Cytotoxic and cytogenetic effects of asbestos on human bronchial epithelial cells in culture.

Asbestos and other mineral fibers elicit responses in several rodent cell transformation systems. The mechanism of this transformation has been hypothesized to involve specific chromosome alterations, especially changes in chromosome number. However, the cytogenetic effects of asbestos fibers in cultured human respiratory epithelium have not been well characterized. The present study examined the effects of chrysotile and crocidolite asbestos fibers on cultures of human bronchial epithelial (HBE) cells growing in serum-free medium. HBE cells were continuously treated with chrysotile (0-4 micrograms/cm2) or crocidolite (0-300 micrograms/cm2) asbestos and examined after 24, 48, 72 or 96 h for cytotoxic and cytogenetic effects. Both asbestos fiber types induced a concentration-dependent inhibition of cell proliferation and colony-forming efficiency; however, in these assays chrysotile was 100-300 times more toxic than crocidolite. Concentrations of asbestos that inhibited growth had little effect upon trypan blue exclusion or intracellular esterase activity, suggesting that the majority of asbestos-exposed cells were still viable. A 2.7-fold increase in binuclei and a 1.6-fold increase in micronuclei were observed 72 h after treatment with 4 micrograms/cm2 chrysotile. A 1.9-fold increase in binuclei was observed 72 h after treatment with 300 micrograms/cm2 crocidolite, but crocidolite did not increase the incidence of micronuclei. Chrysotile asbestos failed to induce significant numerical chromosome changes in HBE cells and increased structural aberrations only at the 24 h time point. These findings contrast with the relatively high incidences of asbestos-induced chromosome changes previously observed in some rodent cell cultures and suggest the existence of species-specific or cell-type-specific differences in either chromosome stability or mechanism(s) of asbestos-induced toxicity.

Analysis of prospective epidemiologic studies on the neurobehavioural effects of lead.

High-level lead exposure can have serious effects on the intellectual and behavioural development of young children. There has been much controversy in the last decade concerning the possible impact of low-level lead exposure upon the neurobehavioural and psychomotor development of children. Five longitudinal studies (Boston, Cincinnati, Cleveland, Port Pirie and Sydney) examining lead effects on child development were initiated in the early 1980s. These studies share multiple design features and include data on blood lead and neurobehavioural measurements from birth, six months, or annual intervals to seven years. All the studies use multivariate analysis to take into account possible confounding covariates with outcome measures.The studies tend to have varying results based on the covariates used and type of subject population. An analysis of the results of the five studies with regard to effects associated with prenatal and postnatal lead exposure and pregnancy outcome has been carried out and reveals inconsistencies in the onset, stability, and nature of neurobehavioural effects correlated with different indices of lead exposure. it is possible that the variation in reported results may be due to the use of different covariates and analyses among studies. A common analysis should be carried out among ail studies to further determine consistency of results. Although individual studies may show some effects, taken as a whole, the current published data from the five studies in this review are inconsistent and do not lend support to the concept that low level lead exposure resulting in blood lead levels below 25 µg dL(-1) is associated with neurobehavioural deficits in children.

The use of tracheal implants in toxicology and carcinogenesis research.

Tracheal implants have served as an important experimental pathology tool with which to study the toxic and/or carcinogenic effects of chemicals upon upper respiratory tract epithelium. Initial studies with this method utilized heterotopic rat tracheal transplants which were exposed to compounds of interest, and assessed for toxic and/or carcinogenic endpoints. Grafts containing rodent tissue have proved useful for studying the cellular and biochemical features of neoplastic progression at different time intervals following in vivo exposure to carcinogens. More recent studies have utilized epithelial denuded tracheal implants inoculated with respiratory cell populations, and xenografted into immunodeficient nu/nu mice. This technique permits the study of airway epithelium from a variety of species, including man. The advent of molecular pathology techniques such as in situ hybridization will further expand the uses of tracheal implant technology for studies with xenografted human tissues. Such implants should prove useful for the examination of species- and tissue-specific characteristics of growth and differentiation by providing a bridge between cell culture and whole animal studies.

Development of a tracheal implant xenograft model to expose human bronchial epithelial cells to toxic gases.

A tracheal implant model was developed which enabled exposure of differentiated normal human bronchial epithelial cells (NHBE) to a single exposure of a model toxic gas (formaldehyde). NHBE cells were grown in vitro in explant culture under defined serum-free conditions from previously frozen bronchial segments, harvested, and used to repopulate de-epithelialized rabbit tracheas. Rabbit tracheal segments repopulated with NHBE cells were implanted into the subcutis of congenitally athymic nude mice. Following graft vascularization, the xenografted NHBE cells differentiated and formed a mucociliary epithelial surface which lined approximately 50% of the surface of the implant lumen. Eight weeks post-implantation both ends of the implanted grafts were cannulated, and formaldehyde (HCHO) vapor (0, 6, or 15 ppm) in humidified air was passed through the tracheal lumens. Representative epithelial surfaces were examined by light and scanning electronmicroscopy, and autoradiography prior to, immediately after, and 48 hr following a 1-hr exposure to the test vapors. Light microscopic examination of implant sections immediately following exposure to 6 and 15 ppm HCHO detected cessation of ciliary activity, which recovered by 48 hr post-exposure. Scanning electron microscopic examination of the epithelial surface demonstrated mild morphologic changes, restricted to those implants exposed to 15 ppm. Findings immediately following HCHO exposure included swelling and exfoliation of individual cells, deciliation, and mucus release. Changes present after 48 hr included presence of flattened cells with few short microvilli and focal increase in the number of S-phase nuclei in the basal epithelium. These results demonstrate the utility of tracheal implants for single short-term exposure of differentiated human bronchial epithelial cells to gaseous agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetic analysis of spontaneous and 2-cyanoethylene oxide-induced tk-/- mutants in TK6 human lymphoblastoid cultures.

Two phenotypic classes of selectable tk-/- mutants have been isolated from the TK6 human lymphoblast cell line; one has a normal growth rate (tkn) relative to the parental cell line; the other has a slow growth rate (tks). Complete karyotypes of metaphase chromosomes were prepared and analyzed from 16 tks mutants (eight spontaneous and eight 2-cyanoethylene oxide [CNEtO]-induced), two spontaneous tkn mutants, and the parental TK6 cell line. Southern blot analysis of these tk-/- mutants indicated that all had lost a 14.8 kb polymorphic band corresponding to the active tk allele. No chromosome abnormalities with respect to the parental cell line TK6 were observed in eight spontaneous tks mutants. Chromosome abnormalities that may have been related to CNEtO treatment were observed in four of eight CNEtO mutants. However, chromosome 17, containing the tk locus in man, was cytologically normal with respect to the parental TK6 cell line in 15 of 16 tks mutants. A visible abnormality of chromosome 17 was present in one CNEtO-induced tks mutant. The abnormality was a duplication of the long arm of the chromosome 17, with break points at q11 and q21. The latter break point is close to the site of the tk locus, suggesting that the aberration observed may be associated with tk-/- phenotype. These observations contrast with the relatively high incidence (greater than or equal to 59%) of chromosome 11 abnormalities reported in tks (small colony) mouse lymphoma L5178Y/TK +/- mutants.

Effect of growth state, tumor promoters, and transformation upon intercellular communication between C3H/10T1/2 murine fibroblasts.

Diminished intercellular communication has been associated with heightened sensitivity of cultured cells to morphological transformation and enhancement of transformation by tumor promoters. Microinjection of Lucifer yellow dye was employed to evaluate intercellular communication between transformable C3H/10T1/2 murine fibroblasts under a variety of culture conditions. Intercellular communication assayed by dye transfer from injected cells to surrounding cells in contact occurred in logarithmically growing cultures, declined to very low levels as confluence was attained, and then resumed upon the formation of mature confluent monolayers. Dye-transfer networks of 50 or more cells resulted from injection of single monolayer cells. Freeze-fracture electron microscopy confirmed the presence of gap junction structures in confluent cultures. Treatment with the initiating agent N-methyl-N'-nitro-N-nitrosoguanidine and/or the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit intercellular communication between C3H/10T1/2 cells during 6-week transformation experiments. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate produced a transient inhibition of dye-coupling upon first introduction to cultures and prolonged the period of diminished dye-coupling at the attainment of confluence, but did not inhibit subsequent interactions between monolayer cells. A simple relationship thus could not be established between levels of dye-coupling within monolayers and focus formation events. Curiously, although the cells of foci in early phases of development did not exhibit dye-transfer capacity, dye-coupling was observed in mass cultures of most transformed cell lines cloned from foci. Co-cultivation of communication-competent transformed cells with nontransformed cells to produce reconstructed foci generally resulted in a cessation of dye-transfer by transformed cells. An often reversible loss of communication competence thus accompanies the growth of transformed C3H/10T1/2 cells as foci and may constitute an adaptive response which facilitates focus growth in the presence of intercellular communication between monolayer cells.

Carcinogen-induced unscheduled DNA synthesis in xenotransplanted human tracheobronchial epithelium: comparison with rat tracheal epithelium.

Extrapolation from rodent genotoxicity data to humans is complicated by variables such as interspecies differences in carcinogen metabolism and DNA repair. A xenograft system containing human bronchial epithelial cells was used to assess the induction of unscheduled DNA synthesis (UDS) by carcinogens and to compare the response with that of rat tracheal epithelium. Cells from human bronchus were grown in explant culture, inoculated into de-epithelialized rat tracheas and implanted subcutaneously into nude mice. Within six weeks, a differentiated mucociliary epithelium lined the xenografted tracheas. Fresh rat tracheas and human xenografts were cut into rings and incubated in media containing [3H]thymidine and either the direct-acting carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 3-1000 microM), or a carcinogen requiring metabolic activation, 4-nitroquinoline-1-oxide (4-NQO, 3-100 microM). Tissues were then fixed, sectioned, processed for autoradiography and the number of nuclear grains (NG) determined for 100 epithelial cells lining the trachea in each section. A time- and concentration-dependent increase in NG was observed in both human xenografts and rat tracheas after treatment with MNNG or 4-NQO, indicating induction of UDS by these agents. The UDS response to MNNG in the human xenografts was similar to that observed in the rat tracheas, whereas the response to 4-NQO was greater in rat tracheas. These studies indicate that the human xenograft system should have applications for the study of carcinogen-induced damage in normal bronchial epithelial cells.

Promotion of C3H/10T1/2 morphological transformation by polychlorinated dibenzo-p-dioxin isomers.

Few of the seventy-five chlorinated dibenzo-p-dioxin isomers present in the environment have been adequately characterized for their carcinogenic potential. In previous studies we observed that the carcinogenic dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin promoted cell transformation when continuously applied to C3H/10T1/2 cells initiated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The current study was undertaken to evaluate the response of the C3H/10T1/2 cell transformation system to several other dioxin isomers of known carcinogenic potential. 1,2,3,6,7,8- and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (HCDD) (1-1000 nM) and 2,7-dichlorodibenzo-p-dioxin (0.1-20 microM) failed to transform C3H/10T1/2 cells or to initiate transformation in cultures subsequently treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Continuous exposure of MNNG-initiated cultures to 2,7-DCDD (1-5000 nM) produced elevated but not statistically significant numbers of transformed foci at the highest dose tested. 1,2,3,6,7,8- and 1,2,3,7,8,9-HCDD were promoters of transformation when applied at concentrations greater than or equal to 12 and 40 pM, respectively, to C3H/10T1/2 cultures initiated with MNNG. Maximum responses for both HCDD isomers were attained at concentrations between 120 and 400 pM. These studies suggest that the C3H/10T1/2 cell transformation system may provide a relevant in vitro model for the identification and study of carcinogenic dioxin isomers.

Subpopulations of human bronchial epithelial cells in culture respond heterogeneously to 12-O-tetradecanoylphorbol-13-acetate (TPA) and other modulators of differentiation.

A greater understanding of the processes involved in the control of proliferation and differentiation should provide insight into the mechanisms involved in carcinogenesis. Studies were undertaken to examine the effects of modulators of differentiation on the proliferation, colony forming efficiency (CFE), and cross-linked envelope (CLE) formation of human bronchial epithelial (HBE) cells in culture. Treatment for 24 h with a low concentration of TPA (0.1 ng/ml) induced a 2-fold increase in CLE but little inhibition of CFE, suggesting that these two endpoints might be occurring independently of one another. Continuous culture in a low concentration of TPA (0.1 ng/ml) arrested growth in greater than 99% of the cells, but after 10-14 days, a few colonies were observed that were resistant to TPA. This TPA resistant subpopulation occurred at a frequency of less than 0.1% of the cells seeded into cultures. Short term treatment (24 h) with fetal bovine serum (FBS; 1-8%) or calcium (0.5-2 mM) resulted in 2-4 fold increases in CLE with no significant change in CFE. Continuous treatment with FBS or calcium for up to 5 days produced similar results. These findings suggest that different subpopulations of cells exist within cultures of HBE cultures, perhaps at different states of maturation, and that these subpopulations respond differently to modulators of differentiation.

Alterations of intercellular communication associated with the transformation of C3H/10T1/2 cells.

Cultures of C3H/10T1/2 mouse embryo cells were treated in accordance with several treatment regimens that induced the focal growth of morphologically transformed cells. Intercellular communication between focus cells, and between focus and monolayer cells, was examined in late stages of transformation experiments by microinjection of Lucifer yellow dye into cells and observation of dye transfer to surrounding cells. Transformed foci produced by treatment with 3-methylcholanthrene, by initiation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA), or by initiation with MNNG and promotion with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied. These included focus types thought to possess tumorigenic potential (Types II and III) and those believed to lack such potential (Type I). Cells within all focus types exhibited only limited communication with each other or with surrounding monolayer cells. In contrast, microinjection of monolayer cells typically resulted in dye transfer to an average of approximately 50 other monolayer cells. The presence of the tumor promoters TPA or TCDD did not alter intercellular communication between monolayer cells. These studies demonstrate that alterations in intercellular communication are evident during the growth of transformed foci. These changes are relatively independent of both the treatment regimen used to produce foci and the presumed oncogenic potential of different focus types.

Differential effects of tumour promoters on the growth of normal human bronchial epithelial cells and human lung tumour cell lines.

The effects of the tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) on the colony-forming efficiency and growth of normal human bronchial epithelial (NHBE) cells and five lung squamous carcinoma cell lines were compared in medium containing 1% foetal bovine serum. TPA (0.1-5.0 ng/ml) inhibited the growth of NHBE cells and one carcinoma cell line, while four of the five carcinoma lines were less sensitive to the growth inhibitory properties of TPA but were slightly inhibited at higher TPA concentrations. The responses of NHBE cells and carcinoma cells to TPA, and the related compounds, mezerein, 4-O-methyl TPA, and phorbol were then compared in serum-free medium. In general, the removal of serum from the medium increased the differences in the responses to TPA between normal and tumour cells. Two carcinoma lines inhibited by TPA in 1% serum were stimulated by TPA in the absence of serum. Mezerein and, to a lesser extent, 4-O-methyl TPA also produced differential responses in colony-forming efficiencies between tumour lines and NHBE cells. Phorbol had no effect on either NHBE cells or on carcinoma cell lines. The relative insensitivity of carcinoma cell lines to the growth inhibitory effects of tumour promoters is consistent with the hypothesis that tumour promotion involves selection against normal cells to permit clonal expansion of preneoplastic or neoplastic cell types.

Lack of di-(2-ethylhexyl) phthalate activity in the C3H 10T 1 2 cell transformation system.

The widely used plasticizer and rodent carcinogen di-(2-ethylhexyl) phthalate (DEHP) was examined for activity in the C3H 10T 1 2 murine fibroblast cell transformation system. Treatment with DEHP or its metabolite, mono-(2-ethylhexyl) phthalate, did not produce oncogenic transformation, initiate the process of transformation in cultures treated with a tumour promoter or promote the process of transformation in cultures pretreated with a chemical carcinogen. These findings are consistent with the suggestion that the carcinogenicity of DEHP is mediated by an indirect mechanism and not by covalent interaction of DEHP with DNA.

Reversible expression of morphological transformation in C3H/10T1/2 mouse embryo cultures exposed to 12-O-tetradecanoylphorbol-13-acetate.

Treatment of C3H/10T1/2 mouse embryo fibroblasts with N-methyl-N'-nitro-N-nitrosoguanidine and then 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the production of numerous foci of morphologically transformed cells. When dishes containing foci were provided medium which did not contain TPA, up to 84% of the foci were found to regress. Promotion of morphological transformation by TPA in C3H/10T1/2 cells may thus be a reversible process.

Effect of mouse skin tumor promoters upon [3H]uridine exchange and focus formation in cultures of C3H/10T1/2 mouse fibroblasts.

The abilities of 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (4-O-methyl-TPA) and mezerein to promote the process of transformation were evaluated in cultures of C3H/10T1/2 mouse embryo fibroblasts treated with N-methyl-N'-nitro-N-nitrosoguanidine. Mezerein was found to be as potent as the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) for the promotion of focus formation, eliciting a promotion response at concentrations that ranged from 100 to 2500 ng/ml. 4-O-Methyl-TPA (25-2500 ng/ml) did not promote focus formation, but was mitogenic for confluent cultures. The effects of promoting and non-promoting compounds upon intercellular communication were then evaluated to determine if a rapid assay for the inhibition of communication might serve as a surrogate for the relatively long term, labor-intensive cell transformation assay. Inhibited intercellular communication, as measured by inhibition of [3H]uridine exchange between cells, appeared to correlate with the ability of phorbol related compounds to promote transformation. However, the potent promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin did not inhibit [3H]uridine transfer. Inhibition of intercellular communication may thus be diagnostic of the promoting potential of phorbol-related compounds in C3H/10T1/2 cultures, but may not be an appropriate endpoint for the study of carcinogenic dioxins.

Potential role of treatment artifact in the effect of cell density upon frequencies of C3H/10T1/2 cell transformation.

Reports of unusual increases in transformation frequency in low density cultures of C3H/10T1/2 cells suggest that transformation occurs via an indirect multistage mechanism. The effect of surviving cell density upon subsequent focus production was examined in C3H/10T1/2 cultures treated with acetone, 12-O-tetradecanoylphorbol-13-acetate (TPA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNNG plus TPA, or 3-methylcholanthrene (MCA). Foci developed independent of cell density in 0.5 and 5.9% of all cultures exposed to acetone and TPA (0.25 micrograms/ml), respectively. Transformation after treatment with MNNG (0.5 micrograms/ml) occurred with low frequency (less than or equal to 7 X 10(-4)/surviving cell) and was enhanced by TPA. In MNNG plus TPA treated cultures containing less than or equal to 140 viable cells the fraction of dishes with foci was dependent upon the number of cells present at the time of MNNG treatment. As a result, relatively constant frequencies of focus formation were obtained (less than or equal to 6 X 10(-3) after correction for focus formation in TPA treated solvent controls). Focus frequency declined at cell densities greater than or equal to 350 cells. In contrast, treatment with MCA (1.0 microgram/ml) produced transformed foci with frequencies that varied from 3.3 X 10(-2) at the lowest density (5.5 cells) to 5.4 X 10(-4) at the highest (4400 cells). In low density cultures (5.6-56 cells), the fraction of dishes with foci was independent of the number of cells treated. Thus cell density had differential effects upon the frequency of foci produced by MCA or MNNG plus TPA. However, binding studies demonstrated that 6-7% of the MCA added to cell culture dishes was retained after the termination of carcinogen treatment. This residual MCA possessed biological activity which may be sufficient to elevate transformation frequencies in low density cultures.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) promotes the transformation of C3H/10T1/2 cells.

Continuous treatment of C3H/10T1/2 cells with low concentrations (greater than or equal to 4 pM) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhanced focus production in cultures pretreated with N-methyl-N'-nitro-N-nitrosoguanidine. Maximal enhancement occurred at 40 pM TCDD, a concentration 10 000-fold lower than that required to produce an optimal response with 12-O-tetradecanoylphorbol-13-acetate. Single treatments with 0.06 nM-5 microM TCDD did not transform C3H/10T1/2 cells or initiate the process of transformation in cultures subsequently exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Promotion of transformation is thus the predominant effect of TCDD in the C3H/101/2 cell transformation system.

Initiation and promotion in cultures of C3H10T1/2 mouse embryo fibroblasts.

Studies conducted in numerous laboratories have demonstrated that the transformation of C3H10T1/2 cells can proceed through discrete stages of initiation and promotion. Indeed, multiple operational aspects of initiation and promotion in this system closely mimic the essential characteristics of initiation and promotion on mouse skin. The sensitivity of this system to the effects of different tumor promoters also appears to parallel that of mouse skin, and there is evidence to suggest that the C3H10T1/2 system is most sensitive to agents acting as stage II tumor promoters on mouse skin. Sensitivity to compounds active at other tissue sites in rodents and perhaps man has also been observed. At this time it is difficult to assess the relevance of the C3H10T1/2 system for the study of agents capable of modulating respiratory carcinogenesis. The process of promotion can possess extreme tissue and species specificity and effects observed in murine fibroblasts of embryonic origin may have little practical bearing upon effects to be anticipated in the tracheal epithelium of the rat or the bronchial epithelium of man. This is not to say that the C3H10T1/2 system is irrelevant to respiratory carcinogenesis. However, due recognition must be taken of the probable natural limitations of this system for the study of promoters of respiratory carcinogenesis. As the data base for the use of this system is expanded, the relationship between promotion in C3H10T1/2 cells and the respiratory tract of man and rodents will become better defined. Until such time as this relationship is firmly established, it is perhaps best to regard the C3H10T1/2 system as an interesting model with which results obtained using respiratory tissue can be compared or contrasted.

Mechanistic aspects of initiation and promotion in C3H/10T1/2 cells.

The transformation of C3H/10T1/2 cells can be made to proceed through discrete stages of initiation and promotion. Studies of the effect of cell density upon focus formation in cultures treated with MNNG and TPA suggest that initiation by MNNG is due to a relatively infrequent, irreversible event induced by a single carcinogen treatment. In contrast, promotion appears to be a reversible process requiring multiple treatments with TPA over a protracted period of time. Some evidence suggests that promotion may entail the induction of phenotypic changes which impart a growth advantage to phenotypically unstable "initiated" cell populations. The actual cellular mechanism(s) for most of the phenomena observed in C3H/10T1/2 cultures have eluded precise definition and widely divergent hypotheses have been advanced to explain transformation, initiation, and promotion. Conceivably there are multiple mechanisms responsible for each of these phenomenon. Some agents may transform by a multistage mechanism whereas others may exert their effects in a more direct fashion. Some of the foci produced by promotion may be the result of simple selective processes, others the product of more complex inductive events. Variations would thus be expected between laboratories working with different protocols and agents. As demonstrated by the possible involvement of an MCA residue in transformation, it is also apparent that fundamental technical aspects of this conceptually simple cell transformation system are poorly understood. While it is natural to develop mechanistic models based on quantitative observations of transformation, a limited understanding of the basic cell culture variables which modulate both the induction and expression of transformation dictate that caution be exercised in extrapolating the significance of such models to in vivo carcinogenesis.

Enhanced sensitivity of the C3H/10T1/2 cell transformation system to alkylating and chemotherapeutic agents by treatment with 12-O-tetradecanoylphorbol-13-acetate.

The failure of the C3H/10T1/2 cell transformation system to respond to numerous known carcinogens has limited its applications for the detection and study of cancer-causing substances. Recent studies have found, however, that some carcinogens function as initiating agents for the process of transformation in these cells. Treatment with such agents is generally not sufficient to transform low-density asynchronous cultures of C3H/10T1/2 cells, but morphologic transformation will occur if such cultures are subsequently exposed to the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, the ability of TPA to enhance transformation was examined in cultures treated with a variety of chemical agents. The addition of TPA after chemical treatment enhanced the transformation of these cells by methylmethanesulfonate, ethylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, N-nitrosomethylurea, N-nitrosoethylurea, mitomycin C, 5-fluorodeoxyuridine, and 5-azacytidine. Treatment with amethopterin or benzo(e)pyrene did not produce significant numbers of foci in the presence or absence of TPA. TPA inhibited transformation by high concentrations of 3-methylcholanthrene and benzo(a)pyrene. Thus, numerous carcinogens function as initiating agents for these cells and the presence of TPA can dramatically increase the sensitivity of this cell transformation system.

A critical review of the literature on hydrogen sulfide toxicity.

The information available on the biological activity of hydrogen sulfide has been examined for present status of critical results pertaining to the toxicity of hydrogen sulfide. This review of the literature is intended as an evaluative report rather than an annotated bibliography of all the source material examined on hydrogen sulfide. The information was selected as it might relate to potential toxic effects of hydrogen sulfide to man and summarized, noting information gaps that may require further investigation. Several recommendations are listed for possible consideration for either toxicological research or additional short- and long-term tests. Two bibliographies have been provided to assist in locating references considered in this report: (1) literature examined but not cited and (2) reference citations. The majority of the references in the first bibliography were considered peripheral information and less appropriate for inclusion in this report.