To the Editor:We read with interest the article by Veselik and colleagues in the May issue of Cancer Research (1). Using human breast cancer cells, they show that inorganic nitrate and nitrite mimicked the effects of estradiol on cell growth and gene induction via activation of estrogen receptor-α. The findings add to the growing body of evidence now suggesting important physiologic roles for these supposedly inert anions (2).The observed effects are remarkable, especially when considering the extremely low concentrations that were used. In humans, normal plasma levels of nitrate are ∼20 to 40 μmol/L, and for nitrite, 50 to 500 nmol/L (2, 3). The authors report potent effects at concentrations far below these levels, and in many cases, lower than what is ever seen in vivo. Given the physiologic plasma levels of nitrate, nitrite, and estradiol, this study implies that the three compounds are equally important in regulating estrogen receptor-α in vivo, with a further shift after menopause. Surprisingly, nitrate and nitrite were equipotent, suggesting a complete conversion of nitrate to nitrite. A functional mammalian nitrate reductase activity was indeed described recently, although in that study, the rate of aerobic nitrate reduction was much lower (4).Of concern is that most cell culture media already contain nitrate and nitrite at fairly high concentrations. Using a highly sensitive chemiluminescence method (3, 5), we measured nitrate and nitrite in 10 different culture media with or without the addition of charcoal-stripped fetal bovine serum. Nitrate varied between 0.8 and 100 μmol/L, whereas nitrite levels were 50 and 400 nmol/L. Therefore, it is highly surprising to see significant effects after adding only 1 μmol/L of nitrate. Did the authors measure nitrate and nitrite levels in the medium?The authors suggest a possible endocrine-like role for nitrate and nitrite in breast cancer. Again, we find it hazardous to extrapolate the present results to a discussion regarding dietary nitrate in relation to breast cancer because the effect should already be close to maximal from the normal levels of nitrate and nitrite present in blood and tissues.No potential conflicts of interest were disclosed.