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Possible causes of variability of the adaptive response in human lymphocytes
394
Modulation of heat shock protection against chromatid aberrations induced by chemical mutagens
(Finland) and 1 International Agency for Research on Cancer, Lyon (France)
Nicoloff, H. 1, R. Rieger 2 and A. Michaelis 2, 1 Institute of Genetics, Bulgarian Academy of Science, Sofia (Bulgaria) and 2 Central Institute of Genetics, Academy of Sciences, Gatersleben (G.D.R.)
Styrene has been shown to induce chromosome damage in cultured cells in the presence of erythrocytes, probably through its metabolic activation by oxyhemoglobin to styrene-7,8-oxide. Provided this reaction also occurs in vivo, leukocytes in blood circulation and cells of the erythrocytic series are theoretical targets for the genotoxic action of styrene. In nucleated erythrocytic cells producing hemoglobin the metabolic activation system (hemoglobin) and the biological target (DNA) are in the same cells, which might be important for the development of malignancies of the erythrocytic series. The role of in situ hemoglobin production in the metabolic activation of styrene - measured by the induction of sisterchromatid exchanges (SCEs) - was studied in vitro in Friend murine erythroleukemia cells (cell lines 19-101 and 19-9a) which can be induced to differentiate and to produce hemoglobin. Styrene (treatment for the last 48 h) induced SCEs both in undifferentiated cells (at 2-3 mM; 3-day cultures) and in cells that were allowed to differentiate for 3-5 days (at 1 mM) after induction by hexamethylenebisacetamide (HMBA). Thus the erythroleukemia cells were able to activate styrene regardless of extra hemoglobin production. Nevertheless, styrene induced slightly more SCEs and was also more toxic in the differentiating cells than in the non-differentiating cells, which suggested some hemoglobin-dependent metabolism of styrene. Differentiation of erythroleukemia cells has been reported to be accompanied by increased DNA-repair capacity and loss of membrane-bound P-450, both of which may contribute to the lack of dramatic differences in SCE induction by styrene in response to differentiation.
Heat shock (hs) (10 min at 40°C) prior to challenge treatment with triethylenemelanine (TEM), maleic hydrazide (MH) and bleomycin (Blm) significantly reduced the frequency of induced chromatid aberrations in Vicia faba root tip meristems. A differential expression of hs protection against the clastogenic action of TEM, MH and Blm was, however, observed when novobiocin was applied prior to or after hs induction. Novobiocin treatment before hs did not prevent hs protection against the challenge treatment with the clastogens used, while novobiocin application after hs completely prevented hs protection. A drastically different effect was, however, observed when, instead of novobiocin alone, a combined posttreatment with novobiocin and actinomycin D (Act D) was applied after hs induction. With this treatment sequence, instead of a complete prevention of hs protection, the frequency of metaphases with MH-induced chromatid aberrations proved to be significantly reduced. This means that addition of Act D to novobiocin treatment results in a shift of the effect of novobiocin posttreatment by turning the complete prevention of hs protection to a clearly pronounced protection against aberration induction by MH. This implies, therefore, that Act D could effectively modify the novobiocin action on hs gene expression or structural alterations normally accompanying hs induction and thereby trigger a new protection against MH-clastogenic action.
Influence of cell differentiation on sister-chromatid exchange induction by styrene in murine erythroleukemia cells
Norppa, H., H. Yamasaki 1, H. J~irventaus and H. Vainio, Institute of Occupational Health, Helsinki
Possible causes of variability of the adaptive response in human lymphocytes
Olivieri, G., and A. Bosi, Department of Genetics and Molecular Biology, University 'La Sapienza', Rome (Italy) When human lymphocytes are cultured in triti-
395
ated thymidine (Olivieri et al., 1984; Wiencke et al., 1986) or exposed to very low doses of X-rays (Shadley and Wolff, 1987) they become less susceptible to the induction of chromatid aberrations by subsequent high doses of X-rays. These observations were taken as an indication that low doses of ionizing radiation can induce effects analogous to the adaptive response (AR) reported after treatment with alkylating agents in Escherichia coli (Samson and Cairns, 1977). Recently it has been shown (Sankaranarayanan et al., 1989; Bosi and Olivieri, 1989) that there are variations between individuals with respect to the induction of AR in their lymphocytes. In a sample of 18 different healtly donors we observed that 4 of the 18 donors did not show an adaptive response; in some cases in these individuals a synergistic response of increased, rather than decreased, damage was found. In order to understand whether the lack of A R depends on transient physiological parameters or on some stable constitutional genetically determined trait we repeated, under various e x p e r i m e n t a l conditions, experiments with lymphocytes of donors who showed an absence of AR.
Is the TG r T-Lys BrdUrd assay a useful tool for human population monitoring? Ostrosky-Wegman, P. 1, R. Montero 1, F. Moreno 1 and M. Sandoval 2, 1 Instituto de Investigaciones Biomedicas, UNAM, 04510 Mexico D F (Mexico) and 2 Hospital de Especialidades, La Raza, IMSS (Mexico) The need for a somatic cell gene mutation assay for monitoring genotoxic human exposure has been stressed. Several assays have been proposed to detect damage at that level, but the only relatively widely used system to date is the H G P R T mutation assay which detects thioguanine-resistant Tlymphocytes (TG ~ T-Lys). Some studies have shown that exposure to cytostatics and radiation produces significant increases in the frequency of T G r T-Lys. During the standardization in our laboratory of the H G P R T autoradiographic assay and its BrdUrd modification, we were asked to determine the frequency of T G r T-Lys in individ-
uals suspected of being exposed to radiation during the accidents that had occurred in Cd. Juarez (Mexico) and in Chernobyl (U.S.S.R.). In the same period we were evaluating the genotoxic effects of a mixture of putative mutagenic agents (tomography, anticonvulsants, antibiotics, anesthetics and antiinflammatory drugs) which had been used in the diagnosis and treatment of neurocysticercotic patients and we decided to look for H G P R T point mutations as well. T G r T-Lys frequencies obtained in the individuals suspected of radiation exposure, in the neurocysticercotic patients under putative multiple mutagenic treatments and in individuals not known to be exposed to genotoxic agents show that there is variability in T H r T-Lys frequencies which show a Poisson distribution: the small values correspond to individuals not exposed, while the highest values usually correlate with exposure, although the same exposure does not induce the same T G r frequency, indicating that H G P R T mutant frequency could be caused by odd agents, different to those registered or even by the heterogeneity of the population with regard to inborn sensitivity to the particular genotoxic agent. Although sample sizes are small, others' results and ours indicate that H G P R T autoradiographic or BrdUrd assays are useful to detect exposure, but it should be stressed that for population monitoring validation studies are still needed.
Possible role of placental glutathione-S-transferase (GST-P) during chemical hepatocarcinogenesis in the rat Ouldelhkim, M., M. Martin and F. Decloitre, Institut de Recherches Scientifiques sur le Cancer, U P R 278, BP 8, 94802 Villejuif Cedex (France) Chemical hepatocarcinogenesis implies important biochemical modifications followed by cellular and tissue changes leading to malignant tumor formation. Experimental models showed that when tumor-initiating agents like diethylnitrosamine (DEN) are administered to young adult rats, whether these rats are hepatectomized or not, the resulting preneoplastic lesions are followed by a latency period before neoplastic le-
Modulation of heat shock protection against chromatid aberrations induced by chemical mutagens
(Finland) and 1 International Agency for Research on Cancer, Lyon (France)
Nicoloff, H. 1, R. Rieger 2 and A. Michaelis 2, 1 Institute of Genetics, Bulgarian Academy of Science, Sofia (Bulgaria) and 2 Central Institute of Genetics, Academy of Sciences, Gatersleben (G.D.R.)
Styrene has been shown to induce chromosome damage in cultured cells in the presence of erythrocytes, probably through its metabolic activation by oxyhemoglobin to styrene-7,8-oxide. Provided this reaction also occurs in vivo, leukocytes in blood circulation and cells of the erythrocytic series are theoretical targets for the genotoxic action of styrene. In nucleated erythrocytic cells producing hemoglobin the metabolic activation system (hemoglobin) and the biological target (DNA) are in the same cells, which might be important for the development of malignancies of the erythrocytic series. The role of in situ hemoglobin production in the metabolic activation of styrene - measured by the induction of sisterchromatid exchanges (SCEs) - was studied in vitro in Friend murine erythroleukemia cells (cell lines 19-101 and 19-9a) which can be induced to differentiate and to produce hemoglobin. Styrene (treatment for the last 48 h) induced SCEs both in undifferentiated cells (at 2-3 mM; 3-day cultures) and in cells that were allowed to differentiate for 3-5 days (at 1 mM) after induction by hexamethylenebisacetamide (HMBA). Thus the erythroleukemia cells were able to activate styrene regardless of extra hemoglobin production. Nevertheless, styrene induced slightly more SCEs and was also more toxic in the differentiating cells than in the non-differentiating cells, which suggested some hemoglobin-dependent metabolism of styrene. Differentiation of erythroleukemia cells has been reported to be accompanied by increased DNA-repair capacity and loss of membrane-bound P-450, both of which may contribute to the lack of dramatic differences in SCE induction by styrene in response to differentiation.
Heat shock (hs) (10 min at 40°C) prior to challenge treatment with triethylenemelanine (TEM), maleic hydrazide (MH) and bleomycin (Blm) significantly reduced the frequency of induced chromatid aberrations in Vicia faba root tip meristems. A differential expression of hs protection against the clastogenic action of TEM, MH and Blm was, however, observed when novobiocin was applied prior to or after hs induction. Novobiocin treatment before hs did not prevent hs protection against the challenge treatment with the clastogens used, while novobiocin application after hs completely prevented hs protection. A drastically different effect was, however, observed when, instead of novobiocin alone, a combined posttreatment with novobiocin and actinomycin D (Act D) was applied after hs induction. With this treatment sequence, instead of a complete prevention of hs protection, the frequency of metaphases with MH-induced chromatid aberrations proved to be significantly reduced. This means that addition of Act D to novobiocin treatment results in a shift of the effect of novobiocin posttreatment by turning the complete prevention of hs protection to a clearly pronounced protection against aberration induction by MH. This implies, therefore, that Act D could effectively modify the novobiocin action on hs gene expression or structural alterations normally accompanying hs induction and thereby trigger a new protection against MH-clastogenic action.
Influence of cell differentiation on sister-chromatid exchange induction by styrene in murine erythroleukemia cells
Norppa, H., H. Yamasaki 1, H. J~irventaus and H. Vainio, Institute of Occupational Health, Helsinki
Possible causes of variability of the adaptive response in human lymphocytes
Olivieri, G., and A. Bosi, Department of Genetics and Molecular Biology, University 'La Sapienza', Rome (Italy) When human lymphocytes are cultured in triti-
395
ated thymidine (Olivieri et al., 1984; Wiencke et al., 1986) or exposed to very low doses of X-rays (Shadley and Wolff, 1987) they become less susceptible to the induction of chromatid aberrations by subsequent high doses of X-rays. These observations were taken as an indication that low doses of ionizing radiation can induce effects analogous to the adaptive response (AR) reported after treatment with alkylating agents in Escherichia coli (Samson and Cairns, 1977). Recently it has been shown (Sankaranarayanan et al., 1989; Bosi and Olivieri, 1989) that there are variations between individuals with respect to the induction of AR in their lymphocytes. In a sample of 18 different healtly donors we observed that 4 of the 18 donors did not show an adaptive response; in some cases in these individuals a synergistic response of increased, rather than decreased, damage was found. In order to understand whether the lack of A R depends on transient physiological parameters or on some stable constitutional genetically determined trait we repeated, under various e x p e r i m e n t a l conditions, experiments with lymphocytes of donors who showed an absence of AR.
Is the TG r T-Lys BrdUrd assay a useful tool for human population monitoring? Ostrosky-Wegman, P. 1, R. Montero 1, F. Moreno 1 and M. Sandoval 2, 1 Instituto de Investigaciones Biomedicas, UNAM, 04510 Mexico D F (Mexico) and 2 Hospital de Especialidades, La Raza, IMSS (Mexico) The need for a somatic cell gene mutation assay for monitoring genotoxic human exposure has been stressed. Several assays have been proposed to detect damage at that level, but the only relatively widely used system to date is the H G P R T mutation assay which detects thioguanine-resistant Tlymphocytes (TG ~ T-Lys). Some studies have shown that exposure to cytostatics and radiation produces significant increases in the frequency of T G r T-Lys. During the standardization in our laboratory of the H G P R T autoradiographic assay and its BrdUrd modification, we were asked to determine the frequency of T G r T-Lys in individ-
uals suspected of being exposed to radiation during the accidents that had occurred in Cd. Juarez (Mexico) and in Chernobyl (U.S.S.R.). In the same period we were evaluating the genotoxic effects of a mixture of putative mutagenic agents (tomography, anticonvulsants, antibiotics, anesthetics and antiinflammatory drugs) which had been used in the diagnosis and treatment of neurocysticercotic patients and we decided to look for H G P R T point mutations as well. T G r T-Lys frequencies obtained in the individuals suspected of radiation exposure, in the neurocysticercotic patients under putative multiple mutagenic treatments and in individuals not known to be exposed to genotoxic agents show that there is variability in T H r T-Lys frequencies which show a Poisson distribution: the small values correspond to individuals not exposed, while the highest values usually correlate with exposure, although the same exposure does not induce the same T G r frequency, indicating that H G P R T mutant frequency could be caused by odd agents, different to those registered or even by the heterogeneity of the population with regard to inborn sensitivity to the particular genotoxic agent. Although sample sizes are small, others' results and ours indicate that H G P R T autoradiographic or BrdUrd assays are useful to detect exposure, but it should be stressed that for population monitoring validation studies are still needed.
Possible role of placental glutathione-S-transferase (GST-P) during chemical hepatocarcinogenesis in the rat Ouldelhkim, M., M. Martin and F. Decloitre, Institut de Recherches Scientifiques sur le Cancer, U P R 278, BP 8, 94802 Villejuif Cedex (France) Chemical hepatocarcinogenesis implies important biochemical modifications followed by cellular and tissue changes leading to malignant tumor formation. Experimental models showed that when tumor-initiating agents like diethylnitrosamine (DEN) are administered to young adult rats, whether these rats are hepatectomized or not, the resulting preneoplastic lesions are followed by a latency period before neoplastic le-