Currently, protocols for the dispersal of titanium dioxide (TiO2) nanoparticles are not standardized and often yield non-uniform particles and/or insufficient dispersal in liquid medium. Our study aimed to improve dispersal so that TiO2 nanoparticles are of uniform size, making nanotoxicity testing more reliable. Various combinations of vehicles, sonication durations, and sonication volumes were assessed for optimizing preparations of TiO2 nanoparticles. We tested each of five vehicles: ultrapure water (UPW), 0.2% disodium hydrogen phosphate (DSP), Dulbecco’s phosphate-buffered saline (PBS), 0.9% saline (S), or S containing 0.05% Tween 80 (ST). We also assessed two sonication durations and three sonication volumes. Each suspension underwent ultrasonication and centrifugation; the supernatants were then analyzed. Particle size was measured by dynamic light scattering. P25 nanoparticles (~100 nm; the type of TiO2 nanoparticles used in our study) in UPW and 0.2% DSP were effectively dispersed; however, those in PBS, S, or ST were not. Relevant duration time and volume for sonication were examined with 0.2% DSP. A sonication time of 30 min and volume of 10 mL for each vial were determined to be optimal sonication conditions as determined with our dispersal assay. Under these optimal conditions, P25 nanoparticles sonicated/centrifuged in UPW or 0.2% DSP remained dispersed and exhibited long-term stability (90 days). We thus have developed a reliable procedure for preparing TiO2 nanoparticles in liquid-phase dispersions for toxicity testing.
Stephanthraniline A (STA) is a C21 steroidal aglycone isolated from the stem of Stephanotis mucronata (Blanco) Merr. that exerts growth inhibition in human colon cancer cells. However, the intracellular molecular mechanisms whereby this occurs have not been well characterized. In this study, we found that STA significantly inhibits the growth of HCT116 colon cancer cells in a time- and concentration-dependent manner. The inhibitory effect of STA on cell growth was related to the induction of apoptosis. Activated caspase-3, caspase-8 and caspase-9, along with a decreased Bcl-2/Bcl-x ratio and loss of mitochondrial membrane potential (Δψm), were observed in response to STA treatment. Furthermore, treatment of HCT116 cells with STA resulted in G0/G1 phase cell cycle arrest accompanied by decreased mRNA levels of cyclin-dependent kinase 4 (CDK4), p21 and c-myc. Additionally, the inhibition of Akt signaling and activation of p38 signaling were observed after treatment with STA in HCT116 cells. These findings indicate that STA inhibits HCT116 cell growth by promoting apoptosis, the dysregulation of mitochondrial function, and cell cycle arrest.
Spinocerebellar ataxia type 3 (SCA3) is caused by the expansion of a glutamine-encoding CAG repeat in the ATXN3 gene encoding the protein ataxin-3. The nuclear presence of polyglutamine-expanded ataxin-3 is of critical importance for the pathogenesis of SCA3. Disulfiram, an FDA-approved drug for alcoholism, has also garnered attention in cancer treatment. However, it has shown toxicity in the nervous system. Bearing this in mind, we treated cells expressing ataxin-3 with disulfiram to measure several pathogenic cascades of SCA3, including aggregate formation, soluble ataxin-3 expression and nuclear localization of ataxin-3 and the cytotoxicity, which assess the direct effect of disulfiram on SCA3 cell models. To our knowledge, this is direct evidence that disulfiram elevated the nuclear localization of polyglutamine-expanded ataxin-3 and enhanced the cytotoxicity in a cell model of SCA3. Furthermore, disulfiram did not affect the aggregate formation of polyglutamine-expanded ataxin-3 at least at a single dose. Our findings repurpose disulfiram as a modulator of ataxin-3 nuclear transport that aggravates the pathology of SCA3, which is a new target for disulfiram. This study also represents an important example of determining novel side effects in pre-existing drugs. This study suggests that caution may be warranted when this compound is used to treat alcohol abuse or cancer in patients carrying a SCA3-causing mutation.
Long-term exposure to certain volatile organic compounds is a significant public health concern. A variety of food containers and drinking cups prepared from polystyrene or polystyrene-related plastics could contain styrene monomer. In the current study, the concentrations of styrene in plasma and liver were surveyed and determined after oral doses of 25 mg/kg to rats and 200 mg/kg to control and humanized-liver mice. Plasma concentrations of styrene in rats were still detected 2 hr after 10-25 mg/kg oral doses. In contrast, after an order of magnitude higher oral dose of styrene (200 mg/kg) to mice, styrene in mouse plasma was rapidly cleared within 15 min to the limit-of-detection level. However, unmetabolized styrene was detected in mouse liver 24 hr after oral treatment. A simple physiologically based pharmacokinetic (PBPK) model capable of estimating blood and liver concentrations of styrene was established for rats. A human PBPK model was then set up for styrene by using the same intrinsic hepatic clearances in rats and humans and by applying allometric scaling to rat parameters obtained from the plasma concentrations of styrene in rats. By reverse dosimetry analysis (from concentrations to doses), we found that the 95th percentile values of styrene concentrations (0.132 ng/mL) reported in United States biomonitoring data of more than 1000 human blood samples may imply exposure to repeated oral doses of styrene of 2.89 µg/kg/day. These results suggest that styrene biomonitoring data in human blood samples imply exposures roughly similar to or lower than the established tolerable daily intake level of 7.7 μg/kg/day.
Methylmercury (MeHg) exposure is known to induce neurodegeneration in both the central nervous system (CNS) and peripheral nervous system (PNS). Molecular mechanisms of MeHg-induced neurotoxicity have been well investigated in the CNS, however, it remains unclear in the PNS. In the present study, comprehensive gene expression analysis was performed by analyzing MeHg-exposed adult rat dorsal root ganglion (DRG) by DNA microarray. Methylmercuric chloride (6.7 mg/kg/day) was administered to nine-week-old male Wistar rats for five days, followed by two days without administration; this cycle was repeated once. Rats were anesthetized at 7 or 14 days after commencement of MeHg exposure, and their DRGs were removed and homogenized to make total RNA samples. DNA microarray data from Day 7 samples identified 100 out of 18,513 detected genes as annotated genes with more than two-fold upregulated or downregulated expression compared with controls. Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested strong involvement of immune activation and inflammation pathways in rat DRG exposed to MeHg, and some genes overlapped with previously reported genes affected by MeHg exposure in the cerebellum. The present results suggest that MeHg-induced neurotoxicity is associated with immune activation and inflammatory responses in rat DRG.
Acetaminophen (Paracetamol, APAP) has been widely used for many decades as an analgesic and antipyretic agent but APAP overdose often causes acute adverse reactions, particularly liver damage. The metabolically oxidized form of APAP, N-acetyl-p-benzoquinone imine (NAPQI), is chemically reactive and binds covalently to proteins. Therefore, NAPQI is believed to be the key metabolite that causes hepatotoxicity, especially under conditions of glutathione depletion. Other APAP-induced adverse reactions, such as skin damage, are rare and remain poorly studied. Here, we report a case study of a male patient who presented with an acute swelling skin rash (without hepatotoxicity) caused by therapeutic doses of APAP. Plasma samples were collected at 17 hr after dosing (during the manifestation of symptoms) and at one month (after recovery) and were subjected to LC-MS analysis of NAPQI-adducts. A significant concentration of NAPQI-cysteine adduct (33 pmol/mL) was found together with low concentrations of NAPQI-N-acetylcysteine adduct (2.0 pmol/mL) and NAPQI-glutathione adduct (0.13 pmol/mL). However, the NAPQI-albumin adduct was below the detection limit (below 0.001% modification on albumin) despite a previous report of high concentrations of NAPQI-albumin adduct following acute liver injury. Therefore, the observed APAP-induced skin damage may have had a different cause from APAP-induced liver injury.
Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive deposition of droplets in hepatocytes. Patients with NAFLD can be at risk for nonalcoholic steatohepatitis, which can lead to hepatocellular carcinoma. Autophagy is a cellular pathway that is crucial for survival and homeostasis, and which protects against pathophysiological changes like obesity and cancer. We determined the expression of autophagy markers in preneoplastic hepatic lesions and the effects of an autophagy repressor chloroquine (CQ) or inducer amiodarone (AM) in a steatosis-related hepatocarcinogenesis model. Male F344 rats were fed a control diet or high fat diet (HFD), and subjected to initiation and promotion steps with N-nitrosodiethylamine injection at week 0 and a partial hepatectomy at week 3. Several HFD-fed rats were administered 0.1% CQ and 0.5% AM in their drinking water during week 2 and 8. CQ and AM did not improve HFD-induced obesity. AM, but not CQ, significantly decreased the number of glutathione S-transferase placental form-positive preneoplastic liver foci in the liver. Autophagosome markers LC3 and the LC3-binding protein p62 were heterogeneously expressed in the preneoplastic foci. CQ might inhibit autophagy by significantly increased p62/LC3 ratio, while AM might have a potential of inducing autophagy by showing an increased gene expression of the autophagy regulator, Atg5. These results suggest that preneoplastic lesions express autophagosome markers and that AM might decrease steatosis-related early hepatocarcinogenesis by potentially inducing autophagy in HFD-fed rats, while inhibition of autophagy by CQ did not alter the hepatocarcinogenesis. However, an immunohistochemical trial revealed a technical limitation in detecting autophagosome markers because there were variations in each preneoplastic lesion.