NO ASSOCIATION BETWEEN NAT1*15 AND BREAST CANCER, AND INTERACTION WITH RED MEAT

Acta Medica Mediterranea, 2017, 33: 89 NO ASSOCIATION BETWEEN NAT1*15 AND BREAST CANCER, AND INTERACTION WITH RED MEAT HIBA CHELOUTI *, KAMEL BOUZID **, MALIKA KHELIL * * Département de Biologie Cellulaire et Moléculaire, Faculté des Sciences Biologiques, Université des Sciences et Technologie Houari Boumediene, Alger, Algérie- ** Service Oncologie, Centre Pierre et Marie Curie, CHU-Mustapha Bacha, Alger, Algérie ABSTRACT Introduction: Breast cancer is a complex disease characterized by the accumulation and combination of multiple genetic and environmental factors. Indeed, many studies have suggested that among powerful pre-carcinogens involved in breast carcinogenesis derived from aromatic and heterocyclic amines. These are either directly detoxified or converted into carcinogens by N-acetyltransferases1 and 2. Both highly polymorphic isoenzymes are involved in the increased susceptibility to breast cancer. The objective of our work was to examine the effects of N-acetyltransferase 1*15 (NAT1*15) polymorphism and its relationship with red meat consumption and cigarettes smoking, on breast cancer risk. Materiel and methods: The C and T alleles were identified by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism analysis for 60 Algerian patients with BC and 60 women controls. Results: There was no association between NAT1*15 polymorphisms and Breast cancer. However, the red meat intake increased susceptibility to the disease (p=0.017, OR =5.537). Conclusion: Our results suggest that NAT1*15 polymorphisms would not be involved in the pathogenesis of breast cancer. However, the red meat intake would be a risk factor for breast cancer in our population. This risk can be attributed to the high fat contained in the red meat. Keywords: Genetic polymorphism; N-acetyltransferase 1; Red meat, Risk factor. DOI: 10.19193/0393-6384_2017_1_014 Received September 30, 2016; Accepted November 02, 2016 Introduction Aromatic and heterocyclic amines have been shown to induce tumors of the mammary glands (1, 2). These potent pre-carcinogens molecules, as 4- aminobiphenyl and 2-amino-methyl-6-phenylimidazo [4, 5-b] pyridine (PhlP), are common exposures in modern life, such as smoking, and in the diet, formed when red meat is cooked (3). The results on the association between cigarette smoking and breast cancer (BC) risk have been conflicting (4). Unlike this, several study have shown that red meat intake may be an important risk factor for BC (5). The balance between N and O-acetylation of these compounds depends on several factors, in particular on the N-acetyltransferase (NAT) activity (6-8). In humans, two NAT (NAT1 and NAT2) have been identified (9-11). Both NAT1 and NAT2 gene have been shown to be polymorphic (12). Several polymorphisms affecting NAT1 enzyme activity have been

90 Hiba Chelouti, Kamel Bouzid et Al reported (13). These variations have been implicated to various diseases, including cancer (14). Many epidemiological studies have evaluated the relationship between the rapid activity of NAT1 and BC risk. Indeed, no overall association was observed between NAT1*10 genotype and BC risk for Caucasians and African Americans (15). Conversely, the relationship between the absence of NAT1 activity and BC has not been, much, reported. NAT1*15 is characterized by a single nucleotide polymorphism (SNP) C559T in the NAT1 open reading frame. This mutation changed Arg187 by stop, results in the loss of 104 amino acids from the C-terminus of NAT1. Although the putative active site remains with the truncated protein (residues 47-l11), the C-terminus amino acids (residues 211-250) play an important role in enzyme stability, and residues 112-210 determine specificity (16). Therefore, this polymorphism is likely to severely impair or abrogate enzyme function (17). The aim of our work is to examined the effects of red meat consumption and cigarettes smoking on breast cancer risk and addressed the role of NAT1*15 polymorphism. Materials and methods Selection of population study The sample was composed of 60 patients collected at CPMC-Oncology service at the University Hospital Center Mustapha Bacha. Diagnosis of the disease was confirmed for each of our patients by the oncologists. The control group was composed of 60 healthy individuals. All patients and controls signed an informed consent and completed a questionnaire on their location, habits of consumption of red meat and exposure to cigarette smoke. This study was approved by the local ethics committee. NAT1 genotyping Genomic DNA was extracted from blood samples using the standard salting-out procedures and resuspended in Tris-EDTA buffer (18). The PCR-restriction fragment length polymorphism (RFLP) method described by Dietz et al. (19) was used to determine NAT1*15 polymorphism. The 138 base-pair fragments consisting of polymorphic site investigated was digested overnight at 37 C in a total volume of 20µl with 10 units of ScaI. Fragments were subjected to electrophoresis on a 4% agarose gel and visualized. Statistical analysis Demographic and Clinical characteristics for normally and non-normally distributed data were analyzed using two-tailed Student t and Mann- Whitney U test, respectively. An unconditional logistical regression model was performed to determine the effects of NAT1 polymorphisms and Red meat intake on breast cancer risk with results expressed as Odds Ratios (ORs) and their 95% confidence intervals (CIs) after adjusting for potential confounding variables. Findings were considered statistically significant at P values of less than 0.05. Results Characteristics of the subjects Table 1 shows the background characteristics of BC cases and control subjects. Cases were 2 years older than controls on average. There is no difference in the status of the menaupose in the two groups 32% of cancer patients had family history of cancer and none of the controls group. BC affected both breasts of the same proportion. The tumors presented an estrogen receptor in 83% of the cases. 60% of the patients lived in an urban area. About 52% of the patients are passive smokers, compared with only 33% of the controls. 14% of the patients consumed red meat more than once a week. Information for SNP The primary information for SNP is summarized in Table 2. The NAT1*15 allele contains a base substitution (C559T) in the protein encoding region of the NAT1 gene that introduces a stop codon, leading to the production of a truncated, inactive protein. The gene is localised in chromosome 8, in the position 18124395 and with regulome DB score 6. The minor allele frequency in our controls was similar to minor allele frequency in database. The observed genotype frequency for this polymorphism in the controls was in Hardy- Weinberg equilibrium. Univariate Logistic Regression Association between NAT1*15 polymorphism and BC was investigated first by univariate

No association between NAT1*15 and breast cancer, and interaction with red meat 91 analysis (Table 3). No association was observed between NAT1*15 allele C (OR=1.952, p=0.149), T (OR=1.333, p=0.401) and BC. No significant difference was observed between NAT1*15 genoype CC (OR=0.750, p=0.552), CT (OR=0.422, p=0.076), TT (OR=0.512, p=0.307) and BC. Whereas, Significant difference was noted between patients and controls regarding age (OR= 0.933, p=0.000) and menopause (OR=0.259, p=0.002) indicating a protective role of sexual hormone against BC. Characteristics Cases % Controls % P-value Number of Individuals (n) 60 60 - Age (mean ± SD) 63.31 ± 10.01 61.2± 16.22 0.42 Menopause Pre-menopausal 40.7 45.7 0.27 Post-menopausal 59.3 54.3 Family history of BC Yes 32.1 0 No 67.9 100 Localization of tumor Right breast 48.4 - - Left breast 51.6 - - Estrogen Receptors (ER) ER+ 83 - - ER- 17 - - Geographical location Urban 60.5 37.4 0.045 Rural 39.5 62.6 Smoking status Passive smoker 52.3 33.1 0.069 Genotyped SNPs NAT1 rs5030839 559 C>T Chromosome 8 Gene official symbol Haplotype (NAT1 Allele) Phenotype Amino Acid Change(s) NAT1 NAT1*15 Truncated protein/no enzyme activity R187Stop Chromosome Position (Genome Build 36.3) 18124395 Regulome DB Score a 6 Distance (p b ) 12500 705 TFBS b - RegPotential b 0 Conservation b 0.176 MAF c 0.003 nssnp - Stop codon value for HWEd test 0.736 Genotyping method e Y RFLP Table 2: Primary information for NAT1 rs5030839 C>T polymorphism. a: http://www.regulomedb.org/; b: TFBS: Transcription Factor Binding Site (http://snpinfo.niehs.nih.gov/snpinfo/snpfunc.htm); c: MAF: minor allele frequency for controls; d: HWE: Hardy Weinberg equilibrium; e: RFLP: Restriction Fragment Length Polymorphism; NAT1: N-acetyltransferase 1, SNP: Single Nucleotid Polymorphism, pb: pair base NAT1*15 (559C/T) P-value OR CI (%95) Genotype CC 0.552 0.75 0.290-1.937 CT 0.076 4.222 0.858-20.779 TT 0.307 0.512 0.142-1.850 Non smoker 47.7 66.9 Red Meat intake status Alleles C 0.149 1.952 0.787-4.840 T 0.401 1.333 0.682-2.608 no meat intake 3.4 10 Low meat intake 1 82 81 0.13 High meat intake 2 14.6 9 Table 1: Distribution of selected demographic variables and clinical carateristics of breast cancer cases and controls. Characteristics were compared using two-tailed Student t test (for normally distributed data) or by Mann-Whitney U test (for non-normally distributed data). 1: Less than 3 time a week; 2: More than 1 time per week. Bold values are statistically significant (p<0.05). BC: Breast Cancer, ER: Estrogen Receptor. Age 0 0.933 0.093-0.608 Menopause 0.002 0.259 0.108-0.621 Family history of BC 0.998 2.325 0.000-? Geographical location 0.046 2.523 1.016-6.262 Smoking status 0.07 2.207 0.938-5.194 Red meat intake 0 14.37 5.236-39.440 Table 3: Results of univariate analysis between breast cancer cases and controls according to demographic parameters, NAT1*15 genotypes and alleles. Characteristics were compared using Univariate Regression. p<0.05 was considered to indicate statistical significance. OR: Odd Ratio refers to BC. NAT1*15: N-acetyltransferase 1*15, OR: Odds Ratio, CI: Confidence Interval, BC: Breast Cancer.

92 Hiba Chelouti, Kamel Bouzid et Al In the current study, geographical location (OR= 2.523, p=0.046) and red meat intake (OR=14.370, p=0.000) presented risk factors for BC. When evaluating the parameters of smoking statuts (OR=2.207, p=0.070) and Family history of BC (OR=2.325, p=0.998) according to BC, there was no statistically difference. Multivariate Logistic Regression Multivariate logistic regression analysis was chosen to find the best independent predictors. In this analysis, the red meat intake appear as an independant risk factor for BC (p=0.017, OR=5.337) (Table 4). Discussion p- value OR CI (%95) Red Meat Intake 0.017 5.337 1.342-21.229 Table 4: : Results of multivariate analysis between breast cancer cases and controls according to demographic parameters, NAT1*15 genotypes and alleles. Adjusted for Age, Menopause, Geographical location, Smoker Status, Red meat intake, CC, TT. OR refer to risk of Breast Cancer. NAT1*15: N-acetyltransferase 1*15, OR: Odds Ratio, CI: Confidence Interval. Our study found an association between red meat consumption and BC risk, in accordance with the study of Cho et al. (20) who reports that red meat intake was associated with an increased risk of BC in premenopausal women. However, our population is young and we shown that red meat intake was associated with a higher risk of BC (OR=5.337). This can be explained in part by the fact that red meat is a source of carcinogens, such as heterocyclic amines, N-nitroso-compounds, and polycyclic aromatic hydrocarbons, which increase mammary tumors in animals and have been hypothesized to increase BC risk (21). In the other hand, several other studies (22, 23) attributed the risk factor effect of red meat to the high fat. In our study, we found an association between passive smoking and BC, in univariate regression. This has already been proven by Gamon et al. (24) but he suggests that effect is restricted to selected subgroups of women, such as those with long-term exposure from a smoking spouse. Despite the inconsistent results for this area, several studies have been recognized that cigarette smoking plays (25, 26) an important role in the risk of BC and explained this by the known mammary carcinogenicity of some tobacco smoke constituents, such as polycyclic aromatic hydrocarbons. The multivariate shows that the association between passive smoking and BC disappeared after adjustment for other risk factors, suggesting that passive smoking does not interact with these established risk factors. However, we observed no significant association of the NAT1 C559T polymorphism with risk of BC. This is in agreement with the study of Zheng et al. (27). The C559T substitution in the NAT1*15 allele causes an amino acid change from Arginin to Stop (17). An individual homozygous for C559T (R187stop) present in NAT1*15 showed undetectable NAT1 activity in human blood lysates (28) and recombinant expression of NAT1*15 in bacteria resulted in complete lack of NAT1 catalytic activity (29). The lack of association was similar within strata of age, menopausal status and other BC risk factors. Conclusion Our data showed that the consumption of redmeat is a risk factor for BC. These findings have potential public health implications in preventing BC and should be evaluated further by increasing the sample rate. Lack of association between NAT1*15 polymorphism and BC suggest that other allelic variant should be involved. To the best of our knowledge, this is the first study of the possible association between NAT1*15 and BC in an Algerian population. References 1) Shirai T, Tamano S, Sano M, Masui T, Hasegawa R, Ito N. Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo [4,5b] pyridine (PhIP) in rats: dose response studies. Princess Takamatsu Symp 1995; 23: 232-9. 2) Nagao M, Ushijima T, Wakabayashi K, Ochiai M, Kushida H, Sugimura T. Dietary carcinogens and mammary carcinogenesis. Cancer 1994; 74: 1063-1069. 3) Layton DW, Bogen KT, Knize M, Hatch FT, Johnson VM, Felton JS. Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis 1995; 16: 39-52. 4) Palmer JR, Rosenberg LR. Cigarette smoking and the risk of breast cancer. Epidemiol Rev 1993, 15: 145-162. 5) Zheng W, Gustafson DR, Sinha R, Cerhan JR, Moore D, Hong CP. Well-done meat intake and the risk of breast cancer. J Natl Cancer Inst (Bethesda) 1998; 90: 1724-1729.

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