Manipulation of the phenotype of immortalised rat hepatocytes by different                 culture configurations and by dimethyl sulphoxide

The liver-specific phenotype of immortalised rat hepato-cytes is not irretrievably lost as they age in culture but can be manipulated by modifying the culture environment. Testosterone metabolism was used to investigate the profile of cytochrome P450 isoenzymes present in two immortalised cell lines, P9 and LQC, and in primary cultures of rat hepatocytes, cultured on collagen films, gels and double gel cultures (sandwich configuration). The extent of testosterone metabolism, and the range of metabolites produced, was increased in immortalised cells by the presence of collagen as a substratum film or gel but survival was poorer and the range of metabolites was reduced in sandwich culture. In contrast, testosterone metabolism was retained in primary hepatocytes in sandwich cultures at a higher level than in collagen film or gel cultures. Expression of alpha class glutathione-S-transferases (GSTs) increased and that of GSTP1 decreased (changes which indicate a recovery of normal liver GST phenotype) when the medium of immortalised cell cultures was supplemented with dimethyl sulph-oxide (DMSO). DMSO also improved ethoxyresorufin 0-deethylation (EROD) and testosterone metabolism in immortalised cells. It also markedly inhibited prolifera-tion, DNA, RNA and protein synthesis. Maximal testosterone metabolism was observed in immortalised cells cultured on collagen gels in the presence of l1o (v/v) DMSO. Development of a protocol for treating immortalised liver cells cultured on collagen gels with DMSO to switch between proliferation and differentiation may provide a convenient system expres-sing the xenobiotic metabolising enzymes required for in vitro toxicity testing.

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Preparation of primary cultures of rat hepatocytes suitable for in vitro toxicity testing

Journal of Tissue Culture Methods ◽

10.1007/bf01834627 ◽

1983 ◽

Vol 8 (1) ◽

pp. 1-6 ◽

Cited By ~ 21

Author(s):

Kathryn A. Elliget ◽

Gerald J. Kolaja

Keyword(s):

Primary Cultures ◽

Toxicity Testing ◽

Rat Hepatocytes ◽

In Vitro Toxicity

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MEIC Evaluation of Acute Systemic Toxicity

Alternatives to Laboratory Animals ◽

10.1177/026119299802601s02 ◽

1998 ◽

Vol 26 (1_suppl) ◽

pp. 93-129 ◽

Cited By ~ 2

Author(s):

Cecilia Clemedson ◽

Frank A. Barile ◽

Barbro Ekwall ◽

Maria José Gómez-Lechón ◽

Tony Hall ◽

...

Keyword(s):

Cell Lines ◽

Primary Cultures ◽

Toxicity Testing ◽

Preceding Paper ◽

In Vitro Toxicity ◽

Toxicity Data ◽

Animal Cells ◽

Basal Cytotoxicity

Results from tests on the first 30 MEIC reference chemicals in 16 different systems are presented as a prerequisite to the subsequent in vitro/in vivo comparisons of acute toxicity data, i.e. the final MEIC evaluation of all test results of the study. The study is a supplement to the previously published results from 68 methods (including methods 45B and 46B [old numbers]) used to test the same set of chemicals. The strategies and methods of the preceding paper were employed to enable a comparative cytotoxicity analysis of the results from these 68 methods and from the 16 new methods to be made. Principal components analysis (PCA) of 82 assays demonstrated a dominating first component which described as much as 83% of the variance in the toxicity data. This remarkable similarity of all toxicity data was the main finding of the present study, and confirmed the results of the previous study with a less-extensive database. Also, the influence on the general variability of results of several key methodological factors was evaluated by analysis of selected sets of data, including linear regression of the results of pairs of methods, which were similar in all respects except for the factor under analysis. This analysis of the same 82 assays as before also confirmed previous results from the 68 assay database: a) the toxicities of a third of the chemicals increased considerably with exposure time; b) in general, cytotoxicity for human cells was well predicted by cytotoxicity tests with animal cells; c) this prediction was poor for two chemicals, i.e. digoxin and malathion; d) prediction of human cytotoxicity by ecotoxicological tests was only fairly good; e) 25 comparisons of similar assays employing different cell lines showed strikingly similar toxicities (mean R2 = 0.86); f) 22 comparisons of similar pairs of assays employing different primary cultures and cell lines also revealed similar toxicities (mean R2 = 0.79); and g) 15 comparisons of similar assays with different growth/viability endpoint measurements demonstrated strikingly similar toxicities (mean R2 = 0.89). Results b, e, f and g must be the main causes of the general similarity of results, while results a, c and d, together with other factors, could explain the 20% dissimilarity. These findings support the basal cytotoxicity concept and may assist in guiding and refining in vitro toxicity testing in the future.

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Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

Nanomaterials ◽

10.3390/nano11081929 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1929

Author(s):

Tereza Cervena ◽

Andrea Rossnerova ◽

Tana Zavodna ◽

Jitka Sikorova ◽

Kristyna Vrbova ◽

...

Keyword(s):

Cytochalasin B ◽

Culture Media ◽

Methodological Approach ◽

Micronucleus Assay ◽

Toxicity Testing ◽

In Vitro Toxicity ◽

Testing Strategies ◽

Transmission Electron ◽

The Impact

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

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A survey of aerosol exposure systems relative to the analysis of cytotoxicity: A Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) perspective

Toxicology Research and Application ◽

10.1177/23978473211022267 ◽

2021 ◽

Vol 5 ◽

pp. 239784732110222

Author(s):

David Thorne ◽

Roman Wieczorek ◽

Toshiro Fukushima ◽

Han-Jae Shin ◽

Robert Leverette ◽

...

Keyword(s):

Scientific Research ◽

Toxicity Testing ◽

User Preferences ◽

In Vitro Toxicity ◽

Aerosol Generation ◽

Aerosol Exposure ◽

Comprehensive Survey ◽

Survey Results ◽

System Data

During a Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) meeting, the in vitro toxicity testing Sub-Group (IVT SG) met to discuss the evolving field of aerosol exposure research. Given the diversity of exposure parameters and biological endpoints being used, it was considered a high priority to investigate and contextualise the responses obtained. This is particularly driven by the inability to compare between studies on different exposure systems due to user preferences and protocol differences. Twelve global tobacco and contract research companies met to discuss this topic and formulate an aligned approach on how this diverging field of research could be appropriately compared. Something that is becoming increasingly important, especially in the light of more focused regulatory scrutiny. A detailed and comprehensive survey was conducted on over 40 parameters ranging from aerosol generation, dilution and data analysis across eight geographically independent laboratories. The survey results emphasise the diversity of in vitro exposure parameters and methodologies employed across the IVT SG and highlighted pockets of harmonisation. For example, many of the biological protocol parameters are consistent across the Sub-Group. However, variables such as cell type and exposure time remain largely inconsistent. The next steps for this work will be to map parameters and system data against biological findings and investigate whether the observed inconsistencies translate into increased biological variability. The results from the survey provide improved awareness of parameters and nuances, that may be of substantial benefit to scientists in intersecting fields and in the development of harmonised approaches.

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In Vitro Toxicity Testing: Redefining our Objectives

Alternatives to Laboratory Animals ◽

10.1177/026119299202000411 ◽

1992 ◽

Vol 20 (4) ◽

pp. 571-574

Author(s):

Oliver Flint

Keyword(s):

Toxicity Testing ◽

In Vitro Toxicity

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Toxicity of Glycols and Allyl Alcohol Evaluated by Means of Co-cultures of Microcarrier-attached Rat Hepatocytes and BALB/C 3T3 Mouse Fibroblasts

Alternatives to Laboratory Animals ◽

10.1177/026119299202000216 ◽

1992 ◽

Vol 20 (2) ◽

pp. 266-270

Author(s):

Jens-Uwe Voss ◽

Hasso Seibert

Keyword(s):

Lactate Dehydrogenase ◽

Ethylene Glycol ◽

Allyl Alcohol ◽

Rat Hepatocytes ◽

3T3 Cells ◽

Active Metabolites ◽

Mediated Effects ◽

Cells Cultured

The toxicity of allyl alcohol and several glycols (ethylene glycol, 1,2-propanediol, 1,3-propanediol, methoxyethanol, and the glycol ether dioxane) was studied in cultures of 3T3 cells and in co-cultures of 3T3 cells with microcarrier-attached hepatocytes. Metabolism-mediated effects on the cytotoxicity to 3T3 cells were recorded by differences in the growth of the cultures exposed in the presence or absence of hepatocytes. Hepatocyte viability was determined by depletion of intracellular lactate dehydrogenase and effects on the biotransformation ability of hepatocytes were assessed by determination of O-deethylation of 7-ethoxycoumarin (EOD activity). Allyl alcohol was the only substance more toxic to the hepatocytes than to 3T3 cells cultured in the absence of hepatocytes. Toxicity to 3T3 cells of allyl alcohol, ethylene glycol, and 1,3-propanediol, but not of 1,2-propanediol, methoxyethanol and dioxane, was markedly enhanced when the cells were co-cultured with hepatocytes. The results indicate that the toxicity of allyl alcohol, ethylene glycol, and 1,3-propanediol, to 3T3 cells depends on the formation of active metabolites. For ethylene glycol and 1,3-propanediol, growth of 3T3 cells in co-cultures was reduced at concentrations without effects on hepatocyte viability. Co-culture of 3T3 cells with microcarrier-attached rat hepatocytes represents a suitable approach for the in vitro evaluation of metabolism-mediated cytotoxicity.

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Time-lapse Phase-contrast Microphotography of Cell Populations as a Basis for Improvement of In Vitro Toxicity Assessment

Alternatives to Laboratory Animals ◽

10.1177/026119299202000223 ◽

1992 ◽

Vol 20 (2) ◽

pp. 302-306

Author(s):

Miroslav Červinka

Keyword(s):

Cell Proliferation ◽

Cell Morphology ◽

Video Recording ◽

Toxicity Testing ◽

Time Lapse ◽

Time Dependent ◽

In Vitro Toxicity ◽

Simple Modification ◽

Pertinent Data

Recent trends in the field of in vitro toxicology have centred around the validation of in vitro methods. The ultimate goal is to obtain pertinent data with the minimum of effort. In our laboratory, we have used toxicological methods based on the evaluation of cell morphology and cell proliferation. A method suitable for this purpose is time-lapse microcinematographic (or video) recording of cellular changes, which we used for many years. For practical in vitro toxicity testing, however, this method is far too complicated. Therefore, we have tried to develop a simple modification for the evaluation of cell morphology and cell proliferation, which would still allow for a basic time-dependent analysis. Comparison of detailed microcinematographic analysis with analysis according to our new proliferation assay is demonstrated with cisplatin as the toxicant. We believe that a time-dependent approach could improve the in vitro assessment of toxicity.

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