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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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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 order to investigate the alleged potential of paternally administered urethane to cause foetal abnormalities and heritable tumours, male CD-1 mice were treated with urethane, either acutely by intraperitoneal injection at doses of 1.25 and 1.75 g/kg bodyweight (bwt) or sub-chronically in the drinking water at 1.25 for 10 weeks, and 3.75 mg/ml for 9 weeks or vehicle for the control groups. They were mated to untreated females 1 week later. Uterine contents of half the pregnant females were examined just before full term, while the remaining females were allowed to deliver their litters. The resulting F1 offspring were observed for approximately 18 months and 12 months for acute and sub-chronic exposures respectively and subjected to necropsy examination. Some of the mice treated acutely with 1.75 g/kg bwt exhibited partial infertility but none of those treated with 1.25 g/kg bwt had an adverse effect on their reproductive ability. There was no genetic effect of acute urethane treatment on male germ cells as indicated by dominant lethality. After birth, there was an increase (P < 0.05) in post-implantation deaths possibly due to perinatal mortality. There was an increased incidence and earlier onset of liver tumours induced in F1 male offspring from F0 males treated with 1.75 g/kg bwt, (20.7% vs. 10.1%, P = 0.026) but not in the female offspring. F1 males from both treatment groups had mean bodyweights significantly higher than controls (P < 0.01). Some males from each dose group of the acute study were examined using the restriction site mutation assay involving analysis of exon sequences. No mutations were identified in testes, liver or spleen of DNA isolated from the urethane-treated animals. No reproductive or genetic effects were seen with sub-chronic treatment at either 1.25 or 3.75 mg/ml urethane nor was there any predisposition of F1 animals to tumours although observation times were shorter.

Earlier studies have established that the rodent bladder carcinogen o-anisidine (OA) gives negative results in all of the standard rodent genetic toxicity assays. In the present study, a single oral administration of the maximum tolerated dose level (750 mg/kg) of OA to B6C3F1 mice yielded negative results in 32P-post-labelling assays of bladder and liver DNA (24 h after dosing). Likewise, 14C-ring-labelled OA administered orally to B6C3F1 mice gave no evidence of DNA binding 6, 12 or 24 h later. Administration of OA (750 mg/kg) to transgenic lacI- mice (Big Blue) led to a small increase in mutation frequency (MF) in the bladder, but not in the liver. Increased MFs were observed in the bladder following 1, 3 or 10 daily doses with sampling times of 1 or 2 weeks after the final dose. However, statistical significance (P < 0.01) was only reached 2 weeks after either 3 or 10 daily administrations of OA. The positive control chemical (dimethylnitrosamine) gave a positive result (P < 0.01) in the liver, but not the bladder, 7 days after a single administration of 10 mg/kg. The possibility that OA is mutagenic and carcinogenic to the rodent bladder via formation of radical species is suggested.

The genotoxicities in vitro and in vivo of the mouse-skin carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) have been compared with those of its weakly carcinogenic 4,5-sulphur analogue, 6,11-dimethylbenzo[b]naphtho-[2,3-d]thiophene (S-DMBA). The only datasets that correlated with the relative carcinogenicity of these agents to the skin were those conducted using topically exposed mouse skin. Thus, both chemicals induced lacZ- mutations in the skin of lacZ+ transgenic mice, and both produced DNA adducts on mouse-skin DNA as assessed using the 32P-postlabeling technique. In each case, DMBA gave a stronger response than did S-DMBA. In contrast to these responses, only DMBA was active in the mouse bone-marrow micronucleus assay and in the C3H10T1/2 in vitro cell transformation assay. Both chemicals were mutagenic to Salmonella and of approximately equal potency. The molecular geometry of DMBA and S-DMBA are compared, and divergent CASE predictions of activity in the Salmonella assay and skin-painting bioassay are discussed. The importance of conducting predictive genotoxicity assays in systems close to those in which carcinogenicity is to be assessed is emphasized by these data.

Following exposure to the alkaloid noscapine hydrochloride over a concentration range of 10-120 micrograms/ml immortal cultures of Syrian hamster dermal fibroblasts were shown to undergo morphological transformation. The resultant transformed foci produced cultures which were anchorage independent as confirmed by soft agar tests. Karyotype analysis of a noscapine transformed colony demonstrated an increase in chromosome number compared to the immortal culture and the non-random duplication of a translocated chromosome 9 previously identified in the immortal culture. These data indicate that noscapine, which has previously been shown to be a spindle inhibitor and inducer of polyploidy in cultured cells, is capable of inducing in vitro cell transformation. Such data indicate a carcinogenic potential for this widely used cough suppressant.

The objectives of the European Economic Community's Environmental Research and Development Programmes are to provide a scientific basis for current environmental policy and future environmental management. This paper reviews the work and achievements of the studies carried out by the collaborating laboratories during the period 1981-1985 of the 3rd Programme within the research area 'The Genetic Effects of Environmental Chemicals'. Research efforts supported by the programme may be divided into (i) those involving the validation of current genotoxicity assays and the evaluation of their use in community regulatory activities such as the classification and labelling of chemicals, (ii) those involving the characterization of the metabolic and genotoxic profile of specific chemicals and (iii) those involving the development of new technologies suitable for the hazard identification and risk evaluation of genotoxins. Validation studies have included those of the host mediated assay (and its various modifications), unscheduled DNA synthesis assays and a variety of Drosophila genotoxicity assays. New developments include assays for the detection of chromosome aneuploidy, protocols for cell transformation studies, the detection and quantification of genotoxic lesions, techniques for the study of transplacental genotoxicity, assays for the detection of genotoxic activity in the liver and in germ cells and monitoring techniques for the assessment of both environmental quality and genotoxin exposure in individuals and in populations. The coordination of research effort, such as that described here provides an effective mechanism for the concentration of research on specific problems, for the rapid dissemination of experimental data and the transfer of technology within the European Community.

A range of conjugated and free bile acids were assayed for their ability to induce a variety of genetic endpoints in growing cells of yeast. None of the bile acids showed any activity in assays for the induction of mitotic crossing-over and mutation whereas the free bile acids lithocholic, chenodeoxycholic, deoxycholic and cholic acid were potent inducers of mitotic chromosome aneuploidy. In contrast, both conjugated bile acids, taurodeoxycholic and glycodeoxycholic lacked the ability to induce mitotic aneuploidy. When the potency of the free bile acids were compared, lithocholic and chenodeoxycholic acids showed higher levels of induction of mitotic aneuploidy per lethal event compared with cholic and deoxycholic acids. In view of the previously observed correlation between the ability of a chemical to induce chromosome aneuploidy and tumour promotional activity, the results indicate that the levels of free bile acids in the colon may be significant factors in the etiology of colonic cancer.