Conceived and designed the experiments: JHL GED DRS. Performed the experiments: JHL ZW VL YW. Analyzed the data: JHL GED ZW TCR. Contributed reagents/materials/analysis tools: JHL GED DRS. Wrote the paper: JHL GED DRS.
The authors have declared that no competing interests exist.
Intranasal medications are used to treat various nasal disorders. However, their effects on olfaction remain unknown. Zicam (zinc gluconate; Matrixx Initiatives, Inc), a homeopathic substance marketed to alleviate cold symptoms, has been implicated in olfactory dysfunction. Here, we investigated Zicam and several common intranasal agents for their effects on olfactory function. Zicam was the only substance that showed significant cytotoxicity in both mouse and human nasal tissue. Specifically, Zicam-treated mice had disrupted sensitivity of olfactory sensory neurons to odorant stimulation and were unable to detect novel odorants in behavioral testing. These findings were long-term as no recovery of function was observed after two months. Finally, human nasal explants treated with Zicam displayed significantly elevated extracellular lactate dehydrogenase levels compared to saline-treated controls, suggesting severe necrosis that was confirmed on histology. Our results demonstrate that Zicam use could irreversibly damage mouse and human nasal tissue and may lead to significant smell dysfunction.
As one of the five senses, the ability to smell plays a crucial role in defining the quality of life. Indeed, loss of sense of smell or anosmia can have detrimental consequences as our ability to detect and process noxious chemosensory stimuli is impaired
One possible cause for the development of smell dysfunction is the use of various intranasal medications. Numerous intranasal drugs are available to treat various nasal disorders, including sinusitis, allergic rhinitis and nasal congestion. Although the safety and efficacy of the majority of these agents are well known, their effects on olfaction are not established. Moreover, some intranasal agents classified as “homeopathic” are marketed to treat common nasal disorders such as symptoms associated with the common cold. These agents are gaining popularity with consumers despite the lack of scientific data on their safety and efficacy
In this study, we examined the short-term and long-term effects of several commonly used intranasal agents using mouse and organotypic cultures from human nasal tissue. Specifically, we tested saline, Afrin (Schering-Plough, Kenilworth, NJ), Nasacort (Sanofi-Aventis, Bridgewater, NJ), lidocaine (Hospira, Inc., Lake Forest, IL) and epinephrine (Hospira, Inc., Lake Forest, IL), which are frequently utilized by physicians to treat various nasal disorders. Additionally, we tested one of the “homeopathic”, zinc-based intranasal agents, Zicam (Matrixx Initiatives, Inc., Phoenix, AZ) as it has been previously implicated in smell dysfunction
To determine the effects of various intranasal agents on the functional properties of olfactory sensory neurons (OSN), we performed electro-olfactogram (EOG) analysis on mouse main olfactory epithelium (MOE) 3 and 9 days after intranasal administration of either saline, Afrin, Nasacort, epinephrine, lidocaine or Zicam. Following odorant stimulation, the activation of OSN
(a) Odorant-stimulated EOG responses from mice treated with either saline, Afrin, epinephrine, lidocaine, Nasacort or Zicam, 3 and 9 days following intranasal administration. Red arrowhead indicates the time at which the odorant was delivered to the MOE. (b) Summary of the mean EOG amplitudes in response to odorants on day 3 and 9 following intranasal agent administration. Only Zicam-treated MOE failed to elicit odorant-stimulated EOG response (n = 5 for all groups).
We also examined the MOE for the expression of several proteins expressed in OSN. In all MOE examined, except those treated with Zicam, immunofluorescence staining demonstrated robust expression of AC3 (adenylyl cyclase 3) in the olfactory cilia, neural-specific β-tubulin and OMP (olfactory marker protein) in the cell bodies and processes of OSN (
(a) Immunofluorescence of mouse MOE stained with antibodies specific for AC3, β-tubulin, and OMP. Hoechst counterstain is shown in blue. Scale bar, 10 µm for AC3; 20 µm for β-tubulin and OMP. (b) Analysis of AC3 immunofluorescence signal intensity in MOE (Afrin, Epinephrine, Lidocaine, Nasacort, Zicam, *p = 0.006; n = 4 for all groups). (c) Analysis of β-tubulin immunopositive cells in MOE (Afrin, Epinephrine, Lidocaine, Nasacort, Zicam; *p = 0.003; n = 4 for all groups). (d) Analysis of OMP immunopositive cells in MOE (Afrin, Epinephrine, Lidocaine, Nasacort, Zicam, *p = 0.002; n = 4 for all groups). All tested groups were normalized to saline-treated controls.
Given that MOE maintains regenerative capacity following injury
(a) Appearance of MOE on day 31 in saline or Zicam-treated mice. Black arrowheads depict significant atrophy in endoturbinates of Zicam-treated MOE. (b) Odorant-stimulated EOG responses from saline or Zicam-treated mice, 31 days following intranasal administration. Red arrowhead indicates the time at which the odorant was delivered to the MOE. (c) Summary of the mean EOG amplitudes in response to odorants on day 31 in saline or Zicam-treated mice. Zicam-treated MOE failed to elicit odorant-stimulated EOG response (n = 4, saline; n = 5, Zicam). (d) Appearance of MOE 65 days after intranasal administration of either saline or Zicam. (e) Odorant-stimulated EOG responses from mice treated with either saline or Zicam, 65 days following intranasal administration. (f) Summary of the mean EOG amplitudes in response to odorants 65 days following either saline or Zicam treatment (n = 5 for all groups). (g) Immunofluorescence of mouse MOE stained with antibodies specific for AC3, β-tubulin, and OMP 31 and 65 days after saline or Zicam treatment. Hoechst counterstain is shown in blue. Scale bar, 10 µm for AC3; 20 µm for β-tubulin and OMP. (h) Analysis of AC3 immunofluorescence signal intensity in MOE of Zicam-treated mice normalized to saline-treated controls (p = 0.05, day 31; p<0.005, day 65; n = 4 for all groups). (i) Analysis of β-tubulin (p<0.05, day 31; p<0.0005, day 65) and OMP (p<0.01, day 31; p<0.05, day 65) immunopositive cells in the MOE treated with Zicam normalized to saline-treated controls (n = 4 for all groups).
Although our EOG findings demonstrated the lack of odorant-stimulated electrophysiological response in Zicam-treated MOE, it is nonetheless an
(a) Olfactory habituation assay 7–10 days following saline or Zicam intranasal treatment. The comparison of the ratio of the number of times the mouse sniffed an odorant-soaked cotton swab to the number of times it sniffed a water-soaked cotton swab on initial exposure is an indication of the ability of the animal to detect a specific substance. Cotton swabs were laced with 50 µl of citralva (10 µm), heptanone (50 µm) or mouse male urine (50-fold dilution). Significant differences are noted for the ability of Zicam-treated (n = 8) and saline-treated mice (n = 8) to detect citralva (p<0.05), heptanone (p<0.01) and male urine (p<0.05). (b) Olfactory habituation assay 70–80 days following saline or Zicam intranasal treatment. Again, significant differences are observed for the ability of Zicam-treated (n = 8) and saline-treated mice (n = 8) to detect citralva (p<0.0005), heptanone (p<0.005) and male urine (p<0.0005).
Our experiments in mice indicated the likelihood of developing significant olfactory dysfunction following Zicam treatment. Because a mouse study alone cannot adequately predict the biological effects of intranasal agents on human nasal tissue, we established an organotypic tissue culture system with human nasal explants (
(a) Organotypic tissue culture system setup for human nasal explants. Black arrow depicts newly harvested human middle turbinate tissue. (b) Graphical overview of the LDH assay. (c) Analysis of relative LDH levels from human nasal explants treated with either saline, Afrin, lidocaine, Nasacort, epinephrine or Zicam from various nasal regions over 5 days. LDH levels for each group are normalized to the control growth medium. (d) Summary of mean LDH levels from lidocaine, Zicam (day 2, *p<0.02; day 3,**p<0.002; day 4, **p<0.002), epinephrine, Nasacort and Afrin-treated human nasal explants normalized to saline-treated controls. All LDH levels other than the Zicam day 2, 3 and 4 did not reach statistical significance.
Age (range, mean) | 20–58, 44.6 |
Male | 4 |
Female | 4 |
UPSIT (range, mean) | 13–39, 27 |
LM CT (range, mean) | 1–24, 5.4 |
SNOT (range, mean) | 0–3.15, 1.50 |
Indication for surgery (N) | |
Chronic sinusitis | 5 |
Pituitary tumor | 1 |
Nasal congestion | 2 |
LM CT, Lund-Mackay staging system; SNOT-20, Sino-Nasal Outcomes Test; UPSIT, University of Pennsylvania Smell Identification Test). |
Functional endoscopic sinus surgery.
Transnasal endoscopic approach to the pituitary tumor removal.
Inferior turbinate submucosal resection.
We measured extracellular LDH levels following application of various intranasal agents directly onto the human nasal explants as a measure of cellular damage (
The elevation in extracellular LDH levels following Zicam treatment suggested cell death mediated by necrosis. Histological evaluation of Zicam-treated human nasal tissue indeed confirmed the presence of significant necrosis with the destruction of olfactory epithelium and subepithelial structures (
(a) Hematoxylin and eosin (H&E) stained human nasal explants from superior nasal septum following saline or Zicam application. (b) H&E stained human nasal explants from various nasal regions treated with either saline, lidocaine, Afrin, epinephrine, Nasacort or Zicam. Black arrows depict dead epithelial cells. Red arrows indicate basal surface of the remaining epithelium. Note the infiltration of inflammatory cells and severe necrosis in Zicam treated-nasal explants.
The present study demonstrated that Zicam treatment to both mouse and human nasal tissue induced significant cellular damage. In contrast, treatment with either saline, Afrin, Nasacort, lidocaine and epinephrine did not cause statistically significant tissue damage or olfactory dysfunction. The cytotoxic effects of Zicam were especially profound in the olfactory neuroepithelium. The resultant death of olfactory sensory neurons (OSN) in the MOE contributed to a long-lasting and apparently irreversible olfactory dysfunction in the mouse. Despite the ability of MOE to replace injured OSN via differentiation of stem cells, we found no evidence of functional recovery after two months. In fact, histological analysis of the mouse MOE about two months after Zicam treatment showed complete or near-complete loss of the epithelium and submucosa (
Our findings contradict a previous report on the effects of intranasal treatment of Zicam in mice
Second, Slotnick and colleagues utilized anterograde tracing with wheat germ agglutinin horseradish peroxidase to determine the integrity of olfactory epithelium
Many studies have demonstrated the cytotoxic effects of zinc cation on various cell types, including cultured cortical neurons
The regenerative capacity of MOE will depend on the extent of necrosis. While our study showed a high degree of cellular damage into deep tissue layers of MOE, variability in clinical presentation (i.e., severity and recovery) is possible given the variable anatomy of the human nasal cavity. Indeed, published case reports indicate varying outcomes of smell dysfunction associated with Zicam use
In this paper, we demonstrated the effects of several commonly used intranasal agents on olfaction using mouse and human nasal tissue. Our study is the most comprehensive examination, to date, of the role that intranasal medications have on short-term and long-term olfactory function. Intranasal administration of Zicam, unlike other tested agents, resulted in significant cytotoxicity to both mouse and human nasal tissue. This is a concerning finding given the potential development of long-lasting, and perhaps irreversible smell dysfunction. Because Zicam was previously classified as a homeopathic substance, it was not required to undergo stringent safety or efficacy evaluation as other conventional drugs. However, the Food and Drug Administration (FDA) recently issued a public health advisory cautioning against the use of some Zicam cold remedy nasal products. The FDA also issued a warning letter to the manufacturers of Zicam reclassifying these products as “drugs” that would require additional safety and efficacy testing to continue to market these products. The potential health risks of intranasal Zicam use demonstrated in our study stresses the need for stringent oversight of homeopathic remedies to protect the public from potentially unknown and dangerous side effects.
Adult male C57BL/6 mice (Charles River, Wilmington, MA) were used for all experiments. All work with animals was approved by the University of Washington Institutional Animal Care and Use Committee.
Human subjects were recruited as a consecutive sample from a rhinology clinic at a tertiary medical center. Written consent was obtained from all subjects donating the nasal tissue. Inclusion criteria included any patient over age 18 already scheduled for an endoscopic nasal procedure. Patients were excluded if they had a history of blood borne pathogens. The recruitment of patients and the study protocol was approved by the Institutional Review Board at the University of Washington (#35031).
For EOG and biochemical studies, about 15 µl of above intranasal agents were delivered slowly into the right nasal cavity of an adult mouse using a blunted 27-gauge needle. For behavioral testing, about 15 µl of intranasal agent was administered into both nasal cavities using the blunted 27-gauge needle. Animals were sedated with intraperitoneal administration of 80 mg/kg ketamine and 8 mg/kg xylazine prior to intranasal delivery of various agents.
EOG recordings from the MOE were performed as described previously
The odorant habituation assay of adult male mice was performed as described previously
Mouse MOE and human nasal tissues were immersed in 4% paraformaldehyde overnight at 4C, followed by cryoprotection in 30% sucrose overnight at 4C. Mouse MOE was decalcified in 0.5 M EGTA at 4C for 3–4 days prior to cryoprotection with 30% sucrose. The tissue was subsequently embedded in OCT (Sakura Finetek USA, Inc., Torrance, CA), frozen at −20C, cryosectioned at 30 µM and mounted onto slides.
Immunofluorescence was performed as described before with the following modifications
The tissues were processed as above and air dried for 30 minutes before staining in hematoxylin and eosin (Surgipath Medical Industries, Inc, Richmond, IL) for 1 min each. Tissues were then dehydrated in ascending series of ethanol, cleared in xylene and coverslipped with DPX (Fluka, Milwaukee, WI) and visualized with light microscope.
Subject disease severity was assessed with several instruments including a sinus CT scan (evaluated with Lund-Mackay staging system; LM CT)
The biopsied nasal explants were removed of any tissue debris or blood, and divided into small pieces (approximately 2–4 mm×2–4 mm×1–2 mm) using a 15-blade scalpel and a fine forcep under a dissecting scope. It was then placed on 0.4 µm polytetrafluoroethane (PTFE) membrane (Millicell-CM; Millipore, Cork, Ireland) immersed in 1.7 ml of culture medium consisting of 50% Dulbecco's Modified Eagle Medium High Glucose (Invitrogen, Carlsbad, CA); 25% Hank solution (Invitrogen, Carlsbad, CA); 50 U/mL penicillin G and 40 µg/mL streptomycin (Invitrogen, Carlsbad, CA) in a 60×15 mm tissue culture dish (Corning, Corning, NY). The tissue was placed on the membrane such that epithelial side was exposed to the air, and placed in humidified incubator at 37°C with 5% CO2. The culture media was changed every day.
LDH assay was performed with CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI) with the following modifications. On the day of the assay, 50 µl of culture medium was removed and added to 50 µl of ‘substrate mix’. Following 30 minute incubation at room temperature, the reaction was stopped with 50 µl of ‘stop solution’. Absorbance at 490 nm was obtained with Epx Precision Microplate Reader (Molecular Devices, Sunnyvale, CA). For measuring LDH levels in the human nasal explants, we stabilized the nasal tissue in the cultured environment for approximately 48 hours. We then measured the baseline LDH levels, and applied 2 µl of either 0.9% saline, Afrin (Schering-Plough HealthCare Products, Inc., Kenilworth, NJ), Nasacort (Sanofi-Aventis, Bridgewater, NJ), lidocaine (Hospira, Inc., Lake Forest, IL), 1∶100,000 epinephrine (diluted with 0.9% saline; Hospira, Inc., Lake Forest, IL) or Zicam (Matrixx Initiatives, Inc., Phoenix, AZ) directly onto the tissue. The levels of LDH were again measured 24 hours later. We repeated the addition of the intranasal agent and LDH measurement for five days.
Data are reported as mean +/− S.E.M. We performed statistical analyses using the two-tailed Student t-test. Statistical significance was defined as P<0.05. For the human tissue analysis, a power calculation was performed that showed a sample size of three would give 80% power to detect a significant difference (based on two pilot samples that showed a large effect size). We used linear mixed regression modeling to account for the repeated measures of the LDH assays on successive days for each tissue sample. To account for multiple comparisons, we used Bonferroni adjusted P-values to determine statistical significance (P = 0.05/25 = 0.002 for significance).
Gross appearance of mouse MOE 9 days after various intranasal agent administrations. Note the atrophy of MOE in Zicam-treated mouse. Black arrowheads indicate atrophic endoturbinates.
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Damage to mouse olfactory epithelium following Zicam treatment at various time points. H&E staining of mouse MOE depicting a significant loss of epithelium and submucosal damage 9 days after intranasal administration of Zicam as compared to saline treatment. Much greater damages to the epithelium and submucosal structure are observed without evidence of regeneration 31 and 35 days after intranasal administration of Zicam. Black arrows indicate damaged and remnants of MOE with fibrosis (e.g., days 31 and 65) in Zicam-treated mice. Scale bar, 100 µm.
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Cell death in human nasal explants following Zicam treatment from various regions of nasal cavity. H&E staining is shown. (a) Subject 1, inferior turbinate. (b) Subject 2, middle turbinate. (c) Subject 3, superior nasal septum. (d) Subject 4, middle turbinate.
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We thank Carolyn Bea for excellent technical assistance. We also thank the Storm laboratory for critical reading of this manuscript.