Association between genetic polymorphisms and biomarkers in styrene-exposed workers

A comprehensive approach to evaluate genotoxic effects induced by styrene exposure was employed in 44 hand-lamination workers in comparison with 18 unexposed controls. The acquired data on single-strand breaks in DNA (SSBs), frequency of chromosomal aberrations and HPRT mutant frequency in peripheral blood lymphocytes were compared to the results on genotyping of some of the xenobiotic-metabolising enzymes (CYP1A1, CYP2E1, epoxide hydrolase and GSTM1, GSTP1 and GSTT1). Multifactorial regression analysis indicated that SSB in DNA were significantly associated with styrene exposure and with heterozygosity in CYP2E1 (5'-flanking region and intron 6; r(2)=0.614). The frequency of chromosomal aberrations (CA), as analysed by linear multiple regression analysis, significantly correlated with years of employment (P=0.004) and with combinations of epoxide hydrolase (EPHX) genotypes (exon 3, Tyr/His and exon 4, His/Arg), where individuals with low and medium activity EPHX genotypes exhibited higher frequencies of CA than those with high activity genotypes (P=0.044, r(2)=0.563). Moderately higher HPRT mutant frequencies were detected in styrene-exposed individuals (20.2 +/- 25.8 x 10(-6)) as compared to controls (13.3 +/- 6.3 x 10(-6)), but this difference was not significant. ANOVA (in the whole set of data) revealed that mutant frequencies at the HPRT gene were significantly associated with years of employment (F=6.9, P=0.0001), styrene in blood (F=10.1, P=0.0001), and heterozygosity in CYP2E1 (intron 6; F=13.5, P=0.0008) and GSTP1 (exon 5; F=3.6, P=0.038).In conclusion, our present data suggest that analysed biomarkers of DNA damage may be modulated by polymorphic CYP2E1, EPHX and GSTP1. In our study, styrene-specific DNA and haemoglobin adducts are under investigation. Completing these data with the results of genotyping of metabolising enzymes may provide a useful tool for individual genotoxic risk assessment.     

Relationship between the capacity to repair 8-oxoguanine, biomarkers of genotoxicity and individual susceptibility in styrene-exposed workers

Genotoxic effects related to exposure to styrene have been a matter of investigation for many years by employing markers of exposure, effect and susceptibility. The role of individual DNA-repair capacity in response to exposure to styrene may explain the controversial results so far obtained, but it is still scarcely explored. In the present study, we measured capacity to repair oxidative DNA damage in cell extracts obtained from 24 lamination workers occupationally exposed to styrene and 15 unexposed controls. The capacity to repair oxidative DNA damage was determined by use of a modified comet assay, as follows: HeLa cells, pre-treated with photosensitizer and irradiated with a halogen lamp in order to induce 7,8-dihydroxy-8-oxoguanine, were incubated with cell extracts from mononuclear leukocytes of each subject. The level of strand breaks reflects the removal of 7,8-dihydroxy-8-oxoguanine from substrate DNA by the enzymatic extract. In styrene-exposed subjects a moderate, non-significant increase in oxidative DNA repair was observed. Stratification for sex and smoking habit showed that unexposed males (P=0.010) and unexposed smokers (P=0.037) exhibited higher DNA-repair rates. The repair capacity did not correlate with parameters of styrene exposure and biomarkers of genotoxic effects (DNA strand breaks, N1-styrene-adenine DNA adducts, chromosomal aberrations and mutant frequencies at the HPRT locus). Significantly higher levels of DNA-repair capacity were observed in carriers of GSTM1-plus, compared to those with a deletion in GSTM1. The DNA-repair capacity was significantly lower in individuals with variant Gln/Gln genotype in XRCC1 Arg399Gln than in those with heterozygous Arg/Gln and wild-type Arg/Arg genotypes. Significantly lower repair capacity was also found in individuals with the wild-type Lys/Lys genotype in XPC Lys939Gln as compared with those homozygous for the Gln/Gln variant genotype.     

Biomarkers in styrene-exposed boatbuilders.

14 fiberglass-reinforced plastics (FRP) boatbuilders were compared with 9 unexposed controls with respect to several chemical specific and nonspecific biomarkers measured in peripheral blood. Biomarkers included styrene-hemoglobin adducts (styrene-Hb), sister-chromatid exchanges (SCEs), micronuclei (MN), single-strand breaks (SSBs) and N-acetoxy-2-acetylaminofluorene-induced DNA binding (NA-AAF binding) as a measure of susceptibility to DNA damage. Workers' exposures averaged 11 ppm (8-h TWA; geometric mean) and ranged from 0.6 to 44 p.p.m. Mandelic acid levels were measured in end-of-shift urine samples and reflected an average styrene exposure equivalent to 15 p.p.m. There was a large though not significant difference in levels of styrene-Hb adducts among exposed workers and controls, largely the consequence of a single heavily-exposed individual with an extremely high level of adducts. Significant differences between biomarker levels in exposed workers and controls were observed with MN, SSBs and NA-AAF binding. No significant differences were seen in mean levels of SCEs nor in the incidence of cells with a high frequency of SCEs. The data suggest that exposure to levels of styrene in occupational settings near or below the current OSHA standard (50 p.p.m.) can induce damage at the cellular/molecular level. Appropriately-selected panels of biomarkers can be useful in identifying potentially harmful exposures.     

Analysis of heat-labile sites generated by reactions of depleted uranium and ascorbate in plasmid DNA

The goal of this study was to characterize how depleted uranium (DU) causes DNA damage. Procedures were developed to assess the ability of organic and inorganic DNA adducts to convert to single-strand breaks (SSB) in pBR322 plasmid DNA in the presence of heat or piperidine. DNA adducts formed by methyl methanesulfonate, cisplatin, and chromic chloride were compared with those formed by reaction of uranyl acetate and ascorbate. Uranyl ion in the presence of ascorbate produced U–DNA adducts that converted to SSB on heating. Piperidine, which acted on DNA methylated by methyl methanesulfonate to convert methyl–DNA adducts to SSB, served in the opposite fashion as U–DNA adducts by decreasing the level of SSB. The observation that piperidine also decreased the gel shift for metal–DNA adducts formed by monofunctional cisplatin and chromic chloride was interpreted to suggest that piperidine served to remove U–DNA adducts. Radical scavengers did not affect the formation of uranium-induced SSB, suggesting that SSB arose from the presence of U–DNA adducts and not from the presence of free radicals. A model is proposed to predict how U–DNA adducts may serve as initial lesions that convert to SSB or AP sites. The results suggest that DU can act as a chemical genotoxin that does not require radiation for its mode of action. Characterizing the DNA lesions formed by DU is necessary to assess the relative importance of different DNA lesions in the formation of DU-induced mutations. Understanding the mechanisms of formation of DU-induced mutations may contribute to identification of biomarkers of DU exposure in humans.     

DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).     

Ring test for the determination of N-terminal valine adducts of styrene 7,8-oxide with haemoglobin by the modified Edman degradation technique

A ring-test was organised between three laboratories using different versions of the modified Edman degradation technique for the gas chromatographic-mass spectrometric determination of N-terminal valine adducts of styrene 7,8-oxide. The analyses were performed on a sample of human haemoglobin reacted in vitro with styrene 7,8-oxide and on a set of five haemoglobin samples from mice dosed by i.p. injection of styrene. Strong correlations between the haemoglobin adduct determinations of the different laboratories were observed. However, covariance analysis revealed different slopes for the dose-response curves, indicating differences for the calibration of the reference globin or reference peptide.     

Formation of DNA adducts by microsomal and peroxidase activation of p-cresol: role of quinone methide in DNA adduct formation.

We have investigated the activation of p-cresol to form DNA adducts using horseradish peroxidase, rat liver microsomes and MnO(2). In vitro activation of p-cresol with horseradish peroxidase produced six DNA adducts with a relative adduct level of 8.03+/-0.43 x 10(-7). The formation of DNA adducts by oxidation of p-cresol with horseradish peroxidase was inhibited 65 and 95% by the addition of either 250 or 500 microM ascorbic acid to the incubation. Activation of p-cresol with phenobarbital-induced rat liver microsomes with NADPH as the cofactor; resulted in the formation of a single DNA adduct with a relative adduct level of 0.28+/-0.08 x 10(-7). Similar incubations of p-cresol with microsomes and cumene hydroperoxide yielded three DNA adducts with a relative adduct level of 0.35+/-0.03 x 10(-7). p-Cresol was oxidized with MnO(2) to a quinone methide. Reaction of p-cresol (QM) with DNA produced five major adducts and a relative adduct level of 20.38+/-1.16 x 10(-7). DNA adducts 1,2 and 3 formed by activation of p-cresol with either horseradish peroxidase or microsomes, are the same as that produced by p-cresol (QM). This observation suggests that p-cresol is activated to a quinone methide intermediate by these activation systems. Incubation of deoxyguanosine-3'-phosphate with p-cresol (QM) resulted in a adduct pattern similar to that observed with DNA; suggesting that guanine is the principal site for modification. Taken together these results demonstrate that oxidation of p-cresol to the quinone methide intermediate results in the formation of DNA adducts. We propose that the DNA adducts formed by p-cresol may be used as molecular biomarkers of occupational exposure to toluene.     

Spectrum of styrene-induced DNA adducts: the relationship to other biomarkers and prospects in human biomonitoring

Styrene is an important industrial chemical that has shown genotoxicity in many toxicology assays. This is believed to be related to the DNA-binding properties of styrene-7,8-oxide (SO), a major metabolite of styrene. In this review, we have summarized knowledge on various aspects of styrene genotoxicity, especially in order to understand the formation and removal of primary DNA lesions, and the usefulness of biomarkers for risk assessment. Biological significances of specific DNA adducts and their role in the cascade of genotoxic events are discussed. Links between markers of external and internal exposure are evaluated, as well as metabolic aspects leading to the formation of DNA adducts and influencing biomarkers of biological effect. Finally, we suggest a design of a population study, which may contribute to our understanding genotoxic events in the exposure either to single xenobiotic or complex mixture.     

Measurement of HPRT mutations in splenic lymphocytes and haemoglobin adducts in erythrocytes of Lewis rats exposed to ethylene oxide

Young adult male Lewis rats were exposed to ethylene oxide (EO) via single intraperitoneal (i.p.) injections (10-80 mg kg-1) or drinking water (4 weeks at concentrations of 2, 5, and 10 mM) or inhalation (50, 100 or 200 ppm for 4 weeks, 5 days week-1, 6 h day-1) to measure induction of HPRT mutations in lymphocytes from spleen by means of a cloning assay. N-ethyl-N-nitrosourea (ENU) and N-(2-hydroxyethyl)-N-nitrosourea (HOENU) were used as positive controls. Levels of N-(2-hydroxyethyl)valine (HOEtVal) adducts in haemoglobin (expressed in nmol g-1 globin) were measured to determine blood doses of EO (mmol kg-1 h, mM h). Blood doses were used as a common denominator for comparison of mutagenic effects of EO administered via the three routes. The mean HPRT mutant frequency (MF) of the historical control was 4.3 x 10(-6). Maximal mean MFs for ENU (100 mg kg-1) and HOENU (75 mg kg-1) were 243 x 10(-6) and 93 x 10(-6), respectively. In two independent experiments, EO injections led to a statistically significant dose-dependent induction of mutations, with a maximal increase in MF by 2.3-fold over the background. Administration of EO via drinking water gave statistically significant increases of MFs in two independent experiments. Effects were, at most, 2.5-fold above the concurrent control. Finally, inhalation exposure also caused a statistically significant maximal increase in MF by 1.4-fold over the background. Plotting of mutagenicity data (i.e., selected data pertaining to expression times where maximal mutagenic effects were found) for the three exposure routes against blood dose as common denominator indicated that, at equal blood doses, acute i.p. exposure led to higher observed MFs than drinking water treatment, which was more mutagenic than exposure via inhalation. In the injection experiments, there was evidence for a saturation of detoxification processes at the highest doses. This was not seen after subchronic administration of EO. The resulting HPRT mutagenicity data suggest that EO is a relatively weak mutagen in T-lymphocytes of rats following exposure(s) by i.p. injection, in drinking water or by inhalation.     

Induction and decline of HPRT mutants and deletions following a low dose radiation exposure at Chernobyl

This study was conducted to evaluate the ability of mutation in the hypoxanthine-phosphoribosyltransferase gene (HPRT) to detect radiation-induced mutation in lymphocytes of Russian Chernobyl Clean-up workers, particularly as a function of time after exposure. It is part of a multi-endpoint study comparing HPRT mutation with chromosome translocation and glycophorin A mutation [Radiat. Res. 148 (1997) 463], and extends an earlier report on HPRT [Mutat. Res. 431 (1999) 233] by including data from all 9 years of our study (versus the first 6 years) and analysis of deletion size. Blood samples were collected from 1991 to 1999. HPRT mutant frequency (MF) as determined by the cloning assay was elevated 16% in Clean-up workers (N=300, the entire group minus one outlier) compared to Russian Controls (N=124) when adjusted for age and smoking status (P=0.028). Since exposures occurred over a short relative to the long sampling period, the year of sampling corresponded roughly to the length of time since exposure (correlation coefficient=0.94). When date of blood sample was considered, Control MF was not time dependent. Clean-up worker MF was estimated to be 47% higher than Control MF in 1991 (P=0.004) and to decline 4.4% per year thereafter (P=0.03). A total of 1123 Control mutants and 2799 Clean-up worker mutants were analyzed for deletion type and size by PCR assay for retention of HPRT exons and flanking markers on the X chromosome. There was little difference between the overall deletion spectra of Clean-up workers and Controls. However, there was a decline in the average size of deletions of Clean-up workers as time after exposure at Chernobyl increased from 6 to 13 years (P< or =0.05). The results illustrate the sensitivity of HPRT somatic mutation as a biomarker for populations with low dose radiation exposure, and the dependence of this sensitivity on time elapsed since radiation exposure.     

Biomarkers of air pollution exposure--a study of policemen in Prague

The effect of exposure to organic compounds adsorbed onto respirable air particles (<2.5microm) on DNA adducts in lymphocytes was studied in a group of non-smoking policemen (N=109, aged 35+/-0.9 years) working in the downtown area of Prague and spending >8h daily outdoors. Personal exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) adsorbed on respirable particles was monitored in each subject for 48h before biological sampling. DNA adducts were analyzed by a (32)P-postlabelling assay, and total DNA adduct levels and B[a]P-like spots were determined. Further biomarkers included cotinine levels in urine to control for exposure to tobacco smoke, plasma levels of vitamins A, E and C and polymorphisms of metabolic genotypes (GSTM1, GSTP1, GSTT1, CYP 1A1-Msp I and Ile/Val, MTHFR, MS), DNA repair genotypes (XRCC1, hOGG1 and XPD exons 6 and 23) and the p53 gene (p53 Msp I and BstU I). All the biomarkers of exposure and effect were analyzed repeatedly during a period of one year at 2-3 month intervals (January, March, June, September 2004) to cover periods with high (winter) and low (summer) levels of air pollution. The highest personal exposure to c-PAHs was found in January (8.1+/-8.8ng/m(3)), while the other three sampling periods exhibited 3-4-fold lower c-PAH exposure. The total DNA adducts were only slightly elevated in January (2.08+/-1.60) compared to March (1.66+/-0.65), June (1.96+/-1.73) and September (1.77+/-1.77). B[a]P-like DNA adducts, however, were significantly higher in January than in the March and June sampling periods (0.26+/-0.14 vs. 0.19+/-0.12 and 0.22+/-0.13, respectively; p<0.0001 and p=0.017) indicating that c-PAH exposure probably plays a crucial role in DNA adduct formation in lymphocytes. No effect of individual metabololic or DNA repair genotypes on DNA adduct levels was observed. However, the combination of two genotypes encoding enzymes metabolizing c-PAHs - CYP 1A1 and GSTM1 - was associated with the levels of total and B[a]P-like DNA adducts under conditions of increased exposure to c-PAHs. Our study suggests that DNA adducts in the lymphocytes of subjects exposed to increased c-PAH levels are an appropriate biomarker of a biologically effective dose, directly indicating whether or not the extent of exposure to these compounds is related to an increased mutagenic and carcinogenic risk.     

Measurement of 7 methyl and 7 2 hydroxyethyl guanine DNA adducts in white blood cells of smokers and non smokers

The goal of the present study was to measure the levels of 7-methylguanine and 7-(2- hydroxyethyl)guanine DNA adducts in human white blood cells in relation to smoking. DNA was isolated from samples of 11 smokers and eight non-smokers. The 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts were analysed by thin-layer chromatography (TLC) combined with a high pressure liquid chromatography (HPLC) assay. In smokers the mean 7-methylguanine and 7-(2-hydroxyethyl)guanine levels were 32.3 +/- 7.1 and 6.6 +/- 2.3 adducts per 108 nucleotides respectively. The corresponding values in non-smokers were 25.0 +/- 7.0 and 3.7 +/- 2.4 adducts per 108 nucleotides. There were significantly higher levels of 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts in WBC in smokers than in non-smokers ( p = 0.041; p = 0.018), respectively. A positive correlation between 7-methylguanine and 7-(2-hydroxyethyl)guanine levels was observed.     

Measurement of 7 methyl and 7 2 hydroxyethyl guanine DNA adducts in white blood cells of smokers and non smokers

The goal of the present study was to measure the levels of 7-methylguanine and 7-(2- hydroxyethyl)guanine DNA adducts in human white blood cells in relation to smoking. DNA was isolated from samples of 11 smokers and eight non-smokers. The 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts were analysed by thin-layer chromatography (TLC) combined with a high pressure liquid chromatography (HPLC) assay. In smokers the mean 7-methylguanine and 7-(2-hydroxyethyl)guanine levels were 32.3 +/- 7.1 and 6.6 +/- 2.3 adducts per 108 nucleotides respectively. The corresponding values in non-smokers were 25.0 +/- 7.0 and 3.7 +/- 2.4 adducts per 108 nucleotides. There were significantly higher levels of 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts in WBC in smokers than in non-smokers (p = 0.041; p = 0.018), respectively. A positive correlation between 7-methylguanine and 7-(2-hydroxyethyl)guanine levels was observed.     

The use of biomarkers for improved retrospective exposure assessment in epidemiological studies: summary of an ECETOC workshop

During a scientific workshop the use of biological monitoring in characterization of retrospective exposure assessment was discussed. The workshop addressed currently available methodology and also novel approaches such as in different fields of ‘omics’. For use in epidemiology requiring retrospective exposure assessment, biomarker levels should not vary too much over time. If variability in exposure over time is large and differences in exposure between individuals are relatively small, this may lead to underestimation of the exposure–response relationship. This means that, for a sound assessment of health risk, biomarkers that reflect cumulative exposure over a long period of time are preferred over biomarkers with short half-lives. Most of the existing biomarkers such as metabolites in body fluids usually have rather short half-lives, typically less than 1–2 days. Some adducts to DNA show somewhat longer half-lives. The current limit to persistence of biomarkers reflecting cumulative exposure over time is from adducts to haemoglobin with a half-life of 4 months. Some specific organic substances may be more persistent due to storage in adipose tissue or metals in kidneys, nails and hair. The metabonomics, proteomics and present gene expression profiling approaches do not provide a perspective to the availability of more persistent biomarkers and most approaches discussed to date show that it is difficult to interpret study outcomes in terms of exposure to a specific xenobiotic factor. Research efforts should focus on improvement and validation of currently available approaches in the field of addition products to DNA and proteins. Promising new developments may be phosphotriester DNA adducts and adducts to more long-lived proteins such as histones.     

Haemoglobin adducts as biomarkers of exposure to the herbicides propanil and fluometuron

Abstract

Aromatic amine herbicides, including propanil, fluometuron, alachlor, trifluralin, and pendimethalin, were examined for their ability to form haemoglobin adducts in rats as potential biomarkers of exposure. Many aromatic amines are known to form haemoglobin adducts via conversion to the nitroso metabolite and binding of this metabolite to cysteinyl groups on haemoglobin. Since red blood cells are long lived, adducts formed with these cells may be reliable biomarkers of exposure with the potential for showing progressive accumulation. Gas chromatographic-mass spectrometric analyses of haemoglobin revealed that adducts were formed in rats treated with the rice herbicide propanil and the cotton herbicide fluometuron. Adducts were not detected with the herbicides alachlor, trifluralin, or pendamethalin.     

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.     

Do biomarkers of exposure and effect correlate with internal exposure to PAHs in swine?

Humans are commonly exposed to polycyclic aromatic hydrocarbons (PAHs), a family of compounds present as mixtures in the environment. This study exposed swine to PAH mixtures in single and subacute dose regimens and collected liver and ileum tissue to measure cytochrome P450 mRNA expression and enzyme activity as biomarkers of exposure and DNA adducts and oxidized proteins as biomarkers of effect. Micronucleated reticulocytes were measured as systemic biomarkers of effect. Duration of exposure did not influence biomarkers of exposure, though exposure duration produced significant increases in DNA adducts and oxidative stress. Micronucleated reticulocyte numbers were not affected by exposure length.     

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.     

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.