Aflatoxin B1 (AFB1) is the most potent naturally occurring carcinogen for humans and animals produced by the common fungus Aspergillus flavus (A. flavus). Aflatoxin (AF) contamination in commodities is a global concern related to the safety of food and feed, and it also impacts the agricultural economy. In this study, we investigated the AFB1-inhibiting activity of a new benzaldehyde derivative, 2-[(2-methylpyridin-3-yl)oxy]benzaldehyde (MPOBA), on A. flavus. It was found that MPOBA inhibited the production of AFB1 by A. flavus, with an IC₅₀ value of 0.55 mM. Moreover, the inhibition of conidiation was also observed at the same concentration. The addition of MPOBA resulted in decreased transcript levels of the aflR gene, which encodes a key regulatory protein for the biosynthesis of AF, and also decreased transcript levels of the global regulator genes veA and laeA. These results suggested that MPOBA has an effect on the regulatory mechanism of the development and differentiation of conidia, leading to the inhibition of AFB1 production. In addition, the cytotoxicity study showed that MPOBA had a very low cytotoxic effect on the Madin-Darby canine kidney (MDCK) cell line. Therefore, MPOBA may be a potential compound for developing practically effective agents to control AF contamination.

CMC is the most frequently diagnosed cancer and one of the leading causes of death in non-spayed female dogs. Exploring novel therapeutic agents is necessary to increase the survival rate of dogs with CMC. MPOBA is a BZOP derivative that has a significant anticancer effect in a human cell line. The main goal of this study was to investigate the anticancer properties of MPOBA against two CMC cell lines (REM134 and CMGT071020) using a 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, a wound healing assay, a transwell migration assay, an Annexin V-FITC apoptosis assay with a flow cytometry analysis, a mRNA expression analysis using quantitative real-time PCR (qRT-PCR), and an immunohistochemistry (IHC). According to the accumulated studies, MPOBA caused significant concentration- and time-dependent reductions in cell proliferation and cell migration and induced apoptosis in both CMC cell lines. In gene expression analysis, nine canine genes, including TP53, BCL-2, BAX, epidermal growth factor receptor (EGFR), snail transcription factor (SNAIL), snail-related zinc-finger transcription factor (SLUG), TWIST, E-cadherin, and N-cadherin, were investigated. The mRNA expression results revealed that MPOBA induced upregulation of TP53 and overexpression of the pro-apoptotic gene BAX, together with an inhibition of BCL-2. Moreover, MPOBA also suppressed the mRNA expression levels of SNAIL, EGFR, and N-cadherin and induced upregulation of E-cadherin, crucial genes related to the epithelial-to-mesenchymal transition (EMT). However, there was no significant difference in the IHC results of the expression patterns of vimentin (VT) and cytokeratin (CK) between MPOBA-treated and control CMC cells. In conclusion, the results of the present study suggested that MPOBA exhibited significant anticancer activity by inducing apoptosis in both CMCs via upregulation of TP53 and BAX and downregulation of BCL-2 relative mRNA expression. MPOBA may prove to be a potential candidate drug to be further investigated as a therapeutic agent for CMC.

The increasing prevalence of cancer in Thailand over the past decade has resulted in a substantial rise in the use of anticancer drugs, which are eventually discharged into municipal wastewater through hospital and domestic effluents. The inability of conventional wastewater treatment systems to completely remove these pharmaceuticals has been widely reported. The continuous release of these emerging anticancer agents into aquatic environments reduces water quality and threatens biodiversity. Even at trace levels, these compounds may act as persistent pollutants capable of impairing ecosystem. This study investigated the occurrence and concentration levels of three widely used chemotherapeutic agents including cyclophosphamide (COP), doxorubicin (DOX), and vincristine (VIN) in Bangkok’s municipal wastewater to evaluate their potential environmental risks. Thirty-two influent and effluent wastewater samples were collected from eight large-scale wastewater treatment plants (WWTPs) from October 2024 to January 2025. Samples were processed using solid-phase extraction (SPE) and analyzed by liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS). The analytical method demonstrated high precision and reproducibility, with relative standard deviations (%RSD) below the 20% acceptance limit for all compounds. Method accuracy ranged from 81.84% to 107.21%. Results showed the presence of only COP in almost influent and effluent at levels ranging from 0.26 to 2.06 µg/L. In contrast, DOX and VIN levels remained consistently below the limits of quantitation (LOQ) in all WWTP samples. This study establishes the first baseline for COP, DOX, and VIN contamination in Bangkok’s municipal wastewater. Notably, the residue of COP in wastewater suggests that current wastewater treatment facilities in Thailand are insufficient for its removal, posing a potential long-term risk to local aquatic ecosystems.

Background and Aim: Canine mammary cancer (CMC) is one of the most common malignant neoplasms in female dogs, with high metastatic potential and limited therapeutic options. Natural bioactive compounds derived from medicinal mushrooms have gained increasing attention because of their anticancer properties and low toxicity. Hericium erinaceus (HE) is a medicinal mushroom known for its antioxidant and antitumor activities; however, its anticancer effects in CMC remain poorly understood. Therefore, this study investigated the in vitro antiproliferative, pro-apoptotic, and epithelial–mesenchymal transition (EMT)-inhibitory effects of HE methanolic extract in two CMC cell lines, CHMp-13a and CHMp-5b. Materials and Methods: The anticancer activity of HE methanolic extract was evaluated using CHMp-13a and CHMp-5b CMC cell lines, while Madin-Darby canine kidney cells were used as normal controls. Cell viability was assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Cell migration and invasion were evaluated using wound healing and Transwell assays, respectively. Apoptosis was analyzed using Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry. Relative mRNA and protein expression levels of apoptosis- and EMT-related markers were determined using quantitative real-time polymerase chain reaction and western blotting. The phytochemical profile of the extract was characterized using liquid chromatography quadrupole time-of-flight mass spectrometry. Results: HE extract significantly inhibited proliferation of both CMC cell lines in a dose- and time-dependent manner, with greater selectivity toward CHMp-13a cells and minimal cytotoxicity in normal cells. Morphological analysis revealed apoptotic features, including cell shrinkage, detachment, and cytoplasmic vacuolization. The extract significantly suppressed migration and invasion capacities of both CMC cell lines. Flow cytometric analysis demonstrated increased apoptotic cell populations following treatment. Molecular analyses showed upregulation of the pro-apoptotic marker BAX and downregulation of the anti-apoptotic marker BCL-2. Furthermore, HE extract suppressed EMT progression by increasing E-cadherin expression while reducing N-cadherin expression. Phytochemical screening identified 17 bioactive compounds, including erinacines, hericenones, hericene derivatives, and phenolic compounds, which may contribute to the observed anticancer activities. Conclusion: HE extract demonstrated potent in vitro anticancer activity against CMC cells through synergistic induction of apoptosis and suppression of EMT-associated metastatic behavior. These findings suggest that HE extract may serve as a promising natural adjuvant candidate for the management of CMC and warrants further in vivo and mechanistic investigations. Keywords: apoptosis, canine mammary cancer, epithelial–mesenchymal transition, Hericium erinaceus, medicinal mushroom, metastasis inhibition, phytochemicals, selective cytotoxicity.

Currently, emerging pharmaceutical contaminants (EPCs) pose a significant risk to aquatic biodiversity on both a global and regional scale. Their accumulation in the environment presents several challenges to both human health and ecological integrity. Among EPCs, cyclophosphamide (COP), a widely used alkylating cytotoxic and immunosuppressive drug in human and veterinary oncology, has become one of the most frequently detected environmental contaminants. It has been reported to impair the immune system, induce oxidative stress, and exhibit genotoxic and cytotoxic effects in various fish species. However, limited research has evaluated its toxicity in the Siamese fighting fish (Betta splendens), an endemic significant aquatic species in Thailand. This study evaluated the acute toxicity of COP in Betta splendens by assessing systemic oxidative stress responses, alterations in gene expression, and localized histopathological changes within both hepatic and renal tissues. The 96 h of median lethal concentration (LC₅₀) value for COP in Betta splendens was determined to be 793.4 mg/L. High-concentration exposure (800 mg/L) induced severe systemic oxidative stress, evidenced by a significant reduction in superoxide dismutase (SOD) activity and a marked elevation in malondialdehyde (MDA) levels across both liver and kidney. At the transcriptomic level, acute COP exposure significantly upregulated pro-inflammatory (interleukin-1β, IL-1β and tumor necrosis factor, TNF-α), cellular stress (heat shock protein 70, HSP70), and apoptotic (caspase-3, Casp3) genes. Semi-quantitative histopathological evaluation revealed severe concentration-dependent structural damage. Hepatic lesions peaked at 800 mg/L characterized by diffuse vacuolation, vascular congestion, and extensive necrosis. Similarly, severe renal damage occurred at 800 mg/L, featuring marked vascular congestion, melanomacrophage aggregation, and widespread tubular necrosis. Overall, these findings demonstrate that acute waterborne COP exposure induces severe hepatotoxicity and nephrotoxicity in Betta splendens driven by oxidative stress, pro-inflammatory signaling, and apoptotic pathways. This study provides essential baseline toxicity thresholds and highlights the physiological risks COP spills pose to tropical freshwater labyrinth fish.

Background and Aim: Canine mammary cancer (CMC) is the most frequently diagnosed malignancy in female dogs, sharing significant pathological and molecular similarities with human breast cancer (HBC). Despite the availability of various CMC cell lines, most represent triple-negative or epidermal growth factor receptor 2 (ErbB2)-enriched subtypes, which limit research on hormone receptor-positive cancers. This study aimed to establish and characterize novel CMC cell lines representing luminal A and B subtypes. Materials and Methods: Between 2020 and 2021, 31 canine mammary tumors (CMTs) were collected from clinical cases. Tumor tissues were processed for primary culture, and two cell lines – CMGT_071020 and CMGT_180321 – were successfully established. Immunohistochemistry (IHC) was used to assess expression of estrogen receptor alpha (ERα), progesterone receptor (PR), ErbB2, Ki-67, vimentin, and multi-cytokeratin. Functional assays (wound-healing and transwell migration) assessed metastatic behavior. Gene expression (EGFR, TP53, Bcl-2, PTEN, SNAIL, N-cadherin, and E-cadherin) was analyzed using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Cell line authentication was confirmed through short tandem repeat (STR) profiling and mycoplasma testing. Results: The CMGT_071020 (luminal B) and CMGT_180321 (luminal A) cell lines were derived from malignant epithelial tumors and maintained stable growth over 30 passages. IHC confirmed molecular subtype classifications. CMGT_071020 exhibited a fibroblast-like morphology, a high Ki-67 index (67%), and superior migratory capacity compared to CMGT_180321 and the commercial ErbB2-enriched REM134 cell line. E-cadherin expression was significantly elevated in CMGT_071020 (p < 0.05), whereas the expression levels of other genes were comparable. STR analysis verified their genetic uniqueness, and both lines were free from mycoplasma contamination. Conclusion: This study successfully established and characterized two novel hormone receptor-positive CMC cell lines, representing luminal A and luminal B subtypes. The CMGT_071020 line exhibited higher metastatic potential, offering a promising model for aggressive hormone-responsive CMC. These cell lines provide valuable tools for comparative oncology and may facilitate subtype-specific therapeutic research. Keywords: canine mammary cancer, cell line establishment, comparative oncology, immunohistochemistry, luminal A, luminal B, metastasis, molecular subtype, reverse transcription quantitative real-time polymerase chain reaction.