In vitro effects of preservatives in nasal sprays on human
`nasal epithelial cells
`
`Ching-Yin Ho, M.D., Ph.D.,* Meng-Chuan Wu, M.D.,* Ming-Ying Lan, M.D.,#
`Ching-Ting Tan, M.D., Ph.D.,§ and An-Hang Yang, M.D.¶
`
`ABSTRACT
`Background: The preservatives benzalkonium chloride and potassium sorbate are widely used in nasal drops and sprays. Recently, side
`effects resulting from mucosal damage caused by benzalkonium chloride and potassium sorbate were reported.
`Methods: We investigated the toxicity of benzalkonium chloride and potassium sorbate on human nasal epithelial cells in vitro. Using
`primary human nasal epithelial cells, different concentrations of benzalkonium chloride, potassium sorbate, or phosphate-buffered saline (PBS;
`control group) solutions were cocultured with nasal epithelial cells for 15 minutes. Then, the viability of the cells and the cell morphology
`were assessed.
`Results: Nasal epithelial cells were more severely damaged with use of clinical preparations or higher concentrations of benzalkonium
`chloride than in the control group. In addition, nasal epithelial cell membrane lysis was seen on electronic microscopy in the benzalkonium
`chloride groups. In contrast, there was no significant cell damage seen in the potassium sorbate groups compared with the control group, even
`with higher concentrations than clinically used.
`Conclusion: Potassium sorbate appears to be a relatively safer preservative than benzalkonium chloride for use in nasal sprays and drops
`in vitro study.
`
`(Am J Rhinol 22, 125–129, 2008; doi: 10.2500/ajr.2008.22.3154)
`
`Key words: Adverse effect, benzalkonium chloride, cell viability, nasal epithelial cell, potassium sorbate, preservatives, primary
`cell culture
`
`Local administration of medicine has been adapted to med-
`
`ical treatment for disease in the nasal cavity and para-
`nasal sinuses, including allergic rhinitis and sinusitis. Various
`preservatives are used for preventing bacterial contamination
`of nasal spray preparations. The preservatives should not
`alter the main pharmacologic actions of the drug in the prep-
`aration and should not cause side effects in the body. Among
`the preservatives, benzalkonium chloride and potassium sor-
`bate are the ones we often find in preparations that we use
`daily in nasal drops, eye drops, cosmetics, and food prepara-
`tions. Several studies showed that benzalkonium chloride
`induced alteration of the nasal mucosa in vitro and in vivo.1–7
`Some of these studies reported that benzalkonium chloride
`did not induce nasal cell damage.8,9 The effects of benzalko-
`nium chloride on the nasal passages are controversial. Studies
`of the effects of potassium sorbate on nasal mucosa are lack-
`ing. In 2004, Hofmann et al. found that the isolated potassium
`sorbate did not have negative influence on ciliary beat fre-
`quency in vitro.3 But in vivo, some studies indicated that even
`a low concentration of potassium sorbate can lead to nasal
`lesions, including intraepithelial glandular formation, inflam-
`matory cell infiltration, vascular hyperplasia, and edematous
`
`From the *Department of Otolaryngology, Veterans General Hospital-Taipei and Na-
`tional Yang-Ming University, Taipei, Taiwan, #Institute of Clinical Medicine, Na-
`tional Yang-Ming University and Department of Otolaryngology, Chutung Veterans
`Hospital, Taipei, Taiwan, §Department of Otolaryngology, National Taiwan Univer-
`sity Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, and
`¶Department of Pathology, Veterans General Hospital–Taipei, Taipei, Taiwan
`Address correspondence and reprint requests to Ching-Ting Tan, M.D., Ph.D., De-
`partment of Otolaryngology, National Taiwan University Hospital and National Tai-
`wan University College of Medicine, No. 7, Chung-Shan South Road, Taipei 100,
`Taiwan
`E-mail address: tanct5222@ntu.edu.tw
`Copyright © 2008, OceanSide Publications, Inc., U.S.A.
`
`change.10 Thus, the effects on the nasal mucosa of preserva-
`tives used in nasal sprays remain unclear. We investigated the
`effects of different concentrations of benzalkonium chloride
`and potassium sorbate on primary human nasal epithelial
`cells via measurement of cell viability and cell morphology.
`
`METHODS
`For the benzalkonium chloride group, a 0.01% benzalko-
`nium chloride solution that is commonly used as a preserva-
`tive in nasal drops and 0.1, 0.001, and 0.0001% solutions
`(Sigma Chemical Co., St. Louis, MO) were used for assessing
`dose dependence. For the potassium sorbate group, a 0.1%
`potassium sorbate solution commonly used as a preservative
`in nasal drops and 1, 0.01, and 0.001% potassium sorbate
`solutions were used for assessing dose dependence (Sigma
`Chemical Co.). Phosphate-buffered saline (PBS) was used for
`the control group.
`
`Primary Human Nasal Epithelial Culture with
`Air–Liquid Interface Method
`This study was approved by the Ethics Review Board of the
`hospital and with informed consent from the patients. The
`primary human nasal epithelial cell culture with air– liquid
`interface procedure was modified from methods previously
`described.11,12 Human inferior turbinate cells were obtained
`from 18 patients receiving submucosal resection of inferior
`turbinates due to chronic hypertrophic rhinitis. Each speci-
`men was rinsed several times with Leibovitz’s L-15 medium
`containing penicillin (100 U/mL), streptomycin (100 ␮g/mL),
`and amphotericin B (0.25 ␮g/mL). The tissue was then cut
`into 1- to 2-mm2 pieces, and three or four pieces of tissue were
`planted with the epithelium side facing down onto 6-well
`culture inserts (Falcon, Franklin Lake, NJ), in which the mem-
`
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`brane growth area was 4.2 cm2, and with a pore size of 0.4 ␮m.
`Cells were cultured with Airway Epithelial Cell Growth Me-
`dium (PromoCell Bioscience Alive, Germany) containing bo-
`vine pituitary extract (0.004 mL/mL), human epidermoid
`growth factor (0.5 ng/mL), insulin (5 ␮g/mL), transferrin (10
`␮g/mL), hydrocortisone (0.5 g/mL), epinephrine (0.5 ␮g/
`mL), triiodo-l-thronine (6.7 ng/mL), retinoic acid (0.1 ng/
`mL), and phenol red (0.62 ng/mL). Cultures were maintained
`at 37°C in a humidified atmosphere of 5% CO2. Media were
`changed three times weekly. The airway epithelial cells were
`grown on a porous membrane, on which they formed a con-
`tinuous epithelial sheet, with the basal aspect exposed to the
`medium and the apical surface exposed to air. Cells grown on
`the inserts were confluent after 10 days of incubation. Cells
`were then detached with 0.1% trypsin-EDTA and seeded into
`96- or 24-well culture plates.
`
`Assay of Cell Viability and Morphological
`Observation
`The viability of the cells treated with PBS, benzalkonium
`chloride, or potassium sorbate was quantified by the trypan
`blue exclusion method and 3-(4,5- dimethylthiazol-2-yl)-2,5-
`diphenyltetrazolium bromide (MTT) assay and electron mi-
`croscopy. Trypan blue (Sigma Chemical Co.) chromophore is
`negatively charged and does not interact with cells unless the
`membranes are damaged. Therefore, cells that exclude the
`dye are viable. The cells were washed twice in PBS and
`detached by swirling with 0.01% trypsin-EDTA. The cells
`were collected by centrifugation at 1500 rpm for 6 minutes at
`4°C and the supernatant was decanted. The cell pellet was
`resuspended in growth medium. An aliquot of 0.4% trypan
`blue was mixed with an equal volume of cell suspension for 5
`minutes and viable cells were counted using a hemacytometer.
`
`MTT
`Cell viability was determined on day 13 using the MTT
`test (SigmaChemical Co.). MTT is a pale yellow substrate
`
`that is cleaved by living cells to yield a dark blue formazan
`product. The MTT assay is based on reduction of MTT by
`actively growing cells to produce a blue formazan product
`with absorbance at 570 nm. This process requires active
`mitochondria, and freshly dead cells do not cleave signifi-
`cant amounts of MTT. A low MTT absorbance indicates cell
`death. The colorimetric assay was used for proliferation in
`96-well flat-bottom tissue culture plates of good optical
`quality (Falcon). The final volume of serum-free culture
`medium in each well was 0.1 mL. At the end of the assay,
`0.01 mL of MTT solution added to each well and the cul-
`tured cells were incubated at 37°C for another 4 hours.
`Subsequently, 100 ␮L of isopropanol/0.04 N hydrochloric
`acid solution was added to each well and mixed thor-
`oughly. The absorbance was measured in an enzyme-linked
`immunosorbent assay (ELISA) plate reader at a test wave-
`length of 570 nm within 1 hour.
`
`Transmission Electron Microscopy
`Cells were prefixed with 1.5% glutaraldehyde in 0.2 M of
`phosphate buffer, postfixed in 1% osmium tetroxide, dehy-
`drated through absolute alcohol, transferred to propylene
`oxide, and impregnated overnight in Epon resin. This prepa-
`ration was finally embedded in Epon resin and polymerized
`at 50°C. Sections were cut with an ultramicrotome to a thick-
`ness of 60 nm and stained with uranyl acetate and lead citrate.
`The samples of nasal epithelial cells were observed using a
`transmission electron microscope (JEOL 2300; JEOL Tokyo,
`Japan)
`
`Statistical Analysis
`Results are presented as means ⫾ SE. Statistical compari-
`sons of multigroup data were analyzed using analysis of
`variance followed by the Duncan post test. A value of p ⬍ 0.05
`was considered significant.
`
`a
`
`a
`
`*
`
`B
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`bc
`
`a
`
`*
`
`b
`a
`
`*
`
`a
`
`*
`
`*
`K C
`0 . 0
`
`S
`
`B
`
`P
`
`0 . 1 % B
`
`A
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`Cellviability(%ofPBS)
`
`0
`
`1 % B
`
`K C
`0 . 0
`
`1 % B
`
`0
`
`K C
`
`1 % B
`
`0
`
`0
`
`0
`
`S
`
`B
`
`P
`
`S
`
`1 % P
`
`S
`
`0 . 1 % P
`
`S
`
`1 % P
`
`0 . 0
`
`0
`
`0 . 0
`
`S
`
`1 % P
`
`K C
`0 . 0
`Figure 1. Trypan blue method for cytotoxicity of benzalkonium chloride (BKC) and potassium sorbate (PS). Primary human nasal epithelial
`cells were incubated with various concentrations of PBS, BKC, or PS for 15 minutes. Cell viability was evaluated by trypan blue method.
`Data are means ⫾ SE from three independent experiments. *p ⬍ 0.05 compared with PBS; a, p ⬍ 0.05 compared with (A) 0.1% BKC and
`(B) 1% PS; b, p ⬍ 0.05 compared with 0.01% BKC; c, p ⬍ 0.05 compared with 0.001% BKC.
`
`126
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`b
`a
`
`b
`a
`
`B
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`*
`
`*
`
`A
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`Cellviability(%PBS)
`
`0
`
`S
`
`B
`
`P
`
`0 . 1 % B
`
`C
`
`K
`
`0 . 0
`
`1 % B
`
`K
`
`C
`0 . 0
`
`1 % B
`
`0
`
`C
`
`K
`
`1 % B
`
`0
`
`0
`
`C
`K
`0 . 0
`Figure 2. MTT for cytotoxicity of benzalkonium chloride (BKC) and potassium sorbate (PS). Primary human nasal epithelial cells were
`incubated with various concentration of PBS, BKC, or PS for 15 minutes. Cell viability was evaluated by MTT assay. Data are means ⫾
`SE from three independent experiments. *p ⬍ 0.05 compared with PBS; a, p ⬍ 0.05 compared with (A) 0.1% BKC and (B) 1% PS; b, p ⬍
`0.05 compared with 0.01% BKC.
`
`0
`
`S
`
`B
`
`P
`
`S
`
`1 % P
`
`S
`
`0 . 1 % P
`
`S
`
`1 % P
`
`0 . 0
`
`0
`
`0 . 0
`
`S
`
`1 % P
`
`RESULTS
`
`Cell Viability
`The higher the concentration of benzalkonium chloride or
`potassium sorbate used, the lesser the cell viability via the trypan
`blue and MTT methods (Figs. 1 and 2). The clinically used
`benzalkonium chloride concentration of 0.01% significantly re-
`duced cell viability compared with the control group. Only 14
`and 19% of nasal epithelial cells survived treatment with 0.01%
`benzalkonium chloride via the trypan blue and MTT methods,
`respectively. In contrast, the clinically used concentration of
`potassium sorbate (0.1%) did not induce cell toxicity.
`
`Cell Morphology
`After 15 minutes of incubation, 0.01% benzalkonium chlo-
`ride induced cell swelling (Fig. 3) and loss of cell contact. In
`the trypan blue exclusion test, nucleoli were stained in almost
`90% cells after incubation with 0.01% benzalkonium chloride
`for 15 minutes. There were no morphological changes in the
`potassium sorbate and control groups, and nucleoli were
`stained in only a few cells.
`Electron microscopic analysis of the structural features of
`nasal epithelial cells in the control group and the 0.1% potas-
`sium sorbate–treated group and the 0.01% benzalkonium
`chloride-treated group are shown in Fig. 4. The cells of the
`control and 0.1% potassium sorbate–treated group showed
`microvilli (arrows) and keratin intermediate filaments, both of
`which are specific characters of respiratory epithelial cells; the
`cells are also tightly bound and showed no destruction of the
`cell membrane and were well aligned. Nasal epithelial cells
`incubated with 0.01% benzalkonium chloride for 15 minutes
`showed the loss of microvilli, destruction of cell membranes,
`and poor cytoskeletal alignment.
`
`DISCUSSION
`Benzalkonium chloride and potassium sorbate usually are
`added to nasal sprays for prevention of bacterial contamina-
`
`tion. In our study, the benzalkonium chloride–induced nasal
`epithelial cell damage occurred in a dose-dependent manner.
`Even at the clinically used concentration of benzalkonium
`chloride, cell viability was reduced. In contrast, the clinically
`used concentration of potassium sorbate in nasal spray did
`not induce human nasal epithelial cell damage.
`Preparations of various medicines, such as steroids, anti-
`
`X001
`
`X002
`
`PBS
`
`PS
`
`BKC
`
`Figure 3. Morphological appearances of primary nasal epithelial
`cells after incubation with various concentration of PBS, benzalko-
`nium chloride (BKC), or potassium sorbate (PS) for 15 minutes.
`(Upper Panel) Treated with PBS. The cells are intact with good
`cell–cell contact. (Middle Panel) Treated with 0.1% PS for 15
`minutes. The cell morphology did not change compared with PBS-
`treated cells. (Lower Panel) Treated with 0.01% BKC for 15 min-
`utes. The cell was rounding and loss cell–cell contact.
`
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`*
`
`B
`
`D
`
`*
`
`*
`
`A
`
`C
`
`*
`
`Figure 4. Morphologic appearances of primary nasal epithelial cells
`measured by transmission electron microscopy. Cells of control
`group (A) and 0.1% PS-treated group (B) showed microvilli (*) and
`keratin intermediate filaments (arrow) both of which are specific
`characters of respiratory epithelial cells. Nasal epithelial cells incu-
`bated with 0.01% BKC (C and D) for 15 minutes demonstrated the
`losing of microvilli, the lysis of cell membrane (*) and destruction of
`keratin intermediate filaments (arrow). Scale bar as figure showing
`2 ␮m in A, B and D; 5 ␮m in C).
`
`histamines, and decongestants, are administered into the na-
`sal cavity and paranasal sinuses for allergic rhinitis, sinusitis,
`and chronic hypertrophic rhinitis. In addition to the main
`drug, the other ingredients of these sprays include various
`additives, such as osmoregulators, viscosity regulators, pre-
`servatives, and pH regulators, which are recognized as harm-
`less to humans. The adverse effects that are reported are
`mostly for preservatives.13,14 Benzalkonium chloride and po-
`tassium sorbate are the most commonly used preservatives in
`nasal sprays. Several studies showed that benzalkonium chlo-
`ride induced alteration of the nasal mucosa in vitro and in
`vivo.1–7 In 2004, Hofmann et al.3 reported that benzalkonium
`chloride could cause a decrease in cilia beat frequency of nasal
`mucosa and the change was irreversible if the concentration of
`benzalkonium chloride was high. In 2000, Storaas et al.4 found
`that benzalkonium chloride in dosages commonly used as
`preservatives in nasal decongestant sprays produced short-
`term glandular secretion and nasal pain in vivo. In histological
`studies, benzalkonium chloride induced squamous metapla-
`sia, edematous change, glandular hyperplasia, and inflamma-
`tory cell infiltration in nasal mucosa.10,15,16 In contrast, some
`studies reported that benzalkonium chloride–containing nasal
`sprays did not induce nasal mucosa damage or ciliotoxic
`effects.17,18 Other studies showed that benzalkonium chlo-
`ride–containing nasal sprays had a marked ciliotoxic effect,
`but did not change the biomarker in nasal secretions or in-
`flammatory cell recruitment.1 This probably is because the
`
`mucosa is well protected by a layer of mucus and by the
`continual ciliary beating, which quickly moves the benzalko-
`nium chloride from one site to another. This probably ex-
`plains the discrepancy in results between in vitro and in vivo
`studies. In our in vitro study, primary human nasal epithelial
`cells treated with the clinically used concentration of benza-
`lkonium chloride, or even less than the clinically used con-
`centration, for 15 minutes induced severe nasal epithelial
`damage, including decreased cell viability; loss of cilia; de-
`struction of the cell membrane; and loss of cytoskeleton align-
`ment. Benzalkonium chloride induced great cytotoxicity in
`human nasal epithelial cells in vitro.
`Previous studies of the influence of potassium sorbate on
`nasal mucosa are inconclusive. In 2000, Cho et al. showed that
`preservatives in nasal sprays, including benzalkonium chlo-
`ride and potassium sorbate, induce nasal histological change,
`including intraepithelial glandular formation, inflammatory
`cell
`infiltration, vascular hyperplasia,
`and edematous
`change.10 The histological changes were pronounced with
`prolonged duration of administration. In 2004, Hofmann
`found that potassium sorbate did not influence the ciliary beat
`frequency of human nasal mucosa in vitro.3 Our results
`showed that the clinically used concentration of potassium
`sorbate did not reduce cell viability of human nasal epithelial
`cells, and it did not destroy the nasal epithelial cell morphol-
`ogy.
`
`CONCLUSIONS
`Benzalkonium chloride in nasal sprays reduced the viabil-
`ity of human nasal epithelial cells and induced cell lysis in
`vitro. In contrast, potassium sorbate did not show a significant
`influence on cell viability and cell morphology in vitro. Potas-
`sium sorbate appears to be a safer preservative than benza-
`lkonium chloride in topical nasal sprays. However, additional
`in vivo studies are needed to clear up the side effects of
`preservatives in the human nasal mucosa.
`
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