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.

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.

The effect of insecticides on Chinese hamster cell cultures.

The effect of p,p'-isomers of DDT and its derivatives DDD, DDE and DDA on Chinese hamster cells in culture was studied. At different concentrations and various times of treatment the proliferation rate was inhibited most strongly by DDD and DDT, whereas DDE exhibited a markedly weaker influence. DDA was the least toxic compound of the four. The cytogenetic effects were also different. Again, DDA induced the least damage. Only enhanced gap rates but no chromosome breaks were observed. DDE was more active, and higher break rates occurred. DDD and DDT were by far the most damaging compounds, and they raised the gap and break rates markedly. However, no induction of configuration anomalies was found in any experiment. Chronic treatment of the cells for 3 months with DDT at 8 ppm did not alter the proliferation rate, the sensitivity to acute treatment with higher DDT concentrations or the chromosomal aberration rates. The results are discussed in relation to the relevance of differential pesticide effectivity in organs of higher animals and man.

Busulphan-induced chromosomal aberrations in intestinal cells of Chinese hamster.

Chinese hamsters were given Busulphan, 25 and 50 mg/kg bw. After 18 h, chromosomes of the lining cells of the upper intestinal tract were prepared according to a newly developed method. The aberration rate was enhanced by a factor of about 25 after 25 mg/kg, but 50 mg/kg bw did not induce higher aberration rates. In the same animals the dose-dependent aberration rates were also determined in bone-marrow cells. Furthermore, the formation of SCEs in intestinal cells was observed. In a preliminary experiment, metaphase plates from intestinal cells of mice were prepared, too.

Photodecomposition of 1-nitropyrene and other direct-acting mutagens extracted from diesel-exhaust particulates.

The effect of irradiation with wavelengths of 320-418 nm on direct-acting mutagenicity of 1-nitropyrene (1-NP) and particulate-matter extracts of a direct-injecting diesel engine was examined. The activity of samples in the Ames test with and without addition of S9 mix in Salmonella typhimurium strain TA98, TA100 and TA1538 decreased with increasing irradiation energy. Visible light was sufficient to destroy the mutagenicity of a 0.1-mM 1-NP solution. The same was true for particulate matter crude extracts as well as the transitional and oxygenate subfractions isolated by column chromatography. UV spectroscopy, thin-layer chromatography and GC-MS analysis were performed to characterize the irradiation products of 1-NP. The mechanism of photodecomposition of 1-NP at different wavelengths and the significance of this finding for the evaluation of health risks from diesel vehicles are discussed.

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.

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.

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.

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.

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.

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.

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.

HZE effects on mammalian cells.

In track segment experiments cell survival and chromosome aberrations of mammalian cells have been measured for various heavy ion beams between helium and uranium in the energy range between 0.5 and 960 MeV/u, corresponding to a velocity range of 0.03 to 0.87 C, and an LET spectrum from 10 to 15 000 keV/micrometers. At low LET, the cross section (sigma) for cell killing increases with increasing LET and shows a common curve for all ions regardless of the atomic number. This indicates that in this region the track structure of the different ions is of only a minor influence, and it is rather the total energy transfer, which is important for cell killing. At higher LET values, deviations from a common sigma-LET curve can be observed which indicate a saturation effect. The saturation of the lighter ions occurs at lower LET values than for the heavier ions. These findings are also confirmed by the chromosome data, where the efficiency for the induction of chromosomal aberrations for high LET particles depends on the track structure and is nearly independent of LET. In the heavier beams (Z > or = 10) individual particles cause multiple chromosome breaks in mitotic cells.

Qualitative and quantitative aspects of animal cell in vitro systems.

The evaluation of sublethal cellular damage is shown to be important for the performance of experiments and bioproduction with animal cells. Detailed information on the fate of single cells in colony pedigrees from untreated and irradiated mammalian cells was obtained in vitro with cinematography recordings. It can be demonstrated that in generations two, three, and four the cells are more sensitive because of higher death rates and increased generation times induced by the handling of the cells during passaging or by the damaging agent. It is obvious that chemical agents can act in the same way. Also poor medium quality induces similar effects. Growth curves of single colony cell populations render much better information on the proliferation of a cell population than the determination of cells per ml (= particle counting). Examples are given for such growth curves and for colony size distribution curves based on individual cell countings. The advantages of the use of flow-cytofluorometry for the control of cell cycle phases are shown. As an example for the relevance of qualitative and quantitative data on cell substrate and medium conditions for bioproduction the in vitro mass replication of an insect pathogenic virus (baculovirus) is presented. Baculoviruses are potentially important biological pesticides which can replace chemical insecticides partially.

The replication of insect pathogenic viruses (nuclear polyhedrosis viruses) in cell cultures.

The phenomena and steps of replication of insect pathogenic viruses (baculoviruses : nuclear polyhedrosis viruses) in their natural hosts and in insect cell cultures are described.

The effect of cyclophosphamide on the centromere separation sequence in Chinese hamster spermatogonia.

The sequence of centromere separation in spermatogonial chromosomes of untreated and cyclophosphamide-treated Chinese hamsters is described. Centromeres of chromosome 1 and 2 separated much earlier than all other chromosomes, especially 6-8. Cyclophosphamide significantly inhibits the centromere separation in all chromosome groups but does not alter the sequence of separation.