The genotoxicity and cytotoxicity assessments of andrographolide in vitro.

Andrographolide is a major phytoconstituent present in Andrographis paniculata, a plant used in traditional medicines in Asia for various ailments. This tropical shrub was reported to possess various pharmacological activities and has been marketed around the world including Europe, however the toxicological data especially potential genotoxicity assessment on the phytocompound is still lacking. This study was performed to assess the ability of andrographolide to induce chromosomal changes using the in vitro cytokinesis-blocked micronucleus assay with immunofluorescent labelling of kinetochores in metabolically-competent AHH-1 and MCL-5 human lymphoblastoid cell lines. Various cytotoxicity endpoints were also evaluated in this study. Andrographolide was found to cause a weak increase in micronuclei induction at 10-50 µM in both AHH-1 and MCL-5 cell lines, respectively which were within the historical range. Kinetochore analysis revealed that the micronuclei induced in MCL-5 cells due to andrographolide exposure originated via an aneugenic mechanism that was indicated by the relatively higher but non-significant percentage of kinetochore positive micronuclei compared to negative control. Andrographolide also elicited a dose-dependent cellular cytotoxicity, with cells dying primarily via necrosis compared to apoptosis. Here we report that andrographolide was not genotoxic at the doses tested and it induces dose-dependent necrosis in vitro.

Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing.

The ability of the in vitro mammalian cell tests currently used to identify genotoxins has been shown to be limited by a high rate of false-positive results, triggering further unnecessary testing in vivo. During an European Centre for the Validation of Alternative Methods workshop on how to improve the specificity of these assays, testing at high concentrations was identified as one possible source of false positives. Thus far, Organisation for Economic Co-operation and Development genotoxicity test guidelines have required testing of chemicals using mammalian cells in vitro should be undertaken to concentrations as high as 10 mM (5000 µg/ml). Recently, a draft revision of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use genotoxicity test guidelines has recommended that testing concentrations should be reduced to 1 mM (500 µg/ml). To assess the impact that this lowering would have on the outcome of in vitro genotoxicity testing, we established a database of 384 chemicals classified as rodent carcinogens and reported Ames test results and the test concentrations that produced positive results in the mouse lymphoma assay (MLA), in vitro chromosome aberration (CA) assay and in vitro micronucleus test. Genotoxicity testing results were illustrated for 229 and 338 compounds in the MLA and in vitro CA assay, respectively. Of these test compounds, 62.5% produced positive results in the MLA, of which 20.3% required testing between 1 and 10 mM. A total of 58.0% produced positive results in in vitro CA assays, of which 25.0% required testing between 1 and 10 mM. If the testing concentration limit for mammalian cell assays was reduced to 1 mM, 24 (6.25%) potential carcinogens would not be detected in any part of the standard in vitro genotoxicity test battery (Ames test, MLA and in vitro CA assay). Further re-evaluation and/or retest of these compounds by Kirkland and Fowler [Kirkland, D. and Fowler, P. (2010) Further analysis of Ames-negative rodent carcinogens that are only genotoxic in mammalian cells in vitro at concentrations exceeding 1 mM, including retesting of compounds of concern. Mutagenesis 25, 539-553] suggest that the current 10 mM top concentration can be reduced without any loss of sensitivity in detecting rodent carcinogens.

Special issue on in vitro MN trial.

In this commentary we are addressing some additional thoughts on the in vitro MN test: its predictivity for in vivo MN assays, its sensitivity, and how the choice of the cell line and the protocol (with or without cytochalasin-B) can influence these aspects. These considerations might help to make the in vitro MN test a reliable, toxicologically relevant and sensitive in vitro genotoxicity test covering both clastogenic and aneugenic events, and predictive for in vivo genotoxicity, in humans as well.

Metabolic influences for mutation induction curves after exposure to Sudan-1 and para red.

Sudan-1 and para red are industrial dyes that have been illegally added to some foodstuffs, leading to withdrawal of the adulterated products throughout the UK since 2003. This resulted in international concern that arose because Sudan-1 is classified by International Agency for Research on Cancer as a Category 3 carcinogen. However, little is known about the dose response of this chemical at low, more biologically relevant, doses. The study therefore aimed to characterize the dose response for gene mutation and chromosomal damage induced by two azo dyes, namely Sudan-1 and para red. Gene mutations were analysed using the hypoxanthine phosphoribosyltransferase forward mutation assay and chromosomal damage was measured using the cytokinesis-blocked micronucleus assay. Two cell lines were used in these investigations. These were the AHH-1 cell line, which inducibly expresses CYP1A1, and the MCL-5 cell line derived from a subpopulation of AHH-1 cells that expresses a particularly high level of CYP1A1 activity. The MCL-5 cell line has also been transfected with two plasmids that stably express CYP1A2, CYP2A6 and CYP3A4 and all four of these CYP enzymes are known to metabolically activate Sudan-1. AHH-1 cells were used to investigate the dose response of the azo dyes, and MCL-5 cells were used to see if the dose response changed with increased metabolism. Sudan-1 induced a non-linear dose-response curve for gene mutation and chromosomal damage in AHH-1 cells. The genotoxic activity of Sudan-1 was greatly increased in MCL-5 cells. This indicated that the oxidation metabolites from Sudan-1 were both more mutagenic and more clastogenic than the parent compound. Para red also demonstrated a non-linear dose response for both gene mutation and chromosome damage in AHH-1 cells, and an increase in micronuclei induction was observed after increased oxidative metabolism in MCL-5 cells. Sudan-1 and para red are genotoxic chemicals with non-linear dose responses in AHH-1 but not in MCL-5 cells, and oxidative metabolism increases the genotoxic effect of both compounds.

In vitro genotoxic assessment of xenobiotic diacylglycerols in an in vitro micronucleus assay.

Xenobiotic diacylglycerols (DG) may induce pathological disorders by causing abnormal chromosomal segregation, which could be aneuploid. In this study, seven xenobiotic-diacylglycerols (four of drug origin and three of pesticide origin) were evaluated for their ability to induce aneuploidy in mammalian cultures using in vitro cytokinesis blocked micronucleus (CBMN) assay coupled with kinetochore labeling and interphase fluorescent in situ hybridization. Out of seven xeno-DGs, two (ibuprofen-DG and fenbufen-DG) induced statistically significant (P < 0.001) and dose-dependent increase in micronucleus induction, but this apparent micronucleus induction was very weak in case of fenbufen-DG. These MN were produced predominantly by aneugenic and clastogenic mechanisms, respectively, confirmed by immunofluorescent labeling of kinetochores. Fluorescent in situ hybridization analysis revealed that ibuprofen-DG induced significantly higher nondisjunction for chromosomes 10, 17, and 18. Other xenobiotic diacylglycerols (indomethacin-DG, salicylic acid-DG, 4-(2-methyl-4-chlorophenoxy) butanoic acid-DG (MCPB-DG), 2-(2-methyl-4-chlorophenoxy) propanoic acid-DG (MCPP-DG) and 2-(4-dichlorophenoxy)-butanoic acid-DG (2,4 DB-DG) did not induce micronuclei, but the concentrations tested did not reach levels that caused the marked growth suppression typically required for testing for regulatory testing purposes. However, the levels of growth suppression achieved were similar to that seen with ibuprofen-DG, which was positive. This study shows that xeno-DGs, which have been neglected in the past for their possible link to any pathological disorders, need serious assessment of their mutagenic potential.

The detection and assessment of the aneugenic potential of selected oestrogens, progestins and androgens using the in vitro cytokinesis blocked micronucleus assay.

The use of 17-beta-oestradiol, testosterone, progesterone, zearanol, trenbolone acetate and melengesterol acetate in animal feed as growth promoters has been banned in the European Union since 1989. However, the data available on their genotoxicity is limited. To bridge this gap the present study was carried out with the aim of evaluating these hormones for their ability to induce aneuploidy. Aneuploidy has been recently considered sufficiently important to be included in the routine testing of chemicals and radiation. These types of numerical chromosomal aberrations may arise by at least two mechanisms, chromosome loss and non-disjunction. Over the past few years, the cytokinesis blocked micronucleus (CBMN) technique has evolved into a robust assay for the detection of aneuploidy induction. At the present time, it is the only assay which can reliably detect both chromosome loss and non-disjunction when the basic methodology is coupled with appropriate molecular probing techniques such as immunoflourescent labelling of kinetochores and Fluorescence in situ Hybridisation. In this present study, aneuploidy induction by three groups of hormones was studied using CBMN assay coupled with Fluorescence in situ Hybridisation. The results from the present study demonstrate that 17-beta-oestradiol, diethylstilboestrol, progesterone and testosterone are genotoxic and induce aneuploidy by non-disjunctional mechanism, whereas trenbolone is also genotoxic by a clastogenic mechanism. However, melengesterol acetate and zearanol proved to be non-genotoxic in vitro.

Do oestrogens induce chromosome specific aneuploidy in vitro, similar to the pattern of aneuploidy seen in breast cancer?

The study was concerned with investigating the specific effects of non-DNA reactive oestrogens at low "biologically relevant" doses and the causative role they may play in breast cancer through inducing aneuploidy. A review of previous studies identified a non-random pattern of aneuploidy seen in breast cancers. This information was used to select those chromosomes that undergo copy number changes in breast cancer and chromosomes that appear stable. A panel of centromeric specific probes were selected and centromeric specific fluorescence in situ hybridisation (FISH) was carried out on the human lymphoblastoid cell line, AHH-1, which had been pre-treated with the chemical aneugens 17-beta oestradiol, diethylstilbestrol (DES) and bisphenol-A (BP-A). The results suggest that oestrogens may play a causative role in breast cancer by inducing a specific pattern of aneuploidy similar to that seen in breast carcinomas. 17-beta oestradiol appears to induce changes most similar to those seen in breast tumours, BP-A induces the same pattern but at a lower frequency and DES appears to be less chromosome specific in its act.

Identification of early p53 mutations in clam ileocystoplasties using restriction site mutation assay.

OBJECTIVES: Because a risk of cancer arising in enterocystoplasties exists, it is necessary to identify which patients are most at risk of tumor formation. The aim of this study was to determine whether rare mutated p53 sequences were more common at the enterovesical anastomosis than in the bladder remnant in patients with a clam ileocystoplasty using the restriction site mutation (RSM) assay. METHODS: DNA was extracted from endoscopic biopsies obtained from the ileovesical anastomosis and native bladder remnant (control specimens) of 38 patients with a clam ileocystoplasty. The RSM assay was used to study five known hotspots for mutations of the p53 gene using the restriction enzymes Hha I (codon 175), Taq I (codon 213), Hae III (codon 249/250), and Msp I (codons 248 and 282). The mutational events of p53 were confirmed by sequencing the undigested mutated polymerase chain reaction products identified by RSM analysis. RESULTS: We found p53 mutations at the ileovesical anastomosis in 7 of the 38 patients. The mutations were observed at codon 213 (n = 1), codon 248 (n = 3), and codon 250 (n = 3). No p53 mutations were detected in any control specimen. CONCLUSIONS: The ileovesical anastomosis is genetically unstable in patients with a clam ileocystoplasty. The p53 mutations identified by the RSM assay at the enterovesical anastomosis could possibly be used as markers of genetic instability to identify patients at risk of developing a tumor. Prospective, randomized longitudinal studies are required to substantiate this hypothesis.

Bone morphogenic factor gene dosage abnormalities in prostatic intraepithelial neoplasia and prostate cancer.

Abnormal expression of bone morphogenic proteins (BMP) has been reported in prostate cancer as compared to benign prostatic tissue. Since aberrations in gene expression often result from alterations in gene copy number, we have investigated this possibility in patients with early prostate cancer. Probes for fluorescence in situ hybridization for the BMP, BMP5, BMP7, and UC28 gene loci were developed and applied to archival sections with areas of adjacent benign epithelium, high-grade prostatic intraepithelial neoplasia, and prostate carcinoma. Two hundred nuclei from each region were evaluated. No deletions of the gene loci examined were observed, but gain of BMP2, BMP5, BMP7, and UC28 occurred in 58, 50, 50, and 67% of tumor foci, respectively. These aberrations in copy number may be caused by early events in tumor development because they were also present in 10-30% of high-grade prostatic intraepithelial hyperplasia foci. In addition, one tumor demonstrated a tandem amplification of the UC28 gene locus. Approximately half of the prostate tumors displayed increased copy numbers of the BMP2, BMP5, BMP7, and UC28 gene loci, which may account for their abnormal gene expression patterns in neoplastic prostate tissue.

Comparative genomic hybridization (CGH) of augmentation cystoplasties.

OBJECTIVE: Tumors arising within augmentation cystoplasties are aggressive, have poor prognosis and the majority are not detected at follow-up cystoscopy. Genetic changes in tumors precede morphological abnormalities. Therefore, the aim of this study was to investigate whether genetic abnormalities detected by comparative genomic hybridization (CGH) could be used to identify those patients with augmentation cystoplasties at increased risk of tumorigenesis. METHODS: Bladder biopsy samples were obtained from 16 augmentation cystoplasty patients both distant from and near to the enterovesical anastomosis. CGH was used to detect genetic abnormalities in DNA extracted from the biopsies, archival specimens of two augmentation cystoplasties and two de novo bladder adenocarcinomas. RESULTS: A greater number of amplifications on 2p, 3q, 8q, 9p, 17p, 18pq and 20pq, were observed in bladder biopsies obtained near to the enterovesical anastomosis compared to those taken distant to the suture line. CGH of archival augmentation cystoplasty tumor DNA indicated abnormalities at several loci with amplifications at 2q, 5q, 10p and 21pq, while deletions occurred at 5p and 16p. CONCLUSIONS: The results of this study suggest that the urothelium adjacent to the bladder and/or bowel anastomosis in augmentation cystoplasties is genetically unstable. Furthermore, longitudinal studies are required to establish whether or not patients exhibiting genetic instability following augmentation cystoplasty are at greater risk of developing tumors than those with genetically stable epithelia.

Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens.

A mechanistic understanding of carcinogenic genotoxicity is necessary to determine consequences of chemical exposure on human populations and improve health risk assessments. Currently, linear dose-responses are assumed for DNA reactive compounds, ignoring cytoprotective processes that may limit permanent damage. To investigate the biological significance of low-dose exposures, human lymphoblastoid cells were treated with alkylating agents that have different mechanisms of action and DNA targets: methylmethane sulfonate (MMS), methylnitrosourea (MNU), ethylmethane sulfonate (EMS), and ethylnitrosourea (ENU). Chromosomal damage and point mutations were quantified with the micronucleus and hypoxanthine phosphoribosyltransferase forward mutation assays. MNU and ENU showed linear dose-responses, whereas MMS and EMS had nonlinear curves containing a range of nonmutagenic low doses. The lowest observed effect level for induction of chromosomal aberrations was 0.85 microg/mL MMS and 1.40 microg/mL EMS; point mutations required 1.25 microg/mL MMS and 1.40 microg/mL EMS before a mutagenic effect was detected. This nonlinearity could be due to homeostatic maintenance by DNA repair, which is efficient at low doses of compounds that primarily alkylate N(7)-G and rarely attack O atoms. A pragmatic threshold for carcinogenicity may therefore exist for such genotoxins.

Deoxycholic acid at neutral and acid pH, is genotoxic to oesophageal cells through the induction of ROS: The potential role of anti-oxidants in Barrett's oesophagus.

Bile acids are often refluxed into the lower oesophagus and are candidate carcinogens in the development of oesophageal adenocarcinoma. We show here that the secondary bile acid, deoxycholic acid (DCA), is the only one of the commonly refluxed bile acids tested here, to show genotoxicity, in terms of chromosome damage and mutation induction in the human p53 gene. This genotoxicity was apparent at both neutral and acidic pH, whilst there was a considerable increase in bile-induced toxicity at acidic pH. The higher levels of cell death and low cell survival rates at acidic pH may imply that acid bile exposure is toxic rather than carcinogenic, as dead cells do not seed cancer development. We also show that DCA (at neutral and acid pH) induced the release of reactive oxygen species (ROS) within the cytoplasm of exposed cells. We further demonstrate that the genotoxicity of DCA is ROS mediated, as micronucleus induction was significantly reduced when cells were treated with DCA + the anti-oxidant vitamin C. In conclusion, we show that DCA, is an effective genotoxin at both neutral and acidic pH. As bile acids like DCA can induce DNA damage at neutral pH, suppressing the acidity of the refluxate will not completely remove its carcinogenic potential. The genotoxicity of DCA is however, ROS dependent, hence anti-oxidant supplementation, in addition to acid suppression may block DCA driven carcinogenesis in Barrett's patients.

The use of the in vitro micronucleus assay to detect and assess the aneugenic activity of chemicals.

The successful validation of the in vitro micronucleus assay by the SFTG now provides the opportunity for this highly cost effective assay to be used to screen chemicals for their ability to induce both structural (clastogenic) and numerical (aneugenic) chromosome changes using interphase cells. The use of interphase cells and a relatively simple experimental protocol provides the opportunity to greatly increase the statistical power of cytogenetic studies on chemical interactions. The application of molecular probes capable of detecting kinetochores and centromeres provides the opportunity to classify mechanisms of micronucleus induction into those which are primarily due to chromosome loss or breakage. When a predominant mechanism of micronucleus induction has been shown to be based upon chromosome loss then further investigation can involve the determination of the role of non-disjunction in the induction of aneuploidy. The binucleate cell modification of the in vitro micronucleus assay can be combined with the use of chromosome specific centromere probes to determine the segregation of individual chromosomes into daughter nuclei. The combination of these methods provides us with powerful tools for the investigation of mechanisms of genotoxicity particularly in the low dose regions.

Assessing the potential mutagenicity of pesticides.

Determining mutagenic profiles of pesticides requires tests of high sensitivity and specificity. An effective strategy uses tests that produce reproducible and biologically relevant data based upon three stages. Stage 1, in vitro, uses (i) bacterial gene mutation assays, (ii) assays measuring clastogenicity and aneugenicity, and (iii) assays measuring the induction of gene mutations in cultured mammalian cells. Stage 1 can detect most mutagenic hazards. Stage 2, in vivo testing in somatic cells of rodents, is required to determine whether in vitro positives are reproduced in vivo and to detect activity only produced in intact animals. Decisions on assay selection should be based on the in vitro profile. In most cases in vivo assessment is based on the micronucleus assay in rodent bone marrow. Stage 3, in vivo germ-cell testing, is rarely required for pesticides that have been shown to be mutagenic in somatic cells in vivo.

The detection of genotoxic activity and the quantitative and qualitative assessment of the consequences of exposures.

A wide range of assays are now available which enable the effective detection of the mutagenic (the induction of gene and chromosomal changes) and more generally genotoxic (cellular interactions such as DNA lesion formation) activity of individual chemicals and mixtures. However, when genotoxic activity has been detected and human exposure occurs the critical questions relate to the qualitative and quantitative activity of the agent and the parameters such as routes of exposure, target organs and metabolism. Of major importance in hazard and risk estimation is the nature of the dose response relationship of each chemical and their potential interactions in mixtures. In this paper, we illustrate the methods available to produce quantitative and qualitative data in vitro using the micronucleus assay (as a measure of chromosomal structural and numerical mutations) and the HPRT assay (as a measure of induced gene and point mutations) and the current limitations (such as the large numbers of animals required) for obtaining such information in vivo. We recommend that in vivo studies should primarily focus upon confirmatory mechanistic analysis. For individual chemicals, profiles of the base changes induced can be obtained using the HPRT gene mutation assay and comparisons produced both in vitro and in vivo and thus allow identification of mechanistic differences between different modes of exposure.

Differential expression of the MAD2, BUB1 and HSP27 genes in Barrett's oesophagus-their association with aneuploidy and neoplastic progression.

Chromosomal instability (CIN) leading to aneuploidy is a ubiquitous and early event in the progression of Barrett's oesophagus, but its origins are unknown. Hence, the transcriptional levels of components of the mitotic spindle checkpoint (important in ensuring precise chromosome segregation) were examined in Barrett's lesions and correlated with the degree of aneuploidy present in the tissues. Gene expression levels of the MAD2 and BUB1 mitotic spindle checkpoint genes were assessed in 37 Barrett's patients (with histology ranging from metaplasia to adenocarcinoma) by real-time RT-PCR. In addition, the transcriptional levels of HSP27 were also examined as firstly, its expression is known to be down regulated in Barrett's metaplasia (BM) and thus was included as a positive control for the real-time RT-PCR assay. While, secondly, the expression pattern of this gene during Barrett's neoplastic progression was investigated, as this has not been previously assessed. Both over and under expression of the MAD2 and BUB1 mitotic spindle checkpoint genes were detected at all Barrett's histological stages with no apparent selective trend with neoplastic progression. In addition, no correlation with aneuploidy was established, indicating an alternative mechanism must underlie Barrett's associated chromosomal instability. HSP27 expression was reduced in metaplasia and then significantly increased with progression. Gender-related differences were observed and HSP27 expression was higher in poorly-differentiated adenocarcinomas than in well-differentiated forms. HSP27 transcriptional patterns therefore present potential as a prognostic tool to predict the aggressiveness of oesophageal adenocarcinomas (OA).

Comparative genomic hybridization analysis of N-methyl-N'-nitrosoguanidine-induced rat gastrointestinal tumors discloses a cytogenetic fingerprint.

Exposure to N-nitroso compounds is thought to play a key role in the development of gastric cancer in humans. The alkylating agent N-methyl-N'-nitrosoguanidine (MNNG) is carcinogenic in a number of animal models and its preferential target tissue is the gastrointestinal (GI) tract. The genetic synteny among rats and humans makes the rat a useful model for induced tumorigenesis. However, because of the limited availability of genetic information, cytogenetic and molecular studies are rarely performed in the rat. We report an investigation of eight MNNG-induced rat gastric tumors by comparative genomic hybridization (CGH). The tumors were from forestomach (induced by a single dose of MNNG) and from pylorus (induced by chronic exposure). CGH identified a genetic fingerprint of chromosomal imbalances common to the two types of the tumors. Frequent gains were observed at 9q11-q12, 15q22-25, and Xq11-q12. Forestomach carcinomas were also characterized by gains in 7q11-q12, 20q13, and Yq12. Homology studies between the rat and human genomes indicate the presence of genes within these regions with potential relevance to tumorigenesis in the GI tract. Our findings provide new insights into the location of genes involved in MNNG-induced gastric cancer initiation and/or progression in the rat.

Aneugenic and clastogenic effects of doxorubicin in human lymphocytes.

Doxorubicin, a benzanthroquinone anticancer agent, was examined for its effect on micronucleus induction in cultured human lymphocytes. A statistically significant dose-dependent increase in micronucleus frequency (P<0.001) in binucleated cells was seen and an increase in the kinetochore-positive (P<0.001) and kinetochore-negative micronuclei (P<0.001) was observed. An increase was also observed in the number of necrotic cells, but the frequency of apoptotic cells remained almost constant. This confirms that doxorubicin is both clastogenic and aneugenic.