Mechanisms of antibiotic resistance in bacteria mediated by silver nanoparticles.

Silver nanoparticles (AgNPs) are widely used in industry, consumer products, and medical appliances due to their efficient antimicrobial properties. However, information on environmental toxicity and bacterial impact of these particles is not completely elucidated. Results showed that AgNPs produced growth inhibition and oxidative stress in bacteria Escherichia coli (gram negative) and Staphylococcus aureus (gram positive), with half-maximal inhibitory concentrations (IC50) of 12 and 7 mg/L, respectively. Surprisingly, bacteria pre-exposed to sublethal dose of AgNPs exhibited increased resistance toward antibiotics (ampicillin and Pen-Strep) with IC50 elevated by 3-13-fold. Further, AgNP pre-exposure raised the minimal inhibitory concentration and minimal biocidal concentration by two- to eightfold when cells were challenged with antibiotics with diverse mechanisms of action (penicillin, chloramphenicol, and kanamycin). Interestingly, we found that upon exposure to ampicillin, strains pretreated with AgNPs exhibited lower levels of membrane damage and oxidative stress, together with elevated levels of intracellular ATP relative to untreated cells. Bacterial reverse mutation assay (Ames test) showed that AgNPs are highly mutagenic, consistent with further assays demonstrating abiotic reactive oxygen species (ROS) generation and intrinsic DNA cleavage activity in vitro of AgNPs. Overall, our results suggest that AgNPs enhance bacterial resistance to antibiotics by promoting stress tolerance through induction of intracellular ROS. Our data suggest potential consequences of incidental environmental exposure of bacteria to AgNPs and indicate the need to regulate use and disposal of AgNPs in industry and consumer products.

Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans.

Human CYP3A is the most abundant P450 isozyme present in the human liver and small intestine, and metabolizes around 50% of medical drugs on the market. The human CYP3A subfamily comprises four members (CYP3A4, CYP3A5, CYP3A7, CYP3A43) encoded on human chromosome 7. However, transgenic mouse lines carrying the entire human CYP3A cluster have not been constructed because of limitations in conventional cloning techniques. Here, we show that the introduction of a human artificial chromosome (HAC) containing the entire genomic human CYP3A locus recapitulates tissue- and stage-specific expression of human CYP3A genes and xenobiotic metabolism in mice. About 700 kb of the entire CYP3A genomic segment was cloned into a HAC (CYP3A-HAC), and trans-chromosomic (Tc) mice carrying a single copy of germline-transmittable CYP3A-HAC were generated via a chromosome-engineering technique. The tissue- and stage-specific expression profiles of CYP3A genes were consistent with those seen in humans. We further generated mice carrying the CYP3A-HAC in the background homozygous for targeted deletion of most endogenous Cyp3a genes. In this mouse strain with 'fully humanized' CYP3A genes, the kinetics of triazolam metabolism, CYP3A-mediated mechanism-based inactivation effects and formation of fetal-specific metabolites of dehydroepiandrosterone observed in humans were well reproduced. Thus, these mice are likely to be valuable in evaluating novel drugs metabolized by CYP3A enzymes and in studying the regulation of human CYP3A gene expression. Furthermore, this system can also be used for generating Tc mice carrying other human metabolic genes.

Effects of polystyrene nanoplastic size on zebrafish embryo development.

Polystyrene nanoplastics (PS) require a comprehensive evaluation of their toxicity and potential risks to humans and the environment. The zebrafish model, a well-established animal model increasingly utilized for nanotoxicity assessments, was employed in this study. Our research aimed to explore the toxic effects of PS with sizes of 30, 100, 200, and 450 nm on zebrafish embryos. Exposure experiments were conducted on embryos at 4 h post-fertilization (hpf) using various concentrations of nanoparticles (20, 40, 60, 80, and 100 mg/L) until 96 hpf. Notably, PS ranging from 100 to 450 nm did not adversely affect zebrafish embryo development. However, PS with a size of 30 nm at a concentration of 100 mg/L resulted in embryo mortality but not embryonic malformations. Furthermore, our investigation confirmed the uptake of these nanoparticles by zebrafish larvae following the opening of their mouths, with the particles being found predominantly in the digestive system of the larvae. Additionally, 30 nm PS were found to significantly modulate the expression levels of genes associated with oxidative stress and apoptosis. These findings highlight the developmental impacts of 30 nm PS on zebrafish embryos, raising concerns about potential similar consequences in humans. Considering our findings, it is essential to encourage further research into the management and regulation of PS to mitigate their potential environmental and health impacts.

Pseudotyped zoonotic thogotoviruses exhibit broad entry range in mammalian cells.

Viruses in the thogotovirus genus of the family Orthomyxoviridae are much less well-understood than influenza viruses despite documented zoonotic transmission and association with human disease. This study therefore developed a cell-cell fusion assay and three pseudotyping tools and used them to assess envelope function and cell tropism. Envelope glycoproteins of Dhori (DHOV), Thogoto (THOV), Bourbon, and Sinu viruses were all revealed to exhibit pH-dependent triggering of membrane fusion. Lentivirus vectors were robustly pseudotyped with these glycoproteins while influenza virus vectors showed pseudotyping compatibility, albeit at lower efficiencies. Replication-competent vesicular stomatitis virus expressing DHOV or THOV glycoproteins were also successfully generated. These pseudotyped viruses mediated entry into a wide range of mammalian cell lines, including human primary cells. The promiscuousness of these viruses suggests the use of a relatively ubiquitous receptor and their entry into numerous mammalian cells emphasize their high potential as veterinary and zoonotic diseases.

Impact of a real food matrix and in vitro digestion on properties and acute toxicity of polystyrene microparticles.

Although it is proved that humans ingest microplastics via food, and microplastics were found in human tissues, blood and feces, there needs to be more data on the properties and health-related effects of plastic particles that interact with food and undergo digestion. This study aimed to examine the impact of a real food matrix, milk, on the behavior and gastrointestinal fate of polystyrene microparticles (PSMP). In the presence of the food matrix, the net negative ζ-potential values of PSMP (diameter size of 1.823 µm) decreased significantly due to the formation of the corona, mostly consisting of α and ß-casein fragments. Protein corona profiles and morphologies of particles incubated with whole and skim milk were found to be similar, and the protein profiles were completely altered after in vitro digestion simulation. In vitro and in vivo toxicity studies showed that neither bare PSMP nor food-interacted PSMP pose acute toxicity on the Caco-2 cell line and zebrafish embryos under the chosen experimental conditions. In summary, these results may contribute to a better understanding of changes that microplastics undergo in foods. Further studies on repeated exposure or chronic toxicity are needed to fully reveal the effect of food matrix on microplastic toxicity.

Quaternization of high molecular weight chitosan for increasing intestinal drug absorption using Caco-2 cells as an in vitro intestinal model.

Potential use of a quaternized chitosan (MW 600 kDa) with 65% of 3-chloro-2-hydroxypropyltrimethylammonium (600-HPTChC65) as an absorptive enhancer was investigated in Caco-2 monolayers. 600-HPTChC65 (0.005% w/v) quickly reduced transepithelial electrical resistance (TEER) to the maximum level in 40 min with full recovery within 6 h after removal. Its TEER reduction was corresponded to increased FD4 transport across the monolayers and disrupted localization of tight junction proteins ZO-1 and occludin at the cell borders. 600-HPTChC65 was densely localized at the membrane surface and intercellular junctions. This chitosan (0.08-0.32% w/v) reduced the efflux ratio of [3H]-digoxin by 1.7- 2 folds, suggesting an increased [3H]-digoxin transport across the monolayers. Its binding with P-gp on Caco-2 monolayer increased the signal of fluorescence-labeled anti-P-gp (UIC2) reactivity due to conformational change. 600-HPTChC65 (0.32% w/v) had no effect on P-gp expression in the Caco-2 monolayers. These results suggest that 600-HPTChC65 could enhance drug absorption through tight junction opening and decreased P-gp function. Its interaction with the absorptive barrier mainly resulted in disrupting ZO-1 and occludin organization as well as changing in P-gp conformation.

Development of lung tissue models and their applications.

The lungs are important organs that play a critical role in the development of specific diseases, as well as responding to the effects of drugs, chemicals, and environmental pollutants. Due to the ethical concerns around animal testing, alternative methods have been sought which are more time-effective, do not pose ethical issues for animals, do not involve species differences, and provide easy investigation of the pathobiology of lung diseases. Several national and international organizations are working to accelerate the development and implementation of structurally and functionally complex tissue models as alternatives to animal testing, particularly for the lung. Unfortunately, to date, there is no lung tissue model that has been accepted by regulatory agencies for use in inhalation toxicology. This review discusses the challenges involved in developing a relevant lung tissue model derived from human cells such as cell lines, primary cells, and pluripotent stem cells. It also introduces examples of two-dimensional (2D) air-liquid interface and monocultured and co-cultured three-dimensional (3D) culture techniques, particularly organoid culture and 3D bioprinting. Furthermore, it reviews development of the lung-on-a-chip model to mimic the microenvironment and physiological performance. The applications of lung tissue models in various studies, especially disease modeling, viral respiratory infection, and environmental toxicology will be also introduced. The development of a relevant lung tissue model is extremely important for standardizing and validation the in vitro models for inhalation toxicity and other studies in the future.

Protective effects of Thai silk sericins and their related mechanisms on UVA-induced phototoxicity and melanogenesis: Investigation in primary melanocyte cells using a proteomic approach.

UV radiation causes excess production of melanin as a result of hyperpigmentation and skin disorders. Silk sericin exhibited bioactivities to skin and inhibited UV-induced phototoxicity and melanogenesis in skin cells; however, the mechanism related to sericin against UV-induced melanogenesis has not been investigated. This study aimed to investigate the protective effects of Thai silk sericins against UVA-induced phototoxicity and melanogenesis and their related mechanisms. Thai silk sericins exhibited cytoprotective effects against UV-induced toxicity in human primary melanocytes by attenuation of cytotoxicity, intracellular ROS generation, and mitochondrial potential impairment. Pre- and post-treatment with sericin significantly inhibited melanin synthesis and tyrosinase activity against UVA exposure. In addition, sericin S2 could reduce the basal melanin content in zebrafish embryos. The proteomic analysis demonstrated that Thai silk sericins altered the protein expression in melanocytes especially proteins related to stress, inflammatory, cytokine stimulation, cell proliferation, and cell survival processes that contribute to cytoprotective effect and inhibitory effect on melanogenesis of sericin. Moreover, we demonstrated the novel mechanism of Thai silk sericins in inhibiting UVA-induced melanogenesis via increasing BMP4 expression in MAPK/ERK signaling pathway. These evidences support the potential use of Thai silk sericins in prevention of hyperpigmentation in skin disorders especially after UVA exposure.

Chemical Composition, Sources, and Health Risk Assessment of PM2.5 and PM10 in Urban Sites of Bangkok, Thailand.

Of late, air pollution in Asia has increased, particularly in built-up areas due to rapid industrialization and urbanization. The present study sets out to examine the impact that pollution can have on the health of people living in the inner city of Bangkok, Thailand. Consequently, in 2021, fine particulate matter (PM2.5) and coarse particulate matter (PM10) chemical composition and sources are evaluated at three locations in Bangkok. To identify the possible sources of such particulates, therefore, the principal component analysis (PCA) technique is duly carried out. As determined via PCA, the major sources of air pollution in Bangkok are local emission sources and sea salt. The most significant local sources of PM2.5 and PM10 in Bangkok include primary combustion, such as vehicle emissions, coal combustion, biomass burning, secondary aerosol formation, industrial emissions, and dust sources. Except for the hazard quotient (HQ) of Ni and Mn of PM2.5 for adults, the HQ values of As, Cd, Cr, Mn, and Ni of both PM2.5 and PM10 were below the safe level (HQ = 1) for adults and children. This indicates that exposure to these metals would have non-carcinogenic health effects. Except for the carcinogenic risk (HI) value of Cr of PM2.5 and PM10, which can cause cancer in adults, at Bangna and Din Daeng, the HI values of Cd, Ni, As, and Pb of PM2.5 and PM10 are below the limit set by the U.S. Environmental Protection Agency (U.S. EPA). Ni and Mn pose non-carcinogenic risks, whereas Cr poses carcinogenic risks to adults via inhalation, a serious threat to the residents of Bangkok.

Transparent Anti-SARS COV-2 Film from Copper(I) Oxide Incorporated in Zeolite Nanoparticles.

The high antibacterial and antiviral performance of synthesized copper(I) oxide (Cu2O) incorporated in zeolite nanoparticles (Cu-Z) was determined. Various Cu contents (1-9 wt %) in solutions were loaded in the zeolite matrix under neutral conditions at room temperature. All synthesized Cu-Z nanoparticles showed high selectivity of the cuprous oxide, as confirmed by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis. An advantage of the prepared Cu-Z over the pristine Cu2O nanoparticles was its high thermal stability. The 7 and 9 wt % Cu contents (07Cu-Z and 09Cu-Z) exhibited the best activities to deactivate Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus bacteria. The film coated with 07Cu-Z nanoparticles also had high antiviral activities against porcine coronavirus (porcine epidemic diarrhea virus, PEDV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Specifically, the 07Cu-Z-coated film could reduce 99.93% of PEDV and 99.94% of SARS-CoV-2 viruses in 5 min of contact time, which were higher efficacies and faster than those of any previously reported works. The anti-SARS-CoV-2 virus film was coated on a low-cost PET or PVC film. A very small amount of cuprous oxide in zeolite was used to fabricate the antivirus film; therefore, the film was more transparent (79.4% transparency) than the cuprous oxide film or other commercial products. The toxicity of 07Cu-Z nanoparticles was determined by a toxicity test on zebrafish embryo and a skin irritation test to reconstruct a human epidermis (RhE) model. It was found that the impact on the aquatic environment and human skin was lower than that of the pristine Cu2O.

Effects of Porous Size and Membrane Pattern on Shear Stress Characteristic in Gut-on-a-Chip with Peristalsis Motion.

Dynamic gut-on-a-chip platform allows better recreation of the intestinal environment in vitro compared to the traditional static cell culture. However, the underlying mechanism is still not fully discovered. In this study, the shear stress behavior in a gut-on-a-chip device with porous membrane subjected to peristalsis motion is numerically investigated using CFD simulation for three different pore sizes and two pattern layouts. The results reveal that, in the stationary microchannel, the average shear stress on the porous membrane is approximately 15% greater than that of the flat membrane, regardless of the pore size. However, when subjected to cyclic deformation, the porous membrane with smaller pore size experiences stronger variation of shear stress which is ±5.61%, ±10.12% and ±34.45% from its average for the pore diameters of 10 µm, 5 µm and 1 µm, respectively. The shear stress distribution is more consistent in case of the staggered pattern layout while the in-line pattern layout allows for a 32% wider range of shear stress at the identical pore size during a cyclic deformation. These changes in the shear stress caused by peristalsis motion, porous size and membrane pattern could be the key factors that promote cell differentiation in the deforming gut-on-a-chip model.

Multilayered fibroblasts constructed by accelerated cellular self-assembly and applications for regenerative medicine.

Regenerative medicine research requires animal experiments to evaluate the treatment effects. According to the 3Rs principles, alternative models have been developed and utilized to evaluate the efficacy and safety of new products. Three-dimensional (3D) cell cultures have been recognized for their relevant structures and biological functions akin to native tissues. They can better represent in vivo conditions than two-dimensional (2D) cell cultures. Herein, we present a fast and simple technique for the construction of 3D dermal fibroblasts (3D-DFs) without exogenous scaffolds. The 3D-DFs can be obtained within 3 days by seeding DFs at a level that exceeds their confluent density and culturing in the presence of ascorbic acid. The 3D-DFs had a compact multilayer structure, as revealed from their histology. The collagen content of the resulting 3D-DFs drastically increased compared to in a monolayer. The 3D-DF-derived extracellular matrix can serve for the 3D culturing of other cells. A gap closure assay was performed with the 3D-DFs to represent a 3D-wounded dermal model. Interestingly, the multilayered structure of the 3D-DFs could be regenerated after wounding even when cultured in the absence of ascorbic acid. Moreover, skin grafting of the 3D-DFs was demonstrated in vitro using wounded full-thickness skin models as an alternative to animal experiments. The 3D-DFs will potentially be useful for regenerative medicine or as tissue models for in vitro studies.

Protective effect of Thai silk extracts on drug-induced phototoxicity in human epidermal A431 cells and a reconstructed human epidermis model.

Bombyx mori silk extracts, derived from the cocoon degumming process of draw and dye silk in the textile industry, are mainly composed of sericin protein. To add value to the Thai silk extracts, and hence the silk industry, a simple enrichment process was recently developed and the enriched silk extracts were then applied in nano-cosmeceutical products and nano-delivery systems. In this study, the protective effect of Thai silk extracts from three different strains of Bombyx mori on the drug-induced phototoxicity was evaluated in vitro using chlorpromazine (CPZ), a commonly used antipsychotic drug, as a representative phototoxic drug. The human epidermal A431 cell line and reconstructed human epidermis (RhE) model were used as the in vitro skin model. The silk extracts significantly improved the viability of A431 cells after CPZ exposure and ultraviolet A (UVA) irradiation, as shown by the significantly increased CPZ and UVA IC50 values and the decreased proportion of apoptotic cells. The protective effect of these silk extracts against the CPZ-induced UVA-phototoxicity in A431 cells was associated with the attenuation of intracellular oxidative stress via an increased intracellular glutathione level. Likewise, the silk extracts exhibited a protective effect on the CPZ-induced UVA-phototoxicity in the RhE model, in terms of an improved tissue viability and attenuation of the released inflammatory cytokine, interleukin-1α. These findings support the potential usefulness of silk extracts in novel applications, especially in the protection of drug-induced phototoxicity.

Hepatocyte nuclear factor 1 alpha and 4 alpha are factors involved in interindividual variability in the expression of UGT1A6 and UGT1A9 but not UGT1A1, UGT1A3 and UGT1A4 mRNA in human livers.

UDP-glucuronosyltransferases (UGTs) catalyze phase-II biotransformation reaction of a variety of substances. Among the UGT1A isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9 are predominantly expressed in the liver. Interindividual variability in expression of these isoforms would cause interindividual differences in drug response, toxicity and cancer susceptibility. In the present study, we investigated the interindividual variability in UGT1A mRNA expression and whether hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha were factors responsible for their variability in human livers. The amounts of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, HNF1alpha and HNF4alpha mRNA in 18 human livers were measured by quantitative real-time polymerase chain reaction. The largest and smallest interindividual differences in expression levels were observed in UGT1A1 (8.6-fold) and UGT1A4 (2.5-fold) mRNA, respectively. The amounts of HNF1alpha and HNF4alpha mRNA were strongly correlated with the amount of UGT1A9 mRNA and moderately correlated with that of UGT1A6 mRNA, whereas no significant correlation was found with the amounts of UGT1A1, UGT1A3 and UGT1A4 mRNA. Our results suggest that HNF1alpha and HNF4alpha are the factors involved in the interindividual variability of UGT1A6 and UGT1A9 mRNA expression. Further studies of other transcription factors are needed to clarify the factor(s) determining the interindividual variations in UGT1A1, UGT1A3 and UGT1A4 mRNA expression.

Shape and surface properties of titanate nanomaterials influence differential cellular uptake behavior and biological responses in THP-1 cells.

We investigated cellular uptake behavior and biological responses of spherical and fibrous titanate nanomaterials in human monocyte THP-1 cells. Two titanate nanofibers (TiNFs), namely TF-1 and TF-2, were synthesized from anatase TiO2 nanoparticles (TNPs) via hydrothermal treatment. The synthesized TiNFs and TNPs were thoroughly characterized for their size, crystallinity, surface area and surface pH. TF-1 (∼2 µm in length) was amorphous with an acidic surface, while TF-2 (∼7 µm in length) was brookite with a basic surface. The results demonstrated that none of these titanate nanomaterials resulted in significant cytotoxicity, even at the highest doses tested (50 µg/ml), consistent with an absence of ROS generation and lack of change of mitochondrial membrane potential. While no cytotoxic effect was found in the titanate nanomaterials, TF-2 tended to decrease the proliferation of THP-1 cells. Furthermore, TF-2 resulted in an inflammatory cytokine response, as evidenced by dramatic induction of IL-8 and TNF-α release in TF2 but not TF-1 nor TNPs. These results suggest that shape of titanate nanomaterials plays an important role in cellular internalization, while surface pH may play a prominent role in inflammatory response in THP-1 cells.

Human primary erythroid cells as a more sensitive alternative in vitro hematological model for nanotoxicity studies: Toxicological effects of silver nanoparticles.

Although immortalized cells established from cancerous cells have been widely used for studies in nanotoxicology studies, the reliability of the results derived from immortalized cells has been questioned because of their different characteristics from normal cells. In the present study, human primary erythroid cells in liquid culture were used as an in vitro hematological cell model for investigation of the nanotoxicity of silver nanoparticles (AgNPs) and comparing the results to the immortalized hematological cell lines HL60 and K562. The AgNPs caused significant cytotoxic effects in the primary erythroid cells, as shown by the decreased cell viability and induction of intracellular ROS generation and apoptosis, whereas they showed much lower cytotoxic and apoptotic effects in HL60 and K562 cells and did not induced ROS generation in these cell lines. Scanning electron microcopy revealed an interaction of AgNPs to the cell membrane in both primary erythroid and immortalized cells. In addition, AgNPs induced hemolysis in the primary erythroid cells in a dose-dependent manner, and transmission electron microcopy analysis revealed that AgNPs damaged the erythroid cell membrane. Taken together, these results suggest that human primary erythroid cells in liquid culture are a more sensitive alternative in vitro hematological model for nanotoxicology studies.

Mechanistic study on the biological effects of silver and gold nanoparticles in Caco-2 cells--induction of the Nrf2/HO-1 pathway by high concentrations of silver nanoparticles.

The most commonly used metal nanoparticles (NPs) across diverse applications, including in agro-food applications, include silver (AgNPs) and gold (AuNPs). In the present study, we aimed to investigate the biological responses and possible toxicological effects of AgNPs and AuNPs in the Caco-2 cells as an in vitro human GI tract model. Both AgNPs and AuNPs were internalized into the cytoplasm of Caco-2 cells, but not within the nucleus and only exposure to high concentrations of AgNPs, but not AuNPs, caused acute cytotoxicity and depolarization of the mitochondrial membrane potential. In addition, only AgNPs significantly depleted the total intracellular glutathione level, induced the activation of the stress-responsive gene, Nrf2, and dramatically increased the expression of heme oxygenase-1 (HO-1). Furthermore, siRNA silencing of Nrf2 transcripts significantly reduced the AgNP-induced HO-1 mRNA induction, suggesting a key role for Nrf2 in the control of HO-1 expression. Taken together, AgNPs but not AuNPs induced acute cytotoxicity and cellular responses via the oxidative stress-related activation of Nrf2/HO-1 signaling pathway in Caco-2 cells. The expression of HO-1 transcripts may be useful as a sensitive marker for safety evaluation of AgNPs in the GI tract of humans.

Silver nanoparticles induce toxicity in A549 cells via ROS-dependent and ROS-independent pathways.

Silver nanoparticles (AgNPs) are incorporated into a large number of consumer and medical products. Several experiments have demonstrated that AgNPs can be toxic to the vital organs of humans and especially to the lung. The present study evaluated the in vitro mechanisms of AgNP (<100 nm) toxicity in relationship to the generation of reactive oxygen species (ROS) in A549 cells. AgNPs caused ROS formation in the cells, a reduction in their cell viability and mitochondrial membrane potential (MMP), an increase in the proportion of cells in the sub-G1 (apoptosis) population, S phase arrest and down-regulation of the cell cycle associated proliferating cell nuclear antigen (PCNA) protein, in a concentration- and time-dependent manner. Pretreatment of the A549 cells with N-acetyl-cysteine (NAC), an antioxidant, decreased the effects of AgNPs on the reduced cell viability, change in the MMP and proportion of cells in the sub-G1population, but had no effect on the AgNP-mediated S phase arrest or down-regulation of PCNA. These observations allow us to propose that the in vitro toxic effects of AgNPs on A549 cells are mediated via both ROS-dependent (cytotoxicity) and ROS-independent (cell cycle arrest) pathways.

Titanium dioxide nanoparticles-mediated in vitro cytotoxicity does not induce Hsp70 and Grp78 expression in human bronchial epithelial A549 cells.

Titanium dioxide nanoparticles (TiO(2)NPs) are increasingly being used in various industrial applications including the production of paper, plastics, cosmetics and paints. With the increasing number of nano-related products, the concern of governments and the general public about the health and environmental risks, especially with regard to occupational and other environmental exposure, are gradually increasing. However, there is insufficient knowledge about the actual affects upon human health and the environment, as well as a lack of suitable biomarkers for assessing TiO(2)NP-induced cytotoxicity. Since the respiratory tract is likely to be the main exposure route of industrial workers to TiO(2)NPs, we investigated the cytotoxicity of the anatase and rutile crystalline forms of TiO(2)NPs in A549 cells, a human alveolar type II-like epithelial cell line. In addition, we evaluated the transcript and protein expression levels of two heat shock protein (HSP) members, Grp78 and Hsp70, to ascertain their suitability as biomarkers of TiO(2)NP-induced toxicity in the respiratory system. Ultrastructural observations confirmed the presence of TiO(2)NPs inside cells. In vitro exposure of A549 cells to the anatase or rutile forms of TiO(2)NPs led to cell death and induced intracellular ROS generation in a dose-dependent manner, as determined by the MTS and dichlorofluorescein (DCF) assays, respectively. In contrast, the transcript and protein expression levels of Hsp70 and Grp78 did not change within the same TiO(2)NPs dose range (25-500 µg/ml). Thus, whilst TiO(2)NPs can cause cytotoxicity in A549 cells, and thus potentially in respiratory cells, Hsp70 and Grp78 are not suitable biomarkers for evaluating the acute toxicological effects of TiO(2)NPs in the respiratory system.

Species differences in mechanism-based inactivation of CYP3A in humans, rats and mice.

Mechanism-based inactivation (MBI) of cytochrome P450 3A (CYP3A) often causes serious drug-drug interactions. To examine species differences in MBI of CYP3A between humans and rodents, we compared MBI potencies of five representative CYP3A inhibitors in human, rat and mouse liver microsomes. Among the inhibitors studied, erythromycin and clarithromycin exhibited markedly weaker MBI effects on CYP3A activity in rat and mouse liver microsomes compared to human liver microsomes. Results of spectroscopic experiments showed that erythromycin and clarithromycin form a metabolic intermediate complex with human liver microsomes but not with rat or mouse liver microsomes. In contrast, troleandomycin, diltiazem and nicardipine form a metabolic intermediate complex with rat and mouse liver microsomes, although some differences in MBI potency among species were observed. Parameters for MBI potency (k(inact)/K(I) ratio) and reversible inhibition (IC(50)) were negatively correlated (r=-0.820, p=0.0003), suggesting that the different affinities of CYP3A inhibitor for CYP3A may partly contribute to the different MBI potencies of inhibitor among species. Taken together, the results suggest that there are species differences in MBI of CYP3A in humans, rats and mice, which should be considered when rodents are used as in vivo models for MBI-mediated drug-drug interaction study.