Kristina Sundqvist

Toxicity of Formaldehyde to Human Oral Fibroblasts and Epithelial Cells: Influences of Culture Conditions and Role of Thiol Status

The toxicity of formaldehyde, a monomer released from certain polymeric dental materials, was studied in cultured human oral fibroblasts and epithelial cells. The influences of growth conditions were evaluated for both cell types, as well as the role of the internal and external thiol states. A one-hour exposure to formaldehyde decreased the colony-forming efficiency (CFE) of both cell types in a concentration-dependent manner, although the toxicity varied up to 100-fold with the conditions. Clearly, the presence of serum and the thiol cysteine counteracted the toxicity in fibroblasts. Similarly, pituitary extract and cysteine, or a mixture of amino acids and ethanolamines, counteracted the formaldehyde toxicity in serum-free cultures of epithelial cells. In contrast, a growth-promoting surface matrix of fibronectin and collagen did not influence the formaldehyde toxicity, as shown by both the CFE assay and a dye reduction assay. Further, a short-term change to the various growth media per se with or without the supplements serum or cysteine did not significantly alter the CFE. Analysis of the thiol state demonstrated significant differences between epithelial cells and fibroblasts, i.e., comparatively lower cellular levels of the free low-molecular-weight thiols glutathione and cysteine in fibroblasts. This result correlated to significantly higher formaldehyde toxicity in the fibroblasts than in the epithelial cells. Taken together, the results indicated the cytoprotective function of both intracellular and extracellular thiols toward formaldehyde, as well as the usefulness of thiol-free and chemically defined conditions for toxicity assessments in oral epithelial cells and fibroblasts. We conclude that the combined use of a controlled external milieu and the presumed target cell type may be advantageous in evaluations of oral toxicity mechanisms or the toxic potency of dental materials, particularly those which, like formaldehyde, may react with thiols or amines.

Growth regulation of serum-free cultures of epithelial cells from normal human buccal mucosa

SummaryHuman buccal epithelial cells have been reared from explants maintained in supplemented MCDB 153 medium. Primary epithelial outgrowths show typical structural features and uniformly express keratins; subunit analyses demonstrate expression of keratins 5, 6, 14, 16/17, and 19. The cells exhibit up to 6% colony forming efficiency and divide at about 0.8 population doublings per day on fibronectin/collagen-coated dishes at clonal density. Studies of markers of proliferation and differentiation in buccal epithelial cells indicate that epidermal growth factor, cholera toxin, retinoic acid, and pituitary extract each exhibit a distinctive ability to enhance growth and variably affect cell migration and cell surface area. Transforming growth factorβ-1 inhibits growth and increases surface area without affecting migration, involucrin expression, and cross-linked envelope formation. Moreover, exposure of cells to fetal bovine serum, the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate or an elevated Ca2+ concentration (from 0.1 to 1 mM) inhibits growth and induces squamous differentiation as indicated by inhibition of migration, increases in surface area, involucrin expression, or formation of cross-linked envelopes. The results show that epithelial cells can be reproducibly derived from explant cultures of human buccal mucosa specimens and the cells transferred under serum-free conditions. Buccal epithelial cells in culture undergo a pattern of growth and differentiation that mimics parakeratinization in vivo and variably respond to several agents shown to modulate growth of cells that originate from other types of epithelia.

Serum-free growth and karyotype analyses of cultured normal and tumorous (SqCC/Y1) human buccal epithelial cells.

Epithelial cell cultures were obtained following tryptic digestion of normal human buccal mucosa. Primary cultures exhibited markedly higher colony-forming efficiencies and growth rates using fibronectin/collagen-coated, as compared to non-coated culture dishes and a serum-free MCDB 153 medium developed for epidermal epithelial cells than a similar medium previously developed for buccal explant outgrowth cultures. At the preferred conditions, the cells could be transferred at least 5-fold, divided at about one population doubling per day, and commonly underwent 60 population doublings resulting in yields of 10(8) to 10(11) cells per cm2 mucosal specimen. Moreover, these conditions successfully cultivated a buccal carcinoma cell line (SqCC/Y1) for several months. The carcinoma cells were resistant to factors that inhibited growth or induced differentiation of normal cells, i.e., transforming growth factor type beta 1, Ca2+, or serum. Karyotype analyses of SqCC/Y1 cells showed 63 to 83 chromosomes per metaphase and consistent occurrences of monosomy 1, tetrasomy 19 and 20, as well as trisomy 22, and at least 7 marker chromosomes, whereas cells obtained from non-cancerous donors were diploid. It is concluded that the similarly defined culture conditions may now be applied to study characteristics of both normal and tumorous buccal epithelial cells.

Motivational Interviewing delivered by existing prison staff: a randomized controlled study of effectiveness on substance use after release.

A sample of 296 drug-using inmates in 14 Swedish prisons was randomized during 2004–2006 into three intervention groups; Motivational Interviewing delivered by counselors with workshop-only training, or by counselors with workshop training followed by peer group supervision, and controls. Drug and alcohol use was measured by the Addiction Severity Index (ASI) at intake and at 10 months after release. Complete data from 114 clients were analyzed by a stepwise regression analysis. All three groups reduced alcohol and drug use. Limitations in the study are discussed and future research is suggested. The study is financed by grants from the Research Committee of the National Prison and Probation Administration.