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Possible role of placental glutathione-S-transferase (GST-P) during chemical hepatocarcinogenesis in the rat
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-
396
sions appear. This latency period may be shortened by the use of liver promoters or other hepatocarcinogens like 2-acetylaminofluorene (2AAF). If rats are partially hepatectomized in the presence of 2-AAF, this latency period is even shorter: 2-AAF may be able to initiate other cells and to suppress normal hepatocyte proliferation. In this work we studied the response of 2 preneoplastic markers through 4 experimental models: in the liver we routinely measured 7glutamyltransferase activity (yGT), usually considered as a choice preneoplastic marker in chemical hepatocarcinogenesis and placental type glutathione-S-transferase (GST-P), also involved in chemical hepatocarcinogenesis though its precise role still remains unclear. When young adult rats were submitted to Solt and Farber's model using DEN as the initiating carcinogen, then fed 2-AAF in the diet for 2 weeks and partially hepatectomized at mid-point, both GST-P expression and y G T activity were greatly increased. When partial hepatectomy was not performed (DEN initiation and 2-AAF in the diet only), the levels of the 2 markers were lower. When rats were only initiated with DEN or initiated and partially hepatectomized (without 2AAF in the diet in both models), GST-P expression and 7GT activity were even weaker. These results suggest that GST-P expression could be correlated with the coming of neoplastic lesions and might result from some synergistic mechanism of the carcinogens (DEN and 2-AAF) used. This phenomenon is still more marked when young adult rats are partially hepatectomized after DEN administration and in the presence of 2AAF. Therefore, the GST-P marker would be an interesting tool to study neoplastic development at an early stage.
Effects of chlorpromazine on the structure of nucleic acids and cytogenetic studies in combination with adriamycin in Ehrlich ascites cells in vivo a n d / o r with caffeine in human lymphocytes in vitro
Lialiaris, T. 1, and A. Pantazaki 2, 1 Medical Research Institute of Alexandroupolis and 2 Laboratory of Biochemistry, Department of Chemistry,
Aristotelian University of Thessaloniki, Thessaloniki (Greece) We have studied the action of chlorpromazine (CPZ) upon (a) linear DNA of X bacteriophage, (b) double-stranded DNA isolated from Ehrlich ascites tumor (EAT) cells, (c) RNA and singlestranded DNA isolated from EAT cells and (d) supercoiled plasmid pKS (M13 + ) of E. coli. Our findings indicate that CPZ causes damage and scissions on the nucleic acids at concentrations much higher than the one used in our cytogenetic experiments and the produced damage depends on the concentrations used. EAT cells taken from mice treated in vivo with CPZ alone were found to have increased sister-chromatid exchange (SCE) levels and in combination with adriamycin (ADR) increased cell-division delays. In human lymphocytes in vitro CPZ was found to induce cell-division delays and to have no effect on SCEs and on mitotic indices (MIs). CPZ induces cytogenetic effects: (a) in combination with caffeine (CAF) or with A D R producing cell-division delays and suppression of MIs, (b) in combination with A D R and CAF as can be deduced from the observed synergism on induction of SCEs, cell division-delays and suppressed MIs. It appears that the double combination CPZ + A D R in vivo and the triple one with CAF in vitro induce higher cytogenetic damages and this may be due to CPZ, which acts upon the membranes, allowing CAF and A D R to enter the cells more easily. As a consequence A D R acts more effectively upon DNA since CAF inhibits DNA repair.
Comparative study on cytogenetic damage and on antineoplastic effects induced by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenyi propenoic acid in human lympocytes in vitro and in P ~ leukemia cells in vivo
Papageorgiou, A. 1, L. Boutis 2, D. Mourelatos 2 P. Catsoulacos 3 and J. Dozi-Vassiliades 2 1 Laboratory of Experimental Chemotherapy, Theagenion Cancer Hospital, Thessaloniki, 2 Department of General Biology, Faculty of Medicine, Aristotelian University, Thessaloniki
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-
396
sions appear. This latency period may be shortened by the use of liver promoters or other hepatocarcinogens like 2-acetylaminofluorene (2AAF). If rats are partially hepatectomized in the presence of 2-AAF, this latency period is even shorter: 2-AAF may be able to initiate other cells and to suppress normal hepatocyte proliferation. In this work we studied the response of 2 preneoplastic markers through 4 experimental models: in the liver we routinely measured 7glutamyltransferase activity (yGT), usually considered as a choice preneoplastic marker in chemical hepatocarcinogenesis and placental type glutathione-S-transferase (GST-P), also involved in chemical hepatocarcinogenesis though its precise role still remains unclear. When young adult rats were submitted to Solt and Farber's model using DEN as the initiating carcinogen, then fed 2-AAF in the diet for 2 weeks and partially hepatectomized at mid-point, both GST-P expression and y G T activity were greatly increased. When partial hepatectomy was not performed (DEN initiation and 2-AAF in the diet only), the levels of the 2 markers were lower. When rats were only initiated with DEN or initiated and partially hepatectomized (without 2AAF in the diet in both models), GST-P expression and 7GT activity were even weaker. These results suggest that GST-P expression could be correlated with the coming of neoplastic lesions and might result from some synergistic mechanism of the carcinogens (DEN and 2-AAF) used. This phenomenon is still more marked when young adult rats are partially hepatectomized after DEN administration and in the presence of 2AAF. Therefore, the GST-P marker would be an interesting tool to study neoplastic development at an early stage.
Effects of chlorpromazine on the structure of nucleic acids and cytogenetic studies in combination with adriamycin in Ehrlich ascites cells in vivo a n d / o r with caffeine in human lymphocytes in vitro
Lialiaris, T. 1, and A. Pantazaki 2, 1 Medical Research Institute of Alexandroupolis and 2 Laboratory of Biochemistry, Department of Chemistry,
Aristotelian University of Thessaloniki, Thessaloniki (Greece) We have studied the action of chlorpromazine (CPZ) upon (a) linear DNA of X bacteriophage, (b) double-stranded DNA isolated from Ehrlich ascites tumor (EAT) cells, (c) RNA and singlestranded DNA isolated from EAT cells and (d) supercoiled plasmid pKS (M13 + ) of E. coli. Our findings indicate that CPZ causes damage and scissions on the nucleic acids at concentrations much higher than the one used in our cytogenetic experiments and the produced damage depends on the concentrations used. EAT cells taken from mice treated in vivo with CPZ alone were found to have increased sister-chromatid exchange (SCE) levels and in combination with adriamycin (ADR) increased cell-division delays. In human lymphocytes in vitro CPZ was found to induce cell-division delays and to have no effect on SCEs and on mitotic indices (MIs). CPZ induces cytogenetic effects: (a) in combination with caffeine (CAF) or with A D R producing cell-division delays and suppression of MIs, (b) in combination with A D R and CAF as can be deduced from the observed synergism on induction of SCEs, cell division-delays and suppressed MIs. It appears that the double combination CPZ + A D R in vivo and the triple one with CAF in vitro induce higher cytogenetic damages and this may be due to CPZ, which acts upon the membranes, allowing CAF and A D R to enter the cells more easily. As a consequence A D R acts more effectively upon DNA since CAF inhibits DNA repair.
Comparative study on cytogenetic damage and on antineoplastic effects induced by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenyi propenoic acid in human lympocytes in vitro and in P ~ leukemia cells in vivo
Papageorgiou, A. 1, L. Boutis 2, D. Mourelatos 2 P. Catsoulacos 3 and J. Dozi-Vassiliades 2 1 Laboratory of Experimental Chemotherapy, Theagenion Cancer Hospital, Thessaloniki, 2 Department of General Biology, Faculty of Medicine, Aristotelian University, Thessaloniki