AMP deaminase in vitro inhibition by xenobiotics

Environmental Toxicology and Pharmacology 19 (2005) 291–296

AMP deaminase in vitro inhibition by xenobiotics A potential molecular method for risk assessment of synthetic nitro- and polycyclic musks, imidazolium ionic liquids and N-glucopyranosyl ammonium salts A.C. Składanowskia , P. Stepnowskib,∗ , K. Kleszczy´nskia , B. Dmochowskab a

Intercollegiate Faculty of Biotechnology, Medical University of Gda´nsk and University of Gda´nsk, PL 80-211 Gda´nsk, ul. Debinki 1, Poland b Faculty of Chemistry, University of Gdansk, PL 80-952 Gdansk, ul. Sobieskiego 18, Poland Received 12 January 2004; accepted 3 August 2004 Available online 30 September 2004

Abstract The usefulness of in vitro AMP deaminase inhibition was examined as a potential molecular method in risk assessment of xenobiotics. The enzyme participates in the principal purine nucleotide interconversion and degradation pathways, and its absence caused perturbations in the cellular ATP pool. The compounds selected were synthetic musks with a known negative environmental impact and the toxicologically unknown ionic liquids and N-glucopyranosyl ammonium bromides, which have recently attracted much interest from the chemical and related industries. All the compounds tested demonstrated a dose-dependent inhibition of AMP deaminase activity. IC50 ranged from 0.3 ␮M for polycyclic musks to 500 ␮M for N-glucopyranosyl trimethylammonium bromide. Analysis of Dixon plots showed the inhibition type for all the compounds to be noncompetitive. The results support the choice of such an assay for the prospective risk assessment of these compounds. © 2004 Elsevier B.V. All rights reserved. Keywords: AMP deaminase; Synthetic musk; Ionic liquids; Quaternary amines

1. Introduction When evaluating novel, possible biological responses to pollutant exposure in vivo, a fundamental principle must be taken into consideration, namely, that any such response be essential to the normal functioning of a cell, tissue or organism. Moreover, when an ecotoxicological effect is examined, the effect of exposure should assume a causal relationship between the pollutant concentration in the environment and the response of the target factor (molecule, cell, tissue, etc.), which can be extrapolated to the level of populations and above (Handy et al., 2003).

∗

Corresponding author. Tel.: +48 58 3450 448. E-mail address: [email protected] (P. Stepnowski).

1382-6689/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.08.005

AMP deaminase (AMP-DA, AMP-aminohydrolase, EC 3.5.4.6.) is common in the tissues of invertebrates, higher organisms, and plants. Belonging as it does to the hydrolase class, it is involved in a number of physiological processes, such as the conversion of adenine nucleotide to hypoxanthine or guanine nucleotide, the stabilization of the adenylate energy charge, the metabolism of amino-acid-derived nitrogen, and the reactions of the purine nucleotide cycle. The numerous physiological regulators of this enzyme include nucleotides, inorganic phosphate, sugar phosphates, phospholipids, fatty acid-CoAs and amino acids. AMP deaminase deficiency has been found to influence muscle physiology. Very broad involvement in metabolism as well as its physiological roles provide an excellent opportunity for further toxicological and ecotoxicological evaluation of AMP deaminase inhibition by selected xenobiotics.

292

A.C. Składanowski et al. / Environmental Toxicology and Pharmacology 19 (2005) 291–296

AMP deaminase has already been targeted for inhibition by herbicides: this causes necrosis in selected plant cells through perturbation of the intracellular ATP pool (Dancer et al., 1997). The 8-hydroxy-[1,3]-diazepin ring of coformycin or carbocyclic coformycin and their analogues were found to be the most potent inhibitors mimicking a transition state or high-energy intermediate in reactions catalyzed by AMP-DA (Lindell et al., 1999). For this study we have chosen two general groups of compounds: one comprises synthetic musks, which have been produced for more than a century and are ubiquitous, persistent and bio-accumulative pollutants, the other involves imidazolium ionic liquids and N-glucopyranosyl ammonium salts, which are of growing industrial interest, yet whose toxicological and environmental properties are almost unknown. Synthetic musks are widely used as fragrances and fixatives in a wide range of consumer goods. Musk xylene, musk ketone and musk ambrette are the oldest synthetic nitro musks, which have now come under scrutiny in a number of countries because of their persistence and possible adverse environmental impacts (Daughton and Ternes, 1999). Nitro musk has been found to bio-accumulate in invertebrates, fish, human milk, and has been detected worldwide in river water, where it has resisted biodegradation (Geyer et al., 1994; Suter-Eichenberger et al., 1998). Polycyclic musks, e.g. Galaxolide and Tonalide, are the major musks used today in nearly every commercial fragrance formulation and food additives. They have been identified in fish muscle and fat tissue, as well as in surface waters in the vicinity of wastewater treatment plants (Draisci et al., 1998; Heberer et al., 1999). Ionic liquids are low-melting-point salts representing a new class of non-molecular ionic solvents. They are considered to be highly promising neoteric solvents: they are easily designed in that a variety of anions are attached to the same bulky organic cation (Rogers and Seddon, 2002). Ionic liquids are already associated with the term “green”, but only because they have no measurable vapour pressure, meaning that they emit no volatile organic compounds. Little is known about either their toxicological or their ecotoxicological properties (Jastorff et al., 2003). It is therefore crucial to report on results and propose new methods for evaluating the possible

toxic effects of these compounds before they are put on the market on an industrial scale. With their potential antibacterial and fungicidal properties, N-glucopyranosyl pyridinium and ammonium bromides could also be very attractive to industry, although knowledge of their toxicity in Eukaryota is as scanty as that of ionic liquids.

2. Materials and methods 2.1. Isolation of AMP deaminase The enzyme was isolated from skeletal muscle of rats following the modified method of Smiley et al. (1967). Male Wistar rats weighing 200–250 g were used. After the rats had been decapitated, their leg and back muscles were dissected, damp-dried, minced, and homogenized in ice-cold buffer (0.18 M KCl; 0.054 M KH2 PO4 ; 0.035 M K2 HPO4 ; 0.1 mM DTT; pH 6.5; 25%) for 3 min using MSE WaringBlendor. The homogenate was centrifuged at 3000 rpm at 4 ◦ C for 30 min, and the supernatant was further centrifuged at 16 000 rpm for 30 min. The final supernatant was filtered and incubated for 1h with fresh prepared phosphocellulose PC-11 suspension as in the original Smiley et al. (1967) procedure. After centrifugation at 1000 rpm for 1 min, the suspension was rinsed twice with homogenization buffer and transferred to the column (22 cm length, 2 cm i.d.). AMP deaminase was eluted in a linear gradient from 0.75 to 2.0 M of KCl adjusted to pH 6.5. Samples were analyzed for total protein content using Bradford’s (1976) method (Fig. 1). Bovine serum albumin was used as standard. 2.2. AMP deaminase assay AMP deaminase activity in the eluted fractions was determined using Chaney and Marbach’s (1962) method, in which ammonium derived from AMP reacts with phenol and sodium hypochlorite to produce a relative amount of blue-colored indophenol. In the assay, 10 ␮l of 100 mM AMP substrate in succinate–KOH buffer (pH 6.5) was added to 0.5 ml of the

Fig. 1. Elution profile of AMP deaminase (solid line) and protein concentration (dashed line) from rat skeletal muscle. Elution gradient at a flow of 15 ml/h, from 0.45 to 2 M KCl, pH 6.5, fraction volume 2 ml.

A.C. Składanowski et al. / Environmental Toxicology and Pharmacology 19 (2005) 291–296

reaction mixture and left for 2 min at 30 ◦ C. The reaction was terminated by the addition of 0.5 ml of 2% phenol solution and 0.5 ml of 0.05% sodium hypochlorite solution, after which it was kept for 1 h at 37 ◦ C. The enzymatic reaction rate was quantified using an Epoll-2 colorimeter equipped with a 620 nm filter against a blank without substrate for each activity measurement. The activity was expressed as micromoles of ammonia released from AMP during 1 min by 1 mg protein. One unit of activity is defined as 1 ␮mol of product formed in 1 min of the reaction at 37 ◦ C. During the analysis of the xenobiotic concentration inhibiting 50% of enzyme activity (IC50 ), the same time and temperature assay conditions were applied and the concentration of AMP was 1 mM. All compounds analyzed were checked for possible interference with assay reagents and for ammonia content—no significant interference was found. The xenobiotics were assayed in duplicate series over varying concentration ranges depending on their previously measured inhibiting potency. IC50 values were determined from the lognormal regression of enzyme activity vs. substrate concentration. The type of AMP-DA inhibition was estimated by plotting Dixon’s linear relation of 1/V = k(I) for two AMP concentrations of 0.1 and 0.5 mM. 2.3. Synthetic musks Musk xylene [MX] and musk ketone [MK], Galaxolide [HHCB] and Tonalide [AHTN] were purchased as a reference materials for routine analysis from Promochem (Wesel, Germany). Substances were used as obtained without further purity checks. All compounds, together with their systematic and trade names and chemical formulae are listed in Table 1.

293

glucopyranosyl) pyridinium bromide were synthesized, checked for their high purities (>99.9%) and kindly supplied by Prof. A. Wi´sniewski (Faculty of Chemistry, University of Gda´nsk, Poland). These compounds, their systematic names and chemical formulae are listed in Table 1.

3. Results and discussion AMP deaminase was detected in fractions eluted from the phosphocellulose column where activity and protein content were proportional, which is indicative of high purity (Fig. 1). This was checked afterwards with routine SDS PAGE procedure (Fig. 2) and verified as 80% protein in the main band of the proper molecular weight. The specific activity measured under optimal conditions was ca. 50 units per mg protein. The protein content in the pooled fractions was 165 ␮g ml−1 . The present data show that all the xenobiotics investigated here are capable of inhibiting skeletal muscle AMP deaminase. The results of in vitro inhibition are presented in Table 2. All the compounds analyzed decrease enzymatic activity at different concentration levels. Synthetic musks display the greatest inhibitory capabilities. Both polycylic compounds (Galaxolide and Tonalide) and one of the nitro compounds (musk xylene) inhibit up to 50% of AMP-DA activity at 0.3 ␮M, while the other nitro – musk ketone – at 0.5 ␮M. By way of example, a dose response plot for musk ketone is shown in Fig. 3. Although not as effective, ionic liquids also inhibited AMP deaminase activity. On examination of the 1-n-butyl imidazolium cation is examined, one finds a distinct involvement of counter anions. IC50 values for those containing a flu-

2.4. Ionic liquids The ionic liquids used in these experiments were synthesized and kindly supplied by B. Ondruschka (Institute for Technical and Environmental Chemistry, Jena, Germany). They were the 1-n-butyl-3-methylimidazolium [BMIM] salts of the following anions: hexafluorophosphate, tetrafluorobromate, p-tosylate and chloride. All compounds were checked for their purities by HPLC analysis following a method of Stepnowski et al. (2003). No nonvolatile impurities were found in this assay. Volatile contaminants (up to 300 ◦ C b.p.) of ionic liquids were analyzed by gas chromatography headspace technique during routine checks of synthesized products in the Prof. Bernd Jastorff’s laboratory at the Centre for Environmental Research and Technology, UFT, University of Bremen. They were at the level of 0.1%. The compounds, their systematic names and chemical formulae are listed in Table 1. 2.5. N-Glucopyranosyl ammonium salts N-(2,3,4,6-tetra-O-Acetyl-␤-d-glucopyranosyl) trimethylammonium bromide and N-(2,3,4,6-tetra-O-acetyl-␤-d-

Fig. 2. SDS PAGE of AMP deaminase from rat skeletal muscle, showing sequence of purification (M: marker, H: homogenate, PC: before column, F: fraction studied).

294

A.C. Składanowski et al. / Environmental Toxicology and Pharmacology 19 (2005) 291–296

Table 1 The compounds studied, their systematic and trade names, chemical formulae and relative molecular masses Systematic name

Trade name/abbreviation

Mr

4-Acetyl-1-t-butyl-3,5-dimethyl-2,6-dinitrobenzene

Musk ketone [MK]

294.31

1-t-Butyl-3,5-dimethyl-2,4,6-trinitrobenzene

Musk xylene [MX]

297.27

1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta-␥-2-benzopyran

Galaxolide [HHCB]

258.40

1-(5,6,7,8-Tetrahydro)-3,5,5,6,8,8-hexamethyl-2-naphthalenyl-ethanone

Tonalide [AHTN]

258.40

1-n-Butyl-3-methylimidazolium hexafluorophosphate

[BMIM][PF6 ]

284.18

1-n-Butyl-3-methylimidazolium tetrafluoroborate

[BMIM][BF4 ]

226.03

1-n-Butyl-3-methylimidazolium chloride

[BMIM][Cl]

174.67

1-n-Butyl-3-methylimidazolium p-tosylate

[BMIM][pTS]

311.40

N-(2,3,4,6-tetra-O-Acetyl-␤-d-glucopyranosyl)pyridinium bromide

[TAGPB]

490.30

N-(2,3,4,6-tetra-O-Acetyl-␤-d-glucopyranosyl)trimethylammonium bromide

[TAGMAB]

470.31

Chemical formula

Musks

Ionic liquids

N-glucopyranosyls

orine compartment [PF6 ]− and [BF4 ]− are higher (5 ␮M) than those for chloride and p-tosylate (10 ␮M). The dose response curves for ionic liquids are similar to the corresponding curves for the musks. A plot of AMP-DA activity versus [BMIM][PF6 ] concentration is presented in Fig. 4. The AMP deaminase inhibition constants obtained for Nglucopyranosyls are one order of magnitude higher in com-

parison to those obtained with ionic liquids; IC50 values were equal to 50 and 500 ␮M for [TAGPB] and [TAGMAB], respectively. The only structural difference between the two compounds is the amine fragment of the compound (trimethylammonium versus pyrydinium), which is responsible for the one order of magnitude difference in the inhibition constant.

A.C. Składanowski et al. / Environmental Toxicology and Pharmacology 19 (2005) 291–296 Table 2 AMP deaminase IC50 values (␮M) for synthetic nitro- and policyclic musks, imidazolium ionic liquids and N-glucopyranosyl amines Compound Synthetic musks [MK] [MX] [HHCB] [AHTN] Ionic liquids [BMIM][PF6 ] [BMIM][BF4 ] [BMIM][Cl] [BMIM][pTS] N-glucopyranosyls [TAGPB] [TAGMAB]

IC50 (␮M) 0.5 0.3 0.3 0.3

Concentration range (␮M) 10−6 to 102

5 5 10 10

10−4 to 103

50 500

10−3 to 103

In order to carry out a preliminary evaluation of the nature of AMP deaminase inhibition by the xenobiotics studied here, further experiments were performed in the presence of two close-to-physiological AMP concentrations: 0.1 and 0.5 mM. For all the analyzed compounds the intersection points of the Dixon dependencies (Fig. 5) are close to a negative intercept on the [I]-axis, suggesting that the potential mechanism is rather noncompetitive. This fact is of the greatest importance if the use of AMP-DA as a

295

biomarker is to be considered in future toxicological and ecotoxicological assessment. The given dose response effect should always be independent of the natural substrate concentration. The high susceptibility of the enzyme to all synthetic musks offers good prospects for the use of AMP deaminase inhibition as a reliable assay of the indoor and outdoor exposure of these compounds. The same should apply to ionic liquids, but so far little is known about their toxicity. The use of this potential molecular test to evaluate the ecotoxicological effect of these interesting compounds can only be considered in prospective terms. In the case of N-glucopyranosyls, the dose response effect is also detectable but at a much higher concentration level. The interesting feature of the one-orderof-magnitude inhibition difference obtained by substituting trimethylammonium with pyridinium demonstrates a selective binding preference for heterocyclic moieties similar to that shown by nucleosides or nucleotide-type compounds naturally reacting with AMP-DA. Although this work is still in its infancy, is has provided initial guidelines for the application of AMP deaminase inhibition as a useful assay for potential xenobiotics such as synthetic musks and ionic liquids. It is likely that this test system could be easily applied for assessment of exposure to the structurally related compounds already present in environment, functionalized with nitro groups or possessing het-

Fig. 3. In vivo effect of musk ketone on AMP deaminase inhibition.

Fig. 4. In vivo effect of 1-n-butyl-methylimidazolium hexafluorobromide on AMP deaminase inhibition.

296

A.C. Składanowski et al. / Environmental Toxicology and Pharmacology 19 (2005) 291–296

toxicological and ecotoxicological risk assessment”. Financial support was provided by a grant from the Medical University of Gdansk and by the Polish Ministry of Scientific Research and Information Technology (DS 8000-4-0141-4 and BW 8000-5-0195-4). This funding is gratefully acknowledged.

References

Fig. 5. Dixon plots for selected xenobiotics in the presence of 0.1 and 0.5 mM AMP.

erocyclic moieties in their structure. These are for example trinitrotoluene and its metabolites or quaternary ammonium herbicides such as paraquat, diqat or difenzoquat, both known for their persistence and extreme negative environmental impact. Future work should therefore focus on this group of compounds, which are of highest importance for the environmental chemistry and ecotoxicology. The method itself should be further verified for its utility with in vivo studies either on exposed organisms or specialized cell lines. Acknowledgments The work was supported within the NATO Collaborative Linkage grant (EST) “Designing molecular test systems for

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 7, 248. Chaney, A.L., Marbach, E.P., 1962. Modified reagents for determination of urea and ammonia. Clin. Chem. 8, 130. Dancer, J.E., Hughes, R.G., Lindell, S.D., 1997. Adenosine-5 phosphate deaminase. A novel herbicide target. Plant Physiol. 114, 119. Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107, 907. Draisci, R., Marchiafava, C., Ferretti, E., Palleschi, L., Catellani, G., Anastasio, A., 1998. Evaluation of musk contamination of freshwater fish in Italy by accelerated solvent extraction and gas chromatography with mass spectrometric detection. J. Chromatogr. A 814, 187. Geyer, H.J., Rimkus, G., Wolf, M., Attar, A., Steinberg, C., Kettrup, A., 1994. Synthetic nitro musk fragrances and bromocyclene—new environmental chemicals in fish and mussels as well as in breast milk ¨ and human lipids. Z Umweltchem Okotox 6, 9. Handy, R.D., Galloway, T.S., Depledge, M.H., 2003. A proposal for the use of biomarkers for the assessment of chronic pollution and in regulatory toxicology. Ecotoxicology 12, 331. Heberer, Th., Gramer, S., Stan, H.-J., 1999. Occurrence and distribution of organic contaminants in the aquatic system in Berlin. Part iii: Determination of synthetic musks in Berlin surface water applying solid-phase microextraction (SPME) and gas chromatography– mass spectrometry (GC/MS). Acta Hydrochim. Hydrobiol. 27, 150. Jastorff, B., St¨ormann, R., Ranke, J., M¨olter, K., Stock, F., Oberheitmann, B., Hoffman, W., Hoffmann, J., N¨uchter, M., Ondruschka, B., Filser, J., 2003. How hazardous are ionic liquids? Structure–activity relationships and biological testing as important elements for sustainability evaluation. Green Chem. 5, 136. Lindell, S.D., Moloney, B.A., Hewitt, B.D., Earnshaw, C.G., Dudfield, P.J., Dancer, J.E., 1999. The design and synthesis of inhibitors of adenosine 5 -monophosphate deaminase. Bioorg. Med. Chem. Lett. 9, 1985. Rogers, R.D., Seddon, K.R. (Eds.), 2002. Ionic Liquids: Industrial Applications to Green Chemistry. American Chemical Society. Smiley, K.L., Berry, A., Suelter, C.H., 1967. An improved purification, crystallization and some properties of rabbit muscle 5 -adenylic acid deaminase. J. Biol. Chem. 242, 2502. Stepnowski, P., Muller, A., Behrend, P., Ranke, J., Hoffmann, J., Jastorff, B., 2003. Reverse phase liquid chromatographic method for the determination of selected room temperature ionic liquids cations. J. Chromatogr. A 993, 173. Suter-Eichenberger, S., Altorfer, H., Lichtensteiger, W., Schlumpf, M., 1998. Bioaccumulation of musk xylene (MX) in developing and adult rats of both sexes. Chemosphere 36, 2747.