Next generation risk assessment: an ab initio case study to assess the systemic safety of the cosmetic ingredient, benzyl salicylate, after dermal exposure.

We performed an ab initio next-generation risk assessment (NGRA) for a fragrance ingredient, benzyl salicylate (BSal), to demonstrate how cosmetic ingredients can be evaluated for systemic toxicity endpoints based on non-animal approaches. New approach methodologies (NAMs) used to predict the internal exposure included skin absorption assays, hepatocyte metabolism, and physiologically based pharmacokinetic (PBPK) modeling, and potential toxicodynamic effects were assessed using pharmacology profiling, ToxProfiler cell stress assay, transcriptomics in HepG2 and MCF-7 cells, ReproTracker developmental and reproductive toxicology (DART) assays, and cytotoxicity assays in human kidney cells. The outcome of the NGRA was compared to that of the traditional risk assessment approach based on animal data. The identification of the toxicologically critical entity was a critical step that directed the workflow and the selection of chemicals for PBPK modeling and testing in bioassays. The traditional risk assessment and NGRA identified salicylic acid (SA) as the "toxdriver." A deterministic PBPK model for a single-day application of 1.54 g face cream containing 0.5% BSal estimated the Cmax for BSal (1 nM) to be much lower than that of its major in vitro metabolite, SA (93.2 nM). Therefore, SA was tested using toxicodynamics bioassays. The lowest points of departure (PoDs) were obtained from the toxicogenomics assays. The interpretation of these results by two companies and methods were similar (SA only results in significant gene deregulation in HepG2 cells), but PoD differed (213 µM and 10.6 µM). A probabilistic PBPK model for repeated applications of the face cream estimated the highest Cmax of SA to be 630 nM. The resulting margins of internal exposure (MoIE) using the PoDs were 338 and 16, which were more conservative than those derived from external exposure and in vivo PoDs (margin of safety values were 9,705). In conclusion, both traditional and ab initio NGRA approaches concluded that the daily application of BSal in a cosmetic leave-on face cream at 0.5% is safe for humans. The processing and interpretation of toxicogenomics data can lead to different PoDs, which can subsequently affect the calculation of the MoIE. This case study supports the use of NAMs in a tiered NGRA ab initio approach.

The chemical structure impairs the intensity of genotoxic effects promoted by 1,2-unsaturated pyrrolizidine alkaloids in vitro.

1,2-unsaturated pyrrolizidine alkaloids (PAs) represent a large group of secondary plant metabolites exhibiting hepatotoxic, genotoxic, and carcinogenic properties upon bioactivation. To examine how the degree of esterification affects the genotoxic profile of PA we investigated cytotoxicity, histone H2AX phosphorylation, DNA strand break induction, cell cycle perturbation, micronuclei formation, and aneugenic effects in different cell models. Analysis of cytotoxicity and phosphorylation of histone H2AX was structure- and concentration-dependent: diester-type PAs (except monocrotaline) showed more pronounced effects than monoester-type PAs. Cell cycle analysis identified that diester-type PAs induced a S-phase arrest and a decrease in the occurrence of cells in the G1-phase. The same structure-dependency was observed by flow-cytometric analysis of PA-induced micronuclei in CYP3A4-overexpressing V79 cells. Analysis of centromeres induced by lasiocarpine in the micronuclei by fluorescence in situ hybridization indicated an aneugenic effect in V79h3A4 cells. Comet assays revealed no significant induction of DNA strand breaks for all investigated PAs. Overall, diester-type PAs induced more pronounced effects than monoester-type PAs. Furthermore, our results indicate aneugenic effects upon exposure towards lasiocarpine in vitro. These data improve our understanding how structural features of PA influence the genotoxic profile. Especially, the monoester-type PAs seem to induce less severe effects than other PAs.

Pyrrolizidine alkaloid-induced transcriptomic changes in rat lungs in a 28-day subacute feeding study.

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites synthesized by a wide range of plants as protection against herbivores. These toxins are found worldwide and pose a threat to human health. PAs induce acute effects like hepatic sinusoidal obstruction syndrome and pulmonary arterial hypertension. Moreover, chronic exposure to low doses can induce cancer and liver cirrhosis in laboratory animals. The mechanisms causing hepatotoxicity have been investigated previously. However, toxic effects in the lung are less well understood, and especially data on the correlation effects with individual chemical structures of different PAs are lacking. The present study focuses on the identification of gene expression changes in vivo in rat lungs after exposure to six structurally different PAs (echimidine, heliotrine, lasiocarpine, senecionine, senkirkine, and platyphylline). Rats were treated by gavage with daily doses of 3.3 mg PA/kg bodyweight for 28 days and transcriptional changes in the lung and kidney were investigated by whole-genome microarray analysis. The results were compared with recently published data on gene regulation in the liver. Using bioinformatics data mining, we identified inflammatory responses as a predominant feature in rat lungs. By comparison, in liver, early molecular consequences to PAs were characterized by alterations in cell-cycle regulation and DNA damage response. Our results provide, for the first time, information about early molecular effects in lung tissue after subacute exposure to PAs, and demonstrates tissue-specificity of PA-induced molecular effects.

Metabolic fate and toxicity reduction of aflatoxin B1 after uptake by edible Tenebrio molitor larvae.

The use of insects as food and feed is gaining more attention for ecological and ethical reasons. Despite the high tolerance of edible yellow mealworm (Tenebrio molitor) larvae to aflatoxin B1 (AFB1), the metabolic fate of the toxin along with its toxic potential in the insect is uncertain. The present study aimed at investigating the AFB1 mass balance and the metabolite formation in a feeding trial with AFB1-contaminated grain flour. T. molitor larvae tolerated the AFB1 level of 10,700 µg/kg in the feed, however, weight gain was decreased by 15% over a 4-weeks feeding period. The investigation of the phase I metabolite pattern revealed the formation of AFM1 and a novel presumably monohydroxylated compound in larvae extracts that was not formed by reference incubation with rat, bovine or porcine liver microsomes. Mass balance quantification of ingested AFB1 revealed that 87% of the initial toxin remain undetected in larval body or residue. Analysis of histone H2Ax phosphorylation in human liver cells as a surrogate for genotoxicity showed that extracts from exposed larvae did not exhibit an elevated toxic potential. Although toxicological uncertainties remain due to the undetected transformation products, the resulting mutagenicity of the edible larvae appears to be low.

Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study.

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA.In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios.

The pyrrolizidine alkaloid senecionine induces CYP-dependent destruction of sinusoidal endothelial cells and cholestasis in mice.

Pyrrolizidine alkaloids (PAs) are widely occurring phytotoxins which can induce severe liver damage in humans and other mammalian species by mechanisms that are not fully understood. Therefore, we investigated the development of PA hepatotoxicity in vivo, using an acutely toxic dose of the PA senecionine in mice, in combination with intravital two-photon microscopy, histology, clinical chemistry, and in vitro experiments with primary mouse hepatocytes and liver sinusoidal endothelial cells (LSECs). We observed pericentral LSEC necrosis together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased sinusoidal platelet aggregation in the damaged tissue regions. In vitro experiments showed no cytotoxicity to freshly isolated LSECs up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs with an EC50 of approximately 22 µM. The cytochrome P450 (CYP)-dependency of senecionine bioactivation was confirmed in CYP reductase-deficient mice where no PA-induced hepatotoxicity was observed. Therefore, toxic metabolites of senecionine are generated by hepatic CYPs, and may be partially released from hepatocytes leading to destruction of LSECs in the pericentral region of the liver lobules. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by transcriptional deregulation of hepatic bile salt transporters like Abcb11 or Abcc1. In conclusion, senecionine destroys LSECs although the toxic metabolite is formed in a CYP-dependent manner in the adjacent pericentral hepatocytes.

Human CYP3A4-mediated toxification of the pyrrolizidine alkaloid lasiocarpine.

Pyrrolizidine alkaloids (PA) are widely distributed phytotoxins contaminating food and feed. Hepatic enzymes are considered to bioactivate PA. Previous studies showed differences in the metabolism rate in liver homogenates of different species. Thus, uncertainty remains with respect to the relevance of human metabolism. Our study aimed to analyze whether the PA representative lasiocarpine is toxified by human cytochrome P450 (CYP) enzymes. We compared the metabolic elimination of lasiocarpine in the presence of rat and human S9 fractions and liver microsomes. Experiments with the potent CYP3A/Cyp3a inhibitor ketoconazole and supersomes containing individual human and rat CYPs revealed that enzymes of the CYP3A/Cyp3a family of both species are of major relevance for lasiocarpine metabolism. To assess if metabolism by human CYP3A4 results in a toxification of lasiocarpine we performed experiments with V79 cells. γH2AX and micronucleus formation were analyzed as endpoints for genotoxicity. No effects were observed in the wildtype cells, which lack CYP activity. By contrast, a V79 clone engineered for expression of human CYP3A4 showed concentration-dependent γH2AX and micronucleus formation. Concluding, our results showed the CYP3A4-dependent formation of genotoxic metabolites of lasiocarpine. The results confirm previous data indicating the need to include metabolism of PA for human risk assessment.

Sensitization of Human Liver Cells Toward Fas-Mediated Apoptosis by the Metabolically Activated Pyrrolizidine Alkaloid Lasiocarpine.

SCOPE: Pyrrolizidine alkaloids (PAs) are common phytotoxins. Intoxication can lead to liver damage. Previous studies showed PA-induced apoptosis in liver cells. However, the exact role of the extrinsic apoptotic pathway has not been investigated yet. This study aims to analyze whether the PA representative lasiocarpine sensitizes human liver cells toward extrinsic Fas-mediated apoptosis. METHODS AND RESULTS: HepG2 cells with limited xenobiotic metabolic activity are used to analyze metabolism-dependent effects. External in vitro metabolism is simulated using rat or human liver enzymes. Additionally, metabolically competent HepaRG cells are used to confirm the observed effects in a human liver cell system with internal xenobiotic metabolism. Metabolized lasiocarpine decreases cell viability and induces Fas receptor gene expression in both cell lines. Increased Fas receptor protein expression on the cell surface is demonstrated by flow cytometry. The addition of a Fas ligand-simulating antibody induces apoptosis. Induction of extrinsic Fas-mediated apoptosis is verified by Western blotting for cleaved caspase 8, the initiator caspase of extrinsic apoptosis. All effects are dependent on lasiocarpine metabolism. CONCLUSION: The results demonstrate that metabolically metabolized lasiocarpine sensitizes human liver cells toward Fas-mediated apoptosis. They broaden our knowledge on the hepatotoxic molecular mechanisms of PA as widely distributed food contaminants.

Pyrrolizidine alkaloid-induced alterations of prostanoid synthesis in human endothelial cells.

Pyrrolizidine alkaloids (PA) are a group of secondary plant metabolites belonging to the most widely distributed natural toxins. PA intoxication of humans leads to severe liver damage, such as hepatomegaly, hepatic necrosis, fibrosis and cirrhosis. An acute consequence observed after ingestion of high amounts of PA is veno-occlusive disease (VOD) where the hepatic sinusoidal endothelial cells are affected. However, the mechanisms leading to VOD after PA intoxication remain predominantly unknown. Thus, we investigated PA-induced molecular effects on human umbilical vein endothelial cells (HUVEC). We compared the effects of PA with the effects of PA metabolites obtained by in vitro metabolism using liver homogenate (S9 fraction). In vitro-metabolized lasiocarpine and senecionine resulted in significant cytotoxic effects in HUVEC starting at 300 µM. Initial molecular effect screening using a PCR array with genes associated with endothelial cell biology showed PA-induced upregulation of the Fas receptor, which is involved in extrinsic apoptosis, and regulation of a number of interleukins, as well as of different enzymes relevant for prostanoid synthesis. Modulation of prostanoid synthesis was subsequently studied at the mRNA and protein levels and verified by increased release of prostaglandin I2 as the main prostanoid of endothelial cells. All effects occurred only with in vitro-metabolically activated PA lasiocarpine and senecionine. By contrast, no effect was observed for the PA echimidine, heliotrine, lasiocarpine, senecionine, senkirkine and platyphylline in the absence of an external metabolizing system up to the highest tested concentration of 500 µM. Overall, our results confirm the metabolism-dependent toxification of PA and elucidate the involved pathways. These include induction of inflammatory cytokines and deregulation of the prostanoid synthesis pathway in endothelial cells, linking for the first time PA-dependent changes in prostanoid release to distinct alterations at the mRNA and protein levels of enzymes of prostanoid synthesis.

It takes more than a coating to get nanoparticles through the intestinal barrier in vitro.

Size and shape are crucial parameters which have impact on the potential of nanoparticles to penetrate cell membranes and epithelial barriers. Current research in nanotoxicology additionally focuses on particle coating. To distinguish between core- and coating-related effects in nanoparticle uptake and translocation, two nanoparticles equal in size, coating and charge but different in core material were investigated. Silver and iron oxide nanoparticles coated with poly (acrylic acid) were chosen and extensively characterized by small-angle x-ray scattering, nanoparticle tracing analysis and transmission electron microscopy (TEM). Uptake and transport were studied in the intestinal Caco-2 model in a Transwell system with subsequent elemental analysis. TEM and ion beam microscopy were conducted for particle visualization. Although equal in size, charge and coating, the behavior of the two particles in Caco-2 cells was different: while the internalized amount was comparable, only iron oxide nanoparticles additionally passed the epithelium. Our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. Knowledge about the different roles of the particle coating and core materials in crossing biological barriers will facilitate toxicological risk assessment of nanoparticles and contribute to the optimization of pharmacokinetic properties of nano-scaled pharmaceuticals.

Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells.

Because of the rising application of nanoparticles in food and food-related products, we investigated the influence of the digestion process on the toxicity and cellular uptake of silver nanoparticles for intestinal cells. The main food components--carbohydrates, proteins and fatty acids--were implemented in an in vitro digestion process to simulate realistic conditions. Digested and undigested silver nanoparticle suspensions were used for uptake studies in the well-established Caco-2 model. Small-angle X-ray scattering was used to estimate particle core size, size distribution and stability in cell culture medium. Particles proved to be stable and showed radii from 3.6 to 16.0 nm. Undigested particles and particles digested in the presence of food components were comparably taken up by Caco-2 cells, whereas the uptake of particles digested without food components was decreased by 60%. Overall, these findings suggest that in vivo ingested poly (acrylic acid)-coated silver nanoparticles may reach the intestine in a nanoscaled form even if enclosed in a food matrix. While appropriate for studies on the uptake into intestinal cells, the Caco-2 model might be less suited for translocation studies. Moreover, we show that nanoparticle digestion protocols lacking food components may lead to misinterpretation of uptake studies and inconclusive results.

Examination of the phenolic profile and antioxidant activity of the leaves of the Australian native plant Smilax glyciphylla.

Together with the sweet principle component glycyphyllin A (3), seven phenolic compounds including two new dihydrochalcone rhamnopyranosides, glycyphyllin B (1) and glycyphyllin C (2), and five known flavonoids, catechin (4), kaempferol-3-O-ß-D-glucopyranoside (5), quercetin-3-O-ß-D-glucopyranoside (6), kaempferol-3-O-ß-neohesperidoside (7), and 2R,3R-dihydrokaempferol-3-O-ß-D-glucopyranoside (8), have been isolated from the ethanolic extract of the leaves of Smilax glyciphylla for the first time. The structures of these compounds were characterized by spectroscopic methods including UV, MS, and 1D and 2D NMR. In vitro antioxidant capacity tests employing FRAP and DPPH assays indicated that 1, 4, and 6 exhibited potent antioxidant activity and are the key phenolics responsible for the antioxidant activity of the leaf extract of S. glyciphylla.