The evaluation of developmental toxicity requires new methods that provide an alternative to animal use. In this study, we evaluated the effects of thalidomide exposure [upon nanosecond pulsed electric field (nsPEF) treatment] on the embryonic development of, teratogenic effects in, and gene expression levels in medaka embryos, to determine whether the adverse effects of thalidomide can be detected using medaka. Incorporation of thalidomide into medaka embryos led to malformations in individuals. The results obtained by microinjecting thalidomide into zebrafish embryos could be reproduced by medaka embryo electroporation. Furthermore, the teratogenic mechanism of thalidomide was confirmed at the genetic level. Medaka eggs, in addition to zebrafish, can be used to assess the effects of thalidomide. As the nsPEF method can efficiently incorporate of test chemicals into medaka eggs, it is suggested to have great potential for use as a high-throughput animal alternative evaluation system.

Gavage has been used for many years in mammalian toxicology and experimental animal biology, as a direct oral administration method. It has the advantage of providing an exact dosage per body weight; however, gavage is not considered applicable to small fish like zebrafish (Danio rerio), owing to the difficulty associated with handling. In this study, a new gavage method for zebrafish was developed to minimize stress and damage to their digestive tracts, without the need for anesthesia. This device allows us to hold the zebrafish firmly in a predetermined position without directly touching them. This method involves holding the fish with a novel device and uses a specific syringe for dosing the test substance into the digestive organs. An acute oral toxicity test of potassium dichromate was conducted in adult zebrafish to demonstrate the new gavage method. No mortality was observed in the vehicle (water) control group. Furthermore, the median lethal dose (LD₅₀) values of potassium dichromate in male and female zebrafish were 103 and 195 mg/kg body weight, respectively. These values are directly comparable to the values in rats.

We investigated the expression of miRNAs as a potential biomarker in the colon, rectum, plasma, and feces of the rat dextran sulfate sodium (DSS)-colitis model. A 5% DSS solution with drinking water was administered to male SD rats for 1 week, followed by a 1-week off-dose period. In-life parameters were examined daily, and colon length and pathological changes were assessed post-mortem. A selected panel of miRNAs (miR-16-3p, miR-21-5p, miR-31a-5p, miR-34a-5p, miR-146b-5p, miR-155-5p, miR-181b-5p, miR-221-5p, and miR-223-3p) was also measured in the colon, rectum, plasma, and feces using digital polymerase chain reaction (PCR). A high disease activity index (DAI) and reduction in colon length were observed in DSS-treated rats. Erosion and inflammatory cell infiltration were evident in the colon and rectum after DSS treatment. These parameters tended to recover, and regenerative hyperplasia was observed in the rectum after the recovery period. After the end of the administration period, all nine selected miRNA levels showed lower expression in colonic and rectal tissues. miRNA levels, except miR-21-5p and miR-155-5p, were lower in both plasma and feces at 5% DSS group. In contrast, at the end of the recovery period, miRNA levels tended to increase in all samples. Particularly, a higher expression of miR-31a-5p, miR-181b-5p, and miR-223-3p was observed in feces. We suggest that the miRNAs from colon, rectum, plasma, and feces are potential quantitative and sensitive biomarkers in the rat DSS-colitis model. In particular, miR-31a-5p, miR-181b-5p, and miR-223-3p from feces could be used as non-invasive biomarkers to evaluate the reversibility in DSS-treated rats.

Inhalation exposure systems for small experimental animals are necessary evaluation tools of efficacy, pharmacokinetics, and safety when developing inhaled drugs. However, the development of inhalants is characterized by high technical barriers and costs. This project aimed to develop an aerosol generator specialized for a pressurized metered-dose inhaler (pMDI) formulation of ciclesonide (CIC), a prodrug-type corticosteroid for asthma. Our results showed that the developed aerosol generator achieved approximately 160 mg/m³ in mass concentration, by using 60 bottles of the pMDI within a one-hour inhalation exposure study. The CIC used in the study was 672 mg in total. The mass median aerodynamic diameter (MMAD) was approximately 1 µm, with less than 2 in geometric standard deviation. Although the amount of test article used was less than 1 g, the aerosol generator achieved approximately 160 mg/m³ in mass concentration, and enough of the CIC was delivered to the rat lungs to allow the visualization of its spatial localization by desorption electrospray ionization–time-of-flight mass spectrometry imaging. We concluded that (i) the aerosol generator was able to drive pMDI accurately, and (ii) the CIC aerosol was delivered to the rodent under appropriate MMAD and concentration; the device’s performance as an excellent nonclinical inhalation exposure system was thus demonstrated. Furthermore, as the device is highly versatile, it would be possible to utilize it when conducting nonclinical inhalation studies at the optimal conditions for various pMDIs. In the future, aerosol generators could reduce costs and shorten the development period of inhaled drugs.