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J C Barrett

Publications and source records attributed to J C Barrett.

At least 343 records · Page 19Linked to original sources
PubMed1984

Dependence on exogenous metabolic activation for induction of unscheduled DNA synthesis in Syrian hamster embryo cells by diethylstilbestrol and related compounds.

Diethylstilbestrol (DES) induces morphological and neoplastic transformation of Syrian hamster embryo cells in vitro in the absence of any measurable induction of gene mutations, which is consistent with the lack of genotoxicity of DES in a number of other assays. However, a few reports of a genotoxic activity of DES in certain systems have been published. In order to understand these differences, we have investigated whether DES induces unscheduled DNA synthesis (UDS) in Syrian hamster embryo cells under the conditions which result in cell transformation and have examined the role of an exogenous metabolic activation system on DES-induced UDS. DES, over a concentration range of 1 to 10 micrograms/ml, failed to induce any detectable UDS in the cells, while other known transforming agents, including UV irradiation (6 to 24 J/sq m), benzo(a)pyrene (0.1 to 1.0 micrograms/ml), and aflatoxin B1 (10 to 100 micrograms/ml), induced significant levels of UDS. In contrast, UDS was induced in a dose-dependent manner by DES (1 to 10 micrograms/ml) after addition of an Aroclor-induced rat liver postmitochondrial supernatant fraction and other cofactors for exogenous metabolic activation. In order to probe the basis for this alteration in UDS induction, the ability of structural analogues and metabolites of DES to induce UDS was examined. In the absence of exogenous activation, the only oxidative metabolite of DES detected in the presence of the cells was cis,cis-dienestrol, which did not induce UDS by itself. In the presence of exogenous activation, cis,cis-dienestrol and its trans,trans-isomer induced UDS but not to a greater extent than DES. With the addition of the exogenous metabolizing system, increased metabolism of DES to cis,cis-dienestrol and additional polar derivatives of DES or dienestrol, possibly hydroxylated derivatives, were observed. With exogenous metabolic activation, tetrafluoro-DES and hexestrol, which differ in their ability to be peroxidatively metabolized to quinone and phenoxyradical intermediates, both induced UDS, although tetrafluorodiethylstilbestrol at 10 micrograms/ml stimulated a higher level of UDS. None of the DES-related compounds examined was active in the UDS assay without exogenous metabolic activation, but all of the compounds can potentially form phenoxyradical intermediates by a peroxidase-mediated reaction. The compounds which can be further oxidized to a quinone were most active in inducing UDS. These results are consistent with the hypothesis that this peroxidase-mediated pathway is important in the induction of UDS, although secondary metabolites may also be involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Dependence of asbestos- and mineral dust-induced transformation of mammalian cells in culture on fiber dimension.

The abilities of chrysotile and crocidolite asbestos, glass fibers of differing dimensions, and nonfibrous mineral particulates to induce morphological transformation of Syrian hamster embryo cells in culture were compared. Chrysotile and crocidolite asbestos induced morphologically transformed colonies which were indistinguishable from transformed colonies observed following treatment with known chemical carcinogens. A linear, dose-dependent increase in the frequency of transformed colonies was observed. The slope of the dose-response curve on a log-log scale was approximately 1, which is consistent with a one-hit mechanism for their induction. The transforming potency of chrysotile asbestos was decreased by milling of the fibers but not by extraction with an organic solvent. Chrysotile asbestos was nearly twice as potent in inducing morphological transformation as crocidolite asbestos. Glass fibers were also very active in this assay. Thin glass fibers with an average diameter of 0.1 to 0.2 micrometer were as active as asbestos. In contrast, two nonfibrous particulates, alpha-quartz and Min-U-Sil, were inactive over the same concentration range used for the fibrous dusts; however, both were active at higher doses. The effect of varying fiber dimension on induction of morphological transformation was examined with glass fibers. When compared on a per-weight basis, thick glass fibers [average diameter, 0.8 plus/minus 0.06 micrometer (S.E.)] were 20-fold less potent than thin fibers [average diameter, 0.13 plus/minus 0.005 micrometer] in inducing cell transformation. When the average fiber length of thin glass fibers was reduced from 9.5 to 1.7 micrometer by milling the fibers in a mortar and pestle, a 10-fold decrease in transforming activity resulted. When the average fiber length was reduced to 0.95 micrometer, transforming ability was totally absent. The cytotoxic potencies of the various mineral dusts correlated with their transforming potencies. The varying abilities of the mineral dusts to induce cell transformation in vitro are similar to their abilities to induce mesotheliomas in vivo. Thus, this system provides a unique model for studying the mechanism of mineral fiber tumorigenesis and for comparing the relative risks of mineral dusts.

Animals↗
PubMed1984

Studies on the action of tumour promoters and antipromoters on respiratory-tract epithelium.

Studies were carried out to examine the susceptibility of normal and initiated tracheal epithelial cells of rats to known tumour-promoting agents. The ability of normal rat tracheal epithelial cells to form colonies in cultures was enhanced markedly by addition of known tumour promoters to the medium. Several retinoids were shown to reduce the ability of these cells to form colonies in culture and to inhibit this effect of 12-O-tetradecanoylphorbol-13-acetate (TPA). A transformation assay of tracheal epithelial cells was used to study promotional effects of TPA in cultures initiated with N-methyl-N'-nitro-N-nitrosoguanidine. In this assay, four stages of transformation are recognized. TPA did not affect measurably the first two transformation stages, i.e., the development of transformed colonies (enhanced growth variants) and the 'immortalization' of enhanced growth variant-derived subcultures. However, treatment of cultures with TPA during the early post-initiation period resulted in a marked enhancement of the appearance of the third transformed phenotype, which is characterized by anchorage independence of growth. The findings of these in-vitro initiation-promotion studies paralleled, in all major respects, the results obtained in in-vivo - in-vitro studies. Tracheas exposed in vivo to initiator and promoter were shown to develop the same transformants observed in the in-vitro assay. TPA affected primarily the late anchorage-independence phenotype. Parallel tumour induction studies showed that TPA increased markedly the incidence of tracheal carcinomas following initiation with a low dose of 7,12-dimethylbenz[a]anthracene. The studies thus demonstrate that TPA is an effective tumour promoter for rat tracheal epithelium, causing an increase in tracheal carcinomas. They further suggest that the action of TPA on an early transformed cellular phenotype enhances the development of later phenotypes, including neoplastic cell variants.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1984

Inhibition of transformation of primary rat tracheal epithelial cells by retinoic acid.

The effect of retinoic acid (RA) on N-methyl-N'-nitro-N-nitrosoguanidine-induced transformation of primary cultures of rat tracheal epithelial cells was investigated. RA inhibited transformation of rat tracheal epithelial cells by up to 95% at concentrations of 3.3 to 33 nM which did not substantially affect cell survival. The inhibitory effect of RA on transformation was concentration dependent and was also dependent upon timing and duration of treatment. Treatment with RA for only 1 week following N-methyl-N'-nitro-N-nitrosoguanidine exposure diminished the transformation frequency by 30 to 57%, although longer treatment times were more effective. Because RA was able to inhibit transformation effectively at concentrations which were not substantially inhibitory to colony-forming efficiency of rat tracheal epithelial cells, the mechanism of inhibition of cell transformation does not seem to be related to cytotoxic effects of RA known to occur at high RA concentrations.

Animals↗
PubMed1983

Quantitation of cell proliferation, colony formation, and carcinogen induced cytotoxicity of rat tracheal epithelial cells grown in culture on 3T3 feeder layers.

A cell culture system is described for the growth of rat tracheal epithelial (RTE) cells at clonal density. The system uses normal, early passage RTE cells grown on feeder layers of lethally irradiated 3T3 cells. The RTE cells have a high colony forming efficiency (5 to 10%) in culture, can be passaged up to 5 times, and are capable of more than 20 cumulative doublings per colony forming cell. The epithelial nature of the cells was confirmed by cell and colony morphology, immunoperoxidase staining of intracellular keratin, and cellular ultrastructural studies. The cytotoxic response of RTE cells to a variety of carcinogens, including a direct acting chemical carcinogen, a physical carcinogen, and a series of polycyclic aromatic hydrocarbons, was quantitated. A linear decrease in the logarithm of survival was observed with increasing doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), gamma-irradiation, 7,12-dimethylbenz(a)anthracene, and a diol-ep-oxide of benzo(a)pyrene. No toxicity was observed after treatment with benzo(a)pyrene or 3-methylcholanthrene over the concentration range examined. In contrast, phorbol ester tumor promoters stimulated cell growth markedly. Based on these and other studies, the RTE cell culture system represents a model system that will be useful for quantitative studies of epithelial cell growth, differentiation, and carcinogenesis.

Animals↗
PubMed1983

Inability of diethylstilbestrol to induce 6-thioguanine-resistant mutants and to inhibit metabolic cooperation of V79 Chinese hamster cells.

The mutagenicity of diethylstilbestrol (DES) in V79 Chinese hamster cells was examined under a variety of conditions. DES over a concentration range 0.01-10 micrograms/ml failed to induce any increase above the spontaneous frequency of 6-thioguanine-resistant V79 cells. The effect of varying the expression time after treatment in the mutation assay from 3 to 9 days was studied and DES was nonmutagenic at all time points, while N-methyl-N'-nitro-N-nitrosoguanidine was highly mutagenic with a peak response after a 5-7 day expression time. The mutagenicity of benzo[a]pyrene and DES, both of which induce morphological and neoplastic transformation of Syrian hamster embryo (SHE) cells, was tested by cocultivating V79 cells with SHE cells for possible metabolic activation of the chemicals. Neither compound was mutagenic to V79 cells in the absence of SHE cells. Benzo[a]pyrene, but not DES, was mutagenic to V79 cells cocultivated with SHE cells. These results support the observation that DES can induce cell transformation under conditions that do not result in any measurable gene mutations. Moreover, the ability of DES to enhance the recovery of 6-thioguanine-resistant mutations was studied by determining the ability of DES to inhibit metabolic cooperation of V79 cells. Unlike the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, DES was a weak or inactive inhibitor of metabolic cooperation.

Animals↗
PubMed1983

Role of gene and chromosomal mutations in cell transformation.

To understand the role of mutagenesis in carcinogenesis fully, we must consider all types of mutations including gene, chromosomal, and gene-number mutations and all changes involved in the progressive development of neoplastic cells. We have found that certain known human carcinogens (i.e., DES and asbestos), which were classified as epigenetic carcinogens based on gene-mutation assays, have mutational activity at the chromosomal level that correlates with their ability to induce cell transformation. This should caution against classification of chemicals as genotoxic or epigenetic without a complete understanding of their mechanism of action. Furthermore, our studies indicate that more than gene-mutation assays is needed for carcinogen testing. In particular, chromosomal changes induced by chemicals, both aberrations and aneuploidy, need to be carefully assessed. In addition, the role of all types of mutation in the overall process of neoplastic transformation needs to be determined. This can only be examined by studying each individual change involved in neoplastic progression. Thus, any attempt to assess a chemical's carcinogenic potential should consider not only all types of mutational changes but both early and late changes involved in neoplastic transformation.

2-Aminopurine↗
PubMed1983

Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis.

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.

Aneuploidy↗
PubMed1983

Involvement of prostaglandin synthetase in the peroxidative metabolism of diethylstilbestrol in Syrian hamster embryo fibroblast cell cultures.

The mechanism for induction of tumors by estrogens is still unresolved. Neoplastic transformation of Syrian hamster embryo fibroblasts by diethylstilbestrol (DES) suggests that established principles of chemical carcinogenesis may be involved. The Syrian hamster embryo fibroblast cells provide a system in which now the question can be asked whether the metabolic activation of DES is a prerequisite for its biological activity in this system. In this study, Syrian hamster embryo fibroblast cell cultures were shown to oxidatively metabolize DES to cis,cisdienestrol (Z,Z-DIES) which is a DES metabolite commonly found in vivo. The only other metabolic conversion of DES detectable in these cell cultures was the formation of the glucuronides of DES and Z,Z-DIES. Z,Z-DIES is formed more efficiently in incubations with rapidly growing cells than in cultures approaching confluence. When arachidonic acid was added to the medium, Z,Z-DIES formation was enhanced, whereas indomethacin added to the cell cultures inhibited the formation of this metabolite. These data suggest the involvement of prostaglandin synthetase in the oxidative metabolism of DES by Syrian hamster embryo fibroblast cells in culture and suggest that cooxidation may play a role for its biotransformation in whole cells. Moreover, since many competing metabolic pathways are available to DES in vivo, this present study adds important additional support to the hypothesis that metabolism of DES via a peroxidative route plays a role in its carcinogenicity.

Animals↗
PubMed1983

High frequency of carcinogen-induced early, preneoplastic changes in rat tracheal epithelial cells in culture.

To study the mechanisms of carcinogenesis, we have developed a system that uses normal cells from an environmentally and epidemiologically relevant tissue, respiratory epithelium. The induction of preneoplastic variants of epithelial cells in culture was quantitated on a per-cell basis following exposure of rat tracheal epithelial (RTE) cells in vitro to the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Following treatment of normal RTE cells, large colonies of altered cells exhibiting an enhanced growth potential under selective culture conditions were observed, while normal RTE cells ceased proliferation after several cell doublings. After further growth in culture, these altered cells acquired the ability to grow in semisolid medium and to produce squamous cell carcinomas when injected into nude mice. The induction of enhanced growth variants of RTE cells by MNNG occurred with a high frequency (greater than or equal to 2.6%/colony-forming cell). In addition, a linear dose-response curve with a slope of approximately 1 was observed when the logarithm of MNNG-induced transformation frequency was plotted versus the logarithm of MNNG dose. These results are consistent with a one-hit mechanism for induction of preneoplastic variants of RTE cells by MNNG. Similar frequencies and kinetics of induction of preneoplastic variants in other culture systems using diploid cells have been observed, suggesting a common mechanism for this early step in carcinogenesis. The RTE cell system will be useful for mechanistic studies of early as well as late changes in the development of neoplasia by epithelial cells.

Animals↗
PubMed1982

Measurement of spontaneous mutation rates at the Na+/K+ ATPase locus (ouabain resistance) of human fibroblasts using improved growth conditions.

The mutation rate for the Na+/K+ ATPase locus (ouabain resistance, OuaR) in mammalian cells in culture has been reported to be 10-100-fold lower than the mutation rate of other gene loci in culture, such as the hypoxanthine phosphoribosyl transferase (HPRT) locus. Determination of the mutation rate to ouabain resistance is sensitive to culture conditions and the concentration of ouabain used to select mutants. Our improved growth conditions for human cells have permitted absolute cloning efficiencies of 70-90% and population doubling times of 16-17 h with both normal human diploid fibroblasts, KD, and their chemically induced neoplastic derivative, Hut-11A. Ouabain at 10(-7) M was found to be adequate to select for resistant (OuaR) mutants with an absolute recovery efficiency of 54-102%. Under these conditions, the mutation rates to ouabain resistance for human cells were measured and found to be 1-8.5 X 10(-7)/cell/generation for KD cells and 6-13 X 10(-7)/cell/generation for Hut-11A cells. These rates are 5-25 times higher than previously reported for human cells. Improved growth and the use of a lower concentration of ouabain for selection may allow for the increased recovery of OuaR mutants and an improved estimate of the mutation rate at this locus, which is only 2-10-fold less than the mutation rate at the HPRT locus in the same cells.

Cell Transformation, Neoplastic↗
PubMed1982

Morphologic and neoplastic transformation of Syrian hamster embryo fibroblasts by diethylstilbestrol and its analogs.

A mammalian cell culture system using normal, diploid Syrian hamster embryo fibroblasts was used as a model to study the ability of diethylstilbestrol (DES) and related compounds to induce neoplastic transformation. Like benzo(a)pyrene, a known chemical carcinogen, DES (0.01 to 10 micrograms/ml) induces morphological transformation of Syrian hamster embryo fibroblasts in vitro; the transformed colonies were indistinguishable from colonies of benzo(a)pyrene-transformed cells. The morphologically transformed colonies derived from either benzo(a)pyrene- or DES-treated cells were tumorigenic when injected into newborn hamsters. At doses of 0.01 to 1 microgram/ml for 48 hr, DES did not inhibit or stimulate cell proliferation. Structural analogs of DES were also tested in the Syrian hamster embryo transformation system. Like DES, tetrafluorodiethylstilbestrol, dimethylstilbestrol, and cis,cis-dienestrol morphologically transformed Syrian hamster embryo cells. The transformation frequency of dimethylstilbestrol was much less than that of DES, tetrafluorodiethylstilbestrol, or cis,cis-dienestrol. Hexestrol, dimethoxydiethylstilbestrol, and trans,trans-dienestrol did not transform these cells. No correlation could be demonstrated between reported estrogenic potency of the compounds and their cell-transforming capacity. Structure-activity relationships developed for these chemicals suggest that metabolism via specific pathways plays a role in DES-induced cell transformation.

Animals↗
PubMed1982

Prostaglandin endoperoxide synthetase-dependent cooxidation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in C3H/10T 1/2 clone 8 cells.

(+/-)trans-7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol), the proximate form of the carcinogen benzo(a)pyrene, is cooxidized during the oxidation of arachidonic acid to prostaglandins by prostaglandin endoperoxide synthetase (PES). This enzyme can oxidize BP-7,8-diol to the reactive intermediate (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, which binds covalently to macromolecules, is mutagenic in bacterial test systems, and forms 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP-tetrol) isomers. We have examined the cooxidation of BP-7,8-diol in an intact cell culture system of C3H/10T 1/2 clone 8 mouse embryo fibroblasts, in which both the mixed-function oxidase and PES systems are present. When BP-7,8-diol is incubated for 72 hr with approximately 10(6) confluent cells, high-performance liquid chromatography analysis of the organic extractable products reveals all four pairs of BP-tetrols, with those from (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene predominating. The addition of arachidonic acid (100 microM) produced a 2- to 3-fold increase in the formation of BP-tetrols from (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, while the metabolism of BP-tetrols from (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10 tetrahydrobenzo(a)pyrene was unchanged. The addition of the PES inhibitor indomethacin (100 microM) completely eliminated this stimulation. Cell transformation assays were carried out under the same conditions. The addition of arachidonic acid resulted in a 10-fold increase in foci formation, while indomethacin inhibited the increase in foci formation by 70%. These results suggest that cooxidation of BP-7,8-diol to reactive intermediates by PES can occur in an intact cell system if stimulated with arachidonic acid. In addition to mixed-function oxidase-dependent activation of carcinogens, the cooxidation of chemicals to reactive metabolites during prostaglandin biosynthesis may also play a role in carcinogenesis.

Animals↗