A two-year toxicology study of bisphenol A (BPA) in Sprague-Dawley rats: CLARITY-BPA core study results.

We report the data from the guideline-compliant two-year toxicology study conducted as part of the Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA). BPA (0, 2.5, 25, 250, 2,500, and 25,000 µg/kg body weight (bw)/day) was administered daily by gavage in 0.3% carboxymethylcellulose vehicle to NCTR Sprague-Dawley rats from gestation day 6 through the start of parturition and then directly to pups from the day after birth until postnatal day 21 (stop-dose arm) or continuously until termination at one or two years. The stop-dose arm was included to assess the potential for any BPA effects that were due to developmental exposure. No BPA-related effects were evident in the in-life and non-histopathology data. Neoplastic and nonneoplastic lesions diagnosed in both females and males were common age-associated lesions that were variable across control and BPA-treated groups. The lack of consistent responses within the continuous- and stop-dose arms within and across tissues brought into question the plausible relationship of most of these lesions to BPA treatment. There was a possible relationship between the increased incidences of lesions in the female reproductive tract and the male pituitary and exposure to the 25,000 µg BPA/kg bw/day dose level.

Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats.

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (∼6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-2'-deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 10(8) nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo.

Effects of acrylamide exposure on serum hormones, gene expression, cell proliferation, and histopathology in male reproductive tissues of Fischer 344 rats.

Acrylamide (AA) is a reactive monomer used in many technological applications, but it is the incidental formation during cooking of common starchy foods that leads to pervasive human exposure, typically in the range of 1 µg/kg body weight (bw)/day (d). AA is carcinogenic in multiple organs from both sexes of several rodent models and a consistent body of evidence points to a genotoxic mechanism based on metabolism to a DNA-reactive epoxide, glycidamide (GA). In F344 rats, tumorigenesis occurs in several hormonally regulated tissues (thyroid, mammary gland, and peri-testicular mesothelium), which has prompted speculation about endocrine dysregulation as a possible mechanism. The present study evaluated the effects of a 14 d exposure to AA administered through the drinking water on reproductive tissues and the hypothalamic-pituitary-testes (HPG) axis in male F344 rats. The doses selected encompass a range from approximately 2.5 mg/kg bw/d, which is carcinogenic after lifetime exposure, to 50 mg/kg bw/d, a maximally tolerable dose that causes hind limb paralysis. AA caused significant changes in serum hormones, histopathology, testicular gene expression, and cell proliferation, especially at the highest dose. Despite strong evidence for activation of the HPG axis subsequent to decreases in testosterone levels, and histopathological changes associated with significant effects on Leydig and germ cells, with concomitant mRNA expression changes, the precise mechanism(s) for AA-induced testicular toxicity remains unclear; however, the absence of evidence for increased proliferation of the peri-testicular mesothelium (Ki-67 immunoreactivity) does not support hormonal dysregulation as a contributing factor to the predisposition of this tissue to the carcinogenic effects of AA.

The effects of chronic methylphenidate administration on operant test battery performance in juvenile rhesus monkeys.

Methylphenidate (MPH) is an amphetamine derivative widely prescribed for the treatment of attention deficit-hyperactivity disorder. Recent concern over its genotoxic potential in children [11] spurred a study on the effects of chronic MPH treatment in a nonhuman primate population and the studies reported here were conducted in conjunction with that study in the same animals. Here, the focus was on the ability of juvenile rhesus monkeys to learn how to perform a battery of operant behavioral tasks while being treated chronically with MPH. Performance of the National Center for Toxicological Research (NCTR) Operant Test Battery (OTB) was used to quantify the learning of tasks thought to model specific aspects of cognitive function including learning, motivation, color and position discrimination, and short-term memory. The OTB tasks designed to assess these specific behaviors included Incremental Repeated Acquisition (IRA), Progressive Ratio (PR), Conditioned Position Responding (CPR), and Delayed Matching-to-Sample (DMTS), respectively. Juvenile males (n=10/group) pressed levers and press-plates for banana-flavored food pellets. Subjects were treated orally, twice a day, five days per week (M-F) for 66 weeks with escalating doses (0.15 mg/kg initially, increased to 2.5 mg/kg for the low dose group and to 12.5 mg/kg for the high dose group) and tested in OTB tasks 30 to 60 min after the morning dose. The findings indicate that MPH at doses up to 2.5 mg/kg twice per day were well tolerated (performance was no different than controls) but at doses of 12.5 mg/kg twice per day there was a significant decrement in OTB performance, characterized by decreases in both percent task completed and response rates for all tasks. These effects of MPH seem primarily due to decreases in motivation to perform for food, which is not surprising given the well known appetite suppressing effects of amphetamine-like stimulants. Thus, the current data do not strongly suggest cognitive impairments following chronic MPH administration.

The effects of subchronic acrylamide exposure on gene expression, neurochemistry, hormones, and histopathology in the hypothalamus-pituitary-thyroid axis of male Fischer 344 rats.

Acrylamide (AA) is an important industrial chemical that is neurotoxic in rodents and humans and carcinogenic in rodents. The observation of cancer in endocrine-responsive tissues in Fischer 344 rats has prompted hypotheses of hormonal dysregulation, as opposed to DNA damage, as the mechanism for tumor induction by AA. The current investigation examines possible evidence for disruption of the hypothalamic-pituitary-thyroid axis from 14 days of repeated exposure of male Fischer 344 rats to doses of AA that range from one that is carcinogenic after lifetime exposure (2.5 mg/kg/d), an intermediate dose (10 mg/kg/d), and a high dose (50 mg/kg/d) that is neurotoxic for this exposure time. The endpoints selected include: serum levels of thyroid and pituitary hormones; target tissue expression of genes involved in hormone synthesis, release, and receptors; neurotransmitters in the CNS that affect hormone homeostasis; and histopathological evaluation of target tissues. These studies showed virtually no evidence for systematic alteration of the hypothalamic-pituitary-thyroid axis and do not support hormone dysregulation as a plausible mechanism for AA-induced thyroid cancer in the Fischer 344 rat. Specifically, there were no significant changes in: 1) mRNA levels in hypothalamus or pituitary for TRH, TSH, thyroid hormone receptor alpha and beta, as well 10 other hormones or releasing factors; 2) mRNA levels in thyroid for thyroglobulin, thyroid peroxidase, sodium iodide symporter, or type I deiodinases; 3) serum TSH or T3 levels (T4 was decreased at high dose only); 4) dopaminergic tone in the hypothalamus and pituitary or importantly 5) increased cell proliferation (Mki67 mRNA and Ki-67 protein levels were not increased) in thyroid or pituitary. These negative findings are consistent with a genotoxic mechanism of AA carcinogenicity based on metabolism to glycidamide and DNA adduct formation. Clarification of this mechanistic dichotomy may be useful in human cancer risk assessments for AA.

Effect of dietary genistein on cell replication indices in C57BL6 mice.

The phytoestrogen and isoflavone, genistein, inhibited the activity of the DNA synthesis-related enzyme, topoisomerase-II (topo-II), altered cell-cycle traverse and produced cell death in cell culture models. In order to examine the potential effects of genistein on cell replication and cell death in an animal model, 8-week-old C57BL6 mice were fed either a control diet or one containing one of five doses (100-2000 ppm) of genistein for 28 days. At the end of the feeding period, both male and female mice were sacrificed and the serum isoflavone and aglycone levels determined by liquid chromatography with electrospray tandem mass spectrometry (LC-ES/MS/MS). Immunohistochemistry was utilized to measure the cell replication and cell death rates in the small intestine. Total isoflavone concentration increased from below the limits of detection (0.001 microM) in control animals to 0.28 microM in male and 0.54 microM in female mice fed the 2000 ppm diet. A decrease in the percentage of cells in G(0) and an increase in the percentage of cells in S-phase, consistent with topo-II-induced S-phase arrest, was found in the duodenum and jejunum of the small intestine. Thus, genistein appears to accumulate to a sufficient level to affect topo-II activity in the small intestine.

Degradation of biphenyl by Mycobacterium sp. strain PYR-1.

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.

Analysis of DNA adducts from chemical carcinogens and lipid peroxidation using liquid chromatography and electrospray mass spectrometry.

The identification and dosimetry of DNA adducts are cornerstones of research on cancer etiology in experimental animals and humans. DNA adducts can result from exposure to exogenous chemical carcinogens or through reactions with endogenous by-products of oxidative metabolism. An important research need is high throughput methodology for quantification of any and all adducts that are present at trace amounts in DNA derived from target tissues of animals and humans. This review describes some recent progress made through applications of liquid chromatography coupled with mass spectrometry to structural characterization of unknown DNA adducts and highly sensitive quantitative analysis of target adducts.

Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice.

Previously our laboratory has shown that the soy isoflavone, genistein, stimulates growth of human breast cancer (MCF-7) cells in vivo and in vitro. In this study, the dose-response analysis of genistein at the physiologically achievable concentration range between 125 and 1,000 microg/g in the diet was conducted in ovariectomized athymic nude mice implanted with MCF-7 cells. We hypothesized that genistein at this concentration range can stimulate dose-dependently the breast tumor growth, cell proliferation and an estrogen-responsive pS2 gene induction. Tumor size and body weight were monitored weekly. At completion of the study, we analyzed cellular proliferation of tumors using incorporation of BrdU, pS2 expression of tumors using a Northern blot analysis and total genistein level in plasma using liquid chromatography-isotope dilution mass spectrometry (LC-ES/MS). Dietary genistein (> or = 250 microg/g) increased tumor size in a dose-dependent manner [8.4x the negative control (NC) group in the 250 microg/g group, 12.0x in the 500 microg/g group, 20.2x in the 1,000 microg/g group and 23.2x in the positive control (PC) group]. The percentage of proliferating cells was significantly increased by genistein at and above 250 microg/g (5.3x the NC group in the 250 microg/g, 5.6x in the 500 microg/g, 5.0x in the 1,000 microg/g and 4.8x in the PC group). Expression of pS2 mRNA was also significantly increased with increasing dietary genistein levels (11.25x the NC group in the 500 microg/g group and 15.84x in the 1,000 microg/g group). Total plasma genistein concentrations were between 0.39 and 3.36 micromol/L in mice fed between 125 and 1,000 microg/g genistein. In conclusion, dietary treatment with genistein at physiological concentrations produces blood levels of genistein sufficient to stimulate estrogenic effects, such as breast tumor growth, cellular proliferation and pS2 expression in athymic mice in a dose-responsive manner similar to that seen in vitro.

Metabolism of anthracene by a Rhodococcus species.

A Rhodococcus sp. isolated from contaminated river sediment was investigated to determine if the isolate could degrade high molecular mass polycyclic aromatic hydrocarbons. The Rhodococcus sp. was able to utilize anthracene (53%), phenanthrene (31%), pyrene (13%), and fluoranthene (5%) as sole source of carbon and energy, but not naphthalene or chrysene. In a study of the degradation of anthracene by a Rhodococcus sp., the identification of ring-fission products indicated at least two ring-cleavage pathways. One results in the production of 6,7-benzocoumarin, previously shown to be produced chemically from the product of meta cleavage of 1,2-dihydroxyanthracene, a pathway which has been well established in Gram-negative bacteria. The second is an ortho cleavage of 1,2-dihydroxyanthracene that produces 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, a dicarboxylic acid ring-fission product. This represents a novel metabolic pathway only identified in Gram-positive bacteria.

Effect of soy isoflavone supplementation on markers of oxidative stress in men and women.

Dietary intake of soy has been linked with decreased cancer risk, and the active compounds in soy that have been identified include the isoflavones genistein and daidzein. Since these compounds have antioxidant properties, we examined levels of oxidative damage in blood of six women and six men before and during soy supplementation using Novasoy tablets. Blood samples were obtained at weekly intervals for 3 weeks from the women taking 50-mg isoflavones once daily and the men taking 50-mg isoflavones twice daily. Plasma levels of genistein and daidzein increased after supplementation with maximal levels occurring at 2 weeks for the women while levels in men kept increasing over the 3 weeks of study. There was wide variation between individuals in the levels of isoflavones achieved. Mean levels of 5-hydroxymethyl-2'-deoxyuridine (5-OHmdU) in DNA from nucleated blood cells decreased after 1 week of supplementation in the women, with a decrease of 47% in mean 5-OHmdU levels after 3 weeks. In men, mean 5-OHmdU levels did not decrease until after 3 weeks of supplementation, at which there was 61% decrease. Mean plasma levels of 8-isoprostanes were not changed appreciably in either men or women. These pilot results suggest that soy isoflavone supplementation decreases levels of oxidative DNA damage in humans, and this may be a mechanism behind the cancer-preventive effects of soy isoflavones.

Gender-based differences in rats after chronic dietary exposure to genistein.

Gender-based differences can be observed from pharmacokinetic, behavioral, or anatomical assessments. No single assessment tool will provide a complete answer, but the use of a variety of indices, each with known gender-related outcome differences, can reveal agent-induced gender-based alterations. In a series of initial range-finding studies in rats conducted at the National Center for Toxicological Research (NCTR), the effects of dietary exposure to the weak estrogen, genistein, have been assessed using a number of techniques with validated gender-related outcome measures. The findings indicated that (1) the internal dose of genistein was higher in females than males after equivalent dietary exposure and this was consistent with the faster rate of genistein elimination in males; (2) in behavioral assessments, males and females in the high-dose dietary genistein group consumed more of a sodium-flavored solution; however, no genistein-related changes were observed in open field or running wheel activity, play behavior, or intake of a saccharin-flavored solution; and (3) dose-related alterations of the volume of the sexually dimorphic nucleus of the medial preoptic area were observed in genistein-exposed male rats but not females. These observations describe the utility of a variety of gender-based assessment tools and indicate that dose-related effects of developmental and chronic dietary exposure to genistein can be observed in the rodent. Additional studies, perhaps in nonhuman primates, are necessary to further predict the effect(s) of genistein on human gender-based development.

Biotransformation of malachite green by the fungus Cunninghamella elegans.

The filamentous fungus Cunninghamella elegans ATCC 36112 metabolized the triphenylmethane dye malachite green with a first-order rate constant of 0.029 micromol x h(-1) (mg of cells)(-1). Malachite green was enzymatically reduced to leucomalachite green and also converted to N-demethylated and N-oxidized metabolites, including primary and secondary arylamines. Inhibition studies suggested that the cytochrome P450 system mediated both the reduction and the N-demethylation reactions.

Molecular cloning, nucleotide sequence, and expression of genes encoding a polycyclic aromatic ring dioxygenase from Mycobacterium sp. strain PYR-1.

Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5' to the 3' direction, were a dehydrogenase, the dioxygenase small (beta)-subunit, and the dioxygenase large (alpha)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large alpha subunit did not cluster with most of the known alpha-subunit sequences but rather with three newly described alpha subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits.

Placental transfer of the soy isoflavone genistein following dietary and gavage administration to Sprague Dawley rats.

Genistein, the principal soy isoflavone, has estrogenic activity and is widely consumed by humans for putative beneficial health effects. The goal of the present study was to measure placental transfer of genistein in rats as a possible route of developmental exposure. Pregnant Sprague-Dawley rats were administered genistein orally, either by diet or by gavage. Concentrations of genistein aglycone and conjugates were measured in maternal and offspring serum and brain using HPLC with isotope dilution electrospray tandem mass spectrometry. Although fetal or neonatal serum concentrations of total genistein were approximately 20-fold lower than maternal serum concentrations, the biologically active genistein aglycone concentration was only 5-fold lower. Fetal brain contained predominately genistein aglycone at levels similar to those in the maternal brain. These studies show that genistein aglycone crosses the rat placenta and can reach fetal brain from maternal serum genistein levels that are relevant to those observed in humans.

Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1.

Cultures of Mycobacterium sp. strain PYR-1 were dosed with anthracene or phenanthrene and after 14 days of incubation had degraded 92 and 90% of the added anthracene and phenanthrene, respectively. The metabolites were extracted and identified by UV-visible light absorption, high-pressure liquid chromatography retention times, mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectrometry, and comparison to authentic compounds and literature data. Neutral-pH ethyl acetate extracts from anthracene-incubated cells showed four metabolites, identified as cis-1,2-dihydroxy-1,2-dihydroanthracene, 6,7-benzocoumarin, 1-methoxy-2-hydroxyanthracene, and 9,10-anthraquinone. A novel anthracene ring fission product was isolated from acidified culture media and was identified as 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid. 6,7-Benzocoumarin was also found in that extract. When Mycobacterium sp. strain PYR-1 was grown in the presence of phenanthrene, three neutral metabolites were identified as cis- and trans-9,10-dihydroxy-9,10-dihydrophenanthrene and cis-3,4-dihydroxy-3,4-dihydrophenanthrene. Phenanthrene ring fission products, isolated from acid extracts, were identified as 2,2'-diphenic acid, 1-hydroxynaphthoic acid, and phthalic acid. The data point to the existence, next to already known routes for both gram-negative and gram-positive bacteria, of alternative pathways that might be due to the presence of different dioxygenases or to a relaxed specificity of the same dioxygenase for initial attack on polycyclic aromatic hydrocarbons.

Development of a (32)P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro.

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation in vivo.

Riddelliine is a representative naturally occurring genotoxic pyrrolizidine alkaloid. We have studied the mechanism by which riddelliine induces hepatocellular tumors in vivo. Metabolism of riddelliine by liver microsomes of F344 female rats generated riddelliine N-oxide and dehydroretronecine (DHR) as major metabolites. Metabolism was enhanced when liver microsomes from phenobarbital-treated rats were used. Metabolism in the presence of calf thymus DNA resulted in eight DNA adducts that were identical to those obtained from the reaction of DHR with calf thymus DNA. Two of these adducts were identified as DHR-modified 7-deoxyguanosin-N(2)-yl epimers (DHR-3'-dGMP); the other six were DHR-derived DNA adducts, but their structures were not characterized. A similar DNA adduct profile was detected in the livers of female F344 rats fed riddelliine, and a dose-response relationship was obtained for the level of the total (eight) DHR-derived DNA adducts and the level of the DHR-3'-dGMP adducts. These results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism and the eight DHR-derived DNA adducts are likely to contribute to liver tumor development.

Quantitative analysis of etheno-2'-deoxycytidine DNA adducts using on-line immunoaffinity chromatography coupled with LC/ES-MS/MS detection.

Etheno DNA adducts, including 3,N4-etheno-2'-deoxycytidine (etheno-dC), are promutagenic lesions present in normal animal and human tissues. These DNA adducts are believed to be important in the etiology of cancer. Existing methods for quantifying etheno-dC use 32p. postlabeling. Although highly sensitive, postlabeling requires the use of an energetic radioisotope and considerable time and effort. The new methodology reported here permits automated quantification of trace levels of etheno-dC in crude DNA hydrolysates on the order of 5 adducts in 10(8) normal nucleotides from 100-microg samples of DNA. This was accomplished by using on-line immunoaffinity chromatography, a reverse-phase LC separation on graphitized carbon, tandem mass spectrometric detection, and an isotopically labeled internal standard. The automated procedures permitted analysis of 4 DNA hydrolysates/hr. The sensitivity using immunoaffinity cleanup was approximately 100-fold greater than that observed when using a silica-based trapping system. The validated method was applied to the analysis of etheno-dC in commercial calf thymus DNA, untreated mouse liver, and untreated rat liver DNA. The demonstrated level of performance suggests future applicability of this method in studies of cancer in humans and experimental animals.

Quantification of etheno-DNA adducts using liquid chromatography, on-line sample processing, and electrospray tandem mass spectrometry.

Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 10(8) normal nucleotides from 100 microg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethane-treated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.