Pyrene-Containing Peptide-Based Fluorescent Organogels: Inclusion of Graphene into the Organogel
Formation of pyrene metabolites by the terrestrial isopod Porcellio scaber
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Transcript of Formation of pyrene metabolites by the terrestrial isopod Porcellio scaber
~ ) Pergamon
PII: S0045-6535(96)00316-5
Chemosphere, Vol. 33, No. 10, pp. 1905--1914, 1996 Copyright © 1996 Elsevier Science Ltd
Printed in Great Britain. All rights reserved 0045-6535/96 $15.00+0.00
FORMATION OF PYRENE METABOLITES BY THE TERRESTRIAL ISOPOD
PORCELLIO SCABER
~ l ~ I I ~ Q e ~ t , C. Reuther 2, I. Koz in 3, T.C. van Brummelen 2, C.A.M. van Gestel 2, C. Gooijer 3 and
W.P. Cofino t
tinsitute for Environmental Studies, Vrije Universiteit, De Boelelaan 1115, 1081 HV
Amsterdam, the Netherlands
2Department of Ecology and Ecotoxicology, Vrije Universiteit, De Boelelaan 1087, 1081 HV,
Amsterdam, the Netherlands.
3Department of General and Analytical Chemistry, Vrije Universiteit, De Boelelaan 1083, 1081
HV, Amsterdam, the Netherlands.
(Received in Germany 26 April 1996; accepted 12 August 1996)
Abstract . The formation of pyrene metabolites by the terrestrial isopod Porcelh'o scaber (Latr.) w a s studied
following exposure for 13 days to 100 tag/g pyrene in its food. An alkaline hydrolysis method with subsequent
liquid-liquid extraction was developed to determine the metabolite content, especially 1-hydroxypyrene.
Synchronous Fluorescence Spectroscopy (SFS) was explored as a fast screening method and HPLC was applied
as a complementary method for confirmation of the SFS results. In PorceUio scaber the amount of 1-
hydroxypyrene exceeded that of pyrene, which indicates that pyrene is being metabolized extensively. SFS and
HPLC demonstrated that Porcellio scaber also forms a number of other fluorescent metabolites. These
metabolites exhibit fluorescence at the same excitation wavelength as 1-hydroxypyreue, possibly indicating a
similar fluorophore structure. Determination of 1-hydroxypyrene is considered as a first step towards an
improved environmental risk assessment of PAH exposure, as pyrene is a predominant member of this class of
environmental contaminants and 1-hydroxypyrene is its major metabolite. Copyright © 1996 Elsevier Science Ltd
I n t r o d u c t i o n
Polycyclic Aromatic Hydrocarbons (PAHs) are widely spread throughout the natural enviromnent due to both
natural and antropogenic causes [1-4], As several members of this class of environmental contaminants have
been identified as potent carcinogens, their analysis has been incorporated in monitoring programs of
environmental agencies for quite some time. However, it is generally accepted that toxic and carcinogenic effects
of PAHs are mainly caused by metabolites rather than by the parent compounds [5,6]. Detection and quantitatien
of these metabolites might therefore provide a better method for environmental risk assessment.
1905
1906
As pyrene is encountered in a variety of environmental matrices, the ability to quantitate its metabolites in biota is
considered a first step towards this new approach in risk assessment. The attention is focused on 1-
hydroxypyrene, as numerous studies devoted to occupational PAH exposure have report(~ 1-hydroxypyrene to
be a useful biomarker [7]. Furthermore, 1-hydroxypyrene has been detected in fish bile, showing a good
correlation with the degree of pollution of the area [8,9]. Regardless of the exposure to other PAHs, 1-
hydroxypyrene was found to be the predominant PAH metabolite in bile in those surveys. Although 1-
hydroxypyrene is merely a detoxification product, its occurrence can reflect on both PAH exposure as well as the
metabolic capacity of isopods.
In the present study we investigate the possible formation of pyrene metabolites by the terrestrial isopod
Porcellio scaber. In general, contrary to aquatic invertebrates, little is known about PAIl metabolism by
terrestrial invertebrate species. In the past, invertebrates were regarded as being less capable of metabolizing
PAHs [10]. However, field studies revealed that the PAH content of PorceUio scaber in a contaminated area
deviates from the equilibrium partitioning theory, indicating metabolism and subsequent enhanced excretion
[11]. Furthermore, exposure studies conducted on Porcellio scaber with benzo[a]pyrene rendered high
elimination rates, suggesting metabolism [12].
Previous biomarker studies concerning 1-hydroxypyrene dealt with liquid matrices such as urine and bile, using
enzymes to hydrolyze conjugated metabolites. Unfortunately, for tissue samples of exposed biota this approach
cannot be followed; a more robust extraction method is needed. In the present paper we report on the
development of a method using alkaline hydrolysis -also known as saponification- and subsequent liquid-liquid
extraction as a method to determine the pyrene metabolite content of Porcellio scaber.
Within this context the usefulness of Synchronous Fluorescence Spectroscopy (SFS) for identification and
quantitation purposes is explored. It has been shown in the literature that SFS provides a fast screening method
for the detection of both pyrene mad its metabolites inl for instance, bile samples [9]; for these samples no prior
clean up and chromatographic separation was needed. Fluorescent compounds show selective maxima in both
their excitation and emission spectra. Therefore, it is possible to discriminate between pyrene and pyrene
metabolites by choosing optimal excitation/emission wavelengths. Using SFS the excitation and emission
monochromators are scanned simultaneously, while a fixed wavenumber offset is maintained. Thus, sufficient
spectroscopic separation is achieved in order to detect pyrene and its metabolites during one scan. The spectra
provide information on the effectiveness of the saponification procedure and, furthermore, give an estimate on
the quantity of the metabolites present. In the present explorative investigations, High Performance Liquid
Chromatography (HPLC) with fluorescence detection was used as a complementary method for confirmation of
the SFS results.
Materials and Methods
Chemicals
Pyrene (Gold Marke) was purchased from EGA Chemie KG, Steinheim/Albuch, Germany. 1-Hydroxypyrene
(98%) was acquired from Aldrich Chem. Co. Milwaukee, WI, USA. Acetone (p.a.), ethanol absolute (p.a.),
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ammonium acetate (p.a.) and hydrochloric acid (37%) were obtained from Riedel-de H a ~ AG, Seelze,
Germany. Water (HPLC grade), potassium hydroxide pellets (p.a.) and anhydrous sodiumsulfate (p.a.) were
purchased from J.T. Baker, Deventer, The Netherlands. pH-paper Dual-Tint (range 5.0-8.0) was acquired from
J.T. Baker Inc. Phillipsburg, N J, USA. Dichloromethane (Suprasolv) was obtained from Merck, Darmstadt,
Germany. Acetonitrile, (HPLC grade) was purchased from Promochem, Wesel, Germany.
An/real treatment
A group of 39 isopods of the species Porcellio scaber (Latreille), caught in the Spanderswoud forest near
Hilversum, the Netherlands, were kept in a polystyrene container (15 an ID x 10 era height) with a plaster of
Paris bottom. The container was placed on a tray with a plaster of Paris inlay, which was kept wet in order to
maintain humidity. The lid was a gauze which enabled due aeration and was held in place by a wider polystyrene
ring. The food consisted of 4 g of ground poplar and maple leaves (1:1 w:w) spiked with pyrene at a
concentration of 100 ~,/g DW. Spiked material was prepared by dissolving pyrene in acetone (100 lag/ml) and
mixing 4 ml of this acetone spike with the leaf material. An additional 12 ml of acetone was added to flood the
material and after mixing the acetone was evaporated overnight under a gentle flow of nitrogen. Prior to
transferring the food into the container it was wetted with 12 ml of water. The food was spread out over the
container floor and its amount sufficed for the duration of the exposure period. The container was stored at
18.0i-0.1"C in a climatized room which had a light/dark regime of 12h/12h and a relative humidity of 75%. The
exposure lasted for 13 days. In this period 3 mfimals died, the remaining 36 were killed by flash freezing and
lyophilized for 48h, after which they were ground in a agate mortar.
Saponification and liquid-liquid extraction Saponification conditions were modeled after standard procedures using ethanol/potassium hydroxide solution
(40% w:v) [1:1; (v:v)] at 80"C for 1 hour. Liquid-liquid extractions were performed with dichloromethane,
which showed satisfactory recoveries for both pyrene and 1-hydroxypyrene. The ixocedure was scaled down in
such a fashion as to allow the execution of both the saponification and subsequent liquid-liquid extraction in one
single 15 ml reaction tube. The resulting procedure is described in the results section.
Synchronous Fluorescence Spectroscopy SFS was performed with an I.S 50 Perkin Elmer Luminescence Spectrometer (Beaconsfield, UK) using a 1 cm
quartz cuvette. Spectral bandwidth was 2.5 nm for both excitation and emission. Acetonitrile was used as
solvent. The optimization of wavelength offsets and other experimental parameters is described in the results
section.
High Performance Liquid Chromatography HPLC was performed using a Varian Star liquid chromatography system which consisted of a Star 9012 solvent
delivery system, a Varian Mistral colulrm thermostat, a Star 9100 autosampler (Walnut Creek, CA, USA), a
1908
Separations GT-103 degasser (H.I. Ambacht, The Netherlands), a Vydae RP C18 column [25 em x 4.6 mm ID]
(Hesperia, CA, USA) and a Jasco FP-920 fluorescence detector (Tokyo, Japan).
Results
Saponification and liquid-liquid extraction From the tissue pool, 20 mg -equal to the dry weight of one animal- was weighed on a small piece of a l ~
foil and quantitatively transferred into a 15 ml glass stoppered reaction tube with 1.5 ml of ethanol. Next 1.5 ml
of a potassium hydroxide (40% w:v) solution and a boiling chip were added. After stirring gently, ~ mixture
was hydrolyzed at 80'C in a water bath under reflux. The hydrolysis was ended after 1 hour and the samples
were left to cool Subsequently the hydrolysis mixture was neutralized by adding 1 ml 5 M ammoaium acetate as
a buffer and approximately 1.6 ml of 1:1 (v:v) diluted 37% hydrochloric acid. The final pH range (6.0-7.0) was
checked with pH paper.
For extraction, 2 ml of dichloromethane was added and the mixture was vortexed for 30 seconds, sonieated for 5
minutes, and mechanically shaken for another 15 minutes. The tubes were subsequently centrifuged for 4
minutes at 3000 rpm in order to enhance phase separation and the dichloromethane phase was collected in a glass
vial containing 1.5 g of anhydrous sodiumsulfate. This extraction procedure was repeated two mtx~ times. The
combined dichloromethane fractions were quantitatively transferred into a 15 ml reaction tube and evaporated under a gentle nitrogen flow while being kept in a water bath at 30"C. The residue was dissolved in 2 ml of
acetonitrile and diluted prior to analysis. The samples were stored at 4"C in the dark.
Recoveries were determined by separately spiking uncontaminated isopod tissue with eith~ pyrane or 1-
hydroxypyrene. For this purpose, 20 mg of dried and ground isopod tissue (of uncontaminated animals) was
mixed with 1.5 ml of ethanol. Then 48 # of ethanol (containing 120 ng of compound) was added, gently stirred
and left to stand for 30 minutes before potassium hydroxide solution was added. The saponification and
extraction were performed as described above. Recoveries were determined both by SFS and HPLC. For pyrane
recoveries of 99+9% [SFS] and 97+4% [HPLC] [n=2] were found, while for 1-hydroxypyrene recoveries were
92+9% [SFS] and 91+4% [HPLC] [n=2].
Synchronous Fluorescence Spectroscopy; Optimization Optimum wavenumber offsets were established by recording conventional fluorescence excitation and emission
spectra of standard solutions of both pyrene and 1-hydroxypyrene in aeetonitrile. From the wavelengths
corresponding to the maxima in those spectra, wavenumber offsets were determined and synchronous scans
were performed.
Figure 1 shows the excitation and emission spectra obtained for 1-hydroxypyrene. From these spectra it was
determined that two wavenumber offsets, i.e. 2800 and 10130 cm 1 are suitable for performing SFS. This is
illustrated by the synehronous scans of 1-hydroxypyrene depicted in Figure 1 as well. As the wavelangth soak)
of the synchronous scan is linked with the excitation monochromator, the maxima in the synchronous scans
1909
coincide with the m a n n a in the excitalion specmnn. From measurements in extracts of isopod tissue it was
concluded that 2800 crn "~ was preferred over 10130 cm "~. Using 10130 cm "1, excitation is pedormed Jt relalivety
short wavelengths (250-320 nm) which leads ~o a highe¢ background because more ina~e r~ces IXeJent in the
sample exhibit fluorescence if excited at such shoot wavelengths. At 2800 ¢m "1 the S F S - ~ - l r m n is er~__t_,~_ using
an excitation window at longer wavelengths (320-375 rim), where f l ~ backgnmnd is l eu importa~
d
. S ~ A
10130 cm -1
C
:._ _S
2800 cm -1
D
i
. . . . . . . . . . ,
I I I I I I 250 300 350 400 450 500
Wavelength (rim)
Fig. 1: Spectra of 1-hydroxypyrene (0.1 taM in acetonitrile). A: Excitation spectrum, emission fixed at
407 nm, B: Emission spectrum, excitation fixed at 348 nm, C: SFS spectrum, wavenumber offset 10130
cm "~, D: SFS spectrum, wavenumber offset 2800 cm "~.
Similar spectra were obtained for pyrene resulting in an optimum wavenumber offset of 3109 ern ]. As can be
seen in Figure 1, the excitation band of 1-hydroxypyrene around 348 nm is rather wide. This makes it possible
to use 3109 cm 1 (the optimum offset for pyrene) for 1-hydroxypyrene as well; ~_e excitation band is wide
enough to shift the excitation window without loosing much sensitivity and a narrow SFS-spectrum of 1-
hydroxypyrene is obtained. The SFS-spectra of both pyrene and 1-hydroxypyrene at 3109 a n "~ are shown in
Figure 2. Both excitation and emission monochromator slits were set at 2.5 nm, scans ran from 320 to 375 nm
(based on excitation wavelength).
1910
Synchronous Fluorescence Spectroscopy; Calibration
Calibration curves were made using standard solutions containing both pyrene and 1-hydroxypyrene in
acetonitrile. The curves are shown in Figure 3, displaying results obtained before and after deoxygeaation.
Deoxygenation diminishes the fluorescence quenching caused by oxygen, leading to an enhancement of the
fluorescence signals of pyrene and 1-hydroxypyrene by a factor of 10 and 2, respectively. This was achieved by
passing a gentle flow of nitrogen through the sample in the cuvette for 3 minutes via an uncoated silica capill~y.
320
/
i
t i
330
i
I
333.5 3,~0 344.5 350 360 370
J
Excitation Wavelength (urn)
Fig. 2: SFS spectra with wavenumber offset 3109 cm ~, solvent: aeetonitrile. A: pyrene (6 ng/ml), B: 1-
hydroxypyrene (4 ng/ml), [The overlaying spectrum -obtained by summation of spectra A and B- shows
the resulting spectrum for a mixture of both compounds]. C: SFS spectrum of Porcellio scaber exposed
to pyrene for 13 days.
After removal of the capillary, the cuvette was simply closed with a Teflon stopper and a spectrum reco~ed. The
deoxygenation procedure was sufficient, as after 3 minutes no further increase in signal intensity was achieved.
Speelra were taken within 1 minute after the nitrogen flow was removed. For acetonilrile, purging with nitrogen
during 3 minutes caused no more than 2-3% loss of the sample volume due to evaporation. For quantitatioaa the
spectral peak heights (and not the areas) of pyrene and 1-hydroxypyrene were measured since this procedure
was least influenced by spectral overlap of both spectra, as is shown in Figure 2.
1911
Synchronous Fluorescence Spectroscopy; Results Samples were diluted 12 limes with acetoninile and 2 ml of this solution was transferred into a 1 em quartz
cuvette. Dilution diminishes the interference caused by the sample malrix which, in principle, can absorb both
excitation and emission light and also can induce quenching of analyte fluorescence. The minimal dttution factor
(3x) was established by diluting samples until the reduction of fluorescence signal was equal to the dilution
factor. As both pyrene and 1-hydroxypyrene signals were sufficiently intense, additional dilution still
satisfactory signals for quantitation purposes.
4 A y = 8.2678e-2 + 0 . 4 3 2 2 9 x ~ "
2
1 J y = 9.0583e-3 + 3.4224e-2x /
2 4 6 8 10
y = 2.6349e-3 + 0.33786x
00 2 4 6 8 10
Concentration (ng/ml)
Fig. 3: SFS calibration curves of pyrene (left) and 1-hydroxypyrene (right) made before [@] and after
[O] deoxygenation. Line equations and regression coefficients are represented for each curve.
Figure 2 displays the typical spectrum of the hydrolyzed tissue from the exposed animals. The spectra indicate
that pyrene is present while its spectrum (shape and maximum) seems undisturbed. The maximum in the
spectrum is at the expected position and the peak shape -left of the valley- corresponds to that of the standard
solution. The pyrene content was found to be 2.3_+0.2 b~g/g DW (dry weight) In=5].
Contrary to the left-hand side of the spectrum, the right-hand side -corresponding to the 1-hydroxypyrene peak-
differs in shape. However, although shifted to a shorter wavelength and a distorted left flank its overall
approaches that of the standard 1-hydroxypyrene. Ignoring this spectral difference, the 1-hydroxypyrene content
was established as 5.2_+0.9 lag/g DW [n=5].
High Performance Liquid Chromatography HPLC was performed by applying a programmed gradient of water/acetonitrile. At first, 65% acetonitrile was
pumped which after injection was raised to 70% in 5 minutes time. Next the acetonitrile content was raised to
100% in 10 minutes lime and maintained for another 10 minutes. The column was maintained at 18'C. The
retention times for 1-hydroxypyrene and pyrene were found to be about 6.4 and 12 minutes, respectively. The
fluorescence detector was programmed accordingly, with an excitation wavelength of 346 nm for the first 8
1912
minutes and 334 nm for the remaining part of the chromatogram. The emission detector was set at 383 nm with
an emission band pass of 40 rim. From the -diluted- samples that were analyzed with SFS, 20 pl was injected
(except for one sample which was stored in hexane).
40
30
2O
10
B
. . . . . . . . i o . . . . 1'5 Time (minutes)
Fig 4: HPLC chromatogram after alkaline hydrolysis of Porcellio scaber exposed to pynme for 13 days.
Excitation wavelength; 0-8 minutes: 346 nm, 8-15 minutes: 333 nm. A: 1-hydroxypyrene. B: pyrene.
I, 2 and 3 : unknown compounds.
A typical HPLC chromatogram is depicted in Figure 4. Apart from pyrene and 1-hydroxypyrene, which ~te
unambiguously identified, the other peaks indicate the presence of at least three extra compounds. The cenlral
compound of those three was also found present in saponification extracts of non-exposed isopods (data not
shown). The pyrene content calculated following the alkaline hydrolysis approach was found to be 2.7i'0.2 pg/g
DW [n--4] while the 1-hydroxypyrene content was 3.5+2.3 I.tg/g DW [n--4].
Discuss ion
Previous research on PAH uptake and elimination rates by Porcelh'o scaber was based on measurements of a
parent compound, i.e. benzo[a]pyrene [12]. However, detailed information on the formation of PAH metabolites
is required, as the metabolic activation of PAHs is responsible for the main toxic effect [5,6]. The behavior of
1913
pyrene was studied as a first step towards this objective. The formation of pyrene metabolites by Porcellio scaber
was proven indeed; demonstrating directly the metabolic capability of this terrestrial isopod. The alkaline
saponification procedure is capable of liberating both pyrene and its metabolites from the isopod tissue. As far as
quantitative aspects are considered, the pyrene content calculated was the same regardless of the analytical
procedure. Furthermore, it is fully in line with the amount found while applying soxhlet exlraction on the same
tissue pool (data not shown). This suggests that the saponification procedure and subsequent liqnid-liquld
extraction renders satisfactory quantitative results.
The present study shows that it is possible to reliably determine parent pyrene and also get an estimate on its
metabolites by means of SFS as a rapid screening method. Obviously, more metabolites play a role. The
saponification method as described renders a number of fluorescent compounds which d i s t ~ the 1-
hydroxypyrene SFS spectrum. Therefore, the 1-hydroxypyrene content calculated from the spectrum (Fig. 2)
should be considered as a first approximation. The HPLC chromatogram (Fig. 4) clearly shows the presence of
more compounds, which exhibit fluorescence at the same excitation and emission wavelength settings as 1-
hydroxypyrene; the identity of these fluorescent compounds is currently under investigation. Nevertheless, it is
tempting to speculate about their nature. The fact that they are excited under the same excitation and emission
conditions as 1-hydroxypyrene could indicate that they possess a similar fluorophore structure, as in glucuronide
or sulfate derivatives of 1-hydroxypyrene. The blue shifted emission observed in spectrum 2 is in agreement
with the SFS spectrum of 1-hydroxypyrene glucuronide [9]. If this assumption is valid, it might be possible that
by either changing saponification conditions or supplementing the current method with an enzymatic hydrolysis
procedure, the number of metabolite types could be reduced. This would imply that eventually the 1-
hydroxypyrene content -after hydrolysis- could be regarded as a sum parameter for the total pyrene metabolite
content.
The present results might suggest that HPLC with fluorescence detection is the preferred analytical technique
compared to SFS. However, SFS can still be very useful as a rapid screening method. Furthermore, it should be
taken into account that HPLC analysis of samples with a more complex matrix requires a proper clean up
procedure. Especially when analyzing field samples with a low PAH (metabolite) content, samples need to be
strongly preeoncentrated to enable HPLC analysis. Of course, such strong preconeenlration will cause additional
problems. In such cases SIS will be of great interest as no sample clean up is required.
Finally, the relevance of studying pyrene and its metabolites may be questioned, since PAH contamination in the
field is caused by a wide range of individual PAHs. It should be emphasized that also under such conditions the
analysis of pyrene metabolites can still be quite appropriate, provided the PAH profile is fairly constant. In that
case the 1-hydroxypyrene content can be used as a biomarker, a relative measure of the total PAH exposure. In
fact, 1-hydroxypyrene was identified as the major metabolite present in fish bile [9]. The present study supports
this approach, since the amount of pyrene present in the isopods was exceeded by that of 1-hydroxypyrene.
Thus, screening of other biota and their tissues for 1-hydroxypyrene might constitute a new approach on PAH
exposure studies and subsequent environmental risk assessment.
1914
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