Anne Spickenheuer

Common variants in the UBC9 gene encoding the SUMO-conjugating enzyme are associated with breast tumor grade

UBC9 encodes a protein that conjugates small ubiquitin‐related modifier (SUMO) to target proteins resulting in a change of their localization, activity or stability. Genetic variability may affect expression and activity of UBC9 and may have an impact on breast tumor progression. We investigated associations between UBC9 genotypes and histopathological parameters in 1,021 breast cancer cases of the GENICA collection using a single nucleotide polymorphism (SNP) tagging approach. Genotyping analyses were performed by TaqMan® allelic discrimination. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by ordinal logistic regression. Multiple imputation based on HapMap data was applied to boost the power of the study. The study revealed significant associations of three UBC9 SNPs with histological grade (rs7187167, ptrend = 0.001; rs11248866, ptrend = 0.009; rs8052688, ptrend = 0.008). Model selection identified a recessive penetrance model for rs7187167 as the best representation of tumor grade (global p = 0.001). This model did not improve by inclusion of additional SNPs in linkage disequilibrium. Imputation of SNPs in a 300 kb region around the genotyped SNPs supported rs7187167 as a major contributor to tumor grade. Compared with common allele carriers, rare homozygotes presented less frequently with high grade tumors (G3 vs. G1: OR 0.26, 95% CI 0.11–0.62; G3 vs. G2: OR 0.45, 95% CI 0.23–0.86). In addition to tumor size, nodal status and estrogen receptor status, multivariate analyses confirmed an independent role of rs7187167 as predictor of tumor grade (p = 0.0003). The present results underline the value of genetic variation in UBC9 for breast cancer prognosis.

Development of a Sandwich ELISA to Measure Exposure to Occupational Cow Hair Allergens

Background: Cow hair and dander are important inducers of occupational allergies in cattle-exposed farmers. To estimate allergen exposure in farming environments, a sensitive enzyme immunoassay was developed to measure cow hair allergens. Methods: A sandwich ELISA was developed using polyclonal rabbitantibodies against a mixture of hair extracts from different cattle breeds. To assess the specificity of the assay, extracts from other mammalian epithelia, mites, molds and grains were tested. To validate the new assay, cow hair allergens were measured in passive airborne dust samples from the stables and homes of farmers. Dust was collected with electrostatic dust fall collectors (EDCs). Results: The sandwich ELISA was found to be very sensitive (detection limit: 0.1 ng/ml) and highly reproducible, demonstrating intra- and interassay coefficients of variation of 4 and 10%, respectively. The assay showed no reactivity with mites, molds and grains, but some cross-reactivity with other mammalian epithelia, with the strongest reaction with goat. Using EDCs for dust sampling, high concentrations of bovine allergens were measured in cow stables (4,760–559,400 µg/m2). In addition, bovine allergens were detected in all areas of cattle farmer dwellings. A large variation was found between individual samples (0.3–900 µg/m2) and significantly higher values were discovered in changing rooms. Conclusion: The ELISA developed for the detection of cow hair proteins is a useful tool for allergen quantification in occupational and home environments. Based on its low detection limit, this test is sensitive enough to detect allergens in passive airborne dust.

New Biomarkers of Occupational Exposure to Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAH) are metabolized in a complex manner. Although biological activity is associated with diol-epoxide formation, phenolic metabolites have predominantly been used in human biomonitoring. In this study monohydroxylated and new metabolites were characterized as biomarkers for occupational PAH exposure. In 97 male workers, personal exposure to 16 airborne PAH compounds was measured during shift. In postshift urine, 1-hydroxypyrene and 1,6- and 1,8-dihydroxypyrene (1-OHP, DiOHP) were determined as metabolites of pyrene (P), and the sum of 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrenes (OHPHE), and PHE-dihydrodiols (PHED) as metabolites of phenanthrene (PHE). The referent group comprised 21 nonsmoking construction workers. Median (interquartile range) shift concentrations of airborne P and PHE were 1.46 (0.62–4.05 μg/m3) and 10.9 (3.69–23.77 μg/m3), respectively. The corresponding parameters were 3.86 (2.08–7.44) μg/g creatinine (crn) for 1-OHP, 0.66 (0.17–1.65) μg/g crn for DiOHP, 11.44 (5.21–34.76) μg/g crn for OHPHE, and 12.28 (3.3–97.76) μg/g crn for PHED in PAH-exposed workers. The median levels of 1-OHP and OHPHE were 0.09 (0.08–0.17 μg/m3) and 0.59 (0.45–1.39 μg/m3), respectively, in the referents. PHE correlated significantly with OHPHE and PHED, and P with 1-OHP but not with DiOHP. Under a doubling of PHE, OHPHE increased by a factor of 1.56 and PHED by 1.57. With a doubling of P, 1-OHP rose by 1.31 and DiOHP by 1.27. P is predominantly metabolized into 1-OHP, whereas PHE is metabolized equally into OHPHE and PHED. Thus metabolites of PHE were found as reliable biomarkers for PAH exposure.

Irritative effects of vapours and aerosols of bitumen on the airways assessed by non-invasive methods

Irritative effects caused by vapours and aerosols of bitumen were assessed by non-invasive methods including spirometry, nasal lavage fluid (NALF) and induced sputum (IS) in a cross-shift study comparing 320 bitumen-exposed workers with 118 road construction workers as the reference group. Lung function parameters, forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) were within normal ranges in both the reference and the bitumen-exposed groups pre- and post-shift with marginally lower values in smokers of both groups. During the shift, a slight decline in FEV1 and FVC was observed in the bitumen-exposed group independent of their smoking habits, whereas in the non-smoking reference group, the decline in FEV1 was not observed. No significant differences between bitumen-exposed workers and the reference group and no significant shift effect were observed on the upper airways using NALF analysis. The IS concentrations of interleukin (IL)-8, total protein and matrix metalloproteinase-9 were significantly higher in bitumen-exposed workers than in the reference group. However, the concentration of these three biomarkers in the IS samples, which are indicators of inflammatory effects on the lower airways of bitumen-exposed workers, was already higher in exposed workers before shift and did not show an increase during the shift. Therefore, the key finding of this aspect of the Human Bitumen Study is the detection of potentially (sub-) chronic irritative inflammatory effects in the lower airways of bitumen-exposed workers.

DNA adducts and strand breaks in workers exposed to vapours and aerosols of bitumen: associations between exposure and effect

To study the associations between exposure to vapours and aerosols of bitumen and genotoxic effects, a cross-sectional and cross-shift study was conducted in 320 exposed workers and 118 non-exposed construction workers. Ambient air measurements were carried out to assess external exposure to vapours and aerosols of bitumen. Hydroxylated metabolites of naphthalene, phenanthrene and pyrene were measured in urine, whereas (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide ((+)-anti-BPDE), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8oxodGuo) and DNA strand breaks were determined in blood. Significantly higher levels of 8-oxodGuo adducts and DNA strand breaks were found in both pre- and post-shift blood samples of exposed workers compared to those of the referents. No differences between exposed workers and referents were observed for (+)-anti-BPDE. Moreover, no positive associations between DNA damage and magnitude of airborne exposure to vapours and aerosols of bitumen could be observed in our study. Additionally, no relevant association between the urinary metabolites of PAH and the DNA damage in blood was observed. Overall, our results indicate increased oxidative DNA damage in workers exposed to vapours and aerosols of bitumen compared to non-exposed referents at the group level. However, increased DNA strand breaks in bitumen workers were still within the range of those found in non-exposed and healthy persons as reported earlier. Due to the lack of an association between oxidative DNA damage and exposure levels at the workplaces under study, the observed increase in genotoxic effects in bitumen workers cannot be attributed to vapours and aerosols of bitumen.

Levels and determinants of exposure to vapours and aerosols of bitumen.

Bitumen (referred to as asphalt in the United States) is a widely used construction material, and emissions from hot bitumen applications have been a long-standing health concern. One objective of the Human Bitumen Study was to identify potential determinants of the exposure to bitumen. The study population analysed comprised 259 male mastic asphalt workers recruited between 2003 and 2008. Personal air sampling in the workers’ breathing zone was carried out during the shift to measure exposure to vapours and aerosols of bitumen. The majority of workers were engaged in building construction, where exposure levels were lower than in tunnels but higher than at road construction sites. At building construction sites, exposure levels were influenced by the room size, the processing temperature of the mastic asphalt and the job task. The results show that protective measures should include a reduction in the processing temperature.

N-acetyltransferase 2, exposure to aromatic and heterocyclic amines, and receptor-defined breast cancer.

The role of N-acetyltransferase 2 (NAT2) polymorphism in breast cancer is still unclear. We explored the associations between potential sources of exposure to aromatic and heterocyclic amines (AHA), acetylation status and receptor-defined breast cancer in 1020 incident cases and 1047 population controls of the German GENICA study. Acetylation status was assessed as slow or fast. Therefore, NAT2 haplotypes were estimated using genotype information from six NAT2 polymorphisms. Most probable haplotypes served as alleles for the deduction of NAT2 acetylation status. The risks of developing estrogen receptor &agr; (ER) and progesterone receptor (PR)-positive or negative tumors were estimated for tobacco smoking, consumption of red meat, grilled food, coffee, and tea, as well as expert-rated occupational exposure to AHA with logistic regression conditional on age and adjusted for potential confounders. Joint effects of these factors and NAT2 acetylation status were investigated. Frequent consumption of grilled food and coffee showed higher risks in slow acetylators for receptor-negative tumors [grilled food: ER−: odds ratio (OR) 2.57, 95% confidence interval (CI) 1.07–6.14 for regular vs. rare; coffee: ER−: OR 2.55, 95% CI 1.22–5.33 for ≥4 vs. 0 cups/day]. We observed slightly higher risks for never smokers that are fast acetylators for receptor-positive tumors compared with slow acetylators (ER−: OR 1.32, 95% CI 1.00–1.73). Our results support differing risk patterns for receptor-defined breast cancer. However, the modifying role of NAT2 for receptor-defined breast cancer is difficult to interpret in the light of complex mixtures of exposure to AHA.

Polymorphisms in the UBC9 and PIAS3 genes of the SUMO-conjugating system and breast cancer risk

SUMOylation consists in the covalent conjugation of small ubiquitin-related modifiers to target proteins. SUMOylation participates in processes that are tightly linked to tumorigenesis, and genetic variability in the SUMO-conjugating system may influence the development of breast cancer. We recently reported that variation in the UBC9 gene encoding the SUMO-conjugating enzyme may affect the grade of breast tumors. Following comprehensive in silico analyses for detection of putative functional polymorphisms in 14 genes of the SUMO system, we selected one coding SNP in PIAS3 and seven tag SNPs in UBC9 for association analyses. Results were based on 1,021 cases, and 1,015 matched controls from the population-based GENICA study. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by conditional logistic regression. To explore the association with polymorphisms closely linked to the genotyped variants, multiple imputation based on HapMap data was applied. The study revealed associations of four UBC9 polymorphisms with risk of grade 1 tumors. Comparison of genotype and haplotype models indicated that the best representation of risk solely relied on rs7187167 under dominant penetrance. Women carrying the rare allele showed an increased risk of grade 1 tumors compared with common homozygotes (OR 1.87, 95% CI 1.18–2.95). This effect appeared to be stronger in women with a family history of breast or ovarian cancer. Imputation of polymorphisms in a 300-kb region around the genotyped polymorphisms identified no variants with stronger associations. Our findings suggest that genetic variation in UBC9 may affect the risk of grade 1 breast tumors.

Polymorphic loci of E2F2, CCND1 and CCND3 are associated with HER2 status of breast tumors.

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast tumors is associated with bad prognosis. Therefore, it is highly relevant to further improve understanding of the regulatory mechanisms of HER2 expression. In addition to gene amplification, transcriptional regulation plays a crucial role in HER2 overexpression. In this study, we analyzed 3 polymorphisms E2F2_‐5368_A>G, CCND1_870_A>G and CCND3_‐677_C>T located in genes involved in cell cycle regulation in the GENICA population‐based and age‐matched breast cancer case‐control study from Germany. We genotyped 1,021 cases and 1,015 controls by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Statistical analyses were performed by conditional logistic regression. We observed no differences in genotype frequencies between breast cancer cases and controls. Subgroup analysis showed associations between carriers of the E2F2_‐5368_G allele (OR: 0.60, 95% CI: 0.42–0.85), carriers of the CCND1_870_G allele (OR: 0.66, 95% CI: 0.45–0.96) and carriers of the CCND3_‐677_T allele (OR: 1.72, 95% CI: 1.20–2.49) and HER2 expression in breast tumors. This finding points to an association of an increased expression of these cell cycle regulators with lower expression of HER2. An explanation for this observation might be that low expression of E2F2, CCND1 and CCND3 decrease levels of factors down‐regulating HER2. We conclude that the analyzed polymorphisms located in E2F2, CCND1 and CCND3 are potential markers for HER2 status of breast tumors.

The earwax-associated SNP c.538G>A (G180R) in ABCC11 is not associated with breast cancer risk in Europeans

Genetic polymorphisms of human ABC-transporter genes have been suggested to modulate breast cancer risk in the general population. In particular ABCC11 (MRP8), which is highly expressed in breast cancer tissue and involved in the efflux of conjugated estrogen metabolites such as estrone-3-sulfate and estradiol-17beta-glucuronide, has recently been proposed as a potential risk factor for female breast cancer. The wet earwax-associated G-allele of the c.538G>A polymorphism was associated with an increased risk for breast cancer in Japanese women. In contrast, no evidence for such an association could be observed in Caucasian women. We aimed to confirm/refute the association of the c.538G>A variant in ABCC11 with breast cancer risk and/or histo-pathological tumor characteristics in an independent population-based breast cancer case–control study from Germany comprising 1021 cases and 1015 age-matched controls. No association for allele and genotype frequencies of the 538G>A variant in ABCB11 with breast cancer risk was found. Our data suggest that the c.538G>A variation in ABCC11 does not contribute to breast carcinogenesis in women of European descent.