Function, expression and polymorphism of human alcohol dehydrogenase 3/glutathionedependent formaldehyde dehydrogenase

Alcohol dehydrogenase 3 (ADH3), identical to glutathione-dependent formaldehyde dehydrogenase, plays a vital role in the defense against formaldehyde through the activity for the spontaneously formed adduct between formaldehyde and glutathione, Shydroxymethylglutathione (HMGSH). ADH3 may also participate in the metabolism of Snitrosylated glutathione, GSNO. To further investigate substrate enzyme interactions, Arg115 which has been shown to be crucial for HMGSH binding through charge interactions, was mutated into Ser or Lys. Both ADH3 mutants showed reduced activities for HMGSH and GSNO while only the ADH3-Arg115Ser mutant showed reduced activity for the model substrate 12- hydroxydodecanoate.

Mainly, changes in activities were due to increased K. values. The decreased HMGSH and GSNO activities of the ADH3-Arg115Lys mutant indicate that not only a positive charge but also an exact positioning of the substrate is necessary for efficient catalysis. In addition, an attempt was made to introduce ADH3 characteristics in an ADH1 enzyme through the generation of the double mutant ADH1C2Leu57Asp/Aspll5Arg. This was partly successful since ADH3-like kinetic features for 12hydroxydodecanoate and ethanol was monitored. However, the ADH1C2Leu57Asp/Asp115Arg displayed no HMGSH activity. GSNO was efficiently reduced by the human ADH3 with NAD+ as preferred coenzyme. By electrospray tandem mass spectrometry the major products of GSNO reduction were identified as glutathionesulfinamide and GSSG.

We speculate that ADH3 catalyzed GSNO reduction takes place in vivo. The potential enzymatic defense by ADH3 was characterized in oral buccal tissue specimens and oral epithelial cell lines using RNA hybridization and immunological methods as well as enzyme activity measurements. From mRNA and protein distribution profiles in the intact tissue, substantiated by in vitro experiments, an association of ADH3 mRNA was observed primarily to proliferative keratinocytes while the protein was retained during the entire keratinocyte life span in oral mucosa. Substantial capacity for formaldehyde detoxification was shown from quantitative assessments of alcohol and aldehyde oxidizing activities including Km determinations, demonstrating that ADH3 is the major enzyme involved in formaldehyde oxidation in oral mucosa.

The expression of ADH3 in epithelial in vitro model systems, i.e., monolayer cultures and regenerated epithelia with normal and transformed epithelial cell lines, were characterized. Similar ADH3 expression levels among the various cell lines and tissue like protein distribution in regenerated epithelia indicate preservation of ADH3 during malignant transformation and functionality of the transformed cell lines as in vitro models for studies of formaldehyde metabolism in human oral mucosa. A screen for allelic variants of ADH3 revealed four possible base pair exchanges in the promoter region: GG.197,-196 -4 AA, G-79 -4 A and C+9 g T. The AA-197,-196 allele was relatively common among Chinese, Spaniards and Swedes while the presence of the A-79 allele was restricted to Spaniards and Swedes. The T+9 allele was found only among Swedes with a frequency of 1.5 %. Promoter activity assessments and electrophoretic mobility shift assays demonstrated that the C+9 gT exchange resulted in a significant transcriptional decrease in HeLa cells and possibly also a decreased binding of nuclear proteins.

In summary, the finely tuned substrate specificity of ADH3 offers an enzymatic defense against both formaldehyde and nitrosative stress. In human oral tissue as well as in human epithelial cell lines, ADH3 serves as the prime guardian against formaldehyde. Finally, the ADH3 gene is polymorphic which might influence the protective capacity in certain individuals.

List of scientific papers

I. Hedberg JJ, Stromberg P, Hoog JO (1998). "An attempt to transform class characteristics within the alcohol dehydrogenase family. " FEBS Lett 436(1): 67-70
https://pubmed.ncbi.nlm.nih.gov/9771895

II. Hedberg JJ, Griffiths JW, Nilsson SJF, Hoog JO (2001). "Reduction of S-nitrosoglutathione by human alcohol dehydrogenase 3." (Manuscript)

III. Hedberg JJ, Hoog JO, Nilsson JA, Xi Z, Elfwing A, Grafstrom RC (2000). "Expression of alcohol dehydrogenase 3 in tissue and cultured cells from human oral mucosa. " Am J Pathol 157(5): 1745-55
https://pubmed.ncbi.nlm.nih.gov/11073833

IV. Hedberg JJ, Hansson A, Nilsson JA, Hoog JO, Grafstrom RC (2001). "Uniform expression of alcohol dehydrogenase 3 in epithelia regenerated with cultured normal, immortalized and malignant human oral keratinocytes." Atla 29: (In Print)

V. Hedberg JJ, Backlund M, Stromberg P, Lonn S, Dahl ML, Ingelman-Sundberg M, Hoog JO (2001). "Functional polymorphism in the alcohol dehydrogenase 3 (ADH3) promoter." (Submitted)