Application of cytochrome P450 reactivity on the characterization of chemical compounds and its association with repeated-dose toxicity.

Repeated-dose toxicity (RDT) studies are one of the critical studies to assess chemical safety. There have been some studies attempting to predict RDT endpoints based on chemical substructures, but it remains very difficult to establish such a method, and a more detailed characterization of chemical compounds seems necessary. Cytochrome P450s (P450s) comprise multiple forms with different substrate specificities and play important roles in both the detoxification and metabolic activation of xenobiotics. In this study, we investigated possible use of P450 reactivity of chemical compounds to classify the compounds. A total of 148 compounds with available rat RDT test data were used as test compounds and subjected to inhibition assays against 18 human and rat P450s. Among the tested compounds, 82 compounds inhibited at least one P450 form. Hierarchical clustering analyses using the P450 inhibitory profiles divided the 82 compounds into nine groups, some of which showed characteristic chemical and biological properties. Principal component analyses of the P450 inhibition data in combination with the calculated chemical descriptors demonstrated that P450 inhibition data were plotted differently than most chemical descriptors in the loading plots. Finally, association analyses between P450 inhibition and RDT endpoints showed that some endpoints related to the liver, kidney and hematology were significantly associated with the inhibition of some P450s. Our present results suggest that the P450 reactivity profiles can be used as novel descriptors for characterizing chemical compounds for the investigation of the toxicity mechanism and/or the establishment of a toxicity prediction model.

Construction of the Database of Rat Repeated-dose Toxicity Tests of Pesticides for the Toxicological Characterization of Hepatocyte Hypertrophy.

Liver and hepatocyte hypertrophy can be induced by exposure to chemical compounds, but the mechanisms and toxicological characteristics of these phenomena have not yet been investigated extensively. In particular, it remains unclear whether the hepatocyte hypertrophy induced by chemical compounds should be judged as an adaptive response or an adverse effect. Thus, understanding of the toxicological characteristics of hepatocyte hypertrophy is of great importance to the safety evaluation of pesticides and other chemical compounds. To this end, we have constructed a database of potentially toxic pesticides. Using risk assessment reports of pesticides that are publicly available from the Food Safety Commission of Japan, we extracted all observations/findings that were based on 90-day subacute toxicity tests and 2-year chronic toxicity and carcinogenicity tests in rats. Analysis of the database revealed that hepatocyte hypertrophy was observed for 37-47% of the pesticides investigated (varying depending on sex and testing period), and that centrilobular hepatocyte hypertrophy was the most frequent among the various types of hepatocyte hypertrophy in both the 90-day and 2-year studies. The database constructed in this study enables us to investigate the relationships between hepatocyte hypertrophy and other toxicological observations/findings, and thus will be useful for characterizing hepatocyte hypertrophy.

Effects of heat stress on filamentous actin and prestin of outer hair cells in mice.

When the ear is exposed to traumatic loud noise, outer hair cells (OHCs) are damaged and thus permanent hearing loss occurs. Recently, prior conditioning with heat stress has been reported to protect OHCs from traumatic noise exposure by increasing the stiffness of the OHC soma and has also been reported to enhance distortion product otoacoustic emissions [DPOAEs; Murakoshi, M., Yoshida, N., Kitsunai, Y., Iida, K., Kumano, S., Suzuki, T., Kobayashi, T., Wada, H., 2006. Effects of heat stress on Young's modulus of outer hair cells in mice. Brain Res. 1107, 121-130]. In the present study, to further investigate the heat stress-induced protective mechanism of hearing and such stress-induced DPOAE enhancement mechanism, the amount of filamentous actin (F-actin), which is concerned with cell stiffness, and the amount of prestin, which is concerned with the generation of DPOAEs, were examined in OHCs, with and without heat stress. Heat stress was found to increase the amount of F-actin 6-24 h after heat stress. The greatest increase in the amount of F-actin was observed at the cuticular plate where F-actin anchors the roots of the stereocilia to the cell body. Based on this result, the part of the stereocilia most reinforced and protected by heat stress was concluded to be the roots of the stereocilia. In contrast with F-actin, heat stress did not affect the amount of prestin.

Effects of heat stress on Young's modulus of outer hair cells in mice.

Intense sound exposure causes permanent hearing loss due to hair cell and cochlear damage. Prior conditioning with sublethal stressors, such as nontraumatic sound, heat stress and restraint protects the ear from acoustic injury. However, the mechanisms underlying conditioning-related cochlear protection remain unknown. In this paper, Young's modulus and the amount of filamentous actin (F-actin) of outer hair cells (OHCs) with/without heat stress were investigated by atomic force microscopy and confocal laser scanning microscopy, respectively. Conditioning with heat stress resulted in a statistically significant increase in Young's modulus of OHCs at 3-6 h after application, and such modulus then began to decrease by 12 h and returned to pre-conditioning level at 48 h after heat stress. The amount of F-actin began to increase by 3 h after heat stress and peaked at 12 h. It then began to decrease by 24 h and returned to the pre-conditioning level by 48-96 h after heat stress. These time courses are consistent with a previous report in which heat stress was shown to suppress permanent threshold shift (PTS). In addition, distortion product otoacoustic emissions (DPOAEs) were confirmed to be enhanced by heat stress. These results suggest that conditioning with heat stress structurally modifies OHCs so that they become stiffer due to an increase in the amount of F-actin. As a consequence, OHCs possibly experience less strain when they are exposed to loud noise, resulting in protection of mammalian hearing from traumatic noise exposure.