The Synthetic Progestin Levonorgestrel Is a Potent Androgen in the Three-Spined Stickleback (<i>Gasterosteus aculeatus</i>)

The use of progestins has resulted in contamination of aquatic environments and some progestins have in experimental studies been shown to impair reproduction in fish and amphibians at low ng L^–1 concentrations. The mechanisms underlying their reproductive toxicity are largely unknown. Some progestins, such as levonorgestrel (LNG), exert androgenic effects in mammals by activating the androgen receptor (AR). Male three-spined stickleback (<i>Gasterosteus aculeatus</i>) kidneys produce spiggin, a gluelike glycoprotein used in nest building, and its production is directly governed by androgens. Spiggin is normally absent in females but its production in female kidneys can be induced by AR agonists. Spiggin serves as the best known biomarker for androgens in fish. We exposed adult female sticklebacks to LNG at 5.5, 40, and 358 ng L^–1 for 21 days. Androgenic effects were found at LNG concentrations ≥40 ng L^–1 including induction of spiggin transcription, kidney hypertrophy, and suppressed liver vitellogenin transcription. These are the first in vivo quantitative data showing that LNG is a potent androgen in fish supporting the contention that androgenic effects of certain progestins contribute to their reproductive toxicity

Antibiotics quantified in the water phase over 100 days in microcosms for three microcosms (1x, 10x and 1000x antibiotic concentration).

<p>Error bars indicate the standard error of the mean. The reduction (relative decrease of the initial concentration as compared to day ‘100’) is presented in parenthesis after each line. Sampling points without dots indicate that the antibiotic concentration was below the limit of quantification except for the quantification of oxytetracycline at day ‘28’ in the 1000x microcosm which could not be performed due to sample loss.</p

The experimental set-up consisted of three series of microcosms with varying concentrations of antibiotics added (1x, 10x and 1000x), and additional control microcosms without antibiotic addition.

<p>The experimental set-up consisted of three series of microcosms with varying concentrations of antibiotics added (1x, 10x and 1000x), and additional control microcosms without antibiotic addition.</p

Average concentrations (n = 3) of antibiotics in vicinity of drug formulation facilities (ng L⁻¹).

<p><i>P1a:</i> upstream Kahuta industrial estate (KIE); <i>P1b:</i> downstream KIE; <i>P2–P4:</i> drug formulation facilities in and around Lahore (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062712#pone-0062712-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062712#pone.0062712.s002" target="_blank">Table S2</a>); <i>( )</i>: RSD (relative standard deviation).</p

Quantification and detection of antibiotic resistance genes <i>sulI</i> and <i>ermB</i> in microcosm sediments with values in units of gene copies/10⁶ 16S rRNA gene copies.

<p>‘-’: below limit of detection.</p><p>‘+’: detected, but below quantification limit.</p><p>Quantification and detection of antibiotic resistance genes <i>sulI</i> and <i>ermB</i> in microcosm sediments with values in units of gene copies/10⁶ 16S rRNA gene copies.</p

Levels of antibiotic resistance genes (<i>sulI, dfrA1, erm B, tetA and tetB</i>) and integrons (<i>intl1</i>) at eight sampling sites (n = 1, pooled samples).

<p>Levels of antibiotic resistance genes (<i>sulI, dfrA1, erm B, tetA and tetB</i>) and integrons (<i>intl1</i>) at eight sampling sites (n = 1, pooled samples).</p

Map of the region with sampling sites indicated.

<p>R1–R10 are rivers, D is the Rawal dam, C1–C2 is the Lahore branch canal, SD is the sewage drain nearby hospitals and P1–P4 are the drug formulation facilities; *: sites where sediment samples are also taken along with water samples.</p

Average concentrations (R1–3, R5–7, SD n = 3; C1 and C2 n = 2; R4, R8–10, D n = 1) of antibiotics in rivers, dam, canal and vicinity of hospitals (ng L⁻¹).

<p><i>ATB:</i> antibiotics; <i>R1–R10:</i> river sites; <i>D:</i> dam; <i>C1–C2:</i> lahore branch canal; <i>SD:</i> sewage drain near hospitals (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062712#pone-0062712-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062712#pone.0062712.s003" target="_blank">Table S3</a>); <i>( )</i>: RSD (relative standard deviation); <i>LOQ</i>: Limit of quantification.</p

Sampling locations in Kyoto (R1–R4) and Osaka (R5–R6) prefectures, Japan.

<p>Sampling locations in Kyoto (R1–R4) and Osaka (R5–R6) prefectures, Japan.</p

Average water concentrations (ng L⁻¹) of oseltamivir carboxylate (OC) measured in Kyoto (R1–R4) and Osaka (R5–R6) prefectures, Japan.

<p>Average water concentrations (ng L⁻¹) of oseltamivir carboxylate (OC) measured in Kyoto (R1–R4) and Osaka (R5–R6) prefectures, Japan.</p