Exposure of zebrafish embryos to environmentally relevant nitrate (NO3−) and nitrite (NO2−) downregulates tyrosine hydroxylase (TH) in developing dopaminergic neurons and impairs motor behavior, and pharmacological evidence (reversal by the estrogen receptor antagonist ICI 182,780; attenuation by the flavoprotein oxidoreductase inhibitor diphenyleneiodonium; increased NO staining) attributes these effects to estrogen receptor (ER)-dependent signaling fed by conversion of the anions to nitric oxide (NO). A decade of follow-up work has extended this phenomenology — nitrate and nitrite acting as endocrine disruptors, i.e. chemicals that interfere with hormonal signaling — to the cardiovascular system, retina, and other xenoestrogens (estrogen-mimicking environmental compounds), and a separate fish-physiology literature has characterized enzymatic nitrite-to-NO conversion (deoxyhemoglobin, globins, xanthine oxidoreductase-dependent nitrate reductase activity) in other contexts. No study, however, has identified which ER pathway (nuclear esr1/esr2a/esr2b versus membrane-associated estrogen signaling) mediates the TH downregulation, how NO and ER signaling are molecularly coupled, which enzyme(s) generate the responsible NO from nitrate/nitrite in the embryo, or whether the locomotor phenotype reflects lost dopamine synthesis versus direct NO/peroxynitrite effects on motor neurons. The mechanism of this neuroendocrine disruptor action remains open.

Background

Nitrate (NO3−) and nitrite (NO2−) are the most widespread inorganic nitrogen contaminants of surface water and groundwater, and nitrate is recognized as an endocrine disruptor — a chemical that interferes with hormonal signaling — whose in vivo conversion to nitrite and nitric oxide (NO) produces physiological effects that are dose-dependent and often non-monotonic; a 2018 review concluded that the reproductive endocrinology of environmental nitrate remains internally contradictory and mechanistically incomplete (Edwards and Hamlin 2018). In fish, the chemistry of nitrite-derived NO is comparatively well characterized in adult and hypoxic contexts: deoxyhemoglobin reduces nitrite to NO as blood cycles through oxygenation states (Jensen 2007), crucian carp tissues show low but significant nitrate reductase activity that is partly catalyzed by xanthine oxidoreductase (Hansen et al. 2016), and the globin family (a family of heme proteins comprising hemoglobin, myoglobin, cytoglobins, neuroglobin, and globin X) are candidate nitrite reductases thought to maintain NO homeostasis during fish embryonic development (Rochon and Corti 2020). Physiological nitrite also modulates zebrafish heart regeneration via NO-dependent signaling (Rochon et al. 2020), and excessive nitrite disrupts zebrafish valve formation through NO/cGMP signaling — NO acting through cyclic GMP as a second messenger (Li et al. 2014). By contrast, how nitrate and nitrite act on the developing nervous system is far less understood.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme of dopamine synthesis; zebrafish carry two TH paralogs (th1 and th2), and dopaminergic neurons are routinely visualized by TH immunostaining from 48 hours post-fertilization (hpf). Estrogen signaling in zebrafish involves nuclear estrogen receptors encoded by esr1 (ERα), the teleost-duplicated esr2a and esr2b (ERβ paralogues, i.e. duplicated gene copies produced by the whole-genome duplication in the teleost lineage), and the membrane-associated G-protein-coupled estrogen receptor gper1. Two pharmacological tools frame the candidate problem: ICI 182,780 (fulvestrant), a pan-estrogen-receptor antagonist, and diphenyleneiodonium (DPI), an inhibitor of flavoprotein oxidoreductases — a class that includes both enzymes that reduce nitrate/nitrite (such as molybdenum-containing oxidoreductases) and nitric oxide synthase (NOS, the enzyme that synthesizes NO from arginine), so DPI cannot by itself distinguish NO formed from exogenous nitrite from NO formed by NOS.

Jannat, Fatimah and Kishida (2014) exposed zebrafish embryos to nitrate and nitrite at environmentally relevant concentrations. Both anions decreased TH immunostaining in dopaminergic neurons at 48 hpf; nitrite decreased the tactile response at 72 hpf, and both decreased free-swimming activity at 6 days post-fertilization (dpf). Co-exposure with ICI 182,780 reversed the TH and behavioral effects, implicating estrogen receptors; co-exposure with DPI attenuated them, and staining for NO increased after exposure, implicating NO derived from nitrate/nitrite. The authors summarized their conclusion in deliberately hedged terms — nitrate and nitrite are neurotoxicants acting as endocrine disruptors "possibly through conversion to NO" — leaving the causal molecular chain from exposure to phenotype unspecified, and raising the mechanistic questions that constitute the present problem.

Problem Statement

Determine the molecular pathway by which waterborne nitrate (NO3−) and nitrite (NO2−) downregulate tyrosine hydroxylase (TH, the rate-limiting enzyme of dopamine synthesis) expression in dopaminergic neurons and impair motor behavior during early zebrafish development, as observed by Jannat, Fatimah and Kishida (2014). Because no study has yet established this pathway, the accepted form of answer is an evidence-based mechanistic model. A complete answer ties together all three linked components of the causal chain, giving for each the supporting evidence and the experiment(s) that would discriminate among the alternatives:

  1. The estrogen receptor route: which estrogen receptor pathway — the nuclear receptors esr1, esr2a, esr2b, or membrane-associated estrogen signaling such as gper1 — mediates the downregulation of TH (th1/th2) transcription, and how NO and ER signaling are coupled (whether nitrate/nitrite-derived NO acts upstream of ER activation, or ER signaling is required upstream of the production of the responsible NO).

  2. The enzymatic NO source: which enzyme(s) generate the responsible NO from nitrate and nitrite in the embryo — nitric oxide synthase, the DPI-sensitive nitrate/nitrite-reducing oxidoreductases (for example molybdenum-containing oxidoreductases), or the DPI-insensitive heme-protein nitrite reductases such as the globins.

  3. The behavioral mechanism: whether the locomotor impairments arise from reduced dopamine synthesis/release by the compromised dopaminergic neurons, or from direct effects of NO on motor neurons and neuromuscular development — for example through formation of peroxynitrite (ONOO−, a strong oxidant produced when NO reacts with superoxide, which can oxidize dopamine and damage neurons) — and whether conversion to NO fully accounts for these effects.

References

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