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B Herman

Publications and source records attributed to B Herman.

At least 55 records · Page 3Linked to original sources
PubMed1994

Automatic detection of clustered, fluorescent-stained nuclei by digital image-based cytometry.

Automatic image-based cytometry (IC) can conveniently quantify the distributions of several specific, fluorescence-labeled molecules within individual, isolated cells of slide- or tissue-based specimens. However, many specimens contain clusters of cells or nuclei that are not detected as individual entities by existing automatic methods. We have developed analysis algorithms which detected individual nuclei occurring in clusters or as isolated nuclei. Specimens were labeled with a fluorescent DNA stain, imaged and the images were segmented into regions of nuclei and background. Clusters of nuclei, identified by their size and shape, were divided into individual nuclei by searching for dividing paths between nuclei. The paths, which need not be straight, possessed the highest average gradient per pixel. In addition, both high- and low-pass filtered images of the original image were analyzed. For each individual nucleus, one of the three segmented regions representing the nucleus (from either the original or one of two filtered images) was chosen as the final result, based on the closeness of the regions to average nuclear morphology. The algorithms correctly detected a high proportion of isolated (328/333) and clustered (254/271) nuclei when applied to images of 2 microns prostate and breast cancer sections. Thus, these algorithms should enable much more accurate detection and analyses of nuclei in intact specimens.

Algorithms↗
PubMed1994

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry.

Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical smears.

Algorithms↗
PubMed1994

Additive effect on gas exchange of inhaled nitric oxide and intravenous almitrine bismesylate in the adult respiratory distress syndrome.

OBJECTIVE: To assess the additive effect of inhaled nitric oxide (NO) and intravenous almitrine bismesylate (ALM) on gas exchange. DESIGN: Prospective self-controlled study. SETTING: 3 medico-surgical intensive care units. PATIENTS: 17 patients with severe hypoxemia (PaO2/FIO2 ratio: 88 +/- 30 mmHg, venous admixture: 47 +/- 7%) and elevated mean pulmonary artery pressure (MPAP: 30 +/- 5 mmHg) due to adult respiratory distress syndrome (ARDS). INTERVENTIONS: 5 conditions were studied: 1) baseline, 2) 5 to 10 ppm of NO during 30 min, 3) discontinuation of NO during 30 min, 4) ALM infusion (0.5 mg/kg) during 30 min, 5) ALM infusion (0.5 mg/kg) during 30 min in combination with 5 to 10 ppm of NO. MEASUREMENT AND RESULTS: The PaO2/FIO2 ratio rose from 88 +/- 30 to 98 +/- 37 mmHg (NS) with NO alone, and from 92 +/- 25 to 130 +/- 56 mmHg (p < 0.01) with NO + ALM (p < 0.05 vs NO alone). Seven patients were considered as "NO-responders" (rise in PaO2/FIO2 ratio of 10 mmHg or more with NO); in this subgroup the PaO2/FIO2 ratio rose from 87 +/- 30 to 128 +/- 39 mmHg (p < 0.05) with NO alone, and from 93 +/- 20 to 169 +/- 51 mmHg (p < 0.01) with NO + ALM (p < 0.05 versus NO alone). MPAP decreased from 30 +/- 5 to 26 +/- 5 mmHg (p < 0.01) with NO alone, increased slightly from 28 +/- 5 to 31 +/- 5 mmHg (NS) with ALM alone and decreased to 27 +/- 5 mmHg (p < 0.05) with NO + ALM. CONCLUSIONS: NO + ALM had additive effects on gas exchange while decreasing MPAP in patients with ARDS. The effects of NO alone were small and non significant, except in a subgroup of 7 patients in whom the combination of both therapies had the more pronounced results.

Administration, Inhalation↗
PubMed1994

Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study.

Exploiting the optical sectioning capabilities of laser scanning confocal microscopy and using parameter-specific fluorescent probes, we determined the distribution of pH, free Ca2+, electrical potential, and volume inside cultured adult rabbit cardiac myocytes during ATP depletion and reductive stress with cyanide and 2-deoxyglucose ("chemical hypoxia"). During normoxic incubations, myocytes exhibited a cytosolic pH of 7.1 and a mitochondrial pH of 8.0 (delta pH = 0.9 units). Sarcolemmal membrane potential (delta psi) was -80 mV, and mitochondrial delta psi was as high as -100 mV, yielding a mitochondrial protonmotive force (delta p) of -155 mV (delta P = delta psi - 60 delta pH). After 30 min of chemical hypoxia, mitochondrial delta pH decreased to 0.5 pH units, but mitochondrial delta psi remained essentially unchanged. By 40 min, delta pH was collapsed, and mitochondrial and cytosolic free Ca2+ began to increase. Mitochondrial and sarcolemmal delta psi remained high. as Ca2+ rose, myocytes shortened, hypercontracted, and blebbed with a 30% decrease of cell volume. After hypercontraction, extensive mitochondrial Ca2+ loading occurred. After another few minutes, mitochondrial depolarized completely and released their load of Ca2+. After many more minutes, the sarcolemmal permeability barrier broke down, and viability was lost. These studies demonstrate a sequence of subcellular ionic and electrical changes that may underlie the progression to irreversible hypoxic injury.

Animals↗
PubMed1994

Moderate and vigorous leisure-time physical activity and cardiovascular disease risk factors in West Germany, 1984-1991.

The relationship between leisure-time physical activity (LTPA) and cardiovascular disease (CVD) risk factors was analysed based on data of three cross-sectional National Health Surveys, carried out in West Germany between 1984 and 1991. A total of 7663 males and 7722 females, aged 25-69 years, were examined. A self-administered questionnaire was used to assess LTPA which was defined as the mean duration of time (in minutes) spent during the past week on 14 specified sports during the previous 3 months. The mean time for LTPA per week was 102 minutes for males and 70 minutes for females. No significant differences were observed in LTPA mean duration for the three surveys. Younger age and higher social class were strong predictors for greater LTPA. In multiple linear regression models, after adjustment for age and social class, LTPA was significantly associated with lower systolic and diastolic blood pressure (both sexes), total cholesterol (males only), cigarette smoking (males only) and body mass index (females only), and with higher HDL-cholesterol (both sexes). The data suggest that LTPA plays an important role if aiming at reducing CVD risk factors in the general population.

Adult↗
PubMed1994

ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition.

The importance of ATP depletion and mitochondrial depolarization in the toxicity of cyanide, oligomycin, and carbonyl cyanide m-cholorophenylhydrazone (CCCP), an uncoupler, was evaluated in rat hepatocytes. Oligomycin, an inhibitor of the reversible mitochondrial ATP synthase (F1F0-adenosinetriphosphatase), caused dose-dependent cell killing with 0.1 microgram/ml being the minimum concentration causing the maximum cell killing. Oligomycin also caused rapid ATP depletion without causing mitochondrial depolarization. Fructose (20 mM), a potent glycolytic substrate in liver, protected completely against oligomycin toxicity. CCCP (5 microM) also caused rapid killing of hepatocytes. Fructose retarded cell death caused by CCCP but failed to prevent lethal cell injury. Although oligomycin (1.0 microgram/ml) was lethally toxic by itself, in the presence of fructose it protected completely against CCCP-induced cell killing. Cyanide (2.5 mM), an inhibitor of mitochondrial respiration, caused rapid cell killing that was reversed by fructose. CCCP completely blocked fructose protection against cyanide, causing mitochondrial depolarization and rapid ATP depletion. In the presence of fructose and cyanide, oligomycin protected cells against CCCP-induced ATP depletion and cell death but did not prevent mitochondrial depolarization. In every instance, cell killing was associated with ATP depletion, whereas protection against lethal cell injury was associated with preservation of ATP. In conclusion, protection by fructose against toxicity of cyanide, oligomycin, and CCCP was mediated by glycolytic ATP formation rather than by preservation of the mitochondrial membrane potential. These findings support the hypothesis that inhibition of cellular ATP formation is a crucial event in the progression of irreversible cell injury.

Adenosine Triphosphate↗
PubMed1994

Introduction of a fast and sensitive fluorescent in situ hybridization method for single-copy detection of human papillomavirus (HPV) genome.

At present, in situ hybridization (ISH) is the only method for detection of specific genes in morphologically intact cells or tissue. We have developed a highly sensitive and quantitative fluorescence-based in situ hybridization (FISH) technique that can detect as few as one to five copies of the integrated human papillomavirus (HPV) type 16 genome in cervical cell lines, using digoxygenin tail-labeled oligonucleotides (Method 1). The entire procedure can be carried out in 4.5 hr through the elimination of some of the steps routinely used in other ISH protocols. We also compared the sensitivity of this new FISH method (Method 1) to four other FISH techniques: digoxigenin-labeled DNA probe (Method 2); fluorescein-15-d-ATP-labeled oligonucleotides (Method 3); fluorescein 15-d-ATP labeled DNA probe (Method 4); and biotin-DNA-labeled probe (Method 5), for their ability to detect HPV DNA in the HPV-positive human cervical cell lines CaSki (500 copies) and SiHa (1-5 copies), but not in C33-A and HT-3, which do not contain any copies of HPV. Our results indicate that Method 1 is more sensitive than the other methods employed. Method 1 was the only method that could reliably detect an HPV-16 genome in all SiHa cells. Our data suggest that the Method 1 FISH technique is highly sensitive and may therefore be of general use for detection and quantitation of a variety of viral genomes (including HIV), oncogenes, and drug-resistant genes, in a variety of morphologically intact cells and tissues.

Base Sequence↗
PubMed1993

Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes.

Cell killing, oxygen consumption, and hydroperoxide formation were determined in rat hepatocytes after glycolytic and respiratory inhibition. These conditions model the ATP depletion and reductive stress of anoxia ("chemical hypoxia"). Glycolysis was inhibited with iodoacetate, and mitochondrial electron transfer was blocked with sodium azide, cyanide, or myxothiazol. Cell killing, hydroperoxide formation, and inhibitor-insensitive oxygen consumption were greater after azide than after myxothiazol or cyanide. Desferrioxamine, an inhibitor of iron-catalyzed hydroxyl radical formation, delayed cell killing after each of the respiratory inhibitors. Anoxia also delayed cell killing during chemical hypoxia. However, during anoxic incubations, desferrioxamine did not delay the onset of cell death. These findings indicate that reactive oxygen species participate in lethal cell injury during chemical hypoxia. In isolated mitochondria, previous studies have shown that myxothiazol inhibits Q cycle-mediated ubisemiquinone formation in complex III (ubiquinol-cytochrome c oxidoreductase) and that ubisemiquinone can react with molecular oxygen to form superoxide. Decreased killing of hepatocytes with myxothiazol compared with azide suggests, therefore, that mitochondrial oxygen radical formation by complex III is involved in cell killing during reductive stress. In support of this hypothesis, myxothiazol reduced rates of cell killing and hydroperoxide formation in hepatocytes incubated with azide or cyanide. This mitochondrial mechanism for oxygen radical formation may be important in relative but not absolute hypoxia.

Animals↗
PubMed1993

Intracellular pH and Ca2+ homeostasis in the pH paradox of reperfusion injury to neonatal rat cardiac myocytes.

Ischemia is characterized by anoxia and a large decrease of tissue pH. After a critical period of ischemia, reperfusion precipitates irreversible injury. Previous work showed that reperfusion injury to cultured neonatal myocytes was precipitated by a rapid return to physiological pH, a "pH paradox" (Bond, J., B. Herman, and J. Lemasters. Biochem. Biophys. Res. Commun. 179: 798-803, 1991). The aim of this study was to measure intracellular pH (pHi) and cytosolic free Ca2+ during the pH paradox of reperfusion injury to cultured neonatal rat cardiac myocytes. pHi and free Ca2+ were measured by ratio imaging of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and fura 2 fluorescence. To simulate ATP depletion and acidosis of ischemia, myocytes were incubated with 20 mM 2-deoxyglucose plus 2.5 mM NaCN at pH 6.2. During simulated ischemia, pHi dropped to < 6.5 and subsequently remained constant. During this time, some blebbing but little hypercontraction occurred. After 3 or 4 h of simulated ischemia, inhibitors were removed and cells were incubated at pH 7.4 to simulate reperfusion. pHi began to increase, blebbing accelerated, and myocytes hypercontracted. As pHi increased, viability was lost. The same occurred if pH was increased but metabolic inhibitors were not removed. Monensin, a Na(+)-H+ ionophore, accelerated the increase of pH after reperfusion and hastened cell killing. Hypercontraction, blebbing, and loss of viability did not occur when inhibitors were removed at pH 6.2 or in the presence of dimethylamiloride, an inhibitor of Na(+)-H+ exchange. Protection was associated with maintenance of an acidotic pHi. Free Ca2+ progressively increased during simulated ischemia. After simulated reperfusion, free Ca2+ increased further.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1993

Coronary heart disease case fatality in four countries. A community study. The Acute Myocardial Infarction Register Teams of Auckland, Augsburg, Bremen, FINMONICA, Newcastle, and Perth.

BACKGROUND: Community-based registers participating in the MONICA Project of the World Health Organization show markedly different attack and death rates of coronary heart disease. This variation is a function of both the incidence and case fatality occurring within countries. The contribution of case fatality to the international variation in coronary heart disease mortality rates is not well understood. METHODS AND RESULTS: The register data from eight study populations--Augsburg and Bremen in Germany, Auckland in New Zealand, Perth and Newcastle in Australia, and North Karelia, Kuopio, and Turku/Loimaa in Finland--were compared. All patients with definite myocardial infarction or coronary death aged 35 to 64 years occurring in the study populations in 1985 through 1989 are the basis for the case fatality calculations by different definitions: 28-day case fatality for all cases, for hospitalized cases, and for hospitalized 24-hour survivors; out-of-hospital case fatality; and 24-hour case fatality for hospitalized cases. Differences in case fatality were much smaller than differences in attack and mortality rates in these populations. About two thirds of deaths occurred before the patients reached a hospital. The 28-day case fatality ranged from 37% for men in Perth to 58% for women in Augsburg. Among those who reached the hospital alive, 28-day case fatality was 13% to 27% for men and 20% to 35% for women. In those who survived 24 hours from the onset of symptoms, 28-day case fatality was 8% to 17% for men and 12% to 26% for women. CONCLUSIONS: Differences in case fatality were not associated with differences in coronary mortality rates between these populations. As most deaths occurred before reaching a hospital, opportunities for reducing case fatality through improved hospital care are limited. This emphasizes the primary role of prevention in reducing coronary death rates.

Adult↗
PubMed1993

Noninvasive pressure support ventilation in patients with acute respiratory failure.

To further delineate indications for noninvasive pressure support ventilation (NIPSV), we proposed this noninvasive technique as an alternative to endotracheal intubation in 17 consecutive patients with acute respiratory failure from various causes. Eight patients (47 percent) were successfully ventilated with NIPSV, while in 9 (53 percent), NIPSV failed. Gas exchange values before initiating NIPSV were different between the 2 groups: patients successfully ventilated with NIPSV had a higher PaCO2 (57 +/- 15 mm Hg vs 37 +/- 17 mm Hg; p = 0.01), a lower pH (7.33 +/- 0.03 vs 7.45 +/- 0.08; p = 0.02), and a lower alveolo-arterial oxygen pressure difference (P[A-a]O2) (144 +/- 46 mm Hg vs 265 +/- 18 mm Hg; p = 0.01), suggesting that CO2 retention without major hypoxemia is a better indication for NIPSV than severe hypoxemia alone. Acute respiratory failure occurring after extubation seemed to be a good indication for NIPSV, with an 83 percent rate of success. In both groups of patients, gas exchange improved after 1 h on NIPSV, but such values were not improved on the first day in patients who failed with NIPSV.

Acute Disease↗
PubMed1993

Detection of hepatitis B viral DNA by polymerase chain reaction in dialysis patients.

The polymerase chain reaction (PCR) was used to search for hepatitis B virus (HBV) DNA sequences in the sera of 51 dialyzed patients (26 women, 25 men; mean age 60.5 years, range 35 to 85). Two different sets of specific primers for HBV core and surface gene sequences were synthesized and used for each sample. Controls were 90 HBV negative blood donors. Results were analyzed according to other serological markers of HBV. Among the eight HBsAg positive patients (anti-HBc+: 8/8), seven were positive for HBV DNA. Four of eight patients were vaccinated but later developed acute HBs hepatitis. The presence of HBV DNA was detected in six of 43 HBsAg negative patients (anti-HBc+: 5/6; anti-HBs+: 3 of 6; HBeAg: 0 of 6; anti-HBe: 2 of 6). These six patients were vaccinated and four of six developed mild and transient cytolytic hepatitis (3 before vaccination; 2 later). These results showed that HBsAg seronegative patients can be infectious. The role of HBV vaccination and/or the existence of variations in the structure of the viral genome is discussed.

Adult↗
PubMed1993

Co-localization of the tumor-suppressor protein p53 and human papillomavirus E6 protein in human cervical carcinoma cell lines.

The loss of the tumor-suppressor activity of p53, either by mutation or by interaction with the human papillomavirus (HPV) E6 protein, is considered to be an important mechanism in the carcinogenesis of cervical cancer. We have studied the cytological distribution of these proteins in human cervical carcinoma cell lines using polyclonal anti-p53 and monoclonal anti-E6 antibodies. The antibody specificity was confirmed by immunoblot and immunocompetition analyses. The intracellular localization of p53 and E6 was detected using the techniques of conventional and three-dimensional confocal microscopy. In the HPV-18 or -16 integrated cell lines, HeLa, CaSki and SiHa, viral oncoprotein E6 and endogenous tumor-suppressor protein, p53, were observed by immunofluorescence in the cytoplasm; p53 also had a weak punctate staining in the nuclei of HeLa and CaSki cells. In the HPV-negative cervical carcinoma cell lines, C-33A and HT-3, which have mutated p53, p53 was localized predominantly to the nucleus, with C-33A cells having elevated levels of p53 compared with the other cell lines. High spatial resolution imaging, using confocal microscopy, was performed on the cells after double fluorescence staining for p53 (fluorescein) and E6 (rhodamine). The images showed that both p53 and E6 had similar cytoplasmic distributions, which implied that these two proteins may exist as a cytoplasmic complex. To substantiate this implication, fluorescence resonance energy transfer microscopy was performed, which provided direct evidence of a close association between p53 and E6 within individual HeLa cells. The results from this study support the theory that p53 protein binds HPV-16/18 E6 protein in the cell cytoplasm, thus preventing p53 from exerting its tumor-suppressor function in the nucleus. Hence, inactivation of wild-type p53 by p53-E6 complex formation in cervical cancer may be a critical step in malignant transformation.

Carcinoma↗
PubMed1993

Mitochondrial and glycolytic dysfunction in lethal injury to hepatocytes by t-butylhydroperoxide: protection by fructose, cyclosporin A and trifluoperazine.

In isolated mitochondria, t-butylhydroperoxide (t-BuOOH) and other pro-oxidants cause a permeability transition characterized by increased permeability to small ions, swelling and loss of membrane potential. Cyclosporin A and trifluoperazine inhibit this permeability transition. Here, we investigated the role of the mitochondrial permeability transition in lethal cellular injury from t-BuOOH. Hepatocytes from fasted rats were isolated by collagenase perfusion, and cell viability was assessed by propidium iodide fluorescence. t-BuOOH caused dose- and time-dependent cell killing. Fructose, a substrate for glycolytic ATP formation, protected at lower (< or = 100 microM), but not at higher concentrations of t-BuOOH. In fructose-treated cells, oligomycin (10 micrograms/ml) delayed cell killing after 100 to 300 microM t-BuOOH, whereas cyclosporin A (0.5 microM) plus trifluoperazine (5 microM) even more potently reduced lethal injury. In hepatocyte suspensions, 100 microM t-BuOOH caused mitochondrial depolarization as determined by release of rhodamine 123. Cyclosporin A plus trifluoperazine in the presence of fructose substantially reduced release of rhodamine 123. Similarly, in single cultured hepatocytes viewed by laser scanning confocal microscopy, t-BuOOH caused leakage of rhodamine 123 from mitochondria, an event which preceded cell death and which was delayed by fructose in combination with cyclosporin A plus trifluoperazine. At 1 mM, t-BuOOH inhibited glycolysis, and fructose in combination with either oligomycin or cyclosporin A plus trifluoperazine had only a short-lived protective effect. In conclusion, t-BuOOH toxicity was progressive with increasing dosages. At low t-BuOOH (< or = 50 microM), mitochondrial ATP synthetic capacity was inhibited, but not uncoupled.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Platelet-derived growth factor (PDGF) alpha receptor activation modulates the calcium mobilizing activity of the PDGF beta receptor in Balb/c3T3 fibroblasts.

In order to determine whether distinct platelet-derived growth factor (PDGF) receptors (alpha and beta) can modulate the activity of one another, PDGF isoform (AA, BB, and AB)-stimulated changes in Ca2+i were monitored by digitized video microscopy in single cells upon sequential addition of PDGF isoforms. In Balb/c 3T3 fibroblasts, all PDGF isoforms were capable of stimulating increases in Ca2+i of 200-600% above basal levels, although with different potencies: BB greater than or equal to AB greater than AA. All cells were BB-PDGF-responsive, but only 74% of cells examined responded to AA-PDGF. The Ca2+i response elicited by BB-PDGF was inhibited by 60-75% in cells stimulated 10 min earlier with the AA isoform. The half-life of this inhibition was 22 min. In cells in which the alpha receptor was down-regulated by prolonged incubation with AA-PDGF, BB-induced Ca2+i responses were not inhibited. Pretreatment of cells with phorbol ester did not inhibit BB-PDGF-induced increases in Ca2+i, yet down-regulation of PKC activity prevented the AA-PDGF inhibition of BB-PDGF-induced Ca2+i responses. An increase in Ca2+i induced by AlF(4-)-stimulated IP3 generation did not inhibit a subsequent BB-PDGF Ca2+i response; however, attenuation of AA-PDGF-induced extracellular Ca2+ influx with EGTA prevented the inhibition of BB-PDGF-induced Ca2+i increases. Readdition of Ca2+ to the medium after removal of EGTA restored the inhibition of the BB-PDGF Ca2+i response. The inhibition of the BB-PDGF Ca2+i response by AA-PDGF was not caused by inhibition of PDGF receptor tyrosine autophosphorylation, which was unchanged after pretreatment with AA-PDGF. These results demonstrate: (a) that only a subpopulation of cells possess a functional alpha receptor-mediated response as assessed by AA-PDGF-induced increases in Ca2+i, whereas all cells possess the beta receptor-mediated responses; and (b) AA-PDGF and its associated alpha receptor can modulate the activity of the beta receptor through a mechanism that is dependent upon Ca(2+)-influx which may be controlled in part by PKC activation.

3T3 Cells↗
PubMed1992

A novel cytotoxicity screening assay using a multiwell fluorescence scanner.

A new assay using a multiwell fluorescence scanner was developed for screening cytotoxicity to cells cultured in 96-well microtiter plates. The assay is based on binding of propidium iodide to nuclei of cells whose plasma membranes have become permeable due to cell death. Fluorescence of propidium iodide measured with a multiwell fluorescence scanner increased in proportion to the number of permeabilized cells. After ATP depletion of hepatocytes and neonatal cardiac myocytes with metabolic inhibitors ("chemical hypoxia"), and exposure of Madine Darby canine kidney cells to the toxic chemical, HgCl2, propidium iodide fluorescence progressively increased. Increases of fluorescence were linearly proportional with release of lactate dehydrogenase into the culture medium. Employing this cytotoxicity screening assay, protection by various agents against lethal injury was evaluated in cultured hepatocytes during chemical hypoxia. Inhibitors of cysteine proteases (i.e., antipain, leupeptin, E-64), serine proteases (i.e., PMSF), and aspartic acid proteases (i.e., pepstatin A) did not protect against chemical hypoxia. In contrast, 1,10-phenanthroline, an inhibitor of metalloprotease, markedly protected against the onset of cell death during chemical hypoxia. Half-maximal protection after 60 min occurred at 0.5 microM. Phospholipase inhibitors, chlorpromazine (50 microM) and mepacrine (50 microM), also substantially retarded cell killing. U74006F, an inhibitor of lipid peroxidation, slowed cell killing to a lesser extent during chemical hypoxia and after oxidative stress with t-butyl hydroperoxide. Calciphor, a dimer of prostaglandin B1, did not protect against cell killing during chemical hypoxia or t-butyl hydroperoxide toxicity. In conclusion, this high capacity cytotoxicity assay for cells cultured in 96-well microtiter plates is suitable for rapid screening of potential cytoprotective agents in a variety of cell types.

Animals↗