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.

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)

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.

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.

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.

Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. II. Studies to investigate the effect of bacterial liquid culture preparation conditions on Salmonella mutagenicity test results.

The influence of various parameters and growth conditions in the "overnight culture" of Salmonella typhimurium strains on mutagenicity test results was investigated. A number of factors were first suspected to be of some importance for the quantitative outcome of the mutagenicity test. None of them, however, was found to influence the results to such a marked extent as to be a major source of variability. Only the brand of nutrient broth used for the propagation of the bacteria proved finally to have a certain effect on the number of (spontaneous and induced) revertant colonies, although no precise and quantitative statements can be made with regard to a possible standardization of this experimental segment in the Salmonella mutagenicity test. The occurrence of such unpredictable but noticeable influences is, however, evidence for the importance of an intralaboratory optimization and standardization of all parts of the test procedure.

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.

Viral pesticides : biohazard evaluation on the cytogenetic level.

Promising agents for integrated pest control are naturally occuring insect pathogenic viruses that belong to the group of baculoviruses. We have performed experiments using mammalian cells from different species in vivo and in vitro to test a possible effect of baculoviruses on chromosome aberration rates and sister chromatid exchanges. No cytogenetic effects were found.

Testing propafenone hydrochloride in the Ames Salmonella microsome system and in mammalian cytogenetic systems (bone marrow, spermatogonia).

2'-(2-Hydroxy-3-propylaminopropoxy)-3-phenyl-propiophenone-hydrochloride (propafenone-HCl, Rytmonorm) was tested for mutagenic activity in the Salmonella microsome system (Ames test) and in mammalian cell systems. Neither in the Ames test (62.5-1000 microgram/plate) nor in the bone marrow cells and spermatogonia of Chinese hamster (injected doses: 1.57, 3.13, 6.27 mg/kg) there is any evidence for an enhancement of mutation frequencies or chromosome aberration rates. Also, the micronucleus rates were in the control range. Under the experimental conditions of this investigations propafenone-HCl was not mutagenic.

The effect of cyclophosphamide and isoniazid (INH) alone and in combination on the centromere separation sequence in Chinese hamster bone marrow cells.

X-irradiation of Chinese hamsters with 50 and 200 rad delayed the centromere separation of bone marrow chromosomes. Combination of a treatment with 13.3 mg cyclophosphosphamide/kg body wt. and 50 rad did not result in an enhanced effect compared with the single treatments. Again, the separation sequence was not influenced. No delay was induced by a treatment with 125 mg isoniazid/kg, but a slightly and significantly earlier separation of the chromatids in both groups of metacentric and acrocentric chromosomes occurred. The separation delay seen after irradiation with 50 rad disappeared when the animals were pretreated with isoniazid. Independently of these results the asynchronous separation sequence was not altered in any of the experiments.

Sequence of centromere separation of mitotic chromosomes in Chinese hamster.

Chromosome preparation in late metaphase cells from bone marrow of colcemid treated male Chinese hamsters were used to analyse the sequence of separation of sister centromeres. Chromatids of chromosomes 2 and 1 are the first ones to separate at centromeres, followed by members of group B, D and C. Some acrocentric chromosome is always the last one to separate at the centromere. The data point to a possible correlation between the position of a centromere in the separation sequence in the genome and the amount of centromeric heterochromatin as well as relation to the phenomenon of non-disjunction.

[On the simultaneous effects of ionising radiation and chemical agents on animal cells. 1st communication. Cytogenetic studies on X-rays and isonicotinic acid hydrazide in the Chinese hamster, Cricetulus griseus (author's transl)]. / Uber die gleichzeitige Wirkung von ionisierender Strahlung und chemischen Agentien auf tierische Zellen. 1. Mitteilung: Zytogenetische Untersuchungen mit Röntgenstrahlen und Isonicotinsäurehydrazid (INH) beim Chinesischen Hamster, Cricetulus griseus

In bone marrow cells of Chinese hamsters enhanced chromosome damage is observed after 48 h treatment of the animals with isoniazid (INH) combined with X-rays only at much higher doses than normally applied in human therapy. Compared to X-ray-induced aberration levels, 125 mg/kg INH and 50 R given simultaneously increase the number of gaps and breaks, the latter being increased over-additively. This points to an interaction between both agents. The combined treatment changes the mitotic rate of the bone marrow as well.

[Simultaneous effect of 5-bromodeoxyuridine and cysteamine on x-irradiated diploid cells of Chinese hamster, Cricetulus griseus, in vitro]. / Untersuchungen über die gleichzeitige Wirkung von 5-Bromdesoxyuridin und Cysteamin auf röntgenbestrahlte, diploide Zellen des Chinesischen Hamsters, Cricetulus griseus, in vitro

Cells of the Chinese hamster line B 14 FAF 28 were treated with BUdR and irradiated with X-rays under the presence of cysteamine. The dose-dependent inactivation rates could be determined rather precisely by marking single cells. Daily cell counts in the growing colonies made it possible to establish individual colony growth curves. The results of both analyses show for untreated and treated cells a nearly equal factor with respect to the protecting effect of cysteamine. Also in the "macrocolonies" this effect is clearly measurable even five days after irradiation indicating a still existing very different vitality of the cells at that time. The results are discussed with concern to following irradiations.