Induction of micronuclei in mouse and rat by glycidamide,genotoxic metabolite of acrylamide

Male CBA mice and male Sprague-Dawley rats were treated by i.p. injection of glycidamide (GA), the presumed genotoxic metabolite of acrylamide (AA). GA was obtained through a new way of synthesis. As an endpoint of chromosome damage, micronucleus (MN) induction in erythrocytes was measured. Hemoglobin (Hb) adducts were used as a measure of in vivo dose of GA. GA induced linear dose-dependent increases in adduct levels in both species. Rats exhibit, compared with mice, 30% higher Hb adduct levels per unit of administered amount of GA. The incremental MN frequencies per administered dose of GA in mice showed a linear-quadratic dose-dependent curve. In the rat no positive dose-response relationship was obtained, probably due to toxic effects to the bone marrow. The main result of this study is the finding that after treatment with synthetic GA the MN frequency per unit of the in vivo dose of GA in the mouse is very similar to that obtained in a previous study, where animals were treated with AA and GA as a metabolite. This equality in potency of GA, whether its in vivo dose is established by injection of synthetic GA or through metabolism of AA, supports the view that GA is the predominant genotoxic factor in AA exposure.     

Hemoglobin adducts and micronuclei in rodents after treatment with isoprene monoxide or butadiene monoxide

1,3-Butadiene and isoprene (2-methyl-1,3-butadiene) are chemically related substances that are carcinogenic to rodents. The overall aim of this work is to elucidate the role of the genotoxic action of diepoxide metabolites in the carcinogenesis of the dialkenes. In vivo doses of the diepoxide metabolites were measured through reaction products with hemoglobin (Hb adducts) in studies of induced micronuclei (MN) in rodents. In the reaction with N-terminal valine in Hb, diepoxybutane and isoprenediepoxide form ring-closed adducts, pyrrolidines [N,N-(2,3-dihydroxy-1,4-butadiyl)valine and N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valine, respectively]. The method applied for Hb-adduct measurement is based on tryptic degradation of the protein and liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) analysis. Mice were given single i.p. injections of the monoepoxides of butadiene and isoprene, 1,2-epoxy-3-butene or 1,2-epoxy-2-methyl-3-butene, respectively. Rats were treated in the same way with 1,2-epoxy-3-butene. In mice pyrrolidine adduct levels increased with increasing administered doses of the monoepoxides. The in vivo dose of diepoxybutane was on average twice as high (0.29+/-0.059 mMh) as the in vivo dose of isoprenediepoxide (0.15+/-0.053 mMh) per administered dose (mmol/kg body weight) of the monoepoxides. In mice the genotoxic effects of the two monoepoxides, measured as the increase in the frequencies of micronuclei (MN), were approximately linearly correlated to the in vivo doses of the diepoxides (except at the highest dose of diepoxybutane). In rats the pyrrolidine-adduct levels from diepoxybutane were below the limit of quantification at all administered doses of 1,2-epoxy-3-butene and no significant increase was observed in the frequency of MN. Measurement of the ring-closed adducts to N-termini in Hb by the applied method permits analysis of in vivo doses of diepoxybutane and isoprenediepoxide, which may be further used for the elucidation of the mechanisms of carcinogenesis of butadiene and isoprene.     

The research background for risk assessment of ethylene oxide: aspects of dose

Data for relationships between in vivo doses inferred from levels of hemoglobin (Hb) or DNA adducts and administered (by inhalation or injection) doses of ethylene oxide (EO) in mice, rats and humans are reviewed. At low absorbed doses or dose rates these relationships appear to be linear, whereas at higher dose rates deviations from linearity due to saturation kinetics of detoxification and of DNA repair as well as certain toxic effects have to be allowed for. If these factors are taken into consideration, a rather consistent picture is obtained for animal studies, with a variation by less than a factor 2 between estimates of adduct level increments or in vivo dose increments per unit of administered dose. Although the value for in vivo dose per unit of exposure dose (ppm-hour) in humans is uncertain because of unreliable data for the time-weighted average exposure level, the most likely value for this relationship, supported by data for ethene, agrees with data for the rodents. In the animal species testis doses are approximately one-half of the blood doses inferred from Hb adducts.     

Biomonitoring of exposure to aromatic amines: haemoglobin adducts in humans

Haemoglobin (Hb) adducts from aromatic amines (AAs) are well established biomarkers of exposure. Tobacco smoking and occupational exposure are major sources of AA Hb adducts. The origin of background levels in non-smokers and non-occupationally exposed humans are largely unknown. Here we examine the determination of AA Hb adducts, focussing on the analytical strategies for Hb isolation, removal of unbound AAs from Hb solutions, hydrolysis of the Hb bound AAs, extraction, preconcentration, clean-up and derivatisation of the free amines for determination by gas chromatography-mass spectrometry. Finally, a detailed summary of available results on the determination of AA Hb adducts is given.     

Quantification of aristolochic acid-derived DNA adducts in rat kidney and liver by using liquid chromatography-electrospray ionization mass spectrometry

Aristolochic acid (AA), derived from the herbal genus Aristolochia and Asarum, has recently been shown to be associated with the development of nephropathy. Upon enzyme activation, AA is metabolized to the aristolactam-nitrenium ion intermediate, which reacts with the exocyclic amino group of the DNA bases via an electrophilic attack at its C7 position, leading to the formation of the corresponding DNA adducts. The AA-DNA adducts are believed to be associated with the nephrotoxic and carcinogenic effects of AA. In this study, liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS) was used to identify and quantify the AA-DNA adducts isolated from the kidney and liver tissues of the AA-dosed rats. The deoxycytidine adduct of AA (dC-AA) and the deoxyadenosine-AA adduct (dA-AA) were detected and quantified in the tissues of rats with one single oral dose (5mg or 30mg AA/kg body weight). The deoxyguanosine adduct (dG-AA), however, was detected only in the kidney of rats that were dosed at 30mg AA/kg body weight for three consecutive days. The amount of AA-DNA adducts found in the rats correlated well with the dosage.     

Use of haemoglobin adducts in exposure monitoring and risk assessment

Many industrial bulk chemicals are oxiranes or alkenes that are easily metabolised to oxiranes in mammalian systems. Many oxiranes may react with DNA and are therefore mutagenic in vitro. Some oxiranes have been shown to be carcinogenic in rodents in vivo as well. Despite the very limited evidence of the carcinogenicity of oxiranes in humans, they should be considered potential human carcinogens. As a consequence, exposure to these compounds should be minimised and controlled. Twenty-five years ago, Ehrenberg and co-workers suggested that exposure to oxiranes might be determined through the measurement of the adducts they form with haemoglobin (Hb). Ten years later, a modification of the Edman degradation was developed at Stockholm University that allowed determination of adducts with the N-terminal valine of Hb by GC-MS. In our laboratory, this methodology was modified and adapted for analysis on an industrial scale. Since 1987, exposure of operators in our facilities to ethylene oxide (EO) has been routinely monitored by determination of N-(2-hydroxyethyl)valine in Hb. Biological monitoring programmes for propylene oxide (PO) and 1,3-butadiene (BD) were developed later. In this review, the methodology and its results are discussed as a tool in human risk assessment of industrial chemicals. Two major advantages of Hb adduct determinations in risk assessment are (1) the qualitative information on the structure of reactive intermediates that may be obtained through the mass spectrometry, which may provide insight in the molecular toxicology of compounds such as BD, and (2) the possibility of reliable determination of exposure over periods of several months with limited number of samples for compounds such as ethylene oxide (EO), propylene oxide (PO) and BD which form stable adducts with Hb. Since good correlations between the airborne concentrations of these chemicals with their respective adducts have been established, Hb adducts can also be used to quantitate airborne exposure which is of paramount importance as exposure assessment is usually one of the weaker parameters in risk assessment.     

Adducts with haemoglobin and with DNA in epichlorohydrin-exposed rats

Epichlorohydrin (1-chloro-2,3-epoxypropane; ECH) is an important industrial chemical and a carcinogen in experimental animals. The main aims of the present study were to characterize the adduct formation in female Wistar rats and to identify adducts that could potentially be used in human biomonitoring studies. The total binding of radioactivity to haemoglobin in rats administered 0, 0. 11, 0.22, 0.43, or 0.97 mmol [3H]ECH/kg body weight by i.p. injection, and sacrificed 24 h after treatment, was linearly related to a dose up to 0.43 mmol/kg body weight. The binding at the highest dose was higher than predicted by extrapolation from lower doses, indicating saturation of a metabolic process for elimination of ECH. Ion-exchange chromatography of a globin hydrolysate showed one major radioactivity peak corresponding to S-(3-chloro-2-hydroxypropyl)cysteine. The half-life of this adduct was estimated as about 4 days by analysis of globin from rats administered 0.43 mmol/kg body weight and sacrificed after 1, 2 and 9 days. Crosslinking of the adduct, presumably with glutathione, appeared to be the predominant secondary reaction. Hydrolysis of N-(3-chloro-2-hydroxypropyl)valine, the primary reaction product of ECH with N-terminal valine, would give N-(2,3-dihydroxypropyl)valine. A sensitive gas chromatography/mass spectrometry method for the dihydroxypropyl adduct was used to follow its formation and removal after administration of nonlabelled ECH (0.11 mmol/kg body weight). The level of this adduct reached a maximum of about 20 pmol/g globin after a few weeks, corresponding to about 0.1% of the initial binding of ECH to globin. N-7-(3-Chloro-2-hydroxypropyl)guanine was detected in rats administered 0.97 mmol [3H]ECH/kg body weight and sacrificed 6 h after treatment. The adduct levels in haemoglobin and DNA were compared with previously reported adduct levels in male Fischer 344 rats exposed to propylene oxide. Despite its higher chemical reactivity, the capacity of ECH to alkylate macromolecules in vivo was found to be somewhat lower than that of propylene oxide.     

DNA adducts derived from administration of acrylamide and glycidamide to mice and rats

Acrylamide (AA) is an important industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in chronic rodent bioassays. Recent findings of AA in many common starchy foods have sparked renewed interest in determining toxic mechanisms and in understanding the cancer, neurotoxicity, and reproductive risks from typical human exposures. Dosing mice and rats with AA (50 mg/kg) led to presence of glycidamide (GA) in serum and tissues. Furthermore, GA-derived DNA adducts of adenine and guanine were formed in all tissues examined, including both target tissues identified in rodent carcinogenicity bioassays and in non-target tissues. Dosing rats and mice with an equimolar amount of GA typically produced higher levels of DNA adducts than observed with AA. Kinetics of DNA adduct formation and accumulation were measured following oral administration of a single dose of AA (50 mg/kg) or from repeat dosing (1 mg/kg/day), respectively. The formation of these DNA adducts is consistent with previously reported mutagenicity of AA and GA in vitro, which involved reaction of GA with adenine and guanine bases. These results provide strong support for a genotoxic mechanism of AA carcinogenicity in rodents. The kinetic/biomarker approaches described here may represent a meaningful way to extrapolate cancer risks to actual human exposures from food, which are much lower.     

DNA adducts derived from administration of acrylamide and glycidamide to mice and rats

Acrylamide (AA) is an important industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in chronic rodent bioassays. Recent findings of AA in many common starchy foods have sparked renewed interest in determining toxic mechanisms and in understanding the cancer, neurotoxicity, and reproductive risks from typical human exposures. Dosing mice and rats with AA (50 mg/kg) led to presence of glycidamide (GA) in serum and tissues. Furthermore, GA-derived DNA adducts of adenine and guanine were formed in all tissues examined, including both target tissues identified in rodent carcinogenicity bioassays and in non-target tissues. Dosing rats and mice with an equimolar amount of GA typically produced higher levels of DNA adducts than observed with AA. Kinetics of DNA adduct formation and accumulation were measured following oral administration of a single dose of AA (50 mg/kg) or from repeat dosing (1 mg/kg/day), respectively. The formation of these DNA adducts is consistent with previously reported mutagenicity of AA and GA in vitro, which involved reaction of GA with adenine and guanine bases. These results provide strong support for a genotoxic mechanism of AA carcinogenicity in rodents. The kinetic/biomarker approaches described here may represent a meaningful way to extrapolate cancer risks to actual human exposures from food, which are much lower.     

Development of a competitive immunoassay for the determination of N-(2-hydroxyethyl)valine adducts in human haemoglobin and its application in biological monitoring

Ethylene oxide (EO) is an important industrial compound and a directly acting mutagen. Human exposure to it can be monitored by the determination of haemoglobin (Hb) adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxyethyl)valine in whole blood was developed and its potential usefulness as a tool for biologically monitoring occupational exposure demonstrated. Analytical reliability was confirmed in a comparative study with gas chromatography-mass spectrometry (range 0.040–589?nmol?g^?1 Hb, correlation coefficient 0.98, n=10). The assay was configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay uses a whole blood matrix and has a working range of 10–10?000 pmol N-(2-hydroxethyl)valine?g^?1 Hb. The assay does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results from potentially exposed workers indicate the assay might be a powerful tool for the routine occupational biomonitoring of EO exposure.     

Exposure-dependent accumulation of N-(2-hydroxypropyl)valine in hemoglobin of F344 rats exposed to propylene oxide by the inhalation route

The detection of hemoglobin adducts by mass spectrometry is a very sensitive and specific measurement of the extent of covalent binding of electrophilic chemicals. The exposure-dependent accumulation of N-(2-hydroxypropyl)valine (N-HPVal) in globin of rats exposed to propylene oxide (PO) (0, 5, 25, 50, 300 or 500 ppm) by the inhalation route was measured to assess the utility of Hb adducts as biomarkers of exposure. Analysis of N-HPVal by gas-chromatography tandem mass spectrometry showed a linear exposure-dependent response for adduct accumulation in globin of rats exposed to PO for 3 days (6 h/day). After 20 days of exposure (6 h/day; 5 days/week), the exposure-response curve was slightly sub-linear. DNA adducts had been measured in several organs of the same animals in a companion study. The dose-response for accumulation of DNA adducts was similar to that obtained for Hb adducts. However, the number of DNA adducts varied by 17-fold between different tissues. The highest number of DNA adducts was found in respiratory nasal tissue, followed by lung and then liver. These data demonstrate that hemoglobin adducts provide a sensitive dosimeter for systemic exposure, but cannot be used to predict the extent of DNA binding in individual tissues. Furthermore, the exposure-response curve for both hemoglobin and DNA adduct accumulation does not reflect the tumor incidence curve for PO, providing evidence that the assessment of risk to cancer is more complex than simple biomarker measurements. When the present rat data were compared with recent N-HPVal measurements in humans, similar binding was found.     

A fluorescence-based analysis of aristolochic acid-derived DNA adducts

Aristolochic acids (AAs), major components of plant extracts from Aristolochia species, form (after metabolic activation) pro-mutagenic DNA adducts in renal tissue. The DNA adducts can be used as biomarkers for studies of AA toxicity. Identification of these adducts is a complicated and time-consuming procedure. We present here a fast, nonisotopic, fluorescence-based assay for the detection of AA-DNA adducts in multiple samples. This approach allows analysis of AA adducts in synthetic DNA with known nucleotide composition and analysis of DNA adducts formed from chemically diverse AAs in vitro. The method can be applied to compare AA-DNA adduct formation in cells and tissues.     

Hemoglobin adducts and urinary mercapturic acids in rats as biological indicators of butadiene exposure

Binding of 1,2-epoxy-3-butene, the primary metabolite of butadiene, to hemoglobin (Hb) and excretion of its mercapturic acid in urine were studies as potential indicators of butadiene exposure. Four groups of Wistar rats were exposed to butadiene at 0, 250, 500 and 1000 ppm 6 h/day, 5 days/week, during 2 weeks. Blood was collected at the end of exposure and 17 days later for analysis of hemoglobin adducts and adduct stability. Urine was collected each day during exposure (afternoon samples) and in between exposures (morning samples). Adducts of 1,2-epoxy-3-butene to N-terminal valine in Hb were measured using the N-alkyl Edman procedure and GC/MS of the thiohydantoin derivatives. The corresponding mercapturic acid was analysed, after deacetylation, through derivatization with phthaldialdehyde and HPLC with fluorescence detection. The Hb adducts proved to be stable and are therefore useful for dosimetry of long-term exposure to butadiene. The adduct levels increased linearly with exposure dose up to 1000 ppm (3 nmol/g Hb at 1000 ppm). The increase with exposure dose of the mercapturic acid concentration in urine was also compatible with a linear does response up to 1000 ppm. The sensitivity of both analytical methods needs to be improved for their application to human samples.     

Development of a competitive immunoassay for the determination of N-(2-hydroxyethyl)valine adducts in human haemoglobin and its application in biological monitoring

Ethylene oxide (EO) is an important industrial compound and a directly acting mutagen. Human exposure to it can be monitored by the determination of haemoglobin (Hb) adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxyethyl)valine in whole blood was developed and its potential usefulness as a tool for biologically monitoring occupational exposure demonstrated. Analytical reliability was confirmed in a comparative study with gas chromatography-mass spectrometry (range 0.040-589 nmol g^-1 Hb, correlation coefficient 0.98, n=10). The assay was configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay uses a whole blood matrix and has a working range of 10-10 000 pmol N-(2-hydroxethyl)valine g^-1 Hb. The assay does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results from potentially exposed workers indicate the assay might be a powerful tool for the routine occupational biomonitoring of EO exposure.     

Dose responses for DNA adduct formation in tissues of rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-[(14)C]-butadiene

Male Sprague-Dawley rats and B6C3F1 mice were exposed to either a single 6h or a multiple (5) daily (6h) nose-only dose of 1,3-[2,3-(14)C]-butadiene at exposure concentrations of nominally 1, 5 or 20 ppm. The aim was to compare the results with those from a similar previous study at 200 ppm. DNA isolated from liver, lung and testis of exposed rats and mice was analysed for the presence of butadiene related adducts, especially the N7-guanine adducts. Total radioactivity present in the DNA from liver, lung and testis was quantified and indicated more covalent binding of radioactivity for mouse tissue DNA than rat tissue DNA. Following release of the depurinating DNA adducts by neutral thermal hydrolysis, the liberated depurinated DNA adducts were measured by reverse phase HPLC coupled with liquid scintillation counting. The guanine adduct G4, assigned as N7-(2,3,4-trihydroxybutyl)- guanine, was the major adduct measured in liver, lung and testis DNA in both rats and mice. Higher levels of G4 were detected in all mouse tissues compared with rat tissue. The dose-response relationship for the formation of adduct G4 was approximately linear for all tissues studied for both rats and mice exposed in the 1-20 ppm range. The formation of G4 in liver tissue was about three times more effective for mouse than rat in this exposure range. Average levels of adduct G4 measured in liver DNA of rats and mice exposed to 5 x 6 h 1, 5 and 20 ppm 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 0.79 +/- 0.30, 2.90 +/- 1.19, 16.35 +/- 4.8 adducts/10(8) nucleotides and for mice: 2.23 +/- 0.71, 12.24 +/- 2.15, 48.63 +/- 12.61 adducts/10(8) nucleotides. For lung DNA the corresponding values were for rats: 1.02 +/- 0.44, 3.12 +/- 1.06, 17.02 +/- 4.07 adducts/10(8) nucleotides, and for mice: 3.28 +/- 0.32, 14.04 +/- 1.55, 42.47 +/- 13.12 adducts/10(8) nucleotides. Limited comparative data showed that the levels of adduct G4 formed in liver and lung DNA of mice exposed to a single exposure to butadiene in the present 20 ppm study and earlier 200 ppm study were approximately directly proportional across dose, but this was not observed in the case of rats. From the available evidence it is most likely that adduct G4 was formed from a specific isomer of the diol-epoxide metabolite, 3,4-epoxy-1,2-butanediol rather than the diepoxide, 1,2,3,4-diepoxybutane. Another adduct G3, possibly a diastereomer of N7-(2,3,4-trihydroxybutyl)-guanine or most likely the regioisomer N7-(1-hydroxymethyl-2,3-dihydroxypropyl)-guanine, was also detected in DNA of mouse tissues but was essentially absent in DNA from rat tissue. Qualitatively similar profiles of adducts were observed following exposures to butadiene in the present 20 ppm study and the previous 200 ppm study. Overall the DNA adduct levels measured in tissues of both rats and mice were very low. The differences in the profiles and quantity of adducts seen between mice and rats were considered insufficient to explain the large difference in carcinogenic potency of butadiene to mice compared with rats.     

A novel and specific method for the determination of aristolochic acid-derived DNA adducts in exfoliated urothelial cells by using ultra performance liquid chromatography-triple quadrupole mass spectrometry

Aristolochic acid nephropathy (AAN) is associated with the prolonged exposure to nephrotoxic and carcinogenic aristolochic acids (AAs). DNA adducts induced by AAs have been proven to be critical biomarkers for AAN. Therefore, accurate and specific quantification of AA-DNA adducts is important. In this study, a specific method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and applied for the determination of 7-(deoxyadenosin-N(6)-yl)aristolactam I (dA-AAI) in exfoliated urothelial cells of AA-dosed rats. After the isolation from urine samples, DNA in urothelial cells were subjected to enzymatic digestion and solid-phase extraction on a C(18) Sep-Pak cartridge for the enrichment of DNA adducts. The sample extracts were analyzed by reverse-phase UPLC-MS/MS with electrospray ionization in positive ion mode. The quantification of the AA-DNA adduct was performed by using multiple reaction monitoring with reserpine as internal standard. The method provided good accuracy and precision with a detection limit of 1 ng/ml, which allowed the detection of trace of dA-AAI in exfoliated urothelial cells. After one-month oral dose of AAI at 10 mg/kg/day, 2.1±0.3 dA-AAI per 10(9) normal dA was detected in exfoliated urothelial cells of rats. Compared to the traditional methods such as (32)P-postlabelling and HPLC with fluorescence detection, the developed UPLC-MS/MS method is more specific and rapid with a retention time of 4 min. The outcome of this study may have clinical significance for diagnosing and monitoring AA-associated disease because detection of DNA adducts in exfoliated urothelial cells is non-invasive and convenient.     

Development of a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin and its application in biological monitoring.

Propylene oxide (PO) is an important industrial compound and a directly acting mutagen. Human exposure to PO can be monitored by the determination of haemoglobin adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxypropyl)valine in whole haemoglobin was developed and its potential usefulness as a tool for biologically monitoring occupational exposure was demonstrated. Analytical reliability was confirmed in a comparative study with GC-MS (range 3.7-992 nmol g-1 haemoglobin (Hb), correlation coefficient 0.99, n=10). The assay has been configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay employs a whole blood matrix and has a working range of 2-250 pmol g-1 Hb. It does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results in potentially exposed workers indicate the assay's high potential usefulness in routine occupational biomonitoring of exposure to PO.     

DNA adducts of styrene-7,8-oxide in target and non-target organs for tumor induction in rat and mouse after repeated inhalation exposure to styrene

Styrene by inhalation had been shown to increase the lung tumor incidence in mice at 20 ppm and higher, but was not carcinogenic in rats at up to 1000 ppm. Styrene-7,8-oxide, the major metabolic intermediate, has weak electrophilic reactivity. Therefore, DNA adduct formation was expected at a low level and a 32P-postlabeling method for a determination of the two regioisomeric 2'-deoxyguanosyl-O6-adducts at the alpha(7)- and beta(8)-positions had been established. The first question was whether DNA adducts could be measured in the rat at the end of the 2 years exposure of a bioassay for carcinogenicity, even though tumor incidence was not increased. Liver samples of male and female CD rats were available for DNA adduct analysis. Adducts were above the limit of detection only in the highest dose group (1000 ppm), with median levels of 9 and 8 adducts per 10(7) nucleotides in males and females, respectively (sum of alpha- and beta-adducts). The result indicates that the rat liver tolerated a relatively high steady-state level of styrene-induced DNA adducts without detectable increase in tumor formation. The second question was whether different DNA adduct levels in the lung of rats and mice could account for the species difference in tumor incidence. Groups of female CD-1 mice were exposed for 2 weeks to 0, 40, and 160 ppm styrene (6h per day; 5 days per week), female CD rats were exposed to 0 and 500 ppm. In none of the lung DNA samples were adducts above a limit of detection of 1 adduct per 10(7) DNA nucleotides. The data indicate that species- and organ-specific tumor induction by styrene is not reflected by DNA adduct levels determined in tissue homogenate. The particular susceptibility of the mouse lung might have to be based on other reactive metabolites and DNA adducts, indirect DNA damage and/or cell-type specific toxicity and tumor promotion.     

Dose responses for the formation of hemoglobin adducts and urinary metabolites in rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-(14)C]-butadiene

Blood and urine were obtained from male Sprague-Dawley rats and B6C3F1 mice exposed to either a single 6 h or multiple daily (5 x 6 h) nose-only doses of 1,3-[2,3- (14)C]-butadiene at atmospheric concentrations of 1, 5 or 20 ppM. Globin was isolated from erythrocytes of exposed animals and analyzed for total radioactivity and also for N-(1,2,3-trihydroxybut-4-yl)-valine adducts. The modified Edman degradation procedure coupled with GC-MS was used for the adduct analysis. Linear relationships were observed between the exposures to 1,3-[2,3-(14)C]-butadiene and the total radioactivity measured in globin and the level of trihydroxybutyl valine adducts in globin. A greater level of radioactivity (ca. 1.3-fold) was found in rat globin compared with mouse globin. When analyzed for specific amino acid adducts, higher levels of trihydroxybutyl valine adducts were found in mouse globin compared with rat globin. Average levels of trihydroxybutyl valine adduct measured in globin from rats and mice exposed for 5 x 6 h at 1, 5 and 20 ppM 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 80, 179, 512 pM/g globin and for mice: 143, 351, 1100 pM/g globin. The profiles of urinary metabolites for rats and mice exposed at the different concentrations of butadiene were obtained by reverse phase HPLC analysis on urine collected 24 h after the start of exposure and were compared with results of a previous similar study carried out for 6 h at 200 ppM butadiene. Whilst there were qualitative and quantitative differences between the profiles for rats and mice, the major metabolites detected in both cases were those representing products of epoxide hydrolase mediated hydrolysis and glutathione (GSH) conjugation of the metabolically formed 1,2-epoxy-3-butene. These were 4-(N-acetyl-l-cysteine-S-yl)-1,2-dihydroxy butane and (R)-2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene, 1-(N-acetyl-l-cystein-S-yl)-2-(S)-hydroxybut-3-ene, 1-(N-acetyl-l-cystein-S-yl)-2-(R)-hydroxybut-3-ene, (S)-2-(N-acetyl-l-cystein-S-yl)-1-hydroxybut-3-ene, respectively. The former pathway showed a greater predominance in the rat. The profiles of metabolites were similar at exposure concentration in the range 1-20 ppM. There were however some subtle differences compared with results of exposure to the higher 200 ppM concentrations. Overall the results provide the basis for cross species comparison of low exposures in the range of occupational exposures, with the wealth of data available from high exposure studies.     

Role of malondialdehyde-acetaldehyde adducts in liver injury

Malondialdehyde and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. MAA-protein adducts are composed of two major products whose structures and mechanism of formation have been elucidated. MAA adduct formation, especially in the liver, has been demonstrated in vivo during ethanol consumption. These protein adducts are capable of inducing a potent immune response, resulting in the generation of antibodies against both MAA epitopes, as well as against epitopes on the carrier protein. Chronic ethanol administration to rats results in significant circulating antibody titers against MAA-adducted proteins, and high anti-MAA titers have been associated with the severity of liver damage in humans with alcoholic liver disease. In vitro exposure of liver endothelial or hepatic stellate cells to MAA adducts induces a proinflammatory and profibrogenic response in these cells. Thus, during excessive ethanol consumption, ethanol oxidation and ethanol-induced oxidative stress result in the formation of acetaldehyde and malondialdehyde, respectively. These aldehydes can react together synergistically with proteins and generate MAA adducts, which are very immunogenic and possess proinflammatory and profibrogenic properties. By virtue of these potentially toxic effects, MAA adducts may play an important role in the pathogenesis of alcoholic liver injury.