Caffeine (CFF) is efficiently absorbed after ingestion, and approximately 80% of ingested CFF is metabolized to paraxanthine (PXT). Although PXT has approximately twice the adenosine receptor antagonist activity of CFF, there are few reports measuring the metabolite concentrations during CFF intoxication. Furthermore, no studies have examined the efficacy of hemodialysis (HD) on PXT or the indicators that contribute to treatment strategies for patients with acute CFF intoxication. This study analyzed the association between CFF and PXT blood levels, the blood biochemical data, and the vital signs of 27 cases with information on CFF intake and elapsed time data. It was found that HD was not as effective as CFF against PXT in CFF intoxication; however, HD was effective in cases with relatively high PXT concentrations (>10 μg/mL). Simultaneous analysis of CFF and PXT would make it possible to estimate the time elapsed from CFF intake and the risk of hyperCKemia, which may develop in cases left untreated for a prolonged period after ingestion. Therefore, the measurement of PXT, in addition to CFF, is expected to provide useful information for understanding the pathogenesis of CFF intoxication and the development of treatment strategies of acute CFF intoxication.

Neuroinflammation plays a critical role in various neurological disorders. Oxycodone has anti-inflammatory properties. The purpose of this work was to look into the effect of oxycodone in controlling lipopolysaccharide (LPS)-induced neuroinflammation in microglia.

Resistance to chemotherapeutic medicines complicates and eventually kills people with ovarian cancer. Nafamostat mesylate (NM) has been used as an adjuvant therapy to enhance chemotherapy sensitivity in several cancers. This study aimed to evaluate the effect of NM on ovarian cancer cells susceptible to carboplatin (CBP) and to determine the underlying mechanism involved. Herein, qRT-PCR, western blot, and IHC were used to analyze mRNA and protein expression. Cell viability and proliferation were measured using the MTT and colony formation assays. Cell migration and invasion were examined using the Transwell assay. Flow cytometry was employed to detect cell apoptosis. The interaction between zinc finger protein 24 (ZNF24) and wingless-type MMTV integration site family member 2b (WNT2B) was validated via the dual-luciferase reporter and Chromatin immunoprecipitation assays. A xenograft nude mouse model was used to assess the effect of NM on CBP sensitivity in vivo. Our results showed that NM intervention inhibited the viability, proliferation, migration, and invasion and facilitated the apoptosis of CBP-resistant ovarian cancer cells. Furthermore, NM sensitized ovarian cancer cells to CBP by upregulating ZNF24. ZNF24 inactivated Wnt/β-catenin signaling by inhibiting the transcription of WNT2B. Additionally, NM enhanced the inhibitory effect of CBP on tumor growth in vivo. Taken together, NM enhanced the CBP sensitivity of ovarian cancer cells by promoting the ZNF24-mediated inactivation of the WNT2B/Wnt/β-catenin axis. These findings suggest a viable treatment approach for improving CBP resistance in ovarian cancer.

The octanol/water partition coefficient, P (logP), is a hydrophobicity index and is one of the determining factors of the pharmacokinetics of chemical compounds. LogP values obtained from in silico software, open chemistry databases, and in vitro liquid chromatography retention factors may vary. Some chemicals (boscalid, etoxazole, and permethrin) have up to four-order-magnitude differences in in silico/in vitro P values. This study aimed to evaluate the effects of logP values of these three compounds, along with bisphenol A, 1,2-dibromobenzene, tetrabromobisphenol A, trazodone, and triazolam, on the input parameters and output plasma/hepatic concentration-time profiles of simple physiologically based pharmacokinetic (PBPK) models. Although the blood-to-plasma concentration ratios (~0.9-0.6) were slightly affected by variations in logP values, logarithmic plasma unbound fraction values and liver-to-plasma partition coefficients (K) were, respectively, inversely and linearly correlated with logP values (K was stable at ~6.7 for logP > 4). LogP was among the input parameters for previously established machine learning systems; consequently, the resulting logarithmic intrinsic clearance values were correlated with logP values in the range 2-8. However, the bioavailability, absorption rate constants, and volumes of distribution were not affected. PBPK-modeled plasma and hepatic maximum concentrations and areas under the concentration-time curves after virtual oral doses were mostly within ~0.5- to ~2-fold ranges, except for substances with low in vitro logP values, e.g., etoxazole and permethrin. These results suggest that in silico logP values are generally suitable for pharmacokinetic modeling; nevertheless, caution is needed for compounds with low in vitro logP values of ~2.

To investigate the carcinogenicity of anatase-type nano-titanium dioxide (aNTiO), F344/DuCrlCrlj rats were exposed to aNTiO aerosol at concentrations of 0, 0.5, 2, and 8 mg/m. The rats were divided into 2 groups: carcinogenicity study groups were exposed for two years, and satellite study groups were exposed for one year followed by recovery for 1 day, 26 weeks, and 52 weeks after the end of exposure. In the carcinogenicity groups, bronchiolo-alveolar carcinomas were observed in two 8 mg/m-exposed males, showing an increasing trend by Peto's test. However, this incidence was at the upper limit of JBRC's historical control data. Bronchiolo-alveolar adenomas were observed in 1, 2, 3, and 4 rats of the 0, 0.5, 2, and 8 mg/m-exposed females and were not statistically significant. However, the incidence in the 8 mg/m-exposed females exceeded JBRC's historical control data. Therefore, we conclude there is equivocal evidence for the carcinogenicity of aNTiO in rats. No lung tumors were observed in the satellite groups. Particle-induced non-neoplastic lesions (alveolar epithelial hyperplasia and focal fibrosis) were observed in exposed males and females in both the carcinogenicity and satellite groups. Increased lung weight and neutrophils of bronchoalveolar lavage fluid were observed in the 8 mg/m-exposed carcinogenicity groups. The aNTiO deposited in the lungs of the satellite group rats was decreased at 26 weeks after the end of exposure compared to 1 day after the end of exposure. At 52 weeks after the end of exposure, the decreased level was the same at 26 weeks after the end of exposure.

The indispensability of biometals nickel, copper, and selenium in pharmaceutical, agricultural, and other industrial applications, coupled with their release from mining processes, has made them potent environmental contaminants, especially when present in aquatic ecosystems at levels above the essential range. The toxicity of these biometals in fish embryogenesis, including their toxicity levels, was studied using medaka embryos. Test solutions (0.001-10 ppm) of the biometals, along with an isotonic solution as a control, were introduced into the embryos using a nanosecond pulsed electric field application. The exposed embryos were cultured at 25 ± 1°C and microscopically observed daily for 14 days in an isotonic solution. Developmental abnormalities and toxicity were observed during the 14-day observation period. All biometals caused some abnormalities in developing embryos at all concentrations. Major abnormalities included delayed development; deformities such as curvature of bones or spines; abnormal formation of the hearts, eyes, and circulatory systems; and mortality. The toxicity of the biometals was significantly different (p < 0.05) from that of the control. Gene expression analysis revealed that 4747, 1961, and 1952 genes were affected by copper, nickel, and selenium, respectively. Copper affected the highest number of genes and caused the highest toxicity. These results indicate that nickel, copper, and selenium can cause toxicity in developing fish embryos at concentrations ranging from 0.01 ppb to 10 ppm. Therefore, there is a need to constantly monitor the levels of these biometals, particularly in aquatic ecosystems, to preserve aquatic life.

Numerous studies have confirmed that the apoptosis induced by the methacrylate resin monomers triethyleneglycol-dimethacrylate (TEGDMA), 2-hydroxy ethyl methacrylate (HEMA), etc., in pulp cells and odontoblast-like cells is caused mainly by oxidative stress (OS). Reactive oxygen species (ROS), recognized as the most important risk factor for apoptosis in cells of the pulp-dentin complex, are produced mainly via the mitochondrial respiratory chain. When the free resin monomers in the oral cavity and pulp reach a toxic level, the monomers induce oxidative DNA damage, activate ATM-p53 in the nucleus, and mediate the intrinsic apoptotic pathway in the presence of Bcl-2 family proteins. A vicious cycle is established between OS cellular responses and abnormalities in mitochondrial dynamics that accelerate apoptosis. Despite numerous products generated via iteration, complete polymerization of resin monomers is not currently possible. The cytotoxicity of free monomers may lead to adverse reactions, such as pulp sensitivity. This review is based on the most important papers describing the roles of resin monomers in mediating apoptosis in the pulp-dentin complex and provides an overview of the precise mechanisms related to mitochondrion-mediated cytotoxicity, suggesting ways to reduce or eliminate their cytotoxicity in the future through advancements in material technology.

Nasopharyngeal carcinoma (NPC) originates from the nasopharynx epithelium, and luteolin is recognized as an important anti-cancer agent. This study investigated the effects of luteolin on ferroptosis in NPC cells. NPC cells were cultured and exposed to varying concentrations of luteolin. Cell viability, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, glutathione (GSH) levels, Fe concentration, and glutathione peroxidase 4 (GPX4) protein level were assessed. Additionally, SRY-related high-mobility-group box 4 (SOX4) expression was measured. Subsequently, the binding of SOX4 to the growth differentiation factor-15 (GDF15) promoter and GDF15 mRNA levels were evaluated. The impact of the SOX4/GDF15 axis on luteolin-induced ferroptosis in NPC cells was assayed. Luteolin treatment induced cell ferroptosis, evidenced by decreased cell viability, increased MDA and Fe levels, and reduced SOD, GSH, and GPX4 levels. Furthermore, luteolin downregulated SOX4 expression, while overexpression of SOX4 reversed luteolin's pro-ferroptotic effects in NPC cells. SOX4 was found to up-regulate GDF15 transcription by directly binding to its promoter. Conversely, overexpression of GDF15 mitigated the ferroptotic effects induced by luteolin in NPC cells. Therefore, luteolin induces ferroptosis in NPC cells via modulation of the SOX4/GDF15 axis. In conclusion, luteolin reduces the binding of SOX4 to the GDF15 promoter by suppressing SOX4 expression, thereby down-regulating GDF15 transcription levels and inducing ferroptosis in NPC cells.

Chlorfenapyr is a novel pyrrole compound with the chemical formula C15H11BrClF3N2O, exhibiting potent insecticidal and acaricidal effects. It primarily acts on the multi-functional oxidases in the mitochondria of insects, inhibiting the conversion of adenosine diphosphate to adenosine triphosphate, leading to cellular dysfunction due to energy depletion. With increased production and market availability, the population's exposure to chlorfenapyr has risen, resulting in a growing number of fatal poisoning incidents. This report describes the clinical presentation, disease progression, and treatment outcomes of a 2-year and 11-month-old toddler poisoned with chlorfenapyr. The child exhibited symptoms of nausea and vomiting two hours post-poisoning, received gastric lavage and fluid replacement at the local hospital, and was subsequently transferred to our facility. On admission, the child's vital signs were stable for the first two days, with normal laboratory findings. On the third day, the child showed signs of fatigue and diaphoresis, followed by high fever, profuse sweating, altered consciousness, and muscle tremors on the fourth day. By the fifth day, the child displayed rigid muscles in the limbs and trunk, respiratory and circulatory failure, despite rescue efforts proving futile, leading to eventual demise.

A simple monitoring method has been proposed by measuring uranium (U) concentration and its chemical form in serum. The droplet of the 1 µL rat serum specimen was then examined by subjecting it to high energy synchrotron radiation X-ray fluorescence spectroscopy (SR-XRF) and X-ray absorption fine structure (XAFS) to determine the concentration and chemical form of U. The detection limit of U in 1 µL droplet was calculated to be 0.071 µg/g. The U concentration in the specimen obtained from the rat exposed to U was consistent with that determined by inductively coupled plasma mass spectrometry. Uranium in the rat serum was estimated to be hexavalent U based on the standard specimens of tetravalent and hexavanet U. This method developed might be used for monitoring and decorporation of patients at nuclear disasters and environmental pollution.

Ferroptosis, a mode of cell death involving iron-dependent lipid peroxidation, has attracted widespread attention in the development of anticancer drugs and toxicological studies as a potential mechanism of chemical-induced cytotoxicity. This process is regulated by several antioxidant enzymes, of which the selenium-containing glutathione peroxidase 4 (GPx4) is the prime regulator. However, accurately and reproducibly evaluating ferroptosis in cultured cells is challenging since numerous experimental factors in in vitro setting can influence the results. In the present study, we found that the expression levels of selenoproteins, such as GPx4 and GPx1, fluctuate across several cell lines depending on the selenium content of different origin of fetal bovine serum (FBS). Cells cultured in FBS containing higher selenium concentrations exhibited elevated GPx4 expression, and were resistant to ferroptosis induced by erastin and RSL3. These findings suggest that the variability of selenium content in different FBS batches can significantly influence the susceptibility of cells to ferroptosis, highlighting the importance of standardizing these factors to enhance the reproducibility of ferroptosis-related experiments.

The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization assay adopted by the OECD as Test Guideline 442E. In the h-CLAT, 2,4-dinitrochlorobenzene (DNCB) is used as a positive control; however, DNCB is considered a poisonous substance under the Poisonous and Deleterious Substances Control Act in Japan since 2014 because of its high acute toxicity. Strict control, handling, and storage are required when using DNCB, which is a burden to the users. Although the use of other suitable positive controls with historical data is accepted by the guideline, to our knowledge, there have been no reports of such substances. Therefore, in this study, we investigated suitable positive controls that can be used in addition to DNCB for the h-CLAT. Three candidates that are not considered poisonous substances, imidazolidinyl urea, hydroxycitronellal, and 2,4-dinitrofluorobenzene, were selected. To determine their suitability as positive controls, the h-CLAT was performed repeatedly for each chemical in two laboratories. For imidazolidinyl urea, the results that failed the positive control criteria were observed in both laboratories, indicating that it was inconclusive for the suitability as a positive control at the tested concentration. In contrast, all experiments with hydroxycitronellal and 2,4-dinitrofluorobenzene met the criteria and resulted in relative fluorescence intensity values of CD86/CD54, which were comparable to those for DNCB. Based on these results, hydroxycitronellal and 2,4-dinitrofluorobenzene may be used as positive controls. This study would provide valuable information for users examining other suitable positive controls in the h-CLAT.

A representative surfactant, benzalkonium chloride (BAC) is used as a disinfectant, but sometimes causes serious side effects, including lung disorders such as interstitial pneumonia. However, its pathogenic mechanisms remain unexplained. In this study, we identified a novel mechanism by which BAC initiates inflammatory responses that may be responsible for its side effects. We firstly investigated whether BAC initiates inflammation, and found that BAC promotes the secretion of the pro-inflammatory cytokine interleukin-1β (IL-1β) but not tumor necrosis factor-α (TNF-α) in macrophages. Interestingly, the IL-1β secretion triggered by the surfactants was completely blocked by the K-ATP channel blocker glibenclamide or the calcium chelating agent 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM. Moreover, genetic experiments revealed that BAC-dependent IL-1β secretion is mediated by the NLRP3 inflammasome. These results suggest that derangement of ion fluxes associated with the interfacial effects of BAC triggers NLRP3 inflammasome activation and subsequent inflammation. Thus, the NLRP3-dependent mechanisms triggered by BAC may explain the pathogenesis of surfactant-caused adverse effects.

The application of organoids derived from animal tissues and human-induced pluripotent stem cells to safety assessments of environmental chemicals has been introduced over the last decade. One of the objectives of this approach is to develop an alternative method for animal toxicological studies, while another is to focus on the local reactions of chemicals in each organ/tissue. One of the most important goals is bridging the toxicological properties of chemicals between animals and humans, which may be compared on a level playing field using healthy organoids derived from both animals and humans in vitro, excluding species difference in the absorption, distribution, metabolism, and excretion properties of chemicals in vivo. An overview of the application of organoid systems to safety assessments of environmental chemicals, including general toxicology, developmental toxicology, carcinogenicity, and mutagenicity, was provided herein, and bridging strategies using both animal and human organoids are proposed as a future perspective.

We propose a modified Comparative Thyroid Assay (CTA, USEPA) utilizing a smaller number of Sprague-Dawley rats (N=10/group) that assesses brain thyroid hormone (TH) concentrations and periventricular heterotopia while maintaining assay sensitivity. Our recent findings demonstrated that a prenatal test cohort of the modified CTA detected a dose-dependent decrease in maternal serum T3 (up to -26%) and T4 (up to -44%) with sodium phenobarbital (NaPB) exposure at 1000 ppm and 1500 ppm, equivalent to intakes of 60 and 84 mg/kg/day, respectively. On gestation day (GD) 20, fetuses exhibited reduced serum (-26%) and brain (-29%) TH concentrations, although these reductions were not dose dependent. The present study expanded the treatment in a postnatal test cohort, with maternal exposure to NaPB (81-93 mg/kg/day) from GD6 to lactation day (LD) 21. We assessed serum and brain TH concentrations, and periventricular heterotopia in pups on postnatal days (PND) 4, 21, and 28. While LD21 dams showed significant reductions in serum T3 (up to -34%) and T4 (up to -54%), the pups did not exhibit significant TH suppression or periventricular heterotopia at any test point. Instead, a compensatory increase in T4 was observed in serum and brain of PND21 pups. The present study confirmed that perinatal maternal exposure to high doses of NaPB leads to a moderate decrease in maternal TH concentrations; however, the exposure of maternal rats to a similar dose of NaPB did not significantly reduce serum or brain TH concentrations in their postnatal offspring.

Drug-induced convulsion is a serious concern in drug development, such that the convulsion liability of drug candidates must be evaluated in preclinical safety studies. However, information on the differences among species regarding their sensitivity to convulsions induced by convulsant drugs in humans remains limited. Here, we selected 11 test articles from several pharmacological classes and compared the sensitivities of three types of laboratory animal to convulsion. All 11 test articles were examined in mice via intraperitoneal injection and in rats via intravenous bolus; and 6 of the 11 test articles, selected mainly based on availabilities of data on drug plasma concentrations in humans at convulsion, were examined in non-human primates (NHPs) via intravenous infusion. Plasma concentrations of the test articles shortly after convulsion onset or 5 min after administration were measured. All 11 articles tested in mice, 10 of 11 articles tested in rats, and all 6 articles tested in NHPs induced convulsion with premonitory signs. Although there was a general tendency that rats and NHPs exhibited convulsions at lower plasma drug concentrations than did mice, the plasma concentrations at convulsion onset were generally comparable, within 3-fold differences, across the animal species. We conclude that the mice, rats, and NHPs examined in the present study generally showed similar sensitivities to convulsion induced by the test articles. Thus, each of these laboratory animals can be used for the assessment of convulsion risk in the early stages of drug development, depending on throughput, cost, and test article-specific requirements.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes, of which progression is related to high glucose (HG)-induced oxidative stress in renal mesangial cells. Our study aims to explore the antioxidant activity and the underlying mechanism of Puerarin (Pu) in renal mesangial cells exposed to HG. After the cells finished different treatments, DCFH-DA was used to detect the generation of ROS while the expression of AGE, MDA, SOD, and GSH-PX was measured by the ELISA and corresponding kits. The cell morphology was captured by optical microscopy. The mRNA expressions of RAGE, PKCα, PKCβ, PKCγ, and NOX4 were calculated by RT-PCR assays, while the protein expressions of RAGE, NOX4, and PKCβ were quantified via western blotting. Compared with the normal glucose (NG) group, the ROS level, SOD activity, and GSH-PX expression were markedly reduced in the HG group while the MDA expression was increased in the HG group. Then, Pu treatment was proved to significantly prevent the HG-induced up-regulation of ROS level, MDA expression, and down-regulation of SOD activity and GSH-PX expression. Besides, Pu treatment can notably inhibit the AGE expression and reverse the increased RAGE, PKCβ, and NOX4 expressions by HG environment at both RNA and protein levels. Moreover, the antioxidant effect of Pu against access glucose could not be observed in PKCβ knockdown cells. Pu can alleviate the HG-induced oxidative stress via the RAGE/PKC/NOX4 axis in renal mesangial cells, which innovatively suggests the therapeutic potential of Pu for DN treatment.

We conducted a two-year inhalation study of butyraldehyde using F344/DuCrlCrlj rats. The rats were exposed to 0, 300, 1,000 and 3,000 ppm (v/v) for 6 hr/day, 5 days/ week for 104 weeks using whole-body inhalation chambers. The incidence of squamous cell carcinoma of the nasal cavity was increased in the 3,000 ppm groups of both male and female rats, with Fisher's exact test and the Peto test indicating that the incidence was significant. In addition to squamous cell carcinoma in the nasal cavity, in the 3,000 ppm groups one male had an adenosquamous carcinoma, one male had a carcinosarcoma, one male had a sarcoma NOS (Not Otherwise Specified), and one female had a squamous cell papilloma in the nasal cavity. The combined incidence of squamous cell carcinoma, adenosquamous carcinoma and carcinosarcoma was significantly increased in male rats and the combined incidence of squamous cell papilloma and carcinoma was significantly increased in female. Based on these results, we conclude that there is clear evidence of butyraldehyde carcinogenicity in male and female rats.

N-methyl-N-nitrosourea (MNU) exposure impairs hippocampal neurogenesis in rats. The present study investigated the gene expression profiles that were commonly up or downregulated across different brain substructures in response to repeated MNU administration in rats. Five-week-old rats were orally administered MNU at 0, 5, 15 mg/kg body weight/day for 28 days and subjected to gene expression microarray analysis in the hippocampal dentate gyrus, corpus callosum, cerebral cortex and cerebellar vermis. MNU at 15 mg/kg revealed multiple functional clusters of upregulated genes related to immune and inflammatory responses in all brain regions, and also clusters of up or downregulated genes related to regulation of apoptotic process in several regions. Specifically, the upregulated genes commonly found in all four regions were enriched in clusters of "immune response" and/or "inflammatory response" (Cd74, Ccl3, Fcgr3a, Serping1, Lgals3, Fcgr2b, Hcst, Kcnn4, Tnf, Gpr18, Tyrobp and Cyba) and "metal-binding proteins" (Mt1, Mt2A and Apobec1). Meanwhile, downregulated genes common to all four regions (Bmp4, Vcan and Fhit) were included in clusters of "cell proliferation", "glial cell migration" and "nucleotide metabolism". Immunohistochemical analysis of representative gene products revealed that in all brain regions examined, MNU treatment increased metallothionein-I/II cells and galectin-3 cells co-expressing Iba1, and also increased Iba1 and CD68 cells. These results suggest that repeated MNU administration in rats causes neuroinflammation and oxidative stress accompanied by apoptosis of neural cell components in the brain, as well as concurrent anti-inflammatory responses for neuroprotection from MNU exposure, involving activation of microglia producing metallothionein-I/II and galectin-3 on these responses.

Cadmium is a heavy metal that pollutes the environment and foods and is a risk factor for vascular disorders. We have previously demonstrated that pretreatment of vascular endothelial cells with zinc and copper protects the cells against cadmium cytotoxicity. In contrast, cadmium cytotoxicity was potentiated in cells following exposure to lead, thereby indicating that in vascular endothelial cells, cadmium cytotoxicity can be differentially modified by the co-occurrence of other heavy metals. In this study, we revealed that simultaneous treatment or pretreatment with manganese protects vascular endothelial cells against cadmium cytotoxicity. Intracellular accumulation of cadmium was observed to be reduced by simultaneous treatment with manganese, although not by pretreatment. The mRNA expression of metal transporters that regulate the uptake of both cadmium and manganese (ZIP8, ZIP14, and DMT1) remained unaffected by either simultaneous treatment or pretreatment with manganese, and simultaneous treatment with manganese suppressed the cadmium-induced expression of metallothionein but pretreatment with manganese did not exhibit such suppressive effect. Thus, the protection of vascular endothelial cells against cadmium cytotoxicity conferred by simultaneous treatment with manganese is assumed to be partially attributed to a reduction in the intracellular accumulation of cadmium, whereas the effects of pretreatment with manganese are independent of both the reduced intracellular accumulation of cadmium and the induction of metallothionein. These observations accordingly indicate that the protective effects of manganese are mediated via alternative (as yet unidentified) mechanisms.

In illicit drug markets, the most recently expanding new synthetic opioid subclass is benzimidazoles, also known as nitazenes, which were originally developed as analgesics in the 1950s. The emergence of this classical, potent drug family has attracted extensive research interest in the field of forensic toxicology; however, information on their psychological and physical dependence is very limited. Herein, we evaluated the rewarding effects of four nitazene analogs using a battery of in vivo experiments, with a positive control drug (isotonitazene). The four test materials, metonitazene, etodesnitazene, metodesnitazene, and flunitazene, were administered to male C57BL/6J mice by i.p. administration at 0.5, 2, 20, and 20 mg/kg, respectively. In comprehensive behavioral observation tests, representative opioid-related physiological and behavioral states, including analgesia, stereotypic circling behavior, hyperlocomotion, and Straub tail response, were observed. A set of conditioned place preference tests revealed that all the four analogs induced palatability in mice. Furthermore, measurements of dopamine levels in the nucleus accumbens shell by in vivo microdialysis resulted in significant elevations in all test material-treated groups, suggesting that the nitazenes elicit the rewarding effect through a neural circuit originating from the μ-opioid receptor activation at the ventral tegmental area. Our findings add important data regarding the psychological dependence of nitazenes and highlight the abuse potential of these four materials and other prevailing nitazene analogs.