Comment re: Nitrite Activates Estrogen Receptor-α Free

In Response:

Our study reports that nitrite (10⁻⁷–10⁻⁵ mol/L) activates estrogen receptor-α in MCF-7 breast cancer cells (1). Nitrate (10⁻⁶ mol/L) also activates estrogen receptor-α in the absence, but not in the presence, of an oxidoreductase inhibitor. Although several enzymes reduce nitrate to nitrite, the enzyme(s) in MCF-7 cells has not been identified. Consequently, the flavoprotein inhibitor, diphenyleneiodonium, was used to block the reduction of nitrate. As many culture media have added nitrate (0.05–100 mg/L), the experiments were conducted in improved minimum essential medium (IMEM) containing 5% charcoal-treated calf serum (CCS). IMEM has no added nitrite/nitrate. To address the question of serum nitrite/nitrate, some experiments were repeated in IMEM without serum or containing 1% CCS and similar results were obtained (data not reported).

Although the primary source of circulating nitrites/nitrates is the oxidation of nitric oxide, exogenous exposures contribute to plasma concentrations (2, 3). Occupational exposures occur in the manufacture of explosives, paper, and glass and in the manufacture/use of fertilizers. Diet also influences plasma concentrations (4). Nitrate is low in water (0–18 mg/L) but has increased over the last few decades (4) due to the use of fertilizers, contamination from dumps, oxidation of ammonia from human/animal waste, and treatment of drinking water with chloramines. High amounts of the anions are present in cured and processed meats (nitrites and nitrates; 2.7–945 and 0.2–6.4 mg/kg, respectively) and in some fruits and vegetables (nitrate; 200–2,500 mg/kg) as a result of greenhouse cultivation (5). The total estimated daily dietary exposure to nitrites/nitrates ranges from 1.2 to 3 mg and 39 to 268 mg, respectively (4). Tobacco contains nitrates (0.05–13 mg/g) and smokers have a 2-fold to 3.7-fold higher serum concentration. Medications also contribute to exposure. Although alcohol does not contain significant amounts, alcohol consumption increases serum nitrite/nitrate. Serum concentrations may not reflect tissue concentrations. Some tissues have higher, whereas others have lower, concentrations of nitrite (0.45–22.5 μmol/L) and the amount of soluble anion varies (25–78%) suggesting that a significant fraction of nitrite may not be biologically available.

In animal models, increasing serum nitrite to the upper physiologic range alters signaling pathways, inhibits cytotoxicity after ischemia-reperfusion injury, and increases vasodilation. In humans, increasing serum concentrations has similar beneficial effects on the cardiovascular system. However, environmental exposure to nitrite/nitrate is associated with non–Hodgkins lymphoma, astrocytoma, liver, stomach, pancreatic, and bladder cancers. Breast cancer is associated with farming, tobacco smoke, and alcohol, and patients with breast cancer have elevated serum (246–363 μmol/L) and breast tissue (745 nmol/g) nitrite/nitrate levels. A clear link between environmental exposure to the anions and cancer remains to be established.