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The heterogeneous reactions of ambient particulate matter (PM)-bound polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs (NPAHs) with NO3/N2O5, OH radicals, and O3 were studied in a laboratory photochemical chamber. Ambient PM2.5 and PM10 samples were collected from Beijing, China, and Riverside, California, and exposed under simulated atmospheric long-range transport conditions for O3 and OH and NO3 radicals. Changes in the masses of 23 PAHs and 20 NPAHs, as well as the direct and indirect-acting mutagenicity of the PM (determined using the Salmonella mutagenicity assay with TA98 strain), were measured prior to and after exposure to NO3/N2O5, OH radicals, and O3. In general, O3 exposure resulted in the highest relative degradation of PM-bound PAHs with more than four rings (benzo[a]pyrene was degraded equally well by O3 and NO3/N2O5). However, NPAHs were most effectively formed during the Beijing PM exposure to NO3/N2O5. In ambient air, 2-nitrofluoranthene (2-NF) is formed from the gas-phase NO3 radical- and OH radical-initiated reactions of fluoranthene, and 2-nitropyrene (2-NP) is formed from the gas-phase OH radical-initiated reaction of pyrene. There was no formation of 2-NF or 2-NP in any of the heterogeneous exposures, suggesting that gas-phase formation of NPAHs did not play an important role during chamber exposures. Exposure of Beijing PM to NO3/N2O5 resulted in an increase in direct-acting mutagenic activity which was associated with the formation of mutagenic NPAHs. No NPAH formation was observed in any of the exposures of the Riverside PM. This was likely due to the accumulation of atmospheric degradation products from gas-phase reactions of volatile species onto the surface of PM collected in Riverside prior to exposure in the chamber, thus decreasing the availability of PAHs for reaction.

The heterogeneous reactions of benzo[a]pyrene-d12 (BaP-d12), benzo[k]fluoranthene-d12 (BkF-d12), benzo[ghi]perylene-d12 (BghiP-d12), dibenzo[a,i]pyrene-d14 (DaiP-d14), and dibenzo[a,l]pyrene (DalP) with NO2, NO3/N2O5, and OH radicals were investigated at room temperature and atmospheric pressure in an indoor Teflon chamber and novel mono NO2-DaiP, and mono NO2-DalP products were identified. Quartz fiber filters (QFF) were used as a reaction surface and the filter extracts were analyzed by GC/MS for nitrated-PAHs (NPAHs) and tested in the Salmonella mutagenicity assay, using Salmonella typhimurium strain TA98 (with and without metabolic activation). In parallel to the laboratory experiments, a theoretical study was conducted to rationalize the formation of NPAH isomers based on the thermodynamic stability of OH-PAH intermediates, formed from OH-radical-initiated reactions. NO2 and NO3/N2O5 were effective oxidizing agents in transforming PAHs to NPAHs, with BaP-d12 being the most readily nitrated. Reaction of BaP-d12, BkF-d12 and BghiP-d12 with NO2 and NO3/N2O5 resulted in the formation of more than one mono-nitro isomer product, while the reaction of DaiP-d14 and DalP resulted in the formation of only one mono-nitro isomer product. The direct-acting mutagenicity increased the most after NO3/N2O5 exposure, particularly for BkF-d12 in which di-NO2-BkF-d10 isomers were measured. The deuterium isotope effect study suggested that substitution of deuterium for hydrogen lowered both the direct and indirect acting mutagenicity of NPAHs and may result in an underestimation of the mutagencity of the novel NPAHs identified in this study.

Atmospheric particulate matter with diameter

Assessment of zinc status remains a challenge largely because serum/plasma zinc may not accurately reflect an individual’s zinc status. The comet assay, a sensitive method capable of detecting intracellular DNA strand breaks, may serve as a functional biomarker of zinc status. We hypothesized that effects of zinc supplementation on intracellular DNA damage could be assessed from samples collected in field studies in Ethiopia using the comet assay. Forty women, from villages where reported consumption of meat was less than once per month and phytate levels were high, received 20 mg zinc as zinc sulfate or placebo daily for 17 days in a randomized placebo-controlled trial. Plasma zinc concentrations were determined by inductively coupled plasma mass spectrometry (ICPMS). Cells from whole blood at the baseline and endpoint of the study were embedded in agarose, electrophoresed, and stained before being scored by an investigator blinded to the treatments. Although zinc supplementation did not significantly affect plasma zinc, mean (± SEM) comet tail moment measurement of supplemented women decreased from 39.7 ± 2.7 to 30.0 ± 1.8 (p

Sulforaphane (SFN), an isothiocyanate derived from crucifers, has numerous health benefits. SFN bioavailability from dietary sources is a critical determinant of its efficacy in humans. A key factor in SFN absorption is the release of SFN from its glucosinolate precursor, glucoraphanin, by myrosinase. Dietary supplements are used in clinical trials to deliver consistent SFN doses, but myrosinase is often inactivated in available supplements. We evaluated SFN absorption from a myrosinase-treated broccoli sprout extract (BSE) and are the first to report effects of twice daily, oral dosing on SFN exposure in healthy adults.Subjects consumed fresh broccoli sprouts or the BSE, each providing 200 μmol SFN daily, as a single dose and as two 100-μmol doses taken 12 h apart. Using HPLC-MS/MS, we detected ∼3 x higher SFN metabolite levels in plasma and urine of sprout consumers, indicating enhanced SFN absorption from sprouts. Twelve-hour dosing retained higher plasma SFN metabolite levels at later time points than 24-hour dosing. No dose responses were observed for molecular targets of SFN (i.e. heme oxygenase-1, histone deacetylase activity, p21).We conclude that the dietary form and dosing schedule of SFN may impact SFN absorption and efficacy in human trials.

The application of a simple lag-time assay for antioxidant capacity using myoglobin and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) or ABTS has been studied for its general application conditions. In the presence of an antioxidant, the ABTS(*+) radical-cation-forming chromogenic reaction, catalyzed by myoglobin and initiated by hydrogen peroxide (H(2)O(2)), has a lag period, and its duration is linearly correlated to the concentration of that antioxidant. The high linearity between the lag time and the antioxidant concentration remained unchanged regardless of the assay conditions. It was also found that the linearity was better for antioxidants at lower concentrations. The change of assay condition could significantly affect the relative antioxidant value of a chemical to the standard (ascorbic acid), although not to a large extent. Most of antioxidants investigated were found suitable to be assayed using this method. Some antioxidants, e.g., genistein, however, were not, probably due to their low reactivity toward ferrylmyoglobin or ABTS(*+). In conclusion, the lag-time assay is a reliable method for measuring the antioxidant capacity, provided caution is taken for antioxidants that mainly act through lowering the rate of the chromogenic reaction.

The single cell gel electrophoresis assay (Comet assay) was used to measure DNA damage in peripheral lymphocytes from a group of individuals from The Gambia in order to determine whether such damage could be associated with increased exposure to aflatoxin in this population. Responses obtained were correlated to responses previously obtained [1] in a cross-sectional study in the same individuals of various cytogenetic alterations [chromosomal aberrations, micronuclei (crest positive and negative staining), and sister chromatid exchanges], and aflatoxin-albumin adducts. Analysis of variance methods were used to assess the effects of smoking, GSTM1 genotype, sex, age, and smoking status. A comparison was also made between The Gambian individuals and a group of healthy, non-smoking volunteers in the United Kingdom where aflatoxin exposure would be expected to be low. From the earlier study [1], it was determined that the levels of the sister chromatid exchanges and micronuclei were higher in The Gambian group than in a European group where aflatoxin exposure was lower, but that there were no correlations between the adduct levels and the cytogenetic abnormalities at the individual level. In the present study, DNA damage as measured in the Comet assay was not significantly higher than in the healthy United Kingdom volunteers. In addition, there were no associations between cytogenetic damage, GSTM1 genotype, age, sex, lifestyle factors (smoking and aflatoxin exposure), and Comet response at the individual level. Comet response was higher in females than males in The Gambia if one outlier was excluded from analysis and not taking into account other sources of variability. It would appear that DNA damage as measured in the Comet assay in peripheral blood lymphocytes is not a sensitive genotoxic marker of aflatoxin exposure in this population. Teratogenesis Carcinog. Mutagen. 19:147–155, 1999. © 1999 Wiley-Liss, Inc.

In previous studies. N-(N'-acetyl-L-propyl)-N-nitrosoglycine (APNG) has been shown to be a potent mutagen in a variety of genotoxicity assays and a carcinogen in a limited cancer study. APNG decomposes to a carboxymethyldiazonium ion, which can also be generated from potassium diazoacetate (KDA). KDA is particularly interesting because it is a stable nitrosated derivative of glycine, one of the most common dietary amino acids. KDA has been shown to produce more O 6 carboxymethyl-and O 6 methyl-adducts than APNG, so it was anticipated that it might also be a potent genotoxic agent. Thus in the present study KDA has been investigated in the single cell gel electrophoresis (Comet) assay, which primarily measures DNA strand hreakage. Since KDA has been shown to be formed in the gut, the genotoxic effects of KDA were investigated in vitro in human adenocarcinoma colon Caco-2 cells, and in rat primary colon cells and compared to responses from human peripheral lymphocytes. KDA induced DNA damage in the three cell types, confirming that KDA is genotoxic in a range of mammalian cells.

1,3-Butadiene is a known male mouse germ-cell mutagen, to which humans may either be occupationally or environmentally exposed. Prolonged exposure to moderate or high doses in male mice can cause dominant lethal mutations and one report has indicated that 10 week inhalation administration of low doses can result in the production of malformed foetuses. The present study had dual purposes: (a) to attempt to clarify the suspected ability of sub-chronic (6 h/day, 5 days/wk, 10 weeks) low-dose exposure to 1,3-butadiene to induce heritable mutations in mouse male germ cells; (b) investigation of the relationships between testicular DNA damage, testicular DNA repair and foetal outcome. Adult male mice were exposed to low or moderate doses of 1,3-butadiene by inhalation sub-chronically or for a single 6 h period and either used for mating (sub-chronic exposure only) or for studies of DNA damage and repair. Litter size, dominant lethality and numbers of abnormal foetuses were determined the day preceding the normal day of parturition. Testicular DNA damage and repair were assessed by the Comet assay (for DNA damage) and the unscheduled DNA synthesis assay (for DNA repair). 1,3-Butadiene caused a statistically significant increase in dominant lethality at 125 ppm but not 12.5 ppm. No significant increase in DNA repair was found with either dose level or exposure period while only 6 h exposure to 125 ppm caused a small but significant increase in DNA damage as detected by the Comet assay. These effects demonstrate the reproductive genotoxicity of (125 ppm) 1,3-butadiene but do not confirm its ability to cause abnormalities in the offspring via the sperm. It is suggested that the relationship between 1,3-butadiene-induced DNA damage, DNA repair and heritable defects in the offspring may depend on the pattern of metabolites produced.

Oncogenes are involved with the regulation of cellular proliferation. Ras oncogenes can be activated by chemical treatment and any increased activity could be modulated by further chemical treatment. In the present study, therefore, ras p21 protein expression was examined in in vitro cultures of human lymphocytes treated with mitomycin C and in the human colon adenocarcinoma Caco-2 cell line treated with doxorubicin with and without deferoxamine. Both chemotherapeutic agents act partially through oxygen radical mechanisms. Increases in p21 protein levels were seen with mitomycin C but no clear response was seen with doxorubicin. However, deferoxamine, with and without doxorubicin, altered p21 expression. Deferoxamine is an iron chelator so these results support the hypothesis that oxygen radicals were responsible for the altered p21 protein levels. Modulating responses were confirmed by measuring DNA strand-breakage in the Comet assay after treatment with doxorubicin and deferoxamine. Alterations of ras p21 protein expression in vitro might prove a suitable system for examining modulating effects on chemical carcinogens. Teratogenesis Carcinog. Mutagen. 18:219–230, 1998. © 1998 Wiley-Liss, Inc.

The Comet assay is a rapid and sensitive method for analyzing single cells for DNA damage. Using human lymphocytes, the assay is particularly useful for human monitoring studies, as well as for in vitro genotoxicity testing of chemicals. In such studies, it is not always possible to collect and process matched samples on the same day as the blood is taken. It would be useful if some samples could be stored and examined at a different time, without loss of viability or other factors affecting responses. It is thus important to understand the effects of storage conditions on blood to be used in such studies and how exposure or treatment might modify such responses. In a joint study in two laboratories, blood was taken from various donors and stored under different conditions. It was examined on day 1 (day on which sample was taken) and days 2, 3, 4, 5, or 8 at room temperature, 4°C, or −20°C. Cells were treated after storage (from day 2 onward) with bleomycin (BLM) and ethylnitrosourea (ENU). The data were analyzed either by eye (classifying cells with different categories of damage) or by using a computerized image analysis system (Kinetic Imaging Ltd., Liverpool UK, Software Package Comet 3.0) where the tail moment, which is considered to be a sensitive measurement, has been analyzed. There was no loss of cell viability at 4°C or room temperature up to 8 days when measured by trypan blue dye exclusion. Findings suggest that on days 1–4 for the untreated samples at room temperature or 4°C there were no biologically meaningful changes in both the different categories of cell damage and tail moment data. In treated cultures up to day 4, either at room temperature or at 4°C, responses were only minimally affected and changes were considered not to be of biological significance. However, there was slightly less variability between samples at 4°C than at room temperature in one laboratory. The reverse was true in the other. This would suggest that samples can probably be stored up to day 4 at 4°C or room temperature without any untoward effects. Provided samples can be processed within this 4-day time frame, it would not seem necessary to cryopreserve samples at −196°C. Teratog. Carcinog. Mutagen. 17:115–125, 1997.© 1997 Wiley-Liss, Inc.

A human volunteer study was conducted to test the effect of vitamin C supplementation on biomarkers of oxygen radical-mediated damage in individuals with a range of serum cholesterol levels. A group of 48 non-smokers, 24 men and 24 women, was selected from a panel of over 100 volunteers to give as wide a range of serum cholesterol levels as possible. None of the volunteers was taking medication to control cholesterol levels and they maintained their normal dietary habits so as not to compromise their cholesterol status. Volunteers were allocated to three groups of 16, each consisting of four males with low cholesterol levels ( 6 mmol/L) and eight females matched in the same way. A three-treatment, three-treatment period, cross-over design was adopted to take account of any temporal differences in response. The three treatments given were placebo, 60 mg vitamin C/day (the recommended daily allowance) and 6 g vitamin C/day. Each treatment was given for 14 days with 6 weeks between the treatment periods. All procedures were performed to the standards of Good Clinical Practice. Blood samples were taken at the end of each treatment period. Serum was assayed for cholesterol whilst vitamin C, total antioxidant capacity, lipid peroxidation breakdown products and ras p21 protein levels were measured in plasma. Lymphocytes were examined for DNA damage using the Comet assay and chromosome aberration test. The Comet assay was conducted with and without challenge with hydrogen peroxide and the chromosome aberration test with and without challenge with bleomycin. Vitamin C supplementation caused a statistically significant increase in plasma vitamin C concentrations and total antioxidant capacity but did not affect cholesterol levels or ras p21 protein levels. There was a non-significant dose-related decrease in lipid peroxidation breakdown products with vitamin C supplementation. No effect on DNA damage was observed in the Comet assay, either with or without hydrogen peroxide challenge, or in the chromosome aberration test without bleomycin. However, a statistically significant increase in bleomycin-induced aberrations was found after vitamin C supplementation. This may be due to effects of vitamin C on iron status. Comparison of male and female subjects showed statistically significant differences in plasma vitamin C levels, the antioxidant capacity of the plasma and the number of chromosome aberrations induced by bleomycin challenge of lymphocytes in vitro. The results were the same for both low and high cholesterol subjects. This study provides no evidence of a beneficial effect on any of the biomarkers studied of vitamin C supplementation over a short-term supplementation period of 2 weeks in a population of healthy, non-smoking individuals eating a nutritionally adequate diet.

Histone deacetylases (HDACs) and acetyltransferases have important roles in the regulation of protein acetylation, chromatin dynamics and the DNA damage response. Here, we show in human colon cancer cells that dietary isothiocyanates (ITCs) inhibit HDAC activity and increase HDAC protein turnover with the potency proportional to alkyl chain length, i.e., AITC < sulforaphane (SFN) < 6-SFN < 9-SFN. Molecular docking studies provided insights into the interactions of ITC metabolites with HDAC3, implicating the allosteric site between HDAC3 and its co-repressor. ITCs induced DNA double-strand breaks and enhanced the phosphorylation of histone H2AX, ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase-2 (CHK2). Depending on the ITC and treatment conditions, phenotypic outcomes included cell growth arrest, autophagy and apoptosis. Coincident with the loss of HDAC3 and HDAC6, as well as SIRT6, ITCs enhanced the acetylation and subsequent degradation of critical repair proteins, such as CtIP, and this was recapitulated in HDAC knockdown experiments. Importantly, colon cancer cells were far more susceptible than non-cancer cells to ITC-induced DNA damage, which persisted in the former case but was scarcely detectable in non-cancer colonic epithelial cells under the same conditions. Future studies will address the mechanistic basis for dietary ITCs preferentially exploiting HDAC turnover mechanisms and faulty DNA repair pathways in colon cancer cells vs. normal cells.

Medicinal plants play a major role in the life of Turkish people and of late medicinal plant usage has increased in many countries. Green plants in general contain mutagenic and carcinogenic substances, but there is little information about the biological activities of herbal medicine. In the present study, therefore, various Turkish medicinal herbs were investigated for their genotoxic potential in the Salmonella typhimurium microsomal activation assay and the alkaline single cell gel electrophoresis (COMET) assay. Extracts from these medicinal herbs and some fractions of these extracts were examined. The species investigated were Arctium minus, Ecballium elatterium, Momordica charantia, Plantago major, Urtica dioica, Viscum album, Salvia triloba, Euphorbia rigida, Stachys lavandulifolia, Acteoside, Abies nordmannia. They are used for various immune disorders and are applied either topically or taken orally as a herbal tea. Of the 19 samples of the extracts and fractions investigated, none produced a positive response in strains TA98 and TA100 with or without metabolic activation, but all produced an increase above negative control values in the COMET assay. Some extracts were investigated further and produced dose-related increases. In the case of Urtica and Euphorbia species, where two fractions from these plants were examined, one fraction produced a greater response than the other. It is suggested that the lesser response of the fractions might be due to less DNA strand-breaking agents in the fractions or they may have antigenotoxic properties. The breaks that are detected in the COMET assay could be alkali-labile AP-sites and intermediates in base- or nucleotide-excision repair and are difficult to interpret in terms of hazard for man. Further studies with additional genotoxicity assays would be required to make such a prediction.

Increased consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer. Indole-3-carbinol (I3C) and 3, 3′-diindolylmethane (DIM), phytochemicals derived from crucifero...

The effects of antioxidants and various other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes have been investigated using the COMET assay. Hydrogen peroxide (H2O2) and bleomycin (BLM) have produced clear dose-related responses. In 38 independent experiments, there was consistency between the two donors used in the study for the negative and positive control data. The endogenous antioxidant catalase abolished effects with H2O2, but only slightly affected the response with BLM. Superoxide dismutase did not alter the response with H2O2 and only slightly affected BLM. The exogenous antioxidant vitamin C produced a clear dose-related response on its own. In combination with H2O2, there were small protective effects at low doses and exacerbating effects at high doses, but these were within the inter-experimental variability range. Vitamin E (trolox) produced no effects with either H2O2 or BLM, or on its own. Silymarin protected against the effect due to H2O2. Other modifying agents such as apo-transferrin and deferoxamine mesylate produced a clear dose-related protection of effects due to BLM. This protection was less due to H2O2. In the presence of ferrous chloride, the effect due to BLM was exacerbated. In a small sample of 6 smokers and 6 non-smokers, responses from smokers approached borderline significance (P = 0.054) by comparison with non-smokers. These observations would suggest that the COMET assay is a useful tool for examining issues related to oxidative stress in human lymphocytes.

The antimutagenic activity of spearmint (Mentha spicata), a popular food flavoring agent, was studied in the Salmonella assay. Spearmint leaves were brewed in hot water for 5 min at concentrations up to 5% (w/v), and the water extracts were tested against the direct-acting mutagens 4-nitro-1,2-phenylenediamine (NPD) and 2-hydroxyamino-3-methyl-3H-imidazo[4,5-f]quinoline (N-OH-IQ) using Salmonella typhimurium strain TA98. Nontoxic concentrations of spearmint extract inhibited the mutagenic activity of N-OH-IQ in a concentration-dependent fashion, but had no effect against NPD. These experiments by design focused on the water extract consumed commonly as an herbal tea, but chloroform and methanol extracts of spearmint also possessed antimutagenic activity against N-OH-IQ. Water extract of spearmint inhibited the mutagenic activity of the parent compound, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), in the presence of rat liver S9; however, the concentration for 50% inhibition (IC50) against IQ was approximately 10-fold higher than in assays with N-OH-IQ minus S9. At concentrations similar to those used in the Salmonella assays, spearmint extract inhibited two of the major enzymes that play a role in the metabolic activation of IQ, namely, cytochromes P4501A1 and 1A2, based on ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase assays in vitro. In vivo, rats were given spearmint water extract (2%; w/v) as the sole source of drinking fluid before, during, and after 2-week treatment with IQ; colonic aberrant crypt foci were inhibited significantly at 8 weeks (P < 0.05, compared with rats given IQ alone). Collectively, these findings suggest that spearmint tea protects against IQ and possibly other heterocyclic amines through inhibition of carcinogen activation and via direct effects on the activated metabolite(s).

To determine if 6 weeks of supplementation with antioxidants could alleviate exercise-induced DNA damage, we studied 21 runners during a 50 km ultramarathon. Subjects were randomly assigned to one of two groups: (1) placebos (PL) or (2) antioxidants (AO) (1000 mg vitamin C and 400 IU RRR-alpha-tocopheryl acetate). The comet assay was used to assess DNA damage in circulating leukocytes at selected time points: pre-, mid-, and 2 h postrace and daily for 6 days postrace. All subjects completed the race: run time 7.1 +/- 0.1 h, energy expenditure 5008 +/- 80 kcal for women (n = 10) and 6932 +/- 206 kcal for men (n = 11). Overall, the percentage DNA damage increased at midrace (p

Over 200 agents/factors have been examined in the single cell gel electrophoresis assay, more commonly known as the Comet assay, performed either in vitro or in vivo in a variety of species. Unequivocal carcinogenicity data are available for 119 of them, amongst which unequivocal Comet assay data exist for 95 agents. Of these 95 agents the prevalence of carcinogens was 88% (84/95). The carcinogens that were Comet positive (sensitivity) formed 88% (74/84), the non-carcinogens that were Comet negative (specificity) formed 64% (7/11). This simple analysis of the Comet assay has not taken account of the difference between in vitro and in vivo responses, species differences or organ and tissue differences. Also, limitations as to the conduct of the assay have not been examined in any depth. Thus, at the present time the Comet assay has high sensitivity for carcinogens, but its specificity is uncertain because few non-carcinogens have been tested.

The flavonoids, silymarin, myricetin, quercitin, kaempferol, rutin and kaempferol-3-rutinoside have been examined in combination with the food mutagens, 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp-P-2) and 2-amino-3-methylimidazo-(4,5-f) quinoline (IQ), in the Comet assay in human lymphocytes from donor A and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in lymphocytes and sperm. The assays were performed in the absence of metabolic activation, since when quercetin and kaempferol were examined in blood with metabolic activation, there was little or no difference in response to that obtained in its absence. In the blood, there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm, with silymarin, myricetin and quercitin, antigenotoxic effects only were observed, but with kaempferol, in general, there were no protective effects. The food mutagen, 2-amino-1-methyl-6-phenylimadazo (4,5-b)pyridine (PhIP), was also examined in addition to Trp-P-2 and IQ in combination with silymarin and myricetin in donors A and C in human lymphocytes only. Similar exacerbation of effects were found at low doses of these flavonoids with antigenotoxic effects at high doses. This was confirmed in the Ames test. There were slightly different profiles in lymphocytes and sperm, but antigenotoxic effects were observed over a similar dose range. This would suggest that effects occur in somatic and germ cells on a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.

The main purpose of this study was to determine whether well-known reactive oxygen species (ROS)-generating agents can induce DNA damage in a simple chemical system with or without Fenton reaction components (iron and reducing agents), and to explore whether antioxidants which normally exist in the cellular environment can modify such damage, i.e. to determine chemical reactions of relevance to biological systems. A neutral electrophoresis technique was used to investigate DNA double stranded breaks (DSBs) caused by chemical treatments of lambda-DNA in eppendorf tubes by various ROS-generating compounds and the degree of DNA damage was categorised by analysis of enhanced digital images. Double strand breaks were induced by hydroquinone (HQ), benzoquinone (BQ), benzenetriol (BT), hydrogen peroxide (H2O2), bleomycin (BLM) and sodium ascorbate (Vit C). DNA damage was modulated by various agents including catalase (CAT), superoxide dismutase (SOD), desferoxamine mesylate (DFO), ferrous chloride (FeCl2), reduced glutathione (GSH), trolox, silymarin and myricetin. Individual chemicals (except BLM) at the concentration of 1 mM did not induce large numbers of DSBs without iron [Fe(II) or Fe(III) at 25 microM]. GSH enhanced the damaging effect of HQ, BT and Vit C, did not alter the non-damaging effect of H2O2, but had a small protective effect on BLM. When compared with the non-enzyme protein, bovine serum albumin (BSA), SOD had a protective effect against BT, H2O2 and BLM; in the presence of GSH, SOD diminished the effect of HQ, BQ and Vit C but enhanced the effect of BT, H2O2 and BLM. With both GSH and Fe and compared with BSA, SOD enhanced the effect of HQ, BQ and BLM, ameliorated the effect of H2O2, and did not affect the others. CAT showed a protective effect for almost all examined compounds, but had little effect on BLM. With GSH alone, DFO enhanced the effect of HQ, BQ, H2O2 and ameliorated the effect of BT, BLM and Vit C and trolox was largely protective. With GSH and Fe, DFO was protective for all compounds except doxorubicin (Dox), trolox was protective for all compounds except Dox and BLM, silymarin was protective except that it had little effect on BLM and Dox, but myricetin did not show any protective effect. In conclusion, the results from the present study have further highlighted the adverse potential of reducing agents and redox cycling agents, and also the need for a cautious view of antioxidants.

Since the 1970s there have been conflicting reports of decreasing sperm counts in man and increasing testicular cancer. There is a hypothetical link between apparent adverse trends in several measures of human reproductive health and exposure to endocrine disrupters. Rodent bioassays are not suited for the large-scale screening of such chemicals because of their costs, complexity, and ethical concerns. Various in vitro assays have been used to examine the effects of these chemicals, but none has directly used semen as one of the target tissues in man. The present study has examined in the alkaline Comet assay in human sperm the effect of two estrogens—β-estradiol and the phytoestrogen daidzein—and 1,2-epoxybutene, a metabolite of 1,3-butadiene, and compared them with the effects of the known reprotoxin, dibromochloropropane, in two fertile and two infertile frozen sperm samples and two fresh fertile samples. While differences were detected in the frozen fertile and infertile samples with flow cytometry, in the Comet assay both frozen and fresh samples exposed to the chemicals in vitro from fertile and infertile men produced similar altered responses by comparison with untreated samples. The integrity of DNA is necessary not only for the noncancerous state, but also for the accurate transmission of genetic material to the next generation. Thus this assay may be useful for examination of chemicals in fresh and frozen sperm samples. Teratog. Carcinog. Mutagen. 17:97–102, 1997. © 1997 Wiley-Liss, Inc.

The flavonoids silymarin, myricetin, quercetin, kaempferol, rutin, and kaempferol-3-rutinoside have been examined in combination with the food mutagens 3-amino-1-methyl-5H-pyrido (4,3-b)indole (Trp) and 2-amino-3-methylimidazo-4,5-f)quinoline (IQ) in the Comet assay in human lymphocytes from donors A and B and human sperm from donor B. These compounds alone have been shown to produce positive responses in the Comet assay, as have the food mutagens. However, in combination with the food mutagens, the flavonoids produced antigenotoxic effects since DNA damage was reduced in the Comet assay in human lymphocytes and sperm over a similar dose range in the absence of metabolic activation. Only quercetin and kaempferol were examined in blood with metabolic activation, but there was no difference in response to that obtained without activation. In the blood there was an exacerbation or synergy of response at the lowest doses of the flavonoids. In the sperm this was also the case with silymarin and myricetin. With kaempferol there was no antigenotoxic effect and quercetin protected below baseline levels. Since the effects were observed in lymphocytes and sperm over a similar dose range, it would suggest that the Comet assay responses occur in somatic and germ cells in a one-to-one ratio. These results have implications for man in terms of risk assessment and in the modulation of isolated food constituents.

The effects of the mutagens, cyclophosphamide (CP), ethyl methanesulphonate (EMS), bleomycin (BLM) and the testicular toxin ethylene glycol monomethyl ether (EGME) in bone marrow and testicular cells have been compared in the alkaline COMET assay. Sprague-Dawley rats were administered by gavage with 50, 100 and 150 mg/kg body weight (bw) of CP; 100, 200 and 300 mg/kg bw EMS; 50, 100 and 150 mg/kg bw BLM and 500, 1000 and 1500 mg/kg bw EGME. Effects were examined at week 2 after treatment for CP, EMS BLM and EGME and at weeks 5 and 6 for EGME. Bone marrow cells were removed and separated by aspiration of the femur and testicular cells by decapsulation of the testis, treating with collagenase followed by trypsin. Various statistical methods were used to analyse the data. For CP there was an increase in damage above control values for bone marrow at 50 mg/kg bw which decreased at 100 mg/kg bw, and there was mortality of the animals at 150 mg/kg bw. A similar response was found in the testicular cells. For EMS and BLM, there were only occasional slight increases in damage in bone marrow and testicular cells. Two studies were conducted with EGME. In the first, where effects were examined at week 2 after treatment, there was an increase in damage in bone marrow cells, but a larger response was observed in testicular cells. In the second study where effects were examined at weeks 5 and 6 after treatment, bone marrow and testicular cells were not affected. The overall results showed that damage persisted for 2 weeks after treatment with CP and EGME but not in weeks 5 and 6 for EGME. Various statistical methods were used to analyse the data. Statistically significant responses were produced after treatment with CP and EGME and were dose-related for EGME, but after treatment with EMS and BLM statistical increases were sporadic. These results suggest that the assay is useful for measuring DNA damage and its persistence, and for comparing the sensitivity of different target organs in vivo.

Benzene and five of its known metabolites--muconic acid, hydroquinone, catechol, p-benzoquinone, and benzentriol--were examined for DNA damage in human lymphocytes using the alkaline Comet assay, and conditions were optimised to determine responses. Metabolic activation (S-9 mix) was included in the assay for varying times to try to enhance effects. In addition, the effects of catalase were investigated as it is known to be present in S-9 mix reducing oxidative damage, and some benzene metabolites are known to react through oxygen radical mechanisms. Effects were also examined in cycling cells to determine whether they were more sensitive to damage then noncycling cells. Comets were measured either by eye or by image analysis. Data have been presented according to length of treatments. When Comets were measured by eye after treatment with hydrogen peroxide (H2O2), the positive control, and each compound for 0.5 hr, only H2O2 and benzenetriol induced pronounced DNA damage without metabolic activation. The effect of catechol was moderate compared with that of benzenetriol. There was a very weak effect of benzene in the absence of rat liver S-9 mix. In the presence of S-9 mix, benzene was not activated. The effect of benzenetriol was greatly reduced by the external metabolising system, but p-benzoquinone became activated to some extent. Catalase abolished the effect of benzenetriol, suggesting that H2O2 formed during autoxidation may be responsible for the DNA-damaging ability of this metabolite. The presence of catalase in S-9 mix may explain the detoxification of benzenetriol and the failure to detect consistent benzene responses. Mitogen-stimulated cycling cells were less sensitive to H2O2 and benzenetriol than unstimulated G0 lymphocytes. When comets were measured by image analysis, a 0.5-hr treatment with H2O2 and benzenetriol and catechol confirmed results analysed by eye, with S-9 mix greatly reducing responses. When treatments were increased to 1 hr in the presence and absence of S-9 mix, benzene at a 5-fold increased dose produced a significant positive response but not at the lower dose. When treatment times were increased to 2 and 4 hr, doses were also increased, and muconic acid, hydroquinone, catechol, and benzoquinone in the presence of S-9 mix showed positive time and dose-related responses, and at the highest dose of benzoquinone the morphology of the nucleus was affected. Effects tended to become more pronounced at high doses and after longer exposures, although this was not always consistent from experiment to experiment. In conclusion, benzene and all metabolites investigated gave positive responses. Where altered responses were observed, they were significantly different from the corresponding controls.

The promutagenic arylamines, m-phenylenediamine (mPDA) and 2-aminofluorene (2-AF), were evaluated for their genotoxicity in Salmonella typhimurium strain YG1024 and in human lymphocytes. These agents were assayed with and without TX1MX plant activation mix. Both arylamines without activation were refractory in S. typhimurium, demonstrating that plant activation was required for the generation of their ultimate mutagenic metabolites. However, using the alkaline single-cell gel/Comet assay, both mPDA and 2-AF directly induced DNA damage in human lymphocytes. This effect was reduced when the human cells were treated with the arylamine plus TX1MX. mPDA with or without plant activation was not toxic to the exposed cells. However, at concentrations over 80 microM, 2-AF was toxic to lymphocytes. This toxic response was eliminated by incubation with TX1MX. mPDA and 2-AF were plant-activated into mutagens for S. typhimurium. However, these plant-activated products had a reduced genotoxic potency in human lymphocytes.

Protein acetylation is mediated by histone deacetylases (HDACs) and acetyltransferases, which influence chromatin dynamics, protein turnover, and the DNA damage response. HDACs overexpressed in cancer cells have been implicated in protecting against genotoxic insults, whereas HDAC inhibitors circumvent this protection (Rajendran et al. Clin Epigenetics 2011,3:4). Here, we show in human colon cancer cells that sulforaphane and related isothiocyanates (ITCs) inhibited HDAC activity and increased HDAC protein turnover, with the potency directly proportional to alkyl chain length. Under these conditions, DNA damage signaling was triggered by ATR kinases, leading to increased double-strand breaks and histone (H2AX) phosphorylation. Activation of checkpoint kinase-2 was followed by growth arrest and cell death. HDAC inhibition by ITCs enhanced the acetylation of repair proteins, like CtIP, leading to their degradation. Notably, cancer cells were more susceptible than normal cells, the latter exhibiting efficient double-strand-break processing and repair. Thus, dietary ITCs preferentially exploit the HDAC turnover pathways, faulty DNA repair mechanisms, and genomic instability in cancer cells. Supported by NIH grants CA090890, CA65525, CA122906, CA122959, CA80176, and ES007060. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-184. doi:1538-7445.AM2012-LB-184

Background Histone deacetylase (HDAC) inhibitors are currently undergoing clinical evaluation as anti-cancer agents. Dietary constituents share certain properties of HDAC inhibitor drugs, including the ability to induce global histone acetylation, turn-on epigenetically-silenced genes, and trigger cell cycle arrest, apoptosis, or differentiation in cancer cells. One such example is sulforaphane (SFN), an isothiocyanate derived from the glucosinolate precursor glucoraphanin, which is abundant in broccoli. Here, we examined the time-course and reversibility of SFN-induced HDAC changes in human colon cancer cells. Results Cells underwent progressive G2/M arrest over the period 6-72 h after SFN treatment, during which time HDAC activity increased in the vehicle-treated controls but not in SFN-treated cells. There was a time-dependent loss of class I and selected class II HDAC proteins, with HDAC3 depletion detected ahead of other HDACs. Mechanism studies revealed no apparent effect of calpain, proteasome, protease or caspase inhibitors, but HDAC3 was rescued by cycloheximide or actinomycin D treatment. Among the protein partners implicated in the HDAC3 turnover mechanism, silencing mediator for retinoid and thyroid hormone receptors (SMRT) was phosphorylated in the nucleus within 6 h of SFN treatment, as was HDAC3 itself. Co-immunoprecipitation assays revealed SFN-induced dissociation of HDAC3/SMRT complexes coinciding with increased binding of HDAC3 to 14-3-3 and peptidyl-prolyl cis/trans isomerase 1 (Pin1). Pin1 knockdown blocked the SFN-induced loss of HDAC3. Finally, SFN treatment for 6 or 24 h followed by SFN removal from the culture media led to complete recovery of HDAC activity and HDAC protein expression, during which time cells were released from G2/M arrest. Conclusion The current investigation supports a model in which protein kinase CK2 phosphorylates SMRT and HDAC3 in the nucleus, resulting in dissociation of the corepressor complex and enhanced binding of HDAC3 to 14-3-3 or Pin1. In the cytoplasm, release of HDAC3 from 14-3-3 followed by nuclear import is postulated to compete with a Pin1 pathway that directs HDAC3 for degradation. The latter pathway predominates in colon cancer cells exposed continuously to SFN, whereas the former pathway is likely to be favored when SFN has been removed within 24 h, allowing recovery from cell cycle arrest.