logy 24

Black widow spider venom (BWSV) is known to cause

158 Abstracts / Toxico

A model of xenobiotic efflux via Mrp2 in sandwichcultured primary rat hepatocytes

Katharine Howe 1, Nick Plant 1, Tanya Coleman 2,Kevin Jones 2, Gordon Gibson 1

1 Molecular Toxicology Group, School of Biomedicaland Molecular Sciences, The University of Surrey,Guildford, Surrey GU2 7XH, United Kingdom;2 Department of Discovery DMPK, AstraZeneca R&D,Alderley Park, Macclesfield, Cheshire SK10 4JT,United Kingdom

E-mail address: K.howe@surrey.ac.uk (K. Howe).

Biliary excretion is a predominant route of elimina-tion for many xenobiotics and is often an importantcomponent of their clearance in vivo. Many of thesecompounds and/or their metabolites require active trans-port out of hepatocytes in order to cross the canalicularmembrane. Multidrug resistance-associated protein 2(Mrp2 or Abcc2) is a transporter expressed in thecanalicular membrane of hepatocytes and is responsi-ble for the efflux of bilirubin glucuronide, conjugatedbile acids and a number of xenobiotics. Carboxy-dichlorofluroscein diacetate (CDFDA) is often usedto establish when sandwich cultured hepatocytes formbile canalicular structures during culture (LeCluyseet al., 1994). CDFDA diffuses into cells where it ishydrolysed to carboxydichlorofluroscein (CDF), a flu-orescent substrate of Mrp2. CDF is effluxed into thebile canalicular structures enabling them to be visualisedvia fluorescent microscopy (Liu et al., 1999). Using thispreviously established methodology an assay to mea-

sure efflux via Mrp2 was set up in sandwich cultured rathepatocytes.

The efflux of CDF was saturable, as would beexpected for a transporter-mediated process, with an

Fig. 1. CDF efflux via Mrp2 over a range of CDFDA loading concentrations. Pthen triplicate wells loaded with 0–10 �M CDFDA. Cells were incubated for(A) Representative fluorescent images from cells dosed with 0–10 �M CDFDdemonstrating correct localization of MRP2 within the cells. (B) Fluorescendose response curve using non-linear regression analysis.

0 (2007) 154–163

EC50 of 2.8 �M (Fig. 1); this efflux could be abol-ished by MK571, an inhibitor of Mrp2 efflux, with anIC50 of 1.9 �M. This value is in agreement with a pub-lished IC50 of MK571 via Mrp2 (Leier et al., 2000)demonstrating that CDF efflux into bile cannaliculi wasoccurring via Mrp2. Treatment of cells with the HMGCoA reductase inhibitor pravastatin also prevented CDFefflux, with an IC50 of 55.2 �M, confirming its statusas an Mrp2 substrate. In summary, these data supportthe use of sandwich-cultured hepatocytes in the study ofxenobiotic efflux via Mrp2.

References

LeCluyse, E.L., Audus, K.L., Hochman, J.H., 1994. Am. J. Physiol.266, C1764–C1774.

Liu, X., LeCluyse, U.L., Brouwer, K.R., Gan, L.S., Lemasters, J.J.,Stieger, B., Meier, P.J., Brouwer, K.L., 1999. Am. J. Physiol. 277,G12–G21.

Leier, I., Hummel-Eisenbeiss, J., Cui, Y., Keppler, D., 2000. KidneyInt. 57, 1636–1642.

doi:10.1016/j.tox.2007.06.080

A genetic approach to study the function oflatrophilin in C. elegans

Ademola A. Adenle , D. de Pomerai, David R. BellSchool of Biology, University of Nottingham,

University Park, Nottingham NG7 2RD, UK

E-mail address: Plxaa2@nottingham.ac.uk(A.A. Adenle).

rimary rat hepatocytes were grown in sandwich culture for 5 days and25 min at 37 ◦C and then fluorescence examined using an FITC filter.A. CDF fluorescence can be observed in the bile canalicular spaces,

t intensity within cannaliculi was measured and fitted to a sigmoidal

neurotransmitter release from nerve terminals due to itshigh contents of molecular weight protein called latro-toxins (LTX), and is known to bind with high affinity to

Abstracts / Toxicology 240 (2007) 154–163 159

Table 1Genetic characterisation of B0457 cosmid

Genotype Adults assayed Number of embryos Number of adult F1 Defecation cycle (s) Defecation cycle (n)

N2 wild-type 10 304 ± 14 304 ± 14 45 ± 3 15lat-1(ok1465) 10 180 ± 4 3 ± 2b 80 ± 3b 10B 282

tCtnleko

ggtugpctutiolutdBses

uaai

stpwlpwd

0457, unc-54::C 10 282 ± 6

hree neural proteins in mammals. We have established. elegans as a model organism to study the function of

he binding protein, latrophilin, and its role in regulatingeurotransmitter release by latrotoxins, by showing thatatrophilin is required for lethality of BWSV by RNAixperiments (Mee et al., 2004). However, a latrophilin-nockout worm is required for determining the functionf the latrophilin gene.

The lat-1(ok1465) allele has a deletion of the lat-1ene, and we know that ∼97% of lat-1(ok1465) homozy-ous worms arrest or die before adulthood, with onlyhree adult offspring per animal (Table 1). However, it isnclear if this lethality is due to the deletion in the lat-1ene, or to adventitious mutations introduced during therocess of mutagenesis to create this allele. The B0457osmid contains the full sequence of the lat-1 gene, andhis cosmid, together with the fluorescent marker gene,nc-54::C (with unc-54 driving body wall expression ofhe “Cerulean” green fluorescent protein variant), wasnjected into lat-1(ok1465) worms to create transgenicffspring. The B0457 cosmid rescued the lethality ofat-1(ok1465) worms, and concurrent injections with thenc-54::C plasmid yielded six surviving adults from >80ransgenic embryos, showing that the rescue was notue to the unc-54::C. Five independent stable lines with0457 were obtained, with one exhibiting Mendelian

egregation of the array; this line was characterised formbryonic lethality, brood size and defecation cycle ashown in Table 1.

Lat-1(ok1465) worms were injected with B0457 andnc-54::C DNAs, and an integrated transgenic line char-cterised. P values were calculated between lat-1 and N2,nd between B0457, unc-54::C and lat-1, and P < 0.05s indicated (b).

Lat-1(ok1465) worms produced a significantlymaller brood size (3 ± 2; p < 0.0001) compared to wild-ype (304 ± 14), whereas B0457, unc-54::C (282 ± 6,< 0.0001) restored brood size of lat-1(ok1465) toild-type levels. Defecation cycles were measured,

at-1(ok1465) (80 ± 3, p < 0.0001) showed significantlyrolonged defecation cycle compared to wild-typeorms (45 ± 3), and the B0457 cosmid rescued thisefect.

± 6b 50 ± 7b 20

These results show that the lat-1(ok1465) allelecauses developmental lethality and an increase in defeca-tion cycle timing, and that the B0457 cosmid can rescueboth these defects. This is strong evidence that the lat-1 gene is responsible for these defects. Current workaims to rescue the lat-1(ok1465) defects with a lat-1cDNA construct, and to determine the role of this gene inmediating the toxicity of Black Widow Spider venom.

Reference

Mee, J.C., Tomlinson, R.S., Perestenko, P.V., De Pomerai, D., Duce,R.I., Usherwood, R.N.P., Bell, D.R., 2004. Biochem. J. 378,185–191.

doi:10.1016/j.tox.2007.06.081

Zebrafish assay for automated detection of drugeffects on heart rate and cardiac rhythmicity

Eric Sandberg , Cameron Tran, Audrey White,Temitope Aiyejorun, Delali Blavo, Amy Rubinstein,Tim Baranowski, Thanh Doan Zygogen, LLC 24

Peachtree Center Avenue, 520 Kell Hall, Atlanta, GA30303, United States

E-mail address: eric@zygogen.com (E. Sandberg).

Several drugs have been withdrawn from the market dueto severe cardiac toxicity. The most common reason fordrug withdrawal is Torsade de Pointes (TdP), a life-threatening arrhythmia associated with blockage of theKCNH2 potassium channel (hERG) and prolongation ofthe QT interval. Previous reports showed that drugs caus-ing QT prolongation in humans also slow heart rate inzebrafish (Milan et al., 2003; Langheinrich et al., 2003).However, not all drugs that slow heart rate in zebrafishcause TdP in humans, resulting in false positives in thisassay. Drugs associated with TdP in humans have beenshown to cause arrhythmia in zebrafish (Langheinrich

et al., 2003). Finally, it was shown that zebrafish assaysfor cardiac function are amenable to automation (Burnset al., 2005). The purpose of this study was to developautomated assays for cardiotoxicity that can be used to