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Biomedical subjects

U Saffiotti

Publications and source records attributed to U Saffiotti.

At least 55 records · Page 3Linked to original sources
PubMed1984

Mouse epidermal keratinocytes. Clonal proliferation and response to hormones and growth factors in serum-free medium.

A serum-free medium (LEP-1) has been developed for mouse epidermal keratinocytes. LEP-1 consists of "Ca2+-free" Eagle's MEM with non-essential amino acids and seven added supplements (transferrin, 5 micrograms/ml; epidermal growth factor (EGF), 5 ng/ml; hydrocortisone, 0.5 microM; insulin, 5 micrograms/ml; phosphoethanolamine and ethanolamine, each 50 microM; bovine pituitary extract, 180 micrograms of protein/ml). Although serum-free the culture system was dependent for growth on bovine pituitary extract as the only still undefined supplement. LEP-1 supports sustained multiplication of mouse keratinocytes for 25 or more population doublings. A clonal growth assay was developed to investigate the action of growth factors, hormones and other supplements on keratinocytes. Cells grown in LEP-1 (calcium concentration was 0.03 mM) maintained a high proliferative rate and presented the typical morphology of basal epidermal cells. When the calcium concentration of the medium was raised to 1.0 mM, the cells were triggered to differentiate terminally. The epithelial nature of the cells was demonstrated both by electron microscopy and by immunostaining with anti-keratin antibody. The maturation stage of the keratinocytes was defined by several morphological features during the proliferative phase and in terminally differentiating cultures. This serum-free system supported a useful number of cell divisions while keratinocytes retained the capacity to undergo terminal differentiation when given the appropriate stimulus. It provides, therefore, provides a useful model for investigations on growth, differentiation and malignant transformation of epidermal cells in culture.

Animals↗
PubMed1984

Studies on chemically induced neoplastic transformation and mutation in the BALB/3T3 Cl A31-1-1 cell line in relation to the quantitative evaluation of carcinogens.

Mutagenesis and neoplastic transformation assays on mammalian cells in culture have been extensively used for quantitative estimates of the activity of carcinogens, in spite of the limitations that such in vitro systems have when compared with in vivo systems for tumor induction. In order to assess the validity of these correlations, a series of studies was undertaken in our laboratory with the BALB/3T3 Cl A31-1-1 mouse embryo cell line. Different carcinogens were found to induce dose-dependent frequencies of transformation, including the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and carcinogens that were metabolically activated by these cells through different pathways (benzo[a]pyrene, 3-methylcholanthrene, aflatoxin B1, and benzidine). Their respective level of activity on a molar basis was different from that obtained in standard Salmonella + S9 mutagenesis tests. Studies currently underway indicate the possibility of lowering the serum content in the medium considerably, thereby reducing a major variable in the assay. Methods were established for the induction of ouabain-resistant (ouar) mutants in these cells. Studies were conducted by applying 30-min MNNG exposures to cells that were synchronized by serum deprivation followed by serum-induced release from growth block. While maximal induction of mutants occurred in the S phase, the transformation frequency remained constant for treatments in G1 and early or late S.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Temporal dissociation in the exposure times required for maximal induction of cytotoxicity, mutation, and transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 ClA31-1-1 cell line.

Cytotoxicity, alkali-labile DNA lesions, ouabain resistance mutations, and neoplastic transformation were analyzed concurrently in the BALB/3T3 ClA31 -1-1 cell line treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for different exposure times (15, 30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). The half-life of MNNG in complete medium was approximately 70 min, both without cells and with cell numbers as used in the assays for cytotoxicity (2 X 10(2) cells/60-mm dish), transformation (1 X 10(4) cells/dish), and mutation (1 X 10(5) cells/dish). The cytotoxic effect of MNNG (0.5 or 2 micrograms/ml) appeared to be completed after an exposure time between 100 and 200 min. Maximal frequency of ouabain resistance mutations, however, was reached after a much shorter treatment time (30 to 60 min). Detection of DNA damage by alkaline elution analysis showed maximal increase in single-strand breaks already after treatment for 30 min. Exposures for 30 min followed by posttreatment incubation for 30 or 90 min showed active repair of single-strand breaks during these periods, indicative of an even balance between the additional MNNG-induced damage and its repair. Morphological transformation assays, at the same treatment times and concentrations used in the mutation assays, yielded frequency curves that reached their maxima 1 to 3 hr later than did the mutation frequencies. The ratio of transformation to ouabain resistance mutation frequencies was 3.7 for short treatment times (30 to 60 min), while it increased to more than 20 for exposure times of 240 min or longer. The temporal dissociation in the exposure times for maximal induction of mutation and transformation, observed with MNNG in this cell line, supports the hypothesis that a single gene mutational event is not sufficient to account for the full expression of neoplastic transformation.

Animals↗
PubMed1983

Dose-response studies on neoplastic transformation of BALB/3T3 clone A31-1-1 cells by aflatoxin B1, benzidine, benzo[a]pyrene, 3-methylcholanthrene, and N-methyl-N'-nitro-N-nitrosoguanidine.

The BALB/3T3 clone A31-1-1 mouse embryo cell line at passages 7 to 13 was selected for morphologic studies of neoplastic transformation by carcinogens of different chemical classes, in the absence of any added extracellular metabolic activation. Dose-related transforming activity was demonstrated for the carcinogens aflatoxin B1 (AFB) and benzidine (BZ) not previously reported in this system, and was confirmed for benzo[a]pyrene (BP), 3-methylcholanthrene (MCA), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Spontaneous transformation per cells at risk was low (0.14 type III foci x 10(-4), while chemically induced transformation was 2 to 3 orders of magnitude higher with all compounds. The molar concentration of carcinogens in complete medium, required to induce a transformation frequency of 1.0 type III foci x 10(-3) showed the highest level of activity for BP (0.04 microns), an intermediate level for AFB (0.2 to 1.4 microns), MCA (1.1 micron), and MNNG (2.3 microns), and the lowest level of activity for BZ (30.0 microns). The dose-related induction of morphological transformation in this clone by carcinogens of different classes indicates the potential value of this biological system in quantitative studies of carcinogen combinations, especially at low dose levels.

Aflatoxin B1↗
PubMed1983

Mutagenesis and morphological transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 clone A31-1-1 cell line.

Concomitant induction of ouabain resistant mutations and morphological transformation in the BALB/3T3 ClA31-1-1-c mouse embryo cell line were obtained after a 30 min treatment with the direct alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Parameters affecting mutation frequencies were determined. The optimal expression time (48 h) for ouabain resistance was independent from the dose of the carcinogen. A linear dose-response relationship for mutation induction was found after treatment with increasing doses of MNNG. The ratio of malignant transformation to mutation frequencies induced by the short treatment with MNNG was found to be within the same order of magnitude over a four-fold dose range. The development of a mutational assay for ouabain resistance in the BALB/3T3 ClA31-1-1-c cell line makes quantitative comparisons possible between mutation and neoplastic transformation frequencies induced by chemical carcinogens in this single cellular system.

Animals↗
PubMed1983

Evaluation of mixed exposure to carcinogens and correlations of in vivo and in vitro systems.

The biological evaluation of air pollutants is an example of the difficulties of evaluating the effects of mixed concurrent exposures to multiple agents, such as combinations of carcinogens with other carcinogens of the same or different chemical class, with incomplete carcinogens and cocarcinogens, with particulate materials and other factors that modify tissue distribution and retention, and with modifiers of metabolic pathways of activation and detoxication. A research approach is outlined to investigate such interactions in a series of biological systems of increasing complexity but closely related to each other in a step-by-step sequence, e.g., bacterial mutagenesis; mammalian cell mutagenesis, toxicity and neoplastic transformation, including embryo cells, fibroblasts and epithelial cells; organ cultures of target epithelia; in vivo animal systems for short-term and long-term studies, including animal models closely comparable to human pathology; observational studies of human pathology and histopathogenesis; experimental studies of corresponding human target tissues using organ and cell culture methods for metabolism, toxicity, mutagenicity and possibly neoplastic cell transformation. Respiratory carcinogenesis models were successfully used for studies of mixed exposures to different carcinogens and cofactors. The role of particulates has been found to be important but needs to be further characterized. Quantitative variations in the response to carcinogens and cofactors among different biological test systems and among different individuals in the human population make quantitative risk estimation very difficult, but studies in a sequence of related biological systems including human tissues indicate the importance of qualitative risk evaluation.

Air Pollutants↗
PubMed1979

Review of pesticide carcinogenesis data and regulatory approaches.

Carcinogenicity data are presented for compounds used as pesticides which have been evaluated in the IARC Monograph series. These pesticides have been both poorly and scantily tested for carcinogenicity. Additional tests have been carried out under the NCI Carcinogenesis Program, and detailed results of the bioassays are published in the NCI Carcinogenesis Technical Report Series. A prpcedure was developed whereby 'preliminary reports' could be made in the Federal Register. In the second part of the paper, regulatory approaches to pesticides are described. International activities are outlined; and examples are given of factors, such as public concern, scientific work and governmental response, which are highlights in the development of approaches to such regulations. The problem of risk assessment has been approached from many sides but remains largely unsolved. Besides risks and benefits, an essential component in evaluation is the analysis of technological alternatives. Two other factors are public documentation of the process of evaluation and registration of environmental carcinogens and their uses. Reduction of exposure to environmental carcinogens to a 'minimum feasible level' depends on social, economic and temporal factors and implies the acceptance of a finite level of risk.

Animals↗
PubMed1978

Experimental identification of chemical carcinogens, risk evaluation, and animal-to-animal correlations.

Experimental methods for the identification of chemical carcinogens have been extensively developed, including animal bioassay methods, animal models for cancer induction at major organ sites, models for the study of the effects of carcinogens in cells and tissues in culture and methods for the study of molecular events (metabolic activation, binding and detoxification of carcinogens; DNA damage and repair; mutagenicity). Current sources of documentation on carcinogenicity data are reviewed. The number of "known carcinogens" will vary considerably, depending on the criteria adopted for accepting evidence of carcinogenicity. CRITERIA FOR THE EVALUATION OF RISKS, BENEFITS, AND TECHNOLOGICAL ALTERNATIVES FOR PUBLIC POLICY ON ENVIRONMENTAL CARCINOGENS ARE REVIEWED AND THE FOLLOWING STEPS DISCUSSED: registration of environmental chemical carcinogens and their uses; risk evaluation (considering sources, adequacy, quality and limits of the evidence; quantitative dose-response extrapolation within the same biological system; and species and model conversion factors); benefits evaluation; analysis of technological alternatives; comparative judgment and decision; open public documentation. The problem of animal-to-human correlations is considered, particularly for respiratory carcinogenesis. A laboratory approach is reviewed which includes: development and study of whole animal models for carcinogenesis, analysis of animal tissue responses to carcinogens in vivo and through in vitro culture methods for morphological and biochemical studies, and development of in vitro culture methods for human target tissues. This approach is aimed at providing an experimentally controlled and quantifiable method for the correlation of animal and human observations in carcinogenesis.

Animals↗
PubMed1977

Releasing carcinogenesis test results: timing and extent of reporting.

A carcinogenic in an animal bioassay is usually characterized by gradaul accumulation of a tumor incidence significantly in excess in the treated versus the control animals. In some cases, a definite response is realized relatively early, prior to planned sacrifice. In others, positive results are found at termination. In both cases, a long delay may occur before the information is ready to reach the public due to the time needed to complete pathology, data analysis, publication of a report. Since the finding that a chemical is carcinogenic may have serious implications for public health and technology, reports of carcinogenesis bioassays should be thoroughly and critically reviewed before being issued. A delicate ethical dilemma confronts scientists in selecting the proper timing and extent for the release of carcinogenesis test results. Early findings may not be confirmed and may cause technological and economic problems and unnecessary anxiety. On the other hand, delaying public notification of highly suspicious findings until a final, detailed report is published, may delay preventive actions that could protect exposed populations from unnecessary risks. To meet differing requirements in regard to timing and extent of reporting, procedures were developed to issue reports on National Cancer Institute (NCI) carcinogenesis bioassays when the findings are clearly identified and well documented in the following forms: a) reports of preliminary findings, when the evidence is strongly suggestive; b) summary reports of completed bioassays; and c) detailed technical reports. Availability of such reports, particularly for preliminary findings, will be announced in the Federal Register and in a press release. The reports will be submitted for publication in the scientific literature.

Animals↗
PubMed1976

Cutaneous chemical carcinogenesis: past, present, and future.

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small dose of carinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. When epidermal cells in vitro are exposed to promoting agents, a proliferative response analogous to that in vivo is elicited, apparently mediated through control of polyamine metabolism. Neoplastic transformation has been induced in these cultures by known skin carcinogens.

9,10-Dimethyl-1,2-benzanthracene↗