3 logy 290 (2011) 1–46

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Fig. 1. Dose-dependent regulation of KPNA3 by 1,25-OH vitamin D3. Huh7 cellswere transfected with a KPNA3 reporter gene construct and exposed to 0.1–300 nM1,23-OH vitamin D3 for 48 h before reporter gene activity was measured. Data pointsare the mean ± SEM (n = 5), and representative of three independent experiments.

0 Abstracts / Toxico

hereas most other cellular receptors are located in the plasmaembrane, NRs derive their family name from the early and para-

oxical observation that they are generally located in the nucleus,espite responding to extracellular signals. The aim of the cur-ent study was to use an in silico modelling approach to examinehe design principles that underlie the sub-cellular localisation ofuclear receptors (NR), understanding aspects of the topology ofhese networks that might appear unnecessarily complex or evenunctionally paradoxical [2].

In realistic kinetic models of increasing complexity, calcula-ions showed a number of potentially important design principles,or example: (i) cytosolic ‘nuclear’ receptor may shuttle signal

olecules to the nucleus, (ii) the active export of NRs may ensurehat there is sufficient receptor protein to capture ligand at theytoplasmic membrane, (iii) a two conveyor belts design dissipat-ng GTP free energy, greatly aids response, (iv) the active export ofmportins may prevent sequestration of NRs by importins in theucleus (Fig. 1), and (v) the unspecific nature of the nuclear poreay ensure signal-flux robustness.In conclusion, this work provides the first in-depth analysis of

ucleo-cytoplasmic shuttling of NRs, demonstrating that their sub-ellular localisation acts to maximise both sensitivity and speed ofellular response to chemical stimuli.

eferences

lant, Aouabdi, 2009. Xenobiotica 39, 597.olodkin, et al., 2010. Molecular Systems Biology 6, 446.

oi:10.1016/j.tox.2011.09.055

048

egulation of nuclear import pathways by vitamin D signalling:otential biological implications

upaporn Yimthaing, Nick Plant, Kate Plant ∗

Centre for Toxicology, University of Surrey, United Kingdom-mail address: K.Plant@Surrey.ac.uk (K. Plant).

The 48 members of the human nuclear receptor (NR) family haveeen implicated in a diverse range of regulatory functions, suchs in development, cellular growth, inflammation and metabolismPlant and Aouabdi, 2009). Despite their name, these ligand-ctivated transcription factors are located in both the nucleusnd cytoplasm, shuttling between these compartments to achieveheir functionality (Kolodkin et al., 2010). Key mediators of thisucleo-cytoplasmic shuttling are the karyopherin (importin) fam-

ly of proteins, which act to transport cargo proteins through theuclear pore complex (Plant et al., 2006). At present, little is knownbout how the interaction networks between nuclear receptors anduclear import proteins; the present study aimed at understand-

ng the relationship between the Vitamin D receptor (VDR) andaryopherin alpha 3 (KPNA3), a nuclear import protein known toacilitate VDR nucleo-cytoplasmic shuttling.

Initially, approximately 2Kbp of KPNA3 proximal promoteras identified in silico and cloned into a reporter gene plasmid.

ubsequent transfection into human hepatoma Huh7 revealed aose-dependent regulation of KPNA3 transcriptional activity by.1–300 nM 1,25-OH vitamin D3, an effect that was enhancedy cotransfection with an expression plasmid for VDR (Fig. 1).ite-directed mutagenesis revealed four response elements thatediate this transcriptional response in a complex dose-dependent

anner.Finally, using GFP-VDR fusion constructs we demonstrate that

teady state levels of VDR in Huh7 cells are evenly distributedetween nuclear and cytoplasmic, becoming predominantly

IC50s for transcriptional activation and inhibition are 1–2 nM and 48 nM, respec-tively.

nuclear following addition of the agonist 1,25-OH Vitamin D3.Interestingly, over-expression of KPNA3 results in a steady-stateVDR localisation that is completely nuclear, and realistic kineticmodels of the nucleo-cytoplasmic shuttling network presents sev-eral possible biological scenarios that may explain this observation.

In conclusion, this work demonstrates that ligand-activated VDRis able to regulate expression of its cognate import protein, KPNA3,in a dose-dependent manner. Such regulation is consistent witha complex signal transduction network, allowing VDR-mediatedsignalling to be closely regulated by the cell.

References

Plant, Aouabdi, 2009. Xenobiotica 39, 597.Kolodkin, et al., 2010. Molecular Systems Biology 6, 446.Plant, et al., 2006. Pharmacogenetics Genomics 16, 647.

doi:10.1016/j.tox.2011.09.056

P049

Establishment of an HLA-typed cohort to elucidate the cellularand chemical basis of drug hypersensitivity reactions

Catherine Bell 1,∗, Klara Martinsson 1, Lee Faulkner 1, AnaAlfirevic 1, Neil French 1, Jonathan Tugwood 2, Ina Schuppe-Koistinen 3, Karin Cederbrant 3, Munir Pirmohamed 1, DeanNaisbitt 1, B. Kevin Park 1

1 MRC Centre for Drug Safety Science, University of Liverpool, L69 3GE,United Kingdom2 AstraZeneca R&D, Alderley Park, Macclesfield SK10 4TG, United King-dom3 AstraZeneca R&D, Södertälje, Sweden

Drug hypersensitivity reactions represent a major clinicalproblem. Furthermore, due to the idiosyncratic nature of the major-ity of drug hypersensitivity reactions, they may only becomeapparent post-marketing. Significant genetic associations havebeen identified within the MHC region, for drugs such as aba-cavir (HLA-B*5701), flucloxacillin (HLA-B*5701), carbamazepine(HLA-B*1502), and ximelagatran (HLA-DRB1*0701). An HLA-typed

cohort of frozen human lymphocytes from 400 healthy drug-naivevolunteers has therefore been established to explore drug-specificT-cell responses.

Abstracts / Toxicology 290 (2011) 1–46 31

Fig. 1. Active export of importins is advantageous: using realistic kinetic models, the impact of nucleo-cytoplasmic shuttling on nuclear receptor signalling was examined.Active transport of importins produces a greater transcriptional response to ligand than passive diffusion (A), with active export of importins being favourable at higherimportin concentrations, due to limited sequestration being observed (B).

Table 1Characteristics T-cell clones generated from HLA-B*5701 positive volunteers.

Volunteer HLA-B*5701 status No. clones tested No. ABC-specific No. CD4+ No. CD8+ Stimulation index (range)

1 + 276 14 9 5 1.5–6.52 + 208 5 0 5 2.5–5.33 + 314 2 0 2 5.2–6.3

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Aim: To utilise the strong association between possession of theLA-B*5701 allele and development of abacavir hypersensitivity

eactions to explore the mechanisms underlying this toxicity.Methods: Blood samples from 400 volunteers have been

rocessed and genotyped. A panel of immunological assays (prolif-ration, ELISpot, CD107a) have been utilised following 14-day drugxposure to monitor drug-specific T-cell responses. Furthermore,rug-specific T-cell clones from volunteers expressing appropriateLA molecules have been generated to analyse the cellular pheno-

ype and functionality.Results: The current study has been successful in creating

biobank of both DNA and lymphocytes generated from 400ealthy volunteers to study HLA-associated ADRs. These volun-eers encompass a range of ethnicities. Twenty-six HLA-B*5701ositive individuals were identified (1 homozygote, 25 het-rozygotes). Lymphocytes from these individuals were used toxplore the pathogenesis of abacavir hypersensitivity reactions.bacavir-specific T-cell clones were successfully generated fromHLA-B*5701 positive volunteers but could not be generated fromindividuals lacking this genotype (Table 1). These clones both

roliferated and secreted interferon-�, as determined by ELISpotssay. The clones were stimulated by abacavir but not by dihydrobacavir, which lacks an alkene function on the cyclopentane ring.ome of the clones could also be stimulated by antigen present-ng cells that had been pulsed overnight with the drug and then

ashed extensively prior to co-incubation. This may implicate aole for metabolism.

Conclusion: Abacavir-specific T-cell responses are dependentn possession of HLA-B*5701. The generation of T-cell clones will

nable further exploration of the mechanism(s) of antigen presen-ation, phenotype and functionality.

oi:10.1016/j.tox.2011.09.057

n/a

P050

Relationship between cell toxicity and interleukin (IL) 8 produc-tion for chemical contact allergens

I. Kimber a,∗, C. Portsmouth a, G. Maxwell b, R Pendlington b, R.J.Dearman a

a Faculty of Life Sciences, University of Manchester, M13 9PT, UKb Unilever Safety & Environmental Assurance Centre, Bedford MK441LQ, UKE-mail address: rebecca.dearman@manchester.ac.uk (I. Kimber).

There has been considerable interest in the development of invitro tests for the characterisation of chemical allergens, such as theproduction by dendritic cell (DC)-like cell lines of cytokines. Thesemethods are often limited by toxicity and delivery of chemical aller-gen in culture. We have constructed toxicity and IL-8 secretiondose response curves for a number of human cell lines (DC-like:THP-1, U937; erthyromyeloid: K562). Cells (2 × 105 cells/ml; 0.5 mlaliquots) were incubated in 24well TC plates for 24 h with thecontact allergen dinitrochlorobenzene (DNCB; 3-30 �M) or withthe irritant benzenesulfonic acid (BS; 10-300 mM). Toxicity wasassessed by flow cytometry and propidium iodide (PI) staining andIL-8 content of supernatants was analysed by sandwich enzyme-linked immunosorbant assay (ELISA). Results are displayed for n of3 experiments (mean and SE; Tables 1 and 2).

All cell lines expressed IL-8 constitutively and were >95% viable.Similar toxicity profiles for DNCB were observed for each of thecell lines, with 30 �M causing substantial toxicity. There was morevariation in toxicity to BS, with K562 being markedly more resis-tant than the other cell lines. For each of the 3 cell lines, subtoxicdoses of the contact allergen enhanced IL-8 production, althoughfor the K562 cell line such did not reach statistical significance.In contrast, despite using doses that caused similar toxicity pro-files to those observed for DNCB, there was no increase in IL-8

secretion recorded for BS (10 to 300 mM). Cells (THP-1 and U937)were cultured with two additional allergens (dinitrothiocyanoben-zene [DNTB] and dinitrofluorobenzene [DNFB]) or two irritants(1-bromobutane [1BB] and sodium lauryl sulfate [SLS]) at subtoxic