Developing osteoblasts as an endpoint for the mouse embryonic stem cell test

The mouse Embryonic Stem cell Test (EST) using cardiomyocyte differentiation is a promising in vitro assay for detecting potential embryotoxicity; however, the addition of another differentiation endpoint, such as osteoblasts, may improve the predictive value of the test. A number of variables such as culture conditions and starting cell number were investigated. A 14 day direct plating method of D3 mouse embryonic stem cells (mESCs) was used to test the predictivity of osteoblast differentiation as an endpoint in the EST. Twelve compounds were tested using the prediction model developed in the ECVAM validation study. Eight of the compounds selected from the EST validation study served as model compounds; four additional compounds known to produce skeletal defects were also tested. Our results indicate comparable chemical classification between the validated cardiomyocyte endpoint and the osteoblast endpoint. These results suggest that differentiation to osteoblasts may provide confirmatory information in predicting embryotoxicity.     

Assessment of oxidative stress responses and the cytotoxic and genotoxic potential of the herbicide tembotrione in HepG2 cells

Tembotrione is a triketone herbicide, usually used for post-emergence weed control in corn. Currently, there is little or no published data on its genotoxicity to human cells either in vitro or in vivo. This study evaluated the impact of acute (4 and 24 h) exposure to low concentrations of tembotrione [corresponding to the acceptable daily intake (0.17 μg/mL), residential exposure level (0.002 μg/mL) and acceptable operator exposure level (0.0012 μg/mL)] on human hepatocellular carcinoma cell line HepG2, using biomarkers of oxidative stress, CCK-8 colorimetric assay for cell viability, alkaline comet assay, and cytokinesis-block micronucleus “cytome” assay. Tembotrione applied at concentrations likely to be encountered in occupational and residential exposures induced cytogenetic outcomes in non-target cells despite non-significant changes in the values of oxidative stress biomarkers. We assume that the observed effects were mainly the consequence of impaired metabolic pathways in HepG2 cells due to the inhibition of the enzyme 4-hydroxyphenyl-pyruvate-dioxygenase by tembotrione, which possibly caused a depletion of folate levels leading to excess formation of nuclear buds in the affected cells. Regardless of the fact that tembotrione was previously reported negative for mutations and chromosome aberrations in vitro, our findings call for more precaution in its use.     

Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer,in Sprague Dawley rats and Gottingen minipigs

PurposeExtensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer ¹²³I-CMICE-013 to support applications for clinical trials.MethodsSprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 μg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues.ResultsThe acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain.ConclusionThe lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 μg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.     

Investigation of the mechanism of triclosan induced mouse liver tumors

Chronic dietary exposure to Triclosan (TCS) produced increased incidence of liver tumors in mice. The mechanism for liver tumor induction has been attributed to activation of either peroxisome proliferator activated receptor α (PPARα) or constitutive androstane receptor (CAR). To further define the mechanism of TCS induced liver tumors, male CD-1 and C57BL/6 mice were treated with TCS at 0, 10, 100 and 200 mg/kg diet/day for 14 or 28 days. In addition, a recovery group and positive control groups for CAR or PPARα activation with either phenobarbital or diethylhexyl-phthalate were included in the 14-day study. TCS induced a dose-dependent increase in relative liver weight and centrilobular hypertrophy in both strains of mice. Hepatocyte DNA synthesis (BrdU labeling) was also increased in a dose-related pattern. In comparison with previous studies, TCS induced a significant increase in CAR/PXR (Cyp2b10, Cyp3a11) and PPARα (Cyp4a10) responsive genes in both CD-1 and C57BL/6 mice. The corresponding enzyme activity for CAR (7-pentoxyresorufin-O-dealkylase) and PPARα (peroxisomal Acyl-CoA oxidase) were also significantly increased in a similar fashion. Oxidative stress related genes Gpx1 and Aox1 were increased in the C57BL/6 but not in CD-1 mice. The increases in gene expression and enzyme activities returned to control levels after 14-day recovery. The present results demonstrate that both CAR and PPARα activation are involved in the TCS induced mouse liver tumor.     

A review of toxicity studies on graphene-based nanomaterials in laboratory animals

We summarized the findings of toxicity studies on graphene-based nanomaterials (GNMs) in laboratory mammals. The inhalation of graphene (GP) and graphene oxide (GO) induced only minimal pulmonary toxicity. Bolus airway exposure to GP and GO caused acute and subacute pulmonary inflammation. Large-sized GO (L-GO) was more toxic than small-sized GO (S-GO). Intratracheally administered GP passed through the air-blood barrier into the blood and intravenous GO distributed mainly in the lungs, liver, and spleen. S-GO and L-GO mainly accumulated in the liver and lungs, respectively. Limited information showed the potential behavioral, reproductive, and developmental toxicity and genotoxicity of GNMs. There are indications that oxidative stress and inflammation may be involved in the toxicity of GNMs. The surface reactivity, size, and dispersion status of GNMs play an important role in the induction of toxicity and biodistribution of GNMs. Although this review paper provides initial information on the potential toxicity of GNMs, data are still very limited, especially when taking into account the many different types of GNMs and their potential modifications. To fill the data gap, further studies should be performed using laboratory mammals exposed using the route and dose anticipated for human exposure scenarios.     

Flow cytometry evaluation of in vitro cellular necrosis and apoptosis induced by silver nanoparticles

Particles possess unique properties in the nanoscale, e.g., enhanced catalytic activity, high surface area, and light emission/absorption properties, that might result in interference with colorimetric in vitro cytotoxicity assays such as MTT, XTT or MTS. Alternatively, assays that do not use spectrophotometric detection, such as trypan blue exclusion or flow cytometry (FC) based assays, are less likely to be influenced by nanoparticle interference. The aim of this study was to evaluate FC assays to assess the cytotoxicity of three different sizes (10, 100, or 200 nm) of silver nanoparticles (AgNPs) at different mass concentrations (1, 25, or 50 ug/ml) in L-929 fibroblast cells. After 4 h and 24 h exposure, cell necrosis and apoptosis were assessed using 7-AAD and Annexin V dyes, respectively, with FC. The data indicate that cell necrosis and apoptosis in AgNP-exposed fibroblasts depends on dose, exposure time, and AgNP size. The data indicate that AgNPs produced a dose- and time-dependent decrease in cell viability; however, 10 nm AgNPs were significantly more toxic than larger-sized particles. Thus, standard FC assays can be utilized to assess apoptosis and necrosis in response to nanomaterial exposure.     

A novel quinazolinone chalcone derivative induces mitochondrial dependent apoptosis and inhibits PI3K/Akt/mTOR signaling pathway in human colon cancer HCT-116 cells

We have synthesized a novel quinazolinone chalcone derivative (QC) and first time reported its in-vitro and in-vivo anticancer potential. It inhibited the cell proliferation of different cancer cell lines like PC-3, Panc-1, Mia-Paca-2, A549, MCF-7 and HCT-116. It induces apoptosis as measured by several biological endpoints such as apoptotic body formation, evident by Hoechst and scanning electron microscopy, enhanced annexinV-FITC binding of the cells, increased sub-G0 cell fraction, loss of mitochondrial membrane potential (Δψm), reduction of Bcl-2/Bax ratio, activation of caspase-9, caspase-3 and PARP-1 (poly-ADP Ribose polymerase) cleavage in HCT-116 cells. In spite of apoptosis, QC significantly hammers the downstream and upstream signaling cascade of PI3K/Akt/mTOR pathway and cell cycle regulator Skp-2, p21 and p27. Interestingly, QC induces the S and G2/M phase of HCT-116 cells at experimental doses. QC inhibits the tumor growth of Ehrlich ascites carcinoma (EAC), Ehrlich tumor (ET, solid) and sarcoma-180(solid) mice models. Furthermore, it was found to be non-toxic as no animal mortality (0/7) occurred during experimental doses. The present study provides an insight of anticancer potential of QC, which may be useful in managing and treating cancer.     

Alterations of DNA damage response pathway: Biomarker and therapeutic strategy for cancer immunotherapy

Genomic instability remains an enabling feature of cancer and promotes malignant transformation. Alterations of DNA damage response (DDR) pathways allow genomic instability, generate neoantigens, upregulate the expression of programmed death ligand 1 (PD-L1) and interact with signaling such as cyclic GMP–AMP synthase-stimulator of interferon genes (cGAS–STING) signaling. Here, we review the basic knowledge of DDR pathways, mechanisms of genomic instability induced by DDR alterations, impacts of DDR alterations on immune system, and the potential applications of DDR alterations as biomarkers and therapeutic targets in cancer immunotherapy.     

A CYP2B6-humanized mouse model and its potential applications

CYP2B6 is a human microsomal cytochrome P450 enzyme with broad substrate selectivity. CYP2B6 is the only functional member of the human CYP2B gene subfamily, which differs from the situation in rodents, such as mouse, where multiple functional Cyp2b genes are expressed. Recent studies with Cyp2b knockout or knockdown mouse models have yielded insights into the in vivo roles of mouse CYP2B enzymes in drug disposition and xenobiotic toxicity. A CYP2B6-humanized mouse model (CYP2A13/2B6/2F1-transgenic/Cyp2abfgs-null), which expresses human CYP2B6 in the liver, and human CYP2A13 and CYP2F1 in the respiratory tract, but not any of the mouse Cyp2b genes, has also been established. In the CYP2B6-humanized mouse, the CYP2B6 transgene is expressed primarily in the liver, where it was found to be active toward prototype CYP2B6 substrate drugs. The regulatory elements of the CYP2B6 transgene appear to be compatible with mouse nuclear receptors that mediate CYP2B induction. Therefore, the CYP2B6-humanized mouse is a valuable animal model for studying the impact of CYP2B6 expression or induction on drug metabolism, drug efficacy, drug-drug interaction, and drug/xenobiotic toxicity. In this mini-review, we provide a brief background on CYP2B6 and the Cyp2b-knockout and CYP2B6-humanized mice, and discuss the potential applications and limitations of the current models.     

Okadaic acid is taken-up into the cells mediated by human hepatocytes transporter OATP1B3

Okadaic acid is known as a diarrheal shellfish poison. It is thought that there is no specific target organ for okadaic acid after it has been absorbed into the body. However, the details of its pharmacokinetics are still unknown.In this study, we demonstrated that okadaic acid was more toxic to the hepatocyte-specific uptake transporter OATP1B1- or OATP1B3-expressing cells than control vector-transfected cells. In addition, PP2A activity, which is a target molecule of okadaic acid, was more potently inhibited by okadaic acid in OATP1B1- or OATP1B3-expressing cells compared with control vector-transfected cells. The cytotoxicity of okadaic acid in OATP1B1- or OATP1B3-expressing cells was attenuated by known substrates of OATP1B1- and OATP1B3, but not in control vector-transfected cells. Furthermore, after uptake inhibition study using OATP1B3-expressing cells, Dixon plot showed that okadaic acid inhibited the uptake of hepatotoxin microcystin-LR, which is a substrate for OATP1B1 and OATP1B3, in a competitive manner. These results strongly suggested that okadaic acid is a substrate for OATP1B3 and probably for OATP1B1, and could be involved in unknown caused liver failure and liver cancer. Since okadaic acid possesses cytotoxicity and cell proliferative activity by virtue of its known phosphatase inhibition activity.     

Assessing awareness and attitudes of healthcare professionals on the use of biosimilar medicines: A survey of physicians and pharmacists in Ireland

Increasing numbers of biosimilar medicines are becoming available. The objective of this survey was to assess awareness of and attitudes to biosimilars amongst physicians (medical specialists and General Practitioners (GPs)) and community pharmacists in Ireland. Physicians were invited to complete an online questionnaire during April and May 2016. Community pharmacists received a postal questionnaire in August 2015. Responses from 102 medical specialists, 253 GPs and 125 community pharmacists were analysed. The majority of medical specialists (85%) and pharmacists (77%) claimed to be either very familiar or familiar with the term biosimilar, whereas many GPs (60%) were unable to define or had never heard of the term. One in five (21%) healthcare professionals responded that biosimilars were the same as generic medicines. The majority of medical specialists opposed pharmacist-led substitution of biological medicines but some thought it could be appropriate if agreed with the clinician in advance. Medical specialists who prescribe biosimilars (n = 43) were more likely to do so on treatment initiation (67%), than switch a patient from an originator medicine to a biosimilar (28%). The findings will aid the design of educational initiatives for healthcare professionals and highlight attitudes of healthcare professionals to biosimilars, so informing regulators, policy makers and industry.     

A two-year dietary carcinogenicity study of cyadox in Sprague-Dawley rats

To investigate the potential carcinogenicity of cyadox, an antimicrobial agent, four groups of Sprague-Dawley rats (50 rats/sex/group) were fed diets containing cyadox (0, 200, 600 or 2000 mg/kg) for up to two years. There were significant decreases in body weight, feed intake and feed efficiency in both genders during most of the period in the 2000 mg/kg group. Significant decreases in serum ALT were observed in the 2000 mg/kg group at weeks 52, 78 and 104. For the control, 200, 600, and 2000 mg/kg groups, the tumor incidence in females was 33.3%, 37.2%, 40.0% and 19.0%, while it in males it was 18.9%, 2.6%, 17.1% and 13.6%, respectively. At histopathology, no increases in tumor incidence were attributed to treatment with cyadox. The mild swelling and fatty degeneration in hepatocytes, and mild swelling and tubular necrosis in the kidney were observed in 2000 mg/kg group. The no-observed-effect-level (NOEL) for carcinogenicity of cyadox fed to rats was 2000 mg/kg diet (132.18–156.28 mg/kg b.w./day). In conclusion, cyadox was not carcinogenic to rats with the liver and kidney as the target organs, and the side chain may be involved in toxicity and carcinogenicity mediated by QdNOs.     

Rat and poultry feeding studies with soybean meal produced from imidazolinone-tolerant (CV127) soybeans

The safety and nutritional properties of CV127 soybeans were evaluated in rat and broiler feeding studies. Some episodic differences were observed between rats fed CV127, Conquista, and the standard diet for the endpoints examined. None of these differences were considered treatment related, adverse, or biologically meaningful. In general, birds fed diets containing CV127, Conquista, or Monsoy 8001 showed no significant differences in growth and performance response variables. Chickens fed diets containing Coodetec 217 had lower body weight and weight gain for all developmental periods compared to CV127, but no significant differences were found in feed conversion for the two diets during any development period. The results of both feeding studies demonstrate that CV127 soybeans are as safe, wholesome, and nutritionally valuable as the other soybean meals tested, including those varieties for which histories of safe use have been established and well documented.     

Emerging aspects of nanotoxicology in health and disease: From agriculture and food sector to cancer therapeutics

Nanotechnology is an evolving scientific field that has allowed the manufacturing of materials with novel physicochemical and biological properties, offering a wide spectrum of potential applications. Properties of nanoparticles that contribute to their usefulness include their markedly increased surface area in relation to mass, surface reactivity and insolubility, ability to agglomerate or change size in different media and enhanced endurance over conventional-scale substance. Here, we review nanoparticle classification and their emerging applications in several fields; from active food packaging to drug delivery and cancer research. Nanotechnology has exciting therapeutic applications, including novel drug delivery for the treatment of cancer. Additionally, we discuss that exposure to nanostructures incorporated to polymer composites, may result in potential human health risks. Therefore, the knowledge of processes, including absorption, distribution, metabolism and excretion, as well as careful toxicological assessment is critical in order to determine the effects of nanomaterials in humans and other biological systems. Expanding the knowledge of nanoparticle toxicity will facilitate designing of safer nanocomposites and their application in a beneficial manner.     

Predicting points of departure for risk assessment based on in vitro cytotoxicity data and physiologically based kinetic (PBK) modeling: The case of kidney toxicity induced by aristolochic acid I

Aristolochic acids are naturally occurring nephrotoxins. This study aims to investigate whether physiologically based kinetic (PBK) model-based reverse dosimetry could convert in vitro concentration-response curves of aristolochic acid I (AAI) to in vivo dose response-curves for nephrotoxicity in rat, mouse and human. To achieve this extrapolation, PBK models were developed for AAI in these different species. Subsequently, concentration-response curves obtained from in vitro cytotoxicity models were translated to in vivo dose–response curves using PBK model-based reverse dosimetry. From the predicted in vivo dose–response curves, points of departure (PODs) for risk assessment could be derived. The PBK models elucidated species differences in the kinetics of AAI with the overall catalytic efficiency for metabolic conversion of AAI to aristolochic acid Ia (AAIa) being 2-fold higher for rat and 64-fold higher for mouse than human. Results show that the predicted PODs generally fall within the range of PODs derived from the available in vivo studies. This study provides proof of principle for a new method to predict a POD for in vivo nephrotoxicity by integrating in vitro toxicity testing with in silico PBK model-based reverse dosimetry.     

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.     

Absence of subchronic oral toxicity and genotoxicity of rice koji with Aspergillus terreus

Koji products have been considered as an effective fermented food consumed in East Asia with many health benefits. Particularly, rice koji with Aspergillus terreus (RAT) has been reported to be able to prevent hyperlipidemia and hepatic steatosis through regulating cholesterol synthesis. Despite its biological activities, there is a lack of comprehensive information to give an assurance of its safety. Therefore, the objective of this study was to perform a series of toxicological studies (repeated dose oral toxicity and genotoxicity) according to test guidelines published by the Organization for Economic Cooperation and Development. Along with acute toxicity study using rats and beagle dogs, a 13-week toxicity study revealed no clear RAT-related toxic changes, including body weight, mortality, hematology, serum biochemistry, organ weight, and histopathology after oral administration at doses of 500, 1000, and 2000 mg/kg BW. The no-observed-adverse-effect level of RAT was considered to be more than 2000 mg/kg BW/day in rats of both genders. In addition, potential genotoxicity was evaluated using a standard battery of tests (Ames test, chromosome aberration assay, and micronucleus assay) which revealed that RAT showed no genotoxicity. Accordingly, these results suggest that RAT is a safe and non-toxic functional food for human consumption at proper dose.