Unaltered expression of multidrug resistance transporters in polycyclic aromatic hydrocarbon-resistant rat liver cells

Toxicology 156 (2001) 109 – 117 www.elsevier.com/locate/toxicol

Unaltered expression of multidrug resistance transporters in polycyclic aromatic hydrocarbon-resistant rat liver cells Le´a Payen, Arnaud Courtois, Sophie Langoue¨t, Andre´ Guillouzo, Olivier Fardel * INSERM U456, Detoxication et Reparation Tissulaire, Faculte´ de Pharmacie, 2 A6enue du Pr L. Bernard, 35043 Rennes Cedex, France Received 3 May 2000; received in revised form 12 September 2000; accepted 18 September 2000

Abstract Rat liver epithelial cells resistant to the chemical carcinogen 3MC, termed F258/3MC cells and generated by long-term exposure of parental F258 cells to the PAH, were characterized, especially with respect to expression of multidrug resistance transporters such as P-glycoprotein, MRP1 and MRP2. F258/3MC cells were found to be cross-resistant to other PAHs such as BP and dimethylbenz(a)anthracene but remained sensitive to known substrates of multidrug resistance efflux pumps such as doxorubicin and vincristine. They did not display either decreased cellular PAH accumulation or increased PAH efflux. In addition, P-glycoprotein and MRP2 mRNA levels were not, or only barely detected, in F258/3MC cells and in their parental counterparts whereas these PAH-resistant and sensitive cells showed closed levels of MRP1 mRNAs and activity. Moreover, P-gp- and MRP1-overexpressing cells were shown to display similar accumulation and efflux of BP than those found in P-gp- and MRP1-negative control cells. These data therefore suggest that multidrug resistance transporters do not contribute to PAH resistance in PAH-selected liver cells. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemical carcinogen; Cytochrome P4501A1; Drug transporter; Multidrug resistance proteins; P-glycoprotein; Polycyclic aromatic hydrocarbon; Resistance

1. Introduction Abbre6iations: ABC, ATP-binding cassette; BP, benzo(a)pyrene; CF, carboxy-2%,7%-dichlorofluorescein; CFDA, carboxy-2%,7%-dichlorofluorescein diacetate; CYP1A1, cytochrome P4501A1; 3MC, 3-methylcholanthrene; MRP, multidrug resistance protein; MTT, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate-buffered saline; P-gp, P-glycoprotein. * Corresponding author. Tel.: + 33-2-99336241; fax: + 332-99336242. E-mail address: [email protected] (O. Fardel).

Polycyclic aromatic hydrocarbons such as 3MC and BP are widespread environmental contaminants found in air, water and food. These compounds are considered as powerful procarcinogens for various organs, including lung, intestine and skin (Hall and Grover, 1990). Indeed, metabolism of these chemicals via drug metabolizing enzymes such as CYP1A1 can pro-

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duce cytotoxic electrophile intermediates that can react with DNA to form mutagenic DNA adducts and are thus believed to be directly involved in initial events of carcinogenesis. Interestingly, PAHs usually strongly up-regulate CYP1A1 expression and therefore induce their own metabolic activation in various cell types through interaction with the aryl hydrocarbon receptor (Whitlock, 1999; Hankinson, 1995). Long-term treatment of cells by PAHs has been shown to result in the emergence of cell clones resistant to the cytotoxic effects of the carcinogenic chemicals (Hankinson, 1979). A major mechanism underlying such a resistance has been linked to decreased metabolic activation of PAHs through altered PAH-related regulation of CYP1A1 (Hankinson, 1991). Other putative factors contributing to PAH resistance remain largely to be identified. In this context, it is noteworthy that PAHs have been recently postulated to be transported by P-gp (Yeh et al., 1992; Phang et al., 1993), a membrane drug efflux pump conferring resistance to a wide range of anticancer drugs and belonging to the ABC transporter superfamily (Ambudkar et al., 1999). Moreover, a P-gp-like system has been incriminated in the resistance to some PAHs of the freshwater ciliate Tetrahymena pyriformis (Bamdad et al., 1999). In addition, induction of P-gp levels in response to acute treatment by 3MC has been shown in rat liver cells (Gant et al., 1991; Fardel et al., 1996). Taken together, these data suggest that up-regulation of multidrug transporters such as P-gp or possibly other ABC transporters like MRP1 or MRP2 (Borst et al., 1999) may be a part of the response displayed by liver cells to escape the cytotoxic effects of PAHs. For investigating such a hypothesis, we have isolated and characterized 3MC-resistant rat liver cells in the present study, especially with respect to expression of multidrug transporters.

2. Materials and methods

2.1. Chemicals PAHs (3MC, BP, dimethylbenz(a)anthracene,

benz(a)anthracene, dibenz(a)anthracene and benzo(e)pyrene), doxorubicin, verapamil and probenecid were purchased from Sigma Chemical (St Louis, MO). CFDA and calcein acetomethoxy were provided by Molecular Probes (Eugene, OR) whereas [3H] BP was obtained from Amersham (Les Ulis, France).

2.2. Cell culture A 3MC-resistant variant of the rat liver epithelial cell line F258 was generated by continuous exposure of F258 cells to 5 mM 3MC over a 4-month period. The resistant cell line obtained, termed F258/3MC, and the parental cell line F258 were cultured in Williams’ E medium supplemented with 10% fetal calf serum at 37°C under a 5% CO2 atmosphere and were passaged every week using a 0.1% trypsin solution. F258/3MC cells were routinely maintained in the presence of 5 mM 3MC; the PAH was however discarded at least one week before the use of cells for experiments. P-gp-overexpressing K562R/7 cells, MRP1overexpressing GLC4/Sb30 cells and their parental counterparts K562 and GLC4 cells were cultured in RPMI medium supplemented with 10% fetal calf serum and 2 mM glutamine as previously described (Fardel et al., 1995; Vernhet et al., 1999).

2.3. Cell proliferation assays The effect of PAHs, doxorubicin and vincristine on cell proliferation was analysed using the MTT assay (Carmichael et al., 1987). Briefly, cells seeded at 3000/well were cultured with various concentrations of xenobiotics for 96 h in 96-well microplates; 100 ml of a 1 mg/ml MTT solution was then added to each well for 2 h. The medium was then discarded and the blue formazan product formed was dissolved in dimethyl sulfoxide and further quantified by its absorbance at 540 nm using a Titertek Multiskan MCC/340 (Flow Laboratories, Puteaux, France). Growth inhibition was evaluated as IC50, i.e. the xenobiotic concentration providing a 50% reduction in

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cell number as compared to controls cultured in parallel without xenobiotic.

2.4. Benzo(a)pyrene accumulation and efflux assay For BP accumulation studies, cells were incubated with 5 mM BP in Williams’ E medium for various lengths of time ranging from 30 to 120 min. Cells were then washed three times with ice-cold PBS and lysed in distillated water containing 0.1% triton. Amounts of cellular BP were further quantified by fluorimetry using a Spectra Max Gemini spectrofluorimeter (Molecular Devices, Sunnyvale, CA); excitation and emission wavelengths were 355 and 405 nm, respectively. An aliquot of cell lysate was used in parallel to determine cellular protein content by the Bio-Rad assay (Bradford, 1976). Results were expressed as fluorescence arbitrary units after normalization to cellular protein content. For BP efflux studies, BP-loaded cells were incubated in BP-free medium for 120 min in the presence or absence of 100 mM verapamil or 10 mM probenecid, two known inhibitors of ABC transporters (Ford and Hait, 1990; Norman, 1998). Cells were then washed with PBS and intracellular retained BP was further determined as described above. Results were expressed as percentages of initial BP staining.

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end of the loading period was then determined by fluorimetry whereas cellular CF-related fluorescence was analysed after reincubation in CFDAfree medium in the absence or presence of probenecid for 90 min. Excitation and emission wavelengths used for dye analyses were 485 and 538 nm, respectively.

2.6. RNA isolation and northern blot analysis Total RNA was extracted from cells by the guanidium thiocyanate/cesium chloride method of Chirgwin et al. (1979). 10 mg of total RNAs were subjected to electrophoresis in a denaturing 6% (v/v) formaldehyde/1% (w/v) agarose gel, and transferred onto Hybond-N+ sheets (Amersham, Les Ulis, France). The sheets were prehybridized and then hybridized with 32P-labeled probes, washed, dried and auto-radiographed at −80°C. Equal RNA loading onto the gel and efficiency of the transfer were checked up by methylene blue staining of the membranes and by rehybridizing the blots with an 18S rRNA probe. P-gp and CYP1A1 mRNAs were detected using pCHP1 and CYP1A1 cDNA fragments, respectively (Riordan et al., 1985; Affolter et al., 1986) whereas MRP1 and MRP2 mRNAs were analysed using cDNA probes generated by reverse transcriptasepolymerase chain reaction assays as previously described (Payen et al., 1999).

2.5. E6aluation of P-glycoprotein and multidrug resistance protein 1 acti6ity 2.7. E6aluation of DNA adduct formation Cellular P-gp and MRP1 activity were investigated using dye accumulation and efflux assays as previously described (Fardel et al., 1996; Vernhet et al., 1999). For P-gp function, cells were incubated with the P-gp substrate rhodamine 123 for 90 min; intracellular dye accumulation was then determined by fluorimetry. For MRP1 activity, cells were loaded either with calcein acetomethoxy or with CFDA; these compounds are non-fluorescent and non-polar esterified forms of the dyes that freely diffuse into cells where they are cleaved upon action of esterases to give calcein or CF, fluorescent anionic compounds handled by MRP1. Intracellular calcein accumulation at the

BP-DNA adducts were evaluated by quantification of [3H]BP bound to DNA as previously described (Sharma et al., 1994). Briefly, cells were exposed to 0.25 nM [3H]BP for 12 h. After washing with PBS, cells were then incubated overnight at 37°C in lysis buffer containing 400 mM NaCl, 100 mM Tris–HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, 20 mg/ml RNase T1 and 500 mg/ml proteinase K. DNA was further precipitated from cell lysates using isopropanol and washed with ice-cold 70% ethanol (Wu et al., 1995). Amounts of [3H] BP covalently bound to DNA were then determined by scintillation counting and normal-

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ized to amounts of DNA quantified by its absorbance at 260 nm.

2.8. Statistical analysis Data were analysed by the student’s t-test or by two-way ANOVA followed by a Duncan’s test. The criterion of significance was P B0.05.

3. Results F258/3MC cells, generated by long-term exposure to 3MC, were first examined for their sensitivities to various PAHs using the MTT assay. As indicated in Table 1, F258/3MC were found to be highly resistant to 3MC, BP and dimethylbenz(a)anthracene when compared to parental F258 cells. Other PAHs such as benzo(e)pyrene, dibenz(a)anthracene and benz(a)anthracene were demonstrated to be not cytotoxic towards both F258 and F258/3MC cells at the concentrations tested, therefore precluding the search for a putative cross-resistance of F258/3MC cells to these PAHs. F258/3MC cells and their parental counterparts were also found to display similar sensitivities to the anticancer drugs doxorubicin and Table 1 Sensitivity of F258 and F258/3MC cells to polycyclic aromatic hydrocarbons, doxorubicin and vincristine IC50 (nM)a

3-Methylcholanthrene Benzo(a)pyrene Dimethylbenz(a) anthracene Benzo(e)pyrene Dibenz(a)anthracene Benz(a)anthracene Doxorubicin Vincristine

F258 cells

F258/3MC cells

329 2.4 4091.6 5191.2

\5000 (\150)*,b \5000 (\125)* \5000 (\100)*

\5000 \5000 \5000 15.39 2.1 2.290.6

\5000 \5000 \5000 19 9 3.8 (1.2) 2.49 0.8 (1.1)

* PB0.05 when compared to F258 cells. Drug concentration reducing cell growth by 50% when compared to controls cultured in parallel without drug. b Numbers in parentheses indicate the resistance factor, i.e. the ratio of IC50 for F258/3MC cells versus IC50 for F258 cells. a

Fig. 1. BP-DNA adducts in F258 and F258/3MC liver cells. Cells were incubated with 0.25 nM [3H]BP for 12 h. BP-DNA adducts were then determined as described in Section 2. Data are the mean 9 S.D. of three experiments in triplicate. (*) P B0.05 when compared to the amounts of BP-DNA adducts in F258 cells.

vincristine (Table 1). Cytotoxicity of BP towards F258/3MC and F258 cells was also evaluated through analysis of BP-DNA adducts formed in response to exposure to the PAH. As shown in Fig. 1, the amounts of BP-DNA adducts were found to be statistically lower in F258/3MC than in their parental counterparts. Cellular accumulation of BP in F258/3MC and F258 cells was further investigated by spectrofluorimetry (Fig. 2A). F258/3MC cells were found to display slightly enhanced cellular levels of BP over a 120-min incubation period when compared to those found in F258 cells. In addition, efflux experiments demonstrated that F258 and F258/3MC cells similarly retained BP (Fig. 2B). Moreover, cellular BP retention in the two cell lines was not altered in response to verapamil and probenecid, two compounds known to inhibit P-gp- and MRP1-mediated transport, respectively (Ford and Hait, 1990; Norman, 1998). Expression of multidrug resistance transporters such as P-gp, MRP1 and MRP2 was then compared in F258 and F258/3MC cells through northern blot analysis. As indicated in Fig. 3, F258 cells and their 3MC-resistant counterparts showed no, or only barely, detectable levels of P-gp and MRP2 mRNAs whereas high levels of P-gp and MRP2 mRNAs were detected in primary rat hepatocytes used here as positive con-

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trols (Fardel et al., 1992; Courtois et al., 1999). In addition, acute exposure of F258/3MC cells to 5 mM 3MC for 48 h failed to induce major change of P-gp mRNA amounts (data not shown) whereas up-regulation of P-gp in response to 3MC has been reported in other rat liver cell lines not previously selected by the PAH (Fardel et al., 1996) and in primary rat hepatocytes (Chieli et al., 1994). In contrast to P-gp and MRP2 transcripts, those of MRP1 were present at substantial and similar levels in both F258 and F258/3MC cells whereas they were barely evidenced in

Fig. 3. P-gp, MRP1 and MRP2 mRNA levels in F258 and F258/3MC liver cells. Total RNAs were prepared from F258 cells, F258/3MC cells and primary rat hepatocytes. RNAs (10 mg/lane) were then transferred onto Hybond-N+ sheets after electrophoresis and hybridized with P-gp, MRP1, MRP2 and 18S probes.

Fig. 2. Cellular accumulation (A) and efflux (B) of BP in F258 and F258/3MC liver cells. (A) Cells were incubated with 5 mM BP for 30, 60 or 120 min. Intracellular accumulated BP was then determined by fluorimetry. The data are the mean 9 S.D. of three experiments in triplicate. FAU, fluorescence arbitrary unit. (*) P B0.05 when compared to BP levels in F258 cells. (B) Cells were loaded with BP, washed, and reincubated in BP-free medium for 120 min in the presence of 100 mM verapamil or 10 mM probenecid. Intracellular BP retained was then determined by fluorimetry. Results are expressed as percentages of initial cellular BP staining and are the mean 9 S.D. of three experiments in triplicate.

primary hepatocytes (Fig. 3). F258/3MC and F258 cells also displayed similar MRP1 activity; indeed CF efflux studies indicated nearly identical values of dye retention in these cells and the MRP1 blocker probenecid enhanced cellular levels of CF in F258/3MC cells and in their parental counterparts in a similar manner (Fig. 4). We next analysed expression of CYP1A1 mRNAs in F258 and F258/3MC cells (Fig. 5). Both cell lines were found to display very low, if any, expression of CYP1A1 transcripts. Treatment of F258 cells by 5 mM 3MC for 24 h resulted in an induction of CYP1A1; exposure of F258/3MC cells to the PAH also increased CYP1A1 mRNA levels, although clearly to a lesser extent (Fig. 5). However, expression of CYP1A1 transcripts in

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Fig. 4. MRP1 activity in F258 and F258/3MC liver cells. CF-loaded cells were incubated in dye-free medium for 90 min in the presence or absence of 10 mM probenecid. After washing, intracellular retained CF was then determined by fluorimetry. Results are expressed as percentages of initial cellular CF staining and are the mean 9 S.D. of three experiments in triplicate. (*) PB0.05 when compared to cells maintained in the absence of probenecid.

both F258 and F258/3MC cells exposed to 3MC remains very low when compared to that found in 3MC-treated hepatocytes (Fig. 5).

Fig. 6. BP accumulation (A), BP efflux (B) and rhodamine 123 and calcein accumulation (C) in P-gp- and MRP1-overexpressing cells. (A) P-gp-positive K562R/7 cells, MRP1-positive GLC4/Sb30 cells and corresponding K562 and GLC4 control cells were incubated with 5 mM BP for 90 min. Intracellular accumulated BP was then determined by fluorimetry. The data are the mean 9 S.D. of three experiments in triplicate. FAU, fluorescence arbitrary unit. (B) Cells were loaded with BP, washed, and reincubated in BP-free medium for 120 min in the presence of 100 mM verapamil or 10 mM probenecid. Intracellular BP retained was then determined by fluorimetry. Results are expressed as percentages of initial cellular BP staining and are the mean 9 S.D. of three experiments in triplicate. (C) Cells were loaded with 2 mM rhodamine 123 or 0.5 mM calcein acetomethoxy for 90 min. Intracellular accumulations of the dyes were then determined by fluorimetry. The data are the mean 9 S.D. of three experiments in triplicate. FAU, fluorescence arbitrary unit. (*) P B 0.05.

Fig. 5. CYP1A1 mRNA levels in untreated- and 3MC-treated F258 and F258/3MC liver cells. Total RNAs were prepared from F258 and F258/3MC cells either untreated or treated by 5 mM 3MC for 24 h and from primary rat hepatocytes exposed or not to the PAH. RNAs (10 mg/lane) were then transferred onto Hybond-N+ sheets after electrophoresis and hybridized with CYP1A1 and 18S probes.

Finally, in order to determine whether PAHs such as BP may be actually handled by export pumps such as P-gp or MRP1, we have compared BP accumulation and efflux in P-gp- and MRP1overexpressing cells and in corresponding control cells. As indicated in Fig. 6A, similar accumula-

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tion of BP was found in P-gp-positive K562R/7 cells, in MRP1-positive GLC4/Sb30 cells and in their respective parental K562 and GLC4 counterparts. Efflux experiments also demonstrated the same levels of BP retention in the cells regardless of their expression of P-gp or MRP1 (Fig. 6B). Moreover, inhibitors of efflux pumps such as verapamil or probenecid failed to alter intracellular amounts of the PAH. By contrast, K562R/7 and GLC4/Sb30 cells were found to poorly accumulate rhodamine 123 and calcein, two dyes substrates for P-gp and MRP1, respectively, when compared to their parental counterparts (Fig. 6C), thus indicating that P-gp and MRP1 were fully active in K562R/7 and GLC4/Sb30 cells.

4. Discussion The present study was designed in order to characterize 3MC-resistant rat liver epithelial cells, especially with respect to expression of multidrug resistance transporters like P-gp, MRP1 or MRP2. Indeed, these drug efflux pumps, found at high levels in normal liver (for P-gp and MRP2) (Bu¨chler et al., 1996; Ambudkar et al., 1999) and in tumoral liver (for MRP1) (Payen et al., 1999), have been hypothetized to play a role in resistance to chemical carcinogens, including PAHs (Yeh et al., 1992). Our data however indicate that such transporters are unlikely to be involved in PAH resistance of the liver cells used in this work and that have been generated by long-term exposure to 3MC. Indeed, expression of P-gp and MRP2 mRNAs was not, or only barely, detected, in F258/3MC cells and in their parental counterparts whereas these two kinds of cells displayed similar levels of MRP1 mRNAs and activity. Moreover, expression of P-gp mRNAs was not obviously altered in F258/3MC cells in response to acute exposure to 3MC. By contrast, P-gp levels have been shown to be induced in other 3MC-treated rat liver cell lines (Fardel et al., 1996). Such a discrepancy may be linked to phenotypic differences between the independent liver cell lines used in these studies; alternatively, it may reflect the fact that long-term PAH selection of cells might suppress the induction of multidrug resistance

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transporters. It is noteworthy that F258/3MC cells were not cross resistant to anticancer drugs such as doxorubicin and vincristine, known to be handled by P-gp, MRP1 and MRP2 (Ambudkar et al., 1999; Borst et al., 1999; Cui et al., 1999). This also argues against overexpression of one of these transporters in the 3MC-resistant rat liver cells. Moreover, F258/3MC and F258 cells displayed similar cellular BP efflux, not altered in response to known inhibitors of P-gp or MRP1 activity, and cellular accumulation of the PAH was even slightly enhanced in F258/3MC cells when compared to F258 cells. Taken together, these data make unlikely the overexpression of a PAH export pump in F258/3MC cells. In addition, the lack of involvement of P-gp in PAH resistance of F258/3MC cells is also supported by the fact that the P-gp blocker verapamil was not found to sensitize F258/3MC cells to the cytotoxic effects of 3MC in MTT proliferation assays (data not shown). Interestingly, expression of P-gp has been recently reported to be not induced in BP-selected breast tumor cells (Moore et al., 1994). These data therefore suggest that unaltered expression of multidrug resistance transporters can be observed in various PAH-resistant cell lines regardless of their tissue origin and of the PAH used for their selection. Unlike expression of multidrug resistance transporters, that of CYP1A1, especially its PAH-related regulatory pathway, was found to be altered in F258/3MC cells. Indeed, 3MC treatment was found to induce CYP1A1 mRNAs in F258/3MC cells to a lesser extent than in parental F258 cells. The level of CYP1A1 induction in these latter cells remains however low when compared to that occurring in 3MC-treated hepatocytes, suggesting that even F258 cells are rather poorly responsive to 3MC with respect to CYP1A1 expression. On the other hand, it is noteworthy that a decreased inducibility of CYP1A1 in response to PAHs, that lowers the formation of cytotoxic metabolites of PAHs, has been well recognized as a common mechanism of resistance to PAHs in various cell lines (Hankinson, 1991; Moore et al., 1996; Caruso and Batist, 1999; Chang et al., 1999). Consequently, it may also contribute to the altered sensitivity of F258/3MC cells to 3MC, BP

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and dimethylbenz(a)anthracene. In particular, it may underlie the low formation of BP-DNA adducts in F258/3MC cells. Indeed, PAH-DNA adducts usually result from interactions of DNA with electrophile metabolites of PAHs generated via CYP-dependent reactions. Interestingly, benz(a) anthracene, benzo(e)pyrene and dibenz(a)anthracene were not found to be cytotoxic towards both F258 and F258/3MC cells; these PAHs have also been demonstrated to be weak carcinogens (Cavalieri and Rogan, 1992). Such a relatively low toxicity of these compounds likely reflects that their metabolism does not generate, or only in small amounts, cytotoxic electrophile compounds. In contrast with previous studies (Yeh et al., 1992; Phang et al., 1993), we failed to detect enhanced transport of PAHs such as BP in P-gpoverexpressing cells although P-gp was fully functional in such cells as indicated by rhodamine 123 accumulation assays. Similarly, Schuetz et al. (1998) have reported similar BP transport in MDR1 P-gp transfected cells and in corresponding control cells, indicating that cellular retention of the PAH is not influenced by P-gp. The reason for such a discrepancy with respect to PAH efflux in P-gp-positive cells is unclear. It may be linked to altered functional features of P-gp in the multidrug resistant breast cancer MCF7 cell line used by both Yeh et al. (1992) and Phang et al. (1993). Alternatively, this cell line may overexpress a yet unidentified transporter of PAH in addition to P-gp. We also showed similar accumulation and retention of BP in MRP1-positive and negative cells, likely suggesting that PAHs are not handled by MRP1. By contrast, this efflux pump has been recently demonstrated to transport some carcinogenic compounds such as aflatoxin B1 (Loe et al., 1997). In summary, PAH-resistant F258/3MC liver cells generated through long-term exposure to 3MC were not found to display neither enhanced cellular outwardly-directed transport of PAH nor altered expression of drug efflux pumps such as P-gp, MRP1 and MRP2 when compared to parental cells. These data, therefore, unequivocally argue against a major contribution of drug efflux pumps to the PAH resistance displayed by F258/ 3MC liver cells.

Acknowledgements This work was supported by the Ligue Nationale contre le Cancer (Comite´ d’Ille et Vilaine), the Association pour la Recherche sur le Cancer and the Ministe`re de l’Ame´nagement du Territoire et de l’Environnement. Le´a Payen and Arnaud Courtois are recipients of fellowships from the Ligue Nationale contre le Cancer and the Association pour la Recherche sur le Cancer, respectively.

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