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Abstracts / Toxico

iver cytosol showed different binding characteristics for3H]-TCDD is not clear. However, it is possible that thereere different types of AhR in the recombinant system,

or example some recombinant AhRs might be associ-ted with insect chaperone proteins, and some not (Bellnd Poland, 2000). As a result, the AhR molecules couldave different affinity for [3H]-TCDD; mixture of themay show various Kd values.

cknowledgement

Supported by a contract (To1034) from the Food stan-ard Agency.

eferences

ell, D.R., Poland, A., 2000. J. Biol. Chem. 275, 36407–36414.han, W.K., Chu, R., Jain, S., Reddy, J.K., Bradfield, C.A., 1994. J.

Biol. Chem. 269, 26464–26471.oland, A., Golver, E., Kende, A.S., 1976. J. Biol. Chem. 251,

4936–4946.

oi:10.1016/j.tox.2007.06.047

olecular cloning and expression of the arylydrocarbon receptor of the Charles Riveristar(Han) rat

ao Jiang, Minqi Fan, David R. Bell

School of Biology, The University of Nottingham,niversity Park, Nottingham NG7 2RD, Unitedingdom

-mail address: tao.jiang@nottingham.ac.uk (T. Jiang).

The aryl hydrocarbon receptor (AhR) mediates dioxinoxicity and is also involved in diverse physiologicalunctions. AhR polymorphism can dramatically mod-fy dioxin toxicity in laboratory animals (Okey et al.,005). For example in the Han/Wistar (Kuopio) (H/W)at strain, a polymorphism is associated with a >100-old resistance to lethality from dioxin, compared toong-Evans rats (Pohjanvirta et al., 1998). The H/W

Kuopio) strain has distinct properties from its parentistar(Han) colony (Pohjanvirta and Tuomisto, 1990).lthough Charles River Wistar(Han) (CRL:WI(Han))

at is commonly used in toxicity studies, but the geneticature of its AhR is not characterised. We have clonedhe CRL:WI(Han) AhR and expressed the AhR protein

ariants.

CRL:WI(Han) rats (female, 65–70 days old) weresed. Rat liver cytosol was prepared from 14 differ-nt rats, subjected to SDS PAGE and Western blotted.

(2007) 164–192 169

The AhR was detected by an AhR specific antibodywith chemiluminescent detection. Two rats express-ing either the lower or higher Mr AhR were selectedfor gene cloning. The coding region of the AhRwas obtained in three fragments, with primers as fol-lows: fragment 1: 5′-ATGGCCAGCCGCAAGCGGC-GC (f),5′-GCAAGGGATCCATTATGGGAGAGAAA-GG (r); fragment 2: 5′-CTGGAGAGGCTGTACTGT-ACGAGAT (f), 5′-TGCTGCTGCTGAAGCTGC (r);fragment 3: 5′-TGAATTCAGCTTGCCAGCA (f), 5′-TACAGGAATCCGCTGGGTGT (r). The PCR productswere cloned and the double strand DNA sequenced.Recombinant AhR was expressed in rabbit reticulocytelysate system and in insect cells using the baculovirusexpression system.

The AhR protein detected by Western blotting inCRL:WI(Han) rats has two size variants: the higher MrAhR (∼106 kDa) is the same apparent molecular mass asthat in Sprague–Dawley (SD) rats, and a lower Mr AhR(∼98 kDa). These AhR variants could appear either bythemselves, or as a mixture of the two forms. There wasno evidence of interconversion between the two forms,despite preparation of multiple batches of cytosol fromindividual animals. AhR cDNA was cloned separatelyfrom an animal expressing either the high Mr AhR, orthe low Mr AhR. The high Mr AhR has the same DNAsequence as the SD rat. However, the low Mr AhR vari-ants contain a transition mutation in the second base ofcodon 497 (NT1520) (T to C), resulting in a change of Valto Ala. Additionally, up to five AhR deletion/insertionvariants were identified in the transactivation region ofthe AhR. These included one 129 bp deletion, whichcaused 43 amino acids deletion at protein level and fourinsertions of 134 bp, 129 bp 126 bp and 29 bp. The inser-tion contains a stop codon and causes a 38 amino aciddeletion. The multiple variants arise from differentialsplicing, caused by a point mutation in the intron/exonjunction of exon 10 of the AhR gene. The truncated formsof the AhR were expressed in reticulocyte lysate, and inbaculovirus; both systems showed that the insertion anddeletion variants encode a protein which is shorter thanthe “wild-type” AhR, in agreement with the results fromWestern blotting. The deletion variants were in com-mon with that observed in dioxin-resistant H/W (Kuopio)rats. However, it is unexpected that both variants arepresent in CRL:WI(Han) rats. It is not clear if thesevariants impact on dioxin toxicity in the CRL:WI(Han)rat.

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170 Abstracts / Toxico

Acknowledgement

Supported by a contract (T01034) from the Food Stan-dards Agency.

References

Okey, A.B., Franc, M.A., Moffat, I.D., Tijet, N., Boutros, P.C.,Korkalainen, M., Tuomisto, J., Pohjanvirta, R., 2005. Toxicol.Appl. Pharmacol. 207, 43–51.

Pohjanvirta, R., Tuomisto, J., 1990. Toxicol. Appl. Pharmacol. 105,508–509.

Pohjanvirta, R., Wong, J.M.Y., Li, W., Harper, P.A., Tuomisto, J., Okey,A.B., 1998. Mol. Pharm. 54, 86–93.

doi:10.1016/j.tox.2007.06.048

TCDD is a potent developmental toxin, but fails toaffect spermatogenesis in offspring of chronicallytreated CRL:WI(Han) rats

David R. Bell 1, Sally Clode 2, Ming Qi Fan 1, AlwynFernandes 3, Paul M.D. Foster 4, Tao Jiang 1, GeorgeLoizou 5, Alan MacNicoll 1, Brian Miller 6, MartinRose 3, Lang Tran 6, Shaun White 3

1 School of Biology, University of Nottingham,University Park, Nottingham NG7 2RD, UK;2 Covance Laboratories Ltd., Otley Road, Harrogate,North Yorkshire, UK; 3 Central Science Laboratory,Environmental Contaminants and Trace ElementStudies (FSQ5), Sand Hutton, York YO41 1LZ, UK;4 NIEHS, P.O. Box 12233 (MD E1-06), 111 TWAlexander Drive, Research Triangle Park, NC 27709,USA; 5 Health & Safety Laboratory, Harpur Hill,Buxton, Derbyshire SK17 9JN, UK; 6 Institute ofOccupational Medicine, Research Park North,Riccarton, Edinburgh, EH14 4AP, UK

E-mail address: david.bell@nottingham.ac.uk(D.R. Bell).

One of the most potent adverse effects of2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is on thedeveloping male reproductive system, after exposure ofthe fetus via dosing of the pregnant mother to dose levelsas low as 64 ng dioxin/kg, e.g. (Mably et al., 1992; Faqiet al., 1998). This endpoint has been used to establish aTolerable Daily Intake for dioxins in the UK, as set outin (COT, 2001). We have previously found that a singledose of TCDD causes no decrease in F sperm levels

1(Bell et al., 2005), but caused lethality and a delay inbalano-preputial separation (BPS) at top dose (1 �g/kg).We have now undertaken a study to determine if chronicdosing of TCDD (doses were designed to give a com-

0 (2007) 164–192

parable body burden of TCDD to that seen in the acutestudy) affects the developing rat.

CRL:WI(Han) rats were weaned and exposed todietary TCDD (0, 2.8, 9.3 or 53 ng/kg bwt./day) for 12weeks before pairing, then during pairing until the endof gestation. Animals were killed on weeks 10 and 12 ofdosing, and Gestational Day (GD) 16 and 21 for determi-nation of tissue TCDD levels, leaving ∼30 animals pergroup scheduled for littering >90 F1 animals per group(67 for the high dose group) were taken forward withoutfurther treatment for kills at Post-Natal Day (PND) 70and 120, a Functional Observation Battery, and repro-ductive assessment.

There were no adverse signs of TCDD treatment onmaternal weight. The high dose group had eight ani-mals with total litter loss (versus three in control), therewas an adverse effect on pup viability and a dose-relateddecrease in pup weight at birth. The F1 males showed asignificant, dose-related delay in BPS in all three-dosegroups, and the high dose group were slightly less activein motor activity trials. There were no adverse effectsof maternal treatment on seminology data at PND70or 120, nor any effect on the fertility of the animals.Although there was a ∼15% decrease in testis weight atPND70 in the high dose group, there were no decreasesin the weight of accessory sex organs at PND120 norwere there any significant findings in tissues examinedby light microscopy at PND120.

Developmental exposure to TCDD via acute orchronic dosing did not decrease sperm counts of F1animals in our hands; although our study has sufficientpower, it has not been possible to reproduce the findingof Mably et al. (1992). However, our chronic dosing datareveal delay in BPS to be an extremely sensitive responseto maternal TCDD exposure, and that the dosing regimeis a key determinant of response.

Acknowledgement

This was supported by a contract from the Food Stan-dards Agency (T01034).

References

Bell, D.R., Clode, S., Fernandes, A., Foster, P., Loizou, G., MacNicoll,A., Miller, B.G., Rose, M., Tran, L., White, S., 2005. ToxicologistAbstract 681.

COT, 2001. http://www.food.gov.uk/multimedia/pdfs/cot-diox-full.Faqi, A.S., Dalsenter, P.R., Merker, H.-J., Chahoud, I., 1998. Toxicol.

Appl. Pharmcol. 150, 383–392.Mably, T.A., Bjerke, D.L., Moore, R.W., Gendron-Fitzpatrick, A.,

Peterson, R.E., 1992. Toxicol. Appl. Pharmcol. 114, 118–126.

doi:10.1016/j.tox.2007.06.049