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.

Assessing the transferability and reproducibility of 3D in vitro liver models from primary human multi-cellular microtissues to cell-line based HepG2 spheroids.

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 µg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.

A review on the traditional uses, phytochemistry, and pharmacological activities of clove basil (Ocimum gratissimum L.).

In traditional medicine, Ocimum gratissimum (clove basil) is used in the treatment of various diseases such as diabetes, cancer, inflammation, anaemia, diarrhoea, pains, and fungal and bacterial infections. The present study reviewed the phytochemicals, essential oils, and pharmacological activities of O. gratissimum. The bioactive compounds extracted from O. gratissimum include phytochemicals (oleanolic acid, caffeic acid, ellagic acid, epicatechin, sinapic acid, rosmarinic acid, chlorogenic acid, luteolin, apigenin, nepetoidin, xanthomicrol, nevadensin, salvigenin, gallic acid, catechin, quercetin, rutin, and kaempfero) and essential oils (camphene, ß-caryophyllene, α- and ß-pinene, α-humulene, sabinene, ß-myrcene, limonene, 1,8-cineole, trans-ß-ocimene, linalool, α- and δ-terpineol, eugenol, α-copaene, ß-elemene, p-cymene, thymol, and carvacrol). Various in vivo and in vitro studies have shown that O. gratissimum and its bioactive constituents possess pharmacological properties such as antioxidant, anti-inflammatory, anticancer, hepatoprotective, antidiabetic, antihypertensive, antidiarrhoeal, and antimicrobial properties. This review demonstrated that O. gratissimum has a strong preventive and therapeutic effect against several diseases. The effectiveness of O. gratissimum to ameliorate various diseases may be attributed to its antimicrobial and antioxidant properties as well as its capacity to improve the antioxidant systems. However, despite the widespread pharmacological activities of O. gratissimum, further experiments in human clinical trial studies are needed to establish effective and safe doses for the treatment of various diseases.

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.

In vivo studies on the ameliorative effect of coconut water against carbon tetrachloride induced toxicity in rats.

PURPOSE: Coconut water is used in folklore medicine for oral rehydration, treatment of childhood diarrhoea, gastroenteritis and cholera, and is also known to possess antioxidant properties. OBJECTIVE: In this study, we examined the ameliorative potentials of coconut water on carbon tetrachloride (CCl4) induced toxicity in rats. MATERIALS AND METHODS: Rats were randomly assigned into separate cages according to the sex of 5 groups. Groups 2-5 were intraperitoneally injected a single dose of 1 mL/kg CCl4 diluted in olive oil. Only 3, 4 and 5 were orally given 2, 4, 6 mL/kg coconut water respectively, whereas groups 1 and 2 received distilled water. RESULTS: Treatment with coconut water significantly (p < 0.05) increased red blood cell, packed cell volume, haemoglobin, high-density lipoprotein, glutathione, superoxide dismutase, catalase, total protein, and albumin compared to the negative control in both sexes of the rats. Furthermore, platelets, white blood cells, urea, low-density lipoprotein, triglyceride, total cholesterol, malondialdehyde, bilirubin, alkaline phosphatase, alanine and aspartate transaminases decreased significantly (p < 0.05) compared to the negative control in both male and female rats. CONCLUSION: Thus, coconut water supplementation may reverse CCl4 induced toxicity and distortions on haematological parameters, lipid profile and antioxidant enzymes, liver and kidney biomarkers in rats.

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.

Ameliorative effect of aqueous leaf extract of Solanum aethiopicum on phenylhydrazine-induced anaemia and toxicity in rats.

Solanum aethiopicum is used in ethnomedicine for the treatment of overweight, constipation and anaemia. This study evaluated the ameliorative effect of aqueous leaf extract of S. aethiopicum on phenylhydrazine-induced anaemia in rats. Acute toxicity was determined in male and female rats (n = 5/group/sex) by oral administration of single dose of up to 5000 mg/kg of the S. aethiopicum extract. The experimental rats were randomly grouped into five (5) groups of 6 rats each. Group (i) served as normal control, group (ii) negative control, group (iii) standard drug-5 mg/kg ferrous sulphate, groups (iv) and (v), 200 and 400 mg/kg of S. aethiopicum extract respectively. Phenylhydrazine (PHZ) was administered intraperitoneally at the dose of 50 mg/kg body weight for two consecutive days to groups (ii-v). After 14 days, the rats were sacrificed; blood, liver and kidney were collected. The haematological, lipid profile, liver and kidney function parameters were determined and the histopathology of the liver and kidney were examined. In acute toxicity study, no signs of toxicity or death were recorded. The study shows an observable significant (P < 0.05) increase in packed cell volume, haemoglobin and red blood cell counts at 400 mg/kg S. aethiopicum extract in both the male and female rats when compared to other groups. Solanum aethiopicum extract at the dose of 400 mg/kg reduced aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), urea, creatinine and chloride. The results of this study lent credence to the use of S. aethiopicum leaf as an anti-anaemic tonic with a wide margin of safety and hepato/reno-protective potentials.

Evaluating bioactive constituents and toxicological effects of aqueous extract of fermented Pentaclethra macrophylla seeds in rats.

OBJECTIVE: This study aimed at evaluating the bioactive constituents and the toxicological profile of the aqueous fermented seed extract of P. macrophylla. MATERIALS AND METHODS: The chemical constituents of fermented P. macrophylla were assessed using GC-MS. For acute toxicity study, one-time doses of up to 5000 mg/kg of the extract were orally administered to male and female rats whereas 200, 400 and 800 mg/kg of the P. macrophylla extract were orally administered daily for 14 days in sub-acute toxicity investigation. Biochemical, haematological and lipid profiles were assessed following standard methods. RESULTS: Bioactive compounds such as citronellol and oxirane, tetradecyl- (hexadecylene oxide) were identified in the extract. In acute toxicity test, no death or sign of toxicity was identified. For sub-acute study, ALT decreased significantly (p<0.05) while HDL-C had dose-dependent increases. No effect (p<0.05) on haematological parameters except on platelets was found. No histopathological changes were observed. CONCLUSION: Our results demonstrated that the extract of fermented P. macrophylla caused no toxic effects in the rats at the tested doses. Therefore, they may be termed safe for consumption and therapeutic uses.

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.

Toxicological Evaluation of Phytochemical Characterized Aqueous Extract of Wild Dried Lentinus squarrosulus (Mont.) Mushroom in Rats.

Lentinus squarrosulus (Mont.) is an edible wild mushroom with tough fruiting body that belongs to the family Polyporaceae. It is used in ethnomedicine for the treatment of ulcer, anaemia, cough and fever. Recent studies have demonstrated its anticancer, anti-diabetic and antioxidant properties. However, little or no information is available regarding the bioactive components and toxicological study of wild dried L. squarrosulus. Therefore, this study investigated the bioactive components of aqueous extract of boiled wild dried L. squarrosulus and its toxicological effects in rats. The extract of L. squarrosulus was subjected to GC-MS analysis. The acute toxicity test was performed by oral administration of a single dose of up to 5,000 mg/kg extract of L. squarrosulus. In subacute study, the rats were orally administered extract of L. squarrosulus at the doses of 500, 1,000 and 1,500 mg/kg body weight daily for 14 days. The haematological, lipid profile, liver and kidney function parameters were determined and the histopathology of the liver and kidney were examined. The GC-MS analysis revealed the presence of bioactive compounds; 1-tetradecene, fumaric acid, monochloride, 6-ethyloct-3-yl ester, 9-eicosene, phytol, octahydropyrrolo[1,2-a]pyrazine and 3-trifluoroacetoxypentadecane. In acute toxicity study, neither death nor toxicity sign was recorded. In the sub-acute toxicity study, significant differences (p < 0.05) were observed on creatinine, aspartate aminotransferase, alanine aminotransferase, total cholesterol, triglycerides and high-density lipoprotein cholesterol. Whilst no significant differences (p > 0.05) were observed on packed cell volume, heamoglobin, red blood cell, white blood cell and alkaline phosphatase, in all the tested doses. No histopathological alterations were recorded. Our findings revealed that aqueous extract of L. squarrosulus may have antimicrobial, antinocieptive and antioxidant properties based on the result of GC-MS analysis. Results of the toxicity test showed no deleterious effect at the tested doses, suggesting that L. squarrosulus is safe for consumption at the tested doses.

Safety Evaluation of an Aqueous Extract of Termitomyces robustus (Agaricomycetes) in Wistar Rats.

Termitomyces robustus is an edible and highly nutritious wild Basidiomycetes mushroom. It is used in ethnomedicine for treating malnutrition-related diseases, rheumatism, diarrhea, gonorrhea, anemia, and hypertension. Despite the tremendous use of this delicious edible mushroom as a source of nutrients, no comprehensive literature describes its safety and toxicity profiles. Therefore, this study evaluated the toxicity profile of an aqueous T. robustus extract in rats. In the acute toxicity test, male and female rats were orally administered daily a single dose of up to 10 g/kg extract. In the subacute toxicity test, male rats were orally administered the T. robustus extract at graded doses of 500, 1000, and 1500 mg/kg for 14 days. No mortality or any signs of toxicity were observed in the acute toxicity study, indicating that the median lethal dose (LD50) of T. robustus is greater than 10 g/kg. In the subacute toxicity study, T. robustus had no effect (P > 0.05) on hemoglobin, packed cell volume, red blood cell, white blood cell, alanine aminotransferase, alkaline phosphatase, neutrophil, lymphocyte, or lipid profile parameters in any of the rats. However, significant differences (P < 0.05) were noted in alanine aminotransferase, eosinophils, basophils, monocytes, platelets, urea, creatinine, and electrolytes in the tested groups when compared to values from the control group. No histopathological alterations or changes were observed in the liver or kidneys of the rats. This study established that an aqueous extract of T. robustus is nontoxic and therefore safe for consumption at the tested doses.

Toxicological profile of the aqueous-fermented extract of Musa paradisiaca in rats.

OBJECTIVE: This study was conducted to assess the toxicity profile of the aqueous-fermented extract of Musa paradisiaca in rats. MATERIALS AND METHODS: In acute toxicity test, the rats of different groups were orally administered with a single dose of 500, 1000, 2000 and 5000 mg/kg of fermented extract of M. paradisiaca. The rats were monitored for behavioral changes, toxicity signs and mortality. In sub-acute test, the rats were orally administered with fermented M. paradisiaca extract (200, 400 and 800 mg/kg/day) for 14 days. Haematological and serum biochemical parameters were evaluated and histopathological studies of the liver and kidney were done. The study was performed from June to July 2017. RESULTS: Concerning the acute toxicity, no toxicity signs or death were recorded and an LD50 value of >5 g/kg for fermented extract of M. paradisiaca was observed. Regarding the sub-acute toxicity, ingestion of the fermented extract of M. paradisiaca caused no significant effects (p<0.05) in terms of relative organ weight, body weight percentage, haemoglobin, red blood cells count, electrolytes levels, lymphocytes count, basophils count, and aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels. However, significant differences (p<0.05) were observed in white blood cells, eosinophils, platelets, neutrophils and monocytes counts, and urea, creatinine, alanine aminotransferase (ALT) and high-density lipoprotein (HDL) levels. The histological assessments of the liver and kidney showed normal results. CONCLUSION: The findings of this study has suggested that daily administration of fermented extract of M. paradisiaca at doses up to 800 mg/kg for 14 days, is not toxic and may be considered safe for therapeutic uses.

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.