Utility of an initial D-dimer assay in screening for traumatic or spontaneous intracranial hemorrhage.

OBJECTIVE: To evaluate the sensitivity of a D-dimer assay as a screening tool for possible traumatic or spontaneous intracranial hemorrhage. If adequately sensitive, the D-dimer assay may potentially permit omission of a more expensive computed tomography (CT) scan of the head when such hemorrhage is clinically suspected. METHODS: Prospective, consecutive, blinded study of patients (age > 16 years) requiring a CT scan of the head for suspected intracranial hemorrhage over a five-month period at a university, Level I trauma center. All study patients had a serum D-dimer assay obtained prior to their CT scans. Sensitivity and specificity, with 95% confidence intervals (95% CIs), of the enzyme-linked immunosorbent assay (ELISA) D-dimer assay for the detection of intracranial hemorrhage were calculated. RESULTS: Of the 319 patients entered in the study, 25 (7.8%) had a CT scan positive for intracranial hemorrhage. Patients with intracranial hemorrhage were more likely to have a positive D-dimer assay (chi-square = 13.075, p < 0.001). The D-dimer assay had 21 true-positive and four false-negative tests, resulting in a sensitivity of 84.0% (95% CI = 63.7% to 95.5%) and a specificity of 55.8% (95% CI = 55.5% to 55.9%). The four false-negative cases included one small intraparenchymal hemorrhage, one small subarachnoid hemorrhage, one moderate-sized intraparenchymal hemorrhage with mid-line shift, and one large subdural hematoma requiring emergent surgery. CONCLUSIONS: Due to the catastrophic nature of missing an intracranial hemorrhage in the emergency department, the D-dimer assay is not adequately sensitive or predictive to use as a screening tool to allow routine omission of head CT scanning.

Mechanism of tetrahydroxy-1,4-quinone cytotoxicity: involvement of CA2+ and H2O2 in the impairment of DNA replication and mitochondrial function.

In this work we investigated the toxicity of a polyphenolic p-benzoquinone derivative, the tetrahydroxy-1,4-quinone (THQ) toward V79 Chinese hamster fibroblasts and analyzed the role of H2O2 and Ca2+ in that mechanism. The exposure of exponentially growing cultures to THQ, in the presence of 1.0 mM Ca2+, caused a dose-dependent inhibition of cell growth and DNA synthesis. Complete prevention of those effects by catalase indicated that H2O2-induced damages should underlie both toxic processes. Further detection of a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in cells exposed to THQ plus Ca2+, together with the partial protection conferred by the intracellular Ca(2+)-chelator fura-2 against cell growth inhibition, indicated that a disruption of Ca2+ homeostasis is a determinant event in THQ cytotoxicity. Furthermore, the intracellular accumulation of rhodizonic acid (RDZ), the primary oxidative product of THQ, indicated that THQ, or its corresponding semiquinone form, was entering the cells and undergoing further autoxidation to RDZ. It was also evidenced that mitochondria represent an important target in the development of THQ toxicity, as shown by the disruption of the transmembrane electrical potential (delta psi) of isolated rat liver mitochondria, as well as by the Ca(2+)-release by mitochondria of permeabilized V79 cells. We concluded that disruption of Ca2+ homeostasis and generation of H2O2 are critically involved in THQ-induced impairment of DNA replication and mitochondrial functions, leading ultimately to cell growth inhibition.

Streptozotocin-induced genotoxic effects in Chinese hamster cells: the resistant phenotype of V79 cells.

The genotoxic effects of the methylating agent streptozotocin (STZ) on Chinese hamster cells CHO-9 and V79 were evaluated. The induction of cell killing, chromosomal aberrations, sister-chromatid exchanges (SCEs) and mutations was analyzed. Comparisons were made with the the STZ aglyconic analogue N-methyl-N-nitrosourea (MNU). V79 cells were found to be more resistant than CHO-9 cells to STZ and MNU killing effects, as well as to the induction of chromosomal aberrations and SCEs; however, V79 and CHO-9 cells appeared to be equally sensitive to the induction of 6-thioguanine resistant mutants by STZ. These results suggest that an error-free mechanism that tolerates DNA methylation damage confers a resistant phenotype to V79 cells to the genotoxic effects of methylation damage.

Inhibition of protein synthesis and amino acid transport by crystal violet in Trypanosoma cruzi.

[35S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 microM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [35S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [3H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake.

Streptozotocin-induced chromosomal aberrations, SCEs and mutations in CHO-9 parental cells and in EM-C11 mutant cell line.

The genotoxic effects induced by the monofunctional nitrosourea derivative streptozotocin (STZ) were investigated in Chinese hamster ovary cells, parental (CHO-9) and its mutant hypersensitive to alkylating agents, designated EM-C11. The ability of this compound to induce chromosomal aberrations, cell killing, sister-chromatid exchanges (SCEs) and mutations was evaluated on these two cell lines. The mutant cells were found to be slightly more sensitive to the killing effects of STZ than the parental cell line. EM-C11 cells also showed higher levels of STZ-induced chromosomal aberrations than CHO-9 cells, but appeared to be equally sensitive to induction of SCEs. The frequencies of STZ-induced mutations, measured as resistant Na+/K(+)-ATPase and HPRT mutants, revealed a higher sensitivity of EM-C11 to the mutagenic effects of this compound.

Streptozotocin-induced toxicity in CHO-9 and V79 cells.

The cytotoxicity of streptozotocin (STZ) was investigated in Chinese hamster fibroblast lines (CHO-9 and V79) in comparison to two other alkylating agents, methylnitrosourea (MNU) and ethylnitrosourea (ENU), using cell survival as the endpoint. It was found that V79 cells were far more resistant to methylation induced by STZ and MNU than CHO-9 cells (20 and four times, respectively) but equally sensitive to the ethylating agent ENU. The extent of STZ-induced DNA methylation was estimated by analyzing the extent of O6-metG and N7-metG adducts in the DNA of treated cells through high-performance liquid chromatography (HPLC) with electrochemical detection. The number of adducts as well the efficiencies of their removal from the DNA were similar in both cell lines. The response of these cells to the presence of DNA damage was evaluated by analysis of STZ effects on DNA replication and cell cycle progression. Measurement of [3H]-thymidine incorporation showed a similar pattern of response at the level of inhibition of DNA synthesis in both cell lines. However, analysis of metaphase cells 36 hr after STZ exposure showed an accumulation of cells in the second cycle in the CHO-9 line, indicating induction of a cell cycle arrest. Only a slight effect was observed on cell cycle progression in V79 cells, indicating that the methylation resistance of these cells could be related to their ability to progress through the cell cycle despite the presence of DNA lesions.

Disruption of Ca2+ homeostasis in Trypanosoma cruzi by crystal violet.

We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca2+ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca2+ uptake by digitonin-permeabilized epimastigotes. Crystal violet also induced Ca2+ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca2+ uptake and the (Ca(2+)-Mg2+)-ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca2+ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.

ATP and Ca2+ homeostasis in Trypanosoma cruzi.

Cell viability requires the perfect functioning of the processes controlling ATP and Ca2+ homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with disruption of ATP and Ca2+ homeostasis. Therefore, the study of the mechanisms by which different T. cruzi stages regulate the intracellular Ca2+ distribution and the ATP supply to maintain cell viability could provide new insights into the physiology of these parasites. One important objective of these studies is the identification of possible metabolic differences between host and parasite that could be exploited for the rational design of new and more effective trypanocidal drugs.

ATP and Ca2+ homeostasis in Trypanosoma cruzi

Cell viability requires the perfect functioning of the processes controlling ATP and Ca2+ homeostasis. It is known that cell death caused by a variety of toxins or pathological conditions is associated with disruption of ATP and Ca2+ homeostasis. Therefore, the study of the mechanisms by which different T. cruzi stages regulate the intracellular Ca2+ distribution and the ATP supply to maintain cell viability could provide new insights into the physiology of these parasites. One important objective of these studies is the identification of possible metabolic differences between host and parasite that could be exploited for the rational design of new and more effective trypanocidal drugs

Bacterial chemistry. VI. Biological activities and cytotoxicity of 1,3-dihydro-2H-indol-2-one derivatives.

The biosynthetic pigment from Chromobacterium violaceum BB-78, 1,3-dihydro-2H-indol-2-one and its derivatives exhibit biological activities such as antimicrobial action, low hemolytic effects on red blood cells and in vitro trypanocide activity. A relatively high cytotoxicity on V-79 hamster fibroblast cells of the biosynthetic pigment was found, although with the methylol derivative the toxicity was almost eliminated. The methylol derivative exhibited similar toxicity as Nifurtimox, a known, commercial trypanocide compound.

Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.

Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.

Regulation of intracellular calcium homeostasis in Trypanosoma cruzi. Effects of calmidazolium and trifluoperazine.

Trypanosoma cruzi epimastigotes maintained an intracellular free calcium concentration of about 0.15 microM, as measured with the fluorescent indicator Fura-2. The maintenance of low [Ca2+]i is energy-dependent since it is disrupted by KCN and FCCP. When the cells were permeabilized with digitonin, the steady-state free Ca2+ concentration in the absence of ATP was about 0.7 microM. The additional presence of ATP resulted in a steady-state level close to 0.1-0.2 microM which compares favorably with the concentration detected in intact cells. Intracellular Ca2+ uptake at high levels of free Ca2+ (greater than 1 microM) was due to energy-dependent mitochondrial uptake as indicated by its FCCP-sensitivity. However, as the free Ca2+ concentration was lowered from 1 microM, essentially all uptake was due to the ATP-dependent Ca2+ sequestration by the endoplasmic reticulum as indicated by its stimulation by ATP, and its inhibition by sodium vanadate. High concentrations of the calmodulin antagonist trifluoperazine, inhibited both the Ca2+ uptake by the endoplasmic reticulum and by the mitochondria, while calmidazolium released Ca2+ from both compartments. In addition, trifluoperazine and calmidazolium inhibited respiration and collapsed the mitochondrial membrane potential of T. cruzi, thus indicating non-specific effects unrelated to calmodulin.

1,10 phenanthroline, a metal chelator, protects against alloxan- but not streptozotocin-induced diabetes.

The iron chelator, 1,10-phenanthroline (Phen), which inhibits hydroxyl radical formation, was tested in vitro and in vivo against alloxan and streptozotocin (STZ) cytotoxicity. Phen injection reduced the severity of alloxan-induced diabetes in rats and attenuated alloxan-induced toxicity in human fibroblasts (VA 13 line) in culture. These protective effects were not observed against STZ toxic action. These results are consistent with the hypothesis that hydroxyl radicals, generated via an iron-catalyzed reaction, induce the alloxan but not the STZ diabetogenic effects.

Correlation between cytotoxic effect of hydrogen peroxide and the yield of DNA strand breaks in cells of different species.

The rate of loss of reproductive capacity produced by hydrogen peroxide was shown to be 6-times faster for human fibroblasts than for Chinese hamster fibroblasts. Mouse fibroblasts exhibited an intermediate response. The explanation for that does not lie in the different capacities of these cells to destroy H2O2. The kinetics of repair of single-strand breaks although slightly different for the three cell lines also does not provide a full explanation for the different sensitivity. What was shown to correlate well with the killing effect was the yield of strand breaks produced by H2O2 in the DNA of cells from the three species. A similar H2O2 concentration produced 5-10-times more strand breaks in human DNA than in hamster DNA and 2-4-times more than in mouse DNA. This ratio holds for different cell lines from human and hamster and thus seems to be species-specific. Based on our previous findings we propose that this difference may lie in the amount of chromatin-bound iron and the level of superoxide ion in these cells.

Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron.

Phenanthroline, a strong iron chelator, prevents both the formation of DNA single-strand breaks and the killing of mouse cells produced by H2O2. These results, taken together with our previous findings, indicate that the DNA damage is produced by hydroxyl radicals formed when H2O2 reacts with chromatin-bound Fe2+ and that this damage is responsible for the killing effect.

The damaging action of hydrogen peroxide on DNA of human fibroblasts is mediated by a non-dialyzable compound.

Single-strand breaks were formed in DNA when nuclei from human fibroblasts were incubated with buffered salt solutions containing H2O2. This had not been observed when purified DNA had been exposed to H2O2. The nuclear factor which mediates the action of H2O2 on DNA was removed when nuclei were dialyzed against buffered salt solutions containing EDTA. If this complexing agent was not present during dialysis, the factor remained in the nuclear preparation. The formation of DNA single-strand breaks was considerably diminished when nuclei were incubated in solutions containing H2O2 plus hydroxyl radical scavengers. These facts favor a scheme according to which H2O2 reacts with a metal-macromolecule complex in the nucleus giving rise to a hydroxyl radical which is the DNA-damaging agent. Therefore, hydrogen peroxide seems to be a mimetic chemical of ionizing radiation.

DNA damage during the peroxidase-catalyzed aerobic oxidation of isobutanal.

The aerobic oxidation of isobutanal catalyzed by peroxidase, when carried out in the presence of DNA, produces alkali-sensitive bonds in this macromolecule. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Since triplet acetone has been recently identified as an intermediate in this oxidation (Durán, N., Faria Oliviera, O.M.M., Haun, M. and Cilento, G. (1977) J. Chem. Soc. Chem. Commun., 442--443), this species is a likely candidate for the entity which brings about the lesions, via transfer of its electronic energy to DNA.