In vitro cadmium exposure induces structural damage and endothelial dysfunction in female rat aorta

Cadmium is a heavy metal that is widespread in the environment and has been described as a metalloestrogen and a cardiovascular risk factor. Experimental studies conducted in male animals have shown that cadmium exposure induces vascular dysfunction, which could lead to vasculopathies caused by this metal. However, it is necessary to investigate the vascular effects of cadmium in female rats to understand its potential sex-dependent impact on the cardiovascular system. While its effects on male rats have been studied, cadmium may act differently in females due to its potential as a metalloestrogen. In vitro studies conducted in a controlled environment allow for a direct assessment of cadmium's impact on vascular function, and the use of female rats ensures that sex-dependent effects are evaluated. Therefore, the aim of this study was to investigate the in vitro effects of Cadmium Chloride (CdCl2, 5 µM) exposure on vascular reactivity in the isolated aorta of female Wistar rats. Exposure to CdCl2 damaged the architecture of the vascular endothelium. CdCl2 incubation increased the production and release of O2•−, reduced the participation of potassium (K+) channels, and increased the participation of the angiotensin II pathway in response to phenylephrine. Moreover, estrogen receptors alpha (Erα) modulated vascular reactivity to phenylephrine in the presence of cadmium, supporting the hypothesis that cadmium could act as a metalloestrogen. Our results demonstrated that in vitro cadmium exposure induces damage to endothelial architecture and an increase in oxidative stress in the isolated aorta of female rats, which could precipitate vasculopathies. Graphical Abstract. Own source from Canva and Servier Medical Art servers Graphical Abstract. Own source from Canva and Servier Medical Art servers


Introduction
Cadmium (Cd) is a non-essential metal that, in its ionic form, is considered a highly toxic element to the environment, as it has high transfer rates to the soil (ATSDR 2012;Satarug 2018).Cd is obtained from geogenic processes and anthropogenic sources, including the burning of fossil fuels, soil contamination from improper disposal of electronic waste, and the use of phosphate fertilizers (Jacobson and Turner 1980;Faroon et al. 2012;Sripada and Lager 2022).
Population exposure to Cd primarily occurs through soil and food contamination.In regions with high levels of contamination, vegetables and tobacco leaves tend to accumulate significant amounts of Cd from the soil, thereby becoming sources of Abstract Cadmium is a heavy metal that is widespread in the environment and has been described as a metalloestrogen and a cardiovascular risk factor.Experimental studies conducted in male animals have shown that cadmium exposure induces vascular dysfunction, which could lead to vasculopathies caused by this metal.However, it is necessary to investigate the vascular effects of cadmium in female rats to understand its potential sex-dependent impact on the cardiovascular system.While its effects on male rats have been studied, cadmium may act differently in females due to its potential as a metalloestrogen.In vitro studies conducted in a controlled environment allow for a direct assessment of cadmium's impact on vascular function, and the use of female rats ensures that sex-dependent effects are evaluated.Therefore, the aim of this study was to investigate the in vitro effects of Cadmium Chloride (CdCl 2 , 5 µM) exposure on vascular reactivity in the isolated aorta of female Wistar rats.Exposure to CdCl 2 damaged the architecture of the vascular endothelium.CdCl 2 incubation increased the production and release of O 2 •− , reduced the participation of potassium (K + ) channels, and contamination through the consumption of contaminated food and inhalation of tobacco smoke (Hengstler et al. 2003;Satarug et al. 2003;ATSDR 2012).In fact, smokers have approximately three times the blood concentration of Cd compared to non-smokers (1.58 µg/L for smokers vs. 0.47 µg/L for non-smokers) (Abu-Hayyeh et al. 2001).
Cd is an identified risk factor for cardiovascular diseases in humans (Satarug et al. 2003;Nigra et al. 2016), and the vascular endothelium is a target for Cd toxicity (Almenara et al. 2013(Almenara et al. , 2020(Almenara et al. , 2022;;Angeli et al. 2013;Kukongviriyapan et al. 2014;Oliveira et al. 2019;Vassallo et al. 2020).Several experimental reports have shown that acute and chronic Cd exposure may lead to vascular dysfunction by altering vessel function, contributing to the occurrence of vasculopathies (Prozialeck et al. 2006;Almenara et al. 2013Almenara et al. , 2022;;Angeli et al. 2013;Kukongviriyapan et al. 2014;Sangartit et al. 2014;Oliveira et al. 2019).Regarding the mechanisms of Cd toxicity on the vascular endothelium, it is important to note that cadmium per se does not directly generate ROS.Instead, previous studies have suggested that Cd-induced toxicity is associated with reduced nitric oxide (NO) bioavailability, decreased expression and phosphorylation of endothelial nitric oxide synthase (eNOS), and subsequent increase in oxidative stress (Majumder et al. 2008;Yoopan et al. 2008;Almenara et al. 2013;Angeli et al. 2013;Kukongviriyapan et al. 2014;Oliveira et al. 2019;Santamaria-Juarez et al. 2020).Experimental models conducted on male rats have shown that the increase in oxidative stress is attributed to the production of reactive oxygen species (ROS) and superoxide anions (O 2 •− ), coupled with elevated activity of cyclooxygenase-2 (COX-2) and NADPH oxidase (Almenara et al. 2013;Angeli et al. 2013;Nwokocha et al. 2013).On the other hand, some studies have found that both acute and chronic Cd exposure also appears to decrease the activity of several antioxidant enzymes such as catalase, superoxide dismutase (SOD), glutathione reductase (GSSG-R), glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST) in vitro and in vivo (Casalino et al. 2002;Thévenod 2009;Henkler et al  2010; Jamakala and Rani 2015).Therefore, the reduction in the activity of these enzymes by Cd contributes to the increase in oxidative stress.Thus, it is essential to recognize that the production of radicals by Cd occurs indirectly.However, these studies were conducted in male animals.In a recent study, we demonstrated sex-dependent effects induced by sub-chronic Cd exposure (de Oliveira et al. 2022).In male rats, sub-chronic exposure to Cd increases blood pressure and modifies vascular reactivity, while these effects were not observed in females, although myeloperoxidase activity increased in both sexes In this report, we suggest that the differences in Cd-induced vascular effects between the sexes might be due to the protective effect of estrogen on the vasculature, as estrogen levels were not affected by Cd exposure (de Oliveira et al. 2022).
17beta-estradiol (E2) is the main female hormone and the most common form of circulating estrogen.Studies have shown that circulating E2 levels are inversely proportional to the number of cardiovascular events in postmenopausal women due to its cardioprotective effect, which mainly involves E2-induced vasodilation (Kim et al. 2014;Pérez-Cremades et al. 2018;Aryan et al. 2020).Some heavy metals, such as Cd, can bind to estrogen receptors (ER) and act as endocrine disruptors.These metals are referred to as metalloestrogens (Stoica et al. 2000;Johnson et al. 2003;Brama et al. 2007;Aquino et al. 2012;Byrne et al. 2013;Gaudet et al. 2018).Studies have shown that Cd can activate ERα through high-affinity interactions with the receptor's binding site, leading to alterations in receptor conformation (Stoica et al. 2000;Fechner et al. 2011).
As mentioned above, acute exposure to Cd or in vitro studies induce vascular dysfunction in the aorta of male rats (Tzotzes et al. 2007;Angeli et al. 2013).In vitro studies with Cd provide a controlled environment where the direct effects of Cd on the cardiovascular system can be evaluated, allowing for a more comprehensive understanding of its potential impact.Specifically, in the case of female rats, Cd has the potential to act as a metalloestrogen, producing sex-dependent effects that may differ from those observed in male rats.Understanding the vascular effects of Cd in female rats is critical, as it may have important implications for human health, particularly for populations exposed to high levels of this metal.It is important to highlight the lack of research on the vascular toxicity of cadmium in female vasculature, since most of the experimental studies evaluating the toxic effects of cadmium on the vasculature have been conducted in male animals.Therefore, we aimed to specifically examine the effects of cadmium on female vasculature, addressing a significant research gap in the literature.With this in mind, we investigated the effects of in vitro exposure to CdCl 2 (5 µM) on the vascular reactivity of the isolated aorta from Wistar rats, as well as the role of the endothelium, alpha estrogen receptors (Erα), nitric oxide (NO), COX-derived prostanoids, and reactive oxygen species (ROS) agents, to clarify the effects of in vitro CdCl 2 intoxication in female rats.

Experimental animals
Female Wistar rats (Rattus norvegicus albinus), approximately 12 weeks old and weighing 250 g, were used in this study.These animals were kept in cages under controlled temperature conditions of 23 ºC and a 12-h light-dark cycle, with free access to drinking water and standard rodent chow.The animals were handled in accordance with the ethical principles established by the Brazilian College of Animal Experimentation (COBEA-1991).The project was previously approved by the Ethics Committee on Animal Use of the Federal University of Espírito Santo (UFES-CEUA 10/2020).
The rats were anesthetized with ketamine (50 mg/ kg) and xylazine (10 mg/kg) before being sacrificed.The descending thoracic aorta was carefully removed, and the connective and adipose tissue were dissected away.The thoracic aorta was then divided into five cylindrical segments, each measuring approximately 4 mm in length.The rings were mounted in an organ bath containing modified Krebs solution composed of the following concentrations (in mM): NaCl 127, KCl 4.7, CaCl 2 •2H 2 O 2.5, MgSO 4 •7H 2 O 1.2, KH 2 PO 4 1.17, NaHCO 3 24, glucose 11, and EDTA 0.01.The solution was aerated with a carbogenic mixture containing 5% CO 2 and 95% O 2 .
After assembly and normalization of arterial tension, the aorta rings were incubated with 75 mM KCl twice.The first incubation was conducted to assess the integrity of the vascular smooth muscle, and the second incubation was performed to verify the maximum tension.Following washing and stabilization, the functional integrity of the endothelium was tested by measuring the vasodilation response to 10 μM acetylcholine (ACh) after contraction with 100 nM phenylephrine (Phe).In some rings, the endothelium was mechanically removed, and those that did not relax or showed a maximum relaxation of less than 10% at 10 µM ACh, after pre-contraction with 100 nM Phe, were considered capable of carrying out the experimental protocols.
Subsequently, these rings were subjected to different experimental protocols to study the effects of in vitro exposure to Cd at a concentration of 5 µM on vascular reactivity.The segments were exposed to the following drugs: L-NAME 100 µM (a non-selective nitric oxide synthase inhibitor-NOS), enalapril 10 µM (an angiotensin-converting enzyme inhibitor-ACE), indomethacin 10 µM (a non-specific cyclooxygenase inhibitor-COX), SOD 150 U/ml (a superoxide anion scavenger-O 2 •− ), catalase 1000 U/ ml (a hydrogen peroxide scavenger-H 2 O 2 ), apocynin 30 µM (an NADPH oxidase inhibitor), tetraethylammonium (TEA) 1 mM (a non-specific inhibitor of calcium-activated potassium channels-KCa), and fulvestrant 1 μM (a selective estrogen receptor degrader (SERD).After incubation, a concentration-response curve to Phe (10 −10 to 10 −4 M) was performed in the presence or absence of Cd.
The incubation/exposure time for Cd was 45 min before the start of the concentration-response curve.Endothelium-dependent vasodilator responses were evaluated by constructing concentration-response curves to ACh (10 −4 to 10 −11 M).

"In situ" quantification of nitric oxide and superoxide anion
After dissection, the aortas were immersed in tissue freezing medium (Tissue Tek O.C.T. Composite, Sakura Finetek, Inc., Torrance, CA, USA), and arterial sections of 10 μm thickness were obtained using a cryostat (Leica CM1850 Cryostat Microtome, Heidelberger, Nussloch, Germany).For the detection of nitric oxide (NO) in situ, we used the fluorescent probe 4,5-diaminufluorescein (DAF-2).DAF-2 is a non-fluorescent membrane permeability probe that reacts with nitric oxide to produce DAF-2 T, a fluorescent derivative.The fluorescence observed under a fluorescence microscope indicates the formation of nitric oxide.This method has been previously described by Lobato et al. (2011).
For the quantification of superoxide anion (O 2 •− ) production, we used the fluorescence technique based on the oxidation of dihydroethidium (DHE).DHE permeates the cell membrane, and in the presence of O 2 •− , it is oxidized to ethidium bromide, which binds to DNA and emits a red color when excited.This technique has been previously described by Nunes et al. (2014).
In our experimental protocol, the slides containing aortic segments were incubated with DAF-2 and DAPI ( 4 The slides were analyzed on a Leica DM 2500 inverted fluorescence microscope (40 × objective), using a Leica DFC 310 FX camera.To quantify the emitted fluorescence, 5 to 6 images of aortic segments per animal were used to obtain the sample mean.Mean fluorescence density was calculated using ImageJ software (National Institutes of Health, USA).

Scanning electron microscopy (SEM)
The aortas from the different experimental groups, Ct and Cd, were collected and cut to expose the endothelial surface.They were then fixed in Karnovsky's solution (2.5% glutaraldehyde, 2% paraformaldehyde, and 0.1 M cacodylate buffer).Subsequently, the samples were post-fixed in osmium tetroxide and dehydrated with ethanol at different concentrations (30%, 50%, 70%, 90%, and 100%).After dehydration, they underwent the critical point drying procedure, and a 10 nm gold layer was evaporated onto the tissue surface following the method described by Vol.: ( 0123456789) Friques (Friques et al. 2015).Electron micrographs of both groups were obtained using a scanning electron microscope (JEOL JEM6610 LV, Inc. USA) at 1000 × magnification.

Results expression and statistical analysis
Microsoft Office Excel (Redmond, Washington, USA) and GraphPad Prism Software 8.0 (San Diego, California, USA) were used for data analysis and statistical analysis.Results are presented as mean ± standard error of the mean (SEM).The number of animals used in each experiment is indicated in parentheses in the graphs.The concentration-response curves were analyzed using two-way ANOVA, and Bonferroni post-tests were performed when statistical significance was detected.To compare the effects of incubation on the contractile responses to Phe, the results are expressed as differences in the area under the concentration-response curves (dAUCs) for the control and experimental groups.The AUCs were calculated from the individual concentration-response plots using a computer program (GraphPad Prism 8, GraphPad Software, Inc., San Diego, CA).Differences are expressed as the percentage of the dAUCs of the corresponding control situation.For DAF and DHE fluorescence results, Kolmogorov-Smirnov and Shapiro-Wilk tests were performed and analyzed using 2-way ANOVA with Tukey post-test.Data were analyzed and plotted using GraphPad Prism Software (version 8.0, USA).The results were considered significant for p values of < 0.05.

Endothelial architecture analysis by scanning electron microscopy (SEM)
To assess structural damage to the endothelial surface of the aorta after in vitro exposure to Cd, SEM was performed.The SEM images of the thoracic aorta from Control females showed a normal squamous surface with a continuous endothelial cell layer.In contrast, the Cd group exhibited damage to the structural surface, with evident irregularity and loss of normal endothelial structure (Fig. 1).

Vascular reactivity
ACh-induced vasodilation produced a similar relaxation response in isolated aortic rings in the absence or presence of Cd (Fig. 2a).Contraction responses induced by KCl were comparable in aortic segments from both groups (KCl: Ct 1.70 ± 0.13 g, n = 34; vs. Cd 1.84 ± 0.08 g, n = 35, unpaired t-test) and were used to normalize Phe-induced contractions in the subsequent analyses.The contractile responses to Phe did not change after in vitro exposure to Cd (Fig. 2b).Endothelial removal or L-NAME incubation enhanced the contractile responses to Phe in the absence or presence of Cd, with a similar magnitude between the groups, as indicated by the % dAUC (Fig. 3a-f).Consistent with these findings, the "in situ" detection of NO using a fluorescent DAF-2 probe showed similar basal production of NO between the groups (Fig. 3g).To investigate whether in vitro exposure to Cd increases O 2 •− production, the effects of the superoxide anion scavenger SOD and the NADPH oxidase inhibitor apocynin on the vasoactive responses were analyzed.Additionally, the effect of a hydrogen peroxide scavenger was also examined.SOD did not modify the vasoconstrictor responses to Phe in the absence of Cd but reduced these responses in the Cdincubated rat aortic segments (Fig. 4a-b).However, Apocynin or Catalase did not alter the vasoconstrictor responses to Phe in the absence or presence of Cd (Fig. 4c-f).Consistent with the SOD results, an increase in O 2 •− production after Cd in vitro exposure was observed (Fig. 4g).
Given the importance of K + channels in vascular tone regulation, TEA was used to assess the involvement of these channels in the Phe contractile response.TEA incubation increased vascular reactivity to Phe in the absence and presence of Cd (Fig. 5ab), but this effect was attenuated after metal incubation (Fig. 5c).
To investigate the role of local angiotensin II in the Phe responses, enalapril was incubated.Enalapril reduced the Phe responses in the absence and presence of Cd (Fig. 6a-b), but this effect was enhanced after metal exposure, suggesting an increase in angiotensin II due to Cd (Fig. 6c).
To explore the potential role of prostanoids in the response to Phe, aortic rings were incubated with indomethacin.Indomethacin reduced the Phe response only in control arteries, indicating a role of constrictor prostanoids in the Phe contractile response (Fig. 7a).However, after Cd exposure, the effect of indomethacin was attenuated, suggesting a decrease in constrictor prostanoids and/or an increase in prostacyclin (Fig. 7b).
Considering that Cd is a metalloestrogen, the role of ERα receptors in the Phe contractile response was investigated.Fulvestrant incubation increased vascular reactivity to Phe only after Cd incubation, suggesting negative modulation of ERα receptors in the Phe contractile response after in vitro Cd exposure (Figs.8a-b).

Discussion
The lack of effect of Cd on vascular reactivity to Phe observed in this study contrasts with previous findings in male rats (Tzotzes et al. 2007;Sompamit et al. 2010;Donpunha et al. 2011;Almenara et al. 2013;Oliveira et al. 2019), suggesting that the effects of Cd on the cardiovascular system may be sex-dependent.
The results obtained in the present study demonstrate, for the first time, that in vitro exposure to Cd (5 µM), a concentration below that found in the middle layer of the aorta of smokers (Abu-Hayyeh et al. 2001), was capable of damaging the architecture of the vascular endothelium, which may lead to the occurrence of vasculopathies.However, it is important to consider that the concentration of cadmium found in the middle layer of the aorta in smokers results from a chronic and bioaccumulative effect of the metal in this tissue.In our present study, we employed a concentration of 5 µM in vitro, aimed at assessing the toxicity associated with this specific exposure (Friberg et al. 1992;Abu-Hayyeh et al. 2001).Previous studies have shown that both acute and chronic exposure to mercuric chloride (HgCl 2 ) can also damage the endothelial layer of the aorta in Wistar females, exposing the internal elastic membrane of the vessel (Cordeiro et al. 2019;Schereider et al. 2021).Therefore, our findings demonstrate that, like Hg, Cd is a metal that, even in in vitro exposures, is capable of damaging the structure of the vascular endothelium in females.
In the present study, we showed that in vitro exposure to 5 µM Cd does not change vascular reactivity to Phe nor alter ACh-induced endothelium-dependent relaxation.These results are consistent with those observed in subchronic exposure for 30 days with Cd (Cd 100 ppm) through drinking water in female rats (de Oliveira et al. 2022).However, our results oppose the findings in male rats, where it was found that both in vitro (10 μM) and subchronic exposure to Cd (Cd 100 ppm) increased the vascular contractility of the aorta in these rats (Almenara et al. 2013;Angeli et al. 2013;Oliveira et al. 2019).To verify whether in vitro exposure to Cd could have altered endothelial modulation, vascular reactivity to Phe was compared in isolated aorta in the absence (E-) and in the presence of endothelium (E +).Although the magnitude of the responses was similar in isolated aorta in the presence or absence of Cd, we cannot rule out an imbalance between relaxing and contractile factors induced by metal exposure.The reduction of NO bioavailability after exposure to Cd is already well established in the literature in male animals (Majumder et al. 2008;Gökalp et al. 2009;Almenara et al. 2013;Angeli et al. 2013;Nagarajan et al. 2013;Oliveira et al. 2019).The only work carried out in females, subchronically exposed to Cd, demonstrated the preservation of NO in the isolated aorta (de Oliveira et al. 2022).In the present study, incubation with L-NAME increased vascular reactivity to Phe in both isolated aortas with and without Cd, suggesting that endothelium-derived nitric oxide is an important modulator of vascular reactivity in female rats, regardless of Cd exposure.Confirming this finding, the analysis of basal NO production by DAF-2 indicated similar NO production in both groups.Therefore, unlike what was observed in a previous study in male rats (Almenara et al. 2013;Angeli et al. 2013;Oliveira et al. 2019), in vitro or subchronic exposure to Cd (de Oliveira et al. 2022), NO bioavailability was preserved, reinforcing the notion that this metal acts in a sex-dependent manner.
Studies have shown that acute or prolonged exposure to Cd can lead to an imbalance between the production of reactive oxygen species (ROS) and the body's antioxidant defense mechanisms in male animals (Almenara et al. 2013;Angeli et al. 2013; Vol.: (0123456789) Kukongviriyapan et al. 2014;Oliveira et al. 2019;de Oliveira et al. 2022).This oxidative stress can damage cells and tissues, including those in the vascular system.Furthermore, Cd has been found to impair endothelial function, leading to endothelial dysfunction and an increased risk of cardiovascular diseases.In fact, a previous study demonstrated that male rats subchronically exposed to Cd and obtaining blood concentrations of Cd similar to those found in occupationally exposed individuals had increased release of O 2 •− , associated with NADPH oxidation (NOX2 isoform), and participation of H 2 O 2 as a contractile agent (Almenara et al. 2013).Thus, in male rats, in vitro exposure to Cd (10 µM), higher than that found in the middle layer of smokers (Abu-Hayyeh et al. 2001), also demonstrated an increase in O 2 •− and H 2 O 2 as contractile agents (Angeli et al. 2013).In fact, in the present study, we observed that the catalytic removal of O 2 •− through incubation with SOD reduced the contractile response to Phe only in the Cd group, suggesting a release of O 2 •− .However, we did not find the participation of H 2 O 2 in the modulation of vascular reactivity to Phe in the group exposed to Cd.These data suggest a greater bioavailability of O 2 •− acting as a contractile factor and contributing to the modulation of the response to Phe.This hypothesis was confirmed through the fluorescence protocol with the DHE probe, where we observed greater O 2 •− production in situ in the aorta that was exposed to Cd.The origin of the increased O 2 •− does not appear to be NADPH oxidase, as incubation with apocynin did not alter vascular reactivity in Cd-exposed isolated aorta of female rats.However, it is important to emphasize that apocynin is not a selective inhibitor of NADPH oxidase and may act as a scavenger of ROS (Heumüller et al. 2008;Petrônio et al. 2013).The opposite result was found in rats exposed subchronically to Cd, where no alteration in the bioavailability of O 2 •− was observed in the isolated aorta of female rats alteration in the bioavailability of O 2 •− was observed in isolated aorta of female rats (de Oliveira et al. 2022).It is possible that compensatory mechanisms can act to counteract this effect in subchronic Cd exposure.
According to the literature, there is a strong association between Ang II and increased vascular oxidative stress (Zafari et al. 1998;Dikalov et al. 2014).Ang II, a potent vasoconstrictor, could trigger intracellular signaling pathways that generate ROS, contributing to the progression of cardiovascular diseases.A study where male aortas were exposed to Cd (10 µM) in vitro showed the participation of Ang II in the increase in the contractile response to Phe and an increase in the intracellular production of ROS induced by Ang II, predominantly derived from NADPH oxidase (Angeli et al. 2013).In the present study, we also found involvement of Ang II in modulating the vascular reactivity to Phe in Cd-exposed female rat aortas.Therefore, we cannot rule out the participation of Ang II in the increased production of superoxide anion in Cd-exposed female rat aortas.
K + channels are important regulators of vascular tone as they participate in the membrane potential of excitable cells (Nelson and Quayle 1995).A previous study carried out in VSMCs of bovine mesenteric arteries demonstrated that acute exposure to Cd at concentrations below 1 µM can inhibit high-conductance Ca 2+ -activated K + channels (BKCa) of vascular smooth muscle (Stockand et al. 1993) because Cd, being a bivalent metal, can compete with Ca 2+ for the binding site (Blazka and Shaikh 1991).Our results suggest a reduction in the participation of K + channels in the aorta of female rats after exposure to Cd, indicating that, in a way, one of the vasodilation pathways would be impaired after exposure to the metal.It is also known that ROS can negatively modulate the activity of these channels (Tang et al. 2004;Vassallo et al. 2018), which may also contribute to the reduced participation of K + channels found in the isolated rat aorta.
A metalloestrogen refers to a metal or metal compound that mimics or disrupts the action of natural estrogen in the body.These substances have the ability to bind to estrogen receptors, eliciting estrogenic effects and influencing various physiological processes.Cadmium, a toxic heavy metal, has been identified as a potential metalloestrogen based on studies demonstrating its interaction with estrogen receptors and its promotion of estrogen-like responses in cells and tissues (Aquino et al. 2012).At low concentrations, Cd demonstrates effects comparable to estradiol in transient transfection assays.It binds to the hormone-binding domain of the ERα receptor, preventing the binding of estradiol to the receptor (Stoica et al. 2000).This interaction with the ER likely involves several amino acids, inducing structural changes that mimic those induced by estradiol binding.Similar to estradiol, cadmium increases transcription and expression of estrogen-regulated genes, induces cell proliferation, and activates ERα (Garcia-Morales et al. 1994;Stoica et al. 2000;Martínez-Campa et al. 2006;Brama et al. 2007).The effects of cadmium can be counteracted by an antiestrogen, suggesting that the metal's effects are mediated through the ERα genomic pathway (Byrne et al. 2009).In our present study, while vascular reactivity was maintained following in vitro exposure to Cd, the observed vascular dysfunction in the aortic rings of female rats seems to involve the participation of ERα receptors.This suggests that Cd may act as a metalloestrogen and modulate estrogen receptor signaling in female rats.Estrogen-associated vascular reactivity regulation occurs through the maintenance of normal endothelial function, which, through ER activation, increases the bioavailability of NO and prostacyclin (PGI2) (Sobrino et al. 2010(Sobrino et al. , 2017;;Novella et al. 2013).It is known that the expression of ER in arteries is lower in men than in women during pre and post-menopause, as well as the level of estrogen secretion (Orshal and Khalil 2004;Hayashi et al. 2007).Therefore, the presence of these receptors in the endothelium may protect females from the toxic effects of Cd on the vasculature.A previous study demonstrated that subchronic Cd exposure in female rats did not alter estrogen levels or vascular reactivity to phenylephrine, suggesting a putative role of estrogen's protective effect on the vasculature (de Oliveira et al. 2022).Thus, elucidating Cd's action on estrogen receptors is crucial for identifying the mechanism by which Cd affects females.
As mentioned earlier, estrogen plays a role in the vascular endothelium by increasing the bioavailability of both NO and PGI2 (Sobrino et al. 2010(Sobrino et al. , 2017;;Novella et al. 2013;Yuan et al. 2013).Previous studies have demonstrated that estrogen upregulates the release of PGI2 in ovine fetal pulmonary artery endothelium and human umbilical vein endothelial cells (Sherman et al. 2002;Su et al. 2009).Moreover, the estrogen receptor ERβ has been identified as a mediator of cyclooxygenase (COX-2) expression in human placental endothelial cells (Su et al. 2009).However, limited studies have investigated the COX pathway in the context of Cd exposure.A previous study conducted in male rats reported an increase in the release of vasoconstrictor prostanoids in isolated aortas following in vitro exposure to Cd, using a concentration twice that used in our present study (Angeli et al. 2013).It is worth noting that Cd exposure is associated with increased oxidative stress (Almenara et al. 2013;Angeli et al. 2013;de Oliveira et al. 2022), a condition known to enhance the activity and expression of the COX-2 pathway (Alvarez et al. 2008;Nguyen Dinh Cat et al. 2013).In our study, we observed a reduction in the positive modulation of COX-derived prostanoids in the contractile response to phenylephrine (Phe) in female rats exposed to Cd.These findings suggest the possibility of increased PGI2 release and/or a decrease in constrictor prostanoids, potentially serving as important counterbalancers in the modulation of vascular reactivity in response to Cd.However, further studies are required to investigate this hypothesis and better understand the extent of their involvement.
In conclusion, our findings suggest that Cd may not directly affect vascular reactivity to Phe in the isolated aorta of female rats.However, our data indicate that Cd may modulate vascular reactivity through endothelial pathways and act as a metalloestrogen.Specifically, Cd increased superoxide anion production, enhanced the involvement of the Ang II pathway in the Phe response, and reduced the participation of K + channels.Conversely, Cd also decreased the release of vasoconstrictor prostanoids and/or increased the release of prostacyclin.Furthermore, our results demonstrate that Cd alters the endothelial architecture, which could have implications for the development of cardiovascular diseases.
Overall, these findings provide new insights into the potential mechanisms of in vitro Cd-induced vascular effects in female rats and contribute to the development of new strategies for the prevention and treatment of cardiovascular diseases associated with Cd exposure.Our findings highlight the importance of minimizing Cd exposure and implementing measures to protect against its detrimental effects on cardiovascular health.

Study limitations
In the present study, it is important to acknowledge that the concentration of 5 µM presents a limitation to our research.This concentration was selected for an in vitro duration of 45 min and may be considered relatively high in terms of tissue presence when compared to findings from certain occupational and smoking-related studies.For instance, a study Vol.: (0123456789) involving automotive workshop workers reported significantly elevated serum levels of Cd in mechanics compared to the control group (1.17 ± 0.52 µg/L vs. 0.41 ± 0.09) (Babatunde Ishola et al. 2017).Furthermore, a study investigating plasma levels of cadmium in female smokers found even higher concentrations, with an average of 2.98 ± 0.12 ng/ml (Sadeghi et al. 2014).However, it is important to emphasize that these concentrations are lower than those used in our study and are associated with chronic exposure to the metal.Moreover, it should be noted that vascular effects induced by chronic exposure to cadmium may result from secondary actions of this metal, such as hypertension (Eum et al. 2008;Tellez-Plaza et al. 2008;Donpunha et al. 2011;Almenara et al. 2013), which can also lead to significant vascular alterations.Therefore, conducting in situ studies involving direct exposure to cadmium allows us to elucidate how this metal directly affects vascular function, independent of other hemodynamic factors.
It is important again to highlight the lack of research on cadmium vascular toxicity in female vasculature.This study was designed to address the specific research gap mentioned above.Future studies could consider performing sex-specific comparisons to further explore possible differences in the effects of cadmium on vascular reactivity between males and females.
Author contribution All authors whose names appear on the submission: (1) made substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; (2) drafted the work or revised it critically for important intellectual content; (3) approved the version to be published; and (4) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Declarations
Conflict of interest The authors declare that they have no conflicts of interest.

Ethical approval
The research protocols performed in this study were in accordance with the guidelines recommended by the Brazilian College of Animal Experimentation (COBEA).The project was previously approved by the Ethics Committee on Experimentation and Use of Animals of the Federal University of Espírito Santo (UFES-CEUA 10/2020).
',6-diamidino-2-phenylindole) to evaluate basal NO production and inhibition of nitric oxide synthesis.The slides were distributed into different groups: Control + DAF + DAPI, C o n t r o l + D A F + D A P I + L -N A M E , Cadmium + DAF + DAPI, and Cadmium + DAF + DAPI + L-NAME.+ DAPI + SOD, Cadmium + DHE + DAPI, and Cadmium + DHE + DAPI + SOD.SOD (superoxide dismutase) was used as a negative control to confirm the specificity of the technique.

Fig. 1
Fig.1Analysis of the vascular endothelial surface architecture of aortas from female rats.Representative scanning electron microscopy photomicrograph of the endothelial structure of aortas from the control rats (A at 1000 × magnification),

Fig. 2
Fig. 2 Concentration-response curves to Acetylcholine in Control and Cadmium groups.Results are expressed as mean ± SEM and compared by Two-way ANOVA and Bonferroni's post hoc test.p > 0.05 (A).Concentration-response

Fig. 3 Fig. 4
Fig. 3 Concentration-response curves to Phenylephrine in Control and Cadmium groups and percent differences in areas under the curve for comparison (% dAUC) after endothelium removal (A, B, C) and after L-NAME (N-nitro-L-arginine methyl ester) incubation (D, E, F). Results are expressed as mean ± SEM and compared by Two-way ANOVA and Bonferroni's post hoc test.*p < 0.05 vs Control on corresponding concentration.Unpaired T-test was used in % dAUC, p > 0.05.

Fig. 5 Fig. 6
Fig. 5 Concentration-response curves to Phenylephrine in Control and Cadmium groups and percent differences in areas under the curve for comparison (% dAUC) after TEA incuba-tion (A, B, C). Results are expressed as mean ± SEM and compared by Two-way ANOVA and Bonferroni's post hoc test.

Fig. 7 Fig. 8
Fig. 7 Concentration-response curves to Phenylephrine in Control and Cadmium groups after Indomethacin incubation (A, B).Results are expressed as mean ± SEM and compared by Two-way ANOVA and Bonferroni's post hoc test.*p < 0.05 vs