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Response to: "Regarding detection of hydrogen peroxide in cigarette smoke"

Signal Transduction Laboratory, Department of Internal Medicine, University of California, School of Medicine, Davis, California, USA

¹ Correspondence: E-mail: ttgoldkorn{at}ucdavis.edu

The authors of the paper, "Epidermal growth factor receptor exposed to cigarette smoke is aberrantly activated and undergoes perinuclear trafficking," (1) would like to respond to the comment by Dr. I. B. Chatterjee regarding our methodology for the detection of H₂O₂ from cigarette smoke (CS). Dr. Chatterjee writes that CS contains strong reducing agents which may convert the red ferrithiocyanate complex into colorless ferrothiocyanate, thereby rendering this method inappropriate for detecting H₂O₂ in CS. We believe Dr. Chatterjee’s concerns are not applicable for the following reasons:

1) We did not experience any bleaching of the ferrithiocyanate complex in our assay using cell culture medium exposed to whole CS.

2) Dr. Chatterjee states that CS solution added to a solution of pure H₂O₂ results in no significant change in absorbance at 480 nm. There are no details describing the nature of the CS solution used, and upon reading one of Dr. Chatterjee’s publications (2) , it would appear that their CS solution is dramatically different from our cell culture medium exposed to CS. Their aqueous extract of CS is obtained from an Indian commercial cigarette while we used University of Kentucky 2R4F reference cigarettes. Dr. Chatterjee’s protocol includes adjusting the pH of the solution with 2 N sodium hydroxide, filtering through a 0.22 µm filter, three extractions with an equal volume of diethyl ether, and removal of solvent under vacuum. We assayed our sample immediately following exposure without additional processing.

3) It is well known that aged CS solution or extract (CSE) is very different from fresh CSE. After all the steps of processing the CSE, Dr. Chatterjee may very well have been using aged CSE in the H₂O₂ detection assay. In fact, there are short-lived reactive species in CSE that induce a heat shock response, but this effect is abrogated by the addition of old CSE (made by incubating for 24 hrs at room temperature) and protein oxidation levels were strongly reduced in cells exposed to old CSE (3) .

4) Dr. Chatterjee indicates that there are strong reducing agents in CS, but neglects to identify any specific compound. Nonetheless, there are studies demonstrating that CS depletes reducing agents such as glutathione (GSH) and ascorbate (4 , 5) . We agree with the latter and therefore do not believe that any CS-reducing agents are affecting the very strong ferrithiocyanate complex formed from our CS-exposed medium. Indeed, Fig. 2C of our manuscript demonstrated that only when we pretreated the medium with excess GSH, and thus inhibited the CS-generated hydroperoxides, could we eliminate the ferrithiocyanate response.

5) Furthermore, the conversion of ferrothiocyanate to ferrithiocyanate is a very common assay used to detect the presence of peroxides in unknown chemical mixtures (6) . The combination of the inhibitory effects of CS on reducing agents and the large quantities of H₂O₂ being generated is likely converting any ferrothiocyanate back to the red ferrithiocyanate.

We thank you for the opportunity to respond to Dr. Chatterjee’s comment.

FOOTNOTES

The opinions expressed in editorials, essays, letters to the editor, and other articles comprising the Up Front section are those of the authors and do not necessarily reflect the opinions of FASEB or its constituent societies. The FASEB Journal welcomes all points of view and many voices. We look forward to hearing these in the form of op-ed pieces and/or letters from its readers addressed to journals{at}faseb.org.

REFERENCES

1. Khan, E. M., Lanir, R., Danielson, A. R., Goldkorn, T. (2008) Epidermal growth factor receptor exposed to cigarette smoke is aberrantly activated and undergoes perinuclear trafficking. FASEB J. 22,910-917[Abstract/Free Full Text]
2. Panda, K., Chattopadhyay, R., Ghosh, M. K., Chattopadhyay, D. J., Chatterjee, I. B. (1999) Vitamin C prevents cigarette smoke induced oxidative damage of proteins and increased proteolysis. Free Radic. Biol. Med., 27,1064-1079[CrossRef][Medline]
3. Henderson, B., Csordas, A., Backovic, A., Kind, M., Bernhard, D., Wick, G. (2008) Cigarette smoke is an endothelial stressor and leads to cell cycle arrest. Atherosclerosis In press
4. Eiserich, J. P., van der Vliet, A., Handelman, G. J., Halliwell, B., Cross, C. E. (1995) Dietary antioxidants and cigarette smoke-induced biomolecular damage: a complex interaxtion. Am. J. Clin. Nutr., 62(suppl),1490S-1500S[Medline]
5. van der Toorn, M., Smit-de Vries, M. P., Slebos, D.-J., de Bruin, H. G., Abello, N., van Oosterhout, A. J. M., Bischoff, R., Kauffman, H. F. (2007) Cigarette smoke irreversibly modifies glutathione in airway epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol., 293,L1156-L1162[Abstract/Free Full Text]
6. Shugar, G. J., Ballinger, J. T. (1996) Dawkins, L. M. eds. Chemical Technicians’ Ready Reference Handbook ,485 McGraw-Hill Professional New York.

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