**4. Particular concepts related with EDCs**

For decades, two major interrelated concepts are particularly addressed regarding EDCs: the low dose effect and non-monotonous dose–response relationships (e.g. "inverted U-shapes" of the dose–response curve).

Like hormones, some of EDCs act at low or very low doses, other variable, therefore their blood levels are not reflecting the real activity [42].

The traditional toxicological endpoints are not sufficient to preclude the adverse outcome. Therefore for the endocrine-sensitive endpoints it was suggested to set the NOAEL (no observed adverse effect level) or the LOAEL (lowest observed adverse effect level) from traditional toxicological studies or even below the range of human exposures, as the highest dose in experiments designed to test EDCs. For example, low-dose effects of BPA should be investigated in rodents exposed to 400 µg/kg bw/day BPA or lower, because this concentration produces levels of unconjugated BPA in the range of human blood concentrations [43]; this level is incomparable lower with the classical developmental studies, where LOAEL corre‐ sponds to 50 mg/kg bw/day [44]. Actually, most effects were seen at doses below 50 µg/kg [43], so even lower concentration than those normally detected in humans may induce adverse effects.

The effect of EDCs also depends on the type of tissue and the expression of hormone receptors on those cells, therefore the effect is considered to be tissue specific. Taking into account that some EDCs can exhibit different potencies on different receptors isoforms (e.g ERα or ERβ), the effect is also receptor-selective.

A well-known example is related to methyl-and propylparaben. *In vitro*, both parabens are binding to estrogen receptors (ERα and β), but methylparaben exhibits a weak estrogenic activity, while for propylparaben the estrogenic potential is a stronger [45,46]; *in vivo* parabens estrogenic potencies are comparable [46]. The relative activity of parabens compared with estradiol (E2) is 1000 times lower [47]. Interesting, the estrogenic effects of parabens are not modulated only by estrogen receptors, but are also related to the inhibition of sulfotransferases in skin, elevating the local level of free estrogens [48].

By comparison with native hormones, EDCs exhibits lower affinity for hormone receptors, with some exceptions, such as TBT, which is the most potent agonist of retinoid-X-receptor and PPARγ (peroxisome proliferator activating receptor subtype gamma) in the low nano‐ molar range [49].

The shape of the dose-response curve for EDCs does not follow the usual dose-response curve. The curve can have a sigmoidal shape (relationship between dose and effect occure based on the saturability of the receptors), but in general EDCs do act via a non-monotonic doseresponse relationship [43]. In this case, the slope of the curve changes sign somewhere within the range of the examined doses. In other words, some effects can be seen at very low doses, while slightly higher doses can show no effects and then, at high doses, some different types of effects may be found.

For example, hypoglycaemic or hyperglycaemic effects of TCDD (tetrachlorodibenzo-pdioxin) observed in animal models are dose-dependent. Repeated low-dose of TCDD (500 ng/kg p.o.) reduced glucokinase gene expression in mice [50], while higher dosage (12.8 µg/kg TCDD p.o) induced a significantly reduction of serum glucose levels [51]. More‐ over, a higher dose of TCDD (116 µg/kg i.p.) impaired insulin-stimulated glucose uptake in mice [52].

Another example is BPA. On isolated pancreatic islets of Langerhans BPA induced an increase of insulin content following an inverted U-shape dose response curve, with a significant effect observed at 1 nM and 10 nM BPA compared to vehicle. Higher concentrations of BPA (1 µM) produced no increase in insulin content [53].

Like hormones, some of EDCs act at low or very low doses, other variable, therefore their blood

The traditional toxicological endpoints are not sufficient to preclude the adverse outcome. Therefore for the endocrine-sensitive endpoints it was suggested to set the NOAEL (no observed adverse effect level) or the LOAEL (lowest observed adverse effect level) from traditional toxicological studies or even below the range of human exposures, as the highest dose in experiments designed to test EDCs. For example, low-dose effects of BPA should be investigated in rodents exposed to 400 µg/kg bw/day BPA or lower, because this concentration produces levels of unconjugated BPA in the range of human blood concentrations [43]; this level is incomparable lower with the classical developmental studies, where LOAEL corre‐ sponds to 50 mg/kg bw/day [44]. Actually, most effects were seen at doses below 50 µg/kg [43], so even lower concentration than those normally detected in humans may induce adverse

The effect of EDCs also depends on the type of tissue and the expression of hormone receptors on those cells, therefore the effect is considered to be tissue specific. Taking into account that some EDCs can exhibit different potencies on different receptors isoforms (e.g ERα or ERβ),

A well-known example is related to methyl-and propylparaben. *In vitro*, both parabens are binding to estrogen receptors (ERα and β), but methylparaben exhibits a weak estrogenic activity, while for propylparaben the estrogenic potential is a stronger [45,46]; *in vivo* parabens estrogenic potencies are comparable [46]. The relative activity of parabens compared with estradiol (E2) is 1000 times lower [47]. Interesting, the estrogenic effects of parabens are not modulated only by estrogen receptors, but are also related to the inhibition of sulfotransferases

By comparison with native hormones, EDCs exhibits lower affinity for hormone receptors, with some exceptions, such as TBT, which is the most potent agonist of retinoid-X-receptor and PPARγ (peroxisome proliferator activating receptor subtype gamma) in the low nano‐

The shape of the dose-response curve for EDCs does not follow the usual dose-response curve. The curve can have a sigmoidal shape (relationship between dose and effect occure based on the saturability of the receptors), but in general EDCs do act via a non-monotonic doseresponse relationship [43]. In this case, the slope of the curve changes sign somewhere within the range of the examined doses. In other words, some effects can be seen at very low doses, while slightly higher doses can show no effects and then, at high doses, some different types

For example, hypoglycaemic or hyperglycaemic effects of TCDD (tetrachlorodibenzo-pdioxin) observed in animal models are dose-dependent. Repeated low-dose of TCDD (500 ng/kg p.o.) reduced glucokinase gene expression in mice [50], while higher dosage (12.8 µg/kg TCDD p.o) induced a significantly reduction of serum glucose levels [51]. More‐ over, a higher dose of TCDD (116 µg/kg i.p.) impaired insulin-stimulated glucose uptake

levels are not reflecting the real activity [42].

the effect is also receptor-selective.

in skin, elevating the local level of free estrogens [48].

effects.

224 Treatment of Type 2 Diabetes

molar range [49].

of effects may be found.

in mice [52].

A similar non-monotonic behaviour is exhibited by BPA in animal studies where treatment with high dosage (BPA 100 µg/kg bw/day) twice per day for 4 days increased pancreatic insulin content, produced hyperinsulinemia, and induced insulin resistance in adult male mice [54] while sustained exposure of pregnant mouse dams to lower levels of BPA (10 µg/kg bw/day) from gestation day 9–16 impaired glucose tolerance, increased plasma insulin, triglycerides and leptin concentrations, thus revealing the ability of BPA to alter pancreatic function and metabolic parameters [55].

Also PBBs, especially PBDE-153 (polybrominated diphenyl ether) showed an inverted Ushaped association with metabolic syndrome in epidemiological study in humans [56].

So, the most important effects of EDCs observed in animal models are those that occur at low doses, similar with the level of human environmental exposure, therefore only these toxico‐ logical data should be corroborated with epidemiological studies.

We also should note that for the assessment of EDCs effects, the assumption of an experimental threshold (like a NOAEL) is questionable. A first reason is related to the lack of adversity for some endpoints investigated (e.g., uterotrophic assay). A second reason is connected with the difficulties to establish it. According to Blair et al. [57], a threshold could be established in the absence of endogenous hormone at some life stage, if the endogenous hormone induces no adverse effect or if there is effective homeostatic control. Even if a threshold does exist, for a certain endpoint, taking into account the population variability and the connection with already ongoing biological process (EDCs exhibit additive effects), the threshold will not be observable.
