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- Vicente Carrillo, Alejandro, et al.
(author)
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Boar spermatozoa successfully predict mitochondrial modes of toxicity: Implications for drug toxicity testing and the 3R principles
- 2015
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In: Toxicology in Vitro. - : Elsevier. - 0887-2333 .- 1879-3177. ; 29:3, s. 582-591
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Journal article (peer-reviewed)abstract
- Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker and JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P less than 0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r= 0.559) and proportions of motile (r = 0.55) or progressively motile (r = 0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.
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- Boerma, M, et al.
(author)
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A genetic polymorphism in connexin 37 as a prognostic marker for atherosclerotic plaque development
- 1999
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In: Journal of Internal Medicine. - : Wiley. - 1365-2796 .- 0954-6820. ; 246:2, s. 211-218
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Journal article (peer-reviewed)abstract
- BACKGROUND AND OBJECTIVES: Atherosclerosis is a multifactorial disease, in part characterized by chronic inflammatory changes in the vessel wall and loss of normal physical and biochemical interactions between endothelial cells and smooth muscle cells. Previous studies [Hu J., Cotgreave IA. J Clin Invest; 99: 1-5] have provided molecular links between inflammation and myoendothelial communication via gap junctions, suggesting that these structures may be important in the development of the atherosclerotic vessel phenotype. In order to strengthen this premise, the aim of the present work was to probe for structural polymorphisms in connexin 37, a gap junctional protein uniquely expressed in endothelial cells, and to assess for potential genotypic segregation in individuals displaying atherosclerotic plaque. METHODS AND RESULTS: Computer-based comparisons of Expressed Sequence Tags (ESTs) predicted a polymorphism in the human gap junctional protein connexin 37 (cx37). The C1019-T mutation results in a proline to serine shift at codon 319 (cx37*1-cx37*2). A Restriction Fragment Length Polymorphism (RFLP) assay, involving the insertion of a novel Drd I cleavage site in the proline variant revealed a statistically significant over-representation of the cx37*1 allele in association with atherosclerotic plaque-bearing individuals (Odds-ratio for the homozygote = 2.38, Chi2 = 7.693, P = 0.006), in comparison to individuals lacking plaque, irrespective of a history of hypertension. CONCLUSIONS: These data suggest that the C1019-T polymorphism in cx37 may provide 'single gene marker', which could be useful in assessing atherosclerotic plaque development, particularly in cardiovascular risk groups such as those with borderline hypertension.
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- Burgess, D H, et al.
(author)
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Human skeletal muscle cytosols are refractory to cytochrome c-dependent activation of type-II caspases and lack APAF-1.
- 1999
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In: Cell Death and Differentiation. - : Springer Science and Business Media LLC. - 1350-9047 .- 1476-5403. ; 6:3, s. 256-61
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Journal article (peer-reviewed)abstract
- Apoptotic regulatory mechanisms in skeletal muscle have not been revealed. This is despite indications that remnant apoptotic events are detected following exercise, muscle injury and the progression of dystrophinopathies. The recent elicitation of a cytochrome c-mediated induction of caspases has led to speculation regarding a cytochrome c mechanism in muscle. We demonstrate that cytosols from skeletal muscle biopsies from healthy human volunteers lack the ability to activate type-II caspases by a cytochrome c-mediated pathway despite the confirmed presence of both procaspase-3 and -9. This was not due to the presence of an endogenous inhibitor, as the muscle cytosols enhanced caspase activity when added to a control cytosol, subsequently activated by cytochrome c and dATP. In addition, we demonstrate that muscle cytosols lack the apoptosis protease activator protein-1 (APAF-1), both at the protein and mRNA levels. These data indicate that human skeletal muscle cells will be refractory to mitochondrial-mediated events leading to apoptosis and thus can escape a major pro-apoptotic regulatory mechanism. This may reflect an evolutionary adaptation of cell survival in the presence of the profusion of mitochondria required for energy generation in motility.
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- Malmström, Jonas, et al.
(author)
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The antioxidant profile of 2,3-dihydrobenzo[b]furan-5-ol and its 1-thio, 1-seleno, and 1-telluro analogues
- 2001
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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 123:15, s. 3434-3440
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Journal article (peer-reviewed)abstract
- A novel synthesis of 2,3-dihydrobenzo[b]thiophene-S-of based on intramolecular homolytic substitution on sulfur was reported. The ''antioxidant profile" of the series of 2,3-dihydrobenzo[b] furan-5-ol (2a) its l-thio (2b), I-seleno (2c) and 1-telluro (2d) analogues was determined by studies of redox properties, the capacity to inhibit stimulated lipid peroxidation, the reactivity toward tert-butoxyl radicals, the ability to catalyze decomposition of hydrogen peroxide in the presence of glutathione, and the inhibiting effect on stimulated peroxidation in liver microsomes. The one-electron reduction potentials of the aroxyl radicals corresponding to compounds 2a-2d, E degrees (ArO./ArO-) were 0.49, 0.49, 0.49, and 0.52 V vs NHE, respectively, as determined by pulse radiolysis. With increasing chalcogen substitution the compounds become slightly more acidic (pK(a) 10.6, 10.0, 9.9, and 9.5, respectively, for compounds 2a-2d). By using Hess' law, the homolytic O-H bond dissociation enthalpies of compounds 2a-2d (340, 337, 336, and 337 kJ mol(-1), respectively) were calculated. The reduction potentials for the proton coupled oxidation of compounds 2a-2d (ArOH --> ArO. + H+) as determined by cyclic voltammetry in acetonitrile were 1.35 (irreversible), 1.35 (quasireversible) 1.13 (reversible), and 0.74 (reversible) V vs NHE, respectively. As judged by the inhibited rates of peroxidation, R-inh, in a water/chlorobenzene two-phase lipid peroxidation system containing N-acetylcysteine as a thiol-reducing agent in the aqueous phase, the antioxidant capacity increases (2d > 2c = 2b > 2a) as one traverses the group of chalcogens. Whereas the times of inhibition, T-inh were slightly reduced for the oxygen (2a) and sulfur (2b) derivatives in the absence of the thiol-reducing agent, they were drastically reduced for the selenium (2c) and tellurium (2d) derivatives. This seems to indicate that the organochalcogen compounds are continuously regenerated at the lipid aqueous interphase and that regeneration is much more efficient for the selenium and tellurium compounds. The absolute rate constants for the oxidation of compounds 2a-2b by the tert-butoxyl radical in acetonitrile/di-tert-butyl peroxide (10/1) were the same-2 x 10(8) M-1 s(-1). Whereas the oxygen, sulfur, and selenium derivatives 2a-2c were essentially void of any glutathione peroxidase-like activity, the organotellurium compound 2d accelerated the initial reduction of hydrogen peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide in the presence of glutathione 100, 333, and 213 times, respectively, as compared to the spontaneous reaction. Compounds 2a-2d were assessed for their capacity to inhibit lipid peroxidation in liver microsomes stimulated by Fe(II)/ADP/ascorbate. Whereas the oxygen, sulfur, and selenium compounds showed weak inhibiting activity (IC50 values of similar to 250, 25, and 13 muM, respectively), the organotellurium compound 2d was a potent inhibitor with an IC50 value of 0.13 muM.
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- Staab, CA, et al.
(author)
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Modelling of normal and premalignant oral tissue by using the immortalised cell line, SVpgC2a: a review of the value of the model
- 2004
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In: Alternatives to laboratory animals : ATLA. - : SAGE Publications. - 0261-1929 .- 2632-3559. ; 32:4, s. 401-405
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Journal article (peer-reviewed)abstract
- Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, non-tumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in long-term cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols — for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium.
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- Tornqvist, E., et al.
(author)
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Strategic Focus on 3R Principles Reveals Major Reductions in the Use of Animals in Pharmaceutical Toxicity Testing
- 2014
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In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 9:7
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Journal article (peer-reviewed)abstract
- The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.
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