An analysis of the physiological condition of prepubertal gilts indicates that ZEN acts as both an undesirable substance and an endocrine disruptor (ED) [4
]. Even when ingested at MABEL, NOAEL (highest) and LOAEL (very low) doses, ZEN significantly increases the concentrations of selected hormones and causes hyperestrogenism, i.e., supraphysiological hormone levels [4
], in prepubertal gilts. Zearalenone is also characterized by a non-monotonic dose-response curve (according to the principle of hormesis [43
]). Therefore, the results of research studies investigating the effects of different ZEN doses on tissues [44
], cells [46
] and cell organelles [47
] are difficult to compare.
3.1. Zearalenone and Its Metabolites in the Heart Muscle
In the present study, the carry-over of ZEN and its metabolites in the myocardium of prepubertal gilts was highly individualized (absence of significant differences due to high variation in SD values) (Table 1
). The presence of ZEN and a steady increase in its concentrations, proportional to the administered dose, were noted in the myocardium of gilts in groups E1 and E2 on D1. Zearalenone levels were much lower in group E3 on D1, which is partially consistent with previous findings [44
]. Similar conclusions were drawn by Gajęcka et al. [17
] from a study of female wild boars. The concentrations of α-ZEL (rising trend) and β-ZEL in the myocardium were inversely proportional to each other, which, in our opinion, is a normal response [4
]. The bioavailability of ZEN and its metabolites in the myocardium is affected by biotransformation processes in prepubertal females. Interestingly, the distribution of ZEN and metabolite concentrations in the myocardium was similar to the values reported in blood by Rykaczewska et al. [4
]. In contrast to the results reported by Yan et al. [44
], ZEN metabolites were not detected on D1 (or were below the sensitivity of the method), which could be due to the low supply of endogenous steroid hormones. According to other studies [48
], a deficiency of ovarian hormones in mammals leads to pressure overload, thus compromising cardiac function. Supplementation with 17β-estradiol [50
] or mycoestrogen can reverse these effects or alter the profile of estrogen hormones (by modulating feminization) [4
]. It should also be stressed that the 7th day of exposure (D1) marks the end of adaptive processes, in particular adaptive immunity [52
]. These substances could also be used as substrates that regulate the expression of genes encoding hydroxysteroid dehydrogenase [3
], a molecular switch that enables the modulation of steroid hormone prereceptors. These processes were most visible in group E1, where only the parent mycotoxin was detected (100%). In the remaining groups, the presence of metabolites was noted, and their concentrations increased proportionally to the applied dose. The observations made in group E1 (MABEL dose) indicate that prepubertal females utilize even the smallest amounts of estrogen-like substances (what are they zearalenone and its metabolites—[44
]) to compensate for endogenous estrogen deficiency (inducing supraphysiological hormonal levels in prepubertal females—[4
]), which can increase cardiac automaticity [53
On D2, ZEN concentrations increased proportionally to the administered dose and were higher than on D1. In group E1, the proportions of both metabolites (%) were higher than in groups E2 and E3 (group E1: ZEN—73.62%, α-ZEL—11%, β-ZEL—15.37%; group E2: 91.48%, 5.82% and 2.68%, respectively; group E3: 93.42%, 5.96% and 0.61%, respectively). Similar to D1, the concentrations of α-ZEL (rising trend) and β-ZEL in the myocardium were inversely proportional to each other. The levels of α-ZEL were higher, whereas β-ZEL levels were lower in groups E2 and E3. This could result from the saturation of myocardial tissue with ZEN and its metabolites, e.g., active estrogen receptors [54
] as well as other factors that influence the demand for ZEN and ZEN-like mycotoxins over time of exposure [55
]. Unlike in the current experiment, Yan et al. [44
] did not detect ZEN, but identified both ZEN metabolites in samples of heart muscle tissue. However, the cited study was conducted in vitro, and the animals’ age or sex were not specified, which makes it impossible to directly compare the above results with our findings.
The carry-over of ZEN, an exogenous estrogen-like substance, from the porcine gastrointestinal tract to myocardial tissue via the blood was also analyzed by calculating the CF. The CF values for myocardial tissue in prepubertal gilts have never been determined in the literature, in particular during exposure to three low, monotonic doses of ZEN for 21 consecutive days. Even a cursory analysis of CF values indicates that the accumulation of ZEN and its metabolites was much lower in the myocardium than in the blood [4
]. Mycotoxin concentrations ranged from 1 × 10−1
to 1 × 10−3
in the blood, and from 0 (only in group E1 on D1 for both metabolites) to 1 × 10−7
(in the remaining groups on both D1 and D2) in the myocardium. These observations suggest that differences in carry-over decrease the accumulation of ZEN and its metabolites in the myocardium [48
]. These differences are very difficult to explain. Based on the existing knowledge and the extrapolation of previous results, it could be suggested that by disrupting endocrine processes, EDs exert specific effects on cells and tissues [4
] and modulate the structure and functions of the heart muscle [48
]. Most importantly, EDs can induce different responses, depending on the dose, exposure duration and the stage of growth and development in mammals [15
], in particular females.
Therefore, it can be hypothesized that low doses of undesirable substances (including ZEN) exert minor or much smaller effects on myocardial homeostasis, compared with other cells and tissues in the studied animals due to much lower availability.
3.2. Isometric Tension Analyses
The vasodilatory and vasoconstrictive properties of isolated porcine coronary arteries with an intact endothelium, which regulate vascular smooth muscle contraction, were also analyzed in the study. The blood vessels in various organs and species may respond differently to agonists and antagonists. Potassium chloride and acetylcholine induce vasocontraction, whereas sodium nitroprusside induces vasodilation in porcine coronary arteries (PCAs).
The KCl-induced contraction of the PCAs was enhanced in groups E1 and E2 on D1. However, a decreased response was noted in group E3. On D2, KCl-induced vasoconstriction did not differ in groups E1 and E2, but it decreased further in group E3. These results indicate that the sensitivity of smooth muscles of PCAs to K+ is highly dependent on the concentrations of ZEN in the diet and the duration of exposure to this mycotoxin.
Acetylcholine-induced contraction decreased in group E3, which is similar to the response observed for KCl, so this effect might not be entirely dependent on the muscarinic receptors. Surprisingly, decreased response was also observed in group E1 but not in group E2. Acetylcholine’s effect on vascular tension is dependent on muscarinic receptors [57
], which suggests that ZEN is able to modulate the function of these receptors.
Sodium nitroprusside is a donor of exogenous nitric oxide with the endothelium-independent effect. In this study, the sensitivity of PCAs to the nitric oxide was increased in group E3 after prolonged exposure. Surprisingly, arterial sensitivity to nitric oxide decreased in group E1, which suggests that the sensitivity of smooth muscles to nitric oxide changes in response to dietary ZEN, which is endothelium-independent mechanism. These results also indicate that smooth muscles of PCA are targeted by ZEN and its metabolites, and that ZEN my regulate the mechanism(s) of nitric oxide synthesis, which is dose- and time-dependent. Further investigation is needed to examine the mechanism(s) underlying different responses to ZEN and their potential dependence on the endothelium.
The analysis of the effects of COX and e-NOS inhibitors shed a new light on the properties of ZEN. COX inhibitors potentiated ACh-induced vasoconstriction only in group E3. This response decreased in groups E1 and E2, whereas no significant changes were found in the control group. These findings suggest that ACh-induced vasoconstriction in group E3 was at least partly dependent on the net vasodilator effect of prostanoids, whereas the decreased response in groups E1 and E2 was dependent on the vasoconstrictor effect of prostanoids. e-NOS inhibitors increased vasoconstriction in all groups (C, E1, E2 and E3), which indicates that nitric oxide plays a key role in vascular tone regulation of PCA. However, this effect was more pronounced in groups E3 (3.39-fold) and E1 (2.48-fold) than in group E2 (1.29-fold) and the control (1.36-fold), which suggests that ZEN is able to modulate the bioavailability or sensitivity of nitric oxide. When both COX and e-NOS are blocked, mechanisms other than prostanoids and nitric oxide are engaged in vascular tone regulation. These mechanisms are regulated by hormonal changes and, possibly, ZEN. Acetylcholine’s effects were potentiated in the presence of COX and e-NOS inhibitors in group E3, but not in groups E1 or E2.
These results indicate that a different mechanism is responsible for the net vasoconstrictor effect which was upregulated only in group E3. The vasodilator effect of prostanoids (PGI2
) and nitric oxide was upregulated by the administered ZEN dose. The endothelium-derived hyperpolarizing factor (EDHF) could be yet another mechanism of vascular control. The major routes of EDHF regulation include the metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs), potassium channels, gap junctions and hydrogen peroxide [58
]. However, further research is needed to clarify the exact mechanism(s), including EDHF, by which ZEN acts on PCA.