Volume 57, Number 7, 2003 APPLIED SPECTROSCOPY 8350003-7028 / 03 / 5707-0835$2.00 / 0q 2003 Society for Applied Spectroscopy

FT-IR Spectroscopic Analysis of Rainbow Trout LiverExposed to Nonylphenol

GULGUN CAKMAK, INCI TOGAN, CEVDET UGÆ UZ, and FERIDE SEVERCAN*Department of Biological Sciences, Middle East Technical University, 06531, Ankara, Turkey

Nonylphenol (NP) is a biodegradation product of nonylphenolethoxylates (NPEs) belonging to the alkylphenol ethoxylates (APEs)group. APEs are widely used nonionic surfactants in detergents,herbicides, pesticides, paints, and cosmetics. The present work in-vestigates the effects of NP on rainbow trout (Oncorhynchus mykiss )livers at the molecular level using Fourier transform infrared (FT-IR) spectroscopy. The FT-IR spectra revealed dramatic differencesbetween the NP-treated and control tissues, which mainly indicatedthat the level of triglycerides increased, the lipid order increased,and the protein concentration decreased in the treated samples.Moreover, it was also found that glycogen levels signi� cantly de-creased and the relative content of nucleic acids increased in NP-treated � sh. The 17b-Estradiol-treated � sh liver spectra were foundto be quite similar to those of NP-treated � sh. All these resultsimplied that rainbow trout may offer considerable promise to beused as a bioindicator for NP in the future.

Index Headings: Nonylphenol; Estradiol; Rainbow trout; Fouriertransform infrared; FT-IR spectroscopy; Liver tissue.

INTRODUCTION

Nonylphenol ethoxylates (NPEs) are nonionic poly-meric surfactants (synthetic surface-active substances)belonging to the alkylphenol ethoxylates (APEs) group.APEs are the second largest group of nonionic surfactantsin commercial production.1 Among APEs, NPEs accountfor approximately 80 percent of the world’s production.2

NPEs are widely used as detergents, emulsi� ers, and wet-ting agents in household products as well as in agricul-tural and industrial applications such as in the manufac-turing of paints, plastics, cosmetics, lubricant oils, con-struction materials, and paper and in the processing offuels, metals, petroleum, textiles, and leather.3–7 Nonyl-phenol (NP) is the predominant biodegradation productof NPEs in the environment8 and has been found to affectendocrine systems in various organisms.9

In vivo3,10–12 and in vitro13,14 assay systems demonstrat-ed that NP affects the endocrine systems in � sh as wellas in birds and mammals through weak estrogen-likeproperties.9 For example, NP has been shown to causethe synthesis of vitellogenin in the livers of male andimmature female rainbow trout.3,15 NP also had an inhib-itory effect in testicular growth in rainbow trout.3 Fur-thermore, NP has toxic and carcinogenic effects on ani-mals. For example, it has been shown that NP causedhistopathological changes in rat primary germ and Sertolicell cultures.16 Hughes et al.17 showed that NP caused celldeath by the inhibition of calcium pumps in the endo-plasmic reticulum. Schwaiger et al.12 reported that NPinduces anaemia as a consequence of the interaction be-

Received 21 November 2002; accepted 19 February 2003.* Author to whom correspondence should be sent. E-mail: feride@

metu.edu.tr.

tween NP and erythrocyte membrane. It was also shownthat NP inhibits or reduces the ATP synthesis.18–20 Re-cently, NP was shown to cause breast,21,22 testicular, andovarian cancers.23 Despite several biochemical and clin-ical studies, some of which are reported above, the effectsof NP on tissues at the molecular level have not yet beeninvestigated.

Laboratory studies on the effects of NP on freshwater� sh under controlled conditions have a special impor-tance because these kinds of studies will help us to es-timate the degree and the nature of the adverse effects ofNP. Since the liver has an essential role in detoxi� cationof toxic substances, the present study was conducted todetermine the effects of NP on rainbow trout liver.

In this work, the effects of NP on rainbow trout liverwere investigated at the molecular level using Fouriertransform infrared (FT-IR) spectroscopy. This technique,together with infrared microscopy, is becoming an in-creasingly powerful tool for the study of the compositionand structure of cellular components within intact tis-sues.24–29 It is a nondestructive, rapid, and sensitive tech-nique for structural and functional studies.25–31 Shifts inpeak positions and changes in bandwidths and in inten-sities of the infrared bands are used to obtain valuablestructural and functional information about the system ofinterest.24–31

MATERIALS AND METHODS

Reagents. Nonylphenol (4-NP), 17b-Estradiol (E2),and potassium bromide (KBr) were purchased from Al-drich Company, Sigma, and Merck, respectively.

Fish. Eight-month-old juvenile rainbow trout wereused. The � sh were transferred to the laboratory in plasticsacs with aeration. They were then acclimated for severalhours and put into 35 L tanks. Prior to the experiment,the � sh were allowed to rest for 2 days. During the wholeexperiment the � sh were fed with trout chow.

Experimentation. Experiments were conducted in astatic aquaria system. A charcoal � lter was used to gen-erate water media for the � sh. Prior to use, these mediawere allowed to rest for 24 h with an aerator. The � shwere treated with 0 (control), 66, 220, and 660 mg/L NPfor one and two weeks in 35 L aquaria. The range forthe treatment of NP was chosen in accordance with en-vironmentally observed values.1,6,32 A group of � sh wasalso exposed to 22 mg/L estrogen (17-b-estradiol) for twoweeks. NP and 17-b-estradiol (E2) were dissolved in ab-solute ethanol and then introduced into the aquaria. Thesame amount of ethanol without NP was also added tothe tank of the control group.

Eight � sh were put in each aquarium. Two � sh fromeach aquarium were sampled at the end of the � rst week

836 Volume 57, Number 7, 2003

FIG. 1. The general FT-IR spectrum of the rainbow trout livers in the3600–800 cm21 region.

TABLE I. General band assignments of FT-IR spectrum of livertissue based on the literature.a

Peaknumbers

Wavenumbers(cm21) De� nition of the spectral assignment

1 3307 Mainly N–H stretching (Amide A) of pro-teins with the little contribution from O–Hstretching of polysaccharides and intermo-lecular H bonding

2 3015 Ole� nic5CH stretching vibration: lipids,cholesterol esters

3 2959 CH3 asymmetric stretch: mainly lipids, withthe little contribution from proteins, car-bohydrates, nucleic acids

4 2927 CH2 asymmetric stretch: mainly lipids, andthe little contribution from proteins, car-bohydrates, nucleic acids

5 2876 CH3 symmetric stretch: mainly proteins,with the little contribution from lipids,carbohydrates, nucleic acids

6 2857 CH2 symmetric stretch: mainly lipids, withthe little contribution from proteins, car-bohydrates, nucleic acids

7 1739–1744 Ester C5O stretch: triglycerides, cholesterolesters

8 1657 Amide I (protein C5O stretching): a-helices9 1541 Amide II (protein N–H bend, C–N stretch):

a-helices10 1452 CH2 bending: mainly lipids, with the little

contribution from proteins11 1391 COO2 symmetric stretch: fatty acids12 1236 PO2

2 asymmetric stretch: mainly nucleic ac-ids with the little contribution from phos-pholipids

13 1152 CO–O–C asymmetric stretching: glycogenand nucleic acids

14 1080 PO22 symmetric stretching: nucleic acids

and phospholipidsC–O stretch: glycogen

15 1042 C–O stretching: polysaccharides16 969 C–N1–C stretch: nucleic acids

a See Refs. 24, 27, 28, 31, 34–39, and 41–45.

and 6 � sh from each of the treatment tanks were sampledat the end of the second week of the experiment. Fiveliters of water was siphoned and replaced with the samevolume of water and NP concentrations were adjusted inaccordance with the replacement every day. The temper-ature was 9 6 1 8C in each aquarium. Livers of the sam-pled � sh were removed for FT-IR study and were storedat 222 8C. The FT-IR experiments were performed twoweeks after removal of the livers.

Sample Preparation for FT-IR Studies. The liversamples were dried in a HETO-MAXI dry lyo freeze dri-er for 12 h to remove water. The samples then wereground in an agate mortar containing liquid nitrogen toobtain liver powder. The liver powder was mixed withpotassium bromide (at the ratio of 1/100). The mixturewas then dried again in the lyo freeze drier for 6 h toremove all traces of water. The mixture then was sub-jected to a pressure of ;1100 kg/cm 2 in an evacuated dieto obtain a KBr pellet for use in the FT-IR spectrometer.

FT-IR Studies. Infrared spectra were obtained using aBomem 157 FT-IR Spectrometer equipped with a DTGSdetector. The spectrometer was continuously purged bydry air. The spectra of liver samples were recorded in the4000–800 cm21 region at room temperature. Two hun-dred scans were taken for each interferogram at 4 cm21

resolution.Statistical Test. The difference in the means of the

treated and control � sh were compared by means of a t-test.33 A p-value of less than 0.05 was considered signif-icant.

RESULTSAmong the NP concentrations used in this study, 660

mg/L NP was seen to be 100% lethal for the � sh. All the

� sh treated with this concentration died in 4 days. Forthis reason, experiments were carried out at the 66 and220 mg/L NP concentrations.

A representative FT-IR spectrum of control rainbowtrout liver in the 3600–800 cm21 region is shown in Fig.1 and the band assignment is presented in Table I. Asseen from the � gure and table, it is a complex spectrumconsisting of several bands belonging to the functionalgroups in lipids, carbohydrates, proteins, and nucleic ac-ids.

The spectral analyses were performed in two distinctfrequency ranges, namely 3600–2800 and 1800–800cm21. The spectra of � sh belonging to the speci� c groupsshowed similar characteristics. In the � gures, a represen-tative spectrum from each group is presented. However,in the determination of the average values, the peak po-sitions, the band intensities, and the bandwidth valuesbelonging to each group were considered and their meanvalues were studied comparatively (Tables II and III).

Figures 2A and 2B show representative infrared spec-tra of control and one and two weeks NP-treated rainbowtrout livers at different concentrations, respectively, in the3600–2800 cm21 region. Figures 3A and 3B show thesame spectra in the 1800–800 cm21 region after one andtwo weeks of treatment, respectively. As seen from these� gures, different NP concentrations induced remarkable

APPLIED SPECTROSCOPY 837

FIG. 2. The infrared spectra of rainbow trout livers treated with 0(control), 66, and 220 mg/L NP for (A) one week and (B) two weeksin the 3600–2800 cm21 region (the spectra were normalized with respectto the CH2 asymmetric mode, which is observed at 2927 cm21).

FIG. 3. The infrared spectra of rainbow trout livers treated with 0(control), 66, and 220 mg/L NP for (A) one week and (B) two weeksin the 1800–800 cm21 region (the spectra were normalized with respectto the amide I band, which is observed at 1657 cm21).

time- and dose-dependent changes in the bandwidth, in-tensity, and frequency values of the FT-IR bands. Themost signi� cant changes were observed for the 220 mg/LNP-treated liver spectrum. These � gures also indicate thatthe most considerable differences were observed aftertwo weeks of exposure time in the 220 mg/L NP-treatedliver spectrum. For this reason, the detailed spectral anal-ysis is reported only for two-week-220-mg/L-NP-treatedliver spectrum.

Figures 4A, 4B, and 4C show representative infraredspectra of control rainbow trout liver and 220 mg/L NP-treated liver in the 3600–3050, 3030–2800, and 1800–800 cm21 regions, respectively, after two weeks of treat-ment.

The comparisons of the band intensities of some infra-red bands and p-values are listed in Table II. As seenfrom the table, there are signi� cant ( p , 0.05 to p ,0.001) differences in the band intensities at speci� edwavenumbers between control and treated � sh liver tis-sues.

As can be seen from Fig. 4A, in the treated liver spec-trum there was a dramatic reduction ( p , 0.001) in theintensity of the band at 3307 cm21, which contains strongabsorptions arising mainly from the N–H stretching (am-

ide A) mode of proteins with the contribution of the OHstretching mode of polysaccharides27,34–36 and intermolec-ular H bonding.37

As seen from Fig. 4B, the pronounced differences wereseen in this region whose spectrum is populated by ab-sorptions arising from the C–H stretching vibrations ofole� nic5CH, –CH2, and –CH3 groups. The intensity ofthe band at 3015 cm21, which is due to the CH stretchingmode of HC5CH groups, could be used as a measure ofunsaturation in the acyl chains.24 The intensity of thisband increased slightly and the maximum of this bandshifted to lower frequency values from 3015.8 6 0.854to 3012.4 6 0.287 cm21 ( p , 0.001). It was also ob-served that, as a consequence of NP treatment, the ab-sorption intensity of the CH3 asymmetric stretching vi-bration at 2959 cm21 decreased dramatically ( p , 0.001)(Table II). Moreover, the intensity of the CH2 symmetricstretching vibration band at 2857 cm21 signi� cantly in-creased in the NP-treated liver ( p , 0.001), and the shapeof this band was more symmetric compared to that of thecontrol liver. The frequency of this band also shifted re-markably ( p , 0.001) from 2857.48 6 0.746 to 2854.556 0.238 after NP treatment.

838 Volume 57, Number 7, 2003

FIG. 4. The infrared spectra of rainbow trout livers treated with 0(control) and 220 mg/L NP for two weeks in (A) the 3600–3030 cm21

region, (B) the 3030–2800 cm21 region (C–H stretching region), and(C ) 1800–800 cm 21 region (� nger print region).

TABLE II. The intensity values of the bands exhibiting signi� cantchanges for control and NP-treated rainbow trout after two weeksof NP exposure. The values are the mean 6 standard deviation foreach group.

BandNo

Wavenumber(cm21)

Band intensity

Control(n 5 8)

220 mg/LNP-treated

(n 5 6) p-value

1367

10

33072959285717411452

2.123 6 0.2120.694 6 0.0250.027 6 0.0320.294 6 0.0730.311 6 0.002

0.461 6 0.0420.455 6 0.0130.353 6 0.0140.861 6 0.0140.514 6 0.045

,0.001,0.001,0.001,0.001,0.001

11121314

1395123611521080

0.334 6 0.0170.367 6 0.0250.313 6 0.0310.486 6 0.029

0.455 6 0.0460.502 6 0.0230.451 6 0.0510.415 6 0.046

,0.001,0.001,0.01,0.05

TABLE III. Average bandwidth values (mean 6 standard devia-tion) for amide I and amide II bands of the control and of twoweeks 220 mg/L NP-treated liver tissues.

Wavenumber(cm21)

Bandwidth

Control(n 5 8)

220 mg/L NP-treated(n 5 6) p-value

16571541

0.933 6 0.0510.783 6 0.04

1.125 6 0.151.1 6 0.182

,0.05,0.01

The other frequency range under consideration was1800–800 cm21 (Fig. 4C). The band centered at 1741cm21 is mainly assigned to the .C5O ester stretchingvibration in triglycerides.38,39 The bands at 1657 and 1541cm21 are attributable to amide I and II vibrations of struc-tural proteins.40,41 In addition, the intense band at 1452cm21 can be assigned to the CH2 bending mode of lipidsand proteins.41,42 The 1391 cm21 band is assigned toCOO2 symmetric stretching belonging to fatty acids42

(Table I). The relatively strong bands at 1236 and 1080cm21 are mainly due to asymmetric and symmetricstretching modes of phosphodiester groups in nucleic ac-ids.43,44 However, the symmetric phosphate stretchingband might be masked by spectral contributions of gly-cogen, which exhibits three strong peaks at 1042, 1080,and 1152 cm21.45 The relatively weak band at 1152 cm21

is due to the stretching mode of CO–O–C groups presentin glycogen and nucleic acids.46 The band at 1042 cm21

is assigned mainly to C–O stretching vibrations in poly-saccharides.36 The band centered at 969 cm21 is generallyassigned to the symmetric stretching mode of the dianion-ic phosphate monoester of cellular nucleic acids, espe-cially for DNA.31

As illustrated in Fig. 4C, the intensity of the lipid C5Ostretching vibration increased dramatically in the spec-trum of NP-treated liver ( p , 0.001) (Table II). In ad-dition, the frequency of this band shifted from 1741.326 0.694 to 1745.05 6 0.31 cm21 ( p , 0.001). The av-erage bandwidth values of the amide I and amide II bandsof the control and treated liver tissues are listed in TableIII. As can be easily seen from Fig. 4C and Table III,there was a broadening in the bandwidth of the amide Iband ( p , 0.05) in the NP-treated liver. In addition, thefrequency of this band shifted slightly from 1657.43 60.413 to 1656.5 6 0.708 cm21. The bandwidth of theamide II band located at 1541 cm21 broadened signi� -cantly ( p , 0.01) in the treated liver compared to that ofthe control group. The spectra in Fig. 4C clearly showthat the relative intensities of both the amide I and amideII bands decreased in the NP-treated tissue. Moreover, anincrease in intensity was observed at the 1452 cm21 band,which is due to the bending vibration of the CH2 groupsin the lipids and proteins. A notable increase was ob-served in the intensity of the COO2 symmetric stretchingvibration band at 1391 cm21. As shown in Fig. 4C, thetreated tissue spectrum displayed an increase in the in-tensity of the phosphate asymmetric stretching band lo-cated at 1236 cm21 ( p , 0.001), a decrease in the inten-sity of the symmetric phosphate stretching band at 1080cm21 ( p , 0.01), and a change in the shape of thesebands in comparison to the relevant bands in the spec-trum of the control tissue. The ratio of the absorption at1042 cm21 (the C–O stretching vibration) to that at 1080

APPLIED SPECTROSCOPY 839

FIG. 5. The infrared spectra of rainbow trout livers treated with 0(control), 66, and 220 mg/L NP and 22 mg/L E2 for two weeks (A) inthe 3600–2800 cm21 region and (B) in the 1800–800 cm 21 region.

cm21 (the phosphate symmetric stretching vibration) re-vealed a smaller value for NP-treated samples in com-parison to those of the control samples: the mean valueof this ratio slightly decreased from 1.028 6 0.047 to0.931 6 0.082 for treated tissues. In addition, the fre-quency of these bands shifted in the treated tissues. Theshift in the phosphate asymmetric band was not signi� -cant, while the phosphate symmetric band signi� cantlyshifted from 1080.33 6 0.512 to 1090.05 6 1.55 cm21

( p , 0.001). Additionally, Fig. 4C revealed that, in theCO–O–C asymmetric stretching region, the infrared spec-trum of the control sample consisted of a narrow andsymmetric band around 1152 cm21, while the treatedsamples gave a broader and asymmetric band around1163 cm21.

As seen from Fig. 4C, the peak of the band at 1042cm21, which is assigned mainly to C–O stretching vibra-tions in polysaccharides, was almost lost in the treatedliver spectrum, indicating a change in the types of gly-coproteins in the treated tissues.36

The band at 969 cm21, which is due to nucleic acids,was sharper and stronger in the NP-treated liver spectrumthan that in the control group spectrum.

In order to compare the effects of NP with that ofestrogen at the molecular level, the � sh were treated with22 mg/L 17-b Estradiol (E2) for two weeks and then thelivers of the � sh were examined by FT-IR spectroscopy.The infrared spectra of both E2 and various concentra-tions of NP-treated liver tissues are shown in Figs. 5Aand 5B, respectively. As can be seen from these � gures,the 22-mg/L-E2-treated liver spectrum bandwidth and in-tensity changes were quite similar to those generated byNP-treated spectra. These � gures suggested that the effectof 22 mg/L E2 on rainbow trout liver tissue was equiv-alent to NP concentration between 66 and 220 mg/L.However, there were some differences in the shifts of theband frequencies at wavenumbers 2959, 2927, 1739,1657, 1541, and 1152 cm21.

DISCUSSION

In the present study, the livers of rainbow trout wereinvestigated by FT-IR spectroscopy in the presence andabsence of NP. The NP concentrations were chosen be-tween 66 mg/L and 660 mg/L, which were also encoun-tered in the environment.1,6,32 The upper limit of the rangeproved to be 100% lethal in our system. The experimentsshowed that the differences observed between the controland NP-treated groups in trout were dose-dependent andaffected by the duration of exposure. It was seen that 220mg/L NP induced more signi� cant changes than 66 mg/LNP. These differences were also more signi� cant for lon-ger exposure times (Figs. 2A, 2B, 3A, and 3B). Since theeffects of NP were more profound in the spectra of two-week-220-mg/L-NP-treated � sh, the detailed comparisonswith the treated and untreated � sh were carried out underthese conditions.

In the present study, water was largely removed insample preparation for FT-IR spectroscopy. For this rea-son, its contribution to the 3307 cm21 band could be ne-glected and the contribution can be considered to be dueonly to proteins and polysaccharides. The dramatic re-duction observed in the intensity of the 3307 cm21 band

could have resulted from the reduced contribution fromglycogen OH absorption as reported by Melin et al.27

(Fig. 4A). This argument was strongly supported by thevariation of the 1042 cm21 band, which is assigned main-ly to the C–O stretching vibrations in glycogen.36 Thisband was almost lost in the treated liver spectrum (Fig.4C). These results revealed that glycogen levels de-creased in the NP-treated tissue.

The intensity of the band at 3015 cm21, which is dueto the CH stretching mode of HC5CH groups, increasedfor NP-exposed liver (Fig. 4B). In accordance to Taka-hashi et al.,24 this result indicates that the population ofunsaturated lipids increased in the NP-exposed liver. Themaximum of this band also shifted to lower frequencyvalues in the NP-exposed liver. Thus, the increase in theintensity and the shift in the frequency value might bethe results of a change in lipid metabolism induced byNP. This conclusion was supported by the results ob-tained from the bands in the 1800–800 cm21 region (Fig.4C). The intensity of the lipid C5O stretching vibrationat 1741 cm21 increased remarkably in the spectrum of the220 mg/L NP-treated liver. An increase in the intensityof this band suggested an increased concentration of theester groups belonging to triglycerides within NP ex-posed livers during the two weeks of exposure.38,39 Inaddition, the frequency of the absorption maximum ofthe ester C5O stretching vibration shifted from 1741 to1745 cm21, indicating a difference in the packing of ester

840 Volume 57, Number 7, 2003

groups within the tissue.42 This difference in packing ispresumably the result of the increased proportion of un-saturated acyl chains in triglycerides in the 220 mg/L NP-treated liver,38 as also suggested by the increased intensityof the ole� nic5CH stretching absorption at 3015 cm21.The shift in the C5O stretching band also shows a de-crease in hydrogen bonding involved either with the estergroups or the water molecules in the environment.24

Schwaiger et al.12 demonstrated that alkylphenolic com-pounds exert highly toxic effects on the organisms. Sincethe liver is the site of synthesis, storage, and release oflipids, it stands to reason that interference with these pro-cesses by a toxic compound could lead to an accumula-tion of fats in the liver itself. The exposure of toxic com-pounds can block the secretion of triglycerides, leadingto the development of what is commonly called a ‘‘fattyliver’’ a condition in which anywhere from 5 to 50% ofthe liver’s weight is fat.47 Another interesting observationderived from Fig. 4B was the decreased absorbtion in-tensity of the CH3 asymmetric stretching vibration at2959 cm21 as a consequence of NP treatment for twoweeks. The frequency of the symmetric CH2 stretchingband at 2857 cm21 shifted to a lower value after NP treat-ment, indicating an increase in the order of the acyl chainof the lipids.48–50 In addition to this shifting, the intensityof this band increased signi� cantly in treated tissues. This� nding suggested an increased proportion of the CH2

groups in these tissues. This was con� rmed by an in-creased intensity of the CH2 bending vibration at 1452cm21 in the treated liver40,42 (Fig. 4C). The band at 1391cm21 is generally assigned to the COO2 symmetricstretching vibration in amino acids and fatty acids.42

However, in our case, the dominant mode for protein sidechains around 1600 cm21 was not observed. Thus, themain contribution to the 1391 cm21 band may come fromthe COO2 symmetric stretching of fatty acids. We ob-served a signi� cant increase in the intensity of this band.Consequently, the increase in absorption intensities of the3015, 2857, 1741, 1452, and 1391 cm21 bands and thedecrease in the intensity of the 2959 cm21 band are main-ly the result of accumulation of triglycerides in the treatedliver tissues.

There was a broadening in the bandwidth of the amideI band (Table III) and a shifting in the frequency of thisband to a higher value. As the position of amide I ab-sorptions was sensitive to protein conformation, thisshifting and broadening implied conformational changesin tissue proteins.40,51 The intensities of amide I and theamide II bands decreased slightly in NP-treated liver (Fig.4C), which was consistent with the decrease in amide Aband at 3307 cm21. These results indicate a decrease inprotein concentration in the NP-treated tissues. In addi-tion, the treated sample spectrum gave an asymmetric andrelatively broader band near 1161 cm21, which was ab-sent in the control spectrum. The relatively weak band at1161 cm21 is attributed to C–O stretching modes of theC–OH groups in cell proteins. The contribution to thisband by membrane lipids is negligible.43–46

The frequencies of the asymmetric and symmetricphosphate stretching bands at 1236 and 1080 cm21, re-spectively, shifted in the treated tissues (Fig. 4C). Thesebands originated mainly due to the phosphodiester back-bone of cellular nucleic acids.43,44 The contribution to

these bands by phosphate residues present in membranelipids was negligible.43,44,46 The phosphate asymmetricband shifted slightly to higher frequency values and be-came broader in the treated tissues. In addition, the ratioof absorption at 1042 cm21 (the C–O stretching vibration)to 1080 cm21 (the phosphate symmetric stretching vibra-tion) for NP-treated samples had a much smaller valuethan that of the control group. This result implied an in-crease in the relative content of nucleic acids in NP-treat-ed tissues.31 The band at 969 cm21 in the NP-treated liverspectrum was sharper and stronger than those of the con-trol group spectra. In agreement with Ci et al., this resultalso implied an increase in the relative content of thenucleic acids, especially DNA, in the treated tissues.31

Thus, it was clear that DNA became the dominant com-ponent in NP-treated tissues. The increased DNA contri-bution in NP-treated tissues must originate from the cellnucleus, as reported by Chiriboga et al.45 Therefore, theincreased DNA signal is due to differences in nuclearmorphology, organization, and architecture, for example,increased nuclear/cytoplasm ratio, hyperchromicity, chro-matin aggregation, and cell division, and perhaps de-creased DNA condensation between control and NP-treated cells.45

In � sh, as well as in mammals and avian cells, APsmimic the 17b-estradiol by binding the estrogen recep-tors.14 However, potency in estrogenic activity of NP is100 to 1000 times less than that of 17b-estradiol in in-ducing the vitellogenin synthesis.3 To compare the im-posed changes of NP and E2, at least to a � rst degree ofapproximation, the spectra of liver tissues exposed tothese chemicals were studied. The estrogen concentrationwas 10 fold less than that of NP because at this treatmentlevel (22 mg/L) of E2, the obtained spectrum was in arange comparable with the spectra of 66 mg/L and 220mg/L. The E2 spectral bands related to lipids, carbohy-drates, proteins, and nucleic acids exhibited parallelchanges to those observed in 220 mg/L NP spectra (Figs.5A and 5B). A detailed analysis on the comparative ef-fects of these two chemicals in rainbow trout liver tissueas revealed by FT-IR spectra will be presented in anotherpaper, where the relationship between the estrogeniccompounds and vitellogenin will be discussed.

In conclusion, the results of the present study indicatethat when the rainbow trout were treated by an effectivesublethal dose (220 mg/L) of NP for two weeks, it in-duced signi� cant alterations in the liver tissues. The totalcontent of triglycerides increased in NP-treated liver. Inaddition, NP altered lipid order; it caused a decrease inthe synthesis of the protein. Some of these � ndings sup-port the results of previously conducted biochemical andphysiological studies. Moreover, in the present study, itwas found for the � rst time that, in NP-treated � sh, theglycogen level signi� cantly decreased, the relative con-tent of nucleic acids increased, and especially DNA be-came the dominant component. Furthermore, thesechanges became more prominent with increasing doses.The results of the present study also revealed that thecombination of rainbow trout in conjunction with the useof FT-IR spectroscopy holds promise as a bioindicatorfor detecting the level of NP in fresh water systems. Fi-nally, these primary studies also indicate that FT-IR spec-troscopy has a high potential for monitoring structural,

APPLIED SPECTROSCOPY 841

conformational, and metabolic changes in tissues at themolecular level after in vivo exposure to toxic chemicals.

1. M. Ahel, W. Giger, and M. Koch, Water Res. 28, 1131 (1994).2. S. N. Pedersen, L. B. Christiansen, K. L. Pedersen, B. Korsgaard,

and P. Bjerregaard, Sci. Total Environ. 233, 89 (1999).3. S. Jobling, D. Sheahan, J. A. Osborne, P. Matthiessen, and J. P.

Sumpter, Environ. Toxicol. Chem. 15, 194 (1996).4. S. R. Richardson, S. L. Pierens, K. M. Nichols, V. J. Kramer, E.

M. Snyder, S. A. Snyder, J. A. Render, S. D. Fitzgerald, and J. P.Giesy, Environ. Res., Sec. A 80, 122 (1999).

5. C. G. Naylor, J. P. Mieure, W. J. Adams, J. A. Weeks, F. J. Castaldi,L. D. Ogle, and R. R. Romano, J. Am. Oil Chem. Soc. 69, 695(1992).

6. M. A. Blackburn and M. J. Waldock, Water Res. 29, 1623 (1995).7. J. M. Herrero-Martinez, M. Marti-Fernandez, E. Simo-Alfonso, and

G. Ramis-Ramos, Electrophoresis 22, 526 (2001).8. W. Giger, P. H. Brunner, and C. Schaffer, Science (Washington,

D.C.) 225, 623 (1984).9. R. Thibaut, L. Debrauwer, D. Rao, and J. P. Cravedi, Sci. Total

Environ. 233, 193 (1999).10. N. G. Coldham, S. Sivapathasundaram, M. Dave, A. Ashfeld, T. G.

Pottinger, C. Goodall, and M. J. Sauer, Drug Metab. Dispos. 26,347 (1998).

11. F. Yadeite, A. Arukwe, A. Goksoyr, and R. Male, Sci. Total Envi-ron. 233, 201 (1999).

12. J. Schwaiger, O. H. Spieser, C. Bauer, H. Ferling, U. Mallow, W.Kalbfus, and R. D. Negele, Aquat. Toxicol. 51, 69 (2000).

13. S. Jobling and J. P. Sumpter, Aquat. Toxicol. 27, 361 (1993).14. R. White, S. Jobling, S. A. Hoare, J. P. Sumpter, and M. G. Parker,

Endocrinology 135, 175 (1994).15. J. J. Lech, S. K. Lewis, and L. Ren, Fundam. Appl. Toxicol. 30,

229 (1996).16. L. J. Christensen, B. Korsgaard, and P. Bjerregaard, Aquat. Toxicol.

46, 211 (1999).17. P. J. Hughes, H. McLellan, D. Lowes, A. S. Z. Khan, J. G. Bilmen,

S. C. Towey, R. E. Godfrey, R. H. Michell, C. J. Kirk, and F.Michelangeli, Biochem. Biophys. Res. Commun. 277, 568 (2000).

18. F. Michelangeli, P. Orlowski, P. Champeil, M. East, and A. G. Lee,Biochemistry 29, 3091 (1990).

19. M. Bragadin, G. Perin, A. Iero, S. Manente, V. Rizzoli, and G.Scutari, Chemosphere 38, 1997 (1999).

20. P. Burkhardt-Holm, T. Wahli, and W. Meier, Ecotoxicol. Environ.Saf. 46, 34 (2000).

21. A. Bloom, E. Ekman, A. Johannisson, L. Norrgren, and M. Peso-nen, Arch. Environ. Contam. Toxicol. 34, 306 (1998).

22. J. Legler, C. E. Van den Brink, A. Brouwer, A. J. Murk, P. T. Vander Saag, A. D. Vethaak, and B. Van der Burg, Toxicol. Sci. 48,55 (1999).

23. N. E. Skakkebaek, E. R. D. Meyts, N. Jorgensen, E. Carlsen, P. M.Petersen, A. Giwercman, A. G. Andersen, T. K. Jensen, A. M. An-derson, and J. Muller, APMIS 106, 3 (1998).

24. H. Takahashi, S. M. French, and P. T. T. Wong, Alcohol.: Clin. Exp.Res. 15, 219 (1991).

25. L. H. Kidder, P. Colarusso, S. A. Stewart, I. W. Lewin, N. M. Appel,D. S. Lester, P. G. Pentchev, and E. N. Lewis, J. Biomed. Opt. 4,7 (1999).

26. K. F. M. Fung, M. Senterman, P. Eid, W. Faught, Z. N. Mikhael,and P. T. T. Wong, Gynocologic Oncology 66, 10 (1997).

27. A. Melin, A. Perromat, and G. Deleris, Biopolymers (Biospectros-copy) 57, 160 (2000).

28. F. Severcan, N. Toyran, N. Kaptan, and B. Turan, Talanta 53, 55(2000).

29. K. Yano, S. Ohoshima, Y. Shimizu, T. Moriguchi, and H. Katayama,Cancer Lett. 110, 29 (1996).

30. K. Liu, M. Jackson, M. G. Sowa, H. Ju, I. M. C. Dixon, and H. H.Mantsch, Biochim. Biophys. Acta 1315, 73 (1996).

31. Y. X. Ci, T. Y. Gao, J. Feng, and Z. Q. Guo, Appl. Spectrosc. 53,312 (1999).

32. S. O’Halloran, K. Liber, J. Gangl, and M. L. Knuth, Environ. Tox-icol. Chem. 18, 376 (1999).

33. W. W. Daniel, Biostatistic Foundation for Analysis in the HealthSciences (John Wiley and Sons, New York, Chister Weinheim,1999).

34. N. Jamin, P. Dumas, J. Moncuit, W. Fridman, J. Teillaud, L. G.Carr, and G. P. Williams, Appl. Biol. Sci. 95, 4837 (1998).

35. G. I. Dovbeshko, N. Y. Gridina, E. B. Kruglova, and O. P. Pash-chuk, Talanta 53, 233 (2000).

36. D. J. Lyman and J. Murray-Wijelath, Biomed. Mater. Res. (Appl.Biomater.) 48, 172 (1999).

37. E. Grell, Membrane Spectroscopy (Springer-Verlag Berlin Heidel-berg, Germany, 1981), p. 281.

38. M. Nara, M. Okazaki, and H. Kagi, Chem. Phys. Lipids 117, 1(2002).

39. G. Voortman, J. Gerrits, M. Altavilla, M. Henning, L. Van Bergeijk,and J. Hessels, Clin. Chem. Lab. Med. 40, 795 (2002).

40. P. I. Haris and F. Severcan, J. Mol. Catal. B: Enzymatic 7, 207(1999).

41. R. Manoharan, J. J. Baraga, P. R. Rava, R. R. Dasari, M. Fitz-maurice, and M. S. Feld, Atherosclerosis 103, 181 (1993).

42. M. Jackson, B. Ramjiawan, M. Hewko, and H. H. Mantsch, Cell.Mol. Biol. 44, 89 (1998).

43. P. T. T. Wong, E. D. Papavassiliou, and B. Rigas, Appl. Spectrosc.45, 1563 (1991).

44. J. Wang, C. Chi, S. Lin, and Y. Chern, Anticancer Res. 17, 3473(1997).

45. L. Chiriboga, H. Yee, and M. Diem, Appl. Spectrosc. 54, 480(2000).

46. B. Rigas, S. Morgello, I. S. Goldman, and P. T. T. Wong, Proc. Natl.Acad. Sci. U.S.A. 87, 8140 (1990).

47. K. E. Stine and T. M. Brown, Principles of Toxicology (CRC Press,Boca Raton, FL, 1996), p. 153.

48. F. Severcan, Bioscience Rep. 17, 231 (1997).49. J. L. Casal, D. G. Cameron, I. C. P. Smith, and H. H. Mantsch,

Biochemistry 19, 445 (1980).50. E. N. Lewis, I. W. Levin, and C. J. Steer, Biochim. Biophys. Acta

986, 161 (1989).51. M. Jackson, J. R. Mans� eld, B. Dolenko, R. L. Somorjai, H. H.

Mantsch, and P. H. Watson, Cancer Detect. Prevent. 23, 245 (1999).