Grouping of orally ingested silica nanomaterials via use of an integrated approach to testing and assessment to streamline risk assessment.

BACKGROUND: Nanomaterials can exist in different nanoforms (NFs). Their grouping may be supported by the formulation of hypotheses which can be interrogated via integrated approaches to testing and assessment (IATA). IATAs are decision trees that guide the user through tiered testing strategies (TTS) to collect the required evidence needed to accept or reject a grouping hypothesis. In the present paper, we investigated the applicability of IATAs for ingested NFs using a case study that includes different silicon dioxide, SiO2 NFs. Two oral grouping hypotheses addressing local and systemic toxicity were identified relevant for the grouping of these NFs and verified through the application of oral IATAs. Following different Tier 1 and/or Tier 2 in vitro methods of the TTS (i.e., in vitro dissolution, barrier integrity and inflammation assays), we generated the NF datasets. Furthermore, similarity algorithms (e.g., Bayesian method and Cluster analysis) were utilized to identify similarities among the NFs and establish a provisional group(s). The grouping based on Tier 1 and/or Tier 2 testing was analyzed in relation to available Tier 3 in vivo data in order to verify if the read-across was possible and therefore support a grouping decision. RESULTS: The measurement of the dissolution rate of the silica NFs in the oro-gastrointestinal tract and in the lysosome identified them as gradually dissolving and biopersistent NFs. For the local toxicity to intestinal epithelium (e.g. cytotoxicity, membrane integrity and inflammation), the biological results of the gastrointestinal tract models indicate that all of the silica NFs were similar with respect to the lack of local toxicity and, therefore, belong to the same group; in vivo data (although limited) confirmed the lack of local toxicity of NFs. For systemic toxicity, Tier 1 data did not identify similarity across the NFs, with results across different decision nodes being inconsistent in providing homogeneous group(s). Moreover, the available Tier 3 in vivo data were also insufficient to support decisions based upon the obtained in vitro results and relating to the toxicity of the tested NFs. CONCLUSIONS: The information generated by the tested oral IATAs can be effectively used for similarity assessment to support a grouping decision upon the application of a hypothesis related to toxicity in the gastrointestinal tract. The IATAs facilitated a structured data analysis and, by means of the expert's interpretation, supported read-across with the available in vivo data. The IATAs also supported the users in decision making, for example, reducing the testing when the grouping was well supported by the evidence and/or moving forward to advanced testing (e.g., the use of more suitable cellular models or chronic exposure) to improve the confidence level of the data and obtain more focused information.

Ocimum gratissimum leaf extract ameliorates phenylhydrazine-induced anaemia and toxicity in Wistar rats.

OBJECTIVES: Ocimum gratissimum L. is used in traditional medicine for the treatment of bacterial infections and anaemia. This study was designed to evaluate the effect of O. gratissimum leaf extract on phenylhydrazine (PHZ)-induced anaemia and toxicity in rats. METHODS: The experimental rats were divided into five groups (A-E) (n=6/sex/group). Each rat in groups B-E was intraperitoneally administered 50 mg/kg of PHZ for two consecutive days. Group A (normal control) did not receive any PHZ, group B (negative control), group C received orally 5 mg/kg ferrous sulphate whereas groups D and E received 200 and 400 mg/kg O. gratissimum leaf extract respectively, for 14 days. RESULTS: Red blood cell count, packed cell volume, haemoglobin concentration and high-density lipoprotein increased significantly (p<0.05) whereas low-density lipoprotein and very-low-density lipoprotein decreased in extract-treated groups when compared to the negative control. O. gratissimum (400 mg/kg extract) and standard drug (5 mg/kg ferrous sulphate) significantly (p<0.05) reduced the levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. CONCLUSIONS: The results of this study indicate that O. gratissimum leaf extract has a restorative effect on the phenylhydrazine-induced metabolic distortions in the blood, liver, and kidney, and therefore could be used therapeutically as an anti-anaemic tonic.

Time dependent impact of copper oxide nanomaterials on the expression of genes associated with oxidative stress, metal binding, inflammation and mucus secretion in single and co-culture intestinal in vitro models.

The potential for ingestion of copper oxide nanomaterials (CuO NMs) is increasing due to their increased exploitation. Investigation of changes in gene expression allows toxicity to be detected at an early stage of NM exposure and can enable investigation of the mechanism of toxicity. Here, undifferentiated Caco-2 cells, differentiated Caco-2 cells, Caco-2/HT29-MTX (mucus secreting) and Caco-2/Raji B (M cell model) co-cultures were exposed to CuO NMs and copper sulphate (CuSO4) in order to determine their impacts. Cellular responses were measured in terms of production of reactive oxygen species (ROS), the gene expression of an antioxidant (haem oxygenase 1 (HMOX1)), the pro-inflammatory cytokine (interleukin 8 (IL8)), the metal binding (metallothionein 1A and 2A (MT1A and MT2A)) and the mucus secreting (mucin 2 (MUC2)), as well as HMOX-1 protein level. While CuSO4 induced ROS production in cells, no such effect was observed for CuO NMs. However, these particles did induce an increase in the level of HMOX-1 protein and upregulation of HMOX1, MT2A, IL8 and MUC2 genes in all cell models. In conclusion, the expression of HMOX1, IL8 and MT2A were responsive to CuO NMs at 4 to 12 h post exposure when investigating the toxicity of NMs using intestinal in vitro models. These findings can inform the selection of endpoints, timepoints and models when investigating NM toxicity to the intestine in vitro in the future.

Ocimum gratissimum leaf extract ameliorates phenylhydrazine-induced anaemia and toxicity in Wistar rats.

OBJECTIVES: Ocimum gratissimum L. is used in traditional medicine for the treatment of bacterial infections and anaemia. This study was designed to evaluate the effect of O. gratissimum leaf extract on phenylhydrazine (PHZ)-induced anaemia and toxicity in rats. METHODS: The experimental rats were divided into five groups (A-E) (n=6/sex/group). Each rat in groups B-E was intraperitoneally administered 50 mg/kg of PHZ for two consecutive days. Group A (normal control) did not receive any PHZ, group B (negative control), group C received orally 5 mg/kg ferrous sulphate whereas groups D and E received 200 and 400 mg/kg O. gratissimum leaf extract respectively, for 14 days. RESULTS: Red blood cell count, packed cell volume, haemoglobin concentration and high-density lipoprotein increased significantly (p<0.05) whereas low-density lipoprotein and very-low-density lipoprotein decreased in extract-treated groups when compared to the negative control. O. gratissimum (400 mg/kg extract) and standard drug (5 mg/kg ferrous sulphate) significantly (p<0.05) reduced the levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase. CONCLUSIONS: The results of this study indicate that O. gratissimum leaf extract has a restorative effect on the phenylhydrazine-induced metabolic distortions in the blood, liver, and kidney, and therefore could be used therapeutically as an anti-anaemic tonic.

Using 3D gastrointestinal tract in vitro models with microfold cells and mucus secreting ability to assess the hazard of copper oxide nanomaterials.

BACKGROUND: Copper oxide nanomaterials (CuO NMs) are exploited in many products including inks, cosmetics, textiles, wood preservatives and food contact materials. Their incorporation into these products may enhance oral exposure in consumer, environmental and occupational settings. Undifferentiated and differentiated monocultures of Caco-2 cells are commonly used to assess NM toxicity to the intestine in vitro. However, the integration of other cell types into Caco-2 in vitro models increases their physiological relevance. Therefore, the aim of this study is to evaluate the toxicity of CuO NMs and copper sulphate (CuSO4) to intestinal microfold (M) cell (Caco-2/Raji B) and mucus secreting (Caco-2/HT29-MTX) co-culture in vitro models via assessment of their impact on barrier integrity, viability and interleukin (IL)-8 secretion. The translocation of CuO NMs and CuSO4 across the intestinal barrier was also investigated in vitro. RESULTS: CuO NMs and CuSO4 impaired the function of the intestinal barrier in the co-culture models [as indicated by a reduction in transepithelial electrical resistance (TEER) and Zonular occludens (ZO-1) staining intensity]. Cu translocation was observed in both models but was greatest in the Caco-2/Raji B co-culture. CuO NMs and CuSO4 stimulated an increase in IL-8 secretion, which was greatest in the Caco-2/HT29-MTX co-culture model. CuO NMs and CuSO4 did not stimulate a loss of cell viability, when assessed using light microscopy, nuclei counts and scanning electron microscopy. CuO NMs demonstrated a relatively similar level of toxicity to CuO4 in both Caco-2/Raji B and Caco-2/HT29-MTX co- culture models. CONCLUSIONS: The Caco-2/Raji B co-culture model was more sensitive to CuO NM and CuSO4 toxicity than the Caco-2/HT29-MTX co-culture model. However, both co-culture models were less sensitive to CuO NM and CuSO4 toxicity than simple monocultures of undifferentiated and differentiated Caco-2 cells, which are more routinely used to investigate NM toxicity to the intestine. Obtained data can therefore feed into the design of future studies which assess the toxicity of substances (e.g. NMs) and pathogens to the intestine (e.g. by informing model and endpoint selection). However, more testing with a wider panel of NMs would be beneficial in order to help select which in vitro models and endpoints to prioritise when screening the safety of ingested NMs. Comparisons with in vivo findings will also be essential to identify the most suitable in vitro model to screen the safety of ingested NMs.

Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration.

BACKGROUND: Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. METHODS: Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 µg/cm2 Cu (equivalent to 1.95 to 250 µg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 µg/cm2 for CuO NMs, and 4.25 µg/cm2 for copper sulphate (CuSO4), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (Papp); a measure of barrier permeability to CuO NMs. For all experiments, CuSO4 was used as an ionic control. RESULTS: CuO NMs and CuSO4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO4 translocated across the differentiated Caco-2 cell monolayer. CuO NM mediated IL-8 production was over 2-fold higher in undifferentiated cells. A reduction in cell viability in differentiated cells was not responsible for the lower level of cytokine production observed. Both CuO NMs and CuSO4 decreased TEER values to a similar extent, and caused tight junction dysfunction (ZO-1 staining), suggesting that barrier integrity was disrupted. CONCLUSIONS: CuO NMs and CuSO4 stimulated IL-8 production by Caco-2 cells, decreased barrier integrity and thereby increased the Papp and translocation of Cu. There was no significant enhancement in potency of the CuO NMs compared to CuSO4. Differentiated Caco-2 cells were identified as a powerful model to assess the impacts of ingested NMs on the GI tract.