N Kalfa

P Philibert

L S Baskin

C Sultan
email: c-sultan@chu-montpellier.fr

Charles Pr

Sultan

Hypospadias: interactions between environment and genetics

Keywords: Hypospadias, etiology, Hypospadias, genetics, Environment, Endocrine disruptor, Polymorphism, single nucleotide

Hypospadias is one of the most common congenital malformations. It is considered to be a mild form of the 46,XY disorders of sex development (DSD), but its precise etiology remains to be elucidated. Compromised androgen synthesis or effects can cause this frequent malformation, although the mutational analyses of the genes involved in androgen actions have identified abnormalities in only a very small portion of patients.

The overwhelming majority of cases remain unexplained and hypospadias may be a highly heterogeneous condition subject to multiple genetic and environmental factors.

We here review the recent advances in this field and discuss the potential interactions between the environment and genetics.

A c c e p t e d M a n u s c r i p t

Hypospadias is defined as a defect in the development of the ventral aspect of the penis along with an ectopic opening of the urethral meatus. Its incidence ranges from 1/1000 to 1/100 [START_REF] Manson | Molecular epidemiology of hypospadias: review of genetic and environmental risk factors[END_REF], with significant variations according to ethnic origin, making it one of the most frequent developmental defects. In most cases, the degree of hypospadias is relatively mild and a specific endocrine cause is not sought or is not found [START_REF] Rey | Low risk of impaired testicular Sertoli and Leydig cell functions in boys with isolated hypospadias[END_REF]. However, four main elements are involved in male genital construction and may contribute to this malformation [START_REF] Mouriquand | Etiological Aspects of Hypospadias[END_REF][START_REF] Kalfa | Is hypospadias a genetic, endocrine or environmental disease, or still an unexplained malformation?[END_REF]: [START_REF] Manson | Molecular epidemiology of hypospadias: review of genetic and environmental risk factors[END_REF] the genetic and endocrine background of the child, principally the genes of phallic development, gonadal steroid synthesis (mainly testosterone and its 5α reduced form, dihydrotestosterone, DHT), and the responsiveness to these hormones. The genital tubercle thus grows under the influence of androgens and any alteration in androgen production or receptors may produce a hypospadiac penis; [START_REF] Rey | Low risk of impaired testicular Sertoli and Leydig cell functions in boys with isolated hypospadias[END_REF] the placenta, which orchestrates the hormonal climate, especially during the first part of gestation; (3) the mother, with her own hormonal production and possible disorders; and (4) the environment of mother and child, which may also interfere in this fine balance [START_REF] Yiee | Environmental factors in genitourinary development[END_REF][START_REF] Wang | Endocrine disruptors, genital development, and hypospadias[END_REF].

Over the last 30 years, male reproductive health has been altered, with deterioration in sperm count and an increasing number of cases of undescended testes, testicular cancers and hypospadias [START_REF] Toppari | Cryptorchidism and hypospadias as a sign of testicular dysgenesis syndrome (TDS): environmental connection[END_REF]. This phenomenon has raised some concerns regarding environmental chemicals such as the products of industrial and agricultural development [START_REF] Nelson | The increasing incidence of congenital penile anomalies in the United States[END_REF][START_REF] Kalfa | Hypospadias: etiology and current research[END_REF]. We here review the role of the environment in the occurrence of hypospadias and the interaction between environmental factors and genetics.

A c c e p t e d M a n u s c r i p t I Genetic background

Before evaluating the role of the environment, it should be acknowledged that several arguments are in favor of a predominant role for the genetic background. Familial clustering is seen in about 10% of the cases [START_REF] Chen | Genetic studies on hypospadias in males[END_REF][START_REF] Czeizel | Aetiological studies of hypospadias in Hungary[END_REF][START_REF] Kallen | A joint international study on the epidemiology of hypospadias[END_REF][START_REF] Fredell | Complex segregation analysis of hypospadias[END_REF], and the recurrence risk in the male siblings of an affected patient is about 15% [START_REF] Bauer | Hypospadias: a familial study[END_REF][START_REF] Stoll | Genetic and environmental factors in hypospadias[END_REF][START_REF] Asklund | Increased frequency of reproductive health problems among fathers of boys with hypospadias[END_REF]. Seven percent of the fathers of children with hypospadias are also affected [START_REF] Neto | Hypospadias: an epidemiological study in Latin America[END_REF] The risk ratios of hypospadias for male first-, second-, and third-degree relatives of a hypospadiac case were, respectively, 11.6%, 3.27%, and 1.33%. The risk of recurrence for the next male sibling depends on the severity of the hypospadias [START_REF] Bauer | Hypospadias: a familial study[END_REF]. Segregation analysis suggests that hypospadias might be due to monogenic effects in a small proportion of the families, whereas a multifactorial mode of inheritance was reported to be more likely in the majority of families [START_REF] Svensson | Male hypospadias, 625 cases, associated malformations and possible etiological factors[END_REF]. Finally, some of the 200 syndromes that include hypospadias have known genetic bases and shed light on the molecular mechanisms involved in genital development. [START_REF] Edery | Genetics of Hypospadias[END_REF][START_REF]London Dysmorphology Database[END_REF] The environment may act on the genes that contribute to the occurrence of hypospadias at several levels. 4-Level of androgen action: Mutations in the androgen receptor gene (AR) have been found in patients with either severe forms of hypospadias [START_REF] Kaspar | Characterization of two point mutations in the androgen receptor gene of patients with perineoscrotal hypospadia[END_REF][START_REF] Sultan | Disorders linked to insufficient androgen action in male children[END_REF][START_REF] Holterhus | Mutations in the amino-terminal domain of the human androgen receptor may be associated with partial androgen insensitivity and impaired transactivation in vitro[END_REF] or other signs of undervirilzation, such as cryptorchidism [START_REF] Hiort | Molecular characterization of the androgen receptor gene in boys with hypospadias[END_REF] or micropenis [START_REF] Sultan | Mutations of androgen receptor gene in androgen insensitivity syndromes[END_REF][START_REF] Li | Study of genic mutations of androgen receptor in hypospadias[END_REF]. Mutation of the AR gene in partial androgen insensitivity syndrome is found only in 20% to 30% of cases and the phenotype remains particularly variable [START_REF] Sultan | Mutations of androgen receptor gene in androgen insensitivity syndromes[END_REF][START_REF] Deeb | Correlation between genotype, phenotype and sex of rearing in 111 patients with partial androgen insensitivity syndrome[END_REF].

1-Level of phallus development:

II Environment

II-1 Arguments for an environmental contribution

Several findings in both animal and human studies raise suspicion of an environmental contribution to this malformation. Hypospadias, whether associated with micropenis or not, has been reported in numerous wildlife species when the habitat is contaminated by pesticides [START_REF] Hayes | Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses[END_REF]. The effects of prenatal xenoestrogens on animal male reproductive tract development have been studied by several groups. Male rat pups exposed to DES during gestation (at concentrations similar to those measured in first-trimester human fetal tissues) developed hypospadias [START_REF] Gray | Latent effects of pesticides and toxic substances on sexual differentiation of rodents[END_REF][START_REF] Henry | Direct fetal injections of diethylstilbestrol and 17 beta-estradiol: a method for investigating their teratogenicity[END_REF]. Hypospadias was also found in male rodents after maternal treatment with vinclozolin (dose-response effect) [START_REF] Gray | Effects of environmental antiandrogens on reproductive development in experimental animals[END_REF], and similar findings were recorded for prenatal exposure to polychlorinated biphenyls (PCB), phthalates and dioxin [START_REF] Gray | Effects of environmental antiandrogens on reproductive development in experimental animals[END_REF][START_REF] Baskin | Hypospadias and endocrine disruption: is there a connection?[END_REF][START_REF] Fisher | Human 'testicular dysgenesis syndrome': a possible model using in-utero exposure of the rat to dibutyl phthalate[END_REF].

Despite some inaccurate registers which under-evaluate the number of hypospadias cases and the variable geographical distribution of the malformation [START_REF] Calzolari | Aetiological factors in hypospadias[END_REF], several reports suggest an increase in hypospadias over the last 20 years [START_REF] Moretti | Genitourinary tract anomalies: neonatal medical problems[END_REF]. Boisen [START_REF] Boisen | Hypospadias in a cohort of 1072 Danish newborn boys: prevalence and relationship to placental weight, A c c e p t e d M a n u s c r i p t anthropometrical measurements at birth, and reproductive hormone levels at three months of age[END_REF] performed a A c c e p t e d M a n u s c r i p t prospective cohort study and found a high prevalence (1%) of hypospadias in the Danish population of male newborns, whereas the prevalence was reported to be 0.73% in the Netherlands in a cross-sectional study [START_REF] Avellan | The incidence of hypospadias in Sweden[END_REF]. A prospective case-control study of 1,442 male newborns identified 16 cases of middle and posterior hypospadias (1.1%) in the south of France [START_REF] Jeandel | Environmental disruptors and disorders of the external genitals in male newborns : A case-control study[END_REF]. More recent reports have also described an increased incidence [START_REF] Lund | Prevalence of hypospadias in Danish boys: a longitudinal study, 1977-2005[END_REF][START_REF] Sun | Increasing prevalence of hypospadias associated with various perinatal risk factors in chinese newborns[END_REF] This phenomenon has raised some concerns regarding environmental chemicals, such as industrial and agricultural products. Two epidemiological studies reported a possible relationship between exposure to pesticides and hypospadias. Kristensen reported a moderate increase in the odds ratio (OR) for hypospadias in individuals exposed to farm chemicals (OR = 1.51, 95% confidence interval, 1.00-2.26) [START_REF] Kristensen | Birth defects among offspring of Norwegian farmers, 1967-1991[END_REF], and Weidner [START_REF] Weidner | Risk factors for cryptorchidism and hypospadias[END_REF][START_REF] Weidner | Cryptorchidism and hypospadias in sons of gardeners and farmers[END_REF] concluded that maternal farming or gardening led to a slightly increased risk of hypospadias (OR = 1.27, 95% confidence interval, 1.14-2.47). Residence in the vicinity of hazardous waste-disposal A c c e p t e d M a n u s c r i p t sites has been associated with a high incidence of hypospadias [START_REF] Dolk | Risk of congenital anomalies near hazardous-waste landfill sites in Europe: the EUROHAZCON study[END_REF][START_REF] Elliott | Risk of adverse birth outcomes in populations living near landfill sites[END_REF]. Similarly, an increased rate of hypospadias was reported in boys from parents exposed to dioxin after the Seveso industrial accident [START_REF] Mastroiacovo | Birth defects in the Seveso area after TCDD contamination[END_REF]. A vegetarian diet in pregnant women was reported to carry a significant risk of hypospadias [START_REF] North | A maternal vegetarian diet in pregnancy is associated with hypospadias. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood[END_REF] (OR = 4.99, 95% confidence interval, 2.10-11.88). A study in Western Minnesota [START_REF] Garry | Pesticide appliers, biocides, and birth defects in rural Minnesota[END_REF] found a higher rate of congenital abnormalities in infants conceived in spring when herbicides are usually widely used. Birth rates with urogenital abnormalities, as well as abnormalities in other systems, were significantly increased in highuse areas. Another study [START_REF] Garry | Birth defects, season of conception, and sex of children born to pesticide applicators living in the Red River Valley of Minnesota, USA[END_REF] also suggested a possible gene-environment interaction at work in this agricultural region. A total of 22% of the families in which the father applied the herbicides had more than one child with a birth defect.

Although most investigations of congenital anomalies have focused on major structural defects, recent epidemiology finds subtle developmental defects in genital masculinization. Anogenital distance, a reflection of male reproductive tract development which is reduced in hypospadiac patients [START_REF] Hsieh | Associations among hypospadias, cryptorchidism, anogenital distance, and endocrine disruption[END_REF], was found to be reduced in cases of prenatal exposure to phtalates [START_REF] Hsieh | Associations among hypospadias, cryptorchidism, anogenital distance, and endocrine disruption[END_REF][START_REF] Swan | Decrease in anogenital distance among male infants with prenatal phthalate exposure[END_REF].

Last, some of these substances, like DES (diethylstilbestrol), were observed to increase the hypospadias incidence in the second generation, suggesting a transgenerational effect [START_REF] Klip | Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study[END_REF].

II-2 Substances

Several substances in the environment may potentially interfere with male genital development because of their similarity to hormones. Although there is a long list of suspicious substances contained in herbicides, fungicides, insecticides, and industrial by-A c c e p t e d M a n u s c r i p t products or end-products (plasticizers, cosmetics, paints, etc.), none of them has been clearly identified as responsible for hypospadias. Various pollutants potentially involved in the abnormal development of the genital tubercle include: chlorinated pesticides (DTT, Lindane), polychlorinated biphenyl, methoxychlor, phenolic derivatives, nonylphenol, endosulfan, atrazine, phthalates, dioxine, furans [START_REF] Kogevinas | Human health effects of dioxins: cancer, reproductive and endocrine system effects[END_REF], xenoestrogens, phytoestrogens, and mycoestrogens [START_REF] Santti | Phytoestrogens: potential endocrine disruptors in males[END_REF].

II-3 Contamination routes

Humans are in constant contact with many of these substances [START_REF] Garry | Pesticide appliers, biocides, and birth defects in rural Minnesota[END_REF][START_REF] Brock | Serum levels of several organochlorine pesticides in farmers correspond with dietary exposure and local use history[END_REF] as they are found in water, soil, food and air [START_REF] Olea | Inadvertent exposure to xenoestrogens in children[END_REF][START_REF] Restrepo | Birth defects among children born to a population occupationally exposed to pesticides in Colombia[END_REF]. These pollutants enter the body either by ingestion, inhalation, or adsorption, or they may be conveyed through the placenta. Individual exposure varies with dietary habits, life style, and work. Most of these pollutants are lipophilic and are stored in body fat for a lifetime [START_REF] Kelce | Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist[END_REF]. They are also found in breast milk [START_REF] Sonawane | Chemical contaminants in human milk: an overview[END_REF] and in the amniotic fluid. Since most of these chemicals use the same pathways as natural hormones, they have been named xenoestrogens and/or endocrine disrupting chemicals (EDC).

III Interactions between environment and genetics III-1 Types of action of environmental pollutants

Environmental pollutants exhibit several genomic and non-genomic actions. They bind to the nuclear receptors such as estrogen receptors α and β (ERα/ERβ), inducing transcription activation (or repression) of specific gene expression [START_REF] Miyagawa | Estrogen receptor ESR1 is indispensable for the induction of persistent vaginal change by neonatal 5alpha-dihydrotestosterone exposure in mice[END_REF][START_REF] Couse | Estrogen receptor-alpha mediates the detrimental effects of neonatal diethylstilbestrol (DES) exposure in the murine reproductive tract[END_REF]. Non-genomic actions are mediated by a plasma membrane estrogen or androgen receptor. Xenoestrogens have both A c c e p t e d M a n u s c r i p t estrogenic and antiandrogenic actions and compete with natural androgens for the ligandbinding domain (LBD) of the AR gene [START_REF] Paris | Phenylphenols, biphenols, bisphenol-A and 4-tertoctylphenol exhibit alpha and beta estrogen activities and antiandrogen activity in reporter cell lines[END_REF]. They also induce more potent estrogenic metabolites. In addition to these receptor-mediated actions, EDCs may affect synthesis, metabolism, excretion, and binding of endogenous hormones to SHBG, and they have the capacity to inhibit the transcription of androgen-dependent genes [START_REF] Giwercman | Reproductive hormone levels in men exposed to persistent organohalogen pollutants: a study of inuit and three European cohorts[END_REF][START_REF] Dechaud | Xenoestrogen interaction with human sex hormone-binding globulin (hSHBG)[END_REF]. Finally, an epigenetic action has been demonstrated [START_REF] Bredfeldt | Xenoestrogen-induced regulation of EZH2 and histone methylation via estrogen receptor signaling to PI3K/AKT[END_REF].

A major point regarding the action of environmental toxicants in inducing hypospadias is the cumulative effects of multiple low-dose exposures. The cumulative effects of in utero administration of mixtures of "antiandrogens" on male rat reproductive development has previously been demonstrated [START_REF] Rider | Cumulative effects of in utero administration of mixtures of "antiandrogens" on male rat reproductive development[END_REF]. In this study, the complex mixture behaved in a dose-additive manner, and compounds that acted by disparate mechanisms of toxicity displayed cumulative effects when present in combination. This situation could reflect real environmental conditions, in which several chemicals that do not act via a common cellular mechanism of action are present together and disrupt fetal tissues during sexual differentiation in a dose-additive manner [START_REF] Rider | A mixture of seven antiandrogens induces reproductive malformations in rats[END_REF][START_REF] Main | Genital anomalies in boys and the environment[END_REF].

III-2 Candidate genes implicated in the susceptibility to environment

The dialogue between genes and the environment may include variations in gene expression and variations in receptivity. Genetic variants (polymorphisms) may modulate the individual susceptibility to the external environment.

III-2-1 Dysregulation of gene expression

The expression of gonadal development genes varies according to the exposure to estrogen-like substances. Experimental studies in both rats and mice have demonstrated that A c c e p t e d M a n u s c r i p t estrogens can directly inhibit testicular steroidogenesis in the fetus [START_REF] Guyot | Diethylstilbestrol inhibits the expression of the steroidogenic acute regulatory protein in mouse fetal testis[END_REF]. Consistent with this finding, studies have reported an increase in the incidence of hypospadias in male mice that were exposed in utero to DES or ethinyl estradiol [START_REF] Kim | Induction of hypospadias in a murine model by maternal exposure to synthetic estrogens[END_REF]. In addition to this mechanism, a complete loss of androgen receptor protein expression was found to be a more severe effect of these substances [START_REF] Mckinnell | Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol[END_REF].

Xenoestrogens also act as endocrine disruptors. Molecular analyses in fetal rat testes after in utero exposure to phtalates have shed light on the potential mechanisms via which phtalates suppress testicular testosterone production. Several key genes involved in steroidogenesis were disrupted after in utero exposure to monobutyl phthalate and monoethyl hexyl phthalate, such as StAR, HMG-CoA synthase, SRB1 and the steroidogenic enzymes Cyp11a, 3beta HSD and Cyp 17 [97] [98,99]. Linuron, a urea-based pesticide, acts as an antiandrogen. It antagonizes rat and human AR, inhibits androgen-induced gene expression, and reduces testosterone production by effects on LH receptor expression [START_REF] Cook | Investigation of a mechanism for Leydig cell tumorigenesis by linuron in rats[END_REF] Exogenous administration of estrogens also results in an increased expression of ERα (but not ERβ) [START_REF] Agras | Estrogen receptor-alpha and beta are differentially distributed, expressed and activated in the fetal genital tubercle[END_REF]. Expression of TGF-β1 is also modulated by endocrine disruptors.

Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot studies have

shown that the expression of TGF-β1 is upregulated in DEHP-treated mice along with a significant inhibition of male fetal genital tubercule [START_REF] Liu | Di-(2-ethylhexyl) phthalate upregulates ATF3 expression and suppresses apoptosis in mouse genital tubercle[END_REF].

III-2-2 Gene polymorphisms

Polymorphisms of steroid receptors may modulate the response to toxic substances.

An increased GGN trinucleotide repeat in the AR gene has been found to reduce its transcriptional activity in hypospadiac patients [START_REF] Aschim | Linkage between cryptorchidism, hypospadias, and GGN repeat length in the androgen receptor gene[END_REF][START_REF] Radpour | Association of long polyglycine tracts (GGN repeats) in exon 1 of the androgen receptor gene with cryptorchidism and penile hypospadias in Iranian patients[END_REF]. The role of amplification of the CAG repeats remains to be determined [START_REF] Muroya | Hypospadias and the androgen receptor gene: mutation screening and CAG repeat length analysis[END_REF] and may be associated with undermasculinized genitalia, including hypospadias [START_REF] Lim | Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males[END_REF]. For some authors, the V89L variant of the SRD5A2 gene is a risk factor for hypospadias [START_REF] Thai | The valine allele of the V89L polymorphism in the 5-alpha-reductase gene confers a reduced risk for hypospadias[END_REF], whereas for others it is not [START_REF] Van Der Zanden | Genetics of hypospadias: are single-nucleotide polymorphisms in SRD5A2, ESR1, ESR2, and ATF3 really associated with the malformation?[END_REF]. Polymorphisms of the estrogen receptor may also facilitate the deleterious effects of xenoestrogens since their effects are mainly mediated through this receptor. The AGAGA haplotype of the estrogen receptor 1 (ESR1) gene is strongly associated with hypospadias [START_REF] Watanabe | Haplotype analysis of the estrogen receptor 1 gene in male genital and reproductive abnormalities[END_REF]. The ESR1 C-A haplotype, for ESR1 XbaI and ESR2 2681-4A>G, respectively, increases the risk of malformation, as well [START_REF] Ban | Genetic polymorphisms of ESR1 and ESR2 that may influence estrogen activity and the risk of hypospadias[END_REF]. An increased number of CA repeats (and subsequently increased ER activity) also augments the risk of malformation [START_REF] Beleza-Meireles | Risk factors for hypospadias in the estrogen receptor 2 gene[END_REF], and more recently we identified ATF3 polymorphisms in patients with isolated hypospadias [START_REF] Kalfa | Genomic variants of ATF3 in patients with hypospadias[END_REF]. The data regarding the association of polymorphisms with

IV Limitations and questions

Although numerous studies point toward a major role for the environment in hypospadias, two limitations should be considered before attempting to draw definitive conclusions. First, caution should be exercised when extrapolating from murine experiments to humans. In these experiments, xenoestrogens induced hypospadias in male offspring exposed in utero, but the doses given to animals may not be comparable to environmental exposure. Second, several epidemiological studies have reported contradictory results, as noted above [START_REF] Fisch | Hypospadias rates in new york state are not increasing[END_REF][START_REF] Aho | Is the incidence of hypospadias increasing? Analysis of Finnish hospital discharge data 1970-1994[END_REF][START_REF] Martinez-Frias | Secular decreasing trend of the frequency of hypospadias among newborn male infants in Spain[END_REF]. Epidemiological studies on maternal exposure are also inconclusive.

Contrary to previously cited studies [START_REF] Weidner | Risk factors for cryptorchidism and hypospadias[END_REF] [125], some reports did not confirm any significant risk of hypospadias when mothers were exposed to DTT [START_REF] Fisch | Hypospadias rates in new york state are not increasing[END_REF][START_REF] Longnecker | Maternal serum level of 1,1-dichloro-2,2-bis(pchlorophenyl)ethylene and risk of cryptorchidism, hypospadias, and polythelia among male offspring[END_REF]. Moreover, the critical level of exposure to EDCs was not assessed in any of the epidemiological studies implicating the environment [START_REF] Weidner | Risk factors for cryptorchidism and hypospadias[END_REF] [START_REF] Ormond | Endocrine disruptors in the workplace, hair spray, folate supplementation, and risk of hypospadias: case-control study[END_REF]. No relationship was identified between polybrominated biphenyl (PBB) [START_REF] Small | Maternal exposure to a brominated flame retardant and genitourinary conditions in male offspring[END_REF] or polychlorinated biphenyl (PCB) exposure [START_REF] Mcglynn | Maternal pregnancy levels of polychlorinated biphenyls and risk of hypospadias and cryptorchidism in male offspring[END_REF] and hypospadias. A recent meta-analysis indicated only a modestly increased risk of hypospadias associated with pesticide exposure [START_REF] Rocheleau | Pesticides and hypospadias: a meta-analysis[END_REF].

Conclusion

The study of hypospadias is of interest for several reasons. It is an easily diagnosed malformation and, although minor, it recapitulates the pathophysiology of the disorders of sex development; the investigation of hypospadias thus offers insight into the mechanisms of sex A c c e p t e d M a n u s c r i p t determination and differentiation. Moreover, because hypospadias is at the crossroads of genetics and environment, it is a model for exploring genetic and environmental interactions (Figure 1). The environmental data to date, however, should be interpreted with caution.

Indisputable proof of the detrimental effects of the environment is still pending and no single EDC has been identified as a cause of hypospadias in humans. Hypospadias nevertheless remains a sentinel of the effects of the environment through genetics, both at the present time and for the next generation.

Legend

Table 1: Summary of genetic variants associated with an increased risk of hypospadias

.

  The risk of recurrence is also found to aggregate in more distant relatives. Using Danish health registers, Schnack et al. identified 5,380 boys diagnosed with hypospadias in a cohort of 1,201,790 boys born in the period 1973-2005.

2 - 3 -

 23 Homeobox genes A (HOXA) and D (HOXD) participate in the development of the phallus since knock-out of these genes in mice induces a malformation in the external genitalia consistent with hypospadias[23]. In humans, the hand-foot-genital syndrome (HFGS)[24,25] is related to mutations of HomeoboxA13 (HOXA13). HOXA13 allows the normal expression of fibroblast growth factor (FGF) 8 A c c e p t e d M a n u s c r i p t and bone morphogenetic protein (BMP) 7 in the developing urethral epithelium in mice, thus modulating androgen receptor expression and glans vascularization[3]. The FGF gene family, especially FGF10[26], is also implicated in the development of external genitalia in mice[27]. In humans, polymorphisms of FGF8, FGF10 and FGFR2 may be associated with an increased risk of hypospadias[28]. Level of testicular determination: The genes leading to testicular dysgenesis are a cause of hypospadias. Severe hypospadias along with other genital abnormalities[29,30] can reveal heterozygous mutations of Wilms tumor 1 (WT1). SOX9, DMRT1 and GATA4 encode transcription factors acting immediately before the differentiation of the gonad into testis. Mutations of these genes induce testicular dysgenesis and are associated with 46,XY disorders of sex differentiation (DSD), including severe hypospadias [31-34]. Variation in gene dosage, as shown in 46,XX and 46,XX d17 patients with SOX9 duplication, can also induce penoscrotal hypospadias [31]. Level of androgen biosynthesis: Mutations in the LH receptor gene (inducing a Leydig cell hypoplasia) and the 5α-reductase gene (inducing a defect of dihydrotestoterone synthesis) induce hypospadias, most often in a severe form with associated cryptorchidism and/or micropenis [32,35,36] [37]. MAMLD1 (mastermind-like domain containing gene) is another candidate gene that seems to modulate the synthesis of testosterone around the critical period of sex differentiation. MAMLD1 is expressed in the male gonad in mice, and it augments testosterone production and contains the SF1 target sequence[38]. Fukami et al.[39] identified three nonsense mutations in four individuals with 46,XY DSD including micropenis, bifid scrotum and penoscrotal hypospadias. Genetic variants of MAMLD1 were further shown to be present in patients A c c e p t e d M a n u s c r i p t with isolated hypospadias[40], as confirmed by Chen et al. [41], who identified five nonsynonymous mutations, some of them as polymorphisms.

  , but epidemiological studies nevertheless give divergent results about whether the trend of hypospadias is increasing and thus raise questions about temporal trends [62][63]. Aho et al. [64] identified all patients in the national hospital discharge registry who had been born in the period 1970-1986 and surgically treated for hypospadias before the age of 9 years. They calculated the cumulative prevalence by dividing the number of patients by the number of male births and found that the prevalence of hypospadias in Finland remained constant throughout the study period but appeared to be approximately three times higher than previously reported. Improvements in completing the registration forms may account for a substantial proportion of the higher prevalence of hypospadias, according to these authors. Martinez Frias et al. [65] found similar results using the Spanish Collaborative Study of Congenital Malformations (ECEMC) registry to analyze the prevalence in two different periods.

  [START_REF] Mcintyre | Effects of in utero exposure to linuron on androgen-dependent reproductive development in the male Crl:CD(SD)BR rat[END_REF]. Dioxin also suppresses StAR and CYP17 mRNA expression later in gestation, possibly through suppression of LH secretion[START_REF] Takeda | Maternal exposure to dioxin disrupts gonadotropin production in fetal rats and imprints defects in sexual behavior[END_REF] [START_REF] Taketoh | Suppression of fetal testicular cytochrome P450 17 by maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: a mechanism involving an initial effect on gonadotropin synthesis in the pituitary[END_REF].Three estrogen-responsive genes are suspected to be at the crossroads of environment and genetics in hypospadias for several reasons: ATF3, ERα and TGF-β1. ATF3 is the most upregulated estrogen-dependent gene in the foreskin of hypospadiac patients[START_REF] Wang | Up-regulation of estrogen responsive genes in hypospadias: microarray analysis[END_REF].Immunohistochemical analysis on human foreskin confirmed thatthe majority of the hypospadiac samples were positive for expression of ATF3[105]. Both animal and in vitro studies confirmed these findings. In vitro exposure to ethinyl estradiol increases expression and promoter activity of ATF3 in human foreskin fibroblasts [106]. In a murine model, fetal exposure to estrogen increases the level of ATF3 messenger RNA [107], whereas fetal exposure to DEHP (Di-(2-ethylhexyl) phthalate, a common plasticizer, activates the transcription and transduction of the ATF3 gene [108]. Since A c c e p t e d M a n u s c r i p t ATF3 is implicated in cell cycle suppression, its upregulation may interfere with urethra formation[109], as suggested by the dysregulated apoptosis in the murine model described above [108].

A

  c c e p t e d M a n u s c r i p t hypospadias should nevertheless be interpreted with caution. In a large series of 620 Caucasian hypospadiac patients, Van der Zanden et al. [116] failed to confirm the association of single nucleotide polymorphisms (SNPs) in SRD5A2 and ESR1 with hypospadias. The SNPs in ESR2 and ATF3 were even found to be associated in the opposite direction compared with earlier publications. These divergent results confirm that genetic association approaches need to be replicated in very large samples. III-2-3 Xenoestrogen and epigenetics Changes in the epigenetic background induced by synthetic estrogens could be a significant factor in the susceptibility to disease development. The epigenetics appear to involve altered DNA methylation. The primordial germ cells undergo demethylation during migration and early colonization of the embryonic gonad, followed by remethylation starting at the time of sex determination in a sex-specific manner [121][122,123]. The exposure of the pregnant mother at the time of fetal sex determination may be sufficient to alter the remethylation of the germ line in the male fetus and permanently reprogram the imprinted pattern of DNA methylation in boys[124]. Bredfeldt et al. [90] recently showed that 17beta-estradiol (E2) and the xenoestrogen diethylstilbestrol (DES) reduce levels of trimethylation of the lysine residue 27 on histone H3. Interestingly, the modulation of methylation by these substances is mediated by membrane-activated estrogen receptor signaling-through phosphatidylinositol 3 and kinase/protein kinase B-and occurs during windows of uterine development that are susceptible to developmental reprogramming. Activation of this nongenomic pathway has also been shown to reprogram the expression profile of estrogen-responsive genes in uterine myometrial cells. This mechanism for A c c e p t e d M a n u s c r i p t developmental reprogramming caused by early-life exposure to xenoestrogens may contribute to the modulation of the epigenetic machinery during tissue development.

Figure 1 :

 1 Figure 1: Schematic view of the intrication between environment and actors of genital

Table 1

 1

	Gene	Variants
	AR	-CAG/GGN repeat length polymorphism
		-A49T,
	SRD5A2	-L113V -H231R
		-V89L
	HSD17B3	-S232L
	ESR1	-G allele containing variants of ESR1 Xbal
	ESR2	-G allele containing variants of ESR2 2681-4A>G -(CA)n polymorphism in intron 6
		-Specific ''TTC'' haplotype in intron 1
		-c.536A > G(R90)
		-817C > T in the 3'-UTR.
	ATF3	-L23M,
		-C53070T,
		-C53632A,
		-lns53943A
	MAMLD1/CXorf6	-V432A -CAG10 > CAG13

Table


Roberta J Ward
email: roberta.ward@uclouvain.be

Frederic Lallemand

Philippe De Witte

Robert R Crichton

Jacques Piette

Keith Tipton

Karl Hemmings

Arnaud Pitard

Mike Page

Laura Della Corte

Deanna Taylor

David Dexter

Anti-inflammatory actions of a taurine analogue; ethane β-sultam; in phagocytic cells; in vivo and in vitro

Keywords: taurine, anti-inflammatory action, ethane- sultam, nitric oxide, glutamate Abbreviations, NO-nitric oxide, TauT-taurine transporter, LPS-lipopolysaccharide, ROS-reactive oxygen species, IB-IkappaB, IBa-IkappaBalpha, NFB-nuclear factor kappa B, TNF-tumor necrosis factor alpha

A c c e p t e d M a n u s c r i p t

Introduction

The sulphonated amino acid taurine is widely distributed in mammalian tissues, and is present at high concentrations, ~ 50 mM, in leucocytes, microglia and macrophages, [START_REF] Schuller-Levis | Park E Taurine and its chloramines: modulators of immunity[END_REF], where it plays an important anti-inflammatory role. The mechanisms underlying the cytoprotective actions of taurine appear to depend, to a large extent, on the cellular type. The anti-inflammatory actions of taurine are manifested in a variety of forms, including its reaction with hypochlorous acid, in the presence of myeloperoxidase, to form the more stable and less toxic, taurine chloramine (Tau-Cl) in activated neutrophils, as well as modulation of calcium ion homeostasis [START_REF] Corte | The use of taurine analogues to investigate taurine functions and their potential therapeutic applications[END_REF]. The mode of taurine's anti-inflammatory action remains undefined since most studies have investigated the effects of Tau-Cl in immortalised macrophage cell lines, such as RAW 264 and NR8383 (for example see [START_REF] Barua | Taurine chloramine inhibits inducible nitric oxide synthase and TNF-alpha gene expression in activated alveolar macrophages: decreased NFkappaB activation and IkappaB kinase activity[END_REF]). Since activated phagocytic cells, namely microglia, play an important role in many neurodegenerative conditions, such as Parkinson's disease [START_REF] Wilms | Inflammation in Parkinson's diseases and other neurodegenerative diseases: cause and therapeutic implications[END_REF] and alcohol-induced brain damage [START_REF] Suk | Microglial signal transduction as a target of alcohol action in the brain[END_REF], compounds with anti-inflammatory actions, which are able to traverse the blood brain barrier may have therapeutic actions in the retardation of the disease processes.

Although taurine can be synthesised intracellularly from cysteine and methionine, the diet is the main source. Taurine uptake from the plasma into cells is tightly controlled by the taurine transporter (TauT). TauT controls the influx of taurine within very narrow limits, such that exogenously administered taurine only transiently increases levels within a number of cellular types, including liver [START_REF] Ward | Neuroprotection by taurine and taurine analogues[END_REF] and, to a lesser extent, brain [START_REF] Shimada | Distribution of 35 S-taurine in rat neonates and adults. A whole-body autoradiographic study[END_REF] [8] [START_REF] Ward | Neuroprotection by taurine and taurine analogues[END_REF]. TauT activity can be modified by a number of factors including inflammation, where it is decreased [START_REF] Kim | Ionomycin restores taurine transporter activity in cyclosporine-A treated macrophages[END_REF] [10] [START_REF] Kim | Myo-inositol restores the inflammation-induced down regulation of taurine transport by the murine macrophage cell line, RAW 264.7[END_REF], calcium ions and nitric oxide [START_REF] Kim | Ionomycin restores taurine transporter activity in cyclosporine-A treated macrophages[END_REF], glucocorticoids [START_REF] Kim | Regulation of taurine transporter activity by glucocorticoid hormone[END_REF], protein kinase C activation in rat astroglial cells A c c e p t e d M a n u s c r i p t 4 [START_REF] Tchoumkeu-Nzouessa | Activation of protein kinase C downregulates glial but not neuronal taurine uptake[END_REF] and human glioma GL15 cells [START_REF] Tchoumkeu-Nzouessa | Characterisation of taurine transport in human glioma GL 15 cell line: regulation by protein kinase C[END_REF]. Therefore taurine analogues, which are able to traverse membranes independently of TauT, may enhance intracellular taurine levels thereby promoting anti-inflammatory pathways.

It remains unclear as to how modifications to the taurine molecule might enhance its uptake across the lipid membrane bi-layer into the cell or passage across the blood brain barrier, BBB, since it would be against a concentration gradient. The ability of the taurine analogue, acamprosate, calcium acetyl homotaurinate, to reduce alcohol craving in detoxified alcohol abusers, is attributed to the presence of the calcium moiety and the N-acetyl homotaurine, which is reputed to enhance its passage across the blood brain barrier to bind with the metabotrophic glutamate 5 receptors [START_REF] Witte | Neuroprotective and abstinencepromoting effects of acamprosate elucidating the mechanism of action[END_REF].

Macrophages play an integral role in the development of innate and adaptive immune responses against bacterial pathogens. NFkappaB, one of the most ubiquitous transcription factor, plays a pivotal role when macrophages are activated, partly via increased IL-6 and iNOS expression, [START_REF] Miggin | New insights into the regulation of TLR signalling[END_REF] [START_REF] Vazquez-Torres | Oxygen-dependent anti-Salmonella activity of macrophages[END_REF] [START_REF] Fang | Antimicrobial reactive oxygen and nitrogen species: concepts and controversies[END_REF]. Inducible nitric oxide synthase (iNOS) is responsible for generating high levels of nitric oxide (NO) in activated macrophages.

Under conditions favouring the production of high concentrations of both NO and superoxide anion, the highly reactive peroxynitrite anion is also generated. Among the many inflammatory mediators produced by activated phagocytic cells, i.e. macrophages and microglia, NO production has been widely regarded as representative of inflammatory activation [19] [20]. Therefore inhibitors which, preferentially target molecules that are involved in NFkappaB activation may be of therapeutic value.

In the present studies, a new taurine prodrug has been synthesised, ethane sultam. Its mode of passage across the cellular membrane remains unknown. All tissue culture media and chemicals were purchased from Sigma (Belgium) unless stated otherwise. The pK a of ethane sultam was determined by titration using the reversible chromophoric change at 230nm, while hydrolysis of ethane sultam to the amino acid taurine as a function of pH was followed at 300nm.

2.2.Cell culture studies

A c c e p t e d M a n u s c r i p t 7

Isolation and cultures of alveolar macrophages

Alveolar macrophages were isolated from the rats by pulmonary lavage. In brief, the rats were anaesthetised with Nembutal, a catheter inserted into the trachea, and the lungs lavaged gently with phosphate buffered saline, pH 7.4, approximately 50 ml.

The alveolar macrophages were recovered after centrifugation at 1,200rpm for 10 min, and cell viability measured by trypan blue uptake extrusion (>98%). The cells at densities of 1 x10 5 or 2 x 10 5 cells, were plated in wells in Dubecco media supplemented with 10% foetal calf serum, containing penicillin (100 g /ml) and streptomycin (100g /ml). The cells were left to adhere overnight, washed, and resuspended in culture media to which lipopolysaccharides, LPS, (1 g/ml) or LPS (1g/ml) + interferon-gamma, 100 U/ml were added; and the cells were left for a further 24 h. The supernatants were removed and stored at -20 o C prior to analyses.

Preparation of immortalised N9 glial cells

The N9 microglial cell line was donated by Dr Paola Ricciardi Castagnoli (CNR Cellular and Molecular Pharmacology Centre, Milan, Italy). The cell line was originally derived from embryonic day 13 mouse microglial cultures [START_REF] Corradin | Inducible nitric oxide synthase activity of cloned murine microglial cells[END_REF]. The responses from these cells are very similar to those from primary rat microglia [START_REF] Kingham | Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release[END_REF]: [START_REF] Taylor | Stimulation of microglial metabotropic glutamate receptor mGlu2 triggers tumor necrosis factor alpha-induced neurotoxicity in concert with microglial-derived Fas ligand[END_REF]. N9 cells were maintained in DMEM supplemented with 5% fetal bovine serum, 50 M -mercaptoethanol, 50 U/l penicillin and 50 g/ml streptomycin at 37 o C in 5% CO 2 . The immortilised glial cells, N9 were grown to confluence and cells recovered after centrifugation at 1,200 rpm for 10 minutes. Cell viability was measured by trypan blue uptake extrusion (>98%). The glial cells were then plated at densities of 1 x10 5 or 2 x 10 5 cells, in Dubecco media supplemented with 10% foetal calf serum, containing penicillin (100 g /ml) and streptomycin (100g /ml).

A c c e p t e d M a n u s c r i p t

2.3.Cellular studies of the anti-inflammatory action of taurine and the pro-drug ethane -sultam

In vitro administration of taurine and ethane -sultam with alveolar macrophages Isolated alveolar macrophages were incubated with taurine or ethane -sultam, 10 and 50 mM for 24h. The cells were then stimulated with LPS, 1 g/ml, for a further 24 h. The cell supernatants were then removed and both nitrite and glutamate content assayed.

In vitro studies of taurine and ethane -sultam with immortalised N9 glial cells N9 glial cells were incubated with taurine or ethane -sultam, for 24h. The cells were then incubated with LPS, (1g/ml) for 24h. The supernatants were then removed and assayed for nitrite and glutamate content.

Nitrite assay

Aliquots, 100 l, of the cell culture supernatant were mixed with an equal volume of Griess reagent (1% sulphanilamide, 0.1% naphthalene diamine dihydrochloride, and 2.5% phosphoric acid) to determine nitrite concentration. After incubation at room temperature for 10 minutes, the optical density was measured at 560 mm. Sodium nitrite standards, in the range of 1-50 M, were prepared

Glutamate assay

Aliquots, 250 l, of the cell culture supernatant, + 640 l water, were mixed with Tris (0.1M)-EDTA (0.002M)-hydrazine (64%) to which-nicotinamide adenine dinucleotide (30mM) and adenosine 5'-diphosphate (100mM) had been added. The absorbance was read at 340nm after which glutamic dehydrogenase, 24 U, was added.

The absorbance was re-read after 40 minutes incubation. L-glutamate standards in the range 100-500mM were prepared.

Animal experiments

Male Wistar rats 200-250g, were housed in polypropylene cages (Iffa Credo, Belgium) and allowed ad libitum access to normal diet (Credo, Belgium) and water.

All animal procedures were in strict accordance with the recommendations of EEC (86/609/CEF) and with the Belgian 'projet de loi' (Moniteur Belge 19.02.1992, p.

3437) on the care and use of laboratory animals.

In vivo administration of taurine and ethane -sultam to control rats

Groups of rats, 200-220g, n=6 in each group, were administered, taurine or ethane -sultam, 0.15 mmol/kg/day, by gavage, volume = 1.5ml, at 10.00 am each day for 7 days. After this time, the alveolar macrophages were isolated by pulmonary lavage from each group of rats as well as from control rats that had received water, 1.5 ml, by gavage. The cells were stimulated with LPS, 1 g/ml, for a further 24 h.

The cell supernatants were then removed and nitrite and glutamate content assayed.

In vivo administration of ethane--sultam to a rat model of inflammation

A rat model of neuroinflammation has recently been described where ethanol is administered 3x/day for 2 days which is followed by 5 days of abstinence, for a period of 3 weeks [START_REF] Ward | Neuro-inflammation induced in the hippocampus of 'binge drinking' rats may be mediated by elevated extracellular glutamate content[END_REF]. Rats, 100-150g, n=6 in each group were administered the 1g ethanol /kg regime, supplemented or not with one dose/day of ethane--sultam, 0.15 mmol/kg/day, which was given by gavage, 30 minutes before the first ethanol administration. Ethane--sultam was administered daily for 3 weeks. Alveolar macrophages were isolated, plated at densities of 1 x10 5 , and then stimulated with LPS for 24h. The supernatants were then removed and both NO and IL-6 assayed.

A c c e p t e d M a n u s c r i p t 10

2.5.Mechanism of taurine's anti-inflammatory action

NFKappaB analysis in alveolar macrophages isolated from taurine supplemented rats

Rats received taurine orally, 12.5g/l, in the drinking water for 7 days, daily intake = 500 mg/day. = 15mmol/kg/day. The alveolar macrophages were isolated by pulmonary lavage and stimulated with LPS 1 g/ml and TNF 4 U/ml, for 15, 30 and 60 minutes. Nuclear proteins were then isolated from the alveolar macrophages. In brief, the cells were suspended in ice-cold buffer A (10 mM HEPES/KOH, 2 mM MgCl 2 , 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5 mM phenylmethylsulphonyl fluoride (PMSF), pH 7.9) and left on ice for 20 min before being vortex-mixed and centrifuged at 15,000 x g for 30 s. The nuclear pellets were gently suspended in cold buffer B (50 mM HEPES/KOH, 50 mM KCl; 300 mM NaCl, 0.1 mM EDTA, 10%

(by volume) glycerol, 1 mM DTT, 0.5 mM PMSF, pH 7.9) and left on ice for 20min.

After centrifugation at 15,000x g for 5 min at 4°C, aliquots of the supernatant containing the nuclear fraction were rapidly frozen in two aliquots in liquid nitrogen and stored at -80°C. The protein concentrations were assayed, by the BioRad method, in one of the aliquots prior to the electrophoretic mobility shift assay.

The nuclear fraction containing 20 g protein was incubated for 30 min at room temperature with 0.2 ng 32 P-labelled oligonucleotide probe, GATCAGGGACTTTCCGCTGGGGACTTTCCAG-3' or mutated.

5'-GATCACTCACTTTCCGCTGCTCACTTTCCAG-3' was added in excess (50x) in buffer. In each experiment the band of the DNA-protein complex from the unstimulated and stimulated macrophages was verified by comparison to the band obtained after stimulation of U937 with LPS, 10 g/ml. The intensity of each of the NFkappaB complex was quantitated.

Ikb studies in alveolar macrophages isolated from taurine supplemented rats

Rats received taurine in their drinking water at three different concentrations,

In vivo studies of the anti-inflammatory action of taurine and ethane -sultam in alveolar macrophages isolated from control rats.

A c c e p t e d M a n u s c r i p t

The alveolar macrophages were isolated from rats which had received daily doses of either ethane -sultam or taurine, 0.15 mmol/kg/day, by gavage or water for 7 days. These cells were cultured and then stimulated with LPS for 24h. Nitrite release was significantly decreased by both taurine and ethane-sultam, the latter to a greater extent than taurine, Figure 5.

In vivo studies of the anti-inflammatory action of ethane -sultam in alveolar

macrophages isolated from rats with alcohol-induced inflammation.

Binge drinking over a 3 week period activates the innate immune system which is verified by activated macrophages in the periphery and neuro-inflammation in the brain (Ward et al., 2009). The administration of ethane--sultam during the binge drinking regime reduced the activation of the innate immune system which was confirmed by decreases in the release of both NO and IL-6, Figure 6a and Figure 6b.

Investigation of taurine's anti-inflammatory action

NFkappaB activation in macrophages.

Figure 7a shows the electrophoretic mobility-shift assay for the activation of NFB by LPS and TNF in alveolar macrophages isolated from either taurine supplemented, stimulated at 15, 30 and 60 minutes, or control rats, stimulated at 15, 30, 60 and 120 minutes. The density band values identified the higher NFB activation in the controls at each time interval by comparison to the taurine supplemented rats, Figure 7b. It was noteworthy that NFkB activation was reduced at time 0 and at each of the subsequent time points assayed in the taurine supplemented alveolar macrophages by comparison to the controls.

IkappaB stabilisation.

A c c e p t e d M a n u s c r i p t

Figure 8a shows a Western blot for the macrophage cytosolic extracts, which had been immobilised on the membrane and then probed with IkB protein. The levels of IkB increased in the macrophage cytosolic fraction as the concentrations of taurine administered to the rats increased. The density band intensities showed that there was a significant increase with increasing taurine concentrations, Figure 8b.

A c c e p t e d M a n u s c r i p t

Discussion

In these present studies we have shown that the pro-drug ethane -sultam showed an enhanced ability to diminish the inflammatory response after stimulation with LPS in comparison to taurine. Variable results for the anti-inflammatory action of taurine in vitro, have previously been presented. For example, its protective effect in co-cultures was reported in intestinal Caco-2 cell monolayers from the damage by macrophage-like THP-1 cells, [START_REF] Zhao | Attenuation by dietary taurine of dextran sulfate sodium-induced colitis in mice and of THP-1-A c c e p t e d M a n u s c r i p t 25 induced damage to intestinal Caco-2 cell monolayers[END_REF] while no effect was reported, in peritoneal neutrophils [START_REF] Marcinkiewicz | Taurine chloramine down-regulates the generation of murine neutrophil inflammatory mediators[END_REF], RAW 264.7 cells [START_REF] Kim | The production of superoxide anion and nitric oxide by cultured murine leukocytes and the accumulation of TNFalpha in th conditioned media is inhibited by taurine chloramine[END_REF],or peripheral blood mononuclear cells [START_REF] Chorazy | Taurine chloramine modulates cytokine production by human blood mononuclear cells[END_REF].

In contrast the anti-inflammatory actions of its chlorinated form, taurine chloramine, in vitro, inhibits pro-inflammatory mediators, such as nitric oxide and TNF, in a variety of activated cell lines, including rodent macrophages [START_REF] Park | Taurine chloramine inhibits lymphocyte proliferation and decreases cytokine production in activated human leukocytes[END_REF] NR8383 cells, [START_REF] Barua | Taurine chloramine inhibits inducible nitric oxide synthase and TNF-alpha gene expression in activated alveolar macrophages: decreased NFkappaB activation and IkappaB kinase activity[END_REF] and peripheral blood mononuclear cells [START_REF] Chorazy | Taurine chloramine modulates cytokine production by human blood mononuclear cells[END_REF]. The lack of an effect of taurine alone in such systems may be attributable to its limited ability to enter cells, due to TauT down-regulation, as well as to the specific cell line employed, [START_REF] Ganapathy | Expression and regulation of the taurine transporter in cultured cell lines of human origin[END_REF].

The transcription of TauT is up-regulated by a number of compounds by virtue of the presence of various recognition sites on its promoter region. These sites include the TonE hypertonicity site (part of the TonE (tonicity-responsive element)/TonEBP (TonE-binding protein) system [START_REF] Ito | Expression of taurine transporter is regulated through the TonE (tonicity-responsive element)/TonEBP(TonE-binding protein) pathway and contributes to cytoprotcetion in HepG2 cells[END_REF] the TPA responsive element (TGAGTCAG), which is responsible for gene regulation by the protein kinase C (PKC)-mediated signal transduction pathway [START_REF] Park | Finding of TRE (TPA responsive element) in the sequence of human taurine transporter[END_REF] the glucose tumour suppressor gene, p53 [START_REF] Hartwell | Cell cycle control and cancer[END_REF], TNF and an NFB consensus-like sequence [START_REF] Mochizuki | Tumor necrosis factor  stimulates taurine uptake and transporter gene expression in human intestinal Caco2-cells[END_REF].

It was our intention in these studies to overcome such restrictions on its regulation by the use of taurine analogues and prodrugs that could circumnavigate the homeostatic control exerted by TauT. Previously other taurine pro-drugs have been investigated for their anti-inflammatory action, e.g. 5-aminosalicyltaurine, which

A c c e p t e d M a n u s c r i p t 18 liberates taurine and 5-aminosalicylic acid, and was reported to ameliorate chemically induced colitis after rectal administration [START_REF] Kim | A molecular mechanism for the anti-inflammatort effect of taurine-conjugated 5aminosalicylic acid in inflamed colon[END_REF]. In our recent studies of chronically alcoholised rats, oral administration of Acamprosate (400 mg/kg/day), calcium acetyl homotaurinate, during the chronic ethanol intoxication procedure exhibited an antiinflammatory effect by decreasing the formation of reactive oxygen radical species during the initial detoxification period, in comparison to rats chronically ethanol intoxicated alone [START_REF] Dahchour | Production of reactive oxygen species following acute ethanol or acetaldehyde and its reduction by acamprosate in chronically alcoholized rats[END_REF]. The exact mode of action remains unknown, but the presence of the acetyl and calcium moiety, as well as homotaurine contributed to its protective properties, mediated by metabotrophic GluR5 receptors in various brain regions [START_REF] Witte | Neuroprotective and abstinencepromoting effects of acamprosate elucidating the mechanism of action[END_REF].

In the present studies, we clearly showed that in vitro and in vivo, ethane -sultam exhibited greater anti-inflammatory actions than taurine in both alveolar macrophages and microglial cells after ex vivo LPS stimulation. Furthermore, it was shown that there was an increase in taurine levels in specific brain regions after three weeks of ethane--sultam (Della Corte, Dexter, Ward unpublished data) thereby confirming that the prodrug had indeed traversed cellular membranes. In contrast it is extremely difficult to increase brain taurine levels unless extremely high doses of taurine are administered.

Microglia are the resident immune cells of the central nervous system and play an important role in preserving the neurons. However when microglia are activated, by pro-inflammtory stimuli, substantial levels of glutamate are released [START_REF] Barger | Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation[END_REF]. This is due to the activation of NADPHoxidase which generate superoxide, leading to the formation of hydroxyl radical via Fenton chemistry, resulting in oxidative stress.

Depletion of reduced glutathione will occur, which will be remedied by the synthesis of more of this antioxidant by the influx of cystine via the Xc exchange system which releases glutamate [START_REF] Barger | Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation[END_REF]. In these present studies increases in glutamate release were A c c e p t e d M a n u s c r i p t apparent in microglia, but not macrophages, after bacterial stimulation. Expression of inducible nitric oxide synthase will also be enhanced after microglial activation as was evident in these present studies. Nitric oxide will deplete ATP production, and inhibit cytochrome oxidase in competition with oxygen, thereby inducing hypoxia and activating the hypoxia-inducible factors, which will result in the release of glutamate and excitoxicity [START_REF] Brown | Inflammatory neurodegeneration and mechanisms of microglial killing of neurons[END_REF]. However such glutamate release may also stimulate microglia to produce neurotrophic factors which will support neuronal survival and growth. This is mediated via increases in intracellular calcium mediated partly by the stimulation of NMDA receptors and group III metabotrophic glutamate receptors which induces intracellular calcium release from the endoplasmic reticulum, as well as stimulation of glutamate transporters to increase influxes of extracellular calcium [START_REF] Liang | Glutamate induces neurotrophic factor production from microglia via protein kinase C pathway[END_REF]. Such increases in intracellular calcium will lead to activation of the protein kinase C pathway which induces microglial neurtrotrophic expression and production.

Beta sultams are sulfonyl analogues of -lactams, and activated derivatives are able to inactivate serine enzymes, such as elastase, which is released in response to inflammatory stimuli and plays a major role in protein digestion following phagocytosis, and -lactamase, (which is the main cause of bacterial resistance to antibiotics) by sulfonation of the active site serine [START_REF] Page | Beta-sultams mechanism of reactions and use as inhibitors of serine proteases[END_REF]. The parent unsubstituted sultam does not inhibit serine enzymes but is slowly hydrolysed to taurine. The potential therapeutic use of ethane sultam as a prodrug has several advantages compared to taurine. These include an increased lipophilicity, which may facilitate its uptake by the cell prior to transformation to taurine [START_REF] Gupta | Taurine analogues; a new class of therapeutics: Retrospect and prospects[END_REF]. However it should be emphasised that simple diffusion of taurine or its pro-drugs across a lipid bi-layer against a concentration gradient, (i.e. the high concentration of taurine within the cell), might indicate that such prodrugs have an alternative passage across the NFkappaB is an important transcription factor that regulates genes involved in immunity and inflammation [START_REF] Li | NF-kappaB regulation in the immune system[END_REF]. Under normal conditions NFkappaB is present in the cytoplasm in an inactive state bound to the inhibitory protein IkappaB (IkB). decreases in IL-6 will diminish hepcidin secretion thereby preventing the internaliation of iron via the interaction of hepcidin with ferroportin [START_REF] Ward | Iron and the innate immune system[END_REF].

It has been reported that elderly patients (61-81 years) have significantly lower blood taurine concentrations, compared with younger individuals (20-38 years) [START_REF] Jeevanandam | Effect of major trauma on plasma free amino acid concentration in geriatric patients[END_REF].

Such a decline could exacerbate the oxidative damage that occurs during the ageing process as well as the modulation of various neurotransmitter systems and immune responsiveness. Taurine supplementation might reverse these effects [START_REF] Eppler | Dietary taurine manipulations in aged male Fischer 344 rat tissue: taurine concentrations, taurine biosynthesis and oxidative markers[END_REF]. In this respect, the taurine prodrug, studied here, which may exert their beneficial effect independently of TauT, might be particularly valuable. With an ever-increasing older population, palliative measures to retard the effects of ageing and neurodegenerative processes could be of enormous financial and social advantages.

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 tA c c e p t e d M a n u s c r i p t 6 2.

 6 Intracellularly ethane -sultam will hydrolysed to taurine, Figure1. It's antiinflammatory action has been studied both in vivo and in vitro, in alveolar macrophages isolated from rats, supplemented or not with ethane -sultam as well as in vitro in an immortalised microglial cell line, N9. Material and Methods.

A c c e p t e d M a n u s c r i p t 9 IL- 6 quantification

 96 IL-6 ELISA kit (R&D Systems, Abingdon, UK) was used for the quantitative measurement of this cytokine in the supernatants.

A c c e p t e d M a n u s c r i p t 12 All 1 .Figure 2 .A c c e p t e d M a n u s c r i p t 14 3. 2 .

 1212142 of the results are presented as mean + standard deviation. Statistical analysis was by ANOVA 1. Significance was calculated by GB Stat programme and set at P<0.05 by Fischer test. Hydrolysis of ethane--sultam to taurine.The hydrolysis of the ethane-1,2--sultam occurs with exclusive S-N fission and there is no NMR evidence of any reactions involving either C-S or C-N bond breaking, The pH rate profile for the unsubstituted -sultam shows only reactions that are either first order in hydronium-ion or hydroxide-ion concentration, but in aqueous sodium hydroxide solutions the rate becomes pH independent.The pK a of ethane -sultam was found to be 12.1 by means of a reversible chromophoric change at 230nm. In aqueous sodium hydroxide (NaOH) solutions a slow exponential decay of the chromophore of ethane -sultam was observed at 300 nm and 30 o C. The absorbance change had a very slow rate, which was found to be independent of hydroxide-ion concentration and gave a first order rate constant of 1.00 x 10 -5 s -1 . Above pH 7 ethane 1,2--sultam undergoes attack by hydroxide ion at the sulfonyl centre resulting in ring opening via cleavage of the S-N bond and formation of the -amino sulfonic acid, taurine. As a result, in solutions of pH greater than the pK a of ethane -sultam the rate of hydrolysis becomes pH independent as it depends on the concentration of the minor species, the neutral -sultam, which decreases as the pH is increased. The second order rate constant, k OH , for the alkaline hydrolysis of the ß-sultam is 5.16 x 10 -4 M -1 s -1 which is 20-fold lower than the second-order rate constants for the hydroxide ion hydrolysis, k OH , of simple N-alkyl--sultams e.g. N-methyl (1.41 x 10 -2 M -1 s -1 ). At pH 7 ethane-1,2--sultam is relatively stable and only very slowly hydrolysed to taurine. Below pH 7, ethane-1,2--sultam undergoes an acid catalysed hydrolysis to taurine. In vitro studies of the anti-inflammatory action of taurine or ethane -sultam in alveolar macrophages isolated from control rat and immortalised N9 microglial cells.The release of NO and glutamate from isolated alveolar macrophages after LPS or LPS + IFN stimulation is shown in Figure3a. A threefold increase in NO release was evident after LPS stimulation which increased to 4 fold with LPS + IFN stimulation. No changes in glutamate release were apparent after stimulation with either LPS or LPS + IFNFigure 3a.. In these initial studies it was important to ascertain whether ethane--sultam showed superior anti-inflammatory effects to that of taurine. Figure3bclearly showed that both taurine and ethane--sultam reduced nitrite release in LPS-stimulated alveolar macrophages, the latter to a greater extent than taurine. Levels of glutamate present in the supernatants from un-stimulated and stimulated alveolar macrophages did not alter after LPS stimulation (data not shown).

Figure 4a shows the

  Figure4ashows the release of NO and glutamate from N9 cells after stimulation with

A c c e p t e d M a n u s c r i p t 20 membrane

 20 , possibly via a protein transporter. In the present studies ethane sultam showed superior anti-inflammatory properties, both in vivo and in vitro to those of taurine.

  Stimulation with pro-inflammatory cytokines such as TNF initiates an intracellular signalling cascade, resulting in the phosphorylation and subsequent degradation of IkB by the 26S-proteasome. The degradation of IkB releases NFkappaB allowing its translocation to the nucleus, resulting in the activation of cyclooxygensae-2 (COX-2) cytokines, chemokines, cell surface receptors and adhesion molecules that are pivotal mediators of the immune and inflammatory responses. Therefore, therapeutic intervention against NFkappaB activation might be advantageous in preventing the progression of inflammation-related diseases. Previous studies have focused primarily on the action of taurine chloramine on this transcription factor. Taurine chloramine was shown to depress the migration of NFkappaB to the nucleus of activated NR8383 cells,[START_REF] Barua | Taurine chloramine inhibits inducible nitric oxide synthase and TNF-alpha gene expression in activated alveolar macrophages: decreased NFkappaB activation and IkappaB kinase activity[END_REF] and murine peritoneal macrophages and RAW 264.7 cells[START_REF] Kim | Inhibition of LPS-induced NO production by taurine chloramines in macrophages is mediated through Ras-ERK-NF-kappaB[END_REF]. Attenuation of ERK½ activation was also reported in the latter study.Treatment with taurine chloramine will result in decreased phosphorylation[START_REF] Fan | Tyrosine phosphorylation of IkBa activated NFkB through a redox-regulated and c-Src-dependent mechanism following hypoxis reoxygenation[END_REF] [START_REF] Barua | Taurine chloramine inhibits inducible nitric oxide synthase and TNF-alpha gene expression in activated alveolar macrophages: decreased NFkappaB activation and IkappaB kinase activity[END_REF] and oxidation of IB and a lower activity of IB kinase,[START_REF] Barua | Taurine chloramine inhibits inducible nitric oxide synthase and TNF-alpha gene expression in activated alveolar macrophages: decreased NFkappaB activation and IkappaB kinase activity[END_REF]. The present in vivo studies demonstrated a clear interaction between taurine and IkB, with taurine showing a dose dependent stabilisation of this inhibitory protein. Although these studies were not undertaken with ethane--sultam, the corresponding changes in both NO and IL-6 after the incubation with this taurine pro-drug would confirm indirectly that these changes were occurring via changes in NFkappaB activation. Such changes A c c e p t e d M a n u s c r i p t 21 in IL-6 release are also important in decreasing the iron loading of the tissues, since

Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Rate of hydrolysis of ethane--sultam to taurine from pH2-pH10. . The

Figure 3a .

 3a Figure 3a. Release of nitrite and glutamate from alveolar macrophages after

Figure 3b .

 3b Figure 3b. Release of nitrite from alveolar macrophages after ex vivo incubation

Figure 4a .

 4a Figure 4a. Release of nitrite and glutamate from N9 microglial cells after stimulation

A c c e p t e d M a n u s c r i p tFigure 4b .

 4b Figure 4b. Nitrite release into the culture media from N9 microglial cells after

Figure 4c .

 4c Figure 4c. Glutamate release into the culture media from N9 microglial cells after

Figure 5 .

 5 Figure 5. Release of nitrite from alveolar macrophages isolated from rats, n=6 in each

Figure 6 .

 6 Figure 6. Changes in the release of NO, Figure 6a and IL-6, Figure 6b, from

Figure 7a .

 7a Figure 7a. Electromobility shift showing NFkappaB activation in alveolar

Figure 7b .Figure 8a .

 7b8a Figure 7b. Density intensities for each NFkappaB band

Figure 8b .

 8b Figure 8b. Density intensities for Ikappab three individual bands from each

A c c e p t e d M a n u s c r i p t

Figure 1

 1 Figure 1.

  Figure 2

  Figure 3a

  Figure 3b

  Figure 4a

  Figure 4b.

  Figure 4c

Figure 5 Figure 5 A

 55 Figure 5.

Figure 8

 8 Figure 6a

Acknowledgements

Financial support from IREB, (RJW), ERAB (RJW, DTD and LDC) and COST D34 are gratefully acknowledged