DNA-damaging Potency Research Articles

Sensitive MALDI-TOF MS and ‘turn-on’ fluorescent genosensor for the determination of DNA damage induced by CNS acting drugs

The genotoxic and carcinogenic adverse effects of various drugs should be considered for assessing drug benefit/risk ratio. On that account, the scope of this study is to examine the kinetics of DNA damage triggered by three CNS acting drugs; carbamazepine, quetiapine and desvenlafaxine. Two precise, simple and green approaches were proposed for probing drug induced DNA impairment; MALDI-TOF MS and terbium (Tb3+) fluorescent genosensor. The results revealed that all the studied drugs induced DNA damage manifested by the MALDI-TOF MS analysis as a significant disappearance of the DNA molecular ion peak with the appearance of other peaks at smaller m/z indicating the formation of DNA strand breaks. Moreover, significant enhancement of Tb3+ fluorescence occurred, proportional to the amount of DNA damage, upon incubation of each drug with dsDNA. Furthermore, the DNA damage mechanism is examined. The proposed Tb3+ fluorescent genosensor showed superior selectivity and sensitivity and is significantly simpler and less expensive than other methods reported for the detection of DNA damage. Moreover, the DNA damaging potency of these drugs was studied using calf thymus DNA in order to clarify the potential safety hazards associated with the studied drugs on natural DNA.

Toxicity of combined exposure to acrylamide and Staphylococcus aureus

In the current study, the aim was to examine the toxicity of combined exposure to acrylamide and Staphylococcus aureus. We investigated the effect of staphylococcal enterotoxin A (SEA), a toxin produced by Staphylococcus aureus, on the oxidation induced DNA damaging potency of acrylamide in mouse spleen cells using an Formamidopyrimidine-DNA glycosylase-modified (FPG)-modified comet assay. Parameters like tail moment, tail length, and % tail DNA as indicators of DNA damage were significantly increased in the combined acrylamide and SEA treatment compared with SEA or acrylamide alone. Further, we examined the effects of acrylamide and its epoxide metabolite glycidamide on overall production of SEA, SEA mRNA gene expression, and on the formation of biofilm of S. aureus. Acrylamide significantly increased the SEA expression level and SEA production in S. aureus. Acrylamide also significantly increased biofilm formation in S. aureus without affecting its growth rate. Moreover, the addition of acrylamide significantly increased the expression of S. aureus virulence factors RNAIII and icaA in fetal bovine serum. Our results showed that combined exposure to acrylamide and S. aureus or its toxin enhanced their chemical and biological toxicities.

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds in embryo-fetal chicken livers

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds were assessed in embryo-fetal chicken livers. Formation of DNA adducts and strand breaks were measured using the nucleotide 32P-postlabelling (NPL) and comet assays, respectively. Unsubstituted monocyclic benzene, polycyclic fused ring compound naphthalene, covalently connected polycyclic ring compound biphenyl, and heterocyclic ring compound fluorene did not produce DNA damage. Amino-substituted monocyclic compounds, aniline and p-phenylenediamine, as well as polycyclic 1-naphthylamine were also negative. In contrast, carcinogenic monocyclic methyl-substituted anilines: o-toluidine, 2,6-xylidine, 3,4-dimethylaniline, 4-chloro-o-toluidine; 2 methoxy-substituted methylaniline: p-cresidine; 2,4 and 2,6 diamino- or dinitro- substituted toluenes all produced DNA damage. Genotoxic polycyclic amino-substituted 2-naphthylamine, 4-aminobiphenyl, benzidine, methyl-substituted 3,2′-dimethyl-4-aminobiphenyl and 4-dimethylaminoazobenzene as well as amino- and nitro- fluorenes substituted at the 1 or 2 positions also were positive in at least one of the assays. Overall, the DNA damaging activity of cyclic compounds in embryo-fetal chicken livers reflected the type and position of the substitution on the aromatic ring. Additionally, substituted polycyclic compounds exhibited higher DNA-damaging potency compared to monocyclic chemicals. These results are congruent with in vivo findings in other species, establishing chicken eggs as a reliable system for structure-activity assessment of members of groups of related chemicals.

Investigation of DNA damage of glycidol and glycidol fatty acid esters using Fpg-modified comet assay

Glycidol fatty acid esters (GEs) are food process contaminants detected in edible oils. It has been thought that glycidol is released from GEs by lipase in vivo, and shows genotoxicity. While DNA damage from glycidol has been reported, there is very little information on the DNA damaging potency of GEs in vivo. Therefore, we estimated DNA damage of glycidol and glycidyl oleate, which is one type of GEs, using the standard comet assay and a formamidopyrimidine glycosylase(Fpg)-modified comet assay. ICR male mice were orally administrated glycidol and glycidyl oleate (1.0 and 2.0 mmol/kg body weight) at 24 and 3 hr prior to dissection. In the standard comet assay, DNA damage (tail length and % tail DNA) in liver, kidney and blood samples of glycidol-treated groups were increased in a concentration-dependent manner. In Fpg-modified comet assay, glycidol showed DNA damage with higher sensitivity compared with the standard comet assay. DNA damage was not observed in the administration group of glycidyloleate in the standard comet assay. However, in Fpg-modified comet assay, glycidyl oleate showed significant DNA damage in the liver, kidney and blood samples compared with the standard comet assay. In this study, it was revealed that glycidol and glycidyl oleate induce DNA damage, such as oxidative and alkylation damage, recognized by Fpg protein.

Effect of physicochemical character differences on the genotoxic potency of kaolin

BackgroundKaolin is white clay mineral with the chemical composition Al2Si2O5(OH)4, and many varieties of kaolins having different crystal structures are utilized in industrial, cosmetic and medical fields. To evaluate the effect of physicochemical character differences on the genotoxicity of kaolin, two types of kaolin, kaolin-S with smooth, sphere-shaped crystals, and kaolin-P with clusters of thin pseudohexagonal plates, were used in the study.ResultsICR mice were intratracheally instilled with the kaolins (0.05 and 0.2 mg/mouse), and comet assay was performed on their lungs. Both kaolins showed DNA damage in the lungs of the mice, however the DNA damaging potency was much higher with kaolin-P than that with kaolin-S.In order to clarify the mechanisms for the different genotoxic potency, we examined the incorporation rate and ROS generation of these two types of kaolin in alveolar epithelial A549 and macrophage-like RAW264 cells, using flow cytometric (FCM) analysis. Kaolin-P showed a higher incorporation rate into the mammalian cells and ROS generation than that of kaolin-S. Especially, RAW264 cells aggressively incorporated kaolins, and generated ROS, whereas almost no ROS generation was observed in A549 cells. In addition, inflammatory cytokines were quantified, using the ELISA method, to understand further genotoxic potency differences of kaolins. Concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the media were increased by exposure to both kaolins, but in the case of kaolin-P, these inflammatory cytokines were significantly elevated. Based on these findings, differences of genotoxic potency may contribute to incorporation rates into immune cells. Furthermore, it is likely that immune cells and epithelial cells might closely interact with each other for the appearance of genotoxocity in vivo. In order to clarify the interaction between epithelial and immune cells, A549 and RAW264 were co-cultured and RAW264 cells only were exposed to kaolins, then subsequently A549 was applied to FCM analysis and comet assay. DNA damage observed in the A549 cells markedly increased in the presence of kaolin-exposed RAW264 cells compared to the single culture.ConclusionFrom these observations, it is suggested that mechanisms of kaolin genotoxicity against epithelial cells are through the activation of macrophage cells. Therefore, it is thought that interactions between epithelial and immune cells would be very important for evaluation of the genotoxicity of fine particulate matter. We also showed here that co-culture models of epithelial and immune cells could be used as suitable models for evaluation of lung genotoxicity of fine particulate matter, including nanomaterials, as in vivo mimicking systems.

The potential of combi-molecules with DNA-damaging function as anticancer agents.

DNA-damaging agents, such as methylating agents, chloroethylating agents and platinum-based agents, have been extensively used as anticancer drugs. However, the side effects, high toxicity, lack of selectivity and resistance severely limit their clinical applications. In recent years, a strategy combining a DNA-damaging agent with a bioactive molecule (e.g., enzyme inhibitors) or carrier (e.g., steroid hormone and DNA intercalators) to produce a new 'combi-molecule' with improved efficacy or selectivity has been attempted to overcome these drawbacks. The combi-molecule simultaneously acts on two targets and is expected to possess better potency than the parent compounds. Many studies have shown DNA-damaging combi-molecules exhibiting excellent anticancer activity in vitro and in vivo. This review focuses on the development of combi-molecules, which possess increased DNA-damaging potency, anticancer efficacy and tumor selectivity and reduced side reactions than the parent compounds. The future opportunities and challenges in the discovery of combi-molecules were also discussed.

In vitro cytotoxic and genotoxic evaluation to ascertain toxicological potential of ketoprofen

Analgesic and anti-inflammatory properties of ketoprofen are well documented but little is known about its cytotoxic activity and the potential to damage the DNA. The present study was designed to evaluate the cytotoxic and genotoxic potential of ketoprofen. MTT dye (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was used to assess cytotoxicity in which confluent monolayer of vero cells were incubated in the presence of increasing concentrations of ketoprofen. Genotoxicity was evaluated by single cell gel electrophoresis (SCGE) assay or comet assay. Lymphocytes were separated from the mice blood and treated with different concentrations of ketoprofen. Lymphocytes were incorporated in agarose gel on cavity slides and visualized for strand break to asses DNA damage. Ketoprofen concentrations 8, 6, 4.5, 3.3, 2.5, 1.8, 1.4, 1 and 0.5 mM were used for both cytotoxic and comet assay. The results of cytotoxic assay showed significant (p < 0.001) cytotoxicity at 8 and 6 mM concentrations. The cytotoxic concentration for 50% of cells (CC50) value was calculated at 5.2 mM concentration. In case of the comet assay, ketoprofen presented DNA damaging potency, creating significant (p < 0.001) DNA damage at 8 mM concentration, a moderate damage at 6 mM concentration and a mild damage at 4.5 mM concentration which was evident from the comet tail lengths and changes in head diameter. DNA damage index was calculated for each concentration of ketoprofen and compared with the control. The data advocates that ketoprofen possesses cytotoxic and genotoxic potential at higher concentrations and its dosage should be carefully monitored to avoid its toxicity. Key words: Ketoprofen, cytotoxic, genotoxic, MTT assay (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), comet assay, DNA damage.

Comparison of DNA damage in human-derived hepatoma line (HepG2) exposed to the fifteen drinking water disinfection byproducts using the single cell gel electrophoresis assay

Disinfection of drinking water reduces pathogenic infection, but generates disinfection by-products (DBPs) in drinking water. In this study, the effect of fifteen DBPs on DNA damage in human-derived hepatoma line (HepG2) was investigated by the single cell gel electrophoresis (SCGE) assay. These fifteen DBPs are: four trihalomethanes (THMs), six haloacetic acides (HAAs), three haloacetonitriles (HANs), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and chloral hydrate (CH). Based on the minimal effective concentration (MEC) at which DBPs induced significant increase in olive tail moment (OTM), the rank order of DNA-damaging potency is: bromodichloromethane (BDCM) > dibromochloromethane (DBCM) > tribromomethane (TBM) > trichloromethane (TCM) of the four THMs; iodoacetic acid (IA) > bromoacetic acid (BA) > dibromoacetic acid (DBA) > dichloracetic acid (DCA) > trichloroacetic acid (TCA) of the five HAAs; dibromoacetonitrile (DBN) ≈ dichloroacetonitrile (DCN) > trichloroacetonitrile (TCN) of the three HANs. The DNA damaging potency of MX and CH is similar to TCA and DCA, respectively. IA is the most genotoxic DBP in the fifteen DBPs, followed by BA. Chloroacetic acid (CA) is not genotoxic in this assay. Our findings indicated that HepG2/SCGE is a sensitive tool to evaluate the genotoxicity of DBPs and iodinated DBPs are more genotoxic than brominated DBPs, but chlorinated DBPs are less genotoxic than brominated DBPs.

Investigations on cytotoxic and genotoxic effects of laser printer emissions in human epithelial A549 lung cells using an air/liquid exposure system

Exposure to emissions from laser printers during the printing process is commonplace worldwide, both in the home and workplace environment. In the present study, cytotoxic and genotoxic effects of the emission from five low to medium-throughput laser printers were investigated with respect to the release of ozone (O(3) ), volatile organic compounds (VOC), particulate matter (PM), and submicrometer particles (SMP) during standby and operation. Experiments were conducted in a 1 m(3) emission chamber connected to a Vitrocell® exposure system. Cytotoxicity was determined by the WST-1 assay and genotoxicity by the micronucleus test in human A549 lung cells. The five laser printers emitted varying but generally small amounts of O(3) , VOC, and PM. VOC emissions included 13 compounds with total VOC concentrations ranging from 95 to 280 μg/m(3) (e.g., 2-butanone, hexanal, m,p-xylene, and o-xylene). Mean PM concentrations were below 2.4 μg/m(3). SMP number concentration levels during standby ranged from 9 to 26 particles/cm(3). However, three of the printers generated a 90 to 16 × 10(3) -fold increase of SMP during the printing process (maximum 294,460 particles/cm(3)). Whereas none of the printer emissions were found to cause cytotoxicity, emissions from two printers induced formation of micronuclei (P < 0.001), thus providing evidence for genotoxicity. As yet, differences in biological activity cannot be explained on the basis of the specific emission characteristics of the different printers. Because laser printing technology is widely used, studies with additional cytogenetic endpoints are necessary to confirm the DNA-damaging potency and to identify emission components responsible for genotoxicity.

Assessment of the DNA damaging potency and chemopreventive effects towards BaP-induced genotoxicity in human derived cells by Monimiastrum globosum, an endemic Mauritian plant

Naturally occurring compounds have protective effects towards mutagens and carcinogens. The leaf extract of Monimiastrum globosum (Bois de Clous), a Mauritian endemic plant from the Myrtaceae family, was studied for its potency to induce DNA damage in human HepG2 hepatoma cells using DNA migration as a biological endpoint in the alkaline single cell gel electrophoresis (SCGE) assay. This was contrasted with the ability to modulate the benzo[a]pyrene (BaP)-dependent DNA damage in human hepatoma cells. M. globosum caused genotoxicity in HepG2 cells at concentrations exceeding 3 mg fresh weight (FW) per ml cell culture in the absence of cytotoxicity. Pre-treatment of the cells with 12.2 μg FW/ml to 1.56 mg FW/ml led to a pronounced antigenotoxic effect towards BaP-induced DNA damage. DNA migration (OTM) was reduced by 66%, 81.5% and 74% for 49, 98 and 195 μg FW/ml, respectively. A U-shaped dose-response curve was derived for M. globosum indicating genotoxic effects in high doses and antigenotoxic effects in low doses. M. globosum extract had total phenolics (15 mg/g FW) with flavonoids (aglycones and conjugates: 8 mg/g FW) and proanthocyanidins (3 mg/g FW) as major phenolic subclasses. The hydrolysis of conjugated flavonoids yielded the aglycones quercetin (606 μg/g FW) and kaempferol (117.8 μg/g FW) while HPLC-MS/MS analysis of the total extract revealed free flavonoids such as quercetin (19.2 μg/g FW) and myricetin (2.5 μg/g FW). The antioxidant activity of the extract of M. globosum, assessed by the FRAP and TEAC assays yielded values of 275 ± 3.82 μmol/g FW and 346 ± 4.2 μmol/g FW, respectively.

Correlation between induction of DNA fragmentation in lung cells from rats and humans and carcinogenic activity

Six chemicals, known to induce lung tumors in rats, were examined for their ability to induce DNA fragmentation in primary cultures of rat and human lung cells, and in the lung of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the single-cell gel electrophoresis (Comet) assay, were obtained in primary lung cells from male rats with the following, minimally toxic, concentrations of the six test compounds: N-nitrosodimethylamine (NDMA; 2.5–10 mM), hydrazine (HZ; 0.5–4 mM), cadmium sulfate (CD; 31.2 and 62.5 μM), 4,4′-methylene bis (2-chloroaniline) (MOCA; 31.2–125 μM), isobutyl nitrite (IBN; 7.8–31.2 μM) and tetranitromethane (TNM; 1.9–15.6 μM). Similar degrees of DNA fragmentation were obtained in primary human lung cells; however, due to inter-donor differences, the minimum effective concentrations were in some donors lower and in others higher than in rats, and IBN induced DNA damage only in one of three donors. The DNA-damaging potency of HZ was higher in rats than in humans, and the opposite was true for MOCA. In agreement with these findings, statistically significant increases in the average frequency of DNA breaks were obtained in the lung of rats given a single oral dose (1/2 LD50) of the six test compounds. These findings give evidence that genotoxic lung carcinogens may be identified by use of the DNA fragmentation/Comet assay on rat lung cells as targets cells, and show that the six compounds tested produce in primary cultures of lung cells from human donors DNA-damaging effects substantially similar to those observed in rats.

Bioactivation of Carboxylic Acid Compounds by UDP-Glucuronosyltransferases to DNA-Damaging Intermediates: Role of Glycoxidation and Oxidative Stress in Genotoxicity

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin >> gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 Kruskal-Wallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidation- dependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes.

DNA strand breaking capacity of acrylamide and glycidamide in mammalian cells

We compared the DNA damaging potency of acrylamide (AA) and its metabolite glycidamide (GA) in the comet assay in cell systems differing with respect to species origin and cytochrome P450-depended monooxygenase (CYP2E1) expression (V79, Caco-2, primary rat hepatocytes). Only after 24 h incubation in the highest concentration of AA (6 mM) a slight but significant increase in DNA damage was observed in V79 and Caco-2 cells. In primary rat hepatocytes, however, expressing substantial amounts of CYP2E1, no induction of DNA strand breaks was found. At the end of the incubation time period (24 h), still 67 ± 19% of the CYP2E1 protein was detected by Western blotting. Direct treatment with GA resulted in a significant increase in DNA damage in V79 cells and primary rat hepatocytes at concentrations ≥100 μM (24 h). Caco-2 cells were found to be less sensitive, exhibiting an increase in DNA strand breaks at concentrations ≥300 μM GA. These data confirm the higher genotoxic potential of GA compared to AA but also indicate that high expression of CYP2E1 per se is not necessarily associated with increased genotoxicity of AA. We, therefore, investigated whether the intracellular glutathione (GSH) level might be a critical determinant for the genotoxicity of AA in cells with different CYP2E1 status. Depletion of intracellular GSH by dl-buthionine-[S,R]-sulfoxime (BSO) in rat hepatocytes and V79 cells resulted in a significant induction of DNA strand breaks after incubation with 1 mM AA. However, at higher concentrations (≥1.25 mM) a strong increase in cytotoxicity, resulting in a severe loss of viability, was observed. In summary, the DNA strand breaking effect of AA appeared not to be directly correlated with the CYP2E1 status of the cells. Depletion of GSH is associated with an increase in AA genotoxicity but seems also to lead to a substantial enhancement of cytotoxicity.

DNA fragmentation, DNA repair and apoptosis induced in primary rat hepatocytes by dienogest, dydrogesterone and 1,4,6-androstatriene-17β-ol-3-one acetate

Four steroids that share the 17-hydroxy-3-oxopregna-4,6-diene structure – cyproterone acetate, chlormadinone acetate, megestrol acetate, and potassium canrenoate – have been shown previously to behave with different potency as liver-specific genotoxic agents, the response being markedly higher in female than in male rats, but similar in humans of both genders. In this study, performed to better define the relationship between chemical structure and genotoxicity, dydrogesterone (DGT) with double bonds C4 C5 and C6 C7, dienogest (DNG) with double bonds C4 C5 and C9 C10, and 1,4,6-androstatriene-17β-ol-3-one acetate (ADT) with double bonds C1 C2, C4 C5 and C6 C7, were compared with cyproterone acetate (CPA) for their ability to induce DNA fragmentation and DNA repair synthesis in primary cultures of hepatocytes from three rats of each sex. At subtoxic concentrations, ranging from 10 to 90 μM, all four steroids consistently induced a dose-dependent increase of DNA fragmentation, which in all cases was higher in females than in males; their DNA damaging potency decreased in the order CPA > DNG > ADT > DGT. Under the same experimental conditions, the responses provided by the DNA repair-synthesis assay were positive or inconclusive in hepatocytes from female rats and consistently negative in hepatocytes from male rats. In the induction of apoptotic cells, examined in primary hepatocytes from female rats, CPA was more active than ADT and DGT, and DNG was inactive. Considered as a whole these findings suggest that a liver-specific genotoxic effect more marked in female than in male rats might be a common property of steroids with two or three double bonds.

Novel N-thiolated β-lactam antibiotics selectively induce apoptosis in human tumor and transformed, but not normal or nontransformed, cells

Historically, it has been shown that the β-lactam antibiotics play an essential role in treating bacterial infections while demonstrating selectivity for prokaryotic cells. We recently reported that certain N-methylthio-substituted β-lactam antibiotics had DNA-damaging and apoptosis-inducing activities in various tumor cells. However, whether these compounds affect human normal or nontransformed cells was unknown. In the current study, we first show that a lead compound (lactam 1) selectively induces apoptosis in human leukemic Jurkat T, but not in the nontransformed, immortalized human natural killer (NK) cells. Additionally, we screened a library of other N-methylthiolated β-lactams to determine their structure–activity relationships (SARs), and found lactam 12 to have the highest apoptosis-inducing activity against human leukemic Jurkat T cells, associated with increased DNA-damaging potency. Furthermore, we demonstrate that lactam 12, as well as lactam 1, potently inhibits colony formation of human prostate cancer cells. We also show that lactam 12 induces apoptosis in human breast, prostate, and head-and-neck cancer cells. Finally, lactam 12 induces apoptosis selectively in Jurkat T and simian virus 40-transformed, but not in nontransformed NK and parental normal fibroblast, cells. Our results suggest that there is potential for developing this class of β-lactams into novel anticancer agents.

Correlation between induction of DNA fragmentation in urinary bladder cells from rats and humans and tissue-specific carcinogenic activity

Seven chemicals, six of which are known to induce epithelial neoplasms of the urinary bladder in rats, were assayed for their ability to induce DNA damage in primary cultures of rat and human cells from urinary bladder mucosa, and in urinary bladder, liver and kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, were obtained in cells from both rats and humans with the following concentrations of five test compounds: 2-naphthylamine and N-nitrosodi- n-butylamine 0.5 and 1 mM, phenacetin 2 and 4 mM, cyclophosphamide from 2 to 8 mM, and o-toluidine 16 and 32 mM. Nitrilotriacetic acid (1–4 mM), a rat bladder carcinogen, and 4-aminobiphenyl (0.125–0.5 mM), a bladder carcinogen in humans but not in rats, gave a weak positive response in rats cells and a more marked response in humans cells. In terms of DNA-damaging potency, 4-aminobiphenyl, cyclophosphamide, phenacetin and 4 nitrilotriacetic acid were more active in human than in rat cells, whereas the converse occurred with 2-naphthylamine. Consistently with the results observed in vitro statistically significant dose-dependent increases in the average frequency of DNA breaks were detected in the urinary bladder mucosa of rats given p.o. single doses corresponding to 1 4 and 1 2 LD50 of six of the seven test compounds; the only one which gave a substantially negative response was 4-aminobiphenyl. With the exception of N-nitrosodi- n-butylamine which caused DNA damage in liver and of phenacetin and nitrilotriacetic acid which caused damage in kidney in agreement with their tumorigenic activity, any substantial evidence of DNA lesions in these two organs was absent in rats treated with 1 2 LD50 of the other 4 test compounds. These findings give evidence that urinary bladder genotoxic carcinogens may be identified by the DNA damage/Comet assay using as targets cells of urinary bladder mucosa, and show that the effect may be quantitatively different in cells from rats and from human donors.

Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria

AbstractAdenine deoxynucleosides induce apoptosis in quiescent lymphocytes and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. To explain why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis, several mechanisms have been proposed, including the direct binding of dATP to the pro-apoptotic factor Apaf-1 and the activation of the caspase-9 and -3 pathways. In this study it is shown, by means of several assays on whole cells and isolated mitochondria, that 2-chloro-2′-deoxyadenosine (2CdA) and 2-choloro-2′-ara-fluorodeoxyadenosine (CaFdA) disrupt the integrity of mitochondria from primary chronic lymphocytic leukemia (B-CLL) cells. The nucleoside-induced damage leads to the release of the pro-apoptotic mitochondrial proteins cytochrome c and apoptosis-inducing factor. The other adenine deoxynucleosides tested displayed comparable DNA-damaging potency but did not affect mitochondrial function. Interference with mitochondrial integrity, thus, may be a factor in the potent cytotoxic effects of 2CdA and CaFdA toward nondividing lymphocytes.

Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria

Adenine deoxynucleosides induce apoptosis in quiescent lymphocytes and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. To explain why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis, several mechanisms have been proposed, including the direct binding of dATP to the pro-apoptotic factor Apaf-1 and the activation of the caspase-9 and -3 pathways. In this study it is shown, by means of several assays on whole cells and isolated mitochondria, that 2-chloro-2'-deoxyadenosine (2CdA) and 2-choloro-2'-ara-fluorodeoxyadenosine (CaFdA) disrupt the integrity of mitochondria from primary chronic lymphocytic leukemia (B-CLL) cells. The nucleoside-induced damage leads to the release of the pro-apoptotic mitochondrial proteins cytochrome c and apoptosis-inducing factor. The other adenine deoxynucleosides tested displayed comparable DNA-damaging potency but did not affect mitochondrial function. Interference with mitochondrial integrity, thus, may be a factor in the potent cytotoxic effects of 2CdA and CaFdA toward nondividing lymphocytes.

Correlation between Induction of DNA Fragmentation and Micronuclei Formation in Kidney Cells from Rats and Humans and Tissue-Specific Carcinogenic Activity

Five chemicals, known to induce kidney tumors in rats, were assayed for their ability to induce DNA damage and formation of micronuclei in primary cultures of rat and human kidney cells and in the kidney of intact rats. Significant dose-dependent increases of DNA fragmentation, as measured by the Comet assay, and of micronuclei frequency were obtained in primary kidney cells from both rats and humans with the following concentrations of the five test compounds: lead acetate (not tested for micronuclei induction) and potassium bromate from 0.56 to 1.8 mM, phenacetin from 1 to 3.2 mM, and 1,4-dichlorobenzene and nitrilotriacetic acid from 1.8 to 5.6 mM. In terms of DNA-damaging potency all the five chemicals were more active in rat than in human kidney cells, whereas the potencies in inducing micronuclei formation were similar in the two species with the exception of 1,4-dichlorobenzene, which was slightly more potent in human than in rat cells. Consistently with the results observed in vitro, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were detected in the kidney of rats given po a single (12 LD50) or three successive daily doses (13 LD50) of the five test compounds. 4,4′-Methylenedianiline, a carcinogen which does not induce kidney tumors in rats, gave negative responses in both in vitro and in vivo assays. These findings give evidence that kidney carcinogens may be identified by short-term genotoxicity assays using as target kidney cells and show that the five chemicals tested produce in primary cultures of kidney cells from human donors effects similar to those observed in rats.

Dose–response relationships in chemical carcinogenesis: superposition of different mechanisms of action, resulting in linear–nonlinear curves, practical thresholds, J-shapes

The shape of a carcinogen dose–cancer incidence curve is discussed as the result of a superposition of dose–response relationships for various effects of the carcinogen on the process of carcinogenesis. Effects include direct DNA damage, e.g., by covalent binding, indirect DNA damage, e.g., by increased formation of reactive oxygen species or interaction with DNA replication or chromosome integrity. The `fixation' of a DNA adduct as a heritable mutation depends on its pro-mutagenic potency and on the rates of DNA repair and DNA replication. Endogenous and unavoidable DNA damage is responsible for a background rate of the process of mutagenesis and carcinogenesis and forms the basis of spontaneous cancer incidence. For DNA-reactive carcinogens, linearity of the dose response at the low-dose end is expected. With increasing dose, saturation of DNA repair can introduce a sublinearity (example: dimethylnitrosamine). Stimulation of cell division as a result of high-dose toxicity and regenerative proliferation also results in a sublinear deviation from low-dose linearity. If the DNA-damaging potency of the carcinogen is low in comparison with the high-dose effects, the linear part of the low dose–cancer incidence curve might be hidden within the background variability. Under such conditions, `practical thresholds' could be discussed (formaldehyde). If a carcinogen increases the rate of cell division or the level of oxidative stress at high dose but has an antimitogenic or antioxidative effect at low dose, a J-shaped (or: U-shaped) curve with a decrease of the spontaneous tumor incidence at low dose could result (caffeic acid; TCDD). This phenomenon has been observed even under conditions of a genotoxic contribution (ionizing radiation; diesel exhaust particles). For a mechanism-based assessment of a low-dose cancer risk, information on the various modes of action and modulations should be available over the full dose range, and models should be refined to incorporate the respective information.