Tobacco smoke is a source of toxic reactive glycation products

Abstract

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation in vivo. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products "glycotoxins." Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.

Figure 1

Glycotoxins in tobacco leaf extract and cigarette smoke condensate promote AGE formation in vitro. The in vitro AGE formation assay was performed on aqueous extracts of tobacco leaves from eight different brands of American cigarettes at a concentration equivalent to 90 mg of tobacco/ml (A) and on mainstream tobacco smoke at concentrations ranging from 3 to 0.375 g of tobacco/ml (B). Each bar represents a different brand of American cigarettes. Brand E is the light brand equivalent of brand D. AGE formation was revealed by incubation with a rabbit anti-AGE-specific polyclonal antibody. (C) AGE formation was inhibited by passing the mainstream cigarette smoke through a standard commercial filter + 500 mg of aminoguanidine (open bar) but not by passing it through a standard commercial filter (solid bar) or a standard commercial filter + 500 mg of sodium sulfate (striped bar).

Figure 2

Glycotoxins in cigarette smoke condensate induce fluorescence and covalently cross-link proteins. (A) RNase A was exposed to mainstream cigarette smoke for 0, 5, 8, 24, 48, and 72 h and then assayed for RNase A-AGE-specific fluorescence by measuring emission at 440 nm upon excitation at 370 nm in the presence and absence of aminoguanidine. Circles, RNase A alone; squares, RNase A after incubation with cigarette smoke; triangles, RNase A after incubation with cigarette smoke and 5 mM aminoguanidine; diamonds, RNase A after incubation with cigarette smoke and 50 mM aminoguanidine. (B) RNase A was exposed to mainstream cigarette smoke for 0, 5, 8, 24, 48, and 72 h. To assess the formation of dimers, the samples were subjected to SDS/PAGE under reducing conditions, followed by transfer to nitrocellulose and Western blotting with a rabbit anti-RNase A antibody conjugated to horseradish peroxidase. Circles, RNase A alone; squares, RNase A after incubation with cigarette smoke; triangles, RNase A after incubation with cigarette smoke and 5 mM aminoguanidine; diamonds, RNase A after incubation with cigarette smoke and 50 mM aminoguanidine.

Figure 3

Glycotoxins are mutagenic. Salmonella strain TA98 was incubated with serial dilutions of cigarette smoke (solid bars) and cigarette smoke passed through aminoguanidine (open bars) at concentrations ranging from 250 mg of tobacco/ml to 50 mg of tobacco/ml for 1 h and then plated. Forty-eight hours later, the number of colonies on each plate was counted. Each bar represents the mean of three plates ± SD.

Figure 4

Serum ApoB-AGE (A) and total serum protein-AGE levels (B) were measured in healthy nondiabetic smokers and healthy, nondiabetic nonsmokers. AGE-ApoB levels in smokers (mean ± SD, 247 ± 76 units/ml, n = 17) were significantly higher (P = 0.018, unpaired Student t test) than nonsmokers (mean ± SD, 190 ± 54, n = 17). Serum AGE levels in smokers (210 ± 51 AGE units/ml, n = 17) were significantly higher (P = 0.01) than the serum-AGE levels in nonsmokers (161 ± 53 AGE units/ml, n = 17). AGE units are expressed relative to an ApoB-AGE standard (1 unit = 1 ng of ApoB, ICN Pharmaceuticals, Biochemical Division, Aurora, OH) and a BSA-AGE standard in the respective assays. Subjects were not controlled for age, sex, or number of pack-years smoked.