Ethic statements
The Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (TMDU) (A2021-131A) approved all animal experiments, which were conducted under the Department of Parasitology and Tropical Medicine, TMDU.
Materials
Deferoxamine mesylate salt (Sigma-Aldrich); 1,10-Phenanthroline monohydrate (Tokyo Chemical Industry); 5-Methyl-1,10-phenanthroline (Tokyo Chemical Industry); 4,7-Dimethyl-1,10-phenanthroline (Tokyo Chemical Industry); 3,4,7,8-Tetramethyl-1,10-phenanthroline (Tokyo Chemical Industry); 2,9-Dimethyl-1,10-phenanthroline hydrochloride monohydrate (Tokyo Chemical Industry); 4,7-Diphenyl-1,10-phenanthroline (Sigma-Aldrich); 4,7-Dimethoxy-1,10-phenanthroline (Sigma-Aldrich); 1,10-Phenanthroline-4,7-diamine (Enamine); N4,N4,N7,N7-Tetramethyl-1,10-phenanthroline-4,7-diamine (Enamine); Iron(II) sulfate heptahydrate (FUJIFILM Wako Pure Chemicals). All other reagents obtained from commercial sources were used as received unless otherwise stated.
Maintenance of S. mansoni using animals
According to previously described methods [18, 19], to maintain the life cycle of S. mansoni, Biomphalaria glabrata as an intermediate host (Puerto Rico strain), was individually infected with eight miracidia. The infected snails were kept in a thermostatic chamber at 28 °C for over 2 months and subsequently exposed to light to release the cercariae. Moreover, 6-week-old ICR mice (SLC, Hamamatsu, Japan) were infected by placing their tails in a tube containing 180 cercariae in tap water.
Following the Institutional Animal Care and Use Committee of TMDU (2010002C2), the infected mice were kept in a controlled temperature and humid environment with a 12:12 h light/dark cycle and had free access to food and water. Humane endpoints were considered when severe pain, suffering, excessive distress, or impending death was observed in any animal. All mice were euthanized using CO2 gas inhalation after the experiments. The status of the mice was monitored daily using a composite score, including vitality, fur quality, secretions, mobility, dyspnea, neurological signs, ascites, and their ability to ingest food or water. All mice were handled according to the ARRIVE guidelines and relevant regulations.
Preparation of schistosomula and adult worms
Schistosomula were appropriately prepared as follows: over 1000 cercariae obtained from the infected snails after exposure to light, were collected in tubes through centrifugation. The cercariae were passed through a syringe attached to a 20G needle approximately 10 times to produce mechanically transformed schistosomula. The larvae were subsequently placed in 0.2 mL of RPMI1640 medium in a 96-well plate and incubated for 24 h at 37 °C in an atmosphere containing 5% CO2. Adult worms were collected from euthanized mice at 7-week post-infection and placed in 2 mL of RPMI1640 containing 10% FBS (Thermo Fisher Science, USA), streptomycin, penicillin, and l-glutamine (Thermo Fisher Science) in 12-well plates. After incubation for 24 h at 37 °C in an atmosphere containing 5% CO2, all worms were washed thrice with the conditioned medium and were then used for the experiments.
Assessment of in vitro larvicidal activities of the compounds
The prepared schistosomula were placed in a 96-well plate (20–180 larvae/well) and the number of viable larvae was counted. Next, the compounds were added to individual wells and adjusted to final concentrations of 50, 10, 5, 1, 0.5, 0.1, and 0.05 μM. After incubation for 72 h at 37 °C in an atmosphere containing 5% CO2, the number of larvae was counted using previous methods [20, 21]. Dead larvae were identified based on their characteristic lack of movement and a disintegrated or completely crushed outer tegument. The IC50 and IC90 values were calculated based on the curves obtained from viability plots.
Assessment of in vitro anti-fecundity effects of the compounds
Surviving adult worm pairs were placed in a 24-well plate (three pairs/well), and 1 mL of culture medium was added to each well [18]. Subsequently, the compounds were added at the concentration of 5 µM. To promote egg production, 1 × 108 erythrocytes obtained from mice were added, and the solutions were adjusted to 2 mL by adding more culture medium. After incubation for 72 h at 37 °C in an atmosphere containing 5% CO2, the adult worms were removed, and 1 mL of the supernatant was discarded. Then, 2 mL of distilled water was added to each well, and the samples were incubated at room temperature for 1 h to lyse erythrocytes. After adding 1 mL of 50 mM NaOH and incubation at 4 °C overnight, the number of eggs was counted using inverted microscope to calculate the egg production rates.
Assessment of in vivo bio-activities of the compounds
Female 6-week-old BALB/c mice were infected with 180 cercariae (five mice/group). At 2-, 4-, and 6-week post-infections, 0.1 mL of the solution containing compounds dissolved in olive oil was orally administered to each mouse for three consecutive days. After euthanizing the mice at 8-week post-infection, adult worms were collected through perfusion, and the liver and intestinal tracts were removed. The obtained male and female adult worms were separately counted to determine their survival rates. Simultaneously, the liver and intestinal tracts were completely lysed using 4% KOH solution at 37 °C overnight with shaking. After treating the solution with distilled water, 10 µL of the resulting solution was placed on a glass slide, and the eggs were counted under a microscope to calculate the egg production and fecundity rates.
Observation of the ovaries of female adult worms
Ten female adult worms were collected from the groups of mice treated with or without PHN-(OMe)2 at 6-week post-infection. The adult worms were fixed for 1 week using a solution of 10% formalin, 48% alcohol, and 2% glacial acetic acid. Afterwards, the worms were stained overnight with acetocarmine solution (FUJIFILM Wako, Osaka, Japan). They were then destained in 70% acidic ethanol and were dehydrated in a graded ethanol series (70, 90, and 100%). Subsequently, the worms were cleared in a 50% xylene solution diluted in 100% ethanol for 1 min, and were mounted on slides using Bioleit (Okenshoji Co., Ltd., Japan). Finally, images were acquired using a Leica STELLARIS 5 confocal laser scanning microscopy, equipped with a 40x (NA = 1.25) oil-immersion objective and an argon laser at 488 nm as the excitation source. Images were collected as single stacks from at least ten individuals in each group. The size of the ovaries was determined by measuring the area of the images using Fiji software and calculating the means.
Electrospray ionization mass (ESI–MS) spectroscopic analysis
The reaction of PHN-(OMe)2 and Fe(II) ions was initiated by mixing PHN-(OMe)2 (5.0 mM) and FeSO4 (1.67 mM) in a DMSO solution that has been degassed to remove dissolved dioxygen. The solution containing the generated iron complex was exposed to molecular dioxygen and diluted with MeOH to prepare the sample. The chemical composition of the iron complex was then determined using an ESI–MS spectrophotometer (Q Exactive, Thermo Fisher Scientific).
Statistical analysis
Statistical comparisons were carried out using Student’s t test to evaluate the differences between treated and untreated groups. Differences were considered significant at p < 0.05 at a 95% confidence interval.