Aldehyde toxicity in human oral epithelium

Detta är en avhandling från Stockholm : Karolinska Institutet, Institute of Enviromental Medicine

Sammanfattning: Human oral epithelium is an established target for aldehyde toxicity, including from usage of tobacco, intake of foods and fluids, leaching of dental materials, and from mouth inhalation of aldehydes in the work environment. Following establishment of culture/exposure protocols for oral cell cultures, toxicity mechanisms and risk of malignant transformation from aldehyde exposure was investigated, including the metabolic defense offered by alcohol dehydrogenase 3 (ADH3) against formaldehyde toxicity. Exposure of normal oral keratinocytes (NOK) and fibroblasts to formaldehyde demonstrated loss of viability in dose-dependent manners. The presence of fetal bovine serum as well as thiol and amino supplements influenced viability tip to 100-fold as determined from colony formation and dye exclusion. Higher formaldehyde toxicity in fibroblasts was shown to correlate with comparatively lower intracellular levels of the free low-molecular-weight thiols glutathione and cysteine. Subsequent studies demonstrated distinct toxicity profiles of formaldehyde, acetaldehyde and methylglyoxal in NOK and the SV40 T antigenimmortalized human oral keratinocyte line SVpgC2a. Endogenous and induced acetaldehyde and methyIglyoxal DNA adducts was demonstrated by 32P-postlabeling, including of endogenous origin and from treatment. Adducts were clearly formed in dose-dependent manners already at low toxicity, as assessed by dye exclusion. Transcripts, protein and enzyme activity for ADH3 was then demonstrated in oral tissue specimens and various oral cell lines. Quantitative assessments of alcohol and aldehyde oxidizing activities, including Km-determinations, demonstrated that ADH3 constitutes the primary metabolic defense against formaldehyde toxicity. Transcription of ADH3 in tissue was confined to the basal cell layer of oral epithelium, whereas the resultant protein was highly stable, and expressed throughout the tissue. Establishment of protocols that permitted the selective induction of terminal differentiation of proliferative keratinocytes, demonstrated clearly the association of ADH3 transcription with a proliferative state. Attempts of formaldehyde transformation from repeated exposures were successful in SVpgC2a, but not in NOK, including two cell lines followed for their life span. A resultant novel cell line, termed SVpgC3a, appeared from 40 exposures of SVpgC2a to genotoxic levels of formaldehyde. This line exhibited multi-focal growth and cloning in soft agar, albeit it did not induce tumors in the immuno-deficient host. Micro-array and twodimensional gel electrophoresis demonstrated genetic changes at the transcript and protein levels, respectively, related to adhesion, cell cycle progression, signal transduction, and reactive oxygen detoxification. The current work elucidated the potential for adverse health effects of aldehydes in oral mucosa, including assessments of cytotoxicity and genotoxicity, and as well, the cytoprotective effect offered from thiols and metabolism. The results indicate the cytoprotective function of both intracellular and extracellular thiols towards aldehyde toxicity, as well as the usefulness of thiol-free and chemically defined conditions for toxicity assessments in vitro. Analysis of ADH3 expression demonstrates its role as a primary metabolic defense mechanism against formaldehyde. Transcription of ADH3 serves as a marker of cell proliferation, whereas differential regulation of transcript and protein levels indicate that ADH3 follows a novel expression principle hereto not demonstrated for the ADH family. From acting as a transforming agent, formaldehyde generated a novel cell line termed SvpgC3a that extends an existing battery of cell lines used to model oral cancer progression. Besides for exploring the etiology of oral cancer, such cell lines may serve as alternative models useful to safety assessment of chemicals and consumer products.

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