Expresión de acción colectiva de tipo académica

The sex steroids play an integral part in determining the reproductive status of an animal, and are involved in a complex series of feedback controls. Any influence on hormone concentrations or feedback mechanisms has the potential to influence reproductive cycles, conception opportunities and tissue preparation for pregnancy. The effects of soy and purified isoflavones on gonadotrophins (LH and FSH), progesterone (P 4), sex hormone binding globulin (SHBG), E2, oestrone (E1 ), and testosterone are variable within the literature.

A number of studies have reported a lack of effect on certain hormone profiles (Cassidy et al. 1 994; Anthony et al. 1 996; Awoniyi et al. 1 998; Uesugi et al. 2003 ; Malaivijitnond et al. 2004; Hidalgo et al. 2005 ; Nettleton et al. 2005a). On the other hand, some studies have demonstrated decreased LH (Cassidy et al. 1 994 and 1 99 8 ; Duncan et al. 1 999a; Bennetau-Pelissero et al. 200 1 ; Wu et al. 2005), E2, and P4 concentrations (Awoniyi et al. 1 998; Cassidy et al. 1 998; Duncan et al. 1 999ab; Badger et al. 200 1 ; Bennetau-Pelissero et al. 200 1 ; Wu et al. 2005), as well as dehydroepiandrosterone sulphate concentrations (Cassidy et al. 1 998). Additionally, increased sex hormone binding globulin and a decreased E2 : SHBG ratio were reported by others (Duncan et al. 1 999b; Pino et al. 2000), which may indirectly alter the oestrogenic exposure of reproductive tract tissues. In contrast, modulations of morphological reproductive parameters have been reported to occur in the absence of hormonal changes (Malaivijitnond et al. 2004; Hidalgo et al. 2005), suggesting isoflavones may function locally at the tissue level.

The highly variable diurnal fluctuations in steroid hormones may render them insensitive and relatively unreliable biomarkers for isoflavone action. However, the variable duration of exposure, interindividual variation in metabolic capacity and the form/route of isoflavone administration are also likely to complicate comparisons between studies. Effects on steroid hormone concentrations must be considered in conjunction with histological changes in order to determine if changes are elicited via perturbation at the hypothalamus-pituitary or gonadal level. Ultimately though, if cycles and/or reproductive tissues are unaffected, any alteration in hormone concentrations is of dubious clinical relevance.

1 .5.2.5. Reproductive development

Development of the various components of the reproductive system occurs during critical windows in an animal ' s life. Exposure to compounds with oestrogenic potential during these periods may alter the normal endocrine environment. However, the effect of isoflavone consumption on the development of reproductive parameters appears to vary

according to species, as well as with the type of isoflavone consumed, period of exposure and duration of exposure.

The majority of the literature in rodents points towards the ability of isoflavones to accelerate puberty onset (Whitten and Naftolin 1 992; Hilakivi-Clarke et al. 1 998; Casanova et al. 1 999; Gallo et al. 1 999; B adger et al. 200 1 ; Delclos et al. 200 1 ; Kouki et al. 2003 ; Lewis et al. 2003 ; Nikaido et al. 2004 and 2005 ; Takashima-Sasaki et al. 2006), although one study also demonstrated a delay in puberty (Levy et al. 1 995). Altered development of the neuroendocrine system has occurred after isoflavone exposure (Faber and Hughes 1 993 ; Whitten et al. 1 993 ; Levy et al. 1 995; Zhao et al. 2004), while modulations in ovarian function, cyclicity and aberrant sexual differentiation of the hypothalamus and pituitary cells have also been reported (Polkowska et al. 2004; W6jcik­ Gladysz et al. 2005 ; Patisaul et al. 2006). Contrastingly, there is also strong evidence to support a lack of developmental effects following isoflavone exposure (Awoniyi et al. 1 998; Strom et al. 200 1 : Nagao et al. 200 1 ; Kang et al. 2002 ; Hughes et al. 2004; Pace et al. 2004; Naciff et al. 2004; Ryokkynen et al. 2005 ; Pace et al. 2006; Wilhelms et al. 2006).

The pre-pubescent development of the reproductive system is critical in determining life­ time reproductive capacity and success. Interference during this developmental stage is often irreversible and, depending on the nature and extent of perturbation, may result in altered reproduction later in life. As such, isoflavone-induced changes in normal reproductive development, which may not be detected for significant periods of time following isoflavone exposure, have great potential for the modulation of reproductive parameters in adulthood. Yet, as with other factors discussed for isoflavone bioavailability and reproductive effects, the consequences of isoflavone intake on reproductive development are far from predictable.

1 .5.2.6. Molecular and biochemical activity

Examination of the ability of endocrine disruptors to regulate oestrogen-sensitive genes is thought to provide a reliable method for the characterisation of in vivo oestrogenicity (Diel et al. 2000). Indeed isoflavones have produced changes in gene expression, cell signalling and other molecular level effects in the absence of perturbations in other reproductive endpoints (Diel et al. 2000; Brown and Setchell 200 1 ). Changes have also been observed fol lowing isoflavone exposure that was not predicted from studies with E2 (Adachi et al. 2005).

Gene transcription, apoptosis, cell-cycle regulation, cellular proliferation, anti-oxidant pathways, signal transduction pathways as well as genes concerned with oncogenesis have all shown sensitivity to genistein exposure (Konstantakopoulos et al. 2006). However, these in vitro effects may not translate to detectable effects in vivo, especially considering the high potential for other interacting and confounding variables in an in vivo environment. Yet, although genistein concentrations at the lower end of the physiological range may not exert significant in vitro effects on gene transcription, it is important to consider that the duration of in vivo exposure may be longer than that of in vitro studies (Konstantakopoulos et al. 2006). Additionally, in vitro concentrations are based on circulating isoflavone concentrations in plasma and serum, which may not reflect concentrations occurring at the tissue level where accumulation may occur, especially in target tissues (Konstantakopoulos et al. 2006; Section 1 .3 .6).

Overall, the clinical significance/relevance of altered gene expression in the absence of histological changes is unclear and requires long term studies to fully elucidate. The complexities of animal physiology suggest that findings of a perturbation at the genetic or molecular level should be interpreted with caution.

1 . 5 .2.6.a. Sex steroid receptor and oestrogen-responsive gene regulation

Modulation of ERs and oestrogen-response element (ERE)-dependent gene expression by genistein is one of the primary mechanisms implicated in the reproductive potency of genistein and other isoflavones (Diel et al. 2000; Cotroneo et al. 200 1 ; Lee et al. 2004; Staar et al. 2005; Diel et al. 2006; Seo et al. 2006; Chrzan and Bradford 2007). Interactions between endogenous hormones and their receptors are critical in the control and activation of reproductive processes, and form the basis of endogenous hormone control over reproduction. Therefore, by binding to sex steroid receptors, or altering their expression, isoflavones may modify the activity of endogenous hormones and influence normal reproductive functions.

Changes (up-regulation or down-regulation) in receptor expression have been shown to occur in an ovariectomised (OVX) animal model in which circulating hormones were unaffected by isoflavones (Cotroneo et al. 200 1 ). Therefore, induction of receptor expression is unlikely to occur as a result of changes in circulating steroid hormone concentrations, indicating direct gene expression effects. However, effects are not uni­ directional and both down-regulation (Sathyamoorthy and Wang 1 997; Cotroneo et al. 200 1 and 2005; Diel et al. 2005; Wang et al. 2005; Padilla-Banks et al. 2006; Seo et al. 2006), and up-regulation (Jefferson et al. 2002a; Lee et al. 2004; Petroff et al. 2005 ; Staar et al. 2005) of the ER has been reported.

The effect of isoflavones on the two sub types of the ER (a and �), varies according to species, tissue, cell system and laboratory. Some studies have reported a lack of effect on ER�, but significant effect on ERa (Cotroneo et al. 200 1 ; Lee et al. 2004; Petroff et al. 2005), while others have reported a significant effect on both (Patisaul et al. 200 1 ; Ren et al. 200 1 ; Staar et al. 2005 ; Cappelletti et al. 2006; Padilla-Banks et al. 2006; Takashima­

Sasaki et al. 2006; Chrzan and Bradford 2007). However, ER� ligands (such as genistein) may regulate some oestrogen-responsive components via the ERa pathways, without any modulation of ER� expression (Lee et al. 2004). Furthermore, genistein is capable of modifying the relative distribution of ERa and ER� subtypes, but the effect

changes according to the cell type investigated ( Cotroneo et al. 200 1 ; Cappelletti et al. 2006).

1 .5 .2.6.b. Growth factors, cellular proliferation and enzyme inhibition

The reported isoflavone-induced changes in cellular or tissue growth, development and/or hormone profiles may occur as indirect consequences of isoflavone interference in critical enzyme or growth factor pathways. Studies have shown that growth factors are influenced by isoflavones, each with distinct and variable suppressive or proliferative growth effects. Suppression of cellular growth by isoflavones may be mediated by isoflavone-induced inhibition of proliferating growth factors (e.g. Epidermal Growth Factor, EGF), and/or by production and expression of growth-inhibiting factors (e.g. Transforming Growth Factor � (TGF-�) (Bames et al. 2000). In contrast, the uterotrophic effects of isoflavones may occur as a result of action to increase uterine stromal EGF, reduce uterine and blood vessel EGF receptor and/or decrease TGFa in the glandular and luminal uterine epithelium (Brown and Lamartiniere 2000; Cotroneo et al. 200 1 ). The reported ability of isoflavones to protect against certain inflammatory responses may be the result of their inhibition of cytokines such as Tumour Necrosis Factor a (TNF-a) (Kang et al. 2005).

Isoflavones are known to inhibit protein phosphorylation, and this has been reported as their mechanism of action against the EGF receptor (Peters on and Bames 1 993; Bames et al. 2000). Interestingly, daidzein and equol are both capable of inducing phosphorylation to a 2-fold greater extent than achieved by E2 (Totta et al. 2005). Nonetheless, protein phosphorylation is not the universal mechanism by which isoflavones exert their influence on growth factors (Bames et al. 2000). Transcriptional changes rather than direct activity on tyrosine kinase activity have also been reported, whilst certain tyrosine kinases actually appear unresponsive to isoflavones (Brown et al. 1 998; B ames et al. 2000).

Isoflavones are also capable of modulating the production of other enzyme systems, such as aromatase (Adlercreutz et al. 1 993 ; Edmunds et al. 2005 ; Lacey et al. 2005), which has consequences for oestrogen metabolism. However, inhibition of the enzymatic conversion of pregnalone to P 4, and androstenedione to E2 following eo-incubation of genistein or daidzein with human granulosa-luteal cells were only achievable in vitro at concentrations � 1 0 J.lM (Lacey et al. 2005). This suggests that some enzyme-mediated effects of isoflavones are unlikely to occur in vivo.

Markers for cellular proliferation (e.g. proliferative cellular nuclear antigen, PCNA), and apoptosis are reported to be unaffected by soy consumption (Tansey et al. 1 998; Eason et al. 2005). However, purified genistein appeared to be capable of eliciting significant changes in proliferative endpoints, both in vivo and in vitro at physiological concentrations (Eason et al. 2005 ; Ford et al. 2006; Garcia-Perez et al. 2006). These changes show a concentration-dependent response, with E2-induced proliferation enhanced by genistein at low concentrations ( < 1 0 J.lM), but ERE and cellular proliferation suppressed at higher concentrations (Wang et al. 1 996; Po et al. 2002). This high-concentration suppressive effect was suggested to be due to cytotoxic events, since inhibition was demonstrated in ER-negative cell lines (Wang et al. 1 996). Daidzein has also been suggested to elicit its effects in this way (Lehmann et al. 2005) . However, again caution must be heeded when interpreting findings that cannot be replicated in studies utilising natural dietary forms of genistein.