Cigarette Smoke Extract Produces Superoxide in Aqueous Media by Reacting with Bicarbonate

Abstract

The toxicity of cigarette smoke (CS) is largely attributed to its ability to generate reactive oxygen species (ROS). Reportedly, CS generates superoxide in cell culture systems by stimulating the cells to produce superoxide and through direct chemical reactions with components of the culture media. In this study, we investigated CS-induced superoxide formation in biocompatible aqueous media and its characteristics. Cigarette smoke extract (CSE) and total particulate matter (TPM) were prepared from the mainstream smoke of 3R4F reference cigarettes. CSE and TPM generated superoxide in Hank’s balanced salt solution (HBSS), Dulbecco’s modified Eagle media (DMEM), and blood plasma, but not in distilled water and phosphate-buffered saline. Each constituent of HBSS in solution was tested, and bicarbonate was found to be responsible for the superoxide generation. More than half of the superoxide formation was abolished by pretreating CSE or TPM with peroxidase, indicating that the substrates of peroxidase, presumably peroxides and peroxy acids, mainly contributed to the superoxide production. In conclusion, the presence of bicarbonate in experimental conditions should be considered carefully in studies of the biological activity of CS. Furthermore, the local amount of bicarbonate in exposed tissues may be a determinant of tissue sensitivity to oxidative damage by CS.

Cigarette Smoke-Induced Reactive Oxygen Species Formation: A Concise Review

“…In addition to inherent ROS in CS, CS generates ROS through chemical reactions among its redox-active constituents with biological environments. Redox-active constituents like benzosemiquinones [ 77 ], benzy[a]pyrene [ 85 ], α,β-unsaturated carbonyls [ 86 ], peroxides and peroxy acids [ 87 ], and metal ions [ 88 ] are important examples. Benzosemiquinones, with extended half-lives, can penetrate the blood–air barrier and gain access to the circulation, thereby systemically producing superoxide through quinone redox cycling and forming adducts with biomolecules, such as hemoglobin and albumin [ 71 , 89 , 90 ].…”

“…α,β-Unsaturated carbonyls such as acrolein, crotonaldehyde, and methyl vinyl ketone, also referred to as reactive carbonyl species, are reactive electrophiles that produce singlet oxygen through their interaction with peroxynitrite [ 86 ]. Intriguingly, CS preparations generate superoxide in aqueous media like biological buffer solutions, cell culture media, and blood plasma [ 87 ]. While not all constituents were identified, peroxidase substrates such as peroxides and peroxy acids are postulated to react with bicarbonate anion in aqueous environments to yield superoxide.…”

Repeated intratracheal instillation of whole‐cigarette smoke condensate to assess lung damage in a rat model

“…3,4 Alternatively, most in vitro studies were conducted to confirm the side effects and mechanism of CSC exposure in human lung epithelial cells such as the A549 cell line. 5,6 However, intratracheal instillation (ITI) can evaluate the impact of substances on the respiratory system by direct exposure to the lung via trachea and bronchial tubes with low expense and a simple method. 7,8 Therefore, we attempted to develop a novel method to assess the adverse effects of cigarette smoke via the ITI technique of wholecigarette smoke condensate (WCSC).…”

“…In general, since aerosol inhalation studies require high cost, technical experience, and a large number of test substances, most existing studies using cigarette smoke have been clinical studies showing that cigarette smoke causes lung cancer 3,4 . Alternatively, most in vitro studies were conducted to confirm the side effects and mechanism of CSC exposure in human lung epithelial cells such as the A549 cell line 5,6 . However, intratracheal instillation (ITI) can evaluate the impact of substances on the respiratory system by direct exposure to the lung via trachea and bronchial tubes with low expense and a simple method 7,8 …”