In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances.

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.

Cooperative effects in vitro on fibroblast and keratinocyte functions related to wound healing by transforming growth factor-beta and a low molecular weight fraction from hemolyzed blood.

Blood or its constituents, respectively, contain a.o. substances such as TGF-beta (transforming growth factor-beta), PDGF (platelet-derived growth factor) and other factors, which beneficially influence wound healing. Patients with perivascular disturbances and consequently with inadequate supply of the affected tissue cells often suffer from poor healing of dermal wounds. Here, various cellular functions in situations of poor supply and related to wound healing, such as proliferation, colony formation, and migration of fibroblasts, and of monolayer formation by keratinocytes were emulated in vitro by supplementing cultures with reduced amounts of serum. Computer-aided image analysis allowed to quantify the cellular reactions under normal and serum-deprived medium conditions and under the influence of a low molecular weight fraction manufactured by dialysis of hemolyzed calf blood (HD, Solcoseryl) and of TGF-beta. Both preparations are in use for the treatment of poorly healing wounds. While HD preferentially normalized the reduced viability of fibroblasts, the keratinocyte activity was enhanced by TGF-beta. Restoration of fibroblast and keratinocyte functions proved most effective when combining HD with TGF-beta.

Influence of organophosphorus compounds on different cellular immune functions in vitro.

The effects of two organophosphorus compounds (OP) triphenyl phosphate (TPP) and triphenylphosphine oxide (TPPO) on different immune functions in vitro were investigated using an economical multiple endpoint approach. The test battery was designed as follows: immunocompetent cells (peritoneal cells and splenocytes) were isolated from female C57B1 mice and treated in vitro for 1 hr with the test article. Then the cells were washed and assessed for the following immune functions: Fc-receptor-dependent phagocytosis and lipopolysaccharide-induced release of a cytolytic protein (tumour necrosis factor, TNF) of thioglycollate-elicited peritoneal macrophages; natural killer (NK) cell activity, blastogenesis (T and B lymphocytes), and antibody synthesis (B lymphocytes) of spleen-cell suspensions. As an additional parameter the survival of the cells was determined routinely after treatment during all assays. No substantial effect on macrophage phagocytotic activity was observed after TPP or TPPO treatment. TPP led to a concentration-dependent suppression of macrophage TNF activity as well as NK activity of spleen cells. In addition, a slight reduction of B-lymphocyte antibody synthesis was obtained. TPPO treatment revealed a modulation of TNF activity of macrophages in a complex, non-concentration-related manner. A concentration-related suppression of spleen cell NK activity was observed after TPPO treatment. In summary, TPP and TPPO were found to be immunomodulating agents eliciting adverse effects predominantly towards cells of the innate immunity. The functions of T and B lymphocytes, referred to as adaptive immunity, were not substantially impaired. Our results indicate that the in vitro test battery described may be a suitable tool for the screening of OP-mediating immunotoxicity.

The genotoxicity status of senna.

Genotoxicity tests were performed by several laboratories with the drug fructus sennae, senna extract, sennosides, rhein and aloe-emodin. The drug fructus sennae, the sennosides and rhein did not increase mutation frequencies in the following test systems: bacterial systems (Salmonella reverse mutation test and/or Escherichia coli forward mutation test); mammalian cell cultures [hypoxanthine guanine phosphoribosyl transferase (HGPRT) test; mouse lymphoma test; chromosome aberration test with Chinese hamster ovary cells]; bone marrow (micronucleus test; chromosome aberration test); melanoblast cells (mouse spot test) of rodents. With aloe-emodin mutagenic effects were observed only in vitro in the chromosome aberration test with CHO cells and in the Salmonella reverse mutation test (frameshift mutations in strains TA 1537, TA 1538 and TA 98). In the in vitro gene mutation test with V79 cells (HGPRT test) no mutagenic potential of aloe-emodin was observed. In in vivo studies [micronucleus test with bone marrow cells of NMRI mice, chromosome aberration test with bone marrow cells of Wistar rats, mouse spot test (crossing DBA/2J x NMRI) no indication for a mutagenic activity of aloe-emodin was found. The relevance of the absence of a mutagenic potential in in vivo test systems was strengthened by the fact that aloe-emodin could be found in the blood serum after oral administration. Additional information on the interaction of aloe-emodin with DNA was obtained from an ex vivo unscheduled DNA synthesis test performed with hepatocytes of male Wistar rats: aloe-emodin did not induce unscheduled DNA synthesis as expression of DNA damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnological aspects of the production of human pro-kallikrein using the AcNPV-baculovirus-expression system.

We have investigated large scale production processes (up to 2 liters) of recombinant proteins using the baculovirus expression system in order to optimize the product yields. Experiments using cell lines of Spodoptera frugiperda (Sf9) and Mamestra brassicae (IZD-Mb0503) were performed to show the different production capacities of the cell lines. The influence of the infection at different cell densities is described. Beyond that, TC100-, IPL41- and serum-free IPL41-medium were compared to demonstrate their different capabilities of supporting cell growth and protein expression. Additionally, the inhibitory effect of FCS on the protease activity of kallikrein, which is produced in its zymogenic form, is discussed Improved production parameters are described, which enabled us to produce up to 8000 units of activated pro-kallikrein within 14 days using perfusion cultivation.

Immunotoxicity screening in vitro using an economical multiple endpoint approach.

An economical multiple endpoint in vitro test battery has been developed for screening chemically induced immune dysfunction. Bearing in mind the complexity of the immune system, different types of immunocompetent cells were used. Cofactor-fortified liver homogenate obtained from rats pretreated with Aroclor (S-9 mix) was employed as an in vitro metabolizing system. The following principal screening design was applied. Immunocompetent cells (peritoneal cells and splenocytes) obtained from female C57B(1) mice were treated in vitro for 1 hr. For metabolic activation, chemicals were pretreated with S-9 mix for 2 hr. After the incubation period the cells were washed and different immune function assays (antibody-dependent phagocytosis and lipopolysaccharide-induced release of tumour necrosis factor of thioglycollate-elicited peritoneal macrophages; natural killer cell activity, T- and B-cell blastogenesis, and B-cell antibody synthesis of spleen cell suspensions) were performed. For economy the different spleen cell functions were tested in parallel with aliquots of cells derived from the same chemically treated culture. As an additional parameter the survival of the cells was determined routinely after treatment during all assays. Different chemicals (e.g. tributyltinoxide, 7,12-dimethylbenzanthracene, lead acetate, cyclophosphamide, dexamethasone) were assessed using this system. The results indicate that the in vitro test battery described is a suitable tool for immunotoxicity screening.

Validation study of alternatives to the Draize eye irritation test in Germany: Cytotoxicity testing and HET-CAM test with 136 industrial chemicals.

According to OECD guideline 405 revised in 1987 Draize eye tests need not be performed for severely irritating and corrosive chemicals if results from 'well-validated alternative studies' are presented. In 1988 a validation study on alternatives to the Draize eye test was started in Germany to establish 'well-validated alternative methods' for this purpose. During database development, the last stage of the validation programme, 136 chemicals from the German chemical industry were classified in a blind trial with the 3T3 cell neutral red/kenacid blue cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test using fertile chicken eggs. The major goal of this stage of validation was to demonstrate the feasibility and limitations of the two alternative methods. Chemicals were, therefore, selected as representatives of chemical structural groups as well as of physicochemical and toxicological properties. In addition, some of the chemicals were chosen because they were of interest to the cosmetic and detergent industries. Draize eye testing data in vivo were provided by industry. In contrast to data from a previous interlaboratory assessment trial, it was impossible to correlate cytotoxicity data to the EEC classification for in vivo eye irritation. However, seven of 10 severely irritating chemicals (EEC labelling R-41) could be identified correctly in the HET-CAM assay, whereas test conditions of the study described here did not allow identification of irritating chemicals (EEC labelling R-36). The HET-CAM test is, therefore, fulfilling the criteria of a 'well-validated alternative method' according to OECD guideline 405 and should be incorporated into eye irritation testing at the earliest possible stage to reduce effectively the suffering of rabbits in the Draize eye test. Although an 80% correct prediction of 'non-labelled' chemicals in the HET-CAM test is encouraging, for safety assessment of non-irritant chemicals, for use as cosmetic formulations, for example, both government and industry will accept an in vitro assay only if its prediction of the absence of irritant properties is 100% correct.

Inter-laboratory ring trial of in vitro DNA repair tests using rat hepatocytes: further testing and conclusive remarks.

The results of the extension of a collaborative study for the detection of chemical-induced DNA damage in rat hepatocytes in vitro are presented in this report. Three coded compounds, i.e. 1,4-butanediol dimethanesulphonate, hydrazine sulphate and sodium dichromate, were tested for DNA repair synthesis by seven different laboratories, either using autoradiographic procedures or the liquid scintillation counting technique. Inter-laboratory standardization was intentionally not requested in order to investigate the validity of each study design under routine conditions. 1,4-Butanediol dimethanesulphonate was clearly positive in most laboratories; sodium dichromate was generally positive, while the results on hydrazine sulphate were contradictory.

Report of a comparative study of DNA damage and repair assays in primary rat hepatocytes with five coded chemicals.

We report the results of a collaborative study for the detection of chemical-induced DNA damage in primary cultures of rat hepatocytes. The methods include the detection of unscheduled DNA synthesis (UDS) with either autoradiography (5 laboratories) or liquid scintillation counting (2 laboratories) and the assessment of DNA single-strand breaks with the alkaline elution assay (1 laboratory). Interlaboratory standardization was omitted in order to prove the agreement of the assays under routine conditions. Five coded chemicals were tested. For 4 chemicals (2-acetylaminofluorene, thiourea, glycerine and potassium chloride) the UDS data were consistent in all laboratories, thus indicating a high consensus of the test systems applied in the different laboratories. Those 3 chemicals that were not expected to elicit genotoxic activity (thiourea, glycerine, and potassium chloride) yielded negative results in all laboratories. 2-Acetylaminofluorene, a known DNA-damaging agent in hepatocytes, gave strongly positive responses in all laboratories. In contrast, N-nitrosodiphenylamine led to equivocal responses.

Construction and evaluation of a cea-lacZ gene fusion for the detection of environmental mutagens and carcinogens.

The cea-kil operon of the ColE1 plasmid is negatively regulated by the LexA-repressor and therefore, it is under the control of SOS regulation. We constructed a gene fusion between the cea and lacZ genes. Expression of the translational fusion can be easily detected by monitoring the levels of beta-galactosidase. Since the whole detection system is plasmid-based, it can be used in both Escherichia coli and Salmonella typhimurium strains. The SOS-function-inducing activities of 14 chemical mutagens were investigated in E. coli K12 and in two S. typhimurium Ames-strains and compared with results obtained by the SOS-chromotest and by the Umu-test. To correct for the inhibitory effects of test chemicals on mRNA and/or protein synthesis, the level of the constitutive chloramphenicol acetyl transferase was assayed in parallel.

Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany.

A national interlaboratory study to validate two alternative methods to the Draize rabbit's eye test, co-ordinated by ZEBET at the German Federal Health Office (BGA), is described. The aim of the study is to classify chemicals according to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg chorioallantoic membrane (HET-CAM) test. During the last two years 12 toxicology laboratories from industry, universities and other research institutions have tested 32 substances from a variety of chemical classes, characterized by a broad spectrum of locally irritating properties, using the NR/KB cytotoxicity test and the HET-CAM assay. Intra- and interlaboratory reproducibility of the two methods was investigated under standardized conditions. The so-far limited evaluation of the interlaboratory assessment phase of validation indicates that the results of the Draize rabbit's eye test correlate better with the results of the HET-CAM test than with those of the cytotoxicity test as far as false negative results are concerned. However, the intra- and interlaboratory reproducibility of the cytoxicity test is better than that of the HET-CAM test. The validation project has recently entered the stage of database development during which 150 chemicals will be tested in seven laboratories to provide information on whether and to what extent the NR/KB test and the HET-CAM test can replace the Draize rabbit's eye test for the classification and labelling of chemicals with regard to their eye irritation potential.

Multistage production of Autographa californica nuclear polyhedrosis virus in insect cell cultures.

The aim of our study was to establish an efficient system for the in vitro production of the insect pathogenic Autographa californica nuclear polyhedrosis virus in a Spodoptera frugiperda cell line. We optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. Cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. In order to provide sufficient nutrients during virus synthesis filtration and perfusion devices were connected to the bioreactor. A virus production procedure in a repeated batch mode by using a two stage bioreactor system is described. Stage I was optimized for cell production and stage II for virus production.

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures.

Cell cycle kinetic of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G0) of mammalian cells is characterized by a 2c-DNA content whereas G0-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2'-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.

[Genotoxicity of a standardized Hypericum extract]. / Genotoxizität eines standardisierten Hypericum-Extraktes.

St. John's wort (Hypercum perforatum) contains hypericin and hypericin-like substances as well as flavonoids, of which particularly Quercetin has generated a wide-spread controversial discussion with respect to mutagenic action. The genotoxicity of a standardized aqueous ethanolic Hypericum extract (Hypericum extract Steigerwald, Psychotonin M) was verified in different in-vivo and in-vitro testsystems with mammalian cells. The in-vitro investigations were performed with the HGPRT (hypoxanthine guanidine phosphoribosyl transferase)-test, UDS (unscheduled DNA synthesis)-test and with the cell transformation test using Syrian hamster embryo cells. Both the in-vitro tests as well as the in-vivo tests--fur spot test of the mouse and the chromosome aberration test with the bone marrow cells of the chinese hamster--were negative, giving completely no indication of a mutagenic potential of Hypericum extract. These investigations lend support to the view that results from bacterial short-term tests are of very limited transferability to human.

Cytotoxicity detected by image analysis: A new method for the quantification of survival, mortality, recovery and growth of mammalian cell cultures.

The use of in vitro methods is increasingly recommended for the assessment and evaluation of cytotoxic effects resulting in cell damage. In most cases, cellular response and reaction of cell populations is determined by endpoint measurements. Even if a battery of such test systems is applied, information about the dynamics of cell damage and recovery is obtained only in part. However, phenomena of damage and recovery can be followed by observing the fate of individual cells and their progeny in culture over several days, which means over several cell cycles, by using light microscopy combined with image analysis. We have developed a recording system for such continuous observation and registration of toxic effects, based on image analysis. Growth rate, generation time, delay or shifting of cell cycle, identification of the progeny (pedigrees) and cellular locomotion can be recorded simultaneously.

Flow-cytometric cell-cycle analysis of Chinese hamster cells following exposure to cytotoxicants.

Chinese hamster cell-cycle kinetics were studied following exposure to a cytotoxic agent. Different parameters like dependence on concentration and preparation interval were measured by flow cytometry. The results were compared with data from established methods. DNA histograms showed the cell-cycle effect of EMS (ethyl methanesulfonate) at different time intervals after treatment. The principle of quenching the fluorescence of Hoechst 33258 by staining 5-bromodeoxyuridine (BrdU) substituted DNA was applied and supports these results. Comparison to chromosome-aberration studies demonstrates the suitability of this method to screen quickly for adequate dosing of a cytotoxic substance and also gives information on the appropriate preparation interval.

Cell cycle analysis by flow cytometry in an antibody secreting cell line.

This presentation reports on the routine use of flow cytometric techniques in suspension cultures. As a model system an antibody secreting mouse hybridoma cell line was used to monitor antibody production (a Bromodeoxyuridine specific antibody) and proliferative status as obtained by flow cytometry (FCM) and cell counting.

S-phase cell detection with a monoclonal antibody.

The use of a newly developed monoclonal antibody against Bromodeoxyuridine is demonstrated in a study with V79 Chinese hamster cells. It can replace radioactive material in cell cycle analysis studies in the way that BrdU incorporated into the DNA during S-phase is detected by immunofluorescence. By this technique the proportion of actively synthesizing S-phase cells can be determined. The proportion of such cells is a good measure with regard to the quality of a culture and/or medium in question. The method is fast and offers within 3-5 hours the possibility to analyse growth conditions in monolayers and suspension cultures.

Cycling S-phase cells in animal and spontaneous tumours. I. Comparison of the BrdUrd and 3H-thymidine techniques and flow cytometry for the estimation of S-phase frequency.

Evaluation of the proliferative activities of cell populations has mainly been restricted to the use of autoradiography and flow cytometric measurements. The introduction of a new BrdUrd specific antibody makes it possible to determine exactly the DNA synthesizing cells. The BrdUrd technique is safe with respect to handling and the results are obtained within five hours. The suitability of the BrdUrd labelling procedure has been studied in different cell lines and compared with 3H-thymidine autoradiography and flow cytometry.