone and atractyloside appears to occur rapidly (within 20 min) and is irreversible once mitochondrial damage occurs. The inhibition of the rotenone- and atractyloside-induced apoptosis and mitochondrial dysfunction by cyclosporin A suggests the MPT may be involved in the induction of apoptosis by rotenone.     

Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells.

Diarrhetic Shellfish Poisoning (DSP) results from the consumption of shellfish contaminated with okadaic acid (OA) or one of the dinophysistoxins (DTX). It has been reported that this toxin induces apoptosis in several cell models, but the molecular events involved in this process have not been clarified. In this report we studied intracellular signals induced by OA in Caco-2 cells: mitochondrial membrane potential, F-actin depolymerization, caspases activation, cell proliferation and cell membrane integrity. Results indicate that caspases-8 and -9 increased their activity after 30 min of OA treatment according to their role as initiator caspases. In contrast, activation of the downstream caspase-3 is a later event in the execution phase of apoptosis. Mitochondrial membrane potential changes are detected at 30 min of OA exposure indicating that this is an early response in the apoptotic cascade. F-actin depolymerization occurs after 24h of incubation with OA and this effect is significant at low doses of the toxin. LDH is released into the culture medium, although there is not PI uptake, indicative of a significant cell death in addition to apoptosis. Moreover, OA led to a dose- and time-dependent decrease in cellular proliferation.     

The role of mitochondrial and oxidative injury in BDE 47 toxicity to human fetal liver hematopoietic stem cells.

The polybrominated diphenyl ethers (PBDEs) are a group of flame retardants whose residues have markedly increased in the environment and in human tissues during the last decade. Of the various congeners, BDE 47 (2,2',4,4'-tetrabromodiphenyl ether) is typically the predominant congener observed in fish and wildlife samples, as well as in human tissues. Several studies indicate in utero transfer of PBDEs during pregnancy with residues accumulating in fetal tissues, and thus the potential for BDE 47-mediated injury in utero is of concern. In this study, we examined the mechanisms of BDE 47-mediated injury to primary human fetal liver hematopoietic stem cells (HSCs), which comprise a large proportion of fetal hepatic cells and play a key role in hematopoiesis during fetal development. Incubation of fetal liver HSCs with BDE 47 led to a loss of mitochondrial membrane potential and the onset of apoptosis. These effects were observed in the low micromolar range of BDE 47 exposures. At higher concentrations, BDE 47 elicited a loss of viability, which was accompanied by the generation of reactive oxygen species and peroxidation of HSC lipids. Preincubation of fetal liver HSCs with N-acetylcysteine, a glutathione (GSH) precursor, caused an increase in cellular GSH concentrations, restored mitochondrial redox status, and ameliorated the toxicity of BDE 47. BDE 47-mediated cytotoxicity or oxidative injury was not evident at the lower concentrations (< 1microM). Collectively, these data support a role for oxidative stress in the cytotoxicity of BDE 47 and indicate that oxidative stress-associated biomarkers may be useful in assessing the sublethal effects of BDE 47 toxicity in other models. However, the fact that BDE 47 undergoes a concentration-dependent accumulation in other primary cells in media that can underestimate cellular concentrations (W. R. Mundy et al., 2004, Toxicol. Sci. 82, 164-169) suggests that the HSC cell injury observed in our study may be of less relevance to human in utero PBDE exposures.     

Cell cycle inhibition by sodium arsenite in primary embryonic rat midbrain neuroepithelial cells.

Arsenite (As3+) exposure during development has been associated with neural tube defects and other structural malformations, and with behavioral alterations including altered locomotor activity and operant learning. The molecular mechanisms underlying these effects are uncertain. Because arsenic can cross the placenta and accumulate in the developing neuroepithelium, we examined cell cycling effects of sodium arsenite (As3+ 0, 0.5, 1, 2, and 4 microM) on embryonic primary rat midbrain (gestational day [GD] 12) neuroepithelial cells over 48 h. There was a concentration- and time-dependent As3+-induced reduction in cell viability assessed by neutral red dye uptake assay but minimal apoptosis at concentrations below 4 microM. Morphologically, apoptosis was not apparent until 4 microM at 24 h, which was demonstrated by a marginal but statistically significant increase in cleaved caspase-3/7 activity. Cell cycling effects over several rounds of replication were determined by continuous 5-bromo-2'-deoxyuridine (BrdU) labeling and bivariate flow cytometric Hoechst-Propidium Iodide analysis. We observed a time- and concentration-dependent inhibition of cell cycle progression as early as 12 h after exposure (> or =0.5 microM). In addition, data demonstrated a concentration-dependent increase in cytostasis within all cell cycle phases, a decreased proportion of cells able to reach the second cell cycle, and a reduced cell cycle entry from gap 1 phase (G1). The proportion of affected cells and the severity of the cell cycle perturbation, which ranged from a decreased transition probability to complete cytostasis in all cell cycle phases, were also found to be concentration-dependent. Together, these data support a role for perturbed cell cycle progression in As3+ mediated neurodevelopmental toxicity.     

Hexachlorobenzene-induced Immunopathology in Brown Norway rats is partly mediated by T cells.

Hexachlorobenzene (HCB) is a persistent environmental pollutant with (auto)immune effects in humans and rats. The Brown Norway (BN) rat is very susceptible to HCB-induced immunopathology, and oral exposure causes inflammatory skin and lung lesions, splenomegaly, lymph node (LN) enlargement, and increased serum levels of IgE and anti-ssDNA IgM. The role of T cells in HCB-induced immunopathology is unclear and to elucidate this Cyclosporin A (CsA) was used. BN rats were exposed to either a control diet or a diet supplemented with 450 mg/kg HCB for 21 days. CsA treatment started 2 days prior to HCB exposure and rats were injected daily with 20 mg/kg body weight CsA. Treatment with CsA prevented the HCB-induced immunopathology significantly. The onset of skin lesions was delayed and the severity was also strongly decreased. Furthermore, CsA prevented the HCB-induced increase in spleen weight partly and the increase in auricular LN weight completely. The increase in serum IgE and IgM against ssDNA levels was prevented completely. Macrophage infiltrations into the spleen and lung still occurred but infiltrations of eosinophilic granulocytes into the lung were prevented. Restimulation of spleen cells with the T-cell mitogen ConA and the macrophage activator LPS clearly showed that CsA inhibited T-cell activation, but not macrophage activation. Together, our results show that both T cells and macrophages play a prominent role in HCB-induced immunopathology.     

Induction of cell-proliferation hormesis and cell-survival adaptive response in mouse hematopoietic cells by whole-body low-dose radiation.

Hormesis and a cytogenetic adaptive response induced by low-dose radiation (LDR) have been extensively documented. However, few studies have investigated the induction of an adaptive response by LDR for cell survival in vitro. In the present study, we investigated whether LDR could induce hormesis in hematopoietic cells and the adaptive response of these cells to subsequent high-dose radiation-induced cytotoxic effects. Mice were exposed in whole-body to 0 (as control), 0.05, 0.25, 0.50, 0.75, and 1.00 Gy of X-rays. They were killed 12, 24, 48, and 72 h later to observe the stimulating effect of LDR on total bone marrow cells per femur and bone marrow progenitor, colony-forming unit-granulocyte-macrophage (CFU-GM). Exposure to 0.5 Gy of X-rays resulted in significantly stimulating effects on both parameters with a maximum effect at 48 h, showing a cell-proliferation hormesis. In the next experiment, mice were irradiated by 0.5 Gy X-rays as an adaptive exposure (D1), and 6, 12, 24, 48, and 72 h later, they were exposed to 6 Gy X-rays as a challenging exposure (D2). Forty-eight h after D2, cytotoxic effects were analyzed using peripheral blood cells (red blood cells, white blood cells, and platelets) and bone marrow cells (total bone marrow cells of the femur, and bone marrow progenitors such as CFU-GM and erythroid burst-forming unit, BFU-E). An adaptive response to D2-induced cytotoxic effect, named as the cell-survival adaptive response, was found in both peripheral blood cells and bone marrow cells when D1 and D2 exposures were given at intervals of 24-48 h. These results suggested that LDR could induce both cell-proliferation hormesis and cell-survival adaptive response to subsequent high-dose radiation in bone marrow cells. It may be of potential importance, if this phenomenon is confirmed clinically, since it may be applied to reduce the adverse effect of radiotherapy.     

Human CD34+ hematopoietic progenitor cells are sensitive targets for toxicity induced by 1,4-benzoquinone.

Chronic human exposure to benzene has been linked to several hematopoietic disorders, including leukemia and lymphomas. Certain benzene metabolites, including benzoquinone (BQ), are genotoxic and mutagenic. Bone marrow stem cells are targets for benzene-induced cytotoxicity and DNA damage that could result in changes to the genome of these progenitor cells, thereby leading to hematopoietic disorders and cancers. Human bone marrow CD34(+) hematopoietic progenitor cells (HPC) were exposed in vitro to 1,4-BQ to assess cytotoxicity, genotoxicity, and DNA damage responses and the molecular mechanisms associated with these events. CD34(+) HPC from 10 men and 10 women were exposed to 0, 1, 5, 10, 15, or 20 microM of 1,4-BQ and analyzed 72 h later. Apoptosis and cytotoxicity were dose-dependent, with exposure to 10 microM 1,4-BQ resulting in approximately 60% cytotoxicity relative to untreated controls. A significant increase in the percentage of micronucleated CD34(+) cells was detected in cultures treated with 1,4-BQ. In addition, the p21 mRNA level was elevated in 1,4-BQ-treated cells, suggesting that human CD34(+) cells utilize the p53 pathway in response to 1,4-BQ-induced DNA damage. However, there were no significant changes in mRNA levels of the DNA repair genes ku80, rad51, xpa, xpc, and ape1 as well as p53 following treatment with 1,4-BQ. Although interindividual variations were evident in the cellular response to 1,4-BQ, there was no gender difference in the response overall. These results show that human CD34(+) cells are sensitive targets for 1,4-BQ toxicity that use the p53 DNA damage response pathway in response to genotoxic stress. Human CD34(+) HPC will be useful for testing the toxicity of other benzene metabolites and various hematotoxic chemicals.