Zinc sulfate

Adult zebrafish, Danio rerio, of both sexes were obtained from a regional distributor. All fish were maintained in 10-gallon glass aquaria with aerated, conditioned water at 28.5 °C and fed twice daily with freshwater flake food. Care was taken to minimize animal suffering and limit the number of animals used, and all animal procedures were approved by the Institutional Animal Care and Use Committee (Animal Welfare Assurance #A3252-01; protocol 13-05-01, 5 August 2013).
Before exposure to zinc sulfate, fish were anesthetized in 0.03% MS222 (methane sulfonate salt, Sigma, St. Louis, MO, USA) until unresponsive to a tail pinch. Exposed fish received intranasal administration of 2 μL of 0.2, 0.5, 1, or 5 M zinc sulfate (Sigma, St. Louis, MO, USA) in dH₂O to the right olfactory organ. A thin strip of Vaseline petroleum jelly (Unilever US, Englewood Cliffs, NJ, USA) was applied between the nostrils to prevent leakage to the left side, which was untreated to serve as an internal control. Fish were placed on ice to allow for a 3-min exposure to the zinc sulfate before being returned to a recovery tank and examined after 2, 3, 5, 7, 10, or 14 days.

The gingival tissue collected by surgical excision using a disposable no. 15 carbon steel scalpel blade (Embramac Braz. Com. of Surgical Mat. import and Export Trade Industry Ltd.; Campinas; SP; Brazil) on the lower left molar region was stored in vials containing 2 mL of Tris-HCl buffer. This tissue was homogenized using an Ultra 80 ultrasound device (Ultra Stirrer). Then, 360 µL collected from the homogenate, 360 µL of Tris-HCl buffer solution (pH 7.5) at 4°C with 50-mM zinc sulfate (Merck KgaA; Darmstadt; Hessen; Germany) and 0.05% polyethylene glycol (Sigma-Aldrich Corporate Offices; St Louis; Missouri; USA) were added to the quartz cuvette. After that, the solution was taken to the same fluorometer (Varian Medical Systems; Mulgrave; Victoria; Australia) and 80 µL of 200-µM MMP-8 Substrate was added (Sigma-Aldrich Corporate Offices; St Louis; Missouri; USA)²².
The settings used in the fluorometer (Varian Medical Systems; Mulgrave; Victoria; Australia) for the analysis were 280 nm excitation and 360 nm emission. The enzyme kinetics were evaluated based on the maximum speed for the period of 0-45 minutes. Determination of MMP-8 was carried out fivefold and an average of these values was obtained for all subsequent calculations.

Lithium chloride (Sigma), copper sulfate pentahydrate (Sigma), nickel (II) sulfate hexahydrate (Sigma), zinc sulfate (Merck), aluminum chloride hexahydrate (Sigma), cobalt chloride hexahydrate (Sigma), lead chloride (Sigma) and acrylamide (Sigma) were used for the toxicity tests. Cadmium chloride (Merck), arsenic trioxide (Merck), atrazine (EQ Laboratories), chlorpyrifos (EQ Laboratories), carbofuran (Chem Service), dimethoate (Chem Service), glyphosate (EQ Laboratories), and methoxychlor (Fluka) were provided by Taiwan Agricultural Chemicals and the Toxic Substances Research Institute, Council of Agriculture, Taiwan. Heavy metals, acrylamide, dimethoate and glyphosate were dissolved in sterile distilled water to make stock solutions, whereas other chemicals were either dissolved or diluted with dimethyl sulfoxide (DMSO) to the desired concentration as stock solutions.

The NST 2-hydroxy-N-[2-(1H-indol-3-yl)-ethyl]-benzamide was obtained by “in house” synthesis as previously reported (João Pessoa, PB, Brazil; patent document: BR 200304393-A) in Laboratory of Pharmaceutical Chemistry, UFPI (Teresina, PI, Brazil) [5 (link)]. Carrageenan, acetic acid, Triton X-100, L-methionine, hydroxylamine chloride, riboflavin, thiobarbituric acid (TBA), Griess reagent, sodium dodecylsulfate, O-dianisidine, hexadecyltrimethylammonium bromide (HTAB), sodium phosphate, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and sodium nitrite were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Indomethacin (Indocid®), sodium phosphate, zinc sulfate, hydrogen peroxide, Tris base, sodium hydroxide, and trichloroacetic acid were purchased from Merck (Darmstadt, Germany). Turk's solution was purchased from Newprov (Pinhais, PR, Brazil).

sCT (Limhamn, Sweden) was used as the active ingredient. Poly(methyl vinyl ether-alt-maleic acid), mucin, and Coomassie Brilliant Blue G-250 dye were purchased from Sigma Chemical Company (USA). Zinc sulfate, iron chloride, ethanol, methanol, hydrochloric acid, and NaOH were purchased from Merck Chemical Company (Germany). Orthophosphoric acid 85% w/v was from Panreac (Spain).

Chemical reagents used in this investigation are of analytical grade. These are: glucose, lactose, maltose, sodium chloride, ammonium nitrate, monopotassium phosphate, disodium sulfate, magnesium sulfate, calcium chloride, and zinc sulfate (Merck KGaA, Darmstadt, Germany), agarose, acrylamide, bis-acrylamide, urea, Tween 80, Chlorpyrifos (CP) 99.7% and 3,5,6-trichloro-2-pyridinol (TCP) 99.3% (Sigma-Aldrich Merck KGaA, Darmstadt, Germany).

The active ingredient of Tamsulosin hydrochloride (TMS) was purchased from Tolid Daru Pharmaceutical Co (Tehran, Iran). The citric acid (CA), Ethylenediamine (EDA), Rhodamine B (RhB), l-Cysteine (l-Cys), l-leucine (l-Lue), Creatinin, Sucrose, Glucose, Iron (ɪɪɪ) chloride, Aluminum chloride, Calcium chloride, copper (II) sulfate, Magnesium chloride, Sodium chloride, Potassium chloride, Zinc sulfate, Acetic acid, Phosphoric acid, Boric acid, and Methanol (Me-OH) with the analytical grade were purchased from Merck Co. (Darmstadt, Germany). Deionized (DI) water with a resistivity of more than 18 MΩ has been used in the whole of the experiments.