Subchronic toxicity, mutagenicity and allergenicity studies of a cultured dextrose food product.

MicroGARD(R) 200 is a fermented dextrose product used to extend food shelf-life by inhibiting spoilage due to Gram-negative bacteria, selected yeast and molds. The present studies were conducted to evaluate the safety of this food ingredient for determination of GRAS status. MicroGARD 200 was subjected to a bacterial reverse mutation assay, a subchronic oral toxicity study and an oral antigenicity study. It showed no evidence of mutagenic potential or toxicity in four strains of Salmonella typhimurium or in Escherichia coli strain WP2 uvrA with and without metabolic activation. MicroGARD 200 was orally administered to rats for 13 consecutive weeks at dietary concentrations of 0, 0.5, 2.0 or 5.0%. Water consumption and urinary excretion of sodium were slightly increased in both sexes at the high dose due to the sodium content of the test substance (about 6%). Increases in fasting glucose and decreased plasma creatinine were not accompanied by treatment-related histopathological changes and were within the normal range for historical controls. The potential antigenic properties of MicroGARD 200 were investigated via gavage in guinea pigs using an active systemic anaphylaxis (ASA) challenge [Annals of Allergy 67 (1991) 400; Allergy 49 (1994) 361]. There was no evidence of any anaphylactic sensitizing properties for MicroGARD 200.

Blockade of TGFbeta3 up-regulation of p27Kip1 and p21Cip1 by expression of RasN17 in epithelial cells.

Our previous data demonstrated that Ras activation is necessary and sufficient for transforming growth factor beta (TGFbeta)-mediated Erk1 activation, and is partially required for the inhibition of cyclin-dependent kinase 2 (Cdk2) activity, cyclin A expression and DNA synthesis by TGFbeta (KM Mulder and SL Morris, J. Biol. Chem., 267: 5029-5031, 1992; MT Hartsough and KM Mulder, J. Biol. Chem., 270: 7117-7124, 1995; and MT Hartsough et al., J. Biol. Chem., 271: 22368-22375, 1996). Here, we examined the kinetics and role of Ras in TGFbeta3-mediated effects on specific G1 cell cycle components in TGFbeta-sensitive (4-1) and TGFbeta-resistant (4-6) intestinal epithelial cells (IEC's). Our results indicate that inactivation of Ras by stable, inducible expression of a dominant-negative mutant of Ras (RasN17) completely abrogated the ability of TGFbeta3 to up-regulate both CKI's. In contrast, the ability of TGFbeta3 to up-regulate p27Kip1 and p21Cip1 was maintained in ZnCl2-treated control cells. Inactivation of Ras also completely blocked the rapid TGFbeta-mediated increase in new synthesis of p27Kip1 protein. Moreover, up-regulation of p21Cip1 protein levels and new synthesis of p27Kip1, as well as the association of these CKI's with Cdk2, preceded the decrease in Cdk2 activity by TGFbeta. Collectively, our results suggest that p21Cip1 and p27Kip1 are upstream effectors of the TGFbeta-mediated inhibition of Cdk2 activity in IEC 4-1 cells, and demonstrate that Ras activation is obligatory for TGFbeta-mediated up-regulation of these CKIs in untransformed epithelial cells.

Human malignant mesothelioma cell growth: activation of janus kinase 2 and signal transducer and activator of transcription 1alpha for inhibition by interferon-gamma.

Intrapleural injections of recombinant human IFN-gamma have shown some efficacy in reducing tumor growth in early stages of diffuse malignant mesothelioma (DMM). Here, we have addressed the potential therapeutic effect of IFN-gamma in DMM by investigating the activation of the JAK/STAT signaling pathway in seven human mesothelioma cell lines (HMCLs) that were differentially responsive to the antiproliferative activity of IFN-gamma. We showed that janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1) were phosphorylated on tyrosine residues within 15 min in all the HMCLs in which IFN-gamma (500 units/ml) inhibited proliferation. In addition, STAT1 binding activity to the gamma-activated sites DNA sequence was detected within 15 min in electrophoretic mobility-shift assay analysis, and IFN regulatory factor-1 RNA expression was observed within 6 h in the more responsive cells (72.7-95.2% inhibition of DNA synthesis after 72 h of treatment). Conversely, in several HMCLs, absent or limited growth suppressive effect (less than 22% inhibition of DNA synthesis) was associated with alterations in expression or activation of JAK2 or STAT1 or, downstream, with low induction of IFN regulatory factor-1 RNA expression and/or STAT1 protein expression following IFN-gamma treatment. These data suggest that at least part of the IFN-gamma effect on proliferation of HMCLs is mediated directly through activation of the JAK/STAT1 signaling pathway, and it could account for the antitumoral activity reported in DMM patients treated with IFN-gamma.

Differential responsiveness of human and rat mesothelioma cell lines to recombinant interferon-gamma.

Recombinant human interferon-gamma (r-hu-IFN-gamma) has been found to exert an antitumor action in vivo in early stages of human malignant mesothelioma, and an antiproliferative effect in vitro. In order to study the mechanisms of cytostasis in mesothelioma cells, we examined two IFN-gamma-controlled metabolic pathways known to mediate growth arrest in various cell types, measuring production of the antiproliferative compound nitric oxide (NO) and degradation of tryptophan in nine human mesothelioma cell lines (HMCLs) displaying different sensitivities to the antiproliferative effect of r-hu-IFN-gamma. Two rat mesothelioma cell lines were also studied. IFN-gamma receptor was present and functional in HMCLs, regardless of their sensitivity to the growth-inhibitory effect of r-hu-IFN-gamma. However, no NO synthase activity or the resulting antiproliferative molecule NO were induced in HMCLs treated either with r-hu-IFN-gamma alone or with a combination of r-hu-IFN-gamma and other cytokines, and/or with lipopolysaccharide (LPS). In responsive HMCLs, r-hu-IFN-gamma induced strong indoleamine-2,3-dioxygenase (IDO) activity, which causes rapid degradation of tryptophan; however, the correlation between r-hu-IFN-gamma-mediated growth arrest and IDO induction was not absolute. In rat mesothelioma cells, NO synthase was induced in response to murine IFN-gamma + interleukin-1beta (IL-1beta) treatment, and played a role in the cytokine-mediated antiproliferative activity. However, NO production did not seem to be the unique antiproliferative mechanism induced by cytokines in these cells. Our results indicate that two classical pathways accounting for some of the cytostatic effects of IFN-gamma in rodent cells are not efficient in human mesothelioma cells, and suggest that cytokine-induced growth inhibition is mediated by a different pathway in HMCLs.

Altered transforming growth factor signaling in epithelial cells when ras activation is blocked.

We have previously demonstrated that growth inhibition of untransformed intestinal epithelial cells by transforming growth factor beta1 (TGFbeta) and TGFbeta2 was associated with a rapid activation of both Ras and extracellular signal-regulated kinase 1 (Erk1) (Mulder, K. M., and Morris, S. L. (1992) J. Biol. Chem. 267, 5029-5031; Hartsough, M. T., and Mulder, K. M. (1995) J. Biol. Chem. 270, 7117-7124). In order to determine whether Ras was required for TGFbeta regulation of both Erk1 and downstream components associated with TGFbeta-mediated growth inhibition, the intestinal epithelial cell (IEC) line IEC 4-1 was transfected with a vector containing a dominant-negative mutant of Ras (RasN17) under the control of an inducible metallothionein promoter. Using two different RasN17-transfected clones treated with ZnCl2, we demonstrate here that induction of Ras expression by at least 4-fold completely abrogated the TGFbeta-mediated activation of Erk1. Moreover, the RasN17-mediated reversal of the TGFbeta effect on Erk1 was dependent upon the level of expression of the dominant-negative protein. ZnCl2 treatment of control cells transfected with the empty vector did not alter Ras expression or the activation of Erk1 by TGFbeta. In order to determine whether the activation of Ras by TGFbeta was required for the growth inhibitory effect of TGFbeta, we examined TGFbeta2 effects on Cdk2-associated histone H1 kinase activity, cyclin A protein expression levels, and DNA synthesis in two intestinal epithelial cell clones transfected with RasN17. In cells expressing RasN17, we observed a 50% reversal of the inhibition of Cdk2 activity, a 78% reversal of the down-regulation of cyclin A protein expression, and a 21% reversal of the inhibition of DNA synthesis by TGFbeta. Collectively, these results indicate that Ras activation is obligatory for TGFbeta-mediated activation of Erk1, whereas it is partially required for the growth inhibitory effect of TGFbeta.

Maintenance of growth factor signaling through Ras in human colon carcinoma cells containing K-ras mutations.

Fifty percent of human colon carcinomas contain activating mutations in the K-ras gene. However, whether these alterations in K-ras affect the function of Ras proteins in growth factor (GF) signal transduction is now known. Here we have characterized a previously defined human colon carcinoma cell model system for K-ras gene mutations and for altered levels of Ras protein expression and have examined whether these alterations affect Ras function in GF signal transduction. Sequence analysis of PCR-amplified K-ras gene fragments indicated that among the more aggressive cell lines, four had a normal K-ras sequence, whereas 3 others (isolated from the same human tumor) contained a mutation at codon 13. In contrast, all 7 of the less aggressive cell lines contained a mutation at either codon 12 or 13. In addition to the presence of a K-ras mutation, one cell line expressed higher levels of the K-Ras protein and displayed elevated Ras-GTP loading (in the absence of GF addition) compared with the other cell lines examined. Despite these alterations, the mitogenic GF combination epidermal growth factor + insulin + transferrin resulted in an activation of Ras and extracellular signal-regulated kinase 2. Collectively, our results indicate that the malignant phenotype of the cell lines was not correlated with the presence of K-ras mutations or with higher levels of Ras protein expression. Furthermore, K-ras mutations, high levels of K-Ras protein expression, and elevated Ras-GTP loading, as they occur naturally in human colon carcinomas, do not abolish the function of Ras in GF signaling.

Synthesis of poly(ADP-ribose) in asbestos treated rat pleural mesothelial cells in culture.

To investigate the origin of DNA repair in rat pleural mesothelial cells (RPMC) exposed to asbestos fibers, poly(ADP-ribose) polymerase (PARP) activity was measured in the asbestos-treated cells. As bleomycin has been shown to activate poly(ADP-ribose) synthesis in several cell systems, the response to bleomycin with regard to PARP assay was first investigated. Bleomycin produced a dose-dependent increase of poly(ADP-ribose) synthesis in RPMC. Likewise both chrysotile and crocidolite fibers produced a concentration-dependent PARP activation indicating that the formation of DNA strand breaks is one type of damage produced by asbestos in RPMC. Enhancement of DNA repair, assessed by the measurement of [3H] methylthymidine incorporation in growth arrested cells, was not detectable in the presence of 3-methoxybenzamide (3-MBA), a PARP inhibitor, confirming a relation between PARP activation and DNA repair. The participation of DNA breakage in asbestos toxicity on RPMC was determined by the colorimetric 3-4(5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. There was no relationship between DNA breakage and cytotoxicity since the use of PARP inhibitors did not change cell viability. These results indicate that asbestos produce DNA damage that is repaired in RPMC.

Cell localization and regulation of expression of cytochrome P450 1A1 and 2B1 in rat lung after induction with 3-methylcholanthrene using mRNA hybridization and immunohistochemistry.

In order to characterize the response of various pulmonary cell types to polycyclic aromatic hydrocarbons, the expression of cytochrome P450 (CYP) 1A1 and 2B1 mRNA in the lung of rats, with or without induction by 3-methylcholanthrene (3MC), was analyzed by in situ hybridization using appropriate 35S-labeled riboprobes. The expression of the corresponding proteins was investigated immunohistochemically. Following induction with 3MC, the kinetics of mRNA expression differed considerably between Clara cells and type II pneumocytes and venous endothelial cells. In Clara cells, mRNA expression was detected as early as 1 h after induction, peaked between 2 and 4 h, and was completely undetectable at 14 h. In contrast, venous endothelial cells and type II pneumocytes exhibited permanent mRNA expression of CYP 1A1 in 3MC-pretreated rats. These kinetic results explain the striking absence of correlation between mRNA and protein expression observed in Clara cells 24 h after the end of the induction protocol, as these cells exhibited intense protein expression with no mRNA. In contrast, a good correlation was observed for mRNA and protein expression of CYP 2B1, with similar expressions for Clara cells and type II pneumocytes, but no expression in endothelial cells. This study clearly distinguished the regulation of CYP 1A1 expression in the rat lung from that described in the liver. The differences observed in the various lung cell types, whatever the post-transcriptional mechanisms involved, emphasize that studies must be performed at the cellular level in order to understand the specific response to xenobiotics, not only of this organ as a whole but also of its various anatomic structures.

Expression of cytochrome P450 in rat pleural mesothelial cells in secondary cultures.

Cultured rat pleural mesothelial cells (RPMC) isolated from male Sprague-Dawley rats have been shown to metabolize polycyclic aromatic hydrocarbons to more oxygenated metabolites. This capacity, which is maintained with passages, suggested the presence of monooxygenase enzymes. In order to clarify the enzymatic pathway, we investigated the expression of cytochromes P450 (CYP) in cultured RPMC by Western and Northern blot analyses. Cells were cultured in Ham's F10 medium supplemented with 10% fetal calf serum. The CYP expression was studied from passage 9 to 16 on different cell strains treated for 48 hours with P450 inducers. CYP1A1 apoprotein expression was very low in untreated cells, but was markedly induced after treatment with 1 microM 3-methylcholanthrene or 22 microM beta-naphthoflavone. CYP1A1 mRNA was not detected in untreated cells and appeared after 3-methylcholanthrene treatment. CYP2E1 apoprotein was constitutively expressed in cultured RPMC, and markedly increased by 170 mM ethanol, and 0.1 microM or 1 microM dexamethasone treatments. Unexpectedly, whereas the amount CYP2E1 mRNA was not modified by ethanol treatment, dexamethasone has a marked inductive effect on CYP2E1 mRNA level. The CYP expression pattern was found similar in RPMC issued from different rats, and not dependent on passage number. The CYP expression and the detection of NADPH-P450 reductase, and of epoxide hydrolase, ascertained that RPMC contain the overall enzymatic pathway required for the biotransformation and activation of procarcinogen compounds, such as polycyclic aromatic hydrocarbons and nitrosamines. Both expression and regulation properties are maintained in long-term cultures of RPMC.

Role of oxygen derivatives in the cytotoxicity and DNA damage produced by asbestos on rat pleural mesothelial cells in vitro.

The role of reactive oxygen metabolites in the toxic effects of asbestos on pleural mesothelial cells is not well defined. We exposed rat pleural mesothelial cells (RPMC) to chrysotile and crocidolite fibers (0-40 micrograms/cm2) in the presence or absence of catalase and superoxide dismutase (SOD). Cell injury was measured using the colorimetric 3-4 (5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and DNA damage was evaluated in terms of unscheduled DNA synthesis (UDS). Catalase (100 U/ml) and SOD (250 U/ml) protected RPMC against asbestos-induced cytotoxicity and DNA damage. However, the inactivated enzymes and bovine serum albumin also showed some protection, suggesting that the effect of antioxidant enzymes may be partly related to their protein nature. These results suggest that oxygen derivatives are partly involved in the toxic effects of asbestos on cultures of RPMC. The presence of extracellular proteins may also decrease asbestos-produced toxicity by reducing the degree of RPMC-fiber interaction.

In vitro effects of recombinant human interferon gamma on human mesothelioma cell lines.

Malignant mesothelioma is a tumor arising from serous surfaces and often related to asbestos exposure. Malignant mesothelioma is resistant to various forms of therapy. Radiotherapy, surgery or chemotherapy only slightly improve prognosis. IFN-gamma produces complete or partial responses in stage-I patients with malignant mesothelioma. The in vitro biological effect of IFN-gamma on malignant mesothelioma cells remains poorly elucidated. In the present study, 32 well-characterized human mesothelioma cell lines (HMCL) were treated with r-hu IFN-gamma at 4 doses and cell growth was determined by a colorimetric method (MTT assay). Among the 32 HMCLs tested, II exhibited significant cell-growth inhibition; 16 were insensitive to r-hu IFN-gamma, and 5 were slightly inhibited. The sensitive cell lines were strongly inhibited by r-hu IFN-gamma. Our results show that HMCL exhibit a large range of responses to r-hu IFN-gamma, some of which can be compared with those obtained in vivo in humans.

Developmental changes in enzymatic systems involved in protection against peroxidation in isolated rat brain microvessels.

Activities of 3 enzymes involved in the major detoxification pathway for peroxides were assessed in rat brain microvessels. Between the 7th and 60th day after birth, glutathione peroxidase specific activity remained constant in microvessels, while glutathione reductase specific activity increased from day 14 to day 60. On the other hand, the specific activity of these two enzymes evolved similarly in total brain homogenate: they increased between day 7 and day 30, and then reached a plateau. In contrast, catalase specific activity in microvessels was markedly decreased from day 7 to day 60. A significant decrease in this enzyme specific activity was also observed in brain homogenate during development. However, in microvessels, catalase specific activity remained higher than that of brain homogenate throughout the time period studied. Our results support the idea that enzymatic mechanisms against peroxidative damage are required in early age, and could be potent at the level of the blood-brain barrier.

Use of mesothelial cell cultures to assess the carcinogenic potency of mineral or man made fibers.

Natural mineral fibers may produce pulmonary cancers and mesothelioma. In contrast with lung cancer, the incidence of fiber-induced mesothelioma is not enhanced in smokers compared to non smokers. It is therefore of special interest to use mesothelial cells to study the toxicity of natural or man made mineral fibers. Several years ago, we have developed a method to culture rat pleural mesothelial cells (RPMC). We have first studied the effects of asbestos fibers by the application of in vitro tests formerly developed to determine the genotoxicity and transforming potency of soluble xenobiotics. Moreover, we have determined whether RPMC expressed cytochromes P450 known to metabolize polycyclic aromatic hydrocarbons. This paper reviews the results obtained so far. It has been found that asbestos fibers produced a cell transformation and a genotoxicity characterized by the formation of aneuploid cells, abnormal anaphases, chromosomal aberrations and DNA repair (UDS). In addition, RPMC expressed different forms of cytochromes P450. It is nowadays suggested that the tumorigenic potency of asbestos fibers may be related to the fiber dimensions, to their surface properties and in vivo biopersistence; this term involves the fiber solubility in biological medium and the fiber epuration from the lung by clearance mechanisms. Experiments are now in progress to determine whether the in vitro effects are dependent on the fiber parameters suggested as playing a role in the carcinogenic potency.