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Monophthalates promote IL-6 and IL-8 production in the human epithelial cell line A549
Toxicology in Vitro 18 (2004) 265–269 www.elsevier.com/locate/toxinvit
Monophthalates promote IL-6 and IL-8 production in the human epithelial cell line A549 K.F. Jepsen, A. Abildtrup, S.T. Larsen* Department of Chemical Working Environment, National Institute of Occupational Health, Lersoe Parkalle´ 105, DK-2100 Copenhagen, Denmark Received 24 June 2003; accepted 24 September 2003
Abstract The dramatically increasing prevalence of allergic respiratory diseases may in part be due to the presence of certain immunotoxic xenobiotics in the environment. Recent studies have suggested that some plasticizers belonging to the phthalate family, and metabolites thereof, play a role in the development of allergic respiratory diseases. This is probably due to an adjuvant effect, which in some cases may be combined with an inflammatory process. The scope of the present study was to investigate the inflammatory potential of monophthalates, which are degradation products of phthalate plasticizers. The human epithelial cell line A549 was exposed to 15.6 – 2000 mg/ml, in two-fold dilutions, to either mono-n-butyl phthalate, monobenzyl phthalate, mono-n-octyl phthalate, mono-2-ethylhexyl phthalate, mono-iso-nonyl phthalate or mono-iso-decyl phthalate. Concentrations of the proinflammatory cytokines IL-6 and IL-8 were measured in the cell culture supernatant by ELISA.The study showed that some, but not all, monophthalates could induce a concentration-dependent increase in cytokine production, whereas, at higher concentrations, all phthalates suppressed cytokine production. Both the stimulatory and the suppressive properties were highly dependent on the length of the alkyl side chain of the monophthalate - a structure-activity relationship that is supported by recent observations in mice. # 2003 Elsevier Ltd. All rights reserved. Keywords: Monophthalate; Cytokine; A549
1. Introduction The prevalence of allergic diseases, such as asthma and rhinitis, are increasing in the industrialized countries especially among children and teenagers. The reasons for this are not clear, but attention has been turned towards the environment, both indoor (e.g. dust and tobacco smoke) and outdoor (e.g. diesel exhaust particles). Phthalates, used extensively as softeners in poly vinyl chloride (PVC) materials, such as vinyl flooring, are another source of environmental exposure, which might play a role in relation to the development of allergic respiratory diseases. Thus, Jaakkola and colleagues (1999) found that bronchial obstruction in small children was associated with the exposure to phthalate plasticized PVC. Furthermore, our group has shown that phthalates and their degradation products, the * Corresponding author. Tel.: +45-39-16-5200; fax: +45-39-165201. E-mail address: [email protected] (S.T. Larsen). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.09.008
so-called monophthalates, are capable of enhancing an allergy-related immune response in mice and, hence, possibly in humans also (Larsen et al., 2001, 2002a, 2002b, 2003). Phthalates can make up 40% of the weight in PVC flooring materials. The phthalates migrate from the PVC material and adhere to the surfaces of e.g. dust particles in the indoor environment. The most common plasticizer, di(2-ethylhexyl) phthalate, has been found in concentrations of more than 3000 mg/g dust in Danish schools (Clausen et al., 2003). The inhalation of dust particles may be an important exposure source, as it may cause relatively high local concentrations of phthalate in the respiratory tract. This could be particularly relevant for small children, who can spend much of their time during the first couple of years of their life on the floor in close proximity to vinyl flooring, for example. In the organism, phthalates are degraded to the socalled monophthalates by enzymes, such as esterases and lipases (Takahashi and Tanaka, 1989). With respect
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to most toxicological effects, the monophthalate is considered to be more potent than the parent compound (cf. Larsen et al., 2001) and monophthalates were found to be more potent than the phthalates in the enhancement of IgE production in BALB/c mice (Larsen et al, 2001, 2002a, 2002b, 2003). The aim of the present study was to investigate a possible proinflammatory effect of monophthalates on the type II human lung epithelial cell line, A549. Substances with a proinflammatory potential may be identified by their ability to induce the synthesis in, and release from cells, of relevant cytokines, including IL-8, which appear to play a key role in the regulation of an inflammatory response (Schwarze et al., 2000). IL-8 is released from macrophages and epithelial cell types, as well as from other pulmonary cell types (Schwarze et al., 2000). The well-known environmental adjuvant, diesel exhaust particle (cf. Løvik et al. 1997), has been shown to stimulate IL-8 production in three kinds of epithelial cells (Ohtoshi et al., 1998). It was suggested that proinflammatory agents may induce local and systemic inflammatory responses in normal subjects, which could contribute to the risk of developing asthma and chronic obstructive lung disease (Michel, 2000; Schwarze et al., 2000).
2. Materials and methods
tively (Goggans and Copenhaver, 1939). No melting point was available for MINP. The identities of MnBP, MnOP, MINP and MIDP were further confirmed and the purities determined by 1H NMR spectroscopy. The purities of MnBP, MnOP and MINP were all more than 95%. Extensive carbon-NMR analysis showed that the MIDP was a mixture of at least six isomers. The alcohol used for the synthesis of MIDP was a mixture of several alcohols, comprised of two main components, two components in lower concentrations and even lower concentrations of at least two other alcohols. All alcohols were present in the MIDP synthesized. Additionally, the MIDP contained low concentrations of impurities, which could include phthalic acid and phthalic acid diesters. In order to enhance the solubility of the monophthalates, 100 ml of 99.9% ethanol (Danisco Destiller, Aalborg, Denmark), and 400 ml PEG 400 (MerckSchuchardt, Germany), were added stepwise to 40 mg of pure monophthalate and the suspensions were mixed vigorously for 30 seconds. HAMS+ was added to a final concentration of 10 mg monophthalate per ml and the suspensions were subsequently shaken for 30 min, followed by final dilution. The effect of PEG 400 on A549 cells was tested in parallel with monophthalates. The concentrations of PEG 400 did not exceed 0.2% in any of the test and control solutions.
2.1. Reagents
2.2. A549 bioassay
Cells were grown in HAMS F12 (Gibco, Paisley, Scotland) supplemented with penicillin (100 IU/ml, Gibco) and streptomycin (100 mg/ml, Gibco), l-glutamine (2 mM, Gibco), and fetal bovine serum (FBS, 10%, Gibco) (HAMS+). Phorbol-myristate-acetate (PMA) (Sigma, St. Louis, MO, USA), tumour necrosis factor a (TNF-a, BD Pharmingen, San Diego, CA, USA), and lipopolysaccharide from Escherichia coli (LPS, Sigma) were used as control stimulants in 10-fold dilutions ranging from 5.0 ng/ml to 0.05 ng/ml for PMA, from 10 ng/ml to 0.1 ng/ml for TNF-a, and from 10 mg/ml to 0.01 mg/ml for LPS. The following monophthalates were used as stimulants: Mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBnP), mono-n-octyl phthalate (MnOP), mono-2-ethylhexyl phthalate (MEHP), mono-iso-nonyl phthalate (MINP), and mono-iso-decyl phthalate (MIDP). MBnP (purity > 90%) and MEHP (purity > 98%) were purchased from Tokyo Kasei Organic Chemicals (Tokyo, Japan). MnBP, MnOP, MINP, and MIDP were synthesized in our laboratory, as described by Larsen et al. (2001). The melting points of the dried MnBP, MnOP and MINP were found to be 73.0–73.5 C, 22.5–23.3 C and 50.0–51.0 C, respectively. In the literature, the melting points for MnBP and MnOP are reported to be 73.1–73.5 C and 21.5–22.5 C, respec-
The A549 bioassay is based on the human lung epithelial cell line A549 (ATCC CCL-185, American Type Culture Collection, Rockville, MD, USA). Cells were seeded in 24-well plates (Nunc, Denmark) at 105 cells/ well in 1 ml HAMS F12 medium supplemented with penicillin (100 IU/ml) and streptomycin (100 mg/ml), lglutamine (2 mM) and fetal bovine serum (FBS, 10%, Gibco) (HAMS+) and incubated overnight at 37 C, 5% CO2, and 100% relative humidity. The next day, the medium was aspirated and replaced with 900 ml of HAMS+ plus 100 ml stimulant. All monophthalates were tested in 2-fold dilutions and the final monophthalate concentrations ranged from 2000 mg/ml to 15.6 mg/ml. Cells were stimulated in triplicate and incubated for 24 h at 37 C, 5% CO2, and 100% relative humidity. A total of three stimulation series with monophthalates were performed and, in each series, unstimulated controls were included as 6 triplicates. Furthermore, cells were stimulated with the control substances PMA, TNF-a, and LPS, as well as PEG 400. Supernatants were harvested and kept at 45 C until analysis of IL-6 and IL-8 concentrations. 2.2.1. ELISA for IL-6 and IL-8 For the determination of IL-6 and IL-8 production from A549 cells, OptEIA kits from BD Pharmingen
K.F. Jepsen et al. / Toxicology in Vitro 18 (2004) 265–269
(San Diego, CA, USA) were used according to the manufacturer. However, in order to improve the analytical range, the standard curves were extended from 300 pg/ml to 600 pg/ml for IL-8, and from 250 pg/ml to 500 pg/ml for IL-6.
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slope of the concentration–response curve is referred to as the stimulatory potency, which may be used to compare the potencies of the different monophthalates to induce cytokine production; the steeper the slope, the more potent the monophthalate is in stimulating the production of the cytokine.
2.3. Assessment of cytokine production 2.5. Assessment of suppressive properties The ratio between cytokine concentration in the supernatant from exposed cell culture and the unexposed (negative control) cell culture was defined as the stimulatory index (SI):
SI ¼
ðconcentration of IL from exposed cellsÞ= ðconcentration of IL from the negative controlÞ
Negative controls were included in each exposure series and, thus, the SI is normalised for day-to-day variation, allowing comparison of SI-values achieved on different days. 2.4. Assessment of the potency of monophthalates to induce cytokine production The SI was plotted against the logarithm of the monophthalate concentration (cf. Figs. 1 and 2). The increasing part of these curves, which included from three to five concentrations, represents the concentrations of monophthalate that predominantly induced cytokine production in the A549 cells. Regression analyses were performed on this part of the curve. A statistically significant (P< 0.05) slope was accepted as a concentration-dependent cytokine production. The
Fig. 1. IL-6 production by A549 cells after stimulation with various monophthalates. The IL-6 production is expressed as the stimulatory index (SI), which is the ratio of IL-6 level in the exposure group to the IL-6 level in the corresponding negative control (medium only) group. MnBP: mono-n-butyl phthalate; MBnP: monobenzyl phthalate; MnOP: mono-n-octyl phthalate; MEHP: mono-2-ethylhexyl phthalate; MINP: monoisononyl phthalate; MIDP: monoisodecyl phthalate.
To compare the suppressive properties of the monophthalates, we established a No-Observed Effect Level (NOEL) for this effect. We defined the NOEL as the highest concentration of monophthalate that did not give rise to a reduced cytokine production, which, in most cases, was the same as the concentration that induced maximum cytokine production. The NOEL was assessed visually from Figs. 1 and 2.
3. Results Six monophthalates were tested for their potential to stimulate IL-6 and IL-8 production in A549 cells. In general, the monophthalates tend to stimulate cytokine production at lower concentrations, whereas higher concentrations of monophthalate gave rise to a reduced cytokine production compared to lower monophthalate concentrations. Thus, the concentration–response curves were bell-shaped in most cases (cf. Figs. 1 and 2). 3.1. Mono-n-butyl- and monobenzyl phthalate These compounds gave rise to non-significant or relatively low stimulatory potencies for both IL-6 and IL-8
Fig. 2. IL-8 production by A549 cells after stimulation with various monophthalates. The IL-8 production is expressed as the stimulatory index (SI), which is the ratio of the IL-8 level in the exposure group to the IL-8 level in the corresponding negative control (medium only) group. MnBP: mono-n-butyl phthalate; MBnP: monobenzyl phthalate; MnOP: mono-n-octyl phthalate; MEHP: mono-2-ethylhexyl phthalate; MINP: monoisononyl phthalate; MIDP: monoisodecyl phthalate.
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production. Also, the cytokine suppressive effects of these compounds were relatively weak (cf. Figs. 1 and 2 and Table 1). 3.2. Mono-2-ethylhexyl-, mono-n-octyl-, mono-isononyl- and mono-iso-decyl phthalate All of these compounds showed relatively high stimulatory potencies, i.e. they gave rise to steeper concentration–effect curves. The substances tend to stimulate both IL-6 and IL-8 production, even though the stimulatory potencies in all cases were higher for IL6 (cf. Figs. 1 and 2 and Table 1). At the same time, MEHP, MnOP, MINP, and MIDP, all have more pronounced cytokine suppressive effects than MnBP and MBnP. 3.3. PMA, TNF- and LPS These compounds were included as positive controls. They all stimulated cytokine production in a concentration-dependent manner (data not shown). 3.4. PEG 400 The concentrations of the cosolvents PEG 400 and ethanol used in the present study did not give rise to cytokine suppressive effects (data not shown).
4. Discussion All of the monophthalates studied could stimulate cytokine production, but to varying degrees. Also, at higher concentrations, all of the monophthalates studied gave rise to suppression of cytokine production. The latter effect was most likely due to a toxic effect on the cells; this is supported by light microscopy observations, showing lysis of some of the cell membranes. PEG 400
and ethanol did not cause cell lysis under the conditions of the test. The results showed a prominent change in stimulatory potency among the monophthalates. Monophthalates with fewer than eight carbon atoms in their alkyl side chain were weak cytokine inducers, whereas monophthalates with eight or more carbon atoms in the alkyl side chain were more potent cytokine inducers. These findings are generally in accordance with a study in mice, showing that MEHP, MnOP and MINP promoted the formation of Th2-dependent antibodies, whereas MnBP, MBnP and MIDP did not (Larsen et al., 2001). Taking the results from the present study with those reported by Larsen et al. (2001) together, it is tempting to hypothesize that the promotion of antibody production (adjuvant effect) and the proinflammatory effects are interrelated, even though the adjuvant effect of monophthalates is most likely due to a more specific mechanism than simple inflammation, as MIDP stimulated IL-6 and IL-8 production, but lacked an adjuvant effect. Also, it is obvious from Table 1 that the cytokine suppressive effect of the monophthalates increased with increasing alkyl side chain length. This structure-activity relationship of monophthalates has previously been reported to exist also for suppressive effects on the monocytic cell line, THP-1 (Glue et al., 2002), and in mice (Larsen et al., 2001). It is tempting to speculate that the cytokine stimulatory and suppressive effects are interrelated, as the most powerful cytokine stimulants in the present study were also the most potent cytokine suppressors. At the same time, weak cytokine stimulants were weak cytokine suppressors. The levels of IL-6 and IL-8 seem to be correlated - an observation that is in accordance with other studies in which A549 cells have been exposed to proinflammatory substances (Fu¨nfstu¨ck et al., 2001; Saraf et al., 1999; Strˇ ı´zˇ et al., 2000). Epithelial cell lines produced elevated levels of IL-6 and IL-8 after exposure to diesel exhaust particles (Steerenberg et al., 1998; Ohtoshi et al., 1998),
Table 1 Compound
Mono-n-butyl phthalate Monobenzyl phthalate Mono-2-ethylhexyl phthalate Mono-n-octyl phthalate Monoisononyl phthalate Monoisodecyl phthalate
Cna
4 7d 8 8 9 10
Stimulatory potencyb
NOEL for suppressionc
IL-6
IL-8
IL-6 (mg/ml)
IL-8 (mg/ml)
3.1 NS 6.8 23 9.1 8.6
NS NS 2.4 3.9 3.8 2.8
1000 1000 250 125 125 62.5
1000 2000 250 125 125 62.5
NS: not statistically significant concentration–response relationship (regression analysis). a Cn indicates the number of carbon atoms in the alkyl side chain. b The stimulation potency is the slope of the log concentration–response curve (see Materials and methods for further explanation). c The no-observed effect level (NOEL) is here defined as the highest concentration that does not give rise to a reduced cytokine production. d The seven carbon atoms are located in a benzyl group, i.e. a system that is both aromatic and aliphatic, whereas the other monophthalates studied here have aliphatic side chains.
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a well-recognized environmental adjuvant that selectively enhances the Th2 response (Løvik et al., 1997). Th2 cells produce IL-6 (Seymour et al., 1996), which is able to polarize naı¨ve CD4+ T-cells to effector Th2 cells (Gehring et al., 1998; Rincon et al., 1997). Furthermore, proinflammatory agents may play a role in the exacerbation of asthma by activating an inflammatory cascade, which amplifies the inflammatory response to inhaled antigen, as reported in two field studies (Michel et al., 1996; Rizzo et al., 1997). In conclusion, this study shows that some monophthalates are able to enhance IL-6 and IL-8 synthesis in a human lung epithelial cell line. These cytokines play a role in the initiation of inflammation and possibly contribute to the polarization of a Th2-dominated immune response, which plays a role in the development of allergic respiratory diseases.
Acknowledgements This study has been produced as part of the research activities in the Centre for the Environment and Respiratory System, which is supported by the Danish Environmental Research Program.
References Clausen, P.A., Bille, R.L.L., Nilsson, T., Hansen, V., Svensmark, B., Bøwadt, S., 2003. Simultaneous extraction of di(2-ethylhexyl) phthalate and nonionic surfactants from floor dust. Concentrations in floor dust from 15 Danish schools. J Chromatography A 986, 179–190. Fu¨nfstu¨ck, R., Franke, S., Hellberg, M., Ott, U., Knofel, B., Straube, E., Sommer, M., Hacker, J., 2001. Secretion of cytokines by uroepithelial cells stimulated by Escherichia coli and Citrobacter spp. Int. J. Antimicrob. Agents 17, 253–258. Gehring, S., Schlaak, M., van der Bosch, J., 1998. A new in vitro model for studying human T cell differentiation: T(H1)/T(H2) induction following activation by superantigens. J. Immunol. Methods 219, 85–98. Glue, C., Millner, A., Bodtger, U., Jinquan, T., Poulsen, L.K., 2002. In vitro effects of monophthalates on cytokine expression in the monocytic cell line THP-1 and in peripheral blood mononuclear cells from allergic and non-allergic donors. Toxicology In Vitro 16, 657–662. Goggans, J.F., Copenhaver, E., 1939. Monoalkyl phthalates from normal aliphatic alcohols. J. Am. Chem. Soc. 61, 2909–2910. Jaakkola, J.J., Oie, L., Nafstad, P., Botten, G., Samuelsen, S.O., Magnus, P., 1999. Interior surface materials in the home and the development of bronchial obstruction in young children in Oslo, Norway. Am. J. Public Health 89, 188–192.
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Larsen, S.T., Hansen, J.S., Thygesen, P., Begtrup, M., Poulsen, O.M., Nielsen, G.D., 2001. Adjuvant and immuno-suppressive effect of six monophthalates in a subcutaneous injection model with BALB/c mice. Toxicology 169, 37–51. Larsen, S.T., Lund, R.M., Nielsen, G.D., Thygesen, P., Poulsen, O.M., 2002a. Di-(2-ethylhexyl) phthalate possesses an adjuvant effect in a subcutaneous injection model with BALB/c mice. Toxicol. Lett. 125, 11–18. Larsen, S.T., Lund, R.M., Nielsen, G.D., Thygesen, P., Poulsen, O.M., 2002b. Adjuvant effect of di-n-butyl-, di-n-octyl-, di-isononyl- and di-iso-decyl- phthalate in a subcutaneous injection model using BALB/c mice. Pharmacol. Toxicol. 91, 264–272. Larsen, S.T., Lund, R.M., Thygesen, P., Poulsen, O.M., Nielsen, G.D., 2003. Investigation of the adjuvant and immuno-suppressive effects of benzyl butyl phthalate, phthalic acid and benzyl alcohol in a murine injection model. Food Chem. Toxicol. 41, 439–446. Løvik, M., Hogseth, A.K., Gaarder, P.I., Hagemann, R., Eide, I., 1997. Diesel exhaust particles and carbon black have adjuvant activity on the local lymph node response and systemic IgE production to ovalbumin. Toxicology 121, 165–178. Michel, O., 2000. Systemic and local airways inflammatory response to endotoxin. Toxicology 152, 25–30. Michel, O., Kips, J., Duchateau, J., Vertongen, F., Robert, L., Collet, H., Pauwels, R., Sergysels, R., 1996. Severity of asthma is related to endotoxin in house dust. Am. J. Respir. Crit. Care Med. 154, 1641–1646. Ohtoshi, T., Takizawa, H., Okazaki, H., Kawasaki, S., Takeuchi, N., Ohta, K., Ito, K., 1998. Diesel exhaust particles stimulate human airway epithelial cells to produce cytokines relevant to airway inflammation in vitro. J. Allergy. Clin. Immunol. 101, 778–785. Rincon, M., Anguita, J., Nakamura, T., Fikrig, E., Flavell, R.A., 1997. Interleukin (IL)-6 directs the differentiation of IL-4-producing CD4+ T cells. J. Exp. Med. 185, 461–469. Rizzo, M.C., Naspitz, C.K., Fernandez-Caldas, E., Lockey, R.F., Mimica, I., Sole, D., 1997. Endotoxin exposure and symptoms in asthmatic children. Pediatr. Allergy Immunol. 8, 121–126. Saraf, A., Larsson, L., Larsson, B.M., Larsson, K., Palmberg, L., 1999. House dust induces IL-6 and IL-8 response in A549 epithelial cells. Indoor Air 9, 219–225. Schwarze, P.E., Lag, M., Becher, R., Thrane, E.V., Samuelsen, J.T., Hetland, R.B., Refsnes, M., 2000. Role of signal transduction pathways in lung inflammatory responses. Toxicol. Lett. 112-113, 165–170. Seymour, G.J., Gemmell, E., Kjeldsen, M., Yamazaki, K., Nakajima, T., Hara, K., 1996. Cellular immunity and hypersensitivity as components of periodontal destruction. Oral Dis. 2, 96–101. Steerenberg, P.A., Zonnenberg, J.A., Dormans, J.A., Joon, P.N., Wouters, I.M., Van Bree, L., Scheepers, P.T., Van Loveren, H., 1998. Diesel exhaust particles induced release of interleukin 6 and 8 by (primed) human bronchial epithelial cells (BEAS 2B) in vitro. Exp. Lung Res. 24, 85–100. Strˇ ı´zˇ, I., Mio, T., Adachi, Y., Carnevali, S., Romberger, D.J., Rennard, S.I., 2000. Effects of interferons alpha and gamma on cytokine production and phenotypic pattern of human bronchial epithelial cells. Int. J. Immunopharmacol. 22, 573–585. Takahashi, T., Tanaka, A., 1989. Biochemical studies on phthalic esters V. Comparative studies on in vitro hydrolysis of di-n-butyl phthalate isomers in rats. Arch. Toxicol. 63, 72–74.
Monophthalates promote IL-6 and IL-8 production in the human epithelial cell line A549 K.F. Jepsen, A. Abildtrup, S.T. Larsen* Department of Chemical Working Environment, National Institute of Occupational Health, Lersoe Parkalle´ 105, DK-2100 Copenhagen, Denmark Received 24 June 2003; accepted 24 September 2003
Abstract The dramatically increasing prevalence of allergic respiratory diseases may in part be due to the presence of certain immunotoxic xenobiotics in the environment. Recent studies have suggested that some plasticizers belonging to the phthalate family, and metabolites thereof, play a role in the development of allergic respiratory diseases. This is probably due to an adjuvant effect, which in some cases may be combined with an inflammatory process. The scope of the present study was to investigate the inflammatory potential of monophthalates, which are degradation products of phthalate plasticizers. The human epithelial cell line A549 was exposed to 15.6 – 2000 mg/ml, in two-fold dilutions, to either mono-n-butyl phthalate, monobenzyl phthalate, mono-n-octyl phthalate, mono-2-ethylhexyl phthalate, mono-iso-nonyl phthalate or mono-iso-decyl phthalate. Concentrations of the proinflammatory cytokines IL-6 and IL-8 were measured in the cell culture supernatant by ELISA.The study showed that some, but not all, monophthalates could induce a concentration-dependent increase in cytokine production, whereas, at higher concentrations, all phthalates suppressed cytokine production. Both the stimulatory and the suppressive properties were highly dependent on the length of the alkyl side chain of the monophthalate - a structure-activity relationship that is supported by recent observations in mice. # 2003 Elsevier Ltd. All rights reserved. Keywords: Monophthalate; Cytokine; A549
1. Introduction The prevalence of allergic diseases, such as asthma and rhinitis, are increasing in the industrialized countries especially among children and teenagers. The reasons for this are not clear, but attention has been turned towards the environment, both indoor (e.g. dust and tobacco smoke) and outdoor (e.g. diesel exhaust particles). Phthalates, used extensively as softeners in poly vinyl chloride (PVC) materials, such as vinyl flooring, are another source of environmental exposure, which might play a role in relation to the development of allergic respiratory diseases. Thus, Jaakkola and colleagues (1999) found that bronchial obstruction in small children was associated with the exposure to phthalate plasticized PVC. Furthermore, our group has shown that phthalates and their degradation products, the * Corresponding author. Tel.: +45-39-16-5200; fax: +45-39-165201. E-mail address: [email protected] (S.T. Larsen). 0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2003.09.008
so-called monophthalates, are capable of enhancing an allergy-related immune response in mice and, hence, possibly in humans also (Larsen et al., 2001, 2002a, 2002b, 2003). Phthalates can make up 40% of the weight in PVC flooring materials. The phthalates migrate from the PVC material and adhere to the surfaces of e.g. dust particles in the indoor environment. The most common plasticizer, di(2-ethylhexyl) phthalate, has been found in concentrations of more than 3000 mg/g dust in Danish schools (Clausen et al., 2003). The inhalation of dust particles may be an important exposure source, as it may cause relatively high local concentrations of phthalate in the respiratory tract. This could be particularly relevant for small children, who can spend much of their time during the first couple of years of their life on the floor in close proximity to vinyl flooring, for example. In the organism, phthalates are degraded to the socalled monophthalates by enzymes, such as esterases and lipases (Takahashi and Tanaka, 1989). With respect
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to most toxicological effects, the monophthalate is considered to be more potent than the parent compound (cf. Larsen et al., 2001) and monophthalates were found to be more potent than the phthalates in the enhancement of IgE production in BALB/c mice (Larsen et al, 2001, 2002a, 2002b, 2003). The aim of the present study was to investigate a possible proinflammatory effect of monophthalates on the type II human lung epithelial cell line, A549. Substances with a proinflammatory potential may be identified by their ability to induce the synthesis in, and release from cells, of relevant cytokines, including IL-8, which appear to play a key role in the regulation of an inflammatory response (Schwarze et al., 2000). IL-8 is released from macrophages and epithelial cell types, as well as from other pulmonary cell types (Schwarze et al., 2000). The well-known environmental adjuvant, diesel exhaust particle (cf. Løvik et al. 1997), has been shown to stimulate IL-8 production in three kinds of epithelial cells (Ohtoshi et al., 1998). It was suggested that proinflammatory agents may induce local and systemic inflammatory responses in normal subjects, which could contribute to the risk of developing asthma and chronic obstructive lung disease (Michel, 2000; Schwarze et al., 2000).
2. Materials and methods
tively (Goggans and Copenhaver, 1939). No melting point was available for MINP. The identities of MnBP, MnOP, MINP and MIDP were further confirmed and the purities determined by 1H NMR spectroscopy. The purities of MnBP, MnOP and MINP were all more than 95%. Extensive carbon-NMR analysis showed that the MIDP was a mixture of at least six isomers. The alcohol used for the synthesis of MIDP was a mixture of several alcohols, comprised of two main components, two components in lower concentrations and even lower concentrations of at least two other alcohols. All alcohols were present in the MIDP synthesized. Additionally, the MIDP contained low concentrations of impurities, which could include phthalic acid and phthalic acid diesters. In order to enhance the solubility of the monophthalates, 100 ml of 99.9% ethanol (Danisco Destiller, Aalborg, Denmark), and 400 ml PEG 400 (MerckSchuchardt, Germany), were added stepwise to 40 mg of pure monophthalate and the suspensions were mixed vigorously for 30 seconds. HAMS+ was added to a final concentration of 10 mg monophthalate per ml and the suspensions were subsequently shaken for 30 min, followed by final dilution. The effect of PEG 400 on A549 cells was tested in parallel with monophthalates. The concentrations of PEG 400 did not exceed 0.2% in any of the test and control solutions.
2.1. Reagents
2.2. A549 bioassay
Cells were grown in HAMS F12 (Gibco, Paisley, Scotland) supplemented with penicillin (100 IU/ml, Gibco) and streptomycin (100 mg/ml, Gibco), l-glutamine (2 mM, Gibco), and fetal bovine serum (FBS, 10%, Gibco) (HAMS+). Phorbol-myristate-acetate (PMA) (Sigma, St. Louis, MO, USA), tumour necrosis factor a (TNF-a, BD Pharmingen, San Diego, CA, USA), and lipopolysaccharide from Escherichia coli (LPS, Sigma) were used as control stimulants in 10-fold dilutions ranging from 5.0 ng/ml to 0.05 ng/ml for PMA, from 10 ng/ml to 0.1 ng/ml for TNF-a, and from 10 mg/ml to 0.01 mg/ml for LPS. The following monophthalates were used as stimulants: Mono-n-butyl phthalate (MnBP), monobenzyl phthalate (MBnP), mono-n-octyl phthalate (MnOP), mono-2-ethylhexyl phthalate (MEHP), mono-iso-nonyl phthalate (MINP), and mono-iso-decyl phthalate (MIDP). MBnP (purity > 90%) and MEHP (purity > 98%) were purchased from Tokyo Kasei Organic Chemicals (Tokyo, Japan). MnBP, MnOP, MINP, and MIDP were synthesized in our laboratory, as described by Larsen et al. (2001). The melting points of the dried MnBP, MnOP and MINP were found to be 73.0–73.5 C, 22.5–23.3 C and 50.0–51.0 C, respectively. In the literature, the melting points for MnBP and MnOP are reported to be 73.1–73.5 C and 21.5–22.5 C, respec-
The A549 bioassay is based on the human lung epithelial cell line A549 (ATCC CCL-185, American Type Culture Collection, Rockville, MD, USA). Cells were seeded in 24-well plates (Nunc, Denmark) at 105 cells/ well in 1 ml HAMS F12 medium supplemented with penicillin (100 IU/ml) and streptomycin (100 mg/ml), lglutamine (2 mM) and fetal bovine serum (FBS, 10%, Gibco) (HAMS+) and incubated overnight at 37 C, 5% CO2, and 100% relative humidity. The next day, the medium was aspirated and replaced with 900 ml of HAMS+ plus 100 ml stimulant. All monophthalates were tested in 2-fold dilutions and the final monophthalate concentrations ranged from 2000 mg/ml to 15.6 mg/ml. Cells were stimulated in triplicate and incubated for 24 h at 37 C, 5% CO2, and 100% relative humidity. A total of three stimulation series with monophthalates were performed and, in each series, unstimulated controls were included as 6 triplicates. Furthermore, cells were stimulated with the control substances PMA, TNF-a, and LPS, as well as PEG 400. Supernatants were harvested and kept at 45 C until analysis of IL-6 and IL-8 concentrations. 2.2.1. ELISA for IL-6 and IL-8 For the determination of IL-6 and IL-8 production from A549 cells, OptEIA kits from BD Pharmingen
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(San Diego, CA, USA) were used according to the manufacturer. However, in order to improve the analytical range, the standard curves were extended from 300 pg/ml to 600 pg/ml for IL-8, and from 250 pg/ml to 500 pg/ml for IL-6.
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slope of the concentration–response curve is referred to as the stimulatory potency, which may be used to compare the potencies of the different monophthalates to induce cytokine production; the steeper the slope, the more potent the monophthalate is in stimulating the production of the cytokine.
2.3. Assessment of cytokine production 2.5. Assessment of suppressive properties The ratio between cytokine concentration in the supernatant from exposed cell culture and the unexposed (negative control) cell culture was defined as the stimulatory index (SI):
SI ¼
ðconcentration of IL from exposed cellsÞ= ðconcentration of IL from the negative controlÞ
Negative controls were included in each exposure series and, thus, the SI is normalised for day-to-day variation, allowing comparison of SI-values achieved on different days. 2.4. Assessment of the potency of monophthalates to induce cytokine production The SI was plotted against the logarithm of the monophthalate concentration (cf. Figs. 1 and 2). The increasing part of these curves, which included from three to five concentrations, represents the concentrations of monophthalate that predominantly induced cytokine production in the A549 cells. Regression analyses were performed on this part of the curve. A statistically significant (P< 0.05) slope was accepted as a concentration-dependent cytokine production. The
Fig. 1. IL-6 production by A549 cells after stimulation with various monophthalates. The IL-6 production is expressed as the stimulatory index (SI), which is the ratio of IL-6 level in the exposure group to the IL-6 level in the corresponding negative control (medium only) group. MnBP: mono-n-butyl phthalate; MBnP: monobenzyl phthalate; MnOP: mono-n-octyl phthalate; MEHP: mono-2-ethylhexyl phthalate; MINP: monoisononyl phthalate; MIDP: monoisodecyl phthalate.
To compare the suppressive properties of the monophthalates, we established a No-Observed Effect Level (NOEL) for this effect. We defined the NOEL as the highest concentration of monophthalate that did not give rise to a reduced cytokine production, which, in most cases, was the same as the concentration that induced maximum cytokine production. The NOEL was assessed visually from Figs. 1 and 2.
3. Results Six monophthalates were tested for their potential to stimulate IL-6 and IL-8 production in A549 cells. In general, the monophthalates tend to stimulate cytokine production at lower concentrations, whereas higher concentrations of monophthalate gave rise to a reduced cytokine production compared to lower monophthalate concentrations. Thus, the concentration–response curves were bell-shaped in most cases (cf. Figs. 1 and 2). 3.1. Mono-n-butyl- and monobenzyl phthalate These compounds gave rise to non-significant or relatively low stimulatory potencies for both IL-6 and IL-8
Fig. 2. IL-8 production by A549 cells after stimulation with various monophthalates. The IL-8 production is expressed as the stimulatory index (SI), which is the ratio of the IL-8 level in the exposure group to the IL-8 level in the corresponding negative control (medium only) group. MnBP: mono-n-butyl phthalate; MBnP: monobenzyl phthalate; MnOP: mono-n-octyl phthalate; MEHP: mono-2-ethylhexyl phthalate; MINP: monoisononyl phthalate; MIDP: monoisodecyl phthalate.
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production. Also, the cytokine suppressive effects of these compounds were relatively weak (cf. Figs. 1 and 2 and Table 1). 3.2. Mono-2-ethylhexyl-, mono-n-octyl-, mono-isononyl- and mono-iso-decyl phthalate All of these compounds showed relatively high stimulatory potencies, i.e. they gave rise to steeper concentration–effect curves. The substances tend to stimulate both IL-6 and IL-8 production, even though the stimulatory potencies in all cases were higher for IL6 (cf. Figs. 1 and 2 and Table 1). At the same time, MEHP, MnOP, MINP, and MIDP, all have more pronounced cytokine suppressive effects than MnBP and MBnP. 3.3. PMA, TNF- and LPS These compounds were included as positive controls. They all stimulated cytokine production in a concentration-dependent manner (data not shown). 3.4. PEG 400 The concentrations of the cosolvents PEG 400 and ethanol used in the present study did not give rise to cytokine suppressive effects (data not shown).
4. Discussion All of the monophthalates studied could stimulate cytokine production, but to varying degrees. Also, at higher concentrations, all of the monophthalates studied gave rise to suppression of cytokine production. The latter effect was most likely due to a toxic effect on the cells; this is supported by light microscopy observations, showing lysis of some of the cell membranes. PEG 400
and ethanol did not cause cell lysis under the conditions of the test. The results showed a prominent change in stimulatory potency among the monophthalates. Monophthalates with fewer than eight carbon atoms in their alkyl side chain were weak cytokine inducers, whereas monophthalates with eight or more carbon atoms in the alkyl side chain were more potent cytokine inducers. These findings are generally in accordance with a study in mice, showing that MEHP, MnOP and MINP promoted the formation of Th2-dependent antibodies, whereas MnBP, MBnP and MIDP did not (Larsen et al., 2001). Taking the results from the present study with those reported by Larsen et al. (2001) together, it is tempting to hypothesize that the promotion of antibody production (adjuvant effect) and the proinflammatory effects are interrelated, even though the adjuvant effect of monophthalates is most likely due to a more specific mechanism than simple inflammation, as MIDP stimulated IL-6 and IL-8 production, but lacked an adjuvant effect. Also, it is obvious from Table 1 that the cytokine suppressive effect of the monophthalates increased with increasing alkyl side chain length. This structure-activity relationship of monophthalates has previously been reported to exist also for suppressive effects on the monocytic cell line, THP-1 (Glue et al., 2002), and in mice (Larsen et al., 2001). It is tempting to speculate that the cytokine stimulatory and suppressive effects are interrelated, as the most powerful cytokine stimulants in the present study were also the most potent cytokine suppressors. At the same time, weak cytokine stimulants were weak cytokine suppressors. The levels of IL-6 and IL-8 seem to be correlated - an observation that is in accordance with other studies in which A549 cells have been exposed to proinflammatory substances (Fu¨nfstu¨ck et al., 2001; Saraf et al., 1999; Strˇ ı´zˇ et al., 2000). Epithelial cell lines produced elevated levels of IL-6 and IL-8 after exposure to diesel exhaust particles (Steerenberg et al., 1998; Ohtoshi et al., 1998),
Table 1 Compound
Mono-n-butyl phthalate Monobenzyl phthalate Mono-2-ethylhexyl phthalate Mono-n-octyl phthalate Monoisononyl phthalate Monoisodecyl phthalate
Cna
4 7d 8 8 9 10
Stimulatory potencyb
NOEL for suppressionc
IL-6
IL-8
IL-6 (mg/ml)
IL-8 (mg/ml)
3.1 NS 6.8 23 9.1 8.6
NS NS 2.4 3.9 3.8 2.8
1000 1000 250 125 125 62.5
1000 2000 250 125 125 62.5
NS: not statistically significant concentration–response relationship (regression analysis). a Cn indicates the number of carbon atoms in the alkyl side chain. b The stimulation potency is the slope of the log concentration–response curve (see Materials and methods for further explanation). c The no-observed effect level (NOEL) is here defined as the highest concentration that does not give rise to a reduced cytokine production. d The seven carbon atoms are located in a benzyl group, i.e. a system that is both aromatic and aliphatic, whereas the other monophthalates studied here have aliphatic side chains.
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a well-recognized environmental adjuvant that selectively enhances the Th2 response (Løvik et al., 1997). Th2 cells produce IL-6 (Seymour et al., 1996), which is able to polarize naı¨ve CD4+ T-cells to effector Th2 cells (Gehring et al., 1998; Rincon et al., 1997). Furthermore, proinflammatory agents may play a role in the exacerbation of asthma by activating an inflammatory cascade, which amplifies the inflammatory response to inhaled antigen, as reported in two field studies (Michel et al., 1996; Rizzo et al., 1997). In conclusion, this study shows that some monophthalates are able to enhance IL-6 and IL-8 synthesis in a human lung epithelial cell line. These cytokines play a role in the initiation of inflammation and possibly contribute to the polarization of a Th2-dominated immune response, which plays a role in the development of allergic respiratory diseases.
Acknowledgements This study has been produced as part of the research activities in the Centre for the Environment and Respiratory System, which is supported by the Danish Environmental Research Program.
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