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British Toxicology Society Abstracts, Sp

54

nthracene polyamine conjugates induce cell death in humaneukaemic cells

adiah A. Ghani 1, Navneet Kaur 2, Otto Phanstiel 2, Heather M.allace 1,∗

Department of Applied Medicine, University of Aberdeen, UKDepartment of Chemistry, University of Central Florida, Orlando, USA

-mail address: h.m.wallace@abdn.ac.uk (H.M. Wallace).

One central problem in cancer therapy is the lack of specificityf anti-cancer drugs to cancer cells. A delivery system would tar-et only cancer cells and prevent the severe side effects on normalells. The delivery system we have investigated is the polyamineransport system (PTS). The PTS is upregulated in cancer cellsnd can deliver polyamines and similar molecules to cancer cellshus decreasing the non-specific effects on normal cells (Palmert al., 2009). The PTS can accommodate a wide range of substratesPhanstiel et al., 2000). This led to the hypothesis that the PTS caneliver a cytotoxic agent specifically to cancer cells using one ofhe polyamines as a vector. In an attempt to determine the fea-ibility of this idea, a series of compounds combining the DNAntercalator, anthracene, with a variety of polyamine side chains

as synthesized (Phanstiel et al., 2000).The aim of this study was to investigate the mode of cell death

aused by two of these compounds, namely 9-anthracenylmethyl-utanediamine (Ant 4), N1-anthracenylmethyl-4,4-triamine (Ant4) in human leukaemic (K562) cells. Both Ant 4 and Ant 44emonstrated dose-dependent cytotoxicity (Table 1). Morpholog-

cal staining with 4′,6-diamidino-2-phenylindole (DAPI) identifiedhe cell death as apoptosis (Table 1). Both compounds were showno induce apoptosis via a caspase-dependent pathway (Table 1).n interesting finding was that Ant 4 induced caspase-8 activity in562, while Ant 44 increased the activity of caspase-9 (Table 1). Thisuggests that the compounds induce different pathways of apop-osis, the death receptor (caspase 8) and mitochondrial (caspase 9)athway.

In summary, Ant 4 and Ant 44 induce apoptosis via caspase-ependent pathways. Preliminary data indicates Ant 4 and Ant 44re activating and interacting with DNA or other macromolecules torigger apoptosis. Moreover, the findings provide ‘proof of concept’nd support the development of polyamine conjugates using theTS as a promising approach to the delivery of anti-cancer agentselectively to tumour cells.

able 1ffect of Ant 4 and Ant 44 on K562 cells at 48 h exposure.

Untreated Ant 4 Ant 44

IC50 (�M) – 25.0 ± 1.2 18.0 ± 1.5Apoptosis (%) 7.5 ± 1.0 59.3 ± 1.1* 62.4 ± 2.1*Caspase-3 activity (unit/h/mg protein) 1622 ± 3 9224 ± 4*** 8023 ± 4***Caspase-8 activity (units/h/mg protein) 1265 ± 4 2545 ± 3* 1873 ± 2Caspase-9 activity (units/h/mg protein) 1437 ± 3 1645 ± 3* 3489 ± 4

esults shown are the mean ± SEM for control, Ant 44 and Ant 4 treated cells (n = 4–6)ith 2 replicates per experiment. Results were analysed statistically using a paired

-test (*P < 0.01) and (***P < 0.0001).

eference

almer, A.J., Ghani, R.A., Kaur, N., et al., 2009. Biochem. J. 424 (3), 431–438.hanstiel, O., Price, H.L., Wang, L., et al., 2000. J. Org. Chem. 65 (18), 5590–5599.

oi:10.1016/j.tox.2010.08.134

010 / Toxicology 278 (2010) 341–378 373

P55

Use of minipigs in non-clinical juvenile toxicology studies

Andrew Makin ∗, Christina Skytte, Nanna Grand

LAB Research A/S, Hestehavevej 36A, Ejby, 4623 Lille Skensved,Denmark

E-mail address: am@labresearch.dk (A. Makin).

Current regulations require that during the drug developmentprocess there should be a plan for evaluation of the safety ofthe product in relation to paediatric human populations. In manyinstances this means a requirement to test new products in appro-priate juvenile animal models. Such juvenile models should beappropriate in terms of stage of development and drug-handlingcharacteristics in comparison with the age-appropriate targethuman population.

Rats are commonly used in juvenile animal studies, and aresuitable for a number of reasons. For non-rodent species, useof juvenile animals, particularly pre-weaned animals, presentsa number of practical and logistical challenges. Examplesinclude smaller litter sizes compared with rodents, long gesta-tion periods, long pre-weaning periods, space requirements andcost.

Among the non-rodent species that can be used are minip-igs. Over recent years these animals have been used frequentlyas models of juvenile humans at various stages of development.This has enabled collection of a good deal of both experi-ence and background data. This experience and background inturn allows for optimal study design both to obtain the bestpossible scientific data from the studies and to contribute totwo of the 3Rs i.e. reduction of animal use and refinement ofprocedures.

In our study design, we randomise the allocation of off-spring to mothers at birth (cross-fostering) and then treat allanimals allocated to one mother as a group. This minimises influ-ence of external and environmental factors such as parentageon study outcomes. We have measured all standard parame-ters, including bodyweight, growth indicators (crown-rump length,length of long bones), clinical pathology, electrocardiographyand so on. We have also collected organ weight and pathologydata.

Sex Age(weeks)

Hb(mmol/l)

RBC(1012/l)

Ht(ml/100ml)

WBC(109/l)

APTT(s)

PT(s)

Male 1 6.4 5.18 32 17.3 7.7 9.94 7.8 7.56 39 11.5 26.9 11.1

16+ 8.7 8.70 43 11.7 44.5 11.8Female 1 6.2 4.87 31 12.7 7.9 9.7

4 7.4 7.41 37 12.3 23.9 11.016+ 8.7 8.60 42 11.8 48.8 12.0

The data collected clearly show changes over time that indicatethe importance of having age-matched control animals in studies aswell as good background data for the parameters and timepoints inquestion. Examples of notable findings for haematology are shownin the table above. Other parameters are presented and discussedin the poster. The differences from adults are attributable to thenormal pattern of levels of such factors in growing animals, and areexplained in terms of both the ongoing physiological processes inthe animals and the influence of diet etc.

Study design issues are discussed and the results, includingthose mentioned here are highlighted in relation to the stage ofdevelopment of the animals. The conclusion from our data is thatthe variability of normal background findings in animals of different

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74 British Toxicology Society Abstracts, Sp

ges indicates not only the importance of good control data, but thathere are clear differences between young and adult animals thatustify the need for testing of pharmaceuticals in age-appropriatenimal models.

oi:10.1016/j.tox.2010.08.135

56

n silico discovery of on- and off-target effects of beta-blockers

nton Yuryev, Martin J. Griffies ∗

Ariadne Genomics, 9430 Key West Ave., Rockville, MD 20850, USA

-mail address: mjg@ariadnegenomics.com (M.J. Griffies).

In silico analysis can provide valuable insights about how can-idate drug compounds may modulate protein targets and canredict potential drug effects, both on- and off-target. Using Chem-ffect MedScan cartridge (Yuryev et al., 2009) and Pathway Studioatabase (Novichkova et al., 2003) we conducted a text-miningearch of PubMed and other available sources to find publishednformation on 19 known beta-blockers including Propranolol,revibloc, carvedilol, and Atenolol, to find shared pathways mod-lated by these compounds. Using this information we predictednti-inflammatory and prevention of renal failure drug effects foreta-blockers. Many of these predictions were already confirmedy clinical studies of several beta-blockers. The same methodol-gy also predicted additional new effects of diabetes and herplasiahat were not previously described for these drugs. These predictedffects include both harmful toxicities as well as novel disease indi-ations. Our analysis provides a framework to generate and testypotheses about mechanism of drug action that can be used toefine further experiments and interpret experimental design. Thisnalysis also should help to optimize clinical trial strategy, reduceime and cost of drug development, and may provide informationor repositioning of existing therapies.

eference

uryev, A., Kotelnikova, E., Daraselia, N., 2009. Expert Opin. Drug Discov. 4 (12),1307–1318.

ovichkova, S., Egorov, S., Daraselia, N., 2003. Bioinformatics 19 (September (13)),1699–1706.

oi:10.1016/j.tox.2010.08.136

57

ssessment of hearing dysfunction induced by industrial chem-cals in presence or absence of noise: A weight of evidencepproach

dolf Vyskocil 1,∗, Francois Lemay 1, Tony Leroux 3,4, Franceagnon 1, Martine Gendron 3,4, Amar Boudjerida 1, Ginetteruchon 2, Sophie Lim 1, Claude Viau 1

Groupe de recherche interdisciplinaire en santé - Département deanté environnementale et santé au travail, Université de Montréal,anadaInstitut de recherche Robert-Sauvé en santé et en sécurité du travail,ontréal, CanadaÉcole d’orthophonie et d’audiologie, Université de Montréal, CanadaCentre de recherche interdisciplinaire en réadaptation, Montréal,

anada

-mail address: adolf.vyskocil@umontreal.ca (A. Vyskocil).

There is accumulating epidemiological evidence that exposureo some solvents, metals, asphyxiants and other substances in

010 / Toxicology 278 (2010) 341–378

humans is associated with an increased risk of acquiring hear-ing loss. Furthermore, simultaneous and successive exposure tocertain chemicals along with noise can increase susceptibilityto noise-induced hearing loss (Fechter et al., 2002). This projectwas undertaken to develop a toxicological database allowing theidentification of possible ototoxic substances present in the workenvironment alone or in combination with noise exposure. Criticaltoxicological data were compiled for chemical substances includedin the Quebec Occupational Health Regulation. The data were eval-uated only for noise exposures that can be encountered in theworkplace and for chemical exposure concentrations up to theshort-term exposure limit or ceiling value or five times the 8-htime weighted average occupational exposure limit (TWA OEL) forhuman data. For animal studies, the limit was set at 100 times the8-h TWA OEL or ceiling value. In total, 223 experiments (in 150articles of which 43 evaluate simultaneous exposure to noise and achemical) covering 30 substances were assessed using a weight ofevidence approach.

At first, information from each source was given a weight ofevidence qualifier for ototoxicity or for the ability of a chemicalto exacerbate noise-induced hearing dysfunction: strong, medium,weak, absent or “no study found”. We took in consideration the fol-lowing parameters: studied species, number of subjects or animals,exposure route, characteristics of control groups, exposure levels,auditory function and statistical tests, dose/effect relationship. Aweight of evidence table was built allowing combination of theinformation from both human and animal studies on ototoxicityof chemicals. Human data were given more weight in the over-all assessment. Regarding the final conclusion about the ototoxicpotential of chemical substances, all substances bearing a “strongevidence” of ototoxicity overall were considered “ototoxic”. Thosewith “medium evidence” overall were rated “possibly ototoxic”.We considered the ototoxic potential of those with only “weakevidence” as “non conclusive”. Finally, those for which there wasabsence of evidence overall bore the mention “no evidence”, whichsignifies that we found no indication in scientific literature suggest-ing or demonstrating that the substance is ototoxic. However, thisprovides no assurance that the substance is not ototoxic. A similarlogic was applied regarding the final conclusion about the potentialof a substance to exacerbate noise-induced dysfunction.

For the majority of cases where potential ototoxicity was pre-viously proposed, there is a paucity of toxicological data in theprimary literature. Human and animal studies indicate that lead,styrene, toluene and trichloroethylene are ototoxic and ethyl ben-zene, n-hexane and xylene are possibly ototoxic at concentrationsthat are relevant to the occupational setting. Carbon monoxide andtoluene appear to exacerbate noise-induced hearing loss.

Reference

Fechter, L.D., Chen, G.D., Johnson, D.L., 2002. Toxicol. Sci. 66 (1), 131–138.

doi:10.1016/j.tox.2010.08.137

P58

3D human cell culture: A novel system for testing drug toxicity

Jun Li, Niall Blease, Fiona Saunders ∗, Heather M. Wallace

Division of Applied Medicine, University of Aberdeen, Polwarth Build-ing, Foresterhill, Aberdeen AB25 2ZD, United Kingdom

E-mail address: f.saunders@abdn.ac.uk (F. Saunders).

Traditional two dimensional (2D) cell cultures have been usedfor decades to substitute for animal models. However, the 2D cul-ture environment does not truly replicate the conditions found