Metabolically optimised BHK cell fed-batch cultures

The aim of this work was the optimisation of a fed-batch culture by metabolic confinement of BHK21 cells producing an antibody/cytokine fusion protein with potential application in tumour-targeted therapy. Previous results showed that at very low nutrient concentrations, a metabolic shift towards more efficient metabolic pathways occurs. The application of those results in the optimisation of a fed-batch culture resulted in higher cell growth (0.020 vs. 0.016 h(-1)) and cell viability, higher maximum cell concentration (2.5 vs. 1.1x10(6) cell ml(-1)), longer culture span (17 versus nine days) and higher product titre (60% increase), in relation to batch culture. This was achieved by maintaining glucose at 0.3 mM and glutamine at 0.2 mM through the addition of a concentrated solution based on the estimations of future nutrient consumption and growth rates through off line measurements. The production of toxic metabolites such as lactate and ammonia was reduced, especially the lactate production, which was markedly decreased due to the metabolic confinement of the cells. In conclusion, it was possible to increase the final titre of the recombinant antibody/cytokine fusion protein by confining the metabolism of the cells to an energetically more efficient state.     

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.     

Continuous cultivation of Trypanosoma brucei blood stream forms in a medium containing a low concentration of serum protein without feeder cell layers

Blood stream forms (BSF) of Trypanosoma brucei brucei GUT at 3.1 were propagated in vitro in the absence of feeder layer cells at 37 C, using a modified Iscove's medium (HMI-18). The medium was supplemented with 0.05 mM bathocuproine sulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM 2-mercaptoethanol, 1 mM sodium pyruvate. 0.16 mM thymidine, and 20% (v/v) Serum Plus (SP) (Hazleton Biologics, Lenexa, Kansas). The latter contained a low level of serum proteins (13 micrograms/ml). Each primary culture was initiated by placing 3.5-4 x 10(6) BSFs isolated from infected mice in a flask containing 5 ml of the medium (HMI-9) supplemented with 10% fetal bovine serum (FBS) and 10% SP. The cultures were maintained by replacing the medium every 24 hr for 5-7 days. During this period, many BSFs died. However, from day 4 onward, long slender BSFs increased in number. On days 5-7, trypanosome suspensions were pooled and cell debris was removed by means of diethylaminoethyl cellulose (DE52) column chromatography. Blood stream forms then were collected by centrifugation, resuspended in fresh medium at 7-9 x 10(5)/ml, and transferred to new flasks. Subcultures were maintained by readjusting the BSF density to 7-9 x 10(5)/ml every 24 hr. Concentrations of FBS were reduced gradually at 5-7-day intervals by alternating the amounts of FBS and SP in HMI-9 with 5% FBS and 15% SP, with 2% FBS and 18% SP, and finally with 20% SP (HMI-18). By this method, 2-3 x 10(6) VSFs/ml were obtained consistently every 24 hr. for more than 80 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a renal microchip for in vitro distal tubule models

Current developments in tissue engineering and microtechnology fields have allowed the proposal of pertinent tools, microchips, to investigate in vitro toxicity. In the framework of the proposed REACH European directive and the 3R recommendations, the purpose of these microtools is to mimic organs in vitro to refine in vitro culture models and to ultimately reduce animal testing. The microchip consists of functional living cell microchambers interconnected by a microfluidic network that allows continuous cell feeding and waste removal controls by fluid microflow. To validate this approach, Madin Darby Canine Kidney (MDCK) cells were cultivated inside a polydimethylsiloxane microchip. To assess the cell proliferation and feeding, the number of inoculated cells varied from 5 to 10 x 10(5) cells/microchip (corresponding roughly to 2.5 to 5 x 10(5) cells/cm2) and from four flow rates 0, 10, 25, and 50 microL/min were tested. Morphological observations have shown successful cell attachment and proliferation inside the microchips. The best flow rate appears to be 10 microL/min with which the cell population was multiplied by about 2.2 +/- 0.1 after 4 days of culture, including 3 days of perfusion (in comparison to 1.7 +/- 0.2 at 25 microL/min). At 10 microL/min flow rate, maximal cell population reached about 2.1 +/- 0.2 x 10(6) (corresponding to 7 +/- 0.7 x 10(7) cells/cm(3)). The viability, assessed by trypan blue and lactate deshydrogenase measurements, was found to be above 90% in all experiments. At 10 microL/min, glucose monitoring indicated a cell consumption of 16 +/- 2 microg/h/10(6) cells, whereas the glutamine metabolism was demonstrated with the production of NH3 by the cells about 0.8 +/- 0.4 micromol/day/10(6) cells. Augmentation of the flow rate appeared to increase the glucose consumption and the NH3 production by about 1.5- to 2-fold, in agreement with the tendencies reported in the literature. As a basic chronic toxicity assessment in the microchips, 5 mM and 10 mM ammonium chloride loadings, supplemented in the culture media, at 0, 10, and 25 micaroL/min flow rates were performed. At 10 microL/min, a reduction of 35% of the growth ratio with 5 mM and of 50% at 10 mM was found, whereas at 25 microL/min, a reduction of 10% with 5 mM and of 30% at 10 mM was obtained. Ammonium chloride contributed to increase the glucose consumption and to reduce the NH3 production. The microchip advantages, high surface/volume ratio, and dynamic loadings, coupled with the concordance between the present and literature results dealing with ammonia/ammonium effects on MDCK illustrate the potential of our microchip for wider in vitro chronic toxicity investigations.     

Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.     

Effects of metabolic factors in the diabetic state on the in vitro development of preimplantation mouse embryos

The effects of insulin, glucagon, beta-hydroxybutyrate, and acetoacetate on the in vitro development of preimplantation mouse embryos were studied. In controls, 24% of blastocysts failed to develop successfully when grown for 72 h in Eagle's medium supplemented with 10% fetal calf serum. Insulin at concentrations of 1.0 and 2.0 IU/ml of culture medium interfered with development in 62-63% of the blastocysts. Preimplantation embryos showed a threshold pattern in their reaction to glucagon: its addition in concentrations of 0.0015 mM (5 micrograms/ml) did not significantly inhibit blastocyst development, while concentrations of 0.003 mM (10 micrograms/ml) inhibited 70% of blastocysts. The embryotoxic effects of ketone bodies were manifested only in relatively high doses. beta-hydroxybutyrate was embryotoxic at concentrations greater than 5 mg/ml, and its effects were dose dependent: 48 mM (6 mg/ml) inhibited 45% of blastocysts, while 80 mM (10 mg/ml) arrested 87% of embryos from further development. Acetoacetate at concentrations of 0.1 mM (10 micrograms/ml) inhibited the development of 50% of the blastocysts, and its effects were not dose dependent: concentrations of 1 mM (100 micrograms/ml) inhibited development in 63% of the embryos. The combination of the diabetic metabolic factors in relatively low concentrations was highly embryotoxic, especially when accompanied by hyperglycemia.     

Simultaneous determination of nitroglycerin and dinitrate metabolites in metabolism studies using liquid chromatography-mass spectrometry with electrospray ionization

We have developed a liquid chromatographic-mass spectrometric method for the simultaneous determination of nitroglycerin (NTG) and its active metabolites, glyceryl 1,2-dinitrate (1,2-GDN) and glyceryl 1,3-dinitrate (1,3-GDN), for metabolism studies in cell cultures. 1,2,4-Butanetriol-1,4-dinitrate was chosen as an internal standard. Using a linear gradient of water/methanol containing 0.025 mM NH(4)Cl, the compounds were eluted within 12.5 min on an Allure Aqueous C(18) column (100 mm x 2.1 mm). Detection and quantification was achieved with multiple reaction monitoring in the negative ion mode. Intra- and inter-day variabilities for simultaneous determination of the three nitrates were below 10 and 18%, respectively, over a range of NTG and GDN concentrations of 0.5-15 ng/ml. The lower limit of quantification was found to be about 0.01 ng on column. Application of this method was illustrated through in vitro metabolism studies of NTG in culture media bathing LLC-PK1 cells and human vascular smooth muscle cells (HA-VSMC) at 37 degrees C. The degradation half-life of NTG was found to be 4.5 +/- 0.4 h and 39.2 +/- 0.02 h, respectively, for LLC-PK1 cells versus HA-VSMC. At 5 h, the 1,2-GDN versus 1,3-GDN metabolite distribution ratio in the bathing medium was found to be 1.5 +/- 0.1 and 0.2 +/- 0.02 for LLC-PK1 and HA-VSMC cells, respectively. With this method, the degradation half-life of NTG in rat plasma at 37 degrees C was shown to be 26.8 +/- 1.8 min, consistent with previous values obtained using gas chromatography.     

DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts

Sodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.     

Inhibition of jack bean urease by 1,4-benzoquinone and 2,5-dimethyl-1,4-benzoquinone. Evaluation of the inhibition mechanism

1,4-benzoquinone (BQ) and 2,5-dimethyl-1,4-benzoquinone (DMBQ) were studied as inhibitors of jack bean urease in 50 mM phosphate buffer, pH 7.0. The mechanisms of inhibition were evaluated by progress curves studies and steady-state approach to data achieved by preincubation of the enzyme with the inhibitor. The obtained reaction progress curves were time-dependent and characteristic of slow-binding inhibition. The effects of different concentrations of BQ and DMBQ on the initial and steady-state velocities as well as the apparent first-order velocity constants obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. The rapid formation of an initial BQ-urease complex with an inhibition constant of Ki = 0.031 mM was followed by a slow isomerization into the final BQ-urease complex with the overall inhibition constant of Ki* = 4.5 x 10(-5) mM. The respective inhibition constants for DMBQ were Ki = 0.42 mM, Ki* = 1.2 x 10(-3) mM. The rate constants of the inhibitor-urease isomerization indicated that forward processes were rapid in contrast to slow reverse reactions. The overall inhibition constants obtained by the steady-state analysis were found to be 5.1 x 10(-5) mM for BQ and 0.98 x 10(-3) mM for DMBQ. BQ was found to be a much stronger inhibitor of urease than DMBQ. A test, based on reaction with L-cysteine, confirmed the essential role of the sulfhydryl group in the inhibition of urease by BQ and DMBQ.     

Preliminary investigation on the cytotoxicity of tellurite to cultured HeLa cells.

Cytotoxicity of tellurite to cultured HeLa cells was examined by cell viability, lactate dehydrogenase (LDH) assay, and tellurite uptake. The experimental results show that the toxicity of tellurite depends on its concentrations and exposure time. HeLa cells exposed to tellurite for 2 h at 9.1 x 10(-4) to 4.5 x 10(-3) mmol/L did not exhibit cytotoxic effects as measured by cell viability. Exposure to tellurite for 24 h at the same concentrations markedly reduced the cell viability to 57% of the control during the first 5 minutes. Additionally, HeLa cells incubated at 2.7 x 10(-2) to 0.27 mmol/L of tellurite for 2 h retained 53% to 67% of cell viability. Even after 24 h exposure, the HeLa cells incubated at 9.1 x 10(-4) to 4.5 x 10(-2) mmol/L of tellurite still retained 57% to 66% of cell viability. Furthermore, tellurite toxicity was also demonstrated in supernatant of the culture at 37 degrees C by LDH assay. It was found that exposure to tellurite for 90 minutes did not stimulate LDH activity. However, tellurite uptake seems to be more sensitive than the cell viability and LDH activity release tests, as it significantly increases with the increasing of exposure time.     

Effects of dibutyryl adenosine 3':5'-cyclic monophosphate and other agents on induction of alkaline phosphatase activity in monkey kidney cells

The induction of alkaline phosphatase (ALP) by dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP) was investigated in strain JTC-12 . P3 cells derived from monkey (Maccaca irus) kidney cortex. ALP activity was increased by Bt2cAMP in a dose-dependent manner, reaching a plateau at concentrations higher than 5 mM with the activity being about 4 times that of the controls. The concentration of Bt2cAMP required for half-maximal induction of ALP activity was about 0.8 mM. ALP activity was increased rapidly by Bt2cAMP for the first 5 days and then continued to increase gradually towards a plateau level. Removal of Bt2cAMP from the medium caused a rapid decrease in the activity, suggesting that the induction of ALP activity by Bt2cAMP is reversible. ALP activity was induced synergistically in the presence of 1 mM sodium butyrate together with Bt2cAMP at concentrations from 0.01 to 1 mM. It was also found that in the presence of 1 mM Bt2cAMP, sodium butyrate increased ALP activity in the same manner as Bt2cAMP did in the presence of 1 mM sodium butyrate. Although dexamethasone, a potent glucocorticoid, had no effect on ALP activity in control cells, the hormone suppressed the ALP activity induced by Bt2cAMP in a dose-dependent manner. At concentrations above 0.2 mM, two xanthine derivatives, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX), also inhibited the induction of ALP activity by 1 mM Bt2cAMP. Inhibitors of protein synthesis, cycloheximide (1.5 micrograms/ml) and pactamycin (10 micrograms/ml), as well as inhibitors of RNA synthesis, actinomycin D (2 micrograms/ml) and alpha-amanitin (50 micrograms/ml), suppressed the induction of ALP activity.     

Metabolism of 2-hydroxy-4-methoxybenzophenone in isolated rat hepatocytes and xenoestrogenic effects of its metabolites on MCF-7 human breast cancer cells

The metabolism and cytotoxicity of 2-hydroxy-4-methoxybenzophenone (HMB) in isolated rat hepatocytes and the xenoestrogenic activity of HMB and its metabolites in MCF-7 human breast cancer cells and an estrogen receptor competitive binding assay have been studied, respectively. The incubation of hepatocytes with HMB caused a concentration- and time-dependent decrease in cell viability, accompanied by loss of intracellular ATP and adenine nucleotide pools. HMB at a low-toxic level (0.25 mM) in the hepatocyte suspensions was converted enzymatically to 2,4-dihydroxybenzophenone (DHB) and a hydroxylated intermediate, which was tentatively identified as an isomer of 2,2prime prime or minute-dihydroxy-4-methoxybenzophenone (DHMB) as determined by mass spectroscopy coupled with HPLC. Furthermore, the parent compound and both intermediates were rapidly conjugated to glucuronides, whereas free unconjugated DHMB and 2,3,4-trihydroxybenzophenone (THB) were identified as trace intermediates. In another experiment, DHB and THB displaced competitively 17beta-estradiol bound to the recombinant human estrogen receptor alpha in a concentration-dependent manner: IC(50) of diethylstilbestrol and bisphenol A, which are known xenoestorogenic compounds, and DHB and THB was approximately 1 x 10(-8), 1 x 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. Further, DHB at concentrations from 10(-8) to 10(-6) M caused a concentration-dependent proliferation of MCF-7 cells. DHMB and THB at 10(-7) and 10(-6) M also elicited a slight increase in cell numbers, whereas HMB at concentrations from 10(-9) to 10(-4) M did not affect the cell proliferation. Based on the relative IC50 for the competitive binding and the proliferative effect on MCF-7 cells, it follows that in estrogenic potency, DHB>THB>DHMB. These results indicate that some hydroxylated intermediates such as DHB rather than the parent compound act as a xenoestrogen via biotransformation.     

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa.

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.     

Human 293 cell metabolism in low glutamine-supplied culture: interpretation of metabolic changes through metabolic flux analysis

Metabolic flux analysis is a useful tool to analyze cell metabolism. In this study, we report the use of a metabolic model with 34 fluxes to study the 293 cell, in order to improve its growth capacity in a DMEM/F12 medium. A batch, fed-batch with glutamine feeding, fed-batch with essential amino acids, and finally a fed-batch experiment with both essential and nonessential amino acids were compared. The fed-batch with glutamine led to a maximum cell density of 2.4x10(6) cells/ml compared to 1.8x10(6) cells/ml achieved in a batch mode. In this fed-batch with glutamine, it was also found that 2.5 mM ammonia was produced compared to the batch which had a final ammonia concentration of 1 mM. Ammonia was found to be growth inhibiting for this cell line at a concentration starting at 1 mM. During the fed-batch with glutamine, the flux analysis shows that a majority of amino acid fluxes and Kreb's cycle fluxes, except for glutamine flux, are decreased. This observation led to the conclusion that the main nutrient used is glutamine and that during the batch there is an overflow in the Kreb's cycle. Thus, a fed-batch with glutamine permits a better utilization of this nutrient. A fed-batch with essential amino acid without glutamine was also assayed in order to reduce ammonia production. The maximum cell density was increased further to 3x10(6) cells/ml and ammonia production was reduced below 1 mM. Flux analysis shows that the cells could adapt to a medium with low glutamine by increasing the amino acid fluxes toward the Kreb's cycle. Adding nonessential amino acids during this feeding strategy did not improve growth further and the nonessential amino acids accumulated in the medium.     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: application to cellular metabolism and accumulation studies

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile-50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm x 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9-108.3% for CPT-11 in culture media and 94.3-107.2% for CPT-11 in cell lysates; and 87.7-106.8% for SN-38 in culture media and 90.1-105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.     

Effects of in vitro fertilization conditions on preimplantation development and quality of pig embryos

The present study was to investigate the effects of in vitro fertilization conditions on in vitro development and structural integrity of pig embryos. Porcine oocytes matured in vitro were co-incubated with four different spermatozoa concentrations (0.6 x 10(5), 1.2 x 10(5), 2.5 x 10(5) and 5 x 10(5) cells/ml) for 6 h, and at a spermatozoa concentration (1.2 x 10(5) cells/ml) for 2, 4 and 6 h, respectively. Spermatozoa penetration and blastocyst formation were observed at 10 and 144 h post insemination, respectively. The allocation of a blastocyst to inner cell mass (ICM) and trophectoderm (TE) cells was determined by using a differential staining method. Polyspermy frequency increased with increasing spermatozoa concentrations. The spermatozoa-oocyte co-incubation period of 2 h provided for decreased in vitro development rate than 4 and 6 h groups (P < 0.05), although no difference was detected in polyspermy frequency between spermatozoa-oocyte co-incubation periods. Interestingly, blastocysts derived from the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml) had significantly fewer ICM cell nuclei as compared with those groups with lesser spermatozoa concentrations (0.6 x 10(5) and 1.2 x 10(5) cells/ml). There was no difference in the structural integrity of blastocysts among the co-incubation periods. Blastocysts derived from respective experiments were individually classified into three groups (I: <20%; II: 20-40% and III: >40%) based on the ratio of ICM to total cells. Proportion of blastocysts in Group II, with a presumptive normal range of structural integrity, was slightly decreased in the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml). The results indicate that the spermatozoa concentration during in vitro fertilization may be important for developmental competence and quality of pig embryos.     

Applications of short-chain polydimethylacrylamide as sieving medium for the electrophoretic separation of DNA fragments and mutation analysis in uncoated capillaries

The cytosensor microphysiometer (a biosensing instrument for detecting cellular metabolism) was compared to the established tetrazolium salt assay as a chemosensitivity test. Two coumarin compounds, 7-hydroxycoumarin and esculetin, were examined to determine their effect on the cellular metabolism of A431 cells over a 24-h exposure period. In the tetrazolium salt assay, 7-hydroxycoumarin caused suppression of the succinate dehydrogenase activity at concentrations greater than 10 microg/ml. Esculetin exerted a more serious effect on succinate dehydrogenase, with decreases in activity observed at greater than 1 microg/ml. The observed effect was dose-dependent for both compounds examined. The metabolic activities of cells exposed to 100 microg/ml of drug were 90.37 +/- 2.8 and 71.62 +/- 2.96 (n = 3), of control values, for 7-hydroxycoumarin and esculetin, respectively. Using the cytosensor microphysiometer to assess metabolic activities, a similar pattern of inhibition was observed, with esculetin more detrimental to cellular metabolism than 7-hydroxycoumarin. The effect was dose- and time-dependent for both compounds. 7-Hydroxycoumarin (100 microg/ml) caused the cellular metabolic rate to drop to 44.21 +/- 5.34% (n = 4) of the control metabolic rate, while 100 microg/ml esculetin caused the metabolic rate to fall to 21.5 +/- 4.54% (n = 4) of the control rate. The cytosensor method proved to be superior to the tetrazolium salt assay for a number of reasons, which are discussed in this paper.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.