Fluorescent Approach to Quantitation of Reactive Oxygen Species in Mainstream Cigarette Smoke

Abstract

A novel approach to monitoring of mainstream smoke reactive oxygen species (ROS) has been developed and applied to the quantitation of smoke oxidants. Redox-active fluorescent probe dihydrorhodamine 6G (DHR-6G) was selected as the molecular probe because it is sensitive to typical smoke ROS. The experimental system couples an automatic cigarette smoke machine fiber-optic fluorometer for real-time monitoring of the reaction progress between cigarette smoke and DHR-6G. Quantitation was achieved based on the amount of rhodamine 6G, which is the sole product from DHR-6G oxidation. With the optimization of the trapping efficiency, we detected 391 nmol of ROS/cigarette in the mainstream CS for a standard cigarette 2R4F under standard Federal Trade Commission smoking protocol. Applying this method, we quantified the ROS of selected cigarettes and found that the cigarettes made of burley tobacco have much ( approximately 10 times) higher ROS content in the smoke than that in the tobacco made of bright tobacco. The smokeless cigarette, Eclipse, has comparable ROS with cigarettes made of bright tobacco.

Concentration of Reactive Oxygen Species (ROS) in Mainstream and Sidestream Cigarette Smoke

“…Since there was no mainstream smoke, the sample they collected cannot be simply defined as sidestream smoke since puffing a cigarette could affect both the amount and nature of the smoke produced. Ou and Huang (2006) detected a concentration of 391 nmol of ROS/cigarette in mainstream using 2R4F research cigarette (Flicker and Green 2001). They also report burley tobacco cigarette has 10 times higher ROS content than bright tobacco cigarette.…”

“…Kang et al (2002) reported 0.104 ± 0.068 ppbv of gas-phase hydrogen peroxide in the summer in downtown Seoul, Korea. In the past research, people have studied various combustion sources, e.g., vehicles (Hung and Wang 2001), incense burning (Kao and Wang 2002), and cigarettes (Huang et al 2005;Ou and Huang 2006). Both gas phase and PM (mainly tar) are generated in cigarette smoke and both contain radical species (Pryor 1997).…”

Fluorescence Evaluation of Scavenging Efficiency of Antioxidants Against Reactive Oxygen Species (ROS) in Cigarette Smoke

“…Cigarette smoking was carried out according to ISO 3308 machine smoking standard conditions (35 ml puff, drawn for 2 seconds at 60-second intervals). Quantification of ROS in cigarette smoke was adapted from the reported method with some modification (Ou and Huang 2006). Briefly, five replicates (two cigarettes per replicate) of cigarettes were smoked through a SM-450 smoking machine under ISO regime without using Cambridge filter.…”

“…More recently, several fluorescence probes, such as 2, 7-dichlorofluorescein (DCFH), dihydrorhodamine 123, and dihydrorhodamine 6 G (DHR 6 G) have been applied for the detection of ROS in cigarette smoke (Huang, Lin, and Ma 2005;Ou and Huang 2006;Zhao and Hopke 2012). These compounds are nonfluorescent but when oxidized by typical ROS (e.g., O ÀÁ 2 , HO·, R·, RO·, and ROO·), or H 2 O 2 in the presence of horseradish peroxidase they become highly fluorescent dyes that can be quantitatively determined by a fluorescence spectrophotometer.…”

A Novel Approach for Measurement of Total Reactive Oxidant Species (ROS) In Vivo by A Fluorometric Method

“…Although there are various existing methods to assess oxidative damage caused by ROS, such as measuring lipid peroxidation products and DNA adducts, none of them evaluates ROS directly [3][4][5]. After Ou and Huang first demonstrated the use of DHR6G as a fluorometric assay for ROS in cigarette smoke, it logically followed that the use of the same probe to measure the ROS in biological samples should be investigated [6]. The theory behind using DHR6G is that nonfluorescent DHR6G will emit fluorescence after being oxidized by ROS.…”