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binding to the active site of TrxR or if Se increases de novo synthesisof TrxR maintaining its activity levels.

doi:10.1016/j.toxlet.2012.03.443

120 Abstracts / Toxicology L

as performed to investigate binding of steroids to 11�-HSD2.esults: Inhibitory effects for several AAS were observed, wherebyuoxymesterone was the most potent inhibitor of human 11�-SD2. Measurements using murine and rat 11�-HSD2 revealeduch weaker inhibition by the AAS tested, indicating that the

dverse effects of AAS-dependent 11�-HSD2 inhibition can-ot be investigated in these species. Furthermore, we showedhat human 11�-HSD2 metabolizes fluoxymesterone to 11-xofluoxymesterone. Molecular modeling suggested a competitiveode of inhibition of 11�-HSD2-dependent cortisol oxidation. No

irect modulation of MR function was observed. Conclusions: 11�-SD2 inhibition by fluoxymesterone may cause cortisol-inducedR activation, thereby leading to electrolyte disturbances and con-

ributing to the development of hypertension and cardiovascularisease.

oi:10.1016/j.toxlet.2012.03.440

14-221�-HSD2 inhibition by the pesticide thiram: The role ofysteine residues

rne Meyer, Alex Odermatt

University Basel, Switzerland

Purpose: The dithiocarbamate thiram is a widely used pesticide.ome of the observed toxic effects of thiram may be explained byocal disturbances in glucocorticoid homeostasis. We have earlierhown that dithiocarbamates can inhibit glucocorticoid inactiva-ion by inhibition of human 11�-hydroxysteroid dehydrogenase 211�-HSD2), probably via an irreversible sulfhydryl modification ofcysteine residue. Fish in rivers and lakes in areas of extensive agri-ulture may be exposed to pesticides including thiram. Therefore,e investigated whether thiram might also inhibit 11�-HSD2 from

he aquatic model organism zebrafish. Methods: A sequence com-arison revealed the absence of a cysteine residue, correspondingo Cys264 in human 11�-HSD2, in the zebrafish enzyme. The sen-itivity of wild-type human and zebrafish 11�-HSD2 to inhibitiony thiram was compared. In addition, mutant Cys264Ser of human1�-HSD2 and a substitution of the corresponding Ala253 to Cys inebrafish 11�-HSD2 were analyzed. Results: Zebrafish 11�-HSD2as relatively resistant to inhibition by thiram compared with theigh sensitivity of human 11�-HSD2. Substitution of Ala253Cysesulted in a more than ten-fold higher sensitivity of zebrafish1�-HSD2 to thiram. In contrast, Cys264Ser in human 11�-HSD2esulted in a loss of inhibitory activity. Conclusions: Our resultsemonstrate significant differences in the sensitivity of human andebrafish 11�-HSD2 to inhibition by the dithiocarbamate pesticidehiram. Site-directed mutagenesis revealed a key role of Cys264Sern the substrate binding pocket of human 11�-HSD2 for sensitivityo thiram.

oi:10.1016/j.toxlet.2012.03.441

14-23istopathology vs. inhibition of the thioredoxin system ofercury-exposed fish

ristina Carvalho 1, Vasco Branco 1, Paula Ramos 2, João Canário 2,un Lu 3, Arne Holmgren 3, Cristina Carvalho 1,2,3

Faculty of Pharmacy, Univ. of Lisbon, Portugal, 2 Ipimar/IRNB,ortugal, 3 Karolinska Institutet, Sweden

211S (2012) S43–S216

Mercury compounds interact with enzymes leading to loss ofactivity. We have shown that the thioredoxin system enzymes,thioredoxin (Trx) and thioredoxin reductase (TrxR) are inhibitedby mercury compounds in vitro and in vivo. Both enzymes weremore sensitive to divalent mercury (Hg2+) than to methylmercury(MeHg). Given the importance of this enzymatic system to sev-eral cellular pathways, its inhibition leads to the triggering of celldeath mechanisms. To understand how the inhibition of this sys-tem relates to histopathological changes in liver and kidney, weexposed zeabra-seabreams (Diplodus cervinus) for 28 days to eitherHg2+ or MeHg, followed by 14 days of depuration. Samples werecollected 14, 28 and 42 days after the beginning of the experimentand analyzed for mercury, Trx and TrxR activities and tissues werealso treated for histopathological observation. Results showed thatboth enzymes were inhibited Hg2+ and MeHg, by the same orderof magnitude, despite that Hg2+ accumulation was 5-times lower,in the liver and kidney. Moreover, Hg2+ led to loss of enzymaticactivity in the liver after 14 days, while MeHg effects were onlynoticed at day 28. Histopathology observations showed a good cor-relation between cellular effects and the inhibition of TrxR and Trx.After the depuration period enzymes activity was restored to con-trol levels in the liver, but the organ lesions remained observable.These results suggest that the inhibition of the Trx system might bea mechanism by which mercurial compounds lead to cellular andorgan endpoints of toxicity.

doi:10.1016/j.toxlet.2012.03.442

P14-24Mercury and selenium effects on HepG2 cells: TrxR activity andexpression levels

Cristina Carvalho 1, Vasco Branco 1, João Canário 2, JoãoGoncalves 1, Jun Lu 3, Arne Holmgren 3, Cristina Carvalho 1,2,3

1 Faculty of Pharmacy, Univ. of Lisbon, Portugal, 2 Ipimar/IRNB,Portugal, 3 Karolinska Institutet, Sweden

Mercury compounds interact with selenols and thiols of antiox-idant enzymes leading to loss of activity. Recently, we have shownthat the thioredoxin system is inhibited by mercury compoundsin vitro and in vivo and that the selenoenzyme thioredoxin reduc-tase was particular sensitive to mercurial compounds, due to thehighly reactive selenolthiol in the active center. Also, divalent mer-cury (Hg2+) was shown to be a stronger inhibiter of TrxR than MeHg.Selenium (Se) can reactivate purified TrxR inhibited by Hg2+ in vitro,however, in vivo the beneficial effect of Se appears to be organ spe-cific, with the liver being the organ where a protective effect wasobserved. To understand how Se supplementation interferes withthe interaction between Hg2+ and TrxR we co-exposed HepG2 cellsto different concentrations of Hg2+ and Se. Thioredoxin reductaseactivity was analyzed through the insulin reduction endpoint assayand its expression levels were analyzed through Western Blot andReal-Time PCR. These results will allow to understand if Se protec-tive effect results from a direct interaction with Hg2+ preventing its