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Toxicology 283 (2011) 140–150

Contents lists available at ScienceDirect

Toxicology

journa l homepage: www.e lsev ier .com/ locate / tox ico l

haracterization of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-provokedtrong and rapid aversion to unfamiliar foodstuffs in rats

anna Lensua,b,∗, Jouni T. Tuomistoa, Jouko Tuomistoa, Raimo Pohjanvirtac

Department of Environmental Health, National Institute for Health and Welfare (THL), P.O.B. 95, FI-70701 Kuopio, FinlandFaculty of Health Sciences, The School of Pharmacy, University of Eastern Finland, P.O.B. 1611, FI-70211 Kuopio, FinlandDepartment of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, P.O.B. 66, FI-00014 University of Helsinki, Finland

r t i c l e i n f o

rticle history:eceived 7 February 2011ccepted 12 March 2011vailable online 22 March 2011

eywords:,3,7,8-tetrachlorodibenzo-p-dioxin, TCDDat strainseeding behaviourovel food item avoidance

a b s t r a c t

A conspicuous but scantly studied feature of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is avoid-ance of unfamiliar foodstuffs, which seems to be one of the very few exquisitely sensitive behaviouraleffects in adult laboratory animals. Here we characterized this peculiar response further after low dosesof TCDD. The time-course of the novelty avoidance, the role of nutriment form and dependence of theaversion on the time lag between TCDD exposure and the presentation of a novel food item was deter-mined using rats with different sensitivities to lethality of TCDD. Rats were offered chocolate, liquidnutriment or familiar feed with an unfamiliar texture and the consumptions were measured for vary-ing periods. Aversion to a novel food item (chocolate) emerged within 5.5 h after TCDD exposure. A lagof a week or more between TCDD exposure and the presentation of chocolate abolished the avoidancewhereas simultaneous presentation of chocolate with TCDD treatment rendered the rats oblivious to

the chocolate’s presence for over 40 days. Rats avoided also liquid nutriments when these were coupledwith TCDD administration but this faded much sooner than chocolate aversion. Even a change in feedtexture at the exposure was able to elicit the response. However, habituation was found to interfere withthe aversion. These findings indicate that temporal proximity to TCDD exposure is a requisite for theavoidance response which emerges rapidly and may linger on for extended periods, but is not strictlyconfined to any specific food type. The molecular mechanisms of this tantalizing behavioural alteration

.

remain to be determined

. Introduction

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is acutely the mostoxic synthetic compound known with an LD50 value of only1 �g/kg for the guinea pig (Schwetz et al., 1973). TCDD is theost potent congener of dioxins and dioxin-like chemicals. These

nvironmental contaminants are persistent organic pollutants andause a plethora of adverse effects in laboratory animals includ-ng immunotoxicity, teratogenicity and carcinogenicity [reviewed

.g. in (Birnbaum and Tuomisto, 2000; Pohjanvirta and Tuomisto,994)].

One of the noticeable targets of TCDD toxicity in rats is the feed-ng behaviour. Persistently reduced feed intake has been shown to

∗ Corresponding author at: Department of Biology of Physical Activity, Universityf Jyväskylä, P.O.Box 35, FI-40014 Jyväskylä, Finland. Tel.: +358 50 3011 272;ax: +358 14 260 2071.

E-mail addresses: sanna.lensu@elisanet.fi (S. Lensu), jouni.tuomisto@thl.fiJ.T. Tuomisto), jouko.tuomisto@thl.fi (J. Tuomisto), raimo.pohjanvirta@helsinki.fiR. Pohjanvirta).

300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.tox.2011.03.007

© 2011 Elsevier Ireland Ltd. All rights reserved.

underlie wasting syndrome, the most conspicuous facet of acutetoxicity of TCDD (Seefeld et al., 1984). TCDD-treated rats also dis-play altered responses to various feeding regulatory challenges(Pohjanvirta and Tuomisto, 1990). The impact of TCDD on bodyenergy balance appears to be specific, and because rats exposed tosublethal doses of TCDD have a perpetually lower body weight levelthan that of their age-matched controls and defend this level, theexposure has been suggested to decrease the putative body weightset-point [reviewed in (Linden et al., 2010)].

The aforementioned effects of TCDD on feeding are delayed,emerging days or weeks after TCDD exposure. However, shortlyafter TCDD treatment rats have been shown to exhibit a pecu-liar behavioural change, avoidance of palatable novel food items(Tuomisto et al., 2000). This response was found to take place dur-ing the first night post-exposure. Thus, it appeared to representone of the most immediate responses to TCDD, and importantly, it

occurred in adult animals.

The sensitivity of animals to TCDD lethality varies among speciesand even (sub)strains. Furthermore, there are wide species- andstrain-specific differences in some toxic responses (e.g. weight loss,liver toxicity) – whereas some other toxic responses are more

S. Lensu et al. / Toxicology 283 (2011) 140–150 141

Table 1A synopsis of experiments performed.

Study number;question(s) asked

Rat strain; sex Group size; weight (atthe exposure,mean ± SD)

Dose(s) of TCDD(�g/kg)a;administration route

Consumptionoutcome; measuringperiod (after TCDD)

Main result(s)

1. Time-course of(chocolate) aversion?

Line A; maleb 8; 258.8 ± 20 g 3, i.g. Chocolate; 0.5–12 hperiods for 48 h

Chocolate was rejectedafter TCDD, thedifference fromcontrols reachedsignificance by 5.5 h

2. Effect of time lag(between exposureand access tochocolate: 0, 1, 7 or14 days) onaversion? Durationof aversion?

H/W; malec 5–6; 259.5 ± 16 g 10, i.g. Chocolate; daily for 1week (time lag 14days) or 2 weeks (timelag 0, 1, 7), thereafter24 h on days 27, 34, 41,48, 55, 62, 69 and 76after TCDD

Chocolate avoided ifthe lag 1 day or less.Most persistentaversion if chocolatepresentation coincidedwith TCDD exposure:attenuation of theeffect started in 6weeks

3. Is feed texturecritical? Is feedavoided if the textureis changed at theexposure?

Line C, maled 5–6; 348.6 ± 34 g 3, i.g. Standard chow,pelleted or powdered;daily for 4 days

Novel texture offamiliar feed avoidedafter TCDD exposure

4. The role ofconditioned tasteaversion?

Line A, maleb 6; 257.5 ± 26 g 3, i.p. Powdered feed andchocolate; 24 h on days0 and 7

At subsequentchallenge, the choicecoupled to TCDDexposure avoided

5. Effect ofadministration routeon the avoidance?

Line A, maleb 7; 202.2 ± 20 g 3, i.g. or i.p.; 100 i.p. Chocolate; daily for 3days

Administration routeaffects the response:3 �g/kg orally has thesame effect as100 �g/kg injection

6. Is liquid foodequivalent to solid?Does habituationinterfere?

H/W, malec 4–5; 235.9 ± 26 g 3, i.g. Sucrose (10%) orsaccharin (0.25%)solutions;habituation > a month;follow-up daily for 16days

Also liquid foodavoided. Habituationprevented saccharinaversion

7. Effect of habituationon chocolateaversion?

L–E, femalee 4; 172.1 ± 10 g 3, i.g. Chocolate;habituation > a month;follow-up daily for 10days

Habituationdiminished chocolateaversion, but only for aday

a Controls were treated by equal route and volume (4 ml/kg) with corn oil, the vehicle for TCDD.

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b LD50 > 10,000 �g/kg (Tuomisto et al., 1999).c LD50 > 9600 �g/kg (Unkila et al., 1994).d LD50 = 40 �g/kg (Tuomisto et al., 1999).e LD50 = 7 �g/kg (Pohjanvirta et al., 1993; Tuomisto et al., 1999).

onsistent (e.g. induction of xenobiotic-metabolizing enzymes andhymus atrophy) (Pohjanvirta and Tuomisto, 1994; Simanainent al., 2002). In one extreme, there is a thousand-fold differenceven within a single species, as the LD50 (lethal dose for 50% ofosed animals) for a sensitive inbred Long–Evans rat substrainTurku/AB; L–E) is ∼10–20 �g/kg (Pohjanvirta et al., 1993) whereashat for a resistant, originally outbred Han/Wistar rat substrainat present random-bred strain, Kuopio; H/W) is over 9600 �g/kgUnkila et al., 1994). This difference has been shown to originaterom a polymorphism in the aryl hydrocarbon receptor gene (Ahr)esulting in an altered transactivation domain structure in H/W ratryl hydrocarbon receptor protein (AHR) (Pohjanvirta et al., 1998).he AHR is a key protein in mediation of the effects of TCDD (Lindent al., 2010; Poland and Glover, 1974). Other structural variants,ffecting the ligand-binding domain of the AHR, account for sensi-ivity differences among some mouse strains and birds (Karchnert al., 2006; Poland et al., 1994).

Recently we showed that aversion to a novel food item appearslso in mice being dependent on the presence of the AHR. A

ose–response analysis carried out in the same study on ratsevealed that novelty avoidance is a highly sensitive behaviouralesponse to TCDD emerging independently of the rats’ sensitivityo the lethality of TCDD (Lensu et al., 2011). In the present study,e sought to characterize this phenomenon further in differently

dioxin-sensitive rat strains. We analysed its time-course as well asthe roles of habituation and food properties in it.

2. Material and methods

2.1. Animal husbandry

TCDD-induced alterations in food selection and acceptance of novel food itemswere studied in rats by various experimental settings which are summarized inTable 1. The rats of both sexes were 8–13 weeks old at the exposure, except in oneexperiment (experiment 5) where they were 15–18 weeks old. In addition to H/Wand L–E rats, we used line A and line C rats. These lines were produced from H/W andL–E rats by cross-breeding at the National Public Health Institute, Kuopio (Tuomistoet al., 1999). H/W rats carry a deletion/insertion-type reconstruction in the trans-activation domain of their AHR (Pohjanvirta et al., 1998). This structural alterationis the main reason for their resistance. However, another (currently unidentified)gene “B” also affords some resistance to the toxic effects of dioxin in H/W rats. Thegenotype of line A rats is presumed to be Ahrhw/hwBwt/wt , and they have a highly TCDD-resistant phenotype (LD50 >10,000 �g/kg for males and >2000 �g/kg for females).Line C rats have presumably no resistance alleles in their genotype, and they areTCDD-sensitive with LD50 values of 40 (males) and 19 (females) �g/kg (Tuomistoet al., 1999).

Rats were individually housed in stainless steel wire-mesh cages. Unless other-

wise described, rats were adapted to the conditions and handling for at least 1 weekbefore starting the experiment. In the animal room, temperature (21 ± 1 ◦C) andhumidity (50 ± 10%) were controlled, and artificial lights were on from 7 a.m. to 7p.m. In experiment 1, the animal room was illuminated with a red lamp throughoutthe study, because food consumption measurements were conducted also duringthe dark hours. All the animal experiments were reviewed and approved by the

142 S. Lensu et al. / Toxicology 283 (2011) 140–150

Fig. 1. A low dose (3 �g/kg i.g.) of TCDD affects food selection in male rats of line A (n = 8 per group). Rats were exposed at the beginning of darkness, and the consumptions(mean ± SE) of a novel food item, chocolate (A) as well as the familiar chow (B) were followed for the next 48 h. Both feeds were available ad libitum. Note that measuringperiods varied in length (given in h, bars show the amount eaten between two consecutive time points indicated on the x-axis). The grey color in panels depicts darkness,w thoses TCDDs show*

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hite is the illuminated period. Right panels show the control consumptions and leftum of intakes). Significant differences in the chocolate and chow intakes betweenignificant differences between these two groups in their cumulative intakes are*0.001 < p ≤ 0.01, *0.01< p ≤ 0.05).

nimal Experiment Committee of the University of Kuopio and Kuopio Provincialovernment, and they were conducted in accordance with the Guidelines of theuropean Community Council directives 86/609/EEC.

.2. TCDD exposure

TCDD (UFA-Oil Institute, Ufa, Russia) was >99% pure as determined by gashromatography–mass spectrometry and it was dissolved in corn oil (Sigma, C8267,t. Louis, MO, USA; energy content 36.9 kJ/g) as described previously (Simanainent al., 2002). The rats were dosed 4 ml/kg orally (intragastric gavage, i.g.) unless oth-rwise stated (Table 1). Control animals were treated with an equal volume of cornil.

.3. Feed and additional food items in experiments

The animals were fed on regular feed (R3 or R36, Ewos, produced in Södertäljer in Kimstad, Sweden; energy content 12.6 kJ/g) which was either pelleted or pow-ered depending on the experimental design. If the rats were fed on powderedersion of the feed, they were habituated to eating it for at least a week before thexposure. In experiment 7, female L–E rats were fed Altromin 1314F feed (AltrominmbH, Lage-Im Seelenkamp, Germany; energy content 12.5 kJ/g). Feed and waterere available ad libitum throughout the studies. If the intake of the default feedas restricted or some other type of food or drink was offered, the details of theserocedures are given at the experimental design (Table 1).

The chocolate used in some of the experiments was regular milk chocolate com-ercially available in Finland (Panda, Vaajakoski, Finland; energy content 23 kJ/g).

here were several reasons to use chocolate instead of other alternatives in thesexperiments: (1) chocolate was used as a high-energy test food in previous wastingyndrome studies with good results (Lensu et al., 2011; Tuomisto et al., 2000). (2)hocolate is palatable and rats readily consume it. (3) It is easily available and has atandard, homogeneous and stabile composition at room temperature. (4) The levelf quantification is as low as 10 mg, and it is easy to observe the difference betweensingle bite and no bite. (5) It can be placed into a regular wire-mesh cage withoutny additional equipment, thus making experiments with several foods possible. (6)ats do not spill it, which is often a problem with regular feed, and especially so inCDD-treated rats (Lensu et al., submitted for publication-a).

Saccharin (S-6047, Lot. 075K00071 Sigma; contains no energy) and sucrose (ICNiomedicals, 821713, Lot. 8681C, OH, USA; 10% solution contains 1.7 kJ/g energy)ere dissolved in tap water.

.4. Data analysis and statistics

In some instances, consumptions are shown as energy intake (kJ). Furthermore,o take into account the body weights and the metabolic needs of the rats, metaboliceight was calculated for each rat [body weight (kg)]0.67 (Donhoffer, 1986; Feldman

nd McMahon, 1983) and energy intake was related to it.The data were analysed with MS-Excel and SPSS software version 17.0 using lin-

ar mixed model or generalized estimated equations to model the effect of exposuren feeding responses by taking the repeated nature of the observations for each ratnto account (Zeger and Liang, 1986). The level of statistical significance was set at≤ 0.05.

of TCDD-treated rats; dashed lines depict cumulative values of intakes (cumulative-exposed and control rats during individual periods are shown in the TCDD panels;n in the control panels (Linear mixed model for repeated measures, ***p ≤ 0.001,

The whole original data is available at Opasnet website (http://en.opasnet.org/w/TCDD-provoked aversion to foods in rats).

3. Results

3.1. Temporal evolvement of aversion to a novel food item,chocolate

Chocolate consumption of 12-week-old male line A rats wasmeasured after a low oral dose (3 �g/kg) of TCDD or corn oil admin-istration at the beginning of the dark phase (at 7 p.m.). Chocolatewas offered as a novel food item for all rats in conjunction withthe exposure, in addition to their familiar powdered feed. Almostimmediately after exposure, control rats started to eat chocolateavidly, while TCDD-treated rats only tried small or tiny amounts ofit in the beginning. In the course of the study, controls ate chocolatein increasing amounts while TCDD-treated avoided it. A differ-ence in the cumulative chocolate consumptions between these twogroups was already discernible by 2.5 h but it reached significance5.5 h after the exposure remaining then significant until the endof the measuring period (Fig. 1A, linear mixed model for repeatedmeasures with autoregressive correlation structure). Of individualtime points, from 9.5 h on after TCDD treatment chocolate con-sumption was significantly higher in controls than in TCDD-treatedrats (Fig. 1A), except for daytime on day 1 (36–42 h) when choco-late consumption of the controls was low similarly to their chowintake.

In this experiment the rats were fed on powdered feed. TCDD-treated rats clearly preferred the powdered feed to chocolate. Thedifference in cumulative chow consumption reached significanceduring the first illuminated measuring period in the experiment,after 18 h of exposure (Fig. 1B). From thereon, TCDD-exposed ratsate more chow than controls. Body weights of the rats did not differin the experiment (data not shown).

3.2. Chocolate is avoided if it is offered simultaneously or within1 day from TCDD exposure – effect of lag time length

By offering a novel food item, chocolate to male H/W rats at dif-ferent times after TCDD exposure we sought to verify the necessityof temporal proximity between TCDD exposure and presentationof a novel food item for the neophobia-like response. Simultaneous

S. Lensu et al. / Toxicology 283 (2011) 140–150 143

Fig. 2. Effect of lag time between TCDD exposure and presentation of a novel fooditem on chocolate consumption. Male H/W rats (n = 6 per group, except in one controlgroup n = 5) were exposed to 10 �g/kg (i.g.) TCDD. Chocolate was offered simulta-neously or 1, 7 or 14 days after TCDD. Chocolate consumption (mean ± SE) wasmagt

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Fig. 3. Effect of a change in feed texture at TCDD exposure on feed intake (mean ± SE)in male rats of line C. Upper panels show the feed consumptions of controls [cornoil (i.g.), n = 15] and the lower panels (having a light color) of TCDD-treated rats[3 �g/kg (i.g.), n = 16]. Rats were divided into three groups: (A) pelleted standardfeed was continued after the exposure (n = 5 in both groups); (B) pelleted feed waschanged to powder at the exposure (n = 5 in both groups) and (C) rats were allowedto make a choice between familiar pellets and novel powdered feed (n = 5 in controland n = 6 in TCDD-group). Significant differences in feed intake between TCDD andcontrol groups as well as differences among each group between days are depicted

easured for up to 48 days post-exposure. The differences between TCDD-treatednd control rats were assessed as in Fig. 1. For clarity, significant differences betweenroups are not shown on days 2–13 and on days 3–14 (two uppermost panels), buthe differences remained significant (p ≤ 0.001) throughout these periods.

xposure to TCDD (10 �g/kg, i.g.) and presentation of a novel foodtem, chocolate, caused almost total avoidance of chocolate alreadyn the first day post-exposure (the amount eaten was 0.19 ± 0.07 g,ig. 2). Even if the lag between exposure and chocolate presentationas 1 day, exposed rats avoided the chocolate (although the avoid-

nce was not complete on the first day of chocolate appearance)Fig. 2).

After 2 weeks we presented chocolate for rats for 24 h only onceweek to study the duration of aversive response after TCDD treat-ent. The avoidance of chocolate persisted for several weeks, but

fter a month (if chocolate was offered 1 day after the exposure) orweeks (if chocolate was offered simultaneously with TCDD) also

he exposed rats started to eat small but incremental amounts ofhocolate (Fig. 2). Nevertheless, if chocolate was offered simulta-eously with TCDD, exposed rats kept their chocolate consumptionelow control level (mean level of control rats) at least 76 days (endf observation period, data not shown).

If the rats were exposed 7 or 14 days prior to access to the novelood item, chocolate, there was either no difference among exposednd control rats, or TCDD-treated rats ate even more chocolate thanontrols (Fig. 2). All groups gained a little weight with controlsutperforming the TCDD-treated rats by the end of experimentaleriod (data not shown).

.3. Food texture as a determinant of novelty – can a change inhe form of feed at the exposure trigger the TCDD-inducedvoidance behaviour?

In this experiment on male rats of line C, the form of the rats’eed was either changed from pellets to powdered feed or pow-ered form of feed was offered as an additional choice after TCDDxposure. Rats were divided into six groups and their feed con-umptions were followed for three consecutive days after exposureo TCDD (3 �g/kg, i.g.) or vehicle. The novel and familiar forms ofhe standard feed were available after TCDD exposure either aloner together. The low dose of TCDD did not affect intake of the famil-ar pelleted feed when it remained available as the only choice

Fig. 3A), whereas an abrupt change from pellets to powderedeed at TCDD exposure significantly diminished feed consumptionFig. 3B). TCDD-treated rats preferred the familiar pellets, whenoth pellets and a novel form of the same feed, powder, were avail-

with asterisks as in Fig. 1, assessed by linear mixed model.

144 S. Lensu et al. / Toxicology 283 (2011) 140–150

Fig. 4. The role of conditioned taste aversion in the novelty avoidance induced by TCDD was studied with male rats of line A. Rats (n = 6 in each group) were challenged witha new food item, either powdered feed or chocolate, on the day of TCDD exposure (3 �g/kg i.p.) and selection preferences were tested seven days later. Mean values represent2 shoulda erencd easuT

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4-h energy intakes (mean ± SE) of controls (A) and TCDD-treated rats (B), but itlso regular feed available (but its consumption was not measured). Significant diffifference in intake between TCDD-treated and control rats (in their corresponding mCDD-treated and control rats on day 7 (p ≤ 0.05).

ble (Fig. 3C). Only in the group offered powdered chow aloneFig. 3B) feed intake diminished in comparison with other TCDD-reated groups.

Contrary to TCDD-treated rats, controls accepted powderedeed. The change in feed texture did not affect their intake (Fig. 3And B). If they had both pellets and powdered feed available afterorn oil treatment (Fig. 3C), they shifted their preference to pow-ered feed.

.4. Role of conditioned taste aversion in the novel food itemvoidance: at the second encounter with novel feed items,CDD-treated rats avoided the food coupled with the exposure

Following a peripheral (i.p.) treatment with TCDD (3 �g/kg) ororn oil (at 10 a.m.) male rats of line A were presented with a novelood item, chocolate or powdered chow, in addition to their regularellet chow, for 1 day. Their preferences were tested a week later byffering the same food item as at exposure together with one novelood item. On both occasions, 24-h consumptions were measured.

After the exposure to TCDD (on day 0), energy intake fromowdered chow decreased more than that from chocolate in com-arison with controls (Fig. 4). One week later, in the selectionreference test TCDD-treated rats avoided the food they had beenffered on the exposure day. If they had received chocolate, theyvoided it and chose powdered feed. If they had been offered chow,hey showed aversion to it and chose chocolate (Fig. 4B). In clearontrast, control rats preferred chocolate to powdered chow, irre-pective of whether they were offered powdered chow or chocolateweek previously (Fig. 4A). However, on the seventh day control

ats ate more chocolate if it was already familiar to them (Fig. 4A).Total energy intake from the two choices of food was higher

n day 7 vs. day 0 in all groups save for the TCDD-group providedhocolate at exposure. Furthermore, it was higher in controls thann TCDD-treated rats (Fig. 4).

.5. Effect of administration route on avoidance of a novel foodtem, chocolate: 3 �g/kg orally is comparable to 100 �g/kgnjection

In this experiment the role of administration route in the choco-ate aversion was studied by administering TCDD to 8-week-old

ale rats of line A either as injection (i.p., 3 and 100 �g/kg) or orallyi.g., 3 �g/kg) at 3 p.m., 4 h before the onset of darkness. Control

be noted that this is not equivalent to the total daily intake as the animals hades are depicted with asterisks as in Fig. 1. In addition, letter A denotes a significantrements) and letter a denotes a significant difference in total energy intake between

rats were treated equally with the vehicle, corn oil, and chocolatewas offered as a novel food item to all rats simultaneously withthe exposure. During the day of exposure (day 0, Fig. 5A), choco-late consumption diminished similarly after the 3 �g/kg (i.g.) and100 �g/kg (i.p.) doses compared with controls, while the i.p. doseof 3 �g/kg did not affect chocolate consumption. Hence, there wasa significant difference among the two groups exposed to TCDDi.p. One day later (on day 1) chocolate consumption was signifi-cantly lower in all TCDD-treated groups than in their controls butthe impact was less pronounced at 3 �g/kg (i.p.) vs. the two othertreatments (Fig. 5A).

The controls shifted their feeding from chow to chocolatewhereas the converse was true of all TCDD-treated rats (Fig. 5Aand B). When total energy intake (kJ) was calculated relative tometabolic body weight, it increased on day 0 in controls and in the3 �g/kg (i.p.) TCDD-group while it was slightly reduced in the otherTCDD groups. On day 1 metabolic energy intake diminished also inthe 3 �g/kg (i.p.) group to the level of the other TCDD groups whileit remained elevated in controls (Fig. 5C).

3.6. Novel liquids are avoided after TCDD exposure independentlyof their energy contents – habituation interferes with theavoidance response

In this study we tested whether the TCDD-induced noveltyavoidance extends to liquids with different energy contents andhow habituation might influence the avoidance. At the exposure,the rats (male) were 9 weeks old (n in each group = 4–5). For a periodof 1 month prior to exposure all rats in the experiment were pre-sented with another drink bottle containing either water or 0.25%saccharin (which tastes sweet but yields no energy; offered forhabituated groups, see below). Three hours after TCDD exposureeach rat was given a choice in addition to tap water: either 0.25%saccharin or 10% sucrose solution (ad libitum). TCDD (3 �g/kg, i.g.)exposure diminished drinking of both of these sweet-tasting liquidsif they were novel to rats (Fig. 6A, middle and right panel).

We had also 10 male H/W rats habituated to drinking 0.25% sac-charin solution (ad lib.) for a month before the TCDD or corn oil

treatment. On the day before the exposure, these groups did notdiffer in their saccharin drinking (69.3 ± 16 ml, habituated controlsand 78.1 ± 11 ml, habituated TCDD-group), both groups preferredsaccharin to tap water (Fig. 6A and B). The habituation almost abol-ished the TCDD-provoked avoidance response: there was only a

S. Lensu et al. / Toxicology 2

Fig. 5. Effect of administration route on (A) chocolate and (B) chow intakes. In panel(C), total energy intake per metabolic masses of rats (kJ/kg0.67) is shown. TCDD-resistant line A male rats (n = 35, 7 rats per group) were exposed orally (i.g.) orintraperitoneally (i.p.) to either 3 or 100 �g/kg of TCDD, and consumptions weremonitored for the next 2 days. Bars represent the mean ± SE, statistical significanceswere assessed by generalized estimating equations (***p ≤ 0.001, **0.001 < p ≤ 0.01,*0.01 < p ≤ 0.05).

83 (2011) 140–150 145

non-significant modest decrease in saccharin consumption afterTCDD exposure in comparison with corn oil-treated controls.

In control rats, continuous access to saccharin decreased itsdrinking steadily from about 80 ml/day on day 1 to ca. 50 ml/day onday 11; thereafter its intake stabilized. The consumption of sucrosetook an opposite course increasing from initial 70 ml/day to finalca. 100 ml/day. Non-habituated TCDD-treated rats drank only ca.20 ml/day or less of both of these solutions over the first 4 days.The consumptions then increased reaching control levels by theend of the observation period (day 16).

All rats had access to regular feed and tap water ad libitum,both during the habituation period and in the course of the exper-iment. Water consumption was inversely correlated with drinkingof sucrose or saccharin solution (Fig. 6B). Rats largely adjusted theirfeed consumption according to the energy gained from the liquids(data not shown). However, rats with access to sucrose (especiallythe controls) tended to consume more energy than any of the othergroups (Fig. 6C). All rats in the study grew slightly with no differ-ences among the groups (data not shown).

3.7. Does habituation interfere with chocolate avoidancefollowing TCDD?

The key role of novelty in the TCDD-provoked chocolate avoid-ance behaviour was studied with female L–E rats. Half the rats(n = 8) were habituated to eating chocolate by providing them atiny piece of it on weekdays for 1 month before the exposure(3 �g/kg or the corn oil i.g.). The daily piece (providing about 5%of daily energy intake, 0.2–0.5 g) was aimed at not enhancing bodyweight gain, which was the case (data not shown). At the end of thehabituation period, the rats devoured their daily chocolate treatimmediately. The other half (n = 8) got chocolate as a novel fooditem 3 h after the exposure when all rats were given chocolatead libitum.

TCDD-treated rats ate significantly less chocolate than controlsalready on the day of exposure with the difference remainingsignificant throughout the experiment (Fig. 7A). The 1-monthhabituation procedure counteracted this response to TCDD only onthe day of exposure (Fig. 7A). Rats in both control groups preferredeating chocolate and they ate less chow than TCDD-treated rats(Fig. 7B).

TCDD-treated rats kept their energy intake constant, below thecontrol level, whereas controls increased their energy intake fromthe pre-exposure level (relative to their metabolic needs, Fig. 7C)and gained weight (data not shown). Hence the body weights ofthem were slightly (but significantly) higher than those of TCDD-treated groups during the final days of the experiment.

4. Discussion

2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDD, exerts a number ofadverse effects in laboratory animals. One of those is a wastingsyndrome, which finally leads to the death of the animal if thedose is high enough (reviewed e.g. in (Birnbaum and Tuomisto,2000; Dragan and Schrenk, 2000; Linden et al., 2010; Pohjanvirtaand Tuomisto, 1994)). The wasting mainly results from a dose-dependent hypophagia, and sensitivity to the effect varies amonganimal species and strains (Pohjanvirta and Tuomisto, 1994). Atlethal doses in rodents, the wasting culminates in death in a fewweeks (Birnbaum and Tuomisto, 2000). Control rats pair-fed to

fatally TCDD-treated rats die of cachexia at approximately the sametime as their TCDD-exposed partners (Christian et al., 1986; Weberet al., 1991). However, before the body weight loss occurs, TCDDexposure alters feeding and drinking behaviours. We have previ-ously shown that some alterations in feeding appear quickly, within

146 S. Lensu et al. / Toxicology 283 (2011) 140–150

Fig. 6. Novelty avoidance in the case of liquids and the effect of habituation on it was studied in H/W rats (n = 29, 4–5 per group). At the habituation, rats (n = 10) wereoffered 0.25% saccharin solution ad libitum for a month. After TCDD exposure (3 �g/kg i.g.) or corn oil treatment the rats habituated to drinking saccharin continued drinkingit and the rest of the rats were offered either saccharin (0.25%) or sucrose (10%) solutions as novel drinks; the solutions were freely available. In addition, regular feed andwater were available ad libitum. Daily consumptions (mean ± SE) of (A) offered solutions, (B) water and (C) energy were measured for 16 days after the exposure. Statisticalsignificances are as in Fig. 1.

S. Lensu et al. / Toxicology 2

Fig. 7. Female L–E rats (n = 8) were habituated to chocolate with an amountnot influencing their body weight gain. After exposure to TCDD (3 �g/kg i.g.) orcorn oil (controls) chocolate was offered to all rats (n = 4 in each group) anddaily chocolate (A) and chow (B) consumptions (mean ± SE) were measured. Totaldaily energy intakes in relation to the metabolic needs of the rats are shownin panel C. Between habituated and non-habituated TCDD-treated rats a signif-icant difference in both chocolate (A) and chow (B) consumptions emerged onthe exposure day (day 0), whereas in controls habituation affected only chowconsumption (B) on day 0. On days 5 and 6 the difference between the twocontrol groups was significant in chocolate consumption (A), on day 5 also intotal energy intake (C). Differences between habituated, TCDD-treated and controlgroups are shown as ***p ≤ 0.001, **0.001 < p ≤ 0.01, *0.01 < p ≤ 0.05, and differ-ences between non-habituated, TCDD-treated and control groups are shown as###p ≤ 0.001, ##0.001 < p ≤ 0.01, #0.01 < p ≤ 0.05, assessed by linear mixed model forrepeated measures.

83 (2011) 140–150 147

the first feeding period post-exposure to TCDD (Tuomisto et al.,2000).

One of the most immediate responses to TCDD is a peculiar andconspicuous alteration in feeding preferences: palatable, unfamil-iar food items are rejected if they are presented to the animals inthe context of their TCDD exposure (Lensu et al., 2011; Tuomistoet al., 2000). In the present study, we showed a decrease in cumu-lative chocolate consumption emerging already a few hours afterTCDD exposure when this was timed at the onset of darkness. Thedeparture from control intake level was discernible at 2.5 h andbecame statistically significant at 5.5 h. Our results are the first toshow observable behavioural changes, in adult TCDD-resistant ratsafter a low 3 �g/kg dose, within a few hours after a TCDD exposurein an experimental setting. It is important to note that food energywas not avoided as TCDD-treated rats preferred chow to chocolate,eating it significantly more than controls even during daytime.

Interestingly, human patients have initially suffered from gas-trointestinal symptoms before the appearance of the changes(chloracne and facial inflammation) which finally led them tohospital with a subsequent diagnosis of severe TCDD exposure(Geusau et al., 2001; Sorg et al., 2009). Although rats are unableto tell about gastrointestinal discomfort, in them visceral statemodifies feeding responses: if no sign of malaise emerges withinseveral hours after a meal, food appears palatable (Garcia et al.,1972). Two behavioural phenomena, CTA and neophobia preventingestion of unfamiliar and possibly harmful substances. Of theseCTA is a learned behaviour preventing repeated consumption ofharmful and toxic food items (conditioned stimulus, CS) whichhave at the first encounter caused gastrointestinal discomfort(unconditioned stimulus, US). Neophobia is an innate, organizedand adaptive behaviour preventing consumption of substantialamounts of a new food until it appears to be safe. Differences instimulus properties affect the severity and attenuation of thesebehaviours, and different regulatory pathways are involved inthem (Figueroa-Guzman and Reilly, 2008; Hart, 1988; Núnez-Jaramillo et al., 2010; Reilly and Bornovalova, 2005; Reilly and Tri-funovic, 2001). In our previous study, a positive CTA response wasobserved only at an excessively high but sublethal dose of TCDD(1000 �g/kg) in H/W rats. At a lower dose of 50 �g/kg (which isstill lethal to all L–E rats), neither H/W nor L–E rats exhibited it(Pohjanvirta et al., 1994; Tuomisto et al., 2000). In contrast to thosestudies, here in experiment 4 we found strong evidence of CTA onday 7 in TCDD-resistant line A rats treated with a remarkably lowdose of TCDD (3 �g/kg). Differences in experimental settings (liquidsaccharin vs. solid foodstuffs, presence of the CS for 1 h before vs.24 h after US, TCDD exposure and rechallenge at 3 days vs. 7 days)are likely to account for the disparate outcomes. While these find-ings highlight the difficulties encountered in attempts to categorizethe avoidance response to TCDD, they should not discourage fur-ther efforts in that direction because this information would helpelucidate the biochemical pathways involved.

In general, the present study demonstrates the critical role oftemporal coupling of TCDD exposure and unfamiliar food item inthe aversion response, which may last for extended periods asshown in our previous study as well (Tuomisto et al., 2000). If thenovel food item was presented together with a low dose of TCDDor within a day after exposure, aversion manifested. A change infeed texture sufficed to ignite the response. Rats seemed to mem-orize well the association between TCDD exposure and a givennew food item, because even a week afterwards, they still rejectedthis food when challenged with it. Indeed, evidence exists that in

the case of CTA in rats, aversive behaviour and c-FOS induction (amarker of neuronal activation) in the nucleus of solitary tract (NTS)may persist for at least 6 months (Houpt et al., 1996). Simultane-ous TCDD-exposure with a novel chocolate presentation provokedlong-lasting rejection, if chocolate was constantly available for the

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rst 2 weeks after TCDD. Although the exposed rats started to eatome chocolate in 6 weeks (Fig. 2), they did not reach the controlevel by 76 days (end of observation).

The aversion response was also detectable with sweet liq-ids. Habituation to these liquids as well as the time lag betweenCDD-exposure and the primary presentation of a novel food itemad an alleviating effect on the aversion in male H/W rats. There-exposure habituation of TCDD-sensitive female L–E rats tohocolate yielded a surprising result: it did not attenuate chocolateversion almost at all. This was in a stark contrast with the out-ome in the case of saccharin habituation. At the moment we doot have a definite explanation for these differential effects. How-ver, it is plausible that the tiny amount of chocolate provided dailyuring the habituation period was insufficient to represent all theritical aspects associated with ad libitum availability of the sameommodity. At habituation, the rats devoured the chocolate in annstant whereas in the post-exposure situation it was present all theime. It should be kept in mind that a mere change in texture of theamiliar chow was able to trigger the aversion response, and thataccharin, in contrast to chocolate, was freely available for the ratsuring their habituation. It is known that during pre-exposure stim-lus frequency and duration interact with behavioural responses inversion studies (Delacasa and Lubow, 1995).

The gender may also play a role in the differential outcomesbtained in the habituation experiments. For example, a lethalose of TCDD increased kaolin consumption and reduced consump-ion of high-protein diet in female L–E rats but had no significantffect on kaolin intake and tended to elevate high-protein dietonsumption in male rats of the same strain (Pohjanvirta et al.,994). Overall, in rats many feeding-related behaviours and sen-itivity to alterations in them differ between genders; e.g. tasteensitivity and taste aversion are examples of sexually dimorphicehaviours [reviewed in (Varma and Meguid, 2001)]. Therefore,he issue of possible gender differences in the novelty avoidanceesponse caused by TCDD warrants further studies. Nevertheless,hocolate with its very specific taste and flavour may possess prop-rties that render it particularly effective in igniting this response.n our previous studies, a 4-day pre-exposure habituation periodad libitum) postponed (in male L–E rats) or prevented (in male H/Wats) TCDD-provoked aversion response (after a dose of 50 �g/kg.p., which is lethal to L–E but sublethal to H/W rats) to cheeseut had only a slight or no effect on chocolate aversion. In female/W rats, a 16-day habituation period (ad libitum) abolished the

esponse (after a sublethal dose of 1000 �g/kg TCDD i.p.) to cheesend delayed (apparently also mitigated) it to chocolate (Tuomistot al., 2000).

Administration route was found to affect the aversion to a novelood item, chocolate. The dose of 3 �g/kg administered orally to

ale rats of the TCDD-resistant line A caused an aversive effectimilar to that of the dose of 100 �g/kg administered i.p., with aver-ion being detectable immediately on the first night after TCDDxposure. Injection treatment with 3 �g/kg decreased chocolateonsumption only on the second night after exposure. This find-ng probably bears on the outcome of experiment 4, conditionedaste aversion study, in which rats were dosed i.p. (3 �g/kg) andonsumed rather high amounts of chocolate during the exposureay in comparison with other experiments. The differential impactf orally vs. i.p. administered TCDD may stem from differences inCDD kinetics by the two routes (Wang et al., 2000).

Our previous findings had revealed that this response to TCDDs not related to sensitivity to acute lethality or sensitivity to dimin-

shed feeding and body weight loss after TCDD (Lensu et al., 2011).mportantly, all the doses of TCDD used here were well below theethal values and are not supposed to cause major toxicities indult animals. In keeping with this assumption, although palatablehoices increased weight gain in controls, TCDD did not appreciably

83 (2011) 140–150

affect body weight gain in the experiments and therefore the rapidaversive responses after TCDD cannot be secondary to hypophagiaand lowered body weight gain.

The novel food item avoidance response seems to be the earli-est and one of the very few sensitive behavioural effects of TCDDin adult rats reported so far (Lensu et al., 2011; Tuomisto et al.,2000). In adult animals, changes in gene expression (such as thoseinvolved in metabolism of xenobiotics) are detectable within hoursafter TCDD exposure [e.g. (Korkalainen et al., 2005)]. Interestingly,in our recent study the ED50 values for novel food item aversionwere found to correlate closely with those for induction of EROD (ameasure of CYP1A1 enzyme activity) in differently TCDD-sensitiverats, with no relation to sensitivity to lethality (Lensu et al., 2011;Simanainen et al., 2002; Tuomisto et al., 1999). Furthermore, find-ings in AHR-null mice implied AHR mediation for the aversionresponse (Lensu et al., 2011). Hence AHR activation at TCDD dosescausing enzyme induction might serve as a protective mechanismrestricting the ingestion of possibly toxic food. Because an oral dosewas more effective than an i.p. dose, the crucial site of detectionmight be in the gastrointestinal tract.

Food consumption is linked to the survival of an organism andis strictly balanced and secured. Information of food’s nutritiveand hedonic value, familiarity as well as possible toxic charac-ters is guided by tastes. Hence perception of safe and aversivetastes involves learning and different types of memory storageand extinction processes whereby feeding behaviour is controlledaccordingly. Several anatomical areas in the brain together withdistinct biochemical and molecular mechanisms have been shownto participate in the regulation of neophobia and CTA. At least twodissociable pathways seem to exist for sensing the malaise stimu-lus, via vagus nerve or through the area postrema in the hindbrain.Both pathways lead to the NTS which is the first central synap-tic relay for gustatory information and from which signalling isdirected to the parabrachial nucleus (PBN) [reviewed e.g. in (Núnez-Jaramillo et al., 2010; Reilly and Bornovalova, 2005)]. Gustatorycortex modulates the activities of NTS and PBN through inhibitorypathways, and especially insular cortex has a key role in taste mem-ory formation (Bermudez-Rattoni, 2004) as well as in extinction ofaversive memories (Garcia-DelaTorre et al., 2010). For example, N-methyl-d-aspartate receptors (NMDA-R) have important roles bothin storing memories and in the processing of novel gustatory stim-uli [reviewed in (Núnez-Jaramillo et al., 2010)]. Interestingly, TCDDhas been shown to affect NMDA-R in rat offspring after in uteroexposure as well as in neuronal cells in vitro [reviewed in (Lindenet al., 2010)]. In adult animals, the neural mechanisms involved inmediating the effects of TCDD on feeding have mostly remainedelusive.

In adult animals exposed to TCDD, very few behavioural changesother than those related to feeding have been reported (Sirkkaet al., 1992). The reported impacts of TCDD on other types ofbehaviour mainly involve offspring after perinatal or lactationalexposure [e.g. (Kakeyama et al., 2003; Seo et al., 2000; Widholmet al., 2003)]. In addition to the dose of TCDD, numerous otherfactors contribute to sensitivity differences in impacts of TCDD onadult and developing animals (Bell et al., 2010; Ishida et al., 2010).However, independently of the developmental stage, alterationsin behaviour involve alterations in (neural) regulation. Thereforein-depth understanding of the dysregulated mechanisms underly-ing the behavioural responses to TCDD would be highly important,especially from the risk-assessment point of view. On the otherhand, there is a myriad of studies involving body weight bal-

ance and energy homeostasis due to the increasing concern ofoverweight-related problems in the western society. Hence thestudies of mechanisms by which TCDD imparts its effects on feedingprovide also important physiological information of body weightregulation.

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. Conclusions

Although lethal wasting may arise secondary to other toxicesponses to TCDD (e.g. hepatotoxicity), our results show that TCDDs able to induce a rapid and specific consummatory response afterotably low doses. The relationship of the avoidance response withCDD-induced wasting syndrome, together with their molecularechanisms, warrant further studies.

unding

This work was supported by grants from the Centre of Excellencerogram of the Academy of Finland to the Centre of Environmentalealth Risk Analysis (Grant no. 53307) and from the Academy ofinland (Grant no. 123345 [R.P.]). Financial support [S.L.] from theraduate School in Environmental Health (Ministry of Education,inland) is acknowledged.

onflicts of interest

Authors have no actual or potential conflict of interest includingny financial, personal or other relationships with other people orrganizations within 3 years of beginning the work submitted thatould inappropriately influence (bias) this work.

cknowledgements

The authors want to acknowledge the excellent technical assis-ance throughout the studies of Arja Moilanen, Minna Voutilainennd Ulla Naukkarinen. Janne Korkalainen and Markku Forsman arehanked for their help with animal studies. M.Sc. Pekka Tiittanen iscknowledged for the statistical consulting.

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