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Transformation of mammalian cells in culture by chemical carcinogens
101
shown to require metabolic activation in order to produce a biological effect. This metabolic activation is in many cases associated with the binding to nucleophilic sites in nucleic acids and proteins. The growing experimental evidence linking the carcinogenic activity of numerous chemicals to their capacity to be converted into electrophilic derivatives that may also exert a mutagenic effect led to the suggestion that a relationship between chemical carcinogenesis and mutagenesis may exist. Such a correlation has, so far, been limited to those changes of the genotype which appear as a consequence of structural or functional alterations of nucleic acids and thus are genetically transmissible. Although not all chemical mutagens have been shown to be carcinogenic, most chemical carcinogens, several of which cause cancer in man, have now been found to be mutagens when tested in one of the mutagenicity test procedures that combine microbial, mammalian or other animal-cell systems as genetic targets with an in vitro or in vivo metabolic activation system. Theoretical aspects on the relationship between chemical carcinogenesis and mutagenesis will be discussed in the light of recent results with such appropriate mutagenicity tests. Some practical applications of mutagenicity test systems will be given. Difficulties and limitations in assessing the possible carcinogenic risk of chemicals using results from mutagenicity assays will be illustrated.
2 A. Abbondandolo, Laboratorio di Mutagenesi e Differenziamento del CNR, Pisa (Italy) Prospects for evaluating genetic damage in mammalian cells in culture The aim of this communication is to report the recently published work on mutagenesis in cultured mammalian cells, in order to provide investigators operating in related fields, or in the same field, but with different organisms, with the basic information. Firstly, attention will be called on the various genetic effects that may, actually or potentially, be observed in this material. The talk will subsequently focus on m u t a t i o n and repair, as the genetic effects where research is now particularly active and progress, as a consequence, more rapid. Emphasis will be posed on the genetic systems based on isolation and characterization of clones resistant to antimetabolites, and current tests for studying stimulation of repair by chemicals will be recalled. Finally, the relevance of both m u t a t i o n and repair tests available at present to screening programs of mutagenic or cancerogenic activity of environmental substances will be discussed.
3 J.A. Di Paolo, Biology Branch, National Cancer Institute, NIH, Bethesda
(U.S.A.) Transformation of mammalian cells in culture by chemical carcinogens
102
Quantitative in vitro methods for the study of transformation (T) permit determination of the number of T occurring in a cell population after short exposure to chemicals, unobscured by subsequent cell growth which would be found in mass cultures. The number of cells capable of forming transformed colonies {random criss-cross pattern not seen in controls) represents only a small proportion of those able to bind the carcinogen. Use of freshly isolated diploid Syrian hamster or rat embryonic cells or mouse cell lines treated with carcinogens results in formation of altered colonies which may be isolated and propagated to form transplantable tumors when the cells are injected into animals, Thus, chemical, chemical-chemical, chemical-physical and chemical-viral cell interactions involved in transition to neoplasia at the cellular level can be investigated. Another model utilizes freshly isolated diploid guinea pig fetal cells; although morphologic alteration occurs after carcinogen treatment transformation was n o t coincidental with tumorigenic potential. Carcinogens of diverse chemical classes can transform cells of the species used. Frequency of appearance of altered colonies was related to known potency of compounds tested. Some compounds that require metabolic activation to produce the proximate carcinogen become active with a host-mediated in vivo--in vitro technique in which fetal cells are first exposed in utero after intraperitoneal injection of the chemical. This modification minimizes false negatives. T frequency may be enhanced by prior t r e a t m e n t with X-irradiation, alkylating agents, or compounds that inhibit toxicity. Pretreatment with chemical carcinogen enhances Simian adenovirus transformation. Caffeine post chemical carcinogen or adenovirus results in enhanced transformation. In vitro models can be used to study chemical carcinogen induced carcinogenesis.
4 M. Bauchinger and G. RShr, Gesellschaft ffir Strahlen und Umweltforschung, Neuherberg {Federal l~epublic of Germany) Chromosome analyses in cell cultures of Chinese hamster after application of CdS04 Cell cultures of a Chinese hamster cell line were treated with CdSO4 in concentrations of 5 X 10 -4 - 10 -~ mol/1 for different time periods. The effect on the mitotic apparatus and on the chromosome structure was analysed either immediately after treatment or after 2--33 h recovery. A treatment for 16 h with a 10 -6 mol CdSO4 -solution induced a significant reduction of the mitotic index. Chromosome damage was induced only in a few cells by a 10 -s mol solution, but a quantitative analysis was impossible due to strong stickiness and pycnosis. As compared to controls no significant increase in the aberration yield could be induced by lower concentrations. A t r e a t m e n t for 3 h with 10 -4 and 10 -s tool concentrations w i t h o u t additional application of Colcemid and h y p o t o n i c solution yielded a stathmokinetic effect. The mitotic index was 3 times higher than in controls. Simultaneously "initial-C-mitosis" 110 -s mol) and typical apolar stathmokinesis with chromo-
shown to require metabolic activation in order to produce a biological effect. This metabolic activation is in many cases associated with the binding to nucleophilic sites in nucleic acids and proteins. The growing experimental evidence linking the carcinogenic activity of numerous chemicals to their capacity to be converted into electrophilic derivatives that may also exert a mutagenic effect led to the suggestion that a relationship between chemical carcinogenesis and mutagenesis may exist. Such a correlation has, so far, been limited to those changes of the genotype which appear as a consequence of structural or functional alterations of nucleic acids and thus are genetically transmissible. Although not all chemical mutagens have been shown to be carcinogenic, most chemical carcinogens, several of which cause cancer in man, have now been found to be mutagens when tested in one of the mutagenicity test procedures that combine microbial, mammalian or other animal-cell systems as genetic targets with an in vitro or in vivo metabolic activation system. Theoretical aspects on the relationship between chemical carcinogenesis and mutagenesis will be discussed in the light of recent results with such appropriate mutagenicity tests. Some practical applications of mutagenicity test systems will be given. Difficulties and limitations in assessing the possible carcinogenic risk of chemicals using results from mutagenicity assays will be illustrated.
2 A. Abbondandolo, Laboratorio di Mutagenesi e Differenziamento del CNR, Pisa (Italy) Prospects for evaluating genetic damage in mammalian cells in culture The aim of this communication is to report the recently published work on mutagenesis in cultured mammalian cells, in order to provide investigators operating in related fields, or in the same field, but with different organisms, with the basic information. Firstly, attention will be called on the various genetic effects that may, actually or potentially, be observed in this material. The talk will subsequently focus on m u t a t i o n and repair, as the genetic effects where research is now particularly active and progress, as a consequence, more rapid. Emphasis will be posed on the genetic systems based on isolation and characterization of clones resistant to antimetabolites, and current tests for studying stimulation of repair by chemicals will be recalled. Finally, the relevance of both m u t a t i o n and repair tests available at present to screening programs of mutagenic or cancerogenic activity of environmental substances will be discussed.
3 J.A. Di Paolo, Biology Branch, National Cancer Institute, NIH, Bethesda
(U.S.A.) Transformation of mammalian cells in culture by chemical carcinogens
102
Quantitative in vitro methods for the study of transformation (T) permit determination of the number of T occurring in a cell population after short exposure to chemicals, unobscured by subsequent cell growth which would be found in mass cultures. The number of cells capable of forming transformed colonies {random criss-cross pattern not seen in controls) represents only a small proportion of those able to bind the carcinogen. Use of freshly isolated diploid Syrian hamster or rat embryonic cells or mouse cell lines treated with carcinogens results in formation of altered colonies which may be isolated and propagated to form transplantable tumors when the cells are injected into animals, Thus, chemical, chemical-chemical, chemical-physical and chemical-viral cell interactions involved in transition to neoplasia at the cellular level can be investigated. Another model utilizes freshly isolated diploid guinea pig fetal cells; although morphologic alteration occurs after carcinogen treatment transformation was n o t coincidental with tumorigenic potential. Carcinogens of diverse chemical classes can transform cells of the species used. Frequency of appearance of altered colonies was related to known potency of compounds tested. Some compounds that require metabolic activation to produce the proximate carcinogen become active with a host-mediated in vivo--in vitro technique in which fetal cells are first exposed in utero after intraperitoneal injection of the chemical. This modification minimizes false negatives. T frequency may be enhanced by prior t r e a t m e n t with X-irradiation, alkylating agents, or compounds that inhibit toxicity. Pretreatment with chemical carcinogen enhances Simian adenovirus transformation. Caffeine post chemical carcinogen or adenovirus results in enhanced transformation. In vitro models can be used to study chemical carcinogen induced carcinogenesis.
4 M. Bauchinger and G. RShr, Gesellschaft ffir Strahlen und Umweltforschung, Neuherberg {Federal l~epublic of Germany) Chromosome analyses in cell cultures of Chinese hamster after application of CdS04 Cell cultures of a Chinese hamster cell line were treated with CdSO4 in concentrations of 5 X 10 -4 - 10 -~ mol/1 for different time periods. The effect on the mitotic apparatus and on the chromosome structure was analysed either immediately after treatment or after 2--33 h recovery. A treatment for 16 h with a 10 -6 mol CdSO4 -solution induced a significant reduction of the mitotic index. Chromosome damage was induced only in a few cells by a 10 -s mol solution, but a quantitative analysis was impossible due to strong stickiness and pycnosis. As compared to controls no significant increase in the aberration yield could be induced by lower concentrations. A t r e a t m e n t for 3 h with 10 -4 and 10 -s tool concentrations w i t h o u t additional application of Colcemid and h y p o t o n i c solution yielded a stathmokinetic effect. The mitotic index was 3 times higher than in controls. Simultaneously "initial-C-mitosis" 110 -s mol) and typical apolar stathmokinesis with chromo-