https://orcid.org/0000-0003-1049-5054
Abstract
Accidental occupational bromine (Br>2>) exposures are common, leading to significant morbidity and mortality; however, the specific effects of Br>2> inhalation in female victims are unclear. Our studies demonstrated that acute high-concentration Br>2> inhalation is fatal, and cardiac injury and dysfunction play an important role in Br>2> toxicity in males. In this study, we exposed female Sprague Dawley rats, age-matched to those males from previously studied, to 600 ppm Br>2> for 45 min and assessed their survival, cardiopulmonary injury and cardiac function after exposure. Br>2> exposure caused serious mortality in female rats (59%) 48 h after exposure. Rats had severe clinical distress, reduced heart rates and oxygen saturation after Br>2> inhalation as was previously reported with male animals. There was significant lung injury and edema when measured 24 h after exposure. Cardiac injury biomarkers were also significantly elevated 24 h after Br>2> inhalation. Echocardiography and hemodynamic studies were also performed and revealed that the mean arterial pressure was not significantly elevated in females. Other functional cardiac parameters were also altered. Aside from the lack of elevation of blood pressure, all other changes observed in female animals were also present in male animals as reported in our previous study. These studies are important to understand the toxicity mechanisms to generate therapies and better-equip first responders to deal with these specific scenarios after bromine spill disasters.>
Introduction
Men and women uniformly face the risk of accidental or deliberate exposure to toxic halogen gas exposure, as several accidents during the transport of halogens have been reported to cause significant morbidity and mortality [1–7]. In humans, respiratory and myocardial injuries along with cardiac arrest and cardiocirculatory collapse have been reported after isolated cases of halogen exposure [1, 8]. Computational models in the Cl2 spill after a train accident in Graniteville, NC, place concentration at 100,000 ppm at site and at 670 ppm at 2 miles within 60 min [9]. Accidental bromine (Br2) exposures have occurred, leading to significant morbidity and mortality; however, the specific effect of its inhalation in female victims is unclear [1]. Our previous studies utilizing male rats have demonstrated significant cardiopulmonary injuries after Br2 inhalation. While those studies have helped identify mechanisms that lead to halogen exposure-induced cardiac injury, whether this injury is uniform across cross-sections of a population is a question that is still unanswered.
An earlier report by Schlagbauer and Henschler utilized just female mice to demonstrate toxicity to Br2 and suggested that a dose of 315 ppm for 30 min resulted in 100% mortality in an unspecified time postexposure [10]. Although, in this study, comparison with males was not performed, in another study using mice, it was shown that mortality of female mice was not significantly different than males upon exposure to 600 ppm Br2 for 45 min [11]. However, in that study, the mortality curve demonstrated that females succumb earlier than the males [11]. Exposure to methyl bromide gas, which is a widely used form of Br2 in industry for agricultural fumigation, caused equal mortality in rats of both genders; yet, there was increased tissue damage and disease in the females [12]. More importantly, in this study, the females demonstrated significantly increased myocardial degeneration than the males [12].
Despite the growing number of reports in the literature identifying sex-related differences in cardiac function in both rodents and humans, the underlying mechanisms have yet to be determined. Various studies in which animals were subjected to exercise or surgical procedures, treated with drugs or genetically modified, support the existence of sex differences in the cardiovascular system as observed in clinical studies and showed that females may fare better than males [13]. It is also widely known that gender modifies susceptibility to pro-inflammatory stimuli exemplified by decreased risk of cardiovascular disease or cognitive disorders in pre-menopausal women; these differing risks are lost after menopause, and in some cases, even increased in post-menopausal women. Moreover, it is not always the case that women fare better than men in cardiovascular disease. In cases of idiopathic dilated cardiomyopathy, females have a significantly poorer prognosis than males [14]. Women are also more sensitive to alcohol-induced cardiac disease [15–18]. Female animals have exhibited better survival and were less likely to progress to dilation after coronary artery ligation, while in males, there is an increase in the thickness of non-infarcted regions and restrictive diastolic filling patterns in the same models [19–21]. After ischemia, reperfusion, the infarct area size, percentage of apoptosis and inflammatory response are larger in males than in females, while in females, post-ischemic cardiac function was significantly improved when compared to that of males [22–24]. The similarities of Br2 exposure-induced cardiac injury to that of ischemia reperfusion make it essential for this model to be tested for differences that may exist regarding the extent and severity of injury regarding gender. Due to the previously reported differences in gender susceptibility to cardiac injury after acute insults, we hypothesized that similar differences would occur in female animals exposed to bromine.
Materials and Methods
In vivo exposures to Br2
All animal procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Female Sprague Dawley rats (250–275 g, Envigo, Indianapolis, Indiana), age-matched to those males that were used in previously reported study, were utilized. Animals were exposed (whole body) to 600 ppm Br2 for 45 min as described previously [25]. Rats were then returned to room air and were monitored continuously up to 8 h and every 24 h thereafter for the specified duration (Fig. 1A). Clinical scoring was performed, which is a composite score of activity and respiratory quality as previously described [25]. Briefly, a clinical score of 0 is given to an animal with no stridor and normal respiratory quality and activity, mild stridor and mild respiratory symptoms and activity get a score of 1, moderate symptoms are scored as 2 and severe as 3. An animal demonstrating mild stridor, moderate respiratory quality and moderate activity will have a score of 5 (1 + 2 + 2). So higher the score, worse the condition of the animal. Oxygen saturation and heart rates (HRs) were monitored using the MouseOX small animal oximeter (Starr Life Sciences, Oakmont, PA) as previously described in our laboratory [26]. Two endpoints were used for this study; animals for survival studies were followed for 48 h, and animals for physiological studies were kept for 24 h after exposure, at which point, echocardiography, hemodynamic studies and bronchoalveolar lavage fluid (BALF) and tissue collection were performed. Heart weights and lung wet-to-dry weight ratios were also estimated. Arterial blood gas (ABG from descending aorta) at necropsy was also estimated using the element point of care (EPOC) automated blood-gas analysis reader (Ottawa, ON, Canada). Plasma was analyzed for cardiac injury markers, NT-proBNP and Troponin I, by ELISA. BALF protein content was measured to estimate lung injury.
Br2 inhalation increases mortality and causes respiratory distress in survivors; female rats were exposed to Br2 (600 ppm for 45 min) and returned to room air and observed until 48 h for survival and for 24 h for other endpoints as indicated (A); Kaplan–Meier curves were generated for survival (B); the clinical scores that are a composite of respiratory distress and activity are shown in (C); pulse oximetry to measure HR (D) and oxygen saturation (E) was performed every 2 h for 8 h and then at 24 and48 h; data are mean ± SE (n = 12 in panels B–D), and *demonstrates statistical significance from respective 0 ppm, P < 0.05.
Right ventricular and left ventricular hemodynamics and transthoracic 2D echocardiography
All experiments were performed under 2% isoflurane anesthesia in compressed room air as previously performed in our laboratory [5]. The body temperature was maintained at 37°C during measurements. A polyethylene catheter (PE-50) was placed in the left jugular vein and a 2 F high-fidelity catheter (SPR-407, Millar Institute, Houston, TX, USA) was inserted into the left ventricular (LV) via the right carotid artery. LV and right ventricular (RV) high-fidelity pressures were measured with a Harvard data acquisition system interfaced with a PC with AcqKnowledge III (ACQ 3.2). Transthoracic 2D ECHO/Doppler was performed using a Vevo2100 high-resolution ultrasound system (VisualSonics Inc, Toronto, ON, Canada) as previously described in our laboratory [26]. Parasternal long- and short-axis two-chamber M-mode and B-mode views were obtained at mid-papillary level and were averaged to determine LV dimensions at end-systole and end-diastole. LV volumes, cardiac output (CO), fractional shortening (FS) and ejection fraction were calculated (VisualSonics software). Spectral Doppler was used to determine trans-mitral early (E) and atrial (A) wave peak velocities [27, 31]. Operators blinded to exposure performed image collection and analyses.
Statistical analysis
Data are expressed as mean ± standard error analyzed by one-way analysis of variance with Student’s paired test [28]. A P-value < 0.05 was considered to be significant. Analysis was conducted using Graphpad Prism version 7 software (LaJolla, CA). The overall survival rates were determined using Kaplan–Meier method. All echocardiography analysis and calculations were performed using SPSS version 19.0 (SPSS, Inc, Chicago, IL). Two observers with expertise in echocardiography assessed the studies for intra- and inter-observer reproducibility.
Results and Discussion
The increased industrial use and storage of Br2 has brought with it an increase in potential risk to exposure to this toxic halogen. This exposure can be due to an accidental or deliberate spill. Cardiopulmonary injury and few pathophysiological mechanisms deriving from Br2 exposure have been described. However, the data on gender-based differences in susceptibility to acute Br2 exposure, particularly in context of cardiovascular toxicity, are unknown. Not only toxic inhaled chemicals, but gender-specific toxicity to pharmaceuticals has also been suggested, necessitating further the evaluation of effects of bromine in females [29]. In the past few decades, there has been an increased interest in the role of gender in health sciences [30]. The role of gender differences in cardiovascular morbidity and mortality has been vastly shown in clinical studies [31–34]. Our lab has previously shown that Br2-induced cardiac injury mimics that of an ischemia reperfusion model in male rats [25].
Survival and clinical outcome after Br2 exposure
Acute high concentration Br2 inhalation can cause death within hours [3]. Br2-exposure victims experience lacrimation, stinging and burning in the eyes, upper airway irritation, coughing, suffocation, bronchitis and bronchial asthma [6]. Accidental high concentration exposure to Br2 also causes respiratory and myocardial injury and circulatory collapse [1]. Using animal models, our studies have demonstrated that acute Br2 exposure causes death and cardiac damage and dysfunction in survivors, which is related to the formation of Br2 reactants in the pulmonary bed, which are released in the blood and reach the heart [25, 35, 36]. In this study, Br2 (600 ppm, 45 min) inhalation was fatal for females rats as they presented ~59% (n = 24) mortality rate 48 h after exposure (Fig. 1B) similar to that of male rats, which was previously reported by us [25, 35, 37]. Clinical scores were obtained in survivors by assessing breathing mechanics, respiratory quality and activity. The higher score represents more significant injury to the animal. We observed a marked deterioration of the clinical state of the animals after Br2 exposure, and this is made evident by the statistically significant difference between the clinical score at each timepoint after exposure when compared with unexposed rats (Fig. 1C). Ischemic injury causes a decrease in HR in female rats when compared to male rats [38]. Halogen inhalation-induced myocardial depression has also been reported by us before [26]. We have demonstrated a significant bradycardia in male rats following Br2 inhalation [25]. HR after Br2 inhalation was significantly decreased in female rats at 2, 4, 6, 8 and 24 h after exposure when compared to baseline readings (Fig. 1D), which is differing from other types of injury as a similar response is observed here in both genders. Oxygen saturation was also measured to assess respiratory effects. Br2 inhalation caused significant decrease in oxygen saturation in female animals at all times points as compared to unexposed control rats (Fig 1E), [25, 35]. These findings are similar to previously reported values with male animals. Thus, Br2 inhalation is uniformly toxic to males and females and causes mortality and morbidity at high-concentration exposures for prolonged duration, as these results do not differ from those previously reported in male animals [25, 35].
Br2 inhalation-induced pulmonary injury
Br2 is an irritant gas/vapor inhalation of which releases highly reactive intermediates from the respiratory mucosa, which damage the respiratory and alveolar epithelium and lead to acute respiratory distress [39–42]. Since the oxygen saturations were decreased in Br2-inhaling animals, we collected arterial blood 24 h after exposure from descending aorta and performed ABG analysis (Table 1). We earlier reported decreased oxygenation in male rats after Br2 inhalation [25]. In female animals, we observed a significant reduction in pO2 as well as an increase in pCO2 after exposure, while cHCO3− remained unchanged (Table 1). Arterial blood pH was decreased, indicating metabolic acidosis. Arterial hypoxemia was reported in mice exposed to Br2 [43]. Normal ABG values were also described in a case report of accidental Br2-exposure victim [44].
Br2 exposure causes alterations in hemodynamics and arterial gas composition
| Parameter | Unit | 0 ppm | 600 ppm | P | ||
|---|---|---|---|---|---|---|
| Value ± SE | N | Value ± SE | N | |||
| LV Vol s | μl | 112.9 ± 6.69 | 8 | 78.57 ± 15.76 | 7 | 0.07 |
| LV Vol d | μl | 336.4 ± 10.30 | 8 | 284.7 ± 24.44 | 7 | 0.08 |
| RWT; d | 0.53 ± 0.07 | 8 | 0.62 ± 0.04 | 7 | 0.31 | |
| RWT; s | 1.32 ± 0.07 | 8 | 1.60 ± 0.12 | 7 | 0.08 | |
| Ao ET | ms | 66.42 ± 0.76 | 8 | 52.22 ± 2.08* | 7 | <0.001 |
| PAT | ms | 24.44 ± 1.35 | 8 | 22.28 ± 1.87 | 6 | 0.37 |
| PET | ms | 68.83 ± 3.53 | 8 | 50.85 ± 4.80* | 6 | 0.01 |
| PAT/PET | ratio | 0.34 ± 0.01 | 7 | 0.45 ± 0.03* | 6 | 0.02 |
| PV SV | μl | 234.5 ± 2.52 | 8 | 144.40 ± 6.03* | 6 | <0.001 |
| IVC diameter CI | % | 15 ± 2 | 8 | 38 ± 6* | 7 | 0.006 |
| ABG | ||||||
| pH | 7.45 ± 0.01 | 8 | 7.37 ± 0.02* | 14 | 0.001 | |
| pCO2 | mmHg | 40.48 ± 1.78 | 8 | 49.91 ± 1.99* | 14 | 0.002 |
| pO2 | mmHg | 93.14 ± 2.91 | 8 | 79.04 ± 2.86* | 14 | 0.003 |
| cHCO3− | mmol/l | 27.76 ± 0.85 | 8 | 28.98 ± 0.71 | 14 | 0.29 |
| Parameter | Unit | 0 ppm | 600 ppm | P | ||
|---|---|---|---|---|---|---|
| Value ± SE | N | Value ± SE | N | |||
| LV Vol s | μl | 112.9 ± 6.69 | 8 | 78.57 ± 15.76 | 7 | 0.07 |
| LV Vol d | μl | 336.4 ± 10.30 | 8 | 284.7 ± 24.44 | 7 | 0.08 |
| RWT; d | 0.53 ± 0.07 | 8 | 0.62 ± 0.04 | 7 | 0.31 | |
| RWT; s | 1.32 ± 0.07 | 8 | 1.60 ± 0.12 | 7 | 0.08 | |
| Ao ET | ms | 66.42 ± 0.76 | 8 | 52.22 ± 2.08* | 7 | <0.001 |
| PAT | ms | 24.44 ± 1.35 | 8 | 22.28 ± 1.87 | 6 | 0.37 |
| PET | ms | 68.83 ± 3.53 | 8 | 50.85 ± 4.80* | 6 | 0.01 |
| PAT/PET | ratio | 0.34 ± 0.01 | 7 | 0.45 ± 0.03* | 6 | 0.02 |
| PV SV | μl | 234.5 ± 2.52 | 8 | 144.40 ± 6.03* | 6 | <0.001 |
| IVC diameter CI | % | 15 ± 2 | 8 | 38 ± 6* | 7 | 0.006 |
| ABG | ||||||
| pH | 7.45 ± 0.01 | 8 | 7.37 ± 0.02* | 14 | 0.001 | |
| pCO2 | mmHg | 40.48 ± 1.78 | 8 | 49.91 ± 1.99* | 14 | 0.002 |
| pO2 | mmHg | 93.14 ± 2.91 | 8 | 79.04 ± 2.86* | 14 | 0.003 |
| cHCO3− | mmol/l | 27.76 ± 0.85 | 8 | 28.98 ± 0.71 | 14 | 0.29 |
Note: Animals were subjected to Br2 exposure and after 24h were placed under anesthesia and echocardiographic and hemodynamic analyses were performed; following those analyses, animals were euthanized and arterial blood was collected from descending aorta and arterial blood gas (ABG) analysis was performed; left ventricle volume (LV Vol), right wall thickening (RWT), aortic ejection time (Ao ET), pulmonary acceleration time (PAT), pulmonary ejection time (PET), pulmonary stroke volume (PV SV), inferior vena cava (IVC) diameter were measured; s and d denote systole and diastole, respectively. P-values are specified in the table in bold letters, and * denotes statistical significance <0.05
Br2 exposure causes alterations in hemodynamics and arterial gas composition
| Parameter | Unit | 0 ppm | 600 ppm | P | ||
|---|---|---|---|---|---|---|
| Value ± SE | N | Value ± SE | N | |||
| LV Vol s | μl | 112.9 ± 6.69 | 8 | 78.57 ± 15.76 | 7 | 0.07 |
| LV Vol d | μl | 336.4 ± 10.30 | 8 | 284.7 ± 24.44 | 7 | 0.08 |
| RWT; d | 0.53 ± 0.07 | 8 | 0.62 ± 0.04 | 7 | 0.31 | |
| RWT; s | 1.32 ± 0.07 | 8 | 1.60 ± 0.12 | 7 | 0.08 | |
| Ao ET | ms | 66.42 ± 0.76 | 8 | 52.22 ± 2.08* | 7 | <0.001 |
| PAT | ms | 24.44 ± 1.35 | 8 | 22.28 ± 1.87 | 6 | 0.37 |
| PET | ms | 68.83 ± 3.53 | 8 | 50.85 ± 4.80* | 6 | 0.01 |
| PAT/PET | ratio | 0.34 ± 0.01 | 7 | 0.45 ± 0.03* | 6 | 0.02 |
| PV SV | μl | 234.5 ± 2.52 | 8 | 144.40 ± 6.03* | 6 | <0.001 |
| IVC diameter CI | % | 15 ± 2 | 8 | 38 ± 6* | 7 | 0.006 |
| ABG | ||||||
| pH | 7.45 ± 0.01 | 8 | 7.37 ± 0.02* | 14 | 0.001 | |
| pCO2 | mmHg | 40.48 ± 1.78 | 8 | 49.91 ± 1.99* | 14 | 0.002 |
| pO2 | mmHg | 93.14 ± 2.91 | 8 | 79.04 ± 2.86* | 14 | 0.003 |
| cHCO3− | mmol/l | 27.76 ± 0.85 | 8 | 28.98 ± 0.71 | 14 | 0.29 |
| Parameter | Unit | 0 ppm | 600 ppm | P | ||
|---|---|---|---|---|---|---|
| Value ± SE | N | Value ± SE | N | |||
| LV Vol s | μl | 112.9 ± 6.69 | 8 | 78.57 ± 15.76 | 7 | 0.07 |
| LV Vol d | μl | 336.4 ± 10.30 | 8 | 284.7 ± 24.44 | 7 | 0.08 |
| RWT; d | 0.53 ± 0.07 | 8 | 0.62 ± 0.04 | 7 | 0.31 | |
| RWT; s | 1.32 ± 0.07 | 8 | 1.60 ± 0.12 | 7 | 0.08 | |
| Ao ET | ms | 66.42 ± 0.76 | 8 | 52.22 ± 2.08* | 7 | <0.001 |
| PAT | ms | 24.44 ± 1.35 | 8 | 22.28 ± 1.87 | 6 | 0.37 |
| PET | ms | 68.83 ± 3.53 | 8 | 50.85 ± 4.80* | 6 | 0.01 |
| PAT/PET | ratio | 0.34 ± 0.01 | 7 | 0.45 ± 0.03* | 6 | 0.02 |
| PV SV | μl | 234.5 ± 2.52 | 8 | 144.40 ± 6.03* | 6 | <0.001 |
| IVC diameter CI | % | 15 ± 2 | 8 | 38 ± 6* | 7 | 0.006 |
| ABG | ||||||
| pH | 7.45 ± 0.01 | 8 | 7.37 ± 0.02* | 14 | 0.001 | |
| pCO2 | mmHg | 40.48 ± 1.78 | 8 | 49.91 ± 1.99* | 14 | 0.002 |
| pO2 | mmHg | 93.14 ± 2.91 | 8 | 79.04 ± 2.86* | 14 | 0.003 |
| cHCO3− | mmol/l | 27.76 ± 0.85 | 8 | 28.98 ± 0.71 | 14 | 0.29 |
Note: Animals were subjected to Br2 exposure and after 24h were placed under anesthesia and echocardiographic and hemodynamic analyses were performed; following those analyses, animals were euthanized and arterial blood was collected from descending aorta and arterial blood gas (ABG) analysis was performed; left ventricle volume (LV Vol), right wall thickening (RWT), aortic ejection time (Ao ET), pulmonary acceleration time (PAT), pulmonary ejection time (PET), pulmonary stroke volume (PV SV), inferior vena cava (IVC) diameter were measured; s and d denote systole and diastole, respectively. P-values are specified in the table in bold letters, and * denotes statistical significance <0.05
BALF protein content is an important indicator of pulmonary injury and loss of alveolar capillary barrier integrity [45]. Our studies have demonstrated increased BALF protein following toxic gas inhalations, including halogen gas exposure in male rats [25, 26, 35, 45–49]. Br2 exposure caused BALF protein to be increased in female animals when compared to naive (0.387 ± 0.069 mg/ml in Br2-exposed animals vs. 0.1865 ± 0.010 mg/ml in naive animals) (P = 0.04) (Fig. 2A). Pulmonary injury was also assessed by measuring the lung wet weight-by-dry weight (ww/dw) ratio as a marker of edema and inflammation. Br2 inhalation caused significant pulmonary edema in female animals, with ww/dw ratios of 5.007 ± 0.118 before exposure and 5.492 ± 0.123 after exposure (P = 0.01) (Fig. 2B). All these pulmonary injury assessments do not differ from previously published data on male animals. Pulmonary edema has been reported by us and others in rodents after Br2 inhalation [25, 50, 51]. Pulmonary edema in victims of accidental Br2 inhalation has also been reported [44].
Br2 induces injury to the lungs and heart in female rats. Br2 exposures were performed as described in the text and in Fig. 1A; blood and BALF were collected, followed by a necropsy; pulmonary injury was assessed by measuring BALF protein concentration (A) and ww/dw ratio (B); cardiac weights were taken and HW/BW ratio (C) was calculated, and plasma cardiac troponin I (cTnI) (D) and N-terminal pro-brain natriuretic peptide (NT-proBNP) (E) were measured by ELISA; data are mean ± SE (n = 6–8 for each group), and * demonstrates statistical significance from naive animals, P < 0.05. Black filled circles demonstrate the unexposed or 0 ppm controls and open white circles demonstrate the 600 ppm bromine exposed animals.
Cardiac Structural and functional analysis
We have shown that Br2 inhalation causes acute myocardial injury with significant ultrastructural changes, leading to biventricular cardiac dysfunction in adult male rats [25]. Here, we investigated whether gender has any effect on structural alterations that take place in the heart after Br2 exposure. At the 24-h timepoint, Br2 exposure caused no significant change in cardiac weight in female rats, as evidenced by heart weight-by-body weight ratio, HW/BW (Fig. 2C). Although the trends were toward increased HW/BW in females, significant increased HW/BW ratio was observed in previous studies in male rats at this timepoint [25]. This could be related to the lower body weights in these age-matched females. A mechanism of protection against cardiac hypertrophy in females could also be important in this model, but it is a mere speculation at this point. Increase in blood troponin and N-terminal pro-brain natriuretic peptide, NT-proBNP, are of a great prognostic value for predicting serious cardiac outcomes in patients [52, 53]. We have demonstrated that Br2 inhalation in rats causes increase in these key cardiac injury marker levels soon after exposure, and they tend to remain elevated for prolonged durations [25, 37]. Cardiac troponin, cTnI, was measured in the plasma. Br2 inhalation caused significant increase in the plasma cardiac troponin content in female rats as it did on male rats in previously reported studies (Fig. 2D). Cardiac injury was also measured by evaluating plasma NT-proBNP in the Br2-exposed animals. NT-proBNP was also significantly elevated in females (Fig. 2E) as observed in the male rats in our previous study [25]. These findings suggest significant cardiac injury after Br2 inhalation in both genders.
To assess further the impact of bromine inhalation on the heart, cardiac function analysis was performed by compiling hemodynamic and echocardiography analyses. Mean arterial pressure (MAP) was not altered in females after Br2 exposure (91.29 ± 2.77 mmHg) when comparing to naive animals (92.25 ± 2.12 mmHg) (P = 0.79) (Fig. 3A). This was significantly different from what was observed in male rats where the MAP was significantly elevated starting 3 h post-Br2 exposure and remained elevated until 24 h after exposure. MAP is generally increased in males as compared to females and is known to be increased more rapidly under adverse conditions [54, 55]. While evaluating RV peak pressure, we observed a significant increase in female animals from pressures of 24.86 ± .83 mmHg in naive animals to 33.00 ± 1.29 mmHg in Br2-exposed female rats (P = 0.001) (Fig. 3B). Similar to these findings, both LV and RV peak systolic pressures were significantly elevated in male rats 24 h after bromine exposure [25]. In order to study diastolic function, we analyzed changes in LV end diastolic pressure (LVEDP). LVEDP values were 2.93 ± .46 mmHg in naive rats and 3.44 ± .37 mmHg in exposed animals (P = 0.41) (Fig. 3C). RV end diastolic pressure (RVEDP) was measured to estimate RV diastolic function, and we observed no significant difference, with the pressures of unexposed animals being 1.22 ± .24 mmHg; while in exposed females, pressure was 2.63 ± .93 mmHg (Fig. 3D) (P = 0.18). Intra-ventricular dimensions were measured by evaluating LV end systolic diameter (LVESD). There was no statistically significant difference in LVESD, with values of 3.97 ± .16 mm in naive animals and 3.55 ± .16 mm in exposed animals (P = 0.08). The LV end diastolic diameters (LVEDD) values were 6.45 ± .19 mm in naive animals and 5.78 ± .27 mm in Br2-exposed animals (P = 0.07) (Fig. 3E). However, LVEDD was significantly decreased in male rats exposed to bromine when observed at 24 h postexposure [5]. Interestingly, female rat hearts exhibited diastolic dysfunction upon bromine inhalation as shown by decreased MV E/A, which is similar to that observed in Br2-exposed male rats (Fig. 3F). PW thickening measurements were also obtained, and the values were 32.77 ± 2.35% in naive animals and 35.52 ± 2.57% in Br2-exposed female rats (P = 0.94).
Br2 exposure causes cardiac dysfunction; Br2 exposures were performed as described in the text and in Fig. 1A; at endpoint (24 h), animals were placed under anesthesia, and echocardiographic and hemodynamic studies were performed; graphs were plotted for mean arterial pressure (MAP) (A), RV peak pressure (B), LVEDP (C), RVEDP (D), LVEDD (E) and mitral valve E/A wave ratio (F); data are mean ± SE (n = 8 for each group), and * demonstrates statistical significance from naive, 0-ppm animals (P < 0.05). Black filled circles demonstrate the unexposed or 0 ppm controls and open white circles demonstrate the 600 ppm bromine exposed animals.
Acutely increased LV shortening was observed in male rats that were exposed to bromine. LV FS did not differ from controls, while velocity of circumferential fiber shortening (VCFr) was significantly increased upon Br2 inhalation, with values of 7.138 ± 0.42% in naive animals and 11.60 ± 1.56% in Br2-exposed females (P = 0.03) (Fig. 4B).
Br2 exposure increases cardiac contractility and stress in female rats; Br2 exposures were performed as described in the text and in Fig. 1A, animals were placed under anesthesia and echocardiographic and hemodynamic studies were performed 24 h later; values for FS (A) and VCFr (B) are shown; LV VCFr by end systolic wall stress ratio (VCFr/ESWS) was calculated to assess systolic function (C); end systolic wall stress-to-end systolic volume ratio was calculated to measure systolic function (ESWS/ESV) (D); (E) demonstrates a summary of findings in bromine-exposed male and female animals; data are mean ± SE (n = 8 for each group), and * demonstrates statistical significance from 0 ppm (P < 0.05). Black filled circles demonstrate the unexposed or 0 ppm controls and open white circles demonstrate the 600 ppm bromine exposed animals.
We also measured wall stress to evaluate whether changes in stress were different in female animals when compared to those previously reported in studies carried out in male animals. In systole, wall stress (ESWS) was 49.28 ± 3.78% in naive animals and 34.06 ± 4.28% in exposed females (P = 0.02); this decrease can be explained by the lack of change in MAP, while the contractility increases, as afterload plays a major role in ESWS measurement. While in diastole, stress values were 7.15 ± 1.93% in naive animals and 4.54 ± 1.01% in exposed females, presenting no statistically significant difference (P = 0.26). Neither ESWS nor EDWS significantly differed from values previously reported in studies performed in male animals. VCfr was corrected for stress to account for changes in afterload, and this also indicated increased contractility in female rats after Br2 inhalation with values of 0.14 ± 0.01 in naive animals and 0.29 ± 0.04 in exposed animals (P = 0.01)(Fig. 4C). Finally, we assessed the difference in the change of systolic function by measuring parameters ESWS/ESV and observed values of 0.436 ± 0.031 in unexposed and 0.492 ± 0.073 in bromine exposed animals (P = 0.50)(Fig. 4D). These values were also not different in males. In addition, we also found in females an acceleration in Ao ET (Table 1), which also points toward the hypercontractility that was also reported in previous studies in male animals [25]. A decline in RV function as shown by changes in PET, PAT/PET, RV SV and IVC diameter was also observed (Table 1). RV dysfunction was also reported in male animals in previous studies [25]. All of these acute changes in LV systolic function are mostly similar to our previous findings in male rats. However, it important to note that LV VCFr and other indices of LV systolic function were significantly decreased below controls 7 days after Br2 exposure in male rats, which is consistent with the severe underlying myocardial damage [25]. Such acute LV damage progresses to further cardiac injury and dysfunction in later timepoints, such as 14 and 28 days after exposure, timepoints which were not included in this study [37].
As presented here, our results do not support any gender-specific susceptibility difference to Br2-induced cardiotoxicity. We thoroughly examined several factors that are indicative of cardiac structure and health after Br2 inhalation and found that males and females suffered similar cardiopulmonary injury under our exposure conditions. We did not find any differences in the survival and clinical outcome of female rats following Br2 exposure as well. Br2 exposure has shown to have a different effect on blood pressure when evaluating previously reported data from male animals and the female animals that were utilized in this study, with the values being considerably lower in females. This is not surprising, given the documented higher MAP in males, compared to age-matched pre-menopausal females, which pre-disposes men to higher risk of cardiovascular diseases [56]. FS was also found to be different between the male and female rats after Br2 exposure and could be attributed to the gender-specific adaptability and needs to be further evaluated. We did determine VCFr which was similar across genders, which reveals the characteristic hypercontractility that has been reported to characterize Br2-induced cardiac injury.
Based on our observations, we conclude that exposure to Br2, particularly at the dose tested in this study, equally affects male and female rats. In most of our assays to assess cardiac structure and function, no gender-specific effects of Br2 inhalation were observed, which differs from other similar models like that of cardiac ischemia reperfusion where female animals present lesser injury. Such information can be useful when stratifying patients following accidental or intentional exposure to Br2, with clear implications on clinical outcomes. These studies will also provide important endpoints to evaluate the therapeutic efficacy of potential drug targets.
Funding
CounterACT Program; National Institutes of Health Office of the Director (NIH OD); National Institute of Environmental Health Sciences (NIEHS) (grant numbers U01ES028182 and U01ES028182-02S1 to S.A. and L.J.D.I. and U01ES025069 to A.A.); National Heart Lung and Blood Institute (NHLBI) (R01HL114933 to A.A.).
Conflict of interest
The authors do not have any conflict of interest pertinent to this manuscript.
