Bladder cancer course, four genetic high-risk variants, and histopathological findings.

Urinary bladder cancer, a smoking and occupation related disease, was subject of several genome-wide association studies (GWAS). However, studies on the course of the disease based on GWAS findings differentiating between muscle invasive bladder cancer (MIBC) and non-muscle invasive bladder cancer (NMIBC) are rare. Thus we investigated 4 single nucleotide polymorphisms (SNPs) detected in GWAS, related to the genes coding for TACC3 (transforming, acidic coiled-coil containing protein 3), for FGFR3 (fibroblast growth factor receptor 3), for PSCA (prostate stem cell antigen) and the genes coding for CBX6 (chromobox homolog 6) and APOBEC3A (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A). This study is based on 712 bladder cancer patients and 875 controls from 3 different case control studies in Germany. The 4 SNPs of interest (PSCA rs2294008 and rs2978974, FGFR3-TACC3 rs798766, and CBX6-APOBEC3A rs1014971) were determined by real-time polymerase chain reaction. The distribution of the 4 SNPs does not vary significantly between cases and controls in the entire study group and in the 3 local subgroups, including two former highly industrialized areas and a region without such history. Also, no significant differences in the bladder cancer subgroups of MIBC and NMIBC were observed. The 4 investigated SNPs do not noticeably contribute differently to the bladder cancer risk for the bladder cancer subgroups of MIBC and NMIBC.

A short-term inhalation study to assess the reversibility of sensory irritation in human volunteers.

Sensory irritation is an acute adverse effect caused by chemicals that stimulate chemoreceptors of the upper respiratory tract or the mucous membranes of the outer eye. The avoidance of this end point is of uttermost importance in regulatory toxicology. In this study, repeated exposures to ethyl acrylate were analyzed to investigate possible carryover effects from day to day for different markers of sensory irritation. Thirty healthy subjects were exposed for 4 h on five subsequent days to ethyl acrylate at concentrations permitted by the German occupational exposure limit at the time of study. Ratings of eye irritation as well as eye blinking frequencies indicate the elicitation of sensory irritation. These markers of sensory irritation showed a distinct time course on every single day. However, cumulative carryover effects could not be identified across the week for any marker. The rhinological and biochemical markers could not reveal hints for more pronounced sensory irritation. Neither increased markers of neurogenic inflammation nor markers of immune response could be identified. Furthermore, the performance on neurobehavioral tests was not affected by ethyl acrylate and despite the strong odor of ethyl acrylate the participants improved their performances from day to day. While the affected physiological marker, the increased eye blinking frequency stays roughly on the same level across the week, subjective markers like perception of eye irritation decrease slightly from day to day though the temporal pattern of, i.e., eye irritation perception stays the same on each day. A hypothetical model of eye irritation time course derived from PK/PD modeling of the rabbit eye could explain the within-day time course of eye irritation ratings repeatedly found in this study more precisely.

Study of correlation between the NAT2 phenotype and genotype status among Greenlandic Inuit.

N-acetyltransferase 2 (NAT2) is the main enzyme metabolizing isoniazid and genotype-based treatment has been studied for years without becoming common practice. To investigate whether genotype-based isoniazid treatment is feasible in Greenland, we sequenced the coding sequence of NAT2 and determined the NAT2 enzyme-activity by caffeine test. No additional genetic variants were identified in the coding sequence of NAT2, so that genotype status in 260 study participants could be assessed by a well-established 7-SNP panel. Studying the enzyme activity by the ratio of the two caffeine metabolites AFMU and 1X in 260 participants showed a high rate of slow phenotypes with intermediate or rapid genotype. These misclassifications were mainly observed in urine samples with pH<3, a deviation from the standard protocol due to the field work character of the study, where immediate pH adjustment to pH=3.5 was not possible. We excluded these samples. For the remaining 143 individuals with pH>3, we observed a moderate level of discrepancies (19 of the 116 individuals with intermediate or rapid genotype status having a slow phenotype). Further investigation showed that drinking coffee and not tea or cola was the most important factor for high levels of both metabolites. The concordance between phenotype and genotype status with regard to slow metabolism supported the recommendation of lower isoniazid doses in individuals with slow genotype status in order to avoid liver injury, a frequent side effect. The phenotypical variation observed for individuals with intermediate or rapid genotype status warrants further research before increased dosing of isoniazid can be recommended.

Algorithm for the Automated Evaluation of NAT2 Genotypes.

N-Acetyltransferase 2 (NAT2) genotyping by PCR and RFLP-based methods provides information on seven single nucleotide polymorphisms (SNPs) without deriving the chromosomal phase (haplotype). So genotyping results must be processed to get all possible NAT2 haplotype (or allele) combinations. Here we describe the procedure for genotyping and present a program based on Microsoft® Access® which automatically generates all possible haplotype pairs for a given unphased NAT2 genotype. NAT2 haplotypes are important to predict the NAT2 phenotype.

Identification and replication of the interplay of four genetic high-risk variants for urinary bladder cancer.

Little is known whether genetic variants identified in genome-wide association studies interact to increase bladder cancer risk. Recently, we identified two- and three-variant combinations associated with a particular increase of bladder cancer risk in a urinary bladder cancer case-control series (Leibniz Research Centre for Working Environment and Human Factors at TU Dortmund (IfADo), 1501 cases, 1565 controls). In an independent case-control series (Nijmegen Bladder Cancer Study, NBCS, 1468 cases, 1720 controls) we confirmed these two- and three-variant combinations. Pooled analysis of the two studies as discovery group (IfADo-NBCS) resulted in sufficient statistical power to test up to four-variant combinations by a logistic regression approach. The New England and Spanish Bladder Cancer Studies (2080 cases and 2167 controls) were used as a replication series. Twelve previously identified risk variants were considered. The strongest four-variant combination was obtained in never smokers. The combination of rs1014971[AA] near apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) and chromobox homolog 6 (CBX6), solute carrier family 1s4 (urea transporter), member 1 (Kidd blood group) (SLC14A1) exon single nucleotide polymorphism (SNP) rs1058396[AG, GG], UDP glucuronosyltransferase 1 family, polypeptide A complex locus (UGT1A) intron SNP rs11892031[AA] and rs8102137[CC, CT] near cyclin E1 (CCNE1) resulted in an unadjusted odds ratio (OR) of 2.59 (95% CI = 1.93-3.47; P = 1.87 × 10-10), while the individual variant ORs ranged only between 1.11 and 1.30. The combination replicated in the New England and Spanish Bladder Cancer Studies (ORunadjusted = 1.60, 95% CI = 1.10-2.33; P = 0.013). The four-variant combination is relatively frequent, with 25% in never smoking cases and 11% in never smoking controls (total study group: 19% cases, 14% controls). In conclusion, we show that four high-risk variants can statistically interact to confer increased bladder cancer risk particularly in never smokers.

Occupational bladder cancer: Polymorphisms of xenobiotic metabolizing enzymes, exposures, and prognosis.

Approximately 7% of all bladder cancer cases in males are associated with occupation. The question arises whether the use of genome-wide association studies was able to identify bladder cancer risk factors that may modulate occupational bladder cancer risk and prognosis. One hundred and forty-three bladder cancer cases with suspected occupational bladder cancer and 337 controls were genotyped for the following polymorphisms: N-acetyltransferase 2 (NAT2), glutathione S-transferase M1 (GSTM1), glutathione S-transferase T1 (GSTT1), UDP-glucuronyltransferase 1A rs11892031 (UGT1A), rs9642880 (close to c-MYC), and rs710521 (close to TP63). The most relevant polymorphisms for occupational bladder cancer risk were GSTM1 and UGT1A, especially when co-occurring (GSTM1 negative and rs11892031[A/A]: 48% cases vs. 38% controls, OR 1.47, 95% CI 0.99-2.20). The effect was more pronounced in smokers. GSTM1 negative genotype occurred more frequently in cancer cases exposed to aromatic amines, carbolineum, and in painters and varnishers. UGT1A (rs11892031[A/A]) was found frequently in cases exposed to carbolineum, crack test spray, PAH, and in painters and varnishers. All investigated polymorphisms except rs710521 (TP63) seemed to exert an impact on recurrence risk. Relapse-free times were shorter for NAT2 slow and ultra-slow, GSTT1 positive and GSTM1 negative cases. Occupational bladder cancer cases with a number of risk variants displayed significantly shorter relapse-free times compared to cases with few, less relevant risk alleles as evidenced by median difference 8 months. In conclusion, in the present, suspected occupational bladder cancer cases phase II polymorphisms involved in bladder carcinogen metabolism modulate bladder cancer recurrence. Most relevant for bladder cancer risk were GSTM1 and UGT1A but not NAT2.

Urinary bladder cancer risk factors in an area of former coal, iron, and steel industries in Germany.

This study was performed to investigate the frequency of bladder cancer in patients with an occupational history such as underground hard coal mining and/or painting after the structural change in the local industry. A total of 206 patients with bladder cancer and 207 controls were enlisted regarding occupational and nonoccupational bladder cancer risk factors by questionnaire. The phase II enzymes N-acetyltransferase 2 (NAT2), glutathione S-transferases M1 (GSTM1), and T1 (GSTT1) and the single nucleotide polymorphism (SNP) rs11892031[A/C] reported to be associated with bladder cancer in genome-wide association studies were genotyped. The bladder cancer risk in varnishers and underground hard coal miners was increased as previously shown in a study in this area performed in the 1980s. The occupation of a car mechanic was associated with a significantly elevated bladder cancer risk and higher in the case of underground hard coal miners even though the mine was closed in 1987. The frequency of GSTM1 negative genotype was comparable in cases and controls (53% versus 54%). In the case of NAT2, the slow NAT2 genotype was more frequent (62% versus 58%) and ultra-slow NAT2 genotype (NAT2*6A and/or *7B alleles only) was 23% versus 15%. An occupational history of a varnisher or an underground hard coal miner remains a risk factor for bladder cancer occurrence. Data indicate that in the case of bladder cancer, GSTM1 is a susceptibility factor related to environmental and/or occupational exposure.

Polymorphisms of xenobiotic metabolizing enzymes in bladder cancer patients of the Semmelweis University Budapest, Hungary.

Polymorphic xenobiotic metabolizing enzymes such as N-acetyltransferase 2 (NAT2) or glutathione S-transferase M1 (GSTM1) are known to modulate bladder cancer risk. As no apparent data were available from Hungary, a former member of the eastern European economic organization, a study was performed in Budapest. In total, 182 bladder cancer cases and 78 cancer-free controls were investigated by questionnaire. Genotypes of NAT2, GSTM1, GSTT1, rs1058396 and rs17674580 were determined by standard methods. Current smokers' crude odds ratio (OR) (3.43) and former smokers crude OR (2.36) displayed a significantly increased bladder cancer risk. The risk rose by a factor of 1.56 per 10 pack years. Exposure to fumes was associated with an elevated bladder cancer risk (23% cases, 13% controls). Sixty-four % of the cases and 59% of controls were slow NAT2 acetylators. It was not possible to establish a particular impact of NAT2*6A and *7B genotypes (15 cases, 8%, 5 controls, 7%). GSTT1 exerted no marked influence on bladder cancer (negative 21% cases vs. 22% controls). The portion of GSTM1 negative bladder cancer patients was increased (63% cases vs. 54% controls). The SLC14A1 SNPs rs1058396[AG/GG] and the nearby rs17674580[CT/TT] occurred more frequently in cases (79% and 68%) than controls (77% and 55%). The portion of GSTM1 negative bladder cancer patients is comparable with portions reported from other industrialized areas like Lutherstadt Wittenberg/Germany (58%), Dortmund/Germany (70%), Brescia/Italy (66%) or an occupational case-control series in Germany (56%). Data indicate that GSTM1 is a susceptibility factor for environmentally triggered bladder cancer rather than for smoking-mediated bladder cancer.

N-acetyltransferase 1*10 genotype in bladder cancer patients.

In a large bladder cancer study in the greater Berlin area with 425 cases and 343 controls, the haplotype N-acetyltransferase 1*10 (NAT1*10) was associated with a decreased bladder cancer risk. In a recently published meta-analysis, results of the studies were found to be inconclusive. Therefore, the aim of this study was to investigate the frequency of NAT1*10 in bladder cancer patients and controls recruited in an area without industries reported to be associated with increased bladder cancer risk. Rs1057126 (1088 T > A) and rs15561 (1095 C > A) were determined in 412 bladder cancer patients and 415 controls without a known history of malignancies. With these two single-nucleotide polymorphisms (SNP), it was possible to distinguish between NAT1*4 (wild type), NAT1*3 (1095 C > A), and NAT1*10 (1088 T > A, 1095C > A). The frequencies of the determined NAT1 haplotypes did not differ markedly between cases and controls: NAT1*4: 74%, NAT1*3: 6%, NAT1*10: 20%. Bladder cancer risk was not significantly modulated by NAT1*10/*10 (OR 1.03, 95% CI 0.71-1.48) but was higher for NAT1*3/*3 genotypes (OR 2.05, 95% CI 1.32-3.21). In contrast to the Berlin study from 2001, data in present study demonstrated that NAT1*10 haplotype was not associated with a significantly decreased bladder cancer risk. This may be due to local effects in the greater Berlin area, particularly at the time of investigation. The findings of the present study are in agreement with observations of a recently published meta-analysis which also showed no relevant impact of NAT1*10 haplotype on bladder cancer risk. The impact of the rare NAT1*3/*3 genotype was significant but this may be attributed to rarity without major practical relevance.

Micro-brushing-based technique to gain fresh urothelial cells for gene expression analysis.

The gold standard of saving fresh tissue in liquid nitrogen has some serious disadvantages in that this process is not available in daily medical routine practices even in many tumor centers. Our approach of a new minimally invasive technique is obtaining urothelial cells via micro-brushing the urinary bladder on the occasion of urological routine methods such as transurethral resection (TUR). Urothelial cells were obtained from 25 patients via two different micro-brushes from tumor tissue and from macroscopically healthy tissue during TUR. These cells were immediately transferred into RNA stabilization reagent and stored at -20°C. Later, mRNA was isolated, transcribed into cDNA, and amplified. cDNA was stored at -20°C until analysis. The mean RNA quantity was 99.5 ng/µl from tumor tissues and 66.3 ng/µl from macroscopically tumor-free tissue, enabling a considerable number of analyses. The quality of the gained cDNA allowed semi-quantitative PCR analysis of GSTM1 expression as well as quantitative PCR analysis of c-Myc expression. The new technique presents several important advantages. First, staging and grading of the stained tumor sample can be examined immediately, whereas fresh frozen sample is not examined until some days later. Further, this method can be applied in hospitals with no access to liquid nitrogen or without capability to provide an additional examination of frozen tumor sample by a pathologist. This presented minimally invasive method enables investigation of gene expression in the urinary bladder without disadvantages of the need for storage of fresh tissues in liquid nitrogen.

Glycerol-3-phosphate Acyltransferase 1 Promotes Tumor Cell Migration and Poor Survival in Ovarian Carcinoma.

Glycerophosphodiesterase EDI3 (GPCPD1; GDE5; GDPD6) has been suggested to promote cell migration, adhesion, and spreading, but its mechanisms of action remain uncertain. In this study, we targeted the glycerol-3-phosphate acyltransferase GPAM along with choline kinase-α (CHKA), the enzymes that catabolize the products of EDI3 to determine which downstream pathway is relevant for migration. Our results clearly showed that GPAM influenced cell migration via the signaling lipid lysophosphatidic acid (LPA), linking it with GPAM to cell migration. Analysis of GPAM expression in different cancer types revealed a significant association between high GPAM expression and reduced overall survival in ovarian cancer. Silencing GPAM in ovarian cancer cells decreased cell migration and reduced the growth of tumor xenografts. In contrast to these observations, manipulating CHKA did not influence cell migration in the same set of cell lines. Overall, our findings show how GPAM influences intracellular LPA levels to promote cell migration and tumor growth. Cancer Res; 77(17); 4589-601. ©2017 AACR.

Does seasonal allergic rhinitis increase sensitivity to ammonia exposure?

Allergic inflammation in the upper airways represents a wide-spread health issue: Little is known about whether it increases sensitivity to airborne chemicals thereby challenging established exposure limits that neglect such differences in susceptibility. To investigate the role of pre-existing allergic inflammation, 19 subjects with seasonal allergic rhinitis (SAR) and 18 control subjects with low risk of sensitization were exposed for 4h to ammonia in two concentrations (cross-over design): 2.5ppm (odor threshold) and 0-40ppm (occupational exposure limit: 20ppm TWA). Prior to the whole-body exposure, it was confirmed that subjects with SAR showed persistent inflammation outside the pollen season as indicated by increased exhaled nitric oxide and total immunoglobulin E in serum compared to controls. Despite concentration-dependent increases in chemosensory perceptions and acute symptoms, SAR status did not modulate subjective effects of exposure. Moreover, SAR status did not affect the investigated physiological endpoints of sensory irritation: While eye-blink recordings confirmed weak ocular irritation properties of ammonia at 0-40ppm, this effect was not enhanced in SAR subjects compared to controls. Irrespective of SAR status, exposure to 0-40ppm ammonia did not result in a cortisol stress response, objective nasal obstruction as measured with anterior active rhinomanometry, or an inflammatory response as indexed by substance P, tumor-necrosis-factor α, and high-mobility-group protein 1 in nasal lavage fluid. At least for the malodorous compound ammonia, these results do not support the hypothesis that SAR enhances chemosensory effects in response to local irritants. Before generalizing this finding, more compounds as well as sensitization to perennial allergens need to be investigated.

Prediction of human sensory irritation due to ethyl acrylate: the appropriateness of time-weighted average concentration × time models for varying concentrations.

Human data about the potency of ethyl acrylate to evoke sensory irritation is currently not available. Therefore, we conducted an experimental exposure study and the magnitude of chemosensory effects in healthy human volunteers was mathematically modeled by combining the factors current concentration (c) and duration/time (t). In a repeated-measures design, 19 subjects were exposed for 4 h to constant and varying concentrations (including peaks of 5 and 10 ppm) of ethyl acrylate with either a 2.5 or 5 ppm time-weighted average (TWA) concentration. Clean air served as control condition. Nasal lavage fluid, eye blinking frequencies, and rhinomanometry were used as physiological measures of sensory irritation. Several subjective ratings assessed olfactory and trigeminal perceptions. The blinking frequency was significantly increased during the varying 5 ppm condition. Regardless of the TWA concentration, varying exposures caused stronger effects than constant exposures. Our mathematical modeling showed that olfactory perceptions generally decreased over time while ratings of eye irritation increased over time even under the constant 5 ppm condition. Including the current concentration in the mathematical modeling always increased the goodness of fit substantially. The results showed that the intensity of sensory irritation could be predicted best with a complex c × t model. During the 2.5 ppm conditions, only the current concentration predicted the ratings and time-dependent processes could not be observed. However, in both 5 ppm TWA conditions strong eye irritations and increased blinking frequency, only at the end of the 4-h exposures a dose-dependency of these adverse effects was clearly shown.

Determination of aflatoxin M1 in urine samples indicates frequent dietary exposure to aflatoxin B1 in the Bangladeshi population.

Aflatoxin B1 (AFB1), a hepatocarcinogen and highly toxic mycotoxin, is a contaminant of food commodities, especially in hot and humid climates that favour the growth of aflatoxin-producing fungi. As data on AFB1 contamination of food and feed in Bangladesh are scarce, we conducted an initial screening by ELISA on the occurrence of the metabolite and biomarker aflatoxin M1 (AFM1) in urines from Bangladesh which indicated frequent exposure. This finding led us to conduct a follow-up study where we applied a more sensitive method (IAC clean-up and HPLC-FD analysis) to determine AFM1 concentrations in a larger set of urine samples. To account for possible seasonal and regional differences in mycotoxin exposure, in total 218 urines were collected in two districts of Bangladesh: 164 urines (n=69 in summer, n=95 in winter) from residents of a rural and an urban area in Rajshahi district, among them 62 participants enrolled in both sampling periods, and 54 urine samples obtained from pregnant women in Dhaka district. AFM1 was detected in>40% of all Rajshahi urine samples at a range of 1.7-104pg/mL in summer and at a range of 1.8-190pg/mL in winter season. The mean level of urinary AFM1 was higher in winter (27.7±42.6pg/mL) than in summer (13.6±21.2pg/mL) season, and differences were observed at the mean AFM1 level between the rural and the urban Rajshahi cohort. AFM1 was found less frequently in the Dhaka pregnant women (31% above LOD, mean 13.9±33.3pg/mL), but in a similar concentration range (1.7-141pg/mL) as in the Rajshahi cohort. Urinary AFM1 levels did not show significant associations with the participants food consumption pattern. In conclusion, when compared to biomarker data from other countries, detection frequency and urinary AFM1 levels in our Bangladeshi cohorts raise concerns regarding their exposure to potent carcinogenic aflatoxins.

Interindividual differences in chemosensory perception: Toward a better understanding of perceptual ratings during chemical exposures.

Perceptions that arise from stimulation of olfactory and trigeminal receptors in the nasal cavity guide the evaluation of chemical environment in humans. Strong interindividual differences in these assessments may be attributed to nonsensory factors such as gender, anxiety, and chemical sensitivity. Knowledge regarding the influence of these factors originates mainly from basic odor research using short-term exposure scenarios. In situations with continuous chemical exposures-common in the working environment-their impact is less clear. To investigate their role during the exposure to workplace chemicals, 4-hour experimental exposure studies (total N = 105) using nine different airborne chemicals were summarized. In each study, subjects evaluated a single chemical in a controlled environment by rating five chemosensory perceptions, including odor intensity, disgust, annoyance, pungency, and burning, several times during occupational limit and low exposures. It was investigated whether the effects of trait-like modulators, such as anxiety and self-reported chemical sensitivity, depend on exposure-related factors and gender. Trait-like modulators markedly affected ratings by women, but not men. Highly anxious women reported more intense annoyance and disgust than less anxious women. Stronger self-reported chemical sensitivity was associated with increased ratings of pungency and burning in women exposed to occupational limit concentrations. This study demonstrates that a complex interplay of exposure-related factors, gender, and trait-like individual differences affects perceptual ratings during continuous chemical exposure. It seems necessary to incorporate the assessment of specific as well as general trait-like modulators into future experimental exposure studies.

Occupational risk factors for relapse-free survival in bladder cancer patients.

The influence of occupational risk factors on bladder cancer development is well investigated. However, studies on the influence on bladder cancer prognosis are rare. Therefore, it was of interest to investigate the time to first relapse in the follow-ups of three case-control series from Dortmund, Neuss, and Lutherstadt Wittenberg, Germany. Relapse-free survival of in total 794 urinary bladder cancer patients (Dortmund 174, Neuss 407, Lutherstadt Wittenberg 213) was derived from medical records. Cox regression models were used to determine the impact of profession and exposure to bladder carcinogens if the risk factor was present in at least four cases. One or several relapses were observed in 416 cases (52%). Median time to first relapse was 0.94 yr. Ten professions were observed in at least 4 patients. No significant associations were found. However, workers in the leather industry (n = 4), printing industry (n = 4), transportation (n = 43), and chemical industry (n = 40) and locksmiths/mechanics (n = 44) showed shorter relapse-free times. No trend to shorter relapse-free time was observed for miners (n = 42), agriculturists (n = 18), painters/lacquerers (n = 21), colorant production and processing workers (n = 7), foundry workers (n = 5), and persons exposed to aromatic amines (n = 45). Although the follow-up comprised nearly 800 cases, data on occupations and exposures of interest were not sufficient to obtain significant results. However, first results indicated potential associations that are worth further investigations.

Biomonitoring of Mycotoxins in Urine: Pilot Study in Mill Workers.

Contamination of grains with mycotoxins results in a dietary background exposure of the general population. In occupational settings such as during processing of raw materials as in milling, an additional mycotoxin exposure by inhalation is possible. Biomonitoring is an integrative approach to assess human exposure from various sources and by all routes. To investigate possible workplace exposure to mycotoxins, a pilot study was conducted that compared levels of urinary biomarkers in mill workers to those in a control group with dietary mycotoxin intake alone. Workers (n = 17) from three grain mills in North Rhine Westphalia, Germany, provided spot urines during shift; volunteers (n = 13, IfADo staff) with matched age structure served as control group. The mycotoxins selected for biomarker analysis were citrinin (CIT) deoxynivalenol (DON), ochratoxin A (OTA), and zearalenone (ZEN). Immunoaffinity columns (CIT, DON, ZEN) or liquid-liquid extraction (OTA) was employed for urine sample cleanup prior to targeted analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS) or by high-performance liquid chromatography (HPLC). In addition, mycotoxin metabolites that may be formed in the organism were analyzed, including deepoxy-deoxynivalenol (DOM-1), ochratoxin alpha (OTα), dihydrocitrinone (DH-CIT), and α- and ß-zearalenol (α- and ß-ZEL), as well as phase II metabolites that were hydrolyzed with ß-glucuronidase/arylsulfatase prior to sample cleanup. All analyte concentrations were adjusted for creatinine (crea) content in the spot urine samples. Citrinin, DON, OTA, and ZEN were detected in nearly all urine samples from mill workers and controls. Interestingly, DH-CIT was found at higher mean levels than the parent compound (~0.14 and 0.045 µg/g crea, respectively), suggesting an effective metabolism of CIT in humans. Other metabolites DOM-1, OTα, and α- and ß-ZEL were detected less frequently in urine. Deoxynivalenol was detected at the highest concentrations (mean ~6 µg/g crea), followed by OTA (mean ~0.08 µg/g crea); ZEN (mean ~0.03 µg/g crea) and its metabolites appeared in urine at lower levels. Mycotoxin biomarker levels in urine from mill workers and controls were not significantly different. From these results it is concluded that biomarker levels measured in urine samples from the two cohorts reflect mainly dietary mycotoxin exposure. An additional occupational (inhalational) exposure of mill workers, if any, is apparently low at the investigated workplaces.