Mutagenesis - Advance Access

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line

Feitsma, H., de Bruijn, E., van de Belt, J., Nijman, I. J., Cuppen, E. — 2008-05-09

S_N1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

One-day Meeting for UK Molecular Epidemiology Group in collaboration with ECNIS and NuGO held on 13 December 2007 at Pippard Lecture Theatre, Sherfield Building, South Kensington Campus, Imperial College, London, UK: 'Novel Biomarkers and Techniques for Large Prospective Studies'Young investigators' abstracts

2008-05-01

In vivo role of Escherichia coli single-strand exonucleases in SOS induction by gamma radiation

Serment-Guerrero, J., Brena-Valle, M., Espinosa-Aguirre, J. J. — 2008-04-11

Ionizing radiation causes different types of genetic damage, ranging from base modifications to single- and double-stranded DNA breaks, which may be deleterious or even lethal to the cell. There are different repair or tolerance mechanisms to counteract the damage. Among them is the Escherichia coli SOS system: a set of genes that becomes activated upon DNA damage to confer better opportunities for cell survival. However, since this response is triggered by single-stranded DNA regions, most lesions have to be processed or modified prior to SOS activation. Several genes such as recO, recB and recJ that seem to be required to induce the response have already been reported. The results of this work indicate that the four known E.coli single-strand exonucleases take part in processing gamma radiation damage, though RecJ and ExoI proved to be more important than ExoVII or ExoX. In addition, ExoV as well as glycosylases such as Nth and, to a lesser extent, Fpg are also required. A model intended to explain the role of all these genes in damage processing is presented.

Acrylamide-induced molecular mutation spectra at HPRT locus in human promyelocytic leukaemia HL-60 and NB4 cell lines

Ao, L., Liu, S.-X., Yang, M.-S., Fong, C.-C., An, H., Cao, J. — 2008-04-11

Acrylamide (AA) is a compound widely used in many industries around the world. The recent finding that it is formed naturally in foods by heating raises human health concerns. AA is a proven carcinogen in animals and a probable carcinogen in humans, while its mutagenicity detected using in vitro mammalian gene mutation assays is still inconsistent in different cell systems. In the present study, we investigated the mutagenicity of AA in human promyelocytic leukaemia cells, HL-60 and NB4 cells, by examining the mutations at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) gene locus. In a 6-h treatment without the exogenous activation, AA exerted a weak mutagenic effect at the highest concentration used in the study (700 mg/l) in HL-60 cells (P < 0.01) as well as in NB4 cells (P < 0.05). Molecular analysis of AA-induced mutants revealed a different mutation spectrum, when compared to that of spontaneous mutants. The most frequent spontaneous mutations were point mutations, whereas AA-induced mutations were mainly single exon deletions besides point mutations, and an increase in the proportion of partial deletion was associated with the increase of AA treatment. There was no obvious difference in the mutation spectra observed between the HL-60 and NB4 cell lines. These results showed that AA has a weak mutagenic effect at HPRT gene locus in human promyelocytic leukaemia HL-60 and NB4 cell lines and those molecular mutation spectra (single exon deletions and point mutations) may be related to some specific and precise mechanism.

Up-regulation of the glutathione S-transferase system in human liver by polycyclic aromatic hydrocarbons; comparison with rat liver and lung

Pushparajah, D. S., Umachandran, M., Plant, K. E., Plant, N., Ioannides, C. — 2008-04-03

The cytosolic glutathione S-transferases (GSTs) comprise a pivotal enzyme system protecting the cell from electrophilic compounds. It plays a major role in the detoxication of the primary and dihydrodiol epoxides of polycyclic aromatic hydrocarbons (PAHs), so that modulation of this enzyme system by PAHs will impact on their carcinogenic activity. The potential of six structurally diverse PAHs, namely benzo[a]pyrene (B[a]P), fluoranthene, benzo[b]fluoranthene (B[b]F), dibenzo[a,l]pyrene, dibenzo[a,h]anthracene (D[a,h]A) and 1-methhylphenanthrene, to modulate hepatic GST activity was investigated in human precision-cut slices and compared to rat slices, a species frequently used in long-term carcinogenicity studies; changes were monitored at the activity, using three different substrates, protein and mRNA levels. When activity was monitored using the -class selective 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, B[b]F was the only PAH that caused an increase in activity, which was accompanied by a rise in the Ya immunoreacting band. In rat slices, in addition to B[b]F, B[a]P and D[a,h]A also enhanced activity, being paralleled with increased levels of the Ya immunoreacting band. In the rat, all PAHs elevated mRNA levels. In both human and rat liver slices, only B[b]F enhanced activity when 1-chloro-2,4-dinitrobenzene (CDNB) served as substrate. To investigate tissue differences, similar studies were undertaken in precision-cut rat lung slices, incubated with PAHs under identical conditions, using CDNB, as this was the only substrate for which activity could be detected; none of the PAHs studied stimulated activity. It is concluded that some PAHs have the potential to induce GST activity in human liver tissue and that species and tissue differences exist in the induction of this enzyme system in the rat. However, the extent of induction of GST activity is very modest compared with the effect these compounds have on CYP1 expression, the family responsible for their bioactivation, and it is unlikely to compensate for the enhanced production of reactive intermediates.

Cellular protection from oxidative DNA damage by over-expression of the novel globin cytoglobin in vitro

Hodges, N. J., Innocent, N., Dhanda, S., Graham, M. — 2008-03-18

Cytoglobin is a recently identified member of the mammalian globin family that is expressed in neuronal cells in the central and peripheral nervous system where its physiological role remains to be determined. In the current study, we demonstrate that a cytoglobin–green fluoresecent protein (GFP) fusion protein when expressed in the human neuronal cell line TE671 has a nuclear localization in a subpopulation of transfected cells (~15%). Furthermore, the cytoglobin–GFP fusion protein but not GFP alone significantly reduced the induction of intracellular reactive oxygen species as assessed by oxidation of the redox-sensitive probe dichlorofluorescein following treatment with non-cytotoxic concentrations of the pro-oxidant Ro19-8022. In addition, expression of cytoglobin–GFP also afforded cytoprotection from Ro19-8022-induced oxidative DNA damage as assessed by the Fpg-modified comet assay. In conclusion, the current study provides evidence supportive of a role for cytoglobin in cytoprotection of neuronal cells from oxidative-related damage, for example, during ischaemic reperfusion injury following hypoxia.

Radioprotective effect of sulfasalazine on mouse bone marrow chromosomes

Mantena, S. K., Unnikrishnan, M. K., Uma Devi, P. — 2008-03-18

Sulfasalazine (SAZ), a prescribed drug for inflammatory bowel disease, is a potent scavenger of reactive oxygen species. The present study was undertaken to ascertain its ability to protect against gamma radiation-induced damage. Acute toxicity of the drug was studied taking 24-h, 72-h and 30-day mortality after a single intraperitoneal injection of 400–1200 mg/kg body weight (b.wt.) of the drug. The drug LD₅₀ for 24- and 72-h/30-day survival were found to be 933 and 676 mg/kg b.wt., respectively. The optimum time of drug administration and drug dose-dependent effect on in vivo radiation protection of bone marrow chromosomes was studied in mice. Injection of 30–180 mg/kg SAZ 30 min before gamma irradiation (RT) with 4 Gy produced a significant dose-dependent reduction in the RT-induced percent aberrant metaphases and in the frequency of micronucleated erythrocytes at 24 h after exposure, with a corresponding decrease in the different types of aberrations. The optimum dose for protection without drug toxicity was 120 mg/kg b.wt. At this dose, SAZ produced >60% reduction in the RT-induced percent aberrant metaphases and micronucleated erythrocytes. SAZ also produced a significant increase in the ratio of polychromatic erythrocytes to normochromatic erythrocytes from that of irradiated control. Injection of 120 mg/kg of the drug 60 or 30 min before or within 15 min after 4 Gy whole-body RT resulted in a significant decrease in the percent of aberrant metaphases and in the frequency of micronucleated erythrocytes at 24 h post-irradiation; the maximum effect was seen when the drug was administered 30 min before irradiation. These results show that SAZ protect mice against RT-induced chromosomal damage and cell cycle progression delay. SAZ also protected plasmid DNA (pGEM-7Zf) against Fenton's reactant-induced breaks, suggesting free radical scavenging as one of the possible mechanism for radiation protection.

ECVAM retrospective validation of in vitro micronucleus test (MNT)

2008-03-07

In the past decade several studies comparing the in vitro chromosome aberration test (CAT) and the in vitro micronucleus test (MNT) were performed. A high correlation was observed in each of the studies (>85%); however, no formal validation for the micronucleus in vitro assay had been carried out. Therefore, a working group was established by the European Centre for the Validation of Alternative Methods (ECVAM) to perform a retrospective validation of the existing data, in order to evaluate the validity of the in vitro MNT on the basis of the modular validation approach. The primary focus of this retrospective validation was on the evaluation of the potential of the in vitro MNT as alternative to the standard in vitro CAT. The working group evaluated, in a first step, the available published data and came to the conclusion that two studies [German ring trial, von der Hude, W., Kalweit, S., Engelhardt, G. et al. (2000) In-vitro micronucleus assay with Chinese hamster V79 cells: results of a collaborative study with 26 chemicals. Mutat. Res., 468, 137–163, and SFTG International Collaborative Study, Lorge, E., Thybaud, V., Aardema, M., Oliver, J., Wataka, A., Lorenzon, G. and Marzin, D. (2006) SFTG International Collaborative Study on in-vitro micronucleus test I. General conditions and overall conclusions of the study. Mutat. Res., 607, 13–36] met the criteria for a retrospective validation according to the criteria previously defined by the working group. These two studies were evaluated in depth (including the reanalysis of raw data) and provided the information required for assessing the reliability (reproducibility) of the test. For the assessment of the concordance between the in vitro MNT and the in vitro CAT, additional published data were considered. Based on this retrospective validation, the ECVAM Validation Management Team concluded that the in vitro MNT is reliable and relevant and can therefore be used as an alternative method to the in vitro CAT. Following peer review, these conclusions were formally endorsed by the ECVAM Scientific Advisory Committee.

An improved assay for radiation-induced chromatid breaks using a colcemid block and calyculin-induced PCC combination

Shovman, O., Riches, A. C., Adamson, D., Bryant, P. E. — 2008-03-06

We report on a new method for the study of radiation-induced chromatid breaks in stimulated human peripheral blood T lymphocytes, involving a combination of a 1-h colcemid block and a short (15 min) calyculin A treatment. We find that this procedure eliminates the problem of centromere splitting when calyculin A is used alone for a longer period and produces metaphase spreads with superior quality. By this procedure, the chromosomes and the chromatid breaks are expanded and thereby make for improved break scoring. In a comparison of the new technique with the conventional colcemid block method, we show a close proportionality between the frequencies of chromatid breaks scored with the two methods. The frequency of chromatid breaks with the new method was found to be significantly higher than that with colcemid alone, adding a higher sensitivity to the assay as an additional advantage.

Protective effects of mate tea (Ilex paraguariensis) on H2O2-induced DNA damage and DNA repair in mice

2008-02-27

Yerba mate (Ilex paraguariensis) is rich in several bioactive compounds that can act as free radical scavengers. Since oxidative DNA damage is involved in various pathological states such as cancer, the aim of this study was to evaluate the antioxidant activity of mate tea as well as the ability to influence DNA repair in male Swiss mice. Forty animals were randomly assigned to four groups. The animals received three different doses of mate tea aqueous extract, 0.5, 1.0 or 2.0 g/kg, for 60 days. After intervention, the liver, kidney and bladder cells were isolated and the DNA damage induced by H₂O₂ was investigated by the comet assay. The DNA repair process was also investigated for its potential to protect the cells from damage by the same methodology. The data presented here show that mate tea is not genotoxic in liver, kidney and bladder cells. The regular ingestion of mate tea increased the resistance of DNA to H₂O₂-induced DNA strand breaks and improved the DNA repair after H₂O₂ challenge in liver cells, irrespective of the dose ingested. These results suggest that mate tea could protect against DNA damage and enhance the DNA repair activity. Protection may be afforded by the antioxidant activity of the mate tea's bioactive compounds.

Occupational exposure to mineral fibres. Biomarkers of oxidative damage and antioxidant defence and associations with DNA damage and repair

2008-02-14

In order to study the effect of mineral wool exposure on oxidative DNA damage and lipid peroxidation, an epidemiological study was conducted in a mineral wool factory in Slovakia. Altogether 141 subjects were investigated (21–58 years old), 43 controls (20 men and 23 women: 27 non-smokers, 16 smokers) and 98 exposed (75 men and 23 women: 61 non-smokers, 37 smokers). We found higher malondialdehyde (MDA) levels in the group of all exposed workers (P = 0.025) and in exposed non-smokers (P = 0.003) and a significantly suppressed activity of ceruloplasmin oxidase (P = 0.02, P < 0.02, respectively) and catalase (CAT) (P = 0.04, P = 0.01, respectively) in these groups. The activity of glutathione S-transferase (GST) was affected by exposure to mineral wool; levels were significantly lower in all exposed subjects (P = 0.04), in the exposed non-smokers (P = 0.03) and in exposed men (P < 0.01). Concentrations of vitamin C in plasma and the ferric-reducing activity of plasma (FRAP) were not affected by the mineral wool exposure. There was a significant negative correlation between the activity of glutathione peroxidase (GPX) and MDA in the whole group (P < 0.01) and in the exposed group and between CAT activity and MDA in all subjects (P < 0.01). GST activity correlated inversely with oxidized pyrimidines in lymphocyte DNA, in almost all subgroups. We found significant negative correlations between DNA repair and GPX in all subjects (P = 0.03) as well as in control men (P < 0.03) and between DNA repair and CAT in all control subjects (P < 0.02) and in control men (P < 0.01). Interestingly, we found a positive correlation between DNA repair and MDA in all subjects (P < 0.01) and in all exposed subjects (P < 0.03). The presented results indicate that mineral wool exposure induces an increase in oxidative damage to biomolecules especially in the group of male non-smokers. However, optimal levels of antioxidants could have a protective effect. Biomarkers such as MDA, antioxidant enzymes and antioxidant vitamins measured in blood may be useful biomarkers of oxidative stress and antioxidant protection. We do not recommend FRAP as a marker of antioxidant status as interference from other constituents can provide false or confusing results. Our study supports the idea that there might also be other mechanisms by which antioxidant enzymes (especially GST) protect cells against oxidative DNA damage.