Abstracts / Toxicology Letters 189S (2009) S57–S273 S99

N08Inhibition of male rat hepatic testosterone metabolism by mod-ulation of androgenic signalling and castration

Alexius Freyberger ∗, Ludwig Schladt, Ulrich Schmidt, FrancKrötlinger, Peter Andrews

Bayer Schering Pharma AG, BSP-GDD-GED-GTOX Special Toxicology,Wuppertal, Germany

The expression of certain liver cytochrome P450 isoenzymes (CYP)in male rats is androgen-dependent. We investigated the effectsof treatment with anti-androgens or estrogens, and of castrationon hepatic ex vivo metabolism of testosterone in male rats. Adultmales were treated orally with 1, 10, and 100 mg/kg flutamideor with 0.01, 0.05, and 0.2 mg/kg ethinylestradiol for four weeks.Peripubertal castrated rats subcutaneously received vehicle or0.4 mg/kg testosterone propionate (TP) for 10 days, intact vehiclecontrols were kept in parallel. For comparison, juvenile intactrats were treated with 1 mg/kg TP for 10 days. Treatment of adultanimals with 100 mg/kg flutamide or 0.2 mg/kg ethinylestradiolresulted in prostate atrophy and a considerable decrease of 6�-,16�-, 2�-, and 2�-testosterone hydroxylation and androstene-dione formation. Castration induced a comparable pattern ofchanges in enzyme activities. Interestingly, treatment of castratedrats with TP resulted in considerable restauration of prostate tissueweight, but did not affect enzyme activities, whereas in intactjuvenile males TP more than doubled 16�- and 2�-testosteronehydroxylation. CYP2C11 depends on intact androgenic signallingand encodes for 6�-, 16�- and 2�-testosterone hydroxylation andandrostenedione formation in male rat liver. Thus, changes relatedto treatment with anti-androgen/estrogen and after castrationare in line with a downregulation of CYP2C11. However, changesin 2�-testosterone hydroxylation suggest that other CYPs maybe affected as well. Furthermore, our data point to differences inthe androgenic regulation of CYP activities in castrated and intactjuvenile males.

Acknowledgement: This work was partly sponsored by CEFIC-EMSG.

doi:10.1016/j.toxlet.2009.06.322

N09Microcystin-LR, -LW and -LF induced murine neurite degenera-tion

Daniel Feurstein ∗, Julia Kleinteich, Daniel R. Dietrich

University of Konstanz, Human & Environmental Toxicology,Konstanz, Germany

Microcystins (MCs) are a group of cyclic heptapeptide cyanotoxinsencompassing more than 80 structural variants. MCs are assumedto be causal for the hepato- and neurotoxic effects and subse-quent deaths of dialysis patients infused with MC contaminatedsolutions in Caruaru, Brazil. MCs toxicity is primarily governed bythe potent and irreversible inhibition of serine/threonine-proteinphosphatases (PP, i.e. PP1 and PP2A). However, organ or cell specifictoxicity, e.g. neurotoxicity can only evolve upon active trans-port of MCs into cells (e.g. hepatocytes, neuronal cells). Humanorganic anion transporting polypeptides OATP1A2, 1B1, 1B3, mouseOatp1b2 and scate Oatp1d1 have been proven to transport MC-LR. Recent findings demonstrated uptake of MCs, PP inhibition,

cytotoxicity and caspase dependent apoptosis in primary murineneurons, albeit the latter data also suggested a congener specificpreference of transport efficacy and/or toxicity. To further evaluatethe involvement of Oatp based uptake of MCs into primary neurons,the presence of all known murine Oatps was investigated (mRNA)and saturation kinetics were performed using the Oatp/OATP sub-strates taurocholate (TC) and estron-3-sulfate (E3S). Co-incubationwith MC-LR, -LW, -LF demonstrated a 20–40% reduction of TC andE3S uptake. Furthermore MCs induced congener dependent neuritedegeneration (P < 0.01; 0.5 �M MC-LF, 1 �M MC-LW, 8 �M MC-LR)which co-occurred with a∼50% reduction of total neuronal PP activ-ity and hyperphosphorylation of the microtubule associated Tauprotein. Thus the data presented suggest that Oatp mediated andMC congener dependent transport is responsible for MC inducedneuronal toxicity.

doi:10.1016/j.toxlet.2009.06.323

N10In isolated rabbit renal proximal tubules, the beta-lactamantibiotic cephaloridine does not inhibit the mitochondrialuptake of succinate, a biomarker of its nephrotoxicity: A cellularmetabolomic study with carbon 13 NMR

Guy Martin ∗, Sophia Cadi-Soussi, Gabriel Baverel

NSERM U820, Laennec Faculty of Medicine, Metabolomics andMetabolic Diseases, Lyon, France

Purpose: The beta-lactam antibiotic cephaloridine, which has beenwithdrawn from the market, is used as a model compound innephrotoxicity studies. In isolated rabbit renal mitochondria, it hasbeen shown that cephaloridine (i) inhibits succinate uptake and (ii)reduces respiration. In the present study, we have re-examined thisproblem using isolated rabbit renal proximal tubules.

Methods: For this, tubules were incubated for 4 h in oxygenatedKrebs–Henseleit buffer containing either 1,4-13C-succinate, or2,3-13C-succinate or succinate + 13C-bicarbonate without or withcephaloridine. At the end of incubation, substrate removal andproduct formation were measured by enzymatic and carbon 13NMR methods. Enzymatic fluxes were calculated by combiningthese results with an original mathematical model of renal suc-cinate metabolism.

Results: Succinate was converted at high rates into fumarate,malate, glucose and CO2, and to a lesser extent, into, pyruvate,alanine, lactate and alpha-ketoglutarate. Cephaloridine induced anincrease in the accumulation of fumarate, malate and a decreasein the production of glucose and CO2; it also reduced the cellularlevel of ATP. Surprisingly, it did not change the removal of succinate.Fluxes through malate dehydrogenase, glucose-6-phosphatase andcitrate synthase were also diminished by cephaloridine.

In conclusion, the absence of inhibition of mitochondrial uptakeof succinate in intact rabbit renal proximal tubules strongly sug-gests that results obtained with isolated mitochondria do notnecessarily apply to mitochondria in intact cells and, therefore,should be interpreted with great caution. These results also suggestthat mitochondrial functions should be studied in intact cells.

doi:10.1016/j.toxlet.2009.06.324