Use of Parabens (Methyl and Butyl) during the Gestation Period: Mitochondrial Bioenergetics of the Testes and Antioxidant Capacity Alterations in Testes and Other Vital Organs of the F1 Generation

Abstract

Since the mid-1920s, parabens have been widely used as antimicrobial preservatives in processed foods and beverages, pharmaceuticals, and cosmetic products. Paraben use continues to generate considerable controversy, both in the general population and in the scientific community itself. The primary purpose of our study was to determine whether parabens (methyl and butyl at concentrations of 100 and 200 mg/kg body weight by subcutaneous injection) during pregnancy of adult female Wistar rats can have an impact on the F1 generation. As far as we know, we are the first to demonstrate that using parabens during pregnancy has negative repercussions on the mitochondrial bioenergetics and antioxidant activity of testicular germ cells in the F1 generation. Our study showed that there was a 48.7 and 59.8% decrease in the respiratory control index with 100 and 200 mg/kg of butylparaben, respectively. Cytochrome c oxidase activity was significantly inhibited (45 and 51%) in both groups. In addition, 200 mg/kg butylparaben promoted a marked decrease in citrate synthase activity, indicating that mitochondrial content decreased in the germ cells, especially spermatocytes and spermatids. Mitochondrial ROS production increased in groups exposed to parabens in a concentration-dependent manner, especially the butyl one (102 and 130%). The groups exposed to butylparaben showed an increase in superoxide dismutase (SOD) and catalase (CAT) activities, while glutathione reductase (GR) and glutathione S-transferase (GST) decreased. With methylparaben, only differences in SOD and GR were observed; for the latter, this only occurred with the highest concentration. The glutathione (GSH)/glutathione disulfide (GSSG) ratio did not undergo any significant change. However, there was a considerable increase in hydroperoxide content in animals exposed to butylparaben, with 100 and 200 mg/kg resulting in 98.6 and 188% increase, respectively. Furthermore, several other organs also showed alterations in antioxidant capacity due to paraben use. In summary, our study demonstrates that paraben use during pregnancy will cause severe changes in the mitochondrial bioenergetics and antioxidant capacity of testicular germ cells and the antioxidant capacity of several other F1 generation organs.

Keywords: antioxidant activity; gestational exposure; male infertility; mitochondrial bioenergetics; parabens.

Figure 1

Experimental protocol. D1, first gestational day and beginning of paraben injection; D19–22 last gestational day and ending of paraben injection; D24, last lactational day and separation of males from females; D60, euthanasia; P1, gestational period; P2, lactational period; P3, postlactation period.

Figure 2

Oxygen consumption rates determined with mitochondria isolated from testicles of F1 generation rats. The parameters presented are respiratory state 3 (A), state 4 (B), respiratory control index (RCI) (C), and transmembrane potential (D). Mitochondria (0.8 mg/mL) were incubated at 30 °C in 1 mL of normal respiratory medium supplemented with rotenone (1.5 μg/mL). State 3 was obtained by the addition of 200 nmol of ADP in mitochondria to oxidize succinate (10 mM). State 4 was reached after total phosphorylation of ADP. The RCI was calculated as the ratio between the oxygen consumption rate in state 3 and state 4. Succinate-energized mitochondria ΔΨ was measured by means of a tetraphenylphosphonium-selective electrode. Freshly isolated mitochondria (1 mg) were incubated in 1 mL of the medium supplemented with 2 µM rotenone and 3 µM TPP⁺ and energized with 5 mM succinate. Results are expressed as nmol of oxygen consumed/min/mg of mitochondrial protein and are M ± SD of 7–10 independent experiments performed in duplicate. The values marked with the same letter are not statistically significant as determined by the Tukey’s post hoc test (p < 0.05).

Figure 3

Activities of complexes I and IV of the mitochondrial electron transport chain of testes of F1 generation. The enzymatic activities are expressed as nmol oxidized NADH (A) and nmol cytochrome c oxidized (B). The results are M ± SD of 7–10 independent experiments performed in duplicate. The values marked with the same letter are not statistically significant as determined by the Tukey’s post hoc test (p < 0.05).

Figure 4

Citrate synthase activities in mitochondria isolated from testes of F1 generation. The results are M ± SD of 7–10 independent experiments performed in duplicate and expressed as nmol 2-nitro-5-thiobenzoic acid/min/mg protein. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p < 0.05).

Figure 5

Production of hydrogen peroxide by functional mitochondria isolated from testes of F1 generation. The results are expressed as nmol H₂O₂/mg protein/15 min and are represented as M ± SD of 7–10 independent experiments performed with different mitochondrial fractions performed in duplicate. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p < 0.05).

Figure 6

Activity of oxidative stress enzymes in testes of F1 generation rats: (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) glutathione reductase (GR), and (D) glutathione S-transferase (GST). Results are M ± SD of 7–10 independent experiments. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p < 0.05).

Figure 7

The glutathione (GSH)/glutathione disulfide (GSSG) ratio (A) and lipid hydroperoxide concentration (B) in the testes of the F1 generation rats. Results are M ± SD of 7–10 independent experiments. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p < 0.05).