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In studies of hominin adaptations to fire use, the role of the aryl hydrocarbon receptor (AHR) in the evolution of detoxification has been highlighted, including statements that the modern human AHR confers a significantly better capacity to deal with toxic smoke components than the Neanderthal AHR. To evaluate this, we compared the AHR-controlled induction of cytochrome P4501A1 (CYP1A1) mRNA in HeLa human cervix epithelial adenocarcinoma cells transfected with an Altai-Neanderthal or a modern human reference AHR expression construct, and exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We compared the complete AHR mRNA sequences including the untranslated regions (UTRs), maintaining the original codon usage. We observe no significant difference in CYP1A1 induction by TCDD between Neanderthal and modern human AHR, whereas a 150–1,000 times difference was previously reported in a study of the AHR coding region optimized for mammalian codon usage and expressed in rat cells. Our study exemplifies that expression in a homologous cellular background is of major importance to determine (ancient) protein activity. The Neanderthal and modern human dose–response curves almost coincide, except for a slightly higher extrapolated maximum for the Neanderthal AHR, possibly caused by a 5′-UTR G-variant known from modern humans (rs7796976). Our results are strongly at odds with a major role of the modern human AHR in the evolution of hominin detoxification of smoke components and consistent with our previous study based on 18 relevant genes in addition to AHR, which concluded that efficient detoxification alleles are more dominant in ancient hominins, chimpanzees, and gorillas than in modern humans.

In studies of hominin adaptations to fire use, the role of the aromatic hydrocarbon receptor (AHR) in the evolution of hominin detoxification, especially regarding toxic smoke components, has been highlighted, including statements that the modern human AHR is significantly better at dealing with smoke toxins. We compared the AHR-controlled induction of cytochrome P450 1A1 (CYP1A1) in cultured cells transfected with an Altai-Neanderthal respectively a modern human reference AHR expression construct, and exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We compared the complete AHR mRNA sequences including the untranslated regions (UTRs), maintaining the original codon usage, in HeLa human cervix epithelial adenocarcinoma cells. Our experiments complement a previous study of the AHR coding region optimized for mammalian codon usage and expressed in rat cells. Our results show no significant difference in CYP1A1 induction by TCDD between Neanderthal and modern human AHR (instead of a previously reported 150-1000 times difference range before), demonstrating that expression in a homologous cellular background is of major importance. The two dose-response curves almost coincide, except for a higher extrapolated maximum induction level for the Neanderthal AHR, possibly caused by a 5’-UTR G-variant known from modern humans (rs7796976). Our results are strongly at odds with a major role of the modern human AHR in the evolution of hominin detoxification of smoke components and consistent with our previous study which concluded that efficient detoxification alleles are more dominant in ancient hominins than in modern humans based on 18 relevant genes in addition to AHR.

Surface charge is often hypothesized to influence toxicity of nanoparticles (NPs) including polymeric nanoparticles (PNPs) while oxidative stress is considered to be an important mode of action (MOA) for such toxicity. In order to investigate the role of membrane disturbance and oxidative stress in the MOA of PNPs, the cytotoxicity and a range of related cellular endpoints induced by monodisperse, fluorescent, cationic and anionic polystyrene nanoparticles (PSNPs) of 50 and 100 nm sizes were investigated in vitro in macrophage NR8383 cells. Only amine-terminated cationic PSNPs exhibited cytotoxicity which was accompanied by induction of intracellular reactive oxygen species (ROS), increased levels of cytoplasmic free calcium, a reduced phagocytic index, a reduced mitochondrial membrane potential (ΔΨm) and a decreased intracellular ATP content with the effects being more pronounced for 50 nm than 100 nm PSNPs. Both cationic and anionic PSNPs were found to increase the roughness of the cell membrane with the effect being more profound for cationic PSNPs. The pattern of protection by cellular antioxidants against the effects induced by positive PSNPs was similar to the pattern of protection against effects induced by the mitochondrial electron transport disrupting agent 2,4-dinitrophenol (DNP) and dissimilar to that for protection against the model compound for oxidative stress, i.e. hydrogen peroxide (H2O2). Surface charge influences the cellular interaction for NPs. The results collectively indicated that membrane interaction, and disturbance of the mitochondrial electronic transport chain (ETC) may represent a principal mechanism of toxicity for cationic PSNPs resulting in ROS production and oxidative stress as secondary effects.

Studies of the defence capacity of ancient hominins against toxic substances may contribute importantly to the reconstruction of their niche, including their diets and use of fire. Fire usage implies frequent exposure to hazardous compounds from smoke and heated food, known to affect general health and fertility, probably resulting in genetic selection for improved detoxification. To investigate whether such genetic selection occurred, we investigated the alleles in Neanderthals, Denisovans and modern humans at gene polymorphisms well-known to be relevant from modern human epidemiological studies of habitual tobacco smoke exposure and mechanistic evidence. We compared these with the alleles in chimpanzees and gorillas. Neanderthal and Denisovan hominins predominantly possess gene variants conferring increased resistance to these toxic compounds. Surprisingly, we observed the same in chimpanzees and gorillas, implying that less efficient variants are derived and mainly evolved in modern humans. Less efficient variants are observable from the first early Upper Palaeolithic hunter-gatherers onwards. While not clarifying the deep history of fire use, our results highlight the long-term stability of the genes under consideration despite major changes in the hominin dietary niche. Specifically for detoxification gene variants characterised as deleterious by epidemiological studies, our results confirm the predominantly recent appearance reported for deleterious human gene variants, suggesting substantial impact of recent human population history, including pre-Holocene expansions.

Trinil: Masa lalu, Sekarang dan Masa Depan Sebuah Situs Bersejarah. Dusun Trinil menjadi terkenal dengan ditemukannya Pithecanthropus erectus, sekarang Homo erectus, oleh Dubois pada tahun 1891. Setelah ekskavasi Dubois, pada tahun 1907 sebuah ekspedisi besar-besaran dipimpin oleh E. Selenka berlangsung di lokasi yang sama. Selain fosil-fosil sisa manusia, puluhan ribu fosil vertebrata lain dan moluska ditemukan dalam ekskavasi Dubois dan Selenka antara tahun 1891 dan 1907. Koleksi ini sekarang disimpan di Naturalis di Leiden (Belanda) dan di Museum für Naturkunde di Berlin (Jerman). Studi yang berlangsung saat ini terhadap koleksi-koleksi itu mendorong perlunya penelitian baru di lapangan. Tujuannya selain untuk mengetahui potensi situs juga untuk menjawab pertanyaan-pertanyaan yang muncul dalam studi koleksi. Parit penggalian Dubois dan ekspedisi Selenka dikontekstualisasikan dalam peta geografi modern berdasarkan data historis, bahan fotografi yang masih ada, dan peninjauan lapangan 2014/2015. Potensi untuk menemukan tinggalan pada ‘Hauptknochenschicht’ (HK) cukup besar di tepi kiri sungai Solo, di selatan penggalian Dubois yang asli, termasuk di tepi kiri disebelah timur lokasi yang digali. Pertanyaan yang masih tersisa, antara lain menyangkut stratigrafi situs, umur fauna Trinil dan Homo erectus, dan homogenitas himpunan HK, diharapkan dapat terjawab melalui penelitian baru yang akan dilaksanakan di situs ini. Abstract. Trinil became famous through the discovery of Pithecanthropus erectus, now Homo erectus, by Dubois in 1891. After Dubois’ excavations it was the expedition led by E. Selenka in 1907 performing large scale fieldwork at the location. Apart from the hominin remains, thousands of other vertebrate and molluscan fossils were excavated by both Dubois and Selenka between 1891 and 1908. These collections are currently housed at Naturalis in Leiden (The Netherlands) and the Museum für Naturkunde in Berlin (Germany). Ongoing studies of these collections have raised questions that warrant new fieldwork. This study aimed to establish the site‘s present potential to solve extant research questions. The excavation trenches of Dubois and the Selenka expedition were contextualized within a modern geographical map, based on historical data, extant photographic material and a 2014/2015 field trip. The potential to reach the find bearing Hauptknochenschicht (HK) is high at the left bank of the Solo river, south of Dubois’ original excavations. Also the left bank directly east of the former excavation pits has a good potential. Still remaining questions concerning the site stratigraphy, the age of the Trinil fauna, including the Homo erectus finds, and the homogeneity of the HK assemblage, might be resolved by new fieldwork.

The production of drinking water from river water requires a certain minimal river water quality. The Association of River Rhine Water Works (RIWA), therefore, operates a monitoring network. In vitro mutagenicity studies have shown that the genotoxicity of the River Rhine water steadily decreased from 1981 until 2001. Compared to a study in 1978, a decrease in genotoxicity was also observed in an in vivo genotoxicity study in 2005, in which Eastern mudminnows (Umbra pygmaea) were exposed to River Rhine water, and gill cells were used for the Sister Chromatid Exchange (SCE) test and the Comet assay. In this 2005 study, the in vivo genotoxicity increased upon extending exposure of the fish from 3 to 11 days. Therefore, the objectives of this study were to investigate (i) whether new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978, (ii) whether the Comet assay is a suitable alternative to the SCE assay, and (iii) whether further prolonged exposure results in a further increase in in vivo genotoxicity. The new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978. The Comet assay is a useful addition but does not provide a substitute for the SCE endpoint in these in vivo genotoxicity studies. Prolonging the exposure time of Eastern mudminnows to River Rhine water from 11 to 42 days did not give a significant increase in SCEs and DNA damage (Comet assay) in gill cells. Mol. Mutagen. 2012. (c) 2012 Wiley Periodicals, Inc.

UV/H2O2 treatment can be part of the process converting surface water to drinking water, but would pose a potential problem when resulting in genotoxicity. This study investigates the genotoxicity of samples collected from the water treatment plant Andijk, applying UV/H2O2 treatment with an electrical energy dose of 0.54 kWh/m(3) and a H2O2 dose of 6 mg/l. Genotoxicity was tested in vitro using the Ames and Comet assay. All samples showed negative results. Samples were also tested in in vivo genotoxicity tests in Eastern mudminnow fish (Umbra pygmaea) by a sister chromatid exchange (SCE) and a Comet assay. No significant increases in SCEs were observed, but gill cells isolated from fish exposed to water obtained immediately after UV/H2O2 treatment and to Lake IJsselmeer water showed significantly increased DNA damage in the Comet assay. All other samples tested negative in this Comet assay. This indicates that DNA damaging compounds may result from the UV/H2O2 treatment, but also that these can be efficiently eliminated upon granular activated carbon (GAC) treatment of the water. It is concluded that when combined with this subsequent GAC treatment, UV/H2O2 treatment for the production of drinking water from surface water is not of concern with respect to genotoxicity.

Analisis Deskriptif Alat Batu Palaeolithik dari Sulawesi, Hasil Ekspedisi Indonesia-Belanda pada tahun 1970. Studi ini menganalisis artefak temuan ekspedisi Indonesia-Belanda di tahun 1970 di Marale di hulu dan Beru di hilir Sungai Wallanae; termasuk menguji hipotesis bahwa penghalusan material kasar di hilir terjadi pada artefak kecil. Batu gamping, kersikan, dan rijang merupakan bahan yang dominan. Artefak umumnya mengalami abrasi dan pembundaran dari tingkat moderat hingga kuat. Hampir semua artefak terpatinasi. Teknik ‘ crushing ’ merupakan tipe dominan dari persiapan bidang dorsal dekat dataran pukul. Dataran pukul umumnya datar dan ujung distal tipis. Himpunan serpih Marale yang umumnya lebih lebar dan panjang dibandingkan himpunan serpih Beru mendukung hipotesis tersebut. Kebanyakan alat serpih merupakan serut samping. Sebagai tambahan, berdasarkan klasifikasi morfologi yang baru diperkenalkan, umumnya batu inti (70%) memiliki platform tunggal, berbentuk pyramidal atau polihedral , walaupun ada yang double platform . Perkiraan pertanggalan van Heekeren dari 200 dan 100 ka agaknya tepat, sebagaimana publikasi van den Bergh yang mempertanggal artefak in situ dari ekskavasi di daerah yang sama di antara 194 dan 118 ka. Abstract. This study analysis lithic artefacts collected by the Indonesian-Dutch expedition to Sulawesi in 1970. In addition, the hypothesis was tested that downstream fining of coarse material results in smaller artefacts. The artefacts were collected by surveying in Marale (upstream) and Beru (downstream) along the Walanae River. Most artefacts were abraded and rounded. Almost all artefacts were patinated. Silificied limestone and chert were the predominant raw materials for making stone tools. Crushing was the predominant type of dorsal face preparation near the striking platform. The dominant platform type was plain and the dominant distal end feather. The width and the maximal length of the flakes of Marale were significantly larger than those of Beru, confirming the above hypothesis. Most flake tools were side scrapers. In addition to the functional standard classification also a new morphological classification was introduced. Most cores (70%) were single platformed, pyramidal or polyhedral, but also double platformed cores were present. Dating of the stone tools between 200 and 100 ka as earlier suggested by van Heekeren might be plausible based on a recently published study by van den Bergh (2016) who dated in situ artefacts excavated in the same region between 194 and 118 ka.

Quercetin has been shown to act as an anticarcinogen in experimental colorectal cancer (CRC). The aim of the present study was to characterize transcriptome and proteome changes occurring in the distal colon mucosa of rats supplemented with 10 g quercetin/kg diet for 11 wk. Transcriptome data analyzed with Gene Set Enrichment Analysis showed that quercetin significantly downregulated the potentially oncogenic mitogen-activated protein kinase (Mapk) pathway. In addition, quercetin enhanced expression of tumor suppressor genes, including Pten, Tp53, and Msh2, and of cell cycle inhibitors, including Mutyh. Furthermore, dietary quercetin enhanced genes involved in phase I and II metabolism, including Fmo5, Ephx1, Ephx2, and Gpx2. Quercetin increased PPARα target genes, and concomitantly enhanced expression of genes involved in mitochondrial fatty acid (FA) degradation. Proteomics performed in the same samples revealed 33 affected proteins, of which four glycolysis enzymes and three heat shock proteins were decreased. A proteome-transcriptome comparison showed a low correlation, but both pointed out toward altered energy metabolism. In conclusion, transcriptomics combined with proteomics showed that dietary quercetin evoked changes contrary to those found in colorectal carcinogenesis. These tumor-protective mechanisms were associated with a shift in energy production pathways, pointing at decreased cytoplasmic glycolysis and toward increased mitochondrial FA degradation. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA. Molecular Sequence Numbers: GENBANK: A23126, A24639, A26186, A26468, A35340, A35865, A40452, A54889, AAA18026, AAA40933, AAB23369, AAC52676, AAH62238, AAH64440, AAH78829, AAH87023, AAP13984, BAA28215, CAA65655, I52328, I65237, Q63654, S17189; Chemicals / CAS: mitogen activated protein kinase, 142243-02-5; peroxisome proliferator activated receptor alpha, 147258-70-6; quercetin, 117-39-5; Fatty Acids; Proteome; Quercetin, 117-39-5

Polyunsaturated fatty acid (PUFA) rich micro-algal oil was tested in vitro and compared with fish oil for antiproliferative properties on cancer cells in vitro. Oils derived from Crypthecodinium cohnii, Schizochytrium sp. and Nitzschia laevis, three commercial algal oil capsules, and menhaden fish oil were used in cell viability and proliferation tests with human colon adenocarcinoma Caco-2 cells. With these tests no difference was found between algal oil and fish oil. The nonhydrolysed algal oils and fish oil showed a much lower toxic effect on cell viability, and cell proliferation in Caco-2 cells than the hydrolysed oils and the free fatty acids (FFAs). Eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) were used as samples for comparison with the tested hydrolysed and nonhydrolysed oils. The hydrolysed samples showed comparative toxicity as the free fatty acids and no difference between algal and fish oil. Oxidative stress was shown to play a role in the antiproliferative properties of EPA and DHA, as alpha-tocopherol could partially reverse the EPA/DHA-induced effects. The results of the present study support a similar mode of action of algal oil and fish oil on cancer cells in vitro, in spite of their different PUFA content.

KEYWORDS Fibers; metals; gene expression; oxidative stress; cancer ABSTRACT Exposure to ambient air pollution (particles, fibres) is associated with pulmonary diseases and cancer. The mechanisms of induced health effects are believed to involve inflammation and oxidative stress. Oxidative stress mediated by airborne particles and/or fibres may arise from direct generation of reactive oxygen species (ROS) from the surface of particles/fibres, soluble compounds such as transition metals or organic compounds, and activation of inflammatory cells capable of generating ROS and reactive nitrogen species (RNS). Generation of ROS/RNS can directly cause covalent modifications to DNA or they can initiate the formation of genotoxic lipid hydroperoxides. The resulting oxidative DNA damage can lead to changed gene expression such as upregulation of tumor promoters and downregulation of tumor suppressor genes and thus may be implicated in cancer development. The present review describes the important role of free radicals in particle-/fibre- induced cellular damage, the interaction of ROS with target molecules, especially with DNA, and the modulation of specific genes and transcription factor caused by oxidative stress.

The effect of the flavonoid myricetin on the transport of the pro-carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) through differentiated Caco-2 monolayers, a model for the intestinal epithelium, is described. Myricetin causes an increase of the transport of PhIP from the apical to the basolateral compartment. This effect was observed at physiologically relevant concentrations of PhIP and myricetin. Cyclosporin A (MRP2 inhibitor) but not PSC833 (P-gp inhibitor) showed a similar effect on PhIP transport. The results indicate that myricetin induces an increased basolateral uptake of the pro-carcinogen PhIP, in part through inhibition of the MRP2 mediated excretion of PhIP from the intestinal cells back to the lumen. © 2005 Elsevier Ireland Ltd. All rights reserved.

The present study describes the effect of different flavonoids on the absorption of the pro-carcinogen PhIP through Caco-2 monolayers and the development of an in silico model describing this process taking into account passive diffusion and active transport of PhIP. Various flavonoids stimulated the apical to basolateral PhIP transport. Using the in silico model for flavone, kaempferol and chrysoeriol, the apparent Ki value for inhibition of the active transport to the apical side was estimated to be below 53 muM and for morin, robinetin and taxifolin between 164 and 268 microM. For myricetin, luteolin, naringenin and quercetin, the apparent Ki values were determined more accurately and amounted to 37.3, 12.2, 11.7 and 5.6 microM respectively. Additional experiments revealed that the apical to basolateral PhIP transport was also increased in the presence of a typical BCRP or MRP inhibitor with apparent Ki values in the same range as those of the flavonoids. This observation together with the fact that flavonoids are known to be inhibitors of MRPs and BCRP, corroborates that inhibition of these apical membrane transporters is involved in the flavonoid-mediated increased apical to basolateral PhIP transport. Based on the apparent Ki values obtained, it is concluded that the flavonols, at the levels present in the regular Western diet, are capable of stimulating the transport of PhIP through Caco-2 monolayers from the apical to the basolateral compartment. This points to flavonoid-mediated stimulation of the bioavailability of PhIP and, thus, a possible adverse effect of these supposed beneficial food ingredients.

In the present studies, several hypotheses were tested to explain previously reported differential effects of soy and casein on colon cancer biomarkers like cell proliferation, fecal fat, fecal bile acid, alkaline phosphatase, and magnesium excretion in rats. In Study 1, the effect of methionine, a limiting amino acid in soy protein and an amino acid that is thought to have a marked effect on colonic cell proliferation, was tested. It was concluded that methionine up to 1% in the diet had no effect on cell proliferation, using the 3H-thymidine assay. The same study revealed that fecal alkaline phosphatase excretion is a good marker for colonic epithelial damage and fecal magnesium excretion is not. In Study 2, the hypothesis was tested that soy fractions enriched with isoflavones and saponins may increase fat excretion and so influence colonic cell proliferation in rats. It was indeed shown that soy protein isolate and an ethanolic extract from soy protein isolate slightly increased fecal fat excretion (up to 1.7-fold). However, fecal water bile acid and free fatty acid concentrations were decreased after feeding soy protein-based diets compared with casein, and no difference in fecal alkaline phosphatase excretion was observed. In Study 3, the lytic potential of soy saponins and the interaction between saponins and some lytic bile acids were tested in vitro. Data suggest a protective effect from soy saponins by reducing lytic activity of cholic acid. The overall conclusion is that soy protein compared with casein influences several colon cancer risk parameters, indicating a more protective rather than a stimulating effect on colon cancer risk.

This article comments on some of the basic questions put forward in state-of-the-art discussions on hormesis. There seems to be a need for a better definition of the concept itself and reconsideration of whether all biphasic dose-response curves should be considered representative for hormesis. Hormesis may be restricted to phenomena that proceed by mechanisms that are broadly generalizable and represent possibly beneficial overcompensation in response to an adverse stimulus. Using the concept that hormesis is defined as such, the biphasic effect of quercetin on cell proliferation, but also several other receptor-mediated biphasic dose-response phenomena, should not be related to hormesis. Taking into account hormesis in the procedures for risk assessment on compounds characterised by a threshold for the adverse effect is another matter for considerable debate. In our opinion, this would require the reduction of safety factors, providing the possibility for beneficial hormesis-type effects for some people, at the cost of increased chances on adverse effects for other parts of the population. Whether this is a proper way forward remains to be discussed. Improvement of risk assessment strategies may include taking into account biphasic dose-response curves, but should rather start with the consideration of proper physiologically based pharmacokinetic (PBPK) models for better extrapolation of differences in toxicokinetics going from high- to low-dose exposure, as well as taking into account kinetics for gene repair systems. Without considering in vivo toxicokinetics in the in vitro models, extrapolation from in vitro biphasic dose-response curves on cell proliferation to in vivo cell proliferation is difficult to do. Altogether, it is concluded that hormesis is an important phenomenon, especially from the scientific point of view, but that its consequences for risk assessment and the possibilities for in vitro to in vivo extrapolation may remain limited without additional mechanistic insight.

The nutritional value of raw and popped (similar to popcorn preparation) seed proteins of two amaranth species, Amaranthus caudatus L and A cruentus L, was investigated. After popping, the true protein content in A caudatus and A cruentus decreased by 9 and 13% respectively. Among the amino acids, the loss of tyrosine due to the popping effect was the highest, followed by phenylalanine and methionine. Leucine was the first limiting amino acid in the raw samples, followed by lysine, while the reverse order was observed in the popped samples. The in vivo protein quality of raw and popped seeds was tested with male weanling rats and compared with wheat flour and casein samples. There was no difference between the in vivo digestibility of the raw and the popped seeds, although the in vitro digestibility was slightly higher for the popped samples. The protein efficiency ratio (PER) for all the amaranth seed samples was higher than that for the wheat sample, while the PER for the raw amaranth seed samples was close to that for the casein reference protein. The rat blood serum levels of total cholesterol, triglycerides and high-density lipoprotein cholesterol for all the amaranth samples were lower than those for the reference protein, while the wheat flour sample showed the lowest values. (C) 2004 Society of Chemical Industry.

Optimal in vitro conditions regarding quercetin solubility and stability were defined. Using these conditions, the effect of quercetin on proliferation of the colon carcinoma cell lines HCT-116 and HT29 and the mammary adenocarcinoma cell line MCF-7 was investigated. For the colon carcinoma cell lines, at relatively high concentrations, a significant decrease in cell proliferation was observed, providing a basis for claims on the anti-carcinogenic activity of quercetin. However, at lower concentrations, a subtle but significant stimulation of cell proliferation was observed for all cell lines tested. These results point at a dualistic influence of quercetin on cell proliferation that may affect present views on its supposed beneficial anti-proliferative effect. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

During the past few decades, many studies have been conducted to evaluate the effects of n-6 and n-3 polyunsaturated fatty acids (PUFAs) on colorectal carcinogenesis. This report provides a brief overview of the recent studies that have been performed in cultured colon cells, animal models as well as of the population-based and short-term biomarker studies with humans. No differential effect between n-6 and n-3 PUFAs has been observed in vitro. Results from animal models indicate that n-6 PUFAs have a tumor enhancing effect, predominantly during the post-initiation phase. n-3 PUFAs may protect against colorectal carcinogenesis during both the initiation and post-initiation phase. Population-based human studies show little or no associations between n-6 or n-3 PUFA intake and colorectal cancer. Short-term biomarker studies in humans suggest though that fish oil (FO) supplementation with high amounts of n-3 PUFAs may protect against colorectal carcinogenesis and that n-6 PUFA supplementation may increase the risk. © 2002 Elsevier Science B.V. All rights reserved.

To investigate the effects of repeated exposure to nitrogen dioxide (NO2) on antioxidant enzymes in lung tissue and isolated lung cells, rats were continuously exposed to 20 mg/m3 NO2 (10.6 ppm) for 4 days. The activities of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and glutathione peroxidase (GSHPx) were measured in the cytosolic fraction of lung tissue of both control and NO2-exposed rats as well as in isolated alveolar macrophages (AMs) and type II cells. Qualitative and quantitative changes in AM and type II cells were studied by electron microscopy and by morphometric analyses using enzyme and immunohistochemistry. NO2 exposure resulted in significantly increased pulmonary activities of G6PDH, GR, and GSHPx, both expressed per lung and per gram of lung weight. Morphometric data show that NO2 exposure significantly increased the number of type II cells, predominantly in the centriacinar region, indicating proliferation of epithelium following cellular injury. Type II cells in lungs of NO2-exposed rats had a squamous, less cuboidal appearance with more lamellar bodies compared to type II cells in lungs of control rats. Compared to control lungs, a higher number of macrophages could be isolated from NO2-exposed lungs, while numbers of type II cells isolated from lungs of control and NO2-exposed rats were the same. Isolated type II cells from control and NO2-exposed rats were polymorphic, with a small number of lamellar bodies and without polarity. Isolated macro-phages were rounded and contained many filopodia. NO2 exposure caused increases in the activities of G6PDH and GSHPx in isolated type II cells and of GSHPx in isolated macrophages, when expressed per number of cells. Macrophages and type II cells isolated from control and NO2-exposed rats and re-exposed in vitro to NO2, showed no differences in phagocytosis and viability features. Our results indicate that NO2-induced increases in pulmonary antioxidant enzymes are also reflected in isolated AM and type II cells. Since these lung cells do not display a decreased sensitivities toward an in vitro NO2 exposure, overall increase in antioxidant enzyme activities do not seem to play the most pivotal role in controlling cellular NO2 sensitivity and oxidant defence. Combined data from biochemical, morphological, and morphometric analyses of lungs and lung cells suggest that lung cell and tissue oxidant sensitivity and defence largely depends on the cell and tissue organisation, i.e., cell numbers and morphology as well as the ratio of surface area to cytoplasmic volume.

The antimutagenicity of ovalbumin was investigated and compared to that of sodium caseinate, bovine serum albumin and soy protein. Antimutagenicity was measured against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the E. coli DNA repair liquid suspension assay. Heat-denatured ovalbumin showed a strong increase in antimutagenicity compared to undenatured ovalbumin. The antimutagenicity of heat-denatured ovalbumin was superior to the antimutagenicity found for the other proteins tested. After digestion of native and heat-denatured ovalbumin using the pH-stat method, antimutagenicity remained. Antimutagenicity measurements with samples taken from a gastro-intestinal simulator showed strong comutagenic properties. This turned out to be due to comutagenicity against MNNG caused by bile acids and lipase. It was concluded that food proteins exhibit different antimutagenic properties toward MNNG and that the choice of a particular protein digestion model can influence results to a great extent.

The ADI as a tool for risk management and regulation of food additives and pesticide residues is not readily applicable to inherent food plant toxicants: The margin between actual intake and potentially toxic levels is often small; application of the default uncertainty factors used to derive ADI values, particularly when extrapolating from animal data, would prohibit the utilisation of the food, which may have an overall beneficial health effect. Levels of inherent toxicants are difficult to control; their complete removal is not always wanted, due to their function for the plant or for human health. The health impact of the inherent toxicant is often modified by factors in the food, e.g. the bioavailability from the matrix and interaction with other inherent constituents. Risk-benefit analysis should be made for different consumption scenarios, without the use of uncertainty factors. Crucial in this approach is analysis of the toxicity of the whole foodstuff. The relationship between the whole foodstuff and the pure toxicant is expressed in the `product correction factor' (PCF). Investigations in humans are essential so that biomarkers of exposure and for effect can be used to analyse the difference between animals and humans and between the food and the pure toxicant. A grid of the variables characterising toxicity is proposed, showing their inter-relationships. A flow diagram for risk estimate is provided, using both toxicological and epidemiological studies.

The effects of a vegetables-fruit mixture (19.5% w/w) were studied on hepatic (h) and colonic (c) 1,2-dimethylhydrazine (DMH)-metabolizing enzyme activities (ethoxy- and pentoxyresorufine-O-deethylation, NDMA-demethylase, glutathione-S-transferase, UDP-glucuronyltransferase) in rats fed low- or high-fat diets (20 or 40 energy%). A vegetables-fruit mixture added to the diets resulted in altered enzyme activities in animals either treated or not treated with DMH. Remarkably, the vegetables-fruit mixture given to DMH-treated rats decreased glutathione-S-transferase (h) and increased NDMA-demethylase activities (c), whereas the mixture given to controls increased glutathione-S-transferase (h) and decreased NDMA-demethylase activities (c). The high-fat diets only modulated enzyme activities in animals not treated with DMH.

So far, in most animal experimental studies isolated food components have been tested. However, as components may interact with each other at different mechanistic levels, testing complex food mixtures more representative for human consumption patterns may better predict the ultimate carcinogenic risk. Studies were performed in Wistar rats using human and rat control diets to assess the effect of relevant food factors such as heat processing and the presence of non-nutrients in vegetables and fruit. The complete human diets, containing meat, bread and eggs, with or without vegetables and fruit, were composed according to mean consumption figures, balanced for macro- and micronutrients. Experiments were performed with spontaneous as well as with chemical-induced tumor models. Heat processing had no effect on tumor induction, while vegetables and fruit only exerted a protective effect on chemically induced tumors in rats fed low-fat animal diets. Data suggest interaction between major food factors in the human diet on colon carcinogenesis.

The aim of the present investigation was to study the interaction of dietary fat in combination with a vegetables-fruit mixture on 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis in rats. For this purpose, 120 weanling male. Wistar rats received a semisynthetic diet without (Groups A and C) or with a vegetables-fruit mixture (Groups B and D; vegetables and fruit content 19.5% wt/wt) for 35 weeks. Diets of Groups A and B contained 20 energy percent (20e%) fat, whereas diets of Groups C and D contained 40e% fat. The vegetables and fruit used the amount of fat, and its fatty acid composition were chosen according to the mean consumption values of The Netherlands. After the animals were maintained for four weeks on the respective diets, they were given 10 weekly injections of DMH at 50 mg/kg body wt sc. After sacrifice, their colons were removed and examined macroscopically and microscopically for the presence of tumors. Rats fed high-fat diets developed significantly more tumors than rats fed low-fat diets. Furthermore, although not statistically significant, a lower number of colorectal tumors was observed in rats fed a low- or a high-fat diet containing the vegetables-fruit mixture than in rats fed diets without the vegetables-fruit mixture. No differences were observed in intestinal tumor incidences among all groups. The results suggest that the vegetables-fruit mixture used in this experiment, present in an amount comparable with the mean consumption in The Netherlands, has no significant inhibitory effect on the development of colorectal tumors induced by DMH in rats maintained on diets low or high in fat.

The effect of caseinate and soy protein on the mutagenicity induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was assessed in in-vitro and in-vivo studies using a host-mediated assay. Of the two dietary proteins tested, only casein showed antimutagenic activity. Moreover, evidence is presented that enzymatic digestion of sodium-caseinate greatly improved its antimutagenic potential. It is suggested that the molecular structure of a protein determines its protective effect against mutagens. A stronger protection appears to correspond with a lack of secondary and tertiary structure. These findings shed new light on the possible prevention of mutagenesis and/or carcinogenesis by food proteins, with an unique role for milk proteins.

Although it is frequently hypothesized that surface (like surface charge) and physical characteristics (like particle size) play important roles in cellular interactions of nanoparticles (NPs), a systematic study probing this issue is missing. Hence, a comparative cytotoxicity study, quantifying nine different cellular endpoints, was performed with a broad series of monodisperse, well characterized silicon (Si) and germanium (Ge) NPs with various surface functionalizations. Human colonic adenocarcinoma Caco-2 and rat alveolar macrophage NR8383 cells were used to clarify the toxicity of this series of NPs. The surface coatings on the NPs appeared to dominate the cytotoxicity: the cationic NPs exhibited cytotoxicity, whereas the carboxylic acid-terminated and hydrophilic PEG- or dextran-terminated NPs did not. Within the cationic Si NPs, smaller Si NPs were more toxic than bigger ones. Manganese-doped (1% Mn) Si NPs did not show any added toxicity, which favors their further development for bioimaging. Iron-doped (1% Fe) Si NPs showed some added toxicity, which may be due to the leaching of Fe(3+) ions from the core. A silica coating seemed to impart toxicity, in line with the reported toxicity of silica. Intracellular mitochondria seem to be the target for the toxic NPs since a dose-, surface charge- and size-dependent imbalance of the mitochondrial membrane potential was observed. Such an imbalance led to a series of other cellular events for cationic NPs, like decreased mitochondrial membrane potential (ΔΨm) and ATP production, induction of ROS generation, increased cytoplasmic Ca(2+) content, production of TNF-α and enhanced caspase-3 activity. Taken together, the results explain the toxicity of Si NPs/Ge NPs largely by their surface characteristics, provide insight into the mode of action underlying the observed cytotoxicity, and give directions on synthesizing biocompatible Si and Ge NPs, as this is crucial for bioimaging and other applications in for example the field of medicine.

The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size ~45 nm) and polystyrene nanoparticles (PSNPs/size ~50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

Tri-block copolymer nanoparticles (TCNP) are increasingly utilized, especially in drug delivery and diagnostics platforms. From in vitro studies, surface charge was observed to influence the transport of TCNP across the Caco-2 monolayers grown on transwell inserts. The objective of this study was to investigate the influence of surface charge on bioavailability and biodistribution of TCNP after oral exposure in vivo and to compare with the in vitro data. To attain the set objectives, monodisperse (45 ± 5 nm), fluorescent and differently charged (positive and negative) TCNP were orally administered to inbred Fischer 344 rats. Blood samples were collected at t = 0, 1/2, 1, 2, 4 and 6 h followed by sacrifice of the animals and collection of the major organs (lungs, liver, kidney, spleen, brain, intestine, and tibia). Quantitative assessments of TCNP in blood and organs were performed by fluorescence measurements. TCNP of both surface charges got absorbed and appeared in the blood within 1/2 h of oral administration. No significant difference in bioavailability and biodistribution could be found between positive and negative TCNP. Both the TCNP, irrespective of charge, showed major accumulation in the liver, kidneys and spleen, were detected in the brain and did not cause any significant increase in the serum alkaline phosphatase levels. Contrary to the in vitro data, surface charge was not found to influence the in vivo bioavailability and biodistribution of TCNP after oral exposure. The obtained results encourage further development of such TCNP, especially for drug delivery purposes. © 2013 The Royal Society of Chemistry.

The specific properties of nanoscale particles, large surface-to-mass ratios and highly reactive surfaces, have increased their commercial application in many fields. However, the same properties are also important for the interaction and bioaccumulation of the nonbiodegradable nanoscale particles in a biological system and are a cause for concern. Hematite (alpha-Fe2O3), being a mineral form of Fe(III) oxide, is one of the most used iron oxides besides magnetite. The aim of our study was the characterization and comparison of biophysical reactivity and toxicological effects of alpha-Fe2O3 nano- (d 50 mu g/ml). The nanoscale particles were slightly more potent in causing cyto- and genotoxicity as compared with their microscale counterparts. Both types of particles induced intracellular generation of reactive oxygen species. This study underlines that alpha-Fe2O3 nanoscale particles trigger different toxicological reaction pathways than microscale particles. However, the immediate environment of the particles (biomolecules, physiological properties of medium) modulates their toxicity on the basis of agglomeration rather than their actual size.

BackgroundPolymer nanoparticles (PNP) are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce.ResultsFluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm) and surface charges (positive and negative) were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP45(45 nm) showed a higher cytotoxicity compared to the positive bigger PNP90(90 nm) particles including reduction in mitochondrial membrane potential (ΔΨm), induction of reactive oxygen species (ROS) production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨm), uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM). Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP), caveolin (for negative PNP) and mannose receptors (for hydroxylated PNP) were found with smaller PNP45showing stronger interactions with the receptors than bigger PNP90.ConclusionsThe size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges) play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose) leading to cellular internalization were observed to depend on size and surface properties of the different PNP. These properties of the nanoparticles also dominate their cytotoxicity, which was analyzed for many factors. The effective reduction in the mitochondrial membrane potential (ΔΨm), uncoupling of the electron transfer chain in mitochondria and resulting ATP depletion, induction of ROS and oxidative stress likely all play a role in the mechanisms behind the cytotoxicity of these PNP.

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in human.

The possible antimutagenic effects of five different proteins against the mutagen 4-nitroquinoline 1-oxide (4NQO) were assessed in a mammalian cell system, using the sister chromatid exchange (SCE) test in Chinese hamster cells (V79). For this purpose the proteins casein, bovine serum albumin (BSA), soy protein, total whey protein and beta-lactoglobulin were studied, as well as pepsin-hydrolysed casein. In addition, the effect of casein on 1-methyl-1-nitroso-3-nitroguanidine (MNNG) was studied. The proteins were tested at a concentration of 1.15% (w/v). Casein was studied over a concentration range of 0 to 1.15% (w/v). A non-toxic concentration was used for the mutagens. Casein hydrolysis by pepsin took place in vitro, simulating human stomach conditions, which resulted in 84% non-casein-N and 16% remaining casein-N. Casein significantly inhibited SCE induction by 4NQO (inhibition 78%, at 1.15% casein, P < 0.05) and by MNNG (83%, at 1.15% casein, P < 0.01). BSA also significantly inhibited 4NQO-induced SCEs (94%, at 1.15% BSA; P < 0.01). However, soy protein, the whey protein fraction of milk and beta-lactoglobulin showed no inhibitory effects. Pepsin-hydrolysed casein inhibited SCE induction by 4NQO and MNNG to a similar extent as non-hydrolysed casein. It is concluded that casein, its pepsin hydrolysis products and BSA may protect mammalian cells against certain genotoxic compounds, whereas other milk proteins, such as whey protein, beta-lactoglobulin and soy protein, do not have this protective action. Although the mechanism of antimutagenicity is unknown, it seems plausible that the protein acts as a blocking agent by chemical or physical interaction with the mutagens. The accessibility of protein molecules and the presence of nucleophilic binding sites may be decisive factors in determining antimutagenic properties of proteins.

A series of monodisperse (45 ± 5 nm) fluorescent nanoparticles from tri-block copolymers (polymeric nanoparticles (PNPs)) bearing different surface charges were synthesised and investigated for cytotoxicity in NR8383 and Caco-2 cells. The positive PNPs were more cytotoxic and induced a higher intracellular reactive oxygen species production than the neutral and negative ones. The cytotoxicity of positive PNPs with quaternary ammonium groups decreased with increasing steric bulk. The intracellular uptake and cellular interactions of these different PNPs were also tested in NR8383 cells by confocal laser scanning microscopy, which revealed higher uptake for positive than for negative PNPs. Also positive PNPs were found to interact much more with cell membranes, whereas the negative PNPs were internalised mainly by lysosomal endocytosis. Uptake of positive PNPs decreased with increasing steric bulk around the positive charge. A surface charge-specific interaction of clathrin for positive PNPs and caveolin receptors for negative PNPs was observed. These findings confirm that surface charge is important for the cytotoxicity of these PNPs, while they additionally point to considerable additional effects of the steric shielding around positive charges on PNP cytotoxicity.

Background Surface charge and oxidative stress are often hypothesized to be important factors in cytotoxicity of nanoparticles. However, the role of these factors is not well understood. Hence, the aim of this study was to systematically investigate the role of surface charge, oxidative stress and possible involvement of mitochondria in the production of intracellular reactive oxygen species (ROS) upon exposure of rat macrophage NR8383 cells to silicon nanoparticles. For this aim highly monodisperse (size 1.6 ± 0.2 nm) and well-characterized Si core nanoparticles (Si NP) were used with a surface charge that depends on the specific covalently bound organic monolayers: positively charged Si NP-NH2, neutral Si NP-N3 and negatively charged Si NP-COOH. Results Positively charged Si NP-NH2 proved to be more cytotoxic in terms of reducing mitochondrial metabolic activity and effects on phagocytosis than neutral Si NP-N3, while negatively charged Si NP-COOH showed very little or no cytotoxicity. Si NP-NH2 produced the highest level of intracellular ROS, followed by Si NP-N3 and Si NP-COOH; the latter did not induce any intracellular ROS production. A similar trend in ROS production was observed in incubations with an isolated mitochondrial fraction from rat liver tissue in the presence of Si NP. Finally, vitamin E and vitamin C induced protection against the cytotoxicity of the Si NP-NH2 and Si NP-N3, corroborating the role of oxidative stress in the mechanism underlying the cytotoxicity of these Si NP. Conclusion Surface charge of Si-core nanoparticles plays an important role in determining their cytotoxicity. Production of intracellular ROS, with probable involvement of mitochondria, is an important mechanism for this cytotoxicity.

The chemopreventive effects of high fat microalgal oil diet on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were studied in male Fischer 344 rats following 8 weeks of dietary treatment. These effects were compared to the effects of high fat fish oil and high fat corn oil diets to determine whether microalgal oil is a good alternative for fish oil regarding protection against colorectal cancer. Despite the difference in fatty acid composition and total amount of n-3 polyunsaturated fatty acids (PUFAs) between microalgal oil and fish oil, both these oils gave the same 50% reduction of AOM-induced ACF when compared to corn oil. To determine whether oxidative stress could play a role in the chemoprevention of colorectal cancer by n-3 PUFAs, feces and caecal content were examined using the TBA assay. The results showed that lipid peroxidation does occur in the gastrointestinal tract. As several lipid peroxidation products of n-3 PUFAs can induce phase II detoxifying enzymes by an EpRE-mediated pathway, the in vivo results suggest that this route may contribute to n-3 PUFA-mediated chemoprevention. All in all, n-3 PUFA-rich oil from microalgae is as good as fish oil regarding chemoprevention in the colon of the rat.

This study investigates whether the previous observation that quercetin increases the transport of PhIP through Caco-2 monolayers in vitro could be confirmed in an in vivo rat model. Co-administration of 1.45 μmol PhIP/kg bw and 30 μmol quercetin/kg bw significantly increased the blood AUC(0-8 h) of PhIP in rats to 131 ± 14% of the AUC(0-8 h) for rats dosed with PhIP alone. Significantly increased blood PhIP levels were detected at 15, 30, 45 and 180 min. At 4 and 8 h post-dosing a difference in the PhIP levels in the blood between the two treatment groups was no longer observed. In vitro and in silico modeling of PhIP transport using Caco-2 cells and a previously described kinetic model for PhIP transport revealed that the relative increase in PhIP transport caused by quercetin is dependent on the concentration of the two compounds. When substituting the PhIP and quercetin concentrations used in the in vivo experiment in the kinetic model, an effect of quercetin on PhIP transport was predicted that matches the actual effect of 131% observed in vivo. It is concluded that quercetin increases the bioavailability of the pro-carcinogen PhIP in rats pointing at a potential adverse effect of this supposed beneficial food ingredient. © 2008 Elsevier Ltd. All rights reserved. Chemicals / CAS: 2 amino 1 methyl 6 phenylimidazo[4,5 b]pyridine, 105650-23-5; quercetin, 117-39-5; 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, 105650-23-5; Antioxidants; Carcinogens; Imidazoles; Quercetin, 117-39-5

Surface water used for drinking-water preparation requires continuous monitoring for the presence of toxic compounds. For monitoring of genotoxic compounds fish models have been developed, such as the Eastern mudminnow (Umbra pygmaea L.) because of its clearly visible 22 meta-centric chromosomes. It was demonstrated in the late seventies that Rhine water was able to induce chromosome aberrations and sister chromatid exchange in this fish species. Although in vitro mutagenicity studies of the RIWA (Rhine Water Works, The Netherlands) have shown that the genotoxicity of the river Rhine steadily decreased during the last decades, there is still concern about the presence of some residual mutagenicity. In addition, in most studies the water samples have been tested only in in vitro test systems such as the Salmonella-microsome test. For this reason, and in order to be able to make a comparison with the water quality 27 years ago, a study was performed with the same experimental design as before in order to measure the effect of Rhine water on the induction of SCE in the Eastern mudminnow. As a new test system the single cell gel electrophoresis assay (Comet assay) was performed. Fish were exposed to Rhine water or to groundwater for 3 and 11 days in flow-through aquaria. Fish exposed for 11 days to Rhine water had a significantly higher number of SCE and an increased comet tail-length compared with control fish exposed to groundwater. After exposure for three days to Rhine water there was no difference in SCE and a slightly increased comet tail-length compared with the control. It was concluded that genotoxins are still present in the river Rhine, but that the genotoxic potential has markedly decreased compared with 27 years ago. Furthermore, the Comet assay appears to be a sensitive assay to measure the genotoxic potential of surface waters in fish.

In this study the n−3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid appear to be effective inducers of electrophile-responsive element (EpRE) regulated genes, whereas the n−6 PUFA arachidonic acid is not. These n−3 PUFAs need to be oxidized to induce EpRE-regulated gene expression, as the antioxidant vitamin E can partially inhibit the PUFA induced dose-dependent effect. Results were obtained using a reporter gene assay, real-time RT-PCR and enzyme activity assays. The induction of EpRE-regulated phase II genes by n−3 PUFAs may be a major pathway by which n−3 PUFAs, in contrast to n−6 PUFAs, are chemopreventive and anticarcinogenic.

In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1′-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19, and 2D6 are intrinsically able to 1′-hydroxylate methyleugenol. An additional experiment with Gentest microsomes, expressing the same individual enzymes to roughly average liver levels, indicated that P450 1A2, 2C9, 2C19, and 2D6 contribute to methyleugenol 1′-hydroxylation in the human liver. A study, in which correlations between methyleugenol 1′-hydroxylation in human liver microsomes from 15 individuals and the conversion of enzyme specific substrates by the same microsomes were investigated, showed that P450 1A2 and P450 2C9 are important enzymes in the bioactivation of methyleugenol. This was confirmed in an inhibition study in which pooled human liver microsomes were incubated with methyleugenol in the presence and absence of enzyme specific inhibitors. Kinetic studies revealed that at physiologically relevant concentrations of methyleugenol P450 1A2 is the most important enzyme for bioactivation of methyleugenol in the human liver showing an enzyme efficiency (kcat/Km) that is ∼30, 50, and >50 times higher than the enzyme efficiencies of, respectively, P450 2C9, 2C19, and 2D6. Only when relatively higher methyleugenol concentrations are present P450 2C9 and P450 2C19 might contribute as well to the bioactivation of methyleugenol in the human liver. A 5-fold difference in activities was found between the 15 human liver microsomes of the correlation study (range, 0.89-4.30 nmol min-1 nmol P450-1). Therefore, interindividual differences might cause variation in sensitivity toward methyleugenol. Especially lifestyle factors such as smoking (induces P450 1A) and the use of barbiturates (induces P450 2C) can increase the susceptibility for adverse effects of methyleugenol. © 2006 American Chemical Society.

NAD(P)H:quinone oxidoreductase (NQO1)-mediated detoxification of quinones is suggested to be involved in cancer prevention. In the present study, using transfected CHO cells, it was demonstrated that the relation between NQO1 activity and the resulting protection against the cytotoxicity of menadione shows a steep dose-response curve revealing a 'lower protection threshold' of 0.5mumol DCPIP/min/mg protein and an 'upper protection threshold' at 1mumol DCPIP/min/mg protein. In an additional in vivo experiment it was investigated how both in vitro critical activity levels of NQO1, relate to NQO1 activities in mice and man, either without or upon induction of the enzyme by butylated hydroxyanisol (BHA) or indole-3-carbinol (I(3)C). Data from an experiment with CD1 mice revealed that base-line NQO1 levels in liver, kidney, small intestine, colon and lung are generally below the observed 'lower protection threshold' in vitro, this also holds for most human tissue S-9 samples. To achieve NQO1 levels above this 'lower protection threshold' will require 5-20 fold NQO1 induction. Discussion focuses on the relevance of the in vitro NQO1 activity thresholds for the in vivo situation. We conclude that increased protection against menadione toxicity can probably not be achieved by NQO1 induction but should be achieved by other mechanisms. Whether this conclusion also holds for other electrophiles and the in vivo situation awaits further definition of their NQO1 protection thresholds.

The aim of the present study is to demonstrate the use of controlled bioreactors for toxicological studies. As a model system the effect of linoleic acid on hybridoma cells is studied in two well-controlled continuously operated bioreactors placed in series. In the first reactor the effect on rapid proliferating cells can be studied, while in the second reactor a special steady state is created, which allows studying the effect on apoptotic cells. Experiments are done at 0, 25, and 50 microM linoleic acid. At the end of the experiment with 50 microM linoleic acid, the concentration of linoleic acid is increased stepwise to determine the cytotoxic level. For rapid proliferating cells exposed to 25 and 50 microM stimulation of growth was observed. At 50 microM there was at the same time an increase in cell death through apoptosis. For stressed apoptotic cells linoleic acid caused partial growth inhibition at 25 and 50 microM and arrest of cell proliferation in the G(2)/M phase at 50 microM. For both, rapid proliferating cells and stressed apoptotic cells, complete growth inhibition occurred at 85 microM, with cells being arrested in the G(2)/M phase and dying mainly through necrosis. Cells in the bioreactor system appeared to be more sensitive towards linoleic acid than cells grown in multi-well plates. (IC(50) = 300 microM; IC(100) = 400 microM). Altogether the results of the present study reveal that the biostat experiments allow detailed analysis of the effect of a bioactive ingredient on cell physiology and behavior.

Toxicology historically has been directed at studying the mechanisms of adverse effects of isolated compounds on living organisms at high levels of exposure, forming the basis for risk and safety assessment. One way to refocus and mobilize new research funds would be to better match the priorities in regulatory issues and direct the research within the field of toxicology more to low-dose toxicology and risk--benefit analysis. Low-dose toxicology can only be developed when taking into account mechanistic insight and will require risk-benefit analysis and a definition of interactions between compounds at realistic doses of exposure, especially in the case of dietary constituents. This is because the biological effects at low levels of exposure not only may be adverse but also can be beneficial depending on the target organ, the actual end point studied, the receptors activated, and/or the gene expression, protein, and metabolite patterns affected. Toxicologists have the tools and knowledge to study mechanisms of biological effects of chemicals on living organisms, and they should redirect their focus from looking only at adverse effects at high levels of exposure to characterizing the complex biological effects, both adverse and beneficial, at low levels of exposure. This may even result in the notion that beneficial effects can be the result of reaction pathways that are generally considered adverse and vice versa. Low-dose toxicology not only will provide a significant research challenge for the years ahead but also should contribute to better methods for low-dose risk assessment for complex mixtures of chemical compounds. This refocusing from high- to low-dose effects turns the field from a science focusing on adverse effects into a science studying the biological effects of chemical compounds on living organisms, taking into account the realization that the ultimate biological effect of a chemical may vary with its dose, the end point or target organ considered, and/or the combined exposure with other chemicals. By defining the effects of chemicals on living organisms at physiologically relevant exposure levels, toxicologists may contribute not only to better risk and safety assessment but also to preventive medicine, generating knowledge on possible adverse and also beneficial effects of chemicals. In addition, it will result in an approach for food safety assessment more in line with that for drug safety assessment taking the risk-benefit balance into consideration.

This study investigates the pro-oxidant activity of 3'- and 4'-O-methylquercetin, two relevant phase II metabolites of quercetin without a functional catechol moiety, which is generally thought to be important for the pro-oxidant activity of quercetin. Oxidation of 3'- and 4'-O-methylquercetin with horseradish peroxidase in the presence of glutathione yielded two major metabolites for each compound, identified as the 6- and 8-glutathionyl conjugates of 3'- and 4'-O-methylquercetin. Thus, catechol-O-methylation of quercetin does not eliminate its pro-oxidant chemistry. Furthermore, the formation of these A-ring glutathione conjugates of 3'- and 4'-O-methylquercetin indicates that quercetin o-quinone may not be an intermediate in the formation of covalent quercetin adducts with glutathione, protein and/or DNA. In additional studies, it was demonstrated that covalent DNA adduct formation by a mixture of [4-(14)C]-3'- and 4'-O-methylquercetin in HepG2 cells amounted to only 42% of the level of covalent adducts formed by a similar amount of [4-(14)C]-quercetin. Altogether, these results reveal the effect of methylation of the catechol moiety of quercetin on its pro-oxidant behavior. Methylation of quercetin does not eliminate but considerably attenuates the cellular implications of the pro-oxidant activity of quercetin, which might add to the mechanisms underlying the apparent lack of in vivo carcinogenicity of this genotoxic compound. The paper also presents a new mechanism for the pro-oxidant chemistry of quercetin, eliminating the requirement for formation of an o-quinone, and explaining why methylation of the catechol moiety does not fully abolish formation of reactive DNA binding metabolites.

The present review focuses on the mechanisms of mutagenic action and the carcinogenic risk of two categories of botanical ingredients, namely the flavonoids with quercetin as an important bioactive representative, and the alkenylbenzenes, namely safrole, methyleugenol and estragole. For quercetin a metabolic pathway for activation to DNA-reactive species may include enzymatic and/or chemical oxidation of quercetin to quercetin ortho-quinone, followed by isomerisation of the ortho-quinone to quinone methides. These quinone methides are suggested to be the active alkylating DNA-reactive intermediates. Recent results have demonstrated the formation of quercetin DNA adducts in exposed cells in vitro. The question that remains to be answered is why these genotoxic characteristics of quercetin are not reflected by carcinogenicity. This might in part be related to the transient nature of quercetin quinone methide adducts, and suggests that stability and/or repair of DNA adducts may need increased attention in in vitro genotoxicity studies. Thus, in vitro mutagenicity studies should put more emphasis on the transient nature of the DNA adducts responsible for the mutagenicity in vitro, since this transient nature of the formed DNA adducts may play an essential role in whether the genotoxicity observed in vitro will have any impact in vivo. For alkenylbenzenes the ultimate electrophilic and carcinogenic metabolites are the carbocations formed upon degradation of their 1'-sulfooxy derivatives, so bioactivation of the alkenylbenzenes to their ultimate carcinogens requires the involvement of cytochromes P450 and sulfotransferases. Identification of the cytochrome P450 isoenzymes involved in bioactivation of the alkenylbenzenes identifies the groups within the population possibly at increased risk, due to life style factors or genetic polymorphisms leading to rapid metaboliser phenotypes. Furthermore, toxicokinetics for conversion of the alkenylbenzenes to their carcinogenic metabolites and kinetics for repair of the DNA adducts formed provide other important aspects that have to be taken into account in the high to low dose risk extrapolation in the risk assessment on alkenylbenzenes. Altogether the present review stresses that species differences and mechanistic data have to be taken into account and that new mechanism- and toxicokinetic-based methods and models are required for cancer risk extrapolation from high dose experimental animal data to low dose carcinogenic risks for man.

At present, there is an increasing interest for plant ingredients and their use in drugs, for teas, or in food supplements. The present review describes the nature and mechanism of action of the phytochemicals presently receiving increased attention in the field of food toxicology. This relates to compounds including aristolochic acids, pyrrolizidine alkaloids, beta-carotene, coumarin, the alkenylbenzenes safrole, methyleugenol and estragole, ephedrine alkaloids and synephrine, kavalactones, anisatin, St. John's wort ingredients, cyanogenic glycosides, solanine and chaconine, thujone, and glycyrrhizinic acid. It can be concluded that several of these phytotoxins cause concern, because of their bioactivation to reactive alkylating intermediates that are able to react with cellular macromolecules causing cellular toxicity, and, upon their reaction with DNA, genotoxicity resulting in tumors. Another group of the phytotoxins presented is active without the requirement for bioactivation and, in most cases, these compounds appear to act as neurotoxins interacting with one of the neurotransmitter systems. Altogether, the examples presented illustrate that natural does not equal safe and that in modern society adverse health effects, upon either acute or chronic exposure to phytochemicals, can occur as a result of use of plant- or herb-based foods, teas, or other extracts.

The neem tree, Azadirachta indica, provides many useful compounds that are used as pesticides and could be applied to protect stored seeds against insects. However in addition to possible beneficial health effects, such as blood sugar lowering properties, anti-parasitic, anti-inflammatory, anti-ulcer and hepatoprotective effects, also toxic effects are described. In this study we present a review of the toxicological data from human and animal studies with oral administration of different neem-based preparations. The non-aqueous extracts appear to be the most toxic neem-based products, with an estimated safe dose (ESD) of 0.002 and 12.5 microg/kg bw/day. Less toxic are the unprocessed materials seed oil and the aqueous extracts (ESD 0.26 and 0.3 mg/kg bw/day, 2 microl/kg bw/day respectively). Most of the pure compounds show a relatively low toxicity (ESD azadirachtin 15 mg/kg bw/day). For all preparations, reversible effect on reproduction of both male and female mammals seem to be the most important toxic effects upon sub-acute or chronic exposure. From the available data, safety assessments for the various neem-derived preparations were made and the outcomes are compared to the ingestion of residues on food treated with neem preparations as insecticides. This leads to the conclusion that, if applied with care, use of neem derived pesticides as an insecticide should not be discouraged.

In the present study, the cytochrome P450 mediated bioactivation of safrole to its proximate carcinogenic metabolite, 1′-hydroxysafrole, has been investigated for the purpose of identifying the human P450 enzymes involved. The 1′-hydroxylation of safrole was characterized in a variety of in vitro test systems, including Supersomes, expressing individual human P450 enzymes to a high level, and microsomes derived from cell lines expressing individual human P450 enzymes to a lower, average human liver level. Additionally, a correlation study was performed, in which safrole was incubated with a series of 15 human liver microsomes, and the 1′-hydroxylation rates obtained were correlated with the activities of these microsomes toward specific substrates for nine different isoenzymes. To complete the study, a final experiment was performed in which pooled human liver microsomes were incubated with safrole in the presence and absence of coumarin, a selective P450 2A6 substrate. On the basis of the results of these experiments, important roles for P450 2C9*1, P450 2A6, P450 2D6*1, and P450 2E1 were elucidated. The possible consequences of these results for the effects of genetic polymorphisms and life style factors on the bioactivation of safrole are discussed. Polymorphisms in P450 2C9, P450 2A6, and P450 2D6, leading to poor metabolizer phenotypes, may reduce the relative risk on the harmful effects of safrole, whereas life style factors, such as the use of alcohol, an inducer of P450 2E1, and barbiturates, inducers of P450 2C9, and polymorphisms in P450 2D6 and P450 2A6, leading to ultraextensive metabolizer phenotypes, may increase the relative risk.