Formation of tamoxifen-DNA adducts via O-sulfonation, not O-acetylation, of alpha-hydroxytamoxifen in rat and human livers.

Tamoxifen (TAM) is used as the standard endocrine therapy for breast cancer patients and as a chemopreventive agent for women at high risk for this disease. Unfortunately, treatment of TAM increases the incidence of endometrial cancer; this may be due to the genotoxic damage induced by TAM metabolites. Formation of TAM-DNA adducts in rat liver correlates with the development of hepatocarcinoma. TAM-DNA adducts are proposed to be formed through O-sulfonation and/or O-acetylation of alpha-hydroxylated TAM and its metabolites. However, the role of O-sulfonation and O-acetylation in the formation of TAM-DNA adducts has not been extensively investigated. Rat or human hydroxysteroid sulfotransferases (HST), acetyltransferases, and liver cytosol were incubated with calf thymus DNA, alpha-OHTAM, and either 3'-phosphoadenosine 5'-phosphosulfate (PAPS) or acetyl coenzyme A (acetyl-CoA) as a cofactor and analyzed for TAM-DNA adduct formation, using 32P postlableling/polyacrylamide gel electrophoresis analysis. TAM-DNA adduct was formed when PAPS, not acetyl-CoA, was used. No TAM-DNA adducts were produced using human N-acetyltransferase I and II. HST antibody inhibited approximately 90% of TAM-DNA adduct formation generated by the cytosol or HST, suggesting that HST is primarily involved in the formation of TAM-DNA adducts. The formation of TAM-DNA adducts with rat liver cytosol and HST was much higher than that of human liver cytosol and HST. Our results indicate that TAM-DNA adducts are formed via O-sulfonation, not O-acetylation, of alpha-hydroxylated TAM and its metabolites.     

Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD.

Endometriosis is a debilitating disease found in 10-15% of reproductive-age women and is characterized by the presence of endometrial tissue outside of the uterus. The present study characterizes the expression of AhR and ARNT mRNA in a human endometrial explant culture model in the absence and presence of TCDD exposure. In a parallel, companion study using this model, TCDD exposure was shown to induce CYP1A1 mRNA, CYP1B1 mRNA, EROD (7-ethoxyresorufin-O-deethylase) activity, and CYP1B1 protein in human endometrial explants. Explants were prepared from specimens obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. These specimens were a subset of the specimens used in the parallel study. The explants were cultured in medium containing 10 nM estradiol (E(2)) or 1 nM estradiol plus 500 nM progesterone (E(2) + P(4)) with or without TCDD (first 24 h). After culture, AhR and ARNT mRNA expression were quantified by RT-PCR. TCDD treatment significantly increased the expression of AhR mRNA, but not ARNT mRNA. The expression of both genes was similar for all individual explants and the ratio of AhR:ARNT mRNA expression across all samples was 1.7 to 1.8. Constitutive AhR mRNA expression was donor age dependent (increasing with age), while ARNT mRNA expression was donor age and tissue phase dependent (increased in older and proliferative phase specimens). Similar to results in the parallel study on expression of CYP1A1 mRNA, CYP1B1 mRNA, EROD activity, and CYP1B1 protein, the presence of endometriosis did not affect the expression of AhR or ARNT mRNA, either constitutively or following TCDD exposure. However, the detection of disease-specific change was limited by small sample size and variability in tissue cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase, hormonal exposure, and donor age.     

Identification of tamoxifen-DNA adducts induced by alpha-acetoxy-N-desmethyltamoxifen

Treatment with tamoxifen increased the risk of endometrial cancers in breast cancer patients and women participating in the chemoprevention study. In our laboratory, tamoxifen-DNA adducts, including alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM), were detected in the endometrium of women taking tamoxifen [Shibutani, S., et al. (1999) Chem. Res. Toxicol. 12, 646-653]. On the basis of recent animal studies, deoxyguanosinyl-N-desmethyltamoxifen (dG-N-desmethylTAM) adducts are also suspected to be formed in the liver. In the study presented here, we synthesized alpha-acetoxy-N-desmethyltamoxifen as a model activated metabolite of N-desmethyltamoxifen. The overall yield of alpha-acetoxy-N-desmethyltamoxifen from alpha-hydroxytamoxifen was approximately 42%. alpha-Acetoxy-N-desmethyltamoxifen was highly reactive to 2'-deoxyguanosine, as was similarly observed for tamoxifen alpha-sulfate. The two reaction products were identified as a mixture of epimers of the trans form or cis form of alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG-N(2)-N-desmethylTAM) by mass and proton magnetic resonance spectroscopy. In addition, the trans and cis forms of dG 3'-monophosphate-N(2)-N-desmethylTAM were prepared as standard markers for (32)P-postlabeling/HPLC analysis. Using this technique, dG-N(2)-N-desmethylTAM adducts were detected in calf thymus DNA reacted with alpha-acetoxy-N-desmethyltamoxifen.     

Inefficient repair of tamoxifen-DNA adducts in rats and mice.

A long-term treatment with tamoxifen (TAM) to women increases the risk of developing endometrial cancer. The cancer may result from genotoxic damage induced by this drug. In fact, TAM-DNA adducts were detected in the liver of rats treated with TAM and initiated to develop hepatocellular carcinomas. To explore the distribution and repair rate of TAM-DNA adducts, the level of TAM-DNA adducts in all tissues of rats and mice was monitored for 28 days and 7 days, respectively, after the termination of TAM treatment, using 32P-postlabeling/polyacrylamide gel electrophoresis and 32P-postlabeling/HPLC analyses. TAM-DNA adducts were formed specifically in the liver of rodents. In rats, the level of hepatic TAM-DNA adducts was decreased only to 43% in 28 days, indicating that the half-life of adducts was approximately 25 days. Among trans [fraction (fr)-1 and fr-2]- and cis (fr-3 and fr-4)-isoforms of TAM-DNA adducts, a trans-form (fr-1) was removed much more slowly than other adducts, indicating that the repair rate of TAM-DNA adducts varied depending on the structure of isoforms. The repair rate of TAM-DNA adducts was also compared between nucleotide excision repair-deficient (Xpc knockout) and wild mice. Although the level of hepatic TAM-DNA adducts observed with Xpc knockout mice was slightly higher than that of the wild type, the removal of TAM-DNA adducts in both mice was only 20% in 7 days. Thus, TAM-DNA adducts are not efficiently repaired from the targeted tissue, leading to the development of cancer.     

Formation and removal of DNA adducts in Fischer-344 rats exposed to 2,4-diaminotoluene

  ³²P-Postlabeling was used to examine DNA adduct formation and removal in Fischer-344 rats exposed to the animal carcinogen 2,4-diaminotoluene (DAT). Adduct formation and persistence were compared between target (liver and mammary gland) and non-target organs (kidney and lung) to determine if possible differences could explain the observed organ specificity of DAT induced carcinogenesis. The effects of different exposure conditions on DNA adduct formation and removal were also examined by varying the concentration and frequency of compound administration. DAT produced three distinct DNA adducts. Among the organs examined, DNA binding was highest in the liver, with levels approximately 10 times greater than that of the mammary gland and up to 50 times greater than of the two nontarget sites. Despite the large differences in the initial extent of adduct formation, the persistence of adducts among sites was not significantly different. In the liver, there were dose-dependent differences in DNA adduct formation, but adduct removal following different dosages did not vary significantly. The effects of multiple administration on DNA adduct formation and removal were examined by treating rats with 5 mg/kg DAT daily for 10 consecutive days. Adduct yields from multiple treatment were greater than from a single 50 mg/kg exposure. The persistence of adducts following multiple treatment was also greater than after an equivalent single exposure. The results demonstrated organ-specific and dose-dependent differences in initial extent of DNA adduct formation, but no differences in adduct persistence. However, the results did suggest that adduct formation and persistence may change with repeated administration of DAT. Received: 13 April 1994 / Accepted: 4 May 1994     

Formation of hydroxymethyl DNA adducts in rats orally exposed to stable isotope labeled methanol

Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol's well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD?]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD?]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account.     

Resolution of alpha-hydroxytamoxifen; R-isomer forms more DNA adducts in rat liver cells.

The genotoxic tamoxifen metabolite alpha-hydroxytamoxifen has been resolved into R- and S-enantiomers. This was achieved by preparing its ester with S-camphanic acid, chromatographic separation into two diastereoisomers, and hydrolysis to give (+)- and (-)-alpha-hydroxytamoxifen. The configuration of the (-)-isomer was shown to be S- by degradation of an ester to a derivative of (-)-2-hydroxy-1-phenyl-1-propanone, which has already been shown to have S-configuration. Metabolism of tamoxifen by rat liver microsomes gave equal amounts of the two enantiomers. They have the same chemical properties but, on treatment of rat hepatocytes in culture, R-(+)-alpha-hydroxytamoxifen gave at least eight times as many DNA adducts as the S-(-)-isomer.     

Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.     

Formation of 1,2:3,4-diepoxybutane-specific hemoglobin adducts in 1,3-butadiene exposed workers

1,3-Butadiene (BD) is an important industrial chemical that is classified as a human carcinogen. BD carcinogenicity has been attributed to its metabolism to several reactive epoxide metabolites and formation of the highly mutagenic 1,2:3,4-diepoxybutane (DEB) has been hypothesized to drive mutagenesis and carcinogenesis at exposures experienced in humans. We report herein the formation of DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in BD-exposed workers as a biomarker of DEB formation. pyr-Val was determined in BD monomer and polymer plant workers that had been previously analyzed for several other biomarkers of exposure and effect. pyr-Val was detected in 68 of 81 (84%) samples ranging from 0.08 to 0.86 pmol/g globin. Surprisingly, pyr-Val was observed in 19 of 23 administrative control subjects not known to be exposed to BD, suggesting exposure from environmental sources of BD. The mean ± SD amounts of pyr-Val were 0.11 ± 0.07, 0.16 ± 0.12, and 0.29 ± 0.20 pmol/g globin in the controls, monomer, and polymer workers, respectively, clearly demonstrating formation of DEB in humans. The amounts of pyr-Val found in this study suggest that humans are much less efficient in the formation of DEB than mice or rats at similar exposures. Formation of pyr-Val was more than 50-fold lower than has been associated with increased mutagenesis in rodents. The results further suggest that formation of DEB relative to other epoxides is significantly different in the highest exposed polymer workers compared with controls and BD monomer workers. Whether this is due to saturation of metabolic formation or increased GST-mediated detoxification could not be determined.     

Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothane.

The anesthetic halothane can be bioactivated to the reactive intermediate, trifluoroacetyl chloride, which can covalently bind to liver protein. The product of this reaction is trifluoroacetyl-N-epsilon-lysine which can act as a foreign epitope in altering both protein immunogenicity and antigenicity. An in vitro liver slice system was used to study the formation of protein adducts following exposure to halothane. Liver slices (30-35 mg wet weight, 250-300 microns thick) from adult male Hartley guinea pigs (600-800 g) were exposed to [14C]halothane (0.6-0.9 microCi, 1.0-1.7 mM) in 95% O2/5% CO2 for 1, 6 and 12 h. The slices were homogenized and subcellular fractions prepared. Proteins were resolved by electrophoresis and bound radioactivity was detected by scintillation counting and autoradiography. Greater than 80% of detectable radioactivity to whole liver cell protein was localized in the 20-30-kDa range and increased in a linear fashion over the 12-h incubation period. Covalent binding was localized to two proteins of 27 kDa and 26 kDa present in the cytosolic compartment. Purification followed by N-terminal amino acid sequence analysis of the 27-kDa protein has identified it to be homologous with glutathione-S-transferase b. This cytosolic protein appears to be the major target for trifluoroacetylation in liver slices exposed to halothane.     

Hemoglobin adducts of epoxybutene in workers occupationally exposed to 1,3-butadiene

1,3-Butadiene (BD) is an important chemical widely used in the synthetic rubber industry. Hemoglobin adducts of two of its reactive metabolites have been already investigated as possible parameters for exposure assessment. In this study hemoglobin adducts of epoxybutene (EB) were analyzed in blood samples from 17 workers in a BD monomer production unit and 19 controls in a heat production unit of a petrochemical plant near Prague, Czech Republic. BD exposure was determined by personal air sampling. The median level of exposure was 440 microg/m3 (range < 11-17 mg/m3) for the exposed workers and < 6 microg/m3 (< 5-150 microg/m3) for the controls. The adduct N-(2-hydroxy-3-butenyl)valine (HBVal) formed by the reaction of the N-terminal valine of globin with carbon-1 of EB was measured. The N-alkylated amino acid was analyzed by gas chromatography/mass spectrometry (GC/MS) after degradation by the modified Edman procedure. Using published methods problems arose with high background levels, especially in the negative ion chemical ionization (NCI) mode. In the present study a limit of detection of 0.2 pmol/g globin was achieved by using 400 mg globin, a variation in extraction solvents, an additional purification step and a widely extended GC temperature program. The median hemoglobin adduct level of the Czech BD monomer production workers (0.7 pmol/g globin; n = 17) was significantly higher than that of the controls (0.2 pmol/g globin; n = 19; P<0.05). Smoking controls showed higher hemoglobin adduct levels than nonsmoking controls (P<0.1) and significantly higher BD exposure levels (P<0.01).     

Gene and protein responses of human lung tissue explants exposed to ambient particulate matter of different sizes

Context: Exposure to ambient particulate air pollution is associated with increased cardiovascular and respiratory morbidity and mortality. It is necessary to understand causal pathways driving the observed health effects, particularly if they are differentially associated with particle size.

Objectives: To investigate the effect of different size ranges of ambient particulate matter (PM) on gene and protein expression in an in vitro model.

Materials and methods: Normal human tracheobronchial epithelium (NHTBE) three-dimensional cell constructs were exposed for 24?h to washed ambient PM of different sizes (size 1: 7–615?nm; size 2: 616 nm–2.39 µm; size 3: 2.4–10 µm) collected from a residential street. A human stress and toxicity PCR array was used to investigate gene expression and iTRAQ was used to perform quantitative proteomics.

Results: Eighteen different genes of the 84 on the PCR array were significantly dysregulated. Treatment with size 2 PM resulted in the greatest number of genes with altered expression, followed by size 1 and lastly size 3. ITRAQ identified 317 proteins, revealing 20 that were differentially expressed. Enrichment for gene ontology classification revealed potential changes to various pathways.

Discussion and conclusions: Different size fractions of ambient PM are associated with dysregulatory effects on the cellular proteome and on stress and toxicity genes of NHTBE cells. This approach not only provides an investigative tool to identify possible causal pathways but also permits the relationship between particle size and responses to be explored.     

DNA adducts in carp exposed to artificial diesel-2 oil slicks

In attempts to mimic field exposure, oil slicks prepared from diesel-2 oil/water emulsions were poured onto the surface of water in tanks prepared fresh every day and liver DNA adducts were analyzed by ³²P-postlabeling in carp free-swimming in these tanks. ‘Clusters’ of lipophilic DNA adducts were detected, with five major and numerus minor adducts. Essentially a similar adduct pattern was found in the liver DNA of carp exposed to crude oil-polluted water. Diesel-2 adduct induction was observed slowly with a steady increase to > 3000 amol/μg DNA at day 12. After this time fish were transferred to clean water. Adduct levels continued to increase through day 17 (≈ 10,000 amol/μg DNA) despite the cessation of exposure, but a 30% and 80% decline was evident at day 22 and day 27, respectively. All major adducts were distinct from the known benzo[a]pyrene diolepoxide-dG. These results indicate that diesel-2 oil can cause extensive DNA damage in carp in vivo and the damage accumulates proportionately with time of exposure.     

Formation and persistence of DNA adducts in epidermal and dermal mouse skin exposed to benzo(a)pyrene in vivo.

Tritiated benzo(a)pyrene was applied topically to 35 old male Swiss mice that received 250 nMole (0.63 mCi) per mouse in 150 microliters acetone. At each time point of study, 1, 3 and 7 days, 10 animals were killed, the skin was removed and the susceptible epidermic cells were separated from resistant dermis. The initial level of the total BaP-DNA adduct after 1 day of test was 2 fold higher in epidermic cells; in addition, the concentration of the individual modified deoxyribonucleoside adducts was 4 fold greater in epidermis. However, the nature and the repartition of the modified nucleosides analyzed by high performance liquid chromatography were similar. They were 5 fold more in epidermic cells, except for the (+/-) SynBaP- diolepoxide - deoxyguanosine adduct which was 2 fold more in dermic cells. The major DNA adduct formed in both types of cells was the (+) antiBaP diolepoxide - deoxyguanosine = (+) anti BPDE-dG with 75% of total adduct in epidermic and 55% in dermal cells. The decreased amount of BaP bound to DNA of epidermic and dermic cells may be similar and 90% of (+) anti BPDE-dG was removed after a week of treatment; in addition, a minority that bound with 9OH-BaP was also shown to be persistent. Although the persistence of adduct was 7 fold more important in susceptible epidermal cells than in the resistant dermic population, the nature of adduct repartition and the similar type of excision suggest that other mechanisms are also involved in the biologically different response of epidermis versus dermis to the carcinogenic BaP when it is applied topically.     

Urinary N3 adenine DNA adducts in humans occupationally exposed to styrene

Urine samples from humans occupationally exposed to styrene, with mandelic acid levels ranging from 400 to 1145 mg/g creatinine and from 68 to 400 mg/g creatinine for high and low exposure group, respectively, were analysed for N3 adenine DNA adducts, namely, 3-(2-hydroxy-1-phenylethyl)adenine (N3αA) and 3-(2-hydroxy-2-phenylethyl)adenine (N3βA). A sensitive LC-ESI–MSMS method was developed with the limit of quantification of 1 pg/mL for both analytes. Peaks corresponding to N3αA and/or N3βA were found in seven of nine end-of-shift samples of the high exposure group and in six of 19 end-of-shift samples of the low exposure group. Concentration of N3αA + N3βA amounted to 2.8 ± 1.6 pg/mL (mean ± S.D.; n = 9) and 1.8 ± 1.3 pg/mL (mean ± S.D.; n = 19) in the high and low exposure group, respectively. Of other 10 samples taken the next morning after exposure, two contained low but quantifiable concentrations of N3αA and none contained N3βA. However, interfering peaks were detected also in some control urine samples. Out of 22 controls, six and two samples contained peaks co-eluting with N3αA and N3βA, respectively. Therefore, the method used was found insufficiently specific to be applicable for biological monitoring. Comparing the excretion of N3αA + N3βA to that reported previously in mice it can be estimated that at the same absorbed dose, humans excreted not more than 1/30 of the amount of adenine adducts excreted by mice. As a consequence, the damage to DNA caused by styrene 7,8-oxide (SO), a reactive metabolite of styrene, appears to be much lower in humans than in mice.     

Formation of oligonucleotide adducts in pharmaceutical formulations

During preformulation studies, we observed that oligonucleotide extracted from topical formulations contained considerable amounts of covalently modified oligonucleotide adducts. In this report, we describe the identification and characterization of reaction products that form when PS-oligodeoxyribonucleotide ISIS 2302 (1) is brought into contact with aqueous solutions of glycerol-derived excipients. Compatibility tests showed that the presence of certain glycerides in the formulation lead to adduct formation (1+58x amu, 1+72x amu, 1+58x+72y amu, x, and y are the number of modifications on one oligonucleotide strand). No adduct formation was observed in the presence of triglycerides or propylene glycol-derived excipients used in the study. Using nucleosides as model compounds, two modifications of deoxyguanosine were isolated by preparative reversed phase (RP)-high pressure liquid chromatography (HPLC) and characterized by nuclear magnetic resonance (NMR) and HPLC-mass spectrometry (MS). Modifications were identified as N2-(1-carboxymethyl)- and N2-(1-carboxyethyl) derivatives of 2'-deoxyguanosine. The mechanism of formation of these adducts may involve advanced glycation reactions possibly caused by excipient impurities or degradation products such as glyceraldehyde or glyceraldehyde derivatives.     

Molecular dosimetry of N2-hydroxymethyl-dG DNA adducts in rats exposed to formaldehyde

In this study, both endogenous and exogenous N(2)-hydroxymethyl-dG adducts in nasal DNA of rats exposed to 0.7, 2, 5.8, 9.1, or 15.2 ppm [(13)CD(2)] formaldehyde for 6 h were quantified by a highly sensitive nano-UPLC-MS/MS method. Our data clearly demonstrated that exogenous formaldehyde DNA adducts form in a highly nonlinear fashion, with a 21.7-fold increase in exposure causing a 286-fold increase in exogenous adducts. The ratio of exogenous/endogenous DNA adducts demonstrated that endogenous DNA adducts dominated at low exposures, comprising more than 99%. In contrast, exogenous adducts were not detectable in the bone marrow of rats exposed to 15.2 ppm [(13)CD(2)] formaldehyde.     

Identification of cyclophosphamide-DNA adducts in rat embryos exposed in vitro to 4-hydroperoxycyclophosphamide.

Cyclophosphamide and other bifunctional alkylating agents are potent animal teratogens inducing a variety of malformations. Although cyclophosphamide-induced DNA damage is implicated as a primary mechanism underlying the teratogenesis initiated by cyclophosphamide, additional insights into the complex nature of the teratogenic process have been hampered by the inability to analyze the primary teratogenic lesions, i.e., cyclophosphamide-DNA adducts. Using tandem mass spectrometry, we show that the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (NOR-G) and bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-NOR-G) can be detected in the DNA of organogenesis-stage rat embryos after an in vitro exposure to an embryotoxic concentration of activated cyclophosphamide, i.e., 4-hydroperoxycyclophosphamide.     

Distribution of DNA adducts in the respiratory tract of rats exposed to diesel exhaust

Diesel exhaust, inhaled chronically at high concentrations, was previously found to be a pulmonary carcinogen in rats. The exhaust-induced tumors were located exclusively in the peripheral lung, although all of the respiratory tract tissues were exposed to the exhaust. The purpose of this study was to determine whether there were differences in the level of DNA adducts among the regions of the respiratory tract that paralleled the site of tumors. Groups of male F344/N rats were exposed 7 hr/day, 5 day/week for 12 weeks to diesel engine exhaust at a soot concentration of 10 mg/m3 or were sham-exposed to air. The maxilloturbinates, ethmoturbinates, trachea, left mainstem bronchus (airway generation 1), axial airway (airway generations 2-12), and peripheral lung tissue were dissected from the respiratory tract. DNA was isolated from the dissected samples and analyzed for the presence of adducts using the 32P-postlabeling assay. Chromatographic maps of DNA adducts demonstrated unique patterns of DNA adducts for each of the regions. The highest level of total DNA adducts occurred in peripheral lung tissue (approximately 20 adducts per 10(9) bases). The level of DNA adducts detected in the nasal tissues was about one-fourth to one-fifth that detected in peripheral lung. There were less than three adducts per 10(9) bases in each of the regions of the major conducting airways (i.e., trachea, bronchi, axial airway). In control rats, levels of DNA adducts ranged from one adduct per 10(9) bases (mainstem bronchi, axial airway) to about nine adducts per 10(9) bases (parenchyma). The data from this study indicate that higher levels of total DNA adducts and exhaust-induced adducts (i.e., exposed minus control adducts) were present in tissues where exhaust-induced tumors were located. These data suggest that DNA adduct levels in discrete locations of the respiratory tract may be good measures of the "effective dose" of carcinogenic compounds.     

Hemoglobin adducts in animals exposed to gasoline and diesel exhausts. 1. Alkenes

Blood samples from rats and hamsters exposed to automotive engine exhausts in the Committee of Common Market Automobile Constructors long-term inhalation study at Battelle-Geneva were analysed for the levels of 2-hydroxyethylvaline (HOEtVal) and 2-hydroxypropylvaline (HOPrVal) in hemoglobin (Hb). These adducts to the N-terminus of the Hb chains were determined by gas chromatography-mass spectrometry of derivatives obtained by a modified Edman degradation that specifically cleaves off alkylated N-terminal amino acids (valine in Hb). The adduct levels found correspond to the metabolic conversion of about 5-10% of inhaled ethene and propene to ethylene oxide and propylene oxide, respectively, in agreement with results from earlier studies on mice inhaling radio-labelled alkenes. It is concluded that the alkenes, via epoxides, are the main sources of the observed HOEtVal and HOPrVal. From calculated doses and estimates of genotoxic potency the contribution from ethene in urban air to human cancer risk is discussed.