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Journal of the Anatomical Society of India

Effects of Phenol, Benzalkonium Chloride, Oxymetazoline, Tobacco and Formalin on Nasal Mucosa of Albino Rat

Author(s): M. Azim Khan

Vol. 55, No. 1 (2006-01 - 2006-07)

M. Azim Khan
C/o Mr. M.J. Khan, 65-B, Northern Railway Tank Colony, Raebareli – 229001 (U.P.)

Abstract:

Exposure of the nasal mucosa to a vast number of toxic agents is unavoidable. It may be in the form of air pollutants, industrial effluents or by the indiscriminate use of certain therapeutic agents. In the present study young albino rats received 1% phenol 0.01% benzalkonium chloride and 0.05% oxymetazoline as nasal drops, as well as tobacco and 20% formalin as a vapour. Animals were sacrificed after nine days treatment. Observations were made on haematoxylin and eosin stained paraffin sections. Phenol affected respiratory epithelium more than the olfactory epithelium. The changes ranged from loss of characteristic nuclear staining, vacuolation, oedema and subepithelial inflammatory cell infiltration to coagulative necrosis and sloughing of the epithelium. Benzalkonium chloride affected the nasal mucosa in the form of ciliary loss, epithelial spongiosis, mucosal swelling, hyperemic subepithelium with increased mononuclears in lamina propria. Oxymetazoline produced vascular congestion as the most prominent feature, however patchy ciliary loss were also noticed. Tobacco affected the olfactory epithelium which included mild epithelial disarrangement, vacuolar degeneration, partial ciliary attenuation and vascular congestion with tissue spaces in lamina propria. Formalin had effects similar to that of phenol but in a milder form and it affected both olfactory and respiratory epithelia equally. It is concluded that phenol, benzalkonium chloride, tobacco and formalin are harmful to the nasal mucosa and that the harmful effects of formalin vapour and tobacco fumes are comparable to that of topical application of phenol and bezalkonium chloride. Marked vascular congestion produced by oxymetazoline suggested rebound vasodilatation.

Keywords: Nasal mucosa, coagulative necrosis, vascular congestion, vacuolar degeneration, benzaalkonium chloride.

Introduction

Phenol (Carbolic acid) is one of the earliest used antiseptics and still the standard for comparing other germicides. It is relatively weak agent (static at 0.2%, cidal at > 1%) and is a protoplasmic poison (Tripathi, 2003). It is a coal- tar derivative and has an intense penetrating and characteristic odour referred to as phenolic odour. Although known as an acid, has no acid reaction. It is soluble in water and alcohol and forms one of the constituents of most disinfecting agents. Carbolic acid acts as mild corrosive and anaesthetic upon the skin and mucous membranes (Parikh, 1990).

The preservative benzalkonium chloride (BKC), has a bactericidal effect, because it damages the cell wall of the micro-organisms (Richards and Cavila, 1976). When used in the concentrations ordinarily used in nasal sprays and drops has been shown to be toxic to the nasal cilia (Joki et al, 1996).

Oxymetazoline nasal drops have been shown to constrict nasal resistance vessels. This results into reduction of about 59% mucosal blood flow (Zhen et al, 2003), which is more pronounced in patients with acute rhinitis than in healthy subjects. It is believed that when the vasoconstrictive action disappears, there is a compensatory vasodilation with subsequent swelling of the nasal mucosa (Baldwin, 1977). And that protracted use of oxymetazoline and xylometazoline might induce rebound swelling (Rijntjes, 1985 and Graf and Juto 1994, 1995).

Tobacco is derived by curing of leaves of Nicotiana rustica and Nicotiana tobacum. Tobacco may be chewed, smoked, sucked and sniffed. The carcinogenic role of tobacco is related to the type of tobacco product and the way in which it is used. The various constituents of tobacco include polycyclic aromatic hydrocarbons, nicotine, phenol, benzopyrene, carbon monoxide, formaldehyde and oxides of nitrogen, nitrosamine etc (Mori et al, 1995).

Formaldehyde is widely used in the paper, textile and resin industries as well as for embalming and preservation of tissues. It is present in the environment from sources including automobile-exhaust, photochemical-smog and cigarette smoke. It is almost completely absorbed in the nasal mucosa of rats when inhaled nasally (Dallas et al, 1985). Formaldehyde in small quantities is a normal component of intermediary metabolism in animals, including man (Mashford and Jones, 1982) and has been shown to increase the rate of surface epithelial cell proliferation in the rat (Chang et al, 1983), monkey (Monticello et al, 1989).

The chemicals and therapeutic agents described above are widespread and our nasal mucosa always comes in contact to them directly or indirectly. The present study was conducted to observe the microscopic changes in nasal mucosa exposed to these agents. It was also intended to compare the relative susceptibility of respiratory mucosa and olfactory mucosa to chemicals and therapeutic agents used.

Material and Methods

36 adult albino rats weighing 120 gm (±10 gm) of Charles Foster strain obtained from the central animals house, JN Medical College, AMU, Aligarh were used as experimental animal. The rats were divided into 6 groups. Groups A, B and C were exposed to 1% phenol, 0.01% benzalkonium chloride and 0.5% oxymetazoline as nasal drops, 1 drop bilaterally daily, for 9 days respectively. Group D and E were exposed to tobacco fumes and 20% formalin vapour respectively 6 hrs/ day, daily for 9 days by using inhalational chambers and the concentration of chemicals were maintained.

Group F was used as control and received no medication, but were maintained in similar environment and food. Animals were anaesthetized with ether to decapitate and to remove the skin as well as all the soft tissues surrounding the nasal cavity. Then, the bony – framework of the nasal cavity including nasal septum were nibbled out by bone – nibbler and available nasal mucosa was immersion fixed in Karnovsky’s fixative for further processing. Paraffin – wax embedded tissue samples were serially sectioned (6-8 micrometer thick) by Minot microtome from cranial to caudal end (coronal plane) and every 20th section was stained with Haematoxylin and Eosin to observe under light microscope.

Results:

Control group showed respiratory mucosa and olfactory mucosa. Respiratory mucosa consisted of pseudostratified ciliated columnar epithelium interspersed with goblet and basal cells, lying on a thin basal lamina and beneath the basal lamina were clustered groups of serous and mucous glands (Fig. 1). Olfactory mucosa consisted of thick pseudostratified ciliated columnar epithelium, within its thickness there were many layers of nuclei, although towards the free surface, there was a nucleus free zone. The epithelium was lying on basal lamina, underlying lamina propria contained numerous olfactory nerve fascicles and subepithelial olfactory glands of Bowman (Fig. 2). The changes produced by phenol ranged from loss of characteristic nuclear staining, vacuolation and oedema to coagulative necrosis and atrophied goblet cells with complete sloughing of the epithelium (Fig. 3). Sub epithelial connective tissue and glandular stroma showed inflammatory cell infiltration. Overall phenol affected the respiratory epithelium more than the olfactory epithelium.

Benzalkonium chloride (BKC) exposed nasal mucosa showed ciliary loss, atrophic goblet cells, epithelial spongiosis (inter and intracellular edema), mucosal swelling, hyperemic subepithelium with increased mononuclear cells in lamina propria (Fig. 4). It affected the olfactory mucosa and respiratory mucosa alike.

mucosa of rat showing pseudostratified ciliated columnar epithelium

Fig. 1: Photomicrograph of normal respiratory mucosa of rat showing pseudostratified ciliated columnar epithelium® with subepithelial serous and mucous glands (↔). H and E, X 200.

mucosa of rat showing thick pseudostratified ciliated epithelium

Fig. 2: Photomicrograph of normal olfactory mucosa of rat showing thick pseudostratified ciliated epithelium® with subepithelial nerve fascicles (↔). H and E, X 200.

respiratory mucosa of rat showing coagulative necrosis

Fig. 3: Photomicrograph of respiratory mucosa of rat showing coagulative necrosis® and partial sloughing of epithelial cells (↔) on 9 days exposure to 1% phenol. H and E, X 200.

respiratory mucosa of rat showing epithelial spongiosis

Fig. 4: Photomicrograph of respiratory mucosa of rat showing epithelial spongiosis®, loss of cilia (↔) and hyperemic subepithelium with increased mononuclear cells (5) on 9 days exposure to 0.01% benzalkonium chloride. H and E, X 200.

respiratory mucosa of rat showing destruction of epithelium

Fig. 5: Photomicrograph of respiratory mucosa of rat showing destruction of epithelium® with loss of cilia and homogeneous congestion of subepithelial blood vessels (↔) on 9 days exposure to oxymetazoline. H and E, X 100.

Vascular congestion was the most prominent feature of oxymetazoline exposure (Fig.5). Patches of respiratory epithelium showed changes in the form of loss of cytoplasmic staining of the epithelial cells and destruction of goblet cells. Scattered patches of epithelium showed destruction and ulceration of epithelium with loss of cilia. It showed mild effect on olfactory epithelium (Fig. 6).

Tobacco exposure on the olfactory epithelium showed mild epithelial disarrangement, vacuolar degeneration and partial ciliary attenuation (Fig.7). Vascular congestion and tissue spaces were seen in lamina propria. Olfactory epithelium revealed more changes than respiratory epithelium. Goblet cells showed no remarkable change.

Formalin vapour affected both olfactory and respiratory epithelia equally. The epithelium showed a wide range of changes. Loss of individual cell boundaries and loss of characteristic nuclear staining of epithelium were markedly seen. The lesions like vacuolation, epithelial spongiosis (Fig 8), mucoid pool formation (hydropic degeneration) (Fig. 9 and 10), goblet cell metaplasia and severe mucosal atrophy were noticed. Various lympho-epithelial lesions (pre-malignant lesion in which lymphocytes infilterating and destroying the epithelium) were observed at various sites. The detachment of the epithelium from the lamina propria at various sites was also noticed. Subepithelial connective tissue and glandular stroma were associated with infiltration of inflammatory cells. Vessels in the lamina propria were engorged and dilated.

The observations have been summarized in Table 1.

Table I: Effects of exposure to various chemicals and therapeutic agents on the nasal mucosa of albino rat.

Agent Effects on Respiratory mucosa Effects on Olfactory mucosa
Phenol Vacuolation, edema, coagulative necrosis, sloughing of epithelium. Sub-epithelial inflammatory cell infiltration. Minimal
Benzalkonium chloride Ciliary loss, epithelial spongiosis, mucosal swelling, hyperemic subepithelium with increased mononuclear cells in lamina propria. Very similar to those in respiratory mucosa.
Oxymetazoline nasal drops Marked vascular congestion, loss of cytoplasmic staining of epithelial cells, destruction and ulceration of epithelium with loss of cilia. Minimal, vascular congestion and focal ulceration.
Tobacco fumes Marked vascular congestion only. Mild epithelial disarrangement, vacuolar degeneration, and partial ciliary attenuation, vascular congestion.
Formalin vapour Loss of individual cell boundaries, vacuolation, epithelial spongiosis, mucoid pool formation, severe mucosal atrophy, various lymphoepithelial lesions, detachment of the epithelium and sub-epithelial inflammatory cells infiltration. Very similar to those in respiratory mucosa.

Discussion:

Phenol is a coagulant of protein, having remarkable penetrating quality causes necrosis and sloughing of the tissues (Parikh, 1990). The coagulative necrosis and sloughing of nasal epithelium observed in the present study were comparable to the description made by him. In fact when phenol is applied to the skin and prevented from evaporating, it may cause gangrene even in weak solutions (Parikh, 1990), which can be compared to the findings like vacuolation, oedema and inflammatory cell infiltration of the present study. Tripathi (2003) reported that swallowing of carbolic acid (for suicidal purpose) causes buccal, oesophageal and gastric burns which subsequently cicatrise forming strictures.

Benzalkonium chloride is irritant and is toxic to nasal cilia (Joki et al, 1996; Batts et al, 1989). The preservative, benzalkonium chloride is responsible for the epithelial damage as shown by Tonjum (1975) in corneal epithelium of the rabbit.

nasal mucosa of rat showing olfactoryepithelium

Fig. 6: Photomicrograph of nasal mucosa of rat showing olfactoryepithelium (OE), degenerated respiratory epithelium (RE), ulceration (↓) and dilated, congested subepithelial blood vesels (↔) on 9 days exposure to oxymetazoline. H and E, X 200.

olfactory mucosa of rat showing epithelial disarrangement

Fig. 7: Photomicrograph of olfactory mucosa of rat showing epithelial disarrangement®, Vacuolation (↔) and subepithelial hyperemia on 9 days exposure to tobacco. H and E, X 400.

Oxymetazoline induced vascular congestion was found to be the most prominent feature in the present study, however, this has been shown to induce a pronounced decongestion that lasts for 6 to 8 hours (Akerlund et al, 1989). Graf et al, (1995), reported that healthy subjects complained of nasal stuffiness from the 2nd week of receiving the medication. Marked vascular congestion after exposure to oxymetazoline in the present study is in agreement with the findings of rebound nasal congestion following 3 days of oxymetazoline treatment reported by Morris et al, (1997). The present study is not in full agreement with the findings of Graf et al, (1999) recommending safe use of oxymetazoline for 10 days. Rhinostereometric study by Graf and Juto, (1994) showed that no rebound swelling occurs even after 10 days use of oxymetazoline nasal spray. It means vascular congestion expressed clinically as nasal stuffiness may occur only after one week of using oxymetazoline nasal drops. Recent studies have shown that overuse of these drugs also results in rebound congestion and histologic changes of the nasal mucosa (Akerlund and Bende, 1991).

respiratory mucosa of rat showing epithelial spongiosis

Fig. 8: Photomicrograph of respiratory mucosa of rat showing epithelial spongiosis (↓), epithelial disarrangement (↔) and subepithelial congestion (5) on 9 days exposure to formalin vapour. H and E, X 400.

olfactory mucosa of rat showing swelling of epithelium

Fig. 9: Photomicrograph of olfactory mucosa of rat showing swelling of epithelium and mucoid pool formation (→) on 9 days exposure to formalin vapour. H and E, X 200.

olfactory mucosa showing distended epithelium

Fig. 10: Photomicrograph of olfactory mucosa showing distended epithelium and mucoid pool formation® (hydropic degeneration) on 9 days exposure to formalin vapour. H and E, X 400.

Cigarette smoke, contains toxigenic, mutagenic and even carcinogenic compounds and therefore claimed to be major environmental risk factors of various human diseases (Mori et al, 1995). Mild epithelial disarrangement with partial ciliary attenuation and vascular congestion observed in the present study can be compared with the findings of Cunningham et al (2001) in which they described the metaplastic transformation towards squamous epithelium and symptoms of erosion and inflammation in the tissues of the respiratory tract directly exposed to the cigarette smoke. It was also described by Zeid and Muller (1995) that exposure to cigarette smoke results in inflammation with neoplastic changes of respiratory tract. While Shields and Jeffery (1987) reported the hypertrophic, hyperplastic changes and sometimes keratinized metaplastic foci in pseudostratified epithelium of the trachea on exposure to cigarette smoke. The results of tobacco exposure in the present study were also partially comparable to those of Czekaj et al (2002), in which they observed effects of cigarette smoke on respiratory system in pregnant rats. They found squamous metaplasia, cystic degeneration and inflammatory granulomas (non-specific epitheloid cells with inflammatory cells).

Formalin induced lesions like loss of individual cell boundaries, vacuolation and hydropic degeneration of the present study were consistent with the results of acute (9 day) studies of Chang et al, (1983) and Swenberg et al, (1986). The present study with epithelial spongiosis resembled to those reported by Casanova- Schmitz et al, (1989) for other formaldehyde induced biological effects. The goblet cell metaplasia shown in present study resembled to inhibition of mucociliary function shown by Morgan et al, (1986) in F-344 rats. And the presence of lymphoepithelial lesions resembled to the carcinogenic response reported by Kerns et al, (1983) in rats and mice.

It is concluded that phenol, benzalkonium chloride, tobacco and formalin are harmful to the nasal mucosa, and that the harmful effects of formalin vapour and tobacco fumes are comparable to that of topical application of phenol and benzalkonium chloride. Also, the susceptibility of olfactory mucosa to tobacco fumes, while susceptibility of respiratory mucosa to phenol and oxymetazoline nasal drops suggested differential susceptibility of two types of nasal mucosa to different chemicals. The differential susceptibility of two types of nasal mucosa to different chemicals can be explained on the basis of structural differences of two types of nasal mucosa. The olfactory mucosa is more delicate and less vascular than respiratory mucosa (G.J. Romones 1985). Probably, type of drug and its concentration with mode of exposure also affects the susceptibility as well. It is supposed that particular drug in a fixed concentration binds to specific receptors and probably inhalational drugs reach to posterior parts of nasal cavity more easily than nasal drops. Marked vascular congestion produced by oxymetazoline nasal drop suggested rebound vasodilatory action shown by certain vasoconstrictors (Rhinitis medicamentosa).

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