Grantee Research Project Results
Final Report: Interindividual Variations in Genetic Polymorphisms as Risks for Colorectal Cancer
EPA Grant Number: R825280Title: Interindividual Variations in Genetic Polymorphisms as Risks for Colorectal Cancer
Investigators: Lang, Nicholas P. , Ambrosone, Christine , MacLeod, Stewart , Kadlubar, Fred F. , Stotts, Craig
Institution: University of Arkansas for Medical Sciences
EPA Project Officer: Aja, Hayley
Project Period: November 15, 1996 through November 14, 1999
Project Amount: $538,785
RFA: Role of Interindividual Variation in Human Susceptibility to Cancer (1996) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
The objectives of this study were to investigate whether interindividual differences in susceptibility to colon cancer are related to genetically based differences in polymorphic enzymes responsible for the metabolism of heterocyclic aromatic amines (HAA) and bicyclic aromatic amines (AA). These compounds are found in cooked foods, tobacco smoke and other environmental sources. To exert their carcinogenic effects, HAA and AA must be converted to reactive, electrophilic forms. These bind to DNA, forming mutagenic adducts which are thought to be the initial step in carcinogenesis. The quantity of adducts produced, and the resultant extent of tumor induction, is proportional to the quantity of HAA that are converted to the reactive metabolite, which, in turn, depends on the quantity and activity of the enzymes involved in activation as well as in detoxification of these reactive metabolites.Summary/Accomplishments (Outputs/Outcomes):
The major objective of this study was to determine genotypes for five carcinogen metabolizing enzymes for colorectal cancer cases and control subjects. This information was used to determine whether the polymorphic forms of these enzymes are part of the genetic basis for interindividual differences in colorectal cancer risk. Genotypes for GSTM1, GSTT1, CYP1A1, NAT1 and GSTP1 were determined for study subjects by the use of PCR based assays. In addition, the phenotype for sulfotransferase 1A1 (SULT1A1) was determined and a genotype assay was developed to determine allele frequencies for a polymorphism in the SULT1A1 gene.
GSTM1 Genotype. The genetic polymorphisms in the GSTM1 and the GSTT1 genes consist of gene deletions that result in the existence of a null allele. Individuals who are homozygous for the null allele lack GSTM1 or GSTT1 enzyme function. Although GST activity is usually involved in the detoxification of reactive metabolites, GSTT1 has been implicated in the activation of halogenated alkanes to carcinogenic metabolites. We found that colorectal cancer patients were significantly more likely to have the GSTM1 null allele than healthy control subjects (59.1 percent versus 48.2 percent, respectively, P=0.047). This corresponds to an odds ratio of 1.55 (95 percent CI 1.032-2.40), indicating that individuals with the GSTM1 null allele are 1.55 times more likely to be colorectal cancer cases compared to individuals who have one or both GSTM1 alleles. Allele frequencies for the GSTT1 polymorphism were not significantly different between colorectal cancer cases and control subjects in this study, with 18 percent of cases as well as controls having the GSTT1 null allele.
In a series of related experiments, we used competitive DNA binding experiments to show that most of the known human GSTs including A2-2, P1-1, M1-1 T1-1 and T2-2 have low activity toward the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), however, GSTA1-1 is an efficient catalyst for the detoxification of PhIP. A genetic polymorphism in the 5'-regulatory region of the hGSTA1 gene correlates with expression levels of GSTA1 in human liver. We have determined the genotype for the GSTA1 polymorphism for 100 Caucasian colorectal cancer patients and 226 control subjects. This data demonstrated that colorectal cancer cases were more likely to be homozygous for the hGSTA1*B allele compared to control subjects (24.0 percent versus 13.7 percent, respectively, P=0.04), corresponding to an odds ratio for colorectal cancer risk of 2.0 (95 percent CI 1.0-3.7) compared to heterozygous individuals (Coles et al. in press). Thus, individuals who are homozygous for the hGSTA1*B allele and who would be predicted to have lower levels of hGSTA1 expression have an elevated risk of developing colorectal cancer, presumably as a result of inefficient detoxification of N-acetoxy PhIP.
CYP1A1 Genotype. CYP1A1 catalyzes the bioactivation of a number of environmental and dietary procarcinogens, therefore differences in the activity or expression levels of this enzyme could be an important factor in colorectal cancer risk. In this proposal we explored the effects of a single base pair polymorphism from A to G in exon 7 of the CYP1A1 gene that results in an amino acid change from isoleucine to valine in the CYP1A1 protein. The allele frequencies for 107 colorectal cancer cases and 324 control subjects were determined by a designed RFLP-PCR assay (2). We found no significant differences in CYP1A1 allele distribution between these two groups; 97.2 percent of cases and 93.8 percent of controls were homozygous for the Ile/Ile genotype. Only 2.8 percent of cases and 6.2 percent of controls were heterozygous (ile/val) for this polymorphism. None of the subjects in this study population were homozygous for the variant valine allele. Since the val allele is so rare in this population, its effect on colorectal cancer risk would not be evident in a study of this size. We found that the genotype for CYP1A1 influenced CYP1A2 phenotype. Individuals who were homozygous for the CYP1A1 exon 7 ile allele had mean CYP1A2 activity of 9.02, whereas heterozygous individuals had mean CYP1A2 activity of 7.6. This relationship did not achieve statistical significance because of the small number of individuals who are heterozygous for this polymorphism.
NAT1 Genotype. The NAT1 has been found to contain a polymorphism of the polyadenylation signal in the 3' untranslated region of the gene. The polyadenylation signal sequence variant termed NAT1*10 has been associated with an up to 2-fold higher NAT1 activity compared to the more common NAT1*4 allele. The NAT1*10 allele has been associated with an increased risk for development of colon cancer (3, 4). We have determined the NAT1 genotype for 110 colorectal cancer patients and 326 control subjects. We found that 58.2 percent of cancer cases were homozygous for the most common NAT1*4 allele, as were 54.3 percent of control subjects. 40.9 percent of colorectal cancer patients had one or two high activity NAT1*10 alleles, compared to 45.4 percent of control subjects. We found no significant relationship between NAT1 genotype and colorectal cancer risk in the subjects in this study.
GSTP1 Genotype. The glutathione S-transferases are able to detoxify a large variety of reactive xenobiotics and metabolites that damage DNA. The GSTP1 gene contains a polymorphism consisting of a single base pair change from A to G at nucleotide 313 of the cDNA. This polymorphism results in an amino acid change from valine to isoleucine at codon 105 and causes the valine variant to have altered specific activity and affinity for electrophilic substrates (5). In order to determine whether the this polymorphism influences the risk of colorectal cancer, the GSTP1 genotype was determined for colorectal cancer cases and control subjects by a modification of the method of Harries et al.(6). We found no statistically significant association between GSTP1 genotype and the risk of colorectal cancer. We found that 31.9 percent of colorectal cancer patients and 36 percent of control subjects were homozygous for the GSTP1 ile105 allele. Heterozygotes were nearly equally represented between the groups, with 58 percent of cases and 57.5 percent of controls having both GSTP1 alleles. Ten percent of colorectal cancer patients and 6.5 percent of control subjects were homozygous for the val allele.
Sulfotransferase 1A1 (ST1A3) Phenotype Assay, Genotype-Phenotype Correlation. The role of the sulfation pathway in susceptibility to colorectal cancer has been explored by the development of a SULT1A1 phenotype assay that measures sulfotransferase activity in platelet cytosol. A genetic polymorphism in the SULT1A1 gene has been discovered by our group (8) and others (9). We have developed a genotyping assay for a genetic polymorphism that we found was associated with reduced sulfotransferase activity in platelets. We have determined that the genotype for this polymorphism is correlated with sulfotransferase activity in a population of 124 individuals tested. A dose dependent relationship between SULT1A1 genotype and phenotype was discovered; individuals who were homozygous for the SULT1A1*2/*2 genotype had lower platelet sulfotransferase activity. Individuals homozygous for the SULT1A1*1/*1 allele had the highest activity, while heterozygotes (SULT1A1*1/*2) tended to have intermediate activity (10). This data demonstrates a statistically significant relationship between SULT1A1 genotype and phenotype, however, analysis of variance shows that the genetic polymorphism accounts for approximately 30 percent of the phenotypic variation observed. Also, when probit analysis is conducted on each genotype individually, it shows that the stratified phenotype is not normally distributed, indicating that there may be other genetic determinants of SULT1A1 activity.
Metabolism of 2-Amino-1-Methyl-6-Phenylimidazo{4,5-b}Pyridine(PhIP).
PhIP is the most mass abundant heterocyclic amine in cooked meat and fish, and
has proven to be a potent colon, breast and prostate carcinogen in animals (11,
12). To better understand the interactions of metabolic pathways of HAA
activation and detoxification, we conducted an IRB approved pharmacokinetic
pilot project that involved the administration of 14C-PHiP to subjects scheduled
for colon resection. Five colorectal cancer patients who were scheduled for
colorectal surgery were recruited into the concurrent NIH study (NIH grant
5RO1CA55751) which provides for subject recruitment for this EPA grant.
Phenotypes for CYP1A2, NAT2 and SULT1A1 were determined as well as genotypes for
CYP1A1, NAT1, GSTM1 and GSTT1. The metabolic fate of PhIP was determined by
analysis of tissue and excretion products by GC-Mass spectrometry in
collaboration with James Felton and co-workers at Lawrence Livermore
Laboratories. These experiments showed that PhIP, administered to humans in a
dietary relevant dose, is activated in to a species that can bind albumin,
hemoglobin and lymphocyte DNA. PhIP also is bioavailable to the human colon,
where it can form both DNA and protein adducts (13). The most abundant urinary
metabolite in all subjects tested was N-hydroxy-PhIP-glucuronide (14), which
indicates the importance of CYP1A2 in the activation of PhIP to N-OH-PhIP and
also identifies UGT-glucuronosyl transferases in the detoxification of this
compound. Another major urinary metabolite was identified as 4'-PhIP-SO4,
implicating sulfotransferases in PhIP detoxification. SULT 1A1 phenotype for
these subjects was positively correlated with 4'-PhIP-SO4 levels in urine. CYP1A2 phenotype was negatively
correlated with PhIP serum levels at 1hr post administration, but was positively
correlated with urinary N-OH-PhIP levels at 0-4hrs (15). These data confirm the
importance of CYP1A2 in the activation of PhIP as well as the role of SULT1A1 in
the biotransformation of this metabolite. Continuation of this series of
experiments will allow us to determine the influence of differences in phenotype
and genotype in carcinogen metabolizing enzymes on the metabolism of PhIP and
other dietary HAA. A larger number of study subjects will be necessary to
identify the metabolic differences in HAA metabolism among individuals that are
predictive of individual susceptibility to colorectal cancer caused by exposure
to these dietary carcinogens.
References:
Arand M, Muhlbauer R, Hengstler J, et al. A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms. Analytical Biochemistry 1996;236(1):184-6.
Oyama T, Mitsudomi T, Kawamoto T, et al. Detection of CYP1A1 gene polymorphism using designed RFLP and distributions of CYP1A1 genotypes in Japanese. International Archives of Occupational & Environmental Health 1995;67(4):253-6.
Bell DA, Badawi AF, Lang NP, Ilett KF, Kadlubar FF, Hirvonen A. Polymorphism in the N-acetyltransferase 1 (NAT1) polyadenylation signal: association of NAT1*10 allele with higher N-acetylation activity in bladder and colon tissue. Cancer Research 1995;55(22):5226-9.
Badawi AF, Hirvonen A, Bell DA, Lang NP, Kadlubar FF. Role of aromatic amine acetyltransferases, NAT1 and NAT2, in carcinogen-DNA adduct formation in the human urinary bladder. Cancer Research 1995;55(22):5230-7.
Zimniak P, Nanduri B, Pikula S, et al. Naturally occurring human glutathione S-transferase GSTP1-1 isoforms with isoleucine and valine in position 104 differ in enzymic properties. European Journal of Biochemistry 1994;224(3):893-9.
Harries LW, Stubbins MJ, Forman D, Howard GC, Wolf CR. Identification of genetic polymorphisms at the glutathione S-transferase Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis 1997;18(4):641-4.
Frame LT, Ozawa S, Nowell SA, et al. A simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols. Drug Metabolism & Disposition 2000;28(9):1063-8.
Ozawa S, Tang YM, Yamazoe Y, Kato R, Lang NP, Kadlubar FF. Genetic polymorphisms in human liver phenol sulfotransferases involved in the bioactivation of N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines. Chemico-Biological Interactions 1998;109(1-3):237-48.
Raftogianis RB, Wood TC, Otterness DM, Van Loon JA, Weinshilboum RM. Phenol sulfotransferase pharmacogenetics in humans: association of common SULT1A1 alleles with TS PST phenotype. Biochemical & Biophysical Research Communications 1997;239(1):298-304.
Nowell S, Ambrosone C, Ozawa S, et al. Relationship of phenol sulfotransferase activity (SULT1A1) genotype to sulfotransferase phenotype in platelet cytosol. Pharmacogenetics 2000;10:789-797.
Ito N, Hasegawa R, Sano M, et al. A new colon and mammary carcinogen in cooked food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Carcinogenesis 1991;12(8):1503-6.
Shirai T, Sano M, Tamano S, et al. The prostate: a target for carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) derived from cooked foods. Cancer Research 1997;57(2):195-8.
Dingley KH, Curtis KD, Nowell S, Felton JS, Lang NP, Turteltaub KW. DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Epidemiology, Biomarkers and Prevention 1999;8(6):507-12.
Malfatti MA, Kulp KS, Knize MG, et al. The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans. Carcinogenesis 1999;20(4):705-13.
Lang NP, Nowell S, Malfatti MA, et al. In vivo human metabolism of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Cancer Letters 1999;143(2):135-8.
Conclusions:
Future Objectives. Our experiments concerning the metabolism of PhIP
have demonstrated the importance of GSTs, UDP-glucuronyltransferases and
sulfotransferases in the detoxification of dietary HAA. The data generated by
this research also has identified a number of genetic factors that contribute to
differences in individual susceptibility to colorectal cancer. We plan further
studies to identify the enzymatic isoforms that are responsible for these
activities both in colorectal tissue and in the liver. Many members of these
enzyme superfamilies are highly inducible, therefore greater knowledge of the
mechanism of induction of these chemoprotective enzymes will be useful in future
intervention and cancer prevention studies. A thorough analysis of dietary
factors that function as inducers and repressors of these enzymes will be
necessary for the formulation of future anti-cancer dietary recommendations and
public health policy.
The sample collection and preliminary data from this
grant have allowed the identification of a potential genetic basis for
differences in CYP1A2 phenotype. CYP1A2 activity is presently measured by HPLC
analysis of caffeine metabolites, which requires that subjects receive a dose of
caffeine and collect a urine sample after four hours. This is inconvenient for
study subjects and is quite labor intensive for the laboratory. Sequencing of
the CYP1A2 gene from individuals with both high and low CYP1A2 phenotype has
identified a polymorphism that correlates with CYP1A2 expression levels. A PCR
based assay has been developed for determining individual genotype at this locus
that may be useful in identifying individuals with low or high CYP1A2 activity.
If this CYP1A2 genotype approach is successful, we will be able to determine
CYP1A2 activity indirectly using DNA isolated from a blood sample. Our analysis
of CYP1A2 activity by the HPLC based caffeine phenotype assay has lead to the
discovery that one of the caffeine metabolite ratios corresponds to CYP2A6
activity, and that this activity is polymorphic in this study population
(Nowell, et al.). We plan to collaborate with colleagues at Lawrence Livermore
Laboratories to sequence the CYP2A6 gene from individuals who have high and low
activity in order to discover whether there is a genetic basis for this
polymorphism. This polymorphism in CYP2A6 activity may be a significant new
metabolic variation that may have an impact on the incidence of colorectal
cancer.
Future Analysis. The data from this EPA grant combined with the dietary data generated from the NIH Grant (5RO1CA55751: Acetylation and N-Oxidation in Colorectal Cancer) will allow further analysis to determine the influence of genetic polymorphisms on heterocyclic amine metabolism and colorectal cancer risk. Heterocyclic amine exposure data is currently being analyzed with the help of Rashmi Sinha at NCI. Other dietary data, including the intake of dietary compounds that induce the activity of enzymes that are capable of detoxifying reactive heterocyclic amine metabolites, will be analyzed after the HAA data is completed.
Journal Articles on this Report : 10 Displayed | Download in RIS Format
Other project views: | All 24 publications | 10 publications in selected types | All 10 journal articles |
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Type | Citation | ||
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Coles B, Nowell SA, MacLeod SL, Sweeney C, Lang NP, Kadlubar FF. The role of human glutathione S-transferases (hGSTs) in the detoxification of the food-derived carcinogen metabolite N-acetoxy-PhIP, and the effect of a polymorphism in hGSTA1 on colorectal cancer risk. Mutation Research 2001 482(1-2):3-10. |
R825280 (Final) |
not available |
|
Dingley KH, Curtis KD, Nowell S, Felton JS, Lang NP, Turteltaub KW. DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Cancer Epidemiology, Biomarkers & Prevention 1999;8(6):507-512. |
R825280 (Final) |
not available |
|
Frame LT, Ozawa S, Nowell SA, Chou H-C, DeLongchamp RR, Doerge DR, Lang NP, Kadlubar FF. A simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols. Drug Metabolism and Disposition 2000;28(9):1063-1068. |
R825280 (Final) |
not available |
|
Lang NP, Nowell S, Malfatti MA, Kulp KS, Knize MG, Davis C, Massengill J, Williams S, MacLeod S, Dingley KH, Felton JS, Turteltaub KW. In vivo human metabolism of [2-C-14]2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP). Cancer Letters 1999;143(2):135-138 |
R825280 (Final) |
not available |
|
MacLeod SL, Nowell S, Massengill J, Jazieh A, McClure G, Plaxco J, Kadlubar FF, Lang NP. Cancer therapy and polymorphisms of cytochromes P450. Clinical Chemistry and Laboratory Medicine 2000;38(9):883-887. |
R825280 (Final) |
not available |
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Malfatti MA, Kulp KS, Knize MG, Davis C, Massengill JP, Williams S, Nowell S, MacLeod S, Dingley KH, Turteltaub KW, Lang NP, Felton JS. The identification of [2-C-14]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans. Carcinogenesis 1999;20(4):705-713 |
R825280 (Final) |
not available |
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Nowell SA, Massengill JS, Williams S, Radominska-Pandya A, Tephly TR, Cheng ZQ, Strassburg CP, Tukey RH, MacLeod SL, Lang NP, Kadlubar FF. Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved. Carcinogenesis 1999;20(6):1107-1114 |
R825280 (Final) |
not available |
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Nowell S, Ambrosone CB, MacLeod SL, Williams S, Mrackova G, Plaxco J, Ozawa S, Kadlubar FF, Lang NP. Relationship of phenol sulfotransferase (SULT1A1) genotype to sulfotransferase activity phenotype in platelet cytosol. Pharmacogenetics 2000;10:789-797. |
R825280 (Final) |
not available |
|
Ozawa S., Tang Y.M., Yamazoe Y., Kato R., Lang N.P. and Kadlubar F.F. Genetic polymorphisms in human liver phenol sulfotransferases involved in the bioactivation of N-hydroxy derivatives of carcinogenic arylamines and heterocyclic amines. Chemico-Biological Interactions, Volume 109, Issues 1-3, 20 February 1998, Pages 237-248. |
R825280 (Final) |
not available |
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Turteltaub KW, Dingley KH, Curtis KD, Malfatti MA, Turesky RJ, Garner RC, Felton JS, Lang NP. Macromolecular adduct formation and metabolism of heterocyclic amines in humans and rodents at low doses. Cancer Letters 1999;143(2):149-155. |
R825280 (Final) |
not available |
Supplemental Keywords:
exposure, risk, cancer risk, colorectal cancer risk, risk assessment, human health, bioavailability, metabolism, sensitive populations, carcinogen, population, enzymes, diet, metabolism, genetic predisposition, genetic polymorphisms, susceptibility., Health, Risk Assessments, Disease, Disease & Cumulative Effects, cancer risk, health effects, risk assessment, immune system effects, vulnerability, interindividual variations, colorectal cancer, gene-environment interaction, enzyme systems, human exposure, carcinogens, susceptibility, genetic polymorphisms, environmental stressors, environmental toxicant, harmful environmental agents, cancer prevention, cancer risk assessment, biological markers, exposure assessmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.