Daniel Acosta | Universidad de San Buenaventura Cali (original) (raw)
Papers by Daniel Acosta
Toxicology Letters, 1994
The use of in vitro cytotoxicity assays as potential alternatives in assessing ocular irritation ... more The use of in vitro cytotoxicity assays as potential alternatives in assessing ocular irritation of surfactant mixtures was evaluated in a primary culture system of rabbit corneal epithelial cells. Two groups of surfactant mixtures, each with the same surfactant components in varying proportions, were studied. Cytotoxicity was determined by lactate dehydrogenase (LDH) enzyme leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction in the cell culture system. There was a good correlation between the cytotoxicity in vitro and the reported Draize eye irritation data within each group of the surfactant mixtures studied.
Annual Review of Pharmacology and Toxicology, 1998
Journal of Tissue Culture Methods, 1980
The measurement of cell injury or cytotoxicity produced by chemicals or toxicants in cell culture... more The measurement of cell injury or cytotoxicity produced by chemicals or toxicants in cell culture has routinely been evaluated by such criteria as plating efficiency, cell growth, protein or DNA synthesis, and cell viability. We describe a more sensitive and earlier index of cytotoxicity: leakage of cytoplasmic enzymes from injured cells into the culture medium.
Journal of Toxicology and Environmental Health-part A-current Issues, 1981
Primary cultures of hepatocytes from postnatal Sprague‐Dawley rats were grown in arginine‐deficie... more Primary cultures of hepatocytes from postnatal Sprague‐Dawley rats were grown in arginine‐deficient, ornithine‐supplemented medium to inhibit fibroblastic overgrowth and to selectively isolate relatively pure cultures of parenchymal hepatocytes. This system of primary cultures of rat hepatocytes was utilized to evaluate the cytotoxicity of certain tricyclic antidepressant drugs (TCAs). The compounds tested were chosen to represent two distinct chemical classifications of TCAs: the dibenzazepine derivatives, imipramine (I) and desipramine (D), and the dibenzocycloheptadiene derivatives, amitriptyline (A) and nortriptyline (N). The study also allowed direct comparison of the parent tertiary amines, A and I, and their respective demethylated pharmacologically active metabolites, N and D. The hepatotoxicity of the compounds was determined by measuring leakage of cytoplasmic enzymes, lactate dehydrogenase (LDH) and glutamic‐pyruvic transaminase (GPT), into the culture medium and by assessing cell viability by the trypan blue dye exclusion test LDH leakage was a more sensitive index of early cellular injury in this study. The compounds demonstrated a dose‐ and time‐dependent order of toxicity; their hepatotoxic potency was ranked as A=N > D > I.
Journal of Toxicology and Environmental Health-part A-current Issues, 1982
Primary cultures of rat hepatocytes served as an experimental model to evaluate the cytotoxicity ... more Primary cultures of rat hepatocytes served as an experimental model to evaluate the cytotoxicity of cadmium chloride. Cellular injury was assessed by a series of enzymatic and functional indices in 24-h-old cultures exposed for 1 h to concentrations of cadmium chloride ranging from 50 to 400 muM. In cultures that were evaluated immediately after the 1-h exposure to cadmium, little evidence of toxicity was observed as evaluated by total cellular protein content and cell viability. In similarly treated cultures, leakage of lactate dehydrogenase from the hepatocytes into the culture medium was increased in a dose-dependent manner. The most sensitive indicators of cadmium toxicity proved to be two parameters of metabolic activity: lactate-to-pyruvate (L/P) ratios, and intracellular levels of urea. Cadmium exposure produced substantial increases in L/P ratios and decreases in urea content in the cultured liver cells. If the cultures were allowed to recover by replacing the cadmium-containing medium after 1 h of exposure with fresh medium for 24 h, cellular protein content and cell viability were shown to decrease by 40%. These findings indicate that measures of metabolic integrity of cultured hepatocytes are more sensitive indices of early cadmium cytotoxicity than are routine, nonspecific measures such as cellular protein content and dye-exclusion viability tests, which detect the later stages of cell injury, i.e., cell death.
Biochemical Pharmacology, 1981
in Vitro Cellular & Developmental Biology-plant, 1978
A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed... more A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and norethindrone. Caffeine was evaluated as an agent thought to be relatively nontoxic to liver. Cytotoxicity was evaluated by phase-contrast microscopy of cellular morphology and by measurement of leakage of intracellular enzymes [arginosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP)] into the culture medium. Hepatic cultures were treated with each of the agents in concentrations ranging from 5×10−6 to 1×10−3m and for durations from 1 to 24 hr. ASAL was found to be the most sensitive in predicting early cell injury and AP the least sensitive; the other three enzymes tested were intermittent in value and equally sensitive in evaluating cytotoxicity. Treatment of the cultures with tetracycline (5×10−4m) for 6 hr resulted in ASAL leakage that was 400% of control values; and norethindrone (5×10−4m) for 6 hr caused a 250% increase relative to controls. The hepatotoxic agents demonstrated a dose- and timedependence of cytotoxicity in the cultures. In contrast, caffeine was relatively nontoxic to the cultures.
in Vitro Cellular & Developmental Biology-plant, 1984
Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium... more Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium, and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes subjected to morphometric analysis after scanning electron microscopy.
Biochemical Pharmacology, 1984
Primary cultures of rat myocardial cells were used to evaluate the cellular dynamics of calcium a... more Primary cultures of rat myocardial cells were used to evaluate the cellular dynamics of calcium accumulation after exposure to isoproterenol (ISO). Non-toxic concentrations of ISO (2.4 X 10(-7) M) caused a gradual increase in myocyte calcium uptake. These effects peaked 3 min after exposure and returned to control levels within 2 min. Toxic concentrations of ISO caused a biphasic increase in calcium uptake. The initial phase peaked 1 min after exposure and returned to control levels by 3 min. A second phase was characterized by a progressive increase in calcium uptake that plateaued 10 min after exposure. Ascorbic acid (AA, 5 X 10(-3) M) and sodium bisulfite (SB, 9.6 X 10(-4) M) did not modify the calcium uptake of the initial phase, whereas propranolol (1 X 10(-6) M) and verapamil (1 X 10(-5) M) prevented the initial rise in calcium uptake. In contrast, the antioxidants prevented the the second phase of ISO-induced calcium uptake, whereas verapamil and propranolol did not. The toxic accumulation of calcium induced by ISO may be due to oxidative damage of the sarcolemma. Antioxidants may prevent the formation of oxidative metabolites from ISO and the subsequent calcium overload. Our results show that agents which modify slow calcium-channel transport do not prevent ISO-induced calcium overload in our cell culture system.
in Vitro Cellular & Developmental Biology-plant, 1985
A major goal of our laboratory has been the development of primary culture systems that retain di... more A major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.
in Vitro Cellular & Developmental Biology-plant, 1978
Monolayers of liver cells cultured from postnatal rats were grown in two types of media. One set ... more Monolayers of liver cells cultured from postnatal rats were grown in two types of media. One set of cultures was grown in selective medium which contained ornithine but was deficient in arginine: the other set was grown in nonselective medium which contained arginine but no ornithine. The cultures that were grown in the nonselective medium contained primarily a mixture of two cell types found in the liver: parenchymal hepatocytes and fibroblast-like cells. The fibroblast cells tended to overgrow the hepatocytes after several days in culture. In contrast, fibroblast overgrowth was inhibited in cultures grown in the selective, arginine-deficient medium, thereby resulting in relatively pure cultures of functional parenchymal hepatocytes. Comparative studies of sulfobromophthalein (BSP) uptake showed that the cultures grown in selective medium continued to be active much longer than the cultures grown in the nonselective medium. Pyruvate kinase assays revealed that the cultures grown in selective medium contained primarily the L-isoenzyme type which is characteristic of parenchymal hepatocytes. Cultures grown in nonselective medium contained a mixture of L- and M-isoenzymes which is indicative of nonparenchymal liver cells. The reported results indicate that selective, arginine-deficient medium permits primarily the growth of parenchymal hepatocytes found in neonatal rat liver.
Journal of Tissue Culture Methods, 1980
We describe a perfusion with collagenase followed by dissociation with collagenase to isolate hep... more We describe a perfusion with collagenase followed by dissociation with collagenase to isolate hepatocytes from 7 to 10-d postnatal rat liver. Subsequent culture in an arginine-deficient medium selects for hepatocytes by preventing overgrowth by other cell types that are unable to synthesize arginine.
Toxicology Letters, 1994
The use of in vitro cytotoxicity assays as potential alternatives in assessing ocular irritation ... more The use of in vitro cytotoxicity assays as potential alternatives in assessing ocular irritation of surfactant mixtures was evaluated in a primary culture system of rabbit corneal epithelial cells. Two groups of surfactant mixtures, each with the same surfactant components in varying proportions, were studied. Cytotoxicity was determined by lactate dehydrogenase (LDH) enzyme leakage and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye reduction in the cell culture system. There was a good correlation between the cytotoxicity in vitro and the reported Draize eye irritation data within each group of the surfactant mixtures studied.
Annual Review of Pharmacology and Toxicology, 1998
Journal of Tissue Culture Methods, 1980
The measurement of cell injury or cytotoxicity produced by chemicals or toxicants in cell culture... more The measurement of cell injury or cytotoxicity produced by chemicals or toxicants in cell culture has routinely been evaluated by such criteria as plating efficiency, cell growth, protein or DNA synthesis, and cell viability. We describe a more sensitive and earlier index of cytotoxicity: leakage of cytoplasmic enzymes from injured cells into the culture medium.
Journal of Toxicology and Environmental Health-part A-current Issues, 1981
Primary cultures of hepatocytes from postnatal Sprague‐Dawley rats were grown in arginine‐deficie... more Primary cultures of hepatocytes from postnatal Sprague‐Dawley rats were grown in arginine‐deficient, ornithine‐supplemented medium to inhibit fibroblastic overgrowth and to selectively isolate relatively pure cultures of parenchymal hepatocytes. This system of primary cultures of rat hepatocytes was utilized to evaluate the cytotoxicity of certain tricyclic antidepressant drugs (TCAs). The compounds tested were chosen to represent two distinct chemical classifications of TCAs: the dibenzazepine derivatives, imipramine (I) and desipramine (D), and the dibenzocycloheptadiene derivatives, amitriptyline (A) and nortriptyline (N). The study also allowed direct comparison of the parent tertiary amines, A and I, and their respective demethylated pharmacologically active metabolites, N and D. The hepatotoxicity of the compounds was determined by measuring leakage of cytoplasmic enzymes, lactate dehydrogenase (LDH) and glutamic‐pyruvic transaminase (GPT), into the culture medium and by assessing cell viability by the trypan blue dye exclusion test LDH leakage was a more sensitive index of early cellular injury in this study. The compounds demonstrated a dose‐ and time‐dependent order of toxicity; their hepatotoxic potency was ranked as A=N > D > I.
Journal of Toxicology and Environmental Health-part A-current Issues, 1982
Primary cultures of rat hepatocytes served as an experimental model to evaluate the cytotoxicity ... more Primary cultures of rat hepatocytes served as an experimental model to evaluate the cytotoxicity of cadmium chloride. Cellular injury was assessed by a series of enzymatic and functional indices in 24-h-old cultures exposed for 1 h to concentrations of cadmium chloride ranging from 50 to 400 muM. In cultures that were evaluated immediately after the 1-h exposure to cadmium, little evidence of toxicity was observed as evaluated by total cellular protein content and cell viability. In similarly treated cultures, leakage of lactate dehydrogenase from the hepatocytes into the culture medium was increased in a dose-dependent manner. The most sensitive indicators of cadmium toxicity proved to be two parameters of metabolic activity: lactate-to-pyruvate (L/P) ratios, and intracellular levels of urea. Cadmium exposure produced substantial increases in L/P ratios and decreases in urea content in the cultured liver cells. If the cultures were allowed to recover by replacing the cadmium-containing medium after 1 h of exposure with fresh medium for 24 h, cellular protein content and cell viability were shown to decrease by 40%. These findings indicate that measures of metabolic integrity of cultured hepatocytes are more sensitive indices of early cadmium cytotoxicity than are routine, nonspecific measures such as cellular protein content and dye-exclusion viability tests, which detect the later stages of cell injury, i.e., cell death.
Biochemical Pharmacology, 1981
in Vitro Cellular & Developmental Biology-plant, 1978
A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed... more A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and norethindrone. Caffeine was evaluated as an agent thought to be relatively nontoxic to liver. Cytotoxicity was evaluated by phase-contrast microscopy of cellular morphology and by measurement of leakage of intracellular enzymes [arginosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP)] into the culture medium. Hepatic cultures were treated with each of the agents in concentrations ranging from 5×10−6 to 1×10−3m and for durations from 1 to 24 hr. ASAL was found to be the most sensitive in predicting early cell injury and AP the least sensitive; the other three enzymes tested were intermittent in value and equally sensitive in evaluating cytotoxicity. Treatment of the cultures with tetracycline (5×10−4m) for 6 hr resulted in ASAL leakage that was 400% of control values; and norethindrone (5×10−4m) for 6 hr caused a 250% increase relative to controls. The hepatotoxic agents demonstrated a dose- and timedependence of cytotoxicity in the cultures. In contrast, caffeine was relatively nontoxic to the cultures.
in Vitro Cellular & Developmental Biology-plant, 1984
Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium... more Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium, and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes subjected to morphometric analysis after scanning electron microscopy.
Biochemical Pharmacology, 1984
Primary cultures of rat myocardial cells were used to evaluate the cellular dynamics of calcium a... more Primary cultures of rat myocardial cells were used to evaluate the cellular dynamics of calcium accumulation after exposure to isoproterenol (ISO). Non-toxic concentrations of ISO (2.4 X 10(-7) M) caused a gradual increase in myocyte calcium uptake. These effects peaked 3 min after exposure and returned to control levels within 2 min. Toxic concentrations of ISO caused a biphasic increase in calcium uptake. The initial phase peaked 1 min after exposure and returned to control levels by 3 min. A second phase was characterized by a progressive increase in calcium uptake that plateaued 10 min after exposure. Ascorbic acid (AA, 5 X 10(-3) M) and sodium bisulfite (SB, 9.6 X 10(-4) M) did not modify the calcium uptake of the initial phase, whereas propranolol (1 X 10(-6) M) and verapamil (1 X 10(-5) M) prevented the initial rise in calcium uptake. In contrast, the antioxidants prevented the the second phase of ISO-induced calcium uptake, whereas verapamil and propranolol did not. The toxic accumulation of calcium induced by ISO may be due to oxidative damage of the sarcolemma. Antioxidants may prevent the formation of oxidative metabolites from ISO and the subsequent calcium overload. Our results show that agents which modify slow calcium-channel transport do not prevent ISO-induced calcium overload in our cell culture system.
in Vitro Cellular & Developmental Biology-plant, 1985
A major goal of our laboratory has been the development of primary culture systems that retain di... more A major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.
in Vitro Cellular & Developmental Biology-plant, 1978
Monolayers of liver cells cultured from postnatal rats were grown in two types of media. One set ... more Monolayers of liver cells cultured from postnatal rats were grown in two types of media. One set of cultures was grown in selective medium which contained ornithine but was deficient in arginine: the other set was grown in nonselective medium which contained arginine but no ornithine. The cultures that were grown in the nonselective medium contained primarily a mixture of two cell types found in the liver: parenchymal hepatocytes and fibroblast-like cells. The fibroblast cells tended to overgrow the hepatocytes after several days in culture. In contrast, fibroblast overgrowth was inhibited in cultures grown in the selective, arginine-deficient medium, thereby resulting in relatively pure cultures of functional parenchymal hepatocytes. Comparative studies of sulfobromophthalein (BSP) uptake showed that the cultures grown in selective medium continued to be active much longer than the cultures grown in the nonselective medium. Pyruvate kinase assays revealed that the cultures grown in selective medium contained primarily the L-isoenzyme type which is characteristic of parenchymal hepatocytes. Cultures grown in nonselective medium contained a mixture of L- and M-isoenzymes which is indicative of nonparenchymal liver cells. The reported results indicate that selective, arginine-deficient medium permits primarily the growth of parenchymal hepatocytes found in neonatal rat liver.
Journal of Tissue Culture Methods, 1980
We describe a perfusion with collagenase followed by dissociation with collagenase to isolate hep... more We describe a perfusion with collagenase followed by dissociation with collagenase to isolate hepatocytes from 7 to 10-d postnatal rat liver. Subsequent culture in an arginine-deficient medium selects for hepatocytes by preventing overgrowth by other cell types that are unable to synthesize arginine.