Comment on the: Molecular mechanism of CAT and SOD activity change under MPA-CdTe quantum dots induced oxidative stress in the mouse primary hepatocytes (Spectrochim Acta A Mol Biomol Spectrosc. 2019 Sep 5; 220:117104).

The paper by the authors Hau and Liu (Spectrochim Acta A Mol Biomol Spectrosc. 2019 Sep 5;220:117104) showed the effects of mercaptopropionic acid- CdTe quantum dots to the antioxidant enzymes catalase and superoxide dismutase molecules and then demonstrates the subsequent quantum dots toxic effects at a cellular level, and they proposed a mechanism of QD induced apoptosis and cell death involving oxidative stress, revealing their potential risk in the biomedical applications. QD concentrations were not determined according to the Cd concentrations in the QD that could be measured via ICP-MS. In conclusion, since cell viability above 80% as non-toxic based on ISO 10993-5, CdTe QDs cannot be considered as toxic. Also, according to the literature only CAT and SOD enzyme activities are not enough to claim oxidative stress formation.

Conformational and functional effects of MPA-CdTe quantum dots on SOD: Evaluating the mechanism of oxidative stress induced by quantum dots in the mouse nephrocytes.

The application of quantum dots (QDs) is restricted by the biosafety issue. QDs contribute to the adverse effects of organisms probably because of the ability to induce oxidative stress via changing the activity of antioxidant enzyme, for example, superoxide dismutase (SOD). But the underlying molecular mechanisms still remain unclear. This study investigated the harmful effects of oxidative stress induced by mercaptopropionic acid capped CdTe QDs (MPA-CdTe QDs) on the mouse primary nephrocytes as well as the structure and function of SOD molecule and explored the underlying molecular mechanism. After 24-hour MPA-CdTe QD exposure, the activation level of extracellular regulated protein kinase (ERK) signaling pathway and cysteinyl-directed aspartate-specific proteases (Caspases) significantly increased, which led to the increasing level of reactive oxygen species (ROS) and cell apoptosis; the group pretreated with ROS scavenger N-acetyl-L-cysteine (NAC) significantly reduced the apoptotic cell percentage, indicating that ROS played a critical role in QD-induced cytotoxicity. Further molecular experiments showed that the interacting processes between the MPA-CdTe QDs and SOD were spontaneous which changed the conformation, secondary structure of SOD. The interaction significantly resulted in the tightening of polypeptide chains and the shrinkage of SOD, leading to the inhibition of molecular SOD activity. This study demonstrates the adverse effects of QDs, revealing their potential risk in biomedical applications.

Environmental hazard and risk assessment of thiochemicals. Application of integrated testing and intelligent assessment strategies (ITS) to fulfil the REACH requirements for aquatic toxicity.

REACH requires information on hazardous properties of substances to be generated avoiding animal testing where possible. It is the objective of the present case study with thiochemicals to extract as much information as possible from available experimental data with fish, daphnia and algae and to fill data gaps for analogues to be registered under REACH in 2018. Based on considerations of chemical similarity and common mode of action (MOA) the data gaps regarding the aquatic toxicity of the thiochemicals were largely closed by trend analysis ("category approach") and read-across within the same group, for example, thioglycolates or mercaptopropionates. Among 16 thiochemicals to be registered by 2018 there are only 2 substances with sufficient data. 36 data gaps for 14 thiochemicals were identified. Most of the required data (>60%) could be estimated by in silico methods. Only 14 tests (6 algae, 6 daphnia, 1 limit fish test and 1 acute fish test) were proposed. When the results of these tests are available it has to be discussed whether 2 further fish (limit) tests are required. For two substances (exposure-based) waiving was suggested. The relatively high toxicity of the thiochemicals is manifested in low predicted no-effect concentrations (PNECs). Only preliminary predicted environmental concentrations (PECs) could be derived for the thiochemicals for which a risk assessment has to be performed (production rate >10 t/y). The preliminary PEC/PNEC ratios indicate no risk for the aquatic compartment at the production site. PECs due to down-stream use must not exceed the estimated PNECs.

MPA-modified CdTe quantum dots increased interleukin-1beta secretion through MyD88-dependent Toll-like receptor pathway and NLRP3 inflammasome activation in microglia.

The excellent optical properties of CdTe quantum dots (QDs) make researchers realize their value on the application of biomedicine, especially neuroscience, as an advanced fluorescent probe. Thus, it is important to evaluate the biosafety of CdTe QDs on the central nervous system (CNS). Our previous studies have conducted a systematic biosafety evaluation of CdTe QDs on the CNS and found several toxic endpoints, one of which is the inflammation on the rat hippocampus, but their underlying mechanism remains unclear. In this study, when BV2 microglial cells were exposed to CdTe QDs with doses <20 nM, there was no obviously adverse effect. However, 40 nM CdTe QDs exposure could significantly activate the BV2 cells and increase the pro-inflammatory cytokine IL-1ß secretion. Molecular biology analyses suggested that both TLR2/MyD88/NF-κB pathway and NLRP3 inflammasome participated in the CdTe QD-induced IL-1ß secretion. The former served as the first signal for pro-IL-1ß expression, while the later played a role on the maturation of pro-IL-1ß into IL-1ß. The results, taken together, demonstrated that MPA-modified CdTe QDs exposure with a high concentration was capable of activating microglial cells and promoting IL-1ß secretion, which was highly correlated with the activations of both TLR2/MyD88/NF-κB pathway and ROS-induced NLRP3 inflammasome. These findings provide some mechanistic insights regarding the neuroinflammatory responses to cadmium-based QDs.

Transcriptome analysis of different sizes of 3-mercaptopropionic acid-modified cadmium telluride quantum dot-induced toxic effects reveals immune response in rat hippocampus.

Recently, the increasing number of bio-safety assessments on cadmium-containing quantum dots (QDs) suggested that they could lead to detrimental effects on the central nervous system (CNS) of living organisms, but the underlying action mechanisms are still rarely reported. In this study, whole-transcriptome sequencing was performed to analyze the changes in genome-wide gene expression pattern of rat hippocampus after treatments of cadmium telluride (CdTe) QDs with two sizes to understand better the mechanisms of CdTe QDs causing toxic effects in the CNS. We identified 2095 differentially expressed genes (DEGs). Fifty-five DEGs were between the control and 2.2 nm CdTe QDs, 1180 were between the control and 3.5 nm CdTe QDs and 860 were between the two kinds of CdTe QDs. It seemed that the 3.5 nm CdTe QD exposure might elicit severe effects in the rat hippocampus than 2.2 nm CdTe QDs at the transcriptome level. After bioinformatics analysis, we found that most DEG-enriched Gene Ontology subcategories and Kyoto Encyclopedia of Genes and Genomes pathways were related with the immune system process. For example, the Gene Ontology subcategories included immune response, inflammatory response and T-cell proliferation; Kyoto Encyclopedia of Genes and Genomes pathways included NOD/Toll-like receptor signaling pathway, nuclear factor-κB signaling pathway, tumor necrosis factor signaling pathway, natural killer cell-mediated cytotoxicity and T/B-cell receptor signaling pathway. The traditional toxicological examinations confirmed the systemic immune response and CNS inflammation in rats exposed to CdTe QDs. This transcriptome analysis not only revealed the probably molecular mechanisms of CdTe QDs causing neurotoxicity, but also provided references for the further related studies.

Impairments of spatial learning and memory following intrahippocampal injection in rats of 3-mercaptopropionic acid-modified CdTe quantum dots and molecular mechanisms.

With the rapid development of nanotechnology, quantum dots (QDs) as advanced nanotechnology products have been widely used in neuroscience, including basic neurological studies and diagnosis or therapy for neurological disorders, due to their superior optical properties. In recent years, there has been intense concern regarding the toxicity of QDs, with a growing number of studies. However, knowledge of neurotoxic consequences of QDs applied in living organisms is lagging behind their development, even if several studies have attempted to evaluate the toxicity of QDs on neural cells. The aim of this study was to evaluate the adverse effects of intrahippocampal injection in rats of 3-mercaptopropionic acid (MPA)-modified CdTe QDs and underlying mechanisms. First of all, we observed impairments in learning efficiency and spatial memory in the MPA-modified CdTe QD-treated rats by using open-field and Y-maze tests, which could be attributed to pathological changes and disruption of ultrastructure of neurons and synapses in the hippocampus. In order to find the mechanisms causing these effects, transcriptome sequencing (RNA-seq), an advanced technology, was used to gain the potentially molecular targets of MPA-modified CdTe QDs. According to ample data from RNA-seq, we chose the signaling pathways of PI3K-Akt and MPAK-ERK to do a thorough investigation, because they play important roles in synaptic plasticity, long-term potentiation, and spatial memory. The data demonstrated that phosphorylated Akt (p-Akt), p-ERK1/2, and c-FOS signal transductions in the hippocampus of rats were involved in the mechanism underlying spatial learning and memory impairments caused by 3.5 nm MPA-modified CdTe QDs.

Aqueous Synthesis of PEGylated Quantum Dots with Increased Colloidal Stability and Reduced Cytotoxicity.

Ligands used on the surface of colloidal nanoparticles (NPs) have a significant impact on physiochemical properties of NPs and their interaction in biological environments. In this study, we report a one-pot aqueous synthesis of 3-mercaptopropionic acid (MPA)-functionalized CdTe/CdS/ZnS quantum dots (Qdots) in the presence of thiol-terminated methoxy polyethylene glycol (mPEG) molecules as a surface coordinating ligand. The resulting mPEG-Qdots were characterized by using ζ potential, FTIR, thermogravimetric (TG) analysis, and microscale thermophoresis (MST) studies. We investigated the effect of mPEG molecules and their grafting density on the Qdots photophysical properties, colloidal stability, protein binding affinity, and in vitro cellular toxicity. Moreover, cellular binding features of the resulting Qdots were examined by using three-dimensional (3D) tumor-like spheroids, and the results were discussed in detail. Promisingly, mPEG ligands were found to increase colloidal stability of Qdots, reduce adsorption of proteins to the Qdot surface, and mitigate Qdot-induced side effects to a great extent. Flow cytometry and confocal microscopy studies revealed that PEGylated Qdots exhibited distinctive cellular interactions with respect to their mPEG grafting density. As a result, mPEG molecules demonstrated a minimal effect on the ZnS shell deposition and the Qdot fluorescence efficiency at a low mPEG density, whereas they showed pronounced effect on Qdot colloidal stability, protein binding affinity, cytotoxicity, and nonspecific binding at a higher mPEG grafting amount.

Phototoxicity of CdSe/ZnSe quantum dots with surface coatings of 3-mercaptopropionic acid or tri-n-octylphosphine oxide/gum arabic in Daphnia magna under environmentally relevant UV-B light.

The potential ecotoxicological consequences about semiconductor crystal nanoparticles (NPs) are a growing concern. However, our understanding of the mechanism of toxicity in NPs is very limited, especially under varying environmental conditions such as ultraviolet (UV) light. We performed an in vivo study employing Daphnia magna to evaluate the mechanism involved in toxicity of cadmium selenide/zinc selenide quantum dots (QDs) with two different organic coatings under an environmental level of UV-B light. We used QDs with mercaptopropionic acid (MPA) and tri-n-octylphosphine oxide/gum arabic (GA) and measured their toxicities under an environmental level of UV-B light. Whole-body reactive oxygen species (ROS) generation and mRNA expression level biomarkers, as well as acute toxicity, were measured in D. magna. With UV-B light, both cadmium (Cd) and GA-QD became more toxic in daphnids. The levels of small Cd molecules (<10kDa cutoff) increased for GA-QD under UV-B; however, the observed acute lethal toxicity could not be explained by the measured Cd level. Under UV-B light, both Cd and GA-QD generated more ROS. In addition, the expression pattern of mRNAs specific to Cd exposure was not observed from GA-QD with or without UV-B light. These observations suggest that the phototoxicity of QDs may be explained not only by Cd release from the QD core but also by stability of surface coating characteristics and other potential causes such as ROS generation.

Acute toxicity of two CdSe/ZnSe quantum dots with different surface coating in Daphnia magna under various light conditions.

With an increasing use of quantum dots (QDs) in many applications, their potential hazard is of growing concern. However, little is known about their ecotoxicity, especially in vivo. In the present study, we employed freshwater macroinvertebrate, Daphnia magna, to evaluate toxicity characteristics of cadmium selenide/zinc selenide (CdSe/ZnSe) in relation to surface coatings, e.g., mercaptopropionic acid QD ((MPA)QD), and gum arabic/tri-n-octylphosphine oxide QD ((GA/TOPO)QD), and light conditions, i.e., dark, fluorescent light, environmental level of ultraviolet (UV) light, and sunlight. The results of the present study showed that D. magna was more susceptible to (GA/TOPO)QD exposure compared to (MPA)QD. The surface coating of QD appeared to determine the stability of QDs and hence the toxicity, potentially by size change of or the release of toxic components from QDs. However, (GA/TOPO)QD was still less toxic than the equivalent level of CdCl2. The toxicity of all the tested compounds increased by changing the light condition from dark to white fluorescence to UV-B light, and to natural sunlight. The effect of light condition on QDs toxicity could also be explained by photostability of the QDs, which would affect size of the particle, release of toxic component ions, and generation of reactive oxygen species. Considering increasing use of QDs in various applications, their environmental fates and corresponding toxic potentials deserve further investigation.

Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65- and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro.

The vulnerability of brain cells to neurologic insults varies greatly, depending on their neuronal subpopulation. However, cells surviving pathological insults such as ischemia or brain trauma may undergo structural changes, e.g., altered process growth, that could compromise brain function. In this study, we examined the effect of glutamate excitotoxicity on dendrite growth from surviving cortical GABAergic neurons in vitro. Glutamate exposure did not affect GABAergic neuron viability, however, it significantly reduced dendrite growth from GABAergic neurons. This effect was blocked by the AMPA receptor antagonists NBQX and CFM-2, and mimicked by AMPA, but not NMDA. Glutamate excitotoxicity also caused an NMDA receptor-mediated decrease in the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) immunoreactivity from GABAergic neurons, measured using immunocytochemical and Western blot techniques. GAD is necessary for GABA synthesis; however, reduction of GABA by 3-mercaptopropionic acid (3-MPA), which inhibits GABA synthesis, did not alter dendrite growth. These results suggest that GABAergic cortical neurons are relatively resistant to excitotoxic-induced cell death, but they can display morphological and biochemical alterations which may impair their function.

Cleft palate by picrotoxin or 3-MP and palatal shelf elevation in GABA-deficient mice.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the mammalian central nervous system. Gene targeting of GABA-synthetic glutamic acid decarboxylase (GAD) 67 and GABAA receptor beta(3) subunit induces cleft palate in the mouse. These findings appear to contradict previous pharmacological investigations using benzodiazepines and GABA itself, which indicate that GABA suppresses palatogenesis. Therefore, the effects of picrotoxin and 3-mercaptopropionic acid (3-MP) on palate formation were investigated in the present study. Picrotoxin and 3-MP impair GABA functions by blocking the GABA receptor and synthesis, respectively. Pregnant mice in the critical period [Embryonic Day (E) 11-15] of palatogenesis were administered these substances by subcutaneous injection or continuous infusion at subconvulsive doses, and their fetuses at E17-18 were investigated. A complete cleft in the secondary palate was observed in 15% of 333 embryos in 28 litters. In the remaining fetuses, a complete cleft palate was not observed, but microscopic examination of serial sections revealed partial defects of the palate. Furthermore, rescue from cleft palate in GAD67-deficient mice was attempted by GABA infusion. Horizontal elevation of palatal shelves, which is not observed in nontreated mice, did occur after the infusion in all 14 GABA-infused GAD67-deficient fetuses, although cleft palate still persisted. These results indicate that GABA is required for palatogenesis and is consistent with findings in gene knockout mice.

Diurnal variation in the proconvulsant effect of 3-mercaptopropionic acid and the anticonvulsant effect of androsterone in the Syrian hamster.

GABA is the principal neurotransmitter of the mammalian circadian system, and its activity is subject to diurnal and circadian variations, with maximal values in hypothalamic turnover, content and binding during the night. In this study we have examined rhythms in the proconvulsant effect of inhibition of glutamate decarboxylase (GAD) in hamsters (Mesocricetus auratus) as well as the anticonvulsant effect of androsterone, a neurosteroid that positively modulates the GABA(A) receptor. Administration of 10-60 mg/Kg of 3-mercaptopropionic acid (3-MPA, a GAD inhibitor) induced convulsions that were analyzed by an ad-hoc severity scale, with a lower sensitivity threshold at 24:00 h. Moreover, the latency for first and maximal convulsive response times was significantly lower at night. A similar temporal profile (maximal effect at midnight) was found for picrotoxin-induced seizures. Androsterone (40 mg/Kg) completely inhibited 3-MPA-induced tonic/clonic seizures at 12:00 h, while it had a partial inhibitory effect at 24:00 h. These results support the importance of temporal regulation of GABAergic modulation in the central nervous system.

Dose-related proconvulsant and anticonvulsant activity of isatin, a putative biological factor, in rats.

Isatin (indole-2, 3-dione) is an endogenous compound with anxiogenic properties, which occur within a narrow dose range (15-20 mg/kg, i.p.). Dose increment beyond 50 mg/kg, i.p. leads to the loss of anxiogenesis. Since a link has been postulated between anxiogenic and convulsant activity, the effect of a range of doses of isatin (20-80 mg/kg, i.p.) was investigated on subconvulsant and convulsant doses of two seizure-inducing agents, namely, pentylenetetrazole (PTZ) and 3-mercapto-propionic acid (3MPA) in rats. Isatin was found to induce a dose-related effect on PTZ and 3MPA convulsions. The lower dose (20 mg/kg, i.p.) potentiated PTZ and 3MPA convulsions, a median dose (40 mg/kg, i.p.) had insignificant effect, whereas higher doses (60 and 80 mg/kg, i.p.) of isatin exhibited significant anticonvulsant effect against both PTZ and 3MPA induced clonic convulsions. The investigation, thus, supports the contention that anxiogenic agents increase the susceptibility to chemical seizures. The proconvulsant effect of isatin, may be due to its inhibitory effect on central atrial natriuretic peptide receptors and stimulation of 5-hydroxytryptamine3 (5-HT3) rather than its monoamine oxidase (MAO) B inhibitory action. The anticonvulsant effect on higher doses of isatin, on the contrary, may be induced by its metabolites, including 5-hydroxyisatin.

Toxicity study of the angiotensin converting enzyme inhibitor rentiapril in rats.

A three-months toxicity study of an angiotensin converting enzyme (ACE) inhibitor, rentiapril (CAS 80830-42-8), was performed in Sprague-Dawley rats by oral administration. The dose levels of 0, 30, 125, 500 and 1000 mg/kg were tested in both sexes, in which each experimental group comprised 10 rats. Another ACE inhibitor, captopril, was used as a reference compound. Rentiapril at the highest dose of 1000 mg/kg caused low food consumption and death of some animals with signs of bloody feces and anemia. In males and females receiving 500 and 1000 mg/kg, there were low body weight gain, increases in water intake, urine volume and serum BUN level, and decreases in levels of various erythrocytic parameters. Kidney weight was increased dose-dependently in both sexes. Histopathologically, renal changes in the 500 and 1000 mg/kg groups consisted of proximal tubular degeneration, juxtaglomerular cell hyperplasia and interstitial cell infiltration. Similar, but mild, changes in proximal tubules were present in the female 125 mg/kg group. Dead animals from the highest dose groups further showed gastrointestinal hemorrhagic erosion and/or ulcer, decreased bone marrow erythropoiesis and hepatocytic vacuolar degeneration. There was no pathological alteration in rats from other rentiapril-treated groups, as well as in controls. These results indicate that the no-effect dose of rentiapril in rats by three months oral administration is 30 mg/kg in female and 125 mg/kg in male, and suggest that, like other ACE-inhibitors, this compound also has a toxic potential to affect renal tissues.

[The effect of triorthocresyl phosphate on the toxicity of GABA-lytics for mice]. / Vliianie triortokrezilfosfata na toksichnost' GAMK-litikov dlia myshei.

The toxicity of the picrotoxin bicuculline, but not of 3-mercaptopropionic acid increased in mice pretreated with triorthocresyl phosphate (TOCP). TOCP decreased the affinity of brain GABA receptors of intact mice for [3H] GABA. The binding of [3H]-t-butylcycloorthobenzoate was unchanged. The increased toxicity of GABA-antagonists may be associated with inhibition of blood carboxylesterases. The possibility of direct influence of TOCP and its metabolites on the GABA/benzodiazepine receptor complex cannot be excluded.

Motor and electrocorticographic epileptic activity induced by 3-mercaptopropionic acid in immature rats.

The convulsant action of 3-mercaptopropionic acid (3-MPA), a known inhibitor of glutamate decarboxylase activity, was studied in 7-, 12-, 18- and 25-day-old rats and in adult animals. 3-MPA elicited predominantly clonic, minimal seizures as well as generalized tonic-clonic (major) seizures at all developmental stages studied. The CD50 for major seizures did not change during development; CD50 for minimal seizures was significantly lower in 18-day-old rats than in older animals. Latency to the onset of seizures was shortest in 18-day-old rats and extremely long in 12- and, especially, in 7-day-old rats. This long latency might signify either changing molecular properties of glutamate decarboxylase during development or slow turnover of GABA at early postnatal stages. Electrocorticographic recordings demonstrated sharp EEG components in the frontal region as a first sign of 3-MPA action, and seizure patterns exhibited similar developmental changes as found with other seizure models (a decrease in duration of individual graphoelements and an increase in synchronization among various cortical regions). This indicates the primary importance of brain maturation in the expression of epileptic EEG phenomena. The correlation between EEG and motor phenomena was poor in the youngest animals and it ameliorated with age, but it never became perfectly coincidental.

[Effect of preliminary administration of phenobarbital on the toxicity of GABA-lytics in mice]. / Vliianie predvaritel'nogo vvedeniia fenobarbitala na toksichnost' GAMK-litikov u myshei.

The toxicity of picrotoxin bicuculline, but not of 3-mercaptopropionic acid decreased in mice pretreated during three days with phenobarbital and benzonal. The antidotal effectiveness of diazepam by picrotoxin and bicuculline exposure increased significantly. It has been suggested, that the modulation of detoxication systems may be the cause of increased tolerance against GABA-antagonists.

Time-dependent anticonvulsant activity of melatonin in hamsters.

The objective of the present study was to assess whether the anticonvulsant activity of melatonin displays diurnal variability in hamsters. Convulsions were induced by administering 3-mercaptopropionic acid (3-MP). There was a significant diurnal variation in 3-MP-induced convulsions, hamsters being more prone to exhibit seizures during the night than during the day. Melatonin (50 mg/kg i.p.) had a maximal anticonvulsive effect in the early evening (20:00 h). The administration at 20:00 h of the central-type benzodiazepine antagonist, Ro 15-1788, although unable by itself to modify seizure threshold, blunted the anticonvulsant response to melatonin. The results indicate that the time-dependent anticonvulsant activity of melatonin is sensitive to central-type benzodiazepine antagonism.

3-Mercaptopropionic acid administration into the caudate-putamen of the rat provokes dyskinesia.

The unilateral administration of 3-mercaptopropionic acid (MPA) through an implanted guide cannula into the caudate-putamen produced dyskinesia in the rat. Striatal GABA and dopamine were decreased and the dopamine metabolites 3,4-dihydroxyphenylacetic and homovanillic acid were increased on the MPA-injected side at 2-10 min after the onset of dyskinesia. The dyskinetic movements were blocked by GABA or alpha-aminooxaloacetic acid but not by glycine or haloperidol.