Endocrine disrupter chemicals affect the humoral antimicrobial activities of gilthead seabream males even upon the cease of the exposure

Abstract

17α-ethynilestradiol (EE₂) and tamoxifen (Tmx) are pollutants world-wide distributed in aquatic environments. Gilthead seabream, Sparus aurata L., is highlighted as a species model of intensively culture in anthropogenic disturbed environments. The effects of these pollutants on gilthead seabream reproduction and some immune responses have been described but, the humoral innate antimicrobial activities have never received attention. In this work we analysed the latest in the plasma of gilthead seabream males of different ages and reproductive stages treated with 0, 2.5, 5 or 50 μg EE₂ or 100 μg Tmx g^-1 food during different times of exposure and of reverting to commercial diet (recovery). The peroxidase and protease activities decreased as the spermatogenesis of the first reproductive cycle (RC) proceeded in control fish. However, only protease and antiprotease activities showed different level at different stages of the second RC in control fish, but showed scarce disruption in fish treated with EE₂ or Tmx. Peroxidase and bactericide activities are more sensitive to EE₂, than to Tmx. The effects induced by EE₂ varied depending on the activity analyzed, the dose and the time of exposure and the reproductive stage and the age of the specimens.

Figure 1

Antimicrobial humoral activities in not treated gilthead seabream males at different times and reproductive stages. Fish at two different moments during the resting stage, previous to the first RC, and the spermatogenesis stage of the first RC (a), at different moments during the spermatogenesis stage of the second RC (b) and at post-spawning and testicular involution of the second RC (c). Data represent means ± standard error (n = 6). Different letters denote statistically significant differences between the groups (P < 0.05).

Figure 2

Antimicrobial humoral activities in gilthead seabream males at the resting stage, previous to the first RC, and the spermatogenesis stage of the first RC. The fish were treated with 0, 5 or 50 µg EE₂ g⁻¹ food and sampled at 7 and 28 days of treatment where the peroxidase (a), the bactericide (b), the protease (c) and the antiprotease (d) activities were studied. Data represent means ± standard error (n = 6). Different letters denote statistically significant differences between the groups (P < 0.05).

Figure 3

Antimicrobial humoral activities in gilthead seabream males during the spermatogenesis stage of the second reproductive cycle treated with EE₂ or Tmx during a short term period and a short recovery time. The fish were treated with 0, 5 µg EE₂ or 100 µg Tmx g⁻¹ food during 15 and 30 days and after 6 and 22 days of reverting to the commercial diet. The peroxidase (a), bactericide (b), protease (c) and antiprotease (d) activities were studied. Data represent means ± standard error (n = 6). Different letters denote statistically significant differences between the groups (P < 0.05).

Figure 4

Antimicrobial humoral activities in gilthead seabream males in spermatogenesis stage of the second reproductive cycle treated with EE₂ during a long term period and a long recovery time. The fish were treated with 0, 2.5 or 5 µg EE₂ g⁻¹ food for 83 days (from spermatogenesis stage to post-spawning stage) and after 91 days (testicular involution stage) of reverting to the commercial diet. The peroxidase (a), bactericide (b) protease (c) and antiprotease (d) activities were analyzed. Data represent means ± standard error (n = 6). Different letters denote statistically significant differences between the groups (P < 0.05).

Figure 5

Schematic illustration of the timing of the different experiments related to the reproductive stages and age (years) of the gilthead seabream. RC, reproductive cycle; SG, spermatogenesis stage; S, spawning stage; PS, post-spawning stage; R, resting stage; TI, testicular involution stage; EE₂, 17α-ethynilestradiol; Tmx, tamoxifen; dT, days of treatment; dR, days of recovery. Data obtained from this manuscript and from Chaves-Pozo et al. (2005, 2009)^, and Liarte et al. (2007).