[Björn Ekwall and his contribution to modern cell toxicology].

Dr. Björn Ekwall (1940-2000) was a prominent Swedish scientist--cell toxicologist, who made an outstanding contribution in the field of in vitro toxicology. In the early 80-ties Ekwall formulated so called basal cytotoxicity concept, which served as a basis for modern orientation in the field of cell toxicology: the use of tests on cells in culture for prediction of acute systemic toxicity in humans, instead of the use of tests on experimental animals. To be able to verify his theories, Ekwall organized and led the international toxicological project called MEIC: Multicentre Evaluation of In Vitro Cytotoxicity Programme (1989-1999). In this project, 50 selected chemicals were tested in 100 laboratories worldwide with more than 60 different in vitro tests (laboratories have chosen tests themselves). MEIC project was unique not only because its large scale, but, in particular, because, for the first time, the human peak blood concentrations after acute poisoning with chemicals were used as references, aiming to check predictability of the in vitro assays. The results of the MEIC project have clearly demonstrated a possibility to use in vitro tests for prediction of toxicity of chemicals in humans.

DNA damage induced by gamma-radiation in combination with ethylene oxide or propylene oxide in human fibroblasts.

To estimate the effects of interaction of gamma-rays and an epoxide, cell survival and induction of DNA double-strand breaks (DSBs) following combined exposure to ionizing radiation and ethylene oxide (EtO) or propylene oxide (PO) were studied in human fibroblasts. Two treatment protocols were applied: (a) the cells were pre-exposed to different doses of gamma-rays and then treated with epoxide, and (b) the cells were pretreated with epoxide and then exposed to different doses of gamma-rays. Here we show that order of the treatment did not play a role in cell survival and that the effect of combined exposure on cell killing was additive for both epoxides. As to DNA DSBs induction, however, a difference dependent upon the order of the treatment was observed. While EtO or PO treatment followed by gamma-rays exposure led to an increased number of DSBs at higher gamma-ray doses (2-3 Gy), no significant increase of DSBs was detected after the opposite order of the treatment (gamma-ray exposure followed by EtO or PO treatment).

Combined effects of gamma-radiation and ethylene oxide in human diploid fibroblasts.

Human diploid VH-10 fibroblasts were pre-exposed to gamma-rays and then treated with ethylene oxide (EtO). In the reverse experiment, the cells were pretreated with EtO and then exposed to gamma-rays. Two different dose rates of gamma-rays were used: a low dose rate (LDR, 0.66 Gy/min) and a high dose rate (HDR, 10 Gy/min). Cell killing, mutagenicity and DNA double-strand breakage were studied in both types of experiment. The induction of mutations in the HPRT locus was studied by selection in medium containing 6-thioguanine. DNA double-strand breakage, measured as fraction of activity released (FAR), was investigated using pulsed field gel electrophoresis. Concerning mutagenesis, it was found that pre-exposure of the cells to gamma-radiation (1 Gy) followed by treatment with EtO (2.5 mMh) led to an additive co-interaction, irrespective of dose rate. On the other hand, the reverse experimental procedure (pretreatment with EtO followed by gamma-ray exposure) resulted in an antagonistic effect, which was most pronounced when the HDR was applied. In the latter case, the resultant mutant frequency was two times lower than the sum of the mutant frequencies after the individual treatments. However, the effect of the combined treatment on FAR was different: FAR increased with both combinations of agents used compared with the separate and hypothetically expected effects. Moreover, the HDR exposure led to an additional increase in FAR compared with the LDR one.

Effects of three epoxides--ethylene oxide, propylene oxide and epichlorohydrin--on cell cycle progression and cell death in human diploid fibroblasts.

Ethylene oxide (EtO), propylene oxide (PO), and epichlorohydrin (ECH) strongly influenced the G1/S progression in human diploid fibroblasts, VH-10. However, these epoxides did not affect substantially the G2/M progression. It was found that G1 arrest is induced by these epoxides 6-18 h after the treatment at doses above 5, 3, and 0.5 mMh for EtO, PO, and ECH, respectively. An inhibitory effect on DNA synthesis was also demonstrated at the same doses within the same time interval. On the contrary, the epoxides transiently stimulated DNA synthesis 3-18 h after the treatment with the lower doses (below 5, 3, and 0.5 mMh for EtO, PO, and ECH, respectively). This effect was manifested both as an elevated rate of DNA synthesis and as an increase in the number of cells in S-phase. Among the three studied epoxides EtO was the most effective one: the increases of the rate of DNA synthesis and of cells in S-phase were 35 and 55%, respectively. All the epoxides tested induced significant decrease of intracellular level of reduced glutathione (GSH) shortly after cell exposure. While low and moderate doses induced a transient decrease in GSH level the high doses induced its irreversible depletion. The extensive GSH depletion was related to cell death. Morphological examination of cell nuclei indicated that epoxide-treated cells die via necrosis. This conclusion is supported by the lack of such features of the apoptosis as chromatin condensation and the occurrence of so called 'apoptotic bodies'. The absence of nucleosomal fragmentation of DNA and an increase of the permeability of the plasma membrane after the epoxide treatment also indicated a necrotic form of cell death. ECH is about ten times more toxic than the two other epoxides, and it causes almost 100% necrosis at dose of 3.0 mMh.

Rejoining of DNA strand breaks induced by propylene oxide and epichlorohydrin in human diploid fibroblasts.

The repair kinetics of DNA single- and double-strand breaks (SSBs, DSBs) induced with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH), was studied in human diploid fibroblasts. The methods used were: alkaline DNA unwinding (ADU), the comet assay, and pulsed field gel electrophoresis (PFGE). About 70% of SSBs, measured by ADU, were rejoined after the treatment with 5 mMh and 10 mMh of PO within 20 hr, and the half-life was estimated to be approximately 15 hr. On the other hand, effective rejoining of SSBs after ECH treatment was observed only at a dose of 1 mMh (a half-life of approximately 15 hr), whereas after 2 mMh treatment, only 26% of SSBs could be rejoined within 20 hr. Furthermore, the use of the comet assay demonstrated that DNA strand breaks were effectively rejoined after PO and ECH treatment at doses of 5-10 mMh and 0.5-1 mMh, respectively. About 76% and 83% of DSBs induced by 5 and 10 mMh of PO, respectively, were rejoined within 4 hr after the treatment (a half-life of approximately 2.5 hr), with little further repair thereafter. At lower dose of ECH (1 mMh) a half-life for DSBs rejoining was estimated to be approximately 2 hr; however, only 29% of DSBs were rejoined within 2 hr at the higher dose of 2 mMh. After 18 hr, the rejoining following treatment with a lower dose was negligible. At a higher dose, a rapid accumulation of DSBs was observed, probably as the result of cell death and DNA degradation. The results demonstrate the capability of human diploid fibroblasts to repair DNA SSBs and DSBs at low-to-moderate doses of the epoxides. A weak capacity to rejoin DNA strand breaks induced by higher doses of ECH may be a consequence of its higher DNA alkylation activity and approximately 10 times higher toxicity compared to PO.

Alteration of p53 protein in C3H/10T1/2 cells morphologically transformed by gamma-rays in stationary phase.

The panel of 70 transformed clones was isolated after exposure of C3H/10T1/2 cells in stationary phase to low-moderate doses (1-4 Gy) of 137Cs gamma-rays. Two widely different dose rates were used: high (HDR, 0.66 Gy/min) and low (LDR, 4.8 x 10(-4) Gy/min). Immunohistochemical analyses were performed by cellular staining with three types of monoclonal anti-p53 antibodies, Ab-1 (PAb421), Ab-3 (PAb240) and Ab-4 (PAb246) in order to identify wild-type and altered conformation of the p53 protein in cell nuclei. The gamma-ray exposure led to induction of altered p53 protein in the majority of morphologically transformed clones. For LDR exposure the percentage of clones with changed p53 protein was 79 (11/14), 71 (12/17) and 100 (6/6) for the exposure doses of 2, 3 and 3.6 Gy, respectively. For HDR exposure the percentage of such clones was 60 (3/5), 40 (4/10) and 87 (13/15) for 1, 2 and 3 Gy, respectively. Moreover, in some transformed clones, especially in those induced by higher gamma-ray doses, p53 protein in cell nuclei was not found. It was demonstrated by lack of the staining with Ab-1 antibody which can detect both mutant and wild-type of p53 protein. An altered conformation of p53 protein was detected, using Ab-3 antibody which does not react with its native conformation, in 27% (18/67) of all radiation-induced clones. A native conformation of p53 protein was recognized by Ab-4 antibody in 33% (10/30) of clones induced by HDR, and in 22% (8/37) of clones induced by LDR exposure. Our study shows that alterations of p53 protein in cell nuclei is a frequent feature of morphological transformations induced by ionizing radiation in C3H/10T1/2 cells, and that these alterations occur independently of dose rate.

[Decreased survivability and a DNA repair defect in the cells of patients with xeroderma pigmentosum and Cockayne syndrome under the action of radiation and chemical mutagens]. / Ponizhennaia vyzhivaemost' i defekt reparatsii DNK v kletkakh bol'nykh pigmentnoi kserodermoi i sindromom Kokkeina pri deistvii luchevykh i khimicheskikh mutagenov.

The action of ionizing radiation and chemical mutagens--epoxides (ethylene oxide, propylene oxide, epichlorohydrin)--upon survival and repair processes in xeroderma pigmentosum (XP2SP) and Cockayne syndrome (CS1SP) patients' cells was studied, compared to healthy donor's cells VH-10 and C5RO. Ionizing radiation was demonstrated to enhance significantly higher survival decrease of XP2SP and CS1SP fibroblasts, compared to healthy donor's cells, according to the cloning efficiency criterion. In contrast to this, no significant difference between XP2SP and healthy donor's cells was found, according to cells' ability to replicative DNA synthesis after gamma irradiation. Differences in survival of mutant cells and healthy donor's cells after treatment by epoxides were found significant only following XP2SP being treated by ethylene oxide. DNA single-string breaks in XP2SP and in CS1SP cells treated by mutagens studied were proved to occur with the same frequency as in the DNA of the control cells; however the DNA repair according to this criterion was significantly suppressed in mutant cells.

Propylene oxide and epichlorohydrin induce DNA strand breaks in human diploid fibroblasts.

The induction of DNA strand breaks in human diploid fibroblasts (VH-10) was demonstrated after in vitro exposure with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH). Alkaline DNA unwinding (ADU), pulsed field gel electropharosis (PFGE), and the comet assay were used to measure DNA single. (SSBs) and double-strand breaks (DSBs). A dose-dependent increase of DNA strand breaks, measured by ADU, was observed in the dose range 2.5-20 mMh of PO and 0.25-2 mMh of ECH. The dose-response of ECH was about five times higher compared with that of PO (211 vs. 41 SSBs. 100 Mbp-1.mMh-1). The induction rates of DSBs, measured by PFGE, were found to be 18 times higher for ECH compared to PO (4.8 and 0.27 DSBs.100 Mbp-1.mMh-1 for ECH and PO, respectively). Using these two methods, the SSBs/ DSBs ratio was estimated to be 148 for PO and 44 for ECH. The data obtained by the comet assay also demonstrated a dose-dependent ability of PO and ECH to induce DNA damage. It was found that ECH was about six times more effective as an inducer of DNA strand breaks compared to PO (200 and 32x100 Mbp-1.mMh-1 for ECH and PO, respectively). The SSBs/DSBs ratios calculated using comet assay and PFGE data were 125 for ECH and 41 for PO. In addition, ECH is about 10 times more toxic than PO with respect to survival. These properties of ECH can at least in part be explained by its higher chemical reactivity connected with a higher rate of DNA alkylation.

Relationship between radiation induced adaptive response in human fibroblasts and changes in chromatin conformation.

Chromatin conformation changes in the normal human fibroblasts VH-10 were studied by the method of anomalous viscosity time dependence (AVTD). Gamma-irradiation of cells in a dose range of 0.1-3 Gy caused an increase in maximal viscosity of cell lysates. Conversely, irradiation of cells with low doses of 0.5 or 2 cGy resulted in a decrease in the AVTD peaks with a maximum effect approximately 40 min after irradiation. The same exposure conditions were used to study a possible adaptive effect of low doses, measured by changes in cell survival. A primary dose of 2 cGy caused significant modification of cell response to a challenge dose. Approximately 20% protection to challenge doses of 0.5 Gy (p < 0.003), 2 Gy (p < 0.02) and 2.5 Gy (p < 0.002) was observed. However, the direction of this effect (adaptation or synergism) was found to be dependent on a challenge dose. The combined effect of 2 cGy and 1 Gy was significantly synergistic, while no modification was observed for 1.5 Gy and 3 Gy. A partial correlation was found between the AVTD changes and cell survival when the combined effect of a primary dose of 2 cGy and challenge dose was examined. The dose of 2 cGy alone increased survival by 16% (p < 0.0003). These results suggest that the low-dose induced effects on survival may be related to chromatin reorganization.

Radiation-induced neoplastic transformation of stationary phase C3H/10T1/2 cells in response to different dose rates and to post-irradiation treatment with tumor promoter.

The induction of neoplastic transformation by exposure to high (HDR, 0.66 Gy/min) or very low (LDR, 4.8 x 10(-4) Gy/min) dose rates of 137Cs gamma-rays was studied in C3H/10T1/2 mouse embryo fibroblasts. Cells in stationary phase were exposed in the dose range 1-6 Gy in combination with a post-irradiation treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). The post-irradiation treatment with TPA during 6 weeks of transformation assay did not induce any notable increase in the slope of the dose response curves for transformation frequency, compared to the conditions without TPA treatment. The lack of an enhancing TPA effect at both dose rates applied in this study may be related to the fact that the cells were irradiated in the stationary growth phase. Thus, the results differ from those generally obtained when exponentially growing cells are exposed to gamma-rays and afterwards treated with TPA in the transformation assay. Earlier studies of exponentially growing C3H/10T1/2 cells exposed to different dose rates show a significantly higher transformation frequency for high dose rate. This study, using stationary phase cells, also shows that the slopes of dose response curves for transformed foci were somewhat higher (about 1.5-fold) for HDR exposure compared with LDR exposure. However, the difference was not statistically significant.

Comparison of propylene oxide and epichlorohydrin effects in two transformation tests (C3H/10T1/2 and SHE cells).

The neoplastic cell transformation induced by propylene oxide (PO) and epichlorohydrin (ECH) was studied in two in vitro assays, mouse embryo fibroblasts (C3H/10T1/2) and Syrian hamster embryo (SHE) cells. In C3H/10T1/2 cells treated with PO (2.5-10 mM), the transformation frequencies were enhanced about 2-4 times in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with the transformation frequencies in the absence of TPA. In SHE cells, an even higher increase (about 6-9 times) was reached at concentrations of 2.5-20 mM. The presence of TPA strongly influenced the ability of ECH to induce the morphological transformation at low-moderate concentrations (0.25-1 mM). At the highest concentrations applied, 1 mM in C3H/10T1/2 cells and 0.5 mM in SHE cells, 41- and 4-fold increases, respectively, were observed. In C3H/10T1/2 cells, the rad-equivalence (rad/mMh) of PO and ECH in the presence of TPA was calculated to be 36 +/- 8 and 296 +/- 65 (mean +/- S.E.), respectively.

Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide.

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.

Induction of DNA strand breaks by ethylene oxide in human diploid fibroblasts.

In vitro exposure of normal human diploid fibroblasts (strain VH-10) to ethylene oxide (EtO) induced DNA strand breaks in the dose range of 2.5-30 mMh of EtO. Alkaline DNA unwinding (ADU), neutral filter elution (NFE), pulsed field gel electrophoresis (PFGE), and the comet assay were used to measure DNA single (SSBs) and double strand breaks (DSBs). Different induction rates of SSBs and DSBs, depending on applied method and also on treatment conditions (cells in monolayer or in suspension were used), were found. A dose-dependent increase of DNA strand breaks was found by the ADU method in the dose range of 2.5-20 mMh of EtO when treatment was performed in monolayer and in suspension. DSBs were detected by NFE only when the cells were treated with EtO in suspension (doses 10-30 mMh). The highest induction rate of DSBs (about 4 DSBs per 100 Mbp per 1 mMh of EtO) was detected in suspension with PFGE applied. We have shown that heat-labile sites are formed by EtO. Presumably, the different DSB levels detected by PFGE and NFE result from the conversion of these sites to DSBs during cell lysis at elevated temperature in the PFGE method. The results of the comet assay confirmed that apoptotic processes are not involved in the formation of DSBs in our experimental conditions (less than 1% of apoptotic cells were observed at all doses studied). Possible mechanisms for the induction of DNA strand breaks by EtO-treatment are discussed. The capacity to repair DSBs in EtO-exposed (5-7.5 mMh) cells was studied, and it was found that a considerable part of the damage (about 50%) could be repaired during 18 hr of incubation.

Molecular analysis of ethylene oxide-induced mutations at the HPRT locus in human diploid fibroblasts.

Ethylene oxide (EtO)-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were characterized in 28 independently derived 6-thioguanine-resistant human diploid fibroblast clones using polymerase chain reaction-based techniques and Southern blot analysis. Sequence analysis revealed one single base pair deletion and 13 base substitutions, nine of which were transversions: five AT-->TA, three GC-->TA and one GC-->CG. Four mutants were found to have GC-->AT transitions. Seven of the point mutations caused splicing errors. Six occurred in splice site sequences and one created a new splice acceptor site 16 bp upstream of exon 9. Three splice mutations were localized at the same site in the splice donor sequence of intron 8. Fourteen mutants had large HPRT gene deletions. In seven mutants the entire HPRT gene was deleted. The remaining deletion mutants had a truncated HPRT gene, where one or several exons were lost. These results show that EtO induces many different kinds of HPRT mutations, among which as many as 50% are large deletions.

Inhibition of recA induction by the radioprotector 2-mercaptoethylamine.

Our earlier finding that the radioprotective action of 2-mercaptoethylamine (MEA) is counteracted by ascorbate suggests a biochemical mechanism of action, which is supported by observations that MEA is not radioprotective in Rec- E. coli strains. In this study we show that MEA inhibits the induction of the recA gene by UV- or gamma-irradiation or by nalidixic acid in Escherichia coli strain GE94, which contains a recA-lacZ fusion. This effect, which may be counteracted by cysteine, indicates that in general MEA inhibits the induction of SOS functions.

Mutagenic and cytotoxic action of heated pork meat extracts in human diploid fibroblasts.

Extracts from lean pork heated at 200 degrees C have a strong mutagenic activity in the Ames Salmonella assay (strain TA98 +S9). The formation of mutagenicity is highly temperature dependent, thus an extract heated at 100 degrees C is not mutagenic in this system. This paper shows that the 200 degrees C extract also causes mutations at the hprt locus in normal human fibroblasts, as demonstrated by a dose-dependent increase of 6-thioguanine resistant mutants. The mutation frequencies were increased 6 and 13 times, respectively, for extract concentrations corresponding to 100 and 200 mg meat/ml medium. Heterocyclic amines, previously shown to be present in the 200 degrees C extract are conceivably responsible for at least part of the observed mutagenicity. The extracts prepared at 100 degrees C had no significant effect on the mutant frequency in human fibroblasts. A pronounced, dose-dependent, decrease in cell survival was observed with both the 100 and the 200 degrees C extracts. The nature of the cytotoxic components is not clear and might be different in the two extracts.

Induction of 6-thioguanine-resistant mutants in human diploid fibroblasts in vitro with ethylene oxide.

Human diploid fibroblasts (strain VH-10) were exposed to the direct-acting alkylating agent, ethylene oxide (EtO), in vitro, and the frequency of HPRT mutants was evaluated by selection in medium containing 6-thioguanine. A dose-dependent increase of the mutant frequency was found in the dose range of 2.5-10 mMh of EtO. The EtO-induced mutant frequency increased 5-19 times the background frequency at low or moderately toxic doses, which indicates that EtO is a strong mutagen in human fibroblasts in vitro. The mutagenic potency was 9.8 x 10(-6) per mMh.

Studies of the rad-equivalence of ethylene oxide in the presence and absence of 12-O-tetradecanoylphorbol-13-acetate (TPA) in C3H/10T1/2 cells.

Cell transformation in vitro of C3H/10T1/2 cells, using gamma-radiation and ethylene oxide (EtO), in both the absence and presence of the cancer promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), was studied. TPA promotes transformation of C3H/10T1/2 cells to the same extent. In the dose ranges studied the average enhancement of the transformation frequency was 2.4 and 2.5 for EtO and gamma-radiation, respectively. The rad-equivalence of EtO in the presence of TPA was calculated to be 75 +/- 52 rad/mMh (95% confidence interval) which is consistent with the value 78 +/- 14 rad/mMh (95% confidence interval) obtained without TPA treatment.

Radioprotective effect of extract from Spirulina platensis in mouse bone marrow cells studied by using the micronucleus test.

The radioprotective effect of an extract of Spirulina platensis has been studied using the micronucleus test in polychromatic erythrocytes of bone marrow of mice. In this system the extract caused a significant reduction of the micronucleus frequencies induced by gamma-radiation.

Modifying action of gamma-radiation in mutagenesis of E. coli WU36-10 induced by ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate.

The influence of gamma-radiation pre-exposure on ethylene oxide, ethyl methanesulfonate and methyl methanesulfonate mutagenesis in Escherichia coli WU36-10 was studied. Pretreatment with gamma-radiation resulted, in the case of subsequent treatment with ethylene oxide and ethyl methanesulfonate, in a decrease of the frequency of leu+ revertants, and in the case of subsequent treatment with methyl methanesulfonate, in an increase of this mutation frequency.