Upper urothelial carcinoma in Balkan endemic nephropathy and non-endemic regions: A comparative study of pathological features

Upper urothelial carcinoma (UUC), a rare neoplasm, occurs more frequently in some regions of Balkan countries than in other areas in the world. The aim of this study is to compare phenotypic morphological characteristics of UUC in Balkan endemic nephropathy (BEN) region and control rural and city populations free of BEN, and to determine the characteristic(s) that could discriminate tumors in endemic and non-endemic regions. The authors analyzed biopsies from 88 patients with UUC, 40 patients who live in Balkan endemic (BEN) settlements and 48 control subjects. The histological sections were used to assess morphological variables: histologic grade, pathologic stage (pT), growth pattern, pattern of invasion, lympho-vascular invasion (LVI), presence of necrosis and metaplastic changes (squamous or glandular) within the tumor. Statistically significant differences between the groups were found concerning tumor grade, pattern of invasion, growth pattern and metaplastic changes. High-grade tumors and trabecular/infiltrative patterns of invasion were more frequent in the group of BEN tumors (χ²=4.583, p<0.05; χ²=8.064, p<0.05). Moreover, solid growth and metaplastic changes are significant in BEN tumor, χ²=9.696, p<0.01; χ²=9.35, p<0.01, respectively. Discriminant analysis of morphological variables had indicated that BEN and control tumors are significantly different (Wilks’ lambda=0.833, χ²=15.044 and p<0.05). The best characteristic that differentiated them was growth pattern; i.e., solid growth for BEN tumors and papillary for control tumors.     

Urothelial carcinoma associated with Balkan endemic nephropathy. A worldwide disease

Balkan endemic nephropathy (BEN), a familial chronic tubulo-interstitial disease with a slow progression to terminal renal failure, affects people living in the alluvial plains along the tributaries of the Danube River. One of its most peculiar characteristics is a strong association with upper urothelial cancer. An increased incidence of upper urinary tract (UUT) transitional cell cancer (TCC) was discovered among the inhabitants of endemic settlements and in families affected by BEN. In areas where BEN is endemic, the incidence of upper tract TCC is significantly higher, even 100 times, than in non-endemic regions. A high incidence of urothelial cancer in end-stage BEN patients strongly suggests preventive nephro-ureterectomy in all end-stage patients with BEN treated with either transplantation or dialysis. Better understanding of the molecular mechanisms involved in carcinogenesis and tumor progression, has provided a large number of molecular markers of TCC, with a potential diagnostic and prognostic value. Markers that distinguish among TCC, normal urothelium, and benign urothelial conditions are potentially diagnostic, prognostic, and therapeutic targets. The geographic correlation and presence of AA-DNA adducts in both BEN and associated urothelial cancer, support the speculation that these diseases share a common etiology. Dietary exposure to AA is a significant risk factor for BEN and its attendant transitional cell cancer. These are cases of well-known AA induced urothelial carcinoma, and could be detected worldwide. The presence of more than one risk factors is possible and it is important to test etiological hypotheses in different endemic foci, preferably as a multicentric research.     

Decreasing incidence of urothelial cancer in a Balkan endemic nephropathy region in Serbia. A surgery based study from 1969 to 1998

The incidence of upper urothelial tumors (UUT) and bladder tumors associated to Balkan endemic nephropathy (BEN) in the region of South Morava River in Serbia, in the 30-year period, was studied. A total of 575 urothelial neoplasms during 10-year period (1989-1998) was analyzed and compared to 659 urothelial neoplasms in the period from 1969 to 1988. UUT had 11.2-fold increased incidence in endemic than in nonendemic areas, however, this was far less than in the period from 1969 to 1988, when they were 57.1 times more frequent. Bladder tumors were 2.3 times more frequent in endemic than in nonendemic areas, but 11.9 times in the previous study from 1969 to 1988. Neoplasms from the area of BEN, regardless of their location, were not histologically different from the neoplasms out of endemic areas. Similarly to the previous reports that BEN looks like a disease that disappears, this is demonstrated also for urothelial tumors, which are the most frequent associated diseases.     

Mutagenicity and DNA adduct formation by aristolochic acid in the spleen of Big Blue® rats

Aristolochic acid (AA) is a potent human nephrotoxin and carcinogen. We previously reported that AA treatment resulted in DNA damage and mutation in the kidney and liver of rats. In this study, we have determined the DNA adducts and mutations induced by AA in rat spleen. Big Blue® transgenic rats were gavaged with 0, 0.1, 1.0, and 10.0 mg AA/kg body weight five‐times/week for 3 months. Three DNA adducts, [7‐(deoxyadenosin‐N⁶‐yl)‐aristolactam I, 7‐(deoxyadenosin‐N⁶‐yl)‐aristolactam II and 7‐(deoxyguanosin‐N²‐yl)‐aristolactam I], were identified by ³²P‐postlabeling. Over the dose range studied, there were strong linear dose‐responses for AA‐DNA adduct formation in the treated rat spleens, ranging from 4.6 to 217.6 adducts/10⁸ nucleotides. Spleen cII mutant frequencies also increased in a dose‐dependent manner, ranging from 32.7 to 286.2 × 10^?6 in the treated animals. Mutants isolated from the different treatment groups were sequenced; analysis of the resulting spectra indicated that there was a significant difference between the pattern of mutation in the 10 mg/kg AA‐treated and the vehicle control rats. A:T → T:A transversion was the major type of mutation in AA‐treated rats, whereas G:C → A:T transition was the main type of mutation inthe vehicle controls. These results indicate that AA is genotoxic in the spleen of rats exposed under conditions that result in DNA adduct formation and mutation induction in kidney and liver. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

E-cadherin expression in upper urothelial carcinoma in Balkan Endemic Nephropathy and non-endemic regions

There is a high incidence of upper urothelial carcinoma (UUC) in regions affected by Balkan Endemic Nephropathy (BEN). The aim of this study was to compare E-cadherin expression in UUC, in regions affected by BEN, and in control rural and city populations free of BEN. Another aim was to determine the influence of some morphological parameters on the E-cadherin status. In the samples of 85 UUC patients, of whom 40 lived in BEN settlements and 45 served as control subjects, immunoreactions were performed using monoclonal anti-human E-cadherin antibody. Aberrant expression of E-cadherin was more frequent in BEN tumors than in control tumors (p<0.01). Decreased E-cadherin expression was linked to high grade and solid growth in control and BEN tumors (p<0.0001 and <0.05 versus p<0.05 and <0.05, respectively), and to the stage in control tumors (p<0.01). However, BEN low grade and low stage tumors showed aberrant expression more often than did control tumors (p<0.05 and <0.005, respectively). In control tumors, using univariate analysis, E-cadherin status was found to be influenced by grade, stage, and tumor growth (p=0.001, 0.017, 0.015, respectively). In the same group, only the grade was significant according to multistep logistic regression analysis (Wald=6.429 and p=0.011). The growth pattern had a predominant influence on E-cadherin expression in BEN tumors (p=0.005). A significant influence on normal membranous or abnormal cytoplasmic expression of E-cadherin in UUC is exerted by tumor grade, stage, growth, and metaplastic change (p=0.002, 0.048, 0.019, 0.011, respectively), but only by tumor grade in the multistep logistic regression model. These results suggest that decreased expression of E-cadherin in BEN tumors may be linked to tumor growth, while expression of E-cadherin in control tumors may be associated with tumor grade.     

Aristolochic acid nephropathy: Harbinger of a global iatrogenic disease

This review constitutes an overview of our investigations of aristolochic acid nephropathy, a chronic kidney disease associated with carcinomas of the upper urinary tract. Our studies began by confirming the hypothesis that chronic dietary poisoning by aristolochic acid was responsible for endemic (Balkan) nephropathy. A unique TP53 mutational signature in urothelial tumors and the presence of aristolactam‐DNA adducts in the renal cortex, defined in the course of this research, proved to be robust biomarkers of exposure to this potent nephrotoxin and human carcinogen. Armed with this information, we used molecular epidemiologic approaches and novel mechanistic information to establish the causative role of aristolochic acid in upper urinary tract carcinoma in Taiwan, where one‐third of the population had been prescribed herbal remedies containing Aristolochia, and the recorded incidence of upper urinary tract cancers is the highest in the world. As traditional Chinese medicine is practiced similarly in Taiwan and China, it is likely that upper urinary tract carcinomas and their attendant aristolochic acid nephropathy are prevalent in China and other Asian countries where Aristolochia herbs have been used for centuries in the treatment and prevention of disease, creating a potential public health problem of considerable magnitude. Environ. Mol. Mutagen., 2013. © 2012 Wiley Periodicals, Inc.     

ACB‐PCR measurement of H‐ras codon 61 CAA→CTA mutation provides an early indication of aristolochic acid I carcinogenic effect in tumor target tissues

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen, which induces forestomach and kidney tumors in mice and is associated with development of urothelial cancer in humans. This study sought to gain mechanistic insight into AAI‐induced carcinogenesis through analysis of a tumor‐relevant endpoint. Female Hupki mice were treated daily with 5 mg AAI/kg body weight by gavage for 3, 12, or 21 days. Histopathology and DNA adduct analysis confirmed kidney and forestomach as target tissues for AAI‐induced toxicity. H‐ras codon 61 CAA→CTA mutations were measured in mouse kidney and forestomach, as well as liver and glandular stomach (nontarget organs) by allele‐specific competitive blocker‐PCR (ACB‐PCR), because A→T transversion is the predominant mutation induced by AA and this particular mutation was found previously in AA‐induced rodent forestomach tumors. Treatment‐related differences were observed, with the H‐ras mutant fraction (MF) of mouse kidney and forestomach exposed to 5 mg AAI/kg body weight for 21 days significantly higher than that of vehicle‐treated controls (Fisher's exact test, P < 0.05). Statistically significant correlations between dA‐AAI adduct levels (measured previously in the same animals) and induced H‐ras MFs were evident in forestomach of mice treated for 21 days (linear regression, P < 0.05). The significant increase in H‐ras MF in kidney and forestomach, along with the correlation between DNA adducts, histopathology, and oncogene mutation, provide definitive evidence that AA induces tumors through a directly mutagenic mode of action. Thus, measurement of tumor‐associated mutations is a useful tool for elucidating the mechanisms underlying the tissuespecificity of carcinogenesis. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Urinary creatinine excretion in children from families with Balkan endemic nephropathy: evidence for genetic predisposition to the disease

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial kidney disease prevalent in Serbia, Bosnia, Croatia, Bulgaria and Romania. Genetic studies have supported the genetic predisposition to BEN, and some studies in Bulgaria and in the Kolubara region of Serbia have revealed abnormalities of the urinary tract in up to 46% of children from families with BEN. In the present study, urinary excretion of creatinine, an index of muscle mass, was studied in 703 healthy children from endemic and non-endemic areas around the South Morava River. The survey covered a three-year period, and the children were studied three times a year: in the spring, autumn and winter. A urine sample for the period corresponding to 7-10 a.m. was collected during each study period. Evidence has been presented that children from families with BEN excrete significantly less creatinine than those from families without BEN living in the same area, or than children living in villages outside the endemic region or in the city of Nis. This study supports the view that genetic predisposition to BEN is indicated by a smaller muscle mass, although the effect of living conditions and nutrition may also contribute to this.     

DNA adduct kinetics in reproductive tissues of DNA repair proficient and deficient male mice after oral exposure to benzo(a)pyrene

Benzo(a)pyrene (B[a]P) can induce somatic mutations, whereas its potential to induce germ cell mutations is unclear. There is circumstantial evidence that paternal exposure to B[a]P can result in germ cell mutations. Since DNA adducts are thought to be a prerequisite for B[a]P induced mutations, we studied DNA adduct kinetics by ³²P‐postlabeling in sperm, testes and lung tissues of male mice after a single exposure to B[a]P (13 mg/kg bw, by gavage). To investigate DNA adduct formation at different stages of spermatogenesis, mice were sacrificed at Day 1, 4, 7, 10, 14, 21, 32, and 42 after exposure. In addition, DNA repair deficient (Xpc^?/?) mice were used to study the contribution of nucleotide excision repair in DNA damage removal. DNA adducts were detectable with highest levels in lung followed by sperm and testis. Maximum adduct levels in the lung and testis were observed at Day 1 after exposure, while adduct levels in sperm reached maximum levels at ～1 week after exposure. Lung tissue and testis of Xpc^?/? mice contained significantly higher DNA adduct levels compared to wild type (Wt) mice over the entire 42 day observation period (P < 0.05). Differences in adduct half‐life between Xpc^?/? and Wt mice were only observed in testis. In sperm, DNA adduct levels were significantly higher in Xpc^?/? mice than in Wt mice only at Day 42 after exposure (P = 0.01). These results indicate that spermatogonia and testes are susceptible for the induction of DNA damage and rely on nucleotide excision repair for maintaining their genetic integrity. Environ. Mol. Mutagen. 2010. © 2009 Wiley‐Liss, Inc.     

Metabolic activation of diesel exhaust carcinogens in primary and immortalized human TP53 knock‐in (Hupki) mouse embryo fibroblasts

Approximately 50% of human tumors have a mutation in TP53. The pattern and spectra of TP53 mutations often differ between cancer types, perhaps due to different etiological factors. The Hupki (human TP53 knock‐in) mouse embryo fibroblast (HUF) immortalization assay is useful for studying mutagenesis in the human TP53 gene by environmental carcinogens. Prior to initiating an immortalization assay, carcinogen treatment conditions must be optimized, which can require a large number of cells. As primary HUF cultures senesce within 2 weeks, restricting their use, we investigated whether immortalized HUFs retaining wild‐type TP53 can be surrogates for primary HUFs in initial treatment optimization. DNA damage by eight compounds found in diesel exhaust, benzo[a]pyrene, 3‐nitrobenzanthrone, 1‐nitropyrene, 1,3‐dinitropyrene, 1,6‐dinitropyrene, 1,8‐dinitropyrene, 6‐nitrochrysene, and 3‐nitrofluorene, was assessed by ³²P‐postlabeling and the alkaline comet assay in primary HUFs and in an immortal HUF cell line J201. For most compounds, higher levels of DNA adducts accumulated in J201 cells than in primary HUFs. This difference was not reflected in the comet assay or by cell viability changes. Experiments in three additional immortal HUF cell lines (AAI49, U56, and E2‐143) confirmed strong differences in DNA adduct levels compared with primary HUFs. However, these did not correlate with the protein expression of Nqo1 or Nat1/2, or with gene expression of Cyp1a1 or Cyp1b1. Our results show that using immortal HUFs as surrogates for primary HUFs in genotoxicity screening has limitations and that DNA adduct formation is the best measure of genotoxicity of the nitro‐polycyclic aromatic hydrocarbons tested in HUFs. Environ. Mol. Mutagen. 2012. © 2011 Wiley Periodicals, Inc.     

Impact of p53 function on the sulfotransferase-mediated bioactivation of the alkylated polycyclic aromatic hydrocarbon 1-hydroxymethylpyrene in vitro

The tumor suppressor p53, encoded by TP53, is known as the “guardian of the genome.” Sulfotransferases (SULTs) are involved in the metabolism of alkylated polycyclic aromatic hydrocarbons such as 1-hydroxymethylpyrene (1-HMP), which is a known substrate for SULT1A1. To investigate the impact of TP53 on the metabolic activation of 1-HMP, a panel of isogenic human colorectal HCT116 cells having TP53(+/+), TP53(+/−), or TP53(−/−) were treated with 10 μM 1-HMP for 24 hr. 1-HMP-DNA adduct formation was determined by ultraperformance liquid chromatography-tandem mass spectrometry analysis, which quantified two nucleoside adducts N²-(1-methylpyrenyl)-2′-deoxyguanosine and N⁶-(1-methylpyrenyl)-2′-deoxyadenosine. 1-HMP treatment resulted in significantly (~40-fold) higher DNA adduct levels in TP53(+/+) cells than in the other cell lines. Higher levels of 1-HMP-induced DNA adducts in TP53(+/+) cells correlated with higher basal expression of SULT1A1/3 in this cell line, but 1-HMP treatment showed no effect on the expression of this protein. These results indicate that the cellular TP53 status is linked to the SULT1A1/3-mediated bioactivation of 1-HMP, thereby broadening the spectrum of p53's targets. Environ. Mol. Mutagen., 60:752–758, 2019. © 2019 Wiley Periodicals, Inc.     

Assessment of multiple types of DNA damage in human placentas from smoking and nonsmoking women in the Czech Republic

Three classes of DNA damage were assessed in human placentas collected (2000–2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)‐DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by ³²P‐postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7β,8α‐dihydroxy‐9α,10α‐epoxy‐7,8,9,10‐tetrahydro‐benzo[a]pyrene (BPDE) revealed PAH‐DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49‐312 PAH‐DNA adducts/10⁸ nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH‐DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7–8.6 stable/bulky DNA adducts/10⁸ nucleotides and 0.6–47.2 AB sites/10⁵ nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH‐DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. Environ. Mol. Mutagen. 52:58–68, 2011. © 2010 Wiley‐Liss, Inc.     

The human carcinogen aristolochic acid i is activated to form DNA adducts by human NAD(P)H:quinone oxidoreductase without the contribution of acetyltransferases or sulfotransferases

Ingestion of aristolochic acid (AA) is associated with development of urothelial tumors linked with AA nephropathy and is implicated in the development of Balkan endemic nephropathy-associated urothelial tumors. We investigated the efficiency of human NAD(P)H:quinone oxidoreductase (NQO1) to activate aristolochic acid I (AAI) and used in silico docking, using soft-soft (flexible) docking procedure, to study the interactions of AAI with the active site of human NQO1. AAI binds to the active site of NQO1 indicating that the binding orientation allows for direct hydride transfer (i.e., two electron reductions) to the nitro group of AAI. NQO1 activated AAI, generating DNA adduct patterns reproducing those found in urothelial tissues from humans exposed to AA. Because reduced aromatic nitro-compounds are often further activated by sulfotransferases (SULTs) or N,O-acetlytransferases (NATs), their roles in AAI activation were investigated. Our results indicate that phase II reactions do not play a major role in AAI bioactivation; neither native enzymes present in human hepatic or renal cytosols nor human SULT1A1, -1A2, -1A3, -1E, or -2A nor NAT1 or NAT2 further enhanced DNA adduct formation by AAI. Instead under the in vitro conditions used, DNA adducts arise by enzymatic reduction of AAI through the formation of a cyclic hydroxamic acid (N-hydroxyaristolactam I) favored by the carboxy group in peri position to the nitro group without additional conjugation. These results emphasize the major importance of NQO1 in the metabolic activation of AAI and provide the first evidence that initial nitroreduction is the rate limiting step in AAI activation.     

Gene‐diet interactions in exposure to heterocyclic aromatic amines and bulky DNA adduct levels in blood leukocytes

Heterocyclic aromatic amines (HAAs), carcinogens produced in meat when cooked at high temperatures, are an emerging biologic explanation for the meat‐colorectal cancer relationship. HAAs form DNA adducts; left unrepaired, adducts can induce mutations, which may initiate/promote carcinogenesis. The purpose of this research was to investigate the relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct levels. Least squares regression was used to examine the relationship between dietary HAA exposure and bulky DNA adduct levels in blood measured using ³²P‐postlabeling among 99 healthy volunteers. Gene‐diet interactions between dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair were also examined. No main effects of dietary HAAs on bulky DNA adduct levels was found. However, those with the putative NAT1 rapid acetylator phenotype had lower adduct levels than those with the slow acetylator phenotype (P = 0.02). Furthermore, having five or more 'at‐risk' genotypes was associated with higher bulky DNA adduct levels (P = 0.03). Gene‐diet interactions were observed between NAT1 polymorphisms and dietary HAAs (P < 0.05); among the slow acetylator phenotype, higher intakes of HAAs were associated with an increase in DNA adduct levels compared to lower intakes. This study provides evidence of a biologic relationship between dietary HAAs, genetic susceptibility and bulky DNA adduct formation. However, the lack of a strong main effect of HAAs suggests that dietary HAAs are not a large contributor to bulky DNA adducts in this population; future studies should consider relevant gene‐diet interactions to clarify the role of HAAs in carcinogenesis. Environ. Mol. Mutagen. 56:609–620, 2015. © 2015 Wiley Periodicals, Inc.     

Balkan endemic nephropathy: a decreasing incidence of the disease

Balkan endemic nephropathy (BEN) is a chronic tubulointerstitial kidney disease prevalent in Serbia, Bosnia, Croatia, Bulgaria and Romania. This study investigates the incidence of BEN patients on dialysis with end-stage kidney disease (ESKD), and BEN-associated mortality in endemic areas around the South Morava River in Serbia from 1978 to 1997. In the last 10 years a marked decrease in the incidence of ESRD and BEN-induced mortality has been documented in the region. This study shows a significantly decreased incidence of BEN in endemic areas in South Serbia. Since the effects of etiological agent(s) on renal function in children from families in endemic areas was demonstrated in the early nineties and the disease seems to have an endemic-epidemic profile, the possibility of another epidemic outbreak in the future cannot be excluded.     

Use of multivariate analysis to suggest a new molecular classification of colorectal cancer

Molecular classification of colorectal cancer (CRC) is currently based on microsatellite instability (MSI), KRAS or BRAF mutation and, occasionally, chromosomal instability (CIN). Whilst useful, these categories may not fully represent the underlying molecular subgroups. We screened 906 stage II/III CRCs from the VICTOR clinical trial for somatic mutations. Multivariate analyses (logistic regression, clustering, Bayesian networks) identified the primary molecular associations. Positive associations occurred between: CIN and TP53 mutation; MSI and BRAF mutation; and KRAS and PIK3CA mutations. Negative associations occurred between: MSI and CIN; MSI and NRAS mutation; and KRAS mutation, and each of NRAS, TP53 and BRAF mutations. Some complex relationships were elucidated: KRAS and TP53 mutations had both a direct negative association and a weaker, confounding, positive association via TP53–CIN–MSI–BRAF–KRAS. Our results suggested a new molecular classification of CRCs: (1) MSI⁺ and/or BRAF‐mutant; (2) CIN⁺ and/or TP53^– mutant, with wild‐type KRAS and PIK3CA; (3) KRAS‐ and/or PIK3CA‐mutant, CIN⁺, TP53‐wild‐type; (4) KRAS^– and/or PIK3CA‐mutant, CIN^–, TP53‐wild‐type; (5) NRAS‐mutant; (6) no mutations; (7) others. As expected, group 1 cancers were mostly proximal and poorly differentiated, usually occurring in women. Unexpectedly, two different types of CIN⁺ CRC were found: group 2 cancers were usually distal and occurred in men, whereas group 3 showed neither of these associations but were of higher stage. CIN⁺ cancers have conventionally been associated with all three of these variables, because they have been tested en masse. Our classification also showed potentially improved prognostic capabilities, with group 3, and possibly group 1, independently predicting disease‐free survival. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Influence of arylamine n‐acetyltransferase,sex, and age on 4‐aminobiphenyl‐induced in vivo mutant frequencies and spectra in mouse liver

One model for cancer initiation by 4‐aminobiphenyl (ABP) involves N‐oxidation by cytochrome P450 CYP1A2 followed by O‐conjugation by N‐acetyltransferase(s) NAT1 and/or NAT2 and decomposition to a DNA‐binding nitrenium ion. We recently observed that neonatal ABP exposure produced liver tumors in male but not in female mice, and that NAT deficiency reduced liver tumor incidence. However, ABP‐induced liver tumor incidence did not correlate with liver levels of N‐(deoxyguanosin‐8‐yl)‐ABP adducts 24 hr after exposure. In this study, we compared in vivo ABP‐induced DNA mutant frequencies and spectra between male and female wild‐type and NAT‐deficient Muta?Mouse using both the tumor‐inducing neonatal exposure protocol and a 28‐day repetitive dosing adult exposure protocol. ABP produced an increase in liver DNA mutant frequencies in both neonates and adults. However, we observed no sex or strain differences in mutant frequencies in neonatally exposed mice, and higher frequencies in adult females than males. Neonatal ABP exposure of wild‐type mice increased the proportion of G‐T transversions in both males and females, while exposure of Nat1/2(‐/‐) mice produced increased G‐T transversions in males and a decrease in females, even though females had higher levels of N‐(deoxyguanosin‐8‐yl)‐4‐ABP adducts. There was no correlation of mutant frequencies or spectra between mice dosed as neonates or as adults. These results suggest that observed sex‐ and NAT‐dependent differences in ABP‐induced liver tumor incidence in mice are not due to differences in either mutation rates or mutational spectra, and that mechanisms independent of carcinogen bioactivation, covalent DNA binding and mutation may be responsible for these differences. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.     

Serum levels of epithelial‐derived mediators and interleukin‐4/interleukin‐13 signaling after bronchial challenge with Dermatophagoides pteronyssinus in patients with allergic asthma

Allergens are the main trigger that enhances airway type 2 inflammation, and the epithelium is the first line of defense that reacts to its exposure. Therefore, epithelial‐derived mediators, such as interleukin (IL)‐25, IL‐33, thymic stromal lymphopoietin (TSLP) and ezrin, may play a role as alarmins in IL‐4/IL‐13 signaling in allergic asthma (AA). We investigated the serum levels of IL‐25, IL‐33, TSLP, ezrin, IL‐4 and IL‐13, after bronchial challenge with Dermatophagoides pteronyssinus in patients with AA. We examined 18 subjects: nine steroid‐free stable patients with AA sensitized to D. pteronyssinus and nine non‐atopic healthy subjects (HS). Bronchial allergen challenge was performed using inhaled D. pteronyssinus allergen. IL‐4, IL‐13, IL‐25, IL‐33, TSLP and ezrin levels in serum were measured by ELISA at two time points ‐ before and 24 hours after bronchial allergen challenge. The serum levels of IL‐25, TSLP and ezrin did not differ between AA and HS groups at baseline. However, after allergen exposure, significant increases in serum levels of IL‐25, TSLP and ezrin were observed only in patients with AA. The serum level of IL‐33 at baseline was significantly higher in the AA group compared with HS, but the allergen challenge did not provoke an increase of this cytokine in any group. IL‐4 and IL‐13 levels were significantly higher at baseline in the AA group compared with HS and, after allergen exposure, were significantly increased in the AA group, with no effect on HS. Thus, the epithelial‐derived mediators IL‐25, TSLP and ezrin, via IL4/IL13 signaling, enhance type 2 inflammation after bronchial challenge with D. pteronyssinus in AA.     

Influence of GSTM1 and NAT2 genotypes on placental DNA adducts in an environmentally exposed population

The placenta bulky DNA adducts have been studied in relation to metabolic genotypes for glutathione S-transferase M1 (GSTM1) and N-acetyl transferase 2 (NAT2) in 158 mothers (113 nonsmokers and 45 smokers) living in two regions with different annual average air pollution levels of sulphur dioxide, nitrogen oxides, particulate matter <10 μm, and polycyclic aromatic hydrocarbons. One region was the district of Teplice as the polluted industrial region with mines and brown coal power plants, and the other was the district of Prachatice, an agricultural region without heavy industry. DNA adduct levels were determined by using a butanol extraction enrichment procedure of ³²P-postlabeling. GSTM1 and NAT2 genotypes were studied by using polymerase chain reaction. The total DNA adduct levels included a diagonal radioactive zone (DRZ) and one distinct spot outside DRZ (termed X), which was detected in almost all placenta samples and correlated with DRZ (r = .682; P < .001). We found the total DNA adduct levels 2.12 ± 1.46 (0.04–7.70) and 1.48 ± 1.09 (0.11–4.98) adducts per 10⁸ nucleotides for Teplice and Prachatice districts, respectively, indicating significant differences between both regions studied (P = .004). Elevated DNA adduct levels were found in smoking mothers (10 or more cigarettes per day) by comparison with nonsmoking mothers (3.21 ± 1.39 versus 1.32 ± 0.88 adducts per 10⁸ nucleotides; P < .001). Placental DNA adduct levels in smokers correlated with cotinine measured in plasma (r = .432; P = .003). This relation indicates that cigarette smoking could be predominantly responsible for DNA adduct formation in placentas of smoking mothers. DNA adduct levels were evaluated separately for nonsmokers (1.50 ± 1.00 vs. 1.09 ± 0.66 adducts/10⁸ nucleotides for the Teplice and Prachatice districts, respectively; P = .046) and smokers (3.35 ± 1.47 vs. 2.91 ± 1.20 adducts/10⁸ nucleotides for Teplice and Prachatice districts, respectively; P = .384) to exclude the effect of active cigarette smoking on the district variation. These findings indicate that the effect of the environmental pollution in cigarette smokers is practically overlapped by tobacco exposure. No seasonal variation was observed for DNA adduct levels in the overall population studied and no relation between total DNA adduct levels in placenta and levels of vitamins A, C, and E in venous and cord blood was found. A positive GSTM1 genotype was detected in 78 subjects, while negative GSTM1 genotype was found in 80 subjects. Higher DNA adduct levels were detected in the group with GSTM1-negative genotype by comparison with GSTM1-positive genotype (2.05 ± 1.30 vs. 1.66 ± 1.39 adducts/10⁸ nucleotides; P = .018). This finding is more pronounced in the Teplice district (2.33 ± 1.36 vs. 1.88 ± 1.56 adducts/10⁸ nucleotides; P = .053) than for the Prachatice district (1.61 ± 1.09 vs. 1.36 ± 1.10 adducts/10⁸ nucleotides; P = .248) and for nonsmokers (1.45 ± 0.82 vs. 1.18 ± 0.93 adducts/10⁸ nucleotides; P = .029) more than for smokers (3.45 ± 1.14 vs. 2.95 ± 1.62 adducts/10⁸ nucleotides; P = .085). Significant district and seasonal differences were found in subgroups with GSTM1-negative genotype. DNA adduct levels in placentas of the GSTM1-negative subgroup were higher in mothers living in the polluted district of Teplice than in Prachatice (P = .012). The adduct levels in placentas sampled in the summer period were higher than in the winter period in the GSTM1-negative population (P = .006). No effect of the NAT2 genotype on DNA adduct levels was observed. Environ. Mol. Mutagen. 30:184–195, 1997. © 1997 Wiley-Liss, Inc.