Genotoxicity assessment: opportunities, challenges and perspectives for quantitative evaluations of dose-response data.

Genotoxicity data are mainly interpreted in a qualitative way, which typically results in a binary classification of chemical entities. For more than a decade, there has been a discussion about the need for a paradigm shift in this regard. Here, we review current opportunities, challenges and perspectives for a more quantitative approach to genotoxicity assessment. Currently discussed opportunities mainly include the determination of a reference point (e.g., a benchmark dose) from genetic toxicity dose-response data, followed by calculation of a margin of exposure (MOE) or derivation of a health-based guidance value (HBGV). In addition to new opportunities, major challenges emerge with the quantitative interpretation of genotoxicity data. These are mainly rooted in the limited capability of standard in vivo genotoxicity testing methods to detect different types of genetic damage in multiple target tissues and the unknown quantitative relationships between measurable genotoxic effects and the probability of experiencing an adverse health outcome. In addition, with respect to DNA-reactive mutagens, the question arises whether the widely accepted assumption of a non-threshold dose-response relationship is at all compatible with the derivation of a HBGV. Therefore, at present, any quantitative genotoxicity assessment approach remains to be evaluated case-by-case. The quantitative interpretation of in vivo genotoxicity data for prioritization purposes, e.g., in connection with the MOE approach, could be seen as a promising opportunity for routine application. However, additional research is needed to assess whether it is possible to define a genotoxicity-derived MOE that can be considered indicative of a low level of concern. To further advance quantitative genotoxicity assessment, priority should be given to the development of new experimental methods to provide a deeper mechanistic understanding and a more comprehensive basis for the analysis of dose-response relationships.

Occurrence of Alkenylbenzenes in Plants: Flavours and Possibly Toxic Plant Metabolites.

Alkenylbenzenes are naturally occurring secondary plant metabolites. While some of them are proven genotoxic carcinogens, other derivatives need further evaluation to clarify their toxicological properties. Furthermore, data on the occurrence of various alkenylbenzenes in plants, and especially in food products, are still limited. In this review, we tempt to give an overview of the occurrence of potentially toxic alkenylbenzenes in essential oils and extracts from plants used for flavoring purposes of foods. A focus is layed on widely known genotoxic alkenylbenzenes, such as safrole, methyleugenol, and estragole. However, essential oils and extracts that contain other alkenylbenzenes and are also often used for flavoring purposes are considered. This review may re-raise awareness of the need for quantitative occurrence data for alkenylbenzenes in certain plants but especially in final plant food supplements, processed foods, and flavored beverages as the basis for a more reliable exposure assessment of alkenylbenzenes in the future.

3-MCPD as contaminant in processed foods: State of knowledge and remaining challenges.

3-Chloro-1,2-propanediol (3-MCPD) and its fatty acid esters (FE) are present as contaminants in different processed foods. Based on the available toxicological data the potential risk of 3-MCPD and its FE to human health was assessed by risk assessment authorities, including the European Food Safety Authority (EFSA). Considering the available data, EFSA concluded that 3-MCPD is a non-genotoxic compound exhibiting secondary carcinogenic effects in rodents. A tolerable daily intake of 2 µg/kg body weight and day was derived by EFSA for free and ester-bound 3-MCPD in 2018. However, there are still different pending issues that have remained unclear until now. Here, we summarize the current knowledge regarding 3-MCPD and its FE with a focus on pending issues regarding exposure assessment via biomarkers as well as the identification of (toxic) metabolites formed after exposure to FE of 3-MCPD and their modes of action.

Nrf2 induces interleukin-6 (IL-6) expression via an antioxidant response element within the IL-6 promoter.

IL-6 gene expression is controlled by a promoter region containing multiple regulatory elements such as NF-κB, NF-IL6, CRE, GRE, and TRE. In this study, we demonstrated that TRE, found within the IL-6 promoter, is embedded in a functional antioxidant response element (ARE) matching an entire ARE consensus sequence. Further, point mutations of the ARE consensus sequence in the IL-6 promoter construct selectively eliminate ARE but not TRE activity. Nrf2 is a redox-sensitive transcription factor which provides cytoprotection against electrophilic and oxidative stress and is the most potent activator of ARE-dependent transcription. Using Nrf2 knock-out mice we demonstrate that Nrf2 is a potent activator of IL-6 gene transcription in vivo. Moreover, we show evidence that Nrf2 is the transcription factor that activates IL6 expression in a cholestatic hepatitis mouse model. Our findings suggest a possible role of IL-6 in oxidative stress defense and also give indication about an important function for Nrf2 in the regulation of hematopoietic and inflammatory processes.

Risk assessment of nanomaterials in cosmetics: a European union perspective.

In Europe, the data requirements for the hazard and exposure characterisation of chemicals are defined according to the REACH regulation and its guidance on information requirements and chemical safety assessment (Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), and its guidance documents; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2006:396:0001:0849:EN:PDF ; and at: http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_en.htm ). This is the basis for any related risk assessment. The standard reference for the testing of cosmetic ingredients is the SCCP's 'Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation' (The SCCP's Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation (2006); available at: http://ec.europa.eu/health/ph_risk/committees/04_sccp/docs/sccp_o_03j.pdf ), which refers to the OECD guidelines for the testing of chemicals (The OECD Guidelines for the Testing of Chemicals as a collection of the most relevant internationally agreed testing methods used by government, industry and independent laboratories to assess the safety of chemical products; available at: http://www.oecd.org/topic/0,2686,en_2649_34377_1_1_1_1_37407,00.html ). According to the cosmetics directive [76/768/EEC], compounds that are classified as mutagenic, carcinogenic or toxic to reproduction are banned for the use in cosmetic products. Since December 2010, the respective labelling is based on the rules of regulation (EC) No. 1272/2008 (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal L 353, 31/12/2008, pages 1-1355; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:353:0001:1355:en:PDF ) on classification, labelling and packaging of substances and mixtures (CLP). There is no further impact from the CLP regulation on cosmetic products, because regulation (EC) No. 1223/2009 on cosmetic products defines its own labelling rules (Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic products; available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF ). Special notification procedures are mandatory for preservatives, colourants and UV-filters where a safety approval from the European 'Scientific Committee on Consumer Safety' (SCCS) is needed prior to marketing. The risk assessment of nanomaterials in consumer products still poses a significant challenge as highlighted by the example of UV-filters in sunscreens since nanomaterials cannot be classified as a homogenous group of chemicals but still need to be addressed in risk characterisation on a case by case basis.

Myristicin and Elemicin: Potentially Toxic Alkenylbenzenes in Food.

Alkenylbenzenes represent a group of naturally occurring substances that are synthesized as secondary metabolites in various plants, including nutmeg and basil. Many of the alkenylbenzene-containing plants are common spice plants and preparations thereof are used for flavoring purposes. However, many alkenylbenzenes are known toxicants. For example, safrole and methyleugenol were classified as genotoxic carcinogens based on extensive toxicological evidence. In contrast, reliable toxicological data, in particular regarding genotoxicity, carcinogenicity, and reproductive toxicity is missing for several other structurally closely related alkenylbenzenes, such as myristicin and elemicin. Moreover, existing data on the occurrence of these substances in various foods suffer from several limitations. Together, the existing data gaps regarding exposure and toxicity cause difficulty in evaluating health risks for humans. This review gives an overview on available occurrence data of myristicin, elemicin, and other selected alkenylbenzenes in certain foods. Moreover, the current knowledge on the toxicity of myristicin and elemicin in comparison to their structurally related and well-characterized derivatives safrole and methyleugenol, especially with respect to their genotoxic and carcinogenic potential, is discussed. Finally, this article focuses on existing data gaps regarding exposure and toxicity currently impeding the evaluation of adverse health effects potentially caused by myristicin and elemicin.

A knowledge-based search engine to navigate the information thicket of nanotoxicology.

The risk assessment of nano-sized materials (NM) currently suffers from great uncertainties regarding their putative toxicity for humans and the environment. An extensive amount of the respective original research literature has to be evaluated before a targeted and hypothesis-driven Environmental and Health Safety research can be stipulated. Furthermore, to comply with the European animal protection legislation in vitro testing has to be preferred whenever possible. Against this background, there is the need for tools that enable producers of NM and risk assessors for a fast and comprehensive data retrieval, thereby linking the 3Rs principle to the hazard identification of NM. Here we report on the development of a knowledge-based search engine that is tailored to the particular needs of risk assessors in the area of NM. Comprehensive retrieval of data from studies utilising in vitro as well as in vivo methods relying on the PubMed database is presented exemplarily with a titanium dioxide case study. A fast, relevant and reliable information retrieval is of paramount importance for the scientific community dedicated to develop safe NM in various product areas, and for risk assessors obliged to identify data gaps, to define additional data requirements for approval of NM and to create strategies for integrated testing using alternative methods.

Alkenylbenzenes in Foods: Aspects Impeding the Evaluation of Adverse Health Effects.

Alkenylbenzenes are naturally occurring secondary plant metabolites, primarily present in different herbs and spices, such as basil or fennel seeds. Thus, alkenylbenzenes, such as safrole, methyleugenol, and estragole, can be found in different foods, whenever these herbs and spices (or extracts thereof) are used for food production. In particular, essential oils or other food products derived from the aforementioned herbs and spices, such as basil-containing pesto or plant food supplements, are often characterized by a high content of alkenylbenzenes. While safrole or methyleugenol are known to be genotoxic and carcinogenic, the toxicological relevance of other alkenylbenzenes (e.g., apiol) regarding human health remains widely unclear. In this review, we will briefly summarize and discuss the current knowledge and the uncertainties impeding a conclusive evaluation of adverse effects to human health possibly resulting from consumption of foods containing alkenylbenzenes, especially focusing on the genotoxic compounds, safrole, methyleugenol, and estragole.

Kavalactones protect neural cells against amyloid beta peptide-induced neurotoxicity via extracellular signal-regulated kinase 1/2-dependent nuclear factor erythroid 2-related factor 2 activation.

One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide (AP), which can initiate a cascade of oxidative events that may result in neuronal death. Because nuclear factor erythroid 2-related factor 2 (Nrf2) is the major regulator for a battery of genes encoding detoxifying and antioxidative enzymes via binding to the antioxidant response element (ARE), it is of great interest to find nontoxic activators of Nrf2 rendering neuronal cells more resistant to AP toxicity. Using ARE-luciferase assay and Western blot, we provide evidence that the kavalactones methysticin, kavain, and yangonin activate Nrf2 time- and dose-dependently in neural PC-12 and astroglial C6 cells and thereby up-regulate cytoprotective genes. Viability and cytotoxicity assays demonstrate that Nrf2 activation is able to protect neural cells from amyloid beta-(1-42) induced neurotoxicity. Down-regulation of Nrf2 by small hairpin RNA as well as extracellular signal-regulated kinase 1/2 inhibition abolishes cytoprotection. We further give evidence that kavalactone-mediated Nrf2 activation is not dependent on oxidative stress production. Our results demonstrate that kavalactones attenuate amyloid beta-peptide toxicity by inducing protective gene expression mediated by Nrf2 activation in vitro. These findings indicate that the use of purified kavalactones might be considered as an adjunct therapeutic strategy to combat neural demise in Alzheimer disease and other oxidative stress-related diseases.

Increased brain levels of 4-hydroxy-2-nonenal glutathione conjugates in severe Alzheimer's disease.

In the last decade an important role for the progression of neuronal cell death in Alzheimer's disease (AD) has been ascribed to oxidative stress. trans-4-Hydroxy-2-nonenal, a product of lipid peroxidation, forms conjugates with a variety of nucleophilic groups such as thiols or amino moieties. Here we report for the first time the quantitation of glutathione conjugates of trans-4-hydroxy-2-nonenal (HNEGSH) in the human postmortem brain using the specific and very sensitive method of electrospray ionization triple quadrupole mass spectrometry (ESI-MS-MS). Levels of HNEGSH conjugates calculated as the sum of three chromatographically separated diastereomers were determined in hippocampus, entorhinal cortex, substantia innominata, frontal and temporal cortex, as well as cerebellum from patients with AD and controls matched for age, gender, postmortem delay and storage time. Neither age, nor postmortem delay, nor storage time did correlate with levels of HNEGSH conjugates which ranged between 1 and 500 pmol/g fresh weight in the brain areas examined. The brain specimen from patients with clinically and neuropathologically probable AD diagnosed according to criteria of the consortium to establish a registry for AD (CERAD) show increased levels of HNEGSH in the temporal and frontal cortex, as well as in the substantia innominata. Classification of disease severity according to Braak and Braak, which takes into consideration the amount of neurofibrillary tangles and neuritic plaques, revealed highest levels of HNEGSH in the substantia innominata and the hippocampus, two brain regions known to be preferentially affected in AD. These results substantiate the link between conjugates of glutathione with a product of lipid peroxidation and Alzheimer's disease and justify further studies to evaluate the role of HNE metabolites as potential biomarkers for disease progression in AD.

Formation of 4-hydroxy-2-nonenal protein adducts in the ischemic rat heart after transplantation.

BACKGROUND: Free radicals formed during ischemia and reperfusion can lead to lipid peroxidation (LPO) and the formation of 4-hydroxy-2-nonenal (4-HNE), one of the most toxic products of LPO. Using a heterotopic rat heart transplantation model we investigated endogenous 4-HNE formation as a response to cold storage of the transplant and warm blood reperfusion in the recipient. METHODS: Lewis rat hearts were subjected to 30, 240 or 480 minutes of 4 degrees C cold ischemia in Bretschneider cardioplegic solution without or with transplantation and 240-minute reperfusion in F344 recipients. The amount of 4-HNE modified proteins was quantified in rat heart cryosections with an antibody recognizing cysteine-, histidine- and lysine-4-HNE Michael adducts and image analysis of immunostained tissue. RESULTS: We detected 4-HNE-modified proteins in ischemic rat hearts after transplantation and reperfusion. In hearts submitted to ischemia only, 4-HNE-protein adducts comprised 0.7 +/- 0.3% (30 minutes), 0.7 +/- 0.4% (240 minutes) and 0.2 +/- 0.1% (480 minutes) (mean +/- SEM) of the tissue area. Transplantation and reperfusion in the recipient significantly increased the amount of protein adducts to 6.8 +/- 2.6% (p = 0.041), 5.2 +/- 1.4% (p = 0.009) and 5.7 +/- 0.9% (p = 0.002) in 30-, 240- and 480-minute ischemic hearts, respectively. CONCLUSIONS: Under the conditions applied in the present study, cold ischemia for >30 minutes did not significantly alter the amount of 4-HNE protein adducts. However, because after transplantation and reperfusion, 6% of heart tissue consisted of 4-HNE-modified proteins, it can be assumed that this damage negatively affects long-term survival of the transplant.

Heterotopic rat heart transplantation: severe loss of glutathione in 8-hour ischemic hearts.

BACKGROUND: Tissue damage caused by reactive oxygen species (ROS) formed during ischemia/reperfusion seems to be an important risk factor in the failure of transplanted hearts. Although endogenous anti-oxidants protect the myocardium against free radical attack under physiologic conditions, their capacity may become limited during severe oxidative stress. Thus, we investigated the effect of 8-hour cold ischemia on the myocardial anti-oxidative defense system in a heterotopic rat heart transplantation model. METHODS: Lewis rat hearts were subjected to 30 or 480 minutes of 4 degrees C cold ischemia in Bretschneider cardioplegic solution with or without transplantation and reperfusion (30 or 240 minutes) into F344 recipients. Activity levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase (GST), and concentrations of glutathione (GSH), glutathione disulfide (GSSG) and lipid hydroperoxides (LOOH) were analyzed in heart homogenates. For histology, cross-sections of the ventricles were stained with hematoxylin-eosin. RESULTS: Except for GST, enzyme activities and GSSG concentration increased and the glutathione redox ratio (GSH/GSH + 2GSSG) significantly decreased in 480-minute ischemic hearts compared with those with 30-minute ischemia. Reperfusion dramatically decreased both GSH and GSSG and increased LOOH formation but without severe histopathologic findings in the transplants. Applying a tree-structured classifier technique, GSH and LOOH were identified as significant features indicative of transplantation-induced oxidative stress. CONCLUSIONS: In the present study severe loss of glutathione and formation of LOOH indicated transplantation-induced oxidative stress in the rat heart; therefore, alterations of these parameters may hint at relevant deficits in the myocardial anti-oxidative defense and may also predict subsequent tissue damage.

The relevance of iron in the pathogenesis of Parkinson's disease.

Investigations that revealed increased levels of iron in postmortem brains from patients with Parkinson's disease (PD) as compared to those from individuals not suffering from neurological disorders are reported. The chemical natures in which iron predominates in the brain and the relevance of neuromelanin for neuronal iron binding are discussed. Major findings have been that iron levels increase with the severity of neuropathological changes in PD, presumably due to increased transport through the blood-brain barrier in late stages of parkinsonism. Glial iron is mainly stored as ferric iron in ferritin, while neuronal iron is predominantly bound to neuromelanin. Iron overload may induce progressive degeneration of nigrostriatal neurons by facilitating the formation of reactive biological intermediates, including reactive oxygen species, and the formation of cytotoxic protein aggregates. There are indications that iron-mediated neuronal death in PD proceeds retrogradely. These results are also discussed with respect to their relevance for disease progression in relation to cytotoxic alpha-synuclein protofibril formation.