The Breast Cancer and the Environment Research Centers: Transdisciplinary Research on the Role of the Environment in Breast Cancer Etiology

ObjectivesWe introduce and describe the Breast Cancer and the Environment Research Centers (BCERC), a research network with a transdisciplinary approach to elucidating the role of environmental factors in pubertal development as a window on breast cancer etiology. We describe the organization of four national centers integrated into the BCERC network.Data sourcesInvestigators use a common conceptual framework based on multiple levels of biologic, behavioral, and social organization across the life span. The approach connects basic biologic studies with rodent models and tissue culture systems, a coordinated multicenter epidemiologic cohort study of prepubertal girls, and the integration of community members of breast cancer advocates as key members of the research team to comprise the network.Data extractionRelevant literature is reviewed that describes current knowledge across levels of organization. Individual research questions and hypotheses in BCERC are driven by gaps in our knowledge that are presented at genetic, metabolic, cellular, individual, and environmental (physical and social) levels.Data synthesisAs data collection on the cohort, animal experiments, and analyses proceed, results will be synthesized through a transdisciplinary approach.ConclusionCenter investigators are addressing a large number of specific research questions related to early pubertal onset, which is an established risk factor for breast cancer. BCERC research findings aimed at the primary prevention of breast cancer will be disseminated to the scientific community and to the public by breast cancer advocates, who have been integral members of the research process from its inception

A Dominant X-Linked QTL Regulating Pubertal Timing in Mice Found by Whole Genome Scanning and Modified Interval-Specific Congenic Strain Analysis

BACKGROUND: Pubertal timing in mammals is triggered by reactivation of the hypothalamic-pituitary-gonadal (HPG) axis and modulated by both genetic and environmental factors. Strain-dependent differences in vaginal opening among inbred mouse strains suggest that genetic background contribute significantly to the puberty timing, although the exact mechanism remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We performed a genome-wide scanning for linkage in reciprocal crosses between two strains, C3H/HeJ (C3H) and C57BL6/J (B6), which differed significantly in the pubertal timing. Vaginal opening (VO) was used to characterize pubertal timing in female mice, and the age at VO of all female mice (two parental strains, F1 and F2 progeny) was recorded. A genome-wide search was performed in 260 phenotypically extreme F2 mice out of 464 female progeny of the F1 intercrosses to identify quantitative trait loci (QTLs) controlling this trait. A QTL significantly associated was mapped to the DXMit166 marker (15.5 cM, LOD = 3.86, p<0.01) in the reciprocal cross population (C3HB6F2). This QTL contributed 2.1 days to the timing of VO, which accounted for 32.31% of the difference between the original strains. Further study showed that the QTL was B6-dominant and explained 10.5% of variation to this trait with a power of 99.4% at an alpha level of 0.05.The location of the significant ChrX QTL found by genome scanning was then fine-mapped to a region of approximately 2.5 cM between marker DXMit68 and rs29053133 by generating and phenotyping a panel of 10 modified interval-specific congenic strains (mISCSs). CONCLUSIONS/SIGNIFICANCE: Such findings in our study lay a foundation for positional cloning of genes regulating the timing of puberty, and also reveal the fact that chromosome X (the sex chromosome) does carry gene(s) which take part in the regulative pathway of the pubertal timing in mice

CYP3A4 and CYP3A5 genotyping by Pyrosequencing

BACKGROUND: Human cytochrome P450 3A enzymes, particularly CYP3A4 and CYP3A5, play an important role in drug metabolism. CYP3A expression exhibits substantial interindividual variation, much of which may result from genetic variation. This study describes Pyrosequencing assays for key SNPs in CYP3A4 (CYP3A4*1B, CYP3A4*2, and CYP3A4*3) and CYP3A5 (CYP3A5*3C and CYP3A5*6). METHODS: Genotyping of 95 healthy European and 95 healthy African volunteers was performed using Pyrosequencing. Linkage disequilibrium, haplotype inference, Hardy-Weinberg equilibrium, and tag SNPs were also determined for these samples. RESULTS: CYP3A4*1B allele frequencies were 4% in Europeans and 82% in Africans. The CYP3A4*2 allele was found in neither population sample. CYP3A4*3 had an allele frequency of 2% in Europeans and 0% in Africans. The frequency of CYP3A5*3C was 94% in Europeans and 12% in Africans. No CYP3A5*6 variants were found in the European samples, but this allele had a frequency of 16% in the African samples. Allele frequencies and haplotypes show interethnic variation, highlighting the need to analyze clinically relevant SNPs and haplotypes in a variety of ethnic groups. CONCLUSION: Pyrosequencing is a versatile technique that could improve the efficiency of SNP analysis for pharmacogenomic research with the ultimate goal of pre-screening patients for individual therapy selection

Genetic polymorphisms of N-acetyltransferase 1 and 2 and risk of cigarette smoking-related bladder cancer

Aromatic amines from cigarette smoking or occupational exposure, recognized risk factors for bladder cancer, are metabolized by N-acetyltransferases (NAT). This study examined the association of (NAT) 1 and 2 genotypes with the risk of smoking-related bladder cancer. A total of 74 pathologically confirmed bladder cancer patients and 184 controls were serially recruited from the National Taiwan University Hospital. History of cigarette smoking and other risk factors for bladder cancer was obtained through standardized questionnaire interview. Peripheral blood lymphocytes were collected from each subject and genotyped for NAT1 and NAT2 by DNA sequencing and polymerase chain reaction-restriction fragment length polymorphism methods. Allele frequency distributions of NAT1 and NAT2 were similar between cases and controls. There was a significant dose–response relationship between the risk of bladder cancer and the quantity and duration of cigarette smoking. The biological gradients were significant among subjects carrying NAT1*10 allele or NAT2 slow acetylators, but not among NAT2 rapid acetylators without NAT1*10 allele. The results are consistent with the hypothesis that NAT1 and NAT2 might modulate the susceptibility to bladder cancer associated with cigarette smoking. © 1999 Cancer Research Campaig

Cytochrome P450 1A2 (CYP1A2) activity, mammographic density, and oxidative stress: a cross-sectional study

INTRODUCTION: Mammographically dense breast tissue is a strong predictor of breast cancer risk, and is influenced by both mitogens and mutagens. One enzyme that is able to affect both the mitogenic and mutagenic characteristics of estrogens is cytochrome P450 1A2 (CYP1A2), which is principally responsible for the metabolism of 17β-estradiol. METHODS: In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity, malondialdehyde (MDA) levels, and mammographic density. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Levels of serum and urinary MDA, and MDA–deoxyguanosine adducts in DNA were measured. Mammograms were digitized and measured using a computer-assisted method. RESULTS: CYP1A2 activity in postmenopausal women, but not in premenopausal women, was positively associated with mammographic density, suggesting that increased CYP1A2 activity after the menopause is a risk factor for breast cancer. In premenopausal women, but not in postmenopausal women, CYP1A2 activity was positively associated with serum and urinary MDA levels; there was also some evidence that CYP1A2 activity was more positively associated with percentage breast density when MDA levels were high, and more negatively associated with percentage breast density when MDA levels were low. CONCLUSION: These findings provide further evidence that variation in the activity level of enzymes involved in estrogen metabolism is related to levels of mammographic density and potentially to breast cancer risk

DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity

Oxidative stress and lipid peroxidation (LPO) induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods, These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA, To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters, In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coenzyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites, With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis