Helene Terzidis-Trabelsi

Jean Pierre Lefevre

Jean Bignon

Claude R Lambre

Decreased Sialidase Activity in Alveolar Macrophages of Guinea Pigs Exposed to Coal Mine Dust

The origin of immune dysfunctioiis that are observed in pneumoconiotic miners still remains unknown. There is evidence that the carbohydrate moiety of membrane glycoconjugates is of priiiiary iinportance in many functions ofünmunocompetent cells. The glycosylation, and especially the sialylation level of membrane components of various lymphocyte and macrophage subsets, vary depending on the state ofcellular differentiation and activation. Sialidases, which may regulate the ainolint of sialic acids exposed on the cell membrane, can thus be considered äs ininiunoregulatory enzymes. In this report, the sialidase activity has been measured in alveolar macrophages (AM) and in cell-free bronchoalveolar lavage fluid (BALF) from guinea pigs exposed for 4 months to coal mine dust at a concentration of 300 mg/m . The samples were collected by bronchoalveolar lavage 2 months after cessation of exposure. The sialidase activity in the cell-free fluid and in the purified alveolar macrophages showed a 10-fold decrease (p<ft001). Kinetic parameters ofthe enzyme such äs ä" and Optimum pH did not change. This changed activity was specific for sialidase, äs two other lysosomal glycosidases, iß-galactosidase and W-acetylglueosaminidase, showed unchanged activities. These results suggest the possibility that, by inducing a decreased sialidase activity, exposure to coal mine dust may lead to a modified expression ofAM membraneassociated sialic acids giving rise to altered immune functions (i. e., phagocytosis, antigen processing, response to cytokines, ete.).

Introduction

Pneumoconiosis, a chronic fibrotic reaction in the hing, is caused by the inhalation of dust particies contaüüng silica [START_REF] Harington | Mineral fibers: chemical physicochemical and biological properties[END_REF][START_REF] Über | Immunotoxicology ofsilica[END_REF]. In experimental modeis of chronic exposure to mineral dust, it has been shown that alveolar macrophages (AM) play a critical role in the development and persistence ofpulmonary inflammation [START_REF] Bowden | Macrophages, dust, and pulmonary diseases[END_REF]. This inflammation is thought to be partly due to lysosomal enzymes released from lysed AM. Particulate silica ingested by AM has the potential to damage the integrity ofcell membranes, and studies have suggested that lysosomal enzymes are discharged into the cytoplasm after phagolysosomal membrane disruption [START_REF] Allison | An examination ofthe cytotoxiceffectsofsilicaonmacrophages[END_REF][START_REF] Allison | Pötentiation by silica of the growth of Mycobacterium tuberculosis in macrophage cultures[END_REF][START_REF] Heppleston | Pulmonary toxicology ofsilica, coal and asbestos. Environ[END_REF]. The mechanisms by which particies are recognized and ingested by AM remain unclear. Evidence suggests that particies bind to cells through electrostatic forces [START_REF] Harington | Mineral fibers: chemical physicochemical and biological properties[END_REF]. In that context, due to their exposed position on the cell membrane and their negative Charge, sialic acid (SA) residues have been considered important. For instance, sialic acid has the capacity to mediate the initial binding ofpositively charged particies in vitro [START_REF] Gallagher | Sialicacidmediates the initial binding ofpositively charged inorganic particies to alveolar macrophage membranes[END_REF]; also, it is implicated in the interacrions ofcoal fly ash with mammalian cells [START_REF] Hahon | Effects oflignite fly ash particulates and solublecomponentson the interferon System[END_REF]. Furthermore, sialic acid residures are important in modulating the biochemical properties ofmany membrane glycoconjugates [START_REF] Schauer | Sialic acids and their role äs biological masks[END_REF], expressed by immunocompetent cells (II). The sialylation of membrane sialoglycoconjugates is regulated by two Idnds of enzymes, namely, sialyltransferases and sialidases. Factors that modify or alter sialidase activity can influence a diversity of physiological phenomena within cells, on cell surfaces, and in circulating body fluids (72). Sialidases (E.C.3.2.1.18; acylneuraminyl hydrolases) catalyze the hydrolysis of sialic acid residues a-ketosidically bound to oligosaccharides, glycoproteins, and glycolipids. These enzymes are widely distributed in eukaryotic and prokaryotic cells (13}. A large body of evidence suggests that, in eukaryote, the total sialidase activity is actually a composite ofat least three types of activities differing from each other both in their subsü-ate specificity and in their localization within the cell. After tissue homogenization and differential centrifugation, sialidase activities have been demonstrated in the lysosomal, cytosolic, and membrane subcellular fractions; the lysosome-associated form being the predominant one [START_REF] Corfield | The Substrate specificity ofsialidases from microorganisms and mammals[END_REF].

In a rabbit model, lysosomal enzymes such äs ß-glucuronidase or acid phosphatases are released in the bronchoalveolar lavage fluid (BALF) afier intratracheal mjection ofquartz [START_REF] Stankus | Bronchopulmonary humoral and cellular enhancementinexperimentalsilicosis[END_REF]. Because AM possess lysosomal sialidase activity (75) and because this enzyme may participate in the control of the degree of sialylation ofcell surface glycoconjugates resulting in changed biological properties ofthe cells [START_REF] Landolfi | Activation ofT lymphocytes results in an increase in H-2-encoded neuraminidase[END_REF][START_REF] Landolfi | Activated T-lymphocytes express class I molecules which are hyposialylated compared to other lymphocyte populations[END_REF], we investigated the effect ofcoal mine dust exposure on the sialidase activity in AM and in BALF ofguineapigs.

Materials and Methods

Animals and Dust Exposure

Two-month-old, noninbred, male Hartley guinea pigs (300 g) were divided into a control nontreated group (n = 6) and a coal mine dust-exposed group (n = B). The coal mine dust was frorn Ostricourt (France). The size distribution of the particies äs determined by electron microscopy was 3 p.m< 2.21% < 4.5/un, 2 /xm<6.94% <3 /un, l (aa <25.87% <2 /mi, and 64.98% < l {an. Guinea pigs were housed in a stainless-steel exposure chamber [START_REF] Lebouffant | Etüde quantitative de 1'epurationpulmonairechezle rat. Comparaisonentrepoussieres inertes et poussieresnocives[END_REF]. The controlled dust generators operated 6 hr/day, 5 days/week, for 4 months. The dust concentration was adjusted to 300 mg/m 3 ofrespirable dust. The animals were sacrificed 2 months after the end of exposure.

Bronchoalveolar Lavage

Guinea pigs were sacrificed by IP mjection ofa lethal dose of sodium pentobarbital. The ehest was opened and the blood was collected by cardiac puncture. The trachea was intubated by tracheotomy, and bronchoalveolar lavage (BAL) was then performed with a total lavage volume of 100 mL warmphosphatebuffered saline Dulbecco buffer pH 7.2 (without Ca or Mg 2 ^. Total cell count was performed with a Malassez hemocytometer, and differential cell counts were established on smears stained with May-Grunwald Giemsa (at least 200 cells were counted.)

The BAL was centrifüged at 300g for 10 min at 4°C. The BALF supernatant was concentrated 50-fold by filtration on a Diaflo PM 10 membrane (Amicon) at 4°C and then frozen and stored in 0.4-mL aliquots at -80°C. The cell pellet was resuspended in Hanks'saline solution ata concentration of 0.5 X lO'cells/mL, and 20 mL of the Suspension was transferred into 110-mm diameter plastic petri dishes. After a 2-hr incubation at 37°C in a 5 % CO^ humid atmosphere, nonadherent cells were removed and the petri dishes were washed twice with Hanks' saline solution. Adherent cells were harvested with a Teflon scraper, resuspended at a concentration of 10 7 cells/mL in 3 mM imidazol buffer, pH 7.2, containing 250 mM sucrose and l mM ethylenediamine tetraacetate (EDTA). The cells were pelleted by centrifugation at 10,000g for 5 min at 4°C and immediately frozen at -80°C.

Enzymatic Assays

Alveolar macrophages were homogenized using a glass Teflon homogenizer with 20 strokes and assays were carried out immediately. The sialidase activity was assayed by using a fluorogenic Substrate: sodium 4-methylumbelliferyl-Q:-D-neuraminate (Mu-Neu Ac) (79): 20 /xL of cell homogenate or BALF was mixed with 2.5 p,L of2.5 X 10' 3 M Mu-Neu Ac and 2.5 JUL of l M acetic acid/sodium acetate buffer ranging in pH from 4 to 6 and incubated for 2 hr at 37°C. The reaction was stopped with 2.1 mL of 0. l M NaOH. Fluorescence was measured on a Kontron SFM 25 fluorometer (exdtation at 365 nm, emission at 450 nm). Pure methylumbelliferone was used äs a Standard. All assays were done in duplicate. One enzymatic unit was defmed äs the amount of enzyme that hydrolyzes l nmole of Mu-Neu Ac per hourat37°C.

^v-acetylglucosaminidase and jS-galactosidase activities were assayed at their respective Optimum pH using appropriate Substrate [START_REF] Conchie | Mammalian glycosidases distribution in the body[END_REF]. Protein concentration was determined using Bradford's method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities ofprotein utilizing the principle ofprotein-dye binding[END_REF] with bovine serum albumin äs a Standard.

Statistical Evaluation

All the results are expressed äs the mean ± SD. Statistical significance was determined by the Student's f-test.

Results

The number of bronchoalveolar cells obtained by lavage from the dust-exposed animals was significantly higher man from the controls(Fig. 1),40.07 ± D.6 and 13.10 ±6.3 X 10" cells, respectively. Differential cell counts showed that the increased total cell number in dust-exposed animals was not attributable to a single cell type. In normal control guinea pigs, 77.6 ± 5.7% (Fig. 2) of the lavaged cells were alveolar macrophages, and 16.2 ± 5.0% were eosinophils; polymorphonuclear neutrophils (PMN) and lymphocytes each represented less man 3 %. In dust-exposed animals, the relative proportions of PMN increased (5.6 ± 3.6%), whereas those ofthe other cell types were unchanged. On smears stained with May-Grunwald Giemsa, more multinucleated giant cells were observed in dust-exposed animals (2%) than incontrols (< 0.5%). Six months after the beginning of exposure, the protein concentration in BALF was significantly (p< 0.005) increased: 1.65 ± 0.32 and 2.71 ± 1.03 mg/mL BALF from control and exposed animals, respectively.

Sialidase Activity in Guinea Pig Alveolar Macrophages

Sialidase activity, with an Optimum pH at 4.6, was detected in the crude homogenate of control guinea pig alveolar macro- phages (Fig. 3). The mean specific activity was 21.60 ± B. 49 U/mg protein. The apparent Km value for Mu-Neu Ac at pH 4.6 was 1.08 X 10' 4 ± 0.17 x lO^M. The effectof EDTA and of several divalent cations (Zn 3 ranging from 0.5 to 10 x ,Ca , Cu ) at concentrations M using their respective Chlorides (Fig. 4). Cu^ and Zn"" caused a marked inhibition of the sialidase activity. At a 10 mM concentration, Cu^ was able to inhibit about 60 % ofthe initial activity. On the contrary, at low concentration (0.5 mM), Ca 24 ^ and EDTA slightly activated the sialidase activity.

10-3

2+

Sialidase Activity in Alveolar Macrophages from Dust-Exposed Animals

To determine ifany quantitative and/or qualitative changes in sialidase content occurred after coal mine dust exposure, the sialidase activity was assayed in AM from 13 dust-exposed guinea pigs. The results obtained are summarized in Figure 5. Control and exposed animals formed two distinct groups, the specific AM sialidase activity being significantly (p< 0.025) decreased in all dust-exposed animals. This drop in sialidase activity reachedupto 90% ofthe initial activity: 2.35 ± 1.47U/mg protein and 21.6 ± 13.49 U/mg protein in the exposed and control group, respectively. Although the sialidase activity decreased in the dust-exposed group, kinetic parameters such äs K( 0.51 ± 0.18 x 10' 4 M) or the Optimum pH at 4.6 ofthe enzyme did not change significantly.

The major sialidase activity is known to be associated with lysosomes. One can assess that the decreased sialidase activity was specific for this enzyme because the two other lysosomal glycosidase activities that were assayed, namely, jS-galactosidase and A^-acetylglucosammidase, were not significantly modified.

Sialidase Activity in BALF

In BALF from normal animals, a sialidase activity with an Optimum pH at 4.6 was detected. A BALF sialidase activity with the same Optimum pH was detected in 12 out ofthe 13 exposed animals. The major change in dust-exposed guinea pigs was a decrease in the total BALF sialidase activity (Fig. 6

Discussion

As assessed by a 3-fold increase in the number of cells harvested by bronchoalveolar lavage, the initial response ofthe lung in guinea pigs that had been exposed to coal mine dust was characterized by an acute inflammatory reaction. As a single intratracheal injection ofpure silica ledto the same observation in guinea pigs [START_REF] Dauber | Pulmonary fibrosis: bronchoalveolar cell types and impaired ninction of alveolar macrophages in experimental silicosis[END_REF], silica in coal mine dust is likely to be responsible for this reaction. The local cellular recruitment could be due to the secretion of chemoattractants by dust-loaded AM [START_REF] Miller | Evidence ofa quartz-induced chemotactic factor for guinea pig alveolar macrophages[END_REF][START_REF] Dauber | Experimental silicosis. Morphologie and biochemical abnormalities produced by intratracheal instillation of quartz into guinea pig lungs[END_REF]. The relative proportion ofAM in the bronchoalveolar cell population of dust-exposed animals was similar to that seen in controls. As compared to controls, more PMN were present in BAL from dust-exposed animals. In addition, the fraction of PMN was slightly but significantly increased. PMN seem to play a critical role in early cellular events occurring in several types ofexperimental modeis ofpneumoconiosis [START_REF] Adamson | Role of polymorphonuclear leukocytes in silica-induced pulmonary fibrosis[END_REF][START_REF] Lugano | Acute experimental silicosis. Lung morphology, histology, and macrophage chemotaxin secretion[END_REF]. Acontroversy still exists conceming the precise role ofPMN during pulmonary diseases. Depending on the experimental model, both protective and causal [START_REF] Reiser | Early cellular events in pulmonary fibrosis[END_REF] roles have been attributed to PMN in the development of pulmonary interstitial diseases.

The cellular inflammation was associated with a rise in the BALF protein concentration. In another experimental model where hamsters were exposed to pure silica, a 7-fold increase in the BALF protein concentration was demonstrated [START_REF] Demendez | Peroxidase activities in the hamster bronchoalveolar lining fluid: modifications induced by exposure to silica dust[END_REF]. Proteins appearing in the airways may originale from various sources including serum exudation, cell lysis (29), local protein synthesis [START_REF] Stankus | Bronchopulmonary humoral and cellular enhancementinexperimentalsilicosis[END_REF], and enzyme secretion. For example, increased lysosomal enzyme activities were found in BAL from mice injected intratracheally with silica [START_REF] Adamson | Role of polymorphonuclear leukocytes in silica-induced pulmonary fibrosis[END_REF] and in BAL from silica-exposed workers [START_REF] Larivee | Enzyme activities of lung lavage in silicosis[END_REF].

In AM-crude homogenate from control guinea pigs, the sialidase activity was Optimum at pH 4.6. Similar observations were reported for rabbit AM [START_REF] Pilatte | Lysosomal and cytosolic sialidases in rabbit alveolar macrophages: demonstration of increased lysosomal activity after in vivo activation with bacillus Calmette-Guerin[END_REF][START_REF] Eisen | Acid-active neuraminidases in the growth media from cultures of pathogenic Naegleria ßowleri and in sonicates of rabbit alveolar macrophages[END_REF]. The limited number of cells recovered by bronchoalveolar lavage did not allow us to study the subcellular distribution of this activity. Previous reports have provided evidence that the bulk of sialidase activity displays acidic Optimum pH and is associated with the fraction enriched in lysosomes [START_REF] Pilatte | Lysosomal and cytosolic sialidases in rabbit alveolar macrophages: demonstration of increased lysosomal activity after in vivo activation with bacillus Calmette-Guerin[END_REF][START_REF] Nguyen Hong | Studies on the sialidoses. Properties of human leucocyte neuraminidases[END_REF][START_REF] Verheigen | Human placental neuraminidase. Activation, stabilization and association with ßgalactosidase and itsprotectiveprotein[END_REF][START_REF] Lambre | Modifications of sialidase activity during the monocyte-macrophage differentiation in vitro[END_REF]. A cytosolic sialidase with an Optimum activity at pH 5.4 (75) and a sialidase associated with the plasma membrane, active at pH 4.5 [START_REF] Sagawa | Effects of OK-432 activation on the sialidase activities ofrat peritoneal macrophages[END_REF], have also been described. As already shown [START_REF] Michalski | Properties of human liver lysosomal sialidase[END_REF], we observed that divalent cations such äs Ca 24 ^ on the one hand, and Cu^ and Zn 24 ^ on the other hand, have opposite effects on the sialidase activity.

Sialidase activity is present in normal guinea pig BALF [START_REF] Briend-Sutren | Glycosidase activities in alveolar macrophages from guinea pigs stimulated with a glyco-proteic extract from Klebsiellapneumoniae[END_REF], whereas in humans this enzyme is only present in significant amounts in BALF from patients with interstitial lung diseases such äs idiopathic pulmonary fibrosis or sarcoidosis [START_REF] Lambre | Sialidase activity and antibodies to sialidase-treated autologous erythrocytes in bronchoalveolar lavages from patients with idiopathic pulmonary fibrosis or sarcoidosis[END_REF]. Nevertheless, to date, the cellular origin ofthis enzyme has not been definitively demonstrated. However, because AM are numerous in bronchoalveolar spaces and because sialidase activities ofAM and BALF have the same Optimum pH, one can speculate that BALF sialidase represents an activity released from AM.

In dust-exposed animals, sialidase activities decreased both in AM and in BALF; however, kinetic parameters such äs K^ and Optimum pH were not modified. Because particulate silica has been shown to alter the cellular metabolism, the decreased BALF sialidase activity may result from a decreased synthesis and/or liberation of cellular sialidase into the respiratory secretions. In the absence ofcell lysis, the decreased BALF sialidase activity can also be the consequence ofan enzymatic inhibition or inactivation. Numerous mechanisms including inactivationby proteases [START_REF] Miyagi | Rat-liver lysosomal sialidase. Solubilization, Substrate specificity and comparison with the cytosolic sialidase[END_REF] and inhibition by cations [START_REF] Michalski | Properties of human liver lysosomal sialidase[END_REF] may result in a decreased catalytic function of sialidase in BALF from dustexposed animals.

Macrophage sialidase activity can be up-or downregulated. Treatment ofrat peritoneal macrophages with inactivated Streptococcus pyogenes fragments resulted in a 9-fold increase in sialidase activity [START_REF] Sagawa | Effects of OK-432 activation on the sialidase activities ofrat peritoneal macrophages[END_REF]. In vivo bacille Calmette-Guerin (BCG) Stimulation of rabbits induced an upregulation ofAM lysosomal sialidase (75), and a decreased sialidase activity was found in AM from guinea pigs treated with a glycoproteic extract from Klebsiella pneumoniae [START_REF] Briend-Sutren | Glycosidase activities in alveolar macrophages from guinea pigs stimulated with a glyco-proteic extract from Klebsiellapneumoniae[END_REF]. So far, the precise mechanism of sialidase regulation and its direct effectiveness on the degree of sialylation of sialoglycoconjugates is not fully understood. Modifications of sialidase content with a concurrent decline in membrane-bound sialic acid have been reported. For instance, T-cell activation is accompanied by an increase in the endogenous level of sialidase activity and an hyposialylation of class I major histocompatibility complex antigen molecules [START_REF] Landolfi | Activation ofT lymphocytes results in an increase in H-2-encoded neuraminidase[END_REF][START_REF] Landolfi | Activated T-lymphocytes express class I molecules which are hyposialylated compared to other lymphocyte populations[END_REF]. Previous desialylation of the target cells improves recognition and ingestion by macrophages [START_REF] Jibril | Interaction of rat peritoneal macrophages with homologous, sialidase-treated lymphocytes in vitro[END_REF]. These results point out the importance of membrane sialic acids in cellular differentiation, activation, and biological properties including phagocytosis. It is therefore conceivable that a sialidase-mediated differential expression of sialic acids on the AM cell membrane may be a mechanism through which impaired cellular fünctions may participate in the development ofpneumoconiosis. A study ofthe relative sialylation ofAM membrane sialoglycoconjugates from either normal control or dust-exposed guinea pigs should help to answer this question.

FIGURE 2 .

 2 FIGURE l. Total cell count in bronchoalveolar lavage fluid from control(n = 6) and dust-exposed (n = 13) guinea pigs. The results are expressed äs means ± SD. (*) Difference between dust-exposed and control animals is significant, p < 0.05; (**) difference between dust-exposed and control animals is highiy significant, p < 0.005.

FIGURE 3 .FIGURE 4 .

 34 FIGURE 3. Effect of pH on alveolar macrophage sialidase activities prepared from control (• ) and from dust-exposed guinea-pigs (0).

FIGURE 5 .

 5 FIGURE 5. Sialidase activity in alveolar macrophages from control (• ) and dust-exposed animals (0). The activity is expressed äs enymatic units per milligram of protein.

This work was supported by a grant from the Groupe d'Etudes et de Recherches sur les Pneumoconioses (GERP). The authors also thank P. Sebastien for his help and interest in this work.


ROAD TRANSPORTATION OF DANGEROUS GOODS QUANTITATIVE RISK ASSESSMENT AND ROUTE COMPARISON

Transportation of Dangerous Goods (TDG) by road creates risk for the people present on and along the routes (road users and surrounding population). To minimize them is it

better to have TDG going through a city or on the contrary by a longer detour through less populated areas ? The choice is not always easy. It may become very difficult when the detour goes through tunnels. There, accidents may have developments and consequences very different from what might happen on an open air route. To make the right choice it becomes necessary to use a QRA (Quantitative Risk Assessment) approach dealing with the accident scenarios likely to occur, their probability and possible consequences. It may be useful to identify the DG (Dangerous Goods) classes which contribute most to the risk and examine the interest of non-uniform strategies i.e. different routing for certain DG.

Studies for real sites

INERIS has determined the risks due to TDG for real sites where traffic could go either through a city or an alternative route including a tunnel. Lengths of the tunnels ranked between 2 and 9 km. To perform these studies, INERIS has devised and used a method for determining curves of yearly frequency against number of fatalities corresponding to the risks due to TDG on each route. These curves are hereafter mentioned as « F/N curves ». This method is very flexible and may be used in very various configurations.

To practically use it, it is necessary to gather, during a first stage, data concerning :

• density of population in the concerned geographical area (possibility to take into account diurnal or seasonal variations),

• traffic of all vehicles and foreseeable routes (possibility to take into account diurnal or seasonal variations),

• traffic of DG : nature of contents and holders, global annual traffic, foreseeable evolutions,

• meteorology in the concerned geographical area,

• layout of the open-air routes (which are divided in « segments » with constant probability of occurrence of each scenario),

• if it applies, dispositions taken for the design and equipment of the tunnels.

Generally around ten scenarios are chosen (table 1). Five of them are relative to LPGs (propane has been chosen as a representative DG) because of the different physical phenomena they can generate. Their physical consequences (overpressure, heat flux, toxic concentrations for different meteorological conditions) have been calculated and entered in a data-base.

Nature of scenario

Minimizing risk between several alternative routes

In case where DG may be transported by different routes, the method enables to determine the risk that would result from TDG for each route if it were the only one chosen for the DG traffic (DG traffic forbiden on the other routes). Interpretation of results relies on comparing the various F/N curves pertaining to the routes or on comparing the various associated « mathematical hopes » (Each F/N curve gives a mathematical hope which represents the area located under the F/N curve. It indicates the yearly number of fatalities when the accidents of every severity are summed up and is expressed in fatalities per year).

This comparison leads to a certain conclusion in the case of F/N curves that do not intersect (as shown on figure F1).

It is also possible to get intersecting curves, but with significantly different mathematical hopes (as shown on figure F2), and this even when uncertainties are taken into account. Once more, a conclusion is at hand.

When F/N curves intersect and lead to close mathematical hopes, the method does not help in the decision process except give indication that the comparison of risks linked with DG is not discriminating and decision might be made on other criteria.

Judging of the acceptability of risk on a route.

It is possible to judge of the acceptability of the risks due to TDG on a route by comparing the F/N curve with acceptance criteria in case such criteria are available and accepted (Figure 3 shows a fictitious use of such criteria).

Societal risk may also be judged on a local basis using iso-contours like those appearing in Figure 4 . These contours indicate for each area the statistical probability level to get fatalities. These probabilities take into account the population densities and their eventual daily and seasonal variations.

It is also possible to compare individual risk for the surrounding populations with individual risk acceptance criteria.

Conclusion

QRA of risks due to TDG helps in decision making when routes have to be chosen for DG transit and one wants to minimize resulting risks. It also enables to situate the risks due to TDG against other kind of risks (fatalities due to banal traffic accidents i.e. no 5 DG involved for example). It requires complex studies. A method and practical tools have been produced and used for real site cases.

Nevertheless, further work has still to be carried out in order to make these evaluations easier, more accurate and indicate the magnitude of the uncertainties.

List of figures :

Figure

  Figure F1 Example of non intersecting F/N curves.

Figure

  Figure F2 Example of intersecting F/N curves with different mathematical hopes.

Figure 3

 3 Figure 3 Example of comparison of a F/N curve with an acceptance criterion.

Figure 4

 4 Figure 4 Iso-contours of societal risk due to dangerous goods traffic on an open air route (taking into account the population layout).

FigFigure 3 :

 3 Fig F1 -Example of non intersecting F/N curves

Table 1 -

 1 List of scenarios explored for QRA.

	1	Explosion of a lorry transporting 15 tons of explosives
	2	BLEVE (Boiling Liquid Expanding Vapour Explosion) of a 24 tons vessel of
		LPG
	3	VCE (vapor cloud explosion) after a release through a hole of 5 cm² on a 24 tons
		vessel of LPG
	4	VCE after a release through a hole of 100 cm² on a 24 tons vessel of LPG
	5	Jet fire after a release through a hole of 5 cm² on a 24 tons vessel of LPG
	6	Jet fire after a release through a hole of 100 cm² on a 24 tons vessel of LPG
	7	VCE after a release through a hole of 80 cm² on a 30 000 litres vessel of gasoline
	8	Pool fire after a release through a hole of 80 cm² on a 30 000 litres vessel of
		gasoline.
	9	NH3 dispersion after a release through a hole of 50 cm² on a 24 tons vessel of
		ammonia
	10	Fire of lorry transporting 20 tons of NPK fertilizer

Determination of the occurrence probabilities and effects

For each scenario, the zones of effects are determined. For each scenario and each segment of a route, the occurrence probability is determined. These determinations have been backed up by a review of DG accidents occurred during transportation or in industry.

Two numerical tools have been designed to make possible a practical use of the method.


S Aït-Aïssa

J-M Porcher
email: jean-marc.porcher@ineris.fr

C Kretz-Remy

G Velarde

A-P Arrigo

C Lambre

HSP70 induction by anticancer drugs 1 INDUCTION OF THE HSP70 GENE PROMOTER BY VARIOUS ANTICANCER DRUGS

Keywords: hsp70 promoter, anticancer drug, HeLa cells

HeLa cells containing the chloramphenicol acetyl transferase (CAT) gene under the control of the hsp70 promoter have been exposed in vitro to various anticancer drugs. Cisplatin induced CAT production with a dose-effect relationship at a non-cytotoxic dose, whereas no induction was detected with carboplatin. Etoposid induced a significant response at a cytotoxic concentration. The limited positive response with doxorubicin, daunomycin and mitoxantrone was not statistically significant. These chemicals are known to produce reactive oxygen species and induce apoptosis. No induction of the hsp70 promoter could be detected with the other cytostatic compounds that have been tested such as base analogs (5-fluorouracil, cytosine arabinoside 3'-MP), inhibitors of DNA synthesis (amethopterin, aminopterin), antimitotics (vinblastine, colchicine), and alkylating (streptozotocine, carboplatin, melphalan) or intercalating agents (bleomycin). In addition, the role of the transcription inhibitory activity of doxorubicin in this model is evidenced and the consequent question of the suitability of the reporter gene system is discussed. Our results suggest that specific genotoxic compounds are not able to induce the hsp70 promoter, and are in agreement with the concept that stimulation of HSP70 synthesis occurs through a biochemical process involving proteotoxicity.

INTRODUCTION

HSP70 induction by anticancer drugs 2 A wide range of physical, chemical and biological agents are known to induce a set of cellular stress proteins, the heat-shock proteins (HSP). This occurs through a mechanism of toxicity involving the generation of denatured proteins by the stressors [START_REF] Hightower | Heat shock, stress proteins, chaperones, and proteotoxicity[END_REF] and resulting in the binding of a heat shock factor (HSF) to specific DNA sequences called heat shock elements.

Among the different stress proteins, the HSP70 family is the most abundant and conserved. It has frequently been proposed as a biomarker of cellular toxicity [START_REF] Fishbach | Induction of the human growth hormone gene placed under human hsp 70 promoter control in mouse cells: a quantitative indicator of metal toxicity[END_REF][START_REF] Ryan | Stress proteins as molecular biomarkers for environmental toxicology[END_REF].

Anticancer drugs represent a growing threat in environmental health as reported by occupational exposure monitoring [START_REF] Bos | -fluoro--alanine in urine of workers occupationally exposed to 5-fluorouracil in a 5-fluorouracil-producing factory[END_REF] and sewage water contamination by hospital effluents studies [START_REF] Kümmerer | Hospital effluents as a source for platinum in the environment[END_REF]. Many studies showed expression of stress proteins in response to such agents [START_REF] Bielka | Induction of the small stress protein, hsp25, in Ehrlich ascites carcinoma cells by anticancer drugs[END_REF][START_REF] Ciocca | Biological and clinical implications of heat shock protein 27000 (Hsp27): a review[END_REF][START_REF] Matsumoto | Suppression of heat-induced HSF activation by cDDP in human glioblastoma cells[END_REF]. While in most cases the small HSPs (HSP27) rather than HSP70 are induced in response to these compounds, high intracellular levels of both HSPs induced by heat shock or constitutively overexpressed, are closely related to cellular resistance development against these drugs [START_REF] Ciocca | Biological and clinical implications of heat shock protein 27000 (Hsp27): a review[END_REF][START_REF] Garrido | HSP27 as a mediator of confluence-dependent resistence to cell death induced by anticancer drugs[END_REF][START_REF] Roigas | Effects of combined treatment of chemotherapeutics and hyperthermia on survival and the regulation of heat shock poteins in Dunning R3327 prostate carcinoma cells[END_REF], hence posing problems in cancer therapy.

In this study, we apprehended the use of an established transformed HeLa cell line, obtained by transfection with a plasmid construct carrying the human hsp70 promoter linked to the chloramphenicol acetyl transferase (CAT) gene [START_REF] Kretz-Remy | The kinetics of HIV-1 long terminal repeat transcriptional activation resemble those of hsp70 promoter in heat-shock treated HeLa cells[END_REF], as an in vitro toxicity test to screen various molecules widely used in cancer chemotherapy. The relationship between induction and the mechanism of toxic action of the substances is discussed.

MATERIALS AND METHODS

Chemicals. All chemicals were from Sigma (France) except carboplatin (Aldrich, France) and bleomycin sulfate (R. Bellon, Rhône Poulenc, France).

Cell cultures. Transformed HeLa cells were routinely cultured at 37°C in complete DMEM supplemented with 10% heat inactivated foetal bovine serum (Gibco, France) and 1% antibiotics in a humidified atmosphere containing 5% CO 2 .

Induction of the hsp70 promoter. For the induction experiment, cells were seeded onto 24-well plates (Costar) in 1ml/well of complete prewarmed medium and allowed to grow for 3 or 4 days until they reached 80% confluency. At this stage, various dilutions of each toxicant, made in the solvent (DMSO or saline), were added to the culture medium, 24 hr after the last medium change.

Cells were exposed for 16 hr at 37°C in the presence of serum. Control wells received the same concentration of solvent alone (never exceeding 0.5%). At the end of exposure, cells were washed twice with phosphate buffer saline, lysed and total cellular proteins were analysed using a CAT ELISA kit (Boehringer) according to the manufacturer's instructions. In each experiment, heat treatment (43°C, 1 hr), followed by a 24 hr recovery period, was used as a positive control.

Results are expressed as the ratio between the amount of CAT detected and the total cellular protein content assessed by an adaptation of the Lowry's method (BioRad DC Protein Assay kit).

Background level in control cultures varied from 0 to 20 pgCAT/mg protein, corresponding to the range of sensitivity of the assay.

MTT assay. Cytotoxic concentrations were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction test. Cells were grown in 96-well microtiter plates until subconfluence and then exposed to toxicants as describe above. After chemical exposure, the monolayers were washed and incubated with 100µl of 0.5 mg/ml MTT dissolved in RPMI medium for 3 hr at 37°C, 5% CO 2 . MTT was removed and the formazen salt was solubilized, with mixing, in 100µl of isopropanol. Plates were read at 570 nm on a microtiter reader (BioTek Instruments).

RESULTS

Induction of the hsp70 promoter

In order to set the concentrations to be tested in hsp induction experiments, cytotoxicity of the substances was evaluated in preliminary experiments by using the MTT reduction test. Since the test is carried out on subconfluent cells, cytotoxicity can be the reflect of either growth inhibition or direct cell killing or both. For the HSP experiments, serial dilutions were made including cytotoxic and non cytotoxic concentrations determined by the MTT test. The effect of these concentrations on cell viability (cytotoxicity) was further confirmed by a total protein assay performed at the end of the HSP induction experiments.

The tested toxicants showed different effects on the inducibility of the hsp70 promoter (table 1).

Cisplatin induced the promoter significantly (p<0.05) at a concentration (10 µM) which had no significant effect on the cellular viability as measured by the total protein content and MTT assay (figure 1). Etoposid induced a significant response (p<0.05) at a cytotoxic concentration (400µM). A limited positive response was observed with doxorubicin, daunomycin and mitoxantrone but this appeared non statistically significant. No induction of CAT production was detected after exposure to 5-fluorouracil, cytosine arabinoside, amethopterin, aminopterin, colchicine, streptozotocine, bleomycin, carboplatin, melphalan and vinblastine. The preliminary MTT tests revealed that the first five compounds were the most cytotoxic, achieving 100% cell death at the highest concentrations tested (data not shown). Such a cytotoxicity was never obtained with the other substances at the tested concentrations (up to 10 mM for some toxicants).

Case of inhibitors of transcription

In order to test wether protein synthesis inhibition by some toxicants could mask the detection of CAT production, cells were exposed to a heat shock (43°C for 1 hr) in the presence of various concentrations of doxorubicine (known as a transcription inhibitor) or streptozotocin (alkylating agent, not suspected to inhibit specifically gene expression). Lysis of cells occurred five hours after the heat shock to minimise the toxic effect of the substances. The results (figure 2) showed an inhibition of CAT production by doxorubicin while streptozotocine seemed to have no effect on hsp70 promoter expression after heat shock. Hence, we supposed that the weakness of the observed induction with toxicants like doxorubicin, daunomycin or mitoxantrone (table 1), could be explained by an inhibitory effect on gene transcription by these toxicants. Supplementary experiments (figure 3) including recovery periods of 6 and 24 hours after the exposure time showed a stronger CAT production by doxorubicin and thus confirmed our hypothesis.

DISCUSSION

Only a few data are available concerning HSP70 induction by cytostatics in comparison to HSP25, which seems to play a major role in cellular response to such drugs. For instance, bleomycin has been previously shown to induce the hsp70 promoter up to twice the control in hsp70--galactosidase transfected fibroblasts [START_REF] Moseley | Bleomycin induces the hsp 70 heat shock promoter in cultured cells[END_REF] whereas streptozotocine did not induce any HSP synthesis in murine splenocytes in vitro [START_REF] Albers | Stress proteins (HSP) and chemical-induced autoimmunity[END_REF]. [START_REF] Bielka | Induction of the small stress protein, hsp25, in Ehrlich ascites carcinoma cells by anticancer drugs[END_REF] showed that HSP25, but not HSP70, was induced by cisplatin, doxorubicin, daunomycin, cytosine arabinoside and colchicine at concentrations with 100% cytostatic effect on Ehrlich ascites carcinoma cells. Aminopterin, 5-fluorouracil and amethopterin did not induce either stress protein synthesis. In contrast, 5-fluorouracil, adriamycin (doxorubicin) and cisplatin enhanced the expression of the Drosophila hsp70 promoter stimulated by heat shock in prostate carcinoma cells [START_REF] Roigas | Effects of combined treatment of chemotherapeutics and hyperthermia on survival and the regulation of heat shock poteins in Dunning R3327 prostate carcinoma cells[END_REF]. Cisplatin also stimulated HSP70 synthesis in human glioblastoma cells [START_REF] Matsumoto | Suppression of heat-induced HSF activation by cDDP in human glioblastoma cells[END_REF] whereas in HeLa cells it provoked the migration of the protein to the nucleus without any enhancement of its synthesis [START_REF] Melendez-Zajgla | Subcellular redistribution of HSP72 protein during cisplatin-induced apoptosis in Hela cells[END_REF].

To some extent, our results are correlated with these observations. Among the fifteen anticancer drugs that we have tested, only cisplatin, doxorubicin, daunomycin, mitoxantrone and etoposid were able to induce an activation of the hsp70 promoter (table 1, figure 1). Interestingly, MTT assays revealed that these compounds were the most cytotoxic after a 16 hour exposure (data not shown). In addition to their genotoxic effect by producing DNA damage, such as DNA adducts or DNA intra-and interstrand crosslinks, these compounds have various direct biochemical effects like oxidative action, interaction with cellular proteins or apoptosis induction [START_REF] Calabresi | Chemotherapy of neoplastic diseases[END_REF][START_REF] Chu | Cellular response to cisplatin. The roles of DNA-binding proteins and DNA repair[END_REF]Sacchi and Schiaffonati, 1996).

None of the other substances that we have tested in this study neither induced the hsp70 promoter (table 1) nor appeared to be strongly cytotoxic (data not shown) in our experiments. Cytosine arabinoside and 5-fluorouracil (base analogs) or amethopterin and aminopterin (inhibitors of DNA synthesis by inhibition of dihydrofolate reductase) act essentially as cytostatic agents.

Vinblastine and colchicine act as antimitotic agents by interacting with the microtubule system.

Streptozotocine, carboplatin and melphalan are alkylating agents, similarly to the intercalating agent bleomycin, that have a physical action on DNA. Our negative result with bleomycin is not in agreement with a previous observation [START_REF] Moseley | Bleomycin induces the hsp 70 heat shock promoter in cultured cells[END_REF]. It looks as if substances which exert specific effects, mostly at the DNA level, and which do not interact broadly with cellular proteins, are not able to induce hsp70 expression.

Our findings suggest that, in our cellular model, the observed inductions of the hsp70 promoter are rather due to a cytotoxic action resulting either in protein denaturation and HSF activation or apoptosis, than to a cytostatic effect. This is in agreement with the concept that HSP70 is a marker of proteotoxicity [START_REF] Hightower | Heat shock, stress proteins, chaperones, and proteotoxicity[END_REF] rather than genotoxicity.

A further important point to discuss concerns our experimental conditions. Cell lysis occurred at the end of the 16 hr exposure period. This time is theoretically sufficient to observe a stress protein synthesis after a cellular aggression if the gene expression machinery is not inhibited. The inhibitory action on DNA transcription by doxorubicin but not streptozotocine (figure 2), suggests that the poor induction observed in response to doxorubicin, daunomycin and mitoxantrone might have been strongly attenuated by their inhibitory action on gene expression. This effect is reversible since a significant CAT production was detected 6-hr after exposure to doxorubicin (figure 3) at concentrations where heat shock-induced CAT production is inhibited (figure 2).

These observations showed that an assay based on the synthesis of marker protein is not quite adapted to detecting these types of effect. Thus, examination of toxicant effect on heat shock induced CAT production would be a suitable control test to detect false negatives in the case of gene expression inhibitors.

Sacchi C. M. and Schiaffonati L. (1996) The effects of etoposide

Figure1.Figure 2 .Figure 3 .

 23 Figure1. Induction of the hsp70 promoter and survival of HeLa cells after exposure to cisplatin for 16 hours.* significantly different from control cultures (p<0.05, Dunnetts' test). Values are means of three independent experiments made in triplicate. Bars: pg CAT/mg of total protein, line: MTT results.

Table 1 .

 1 on mouse L fibroblasts: modulation of stress response, growth and apoptosis genes. Anticancer Research 16, Summary of hsp70 promoter induction in HeLa cells after a 16 hour exposure to anticancer drugs.

	3659-3664.
	Toxicants	Maximal induction (pgCAT/mg protein a )	(µM) Concentration	(% of control) Total protein	n	b
	cisplatin	359 55*	40	67 2	3
	etoposid	91 1*	400	83 3	3
	doxorubicin	44 34	1	77 2	2
	daunorubicin	39 10	1	66 6	2
	mitoxantrone	185 107	2.5	80 5	2
	carboplatin	nd c	0.1-400 d	74 15 e	3
	bleomycin	nd	10 -9 -10 -1 (mg/ml)	87 6	3
	melphalan	nd	0.1-1000	76 7	2
	vinblastine	nd	0.001-1	47 1	2
	colchicine	nd	0.001-10	54 1	2
	amethopterin	nd	0.001-10	82 5	2
	aminopterin	nd	0.001-25	87 3	2
	cytarabine	nd	0.01-100	88 4	2
	5-fluorouracil	nd	50-3700	79 10	3
	streptozotocine	nd	0.01-5000	85 12	2

*significantly different from untreated control (p<0.05, Dunnetts' test). a: mean values S.E.M. b: n=number of independent experiments made in triplicate. From carboplatin to streptozotocine: c: no induction detected (values <20 pgCAT/mg protein); d: range of the tested concentrations; e: total protein content at the highest concentration. The total protein content in heat treated cultures was not significantly different from untreated control.

Acknowledgements -This work was supported by a doctoral fellowship from the Agence de l'Environnement et de la Maîtrise de l'Energie (to S.A.) and by the Association pour la Recherche sur le Cancer (9186), the Ligue contre le Cancer and the region Rhône-Alpes (to A.-P.A).