Alcoholic Parotid Sialosis: A Structural and Ultrastructural Study

CARDA C, GÓMEZ DE FERRARIS ME, ARRIAGA A, CARRANZA M, PEYDRÓ A. ALCOHOLIC PAROTID SIALOSIS: A STRUCTUAL AND ULTRASTRUCTURAL STUDY. MED ORAL 2004;9:24-32.

SUMMARY

OBJECTIVES: The purpose of this study is to demonstrate the histopathological differences between the initial and advanced stages of Alcoholic Sialosis, a pathology that generally involves parotid hypertrophy and structurally affects, to diverse degrees, the other salivary glands.
STUDY DESIGN: An analysis and comparison was carried out of the structural and ultrastructural modifications of the parotid glands from the hepatic biopsies of chronic alcoholics with clinical diagnosis of cirrhosis and from autopsies on individuals who had died from alcoholic hepatic cirrhosis. Various samples of normal gland obtained from surgical material were used as a control.
RESULTS: The alterations found in the biopsies corresponded to the modifications discovered in the autopsies of alcoholics. Notable in both cases was the massive accumulation of secretory granules of different size, shape and electrodensity, which occupied the cytoplasm of the acinar cells. In both sample types the excretory ducts were enlarged and the epithelium of the striate ducts presented cells with nuclei and cytoplasm of irregular appearance and arrangement. A moderate adipose infiltration in the stroma and slight periacinal edema was also observed. The biopsies revealed, both at optical and electron microscopical levels, lipid inclusions in the acinar cells and the glandular parenchymal ducts.
CONCLUSIONS: The structural and ultrastructural findings of the parotid biopsies and autopsies, clearly show that alterations are already present in the salivary glands of chronic alcoholics before the terminal phase of hepatic cirrhosis. The enlargement of the ductal system lumens could be the principal cause of glandular hypertrophy.

Key words: Parotid , structural and ultrastructural modifications, sialosis, alcoholism.

INTRODUCTION

Chronic alcoholism is one of the etiological causes of sialosis, a pathology generally characterised by a bilateral enlargement, neither neoplastic nor inflammatory, of the parotid gland (1- 3). Sialosis, however, can have different origins, having been described as a consequence of hormonal, nutritional or metabolic disturbances, medicamentosus or neurohumoral alterations (4-6). Furthermore, the process is not exclusive to the parotid, but also affects, to diverse degrees, the other larger and smaller salivary glands (7-10).

Alcoholic sialosis generally involves glandular hypertrophy, produced either by adipose infiltration or by acinar hypertrophy. There are authors who accept the coexistence of both modifications, while others deny such a possibility (11, 12). In fact acinar hypertrophy is not always present in sialosis, as a consequence some authors centre their attention on the glandular dysfunction. This dysfunction is generally manifested as salivary hypofunction and xerostomia (5), however, a greater flow of stimulated parotid saliva has been demonstrated by sialochemistry in the group of cirrhotic patients with respect to the controls, with no differences existing between the cirrhotic alcoholics and other types of cirrhosis of non-alcoholic etiology (13).

In sialosis of alcoholic origin, 60% of the patients with hepatic damage (cirrhosis) present parotidomegaly (14, 15), the glandular enlargement being observed already in the pre-cirrhotic phases in 12% of cases (16, 17).

In previous studies of parotid glands from individuals who had died of alcoholic hepatic cirrhosis (7, 8), we described heterogeneous accumulations of secretory granules of different sizes, irregularly distributed throughout the cytoplasm of the acinar cells, unlike the Von Ebner serosa glands, where the granules were smaller, homogeneous and preferentially located in the apical region. Likewise, the alterations at the epithelial level of the ductal system were highly evident. The striate ducts exhibited an epithelium of pseudostriated appearance, with elongated nuclei of dense chromatin, together with other nuclei surrounded by loose chromatin. In the excretory ducts, of note, was the increase in ductal diameter, the stasis of the secretory material with desquamated cells, and epithelial atrophy, imunohistochemically heterogeneous for cytokeratins.

The majority of histopathological studies referring to parotid sialosis have been undertaken using samples from human autopsies, where the structural changes described correspond to the terminal phase of this pathology. Cases in which biopsies have been used are very rare. With the aim of establishing possible histopathological differences between the initial and terminal phases of alcoholic sialosis, the structural and ultrastructural modifications of the parotid gland were analysed and compared using both biopsies of chronic alcoholics with hepatic cirrhosis and autopsies of individuals who had died from alcoholic hepatic cirrhosis.

MATERIALS AND METHODS

The study was carried out with material from the archives of the Departamento de Patología de la Universidad de Valencia (España), and samples obtained from autopsies from the Instituto Médico Forense de la Provincia de Córdoba (Argentina). 12 biopsies of parotid glands were used from patients with clinical and anatomicopathological diagnosis of hepatic cirrhosis of alcoholic etiology, with clinical signs of non-inflammatory parotid gland hypertrophy. The cases consisted of 8 men and 4 women between 36 and 74 years of age. Part of the biopsy material was fixed in formol of pH 7 and embedded in paraffin for the optical microscope studies; another part was fixed in glutaraldehyde, post fixed in osmic acid and embedded in acrylic resin (Epon) for the electron microscope study. Semi-fine cuts stained with blue toluidine were made for the control and selection of the most representative areas. Ultrafine cuts were contrasted with uranyl acetate and lead citrate, the ultrastructural analysis being carried out with an electron transmission microscope (Joel JEM 1010).

As a control, a portion of normal gland from a parotid removed for oncological reasons from a 60-year-old patient was used. This sample was fixed immediately and processed in a similar manner.

For the comparative study, 6 samples of parotid gland fixed in formal at pH 7 were used, coming from autopsies of individuals of 35 to 65 years of age, who had died of alcoholic hepatic cirrhosis. This material was processed for embedding in paraffin and later processing with the coloration techniques H-E, PAS and blue toluidine pH 3.8. Small portions of parotid were obtained from the previously selected paraffin blocks, which were then reutilized for the study with electron microscope, carrying out deparaffinisation, re-embedding in acrylic resin and the other steps of the above described processes.

RESULTS

Study of the parotid biopsies:
The optical microscopy revealed the structural organisation of the glands to be generally well preserved. The majority of the lobulets were composed of typical serous acini; however, in certain areas signs of acinar hypertrophy were evident, with large cells full of secretory granules widely distributed throughout the cytoplasm. We were able to distinguish large infiltrated adipocytes as well as small intracytoplasmatic lipid drops in the parenchymal cells, especially at acinar level.

In all the glands studied, acinar cells of mucosal appearance were observed, combined, to a greater or lesser degree, with typical serous secretory cells of high electron density. Histochemically, the content of these cells presented a positive reaction to PAS, and light metachromasia with blue toluidine
The ductal system was well developed, in some cases stasis of secretory material was identified in the lumen of the striate and secretory ducts.

The stroma presented a moderate adipose infiltration, represented by large adipocytes with the presence also of isolated lymphocytic foci (Fig 1).

At ultrastructural level, noticeable modifications with respect to the norm were observed in the parenchyma, particularly in the acinar cells. In general these were characterised by the accumulation of intracytoplasmatic granules of diverse typology in shape, size and electrodensity.

The cytoplasm of the typical acinar cells was shown to be full of electrodense granules of circular or polyhedral contour owing to their grouping, occupying the full extent of the cytoplasm, including the basal region where the endoplasmic reticule appeared well developed (Fig 2). Also, in some acini, granules of large, pale, vesicular appearance and with a centre of greater electrodensity were identified. Likewise, a third type of larger granule was observed having a finely fibrillar and low electron-density content, with a tendency to coalesce and whose appearance was similar to the mucosal type vesicles (Fig.·3A-B). In some samples, certain acini were totally constituted of cells with these characteristics.

With respect to the rest of the organelles large Golgi cisterns as well as lisosomes were notable.

Other acinar cells were found which exhibited nuclei of irregular shape, with a different degree of heterochromatic content, while other cells showed evidence of structural alteration, with either pycnotic or degenerating nuclei and a cytoplasm of greater electrodensity, with enlarged endoplasmic reticule cisterns and either scarce or no granular content.

The lipid inclusions arranged in different areas of the cytoplasm were observed in practically all the acinar cells, identifying acini whose cells were completely occupied by lipid drops (Fig. 4).
Between the acinar cells, extensive intercellular caniculi with abundant interdigitations were frequent. Particularly noticeable in some acini were the parallel desmosomic unions.

In the basal region of certain acini, cells of undifferentiated appearance were distinguished, joined by desmosomes to the neighbouring acinar cells that were characterised by their clearer and scarcer cytoplasm with few organelles and no secretory granules. No mitotic figures were found in these areas. In the basal areas of both the acini and the beginning of the ductal system, the degree of development of the mioepithelial cells was notable.

In some glandular regions, small acini existed with a dysfunctional appearance of atrophic type. Likewise, cells with an obvious oncocytic transformation were observed in isolation.
In the striate ducts the alternation between dark and clear cells, with atypical, heterochromatic nuclei, of irregular contour and located at different levels was notable. These cells contained abundant mitochondria and some presented large heterogeneous lisosomic bodies, as well as lipidic inclusions.

The cells of the intercalary ducts also exhibited some lipidic drops.

The appearance of the stroma was normal in some regions, in others a slight edema appeared, more noticeably in the periacinal area, in addition to a certain degree of fibrosis and adipose infiltration.

-Study of autopsy material:

At optical microscope level, the parotid gland presented acini with granular accumulation atypical in size and location while some sectors exhibited acini of mucosal appearance.

The epithelium of the striate ducts showed a clear nuclear heterogeneity and pseudostratification, while in the secretory ducts a bistratal epithelial arrangement was observed, with moderate atrophy and accumulation of secretory material. There was a moderate adipose infiltration and slight periacinal edema in the stroma.

The material analysed after re-embedding for electron microscopy showed notable similarities to the material from the biopsies, although with fixation defects owing to the initial use of formol and to the processing for embedding in paraffin. In the acinar cells, accumulations of electrodense granules of different size and distributed at random throughout the cytoplasm were found (Fig. 5). In certain areas, glandular cells charged with granules of low electrodensity coexisted with mucous material.

At ductal level the optical and ultrastructural findings were also alike, the cellular heterogeneity and the tendency to stratify being a finding in common with the studies on parotid biopsies.

The principal structural and ultrastructural similarities in alcoholic parotid biopsies and autopsies are summarised in Table 1

Table 1. Structural and Ultrastructural Similarities of the Alcoholic Parotid in Biopsies and Autopsies.

DISCUSSION AND CONCLUSIONS

The existence of an increase in size of the salivary glands of neither inflammatory nor neoplastic origin has been widely studied and discussed both in relation to its structural pathogenesis and the terminology to use: sialosis or sialodenosis. In this study we use the term sialosis to refer to this pathology, in concordance with that indicated by Marco and collaborators (12).

The finding of parotid sialosis in our alcoholic cirrhotic patients, was coherent with that described by other authors with respect to the age of appearance, 50 years of age on average, and gender distribution, being equal in both sexes (18). It has been suggested that the size of the parotidomegaly could be in proportion to the quantity of alcohol consumed (19).

The ultrastructural alterations observed at parenchymal glandular level in the biopsy material were comparable to the structural modifications previously found in the parotid glands from autopsies of chronic alcoholics (7, 8), particularly with reference to the accumulation of intracytoplasmatic granules of diverse shape and size, of different electrodensity and occupying, in general, the whole of the cytoplasm. These findings are different from those cited by Gorlin and Goldman (19), who stated that the granules are only observed in the initial phases of this pathology and postulate that the accumulation could be produced by an alteration in the membrane which inhibits exocytosis. On the other hand, Neville (3), as other authors (6, 18), suggests that the accumulation of intracytoplasmatic granules could take place through a functional deficiency in the autonomous innervation of the gland.

With respect to the existence of isolated acini of mucosal type that were found both in the preliminary and final phases of the cirrhotic hepatopathy, this coincides with that described by Rodrigo (11) in parotid biopsies, who is of the opinion that a mucosal transformation could be produced in the pure serosa gland in those patients with a diagnosis of hepatic cirrhosis. Gorlin and Goldman (19) state that in parotid biopsies with cirrhotic sialosis, the acinar cells present cytoplasm 'of almost water-like clarity', a characteristic that they consider as typical in this variety of pathology.

The finding of dilation in the Golgiano system and the accumulation of lisosomes in the acinar cells of alcoholic parotid biopsies, is compatible with the descriptions by Tirapelli (16) of animal experiments, the desmosomic unions in parallel and the enlargement of the intercellular space by way of canaliculi with interdigitations, occurring in the same fashion. The acinar cells, with evident signs of morphofunctional alteration, as well as the oncocytes in the acini and small ducts, have also been mentioned by Banderas in parotids from rats subjected to the effects of ethanol (20).

On the other hand, the initial studies of the alcoholic hepatic steatosis is characterised by the presence of lipid inclusions in the hepatocytes, where the accumulation of lipids is produced by an alteration in the metabolism of the fatty acids and the liberation of lipoproteins owing to an interference in the protein synthesis caused by the effect of alcohol. The hepatic steatosis in acute or persistent alcoholic hepatitis often coexists with cirrhosis (21). In this sense, we consider that alcohol or its metabolites could have provoked the formation of lipid inclusions in the acinar and ductal cells, observed at electronic and optical level in the alcoholic parotid biopsies, coinciding with that indicated by Rodrigo (11).

Contrary to other authors (11), who did not find alterations in the ductal system of the parotids of alcoholic cirrhotic patients, we observed modifications in the striate ducts of biopsies and autopsies, evidenced at electron and optical microscope level respectively. Furthermore, these samples are characterised principally by the presence of very enlarged excretory ducts and with stasis of secretory material.

Scott (10), in their structural studies of salivary glands of cirrhotic patients, did not detect glandular dilation or acinar hypertrophy, in spite of finding abundant adipose infiltration in the stroma. On the other hand, other authors (1, 3, 22) state that the acini of alcoholic parotids are enlarged by hypertrophy and an increase in the acinar function.

In accordance with our results, we suggest that the principal cause of the increase in glandular size could be the notable enlargement of the lumen of the ductal system and not the slight interstitial edema nor the minimal acinar hypertrophy found. Neither was there evidence of inflammatory processes that could justify the parotid hypertrophy.

The morphological modifications in the parotid of chronic alcoholics could be extrapolated to the serosal portion of the minor salivary glands, given that these show alterations to diverse degrees, including changes in the transcriptional activity of the acinar and ductal cells of those individuals who have died of alcoholic hepatic cirrhosis (7, 8,23).

These findings clearly show that:

* The structural and ultrastructural alterations at ductal and acinar level are present in the parotid glands of chronic alcoholics even before the terminal phase of alcoholic hepatic cirrhosis.

* The mucosal appearance of some areas of the parotid at ultrastructural level, could explain the tinctorial and histoche-mical heterogeneity of the samples observed by optical microscope, and could be evidence of morphofunctional alterations in the glands.

* The nuclear ultrastructural modifications in shape, size and chromatic appearance of the ductal cells could correspond to the structural changes previously observed in the parotids of individuals who have died of alcoholic cirrhosis.

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