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Chromosome studies in women who had used oral contraceptives and in their progeny
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46 N.P. Bishun and J. Mills, Research Dept., Marie Curie Memorial Foundation, the Chart (U.K.) Chromosome studies in w o m e n w h o had used oral contraceptives and in their progeny A study has been made of the chromosome numbers and characteristics of leucocytes cultured from mothers (a total of 185) who had taken one or more types of oral contraceptive, and their babies (185}, and compared with similar numbers of control subjects. When the total cytogenetic results from test and control mothers were compared, no significant differences in frequency or type of chromosome abnormalities was observed. However, with certain types of oral contraceptive, minor differences in the numbers of chromosome significance of these findings are discussed. The only significant abnormalities observed in the babies from mothers taking oral contraceptives were slight increases in the numbers who had cells with less than 46 chromosomes and who exhibited chromosomal gaps. The numbers of abnormal cells was significantly higher in babies born to mothers who had been taking norethynodrel (Conovid) {4.6% abnormal cells in test babies; 2.0% in controls).
47 B. Johansson, G. AhnstrSm and K. Ericson, Wallenberg Laboratory, University of Stockholm (Sweden) Rate of strand separation in alkaline solution -- a sensitive m e t h o d for measuring D N A repair in mammalian cells In our laboratory we have developed a simple technique to study induction and repair of strand breaks in DNA. The m e t h o d is based on the principle t h a t single strand breaks serve as untwisting points during strand separation in alkaline solution and that DNA, for a time limited treatment in alkali, is transformed into single stranded form in proportion to the number of strand breaks present initially. The single- and double-stranded DNA are separated by means of h y d r o x y l apatite chromatography. In h u m a n embryonic fibroblast and chinese hamster V-79 cells alkali labile sites are induced by different agencies such as X-ray and N-methyl-N-nitrosourea (MNU). A b o u t 50% of this damage is repaired in 10 min. After UV irradiation t h y m i n e dimers are formed which, in h u m a n cells, are removed by excision repair. The single stranded breaks induced by the cells own endonuclease can be followed as a function of the UV dose. The number of breaks increases with dose up to 40--50 ergs/sec, m m 2 where it levels off in a plateau. The n u m b e r of breaks can be enhanced by t r e a t m e n t with 3 × 10 -3 M h y d r o x y u r e a but the plateau is reached at the same dose. The time for the disappearance of the endonuclease induced breaks varies
128 with the dose. At 10 ergs/sec, m m : the control level is reached within one hour but for 75 ergs/sec, mm 2 it takes about three hours. Post replication repair is studied in hamster cells, in which no endonuclease induced breaks have been detected with this method. It is based on the principle that after UV irradiation, the newly synthesized DNA will contain strand interruptions probably opposite thymine dimers. These breaks are slowly removed after post incubation in medium but the process is inhibited by 0.2 mg/ml of caffeine, a concentration that does n o t affect DNA synthesis. The induction of excision and post replication repair after treatment with the alkylating substances methyl methanesulphonate (MMS) and N-methyl-N-nitrosourea (MNU) will be discussed.
48 R. Di Lernia, G. Della Valle, L. De Carli, O. Temelcou and A. Barbieri, Istituto di Biologia Generale, Facolt~ di Medicina, Universit~ di Milano, and Istituto Sieroterapico Milanese S. Belfanti, Milano (Italy) Relationship between the inhibition of cell survival and growth and the effects on macromolecular syntheses of meta-L-sarcolysin peptide derivatives through the cell cycle of EUE cells Different methods, based either on biological or on biochemical assays, have been assessed to determine the effects of chemical agents on cell cycle progression. Assays were carried o u t on EUE cells (from a human heteroploid line) treated with m-(di-(2-chloroethyl)-amino)-L-phenylalanine peptide derivatives (peptichemio or PTC) at a concentration near to CLDs0 (cellular lethal dose 50). The cells were synchronized by a reversible mitotic arrest with a single Colcemid treatment (0.02 pg/ml), followed by selection of mitoses using the shake method. The degree of synchronisation was monitored by scoring a mitotic index and measuring DNA synthesis in 30-min pulse labeling at successive times in the cell cycle with TdR[3H] (0.5 pCi/ml). The biological parameters examined were the following: 1) plating efficiency (PE) i.e., the ratio between the number of cell colonies formed and the number of single cells inoculated; 2) colony growth (CG), measured by the average number of cells per colony after a fixed time. Treatments with PTC were performed every two hours througho u t the cell cycle. Determinations of PE and CG on treated cultures were performed after a period of growth in the medium w i t h o u t the drug, which corresponded to a b o u t five cell doublings in the control culture. On treated cultures, incorporation rates of labelled precursors of DNA, R N A and proteins were determined by 30-rain pulses with TdR[3H] (0.5 pCi/ml), UR[3H] (0.5 pCi/ml) and Leu[3H] (0.5 pCi/ml). No effect on protein and RNA synthesis was detected, whereas a slight decrease of DNA synthesis was noted in the early " S " phase. More appreciable inhibition values of PE and CG were found for the same period and clear inhibition was also revealed in the pre-synthetic and post-synthetic periods. It can be concluded that, at the drug concentrations and exposure times used in the cell cycle analysis, biological parameters prove to be more suitable than biochemi-
46 N.P. Bishun and J. Mills, Research Dept., Marie Curie Memorial Foundation, the Chart (U.K.) Chromosome studies in w o m e n w h o had used oral contraceptives and in their progeny A study has been made of the chromosome numbers and characteristics of leucocytes cultured from mothers (a total of 185) who had taken one or more types of oral contraceptive, and their babies (185}, and compared with similar numbers of control subjects. When the total cytogenetic results from test and control mothers were compared, no significant differences in frequency or type of chromosome abnormalities was observed. However, with certain types of oral contraceptive, minor differences in the numbers of chromosome significance of these findings are discussed. The only significant abnormalities observed in the babies from mothers taking oral contraceptives were slight increases in the numbers who had cells with less than 46 chromosomes and who exhibited chromosomal gaps. The numbers of abnormal cells was significantly higher in babies born to mothers who had been taking norethynodrel (Conovid) {4.6% abnormal cells in test babies; 2.0% in controls).
47 B. Johansson, G. AhnstrSm and K. Ericson, Wallenberg Laboratory, University of Stockholm (Sweden) Rate of strand separation in alkaline solution -- a sensitive m e t h o d for measuring D N A repair in mammalian cells In our laboratory we have developed a simple technique to study induction and repair of strand breaks in DNA. The m e t h o d is based on the principle t h a t single strand breaks serve as untwisting points during strand separation in alkaline solution and that DNA, for a time limited treatment in alkali, is transformed into single stranded form in proportion to the number of strand breaks present initially. The single- and double-stranded DNA are separated by means of h y d r o x y l apatite chromatography. In h u m a n embryonic fibroblast and chinese hamster V-79 cells alkali labile sites are induced by different agencies such as X-ray and N-methyl-N-nitrosourea (MNU). A b o u t 50% of this damage is repaired in 10 min. After UV irradiation t h y m i n e dimers are formed which, in h u m a n cells, are removed by excision repair. The single stranded breaks induced by the cells own endonuclease can be followed as a function of the UV dose. The number of breaks increases with dose up to 40--50 ergs/sec, m m 2 where it levels off in a plateau. The n u m b e r of breaks can be enhanced by t r e a t m e n t with 3 × 10 -3 M h y d r o x y u r e a but the plateau is reached at the same dose. The time for the disappearance of the endonuclease induced breaks varies
128 with the dose. At 10 ergs/sec, m m : the control level is reached within one hour but for 75 ergs/sec, mm 2 it takes about three hours. Post replication repair is studied in hamster cells, in which no endonuclease induced breaks have been detected with this method. It is based on the principle that after UV irradiation, the newly synthesized DNA will contain strand interruptions probably opposite thymine dimers. These breaks are slowly removed after post incubation in medium but the process is inhibited by 0.2 mg/ml of caffeine, a concentration that does n o t affect DNA synthesis. The induction of excision and post replication repair after treatment with the alkylating substances methyl methanesulphonate (MMS) and N-methyl-N-nitrosourea (MNU) will be discussed.
48 R. Di Lernia, G. Della Valle, L. De Carli, O. Temelcou and A. Barbieri, Istituto di Biologia Generale, Facolt~ di Medicina, Universit~ di Milano, and Istituto Sieroterapico Milanese S. Belfanti, Milano (Italy) Relationship between the inhibition of cell survival and growth and the effects on macromolecular syntheses of meta-L-sarcolysin peptide derivatives through the cell cycle of EUE cells Different methods, based either on biological or on biochemical assays, have been assessed to determine the effects of chemical agents on cell cycle progression. Assays were carried o u t on EUE cells (from a human heteroploid line) treated with m-(di-(2-chloroethyl)-amino)-L-phenylalanine peptide derivatives (peptichemio or PTC) at a concentration near to CLDs0 (cellular lethal dose 50). The cells were synchronized by a reversible mitotic arrest with a single Colcemid treatment (0.02 pg/ml), followed by selection of mitoses using the shake method. The degree of synchronisation was monitored by scoring a mitotic index and measuring DNA synthesis in 30-min pulse labeling at successive times in the cell cycle with TdR[3H] (0.5 pCi/ml). The biological parameters examined were the following: 1) plating efficiency (PE) i.e., the ratio between the number of cell colonies formed and the number of single cells inoculated; 2) colony growth (CG), measured by the average number of cells per colony after a fixed time. Treatments with PTC were performed every two hours througho u t the cell cycle. Determinations of PE and CG on treated cultures were performed after a period of growth in the medium w i t h o u t the drug, which corresponded to a b o u t five cell doublings in the control culture. On treated cultures, incorporation rates of labelled precursors of DNA, R N A and proteins were determined by 30-rain pulses with TdR[3H] (0.5 pCi/ml), UR[3H] (0.5 pCi/ml) and Leu[3H] (0.5 pCi/ml). No effect on protein and RNA synthesis was detected, whereas a slight decrease of DNA synthesis was noted in the early " S " phase. More appreciable inhibition values of PE and CG were found for the same period and clear inhibition was also revealed in the pre-synthetic and post-synthetic periods. It can be concluded that, at the drug concentrations and exposure times used in the cell cycle analysis, biological parameters prove to be more suitable than biochemi-