Length-dependent cruciform extrusion in d(GTAC)n sequences

pBR322-derived plasmids have been constructed carrying d(GTAC)n.d(GTAC)n inserts of different lengths, in order to investigate the effect of insert size on cruciform extrusion and/or the B-Z transition. Plasmids with n ranging from 4 to 12 are hypersensitive to cleavage by the single-strand specific nucleases, S1 nuclease and Bal31 nuclease. Hypersensitive sites associated with the smaller alternating purine-pyrimidine tracts, however, coexist with the major pBR322 sites. Site-selective cleavage of these plasmids with the resolvase, T7 endonuclease I, demonstrates that all the inserts form cruciform structures when stably integrated into negatively supercoiled plasmids. An increase in the negative superhelical density of the DNA's induces cruciform formation within the insert region, resulting in a reduction in torsional stress consistent with the size of the insert. Moreover, as n decreases, the superhelical density required to stabilise the cruciform state increases. Therefore, the cruciform geometry is the favoured conformation of these d(GTAC)n.d(GTAC)n sequences under torsional stress. The stability of these cruciforms increases as n increases, with cruciformation occurring at lower superhelical densities and to the exclusion of the other pBR322 cruciforms.     

Length-Dependent Cruciform Extrusion in d(GTAC)n Sequences

Abstract

pBR322-derived plasmids have been constructed carrying d(GTAC)_n·d(GTAC)_n inserts of different lengths, in order to investigate the effect of insert size on cruciform extrusion and/or the B-Z transition. Plasmids with n ranging from 4 to 12 are hypersensitive to cleavage by the single-strand specific nucleases, S1 nuclease and Bal31 nuclease. Hypersensitive sites associated with the smaller alternating purine-pyrimidine tracts, however, coexist with the major pBR322 sites. Site-selective cleavage of these plasmids with the resolvase, T7 endonuclease I, demonstrates that all the inserts form cruciform structures when stably integrated into negatively supercoiled plasmids. An increase in the negative superhelical density of the DNA's induces cruciform formation within the insert region, resulting in a reduction in torsional stress consistent with the size of the insert. Moreover, as n decreases, the superhelical density required to stabilise the cruciform state increases. Therefore, the cruciform geometry is the favoured conformation of these d(GTAC)_n·d(GTAC)_n sequences under torsional stress. The stability of these cruciforms increases as n increases, with cruciformation occurring at lower superhelical densities and to the exclusion of the other pBR322 cruciforms.     

In vitro genotoxicity of danthron and its potential mechanism

To ascertain the in vitro genotoxicity of danthron and its potential mechanism of action, we performed an Ames test, a cytokinesis-block micronucleus assay and a comet assay in Balb/c 3T3 cells. The Ames test revealed that danthron was mutagenic only toward Salmonella typhimurium strain TA102 in the presence of an exogenous metabolic activation system (S9 mix). Danthron (25, 50 and 100μg/ml) increased the frequencies of micronuclear cells with or without S9 mix, and the comet length, tail length and Olive tail moment in comet assays without S9 mix in a dose-dependent manner. These results demonstrated the in vitro genotoxicity of danthron and that 3T3 cells are capable of activating danthron. When NADP was replaced by NAD in the S9 mix, danthron remained mutagenic toward strain TA102. The addition of dicoumarol, a DT-diaphorase inhibitor, decreased the number of danthron-induced histidine revertants by 35-39%, indicating that DT-diaphorase is involved in the metabolic activation of danthron in the presence of NADH as an electron donor. In 3T3 cells, increases in reactive oxygen species (ROS) formation and 8-hydroxydeoxyguanosine levels as well as a reduction in GSH levels were induced by danthron in a dose-dependent manner, indicating that oxidative stress may be a major contributing pathway in the genotoxicity of danthron.     

Factors influencing production of cationic starches

The main factors – the amount of catalyst NaOH, the temperature and composition of reaction mixture – influencing the effectiveness of starch cationization with glycidyltrimethylammonium chloride (GTAC) were investigated. It was found that cationic or cross-linked cationic starches with preserved microgranules, a degree of substitution from 0.2 to 0.85 and reaction efficiency from 82% to 93% could be obtained during etherification of starch or cross-linked starch with a mixture containing GTAC, “free” water and 0.04–0.08 mol/AGU sodium hydroxide in heterogeneous condition. The activation energy of the GTAC main reaction is lower than that of the side reactions, thus starch cationization at a lower temperature proceeds with higher reaction efficiency.The amount of NaOH in the cationization mixture has a great influence on the rate of the main and side reactions of GTAC. Only the main reaction – cationization of starch – proceeds when all the NaOH present in the reaction mixture is adsorbed from the liquid phase by the starch. The duration of this “first reaction stage” decreases with increasing reaction temperature and the amount of NaOH in the reaction mixture. NaOH present in the liquid phase of the reaction mixture catalyzes the side reactions of GTAC and changes their character.     

Estragole: a weak direct-acting food-borne genotoxin and potential carcinogen

We evaluated the genotoxicity of the food-flavouring agent estragole in V79 cells using the sister chromatid exchange (SCE) assay and the alkaline comet assay. Unexpectedly, we observed an increase in SCE without an exogenous biotransformation system (S9) and a decrease in its presence. Positive results were also observed in the alkaline comet assay without S9, indicating DNA strand breakage. To ascertain repair of damage, we performed the comet assay in V79 cells after two hours of recovery, and observed a reduction of the genotoxic response. Estragole did not produce strand breaks in plasmid DNA in vitro. We then evaluated the formation of DNA adducts in V79 cells by use of the (32)P-postlabelling assay and detected a dose-dependent formation of DNA adducts, which may be responsible for its genotoxicity. We then assayed estragole in the comet assay with two CHO cell lines, a parental AA8 cell line, and an XRCC1-deficient cell line, EM9. Results confirmed the genotoxicity of estragole without biotransformation in both cell lines, although the genotoxicity in EM9 cells compared with that in AA8 cells was not significantly different, suggesting that the XRCC1 protein is not involved in the repair of estragole-induced lesions. Estragole induces apoptosis, but only with high doses (2000μM), and after long treatment periods (24h). Overall, our results suggest that estragole, besides being metabolized to genotoxic metabolites, is a weak direct-acting genotoxin that forms DNA adducts.     

Comparative genotoxicity of nitrosamine drinking water disinfection byproducts in Salmonella and mammalian cells

Nitrosamine water disinfection byproducts (DBPs) are an emerging class of non-halogenated, nitrogen-containing water contaminants. Five nitrosamine DBPs were analyzed for genotoxicity (N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) and N-nitrosodiphenylamine (NDPhA). Using Salmonella typhimurium strain YG7108 the descending rank order of mutagenicity was NDMA>NPIP>NMOR>NPYR; NDPhA was not mutagenic. We developed and calibrated an exogenous S9 mix that was highly effective in activating NDMA in Chinese hamster ovary (CHO) cells using the SCGE (Comet) assay. The descending rank order for genotoxicity was NDMA>NPIP>NMOR. NDPhA was genotoxic only at one concentration and NPYR was not genotoxic. The genotoxic potencies in S. typhimurium and CHO cells were highly correlated. Based on their comparative genotoxicity attention should be focused on the generation and occurrence of NDMA, NPIP and NMOR. Current drinking water disinfection processes may need to be modified such that the generation of nitrosamine DBPs is effectively limited in order to protect the environment and the public health.     

Hydrogen sulfide induces direct radical-associated DNA damage

Hydrogen sulfide (H(2)S) is produced by indigenous sulfate-reducing bacteria in the large intestine and represents an environmental insult to the colonic epithelium. Clinical studies have linked the presence of either sulfate-reducing bacteria or H(2)S in the colon with chronic disorders such as ulcerative colitis and colorectal cancer, although at this point, the evidence is circumstantial and underlying mechanisms remain undefined. We showed previously that sulfide at concentrations similar to those found in the human colon induced genomic DNA damage in mammalian cells. The present study addressed the nature of the DNA damage by determining if sulfide is directly genotoxic or if genotoxicity requires cellular metabolism. We also questioned if sulfide genotoxicity is mediated by free radicals and if DNA base oxidation is involved. Naked nuclei from untreated Chinese hamster ovary cells were treated with sulfide; DNA damage was induced by concentrations as low as 1 micromol/L. This damage was effectively quenched by cotreatment with butylhydroxyanisole. Furthermore, sulfide treatment increased the number of oxidized bases recognized by formamidopyrimidine [fapy]-DNA glycosylase. These results confirm the genotoxicity of sulfide and strongly implicate that this genotoxicity is mediated by free radicals. These observations highlight the possible role of sulfide as an environmental insult that, given a predisposing genetic background, may lead to genomic instability or the cumulative mutations characteristic of colorectal cancer.     

Genotoxicity and DNA adduct formation of incense smoke condensates: comparison with environmental tobacco smoke condensates

Indoor air pollution has now been recognized as a potentially important problem for public health, since people spend most of their day in closed environments. Incense burning is possibly associated with elevated risks of leukemia and brain tumor in children from the epidemiological studies. Thus, evaluation of the genotoxicity of smoke condensates from incense burning is needed. We examined the genotoxicity of incense smoke condensates (ISC) using the Ames test in S. typhimurium strains with different mutagenic specificity and level of metabolic enzyme, the SOS chromotest in E. coli PQ37, and sister chromatid exchange assay in Chinese hamster ovary cells (SCE/CHO). The genotoxicity of environmental tobacco smoke condensates (TSC) was also evaluated by the three assays to compare with the genotoxicity of ISC, ISC showed a positive response in TA98, but not in TA100. It suggested that ISC only contained frame shift mutagens. The mutagenicity of ISC in both strains of TA98NR with deficient nitroreductase and TA98/1,8-DNP₆ with deficient O-acetyl-transferase was markedly decreased compared to that in TA98 strain. However, the mutagenicity was enhanced in YG1024 with overexpression of O-acetyltransferase activity. Thus, nitroarenes seemed to be responsible in part for the mutagenicity of ISC. Interestingly, all of the four ISC and two TSC samples showed a dose-dependent genotoxic response in the SOS chromotest with E. coli PQ37 but a low SCE induction of those samples were observed in CHO cells. When the genotoxicity was analyzed based on the condensates per one gram of original samples, the genotoxicity of two TSC condensates in prokaryotic cells was higher than that of four ISC samples except for the genotoxicity of TSC-2 in TA98 strain. However, the genotoxicity of certain ISC in eukaryotic cells based on the SCE/CHO assay was higher than that of TSC. To compare the covalent binding of DNA reactive intermediates of ISC and TSC to S. typhimurium TA98, the DNA adducts were evaluated by the ³²P-postlabeling method with butanol extraction version. Similar diagonal radioactive zone (DRZ) was observed between ISC and CSC. However, DNA adduct levels induced by TSC were much greater than that of ISC.     

The genotoxicity of enantiomeric aliphatic epoxides

The (R)- and (S)-optical isomers of 9 epoxides, benzyloxymethyloxirane, epichlorohydrin, glycidol, glycidyl 3-nitrobenzenesulfonate, glycidyl 4-nitrobenzoate, glycidyl tosylate, styrene oxide, glycidyl 1-naphthyl ether and glycidyl 4-nitrophenyl ether, have been compared for their in vivo and in vitro genotoxicity. The in vitro short-term test employed was the Ames mutagenicity assay with Salmonella strain TA100. The in vivo tests were chromosomal aberrations (CA) as well as sister-chromatid exchange (SCE) in bone-marrow cells of mice following intraperitoneal administration of these epoxides. Differences in mutagenicity between isomers were established with TA100 for all the compounds. While 13 of the isomers were genotoxic compared to a negative control by CA measurements, only in the case of glycidyl 4-nitrobenzoate could a significant difference be found between isomers by this test. However, with SCE evaluations, differences were detected between the (R)- and (S)-isomers for all the pairs of compounds with the exception of those for benzyloxymethyloxirane and glycidyl 4-nitrophenyl ether. At least in part, differences in the patterns of genotoxicity among compounds can be related to their differences in reaction pathways.     

Structure-activity relationships of epoxides: induction of sister-chromatid exchanges in V79 cells by enantiomeric epoxides.

Analysis of SCE frequencies in Chinese hamster V79 cells was used to investigate the influence of the stereoisomeric forms of epoxides in mammalian genotoxicity tests. The SCE-inducing potency of 12 pairs of (R)- and (S)-enantiomeric epoxides which differed in the degree of substitution of the oxirane ring was determined. Of these, 2 pairs of epoxides failed to induce SCE. Different SCE-inducing potencies between the (R)- and (S)-enantiomers were shown for 5 epoxides. This study demonstrates that stereoselectivity might play an important role in genotoxicity testing of chemicals with asymmetric C atoms.     

Chromosome aberrations in cultured central mudminnow heart cells and Chinese hamster ovary cells exposed to polycyclic aromatic hydrocarbons and sediment extracts

1. Genotoxicity experiments were conducted with cultured fish cells to determine if the high frequency of epidermal papillomas observed in lemon sole from Sturgeon Bank, where a sewage treatment plant discharges, could be correlated with contamination of the sediments with chemicals such as 3,4-benzopyrene. 2. The frequency of chromosome aberrations was measured in cultured Umbra limi heart (U1-H) and Chinese hamster ovary (CHO) cells following exposure to the polycyclic aromatic hydrocarbons (PAH) 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA), and pyrene (PY), activated using S9 prepared from rainbow trout liver. 3. An increase in the chromosome aberration frequency was only observed following exposure to fish S9-activated BP in both cell lines. 4. Following exposure of the cells to both Sturgeon Bank and Spanish Bank sediment extracts, it was determined that a higher level of toxic and genotoxic activity was associated with the Sturgeon Bank sediments. 5. Since the detection of PAH genotoxicity requires the presence of S9, and since a higher level of genotoxic activity was noted following sediment extract exposures with no S9 present, this suggests that the extracts contain a complex mix of chemicals, some of which express genotoxic activity. 6. An assessment using the micronucleus test failed to indicate in vivo genotoxicity in fish collected from Sturgeon and Spanish Banks. 7. It was, therefore, difficult to associate the observed sediment genotoxicity with the previously noted high incidence of epidermal papillomas in lemon sole from this area.     

Genotoxicity of acrylamide and glycidamide in human lymphoblastoid TK6 cells

The recent finding that acrylamide (AA), a potent carcinogen, is formed in foods during cooking raises human health concerns. In the present study, we investigated the genotoxicity of AA and its metabolite glycidamide (GA) in human lymphoblastoid TK6 cells examining three endpoints: DNA damage (comet assay), clastogenesis (micronucleus test) and gene mutation (thymidine kinase (TK) assay). In a 4 h treatment without metabolic activation, AA was mildly genotoxic in the micronucleus and TK assays at high concentrations (> 10 mM), whereas GA was significantly and concentration-dependently genotoxic at all endpoints at > or = 0.5 mM. Molecular analysis of the TK mutants revealed that AA predominantly induced loss of heterozygosity (LOH) mutation like spontaneous one while GA-induced primarily point mutations. These results indicate that the genotoxic characteristics of AA and GA were distinctly different: AA was clastogenic and GA was mutagenic. The cytotoxicity and genotoxicity of AA were not enhanced by metabolic activation (rat liver S9), implying that the rat liver S9 did not activate AA. We discuss the in vitro and in vivo genotoxicity of AA and GA.     

ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact.

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, 'Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, 'Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.     

Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice

The genotoxicity of an acetone extract of locally collected airborne particles was evaluated both in vitro and in vivo using the sister-chromatid exchange (SCE) assay in mice. At the highest concentration (5.36 mg/5 ml culture), the extract caused approximately a 3-fold increase in SCEs over controls in mouse bone marrow and spleen primary cells in vitro. However, the same airborne particle extract did not induce a significant increase in the SCE level over controls in vivo in mouse bone marrow and spleen cells when administered intraperitoneally or through oral gavage. This indicates that bone marrow and spleen primary cell cultures can be used in in vitro genotoxicity studies of complex mixtures, and that the genotoxicity of airborne particles detected in the in vitro system cannot always be detected in vivo with the same cell types. In addition, the same acetone extract of airborne particles caused dose-related his+ revertants in the strain TA98 of Salmonella typhimurium, both with and without S9 activation. The significant finding of this study is that the in vitro genotoxicity results of airborne particle extract may not be very meaningful in an in vivo situation.     

Standardization of conditions for the metabolic activation of N-nitrosodiethylamine in mutagenicity tests

Like all nitrosamines, N-nitrosodiethylamine (NDEA) requires metabolic activation in order to exert its carcinogenic effects. This activation involves cytochrome P450s (CYP), which generates unstable metabolites that react with the DNA of cells in the immediate vicinity of metabolite formation. Although NDEA is carcinogenic, it has been considered a weak mutagen in classic genotoxicity assays. We used optimized Salmonella/mammalian microsome genotoxicity assays to assess the mutagenicity and toxicity of low concentrations of NDEA. Using a fixed concentration of NDEA (36.5 mg/ml), we varied the length of preincubation in the presence of different concentrations of an S9 metabolic activation mixture. Salmonella typhimurium strains TA97 and TA102 were resistant to NDEA-induced mutagenesis, even after a preincubation of up to 120 min and the use of different concentrations of the S9 mix. Strain TA98 was susceptible to mutagenesis by NDEA in the absence of the S9 mix and after preincubation with NDEA for 90 min. When bacteria of this strain were preincubated with NDEA for 60 min, mutagenesis was detected at an S9 mix concentration >9.55 mg/ml. NDEA also induced mutagenesis in strain TA100 after preincubation for 90 or 120 min, and this effect was dependent on the S9 concentration. E. coli strain BH990 also showed a concentration-dependent response, with only 60% of the cells surviving after a 120-min preincubation with NDEA in the presence of 19.1 mg S9 mix/ml.     

Induction of chromosomal aberrations by phenolic compounds: possible role of reactive oxygen species

Phenolic molecules are widely present in the environment and some of them are well known carcinogens. Some phenolic molecules are also genotoxic but the mechanisms involved in this process are not fully understood. We have studied the induction of chromosomal aberrations by phenol, catechol and pyrogallol in V79 cells at different pH values (6.0, 7.4 and 8.0). At the same pH values, the production of hydroxyl radicals was assessed by measuring the degradation of deoxyribose. Apart from phenol, which only induces a non-significant increase in chromosomal aberration in this experimental system, catechol and pyrogallol showed clear clastogenic effect in a pH-dependent way. Experiments carried out at pH 7.4 in the presence of S9 Mix, SOD, catalase and catalase + SOD suggest that the formation of reactive oxygen species is not the main mechanism involved in the genotoxicity of catechol. However, concerning pyrogallol, our results suggest that its genotoxicity is almost exclusively mediated by reactive oxygen species. Taken together, these results suggest that, in spite of the structural similarity between the different molecules studied, the mechanisms of genotoxicity of these molecules could be considerably different. The existence of several mechanisms of genotoxicity, partially shared by this class of compounds, could explain the synergistic effects observed between these compounds in several genotoxicity test systems. Accurate knowledge of their mechanisms of genotoxicity could improve considerably the assessment of their relevance to human health, since these compounds, once absorbed, are subject to a wide range of pH values in vivo.     

The induction of forward gene mutation and gene conversion in yeasts by treatment with cyclophosphamide in vitro and in vivo

The present paper assesses the most suitable conditions for metabolic activation with yeasts in vitro, at least as far as cyclophosphamide (Cy) is concerned. These include treatment time, incubation temperature, the amounts of S9 and cofactors. Particular attention is devoted to the use of various solvents, showing that their use can considerably affect the mutagenic response of the chemical being tested. It also examines the effects of enzyme inducers (by using S9 from rats and mice) such as phenobarbital (PB) and 5,6-benzoflavone (BF) administered separately or together. The metabolizing capability of other organs such as the lungs and kidneys is also determined. All these data are compared with Cy genotoxicity (in vivo) evaluated by the intrasanguineous host-mediated assay and by recovering the yeast target cells from the liver, lungs and kidneys. The most striking effects are that, in vitro, PB greatly enhances Cy genotoxicity, whilst in vivo it substantially reduces it.     

ICH-Harmonised guidances on genotoxicity testing of pharmaceuticals: evolution,reasoning and impact

The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) has convened an expert working group which consisted of the authors of this paper and their respective committees, consulting groups and task forces. Two ICH guidances regarding genotoxicity testing have been issued: S2A, `Guidance on Specific Aspects of Regulatory Genotoxicity Tests' and S2B, `Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.' Together, these guidance documents now form the regulatory backbone for genotoxicity testing and assessment of pharmaceuticals in the European Union, Japan, and the USA. These guidances do not constitute a revolutionary new approach to genotoxicity testing and assessment, instead they are an evolution from preexisting regional guidelines, guidances and technical approaches. Both guidances describe a number of specific criteria as well as a general test philosophy in genotoxicity testing. Although these guidances were previously released within the participating regions in their respective regulatory communiqués, to ensure their wider distribution and better understanding, the texts of the guidances are reproduced here in their entirety (see Appendix A) and the background for the recommendations are described. The establishment of a standard battery for genotoxicity testing of pharmaceuticals was one of the most important issues of the harmonisation effort. This battery currently consists of: (i) a test for gene mutation in bacteria, (ii) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma tk assay, (iii) an in vivo test for chromosomal damage using rodent hematopoietic cells. A major change in testing philosophy is the acceptance of the interchangeability of testing for chromosomal aberrations in mammalian cells and the mouse lymphoma tk assay. This agreement was reached on the basis of the extensive review of databases and newly generated experimental data which are in part described in this publication. The authors are fully aware of the fact that some of the recommendations given in these ICH guidances are transient in nature and that the dynamic qualities and ongoing evolution of genetic toxicology makes necessary a continuous maintenance process that would serve to update the guidance as necessary.