Development of resistance to the growth inhibitory effects of transforming growth factor beta 1 during the spontaneous transformation of rat liver epithelial cells.

The temporary maintenance of a rat liver epithelial cell population at confluence before passaging followed by periods of rapid proliferation resulted in the generation of spontaneous transformants after about 108 population doublings. The appearance of morphologically aberrant transformants correlated directly with an increased resistance of the population to the growth inhibitory effects of transforming growth factor beta 1 (TGF-beta 1). Clonal cell lines derived from the transformants were resistant to TGF-beta 1 dependent inhibition of DNA synthesis. These cell lines were also highly tumorigenic and aneuploid, with characteristic gross chromosomal abnormalities, and they expressed a number of phenotypic markers common to rat liver epithelial cells transformed by oncogenes or chemicals. In contrast, apparently normal looking cell lines cloned from the same population were nontumorigenic and near diploid, with few chromosomal abnormalities, and they were as sensitive to TGF-beta 1 as early passage normal rat liver epithelial cells. Morphologically normal late passage rat liver epithelial cells were sensitive to transformation by the DNA hypomethylating agent 5-aza-2-deoxycytidine, in contrast to earlier passage cells, and this transformation was accompanied by the development of resistance to the growth inhibitory effects of TGF-beta 1. These findings suggest that acquisition of resistance to the effects of growth inhibitors such as TGF-beta 1 is an important and possibly essential stage in the spontaneous transformation of rat liver epithelial cells.

Altered responsiveness of rat liver epithelial cells to transforming growth factor beta 1 following their transformation with v-raf.

The effects of transforming growth factor beta (type 1) (TGF-beta 1) on DNA synthesis, cell proliferation, and protein synthesis were examined in a series of v-raf-transformed rat liver epithelial (RLE) cells, which exhibit a range of transformed phenotypes. All of the transformed cells were relatively resistant to the growth-inhibitory effects of TGF-beta 1, compared to normal RLE cells and control cells infected with a helper virus. The more tumorigenic cell lines had very few surface receptors for TGF-beta 1 and showed no increase in the secretion of a number of specific proteins, including fibronectin, following TGF-beta 1 treatment. In contrast, the more normal-looking, less tumorigenic v-raf-transformed cells bound similar amounts of TGF-beta 1 as normal RLE and control cells and showed a similar pattern of TGF-beta 1-stimulated protein secretion. These findings suggest that the effects of TGF-beta 1 on cell proliferation and on the expression of certain secreted proteins are mediated through different mechanisms. Following transformation of RLE cells with v-raf, the signalling pathways controlling TGF-beta 1 growth inhibition are perturbed, while those involved in regulating the synthesis of certain proteins may remain intact. Thus, the escape from the various distinct biological effects of TGF-beta 1 may be an important stage in the progression of neoplastic transformation of RLE cells in vitro.

Expression of growth-related genes during tumor progression in v-raf-transformed rat liver epithelial cells.

Clonal cell lines were derived from rat liver epithelial cells following their transformation with either v-raf or v-raf/v-myc. Cells transformed with v-raf alone showed reduced tumor incidence and tumor growth rates when implanted into nude mice, compared to cells also expressing the v-myc oncogene. A series of additional clones isolated from a tumor obtained following inoculation of an athymic nude mouse with the v-raf-transformed rat liver epithelial cells displayed an intermediate range of tumor aggressiveness. These findings indicate that unknown genotypic and/or phenotypic changes occur during tumor formation in vivo, which are required in addition to raf activation for complete expression of the malignant phenotype. This in vitro model of tumor progression was used to examine alterations in the expression of genes related to the growth control of liver epithelial cells, which may be involved in the malignant conversion of the preneoplastic cells. A close association was observed between the increased level of expression of the transforming growth factors alpha and beta 1, the decreased expression of extracellular matrix proteins fibronectin and collagen I, and the tumor aggressiveness (latency/growth rate), suggesting a causal role for these factors in the progression of v-raf-transformed rat liver epithelial cells to the fully malignant phenotype.

Isolation and characterization of a rat liver epithelial cell line resistant to the antiproliferative effects of transforming growth factor beta (type 1).

Rat liver epithelial cells resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF-beta 1) were isolated after 3 h exposure to 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine followed by continuous treatment with 1 ng/ml TGF-beta 1 for 6 weeks. In comparison to the parental or N-methyl-N'-nitro-N-nitrosoguanidine-exposed rat liver epithelial cells (concentration causing 50% inhibition of the rate of DNA synthesis, 0.25 ng/ml), these cells were 10-fold more resistant to the antiproliferative effects of TGF-beta 1 and exhibited resistance to growth inhibition by a highly purified liver-derived growth inhibitor, recombinant human tumor necrosis factor, and transforming growth factor beta 2. Single cell cloning of these resistant cells led to the isolation of a nontransformed clonal cell population (clone 11) which maintained stable resistance in the absence of TGF-beta 1 treatment. Binding of 125I-labeled TGF-beta 1 to rat liver epithelial cells and clone 11 cells was similar. Clone 11 cells exhibited a 5-10-fold resistance to the cytotoxins Adriamycin and vinblastine as assessed by a clonogenic assay. This drug resistance was accompanied by an increase in the steady state levels of the mRNAs for multidrug resistance gene (MDR-1), glutathione S-transferase-P, TGF-beta 1, and c-myc genes. The data presented here suggest an association between resistance to the growth-inhibitory effects of TGF-beta 1- and MDR-1-mediated multidrug resistance.

Polymeric nutrition as the primary therapy in children with small bowel Crohn's disease.

BACKGROUND: Recent studies in adults have shown that polymeric (whole protein) diets are as effective as semi-elemental and elemental formulae for the induction of remission in small bowel Crohn's disease. Whole protein diets are more palatable and cheaper. There have been no studies confirming efficacy in children. PATIENTS AND METHODS: We report our experience with seven children with active small bowel Crohn's disease given a casein-based, polymeric feed rich in TGF-beta 2 (Specific Polymeric Diet; Nestle-Clintec; Vevey, Switzerland) as complete nutrition for 8 weeks. RESULTS: Initial and follow-up assessments were performed. All children showed a significant improvement in disease activity, with C-reactive protein returning to normal, an increase in serum albumin and a good weight gain. Initial and follow-up ileal biopsies were assessed and showed reduced mucosal inflammation in six of seven children, with complete healing in two. CONCLUSION: In an uncontrolled descriptive study we have shown that a polymeric (whole protein) diet is a therapeutic option for small bowel Crohn's disease in children. By comprehensive follow-up we have demonstrated clinical and biochemical remission, with an improved endoscopic appearance and a reduction of mucosal inflammation in the terminal ileum.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

Analysis of the content of the diterpenes cafestol and kahweol in coffee brews.

The diterpenes cafestol and kahweol have been implicated as the components in boiled coffee responsible for its hypercholesterolaemic effects. These particular coffee constituents have also been shown to possess anticarcinogenic effects. A simple and sensitive reverse-phase HPLC method using solid-phase extraction has been developed for the analysis of cafestol and kahweol in coffee brews. This method was used to confirm that the method of coffee brewing is a major determinant of the cup content and hence level of consumption of these diterpenes. Scandinavian-style boiled coffee and Turkish-style coffee contained the highest amounts, equivalent to 7.2 and 5.3 mg cafestol per cup and 7.2 and 5.4 mg kahweol per cup, respectively. In contrast, instant and drip-filtered coffee brews contained negligible amounts of these diterpenes, and espresso coffee contained intermediate amounts, about 1 mg cafestol and 1 mg kahweol per cup. These findings provide an explanation for the hypercholesterolaemic effect previously observed for boiled coffee and Turkish-style coffee, and the lack of effect of instant or drip-filtered coffee brews. This methodology will be of value in more correctly assessing the human exposure to these diterpenes through the consumption of coffee, and hence the potential physiological effects of different brews.

Risk management-an industry approach.

An effective risk management system covering the whole process of food production from "farm to fork" is required by the food industry in order to assure that the food provided to consumers is safe. Food safety and quality assurance begins with the design and development of food products starting with product conceptualisation and continuing with the selection, purchasing, and evaluation of raw materials and with the specifications for processing, packaging and distribution. Within a larger quality management framework a number of tools have been developed by the food industry, which when used in an integrated fashion facilitate the management of food safety. These include good manufacturing practice (GMP), good hygiene practice (GHP) and HACCP (hazard analysis critical control point) as well as quality systems which allow the verification that all factors affecting the safety of a product are under control. Finally, regulations and systems can only function if they are applied. Everyone, from the farmer, the line operator in the manufacturing plant, to the person handling the food in distribution and sales, needs to be aware of his influence with regards safety. The effectiveness of safety awareness programs specific to each area is key to an industry approach to risk management.

The ADI as a basis to establish standards for pesticide residues in food products for infants and children.

Pesticides are widely used in agriculture to control a variety of detrimental organisms. As a consequence, low but measurable amounts of residues may be found in the food supply including food intended for infants and young children. This has been the cause of some alarm since it is difficult for the general public to understand the magnitude of health risk associated with the consumption of food contaminated with low levels of potentially toxic chemicals. In this context safety-based regulations for pesticide residues that ensure the protection of infants and young children are of crucial importance. In this article we discuss the applicability of the ADI to infants and children with regards to pesticides and outline a proposal which has been devised to establish residue limits for finished baby food products.

Expression of MHC antigens by intestinal epithelial cells. Effect of transforming growth factor-beta 2 (TGF-beta 2).

The effect of interferon-gamma (IFN-gamma) and TGF-beta 2 on expression of MHC antigens by the human intestinal epithelial cell line HT-29 was examined by immunohistochemistry and flow cytometry. Untreated HT-29 cells constitutively expressed HLA-ABC but little HLA-DR. Expression of both molecules was increased by IFN-gamma (100 U/ml, 24 h). TGF-beta 2 at concentrations > or = 0.5 ng/ml given before or simultaneously with IFN-gamma, inhibited the IFN-induced expression of HLA-DR. Small increases in HLA-ABC expression by IFN-gamma were further increased by pretreatment with TGF-beta 2, while a strong induction of HLA-ABC was inhibited by the TGF-beta 2 pretreatment. Our results suggest that the inhibitory action of the TGF-beta 2 on both HLA-ABC and HLA-DR correlates with the degree of induction following IFN-gamma treatment. Since TGF-beta 2 is present in milk and is produced by gut epithelial cells, one of its possible functions may be to regulate expression of HLA antigens in the neonatal intestine and/or diseased intestine.

Pathology of spontaneous and oncogene transformed rat liver epithelial cells and derived tumours in nude mice.

The histological and ultrastructural features of oncogene transformed rat liver epithelial (RLE) cells in culture and spontaneously transformed RLE cells were studied. The tumours produced in nude mice by all the transformed cells were either moderately well differentiated carcinomas or sarcomatous tumours. Epithelial tumours were produced in the RLE cells after transduction of both v-raf and v-myc oncogenes whereas sarcomatous tumours were produced after transduction of v-raf alone. These data suggested that transformation of RLE cells with a single oncogene, v-raf, produced malignant tumours which were consistent with sarcomas while a combination of two oncogenes, v-raf and v-myc, produced an epithelial tumour, consistent with a carcinoma. The effects of these oncogenes on RLE cells indicate that they were able to differentiate and were capable of producing two morphologically distinct tumour types. The possible role of v-myc in switching the sarcomatous lineage to an epithelial tumour lineage is considered to be significant and worthy of further studies. The epithelial tumour produced in RLE cells by combination of v-raf and v-myc is consistent with an embryonal type of hepatoblastoma. The trabecular type of liver cell carcinoma which resulted from spontaneous transformation of RLE cells illustrates the inherent potential of the RLE cell to undergo malignant change and strongly suggests that the RLE cells may be the precursor cells in the development of hepatocellular carcinoma in the rat.

Characterization of a hepatic proliferation inhibitor (HPI): effect of HPI on the growth of normal liver cells--comparison with transforming growth factor beta.

Improvements in the purification of a hepatic proliferation inhibitor (HPI) from adult rat liver have yielded a product that has an inhibitory activity 1,000-fold greater than previously reported. The growth inhibitory activity, which could be eluted from SDS-PAGE at 17-19 kilodaltons (kD), was compared to that of transforming growth factor beta (TGF-beta). The ID50 of the HPI preparation in Fischer rat liver epithelial cells was 50 pg/ml (2.5 pM) compared to a value of 260 pg/ml (10.4 pM) obtained for pure human TGF-beta. Both inhibitors also modulated the stimulation of DNA synthesis in primary hepatocytes by either epidermal growth factor or a growth stimulatory activity prepared from serum of hepatectomized rats. The ID50s of HPI and TGF-beta in these cells were 250 pg/ml and 40 pg/ml, respectively. In contrast to TGF-beta the growth inhibitory activity of HPI was unaltered in the presence of an antibody raised against TGF-beta. The possible mechanism of action of HPI is discussed.

Effects of interleukin-6 on the growth of normal and transformed rat liver cells in culture.

Recombinant human interleukin-6 produced a dose-dependent inhibition of DNA synthesis in both growing and mitogen-stimulated cultures of normal rat liver epithelial cells and also in primary rat hepatocytes. A significant inhibition of DNA synthesis (P less than 0.001) was obtained with 1 ng/ml (10 Units/ml) interleukin-6 in normal rat liver epithelial cells. The ID50 for inhibition of DNA synthesis in primary rat hepatocytes was 1 ng/ml. In contrast to the effects of transforming growth factor beta (Type I), where an almost complete inhibition of DNA synthesis could be achieved with either cell type, the maximal inhibition observed with interleukin-6 for both of these cell types was about 45%. Thus distinct mechanisms are involved in the inhibition of liver cell growth by these growth modulators. Transformed liver-derived cell lines were relatively resistant to the growth inhibitory effects of both interleukin-6 and TGF-beta 1 compared with the normal cells. However, human Hep G2 cells, which were completely resistant to the growth inhibitory effects of TGF-beta 1, were moderately inhibited by interleukin-6, indicating that the mechanisms responsible for the acquired resistance to growth inhibition is different for these growth inhibitors. The ability of interleukin-6 to function as a growth inhibitor in vitro was confirmed using normal rat liver epithelial cells. Interleukin-6 at a concentration of 10 ng/ml produced a significant decrease (P less than 0.05) in the proliferation of these cells. These data demonstrate that interleukin-6 may have the capability of functioning as a growth regulatory polypeptide for liver cells in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Placental glutathione S-transferase (GST-P) induction as a potential mechanism for the anti-carcinogenic effect of the coffee-specific components cafestol and kahweol.

The coffee specific diterpenes cafestol and kahweol (C + K) have been reported to be anti-carcinogenic in several animal models. It has been postulated that this activity may be related to their ability to induce glutathione S-transferases (GSTs). We investigated the influence of a mixture of C + K, incorporated at various levels in the diet of Sprague-Dawley rats, on the expression of different hepatic GST iso-enzymes. Liver samples were examined using isoform-specific GST substrates and antibodies, and highly selective oligomers were employed to determine effects at the RNA level. A dose-dependent increase in general GST activity was observed in male and female animals following 28 or 90 days of treatment. A time-course study demonstrated that the maximal effect was observed within 5 days of treatment. Little or no effect was found on the activity of GST alpha and mu iso-enzymes. The most striking observation was a dose-dependent induction of placental glutathione S-transferase (GST-P) which could be demonstrated at the mRNA, protein and enzymatic levels. This effect was observed in both male and female rats. The maximal induction was attained within 5 days of treatment with C + K, remained elevated with continued treatment, but was reversible on withdrawal of treatment. Immunohistochemical examination of liver slices revealed a strong even distribution of GST-P expression throughout the acinus at the highest dose of C + K, while at lower doses the induction of GST-P occurred predominantly in periportal hepatocytes. There was no indication of the presence of preneoplastic foci and, furthermore, the effect of C + K on the GST-P was completely reversible. These findings indicate that the anticarcinogenic mechanism of C + K may involve a specific induction of GST-P and suggest a potential role for GST-P in detoxifying carcinogenic compounds.

Limits for pesticide residues in infant foods: a safety-based proposal.

A review of safety procedures for regulating pesticide residues in food commodities has demonstrated that maximum residue levels established by national and international bodies may not be suitable for direct application to finished infant products. We have developed a safety-based strategy specifically aimed at setting limits for pesticide residues in baby foods. The approach is based on the acceptable daily intake (ADI) together with the application, when necessary, of an additional uncertainty factor to account for the potential higher sensitivity of infants to toxicants. The need for this extra factor is dependent on the toxicological information available and may be particularly important for pesticides with a neurotoxic potential. Using this strategy we have evaluated safety-based residue limits for finished products based on a standard food intake pattern of a 4-month-old infant. For most of the pesticides examined the estimated limits fell within a range which can be controlled through existing quality assurance systems. This scientific approach appears usable in practice and is intended as a basis for stimulating discussions aimed at evaluating the need for new limits to ensure the optimal quality of infant products with respect to pesticide safety. This assessment demonstrates that for a number of pesticides there is a need for adequate developmental studies to provide assurance that the current ADI is appropriate for infants. A consequence may be the necessity for enhanced analytical sensitivity for the determination of pesticide residues in infant foods which would be compatible with limits based on an infant-adjusted ADI.

Dexamethasone prevents the growth inhibitory effects of recombinant tumor necrosis factor in a rat hepatoma cell line Reuber-RC-3: an association with the changes in the messenger RNA levels for multidrug resistance gene.

Two days exposure to recombinant tumor necrosis factor (rTNF-alpha) produced a dose-dependent reduction in (methyl-3H) thymidine incorporation in RC-3 cells (ID50 = 25 units/ml). Prolonged treatment with rTNF-alpha further resulted in a significant reduction in colony formation (ID50 = 200 units/ml), which was reversed upon removal of the agent. Interferon levels were undetectable in the supernatants of the rTNF-alpha treated cells. Simultaneous exposure to dexamethasone prevented the growth inhibition in rTNF-alpha-treated RC-3 cells. Significant dose-dependent increase in the steady state levels of the mRNA for multidrug resistance (MDR1) gene was observed after rTNF-alpha treatment while simultaneous exposure to dexamethasone produced a substantial reduction in the mRNA levels for MDR1 gene. These data suggest that growth inhibitory effects of TNF are regulated by dexamethasone and are associated with changes in MDR1 mRNA levels in hepatoma-derived cells.

Growth modulatory effects of a liver-derived growth inhibitor, transforming growth factor beta 1, and recombinant tumor necrosis factor alpha, in normal and neoplastic cells.

The growth modulatory effects of a rat liver-derived growth inhibitor (LDGI), transforming growth factor beta 1 (TGF-beta 1), and recombinant tumor necrosis factor (rTNF-alpha) were examined in a variety of liver-derived and nonliver-derived normal and neoplastic cell culture systems. Normal rat liver epithelial (RLE) cells were highly sensitive to the growth inhibitory effects of LDGI (ID50 = 0.2 ng/ml) and TGF-beta 1 (ID50 = 0.25 ng/ml) but were less sensitive to rTNF-alpha (ID40 = 5000 Units/ml). Aflatoxin B1-transformed RLE cells showed sensitivity to the cytostatic effects of LDGI (ID50 = 1.5 ng/ml); however, these cells were completely resistant to the antiproliferative effects of TGF-beta 1 and rTNF-alpha. Clones isolated from these transformed cells, exhibited a wide range of sensitivities to LDGI but all of the clones were resistant to the growth inhibitory effects of both TGF-beta 1 and rTNF-alpha. Rat hepatoma Reuber cells were extremely sensitive to the antiproliferative effects of rTNF-alpha (ID50 = 10 Units/ml) but exhibited sensitivity to LDGI only at concentrations above 1.5 ng/ml and were resistant to the antiproliferative effects of TGF-beta 1. Rat hepatoma UVM 7777 cells and human hepatoma HepG2 cells, however, were insensitive to the growth inhibitory effects of all three factors. Among the nonliver-derived cells, human breast carcinoma (MCF-7) cells were extremely sensitive to rTNF-alpha (ID50 = 20 Units/ml, exhibited some sensitivity to LDGI (ID50 = 1 ng/ml), and were resistant to the antiproliferative effects of TGF-beta 1. In contrast, the rate of DNA synthesis is rat kidney fibroblasts and human foreskin fibroblasts was significantly stimulated in response to TGF-beta 1, LDGI, and rTNF-alpha. These data demonstrate that LDGI, TGF-beta 1, and rTNF-alpha exert positive and negative modulations of growth in different cell systems and that the growth regulatory effects of LDGI differ from those of TGF-beta 1 and rTNF-alpha in some cell types.