Explants of corpus luteum were washed in PBS containing 100 IU of penicillin and 100 μg/mL of streptomycin. Afterwards, explants were placed in 24-well plates and pre-incubated in 1 mL of phenol red free culture medium (M-199, Sigma Aldrich, Saint Louis, MO, USA) with 5% FBS, 1% BSA and 100 IU/mL of penicillin, and 100 μg/mL of streptomycin for 1.5 h. Subsequently, explants were incubated in medium (M-199 + 1%BSA + antibiotics) with PGF2α (1 µM; Sigma-Aldrich), E2 (10 nM; Sigma-Aldrich), Triclosan (10 nM, 100nM, 1µM and 10 µM; Sigma-Aldrich), and a combination of PGF2α + Triclosan, E2 + Triclosan, and PGF2α + E2 + Triclosan for 24 h at 37 °C in a humidified 5% CO2 atmosphere. Doses of PGF2α and E2 were taken from the literature [10 (link)]. After incubation, medium was collected and frozen at −20 °C until assayed for hormone concentration. Corpus luteum explant viability was determined by lactate dehydrogenase (LDH) activity in the culture medium, measured spectrophotometrically at 340 nm utilizing the principle of Wartburg’s optical test [74 ].
Triclosan
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe
Triclosan is a broad-spectrum antimicrobial agent used in various laboratory applications. It is effective against a wide range of bacteria, fungi, and some viruses. Triclosan is commonly used in disinfectants, antiseptics, and other products to inhibit the growth of microorganisms.
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81 protocols using triclosan
1
Corpus Luteum Explant Hormone Assay
2
Evaluation of Antibacterial Agents Against Bacterial Strains
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Bacterial strains were cultured in 1:20 diluted trypticase soy broth (1:20 TSB) at 37°C, shaking at 200 rpm. For solid medium, TSB was supplemented with 1.5% agar. The following antibacterials were used: ofloxacin, ciprofloxacin, erythromycin, polymyxin B, rifampicin (Sigma – Aldrich), fosfomycin, meropenem (TCI Europe), triclosan (Merck Chemicals), melittin (Bachem), and SPI009 (Figure 1 ). Concentrations are indicated throughout the text. Bacterial strains used in this study are listed in Table 1 .
3
Triclosan Exposure Effects on EpiDerm
4
Triclosan-Induced Oxidative Stress and STAT3 Inhibition
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Triclosan was obtained from Shanghai Baidi Biody-Bio Co., Ltd. Hoechst, Cal-AM, Eth-1, Fluo-3/AM, mito-Tracker, mito-SOX, tetramethylrhodamine methyl ester (TMRM) and DAPI were purchased from Thermo Fisher Scientific, Inc. Dihydroethidium dye was purchased from the Beyotime Institute of Biotechnology. Tempol, 3-MA and acetylcysteine (NAC) were purchased from Sigma Aldrich; Merck KGaA. Tempol (0.5 and 1 mM) is a radical scavenger that was used to test the effect of ROS levels on cytotoxicity induced by Triclosan (20 µM) in the lactate dehydrogenase (LDH) release assay. S3I-201 was purchased from EMD Millipore. S3I-201 (10 and 20 µM) is a STAT3 inhibitor that was used to detect the effect of STAT3 activity change on cytotoxicity induced by Triclosan (20 µM) in the LDH release assay. Anti-p-STAT3 (Y705, #9131, 1:1,000), anti-STAT3 (#4904, 1:1,000), anti-p-AMPK (Thr172, #2535, 1:1,000), anti-AMPK (#2532, 1:1,000) and anti-p62 (#88588, 1:1,000) were purchased from Cell Signaling Technology, Inc., Bcl-2 (ab196495, 1:1,000) antibody was purchased from Abcam and LC3 (L7543, 1:1,000) antibody was purchased from Sigma Aldrich; Merck KGaA.
5
Hospital Wastewater Characterization and Analysis
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Diclofenac sodium salt (NaDCF, 98%+), fluconazole (98%+), hydrochlorothiazide (98%+), chlorhexidine (99%+), ketoconazole (98%+), triclosan (Irgasan, 97%+), atrazine (analytical standard), aluminum–nickel alloy (purum, 50% Al basis, 50% Ni basis) and an aqueous solution of NaBH4 (12 wt.% in 14 M NaOH) were delivered by Merck Co. (Prague, Czech Republic). Deuterated chloroform (CDCl3) was purchased from Merck Co. (Prague, Czech Republic). Additional chemicals and solvents in p.a. quality were obtained from a local supplier (Lach-Ner Co., Neratovice, Czech Republic).
A sample of hospital wastewater was stabilized with HNO3 for AOX (adsorbable organically bound halogens) determination [36 ]. The parameters of hospital wastewater stabilized with HNO3 are: pH = 2.07; [NH4+] = 32.4 mg L−1; [Cl−] = 820.7 mg L−1; [Ca+2 + Mg+2] = 4.5 mM; [NO3−] = 1213.6 mg L−1; CODCr = 349 mg O2 L−1; AOX = 1.44 mg Cl L−1. The experiments were carried out using deionized water, except where noted otherwise. All operations and manipulations were conducted in the air.
A sample of hospital wastewater was stabilized with HNO3 for AOX (adsorbable organically bound halogens) determination [36 ]. The parameters of hospital wastewater stabilized with HNO3 are: pH = 2.07; [NH4+] = 32.4 mg L−1; [Cl−] = 820.7 mg L−1; [Ca+2 + Mg+2] = 4.5 mM; [NO3−] = 1213.6 mg L−1; CODCr = 349 mg O2 L−1; AOX = 1.44 mg Cl L−1. The experiments were carried out using deionized water, except where noted otherwise. All operations and manipulations were conducted in the air.
6
Oral Gavage of Bacterial Strains in Mice
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Yp and Ye were cultured to stationary phase at 28°C and 250 rpm shaking for 16 hours in 2xYT broth supplemented with 2 μg/ml triclosan (Millipore Sigma). Mice of either sex between 8-12 weeks of age were fasted for 16 hours and subsequently inoculated by oral gavage with 100-200 μl phosphate-buffered saline (PBS). All preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. bacterial strains were administered at 2x10 8 colony-forming units (CFU) per mouse with the exception of pYV-which was administered at 20x10 8 CFU per mouse.
7
Oral Gavage Infection Assay
8
Triclosan-Treated Oral Gavage in Mice
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Yp was cultured to stationary phase at 28°C and 250 rpm shaking for 16 hours in 2xYT broth supplemented with 2 μg/ml triclosan (Millipore Sigma). Mice were fasted for 16 hours and subsequently inoculated by oral gavage with 200 μl phosphate-buffered saline (PBS) as previously25 (link) All bacterial strains were administered at 2×108 colony-forming units (CFU) per mouse.
9
Preparation of Triclosan and NADP+ Stocks
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Triclosan (Millipore Sigma,
St. Louis, MO)
was weighed out and dissolved in DMSO to give 100 mM stock solutions.
NADP+ (Millipore Sigma; St. Louis, MO) was weighed out
and dissolved in 50 mM Tris buffer at pH 8 with 100 mM NaCl to give
100 mM stock solutions. Both Triclosan and NADP+ stock
solutions were stored as frozen aliquots at −80 °C, and
each aliquot was discarded after a single use to avoid multiple freeze-thaw
cycles.
St. Louis, MO)
was weighed out and dissolved in DMSO to give 100 mM stock solutions.
NADP+ (Millipore Sigma; St. Louis, MO) was weighed out
and dissolved in 50 mM Tris buffer at pH 8 with 100 mM NaCl to give
100 mM stock solutions. Both Triclosan and NADP+ stock
solutions were stored as frozen aliquots at −80 °C, and
each aliquot was discarded after a single use to avoid multiple freeze-thaw
cycles.
10
Inhibition Kinetics of InhA Enzyme
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Triclosan and NADH were obtained from Sigma-Aldrich. Stock solutions of all compounds were prepared in DMSO such that the final concentration of this co-solvent was constant at 5% v⁄v in a final volume of 1 mL for all kinetic reactions. Kinetic assays were performed using trans-2-dodecenoyl-coenzyme A (DDCoA) and wild type InhA as previously described.
Briefly, pre-incubation reactions were performed in an aqueous buffer (30 mM PIPES and mM NaCl pH 6.8) containing additionally 100 nM InhA, 250 µM cofactor (NADH) and the tested compound (50 µM or 10 µM). After 5 min of pre-incubation at 25 °C, addition of 50 µM DDCoA initiated the reaction which was followed at 340 nm (oxidation of NADH).
The inhibitory activity of each derivative was expressed as the percentage of inhibition of InhA activity (initial velocity of the reaction) with respect to the control reaction without inhibitor. Triclosan was used as a positive control. All activity assays were performed in triplicate.
Briefly, pre-incubation reactions were performed in an aqueous buffer (30 mM PIPES and mM NaCl pH 6.8) containing additionally 100 nM InhA, 250 µM cofactor (NADH) and the tested compound (50 µM or 10 µM). After 5 min of pre-incubation at 25 °C, addition of 50 µM DDCoA initiated the reaction which was followed at 340 nm (oxidation of NADH).
The inhibitory activity of each derivative was expressed as the percentage of inhibition of InhA activity (initial velocity of the reaction) with respect to the control reaction without inhibitor. Triclosan was used as a positive control. All activity assays were performed in triplicate.
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