International Commission for Protection Against Environmental Mutagens and Carcinogens. Cyclosporine A: review of genotoxicity and potential for adverse human reproductive and developmental effects. Report of a Working Group on the genotoxicity of cyclosporine A, August 18, 1993.

Cyclosporine is an important therapeutic agent for transplant recipients and for a growing number of autoimmune diseases. Experimental animal and human data has indicated that cyclosporine is unlikely to be genotoxic. In contrast, azathioprine, an agent often given with cyclosporine, is considered to be genotoxic making the assessment of the independent effects of cyclosporine difficult. Cyclosporine does appear to be related to the development of tumors, primarily lymphomas, in animals and humans, but the basis of its potential carcinogenicity is not completely understood. In terms of reproductive and developmental toxicity, cyclosporine produces some adverse effects in both experimental animals and humans. In animals, the effects are seen at high doses sufficient to cause maternal toxicity. In humans, outcomes such as growth retardation have been noted, but the confounding effects of renal toxicity and resultant pregnancy complications cloud the interpretation. An increase in congenital anomalies and genetic disease have not been found reported in human studies that are limited in sample size. Given that the present data indicate the lack of genotoxicity, a mutation epidemiology study of cyclosporine is not recommended. As indicated above, such a study is probably impractical for many genetic endpoints of interest. However, well-conducted, large multicenter studies of transplant patients and their offspring would allow for routine monitoring of an increased risk for some reproductive and developmental (possibly non-genetic) endpoints that are of public health importance.

No cyclosporin-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro.

Continuous in vitro treatment of human peripheral blood lymphocytes with cyclosporin A for 20 h and 44 h in the absence of metabolic activation decreased the mitotic index in a concentration-dependent way (MI about 35% at 50 micrograms/ml). In the presence of 10% S9 lymphocytes were exposed to cyclosporin A at concentrations which formed cloudy suspension, i.e., above 100 micrograms/ml. A 2 h treatment in the presence of metabolic activation showed a depression of the mitotic index at the 44 h fixation time only. Based upon results concerning the depression of the mitotic index, slides of the 5, 10, 25 and 50 micrograms/ml (-S9, 20 h and 44 h continuous treatment) and 5, 150, 250 and 300 micrograms/ml (+S9, 2 h pulse treatment) treatment groups were selected to be analyzed in the chromosomal aberration assay. At no concentration and at no fixation time did cyclosporin A increase the frequency of cells with chromosomal aberrations.

Induction of mitotic aneuploidy using Chinese hamster primary embryonic cells. Test results of 10 chemicals.

Using primary Chinese hamster embryonic cells, 10 known or suspected aneugens supplied as a part of the EC 4th Environmental Research and Development Programme were evaluated by the technique described by Dulout and Natarajan (1987). The chemicals included cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vincristine. All chemicals except pyrimethamine gave clearly positive effect at most of the doses tested. The ease with which the assay is performed and reproducible results that are obtained with the suspected compounds indicate that this in vitro test using primary embryonic fibroblasts is a promising one for routine screening.

Comparative analysis of the clastogenicity and cytotoxicity of airborne particulate matter generated during the fire at Schweizerhalle on November 1, 1986.

Methanol extracts from 4 pairs of airconditioner filters (one fire-exposed and one control) from various locations (A, B, C and D) at various distances from the site of the fire were examined for their capacity to induce structural chromosomal aberrations and/or cytotoxicity in Chinese hamster V79 cells. Extracts from 2 additional sets of 3 filters which were exposed to urban air for 3 consecutive periods of 10 or 11 days some 4 months after the fire were also tested. Chromosomal aberrations were induced by all filter extracts from location B, as well as by an unused (non-exposed) filter, in a dose-dependent manner. Without the addition of metabolizing enzymes, aberrations were induced only at concentrations which caused more than 95% cell killing. This was not taken as an indication for clastogenic activity of the filter extracts, but was assumed to represent the chromosomal expression of metabolic changes in dying cells. Upon the addition of S9, chromosomal aberrations were induced at biologically relevant survival rates. Under metabolizing conditions, the ranking of the potential of the filter extracts from location B to induce chromosomal aberrations and to cause cell killing was identical. The remaining extracts (locations A, C and D) were therefore tested for cytotoxicity only. The toxicity data indicated that, of 3 pairs of filters, the fire-exposed one was not different from the control. Of the fourth pair (location B), the fire-exposed filter was 2.0-2.5 times more cytotoxic and clastogenic than the control. However, extracts of urban air-exposed filters from this location (exposed in March and April 1987) showed a large variation in toxicity and clastogenicity as well. One was clearly more active than the control (but less than the fire-exposed filter), while the other 2 were either somewhat more or less clastogenic than the control filter. In addition, 4 out of 5 filters from this location were more polluted (as indicated by cytotoxicity) than all the filters from the other locations, irrespective of whether they were fire-exposed or not. It is concluded that the results of this V79 cytotoxicity/clastogenicity test did not confirm the hypothesis that the fire at Schweizerhalle produced clastogenic material at quantities detectable under the conditions employed.

3-Aminobenzamide sensitizes cultured Chinese hamster cells with bromodeoxyuridine-substituted DNA to genetic damage induced by long-wave UV.

Chinese hamster cells, containing BrdUrd-substituted DNA, were irradiated in the presence of 3-aminobenzamide with short-wave UV, long-wave UV or X-rays and analyzed for induced SCEs or chromosomal aberrations. The data presented in this paper show that when BrdUrd-substituted cells are irradiated with lw-UV in the presence of 3-aminobenzamide, genetic damage is increased. Biochemical analysis shows that the molecular weight of BrdUrd-substituted DNA is reduced by this treatment. The sensitization is due to the combined action of lw-UV, incorporated BrdUrd and 3-aminobenzamide, without any involvement of inhibition of poly(ADP-ribose) synthetase. No potentiation occurs when cells are irradiated with X-rays and genetic damage is decreased when cells are irradiated with UV light of 254 nm in the presence of 3AB. This decrease coincides with a reduction in the amount of induced pyrimidine dimers, detected as T4 endonuclease-sensitive sites in DNA.

Effects of 3-aminobenzamide on Chinese hamster cells treated with thymidine analogues and DNA-damaging agents. Chromosomal aberrations, mutations and cell-cycle progression.

The nuclear enzyme, poly(ADP-ribose) synthetase is involved in the repair of damaged DNA. We report here the results obtained with 3-aminobenzamide (3AB), an inhibitor of this enzyme, on induced biological effects. 3AB increases the frequency of chromosomal aberrations induced by DMS, EMS, ENU, bleomycin and CldUrd. The magnitude of the effect is dependent on the type of chemical used, the combinations with DMS and EMS being the most potent ones. No potentiation was observed after treatment of cells with MMC. Mutation frequencies were determined on the HPRT locus and showed that 3AB did not increase the frequency of gene mutations induced by EMS, ENU and CldUrd. Cell-cycle progression is affected when cells are grown in medium containing CldUrd and 3AB, primarily when the inhibitor is present during the second cell cycle when substituted DNA becomes replicated. The extent of the effect depends on the amount of analogue incorporated and is independent of the presence of the analogue in the medium during the second cell cycle. Analysis of chromosomal aberrations in delayed G2 cells with the aid of the premature chromosome-condensation technique revealed numerous aberrations after incorporation of CldUrd and treatment with 3AB.

Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells.

Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations. In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor. The significance of these results is discussed.

DNA lesions, chromosomal aberrations and G2 delay in CHO cells cultured in medium containing bromo- or chloro-deoxyuridine.

Experiments have been performed to investigate whether BrdUrd- and CldUrd-substituted DNA contains lesions causing a delay in cell-cycle progression and induction of chromosomal aberrations. The presence of lesions has been determined directly by alkaline sucrose gradient and nucleoid sedimentation analysis and indirectly by screening for induced chromosomal aberrations. The influence of inhibitors of DNA repair (caffeine and 3-aminobenzamide) or DNA synthesis (hydroxyurea) on the frequencies of such aberrations has been estimated. It is found that BrdUrd and CldUrd are cytotoxic when present in DNA. No randomly located DNA breaks could be detected under neutral conditions, but BrdUrd-substituted DNA was found to contain numerous alkali-labile sites. CldUrd at high concentrations causes G2 delay, similar to the action of known DNA-damaging agents. The extent of delay depends on the pattern of incorporation of the analogue, i.e., incorporation for two cell cycles causes the longest delay, growth for 12 h in CldUrd followed by 12 h in dThd-containing medium causes a lesser delay and the delay is not significant when the cells are incubated in the analogue for only 12 h prior to fixation. Numerous chromatid type aberrations are present in cells incubated at the highest CldUrd concentration, and their induction follows the pattern of induction of G2 delay, indicating the sharing of a common lesion. Caffeine, 3-aminobenzamide and hydroxyurea increase the number of chromosomal aberrations when added 2 h before fixation. The significance of these results is discussed.

3-Aminobenzamide-induced sister chromatid exchanges are dependent on incorporated bromodeoxyuridine in DNA.

Data are presented establishing a direct correlation between 3-aminobenzamide-induced sister chromatid exchange (SCE) frequency and the level of bromodeoxyuridine (BrdU) incorporated into DNA. In addition, it is shown that most of the SCEs are induced in the second cell cycle, in which BrdU-containing DNA is used as the template for replication.

Induction of DNA lesions, chromosomal aberrations, and G2 delay by bromo- and chlorodeoxyuridine.

The presence of lesions in DNA of CHO cells, substituted with BrdUrd or CldUrd, has been investigated in a direct way by alkaline sucrose gradient and nucleoid sedimentation analysis and indirectly by screening for induced CAs. The influence of inhibitors of DNA repair (Caff and 3AMB) or DNA synthesis (HU) on the frequencies of such aberrations has been estimated. No randomly located DNA breaks could be detected under neutral conditions, but BrdUrd-substituted DNA was found to contain numerous alkali labile sites. At high concentrations, CldUrd causes G2 delay, similar to the action of known DNA-damaging agents. The extent of delay depends on the pattern of incorporation of the analog, i.e., incorporation for 2 cell cycles causes the longest delay, growth for 12 hr in CldUrd followed by 12 hr in dThd- containing medium gives less delay, and delay is not significant when the cells are incubated in the analog for only 12 hr prior to fixation. Numerous chromatid-type aberrations are present in cells incubated at the highest CldUrd concentration and their induction follows the pattern of induction of G2 delay, indicating the involvement of a common lesion. 3AMB, and HU increase the number of CAs when added 2 hr before fixation. The significance of these results is discussed.

Standardized shake-off to synchronize cultured CHO cells.

A protocol to obtain CHO cells synchronized in mitosis is described. The synchronized population contained hardly any S-phase cells as judged by the incorporation of 3H-TdR. In these cells a very low frequency of chromatid-type aberrations compared with chromosome-type aberrations was induced by X-rays at 1.5 h after the synchronization.

Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair, chromosomal alterations, and mutations.

The influence of inhibitors of poly(ADP-ribose) polymerase such as 3-aminobenzamide (3AB) and benzamide (B) on the spontaneously occurring as well as mutagen induced chromosomal aberrations, sister chromatid exchanges (SCEs) and point mutations has been studied. In addition, we have measured the influence of 3AB on DNA repair following treatment with physical and chemical mutagens. Post treatment of X-irradiated mammalian cells with 3AB increases the frequencies of induced chromosomal aberrations by a factor of 2 to 3. Both acentric fragments and exchanges increase indicating that the presence of 3AB slows down the repair of DNA strand breaks (probably DNA double strand breaks), thus making breaks available for interaction with each other to give rise to exchanges. 3AB, when present in the medium containing bromodeoxyuridine(BrdUrd) during two cell cycles, increases the frequencies of SCEs in Chinese hamster ovary cells (CHO) in a concentration dependent manner leading to about a 10-fold increase at 10 mM concentration. Most 3AB induced SCEs occur during the second cell cycle, in which DNA containing bromouridine (BU) is used as template for replication. BU containing DNA appears to be prone to errors during replication. The extent of increase in the frequencies of SCEs by 3AB is correlated with the amount of BU incorporated in the DNA of the cells. The frequencies of spontaneously occurring DNA single strand breaks in cells grown in BrdUrd containing medium are higher than in the cells grown in normal medium and this increase depends on the amount of BU incorporated in the DNA of these cells. We have studied the extent of increase in the frequencies of SCEs due to 1 mM 3AB in several human cell lines, including those derived from patients suffering from genetic diseases such as ataxia telangiectasia (A-T), Fanconi's anemia (FA), and Huntington's chorea. None of these syndromes showed any increased response when compared to normal cells. 3AB, however, increased the frequencies of spontaneously occurring chromosomal aberrations in A-T and FA cells. 3AB does not influence the frequencies of SCEs induced by UV or mitomycin C (MMC) in CHO cells. However, it increases the frequencies of SCEs induced by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS). Under the conditions in which 3AB increases the frequencies of spontaneously occurring as well as induced SCEs, it does not increase the frequencies of point mutations in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus. 3AB does not influence the amount of repair replication following dimethylsulphate (DMS) treatment of human fibroblasts, or UV irradiated human lymphocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms for chromosomal aberrations in mammalian cells.

Experimental evidence is presented for the involvement of DNA double-strand breaks in the formation of radiation-induced chromosomal aberrations. When X-irradiated cells were post-treated with single-strand specific Neurospora crassa endonuclease (NE), the frequencies of all classes of aberration increased by about a factor 2. Under these conditions, the frequencies of DNA double-strand breaks induced by X-rays (as determined by neutral sucrose-gradient centrifugation), also increased by a factor of 2. The frequency of chromosomal aberrations induced by fast neutrons (which predominantly induce DNA double-strand breaks) was not influenced by post-treatment with NE. Inhibition of poly(ADP-ribose) polymerase, an enzyme that uses DNA with double-strand breaks as an optimal template, by 3-aminobenzamide also increased the frequencies of X-ray-induced chromosomal aberrations, which supports the idea that DNA double-strand breaks are important lesions for the production of chromosomal aberrations induced by ionizing radiation.

Clonal analysis of radiation-induced translocations in stem-cell spermatogonia of normal and T70H translocation heterozygous mice.

7 T(1;13)70H/+ and 13+/+ male mice were given 2 doses of 250 rad acute X-rays separated by 24 h. The +/+ mice were analysed in 2 groups during the first meiotic division for induced translocations, on average 177 and 233 days after irradiation, and the T70H/+ mice were analysed in parallel with the second group of +/+ males. One testis was treated with normal air-drying procedures yielding a random sample of cells. The other testis was processed according to a new technique, which enable separate analysis of the various locations along the seminiferous epithelium where groups of cells are synchronously in the diakinesis-metaphase I stage of meiosis. The number of cells in such groups was estimated. Both capita epididymes were used for a sperm count. In agreement with an earlier finding, fewer induced translocations were recovered from the T70H/+ mice than from +/+ mice (10.6 versus 19.2%, air-drying technique). Estimates of the group sizes in combination with the occurrence of induced translocations yielded the following information. A synchronously moving group of diakinesis-metaphase I cells originates from, on average, 1.25 stem cells (Appendix). We found an indication for a reduction in group size by 33% when a clone originated from a stem cell carrying an induced translocation compared with a wild-type clone (see Appendix). Both, the data on group size and the sperm counts indicate that, 7 months after the irradiation, the seminiferous epithelium has not totally recovered. Final recovery seems to be slower or absent in the T70H/+ males. The data obtained from the T70H/+ heterozygotes indicate the stem-cell spermatogonia to be responsible for the reduction of the rate or translocation induction with this karyotype, either due to a reduced formation rate or due to a diminished capacity of some of the induced translocation-carrying stem cells to proliferate into a clone reaching the meiotic divisions.