Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures.

Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes (endonucleases, DNase I, NUC 18), which finally cut the chromatin into specific formations of 180-200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISA(plus) (CDDE). In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant. To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISA(plus) and the ToxiLight Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300 microM for 30 min, Lewisite (L) 60 microM for 5 min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent. This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients.

Molecular toxicology of sulfur mustard-induced cutaneous inflammation and blistering.

Sulfur mustard (SM) is a strong alkylating agent, which produces subepidermal blisters, erythema and inflammation after skin contact. Despite the well-described SM-induced gross and histopathological changes, the exact underlying molecular mechanisms of these events are still a matter of research. As part of an international effort to elucidate the components of cellular signal transduction pathways, a large body of data has been accumulated in the last decade of SM research, revealing deeper insight into SM-induced inflammation, DNA damage response, cell death signaling, and wound healing. SM potentially alkylates nearly every constituent of the cell, leading to impaired cellular functions. However, SM-induced DNA alkylation has been identified as a major trigger of apoptosis. This includes monofunctional SM-DNA adducts as well as DNA crosslinks. As a consequence, DNA replication is blocked, which leads to cell cycle arrest and DNA single and double strand breaks. The SM-induced DNA damage results in poly(ADP-ribose) polymerase (PARP) activation. High SM concentrations induce PARP overactivation, thus depleting cellular NAD(+) and ATP levels, which in consequence results in necrotic cell death. Mild PARP activation does not disturb cellular energy levels and allows apoptotic cell death or recovery to occur. SM-induced apoptosis has been linked both to the extrinsic (death receptor, Fas) and intrinsic (mitochondrial) pathway. Additionally, SM upregulates many inflammatory mediators including interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha) and others. Recently, several investigators linked NF-kappaB activation to this inflammatory response. This review briefly summarizes the skin toxicity of SM, its proposed toxicodynamic actions and strategies for the development of improved medical therapy.

Acute effects of sulfur mustard injury--Munich experiences.

Sulfur mustard (SM) is a strong vesicant agent which has been used in several military conflicts. Large stockpiles still exist to the present day. SM is believed to be a major threat to civilian populations because of the persistent asymmetric threat by non-state actors, such as terrorist groups, its easy synthesis and handling and the risk of theft from stockpiles. Following an asymptomatic interval of several hours, acute SM exposure produces subepidermal skin blisters, respiratory tract damage, eye lesions and bone marrow depression. Iranian victims of SM exposure during the Iran-Iraq (1984-1988) war were treated at intensive care units of 3 Munich hospitals. All 12 patients were injured following aerial attacks with SM filled bombs, which exploded in a distance between 5 and 30m. All patients soon noted an offensive smell of garlic, addle eggs or oil roasted vegetables. No individual protective equipment was used. Eye itching and skin blistering started 2h after SM exposure. Some patients complained of nausea, dizziness and hoarseness. 4h after exposure, most patients started vomiting. Eye symptoms worsened and most patients suffered from temporary blindness due to blepharospasm and lid oedema. Additionally, pulmonary symptoms such as productive cough occurred. Patients were transferred to Munich 4-17 days after SM exposure. On admission all patients showed significant skin blistering and pigmentation. Conjunctivitis and photophobia were the major eye symptoms. Pulmonary symptoms, including productive cough were persistent. Bronchoscopy revealed massive inflammation of the trachea with signs of necrosis. 3 patients needed tracheotomy. Chest X-ray did not yield abnormal observations. This presentation summarizes the experience of treating SM victims in Munich and discusses therapeutic implications.

Sulphur mustard induces time- and concentration-dependent regulation of NO-synthesizing enzymes.

UNLABELLED: Sulphur mustard (SM) is a chemical warfare agent that causes erythema and blistering of the skin with a latency of several hours. Although SM is known for almost 200 years the cellular mechanisms involved in the damaging process are not fully understood. There is evidence that changes in nitric oxide (*NO), reactive oxygen species (ROS) and reactive nitrogen species (RNS) might be involved in the damaging process. AIM: To find out more about the pathophysiology of SM, we investigated the initial formation of biochemical markers of nitrosative and oxidative stress as well as activation (translocation from plasma-membrane) and upregulation of eNOS and iNOS, respectively. METHODS: Human immortalized keratinocytes (HaCaT cell line) were exposed to SM (100 microM or 300 microM) for 30 min. Cells were fixed after 1h, 3h or 6h of incubation in SM-free medium and immunostained. Live cell experiments were performed using the NO-sensitive dye DAF2-DA. In order to assess cell viability after BH(4) supplementation, we analyzed apoptosis using CDD-ELISA. RESULTS: SM significantly increased biochemical markers of nitrosative and oxidative stress already 1h after exposure. Moreover, the NO producing enzymes eNOS and iNOS showed concentration- and time-dependent changes in their activation or expression levels. Initially, live cell imaging experiments could not confirm NO production after SM exposure. Only when cells were supplemented with tetrahydrobiopterine, stable NO production was detectable. Apoptotic activity was increased due to SM exposure and could be reduced after BH(4) treatment. CONCLUSION: Our data point towards concentration- and time-dependent formation of iNOS and activation of eNOS due to translocation from plasma-membrane. Live cell experiments yielded first indications of catalytic decoupling of NOS that could be reversed by supplementation with tetrahydrobiopterin (BH(4)). Addition of BH(4) 1h after SM exposure significantly decreased apoptosis compared to the unsupplemented control.

Distribution and excretion of BisGMA in guinea pigs.

Bisphenol-A-glycidyldimethacrylate (BisGMA) is used in many resin-based dental materials. It was shown in vitro that BisGMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of [(14)C]BisGMA applied via gastric and intravenous administration (at dose levels well above those encountered in dental care) were examined in vivo in guinea pigs to test the hypothesis that BisGMA reaches cytotoxic levels in mammalian tissues. [(14)C]BisGMA was taken up rapidly from the stomach and intestine after gastric administration and was widely distributed in the body following administration by each route. Most [(14)C] was excreted within one day as (14)CO(2). The peak equivalent BisGMA levels in guinea pig tissues examined were at least 1000-fold less than known toxic levels. The peak urine level in guinea pigs that received well in excess of the body-weight-adjusted dose expected in humans was also below known toxic levels. The study therefore did not support the hypothesis.

Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells.

Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.

Nebivolol induces eNOS activation and NO-liberation in murine corpus cavernosum.

Erectile function is critically dependent upon the activation of the endothelial nitric oxide synthase (eNOS) in the smooth muscle cells of penile corpus cavernosum tissue. Nebivolol is a beta(1)-selective beta-adrenoceptor blocker (beta-ARB) with additional vasodilating properties, which have been attributed to eNOS-activation. Our study investigated whether nebivolol is able to increase eNOS activity in erectile tissue. Murine penile tissue was incubated in an organ bath under control conditions and in the presence of nebivolol or metoprolol. Immunofluorescence staining was performed using specific antibodies against eNOS-activation or eNOS-serine 1177 phosphorylation. Corpus cavernosum smooth muscle tissue was identified using a smooth muscle actin antibody. In addition, slices of murine erectile tissue were incubated with diaminofluorescein (DAF), a specific fluorescence marker for NO-liberation. Under control conditions and after application of metoprolol, we observed a small eNOS-activation and serine 1177-phosphorylation in murine corpus cavernosum tissue. A significant increase in eNOS-activation and serine 1177-phosphorylation of eNOS was observed only in the presence of nebivolol (10 muM). These alterations of the eNOS protein induced after application of nebivolol were associated with a time-dependent increase in DAF fluorescence in murine erectile tissue. We conclude that beta-adrenoceptor blockers differentially influence erectile tissue. Since cardiovascular diseases are often associated with the development of erectile dysfunction, the nebivolol-induced eNOS-activation in corpus cavernosum may be beneficial when treating patients suffering from cardiovascular disease.

Apoptosis in sulfur mustard treated A549 cell cultures.

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.

In vitro embryotoxicity assessment with dental restorative materials.

OBJECTIVES: Resin (co)monomers may be released from restorative dental materials and can diffuse into the tooth pulp or the gingiva, and can reach the saliva and the circulating blood. Genotoxic potential of some dental composite components has been clearly documented. The genotoxic effects of xenobiotics can represent a possible step in tumor initiation and/or embryotoxicity/teratogenesis. A modified fluorescent mouse embryonic stem cell test (R.E.Tox) was used to test the embryotoxic potential of following dental restorative materials: Bisphenol A glycidylmethacrylate (BisGMA), urethanedimethacrylate (UDMA), hydroxyethylmethacrylate (HEMA), and triethyleneglycoldimethacrylate (TEGDMA), as well as some of their metabolic intermediates 2,3-epoxy-2-methyl-propionicacid-methylester (EMPME), methacrylic acid (MA), and 2,3-epoxy-2-methylpropionic acid (EMPA). METHODS: Mouse embryonic stem (ES) cells stably transfected with a vector containing the gene for the green fluorescent protein under control of the cardiac alpha-myosin heavy chain promoter were differentiated in the presence of various concentrations of the test compounds for 12 days. Fluorescence was measured using the TECAN Safire and values were expressed as percent of control values. To distinguish between cytotoxic and embryotoxic effects, all compounds were tested in a standard MTT assay. RESULTS: HEMA, TEGDMA and EMPME did not influence the differentiation process of ES cells towards cardiac myocytes. No cytotoxic effects were observed at any of the concentration levels tested. Exposure to BisGMA resulted in a 50% decrease in cell survival and a very strong inhibition of cell differentiation at 10(-5)M (p<0.01). Embryotoxic effects were also present at 10(-6) and 10(-7)M (p<0.05). EMPA induced a decrease in ES cell differentiation at 10(-5)M (p<0.01) without cytotoxic effects. No embryotoxic effects were induced at lower concentrations. Exposure to UDMA resulted in a slight decrease of cell differentiation at 10(-5)M (p<0.05). Exposure of cells to MA resulted in an increase of cardiac differentiation up to 150% (p<0.05) at 10(-5)M without cytotoxic effects. CONCLUSIONS: BisGMA induced a significant high embryotoxic/teratogenic effect over a large range of concentration. Therefore attention should be focused on this dental monomer, which should be investigated further by in vivo experiments.

Cytotoxicity of ingredients of various dental materials and related compounds in L2- and A549 cells.

Various ingredients of dental materials and related compounds were tested for cytotoxicity in two alveolar epithelial cell lines (L2 and A549 cells). Release of lactate dehydrogenase (LDH) from cells was measured after incubation with the test substances for time intervals up to 48 h and expressed as percentage of total LDH content of lysed cells. Furthermore, the glutathione content of cells was determined in the nonmalignant L2 cells. Additionally, cell viability was assessed by microscopic examination. The highest cytotoxicity was observed with mercury compounds (methylmercuric chloride and mercury dichloride) in the range of 5-20 micromol/l. The composite components 2-hydroxyethylmethacrylate (HEMA) and triethleneglycoldimethacrylate (TEGDMA) showed time- and concentration-dependent effects of cytotoxicity at high concentrations (about 1-5 mmol/l). A time dependence for GSH decrease was mainly found for the composite components up to 12 h of cellular exposure. L2 cells were more sensitive to both mercury and composite compounds than A549 cells. Gold compounds (sodiumaurothiomalate and gold particles < 1.5 microm) did not produce any sign of toxic reactions. A time-dependent increased toxicity in pulmonary cell lines was found for the composite components HEMA and TEGDMA, but not for mercury and gold compounds.

Biological clearance of HEMA in guinea pigs.

No toxicokinetic data are available about the dental composite component 2-hydroxyethylmethacrylate (HEMA) in vivo in the literature. Therefore, the excretion of HEMA in feces and urine in vivo and, using the pendular perfusion technique with segments of jejunum and colon, in the biliary and enteric excretion in situ were investigated in anesthetized guinea pigs. In the in situ experiments, guinea pigs (n = 4) received HEMA (0.02 mmol/kgbw labelled with a tracer dose 14C-HEMA 0.3 kBq/gbw) injected into the jugular vein. In the in vivo experiments, guinea pigs (n = 4) received HEMA (+ 14C-HEMA, same dose as above) via gastric tube. Urine and feces were collected for 24h. In the in situ experiments, organs from guinea pigs were removed 60 min after the beginning of the experiment, and then the 14C-radioactivity was measured. During the 60 min perfusion period the calculated amount of 14C-activity excreted into the total jejunum and colon was 6.0 +/- 1.0% and 2.7 +/- 0.7% of the dose administered, respectively (mean +/- sem). Of the 14C-HEMA dose, 5.3 +/- 0.3% was found in the bile. Significantly (p < 0.05) higher bile/blood concentration ratios were found at 10-40 min after the injection of HEMA, as compared to the ratio at 60 min. The total 14C-recovery in all organs tested was 20.0 +/- 2.6%. During 24h the amounts of 14C-activity excreted in the feces and urine were 1.1 +/- 0.1% or 17.1 +/- 1.50% of the dose administered, respectively (mean +/- sem). The total 14C-recovery in all organs tested was 11.6 +/- 0.6%. In a second series of in vivo experiments, exhaled air from the animals was captured during the 24h experimental period. 14C was exhaled to 63.6 +/- 2.11% of the administered 14C-HEMA dose (mean +/- sem; n = 4) as 14C-carbondioxide. The results indicate a rapid clearance of 14C-HEMA and/or 14C-HEMA metabolite(s) from the organism, exhalation being the major route of elimination.

Toxicokinetic of HEMA in guinea pigs.

OBJECTIVE: Unconverted 2-hydroxyethylmethacrylate (HEMA) can be released from dental resin materials and can enter the body in humans. In the present study the uptake, distribution and excretion of 14C-HEMA applied via different routes were examined in vivo in guinea pigs. METHODS: HEMA (0.02 mmol/kg bw labelled with a tracer dose 14C-HEMA 0.3 Bq/g bw) was administered by gastric tube or by subcutaneous injection. Urine, feces, and exhaled carbon dioxide were collected for 24 h after administration. Guinea pigs were killed 24 h after the beginning of the experiment and various organs removed and 14C radioactivity measured. RESULTS: Low fecal 14C levels (about 2% of the dose) and urinary levels of about 15% after 24 h were noted with either route of administration. Direct measurement of exhaled CO(2) showed that about 70% of the dose left the body via the lungs. Two pathways for the metabolism of 14C-HEMA can be described. It is likely that 14C-pyruvate is formed in vivo resulting in the formation of toxic 14C-HEMA intermediates. 14C-HEMA was taken up rapidly from the stomach and small intestine after gastric administration and was widely distributed in the body following administration by each of the routes. CONCLUSIONS: Clearance from most tissues following gastric and intradermal administration was essentially complete within one day. The peak HEMA levels in all tissues examined after 24 h were at least onemillion-fold less than known toxic levels.

Effects of Lewisite on cell membrane integrity and energy metabolism in human keratinocytes and SCL II cells.

Lewisite is a highly toxic arsenic compound which can cause skin damage. In the present study effects of Lewisite on cell membrane integrity and energy metabolism as well as antidotal effects of DL-2,3-dimercaptopropanesulfonate (DMPS), and meso-2,3-dimercaptosuccinic acid (m-DMSA) were investigated in a keratinocyte derived cell line (SCL II) and primary human keratinocytes (HK). Cells were incubated in Lewisite (60 microM) containing medium for 5 min. During the following 6 h lactate dehydrogenase (LDH) activity in the supernatant, intracellular ATP content, tetrazolium reduction, glucose consumption and lactate formation were measured. Glucose consumption and lactate production were decreased in both cell lines after Lewisite exposure. In SCL II cells an increase of LDH activity in the supernatant, a decrease of ATP content, and an impaired ability to reduce tetrazolium was found 3 h after Lewisite exposure. In HK cultures tetrazolium reduction was significantly decreased already after 2 h, whereas LDH increase in the supernatant and ATP content decrease occurred only at 6 h after Lewisite exposure. When DMPS or m-DMSA was added directly after Lewisite exposure to SCL II cells, glucose consumption and lactate formation were restored and LDH leakage was prevented. SCL II cells might be more prone to membrane damage whereas in keratinocytes mitochondrial impairment seems to be the predominant effect of Lewisite.

Distribution and excretion of TEGDMA in guinea pigs and mice.

The monomer triethyleneglycoldimethacrylate (TEGDMA) is used as a diluent in many resin-based dental materials. It was previously shown in vitro that TEGDMA was released into the adjacent biophase from such materials during the first days after placement. In this study, the uptake, distribution, and excretion of 14C-TEGDMA applied via gastric, intradermal, and intravenous administration at dose levels well above those encountered in dental care were examined in vivo in guinea pigs and mice as a test of the hypothesis that TEGDMA reaches cytotoxic levels in mammalian tissues. 14C-TEGDMA was taken up rapidly from the stomach and small intestine after gastric administration in both species and was widely distributed in the body following administration by each route. Most 14C was excreted within one day as 14CO2. The peak equivalent TEGDMA levels in all mouse and guinea pig tissues examined were at least 1000-fold less than known toxic levels. The study therefore did not support the hypothesis.

Biological clearance of TEGDMA in guinea pigs.

The excretion of the dental composite component triethylene glycol dimethacrylate (TEGDMA) in feces and urine in vivo and, using the pendular perfusion technique with segments of jejunum and colon, the biliary and enteric excretion in situ were investigated in anesthetized guinea pigs. In the in situ experiments guinea pigs (n = 4) received TEGDMA (0.02 mmol/kg body weight labelled with a tracer dose 14C-TEGDMA 0.7 kBq/g body weight) injected into the jugular vein. In the in vivo experiments guinea pigs (n = 4) received TEGDMA (+14C-TEGDMA; same dose as above) via a gastric tube. Urine and feces were collected for 24 h. In the in situ experiments organs were removed from the guinea pigs 60 min after the beginning of the experiment, and the 14C radioactivity measured. During the 60-min perfusion period the calculated amount of 14C radioactivity excreted into the total jejunum and colon was 0.9 +/- 0.2% and 1.9 +/- 0.1% of the dose administered, respectively (means +/- SEM). Of the 14C-TEGDMA dose, 3.7 +/- 0.2% was found in the bile. A significantly (P < 0.05) higher bile/blood concentration ratio was found 10 min after injection of TEGDMA as compared with the ratios at 20 to 60 min. The following 14C activities (percent of the dose) per total organ were found in guinea pigs (in situ experiment; means +/- SEM): 6.9 +/- 1.7 (muscle), 3.9 +/- 0.5 (kidney), 3.3 +/- 0.1 (skin), 1.4 +/- 0.1 (blood), and 1.2 +/- 0.1 (liver). The 14C activity in all other organs was < 0.4%. The total 14C recovery in all organs tested was 17.5 +/- 1.8%. Over 24 h the amounts of 14C activity excreted in the feces and urine were 0.5 +/- 0.1% and 14.7 +/- 1.8% of the dose administered, respectively (means +/- SEM). The following 14C activities (percent of the dose) per total organ or contents of organs were found (means +/- SEM): 1.4 +/- 0.3 (liver), 0.8 +/- 0.3 (muscle), 0.5 +/- 0.1 (skin), and 0.5 +/- 0.1 (contents of cecum). The 14C activity in all other organs was < 0.2%. The total 14C recovery in all organs tested was 3.9 +/- 0.9%. In a second series of in vivo experiments exhaled air from the animals was captured during the 24-h experimental period. Of the administered dose, 61.9 +/- 4.6% of the 14C (means +/- SEM; n = 4) was exhaled as 14C-carbon dioxide. The results indicate a rapid clearance of 14C-TEGDMA and/or 14C-TEGDMA metabolite(s) from the organism and exhalation is the major route of elimination.

Cytotoxicity of dental composite components and mercury compounds in pulmonary cells.

The cytotoxic potentials of the dental composite components triethyleneglycoldimethacrylate (TEGDMA) and 2-hydroxy-ethylmethacrylate (HEMA) as well as mercuric chloride (HgCl2) and methyl mercury chloride (MeHgCl) were investigated. Proliferating A549 and L2 cell monolayers were cultured in the absence or presence of composite components or mercurials. Twenty-four hours later the tetrazolium salt XTT (sodium 3'-[1-phenyl-aminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzenesulphonic acid) was added. Formazan formation was quantified using a microtiter plate reader. EC50 values were obtained as half-maximum-effect concentrations from fitted curves. EC50 values were in A549 cells (mean values +/- standard deviation; n = 12; micromol/l); HEMA 8854+/-1882; TEGDMA 1821+/-529; HgCl2 41+/-7 and MeHgCl 27+/-3. EC50 values in L2 cells were: HEMA 191+/-28; TEGDMA 112+/-16; HgCl2 25+/-6 and MeHgCl 8+/-6. All tested substances induced a dose-dependent loss of viability in A549 and L2 cells after 24 h. The EC50 values of both mercurials were significantly (p < 0.05) lower compared to the values of both composite components. TEGDMA was about 5-fold (A549 cells) and about 2-fold (L2 cells) more toxic compared to HEMA. It is to be assumed that the risk of lung cell damage by dental composite components is even more unlikely.

Cytotoxicity of dental composite components and mercury compounds in lung cells.

OBJECTIVE: The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA), as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) was investigated on the release of lactatedehydrogenase (LDH) from alveolar epithelial lung cell lines in vitro. METHODS: The confluent cell layers from the A549 (human, malignant) and the L2 cells (rat) were incubated with various concentrations of HEMA, TEGDMA, MeHgCl and HgCl2 at 37 degrees C in 2% (v/v) CO2 atmosphere for 8h. In further experiments the L2 cells were incubated with the same compounds for 6-48 h. LDH release was measured and the values were expressed as percentage of the LDH content. The values were plotted on a concentration log-scale and the substance concentration at the maximum slope was assessed as effective concentration (EC50). RESULTS: A significant (p<0.05) increase in the LDH release was found in the L2 cells after 8-h incubation with HEMA (4 mmol/l), TEGDMA (2 mmol/l), MeHgCl (0.01 mmol/l) and HgCl2 (0.015 mmol/l), and in A549 cells with HEMA (14 mmol/l), TEGDMA (15 mmol/l), MeHgCl (0.15 mmol/l) and HgCl2 (0.05 mmol/l), compared to controls. The EC50 values from compounds in the L2 cells are shown in the following table (mean; sem in parentheses; n=3-6; #n=1): [see text]. SIGNIFICANCE: The toxic effect of HgCl2 and MeHgCl from the L2 cells was about 100-700-fold higher than of the dental composite components. A significant (p<0.05) time dependent increase of toxicity was observed with TEGDMA, HEMA and MeHgCl.

Nuclear colocalization and complex formation of insulin with retinoblastoma protein in HepG2 human hepatoma cells.

Previous structural and biochemical evidence had suggested that insulin may bind to the nuclear tumor suppressor retinoblastoma protein (RB). The present study is now the first to unravel the physical and functional interaction between a growth factor and an anti-oncoprotein, specifically demonstrating the association between insulin and RB in living cells and finding that this complex formation is relevant for cell division. Our immunofluorescence microscopy data suggest that insulin colocalizes with RB in the cell nuclei of HepG2 human hepatoma cells and that contacts the B-region of the RB pocket. Furthermore, these events were found to correlate with an enhancement of cell proliferation. These results are in line with the initial structure-based predictions and, moreover, provide a suitable starting point for the further understanding as well as the pharmacological modulation of nucleocrine interactions between growth factors and tumor suppressors, in physiology and disease.

Correlation of micronucleus and apoptosis assays with reproductive cell death can be improved by considering other modes of death.

PURPOSE: To evaluate, using 10 cell lines, the already shown improved correlation between the sum of micronucleated plus apoptotic cells and reproductive cell death. MATERIALS AND METHODS: The cell lines were X-irradiated with doses selected to obtain comparable survival levels. Micronucleated and apoptotic cells as well as abnormal cells (multinucleated and necrotic cells) were counted over several days. Apoptosis was also confirmed by gel electrophoresis. RESULTS: The data confirmed the already shown improved relationship over the solely performed micronucleus or apoptosis assays with reproductive cell death and radiation dose. However, even this approach revealed a saturation after irradiation in certain cell lines. Including other modes of cell death such as mitotic failure or necrosis could eliminate this effect. CONCLUSION: Multiple parameters should be considered when evaluating the use of a predictive assay for ionizing radiation-induced cell killing or biological dosimetry.

Sulfur mustard induces apoptosis and necrosis in SCL II cells in vitro.

Sulfur mustard (bis(2-chloroethyl) sulfide, HD) is an alkylating agent causing erythema and blistering with a latency of several hours after skin exposure. In the present in vitro study the influence of HD (1 microM-1 mM for 30 min or 4 h) on the viability and growth of SCL II cells was investigated. No significant differences in cytotoxicity were observed as assessed by formazan formation from XTT tetrazolium salt at 24, 48 and 72 h after exposure. Sulfur mustard concentrations of >500 microM were associated with an increasing portion of apoptotic cells without change in necrosis rate as assessed by nuclear morphology and gel electrophoresis of the DNA. The ATP levels were not affected up to 6 h after HD exposure (< or =1 mM). Twelve hours later, ATP depletion was observed at HD concentrations of >500 microM. Colony-forming ability was impaired at concentrations of <1 microM. Cell growth studies in comparison with nuclear morphology indicated late apoptotic death predominating at lower concentrations of HD. In summary, the data show that HD may inhibit cell growth already at concentrations where viability parameters and cell metabolism are not yet affected.