MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 58 (2004) 215–219 www.elsevier.com/locate/marenvrev
Fatal attraction: Synthetic musk fragrances compromise multixenobiotic defense systems in mussels Till Luckenbach b
a,*
, Ilaria Corsi b, David Epel
a
a Hopkins Marine Station of Stanford University, Oceanview Blvd, Pacific Grove, CA 93950, USA Dipartimento di Scienze Ambientali ‘‘G. Sarfatti’’, Universita degli Studi di Siena, via delle Cerchia 3, 53100 Siena, Italy
Abstract We studied interactions of nitromusk compounds musk ketone and musk xylene and polycyclic musks Galaxolidee (HHCB), Celestolidee (ADBI), Tetralidee (AHTN), and Traseolidee (AITI) with multixenobiotic resistance (mxr) transporters in gill tissue of the marine mussel Mytilus californianus (Conrad, 1837). A competitive substrate transport test with rhodamine B was used to assay modulation of transport activity by musks. All tested musks inhibited the transport activity in the low lm range as indicated by increased accumulation of rhodamine B in the tissue. Compared to known substrates of mxr transporters, the effective concentration range was similar to quinidine and about 100 times higher than verapamil. Musk ketone and musk xylene also inhibited efflux of rhodamine B from gill tissue which was loaded with the dye and subsequently incubated with these compounds. Synthetic musk compounds are persistent environmental pollutants in aquatic environments with a high potential to bioaccumulate. As potent inhibitors of mxr transporters they may also play a role as chemosensitizers that enable toxic mxr substrates to accumulate in cells of aquatic organisms. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Nitromusks; Polycyclic musks; Multixenobiotic resistance; Mxr; Chemosensitizer; Mytilus californianus
*
Corresponding author. Tel.: +1-831-655-6227; fax: +1-831-375-0793. E-mail address:
[email protected] (T. Luckenbach).
0141-1136/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2004.03.017
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Pharmaceutical and personal care products (PPCP) have received increasing attention as environmental pollutants in the last years. Some of these chemicals, as synthetic musk compounds, show typical persistent organic pollutants (POP) like properties: they are persistent and bioaccumulate. Synthetic musk compounds are widely used as inexpensive fragrances in personal care products, as in soaps, detergents, washing powders, etc. They are released into the environment through sewage effluents and appear in aquatic environments in concentrations of 109 – 1012 M. Despite evidence of low acute toxicity, high bioconcentration factors of 1000–5000 and chronic toxic effects have raised concern (Kafferlein, Goen, & Angerer, 1998; Rimkus, 1999; Wollenberger, Breitholtz, Kusk, & Bengtsson, 2003; Yamagishi, Miyazaki, Horii, & Akiyama, 1983). Indirect toxic actions, as by chemosensitizers that compromise toxicant-defense systems, have not been studied previously. Synthetic musks were tested for their ability to interact with multixenobiotic (mxr) transporters in the marine mussel Mytilus californianus (Conrad, 1837). Mxr transporters are ATP dependent efflux pumps that remove a broad spectrum of chemically unrelated xenobiotics from the cell (Kartner, Riordan, & Ling, 1983). These membrane proteins have been found in a variety of aquatic organisms where they work as a ‘‘first line of defense’’ against toxic agents (Bard, 2000; Epel, 1998). Also, anthropogenic pollutants have been shown to have substrate properties (Bard, 2000; Epel, 1998; Kurelec, 1992). A competitive substrate transport test using rhodamine B (RB), a fluorescent substrate of mxr transporters, was used to assay modulation of transport activity by musks. If the efflux of RB is inhibited by a test compound, RB accumulates in the cell to a higher degree which is indicated by increased fluorescence. The test compounds were: nitromusks musk ketone (MK) and musk xylene (MX), polycyclic musks Galaxolidee (HHCB) (GAL), Celestolidee (ADBI) (CEL), Tetralidee (AHTN) (TET), and Traseolide (AITI) (TRA), and known mxr substrates ()verapamil hydrochloride (VER) and quinidine (QUI). MK, MX, GAL, CEL were gifts from IFF, Union Beach, NJ, TET from Bush Boake Allen Inc., Jacksonville, FL, and TRA from Quest International, Mount Olive, NJ. RB, VER, and QUI were purchased from Sigma, St. Louis, MO. Ethanol stocks of the musks, VER and QUI were stored at 4 °C in the dark. Mussels were collected from the rocky intertidal at Hopkins Marine Station in Pacific Grove, CA (valve length: 81 5 mm). Discs (diameter 7 mm) were cut out from gill tissue with a biopsy punch (Acuderm, Fort Lauderdale, FL) and mucus was removed with forceps. The tissue discs were incubated under slight rocking in filtered seawater (FSW) with 1 lM RB and different concentrations of the test compounds or solvent only (final concentration of solvent: 1%) for 90 min at 14 °C in the dark (n ¼ 2–4 tissue discs per tested concentration of each compound). Thereafter, the tissue discs were briefly washed in FSW and frozen. 550 lL of butanol were added as a solvent and the tissue was sonicated for 15–30 s to extract intracellular RB and centrifuged for 10 min at 14,000g. The amount of dye in the supernatant was determined with a spectrofluorometer (Perkin–Elmer; emission: 545 nm, excitation: 575 nm). Data were standardized by dividing values for the tested compounds by the control values from the respective experiment. The ex-
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periments were repeated 3 times for each compound and mean values and standard deviations were determined. All tested musks caused increased accumulation of RB in a dose-dependent way compared to the control levels (Fig. 1). The increase of fluorescence averaged over all musks was 1.4- (1 lM) and 1.9-fold (10 lM), respectively. Inhibitory effects of nitromusks appeared to be slightly higher compared to the polycyclic musks (mean fluorescence increase of nitromusks vs. polycyclic musks: 1.5- vs. 1.3-fold (1 lM), 2.0- vs. 1.8-fold (10 lM). Effective inhibitory concentrations of musks were similar to QUI and approximately 100 times higher than for VER. Nitromusks MK and MX were additionally tested for inhibiting efflux of RB after loading the cells from gill tissue with the dye. After incubating tissue discs in FSW with 1 lM RB for 90 min they were transferred to plain FSW or FSW with either 10 lM MK, 10 lM MX, or 10 lM VER. Discs were frozen directly after exposure to RB and after 10 min, 30 min and 60 min of washing and the amount of intracellular RB was determined in the above described way.
relative fluorescence (fold increase vs. control)
3.0 1 µM (*0.01 µM) 10 µM (*0.1 µM)
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Fig. 1. Inhibition of mxr transporters in mussel gill tissue by nitromusk and polycyclic musk compounds (1 and 10 lM) and known mxr substrates quinidine (1 and 10 lM) and verapamil (0.01 and 0.1 lM) as indicated by relative fluorescence levels (mean SD, n ¼ 3) of rhodamine B. Rhodamine B, a substrate of mxr transporters, was applied together with test compounds in a competitive substrate transport test.
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35 control 10 µM musk xylene 10 µM musk ketone 10 µM verapamil
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absolute fluorescence
25 20 15 10 5 0
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t (min) Fig. 2. Efflux of rhodamine B as indicated by fluorescence levels (mean SD, n ¼ 3 (t ¼ 0, 30 and 60 min), n ¼ 2 (t ¼ 10 min)) after 0, 10, 30 and 60 min of washing of rhodamine B preloaded mussel gill tissue discs in 10 lM of nitromusks musk xylene and musk ketone and verapamil.
Both MK and MX inhibited efflux of RB, but were less effective than VER (Fig. 2). After 60 min of washing fluorescence was 1.8-fold (MX) and 2.4-fold (MK) higher compared to the control. The increase in the VER treatment was 4.5-fold. Our data show that synthetic musks are effective inhibitors of mxr transporters in marine mussels. As such they may enhance the toxic potential of other mxr substrates by enabling them to accumulate in the cell. Their potential role as chemosensitizers should be considered in future evaluations of synthetic musk compounds as environmental pollutants.
Acknowledgements Our research was supported by the German Academic Exchange Service (DAAD), US. Fulbright Program 2002–2003, California Sea Grant Program.
References Bard, S. M. (2000). Aquatic Toxicology, 48, 357–389. Epel, D. (1998). Comparative Biochemistry and Physiology A, 120, 23–28.
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