Further development of CometChip technology to measure DNA damage in vitro and in vivo: Comparison with the 2 gels/slide format of the standard and enzyme-modified comet assay.

DNA damage plays a pivotal role in carcinogenesis and other diseases. The comet assay has been used for more than three decades to measure DNA damages. The 1-2 gels/slide format is the most used version of the assay. In 2010, a high throughput 96 macrowell format with a spatially encoded array of microwells patterned in agarose was developed, called the CometChip. The commercial version (CometChip®) has been used for the in vitro standard version of the comet assay (following the manufacturer's protocol), although it has not been compared directly with the 2 gels/slide format. The aim of this work is to developed new protocols to allow use of DNA repair enzymes as well as the analysis of in vivo frozen tissue samples in the CometChip®, to increase the throughput, and to compare its performance with the classic 2 gels/slide format. We adapted the manufacturer's protocol to allow the use of snap frozen tissue samples, using male Wistar rats orally dosed with methyl methanesulfonate (MMS, 200 mg/kg b.w.), and to detect altered nucleobases using DNA repair enzymes, with TK6 cells treated with potassium bromate (KBrO3, 0-4 mM, 3 h) and formamidopyrimidine DNA glycosylase (Fpg) as the enzyme. Regarding the standard version of the comet, we performed thee comparison of the 2 gel/slide and CometChip® format (using the the manufacturer's protocol), using TK6 cells with MMS (100-800 µM, 1 h) and hydrogen peroxide (H2O2, 7.7-122.5 µM, 5 min) as testing compounds. In all cases the CometChip® was performed along with the 2 gels/slide format. Results obtained were comparable and the CometChip® is a good alternative to the 2 gels/slide format when a higher throughput is required.

Mutagenicity and genotoxicity assessment of the emerging mycotoxin Versicolorin A, an Aflatoxin B1 precursor.

Aflatoxin B1 (AFB1) is the most potent natural carcinogen among mycotoxins. Versicolorin A (VerA) is a precursor of AFB1 biosynthesis and is structurally related to the latter. Although VerA has already been identified as a genotoxin, data on the toxicity of VerA are still scarce, especially at low concentrations. The SOS/umu and miniaturised version of the Ames test in Salmonella Typhimurium strains used in the present study shows that VerA induces point mutations. This effect, like AFB1, depends primarily on metabolic activation of VerA. VerA also induced chromosomal damage in metabolically competent intestinal cells (IPEC-1) detected by the micronucleus assay. Furthermore, results from the standard and enzyme-modified comet assay confirmed the VerA-mediated DNA damage, and we observed that DNA repair pathways were activated upon exposure to VerA, as indicated by the phosphorylation and/or relocation of relevant DNA-repair biomarkers (γH2AX and 53BP1/FANCD2, respectively). In conclusion, VerA induces DNA damage without affecting cell viability at concentrations as low as 0.03 µM, highlighting the danger associated with VerA exposure and calling for more research on the carcinogenicity of this emerging food contaminant.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

European Regulatory Framework and Safety Assessment of Food-Related Bioactive Compounds.

A great variety of functional foods, nutraceuticals, or foods with bioactive compounds are provided nowadays to consumers. Aware of the importance of the safety aspects, the food industry has to comply with different legal requirements around the world. In this review, the European regulatory framework for food-related bioactive compounds is summarized. The term 'bioactive compound' is not defined in the European regulations, however, since they can be part of food supplements, fortified foods, or novel food, they are included within the legal requirements of those corresponding types of foods or supplements. Lists of authorized compounds/foods appear in the correspondent regulations, however, when a new compound/food is going to be launched into the market, its safety assessment is essential. Although the responsibility for the safety of these compounds/foods lies with the food business operator placing the product on the market, the European Food Safety Authority (EFSA) carries out scientific evaluations to assess the risks for human health. To facilitate this procedure, different guidelines exist at the European level to explain the tier toxicity testing approach to be considered. This approach divides the evaluation into four areas: (a) toxicokinetics; (b) genotoxicity; (c) subchronic and chronic toxicity and carcinogenicity; and (d) reproductive and developmental toxicity.

Genotoxicity of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are widely used in diverse sectors such as medicine, food, cosmetics, household items, textiles and electronics. Given the extent of human exposure to AgNPs, information about the toxicological effects of such products is required to ensure their safety. For this reason, we performed a bibliographic review of the genotoxicity studies carried out with AgNPs over the last six years. A total of 43 articles that used well-established standard assays (i.e., in vitro mouse lymphoma assays, in vitro micronucleus tests, in vitro comet assays, in vivo micronucleus tests, in vivo chromosome aberration tests and in vivo comet assays), were selected. The results showed that AgNPs produce genotoxic effects at all DNA damage levels evaluated, in both in vitro and in vivo assays. However, a higher proportion of positive results was obtained in the in vitro studies. Some authors observed that coating and size had an effect on both in vitro and in vivo results. None of the studies included a complete battery of assays, as recommended by ICH and EFSA guidelines, and few of the authors followed OECD guidelines when performing assays. A complete genotoxicological characterization of AgNPs is required for decision-making.

Novel antimalarial chloroquine- and primaquine-quinoxaline 1,4-di-N-oxide hybrids: Design, synthesis, Plasmodium life cycle stage profile, and preliminary toxicity studies.

Emergence of drug resistance and targeting all stages of the parasite life cycle are currently the major challenges in antimalarial chemotherapy. Molecular hybridization combining two scaffolds in a single molecule is an innovative strategy for achieving these goals. In this work, a series of novel quinoxaline 1,4-di-N-oxide hybrids containing either chloroquine or primaquine pharmacophores was designed, synthesized and tested against both chloroquine sensitive and multidrug resistant strains of Plasmodium falciparum. Only chloroquine-based compounds exhibited potent blood stage activity with compounds 4b and 4e being the most active and selective hybrids at this parasite stage. Based on their intraerythrocytic activity and selectivity or their chemical nature, seven hybrids were then evaluated against the liver stage of Plasmodium yoelii, Plasmodium berghei and Plasmodium falciparum infections. Compound 4b was the only chloroquine-quinoxaline 1,4-di-N-oxide hybrid with a moderate liver activity, whereas compound 6a and 6b were identified as the most active primaquine-based hybrids against exoerythrocytic stages, displaying enhanced liver activity against P. yoelii and P. berghei, respectively, and better SI values than primaquine. Although both primaquine-quinoxaline 1,4-di-N-oxide hybrids slightly reduced the infection of mosquitoes, they inhibited sporogony of P. berghei and compound 6a showed 92% blocking of transmission. In vivo liver efficacy assays revealed that compound 6a showed causal prophylactic activity affording parasitaemia reduction of up to 95% on day 4. Absence of genotoxicity and in vivo acute toxicity were also determined. These results suggest the approach of primaquine-quinoxaline 1,4-di-N-oxide hybrids as new potential dual-acting antimalarials for further investigation.

Structure-activity relationship of new antimalarial 1-aryl-3-susbtituted propanol derivatives: Synthesis, preliminary toxicity profiling, parasite life cycle stage studies, target exploration, and targeted delivery.

Design, synthesis, structure-activity relationship, cytotoxicity studies, in silico drug-likeness, genotoxicity screening, and in vivo studies of new 1-aryl-3-substituted propanol derivatives led to the identification of nine compounds with promising in vitro (55, 56, 61, 64, 66, and 70-73) and in vivo (66 and 72) antimalarial profiles against Plasmodium falciparum and Plasmodium berghei. Compounds 55, 56, 61, 64, 66 and 70-73 exhibited potent antiplasmodial activity against chloroquine-resistant strain FCR-3 (IC50s < 0.28 µM), and compounds 55, 56, 64, 70, 71, and 72 showed potent biological activity in chloroquine-sensitive and multidrug-resistant strains (IC50s < 0.7 µM for 3D7, D6, FCR-3 and C235). All of these compounds share appropriate drug-likeness profiles and adequate selectivity indexes (77 < SI < 184) as well as lack genotoxicity. In vivo efficacy tests in a mouse model showed compounds 66 and 72 to be promising candidates as they exhibited significant parasitemia reductions of 96.4% and 80.4%, respectively. Additional studies such as liver stage and sporogony inhibition, target exploration of heat shock protein 90 of P. falciparum, targeted delivery by immunoliposomes, and enantiomer characterization were performed and strongly reinforce the hypothesis of 1-aryl-3-substituted propanol derivatives as promising antimalarial compounds.

Is oxidative stress involved in the sex-dependent response to ochratoxin A renal toxicity?

Ochratoxin A (OTA) is a mycotoxin considered the most powerful renal carcinogen in rodents and classified as a possible human carcinogen. Though its mechanism of action is still unknown, indirect DNA reactivity mediated by oxidative stress has been hypothesized to play an important role. Moreover, large sex-differences have been observed in carcinogenicity studies, male rats being more sensitive than females. Male and female F344 rats were administered (p.o.) with bicarbonate or 0.5 mg OTA/kg b.w. for 7 days; or with bicarbonate, 0.21 or 0.5 mg OTA/kg b.w. for 21 days. Total glutathione (tGSH) and oxidized glutathione (GSSG) levels, glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were analysed in kidneys. The standard alkaline comet assay was used in combination with Formamidopyrimidine-DNA glycosylase (Fpg) to detect oxidized DNA bases in kidney. No biologically relevant sex-differences were observed in all the oxidative-stress related parameters analysed. Indeed, no relevant oxidative-stress related response was observed between treated animals and controls. In accordance with the similar OTA levels and histopathological changes between both sexes observed previously in the same animals, and with other oxidative-stress related parameters measured by others, results support that there are no differences between sexes in the oxidative stress response to OTA.

Cytotoxic activity of fucoxanthin, alone and in combination with the cancer drugs imatinib and doxorubicin, in CML cell lines.

In the present study, we evaluate the in vitro cytotoxicity of fucoxanthin (Fx) on two human leukemia cell lines, K562 and TK6, alone and in combination with the conventional anticancer drugs imatinib (Imat) and doxorubicin (Dox). For the purpose, we assessed the cytotoxic and proliferation effects by cell count, induction of DNA damage by comet assay, and cell death by nuclear condensation, annexin V staining, coupled with propidium iodide uptake, and protein expression of Bax, caspase-3, and Bcl-2 (western blot). Our results show that Imat increased cytotoxicity in TK6 cells and inhibited proliferation in K562 cells, while Dox decreased cell viability and proliferation in both cell lines. Fx per se increased cytotoxicity against K562 cells and decreased cell proliferation of K562 and TK6 cells. The effects were confirmed by phase contrast microscopy. However, the antiproliferative effects are not explained by induction of DNA damage or cell death. In co-incubation, Fx increased antiproliferative effects of both drugs in the cell lines tested, however no differences where observed relative to Fx alone. This study unveiled in vitro cytotoxicity of Fx by inhibition of cell proliferation in both cell lines. Further studies are needed to elucidate the signal transduction pathways and molecular targets involved in that effect.

Sex differences in ochratoxin a toxicity in F344 rats after 7 and 21 days of daily oral administration.

Ochratoxin A (OTA) is a potent renal carcinogen in male rats but not in females. The mechanisms underlying these differences are unknown. The sex-dependent response of F344 rats after a repeated OTA oral administration for 7 (0.50 mg/kg bw) or 21 days (0.21 and 0.50 mg/kg bw) was evaluated. General toxicity, sex and thyroid hormones and histopathology were studied. OTA was quantified (HPLC-FLD) in plasma, kidney and liver and the expression of kidney transporters (RT-qPCR) was studied. After 7 days, kidney histopathology showed more pronounced signs of toxicity in males than in females. After 21 days, a higher toxicity was observed but sex differences disappeared. OTA concentration in plasma and tissues was similar in both sexes. Downregulation was the general OTA-induced effect. Oats' downregulation was slow in males and Oat3 did not change in females. Oatp1 was strongly downregulated in males after 21 days. An opposite effect was observed in Bcrp after 21 days: downregulation in males and upregulation in females. Females showed a dose- and time-dependent decrease of progesterone. Despite the sex differences, the final balance in OTA toxicokinetics at renal cell level does not seem to support a higher accumulation of OTA in male kidneys.

Gene expression kinetics of renal transporters induced by ochratoxin A in male and female F344 rats.

Ochratoxin A (OTA) is a mycotoxin that contaminates foodstuffs. The most relevant concern is its high kidney carcinogenicity in male rats and its unclear mechanism of action. It has been hypothesized that variations in transport mechanisms in kidney cells may be the reason of different sex-dependent sensitivities towards OTA. The aim of this study was to analyze, by RT- qPCR, renal transporters expression in 15-week-old male (M) and female (F) F344 rats at basal level and after single oral OTA administration (0.50 mg/kg bw). Temporal profiles (24h, 48h, 72h, 96h, 1 and 2 months) were studied per sex and transporter. The reference gene for all comparisons was Ppia. At basal level, sex differences were confirmed for Oatp1, Bcrp (M>F) and Oat2 (F>M). OTA tended to inhibit the expression of almost all transporters in both sexes, but clearly induced the expression of Oat2 in males. Regarding time profiles, the highest sex differences involved Oat (Slc22) transporters: Oat2, Oat3 and Oat5 expression showed a significant increase in males (24h) while Oat1, Oat2 and Oat5 level decreased in females (48h). Overall, basal sex differences in F344 rats and the specific sex-dependent response to OTA of Oat2 might contribute to high kidney damage in male rats.

A review on ochratoxin A transcriptomic studies.

The mycotoxin Ochratoxin A (OTA) is a potent renal carcinogen in male rats. Transcriptomic studies on OTA (4 in vitro, 6 in vivo, 2 in vitro/in vivo) have been reviewed. The aim of 6 of them was mainly mechanistic whereas the rest had mostly predictive (1) or evaluation (5) purposes. An overall tendency towards gene expression downregulation was observed, probably as a result of protein synthesis inhibition. DNA damage response genes were not deregulated in most of the studies. Genes involved in acute renal injury, cell survival and cell proliferation were upregulated in several in vivo studies. Apoptosis genes were deregulated in vitro but less affected in vivo; activation of several MAPKs has been observed. Many genes related to oxidative stress or involved in cell-to-cell interaction pathways (Wnt) or cytoskeleton structure appeared to be deregulated either in vitro or in vivo. Regucalcin was highly downregulated in vivo and other calcium homeostasis genes were significantly deregulated in vitro. Genes related to OTA transport (OATs) and metabolism (CYPs) appeared downregulated in vivo. Overall, the mechanism of action of OTA remains unclear, however transcriptomic data have contributed to new mechanistic hypothesis generation and to in vitro-in vivo comparison.

Changes in male rat urinary protein profile during puberty: a pilot study.

BACKGROUND: Androgen-dependent proteins (lipocalins) circulate in blood of male rats and mice and, being small (~ 18 kDa), pass freely into glomerular filtrate. Some are salvaged in proximal nephrons but some escape in urine. Several organic molecules can bind to these proteins causing, where salvage occurs, nephropathy including malignancy in renal cortex. In urine, both free lipocalins and ligands contribute to an increasingly-recognised vital biological role in social communication between adults, especially in the dark where reliance is on smell and taste. Crystal structure of the first-characterised lipocalin of male rats, α2u-globulin, has been determined and peptide sequences for others are available, but no study of occurrence during early puberty has been made. We have followed temporal occurrence in urine of juveniles (n = 3) for non-invasive pilot study by high resolution gradient mini-gel electrophoresis, tryptic digest of excised protein bands, and LC-MS/MS of digest to identify peptide fragments and assign to specific lipocalins. Study objective refers directly to external availability for social communication but also indirectly to indicate kinetics of circulating lipocalins to which some xenobiotics may bind and constitute determinants of renal disease. RESULTS: Mini-gels revealed greater lipocalin complexity than hitherto recognised, possibly reflecting post-translational modifications. Earliest patterns comprised rat urinary protein 1, already evident in Sprague-Dawley and Wistar strains at 36 and 52 days, respectively. By 44 and 57 days major rat protein (α2u-globulin) occurred as the progressively more dominant protein, though as two forms with different electrophoretic mobility, characterised by seven peptide sequences. No significant change in urinary testosterone had occurred in Wistars when major rat protein became evident, but testosterone surged by 107 days concomitant with the marked abundance of excreted lipocalins. CONCLUSIONS: Qualitative temporal changes in the composition of excreted lipocalins early in puberty, and apparent increase in major urinary protein as two resolvable forms, should catalyse systematic non-invasive study of urinary lipocalin and testosterone dynamics from early age, to illuminate this aspect of laboratory rodent social physiology. It could also define the potential temporal onset of nephrotoxic ligand risk, applicable to young animals used as toxicological models.

Simple high-performance liquid chromatography-fluorescence detection method for plasma, kidney and liver of rat as a tool for toxicology studies.

A fast and simple HPLC-FLD (high-performance liquid chromatography-fluorescence detection) analytical method has been developed and validated for the determination of ochratoxin A in rat plasma, kidney and liver. The extraction method, calibration curves and chromatographic conditions are common for the three matrices. Plasma and homogenized tissue samples (250 microL) were extracted with ethanol (400 microL) and trichloroacetic acid 20% (w/v) (50 microL). Supernatants were directly injected into the HPLC system, analyzed on a 5-microm (25 cm x 0.4 cm) Tracer Extrasil ODS2 column using FLD (excitation wavelength=225 nm, emission wavelength=461 nm). The mobile phase was 29:29:42 (v/v) methanol-acetonitrile-sodium acetate. The small volume of sample needed which allows the obtaining of ochratoxin A levels in individual tissue samples from small animals and the wide range of concentrations that could be analyzed make this method easy to apply in toxicology and toxicokinetic studies of this mycotoxin, even in low dose carcinogenic studies. This method was linear and selective for all the matrices. Precision and accuracy were always <10% and recovery was very efficient in each case. Limits of detection and quantification were also calculated in plasma (1 and 8.4 microg/L), kidney (14.3 and 55.8 microg/kg) and liver (4.1 and 52.8 microg/kg). Stability of the tissue homogenates was assured for at least 10 months at -80 degrees C. The method has been successfully applied to the analysis of rat samples after 7 days of ochratoxin A (0.5mg/kg b.w. dissolved in an aqueous NaHCO(3) solution) administration by oral gavage.

Gene expression changes induced by ochratoxin A in renal and hepatic tissues of male F344 rat after oral repeated administration.

Ochratoxin A (OTA), a naturally occurring mycotoxin, is nephrotoxic in all animal species tested and is considered a potent renal carcinogen, particularly in male rats. Its mechanism of toxicity is still unknown, although oxidative stress appears to be a plausible mechanism. Therefore, the objective of this study was to identify the biological pathways that are modulated in vivo by OTA in male F344 rats in order to gain further insight into its mechanism of renal toxicity. Rats were gavaged daily with OTA (500 microg/kg bw) and gene expression profiles in target and non-target organs were analyzed after 7 and 21 days administration. As was expected, a time-dependent increase of OTA concentrations was found in plasma, kidney and liver, with the concentrations found in both tissues being quite similar. However, histopathological examinations only revealed changes in kidney; signs of nephrotoxicity involving single cell necrosis and karyomegalic nuclei were observed in the treated rats. The number of differentially expressed genes in kidney was much higher than in liver (541 versus 11 at both time points). Several similarities were observed with other in vivo gene expression data. However, great differences were found with previous in vitro gene expression data, with the exception of DNA damage response which was not observed at mRNA level in any of our study conditions. Down-regulation was the predominant effect. Oxidative stress response pathway and genes involved in metabolism and transport were inhibited at both time points. RGN (regucalcin) - a gene implicated in calcium homeostasis - was strongly inhibited at both time points and genes implicated in cell survival and proliferation were up-regulated at day 21. Moreover, translation factors and annexin genes were up-regulated at both time points. Apart from oxidative stress, alterations of the calcium homeostasis and cytoskeleton structure may be present at the first events of OTA toxicity.