Susana Novella

Ana Paula Dantas

Gloria Segarra

Laura Novensà

Carlos Bueno

Magda Heras

Carlos Hermenegildo

Dr Pascual Medina
email: pascual.medina@uv.es

GATHERING OF AGING AND ESTROGEN WITHDRAWAL IN VASCULAR DYSFUNCTION OF SENESCENT ACCELERATED MICE

Keywords: senescence-accelerated mice, aging, menopause, vascular reactivity, estrogen

A C C E P T E D

M A N U S C R I P T

ABSTRACT

The aim of this work was to characterize a mouse model of experimental menopause and cardiovascular aging that closely reflects menopause in women. Senescence accelerated mouse (SAM)-Resistant type 1 (SAMR1, n=30) and SAM-Prone type 8 (SAMP8, n=30) were separated at 5 months of age into three groups: 1) sham-operated (Sham); 2) ovariectomized (Ovx); and 3) ovariectomized chronically-treated with estrogen (Ovx+E2). Contractile responses to KCl (60 mM) and thromboxane A 2 were greater in aorta from SAMP8 mice compared with SAMR1 in all groups. Neither ovariectomy nor estrogen replacement modified the contractile responses from SAMR1 mice. Conversely, in Ovx SAMP8 increased the maximal contractions that were reversed by estrogen treatment. Rings with endothelium from all SAMR1 groups showed a greater relaxation to acetylcholine than SAMP8 groups. In SAMR1, endothelium-dependent relaxation was not altered in Ovx or Ovx+E2 groups. Rings from Ovx SAMP8 showed a decreased maximal response to acetylcholine compared to Sham SAMP8. Estrogen replacement restored the response to acetylcholine altered by ovariectomy. Nitric oxide inhibition by L-NAME marked reduced acetylcholine responses in all groups, but this effect was more pronounced in SAMP8 and Ovx groups (determined by area under the curve reduction). These results indicate that SAMP8 exhibit a significant decreased endothelium-dependent and NO-mediated relaxation and increased vasoconstrictor responses that are potentiated by the lack of estrogen. Because these responses are closely in agreement with vascular dysfunction observed in menopausal women, we propose SAMP8 Ovx as a new model to concomitantly study the effects of aging and menopause in female mice.

INTRODUCTION

Aging is widely recognized as a risk factor for vascular disease and involves structural and functional alterations in vasculature. Vascular aging is associated with endothelial dysfunction, arterial stiffening and remodeling, impaired angiogenesis, defective vascular repair, and with an increasing prevalence of atherosclerosis [START_REF] Erusalimsky | Vascular endothelial senescence: from mechanisms to pathophysiology[END_REF][START_REF] Lakatta | Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part I: aging arteries: a "set up" for vascular disease[END_REF].

In woman, besides aging a decline of sex hormones production by menopause has been associated to increased risk of cardiovascular disease. Estrogen has been largely described as cardioprotective in both experimental and observational studies [START_REF] Cano | The risk for cardiovascular disease in women: from estrogens to selective estrogen receptor modulators[END_REF][START_REF] Dantas | Does 2-methoxyestradiol represent the new and improved hormone replacement therapy for atherosclerosis?[END_REF]. Cardiovascular protection by estrogen has been mostly associated to an increase of endothelium-derived vasodilator factors, including nitric oxide (NO) [START_REF] Mendelsohn | Estrogen actions in the cardiovascular system[END_REF] and prostacyclin [START_REF] Sobrino | Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-α[END_REF] .

V asoconstrictor prostanoids, such as thromboxane A 2 (TXA2), have also been implicated in the phatophysiology of vascular dysfunction during estrogen withdrawn [START_REF] Dantas | Influence of female sex hormones on endothelium-derived vasoconstrictor prostanoid generation in microvessels of spontaneously hypertensive rats[END_REF][START_REF] Dantas | Relative contribution of estrogen withdrawal and gonadotropins increase secondary to ovariectomy on prostaglandin generation in mesenteric microvessels[END_REF] and aging [START_REF] Briones | Ageing affects nitric oxide synthase, cyclooxygenase and oxidative stress enzymes expression differently in mesenteric resistance arteries[END_REF][START_REF] Vanhoutte | COX-1 and vascular disease[END_REF] and play an important role in the regulation of vascular tone in the normal systemic female vasculature [START_REF] Fulton | Sexual dimorphism in prostanoid-potentiated vascular contraction: roles of endothelium and ovarian steroids[END_REF] , which involves the TXA2 receptor (TXA2R) [START_REF] Li | Estrogen potentiates constrictor prostanoid function in female rat aorta by upregulation of cyclooxygenase-2 and thromboxane pathway expression[END_REF] .

Senescence accelerated mice (SAM) is a murine model that has been widely used to study age-associated diseases. This model consists on a family of inbred strains with a common genetic background, originally derived from a colony of AKR/J mice, created as a result of selective breeding of mice showing a phenotype of severe exhaustion (SAM-Prone) and inbreeding of a normal phenotype (SAM-Resistant) [START_REF] Takeda | A new murine model of accelerated senescence[END_REF]. Most of the clinical features exhibited by SAMP mice correspond to pathophysiological states found in aging humans. Vascular studies using these models

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are not abundant, but few studies in male mice have shown morphological alterations, mechanical and endothelial dysfunction [START_REF] Llorens | The senescence-accelerated mouse (SAM-P8) as a model for the study of vascular functional alterations during aging[END_REF].

In general, the animal models used to study the cardiovascular effects of estrogen withdrawn are often not associated to aging. In these studies, ovariectomy -the most used surgical model to induce menopause in mice -are performed at early age and may not reflects the physiological state of menopause in women. A C C E P T E D M A N U S C R I P T

MATERAL AND METHODS

Experimental animals

Surgical Procedures and Hormonal Treatment

Ovariectomy was performed under isoflurane anesthesia. Briefly, a single 1-cm dorsal midline incision was made in the skin and the ovaries were removed. At the moment of the surgery, half of the Ovx mice received estrogen replacement by subcutaneous implant of an osmotic pump (Model 2004, Alzet Osmotic Pumps®; Durect Corporation, Cupertino, CA, USA) containing 17β-estradiol in 50% DMSO at a dose of 10 µg/Kg/day (Ovx + E2). Untreated Ovx was implanted with a pump filled with vehicle only (50% DMSO in saline). Sham female mice were also included. To control the efficacy of ovariectomy and estrogen treatments, uterine weight was evaluated. Four weeks after surgical procedures, body weight was measured, and mice were euthanized.

Isolated mouse aorta preparation

A C C E P T E D M A N U S C R I P T diameter by repeatedly challenging them to 60 mM KCl. Basal tension was increased gradually until contractions reaches its maximal level (data not shown). Once the optimal resting tension was reached (1 g), aortic rings were allowed to attain a steady level of tension during a 2-hour equilibration period before testing. Functional integrity of the endothelium was confirmed routinely by the presence of relaxation induced by acetylcholine (10 -6 M) during contraction obtained with serotonin (10 -5 M).

Following the equilibration period, arterial segments were exposed to receptorindependent depolarizing agent KCl (60 mM) until the contraction reached a stable plateau (10 to 20 min). After washout and return to stable baseline, contractile responses were determined by cumulative concentration-response curves to the TXA2 mimetic U46619 (10 -9 to 3x10 -7 M). To test the endothelium-dependent responses, aortic rings were precontracted with serotonin (10 -5 M) and cumulative relaxation curves to acetylcholine (10 -9 to 3x10 -6 M) were obtained in each ring. The responses to

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acetylcholine were carried out in rings with and without endothelium. In another series of experiments, the preparations with endothelium were preincubated with L-NAME (10 -4 M) 15 min prior to the concentration-responses curves to acetylcholine.

Immunofluorescence

Thoracic aorta from all groups of mice were excised and mounted in an optimum cutting temperature Tissue-Tek ® OCT compound, mould, snap frozen in dry ice, and stored at -80°C. Arteries were sectioned (4 µm) and thaw mounted onto polylysine covered slides. Sections were fixed in acetone (15 min) and blocked for 30 min with horse serum. Sections were then incubated overnight at 4°C with 1:200 anti-TXA2R rabbit polyclonal antibody (sc-30036, Santa Cruz Biotech). The following day, the tissues were washed with PBS (3 x 5 min) and costained with phalloidin (10µM) and 1:500 Alexa Fluor 488-conjugated goat anti-rabbit (Invitrogen) at room temperature.

The tissues were then washed with PBS (3 x 5 min) and coverslips were mounted on slides using ProLong Gold antifade reagent with DAPI (Invitrogen). Aortic sections were visualized through a fluorescent microscope (Olympus SX-31) with a 40X

objective lens (Olympus) by using a digital color charge-coupled device (CCD) camera (Olympus). To elicit fluorescent images, tissue-mounted slides were illuminated with a 200-W mercury lamp. For each image, the light was passed through a different excitation filter: 1) 350nm (for DAPI); 2) 490 nm (for Alexa 488); and 3) 590 nm (for phalloidin). Each aorta was photographed in 3 different regions and results were expressed as an average of fluorescence elicited (using Mac Biophotonic ImageJ Software).

Drugs

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The following drugs were used: serotonin hydrochloride, acetylcholine chloride, N

Morphometry

Body weight of SAMP8 mice was significantly lower than that of SAMR1 (Figure 1A).

In SAMR1, ovariectomy significantly increased body weight (P<0.05), an effect that was attenuated by estrogen treatment. On the other hand, neither ovariectomy or estrogen replacement affected body weight in SAMP8. Changes on uterine weight by ovariectomy and estrogen treatment were similar in both strains (Figure 1B). As expected, uterine weight decreased with ovariectomy (P<0.05), assuring the efficacy of the surgical process. Estrogen treatment prevented the loss in uterine mass by ovariectomy to similar levels as seen in Sham females. These data demonstrate the effectiveness of estrogen dose used to treat Ovx mice.

Effects of ovariectomy and estrogen treatment on contractile responses

Contractile responses to KCl (60 mM) were greater in aorta from SAMP8 mice compared with SAMR1 in all groups studied (Figure 2A). Neither ovariectomy nor estrogen treatment modified the contractile responses to KCl in aortic rings from SAMR1 mice. Conversely, in SAMP8, ovariectomy significantly increased the maximal contraction to KCl (Sham: 669 ± 25 vs Ovx: 831 ± 44 mg; P<0.05), an effect that was completely reversed by estrogen treatment (Figure 2A). Similarly, contractile responses to the TXA2 analogue U46619 were marked increased in all groups of SAMP8 in comparison to SAMR1 (Figure 2B and Table 1). In Sham SAMR1 mice, U46619 caused concentration-dependent contractions with a pD 2 of 7.92 ± 0.08 and maximal contraction of 1182 ± 67 mg (Figure 2B and Table 1). In SAMR1, neither maximal contraction nor pD 2 values to U46619 were affected by ovariectomy and estrogen treatment (Figure 2B and Table 1). In aortic rings of SAMP8 mice, maximal contraction

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to U46619 was marked increased in Ovx group compared to Sham and Ovx + E2 groups (Figure 2B and Table 1).

Effects of ovariectomy and estrogen treatment on endothelium-dependent and nitric oxide (NO)-mediated relaxation.

In aortic rings with endothelium, all SAMR1 groups showed a greater relaxation to acetylcholine than SAMP8 groups (Figure 3A and Table 2). Despite that most groups have shown similar maximal relaxation, the sensitivity to acetylcholine (evidenced by pD 2 values) was marked increased in aortic rings from SAMR1 (Table 2). In SAMR1, endothelium-dependent relaxation was not altered by ovariectomy or estrogen treatment. On the other hand, aortic rings from Ovx SAMP8 showed a significantly decrease on maximal response to acetylcholine compared with those from shamoperated SAMP8 (Figure 3A and Table 2). Estrogen replacement restored the vasodilator response to acetylcholine in Ovx SAMP8 (Figure 3A and Table 2). The removal of endothelium and nitric oxide production blunted acetylcholine-induced relaxation in all groups studies (Table 2). However, when the areas under the curve were analyzed, there was a greater attenuation of the NO-mediated relaxation in all groups of SAMR1 mice in comparison to the respective SAMP8 groups (Figure 3B), suggesting a more pronounced NO production by young females. Ovariectomy aggravates the responses observed in aged animals, an effect that is improved by estrogen treatment. Interestingly, ovariectomy also induced an attenuation in NOmediated responses in SAMR1, but to a much lower extent than the responses observed in SAMP8 (Figure 3B).

Effects of ovariectomy and estrogen treatment on aortic expression of thromboxane A 2 receptors (TXA2R).

Changes in the expression of TXA2R were determined in aortic rings by immunofluorescence analysis. Figure 4A shows representative immunofluorescent images of thoracic aorta from Sham SAMR1 (top) and Sham SAMP8 (bottom). Images represent staining of nucleus (blue, DAPI), actin fibers (red, phalloidin), TXA2R

(green), and merged images. No significant differences were observed on phalloidininduced fluorescence. As illustrated on Figure 4B, TXA2R expression is marked increased in aortic rings from SAMP8 in comparison to respective SAMR1 groups, although no significant differences were observed on Ovx+E2 groups. Ovariectomy increases TXA2R fluorescence in SAMP8, but not in SAMR1 mice. Despite a trend of increase observed in aortas from Ovx SAMR1, there was no statistical significance (Figure 4B). Estrogen treatment completely abolished ovariectomy-induced TXA2R upregulation in SAMP8 and induced a slightly (yet not significant) decrease in SAMR1.

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DISCUSSION

In these studies we provide a suitable murine model to simultaneously determine the effects of aging and estrogen withdrawn on vascular function, conditions that closely mimic menopause in women. As we have demonstrated, aging is associated with a decline in endothelium-dependent relaxation and an increase of vasoconstrictor responses in aorta of female senescent mice. Moreover, our findings support an important role for estrogen to regulate vascular function in aged, but not in young, blood vessels, suggesting that the protective effects of estrogen can be more noticeable in certain pathophysiological conditions in which some level of vascular damage has already occurred. This must be the case with aging.

Aging, per se, is known to cause a series of alterations in the endogenous mechanisms that control cardiovascular function leading to subsequent increase of risk of cardiovascular disease [START_REF] Ferrari | Invited review: aging and the cardiovascular system[END_REF][START_REF] Lakatta | Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part III: cellular and molecular clues to heart and arterial aging[END_REF][START_REF] Lakatta | Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: Part I: aging arteries: a "set up" for vascular disease[END_REF]. In women, aging includes an aggravating risk factor in comparison to men. The decrease in estrogen production by menopause is thought to contribute to increased cardiovascular risk [START_REF] Kannel | The Framingham Study: historical insight on the impact of cardiovascular risk factors in men versus women[END_REF]. For this reason it is particularly difficult to distinguish what would be the contribution of aging and the lack of estrogen in the control of vascular function in menopausal women.

Although reproductive senescence and loss of ovarian function is a common phenomena among mammals, there are relevant differences between species [START_REF] Wu | Ovarian aging and menopause: current theories, hypotheses, and research models[END_REF].

Contrary to women, the beginning of ovarian decline in rodents is characterized by periods of constant estrous, elevated levels of estrogen and lack of ovulation [START_REF] Felicio | Longitudinal studies of estrous cyclicity in aging C57BL/6J mice: II. Cessation of cyclicity and the duration of persistent vaginal cornification[END_REF][START_REF] Lu | Chronological changes in sex steroid, gonadotropin and prolactin secretions in aging female rats displaying different reproductive states[END_REF]. Only later in life, old rodents will experience low levels of estrogen and progesterone and little or no remaining ovarian follicle [START_REF] Felicio | Longitudinal studies of estrous cyclicity in aging C57BL/6J mice: II. Cessation of cyclicity and the duration of persistent vaginal cornification[END_REF][START_REF] Lu | Chronological changes in sex steroid, gonadotropin and prolactin secretions in aging female rats displaying different reproductive states[END_REF]. In this regard, the cardiovascular effects of estrogen in

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experimental studies have been determined mostly in young ovariectomized females, while the increase of cardiovascular risk during estrogen withdrawn is observed mostly in middle aged women.

Unfortunately, little information is available on whether the vascular effects of estrogen are modified with aging in females. Few recent studies have addressed this issue using rodent models at middle and advanced age. In female rodents that normally do not exhibit cardiovascular complications, aging-associated changes in vascular function are not evident in middle aged animals, becoming more apparent only when animals are senile [START_REF] Leblanc | Estrogen replacement restores flow-induced vasodilation in coronary arterioles of aged and ovariectomized rats[END_REF][START_REF] Lekontseva | Ovariectomy in aged versus young rats augments matrix metalloproteinase-mediated vasoconstriction in mesenteric arteries[END_REF][START_REF] Moien-Afshari | Long-term effects of ovariectomy and estrogen replacement treatment on endothelial function in mature rats[END_REF].

Others, conversely, have described significant effects of aging on vascular function in middle aged female rats with risk for cardiovascular disease [START_REF] Fortepiani | Characterization of an animal model of postmenopausal hypertension in spontaneously hypertensive rats[END_REF][START_REF] Wynne | Age-related reduction in estrogen receptor-mediated mechanisms of vascular relaxation in female spontaneously hypertensive rats[END_REF]. However, the fact that those animal models normally display increased risk for cardiovascular disease at early stage, when they are young adults, they may not represent a good model to determine aging-and menopause-associated vascular modifications in women.

In our studies we have used female senescent-accelerated mouse (SAM) model to determine the progressive effects of aging into the female vasculature. SAM is a murine model that has been increasingly used to study aging-associated diseases, as it ages fast and predictably, allowing experimental work to be performed in a convenient and standard time course [START_REF] Llorens | The senescence-accelerated mouse (SAM-P8) as a model for the study of vascular functional alterations during aging[END_REF]. Moreover, the absence of vascular damage at early age [START_REF] Butterfield | The senescence-accelerated prone mouse (SAMP8): a model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in Alzheimer's disease[END_REF] establishes SAM as an optimal model to study vascular aging. Functional studies on vascular function system and the role of NO, the key component of cardiovascular regulation, have been recently performed in male SAM [START_REF] Llorens | The senescence-accelerated mouse (SAM-P8) as a model for the study of vascular functional alterations during aging[END_REF]. Our studies provide, for first time, a description of changes in vascular reactivity in female, using SAM as a model of aging.

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Like other rodents, SAM mice do not reach reproductive senescence until later in life [START_REF] Yuan | Age-related changes in the oestrous cycle and reproductive hormones in senescence-accelerated mouse[END_REF]. According to uterine weight data in Sham animals, we can suggest that at 6 months SAMR1 and SAMP8 exhibit similar hormonal status. Besides, vaginal smear performed for one week prior sacrifice have revealed no significant modification on estrous cycle on both strains (data not shown), confirming this hypothesis. For this reason, the SAM model still could not be considered, as such, as a good model to study natural menopause, although it is a good model to study progressive aging. In this regard, we have performed a surgical menopause on SAM mice by the time they start to be at middle age and to present impairment of vascular function (5-6 months).

Menopause in women has been largely associated with decreased endotheliumdependent and NO-mediated vasodilation [START_REF] Taddei | Menopause is associated with endothelial dysfunction in women[END_REF][START_REF] Teede | Sex hormones and the cardiovascular system: effects on arterial function in women[END_REF][START_REF] Virdis | Mechanisms responsible for endothelial dysfunction associated with acute estrogen deprivation in normotensive women[END_REF]. Because both aging [START_REF] Erusalimsky | Vascular endothelial senescence: from mechanisms to pathophysiology[END_REF] and changes on hormonal status during menopause [START_REF] Miller | Vascular actions of estrogens: functional implications[END_REF] has been described to modulate both NO production and endothelial function, it remains unclear the specific contribution of each factor to endothelial dysfunction during postmenopause. Our data show that aging per se induces a slightly decrease on the sensitivity to acetylcholine, an effect that is potentiated by the removal of ovaries. NO production was also decreased by aging, as evidenced by the degree in reduction in the area under the curve following L-NAME treatment. The fact that those effects were not as evident on SAMR1 Ovx, suggests an overlap between aging and hormonal status to control endothelial function.

Besides NO, vasoconstrictor prostaglandins have also been implicated in the phatophysiology of vascular dysfunction during aging [START_REF] Briones | Ageing affects nitric oxide synthase, cyclooxygenase and oxidative stress enzymes expression differently in mesenteric resistance arteries[END_REF][START_REF] Vanhoutte | COX-1 and vascular disease[END_REF] and estrogen withdrawn [START_REF] Dantas | Influence of female sex hormones on endothelium-derived vasoconstrictor prostanoid generation in microvessels of spontaneously hypertensive rats[END_REF][START_REF] Dantas | Relative contribution of estrogen withdrawal and gonadotropins increase secondary to ovariectomy on prostaglandin generation in mesenteric microvessels[END_REF]. In our studies we have observed a significant increase on vasoconstriction induced by the TXA2 analog U-46619 in SAMP8 that was aggravated by ovariectomy. In a series of Estrogen has shown to have protective effects by modulating NO production and prostaglandin-mediated contraction in several experimental and clinical studies -for review see [START_REF] Tostes | Effects of estrogen on the vascular system[END_REF]. In contrast, recent clinical trials have questioned the value of estrogen replacement therapy (ERT) in protecting against vascular disease [START_REF] Rossouw | Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial[END_REF]. The surprising results of those trials have been in part explained by the fact that those studies were primarily carried out in aged women, who were on average, 10 years past the onset of menopause, and therefore may not benefit from ERT anymore. The so-called "Timing Hypothesis" suggests that estrogen-mediated benefits to the cardiovascular system may occur only before the detrimental effects of aging are established in the vasculature [START_REF] Harman | Estrogen replacement in menopausal women: recent and current prospective studies, the WHI and the KEEPS[END_REF]. In order to address this important issue we have further treated our menopause model (SAM-Ovx) with estrogen for 4 weeks. In those studies, we have found that estrogen replacement restored both vasodilator and vasoconstrictor responses altered by ovariectomy, but did not improve aging-associated responses. Our data are not in agreement with few studies describing that aging

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negatively affects estrogen-mediated responses in distinct rodent models [START_REF] Berezan | Ovariectomy, but not estrogen deficiency, increases CYP4A modulation of alpha(1)-adrenergic vasoconstriction in aging female rats[END_REF][START_REF] Fortepiani | Characterization of an animal model of postmenopausal hypertension in spontaneously hypertensive rats[END_REF][START_REF] Wynne | Age-related reduction in estrogen receptor-mediated mechanisms of vascular relaxation in female spontaneously hypertensive rats[END_REF]. These discrepancies may be explained by the fact that those animals were studied at a more advanced age or in the presence of an established cardiovascular disease, while we have determined the effects of estrogens on middle aged female that have not reached a high level of vascular damage. With our data so far, we can not determine if estrogen-mediated responses could be affected by a more advanced age in SAMP8 animals. This hypothesis is of great interest in the field and must be addressed in further studies.

Conclusions

In summary, our studies have established a role of aging and estrogen withdrawn to sham-operated groups, and § P < 0.05 vs. Ovx groups.

  It is still unclear to what extent the protective effects of estrogen replacement described in young females can be extrapolated to older ones. Because SAM model offers several advantages to study cardiovascular aging in a convenient and standard time course, we have use female SAM to characterize a model of experimental menopause that closely reflects menopause in women. The induction of surgical menopause in an animal model of senescence will allow us to determine how aging affects vascular function and estrogenmediated effects on nitric oxide and TXA2 responses in females. In this regard, changes in aortic responses from female SAMR (type 1 -SAMR1) and SAMP (type 8 -SAMP8) were determined in an attempt to assess the adequacy of these strains as experimental model to study vascular changes during aging and menopause.

2. 6

 6 Data analysis Data are expressed as means ± SEM. Contractions to KCl and U46619 are shown as absolute tension (mg). Relaxation is expressed as the percentage to the precontraction in response to serotonin (10 -5 M). pD 2 (negative logarithm of the molar concentration at which half-maximum contraction occurs) was determined from individual concentration-response curves by non-linear regression analysis. Area under the concentration-response curve (AUC) was calculated from each individual concentration-response curve to acetylcholine and was expressed as arbitrary units. The contribution of NO to the vascular relaxation induced by acetylcholine was calculated by subtracting from the AUC for acetylcholine, the AUC for acetylcholine in the presence of L-NAME. In each experimental group n indicates the number of animals. Differences between mouse strains (i.e SAMR1 vs. SAMP8) and by experimental treatment groups (i.e, Sham vs. Ovx vs. Ovx + E2) were analyzed by two-way ANOVA, and then Bonferroni's test was performed. Statistical significance was accepted at P < A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

  to determine the expression of TXA2R, we could observe pattern of modulation of these receptors by both aging and ovariectomy in SAM aorta that corroborates the data on vascular reactivity. However, the fact that contractions to KCl showed similar pattern of responses indicate that intrinsic mechanism, that are not dependent on TXA2R up-regulation, also contribute to the altered vasoconstrictor responses observed herein. KCl-induced constriction is mediated primarily via depolarization-induced opening of voltage-gated Ca 2+ channels and influx of extracellular Ca 2+ . Accordingly, the ability of accelerated senescence and ovariectomy in SAMP8 mice to increase constrictor responses to KCl is suggestive of an action on voltage-gated Ca 2+ channels or distal mechanisms that respond to this influx of Ca 2+ .

Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. Contractile responses in aortic rings from sham-operated (Sham), ovarectomized (Ovx) and ovarectomized treated with estradiol (Ovx + E2) SAMR1 and SAMP8 mice. (A) represents maximal contraction to KCl 60mM and (B) concentrationresponse curves to U-46619. Bars graphs (A) indicate mean ± SEM of maximal contraction to KCl (n = 10 animals per group); each data point (B) show mean ± SEM from n=10 mice for each group. * P < 0.05 vs. same treatment of SAMR1 mice, and # P < 0.05 vs. sham-operated and Ovx + E2 SAMP8 groups.

Figure 3 .Figure 4 .

 34 Figure 3. Concentration-response curves to acetylcholine (A) and NO-dependent relaxation to acetylcholine (B) in aortic rings from sham-operated, Ovx, and Ovx + E2 groups from SAMR1 and SAMP8 mice. Each data point (A) show mean ± SEM from n=10 mice for each group. Bars graphs (B) indicate mean ± SEM of differences between area under curve (AUC) for acetylcholine-induced relaxation in the absence and AUC in the presence of L-NAME 10 -4 M (n = 10 animals per group). * P < 0.05 vs.same treatment of SAMR1 mice, and # P < 0.05 vs sham-operated groups.

Table 1 .

 1 pD 2 values and maximal contractions (Emax) to U46619 in aortic rings from sham-operated (Sham), ovariectomized (Ovx) and ovariectomized plus estradiol (Ovx Values are means ± SEM. n number of mice. * p < 0.05 vs. SAMR1 group with same treatment, and # p < 0.05 vs. Sham and Ovx + E2 groups of SAM-P8 mice.

	+E2) groups
		SAMR1	SAMP8
		pD 2	Emax (mg)	pD 2	Emax (mg)
	Sham (n=10)	7.92 ± 0.08	1182 ± 67	8.16 ± 0.08*	1626 ± 69*
	Ovx (n=10)	7.86 ± 0.04	1213 ± 76	8.13 ± 0.02*	1953 ± 46*#
	Ovx+E2 (n=10)	7.89 ± 0.05	1176 ± 81	8.20 ± 0.05*	1548 ± 44*

Table 2 .

 2 pD 2 values and maximal responses (Emax) to acetylcholine in aortic rings with endothelium, without endothelium and with endothelium pretreated with L-NAME (10 -4 M) from sham-operated (Sham), ovariectomized (Ovx) and ovariectomized plus estradiol (Ovx+E2) groups.Values are means ± SEM. n number of mice. Relaxation was expressed as a percentage of the serotonin-induced contraction. † pD 2 value was not calculated as the maximum relaxation to acetylcholine was less than 10%. * p < 0.05 vs. SAMR1 group with same treatment, and # p < 0.05 vs. Sham and Ovx + E2 groups of SAMP8 mice.

		SAMR1	SAMP8
		pD 2	Emax (%)	pD 2	Emax (%)
	Sham
	With endothelium (n=10)	8.3 ± 0.1	95 ± 3	7.8 ± 0.3*	94 ± 3
	Without endothelium (n=6)	†	4 ± 1	†	3 ± 1
	With L-NAME (10 -4 M) (n=6)	†	2 ± 1	†	3 ± 1
	Ovx
	With endothelium (n=10)	8.1 ± 0.1	96 ± 2	7.2 ± 0.2*#	84 ± 4*#
	Without endothelium (n=6)	†	6 ± 2	†	5 ± 1
	With L-NAME (10 -4 M) (n=6)	†	4 ± 1	†	3 ± 1
	Ovx + E2
	With endothelium (n=10)	8.1 ± 0.1	97 ± 2	7.7 ± 0.1*	96 ± 2
	Without endothelium (n=6)	†	7 ± 1	†	6 ± 1
	With L-NAME (10 -4 M) (n=6)	†	5 ± 1	†	5 ± 1

0.05. The statistical analysis was carried out using the Prism 4 software (GraphPad Software Inc., San Diego, CA, USA).

ACKNOWLEDGMENTS

We are indebted to Drs. Inmaculada Noguera and Ana Diaz for their excellent veterinary assistance, and to Nadia Castillo for her exceptional technical assistance.

GRANTS

Supported by the Spanish Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III (grants FIS 06/0589, FIS PI080176 and Red HERACLES RD06/0009/0005 and RD06/0009/0008), Consellería de Sanidad (grants GE-021/10, AP-131/10, AP-117/10).

Research Highlights


Keywords: Child abuse, Psychological and physical abuse, Developing countries, Transitional countries child abuse, psychological and physical abuse, developing countries, transitional countries

Background

Child abuse is a worldwide recognized public health and social problem. Using data from the Multiple Indicator Cluster Surveys (MICS) we aimed to a) compare different forms of child abuse across countries and regions, and b) examine factors associated with different forms of child abuse.

Methods

Information on child abuse was available in 28 developing and transitional countries from the third round of MICS conducted in 2005 and 2006 (n=124 916 children aged between 2 and 14 years). We obtained prevalence of psychological, moderate and severe physical abuse for the preceding month and examined correlates of different forms of child abuse with multilevel logistic regression analysis.

Results

A median 83%, 64% and 43% of children in the African region experienced psychological, moderate and severe physical abuse, respectively. A considerably lower percentage of children in transitional countries experienced these forms of abuse (56%, 46% and 9%, respectively). Parental attitudes towards corporal punishment were the strongest variable associated with all forms of child abuse. The risk of all forms of child abuse was also higher for male children, those living with many household members and in poorer families.

Conclusions

Child abuse was a very common phenomenon in many examined countries. We found substantial variations in prevalence across countries and regions, with the highest prevalence in African countries.

Population-based interventions (e.g. educational programs) should be undertaken to increase public awareness of this problem. Actions on changing parental attitudes towards corporal punishment of children may help to prevent child abuse. The specific local situation in each country should be considered when selecting intervention strategies.

Introduction

Child abuse undertaken by their parents or other guardians is a recognized public health and social problem both in developed and developing countries. According to the WHO, child abuse includes all forms of physical and/or emotional ill-treatment, sexual abuse, neglect and negligent treatment and exploitation. [START_REF]Preventing Child Maltreatment -a guide to taking action and generating evidence[END_REF] In general, one distinguishes four forms of child abuse: emotional or psychological abuse, physical abuse, sexual abuse and neglect. Previous research has mainly focused on issues like prevalence [START_REF] Wong | To unfold a hidden epidemic: prevalence of child maltreatment and its health implications among high school students in Guangzhou, China[END_REF] , determinants [START_REF] Fuhua | Child maltreatment among Asian Americans: characteristics and explanatory framework[END_REF] , and health [START_REF] Bordin | Severe physical punishment: risk of mental health problems for poor urban children in Brazil[END_REF] and social consequences [START_REF] Gershoff | Corporal punishment by parents and associated child behaviors and experiences: a meta-analytic and theoretical review[END_REF] of the child abuse in separate countries or small regions. To our knowledge, comparison of child abuse across different countries and regions have not been done till now. In particular, little is known about these issues in the former Soviet countries and other post communist countries, which experienced economic, social and political transition after the breakdown of the Soviet system at the beginning of the 1990s. In a time of transition, it is important to examine the extent to which children experience different forms of child abuse through their parents or guardians in these countries. It is also important to know which factors contribute to a higher risk of child abuse in order to undertake adequate actions towards reducing child abuse.

The WHO estimates that about 31 000 children under 15 years of age died due to homicide worldwide in 2002. [START_REF]Preventing Child Maltreatment -a guide to taking action and generating evidence[END_REF] A recently published paper reported the yearly prevalence of child abuse in some developed countries, where the prevalence of severe physical abuse varied from 4% to 16%. [START_REF] Gilbert | Burden and consequences of child maltreatment in high-income countries[END_REF] Population-based studies from developing countries show much higher prevalence rates of physical abuse. [START_REF] Catani | Beyond individual war trauma: domestic violence against children in Afghanistan and Sri Lanka[END_REF][START_REF] Leung | Prevalence and determinants of child maltreatment among high school students in Southern China: A large scale school based survey[END_REF] Little attention has been devoted to psychological abuse as compared to physical abuse, although it is known that this type of abuse may have serious health (e.g. mental disorders) and social (e.g., alcohol and drug abuse, poor school performance) consequences. [START_REF] Trickett | Emotional abuse in a sample of multiply maltreated, urban young adolescents: issues of definition and identification[END_REF] The prevalence of psychological abuse varied substantially across countries (about 10% in some developed countries and 12-33% in Eastern European countries). [START_REF] Gilbert | Burden and consequences of child maltreatment in high-income countries[END_REF][START_REF] Sebre | Cross-cultural comparisons of child-reported emotional and physical abuse: rates, risk factors and psychosocial symptoms[END_REF] Among known individual risk factors of child abuse are the child's sex and age, socio-economic status of the family, the parents' education level, mental health and behavior (e.g. alcohol drinking). Parental attitudes towards corporal punishment as a method of child rearing were found to be associated with child abuse. [START_REF] Gagne | Predictors of adult attitudes toward corporal punishment of children[END_REF][START_REF] Vittrup | Attitudes predict the use of physical punishment: a prospective study of the emergence of disciplinary practices[END_REF] However, little research has investigated the interactions between parental attitudes towards corporal punishment and different socio-demographic factors with respect to different forms of child abuse.

Country-specific comparisons of child abuse are difficult to do due to differences in the definition of child abuse. The aims of the present study were to make country-specific and regional comparisons of different forms of child abuse. In particular, we focused on differences in child abuse between developing and transitional countries (in the latter group we included the former Soviet countries and countries of the former Yugoslavia). The second aim of the study was to examine correlates of different forms of child abuse with particular focus on the effect of parental attitudes towards corporal punishment and its interaction with socio-demographic factors.

Material and Methods

The first and second rounds of the MICS surveys were conducted in the middle of 1990s and beginning of 2000, respectively. MICS used standardized questionnaires in all countries. Questions regarding discipline methods applied by parents or guardians to their children in the past month were included in the child discipline module which was used in 29 countries (Table 1). The child discipline module adapted questions from the Parent-Child Conflict Tactics Scale developed by Murray A. Straus, a questionnaire to assess domestic violence, including domestic violence against children. [START_REF] Straus | Identification of child maltreatment with the Parent-Child Conflict Tactics Scales: development and psychometric data for a national sample of American parents[END_REF] The module was applied to children aged between 2 and 14 years; if there was more than one child in this age group, only one child was selected randomly. One survey (Mongolia) was excluded because the dataset was incomplete with respect to the child discipline module. All remaining datasets were checked for consistency and merged.

[Table 1 here]

For the purpose of the analysis three binary outcome variables were created (psychological abuse, moderate and severe physical abuse) by combining the questions from the discipline module: those respondents who responded "No" to all questions were classified as "no abuse" and those who responded "Yes" to any of the questions were classified as practicing specific type of abuse (Table 1).

For this the widely accepted definitions of child abuse were adopted. [START_REF] Krug | World report on violence and health[END_REF] Parental attitudes towards corporal punishment were assessed by the question "Do you believe that in order to bring up (raise, educate) your child properly, you need to physically punish him/her?". Basic socio-demographic data were collected in all countries (child's age and sex, place of residence, and number of household members). Economic status of respondents was assessed by the wealth index, which was developed by the World Bank and the Demographic and Health Survey and is comparable across countries. [START_REF] Rutstein | The DHS Wealth Index[END_REF] The score of the wealth index was created by means of the principal component method and divided into five percentiles (from the poorest 20% to the richest 20%).

Statistical analysis

F o r R e v i e w O n l y 6
Initially, the frequency of different forms of child abuse was calculated by countries and group of countries. Three country groups were created: 1) African countries, 2) transitional countries, which included the former Soviet and former Yugoslavian countries and 3) all remaining countries.

Furthermore, the chi-square test was applied to examine bivariate associations between outcome variables and variables of interest. As a next step, odds ratios and 95% confidence intervals were obtained for different forms of child abuse with adjustment for the child's sex and age, place of residence (urban vs. rural), socio-economic status (assessed by wealth index and number of household members) and respondents' attitudes towards corporal punishment using multiple logistic regression analysis. All two-way interactions between parental attitudes towards corporal punishment and other variables were examined in separate models and were included into the multivariable model when significant on the level p≤0.001. [START_REF] Hosmer | Applied Logistic Regression[END_REF] Three separate models were examined (psychological abuse, moderate and severe physical abuse). The final models with main effects and interaction terms were analyzed by using multilevel logistic regression analysis (the variable country was included as random effects). Data analysis was conducted using the statistical program SAS for Windows, version 9.2. The procedure Proc Glimmix was used for multilevel logistic regression. 19

Results

Information from the child discipline modules was available for 124 916 children aged between 2 and 14 years. The median sample size of the surveys was 3909 (IQR 2854 -5038, range 1017 -13 000).

Table 2 shows the characteristics of children for whom data from the child discipline module were available. About half of the children in all regions were males, the median percentage of children living in urban areas was higher in transitional countries (53%) compared to other countries (about 40%). The results of multilevel multiple logistic regression analyses revealed a similar pattern of association for all forms of child abuse (Table 3). The risk of all forms of child abuse was slightly higher among male children and those living with many household members. A higher risk of psychological and moderate physical abuse was observed among children living in urban areas. The risk of all forms of child abuse was higher among children with poorer economic status (based on wealth index).

Parental attitudes towards corporal punishment

F o r R e v i
However, this effect differs by parents with different attitudes towards corporal punishment: A considerably higher risk of all forms of child abuse was observed among parents who believed that physical punishment is necessary for child rearing, and the risk increased with poorer economic status (Figure 1).

[Table 3 here]

[Figure 1 here]

Discussion

We used population-based cross-sectional data on child discipline methods applied by parents or guardians in 28 developing and transitional countries. The present study showed that child abuse is still a common practice in both developing and transitional countries, although there were considerable variations in prevalence of different forms of abuse across countries and regions. The most frequent form of child abuse used by parents in all countries was psychological abuse (except Albania, where psychological abuse was lower than moderate physical abuse), followed by moderate physical abuse and the less frequent form was severe physical abuse. Overall, the highest prevalences of all forms of child abuse were observed in African countries and the lowest in transitional countries. This finding is concordant with other studies from African [START_REF] Saidi | Child maltreatment at a violence recovery centre in Kenya[END_REF][START_REF] Pierce | Child abuse in South Africa: an examination of how child abuse and neglect are defined[END_REF] and Middle East countries [START_REF] Catani | Beyond individual war trauma: domestic violence against children in Afghanistan and Sri Lanka[END_REF][START_REF] Alyahri | Harsh corporal punishment of Yemeni children: occurrence, type and associations[END_REF] , which reported high prevalence rates of child abuse. Although the prevalence of all forms of child abuse was lowest in transitional countries compared to other countries, a considerable proportion of parents in these countries used psychological (median 58%, range 12-70%) or moderate physical abuse (median 45%, range 20-54%). However, severe physical abuse was at low levels in transitional countries (median 9%, range 2.2-21.2%). A similar finding with respect to severe physical abuse was found in some developed countries (Finland, Italy, New Zealand, Portugal, UK, and USA) where this type of abuse varied between 3.7% and 16.3%. [START_REF] Gilbert | Burden and consequences of child maltreatment in high-income countries[END_REF] Findings from another study in post-communist countries reported a somewhat higher prevalence of severe physical abuse (e.g. 12% in Macedonia and 29% in Moldova).

10

In some African cultures violent behaviors including child abuse are more common and widely accepted as compared to other cultures (e.g. in more developed countries), which may be attributed to the cultural beliefs and existing norms of the societies. Higher child mortality rates due to consequences of child abuse in African countries may support the higher acceptance of abusive behaviors in this region. In this analysis higher positive attitudes towards corporal punishment as a method of child rearing -which may serve as proxy for the acceptance of violent behavior -in African countries than in transitional countries also support it.

The present study showed that male children were at higher risk of all forms of child abuse with an increased risk of severe physical abuse. This finding is consistent with other studies, mainly from developing countries. [START_REF] Wong | To unfold a hidden epidemic: prevalence of child maltreatment and its health implications among high school students in Guangzhou, China[END_REF][START_REF] Shumba | Epidemiology and etiology of reported cases of child physical abuse in Zimbabwean primary schools[END_REF] It is not clear why boys are at a higher risk of physical abuse. A higher risk can be explained by higher expectations from boys as compared with girls because boys are seen as future family breadwinners, especially in many developing countries. This may result in stricter physical disciplining of boys. Regarding age differences, we found that younger children (age groups 2-5 and 6-10 years) were more likely to experience moderate and severe physical abuse compared to older children (11-14 years). This finding is concordant with findings from other studies in developing countries [START_REF] Fuhua | Child maltreatment among Asian Americans: characteristics and explanatory framework[END_REF][START_REF] Tang | The rate of physical child abuse in Chinese families: a community survey in Hong Kong[END_REF] and can be explained by a higher vulnerability and dependency of younger children. We found a slightly higher risk of psychological and moderate physical abuse among children residing in urban areas and no difference in severe physical abuse (OR 1.01). Living in urban areas especially in developing countries is associated with a higher level of social problems which may increase the risk of child abuse. For example, people may experience more stress or depression which could lead to an abusive behavior. [START_REF] Park | The factors of child physical abuse in Korean immigrant families[END_REF] Higher risky behaviors in urban areas (e.g. drug or alcohol abuse) could also contribute to the higher risk of child abuse. [START_REF] Sebre | Cross-cultural comparisons of child-reported emotional and physical abuse: rates, risk factors and psychosocial symptoms[END_REF] Findings of the present study revealed socio-economic differences in child abuse; a higher risk of all forms of child abuse was associated with a higher number of household members and poorer economic status (based on wealth index). A similar finding regarding family size was observed in several studies. [START_REF] Van Ijzendoorn | Elevated risk of child maltreatment in families with stepparents but not with adoptive parents[END_REF][START_REF] Youssef | Children experiencing violence. I: Parental use of corporal punishment[END_REF] Large families are often accompanied with overcrowding, which per se can increase the risk of child abuse. [START_REF] Youssef | Children experiencing violence. I: Parental use of corporal punishment[END_REF] Also, a higher risk of child abuse can be explained by a frequent change in family composition and a presence of distant relatives or even unrelated adults. [START_REF] Schnitzer | Household composition and fatal unintentional injuries related to child maltreatment[END_REF] There is controversial evidence regarding the association between socio-economic status and child abuse. Several studies found a higher risk of child abuse among children from poorer families. [START_REF] Youssef | Children experiencing violence. I: Parental use of corporal punishment[END_REF][START_REF] Wong | Individual, familial and community determinants of child physical abuse among high-school students in China[END_REF] In contrast, Maker et al. showed that socio-economic factors did not predict child abuse. According to them, other factors such as parental violence experience during childhood and cultural beliefs about the need of physical punishment play more important role in determining child abuse. [START_REF] Maker | Child physical abuse: prevalence, characteristics, predictors, and beliefs about parent-child violence in South Asian, Middle Eastern, East Asian, and Latina women in the United States[END_REF] We found that children from poorer families are at higher risk of child abuse (a constant increase of risk of child abuse by worsening of economic status). However, this effect was considerably higher by parents with positive attitudes towards corporal punishment compared to those with negative attitudes. This finding is consistent with previous research which showed that parental attitudes towards corporal punishment were strongly associated with child abuse. [START_REF] Vittrup | Attitudes predict the use of physical punishment: a prospective study of the emergence of disciplinary practices[END_REF]

Strengths and limitations

Our study has several strengths; namely, a) MICS surveys provide nationally representative data, b) standardized sampling methods, including standardized questionnaire on child discipline module were used in all countries, and c) all surveys of the third round of the MICS were conducted in a short period of time (2005 and 2006). All these make it possible to compare different forms of child abuse across countries and regions and to conduct a pooled analysis of different forms of child abuse. Limitations of the study include cross-sectional nature of the study design meaning that causal associations cannot be drawn. We were also restricted to variables available in datasets; information on other predictors of child abuse was not available.

Another limitation of the study was the possible underreporting of child abuse, especially severe forms of physical abuse. This may happen because information on child abuse was collected from parents or adult guardians, who might not be willing to report abusive behaviors towards their children.

Moreover, the extent of not willing to report child abuse may vary across different countries (with a greater extent in more developed countries). Children information about child abuse may reduce a selfreport bias; however, this was probably not possible due to an inclusion of younger children in the surveys (beginning from the age of two years). An opposite problem, over-estimation of child abuse might also take place in certain countries; some parents might overstate their abusive behaviors [START_REF] Milner | Impact and detection of response distortions on parenting measures used to assess risk for child physical abuse[END_REF] , in particular, with regard to psychological and moderate physical abuse, if they think that they need help or as a result of intervention programs. However, the child discipline module which was based on the Parent-Child Conflict Tactics Scale 15 tried to overcome the problems of underreporting and overreporting. For example, the module collects information on the frequency of different ways of corporal punishment rather than on physical consequences of corporal punishment, which believed to increase respondent's response to the module. The items of the module were ordered randomly, which also enhances the respondent's response.

In conclusion, child abuse was very common in the examined countries. However, the prevalence of child abuse varied substantially by countries and regions; all forms of child abuse were higher in African countries compared to transitional countries. We showed that both parental attitudes towards corporal punishment and individual factors like child's age and sex, and socio-economic factors are associated with different forms of child abuse.

Different prevention strategies for child abuse are already developed. However, their effectiveness was not adequately evaluated. In developed countries, school-based education programs involving both children and parents are found to be effective in preventing child abuse. [START_REF] Zwi | School-based education programmes for the prevention of child sexual abuse[END_REF] high or even universal rates of school enrolment were achieved. In countries with lower school enrolment rates such approaches may not be appropriate. In those countries other approaches like family support approach (e.g. home visiting, parent training) may be more effective although the effectiveness of these programs in less developed countries should be considered with caution. [START_REF] Mikton | Child maltreatment prevention: a systematic review of reviews[END_REF] Home visiting was also applied in several developed countries, including USA, Australia, and New Zealand; however, this strategy was found to be less effective in preventing child abuse. [START_REF] Howard | The role of home-visiting programs in preventing child abuse and neglect[END_REF] In countries with high prevalences of child abuse, community-based intervention programs, e.g., educational campaigns should be introduced to increase awareness of this problem among the general population. [START_REF] Hoefnagels | On the threshold of disclosure. The effects of a mass media field experiment[END_REF] In any case, intervention programs should be developed by considering specific local situation in each country.

Funding

No funding.

F

Key messages

• Child abuse is still a common phenomenon in many countries with a considerable variation in prevalence across countries and regions; the highest prevalence was in African countries and the lowest in transitional countries.

• Parental attitudes towards corporal punishment were strongly associated with all forms of child abuse.

• The findings of the present study emphasize the need for intervention programs, especially in African countries, to increase awareness of this problem among the general population.

Intervention programs should be designed by considering specific local situation in each country.

  Child abuse in 28 developing and transitional countriesresults from the Multiple Indicator Cluster Surveys Child abuse in developing and transitional countries Title: Child abuse in 28 developing and transitional countries -results from the Multiple

Figure 1

 1 Figure 1 Association between parental attitudes towards corporal punishment and economic status of the family (based on wealth index) [references -richest group with negative attitudes]

  A considerably higher percentage of parents in African countries than in transitional countries believed that physical punishment should be used as a method of child rearing; the median percentage of positive attitudes towards corporal punishment was around 40% (IQR 33.4%-43.2%) in the African region and 7.7% (IQR 6.8%-14.6%) in transitional countries (Table2, sixth column). More than 90% of respondents in Syria believed that children should be punished physically. The lowest proportion of parents (less than 10%) who believed that physical punishment is necessary was observed in all countries of the former Yugoslavia and in two Central Asian countries, Kazakhstan and Kyrgyzstan.

	F o r
	R
	e v
	e i e
	w w
	O n l O n l
	y y
	7 8

Last month's prevalence of psychological, moderate and severe physical abuse

Table

2

shows prevalences of different forms of child abuse by countries and groups of countries in the month preceding the survey. Psychological abuse was at high levels in almost all countries, with the highest frequency in African countries (median percentage 83.2%, IQR 80.5%-84.4%) and in other countries (75.9%, 64.2%-83.1%) and lowest in transitional countries (57.8%, 44.4%-63.6%). The highest prevalence of psychological abuse was observed in Yemen (92%), Viet Nam (90%), Côte d'Ivoire (87%) and Cameroon (86%) and the lowest in Albania (12%) and Bosnia and Herzegovina (27%). The median percentage of moderate physical abuse was also highest in African countries (64.3%, 62.5%-66.4%) and lowest in transitional countries (45.5%, 35.4%-49.4%). A similar situation was observed for severe physical abuse with a more pronounced difference in median percentage between African (43.1%, 37.1%-52.0%) and transitional countries (8.9%, 6.5%-13.7%). The lowest prevalence of severe physical abuse was in two Central Asian countries, Kyrgyzstan (5%) and Kazakhstan (2%) and the highest percentage in Yemen (61%) and Cameroon (60%).

[Table

2

here] Factors associated with psychological, moderate and severe physical abuse

Table 1

 1 Questions from the child discipline module related to psychological and physical abuse

	Psychological abuse
	Shouted, yelled at him/her?
	Called him/her dumb, lazy or another name like that?
	Moderate physical abuse
	Shook him/her?
	Spanked, hit or slapped him/her on the bottom with bare hand?
	Hit or slapped him/her on the hand, arm or leg?
	Severe physical abuse
	o r Hit or slapped him/her on the face, head or ears?
	Hit him/her on the bottom or elsewhere on the body with something hard objects like a belt? R Beat him/her up with an implement (hit over and over as hard as one could)?
	e v
	i e
	w
	O n l
	y
	13

Table 2

 2 Characteristics of the samples, parental attitudes towards corporal punishment and different forms of child abuse by countries and country groups

	Countries by groups	Year of	Sample	Boys	Urban	Attitudes towards corporal	Psychological abuse	Moderate physical	Severe physical abuse
		survey	size	(%)	areas	punishment (% of positive	(%)	abuse (%)	(%)
					(%)	responses)
	African countries
	Burkina Faso	2006	4233	48.6	25.7	39.9	83.4	51.9	37.1
	Cameroon	2006	5704	49.5	47.9	44.2	86.1	63.2	60.1
	Côte d'Ivoire	2006	6321	50.4	42.1	39.9	87.1	69.9	36.9
	Gambia	2005-06	4703	47.1	42.7	31.7	75.0	64.7	52.6
	Ghana	2006	3915	51.1	41.5	42.9	83.8	60.5	43.3
	Guinea-Bissau	2006	4695	50.4	36.2	26.0	66.6	65.2	42.9
	Sierra Leone	2005-06	6016	49.3	72.2	56.5	82.3	63.8	51.8
	Togo	2006	4570	49.4	38.5	33.9	83.0	71.0	36.5
	Median		4699	49.4	41.8	39.9	83.2	64.3	43.1
	Transitional countries
	Albania	2005	2478	53.6	38.6	6.3	11.8	46.3	8.9
	Belarus	2005	3090	51.3	65.6	15.6	77.4	47.5	10.6
	Bosnia and Herzegovina	2006	2857	50.4	35.6	6.7	27.1	20.0	6.1
	Georgia	2005	4306	53.6	51.4	13.6	58.2	45.5	21.2
	Kazakhstan	2006	6864	53.1	55.0	7.7	47.8	22.6	2.2
	Kyrgyzstan	2005-06	3335	52.3	38.1	8.2	40.9	35.5	4.9
	Macedonia	2005	3557	50.0	51.6	7.6	57.8	53.3	16.8
	Montenegro	2005-06	1177	53.3	62.9	5.2	55.0	41.7	7.6
	Serbia	2005-06	3903	51.4	56.1	6.8	62.2	51.3	10.1
	Tajikistan	2005	5178	52.1	29.4	15.8	69.4	54.3	19.2
	Ukraine	2005	2935	50.9	63.5	17.1	64.9	35.3	6.8
	Median		3335	52.1	51.6	7.7	57.8	45.5	8.9
	Other countries
	Belize	2006	1017	49.1	49.8	26.2	51.0	48.2	31.3
	Guyana	2006	3001	52.3	27.5	24.2	64.2	51.5	40.7
	Jamaica	2005	2118	50.5	58.7	34.5	75.9	70.6	41.6
	Trinidad and Tobago	2006	2040	52.0	-	26.4	65.8	46.9	23.3
	Iraq	2006	13 000	50.0	64.5	25.5	79.7	66.8	32.6
	Syria	2006	12 663	51.1	54.8	92.1	83.1	74.2	28.9
	Yemen	2006	2845	51.3	31.0	45.4	92.3	81.4	61.0
	Lao People's DR	2006	4898	51.6	24.8	18.8	62.5	41.9	14.6
	Viet Nam	2006	2433	50.9	24.4	35.6	89.7	55.4	29.3
	Median		2845	51.1	40.4	26.4	75.9	57.6	30.6

Table 3

 3 Factors associated with different forms of child abuse (results of three multilevel logistic regression analyses, variable country was included as random effects) §

	Variables	Psychological	Moderate	Severe physical
		abuse	physical abuse	abuse
		OR (95% CI)	OR (95% CI)	OR (95% CI)
	Gender
	Male	1.15 (1.12-1.19) 1.19 (1.16-1.22) 1.22 (1.18-1.25)
	Female	1	1	1
	Age of child
	2-5 years	0.76 (0.74-0.79) 1.70 (1.64-1.75) 1.04 (0.99-1.08)
	6-10 years	1.12 (1.08-1.17) 1.61 (1.56-1.67) 1.30 (1.26-1.35)
	11-14 years	1	1	1
	Place of residence
	Urban	1.08 (1.04-1.12) 1.15 (1.11-1.18) 1.01 (0.97-1.05)
	Rural	1	1	1
	Number of household members
	<5 members	0.81 (0.78-0.84) 0.81 (0.78-0.84) 0.79 (0.76-0.82)
	6-7 members	0.89 (0.85-0.93) 0.93 (0.89-0.97) 0.89 (0.85-0.92)
	>7 members	1	1	1
	Parental attitudes towards corporal
	punishment
	Positive	2.44 (2.24-2.66) 3.25 (3.02-3.50) 3.22 (2.98-3.47)
	Negative	1	1	1
	Wealth index
	Poorest group	1.17 (1.11-1.24) 1.17 (1.11-1.24) 1.21 (1.13-1.29)
	Second group	1.10 (1.03-1.16) 1.16 (1.09-1.22) 1.18 (1.10-1.27)
	Middle group	1.10 (1.04-1.16) 1.16 (1.10-1.22) 1.08 (1.01-1.16)
	Fourth group	0.99 (0.93-1.04) 1.07 (1.02-1.13) 1.03 (1.03-1.17)
	Richest group	1	1	1
	Parental attitudes towards corporal
	punishment*wealth index
	Positive attitudes by poorest group	1.52 (1.38-1.69) 1.26 (1.16-1.37) 1.41 (1.31-1.52)
	Positive attitudes by second group	1.45 (1.31-1.60) 1.18 (1.09-1.29) 1.39 (1.29-1.50)
	Positive attitudes by middle group	1.32 (1.20-1.46) 1.14 (1.05-1.24) 1.23 (1.14-1.33)
	Positive attitudes by fourth group	1.19 (1.08-1.32) 1.13 (1.04-1.23) 1.16 (1.08-1.26)
	Positive attitudes by richest group	1	1	1
	§ Adjusted for all other variables in the table

Acknowledgments

I thank the UNICEF for providing me with MICS datasets. I thank Dr. Frank Pessler and Dr. Mobarak Hossain Khan for a critical reading of the manuscript and helpful discussion.

Conflict of interest

None declared.


Awantha Dissanayake
email: awantha@nagasaki-u.ac.jp

Camilla Piggott

Christopher Baldwin

Katherine A Sloman

Elucidating cellular and behavioural effects of contaminant impact (Polycyclic aromatic hydrocarbons, PAHs) in both laboratory-exposed and field-collected shore crabs, Carcinus maenas (Crustacea: Decapoda)

Keywords: Carcinus maenas, PAH, pyrene, behaviour, physiology

published or not. The documents may come

Elucidating cellular and behavioural effects of contaminant impact (Polycyclic aromatic hydrocarbons, PAHs) in both laboratory-exposed and field-collected shore crabs, Carcinus maenas (Crustacea: Decapoda)

INTRODUCTION

Environmental contaminants, such as polyaromatic hydrocarbons (PAHs), are ubiquitous pollutants in the aquatic environment and pose a serious risk to aquatic organisms [START_REF] Blaxter | The effect of pollutants on sensory systems and behaviour of aquatic animals[END_REF]OSPAR 2001). PAHs are a group of chemical compounds, containing two or more fused benzene rings, which are highly lipophilic (log K ow = 3 -8) [START_REF] Readman | Petroleum and PAH contamination of the Black Sea[END_REF]. In the marine environment, PAHs are a product of incomplete combustion processes and petrochemical pollution from anthropogenic sources, such as boat traffic which contributes to high concentrations of fuel in water systems and sediment [START_REF] King | Dynamic behaviour of polycyclic aromatic hydrocarbons in Brighton marina, UK[END_REF].

Examples of PAHs include pyrene, a 4-ringed PAH that is included in the priority pollutant list (OSPAR 2001). The toxic effect of contaminants, including PAHs, originates from chemical processes at the molecular level when the contaminant impact exceeds the effects of compensatory physiological responses; effects are expressed through the hierarchical levels of biological organisation i.e. biochemical, cellular and physiological levels [START_REF] Fossi | The use of nondestructive biomarkers in the hazard assessments of vertebrate populations[END_REF]. Polyaromatic hydrocarbons, such as pyrene, cause physiological impairments such as DNA damage leading to neoplasia in fish [START_REF] Van Der Oost | Bioaccumulation, biotransformation and DNA-binding of PAHs in feral eel (Anguilla anguilla) exposed to polluted sediments -a field survey[END_REF][START_REF] References Aas | PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod[END_REF], and alteration of cellular function (viability and immune function) in various invertebrate species, such as bivalves [START_REF] Lowe | Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: An in vitro study[END_REF]Grundy et al., 1996a;[START_REF] Lowe | The influence of environmental contaminants on lysosomal activity in the digestive cells of mussels (Mytilus galloprovincialis) from the Venice Lagoon[END_REF] and decapod crustaceans (Dissanayake et al., 2008a;2008b).

The shore crab Carcinus maenas, is a common crab species found along European shores and estuaries [START_REF] Hunter | Intertidal migration by the shore crab Carcinus maenas[END_REF][START_REF] Hayward | The marine fauna of the British Isles & North-west Europe[END_REF] The shore crab Carcinus maenas, is a common crab species found along European shores and estuaries [START_REF] Hunter | Intertidal migration by the shore crab Carcinus maenas[END_REF][START_REF] Hayward | The marine fauna of the British Isles & North-west Europe[END_REF] and may be exposed to PAHs both by waterborne (through the gills) and trophic (feeding) exposure as it is a durophagous predator [START_REF] Crothers | The biology of the shore crab Carcinus maenas (L). 2. The life of the adult crab[END_REF]). Predicting impacts of anthropogenic activities and the consequences for marine organisms is essential if we are to conserve species diversity. Changes in organism functions, including behavioural and physiological alterations, facilitate monitoring of contaminant effects [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF][START_REF] Qhm | Queens Harbour Master (R. Avon) QHM[END_REF]. In particular, behavioural indicators may provide a particularly sensitive insight into various levels of molecular, physiological, and ecological aspects of toxicology by providing ecological relevance to routine physiological measures of toxicity [START_REF] Mcgaw | Behavioural physiology of four crab species in low salinity[END_REF][START_REF] Scott | The effects of environmental pollutants on complex fish behaviour: integrating behavioural and physiological indicators of toxicity[END_REF]. Laboratory studies allow for mechanistic understanding of effects of contaminant exposure at lower levels of biological organisation (i.e. biochemical and cellular) [START_REF] Dam | Intermoult duration affects the susceptibility of shore crabs Carcinus maenas (L.) to pyrene and their ability to metabolise it[END_REF]Dissanayake et al., 2008a;2009;[START_REF] Scarlett | Behavioural, physiological and cellular responses following trophic transfer of toxic monoaromatic hydrocarbons[END_REF], however, when compared with behavioural indicators, physiological assessments may not be sensitive enough to signal effects of environmentallyrealistic contaminant exposure in field-collected organisms. The aim of this study was therefore to assess potential physiological and behavioural effects of both laboratory and field exposure to PAH (pyrene) in the shore crab, Carcinus maenas. To achieve this aim, cellular, physiological and behavioural endpoints were measured in crabs collected from three field sites [a 'relatively' PAH contaminated site (Plym Estuary) and two 'comparatively clean' field sites (Avon and Yealm Estuaries)] and compared with responses of laboratory-exposed crabs exposed to an environmentally-realistic sublethal concentration of the model PAH pyrene (200 µg l -1 for 28 days).

METHODS

Collection and maintenance of animals

Intermoult adult male Carcinus maenas (66.3 ± 5.07 mm) were collected using mackerelbaited traps from three estuaries in South-West England (Fig. 1): the Avon Estuary, Bantham (50° 16'42 N, 3° 52' 18 W), the Yealm Estuary, Newton Ferrers (50° 18'49 N, 4° 03' 08 W), and the Plym Estuary, Sutton Harbour (50° 21'54 N, 4° 08'02 W). There is an apparent lack of data concerning environmental quality with reference to specific types of contaminants i.e.

PAHs; PAH metabolites, however, have been shown to be a useful surrogate indicator of PAH contamination in field sites [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]. Pyrene levels can be indicative of PAH contamination (4-ringed PAHs) e.g. 17-20 µg l-1 is indicative of relatively clean coastal sites and > 40 indicative of PAH-contaminated sites; > 200 µg l-1 has been recorded in coastal harbour sites [START_REF] Dissanayake | Detecting polyaromatic hydrocarbon (PAH) exposure in the Bay of Gibraltar, using fluorescence spectrophotometry as a biomonitoring tool of environmental contamination[END_REF]. Based on this lack of knowledge, sites were ranked based on the degree of maritime activity (thus PAH contamination). The Avon Estuary receives < 20 vessels yr -1 (QHM 2006a, personal communication), has low levels of contamination [START_REF] Hagger | Application of biomarkers for improving risk assessments of chemicals under the Water Framework Directive: A case study[END_REF] and is regarded as 'not at risk' from both point source and diffuse pollution (Environment Agency 1996); the Yealm Estuary supports < 250 vessels yr -1 (QHM 2006c, personal communication), has low levels of contamination [START_REF] Hagger | Application of biomarkers for improving risk assessments of chemicals under the Water Framework Directive: A case study[END_REF]. In contrast, the Plym Estuary receives > 1500 vessels yr -1 (QHM 2006b, personal communication). The influence of other contaminants cannot be ruled out in field-exposed crabs as the presence of copper and tributyltin (TBT), a component of antifouling paint has been found previously in both the Yealm and Plym Estuaries and have been listed as 'at risk' from diffuse pollution (Environment Agency 2006) (for details see Environment Agency 1996; [START_REF] Scarlett | Occurrence of the marine antifouling agent irgarol 1051 within the Plymouth Sound locality: Implications for the green macroalga Enteromorpha intestinalis[END_REF][START_REF] Scarlett | Risk posed by the antifouling agent Irgarol 1051 to the seagrass, Zostera marina[END_REF] and is linked with maritime activity.

The experimental series consisted of two parts. In the first experiment, crabs were exposed to PAH contamination in the laboratory and cellular, physiological and behavioural measurements were made at the end of this time (n = 9). In the second experiment, crabs were collected from the three field sites (n = 9 individuals from each site) and cellular, physiological and behavioural measurements were performed soon after collection (see below). During the first experiment, Carcinus maenas individuals collected from the Avon Estuary (a 'comparatively' clean site) were exposed individually to 200 µg l -1 pyrene (nominal concentration) [a known sublethal concentration that induces sublethal physiological and behavioural effects in several invertebrate species [START_REF] Christensen | Metabolism of pyrene by the polychaetes Nereis diversicolor and Arenicola marina[END_REF]Dissanayake et al., 2008a;2009) or placed into clean filtered (10 µm carbon-filtered) aerated seawater (salinity of 34, 15 ± 1 °C) under a 12 h light: 12 h dark photoperiod (control) (n = 9 per treatment). Solvent used to prepare the pyrene exposure water (acetone, 0.005%) does not contribute to PAH metabolite fluorescence or cause any negative effects on cellular or behavioural parameters as shown previously (Dissanayake et al., 2008b). Crabs were held in the laboratory for two weeks prior to the exposure to acclimate to laboratory conditions.

During the 28 day exposure, crabs were fed individually on 1 g of γ-irradiated cockle (Cerastoderma edule, Tropical Marine Centre, Bristol, UK) flesh every two days. Water was changed within 18 h of feeding. At the end of the 28 day period, crabs were sampled in the same way as field-collected crabs (see below).

Sampling Procedure

Within 24 h of the end of the laboratory exposure or field collection, crabs were sampled (n = 9 per treatment) for urine to determine the presence of PAH metabolites. After a further 24 h, haemolymph was sampled from the same crabs for cellular assays. Physiological assessments (heart rate) and behavioural observations were performed on these individuals following a 72 h starvation period [to standardise hunger levels and induce feeding behaviour [START_REF] Thorpe | How costly is fighting? Physiological effects of sustained exercise and fighting in swimming crabs, Necora puber (L) (Brachyura, Portunidae)[END_REF][START_REF] Rovero | When time is of the essence: choosing a currency for prey-handling costs[END_REF][START_REF] Smallegange | Why do shore crabs not prefer the most profitable mussels?[END_REF]]. Urine and haemolymph sampling techniques are non-destructive. The periods between sampling the same individual for different physiological and behavioural parameters have been shown to allow sufficient recovery in previous studies (Bamber & Depledge 1997a;1997b). Heart rate levels, for example, have been observed to decrease from 'active' (i.e. handling stress) to 'resting' levels within 30 min (Dissanayake et al., 2008b). During periods between sampling, crabs were kept in static holding tanks containing filtered seawater. At the end of the experiment crabs were held for a further two weeks to ensure that they were not in proecdysis, which could potentially affect physiological condition and behaviour.

Urine sampling and analysis

To collect urine samples, crabs were restrained on a board with the third maxillipeds separated. Urine was collected using a 1 ml syringe with an attached 10 µl micropipette tip by prising open each opercula of the antennal gland [START_REF] Bamber | Sites of release of putative sex pheromone and sexual behaviour in female Carcinus maenas (Crustacea: Decapoda)[END_REF]. Urine samples were stored at -20 °C prior to analysis, when as a surrogate indicator of contaminant exposure, pyrene metabolites in urine were determined by fluorescence analysis (λ 345/382 ; BIOTEK FL-600) for 1-hydroxylpyrene-type metabolites (Dissanayake & Galloway 2004;[START_REF] Watson | Responses of the decapod crustacean, Carcinus maenas, following exposure to environmental contaminants. Development and application of multiple biomarkers[END_REF]).

Urine sample were diluted with 50 % ethanol (ratio 1:20).

Haemolymph sampling and analysis

Haemolymph samples (500 µl) from individual crabs were extracted by puncture of the arthrodial membrane at the base of the 4 th walking leg using an ice-chilled 1 ml syringe and 21 gauge needle (Sigma-Aldrich, U.K). Cellular techniques were conducted immediately after haemolymph sampling. Cellular viability was determined in vitro using the microplate neutral red dye uptake method of [START_REF] Babich | Cytotoxicity of T-2 toxin and its metabolites determined with the neutral red cell viability assay[END_REF], whereby, incorporation of the dye within the lysosomes of cells indicates viable cells, whereas decreased uptake and dye retention indicates dead or damaged cells. Neutral red solution (200 µl; 0.004 %) was added to haemolymph samples (30 µl) in microtitreplates. After 3 h incubation (10 °C), cells were washed using phosphate buffered saline (phosphate buffer 0.01 M, KCl 0.0027 M, NaCl 0.137 M) and lysed with 1 % acidified ethanol solution (1 % acetic acid/ 50 % ethanol).

Residual dye was determined spectrophotometrically at 550 nm [START_REF] Galloway | A multibiomarker approach to environmental assessment[END_REF]. The phagocytic capability of haemocytes was evaluated by measuring the active uptake of neutral red-stained zymosan particles (from Saccharomyces cerevisiae, Sigma, U.K) in vitro.

Haemolymph samples (30 µl) were incubated (10 °C for 30 min) with dye-stained particles

(1 x 10 8 particles ml -1 ) and washed using phosphate buffered saline to remove excess particles. Cells were lysed with 1 % acidified ethanol solution and the amount of particles taken up was determined spectrophotometrically at 550 nm as described by [START_REF] Parry | Interactive effects of temperature and copper on immunocompetence and disease susceptibility in mussels (Mytilus edulis)[END_REF], and employed by Dissanayake et al.(2008b;2008a).

Foraging behaviour and heart rate monitoring

Prior to measuring foraging behaviour, individuals were starved for 72 h in order to standardise hunger levels. All tanks were oxygenated for 10 min prior to the addition of test individuals. Dissolved Oxygen (D.O.) levels were measured at the start (120 -130 %) and end (112-120 %) of each trial to ensure that individuals were not exposed to hypoxic conditions. Cardiac activity was recorded using an infrared phototransducer attached to the carapace using cyanoacrylate glue to facilitate heart rate monitoring using the Computer-Aided Physiological MONitoring system (CAPMON) [START_REF] Depledge | A computer-aided physiological monitoring-system for continuous, long-term recording of cardiac activity in selected invertebrates[END_REF]). Once the phototransducer had been attached to the carapace, individuals were placed in one end of an observation chamber [L x H x W = 46 x 25 x 25 (cm)] and given five minutes acclimation time. Cardiac activity was then recorded to obtain basal heart rate. The chamber was blacked out on three sides to limit external visual stimuli. A whole cockle within its shell (0.35 ± 0.05 g) [weight class readily accepted by adult shore crabs) [START_REF] Mascaro | Choice of prey size and species in Carcinus maenas (L.) feeding on four bivalves of contrasting shell morphology[END_REF] and abundant prey item at all tested field sites] was placed in the opposite end of the chamber, separated from the crab by a vertically sliding blackened glass divider. Foraging behaviour and cardiac activity was then observed and recorded for 15 min when the divider was raised.

Foraging activity and cardiac activity was recorded for 15 min as this time period allowed sufficient time to record both activities; consumed food passes from the oesophagus to the stomach region within 15 minutes and any further recording of cardiac activity (i.e. past 15 min) may result in elevated heart rates associated with digestion and the specific dynamic action of food [START_REF] Houlihan | Protein synthesis and oxygen consumption of the shore crab Carcinus maenas after a meal[END_REF][START_REF] Mcgaw | Integrated physiological responses to feeding in the blue crab Callinectes sapidus[END_REF]. A video camera (Canon MV830) was used to record behaviour. Foraging behaviour was divided into a series of behaviours that encompass prey search, prey handling and prey consumption, quantified by direct observation and recording of the following behavioural categories [START_REF] Rovero | When time is of the essence: choosing a currency for prey-handling costs[END_REF][START_REF] Smallegange | Why do shore crabs not prefer the most profitable mussels?[END_REF].

T h (s) = T b (s) + T e (s)

Breaking time (T b ) = time taken from individual's first attempt to open cockle, to first consumption of flesh.

Eating time (T e ) = time taken for crab to consume cockle flesh and discard shell.

Handling time (T h ) = Sum of both breaking time and eating time.

Statistical analysis

One-way Analysis of variance tests (ANOVA) were performed on the chemical (PAH tests were used to isolate differences between experimental groups (field-collected and laboratory-exposed crabs).

RESULTS

Significant differences were observed between experimental groups, with the highest metabolite concentrations observed in urine of laboratory pyrene-exposed crabs and Plymcollected crabs (square root-transformed; F 4,40 = 13.82, P < 0.001, Fig. 2). Evaluation of potential contaminant effects at the cellular level in crab haemocytes, revealed significantly lower cellular viability (square root-transformed F 4,40 = 4.91, P < 0.01, Fig. 3A) and phagocytosis (F 4,40 = 3.17, P < 0.01, Fig. 3B) in laboratory-exposed crabs. With regard to foraging behavior, Plym Estuary-collected crabs had significantly higher breaking times compared to all other groups (F 4,40 = 3.64, P < 0.05; Fig 4A). Significant differences in handling time were observed between groups, with Plym Estuary-collected crabs and laboratory pyrene-exposed crabs displaying longer prey handling times compared to all other groups of crabs (F 4, 40 = 2.83, P < 0.05; Fig. 4C). No significant differences were observed in eating time between shore crabs from each of the treatment groups (log-transformed F 4, 40 = 1.23, P = 0.31; Fig. 4B). Physiological monitoring demonstrated that Avon-collected crabs displayed significantly lower heart rates while handling prey than crabs from all the other treatments (square root-transformed; F 4, 40 = 8.03, P < 0.001, Fig. 4D).

DISCUSSION

This study compared the sensitivity of cellular, physiological and behavioural measures using a combination of laboratory-exposed and field-collected shore crabs. Contaminant exposure was validated by spectrofluorimetric determination of pyrene-type metabolites in C. maenas urine with the highest concentrations observed in laboratory pyrene-exposed crabs and Plym Estuary-collected crabs. Although PAH levels (pyrene) were not fully quantified, a screening method for pyrene metabolites was used as a surrogate indicator of the bioavailable pyrene levels at each site and can be used to discern between sites of varying PAH exposure (Dissanayake & Galloway 2004). Pyrene metabolite levels measured in urine from the laboratory-exposed crabs were consistent with levels previously reported for 200 µg L -1 pyrene-exposed crabs [START_REF] Watson | Rapid assessment of polycyclic aromatic hydrocarbon (PAH) exposure in decapod crustaceans by fluorimetric analysis of urine and haemolymph[END_REF]Dissanayake et al., 2008a;2008b). Pyrene metabolite levels measured in crabs from the Plym Estuary were (on average) 30 % less than those found in laboratory-exposed individuals and, therefore, may equate to exposures of approximately 67 µg l -1 of pyrene. Pyrene metabolite concentrations found in the Plym Estuary-collected and laboratory-exposed crabs are consistent with levels reported previously in coastal waters [START_REF] Law | Polycyclic aromatic hydrocarbons (PAH) in seawater around England and Wales[END_REF][START_REF] Kirby | Assessment of water quality in estuarine and coastal waters of England and Wales using a contaminant concentration technique[END_REF][START_REF] Woodhead | Polycyclic aromatic hydrocarbons in surface sediments around England and Wales, and their possible biological significance[END_REF], and within organisms [shore crabs [START_REF] Watson | Rapid assessment of polycyclic aromatic hydrocarbon (PAH) exposure in decapod crustaceans by fluorimetric analysis of urine and haemolymph[END_REF] and in eels Anguilla Anguilla [START_REF] Ruddock | Bile metabolites of polycyclic aromatic hydrocarbons (PAHs) in European eels Anguilla anguilla from United Kingdom estuaries[END_REF]] that are known to result in physiological impairment, such as alteration in antioxidant levels, altered cellular function (viability and immune function), elevated heart rates and decreased oxygen consumption [START_REF] Fossi | Biomarker responses at different levels of biological organisation in crabs (Carcinus aestuarii) experimentally exposed to benzo(alpha)pyrene[END_REF]Dissanayake et al., 2008a;2008b).

Impacts at the cellular level were evaluated in shore crab haemocytes by assessing membrane integrity (cellular viability) and immune function (phagocytosis) which were negatively impacted (decreased cellular viability and phagocytosis) by contaminant exposure. However, only those crabs exposed to pyrene during lab exposures showed significant decreases in cellular viability and phagocytosis index. In C. maenas, lysosomes are found within the hyaline cells which are important in terms of phagocytic activity (the innate immune function in crustaceans) [START_REF] Ratcliffe | A comparative synopsis of the structure and function of the blood cells of insects and invertebrates[END_REF]. There are ca. 2.5 x 10 8 circulating haemocytes in C. maenas, and around 80 % of these cells are capable of phagocytosis, thereby underscoring the phagocytic ability of this species [START_REF] Smith | Host defence reactions of the shore crab, Carcinus maenas (L.), in vitro[END_REF]. Pyrene is biotransformed by shore crabs into more hydrophilic compounds for excretion by phase I metabolism [START_REF] Dam | Intermoult duration affects the susceptibility of shore crabs Carcinus maenas (L.) to pyrene and their ability to metabolise it[END_REF], however, PAHs exert their toxic effect by either the parent compound or subsequently, during metabolism resulting in production of reactive oxygen species (ROS) [START_REF] Livingstone | Organic xenobiotic metabolism in marine invertebrates[END_REF][START_REF] Livingstone | The fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish[END_REF]. Resulting toxic effects in shore crab haemocytes include, membrane destabilisation and immune function alteration (Dissanayake et al., 2008a;2008b).

Although, significant impact at the cellular level was only observed in laboratory pyreneexposed individuals, it provides an insight into the mechanism of acute contaminant exposure.

Pyrene, like other PAHs which are polar hydrophobic contaminants, are sequestered in the lysosomal compartment of cells. Lysosomal damage arising from the accumulation of contaminants and formation of oxyradicals causes increased membrane permeability and subsequent release of acid hydrolases into the cytoplasm, resulting in cellular damage/cell death (as shown by decreased cellular viability) and, subsequently, resulting in a reduced immune function (as shown by reduced phagocytic ability) [START_REF] Lowe | Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: An in vitro study[END_REF]Grundy et al., 1996a;1996b).

To assess behavioural impacts, foraging behaviour was divided into two behavioural components; breaking time and eating time. Significant differences were observed in breaking time, with Plym Estuary-collected crabs taking significantly longer than other fieldcollected and laboratory-exposed crabs to approach the cockle and break into the shell, thereby, exposing the flesh (i.e. possibly due to problems in orientating and breaking the shell). No significant differences were observed, however, in the time required to consume the prey (eating time). This resulted in differences in overall prey handling time with both PAH-contaminated groups of shore crabs (Plym Estuary-collected and laboratory pyreneexposed) displaying significantly longer handling times, corroborating previous findings of increased prey handling times in shore crabs fed with hydrocarbon-exposed mussels (mean tissue concentration of approximately 50 µg/g dry weight; [START_REF] Scarlett | Behavioural, physiological and cellular responses following trophic transfer of toxic monoaromatic hydrocarbons[END_REF]. In terms of physiological disturbance (assessed here as cardiac activity), no difference was observed in cardiac activity between contaminant-exposed or field-collected crabs, except for Avoncollected crabs displaying significantly lower heart rates. Cardiac activity measurement was chosen as a surrogate indicator of the metabolic costing to the individual as a result of foraging behaviour. It is not clear why Avon-collected crabs displayed this lower heart rate, but based on measured PAH metabolites in the urine, there is no evidence that it is linked to the presence or absence of PAH contamination. To standardise foraging behaviour, crabs from all treatments were starved for a period of 72 h in clean water. Therefore, it is possible that behavioural effects of PAHs in the present study were underestimated with these concentrations of PAH possibly exerting much greater effects immediately after exposure.

Hydrocarbon contaminant exposure disrupts various behaviours in crustaceans such as mate search behaviour in amphipods [START_REF] Krang | Naphthalene disrupts pheromone induced mate search in the amphipod Corophium volutator (Pallas)[END_REF]) and locomotor and agonistic behaviour in decapods [START_REF] Pearson | Threshold for detection of naphthalene and other behavioural responses by the blue crab Callinectes sapidus[END_REF][START_REF] Culbertson | Long-term biological effects of petroleum residues on fiddler crabs in salt marshes[END_REF][START_REF] Dissanayake | Physiological condition and intraspecific agonistic behaviour in Carcinus maenas (Crustacea: Decapoda)[END_REF]. Here, water-borne contaminant exposure resulted in disruption of foraging behaviour in Carcinus maenas, contributing to existing evidence of behavioural disruption reported previously in this species [START_REF] Dissanayake | Physiological condition and intraspecific agonistic behaviour in Carcinus maenas (Crustacea: Decapoda)[END_REF][START_REF] Scarlett | Behavioural, physiological and cellular responses following trophic transfer of toxic monoaromatic hydrocarbons[END_REF]. As marine coastal environments continually receive anthropogenic inputs that are potentially toxic to aquatic organisms [START_REF] Galloway | The ECOMAN project: A novel approach to defining sustainable ecosystem function[END_REF], it is imperative to measure appropriate biological endpoints that signal long-term ecological consequences [START_REF] Hebel | Responses of crustaceans to contaminant exposure: A holistic approach[END_REF]. Behavioural measures have been advocated as a subtle approach to potentially link physiological function of the individual (internal biological processes) with important ecological processes [START_REF] Hebel | Responses of crustaceans to contaminant exposure: A holistic approach[END_REF][START_REF] Mcgaw | Behavioural physiology of four crab species in low salinity[END_REF][START_REF] Scott | The effects of environmental pollutants on complex fish behaviour: integrating behavioural and physiological indicators of toxicity[END_REF]. Foraging behaviour as an indicator of environmental quality has been employed successfully in fish, as observed by prey capture rates of shrimp by mummichogs (Fundulus heteroclitus). Fish from contaminated sites generally had significantly higher contaminant body burdens and lower prey capture rates compared to individuals from comparatively uncontaminated sites [START_REF] Weis | Prey capture ability of mummichogs (Fundulus heteroclitus) as a behavioral biomarker for contaminants in estuarine systems[END_REF].

Previous laboratory studies have shown perturbed behaviours (e.g. decreased feeding rates)

resulting from contaminant exposure (reviewed in Maltby 1999), and when feeding rate is

considered in field studies it can significantly reflect environmental quality and contamination, as shown in various invertebrates, including decapods (Moreira et al., 2006a;[START_REF] Moreira | An in situ postexposure feeding assay with Carcinus maenas for estuarine sediment-overlying water toxicity evaluations[END_REF][START_REF] Culbertson | Long-term biological effects of petroleum residues on fiddler crabs in salt marshes[END_REF]. While laboratory exposures may help elucidate the mechanisms of contaminant-mediated toxicity by allowing detection of cellular and physiological impacts, by incorporating behavioural measures, in addition to cellular techniques, thereby allows for a more holistic, ecologically-relevant determination of contaminant impact when determining effects in field-exposed animals. Disruption of important behaviours in this common estuarine and marine organism will, ultimately, have potential knock-on implications for the fitness and survival of the individual. Therefore, studies considering the effects of contaminants across the breadth of biological organisation utilising both laboratory and field exposures are necessary to truly determine the effects of anthropogenic inputs on marine ecosystems.

List of Figures

  metabolites) and biological (cellular viability, immune function and series of foraging behaviour measurements) endpoints (transformed where necessary to meet parametric assumptions) (GMAV 5 for Windows®) (Underwood 2005); Student-Newman-Keuls (SNK)

Fig. Location

  Fig. Location of field sites; Avon and Yealm ('comparatively clean') and Plym ('relatively' contaminated) (SW England, UK) (adapted from Amaral et al., 2009).

  Fig. Location of field sites; Avon and Yealm ('comparatively clean') and Plym ('relatively' contaminated) (SW England, UK) (adapted from Amaral et al., 2009).

Fig.

  Fig. Pyrene-type metabolites in the urine (mean ± SE) of adult Carcinus maenas. (n = 9 per treatment, different letters indicate significant treatment differences).

Fig.

  Fig. Cellular endpoints (mean ± SE) evaluated in Carcinus maenas haemocytes. A) Cellular viability and B) Phagocytosis index.(n = 9 per treatment, different letters indicate significant treatment differences).

Fig.

  Fig. Foraging behaviour (s) (mean ± SE) observed in adult Carcinus maenas. A) breaking time of shelled-prey (T b ), B) eating time of flesh (T e ), C) handling time (T h ) (breaking time + eating time) and D) cardiac activity during prey handling time (beats/minute). (n = 9 per group, different letters indicate significant treatment differences).

Fig

  Fig. 1.