Genotoxicity investigations on nanomaterials: Methods,preparation and characterization of test material,potential artifacts and limitations—Many questions,some answers

Nanomaterials display novel properties to which most toxicologists have not consciously been exposed before the advent of their practical use. The same properties, small size and particular shape, large surface area and surface activity, which make nanomaterials attractive in many applications, may contribute to their toxicological profile. This review describes what is known about genotoxicity investigations on nanomaterials published in the openly available scientific literature to-date. The most frequently used test was the Comet assay: 19 studies, 14 with positive outcome. The second most frequently used test was the micronucleus test: 14 studies, 12 of them with positive outcome. The Ames test, popular with other materials, was less frequently used (6 studies) and was almost always negative, the bacterial cell wall possibly being a barrier for many nanomaterials. Recommendations for improvements emerging from analyzing the reports summarized in this review are: Know what nanomaterial has been tested (and in what form); Consider uptake and distribution of the nanomaterial; Use standardized methods; Recognize that nanomaterials are not all the same; Use in vivo studies to correlate in vitro results; Take nanomaterials specific properties into account; Learn about the mechanism of nanomaterials genotoxic effects. It is concluded that experiences with other, non-nano, substances (molecules and larger particles) taught us that mechanisms of genotoxic effects can be diverse and their elucidation can be demanding, while there often is an immediate need to assess the genotoxic hazard. Thus a practical, pragmatic approach is the use of a battery of standard genotoxicity testing methods covering a wide range of mechanisms. Application of these standard methods to nanomaterials demands adaptations and the interpretation of results from the genotoxicity tests may need additional considerations. This review should help to improve standard genotoxicity testing as well as investigations on the underlying mechanism and the interpretation of genotoxicity data on nanomaterials.     

Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.     

The comet assay as an indicator test for germ cell genotoxicity

The in vivo comet assay is a well-established genotoxicity test. It is currently mainly performed with somatic cells from different organs to detect a genotoxic activity of potential carcinogens. It is regarded as a useful test for follow-up testing of positive or equivocal in vitro test results and for the evaluation of local genotoxicity. However, the comet assay also has the potential to detect germ cell genotoxicity and may be used for demonstrating the ability of a substance or its metabolite(s) to directly interact with the genetic material of gonadal and/or germ cells. Such results are important for the classification of germ cell mutagens, e.g. in the context of the "Globally Harmonized System of Classification and Labelling of Chemicals" (GHS). This review summarizes and discusses available information on the use of the comet assay with germ cells and cells from the gonads in genetic toxicology. The literature contains results from in vitro studies, ex vivo studies and in vivo studies. With regard to the assessment of germ cell genotoxicity, only in vivo studies are relevant but the other kind of studies provided important information on various aspects of the methodology. Many comet assay studies with human sperm have been performed in the context of male infertility and assisted fertilization. The results of these studies are not reviewed in detail here but various aspects of the assay modifications used are discussed. Measuring DNA effects by the comet assay in sperm requires additional steps for chromatin decondensation. Many different modifications of the alkaline and the neutral comet assay are in use but a standard protocol has not been established yet. High and variable background levels of DNA effects were reported and there is still need for standardization and validation of the comet assay with sperm. Some human biomonitoring studies with human sperm were published, but it seems to be premature to use these data for hazard identification and classification of chemicals. In contrast, the standard alkaline in vivo comet assay can easily be adapted to investigations with cells from reproductive organs. Tests with cells from the gonads (testis and ovary) seem to be most appropriate and a promising tool for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells. However, studies to standardize and validate these methods are necessary before the comet assay can be usefully applied in risk assessment of germ cell mutagens.     

Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals

This survey is a compendium of genotoxicity and carcinogenicity information of 838 marketed drugs, whose expected clinical use is continuous for at least 6 months or intermittent over an extended period of time. Of these 838 drugs, 366 (43.7%) do not have retrievable genotoxicity or carcinogenicity data. The remaining 472 (56.3%) have at least one genotoxicity or carcinogenicity test result. Of the 449 drugs with at least one genotoxicity test result, 183 (40.8%) have at least one positive finding. Of the 338 drugs with at least one carcinogenicity test result, 160 (47.3%) have at least one positive result. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, of the 315 drugs which have both genotoxicity and carcinogenicity data 116 (36.8%) are neither genotoxic nor carcinogenic, 50 (15.9%) are non-carcinogens which test positive in at least one genotoxicity assay, 75 (23.8%) are carcinogenic in at least one sex of mice or rats but test negative in genotoxicity assays, and 74 (23.5%) are both genotoxic and carcinogenic. Only 208 (24.8%) of the 838 drugs considered have all data required by current guidelines for testing of pharmaceuticals. However, it should be noted that a large fraction of the drugs considered were developed and marketed prior to the present regulatory climate. Although the laws do not require re-testing based on revised standards, in the absence of epidemiological studies excluding a carcinogenic risk to humans, a re-evalutation would be appropriate.     

Genetics and marine pollution

The development of cytogenetic methods applied to cells and tissues of marine invertebrates has been hampered by (1) a lack of in vitro cell lines, (2) inadequate karyotypic information (partly as a result of too few workers chasing too many organisms), and (3) the failure of their chromosomes to band satisfactorily. Compared to mammalian cytogenetics, our knowledge of marine invertebrates lags behind by several decades. With the current concern about mutagens in the marine environment, and the recognition that the cells of marine species have sensitivities to DNA-damaging agents similar to those of higher organisms, there is a need for methods which can be used (a) in environmental monitoring and (b) to screen potentially harmful substances in the laboratory. In the absence of in vitro cell lines, embryos and larvae have been used to provide a supply of dividing cells for mutation studies, although the advent of molecular methods has now brought with it the means to detect DNA damage without any need for the cells to be in a dividing state. Moreover, the use of FISH (Fluorescence In Situ Hybridisation) now makes it possible to study numerical and structural chromosomal aberrations with far greater accuracy than was previously possible. A new marine genotoxicity assay is described, based on the embryos and larvae of a tube-dwelling polychaete worm (Pomatoceros lamarkii), suitable for both laboratory studies and field monitoring. This new Pomatoceros assay provides, at the same time, a useful model for studying the consequences of adult exposure on the offspring. A novel application of marine cytogenetic research is the study of the evolutionary adaptations of invertebrates living in naturally polluted extreme environments viz. deep sea hydrothermal vents, which are typified by high levels of toxic heavy metals and radionuclides, substances known to inflict damage to DNA. Given these new methodological and conceptual advances, it is predicted that our understanding of the role played by mutation in the marine environment, both in an evolutionary and toxicological context, will increase dramatically over the next decade.     

Genotoxicity assessment using comet assay in Behcet's disease patients

Behcet's disease (BD) was originally described by Turkish dermatologist, Hulusi Behcet in 1937. BD is an inflammatory disorder of unknown cause, characterized by recurrent oral aphthous ulcers, genital ulcers, uveitis, and skin lesions. All these common manifestations are self-limiting except for ocular attacks. The aims of this study were to assess whether BD patients have more genotoxicity than healthy controls and whether colchicine (COL) treated BD patients are different from those not using COL in terms of genotoxicity. A few dozens of methods have been developed and used for the assessment of genotoxicity. The most popular method is based on single cell gel electrophoresis (COMET assay) in alkaline condition. After electrophoresis, captured images are subjected to digital image analysis to find the values for percent tail DNA from comet assay parameters consistent with genotoxicity. COMET assay was performed in isolated lymphocytes from 42 COL treated Behcet's disease patients, 9 BD patients not using COL, and 36 healthy controls. In the COL-BD patients and non-COL-BD patients, the mean age (range 14-56 years) and mean disease duration (range 0.5-24 years) did not differ between the two groups. We found statistical differences in percent tail DNA between BD and the healthy controls (13.38+/-9.58 versus 2.77+/-1.45, P<0.0001). No difference in percent tail DNA was observed between users and non-users of COL, whereas it was more different in inactive BD patients than active ones (19.75+/-10.49 versus 11.83+/-8.79, P<0.05, respectively). Genotoxicity, as assessed by COMET assay, is increased in BD patients. These results suggest that genotoxicity is associated with BD itself rather than COL use.     

The in vitro micronucleus technique

The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.     

Combined histomorphometric and gene-expression profiling applied to toxicology

We have developed a unique methodology for the combined analysis of histomorphometric and gene-expression profiles amenable to intensive data mining and multisample comparison for a comprehensive approach to toxicology. This hybrid technology, termed extensible morphometric relational gene-expression analysis (EMeRGE), is applied in a toxicological study of time-varied vehicle- and carbon-tetrachloride (CCl₄)-treated rats, and demonstrates correlations between specific genes and tissue structures that can augment interpretation of biological observations and diagnosis.     

Study of oxidative DNA damage in TK6 human lymphoblastoid cells by use of the thymidine kinase gene-mutation assay and the in vitro modified comet assay: determination of No-Observed-Genotoxic-Effect-Levels

Nowadays, there is clear progress in using the threshold concept in genetic toxicology, but its demonstration and acceptance in risk assessment is still under debate. Although it has been accepted for some non-DNA-reactive agents for which mechanisms of action were demonstrated, there is a growing weight of evidence to also support the existence of thresholded dose-responses for DNA-reactive agents. In this context, we have recently shown in human TK6 lymphoblastoid cells, that DNA-oxidizing agents [potassium bromate, bleomycin and hydrogen peroxide (via glucose oxidase)] produced non-linear dose-responses in the in vitro micronucleus test, thus allowing the determination of No-Observed-Genotoxic-Effect-Levels (NOGELs). Therefore, the aim of the present study was to focus on the analysis of thresholded dose-response curves in order to further investigate the existence of NOGELs for these same directly DNA-damaging agents, by use of other genotoxicity endpoints. Mutation frequency was determined after a 1-h treatment in the thymidine kinase (TK) gene-mutation assay. Primary DNA damage, especially oxidative DNA damage, was also assessed after 1h of treatment, followed - or not - by a 23-h recovery period, with the modified version of the comet assay (i.e. with the glycosylases Fpg and hOgg1). Overall, our analysis demonstrates that there is convincing evidence to support the existence of thresholded dose-responses for DNA-oxidizing agents. The determination of NOGELs depends on the genotoxic endpoint studied and consequently requires different genotoxicity assays performed concurrently. NOGELs could only be defined for the induction of chromosomal aberrations and gene mutations, i.e. for an effect-endpoint but not for primary DNA damage, i.e. for an exposure-endpoint. Further statistical analyses of these data are now required in order to draw conclusions on the exact level of the thresholds.     

Development of new structural alerts suitable for chemical category formation for assigning covalent and non-covalent mechanisms relevant to DNA binding

The need to assess the ability of a chemical to act as a mutagen is one of the primary requirements in regulatory toxicology. Several pieces of legislation have led to an increased interest in the use of in silico methods, specifically the formation of chemical categories and read-across for the assessment of toxicological endpoints. One of the key steps in the development of chemical categories for mutagenicity is defining the mechanistic organic chemistry associated with the formation of a covalent bond between DNA and an exogenous chemical. To this end this study has analysed, by use of a large set of mutagenicity data (Ames test), the mechanistic coverage of a recently published set of in silico structural alerts developed for category formation. The results show that the majority of chemicals with a positive result in the Ames test were assigned at least one covalent binding mechanism related to the formation of a DNA adduct. The remaining chemicals with positive data in the Ames assay were subjected to a detailed mechanistic analysis from which 26 new structural alerts relating to covalent binding mechanisms were developed. In addition, structural alerts for radical and non-covalent intercalation mechanisms were also defined. The structural alerts outlined in this study are not intended to predict mutagenicity but rather to identify mechanisms associated with covalent and non-covalent DNA binding. This mechanistic profiling information can then be used to form chemical categories suitable for filling data gaps via read-across. A strategy for chemical category formation for mutagenicity is also presented.     

Targeting and non-targeting effects of nanomaterials on DNA: challenges and perspectives

Due to their large-scale manufacture and widespread application, there have been a number of studies related to toxicological assessment of nanomaterials (NMs) over the past decade. Although there has been extensive research on the cytotoxicity of NMs, concerns have been raised about their possible genotoxicity. The genome is constantly exposed to genotoxic insults that can lead to DNA damage, which in turn can have consequences for health, such as the induction of carcinogenesis. This comprehensive review focuses on the direct and indirect interactions of NMs with DNA. Factors influencing the genotoxicity of NMs, such as their physicochemical characteristics, are also discussed. The mechanisms involved in the direct and indirect interactions of NMs with DNA are also reviewed. Many studies have shown that ENMs have genotoxic effects, such as chromosomal fragmentation, DNA strand breaks, point mutations, oxidative DNA adducts, apoptosis, hypoxic responses, mitochondrial dysfunction, and epigenetic modifications. As the data reported to date are inconsistent, it is difficult to draw definitive conclusions regarding the features of NMs that promote genotoxicity. Therefore, challenges and future research perspectives are discussed. This review provides insights into the genotoxic effects of NMs and their consequences for human health.

     

In-vitro cytotoxic and genotoxic effects of arsenic trioxide on human leukemia (HL-60) cells using the MTT and alkaline single cell gel electrophoresis (Comet) assays

Although arsenic trioxide (ATO) has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by ATO in a human leukemia (HL-60) cell line using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. HL-60 cells were treated with different doses of ATO for 24 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that ATO significantly (P < 0.05) reduced the viability of HL-60 cells in a dose-dependent manner, showing a LD₅₀ value of 6.4 ± 0.6 μg/mL. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HL-60 cells associated with ATO exposure. We observed a significant increase (P < 0.05) in comet tail-length, tail arm and tail moment, as well as in percentages of DNA cleavage at all doses tested, showing an evidence of ATO-induced genotoxic damage in HL-60 cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by heavy metals like arsenic. Taken together, our findings suggest that ATO exposure significantly (P < 0.05) reduces cellular viability and induces DNA damage in HL-60 cells as assessed by MTT and alkaline single cell gel electrophoresis assays, respectively.     

Genotoxicity and carcinogenicity studies of antihypertensive agents

This survey is a compendium of genotoxicity and carcinogenicity information of antihypertensive drugs. Data from 164 marketed drugs were collected. Of the 164 drugs, 65 (39.6%) had no retrievable genotoxicity or carcinogenicity data; this group was comprised largely of drugs marketed in a limited number of countries. The remaining 99 (60.4%) had at least one genotoxicity or carcinogenicity test result. Of these 99, 48 (48.5%) had at least one positive finding: 32 tested positive in at least one genotoxicity assay, 26 in at least one carcinogenicity assay, and 10 gave a positive result in both at least one genotoxicity assay and at least one carcinogenicity assay. In terms of correlation between results of the various genotoxicity assays and absence of carcinogenic activity in both mice and rats 2 of 44 non-carcinogenic drugs tested positive in the in vitro bacterial mutagenesis assay, 2 of 9 tested positive in the mouse lymphoma assay, none of 14 tested positive for gene mutation at the hprt locus, 5 of 25 tested positive in in vitro cytogenetic assays, none of 31 in in vivo cytogenetic assays, and none of 14 in inducing DNA damage and/or repair in in vitro and/or in vivo assays. Concerning the predictivity of genetic toxicology findings for long-term carcinogenesis assays, 75 drugs had both genotoxicity and carcinogenicity data; of these 37 (49.3%) were neither genotoxic nor carcinogenic, 14 (18.7%) were non-carcinogens which tested positive in at least one genotoxicity assay, 14 (18.7%) were carcinogenic in at least one sex of mice or rats but tested negative in genotoxicity assays, and 10 (13.3%) were both genotoxic and carcinogenic. Only 42 of the 164 marketed antihypertensives (25.6%) had all data required by the guidelines for testing of pharmaceuticals.     

Induction of Excision Repairable DNA Lesions in Lymphocytes Exposed to Lead and ALA In Vitro

Numbers of studies have been carried out on the potential of lead genotoxicity. The mechanisms of lead genotoxicity are not fully known but partly attributed to the formation of highly reactive oxygen metabolites (ROM). However, lead ions have no ability to generate ROM. In this study, we have investigated the ability of lead and ALA to induce excision repairable DNA lesions by using cytosine arabinoside or cytokinesis block micronucleus (ARA-C/CBMN) assay. N-methyl-N-nitrosourea was used as a positive control which is a mutagen and known to induce excision repair. The results of the ARA-C/CBMN assay show that ALA exposures have significantly (p < 0.01) increased the ratio of excision repairable DNA lesions in peripheral blood lymphocytes; however, lead have not. Accordingly, accumulation of ALA should be considered as an effective partner of lead induced DNA damage in lead exposure.     

The comet assay in male reproductive toxicology

Due to our lifestyle and the environment we live in, we are constantly confronted with genotoxic or potentially genotoxic compounds. These toxins can cause DNA damage to our cells, leading to an increase in mutations. Sometimes such mutations could give rise to cancer in somatic cells. However, when germ cells are affected, then the damage could also have an effect on the next and successive generations. A rapid, sensitive and reliable method to detect DNA damage and assess the integrity of the genome within single cells is that of the comet or single-cell gel electrophoresis assay. The present communication gives an overview of the use of the comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation vs the use of fresh sperm, viability and statistics. It further focuses on in vivo and in vitro comet assay studies with sperm and a comparison of this assay with other assays measuring germ cell genotoxicity. As most of the de novo structural aberrations occur in sperm and spermatogenesis is functional from puberty to old age, whereas female germ cells are more complicated to obtain, the examination of male germ cells seems to be an easier and logical choice for research and testing in reproductive toxicology. In addition, the importance of such an assay for the paternal impact of genetic damage in offspring is undisputed. As there is a growing interest in the evaluation of genotoxins in male germ cells, the comet assay allows in vitro and in vivo assessments of various environmental and lifestyle genotoxins to be reliably determined.     

三种兽药添加剂对土壤赤子爱胜蚓的毒理学研究

喹乙醇、阿散酸和土霉素是应用极广的兽药添加剂,在畜牧业的发展中起着特殊的作用,但其使用不当或过量使用会给环境带来很大危害.采用土壤中的重要生物赤子爱胜蚓作为生物材料,分别研究了喹乙醇、阿散酸和土霉素对赤子爱胜蚓的急性毒性和对赤子爱胜蚓体腔细胞DNA的损伤.结果表明,喹乙醇溶液法LC₅₀>2000mg·L^-1,滤纸法LC₅₀>5.71×10^-2mg·cm^-2,而阿散酸和土霉素在所设最大受试剂量内没有毒性.单细胞凝胶电泳试验结果表明,阿散酸没有引起蚯蚓体腔细胞DNA明显损伤;喹乙醇和土霉素均能引起一定程度的DNA损伤,中、高剂量组与阴性对照相比有极显著差异(P<0.01).因此,有必要加强对以上兽药添加剂,特别是喹乙醇和土霉素的管理,控制用药时间和剂量,以确保人体健康和环境安全.     

A novel and simple method of screening compounds for interaction with DNA: A validation study

We report the development of a simple, cost-effective assay for detecting compounds that have the ability to interact with and modify DNA. Potential uses for the assay lie in the areas of early genotoxicity testing of drug candidates, anticancer and antibiotic drug discovery, environmental monitoring and testing in the food, beverage and cosmetics industries. At present the assay has been used to assess direct-acting compounds only and it is yet to be established whether the assay is compatible with bio-activation. The methodology is based on the oxidative reaction of potassium permanganate with pyrimidine bases, which have become perturbed and more reactive by the agent under test. Results are recorded by use of UV/vis spectroscopy. The adaptation to a multi-well plate format provides the capacity for high throughput utilizing small amounts of compounds. Over 100 compounds, comprising different classes of DNA-binding chemicals as well as non-binding controls, have been put through the assay and the results compared with existing genotoxicity testing data from other methods. The assay has shown to be predictive of the results of other genotoxicity testing methods. We have found that the method is overall predictive of 71% of Ames bacterial reverse-mutation test results (where data are given) encompassing both negative and positive results.     

Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate.

Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.     

The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review

More than 9000 papers using the random amplified polymorphic DNA (RAPD) or related techniques (e.g. the arbitrarily primed polymerase chain reaction (AP-PCR)) have been published from 1990 to 2005. The RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. Despite their extensive use, these techniques have also attracted some criticisms, mainly for lack of reproducibility. In the light of their widespread applications, the objectives of this review are to (1) identify the potential factors affecting the optimisation of the RAPD and AP-PCR assays, (2) critically describe and analyse these techniques in genotoxicity and carcinogenesis studies, (3) compare the RAPD assay with other well used methodologies, (4) further elucidate the impact of DNA damage and mutations on the RAPD profiles, and finally (5) provide some recommendations/guidelines to further improve the applications of the assays and to help the identification of the factors responsible for the RAPD changes. It is suggested that after proper optimisation, the RAPD is a reliable, sensitive and reproducible assay, has the potential to detect a wide range of DNA damage (e.g. DNA adducts, DNA breakage) as well as mutations (point mutations and large rearrangements) and therefore can be applied to genotoxicity and carcinogenesis studies. Nevertheless, the interpretation of the changes in RAPD profiles is difficult since many factors can affect the generation of RAPD profiles. It is therefore important that these factors are identified and taken into account while using these assays. On the other hand, further analyses of the relevant bands generated in RAPD profile allow not only to identify some of the molecular events implicated in the genomic instability but also to discover genes playing key roles, particularly in the initiation and development of malignancy. Finally, to elucidate the potential genotoxic effects of environmental contaminants, a powerful strategy could be firstly to use the RAPD assay as a screening method and secondly to apply more specific methods measuring for instance DNA adducts, gene mutations or cytogenetic effects. It is also envisaged that these assays (i.e. RAPD and related techniques), which reflect effects at whole genome level, would continue to complement the use of emerging technologies (e.g. microarrays which aim to quantify expression of individual genes).