Interactive toxicity of usnic acid and lipopolysaccharides in human liver HepG2 cells

Usnic acid (UA), a natural botanical product, is a constituent of some dietary supplements used for weight loss. It has been associated with clinical hepatotoxicity leading to liver failure in humans. The present study was undertaken to evaluate the interactive toxicity, if any, of UA with lipopolysaccarides (LPS), a potential contaminant of food, at low non‐toxic concentrations. The human hepatoblastoma HepG2 cells were treated with the vehicle control and test agents, separately and in a binary mixture, for 24 h at 37 ⁰C in 5% CO₂. After the treatment period, the cells were evaluated by the traditional biochemical endpoints of toxicity in combination with the toxicogenomic endpoints that included cytotoxicity, oxidative stress, mitochondrial injury and changes in pathway‐focused gene expression profiles. Compared with the controls, low non‐toxic concentrations of UA and LPS separately showed no effect on the cells as determined by the biochemical endpoints. However, the simultaneous mixed exposure of the cells to their binary mixture resulted in increased cytotoxicity, oxidative stress and mitochondrial injury. The pathway‐focused gene expression analysis resulted in the altered expression of several genes out of 84 genes examined. Most altered gene expressions induced by the binary mixture of UA and LPS were different from those induced by the individual constituents. The genes affected by the mixture were not modulated by either UA or LPS. The results of the present study suggest that the interactions of low nontoxic concentrations of UA and LPS produce toxicity in HepG2 cells. Published 2012. This article is a US Government work and is in the public domain in the USA.     

Toxicology of chemical mixtures: a challenging quest along empirical sciences

This paper describes the “quest” of our institute trying to assess the toxicology of chemical mixtures. In this overview, we will discuss some critical developments in hazard identification and risk assessment of chemical mixtures during these past 15 years. We will stand still at empirical and mechanistic modeling. “Empirical” means that only information on doses or concentrations and effects is available in addition to an often empirically selected quantitative dose–response relationship. Empirical models have played a dominant role in the last decade to identify health and safety characteristics of chemical mixtures. Many of these models are based on the work of pioneers in mixture toxicology who defined three basic types of action for combinations of chemicals: simple similar action, simple dissimilar action and interaction. Nowadays, empirical models are mainly based on response-surface analysis and make use of advanced statistical designs. However, possible interactions between components in a mixture can also be given in terms of mechanistic models. In terms of “mechanistic” (or biological) understanding, interactions between compounds may occur in the kinetic phase (processes of uptake, distribution, metabolism and excretion) or in the dynamic phase (effects of chemicals on the receptor, cellular target or organ). A biological phenomenon such as competitive agonism as described for mixtures of drugs (biotransformation enzymes) or sensory irritants (nerve receptors) can accurately predict the effect of any of these mixtures. Thus, far mechanistic and empirical analyses of interactions are usually unrelated. It is one of the future challenges for mixtures research to combine information from both approaches. Also, our current biology-based models have their limitations, since they cannot integrate every relevant biological mechanism. In this respect, mechanistic modeling of mixtures may benefit from the developments coming from the arena of molecular biology (toxicogenomics) which offers an in-depth analysis of several involved enzymatic pathways in parallel through the use of a systems biology approach. This was illustrated with mixtures of food additives known to affect the liver. Key to further maturation of mixture toxicology is collaboration of experimental toxicologists, biomathematicians, biologists, pharmacologists, model developers, molecular biologists and bioinformaticians to ensure parallel and coordinated research in this challenging area of toxicology.

For this reason, the next sequel will be even more challenging and exciting to that first 15 years of empirical testing.     

Effect of temperature on heavy metal toxicity to earthworm Lumbricus terrestris (Annelida: Oligochaeta)

Earthworms (Lumbricus terrestris) acclimated at 2 degrees C above their habitat temperature (10-12 degrees C) showed about 5% increase in basal rate of oxygen consumption, which increased to about 38% in 14-16 degrees C- and 40% in 16-18 degrees C-, but decreased by 84% in 20-22 degrees C-acclimated worms. Temperature also increased the blood hemoglobin (Hb) concentration, which decreased slightly in 20-22 degrees C-acclimated worms. The worms acclimated at 20-22 degrees C showed their blood to be hypovolemic than that of 10-12 degrees C worms indicating dehydration. Pre-exposure of 10-14 degrees C-acclimated worms to sublethal concentrations of zinc, copper, and lead did not significantly affect the rate of respiration. However, at higher temperatures all these metals inhibited oxygen consumption; zinc, lead, and cadmium by approximately 11% and copper by approximately 18% of that at 14-16 degrees C. At 20-22 degrees C, the respiration was further inhibited, 36% by copper, 18% by cadmium, and approximately 10% by lead and zinc. Copper, lead, and zinc decreased the temperature-enhanced increase in blood Hb concentration at all temperatures. In 20-22 degrees C-acclimated worms heavy metal exposure slightly lowered the oxygen affinity of Hb as well as caused shifts in carbon monoxide difference spectra. The acute toxicity of these metals was not affected by a 2 degrees C rise in acclimation temperature but increased by 17% (lead), 33% (copper), and 5% (zinc) in 14-16 degrees C- and by 40% (lead), 149% (copper), and 132% (zinc) in 20-22 degrees C-acclimated worms. The increase in toxicity of metals caused by high temperatures may be due to limiting the scope of aerobic metabolism (oxygen extraction, transport, and utilization) via quantitative and qualitative effects on Hb. This terrestrial species appears to be tolerant of slight increases in habitat temperature, such as that expected with current global climate change.     

Effect of temperature on heavy metal toxicity to juvenile crayfish, Orconectes immunis (Hagen)

The acute toxicity of four selected heavy metals to juvenile crayfish Orconectes immunis (Hagen) (1-2 g wet body wt. each) at room temperature increased in the following order: cadmium (x3) < copper (x10) < zinc (x2) < lead. The toxicity of these metals to crayfish acclimated at 17, 20, 23/24, and 27 degrees C increased with temperature (by 7-20% between 20 and 24 degrees C and 14-26% between 20 and 27 degrees C) as judged by the lowering of LT(50) (time to kill 50% of test animals at a fixed concentration) values. A 4 degrees C rise in temperature (from 20 to 24 degrees C), which increased the toxicity of copper by about 7%, increased the rate of oxygen consumption by about 34%. Heavy metals inhibited the rate of oxygen consumption at all temperatures. In 20 degrees C-acclimated crayfish, copper caused about 17% inhibition of oxygen consumption compared to about 7-12% by other metals including the most toxic cadmium. A 3-4 degrees C rise in temperature tripled the inhibitory effect of copper (20%), cadmium and zinc (26 and 18%, respectively), but not of lead, on oxygen consumption. A 7 degrees C-rise in temperature (from 20 to 27 degrees C) increased the inhibitory effect of heavy metals, including lead, on oxygen consumption by up to 54% in the case of copper. The data indicate that rising global temperatures (currently 0.60 degrees C) associated with climate change can have the potential to increase the sensitivity of aquatic animals to heavy metals in their environment.     

A practical approach to assess inhalation toxicity of metal oxide nanoparticles in vitro

Exposure of humans to metal oxide nanoparticles (NPs) occurs mainly via air, and inhaled metal oxide NPs may generate inflammation. The aim of this study was to investigate the proinflammatory potential of six metal oxide NPs (CeO₂, Mn₂O₃, CuO, ZnO, Co₃O₄ and WO₃; 27–108 μg ml^?1) using human primary 3‐dimensional airway epithelium (MucilAir?) and dendritic cell (DC) models. Metal oxide NPs were mainly aggregated/agglomerated in the cell media, as determined by dynamic light scattering, scanning electron microscopy and differential centrifugal sedimentation. WO₃ and ZnO were highly soluble, both with and without respiratory mucus. Proinflammatory signalling by the epithelium was evaluated after a 24 hour exposure by increased interleukin‐6 and ‐8 and monocyte chemoattractant protein 1 cytokine release, which occurred only for CuO. Moreover, maturation of immature human DCs, which play a key role in the lung immune system, were evaluated by expression of surface markers HLA‐DR, CD80, CD83 and CD86 after a 48 hour exposure. Only Mn₂O₃ consistently upregulated DC maturation markers. Furthermore, by addition of medium from metal oxide NP‐exposed 3‐dimensional airway cultures to metal oxide NP‐exposed DC cultures, the interplay between lung epithelium and DCs was studied. Such an interplay was again only observed for Mn₂O₃ and in one of five DC donors. Our results show that, even when using dosages that represent very high in vivo exposure levels, up to 27 hours of constant human airway exposure, metal oxide NPs cause minimal proinflammatory effects and that epithelial cells not necessarily interfere with DC maturation upon metal oxide NP exposure. The present approach exemplifies a relevant translation towards human safety assessment.     

Acute toxicity of organic chemical mixtures to the fathead minnow

The acute joint toxicity of industrial organic chemicals to the fathead minnow was determined for binary and equitoxic multiple chemical mixtures. Results from binary tests were used to define isobole diagrams. The degree of joint toxic action was determined among 27 chemicals from seven different chemical classes. The slopes of the acute concentration response relationships were quite similar for all test chemicals. This suggests that the mode of acute toxic action for these chemicals is alike though it may not be identical. Intoxication signs of fish exposed to nearly all test chemicals were also similar and indicative of an anesthetic like effect. The results of isobole diagrams for binary mixtures, with 1-octanol as the reference chemical, demonstrated a near concentration additive acute joint action over a wide range of mixture ratios for each chemical from 7 different classes. Tests conducted with mixtures containing equitoxic levels of two to 21 chemicals also displayed a concentration additive acute joint action. All test chemicals can be modeled by a structure-toxicity relationship characteristic of a narcosis type of toxic action. These results are consistent with those of other investigators and are of particular importance when one realizes that numerous industrial chemicals are likely to cause lethality to aquatic organisms through similar toxic action.     

Autophagy in drug-induced liver toxicity

Liver injury resulting from exposure to drugs and chemicals is a major health problem. Autophagy is an important factor in a wide range of diseases, such as cancer, liver disease, muscular disorder, neurodegeneration, pathogen infection, and aging, and emerging evidence indicates that autophagy makes a substantial contribution to the pathogenesis of drug- and chemical-induced liver toxicity. In this review, we summarize current knowledge on autophagy triggered by toxicants/toxins, the protective role of autophagy in liver toxicity, and the underlying molecular mechanisms. We also highlight experimental approaches for studying autophagy.     

The role of early in vivo toxicity testing in drug discovery toxicology

The cost impact of late-stage failures of drug candidates has motivated the pharmaceutical industry to develop, validate, and implement a more proactive testing paradigm, including an emphasis on conducting predictive in vitro and in vivo studies earlier. The goal of drug discovery toxicology is not to reduce or eliminate attrition, as is often mis-stated as such, but rather to reprioritize efforts to shift attrition of future failing molecules upstream in discovery. This shift in attrition requires additional studies and investment earlier in the candidate evaluation process in order to avoid spending resources on molecules with soon-to-be-discovered development-limiting liabilities. While in silico and in vitro models will continually be developed and refined, in vivo preclinical safety models remain the gold standard for assessing human risk. For in vivo testing to influence early discovery effectively, it must: i) require low amounts of compound; ii) provide rapid results to drive decision-making and medicinal chemistry efforts; and iii) be flexible and provide results relevant to the development plan tailored to each target, drug class, and/or indication.     

Immunohistochemical characterization of hepatic stem cell-related cells in developing human liver

Little is known about the expression characteristics of the various kinds of possible markers in hepatic stem cells (HSCs) and other HSC-related cells in human fetal liver in various developmental stages. It is significant to investigate the immunohistochemical expression for better understanding of the origin, differentiation and migration of HSCs in the developing human liver. H-E staining and immunohistochemical methods were used to observe the expression of hepatic/cholangiocellular differentiation markers (AFP, GST-π, CK7, CK19) and hematopoietic stem cell markers(CD34 and c-kit) in several kinds of HSC-related cells in thirty cases of fetal liver samples (4–35 weeks after pregnancy). AFP expression appears in fetal hepatocytes at four weeks’ gestation. It peaks at 16–24 weeks’ gestation and decreases gradually afterwards. Finally, weak signals were only found in some ductal plate cells and a few limiting plate cells. GST-π was detected in hepatic cord cells from the sixth week and in the ductal plate cells from the eighth week. Twenty-six weeks later, only some ductal plate cells and a few limiting plate cells show positive signals. CK19 expression peaks during the 6th–11th weeks in hepatic cord cells and decreases gradually afterwards, except for the ductal plates. CK7 expression was limited in the ductal plate cells and bile ducts cells from the 14th week. CD34 and c-kit were detected at the eighth week in some ductal plate cells and a few mononuclear cells in the hepatic cords/mesenchymal tissue of portal areas. After 21 weeks, CD34 and c-kit were found only in ductal plate cells and a few mononuclear cells in the hepatic mesenchymal tissue of portal areas. Fetal hepatocytes at 4–16 weeks’ gestation are mainly constituted by HSCs characterized with bi-potential differentiation capacity. At 16 weeks’ gestation, most hepatic cord cells begin to differentiate into hepatocytes and abundant HSCs remain in ductal plate (the origin site of Hering canals). It is also indicated that the hematopoietic stem cells may give rise to some HSCs in embryonic liver. These indirectly support the hypothesis about the location and origin of HSCs in “liver valley hypothesis” reported previously. Translated from Academic Journal of Second Military Medical University, 2007, 28(2): 117–121 [译自: 第二军医大学学报]     

Cell biology of heavy metal toxicity in vascular tissue

Cadmium and lead are heavy metals that have been shown to induce vascular disorders such as atherosclerosis in experimental animals. However, little is known about the mechanisms by which cadmium and lead induce vascular toxicity. The toxicity was investigated using a culture system of vascular endothelial and smooth muscle cells. Cadmium destroys the monolayer of endothelial cells and the cytotoxicity is protected by zinc and copper without metallothionein induction. On the other hand, lead does not exhibit cytotoxicity but inhibits the repair of endothelial monolayers after wounding by a lower response to endogenous basic fibroblast growth factor mediated by suppression of the synthesis of perlecan, a large heparan sulfate proteoglycan. In addition, cadmium and lead reduce endothelial fibrinolytic activity by induction of plasminogen activator inhibitor type 1 synthesis and by inhibition of tissue-type plasminogen activator, respectively. In vascular smooth muscle cells, cadmium and lead can promote their proliferation and influence proteoglycan synthesis and fibrinolysis in different manners. These results indicate that cadmium and lead have specific toxicities in the proliferation, fibrinolysis, and extracellular matrix formation of vascular endothelial and smooth muscle cells.     

Cytotoxicity of cyclophosphamide and acrylamide in glioma and neuroblastoma cell lines cocultured with liver cells

A method for the cocultivation of nervous system cells with liver cells has been developed. When acrylamide was tested using the cocultivation procedure no significant liver metabolism-mediated changes in the cytotoxicity in C6 culture were detected. In neuroblastoma NIE115 cultures the toxicity was increased by induced chick hepatocytes at high acrylamide concentrations.     

Quantitative structure-activity relationships and toxicity studies of mixtures of chemicals with anaesthetic potency: Acute lethal and sublethal toxicity to Daphnia magna

In this study quantitative structure-activity relationships (QSARs) were calculated between hydrophobicity of a group of organic chemicals with anaesthetic potency and toxicity (immobilization, mortality and inhibition of reproduction) to Daphnia magna. Differences in slopes of the high quality QSARs might be explained in terms of possible different sites of action for the three criteria of effect.The combined effects of mixtures of 5–50 chemicals on immobilization and mortality did not deviate from additivity, while the effect on reproduction deviated somewhat from it.     

Evaluation of cytotoxicity and genotoxicity of pesticide mixtures on lymphocytes

The cytotoxicity and genotoxicity of pesticide mixtures viz. endosulfan?+?chlorpyrifos, chlorpyrifos?+?profenofos, and endosulfan?+?profenofos were evaluated on cultured human peripheral blood lymphocytes using assays for cell viability, and genotoxicity using chromosomal aberrations test and comet assay. The LC₅₀ values for cytotoxicity were 3.50?μM, 4.18?μM, and 10.5?μM for profenofos, endosulfan, and chlorpyrifos respectively. When combined in equimolar concentrations, the LC₅₀ values for cytotoxicity were 1.4?μM, 1.8?μM, and 2.0?μM for endosulfan?+?chlorpyrifos, chlorpyrifos?+?profenofos, and endosulfan?+?profenofos, respectively. Higher concentrations of individual pesticides (0.5–4.0?μM) but very low concentrations of pesticide mixtures caused significant DNA damage. Additive index values indicated a synergistic effect of toxicity for endosulfan?+?chlorpyrifos combination (1.12 TTU). The binary mixture of chlorpyrifos?+?profenofos showed an additive toxicity (0.46 TTU) while an antagonistic effect was observed for endosulfan?+?profenofos combination. Synergism could be due to these complementary pesticides simultaneously acting in different ways, magnifying their efficacy, whereas an additive interaction would imply that the chemicals are acting by the same mechanism and at the same target. Analysis of toxicity of pesticide mixtures may serve as important biomarker for occupational and household exposure to pesticides, with different modes of action.     

小鼠伍用氯胺酮与赛拉嗪翻正反射和毒性的影响

观察了氰胺酮（Ｋｅｔ）与赛拉嗪（Ｘｙ１）以１：１和３：１配伍对小鼠急性毒性和翻正反射消失效应的影响。用Ｑ（ｘ）统计检验合用后的联合作用性质，并绘制出Ｑ曲线及其９５％可信限图．可见Ｋｅｔ与Ｘｙｌ以１：１和３：１伍用后毒性分别为相加和轻微协同，翻正反射消失效应均呈协同性增强．配伍后的治疗指数，安全指数及可靠安全系数都较单药提高．大鼠和大的实验结果也表明Ｋｅｔ与Ｘｙｌ伍用后效应增强．     

The C. elegans model in toxicity testing

Caenorhabditis elegans is a small nematode that can be maintained at low cost and handled using standard in vitro techniques. Unlike toxicity testing using cell cultures, C. elegans toxicity assays provide data from a whole animal with intact and metabolically active digestive, reproductive, endocrine, sensory and neuromuscular systems. Toxicity ranking screens in C. elegans have repeatedly been shown to be as predictive of rat LD₅₀ ranking as mouse LD₅₀ ranking. Additionally, many instances of conservation of mode of toxic action have been noted between C. elegans and mammals. These consistent correlations make the case for inclusion of C. elegans assays in early safety testing and as one component in tiered or integrated toxicity testing strategies, but do not indicate that nematodes alone can replace data from mammals for hazard evaluation. As with cell cultures, good C. elegans culture practice (GCeCP) is essential for reliable results. This article reviews C. elegans use in various toxicity assays, the C. elegans model's strengths and limitations for use in predictive toxicology, and GCeCP. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Journal of Applied Toxicology published by John Wiley & Sons Ltd.     

Detection of exposure effects of mixtures of heavy polycyclic aromatic hydrocarbons in zebrafish embryos

In this study we evaluated the exposure effects of mixtures of five polycyclic aromatic hydrocarbons (PAHs); namely, benzo[a ]anthracene, benzo[a ]pyrene, benzo[b ]fluoranthene, benzo[k ]fluoranthene and chrysene on zebrafish embryos. Supplementation of the exposure media with 0.45% dimethyl sulfoxide and 50 ppm of Tween 20 could guarantee the solubilization and stabilization of the PAHs up to 24 h without affecting the embryos development. The exposure effects were tested by detecting the differential expression of a number of genes related to the aryl hydrocarbon receptor gene battery. Effects were detectable already after 6 h of exposure. After 24 h of exposure, all PAHs, except for benzo[a ]anthracene, acted as potent inducers of the gene cyp1a1 . Benzo[k ]fluoranthene was the major inducer; the effect caused by the mixture at the lower concentration tested (1 ng ml⁻¹) was dominated by its presence. However, in the mixture at the highest concentration tested (10 ng ml⁻¹) it caused less induction and was not dominant. No significant bioaccumulation values were detected on embryos exposed to the PAHs tested in this study; however, the results obtained, indicated that PAHs undergo a very rapid metabolization inside the embryos, and that those biotransformation products yield changes on the expression of genes involved in the aryl hydrocarbon receptor pathway. Future work should focus on identification of the PAH metabolization products and on the effect of these metabolites on toxicity. Copyright © 2016 John Wiley & Sons, Ltd.     

Airway irritation,inflammation, and toxicity in mice following inhalation of metal oxide nanoparticles

Metal oxide nanoparticles are used in a broad range of industrial processes and workers may be exposed to aerosols of the particles both during production and handling. Despite the widespread use of these particles, relatively few studies have been performed to investigate the toxicological effects in the airways following inhalation. In the present study, the acute (24?h) and persistent (13 weeks) effects in the airways after a single exposure to metal oxide nanoparticles were studied using a murine inhalation model. Mice were exposed 60?min to aerosols of either ZnO, TiO₂, Al₂O₃ or CeO₂ and the deposited doses in the upper and lower respiratory tracts were calculated. Endpoints were acute airway irritation, pulmonary inflammation based on analyses of bronchoalveolar lavage (BAL) cell composition, DNA damage assessed by the comet assay and pulmonary toxicity assessed by protein level in BAL fluid and histology. All studied particles reduced the tidal volume in a concentration-dependent manner accompanied with an increase in the respiratory rate. In addition, ZnO and TiO₂ induced nasal irritation. BAL cell analyses revealed both neutrophilic and lymphocytic inflammation 24-h post-exposure to all particles except TiO₂. The ranking of potency regarding induction of acute lung inflammation was Al₂O₃ = TiO_2?<_?CeO₂ ? ZnO. Exposure to CeO₂ gave rise to a more persistent inflammation; both neutrophilic and lymphocytic inflammation was seen 13 weeks after exposure. As the only particles, ZnO caused a significant toxic effect in the airways while TiO₂ gave rise to DNA-strand break as shown by the comet assay.     

Investigation of heavy metal contents in infusion tea samples of Iran

The current study aimed to determine the concentrations of five heavy metals in samples of infused tea from teas available in Iran. Eighty-one samples were divided into the three groups of bulk, packed, and tea bag samples. The investigation was performed using the inductively coupled plasma-optic emission spectrometry technique. The estimated daily intake of heavy metals was compared with the allowable daily intake (ADI), and the means were compared with the limits allowed by national Iranian standards. Results showed that infusion time had no effect on heavy metal content, except for Pb in bulk samples, and all means were within standard limits. Monitoring heavy metal content is still very important, because these metals are present in soils due to increased industrial activity and the use of pesticides and fertilizers.     

Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS-30D cells

The mechanisms of toxic effects of divalent cations of three heavy metals Hg, Cd and Cu in rat ascites hepatoma AS-30D cells cultivated in vitro were compared. It was found that the toxicity of these ions, applied in the micromolar range (10-500 microM), decreased from Hg(2+) (most toxic) to Cu(2+) (least toxic). Hg(2+) and Cd(2+) produced a high percentage of cell death by both necrosis and apoptosis, whereas Cu(2+) at concentrations up to 500 microM was weakly effective. Hg(2+) at concentration of 10 microM appeared slightly uncoupling (i.e., stimulated resting state respiration and decreased the mitochondrial transmembrane potential), whereas it exerted a strong inhibitory effect on the respiratory chain and rapid dissipation of the membrane potential at higher concentrations. Cu(2+) had inhibitory effect on cell respiration only at 500 microM concentration and after incubation of 48 h but produced a significant uncoupling effect at lower concentrations. Cu(2+) induced an early and sharp increase of intracellular production of reactive oxygen species (ROS). The action of Hg(2+) and Cd(2+) on ROS generation was biphasic. They stimulated ROS generation within the cells at low concentrations and at short incubation times but decreased ROS generation at higher concentrations and at longer incubation. It is concluded that mitochondria are an important target for toxic effects of Hg(2+), Cd(2+) and Cu(2+) in AS-30D rat hepatoma cells.     

炎症细胞和细胞因子在肝纤维化中的作用研究进展

肝纤维化是肝脏受到损伤后,引起肝脏的炎症免疫反应,继而引起组织自我修复的过程。因促炎介质在肝纤维化的发生及发展中扮演了一个重要的角色,且炎症反应是纤维化的始发阶段,可知炎症细胞不可避免地参与到了肝纤维化中。故本文对巨噬细胞、肥大细胞、中性粒细胞等炎症细胞及转化生长因子β（TGF-β）、血小板源性生长因子（PDGF）、肿瘤坏死因子α （TNF-α）等细胞因子在肝纤维化中的作用研究进展进行综述。