Initial characterization of the complement activating compounds in extracts of smokeless tobacco

Aqueous extracts of smokeless tobacco (ST) have been shown to be potent activators of complement. However, the mechanisms by which smokeless tobacco activates complement are not well understood. This study was undertaken to identify the complement activating compounds in ST extracts. The approximate molecular size of the activating agent(s) in smokeless tobacco was determined by dialyzing aqueous extracts of loose leaf chewing tobacco (1S1), dry snuff (1S2), and moist snuff (1S3). Following dialysis (total dilution effect of 1:10(9)), using a membrane with a molecular weight retention limit of 12-14 kDa, all extracts retained full capacity to activate serum complement as determined by a hemolytic assay. Fractionation of the extracts by gel filtration chromatography revealed that the complement activating agents in ST were high molecular weight compounds that eluted between 400 kDa and the void volume (1500 kDa) of a Sephacryl S300 column. The high molecular weight complement-activating peak was isolated and found to be a more potent complement activator than the unfractionated extract. The chemical nature of the complement activating compounds was determined by subjecting the extracts to boiling for 30 min, an organic extraction with chloroform/methanol 2:1, or treatment with a DNAse/RNAse enzyme cocktail. None of these treatments destroyed the capacity of ST extracts to activate complement, suggesting that the activating agents may be carbohydrate-like. Finally, an extraction protocol designed to remove polyphenols significantly diminished the complement activating capacity of the ST extracts. These results clearly demonstrate that the complement activating substances in smokeless tobacco extracts may be large (>400 kDa) polyphenol-containing compounds (i.e. tannins). Identification of this agent(s) will be important for distinguishing the mechanism of smokeless tobacco-induced complement activation.