Differential Gene Expression in Bladder Tumors from Workers Occupationally Exposed to Arylamines.

Occupational exposure to the arylamines benzidine and ß-naphthylamine increase bladder cancer risk up to 100-fold, making them some of the most powerful human carcinogens. We hypothesize that tumors arising in people with occupational exposures have different patterns of gene expression than histologically similar tumors from people without such exposures. In a case-case study, we compare gene expression in 22 formalin-fixed paraffin-embedded (FFPE) bladder tumors from men with high-level occupational exposure to arylamines to that in 26 FFPE bladder tumors from men without such exposure. Gene expression analysis was performed on the NanoString nCounter system using a PanCancer Progression Panel comprised of 740 cancer progression-related genes and a custom panel of 69 arylamine- and bladder cancer-related genes which were chosen from in vitro studies. Although fold differences were small, there was evidence of differential expression by exposure status for 17 genes from the Progression Panel and 4 genes from the custom panel. In total, 10 genes showed dose-response association at a p < 0.01, of which 4 genes (CD46, NR4A1, BAX, and YWHAZ) passed a false discovery rate (FDR) q value cutoff of 0.05 but were not significant after Bonferroni correction. Overall, we find limited evidence for differentially expressed genes in pathways related to DNA damage signaling and epithelial-to-mesenchymal transition (EMT).

Novel benzidine and diaminofluorene prolinamide derivatives as potent hepatitis C virus NS5A inhibitors.

Our study describes the discovery of a series of highly potent hepatitis C virus (HCV) NS5A inhibitors based on symmetrical prolinamide derivatives of benzidine and diaminofluorene. Through modification of benzidine, l-proline, and diaminofluorene derivatives, we developed novel inhibitor structures, which allowed us to establish a library of potent HCV NS5A inhibitors. After optimizing the benzidine prolinamide backbone, we identified inhibitors embedding meta-substituted benzidine core structures that exhibited the most potent anti-HCV activities. Furthermore, through a battery of studies including hERG ligand binding assay, CYP450 binding assay, rat plasma stability test, human liver microsomal stability test, and pharmacokinetic studies, the identified compounds 24, 26, 27, 42, and 43 are found to be nontoxic, and are expected to be effective therapeutic anti-HCV agents.

[Epidemiology and risk factors of upper urinary tract tumors: literature review for the yearly scientific report of the French National Association of Urology]. / Épidémiologie et facteurs de risque des tumeurs de la voie excrétrice urinaire supérieure: revue de la littérature pour le rapport annuel de l'Association française d'urologie.

AIM: To describe the epidemiology, the risk and genetic factors involved in carcinogenesis pathways of upper urinary tumors UTUCs. MATERIAL: A systematic review of the scientific literature was performed from the database Medline (National Library of Medicine, PubMed) and websites of the HAS and the ANSM using the following keywords: epidemiology; risk factor; tobacco; aristolochic acid; urothelial carcinoma; ureter; renal pelvis. The search was focused on the characteristics, the mode of action, the efficiency and the side effects of the various drugs concerned. RESULTS: The estimated UTUC incidence is 1.2 cases/100,000 inhabitant per year in Europe. The incidence of renal pelvis tumor has been stable for 30years, while the frequency of ureteric locations has increased over time. Locally advanced stage and high grade are more frequent at the time of diagnosis. The median age for diagnosis is 70-years-old. Male-to-female ratio is nearly 2. Main carcinogenic factors are tobacco consumption and occupational exposure. There are specific risk factors for UTUC such acid aristolochic (balkan's nephropathy and Chinese herbs nephropathy). Familial cases are distinct from sporadic cases. UTUCs belong to the HNPCC syndrome and they rank third in its tumor spectrum. CONCLUSION: UTUCs are scarce tumors with specific epidemiologic characteristics. UTUCs share common risk factors with other urothelial carcinomas such as bladder tumors but have also specific risk factors that clinicians should know.

Cancer incidence and mortality among workers exposed to benzidine.

BACKGROUND: A historical cohort study was conducted among 997 individuals employed at a chemical production facility to investigate whether occupational exposures to benzidine and other arylamines were associated with the increased risk of cancer. METHODS: Cancers were identified from cancer registries, death certificates, and medical records. Exposures were evaluated using a job-exposure matrix. Workers were categorized into exposure groups to calculate cancer-specific standardized incidence ratios (SIRs) and perform survival analyses. RESULTS: SIRs for cancer of the bladder (SIR = 3.5; CI 1.7, 6.4), small intestine (SIR 18.4; CI 2.2, 66.4), and soft tissue including heart (SIR = 11.9; CI 1.4, 42.8) were elevated among workers with the highest exposures and risk increased with increasing exposures. SIRs for several additional cancers were also elevated. CONCLUSION: Our results support previous findings of increased risk of bladder cancer among individuals exposed to benzidine and other arylamines. Workers may also have been at increased risk for cancers other than cancer of the bladder.

Mechanisms of oxidative DNA damage induced by carcinogenic arylamines.

Most arylamines are pro-carcinogens, and require metabolic activation to yield ultimate carcinogen metabolites. O-Acetylation of the N-hydroxy form of an arylamine yields an acetoxyarylamine, which can form a highly reactive arylnitrenium ion, the ultimate metabolite responsible for DNA adduct formation. However, we demonstrate here that the N-hydroxy and nitroso forms of arylamines can also induce DNA damage, including 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesions, via reactive oxygen species formation. The N-hydroxy and nitroso derivatives of carcinogenic arylamines may contribute to the carcinogenic process through H2O2 formation. N-Hydroxy derivatives induce metal-mediated DNA damage, with remarkable enhancement by NADH. Nitroso derivatives induce NADH-dependent DNA damage in the presence of metal ions. Hydroxy derivatives of arylamines formed by enzymatic hydroxylation or as o- or p-aminophenols can also induce DNA damage in the presence of metal ions. The autoxidation of o-phenylenediamine and several arylamine metabolites is accelerated in the presence of SOD or manganese, resulting in the enhancement of metal-mediated DNA damage. The oxidative DNA damage induced by arylamine compounds may participate in chemical carcinogenesis, in addition to DNA adduct formation.

Biological activity resulting from exposure to aquatic environmental genotoxic pollutants in northern Egypt.

We estimated pollution in Lake Edku and the Mediterranean Sea, El-Maadiya Region, with 3 aromatic amines (1-naphthylamine, 2-naphthylamine and benzidine) in the muscle tissue of fish. There were marked seasonal variations in the aromatic amine levels. We also determined oxidative stress (blood glutathione, and catalase activity) and genotoxic effects (chromosomal aberrations and urinary metabolites) in fishermen from each area. The fishermen suffered from oxidative stress and had high levels of the urinary metabolite sulfanilamide [mean (microg/mg creatinine): Lake Edku 20.7, Mediterranean 14.5, controls 5.3]. Frequencies for total chromosomal aberrations were significantly raised in the peripheral blood lymphocytes of fishermen in both areas [frequency (per 100 metaphases): Mediterranean 67, Lake Edku 45, controls 14].

Occupational exposure to chemical and petrochemical industries and bladder cancer risk in four western Canadian provinces.

Occupational factors have been proposed to play a critical role in bladder cancer. This population-based case-control study was conducted to confirm the association between selected occupational and non-occupational risk factors and risk of bladder cancer using data collected from the four western Canadian provinces. Unconditional logistic regression analyses were based on 549 histologically confirmed bladder cancer cases and 1099 controls. Bladder cancer risk was found to increase with increasing pack-years of cigarette smoking with an odds ratio (OR) in the highest quartile of 3.32 (95% confidence interval [CI], 2.28-4.82). A dose-response relationship was demonstrated between bladder cancer and pack-years of smoking (p < 0.0001). A positive trend was observed with coffee consumption in men (p < 0.0001), with the highest risk in the highest category of exposure: drinkers of four cups or more per day had an OR of 1.77 (95% CI 1.11-2.82). Increased bladder cancer risk was associated with self-reported exposure at work to several chemicals: asbestos (OR 1.69 [95% CI 1.07-2.65]); mineral, cutting or lubricating oil (1.64 [95% CI 1.06-2.55]); benzidine (2.20 [95% CI 1.00-4.87]). The population attributable fraction (PAF) estimates were 51% for cigarette smoking, 17% for heavy coffee consumption, 10% for mineral, cutting or lubricating oil exposure, 6% for asbestos exposure, and 1% for benzidine exposure. Although self-reported chemical exposures have important limitations, the findings are suggestive of increased risk for several associations previously reported between chemical agents or industries and risk of bladder cancer.

Polymorphism in the N-acetyltransferase 1 alleles NAT1*10 and NAT1*14A and cytological gradings of exfoliated urothelial cells in benzidine-exposed Chinese workers: discussion of ethnic differences.

N-acetyltransferase 1 (NAT1) modifies bladder cancer risk in European populations exposed to aromatic amines in cigarette smoke. The present study was performed to investigate a possible association between NAT1*10 and NAT1*14A genotypes and bladder cancer risk in benzidine-exposed Chinese workers. Based on the cytological gradings of exfoliated urothelial cells according to Papanicolaou, an exposed research cohort was stratified into subgroups. An allele-specific PCR-based procedure was used to detect the polymorphism in the polyadenylation signal at the locus NAT1 T(1088)A. A nested PCR-RFLP procedure was conducted to differentiate NAT1*14A (T(1088)A, C(1095)A, and G(560)A) from NAT1*10 (T(1088)A, C(1095)A). No significantly different frequencies of homozygous and heterozygous NAT1*10 alleles were found among the subgroups with (i) gradings according to Papanicolaou < or = II (18.3 and 40.2%, respectively), (ii) higher gradings according to Papanicolaou (> II; 28.0 and 34.1%, respectively), and (iii) with bladder cancer (26.3 and 34.2%, respectively). The present data show that NAT1*10 neither displayed an association with an elevated grading of urothelial cells nor a clear impact on the risk for bladder cancer in benzidine-exposed Chinese workers. Discrepancies with the findings in European populations could point to ethnic differences in the disposition of aromatic amines.

Polymorphism of glutathione S-transferase T1, M1 and P1 genes in a Shanghai population: patients with occupational or non-occupational bladder cancer.

OBJECTIVE: Glutathione S-transferases are involved in the conjugation of xenobiotics. To explore whether GSTs polymorphisms are involved in the development of occupational or non-occupational bladder cancer, polymorphism frequencies of GSTT1, M1 and P1 were investigated in a normal population, which had been settled in a rural area in Shanghai suburb for at least 5 generations as well as in a group of patients with benzidine exposure related occupational bladder cancer in Shanghai dyestuff industry and a group of patients with non-occupational bladder cancer. METHODS: PCR based procedures were performed in the study populations to confirm the genotypes of GSTT1, M1 and P1. RESULTS: The polymorphisms at locus of GSTP1-A1578G in the normal population differed significantly from those in Caucasians or African Americans. All the subjects genotyped so far (n = 118) bore only homogenous wild genotype (C2293/C2293) at GSTP1-C2293T locus. This locus seemed to be a monomorphic in Shanghai population. No significant difference in GSTT1 and GSTM1 polymorphic form frequencies could be confirmed among three groups of subjects. An overrepresentation of GSTP1 AG or GG genotype corresponding a less stable and less effective isozyme protein was detected in patients with benzidine related occupational bladder cancer, compared with that in the normal population though a statistical significance was not yet reached (P = 0.09, OR = 1.96, 95% CI 0.89-4.32). CONCLUSION: This study suggests that GSTM1 or GSTT1 homozygous deficiency genotypes and their combination do not have a clear impact on bladder cancer incidence in a Shanghai population. It seems that GSTP1 polymorphism is not associated with non-occupational bladder cancer. GSTP1 AG or GG genotype has a higher frequency in the patients with benzidine related occupational bladder cancer, and further work is needed to confirm if GSTP1 AG or GG genotype plays a role in the development of occupational bladder cancer.

Carcinogen biomonitoring in human exposures and laboratory research: validation and application to human occupational exposures.

A multiple biomarker approach is required to integrate for metabolism, temporal response and exposure-response kinetics, biological relevance, and positive predictive value. Carcinogen DNA adduct analysis can be used in animal and in vitro studies to detect absorption permutations caused by mixture interactions, and to control metabolic variation when specific CYP450 genes (1A1 or 1A2) are knocked out. These enzymes are not critical to the metabolic activation of model Polycyclic Aromatic Compounds (PAC) and aromatic amines, respectively, as suggested by in vitro analysis. Several human studies have been carried out where multiple biomarkers have been measured. In a study of benzidine workers, the similarities in elimination kinetics between urinary metabolites and mutagenicity is likely responsible for a better correlation between these markers than to BZ-DNA adducts in exfoliated cells. In a study of rubber workers, the relationship between specific departments, urinary 1 HP and DNA adducts in exfoliated cells coincided with the historical urinary bladder cancer risk in these departments; the same relationship did not hold for urinary mutagenicity. In a study of automotive mechanics, biomarkers were used to monitor the effectiveness of exposure interventions. These data reinforce the notion that carcinogen biomarkers are useful to monitor exposure, but that a complementary approaches involving effect and perhaps susceptibility biomarkers is necessary to obtain the necessary information.

Nonassociation of aryl hydrocarbon receptor genotypes with susceptibility to bladder cancer in Shanghai population.

AIM: To assess two polymorphic forms of aryl hydrocarbon receptor (AHR) gene, G1721A (R554K), and G1768A (V570I) in Chinese population and to explore the possible association of human AHR gene polymorphism with elevated incidence of bladder cancer among Chinese Han subjects in east of China. METHODS: An allele specific PCR-based procedure for AHR gene polymorphism genotyping was developed by this work. Genotyping on three groups of subjects in Shanghai area had been performed: a bladder cancer group with the occupational exposure to benzidine, a non-occupational bladder cancer patient group whose members lack an obvious aromatic amine exposure record, and a normal population in the same city as controls. RESULTS: A significant difference (P < 0.01) in frequency distribution at locus G1721A between normal population in Shanghai and a Caucasian population reported by other authors was observed. No mutant allele(A1768) at locus G1768A had ever been detected in our study. The observed frequencies were similar between both genders in the normal population (P = 0.54), and there were no significant difference confirmed between the case group and the control group. CONCLUSION: The locus G1768 of human AHR gene seems to be monomorphic among Chinese in this area. The significant distribution difference at locus G1721A of human AHR gene between Chinese Han and Caucasian was confirmed. This study did not support the association of AHR G1721A polymorphism with higher risk to bladder cancer among the residents in this area, either in a group of occupationally benzidine-exposed individuals or among the persons who never have an obvious aromatic amine exposure record.

Re-evaluation of the latent period of bladder cancer in dyestuff-plant workers in Japan.

BACKGROUND: The carcinogenesis of benzidine (BZ) and beta-naphthylamine (BNA) for bladder is well known. Although it was thought to be rare to develop occupational bladder cancer more than 20 years after the exposure to these chemicals, there are still new clinical cases even 30 years after exposure. The purpose of this study was to re-evaluate the latent carcinogenic period of BZ and BNA, in order to set the safety period after exposure for the health surveillance system. METHODS: The subjects were 236 dyestuff-plant workers in Tokyo, who had been exposed to these dyestuffs. The incidence of bladder cancer and its histopathology in this group was surveyed in the period from 1962 to 1996. RESULTS: Nineteen workers (8.1%) were found to have bladder cancers. The exposure period for these 19 patients was 82.0 +/- 50.2 months. The mean +/- SD latent period from the subjects' initial and final exposure until tumor development was 29.5 +/- 8.2 years and 20.1 +/- 10.6 years, respectively. Significantly, a negative correlation (Pearson) was observed between the exposure period and the latent period from the end of exposure to cancer onset (R = -0.544, P < 0.05). All tumors except one were transitional cell carcinoma. Flow cytometric analysis was performed in 11 patients and all of these patients had DNA aneuploidy. CONCLUSIONS: The latent periods of bladder cancer caused by BZ and BNA were longer than previously expected. It is necessary to survey the onset of bladder cancer in exposed workers more than 30 years after the initial exposure.

Biomarker risk assessment and bladder cancer detection in a cohort exposed to benzidine.

BACKGROUND: Cancer screening with highly sensitive, specific biomarkers that reflect molecular phenotypic alterations is an attractive strategy for cancer control. We examined whether biomarker profiles could be used for risk assessment and cancer detection in a cohort of Chinese workers occupationally exposed to benzidine and at risk for bladder cancer. METHODS: The cohort consisted of 1788 exposed and 373 nonexposed workers, followed from 1991 through 1997. We assayed urothelial cells from voided urine samples for DNA ploidy (expressed as the 5C-exceeding rate [DNA 5CER]), the bladder tumor-associated antigen p300, and a cytoskeletal protein (G-actin). Workers were stratified into different risk groups (high, moderate, and low risk) at each examination based on a predefined biomarker profile. For workers who developed bladder cancer, tumor risk assessment was analyzed from samples collected 6-12 months before the cancer diagnosis. The associations between risk group and subsequent development of bladder cancer were analyzed by Cox proportional hazards regression analysis and logistic analysis, after adjustment. All statistical tests were two-sided. RESULTS: Twenty-eight bladder cancers were diagnosed in exposed workers and two in nonexposed workers. For risk assessment, DNA 5CER had 87.5% sensitivity, 86.5% specificity, an odds ratio (OR) of 46.2 (95% confidence interval [CI] = 8.1 to 867.0), and a risk ratio (RR) of 16.2 (95% CI = 7.1 to 37.0); p300 had 50.0% sensitivity, 97.9% specificity, an OR of 40.0 (95% CI = 9.0 to 177.8), and an RR of 37.9 (95% CI = 16.8 to 85.3). The risk of developing bladder cancer was 19.6 (95% CI = 8.0 to 47.9) times higher in workers positive for either the DNA 5CER or p300 biomarkers than in workers negative for both biomarkers and 81.4 (95% CI = 33.3 to 199.3) times higher in workers positive for both biomarkers. G-actin was a poor marker of individual risk. CONCLUSIONS: Occupationally exposed workers at risk for bladder cancer can be individually stratified, screened, monitored, and diagnosed based on predefined molecular biomarker profiles.

[Occupational experience, epidemiologic observations, and scientific evidence in an illustrative case: adverse effects of the production and use of aromatic amines in Italy]. / Esperienza operaia, osservazione epidemiologica ed evidenze scientifiche in un caso emblematico: gli effetti nocivi della produzione e dell'impiego di amine aromatiche in Italia.

Economical, social and technical interrelations between synthetic dyes' production and bladder cancer in workmen engaged in the manufacture are reviewed. International situation is the background of Italian situation, mainly of the Lombardy region and Turin area. For decades social actors, individuals and institutions, acted as separate bodies, contributed to delay and omit the protection of workers' health. The situation changed when scientific knowledge, technical developments and workers' commitment prioritized workers' health as a main goal. The epidemic of bladder cancers among dyestuff workers was a cornerstone in the development of a different scenario in the production responsibility, workers' participation and eventually scientific community. The authors hypothesise that in our country these events stimulated the growth of epidemiology and epidemiologists; furthermore since then institutions promulgated a new and up-to-date wave of protective legislation in the workers' H&S field.

N-glucuronidation of benzidine and its metabolites. Role in bladder cancer.

Workers exposed to high levels of benzidine have a 100-fold increased incidence of bladder cancer. This review evaluates the overall metabolism of benzidine to determine pathways important to initiation of bladder cancer. Upon incubation of benzidine with liver slices from rats, dogs, and humans, different proportions of this diamine were N-acetylated and N-glucuronidated. With dogs, a non-acetylator species, N-glucuronidation was the major pathway. In contrast, little glucuronidation was observed in rats with N, N'-diacetylbenzidine, the major metabolite of benzidine. Human liver slices demonstrated both extensive N-acetylation and N-glucuronidation. Differences between rats and humans were attributed to rapid deacetylation by human liver with N-acetylbenzidine rather than an accumulation of N, N'-diacetylbenzidine. N-Acetylbenzidine oxidative metabolism was also observed. The acid lability of glucuronide products of benzidine, N-acetylbenzidine, and oxidation products of N-acetylbenzidine metabolism was assessed. N-Glucuronides of benzidine, N-acetylbenzidine, and N'-hydroxy-N-acetylbenzidine were acid-labile, with the latter having a much longer half-time than the former two glucuronides. Because bladder epithelium contains relatively high levels of prostaglandin H synthase and not cytochrome P450, the peroxidative metabolism of N-acetylbenzidine was assessed. N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was the only DNA adduct detected. This adduct is also the major adduct detected in bladder cells from workers exposed to benzidine. In urine from these workers, an inverse relationship between urine pH and levels of free (unconjugated) benzidine and N-acetylbenzidine was observed. A similar inverse relationship was observed for urine pH and levels of bladder cell N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. These results suggest multiple pathways (acetylation, glucuronidation, peroxidation) in multiple organs (liver, blood, kidney, bladder) are important in benzidine-induced bladder cancer.