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PubMed2001

Production of DNA strand breaks in vitro and reactive oxygen species in vitro and in HL-60 cells by PCB metabolites.

PCBs are industrial chemicals that continue to contaminate our environment. They cause various toxic effects in animals and in exposed human populations. The mechanisms of toxicity, however, are not completely understood. PCBs are metabolized by cytochromes P450 to mono- and dihydroxylated compounds. Dihydroxy-PCBs can potentially be oxidized to the corresponding quinones. We hypothesized that reactive oxygen species (ROS) are produced by redox reactions of PCB metabolites. We tested several synthetic dihydroxy- and quinoid-PCBs with 1-3 chlorines for their potential to produce ROS in vitro and in HL-60 human leukemia cells, and DNA strand breaks in vitro. All dihydroxy-PCBs tested produced superoxide. The quinones generated superoxide only in the presence of GSH, probably during the autoxidation of the glutathione conjugates. We observed increased superoxide production with decreasing halogenation. Incubation of dihydroxy-PCBs or PCB quinones + GSH with plasmid DNA resulted in DNA strand break induction in the presence of Cu(II). Tests with various ROS scavengers indicated that hydroxyl radicals and singlet oxygen are likely involved in this strand break induction. Finally, dihydroxy- and quinoid PCBs also produced ROS in HL-60 cells in a dose- and time-dependent manner. We conclude that dihydroxylated PCBs, and PCB quinones after reaction with GSH, produce superoxide and other ROS both in vitro and in HL-60 cells, and oxidative DNA damage in the form of DNA strand breaks in vitro. The reactions seen in vitro and in cells may well be a predictor of the toxicity of PCBs in animals.

Cell Survival↗
PubMed1978

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.

Coliphages↗
PubMedDate not supplied

In vitro remineralization of in vivo and in vitro formed enamel lesions.

Thin sections of natural white spot enamel lesions (WS) and of artificial in vitro lesions (VL) were remineralized simultaneously in vitro. The sections, clamped in a PMMA holder, were microradiographed at baseline and after remineralization in a calcium- and phosphate-containing solution (pH = 7.0; 1 ppm F) after 2 and 4 weeks. All data were analyzed with respect to baseline. The results show that the lesion depth values did not change significantly during 2 and 4 weeks of remineralization. The mineral accumulation (change in DeltaZ), however, was substantial and significant in WS and VL. In WS the change in mineral accumulation was roughly proportional to the amount of mineral at baseline. The WS accumulated more than two times the amount of mineral than VL in the same periods of remineralization. After 4 weeks of remineralization the maximum mineral value Vmax in the surface layer of the WS was nearly up to the sound enamel level thick approximate87 vol%. This study shows that the technique and calculation procedure described make this single section method attractive for longitudinal demineralization-remineralization studies in vitro or in situ. Both WS and VL samples obviously remineralized in vitro similarly with respect to the baseline. Furthermore, this in vitro work indicates that remineralization inhibitors present in saliva, and previously penetrated into the enamel tissue, do not influence remineralization later on.

Absorptiometry, Photon↗
PubMed2006

The impact of in vitro binding on in vitro-in vivo extrapolations, projections of metabolic clearance and clinical drug-drug interactions.

This review provides a vista of the current opportunities and remaining challenges in the area of in vitro-in vivo extrapolation, with particular emphasis on drug binding terms in predictive models, which has been the source of much controversy. Although the importance of fu(inc) (fraction unbound in in vitro incubations) has been acknowledged for decades, it is not always applied in practice. This is somewhat disappointing, since although it may be onerous to measure this term for large numbers of compounds, algorithms to estimate the term from logD(7.4) or logP have been detailed in the literature. These are sufficiently robust to negate routine measurement in early drug discovery. Several groups have recently established convincing relationships between unbound in vivo and in vitro metabolic intrinsic clearance (CL(int)). In the authors' laboratory, correlations of this type have been constructed for rat, dog and Man. The use and interpretation of these models within a drug discovery setting is discussed. The quantitative prediction of drug-drug interactions from in vitro cytochrome P450 (CYP) inhibition data remains a challenge. Although extensive literature databases are at last emerging, apparent ad hoc use of terms for in vivo inhibitor concentrations and only occasional consideration of fu(inc) may only have confused matters. The effect of accounting for drug binding on the accuracy of predictions is reviewed. Other themes including the impact of fu(inc) on relative activity factors (RAFs) and how in vitro data quality and inter-laboratory differences can confound quantitative human pharmacokinetic predictions are also developed.

Animals↗
PubMed1989

Transport across rat trachea in vitro after exposure to cytoskeleton-active drugs in vitro or to ozone in vivo.

Full-length tracheas from Sprague-Dawley rats were exposed to cytoskeleton-active drugs in short-term organ culture, and the permeability of the tracheal epithelium was measured by instilling radiotracers into the lumen and assay of the radioactivity appearing in the external bathing medium. In vitro treatment with cytochalasin D (cyto D, 2-10 x 10(-6) M) increased the rate of movement of [14C]mannitol across the epithelium. Exposure to vinblastine (VB, 10(-4) M) alone had no significant effect. However, VB in combination with cyto D increased the permeability in a dose-dependent manner. In vivo exposure to ozone (O3, 0.8 or 2.0 ppm, 2 h) had only a slight effect on the rate of movement of the tracer as measured in vitro immediately after exposure. At 24 h postexposure there was no significant difference in permeability between ozone- and air-exposed tracheas. Prior in vivo O3 exposure sensitized the tracheas to the in vitro effects of cyto D; treatment of O3-exposed tracheas with cyto D immediately after O3 exposure produced a greater than additive effect on permeability measured in vitro. VB at concentrations up to 10(-4) M had no enhancing effect on permeability in O3-exposed tracheas. Sham exposure to clean air did not affect permeability compared to untreated (shelf) controls. Electron microscopic studies demonstrated penetration of horseradish peroxidase into intercellular spaces in the tracheas treated in vitro with cyto D or cyto D plus VB. Cyto D is known to affect intracellular microfilaments that have attachments at or near the cell surface, while VB affects microtubules associated with internal cellular structures. Therefore, the synergistic effect on tracheal permeability observed with O3 and cyto D, but not with O3 and VB, suggests that O3 may change cell surface structures associated with the microfilamentous cytoskeleton.

Animals↗
PubMed2006

Functional evaluation of a new bioartificial liver system in vitro and in vitro.

AIM: To evaluate the functions of a new bioartificial liver (BAL) system in vitro and in vitro. METHODS: The BAL system was configured by inoculating porcine hepatocyte spheroids into the cell circuit of a hollow fiber bioreactor. In the experiments of BAL in vitro, the levels of alanine aminotransferase (ALT), total bilirubin (TB), and albumin (ALB) in the circulating hepatocyte suspension and RPMI-1640 medium were determined during 6 h of circulation in the BAL device. In the experiments of BAL in vitro, acute liver failure (ALF) model in canine was induced by an end-side portocaval shunt combined with common bile duct ligation and transaction. Blood ALT, TB and ammonia levels of ALF in canines were determined before and after BAL treatment. RESULTS: During 6 h of circulation in vitro, there was no significant change of ALT, whereas the TB and ALB levels gradually increased with time both in the hepatocyte suspension and in RPMI-1640 medium. In the BAL treatment group, blood ALT, TB and ammonia levels of ALF in canines decreased significantly. CONCLUSION: The new BAL system has the ability to perform liver functions and can be used to treat ALF.

Alanine Transaminase↗
PubMed1988

Specific antibody synthesis in vitro. IV. The correlation of in vitro and in vivo antibody response to influenza vaccine in rhesus monkeys.

Fourteen female monkeys (Macaca mulatta) received a trivalent influenza vaccine and antibody response was determined by a change in plasma antibody content (ELISA) before and after vaccine. Lymphocyte cultures were also established from these monkeys and the level of antibody response did not correlate with mitogen-induced lymphocyte blastogenesis or natural killer cell function. In vitro anti-influenza antibody synthesis, however, was found to correlate well with the in vivo response. That is, monkeys who were non-responders, as determined by lack of change in plasma antibody content, were also non-responders in vitro. Accordingly, we believe that vaccine response is not necessarily a measure of immune competence but its measurement may, none the less, have clinical utility. The excellent correlation of in vivo and in vitro response provides predictive value for the in vitro test. Furthermore, because the correlation is good, the in vitro test may be useful as a tool in immunopharmacology and toxicology.

Animals↗
PubMed1995

The in vitro HL-60 cell--Trypanosoma brucei rhodesiense culture system: a rapid in vitro drug screen.

The in vitro culture system is described in which Trypanosoma brucei rhodesiense (LOUTat.1) was grown with the human feed layer cell HL-60. The use of this system in determining the 50% growth Inhibitory Concentration (IC50) of unknown compounds for both the trypanosomes and the host cell was demonstrated. The data shows that several analogues of pentamidine have significantly reduced host cell toxicity but maintain or have increased typanocidal activity. The value of the trypanosome/HL-60 in vitro culture system as a rapid primary in vitro drug screen is discussed. Based upon the ability of this primary screen to predict potential drug efficacy, several analogues screened in vitro were then tested in vivo. The results of the in vivo tests confirmed the ability of the in vitro screen to predict drug efficacy, and also suggests that better analogues of pentamidine (less host toxicity and greater trypanocidal activity) can be obtained to treat human trypanosomiasis.

Animals↗
PubMed1997

Inhibition by omeprazole of proguanil metabolism: mechanism of the interaction in vitro and prediction of in vivo results from the in vitro experiments.

Both the antimalarial prodrug proguanil and the gastric proton pump inhibitor omeprazole are substrates for cytochrome P450 (CYP)2C19 and CYP3A. However, the relative contribution of each enzyme to proguanil bioactivation to cycloguanil and to the metabolism of omeprazole, as well as their potential to interact, remains to be examined. The bioactivation of proguanil to its active metabolite cycloguanil was studied in vitro in human liver microsomes and in vivo in 12 healthy subjects, in the absence and in the presence of omeprazole. The formation of cycloguanil from proguanil exhibited biphasic kinetic behavior in four of six human livers, indicating that at least two enzymes are responsible for this metabolic step. Cycloguanil formation activity did not correlate with immunoreactive CYP3A4 content or with CYP3A4 activity, as measured by testosterone 6beta-hydroxylation, suggesting that CYP3A4 plays a limited role in cycloguanil formation. Furthermore, troleandomycin (10 microM) inhibited only 10 to 17% of cycloguanil formation at proguanil concentrations of 100 and 500 microM. At a proguanil concentration of 20 microM, omeprazole at 10 microM inhibited cycloguanil formation in vitro by 47 +/- 59%. These in vitro results were consistent with the results of our in vivo study in healthy subjects, which showed a 32 +/- 11% decrease in proguanil apparent oral clearance and a 65 +/- 8% decrease in proguanil partial metabolic clearance to cycloguanil in the presence of omeprazole (both P < .001). We conclude that in vitro studies of proguanil metabolism and interactions are predictive of in vivo situations, that CYP2C19 is the main enzyme responsible for proguanil bioactivation to cycloguanil and that omeprazole inhibits this biotransformation in vitro and in vivo by inhibiting this enzyme.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

Immunomodulation in vitro. The predictive value of in vitro testing of lung cancer patients' lymphocyte responsiveness to stimulation by Thymex L. A preliminary report.

Eleven lung cancer patients were selected for combined radio and immunotherapy with a thymic agent-Thymex L. The selection criteria included the pre-therapy testing of patients' immunocompetence and the responsiveness of their lymphocytes to the in vitro addition of Thymex L: only patients with a significant degree of immunodepression, whose depressed cellular immunity parameters (the number of total and active T cells and their mitogen-induced lymphoproliferative response) were significantly increased upon this agent's action in vitro, entered the study. The results of the pre-therapy in vitro stimulation correlated with those obtained after completion of radioimmunotherapy: the administration of Thymex L along with radiotherapy seances prevented iatrogenic deterioration of initial depression of general immunocompetence and enabled to overcome it to a certain degree. This indicates that pre-therapy in vitro testing has a true predictive value. However, the initial immune disturbances were not normalized by immunotherapy; the post-therapy testing of the patients' lymphocytes to the addition of Thymex L in vitro showed that these cells possessed a residual potential to respond by a significant increase of the active T cell number and proliferative capacity, suggesting that immunotherapy could be prolonged in order to potentiate cellular immunity in immunodepressed lung cancer patients.

Adjuvants, Immunologic↗
PubMed1997

Effect of oxygen concentration and free radicals on in vitro development of in vitro-produced bovine embryos.

The effects of free radicals and hypotaurine on the development of bovine embryos produced by in vitro fertilization of in vitro-matured oocytes were examined. Embryos that developed to the 4- to 6-cell stage after in vitro fertilization were cultured without feeder cells in TCM199 medium supplemented with 1% calf serum (CS) under either 5% CO2 in air or 5% O2, 5% CO2 and 90% N2. The percentages of blastocysts (including early, expanding, and hatched stages) that developed under 5% O2 was higher (P < .01) than the percentage of those that developed under 20% O2. The respective percentages in 5% vs 20% O2 concentration were as follows: blastocysts (d 8), 49% vs 17%; expanded blastocysts (d 8), 19% vs 6%; hatched blastocysts (d 10), 16% vs 0%. The development of embryos to blastocysts was suppressed (P < .05) when oxygen radicals were generated in culture medium by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) under both 5% and 20% O2. The addition of hypotaurine permitted the development of embryos to blastocyst stage in medium with AAPH only under 5% O2 (P < .05), but superoxide dismutase (SOD) did not permit the development of embryos to blastocysts. Alternatively, when embryos were cultured in medium without AAPH, the rates of development into blastocysts under 20% O2 increased (P < .05) by the addition of hypotaurine and SOD. However, under 5% O2, the rates of blastocyst formation were not improved by addition of hypotaurine and SOD. Moreover, the cell numbers of blastocysts cultured in medium containing hypotaurine were greater (P < .01) than those of blastocysts cultured in medium without hypotaurine. It is concluded that hypotaurine may exert beneficial effects on in vitro development of bovine embryos under both 20% O2 and 5% O2 of gaseous conditions.

Amidines↗
PubMed1975

In vitro fertilization of rat and mouse eggs by ejaculated sperm and the effect of energy sources on in vitro fertilization of rat eggs.

In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8%) in the rat, but 11 to 41% of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5%) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs.

Animals↗
PubMed1984

Comparative in vivo and in vitro studies of phenytoin protein binding and in vitro lipolysis in plasma of pregnant and nonpregnant rats.

This investigation was designed to determine the cause of the changes in drug protein binding that occur in rat plasma, particularly in plasma from pregnant animals, during in vitro drug-protein binding measurements. In vivo estimates of phenytoin binding in plasma were obtained from steady-state CSF-plasma concentration ratios in pregnant and nonpregnant rats. Immediate ultrafiltration of heparin- or EDTA-anticoagulated plasma yielded phenytoin free fraction values that were in good agreement with in vivo estimates for nonpregnant rats but that were about one-third higher than in vivo estimates for pregnant animals. In vitro free fraction values tended to increase during incubation of plasma and/or during equilibrium dialysis. The concentrations of the four major endogenous free fatty acids were similar in plasma of pregnant and nonpregnant rats if determined immediately after blood collection. Six hours of incubation at 37 degrees C caused fatty acid concentrations to increase about fivefold and twofold in heparin-anticoagulated plasma from pregnant and nonpregnant animals, respectively. The corresponding increases in EDTA-anticoagulated plasma were only about twofold and 1.14-fold, respectively. These changes were associated with decreased plasma protein binding of phenytoin. The in vivo differences between pregnant and nonpregnant rats with respect to phenytoin binding in plasma are not due to differences in fatty acid concentrations, but the in vitro differences are due primarily to corresponding differences in free fatty acid concentrations if extensive in vitro lipolysis occurs.

Animals↗
PubMed1989

Follicular fluid steroid content and in vitro steroid secretion by granulosa-lutein cells from individual follicles among different stimulation protocols for in vitro fertilization-embryo transfer.

The in vitro steroidogenic capacity of granulosa-lutein (G-L) cells aspirated from individual follicles during cycles of in vitro fertilization-embryo transfer was examined and compared among three different stimulation protocols: human menopausal gonadotropins (hMG), clomiphene citrate (CC) and hMG, and follicle stimulating hormone (FSH). In addition, the clinical outcome of the patients in each protocol was examined. After 3 days of culture in basal medium, fresh medium with or without androstenedione (A) (10(-7) M) was added for 24 hr, at which time medium was obtained for measurement of progesterone (P) and estradiol (E) content. Follicular fluid (FF) P, E, and A were measured in each follicle and compared among protocols. FF from individual follicles in patients stimulated with FSH contained higher levels of P compared to FF from patients stimulated with hMG or CC/hMG, while E was higher in patients stimulated with CC/hMG compared to FSH or hMG. FF levels of A were not significantly different among the protocols. In vitro steroid secretion revealed a progressive increase in P secretion in contrast to decreasing E secretion when one compares CC/hMG, hMG, and FSH. Patients undergoing ovarian hyperstimulation with FSH had significantly more atretic oocytes identified at the time of oocyte harvest compared to patients undergoing ovarian hyperstimulation with CC/hMG or hMG. The hMG protocol yielded significantly fewer fertilized oocytes, cleaved embryos, and transferred embryos, compared to the CC/hMG and FSH protocol, however, there was no significant difference in pregnancy rate among the three protocols. These data demonstrate that individual follicles contain G-L cells with markedly different abilities to luteinize in vitro as assessed by steroid secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗
PubMed1987

Preimplantation development following in vitro fertilization of mouse oocytes: effects of timing of superovulation and preincubation in vitro.

The early embryonic development of in vitro fertilized oocytes was assessed following superovulation in F1 hybrid (C57BL/6 X CBA/Ca) mice. Decreasing the time interval between the administration of constant doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) resulted in decreases in the frequency of development to the blastocyst stage but had no significant effect on development to the two-cell stage. Preincubation of postovulatory oocytes in vitro prior to insemination did not compensate for the reduced preovulatory development in vivo but resulted in decreases in the frequency of development to the blastocyst stage. The results indicate that inadequate preovulatory development of superovulated mouse oocytes can adversely affect the preimplantation development of in vitro fertilized embryos in the absence of a visible inhibitory effect on development to the two-cell stage and also that preincubation of postovulatory oocytes in vitro prior to fertilization reduces subsequent developmental capacity.

Animals↗
PubMed1995

Cigarette smoking at time of in vitro fertilization cycle initiation has negative effect on in vitro fertilization-embryo transfer success rate.

PURPOSE: To assess cigarette smoking of female patients prior to starting in vitro fertilization cycle and possible affect on subsequent in vitro fertilization-embryo transfer outcome. METHODS: Retrospective study involving 340 consecutive patient questionnaires filled out at time of in vitro fertilization program entry. Only cycles resulting in embryo transfer after transvaginal ultrasound directed ovum retrieval (n = 253) were considered. The three patient pregnancy outcomes of not pregnant, spontaneous abortion, and live birth, were cross-referenced with smoking and nonsmoking patients. RESULTS: No significant difference found in overall pregnancy rate per embryo transfer for smokers (35%) vs nonsmokers (31%). However, the abortion rate was significantly higher for the smokers (73%) vs nonsmokers (24%) with a P value < 0.001. CONCLUSIONS: Results suggest preentry in vitro fertilization cycle cigarette smoking has adverse affect on potential pregnancy outcome by increasing spontaneous abortion. Preconception health consultation concerning adverse cigarette smoking effects should be implemented prior to program entry.

Abortion, Spontaneous↗
PubMed1989

Liver mitochondrial aldehyde dehydrogenase: in vitro expression, in vitro import, and effect of alcohols on import.

An in vitro expression plasmid (pGRAP) that contained the cDNA coding for the rat mitochondrial aldehyde dehydrogenase precursor was constructed, mRNA was synthesized then translated, and the in vitro synthesized precursor of aldehyde dehydrogenase was used in an in vitro import assay. As expected the 19 amino acid signal peptide of the precursor allowed import of the precursor into rat liver mitochondria. This in vitro system was used to examine the effect of alcohols on import. It was found that the alcohols (ethyl, butyl, hexyl, and octyl) tested inhibited the import of the aldehyde dehydrogenase precursor. Pretreatment of the mitochondria with alcohol was responsible for the inhibition. The inhibition appeared to be relatively specific for pre-aldehyde dehydrogenase as the precursor of ornithine transcarbamylase was still imported in the presence of alcohols. Of potential physiological significance was finding that ethanol inhibited import in a dose-response fashion; 50% inhibition occurred at 75 mM, a concentration achievable during the ingestion of alcohol. In addition, the concentrations of alcohols required to produce an inhibitory effect on import decreased as the hydrocarbon chain length of alcohols increased. The inhibitory effect of alcohols appeared to be specific as other solvents examined did not inhibit import. We postulate that alcohols may perturb the mitochondrial membrane and affect the receptor-translocator necessary for the import of the aldehyde dehydrogenase precursor.

Aldehyde Dehydrogenase↗
PubMed1985

In vitro packaging of plasmid DNA oligomers by Salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro.

In vitro packaging experiments with phage P22 using artificially ligated plasmid concatemers have shown that the pac site is not necessary for DNA packaging although in vivo this initiation signal is indispensable. This indicates that the phage-coded protein gp3 also executes other important functions during phage maturation in addition to the recognition of pac, or that its site specificity is lost in vitro. It has been shown previously that gp3 is necessary for in vitro packaging. Further, it was demonstrated that DNA which is only 74% of headful size cannot be packaged. Oversized DNA, however, is cut in vitro to unit length.

DNA Transposable Elements↗