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PubMed1975

Mutagenesis of certain activated carcinogens in vitro associated with genetically mediated increases in monooxygenase activity and cytochrome P 1-450.

A bacterial mutagenesis assay and genetic differences in microsomal CO-binding cytochromes were combined in vitro to evaluate the metabolic activation of several known carcinogens to frameshift mutagens. With the use of liver fractions from C57BL/6N and DBA/2N control mice and mice treated in vivo with 3-methylcholanthrene, beta-naphthoglavone, phenobarbital, or 2,3,7,,-tetrachlorodibenzo-p-dioxin, the in vitro mutagenicity of 3-methylcholanthrene, 6-aminochrysene, and 2-acetylaminofluorene --but not benzo[a]pyrene==is closely associated with the genetically mediated difference in both aromatic hydrocarbon-inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity and new cytochrome P1-450 formation; such an association between 7,12-dimethylbenz[a]anthracene or benz[a]anthracene activation to mutagens in vitro and these genetic differences between C57BL/6N and DBA/2N mouse strains in uncertain. The Salmonella typhimurium histidine mutant TA1538 is more effective than tester strains TA1537 and TA1535 in the determination of 3-methylcholanthrene mutagenesis in vitro. The relationships between the histidine revertant rate as a function of both liver protein concentration per plate and mutagen concentration per plate are illustrated for 3-methylcholanthrene, benzo[a]pyrene, 6-aminochrysene, and 2-acetylaminofluorene. With the use of offspring from the appropriate genetic crosses, the aromatic hydrocarbon-inducible hydroxylase activity appears to be expressed as an autosomal dominant trait, whereas the mutagenesis of 3-methylcholanthrene in vitro appears to be expressed additively; this apparent discrepancy probably reflects different proportional amounts of phenolic benzo[a]pyrene, compared with mutagenic 3-methylcholanthrene metabolites, formed by the monooxygenase(s). 3-Methylcholanthrene, 6-aminochrysene, and 2-acetylaminofluorene--but not benzo[a]pyrene--are each more mutagenic in vitro per molecule of cytochrome P1-450 than per molecule of CO-binding cytochrome other than P1450. Diethylmaleate, a compound which depletes flutathione content in liver, and 1,1,1-trichloropropene-2,3-epoxide, an inhibitor of epoxide hydrase (EC 4.2.1.63), were also studied in vitro. Diethylmaleate, and especially 1,1,1-trichloropropene-2,3-epoxide, increases the mutagenicity of benzo[a]pyrene, whereas no increases occur with 3-methylcholanthrene, 6-aminochrysene, or 2-acetylaminofluorene activation to mutagens in vitro. Both diethylmaleate and 1,1,1-trichloropropene-2,3-epoxide cause decreases in 2-acetylaminofluorene mutagenesis in vitro when liver fractions from phenobarbital-treated mice are used.

2-Acetylaminofluorene↗
PubMed2002

Microdensitometric assay of enzymatic activities in parthenogenetically activated and in vitro fertilized bovine oocytes.

To examine the paternal genome's role in reprogramming metabolic activity in one-cell embryos, we investigated metabolic aspects of bovine oocytes after in vitro maturation and in vitro fertilization and after in vitro parthenogenetic activation with a Ca2+ ionophore and 6-dimethylaminopurine. We assayed succinate dehydrogenase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities by microspectrophotometry in immature oocytes and oocytes after maturation, in vitro fertilization and parthenogenetic activation. Succinate dehydrogenase activity significantly increased after in vitro maturation, significantly decreased after Ca2+ ionophore activation and further decreased after 6-dimethylaminopurine treatment. Lactate dehydrogenase activity showed a significant decrease in bovine oocytes after in vitro maturation, remained unchanged in Ca2+ ionophore-treated oocytes and rose significantly after 6-dimethylaminopurine treatment. This activity was dramatically reduced after in vitro fertilization, reaching absorbance levels that were not different from those in mature and Ca2+ ionophore-treated oocytes. Glucose-6-phosphate dehydrogenase activity was significantly lower in matured oocytes as compared to immature oocytes, was significantly higher after artificial activation with Ca2+ ionophore and remained constant after 6-dimethylaminopurine treatment or after in vitro fertilization. We suggest that metabolic changes involved in parthenogenetic activation are similar to those occurring after fertilization.

Adenine↗
PubMed1996

Reduced proliferation rate of hypoxanthine-phosphoribosyl transferase mutant human T-lymphocytes in vitro.

The clonal hprt mutation assay in human T-cell is based on the assumption that wild-type cells and hypoxanthine-phosphoribosyl transferase (hprt) mutant cells survive and proliferate at the same rate during the expression phase which is required for the expression of in vitro-induced mutants. We have tested this assumption in a study of mutant frequency (MF) and proliferation rate at different time points during in vitro expansion of human T-cells in non-selective medium. Peripheral blood lymphocytes from 11 individuals were studied using standard cloning procedures to determine the cloning efficiency (CE) and the hprt MF by 6-thioguanine (TG) selection. Another cell portion from each individual was allowed to proliferate in bulk culture for 8 days in vitro, before measuring CE and MF as above. In the directly plated cell population the CE was 45% and the MF 18.7 +/- 15.3 x 10(-6) (mean +/- S.D.), whereas the in vitro expanded cell population showed a CE of 38% and a significantly reduced MF of 8.3 +/- 6.9 x 10(-6) (P = 0.0033). Thus, the mean MF was 56% lower in the in vitro expanded than in the directly plated cell population. The experiment was repeated in another group of ten individuals with essentially the same result. In a third experiment, freshly prepared cells from two donors were allowed to grow for up to 15 days in bulk culture in vitro. Cell growth, CE, and MF were determined every third day. The MF decreased gradually, and at day 12-15 it was only 25% of the initial value. The total number of clonable cells increased 13-fold during the 15 days of in vitro expansion, while the mutant, TG-resistant cell population increased only 3-fold. These results suggest that human hprt mutant T-lymphocytes have a reduced proliferation rate compared to wild-type cells during in vitro proliferation. Thus, measurements of chemical and radiation induced MF with the T-cell clonal assay may underestimate the true MF by a factor of 2 or more.

Adult↗
PubMed1993

An in vivo and in vitro trial of aclarubicin in metastatic breast cancer: a novel approach to the study of analogs.

Aclarubicin is an anthracycline antibiotic that differs from doxorubicin in its structure, mechanism of action, and preclinical toxicity profile, especially its reduced cardiotoxicity. We therefore conducted a side-by-side in vivo and in vitro trial of this agent in metastatic breast-cancer patients and their biopsied tumor specimens, respectively. Aclarubicin (100 mg/m2) was given by intravenous infusion every 3 weeks to 22 patients with objectively measurable metastatic breast cancer, 15 of whom had not previously received doxorubicin. The dose-limiting toxicity consisted primarily of leukopenia and severe nausea and vomiting. No objective response was observed in the 19 evaluable patients. After disease progression, 10 of the 15 doxorubicin-naive patients were treated with doxorubicin; 6 patients achieved a partial response, including 4 who responded to doxorubicin alone and 2 who responded to doxorubicin in combination with thiotepa and vinblastine. Tumor specimens were obtained from 14 of the 22 patients prior to the start of therapy and were tested for in vitro sensitivity to aclarubicin and doxorubicin using a soft agar colony-forming assay. Adequate colony growth occurred in 9 of 14 cultured tumor specimens. All 9 specimens, including 3 obtained from doxorubicin-naive patients, demonstrated in vitro resistance to aclarubicin. In all, 1 of 3 specimens taken from doxorubicin-naive patients demonstrated in vitro sensitivity to doxorubicin, whereas 6 tumor specimens obtained from patients who had undergone prior doxorubicin therapy demonstrated in vitro resistance. The patient whose tumor demonstrated in vitro doxorubicin sensitivity responded to a doxorubicin regimen after failing aclarubicin treatment; in vitro doxorubicin resistance correlated with clinical resistance in all cases. We conclude that aclarubicin is inactive in metastatic breast cancer at the dose and schedule used. Side-by-side in vivo and in vitro trials are feasible and could be useful in the development of investigational agents with activity greater than that of aclarubicin and, particularly, in the evaluation of analogs of clinically active drugs.

Aclarubicin↗
PubMed1993

Origin of intracellular Ca2+ elevation induced by in vitro ischemia-like condition in hippocampal slices.

Microfluorometry was used to investigate the origin of intracellular Ca2+ ([Ca2+]i) elevation in field CA1 of gerbil hippocampal slices perfused with a glucose-free physiological medium equilibrated with a 95% N2/5% CO2 gas mixture (standard in vitro ischemia-like condition). Large [Ca2+]i elevation was detected about 4 min after the beginning of standard in vitro ischemia-like condition, which was accompanied with a negative shift of extracellular DC potential. When slices were perfused with Ca(2+)-free in vitro ischemia-like medium, large [Ca2+]i elevation was observed about 3.5 min after the beginning of Ca(2+)-free in vitro ischemia-like condition, however, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the standard in vitro ischemia-like medium that contained Ca2+. When slices were perfused with the Ca(2+)-free in vitro ischemia-like medium that contained dantrolene (50 microM) which is known to prevent Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the Ca(2+)-free in vitro ischemia-like medium that did not contain dantrolene. These results indicate that large [Ca2+]i elevation induced by in vitro ischemia-like condition in field CA1 of the hippocampus was caused by both Ca2+ influx from extracellular space and Ca2+ release from intracellular Ca2+ stores, and that a part of the Ca2+ release was due to Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores.

Animals↗
PubMed1985

In vitro synthesis of rat brain hexokinase.

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)+ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipitation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical 'soluble' enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.

Animals↗
PubMed1993

In vitro tests for teratogens: desirable endpoints, test batteries and current status of the micromass teratogen test.

Information from in vitro tests can be usefully used as a component of the risk/hazard assessment process. In vivo studies will be required to confirm the in vitro data. If the in vitro test system is designed around endpoints that reflect changes following in vivo toxic insult then it may be possible to modify the in vitro system to account for some of the discrepancies observed between in vivo and in vitro outcomes. When the discrepancy can be accounted for by low bioavailability in vivo, pharmacokinetic studies may be required to determine the relevance of the in vitro toxic concentrations. Reproductive hazard, especially teratogenicity, has been the subject of intensive in vitro test development. The observation of teratogenicity may affect the development of new products more significantly than any other type or category of reproductive toxicity. The micromass test, involving culture of differentiating rat embryo limb and midbrain cells exposed to test agents, may be useful as part of a battery of in vitro tests for teratogens. The most recent protocol for the micromass test is described, followed by a summary of validation and mechanistic studies confirming its usefulness. The test is robust in its transfer to new laboratories. Interlaboratory variability is small.

Abnormalities, Drug-Induced↗
PubMed2004

Post-warming hatching and birth of live calves following transfer of in vitro-derived vitrified water buffalo (Bubalus bubalis) embryos.

Viability of in vitro-derived vitrified-warmed preimplantation stage buffalo embryos were assessed in vitro and in vivo. Oocytes were collected from ovaries of slaughtered riverine buffaloes, matured and fertilized in vitro with frozen semen from riverine buffalo bull and cultured on cumulus cell monolayers. Resultant preimplantation stage embryos were cryopreserved by vitrification with ethylene glycol, ficoll and sucrose. Seventy-one frozen embryos were warmed in 0.5M sucrose and were further cultured in vitro for 72 h to assess hatching rate. On the other hand, 95 embryos were transferred non-surgically to riverine buffalo recipients to assess development competence in vivo through detection of pregnancy and birth of live calves. Hatching rate of 83.10% (59/71) was noted among embryos cultured in vitro. Pregnancy rate was 16.36% (9/55) while calving rate was 10.91% (6/55) after transfer of in vitro-derived vitrified-warmed embryos to recipient animals. Six healthy and normal calves with average birth weight of 38.75+/-3.55 kg were born from the transferred embryos. These results indicate the viability of vitrified in vitro-derived buffalo embryos and the potential application of in vitro embryo production and vitrification techniques for production and transport of buffalo embryos from germplasm-rich sources to guarantee genetic improvement in many parts of the world.

Animals↗
PubMed1992

Obstetric outcome of in vitro fertilization pregnancies compared with normally conceived pregnancies.

OBJECTIVE: To compare the obstetric outcome of in vitro fertilization pregnancies with normally conceived pregnancies. STUDY DESIGN: The obstetric outcome of in vitro fertilization pregnancies achieved in 763 British residents at two in vitro fertilization clinics resulting in the births of 961 babies were compared by means of the relative risk statistic with a control group of naturally conceived primiparous pregnancies matched by maternal age and multiplicity of pregnancy. RESULTS: Twenty-five percent of in vitro fertilization pregnancies were multiple pregnancies. The incidence of singleton term breech presentation was similar to that among controls. As compared with controls there was an increased incidence among in vitro fertilization pregnancies of vaginal bleeding and hypertension requiring hospitalization (p less than 0.001) and cesarean births (p less than 0.001) and, among in vitro fertilization singleton pregnancies, an increased incidence of intrauterine growth retardation (p less than 0.05), placenta previa (p less than 0.05), and preterm delivery (p less than 0.001). The congenital malformation, stillbirth, and perinatal mortality rates were comparable with maternal age-standardized national rates. CONCLUSIONS: Although the majority of in vitro fertilization pregnancies have a satisfactory obstetric outcome, there are a number of increased obstetric risks that may reflect the history of infertility, the relatively high incidence of poor obstetric history, and the lower threshold for obstetric intervention in in vitro fertilization patients.

Adult↗
PubMed2000

Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development.

Recent advances in molecular technology and in vitro production of bovine embryos have enabled studies of gene transcription in preimplantation embryos. On the basis of knowledge of the sequence of the selected gene, various modifications of Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) technology have been employed. Several lines of evidence in mouse and cattle indicate that expression patterns of genes from in vitro-produced embryos are not necessarily representative of those of in vivo embryos. An important gene that has been found to be expressed by in vivo-derived bovine blastocysts, but not in their in vitro-produced counterparts, is the Connexin43 gene that is crucial for maintenance of compaction. The bovine leukemia inhibitory factor (bLIF) and LIF-receptor-beta (LR-beta) genes were expressed by in vitro-produced embryos, but not in their in vivo counterparts. The heat shock protein gene 70.1 (Hsp 70.1) was upregulated by blastocysts produced in vitro compared to in vivo embryos, while the glucose transporter-1 mRNA (Glut-1) was downregulated by morulae produced in vitro as compared to in vivo-derived morulae. Furthermore, mRNA expression levels of a set of "marker genes" were shown to be affected by the presence or absence of serum in the culture medium. Most embryos grown under serum-free conditions had higher mRNA abundances than those cultured in serum-enriched medium. It is hypothesized that persistent alterations of the normal gene expression patterns may be responsible for the large offspring syndrome that is observed in approximately one third of the calves resulting from the transfer of in vitro-produced embryos. A primary candidate for such deviations may be an altered methylation pattern that can either lead to silencing or induction of a specific gene. Messenger RNA phenotyping of genes essential in early development provides a useful tool to assess the normality of the produced embryos and a tool to optimize in vitro culture conditions for bovine embryos.

Animals↗
PubMed2003

Utility of in vitro closure time test for evaluating platelet-related primary hemostasis in dialysis patients.

BACKGROUND: The platelet aggregation and skin bleeding time (SBT) tests currently used for assessment of hemostasis impairment in dialysis patients have important disadvantages. The authors explored the utility of a novel in vitro closure time test (PFA-100, platelet function analyzer) in which the process of platelet adhesion and aggregation after vascular injury is simulated in vitro in dialysis patients. METHODS: Thirty-four long-term dialysis patients were included in the study with 30 healthy volunteers as the control group. In vitro closure time was compared with results from the platelet aggregation and SBT tests. RESULTS: In vitro closure time identified more patients and fewer controls with hemostasis impairment. In the patient group, 60%, 40%, and 20%, and in the control group, 0%, 10% and 3% of persons were found to have hemostasis impairment as determined by in vitro closure time, platelet aggregation, and SBT, respectively. In addition, values for patients and controls were significantly different for in vitro closure time (P < 0.05) but not for platelet aggregation or SBT. Thus, closure time appears to be more sensitive and specific than the other 2 tests. No correlation was found between the 3 tests, either in patients or in controls. However, a high correlation (r = 0.73; P < 0.0001) was found between the 2 types of in vitro closure time test (collagen/epinephrine [CEPI] and collagen/adenosine diphosphate [CADP]) in patients and controls. CONCLUSION: These results indicate that in vitro closure time can be a useful test for detecting platelet-related primary hemostasis defects in dialysis patients.

Aged↗
PubMed2003

The contribution of the male to ovine embryogenesis in an in vitro embryo production system.

The differences in the embryo production potential of four rams used in a commercial embryo transfer program were examined in both in vivo and in vitro embryo production systems. Processing frozen-thawed spermatozoa through Percoll density gradients prior to in vitro insemination eliminated differences in the estimates of sperm viability between the four rams, and yet, differences in embryo production persisted throughout the in vitro culture period. However, there was no effect of ejaculate within ram on embryo production rates. In addition, the timing of the onset of the differences between the rams at each stage of in vitro embryo development were revealed. Ram 2 differed from ram 4 in the proportion of fertilized oocytes at 17 h post-insemination (pi) and by 52 h, ram 3 differed from ram 4 in the proportion of cleaved embryos, and the observed differences between ram 1 and ram 2 in their blastocyst production were initiated prior to activation of the embryonic genome. Once differences in embryo development rates were detected among the four rams, they persisted throughout the in vitro culture period. The reduced in vitro fertilization (IVF) rates from ram 2 compared with the other rams was paralleled in vivo by the significantly lower proportion of embryos recovered from ewes mated to ram 2, and this was further exacerbated by a significantly lower embryo survival rate after transfer. However, the subtle differences observed in the timing of the contribution of each sire to embryo development during in vitro culture were not able to be detected in vivo. However, the higher proportions of transferable quality blastocysts obtained from ewes mated to ram 4 did not result in increased embryo survival throughout the remainder of gestation. Therefore, in this study, the blastocyst production potential for a particular sire, either in vitro or in vivo, does not necessarily reflect the potential for the production of live offspring.

Animals↗
PubMedDate not supplied

Lessons learned from validation of in vitro toxicity test: from failure to acceptance into regulatory practice.

As no scientific approach or regulatory guidelines existed for the experimental validation of in vitro toxicity tests, in 1990 a US/European validation workshop agreed in Amden (Switzerland) on a simple definition of the validation process. Several international validation studies failed, although they were conducted according to these recommendations. Taking into account the lessons learned from this experience, a second validation workshop was held by ECVAM in Amden in 1994 to develop a more precisely defined validation concept. Prevalidation and the development of biostatistically defined prediction models were added as essential elements to the validation process. In 1995/1996 the ECVAM validation procedure was officially accepted by EU member countries and at the international level by the US regulatory agencies and the OECD. The improved validation concept was immediately introduced into ongoing validation studies. In 1996 the ECVAM/COLIPA validation study of the in vitro phototoxicity test, which was conducted according to the ECVAM/OECD validation concept, was finished successfully and in 1998 a supporting study on UV-filter chemicals was undertaken. In 1998 the 3T3 NRU PT in vitro phototoxicity test was the first experimentally validated in vitro toxicity test that was recommended for regulatory purposes by ESAC, the ECVAM Scientific Advisory Committee, and by the DG ENV of the EU Commission. Meanwhile, two in vitro skin corrosivity tests have successfully been validated by ECVAM. Finally, in June 2000 the three experimentally validated tests were accepted by EU member states for regulatory purposes as the first in vitro toxicity tests. In addition, ECVAM has funded a successful validation study of three in vitro embryotoxicity tests, which was conducted in 12 European laboratories and finished in July 2000. The three tests validated in this study were the whole embryo culture (WEC) test applied to rat embryos, the micromass (MM) test employing primary cultures of dissociated mouse limb bud cells and the mouse embryonic stem cell test (EST). Examples will be given of successful validation studies during the past decade with particular reference to in vitro toxicity tests that were evaluated for regulatory purposes either by the US validation centre ICCVAM or ECVAM in the fields of sensitisation, phototoxicity and embryotoxicity

Animal Testing Alternatives↗
PubMedDate not supplied

Present and future in vitro approaches for drug metabolism.

The 1980s through 1990s witnessed the widespread incorporation of in vitro absorption, distribution, metabolism, and excretion (ADME) approaches into drug development by drug companies. This has been exemplified by the integration of the basic science of cytochrome P450s (CYPs) into most drug metabolism departments so that information on the metabolic pathways of drugs and drug-drug interactions (DDIs) is no longer an academic exercise, but essential for regulatory submission. This has come about due to the application of a variety of new technologies and in vitro models. For example, subcellular fractions have been widely used in metabolism studies since the 1960s. The last two decades has seen the increased use of hepatocytes as the reproducibility of cell isolations improved. The 1990s saw the rejuvenation of liver slices (as new slicers were developed) and the utilization of cDNA expressed enzymes as these technologies matured. In addition, there has been considerable interest in extrapolating in vitro data to in vivo for parameters such as absorption, clearance and DDIs. The current philosophy of drug development is moving to a 'fail early--fail cheaply' paradigm. Therefore, in vitro ADME approaches are being applied to drug candidates earlier in development since they are essential for identifying compounds likely to present ADME challenges in the latter stages of drug development. These in vitro tools are also being used earlier in lead optimization biology, in parallel with approaches for optimizing target structure activity relationships, as well as identification of DDI and the involvement of metabolic pathways that demonstrate genetic polymorphisms. This would suggest that the line between discovery and development drug metabolism has blurred. In vitro approaches to ADME are increasingly being linked with high-throughput automation and analysis. Further, if we think of perhaps the fastest available way to screen for successful drugs with optimal ADME characteristics, then we arrive at predictive computational algorithms, which are only now being generated and validated in parallel with in vitro and in vivo methods. In addition, as we increase the number of ADME parameters determined early, the overall amount of data generated for both discovery and development will increase. This will present challenges for the efficient and fast interpretation of such data, as well as incorporation and communication to chemistry, biology, and clinical colleagues. This review will focus on and assess the nature of present in vitro metabolism approaches and indicate how they are likely to develop in the future.

Animals↗
PubMed1997

Static elastic wall properties of the abdominal porcine aorta in vitro and in vivo.

OBJECTIVES: To characterise possible differences in vascular mechanics between in vitro and in vivo settings. DESIGN: Experimental animal study. MATERIAL: Segments of abdominal aorta from five 30kg pigs were studied in vitro and five porcine abdominal aortas were investigated in vivo. METHODS: Vessel luminal cross-sectional area (CSA) was assessed in vitro and in vivo by measuring the electrical impedance of fluid inside a distensible balloon mounted on a catheter. Wall thickness was measured in vitro by means of 20 MHz A-mode ultrasound. The obtained values in vitro were used to calculate wall thickness in vivo. RESULTS: The CSAs were larger in vitro than in vivo (p < 0.05). All segments showed stress-strain relations that accorded well with the exponential equation: Stress = a.exp(b. Strain). The y-axis intercepts (a) and the slope of the curves (b) were different in vitro and in vivo (p < 0.05). Differences were found between the aorta in vivo and in vitro regarding the circumferential stress-strain relations, i.e. the stiffest abdominal aorta was found in vivo. CONCLUSIONS: The differences in dimensions and wall stiffness may be attributed to the tethering to the surrounding tissues in vivo. Impedance planimetry combined with high frequency ultrasound is an accurate way of measuring segmental vascular elasticity in vivo.

Animals↗
PubMed1999

Assessing the mechanism of metabolism-dependent valproic acid-induced in vitro cytotoxicity.

This study was designed to distinguish and evaluate the contribution of reactive metabolite and reactive oxygen species as the mechanism of metabolism-dependent valproic acid-induced in vitro cytotoxicity. The involvement of reactive oxygen species in the mechanism of in vitro cytotoxicity was examined by the addition of a series of antioxidant enzymes and iron chelators to the reaction mixture. Addition of catalase to the reaction mixture resulted in a complete prevention of valproic acid-induced cytotoxicity. Co-incubation of a cell impermeable iron chelator deferoxamine did not effect cytotoxicity, whereas 1,10-phenanthroline, a chelator with the ability to traverse cell membranes at low concentrations, afforded significant protection against valproic acid-induced cytotoxicity. A possible inhibitory effect of catalase and 1,10-phenanthroline on the microsomal metabolism of valproic acid was disproved by the quantification of valproic acid metabolites in the presence and absence of these compounds. To assess the specificity of the mechanism of in vitro valproic acid-induced cytotoxicity, prevention of in vitro acetaminophen-induced cytotoxicity by antioxidant enzymes and iron chelators was also evaluated. Addition of catalase to the reaction mixture in the presence of acetaminophen resulted in a moderate reduction in the level of but a lack of complete protection of cytotoxicity. Addition of 1,10-phenanthroline to the reaction mixture in the presence of acetaminophen did not result in a detectable change in acetaminophen-induced cytotoxicity. These data suggest the involvement of reactive oxygen species in the mechanism of toxicity of valproic acid and perhaps reactive metabolites as the major cause of cytotoxicity in the case of acetaminophen in the in vitro model investigated. Inhibition of poly(ADP-ribose) polymerase activity by various antagonists resulted in complete prevention of valproic acid-induced in vitro cytotoxicity. The cytoprotective effects of known poly(ADP-ribose) polymerase antagonists implicate poly(ADP-ribose) polymerase in the mechanism of in vitro metabolism-dependent valproic acid-induced cytotoxicity under these conditions. These results further point to nuclear DNA as the intracellular site of insult by the generated oxygen radicals. Overall, the data obtained support the hypothesis that the metabolism-dependent valproic acid-induced in vitro cytotoxicity is the result of generation of hydrogen peroxide in the medium that can readily cross cell membranes and subsequently interact intracellularly with iron to produce the highly reactive hydroxyl free radicals.

Acetaminophen↗
PubMed2000

Drug distribution in human skin using two different in vitro test systems: comparison with in vivo data.

PURPOSE: Two in vitro test systems used to study drug penetration into human skin--the Franz diffusion cell (FD-C) and the Saarbruecken penetration model (SB-M)--were evaluated, and the results were compared with data gained under analogous in vivo conditions. METHODS: Excised human skin was used in all in vitro experiments. Flufenamic acid dissolved in wool alcohols ointment, was chosen as a model drug, and the preparation was applied using 'infinite dose' conditions. To acquire quantitative information about the drug penetration, the skin was segmented into surface parallel sections at the end of each experiment, first by tape stripping the stratum corneum (SC), and second by cutting the deeper skin layers with a cryomicrotome. The flufenamic acid was extracted from each sample and assayed by high performance liquid chromatography (HPLC). For in vivo experiments, only the tape stripping technique was used. RESULTS: a) Drug penetration into the SC: In both in vitro test systems the total drug amounts detected in the SC were found to increase over the different incubation times. Similar conditions were obtained in vivo, but on a lower level. Using Michaelis-Menten kinetics, the m(max) value was calculated for the skin of two donors. The relations of the m(max) values for the FD-C and the SB-M closely correspond (1.26 [donor 1] and 1.29 [donor 2]). A direct linear correlation of the drug amount in the SC and the time data were found for in vivo with both in vitro test systems. b) Drug penetration into the deeper skin layers: The detected drug amounts in the deeper skin layers continuously increased with the incubation time in the SB-M, while in the FD-C, only very small drug amounts were observed after incubation times of 30 and 60 minutes. It was also noticed, that the drug amounts rose steeply at time points 3 and 6 hours. Additional studies showed a remarkable penetration of water into the skin from the basolateral acceptor compartment in the FD-C. This could explain the different drug transport into the deeper skin layers between the two in vitro test systems. CONCLUSIONS: Both in vitro models showed comparable results for the drug penetration into the SC and a robust correlation with in vitro data. Different results were obtained for the deeper skin layers. Whether a correlation between in vitro and in vivo data is also possible here has to be investigated by further experiments.

Administration, Topical↗
PubMedDate not supplied

Current progress on assisted reproduction in dogs and cats: in vitro embryo production.

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.

Animals↗