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Discusses the use of genomics in toxicology and epidemiology. Influence of the ethical, legal and regulatory issues on the direction and application of genomics technologies to environmental health research; Need to require suitable biologic samples from large and diverse population groups at the relevant period of exposure to ensure the effectiveness of the application of various technologies.

We have investigated the effects of modification of the growth media pH on Syrian hamster embryo (SHE) cellular lifespan and transformation. Culturing SHE cells in Dulbecco's modified Eagle's medium (DMEM) at pH 6.7 relative to pH 7.3 results in a 4-fold increase in the lifespan of SHE cells before senescence occurs. This effect was observed both with the wild type SHE cell population and with the morphological transformation sensitive subpopulations derived from the wild type SHE cell mixture (c19 and cl17). The increase in SHE cell lifespan at pH 6.7 was observed both as an increase in days in culture and an increase in the number of total population doublings before cellular senescence occurred. The tumor promoting agent TPA increased the lifespan of the SHE cell population and one of the MT sensitive subpopulations when these cells were cultured in pH 7.3 medium. Interestingly, treatment of the SHE cell population, and the two MT sensitive SHE cell clones with TPA at culture medium pH of 6.7 reduced the lifespan of each cell type. Finally, culturing SHE cells in medium of pH 6.7 increased the potential of the cells to undergo morphological transformation in response to treatment with carcinogens, demonstrating a potential connection between SHE cell lifespan and carcinogen induced morphological transformation, probably through a mechanism involving a pH induced block in cellular differentiation. We believe this is the first report demonstrating the role of pH in controlling cellular lifespan. [ABSTRACT FROM PUBLISHER]

Investigations were carried out designed to isolate and biologically characterize the subpopulation of cells within the Syrian hamster embryo (SHE) cell population which are sensitive to morphological transformation (MT). Biological cloning of MT-sensitive cells demonstrated that within the complex SHE cell population, MT-sensitive cells comprise ∼27% of the clonally plateable cellular population. Importantly, MT-sensitive cells display MT rates of ∼7% following benzo[a]pyrene exposure, a rate which falls to <1% with additional passage of the cells, indicating that the ability to undergo MT is a transient phenomenon. Biological characterization of the clonal MT-sensitive cells indicates that these cells are relatively undifferentiated, since they express both cytokeratins and vimentin and respond to a variety of stem cell growth and differentiation factors, although the majority appear to be committed progenitor cells, since they express either mesenchymal or epithelial cell characteristics. Together these data demonstrate that MT-sensitive cells comprise a subpopulation of the cells within the complex SHE cell population, that the ability to undergo MT is a transient phenomenon and that MT-sensitive cells are relatively undifferentiated committed progenitor stem-like cells, all of which gives rise to the hypothesis that MT is an alteration in the cellular differentiation state. [ABSTRACT FROM PUBLISHER]

Syrian hamster embryo (SHE) cells were investigated for their growth factor responsiveness as well as changes in growth factor homeostasis, including alterations in autocrine growth factor production and growth factor responsiveness, during in vitro transformation. For wild-type SHE cells, fetal bovine serum (FBS), epidermal growth factor (EGF) family members, platelet derived growth factor (PDGF) family members, fibroblast growth factor family members, inter leukin-4, interleukin-9, oncostatin M, hepatocyte growth factor, erythropoietin and pituitary extract were found to be mitogenic. SHE cell mitogenesis was inhibited in response to transforming growth factor β (TGF-β) family members, interleukin-1α, interleukin-1β and nerve growth factor. Additional experiments were conducted to study alterations In growth factor responsiveness to three SHE cell mitogens (FBS, EGF and PDGF) and one inhibitor of mitogenesis (TGF-β) during SHE cell in vitro transformation. Alterations in either EGF, PDGF or TGF-β responsiveness were observed in 7/8 SHE transformed lineages during the stepwise transformation process. Finally, 6/8 lineages underwent alterations which resulted in the production of autocrine growth factors during the transformation process. These results indicate that multiple alterations in growth factor homeostasis occur during the transformation process. [ABSTRACT FROM PUBLISHER]

Studies were conducted to determine the effect of culture medium pH on gap junctional intercellular communication (GJIC) in early passage Syrian hamster embryo (SHE) cells. Previous studies have demonstrated that SHE cells cultured at a clonal density at pH 6.70 are morphologically transformed by carcinogens at a significantly higher frequency than cells cultured in media of higher pH. Several other cell characteristics consistent with promotion-like effects are observed with pH 6.70 culture of SHE cells. It was postulated that the promotion-like effects observed in SHE cells cultured at acidic pH are mediated in part by a reduction of GJIC. In this study, we evaluated GJIC in SHE cells by fluorescent dye coupling. Results from this study indicate that GJIC decreased as a function of decreased extracellular pH. Cells cultured at pH 6.70, 7.15 or 7.35 exhibited 47, 75 and 85% coupled cells respectively. The decrease in dye coupling occurred by 24 h after switching the cells from pH 7.15 to 6.70 medium. The decreased GJIC observed at pH 6.70 was not due to changes in cell proliferation and was reversible within 24 h when pH 6.70 cultures were refed with pH 7.15 medium. The phorbol ester 12--tetradecanoylphorbol-13-acetate inhibited SHE cell GJIC in a pH-dependent manner with cells at pH 6.70 exhibiting the greatest inhibition by TPA and cells at pH 7.35 being unresponsive. The effect of pH on GJIC in SHE cells is consistent with the pH-dependent response to chemically induced morphological transformation and may be mechanistically related to this phenomenon. [ABSTRACT FROM PUBLISHER]

Recent studies from our laboratory have demonstrated that clonal cell proliferation of early passage Syrian hamster embryo (SHE) cells is optimal at a bicarbonate concentration in the culture medium of 8.9 mM (pH 6.65–6.75) under the experimental conditions reported. The purpose of the studies reported here was to examine whether morphological transformation induced by benzo[a]pyrene (BP) was enhanced under optimal culture conditions for SHE cell proliferation. Culture media of pH 6.70, 7.11 and 7.34 under incubator conditions of 10% CO in air were obtained by the addition of 0.75 (8.9 mM), 2.25 (26.8 mM) and 3.75 g/l (44.6 mM) of NaHCO respectively to a modified formulation of Dulbecco's modified Eagles medium. The frequency of morphological transformation of SHE cells was increased at 8.9 mM bicarbonate (pH 6.70) relative to media containing 26.8 or 44.6 mM bicarbonate (pH 7.11 and 7.34 respectively). Additionally, the isolate of embryo cells and lot of fetal bovine serum used supported transformation induced by BP at 8.9 mM bicarbonate (pH 6.70), but did not with media of higher bicarbonate concentration and pH. The duration of cell culture and the no. of colonies per plate influenced the amount of increase of morphological transformation observed at 8.9 mM bicarbonate relative to media of higher bicarbonate concentration. Initial studies have shown that a fraction of morphologically transformed colonies generated at reduced bicarbonate concentration were tumorigenic in newborn hamsters. These results are discussed in terms of the potential utility of low bicarbonate concentration cultured SHE cells for transformation studies. [ABSTRACT FROM PUBLISHER]

The effect of the pH, Na concentration and osmolality of the culture medium on early passage Syrian hamster embryo (SHE) clonal cell proliferation was examined. The pH of the medium was adjusted from 6.49 to 7.45 by addition of different amounts of NaHCO to the medium and incubating the cell cultures in a fixed atmosphere of 10% CO/90% air. Our results indicate that clonal SHE cell proliferation is optimal at pH 6.65– 6.80 while plating efficiency is independent of pH between 6.65 and 7.45. Adjustment of Na to that concentration in the medium (3450 p.p.m., 0.15 M) of the greatest NaHCOaddition caused a moderate depression of cell proliferation over the entire pH series. Adjusting the osmolality of the culture medium to a constant value of 338 mOsm/kg did not alter the pH effect on cell proliferation. The pH of the medium also affected cellular and colony morphology. Below pH 6.90 there was an increase in the number of colonies which exhibited a transformed-like morphology (‘altered’ colonies). The ‘altered’ phenotype was characterized by a multilayered, criss-cross pattern of growth throughout the colony. This phenotype was stable upon sub-cloning into pH 6.65 medium but was reversible if sub-cloned into pH 736 medium. The induction of ‘altered’ colonies at low pH could be partially suppressed by Na or osmolality adjustment. These results are discussed in terms of optimizing growth conditions for SHE cells in order to enhance their usefulness for cell transformation studies. The induction of ‘altered’ colonies by low pH is also discussed relative to the involvement of pH regulation in tumor-promoter and growth-factor action on cells in culture. [ABSTRACT FROM PUBLISHER]

We have recently identified a new suppressor molecule we named suppressin (SPN) that has all the characteristics of a global negative regulator of the immune system. SPN is a unique 63-kD monomeric polypeptide with a pI of 8.1 that is produced and secreted under basal conditions by murine splenocytes, human peripheral mononuclear cells, and hormone-secreting pituitary cells. The biological actions of SPN in vitro include the inhibition of mitogen-induced proliferation and immunoglobulin synthesis of lymphocytes and the suppression of interleukin-2-dependent CTLL-2 cell proliferation. In addition, SPN enhances natural killer cell activity by eliciting interferon-α and-β synthesis and secretion. SPN effects are reversible, nontoxic, and require the continuous presence of exogenous SPN. T lymphocytes stimulated with concanavalin A or phytohemagglutinin are more sensitive to SPN (90% inhibition) than are lipopolysaccharide-stimulated B cells (60% inhibition). SPN arrests lymphocytes in the G/G phase of the cell cycle after reduction of their RNA, protein and DNA synthesis, suggesting that SPN inhibits the processes required for G transition to G. SPN is found intracellularly in all unstimulated lymphocyte subsets, monocytes, and in phytohemagglutinin-activated T lymphocytes immunopositive for the low affinity interleukin-2 receptor. These results suggest that SPN may be a major negative regulator of cell proliferation in the immune system. All SPN-producing cell types are also sensitive to SPN. Collectively, the results of these experiments provide the foundations for a model in which SPN regulates lymphocyte proliferation in an autocrine and/or paracrine manner. Additional evidence supporting this hypothesis was provided by experiments showing that by blocking endogenous SPN activity in vitro with neutralizing anti-SPN antibodies unstimulated lymphocyte proliferation is induced. Lastly, SPN also inhibits the proliferation of leukemia and lymphoma cells in vitro, suggesting a potential role for SPN in regulating tumor cell proliferation. [ABSTRACT FROM AUTHOR]

We have investigated the molecular phenotypic differences between Syrian hamster embryo (SHE) cells cultured at pH 6.7 or 7.3. Multiple pH-sensitive phenotypic differences were noted including changes in cellular morphology, a unique charge differential in a major cellular protein, nine uniquely expressed proteins, two unique phosphoserine/threonine phosphoproteins, one unique phosphotyrosine phosphorprotein, and the pH dependent mRNA level of a gap junctional gene (connexin 43). These differences, combined with previously described pH-specific differences (differential transformation rates and gap junctional communication), illustrate that culturing SHE cells in media that differ by 0.6 pH units (0.3 units on either side of pH 7.0) can have a profound influence on the cellular phenotype. [ABSTRACT FROM AUTHOR]

Studies have been performed to understand the interactions and the role which intracellular calcium and intracellular pH have been in mediating mitogen-stimulated cellular proliferation. Stimulation of Syrian hamster embryo (SHE) cells with the mitogen platelet-derived growth factor A/B (PDGF) results in the inositol trisphophate γ receptor calcium channel, followed by an extracellular influx of calcium through a dihydropyridine-sensitive plasma membrane calcium channel. Chronic extracellular/intracellular acidification results in the inactivation of both these calcium channels due to slowly reversible protein alterations. Paradoxically, transient intracellular acidification, like that following PDGF-induced calcium signal did not result in the loss of the PDGF-induced transient intracellular acidification. Importantly with regard to the role intracellular calcium and pH have in mediating the mitogenic signal leading to cellular proliferation, chronic extracellular/intracellular acidification, which leads to a complete loss of the PDGF-induced calcium signal, did not result in the loss of PDGF-induced mitogenesis. These results indicate that the PDGF-induced calcium signal is not essential for PDGF-stimulated mitogenesis in Syrian hamster embryo cells. In contrast, blocking the PDGF-induced transient intracellular acidification completely blocks PDGF-induced mitogenesis, indicating that the mitogen-induced transient intracellular acidification, unlike the intracellular calcium ion signal, is indispensable for cellular proliferation in Syrian hamster embryo cells. [ABSTRACT FROM AUTHOR]

The pH 6.7 Syrian hamster embryo (SHE) cell transformation assay was used to test the morphological transformation potential of 5 rodent carcinogenic single ring aromatic/nitroaromatic amine compounds: 2-amino-4-nitrotoluene, 2,4-diaminotoluene, 2,4-dinitrotoluene, o-anisidine hydrochloride, and o-toluidine; and 5 noncarcinogenic single ring aromatic/nitroaromatic amine compounds: 2,6-diaminotoluene, 2,4-dimethoxyaniline hydrochloride, 4-nitro-o-phenylenediamine, p-phenylenediamine dihydrochloride, and HC Blue No. 2. All 5 rodent carcinogens produced significant morphological transformation in a dose-responsive manner. None of the 5 noncarcinogens yielded significant transformation at any of the doses tested. Therefore, the concordance between the pH 6.7 SHE cell transformation assay and rodent carcinogenicity for these 10 single ring aromatic/nitroaromatic amine compounds is 100%. In contrast, the concordance between the standard SHE cell transformation assay and rodent carcinogenicity for 13 single ring aromatic/nitroaromatic amine compounds was 62%. For 5 aromatic/nitroaromatic amine compounds which were tested in both standard and pH 6.7 SHE cell transformation assays (i.e., a subset of the above two databases), the concordance between the standard SHE cell transformation assay and the rodent bioassay was 40%, while the concordance between the pH 6.7 SHE cell transformation assay and the rodent bioassay was 100%. This relatively high concordance between the pH 6.7 SHE cell transformation assay and rodent bioassay results demonstrates the utility of the pH 6.7 SHE cell transformation assay for predicting the rodent carcinogenic potential of single ring aromatic/nitroaromatic amine compounds. [ABSTRACT FROM PUBLISHER]

Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, vanadium pentoxide, and nickel sulfate heptahydrate were tested in the Syrian hamster embryo (SHE) assay in order to increase the SHE assay database for heavy metals. All five compounds produced significant morphological transformation at one or more doses in a dose-responsive manner. Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, and nickel (II) sulfate heptahydrate were all positive with a 24-hr exposure, suggesting direct DNA perturbation. Vanadium pentoxide was negative with a 24-hr exposure, but positive with a 7-day exposure. This pattern of response (24-hr SHE negative/7-day SHE positive) has been seen with other chemicals which have tumor promotion-like characteristics. Since the inception of the use of the SHE cell transformation assay for detecting the neoplastic transformation potential of chemicals, over 42 heavy metal compounds have been tested in this assay. Based on the 24 metal compounds which have been tested in the SHE, Salmonella, and some type of rodent bioassay, the SHE assay is 92% concordant with rodent bioassay carcinogenicity results, including a sensitivity of 95% (21/22) and a specificity of 50% (1/2). At this time, the measure of SHE assay specificity for rodent carcinogenicity of metals is limited by the paucity of metal compounds which are rodent noncarcinogens. The Salmonella assay results are only 33% concordant with the rodent bioassay for these same chemicals. This relatively high concordance between the SHE assay and the rodent bioassay carcinogenicity results demonstrates the utility of the SHE assay for determining the carcinogenic potential of heavy metal compounds in rodent Cancer bioassays. [ABSTRACT FROM PUBLISHER]

The estimated single-dose oral toxicity (50% lethality) of succinate tartrates (ST) was 2–3 g/kg in rats. ST produced minimal to moderate dermal irritation but no evidence of systemic toxicity in a standard acute percutaneous toxicity test in rabbits. ST was not an eye irritant in a standard rabbit low-volume eye irritation test ST was not genotoxic in a series of six genotoxicity tests. A 14-day oral gavage study in rats at a dose range of 0.05–1.0 g ST/kg/day produced only gastric irritation. The no-observed-effect level (NOEL) for gastric irritation was 0.1 g/kg for males and 0.05 g/kg for females. A 28-day percutaneous toxicity study in rabbits produced minimal to moderate dermal irritation and no adverse systemic effects at a high dose of 450 mg ST/kg/day. Single-dose absorption, distribution, and elimination (ADE) studies in male rats showed that 10–15% of an oral dose and 1–3% of a dermal dose were absorbed. Approximately 98% of the orally administered ST was eliminated as C in urine, feces, or expired CO after 72 hr. Approximately 80% of the dermally absorbed C dose was eliminated in urine, feces, or expired CO after 72 hr. In conclusion, no adverse effects were noted in acute toxicity, genotoxicity, or subchronic toxicity studies conducted with ST. [ABSTRACT FROM PUBLISHER]