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O P Flint

Publications and source records attributed to O P Flint.

At least 19 recordsLinked to original sources
PubMed1998

Workshop overview: scientific and regulatory challenges for the reduction, refinement, and replacement of animals in toxicity testing.

Public concern for animal welfare has been expressed through legislative control of animal use for experimental purposes since the first legislation was introduced in 1876 in the United Kingdom. Legislative control of animal use has been introduced in virtually every developed country, with major initiatives in Europe (1986) and the United States (1966 and 1985). Advances in scientific thinking resulted in the development of the concept of the three Rs--refinement, reduction, and replacement--by Russell and Burch in 1959. The field has expanded substantially since, with specialist scientific journals dedicated to alternatives, World Congresses organized to discuss the scientific and philosophical issues, and European and U.S. validation organizations being launched. Current scientific attention is focused on validation of alternative methods. The underlying scientific principles of chemical toxicity are complicated and insufficiently understood for alternative methods for all toxicity endpoints of importance in protecting human health to be available. Important lessons have been learned about how to validate methods, including the need to have prediction models available before the validation is undertaken, the need to understand the variability of the animal-based data which is to be used as the validation standard, and the need to have well-managed validation programs. Future progress will depend on the development of novel methods, which can now be validated through international collaborative efforts.

Animal Testing Alternatives↗
PubMed1997

Inhibition of cholesterol synthesis by squalene synthase inhibitors does not induce myotoxicity in vitro.

The cholesterol-lowering HMG CoA reductase inhibitors (HMGRI), pravastatin and lovastatin, have been associated with skeletal myopathy in humans and in rats. In a previous in vitro study, HMGRI-induced changes in neonatal rat skeletal muscle cells were characterized by reversible inhibition of protein synthesis and loss of differentiated myotubes at concentrations markedly lower than those inducing enzyme leakage. Myotoxicity was determined to be directly related to inhibition of HMG CoA reductase, since mevalonate, the immediate product of HMG CoA reductase metabolism, abrogated the drug-induced changes. Farnesol, geranylgeraniol, and squalene are metabolites of mevalonate. Squalene, formed from farnesol by squalene synthase, is the first metabolite solely committed to cholesterol synthesis. In contrast, geranylgeraniol, formed by the addition of an isoprene group to farnesol, is the first metabolite uncommitted to cholesterol synthesis. The objective of the present study was to determine the role of inhibition of cholesterol synthesis in HMGRI-induced in vitro myotoxicity. HMGRI-treated neonatal rat skeletal muscle cultures were supplemented with farnesol and geranylgeraniol, and in another study, muscle cultures were exposed to two squalene synthase inhibitors (SSI), BMS-187745 and its prodrug ester, BMS-188494. Endpoints evaluated for both studies included protein synthesis ([3H]leucine incorporation), total cellular protein (a measure of cell loss), intra- and extracellular lactate dehydrogenase activity (a measure of membrane integrity), cholesterol biosynthesis ([14C]acetate incorporation), and morphology. HMG CoA reductase inhibitor-induced morphologic changes and inhibition of protein synthesis were significantly ameliorated by supplementation with farnesol and geranylgeraniol. In contrast to HMGRI-induced in vitro myotoxicity, SSI induced an irreversible, minimal cytotoxicity at close to maximum soluble concentrations. These results indicate that depletion of metabolites of geranylgeranyl pyrophosphate, and not inhibition of cholesterol synthesis, is the primary cause of HMG CoA reductase-induced myotoxicity.

Analysis of Variance↗
PubMed1997

HMG CoA reductase inhibitor-induced myotoxicity: pravastatin and lovastatin inhibit the geranylgeranylation of low-molecular-weight proteins in neonatal rat muscle cell culture.

In previous studies, inhibition of cholesterol synthesis by HMG CoA reductase inhibitors (HMGRI) was associated with myotoxicity in cultures of neonatal rat skeletal myotubes, and rhabdomyolysis in rats, rabbits, and humans in vivo. In vitro myotoxicity was directly related to HMGRI-induced depletion of mevalonate, farnesol, and geranylgeraniol, since supplementation with these intermediate metabolites abrogated the toxicity. Both farnesol and geranylgeraniol are required for the posttranslational modification, or isoprenylation, of essential regulatory proteins in mammalian cells. The objective of the present study was to measure changes in protein isoprenylation in cultured neonatal rat skeletal muscle cells exposed for 24 hr to increasing concentrations of pravastatin or lovastatin. Proteins were labeled with [3H]mevalonate, [3H]farnesyl pyrophosphate (FPP), or [3H]geranylgeranyl pyrophosphate (GGPP), and then separated by SDS-PAGE and quantitated by scintillation counting and densitometry of autoradiographs. Mevalonate and FPP labeling of the majority of proteins increased in a concentration-dependent manner, even at concentrations greater than 2 microM lovastatin and 25 microM pravastatin that completely inhibited cholesterol synthesis. In contrast, mevalonate and FPP labeling of three protein bands with molecular weights of 26.6, 27.7, and 28.9 kDa was markedly inhibited at concentrations higher than 1 microM lovastatin and 400 microM pravastatin, which inhibited protein synthesis and disrupted myotube morphology after longer exposures in a previous study. In contrast, these proteins were equally well labeled by GGPP at all HMGRI concentrations tested, suggesting that isoprenylation of the 26.9-, 27.8-, and 28.9-kDa proteins requires geranylgeraniol. The results of this study indicate that HMGRI-induced myotoxicity is most likely related to reduced posttranslational modification of specific regulatory proteins by geranylgeraniol.

Animals↗
PubMed1995

In vitro myotoxicity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, pravastatin, lovastatin, and simvastatin, using neonatal rat skeletal myocytes.

Pravastatin, lovastatin, and simvastatin, drugs which lower cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, have been linked to skeletal myopathies in humans and rats. The myotoxicity of these three drugs was compared, after 48 hr exposure, in cultures of primary neonatal rat skeletal myotubes. Measurements included HMG CoA reductase activity ([14C]acetate incorporation into cholesterol), indicators of membrane damage (CPK, LDH, and AST), cell viability (mitochondrial dehydrogenase metabolism of MTT), protein synthesis ([3H]leucine incorporation), and energy status (ATP). All three drugs inhibited cholesterol synthesis to the same extent in rat hepatocytes (IC50s approximately 0.07 microM). Lovastatin- and simvastatin-induced inhibition of cholesterol synthesis in myotubes was unchanged compared to that of hepatocytes, but pravastatin was 85-fold less potent (IC50 = 5.9 microM). Protein synthesis and ATP levels were the most sensitive indicators of toxicity. Pravastatin (IC50 = 759 microM) was > 100-fold less inhibitory of protein synthesis than lovastatin (IC50 = 5.4 microM) or simvastatin (IC50 = 1.9 microM). Addition of mevalonic acid (the immediate product of the HMG CoA reductase reaction), as 100 microM mevalonic acid lactone, reversed the toxicity of all three drugs. Removal of serum for 24-72 hr did not alter the toxicity of any of the drugs compared to cultures containing 10% serum, suggesting that differences in protein binding did not account for the differences in toxicity of the drugs. These results indicate that pravastatin is less myotoxic than lovastatin or simvastatin in this in vitro system using neonatal rat skeletal muscle cells, and this differential toxicity is correlated with the selective decrease in inhibition of HMG CoA reductase by pravastatin in nonhepatic tissues.

Animals↗
PubMed1995

Analysis of the steady-state dynamics of organelle motion in cultured neurites: putative indicator of neurotoxic effect.

1. The objective of this study was to develop a physiologically based method to evaluate the neurotoxic potential of drug candidates in vitro. Rat embryo midbrain cells were grown in micromass culture, and the movement of mitochondria labelled with the fluorescent dye rhodamine 123 was quantified in fasciculated neurites, using a laser cytometer. 2. The rhodamine 123 signal in a defined region of fascicle was quantified and photobleached with the laser. A series of post-photobleach scans revealed the movement of fluorescent-labelled mitochondria into the bleached region from adjacent unbleached regions. Recovery of fluorescence is a measure of the size of the mobile pool of mitochondria relative to the total (moving plus stationary) pool. 3. The steady-state levels of fluorescence recovery was dependent on intracellular calcium and magnesium concentrations, energy status (ATP), and microtubule integrity (post taxol or vinblastine treatment). 4. This technique may be a useful indicator of neurotoxic effect.

Adenosine Triphosphate↗
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↗
PubMed1989

Teratogenicity of phenylhydantoins in an in vitro system: molecular orbital-generated quantitative structure-toxicity relationships.

1. The ability of 20 mono- and di-phenylhydantoin derivatives to inhibit differentiation of rat embryo mid-brain and limb bud cells in culture has been used as an index of the teratogenic hazard represented by these compounds. 2. Molecular orbital calculations on these compounds, using the MINDO-3 (modified intermediate neglect of differential overlap) and CNDO-2 (complete neglect of differential overlap) methods, were combined with indices of teratogenicity in the two cell types, to generate a coherent structure-toxicity relationship. 3. Teratogenicity correlated with frontier orbital electron density of the N1 hydantoin ring atom (HOMO-N1) in a sub-series of 12 monophenylhydantoins, whereas the corresponding toxicity for both mono- and di-phenylhydantoins related more to the molecular polarizability (alpha mol) of the molecule. 4. Furthermore the same structural parameter (alpha mol) exhibited a parallelism with log P values of these 20 compounds, indicating the importance of lipophilicity in the toxicity of these compounds. 5. Overall, the data emphasize the ability of electronic structural calculations to identify chemical descriptors of toxicity.

Animals↗
PubMed1988

In vitro toxicology: a commercial proposition?

1. The commercial value of any test system must be equated with its scientific value. In vitro toxicity tests only have scientific value when they are accurate models of toxicity in vivo. 2. Toxicity is a complex event so that the simple tests of cell viability are unlikely to be useful models as these tests often use cell lines which are functionally remote from cells in tissues of the whole animal. 3. Primary cultures retain their differentiated function for hours (hepatocytes) or days (nerve cells) and may acquire differentiated functions in vitro (embryo cells) and are the models of choice for predicting toxic hazard. 4. When the in vitro test is a satisfactory predictive model of toxicity there are sound commercial reasons for its use. It saves time and effort and can be used at an early stage in compound development to sort out the toxic from the non-toxic candidate compounds. Large numbers of structurally similar compounds can be tested (QSAR studies) and a greater understanding of the mechanism of toxic action can often be achieved than in vivo because of the numbers of animals required and the interplay of different organ systems in the toxic events that make interpretation difficult. Thus the in vitro test can not only save animals but can also save compounds.

Animals↗
PubMed1984

Differentation of rat embryo cells in culture: response following acute maternal exposure to teratogens and non-teratogens.

An in vivo-in vitro test system with high sensitivity to teratogens has been developed and validated. A single acute intra-peritoneal injection of teratogens (18) and non-teratogens (13) was administered to pregnant rats on the 12th day after fertilisation, and uteri were removed after 16 h by laparotomy. 34-36 Embryos somites were selected, and mid-brain (CNS) and fore-limb buds (LB) were dissected free and dispersed as single-cell suspensions in Ham's F12 culture medium. The cells were cultured as micromass cell islands for 5 days, and discrete foci of neuronal cells differentiated in CNS cultures and chondrocytes in LB cultures. After 5 days, differentiation as determined by number of stainable foci of differentiated cells and 3H-GABA incorporation in CNS or 35SO4 incorporation in LB and growth (as determined by total protein) were measured. Both differentiation and growth of CNS and LB cultures were markedly reduced following exposure of the dam to teratogens, whereas no significant effect was observed with non-teratogens. One teratogen (amaranth) and one non-teratogen (nitrilotriacetic acid) were classified as false negative and positive, respectively; the sensitivity of the test (proportion of teratogens correct) was therefore 92% and the specificity (proportion of non-teratogens correct) was 94%. Inhibition of growth and differentiation in the rat embryo cell cultures following maternal exposure forms the basis of a short-term in vitro test for teratogens.

Animals↗
PubMed1984

An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells.

A short-term in vitro assay for teratogens has been evaluated and shown to have a high predictability (greater than 90%). Cultured cells derived from midbrain (CNS) and limb buds (LB) of 34 to 36 somite rat embryos were exposed to 46 compounds (27 teratogens, 19 nonteratogens) in a blind trial. Rat liver post mitochondrial supernatant fraction plus cofactors were included in the cultures to provide metabolizing enzymes. Differentiation of neurons from CNS cells and chondrocytes from LB cells was measured after 5 days of culture. Inhibition of differentiation (assessed by reduction of number of foci) was the indicator of potential teratogenicity. Variation between experiments was limited. In repeat experiments with two direct-acting teratogens, aldrin and diphenylhyantoin, interexperiment variability was low (coefficient of variation; range 7 to 24%). Of 27 teratogens 25 (93%) and only 2 of 19 nonteratogens (11%) inhibited differentiation (CNS or LB). Inhibition of differentiation in one cell type alone was less predictive (CNS: 85%, LB: 82%).

Animals↗
PubMed1984

Myogenic cell movement in the developing avian limb bud in presence and absence of the apical ectodermal ridge (AER).

Fragments of quail wing bud containing myogenic cells of somitic origin and fragments of quail sphlanchopleural tissue were introduced into the interior of the wing bud of fowl embryo hosts. No movement of graft into host tissue occurred in the control, but myogenic cells from the quail wing bud fragments underwent long migrations in an apical direction to become incorporated in the developing musculature of the host. When the apical ectodermal ridge (AER), together with some subridge mesenchyme, was removed at the time of grafting, no such cell migration occurred. The capacity of grafted myogenic cells to migrate in the presence of AER persists to H.H. stage 25, when myogenesis has begun, but premyogenic cells in the somites, which normally migrate out into the early limb bud, do not migrate when somite fragments are grafted into the wing bud. Coelomic grafts of apical and proximal wing fragments showed that apical sections of quail wing buds become invaded by myogenic cells of the host, but grafts from proximal wing bud regions do not.

Animals↗
PubMed1983

A micromass culture method for rat embryonic neural cells.

A method of culturing early (13-day) rat embryo neural cells is described. Undifferentiated neural epithelium is disaggregated and cultured in small discrete islands. Cells that destined to differentiate as neurons actively segregate from the other cells in the island and aggregate together into small clumps. Other cells flatten and attach to the substrate and resemble typical fibroblasts throughout the culture period. The clumps of preneuron cells spread out forming large irregular foci. Spreading is mediated by active cell movements. Cells in the foci differentiate as a pure population of neurons identifiable by specific inhibition of 3H-labelled gamma-amino butyric acid incorporation or by labelling with a monoclonal antibody to GQ-ganglioside. The ganglioside is not found on the cell surface at the start of culture after trypsinization, but emerges during the 5 days of culture. The antigen is similarly not present in the embryonic mesencephalon in vivo at 13 days post coitum, only emerging later in the differentiated midbrain. There is thus an apparent de novo synthesis, which is paralleled in vivo and in vitro.

Animals↗
PubMed1982

Cell interactions in the developing somite: in vitro comparisons between amputated (am/am) and normal mouse embryos.

Facial, axial and limb development are all abnormal in the homozygous mutant mouse embryo (amputated). An interpretation of cell behaviour in vivo based on sectioned material which may explain these abnormalities has been previously suggested. In this study, somite cells cultured in vitro were found to behave exactly as predicted in this interpretation: they clump together, forming extensive areas of cell contact, and this has a profound effect on their mobility as measured by time-lapse cinemicrography. The similarity of cell behaviour in vitro and in vivo under two distinct sets of environmental conditions suggests that the abnormal cell behaviour is intrinsic to the cell, and directly linked to the mutation. The more extensive areas of cell contact formed between mutant cells suggests that the mutation changes the adhesive properties of the cell surface, but it cannot be excluded that the cells' motile apparatus is also affected.

Animals↗
PubMed1980

Cell behaviour and cleft palate in the mutant mouse, amputated.

Cleft palate with a genetic origin normally arises because of a failure of the palatal shelves to elevate or fuse. Until now attention in studies of palatal development development has been focused on two critical phases, those of elevation and fusion. In the mutant mouse, amputated, however, cleft palate arise because of a failure of the palatal shelves to make any significant outgrowth between the 12th day after conception when the palatal shelves are first observed and the 14th day when elevation and fusion normally occur. When cell proliferation (mitotic index) was measured in the palatal shelves on days 12, 13 and 14 no difference was found between mutant and normal. The failure of the mutant palate to grow cannot be accounted for on grounds of reduced cell proliferation. For this reason the palatal mesenchyme in 12.5-day and 14.5-day normal and amputated mice has been studied wit the scanning electron microscope. This work shows that the mesenchymal cells in the mutant palate are clumped together and have much greater areas of cell contact than are observed in the normal palate. The abnormal cell behaviour described in mutant palatal mesenchyme is typical of amputated embryonic mesenchyme in general, and in other cases has been shown to cause abnormal morphogenesis. We propose that aberrant cell behaviour causing an aggregation through increased cell adhesion inhibits palatal outgrowth in the mutant, and for this reason the palatal shelves subsequently fail to elevate and fuse.

Animals↗