Shoji Asakura

Toxicity testing in the 21st century beyond environmental chemicals

Summary After the publication of the report titled Toxicity Testing in the 21st Century – A Vision and a Strategy, many initiatives started to foster a major paradigm shift for toxicity testing – from apical endpoints in animal-based tests to mechanistic endpoints through delineation of pathways of toxicity (PoT) in human cell based systems. The US EPA has funded an important project to develop new high throughput technologies based on human cell based in vitro technologies. These methods are currently being incorporated into the chemical risk assessment process. In the pharmaceutical industry, the efficacy and toxicity of new drugs are evaluated during preclinical investigations that include drug metabolism, pharmacokinetics, pharmacodynamics and safety toxicology studies. The results of these studies are analyzed and extrapolated to predict efficacy and potential adverse effects in humans. However, due to the high failure rate of drugs during the clinical phases, a new approach for a more predictive assessment of drugs both in terms of efficacy and adverse effects is getting urgent. The food industry faces the challenge of assessing novel foods and food ingredients for the general population, while using animal safety testing for extrapolation purposes is often of limited relevance. The question is whether the latest paradigm shift proposed by the Tox21c report for chemicals may provide a useful tool to improve the risk assessment approach also for drugs and food ingredients.

Assessment of potential mutagenic activities of a novel benzothiazole MAO-A inhibitor E2011 using Salmonella typhimurium YG1029.

The potential initiation activities of a novel monoamine oxidase type-A (MAO-A) inhibitor E2011, which induced preneoplastic foci in the rat liver, were investigated by comparing the mutagenic activity of E2011, 6-aminobenzothiazole (6-ABT, a structural scaffold of E2011) and its derivatives, which are suggested primary reactive metabolites for E2011-induced hepatotoxicity in the rats in vivo, in the Ames assay system employing two Salmonella tester strains, TA100 and YG1029, a bacterial O-acetyltransferase-overproducing strain of TA100. E2011, a tertiary amine, showed no mutagenic activity both in the Salmonella typhimurium TA100 and YG1029 with and without S9 mix. On the other hand, a secondary aromatic amine ER-174238-00, a typical decarbonated metabolite of E2011, showed weak but significant mutagenicity in YG1029 in the presence of S9 mix, and a primary aromatic amine ER-174237-00, an N-dealkylated derivative of ER-174238-00, exhibited S9-dependent potent mutagenicity in YG1029. Thus, it appears that primary and secondary amino moieties of benzothiazole derivatives at C(6)-position are the specific structures contributing to their mutagenic activity. In addition, the alkyl group at C(2)-position of E2011, ER-174237-00 and ER-174238-00 is suggested to intensify the mutagenic activity, since the mutagenicity of ER-174237-00 is approximately two-fold higher than that of 6-ABT, which has hydrogen at C(2)-position in the place of the alkyl group. These results strongly suggest that E2011 has potential initiation activities in the rat liver in vivo after undergoing decarbonation, one of the metabolic pathways, at the carbonyl moiety of oxazolidinone ring to form mutagenic amine(s).

Combination of circulating microRNAs as indicators of specific targets of retinal toxicity in rats

Circulating miR-96-5p, -124-3p, and 183-5p have been reported as safety biomarkers for retinal toxicity. In the present research, 5 serum microRNAs (miRNAs), which are highly specific to and abundant in the retina, including the 3 miRNAs previously mentioned, were assessed in 3 different models of retinal toxicity. Distinct types of retinal lesions were induced in rats by a single dose of N-methyl-N-nitrosourea (MNU: 10, 30, and 50 mg/kg, i.p.), N-methyl-d-aspartate (NMDA: 200 nmol/eye, intravitreal injection), or sodium iodate (NaIO3: 30 mg/kg, i.v.). Time-course change of serum miRNAs was evaluated by RT-PCR for up to 1 week after administration. Ophthalmologic and histologic examinations and electroretinogram recording were also performed. MNU at 50 mg/kg induced photoreceptor cell death, with elevation in serum miR-96-5p, -124-3p, and -183-5p levels. NMDA induced retinal ganglion and inner nuclear layer cell death, with elevation in serum miR-124-3p. In both models, serum miRNA elevations occurred in parallel with the onset of neuroretinal cell death and retinal dysfunction. NaIO3 induced retinal pigment epithelial cell death without changes in neuroretinal cell or serum miRNAs. In the present research, circulating miR-124-3p was elevated in a case of retinal ganglion and inner nuclear layer cell death as well as photoreceptor cell death. Our data suggest that different patterns of circulating miRNA elevations correspond to death of a specific neuroretinal cell. A miRNA panel consisting of miR-96-5p, -124-3p, and -183-5p may be used as a biomarker to detect neuroretinal cell death and identify the specific target cell.