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

doi:10.21203/rs.3.rs-3044730/v1

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

https://doi.org/10.21203/rs.3.rs-3044730/v1

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published 31 Aug, 2023

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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 gender-specific 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 gender-specific effects are evaluated. Therefore, the aim of this study was to investigate the in vitro effects of Cadmium Chloride (ClCd2, 5µM) exposure on vascular reactivity in the isolated aorta of female Wistar rats. Exposure to ClCd2 damaged the architecture of the vascular endothelium. ClCd2 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.

Cadmium

Vascular reactivity

Endothelial dysfunction

Female wistar

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 O, Ashizawa A, Wright S 2012; Sripada and Lager 2022).

Population exposure to Cd mainly occurs through soil and food contamination. Vegetables and tobacco leaves accumulate high levels of Cd from the soil, making them sources of contamination through 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, 2020, 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. 2013, 2022; Angeli et al. 2013; Kukongviriyapan et al. 2014; Sangartit et al. 2014; Oliveira et al. 2019). 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 gender-specific 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. With this in mind, we investigated the effects of in vitro exposure to ClCd2 (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 CdCl2 intoxication in female rats.

2.1 EXPERIMENTAL ANIMALS

Female Wistar rats (Rattus norvegicus albinus), approximately 12 weeks old and weighing 250g, were used in this study. These animals were kept in cages under controlled temperature conditions of 23ºC and a 12-hour 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, CaCl2·2H2O 2.5, MgSO4·7H2O 1.2, KH2PO4 1.17, NaHCO3 24, glucose 11, and EDTA 0.01. The solution was aerated with a carbogenic mixture containing 5% CO2 and 95% O2.

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 – O2•-), catalase 1000 U/ml (a hydrogen peroxide scavenger – H2O2), 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 (an estrogen α receptor degrader). 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 minutes 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).

2.3 “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). To exclude autofluorescence and unspecific fluorescence during the evaluation of NO and O2•−, the protocols were also conducted in the presence of L-NAME (100 µM) and an O2•− superoxide scavenger, Tiron (1 mM), respectively. Images were captured using a Leica DM 2500 fluorescence microscope with a Leica DFC 310 FX camera (Eclipse Ti2-E, Nikon Instruments Inc., Melville, NY, USA), and the same imaging settings were applied to both groups. To quantify the average fluorescence density produced by DAF-2 and DHE in the target region, four images were analyzed for each animal using MetaMorph and ImageJ software.

2.4 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 Friques (Friques et al. 2015). Electron micrographs of both groups were obtained using a scanning electron microscope (JEOL JEM6610 LV, Inc. USA) at 1000x magnification.

2.5 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. The effect of endothelium removal or certain drugs on the vasoconstrictor response to Phe in the studied groups was evaluated by expressing the results as differences in the area under the curve (dAUC) in the control and experimental situations. Differences were considered statistically significant when P < 0.05.

3.1 ENDOTHELIAL ARCHITECTURE ANALYSIS BY SCANNING ELECTRONIC 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).

3.2 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 O2•- 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 Cd-incubated 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 O2•- 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. 5a-b), 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 (Fig. 8a-b).

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 gender-specific.

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. Previous studies have shown that both acute and chronic exposure to mercuric chloride (Cl2Hg) 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 gender-specific 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; 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 O2•-, associated with NADPH oxidation (NOX2 isoform), and participation of H2O2 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 O2•- and H2O2 as contractile agents (Angeli et al. 2013). In fact, in the present study, we observed that the catalytic removal of O2•- through incubation with SOD reduced the contractile response to Phe only in the Cd group, suggesting a release of O2•-. However, we did not find the participation of H2O2 in the modulation of vascular reactivity to Phe in the group exposed to Cd. These data suggest a greater bioavailability of O2•- 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 O2•- production in situ in the aorta that was exposed to Cd. The origin of the increased O2•- 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 O2•- was observed in the isolated aorta of female rats alteration in the bioavailability of O2•- 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; Nazarewicz 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 Ca2+-activated K + channels (BKCa) of vascular smooth muscle (Stockand J, Sultan A, Molony D, Dubose T 1993) because Cd, being a bivalent metal, can compete with Ca2 + 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 is a term used to describe a metal or metal compound that mimics or disrupts the action of natural estrogen in the body. These substances could bind to estrogen receptors and elicit estrogenic effects, influencing various physiological processes. Cadmium, a toxic heavy metal, has been identified as a potential metalloestrogen based on studies demonstrating its ability to interact with estrogen receptors and promote estrogen-like responses in cells and tissues (Aquino et al. 2012). In the present study, despite the maintenance of vascular reactivity after in vitro exposure to Cd, the vascular dysfunction observed in the aortic rings of female rats seems to also involve the participation of ERα receptors, reinforcing that Cd may act as a metalloestrogen and modulate estrogen receptor signaling in female rats. The regulation of estrogen-associated vascular reactivity occurs through the maintenance of normal endothelial function, which, by activating the ER, increases the bioavailability of NO and prostacyclin (Sobrino et al. 2010, 2017; Novella et al. 2013). It is known that the expression of ER in the arteries is lower in men than in women in 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 has demonstrated that in female rats, subchronic Cd exposure did not modify the estrogen levels and the vascular reactivity to Phe, suggesting a putative role of estrogen's protective effect on the vasculature (de Oliveira et al. 2022). Therefore, the elucidation of this action of Cd on estrogen receptors is important to identify the mechanism of action of Cd in females.

As mentioned above, estrogen might act in the vascular endothelium increasing the bioavailability of NO and prostacyclin (Sobrino et al. 2010, 2017; Novella et al. 2013). Moreover, a previous study identified an increase in the release of vasoconstrictor prostanoids in the isolated rat aorta after in vitro exposure to Cd, with twice the concentration used in the present study (Angeli et al. 2013). Studies that have analyzed the COX pathway in exposure to Cd are scarce. However, exposure to Cd is associated with increased oxidative stress (Almenara et al. 2013; Angeli et al. 2013; de Oliveira et al. 2022), a condition that increases activity/expression of the COX-2 pathway (Briones et al. 2008; Nguyen Dinh Cat et al. 2013). Our results suggest a reduction in the positive modulation of COX-derived prostanoids in the contractile response to Phe in female rats exposed to Cd. These data suggest that there may be an increase in the release of PGI2 and/or a reduction in constrictor prostanoids, contributing as one of the important counterbalancers of the response to Cd in the modulation of vascular reactivity. However, to confirm this hypothesis, more studies are needed to investigate such participation.

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 could act as a metalloestrogen. Moreover, our results also indicate that Cd alters the endothelial architecture, which may have implications for the development of cardiovascular diseases. Overall, these findings provide new insights into the potential mechanisms of Cd-induced vascular effects in female rats and may contribute to the development of new strategies for the prevention and treatment of cardiovascular diseases associated with Cd exposure.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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.

FUNDING INFORMATION

This study was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES – Financing code 001); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – 4/2021) and Fundação de Amparo à Pesquisa do Espírito Santo (FAPES/CNPq – nº 25/2022; 80600115). The funders had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.

COMPLIANCE WITH ETHICAL STANDARDS

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).

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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

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Abstract

Figures

1 INTRODUCTION

2 MATERIALS AND METHODS

2.1 EXPERIMENTAL ANIMALS

2.3 “IN SITU” QUANTIFICATION OF NITRIC OXIDE AND SUPEROXIDE ANION

2.4 SCANNING ELECTRON MICROSCOPY (SEM)

2.5 RESULTS EXPRESSION AND STATISTICAL ANALYSIS

3 RESULTS

3.1 ENDOTHELIAL ARCHITECTURE ANALYSIS BY SCANNING ELECTRONIC MICROSCOPY (SEM)

3.2 VASCULAR REACTIVITY

4 DISCUSSION

STATEMENTS AND DECLARATIONS

REFERENCES

Additional Declarations

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