The role of oxidative and conjugative pathways in the activation of 1,2-dibromo-3-chloropropane to DNA-damaging products in rat testicular cells

The ability of 1,2-dibromo-3-chloropropane (DBCP), several methylated analogs of DBCP and perdeuterated DBCP (DBCP-D5) to cause DNA damage in isolated testicular cells from rats was measured by the alkaline elution technique. Of the methylated analogs studied, only the C3-methyl analog was capable of causing significant DNA damage at concentrations of 0-50 microM. In both time- (0-60 min) and concentration- (0-10 microM) dependent experiments, the testicular cell DNA damage caused by the perdeuterated analog of DBCP closely mimicked the damage resulting from DBCP itself. The lack of an isotope effect between DBCP-D5 and DBCP strongly suggests that metabolism via a cytochrome P-450-dependent pathway is not involved in the DNA-damaging effects of DBCP in rat testicular cells. In contrast, preincubation for 1 hr with diethylmaleate (DEM) inhibited DBCP-induced (10 microM) DNA damage in a concentration-dependent manner (0-500 microM DEM). The decrease in testicular DNA damage was proportional to the decrease in cellular nonprotein sulfhydryl levels. Similarly, it was shown that 1,2-dibromoethane (EDB), a structurally related halogenated alkane, produced DNA damage in isolated testicular cells in both a time- (0-60 min) and concentration- (0-600 microM) dependent fashion. The DNA damage produced by EDB (600 microM) was also inhibited by pretreatment of testicular cells with DEM (1 mM). The testicular genotoxicity induced by EDB is thought to involve its initial conjugation to glutathione and the subsequent formation of a reactive episulfonium ion. The data presented indicate that similar events may be occurring in DBCP-induced DNA damage in rat testicular cells.     

Cellular distribution of cytochrome P-450 loss in rats of different ages treated with alkyl halides

Challenge of male rats with a single dose of the alkyl halides, 1,2-dibromo-3-chloropropane (DBCP), ethylene dibromide (EDB), carbon tetrachloride (CCl₄), and epichlorohydrin (EPI), resulted in significant decreases in cytochrome P-450 in microsomes isolated from liver, kidney, testis, lung, and small intestinal mucosa 48 hr after treatment. Treatment with CCl₄, but not DBCP, EDB, or EPI, was characterized by rapid loss of cytochrome P-450, detectable within 4 hr. Evidence of lipid peroxidation was found only in hepatic microsomes from rats treated with CCl₄, but not in hepatic or extrahepatic microsomes from rats treated with other compounds. In liver tissue, treatment with DBCP and CCl₄ resulted in a decrease in cytochrome P-450 in both rough and smooth microsomal fractions and nuclei, but not in mitochondrial fractions. Mixed-function oxidase (MFO) activities in hepatic microsomes decreased in parallel with cytochrome P-450 content after treatment with DBCP and EDB. In microsomes and nuclei after treatment with CCl₄ and in nuclei after treatment with DBCP, however, the response of the MFO depended on the substrate tested. Microsomal cytochrome P-450, which is susceptible to proteolytic cleavage, and microsomal and nuclear cytochrome P-450, which increased with maturation and decreased with aging of the rat, appeared to be the most responsive of the forms of cytochrome P-450 to alkyl halide treatment. These results suggest that treatment with alkyl halides may preferentially affect specific isozymes of cytochrome P-450.     

A comparative study of chemically induced DNA damage in isolated human and rat testicular cells

Testicular cells prepared from human organ transplant donors or from Wistar rats were used to compare 15 known reproductive toxicants with respect to their ability to induce DNA damage, measured as single-strand DNA breaks and alkali labile sites (ssDNA breaks) with alkaline filter elution. The compounds tested included various categories of chemicals (i.e., pesticides, industrial chemicals, cytostatics, and mycotoxins) most of which are directly acting genotoxicants (i.e., reacting with DNA either spontaneously or via metabolic activation). In addition, a few indirect genotoxic and nongenotoxic reproductive toxicants were included. Six of the chemicals induced no significant levels of ssDNA breaks in human and rat testicular cells: methoxychlor (10 to 100 μM, human and rat), benomyl (10 to 100 μM, human and rat), thiotepa (10 to 1000 μM, human and rat), cisplatin (30 to 1000 μM, human; 100 to 1000 μM, rat), Cd²⁺ (30 to 1000 μM, human; 100 to 1000 μM, rat), and acrylonitrile (30 to 1000 μM, human; 30 to 300 μM, rat). Four chemicals induced significant levels of ssDNA breaks in testicular cells from both species: styrene oxide (≥ 100 μM, rat and human), 1,2-dibromoethane (EDB) (≥ 100 μM, rat; 1000 μM human), thiram (≥ 30 μM, rat; ≥ 100 μM, human), and chlordecone (300 μM, rat; ≥ 300 μM, human). Finally, five chemicals induced ssDNA breaks in one of the two species. Four chemicals induced significant ssDNA breaks in rat testicular cells only: 1,2-dibromo-3-chloropropane (DBCP) (≥ 10 μM), 1,3-dinitrobenzene (1,3-DNB) (≥ 300 μM), Cr⁶⁺ (1000 μM), and aflatoxin B₁ (≥ 100 μM), the last two of these produced only a minor positive response. One chemical, acrylamide, induced a marginal increase in ssDNA breaks in human at 1000 μM, but not in rat testicular cells. Although based on a limited number of donors, the data indicate a close correlation between the induction of DNA damage in human and rat testicular cells in vitro. For some chemicals, however, there appears to be differences in the susceptibility to chemically induced ssDNA breaks of isolated testicular cells from the two species. The data indicate that the parallel use of human and rat testicular cells provides a valuable tool in the assessment of human testicular toxicity.     

Detection of xenobiotic-induced DNA damage by the comet assay applied to human and rat precision-cut liver slices.

The comet assay is a simple and sensitive method for measuring DNA damage at the level of individual cells and is extensively used in genotoxicity studies. It is commonly applied to cultured cells. The aim of this study was to apply the comet assay for use in fresh liver tissue, where metabolic activity, all cell types and tissue architecture are preserved. The response of liver slices to genotoxic agents was tested with the reactive oxygen species generating tert-butyl hydroperoxide (tBOOH, 0.1-2 mM), [corrected] and the pro-carcinogens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ, 0.5-2 mM) and benzo(a)pyrene (BaP, 10-100 microM). Dose-dependent DNA damage was observed and compared to HepG2 cells. At non-cytotoxic concentrations of carcinogens, human liver slices were more sensitive to tBOOH than rat liver slices, while no significant difference was found for BaP and IQ. Human liver slices were more sensitive to IQ than HepG2 cells, equally sensitive to BaP and less to tBOOH. Control slices showed low levels of DNA damage, which did not increase during 24 h preservation (0 degrees C) or 48 h culturing (37 degrees C). In conclusion, the comet assay that we applied for measuring DNA damage in precision-cut liver slices is an useful tool to study genotoxic effects induced by various potential genotoxicants, allowing for detection of species differences in susceptibility to carcinogens.     

Species differences in testicular necrosis and DNA damage, distribution and metabolism of 1,2-dibromo-3-chloropropane (DBCP)

The human testicular toxicant 1,2-dibromo-3-chloropropane (DBCP) was studied for the same end-point in 4 different species of laboratory animals. Marked necrosis and atrophy of the seminiferous epithelium were observed in rats and guinea pigs 10 days after a single i.p. administration of DBCP (170-340 mumol/kg), whereas significantly less damage was observed in hamsters and mice. The testicular concentrations of DBCP measured at various time-points after the i.p. injection of DBCP indicated that factors in addition to tissue concentration were of importance for the observed species differences in sensitivity towards DBCP-induced testicular damage. Also, there did not seem to be any direct correlation between DBCP-induced in vivo testicular toxicity and in vitro GSH-dependent dehalogenation, inasmuch as the rate of bromide release from DBCP with hamster testicular cytosol was as fast as that with rat cytosol. Testicular DNA damage, as determined by alkaline elution 60 min after in vivo administration of 170 mumol/kg DBCP, was observed only in rats and guinea pigs. Thus, induction of DNA damage correlates with the relative susceptibilities of the species towards DBCP-induced testicular necrosis. To further study species differences in testicular activation of DBCP to DNA-damaging intermediate(s), cells isolated from the testes of the 4 species were incubated with DBCP. Testicular cells from rats and guinea pigs were the only preparations developing substantial DNA damage after 60 min incubation with low concentrations of DBCP (5-50 microM). The findings indicate that rats are sensitive towards DBCP-induced testicular necrosis because rat testicular cells easily activate DBCP to a DNA-damaging intermediate(s). The relative high testicular DBCP concentration as well as the ability to activate DBCP may explain the sensitivity of guinea pigs towards DBCP-induced testicular toxicity.     

Analysis of nicotine-induced DNA damage in cells of the human respiratory tract

Epithelium of the upper and lower airways is a common origin of tobacco-related cancer. The main tobacco alkaloid nicotine may be associated with tumor progression. The potential of nicotine in inducing DNA mutations as a step towards cancer initiation is still controversially discussed. Different subtypes of nicotinic acetylcholine receptors (nAChR) are expressed in human nasal mucosa and a human bronchial cell line representing respiratory mucosa as a possible target for receptor-mediated pathways. In the present study, both cell systems were investigated with respect to DNA damage induced by nicotine and its mechanisms.Specimens of human nasal mucosa were harvested during surgery of the nasal air passage. After enzymatic digestion over night, single cells were exposed to an increasing nicotine concentration between 0.001 mM and 4.0 mM. In a second step co-incubation was performed using the antioxidant N-acetylcysteine (NAC) and the nAChR antagonist mecamylamine. DNA damage was assessed using the alkali version of the comet assay. Dose finding experiments for mecamylamine to evaluate the maximal inhibitory effect were performed in the human bronchial cell line BEAS-2B with an increasing mecamylamine concentration and a constant nicotine concentration. The influence of nicotine in the apoptotic pathway was evaluated in BEAS-2B cells with the TUNEL assay combined with flow cytometry.After 1 h of nicotine exposure with 0.001, 0.01, 0.1, 1.0 and 4.0 mM, significant DNA damage was determined at 1.0 mM. Further co-incubation experiments with mecamylamine and NAC were performed using 1.0 mM of nicotine. The strongest inhibitory effect was measured at 1.0 mM mecamylamine and this concentration was used for co-incubation. Both, the antioxidant NAC at a concentration of 1.0 mM, based on the literature, as well as the receptor antagonist were capable of complete inhibition of the nicotine-induced DNA migration in the comet assay. A nicotine-induced increase or decrease in apoptosis as assessed by the TUNEL assay in BEAS-2B could not be detected.These results support the hypothesis that oxidative stress is responsible for nicotine-induced DNA damage. Similar results exist for other antioxidants in different cell systems. The decrease in DNA damage after co-incubation with a nAChR antagonist indicates a receptor-dependent pathway of induction for oxidative stress. Further investigations concerning pathways of receptor-mediated DNA damage via nAChR, the role of reactive oxygen species and apoptosis in this cell system will elucidate underlying mechanisms.     

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 14 and 12 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 12 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.     

Respiratory pathology in rats and mice after inhalation of 1,2-dibromo-3-chloropropane or 1,2 dibromoethane for 13 weeks

Seventy F344 rats and 144 B6C3F1 mice were subdivided into seven groups. Three groups were each exposed via inhalation to 1, 5, or 25 ppm of 1,2-dibromo-3-chloropropane (DBCP) for 6 h per day, 5 days per week for 13 weeks. Three additional groups were each similarly exposed to 3, 15, or 75 ppm of 1,2-Dibromoethane (EDB). The remaining group was exposed to room air under the same conditions. At 13 weeks, rats and mice showed severe necrosis and atrophy of the olfactory epithelium in the nasal cavity after inhalation of 5 or 25 ppm DBCP and 75 ppm EDB. Lower concentrations induced squamous cell metaplasia, hyperplasia and cytomegaly of the epithelium of the respiratory nasal turbinals. Squamous metaplasia, hyperplasia and cytomegaly of the epithelium was also seen in larynx, trachea, bronchi and bronchioles. Other compound related toxic lesions in rats were seen in the liver, kidney and testes.     

Depletion by 1,2-dibromoethane, 1,2-dibromo-3-chloropropane, tris(2,3-dibromopropyl)phosphate, and hexachloro-1,3-butadiene of reduced non-protein sulfhydryl groups in target and non-target organs

the abilities of several aliphatic organohalide compounds to deplete reduced non-protein sulfhydryl (NPS)concentrations in rodent kidney, liver, lung, stomach, and testis were measured. the sites of NPS loss were compared to the sites of tissue injury as documented in previous reports. Single intraperitoneal (i.p.) injections of 1,2-dibromo-3-chloropropane (DBCP), 1,2-dibromoethane (ethylene dibromide, EDB), or hexachloro-1,3-butadiene (HCBD) decreased hepatic and renal NPS concentrations in mice in a dose-related manner. Small NPS losses were produced in lung, testis and stomach, but oniy at high organohalide doses. Administration of tris(2,3-dibromopropyl)phosphate (TRIS) decreased NPS content in liver only. Pretreatments with the enzyme inducer polybrominated biphenyls (PBB) or the enzyme inhibitor piperonyl butoxide quantitatively decreased the renal and hepatic NPS-depleting actions of DBCP or EDB in vivo, but the enzyme inhibitor β-diethylaminoethyl diphenylpropylacetate (SKF 525-A) was without demonstrable effect. the acute, i.p. LD₅₀ value of DBCP was increased by prior treatment of mice with PBB. the LD₅₀ values of DBCP and EDB, however, were unaffected by prior treatment with piperonyl butoxide. DBCP or EDB enhanced the loss of reduced NPS groups from rat hepatic homogenates in vitro; the reaction was time dependent and appeared to be enzyme mediated. Although the primary target organs for DBCP or TRIS toxicity are reported to be the kidney and the testis, the major site of NPS depletion was found to be the liver. Also, HCBD depleted renal NPS in mice only (not rats), though HCBD is nephrotoxic in rats as well as in mice. the correlations between tissue depletion of NPS and sites of organohalide injury, therefore, were poor.     

Deuterium isotope effect on the metabolism and toxicity of 1,2-dibromoethane

The metabolism, hepatotoxicity, and hepatic DNA damage of 1,2-dibromoethane (EDB) and tetradeutero-1,2-dibromoethane (d₄EDB) were compared in male Swiss-Webster mice. In vitro studies that measured bromide ion released from the substrate to monitor the rate of metabolism showed that the hepatic microsomal metabolism of EDB was significantly reduced by deuterium substitution, while metabolism by the hepatic glutathione S-transferases was unaffected. Three hours after ip administration of EDB or d₄EDB (50 mg/kg), there was 42% less bromide in the plasma of d₄EDB-treated mice than in the plasm of EDB-treated mice. This difference demonstrates a significant deuterium isotope effect on the metabolism of EDB in vivo. Although the metabolism of d₄EDB was less than that of EDB 3 hr after exposure, the DNA damage caused by both analogs was not significantly different at this time point. At later time points (8, 24, and 72 hr), d₄EDB caused significantly greater DNA damage than EDB. Since the decreased metabolism of d₄EDB was apparently due to a reduced rate of microsomal oxidation, these data support the hypothesis that conjugation with GSH is responsible for the genotoxic effects of EDB.     

Vanadyl sulfate can differentially damage DNA in human lymphocytes and HeLa cells

Using the comet assay, we showed that vanadyl sulfate induced DNA damage in human normal lymphocytes and in HeLa cells. Vanadyl at 0.5 and 1 mM produced DNA single- and double-strand breaks (SSBs and DSBs) in lymphocytes, whereas in HeLa cells we observed only SSBs. Post-treatment of vanadyl-damaged DNA from lymphocytes with formamidopyrimidine-DNA glycosylase (Fpg), an enzyme recognizing oxidized purines, gave rise to a significant increase in the extent of DNA damage. A similar effect was observed in HeLa cells, but, using endonuclease III, we also detected oxidized pyrimidines in DNA of these cells. There were no differences in the extent of DNA damage in the lymphocytes and HeLa cells in the pH >13 and pH 12.1 conditions of the comet assay, which indicates that strand breaks, and not alkali-labile sites, contributed to the measured DNA damage. Study of DNA repair, determined in the comet assay as an ability of cells to decrease of DNA damage, revealed that HeLa cells retained the ability to repair vanadyl-damaged DNA induced at a ten-fold higher concentration than that in lymphocytes. Incubation of the cells with nitrone spin traps DMPO, POBN and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by vanadyl sulfate. The presence of vitamins A, C or E caused an increase of DNA damage in HeLa cells whereas in lymphocytes such an increase was observed only for vitamin C. Our data indicate that vanadyl sulfate can be genotoxic for normal and cancer cells. It seems to have a higher genotoxic potential for cancer cells than for normal lymphocytes. Vitamins A, C and E can increase this potential.     

Bisphenol A-glycidyl methacrylate induces a broad spectrum of DNA damage in human lymphocytes

Bisphenol A-glycidyl methacrylate (BisGMA) is monomer of dental filling composites, which can be released from these materials and cause adverse biologic effects in human cells. In the present work, we investigated genotoxic effect of BisGMA on human lymphocytes and human acute lymphoblastic leukemia cell line (CCRF-CEM) cells. Our results indicate that BisGMA is genotoxic for human lymphocytes. The compound induced DNA damage evaluated by the alkaline, neutral, and pH 12.1 version of the comet assay. This damage included oxidative modifications of the DNA bases, as checked by DNA repair enzymes EndoIII and Fpg, alkali-labile sites and DNA double-strand breaks. BisGMA induced DNA-strand breaks in the isolated plasmid. Lymphocytes incubated with BisGMA at 1 mM were able to remove about 50% of DNA damage during 120-min repair incubation. The monomer at 1 mM evoked a delay of the cell cycle in the S phase in CCRF-CEM cells. The experiment with spin trap—DMPO demonstrated that BisGMA induced reactive oxygen species, which were able to damage DNA. BisGMA is able to induce a broad spectrum of DNA damage including severe DNA double-strand breaks, which can be responsible for a delay of the cell cycle in the S phase.     

Lack of genotoxicity of potassium iodate in the alkaline comet assay and in the cytokinesis-block micronucleus test. Comparison to potassium bromate.

Iodine could be added to the diet of human population in the form of iodide or iodate but iodate had not been adequately tested for genotoxicity and carcinogenicity. In the present study, genotoxic effects of potassium iodate were evaluated in vitro using the alkaline comet assay and the cytokinesis-block micronucleus assay on CHO cells and compared to halogenate salt analogues potassium bromate and chlorate and also to their respective reduced forms (potassium iodide, bromide and chloride). The results showed that the comet assay failed to detect the presence of DNA damage after a treatment of cells by potassium iodate for concentrations up to 10 mM. This absence of primary DNA damage was confirmed in the cytokinesis-block micronucleus assay. In the same way, results showed that potassium chlorate as well as potassium iodide, bromide and chloride did not induced DNA damage in the alkaline comet assay for doses up to 10 mM. By contrast, potassium bromate exposure led to an increase in both DNA damage and frequency of micronucleated cells. The repair of bromate-induced DNA damage was incomplete 24 h after the end of treatment. These results seem to indicate that potassium bromate would induce DNA damage by several mechanisms besides oxidative stress.     

Genotoxic damage in the oral mucosa cells of subjects carrying restorative dental fillings

A large proportion of the population carries restorative dental fillings containing either classic Hg-based amalgams and/or the more frequently used methacrylates. Both Hg- and resin-based materials have been shown to be released into the buccal cavity and to be spread systemically. In addition, they induce toxic and genotoxic alterations in experimental test systems. Using the comet assay, we previously demonstrated that circulating lymphocytes of subjects with dental fillings have an increased DNA damage. Here, we analyzed the oral mucosa cells of 63 young subjects of both genders, by using both the comet assay and the micronucleus (MN) test and by monitoring cell death markers. The results obtained show that both amalgams and resin-based composite fillings can induce genotoxic damage in human oral mucosa cells, as convincingly and dose-dependently inferred from the results of the MN test and, more marginally, from comet assay data. Lifestyle variables, also including alcohol intake and smoking habits, did not affect the genotoxic response and did not act as confounding factors. Thus, we provide unequivocal evidence for the genotoxicity of both amalgams and resin-based dental fillings in humans not only by testing circulating lymphocytes but also by analyzing oral mucosa cells. These findings are of particular relevance due to the circumstance that subjects with restorative materials are exposed continuously and for long periods of time.     

Effects of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites on DNA damage and repair under in vitro conditions

3-monochloropropane-1,2-diol (3-MCPD) is a food contaminant that occurs during industrial production processes and can be found mainly in fat and salt containing products. 3-MCPD has exhibited mutagenic activity in vitro but not in vivo, however, a genotoxic mechanism for the occurrence of kidney tumors has not so far been excluded. The main pathway of mammalian 3-MCPD metabolism is via the formation of β – chlorolactatic acid and formation of glycidol has been demonstrated in bacterial metabolism. The aim of this study was to investigate genotoxic and oxidative DNA damaging effects of 3-MCPD and its metabolites, and to provide a better understanding of their roles in DNA repair processes. DNA damage was assessed by alkaline comet assay in target rat kidney epithelial cell lines (NRK-52E) and human embryonic kidney cells (HEK-293). Purine and pyrimidine base damage, H₂O₂ sensitivity and DNA repair capacity were assessed via modified comet assay. The results revealed in vitro evidence for increased genotoxicity and H₂O₂ sensitivity. No association was found between oxidative DNA damage and DNA repair capacity with the exception of glycidol treatment at 20 μg/mL. These findings provide further insights into the mechanisms underlying the in vitro genotoxic potential of 3-MCPD and metabolites.     

Testicular necrosis and DNA damage caused by deuterated and methylated analogs of 1,2-dibromo-3-chloropropane in the rat

To study the role of metabolism in 1,2-dibromo-3-chloropropane (DBCP)-induced testicular damage in rats, selectively deuterated and methylated analogs of DBCP were given as a single ip dose of 340 mumol/kg and testicular toxicity was determined 10 days after treatment. None of the four deuterated analogs C1-D2-, C2-D1-, C3-D2-, or C1-C2-C3-D5-DBCP reduced the degree of testicular damage compared to DBCP, indicating that metabolic cleavage of a C-H bond was not rate-limiting in DBCP-induced testicular toxicity. Of the five methylated analogs, C1-methyl-, C1-dimethyl-, C2-methyl-, and C3-methyl-DBCP and 1,2-dibromo-4-chlorobutane, only C3-methyl-DBCP caused testicular toxicity. DBCP treatment resulted in increased testicular DNA damage at doses of 85-170 mumol/kg as measured by alkaline elution of DNA from testicular cells isolated 3 hr after in vivo treatment. The perdeutero-DBCP analog induced testicular DNA damage that was at least as extensive as that induced by DBCP. Of the methylated analogs tested, only C3-methyl-DBCP gave a marked dose-dependent increase in testicular DNA damage between 170 and 540 mumol/kg. There were no significant differences in the testicular tissue distribution between DBCP, perdeutero-DBCP, and the methylated DBCP analogs. Furthermore, in distribution studies with DBCP, C1-methyl- and C3-methyl-DBCP, and 1,2-dibromo-4-chlorobutane, the highest tissue concentrations were found in the kidneys, followed by the liver and then the testes. The fact that testicular DNA damage of DBCP and its deuterated and methylated analogs paralleled their ability to cause testicular necrosis and atrophy makes measurement of DNA damage a very useful correlate in mechanistic studies of DBCP-induced testicular cell death.     

Evaluation of the DNA damaging effect of the heat-induced food toxicant 5-hydroxymethylfurfural (HMF) in various cell lines with different activities of sulfotransferases

5-Hydroxymethylfurfural (HMF), a heat-induced food toxicant present in a vast number of food items, has been suggested to be genotoxic after being bioactivated by the sulfotransferase SULT1A1. The comet assay was used to evaluate the DNA damaging effect of HMF in cell lines with different activities of SULT1A1: two human cell lines (Caco-2, low activity; and HEK293, higher activity), one cell line from mouse (L5178Y, no activity) and two cell lines from Chinese hamster (V79, negligible activity; and V79-hP-PST, high activity of human SULT1A1). HMF induced significant DNA damage in all cell lines after 3 h exposure to 100 mM. Most sensitive were V79 and V79-hP-PST where HMF induced significant DNA damage at 25 mM. Consequently, in the present study we have shown that HMF is a DNA damaging agent in vitro independent of the activity of SULT1A1 in the cells. The HMF-induced DNA damage was only observed at rather high concentrations which usually was associated with a concomitant decrease in cell viability.     

Carboxylic acid drug-induced DNA nicking in HEK293 cells expressing human UDP-glucuronosyltransferases: role of acyl glucuronide metabolites and glycation pathways

Glucuronidation of carboxylic-acid-containing drugs can yield reactive acyl (ester-linked) glucuronide metabolites that are able to modify endogenous macromolecules. Previous research has shown that several carboxylic acid drugs are genotoxic in isolated mouse hepatocytes, and that DNA damage is prevented by the glucuronidation inhibitor, borneol. Whether these species induce comparable genetic damage in human cells is unknown. In this study, we investigated the mechanisms of clofibric acid-induced genotoxicity in HEK293 cells expressing the human UDP-glucuronosyltransferases UGT1A3, UGT1A9, or UGT2B7, and screened three other carboxylic acid drugs for UGT-dependent genotoxicity. DNA damage was detected using the alkaline version of the comet assay. HEK293 cells were incubated for 18 h with vehicle (2.5 mM NaOH), 0.1-2.5 mM clofibric acid or 0.1-1.0 mM benoxaprofen, bezafibrate, or probenecid. To identify mechanisms underlying any observed genotoxicity, we treated UGT2B7 transfectants with 10 mM aminoguanidine, 1 mM borneol, or 2 mM desferrioxamine mesylate prior to co-incubation with 1 mM clofibric acid for 18 h. Compared to vehicle, clofibric acid, benoxaprofen, and probenecid produced significant DNA damage in all three UGT-transfected HEK293 cell lines, detectable from the lowest concentration tested. Bezafibrate caused DNA damage only at higher concentrations (1.0 mM) in UGT2B7- and UGT1A9-, but not UGT1A3-transfected cells. No drug-induced DNA damage was detected in untransfected cells, consistent with the limited glucuronidation capacity of these cells. The glycation/glycoxidation inhibitor aminoguanidine and the glucuronidation inhibitor borneol significantly decreased clofibric-acid-mediated DNA damage in UGT2B7 transfected cells by 73.5 and 94.8%, respectively. The inhibitor of transition-metal-catalyzed oxidation, desferrioxamine mesylate, had no significant effect on DNA damage. This study demonstrates the substrate-dependent role of human UGTs in the bioactivation of carboxylic acid drugs to genotoxic acyl glucuronide metabolites that are able to damage nuclear DNA via glycation and/or glycoxidation mechanisms.     

Endogenous antioxidants and nasal human epithelium response to air pollutants: genotoxic and inmmuno-cytochemical evaluation

Nasal epithelium is a source for identifying atmospheric pollution impact. Antioxidants play a relevant role in the protection of the cells from environmental injury, but scarce information is available about the interaction of endogenous antioxidants and genotoxic damage in nasal epithelium from urban populations highly exposed to traffic-generated air pollutants. An immunocytochemical and genotoxic evaluation was implemented in nasal cell epithelium in a population chronically exposed to atmospheric pollution from autumn 2004 to autumn 2005. Superoxide dismutase (SOD) and Catalase (CAT) were evaluated in nasal scrapings by morphometry and genotoxicity by comet assay. An increase in DNA damage correlates with a decrease in SOD and CAT in nasal cells during autumn and the inverse result was observed during summer (R = 0.88). Not only should exogenous antioxidant supplements be encouraged, but also a healthy diet to strengthen intracellular defenses against oxidative stress induced by exposure to air pollutants.