Nrf2-mediated induction of antioxidant response element-driven gene expression by flavonoids is dependent on their chemical structure

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Abstracts / Toxicology 290 (2011) 1–46

Table 1 Relative area of major simvastatin metabolites in the presence or absence of gentamicin. Mean relative area of metabolite/parent compound (%)

+NADPH Gentamicin 0.1 mg/mL 0.5 mg/mL 1.0 mg/mL +NADPH +ABT

M2

M4

M7

M8

M10

M11

Simvastatin

4.83 ± 2.93

8.05 ± 2.41

3.88 ± 0.49

30.11 ± 5.05

16.87 ± 7.26

2.00 ± 0.59

16.41 ± 6.78

2.69 3.98 2.98 0.27

± ± ± ±

3.18 2.77 3.21 0.29**

7.26 7.18 7.25 2.04

± ± ± ±

3.52 3.68 3.81 1.98*

3.95 4.14 4.12 0.08

± ± ± ±

0.39 0.25 0.44 0.16**

30.76 32.24 32.26 6.40

± ± ± ±

4.45 6.53 5.69 3.62*

17.33 15.97 15.81 10.35

± ± ± ±

7.21 7.95 8.26 6.42

2.23 2.07 2.01 0.58

± ± ± ±

0.69 0.75 0.78 0.15*

18.17 15.89 16.96 20.91

± ± ± ±

6.45 4.65 6.65 6.76

Student’s t test, comparison with +NADPH. * P < 0.01. ** P < 0.001

megalin activity. However statins are associated with serious clinical adverse events partly caused by drug–drug interactions (DDIs), principally via alterations in hepatic metabolism and pharmacokinetics. The purpose of this pilot investigation was to assess the potential for statin/aminoglycoside drug-drug interactions, specifically looking at the CYP450 metabolism of simvastatin in the presence of gentamicin. Simvastatin (200 ␮M) was incubated with and without gentamicin (0.1–1.0 mg/mL) or 1-aminobenzotriazole (1 mM; ABT, non-specific CYP450 inhibitor) in rat liver microsomes (RLM; 1 mg/mL) with NADPH (2 mM) for 60 minutes at 37 ◦ C (n = 9 ± SD). Samples were analysed by HPLC–UV where simvastatin and its metabolites were quantified by peak area in the absence or presence of gentamicin (Table 1). Gentamicin did not have a significant effect on the number or relative area of simvastatin metabolites in RLM in the presence of NADPH and at supratherapeutic concentrations. ABT inhibited the production of all simvastatin metabolites by more than 90%, excluding M10, which is proposed to be simvastatin lactone (a metabolite that is not the product of CYP-mediated metabolism). Future studies will utilise LC-MS for metabolite identification. These data support the further investigation of statin/AG DDIs in vivo.

References Antoine, D.J., Srivastava, A., Pirmohamed, M., Park, B.K., 2009. Biochem. Pharmacol. 79, 647–654. Khwaja, A., O’Connelly, J., Hendry, B.M., 2000. Lancet 355, 741–744. Lopez-Novoa, J.M., Quiros, Y., Vicente, L., Morales, A.I., Lopez-Hernandez, F.J., 2010. Kidney Int. 79, 33–45. Schmitz, C., Hilpert, J., Jacobsen, C., Boensch, C., Christensen, E.I., Luft, F.C., Willnow, T.E., 2002. J. Biol. Chem. 277, 618–622.

are known for their antioxidant properties and it is probable that a major contributor to these properties is the transcription factor Nrf2, which upregulates a battery of approximately 100 cytoprotective genes (Tanigawa et al., 2007). Nrf2 is stabilised through cysteine modification of its negative regulator Keap1, leading to loss of Nrf2 ubiquitination and degradation. The flavonoids are a highly diverse family of phytochemicals and little is know about which structural features are most important for their stabilisation and activation of Nrf2. To this end, we screened a wide array of structurally diverse flavonoids, using an AREc32 luciferase reporter cell line (Fig. 1), to determine which structural features are critical for inducing Nrf2 and which can enhance this activation. We identified quercetin, a flavonol, as a strong inducer for AREdriven genes. Quercetin up-regulated the ARE-driven gene NQO1 at enzyme activity, protein and mRNA level. In addition, mutagenesis experiment suggested that modification of Cys151, His225, Cys226, and cys613 in Keap1, is responsible for the activation of Nrf2 protein by quercetin and of these residues, Cys151 was the most critical. However, by what mechanism quercetin modifies these cysteines has yet to be determined. The structural features of the flavonoids greatly influences their ability to activate Nrf2 and are therefore likely to impact on their potential as agents to induce endogenous antioxidant genes. Quercetin is a strong inducer of ARE-driven gene expression through modification of cys151 in Keap1.

doi:10.1016/j.tox.2011.09.045 P038 Nrf2-mediated induction of antioxidant response elementdriven gene expression by flavonoids is dependent on their chemical structure Han Xiao 1 , John M. Hourihan 1 , Laura J. Brown 1,∗ , Michael McMahon 1 , Derek Stewart 2 , John D. Hayes 1 1

Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom 2 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, United Kingdom E-mail address: [email protected] (L.J. Brown). Flavonoids are a family of polyphenolic compounds, which are found ubiquitously in fruit and vegetables and have been linked with the reduction in degenerative diseases associated with consuming a diet high in these foods (Manach et al., 2004). Flavonoids

Fig. 1. Relative luciferase activity in an AREc32 luciferase reporter cell lines in response to flavanoids.

Abstracts / Toxicology 290 (2011) 1–46

References Manach, C., Scalbert, A., Morand, C., Rémésy, C., Jiménez, L., 2004. Am. J. Clin. Nutr. 79 (May (5)), 727–747. Tanigawa, S., Fujii, M., Hou, D.X., 2007. Free Radic. Biol. Med. 42 (June (11)), 1690–1703.

doi:10.1016/j.tox.2011.09.046 P039 Development and optimisation of a suitable method to generate soluble recombinant high mobility group box 1 protein

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were purified on a 5 ml HisTrap FF affinity column and fractions of interest were analysed on a SDS gel (Fig. 1A). Two protein bands (approx. 30 and 35 kDa) were over-expressed within our cell culture system, consistent with the results reported by Li et al. (2004), who generated full length and truncated HMGB1 using a similar conventional cloning strategy. The protein bands were separated using ion-exchange chromatography (Fig. 1B) and a trypsin digest revealed that both protein bands contain HMGB1. We report the successful production of recombinant human HMGB1 protein, which will be utilised in functional studies. Full characterisation of HMGB1, its A box and HMGB1 with a C106S mutation will be presented.

Hannah L. Aucott 1,∗ , Daniel J. Antoine 1 , Robert Gibson 2 , Dominic P. Williams 1 , Lu-Yun Lian 2 , B. Kevin Park 1 1

MRC Centre for Drug Safety Science, Department of Pharmacology and Therapeutics, Institute of Translational Medicine, University of Liverpool, Liverpool L69 3GE, UK 2 School of Biological Sciences, Biosciences Building, University of Liverpool, Liverpool L69 7ZB, UK E-mail address: [email protected] (H.L. Aucott).

The high mobility group box 1 (HMGB1) protein, a ubiquitous and highly conserved damage associated molecular pattern (DAMP) molecule, may act as a novel and functional biomarker of hepatic stress or injury, with serum HMGB1 levels known to be elevated during APAP-induced hepatotoxicity (Antoine et al., 2009). To further elucidate the role of HMGB1 in the development and progression of hepatotoxicity we have developed and optimised a method for the generation of soluble human recombinant HMGB1. This will be a vital tool for future work investigating the interactions of HMGB1 with its multiple binding partners and receptors and for determining the importance of anti-HMGB1 antibodies in various disease states. HMGB1 DNA was amplified from a cDNA clone, obtained from a human embryonic testis carcinoma cell line by PCR. The PCR method was modified for maximum efficiency, including the redesign of the initial forward primer, addition of betaine [100 ␮M] and optimisation of the PCR cycling conditions. Purified DNA was digested with Nco1 and EcoR1 for 1hr, and inserted into a pETM11 vector. Successful ligation was verified using a double enzyme digest protocol, whilst correct orientation in the positive clones was confirmed by DNA sequencing of both strands. The recombinant plasmid was transformed into E. coli strain BL21 (DE3) cells that had reduced protease activity. Protein expression was induced by the addition of Isopropyl-D-thiogalactopyranoside (IPTG) [1 mM] when the optimal density at A600 achieved 0.7–0.8. Lysed cells

Fig. 1. Two step HMGB1 purification method. Fractions of interest (A: 1–2, 4–5 & 6–7, and B: 6–15), column waste and protein load were resolved by SDS gel electrophoresis and the proteins were visualised by staining with coomassie. (A) Isolation of HMGB1 proteins from the lysed cell culture using affinity column chromatography. Two over expressed protein bands were isolated to fractions 6–7. (B) Separation of upper and lower HMGB1 bands using ion-exchange chromatography. The lower band was successfully isolated to fractions 7–9 and the upper band to fractions 14–15. Fractions 10–13 contained a mixture of both bands.

References Antoine, D.J., Williams, D.P., Kipar, A., Jenkins, R.E., Regan, S.L., Sathish, J.G., Kitteringham, N.R., Park, B.K., 2009. Toxicol. Sci. 112 (2), 521–531. Li, J., Wang, H., Mason, J.M., Levine, J., Yu, M., Ulloa, L., Czura, C.J., Tracey, K.J., Yang, H., 2004. J. Immunol. Methods 289 (1–2), 211–223.

doi:10.1016/j.tox.2011.09.047 P040 Transporter mediated uptake and efflux of antiretroviral drugs: Potential for drug–drug interactions Zoe Riches ∗ , Gary A. Cameron, Gabrielle M. Hawksworth University of Aberdeen, Division of Applied Medicine, Foresterhill, Aberdeen AB25 2ZD, United Kingdom E-mail address: [email protected] (Z. Riches). Nucleos(t)ide reverse transcriptase inhibitors are used as antiretroviral drugs and can cause renal toxicity (Zimmermann et al., 2006). HAART is a combination therapy used to treat HIV and due to co-administration of drugs there is potential for drug–drug interactions. Transport related uptake and efflux is thought to contribute to the adverse drug reactions reported (Ray et al., 2006). Indeed, cidofovir is co-administered with probenecid, an organic anion transporter inhibitor, to reduce renal exposure and nephrotoxicity. The cellular transport of six NRTIs, with varying degrees of toxicity, was compared to investigate the significance of drug transport in relation to drug-induced toxicity. HEK293 cells transfected with human uptake transporters, organic anion transporters (hOATs) -1, -3 and -4 or human organic cation transporters-1 and -2 (hOCTs), were used. The multidrug resistance associated proteins-2 and -4 (MRPs) were expressed in Sf9 inside-out vesicles and could be used to determine if the transporters were involved in drug efflux. The transfected cell lines or Sf9 inside-out vesicles were incubated with substrates for 5 min at 37 ◦ C, washed with PBS or transport buffer and any internalised substrate measured by scintillation counting or LC–MS–MS. Tenofovir was taken up by hOAT1- and hOAT3-expressing cells compared with untransfected controls (Table 1, Student’s t-test, p < 0.001), whereas adefovir and cidofovir were substrates for hOAT1 but not hOAT3. Abacavir, lamivudine and zidovudine were not significantly transported by hOAT1-expressing cells compared with untransfected controls. However they all had a higher rate of passive diffusion, between 213 and 351 pmol/min/mg, at 100 ␮M, compared with 12 and 27 pmol/min/mg for the other NRTIs tested. There was no significant uptake of the six NRTIs into hOCT1or hOCT2-expressing cells compared with controls, although tetraethylammonium bromide, a positive control, was transported into transfected cells at a rate 15- and 10-fold greater than controls, showing that the expressed transporters were functional. Only tenofovir and abacavir were tested with MRP2- and MRP4expressing membranes. Tenofovir uptake was 5-fold greater than