Significant Increase In Sister Chromatid Exchanges Research Articles

Genotoxic Evaluation of Duloxetine II. The Effect on the Number of Sister Chromatid Exchanges, the Mitotic Index, and the Proliferation Kinetics in Mouse Bone Marrow.

Duloxetine is an antidepressant which has showed valuable results, particularly in patients with major depression. This type of drugs is known to require genotoxic studies as part of their preclinical safety evaluation. In the case of duloxetine, however, there have been controversial results. Therefore, we considered it worthwhile to extend studies on the matter in an attempt to reach a conclusion. The present assay was made in mouse bone marrow to evaluate the capacity of the drug to induce sister chromatid exchanges (SCE), as well as to modify the proliferation kinetics and the mitotic index. Three doses of the antidepressant were tested (2, 20, and 200 mg/kg), besides the control mice were administered with purified water, and the positive treated animals administered with 1 mg/kg of doxorubicin. The results indicated a moderate but significant increase of SCE with the three tested doses, no effect regarding the mitotic index and a small reduction in the proliferation kinetics. Although in our assay the drug showed a lower effect, the present study agreed with a previous report that analyzed the amount of micronuclei in mouse peripheral blood, and it confirmed the relevance of evaluating the genotoxic effect of antidepressants, specifically duloxetine by applying diverse tests.

Internal Tandem Duplication Mutation of FLT3 (FLT3/ITD) Significantly Increases Inter Chromosomal Homologous Recombination Providing a Direct Link Between Constitutive FLT3-ITD Activity and Propagation of Copy Neutral Loss of Heterozygosity (CN-LOH) in Acute Myeloid Leukaemia

The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through up-regulation of inter chromosomal (between maternal and paternal chromosomes) HR DNA repair. We have showed previously that constitutional FLT3-ITD kinase activity increased reactive oxygen species resulting in elevated double strand breaks. Moreover, FLT3-ITD mutation increases homologous recombination activity through transcriptional augmentation of HR factor, RAD51 expression.Firstly, to evaluate FLT3-ITD induced genomic instability we measured sister chromatid exchanges (SCE) that result from HR mediated chromosome cross-over. It has been shown previously in the pre-leukaemic chromosomal instability disorder, Blooms syndrome is characterised by increased SCE and an increased propensity for CN-LOH. FLT3/ITD primary AML (n=17), MOLM-13 and FLT3-ITD transfected U937 cells show a significant increase in SCE compared to WT FLT3 primary AML cells, U937 cells transfected with empty vector, (10 vs. 6 SCE per metaphase, p<0.001) (Figure 1A-B). Importantly, primary AML with CN-LOH at 13q + FLT3-ITD mutation gives significantly higher HR activity and SCE than primary AML + FLT3-ITD mutation without CN-LOH (p<0.05). Moreover, treatment of FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected U937 cells with the FLT3 inhibitor, CEP701 and the anti-oxidant agent, N-acetyl cysteine restored SCE to levels observed for WT FLT3. Next, we investigated whether inappropriate HR activity at G0 rather than at S phase of the cell cycle could result in illegitimate HR resulting in inter chromosomal HR. Western blot studies showed that G0 primary AML with FLT3/ITD, synchronised G0 MOLM 13 and FLT3-ITD transfected U937 cells showed augmented RAD51 expression compared to non-mutated FLT3 primary AML cells, cell lines and KG-1 transfected with empty vector (Figure 1C). Lastly, we used a thymidine kinase (TK) gene mutation assay system to screen for FLT3-ITD derived CN-LOH. The human lymphoblastoid cell line, TK6 is heterozygous for TK and becomes resistant to trifluorothymidine (TFT) upon loss of the wild type TK allele on 17q. FLT3-ITD transfected TK6 cells (using 2 different expression constructs) demonstrated significantly increased mutation frequency (grey bars) and mutation ate (Black bars) and TFT resistance compared to WT FLT3 and emptor vector control transfection (p<0.001) (Figure 1D). Colony PCR of TFT resistant colonies revealed that resistance was due primarily (80%) to inter chromosomal HR at 17q in contrast to empty vector (20%) and cisplatin treated (45%) TK6 cells (Figure 1E). The findings that FLT3-ITD mutation induces increased ROS production inducing DNA double strand breaks and the concomitant increase in the rad51 dependent inter homologous recombination provide a strong proof of principle that constitutive FLT3-ITD kinase activity directly confers CN-LOH that furthers our understanding of leukaemic progression and relapse in NK-AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Health assessment of gasoline and fuel oxygenate vapors: Micronucleus and sister chromatid exchange evaluations

Micronucleus and sister chromatid exchange (SCE) tests were performed for vapor condensate of baseline gasoline (BGVC), or gasoline with oxygenates, methyl tert-butyl ether (G/MTBE), ethyl tert butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), t-butyl alcohol (TBA), or ethanol (G/EtOH). Sprague Dawley rats (the same 5/sex/group for both endpoints) were exposed to 0, 2000, 10,000, or 20,000mg/m3 of each condensate, 6h/day, 5days/week over 4weeks. Positive controls (5/sex/test) were given cyclophosphamide IP, 24h prior to sacrifice at 5mg/kg (SCE test) and 40mg/kg (micronucleus test). Blood was collected from the abdominal aorta for the SCE test and femurs removed for the micronucleus test. Blood cell cultures were treated with 5μg/ml bromodeoxyuridine (BrdU) for SCE evaluation. No significant increases in micronucleated immature erythrocytes were observed for any test material. Statistically significant increases in SCE were observed in rats given BGVC alone or in female rats given G/MTBE. G/TAME induced increased SCE in both sexes at the highest dose only. Although DNA perturbation was observed for several samples, DNA damage was not expressed as increased micronuclei in bone marrow cells. Inclusion of oxygenates in gasoline did not increase the effects of gasoline alone or produce a cytogenetic hazard.

Internal Tandem Duplication Mutation Of FLT3 (FLT3/ITD) Induces Increased Homologous Recombination DNA Repair Activity, Drug Resistance and Sister Chromatid Exchanges In Acute Myeloid Leukaemia (AML)

The majority of patients with adult acute myeloid leukaemia (AML) that present with an apparently normal karyotype (NK-AML) are grouped together in the “intermediate” risk category and constitute 40-45% of all adult AML patients. Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in NK-AML. The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH in 20% and 40% of newly diagnosed and relapsed AML respectively. CN-LOH at 6p or at the FLT3 locus 13q associated with a FLT3-ITD mutation in NK-AML results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It is has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined.To elucidate the mechanisms that produce CN-LOH in NK-AML we determined whether oncogenes such as FLT3-ITD have the propensity to generate CN-LOH through production of excessive DNA double strand breaks (DSB) and up-regulation of HR DNA repair. Primary NK-AML (n=17) were initially characterised by SNP-A and DNA sequencing. 12/17 primary NK-AML had FLT3/ITD mutations with a clone size ≥ 25%, but were wild type (WT) for RAS. From this cohort 6/12 possessed CN-LOH with 3/12 having CN-LOH at 13q. 5/17 primary NK-AML were WT for FLT3 and RAS, but had nucleophosmin (NPM) and DNA methyltransferase 1 (DNMT1) mutations. From this cohort, 1/5 primary NK-AML + WT FLT3 had CN-LOH.We showed that primary AML + FLT3/ITD cohort, the MOLM 13 cell line that possesses a FLT3/ITD mutation and WT FLT3 KG-1 cells transfected with a FLT3-ITD expression vector demonstrate increased ROS compared to the WT FLT3 primary NK-AML cohort, the WT FLT3 cell line, U937 and KG-1 transfected with empty vector. Immunofluorescence studies showed that primary AML with FLT3/ITD, MOLM 13 and FLT3-ITD transfected KG-1 cells have increased mobilization of phospho-γH2AX (a DNA DSB intermediate) and RAD51 (a HR factor) to sites of DNA DSB in response to mitomycin C (MMC) induced DNA damage compared to non-mutated FLT3 primary AML cells, cell lines and KG-1 transfected with empty vector (phospho-γH2AX, 29% vs. 21%, p<0.005 and RAD51, 25% VS 19%, p<0.01). Moreover, we show that HR is augmented in FLT3-ITD transfected cells and MOLM-13 with and without exogenous DNA damage using DR-GFP gene reporter assays. Increased HR activity has been associated with increased resistance to genotoxic agents. Using soft agar clonogenic assays, MOLM 13 and FLT3-ITD transfected KG-1 showed considerably increased resistance to MMC and the radiomimetic agent, bleomycin compared to U937 cells and KG-1 + empty vector. Finally, to evaluate FLT3-ITD induced genomic instability we measured sister chromatid exchanges (SCE) that result from HR mediated chromosome crossing over. FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected KG-1 cells show a significant increase in SCE compared to WT FLT3 primary AML cells, U937 cells and KG-1 transfected with empty vector, (10 vs. 6 SCE per metaphase, p<0.001). Importantly, primary AML with CN-LOH at 13q + FLT3-ITD mutation gives significantly higher HR activity and SCE than non-13q CN-LOH primary AML + FLT3-ITD mutation (p<0.05). Moreover, treatment of FLT3/ITD primary AML, MOLM-13 and FLT3-ITD transfected KG-1 cells with the FLT3 inhibitor, CEP701 and the anti-oxidant agent, N-acetyl cysteine restored ROS levels, HR activity, sensitivity to MMC and SCE to levels observed for WT FLT3. These findings not only provide a strong proof of principle that FLT3-ITD derived DNA damage is the trigger for CN-LOH in NK-AML, but further our understanding of leukaemic progression and relapse. Disclosures:No relevant conflicts of interest to declare.

Genotoxicity assessment of vaccine adjuvant squalene

The genotoxic potential of the vaccine adjuvant Squalene was assessed by the chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and micronucleus (MNs) tests in human lymphocytes and comet assay in both human and rat lymphocytes. Five different concentrations of squalene (1250-20,000 μg/ml for human lymphocytes and 0.07-1.12 mg/kg for rat lymphocytes) were studied. Squalene did not affect the CAs and MN frequency, in all treatments in vitro. A significant increase in SCEs was observed in almost all concentrations at 24 h treatment. Squalene did not affect significantly the comet tail length (CTL) (except 2500 μg/ml) and comet tail intensity (CTI) at all treatments in vitro. In rats, squalene significantly increased and decreased CTL and CTI in some doses. Although there are increasing and reduction in the effect, squalene cannot be regarded as genotoxic in human lymphocytes. However, further in vivo studies are required to be sure on the effect.

Genotoxicity testing of samples generated during UV/H2O2 treatment of surface water for the production of drinking water using the Ames test in vitro and the Comet assay and the SCE test in vivo

UV/H2O2 treatment can be part of the process converting surface water to drinking water, but would pose a potential problem when resulting in genotoxicity. This study investigates the genotoxicity of samples collected from the water treatment plant Andijk, applying UV/H2O2 treatment with an electrical energy dose of 0.54 kWh/m(3) and a H2O2 dose of 6 mg/l. Genotoxicity was tested in vitro using the Ames and Comet assay. All samples showed negative results. Samples were also tested in in vivo genotoxicity tests in Eastern mudminnow fish (Umbra pygmaea) by a sister chromatid exchange (SCE) and a Comet assay. No significant increases in SCEs were observed, but gill cells isolated from fish exposed to water obtained immediately after UV/H2O2 treatment and to Lake IJsselmeer water showed significantly increased DNA damage in the Comet assay. All other samples tested negative in this Comet assay. This indicates that DNA damaging compounds may result from the UV/H2O2 treatment, but also that these can be efficiently eliminated upon granular activated carbon (GAC) treatment of the water. It is concluded that when combined with this subsequent GAC treatment, UV/H2O2 treatment for the production of drinking water from surface water is not of concern with respect to genotoxicity.

Genotoxic effects in the Eastern mudminnow (Umbra pygmaea) after prolonged exposure to River Rhine water, as assessed by use of the in vivo SCE and Comet assays

The production of drinking water from river water requires a certain minimal river water quality. The Association of River Rhine Water Works (RIWA), therefore, operates a monitoring network. In vitro mutagenicity studies have shown that the genotoxicity of the River Rhine water steadily decreased from 1981 until 2001. Compared to a study in 1978, a decrease in genotoxicity was also observed in an in vivo genotoxicity study in 2005, in which Eastern mudminnows (Umbra pygmaea) were exposed to River Rhine water, and gill cells were used for the Sister Chromatid Exchange (SCE) test and the Comet assay. In this 2005 study, the in vivo genotoxicity increased upon extending exposure of the fish from 3 to 11 days. Therefore, the objectives of this study were to investigate (i) whether new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978, (ii) whether the Comet assay is a suitable alternative to the SCE assay, and (iii) whether further prolonged exposure results in a further increase in in vivo genotoxicity. The new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978. The Comet assay is a useful addition but does not provide a substitute for the SCE endpoint in these in vivo genotoxicity studies. Prolonging the exposure time of Eastern mudminnows to River Rhine water from 11 to 42 days did not give a significant increase in SCEs and DNA damage (Comet assay) in gill cells.

Effects of boric acid and borax on titanium dioxide genotoxicity

Titanium dioxide (TiO(2)) is a potential carcinogenic/mutagenic agent although it is used in many areas including medical industries and cosmetics. Boron (as boric acid and borax) has also well-described biological effects and therapeutic benefits. In a previous study, sister-chromatid exchanges (SCEs) and micronuclei (MN) rates were assessed in control and TiO(2)-treated (1, 2, 3, 5, 7.5 and 10 microm) human whole blood cultures. The results showed that the rates of SCE (at 2, 3, 5, 7.5 and 10 microm) and MN (at 5, 7.5 and 10 microm) formation in peripheral lymphocytes were increased significantly by TiO(2) compared with the controls. The present study also investigated the genetic effects of boric acid and borax (2.5, 5 and 10 microm) on cultures with and without TiO(2) addition. No significant increase in SCE and MN frequencies were observed at all concentrations of boron compounds. However, TiO(2)-induced SCE and MN could be reduced significantly by the presence of boric acid and borax. In conclusion, this study indicated for the first time that boric acid and borax led to an increased resistance of DNA to damage induced by TiO(2).

Investigation of genotoxic effect of ultrasound in cases receiving therapeutic ultrasound by using micronucleus method

In 1991, Miller et al. (1991) reported that therapeutic ultrasound (US) did not induce sister chromatid exchanges (SCEs) in patients whereas, in 1984, Stella et al. (1984) reported that each of 10 patients exposed to therapeutic US had a statistically significant increase in SCEs. The present study was planned to investigate if there was chromosomal damage resulting from therapeutic US by using a micronucleus (MN) method, and to counter the lack of reports in this area over the past 10 years. A total of 20 female volunteers were included in the study; 10 of them with low back pain (mechanical low back pain and facet syndrome) were treated with US and 10 healthy cases constituted the control group. Patients with low back pain received 10 sessions of US therapy at an intensity of 2 W/cm 2 and a frequency of 1 MHz for 10 min and patients in the control group received sham US therapy for 10 min. Peripheral blood taken before and after the fifth and tenth applications of US therapy was cultured for MN frequencies both for the treatment and the control groups. The scores of MN assessed before the therapy were compared with those at the end of the fifth session and the end of the tenth session in the treatment and the control groups. Pretreatment, end of the fifth session and end of the tenth session MN frequencies were compared between the treatment and the control groups. There was no statistically significant difference in MN frequencies between pretreatment and fifth session or pretreatment and tenth session in both groups. Nor was there any significant difference in the MN frequencies of the treatment and control groups between pretreatment, fifth session and tenth session evaluations. In conclusion, we observed that therapeutic US did not induce increases in MN frequency, which are a sign of cytogenetic damage. (E-mail: donmezh@erciyes.edu.tr)

A new approach for evaluating in vivo anti-leukemic activity using the SCE assay: An application on three newly synthesised anti-tumour steroidal esters

Three newly synthesised steroidal esteric derivatives of nitrogen mustard (compounds 1– 3) were comparatively studied on a molar basis regarding their ability to induce sister chromatid exchanges (SCEs) in normal human lymphocytes in vitro and therapeutic effects on leukemia P388 bearing mice. Compounds 1 and 3 are modified steroidal esters of p-methyl- m- N, N-bis(2-chloroethyl)amino benzoic acid, and compound 2 is a modified steroidal ester of chlorambucil. All compounds induced statistically significant increases in SCEs and decreases in proliferation rate indices (PRIs) of cultured human lymphocytes and significantly increased the life span of P388 bearing mice. In this study, the doses applied for therapeutic purposes upon leukemia P388 bearing mice in vivo were derived from cytogenetic observations in normal human lymphocytes in vitro. A substantially better therapeutic effect was obtained compared to the effect achieved after the use of quite higher doses related with LD 10 values. We have demonstrated that the order of anti-tumour effectiveness of the treatment schedules of the three newly synthesised compounds tested (at doses derived from cytogenetic observations) coincides with the order of the cytogenetic effects they induce. The SCE assay appears to have an application in the clinical prediction of tumour sensitivity to potential chemotherapeutics.

Squalene but not n-3 fatty acids protect against hydrogen peroxide-induced sister chromatid exchanges in Chinese hamster V79 cells

Fish-oil derived n-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been shown to exhibit anti-carcinogenic effects in vivo and in vitro. Squalene, found in shark liver oil and olive oil, can effectively inhibit chemically induced tumorigenesis in rodents. The aim of the present study was to investigate whether supplementation with EPA (50 μmol/l), DHA (50 μmol/l) or squalene (50 μmol/l) for 24 h would protect Chinese hamster V79 fibroblast cells against oxidant-induced DNA damage. EPA and DHA were delivered to the cells either complexed to bovine serum albumin (BSA) or dissolved in ethanol. DNA damage was assessed using the sister chromatid exchange (SCE) assay. V79 cells exposed to hydrogen peroxide (H 2O 2) alone showed a significant increase in SCE ( P < 0.05) when compared with control levels. Pre-incubation with either EPA or DHA did not significantly affect H 2O 2-induced SCE regardless of the delivery vehicle employed. However, pre-treatment with squalene significantly decreased the frequency of SCE induced by H 2O 2 ( P < 0.05). Results indicate that squalene was more effective than EPA and DHA in protecting against H 2O 2-induced SCE.

In vitro testing for genotoxicity of the herbicide terbutryn: cytogenetic and primary DNA damage

Terbutryn is a widely used preemergence and postemergence s-triazine herbicide. This pesticide is used in agriculture as a control agent for most grasses and many annual broadleaf weeds in cereal and legume fields, and under fruit trees. Unexpectedly, this compound was found to persist in the environment (240 and 180 days in pond and river sediment, respectively) and to have the tendency to move from treated soils to water compartments through water runoff and leaching. However, only scant information is available about the genotoxic properties of terbutryn. In the present in vitro study, we investigated the relationship between cytogenetic damage, as evaluated in the sister-chromatid exchange (SCE) assay and the micronucleus (MN) test, and primary DNA damage (as evaluated by the “comet” assay). Cytogenetic and primary DNA damage were recorded in vitro in freshly isolated human peripheral blood leukocytes. Our results showed that the tested compound failed to produce any significant increases in SCE or MN, neither in the absence nor in the presence of S9-mix. However, terbutryn was found to induce primary DNA damage, more pronounced without S9 mix, even though in the absence of a clear trend for dose-dependence and in the presence of a concomitant mild cytotoxic effect.

Genotoxic effects after in vivo exposure of vegetable extracts containing heavy metals from the Dhapa area of Calcutta, India. I. Effects of cauliflower, spinach and radish

Several reports have indicated that the sewage-fed vegetables of the Dhapa area, near the city of Calcutta, contain a very high amount of heavy metals. Currently 800 ha of land is being utilised throughout the year to cultivate more than eight types of vegetables, with a production of about 147 tonnes per day. A major population of Calcutta consumes these vegetables grown in the Dhapa area. Recently there has been huge pressure on the State Government to ban vegetables grown in the Dhapa area for human consumption. For this reason, we have studied the genotoxic effects of some of the most commonly used vegetable extracts from the Dhapa area after in vivo acute exposure in mice as measured by chromosomal aberrations (CA) and sister chromatid exchanges (SCE) to find out the minimum threshold dose to induce CA and SCE. Three different concentrations of the three most commonly used vegetable extracts (cauliflower, spinach, radish) were fed by gavage to mice for the study of CA and SCE. A significant increase in CA was observed only at the highest concentration of all the vegetable extract-treated groups when compared with the solvent control. A significant increase in SCE were observed in the middle and high doses of spinach and only the high dose of cauliflower and radish extract-treated series when compared with distilled water control. The lowest dose was equivalent to approximately 1 kg of vegetables consumed by a human (60 kg body weight) in a day. The middle and high doses of each vegetable extract were much higher than the normal amount of vegetables that a human can consume per day. So the minimum dose for inducing SCE and CA was much higher than the amount a human can consume in a day. Therefore this study indicates that these vegetables are safe for human consumption up to a certain limit, and attention should be given to reducing the heavy metal contents in the soil and sewage of the Dhapa area to thus reduce the heavy metal concentrations in the vegetables.

Cytogenetic studies of three triazine herbicides: I. In vitro studies

Atrazine, simazine, and cyanazine are widely used pre-emergence and post-emergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Because of this and the prevalence of contradictory cytogenetic studies in the literature on atrazine, simazine, and cyanazine, a series of in vitro experiments was performed to investigate the ability of these three triazines to induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in human lymphocyte cultures. Our results showed that all three triazines failed to produce any significant increases in SCEs or CAs up to the limits of solubility [using 0.5% dimethyl sulfoxide (DMSO)]. Our results are discussed in light of contradictory results in the literature.

Cytogenetic damage and ras p21 oncoprotein levels from patients with chronic obstructive pulmonary disease (COPD), untreated lung cancer and healthy controls

The purpose of the present communication was to determine in patients with chronic obstructive pulmonary disease (COPD), untreated lung cancer and healthy controls if there was a possible association between the disease state and biomarkers of cytogenetic damage and ras p21 oncoprotein levels, and if various exogenous confounding factors such as smoking habit and endogenous ones (sex, cancer in the immediate family) could affect these biomarkers. The individuals in all groups were as well-matched as possible for age to determine if this could be eliminated as a confounder. Peripheral blood and plasma were collected from 20 COPD patients, 31 cancer patients and 20 healthy controls. Chromosomal aberrations (CA), sister chromatid exchanges (SCE) and high frequency SCE cells (HFC) were examined from the blood and ras p21 oncoproteins from the plasma. These parameters were used as biomarkers of genotoxic anomalies. All the biomarkers were examined for their relationship to the confounding factors. Results were analysed by a t-test, analysis of variance (ANOVA) and stepwise multivariate regression analysis. There was an increase in CA, although not statistically so, in COPD and cancer patients by comparison with healthy controls, but there was a statistically significant increase in SCE, HFC and ras p21 oncoproteins. There was also a statistically significant difference between respiratory volume parameters in COPD patients and controls. Respiratory parameters were not measured in cancer patients. Ras p21 oncoproteins were also statistically significantly increased in the COPD and cancer patients, suggesting that the disease state alone might be sufficient to increase the oncoproteins, or that some of the COPD patients were in the process of developing cancer or perhaps some would die from COPD before cancer developed. Smoking was shown to have a marked effect on all parameters investigated. Ex-smokers showed less effects. Since age was very well controlled, there was little effect due to age. There was an effect due to sex, but cancer in the immediate family had little effect on any of the parameters.

Genotoxic effects of triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo

Two organotin pesticides, triphenyltin acetate (TPTA) and triphenyltin hydroxide (TPTH), were evaluated for their ability to induce micronuclei (MN) and sister chromatid exchange (SCE) in vitro using cultured Chinese hamster ovary (CHO) cells and in vivo BALB/c mouse erythrocytes. Both pesticides induced a dose-dependent increase but only TPTH induced a significant increase in MN at the highest dose (150 ng/ml) tested in CHO cells. With adding S9 microsomal fractions, both pesticides induced a meaningful MN induction at 150 ng/ml and a dose-dependent significant increase in SCE. In vivo MN induction in erythrocytes was conducted by treating BALB/c mice orally or intraperitoneally with these pesticides either in a single or triple treatments. Oral gavage (p.o.) of TPTA resulted in a dose-related significant increase of MN induction in peripheral blood and of TPTH induced a significant increase in micronucleated reticulocyte (MNRETs) only in a single treatment. Intraperitoneal administration of TPTA or TPTH, however, resulted in meaningless random increases in MN though these increases might be attributable to toxic effects. The MNRETs levels in the treatment with both pesticides were independent to the sampling time. This study demonstrated that TPTA and TPTH was potential chromosome mutagens.

An evaluation of the feasibility of using cytogenetic damage as a biomarker for alachlor exposure

Alachlor is a widely used herbicide for which there is significant human exposure, principally through groundwater contamination and inhalation. Because alachlor is purported to be carcinogenic and mutagenic, we initiated studies to determine if induced cytogenetic damage could be used as a biomarker for exposure to this herbicide. Both isolated and whole blood human lymphocytes were exposed to alachlor using several protocols. The lymphocytes were cultured for analysis of sister chromatid exchange (SCE), chromosome aberrations (CAs), micronuclei (MN) in cytochalasin B-induced binucleated cells, and proliferation kinetics using the replicative index (RI). In addition, CD rats were injected with either 10 or 50 mg kg −1 of alachlor, 2-chloro- N-(2,6-diethylphenyl) acetamide (CDEPA) or 2,6-diethylanaline (DEA). After 24 h, the peripheral blood lymphocytes were removed and cultured for SCE and RI analysis. Alachlor did induce a concentration-related increase in SCE in vitro, but neither it nor its metabolites (CDEPA or DEA) induced a significant increase in SCEs or an alteration of RI in vivo. At the highest in vitro concentration tested, alachlor induced a statistically-significant increase in MN, but no concomitant increase in CAs was seen. From analyses of our data and the literature on alachlor clastogenicity and exposure levels, we concluded that cytogenetic damage may not be an adequately sensitive marker for evaluating human exposure to alachlor.

Exposure to polycyclic aromatic hydrocarbons and genotoxic effects on nonsmoking Swedish road pavement workers.

The objective of this study was to investigate exposure to polycyclic aromatic hydrocarbons (PAH) from asphalt fumes among Swedish road pavement workers and determine whether any effects could be detected with genotoxic tests. The study included 28 nonsmoking road pavers and 30 nonsmoking referents. The concentration of PAH was determined in the breathing zone of the road pavers. 1-Hydroxypyrene was analyzed before and after shifts of asphalt work and during the afternoon for referents. Sister chromatid exchanges (SCE) and micronuclei (MN) were determined in peripheral lymphocytes. Several 3- or 4-ring PAH were found, and the analysis indicated that they occurred in bitumen fumes rather than in traffic fumes. The average total concentration of PAH was 2.3 (range 0.2-23.8) microg/m3. The concentration of 1-hydroxypyrene in urine was higher for the road pavers than for the referents, but there was no significant difference between the pre- and postshift values of the road pavers. The road pavers had no significant increase in SCE or MN. The study showed that Swedish road pavers have an increased exposure to PAH from bitumen fumes, but no genotoxic effects could be detected by SCE or MN tests.

Low sensitivity of the sister chromatid exchange assay to detect the genotoxic effects of radioiodine therapy.

To assess the genotoxic risk associated with 131I therapy, sister chromatid exchanges (SCEs) and cells with unusually high SCE counts (HFC) were determined in a follow-up study performed with 46 hyperthyroidism and 39 thyroid cancer patients treated with 131I. In addition, a cross-sectional study was also carried out with 78 control persons and 51 thyroid cancer patients that had completed radioiodine therapy from 1 to 6 years prior to the current investigation. In the follow-up analysis, the study was conducted over time and four blood samples were drawn from each patient: the first one prior to the radioiodine treatment, with the remaining three taken sequentially over the year after therapy. Concerning the results obtained for the whole population in the follow-up study, the SCE and HFC values found after radioiodine therapy did not show any significant increase, neither in the hyperthyroidism nor thyroid cancer groups. Unlike the results mentioned above, when the effect of smoking habit was considered, there was a slight but significant increase in SCE in the samples taken 3 months and 1 week after 131I therapy in the hyperthyroidism and thyroid cancer non-smokers, respectively. The data obtained in the cross-sectional study did not show differences in SCE and HFC between the control group and the cancer group treated with 131I. It is noteworthy that among the different parameters analysed, smoking habit is the only factor that showed a direct relationship with SCE and HFC and, as a consequence, smokers had significantly more SCE and HFC than non-smokers. Taking into account our previous investigations showing a highly significant increase in the frequency of micronuclei for the same patients and sampling times, the outcomes obtained would suggest that the eventual genotoxic effect of 131I therapy could not be clearly detected by the SCE assay. This would reinforce the view that ionizing radiation appears to be a poor inducer of SCEs.

Mutagenicity assay in Salmonella and in vivo sister chromatid exchange in bone marrow cells of mice for four pyrazolone derivatives

Phenylbutazone (PB), oxyphenbutazone (OPB), antipyrine (AP) and dipyrone (DP) are four important pyrazolone derivatives mainly used as anti-inflammatory, antipyretic and analgesic drugs. At present these are the most widely used pyrazolone derivatives throughout the world. The widespread use of these drugs are of great concern for human health problems. In the present study these four drugs were tested in mutagenicity assays in Salmonella strains TA97a, TA98, TA100 and TA102 using a plate incorporation assay both with and without S-9 mix and for in vivo sister chromatid exchanges (SCE) in bone marrow cells of mice. The first three drugs were negative in all the tester strains but dipyrone showed a weak mutagenic activity at higher concentrations in all four strains both with and without metabolic activation. In the in vivo SCE assay in male mice, all four drugs showed a statistically significant increase in SCE in bone marrow cells when compared with control.