Proton NMR Spectroscopic Studies on Tissue Extracts of Invertebrate Species with Pollution Indicator Potential

Comp. Biochem. Physiol. Vol. 118B, No. 3, pp. 587–598, 1997 Copyright  1997 Elsevier Science Inc. All rights reserved.

ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00063-1

Proton NMR Spectroscopic Studies on Tissue Extracts of Invertebrate Species with Pollution Indicator Potential J. O. T. Gibb,1 E. Holmes, J. K. Nicholson,1 and J. M. Weeks 2 1 Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London, WC1H OPP, U.K. and 2 Institute of Terrestrial Ecology, Monks Wood, Abbots Ripton, Huntingdon, Cambridgeshire, PE17 2LS, U.K.

ABSTRACT. High resolution proton nuclear magnetic resonance (1 H NMR) spectroscopic methods have been used to characterise tissue extracts of a series of common British invertebrate species with pollution indicator potential. These include two earthworm species Lumbricus rubellus (Hoffmeister) and Eisenia andrei (Savigny), two terrestrial isopods, Oniscus asellus (L.) and Porcellio scaber (Latreille), the diplopodous arthropod, Glomeris marginata (Villers) and a pulmonate gastropod, Arion subfuscus (Draparnaud). One and two-dimensional NMR techniques including 1 H-1 H homonuclear correlation and 1 H J-Resolved NMR spectroscopic methods were applied to allow characterisation of the major organic components in the tissue extracts. The extracts gave characteristic low molecular weight metabolite NMR fingerprints for each species studied. Endogenous metabolites identified included glucose and trehalose, a range of free amino acids and organic acids and bases. The presence or absence of metabolites observed in the NMR spectra was examined by cluster analysis to investigate species similarity and differences in metabolite profiles. The use of Principal Component Analysis to interrogate NMR data-reduced spectra of tissue extracts allowed for distinct separation of the two morphologically similar earthworm species L. rubellus and E. andrei. The work indicates that 1 H NMR spectroscopic methods provide a rapid means of profiling invertebrate biochemistry and may be of value in studies on the comparative toxicology of invertebrate species. comp biochem physiol 118B;3:587–598, 1997.  1997 Elsevier Science Inc. KEY WORDS. 1 H NMR spectroscopy, endogenous metabolites, tissue extracts, pollution indicators, Eisenia andrei, Lumbricus rubellus, Oniscus asellus, Porcellio scaber

INTRODUCTION High resolution 1 H NMR spectroscopy has been widely applied to investigate the composition of vertebrate biofluids in a variety of studies on endogenous and xenobiotic metabolism (17). 1 H NMR spectroscopy has proved to be particularly successful as a biomedical probe as it provides a rapid, specific but nonselective and nondestructive detector for a wide range of low molecular weight compounds in small samples of untreated biological fluid or tissue extract. Biochemical studies involving NMR spectroscopy have led to the discovery of novel metabolic ‘‘biomarkers’’ of toxicity and disease in mammals especially with the aid of pattern recognition (PR) methods (5,16,17). A prerequisite of toxicological investigations is knowledge of the basal metabolic status and biochemical composition of potential pollution indicator species. Furthermore, Address reprint requests to: J. K. Nicholson, Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London, WC1H OPP, U.K.; Tel./Fax 0171-380-7468; E-mail: [email protected]. Received 7 November 1996; revised 20 March 1997; accepted 14 April 1997.

such knowledge must include the degree of intraspecific variation that is to be expected in an unperturbed population. However, relatively little work has been performed on the NMR characterisation of low molecular weight metabolites in the tissues of invertebrate species. Where NMR spectroscopic techniques have been applied to invertebrate extracts, they have typically involved the structural investigation of a small range of steroids or proteins in highly purified preparations (4,7,10,12). The aim of our current work was to use 1 H NMR spectroscopic methods to characterise the low molecular weight components in tissue extracts of whole organisms found in terrestrial environments and with known pollution indicator potential. This is with a long-term view to developing NMR-pattern recognition (PR) techniques for investigating metabolite profile perturbations in ecotoxicological studies. As high resolution 1 H NMR spectra of biofluids are extremely complex, automatic data reduction, prior to the application of multivariate analysis techniques, such as PR (which include principal components analysis and nonlinear mapping), can be used to simplify spectral interrogation.

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TABLE 1. Summary of metabolites in tissue extracts (pH 7.3 6 0.2) observed by 1 H NMR in the species in which they

were found Metabolite Adenine Acetate Acetoacetate Alanine Betaine Choline Citrate Dimethylamine Ethanol Fumarate Glucose Glutamate Glutamine Histidine Isoleucine Lactate Leucine Lipid Lysine Phenylalanine Succinate Sucrose Threonine Trehalose Tyrosine Tryptophan Uridine Valine

L. rubellus

E. andrei

A. subfuscus

O. asellus

P. scaber

G. marginata

1

1

1 1

1 1 1 1

1 1

1

1 1

1

1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1

1 1

1 1

1

1 1

1 1 1 1

1 1 1 1

1 1 1

1 1 1 1

1 1 1 1 1 1 1 1 1 1

1 1 1

1

1

1

1 1 1

1 1 1

1 1 1

1 1 1

1

1

1 1

1 1

1 1

1 1 1

1

1 1

1

1

1

1 1

1: Metabolites observed in each replicate of the six invertebrate species (N 5 3, with the exception of A. subfuscus where N 5 1).

The use of terrestrial invertebrates as monitors of pollutants is well reviewed in the literature [e.g., (9)] and earthworms in particular are routinely used as ecotoxicological test organisms by regulatory bodies (18). The advantages inherent in the use of invertebrates in ecotoxicological studies are numerous, not least that they represent 95% of all animals species, that they are typically abundant and they are found at all trophic levels and in all ecosystems (3). There is considerable interest in the development of novel biochemical markers of pollutant exposure and NMR spectroscopy potentially represents a powerful approach to the study of perturbed biochemistry and toxicological biomarkers. MATERIALS AND METHODS Sample Preparation Individuals of the following species were collected from noncontaminated locations in the U.K., were allowed to depurate for a minimum of 24 h at 17°C (63) and 54% humidity (65): Arion subfuscus (Draparnaud), Eisenia andrei (Savigny), Glomeris marginata (Villers), Lumbricus rubellus (Hoffmeister), Oniscus asellus (L.), and Porcellio scaber (La-

treille). They were rinsed in distilled water, blot-dried, and snap-frozen in LN 2. After removal from LN 2 samples were homogenised in a 1 :2 ratio of body volume to volume of physiological Ringer solution (pH 7.3; 14). The homogenates were centrifuged (30 min/4,000 rpm) at 5°C, and the supernatant ultrafiltered (Sartorius Centrisart I) to a MW cut-off point of 10 kD. The extract was then either freezedried and reconstituted in D 2 O or 100 µl of D 2O was added to provide a spectrometer field frequency lock prior to NMR measurement. 1

H NMR Spectroscopy of Tissue Extracts

Single pulse 1 H NMR spectra were obtained using either a JEOL GSX500 or a Bruker AMX600 spectrometer operating at 500.14 and 600.13 MHz observation frequencies, respectively. Spectra were measured at ambient probe temperature, using 45° pulses and a 6000 Hz spectral width, typically with 256 free induction decays (FIDs) collected for each sample into 32,768 computer points. An acquisition time of 2.73 sec was used with a further delay of 2.27 sec to ensure that the spectra were obtained under T1-relaxed conditions. Where appropriate, the water 1 H signal was suppressed during acquisition by a gated secondary irradiation

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FIG. 1. Single-pulse 500 MHz 1H NMR spectra of tissue extracts of (A) Lumbricus rubellus, (B) Eisenia andrei, (C) Arion

subfuscus, (D) Oniscus asellus, (E) Porcellio scaber, and (F) Glomeris marginata. Many of the major metabolites are assigned.

field at the water resonance frequency. The FIDs were multiplied by an exponential weighting function corresponding to a 0.19 Hz line broadening prior to Fourier transformation. Two-Dimensional 1 H-1 H Correlation Spectroscopy (COSY) Two-dimensional COSY45 spectra (2) were obtained using a Varian VXR600 Spectrometer operating at 599.95 MHz 1 H resonance frequency and at ambient probe temperature (298 K). The following pulse sequence was used: [D 2 90° 2 t 1 2 45° 2 collect FID] Where D was a 2-sec relaxation delay, while t 1 was an incremented delay to allow modulation of the spin-spin coupling. Five hundred and twelve increments, with 16 FIDs were

collected into 4096 computer points with a spectral width of 6500 Hz and an acquisition time of 0.32 sec.

Homonuclear 1 H Two-Dimensional J-Resolved Spectroscopy (JRES) JRES spectra (1) were measured at 600.13 MHz using a Bruker AMX600 Spectrometer. The following pulse sequence was used: [D 2 90° 2 t 1 2 180° 2 t 1 2 collect FID for time t 2] D was a 2-sec relaxation delay, the t 2 acquisition time was 0.63 sec, while t 1 was an incremented delay to allow modulation of the spin-spin coupling. The F 2 domain was collected into 8192 computer points using a spectral width of 6550 Hz and the F1 domain used a 30 Hz spectral width

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FIG. 1. (continued)

with 64 increments. Eight FIDs were collected for each t 1 increment.

all other species N 5 3, and only metabolites observed in each replicate were included.

Statistical Analysis of Scored NMR Spectroscopic Data

Automated Data Reduction of 1 H NMR Spectra

Hierarchical clustering of scored data, to investigate the similarities of the 1 H NMR metabolite profiles of the species investigated, was performed using Minitab 10 statistical software. Cluster analysis was carried out on the data to eliminate the effects of variability in a population and different units of the observations (see 6 for overview). Metabolites were scored as either present or absent in the singlepulse 1 H NMR spectra of tissue extracts (Table 1). Each species was treated as a cluster and Euclidean distance was used to measure the distance between each cluster according to the single-linkage algorithm. The closest clusters were then merged to form a new cluster and the resulting hierarchy is shown as a dendogram. As only one tissue extract of A. subfuscus was analysed by 1 H NMR the results for it have been included for comparison purposes only. For

NMR spectral data reduction was carried out after analysis of tissue extracts from the two earthworm species, L. rubellus (N 5 5) and E. andrei (N 5 4), using the AMIX program (Bruker Analytische Messtechnik GmbH, Silberstreißen, Germany, 1996). The spectra between δ 0.2–10.0 was segmented into 256 regions of 0.04 ppm width each. The area under the spectrum was calculated for each region and expressed as an integral value (8). The area of the spectrum, which included water (δ 4.2–6.0), was excluded from the analysis to eliminate the variation in water suppression. The resulting data sets were tabulated using the SAS software suite (version 6.10) on a Silicon Graphics Power Indigo2  R8000 computer. A mean ‘‘noise value’’ was calculated from the first 15 segments (δ 10.00–9.60), which were known to contain no detectable metabolite signals in any samples.

Proton NMR Spectroscopic Studies on Invertebrate Pollution Indicator Species

591

FIG. 1. (continued)

The noise contribution to the spectral integral values was removed by subtraction of the calculated mean noise value from each segment. In order to reduce interference from artifacts, integral values less than five times the calculated mean noise value for all of the tabulated spectra were discarded. The remaining segments were scaled to the total integrated area of the spectrum. Principal Components Analysis of 1 H NMR Spectroscopic Data In order to establish the presence of any intrinsic speciesrelated patterns or clusters in the basic NMR data, Principal Component (PC) Analysis was performed on these data without any inclusion of information concerning the classification of samples on the basis of species. Each integral value was used as a descriptor for PC Analysis, which was performed on the correlation (the standardised covariance) matrix. PC Analysis methods involve the cal-

culation of linear combinations of the original descriptors, the PCs, such that each PC is orthogonal to all others with the first PC (PC1) containing the largest amount of variance (subsequent PCs contain progressively less variance) (11). Thus, a plot of PC1 versus PC2 provides the most efficient 2D representation of the information contained in the data set. A two-dimensional PC plot of PC3 versus PC2 was constructed in order to establish the presence of any intrinsic class-related patterns or clusters in these data. Student’s t-tests on the integral values and analyses of the eigenvectors were used to locate the spectral regions that contained metabolite resonances that showed significant variation between the two species and were therefore responsible for classification. In addition, to visualise differences in ratios and occurrence of metabolites, difference spectra were generated where a representative NMR spectra of L. rubellus tissue extract was subtracted from a representative NMR spectra of E. andrei tissue extract. The resulting

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FIG. 1. (continued)

figure displays the gross differences in the metabolic fingerprints of the two earthworm species. RESULTS A variety of endogenous metabolites were identified in each invertebrate species by use of one- and two-dimensional NMR methods. The compounds identified include many of the free primary amino acids, sugars such as glucose and trehalose, citric acid cycle intermediates such as citrate and fumarate, the organic bases choline and DMA, and several other organic acids including lactate and acetate (Fig. 1A– F). Indeed, at least 150 peaks were resolved in each of the tissue extracts including many as yet unassigned peaks. Glycerol signals seen in some samples were identified as a contaminant from the ultrafiltration process (despite extensive washing of ultrafilters). Metabolites that were observed in the single-pulse 1 H NMR spectra from all species are displayed in Table 1. The assignments are detailed in Table 2 which include those of resonances not assignable in the tissue extracts. The use of two-Dimensional NMR experiments allowed

detailed spectral assignments to be made and confirmed. COSY experiments mainly provide information on coupled spin systems with adjacent CH, CH 2, and CH 3 groups through their three bond 1 H-1 H connectivities, as is shown for lactate in Fig. 3. Homonuclear 1 H JRES spectroscopy show the scalar coupling patterns and coupling constant data dispersed in an orthogonal frequency domain to the chemical shift, as shown in Fig. 4 for the earthworm L. rubellus. With the use of such techniques more than 170 resolved 1 H NMR signals were observed, for example, in the tissue extract of L. rubellus (Figs 1A, 3, and 4). The 1 H NMR spectra of the tissue extracts showed characteristic metabolite profiles for each species examined. The metabolite profiles reflect the dominant extracelluar and intracellular low molecular weight components present in a variety of tissue compartments. Macromolecular components do not contribute to the spectra. The variation in low molecular weight components observed is likely to be influenced by a range of physiological variations, dietary differences and constitutive enzymatic differences between species. In order to make a qualitative comparison of the NMR spectra from the various species studied cluster analy-

Proton NMR Spectroscopic Studies on Invertebrate Pollution Indicator Species

593

FIG. 1. (continued)

sis was performed on a simple presence or absence score of each identified low molecular weight metabolite. Cluster analysis showed greatest similarity between the two woodlice species, O. asellus and P. scaber, and the two earthworms L. rubellus and E. andrei. Furthermore, the slug, A. subfuscus, was found to have greatest similarity to the earthworm cluster, whilst the millipede G. marginata had greatest similarity with the woodlouse cluster. Principal Component Analysis of NMR spectral data showed both metabolite clustering of individuals in a particular earthworm species and separations between the mapping position of the two species (Figs. 5a and b). Greatest separation of the two species was described by PC1 (66.3% of the variance) (Fig. 5a). PC2 described a further 17.2% of the total variance and PC3 a further 6.9%. Both the ttest and eigenvectors detected significant variance in the spectral region containing the glutamate resonances. In addition, the t-test detected significant variance in regions of the spectra containing the glucose α-4 and β-4, α-3, β-6, and α-5 1 H NMR signals at δ3.40, 3.70, 3.71, and 3.85, respectively. Eigenvectors additionally detected that sig-

nificant variance in two regions containing singlets from unassigned metabolites at δ2.62 and 2.65, which were found at higher ratios in L. rubellus tissue extracts. These and other differences can be visualised in difference spectra (Fig. 6). Resonances projecting above the baseline were either at relatively higher concentrations in E. andrei, such as those from glucose, glutamine and alanine, or were absent in L. rubellus extracts, such as betaine and choline. Resonances projecting below the baseline, including lactate and unassigned metabolites at δ1.54 and 2.42 were at higher ratios in L. rubellus. For the majority of resonances there were no differences in metabolites, for example lysine (shown in Fig. 6), where projections are equal above and below the baseline. DISCUSSION Single pulse high resolution 1 H NMR spectroscopy has shown that the tissue extracts investigated contain highly complex mixtures of low molecular weight metabolites (Fig. 1A-F). Despite the high degree of signal overlap observed

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FIG. 1. (continued)

in most of the spectra, many resonances can be assigned (particularly with the aid of two-dimensional NMR methods). Obvious differences were observed between the tissue extracts of the different species, not only in the ratios of the metabolites, but also in the absence or presence of certain metabolites (Table 1). The absence or presence of low molecular weight metabolites, detected by single pulse 1 H NMR, have been statistically analysed to investigate species similarity. As the isopods, O. asellus and P. scaber, and the diplopod, G. marginata all belong to the Phylum Arthropoda, one expects an inherent degree of phylogenetic similarity. Furthermore, there is notable evolutionary convergence of the land isopod (woodlice) and the myriapod diplopods (millipedes) (20). Their morphological and indeed behavioral and lifestyle similarities are, to some extent, reflected in their biochemistry: similarities include the presence of lipids, the 1 H NMR signal of the terminal CH 3 and long-chain (CH 2 ) n groups, which can be observed at between δ 0.80–0.95 and δ 1.2–1.5, respectively, and the citrate AB spin system resonances, which were not detected in spectra from the other

FIG. 2. Dendogram showing species similarity, using the

presence or absence of the major constitutive metabolites identified by single-pulse 1 H NMR in tissue extracts as observations, of six terrestrial invertebrate species.

Proton NMR Spectroscopic Studies on Invertebrate Pollution Indicator Species

TABLE 2. Full list of 1 H NMR resonance assignments of me-

tabolites observed in invertebrate tissue extracts (pH 7.3 6 0.2) at 500 MHz

Metabolite Adenine Acetate Acetoacetate Alanine Betaine Choline Citrate Dimethylamine Ethanol Fumarate α-glucose

β-glucose

Glutamate Glutamine Histidine

Isoleucine

Lactate Leucine

Lipid Lipid (CH 2 )n

Group cyclic C8H cyclic C2H β-CH 2 γ-CH 3 β-CH 3 α-CH N-(CH 3 ) 3 CH 2 N-(CH 3 ) 3 β-CH 2 α-CH 2 α -, γ-CH α ′-, γ ′-CH CH 3 CH 3 CH 2 α-, β-C5C H4 H2 H3 1/26CH 1/26CH H1 H2 H4 H5 H3 1/26CH 1/26CH H1 β-CH 2 γ-CH 2 α-CH β-CH 2 γ-CH 2 α-CH β-CH β ′-CH α-CH cyclic 4H cyclic 2H δ-CH 3 γ ′-CH 3 γ-CH γ ′-CH β-CH α-CH β-CH 3 α-CH δ ′-CH 3 δ-CH 3 γ-CH β-CH 2 α-CH ω-CH 3 CH 2-CH 3

1

H Multiplicity

d1H Chemical shifts*

s s s s d q s s s s s d d s t q s dd dd m m m d dd m ddd m dd dd d dt t t m m t dd dd dd s s t d m m m m d q d d m m t m m

8.22 8.36 1.93 2.23 1.48 3.77 3.30 3.90 3.21 3.43 3.96 2.52 2.68 2.75 1.16 3.66 6.51 3.42 3.54 3.71 3.76 3.84 5.25 3.24 3.40 3.47 3.48 3.72 3.90 4.65 2.09 2.34 3.75 2.14 2.41 3.77 3.20 3.25 4.00 6.99 7.71 0.93 1.00 1.25 1.45 1.96 3.65 1.33 4.12 0.94 0.95 1.70 1.72 3.69 0.83 1.28

595

TABLE 2. (continued)

Metabolite Lysine

Phenylalanine

Succinate Sucrose Threonine Trehalose

Tyrosine

Tryptophan

Uridine Valine

Group

γ-CH 2 δ-CH 2 β-CH 2 ε-CH 2 α-CH β ′-CH β-CH α-CH cyclic C2, 6 cyclic C4 cyclic C3, 5 α-, β-CH 2 G1H β-CH γ-CH 3 α-CH H4 H5 H6 H2 H3 H1 β ′-CH β-CH α-CH cyclic 5H cyclic 6H β-CH β ′-CH α-CH cyclic 5H cyclic 6H cyclic 2H cyclic 7H cyclic 4H Ribose H1 cyclic H5 cyclic H6 γ-CH 3 γ ′-CH 3 β-CH α-CH

1

H Multiplicity m m m t t dd dd dd m d m s m m d d t dd m m m t dd dd dd d d dd dd dd m m s m m d d d d d m d

d1H Chemical shifts* 1.45 1.70 1.90 3.03 3.74 3.13 3.26 3.98 7.33 7.38 7.43 2.40 5.40 4.25 1.32 3.58 3.44 3.64 3.75, 3.85 3.83 3.85 5.19 3.05 3.15 3.93 6.90 7.20 3.31 3.48 4.05 7.20 7.29 7.38 7.51 7.72 5.82 5.90 7.90 0.99 1.03 2.27 3.61

s, singlet; d, doublet; t, triplet; q, quartet; m, second order multiplet; dd, doublet of doublets; ddd, doublet of doublets of doublets. *Bold chemical shifts indicate those resonances observable in the 1D spectra.

three species. The arthropod tissue extracts, and in particular those from two isopod species, were also notable in their absence of amino acids with aromatic side chains. Singlelinkage Euclidean cluster analysis supported such observations, suggesting, on the basis of metabolites observed by single-pulse 1 H NMR spectroscopy, that there is greatest similarity between the two woodlice species, O. asellus and P. scaber (Fig. 2). Furthermore, that G. marginata is closest to the two species of woodlice. Similarity between the two

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FIG. 3. 600 MHz COSY45 1 H NMR spectra of earthworm

(L. rubellus) tissue extract aliphatic region, d 0.4 to 4.9. The major metabolites have been assigned and the abbreviations are as follows: Ala, alanine; Asn, asparagine; Asp, aspartate; Chol, choline; b-glu, H1 anomeric proton of b-glucose; Gln, glutamine; Glu, glutamate; His, histidine; Iso, isoleucine; Lac, lactate; Leu, leucine; Lys, lysine; Phe, phenylalanine; Tau, taurine; Val, valine. The dashed line indicates three bond 1 H-1 H connectivity within the lactate molecule.

rubellus) tissue extract aliphatic region, d0.7 to 4.7 showing contour plot and the F2 skyline projection. In addition to those in Fig. 3 abbreviations: Ace, acetone; Arg, Arginine; DMA, dimethylamine; Eth, ethanol; a4G, H4 proton of the a-glucose anomer; b1G-b6G, the appropriate proton signals from the b-glucose anomer; Gly, glycerol; Ins, myo-inositol; Meth, methionine; Thr, threonine; Tyr, tyrosine.

earthworm species was high when assessed by these criteria, whilst the earthworm subset displayed least similarity to the woodlice subset (Fig. 2). The evolutionary origins of the earthworms and of the woodlice are thought to be freshwater and marine habitats, respectively (13), a difference possibly reflected by the contrasting low molecular weight metabolite profiles observed by 1 H NMR spectroscopy. The evolutionary origin of the terrestrial pulmonates (such as A. subfuscus) is less certain, although a marine origin is suspected (13). Cluster analysis suggests that the 1 H NMR spectroscopic metabolite profile of A. subfuscus is closer to that of the earthworms than to the isopods (Fig. 2), although this is only a tentative observation as N 5 1 for this species. It should be noted that replicate tissue extracts show a high degree of intraspecific similarity and for each species only metabolites observed in all samples were included in Table 1 and subsequently used in the cluster analysis. When the NMR spectral data from the two earthworm species is analysed both qualitatively and quantitatively, obvious separation of the two groups is observed, with the exception of an outlier of the species L. rubellus (Fig. 5). It is also apparent that the interspecific variance is greater than the intraspecific variation. The key spectral regions that significantly differed between the two species, and hence

caused separation of the clusters, can be seen in the difference spectra (Fig. 6). However, more subtle differences between the species NMR spectral profiles, which are not immediately apparent from the difference spectra, can be detected by PC Analysis. These include the elevation of unassigned low molecular weight metabolites resonating at δ2.62 and 2.65. In this study, the destructive preparation of tissue extract samples was required as 1 H NMR analysis of invertebrate coelomic fluid, extracted nondestructively, failed to provide the wealth of information typically yielded from the destructively sampled tissue extracts. In fact, the absence of amino acids and many other metabolites from invertebrate and, in particular, earthworm coelomic fluids has been noted in previous work (15). Destructive sampling, however, is inherent in the use of invertebrates for biochemical analysis due to their small size (3) and due to the analytical methods typically employed. With this sampling method metabolic changes will occur if compared to the in vivo biochemistry (21) due to enzymatic activity. The biochemical pattern is thereafter stable and indeed with a consistent sample preparation technique, changes will be constant between samples. NMR spectroscopy, in comparison, is nondestructive as the extracts themselves are not destroyed. Thus if a nondestructive extraction method is developed,

FIG. 4. 600 MHz JRES 1 H NMR spectra of earthworm (L.

Proton NMR Spectroscopic Studies on Invertebrate Pollution Indicator Species

FIG. 5. Principal components maps of (a) PC1 versus PC2

and (b) PC3 vs PC2. ■ 5 L. rubellus and h 5 E. andrei.

metabolic profiling can be achieved without necessitating the sacrifice of individuals. Indeed, this would allow for a series of samples to be collected and analysed over a timecourse from the same individual. Traditionally, the use of pollution indicator species has involved first, the measurement of accumulated pollutant residue and second, the determination of lethal dose concentrations (19). Subsequent to this, critical dose levels are assessed to investigate the more realistic sublethal effects of pollutants. It is, however, at the cellular and subcellular levels that xenobiotics have their effects and a more diagnostic

597

approach would be to investigate biochemical changes resulting from exposure to pollutants. Homeostasis maintains metabolites within certain ratios, however, with toxic insult, there is often loss of this control and a subsequent increase or decrease in particular metabolites (17). Characterisation of such metabolite ‘‘biomarkers’’ has been classically achieved by specific biochemical assaying. This approach tends to be time consuming, labour intensive and involves extensive methodological development for single assays. In contrast, NMR spectroscopy requires little sample pretreatment, is rapid and is not preselective. Admittedly, the cost of a high field spectrometer may appear prohibitive, however, they are widely available in major academic institutions and the cost per sample analysed is minimal. As it is expected that the NMR fingerprints of the pollution indicator species presented here closely reflect the metabolic status of the individual, then exposure to environmental toxins in the soil matrix would results in biochemical perturbations that are observable in the tissue extracts of these invertebrates (Gibb et al., unpublished data). This is particularly useful as we have seen that 1 H NMR spectroscopy has provided us with unique and characteristic fingerprints of endogenous metabolites for species in their basal metabolic state. As a wide range of biochemicals in important intermediary pathways can be measured simultaneously by 1 H NMR spectroscopy the potential for the de-

FIG. 6. High frequency region of difference spectra where the 600 MHz 1H NMR spectra of L. rubellus has been subtracted

from that of E. andrei. U 5 unassigned.

598

tection of biochemical perturbations is high. Comprehensive assignment of resonances is a prerequisite for any such 1 H NMR investigations. The establishment of biomarkers may be aided by the application of computational pattern recognition techniques, which have been extensively developed for toxicological biomarker exploration in vertebrate species (5) and have been used here for the first time to separate different invertebrate species. As pointed out by Weeks (22), the future of biomarkers lies in their ability to predict disturbances at higher levels of organisation (communities and ecosystems) rather than their present usage as simple measures of response. The authors would like to thank NERC, the EC BIOPRINT project and Hoechst Marion Roussel for financial support, Dr J. Parkinson of the EPSRC 600MHz NMR centre at the University of Edinburgh, the ULIRS 600MHz NMR service at Queen Mary and Westfield College and T. H. Sparks for statistical advice.

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