Induction of sister-chromatid exchanges by direct and indirect agents in a human teratoma cell line.

We have extended the characterization of the P3 cell line, derived from a human epithelial teratocarcinoma, by studying the induction of sister-chromatid exchanges (SCEs) by direct and indirect carcinogens. Several direct acting carcinogens produce a dose-dependent increase in SCEs. Most notably, N-methyl-N'-nitro-N-nitrosoguanidine and 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene produce increases in SCEs at doses comparable to those used to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. The indirect carcinogens elicit SCEs only when the P3 cells are cocultured with cells capable of metabolizing the indirect carcinogens to the active form. Human breast carcinoma (BJ-015) and rat hepatoma (RL-12) cells are equally efficient in activating polycyclic aromatic hydrocarbons to the active form. This cell-mediated induction of SCEs is obtained when the P3 cells are incubated with live, X-irradiated, or UV-irradiated BJ or RL cells. This P3 cell line is thus equally suitable to study the induction of mutations or the induction of SCEs with direct and indirect carcinogens.

Genotoxicity of 1-nitropyrene and 2,4,7-trinitro-9-fluorenone to mammalian cells.

Nitroarenes are ubiquitous environmental pollutants displaying potent genotoxicity in bacterial and mammalian cells. In this study, 2,4,7-trinitro-9-fluorenone (TNF) was more potent than 1-nitropyrene (1-NP) in eliciting genotoxic responses in 4 mammalian cell lines. All 4 cell types were capable of activating the nitroarenes, since no special incubation conditions were required. Inhibition of normal DNA synthesis and cytotoxicity were significantly increased with TNF in a dose range of 0.2-5 micrograms/ml for human teratocarcinoma (PA1) cells, mouse Sertoli (TM4) cells, rat hepatoma (RL12) cells, and human-Chinese hamster ovary (CHO-K1) cells. For 1-NP, a dose range of 10-20 micrograms/ml was required to achieve comparable results for the respective cell lines. Only the RL12 and CHO-K1 cells showed recovery of normal DNA synthesis when TNF or 1-NP was removed from the medium. The other cell types showed little or no recovery up to 42 h after removal of the nitroarene. In exclusively studying TNF, the induction of sister-chromatid exchanges (SCEs) and a delay in cell cycle as monitored by harlequin chromosomes, were observed at a concentration range of 0.003-0.2 microgram/ml in PA1, TM4, and RL12 cells. In CHO-K1 cells, TNF at 0.001-1 microgram/ml was clearly mutagenic at the hprt locus.

Metabolic activation of procarcinogens to genotoxic products in cultured rat liver cells.

A recently established rat liver cell line, RL-12, exhibits an unusually high sensitivity to the genotoxic effects of a number of selected procarcinogens. Significant reductions in cell survival (D37%) and induction of sister chromatid exchanges were obtained with 1 X 10(-9) M benzo[a]pyrene and 2 X 10(-8) M 7,12-dimethylbenz[a]anthracene. This rat liver epithelial cell line may serve as a useful model system to study the metabolic activation of procarcinogens to their ultimate genotoxic form.

Human CD4- CD8- alpha beta+ T cells express a functional T cell receptor and can be activated by superantigens.

The CD4 and CD8 molecules play an important role in the stimulation of T cells and in the process of thymic education. Most mature T cells express the alpha beta TCR and either CD4 or CD8; however, there is a small population of alpha beta+ TCR T cells that lack both CD4 and CD8. Little is known of the biology of the CD4- CD8- (double-negative) alpha beta+ TCR T cells or the nature of the Ag to which they may respond. These cells not only represent a novel population of T cells but also provide useful biologic tools to study the roles that CD4 and CD8 play in T cell activation. In this study we have addressed two questions. Firstly, whether CD4- CD8- alpha beta+ TCR T cells have functionally active TCR and, secondly, whether CD4 or CD8 is required for the activation of T cells by bacterial enterotoxins. Six double-negative alpha beta+ TCR T cell clones, propagated from two healthy donors, were challenged with a panel of nine bacterial enterotoxins. The V alpha and V beta usage of their TCR was determined by polymerase chain reaction. All of the CD4-CD8- clones proliferated in response to at least one of the enterotoxins, in a V beta-specific manner. The proliferative response of the CD4-CD8- alpha beta+ TCR T cell clones was similar in magnitude to that exhibited by CD4+ T cell clones of known V beta expression. These data clearly show that the CD4 and CD8 molecules are not required for the activation of untransformed human T cells by bacterial enterotoxins. Furthermore, these results indicate that CD4-CD8- alpha beta+ TCR T cells, normally present in all individuals, are not functionally silent, because they can be stimulated via their TCR. Their physiologic role, like that of gamma delta T cells, remains to be elucidated.

Experimentally produced synovial sarcoma in mice.

BALB/c and C57 B1/6 mice which received weekly subcutaneous injections of a cannabinoid developed tumors at the points of injection. In the groin the tumors were fibrosarcomas, while in the interscapular region they were anaplastic sarcomas and synovial sarcomas. The latter developed faster and were larger in cannabinol - and cannabidiol-treated C57 B1/6 mice than in delta 9-tetrahydrocannabinol-treated BALB/c mice. This difference could not be attributed to strain differences. 2 cannabidiol- and 1 cannabinol-treated C57 B1/6 females with the fastest growing synovial sarcomas were pregnant and, in addition, 2 of them developed mammary adenocarcinomas, indicating that they were rich in estrogen. On the other hand, it is known that delta 9-tetrahydrocannabinol causes a decrease in the sex hormone. It is consequently thought that the cannabinoids are only initiators of synovial sarcoma, while growth or promotion of the latter is influenced partially or entirely by the sex hormone.

Cannabinoids induce incomplete maturation of cultured human leukemia cells.

Monocyte maturation markers were induced in cultured human myeloblastic ML-2 leukemia cells after treatment for 1-6 days with 0.03-30 microM delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana. After a 2-day or longer treatment, 2- to 5-fold increases were found in the percentages of cells exhibiting reactivity with either the murine OKM1 monoclonal antibody or the Leu-M5 monoclonal antibody, staining positively for nonspecific esterase activity, and displaying a promonocyte morphology. The increases in these differentiation markers after treatment with 0.03-1 microM THC were dose dependent. At this dose range, THC did not cause an inhibition of cell growth. The THC-induced cell maturation was also characterized by specific changes in the patterns of newly synthesized proteins. Pronounced among these changes was an increase in the synthesis of at least 10 proteins that are found abundantly in monocytes. The THC-induced differentiation did not, however, result in cells with a highly developed mature monocyte phenotype; the THC-treated cells failed to exhibit other monocyte markers such as attachment to the surface of tissue culture dishes or morphological maturation beyond the promonocyte stage. However, treatment of these "incompletely" matured cells with either phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxycholecalciferol, which are inducers of differentiation in myeloid leukemia cells (including ML-2 cells), produced cells with a mature monocyte morphology. Two other cannabinoids, cannabidiol and cannabinol, which were more cytotoxic than THC at comparable doses, also caused an increase in the expression of maturation markers, but at doses higher than those required for THC. The ML-2 cell system described here may be a useful tool for deciphering critical biochemical events that lead to the cannabinoid-induced "incomplete" cell differentiation of ML-2 cells and other related cell types. Findings obtained from this system may have important implications for studies of cannabinoid effects on normal human bone-marrow progenitor cells.

Human in vitro immune responses to Mycobacterium tuberculosis.

SETTING: T helper cells can be divided into 2 subsets on the basis of their cytokine generation. T helper 1 cells secreting gamma interferon and interleukin 2 appear to be more prominent in patients with limited tuberculous disease. OBJECTIVE: The purpose of this study was to evaluate human T helper cell immune responses to mycobacterial antigens in vitro and correlate these with the clinical features of patients with tuberculous infection or disease. DESIGN: We studied 51 subjects and 11 controls who were grouped according to disease involvement as follows: 1) Mantoux negative, BCG negative, no disease; 2) Mantoux positive, no disease; 3) localized extrapulmonary; 4) healed pulmonary; 5) active pulmonary; and 6) miliary/disseminated. Peripheral blood mononuclear cells were cultured with PHA, PPD or Tetanus Toxoid, proliferation assessed and the supernatant analysed using an ELISA for IFN gamma. ELISA was also used to measure M. tuberculosis specific antibodies in the serum. RESULTS: Mantoux size correlated with PPD proliferation r = 0.5, P = 0.005 and gamma IFN production r = 0.36, P < 0.01. All groups produced abundant gamma IFN although there was a trend toward higher production in groups 3 and 4. M. tuberculosis specific IgA (P = 0.003) and IgG1 (P = 0.002) was higher in groups 5 and 6. Those patients with limited disease (groups 2-4) had significantly lower levels of IgG4 than patients with severe disease (groups 5 & 6) (P < 0.02). CONCLUSION: In conclusion patients with healed or extrapulmonary disease have immune responses in vitro suggestive of a TH1 (cell mediated immune) response, whereas patients with miliary/disseminated disease have antibody production suggestive of a TH2 response, together with high gamma IFN production. Both TH1 and TH2 responses may be necessary for host protection if there is a high bacillary load.