Development of a genotoxicity/carcinogenicity assessment method by DNA adductome analysis.

Safety evaluation is essential for the development of chemical substances. Since in vivo safety evaluation tests, such as carcinogenesis tests, require long-term observation using large numbers of experimental animals, it is necessary to develop alternative methods that can predict genotoxicity/carcinogenicity in the short term, taking into account the 3Rs (replacement, reduction, and refinement). We established a prediction model of the hepatotoxicity of chemicals using a DNA adductome, which is a comprehensive analysis of DNA adducts that may be used as an indicator of DNA damage in the liver. An adductome was generated with LC-high-resolution accurate mass spectrometer (HRAM) on liver of rats exposed to various chemicals for 24 h, based on two independent experimental protocols. The resulting adductome dataset obtained from each independent experiment (experiments 1 and 2) and integrated dataset were analyzed by linear discriminant analysis (LDA) and found to correctly classify the chemicals into the following four categories: non-genotoxic/non-hepatocarcinogens (-/-), genotoxic/non-hepatocarcinogens (+/-), non-genotoxic/hepatocarcinogens (-/+), and genotoxic/hepatocarcinogens (+/+), based on their genotoxicity/carcinogenicity properties. A prototype model for predicting the genotoxicity/carcinogenicity of the chemicals was established using machine learning methods (using random forest algorithm). When the prototype genotoxicity/carcinogenicity prediction model was used to make predictions for experiments 1 and 2 as well as the integrated dataset, the correct response rates were 89 % (genotoxicity), 94 % (carcinogenicity) and 87 % (genotoxicity/carcinogenicity) for experiment 1, 47 % (genotoxicity), 62 % (carcinogenicity) and 42 % (genotoxicity/carcinogenicity) for experiment 2, and 52 % (genotoxicity), 62 % (carcinogenicity), and 48 % (genotoxicity/carcinogenicity) for the integrated dataset. To improve the accuracy of the toxicity prediction model, the toxicity label was reconstructed as follows; Pattern 1: when +/+ and -/- chemicals were used from the toxicity labels +/+, +/-, -/+ and -/-; and Pattern 2: when +/+, +/-, and -/+ other than -/- were replaced with the label "Others". As a result, chemicals with only +/+ and -/- toxicity labels were used and the correct response rates were approximately 100 % for the measured data in experiment 1, 53 %-66 % for the data in experiment 2, and 59-73 % for the integrated data, all of which were 10 %-30 % higher compared with the data before the label change. In contrast, when the toxicity labels were replaced with -/- and "Others", they reached nearly 100 % in the measured data from experiment 1, 65 %-75 % in the data from experiment 2, and 70 %-78 % in the integrated data, all of which were 10 %-50 % higher compared with the data before the label change.

Stimulation of nuclear receptor REV-ERBs alleviates monosodium iodoacetate-induced osteoarthritis pathology of mice and the induction of inflammatory molecules expression in primary cultured chondrocytes.

Because inflammation in chondrocytes contributes to the induction of osteoarthritis (OA), regulation of their activity is essential. A previous study showed that stimulation of the reverse erythroblastosis virus (REV-ERB) nuclear receptors in spinal glial cells elicits anti-inflammatory and antinociception effects in animal models of chronic pain. However, the involvement of REV-ERBs in chondrocyte functions and OA pathologies remains to be elucidated. In the current study, we found that pretreatment with the REV-ERB agonist SR9009 significantly blocked the increases in inflammatory molecules [(matrix metalloproteinase (MMP) 3, MMP9, and MMP13] and cytokines (interleukin-1ß and tumor necrosis factor) in primary cultured chondrocytes following treatment with lipopolysaccharide. Furthermore, repeated intra-articular treatment with SR9009 significantly prevented monosodium iodoacetate-induced mechanical hypersensitivity and tended to partially reduce knee joint damage in mice. In conclusion, our findings suggest that REV-ERBs have a critical role in alleviating nociceptive hypersensitivity in OA pathologies by negatively regulating inflammation in chondrocytes.

Environmental detection of eumycetoma pathogens using multiplex real-time PCR for soil DNA in Sennar State, Sudan.

BACKGROUND: Mycetoma is a chronic disease affecting the skin and subcutaneous tissue endemic in the tropical and subtropical regions. Several bacteria and fungi can cause mycetoma, but fungal mycetoma (eumycetoma) is challenging because the treatment requires a combination of a long-term antifungal agent and surgery. Although the transmission route has not yet been elucidated, infection from the soil is a leading hypothesis. However, there are few soil investigation studies, and the geographical distribution of mycetoma pathogens is not well documented. Here, we used multiplex real-time PCR technology to identify three fungal species from soil samples. METHODS: In total, 64 DNA samples were extracted from soil collected in seven villages in an endemic area in Sennar State, Sudan, in 2019. Primers and fluorescent probes specifically targeting the ribosomal DNA of Madurella mycetomatis, Falciformispora senegalensis, and F. tompkinsii were designed. RESULTS: Multiplex real-time PCR was performed and identified the major pathogen, M. mycetomatis that existed in most sites (95%). In addition, two other pathogens were identified from some sites. This is the first report on the use of this technique for identifying the eumycetoma causative microorganisms. CONCLUSIONS: This study demonstrated that soil DNA investigation can elucidate the risk area of mycetoma-causative agents. The results will contribute to the design of prevention measures, and further large-scale studies may be effective in understanding the natural habitats of mycetoma pathogens.

Evaluation of intracellular signal molecules that regulate TLR4-stimulated inflammatory mediator expression in cultured rat chondrocytes.

Osteoarthritis (OA) is characterized by inflammation of joints and degradation of articular cartilage matrix. As involvement of damage-associated molecular patterns (DAMPs) in the pathogenesis of OA has been reported, the present study comprehensively investigated the regulation of inflammatory mediator expression in chondrocytes mediated by Toll-like receptor 4 (TLR4), a receptor for DAMPs. Treatment of cultured rat chondrocytes with lipopolysaccharide (LPS) induced the mRNA expression of proinflammatory cytokines (interleukin [IL]-1ß, IL-6, tumor necrosis factor [TNF]), matrix degradation enzymes (metalloproteinase [MMP] 3, MMP13), and inducible nitric oxide synthase (iNOS) through TLR4. Transforming growth factor ß-activated kinase-1 (TAK1) and nuclear factor-κB (NF-κB) were crucial for the upregulated expression of these inflammatory mediators. The induction of IL-1ß and TNF was regulated by extracellular signal-regulated kinase (ERK), while the induction of IL-6 was mediated by Tank-binding kinase 1 (TBK1) and c-Jun N-terminal kinase (JNK). The induction of MMP3 and MMP13 was regulated by TBK1, ERK, and JNK, while the induction of iNOS was mediated by ERK and JNK. In summary, some of the regulatory mechanisms underlying the expression of key inflammatory mediators for OA pathogenesis have been demonstrated. Further clarification may allow these signaling molecules to become new therapeutic targets for OA treatment strategies.

Detection of multiple mycetoma pathogens using fungal metabarcoding analysis of soil DNA in an endemic area of Sudan.

Mycetoma is a tropical disease caused by several fungi and bacteria present in the soil. Fungal mycetoma and eumycetoma are especially challenging to treat; therefore, prevention, early diagnosis, and early treatment are important, but it is also necessary to understand the geographic distribution of these pathogenic fungi. In this study, we used DNA metabarcoding methodology to identify fungal species from soil samples. Soil sampling was implemented at seven villages in an endemic area of Sennar State in Sudan in 2019, and ten sampling sites were selected in each village according to land-use conditions. In total, 70 soil samples were collected from ground surfaces, and DNA in the soil was extracted with a combined method of alkaline DNA extraction and a commercial soil DNA extraction kit. The region for universal primers was selected to be the ribosomal internal transcribed spacer one region for metabarcoding. After the second PCR for DNA library preparation, the amplicon-based DNA analysis was performed using next-generation sequencing with two sets of universal primers. A total of twelve mycetoma-causative fungal species were identified, including the prime agent, Madurella mycetomatis, and additional pathogens, Falciformispora senegalensis and Falciformispora tompkinsii, in 53 soil samples. This study demonstrated that soil DNA metabarcoding can elucidate the presence of multiple mycetoma-causative fungi, which may contribute to accurate diagnosis for patient treatment and geographical mapping.

Stimulation of nuclear receptor REV-ERBs suppresses inflammatory responses in spinal microglia.

As spinal microglia have a critical role in the development of chronic pain, regulation of their activity is essential for pain relief. Previous study has shown that stimulation of the REV-ERB nuclear receptors in the spinal dorsal horn produces antinociception in animal models of both inflammatory and neuropathic pain. However, the involvement of spinal microglia in the antinociceptive action of REV-ERBs remains to be elucidated. In the current study, we found that intrathecal treatment with the REV-ERB agonist SR9009 significantly blocked the increase in ionized calcium-binding adaptor molecule immunoreactivity in the spinal dorsal horn of mice following intrathecal administration of lipopolysaccharide and peripheral sciatic nerve ligation. Furthermore, both Rev-erbα and Rev-erbß mRNAs were expressed in cultured rat spinal microglia. Treatment of cultured rat spinal microglia with SR9009 significantly blocked the lipopolysaccharide-induced increase in interleukin (IL)-1ß and IL-6 mRNA expression. In conclusion, the current findings suggest that REV-ERBs negatively regulate spinal microglial activity and might contribute to the REV-ERB-mediated antinociceptive effect in the spinal dorsal horn.

Identification of an isoform catalyzing the CoA conjugation of nonsteroidal anti-inflammatory drugs and the evaluation of the expression levels of acyl-CoA synthetases in the human liver.

Nonsteroidal anti-inflammatory drugs (NSAIDs) containing carboxylic acid are conjugated with coenzyme A (CoA) or glucuronic acid in the body. It has been suggested that these conjugates are associated with toxicities, such as liver injury and anaphylaxis, through their binding via trans-acylation to cellular proteins. Although studies on glucuronidation have progressed, studies on CoA conjugation of drugs catalyzed by acyl-CoA synthetase (ACS) enzymes are still in the early stages. This study aimed to clarify the human ACS isoforms responsible for CoA-conjugation of NSAIDs through consideration of the hepatic expression levels of ACS isoforms. We found that among 10 types of NSAIDs, propionic acid-class NSAIDs, namely, alminoprofen, flurbiprofen, ibuprofen, ketoprofen, and loxoprofen, were conjugated with CoA in the human liver, whereas NSAIDs in the other classes, including diclofenac and mefenamic acid, were not. qRT-PCR revealed that among the 26 ACS isoforms, ACSL1 was the most highly expressed in the human liver, followed by ACSM2B. The propionic acid-class NSAIDs were conjugated with CoA by recombinant human ACSL1. The protein binding abilities of the CoA conjugates and the glucuronide forms of propionic acid-class NSAIDs were compared as an index of toxicity. The CoA conjugates had stronger adduct formation with liver microsomal proteins than glucuronides for all 5 propionic acid-class NSAIDs. In conclusion, we found that propionic acid-class NSAIDs could be conjugated to CoA by ACSL1 in the human liver to form CoA conjugates, which likely cause toxicity by protein adduct formation.

Application of environmental DNA analysis for the detection of Opisthorchis viverrini DNA in water samples.

Opisthorchiasis, which can lead to cholangiocarcinoma in cases of chronic infection, is a major public health problem in Southeast Asian countries. The trematode, Opisthorchis viverrini, is the causative agent of the disease. Accurate and rapid monitoring of O. viverrini is crucial for disease prevention and containment. Therefore, in this study we sought to develop a novel species-specific real-time PCR assay for detecting O. viverrini using environmental DNA (eDNA). The diagnostic sensitivity of the newly developed real-time PCR assay was similar to that of the traditional PCR assay for 50 fecal samples collected in Lao PDR (21 and 19 samples were positive by real-time PCR and traditional PCR, respectively). The efficacy of eDNA analysis and its applicability in the field were tested using a total of 94 environmental water samples collected from 44 sites in Savannakhet, Lao PDR during May and October 2015 and February 2016. O. viverrini eDNA was detected in five samples by real-time PCR, indicating the presence of the fluke in the area and the risk of infection for individuals consuming fish from these water sources. The application of eDNA analysis would facilitate the identification of O. viverrini endemic hotspots and contribute to the ecological control of opisthorchiasis, and this strategy can be applied to other eukaryotic water pathogens.

Environmental DNA as a 'Snapshot' of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay, Sea of Japan.

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R(2) value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10-150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a 'snapshot' of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.