Toxicological Compounds Research Articles

Unmodified Polystyrene Nanoparticles Induce Inflammatory and Oxidative Stress Responses in Human Lung Epithelial Cells

Polystyrene (PS) is one of the commercially used polymers considered as a pollutant in both aquatic and terrestrial environments. Moreover, PS-NPs are used in nanosensors and nanocarrier for the drug. Thus, it is crucial to determine the potential toxicological effect of PS-NPs to cells. Here we used varying concentrations of unmodified PS-NPs of 60±12 nm in size and analyzed their cyto-toxic effect and stress and toxicity gene expressions in NCI-H292 cells. NCI-H292 cells were ex-posed to minimum of 5 µg/mL and maximum of 50 µg/mL of PS-NPs; 50 µg/mL of PS-NPs showed about 50% cell viability. Moreover, unmodified PS-NPs and induce expressions of stress-related genes, such as those encoding interleukin 6 (IL 6), colony-stimulating factor 2 (CSF 2), C-X-C motif chemokine 10 (CXCL 10) responsible for inflammation and heme oxygenase 1 (HMOX 1) responsible for oxidative stress in NCI-H292 cell lines. Our findings suggest that PS-NPs are considered as a potential toxicological compound to the human cell line.

In Silico Taste–Toxicity Study of New Hypothetical Hetero-Abiraterone Derivatives

First Iraqi attempt to study drug used for prostate cancer treatment (Abiraterone) that hypothetically reacted with known chemicals classified as Anticancer drug: Tirapazamine (T) and 5-Fluorouracil (F), food additive and preservative: Butylated Hydroxy Toluene (B) and Ethoxyquin (E), and sweeteners: P-4000 (P), Sodium Cyclamate (CS), Alitame (AT), and Saccharin (SA). The second step in this work was computational study of all reactants and the formed products having newly ether, amine, and carboxylic acid ester, and sulphonate bonds by online websites. Taste, toxicity, and ADMET were calculated by three online websites related to Charite University of Medicine, Institute for Physiology, Germany and University of Melbourne, Australia. SMILES of the reactants were obtained from National Library of Medicine/ National Center for Biotechnology Information websites while products were drawn by the molecular editor CS ChemDraw Ultra and rechecked by MarvinSketch program. This in Silico study showed various results of the formed products compared to Abiraterone (A) that predicated it as sour chemical belongs to Class 4 as a harmful substance if swallowed. Abiraterone (A) toxicity on liver organ was 61% probability percentage as hepatotoxicity while carcinogenicity, Immunotoxicity, Mutagenicity, cytotoxicity, AhR, AR, Aromatase, ER, HSE, and p53 were more than 70 % to bind Progesterone or Androgen. Also, Abiraterone (A) has a poor water solubility leading to high intestinal absorption, moderate total clearance, and giving inhibition reaction to Cytochrome P450 type CYP2C19, hERG II, and Ames test. These results confirmed that Abiraterone is structurally less harmful acute class with highly chance to interact with cell components resulting lethal response. All Abiraterone hypothetical products had a harmful reaction if swelled (Class 4), sour taste. All toxicological characters may be highly affected by its water solubility and intestinal absorption towards CNS, BBB, and CaCO2 permeability, skin sensation, and Ames test issues. For example, this in Silico- QSAR foundations about Abiraterone – Saccharin (A-SA) suggest that A-SA is structurally safe and there are several possibilities of becoming an active–multiple toxicological compound.

A Genome-Edited ERα-HiBiT Fusion Reporter Cell Line for the Identification of ERα Modulators Via High-Throughput Screening and CETSA.

The estrogen receptor α (ERα) is a target of intense pharmacological intervention and toxicological biomonitoring. Current methods to directly quantify cellular levels of ERα involve antibody-based assays, which are labor-intensive and of limited throughput. In this study, we generated a post-translational reporter cell line, referred to as MCF7-ERα-HiBiT, by fusing a small pro-luminescent nanoluciferase (NLuc) tag (HiBiT) to the C-terminus of endogenous ERα in MCF7 cells. The tag allows the luminescent detection and quantification of endogenous ERα protein by addition of the complementary NLuc enzyme fragment. This MCF7-ERα-HiBiT cell line was optimized for quantitative high-throughput screening (qHTS) to identify compounds that reduce ERα levels. In addition, the same cell line was optimized for a qHTS cellular thermal shift assay to identify compounds that bind and thermally stabilize ERα. Here, we interrogated the MCF7-ERα-HiBiT assay against the NCATS Pharmacological Collection (NPC) of 2,678 approved drugs and identified compounds that potently reduce and thermally stabilize ERα. Our novel post-translational reporter cell line provides a unique opportunity for profiling large pharmacological and toxicological compound libraries for their effect on ERα levels as well as for assessing direct compound binding to the receptor, thus facilitating mechanistic studies by which compounds exert their biological effects on ERα.

Using a micro-physiological system to prolong the preservation of ex vivo lung tissue

Abstract Current in vitro and in vivo disease models have been reported to lack sufficient translation to human. Precision-Cut Lung Slices (PCLS) are viable sections of lung tissue and have been described to be a translational model for the ex vivo assessment of pharmacological and toxicological compounds. In most studies PCLS were cultured under static conditions. These lung sections, however, suffer from the limited viability. Here we present a novel modular microphysiological system (MPS) to prolong the cultivation of ex vivo lung tissue. A tailored MPS setup was designed using the PDMS free modular plug&play MPS construction kit. PCLS from mice were cultivated for up to one week under static versus perfused conditions. Using the MPS technology enabled a prolonged culture period with improved viability as shown by lowered lactate dehydrogenase release and improved membrane integrity. Using this technology might allow us to use PCLS for longer culture periods such as e.g. repeated dose toxicity or pharmacology studies.

Assessment of physicochemical properties of water and their seasonal variation in an urban river in Bangladesh

To assess the magnitude of water quality decline in the Turag River of Bangladesh, this study examined the seasonal variation of physicochemical parameters of water, identified potential pollution sources, and clustered the monitoring months with similar characteristics. Water samples were collected in four distinct seasons to evaluate temperature, pH, dissolved oxygen (DO) concentration, five-day biochemical oxygen demand (BOD5), chemical oxygen demand (COD), electrical conductivity (EC), chloride ion (Cl−) concentration, total alkalinity (TA), turbidity, total dissolved solids (TDS) concentration, total suspended solids (TSS) concentration, and total hardness (TH) using standard methods. The analytical results revealed that 40% of water quality indices were within the permissible limits suggested by different agencies, with the exception of EC, Cl− concentration, TA, turbidity, DO concentration, BOD5, and COD in all seasons. Statistical analyses indicated that 52% of the contrasts were significantly different at a 95% confidence interval. The factor analysis presented the best fit among the parameters, with four factors explaining 94.29% of the total variance. TDS, BOD5, COD, EC, turbidity, DO, and Cl− were mainly responsible for pollution loading and were caused by the significant amount of industrial discharge and toxicological compounds. The cluster analysis showed the seasonal change in surface water quality, which is usually an indicator of pollution from rainfall or other sources. However, the values of different physicochemical properties varied with seasons, and the highest values of pollutants were recorded in the winter. With the change in seasonal temperature and increase in rainfall, the seasonal Turag River water followed a self-refining trend as follows: rainy season > pre-winter > summer > winter.

Effect of Zolpidem Tartrate on the Developmental Rate of Forensically Important Flies Chrysomya megacephala (Diptera: Calliphoridae) and Chrysomya saffranea.

Entomotoxicology is an emerging science in the forensic investigation of death; it now influences judicial activities. Briefly, carrion flies ingest various chemicals as they feed on a cadaver. In entomotoxicology, such toxicological compounds are qualitatively and quantitatively analyzed in insects recovered from carrion. Some of these chemicals can disrupt flies' cellular physiology, leading to acceleration or delay in their developmental rate. In this study, the effect of zolpidem-contaminated tissue on the dipteran flies Chrysomya megacephala (Fabricius 1794) and Chrysomya saffranea (Bigot 1877) was assessed. Zolpidem tartrate is a sedative-hypnotic compound commonly used to treat anxiety and sleeping disorders, but it is associated with death from suicide. The effects were tested by providing the flies with liver tissue treated with various concentrations of zolpidem tartrate. The morphological parameters and development rate for all life cycle stages of treated insects were determined and used to estimate postmortem intervals. Results showed that the morphological parameters, i.e., weight, width, and length and rate of development of C. megacephala and C. saffranea was negatively associated with zolpidem tartrate concentration of C. saffranea, were negatively associated with the concentration of zolpidem tartrate, indicating that arthropod evidence evaluation can help establish the cause and time of death.

Analysis, formation and inhibition of polycyclic aromatic hydrocarbons in foods: An overview

Polycyclic aromatic hydrocarbons (PAHs), formed through incomplete combustion or pyrolysis of wood or gasoline, represents an important class of toxicological compounds because of its wide distribution in the environment and possible contamination of foods. This paper deals with an overview of analysis, formation and inhibition of PAHs in foods. Extraction of PAHs from food is routinely conducted by saponification of lipid with Soxhlet method, followed by purification with a Sep-Pak Florisil cartridge and partition. This method can remove more impurities than the sonication method. With HPLC, all 16 PAHs can be simultaneously separated by a gradient solvent system and detected by UV at 254 nm or fluorescence employing programmable wavelength with seven settings of excitation/emission. With GC, all 16 PAHs can also be simultaneously separated by a temperature programming method and detected by flame ionization detector (FID) or ion-trap mass detector (ITD). Although HPLC can provide adequate separation of 16 PAHs, nevertheless, the presence of impurities in food sample can interfere with the subsequent identification and separation. For GC, a number of isomeric PAHs partially overlap. Nonetheless, the application of GC-ITD can readily identify the various PAHs in foods through reconstructed ion chromatograms even in the presence of fat- or PAH-like impurities. Charcoal-grilling and smoking are the two major processing methods which can result in the formation of high amount of total carcinogenic PAHs. No carcinogenic PAHs are detected in steaming and liquid smoke flavoring. The amount of PAHs in food samples can also be greatly reduced by sorption into low-density polyethylene bags. Further research in necessary to determine how the formation of PAHs during processing can be directly retarded or eliminated.

Analysis, Formation and Inhibition of Heterocyclic Amines in Foods: An Overview

Heterocyclic amines (HAs), an important class of toxicological compounds, can be formed through heating of four precursors, amino acids, creatinine, creatine and sugars. Numerous studies have shown that most HAs are mutagenic and carcinogenic, and the safety of HAs-containing foods has become a major concern for the public. This paper deals with an overview of analysis, formation and inhibition of HAs in foods. Analysis of HAs from foods is routinely conducted by protein precipitation, followed by acid-base solvent partition, purification with column chromatography and identification and quantification by high-performance liquid chromatography (HPLC), or gas chromatography-mass spectrometry (GC-MS). However, most of these methods result in low recovery. With HPLC, all 16 HAs can be simultaneously separated by a binary or ternary gradient solvent system and detected by UV. Using fluorescence detection, only nine HAs can be detected. For GC-MS, only a number of HAs are separated and identified by employing a temperature programming method. GC-MS provides rapid identification and quantification of HAs in foods, however, this technique requires a time-consuming derivatization and the number of HAs separation is limited. Also, some HAs may degrade and result in peak tailing. The application of HPLC with photodiode-array detection (HPLC-DAD) can not only permit rapid identification and quantification of HAs, but also enhance sensitivity and selectivity. Broiling and frying are two major processing methods causing the formation of high amount of HAs. The formation of HAs in foods during cooking can be inhibited by incorporating additives such as antioxidants and amino acids. However, the results have been controversial. Further research is necessary to study the effect of various additives on inhibition of HAs formation in foods.

Demography of avian scavengers after Pleistocene megafaunal extinction

The late Quaternary megafauna extinctions reshaped species assemblages, yet we know little about how extant obligate scavengers responded to this abrupt ecological change. To explore whether obligate scavengers persisted by depending on contemporary community linkages or via foraging flexibility, we tested the importance of the trophic interaction between pumas (Puma concolor) and native camelids (Vicugna vicugna and Lama guanicoe) for the persistence of Andean condors (Vultur gryphus) in southern South America, and compared the demographic history of three vultures in different continents. We sequenced and compiled mtDNA to reconstruct past population dynamics. Our results suggest that Andean condors increased in population size >10 KYA, whereas vicuñas and pumas showed stable populations and guanacos a recent (<10 KYA) demographic expansion, suggesting independent trajectories between species. Further, vultures showed positive demographic trends: white-backed vultures (Gyps africanus) increased in population size, matching attenuated community changes in Africa, and California condors (Gymnogyps californianus) exhibited a steep demographic expansion ~20 KYA largely concurrent with North American megafaunal extinctions. Our results suggest that dietary plasticity of extant vulture lineages allowed them to thrive despite historical environmental changes. This dietary flexibility, however, is now detrimental as it enhances risk to toxicological compounds harbored by modern carrion resources.

Biostatistical Assessment of Mutagenicity Studies: A Stepwise Confidence Procedure

The paper addresses the issue of identifying the maximum safe dose in the context of noninferiority trials where several doses of toxicological compounds exist. Statistical methodology for identifying the maximum safe dose is available for three-arm noninferiority designs with only one experimental drug treatment. Extension of this methodology for several experimental groups exists but with multiplicity adjustment. However, if the experimental or the treatment groups can be ordered a priori according to their treatment effect, then multiplicity adjustment is unneeded. Assuming homogeneity of variances across dose group in normality settings, we employed the generalized Fieller’s confidence interval method in a multiple comparison stepwise procedure by incorporating the partitioning principle in order to control the familywise error rate (FWER). Simulation results revealed that the procedure properly controlled the FWER in strong sense. Also, the power of our procedure increases with increasing sample size and the ratio of mean differences. We illustrate our procedure with mutagenicity dataset from a clinical study.

P-34 - Kinetics and biological consequences of quinone-induced protein adduction

Quinones are a major class of toxicological compounds, that can both generate radicals, and act as Michael acceptors with nucleophiles including Cys residues on proteins. This study aimed to quantitatively assess quinone-protein reactions and examine the relationship between quinone adduction and protein dysfunction. A range of quinones were incubated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), creatine kinase (CK), papain, bovine serum albumin (BSA), and human serum albumin (HSA), with both the kinetics of adduction and effects on protein structure and activity determined. Adduction rate constants at Cys residues, which were dependent on the quinone and protein structures, and thiol pKa, were in the range 103 -105 M-1s-1. p-Benzoquinone (BQ) induced dimerization of GAPDH and CK (but not BSA, HSA, or papain) in a dose- and time-dependent manner, as well as a loss of enzyme activity. Glutathione (GSH) reacts competitively with BQ, and can thereby reverse the activity loss and dimerization of GAPDH and CK. Mass spectrometry, after digestion to peptide, showed that BQ preferentially forms adducts with GAPDH at Cys149 and Cys244. These data suggest that quinones can induce toxicity by rapidly and selectively forming adducts with Cys residues in proteins.

Antioxidant Effect of Clorella vulgaris on Wistar Rat Kidney Induced by CCl4: A Histopathological Review

Kidney is very susceptible to damage by toxicological compounds such as carbon tetrachloride (CCl­4). CCl4 produce free radicals, which cause lipid peroxidation and kidney damage and free radical release, which can be prevented by the administration of exogenous antioxidants, such as Chlorella vulgaris.The aim of study was to determine an effect of antioxidant of C. vulgaris on the histopathological features of Wistar rat kidney which is induced by CCl4. Experimental study with completely randomized design. The variable was histopathology features of the kidneys. The doses of C. vulgaris extract were 3 mg, 4 mg, and 5 mg per 100 grams of rat body weight (BW). The administration of C. vulgaris extract was performed within 30 days, while the CCl4 induction (0.25 ml / 100 g BW) was administered orally on the day 9, 12, 16, 19, 23, and 26. Parameters were histopathology features of renal damage, proportion of tubular cell damage, and Bowman's space diameter. The results showed the administration of C. vulgaris extract was able to reduce the impact of damage caused by CCl4 (p&lt;0.05). This was supported by histologic observations, which was showing a decrease of picnotic and vacuolated cells, normal brush border, and decrease of Bowman's space. In conclusion, 5 mg / 100 g BW of C. vulgaris extract can effectively protect the kidney from damage caused by CCl4. The results of this study strongly support further research on immunostimulant content test C. vulgaris and determine the efficient dose for representative animals mammals also in humans.

An automated medium scale prototype for anaerobic co-digestion of olive mill wastewater

Olive oil production constitutes one of the most important agro-industrial business for Mediterranean countries, where 97% of the international production is focused. Such an activity, mainly carried out through three phase olive oil mill plants, generates huge amounts of solid and liquid by-products further than olive oil. Physico-chemical features of these by-products depend on various factors such as soil and climatic conditions, agricultural practices and processing. As currently carried out, the disposal of these by-products may lead to numerous problems taking into account management, economic and particularly environmental aspects. Indeed, olive mill wastewater is not easily biodegradable due to its high chemical and biochemical oxygen demand, its high content in phenolic compounds, high ratio C/N and low pH, leading consequently to soil and water source pollution. Considering, the above-mentioned statements, olive mill waste disposal constitutes nowadays a challenge for oil industry stakeholders. It becomes necessary to look for alternative solutions in order to overcome environmental problems and ensure the sustainability of oil industry. Anaerobic co-digestion of olive mill wastewater with other agro-industrial matrices could be one of these solutions; since it offers the possibility to produce green energy and break down toxicological compounds contained in these wastewater for a better disposal of the digested matrices as soil conditioner. In this contest, this note reports the functioning principle of an automated medium scale plant for anaerobic co-digestion of olive mill wastewater.

Efficient forensic toxicological screening and quantitation workflow using QTOF LC–MS/MS system

Quadrupole time-of-flight (QTOF) mass spectrometry is becoming the desired technology for sensitive and selective screening workflows in a forensic toxicological setting. The technology overcomes many challenges faced when using traditional techniques and more significantly captures all information about the sample in one injection to allow for retrospectively mining the data. Here we describe a new benchtop QTOF system with revolutionary N-optic designed flight path and new, intuitive software for easy adoption of accurate mass technology to forensic testing. Blank human urine was spiked with more than 50 common drugs found in forensics setting at different concentration levels. HPLC separation was performed using the SCIEX ExionLC AC system on a reverse-phase column. Data was collected on a SCIEX X500R QTOF mass spectrometer with SCIEX OS software: TOF – MS survey scan with Information Dependent Acquisition (IDA) – triggering of up to 16 product ion scans; or SWATH® acquisition. For SWATH® acquisition, the precursor isolation window width was varied for each MS/MS experiment. Data processing was also performed in SCIEX OS software with simultaneous identification and quantification being accomplished in single software. Screening: multiple confidence criteria including mass accuracy, retention time, isotope, and most importantly, library matching were used. The availability of MS/MS information for identification was required in this study through library searching the acquired spectra against a SCIEX high resolution forensic library containing MS/MS spectra of 1700 compounds. Excellent true positive rates were accomplished for both acquisition modes (89% with IDA and 98% with SWATH® acquisition). This result is consistent with the expectation that the IDA-MS/MS data acquisition has the risk of occasionally missing acquiring MS/MS. The acquisition of MS/MS data in the screening approach allowed the confident identification of structural isomers that have similar retention times through unique fragment ions and their ratios. Simultaneous Quantification Linearity on the new QTOF system for the majority of compounds tested was four orders linear dynamic range covering 0.1 to 1000 ng/mL. SWATH® acquisition ensures that all data, including MS/MS, is acquired at all times. Due to the fast scanning speed (up to 100 Hz at 35 K resolution) in MS/MS mode, a fast cycletime (0.5 sec) was used for the 6.5-min LC method that allowed for one TOF-MS scan and 16 MS/MS scans that covered 120 to 500 m/z range. A sensitive and selective workflow was developed for toxicological compound screening in a forensic setting using the new X500R QTOF system. The newly designed software that accompanies the new benchtop QTOF instrument allows for simultaneous compound identification and quantification in one step all in one place. SCIEX OS software allows the easy adoption of accurate mass technology for routine forensic workflows.

Chronic fluoxetine treatment induces anxiolytic responses and altered social behaviors in medaka, Oryzias latipes

Medaka (Oryzias latipes) is a small freshwater teleost that is an emerging model system for neurobehavioral research and toxicological testing. The selective serotonin reuptake inhibitor class of antidepressants such as fluoxetine is one of the widely prescribed drugs, but little is known about the effects of these drugs on medaka behaviors. To assess the behavioral effects of fluoxetine, we chronically administrated fluoxetine to medaka adult fish and analyzed the anxiety-related and social behaviors using five behavioral paradigms (diving, open-field, light–dark transition, mirror-biting, and social interaction) with an automated behavioral testing system. Fish chronically treated with fluoxetine exhibited anxiolytic responses such as an overall increased time spent in the top area in the diving test and an increased time spent in center area in the open-field test. Analysis of socially evoked behavior showed that chronic fluoxetine administration decreased the number of mirror biting times in the mirror-biting test and increased latency to first contact in the social interaction test. Additionally, chronic fluoxetine administration reduced the horizontal locomotor activity in the open-field test but not the vertical activity in the diving test. These investigations are mostly consistent with previous reports in the other teleost species and rodent models. These results indicate that behavioral assessment in medaka adult fish will become useful for screening of effects of pharmaceutical and toxicological compounds in animal behaviors.

A new software tool for confident identification and quantitation of drugs in urine and serum

Aim High-resolution time-of-flight mass spectrometry has become an excellent tool in forensic toxicology. The accurate mass based inherent characteristics like sensitive wide scope screening together with retrospective and general unknown analysis capabilities make it an ideal tool for this work. We describe the development and evaluation of a new software tool allowing screening and quantification using ultra-high-resolution LC-Q-ToF, accurate mass analysis. The new software allows applying the diagnostic ion concept, using enhanced confirmation criteria such as isotopes, adducts, fragments and qualifier ratios for reducing false positive detection rates. Methods Sixty-one compounds covering several compound classes based on their relevance in post-mortem and routine drug screening were used in this study. After acetonitrile precipitation, urine and serum samples were spiked with the toxicological compound mixes at four levels (between 10–500 ng/mL) and analyzed in ESI(+) mode by LC-Q-ToF-MS (Impact II, Bruker Daltonics, USA), in full scan and bbCID modes using a 14 minute gradient, reverse phase UHPLC separation. The data acquired in bbCID mode were processed with the new software package including a database with more than 2000 compounds of forensic relevance. Results In the full scan ToF-MS channel, all compounds were detected at all concentration levels, no false negatives were encountered. In urine, the high matrix load produced a high number of false positives (547 compared to the 276 expected positives). Only seven compounds were causing ∼ 75% of all -false positives. These false positives typically arose due to a very low detection threshold. In total 35 different compounds appeared as false positives. After applying the enhanced diagnostic ion detection criteria the false positives were completely removed. A challenge was tramadol in the presence of o-desmethylvenlafaxine as these compounds have the same retention time, same sum-formula and the same main fragment ion. However, two smaller characteristic fragments allowed the unambiguous identification of o-desmethylvenlafaxine. Conclusion The screening method with the new software is robust and has been shown to work reproducibly for forensic toxicological screening of blood and urine samples. The application of the ‘diagnostic ion’ concept has proven to be a very powerful tool to eliminate false positive findings.

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.

Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data.

Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2 . A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research.

The isolation and characterization of a telomerase immortalized goat trophoblast cell line

Trophoblast cells play vital roles in the processes of embryonic implantation and placentation. Many toxicological compounds can induce the malfunction of trophoblast cells, resulting in implantation failure or early embryonic loss. The finite lifespan of primary trophoblast cells limits investigation of the long-term effects of some toxicological compounds on trophoblast cells in vitro. In this study, primary goat trophoblast cells were purified by density gradient centrifugation and specific immuno-affinity purification. Then, the purified cells were immortalized through transfection of a plasmid containing the human telomerase reverse transcriptase (hTERT) gene. hTERT-transfected goat trophoblast cells (hTERT-GTCs) could steadily express hTERT gene and exhibit higher telomerase activity, and persistently proliferate without any signs of senescence up to 50 passages. The immortalized goat trophoblast cells still possessed the basic and key properties of normal primary goat trophoblast cells to express the specific intracellular marker cytokeratin 7 (CK-7) and secrete chorionic gonadotrophin β-subunit (CG-β) and placental lactogen (PL). Further studies showed that the immortalized goat trophoblast cells expressed vimentin and non-classical MHC class I antigen and exhibited invasive phenotype, suggesting that the immortalized goat trophoblasts resembled human extravillous trophoblasts. In addition, this cell line did not show neoplastic transformation either in vivo or in vitro. We concluded the immortalized goat trophoblast cells by hTERT transfection retained the basic and key characteristics of primary trophoblast cells and may provide a useful model to study the effects of some toxicological compounds on trophoblast cells.

A comprehensive profiling of toxicological semivolatile compounds using GC-MS

A comprehensive profiling of toxicological semivolatile compounds using GC-MS