Ultrastructural changes in human gingival fibroblasts in vitro after exposure to vapour phase smoke components

ANNALS OF ANATOMY

Ultrastructural changes in human gingival fibroblasts in vitro after exposure to vapour phase smoke components Paola Poggi, Massimo T. Rota, Maria Gabriella Cusella De Angelis, and Renata Boratto Department of Experimental Medicine, Section of Human Normal Anatomy, Faculty of Medicine, University of Pavia, Via Forlanini 8, 1-27100 Pavia, Italy

Summary. Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including gingival fibroblasts. Because normal gingival fibroblast functioning is fundamental to the maintenance of the oral connective tissue as well as to wound healing, we examined the effect of two vapour phase smoke components (acrolein and acetaldehyde) on proliferation and ultrastructure of human gingival fibroblasts (HGFs) in culture. A human gingival fibroblast strain derived from healthy individuals was used in this study. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde and cell proliferation and fine morphology were evaluated. The results show that acrolein and acetaldehyde produced dose dependent inhibition of H G F viability and alteration of cytoplasmic organelles. The main ultrastructural finding for the H G F cytoplasm was the presence of vacuoles and lysosomal structures which became prominent with increasing concentration of acrolein and acetaldehyde. Our results suggest that the ultrastructural alterations we observed in H G F s may be due to the uptake and storage of acrolein and acetaldehyde by the cells.

blast functioning is fundamental to the maintenance of the oral connective tissue as well as to wound healing responses, but during inflammatory processes which may occur with tobacco exposure (Payne et al. 1998), the substances contained in tobacco smoke may affect their survival. Most in vitro studies have described the effects of nicotine, a major component of the particulate phase of tobacco smoke, on oral fibroblasts. In fact, tobacco products affected the cell adhesion and growth (Peacock et al. 1993; Tipton et al. 1995; James et al. 1999) and they produced modifications of cytoplasmic structures according to the concentrations of the toxic substances (Chamson et al. 1980; Alpar et al. 1998). However, other substances in tobacco smoke, of which there are at least 4 000, are thought to have a toxic effect. Among these, some volatile components, like acrolein and acetaldehyde, are present in large amounts (Newsome et al. 1965; Commitee on aldehydes, 1982). In order to better define the effects of cigarette smoking on oral tissue, we studied the nature of ultrastructural modifications appearing in cultured gingival fibroblasts after exposure to various concentration of acrolein and acetaldehyde.

Key words: H u m a n gingival fibroblasts - Culture - Acrolein - Acetaldeyde - Electron microscopy

Material and methods Introduction Oral and dental integrity depends, among other things, on normal functioning of gingival epithelial and connective tissue cells such as fibroblasts. So normal gingival fibro-

Correspondence to: R Poggi

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Ann Anat (2000) 182:427-432 © Urban & FischerVerlag http:llwww.urbanfischer.deljournalslannanat

Cell cultures and treatments. Human gingival fibroblasts (HGFs) were obtained from gingival biopsies taken during routine surgical procedures on healthy subjects with non-inflamed gingiva. Procedures for growing such cell and concentration ranges of acrolein and acetaldehyde used in our study refer to the previous experiments by Nakamura et al. (1995). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and maintained at 37 °C. In order to obtain subcultures, the primary HGF cultures were released with

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0.25% trypsin, resuspended in DMEM and placed in new culture tubes at 37 °C in an atmosphere of 5% CO2 in air. On the third day from seeding in Petri dishes (cell density 50 000 per dish), subcultures were incubated in the presence of different concentrations of acrolein (10 -6, 3x10 6, 10-5, 3x10 -5, 10-4M) and acetaldehyde (10-~, 3x10 ~*, 10-3, 3x10 -3, 10-2M). Untreated subcultures were utilized as controls. After 5 days' incubation some treated and untreated subcultures were tripsynized, and centrifuged in order to evaluate the cell proliferation by a Burker's counting chamber (Jonsson et al. 1997). Electron microscopy. For transmission electron microscopic (TEM) examination, untreated and treated cell cultures were fixed in situ in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for i h at 4 °C, postfixed in 1% OsO4 collidine buffer for 1 h at 4 °C, dehydrated and embedded in epoxy resin. Semithin sections (0.5 ~tm) stained with toluidine blue were examined and photographed under the light microscope. Ultrathin sections (80 nm) contrasted with uranyl acetate and lead citrate were examined and photographed with a Zeiss EM 109.

Statistical evaluation. Statistical evaluation was performed with Student's unpaired t-test (two tailed). Comparisons were statistically significant at the p < 0.05 level. All data were expressed as mean + SD.

Results H u m a n gingival fibroblast p r o l i f e r a t i o n was e v a l u a t e d five days after exposure to acrolein and acetaldehyde. C o n c e n t r a t i o n s of 10-6M, 3 x 10 .6 M, 10-5M for acrolein and of 1 0 m M and 3 x 1 0 - 4 M for a c e t a l d e h y d e did not significantly affect cell viability. Significant inhibition of cell p r o l i f e r a t i o n was o b s e r v e d at a c o n c e n t r a t i o n of 3 x 10-5M for acrolein and of 10-3M for acetaldehyde. A t the highest c o n c e n t r a t i o n of acrolein (10 4M) and of a c e t a l d e h y d e (10 .2 M) cell p r o l i f e r a t i o n was highly inhib-

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Fig. 1. Dose related effects on H G F viability after exposure to acrolein (a) and acetaldehyde (b) for 5 days. The values were significantly different from the 100% control values (p < 0.05) at the concentrations of 3 x 10 5M for acrolein and 3 x 10-3M for acetaldehyde.

Fig. 2. Normal control fibroblasts: the cells show a normal spindle-shape and parallel alignment (a) Fibroblast treated with acetaldehyde at the dose of 3 x 10-3: the cells show oval shape and large vacuoles fill the cytoplasm. Cellular debris were also seen (b). (semithin sections stained with toluidine blue. a: x 1 000; b: x 2 000).

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Fig. 3. Normal control fibroblast: intracellular components such as mitochondria, ribosomes have normal appearance. Bundles of microfilaments are uniformly distributed throughout the cytoplasm (electron-micrograph from ultrathin sections: x 14 000).

ited. Cell death increased up to 80% of the total control cells (Fig. i a, b). Under the light microscope, normal control fibroblasts exhibited a normal spindle-shape with oval nuclei supplied with one or more nucleoli. Cell arrangement was generally uniform, with parallel alignment of elongated fibroblasts with minimal overlapping. (Fig. 2 a). At the ultrastructural level intracellular components had a normal appearance. Bundles of microfilaments were uniformly distributed throughout the cytoplasm. Ribosomes were dispersed everywhere as electron-dense dots within the cytoplasm: rosette formations of polysomes were also found. Elongated mitochondria were also abundant and most of them had an electron-dense matrix. Few vacuoles were seen within the cytoplasm. A few empty vesicles were seen among the cytoplasmic organelles (Fig. 3). Morphological changes were observed in acrolein and acetaldehyde treated fibroblasts, both substances having the same effects. At low concentrations of both acrolein (10-6M) and acetaldehyde (10-4M), cell orientation was moderately disrupted. In contrast to the generally parallel orientation seen in the controls, fibroblasts were oriented in many directions with frequent overlapping. At the ultrastructural level the presence of vacuoles and lysosomes were the main features characterizing the cytoplasm of the cells and they became more and more prominent with increasing concentrations of acrolein and acetaldehyde. At doses of acrolein of 3 x 1 0 5 M and acetaldehyde of 3 x 10-3M, cellular changes were more distinct. The cells often had a round or oval shape and large vacuoles filled the cytoplasm. Cellular debris were also seen among living cells. Nucleoli appeared more prominent and numerous (Fig. 2b). At the ultrastructural level, vacuoles

became larger and lysosomes spread progressively throughout the cytoplasm. The latter contained highly electron-dense material, lamellated membranes or tubular structures. R E R cisternae were less prominent as free ribosomes, polysomes and rosette formations increased in number (Fig. 4 a, b). No changes were seen in mitochondria which maintained their elongated shape whatever the concentration of acrolein and acetaldehyde. At the highest doses of acrolein (10-4M) and of acetaldehyde (10-2M) vacuoles and lysosomes spread throughout the cell cytoplasm that was approaching disruption (Fig. 5).

Discussion Cigarette smoke contains several chemical species (Huber et al. 1991) that affect on the development and progression of inflammatory processes of oral tissues in different ways (Martinez-Canut et al. 1991; Genco 1996). The enhanced periodontal destruction observed in smokers may be caused by a defective response on the part of oral and peripheral leucocytes (Kenney et al. 1977; Hughes et al. 1985; Lannan et al. 1992; Selby et al. 1992; Bouclin et al. 1997). However, which substances are responsible for cytotoxicity and in what doses remains undefined. It has been documented that nicotine, a particulate phase of tobacco smoke, heavily contributes to the pathogenesis of oral diseases by affecting the regenerative and reparative processes in the gingival and periodontal cells. Some in vitro studies (Raulin et al. 1988; Tipton and Dabbous 1995; Atpar et al. 1998), demonstrated that nicotine caused alterations in reproduction, attachment and conse-

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Fig. 4. Details from fibroblasts treated with acrolein at the dose of 3 x 10-5 M. a, b: Polysomes, rosette formations and some R E R cisternae spread throughout the cell cytoplasm. Residual bodies containing electron-dense material and variously shaped structures are seen (electron-micrographs from ultrathin sections: a: x 14 000; b: x 24 000).

Fig. 5. Fibroblast culture treated with acetaldehyde at the dose of 10-2M. The whole cell cytoplasm is filled by numerous and large vacuolizations, and the cytoplasmic organelles are completely disrupted (electron-micrograph from ultrathin section, x 6 000).

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quently the morphology of human gingival fibroblasts. In contrast, Peacock et al. (1993) have showed that nicotine at low concentrations can even enhance H G F attachment and reproduction. Tobacco smoke extract (CSE), on the other hand, inhibits the proliferation and migration of lung fibroblasts at non-cytotoxic concentrations, but when the volatile components were removed from CSE, it had less toxic effects on fibroblast proliferation (Nakamura et al. 1995). Because of these conflicting data, we focused our attention on acrolein and acetaldehyde, two volatile components of tobacco smoke, in order to verify their toxicity on gingival fibroblasts. Acrolein and acetaldehyde are well-known to have a dose dependent cytotoxic effect on other cells, inhibiting D N A replication, on skin fibroblasts and epithelial cells (Dypbukt et al. 1993; Grafstrom et al. 1994). We found that acrolein and acetaldehyde produced dose dependent inhibition of human gingival fibroblast proliferation, the critical doses measured at 3 x 10-5M for acrolein and 3 x 10-3 M for acetaldehyde, being similar to those evaluated as critical for lung fibroblast proliferation (Nakamura et al. 1995; Carnevali et al. 1998). These critical doses for inhibition of cell proliferation were different for the two substances: a difference that could be explained by the fact that acetaldehyde, although generated in large quantities, at an estimate 980 mg/cigarette, is a less potent toxin (Huber et al. 1991). Acrolein is generated in much smaller amounts, at 85 mg/cigarette (Huber et al. 1991), but is much more potent. Hence acrolein is much more toxic than acetaldehyde at the same concentrations. These findings are also supported by morphological evidence. Under the light microscope we have observed a dose dependent disruption of the normal orientation of the cells and an increase in the size and number of intracellular vacuoles in the H G F s after exposure to progressive concentrations of acrolein and acetaldehyde. Similar changes due to nicotine and other ingredients of tobacco smoke have also been described by several authors (Chamson et al. 1980; Raulin et al. 1988; Tipton et al. 1995). Similarly, it can also be hypothesized that the presence of vacuoles reflects the retention of secretory vesicles and impaired synthesis of proteins by the cells exposed to acrolein and acetaldehyde (Hanes et al. 1991). Possibly, the vacuolization of fibroblasts could be a response to toxic agents (Freshney et al. 1987; Raulin et al. 1988). At the ultrastructural level we also observed numerous residual bodies filled with electron dense material close to the vacuoles. This finding can be related to the storage of acrolein and acetaldehyde into lysosomes that are able to inactivate toxic substances with their enzymes, and consequently the electron dense material stored within lysosomes may indeed be a metabolic product induced by the two toxic substances (Chamson et al. 1980; Corsetti et al. 1998). Much greater lysosomal enzyme production was also confirmed by the presence of nuclei containing two or more nucleoli and free ribosomes and rosettes that were numerous with increasing concentrations of acrolein

and acetaldehyde. We can hypothesize that the cells dedicate themselves to synthetizing the endogenous proteins required to neutralize the toxic agents, so losing their capacity to produce macromolecules needed for cell proliferation (Olivares et al. 1997) and to synthetize other proteins essential for maintenance of the gingival extracellular matrix. In addition the presence of residual bodies containing concentric lamellae associated with granular material could reflect an autophagocytic response to acrolein and acetaldehyde treatment which disrupts the membrane system (Smith et al. 1991). From our in vitro studies we conclude that the volatile components of cigarette smoke, like acrolein and acetaldehyde significantly affect the proliferation and morphology of human gingival fibroblasts in a dose dependent manner. In addition, the association of both substances with cytoplasmic structures could be the result of an accumulation of high intracellular levels of acrolein and acetaldehyde which may interfere with normal cellular functions. These detrimental effects of the volatile components of tobacco smoke may also occur in vivo and thus might impair the reparative and regenerative potential of oral tissue.

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Accepted February 7, 2000

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