Genetic toxicity assessment: employing the best science for human safety evaluation part III: the comet assay as an alternative to in vitro clastogenicity tests for early drug candidate selection.

Early screening of drug candidates for genotoxicity typically includes an analysis for mutagenicity in bacteria and for clastogenicity in cultured mammalian cells. In addition, in recent years, an early assessment of photogenotoxicity potential has become increasingly important. Also, for screening purposes, expert computer systems can be used to identify structural alerts. In cases where structural alerts are identified, mutagenicity testing limited to bacteria can be conducted. The sequence of computer-aided analysis and limited testing using bacteria allows for screening a comparatively large number of drug candidates. In contrast, considerably more resources, in terms of supplies, technical time, and the amount of a test substance needed, are required when screening for clastogenic activity in mammalian cells. In addition, the relatively large percentage of false positive results for rodent carcinogenicity associated with clastogenicity assays is of considerable concern. As a consequence, mammalian cell-based alternatives to clastogenicity assays are needed for early screening of mammalian genotoxicity. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage in mammalian cells. This assay seems especially useful for screening purposes because false positives associated with excessive toxicity appear to occur less frequently, only relatively small amounts of a test compound are needed, and certain steps of the test procedure can be automated. Therefore, the in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates.     

Genetic toxicity assessment: employing the best science for human safety evaluation. Part II: Performances of the in vitro micronucleus test compared to the mouse lymphoma assay and the in vitro chromosome aberration assay.

The in vitro micronucleus test is commonly used in the early stages of pharmaceutical development as a predictive tool for the regulatory mouse lymphoma assay or in vitro chromosome aberration test. The accumulated data from this assay leads to the suggestion that it could be used as an alternative to the chromosome aberration test or the mouse lymphoma assay in the regulatory genotoxicity battery. In this paper, we present the results of the in vitro micronucleus test on L5178Y mouse lymphoma cells with 25 compounds from Servier research and have compared these results to those obtained in the genotoxicity regulatory battery. All the negative compounds were also negative in the in vitro micronucleus assay. Among the 14 positive compounds, two of them, positive in the mouse lymphoma assay, were found negative in the in vitro micronucleus test. However, this apparent discordance was likely to be due to cytotoxicity- or high concentration-related false positive responses in the mouse lymphoma assay. In addition, we confirmed that the in vitro micronucleus assay is useful for detecting aneugens, especially, when cells in metaphasis and multinucleated cells are also scored and when cells are allowed to recover after the long treatment. On this series of compounds, the in vitro micronucleus assay showed high sensitivity and possibly a better specificity than the mouse lymphoma assay. Thus, the in vitro micronucleus assay was shown to be at least as adequate as the mouse lymphoma assay or the in vitro chromosome aberration test to be used in the standard genotoxicity battery.     

Genotoxicity assessment of NIM-76 and its formulation (pessary) in an in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test

Abstract

The possible genotoxic potential of NIM-76, a volatile fraction obtained from neem oil, having promising contraceptive activity, as well as its formulation product, called pessary (7.5% NIM-76 in polyethylene glycol), were evaluated in the Ames assay and mouse bone marrow micronucleus (MN) assay. Genotoxicity of NIM-76 (0.1–1000?µg/plate) and pessary (0.1–10?000?µg/plate) were studied using the liquid preincubation protocol of the Ames assay both in the presence and absence of S9. Likewise, the ability of NIM-76 [1–1000?mg/kg body weight (b.w.)] and its formulation product (18.75–300?mg/kg b.w.) to induce clastogenic effects were studied in the female mouse bone marrow MN test by using a two-dose intraperitoneal treatment protocol. There was no increase in the number of revertant colonies resulting from NIM-76 or pessary at any of their doses over the respective negative control plates, either in the presence or absence of S9. Similarly, in the MN assay, neither of them showed any clastogenic activity because there was no significant increase in the frequency of micronucleated polychromatic erythrocytes, over the negative control group of animals. The use of this compound in humans is therefore not likely to have mutagenic effects and may be considered as safe with regard to genotoxic potential.     

Genotoxic evaluation of Halfenprox using the human peripheral lymphocyte micronucleus assay and the Ames test

The genotoxicity and mutagenicity of Halfenprox, a synthetic pyrethroid insecticide and acaricide, was assessed using two standard genotoxicity assays of the Salmonella typhimurium mutagenicity assay (Ames test) and in vitro micronucleus (MN) assay in human peripheral lymphocytes. In the Ames test, Salmonella strains TA98 and TA100 were treated with or without S9 fraction. The doses of Halfenprox were 6.25, 12.5, 25, 50, and 100?μg/plate and test materials were dissolved in DMSO. The concentrations of Halfenprox did not show mutagenic activity on both strains with and without S9 fraction. The MN assay was used to investigate the genotoxic effects of Halfenprox in human peripheral lymphocytes treated with 250, 500, 750, and 1000?μg/ml concentrations of Halfenprox for 24 and 48?h, and at 1000?μg/ml the concentration was significantly increased and the MN formation was compared with the negative control for both treatment periods. In addition, a significant decrease of the nuclear devision index (NDI) values at the higher concentrations of Halfenprox and at both treatment periods was observed.     

Evaluation of genotoxic potential of avarol,avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3′-methoxyavarone, 4′-(methylamino)avarone and 3′-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3′-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3′-methoxyavarone and 3′-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.     

In vivo genotoxicity and cytotoxicity assessment of a novel quinoxalinone with trichomonacide activity

The compound VAM2‐6 (1‐methyl‐7‐nitro‐4‐(5‐(piperidin‐1‐yl)pentyl)‐3,4‐dihydroquinoxalin‐2(1H)‐one) has previously been shown to have an in vitro efficacy of 100% at a concentration of 100 µg ml^–1 against Trichomonas vaginalis, a protozoon parasite that causes the sexually transmitted disease trichomoniasis. Because VAM2‐6 is a quinoxaline derivative and given the lack of studies on the genotoxic activity of this compound, the present study was undertaken to evaluate its ability to induce DNA damage in the peripheral blood of mice using single‐cell gel electrophoresis (SCGE or comet assay) and the micronucleus (MN) assay. Cell viability was assessed using a fluorochrome‐mediated viability test. The compound was tested on CD1 mice at 60, 40 and 10 mg kg^–1 body weight administrated intraperitoneal (i.p.) in a single dose. Peripheral blood samples were collected 24 and 48 h after treatment. N‐Ethyl‐N‐nitrosourea (ENU) was used as a positive control for the comet and micronucleus assays. The results showed that i.p. VAM2‐6 induced single‐strand DNA breaks and increased the average number of micronuclei in the treated mice in a dose‐dependent manner at 60, 40 and 10 mg kg^–1. Cell viability decreased at 24 h but recovered at 48 h for all three evaluated doses. Therefore, the chemical structure of VAM2‐6 should be modified to reduce its genotoxic potential. Copyright © 2012 John Wiley & Sons, Ltd.     

Developmental toxicity and DNA damage to zebrafish induced by perfluorooctane sulfonate in the presence of ZnO nanoparticles

Perfluorooctane sulfonate (PFOS) and ZnO nanoparticles (ZnO–NPs) are frequently detected in the environment, but few studies have assessed their joint toxicity. In this study, the acute toxicity and chronic toxicity to zebrafish (Danio rerio) induced by PFOS in the presence of ZnO–NPs were investigated, including developmental toxicity and DNA damage. The embryos were exposed to PFOS (only) (0.4, 0.8, and 1.6 mg/L) and PFOS plus ZnO–NPs (0.4 + 50, 0.8 + 50, and 1.6 + 50 mg/L) solutions to evaluate mortality (96 h), body length (96 h), hatch rate (72 h), heart rate (48 h),and malformation rate (96 h). The results revealed that the co‐treatment could cause more severe developmental toxicity compared with the control and single‐treatments, and the toxic effects generally increased in a dose–response manner. In addition, adult zebrafish were exposed to single and mixed solutions of PFOS and ZnO–NPs (at the concentrations mentioned above) for 30 days. DNA damage to zebrafish was evaluated by the comet assay and micronucleus test. We found that the PFOS single‐treatment at all doses (0.4, 0.8, and 1.6 mg/L) could strongly induce DNA damage to peripheral blood cells and that ZnO–NPs could aggravate the formation of DNA damage in co‐treatments. Histological examination of liver, testicle, and ovary showed that the presence of ZnO–NPs (50 mg/L) could also cause more serious histological damage to adult zebrafish than PFOS alone. As a result, the synergistic effects played an important role during joint exposure. Our observations provide a basic understanding of the joint toxicity of PFOS and ZnO–NPs to aquatic organisms. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 360–371, 2016.     

Preservation: past, present and future

Foods deteriorate in quality due to a wide range of reactions including some that are physical, some that are chemical, some enzymic and some microbiological. The various forms of spoilage and food poisoning caused by micro-organisms are preventable to a large degree by a number of preservation techniques, most of which act by preventing or slowing microbial growth. These include freezing, chilling, drying, curing, conserving, vacuum packing, modified atmosphere packing, acidifying, fermenting, and adding preservatives. In contrast, a smaller number of techniques act by inactivating micro-organisms, predominantly heating (pasteurization and sterilization). Complementary techniques restrict access of micro-organisms to food products, e.g. aseptic processing and packaging. New and 'emerging' preservation techniques include more that act by inactivation. They include the application of ionizing radiation, high hydrostatic pressure, high voltage electric discharges, high intensity light, ultrasonication in combination with heat and slightly raised pressure ('manothermosonication'), and the addition to foods of bacteriolytic enzymes, bacteriocins, and other naturally-occurring antimicrobials. Major trends, reacting to consumers' needs, are towards the use of procedures that deliver food products that are less 'heavily' preserved, higher quality, more convenient, more 'natural', freer from additives, nutritionally healthier, and still with high assurance of microbiological safety.     

Pharmacogenetics: past, present and future

The subject area of pharmacogenetics, also known as pharmacogenomics, has a long history. Research in this area has led to fundamental discoveries, which have helped our understanding of the reasons why individuals differ in the way they handle drugs, and ultimately in the way they respond to drugs, either in terms of efficacy or toxicity. However, not much of this knowledge has been translated into clinical practice, most drug-gene associations that have some evidence of clinical validity have not progressed to clinical settings. Advances in genomics since 2000, including the ready availability of data on the variability of the human genome, have provided us with unprecedented opportunities to understand variability in drug responses, and the opportunity to incorporate this into patient care. This is only likely to occur with a systematic approach that evaluates and overcomes the different translational gaps in taking a biomarker from discovery to clinical practice. In this article, I explore the history of pharmacogenetics, appraise the current state of research in this area, and finish off with suggestions for progressing in the field in the future.     

Valproate: past,present, and future

Preclinical studies have been carried out during the past four decades to investigate the different mechanisms of action of valproate (VPA). The mechanisms of VPA which seem to be of clinical importance include increased GABAergic activity, reduction in excitatory neurotransmission, and modification of monoamines. These mechanisms are discussed in relation to the various clinical uses of the drug. VPA is widely used as an antiepileptic drug with a broad spectrum of activity. In patients, VPA possesses efficacy in the treatment of various epileptic seizures such as absence, myoclonic, and generalized tonic-clonic seizures. It is also effective in the treatment of partial seizures with or without secondary generalization and acutely in status epilepticus. The pharmacokinetic aspects of VPA and the frequent drug interactions between VPA and other drugs are discussed. The available methods for the determination of VPA in body fluids are briefly evaluated. At present, investigations and clinical trials are carried out and evaluated to explore the new indications for VPA in other conditions such as in psychiatric disorders, migraine and neuropathic pain. Furthermore, the toxicity of VPA, both regarding commonly occurring side effects and potential idiosyncratic reactions are described. Derivatives of VPA with improved efficacy and tolerability are in development.     

Sublethal toxicity of esbiothrin relationship with total antioxidant status and in vivo genotoxicity assessment in fish (Cyprinus carpio L., 1758) using the micronucleus test and comet assay

Esbiothrin, synthetic pyrethroid with quick activity against insects, is widely used against household pests and in public health. Despite widespread use, data on ecotoxicity and genotoxic effects are extremely scarce. The aim of the present study is to evaluate the genotoxic potential of esbiothrin on a model fish species Cyprinus carpio L., 1758 (Pisces: Cyprinidae, koi) using the micronucleus test and comet assay in peripheral blood erythrocytes. Effects of two sublethal exposure concentrations on plasma total antioxidant status (TAS mmol/L), and Hct values were examined. On the basis of the 96 h LC₅₀ data from U.S. EPA ecotox database (32 μg/L) two sublethal exposure concentrations (5 and 10 μg/L) were used together with ethyl methanesulfonate (EMS) (5 mg/L) as positive control. Five fish were used for each dose/duration group (24, 48, and 72 h) under controlled laboratory conditions. The fish showed behavioral changes at the higher dose. Plasma TAS (mmol/L) levels decreased in 24 h; an increase was observed slightly for 48 and obviously for 72 h in both exposure doses. Similarly, hematocrit (Hct) values differed between exposure duration but no significant differences in mean values were found between groups of the same exposure time. The general trend was a rise after 48 h, which decreased afterwards. Our results revealed significant increases in the frequencies of micronuclei and levels of DNA strand breaks and thus demonstrated the genotoxic potential of this pesticide on fish, a nontarget organism of the aquatic ecosystem. To our knowledge this is the first study to report observable genotoxic effects of esbiothrin on fish. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28:644–651, 2013.     

Evaluation of cytotoxic and genotoxic effects of Benodanil by using Allium and Micronucleus assays

The aim of this study was to evaluate the potential cytotoxic effects of Benodanil fungicide by employing both mitotic index (MI) and mitotic phases on the root meristem cells of Allium cepa and genotoxic effects by using in vitro micronucleus assay (MN) in human peripheral blood lymphocyte. In the Allium root growth inhibition test, the EC₅₀ value was first determined as 25?ppm. Then, 2?×?EC₅₀ value (50?ppm), EC₅₀ value (25?ppm), and 1/2?×?EC₅₀ value (12.5?ppm) were tested with different treatment periods (24, 48, and 72?h). Both negative and positive controls were also used in parallel experiments. We obtained that mitotic index and prophase index decreased when compared with the control in all concentrations. In the micronucleus assay, lymphocytes were treated with various concentrations (250, 500, 750, and 1000?µg/ml) of Benodanil for 24 and 48?h. The results showed that Benodanil did not induce MN frequency in all concentrations of both treatment periods. Additionally, it was determined that this pesticide decreased nuclear division index (NDI) significantly. It was concluded that Benodanil has a cytotoxic effects depending on decreasing of MI and NDI.     

Differential effects of silver nanoparticles on DNA damage and DNA repair gene expression in Ogg1-deficient and wild type mice

Due to extensive use in consumer goods, it is important to understand the genotoxicity of silver nanoparticles (AgNPs) and identify susceptible populations. 8-Oxoguanine DNA glycosylase 1 (OGG1) excises 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), a pro-mutagenic lesion induced by oxidative stress. To understand whether defects in OGG1 is a possible genetic factor increasing an individual’s susceptibly to AgNPs, we determined DNA damage, genome rearrangements, and expression of DNA repair genes in Ogg1-deficient and wild type mice exposed orally to 4?mg/kg of citrate-coated AgNPs over a period of 7?d. DNA damage was examined at 3 and 7?d of exposure and 7 and 14?d post-exposure. AgNPs induced 8-oxoG, double strand breaks (DSBs), chromosomal damage, and DNA deletions in both genotypes. However, 8-oxoG was induced earlier in Ogg1-deficient mice and 8-oxoG levels were higher after 7-d treatment and persisted longer after exposure termination. AgNPs downregulated DNA glycosylases Ogg1, Neil1, and Neil2 in wild type mice, but upregulated Myh, Neil1, and Neil2 glycosylases in Ogg1-deficient mice. Neil1 and Neil2 can repair 8-oxoG. Thus, AgNP-mediated downregulation of DNA glycosylases in wild type mice may contribute to genotoxicity, while upregulation thereof in Ogg1-deficient mice could serve as an adaptive response to AgNP-induced DNA damage. However, our data show that Ogg1 is indispensable for the efficient repair of AgNP-induced damage. In summary, citrate-coated AgNPs are genotoxic in both genotypes and Ogg1 deficiency exacerbates the effect. These data suggest that humans with genetic polymorphisms and mutations in OGG1 may have increased susceptibility to AgNP-mediated DNA damage.     

Antimalarial quassinoids: past,present and future

This review is a compilation of the investigations reported to date on the sources, isolation, chemistry and antimalarial activities of natural quassinoids and their synthetic and semisynthetic analogs. It also provides an analysis of the in vitro structure–activity relationship of quassinoids for further evaluation in animal models. The introduction of non-nitrogenous antimalarial drugs has created a new era of malaria chemotherapy to treat Plasmodium falciparum strains that are resistant to existing nitrogenous drugs and the rising incidence of the deadly cerebral malaria. Many antimalarial quassinoids are discovered from simaroubaceous plants that are used traditionally to treat fever and malaria, thereby reiterating the critical role of ethnopharmacology as a rich source of novel drug discovery.