Joana D Amaral

Ana R Correia

Clifford J Steer

Cláudio M Gomes

Cecilia Mp Rodrigues
email: cmprodrigues@ff.ul.pt

Cecília M P Rodrigues

No evidence of direct binding between ursodeoxycholic acid and the p53 DNA-binding domain

Keywords: Apoptosis -Binding -Circular dichroism -Mdm-2 -p53 -UDCA CD, circular dichroism, DBD, DNA binding domain, DTT, dithiothereitol, FITC, fluorescein isothiocyanate, IPTG, isopropyl-β-D-thiogalactoside, Mdm-2, mouse double minute 2, NSR, nuclear steroid receptors

INTRODUCTION

The tumor suppressor protein p53 is a transcription factor that functions to maintain genome integrity. It exerts its role by inducing or repressing the expression of a network of genes involved in cell cycle control, senescence, and apoptosis, in response to diverse stress stimuli [START_REF] Riley | Transcriptional control of human p53-regulated genes[END_REF]. Although there is evidence that p53 can mediate apoptosis by transcription-independent mechanisms [START_REF] Schuler | p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release[END_REF][START_REF] Speidel | Dissection of transcriptional and nontranscriptional p53 activities in the response to genotoxic stress[END_REF][START_REF] Marchenko | Monoubiquitylation promotes mitochondrial p53 translocation[END_REF], numerous apoptotic genes are transcriptionally activated by this tumor suppressor. These include members of the Bcl-2 family, such as Bax, or the BH3-only proteins Noxa and Puma [START_REF] Miyashita | Tumor suppressor p53 is a direct transcriptional activator of the human bax gene[END_REF][START_REF] Oda | Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis[END_REF][START_REF] Nakano | PUMA, a novel proapoptotic gene, is induced by p53[END_REF]. The regulatory functions of p53 are usually under tight control. In normal cells, due to p53 constitutive ubiquitination by Mdm-2, an E3 ligase that targets p53 to proteasomal degradation its levels are almost undetectable [START_REF] Brooks | Mechanistic studies of MDM2-mediated ubiquitination in p53 regulation[END_REF].

Posttranslational modifications, such as phosphorylation, acetylation, sumoylation, among others are also important mechanisms for p53 regulation [START_REF] Ashcroft | Regulation of p53 stability[END_REF].

p53 complexity starts in its structure. The protein is only active when it forms a tetramer and the structure and dynamics of this oligomeric form is crucial to understand its function. In addition, the monomer itself is already complex. It has 393 aminoacid residues that can be divided into 3 functional domains: the N-terminal domain (1-93), an intrinsically disordered region that includes the transactivation domain (TAD) and the proline-rich region, the DNA-binding domain (DBD) (102-292), a structured region that constitutes the proteins core, and finally the C-terminal domain that includes two small domains, a tetramerization domain that regulates the oligomerization state of p53 (320-356), and a natively unfolded regulatory domain (363-393) that binds DNA nonspecifically [START_REF] Joerger | Structural Biology of the Tumor Suppressor p53[END_REF].

Ursodeoxycholic acid (UDCA) is an endogenous hydrophilic bile acid, widely used in the treatment of certain cholestatic disorders of the liver. The therapeutic effects of UDCA may result, in part, from its ability to inhibit liver cell apoptosis. It has been demonstrated that this bile acid plays a unique role in modulating the apoptotic threshold in both hepatic and nonhepatic cells, in response to a variety of agents acting through different apoptotic pathways [START_REF] Rodrigues | A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation[END_REF]. The precise mechanism(s) by which UDCA prevents cell death remains speculative and involves molecular targets other than mitochondria. Previous studies indicated that UDCA inhibits the E2F-1/Mdm-2/p53 apoptotic cascade induced by transforming growth factor β1 (TGF-β1) [START_REF] Solá | Ursodeoxycholic acid modulates E2F-1 and p53 expression through a caspase-independent mechanism in transforming growth factor β1-induced apoptosis of rat hepatocytes[END_REF][START_REF] Solá | Modulation of nuclear steroid receptors by ursodeoxycholic acid inhibits TGF-β1-induced E2F-1/p53-mediated apoptosis of rat hepatocytes[END_REF]. The ability of E2F-1 to induce apoptosis involves stabilization of p53 via transcription of p14ARF, which markedly inhibits the p53 repressor, Mdm-2 [START_REF] Phillips | E2F-1 induced apoptosis[END_REF]. UDCA, in turn, counteracts TGF-β1 effect modulating the expression of apoptosis-related p53 target genes. Further, we demonstrated that UDCA protects hepatocytes from p53-mediated apoptosis, by reducing p53 transcriptional and DNA-binding activities, possibly through mechanisms involving Mdm-2 [START_REF] Amaral | p53 is a key molecular target of ursodeoxycholic acid in regulating apoptosis[END_REF]. Importantly, UDCA can reach the nucleus where it may interfere with several nuclear proteins, such as E2F-1 or p53 itself, a mechanism that appears to be dependent on nuclear steroid receptors (NSR) [START_REF] Solá | Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-β1-induced apoptosis in rat hepatocytes[END_REF]. In fact, we and others have shown that UDCA and its taurine-conjugated derivative, tauroursodeoxycholic acid, interact with NSR [START_REF] Solá | Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-β1-induced apoptosis in rat hepatocytes[END_REF][START_REF] Tanaka | Ursodeoxycholic acid-dependent activation of the glucocorticoid receptor[END_REF][START_REF] Solá | Functional modulation of nuclear steroid receptors by tauroursodeoxycholic acid reduces amyloid {β}-Peptide-induced Apoptosis[END_REF]. More importantly, these bile acids promote dissociation of NSR from their cytosolic chaperone, Hsp90, and translocate into the nucleus as a ligand-receptor complex to reduce apoptosis.

In the present study, we have used complementary biophysical techniques to determine whether UDCA binds to or alters the structural stability and folding properties of the p53 DBD. UDCA could compromise p53 function either by destabilizing its structure or by inducing structural fluctuations that could inhibit its DNA-binding specificity. Previously, this approach has been successfully used to Chemicals, reagents and enzymes were of the highest purity grade commercially available. Chromatography materials were from Amersham Pharmacia Biotech (Uppsala, Sweden). For protein expression, p53 DBD plasmid coding for amino acids 94-312 of human wild-type p53 was transformed into E.coli BL21 competent cells.

Gene expression and protein

The DBD of p53 was expressed as soluble protein as described by Bell et al. [START_REF] Bell | p53 contains large unstructured regions in its native state[END_REF]. In brief, bacteria were grown in Luria-Bertani medium supplemented with 100 μg/ml ampicilin, at 37ºC, to an A 600 of 0.5 followed by overnight induction at 22ºC with 1 mM isopropyl-β-D-thiogalactoside (IPTG). After induction, cells were harvested by centrifugation, resuspended in 50 mM Tris-HCl, pH 6.8, 5 mM dithiothereitol (DTT), 1 mM benzamidine, complete protease inhibitor tablets (Complete TM ; Roche Applied Science, Mannheim, Germany), 200 units of benzonase (Merck, Darmstadt, Germany), and disrupted by high-pressure dispersion using a Thermo IEC French Press (Needham Heights, MA). Purification of p53 DBD was performed at 4ºC using a combination of cation exchange chromatography on a HiTrap SP Fast Flow column and affinity chromatography on a Heparin HiTrap column (GE Healthcare Life Sciences, Freiburg, Germany) in 50 mM sodium phosphate, pH 7,0, 150 mM KCl, and 5 mM DTT. The purity of the protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Figure 1). p53 DBD was found to be stable in solution and maintained its spectroscopic properties and melting temperature after flash-freezing in liquid nitrogen. Protein concentration was determined spectrophotometrically by using the extinction coefficient of 0.649 for p53 DBD calculated for a 1 mg/ml solution in a 1 cm cuvette at 280 nm.

Spectroscopic methods

Anisotropy measurements were performed at 10ºC, using a SLM Aminco spectrofluorimeter (Spectronic Instruments, Inc., Rochester, NY) with cell stirring. Far-UV circular dichroism (CD) spectra were recorded typically at 0.2 nm resolution on a Jasco J-815 spectropolarimeter (Jasco, Tokyo, Japan) fitted with a cell holder thermostatically controlled by a Peltier.

Thermal denaturation

Thermal unfolding was followed by monitoring the intrinsic tryptophan fluorescence (λ em = 340) and the ellipticity (Δε mrw at 222 nm) variations. In all experiments, a heating rate of 1ºC/min was used, and the temperature was changed from 10 to 90ºC. Data were analyzed according to a two-state model described by the following equation: where y is the spectroscopic signal observed, m f and m u are the slopes of the pre-and post-transition baselines, y f and y u correspond to the value of y for folded and unfolded forms, T m is the midpoint of the thermal unfolding curve, and ΔH m is the enthalpy change for unfolding at T m . The fits to the unfolding transitions were made using Origin (MicroCal Software Inc, Northampton, MA)

Binding studies

All measurements were performed with fluorescein isothiocyanate (FITC)-tagged UDCA. The interaction between p53 DBD and FITC-UDCA was evaluated by using both far-UV CD and fluorescence anisotropy spectroscopy, at a constant temperature of 10ºC, in buffer containing 20 mM NaP, 50 mM NaCl, and 2 mM DTT, pH 7.5. Far-UV CD measurements were performed with 100 µM p53 DBD, before and after the addition of the bile acid at stoichiometric ratio 1:1, using a 1 mm cell path length (bandwidth 2 nm). The spectra were recorded from 260 to 190 nm and accumulated 10 times. For titration experiments, aliquots of 3 µl of titrant (500 µM FITC-UDCA) were sequentially added to p53 DBD peptide in solution (0.22 mg/ml), until the concentration reached p53 DBD 1:54 UDCA. All spectra were corrected for buffer contributions and protein concentration. Ellipticities were followed at 208 and 222 nm.

For anisotropy experiments, p53 DBD (initial concentration 30 µM) was titrated with FITC-UDCA (500 µM) with increments of 5 µl, waiting ~ 5 min between each addition. Similarly, FITC-UDCA (initial concentration 5 µM) was titrated with p53 DBD (50 µM), additions of 10 µl every 5 min. When the FITC-UDCA was titrated, fluorescence anisotropy was measured on excitation at 480 nm (bandwith 8 nm) and emission at 520 nm (bandwith 8 nm). When the protein was titrated fluorescence anisotropy was measured on excitation at 280 nm (bandwith 8 nm) and emission at 340 nm (bandwith 8 nm).

RESULTS

Characterization of p53 DNA-binding domain

p53 DBD was expressed in E.coli with a low yield of ~ 3 mg of purified protein per 3 g of wet culture (Figure 1). The fold and the stability of the purified p53 DBD was evaluated by far-UV CD to confirm that the protein used in the binding assays was correctly folded. The far-UV CD spectrum is shown in Figure 2A and is in agreement with previously published data by Klein et al. [START_REF] Klein | High thermostability and lack of cooperative DNA binding distinguish the p63 core domain from the homologous tumor suppressor p53[END_REF]. As expected, the far-UV CD spectrum of the DNA binding domain of p53 showed two negative peaks (208 and 222 nm), which reflect its α-helical content. As a result of the β-sheet contributions, the 208 nm peak was broader and the 222 nm peak was less intense. Thermal denaturation following far-UV CD signal at 222 nm, allowed to evaluate the p53 DBD stability (Figure 2B). In agreement with previously published data [START_REF] Bell | p53 contains large unstructured regions in its native state[END_REF], a melting temperature (T m ) of 41.5 ± 0.3ºC was determined.

Evaluation of UDCA interaction with p53 DNA-binding domain

To investigate a possible interaction between UDCA and p53 DBD, we used both far-UV CD and fluorescence anisotropy to monitor structural changes that might occur as a consequence of bile acid presence. First, we followed the titration of p53 DBD with FITC-UDCA by far-UV CD (208 nm and 222 nm). Adding the bile acid has no effect on the protein spectrum, even for a stoichiometry of 1 p53 DBD to 54 FITC-UDCA (Figure 2A and 3A), meaning that FITC-UDCA does not affect the p53 DBD secondary structure. Similarly, the thermal stability of p53 DBD is not significantly affected by the presence of FITC-UDCA. In case of a specific interaction of UDCA with the native conformation of the p53 DBD, a positive T m shift would be expected. Our results indicate that the presence of this bile acid induces a ΔT m -3.3 ± 1ºC (Figure 2B), which may be considered negligible since FITC-UDCA also significantly reduces the signal/noise, thus increasing the uncertainty in the T m determination. Alternatively, this shift may result from weak unspecific interactions of UDCA with p53 DBD, given the mild detergent properties of UDCA. Similar titrations were performed following changes in fluorescence anisotropy. Initially, p53 DBD (30 µM) was titrated with FITC-UDCA (500 µM); however, no changes in p53 fluorescence anisotropy were detected (Figure 3B). The same result was obtained when titrating FITC-UDCA with increasing amounts of p53 DBD (data not shown).

Under the conditions used, it was not possible to detect a direct interaction between anti-apoptotic UDCA and the DNA-binding domain of human p53, in vitro.

DISCUSSION

The mechanisms of bile acid effects on cell survival and apoptosis are not entirely understood. We have previously reported that UDCA prevents apoptosis in several cell types by inhibiting the mitochondrial pathway [START_REF] Rodrigues | A novel role for ursodeoxycholic acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation[END_REF][START_REF] Botla | Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism of UDCA cytoprotection[END_REF][START_REF] Rodrigues | Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation[END_REF] and modulating the E2F-1/Mdm-2/p53 apoptotic cascade [START_REF] Solá | Ursodeoxycholic acid modulates E2F-1 and p53 expression through a caspase-independent mechanism in transforming growth factor β1-induced apoptosis of rat hepatocytes[END_REF][START_REF] Amaral | p53 is a key molecular target of ursodeoxycholic acid in regulating apoptosis[END_REF]. In addition, bile acids have been detected in nuclei of several cell types [START_REF] Solá | Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-β1-induced apoptosis in rat hepatocytes[END_REF][START_REF] Setchell | Bile acid concentrations in human and rat liver tissue and in hepatocyte nuclei[END_REF][START_REF] Kren | Modulation of steadystate messenger RNA levels in the regenerating rat liver with bile acid feeding[END_REF][START_REF] Monte | Changes in the pool of bile acids in hepatocyte nuclei during rat liver regeneration[END_REF]. We have suggested that UDCA, as a cholesterol-derived molecule, could reach the nucleus through mechanisms similar to those used by steroid hormones. It appears that NSR are specifically required for the nuclear trafficking of UDCA, ultimately protecting cells from undergoing apoptosis [START_REF] Solá | Nuclear translocation of UDCA by the glucocorticoid receptor is required to reduce TGF-β1-induced apoptosis in rat hepatocytes[END_REF][START_REF] Solá | Functional modulation of nuclear steroid receptors by tauroursodeoxycholic acid reduces amyloid {β}-Peptide-induced Apoptosis[END_REF]. Therefore, it is conceivable that bile acids may act as molecular modulators of apoptosis-related gene expression, by interfering directly with DNA, or by interacting with transcription factors and other nuclear proteins involved in the apoptotic machinery.

p53 has a multidomain structure comprising a folded region with DNA-binding and tetramerization domains, and natively unfolded regions with molecular recognition features that provide binding promiscuity [START_REF] Joerger | Structural Biology of the Tumor Suppressor p53[END_REF]. Since specific binding to p53responsive genes occurs through the DNA-binding domain of p53, and based on our previous data indicating that UDCA is capable of reducing p53 transcriptional and DNA-binding activities [START_REF] Amaral | p53 is a key molecular target of ursodeoxycholic acid in regulating apoptosis[END_REF], here we have focused on the effect of UDCA in the structure and conformation of p53 DBD.

Two complementary biophysical techniques were used: far-UV CD and fluorescence anisotropy-Far-UV CD spectroscopy is widely used in the study of molecular interactions due to its remarkable sensitivity. Far-UV CD bands derive mainly from peptide bond absorption, reflecting the secondary structure of the protein [START_REF] Martin | Calcium-Binding Protein Protocols[END_REF]. Fluorescence anisotropy, in turn, detects the rotational mobility of a fluorophore, which is also a reliable and sensitive spectroscopic method to locally evaluate the existence of physical interactions. Unbounded molecules can freely rotate in solution, while formation of binding structures restricts their motion, thereby increasing anisotropy [START_REF] Royer | Fluorescence Spectroscopy. Protein Stability and Folding: Theory and Practice[END_REF].

Under the conditions tested, neither CD nor fluorescence anisotropy supported the hypothesis of a direct interaction between UDCA and the p53 DBD. CD experiments have shown that the presence of UDCA does not induce changes on the secondary structure of p53 DBD. Similarly, data on fluorescence anisotropy showed no alterations in the mobility of either p53, or UDCA, when together.

UDCA could be interacting with other regions of p53. The N-terminal region of p53 contains the TAD, considered to be a binding site for a multitude of interacting proteins, such as components of the transcription machinery [START_REF] Lu | Human TAFII31 protein is a transcriptional coactivator of the p53 protein[END_REF][START_REF] Di Lello | Structure of the Tfb1/p53 complex: Insights into the interaction between the p62/Tfb1 subunit of TFIIH and the activation domain of p53[END_REF], the transcriptional coactivators p300/CBP (CREB-binding protein) [START_REF] Antonsson | Inhibition of Bax channel-forming activity by Bcl-2[END_REF][START_REF] Teufel | Four domains of p300 each bind tightly to a sequence spanning both transactivation subdomains of p53[END_REF], and the negative regulator Mdm-2 [START_REF] Kussie | Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain[END_REF][START_REF] Schon | Molecular mechanism of the interaction between MDM2 and p53[END_REF]. Upon binding, intrinsically disordered regions of the p53 TAD rigidify and become fully folded. This is the case for the p53 fragment comprising residues 15-29 after binding to a hydrophobic cleft in the N-terminus of Mdm-2 [START_REF] Popowicz | Molecular basis for the inhibition of p53 by Mdmx[END_REF]. Moreover, it has been reported that Mdm-2 also undergoes extensive conformational changes along its N-terminal region, upon binding to p53 [START_REF] Schon | Molecular mechanism of the interaction between MDM2 and p53[END_REF]. Interestingly, the Mdm-2 protein is the major repressor of p53, and UDCA has been shown to increase its levels in rat hepatocytes undergoing TGF-β1-induced apoptosis [START_REF] Solá | Ursodeoxycholic acid modulates E2F-1 and p53 expression through a caspase-independent mechanism in transforming growth factor β1-induced apoptosis of rat hepatocytes[END_REF]. More importantly, the bile acid was shown to play a role in p53/Mdm-2 association during p53-induced hepatocyte apoptosis [START_REF] Amaral | p53 is a key molecular target of ursodeoxycholic acid in regulating apoptosis[END_REF]. Thus, the N-terminus of p53, or more specifically, the TAD domain, could also be a potential target region for UDCA binding/interaction. The bile acid may additionally interact with Mdm-2 itself, in the p53-binding cleft of bounded or A c c e p t e d M a n u s c r i p t unbounded Mdm-2, to somehow stabilize the complex. The p53-binding cleft is the most flexible region of the Mdm-2 protein, presenting enough plasticity to adapt to diverse topologies of incoming ligands [36]. In fact, the number of newly discovered small-molecules that bind the Mdm-2-p53 binding cleft is continuously increasing over the years [START_REF] Vassilev | In vivo activation of the p53 pathway by small-molecule antagonists of MDM2[END_REF][START_REF] Grasberger | Discovery and cocrystal structure of benzodiazepinedione HDM2 antagonists that activate p53 in cells[END_REF][START_REF] Dudkina | Small molecule protein-protein inhibitors for the p53-MDM2 interaction[END_REF][START_REF] Dey | Updates on p53: modulation of p53 degradation as a therapeutic approach[END_REF].

Interaction between UDCA and the extreme C-terminus of p53 should also be analyzed. The C-terminal region of p53, called regulatory domain, is subjected to extensive posttranslational modifications to regulate p53 function, including acetylation, ubiquitination, phosphorylation, and methylation. Finally, three putative nuclear localization signals [START_REF] Liang | A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain[END_REF] and one nuclear export signal [START_REF] Stommel | A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking[END_REF] sequences have been identified within the C-terminal region of p53. Subcellular localization of p53 is determinant for its activity, since stabilized p53 remains active in the nucleus, whereas translocation to the cytoplasm is required for Mdm-2-mediated p53 degradation [START_REF] O'keefe | Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination[END_REF]. Thus, UDCA could inhibit the p53 apoptotic pathway not by directly binding to p53 but by interfering with other proteins and molecular pathways involved in the regulation of this complex protein.

The multifaceted network of p53 regulation opens the door for an endless world of hypothesis in which UDCA could participate and play a role. Although, we could not detect direct binding between the p53 DBD and UDCA, our findings contributed to the characterization of the interaction between these two molecules. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 07 Oct 2009 as manuscript BSR20090107 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090107 A c c e p t e d M a n u s c r i p t EXPERIMENTAL Materials and plasmids

  Bioscience Reports Immediate Publication. Published on 07 Oct 2009 as manuscript BSR20090107 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090107 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 07 Oct 2009 as manuscript BSR20090107 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090107A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 07 Oct 2009 as manuscript BSR20090107 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090107

Figure 1

 1 Figure 1 Purity of p53 DNA-binding domain. p53 DBD purified from E.coli bacteria was subjected to electrophoresis on 12.5% polyacrylamide gels and stained with Coomassie brilliant blue.

Figure 2

 2 Figure 2 Effect of UDCA in the structural properties of p53 DNA-binding domain. (A) Far-UV CD spectra of p53 DBD (0.22 mg/ml), with or without UDCA. (B) Far-UV CD analysis of thermal denaturation transitions of p53 DBD alone (■), or in the presence of UDCA (□). See "Experimental" for details.

Figure 3

 3 Figure 3 Titration of p53 DBD with FITC-UDCA. Titration of p53 DBD protein with increasing amounts of FITC-UDCA, analyzed by far-UV CD (A) or fluorescence anisotropy spectroscopy (B). See "Experimental" for details.

FIGURES Figure 1

 1 FIGURES

Figure 2 Bioscience

 2 Figure 2

Figure 3 Bioscience

 3 Figure 3

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We thank Dr. Christian Klein, Roche Diagnostics GmbH, Penzberg, Germany, for providing the human recombinant p53 DBD expression plasmid, and Dr. Jesse D. Martinez, University of Arizona, Tucson, USA the FITC-UDCA.

FUNDING

Supported by grants PPCDT/SAU-FCF/62479/2004 and PTDC/SAU-FCF/67912/2006 from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal (to C.M.P.R.). J.D.A. and A.R.C. were recipients of Ph.D. fellowships (BD/17799/2004 and BD/24949/2005, respectively) from FCT. Bioscience Reports Immediate Publication. Published on 07 Oct 2009 as manuscript BSR20090107 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090107

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


MD, MPH Ana M Novoa

PhD Katherine Pérez
email: cperez@aspb.cat

MPH Elena Santamariña-Rubio

Marc Marí-Dell'olmo

MD Rogelio Cozar

MD, PhD Josep Ferrando

Rosana Peiró-Pérez

PhD Aurelio Tobías

Pilar Zori

MD, PhD Carme Borrell

MPH Rosana Peiró

Road Safety in the Political Agenda: the Impact on Road Traffic Injuries

Keywords: accidents, traffic, effectiveness, evaluation studies, intervention studies, policy making, wounds and injuries

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

BACKGROUND

Road traffic injuries cause great mortality and morbidity worldwide. [START_REF] Peden | World report on road traffic injury prevention[END_REF][START_REF] Lopez | Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data[END_REF] In Spain, they are the primary cause of death among individuals 1-39 years, the third for individuals 40-59 years and the fifth for individuals 60-69 years. [START_REF] Peiró | Lesiones por tráfico, de ocio y domésticas y laborales. Descripción de la situación en España. Informe SESPAS[END_REF] In addition, they are the primary cause of potential years of life lost in men, the second in women. [START_REF] Cubí | Evaluación de riesgos y del impacto de los accidentes de tráfico sobre la salud de la población española, 1964-2004[END_REF][START_REF] Llàcer | Mortalidad en España en 1998. Evolución en la década. (I) Mortalidad General, principales causas de muerte y años potenciales de vida perdidos[END_REF] To improve road safety, the White Paper on European transport policy established the target of reducing road fatalities by 50% by the year 2010 (compared to 2001).[6] The Road Safety Action Programme describes specific measures -related to road user behaviour (mainly based on police enforcement), vehicle safety and road infrastructureaimed at achieving this target. [START_REF]Saving 20 000[END_REF] A strong and sustained political will is required to achieve these targets and ensure that road safety is given enough priority, including appropriate funding, necessary legislative changes and a capable bureaucracy. [START_REF]Achieving ambitious road safety targets[END_REF][START_REF]Impact assessment road safety action programme[END_REF][START_REF]A methodological approach to national road safety policies[END_REF] Following the approval of the Road Safety Action Programme, the Spanish government established road safety as a political priority, and created the Road Safety Special Measures 2004-2005 [START_REF][END_REF] and the Road Safety Strategic Programme 2005-2008, [START_REF][END_REF] the main goal of which is to achieve a 40% reduction in road fatalities by the year 2008 (compared to 2003), and lists 182 actions -to be progressively implemented -included in 10 strategic areas: 1) road safety education, 2) road safety awareness, 3) surveillance and control, 4) vehicle safety, 5) road infrastructures and improvement in road safety information and management, 6) road safety in the field of transport and labour, 7) attention to the victims and their families, 8) road safety research and analysis, 9) participation of society, and 10) coordination between administrations.

Prior to the year 2004 the implemented interventions were mostly based on road safety normative, such as the establishment of illegal blood alcohol concentration levels, speed limits, or making compulsory to use passive safety devices. Also, road infrastructure and health care delivery were improved. [START_REF] Villalbí | Evaluation of regulatory policies: the prevention of traffic accidents in Spain[END_REF] However, road safety enforcement measures were mostly implemented from the year 2004 on (e.g. the number of new speed cameras installed increased from 4 during the year 2004 up to 197 during 2006; the number of alcohol checkpoints performed over the number of registered drivers increased from an 11.1% in 2003 to a 15.8% in 2006).[14,15] Further road safety normative was also approved: on 2004 standardized child safety seats and bicycle helmets on non-urban roads were made compulsory, and on 2006 the penalty points system was implemented and the life period for school buses was set at a maximum of ten years. [START_REF] Rodríguez | Del viejo código al permiso por puntos[END_REF] Although other countries have undertaken similar initiatives to those implemented in Spain, to date, the overall effect of the actions implemented following road safety prioritisation has not yet been assessed.

The objective of this paper is to assess the impact of road safety prioritisation in the year 2004 on the number of traffic injured people in Spain. Specifically, the effectiveness of the package of road safety interventions implemented after the introduction of road safety in the political agenda will be assessed. Differences in the effectiveness with respect to gender, age, injury severity and type of road user will also be assessed.

MATERIAL AND METHODS

Study design and population

An evaluation study was performed using an interrupted time-series design. The study population were people injured (fatal and non-fatal) in traffic crashes in Spain between January 2000 and December 2006.

Sources of information

The road traffic crashes database of the Dirección General de Tráfico (DGT) (General Directorate for Traffic) was used, which contains data -collected by police officers -for injury collisions: the characteristics of the collision, the vehicle and the subjects involved.

Vehicle fleet and the number of new vehicle registrations, used as proxies for exposure, were available at the DGT homepage.[17]

Variables

The dependent variable was the number of people injured in traffic collisions. This variable was stratified according to sex, age (0-13 years, 14-15 years [allowed to ride mopeds], 16-17 years [also allowed to ride motorcycles ≤125 cc], 18-29 years [allowed to drive any type of vehicle], 30-44 years, 45-64 years, 65-74 years, >74 years), type of road user (car user, motorcycle user, moped user, pedestrian), and severity (slight, serious, fatal -in 24 hours -). The police classifies as seriously injured those who are hospitalised more than 24 hours.

The explanatory variable was the intervention, which in this case includes overall interventions implemented after road safety was included in the political agenda (compared to those implemented prior to it). A dummy variable was created to compare the post-intervention period (January 2004 -December 2006) with the pre-intervention period (January 2000 -December 2003).

An exhaustive database containing all of the implemented road safety measures in Spain is not available. Moreover, whereas the national government is in charge of road safety normative, the specific road safety actions performed also depend on the local governments and vary greatly depending on the region. Consequently, given that it is not possible to clearly define the implemented interventions before and after road safety prioritisation, these two periods have to be thought of as two black boxes that mainly differ in that the intensity of road safety enforcement was much higher in the postintervention period.

Several socio-economic variables were accounted for as potential confounding factors: gasoline and gas-oil consumption, unemployment rate, and the gross national product.

Since July 2005, there is a new protocol for police data collection aimed at improving the reporting of traffic crashes in Spain. This has probably involved an increase in the number of collisions registered, mostly involving slight injuries, but also serious injuries. To account for the effect of this protocol, certain analyses were repeated including a dummy variable in the model which compared the period before (January 2000 -June 2005) and after (July 2005 -December 2006) its approval.

Statistical analysis

Monthly time-series analyses were carried out using Poisson regression models adjusted for over-dispersion (quasi-Poisson). [START_REF] Yannis | Road casualties and enforcement: distributional assumptions of serially correlated count data[END_REF] The absolute number of people injured was compared throughout the time series.

Potential confounding by time trend and seasonal patterns were controlled for using a linear trend and sine and cosine functions. [START_REF] Stolwijk | Studying seasonality by using sine and cosine functions in regression analysis[END_REF] Thus, the model can be summarized as follows:

( ) [ ] ( )

∑ ∑ + + + + ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ + + = = j t jt j t t k k k t Z t X X T t k T t k t Y E ε β β β π β π β β β 6 5 4 3 2 6 1 1 0 2 cos 2 sin ln
where t is the time period (t=1 for the first month of the series, t=2 for the second month, etc.), k takes values between 1 and 6 (for example, k=1 for annual seasonality; k=2 for six-monthly seasonality), T is the number of periods described by each sinusoidal function (for example, T=12 months), X t identifies the pre-and postintervention periods (X t =1 for the post-intervention period), Z jt other co-variables introduced (socio-economic variables, new protocol for police data collection), j the number of co-variables introduced, and ε the error term. Only statistically significant terms were included in the final model.

Relative risks (RR) and their 95% confidence intervals (95% CI) were derived from the adjusted models. Two different RRs were obtained, one corresponding to mean change (ß 4 + ß 5 t 0 , where t 0 identifies the month in which the intervention took place), which indicates the change in the mean number of people injured during the first month of the post-intervention period compared with the previous month (adjusting for time trend and seasonality) (short-term effect), and another corresponding to trend change (ß 5 ), which indicates the change in the time trend between the two periods (long-term effect). [START_REF] Langbein | The quasi experiment[END_REF] The RR for the mean change should not be confused with the mean change in the overall number of people injured throughout the whole post-intervention period.

The number of people injured prevented by road safety prioritisation was calculated as the difference between the observed and expected numbers of people injured throughout the whole post-intervention period -thus summarizing the short-and long-term effect -.

The expected numbers were predicted with the statistical models.

Statistical analyses were carried out using Stata statistical software, release 10. [START_REF] Statacorp | Stata Statistical Software: Release 10[END_REF] Exposure-adjusted analyses The analysis of the absolute number of people injured assumes that exposure has remained stable throughout the study period. Since this assumption might be unrealistic, analyses were also performed using as denominators the monthly car, motorcycle and moped fleet and the monthly number of new car, motorcycle and moped registrations.

However, appropriate exposure denominators (i.e. kilometres travelled by vehicle) were not available (information is only available for non-urban roads). These denominators where included in the models as an offset.

Given that moped fleet was not available for the whole study period and that similar results were obtained using vehicle fleet and vehicle registrations, only registrationsadjusted results will be shown (Figure 1).

RESULTS

During the study period 1,046,900 people were injured in traffic collisions (annual median of 152,264), 66.8% of them being men, and 65.1% between 18 and 44 years of age (Table 1).

The type of road user varied with the age and sex of the person injured (Figure 2): subjects were mostly car users (54.2% in men, 67.1% in women), with the exception of those aged 14-15 and 16-17 years old, who were mostly moped users (66.0% and 41.2% in boys and girls from 14-15 years, respectively; 76.8% and 50.7% from 16-17 years). Also, the proportion of pedestrians was higher among individuals from 0-13 years (31.9% in boys, 24.2% in girls), decreasing with age, and increasing again from 45 years on, up to a 37.3% in men and 50.2% in women aged >74 years. The proportion of injured motorcycle or moped users was higher in men than in women, whereas the proportion of injured car users and pedestrians was higher in women (p<0.05).

Sex and severity

In men, a reduction in the risk of being injured was observed during the first month of the post-intervention period (mean change) (RR=0.91; 95%CI: 0.87-0.95). However, a 0.27% significant increase in the risk of being injured was observed regarding the time trend (Table 2).

Taking injury severity into account, results among slightly injured men followed a similar pattern compared to overall results. A statistically significant reduction in the risk of being injured in the post-intervention period was observed in the mean number for seriously injured men, and in the time trend for male fatalities (Table 2 and Figure 3).

Results for the type of road user showed a statistical significant reduction in the risk of being injured among male car users in both the mean number and the time trend.

Conversely, the time trend for motorcycle and moped users showed a significant risk increase. The number of injured men pedestrians stayed stable throughout the pre-and post-intervention periods (Table 2 and Figure 4).

In women, a reduction in the risk of being injured was observed during the first month of the post-intervention period (RR=0.89; 95%CI: 0.85-0.94), along with a nonsignificant 0.07% increase in the risk of being injured regarding the time trend. Results for injury severity and type of road user were similar to those observed in men (Table 2 and Figures 3 and4), although with some differences: the time trend for overall and slightly injured women remained stable throughout the pre-and post-intervention periods, and a significant increase was observed in the time trend for women pedestrians injured. On the whole, larger risk reductions were observed among women than among men.

Similar results were obtained when adjusting the approval of the new protocol for police data collection in the models.

Age

Since the distribution of type of road user varied between age groups (Figure 2), the age analyses were stratified by type of road user. There were no notable differences between age groups in any of the road user categories considered (data not shown).

Prevented number of people injured

Almost 25,000 people injured and more than 1,500 deaths were prevented during the post-intervention period attributable to road safety prioritisation, representing a 5.6% and 12.5% reduction with respect to the expected numbers, respectively (Table 3). Larger reductions were observed among fatalities and seriously injured people compared to slightly injured people, and among women compared to men. The number of people injured was greater than expected among motorcycle users, moped users, and women pedestrians.

Exposure adjusted results

When the number of new vehicle registrations was used as an exposure denominator, in both men and women motorcycle users, significant risk reductions were observed for both the mean number (RR=0.64 in men, RR=0.60 in women) and the time trend (1.21% and 1.05% monthly reductions, respectively). Similar results were observed for moped users (RR=0.68 and RR=0.71; 2.68% and 2.90% monthly reductions) and for car users (RR=0.61 and RR=0.60; 3.61% and 3.45% monthly reductions) (Table 4).

DISCUSSION

The present study suggest that overall road safety interventions implemented following road safety political prioritisation reduced road traffic people injured in Spain.

Effectiveness was shown across all injury severity categories, age groups and road users, except for pedestrians.

Although a large number of studies have assessed the effectiveness of single or combined interventions, to our knowledge, this is the first study to assess the effectiveness of prioritising road safety.

Effectiveness of road safety prioritisation

Sex and severity

Larger risk reductions were observed among women. This could be explained by their higher willingness for behaviour change. [START_REF] Jonah | Sensation seeking and risky driving: a review and synthesis of the literature[END_REF][START_REF] Laapotti | Differences in fatal loss-of-control accidents between young male and female drivers[END_REF] The severely injured or killed people showed larger risk reductions than the slightly injured, which could be partly explained because, among the implemented interventions, some aimed at reducing injury severity. These interventions are expected to reduce the proportion of collisions that result in fatal or serious injuries, thereby increasing the number of slightly injured people and, to a lesser extent, the number of seriously injured people. Also, it could be due to the new protocol for police data collection. However, the models adjusted and unadjusted by the new protocol showed limited differences, which could be explained by a lag-time effect with respect to the consequences of the protocol, or because the variable also includes the effect of other events such as the penalty points system.

Type of road user and exposure-adjusted results

Unadjusted results showed risk reductions among car users. However, large risk increases were observed regarding time trend among motorcycle and moped users, although they are probably due to exposure variations, since the exposure-adjusted models showed significant risk reductions among these road users. In fact, the series for injured motorcycle and moped users is very similar to that for motorcycle and moped registrations. Moreover, similar figures have been observed in Europe: motorcycle fatalities increased a 5.3% in 2006 (compared to 2000), and the proportion of injured moped users decreased substantially during 2000-2003, and later stabilized. [START_REF]Annual Statistical Reports[END_REF] Nonetheless, exposure-adjusted results should be interpreted with caution because this denominator -although the best available -does not reflect changes in road users' mobility, but only in the number of available vehicles. Finally, no effect was observed among pedestrians. This seems reasonable, since road safety interventions were mainly focused on vehicles (e.g. speed cameras, alcohol sobriety checkpoints) and on non-urban roads.

Short-and long-term effectiveness

Both short-and long-term effectiveness was observed. This reveals that there were effective measures implemented at the beginning of the year 2004 (short-term effect). In addition, other interventions implemented throughout the post-intervention period were also effective, progressively reducing the number of people injured (time trend) (longterm effect).

Limitations and strengths

The number of vehicle-kilometres could not be used. However, vehicle fleet and new vehicle registrations were used as proxies for exposure. Also, the increased effort made from the police department to improve the reporting of collisions probably affected the results.

No comparison group was available, since the evaluation was nationwide. Nonetheless, although it may add evidence to the results, it is not compulsory when using time series analysis, as percent change is only compared among time points in the same series.

Uncontrolled factors could be influencing the results. However, several socio-economic variables were accounted for as potential confounding factors. Only fuel consumption was statistically associated with the series of people injured and did not modify the results noticeably (data not shown).

The validity of the results are subject to data quality. Misclassification among mopeds and motorcycles has been observed previously in the police database, [START_REF] Pérez | Impact of relaxing licensing requirements for motorcycle driving on traffic injuries: a time series analysis[END_REF] and also with injury severity data: one third of seriously injured people are classified as being slightly injured. [START_REF] Pérez | Plasencia A on behalf of the work group of the Spanish Society of Epidemiology on the Measuring of the Impact on health of road traffic accidents in Spain. Motor vehicle crash fatalities at 30 days in Spain[END_REF] The small number of missing values regarding sex (3.4%) are not expected to have significantly affected the results. Moreover, this number was approximately 4% between 2000-2004, and dropped to 1.8% and 1% in 2005 and 2006, respectively, which goes against our hypothesis.

Among the strengths of the study, the design and the statistical analysis performed allowed to control for the main confounding factors that usually affect road safety evaluation studies, such as regression to the mean, and general trends in the number of crashes. [START_REF] Hauer | Observational Before-After Studies in Road Safety: Estimating the Effect of Highway and Traffic Engineering Measures on Road Safety[END_REF] Although other authors suggest using ARIMA models, [START_REF] Dupont | Multilevel modeling and time series analysis in traffic research -Methodology[END_REF] Poisson regression has been observed to yield similar estimates with a similar goodness of fit of the models. Moreover, their coefficients can be interpreted in terms of relative risks, which provide a straightforward interpretation of the effectiveness of an intervention. [START_REF] Tobias | Use of poisson regression and box-jenkins models to evaluate the short-term effects of environmental noise levels on daily emergency admissions in Madrid, Spain[END_REF][START_REF] Kuhn | Use of Poisson regression and time series analysis for detecting changes over time in rates of child injury following a prevention program[END_REF] In addition, the use of two different RRs (one for mean change and another for time trend), compared to the use of only one RR, allows to distinguish between the short-and long-term effects. Also, the long pre-and post-intervention periods available provide stability to the analysis. In addition, a large sample size was available, allowing for subgroup analyses. Finally, the hospital discharge registry was also analysed to improve the validity of the results among seriously injured people, [START_REF] Lyons | Using multiple datasets to understand trends in serious road traffic casualties[END_REF] yielding similar results to those obtained with the police database, adding consistency to the results: the risk of being admitted to hospital due to traffic collisions in the postintervention period was close to one regarding the mean number for both men (RR=0.98; 95%CI: 0.93-1.04) and women (RR=0.98; 95%CI: 0.91-1.05), and a statistically significant reduction was observed regarding the time trend (a 0.41% and 0.82% monthly reduction, respectively).

Conclusions

In conclusion, overall road safety interventions implemented following the inclusion of road safety on the political agenda reduced the number of traffic people injured, thus suggesting the effectiveness of road safety prioritisation. Further studies should assess the differences in the effectiveness between road types, geographical area, and type of measures implemented, which would help resolve which combinations of road safety strategies are the most effective in reducing traffic injuries.

WHAT THIS PAPER ADDS

What is already known on this subject -A strong political will is required to reduce road traffic injuries.

-However, to date, the overall effect of the actions implemented following the prioritisation of road safety has not been assessed.

What this study adds

-The present study suggests that the inclusion of road safety on the political agenda (and the subsequent implementation of several road safety interventions) reduces the number of people injured in road traffic collisions.

-In Spain, 24,629 people injured and 1642 deaths due to road traffic collisions were prevented three years after road safety prioritisation.

  05 a. Time trends in the pre [Pre(%)] and post-intervention [Post(%)] periods are expressed as the mean percentage increase/decrease in the number of people injured per month, being statistically significant trends indicated with an asterisk (*). Relative change is expressed as the mean percentage increase/decrease in the number of people injured per month in the post-intervention period compared with that in the pre-intervention period. Pre-intervention period: 1 st January 2000 -31 st December 2003; Post-intervention period: 1 st January 2004 -31 st December 2006.

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Distribution of type of road user involved in road traffic crashes in Spain by age and gender. Spain 2000-2006.

Table 1 . Distribution of road traffic injured people by age, injury severity and type of road user. Spain 2000-2006.

 1

		Police data a
	Men (n=665,788)	Women (n=345,312)
	%	Monthly median	%	Monthly median

Table 2 . Adjusted relative risks for people being injured in traffic collisions in the post-intervention period compared with the pre-intervention period, regarding mean change and time trend change a between the pre-and post-intervention period, according to injury severity, age and type of road user. Spain 2000-2006.

 2

	MEN

RR: relative risk; 95% CI: 95% Confidence Intervals; Pre: pre-intervention period; Post: post-intervention period. *p<0.05 a. Time trends in the pre [Pre(%)] and post-intervention [Post(%)] periods are expressed as the mean percentage increase/decrease in the number of people injured per month, being statistically significant trends indicated with an asterisk (*). Relative change is expressed as the mean percentage increase/decrease in the number of people injured per month in the post-intervention period compared with that in the pre-intervention period. Pre-intervention period: 1 st January 2000 -31 st December 2003; Post-intervention period: 1 st January 2004 -31 st December 2006.

Table 3 . Prevented a numbers of men and women injured in road traffic collisions in the post-intervention period. Spain 2000-2006.

 3 Negative numbers indicate an excess of people injured in the post-intervention period compared to the expected, according to the numbers observed in the pre-intervention period. b. Calculated as the number of prevented people injured over the expected number of people injured in the post-intervention period.

		MEN		WOMEN
		n	% b	n	% b
	Overall	9,850	3.5	14,779*	9.3
	Injury severity
	Slight	3,311	1.5	10,136*	7.5
	Serious	5,414*	10.2	4,299*	19.2
	Fatal (24 hours)	1,167*	11.5	475*	16.1
	Type of vehicle
	Car	28,405*	16.9	19,230*	16.7
	Motorcycle	-9,617*	-39.3	-1,445*	-31.8
	Moped	-4,847*	-12.0	-1,521*	-10.8
	Pedestrian	1	0.0	-921	-5.9
	*p<0.05
	a.

Table 4 . Adjusted relative risks for people being injured in traffic collisions in the post-intervention period compared with the pre-intervention period, regarding mean change and time trend change a between the pre-and post-intervention period, according to type of vehicle. Comparison of the results obtained from the original models with those from models which include the number of new vehicle registrations as an exposure denominator. Spain 2000-2006.

 4

	MEN

RR: relative risk; 95% CI: 95% Confidence Intervals; Pre: pre-intervention period; Post: post-intervention period. *p<0.

FUNDING

This work was supported by the Agencia Española de Tecnologías Sanitarias (Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+I) e Instituto de Salud Carlos III-Subdirección General de Evaluación y Fomento de la Investigación) [PI07/90157]. The funding source has not had any involvement in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

NOTE

This paper will be included in the thesis of one of the authors (Ana M. Novoa), performed at the Pompeu Fabra University (UPF).

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees, to permit this article (if accepted) to be published in JECH and any other BMJPG products to exploit all subsidiary rights, as set out in our licence (http://jech.bmj.com/ifora/licence.pdf).

CONFLICTS OF INTEREST

All of the authors declare that they have no conflicts of interest.