IJCS | Volume 32, Nº2, May/June 2019

249 Negrão et al. Effect of herbicide 2,4D inhalation on cardiac remodeling Int J Cardiovasc Sci. 2019;32(3)247-252 Original Article ingredient per hectare (g.i.a/ha) of 2,4D, diluted in 10 mL of 0.9% sodium chloride. The time of exposure consisted of 15 minutes/day on 3 consecutive days. - HCG: Solution consisting of 9.28 x 10-3 grams of active ingredient per hectare (g.i.a/ha) of 2,4-D diluted in 10 mL of 0.9% sodium chloride. The time of exposure was 15 minutes/day for 3 consecutive days. On the first day of exposure, all 15 animals were submitted to the nebulization with the recommended concentration for each group. After the 3 consecutive days of nebulization, they were anesthetized with Thiopental Sodium, at a dose of 100 mg/kg of weight, administered in the peritoneal cavity. Soon after being euthanized, the animals had their hearts fixed in 10% buffered formalin for 72 hours. Histological and histomorphometric analyses After being fixed in formalin, the tissue was embedded in paraffin blocks to obtain 4-micrometer thick coronal histological sections. The histological sections were stained on a slide with the Hematoxylin-Eosin (HE) solution to measure cardiomyocyte cross-sectional areas using a LEICA microscope (model DM750, Germany), which sends digital images to the computer using the LeicaApplication Suite LAS 4.2.0 image analysis system (Media Cybernetics, Silver Spring, Maryland, USA). 21,22 The images were obtained using a binocular optical microscope. All images were captured by video camera at 400x magnification (40x objective). Four LV sections were obtained from each animal and different field captures were analyzed, which were chosen according to the place where more cells could be visualized in a cross-sectional view. 22,23 Fifty cells were measured per analyzed ventricle. The selected cardiomyocytes were transversely sectioned and exhibited a round shape, with a nucleus visible in the center of the cell andwere located in the subendocardial layer of the LVmuscle wall. 22,23 The mean sectional areas obtained for each group were used as indicators of cell size. 23 We also performed the fractal dimension analysis through the box-countingmethod, in which 3 images were captured per each analyzed slide; after that, the ImageJ program was used to calculate the fractal dimension of each group (Figure 1). This careful technique aims to standardize the set of cardiomyocytes from the different groups to the maximum. The mean sectional areas obtained for each group were used as indicators of cell size. 23 Statistical analysis The softwareGraphPadPrismwas used for the statistical analysis. The Shapiro-Wilk test was performed to analyze data normality. The data showed normal distribution and were presented as mean ± standard deviation. One-Way ANOVA followed by Tukey test (p< 0.05) were performed for comparison between the groups. Results There was no alteration in the fractal dimension parameter between the CG = 1.37 ± 0.02, LCG = 1.33 ± 0.04 and HCG = 1.33 ± 0.07 groups (p > 0.05). The animals submitted to a high dose of the herbicide had cardiomyocyte hypertrophy (HCG = 303.9 ± 38.80 μm 2 ) when compared to the control group (CG = 236.9 ± 61.71 μm 2 ; p = 0.034). (Figure 2). Discussion To the best of our knowledge, this is the first study showing that the inhalation of 2,4D herbicide at high concentrations for a short time in mice causes cardiac remodeling. The 2,4D herbicide, when inhaled, is absorbed and distributed by the lungs, kidney and circulatory system. Enzymes promote the metabolism of the herbicide in these organs and tissues, which results in the formation of free radicals, which can be often more toxic than the herbicide itself. 24 Oxidative stress is caused by the release of free radicals, and it is an important mechanism that causes cell mutations. 24-26 There is strong evidence that oxidative stress plays a prominent pathophysiological role in cardiac remodeling. 27-28 The remodeling process is associated with changes in different mechanisms related to cardiac dysfunction, such as alterations in LV geometry, wall thickness, cavity diameter and normal configuration. 24-26 The animals of this study that inhaled 2,4D herbicide at a high concentration showed left ventricular hypertrophy, which suggests the development of cardiotoxicity and confirms the action of the 2,4D in the development of cardiac remodeling. Other studies using the same experimental model in rats indicate that the ingestion of a dose equivalent to 10% of the lethal dose (LD50) of oral pyrethroid insecticides causes changes in ventricular depolarization and repolarization, suggesting cardiotoxicity. 27,28