Annual Meeting of the Japanese Society of Toxicology

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The central nervous system is an important target for air pollution to cause adverse health effects such as neurodevelopmental and neurodegenerative disorders like autism spectrum disorder and Alzheimer’s disease. Both epidemiological and animal studies have indicated that exposure to air pollution may lead to neurotoxicity. Previously, our research group has shown that the effects of primary particles such as carbon black nanoparticles and nanoparticle-rich diesel exhaust on brain inflammatory mediators, neurotransmitter system, memory function-related gene expression and learning performance in adult mice. We have demonstrated that exposure to carbon black nanoparticles via airways caused neuroinflammatory and neurotoxic effects by measuring excitatory amino acid neurotransmitter levels in the hippocampus of adult mice. Furthermore, we have also shown that the effects of nanoparticle-rich diesel exhaust exposure on brain neurotransmitter, inflammatory biomarkers and learning ability in adult mice. Recently, we generated secondary organic aerosol (SOA) by adding ozone to diesel exhaust (DE-SOA) and developed a method for SOA inhalation exposure in our Research Institute. Using the SOA inhalation chambers, we have shown that SOA exposure for three months caused learning and memory impairment in adult male mice and SOA exposure for one month in female mice may cause changes in maternal behavior. More recently, we have established a neonatal animal model for early detection of environmental pollutant-induced learning disability and reported that exposure to DE-SOA may impair olfactory-based spatial learning activity in preweaning mice. In this talk, we will share research information based on our original data regarding assessment of neurotoxicity caused by exposure to air pollutants using various exposure systems, behavioral tests and biomarkers in animal models.

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Drug-induced liver injury (DILI) with idiosyncratic nature has led to the market withdrawal of many drugs in the past. Since animal experiments are not predictive of this type of toxicity, the pharmaceutical industry continues to seek new methodologies for the prevention of such toxicity. Although the mechanism of the idiosyncratic drug toxicity including DILI remains unclear, immune reactions are likely involved. Drugs with low molecular weights, generally thought non-immunogenic, could become haptens after being converted to chemically reactive metabolites and binding covalently to proteins. Therefore, screening tests to detect chemically reactive metabolites, most frequently by trapping with glutathione, are carried out at early stages of drug development. More quantitative methods are needed in later stages of drug development; radioassays for covalent binding (using ¹⁴C- or ³H-labeled compounds) are employed. A zone classification system could assess a risk of drug candidates for idiosyncratic DILI by plotting the clinical dose levels against the amount of the chemically reactive metabolites bound to proteins in vitro. We propose a mechanism for idiosyncratic DILI by analogy to virus-induced hepatitis, where cytotoxic T lymphocytes play an important role. We suggest that idiosyncrasy reflects the involvement of polymorphisms in the human leucocyte antigen-encoding loci. In fact, a strong correlation has been found between idiosyncratic DILI and specific human leucocyte antigen genotypes. Therefore, screening of patients for gene biomarkers is expected to reduce the clinical risk of idiosyncratic DILI, thereby prolonging the life cycle of otherwise useful drugs.

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Acetaminophen (APAP) is a widely used analgesic and anti-pyretic drug; an overdose can cause severe hepatotoxicity and even acute liver failure in humans. The liver injury observed in humans can be reproduced in mice but not in rats. In addition, since APAP is mainly metabolized in hepatocytes, primary hepatocytes from mice or humans and metabolically competent hepatoma cell lines, e.g. HepaRG cells, were used to study the signaling mechanisms of cell death. Critical for the toxicity is the metabolism of APAP by P450 enzymes such as Cyp2E1 to form a reactive metabolite, which depletes glutathione and binds to sulfhydryl groups of proteins, in particular mitochondrial proteins. This causes an impairment of the mitochondrial electron transport with enhanced formation of reactive oxygen and peroxynitrite. The initial oxidant stress is amplified through MAP Kinases ultimately resulting in phosphorylation and mitochondrial translocation of c-Jun N-terminal kinase. The enhanced oxidant stress triggers the mitochondrial membrane permeability transition pore (MPTP) opening. The MPTP-induced matrix swelling with rupture of the outer membrane results in release of intermembrane proteins and nuclear translocation of endonuclease G, which trigger nuclear DNA fragmentation. Together, these events result in necrotic cell death. Adaptive mechanisms such as induction of antioxidant genes and removal of damaged mitochondria by autophagy limit cell death. Assessment of mechanistic biomarkers in serum, e.g. mitochondrial DNA and nuclear DNA fragments support these mechanisms in APAP overdose patients. The extensive necrosis leads to a sterile inflammatory response, which is critical for regeneration and recovery.

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　Unpredictable drug-induced liver injury (DILI) is currently the major cause for discontinuations of new candidates in development or for withdrawals of drugs from the market, but it is difficult to predict DILI risk in humans based on preclinical safety evaluations using conventional experimental animals. On the basis of the accumulating recent knowledge of immune systems in hepatic pathology, immune-mediated DILI is recognized to cause an imbalance of differentiated T cells. Recently, we reported the relationship between expression changes of Th cell differentiation- and inflammation-related factors in our established mouse DILI models of halothane, phenytoin, dicloxacillin, diclofenac, carbamazepine, flutamide, and methimazole.
　Little is known about the role of individual miRNA and its target involved in immune- and inflammation-related response in DILI. Th17-type immune responses had been reported in halothane (HAL)-induced liver injury in mice. Although the peak of ALT level is shown 24 h after the HAL administration, the miRNA gene down regulation that occurred 1 h after HAL administration was primarily related to inflammation- and immune-related DILI. Up-regulation of STAT3 by miR-106b was critical for the pathogenesis of HAL-induced liver injury. The similar miRNA story was demonstrated in Th2-type immune response using a methimazole (MTZ)-induced liver injury mouse model. Negative regulations of the expression of SRY-related HMG-box 4 (SOX4) by miR-29b-1-5p and that of lymphoid enhancer factor-1 (LEF1) by miR-449a-5p were suggested to play an important role for development of Th2 bias in the early phase of MTZ-induced liver injury. Key immune-related regulatory events occur during the early phase of toxicity. MiRNA involved in regulatory mechanisms of toxicological signal cascades and could be predictive biomarkers in the early phase of DILI.

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