Intracellular Lactate Dehydrogenase Activity Research Articles

Metabolism and toxicity of coumarin on cultured human, rat, mouse and rabbit hepatocytes.

We compared the cytotoxic effect of coumarin and its derivatives, 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC), o-hydroxyphenyl acetic acid (OHPAA) and o-coumaric acid (CA), on cultured hepatocytes from human, rat, mouse and rabbit liver. At 10(-5) and 5 x 10(-5) M, coumarin and its derivatives did not give rise to any signs of toxicity on cultured hepatocytes of the four species. At 10(-4) M, coumarin, but not its derivatives, induced release of lactate dehydrogenase (LDH) into the medium, especially in rat hepatocyte cultures. Intracellular LDH activities were correspondingly reduced. The cytotoxic effect of coumarin in cultured rat hepatocytes was evidenced on morphological examination and from the results of the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) reduction test. At higher concentrations (5 x 10(-4) M), 7-OHC and CA were also found to be cytotoxic in cultured rat hepatocytes. The cytotoxic effect of coumarin (5 x 10(-4) M) was decreased in the presence of SKF 525-A, a cytochrome P450 inhibitor. Interspecies comparisons showed that rat hepatocytes were the most sensitive to the toxicity of coumarin and its derivatives, whereas human hepatocytes were the most resistant. Our results suggest that the cytotoxicity of coumarin is metabolism and species-dependent. Thus, the rat may not be a suitable model for evaluating the pharmacological hazards of coumarin in humans.

Reactive Oxygen Species and DNA Damage in 2-Bromo-(glutathione-S-yl) hydroquinone-Mediated Cytotoxicity

Exposure of renal proximal tubular epithelial cells (LLC-PK1) to the nephrotoxicants 2-bromo-6-(glutathion-S-yl)hydroquinone, 2-bromo-3-(glutathion-S-yl)-hydroquinone, and 2-bromo-(diglutathion-S-yl)hydroquinone caused DNA fragmentation and cytotoxicity. Viability measured by lysosomal neutral red accumicrolation was the most sensitive parameter of cytotoxicity, and preceded toxicity determined by either the mitochondrial MTT assay or by measuring intracellular lactate dehydrogenase activity. DNA fragmentation was detected as early as 15 min after exposure to 2-bromo-6-(glutathion-S-yl)hydroquinone (100 μM), 2-bromo-3-(glutathion-S-yl)hydroquinone (200 μM), and 2-bromo-(diglutathion-S-yl) hydroquinone (400 μM) and prior to other indices of toxicity. The ability of the cells to repair DNA damage was evident by the decrease in the extent of single strand breaks following removal of 2-bromo-3-(glutathion-S-yl)hydroquinone from the incubation medium. Moreover, inhibition of poly(ADP-ribose)polymerase with 3-amino-benzamide (10 nM), following exposure of LLC-PK1 cells to 0.5 mM 2-bromo-6-(glutathion-S-yl)hydroquinone or 2-bromo-(diglutathion-S-yl)hydroquinone, decreased cytotoxicity, indicating that DNA repair processes, activated in response to DNA damage, exacerbate toxicity. Treatment with the endonuclease inhibitor, aurintricarboxylic acid did not decrease cytotoxicity. A decrease in the cytotoxicity caused by 2-bromo-6-(glutathion-S-yl)hydroquinone and 2-bromo(diglutathion-S-yl)hydroquinone was observed when cells were incubated with catalase or pretreated with deferoxamine (10 mM). The data suggest a mechanism whereby the conjugates generate hydrogen peroxide, and the subsequent iron-catalyzed generation of hydroxyl radicals causes DNA fragmentation and cytotoxicity.

Growth kinetics of animal cells immobilized within porous support particles in a perfusion culture

The growth kinetics of anchorage-independent animal cells [mouse myeloma MPC-11 (ATCC CCL 167)] immobilized within porous polyvinyl formal resin biomass support particles (BSPs; 3 x 3 x 3 mm cubes; mean pore diameter, 60 microns) was analyzed by measuring intracellular and extracellular lactate dehydrogenase (LDH) activities in a perfusion culture. First, the intracellular LDH content of cells immobilized within the BSPs was assayed in a shake-flask culture and found to remain almost comparable to that of non-immobilized cells in the exponential growth phase under static culture. Then, cells inoculated in the BSPs were grown in a continuous stirred-tank bioreactor. Using the LDH content of non-immobilized cells, the density of immobilized cells and the numbers of leaked and dead cells were evaluated, respectively, by the intracellular LDH activity of immobilized and leaked cells and the LDH activity in cell-free culture supernatant. In the initial period, immobilized cells exhibited exponential growth at a constant apparent specific growth rate; however, the actual specific growth rate, which takes into consideration cell death and cell leakage, decreased significantly. In the stationary phase, the actual specific growth rate maintained a constant but markedly lower value than during exponential growth.

Growth characteristics in fed‐batch culture of hybridoma cells with control of glucose and glutamine concentrations

An online system using HPLC was developed for the measurement of glucose, glutamine, and lactate in a culture broth. Using the system, the glucose and glutamine concentrations were controlled simultaneously by an adaptive-control algorithm within the ranges of 0.2 to 2.0 and 0.1 to 0.6 g/L, respectively. When the glucose concentration was controlled at the low level of 0.2 g/L, the intracellular lactate dehydrogenase activity decreased by one-half and the lactate concentration by one-third, whereas the uptake rates of serine and glycine were about twice as high, compared with the amounts when the glucose concentration was controlled at 1.0 g/L. On the other hand, ammonia production increased when the glucose concentration was kept low. To reduce the production of inhibitory metabolites such as ammonia and lactate and improve the antibody production rate in a hybridoma cell culture, the concentrations of glucose and glutamine were controlled at 0.2 and 0.1 g/L, respectively. With these low concentrations of glucose and glutamine, the cell concentration (4.1 x 10(6) cells/mL) and antibody production (172 mg/L) both increased about twofold compared with the amounts when the glucose was controlled at higher levels. From these results, simultaneous control of the glucose and glutamine concentrations was shown to be useful in the production of antibody by hybridoma cell cultivation.

Effect of periportal- and centrilobular-equivalent oxygen tension on liver specific functions in long-term rat hepatocyte cultures

The influence of periportal- and centrilobular-equivalent oxygen tensions on cellular functions and xenobiotic metabolism was investigated in rat hepatocytes cultured under 20% (air/CO 2), 13% or 4% O 2 for up to 9 days on teflon membrane dishes, coated with crude membrane fractions and collagen. Protein content, total cytochrome P-450 content, 7-ethoxy-resorufin- O-deethylase (EROD) and the profile of hydroxytestosterone metabolites were not significantly affected by the oxygen tension, whereas intracellular lactate dehydrogenase activity and albumin secretion were increased at 4% O 2 in comparison with 13 and 20% O 2 cultures. The induction of P-450 isoenzymes and corresponding catalytic activity was determined on days 4 and 9, following a 3-day exposure to phenobarbital (PB, 0.75 m m) or 3-methylcholanthrene (3-MC, 6.25 μ m). On day 4, PB increased CYP2B1/B2 content 19-fold (4% O 2) or 27-fold (13% O 2), CYP2C6 six-fold at both oxygen tensions, and CYP3A2 two-three-fold only in 4% O 2 cultures. On day 9, CYP2B1/B2 was still significantly increased. A clear correlation was found between P-450 isoenzyme content and corresponding isoenzyme activities, as assayed by testosterone hydroxylation and EROD activity on day 4. 3-MC increased CYP1A1/2 11-fold and eight-fold in 4 and 13% O 2 cultures, respectively, and the EROD activity 37-fold (4% O 2) and 30-fold (13% O 2) on day 4. Activities were still measurable on day 9, whereas the corresponding apoproteins CYP1A1 was not detectable. These results suggest that hepatocytes are able to adapt their xenobiotic metabolism to different tissue oxygen tensions and confirm the likelihood that oxygen tension is an important modulator of the region-specific expression of xenobiotic metabolism in liver.

Cocaine hepatotoxicity: Two different toxicity mechanisms for phenobarbital-induced and non-induced rat hepatocytes

Hepatocytes isolated from both phenobarbital-induced and control rats were short-term cultured and exposed to cocaine (8–2000 μM) for varying times. Intracellular lactate dehydrogenase activity, free calcium levels ([Ca 2+] i), reduced glutathione (GSH) and lipid peroxidation were investigated to evaluate the toxic effect of cocaine on hepatocytes. Cytochrome P450 induction by phenobarbital potentiated the in vitro cytotoxicity of cocaine by a factor of 13 (IC 50 = 84 μM induced cells vs 1100 μM in non-induced cells). This difference in the susceptibility of the two types of hepatocytes to cocaine correlated well with the activity of cytochrome P450 2 B 1 2 . Rapid depletion of GSH, reaching 30% of the control levels, and massive lipid peroxidation thereafter were the two most remarkable phenomena preceding cell death in phenobarbital-induced hepatocytes. On the other hand, a sustained rise in [Ca 2+] i starting 2 hr after incubation with cocaine was the most noteworthy finding in non-induced liver cells. We suggest two different pathways for cocaine hepatotoxicity: in phenobarbital-induced hepatocytes oxidative metabolism of the drug causes GSH depletion, subsequent extensive lipid peroxidation and cell death, at low concentrations of cocaine. In non-induced hepatocytes these changes are less relevant, and the major alteration caused by cocaine is a non-transient rise in [Ca 2+] 1 that is evident at higher concentrations of the drug.

Neuron and glial cell marker proteins as indicators of heavy metal-induced neurotoxicity in neuroblastoma and glioma cell lines.

The sensitive and specific biochemical indicators for assessing chemical-induced neurotoxic insults in cell culture models have not been sufficiently explored. This study was designed to assess the usefulness of glia-specific beta-S100 protein and neuron-specific enolase (NSE) as indices of in vitro neurotoxicity of heavy metals. Glioma C6 and neuroblastoma N18TG-2 cells were grown in Dulbecco's modified Eagle's medium containing various concentrations of mercuric chloride (HgCl2) or cadmium chloride (CdCl2) for 5 days. Toxic response patterns of the neurospecific endpoints (beta-S100 and NSE), which were monitored with enzyme immunoassays, were compared with those of the non-neurospecific endpoints such as cell viability, total cellular protein, lactate dehydrogenase (LDH) activity, and cumulative glucose consumption in the two cell lines. Both HgCl2 and CdCl2 produced dose-dependent inhibition of neurospecific endpoints and non-specific endpoints. However, by ranking the EC50 values (effective concentration producing half-maximal inhibition) for various endpoints, the lowest values were found for beta-S100 in C6 cells, and for NSE in N18TG-2 cells. In lower and intermediate concentrations, the inhibitory effects of the heavy metals on the content of beta-S100 and NSE occurred in the absence of any detectable effect on intracellular LDH activity, and independently of total cellular protein inhibition. The sensitive and excess responses of the neurospecific endpoints relative to that of the non-specific endpoints may reflect the specific neurotoxic insults of the heavy metals on the cultured cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration- and time-related effects of sulphur mustard on human epidermal keratinocyte function

The concentration- and time-dependent effects of sulphur mustard on cultured human epidermal keratinocyte function were investigated with respect to cell proliferation, DNA and protein synthesis, the level of NAD + and ATP, and the intracellular activity of lactate dehydrogenase. If human epidermal keratinocytes were stimulated to proliferate by replating following exposure to sulphur mustard, inhibition of cell growth was observed after treatment of cells with 0.5 μ m-sulphur mustard or more. Similarly, DNA synthesis was reduced in a concentration-dependent manner following exposures to 0.5 μ m-sulphur mustard or higher. Inhibition of protein synthesis was observed only on exposure to concentrations higher than 10 μ m. These effects could be detected immediately after exposure and were constant for at least 24 hr. In contrast, parameters for cellular energy decreased with time following exposure. At 24 hr post-exposure a concentration-dependent depletion of NAD + and ATP was observed, starting at exposure levels of 50 μ m-sulphur mustard. Decrease of intracellular lactate dehydrogenase activity also occurred with exposures to 50 μ m-sulphur mustard or more, although it was not possible to cause a reduction of more than 50% of control values. Furthermore, the results showed that sulphur mustard induces either persistent damage or damage that develops within 24 hr following treatment. The concurrent inhibition of DNA and protein synthesis combined with a decreased energy supply may have an important role in sulphur mustard-induced blister formation in skin.

Cyclodextrins as nasal absorption promoters of insulin: mechanistic evaluations.

The safety and effectiveness of cyclodextrins (CD) as nasal absorption promoters of peptide-like macromolecules have been investigated. The relative effectiveness of the cyclodextrins in enhancing insulin nasal absorption was found to be in the descending order of dimethyl-beta-cyclodextrin (DM beta CD) greater than alpha-cyclodextrin (alpha-CD) greater than beta-cyclodextrin (beta-CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) greater than gamma-cyclodextrin (gamma-CD). A direct relationship linking absorption promotion to nasal membrane protein release is evident, which in turn correlates well with nasal membrane phospholipid release. The magnitude of the membrane damaging effects determined by the membrane protein or phospholipid release may provide an accurate, simple, and useful marker for predicting safety of the absorption enhancers. In order to estimate further the magnitude of damage and specificity of cyclodextrin derivatives in solubilizing nasal membrane components, the enzymatic activities of membrane-bound 5'-nucleotidase (5'-ND) and intracellular lactate dehydrogenase (LDH) in the perfusates were also measured. HP beta CD at a 5% concentration was found to result in only minimal removal of epithelial membrane proteins as evidenced by a slight increase in 5'-ND and total absence of LDH activity. On the other hand, 5% DM beta CD caused extensive removal of the membrane-bound 5'-ND. Moreover, intracellular LDH activity in the perfusate increased almost linearly with time. The cyclodextrins are also capable of dissociating insulin hexamers into smaller aggregates, and this dissociation depends on cyclodextrin structure and concentration. Enhancement of insulin diffusivity across nasal membrane through dissociation may provide an additional mechanism for cyclodextrin promotion of nasal insulin absorption.

Measurement of intracellular LDH activity in 96-well cultures: A rapid and automated assay for cytotoxicity studies

The present paper describes a rapid, easy, sensitive, and automated spectrophotometric assay for intracellular lactate dehydrogenase (LDH) measurement in 96-well plates of adherent cells for cytotoxicity studies. The procedure involves “in situ” homogenization of cells, followed by measurement of LDH activity with a colorimetric method based on the reduction of a tetrazolium salt to a violet formazan by the NADH formed by LDH. Color intensity can be measured in conventional ELISA readers, and the data can be fed to an “on line” computer for rapid processing. The color absorbance measured is time- and enzyme-concentration dependent. LDH activity measured with this micromethod is coincident with that measured in larger culture plates after individual homogenization and conventional LDH measurement. The advantages of this method are the smaller number of cells required, easy automation, drastic reduction of time for processing individual wells, and the possibility of examining multiple variables in the same experiment.

The use of pig hepatocytes for cytotoxicity studies of veterinary drugs: A comparative study with furazolidone and other nitrofurans

The possible use of primary cultures of pig hepatocytes for cytotoxicity studies of veterinary drugs was investigated using some nitrofurans. The substances tested were furazolidone, furaltadone, nitrofurazone, nitrofurantoin (at concentrations of 15–500 μ m) and nitrovin (5–50 μ m); acetaminophen (1–10 m m) was used as a positive control. The leakage of lactate dehydrogenase (LDH) from the cells into the medium could be measured at high concentrations of nitrofurantoin, nitrovin, nitrofurazone and acetaminophen. After incubation of the cells with high concentrations of any of the drugs, intracellular LDH activities were lower than in controls. The incorporation of [ 14C]leucine into proteins, especially those excreted into the medium, was shown to be a more sensitive parameter of nitrofuran toxicity than LDH leakage, and was decreased by all of the drugs at most concentrations. Intracellular levels of oxidized glutathione were increased upon exposure to any of the nitrofurans. However, only incubation with 500 μ m-nitrofurantoin decreased intracellular levels of reduced glutathione (GSH). In all other cases GSH levels were unchanged or even elevated upon exposure. The most sensitive parameter measured was the accumulation of pyruvate and lactate in the medium after treatment with any of the nitrofurans except nitrovin. Experiments with furazolidone showed that this effect did not disappear immediately after the end of exposure and also that the effect accumulated upon repeated treatment of the cells, especially at low doses. The results of the study clearly demonstrate that primary cultures of pig hepatocytes can be used for cytotoxicity studies of veterinary drugs.

The effects of buprenorphine on the metabolism of human hepatocytes

Adult human hepatocytes cultured in chemically defined conditions were used as a biological model to examine the metabolic effects of buprenorphine on the human liver. Cell extension and monolayer formation of human hepatocytes were affected in a dose-dependent manner after 24 hr of exposure to the drug. According to the several endpoints evaluated (cellular protein, intracellular lactate dehydrogenase activity and the MTT test), the half-maximal cytotoxic effect (IC 50) of buprenorphine was close to 100 μ m. Longer exposure of hepatocytes to buprenorphine (72 hr) increased its cytotoxicity, and the IC 50 of the drug was reduced to 50 μ m. Lower concentrations of the drug (in the 5–50-μ m range) significantly impaired the metabolic functions of the hepatocytes. Incubation of the cells with 40 μ m-buprenorphine for 24 hr reduced the glycogen content to 60% of the initial content and 50 μ m-buprenorphine inhibited glycogen synthesis in glucagon-depleted human hepatocytes by about 40%. Albumin synthesis was the most sensitive metabolic parameter, and 24-hr exposure of hepatocytes to 10 μ m-buprenorphine, a concentration with no apparent cytotoxic effects, reduced albumin synthesis to 50%. Urea synthesis was moderately affected by buprenorphine. Glutathione content was reduced in a dose-dependent manner by the drug, reaching a minimum value (60% of control values) after 6 hr of exposure to 50 μ m of the opiate. Analysis of the data on the therapeutic dosage of buprenorphine and other opiates showed that the toxicity risk index of buprenorphine, like that of meperidine, lies somewhere between that of morphine and methadone.

Cellular responses to mechanical loading in vitro

A technique has been established in which cancellous bone biopsies may be simultaneously perfused and subjected to mechanical load bearing. Assessments of cell viability over a period of 24 h were based on the cAMP response to parathyroid hormone, intracellular lactate dehydrogenase activity, and electron micrograph morphology. Two cellular responses to mechanical loading were demonstrated similar to those that follow "osteogenic" loading in vivo, as reported previously. These were (1) a rise in intracellular G6PD in lining cells immediately after loading, and (2) an increase in RNA synthesis measured in osteocytes 6 h after loading. In vivo the osteogenic response to loading was modulated by indomethacin. In these in vitro experiments, addition of indomethacin inhibited both the loading-related G6PD and the RNA responses.

Effects of Halothane on Human and Rat Hepatocyte Cultures

The aim of this study was to investigate direct cytotoxicity to human and rat hepatocytes in primary culture from halothane and compare it with that of isoflurane, which is known to be minimally metabolized and less toxic in vivo. Both human and rat parenchymal cells were isolated by the two-step collagenase perfusion method and after attachment to plastic were incubated with either volatile anesthetic for 24 h. All the cultures were maintained in 20% O2 condition and were not induced prior to anesthetic treatment. Temperature, atmosphere conditions, and anesthetic concentrations were kept constant during the study period. Evaluation of cytotoxicity was based on morphologic, biologic (determination of both extracellular and intracellular lactate dehydrogenase activity), and metabolic (protein synthesis and secretion) end points. Protein synthesis and secretion rates were found to be the most sensitive parameters in hepatocyte cultures from both species. Protein synthesis was inhibited by 18% and protein secretion by 50% in the presence of 1 and 1.25 mM halothane, respectively, in human cell cultures (P less than 0.05). With 1.25 mM halothane intracellular lactate dehydrogenase was also decreased; lactate dehydrogenase leakage and morphologic alterations were detected only beyond 5 mM halothane. By contrast, in rat hepatocyte cultures protein secretion was inhibited by 26% and protein synthesis by 20% in the presence of 0.1 and 0.75 mM halothane, respectively, whereas morphologic alterations and a 37% lactate dehydrogenase leakage increase were observed with the concentration of 1 mM (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic and enzymatic characteristics of adult rat liver parenchymal cells in non-proliferating primary monolayer cultures

Parenchymal cells from normal adult rat liver, prepared with high yield (30 × 10 6 cells/g liver) and viability index (>96%) by a non-perfusion method, were maintained in non-proliferating monolayer culture. Several metabolic functions were investigated for 7 days to evaluate functional integrity of the cultured hepatocytes. Leucine was linearly incorporated into protein for 4.5 h at each day of cultivation and the incorporation rate increased up to 2-fold after 3 days. Urea production was maintained at a rate of 0.5 μmoles/mg protein × h for at least 7 days, and its amount was enhanced 2-fold within 24 h by the addition of 3 mM NH 4Cl. Glucose was formed during the first days by the hepatocytes and was then taken up with increasing amount from the surrounding medium. Lactate consumption, on the other hand, was replaced by lactate production after one day of cultivation. Variations in enzyme levels of lactate dehydrogenase, arginase, glutamine synthetase and glucose-6-phosphatase were also studied during the whole culture period. Cell leakage, which was detected only in the case of lactate dehydrogenase (LDH), occurred through the 4th day along with a concomitant loss of intracellular LDH activity. After 4 days, however, the enzyme activity returned to the initial level. Arginase was maintained throughout the cultivation period and was stimulated 2- to 3-fold within 24 h by NH 4Cl. Glutamine synthetase declined within the first 4 h of cultivation and then remained in the hepatocytes with a transitory rise after 2 days. Its activity was also found to be inversely related to the concentration of glutamine in the culture medium up to 4 mM. Glucose-6-phosphatase gradually decreased during the cultivation period, the enzyme activity, however, was stimulated by glucagon within 24 h.

On the variation in enzyme activities during the growth of burkitts lymphoma cells in suspension culture

1. 1. The growth characteristics of Burkitt's lymphoma cells in suspension culture have been studied, and a mean population doubling time of 20–21 hr established for this cell line under a range of nutritional and physical conditions; data which have provided a basis for the assessment of the reproducibility of the culture techniques and conditions which were employed in the subsequent studies. 2. 2. Activities of lactate dehydrogenase (LDH), aldolase and esterase, as well as the cellular content of total soluble protein, and the isoenzyme pattern of LDH, were monitored in randomly growing Raji cells for the duration of a complete growth cycle. 3. 3. In this period, the temporal pattern of variation in the levels of total soluble protein were seen to reflect alterations in LDH activity during a single growth cycle. 4. 4. The fluctuations observed in LDH activity were greater than those observed for either aldolase or esterase activity, and, from the data considered, the maximum degree of variation appeared to be confined to the initial stages of growth. 5. 5. Extracellular levels of LDH activity remained relatively constant throughout the growth cycle. so that the large fluctuations in intracellular LDH activity could not be attributed to either secretion or leakage of the enzyme into the culture medium. 6. 6. No gross changes in the pattern of LDH isoenzymes in these Raji cells were detected during the course of a single growth cycle.