When a comet assay, an increasingly popular in vivo genotoxicity test, shows a positive test result, interpretation of that response requires ruling out any confounding tissue site toxicity. Since the comet assay typically uses only two or three daily doses of test agent, precursor tissue changes indicative of toxicity may be easily overlooked. Using case examples for two flavoring agents, perillaldehyde and 4,5-epoxydec-2(trans)-enal, we highlight the role of pathology peer review in verifying precursor tissue changes indicative of tissue site toxicity, thereby increasing confidence in final interpretation of comet assay results. Given global deliberation regarding safety assessment of compounds entering the marketplace, we recommend consideration of pathology peer review for equivocal and positive comet assays so that interpretations are universally consistent.

It is known that γH2AX, which is formed when there is a double-strand break in DNA, can act as a sensitive marker of genomic instability. In this experiment, the time-course manner of the expression of γH2AX in the lung was examined in the early phase after treatment with a lung carcinogen, N-bis (2-hydroxypropyl) nitrosamine (DHPN). The expression of γH2AX is expected to be one of the useful markers for lung carcinogenesis in early stages. Rats were separated into 10 groups of 5 rats. The DHPN groups were administered 0.1% DHPN in drinking tap water for two weeks, while the control group received drinking tap water. At 0, 1, 3, 7, and 14 days after finishing DHPN treatment, one group each from the DHPN and control groups was sacrificed. The removed lung tissues were examined for immunostaining of γH2AX and PCNA, and positive cells were counted. The γH2AX levels of the DHPN-treated groups were found to be increased significantly at 0, 1, 3, and 7 days (4.4 ± 1.4, 5.1 ± 2.7, 3.3 ± 1.0, and 4.1 ± 1.3%, respectively), and they dropped significantly on day 14 (1.1 ± 0.4%). The experiment showed that the γH2AX-positive score could be effectively measured for up to 7 days after exposure, as a significance difference was observed between the treated group and the control group. It can be deduced that γH2AX is an effective marker for DHPN-induced double-strand breaks in pulmonary epithelial cells.

Despite its antimicrobial activity, nitrofurantoin (NFT) is a renal carcinogen in rats. Oxidative stress induced by reduction of the nitro group of NFT may contribute to its genotoxicity. This is supported by our recent results indicating that the structure of the nitrofuran plays a key role in NFT-induced genotoxicity, and oxidative DNA damage is involved in renal carcinogenesis. Nuclear factor erythroid 2-related factor 2 (NRF2) regulates cellular responses to oxidative stress. To clarify the role of oxidative stress in the chemical structure-related genotoxic mechanism of NFT, we performed reporter gene mutation assays for NFT and 5-nitro-2-furaldehyde (NFA) using Nrf2-proficient and Nrf2-deficient gpt delta mice. NFT administration for 13 weeks resulted in a significant increase in 8-hydroxydeoxyguanosine (8-OHdG; a marker of oxidative stress) and gpt mutant frequency only in the kidneys of Nrf2^−/− mice. The mutation spectrum, characterized by increased substitutions at guanine bases, suggested that oxidative stress is involved in NFT-induced genotoxicity. However, NFA did not increase the mutation frequency in the kidneys, despite the increased 8-OHdG in NFA-treated Nrf2^−/− mice. Thus, it is unlikely that oxidative stress is involved in the genotoxic mechanism of NFA. These results imply that nitro reduction plays a key role in the genotoxicity of NFT, but the lack of a role of oxidative stress in the genotoxicity of NFA indicates a potential role of side chain interactions in oxidative stress caused by nitro reduction. These findings provide a basis for the development of safe nitrofurans.

Oxidative stress is well known as a key factor of chemical carcinogenesis. However, the actual role of oxidative stress in carcinogenesis, such as oxidative stress-related in vivo mutagenicity, remains unclear. It has been reported that 8-hydroxydeoxyguanosine (8-OHdG), an oxidized DNA lesion, might contribute to chemical carcinogenesis. Potassium bromate (KBrO₃) and nitrofurantoin (NFT) are known as renal carcinogens in rats. Our previous studies showed an increase in mutant frequencies accompanied by an increased level of 8-OHdG in the kidneys of rodents following KBrO₃ or NFT exposure. Furthermore, KBrO₃ and NFT induced different types of gene mutations. Thus, in the present study, we performed reporter gene mutation assays and 8-OHdG measurements following KBrO₃ or NFT exposure using Nrf2-proficient and Nrf2-deficient mice to clarify the relationship between KBrO₃- or NFT-induced oxidative stress and subsequent genotoxicity. Administration of 1,500 ppm of KBrO₃ in drinking water resulted in an increase in deletion mutations accompanied by an increase in 8-OHdG level, and administration of 2,500 ppm of NFT in diet induced an increase in guanine base substitution mutations without elevation of the 8-OHdG level in Nrf2-deficient mice. These results demonstrated that the formation of 8-OHdG, which resulted from the oxidizing potential of KBrO₃, was directly involved in the increase in deletion mutations, although factors related to oxidative stress other than 8-OHdG might be crucial for NFT-induced guanine base substitution mutations. The present study provides new insight into oxidative stress-related in vivo mutagenicity.

A 3-year-old female Richardson’s ground squirrel developed a subcutaneous mass at the left oral angle. Seven days after removal of the mass, the mass recurred and metastasized to the cervical lymph node. Histologically, the primary mass was subdivided by fibrous trabeculae into various-sized neoplastic cell lobules showing a solid growth pattern with frequent mitoses and sometimes forming intracytoplasmic lumina. Large to medium-sized lobules formed a central cyst plugged by comedo necrosis. Neoplastic cells showed infiltrative subcutaneous growth. In the recurrent tumor, tubular structures lacking apparent apocrine secretion appeared within the solid growth portion. Neutrophil infiltration was evident within the tubules and intracytoplasmic lumina. Neoplastic cells were diffusely immunopositive for AE1/AE3 pan-cytokeratin (CK) in all lobules and focally positive for CAM5.2 CK in the lobules forming a central cyst and/or tubular structures, but they entirely lacked positivity for the periodic acid Schiff reaction. Ki-67-positive proliferating neoplastic cells were higher in numbers with the recurrent tumor than with the primary tumor. In addition, phosphorylated c-MYC immunoreactivity was observed in neoplastic cell nuclei, distinctly at the portion of invasive growth. Thus, the present case was diagnosed as apocrine ductal carcinoma originating from the oral scent gland, which typically shows highly aggressive biological behavior.

Murine hepatoblastoma (HB) is a rare spontaneous tumor with controversial histogenesis. It mainly occurs in aged males, frequently in close association with preexisting hepatocellular neoplasms. The present work describes a spontaneous HB arising within a hepatocellular carcinoma (HCC) in a 22-month-old male C57BL/6J mouse. The mouse also developed pulmonary co-metastases with either tumor components physically associated within the same metastatic foci. Microscopically, the HB consisted of a densely cellular neoplastic growth composed of palisades and perivascular pseudorosettes of poorly differentiated primitive cells, with a scant amount of cytoplasm, elongated hyperchromatic nuclei, and a high mitotic rate, whereas the hepatocellular carcinoma was composed of solid areas of neoplastic hepatocytes. Both in primary tumors and their metastases, β-catenin immunohistochemistry revealed a strong nucleocytoplasmic signal in HB cells, while neoplastic hepatocytes displayed a delicate membranous staining pattern. These findings suggest that the Wnt/β-catenin oncogenic pathway is upregulated in murine HB but not in the co-existing HCC, thus providing some insights into their divergent pathogenesis. Coexisting murine HB and HCC have been demonstrated to be completely distinct entities including origin, mutational landscape, and molecular profile. In this context, they might be regarded as collision tumors because of their intimate association, unique histologic features, and distinct immunohistochemical patterns. Nevertheless, the nature of their coevolution and progression to a co-metastatic phenotype reflects a close interdependence and support the overall idea that HB’s origin and progression might be promoted by not otherwise specified paracrine stimuli provided by the concurrent hepatocellular tumor (the so called “interaction theory”).

To prevent fixation defects or artifacts in the whole bodies of fish caused by conventional fixatives, such as formalin solution, Bouin’s fluid (BF), and Davidson’s fluid (DF), the optimal fixatives and fixing method were examined. An improved method of fixing the whole bodies of fish was examined that makes use of a combination of 20% formalin and BF or DF. The fixatives were examined with four representative tissues, i.e., the gill, liver, intestinal tract, and kidney, to evaluate end points including the appearance of degraded tissues and artifacts caused by each fixative, overall morphological clarity of nuclei, staining intensity, and integrity of the other tissues. The best results were obtained when the fresh whole bodies were initially fixed in 20% formalin (primary fixation) at 4°C for 1 h and subsequently fixed in BF for 5 h at 4°C (secondary fixation). Therefore, the current findings led the authors to conclude that the combination of primary fixation with 20% formalin at 4°C for 1 h and secondary fixation with BF at 4°C for 5 h was suitable for fixation of the whole bodies of fish.

Macrophages are required during kidney development and appear in the initiation and propagation of renal injury. To establish baseline data, we analyzed the kinetics of the macrophage with different immunophenotypes in the developing rat kidney (fetus at 18 and 20 days, neonate at 1-21 days, and adult at 7-weeks old). Macrophages reacting to CD68, CD163, and MHC class II were identified in the cortex and medulla of the developing rat kidney. CD68⁺ macrophages appeared in the fetal kidney as early as fetal day 18, and the number increased gradually in the neonatal kidney, whereas MHC class II⁺ and CD163⁺ macrophages first appeared on neonatal days 4 and 8, respectively. Apoptotic bodies were seen in the fetal kidney and early stages of the neonatal kidney (days 1–4), and simultaneously CD68⁺ macrophages appeared, indicating that CD68⁺ macrophages may have roles in phagocytosis of apoptotic bodies and contribute to renal tissue maturation. Colony stimulating factor 1 and insulin growth factor 1 mRNAs were increased in the late stage of renal development (neonatal day 12 or later), and simultaneously CD163⁺ and MHC class II⁺ cells appeared, suggesting that these cells may be a source of these growth factors and participate in renal tissue modeling. Generally, the CD163⁺ and MHC class II⁺ cell number was much smaller than that of CD68⁺ cells in the developing neonatal kidney. Therefore, the obtained findings provide valuable information on the participation of macrophages in the developing rat kidney. This information may be useful for evaluation of renal toxicity when macrophages are involved in the development of renal injury.

Assessing how gene expression analysis by RNA sequencing (RNA-Seq) correlates to a unique morphology is increasingly necessary, and laser capture microdissection (LCM) is a critical research tool for discovering the genes responsible in a region of interest (ROI). Because RNA-Seq requires high-quality RNA, a sample preparation procedure that can preserve morphology and give the required quality of RNA is essential. A PAXgene®-fixed paraffin-embedded (XFPE) block can satisfy the need for high-quality RNA, but there are few reports on adapting the method for LCM, such as how small an ROI is analyzable by RNA-Seq. In this study, we confirmed the morphology and preservation of RNA in XFPE and then assessed the relationship between the size of pieces cut by LCM and their RNA quality. In XFPE, the morphology was similar to that in alcohol-based fixed samples, the quality of the RNA extracted from a whole sample was excellent, that is equivalent to that of a fresh frozen sample, and the quality was maintained over one year later. Three sizes of pieces—large (25,000 µm²), medium (5,000 µm²), and small (1,000 µm²)—were cut by LCM so that the total areas of the sections cut per size were the same. RNA quality was found to be best preserved when tissue was cut into pieces of over 5,000 µm². In summary, XFPE exhibits good morphology and excellent preservation of RNA quality. Furthermore, it can be a good tool when used with LCM and RNA-Seq, giving well-balanced RNA quality and tissue morphology in the ROI.

To evaluate the usefulness of imaging mass spectrometry (IMS) technology for assessing drug toxicity, we analyzed animal tissues in an amiodarone (AMD)-induced phospholipidosis model by IMS and confirmed the relationship between the distribution of AMD, its metabolites, and representative phospholipids (phosphatidylcholine, PC) and histological changes. AMD was administered to rats for 7 days at 150 mg/kg/day. The lung, spleen, and mesenteric lymph node were histologically examined and analyzed using IMS. The detection intensities of AMD, its metabolites, and typical PCs were higher in regions infiltrated by foamy macrophages compared with normal areas. This tendency was common in all three organs analyzed in this study. For the spleen, signals for AMD, its metabolites, and typical PCs were significantly more intense in the marginal zone, where foamy macrophages and vacuolated lymphocytes are abundant, than in the other areas. These results indicate that AMD, its metabolites, and PCs accumulate together in foamy or vacuolated cells, which is consistent with the mechanism of AMD-induced phospholipidosis. They also indicate that IMS is a useful technique for evaluating the distribution of drugs and biological components in the elucidation of toxicity mechanisms.