Polycyclic aromatic hydrocarbon-DNA adducts in human sperm as a marker of DNA damage and infertility

Severe DNA damage, which might prevent egg fertilization or the development of the embryo, could be a cause of infertility. In order to assess whether polycyclic aromatic hydrocarbon (PAH)-DNA adducts are an early marker of sperm genotoxicity and infertility, we studied 205 men consecutively recruited from 1 January to 30 May 2001 through the Infertility Clinic of the University of Milan (Italy), with morphological abnormalities in the sperm. No known causes of infertility were present in their female partners. Sperm were collected after 3-5 days of abstinence, fixed on polylysine slides, and frozen at -20 degrees C. PAH-DNA adducts were measured by immunofluorescence using a polyclonal antiserum. A questionnaire was filled out at the time of the visit, with demographic information, smoking and drinking habits, and occupational history. Data on PAH-DNA adducts were available for 182 men. The mean age of the subjects was 35.5+/-5.0 years; 38.6% of them were current smokers. PAH-DNA adducts were negatively correlated with the percentage of physiologic forms (r=-0.18; P=0.016) and with abnormalities of the neck of the sperm cell (r=-0.21; P>/=0.009), while they were positively correlated with morphological abnormalities of the head (r=0.30; P>0.0001). Occupational exposure to PAH, but not smoking, was significantly associated with higher levels of PAH-DNA adducts. A significant negative association was observed between daily alcohol consumption and PAH-DNA adducts in sperm (P=0.01). PAH-DNA adducts levels were significantly higher in infertile versus fertile men (P=0.04). These results suggest a role for DNA damage in infertility.     

Genotoxicity of 3'-azido-3'-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT.

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10(6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 x 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.     

Bulky DNA adducts in human sperm: relationship with fertility,semen quality,smoking, and environmental factors

The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.     

Immunoperoxidase detection of 4-aminobiphenyl- and polycyclic aromatic hydrocarbons-DNA adducts in induced sputum of smokers and non-smokers

Tobacco smoke constituents, 4-aminobiphenyl (4-ABP) and polycyclic aromatic hydrocarbons (PAH) possess carcinogenic properties as their reactive metabolites form DNA adducts. We studied the formation of 4-ABP- and PAH-DNA adducts in induced sputum, a non-invasively obtainable matrix from the lower respiratory tract, of smokers (n=20) and non-smokers (n=24) utilizing a semi-quantitative immunohistochemical peroxidase assay. Smokers had significantly higher levels of 4-ABP-DNA adducts as compared to non-smokers (0. 08+/-0.02 versus 0.04+/-0.01, P=0.001, density of immunohistochemical staining), and the levels of adducts were related to current smoking indices (cigarettes/day: r=0.3, P=0.04 and tar/day: r=0.4, P=0.02). Likewise, smokers had elevated levels of PAH-DNA adducts as compared to non-smokers, however, the differences was not statistically significant (0.13+/-0.02 versus 0. 08+/-0.02, P=0.07). The levels of PAH-DNA adducts were only significantly related to the amount of tar consumed per day (r=0.4, P=0.04) but not to the number of cigarettes smoked per day. Neither the levels of 4-ABP-DNA adducts nor those of PAH-DNA adducts were related to smoking history index (pack years). Further, the levels of 4-ABP-DNA adducts were correlated with those of PAH-DNA adducts (r=0.4, P=0.02). We conclude that immunohistochemistry of 4-ABP-DNA adducts in induced sputum is a specific approach to assess current exposure to tobacco smoke in the lower respiratory tract, however, in the case of PAH-DNA adducts, such analysis is less specific as it does not explicitly reflect the magnitude of the exposure.     

Lipid peroxidation-derived etheno-DNA adducts in human atherosclerotic lesions

Atherosclerosis and cancer are characterized by uncontrolled cell proliferation and share common risk factors, such as cigarette smoking, dietary habits and ageing. Growth of smooth muscle cells (SMCs) in atherosclerotic plaques may result from DNA damage, caused either by exogenous mutagens or by agents endogenously generated due to oxidative stress and lipid peroxidation (LPO). Hydroxy-2-nonenal (HNE), a major LPO product, binds covalently to cellular DNA to form the exocyclic etheno-DNA-base adducts, 1,N(6)-ethenodeoxyadenine (varepsilondA) and 3,N(4)-ethenodeoxycytosine (varepsilondC). By applying an ultrasensitive (32)P-postlabeling-immunoaffinity method, varepsilondA and varepsilondC were quantified in abdominal aorta SMCs from 13 atherosclerotic patients and 3 non-smoking subjects without atherosclerotic lesions. The levels of etheno-adducts ranged for varepsilondA from 2.3 to 39.6/10(8)dA and for varepsilondC from 10.7 to 157.7/10(8)dC, with a high correlation between varepsilondA and varepsilondC (r=0.84, P=0.0001). Etheno-adduct levels were higher in atherosclerotic smokers than in ex-smokers for both varepsilondA (means 15.2 versus 7.3, P=0.06) and varepsilondC (71.9 versus 51.6, not significant). varepsilondC levels were higher in either ex-smokers (P=0.03) or smokers (P=0.07) than in non-smokers. There was a poor correlation between either varepsilondA or varepsilondC and 8-hydroxy-2'-deoxyguanosine, whereas significant positive correlations were detected with the levels of several postlabeled bulky aromatic DNA adducts. In conclusion, two different types of DNA damage may be involved in atherosclerotic plaque formation and progression: (i) bulky aromatic compounds, to which aorta SMCs are chronically exposed in smokers, can either covalently bind to DNA, induce redox-cycling via quinone intermediates and/or activate local chronic inflammatory processes in the arterial wall; ii) this in turn leads to a self perpetuating generation of reactive oxygen species, LPO-products and increasing DNA-damage, as documented by the presence of high levels of miscoding etheno-DNA adducts in human aorta SMCs.     

Polymorphisms of phase II xenobiotic-metabolizing and DNA repair genes and in vitro N-ethyl-N-nitrosourea-induced O6-ethylguanine levels in human lymphocytes

This study tested the hypothesis that genetic variants of phase II detoxification enzymes and DNA repair proteins affect individual response to DNA damage from alkylating agents. In 171 healthy individuals, an immunoslot blot assay was used to measure O6-ethylguanosine (O6-EtGua) adduct levels in peripheral blood lymphocytes treated with N-ethyl-N-nitrosourea (ENU) in vitro. The genotypes of GSTM1, GSTT1, GSTP1 I(105)V and A(114)V, MGMT L(84)F and I(143)V, XPD D(312)N and K(751)Q, and XRCC3 T(241)M were determined. Demographic and exposure information was collected by in-person interview. Student's t-test, analysis of (co)variance, and multiple linear regression models were used in statistical analyses. The mean and median (range) O6-EtGua levels were 94.6 and 84.8 (3.2-508.1)fmol/g DNA, respectively. The adduct level was significantly lower in people who smoked >or=25 years than that in never-smokers (square-root transformed mean values 8.20 versus 9.37, P=0.03). Multiple linear regression models revealed that GSTT1 (beta=-2.36, P=0.009) polymorphism was a significant predictor of the level of adducts in 82 never-smokers, whereas the number of years smoked (beta=-0.08, P=0.005) and XRCC3 T(241)M (beta=2.22, P=0.007) in 89 ever-smokers. The association between GSTP1 I(105)V, MGMT I(143)V, and XPD D(312)N with the level of adducts was not conclusive. Each polymorphism could explain 2-10% of the variation of the adduct level. These observations suggest that GSTT1 null and XRCC3 T(241)M polymorphism may have some functional significance in modulating the level of ENU-induced DNA damage and these effects are smoking-dependent. Results from this exploratory study need to be confirmed in other experimental systems.     

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).     

A variant estrogen receptor messenger ribonucleic acid is associated with reduced levels of estrogen binding in human mammary tumors

An analysis of the human estrogen receptor (ER) mRNA was performed on 71 human breast tumors using an RNase protection assay. Complementary DNA clones to the human estrogen receptor (lambda R8 and lambda R3) were used to generate small antisense 32P-labeled RNA molecules that were hybridized to the tumor RNA. We determined the relative amounts of ER mRNA in each tumor by measuring the amount of RNases A and T1 resistant hybrids. Moreover, because RNase A has the ability to cleave single-base mismatches within RNA/RNA duplexes, we were able to use the assay to screen for possible mutations or deletions in the ER mRNA. A significant correlation was found between the ER mRNA levels and the estrogen binding concentrations determined by a dextran-coated charcoal assay (r = 0.68; P less than 0.0001; n = 58). We also identified a subpopulation of tumors in which a mismatch in the ER mRNA was detected. This message modification, in the B region of the message, significantly correlated with low levels of estrogen binding. This result suggests that the observed B variant might lead to the production of receptors with altered properties.     

AZT induces oxidative damage to cardiac mitochondria: protective effect of vitamins C and E

AZT (zidovudine) is a potent inhibitor of HIV replication and a major antiretroviral drug used for AIDS treatment. A major limitation in the use of AZT is the occurrence of severe side effects. The aim of this work was to test whether AZT causes oxidative damage to heart mitochondria and whether this can be prevented by supranutritional doses of antioxidant vitamins. An experimental animal model was used in which mice were treated with AZT for 35 days (10 mg/kg/day) in drinking water. Animals treated with antioxidant vitamins were fed the same diet as controls but supplemented with vitamins C (ascorbic acid, 10 g/ kg diet) and E (alpha-dl-tocopherol, 0.6 g/kg diet) for 65 days before sacrifice. This resulted in a daily intake of 1250 mg/kg/day (vitamin C) and 75 mg/kg/day (vitamin E). Cardiac mitochondrial DNA (mtDNA) of mice treated with AZT had over 120% more oxo-dG (8-oxo-7,8-dihydro-2'-deoxyguanosine, which is a biomarker of oxidative damage to DNA) in their mitochondrial DNA than untreated controls. AZT treatment also caused an increase in mitochondrial lipid peroxidation and an oxidation of mitochondrial glutathione. Dietary supplementation with supranutritional doses of the antioxidant vitamins C and E protected against these signs of mitochondrial oxidative stress. The oxidative effects of AZT are probably due to an increase in production of reactive oxygen species by mitochondria of AZT-treated animals, raising the possibility that oxidative stress may play an important role in the cardiotoxicity of AZT.     

A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of N-methyl-4-aminoazobenzene

N-Methyl-4-aminoazobenzene (MAB) is believed to be metabolized in the liver to an electrophilic N-sulfonyloxy ester which binds covalently to cellular macromolecules, resulting in the induction of hepatic neoplasia. Previous in vivo studies in the rat detected only two hepatic MAB-DNA adducts, 3-(deoxyguanosin-N²-yl)-MAB(N²-dG) and N-(deoxyguanosin-8-yl)-MAB(C8-dG), which respectively accounted for 25% and 70% of the total MAB bound to DNA at 8 h after a single dose of the carcinogen. Subsequently, the C8-dG adduct was shown to be rapidly lost from the DNA while the N²-dG adduct was a persistent lesion. Since a single dose of MAB is not sufficient for complete carcinogenic activity, we sought to identify the MAB-DNA adducts present in rat liver after multiple oral doses of [³H]MAB. The MAB was administered by intubation at a level of 0.2 mmol/kg for 1, 3 or 4 doses and animals were sacrificed at 8 h after the last dose. Hepatic DNA was isolated by extraction and hydroxylapatite chromatography and was enzymatically hydrolyzed to MAB-mononucleoside adducts, which were quantitated by high pressure liquid chromatography (HPLC). After 3 doses, N²-dG, C8-dG, and an unknown adduct were detected. By 4 doses, these accounted for 51%, 25% and 23% of the total adducts. This data is consistent with rapid removal of the C8-dG derivative and the relative persistence of the N²-dG and the unknown adduct. The latter was shown to exhibit chromatographic and pH-dependent solvent partitioning properties that were identical to a product also present in DNA treated with the synthetic ultimate carcinogen, N-benzoyloxy-MAB. Analysis of this adduct by field desorption mass spectrometry (M⁺ = 460) and, after perdeuteromethylation, by electron impact mass spectrometry (M⁺ = 528; M-N(CH₃)(CD₃) = 481) indicated the structure to be a deoxyadenosin-N⁶-yl derivative substituted through an aromatic ring of MAB. Further analysis by 270 MHz ¹H-NMR spectroscopy allowed complete assignment of the MAB and adenyl resonances and was uniquely consistent with a 3-(deoxyadenosin-N⁶-yl)-MAB structure. Since this persistent adduct is potentially mutagenic due to possible tautomeric equilibria between the N⁶-amino and N⁶-imino structures, it may represent an initiating lesion in MAB hepatocarcinogenesis.     

Detection of DNA adducts using a quantitative long PCR technique and the fluorogenic 5′ nuclease assay (TaqMan)

The detection of DNA adducts is an important component in assessing the mutagenic potential of exogenous and endogenous compounds. Here, we report an in vitro quantitative long PCR (XL-PCR) assay to measure DNA adducts in human genomic DNA based on their ability to block and inhibit PCR amplification. Human genomic DNA was exposed to test compounds and then a target sequence was amplified by XL-PCR. The amplified sequence was then quantified using fluorogenic 5′ nuclease PCR (TaqMan^®) and normalized to a solvent-treated control. The extent of DNA adduction was determined based on the reduction in amplification of the target sequence in the treated sample. A 17.7 kb β-globin fragment was chosen as the target sequence for these studies, since preliminary experiments revealed a two-fold increased sensitivity of this target compared to a 10.4 kb HPRT fragment for detecting hydrogen peroxide-induced DNA damage. Validation of the XL-PCR assay with various compounds demonstrated the versatility of the assay for detecting a wide range of adducts formed by direct acting or S9-activated mutagens. The same DNA samples were also analyzed using ³²P-postlabeling techniques (thin-layer chromatography or high-performance liquid chromatography) to confirm the presence of DNA adducts and estimate their levels. Whereas ³²P-postlabeling with nuclease P₁ enrichment was more sensitive for detecting bulky adducts induced by the compounds benzo[a]pyrene, dimethylbenzanthracene, 3-methylindole, indole 3-carbinol, or 2-acetylaminofluorene, the XL-PCR procedure was more sensitive for detecting smaller or labile DNA adducts formed by the compounds methyl methanesulfonate, diethyl nitrosamine, ethylnitrosourea, diepoxybutane, ICR-191, styrene oxide, or aflatoxin B₁. Compounds not expected to form adducts in DNA, such as clofibrate, phenobarbital, chloroform or acetone, did not produce a positive response in the XL-PCR assay. Thus, quantitative XL-PCR provides a rapid, high-throughput assay for detecting DNA damage that complements the existing ³²P-postlabeling assay with nuclease P₁ enrichment.     

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.     

Repair kinetics of trans-4-hydroxynonenal-induced cyclic 1,N2-propanodeoxyguanine DNA adducts by human cell nuclear extracts

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [(32)P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were approximately 1200/10(6) dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of alpha[(32)P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [(32)P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 degrees C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.     

Influence of GSTM1 genotype on association between aromatic DNA adducts and urinary PAH metabolites in incineration workers

Waste incinerating workers are exposed to various pyrolysis products including polycyclic aromatic hydrocarbons (PAHs). We examined their PAH exposure by assessing urinary 1-hydroxypyrene glucuronide (1-OHPG), as a measure of internal dose, and aromatic DNA adducts in peripheral white blood cells (WBCs), as a measure of biological effect dose. The potential effect of genetic polymorphisms of three enzymes involved in PAH metabolisms (i.e., CYP1A1, GSTM1, and GSTT1) on these exposure markers was also investigated.Twenty-nine employees including workers incinerating industrial wastes and 21 non-exposed on-site controls were recruited from a company handling industrial wastes in South Korea. Sixteen ambient PAHs were determined by GC/MSD (NIOSH method) from personal breathing zone samples of nine subjects working near incinerators. Urinary 1-OHPG was assayed by synchronous fluorescence spectroscopy (SFS) after immunoaffinity purification using monoclonal antibody 8E11. Aromatic DNA adducts in peripheral WBC were measured by the nuclease P1-enhanced post-labelling assay. Genotypes were assessed by PCR-based methods. Information on smoking habits and use of personal protective equipment were collected by self-administered questionnaire.Urinary 1-OHPG levels were significantly higher in workers handling industrial wastes than in those with presumed lower exposure to PAHs (P=0.006, by Kruskal-Wallis test). A statistically significant dose-response increase in 1-OHPG levels was seen with the number of cigarettes consumed per day (r=0.686, P<0.001). Smoking and GSTM1 genotype were significant predictors for log-transformed 1-OHPG by multiple regression analysis (overall model R(2)=0.565, P<0.001), whereas smoking was the only significant predictor for log-transformed aromatic DNA adducts (overall model R(2)=0.249, P=0.201). Aromatic DNA adducts were significantly correlated with log-transformed urinary 1-OHPG level (r=0.31, P=0.04). However, the partial correlation coefficient adjusting for age, sex, and cigarette consumption was not significant (r=0.15, P=0.17). The significant association exists only in individuals with the GSTM1 null genotype (Pearson's correlation coefficient, r=0.52, P=0.01; partial correlation coefficient adjusting for age, sex, and cigarette consumption, r=0.36, P=0.04).Our results suggest that the significant increase in urinary 1-OHPG in the exposed workers is due to higher prevalence of smokers among them, and that the association between urinary PAH metabolites and aromatic DNA adducts in workers of industrial waste handling may be modulated by GSTM1 genotype. These results remain to be confirmed in future larger studies.     

Investigating DNA adduct-targeted mutagenicity of tamoxifen: preferential formation of tamoxifen-DNA adducts in the human p53 gene in SV40 immortalized hepatocytes but not endometrial carcinoma cells

Tamoxifen is a widely used drug for chemotherapy and chemoprevention of breast cancer worldwide. Tamoxifen therapy is, however, associated with an increased incidence of endometrial cancer. The carcinogenicity of tamoxifen is ascribed to its genotoxic and estrogen agonist effects. We investigated DNA adduct-targeted mutagenicity of tamoxifen as a function of its genotoxicity in the cII transgene in Big Blue mouse embryonic fibroblasts and mapped the formation of tamoxifen-induced DNA adducts in the p53 tumor suppressor gene in SV40 immortalized human hepatocytes and human endometrial carcinoma cells. We used the terminal transferase-dependent polymerase chain reaction for mapping of DNA adducts in the cII and p53 genes. We utilized a lambda phage-based assay and DNA sequencing for determining cII mutant frequency and mutation spectrum, respectively. Tamoxifen treatment yielded polymerase-blocking DNA adducts at multiple nucleotide positions along the cII transgene. The treatment significantly and dose-dependently increased the cII mutant frequency (p < 0.01), leaving a unique mutation spectrum (p < 0.0001) and a signature mutation of G:C --> T:A transversions (p < 0.03), relative to the control. Tamoxifen treatment of the immortalized human hepatocytes but not endometrial carcinoma cells, even in the presence of an external activation system, i.e., rat liver S9 mix, induced DNA adducts at specific codons along exons 6 and 8 of the p53 gene. These data suggest a proficient metabolic activation of tamoxifen in human liver and an inefficient activation and/or efficient detoxification of tamoxifen in human endometrium. Because the liver is essentially a mitotically quiescent organ, tamoxifen-DNA adduction in the liver may, at least partially, prevent its reactants from reaching highly proliferative organs via, e.g., circulating blood. Thus, tamoxifen-DNA adduction in the liver may not have as significant biological consequences as it might have in highly proliferative organs. Our findings favor an involvement of a nongenotoxic mechanism in tamoxifen-associated human endometrial cancer.     

Mannose binding lectin: a biomarker of systemic lupus erythematosus disease activity

Introduction

A role for mannose binding lectin (MBL) in autoimmune diseases has been demonstrated earlier and elevated level of MBL has been shown in systemic lupus erythematosus (SLE) patients. In the current study, we investigated MBL as a potential biomarker for disease activity in SLE.

Methods

In a case control study SLE patients (93 females) and 67 age, sex, ethnicity matched healthy controls were enrolled. Plasma MBL levels were quantified by enzyme linked immunosorbent assay (ELISA). Clinical, serological and other markers of disease activity (C3, C4 and anti-dsDNA) were measured by standard laboratory procedures.

Results

Plasma MBL levels were significantly high in SLE patients compared to healthy controls (P < 0.0001). MBL levels were variable in different clinical categories of SLE. Lower levels were associated with musculoskeletal and cutaneous manifestations (P = 0.002), while higher and intermediate MBL levels were significantly associated with nephritis in combination with other systemic manifestations (P = 0.01 and P = 0.04 respectively). Plasma MBL correlated with systemic lupus erythematosus disease activity index (SLEDAI) (P = 0.0003, r = 0.36), anti-dsDNA (P < 0.0001, r = 0.54), proteinuria (P < 0.0001, r = 0.42) and negatively correlated with C3 (P = 0.007, r = -0.27) and C4 (P = 0.01, r = -0.29).

Conclusions

Plasma MBL is a promising marker in the assessment of SLE disease activity.     

Sensitivity of CFU-GM from normal human bone marrow and leukaemic clonogenic cells (CFU-L) from blood of patients with myelogenous leukaemia to 3'-deoxy-3'-fluorothymidine in comparison to 3'-azido-3'-deoxythymidine

3'-Deoxy-3'-fluorothymidine (FT), a thymidine analogue highly effective against HIV 1 in vitro was investigated as to its in vitro effect on normal human bone marrow CFU-GM (agar colony assay) and on human peripheral myeloid leukaemic clonogenic cells (CFU-L, colony assay in methylcellulose). For comparison, 3'-azido-3'-deoxythymidine (AZT), structurally related and used in AIDS treatment, was included in the study. Both compounds inhibit the formation of clusters and colonies from bone marrow stem cells with an [IC]50 between 10(-6) and 10(-5)M. In concentrations only 5-10 times lower than the [IC]50, FT begins to stimulate cluster and colony formation. AZT and FT also inhibit the formation of clusters and colonies from CFU-L. Compared to CFU-GM, CFU-L were more sensitive to FT, and a stimulation was not seen. It is concluded that similar side effects on bone marrow could be expected for possible use of FT against AIDS as have been found for AZT and that both compounds are potential candidates for anti-leukaemic drugs.